Synthesis, DNA-binding, and antiviral activity of certain pyrazolo[3,4- d]pyrimidine derivatives

Article abstract of DOI:10.1002/(SICI)1521-4184(20004)333:4<99::AID-ARDP99>3.0.CO;2-O

Some new pyrazolo[3,4-d]pyrimidine derivatives have been prepared and tested for their antiviral and DNA-binding activities. Compounds 4, 5, and 6 showed high binding affinity to DNA at concentrations of 19, 27, and 28 μg/ml, respectively. On the other hand, compounds 6 and 10 reduced the number of viral plaques of Herpes simplex type-1 (HSV-1) by 66 and 41%, respectively. The detailed synthesis and spectroscopic and biological data are reported.

Full text of DOI:10.1002/(SICI)1521-4184(20004)333:4<99::AID-ARDP99>3.0.CO;2-O

Pyrazolo[3,4-d]pyrimidine Derivatives
99
Synthesis, DNA-binding, and Antiviral Activity
of Certain Pyrazolo[3,4-d]pyrimidine Derivatives
a)
b)
E. R. El-Bendary* and F. A. Badria
Departments of a) Medicinal Chemistry and b) Pharmacognosy, Faculty of Pharmacy, University of Mansoura, Mansoura, 35516 Egypt
Key Words: Pyrazolo[3,4-d]pyrimidines; antiviral activity; DNA-binding affinity
compound 3 in 47% yield. The synthetic pathways employed
in the preparation of compounds 4–8 are reported in
Scheme 1. Reaction of pyrazolo[3,4-d]pyrimidin-4-one 3
with either triethylorthoformate or triethylorthoacetate, in the
presence of p-toluenesulfonic acid, yielded 1,8-diphenyl-
pyrazolo[3,4-d][1,2,4]triazolo[2,3-a]pyrimidin-4-one 4 and
7-methyl-1,8-diphenylpyrazolo[3,4-d][1,2,4]triazolo[2,3-a]-
pyrimidin-4-one 5, whereas 7-thioxo derivative 6 was ob-
tained by the reaction of 3 with carbon disulphide and potas-
sium hydroxide. 1-Phenyl-6-phenylamino-5-(2-thienyl-
carboxamido)pyrazolo[3,4-d]pyrimidin-4-one 7 was pre-
pared through the reaction of 2-thenoylchloride, in presence
of triethylamine, with the starting material 3. Furthermore,
reaction of 3 with phenyl isothiocyanate yielded 1-phenyl-6-
phenylamino-5-(N-phenylthiourido)pyrazolo[3,4-d]pyrim-
idin-4-one 8.
Summary
Some new pyrazolo[3,4-d]pyrimidine derivatives have been pre-
pared and tested for their antiviral and DNA-binding activities.
Compounds 4, 5, and 6 showed high binding affinity to DNA at
concentrations of 19, 27, and 28 µg/ml, respectively. On the other
hand, compounds 6 and 10 reduced the number of viral plaques of
Herpes simplex type-1 (HSV-1) by 66 and 41%, respectively. The
detailed synthesis and spectroscopic and biological data are re-
ported.
Introduction
Pyrimidines and condensed pyrimidines have received
Condensation of 3 with certain aromatic aldehydes, gave
the desired 5-(4-halobenzylideneamino)pyrazolo[3,4-d]-
pyrimidin-4-one derivatives 9–11 (Scheme 2). Compound 3
was also utilized by allowing it to react with certain α-bro-
moacetophenones in ethanol in the presence of p-toluenesul-
fonic acid as a catalyst for prolonged time to afford the
cyclized product 1,9-diphenyl-7-(4-halophenyl)-8,9-dihy-
much attention over the years because of their interesting
[1–6]
pharmacological properties
. In our laboratory consider-
able effort has recently been invested in the preparation and
evaluation of heterocyclic compounds containing the
pyrimidine nucleus, leading to numerous compounds with
[7–10]
promising antiviral and antitumor activities
. Moreover,
some substituted pyrazolopyrimidines have been docu-
[1–3]
mented as adenosine antagonists
. These facts, in addi- dropyrazolo[3,4-d]pyrimidino[1,2-b][1,2,4]triazin-4-ones
12–14 (Scheme 2).
tion to our previously obtained results about the remarkable
[11]
activity of thiazolotriazolopyrimidines
(structure A), en-
couraged the synthesis of new derivatives of the pyrazolo-
[3,4-d]pyrimidine nucleus as purine isosteres and the Biology
evaluation of their DNA-binding and antiviral activities.
