A novel and efficient synthesis of pyrazolo[3,4-d]pyrimidine derivatives and the study of their anti-bacterial activity

Article abstract of DOI:10.1016/j.cclet.2013.04.035

In this work 4-amino-6-aryl-2-phenyl pyrimidine-5-carbonitrile derivatives were synthesized through a one-pot, three-component reaction of an aldehyde, malononitrile and benzamidine hydrochloride, in the presence of magnetic nano Fe₃O₄ particles as a catalyst under solvent-free conditions. 3-Amino-6-aryl-2-phenylpyrazolo[3,4-d]pyrimidine derivatives were prepared through an efficient and environmentally friendly reaction between 4-amino-6-aryl-2-phenylpyrimidine-5-carbonitrile derivatives and hydrazine hydrate and their antibacterial activity has been evaluated.

Full text of DOI:10.1016/j.cclet.2013.04.035

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Chinese Chemical Letters xxx (2013) xxx–xxx
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Original article
A novel and efficient synthesis of pyrazolo[3,4-d]pyrimidine derivatives
and the study of their anti-bacterial activity
a
b
c,d
Shahnaz Rostamizadeh a, , Masoomeh Nojavan , Reza Aryan , Hamid Sadeghian
,
*
Mahdieh Davoodnejad d
a Department of Chemistry, Faculty of Science, K. N. Toosi University of Technology, P.O. Box 15875-4416, Tehran, Iran
b Department of Chemistry, Faculty of Science, University of Zabol, Zabol, Iran
c Resistance Research Center, Buali Research Institute, Mashhad University of Medical Sciences, Mashhad 91967-73117, Iran
d Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences, Mashhad 91857-63788, Iran
A R T I C L E I N F O
A B S T R A C T
Article history:
In this work 4-amino-6-aryl-2-phenyl pyrimidine-5-carbonitrile derivatives were synthesized through a
one-pot, three-component reaction of an aldehyde, malononitrile and benzamidine hydrochloride, in the
presence of magnetic nano Fe3O4 particles as a catalyst under solvent-free conditions. 3-Amino-6-aryl-
2-phenylpyrazolo[3,4-d]pyrimidine derivatives were prepared through an efficient and environmentally
friendly reaction between 4-amino-6-aryl-2-phenylpyrimidine-5-carbonitrile derivatives and hydra-
zine hydrate and their antibacterial activity has been evaluated.
Received 20 February 2013
Received in revised form 7 April 2013
Accepted 11 April 2013
Available online xxx
Keywords:
ß 2013 Shahnaz Rostamizadeh. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All
rights reserved.
Pyrazolo[3,4-d]pyrimidine derivatives
Pyrimidine-5-carbonitrile derivatives
Antibacterial activity
Staphylococcus aureus
Enterococcus raffinosus
1. Introduction
has some advantages, including benign reaction conditions, fewer
synthetic steps to reach pyrazolo[3,4-d]pyrimidine derivatives
Fused pyrimidine derivatives have shown diverse biological
activities. Among them, pyrazolo[3,4-d]pyrimidines as purine
analogs have attracted considerable interest due to their remark-
able pharmacological properties. These compounds were designed
and synthesized as potent and selective kinase inhibitors [1,2],
antileishmanial, antitrypanosomal [3], antibacterial [4] and
antiviral agents [5] and adenosine A2A receptor antagonists [6].
Some literature examples exist for the synthesis of pyrazolo[3,4-
d]pyrimidines [7–13]. These methods suffer from some drawbacks
such as lengthy reaction sequences, expensive reagents, and/or
tedious workup procedures. These interesting bioactivity of
pyrazolo[3,4-d]pyrimidines motivated us to search for a more
facile synthetic route for the 3-amino-6-aryl-2-phenylpyra-
zolo[3,4-d]pyrimidine derivatives. Herein, we wish to report a
straightforward synthesis of novel pyrazolo[3,4-d]pyrimidines
derivatives using 6-amino-4-aryl-2-phenylpyrimidine-5-carboni-
trile derivatives as the starting materials. This synthetic protocol
compared to other reports from readily available materials, simple
work-up, high overall yields of the products and the simultaneous
conversion of nitro to amino groups, which provides an
opportunity to synthesize more complex structures with addi-
tional reaction steps.
2. Experimental
Melting points were recorded on a Buchi B-540 apparatus. IR
spectra were recorded on an ABB Bomem Model FTLA200-100
instrument. 1H NMR and 13C NMR spectra were measured on a
Bruker DRX-300 spectrometer at 300 MHz and 75 MHz using TMS
as an internal standard. Chemical shifts (d) were reported relative
to TMS, and coupling constants (J) were reported in hertz (Hz).
Mass spectra were recorded on a Shimadzu QP 1100 EX mass
spectrometer with 70 eV ionization potential. Elemental analyses
of new compounds were performed with a Vario EL III 0 Serial No.
11024054 instrument and their results favorably agreed with the
calculated values. The bacterial strains were isolated from different
organs of patients at the Microbiological Laboratory of Ghaem
Hospital of Medical University of Mashhad, Iran.
* Corresponding author.
E-mail addresses: shrostamizadeh@yahoo.com, rostamizadeh@hotmail.com
(S. Rostamizadeh).
1001-8417/$ – see front matter ß 2013 Shahnaz Rostamizadeh. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.04.035
Please cite this article in press as: S. Rostamizadeh, et al., A novel and efficient synthesis of pyrazolo[3,4-d]pyrimidine derivatives and
the study of their anti-bacterial activity, Chin. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.cclet.2013.04.035

