Synthesis, anti-microbial evaluation, and molecular modeling of new pyrazolo[3,4-d]pyrimidine derivatives

Article abstract of DOI:10.1007/s00044-011-9911-y

Synthesis of some new pyrazolo[3,4-d]pyrimidine derivatives using readily available starting materials are described. A one-pot multi component cyclocondensation reaction was used to prepare the novel 3-methyl-4-Aryl-1- phenyl-1H-pyrazolo[3,4-d]pyrimidine-6-thiol which served as a new starting material for all new compounds in this research. The anti-microbial activities of the selective synthesized compounds have been evaluated. Some of the newly prepared compounds were found to have moderate to strong anti-microbial activity, e.g., compound 4a, 6a, and 8, in comparison to the reference drugs. Molecular modeling of the most three biologically active new compounds 4a, 6a, and 8 compared to the reference drugs tobramycin and fluconazole was carried out using Fieldalign 2.0 software. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9911-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3848–3857
DOI 10.1007/s00044-011-9911-y
ORIGINAL RESEARCH
Synthesis, anti-microbial evaluation, and molecular modeling
of new pyrazolo[3,4-d]pyrimidine derivatives
•
Ahmad F. Eweas S. A. Swelam O. A. Fathalla
•
•
•
N. M. Fawzy Sh. I. Abdel-Moez
Received: 8 April 2011 / Accepted: 16 November 2011 / Published online: 10 December 2011
ꢀ Springer Science+Business Media, LLC 2011
Abstract Synthesis of some new pyrazolo[3,4-d]pyrimi-
dine derivatives using readily available starting materials
are described. A one-pot multi component cycloconden-
sation reaction was used to prepare the novel 3-methyl-4-
aryl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine-6-thiol which
served as a new starting material for all new compounds in
this research. The anti-microbial activities of the selective
synthesized compounds have been evaluated. Some of the
newly prepared compounds were found to have moderate
to strong anti-microbial activity, e.g., compound 4a, 6a,
and 8, in comparison to the reference drugs. Molecular
modeling of the most three biologically active new com-
pounds 4a, 6a, and 8 compared to the reference drugs
tobramycin and fluconazole was carried out using Field-
align 2.0 software.
Keywords Pyrazolopyrimidines Á Hydrazino derivatives Á
Anti-microbial
Introduction
In the past few decades, the development of resistant
microbes has been greatly accelerated by several concur-
rent trends which resulted in increasing the number of
infections and which expands the need for new anti-
microbial drugs.
In recent years pyrazolo[3,4-d]pyrimidines and related
fused heterocyclic have attracted a great interest in
medicinal chemistry and drug discovery in part this is
attributed to their structure similarity to purines (Bakavoli
et al., 2010). In this aspect a wide array of biological
activity of pyrazolo[3,4-d]pyrimidines have been recently
reported including CNS, metabolic diseases, oncology,
selective cox-2 inhibitor, CCK1 receptor antagonist, non-
steroidal anti-inflammatory, and anti-proliferative agents
(Genin et al., 2000; Lyga et al., 1994; Jorda et al., 2011;
Lee et al., 2003; Huang et al., 2000; Gomez et al., 2007) as
well as blockbuster drugs, such as celecoxib (celebrex) and
sildenafil (Viagra) (Penning et al., 1997; Terrett et al.,
1996). Recently, extensive studies have been devoted to the
synthesis of pyrazolo[3,4-d]pyrimidines (Liu et al., 2007;
Schenone et al., 2008; Heravi et al., 2007; Quiroga et al.,
2008). Multicomponent reactions (MCRs) have recently
gained tremendous importance in organic and medicinal
chemistry. The main contributing factors are the high atom
economy, wide application in combinatorial chemistry and
A. F. Eweas (&) Á O. A. Fathalla
Medicinal Chemistry Department, National Research Centre,
Dokki, Cairo, Egypt
e-mail: a.eweas@tu.edu.sa
A. F. Eweas
Pharmaceutical Chemistry Department, College of Pharmacy,
Taif University, Taif, Kingdom of Saudi Arabia
S. A. Swelam
Photochemistry Department, National Research Centre,
Dokki, Cairo, Egypt
N. M. Fawzy
Department of Natural and Microbial Products,
National Research Centre, Dokki, Cairo, Egypt
´
diversity–oriented synthesis (Ramon et al., 2005; Andreana
et al., 2004; Denmark et al., 2003; Ramachary et al., 2004;
Cozzi et al., 2005; Armstrong et al., 1996; Burke et al.,
2004).
