Synthesis and in vitro antiproliferative activity of novel pyrazolo[3,4-d]pyrimidine derivatives

Article abstract of DOI:10.1039/c5md00127g

A novel series of pyrazolo[3,4-d]pyrimidine derivatives were designed, synthesized and evaluated for their antiproliferative activity. Among the five compounds selected by NCI, compound 11a showed a distinctive pattern of selectivity on cell line panels and was further screened for a 5-log dose range, where it showed potent antiproliferative activity with median growth inhibition (GI₅₀) equal to 1.71 μM against the CNS cancer SNB-75 cell line. The tested derivative showed remarkably the highest cell growth inhibition against non-small cell lung cancer HOP-62, CNS cancer SNB-75, breast cancer HS578T, and melanoma MALME-3M cell lines. Flow cytometric analysis revealed that compound 11a could significantly induce apoptosis in A549 cells in vitro at low micromolar concentrations. Further investigation showed that compound 11a induced significant cell cycle arrest at G0/G1 phase partly due to its ability to downregulate cyclin D1 and upregulate p27^kip1 levels.

Full text of DOI:10.1039/c5md00127g

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CONCISE ARTICLE
Synthesis and in vitro antiproliferative activity of
novel pyrazoloij3,4-d]pyrimidine derivatives†‡
Cite this: DOI: 10.1039/c5md00127g
a
Nermin S. Abdou, Rabah A. T. Serya, a Ahmed Esmat,b Mai F. Tolba,bc
*
Nasser S. M. Ismaila and Khaled A. M. Abouzida
A novel series of pyrazoloij3,4-d]pyrimidine derivatives were designed, synthesized and evaluated for their
antiproliferative activity. Among the five compounds selected by NCI, compound 11a showed a distinctive
pattern of selectivity on cell line panels and was further screened for a 5-log dose range, where it showed
potent antiproliferative activity with median growth inhibition (GI50) equal to 1.71 μM against the CNS can-
cer SNB-75 cell line. The tested derivative showed remarkably the highest cell growth inhibition against
non-small cell lung cancer HOP-62, CNS cancer SNB-75, breast cancer HS578T, and melanoma MALME-
3M cell lines. Flow cytometric analysis revealed that compound 11a could significantly induce apoptosis in
A549 cells in vitro at low micromolar concentrations. Further investigation showed that compound 11a
induced significant cell cycle arrest at G0/G1 phase partly due to its ability to downregulate cyclin D1 and
upregulate p27kip1 levels.
Received 28th March 2015,
Accepted 8th June 2015
DOI: 10.1039/c5md00127g
www.rsc.org/medchemcomm
5 (PDE-5),5 modulation of the human adenosine receptor,6
1 Introduction
antiviral,7 anticoccidial,8 antimicrobial,9 etc. These investiga-
Cancer is an enormous global health burden, touching every
region and socio-economic level. It is the second major cause
of death worldwide, exceeded only by heart disease.1 Exten-
sive efforts are being carried out in order to discover new
treatments but despite improved imaging, molecular diagnos-
tic techniques, and advances in prevention and chemothera-
peutic management, the disease still affects many millions of
patients worldwide2,3 and no complete cure has been discov-
ered. Apart from surgical treatment and irradiation tech-
niques, chemotherapy still remains an important option for
cancer therapy; unfortunately, the treatment of most types of
solid tumors (e.g. breast and ovarian) is still a problem and
survival rates remain significantly low.4
tions revealed that substitution of various groups on the ring
imparts different activities.5–9
The pyrazoloij3,4-d]pyrimidine scaffold can be considered
as a bioisostere of the ATP purine ring, and hence may
exhibit promising antitumor activity by acting as a competi-
tive ATP inhibitor of many kinase enzymes. Fig. 1 illustrates
the similarity between the ATP structure and different
reported pyrazoloij3,4-d]pyrimidine derivatives as antitumor
agents.10–13
Angiogenesis is the formation of new blood vessels, and is
an essential process for tumor growth. The selective inhibi-
tion of tumor angiogenesis might be a useful strategy for
inhibiting tumor growth and metastasis. Vascular endothelial
growth factor (VEGF) is an important regulator of vascular
growth and permeability. Overexpression of VEGF in solid
tumors has been shown to promote angiogenesis, thereby
facilitating tumor growth. VEGF acts on VEGF-R2, a receptor
tyrosine kinase present on the surface of endothelial cells.
The inhibition of VEGF-R2 by small molecules is a validated
therapeutic approach for treatment of cancer and has
received much attention in the literature.14
Finding safe and effective anticancer agents is still one of
the hardest challenges in the field of drug discovery.
