A facile and novel synthesis of N2-, C6-substituted pyrazolo[3,4- d ]pyrimidine-4 carboxylate derivatives as adenosine receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2015.01.046

An efficient synthetic procedure was adopted to synthesize a series of new molecules containing the pyrazolo[3,4-d]pyrimidine (PP) scaffold, which have been evaluated as promising human adenosine receptor (AR) antagonists. The effect of substitutions at t

Full text of DOI:10.1016/j.ejmech.2015.01.046

European Journal of Medicinal Chemistry 92 (2015) 784e798
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
2
6
A facile and novel synthesis of N -, C -substituted pyrazolo[3,4-d]
pyrimidine-4 carboxylate derivatives as adenosine receptor
antagonists
G. Venkatesan , P. Paira , S.L. Cheong , S. Federico , K.N. Klotz , G. Spalluto c
G. Pastorin
a
a
b
a
c
,
*
d
, *
,
a, *
Department of Pharmacy, National University of Singapore, 3 Science Drive 2, Singapore 117543, Singapore
b
c
Pharmaceutical Chemistry Division, School of Advance Science, Vellore Institute of Technology University, Vellore 632014, Tamilnadu, India
Dipartimento di Scienze Chimiche e Farmaceutiche, Universit aꢀ degli Studi di Trieste, Piazzale Europa 1, Trieste 34128, Italy
Institut für Pharmakologie und Toxikologie, Universit a€ t Würzburg, Versbacher Strasse 9, Würzburg 97078, Germany
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 November 2014
Received in revised form
An efficient synthetic procedure was adopted to synthesize a series of new molecules containing the
pyrazolo[3,4-d]pyrimidine (PP) scaffold, which have been evaluated as promising human adenosine
receptor (AR) antagonists. The effect of substitutions at the N , C and C6 positions of PPs on the affinity
2
4
21 January 2015
and selectivity towards the adenosine receptors were explored. Most of the pyrazolo[3,4-d]pyrimidine-4-
Accepted 22 January 2015
Available online 22 January 2015
carboxylates displayed from moderate to good affinity at the human A
low micromolar range of K values (K hA M). In particular, compounds 60 and 62 dis-
AR ¼ 0.7e34
played good affinity at the hA AR (60, K hA M and 62, K hA M) and selectivity
AR ¼ 2.2
3 3
AR (hA AR), as indicated by the
i
i
3
m
3
i
3
m
i
3
AR ¼ 2.9
m
Keywords:
towards the other AR subtypes (60, >46-fold selective and 62, >34-fold selective, respectively). In view of
Pyrazolo[3,4-d]pyrimidine
Carboxylate derivatives
Adenosine receptor antagonists
Homology modeling
these results, these novel PP analogues were docked both in the crystallographic structure of the hA2AAR
and in a homology model of the hA AR in order to support the structure activity relationship (SAR)
3
analysis. These preliminary results demonstrated that pyrazolo[3,4-d]pyrimidine can be considered a
Molecular docking
Structure-affinity relationship
promising scaffold to obtain new molecules with potent hA AR antagonist activity.
3
©
2015 Elsevier Masson SAS. All rights reserved.
1
. Introduction
Nevertheless, the ubiquitous distribution of ARs in mammalian cell
types, the existence of four distinct subtypes together with the
variability of physiological responses indicate the importance of
obtaining potential ligands that are highly selective in their action
[6,7].
Adenosine is a nucleoside made up of adenine attached to a
9
ribose moiety through a
b-N -glycosidic linkage. In our body,
adenosine acts as an endogenous modulator that regulates many
physiological processes through activation of specific human
adenosine receptors (hARs) [1e3]. The ARs are generally catego-
3
Among the four AR subtypes, the A receptor has been shown to
be involved in several patho-physiological conditions that occur in
the central nervous and cardiovascular systems, as well as in in-
flammatory disorders and cancer [8]. Several research groups have
made intense efforts in exploring potent and selective ligands for
1 3
rized into four subtypes, namely A , A2A, A2B and A ARs [4,5]. All of
these ARs possess their own distinct tissue localization, biochem-
istry and pharmacological function. The diverse physiological
functions regulated by the ARs highlight the significant benefits of
developing new therapeutics to modulate these receptors.
the A
classes of compounds with various chemical scaffolds have been
successfully synthesized and tested as A AR antagonists [8,9]. In
3
AR subtype over the past two decades. To date, different
3
particular, our group has focused on the design and synthesis of
derivatives bearing the tricyclic pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]
pyrimidine (PTP) scaffold (Compound 1, Fig. 1), which has resulted
in series of compounds with very good binding affinity at the
3 3
Abbreviations: hA AR, human A adenosine receptor; SAR, structure-activity
relationship; PTP, pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine; PP, pyrazolo[3,4-
d]pyrimidine.
3
hA AR and high selectivity against the other AR subtypes [10e17].
*
Corresponding authors.
E-mail address: phapg@nus.edu.sg (G. Pastorin).
However, in the case of the PTP scaffold and also in most of the
http://dx.doi.org/10.1016/j.ejmech.2015.01.046
223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
0

G. Venkatesan et al. / European Journal of Medicinal Chemistry 92 (2015) 784e798
785
3
Fig. 1. Examples of hA AR antagonists.
tricyclic heterocyclic derivatives described in the literature, they are
associated with a number of limitations such as complex synthetic
schemes, time-consuming reactions, low yields, as well as high
molecular weight and lipophilicity which affect the ADME prop-
erties [10e13,18]. As such, these observations suggest the need to
extend further investigations on different heterocyclic structures in
order to overcome these limitations while maintaining or
improving the affinity and selectivity towards the hA
In our initial attempt to introduce some structural diversity in
the main PTP scaffold without loosing affinity at the hA AR, we
3
AR.
3
II
investigated a series of Ru -arene complexes containing the (PP)
scaffold (Compound 2, Fig. 1) as chelating ligand, with the aim to
explore the effect of altering bulky groups at the 2-aryltriazole
3
Fig. 2. Pyrazolo[3,4-d]pyrimidines (PP) as hA AR antagonists.
position of the PTP scaffold towards binding affinity at the hA
and to overcome the solubility limitations of the PTP analogues
19]. From the study, it was noticed that the newly synthesized PP-
3
AR,
with easier synthetic procedures and better ADME properties than
the potent tricyclic hA AR antagonists reported in the literature
18].
3
[
[
II
Ru -arene complexes displayed good aqueous solubility and sta-
II
bility. However, the sterically hindered Ru -arene complexes
exhibited affinity in the high micromolar range at the hA
3
AR and
2. Results and discussion
poor selectivity towards the other AR subtypes. In a more recent
study we have applied a different molecular simplification strategy
2.1. Chemistry
[20]. The pyrazolo[3,4-d]pyrimidine scaffold was maintained
because it represents the core domain of PTP, which interacts with
the AR binding pocket. Furthermore, we have introduced various
substituents with different lipophilicity and steric hindrance at the
In this new series of pyrazolo[3,4-d]pyrimidine (PP) derivatives,
we have rationally designed and incorporated various substituents
2
6
at the N (lipophilic alkyl and aralkyl chains), C (amino, substituted
benzamido and phenylacetamido groups) and C4 (ester sub-
stituents) positions of the bicyclic scaffold in order to examine their
effects on the binding affinity and selectivity profiles. Based on the
synthetic steps illustrated in Schemes 1 and 2, the new series of 6-
substituted ethyl 2-(ar)alkyl-2H-pyrazolo[3,4-d]pyrimidine-4-
carboxylate derivatives (42e69) have been successfully synthe-
sized and characterized.
2
8
N
position, which corresponds to the N position of the PTP nu-
cleus and where small alkyl groups gave good affinity at the hA AR
21]. Benzamido or phenylacetamido moieties were inserted at the
3
[
C
6
5
position, such as at the C position of the PTP scaffold [22] in
AR. Finally, at the
order to confer potency and selectivity at the hA
3
4
C position, a chlorine atom was chosen for its restricted rotation,
with the aim to enable the pyrazolo[3,4-d]pyrimidine to attain the
same binding pose of PTP (Compound 3, Fig. 1) [20]. However, also
in this case we obtained derivatives with affinity in the high
micromolar range and displaying a poor selectivity profile. Sur-
prisingly, a structural modification on the 4-chloro group through
substitution with the rotatable ethyl ester substituent successfully
As reported in literature, the alkylation of 3-amino-4-pyrazole
carbonitrile was carried out with alkyl or aralkyl iodide or bro-
mide in dry DMF using dry potassium carbonate as a weak base,
1
which led to N substituted regioisomer (6e9) as a major product
[10,19,20].
1
improved the binding affinity and selectivity profiles at the hA
of the new derivatives [20]. In fact, compounds 4 and 5 displayed
affinities at the hA AR in the low or even submicromolar range (4,
hA M and 5, K hA AR ¼ 0.9 M) (Fig. 2).
AR ¼ 1.3
In view of the good results obtained in terms of affinity and
selectivity at the hA AR, in the present study we decided to expand
3
AR
The 3-amino-N -substituted-4-pyrazolo carbonitriles (6e9)
were then hydrolysed to corresponding carboxamides (10e13)
under acidic conditions at low temperature for 30 min, followed by
stirring at ambient conditions for 5 h (Scheme 1). The conversion of
3
K
i
3
m
i
3
m
1
nitrile to amide was confirmed by H NMR analysis and two broad
3
singlets were observed for CONH proton as specified in the liter-
2
the ethyl 4-carboxylate-PP series (46e69). We have synthesised
ature [23e26]. Among the carboxamide derivatives (10e13), com-
new PP analogues, where the ethyl ester moiety was maintained at
pound 10 has been reported in the literature [27] and compound 11
is commercially available.
4
2
6
the C position, while N alkyl and C amido substituents (that were
8
5
present at the equivalent N and N position of PTP derivatives
21,22] were introduced in order to explore their effect in terms of
affinity and selectivity at the hA AR. Overall, the main aim of the
project was to synthesize PP derivatives with good affinity and
selectivity at the hA subtype in order to obtain hA AR antagonists
Yamazaki and collaborators have described the synthesis of
guanosine derivatives by condensation of 4-cyanamido-1H-imid-
azole-5-carboxamide with benzoyl isothiocyanate, followed by
methylation and ring closure [28]. Similar synthetic steps were
adopted in our scheme for the synthesis of benzoylthiouriedo
[
3
3
3

786
G. Venkatesan et al. / European Journal of Medicinal Chemistry 92 (2015) 784e798
ꢀ
Scheme 1. General procedure for the synthesis of the 6-substituted ethyl 2-(ar)alkyl-2H-pyrazolo[3,4-d]pyrimidine-4-carboxylate derivatives. Reagents: (a) conc. H
2
SO
4
, 0 C to rt,
ꢀ
5
2
h; (b) C6H5CONCS, acetone, reflux, 12 h; (c) CH
4 h; (g) NaCN, p-toluene sulfinate sodium, DMF, 80 C, 2 h; (h) K
3
I, 0.1 N NaOH, rt, 3 h; (d) 2% aq. NH
3
, DMF, 120 C sealed tube, 3 h; (e) 1 N NaOH, reflux, 12 h; (f) POCl
3
, dimethylaniline, reflux,
ꢀ
2
CO
3
, 30% H
2
O
2
, rt, 1 h; (i) EtOH, conc. H
2
4
SO , reflux, 12 h.
Scheme 2. General procedure for the synthesis of the 6-substituted ethyl 2-(ar)alkyl-2H-pyrazolo[3,4-d]pyrimidine-4-carboxylate derivatives. Reagents : (a) substituted benzoyl
ꢀ
ꢀ
chloride, DIPEA, toluene, 120 C, reflux, 24 h ; (b) phenyacetyl chloride, DIPEA, toluene, 120 C, reflux, 24 h.

