Synthesis of novel 2-alkyl-4-substituted-amino-pyrazolo[3,4-d]pyrimidines as new leads for anti-bacterial and anti-cancer activity

Article abstract of DOI:10.1007/s00044-012-0084-0

Treatment of 6-alkyl-1-phenyl-4-chloro-(1H)-pyrazolo[3,4-d]pyrimidines, with different amines afforded a series of compounds whose identity and purity were confirmed by spectral and analytical means. The compounds were tested for antibacterial activity against four organisms viz. Staphylococcus aureus (Gram positive), S. epidermidis (Gram positive), Bacillus subtilis (Gram positive), Escherichia coli (Gram negative) using amoxicillin as standard control. Compounds 4d, 6b, 6c have shown best antibacterial activity in the series. The antiproliferative activity was tested against human skin cancer cell line G361. The compounds 3d, 4d, 5b, 5d, 5e, 6c, 7a were found to be the best of the series and showed the activity at micromolar concentration.

Full text of DOI:10.1007/s00044-012-0084-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:1090–1101
DOI 10.1007/s00044-012-0084-0
ORIGINAL RESEARCH
Synthesis of novel 2-alkyl-4-substituted-amino-pyrazolo
[3,4-d]pyrimidines as new leads for anti-bacterial
and anti-cancer activity
•
Murty Devarakonda Raghava Doonaboina
•
•
•
•
Sreedher Vanga Jahnavi Vemu Sathoshini Boni
Raghu Prasad Mailavaram
Received: 1 November 2011 / Accepted: 3 May 2012 / Published online: 4 June 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Treatment of 6-alkyl-1-phenyl-4-chloro-(1H)-
pyrazolo[3,4-d]pyrimidines, with different amines afforded
a series of compounds whose identity and purity were
confirmed by spectral and analytical means. The com-
pounds were tested for antibacterial activity against four
organisms viz. Staphylococcus aureus (Gram positive),
S. epidermidis (Gram positive), Bacillus subtilis (Gram
positive), Escherichia coli (Gram negative) using amoxi-
cillin as standard control. Compounds 4d, 6b, 6c have
shown best antibacterial activity in the series. The anti-
proliferative activity was tested against human skin cancer
cell line G361. The compounds 3d, 4d, 5b, 5d, 5e, 6c, 7a
were found to be the best of the series and showed the
activity at micromolar concentration.
CNS modulating (Neustadt et al., 2007), antiviral (Chern
et al., 2004), antifungal (Holla et al., 2006), anticancer (Abd
Razik and Abdel Wahab, 2011) and anti-inflammatory
(Quintela et al., 2003) activities. These molecules have shown
affinity towards adenosine receptors (Quinn and Poulsen,
1996), 5HT-6 and CRF-1 (Gilligan et al., 2000) receptors.
Further, it has been revealed that the replacement of 1H of
pyrazole of the pyrazolo[3, 4-d]pyrimidine ring system with
other bioactive moiety shows good pharmacological proper-
ties probably by increasing the p staking (Holla et al., 2006).
In the present study, proton at position 1 on pyrazole ring has
replaced with phenyl group. In view of developing molecules
with potentantibacterial and anticanceractivities, weaimed at
the synthesis of 6-alkyl-1-phenyl-4-substituted-amino-(1H)-
pyrazolo[3,4-d]pyrimidines. As a part of our research, we
synthesized the title compounds, with phenyl group at 1H
position and amine substitution at 4H position of 6-alkyl
pyrazolo[3,4-d]pyrimidines. The compounds were tested for
antibacterial activity against four organisms viz. Staphylo-
coccus aureus, S. epidermidis, Bacillus subtilis, Escherichia
coli. The antiproliferative activity was tested against human
skin cancer cell line G361.
Keywords Antibacterial activity ꢀ Anticancer activity ꢀ
G361 cell line ꢀ Skin cancer ꢀ Sulforhodamine B assay
Introduction
The synthetic heterocyclic chemistry has provided means
for the invention of various drugs and pyrazolopyrimidines
are one such. These derivatives, being purine analogs, are
of considerable chemical and pharmacological importance.
Literature survey reveals that pyrazolopyrimidines have
antibacterial (Bakavoli et al., 2010; Bazgir et al., 2008),
antiproliferative (Ahmed et al., 2009; Krystof et al., 2006),
Results and discussion
Chemistry
The general procedure for synthesis of 4-chloro-6-alkyl-1-
phenyl-1H-pyrazolo[3,4-d]pyrimidine (3a–7a) were syn-
thesized by route shown in Scheme 1. The starting mate-
rial, 1-phenyl-4-cyano-5-aminopyrazole 1 was synthesized
as described in the literature (Das et al., 2008). The key
intermediate 5-amino-1-phenyl-1H-pyrazole-4-carboxam-
ide 2 was obtained by acid hydrolysis of 1-phenyl-4-cyano-
M. Devarakonda ꢀ R. Doonaboina ꢀ S. Vanga ꢀ J. Vemu ꢀ
S. Boni ꢀ R. P. Mailavaram (&)
Department of Pharmaceutical Chemistry, Shri Vishnu College
of Pharmacy (Affiliated To Andhra University, Visakhapatnam),
Vishnupur, Bhimavaram 534202, Andhra Pradesh, India
e-mail: raghumrp@mailcity.com; raghumrp@rediffmail.com
123

Med Chem Res (2013) 22:1090–1101
1091
CN
N
NH2
(1)
N
A
Cl
N
O
N
O
Cl
N
O
N
N
NH
CH2Cl
NH2
NH2
C
N
NH
C
B
N
N
N
N
N
N
CH2Cl
N
N
N
N
X
(7a)
(2)
(7)
(3)
(3a)
R-NH
D
R
2
O
B
B
O
B
R
N
NH
CH3
N
N
NH
N
CH3
CH3
N
N
N
CH2R
N
N
N
N
N
(7b-7f)
O
N
N
(4)
(6)
(3b-3f)
NH
C
N
CH3
N
N
C
Cl
Cl
(5)
N
N
C
N
N
CH3
CH3
N
N
N
CH3
N
Cl
(6a)
N
(4a)
N
CH3
N
D
N
D
R
(5a)
R
D
R
N
N
N
N
N
N
CH3
CH3
N
N
CH3
N
N
N
(4b-4k)
CH3
N
(6b-6d
)
(5b-5g)
Scheme 1 Synthesis of 6-alkyl-1-phenyl-4-substituted-(1H)-pyrazolo[3,4-d]pyrimidin-amines. A H2SO4/room temp/1 h, B sodium ethoxide/
ethanol reflux/MWI, 100 W, 80 °C, 45 s, 20 bar, X sodium ethoxide/ethanol/room temp/1 h, C phosphorous oxychloride/MWI, 100 W, 120 °C,
10 min 30 bar; D toluene/RH/room temp/18–36 h, R –NHCH2CH2CH3, –NHCH(CH3)2, –NHCH2CH2CH2CH3,
O
N
O
N
H
,
5-aminopyrazole 1. The amide derivative 2 was reacted
with various esters by irradiating in a CEM Discover
microwave oven at 80 °C to give pyrazolo[3,4-d]pyrimi-
din-ones 3–7 (Miyashita et al., 1990; Daniels et al., 2008).
These compounds were chlorinated with phosphorous
oxychloride (Jung et al., 2005) by irradiating in a micro-
wave oven to give 3a–7a and finally treated with various
amines at room temperature in the presence of dry toluene
(Schenone et al., 2004) to give the target compounds. The
intermediate compounds 3–6, 3a–6a were synthesized both
by conventional and microwave irradiations. By micro-
wave irradiation, the reaction times drastically reduced and
compound yields were also improved. Table 1 gives
comparison data of reaction time and compound yields by
conventional of synthesis and microwave irradiation
methods. The synthetic route has been outlined in
Scheme 1. The newly synthesized compounds were ana-
1
lysed and their structures were confirmed by IR, H NMR,
mass spectral studies and elemental analysis.
