Design and Synthesis of Novel Pyrazolo[3,4-d]Pyrimidines: In Vitro Cytotoxic Evaluation and Free Radical Scavenging Activity Studies

Article abstract of DOI:10.1007/s11094-020-02190-2

With evident biological importance, a new series of pyrazolo[3,4-d]pyrimidines 3a,3b and 4a,4b were synthesized via the formation of pyrazol-3-one 2a and 2b substrates. All compounds were evaluated for in vitro cytotoxic activity against MCF-7 (breast ade

Full text of DOI:10.1007/s11094-020-02190-2

DOI 10.1007/s11094-020-02190-2
Pharmaceutical Chemistry Journal, Vol. 54, No. 3, June, 2020 (Russian Original Vol. 54, No. 3, March, 2020)
DESIGN AND SYNTHESIS OF NOVEL PYRAZOLO[3,4-d]PYRIMIDINES:
IN VITRO CYTOTOXIC EVALUATION AND FREE RADICAL
SCAVENGING ACTIVITY STUDIES
Rima D. Alharthy*
Original article submitted December 09, 2019.
With evident biological importance, a new series of pyrazolo[3,4-d]pyrimidines 3a,3b and 4a,4b were synthe-
sized via the formation of pyrazol-3-one 2a and 2b substrates. All compounds were evaluated for in vitro
cytotoxic activity against MCF-7 (breast adenocarcinoma) and A549 (lung cancer) cell lines. The obtained re-
sults showed that pyrazolo[3,4-d] pyrimidin-4-ol 3a bearing phenyl group at N-1 and p-C H at C-6, and 4b
6
4
with dinitrophenyl at N-1 and furanyl moiety at C-6 had better inhibitory activity against MCF-7 with IC50
values in a micromolar range as compared to other substrates. The synthesized compounds can be considered
as new candidates for further optimization as anticancer agents.
Keywords: pyrazolo[3,4-d]pyrimidines; pharmacophore; cytotoxicity; anticancer; radical scavenging activity.
1
. INTRODUCTION
Cancer is the second leading cause of death, and by 2020
The synthesis and anticancer activity studies of
pyrazolo[3,4-d]pyrimidine structures inhibiting different key
enzymes was described in several reports [18 – 20]. Some
examples of pyrazolo[3,4-d]pyrimidine derivatives (A – D)
with potential anticancer activity related to the inhibition of
various protein kinases are shown in Fig. 1 [19, 21, 22].
Pyrazolo[3,4-d]pyrimidine derivatives A and B were synthe-
sized bearing chained substrates at various positions and bio-
logical evaluation against cyclic dependent kinases (CDKs)
were envisaged and reported. Other pyrazolo[3,4-d]pyrimi-
dine series C and D endowed with many functionalities at
the fused ring system were evaluated against Ehrlich ascites
carcinoma (EAC) cell line and showed mild anticancer activ-
its trend is expected to attribute to 15 million death cases per
year [1]. Although there have been great developments in the
discovery and treatment of cancer, it remains a challenging
health concern [2]. Current treatments available for cancer,
including surgery, chemotherapy, and radiotherapy [3] are
associated with severe side effects. This situation demands
for a search of new molecules to improve the selectivity,
pharmacokinetic profiles, and in vivo efficacy of anticancer
treatment [2, 4, 5]. In this context, fused pyrimidine scaffolds
gained considerable interests of both research and practical
pharmacy as attractive therapeutic targets for many diseases
particularly cancer [1, 6, 7]. Pyrazolo[3,4-d] pyrimidine, an
isostere of purines, is known as a versatile drug-like frag-
ment that has drawn much attention as pharmacophore
ity compared to doxorubicin.
Motivated by these findings and in continuation of our
previous studies for identification of new anticancer mole-
cules, the present investigation was directed toward the de-
sign of more active anticancer compounds derived from the
class of pyrazolo[3,4-d]pyrimidines. The synthetic approach
was aimed to incorporate various substituents at N-1 and C-6
positions of pyrazolo[3,4-d]pyrimidine ring (Fig. 2). The
cytotoxic activity of the synthesized pyrazolopyrimidine de-
rivatives was evaluated in two cancer cell lines: MCF-7
breast adenocarcinoma cells and A549 lung cancer cells. The
cytotoxic activity results were supported by colony forma-
tion inhibition assay.
