Synthesis and in vitro antibacterial evaluation of 6-substituted 4-amino-pyrazolo[3,4-d]pyrimidines

Article abstract of DOI:10.1007/s11696-017-0163-2

Pyrazolo[3,4-d]pyrimidines are one of the most important classes of fused heterocyclic compounds which exhibit a broad range of biological and medicinal properties. They are known as anticancer, antifungal, antibacterial, antiviral and anti-inflammatory agents. In this study, some new 6-substituted 4-amino-pyrazolo[3,4-d]pyrimidine derivatives were prepared via reaction of 5-amino-3-methyl-1-phenyl-1H-pyrazole-4-carbonitrile with various nitriles in the presence of sodium ethoxide as catalyst. The inhibitory properties of synthesized compounds were studied according to CLSI guidelines against some pathogenic bacteria including four gram-positive strains (Streptococcus pyogenes, Staphylococcus aureus, Bacillus cereus and Bacillus subtilis subsp. spizizenii) and three gram-negative strains (Pseudomonas aeruginosa, Shigella flexneri and Salmonella enterica subsp. enterica). The antibacterial effects of all derivatives were compared with those of antibiotics belonging to different classes. The values were reported as inhibition zone diameter (IZD), minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The effect of substituents on the biological activity of derivatives was discussed as well. The inhibitory effect of compound 6a, was shown to be the most, with MIC values in the range of 32-4096 μg/mL. Since most of the synthesized compounds were effective against Streptococcus pyogenes and Pseudomonas aeruginosa, they can be considered as inhibitors of these two bacteria.

Full text of DOI:10.1007/s11696-017-0163-2

Chem. Pap.
DOI 10.1007/s11696-017-0163-2
ORIGINAL PAPER
Synthesis and in vitro antibacterial evaluation of 6-substituted
4-amino-pyrazolo[3,4-d]pyrimidines
Hamid Beyzaei1 Mohammadreza Moghaddam-Manesh2 Reza Aryan1
•
•
•
Behzad Ghasemi3 Alireza Samzadeh-Kermani1
•
Received: 2 January 2017 / Accepted: 14 March 2017
Ó Institute of Chemistry, Slovak Academy of Sciences 2017
Abstract Pyrazolo[3,4-d]pyrimidines are one of the most
important classes of fused heterocyclic compounds which
exhibit a broad range of biological and medicinal proper-
ties. They are known as anticancer, antifungal, antibacte-
rial, antiviral and anti-inflammatory agents. In this study,
some new 6-substituted 4-amino-pyrazolo[3,4-d]pyrim-
idine derivatives were prepared via reaction of 5-amino-3-
methyl-1-phenyl-1H-pyrazole-4-carbonitrile with various
nitriles in the presence of sodium ethoxide as catalyst. The
inhibitory properties of synthesized compounds were
studied according to CLSI guidelines against some patho-
genic bacteria including four gram-positive strains (Strep-
tococcus pyogenes, Staphylococcus aureus, Bacillus cereus
and Bacillus subtilis subsp. spizizenii) and three gram-
negative strains (Pseudomonas aeruginosa, Shigella flex-
neri and Salmonella enterica subsp. enterica). The
antibacterial effects of all derivatives were compared with
those of antibiotics belonging to different classes. The
values were reported as inhibition zone diameter (IZD),
minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC). The effect of sub-
stituents on the biological activity of derivatives was dis-
cussed as well. The inhibitory effect of compound 6a, was
shown to be the most, with MIC values in the range of
32–4096 lg/mL. Since most of the synthesized compounds
were effective against Streptococcus pyogenes and Pseu-
domonas aeruginosa, they can be considered as inhibitors
of these two bacteria.
Keywords Antibacterial effects Á Pyrazolo[3,4-
d]pyrimidines Á Nitriles Á Cyclocondensation Á Sodium
ethoxide catalyst
Introduction
Pyrazolo[3,4-d]pyrimidine core is founded in many natural
products and biologically active compounds. A vast num-
ber of studies in the field of cancer showed the inhibitory
activity of pyrazolo[3,4-d]pyrimidines against protein
kinase enzymes (Laurenzana et al. 2016; Verschueren et al.
2017). Allopurinol (1,5-dihydro-4H-pyrazolo[3,4-d]pyrim-
idin-4-one) was known as an anticancer agent and inhibitor
of xanthine oxidase (Irwig and Krakoff 1966; Kumar et al.
2011). It was also used to treat gout and hyperuricemia.
The derivatives of pyrazolo[3,4-d]pyrimidin-4-one were
described as adenosine deaminase inhibitors by La Motta
et al. (2009). Anti-HSV-1 activity of some pyrazolo[3,4-
d]pyrimidine derivatives was examined using plaque
reduction infectivity assay by Rashad et al. (2009). In a
research project, the specific absorption rate (SAR) and
activity of substituted 4-amino-1H-pyrazolo[3,4-d]pyrim-
idines were measured against insulin-like growth factor-1
receptor (IGF1R), epidermal growth factor receptor
(EGFR) and receptor tyrosine-protein kinase 2 (ErbB2) by
Wang et al. (2010).