The condensation of 5-amino-1-phenyl-1H-pyrazole-4-
carboxamide 2 with methyl chloro acetate afforded 6-chlo-
romethyl-1-phenyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimi-
1
din-4-one 7. The H NMR of 7 showed the signal at d
4.63 ppm as singlet corresponding to two protons indicating
the presence of chloro methyl (–CH2Cl) group at sixth
position of pyrazolopyrimidines. The compounds 3–7 were
chlorinated with phosphorous oxychloride, and preceded to
the next step immediately without any purification as they
123

1092
Med Chem Res (2013) 22:1090–1101
were unstable. The chlorinated compounds were treated with
various amines, in dry toluene to produce the title com-
pounds. The obtained compounds showed a prominent peak
at 3,500–3,200 cm-1 which is a characteristic of secondary
amino group in IR spectrum, which indicated formation of
Table 1 Results obtained by conventional and microwave methods
Compound MWI
code
Conv
(conditions) (time, h) (time) (yield %) (yield %)
MWI
Conv
MWI
3
B
B
B
B
C
C
C
C
C
14
14
14
14
12
12
12
12
12
45 s
45 s
45 s
45 s
80
78
80
82
91
96
92
93
44
45
42
43
41
1
4
the expected product. The H NMR of compounds 3b, 4b,
5
5b, 7b showed signal at d 0.94–0.98 ppm, a triplet, inte-
grating for three protons indicating the terminal protons of
the propyl amine substituent. It also showed signals at d
1.62–1.69 ppm, a sextet and a peak at d 3.47–3.52, a quartet
integrating the protons of the second and first carbon of the
propyl amine substituent at fourth position of pyrazolopyr-
imidines. The compounds 3c, 4c, 5c, 6c, 7c showed a heptet
at d 4.43–4.47 ppm confirms the isopropyl group. The
compounds 3e, 4e, 5e showed multiplet at d 7.40 ppm and
doublet at d 7.48–7.52 ppm confirming the fourth and third,
6
3a
4a
5a
6a
7a
5 min 40
5 min 35
7 min 37
6 min 33
6 min 35
B sodium ethoxide/ethanol reflux/MWI, 100 W, 80 °C, 45 s, 20 bar,
C phosphorous oxychloride/MWI, 100 W, 120 °C, 10 min 30 bar,
Conv conventional, MWI microwave irradiation
Table 2 Antibacterial activity
data of 3b–3f, 4b–4k, 5b–5g,
6b–6d, 7b–7f at 20 lg/mL
Code
X
R
E. coli
B. s
S. a
S. e
3b
3c
3d
3e
3f
H
Propyl
13.2 0.060
13.3 0.050
13.6 0.050
12.8 0.060
13.3 0.050
12.7 0.062
13.0 0.070
14.4 0.101
13.6 0.050
12.7 0.044
13.3 0.050
13.6 0.100
12.6 0.050
13.4 0.050
13.5 0.052
13.6 0.050
13.3 0.050
12.7 0.066
12.7 0.044
13.0 0.00
13.3 0.050
13.3 0.050
14.6 0.111
12.8 0.078
13.3 0.050
1.30 0.00
13.3 0.050
13.6 0.050
13.3 0.050
18.7 0.066
13.2 0.080
13.3 0.050
13.0 0.050
13.6 0.050
12.8 0.060
13.0 0.707
13.6 0.707
13.8 0.120
12.5 1.052
13.6 0.050
13.6 0.050
13.1 0.060
13.3 0.050
13.3 0.050
13.4 0.057
13.0 0.00
13.3 0.050
12.8 0.060
13.6 0.050
13.3 0.050
13.3 0.050
12.8 0.060
14.4 0.113
13.5 0.050
13.6 0.050
13.6 0.050
12.7 0.083
1.30 0.00
13.6 0.050
19.0 0.086
12.7 0.040
13.6 0.050
13.3 0.050
13.0 0.086
13.6 0.050
13.6 0.050
13.3 0.050
14.6 0.120
12.7 0.060
13.3 0.050
13.4 0.072
13.7 0.044
13.1 0.060
13.3 0.050
13.3 0.050
13.3 0.050
13.6 0.050
13.3 0.050
13.3 0.050
13.0 0.00
13.6 0.050
14.4 0.101
14.2 0.109
13.3 0.050
1.33 0.050
1.30 0.000
12.6 0.050
13.6 0.050
13.2 0.044
17.3 0.132
13.3 0.050
13.6 0.050
13.3 0.050
13.6 0.050
12.7 0.083
13.6 0.050
13.7 0.092
14.5 0.087
13.3 0.050
13.6 0.050
13.6 0.050
13.3 0.050
12.7 0.066
13.3 0.050
13.6 0.050
13.3 0.050
13.6 0.050
13.0 0.00
13.6 0.050
13.0 0.00
13.6 0.050
14.1 0.105
14.3 0.111
13.3 0.050
1.33 0.050
1.33 0.050
13.6 0.050
13.3 0.050
13.0 0.000
16.6 0.05
H
Isopropyl
Butyl
H
H
Furfuryl
Morpholine
Propyl
H
4b
4c
4d
4e
4f
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
i-C3H7
i-C3H7
i-C3H7
CH2Cl
CH2Cl
CH2Cl
CH2Cl
CH2Cl
–
Isopropyl
Butyl
Furfuryl
Morpholine
Aniline
Diethyl
Benzyl
4g
4h
4i
4j
Ethyl
4k
5b
5c
5d
5e
5f
o-Anisidine
Propyl
Isopropyl
Butyl
Furfuryl
Morpholine
Aniline
Butyl
5g
6b
6c
6d
7b
7c
7d
7e
7f
Morpholine
Aniline
Propyl
Zone of inhibition in
millimeters (average SEM)
(n = 9)
E. coli Escherichia coli (NCIM-
2803), B. s Bacillus subtilis
(NCIM-2545),
S. a Staphylococcus aureus
(NCIM-5021),
Isopropyl
Ethyl
Morpholine
Aniline
–
S. e Staphylococcus epidermidis
(NCIM-2493)
AMOX
123

Med Chem Res (2013) 22:1090–1101
1093
Table 3 Antibacterial activity selected compounds at 10 lg/mL
Compound
X
R
E. coli
B. s
S. a
11.3 0.050
S. e
4d
CH3
Butyl
Butyl
Morpholine
–
11.0 0.382
0.0
0.0
11.1 0.033
11.0 0.0
11.0 0.0
13.0 0.30
6b
i-C3H7
i-C3H7
–
0.0
11.0 0.0
10.33 0.10
16.6 0.05
6c
10.6 0.050
15.5 0.072
11.3 0.050
16.3 0.05
AMOX
Zone of inhibition in millimeters (average SEM) (n = 9)
E. coli Escherichia coli (NCIM-2803), B. s Bacillus subtilis (NCIM-2545), S. a Staphylococcus aureus (NCIM-5021), S. e Staphylococcus
epidermidis (NCIM-2493), i-C3H7 isopropyl
Table 4 Results of in vitro anticancer activity of testing of 3b–3f,
4b–4k, 5b–5g, 6b–6d, 7b–7f
Table 5 Parameters of anticancer activity calculated from graph
Drug code
Molar drug concentration
Drug code
Human skin cancer cell line G361
% Growtha
LC50
TGI
GI50
3b
3d
3e
3f
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[10-4
[[10-4
[10-4
1.7 9 10-7
[10-4
[10-4
Molar drug concentration
3.7 9 10-5
[10-4
2.1 9 10-6
[10-4
3b
3d
3e
3f
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
99.7
100.0
90.8
99.7
43.4
64.8
-4.5
51.7
[10-4
[10-4
4b
4c
4d
4e
4f
[10-4
3.6 9 10-5
3.2 9 10-5
2.4 9 10-6
3.9 9 10-5
[10-4
97.5
92.3
[10-4
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
94.7
100.0
100.0
100.0
100.0
100.0
99.8
61.5
[10-4
4b
4c
4d
4e
4f
25.3
[10-4
13.5
[10-4
-30.0
30.1
4g
4h
4i
[10-4
[10-4
[10-4
[10-4
43.9
[10-4
[10-4
4g
4h
4i
100.0
100.0
100.0
99.6
65.6
4j
[10-4
3.87 9 10-5
2.2 9 10-6
3.8 9 10-5
2.02 9 10-6
2.04 9 10-6
2.8 9 10-5
[10-4
61.8
5b
5c
5d
5e
5f
3.7 9 10-5
[10-4
45.5
4j
29.2
3.2 9 10-5
3.2 9 10-5
[10-4
5b
5c
5d
5e
5f
84.7
-34.7
40.0
96.4
83.3
96.5
84.8
-62.5
-61.0
30.9
6b
6c
6d
7
[10-4
96.5
86.3
[10-4
2.6 9 10-6
3.2 9 10-5
2.08 9 10-6
[10-4
85.5
65.9
[10-4
6b
6c
6d
7a
7b
7c
7d
7e
7f
100.0
92.5
100.0
77.6
45.4
3.32 9 10-5
[10-4
6.9
7b
7c
7d
7e
7f
100.0
100.0
99.6
91.9
19.2
[10-4
[10-4
90.9
-59.3
75.1
[10-4
[10-4
100.0
99.7
[10-4
[10-4
100.0
100.0
99.6
56.5
[10-4
[10-4
100.0
94.3
87.7
ADR
\10-7
\10-7
47.6
100.0
-24.0
100.0
-62.5
99.7
48.5
ADR
-64.2
-70.7
a
Average value of 3 experiments
group is confirmed by the doublet signals of two protons CH2
at d 4.78–4.79 ppm, thus differing from aniline substituent.