G. Venkatesan et al. / European Journal of Medicinal Chemistry 92 (2015) 784e798
787
derivatives 14e17 with some modifications. As per methods re-
ported in the literature, the carboxamide (10e13) was initially
reacted with benzoyl isothiocyanate in water at room temperature.
However, the corresponding benzoylthioureido derivatives were
Instead, disubstituted derivatives (61e65) were prepared under
standard conditions, using 6 equivalents of substituted benzoyl
1
chlorides and DIPEA. Final derivatives were characterized by
NMR, C NMR, HMQC NMR and ESI-Mass analysis.
H
13
not obtained. Nevertheless, when the reaction was carried out in
ꢀ
5
0% aqueous acetone at high temperature (80e100 C), thioureido
2.2. Structure-affinity relationship study
derivatives were obtained with low yield, probably due to partial
decomposition of benzoyl isothiocyanate in aqueous conditions. As
such, the reaction was performed in dry acetone, followed by
condensation with benzoyl isothiocyanate under reflux conditions
for 12 h. Upon cooling, the benzoylthioureido derivatives (14e17)
were obtained as yellow solids with good yields.
It is known that the methylmercapto group activated by the
electron-withdrawing nature of benzoyl group would be more
reactive. Hence, the methylation reaction was performed by treat-
ing intermediate compounds 14e17 with methyliodide in aqueous
sodium hydroxide solution at ambient temperature for 3 h to
obtain methylthio derivatives (18e21). After adjusting the pH to 6,
the corresponding carbamidothioates (18e21) were obtained as
white solids.
Pharmacological evaluation was performed for the new series of
pyrazolo[3,4-d]pyrimidine-4-carboxylate to examine their affinity
at all four AR subtypes. The receptor binding affinities of com-
1 3
pounds 38e69 were determined at the human A , A2A, A receptors,
while the inhibition of adenylyl cyclase activity was determined for
the human A2B receptors (Table 1). Also compounds 4 and 5,
recently reported in our work on the PP series [20] were included in
Table 1 as they are structurally related to the new synthesised de-
rivatives of this study and could help to make a more detailed SAR
profile of PP derivatives.
Most of the synthesized derivatives showed affinity in the low
micromolar range for at least one of the AR subtypes. Their selec-
2
4
tivity profile is regulated by substituents present at the N , C and
6
Subsequently, the nucleophilic substitution of methylmercapto
C
position. Indeed, most of the pyrazolo[3,4-d]pyrimidine-4-
carboxylates displayed from moderate to good affinity at the hA
receptors as indicated by the low micromolar range of K values (K
hA M) and with some compounds displaying high
AR ¼ 0.7e34
selectivity (e.g. 60, hA /hA and hA2A/hA >46) towards other AR
group by NH
vigorous heating in a sealed tube. As expected, the electron-poor
isothiourea carbon was readily attacked by the nucleophile NH
2
was carried out in DMF containing 2% ammonia with
3
i
i
2
3
m
to afford benzoylguanidino derivatives 22e25. The completion of
the reaction was confirmed by the odour of methyl mercaptan from
the reaction vessel. Unfortunately, the synthesized compounds
1
3
3
subtypes. Besides that, all the PP-4-carboxylates were found inac-
tive at the hA2BAR. The binding assay results of the analogues
bearing various alkyl substitutions (i.e. methyl, isopropyl or neo-
2
2e25 were insoluble in any solvent. Hence, they were directly
2
taken to the next reaction. Afterwards, benzoyl guanidines 22e25
were easily cyclized to 6-amino-2-substituted-2H-pyrazolo[3,4-d]
pyrimidin-4(5H)-one derivatives (26e29) upon treatment with 1 N
sodium hydroxide under reflux conditions. Upon cooling, the pH of
the reaction mixture was adjusted to 6 to obtain a precipitate. For
this reaction, the nitrogen atom of the carbamoyl group attacked
the carbon atom of the methenamine group, followed by elimina-
tion of ammonia and benzoic acid. The traces of benzoic acid were
easily separated by stirring the product in hot ethanol. Compounds
pentyl) or a phenylethyl group at N position were compared to
examine the impact of those substitutions on the binding affinity
and selectivity profiles at the four AR subtypes (Table 1).
6
2.2.1. Effect of C free aminosubstitution on binding affinity of PP
scaffold
4
Binding affinity of C amide substituted derivatives (38e41)
bearing a C free amino group were compared to that of the cor-
responding C ester substituted compounds (42e45). All of the C -
amido substituted compounds (38e41) were shown to exhibit
6
4
4
2
6e29 were obtained in higher yields (ranging from 68 to 81 %)
20].
Chlorination of the 6-amino-2-alkyl-2H-pyrazolo[3,4-d]pyr-
imidin-4(5H)-ones (26e29) was carried out with phosphoryl tri-
chloride (POCl ) in the presence of N,N-dimethyl aniline under
reflux condition for 24 h [20]. After completion of the reaction, the
excess of POCl was carefully removed and the crude product was
[
weak affinity towards the hA
3
AR (K
i
hA
compounds
mM). However, C -amido compounds bearing little
3
AR ¼ 16.9e>100
mM) as
4
compared
hA
AR ¼ 0.7e30
alkyl moieties at the N position, such as a methyl (38) or a propyl
(39) group, propelled the affinity towards hA2AAR and hA AR and
displayed up to 11-fold selectivity over the hA AR. Conversely,
among the C -ester derivatives (42e45), compound 42, with a
to
the
C -ester
(42e45,
K
i
4
3
2
3
1
3
3
4
purified by column chromatography to obtain the corresponding
chloro analogues (30e33) as solids. Compounds 34e37 were pre-
pared by treating chloro derivatives 30e33 with sodium cyanide in
2
methyl group at N , displayed the best affinity at the hA
hA
AR ¼ 0.7
with moderate affinity towards the other receptor subtypes (K
hA2AAR ¼ 2 M and K hA AR ¼ 9.3
compound 43, with a N isopropyl group, displayed a sub-
micromolar affinity towards the hA AR (K hA M), and
AR ¼ 0.9
35-fold selectivity over the hA AR. So, excepted for the hA2BAR, C -
3
AR (K
i
3
mM) in the whole series of PP derivatives, together
dry DMF in the presence of sodium p-toluene sulfinate as catalyst at
i
ꢀ
8
0 C for 2 h. The nitrile groups were detected by the presence of CN
m
i
1
mM). On the other hand,
13
2
peak at 115 ppm in C NMR spectra and also confirmed by the
ꢁ1
presence of CN peak between 2250 and 2260 cm in the IR spectra.
The following hydrolysis of the nitrile group was carried out based
on the literature method called “Radziszewski reaction”, by using
aqueous hydrogen peroxide and aqueous potassium carbonate in
cold or hot conditions [29]. However, the product was obtained in
low yields. Conversely, when the reaction was carried out at room
temperature, the resulting carbamides (38e41) were obtained in
good yield. Subsequently, these intermediate compounds (38e41)
1
i
1
m
4
3
ester compounds displayed affinity in the micromolar range at all
the AR subtypes.
6
2.2.2. Effect of C acyl substitution on binding affinity of PP scaffold
2
2.2.2.1. N -methyl series. Introduction of a benzoyl or substituted
6
benzoyl group at the C amino moiety of compound 42, which
2
were esterified in the presence of conc. H
2
SO
4
with absolute
bears a methyl group at N position, gave rise to compounds with
ethanol under reflux conditions to afford the corresponding car-
boxylates 42e45 as solids.
drastically decreased affinity towards the hA
3
AR (46e49, 61 and 63,
with K hA AR >100 M). The only exception was observed with
i
3
m
Acylation of compounds 42e45 was carried out as described in
Scheme 2. 3 equivalents of substituted benzoyl chlorides or phe-
nylacetyl chloride and diisopropylethylamine (DIPEA) were used to
obtain monoacylated compounds 46e60, and 66e69, respectively.
compound 62 bearing a 4-bromo-N-(4-bromobenzoyl)-benzamido
6
substitution at C position: this compound, in fact, presented the
2
best combined affinity and selectivity profile among the N small
alkyl substituted derivatives (62, K
i
3
hA AR ¼ 2.9
1 3
mM, hA /A >34,

788
G. Venkatesan et al. / European Journal of Medicinal Chemistry 92 (2015) 784e798
Table 1
i 1 3 1
Binding affinity (K ) of synthesized compounds at hA AR, hA2AAR, and hA AR; adenylyl cyclase activity at the hA2BAR and selectivity against hA AR and hA2AAR.
Compd
R
R1
R2
R3
K
i
hA
1
(
m
M)a
K
i
hA2A
(
m
M)b
K
i
hA2B
(m
M)c
K
i
hA
3
(m
M)d
Selectivity
hA /hA
1
3
hA2A/hA
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
4
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
CH
CH(CH )
3 2
3
e
e
e
e
e
e
e
e
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
e
e
e
e
e
e
e
e
H
F
e
e
e
e
e
e
e
e
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
F
10.2 (6.2e18.2)
8.2 (5.9e11.3)
>90
40.2 (23.6e68.6)
8.8 (5.4e14.3)
0.9 (0.7e1.1)
4.0 (3.2e5.2)
3.5 (2.0e5.9)
14.0 (6.3e31.1)
>100
3.0 (1.8e4.9)
9.2 (7.4e11.5)
>80
>20
>20
>20
>20
>20
>10
>15
~60
>20
>20
>20
>20
>10
>20
>20
>20
>20
>20
>20
>10
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>10
>20
>10
>100
>100
>0.10
>0.08
4.6
1.2
9.7
0.06
0.38
0.19
<0.14
>0.03
>0.09
4.1
0.47
2.2
0.18
0.48
0.26
<0.16
<0.14
e
CH
2
CH
2
CH
3
C(CH
CH
3
)
3
19.7 (10.4e37.3)
32.9 (21.5e50.3)
0.9 (0.5e1.6)
14.0 (3.2e61.2)
10.4 (5.1e21.0)
18.1 (10.7e30.5)
>100
>100
>100
~100
2
C
6
H
5
15.7 (9.6e25.8)
2.0 (1.6e2.4)
2.6 (1.9e3.6)
5.0 (3.0e8.7)
4.8 (3.4e6.7)
16.1 (13.8e18.7)
11.5 (6.9e19.0)
>100
11.8 (8.2e16.9)
3.3 (2.9e3.8)
2.8 (1.4e5.5)
>100
CH(CH
3
)
2
CH
2
CH
2
CH
3
CH
3
CH
3
CH
3
C(CH
CH
3 3
)
2
C
6
H
5
e
e
CF
CH
H
3
>100
>100
3
>1
0.5
0.5
0.12
0.4
0.5
CH(CH
CH(CH
CH(CH
CH(CH
3
)
3
)
3
)
3
)
2
4.3 (2.3e8.1)
2.8 (1.8e4.4)
>100
0.7 (0.5e0.9)
2.7 (1.8e3.9)
>100
8.6 (3.3e22.3)
5.7 (5.5e5.9)
>100
2
2
2
F
CF
CH
F
CF
CH
H
3
e
e
3
2.3 (1.2e4.3)
5.7 (3.7e8.7)
>100
28.0 (20.5e38.3)
4.3 (2.9e6.5)
1.3 (1.3e1.4)
0.9 (0.7e1.0)
12.2 (3.5e42.6)
6.8 (4.6e10.1)
5.8 (2.5e13.7)
3.0 (2.7e3.4)
3.6 (3.5e3.7)
2.2 (1.4e3.1)
>100
2.9 (1.9e4.2)
>100
5.4 (3.5e7.6)
>100
14.6 (9.5e22.3)
13.3 (6.5e27.3)
4.3 (2.9e6.5)
10.4 (4.3e24.8)
0.02
0.62
>77
>111
0.41
0.73
0.5
>33
0.72
>46
0.1
1.3
>77
>111
0.3
4
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3
CH
3
CH
3
C(CH
C(CH
C(CH
)
3 3
)
3 3
)
3 3
e
3
e
3
>100
>90
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
CH
CH
CH
CH
CH
CH
2
C
2
C
2
C
2
C
2
C
2
C
6
6
6
6
6
6
H
5
5
5
5
5
5
5.1 (3.8e6.8)
5.0 (3.5e7.2)
2.5 (1.4e4.3)
>100
2.6 (2.2e2.9)
>100
>100
>100
>100
17.7 (11.3e25.7)
>100
3.7 (3.1e4.4)
6.1 (4.5e7.0)
7.5 (5.6e9.7)
6.0 (4.4e7.9)
4.2 (2.4e7.2)
>100
>100
>100
>100
>100
H
H
H
H
H
F
0.8
1.3
2
1.2
CF
CH
Br
Cl
H
Br
F
H
3
3
>46
e
C
6
H
5
CO
(4Br)C
(3,4F)C
e
6
H
5
CO
CO
>34
>34
e
6
H
5
e
CH(CH
3
)
2
C
C
6
H
H
5
CO
CO
H
H
e
e
e
e
3.2
>18.5
e
CH
CH
2
CH
2
C
6
H
5
6
5
H
e
>100
e
3
e
e
e
e
5.9 (4.4e7.3)
0.6 (0.3e1.2)
2.7 (1.8e3.9)
0.6 (0.6e0.7)
7.3 (6.2e8.5)
8.8 (7.8e9.9)
5.7 (3.7e8.7)
2.4 (1.2e4.5)
0.4
0.5
0.66
1.3
CH(CH
3
)
2
e
0.04
0.62
0.05
CH
CH
2
C(CH
CH
)
3 3
e
2
2
C
6
H
5
e
0.2
a
b
c
3
Displacement of specific [ H]-2-chloro-6-cyclopentyl adenosine (CCPA) binding at hA
1
AR expressed in Chinese Hamster Ovary (CHO) cells (n ¼ 3e6).
3
0
Displacement of specific [ H]-5 -N-ethylcarboxamido adenosine (NECA) binding at hA2AAR expressed in CHO cells (n ¼ 3e6).
K
i
values for inhibition of NECA-stimulated adenylyl cyclase activity in CHO cells (n ¼ 3e6).
d
e
3
6
Displacement of specific [ H]-2-(1-hexynyl)-N -methyl adenosine (HEMADO) binding at hA
20].
3
AR expressed in CHO cells (n ¼ 3e6).
[
6
hA2A/hA
clear, since similar benzoylbenzamido compounds 61 and 62 were
shown to be inactive at the hA AR (K hA AR >100 M). This sug-
gests that a relatively big group at para position of dibenzoyl chain,
such as a bromo atom, might play an essential role for the
3
>34). The reason behind such affinity profile is still un-
CH
the hA
hA
¼ 0.7
3
benzamido chain at C position, while was almost inactive at
3
AR, showed submicromolar affinity towards the hA
1 i
AR (K
3
i
3
m
1
mM).
2
2
2.2.2.3. N -neopentyl series. Notably, N neopentyl analogues with
6
improvement of affinity at the hA
3
AR.
C -benzamido substitution displayed the overall best hA
hA M) and selectivity among the other N alkyl
AR ¼ 0.9e8.1
and phenylethyl substituted derivatives. The presence of para-F
group in the benzamido chain led to compound 54, with hA af-
M) comparable to its N isopropyl coun-
AR ¼ 5.7 M).
When the para-F group of benzamide was replaced by a bigger
electron withdrawing CF group, the affinity was increased by
about 4-fold (4, K hA M) [20] in comparison to com-
AR ¼ 1.3
pound 54. In addition, the selectivity towards both hA2AAR and
hA AR was remarkably improved (4, hA2A/hA > 77). Similarly,
when para-CH group was introduced at the benzamide chain, the
hA affinity was further improved (5, K hA M) by 5-fold
AR ¼ 0.9
3
affinity
2
(K
i
3
m
2
2
.2.2.2. N -isopropyl series. On the other hand, benzamido substi-
6
2
tution at C position of N isopropyl derivatives (50e53) showed
much improved hA affinity (K hA M) as compared
AR ¼ 5.3e28.7
to its N -methyl counterpart derivatives (46e49,
hA M), with the exception of compound 52, which was
AR ꢂ 100
inactive at all the AR subtypes. Nevertheless, these derivatives
50e53) showed better affinity at the hA2AAR and hA AR, thus
resulting in lower selectivity of the compounds. Compound 64,
3
2
3
i
3
m
finity (K
i
hA
3
AR ¼ 4.5
hA
m
2
K
i
terpart (51, K
i
3
m
3
m
3
(
1
i
3
m
6
with a N-benzoylbenzamide at the C position, displayed good hA
affinity (K hA M) and high selectivity (hA2A/hA >19)
AR ¼ 5.4
towards the hA2AAR subtype. In contrast, compound 53, with para-
3
1
3
i
3
m
3
3
3
i
3
m