& Hamid Beyzaei
hbeyzaei@yahoo.com; hbeyzaei@uoz.ac.ir
1
Present Address: Department of Chemistry, Faculty of
Science, University of Zabol, Zabol, Iran
2
A wide range of pharmacological properties such as
anti-inflammatory and analgesic (Kadry 2014), anti-tuber-
cular (Trivedi et al. 2012) antitumor and antiparasitic
(Kandeel et al. 2013; Ma et al. 2016) and herbicidal
Department of Chemistry, Faculty of Science, Kerman
Branch, Islamic Azad University, Kerman, Iran
3
Young Researchers and Elite Club, Neyshabur Branch,
Islamic Azad University, Neyshabur, Iran
123

Chem. Pap.
activities (Liu et al. 2007) were also described in the
literatures.
and 100 MHz, respectively, in DMSO-d6 with SiMe4 (1H
and 13C) internal standard. Elemental analyses were per-
formed for C, H and N on a Thermo Finnigan Flash EA
microanalyzer.
There are several approaches reported in the synthesis of
pyrazolo[3,4-d]pyrimidines, for instance: reaction of
5-amino-pyrazole-4-carbohydrazides with formamide and
hydrazine hydrate under microwave irradiation (Todorovic
et al. 2011), or with urea and decaline (Ghorab et al. 2010)
or with benzoyl chloride derivatives and triethylorthofor-
mate (Daidone et al. 2002). There are reports on the
reaction of ethyl 5-amino-1H-pyrazole-4-carboxylates with
hydrazine hydrate and triethylorthoformate (Ghorab et al.
2010) and also with formamide or benzoyl isothiocyanate
(Carraro et al. 2006) as well as reaction of 4-hy-
drazinylpyrimidine derivatives with isocyanates (Bhuyan
et al. 2002). It is also observed that this ring system could
form in the reaction of t-butyl carbazate with 4-chloropy-
rimidine-5-carbonitrile (Soth et al. 2011) or treatment of
5-aminopyrazole derivatives with formamide in the pres-
ence of phosphorous tribromide (Huang et al. 2012).
One of the common starting materials in the synthesis of
pyrazolo[3,4-d]pyrimidines is 5-amino-4-cyanopyrazoles
(Quintela et al. 2001; Song et al. 2011; Rahmouni et al.
2014; Agrebi et al. 2014; Tintori et al. 2015; Siebeneicher
et al. 2016). Reaction of these compounds with nitriles
under various conditions is reported (Hanefeld et al. 1996;
Smith et al. 2007; Davoodnia et al. 2012). Among which,
EtONa(or K) in ethanol is less costly and more appropriate
in comparison to other available methods (Hanefeld et al.
1996; Salaheldin et al. 2009).
Synthesis of 5-amino-3-methyl-1-phenyl-1H-pyrazole-4-
carbonitrile (4)
Compound 4 was prepared according to the procedure
reported in the literature (Allouche et al. 2013), as follows:
two drops of acetic acid was added to a solution of
malononitrile (1) (0.01 mol, 0.66 g), triethyl orthoacetate
(2) (0.01 mol, 1.62 g) and phenylhydrazine (3) (0.01 mol,
1.07 g) in ethanol (10 mL), and the solution was heated to
reflux for 12 h. After cooling the reaction mixture to room
temperature, the precipitated product was collected by fil-
tration, washed sequentially with water (10 mL) and cold
ethanol (10 mL). The crude product was recrystallized
from ethanol to afford pure pyrazole 4. White crystals,
1
yield (1.68 g, 85%), mp 128–129 °C. H NMR (Acetone-
d6), d: 2.13 (s, 3H, Me), 6.04 (br, 2H, NH2), 7.32–7.51 (m,
5H, Ph). 13C NMR (Acetone-d6), d: 11.47 (Me), 76.82
[C(4)], 115.34 (C:N), 123.76 [C(20), C(60)], 126.87
[C(40)], 128.93 [C(30), C(50)], 139.04 [C(10)], 148.79
[C(3)], 150.45 [C(5)]. IR (m/cm-1): 3337, 3232 (NH2),
2208 (C:N), 1644 (C=N). Anal. Calcd. for C11H10N4 (%):
C, 66.65; H, 5.09; N, 28.26. Found (%): C, 66.69; H, 5.07;
N, 28.24.
Due to the biologically importance of the pyrazolo[3,4-
d]pyrimidine skeleton especially as antimicrobial agents
(Burch 1968; Eweas et al. 2012; Khobragade et al. 2010),
we focused on the synthesis of some novel 6-substituted
4-amino-pyrazolo[3,4-d]pyrimidines as potential prodrugs
(Vignaroli et al. 2013) using ethanolic sodium ethoxide
solution. In vitro inhibitory effects of the synthesized
compounds against seven gram-positive and -negative
bacterial strains were evaluated via measuring inhibition
zone diameter (IZD), and determining minimum inhibitory
concentration (MIC) and minimum bactericidal concen-
tration (MBC). The effects of substituent variation on the
antibacterial activity of derivatives were discussed as well.