Sh. I. Abdel-Moez
Department of Microbiology and Immunology,
National Research Centre, Dokki, Cairo, Egypt
123

Med Chem Res (2012) 21:3848–3857
Results and discussion
Chemistry
3849
The hydrazino derivative 10 was prepared by heating of
4a with hydrazine hydrate in dioxane at refluxing temper-
1
ature (Scheme 2). HNMR (DMSO-d6) of 10 showed sig-
nals at d 2.15 (s, 3H, Me), d 2.03 (bs, 1H, exchangeable
with D2O, NH), d 4.02 (s, 2H, exchangeable with D2O,
NH2). The resultant hydrazino product 10 underwent a
series of cyclization reactions (Scheme 3).
In continuation to our program directed to the synthesis and
biological evaluation of fused heterocycles, we herein
report the synthesis of new derivatives based on pyrazol-
opyrimidine moiety (Zaki et al., 1999; Swelam et al., 2008;
Swelam et al., 2004; Swelam & Fawzy, 2004).
First with triethylorthoformate, triethylorthoacetate to give
the corresponding triazole derivatives 11a, b (Scheme 3).
Pyrazolo[3,4-d]pyrimidin-6-yl]-5-methyl-1,2-dihydro-
pyrazol-3-one derivative 12 was prepared via reaction of
10 with active methylene compound namely ethyl aceto-
acetate (Scheme 3). 1HNMR spectrum of 12 showed signal
at d 6.54 (s, 1H) specific for pyrazolone ring.
A one-pot cyclocondensation of pyrazolone 1, aromatic
aldehyde, and thiourea under acidic conditions afforded the
corresponding pyrazolopyrimidine 4a–c in good yields,
compounds 4a–c served as a new starting material
throughout this study (Scheme 1).
The structures of 4-(4-aryl)-3-methyl-1-phenyl-1,7-di-
hydropyrazolo[3,4-d]pyrimidine-6-thione (4a–c) were con-
The hydrazino derivative 10a interact with a, b-unsatu-
rated nitrite derivatives 13a, b and 15a, b afforded com-
pounds 14a, b and 16a, b. The structures of the formed
products were confirmed from their correct values in ele-
mental analyzes and spectral data. IR (KBr, m cm-1) for
compounds 14a, b revealed peaks at 3,425–3,100 (NH2),
2,210 (CN). IR (KBr, m cm-1) for compounds 16a, b revealed
peaks at 2,210 (CN), 1,653 (CO), respectively (Scheme 3).
The synthesis of ketene dicyanoacetals (Tominaga et al.,
1985), have attracted considerable attention as starting
materials for synthesis of wide variety of heterocyclic
compounds. Thus, compound 17 was formed upon heating
of 10 with 2-((methylthio)methylene)malononitrile in basic
medium. The structure of 17 was confirmed by its correct
values in elemental analyzes and their agreeable spectral
data. IR (KBr, m cm-1) of compound 17 revealed peaks at
3,401–3,010 (NH2), 2,215 (CN) (Scheme 3). 1H-NMR
(DMSO-d6) (d, ppm) of compound showed signal at 4.25
(s, 3H, SCH3) (Scheme 3, exp.).
1
firmed via elemental analysis and spectral data. H-NMR
(DMSO-d6) (d, ppm) of 4a–c showed signals at 3.00–3.20 (s,
1H, SH, exchangeable with D2O) and d 7.25–8.32 (m, Ar–H).
Cyclocondensation of 4a with the appropriate aldehydes
and chloroacetic acid in the presence of sodium acetate in ace-
tic acid medium were carried out to produce the corresponding
adducts 6a, b. In addition to spectral prove, the structure
assignments of the formed products were further confirmed
chemically by alternative synthesis of 6a, b via cyclocon-
densationof4a with chloroacetic acid then condensation of the
products obtained with the appropriate aldehyde (Scheme 2).
The S-alkylation of 4a with a- halo compounds namely
chloroacetyl chloride and P-methoxy a-bromoacetophe-
none affording the corresponding ethanone derivatives 7, 8.
The structures of the 7, 8 were confirmed via micro ana-
lyzes and spectral data in addition to chemical confirmation
1
reaction. H-NMR (DMSO-d6) (d, ppm) showed peaks at
4.52 and 5.02 specific to (s, 2H, SCH2).