The mass spectrum showed molecular ion peak [M?] as the
base peak and fragmentation pattern characteristic to its
structure. The elemental analyses of all the newly synthe-
sized compounds confirmed their formation.
fifth proving of furfuryl group. The compounds 3f, 4f, 5f, 6c,
7e showed triplets at d 3.77–3.79 ppm and d 3.96–3.98 ppm
which indicates the protons of morpholine group. The benzyl
123

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Med Chem Res (2013) 22:1090–1101
Antibacterial activity
Conclusions
The compounds 3b–3f, 4b–4k, 5b–5g, 6b–6d, 7b–7f were
screened for their antibacterial activity against Gram positive
[B. subtilis (NCIM-2545), S. epidermidis (NCIM-2493),
S. aureus (NCIM-5021)] and Gram negative [E. coli (NCIM-
2803)] bacteria by agar diffusion method and the results are
summarized in Tables 2 and 3. All the compounds were found
to be active against all the four bacterial strains at 20 lg/mL
concentration. The activity of these compounds varied with
the kind of organism. The compounds 4d, 6b, 6c showed
higher zone of inhibitions than the rest thus being the best of
the series. The above studies also showed that 6-isopropyl-4-
(morpholino-4-yl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine
6c was the best of the series, showed activity against all the
four organisms, at a concentration of 10 lg/mL. The com-
pound N-butyl-6-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyr-
imidin-4-amine 4d exhibited activity against E. coli,
S. aureus and S. epidermidis at a concentration of 10 lg/
mL. The compound N-butyl-6-isopropyl-1-phenyl-1H-pyr-
azolo[3,4-d]pyrimidin-4-amine 6b showed activity against
S. aureus and S. epidermidis.
We have successfully synthesized 6-alkyl-1-phenyl-4-sub-
stituted-1H-pyrazolo[3,4-d]pyrimidines. Basically pyrazol-
o[3,4-d]pyrimidines were reported to be good antibacterial
agents, and in our study we have replaced the 1H atom of
pyrazole ring with phenyl group as an active group compared
to the non substituent group. And the final compounds which
are substituted with various amines were found to be more
active towards the tested organisms. The antibacterial
activity data showed that 4d, 6b, 6c are the compounds found
to have best antibacterial activity of the series. The com-
pounds 7, 3d, 4d, 5b, 5d, 5e, 5f, 6c showed antiproliferative
activity against human skin cancer cell lines. The presence of
ethyl group at sixth position was found to be responsible for
anticancer property. The presence of isopropyl group at sixth
position in 6b, 6c and the presence of butyl amine group at
fourth position in 4d, 6b might be the reason for both anti-
bacterial and anticancer activities. The replacement of ali-
phatic amines at fourth position with bioactive groups such
as furfuryl amine and morpholine groups showed anticancer
activity.
Anticancer activity
Experimental
The compounds were 3b–3f, 4b–4k, 5b–5g, 6b–6d, 7b–7f
screened for their anticancer activity against Human Skin
Cancer Cell Line G361 by sulforhodamine B (SRB) assay
and the results are summarized in Tables 4 and 5. The
compounds 3d, 4d, 5b, 5d, 5e, 6c, 7a showed antiprolif-
erative activity. The % growth inhibition was found to be
considerable at a concentration of 10-4 M, while the ref-
erence drug doxorubicin has its effect at 10-7 M. TGI50
(growth inhibition of 50 % cells, calculated from drug
concentration resulting in a 50 % reduction in the net
General techniques
Melting points were determined on VEEGO-programmable
melting point apparatus and are uncorrected. The micro-
wave-assisted synthesis was carried out on microwave
(CEM Discover). The IR spectra (KBr pellet method) were
recorded on a Bruker-Alpha IR spectrophotometer. 1HNMR
spectra were recorded on Bruker NMR spectrophotometer
400 MHz in DMSO-d6, CDCl3 as solvents. Chemical shift
values are reported in d ppm relative to tetramethylsilane
(TMS) as relative standard. The mass spectra [EI–MS] of the
compounds were recorded on APEX instrument. Elemental
analyses for C, H, N and O were performed using a Carlo
Erba 1108 elemental analyser and the % of C, H, N was found
to be within 0.4 % of the theoretical values. The purity of
compounds was checked by thin layer chromatography
(TLC, Merck, silica F254) plates using chloroform:ethyl
acetate (7:3) as mobile phase. All the compounds were
characterized by the above techniques. Amoxicillin and
doxorubicin were obtained commercially.
protein increase) values are in the range 2.08–2.8 9 10-6
.
Based on structural features and the activity shown by
the synthesized compounds, structure and activity rela-
tionship may be obtained. As the SAR is concerned, the
isopropyl amine group at sixth position of pyrazolopyr-
imidine ring of compounds 6b, 6c might be contributing to
the best antibacterial activity. Similarly, the presence of
butyl amine group at fourth position in 4d, 6b might be the
reason for the antibacterial activity at low concentration.
The presence of butyl amine group at fourth position may
be responsible for the anticancer activity for 3d, 4d, 5d, 6b.
The presence of bioactive groups such as furfuryl amine
and morpholine groups at fourth position may be the reason
for anticancer activity in 5e, 6c compounds. The presence
of ethyl group at sixth position may contribute to anti-
cancer activity as that series produced greater number of
compounds 5b, 5d, 5e, 5f with anticancer property.
General procedure for synthesis of 6-alkyl-1-phenyl-1,
5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-ones (3–6)
Conventional synthesis Sodium ethoxide was prepared by
dissolving sodium metal (4 g, 100 mmol) in ethanol
(150 mL) under nitrogen atmosphere. The compound
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1095
5-amino-1-phenyl-1H-pyrazole-4-carboxamide
2
(3.4 g,
mixture and neutralized acid using sodium bicarbonate
solution to precipitate the product. The product obtained
was filtered, dried and taken to next step immediately
without further purification as they were unstable (3a–7a).
20 mmol) obtained in the previous step and various esters
(200 mmol) were added to the prepared sodium ethoxide
and heated to reflux at 80 °C for 14 h to get the products
pyrazolo[3,4-d]pyrimidin-ones 3–6.
The compound 5-amino-1-phenyl-1H-pyrazole-4-car-
boxamide 2 (3.4 g, 20 mmol) obtained in the previous step
and various esters (200 mmol) were added to the prepared
sodium ethoxide and stirred the reaction mixture in a
round-bottomed flask at room temperature for 1 h to get the
compounds 6-chloromethyl-1-phenyl-1,5-dihydro-4H-pyr-
azolo[3,4-d]pyrimidin-4-one 7.
General procedure for synthesis of 4-substituted-amino-6-
alkyl/aryl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidines (3b–3f,
4b–4k, 5b–5g, 6b–6g, 7b–7f)
The series of compounds prepared in the previous step (3a–
7a) (20 mmol) was dissolved in anhydrous toluene
(10 mL) and corresponding amines (30 mmol) was added
and the reaction mixture was stirred at room temperature
for 18–36 h. The reaction mixture was washed with water
(10 mL), and the organic phase was dried under reduced
pressure. The oil residue was crystallized by absolute
ethanol to give the title compounds.
Microwave method of synthesis The compound 5-amino-
1-phenyl-1H-pyrazole-4-carboxamide 2 (3.4 g, 20 mmol)
obtained in the previous step and various esters (200 mmol)
were added to the prepared sodium ethoxide and irradiated at
80 °C in CEM microwave for 45 s at 100 W, 20 bar for
compounds pyrazolo[3,4-d]pyrimidin-ones 3–6.