General procedure for the preparation of 6-substituted
4-amino-pyrazolo[3,4-d]pyrimidines 6a–g
Clean metallic sodium (0.01 mol, 0.23 g) was added por-
tion wise during at least 5 min to a stirred and cooled (ice-
water bath) absolute ethanol (20 mL). Aminopyrazole 4
(0.01 mol, 1.98 g) and nitriles 5a–g (5a: 0.02 mol, 0.41 g;
5b: 0.01 mol, 1.03 g; 5c: 0.01 mol, 1.17 g; 5d: 0.01 mol,
1.82 g; 5e: 0.01 mol, 1.48 g; 5f: 0.01 mol, 1.38 g; 5g:
0.01 mol, 1.72 g) were then added to a sodium ethoxide
solution. The resulting solution or mixture was heated to
reflux for 8–12 h (6a: 12 h, 6b: 11 h, 6c: 12 h, 6d: 10 h,
6e: 8 h, 6f: 10 h, 6g: 8 h). Let, the reaction mixture was
cooled to room temperature, the precipitated product was
filtered off, and washed, respectively, with water (10 mL)
and ethanol (10 mL). The desired product was dried in air
and recrystallized from methanol to provide analytically
pure derivatives 6a–g.
Experimental
Materials
Melting points were recorded on a Kruss type KSP1N
melting point meter and are uncorrected. The IR spectra
were determined on Bruker Tensor-27 FT-IR spectrometer
in KBr pellets. The 1H and 13C NMR spectra were recorded
on a Bruker FT-NMR Ultra Shield-400 spectrometer at 400
3,6-Dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
amine (6a) Bright yellow powder, yield (1.79 g, 75%),
1
mp 268–270 °C. H NMR, d: 2.37 (s, 3H, Mepyrimidine),
2.50 (s, 3H, Mepyrazole), 7.32 (t, 2H, J = 7.4 Hz, NH2),
7.52 (t, 3H, J = 7.7 Hz, 30-H, 40-H, 50-H), 8.07 (d, 2H,
123

Chem. Pap.
J = 8.0 Hz, 20-H, 60-H). 13C NMR, d: 13.89 (Mepyrazole),
22.80 (Mepyrimidine), 98.91 [C(3a)], 120.63 [C(20), C(60)],
125.79 [C(40)], 129.47 [C(30), C(50)], 139.26 [C(10)],
143.11 [C(3)], 154.10 [C(7a)], 160.43 [C(4)], 166.10
[C(6)]. IR (m/cm-1): 3494 (NH2), 1676 (C=N). Anal. Calcd.
for C13H13N5 (%): C, 65.25; H, 5.48; N, 29.27. Found (%):
C, 65.17; H, 5.49; N, 29.34.
3-Methyl-6-(4-nitrophenyl)-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (6e) Orange powder, yield (2.66 g,
77%), decomp. 264–266 °C. 1H NMR, d: 2.68 (s, 3H, Me),
7.55–7.62 (m, 3H, 30-H, 40-H, 50-H), 7.60 (t, 2H,
J = 7.6 Hz, NH2), 8.05 (d, 2H, J = 8.6 Hz 200-H, 600-H),
8.10–8.14 (m, 2H, 20-H, 60-H), 8.35 (d, 2H, J = 8.6 Hz, 300-
H, 500-H). 13C NMR, d: 14.98 (Me), 9,9.80 [C(3a)], 120.77
[C(20), C(60)], 125.86 [C(40)], 129.68 [C(30), C(50)], 133.69
[C(200), C(600)], 135.36 [C(300), C(500)], 139.46 [C(10)],
140.44 [C(100)], 143.47 [C(3)], 149.58 [C(400)], 153.75
[C(7a)], 159.06 [C(4)], 167.43 [C(6)]. IR (m/cm-1): 3373,
3178 (NH2), 1657 (C=N). Anal Calcd. for C18H14N6O2
(%): C, 62.42; H, 4.07; N, 24.27; O, 9.24. Found (%): C,
62.37; H, 4.01; N, 24.21; O, 9.32.
3-Methyl-1,6-diphenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
amine (6b) Yellow powder, yield (1.66 g, 55%), mp
1
227–228 °C. H NMR, d: 2.67 (s, 3H, Me), 7.32 (t, 2H,
J = 7.3 Hz, NH2), 7.52 (m, 3H, 300-H, 400-H, 500-H), 7.58 (t,
3H, J = 7.7 Hz, 30-H, 40-H, 50-H), 8.32 (d, 2H,
J = 7.9 Hz, 20-H, 60-H), 8.45 (m, 2H, 200-H, 600-H). 13C
NMR, d: 15.00 (Me), 99.63 [C(3a)], 120.61 [C(20), C(60)],
125.90 [C(40)], 128.56 [C(200), C(600)], 128.80 [C(300),
C(500)], 129.61 [C(30), C(50)], 130.88 [C(400)], 138.39
[C(100)], 139.62 [C(10)], 143.39 [C(3)], 155.94 [C(7a)],
158.97 [C(4)], 162.12 [C(6)]. IR (m/cm-1): 3497, 3312
(NH2), 1650 (C=N). Anal. Calcd. for C18H15N5 (%): C,
71.74; H, 5.02; N, 23.24. Found (%): C, 71.64; H, 5.04; N,
23.32.