The antitumor activity of the newly synthesized compounds
[14]
was determined using DNA binding assay
and methyl
[15]
green DNA displacement assay
.
DNA Binding Assay
In this method, a fixed amount of the ligand is spotted on
the RP-18 TLC plates followed by addition of known amount
of DNA on the same spot. The plate was then developed and
the position of unbound DNA was determined by spraying
the plates with anisaldehyde reagent. The free DNA was
Results and Discussion
Chemistry
The preparation of 5-amino-4,5-dihydro-1-phenyl-6-
phenylamino-pyrazolo[3,4-d]pyrimidin-4-one 3 necessary
for this study is shown in Scheme 1. 5-Amino-1-phenyl-1H-
pyrazole-4-carboxylic acid ethyl ester 1 was prepared
detected as a blue spot (R , MeOH–H O, 8:2) on RP-18 TLC.
f
2
It was demonstrated that, when DNA was mixed with com-
pounds known to interact with it, e.g. ethidium bromide, the
complex was retained at the origin. Compounds with high
binding affinity to DNA remained on the base line or mi-
grated for a very short distance, while compounds with poor
binding affinity did not cause DNA to be retained at the
through the reaction of ethyl (ethoxymethylene) cyanoacetate
[3,12]
with phenylhydrazine
. Treatment of 1 with phenyl iso-
thiocyanate afforded ethyl 5-(phenylthiourido)-1-phenyl-
pyrazole-4-carboxylate 2
[13]
, which was then cyclized by
refluxing in neat hydrazine hydrate to produce the target origin.
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0365-6233/00/0404/0099 $17.50 +.50/0

100
El-Bendary and Badria
Scheme 1
Scheme 2
Arch. Pharm. Pharm. Med. Chem. 333, 99–103 (2000)

Pyrazolo[3,4-d]pyrimidine Derivatives
101
Methyl Green-DNA Displacement Assay
Experimental Part
Methyl green reversibly binds polymerized DNA forming
a stable complex at neutral pH. The absorption maximum for
the DNA/methyl green complex is 642.5–645 nm. This assay
was used to measure the displacement of methyl green from
DNA by compounds having ability to bind with DNA. The
degree of displacement was determined spectrophotometri-
cally by measuring the change in initial absorbance of the
DNA/methyl green solution in the presence of reference
compound. The activity of compounds which showed high
affinity for DNA have been determined and expressed as
Chemistry
The melting points (°C, uncorrected) were determined in open glass
capillaries on a Mel-temp II melting point apparatus. 1H-NMR spectra were
recorded on a varian EM 360 (90 MHz) instrument using TMS as an internal
standard (Chemical shift in δ ppm). Microanalytical data (C,H,N) were in
agreement with the proposed structures within ±0.4% of the theoretical
values were performed at the Microanalytical Center, Cairo University,
Egypt.
5-Amino-4,5-dihydro-1-phenyl-6-phenylaminopyrazolo[3,4-d]pyrimidin-
4-one (3)
CC (Concentration required for 50% decrease in the initial
50
absorbance of the DNA/methyl green solution). These results
indicated that compounds 4,5, and 6 showed high binding
affinity for DNA and their structures are characterized by a
pyrazolotriazolopyrmidine moiety. On the other hand, re-
placement of the triazole nucleus by 1,2,4-triazine led to
compounds 12, 13, and 14 that exhibited moderate affinity
for DNA (Table 2).
A mixture of ethyl 5-(phenylthiourido)-1-phenylpyrazole-4-carboxylate 2
(3.66 g, 0.01 mol) and hydrazine hydrate (10 g, 0.2 mol) was heated under
reflux for 10 h. The solid that separated upon cooling was filtered, washed
with water and recrystallized from acetic acid to yield 1.5 g (47%) of 3, mp:
173–175°C. Analysis for (C17H14N6O): C,H,N. 1H-NMR (DMSO-d6):
6.05(s, 2H, NH2, exchangeable), 7.15–7.45 (m, 10 H, ArH), 7.65 (s, 1H,
pyrazole), 8.8 (br s, 1H, NH, exchangeable).