G. Venkatesan et al. / European Journal of Medicinal Chemistry 92 (2015) 784e798
789
4
in relative to compound 54. Moreover, compound 5 showed the
second best hA affinity and remained the most selective (111-fold
against hA2AAR and hA AR) in the whole series of PP derivatives
20]. This result suggests that both electron-withdrawing CF and
i) C ester for conferring affinity;
2
ii) lipophilic neopentyl group at N position for conferring
3
affinity;
1
6
[
3
iii) substitution at the aromatic ring of the benzamide at C for
electron-donating CH
for the affinity at hA
subtypes.
3
groups with steric hindrance are essential
AR and selectivity towards the other AR
conferring selectivity;
iv) N methyl with free amino group or dibenzamide substitu-
2
3
6
tion with steric groups (e.g. Br) at C for conferring affinity.
2
.3. Molecular docking studies
2
.2.2.4. N2 phenylethyl series. Benzamido substitution in the N2
phenylethyl series (55e60) improved the hA affinity (K
hA M) as compared to the free amino counterpart
AR ¼ 2.2e9.0
5 (K hA M). On the contrary, dibenzoyl substitution of
AR ¼ 17
3
i
The molecular docking studies were performed on newly syn-
3
m
m
thesized pyrazolopyrimidine-4-carboxylate derivatives (38e69)
and on the previously reported, structurally related, compounds 4
and 5 [20] to identify hypothetical binding modes and interactions
4
i
3
6
the amino group at the C position led to compound 65, which was
not active at any of the receptor subtypes. Among these derivatives,
unsubstituted benzamide (55) showed moderate affinity at the
3
at the hA AR, as well as to rationalize the results of the binding
data. Docking studies were carried out using molecular operating
environment (MOE) programme and all pyrazolo[3,4-d]pyrimi-
dine-4-carboxylate derivatives were docked into the orthosteric
transmembrane (TM) binding cavities of both hA
Docking simulation at the hA AR demonstrated that the binding
pattern of the new derivatives varied depending on the sub-
stituents at the N and C position of the PP scaffold. Ligand-
recognition occurred in the upper region of the TM bundle, and
the pyrazolo[3,4-d]pyrimidine scaffold was surrounded by TMs 3, 5,
6 and 7. Four different types of interactions were observed and
among them hydrophobic interactions and hydrogen bonding
interaction seemed to play an essential role for the binding affinity
of ligands.
hA
substitution such as F (56, K
hA M) failed to make any additional impact on the af-
AR ¼ 9.0
finity at hA AR. Conversely, para-CH benzamide (58,
hA M) improved the hA AR affinity by around 2.5-fold as
AR ¼ 3.0
compared to the unsubstituted benzamide derivative (55) and also
strengthened the selectivity against hA AR (hA2A/hA > 33). In
addition, introduction of a bulky Br group at the para position of
benzamide (59) led to a 2-fold increment in the hA AR affinity (K
hA M) in relative to 55. Similarly, steric dichloro sub-
AR ¼ 3.6
stitution at 3- and 4-position of benzamide (60) ameliorated the
affinity by >3-fold (K hA M) as compared to 55. This
AR ¼ 2.2
compound (60) was at least 46 times more selective for hA AR
against the hA2AAR and hA AR.
Throughout the whole N
lengthy phenylacetamide substitution (66e69) at the C position
did not cause any significant improvement to the affinity (K
hA M) and selectivity at the hA AR. This suggests
AR ¼ 7.1e15.2
that phenylacetamido moiety was tolerated in all the AR subtypes
in a similar manner. Moreover, compounds 67 (K hA M)
AR ¼ 0.7
and 69 (K hA M) showed higher affinity towards the
AR ¼ 0.6
hA AR.
As a whole, further ligand modification of the ethyl 4-
carboxylate-PP analogues led to the new series of PP derivatives
with affinity values towards hA AR in the micromolar range. The
structure-affinity relationship study showed that substituents at
3
AR (K
i
hA
3
AR ¼ 7.4
m
M). Surprisingly, electron withdrawing
i
hA M) and CF (57, K
3
AR ¼ 7.0
m
3
i
3
m
3
and hA2A ARs.
3
3
K
i
3
3
m
3
2
6
1
3
3
i
3
m
i
3
m
6
3
Among the C free amino derivatives, compound 42 was posi-
1
tioned at the centre of the binding pocket on the top of the TM helix
(Fig. 3-A). This could be due to the unrotatable nature of the N -
methyl group, which assisted the ligand to obtain a stable confor-
mation at the centre of the binding pocket.
Moreover, compound 42 also made a stabilizing hydrogen
bonding interaction with Asn250, whereas pyrimidine ring of the
2
2
(ar)alkyl series, the presence of
6
i
3
m
3
i
1
m
PP scaffold made pep stacking interaction with Phe168. On the
2
i
1
m
other hand, other compounds (43e45) with bulky chains at N
position, due to steric hindrance, orientated in a different fashion,
which shifted away from the centre and moved towards the TM4.
The binding mode of compound 42 inside the hA2AAR was also
analysed. Interestingly, the compound occupied the same position
as hA
with Asn253 and other hydrophobic interactions with hA2AAR. This
has led to the poor selectivity of compound 42 over the hA2AAR (K
hA AR ¼ 0.7 M, K hA2AAR ¼ 2.0 M).
In contrast, acylated compounds of N methyl derivatives
(46e49, 61) displayed different binding pose at the hA AR as
1
3
3
AR and it retained the vital hydrogen bonding interaction
2
4
6
N , C and C positions play a key role in modulating the binding
2
affinity at the hA
substitution showed favourable effect on the hA
hA M). The previously reported compounds 4 and 5 [20]
AR ¼ 0.9
were the best derivatives of the series, showing a good affinity and
selectivity towards the hA AR. In particular, para-CF and para-CH
groups on the C -benzamido moiety seem to be important for the
3
AR. In particular, at the N position, neopentyl
i
3
AR affinity (5, K
i
3
m
i
m
2
3
m
3
3
3
3
compared to compound 42. The presence of a benzoyl group on the
amino group at the C position of the analogues caused the ligands
6
6
hA
3
AR selectivity (5; hA
1
/hA
3
>111, hA2A/hA
3
>111 and 4; hA
1
/hA
3
2
3
to move away from the centre of the hA AR binding pocket, and to
orient slightly to the interior towards TM4. This caused the ligands
to lose an essential hydrogen bonding interaction with Asn250 and
6
>
77, hA2A/hA
3
>77). Additionally, C -free amino group with N
methyl substitution was demonstrated to be vital for the hA
finity (42, K hA M) but not for selectivity, whereas
AR ¼ 0.7
subsequent acylation was detrimental to the affinity of the methyl
derivatives (46, K hA M). In contrast, dibenzamide
AR ꢂ 100
substituents with steric groups at the para position in the N methyl
series was essential for hA affinity (62, K hA M) and
AR ¼ 2.9
selectivity (hA /hA >34, hA2A/hA >34) against the other AR
subtypes. N -phenylethyl derivatives (55e60) displayed good af-
finity at the hA AR, but selectivity was poor, excepted for com-
pound 60. In fact, the 3,4-dichlorobenzamido substitution led to an
enhancement of affinity (60, K hA M) and selectivity
AR ¼ 2.2
hA /hA >46, hA2A/hA >46) towards hA AR. Overall, SAR results
3
af-
i
3
m
also a
p
e
p
stacking interaction with Phe168, which could easily
AR subtype.
explain the inactivity of these compounds at the hA
3
6
i
3
m
Whereas compound 68, with lengthy phenylacetamide chain at C
position, was able to interact with some of the hydrophobic resi-
dues because of the rotatable methylene group, which assisted the
ligand to attain a slightly different binding pattern. In fact, com-
2
3
i
3
m
1
3
3
2
pound 68 showed some affinity at the hA
3
AR (K
i
hA
3
AR ¼ 15.2
mM).
3
In addition, compound 62, with 4-bromo-N-(4-bromobenzoyl)-
6
benzamido substituent at C position, was positioned at the centre,
i
3
m
due to broaden and rotatable nature of the dibenzamide chain
(Fig. 3-B). Moreover, this substituted N-benzoylbenzamido chain
interacted with hydrophobic residues such as Val72, Ser73, Leu68
and Tyr265. Interestingly, these interactions were not seen with the
(
1
3
3
3
confirmed the importance of the following substitutions for the hA
affinity and selectivity:
3

790
G. Venkatesan et al. / European Journal of Medicinal Chemistry 92 (2015) 784e798
3
Fig. 3. Hypothetical binding mode of compound 42 (A) and compound 62 (B) obtained after docking simulations inside the hA AR binding site. Poses are viewed from the
membrane side facing TM6, TM7, and TM1.
para unsubstituted N-benzoylbenzamido derivatives (compound
(4, 5, 54) displayed a slightly different binding pose in comparison
to compound 42. Docking analysis of compound 4 and 5 was also
reported in our previous work on PP derivatives [20] and, regarding
compound 5, (Fig. 4-A), which was the most potent analogue of the
4
6
1). Similarly, the ethyl group of the C -ester chain of compound 62
interacted with Met177, Leu246, and Leu264, while the basic nu-
cleus of PP made stacking interactions with Phe168. Most
p-p
4
importantly, the carbonyl group of the ester substituent made
hydrogen bonding interactions with Asn250. This proves the
speculation that hA AR binding site possesses the maximum spatial
3
C -ester series, it was observed that this compound was placed
slightly 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 towards the ECL2.
Due to this reason, compound 5 was able to make three stabilizing
hydrogen bonding interactions with Asn250. Similarly, compound
4 shared a close resemblance of the binding pose and was also
involved in similar binding interactions. Besides the hydrogen
bonding with Asn250, we could observe hydrophobic interactions
with residues such as Val169, Ile253, Val259 and Leu264. In
particular, Val169, which was not conserved among the AR sub-
types, was deemed to influence not only the affinity of a ligand but
also its selectivity towards the other AR subtypes [30]. In addition,
the neopentyl chain enabled the hydrophobic interaction with
Trp243, Leu91, Ser181 and Ser247. The central PP scaffold was
tolerability, which can accommodate the broaden diacyl chain with
even bulky steric groups (e.g. Br).
2
Generally, acylated N isopropyl ligands (50e53, 64, 67) moved
towards the centre of the binding pocket due to the steric hin-
drance produced by the isopropyl group, which extended towards
the TM4. Ligands were bound in a vertical direction at the centre of
the binding pocket and these binding poses are quite different from
2
the one observed for the benzamide derivatives (46e49) of N
methyl group series. The basic scaffold was situated at the centre of
the binding pocket and the benzamide chains are extended towards
the TM6 and TM7. Due to this close contact with TM helix, the
carbonyl group of the ester chains was able to make a hydrogen
bonding interaction with Asn250, with exception of compound 52
involved in a
p-p stacking interaction with Phe168, which is
with steric CF
3
group that created a steric hindrance and made the
3
considered to be crucial for the hA AR but also for the hA2AAR
chain unavailable for the interaction with Asn250 residue.
antagonist activity [31,32].
2
2
Interestingly, N neopentyl substituted benzamide compounds
In addition, N phenylethyl benzamide substituted derivatives
Fig. 4. A. Hypothetical binding mode of compound 5 obtained after docking simulations inside the hA
3
AR binding site. B. Compound 5 inside the hA2AAR binding site. C. Hy-
pothetical binding mode of compound 60 obtained after docking simulations inside the hA AR binding site. Poses are viewed from the membrane side facing TM6, TM7, and TM1.
3