6-(4-Chlorophenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (6f) White powder, yield (1.71 g,
51%), mp 195–196 °C. 1H NMR, d: 2.66 (s, 3H, Me), 7.33
(t, 2H, J = 7.4 Hz, NH2), 7.56–7.62 (m, 5H, 30-H, 40-H, 50-
H, 300-H, 500-H), 8.27–8.29 (m, 2H, 20-H, 60-H), 8.42–8.45
(m, 2H, 200-H, 600-H). 13C NMR, d: 14.97 (Me), 99.64
[C(3a)], 120.71 [C(20), C(60)], 126.01 [C(40)], 128.90
[C(200), C(600)], 129.62 [C(30), C(50)], 130.24 [C(300),
C(500)], 135.68 [C(400)], 137.24 [C(100)], 139.51 [C(10)],
143.42 [C(3)], 155.76 [C(7a)], 158.96 [C(4)], 161.04
[C(6)]. IR (m/cm-1): 3497, 3311 (NH2), 1650 (C=N). Anal
Calcd. for C18H14ClN5 (%): C, 64.38; H, 4.20; N, 20.86;
Cl, 10.56. Found (%): C, 64.32; H, 4.13; N, 20.90; Cl,
10.65.
3-Methyl-1-phenyl-6-p-tolyl-1H-pyrazolo[3,4-d]pyrimidin-
4-amine (6c) White powder, yield (2.21 g, 70%), mp
1
218–219 °C. H NMR, d: 2.40 (s, 3H, Mebenzene), 2.66 (s,
3H, Mepyrazole), 7.31 (t, 2H, J = 8.2 Hz, NH2), 7.34 (d, 2H,
J = 8.1 Hz, 300-H, 500-H), 7.58 (t, 3H, J = 7.7 Hz, 30-H, 40-
H, 50-H), 8.31 (d, 2H, J = 7.9 Hz, 20-H, 60-H), 8.34 (d, 2H,
J = 8.1 Hz, 200-H, 600-H). 13C NMR, d: 14.99 (Mepyrazole),
21.52 (Mebenzene), 99.53 [C(3a)], 120.58 [C(20), C(60)],
125.85 [C(40)], 128.56 [C(200), C(600)], 129.41 [C(30),
C(50)], 129.61 [C(300), C(500)], 135.71 [C(400)], 139.65
[C(10)], 140.58 [C(100)], 143.35 [C(3)], 156.00 [C(7a)],
158.99 [C(4)], 162.20 [C(6)]. IR (m/cm-1): 3497, 3313
(NH2), 1655 (C=N). Anal Calcd. for C19H17N5 (%): C,
72.36; H, 5.43; N, 22.21. Found (%): C, 72.27; H, 5.46; N,
22.27.
6-(2,4-Dichlorophenyl)-3-methyl-1-phenyl-1H-pyra-
zolo[3,4-d]pyrimidin-4-amine (6g) Yellow powder, yield
1
(2.37 g, 64%), mp 192–194 °C. H NMR, d: 2.68 (s, 3H,
Me), 7.29 (t, 2H, J = 7.3 Hz, NH2), 7.49–7.57 (m, 4H, 30-
H, 40-H, 50-H, 300-H), 7.74-7.79 (m, 2H, 500-H, 600-H), 8.21
(d, 2H, J = 7.9 Hz, 20-H, 60-H). 13C NMR, d: 14.97 (Me),
99.39 [C(3a)], 120.70 [C(20), C(60)], 126.11 [C(40)], 127.62
[C(500)], 129.55 [C(30), C(50)], 129.95 [C(600)], 133.04
[C(300)], 133.20 [C(200)], 134.40 [C(400)], 137.97 [C(100)],
139.31 [C(10)], 143.47 [C(3)], 154.98 [C(7a)], 158.95
[C(4)], 162.08 [C(6)]. IR (m/cm-1): 3491, 3308 (NH2),
1647 (C=N). Anal Calcd. for C18H13Cl2N5 (%): C, 58.39;
H, 3.54; N, 18.92; Cl, 19.15. Found (%): C, 58.32; H, 3.62;
N, 20.90; Cl, 18.84.
6-(4-Bromophenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (6d) White powder, yield (2.77 g,
73%), mp 212–213 °C. 1H NMR, d: 2.66 (s, 3H, Me), 7.33
(t, 2H, J = 7.4 Hz, NH2), 7.57 (t, 3H, J = 7.6 Hz, 30-H, 40-
H, 50-H),7.73 (d, 2H, J = 8.5 Hz, 300-H, 500-H), 8.27 (d, 2H,
J = 7.7 Hz, 20-H, 60-H), 8.37 (d, 2H, J = 8.5 Hz, 200-H,
600-H). 13C NMR, d: 14.97 (Me), 99.68 [C(3a)], 120.72
[C(20), C(60)], 124.63 [C(400)], 125.99 [C(40)], 129.61
[C(30), C(50)], 130.51 [C(200), C(600)], 131.81 [C(300),
C(500)], 137.62 [C(100)], 139.51 [C(10)], 143.42 [C(3)],
155.76 [C(7a)], 158.96 [C(4)], 161.15 [C(6)]. IR (m/cm-1):
3497, 3318 (NH2), 1653 (C=N). Anal Calcd. for C18H14-
BrN5 (%): C, 56.86; H, 3.71; N, 18.42; Br, 21.01. Found
(%): C, 56.91; H, 3.77; N, 18.38; Br, 20.94.
