Synthesis, characterization, antiamoebic activity and cytotoxicity of new pyrazolo[3, 4-d]pyrimidine-6-one derivatives

Article abstract of DOI:10.3109/14756366.2010.528414

A new series of pyrazolo[3,4-d]pyrimidine-6-one derivatives (2a2j) were prepared by using the Biginelli multicomponent cyclocondensation of 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one (1a), different aromatic aldehydes, and urea with a catalytic amount of HCl

Full text of DOI:10.3109/14756366.2010.528414

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(4): 472–479
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2010.528414
ORIGINAL ARTICLE
Synthesis, characterization, antiamoebic activity and
cytotoxicity of new pyrazolo[3, 4-d]pyrimidine-6-one
derivatives
Faisal Hayat, Attar Salahuddin, and Amir Azam
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
Abstract
A new series of pyrazolo[3,4-d]pyrimidine-6-one derivatives (2a–2j) were prepared by using the Biginelli
multicomponent cyclocondensation of 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one (1a), different aromatic aldehydes,
and urea with a catalytic amount of HCl at reflux temperature. These compounds were characterized by IR, 1H NMR,
13C NMR, and Mass spectral data. In vitro antiamoebic activity was performed against HM1:IMSS strain of Entamoeba
histolytica. The results showed that the compounds 2b, 2i, and 2j with IC50 values of 0.37 µM, 0.04 µM, and 0.06
µM, respectively, exhibited better antiamoebic activity than the standard drug metronidazole (IC50 =1.33 µM). The
toxicological studies of these compounds on human breast cancer MCF-7 cell line showed that the compounds 2b,
2i, and 2j exhibited >80% viability at the concentration range of 1.56–50 µM.
Keywords: 3-Methyl-1-phenyl-1H-pyrazol-5(4H)-one, Pyrazolo[3;4-d]pyrimidine-6-one derivatives, Entamoeba
histolytica, MTT assay
Introduction
failures in the treatment of several intestinal protozoan
Parasitic infections constitute one of the most wide-
spread human health problems, and most of them
occur through contaminated food or water. e human
intestine is a major target of these ingested pathogenic
microorganisms, resulting in severe infections, of which
amoebiasis (potential life-threatening dysentery) is one.
Amoebiasis is the second leading cause of death from a
protozoan parasite, Entamoeba histolytica, and remains
as a major health problem in ird World countries
(1). It affects more than 10% of the world’s population,
and untreated infection may lead to severe complica-
tions including hepatic amoebiasis and intestinal tissue
destruction (2). Globally, amoebiasis accounts for 50 mil-
lion clinical cases and is responsible for approximately
110,000 deaths annually (3,4). Metronidazole (MNZ) is
known to be highly effective amoebicide and is consid-
ered to be the drug of choice for the treatment of amoe-
biasis, but recent studies have shown that this drug have
several toxic effects such as genotoxicity, gastric mucus
irritation, and spermatozoid damage (5,6). Furthermore,
parasites may result from drug resistant to parasites (7,8).
According to the International Agency for Research on
Cancer (IARC), MNZ is classified in the 2B group, that
is, potentially carcinogenic to humans and proved to be
carcinogenic to animals (9). is prompted us to search
for new antiamoebic agents.
Pyrimidines are of great importance in fundamental
metabolism, being an integral part of DNA and RNA and
found as core structure in a large verity of compounds
that exhibited important biological activities such as anti-
cancer, antimicrobial, antioxidant, and antiviral activities
(10–14). In the recent years, a lot of attention has been
drawn by the pyrimidines derivatives due to their diverse
range of activities, especially calcium channel blocker
property (15). Moreover, several alkaloids containing the
dihydropyrimidine core unit that have been isolated from
marine sources also showed interesting biological prop-
erties. In particular, the batzelladine alkaloids have been
found to be potent HIV gp-120-CD4 inhibitors (16,17).
According to the literature, pyrazolones are also used as
Address for Correspondence: Amir Azam, Tel.: +91 11 2698 1717/3254; Fax: +91 11 2698 0229. E-mail: amir_sumbul@yahoo.co.in
(Received 31 March 2010; accepted 13 September 2010)
472

Synthesis of pyrazolo[3, 4-d]pyrimidine-6-one derivatives 473
starting materials for the synthesis of biologically active
pyrazolo[3,4-d]pyrimidine derivatives (18,19).
cold ethanol and then recrystallized from appropriate
solvent.