[
8 – 11]. These structures exhibit promising pharmacological
properties including anti-proliferative [12] and antitumor ac-
tivity, highlighting their immense contribution for develop-
ing target-specific cancer chemotherapeutics [13 – 15]. Pre-
vious findings outlined some potent pyrazolo[3,4-d]pyrimi-
dine structures that reduced cell division and/or induced
apoptosis in tumor [16] and gastric cancer [17].
1
Department of Chemistry, Science and Arts College, Rabigh Campus,
King Abdulaziz University, Jeddah, Saudi Arabia
*
e-mail: iaaalharte@kau.edu.sa
273
0
091-150X/20/5403-0273 © 2020 Springer Science+Business Media, LLC

2
74
Rima D. Alharthy
Fig. 1. Chemical structures of pyrazolo[3,4-d]pyrimidine derivatives A – D as anticancer agents: (A) IC against CDK2, 0.5 µM; [19]; (B)
5
IC against CDK9, 17 nM) [21]; (C) IC against EAC, 100 mg/ mL; (D) IC against EAC, 90 mg/ mL [22].
0
5
0
50
50
Fig. 2. Structural modifications of pyrazolo[3,4-d]pyrimidine scaffold.
2
. EXPERIMENTAL
2.2. Chemical Synthesis
2
.1. Materials and methods
General procedure for preparation of pyrazolone de-
rivatives 2a and 2b. Phenyl hydrazine derivatives 1a and 1b
Starting materials were obtained from commercial sup-
(
0.01 mol) were added to ethyl acetoacetate (0.01 mol) in
pliers and used without further purification unless otherwise
stated. Melting points were determined with a Kofler block
apparatus and are uncorrected. Thin layer chromatography
ethanol (30 mL). The reaction mixture was heated at reflux
for 18 h and then cooled, filtered, and recrystallized from
ethanol to give target compounds 2a and 2b.
(
TLC) was carried out on Merck silica gel pre-coated sheets
3
-Methyl-1-phenyl-pyrazol-5-one (2a). Brown powder
SIL G/UV254. Patterns were visualized by exposure to UV
light (254 nm) followed by staining with basic potassium
permanganate and/or vanillin as appropriate. Infrared spectra
were recorded on a Perkin-Elmer 1720 FTIR spectrometer,
using KBr discs. Elemental analyses were performed on a
microanalytical data center at the Faculty of Science, Cairo
University, Egypt. NMR spectra were obtained as dilute so-
lutions in the appropriate solvent at 25°C unless otherwise
(
90%); m.p. 98 – 100 (C; R = 0.72 (10% MeOH in CHCl );
f
3
-1
IR spectrum (KBr), n/cm : 3092 (CH aromatic), 1645
1
C=O), 1460 and 1375; H NMR (400 MH , (CD ) SO) 7.72
(
Z 3 2
(
2H, d, J 2.5, ArCH), 7.48 – 7.42 (1H, m, ArCH),
7
.37 – 7.22 (2H, m, ArCH), 3.07 (2H, s, CH , pyrazolone)
1
and 1.94 (3H, s, CH , pyrazolone); C NMR (100 MHz,
2
3
3
(
CD ) SO) 174.5 (C, C=O pyrazolone), 162.8 (C,
3 2
1
13
stated. The H NMR and C spectra were recorded on
Brucker DPX 400 MHz spectrometer. The chemical shifts
were determined on the d-scale using residual solvent as an
internal standard (CD ) SO (dH, 2.50; dC, 39.43). All cou-
pyrazolone), 138.9 (C, ArCH), 128.9 (CH), 128.0 (CH),
127.4 (CH), 124.6 (CH), 123.9 (CH) (5C, ArCH), 42.5 (CH2,
pyrazolone) and 16.7 (CH , pyrazolone); Anal. Calcd for
3
C H N O: C, 68.95; H, 5.79; N, 16.08. Found C, 69.12; H,
10 10
2
3
2
+
+
.54; N, 15.75; HRMS m/z (ES ): Found 174.08 (M ,
5
pling constants are reported in Hertz units (Hz) and multi-
plicities denoted conventionally: s (singlet), d (doublet), and
m (multiplet), with prefixes br (broad) and app (apparent).