1,8-Diphenylpyrazolo[3,4-d][1,2,4]triazolo[2,3-a]pyrimidin-4-one (4) and
7-methyl-1,8-diphenylpyrazolo[3,4-d][1,2,4]triazolo[2,3-a] pyrimidin-
4-one (5)
In vitro Anti-Herpes simplex-1 Virus (HSV-1)
The synthesized compounds were tested in vitro for their
antiviral activity using Herpes simplex type-1 (HSV-1) as a
viral model. The antiviral testing was performed using Vero
cells (cells isolated from the kidneys of green monkeys),
HSV-1, and Aphidicolin as a positive control
culture and test solutions were incubated for 66 h. At the end
of the incubation period, the percentage reduction in the
number of virus plaques were recorded and compared to the
positive control. Compounds 6 and 10 showed the highest
activity among the tested compounds and reduced the number
of HSV-1 plaques by 66 and 41% respectively while com-
pounds 4, 5, 11, 9, 12, 14, and 13 showed 33, 31, 30, 22, 21,
20, and 15% reduction, respectively. Compounds 7 and 8
proved to lack any antiviral activity (Table 3).
A mixture of 3 (3.8 g, 0.012 mol), p-toluenesulfonic acid (3.8 g, 0.02 mol)
and triethylorthoformate or triethylorthoacetate (45 ml) was heated under
reflux for 20 h. The solution was concentrated in vacuo and the obtained
residue was triturated with ice-cold water, filtered, dried and recrystallized
from aqueous ethanol (Table 1). 1H-NMR (CDCl3) 4: δ 7.08–7.40 (m, 6H,
ArH), 7.71–7.90 (m, 4H, ArH), 8.05 (s, 1H, pyrazole), 8.5 (s, 1 H, triazole).
5: δ 2.40 (s, 3H, CH3), 7.10–7.41 (m, 6H, ArH), 7.90–8.0 (m, 4H, ArH), 8.07
(s, 1H, pyrazole).
[16]
. The viral
1,8-Diphenylpyrazolo[3,4-d][1,2,4]triazolo[2,3-a]pyrimidin-4-one-
7-thione (6)
Carbon disulphide (3 ml, 0.05 mol) was added dropwise to a mixture of 3
(1.6 g, 0.005 mol) and potassium hydroxide (0.28 g, 0.005 mol) in ethanol
(15 ml). After complete addition, the reaction mixture was stirred at room
temperature for 1 h. followed by heating under reflux for 8 h. The solvent
was evaporated in vacuo. Water was added to the residue and the alkaline
solution was filtered. The clear filtrate was acidified with dilute hydrochloric
acid and the separated solid was collected and recrystallized from chloro-
form-ethanol to give 0.7 g (46%) of 6; mp: 255–257 °C. Analysis
(C18H12N6OS); C,H,N. 1H-NMR (DMSO-d6),: δ 7.20–7.50 (m, 10 H, ArH),
7.7 (s, 1H, pyrazole), 14.08 (br s, 1H, NH, exchangeable).
Cytotoxicity Assay
Cultured mammalian cells have been utilized to determine
the response of tumor cells isolated from cancer patients to
[17,18]
various chemotherapeutic agents
as well as the antiviral
potency of the synthesized compounds using Herpes Simplex
[16]
as a viral model . CC (the compound concentration that
50
1-Phenyl-6-phenylamino-5-(2-thienylcarboxamido)pyrazolo-
[3,4-d] pyrimidin-4-one (7)
cause 50% reduction in cell density) was recorded and com-
pared to the positive control. Regarding the cytotoxicity of
the tested compounds, the results obtained revealed that these
compounds have weak cytotoxicity which demonstrated a
selective DNA binding and consequently a safe antiviral
activity.
2-Thenoylchloride (0.73 g, 0.005 mol) was added dropwise to a stirred
solution of 3 (1.6 g, 0.005 mol) and triethylamine (0.51 g, 0.005 mol) in
dichloromethane (10 ml). The reaction mixture was heated at 70 °C for 3h.
After cooling, the separated solid was collected by filteration, washed with
ice-water, dried and recrystallized from chloroform to produce 0.86g (40%)
of 7; mp: 128–130 °C. Analysis (C22H16N6O2S); C,H,N. 1H-NMR (DMSO-
D6): δ 6.55 (s, 1H, NH-CO exchangeable), 6.85 – 7.70 (m, 13 H, ArH), 7.8
(s, 1H, NH exchangeable), 8.06(s, 1H, pyrazole).