N-Propyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
(3b) Yield 58.65 %, mp 188 °C. IR (KBr, cm-1): 3444.54
(–NH), 3218.10 (=C–H, aromatic), 1589.49 (C=C, aromatic),
770.88 (–CH oop); 1H NMR (400 MHz, DMSO-d6) d:
0.94–0.98 (t, J = 7.4 Hz, 3H, –CH2CH2CH3), 1.62–1.69
(sextet, J = 7.2 Hz, 2H, –CH2CH2CH3), 3.47–3.52 (q,
J = 6.6 Hz, 2H, –CH2CH2CH3), 7.32–7.36 (t, J = 8.0 Hz,
1H, H-40 Ph), 7.52–7.56 (d, J = 8.0 Hz, 2H, H-20 and 60 Ph),
8.19–8.21 (d, J = 7.6 Hz, 2H, H-30 and 50 Ph), 8.37 (s, 1H,
H-3), 8.4 (s, 1H, H-6). MS m/z 253 (M?1), 254 (M?2). Anal.
Calc. forC14H15N5: C66.38,H5.97, N27.65.Found:C 65.38,
H 4.89, N 26.65.
Reaction workup The reaction mixture was added into cold
water and neutralized with dropwise addition of concentrated
hydrochloric acid. The precipitate formed was filtered, dried
and recrystallized from chloroform to give compounds 3–7.
6-Chloromethyl-1-phenyl-1,5-dihydro-4H-pyrazolo[3,4-
d]pyrimidin-4-one (7)
Yield 45.89 %, mp 230 °C. IR (cm-1) (KBr): 3442.7 (–NH,
2° amide), 2971.9 (–CH2), 1693.4 (–C=O), 1594.1 (C=C,
aromatic), 779.1 (–CH oop); H NMR (400 MHz, DMSO-
1
d6) d: 4.63 (s, 2H, –CH2 –Cl), 7.41–7.44 (t, J = 7.4 Hz, 1H,
H-40 Ph), 7.56–7.60 (t, J = 7.8 Hz, 2H, H-20 and 60 Ph),
8.03–8.05 (d, J = 8.0 Hz, 2H, H-30 and 50 Ph), 8.35 (s, 1H,
H-3). MS m/z 260 (M?1), 261 (M?2). Anal. Calc. for
C12H9ClN4O: C 55.29, H 3.48, Cl 13.60, N 21.49, O 6.14.
Found: C 55.09, H 3.28, Cl 13.10, N 21.09, O 6.04.
N-Isopropyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
(3c) Yield 69.93 %, mp 137 °C. IR (KBr, cm-1):
3446.29 (–NH), 3087.90 (=C–H, aromatic), 2970.40
1
(–CH), 1585.79 (C=C, aromatic), 747.31 (–CH oop); H
NMR (400 MHz, DMSO-d6) d: 1.26–1.28 (d, J = 6.8 Hz,
6H, –CH–[CH3]2), 4.43–4.47 (heptet, J = 5.2 Hz, 1H,
–CH–[CH3]2), 7.32–7.35 (t, J = 7.4 Hz, 1H, H-40 Ph),
7.52–7.56 (d, J = 8.0 Hz, 2H, H-20 and 60 Ph), 8.21–8.23
(d, J = 8.0 Hz, 2H, H-30 and 50 Ph), 8.37 (s, 1H, H-3), 8.4
(s, 1H, H-6). MS m/z 253 (M?1), 254 (M?2). Anal. Calc.
for C14H15N5: C 66.38, H 5.97, N 27.65. Found: C 65.38, H
4.97, N 26.65.
General procedure for synthesis of 4-chloro-6-alkyl-1-
phenyl-1H-pyrazolo[3,4-d]pyrimidines (3a–7a)
Conventional synthesis Compounds 3–7 and phosphorous
oxychloride in 1 g:10 mL ratio were taken in the reaction
vessel and heated to reflux for 10 min at 120 °C for 12 h to
get the product.
N-Butyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (3d)
Yield 60.34 %, mp 102 °C. IR (KBr, Cm-1): 3446.21
(–NH), 3077.47 (=C–H, aromatic), 2931.03 (–CH2), 1591.60
(C=C, aromatic), 750.70 (–CH oop); 1H NMR
(400 MHz, DMSO-d6) d: 0.97–1.01 (t, J = 7.4 Hz, 3H,
–CH2–CH2–CH2–CH3), 1.40–1.49 (sextet, J = 7.3 Hz, 2H,
–CH2–CH2–CH2–CH3), 1.63–1.71 (pentet, J = 7.2 Hz, 2H,
–CH2–CH2–CH2–CH3), 3.56–3.61 (q, J = 6.5 Hz, 2H,
–CH2–CH2–CH2–CH3), 7.37–7.41 (t, J = 7.4 Hz, 1H, H-40
Microwave method of synthesis Compounds 3–7 and
phosphorous oxychloride in 1 g:10 mL ratio were taken in
the reaction vessel and irradiated in microwave at 120 °C
for 5–7 min at 100 W, 30 bar to get the products.
Reaction workup Excess phosphorous oxychloride was
distilled off then crushed ice was added to the reaction
123

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Med Chem Res (2013) 22:1090–1101
Ph), 7.57–7.61 (d, J = 7.8 Hz, 2H, H-20 and 60 Ph),
8.24–8.26 (d, J = 7.6 Hz, 2H, H-30 and 50 Ph), 8.42 (s, 1H,
H-3), 8.44 (s, 1H, H-6). MS m/z 267 (M?1), 268 (M?2).
Anal. Calc. for C15H17N5: C 67.39, H 6.41, N 26.20. Found:
C 66.05, H 5.31, N 25.20.
(t, J = 7.4 Hz, 1H, H-40 Ph), 7.52–7.56 (d, J = 7.8 Hz, 2H,
H-20 and 60 Ph), 8.20–8.22 (d, J = 8.0 Hz, 2H, H-30 and 50
Ph), 8.35 (s, 1H, H-3). MS m/z 267 (M?1), 268 (M?2). Anal.
Calc. for C15H17N5: C 67.39, H 6.41, N 26.20. Found: C
66.13, H 5.21, N 25.10.
N-(Furan-2-yl-methyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrim-
idin-4-amine (3e) Yield 55.37 %, mp 160 °C. IR (KBr,
cm-1): 3451.14 (–NH); 3071.76 (=C–H, aromatic),
2911.84 (–CH2), 1591.64 (C=C, aromatic), 739.76 (–CH
oop); 1H NMR (400 MHz, CDCl3) d: 4.86–4.87 (d,
J = 5.2 Hz, 2H, –NH–CH2–), 5.85 (s, 1H, NH), 6.34–6.36
(d, J = 3.2 Hz, 2H, H-20 and 60 Ph), 7.30–7.34 (t,
J = 7.4 Hz, 1H, H-40 Ph), 7.40 (m, 1H, H-400), 7.48–7.52
(d, J = 8.0 Hz, 2H, H-300 and 500), 8.04 (s, 1H, H-3),
8.13–8.15 (d, J = 8.0 Hz, 2H, H-30 and 50 Ph), 8.51 (s, 1H,
H-6). MS m/z 291 (M?1), 292 (M?2). Anal. Calc. for
C16H13N5O: C 65.97, H 4.50, N 24.04, O 5.49. Found: C
64.35, H 3.50, N 22.69, O 4.51.
N-Butyl-6-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
amine (4d) Yield 76.25 %, mp 110 °C. IR (KBr, cm-1):
3382.9 (–NH), 2981.0, 2966.8, 2925.2 (–CH3), 1597.1
1
(C=C, aromatic), 787.2 (–CH oop); H NMR (400 MHz,
DMSO-d6) d: 0.93–0.96 (t, J = 7.4 Hz, 3H, –CH2–CH2–
CH2–CH3), 1.36–1.43 (pentet, J = 7.4 Hz, 2H,–CH2–
CH2–CH2–CH3), 1.58–1.65 (pentet, J = 7.1 Hz, 2H,
–CH2–CH2–CH2–CH3), 2.48 (s, 3H, –CH3), 3.50–3.55 (q,
J = 6.5 Hz, 2H, –CH2–CH2–CH2–CH3), 7.30–7.34 (t,
J = 7.4 Hz, 1H, H-40 Ph), 7.52–7.56 (t, J = 8.0 Hz, 2H,
H-20 and 60 Ph), 8.20–8.22 (d, J = 7.6 Hz, 2H, H-30 and 50
Ph), 8.33 (s, 1H, H-3). MS m/z 281 (M?1), 282 (M?2).