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Ar
O
N
N
N
Nano Fe3O4
CN
NH2
+
+
Ar
H
100 oC
H2N
NH2Cl
N
1
4
3
2
Scheme 1. Reaction used for the synthesis of pyrimidine-5-carbonitrile derivatives.
2.1. General procedure for the synthesis of 4a–4m
compound that prevented the bacteria growth (<99%) was
designated as the minimum inhibitory concentration (MIC).
Growth was present in the medium control but was absent from
the inoculum control.
Malonitrile (1 mmol), aldehyde (1 mmol), benzamidine hydro-
chloride (1 mmol), magnetic nano Fe3O4 (0.06 g), were ground and
placed in a test tube. The reaction mixture was stirred under
heating for 1–1.5 h at 100 8C and monitored by TLC, (EtOAc/
petroleum ether, 1:2). After the completion of the reaction, the
reaction mixture was cooled to ambient temperature, EtOAc was
added, and the nano Fe3O4 was separated using an external
magnet, then the mixture was concentrated in vacuo to obtain a
solid product. The residues were further purified by crystallization
from EtOH. It is worth to mention that the nano Fe3O4 could be
reused three times without any significant loss of activity.
3. Results and discussion
Initially, aromatic aldehyde 1, malononitirile 2, and benzami-
dine hydrochloride 3, were mixed and heated in the presence of
magnetic Fe3O4 nanoparticles as a catalyst under solvent-free
conditions at 100 8C (Scheme 1, Table 1). The products 4a–m were
all prepared with excellent yields at 100 8C in 1–1.5 h. Both
aromatic aldehydes with electron donating substituents (Table 1,
entries 7 and 13) and electron-withdrawing substituents (Table 1,
entries 6, 9 and 10) showed significant reactivity in this process.
Then, 6-amino-4-(4-chlorophenyl)-2-phenylpyrimidine-5-car-
bonitrile (1 mmol) (Table 1, entry 2, 4b), hydrazine hydrate (1 mL)
and EtOH (5 mL) were mixed and refluxed for 8 h. The reaction was
completed and only one new spot appeared on the TLC plate. After
aqueous work-up, a pure pale yellow solid was obtained from the
reaction mixture whose full characterization by spectroscopic
methods (IR, 1H NMR, 13C NMR, and mass spectra) revealed that
pyrazolo[3,4-d]pyrimidine 5b was obtained. In order to determine
the amount of hydrazine needed in this process, the reaction of
pyrimidine 4b was performed using 0.5 mL of hydrazine hydrate
and the same product was again obtained.
2.2. General procedure for the synthesis of 5a–5i
To a solution of 4-amino-6-aryl-2-phenyl pyrimidine-5-carbo-
nitrile derivatives (1 mmol) in EtOH (5 mL) was added hydrazine
hydrate (0.5 mL) and the mixture was heated under reflux. After
the completion of the reaction (monitored by TLC, EtOAc:
petroleum ether, 4:1) the reaction mixture was cooled to ambient
temperature and water (10 mL) was added. The solid materials
were filtered off and filtrate was concentrated and recrystallized
from ethanol.
Selected analytical data: 3-Amino-6-(4-chlorophenyl)-2-phenyl-
pyrazolo[3,4-d]pyrimidine (5b): Pale yellow powder; mp: 267 8C; IR
(KBr, cmÀ1):
n d
3449, 3325, 3307; 1H NMR (300 MHz, DMSO-d6):
However, using 0.065 mL (1 mmol) of hydrazine hydrate did
not result in the formation of product 5b and only the starting
material was recovered. So, the amount of 0.5 mL was chosen as
the optimal amount of hydrazine hydrate for each mmol of
pyrimidine derivatives. In the next step a model reaction (Scheme
1, Ar = 4-Cl–Ph) was performed in different solvents under
different conditions and the results showed that using EtOH as
solvent under reflux is the best condition. In order to examine the
scope and generality of this protocol for the synthesis of other
fused pyrazolo[3,4-d]pyrimidine derivatives, the pyrimidine deri-
vatives shown in Table 1 were also subjected to this process
12.82 (s, 1H, NH), 8.48–8.49 (m, 2H), 8.04–8.07 (d, 2H, J = 8.3 Hz),
7.66–7.69 (d, 2H, J = 8.3 Hz), 7.51–7.53 (m, 3H), 5.19 (s, 2H, NH2);
13C NMR (75 MHz, DMSO-d6):
d 159.9, 159.8, 156.1, 147.8, 137.6,
136.0, 135.3, 131.2, 130.6, 128.8, 128.6, 128.1, 100.7; MS (m/z): 321
(54); Anal. Calcd. for C17H12ClN5: C, 63.46; H, 3.76; Cl, 11.02; N,
21.77; Found: C, 63.58; H, 3.75; N, 21.65. 3-Amino-6-(3-amino-
phenyl)-2-phenylpyrazolo[3,4-d]pyrimidine (5g): Pale yellow pow-
der; mp: 311.5 8C; IR (KBr, cmÀ1):
n
3430, 3416, 3341; 1H NMR
(300 MHz, DMSO-d6): 12.00 (s, NH, br), 8.47–8.50 (m, 2H), 7.50–
d
7.52 (m, 3H), 7.23–7.28 (m, 1H), 7.13 (s, 1H), 7.00–7.03 (d, 1H,
J = 7.5 Hz), 6.77–6.80 (d, 1H, J = 7.4 Hz), 5.42 (s, 2H, NH2), 5.09 (s,
2H, NH2); 13C NMR (75 MHz, DMSO-d6):
d 162.1, 160.0, 155.8,
149.2, 147.6, 138.0, 137.7, 130.4, 129.3, 128.5, 128.0, 116.1, 115.7,
113.7,100.4; MS (m/z): 302 (100), 301 (64); Anal. Calcd. for
Table 1
Synthesis of pyrimidines 4a–m under solvent free conditions catalyzed by magnetic
C
17H14N6: C, 67.54; H, 4.67; N, 27.80; Found: C, 67.47; H, 4.56; N,
Fe3O4 nanocatalyst.
28.18.
Entry
Product
Ar
Yielda (%)
Mp (8C)
Lit. mp (8C)
1
2
4a
4b
4c
4d
4e
4f
Ph
98
96
94
96
96
98
96
96
96
96
90
95
95
214
210–212 [15]
222 [15]
236[14]
–
2.3. Determination of minimum inhibitory concentration (MICs)
4-Cl–Ph
222
3
4-Br–Ph
234–236
231–234
200–202
299–300
211
The MICs were assayed by the tube dilution test method,
introduced by National Committee for Clinical Laboratory Stan-
dards (NCCLS). A serial of dilutions of tested compounds (final
4
2,3-diCl–Ph
2-Cl–Ph
5
196 [16]
–
6
4-CN–Ph
4-Me–Ph
2,4-diCl–Ph
3-NO2–Ph
4-NO2–Ph
3-indolyl
4-CH3CONH–Ph
4-MeO–Ph
7
4g
4h
4i
210 [15]
170–17 [18]
–
concentration of 300–0.8
mmol/L) were added to the tested
8
174
bacteria in Mueller–Hinton broth (0.5 mL) in 24-well plates and
were incubated at 37 8C for 24 h (5 Â 105 CFU/mL). After sufficient
amount of incubation (24 h), the wells were examined for turbidity
as an indicator for growth of the bacteria. For further confirmation,
an aqueous solution of 2,3,5-triphenyltetrazolium chloride (0.5%;
9
201–202
215–217
230–232
245
10
11
12
13
4j
215 [16,17]
–
4k
4l
243–244 [17]
213 [15]
4m
213
a
100
m
L) was added to the wells. The lowest concentration of the
Isolated yield.
Please cite this article in press as: S. Rostamizadeh, et al., A novel and efficient synthesis of pyrazolo[3,4-d]pyrimidine derivatives and
the study of their anti-bacterial activity, Chin. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.cclet.2013.04.035