In view of the number of pharmacological signifi-
cances of pyrimidine derivatives and as a part of our
continuous efforts toward the development of more
potent amoebicidal agents, we herein report the synthe-
sis, characterization, in vitro antiamoebic activity, and
cytotoxicity of a new series of pyrazolo[3,4-d]pyrimidine-
6-one derivatives (2b–2j), based on modification of the
classical one-pot Biginelli reaction (18,20).
4, 5-Dihydro-3-methyl-1,4-diphenyl-1
H-pyrazolo[3,4-d]pyrimidin-6(7H)-one (2a)
Yield 35%; Solid; (Ethanol) mp: 167°C; Anal. calc. for
C18H16N4O: C 71.04, H 5.30, N 18.41%; found: C 71.02, H
5.23, N 18.36%; IR νmax (cm−1): 3233 (N-H), 3064 (Ar-H),
1
2923 (C-H), 1689 (C=O); H NMR (DMSO-d6) δ(ppm):
8.22 (s, 1H, NH pyrimidine), 8.14 (s, 1H, NH pyrimidine),
7.37–7.08 (m, 10H, Ar-H), 5.13 (s, 1H, -CH pyrimidine),
2.48 (s, 3H, CH3 pyrazole ring); 13C NMR (DMSO-d6)
δ(ppm): 162.5 (C=O), 151.8 (pyrazolopyrimidine ring),
147.1 (pyrazole ring), 139.5, 134.7, 131.7, 129.5, 124.5
(Ar-C), 117.8 (pyrazolopyrimidine ring), 50.1 (-CH
pyrimidine), 13.6 (CH3 pyrazole ring); FAB-MS (m/z):
[M++1] 305.44.
Materials and methods
All the required chemicals were purchased from Merck
and Aldrich Chemical Company (USA). Precoated alu-
minum sheets (silica gel 60 F254, Merck Germany) were
used for thin-layer chromatography (TLC) and spots
were visualized under UV light. Elemental analysis was
carried out on CHNS Elementar (Vario EL-III), and the
results were within 0.3% of the theoretical values. IR
spectra were recorded on Perkin-Elmer model 1600 FT-IR
4-(4-Chlorophenyl)-4,5-dihydro-3-methyl-1-phenyl-
1H-pyrazolo[3,4d]pyrimidin-6 (7H)-one (2b)
Yield 37%; Solid; (Ethanol) mp: 237°C; Anal. calc. for
C18H15N4OCl: C 63.81, H 4.46, N 16.54%; found: C 63.72,
H 4.39, N 16.48%; IR νmax (cm−1): 3255 (N-H), 3088 (Ar-H),
2846(C-H), 1678(C=O);1HNMR(DMSO-d6)δ(ppm):8.24
(s, 1H, NH pyrimidine), 8.01(s, 1H, NH pyrimidine), 7.65
(d, 2H, J = 7.8 Hz, Ar-H), 7.47–7.42 (t, 2H, J = 7.8 Hz, Ar-H),
7.33–7.21 (m, 5H, Ar-H) 4.99 (s, 1H, -CH pyrimidine), 2.48
(s, 3H, CH3 pyrazole ring); 13C NMR (DMSO-d6) δ(ppm):
161.8 (C=O), 152.4 (pyrazolopyrimidine ring), 148.8
(pyrazole ring), 139.5, 135.7, 130.2, 122.4 (Ar-C), 113.2
(pyrazolopyrimidine ring), 52.65 (-CH pyrimidine), 14.8
(CH3 pyrazole ring); FAB-MS (m/z): [M++1] 339.38.
1
RX1 spectrophotometer as KBr discs. H NMR and 13C
NMR spectra were recorded on Bruker Spectrospin DPX
300 MHz and Bruker Spectrospin DPX 75 MHz spectrom-
eter, respectively, using CDCl3 and DMSO-d6 as a sol-
vent and trimethylsilane (TMS) as an internal standard.
Splitting patterns are designated as follows; s, singlet; d,
doublet; t, triplet; m, multiplet. Chemical shift values are
given in ppm. e FAB mass spectra of the compounds
were recorded on JEOL SX 102/DA-6000 mass spectrom-
eter using Argon/Xenon 6KV (10 mA) as the FAB gas, and
m-nitrobenzyl alcohol (NBA) was used as the matrix.
Preparation of 5-methyl-2-phenyl-2,4-dihydro-3H-
pyrazol-3-one (1a)
4-(3-Chlorophenyl)-4,5-dihydro-3-methyl-1-phenyl-
1H-pyrazolo[3,4-d]pyrimidin-6 (7H)-one (2c)
A mixture of phenyl hydrazine (0.1 mol) and ethyl ace-
toacetate (0.1 mol) in 50 ml ethanol was heated under
reflux for 5 h. After cooling, the reaction mixture was
poured into 200 ml of ice water. e crude product was
filtered, dried, and recrystallized with 95% ethanol.