Assignments were made based on the chemical shift and,
where appropriate, with the benefit of DEPT sequences. The
GC-MS spectra were recorded on Agilent 7000 Triple Quad
series gas chromatograph interfaced to a mass spectrometer.
C H N O requires 174.08).
10 10
2
1-(2,4-Dinitrophenyl)-3-methyl-pyrazol-5-one (2b).
Brown powder (86%); m.p. 88 – 90 (C; R = 0.72 (10%
f
-1
MeOH in CHCl ); IR spectrum (KBr), n/cm : 3056 (CH aro-
3
1
matic), 1640 (C=O), 1511, 1336 (NO ), 1450 and 1375; H
2
NMR (400 MH , (CD ) SO) 7.90 (1H, d, J 2.0, ArCH), 7.84
Z
3 2

Design and Synthesis of Novel Pyrazolo[3,4-d]pyrimidines
275
(
1H, d, J 2.0, ArCH), 7.46 (1H, s, ArCH), 3.07 (2H, s, CH2
6-(Furan-2-yl)-3-methyl-1-phenyl-tetrahydro-1H-py-
razolo[3,4-d]pyrimidin-4-ol (4a). Red crystals (87%); m.p.
136 – 138 (C; R = 0.45 (7% MeOH in CHCl ); IR spectrum
13
pyrazolone) and 1.94 (3H, s, CH pyrazolone); C NMR
3
(
100 MHz, (CD ) SO) 174.5 (C, C=O pyrazolone), 162.8 (C,
3 2
f
3
-
1
pyrazolone), 138.9 (C, ArCH), 128.9 (CH), 128.0 (CH),
24.6 (CH) (3C, ArCH), 42.5 (CH , pyrazolone) and 16.7
(KBr), n/cm : 3437 (OH), 3250 (NH), 3095 (CH aromatic),
1
1375; H NMR (400 MH , (CD ) SO) 8.86 (1H, s, CH, py-
1
2
Z
3 2
(
CH , pyrazolone); Anal. Calcd for C H N O : C, 45.46; H,
rimidine), 7.90 (2H, d, J 2.0, ArCH), 7.56 (1H, s, ArCH),
7.46 (2H, d, J 2.0, ArCH), 7.32 (1H, s, ArCH), 6.39 (1H, s,
ArCH), 6.29 (1H, s, ArCH), 5.31 (1H, br s, OH), 4.70 (1H, s,
3
10
8
4
5
3
.05; N, 21.21. Found C, 45.87; H, 2.59; N, 22.06; HRMS
+
m/z (ES ): Found 264.05 (M , C H N O requires 264.05).
+
10
8
4
5
General procedure for preparation of pyrazolopyri-
midine derivatives 3a,3b and 4a,4b.
CHOH), 1.94 (3H, s, CH ) 1.91 (1H, s, NH) and 1.50 (1H, s,
3
1
CH, pyrazolopyrimidine); C NMR (100 MHz, (CD ) SO)
3
3
2
Pyrazolones 2a and 2b (0.01 mol) were dissolved in ab-
solute ethanol (40 mL), corresponding aldehydes (0.01 mol)
were added to the reaction mixture followed by urea
166.0 (C), 155.6 (C), 152.6 (C), 146.2 (C), (4C, ArCH),
142.1 (CH), 129.5 (CH), 128.5 (CH), 128.3 (CH), 128.0
(CH), 122.4 (CH), 110.6 (CH), 106.7 (CH) (8CH, ArCH),
(
0.01 mol). The reaction mixture was heated at reflux for
8 h. The reaction mixture was cooled in refrigerator over-
night, filtered, dried and recrystallized from ethanol to afford
compounds 3a,3b and 4a,4b.
-(4-Chlorophenyl)-3-methyl-1-phenyl-tetrahydro-1H-
74.1 (CH, pyrimidinol), 69.3 (CHOH), 44.3 (CH pyrazole)
,
1
and 15.3 (CH3, pyrazole); Anal. Calcd for C H N O :
16
16
4
2
Calcd: C, 64.85; H, 5.44; N, 18.91. Found C, 65.10; H, 5.73;
+
N, 16.53; HRMS m/z (ES ): Found 296.09 (M , C H N O
+
1
6
16
4
2
6
requires 296.09).
pyrazolo[3,4-d]pyrimidin-4-ol (3a).