Anal. Calc. for C16H19N5: C 68.30, H 6.81, N 24.89.
Found: C 67.05, H 5.51, N 23.69.
4-(Morpholin-4-yl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine
(3f) Yield 51.21 %, mp 156 °C. IR (KBr, cm-1): 3018.2
(=C–H, aromatic), 2917.65(–CH2), 1570.75 (C=C, aromatic),
771.58 (–CH oop); 1H NMR (400 MHz, DMSO-d6) d:
3.77–3.79 (t, J = 4.8 Hz, 4H, H-200 and 600 morph), 3.96–3.98
(t, J = 4.8 Hz, 4H, H-300 and 500 morph), 7.35–7.39 (t,
J = 7.4 Hz, 1H, H-40 Ph), 7.54–7.58(d, J = 7.6 Hz, 2H, H-20
and60 Ph), 8.17–8.19(d, J = 8.4 Hz, 2H, H-30 and50 Ph),8.41
(s, 1H, H-3), 8.60 (s, 1H, H-6). MS m/z 281 (M?1), 282
(M?2). Anal. Calc. for C15H15N5O: C 64.04, H 5.37, N 24.90,
O 5.69. Found: C 63.35, H 5.00, N 22.69, O 4.51.
N-(Furan-2-ylmethyl)-6-methyl-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (4e) Yield 73.29 %, mp 116 °C. IR
(KBr, cm-1): 3241.1 (–NH), 2957.3 (–CH2), 1580.4 (C=C,
1
aromatic), 784.0 (–CH oop); H NMR (400 MHz, CDCl3)
d: 3.32 (s, 3H, –CH3) 4.77–4.78 (d, J = 4.8 Hz, 2H, –NH–
CH2–), 6.39–6.44 (dd, J = 8.0 Hz, 2H, H-300 and 500),
7.32–7.35 (t, J = 7.2 Hz, 1H, H-40 Ph), 7.53–7.57 (t,
J = 7.8 Hz, 2H, H-20 and 60 Ph), 7.63 (s, 1H, H-400),
8.20–8.22 (d, J = 8.0 Hz, 2H, H-30 and 50 Ph), 8.37 (s, 1H,
H-3). MS m/z 305 (M?1), 306 (M?2). Anal. Calc. for
C17H15N5O: C 66.87, H 4.95, N 22.94, O 5.24. Found: C
65.47, H 3.55, N 21.34, O 4.04.
N-Propyl-6-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
amine (4b) Yield 70.68 %, mp 113 °C. IR (KBr, cm-1):
3447.9 (–NH), 2955.7, 2922.8 (–CH3), 1606.2 (C=C, aro-
6-Methyl-4-(morpholin-4-yl)-1-phenyl-1H-pyrazolo[3,4-d]pyrim-
idine (4f) Yield 85.5 %, mp 116 °C. IR (KBr, cm-1):
2960.7 (–CH3), 1590.6 (C=C, aromatic), 787.6 (–CH oop);
1H NMR (400 MHz, DMSO-d6) d: 3.68 (s, 3H, –CH3),
3.76–3.78 (t, J = 4.0 Hz, 4H, H-200 and 600 morph),
3.94–3.95 (d, J = 4.4 Hz, 4H, H-300 and 500 morph),
7.34–7.37 (t, J = 7.2 Hz, 1H, H-40 Ph), 7.54–7.58 (t,
J = 7.4 Hz, 2H, H-20 and 60 Ph), 8.18–8.20 (d, J = 8.0 Hz,
2H, H-30 and 50 Ph), 8.52 (s, 1H, H-3). MS m/z 295 (M?1),
296 (M?2). Anal. Calc. for C16H17N5O: C 65.07 H 5.80, N
23.71, O 5.42. Found: C 64.07, H 4.27, N 22.41, O 4.16.
1
matic), 784.9 (–CH oop); H NMR (400 MHz, DMSO-d6)
d: 0.94–0.98 (t, J = 7.4 Hz, 3H, –CH2CH2CH3), 1.60–1.69
(sextet, J = 7.2 Hz, 2H, –CH2CH2CH3), 2.48 (s, 3H,
–CH3), 3.46–3.51 (q, J = 6.5 Hz, 2H, –CH2–CH2–CH3),
7.30–7.34 (t, J = 7.4 Hz, 1H, H-40 Ph), 7.52–7.56 (t,
J = 7.8 Hz, 2H, H-20 and 60 Ph), 8.20–8.22 (dd,
J = 8.0 Hz, 2H, H-30 and 50 Ph), 8.33 (s, 1H, H-3). MS
m/z 267 (M?1), 268 (M?2). Anal. Calc. for C15H17N5: C
67.39, H 6.41, N 26.20. Found: C, 66.39, H 5.41, N 25.20.
N-Isopropyl-6-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimi-
din-4-amine (4c) Yield 80.2 %, mp 114 °C. IR (KBr,
cm-1): 3382.9 (–NH), 2981.0, 2966.8, 2925.2 (–CH3),
1597.1 (C=C, aromatic), 787.2 (–CH oop); 1H NMR
(400 MHz, DMSO-d6) d: 1.25–1.27 (d, J = 6.8 Hz, 6H,
–CH–[CH3]2), 2.49–2.51 (dd, J = 8.0 Hz, 3H, –CH3),
4.46–4.48 (d, J = 7.2 Hz, 1H, –CH–[CH3]2), 7.31–7.34
6-Methyl-N,1-diphenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
(4g) Yield 75.5 %, mp 115 °C. IR (KBr, cm-1): 3449.7
(–NH), 2909.3 (–CH2), 1596.5 (C=C, aromatic), 784.49
(–CH oop);1H NMR (400 MHz, DMSO-d6) d: 2.51–2.58 (d,
J = 3.0 Hz, 3H, –CH3), 7.05–7.14 (dd, J = 4.4 Hz, 2H,
,
H-40 and 400 Ph), 7.31–7.42 (m, J = 6.8 Hz, 4H, H-200 300, 500
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Med Chem Res (2013) 22:1090–1101
1097
and 600 Ph), 7.91–7.92 (d, J = 6.0 Hz, 2H, H-20 and 60 Ph),
8.21–8.23 (d, J = 7.2 Hz, 2H, H-30 and 50 Ph), 8.48 (s, 1H,
H-3). MS m/z 301 (M?1), 302 (M?2); Anal. Calc. for
C18H15N5: C 71.74, H 5.02, N 23.24. Found: C 70.34, H 4.13,
N 22.04.
C19H17N5O: C 68.87, H 5.17, N 21.13, O 4.83. Found: C
68.87, H 4.13, N 21.13, O 4.83.
N-Propyl-6-ethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
amine (5b) Yield 75.21 %, mp 132 °C. IR (KBr, cm-1):
3422.2 (–NH), 2958.9, 2924.8 (–CH2), 1596.2 (C=C, aro-
1
N,N-Diethyl-6-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimi-
din-4-amine (4h) Yield 79.04 %, mp 145 °C. IR (KBr,
cm-1): 2982.0 (–CH2), 1592.4 (C=C, aromatic), 780.8
matic), 757.6 (–CH oop); H NMR (400 MHz, DMSO-d6)
d: 0.94–0.98 (t, J = 7.4 Hz, 3H, –NH–CH2–CH2–CH3),
1.27–1.31 (t, J = 7.4 Hz, 3H, –CH2–CH3), 1.63–1.70
(pentet, J = 7.1 Hz, 2H, –NH–CH2–CH2–CH3), 2.72–2.78
(q, J = 7.3 Hz, 2H, –CH2–CH3), 3.47–3.52 (q, J =
6.6 Hz, 2H, –NH–CH2–CH2–CH3), 7.30–7.33 (t, J =
7.4 Hz, 1H, H-40 Ph), 7.52–7.56 (t, J = 7.8 Hz, 2H, H-20
and 60 Ph), 8.24–8.25 (d, J = 7.6 Hz, 2H, H-30 and 50 Ph),
8.32 (s, 1H, H-3). MS m/z 281 (M?1), 282 (M?2). Anal.
Calc. for C16H19N5: C 68.30, H 6.81, N 24.89. Found: C
67.30, H 5.81, N 23.89.