Yield 75%; Solid; m.p.: 123°C Anal. calc. for C10H10N2O:
C68.95, H5.79, N16.08%;found:C68.88, H5.73, N18.03%;
IR νmax (cm−1): 3044 (Ar-H), 2976 (C-H), 1709 (C=O), 1588
Yield 40%; Solid; (Ethanol:Methanol) mp: 133°C; Anal.
calc. for C18H15N4OCl: C 63.81, H 4.46, N 16.54%; found:
C 63.74, H 4.41, N 16.47 %; IR νmax (cm−1): 3257 (N-H),
1
3041 (Ar-H), 2853 (C-H), 1653 (C=O); H NMR (DMSO-
d6) δ(ppm): 9.19 (s, 1H, NH pyrimidine), 8.22 (s, 1H,
NH pyrimidine), 7.69 (d, 2H, J = 7.8 Hz, Ar-H), 7.49–7.19
(m, 7H, Ar-H), 4.97 (s, 1H, -CH pyrimidine), 2.48 (s, 3H,
CH3 pyrazole ring); 13C NMR (DMSO-d6) δ(ppm): 162.3
(C=O), 151.7 (pyrazolopyrimidine ring), 147.6 (pyrazole
ring), 135.7, 132.2, 131.1, 130.3, 122.4 (Ar-C), 118.4 (pyra-
zolopyrimidine ring), 50.97 (-CH pyrimidine), 14.04 (CH3
pyrazole ring); FAB-MS (m/z): [M++1] 339.21.
1
(C=N), 1340 (C-N); H NMR (CDCl3) δ(ppm): 7.86 (d,
2H, J = 7.8Hz, Ar-H), 7.40–7.35 (t, 2H, J = 7.8 Hz, Ar-H),
7.19 (t, 1H, J = 7.2Hz, Ar-H), 3.39 (s, 2H, -CH2 pyrazolone
ring), 2.16 (s, 3H, CH3 pyrazolone ring), 13C NMR (CDCl3)
δ(ppm): 170.48 (C=O), 147.1 (pyrazole), 139.5, 134.7,
131.7, 129.5, 124.5, (Ar-C), 13.6 (CH3 pyrazolopyrimidine
ring); FAB-MS (m/z): [M++1] 175.19.
4, 5-Dihydro-3-methyl-1-phenyl- 4-p-tolyl-1H-
pyrazolo [3,4-d] pyrimidin-6(7H)-one (2d)
Yield 45%; Solid; (Ethanol) m.p: 188°C; Anal. calc. for
C19H18N4O: C 71.68, H 5.70, N 17.60%; found: C 71.59, H
5.62, N 17.54%; IR νmax (cm−1): 3250 (N-H), 3086 (Ar-H),
Synthesis of pyrazolo[3,4-d]pyrimidine-6-ones (2a–2j)
General procedures
1
Amixtureofurea(0.01mol), aromaticaldehyde(0.01 mol)
and3-methyl-1-phenyl-1H-pyrazol-5(4H)-one(0.01 mol)
in absolute ethanol (30 ml) contain 2–3 drops of 37% HCl
as catalyst was refluxed for 5 h. e crude product which
precipitated on cooling was filtered and washed with
2857 (C-H), 1698 (C=O); H NMR (DMSO-d6) δ(ppm):
8.11 (s, 1H, NH pyrimidine), 8.01 (s, 1H, NH pyrimidine),
7.65 (d, 2H, J = 7.8 Hz, Ar-H), 7.49 (t, 2H, J = 7.5 Hz, Ar-H),
7.33–7.05 (m, 5H, Ar-H), 4.99 (s, 1H, -CH pyrimidine),
2.48 (s, 3H, CH3 pyrazole ring), 2.37 (s, 3H, CH3 phenyl);
© 2011 Informa UK, Ltd.

474 Hayat et al.
13C NMR (DMSO-d6) δ(ppm): 162.6 (C=O), 155.3 (pyra-
zolopyrimidine ring), 148.3 (pyrazole ring), 140.5, 139.7,
132.4, 130.3, 124.3 (Ar-C), 113.4 (pyrazolopyrimidine
ring), 50.07 (-CH pyrimidine), 21.5 (CH3 Aromatic), 14.1
(CH3 pyrazole ring); FAB-MS (m/z): [M++1] 319.54.