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3
Table 2
Synthesis of 5a–i in EtOH under reflux condition.
Entry
1
Pyrimidine
CN
Product
H2N
Time (h)
8
Yield (%)a
97
Mp (8C)
261–264
N
NH
NH2
N
N
N
N
Ph
Ph
5a
2
3
4
8
8
1
95
91
98
267
271
212
H2N
N
Cl
Br
Cl
Br
CN
N
NH
NH2
N
N
N
N
Ph
Ph
5b
H2N
N
CN
N
NH
NH2
N
N
Ph
Ph
5c
H2N
N
CN
N
NH2
NH
Cl
N
N
Cl
N
Ph
Ph
5d
5e
5
3
85
261
215
NC
H2N
N
CN
N
NH
NH2
N
N
N
Ph
Ph
6
7
2
5
97
90
H2N
CN
N
NH
NH2
N
N
N
N
Ph
Ph
5f
311.5
H2N
N
CN
N
NH
NH2
O2N
O2N
H2N
H2N
N
N
N
Ph
Ph
5g
8
5
8
88
95
223–229
H2N
N
CN
N
NH
NH2
N
N
N
Ph
Ph
5h
9
211
O
H2N
N
O
CN
N
NH2
NH
N
N
N
Ph
Ph
5i
a
Isolated yield.
(Scheme 2, Table 2). The results of the synthesis of the fused
pyrimidine derivatives are presented in Table 2. Some unantici-
pated observations are made from the data in Table 2.
When the pyrimidine derivatives with a 4-(3-nitrophenyl) or a
4-(4-nitrophenyl) group (Table 1, entries 9 and 10) were used, the
reaction led to the formation of pyrazolopyrimidine derivatives
with the nitro group on the phenyl ring having been reduced to an
amino group (Table 2, entries 7 and 8). Such a transformation has
been reported before in the literature for the reduction of nitro to
amino group using hydrazine as reductant [19]. Similarly, when a
pyrimidine derivative with a 4-(4-cyanophenyl) substituent
(Table 1, entry 6) was used, we again obtained a pyrazolopyr-
imidine derivative lacking a cyano group on the phenyl ring as
can be seen in product 5a (Table 2, entry 5). When bulky aryl
substituents were placed at position 4 of the pyrimidine ring
(Table 1, entries 4, 8, 11 and 12), the addition of hydrazine
hydrate led to decomposition of the pyrimidine to the
benzylidiene malononitrile and benzamidine intermediates
rather than the formation of related pyrazolo[3,4-d]pyrimidine
derivatives.
Please cite this article in press as: S. Rostamizadeh, et al., A novel and efficient synthesis of pyrazolo[3,4-d]pyrimidine derivatives and
the study of their anti-bacterial activity, Chin. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.cclet.2013.04.035