Antibacterial activity
Solution of all derivatives and antibiotics were, respec-
tively, prepared in 10% DMSO and double-distilled water
at initial concentrations of 9011 and 17.6 lg/mL. All tests
were repeated three times and the results were expressed as
the average of three independent experiments.
123

Chem. Pap.
Preparation of the bacterial suspension
Gram-negative bacterial strains including Pseudomonas
aeruginosa (PTCC 1310), Shigella flexneri (PTCC 1234),
Salmonella enterica subsp. enterica (PTCC 1709), and
gram-positive bacterial strains including Streptococcus
pyogenes (PTCC 1447), Staphylococcus aureus (PTCC
1189), Bacillus cereus (PTCC 1665) and Bacillus subtilis
subsp. spizizenii (PTCC 1023) were prepared from the
Persian Type Culture Collection (PTCC), Tehran, Iran. All
bacteria were cultured on nutrient agar (HiMedia, India),
and then incubated for 24 h at 37 °C under normal atmo-
spheric air. Finally, in sterile conditions, a bacterial sus-
pension with concentration of 0.5 McFarland
(1.5 9 108 CFU/ml) in Mu¨ller-Hinton broth (HiMedia,
India) was obtained spectrophotometrically, which used as
a storage source. Antibacterial activities were tested
according to Clinical and Laboratory Standards Institute
(CLSI) broth microdilution and disk diffusion methods
described in guidelines M07-A9, M26-A and M02-A11
(Balouiri et al. 2016) with a slight modification.
Scheme 1 Synthesis of pyrazolo[3,4-d]pyrimidines 6a–g
were incubated at 37 °C for 24 h under the conditions
listed previously. The MBC was identified as the lowest
concentration of derivatives or antibiotics at which no
bacteria survived.
Results and discussion
Chemistry
In this study, a pyrazole core was used as the starting material
for the preparation of pyrazolo[3,4-d]pyrimidines 6a–g.
5-Amino-3-methyl-1-phenyl-1H-pyrazole-4-carbonitrile
(4) was synthesized in one-pot procedure from thereactionof
malononitrile (1), triethyl orthoacetate (2) and phenylhy-
drazine (3). Then, compound 4 was reacted with various
nitriles 5a–g under reflux conditions in EtONa/EtOH which
afforded products 6a–g (Scheme 1).
Measurement of inhibition zone diameters
100 lL of a bacterial suspension of the storage source was
spread on Mu¨ller-Hinton agar (HiMedia, India). Some
sterile blank discs were placed on this culture medium at
appropriate intervals. 10 lL of initial concentrations for
derivatives and antibiotics were poured onto these disks,
IZD values were measured by calliper after incubation at
37 °C for 24 h under mentioned conditions.
According to the results recorded in the experimental
section, there is no significant relationship between yields
and reactivity of reagents.
The reaction is carried out in the presence of sodium
ethoxide as the catalyst. Probably, formation of strong
hydrogen bond between the amine hydrogens on pyrazole
ring and the oxygen of sodium ethoxide increased the
nucleophilicity and reactivity of the amino group. Unfor-
tunately, all our efforts to use potassium carbonate and
glycerol as a basic deep eutectic media were unsuccessful
(Naser et al. 2013).
Determination of the minimum inhibitory concentration
(MIC)
100 lL of Mu¨ller-Hinton broth medium was added to all the
wells in each row. Then, 100 lL of initial solution of
derivatives in 10% DMSO as a solvent was added to the first
well. After mixing, serial twofold dilutions were continued
to the eighth well. Finally, 10 lL of bacterial suspension was
added to each well. The same procedure was applied to the
antibiotics. As a result, the concentration range of derivatives
and antibiotics were, respectively, 4096–32 and 8–0.063 lg/
mL. The plates were incubated with shaking at 100 rpm at
37 °C for 24 h under similar conditions. The MICs were
determined as the lowest concentration of derivatives or
antibiotics which resulted in no visible bacterial growth.
Antibacterial effects
Antibacterial effects of synthesized derivatives and the
antibiotics (penicillin, ampicillin, cefazolin, gentamicin,
tetracycline, azithromycin and nalidixic acid) were evalu-
ated against seven pathogenic bacteria from different genera
and presented as IZD, MIC and MBC values in Table 1.
Most of the compounds have the ability to inhibit the
growth of gram-positive bacterium Streptococcus pyoge-
nes and gram-negative bacterium Pseudomonas aerugi-
nosa. On the basis of the spread of inhibitory properties
and the MIC values, derivatives were ranked in the fol-
lowing order 6a [ 6f [ 6c [ 6d [ 6e [ 6g [ 6b, and
based on the substituents at 6-position were arranged by
Determination of the minimum bactericidal concentration
(MBC)
Samples of all wells that showed no growth in the MIC test,
were cultured in Mu¨ller-Hinton agar medium, which then,
123

Chem. Pap.