G. Venkatesan et al. / European Journal of Medicinal Chemistry 92 (2015) 784e798
791
(
55e60) oriented in the same fashion as N2 isopropyl derivatives
positional effects and rotatable nature. This resulted in compounds
showing common interactions (which include the hydrogen
bonding interactions with Asn253) and other hydrophobic in-
teractions essential for hA2AAR activation. Thus, some compounds
showed good affinity towards the hA2AAR as well and subsequent
poor selectivity.
and were involved in common hydrophobic interactions and lone
hydrogen bonding interactions with Asn250. However, compound
6
interactions with Asn250 (Fig. 4-C). Similarly, compound 60 mimics
the binding pose of compound 5 of previous work and was also
involved in similar binding interactions such as hydrophobic in-
teractions (Val169, Ile253, Val259 and Leu264), hydrogen bonding
0 of this series made 3 additional stabilizing hydrogen bonding
interaction (Asn250) and
p-p
stacking interaction (Phe168). In
3. Conclusions
addition, the phenylethyl chain enabled the essential hydrophobic
interactions with Trp243, Leu90, and Ser181. In fact, compound 60
displayed an affinity value of 2.2 mM at the hA AR.
3
In summary, we have synthesized and characterized a new se-
ries of simplified pyrazolo[3,4-d]pyrimidine-4-carboxylate de-
rivatives using an efficient synthetic procedure. The previously
Generally, dibenzamide substituted compounds (61, 63 & 65)
moved away from the binding site, and oriented deeply into the
intracellular loop while the ligands were flipped to opposite di-
rection as of ligand 60, i.e. the pyrazole towards the TM6 (instead of
TM3) and the pyrimidine towards the TM3 (instead of TM6).
We also docked the newly synthesized compounds inside the
hA2A binding pocket. We noticed that most of the compounds
shared similar binding pose inside the binding pocket. In addition,
the most potent and selective compound of the series, compound 5,
showed quite a opposite binding orientation inside the hA2A
2
reported compounds 4 and 5, bearing the N -neopentyl moiety and
6
a para-CF
3
and para-CH
3
benzamido groups at the C position,
remained the most potent and selective hA
3
AR antagonists of the
2
PP series. In addition, the exploration of new substituents at the N
6
and C position of the nucleus led to the discovery of new de-
rivatives, such as compounds 60 and 62, which displayed good
affinity at the hA
3
AR (60, K
i
hA
3
AR ¼ 2.2
m
M; 62, K
i
hA
3
AR ¼ 2.9
mM)
and a significant selectivity towards the other AR subtypes (60,
>46; 62, >34). Even if the binding data make it difficult to elaborate
a clear SAR profile, we can conclude that the presence of an ethyl
ester and of a substituted benzamido group at the C and C posi-
tions, respectively, in combination with a bulky group at the N
3
binding pocket (Fig. 4-B) as compared to hA AR and failed to make
4
6
hydrophobic interaction with essential residues such as Glu169 and
barely made contact with Asn253, which is essential for the hA2A
antagonist activity. Some of the common hydrophobic residues
observed around the ligands are Ala63, Ile80, Val84, Leu85, Trp246
and Tyr271.
The docking pose of the compound 5 was also compared with
the most potent PTP compound (Figure S-1). The binding orienta-
tions of both the ligands are slightly different. However, compound
2
position (such as neopentyl (4, 5, 54) or phenylethyl (57e60)), led
to good affinity towards hA
3
AR. In some cases (e.g. 4, 5 & 60) the
AR. A little
compounds showed good selectivity towards the hA
3
2
alkyl group at the N position, such as methyl, was tolerated only
when a sterically hindered moiety was concurrently present at the
6
C
position (62). Docking studies confirmed the pharmacological
5
is oriented in the same plane as PTP. Interestingly, there is a trend
data: compounds 4, 5, 60 and 62 inside the binding pocket of the
hA AR displayed interactions with key residues, such as Asn250 (4,
5, 60, 62), Phe168 (4, 5, 62), Val169 (4, 5) and Leu264 (4, 5, 62).
Moreover, these simplified analogues were able to overcome the
limitations typically associated with the tricyclic PTP structures,
such as high molecular weight and lipophilicity.
observed between the binding pose of the PTP and compound 5.
The phenyl ring of the benzamide linkage in the PP is aligned over
the phenyl ring of the PTP, thereby the PP has retained the essential
hydrophobic interactions with residues such as Met177, Leu246 and
Leu264 and Trp243, which are observed at the phenyl ring of PTP.
3
8
Moreover, the ester chain of 5 was aligned over N methyl group of
the PTP counterpart, thus retaining the interaction with residues
8
2
associated with N methyl of PTPs. Finally, the N -neopentyl group
in PP was aligned over the phenylacetamide chain of the PTP and
involved in the hydrophobic interactions common to the phenyl-
acetamide chain of PTP. These findings signify that most of the
hydrophobic interactions associated with PTP are retained in the PP
ligands as well.
4. Experimental
4.1. Chemistry
Reactions were routinely monitored by thin-layer chromatog-
raphy (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 Gal-
lenkamp 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 Fin-
nigan MAT mass spectrometer (source of ionization: Electrospray
ionization (ESI) probe). H and C NMR spectra were determined in
the deuterated dimethylsulfoxide (DMSO-d ) solutions on Bruker
DPX ultrashield NMR (400 MHz) spectrometers, with chemical
shifts given in parts per million ( ) downfield relative to tetrame-
thylsilane (TMS) as internal standard, and J values (coupling con-
stants) given in hertz. The following abbreviations were used: s,
singlet; d, doublet; t, triplet; sep, septet; m, multiplet; br, broad.
HPLC Studies: The purity of the compounds was determined via
analytical HPLC using HITACHI, version 3.1.8b on a Phenomenex
In summary, docking evaluations were carried out on both
homology-based hA AR and crystallographic hA2AAR. Different
3
binding orientation was observed for the ligands in the both
binding pockets, which differ based on the substituents at C and
6
2
N
positions. As mentioned earlier, the binding pattern of com-
2
pounds bearing N -neopentyl derivatives are slightly different from
2
1
13
the other N substituted analogues because of the steric hindrance
produced by the neopentyl groups inside the hA
hA2AAR.
Generally, docking studies confirmed that the most potent
compounds of the PP-4-carboxylate series possessed better hA AR
affinity due to stabilizing hydrogen bonding interactions with the
Asn250, and displayed some specific hydrophobic interactions with
Val169, Val259 and Ile253. These hydrogen bonding and hydro-
3
AR model and
6
d
3
3
phobic interactions have contributed to the hA AR affinity and
selectivity to a considerable extent. In addition, most of the newly
synthesized derivatives showed a common binding orientation
pattern throughout the series inside the hA2AAR, with the excep-
tion of some compounds, which were bound in a slightly different
fashion due to the substituents effects such as electronic properties,
Gemini 5U C18 column (5
m
M, 110 Å, 150 mm ꢃ 4.60 mm i.d.) at
room temperature at a flow rate of 1.0 mL/min with two different
mobile phase eluent systems at 254 nm UV detection. The isocratic
elution was followed and analysis was carried out using mobile
2
phase eluent systems ACN (65%): H O.

792
G. Venkatesan et al. / European Journal of Medicinal Chemistry 92 (2015) 784e798
4
.1.1. General procedure for the synthesis of 3-amino-1-alkyl or
DMSO-d
6
):
d
¼ 0.90 (s, 9H, 3CH
3 2
), 3.64 (s, 2H, CH ), 5.32 (s, 2H,
arylalkyl-1H-pyrazole-4-carbonitriles (6e9)
NH ), 6.72 (br s, 1H, NH), 7.21 (br, 1H, NH), 7.85 (s, 1H, pyrazole-H).
2
To a solution of 3-amino-1H-pyrazole-4-carbonitrile (1.08 g,
LCeMS (ESI) analysis (m/z) calcd for C
[MþH] .
9
H
16
N
4
O (196.25): found 197.1
þ
0
.01 mol) in anhydrous DMF (5 ml), anhydrous potassium car-
ꢀ
bonate (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
4.1.2.4. 3-Amino-1-phenethyl-1H-pyrazole-4-carboxamide
(13).
White solid, mp 166e168 C (5.73 g, 90%). H NMR (400 MHz,
DMSO-d ): ), 4.12 (t, J ¼ 7.16 Hz, 2H,
¼ 3.08 (t, J ¼ 7.16 Hz, 2H, CH
CH ), 5.44 (s, 2H, NH ), 6.24 (br s, 1H, NH), 6.74 (br s, 1H, NH),
ꢀ
1
added slowly over 15 min and the reaction mixture was heated at
6
d
2
ꢀ
9
0
C for 10 h. Then the resulting reaction mixture was cooled,
2
2
poured over ice cold water and the aqueous phase was extracted
7.19e7.35 (m, 5H, AreH), 7.80 (s, 1H, pyrazole-H). LCeMS (ESI)
þ
with EtOAc (3 ꢃ 10 ml), the combined organic layers were dried
analysis (m/z) calcd for C12
H
14
N
4
O (230.27): found 231.0 [MþH] ,
þ
over Na
2
SO
4
, filtered, and the solvent was removed under reduced
254.2 [MþNa] .
pressure to give an oily residue that was purified via column
chromatography eluting with a mixture of hexane/ethylacetate
4.1.3. General procedure for synthesis of 3-(3-benzoylthioureido)-1-
(alkyl or aryl alkyl)-1H-pyrazole-4-carboxamide (14e17)
(
1:1) to obtain the desired products (6e9) as solids.
To a solution of carboxamides 10e13 (0.015 mol) in dry acetone
(10 ml), benzoyl isothiocyanate (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 dried
under vacuum. The crude product was further recrystallized from
methanol to obtain a pure solids.
4
.1.1.1. 3-Amino-1-methyl-1H-pyrazole-4-carbonitrile (6). Pale yel-
ꢀ
1
ꢀ
low solid, mp 125e127 C (0.723 g, 67%). H NMR (400 MHz, DMSO-
): 3.52 (s, 3H, N-CH ), 6.53 (s, 2H, NH ), 7.51 (s, 1H, pyrazole-H).
C NMR (400 MHz, DMSO-d ): 35.0 (NeCH ), 72.6 (CeCN), 115.7
CN), 140.3 (CH), 151.9 (CeNH
for C
d
6
d
3
2
1
3
6
d
3
(
2
). LCeMS (ESI) analysis (m/z) calcd
(122.13): found 122.8 [MþH] .
þ
5
H
6
N
4
4
.1.3.1. 3-(3-Benzoylthioureido)-1-methyl-1H-pyrazole-4-
ꢀ
1
4
.1.1.2. 3-Amino-1-isopropyl-1H-pyrazole-4-carbonitrile
(7).
Pale yellow solid, mp 120e122 C (0.745 g, 69%). H NMR (400 MHz,
DMSO-d ):
H, CH), 5.52 (s, 2H, NH
analysis (m/z) calcd for C
carboxamide (14). Yellow solid, mp 218e220 C (1.28 g, 61%).
NMR (400 MHz, DMSO-d ): ), 7.17 (br s, 1H,
¼ 3.61 (s, 3H, NeCH
NH), 7.46e7.60 (m, 5H, AreH), 7.73 (d, 2H, CONH ), 7.78 (s, 1H,
H
ꢀ
1
6
d
3
6
d
1.33 (d, J ¼ 6.64 Hz, 6H, 2CH
3
), 4.24 (sep, J ¼ 6.64 Hz,
2
1
2
), 8.10 (s, 1H, pyrazole-H). LCeMS (ESI)
pyrazole-H), 11.47 (br s, 1H, NH). LCeMS (ESI) analysis (m/z) calcd
þ
þ
7
H
10
N
4
(150.18): found 151.0 [MþH] .
for C13H
13
N
5
O
2
S (303.34): found 304.0 [MþH] .
4
.1.1.3. 3-Amino-1-neopentyl-1H-pyrazole-4-carbonitrile
(8).
Pale yellow solid, mp 123e125 C (0.766 g, 71%). H NMR (400 MHz,
DMSO-d ): 0.89 (s, 9H, 3CH ), 3.67 (s, 2H, CH ), 5.50 (s, 2H, NH ),
.04 (s, 1H, pyrazole-H). LCeMS (ESI) analysis (m/z) calcd for
4.1.3.2. 3-(3-Benzoylthioureido)-1-isopropyl-1H-pyrazole-4-
ꢀ
1
ꢀ
1
carboxamide (15). Yellow solid, mp 212e214 C (1.58 g, 63%).
NMR (400 MHz, DMSO-d ):
¼ 1.42 (d, J ¼ 5.76 Hz, 6H, 2CH
4.44e4.50 (m, 1H, CH), 7.07 (br, 1H, NH), 7.57e7.68 (m, 5H, AreH),
8.00 (d, 2H, CONH ), 8.31 (s, 1H, pyrazole-H), 11.06 (br s, 1H, NH).
H
6
d
3
2
2
6
d
3
),
8
C
þ
H
9 14
N
4
(178.23): found 179.2 [MþH] .
2
LCeMS (ESI) analysis (m/z) calcd for C15
332.0 [MþH] .
17
H N
5
O
2
S (331.39): found
þ
4
.1.1.4. 3-Amino-1-phenethyl-1H-pyrazole-4-carbonitrile
(9).
Pale yellow solid, mp 132e134 C (0.820 g, 76%). H NMR (400 MHz,
DMSO-d ): ), 4.11 (t, J ¼ 7.16 Hz, 2H,
3.03 (t, J ¼ 7.16 Hz, 2H, CH
CH ), 5.57 (s, 2H, NH ), 7.13e7.29 (m, 5H, AreH), 7.92 (s, 1H, pyr-
azole-H). LCeMS (ESI) analysis (m/z) calcd for C12 (212.25):
ꢀ
1
6
d
2
4.1.3.3. 3-(3-Benzoylthioureido)-1-neopentyl-1H-pyrazole-4-
ꢀ
1
2
2
carboxamide (16). Yellow solid, mp 215e217 C (1.76 g, 60%).
NMR (400 MHz, DMSO-d ): ), 3.91 (s, 2H, CH
¼ 0.91 (s, 9H, 3CH
7.09 (brs, 1H, NHCO), 7.57e7.70 (m, 5H, AreH), 8.00 (d, 2H, CONH
H
),
),
H
12
N
4
6
d
3
2
þ
þ
found 213.0 [MþH] , 235.0 [MþNa] .
2
8
C
.18 (brs, 1H, pyrazole-H). LCeMS (ESI) analysis (m/z) calcd for
þ
4
.1.2. General procedure for synthesis of 3-amino-1-(alkyl or aryl
17
H
21
N
5
O
2
S (359.45): found 360.2 [MþH] .
alkyl)-1H-pyrazole-4-carboxamide (10e13)
Carbonitriles 6e9 (0.03 mol) were dissolved in 7 ml conc. H
at 0 C and stirred at room temperature for 5 h. Then the reaction
mixtures were poured into ice cold water and neutralized to pH 7
2
SO
4
4.1.3.4. 3-(3-Benzoylthioureido)-1-phenethyl-1H-pyrazole-4-
ꢀ
ꢀ
1
carboxamide (17). Yellow solid, mp 208e210 C (2.44 g, 71%). H
NMR (400 MHz, DMSO-d ): ), 4.34 (br s, 2H,
¼ 3.07 (br d, 2H, CH
CH ), 7.21e7.27 (m, 5H, AreH), 7.49e7.69 (m, 5H, AreH), 7.99 (s, 2H,
CONH ), 8.01 (s, 1H, pyrazole-H), 11.51 (br s, 1H, NHCO), 12.7 (br s,
H, NH). LCeMS (ESI) analysis (m/z) calcd for C20
6
d
2
with 28% NH
3
solution. The white precipitates were formed which
2
were further washed with cold water and dried under vacuum.
2
1
19 5 2
H N O S
þ
4
.1.2.1. 3-Amino-1-methyl-1H-pyrazole-4-carboxamide
(10).
White solid, mp 155e157 C (3.0 g, 83%). H NMR (400 MHz, DMSO-
): ), 6.12 (s, 2H, NH ), 6.64 (br s, 1H, NH),
¼ 3.50 (s, 3H, NeCH
.16 (br s, 1H, NH), 7.59 (s, 1H, pyrazole-H). LCeMS (ESI) analysis (m/
(393.46): found 393.9 [MþH] .
ꢀ
1
d
6
d
3
2
4.1.4. General procedure for synthesis of (E)-methyl N'-benzoyl-N-
(4-carbamoyl-1-(alkyl or aryl alkyl)-1H-pyrazol-3-yl)
carbamimidothioate (18e21)
7
þ
z) calcd for C
5
H
8
N
4
O (140.14): found 140.9 [MþH] .
To a solution of benzoylthioureidos 13e17 (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
.1.2.2. 3-Amino-1-isopropyl-1H-pyrazole-4-carboxamide
(11).
White solid, mp 159e161 C (3.82 g, 85%). 1H NMR (400 MHz,
DMSO-d ): ), 4.18 (sep, J ¼ 6.64 Hz,
¼ 1.34 (d, J ¼ 6.52 Hz, 6H, 2CH
H, CH), 5.34 (s, 2H, NH ), 6.69 (br s,1H, NH), 7.16 (br s,1H, NH), 7.94
ꢀ
6
d
3
1
2
(
(
s, 1H, pyrazole-H). LCeMS (ESI) analysis (m/z) calcd for C
168.20): found 169.0 [MþH] .
7
H
12
N
4
O
þ
4
.1.2.3. 3-Amino-1-neopentyl-1H-pyrazole-4-carboxamide
(12).
4.1.4.1. (E)-Methyl-N'-benzoyl-N-(4-carbamoyl-1-methyl-1H-pyr-
ꢀ
1
ꢀ
White solid, mp 161e163 C (4.17 g, 78%). H NMR (400 MHz,
azol-3-yl) carbamimidothioate (18). White solid, mp 196e198
C