Coupling of compound 8 with appropriate to the aro-
matic aldehyde in basic medium afforded 3-(4-chloro-
phenyl)-2-((4-(4-chlorophenyl)-3-methyl-1-phenyl-1H-pyraz-
olo[3,4-d]pyrimidin-6-yl)thio)-1-(4-methoxyphenyl)prop-
2-en-1-one 9a and 3-(4-bromophenyl)-2-((4-(4-chloro-
phenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)
thio)-1-(4-methoxyphenyl)prop-2-en-1-one 9b in good
yields (Scheme 2).
Anti-microbial activities
Anti-microbial activity of the ten new compounds was eval-
uated against the following tested strains; bacterial strains
Escherichia coli O157:H7, S. typhimurium, Ps. areginosus,
S. aureus, L. monocytogenes, and B. cereus. Fungal strains
Candida albicans and Aspergillus flavus. Tobramycin (10 lg/
ml) was used as standard control positive anti-bacterial while
Ar
H
Ar
N
NH
NH2
NH2
AcOH
N
+
Ar-CHO
S
+
N
N
N
N
N
N
H
O
S
N
SH
Ph
Ph
3a-c
Ph
2a-c
1
4a-c
a= P-Cl C6H4
b= 1-napthyl
c= 2-thiophyl
Scheme 1
123

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Med Chem Res (2012) 21:3848–3857
Cl
Cl
O
O
H
N
N
a, Ar= P-CNC6H4
b, Ar= 2-thiophyl
ArCHO/AcOH
N
S
N
S
N
Ar
N
N
N
6a ,b
5
Cl
Cl
Cl
ArCHO/AcOH
ClCH2CO2H/AcONa
CLCH2CO2H
AcOH
OMe
ClCOCH2Cl/drybenzene
stirr/rt
N
N
N
P-OMeC6H4-COCH2Br/pyridine
N
N
Cl
N
N
N
S
stirr/rt
N
Ph
N
S
SH
N
Ph
N
Ph
O
O
N2H4/diox.
8
7
4a
p-X-C6H4CHO/EtOH/pipyridine.
Cl
Cl
N
X
N
N
N
NHNH2
N
Ph
N
O
N
N
S
10
Ph
9a,b
a, X=Cl
b, X=Br
OMe
Scheme 2
fluconazole (25 lg/ml) was used as standard anti-fungal, on
the other hand dimethyl sulphoxide (DMSO) was used as
control negative for both anti-bacterial and anti-fungal activity.
Results (Table 1) revealed that compound 6a (100 lg/ml)
gives the highest anti-bacterial activity against all tested
strains with a mean zone of inhibition of 17.25 mm followed
by 8 (100 lg/ml), 16.63 mm then 4a (100 lg/ml), 16.13 mm
then 10a (100 lg/ml), 14.13 mm, and 7, 4c and 16a (100 lg/
ml), 14.00 mm finally compounds 16b, 11a, and 14a showed
the least zone of inhibition against all tested bacterial and
fungal pathogenic strains 13.88, 13.60, and 8.63, respectively.
ribosome for tobramycin and the fungal cytochrome P450
14a-demethylase for fluconazole. Two molecules which
both bind to a common active site tend to make similar
interactions with the protein and hence have highly similar
field properties like electrostatic interactions, H-bonding,
and van der Waals interactions. Accordingly, aligning and
scoring molecules based on the similarity in these properties
compared to the reference drugs (tobramycin and fluconaz-
ole) is considered as a powerful tool as it concentrates on the
aspects of the molecules that are important for biological
activity. The alignments provided give ideas on how mole-
cules with different structures could interact with the same
protein, and the scores for those alignments provide insights
into SAR and ideas for further synthesis.
Molecular modeling study
Introduction
Results
Assuming that the tested compound interact with the same
targets as the reference drugs used for both anti-bacterial and
anti-fungal activity, i.e., 16S rRNA on the bacterial 30S
The three dimensional structures of both reference drugs
tobramycin and fluconazole in their bioactive conformations
123

Med Chem Res (2012) 21:3848–3857
3851
Cl
CHR(OC2H5)3 /Ac2O
N
N
N
N
N
11, a, R=H
b, R=CH 3
Ph
N
R
11a,b
Cl
O
O
N
N
EtOH/pip.