1-(2,4-Dinitrophenyl)-6-(furan-2-yl)-3-methyl-tetrahyd-
ro-1H-pyrazolo[3,4-d]pyrimidin-4-ol (4b). Yellow powder
(90%); m.p. 102 – 104(C; R = 0.45 (5% MeOH in CHCl );
Red powder (73%); m.p. 112 – 114 (C; R = 0.45 (7%
f
-1
MeOH in CHCl ); IR spectrum (KBr), n/cm : 3421 (OH),
3
f
3
1
283 (NH), 3085 (CH aromatic) and 1367; H NMR (400
-1
IR spectrum (KBr), n/cm : 3434 (OH), 3311 (NH), 3101
3
1
(CH aromatic), 1512, 1338 (NO ), 1425; H NMR (400
MH , (CD ) SO) 8.23 (1H, s, CH, pyrimidine), 7.91 (2H, d,
Z
3 2
2
J 2.0, ArCH), 7.77 (2H, s, ArCH), 7.45 (2H, app t, J 2.0,
ArCH), 7.24 – 7.18 (2H, m, ArCH), 7.32 (1H, s, ArCH), 5.21
1H, br s, OH), 4.70 (1H, s, CHOH), 1.94 (3H, s, CH ) 1.88
MH , (CD ) SO) 8.99 (1H, s, CH, pyrimidine), 8.85 (3H, s,
Z
3 2
ArCH), 7.88 – 7.80 (3H, m, ArCH), 4.31 (1H, br s, OH),
(
4.70 (1H, s, CHOH), 1.94 (3H, s, CH ) 1.91 (1H, s, NH) and
13
1.50 (1H, s, CH, pyrazolopyrimidine); C NMR (100 MHz,
3
3
1
3
(
1H, s, NH) and 1.50 (1H, s, CH, pyrazolopyrimidine);
C
NMR (100 MHz, (CD ) SO) 166.0 (C, pyrazolopyrimidine),
(CD ) SO) 166.0 (C), 147.9 (C), 140.2 (C), 140.0 (C), 138.8
3
2
3 2
1
55.6 (C, pyrazolopyrimidine), 146.2 (C), 140.2 (C), 132.6
C), (3C, ArCH), 129.5 (CH), 129.3 (CH), 128.6 (CH), 128.0
CH) 127.7 (CH), 126.9 (CH), 126.0 (CH), 122.4 (CH),
19.3 (CH) (9CH, ArCH), 72.9 (CH, pyrimidinol), 71.7
CHOH), 44.9 (CH pyrazole) and 15.3 (CH3, pyrazole);
(C), 132.6 (C) (6C, ArCH), 130.8 (CH), 129.3 (CH), 128.6
(CH), 128.4 (CH), 120.8 (CH), 118.1 (CH) (6CH, ArCH),
(
(
72.9 (CH, pyrimidinol), 71.7 (CHOH), 44.9 (CH pyrazole)
,
1
and 15.3 (CH3, pyrazole); Anal. Calcd for C H N O :
16
14
6
6
(
Calcd: C, 49.74; H, 3.65; N, 21.75. Found C, 50.17; H, 3.42;
,
+
N, 21.38; HRMS m/z (ES ): Found 386.07 (M , C H N O
+
Anal. Calcd for C H ClN O: C, 63.44; H, 5.03; N, 16.44.
+
Found C, 63.12; H, 4.85; N, 16.93; HRMS m/z (ES ): Found
1
8
17
4
16 14 6 6
requires 386.07).
.3. Cell Culture
MCF-7 breast cancer cells and A549 lung cancer cells
+
40.09 (M , C H ClN O requires 340.09).