G. Venkatesan et al. / European Journal of Medicinal Chemistry 92 (2015) 784e798
793
1
(
3
0.682 g, 75%). H NMR (400 MHz, DMSO-d
.57 (s, 3H, NCH ), 7.42e7.52 (m, 5H, AreH), 7.70 (br s,1H, pyrazole-
H), 7.78 (d, 2H, CONH
m/z) calcd for C14
6
):
d
¼ 2.44 (s, 3H, SCH
3
),
evaporated to dryness to obtain a pure desired product as white
solid.
3
2
N
), 11.52 (br s, 1H, NH). LCeMS (ESI) analysis
þ
(
H
15
5
O
2
S (317.37): found 318.1 [MþH] .
4.1.6.1. 6-Amino-2-methyl-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
ꢀ
1
(
26). White solid, mp > 300 C (0.721 g, 72%). H NMR (400 MHz,
4
.1.4.2. (E)-Methyl-N'-benzoyl-N-(4-carbamoyl-1-isopropyl-1H-pyr-
DMSO- d ): ), 6.69 (s, 2H, NH ), 7.73 (s, 1H,
6
d
¼ 3.69 (s, 3H, NCH
3
2
ꢀ
azol-3-yl)carbamimidothioate (19). White solid, mp 223e225
C
pyrazole-H), 10.58 (s, 1H, NHCO). LCeMS (ESI) analysis (m/z) calcd
0.665 g, 67%). 1H NMR (400 MHz, DMSO-d
):
), 4.59 (br s, 1H, CH),
), 8.24 (s, 1H, pyrazole-
d
¼ 1.45 (d,
for C
O (165.16): found 166.1 [MþH] .
þ
(
6
6
7
H N
5
J ¼ 6.52 Hz, 6H, 2CH
3 3
), 2.41 (s, 3H, SCH
7.39e7.91 (m, 5H, AreH), 8.02 (d, 2H, CONH
2
4.1.6.2. 6-Amino-2-isopropyl-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-
ꢀ
1
H), 13.09 (br s, 1H, NH). LCeMS (ESI) analysis (m/z) calcd for
C
one (27). White solid mp > 300 C (0.825 g, 75%). H NMR
(400 MHz, DMSO- d ): ), 4.50 (sep,
¼ 1.42 (d, J ¼ 6.52 Hz, 6H, 2CH
J ¼ 6.52 Hz, 1H, CH), 6.90 (s, 2H, NH ), 8.11 (s, 1H, pyrazole-H), 10.61
(s, 1H, NHCO). LCeMS (ESI) analysis (m/z) calcd for C
þ
H
16 19
N
5
O
2
S (345.42): found 346.0[MþH] .
6
d
3
2
4
.1.4.3. (E)-Methyl-N'-benzoyl-N-(4-carbamoyl-1-neopentyl-1H-
8 11 5
H N O
ꢀ
þ
þ
pyrazol-3-yl)carbamimidothioate (20). White solid, mp 219e221 C
(193.21): found 194.2 [MþH] , 216.0 [MþNa] .
1
(
2
(
0.600 g, 56%). H NMR (400 MHz, DMSO-d
.42 (s, 3H, SCH ), 4.02 (s, 2H, CH ), 7.40e7.95 (m, 5H, AreH), 8.02
d, 2H, CONH
6
):
d
¼ 0.90 (s, 9H, 3CH
3
),
3
2
4.1.6.3. 6-Amino-2-neopentyl-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-
ꢀ
1
2
), 8.17 (s, 1H, pyrazole-H). LCeMS (ESI) analysis (m/z)
one (28). White solid, mp > 300 C (0.815 g, 68%). H NMR
(400 MHz, DMSO-d ): ), 3.70 (s, 2H, CH ), 6.24
¼ 0.96 (s, 9H, 3CH
(s, 2H, NH ), 7.67 (s, 1H, pyrazole-H). LCeMS (ESI) analysis (m/z)
calcd for C10
þ
calcd for
C
H
18 23
N
5
O
2
S
(373.47): found 374.1[MþH] , 396.2
6
d
3
2
[
MþNa]þ.
2
þ
þ
H
15
N
5
O (221.26): found 222.0 [MþH] , 244.0 [MþNa] .
4
.1.4.4. (E)-Methyl-N'-benzoyl-N-(4-carbamoyl-1-phenethyl-1H-
ꢀ
pyrazol-3-yl)carbamimidothioate (21). White solid, mp 235e237 C
4.1.6.4. 6-Amino-2-phenethyl-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-
1
ꢀ
1
(
3
0.743 g, 63%). H NMR (400 MHz, DMSO-d
.13 (t, J ¼ 7.04 Hz, 2H, CH ), 4.45 (t, J ¼ 7.04, Hz, 2H, CH
m, 5H, AreH), 7.39e7.87 (m, 5H, AreH), 8.00 (d, 2H, CONH
6
):
d
¼ 2.41 (s, 3H, SCH
), 7.11e7.23
), 8.12
3
),
one (29). White solid, mp > 300 C (1.067 g, 81%). H NMR
(400 MHz, DMSO-d ): ), 4.36 (t,
¼ 3.10 (t, J ¼ 6.64 Hz, 2H, CH
J ¼ 6.64 Hz, 2H, CH ), 7.34 (s, 2H, NH ), 7.14e7.30 (m, 5H, CH), 7.89
(s, 1H, pyrazole-H), 10.62 (s, 1H, NHCO). LCeMS (ESI) analysis (m/z)
2
2
6
d
2
(
(
2
2
2
s, 1H, pyrazole-H), 13.10 (br s, 1H, NH). LCeMS (ESI) analysis (m/z)
þ
þ
þ
calcd for C21
21
H N
5
O
2
S (407.49): found 408.2 [MþH] , 430.2
calcd for C13H
13
N
5
O (255.28): found 256.0 [MþH] , 278.0 [MþNa] .
þ
[MþNa] .
4.1.7. General procedure for synthesis of 4-chloro-2-(alkyl or
4.1.5. General procedure for synthesis of (Z)-3-(2-
arylalkyl)-2H-pyrazolo[3,4-d]pyrimidin-6-amine (30e33)
benzoylguanidino)-1-(alkyl or aryl alkyl)-1H-pyrazole-4-
carboxamide (22e25)
A mixture of 6-amino-2-(alkyl or arylalkyl)-2H-pyrazolo[3,4-d]
pyrimidin-4(5H)-ones (26e29) (0.005 mol), phosphoryl trichloride
(0.1 mol, 20 equiv) and N,N-dimethylamine (0.005 mol, 1 equiv)
Carbamimidothioate derivatives 18e21 (0.003 mol) were dis-
solved in dry DMF (5 ml) containing 2% ammonia (5 ml) and heated
ꢀ
was refluxed at 110 C for 24 h. Then the reaction mixture was
ꢀ
to 120 C for 3 h in a sealed tube. At the end of the reaction the
cooled and the excess phosphoryl trichloride was removed under
reduced pressure. The resulting red oil was poured onto ice mixture
slowly, stirred for 10 min and the aqueous part was extracted with
EtOAc (3 ꢃ 10 ml). The combined organic layers were dried over
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.
2 4
Na SO , filtered, and the solvent was removed under reduced
4
.1.5.1. (Z)-3-(2-Benzoylguanidino)-1-methyl-1H-pyrazole-4-
pressure to give an oily residue that was purified via column
chromatography eluting with a mixture of hexane/ethylacetate
(1:1) to obtain pale yellow solids as desired products (30e33).
ꢀ
carboxamide (22). White solid, mp > 300 C (0.504 g, 53%).
Insoluble.
4
.1.5.2. (Z)-3-(2-Benzoylguanidino)-1-isopropyl-1H-pyrazole-4-
4.1.7.1. 4-Chloro-2-methyl-2H-pyrazolo[3,4-d]pyrimidin-6-amine
ꢀ
ꢀ
1
carboxamide (23). White solid, mp > 300 C (0.642 g, 62%).
Insoluble.
(30). Pale yellow solid, mp 247e249 C (0.536 g, 65%). H NMR
(400 MHz, DMSO-d ), 6.90 (s, 2H, NH ), 8.43
): ¼ 4.00 (s, 3H, NeCH
s, 1H, pyrazole-H). C NMR (400 MHz, DMSO-d ):
¼ 33.9, 106.5,
132.2, 153.8, 156.1, 161.8. LCeMS (ESI) analysis (m/z) calcd for
6
3
2
1
3
(
6
d
4
.1.5.3. (Z)-3-(2-Benzoylguanidino)-1-neopentyl-1H-pyrazole-4-
ꢀ
þ
þ
carboxamide (24). White solid, mp > 300 C (0.627 g, 56%).
Insoluble.
C
6
H
6
ClN
5
(183.60): found 184.3 [MþH] , 206.1 [MþNa] . HPLC
purity (254 nm); 98.2%; eluent: 60% ACN:H
2
O, t
R
¼ 2.2 min.
4
.1.5.4. (Z)-3-(2-Benzoylguanidino)-1-phenethyl-1H-pyrazole-4-
4.1.7.2. 4-Chloro-2-isopropyl-2H-pyrazolo[3,4-d]pyrimidin-6-amine
(31). Pale yellow solid, mp 151e153 C (0.647 g, 67%). H NMR
ꢀ
ꢀ
1
carboxamide (25). White solid, mp > 300 C (0.646 g, 53%).
Insoluble.
(400 MHz, DMSO-d
J ¼ 6.64 Hz, 1H, CH), 6.18 (s, 2H, NH
NMR (400 MHz, DMSO-d ):
6
):
d
¼ 1.48 (d, J ¼ 6.64 Hz, 6H, 2CH
3
), 4.53 (sep,
13
2
), 8.30 (s,1H, pyrazole-H).
C
4.1.6. General procedure for synthesis of 6-amino-2-(alkyl or aryl
6
d
¼ 22.0, 48.4, 106.7, 132.1, 153.7, 155.1,
alkyl)-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (26e29)
166.6. LCeMS (ESI) analysis (m/z) calcd for C
8
H10ClN
5
(211.65):
þ
A
suspension of benzoylguanidino derivatives 22e25
found 212.1 [MþH] . HPLC purity (254 nm); 96.8%; eluent: 60%
ꢀ
(
3.5 mmol) in 1 N NaOH (10 ml) was refluxed at 110 C for 12 h. The
ACN:H
2
O, t
R
¼ 3.3 min.
resulting milky white mixture was adjusted to pH 6 with acetic
acid. A mixture of benzoic acid and free amine derivatives were
obtained which was washed with water and dried. Finely divided
powder was suspended in hot ethanol with stirring to remove
benzoic acid and filtered hot. The resulting ethanolic portion was
4.1.7.3. 4-Chloro-2-neopentyl-2H-pyrazolo[3,4-d]pyrimidin-6-amine
ꢀ
1
(32). Pale yellow solid, mp 154e156 C (0.815 g, 77%). H NMR
(400 MHz, DMSO-d ): ), 4.01 (s, 2H, CH ), 7.29
¼ 0.96 (s, 9H, 3CH
(s, 2H, NH ), 8.02 (s, 1H, pyrazole-H). C NMR (400 MHz, DMSO-d ):
6
d
3
2
1
3
2
6