/
O
H
N
N
N
N
O
Ph
12
Cl
Ar
CN
CN
EtOH/ip.
a, Ar=p-Cl-C6H4
b, Ar=thiophene
14,
H
10
N
N
13a,b
N
N
N
N
NH2
Ph
Ar
14a,b
CN
Cl
Ar
COOC2H5
CN
15a,b
H
EtOH/ip.
N
H
N
N
N
N
N
O
CN
Ph
16, a, Ar=p-Cl-C6H4
b, Ar=thiophene
Ar
16a,b
Cl
H3CS CN
H
CN
MeOH/ip.
N
N
N
N
N
N
NH2
CN
Ph
SCH3
17
Scheme 3
were extracted from their X-ray crystallography PDB files
while bound to their receptors 16S RNA PDB code 1LC4 for
tobramycin and 14a-sterol demethylase (CYP51) for fluco-
nazole PDB code 2WV2.
minimized by using MOPAC with 100 iterations and mini-
mum RMS gradient of 0.10. The aforementioned compounds
were thenalignedtothereferencedrugmolecules tobramycin
and fluconazole, used as reference drugs in the anti-microbial
testing based on their molecular fields using Fieldalign soft-
ware (v 2.0.1). The interaction between a ligand and a protein
involves electrostatic fields and surface properties (e.g.,
hydrogen bonding, hydrophobic surfaces… etc.).
The most biologically active tested compounds 6a, 8, and
4a were constructed using Chem3D ultra 12.0 software
[chemical structure drawing standard; Cambridge soft cor-
poration, USA (2010)], then they were energetically
123

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Med Chem Res (2012) 21:3848–3857
Table 1 The anti-microbial
activity of the tested compounds
against bacterial and fungal
strains isolated from food and
milk of animal origin
Compound no.
E. coli O157
S. typhimurium
L. monocytogenes
S .aureus
4a
0
14
18
9
12
14
15
11
13
13
13
9
24
16
20
29
14
16
20
18
29
32
20
-
18
14
28
14
16
16
14
12
0
4c
6a
7
8
16
10
10
0
10a
11a
14a
16a
0
9
16b
0
10
18
-
14
19
-
Tobramycin (10 lg/ml)
Flucanazole (25 lg/ml)
DMSO
20
-
-
-
-
-
Compound no.
Ps. Arginosus
B. cereus
C. albicans
A .flavus
Mean zone
of inhibition
4a
24
16
18
22
18
16
12
9
22
18
22
10
18
26
22
12
24
9
29
10
9
0
10
8
16.13
14.00
17.25
14.00
16.63
14.13
13.60
8.63
4c
6a
7
8
9
8
26
16
18
9
12
0
10a
11a
0
14a
0
16a
14
14
18
-
24
20
-
12
12
-
14.00
13.88
19
16b
Tobramycin (10 lg/ml)
Flucanazole (25 lg/ml)
DMSO
19
-
17
-
16
-
16.5
-
-
-
The result of alignments reveals that the tested com-
pounds filed similarity compared to tobramycin ranged
from 0.427 to 0.518 where maximum similarity value is
1.00 (Table 2). While their filed similarity compared to
fluconazole is ranged from 0.478 to 0.517 (Table 3).
Compound 8 showed the highest filed similarity com-
pared to tobramycin. On the other hand, compound 4a
showed the highest filed similarity compared to fluconaz-
ole. The alignment results of the tested compounds are
shown in Tables 2, 3.
Laboratory of the National Research Centre, Cairo, Egypt
(satisfactory micro analyzes were obtained C 0.40, H
0.02, Cl 0.21, N 0.30 and S 0.23). The IR spectra
were recorded in KBr disks, on a Jasco Fourier Transform
Infrared Spectrophotometer Model FT/IR3000E. The
1H-NMR were recorded (DMSO-d6), on JOEL-JNM-EX
270 FTNMR system (NRC) and chemical shifts (d) are
expressed in ppm using TMS as an internal standard.
Splitting patterns are indicated as s singlet, d doublet, m
multiple, b broad signal. The MS were performed at 70 eV
on a Finnegan MAT SSQ7000 spectrometer.
Experimental
3-Methyl-1-phenyl-4-(aryl)-1H-pyrazolo[3,4-d]pyrimidine-
6-thiol (4a–c)
Chemistry
A mixture of equimolecular amounts of 3-methyl-1-phenyl-
5-pyrazolone 1, appropriate aromatic aldehyde (namely,
p-chlorobenzaldehyde, 1-naphthalene aldehyde, and 2-thi-
ophene aldehyde, respectively), and thiourea in acetic acid
(20 ml) was heated to 80–85ꢁC for 8 h with stirring, after
reaction completion, the reaction mixture was cooled and
The purity of the newly synthesized compounds was evi-
denced by TLC. All solvents which were used for crys-
tallization were of analytical grade. All melting points were
determined on a Gallenkamp apparatus and uncorrected.