Conclusion
The basic structure pyrazolo[3,4-d]pyrimidine 9–11 pos-
sesses promising antiviral activity with lower affinity for
DNA, while the pyrazolotriazolopyrimidine compounds 4–6
showed higher potency towards HSV-1 and high binding
affinity for DNA. On the other hand, formation of pyraz-
olopyrimidinotriazines moieties reduced the antiviral po-
tency and afforded compounds 12–14 with moderate affinity
to DNA.
1-Phenyl-6-phenylamino-5-(N-phenylthiourido)pyrazolo[3,4-d]pyrimidin-
4-one (8)
Phenylisothiocynate (1.35 g, 0.01 mol) was added dropwise to a solution
of 3 (1.6 g, 0.005 mol) in ethanol (20 ml). The mixture was heated under
reflux for 6 h. After cooling, the mixture was poured into water. The
precipitated solid was collected by filtration and recrystallized from metha-
nol to yield 1.2 g (53%) of the title compound 8; mp: 105–107 °C. Analysis
(C24H19N7OS); C,H,N. 1H-NMR (DMSO-d6): δ 6.90 (s, 1H, NH exchange-
Arch. Pharm. Pharm. Med. Chem. 333, 99–103 (2000)

102
El-Bendary and Badria
able) 7.2–7.55 (m, 15H, ArH), 7.80 (s, 1H, pyrazole), 7.90 (s, 1H, NH
exchangeable), 9.4 (s, 1H, NH exchangeable).
Colorimetric Assay for Compounds that Bind to DNA
(Methyl Green DNA Displacement Assay)
DNA methyl green (20 mg, Sigma. St. Louis, MO, USA) was suspended
in 100 ml of 0.05 M Tris-HCl buffer, pH 7.5, containing 7.5 mM MgSO4 and
stirred at 37 °C for 24 h. Unless otherwise indicated, samples to be tested
were dissolved in EtOH in Eppendorff tubes. Solvent was removed in vacuo,
and 200 µl of the DNA/methyl green solution was added to each tube. The
absorption maximum for the DNA/methyl green complex is 642.5–645 nm.
Samples were incubated in the dark at ambient temperature. After 24 h, the
final absorbance of samples was determined. Readings were corrected for
initial absorbance and normalized as a % of the untreated DNA/methyl green
absorbance value. CC50 were determined for each compound as shown in
Table 2.
1-Phenyl-6-phenylamino-5-(4-halobenzylideneamino)pyrazolo-
[3,4-d]pyrimidin-4-one (9–11)
A suspension of 3 (1.6 g, 0.005 mol) in acetic acid (12 ml) was heated at
reflux with the appropriate aldehyde (0.005 mol) for 4 h. The solid separated
upon cooling was collected by filtration, dried and recrystallized [9 (HOAc)
and 10,11 (HOAc-H2O] (Table 1). 1H-NMR (DMSO-d6) 9: δ 7.21–7.60 (m,
14 H, ArH), 7.80 (s, 1H, pyrazole), 10.09 (s, 1H, CH), 11.18 (s, 1H, NH
exchangeable). 1H-NMR (DMSO-d6) 10: δ 7.28–7.56 (m, 14H, ArH), 7.85
(s, 1H, pyrazole), 9.9 (s, 1H, CH), 10.09 (s, 1H, NH exchangeable). 1H-NMR
(DMSO-d6) 11: δ 7.28–7.59 (m, 14H, ArH), 8.0 (s, 1H, pyrazole), 10.0 (s,
1H, CH), 11.18 (s, 1H, NH exchangeable).
Antiviral Screening
1,9-Diphenyl-7-(4-halophenyl)-8,9-dihydropyrazolo[3,4-d]pyrimidino-
[1,2-b] [1,2,4]triazin-4-one (12–14)
Microtiter trays with confluent cultures of vero cells were inverted and the
medium was shaken out. A serial dilution of sterile samples dissolved in the
medium was then added (100 µl in each well).Tray wells were then inocu-
lated with 30 plaque forming units of Herpes simplex-type 1 (HSV-1) virus
A mixture of 3 (3.8 g, 0.012 mol ), 4-halo-α-bromoacetophenone
(0.012 mol) and p-toluenesulfonic acid (3.8 g, 0.2 mol) in ethanol (20 ml)
was heated at reflux for 24 h. After cooling, the mixture was poured into
ice-water and the obtained product was filtered, dried and recrystallized from
Table 2. Activity of compounds on DNA-methyl
green assay.