794
G. Venkatesan et al. / European Journal of Medicinal Chemistry 92 (2015) 784e798
d
¼ 28.1, 33.7, 57.3, 106.2, 132.2, 153.9, 156.9, 161.8. LCeMS (ESI)
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 (38e41) as solids.
þ
analysis (m/z) calcd for C10
HPLC purity (254 nm); 100%; eluent: 60% ACN:H
H14ClN
5
(239.70): found 240.0[MþH] .
2
O, t
R
¼ 6.0 min.
4
.1.7.4. 4-Chloro-2-phenethyl-2H-pyrazolo[3,4-d]pyrimidin-6-amine
4
.1.9.1. 6-Amino-2-methyl-2H-pyrazolo[3,4-d]pyrimidine-4-
ꢀ
1
(
(
33). Pale yellow solid, mp 146e148 C (0.918 g, 72%). H NMR
400 MHz, DMSO-d ): ), 4.38 (t,
¼ 3.10 (t, J ¼ 6.95 Hz, 2H, CH
), 7.10e7.22 (m, 5H, AreH), 7.26 (s, 2H, NH ),
¼ 35.0,
7.8, 106.5, 126.8, 128.8, 129.0, 132.4, 138.6, 153.8, 156.1, 161.7.
ꢀ
1
carboxamide (38). White solid, mp > 300 C (78%). H NMR
400 MHz, DMSO-d ): ), 7.03 (s, 2H, NH ),
¼ 3.83 (s, 3H, NeCH
.84 (br s, 1H, NH), 7.96 (br s, 1H, NH), 8.16 (s, 1H, pyrazole-H).
6
d
2
(
7
6
d
3
2
J ¼ 6.95 Hz, 2H, CH
2
2
13
7
4
.97 (s,1H, pyrazole-H). C NMR (400 MHz, DMSO-d ): d
6
LCeMS (ESI) analysis (m/z) calcd for C
7
H
8
N
6
O (192.18): found 191.4
ꢁ
[MꢁH] .
LCeMS (ESI) analysis (m/z) calcd for C13
H12ClN
5
(273.72): found
O,
ꢁ
2
72.0 [MꢁH] . HPLC purity (254 nm); 99.3%; eluent: 60% ACN:H
2
4
.1.9.2. 6-Amino-2-isopropyl-2H-pyrazolo[3,4-d]pyrimidine-4-
t
R
¼ 5.5 min.
ꢀ
1
carboxamide (39). White solid, mp 177e179 C (70%). H NMR
400 MHz, DMSO-d ): ), 4.93 (sep,
¼ 1.48 (d, J ¼ 6.8 Hz, 6H, 2CH
), 7.90 (br s, 1H, NH), 7.98 (br s,
H, NH), 8.23 (s, 1H, pyrazole-H). LCeMS (ESI) analysis (m/z) calcd
(
6
d
3
4
.1.8. General procedure for synthesis of 6-amino-2-(alkyl or
J ¼ 6.8 Hz, 1H, CH), 7.03 (s, 2H, NH
2
aralkyl)-2H-pyrazolo[3,4-d]pyrimidine-4-carbonitrile (34e37)
A mixture of 4-chloro-2-(alkyl or aralkyl)-2H-pyrazolo[3,4-d]
pyrimidin-6-amines (30e34) (0.00054 mol), sodium cyanide
1
for C
þ
9
H
12
N
6
O (220.23): found 219.0 [MꢁH] .
(
0
8
0.0011 mol, 2 equiv), sodium p-toluene sulfinate (0.00016 mol,
.3 equiv) in 5 mL of anhydrous DMF was heated on an oil bath at
4.1.9.3. 6-Amino-2-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4-
ꢀ
1
ꢀ
carboxamide (40). White solid, mp 174e176
400 MHz, DMSO-d ): ), 4.07 (s, 2H, CH
¼ 0.99 (s, 9H, 3CH
(s, 2H, NH ), 7.89 (br s, 1H, NH), 8.00 (br s, 1H, NH), 8.24 (s, 1H,
C (79%). H NMR
0
C for 2 h. The resultant mixture was cooled, poured into ice
(
6
d
3
2
), 7.03
water mixture and stirred for 10 min. The solid was collected by
filtration, washed with water and recrystallized from dime-
thylformamide and methanol mixture to obtain the desired prod-
ucts (34e37) as solids.
2
pyrazole-H). LCeMS (ESI) analysis (m/z) calcd for C11
(248.28): found 247.0 [MꢁH] .
H
16
N
6
O
ꢁ
4
.1.8.1. 6-Amino-2-methyl-2H-pyrazolo[3,4-d]pyrimidine-4-
4.1.9.4. 6-Amino-2-phenethyl-2H-pyrazolo[3,4-d]pyrimidine-4-
ꢀ
1
ꢀ
1
carbonitrile (34). Pale yellow solid, mp 131e133 C (65%). H NMR
400 MHz, DMSO-d ): ), 7.51 (s, 2H, NH ),
¼ 3.88 (s, 3H, NeCH
.24 (s, 1H, pyrazole-H). C NMR (400 MHz, DMSO-d ):
¼ 33.7,
carboxamide (41). Pale yellow solid, mp 182e184 C (84%). H NMR
(400 MHz, DMSO-d ):
(
8
6
d
3
2
d
¼ 3.16 (br s, 2H, CH ), 4.44 (br s, 2H, CH ),
6
2
2
13
6
d
7.00 (s, 2H, NH ), 7.16e7.25 (m, 5H, AreH), 7.85 (br s, 1H, NH), 7.96
2
108.7, 115.1, 131.9, 135.3, 155.7, 162.2. LCeMS (ESI) analysis (m/z)
(br s, 1H, NH), 8.19 (s, 1H, pyrazole-H). LCeMS (ESI) analysis (m/z)
ꢁ
þ
calcd for C
H
7 6
N
6
(174.16): found 173.1 [MꢁH] .
calcd for C14H14N O (282.30): found 281.3 [MꢁH] .
6
4
.1.8.2. 6-Amino-2-isopropyl-2H-pyrazolo[3,4-d]pyrimidine-4-
4
.1.10. General procedure for synthesis of ethyl 6-amino-2-(alkyl or
ꢀ
1
carbonitrile (35). Pale yellow solid, mp 125e127 C (77%). H NMR
aralkyl)-2H-pyrazolo[3,4-d]pyrimidine-4-carboxylate (42e45)
To a solution of 6-amino-2-(alkyl or aralkyl)-2H-pyrazolo[3,4-d]
pyrimidine-4-carboxamides 38e41 (0.00052 mol) in absolute
(
400 MHz, DMSO-d
J ¼ 6.64 Hz, 1H, CH), 7.46 (s, 2H, NH
NMR (400 MHz, DMSO):
6
):
d
¼ 1.47 (d, J ¼ 6.64 Hz, 6H, 2CH
3
), 4.91 (sep,
13
2
), 8.23 (s,1H, pyrazole-H).
C
d
¼ 21.9, 48.3, 109.1, 115.1, 131.8, 135.3,
ethanol (30 mL), conc. H
2
SO
4
(3 mL) was added slowly and the
154.8, 162.0. LCeMS (ESI) analysis (m/z) calcd for
C
9
H
10
N
6
ꢀ
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
ꢁ
(
202.22):found 201.1 [MꢁH] .
4
.1.8.3. 6-Amino-2-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4-
1
0 min. The solid was collected by filtration, washed with water and
recrystallized from methanol to obtain the desired products
42e45) as solids.
ꢀ
1
carbonitrile (36). Pale yellow solid, mp 148e150 C (62%). H NMR
(
400 MHz, DMSO-d
6
):
d
¼ 0.98 (s, 9H, 3CH
3
), 4.06 (s, 2H, CH
2
), 7.48
):
(
13
(
s, 2H, NH ), 8.27 (s, 1H, pyrazole-H). C NMR (400 MHz, DMSO-d
2
6
d
¼ 28.1, 33.7, 57.2, 108.5, 115.1, 131.8, 135.4, 156.6, 162.3. LCeMS
4
.1.10.1. Ethyl-6-amino-2-methyl-2H-pyrazolo[3,4-d]pyrimidine-4-
(
[
ESI) analysis (m/z) calcd for C11
MꢁH] .
H
14
N
6
(230.27): found 229.2
ꢀ
1
ꢁ
carboxylate (42). Pale brown solid, mp 155e156 C (72%). H NMR
400 MHz, DMSO-d ): ), 3.83 (s, 3H,
¼ 1.37 (t, J ¼ 7.16 Hz, 3H, CH
NeCH ), 7.26 (s, 2H, NH ), 8.09 (s, 1H,
), 4.42 (q, J ¼ 7.10 Hz, 2H, CH
pyrazole-H). C NMR (400 MHz, DMSO-d ):
¼ 14.5, 33.6, 62.3,
(
6
d
3
3
2
2
4
.1.8.4. 6-Amino-2-phenethyl-2H-pyrazolo[3,4-d]pyrimidine-4-
13
ꢀ
1
6
d
carbonitrile (37). Pale yellow solid, mp 135e137 C (81%). H NMR
400 MHz, DMSO-d ): ), 4.44 (t,
¼ 3.14 (t, J ¼ 7.16 Hz, 2H, CH
J ¼ 7.16 Hz, 2H, CH ), 7.12e7.26 (m, 5H, AreH), 7.43 (s, 2H, NH ),
.21 (s, 1H, pyrazole-H). C NMR (400 MHz, DMSO-d
¼ 34.9,
7.6, 108.8, 115.0, 126.9, 128.8, 129.0, 132.1, 135.3, 138.5, 155.8, 162.2.
106.4, 133.9, 150.8, 156.9, 162.4, 163.8. LCeMS (ESI) analysis (m/z)
(
6
d
2
þ
calcd for C
(254 nm) 99.7%; t ¼ 2.8 min.
9
11
H N
5
O
2
(221.22): found 222.0 [MþH] . HPLC: purity
2
2
13
R
8
4
6
): d
LCeMS (ESI) analysis (m/z) calcd for C14
H
12
N
6
(264.29): found 263.4
4.1.10.2. Ethyl-6-amino-2-isopropyl-2H-pyrazolo[3,4-d]pyrimidine-
ꢁ
ꢀ
1
[MꢁH] .
4-carboxylate (43). Pale yellow solid, mp. 139e141 C (75%).
NMR (400 MHz, DMSO-d ): ), 1.43 (d,
¼ 1.37 (t, J ¼ 7.16 Hz, 3H, CH
J ¼ 6.76 Hz, 6H, 2CH ), 4.42 (q, J ¼ 7.16 Hz, 2H, CH ), 4.89 (sep,
J ¼ 6.76 Hz, CH), 7.22 (s, 2H, NH ), 8.11 (s,1H, pyrazole-H). C NMR
(400 MHz, DMSO-d ):
155.9, 162.4, 163.9. DEPT (400 MHz, DMSO-d
(CH ), 22.0 (CH ), 47.9 (CH-alkyl), 62.3 (CH
H
6
d
3
4.1.9. General procedure for synthesis of 6-amino-2-(alkyl or
3
2
13
aralkyl)-2H-pyrazolo[3,4-d]pyrimidine-4-carboxamide (38e41)
To a stirred solution of 6-amino-2-(alkyl or aralkyl)-2H-pyrazolo
2
6
d
¼ 14.5, 22.0, 47.9, 62.3, 106.6, 133.7, 151.0,
[
(
3,4-d] pyrimidine-4-carbonitriles 34e37 (0.00057 mol) in DMSO
3 mL) at cold condition, 30% H (0.0023 mol, 4 equiv) and
CO (0.0011 mol, 2 equiv) were added. The mixture
was stirred at room temperature for 1 h. Then the resultant mixture
6
):
d
14.5.
2
O
2
3
3
2
), 133.7 (CH-pyr-
anhydrous K
2
3
azole). LCeMS (ESI) analysis (m/z) calcd for C11
found 250.1 [MþH] . HPLC: purity (254 nm) 100%; t
H
15
N
5
O
2
(249.27):
þ
R
¼ 2.7 min.