Elemental analyzes were carried out at the Micro analytical
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Med Chem Res (2012) 21:3848–3857
3853
Table 2 Field alignment of compound 4a, 6a, and 8 against reference drug tobramycin
Compound no.
Filed align picture
Field align score
0.518
8
4a
0.499
6a
0.427
4-(4-Chlorophenyl)-3-methyl-1-phenyl-4,7-
dihydropyrazolo[3,4-d]thiazolo[3,2-a]pyrimidin-6(1H)-
one (5)
added to ice water (100 ml). The solid precipitated was fil-
tered off, air dried, and crystallized from ethanol affording
(4a–c).
4a: Yield 75%, m.p. 221–223ꢁC, M.wt. C18H13ClN4S
(352.84), molecular formula C18H13ClN4S; 1H NMR
(DMSO-d6) d 2.16 (s, 3H, Me), 3.00 (s, 1H, SH,
exchangeable with D2O), d 7.25–8.32 (m, 9H, Ar–H); MS
m/z (353, 32%) (355, 10%) (77, 100%).
To a warm solution of compound 4a (3.52 g, 10 mmol) in
(30 ml) acetic acid, chloroacetic acid (0.95 g, 10 mmol),
anhydrous sodium acetate (1.64 g, 20 mmol) was added.
The reaction mixture was heated gently with stirring on
water bath at 60ꢁC under reflux for 12 h. The reaction
mixture was allowed to cool to room temperature, poured
into ice water (100 ml), the precipitated solid filtered off,
air dried, and crystallized from ethanol affording com-
pound 5.
4b: Yield 77%, m.p. 185–187ꢁC, M.wt. (368.47),
molecular formula C22H16N4S; 1H NMR (DMSO-d6) d
2.19 (s, 3H, Me), 3.01 (s, 1H, SH, exchangeable by D2O), d
7.31–8.02 (m, 12H, Ar–H); MS m/z (368, 40%), (370,
14%), (77, 100%).
5: Yield 70%, m.p. 164–166ꢁC, M.wt. (494.890),
molecular formula C20H15ClN4OS; 1H NMR (DMSO-d6) d
2.21 (s, 3H, Me), 3.82 (s, 2H, thiazolo), d 7.02 (s, 1H,
pyrimidine), d 7.31–8.03 (m, 9H, Ar–H); MS m/z (495
HNCS, 65%), (497 HNCS, 22%), (77, 100%).
4c: Yield 74%, m.p. 148–150ꢁC, M.wt. (324.43),
molecular formula C16H15N4S2; H NMR (DMSO-d6) d
2.21 (s, 3H, Me), 3.20 d (s, 1H, SH, exchangeable by D2O),
d 7.31–7.98 (m, 8H, Ar–H); MS m/z (324, 12%), (65,
100%).
1
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Med Chem Res (2012) 21:3848–3857
Table 3 Field alignment of compound 4a, 6a, and 8 against reference drug fluconazole
Compound no.
Filed align picture
Field align score
0.517
4a
6a
0.500
8
0.478
Color key
Reference compounds tobramycin and fluconazole are shown in red color while tested compounds shown in gray color
Filed points color key
Blue negative field points
Red positive field points
Yellow van der Waals surface field points
Gold/Orange hydrophobic field points
(Z)-4-(4-Chlorophenyl)-3-methyl-1-phenyl-7-
(arylmethylene)-4,7-dihydropyrazolo[3,4-d]thiazolo[3,2-
a]pyrimidin-6(1H)-one (6a, b)
The formed solid was collected by filtration, air dried, and
crystallized from appropriate solvent affording 6a, b.
Method B A mixture of compound 5 (4.94 g, 10 mmol)
appropriate aldehyde (10 mmol) (4-cyanobenzaldehyde,
2-thiophene aldehyde) and anhydrous sodium acetate
(1.64 g, 20 mmol) in a mixture of acetic acid and acetic
anhydride (30 ml, 1:1) was stirred under refluxing temper-
ature for 8 h. The deposited solid after cooling was filtered
off, air dried, and crystallized from appropriate solvent
affording 6a, b.