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S. Rostamizadeh et al. / Chinese Chemical Letters xxx (2013) xxx–xxx
H2N
N
X
Y
N
NH
CN
NH2
EtOH
NH2NH2.H2O
+
N
N
N
reflux
Ph
Ph
4
5
Scheme 2. Synthesis of 3-amino-6-aryl-2-phenylpyrazolo[3,4-d]pyrimidine derivatives under optimized reaction conditions.
References
Table 3
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The inhibitory property of the synthetic compounds against
two Gram-negative strains of bacteria, Pseudomanas aeruginosa
and Klebsiella pneumoniae, and two Gram-positive bacteria
Staphylococcus aureus and Enterococcus raffinosus were evaluated.
The results are presented in Table 3 and penicillin G was used as a
reference antibacterial agent. Compounds 5a–i exhibited weak to
moderate antibacterial activity only against the Gram-positive
pathogens (MIC = 3.8–300
tested compounds against E. raffinosus (MIC = 3.8–202.5
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most active agents. The MIC values of 3.8 mol/L and 4.2 mol/L
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In summary, we have developed a novel, efficient protocol for
the synthesis of fused pyrazolo[3,4-d]pyrimidine derivatives from
the corresponding pyrimidine-5-carbonitrile derivatives. The
spontaneous conversion of a nitro to an amino group that provides
an opportunity to synthesize more complex structures with
additional reaction steps is a unique advantage of this protocol.
Also these compounds were evaluated for biological activity and 5c
and 5i showed interesting antibacterial activity compared to the
reference penicillin G.
Please cite this article in press as: S. Rostamizadeh, et al., A novel and efficient synthesis of pyrazolo[3,4-d]pyrimidine derivatives and
the study of their anti-bacterial activity, Chin. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.cclet.2013.04.035