4.93, N 20.32%; IR νmax (cm−1): 3249 (N-H), 3067 (Ar-H),
2891 (C-H), 1653 (C=O); H NMR (DMSO-d6) δ(ppm):
1
12.66 (s, 1H, NH, Indole), 9.80 (s, 1H, NH pyrimidine),
8.16 (s, 1H, NH pyrimidine), 7.97 (d, 2H, J= 7.8 Hz, Ar-H),
7.59–7.12 (m, 8H, Ar-H), 5.22 (s, 1H, -CH pyrimidine),
2.47 (s, 3H, CH3 pyrazole ring); 13C NMR (DMSO-d6) δ
(ppm): 162.5 (C=O), 153.6 (pyrazolopyrimidine ring),
148.9 (pyrazole ring), 140.1, 134.7, 132.2, 131.6, 130.8,
123.4, (Ar-C), 115.3 (pyrazolopyrimidine ring), 50.34
(-CH pyrimidine), 13.84 (CH3 pyrazole ring); FAB-MS
(m/z): [M++1] 344.25.
4,5-Dihydro-4-(4-isopropylphenyl)-3-methyl-1-phenyl-
1H-pyrazolo[3,4-d]pyrimidin-6(7H)-one (2e)
Yield 48%; Solid; (Ethylacetate) m.p: 192°C; Anal. calc. for
C21H22N4O: C 72.81, H 6.40, N 16.17%; found: C 72.76, H
6.33, N 16.19%; IR νmax (cm−1): 3293 (N-H), 3031 (Ar-H),
1
2921 (C-H), 1698 (C=O); H NMR (DMSO-d6) δ(ppm):
8.02 (s, 1H, NH pyrimidine), 7.97 (s, 1H, NH pyrimidine),
7.86 (d, 2H, J = 8.1 Hz, Ar-H), 7.79 (s, 1H, J = 8.1 Hz, Ar-H),
7.46–7.35 (m, 3H, Ar-H), 7.21–6.90 (m, 3H, Ar-H), 5.17 (s,
1H, -CH pyrimidine), 3.77 (m, 1H, -CH (CH3)2), 2.48 (s,
3H, CH3 pyrazole ring), 2.16 (s, 3H, CH3 isopropylphenyl),
2.11 (s, 3H, CH3 isopropylphenyl); 13C NMR (DMSO-d6)
δ(ppm): 161.5 (C=O), 153.8 (pyrazolopyrimidine ring),
148.8 (pyrazole ring), 138.6, 135.3, 130.8, 123.4 (Ar-C),
118.8 (pyrazolopyrimidine ring), 51.47 (-CH pyrimidine),
13.8 (CH3 pyrazole ring); FAB-MS (m/z): [M++1] 347.56.
4-(2-Chloroquinoline-3-yl)-4,5-dihydro-3-methyl-1-
phenyl-1H-pyrazolo[3,4-d] pyrimidine-6(7H)-one (2i)
Yield 28%; Solid; (Ethanol) m.p: 168°C; Anal. calc. for
C21H16N5OCl: C 64.70, H 4.14, N 7.96%; found: C 64.63, H
4.08, N 7.87%; IR νmax (cm−1): 3298 (N-H), 3061 (Ar-H), 2955
1
(C-H), 1668 (C=O); H NMR (DMSO-d6) δ(ppm): 12.3 (s,
1H, NH pyrimidine), 10.0 (s, 1H, NH pyrimidine), 8.49 (s,
1H, quinoline), 7.97–7.17 (m, 9H, Ar-H & quinoline), 5.13
(s, 1H, -CH pyrimidine), 2.48 (s, 3H, CH3 pyrazole ring);
13C NMR (DMSO-d6) δ(ppm): 161.6 (C=O), 152.3 (pyra-
zolopyrimidine ring), 147.3 (pyrazole ring), 139.6, 133.7,
132.2, 131.6, 128.3, 121.4 (Ar-C), 115.3 (pyrazolopyrimi-
dine ring), 52.80 (-CH pyrimidine), 14.67 (CH3 pyrazole
ring); FAB-MS (m/z): [M++1] 390.37.