G. Venkatesan et al. / European Journal of Medicinal Chemistry 92 (2015) 784e798
795
4
4
.1.10.3. Ethyl-6-amino-2-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-
4.1.11.5. Ethyl-6-benzamido-2-isopropyl-2H-pyrazolo[3,4-d]pyrimi-
ꢀ
1
ꢀ
1
-carboxylate (44). Pale yellow solid, mp. 120e122 C (84%).
): ), 1.43 (t, J ¼ 7.0 Hz,
¼ 0.99 (s, 9H, 3CH
), 4.47 (q, J ¼ 7.0 Hz, 2H, CH ), 7.27 (s, 2H,
), 8.18 (s, 1H, pyrazole-H). C NMR (400 MHz, DMSO-d ):
H
dine-4-carboxylate (50). White solid, mp 115e117 C (49%). H NMR
(400 MHz, DMSO-d ): ), 1.51 (d,
¼ 1.42 (t, J ¼ 7.12 Hz, 3H, CH
), 5.07 (sep,
NMR (400 MHz, DMSO-d
H, CH ), 4.07 (s, 2H, CH
NH
6
d
3
6
d
3
3
3
2
2
J ¼ 6.64 Hz, 6H, 2CH
3
), 4.50 (q, J ¼ 7.12 Hz, 2H, CH
2
13
2
6
J ¼ 6.64 Hz, 1H, CH), 7.51e8.02 (m, 5H, AreH), 8.45 (s, 1H, pyrazole-
d
¼ 14.5, 28.2, 33.8, 57.0, 62.3, 106.0, 133.7, 151.0, 157.7, 162.7, 163.9.
H), 11.44 (s, 1H, NH). LCeMS (ESI) analysis (m/z) calcd for
þ
LCeMS (ESI) analysis (m/z) calcd for C13
2
H
19
N
5
O
2
(277.32): found
C
18
H
19
N
5
O
3
(353.38): found 354.1 [MþH] . HPLC: purity (254 nm)
þ
78.1 [MþH] . HPLC: purity (254 nm) 97.9%; t
R
¼ 3.6 min.
100%; t
R
¼ 4.0 min.
4
.1.11.6. Ethyl-6-(4-fluorobenzamido)-2-isopropyl-2H-pyrazolo[3,4-
4
4
.1.10.4. Ethyl-6-amino-2-phenethyl-2H-pyrazolo[3,4-d]pyrimidine-
ꢀ
1
d]pyrimidine-4-carboxylate (51). White solid, mp 107e109
ꢀ
C
),
-carboxylate (45). Yellowish brown solid, mp 131e132 C (88%). H
): ), 3.15 (t,
¼ 1.37 (t, J ¼ 6.96 Hz, 3H, CH
), 4.39e4.46 (m, 4H, 2CH ), 7.14e7.24 (m, 7H,
), 8.11 (s, 1H, pyrazole-H). C NMR (400 MHz, DMSO-d ):
¼ 14.5, 35.1, 47.5, 62.3, 106.4, 126.8, 128.8, 129.1, 133.9, 138.7, 151.0,
56.9, 162.6, 163.9. LCeMS (ESI) analysis (m/z) calcd for C16
1
(42%). H NMR (400 MHz, DMSO-d
6
):
d
¼ 1.41 (t, J ¼ 7.12 Hz, 3H,
NMR (400 MHz, DMSO-d
J ¼ 6.84 Hz, 2H, CH
AreH, NH
d
1
6
d
3
CH
5
3
), 1.51 (d, J ¼ 6.64 Hz, 6H, 2CH
3
), 4.50 (q, J ¼ 7.12 Hz, 2H, CH
2
2
2
13
.07 (sep, J ¼ 6.64 Hz, 1H, CH), 7.33e8.10 (m, 4H, AreH), 8.45 (s, 1H,
2
6
13
pyrazole-H), 11.49 (s, 1H, NH). C NMR (400 MHz, DMSO-d
d
6
):
¼ 14.5, 22.1, 49.3, 62.7, 110.2, 115.6, 115.8, 131.6, 131.7, 133.9, 150.8,
17 5 2
H N O
þ
154.5, 155.8, 163.4, 165.3. LCeMS (ESI) analysis (m/z) calcd for
(
311.34): found 312.1 [MþH] . HPLC: purity (254 nm) 100%;
þ
C
18
H
18FN
5
O
3
(371.37): found 372.1 [MþH] . HPLC: purity (254 nm)
t
R
¼ 3.1 min.
97.7%; t
R
¼ 4.3 min.
4
.1.11. General procedure for synthesis of ethyl 6-benzamido-(alkyl
4
.1.11.7. Ethyl-2-isopropyl-6-(4-(trifluoromethyl)benzamido)-2H-
or aralkyl)-2H-pyrazolo[3,4-d]pyrimidine-4-carboxylate (46e60)
To a suspension of ethyl 6-amino-2-(alkyl or aralkyl)-2H-pyr-
azolo[3,4-d]pyrimidine-4-carboxylates 42e45 (0.00045 mol) in
anhydrous toluene (5 mL), diisopropylethylamine (0.00135 mol,
pyrazolo[3,4-d]pyrimidine-4-carboxylate (52). White solid, mp
ꢀ
1
1
28e130 C (53%). H NMR (400 MHz, DMSO-d
J ¼ 7.04 Hz, 3H, CH ), 1.49 (d, J ¼ 6.64 Hz, 6H, 2CH
J ¼ 7.04 Hz, 2H, CH
H, AreH), 8.46 (s, 1H, pyrazole-H), 11.72 (s, 1H, NH). LCeMS (ESI)
6
):
d
¼ 1.41 (t,
3
3
), 4.49 (q,
2
), 5.01 (sep, J ¼ 6.64 Hz, 1H, CH), 7.88e8.15 (m,
3
equiv) and substituted benzoyl chlorides (0.00135 mol, 3 equiv)
4
were added. The mixture was heated under reflux for 24 h. The
solvent was removed under reduced pressure and the resulting
residue was purified by column chromatography (hexane: EtOAc,
analysis (m/z) calcd for C19
H F
18 3
N
5
O
3
(421.37): found 422.1
¼ 6.9 min.
þ
[MþH] . HPLC: purity (254 nm) 98.1%; t
R
4
:6) to obtain the desired products (46e60) as solids.
4
.1.11.8. Ethyl-2-isopropyl-6-(4-methylbenzamido)-2H-pyrazolo[3,4-
ꢀ
d]pyrimidine-4-carboxylate (53). White solid, mp. 131e133
C
4
.1.11.1. Ethyl-6-benzamido-2-methyl-2H-pyrazolo[3,4-d]pyrimi-
1
(
51%). H NMR (400 MHz, DMSO-d
CH
), 1.51 (d, J ¼ 6.76 Hz, 6H, 2CH
J ¼ 7.16 Hz, 2H, CH ), 5.08 (sep, J ¼ 6.76 Hz, 1H, CH), 7.32e7.94 (m,
6
):
d
¼ 1.42 (t, J ¼ 7.16 Hz, 3H,
ꢀ
1
dine-4-carboxylate (46). White solid, mp 107e109 C (51%).
NMR (400 MHz, DMSO-d ): ), 4.03 (s,
), 4.50 (q, J ¼ 7.16 Hz, 2H, CH ), 7.51e8.03 (m, 5H, AreH),
.45 (s, 1H, pyrazole-H), 11.47 (s, 1H, NH). C NMR (400 MHz,
):
¼ 14.5, 34.2, 62.8, 109.9, 128.8, 128.9, 129.7, 132.7,
H
3
3
), 2.40 (s, 3H, CH
3
), 4.50 (q,
6
d
¼ 1.42 (t, J ¼ 7.16 Hz, 3H, CH
3
2
3
8
H, NeCH
3
2
4
H, AreH), 8.45 (s, 1H, pyrazole-H), 11.36 (s, 1H, NH). LCeMS (ESI)
13
þ
analysis (m/z) calcd for C19
HPLC: purity (254 nm) 98.1%; t
H
21
N
5
O
3
(367.40): found 368.1 [MþH] .
DMSO-d
6
d
R
¼ 5.0 min.
134.0, 150.8, 155.6, 156.2, 163.4, 166.3. LCeMS (ESI) analysis (m/z)
þ
calcd for C16
H
15
N
5
O
3
(325.32): found 326.1 [MþH] . HPLC: purity
4.1.11.9. Ethyl-6-(4-fluorobenzamido)-2-neopentyl-2H-pyrazolo[3,4-
(
254 nm) 100%; t
R
¼ 3.0 min.
ꢀ
d]pyrimidine-4-carboxylate (54). Pale yellow solid, mp 112e114 C
1
(
57%). H NMR (400 MHz, DMSO-d
J ¼ 7.16 Hz, 3H, CH ), 4.21 (s, 2H, CH
.33e8.09 (m, 4H, AreH), 8.48 (s, 1H, pyrazole-H), 11.49 (s, 1H, NH).
6
):
d
¼ 0.96 (s, 9H, 3CH
3
), 1.43 (t,
4
.1.11.2. Ethyl-6-(4-fluorobenzamido)-2-methyl-2H-pyrazolo[3,4-d]
3
2
), 4.49 (q, J ¼ 7.16 Hz, 2H, CH
2
),
ꢀ
pyrimidine-4-carboxylate (47). White solid, mp 103e105 C (53%).
7
1
H NMR (400 MHz, DMSO-d
s, 3H, NeCH
6
):
d
¼ 1.42 (t, J ¼ 7.04 Hz, 3H, CH
3
), 4.03
LCeMS (ESI) analysis (m/z) calcd for C20
4
H
22FN
5
O
3
(399.42): found
(
3
), 4.50 (q, J ¼ 7.04 Hz, 2H, CH
2
), 7.34e8.11 (m, 4H,
þ
00.1[MþH] . HPLC: purity (254 nm) 98.9%; t
R
¼ 6.6 min.
AreH), 8.45 (s, 1H, pyrazole-H), 11.52 (s, 1H, NH). LCeMS (ESI)
analysis (m/z) calcd for C16
HPLC: purity (254 nm) 95.6%; t
þ
H
14FN
5
O
3
(343.31): found 344.1 [MþH] .
4
.1.11.10. Ethyl-2-neopentyl-6-(4-(trifluoromethyl)benzamido)-2H-
R
¼ 3.0 min.
pyrazolo[3,4-d]pyrimidine-4-carboxylate (4). Pale yellow solid, mp
ꢀ
1
1
3
16e118 C (61%). H NMR (400 MHz, DMSO-d
CH ), 4.20 (s, 2H, CH
), 1.47 (t, J ¼ 7.0 Hz, 3H, CH
), 7.93e8.20 (m, 4H, AreH), 8.53 (s, 1H, pyr-
6
):
d
¼ 0.99 (s, 9H,
4
.1.11.3. Ethyl-2-methyl-6-(4-(trifluoromethyl)benzamido)-2H-pyr-
3
3
2
), 4.55 (q,
azolo[3,4-d]pyrimidine-4-carboxylate
(48). White solid,
):
¼ 1.42 (t,
), 4.50 (q, J ¼ 7.16 Hz, 2H,
), 7.89e8.18 (m, 4H, AreH), 8.46 (s, 1H, pyrazole-H), 11.75 (s, 1H,
mp
J ¼ 7.02 Hz, 2H, CH
2
14e116 C (55%). 1H NMR (400 MHz, DMSO-d
ꢀ
d
1
6
azole-H), 11.76 (s, 1H, NH). LCeMS (ESI) analysis (m/z) calcd for
þ
J ¼ 7.16 Hz, 3H, CH
3 3
), 4.02 (s, 3H, NeCH
C
H
21 22
F
3
N
5
O
3
(449.43): found 450.1[MþH] . HPLC: purity
CH
2
(254 nm) 100%; t
R
¼ 8.0 min.
NH). LCeMS (ESI) analysis (m/z) calcd for C17
found 394.1 [MþH] . HPLC: purity (254 nm) 98.4%; t
H F
14 3
N
5
O
3
(393.35):
¼ 5.9 min.
þ
R
4
.1.11.11. Ethyl-6-(4-methylbenzamido)-2-neopentyl-2H-pyrazolo
ꢀ
[3,4-d]pyrimidine-4-carboxylate (5). Pale yellow solid, mp 97e99 C
1
4.1.11.4. Ethyl-2-methyl-6-(4-methylbenzamido)-2H-pyrazolo[3,4-d]
(48%). H NMR (400 MHz, DMSO-d
J ¼ 7.16 Hz, 3H, CH ), 2.45 (s, 3H, CH
J ¼ 7.16 Hz, 2H, CH ), 7.36e7.98 (m, 4H, AreH), 8.51 (s,1H, pyrazole-
H), 11.39 (s, 1H, NH). C NMR (400 MHz, DMSO-d
6
):
d
¼ 1.01 (s, 9H, 3CH
3
), 1.48 (t,
), 4.25 (s, 2H, CH ), 4.55 (q,
ꢀ
pyrimidine-4-carboxylate (49). White solid, mp 109e111 C (58%).
3
3
2
1
H NMR (400 MHz, DMSO-d
6
):
d
¼ 1.42 (t, J ¼ 7.16 Hz, 3H, CH
3
), 2.40
),
2
13
(
s, 3H, CH ), 4.03 (s, 3H, NeCH
3
3
), 4.50 (q, J ¼ 7.16, 2H, CH
2
6
):
d
¼ 14.5, 21.5,
7.33e7.95 (m, 4H, AreH), 8.44 (s, 1H, pyrazole-H), 11.38 (s, 1H, NH).
28.1, 33.8, 57.8, 62.7, 109.4, 128.9, 129.3, 129.9, 131.7, 133.9, 142.8,
LCeMS (ESI) analysis (m/z) calcd for C17
H
17
N
5
O
3
(339.35): found
150.9, 156.3, 163.5, 166.3. LCeMS (ESI) analysis (m/z) calcd for
þ
þ
þ
3
40.1 [MþH] , 362.1 [MþNa] . HPLC: purity (254 nm) 100.0%;
C H
21 25
N
5
O
3
(395.45): found 396.2 [MþH] . HPLC: purity (254 nm)
t
R
¼ 3.7 min.
100%; t
R
¼ 7.1 min.