3
18 17 4
2
6-(4-Chlorophenyl)-1-(2,4-dinitrophenyl)-3-methyl-tet-
rahydro-1H-pyrazolo[3,4-d]pyrimidin-4-ol (3b). Yellow
powder (65%); m.p. 78 – 80 (C; R = 0.45 (5% MeOH in
were purchased from ATCC (Manassas, VA). The cells were
grown in Dulbecco’s modified Eagle’s medium (DMEM)
containing 10% fetal bovine serum (FBS), 1% penicil-
f
-1
CHCl ); IR spectrum (KBr), n/cm : 3432 (OH), 3309 (NH),
3
1
102 (CH aromatic), 1512, 1337 (NO ), 1420; H NMR (400
3
2
MH , (CD ) SO) 8.88 (1H, s, CH, pyrimidine), 8.42 (2H, d,
lin/streptomycin at 37°C under 5% CO . The culture medium
Z
3 2
2
J 2.0, ArCH), 8.38 (2H, d, J 2.0, ArCH), 7.85 (2H, d, J 2.0,
ArCH), 4.20 (1H, br s, OH), 4.70 (1H, s, CHOH), 1.94 (3H,
was replaced with fresh medium every two days. After
reaching 80 – 90% confluence, cells were treated with 0.25%
trypsin-EDTA for further passages. In all the experiments,
ells were used at passages 4 – 6.
s, CH ) 1.89 (1H, s, NH) and 1.50 (1H, s, CH,
3
13
pyrazolopyrimidine); C NMR (100 MHz, (CD ) SO) 166.0
3
2
(
C, pyrazolopyrimidine), 147.9 (C, pyrazolopyrimidine),
40.2 (C), 138.8 (C), 132.6 (C), 132.4 (C), 132.2 (C), (5C,
ArCH), 130.8 (CH), 129.3 (CH), 128.6 (CH), 128.3 (CH),
28.0 (CH), 120.8 (CH), 118.1 (CH), (7CH, ArCH), 72.9
CH, pyrimidinol), 71.7 (CHOH), 44.9 (CH pyrazole) and
Colony formation inhibition assay. Human cancer cells
lines (MCF-7 and A549) at the exponential phase were
plated into 24-well culture plates at a single cell density
(200 cells/well) and allowed to adhere for 24 h before treat-
ment. Cells were incubated with culture medium containing
different concentrations (10, 50, 100 and 200 mg/ mL) of
synthesized derivatives. After treatment with extracts for
24 h, the medium was removed and cells were washed with
1X PBS (pH 7.4), fixed with 4% paraformaldehyde, and
1
1
(
,
1
5.3 (CH pyrazole); Anal. Calcd for C H ClN O : Calcd:
3, 18 15 6 5
C, 50.18; H, 3.51; N, 19.51. Found C, 51.02; H, 3.65; N,
+
9.68; HRMS m/z (ES ): Found 430.05 (M , C H N O re-
+
1
18 14 6 6
quires 430.05).

2
76
Rima D. Alharthy
Scheme 1. Synthesis of pyrazol-3-ones 2a,2b and phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-ol derivatives 3a,3b and 4a,4b.
stained with neutral red solutions. Plates were photographed
using a Leica DFC420C camera connected to Leica DMI300
B microscope.
(Scheme 1). The IR spectra of compounds 2a and 2b con-
firmed the presence of carbonyl group (C=O) at 1645 and
-
1
1
640 cm , and nitro group (NO ) in compound 2b at 1511
2
-1
and 1336 cm . Subsequent multicomponent condensation of
pyrazol-3-ones 2a and 2b with aromatic aldehydes and urea
furnished the desired corresponding pyrazolo[3,4-d]pyrimi-
din-4-ol derivatives 3a,3b and 4a,4b in good yields, as
shown in Scheme 1. The IR spectra indicated the formation
of pyrazolo[3,4-d]pyrimidin-4-ol derivatives 3a,3b and
4a,4b by new peaks corresponding to (-OH) and (-NH)
groups.
3
. RESULTS AND DISCUSSION
3
.1. Chemistry
The synthesis of phenyl-1H-pyrazolo[3,4-d]pyrimidin-
-ol derivatives 3a,3b and 4a,4b started with the condensa-
4
tion of phenyl hydrazines 1a and 1b with ethyl acetoacetate
as b-keto ester, resulting in phenyl-3H-pyrazol-3-ones 2a
and 2b in very good yields 90% and 86%, respectively
1 13
The H and C NMR spectra of the synthesized products
supported the proposed structures 3a,3b and 4a,4b. In the
1
H-NMR spectra, the characteristic signals were identified
for OH group (at 5.21 and 5.31 ppm of compounds 3a and
3
b, and at 4.20 and 4.31 ppm of compounds 4a and 4b, re-
spectively); same was observed for NH protons (at 1.88 and
.91 ppm of substrates 3a and 3b, and at 1.89 and 1.91 ppm
TABLE 1. Cytotoxic Effect of Pyrazolones 2a,2b and pyrazolopy-
rimidines 3a,3b and 4a,4b against Human Breast Cancer (MCF-7)
and Lung Cancer (A549) Cell Lines
1
of compounds 4a and 4b, respectively).