1
(–CH oop); H NMR (400 MHz, DMSO-d6) d: 1.16–1.21
(t, J = 7.6 Hz, 3H, –N (–CH2–CH3)2), 2.51 (s, 3H, –CH3),
2.86–2.91 (q, J = 7.2 Hz, 2H, –N (–CH2–CH3)2),
7.30–7.34 (t, J = 7.4 Hz, 1H, H-40 Ph), 7.52–7.56 (t,
J = 7.8 Hz, 2H, H-20 and 60 Ph), 8.20–8.22 (d, J = 8.0 Hz,
2H, H-30 and 50 Ph), 8.31 (s, 1H, H-3). MS m/z 281 (M?1),
282 (M?2). Anal. Calc. for C16H19N5: C 68.30, H 6.81, N
24.89. Found: C 67.38, H 5.61, N 23.59.
N-Benzyl-6-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-
4-amine (4i) Yield 56.78 %, mp 139 °C. IR (KBr, cm-1):
3449.7 (–NH), 2909.3 (–CH2), 1596.5 (C=C, aromatic),
784.49 (–CH oop); 1H NMR (400 MHz, DMSO-d6) d:
2.50–2.51 (m, J = 1.7 Hz, 3H, –CH3), 4.78–4.79 (d, J =
5.6 Hz, 2H, –NH–CH2–C6H5), 7.28–7.31 (t, J = 6.8 Hz,
N-Isopropyl-6-ethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4
-amine (5c) Yield 72.9 %, mp 122 °C. IR (KBr, cm-1):
3397.1 (–NH), 2948.2 (–CH2), 1566.6 (C=C, aromatic),
784.9 (–CH oop); 1H NMR (400 MHz, DMSO-d6) d:
1.26–1.31 (q, J = 6.6 Hz, 9H, –CH–(CH3)2 and –CH2–
CH3), 2.73–2.78 (q, J = 7.1 Hz, 2H, –CH2–CH3), 4.47 (s,
1H, –NH), 7.30–7.33 (t, J = 7.2 Hz, 1H, H-40 Ph), 7.52–7.56
(t, J = 7.8 Hz, 2H, H-20 and 60 Ph), 8.23–8.25 (d,
J = 7.6 Hz, 2H, H-30 and 50 Ph), 8.35 (s, 1H, H-3). MS m/
z 281 (M?1), 282 (M?2). Anal. Calc. for C16H19N5: C
68.30, H 6.81, N 24.89. Found: C 67.30, H 5.81, N 23.89.
,
1H, H-40 Ph), 7.33–7.41 (m, J = 7.2 Hz, 5H, H-200 300, 500,
600 and 40 Ph), 7.52–7.56 (t, J = 7.8 Hz, 2H, H-20 and 60
Ph), 8.20–8.22 (d, J = 8.0 Hz, 2H, H-30 and 50 Ph), 8.37 (s,
1H, H-3). MS m/z 315 (M?1), 316 (M?2). Anal. Calc. for
C19H17N5: C 72.36, H 5.43, N 22.21. Found: C 71.06, H
4.13, N 21.01.
N-Butyl-6-ethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
amine (5d) Yield 65.7 %, mp 126 °C. IR (KBr, cm-1):
3442.7 (–NH), 2961.6, 2926.5 (–CH2), 1583.5 (C=C, aro-
N-Ethyl-6-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-
4-amine (4j) Yield 56.89 %, mp 140 °C. IR (KBr, cm-1):
3274.1 (–NH), 2957.5 (–CH2), 1616.6 (C=C, aromatic),
786.6 (–CH oop); 1H NMR (400 MHz, DMSO-d6) d:
1.22–1.25 (t, J = 7.2 Hz, 3H, –NH–CH2–CH3), 2.49–2.51
(d, J = 6.8 Hz, 2H, –CH3), 3.52–3.59 (pentet, J = 6.6 Hz,
2H, –NH–CH2–CH3), 7.30–7.34 (t, J = 7.4 Hz, 1H, H-40
Ph), 7.52–7.56 (t, J = 7.8 Hz, 2H, H-20 and 60 Ph), 8.20–8.22
(d, J = 8.0 Hz, 2H, H-30 and 50 Ph), 8.31 (s, 1H, H-3). MS
m/z 253 (M?1), 254 (M?2). Anal. Calc. for C14H15N5:
C 66.38, H 5.97, N 27.65. Found: C 65.38, H 4.27, N 26.65.
1
matic), 753.6 (–CH oop); H NMR (400 MHz, DMSO-d6)
d: 0.92–0.96 (t, J = 7.4 Hz, 3H,–NH–CH2–CH2–CH2–
CH3), 1.27–1.31 (t, J = 7.4 Hz, 3H, –CH2–CH3),
1.37–1.44 (pentet, J = 7.2 Hz, 2H,–NH–CH2–CH2–CH2–
CH3), 1.59–1.66 (pentet, J = 6.8 Hz, 2H, –NH–CH2–
CH2–CH2–CH3), 2.72–2.78 (q, J = 7.3 Hz, 2H, –CH2–
CH3), 3.52–3.57 (q, J = 6.2 Hz, 2H, –NH–CH2–CH2–
CH2–CH3), 7.30–7.33 (t, J = 7.4 Hz, 1H, H-40 Ph),
7.52–7.56 (t, J = 7.8 Hz, 2H, H-20 and 60 Ph), 8.24–8.26
(d, J = 8.0 Hz, 2H, H-30 and 50 Ph), 8.32 (s, 1H, H-3). MS
m/z 295 (M?1), 296 (M?2). Anal. Calc. for C17H21N5: C
69.12, H 7.17, N 23.71. Found: C 68.12, H 6.17, N 22.31.
N-(2-Methoxyphenyl)-6-methyl-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (4k) Yield 62.65 %, mp 150 °C. IR
(KBr, cm-1): 3423.2 (–NH), 2909.3 (–CH2), 1628.3 (C=C,
1
aromatic), 746.3 (–CH oop); H NMR (400 MHz, DMSO-
N-Furfuryl-6-ethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-
4-amine (5e) Yield 56.7 %, mp 124 °C. IR (KBr, cm-1):
3442.2 (–NH), 2975.5, 2923.7 (–CH2), 1583.6 (C=C, aro-
d6) d: 2.89 (s, 3H, –CH3), 3.90 (s, 3H, –O–CH3), 7.31–7.42
(m, J = 6.8 Hz, 4H, H-300, 400, 500 and 600 Ph), 7.91–7.92 (d,
J = 6.0 Hz, 2H, H-20 and 60 Ph), 8.21–8.23 (d, J = 7.2 Hz,
2H, H-30 and 50 Ph), 8.48 (s, 1H, H-3), 9.90 (s, 1H, –NH).
MS m/z 331 (M?1), 332 (M?2); Anal. Calc. for
1
matic), 784.2 (–CH oop); H NMR (400 MHz, DMSO-d6)
d: 1.29–1.33 (t, J = 7.6 Hz, 3H, –CH2–CH3), 2.76–2.81 (q,
J = 7.4 Hz, 2H, –CH2–CH3), 4.77–4.78 (d, J = 5.6 Hz,
123

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Med Chem Res (2013) 22:1090–1101
2H, –NH–CH2–), 6.39–6.43 (dd, J = 8.0 Hz, 2H, H-300 and
500furfuryl), 7.31–7.34 (t, J = 7.2 Hz, 1H, H-40 Ph),
7.53–7.57 (t, J = 8.0 Hz, 2H, H-20 and 60 Ph), 7.62 (s, 1H,
H-400furfuryl), 8.23–8.25 (d, J = 8.0 Hz, 2H, H-30 and 50
Ph), 8.36 (s, 1H, H-3). MS m/z 319 (M?1), 320 (M?2).
Anal. Calc. for C18H17N5O: C 67.70, H 5.37, N 21.93, O
5.01. Found: C 66.47, H 4.21, N 20.53, O 4.01.
6-Isopropyl-4-(morpholino-4-yl)-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidine (6c) Yield 69.9 %, mp 129 °C. IR (KBr,
cm-1): 2959.8, 2927.0 (–CH3), 1592.6 (C=C, aromatic),
756.7 (–CH oop); 1H NMR (400 MHz, DMSO-d6) d:
1.28–1.30 (d, J = 6.8 Hz, 6H, –CH–(CH3)2), 2.94–3.05
(heptet, J = 6.8 Hz, 1H, –CH–(CH3)2), 3.76–3.79 (t,
J = 4.8 Hz, 4H, H-200 and 600 morph), 3.94–3.97 (t,
J = 4.6 Hz, 4H, H-300 and 500 morph), 7.32–7.36 (t,
J = 7.4 Hz, 1H, H-40 Ph), 7.54–7.58 (t, J = 8.0 Hz, 2H,
H-20 and 60 Ph), 8.24–8.26 (d, J = 7.6 Hz, 2H, H-30 and 50
Ph), 8.51 (s, 1H, H-3). MS m/z 323 (M?1), 324 (M?2).