General procedure Method A A mixture of compound 4a
(3.52 g, 10 mmol), chloroacetic acid (0.95 g, 10 mmol),
appropriate aldehydes (10 mmol) (4-cyanobenzaldehyde,
2-thiophenealdehyde), andanhydroussodiumacetate(1.64 g,
20 mmol) in a mixture of acetic acid and acetic anhydride
(30 ml, 1:1) was stirred under refluxing temperature for 8 h.
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Med Chem Res (2012) 21:3848–3857
3855
6a: Yield 55%, m.p. 148–150ꢁC, crystallized from ben-
zene, M.wt. (508.01) molecular formula C28H18ClN5OS, IR
(KBr, m cm-1) 2,216 (CN); 1H NMR (DMSO-d6) d 2.16 (s,
3H, Me), d 7.42 (s, 1H, pyrimidine), d 7.31–8.03 (m, 13H,
Ar–H), d 8.54 (s, 1H, Ar–CH); MS m/z (508, 65%), (510,
21%), (96, 100%).
d 2.23 (s, 3H, Me), d 4.12 (s, 3H, OCH3), d 7.35–8.23 (m,
17H, Ar–H), d 8.01 (s, 1H, Ar–CH).
9b: Yield 48%, m.p. 310–312ꢁC, M.wt. (668.02),
molecular formula C34H24BrClN4O2S; H NMR (DMSO-
1
d6) d 2.23 (s, 3H, Me), d 4.12 (s, 3H, OCH3), d 7.35–8.23
(m, 17H, Ar–H), d 8.01 (s, 1H, CH).
6b: Yield 51%, m.p. 196–198ꢁC, crystallized from meth-
anol, M.wt. (488.02), molecular formula C25H17ClN4OS2;
1H NMR (DMSO-d6) d 2.25 (s, 3H, Me), d 6.42 (s, 1H,
pyrimidine), d 7.31–8.03 (m, 12H, Ar–H), d 9.02 (s, 1H, Ar–
CH); MS m/z (488, 25%), (490, 8%), (65, 100%).
2-[4-(4-Chlorophenyl)-3-methyl-1-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-6-yl]-hydrazine (10)
A solution of compound 4a (3.52 g, 10 mmol) in dioxane
(25 ml) and hydrazine hydrate (0.05 g, 10 mmol) was
heated at refluxing temperature for 10 h. Then, the white
crystals formed was collected by filtration, washed with
cold water then dried under suction and crystallized from
ethanol affording 10.
2-((4-(4-Chlorophenyl)-3-methyl-1-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-6-yl)thio)acetyl chloride (7)
An equimolecular amounts of compound 4a (3.52 g,
10 mmol) and chloroacetylchloride (1.12 g, 10 mmol) in dry
benzene (20 ml) was stirred at room temperature for 2 h. The
solid formed was filtered off and crystallized from benzene.
7: Yield 56%, m.p. 180–182ꢁC, M.wt. (393.88),
molecular formula C20H14ClN4OS; 1H NMR (DMSO-d6) d
2.18 (s, 3H, Me), d 4.52 (s, 2H, SCH2), d 7.31–8.03 (m, 9H,
Ar–H), MS m/z (393, SCHCOCl, 55%), (395, SCHCOCl,
18%), (65, 100%).
10a: Yield 80%, m.p. 208–210ꢁC, M.wt. (350.81),
molecular formula C18H15ClN6, IR (KBr, m cm-1) 3,310,
1
3,102 (NH2 NH); HNMR (DMSO-d6) d 2.15 (s, 3H, Me),
d 2.03 (bs, 1H, exchangeable with D2O, NH), d 4.02 (s, 2H,
exchangeable with D2O, NH2), d 7.35–8.23 (m, 9H, Ar–H);
MS m/z (351, 71%), (353, 23%), (65, 100%).
Preparation of 11a, b An equimolecular amounts of
compound 10a (3.50 g, 10 mmol) and triethylorthoformate
or triethylorthoacetate (10 mmol) in AcOH (20 ml) was
stirred at refluxing temperature for 8 h. The solid formed
on cooling was filtered off and crystallized from ethanol.
2-((4-(4-Chlorophenyl)-3-methyl-1-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-6-yl)thio)-1-(4-
methoxyphenyl)ethanone (8)
An equimolecular amounts of compound 4a (3.52 g,
10 mmol) and P-methoxy b-acetophenone (10 mmol) in
dry pyridine (20 ml) was stirred at room temperature for
12 h. The precipitated solid was filtered off and crystallized
from ethanol.