123

Chem. Pap.
the order alkyl [ monosubstituted phenyl [ disubstituted
phenyl [ phenyl. Compound 6a was the most broad-
spectrum antibacterial agent and showed inhibitory effects
against six bacterial strains. Pyrimidine ring in derivative
6a unlike others contains an aliphatic substituent (CH3) at
6-position. Among aryl-substituted derivatives 6b–g,
inhibitory properties of compound 6f including 4-chlor-
ophenyl substituent at the same position was more sig-
nificant. Such effect is probably due to the presence of a
chlorine as an antimicrobial agent. Unexpectedly, the
presence of residual chlorine in product 6g reduced
antibacterial activities. A more remarkable reduction was
also observed in 6-phenyl substituted heterocycle 6b,
which was the only effective against Pseudomonas
aeruginosa. In this study, some common antibiotics
including penicillin, ampicillin, cefazolin, gentamicin,
tetracycline, azithromycin and nalidixic acid from peni-
cillin, penicillin combination, cephalosporin, aminogly-
coside, tetracycline, macrolide and quinolone classes were
used as positive controls. No inhibitory activity against
Pseudomonas aeruginosa and Bacillus cereus was
observed from b-lactam antibiotics (the first three
medicines) unlike other antibiotics. Gentamycin was
effective on all bacterial strains tested. It seems that
tetracycline and nalidixic acid, which blocked the growth
of only two pathogens, were not proper options for the
treatment of infections caused by bacteria listed above.
References
Agrebi A, Allouche F, Fetoui H, Chabchoub F (2014) Synthesis and
biological evaluation of new pyrazolo[3,4-d]pyrimidine deriva-
tives. Mediterr J Chem 3:864–876. doi:10.13171/mjc.3.2.2014.
13.05.23
Allouche F, Chabchoub F, Carta F, Supuran CT (2013) Synthesis of
aminocyanopyrazoles via a multi-component reaction and anti-
carbonic anhydrase inhibitory activity of their sulfamide deriva-
tives against cytosolic and transmembrane isoforms. J Enzyme
Inhib Med Chem 28:343–349. doi:10.3109/14756366.2012.
720573
Balouiri M, Sadiki M, Ibnsouda SK (2016) Methods for in vitro
evaluating antimicrobial activity: a review. J Pharm Anal
6:71–79. doi:10.1016/j.jpha.2015.11.005
Bhuyan PJ, Borah HN, Sandhu JS (2002) Studies on uracils: a facile
one-pot synthesis of pyrazolo[3,4-d]pyrimidines. Tetrahedron
Lett 43:895–897. doi:10.1016/S0040-4039(01)02285-7
Burch HA (1968) Nitrofuryl heterocycles. VII. 4-Amino-6-(5-nitro-2-
furyl)-1H-pyrazolo[3,4-d]pyrimidines. J Med Chem 11:79–83.
doi:10.1021/jm00307a017
Carraro F, Naldini A, Pucci A, Locatelli GA, Maga G, Schenone S,
Bruno O, Ranise A, Bondavalli F, Brullo C, Fossa P, Menozzi G,
Mosti L, Modugno M, Tintori C, Manetti F, Botta M (2006)
Pyrazolo[3,4-d]pyrimidines as potent antiproliferative and
proapoptotic agents toward A431 and 8701-BC Cells in culture
via inhibition of c-Src phosphorylation.
49:1549–1561. doi:10.1021/jm050603r
Daidone G, Raffa D, Plescia F, Maggio B, Roccaro A (2002)
Synthesis of pyrazole-4-carbohydrazide derivatives of pharma-
ceutical interest. Arkivoc 11:227–235. doi:10.3998/ark.5550190.
0003.b20
J Med Chem
Davoodnia A, Vahedinia A, Tavakoli-Hoseini N (2012) Synthesis of
some new pyrazolo[3,4-d]pyrimidin-4-amines. Asian J Chem
24:3433–3435
Eweas AF, Swelam SA, Fathalla OA, Fawzy NM, Abdel-Moez SI
(2012) Synthesis, anti-microbial evaluation, and molecular
modeling of new pyrazolo[3,4-d]pyrimidine derivatives. Med
Chem Res 21:3848–3857. doi:10.1007/s00044-011-9911-y
Ghorab MM, Ragab FA, Alqasoumi SI, Alafeefy AM, Aboulmagd SA
(2010) Synthesis of some new pyrazolo[3,4-d]pyrimidine
derivatives of expected anticancer and radioprotective activity.
Eur J Med Chem 45:171–178. doi:10.1016/j.ejmech.2009.09.039
Hanefeld U, Rees CW, White AJP, Williams DJ (1996) One-pot
synthesis of tetrasubstituted pyrazoles-proof of regiochemistry.