Anal. Calc. for C18H21N5O: C 66.85, H 6.55, N 21.66, O
4.95. Found: C 65.35, H 5.10, N 20.24, O 3.56.
6-Ethyl-4-(morpholin-4-yl)-1-phenyl-1H-pyrazolo[3,4-d]pyrim-
idine (5f) Yield 75.8 %, mp 123 °C. IR (KBr, cm-1):
2946.4 (–CH2), 1566.3 (C=C, aromatic), 784.2 (–CH oop);
1H NMR (400 MHz, DMSO-d6) d: 1.27–1.31 (t, J =
7.4 Hz, 3H, –CH2–CH3), 2.74–2.79 (q, J = 7.4 Hz, 2H,
–CH2–CH3), 3.76–3.78 (t, J = 4.8 Hz, 4H, H-200 and 600
morph), 3.93–3.96 (t, J = 4.8 Hz, 4H, H-300 and 500 morph),
7.32–7.36 (t, J = 7.4 Hz, 1H, H-40 Ph), 7.53–7.57 (t,
J = 7.8 Hz, 2H, H-20 and 60 Ph), 8.22–8.24 (d, J = 8.0 Hz,
2H, H-30 and 50 Ph), 8.50 (s, 1H, H-3). MS m/z 309 (M?1),
310 (M?2). Anal. Calc. for C17H19N5O: C 66.00, H 6.19,
N 22.64, O 5.17. Found: C 65.35, H 5.10, N 21.24, O 4.11.
6-Isopropyl-N,1-diphenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
amine (6d) Yield 72.3 %, mp 127 °C. IR (KBr, cm-1):
3370.1 (–NH), 2966.3, 2925.1 (–CH3), 1597.5 (C=C, aro-
1
matic), 754.1 (–CH oop); H NMR (400 MHz, DMSO-d6)
d: 1.34–1.36 (d, J = 6.8 Hz, 6H, –CH2–(CH3)2), 3.06–3.13
(pentet, J = 6.9 Hz, 1H, –CH–(CH3)2), 7.11–7.15 (t,
J = 7.4 Hz, 1H, H-400 Ph), 7.34–7.37 (t, J = 7.4 Hz, 1H,
H-40 Ph), 7.40–7.44 (t, J = 7.8 Hz, 2H, H-200 and 600 Ph),
7.56–7.60 (t, J = 8.0 Hz, 2H, H-20 and 60 Ph), 7.94–7.96
(d, J = 8.0 Hz, 2H, H-300 and 500 Ph), 8.27–8.29 (d,
J = 8.0 Hz, 2H, H-30 and 50 Ph), 8.48 (s, 1H, H-3), 10.10
(s, 1H, –NH), MS m/z 329 (M?1), 330 (M?2), Anal. Calc.
for C20H19N5: C 72.93, H 5.81, N 21.26. Found: C 71.16, H
4.13, N 20.04.
6-Ethyl-N,1-diphenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
amine (5g) Yield 74.6 %, mp 125 °C. IR (KBr, cm-1):
3423.2 (–NH), 2935.2 (–CH2), 1577.9 (C=C, aromatic),
758.5 (–CH oop). 1H NMR (400 MHz, DMSO-d6) d:
1.33–1.37 (t, J = 7.4 Hz, 3H, –CH2–CH3), 2.84–2.89 (q,
J = 6.9 Hz, 2H, –CH2–CH3), 7.12–7.15 (t, J = 7.4 Hz,
1H, H-400 Ph), 7.34–7.38 (t, J = 7.4 Hz, 1H, H-40 Ph),
7.40–7.44 (t, J = 7.0 Hz, 2H, H-200 and 600 Ph), 7.56–7.59
(t, J = 7.0 Hz, 2H, H-20 and 60 Ph), 7.92–7.94 (d,
J = 8.0 Hz, 2H, H-300 and 500 Ph), 8.25–8.27 (d,
J = 7.6 Hz, 2H, H-30 and 50 Ph), 8.47 (s, 1H, H-3). MS m/
z 315 (M?1), 316 (M?2). Anal. Calc. for C19H17N5: C
72.36, H 5.43, N 22.21. Found: C 71.16, H 4.13, N 21.04.
N-Propyl-6-[(propylamino) methyl]-1-phenyl-1H-pyrazol-
o[3,4-d]pyrimidin-4-amine (7b) Yield 67.7 %, mp 113 °C.
IR (KBr, cm-1): 3449.1 (–NH), 2959.6, 2924.7 (–CH2),
1592.4 (C=C, aromatic), 758.4 (–CH oop); 1H NMR
(400 MHz, DMSO-d6) d: 0.48–0.51 (t, J = 4.4 Hz, 3H,
–NH–CH2–CH2–CH3), 0.94–0.98 (t, J = 7.4 Hz, 3H,
–CH2–NH–CH2–CH2–CH3), 1.60–1.69 (sextet, J = 7.2 Hz,
2H, –NH–CH2–CH2–CH3), 1.89–1.99 (pentet, J = 7.8 Hz,
2H, –CH2–NH–CH2–CH2–CH3), 3.47–3.52 (q, J = 6.6 Hz,
2H, –NH–CH2–CH2–CH3), 3.96–4.05 (q, J = 6.2 Hz, 2H,
–CH2–NH–CH2–CH2–CH3), 4.48 (s, 2H, –CH2–NH–CH2–
CH2–CH3), 7.30–7.34 (t, J = 7.4 Hz, 1H, H-40 Ph),
7.52–7.56 (t, J = 7.8 Hz, 2H, H-20 and 60 Ph), 8.20–8.22 (d,
J = 8.0 Hz, 2H, H-30 and 50 Ph), 8.31 (s, 1H, H-3). MS m/
z 324 (M?1), 325 (M?2). Anal. Calc. for C18H24N6: C
66.64, H 7.46, N 25.90. Found: C 65.43, H 6.21, N 24.60.
N-Butyl-6-isopropyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
amine (6b) Yield 65.4 %, mp 125 °C. IR (KBr, cm-1):
3315.5 (–NH), 2962.7, 2929.0 (–CH3), 1594.6 (C=C, aro-
matic), 753.3 (–CH oop); 1H NMR (400 MHz, DMSO-d6) d:
0.93–0.97 (t, J = 7.0 Hz, 3H,–NH–CH2–CH2–CH2–CH3),
1.29–1.31 (d, J = 6.8 Hz, 6H, –CH–(CH3)2), 1.34–1.36 (d,
J = 6.0 Hz, 1H, –CH–(CH3)2), 1.38–1.43 (q, J = 7.3 Hz,
2H, –NH–CH2–CH2–CH2–CH3), 1.63–1.66 (t, J = 7.0 Hz,
2H, –NH–CH2–CH2–CH2–CH3), 3.59–3.60 (d, J = 4.8 Hz,
2H, –NH–CH2–CH2–CH2–CH3), 3.69 (s, 1H, –NH),
7.33–7.36 (t, J = 7.0 Hz, 1H, H-40 Ph), 7.54–7.58 (t,
J = 7.6 Hz, 2H, H-20 and 60 Ph), 8.21–8.23 (d, J = 7.2 Hz,
2H, H-30 and 50 Ph), 8.44 (s, 1H, H-3). MS m/z 309 (M?1),
310 (M?2). Anal. Calc. for C18H23N5: C 69.87, H 7.49, N
22.63. Found: C 68.12, H 6.17, N 21.31.
N-Isopropyl-6-[(isopropylamino) methyl]-1-phenyl-1H-pyrazolo
[3,4-d]pyrimidin-4-amine (7c) Yield 72.7 %, mp 115 °C.