4-(4-Chlorophenyl)-3-methyl-1-phenyl-1H-pyrazolo[4,3-
e][1,2,4]triazolo[4,3-a]pyrimidine (11a)
11a: Yield 60%, m.p. 180–182ꢁC, M.wt. (362.82),
1
molecular formula C19H13ClN6; H-NMR (DMSO-d6), d
8: Yield 63%, m.p. 160–162ꢁC, M.wt. (500.97),
molecular formula C27H21ClN4O2S; H NMR (DMSO-d6)
2.23 (s, 3H, Me), d 6.41 (s, 1H, triazolo), d 7.42–7.99 (m,
9H, Ar–H); MS m/z (363, 25%), (365, 8%), (75, 100%).
1
d 2.23 (s, 3H, Me), d 5.02 (s, 2H, SCH2), d 4.12 (s, 3H,
OCH3), d 7.35–8.23 (m, 13H, Ar–H), MS m/z (500 SCH-
COCl, 15%), (502, SCHCOCl, 5%), (75, 100%).
4-(4-Chlorophenyl)-3,8-dimethyl-1-phenyl-1H-
pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidine (11b)
3-(4-Halophenyl)-2-((4-(4-chlorophenyl)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)thio)-1-(4-
methoxyphenyl)prop-2-en-1-one 9a, b
11b: Yield 51%, m.p. 130–133ꢁC, M.wt. (374.83),
molecular formula C20H15ClN6; H NMR (DMSO-d6) d
1.19, 2.23 (2s, 6H, 2Me), d 7.42–7.99 (m, 9H, Ar–H); MS
m/z (375, 27%), (377, 9%), (75, 100%).
1
A mixture of equimolecular amounts of compound 8
(5.01 g, 10 mmol) and appropriate aromatic aldehyde
(p-chlorobenzaldehyde and p-bromobenzaldehyde, respec-
tively) (10 mmol) in ethanol (20 ml) in the presence of
catalytic amount of pyrimidine (1 ml) was heated under
refluxing temperature for 12 h. The solid formed on cool-
ing was filtered off, air dried, and crystallized from ethanol.
9a: Yield 52%, m.p. 294–296ꢁC, M.wt. (623.57),
molecular formula C34H24Cl2N4O2S; 1H NMR (DMSO-d6)
1-(4-(4-Chlorophenyl)-3-methyl-1-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-6-yl)-5-methyl-1H-pyrazol-
3(2H)-one (12)
An equimolecular amounts of compound 10a (3.50 g,
10 mmol), ethylacetoacetate (1.31 g, 10 mmol), and cata-
lytic amount pyrimidine (1 ml) in ethanol (20 ml) was
stirred at refluxing temperature for 8 h. After cooling to
123

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Med Chem Res (2012) 21:3848–3857
room temperature the solid formed was filtered off, air
dried, and crystallized from ethanol.
diffusion test (Sgouras et al., 2004; Wiart, 2007) using two
different concentration of the compounds (100 lg/ml)
dissolved in (1 ml) DMSO as a qualitative method for
studying the anti-microbial activity of the tested com-
pounds against the following tested stains; bacterial strains
E. coli O157, S. typhimurium, Ps. areginosus, S. aureus,
L. monocytogenes, B. cereus. Fungal strains are C. albicans
and A. flavus. As control positive tobramycin (10 lg/ml)
was used as standard anti-bacterial while fluconazole
(25 lg/ml) was used as standard anti-fungal while DMSO
was used as control negative.
12: Yield 49%, m.p. 295–297ꢁC, M.wt. (416.87),
1
molecular formula C22H17ClN6O; H NMR (DMSO-d6), d
2.20, 2.09 (2s, 6H, 2Me), d 6.54 (s, 1H, pyrazolo), d
7.42–7.99 (m, 9H, Ar–H), d 8.02 (bs, 1H, exchangeable
with D2O, NH); MS m/z (417, 16%), (419, 5%), (77,
100%).
General procedure for preparation of 14a, b, 16a, b and 17
A mixture of compound 10a (10 mmol) and ethyl(Z)-2-
cyano-3-substituted phenylacrylate derivatives 13a, b, 15a,
b or 2-((methylthio)methylene) malononitrile (10 mmol) in
ethanol (20 ml) was heated under reflux for 12–14 h. The
reaction completion was followed by TLC. The reaction
mixture was cooled, poured into ice water and the solid
formed was filtered off, dried under air suction, and crys-
tallized from methanol.