4,5-Dihydro-4-(3-hydroxyphenyl)-3-methyl-1-phenyl-
1H-pyrazolo[3,4-d]pyrimidin-6(7H)-one (2f)
Yield 35%; Solid; (Ethanol) m.p: 176–177°C; Anal. calc.
for C18H16N4O2: C 67.49, H 5.03, N 17.49%; found: C 67.43,
H 5.05, N 17.42%; IR νmax (cm−1): 3408 (O-H), 3192 (N-H)
3071 (Ar-H), 2917 (C-H), 1665 (C=O); 1H NMR (DMSO-d6)
δ(ppm): 10.2 (s, 1H, NH pyrimidine), 10.0 (s, 1H, Ar-OH),
9.74 (s, 1H, NH pyrimidine), 7.65–6.52 (m, 9H, Ar-H), 5.11
(s, 1H, -CH pyrimidine), 2.48 (s, 3H, CH3 pyrazole ring);
13C NMR (DMSO-d6) δ(ppm): 162.5 (C=O), 159.3 (C-OH),
154.9 (pyrazolopyrimidine ring), 147.8 (pyrazole ring),
139.3, 137.1, 135.42, 130.8, 122.4, (Ar-C), 112.4 (pyrazo-
lopyrimidine ring), 52.44 (-CH pyrimidine), 13.9 (CH3
pyrazole ring); FAB-MS (m/z): [M++1] 321.11.
4-(2-Chloro-7-methylquinoline-3-yl)-4, 5-dihydro-3-
methyl-1-phenyl-1 H-pyrazolo[3,4-d]pyrimidin-6(7H)-
one (2j)
Yield 35%; Solid; (Ethanol) m.p: 194°C Anal. calc. for
C22H18N5OCl: C 65.43, H 4.49, N 17.34%; found: C 65.38,
H 4.43, N 17.27%; IR νmax (cm−1): 3296, (N-H) 3048 (Ar-H),
1
2977 (C-H), 1665 (C=O); H NMR (DMSO-d6) δ(ppm):
12.2 (s, 1H, NH pyrimidine), 10.1 (s, 1H, NH pyrimidine),
8.40 (s, 1H, quinoline), 8.13 (s, 1H, quinolne), 7.67 (d, 1H,
J= 8.1 Hz, quinoline), 7.59–7.20 (m, 6H, Ar-H & quino-
line), 5.10 (s, 1H, -CH pyrimidine), 2.48 (s, 3H, CH3 pyra-
zole ring), 2.38 (s, 3H, quinoline); 13C NMR (DMSO-d6)
δ(ppm): 162.4 (C=O), 153.6 (pyrazolopyrimidine ring),
148.4 (pyrazole ring), 140.6, 133.7, 132.1, 131.9, 129.3,
123.1 (Ar-C), 114.3 (pyrazolopyrimidine ring), 50.13 (-CH
pyrimidine), 14.12 (CH3 pyrazole ring); FAB-MS (m/z):
[M++1] 404.42.
4,5-Dihydro-4-(3,4-dimethoxyphenyl)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-d]pyrimidin-6 (7H)-one (2g)
Yield 30%; Solid; (Ethanol:Methanol) m.p: 183°C; Anal.
calc. for C20H20N4O3: C 5.92, H 5.53, N 15.38%; found: C
5.87, H 5.47, N 15.33%; IR νmax (cm−1): 3263 (N-H), 3076
(Ar-H), 2853 (C-H), 1663 (C=O); 1H NMR (DMSO-d6)
δ(ppm): 8.81 (s, 1H, NH pyrimidine), 8.04 (s, 1H, NH
pyrimidine), 7.90 (d, 2H, J = 8.1 Hz, Ar-H), 7.90 (d, 2H,
Ar-H), 7.69 (s, 1H, Ar-H), 7.43–7.11 (m, 3H, Ar-H), 5.10 (s,
1H, -CH pyrimidine), 3.86 (s, 6H, 2xOCH3), 2.48 (s, 3H,
CH3 pyrazole ring); 13C NMR (DMSO-d6) δ(ppm): 161.9
(C=O), 152.9 (pyrazolopyrimidine ring), 147.2 (pyrazole
ring), 140.7, 135.4, 131.6, 130.3, 122.4 (Ar-C), 116.3 (pyra-
zolopyrimidine ring), 52.08 (-CH pyrimidine), 14.64 (CH3
pyrazole ring); FAB-MS (m/z): [M++1] 365.17.
Pharmacology
All the pyrazolo[3, 4-d]pyrimidine-6-one derivatives
(2a–2j) were screened in vitro against HM1:IMSS strain
of E. histolytica by microdilution method (21). All the
experiments were carried out in triplicate at each con-
centration level and repeated thrice. Cytotoxicity of active
compounds has been studied by MTT assay on breast
cancer MCF-7 cell line. e results of biological activity
and cytotoxicity are summarized in Table 1.