IR (KBr, cm-1): 3447.8 (–NH), 2961.4, 2925.0 (–CH2),
1593.5 (C=C, aromatic), 759.2 (–CH oop); 1H NMR
(400 MHz, DMSO-d6) d: 1.26–1.28 (d, J = 6.8 Hz, 6H,
–NH–CH–(CH3)2), 1.48–1.50 (d, J = 6.4 Hz, 6H,
CH2–NH–CH–(CH3)2), 3.04–3.11 (q, J = 8.0 Hz, 1H,
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Med Chem Res (2013) 22:1090–1101
1099
Antibacterial testing
CH2–NH–CH–(CH3)2), 4.43–4.47 (q, J = 6.7 Hz, 6H,
–NH–CH–(CH3)2), 4.62 (s, 2H, –CH2–NH–CH–(CH3)2),
7.32–7.35 (t, J = 7.4 Hz, 1H, H-40 Ph), 7.52–7.56 (t,
J = 7.8 Hz, 2H, H-20 and 60 Ph), 8.21–8.24 (d, J = 8.0 Hz,
2H, H-30 and 50 Ph), 8.37 (s, 1H, H-3). MS m/z 324 (M?1),
325 (M?2). Anal. Calc. for C18H24N6: C 66.64, H 7.46, N
25.90. Found: C 65.43, H 6.21, N 24.60.
Test microorganisms
The newly synthesized compounds were screened for their
antibacterial activity against Gram positive [B. subtilis
(NCIM-2545), S. epidermidis (NCIM-2493), S. aureus
(NCIM-5021)], and Gram negative [E. coli (NCIM-2803)]
bacteria. All the strains were maintained by weekly sub-
culturing on nutrient agar slant stored at 4 °C after previous
24 h incubation at 37 °C. Before each experiment, the
organism was activated by successive subculturing and
incubation.
N-Ethyl-6-[(ethylamino) methyl]-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidin-4-amine (7d) Yield 69.9 %, mp 116 °C. IR
(KBr, cm-1): 3441.5 (–NH), 2966.3, 2924.3 (–CH2),
1631.4 (C=C, aromatic), 758.9 (–CH oop); 1H NMR
(400 MHz, DMSO-d6) d: 0.69–0.73 (t, J = 7.4 Hz, 3H,
–CH2–NH–CH2–CH3), 1.22–1.25 (t, J = 7.2 Hz, 3H,
–NH–CH2–CH3), 2.67–2.73 (pentet, J = 6.8 Hz, 2H,
–CH2–NH–CH2–CH3), 3.52–3.59 (pentet, J = 6.8 Hz, 2H,
–CH2–NH–CH2–CH2–CH3), 3.47–3.52 (q, J = 6.6 Hz,
2H, –NH–CH2–CH2–CH3), 3.96–4.05 (q, J = 6.2 Hz, 2H,
–CH2–NH–CH2 –CH3), 4.47 (s, 2H, –CH2–NH–CH2–
CH3), 7.30–7.34 (t, J = 7.4 Hz, 1H, H-40 Ph), 7.52–7.56 (t,
J = 7.8 Hz, 2H, H-20 and 60 Ph), 8.20–8.22 (d, J = 8.0 Hz,
2H, H-30 and 50 Ph), 8.31 (s, 1H, H-3). MS m/z 296 (M?1),
297 (M?2); Anal. Calc. for C16H20N6: C 64.84, H 6.80, N
28.36. Found: C 63.54, H 5.52, N 27.15.
Standardization of test microorganisms
To standardize the inoculums density for a susceptibility
test, a BaSO4, turbidity standard, equivalent to a 0.5
McFarland standard is used. The BaSO4, Mcfarland 0.5
standard is prepared as follows. A 0.5 mL of 1.175 % w/v
of BaCl2ꢀ2H2O is added to 99.5 mL of 1 % w/v of H2SO4
with constant stirring to maintain suspension. The correct
density of the turbidity standard is verified using a UV
spectrophotometer by determining the absorbance. The
absorbance at 625 nm is 0.08–0.10 for this standard. This
suspension is used to standardize the inoculums density. A
10-mL volume of sterile water was added to the agar slant
containing a 24-h-old culture of purified test microorgan-
ism and shaken carefully to harvest the organism. Subse-
quently, dilutions were carried out to get microbial
population of 105 cfu/mL by comparing with BaSO4,
equivalent to Mcfarland 0.5 standard.
6-(Morpholin-4-ylmethyl)-4-(morpholino-4-yl)-1-phenyl-1H-
pyrazolo[3,4-d]pyrimidine (7e) Yield 59.8 %, mp 120 °C.
IR (KBr, cm-1): 2966.3, 2924.3 (–CH2), 1631.4 (C=C,
1
aromatic), 758.9 (–CH oop); H NMR (400 MHz, DMSO-
d6) d: 2.32–2.34 (t, J = 5.2 Hz, 4H, H-2000 and 6000 morph),
3.53–3.55 (t, J = 4.8 Hz, 4H, H-3000 and 5000 morph),
3.77–3.79 (t, J = 4.8 Hz, 4H, H-200 and 600 morph),
3.96–3.98 (t, J = 4.8 Hz, 4H, H-300 and 500 morph), 4.47 (s,
2H, –CH2–), 7.35–7.39 (t, J = 7.4 Hz, 1H, H-40 Ph),
7.54–7.58 (t, J = 7.6 Hz, 2H, H-20 and 60 Ph), 8.17–8.19 (d,
J = 8.0 Hz, 2H, H-30 and 50 Ph), 8.41 (s, 1H, H-3). MS m/
z 380 (M?1), 381 (M?2). Anal. Calc. for C20H24N6O2: C
63.14, H 6.36, N 22.09, O 8.41. Found: C 62.01, H 5.16, N
21.00, O 7.32.
Preparation of stock solution and determination of zones
of inhibition
All the compounds to be tested were dissolved in DMSO to
obtain a stock concentration of 1 mg/mL. The final con-
centrations were made from the stock solution and the
zones of inhibition measured. Inoculums of 100-lL solu-
tion from the standardized bacterial suspension was added
to the Muller Hinton broth and plated. The cups for each
compound were made in three Petri dishes so as to make
(n = 9) and the bacterial suspension used is a 24-h culture.
Cups were made in the agar plates, which were filled with
the specific concentration (20 lg/mL) of the prepared drug
solution. The plates were incubated for 24 h at 35 °C in an
ambient air incubator. Solvents and growth controls were
kept and the zones of inhibition were noted. These results
were compared with amoxicillin as standard and are given
in Table 1. The above studies showed that 6c was best of
the series of compounds, showed activity against all the
four organisms. The compound 4d, exhibited against
N,1-Diphenyl-6-[(phenylamino)methyl]-1H-pyrazolo[3,4-d]
pyrimidin-4-amine (7f) Yield 72.4 %, mp 158 °C. IR
(KBr, cm-1): 3397.2 (–NH), 2926.1 (–CH2), 1575.7 (C=C,
1
aromatic), 787.1 (–CH oop); H NMR (400 MHz, DMSO-
d6) d: 4.47 (s, 2H, –CH2–NH–C6H5), 6.58–6.62 (t,
J = 7.0 Hz, 1H, H-4000 Ph), 6.76–6.62 (d, J = 7.6 Hz, 2H,
H-2000 and 6000 Ph), 7.10–7.14 (t, J = 7.2 Hz, 3H, H-3000,5000
and 400 Ph), 7.34–7.38 (t, J = 6.8 Hz, 3H, H-200,600 and 40
Ph), 7.50–7.54 (t, J = 7.8 Hz, 2H, H-300 and 500 Ph),
7.83–7.85 (d, J = 7.6 Hz, 2H, H-20 and 60 Ph), 8.17–8.19
(d, J = 8.0 Hz, 2H, H-30 and 50 Ph), 8.51 (s, 1H, H-3), MS
m/z 392 (M?1), 393 (M?2), Anal. Calc. for C24H20N6: C
73.45, H 5.14, N 21.41. Found: C 72.17, H 4.13, N 20.01.
123

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Med Chem Res (2013) 22:1090–1101
Tata Memorial Centre [Advanced Centre for Treatment Research and
Education in Cancer (ACTREC)], Mumbai to carry out anticancer
activity.
E. coli, S. aureus and S. epidermidis. The compound 6b
showed activity against S. aureus and S. epidermidis. The
remaining compounds were found to have antibacterial
activity against the tested organisms.
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Acknowledgments Dr. MRP gratefully acknowledges the support
rendered by Department of Science and Technology, New Delhi in
the form of Fast Track Fellowship for Young Scientists [SR/FTP/CS-
59/2007]. Authors thank Shri K. V. Vishnu Raju, Chairman and Shri
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