IC50 ± SD (mM)
*
Compound
Log P
3.2. In Vitro Cytotoxic Activity
MCF-7
32 ± 3.1
1257 ± 2.9
74 ± 4.3
571.5 ± 2.1
-
A549
The synthesized pyrazol-3-ones 2a,2b and pyrazolopy-
rimidine derivatives 3a,3b and 4a,4b were evaluated for
their cytotoxicity against human breast cancer cell line
(MCF-7) and lung cancer cell line (A549) using neutral red
uptake assay methods. This methodology offers a measur-
able evaluation of the number of viable cells in any culture
and is commonly used in cytotoxicity tests. This test is based
on the ability of viable cells to incorporate and bind the
supravital dye neutral red in the lysosomes. This experiment
could allow further direct comparisons between target mole-
cule bearing functionalities at the fused ring system and frag-
2
a
121 ± 2.9
233 ± 2.1
11.5 ± 2.1
460 ± 1.8
179 ± 1.5
150 ± 2.6
9.80 ± 8.7
1.282
1.289
1.987
2.063
0.863
0.969
-
2
b
3
a
3
b
4
a
4
b
291 ± 5.3
35.2 ± 4.2
Doxorubicin
*
Log P calculated with MedChem Designer.

Design and Synthesis of Novel Pyrazolo[3,4-d]pyrimidines
277
(
a) MCF-7; positive control, compounds 2a, 3a ,4b
(
b) A549; positive control, compounds 2a, 3a ,4b
Fig. 3. Colony formation inhibition assay for (a) MCF-7 and (b) A549 cancer cells lines. Cells were treated with pyrazol-3-one 2a or
pyrazolopyrimidine derivatives 3a and 4b at final concentration of 200 mg/mL for 24 h. Then, medium was removed, and cells were incubated
in fresh medium for 24 h. The viable cells formed colonies, which were stained with neutral red solution.
ment before chemical modification. The results obtained for
pyrazol-3-ones 2a,2b and pyrazolopyrimidine derivatives
a,3b and 4a,4b were compared with doxorubicin (standard
remarkably reduced by treatment with derivatives 2a and 3a
as compared to untreated cells and the positive control, as
shown in Fig. 3.
3
anticancer agent) used as positive control. The results, as
summarized in Table 1 for all examined derivatives, showed
variable effects ranging from high to low activity against the
MCF-7 and A549 cells tested.
The aforementioned findings promote further studies to-
ward the optimum design of pyrazolo[3,4-d]pyrimidines as
potent anticancer compounds.
For MCF-7 cells, pyrazol-3-one 2a and pyrazolopyrimi-
dine derivatives 3a and 4b exhibited high to moderate cyto-
toxic activity. Pyrazol-3-one 2a showed the highest growth
inhibitory activity against MCF-7 cell line with
ACKNOWLEDGEMENTS
The authors are thankful to the Deanship of Scientific
Research, King Abdulaziz University, Jeddah, Saudi Arabia
(
grant number, G-1436-363-328) for great support of this
(IC50 = 32 ± 3.1 mg/mL) better than that for doxorubicin
(IC50 = 35.2 ± 4.2 mg/mL), followed by compound 3a
(IC50 = 74 ± 4.3 mg/mL) and finally 4b (IC50 = 291 ±
work.
CONFLICT OF INTEREST
5
.3 mg/mL). The remaining compounds including 2b
IC50 = 1257 ± 2.9 mg/mL) and 3b (IC50 = 571.5 ±
.1 mg/mL) had low growth inhibitory activity compared to
(
Author declares no conflicts of interest.
2
the positive control. The structural modifications at N-1 and
C-6 of pyrazol-3-ones 2a,2b and pyrazolopyrimidine deriva-
tives 3a,3b and 4a,4b may play a role for different cytotoxic
activities (Fig. 1; Table 1). Probably, the presence of
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