4,5-Dihydro-4-(1H-indol-3-yl)-3-methyl-1-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-6(7H)-one (2h)
Yield 45%; Solid; (Ethylacetate) m.p: 238°C; Anal. calc. for
C20H17N5O: C 69.96, H 4.99, N 20.40%; found: C 69.91, H
In vitro antiamoebic assay
All the compounds (2a–2j) were screened in vitro for anti-
amoebic activity against HM1:IMSS strain of E. histolytica
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of pyrazolo[3, 4-d]pyrimidine-6-one derivatives 475
by microdilution method (21). E. histolytica trophozoites
were cultured in wells of 96-well microtiter plate by using
Diamond TYIS-33 growth medium (22). e test com-
pounds (1 mg) were dissolved in 40 μl DMSO and the
final solutions in each well contained 0.1% DMSO or less,
which did not affect viability (23,24). e stock solutions
of the compounds were prepared freshly before use at a
concentration of 1 mg/ml. Twofold serial dilutions were
made in the wells of 96-well microtiter plate (costar).
Each test includes MNZ as a standard amoebicidal drug,
control wells (culture medium plus amoebae) and a
blank (culture medium only). All the experiments were
carried out in triplicate at each concentration level and
repeated thrice. e amoeba suspension was prepared
from a confluent culture by pouring off the medium at
37°C and adding 5 ml of fresh medium, chilling the cul-
ture tube on ice to detach the organisms from the side of
the flask. e number of amoeba/ml was estimated with a
haemocytometer, using trypan blue exclusion to confirm
the viability. e suspension was diluted to 105 organism/
ml by adding fresh medium and 170 μl of this suspension
was added to the test and control wells in the plate so that
the wells were completely filled (total volume, 340 μl). An
inoculum of 1.7 × 104 organisms/well was chosen so that
confluent, but not excessive growth, took place in con-
trol wells. Plates were sealed and gassed for 10 min with
nitrogen before incubation at 37°C for 72 h. After incu-
bation, the growth of amoeba in the plate was checked
with a low-power microscope. e culture medium was
removed by inverting the plate and shaking gently. Plate
was then immediately washed with sodium chloride
solution (0.9%) at 37°C. is procedure was completed
quickly and the plate was not allowed to cool to prevent
the detachment of amoebae. e plate was allowed to dry
at room temperature, and the amoebae were fixed with
methanol and when dried, stained with (0.5%) aqueous
eosin for 15 min. e stained plate was washed once with
tap water, then twice with distilled water and allowed to
dry. A 200-μl portion of 0.1N sodium hydroxide solution
was added to each well to dissolve the protein and release
the dye. e optical density of the resulting solution in
each well was determined at 490 nm with a microplate
reader. e percent inhibition of amoebal growth was
calculated from the optical densities of the control and
test wells and plotted against the logarithm of the dose
of the drug tested. Linear regression analysis was used to
determine the best fitting line from which the IC50 value
was found. e IC50 values in μM are reported in Table 1.
MTT assay
MCF-7 cells were cultured and maintained as mono-
layer in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% of fetal calf serum (FCS),
antibiotics: 100 IU/ml of penicillin and 100 μg/ml of
streptomycin. All cells were cultured in flasks at 37°C
in the 100%-humidity atmosphere and 5% of CO2 (25).
Only viable cells were used in the assay. Exponentially
growing cells were plated at 1.2 × 104 cells per well into
96-well plates and incubated for 48h before the addition
of drugs. Stock solutions of compounds were initially
dissolved in 20% (v/v) DMSO and further diluted with
fresh complete medium. e growth-inhibitory effects
of the compounds were measured using standard tet-
razolium MTT assay. After 48 h of incubation at 37°C,
the medium was removed and 25µl of MTT reagent
(5 mg/ml) in serum-free medium was added to each
well. e plates were incubated at 37°C for 4 h. At the end
of the incubation period, the medium was removed and
pure DMSO (100 µl) was added to each well. e metabo-
lized MTT product dissolved in DMSO was quantified
by reading the absorbance at 570 nm. All assays were
performed in triplicate. Percent viability was defined as
the relative absorbance of treated versus untreated con-
trol cells. Plates were analyzed in an ELISA plate reader
(Labsystems Multiskan RC, Helsinki, Finland) at 570 nm
with a reference wavelength of 655 nm.
CH
3
NH
2
N
O
HN
N
O
O
i
+
H C
O
CH
3
3
ii
(1a)
H
N
O
N
N
NH
H C
3
R
(2a-2j)
Scheme 1. General method for the synthesis of pyrimidine derivatives (2a–2j). Reagent and conditions: (i) C2H5OH, 78°C reflux 5–6h,
(ii) Urea, HCl (2–3 drops), different aromatic aldehydes, where R=aryl group of different aldehydes.