Conclusions
In this research project, some novel 6-substituted 4-amino-
pyrazolo[3,4-d]pyrimidine derivatives were synthesized as
potential antibacterial agents in a two-step procedure from
cyclocondensation of 5-amino-3-methyl-1-phenyl-1H-
pyrazole-4-carbonitrile with different nitriles in EtONa/
EtOH. Antimicrobial activities of derivatives were evalu-
ated against a variety of gram-positive and -negative
pathogenic bacteria. Finally, the inhibitory effects of
derivatives against some bacteria especially Streptococcus
pyogenes and Pseudomonas aeruginosa were shown.
Based on the broad-spectrum antibacterial activities of
derivatives specially heterocycle 6a, the effect of sub-
stituent changes at positions 1, 3 and 6 on the antimicrobial
or other biological properties of these prodrugs can be
studied in future experiments.
J Chem Soc Perkin Trans 1(13):1545–1552. doi:10.1039/
P19960001545
Huang Y-Y, Wang L-Y, Chang C-H, Kuo Y-H, Kaneko K, Takayama
H, Kimura M, Juang S-H, Wong FF (2012) One-pot synthesis
and antiproliferative evaluation of pyrazolo[3,4-d]pyrimidine
derivatives. Tetrahedron 68:9658–9664. doi:10.1016/j.tet.2012.
09.054
Irwig H, Krakoff IH (1966) Use of allopurinol in preventing
hyperuricemia in leukemia and lymphoma. Cancer
19:1489–1496.
doi:10.1002/1097-0142(196611)19:11\1489:
AID-CNCR2820191105[3.0.CO;2-F
Kadry HH (2014) Synthesis, biological evaluation of certain
pyrazolo[3,4-d]pyrimidines as novel anti-inflammatory and
analgesic agents. Med Chem Res 23:5269–5281. doi:10.1007/
s00044-014-1079-9
Kandeel MM, Roshdy SM, Abdelall EKA, Abdelgawad MA, Lamie
PF (2013) Design, synthesis and cytotoxic activity of some novel
compounds containing pyrazolo[3,4-d]pyrimidines nucleus.
J Chem Sci 125:1029–1043. doi:10.1007/s12039-013-0485-8
Acknowledgements The authors would like to thank members of the
University of Zabol especially Dr Mohammad Allahbakhsh, Deputy
of Research and Technology, and Dr Mansour Ghaffari-Moghaddam,
Dean of the Faculty of Science, for their support and assistance at the
various stages of this project.
123

Chem. Pap.
Khobragade CN, Bodade RG, Konda SG, Dawane BS, Manwar AV
(2010) Synthesis and antimicrobial activity of novel pyra-
Smith CJ, Iglesias-Sigu¨enza FJ, Baxendale IR, Ley SV (2007) Flow
and batch mode focused microwave synthesis of 5-amino-4-
cyanopyrazoles and their further conversion to 4-aminopyra-
zolopyrimidines. Org Biomol Chem 5:2758–2761. doi:10.1039/
B709043A
Song XJ, Shao Y, Dong XG (2011) Microwave-assisted synthesis of
some novel fluorinated pyrazolo[3,4-d]pyrimidine derivatives
containing 1,3,4-thiadiazole as potential antitumor agents. Chin
Chem Lett 22:1036–1038. doi:10.1016/j.cclet.2011.05.012
Soth M, Abbot S, Abubakari A, Arora N, Arzeno H, Billedeau R,
Dewdney N, Durkin K, Frauchiger S, Ghate M, Goldstein DM,
Hill RJ, Kuglstatter A, Li F, Loe B, McCaleb K, McIntosh J,
Papp E, Park J, Stahl M, Sung ML, Suttman R, Swinney DC,
Weller P, Wong B, Zecic H, Gabriel T (2011) 3-Amino-
pyrazolo[3,4-d]pyrimidines as p38a kinase inhibitors: Design
and development to a highly selective lead. Bioorg Med Chem
Lett 21:3452–3456. doi:10.1016/j.bmcl.2011.03.098
Tintori C, La Sala G, Vignaroli G, Botta L, Fallacara AL, Falchi F,
Radi M, Zamperini C, Dreassi E, Dello Iacono L, Orioli D,
Biamonti G, Garbelli M, Lossani A, Gasparrini F, Tuccinardi T,
Laurenzana I, Angelucci A, Maga G, Schenone S, Brullo C,
Musumeci F, Desogus A, Crespan E, Botta M (2015) Studies on
the ATP binding site of Fyn kinase for the identification of new
inhibitors and their evaluation as potential agents against
tauopathies and tumors. J Med Chem 58:4590–4609. doi:10.
1021/acs.jmedchem.5b00140
Todorovic N, Awuah E, Shakya T, Wright GD, Capretta A (2011)
Microwave-assisted synthesis of N1- and C3-substituted pyra-
zolo[3,4-d]pyrimidine libraries. Tetrahedron Lett 52:5761–5763.
doi:10.1016/j.tetlet.2011.08.103
zolo[3,4-d]pyrimidin derivatives. Eur
45:1635–1638. doi:10.1016/j.ejmech.2009.12.040
J
Med Chem
Kumar R, Darpan Sharma S, Singh R (2011) Xanthine oxidase
inhibitors: a patent survey. Exp Opin Ther Pat 21:1071–1108.