Strains selected for being tested are isolated, and feed by
products of animal origin including poultry (E. coli O157
and S. typhimuruim) as well as from mastitic cow milk,
(L. monocytogenes, S. and B. cereus) and from minced cow
meet (S. aureus, Ps. aerigenosus). These strains are com-
monly accused of being a cause of food intoxication.
Muller–Hinton agar plates were inoculated with bacterial
strains while sabouraud dextrose agar plates were inoculated
with fungal strains prepared in conc. equivalent with 0.5
MacFerland for bacterial strains and 2 9 105 and streaked
onto the agar plates using sterile swabs, and then 50 lg of the
dissolved compound in DMSO were placed into the wells
under sterile conditions. All plates were incubated at 37ꢁC/
24 h for bacterial growth and at 28ꢁC/48–72 h for fungal
growth. Zone of inhibition were measured in mm using a
ruler. The experiment was carried out in duplicate and the
mean of the zone of inhibition was tabulated in (Table 1).
14a: Yield 49%, m.p. 275–277ꢁC, M.wt. (537.42),
molecular formula C28H18Cl2N8; IR (KBr, m cm-1),
1
3,425–3,100 (NH2), 2,210 (CN); H-NMR (DMSO-d6), d
2.29 (s, 3H, Me), d 4.54 (s, 2H, exchangeable with D2O,
NH2), d 6.98–8.01 (m, 13H, Ar–H); MS m/z (538 HCN,
16%), (540 HCN, 16%), (542 HCN, 5%), (77, 100%).
14b: Yield 42%, m.p. 295–297ꢁC, M.wt. (509.0),
molecular formula C26H17ClN8S; IR (KBr, m cm-1),
1
3,421–3,210 (NH2), 2,210 (CN); H-NMR (DMSO-d6), d
2.29 (s, 3H, Me), d 4.34 (s, 2H, exchangeable with D2O,
NH2), d 6.98–7.94 (m, 12H, Ar–H); MS m/z (510 HCN,
16%), (512 HCN, 5), (77, 100%).
Conclusion
16a: Yield 54%, m.p. 268–270ꢁC, M.wt. (584.47);
molecular formula C30H23Cl2N7O2; IR (KBr, m cm-1),
2,210 (CN), 1,653 (CO); 1H-NMR (DMSO-d6), d 1.05,
2.29 (2s, 6H, 2Me), d 2.54 (s, 2H, exchangeable with D2O,
NH2), d 7.34–7.84 (m, 13H, Ar–H); MS m/z (584 HCN,
25%), (586 HCN, 25%), (588 HCN, 8%), (77, 100%).
16b: Yield 34%, m.p. [ 310ꢁC, M.wt. (558.05),
molecular formula C28H22ClN7O2S; IR (KBr, m cm-1),
2,210 (CN), 1,653 (CO); 1H-NMR (DMSO-d6), d 1.35,
2.19 (2s, 6H, 2Me), 4.54 (s, 2H, exchangeable with D2O,
NH2), d 7.34–7.84 (m, 12H, Ar–H); MS m/z (559 HCN,
25%), (561 HCN, 8%), (77, 100%).
In conclusion, we have developed a simple and efficient
method for the synthesis of substituted pyrazolopyrimidines.
We also believe that the procedural simplicity, the efficiency
and the easy accessibility of the reaction partners gives
access to a wide array of heterocyclic frameworks containing
a pyrazolopyrimidine unit. The anti-microbial activity of the
tested compounds against bacterial and fungal strains iso-
lated from food and milk of animal origin was evaluated. The
molecular modeling of the most biologically active com-
pounds among the newly synthesized compounds was car-
ried out using Fieldalign software revealed that these
compounds showed moderate field similarity compared to
the reference drugs used in the anti-microbial testing.
17: Yield 39%, m.p. 245–247ꢁC, M.wt. (472.96),
molecular formula C23H17ClN8S; IR (KBr, m cm-1),
1
3,401–3,010 (NH2), 2,215 (CN); H-NMR (DMSO-d6), d
2.20 (s, 3H, Me), d 3.74,3.9 (2s, 2H, exchangeable with D2O,
NH2), d 4.25 (s, 3H, SCH3), d 6.56–7.94 (m, 9H, Ar–H).
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