© 2011 Informa UK, Ltd.

476 Hayat et al.
of 1653–1698 cm−1 due C=O stretching vibration also
confirmed the formation of desired compounds (2a–2j)
containing pyrazolo[3,4-d]pyrimidine ring.
Results and Discussion
Chemistry
e synthesis of pyrazolo[3,4-d]pyrimidine-6-one deriv-
atives (2a–2j) was performed in a manner as outlined
in Scheme 1. e reaction of 3-methyl-1-phenyl-1H-
pyrazol-5(4H)-one, urea and various aromatic aldehydes
in absolute ethanol using HCl as a catalyst and refluxed
for 5 h gave pyrazolo[3,4-d]pyrimidine-6-one derivatives
(2a–2j). e compounds were stable in solid state and
were soluble in methanol and DMSO. Melting points
were recorded on KSW melting point apparatus and are
uncorrected.
Moreover the structure of 5-methyl-2-phenyl-2,4-
dihydro-3H-pyrazol-3-one (1a) was further confirmed
1
by H NMR spectra. e appearance of singlet for two
protons at 3.39 due to CH2 group showed the forma-
tion of desired pyrazolon ring in the structure (1a). e
appearance of singlet at 2.16 ppm corresponds to the
methyl group of pyrazolon nucleus also confirmed the
formation of cyclized structure (1a). e structure of
5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (1a)
was further supported by 13C NMR spectra. e appear-
ance of signal at 170 ppm due to (C=O) group of desire
pyrazolon nucleus confirmed the formation of 5-methyl-
2-phenyl-2,4-dihydro-3H-pyrazol-3-one.
In case of pyrazolo[3,4-d]pyrimidine-6-ones (2a–2j),
NH protons were appeared in the range of 7.97–12.6 ppm
onlyasasingletduetopoorresolutionofitssignalrevealed
thepresenceofpyrimidinesnucleusinallthecompounds.
A sharp peak representing the methine proton of the
pyrimidine was observed in the range of 4.97–5.22 ppm
also confirming the formation of the pyrazolo[3,4-d]
pyrimidine nucleus in all the synthesized compounds
(2a–2j) (18). e appearance of signals at 8.11–8.13 and
8.40–8.49 ppm were found in compounds (2i) and (2j) for
chloroquinoline rings, a singlet at 10.0 ppm for OH pro-
ton was observed in compound (2f). For methyl group
of pyrazol ring, a singlet at 2.47–2.48 ppm was found in
Synthesis
In the present work, Biginelli-type cyclocondensation
reaction was studied for the preparation pyrazolo[3,4-d]
pyrimidine-6-ones (2a–2j). e classical three-compo-
nents urea, aromatic aldehyde, and 3-methyl-1-phenyl-
1H-pyrazol-5(4H)-one were carried out in alcoholic
solution containing a few drops of concentrated hydro-
chloric as catalyst, and refluxed for 5 h, as shown in
Scheme 1. All the compounds (2a–2j) were recrystallized
from appropriate solvents and the yield was in the range
of 36–48%. All the compounds were highly soluble in
1
methanol and DMSO and were characterized by IR, H
and 13C NMR, and Mass spectra. e purity of the com-
pounds was confirmed by elemental analysis and data
were found in accordance with 0.3%.
Characteristics IR bands provides significance
indication for the formation of 5-methyl-2-phenyl-2-
,4-dihydro-3H-pyrazol-3-one (1a). e appearance of
absorption bands at 1709, 1588, and 1340 cm−1 corre-
sponding to the C=O, C=N, C-N stretching vibrations,
confirmed the formation of cyclized product (1a). In
cases of pyrazolo[3,4-d]pyrimidine-6-ones (2a–2j), a
sharp band was observed for NH-stretching vibrations
in the range 3233–3298 cm−1 showed the formation of
pyrazolo[3,4-d]pyrimidine ring in all the compounds
(2a–2j). e appearance of strong band in the range
H
N
O
N
N
NH
H3C
R
Figure 1. Pyrazolo[3,4-d]pyrimidine-6- one derivatives (2a–2j).
120
100
80
60
40
20
0
Control
MNZ
2b
2i
2j
0
1.56
3.14
6.25
12.5
25
50
100
Concentration (µM)
Figure 2. Percentage of viable cells after 48 h. pre-treatment of human breast cancer MCF-7 cell line with compounds 2b, 2i, 2j and
metronidazole (MNZ), evaluated by MTT assay.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of pyrazolo[3, 4-d]pyrimidine-6-one derivatives 477
Table 1. In vitro antiamoebic activity of pyrazolo[3,4-d] pyrimidine-6-one derivatives (2a–2j) against HM1:IMSS strain of Entamoeba
histolytica and cytotoxicity profile of compounds 2b, 2i, 2j and metronidazole.