796
G. Venkatesan et al. / European Journal of Medicinal Chemistry 92 (2015) 784e798
4
.1.11.12. Ethyl-6-benzamido-2-phenethyl-2H-pyrazolo[3,4-d]pyrim-
pyrazolo[3,4-d]pyrimidine-4-carboxylates 42e45 (0.00045 mol) in
ꢀ
idine-4-carboxylate (55). Pale yellow solid, mp 106e108 C (57%).
anhydrous toluene (5 mL), diisopropylethylamine (0.0027 mol,
6 equiv) and benzoyl chloride or substituted benzoyl chlorides
(0.0027 mol, 6 equiv) were added. The mixture was heated under
reflux for 24 h. The solvent was removed under reduced pressure
and the resulting residue was purified by column chromatography
(hexane: EtOAc, 4:6).
1
H NMR (400 MHz, DMSO-d
t, J ¼ 7.28 Hz, 2H, CH ), 4.56 (q, J ¼ 7.16 Hz, 2H, CH
), 7.22e7.32 (m, 5H, AreH), 7.57e8.08 (m, 5H,
6
):
d
¼ 1.48 (t, J ¼ 7.16 Hz, 3H, CH
3
), 3.34
), 4.72 (t,
(
2
2
J ¼ 7.28 Hz, 2H, CH
2
AreH), 8.49 (s, 1H, pyrazole-H), 11.51 (s, 1H, NH). LCeMS (ESI)
þ
analysis (m/z) calcd for C23
HPLC: purity (254 nm) 99.7%; t
H
21
N
5
O
3
(415.44): found 416.1 [MþH] .
R
¼ 3.3 min.
4
.1.12.1. Ethyl-6-(N-benzoylbenzamido)-2-methyl-2H-pyrazolo[3,4-
ꢀ
4
.1.11.13. Ethyl-6-(4-fluorobenzamido)-2-phenethyl-2H-pyrazolo
d]pyrimidine-4-carboxylate (61). White solid, mp. 105e107
(56%). H NMR (400 MHz, DMSO-d
CH ), 3.87 (s, 3H, NeCH
(m, 10H, AreH), 8.53 (s, 1H, pyrazole-H). LCeMS (ESI) analysis (m/z)
calcd for C23
C
1
[
3,4-d]pyrimidine-4-carboxylate (56). Pale yellow solid, mp
6
):
d
¼ 1.36 (t, J ¼ 7.16 Hz, 3H,
12e113 C (56%). 1H NMR (400 MHz, DMSO-d
ꢀ
):
), 3.25 (t, J ¼ 7.20 Hz, 2H, CH
), 4.65 (t, J ¼ 7.20 Hz, 2H, CH
AreH), 7.33e8.10 (m, 4H, AreH), 8.42 (s, 1H, pyrazole-H), 11.49 (s,
d
¼ 1.42 (t,
), 4.43 (q, J ¼ 7.16 Hz, 2H, CH
), 7.47e7.83
1
6
3
3
2
J ¼ 7.12 Hz, 3H, CH
3
2
), 4.49 (q,
þ
J ¼ 7.12 Hz, 2H, CH
2
2
), 7.16e7.25 (m, 5H,
H
19
N
5
O
4
(429.43): found 430.1 [MþH] . HPLC: purity
(254 nm) 98.0%; t
R
¼ 5.4 min.
1
H, NH). LCeMS (ESI) analysis (m/z) calcd for C23
H
20FN
5
O
3
þ
(
433.44): found 434.1 [MþH] . HPLC: purity (254 nm) 100%;
4.1.12.2. Ethyl-6-(4-bromo-N-(4-bromobenzoyl)benzamido)-2-
t
R
¼ 6.5 min.
methyl-2H-pyrazolo[3,4-d]pyrimidine-4-carboxylate (62).
White solid, mp. 128e130 C (53%). H NMR (400 MHz, DMSO-d ):
), 4.43 (q,
), 7.40e8.05 (m, 8H, AreH), 8.56 (s, 1H, pyr-
ꢀ
1
6
4
.1.11.14. Ethyl-2-phenethyl-6-(4-(trifluoromethyl)benzamido)-2H-
d
¼ 1.36 (t, J ¼ 7.08 Hz, 3H, CH
3 3
), 3.92 (s, 3H, NeCH
pyrazolo[3,4-d]pyrimidine-4-carboxylate (57). Pale yellow solid, mp
J ¼ 7.08 Hz, 2H, CH
2
05e107 C (52%). 1H NMR (400 MHz, DMSO-d
ꢀ
):
), 3.31 (t, J ¼ 7.26 Hz, 2H, CH
), 4.72 (t, J ¼ 7.26 Hz, 2H, CH
AreH), 7.39e8.16 (m, 4H, AreH), 8.49 (s, 1H, pyrazole-H), 11.56 (s,
d
¼ 1.48 (t,
azole-H). LCeMS (ESI) analysis (m/z) calcd for C23
H
O
4
1
6
2
17Br N
5
þ
J ¼ 7.16 Hz, 3H, CH
3
2
), 4.56 (q,
(586.26): found 587.2 [Mþ] . HPLC: purity (254 nm) 95.5%;
J ¼ 7.16 Hz, 2H, CH
2
2
), 7.24e7.31 (m, 5H,
t
R
¼ 13.0 min.
1
H, NH). LCeMS (ESI) analysis (m/z) calcd for C24
H
20
F
3
N
5
O
3
4.1.12.3. Ethyl-6-(N-(3,4-difluorobenzoyl)-3,4-difluorobenzamido)-
þ
(
483.44): found 483.1 [Mþ] . HPLC: purity (254 nm) 100%;
2-methyl-2H-pyrazolo[3,4-d]pyrimidine-4-carboxylate (63).
White solid, mp. 169e171 C (59%). H NMR (400 MHz, DMSO-d ):
), 4.43 (q,
), 7.52e8.00 (m, 6H, AreH), 8.55 (s,1H, pyrazole-
ꢀ
1
t
R
¼ 6.7 min.
6
d
¼ 1.36 (t, J ¼ 7.16 Hz, 3H, CH
3 3
), 3.92 (s, 3H, NeCH
4
.1.11.15. Ethyl-6-(4-methylbenzamido)-2-phenethyl-2H-pyrazolo
J ¼ 7.16 Hz, 2H, CH
2
[
1
3,4-d]pyrimidine-4-carboxylate (58). Pale yellow solid, mp
H). LCeMS (ESI) analysis (m/z) calcd for C23
H
15
F
4
N
5
O
4
(501.39):
43e144 C (50%). 1H NMR (400 MHz, DMSO-d
ꢀ
):
), 3.30 (t, J ¼ 7.28 Hz, 2H, CH
), 4.71 (t, J ¼ 7.28 Hz, 2H, CH ), 7.21e7.30
m, 5H, AreH), 7.37e7.99 (m, 4H, AreH), 8.47 (s, 1H, pyrazole-H),
1.40 (s, 1H, NH). LCeMS (ESI) analysis (m/z) calcd for C24
d
¼ 1.47 (t,
found 502.1 [MþH] . HPLC: purity (254 nm) 100.0%; t
þ
¼ 11.6 min.
6
R
J ¼ 7.16 Hz, 3H, CH
3
), 2.45 (s, 3H, CH
3
2
),
4
(
1
.54 (q, J ¼ 7.16 Hz, 2H, CH
2
2
4.1.12.4. Ethyl-6-(N-benzoylbenzamido)-2-isopropyl-2H-pyrazolo
ꢀ
[3,4-d]pyrimidine-4-carboxylate (64). White solid, mp 140e142
C
1
H
23
N
5
O
3
(53%). H NMR (400 MHz, DMSO-d
6
):
d
¼ 1.25 (d, J ¼ 6.64 Hz, 6H,
þ
(
429.47): found 430.1 [MþH] . HPLC: purity (254 nm) 100%;
2CH ), 4.45 (q, J ¼ 7.04 Hz, 2H, CH
3
), 1.37 (t, J ¼ 7.04 Hz, 3H, CH
3
2
),
t
R
¼ 7.2 min.
4.79 (sep, J ¼ 6.64 Hz,1H, CH), 7.47e7.97 (m,10H, AreH), 8.51 (s,1H,
pyrazole-H). LCeMS (ESI) analysis (m/z) calcd for C25
23
H N
5
O
4
þ
4
.1.11.16. Ethyl-6-(4-bromobenzamido)-2-phenethyl-2H-pyrazolo
(457.48): found 458.1 [MþH] . HPLC: purity (254 nm) 99.7%;
[
3,4-d]pyrimidine-4-carboxylate (59). Pale yellow solid, mp
t
R
¼ 8.0 min.
39e141 C (67%). 1H NMR (400 MHz, DMSO-d
ꢀ
):
), 3.25 (t, J ¼ 7.16 Hz, 2H, CH
), 4.65 (t, J ¼ 7.16 Hz, 2H, CH
AreH), 7.81e8.19 (m, 4H, AreH), 8.43 (s, 1H, pyrazole-H), 11.59 (s,
d
¼ 1.42 (t,
1
6
J ¼ 7.04 Hz, 3H, CH
3
2
), 4.49 (q,
4.1.12.5. Ethyl-6-(N-benzoylbenzamido)-2-phenethyl-2H-pyrazolo
J ¼ 7.04 Hz, 2H, CH
2
2
), 7.16e7.52 (m, 5H,
[3,4-d]pyrimidine-4-carboxylate (65). Pale yellow solid, mp
ꢀ
1
157e159 C (55%). H NMR (400 MHz, DMSO-d
6
):
d
¼ 1.41 (t,
1
H, NH). LCeMS (ESI) analysis (m/z) calcd for C23
H
20BrN
5
O
3
J ¼ 7.04 Hz, 3H, CH
3
), 2.87 (t, J ¼ 6.92 Hz, 2H, CH
2
), 4.48 (q,
þ
(
493.07): found 494.0 [MþH] . HPLC: purity (254 nm) 97.9%;
J ¼ 7.04 Hz, 2H, CH
2
), 4.57 (t, J ¼ 6.92 Hz, 2H, CH
2
), 6.92e7.25 (m,
t
R
¼ 10.3 min.
5H, AreH), 7.54e8.02 (m, 10H, AreH), 8.50 (s, 1H, pyrazole-H).
LCeMS (ESI) analysis (m/z) calcd for C30
520.19 [MþH] . PLC purity (254 nm); eluent: 95.8%; t
H
25
N
5
O
4
(519.55): found:
¼ 9.4 min.
þ
4.1.11.17. Ethyl-6-(3,4-dichlorobenzamido)-2-phenethyl-2H-pyrazolo
R
[
3,4-d]pyrimidine-4-carboxylate (60). Pale yellow solid, mp
47e149 C (64%). 1H NMR (400 MHz, DMSO-d
ꢀ
):
), 3.25 (t, J ¼ 7.28 Hz, 2H, CH
), 4.65 (t, J ¼ 7.28 Hz, 2H, CH
AreH), 7.77e8.26 (m, 3H, AreH), 8.43 (s, 1H, pyrazole-H), 11.66 (s,
H, NH). 13C NMR (400 MHz, DMSO-d
):
¼ 14.5, 34.9, 48.3, 62.8,
10.0, 126.9, 128.8, 129.1, 130.9, 131.1, 131.4, 131.7, 134.2, 135.0, 138.4,
d
¼ 1.42 (t,
4.1.13. General procedure for synthesis of ethyl 2-(alkyl or aralkyl)-
6-(2-phenylacetamido)-2H-pyrazolo[3,4-d]pyrimidine-4-
carboxylate (66e69)
To a suspension of ethyl-6-amino-2-(alkyl or aralkyl)-2H-pyr-
azolo[3,4-d]pyrimidine-4-carboxylates 42e45 (0.00045 mol) in
anhydrous toluene (5 mL), diisopropylethylamine (0.00135 mol,
3 equiv) and phenylacetyl chloride (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
purified by column chromatography (hexane: EtOAc, 4:6).
1
6
J ¼ 7.16 Hz, 3H, CH
3
2
), 4.49 (q,
J ¼ 7.16 Hz, 2H, CH
2
2
), 7.14e7.25 (m, 5H,
1
1
6
d
150.8, 155.4, 155.7, 163.3, 164.3. LCeMS (ESI) analysis (m/z) calcd for
þ
C
23
H
19Cl
2
N
5
O
3
(483.09): found 484.1 [Mþ] . HPLC: purity (254 nm)
97.7%; t
R
¼ 11.1 min.
4.1.12. General procedure for synthesis of ethyl 6-(N-
benzoylbenzamido)-2-methyl-2H-pyrazolo[3,4-d]pyrimidine-4-
carboxylate (61e65)
4.1.13.1. Ethyl-2-methyl-6-(2-phenylacetamido)-2H-pyrazolo[3,4-d]
ꢀ
pyrimidine-4-carboxylate (66). White solid, mp 168e170 C (49%).
1
To a suspension of ethyl 6-amino-2-(alkyl or aralkyl)-2H-
H NMR (400 MHz, DMSO-d
6
):
d
¼ 1.42 (t, J ¼ 7.16 Hz, 3H, CH
3
), 3.87

G. Venkatesan et al. / European Journal of Medicinal Chemistry 92 (2015) 784e798
797
(
7
s, 2H, CH
2
), 3.98 (s, 3H, NeCH
3
), 4.49 (q, J ¼ 7.16 Hz, 2H, CH
2
),
4.4. Homology modeling
.24e7.38 (m, 5H, AreH), 8.39 (s,1H, pyrazole-H), 11.29 (s, 1H, NH).
LCeMS (ESI) analysis (m/z) calcd for C17
H
17
N
5
O
3
(339.35): found:
The crystallographic structure of human A2A AR complexed with
ZM-241385 as high affinity antagonist (PDB code: 3EML) [39] was
employed to build up a homology model of the hA AR by using
3
þ
3
40.1 [MþH] . HPLC: purity (254 nm) 100%; t
R
¼ 3.8 min.
4
.1.13.2. Ethyl-2-isopropyl-6-(2-phenylacetamido)-2H-pyrazolo[3,4-
software Modeller version 9.11 [40e43]. The tip of the second
extracellular loop (Gln148 to Ser156) is absent in the crystal
structure of A2A AR (PDB code: 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 re-
ported in Jaakola et al. [39]. The refined model was validated using
PROCHECK program in Protein Structure Validation Suite (PSVS)
ꢀ
d]pyrimidine-4-carboxylate (67). White solid, mp 120e122
C
1
(
45%). H NMR (400 MHz, DMSO-d
CH
), 1.49 (d, J ¼ 6.64 Hz, 6H, 2CH
J ¼ 7.16 Hz, 2H, CH ), 5.04 (sep, J ¼ 6.64 Hz, 1H, CH), 7.25e7.37 (m,
6
):
d
¼ 1.41 (t, J ¼ 7.16 Hz, 3H,
3
3
), 3.87 (s, 2H, CH
2
), 4.49 (q,
2
5
H, AreH), 8.39 (s, 1H, pyrazole-H), 11.28 (s, 1H, NH). LCeMS (ESI)
þ
analysis (m/z) calcd for C19
HPLC: purity (254 nm) 100%; t
H
21
N
5
O
3
(367.40): found 368.1 [MþH] .
R
¼ 4.1 min.
[44].
4
.1.13.3. Ethyl-2-neopentyl-6-(2-phenylacetamido)-2H-pyrazolo[3,4-
ꢀ
d]pyrimidine-4-carboxylate (68). Pale yellow solid, mp 112e114 C
4.5. Molecular docking simulations
1
(
47%). H NMR (400 MHz, DMSO-d
J ¼ 7.16 Hz, 3H, CH ), 3.92 (s, 2H, CH
), 7.28e7.41 (m, 5H, AreH), 8.45 (s, 1H, pyrazole-
6
):
d
¼ 0.97 (s, 9H, 3CH
3
), 1.46 (t,
), 4.21 (s, 2H, CH ), 4.53 (q,
3
2
2
Ligand structures were built using MOE-builder tool [36] and
were subjected to MMFF94x energy minimization until the rms of
J ¼ 7.16 Hz, 2H, CH
2
ꢁ
1
ꢁ1
. Partial charges for the
H), 11.30 (s, 1H; NH). LCeMS (ESI) analysis (m/z) calcd for
conjugate gradient was <0.05 kcal mol
Ǻ
þ
C
9
21
H
25
N
5
O
3
(395.45): found 396.2 [MþH] . HPLC: purity (254 nm)
ligands were calculated using PM3/ESP methodology.
All ligands were docked into the hypothetical TM binding site of
3
the hA AR model and that of the hA2AAR crystal structure by using
9.9%; t
R
¼ 5.7 min.
4
.1.13.4. Ethyl-2-phenethyl-6-(2-phenylacetamido)-2H-pyrazolo[3,4-
the docking tool of the MOE suite [36]. In the MOE-Dock, the Alpha
PMI placement method, followed by force field refinement and
London dG scoring, were used for the docking runs. MOE-Dock
performed 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
subjected to MMFF94x energy minimization until the rms of con-
ꢀ
d]pyrimidine-4-carboxylate (69). Pale yellow solid, mp 141e143 C
1
(
53%). H NMR (400 MHz, DMSO-d
CH
), 3.26 (t, J ¼ 7.16 Hz, 2H, CH
J ¼ 7.16 Hz, 2H, CH ), 4.65 (t, J ¼ 7.16 Hz, 2H, CH
AreH), 7.35e7.41 (m, 5H, AreH), 8.41 (s, 1H, pyrazole-H), 11.28 (s,
6
):
), 3.92 (s, 2H, CH
), 7.16e7.31 (m, 5H,
d
¼ 1.44 (t, J ¼ 7.16 Hz, 3H,
3
2
2
), 4.51 (q,
2
2
1
H; NH). LCeMS (ESI) analysis (m/z) calcd for C24
H
23
N
5
O
3
(429.47):
þ
ꢁ1
ꢁ1
found 430.1 [MþH] . HPLC: purity (254 nm) 100%; t
R
¼ 7.7 min.
jugate gradient was <0.1 kcal mol
Ǻ . Prediction of antago-
nistereceptor complex stability (in terms of corresponding pK
i
4
.2. Binding assay and adenylyl cyclase activity assay
value) and the quantitative analysis for non bonded intermolecular
interactions (H-bonds, hydrophobic, electrostatic) were calculated
and visualized using tools implemented in MOE suite [36].
Binding experiments with respective tritiated radioligands at
AR, hA2AAR and hA AR were carried out for 3 h at room tem-
hA
1
3
perature as described previously [33]. Nonspecific binding was
determined in the presence of 1 mM theophylline for hA AR and
M (R)-N -phenyliso-propyladenosine (R-PIA) for both hA2AAR
and hA AR. Bound and free radioactivity was separated by filtering
the assay mixture through Whatman GF/B glass-fibre filters using a
Micro-Mate 196-cell harvester (Packard Instrument Company). The
filter bound radioactivity was counted on Top Count (efficiency of
Acknowledgements
1
6
100
m
This work was supported by the National University of
Singapore, and Singapore Ministry of Education (MOE2009-T2-2-
011 and R-398-000-068-112; NUS-FRC R-148-000-164-112 and
NUS NUSAGE N-148-000-009-001). The authors would like to
thank Sonja Kachler for her assistance and expertise in the phar-
macological evaluation of the compounds.
3
i
57%) with Micro-Scint 20. The K values were calculated from
competition curves by nonlinear curve fitting with the program
SCTFIT [34]. In the case of hA2BAR, adenylyl cyclase experiments
were carried out as described previously with minor modifications
Appendix A. Supplementary data
[
33e35]. Membranes transfected with hA2B receptor were incu-
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.01.046.
3
2
bated with 100 nM NECA, as well as 150,000 cpm of [a- P]ATP and
tested compounds in different concentrations for 20 min in the
incubation mixture without EGTA (ethylene glycoltetraacetic acid)
References
i
and NaCl. The K values for concentration-dependent inhibition of
NECA-mediated adenylyl cyclase activity caused by tested antago-
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