doi:10.1517/13543776.2011.577417
La Motta C, Sartini S, Mugnaini L, Salerno S, Simorini F, Taliani S,
Marini AM, Da Settimo F, Lavecchia A, Novellino E, Antonioli
L, Fornai M, Blandizzi C, Del Tacca M (2009) Exploiting the
pyrazolo[3,4-d]pyrimidin-4-one ring system as a useful template
to obtain potent adenosine deaminase inhibitors. J Med Chem
52:1681–1692. doi:10.1021/jm801427r
Laurenzana I, Caivano A, La Rocca F, Trino S, De Luca L, D’Alessio
F, Schenone S, Falco G, Botta M, Del Vecchio L, Musto P
(2016) A Pyrazolo[3,4-d]pyrimidine compound reduces cell
viability and induces apoptosis in different hematological
malignancies. Front Pharmacol 7:416. doi:10.3389/fphar.2016.
00416
Liu H, Wang HQ, Liu ZJ (2007) Synthesis and herbicidal activity of
novel pyrazolo[3,4-d]pyrimidin-4-one derivatives containing
aryloxyphenoxypropionate moieties. Bioorg Med Chem Lett
17:2203–2209. doi:10.1007/s00044-011-9911-y
Ma S, Zhou S, Lin W, Zhang R, Wu W, Zheng K (2016) Study of
novel pyrazolo[3,4-d]pyrimidine derivatives as selective
TgCDPK1 inhibitors: molecular docking, structure-based 3D-
QSAR and molecular dynamics simulation. RSC Adv
6:100772–100782. doi:10.1039/C6RA20277B
Naser J, Mjalli F, Jibril B, Al-Hatmi S, Gano Z (2013) Potassium
carbonate as a salt for deep eutectic solvents. Int J Chem Eng
Appl 4:114–118. doi:10.7763/IJCEA.2013.V4.275
Quintela JM, Peinador C, Moreira MJ, Alfonso A, Botana LM,
Riguera R (2001) Pyrazolopyrimidines: synthesis, effect on
histamine release from rat peritoneal mast cells and cytotoxic
activity. Eur J Med Chem 36:321–332. doi:10.1016/j.ejmech.
2012.08.007
Trivedi AR, Dholariya BH, Vakhariya CP, Dodiya DK, Ram HK,
Kataria VB, Siddiqui AB, Shah VH (2012) Synthesis and anti-
tubercular evaluation of some novel pyrazolo[3,4-d]pyrimidine
derivatives. Med Chem Res 21:1887–1891. doi:10.1007/s00044-
011-9712-3
Verschueren K, Cobbaut M, Demaerel J, Saadah L, Voet ARD, Van
Lintb J, De Borggraeve WM (2017) Discovery of a potent
protein kinase D inhibitor: insights in the binding mode of
pyrazolo[3,4-d]pyrimidine analogues. Med Chem Commun.
doi:10.1039/C6MD00675B
´
Rahmouni A, Romdhane A, Ben said A, Guerineau V, Touboul D,
Ben Jannet H, (2014) Synthesis of novel isoxazolines and
isoxazoles of N-substituted pyrazolo[3,4-d]pyrimidin-4(5H)-one
derivatives through [3?2] cycloaddition. Arabian J Chem.
doi:10.1016/j.arabjc.2014.10.053
`
Vignaroli G, Zamperini C, Dreassi E, Radi M, Angelucci A, Sanita P,
Rashad AE, Hegab MI, Abdel-Megeid RE, Fathalla N, Abdel-Megeid
FME (2009) Synthesis and anti-HSV-1 evaluation of some
pyrazoles and fused pyrazolopyrimidines. Eur J Med Chem
44:3285–3292. doi:10.1016/j.ejmech.2009.02.012
Crespan E, Kissova M, Maga G, Schenone S, Musumeci F, Botta
M (2013) Pyrazolo[3,4-d]pyrimidine prodrugs: strategic opti-
mization of the aqueous solubility of dual Src/Abl inhibitors.
Med Chem Lett 4:622–626. doi:10.1021/ml4000782
Salaheldin AM, Oliveira-Campos AMF, Rodrigues LM (2009)
Heterocyclic synthesis with nitriles: Synthesis of pyrazolopy-
rimidine and pyrazolopyridine derivatives. Synth Commun
39:1186–1195. doi:10.1080/00397910802517814
Siebeneicher H, Bauser M, Buchmann B, Heisler I, Mu¨ller T,
Neuhaus R, Rehwinkel H, Telser J, Zorn L (2016) Identification
of novel GLUT inhibitors. Bioorg Med Chem Lett
26:1732–1737. doi:10.1016/j.bmcl.2016.02.050
Wang GT, Mantei RA, Hubbard RD, Wilsbacher JL, Zhang Q,
Tucker L, Hu X, Kovar P, Johnson EF, Osterling DJ, Bouska J,
Wang J, Davidsen SK, Bell RL, Sheppard GS (2010) Substituted
4-amino-1H-pyrazolo[3,4-d]pyrimidines as multi-targeted inhi-
bitors of insulin-like growth factor-1 receptor (IGF1R) and
members of ErbB-family receptor kinases. J Bioorg Med Chem
Lett 20:6067–6071. doi:10.1016/j.bmcl.2010.08.052
123