H
N
O
N
N
NH
H C
3
R
Antiamoebic activity
Cytotoxicity profile
IC50 (μM)
SDa
IC50 (μM)
SDa
Compound
R
2a
4.24
0.18
ND
ND
2b
0.37
0.13
>100
0.18
Cl
2c
9.23
7.25
0.34
0.16
ND
ND
ND
ND
Cl
2d
CH3
CH3
2e
2f
15.5
0.20
0.26
ND
ND
ND
ND
CH3
7.99
OH
2g
2h
14.3
0.33
0.16
ND
ND
ND
ND
OH
OCH3
OCH3
8.51
N
H
2i
0.04
0.06
1.33
0.21
0.19
0.11
68.49
>100
>100
0.16
ND
Cl
N
CH3
2j
Cl
N
Metronidazole
0.28
aStandard deviation.
ND, not done.
© 2011 Informa UK, Ltd.

478 Hayat et al.
all the compounds (2a–2j), whereas for compounds (2e)
and (2j), the appearance of singlet for methyl group was
found in the range of 2.11–2.38ppm. A singlet at 3.86 ppm
for two OCH3 protons was found in compound (2g).e
structure of the compounds (2a–2j) was further sup-
ported by 13C NMR spectra. e appearance of signals
in the range of 161.5–162.8 ppm and 50.1–52.8 ppm due
to C=O and methine carbon atom (pyrimidine nucleus)
confirmed the formation of pyrazolo[3,4-d]pyrimidine-6-
ones (2a–2j) (Figure 1). e signal due to aromatic and
aliphatic carbons resonates at their usual positions and
the data shown in the experimental section.
MCF-7 cell line. The cytotoxicity IC50 values along with
the standard deviation values of 2b, 2i, 2j and MNZ are
given in Table 1.
Conclusion
A new series of pyrazolo[3,4-d]pyrimidine-6-one deriva-
tives (2a–2j) was prepared by simple one-pot Biginelli
method. Compounds 2b, 2i, and 2j with IC50 values of
0.37 µM, 0.04 µM, and 0.06 µM, respectively, exhibited
higher antiamoebic activity than the standard drug MNZ
(IC50 = 1.33 µM). Cytotoxicity studies were performed on
human breast cancer MCF-7 cell line and results showed
that the compounds 2b, 2i, 2j and MNZ offered remark-
able viability (>80% at 50 μM).
Antiamoebic activity
Preliminary experiments were carried out to determine
the in vitro antiamoebic activity of all the compounds
(2a–2j) by microdilution method using HM1:IMSS strain
of E. histolytica. e results are summarized in Table 1.
e data are present in terms of percent growth inhibi-
tion relative to untreated controls and plotted as probit
values as a function of drug concentration. e anti-
amoebic effect was compared with the most widely used
antiamoebic medication MNZ which had a 50% inhibi-
tory concentration (IC50) of 1.33 μM in our experiments.
e results showed that the synthesized pyrazolo[3,4-d]
pyrimidine-6-one derivatives (2a–2j) and their structural
activity relationship (SAR) established on the basis of
substituted and unsubstituted aromatic ring attached
with the methine carbon atom of pyrimidine nucleus.
e pyrazolo[3,4-d]pyrimidine-6-one derivatives with
substituted and unsubstituted phenyl ring (2a–2h) and
quinoline ring (2i, 2j) attached with the pyrimidine
ring showed IC50 for antiamoebic activity in the range of
0.04–14.3 μM. erefore out of ten compounds screened
in vitro antiamoebic activity, three compounds (2b, 2i,
and 2j) were found to be more active against E. histolyt-
ica as compared with reference drug MNZ (IC50 = 1.33
μM). Among these three compounds, two compounds
2i (IC50 = 0.04 µM) and 2j (IC50 = 0.06 µM) substituted by
chloroquinoline rings showed excellent antiamoebic
activity and the rest one compounds 2b (IC50 = 0.37 µM)
substituted by hydroxyl phenyl showed moderatively
active than the standard drug MNZ.
Declaration of interest
is work was supported by Council of Scientific and
Industrial Research [grant no. 01(2278)/08/EMR-II New
Delhi, India]. F.H. is thankful to the University Grant
commission (UGC), India for the financial support.
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