———————————————————
1
a chloroform-pet. ether mixture (Table 1). H-NMR (CDCl3) 12: δ 5.3 (s,
2H, CH2), 6.98–7.75 (m, 14 H, ArH), 7.95 (s, 1H, pyrazole). 13: δ 5.28 (s, 2
H, CH2), 7–7.73 (m, 14H, ArH), 8.06 (s, 1H, pyrazole). 14: δ 5.3 (s, 2H,
CH2), 7.0–7.74 (m, 14H, ArH), 8.05(s, 1H, pyrazole).
Compound
CC50
(µg/ml)
———————————————————
Ethidium bromide
79
19
27
28
40
61
52
41
30
38
Biological Investigation
4*
5*
6*
9
The following instruments and materials were used in the biological
evaluation; hemocytometer (Bright line, AO instrument Co. Buffalo, NY,
USA), inverted microscope (Nikon Inc., Instrument Division, Garden City,
NY, USA), Dulbecco’s modified Eagles medium (Gibco, Grand Island, NY,
USA), supplement with 10% calf serum (Gibco), antibiotic and trypsin
(Sigma, St. Louis, MO, USA), Giemsa stain (Fisher Scientific Co., Fair
Lawn, NY, USA). TLC plates (RP-18F254; 0.25 mm, Merck), DNA (calf
thymus type I, Sigma, 100 µg/ml), DNA/methyl green (Sigma, St. Louis,
MO, USA).
10
11
12
13
14
———————————————————
Values represented the concentration required for
50% decrease in the initial absorbance of the
DNA-methyl green solution.
DNA Binding Assay
Analysis of the DNA binding affinity of the tested compounds was
performed using RP-TLC. TLC plates (RP-18 F254; 0.25 mm; Merck) were
predeveloped with MeOH-H2O (8:2). Test compounds were then applied
(5 mg/ml in MeOH) at the origin, followed by the addition of DNA (1 mg/ml
in H2O-MeOH mixture) at the same positions at the origin. The plates were
then developed with the same solvent system and the position of the DNA
was determined by the spraying with anisaldehyde reagent. The reagent
yields a blue color spot with DNA, and the intensity of the color was
proportional to the quantity of DNA added to the plate. Ethidium bromide
was used as a positive control.
* Compounds which possess high DNA affinity.
Table 3. Antiviral and cytotoxic activity of tested compounds.
——————————————————————————————
Compound % reduction in number of plaques
bCC50 (µg/ml)
——————————————————————————————
Aphidicolina
4
5
100
33
31
220
220
100
220
220
220
150
220
100
220
100
100
Table 1. Melting points, yield percentages, and molecular formulae of the
new compounds.
——————————————————————————————
6
66
7
8
9
None
None
22
Compound
R
Mp (°C)
Yield %
Molecular formulae
——————————————————————————————
10
11
12
13
14
41
30
21
15
4
5
H
173–175
115–117
210–212
145–147
198–200
83–85
56
65
71
78
86
39
42
48
C18H12N6O
C19H14N6O
C24H17FN6O
C24H17ClN6O
CH3
F
9
10
11
12
13
14
Cl
Br
F
20
C
24H17BrN6O
C25H17FN6O
25H17ClN6O
C25H17BrN6O
——————————————————————————————
a Positive control. The minimum antiviral concentration of Aphidicolin is
5 µg/ml and of the tested compounds is 100 µg/ml.
Cl
Br
87–89
C
b CC50 values (Concentration of compound resulting in 50% reduction in cell
density) were estimated visually from triplicate cultures.
96–98
——————————————————————————————
Arch. Pharm. Pharm. Med. Chem. 333, 99–103 (2000)

Pyrazolo[3,4-d]pyrimidine Derivatives
103
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but not test compounds. The trays were incubated under normal culture
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onto a pad of paper towels and the remaining cells were rinsed carefully with
the medium and fixed with 3.7% (v/v) formaldehyde in saline solution for
20 min, then rinsed with water, stained with 0.5% (w/v) crystal violet solution
in 20% aqueous ethanol for 30 min and rinsed with water again. The trays
were examined visually, antiviral activity was estimated as the % reduction
in the number of virus plaques (Table 3).
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Received: October 8, 1999 [FP431]
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Arch. Pharm. Pharm. Med. Chem. 333, 99–103 (2000)