Pyrazoloij3,4-d]pyrimidine derivatives demonstrate various
biological activities, such as inhibition of phosphodiesterase-
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams
University, Cairo 11566, Egypt. E-mail: rabahat2003@yahoo.com,
khaled.abouzid@pharma.asu.edu.eg; Tel: +20 2 01229415877
b Department of Pharmacology & Toxicology, Faculty of Pharmacy, Ain Shams
University, Cairo 11566, Egypt
c Biology Department, School of Sciences and Engineering, The American
University in Cairo, New Cairo, Egypt
In this study, we report the design, synthesis and biologi-
cal evaluation of pyrazoloij3,4-d]pyrimidine derivatives as tar-
get antiproliferative agents.
Our basic design strategy for new scaffolds acting as anti-
proliferative agents was based on the following:
† The authors declare no conflict of interest.
1. The pyrazoloij3,4-d]pyrimidine scaffold was chosen as it
is considered to be an isostere of purine and hence may
‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5md00127g
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Fig. 1 Structures of ATP and different reported biologically active compounds containing the pyrazoloij3,4-d]pyrimidine scaffold imparting
different mechanisms as antitumor agents.
exhibit promising antitumor activity by acting as a competi-
tive ATP inhibitor of many kinase enzymes.
prepared in moderate to high yields from the reaction of the
chloro derivatives 1a and b with the appropriate amines
2b–e.
4-Hydroxy diphenyl urea derivatives were synthesized from
phenyl isocyanate by reaction with 4-aminophenol in dioxane
according to the reported procedures,26 and then the hydroxy
derivative was reacted with the chloro derivatives 1a and b to
give target compounds 4a and b.
2. Exploration of previously revealed SAR studies and bio-
isosteric modifications of reported lead compounds.15–20
The target compounds were originally designed to act as
VEGFR inhibitors through the following considerations:
1. The main scaffold occupying the ATP binding site can
be replaced with
scaffold.
a substituted pyrazoloij3,4-d]pyrimidine
The synthetic route which was attempted to synthesize
6-nitro-2,3-dihydrobenzoijd]thiazol-2-one started with cyclization
of 4-nitroaniline to 6-nitro-2-amino-2,3-dihydrobenzoijd]thiazole
under standard conditions,27 followed by diazotization and
hydrolysis in acidic medium28 to give the intermediate
6-nitro-3H-benzothiazol-2-one in an overall low yield (26%).
Alkylation of 6-nitro-3H-benzothiazol-2-one to give the corre-
sponding N-alkylated intermediates 5a–d was conducted in a
KOH/acetone/water mixture using appropriate aralkyl halides
under reflux conditions29 to obtain the desired intermediates
in an excellent yield.
Reduction of the N-alkylated intermediates 5a–d was car-
ried out under standard conditions reported by Abdelaal
et al.28 using 10% Pd/C and hydrogenation at 35 psi to give
the corresponding amines 6a–d. Carrying out the reduction
in ethanol/THF at a ratio of 3 : 1 improved the yield dramati-
cally. The target compounds 7a–e were finally obtained from
the reaction of the amines 6a–d and the chloro intermediates
1a and b in ethanol under reflux with the addition of few
drops of triethylamine.
2. The linker can be urea, amide, or ether.
3. The aryl moiety, essential for allosteric site binding, is
to be conserved either substituted or not.
The target compounds (series 1–5) were rationally
designed based on the bioisosteric modifications of the
reported lead VEGFR inhibitors as illustrated in Fig. 2.
2 Results and discussion
2.1. Chemistry
The synthetic approaches adopted to obtain the different
derivatives (7a–e, 9a–d, 11a–f, and 13a–d) are outlined in
Schemes 1–5.
The general synthetic route to 4-chlorophenyl-1H-
pyrazoloij3,4-d]pyrimidine 1a and 4-chloro-1-chlorophenyl-1H-
pyrazoloij3,4-d]pyrimidine 1b has been outlined in Scheme S1
(c.f. ESI‡). The pyrazoles were cyclized to pyrazoloij3,4-d]pyrimidine
by refluxing in formamide,21,22 then the 4-chloro derivatives
were prepared by chlorination with phosphorus oxychloride.23
In Scheme 1, the 4-amino diphenyl urea derivatives 2b–e
were synthesized in 2 steps first by reacting 4-nitroaniline
with the appropriate aryl isocyanate in methylene chloride,24
then reduction of the produced 4-nitro derivative using stan-
nous chloride dihydrate.25 The target products 3a–e were
Compounds 9a–d were also prepared in the same way
from the reaction of amines 8a and b and the chloro interme-
diates 1a and b under the same conditions. The synthesis of
these compounds was outlined in Scheme 3.25
In order to synthesize compounds 11a–f, the steps illus-
trated in Scheme 4 were adopted. First, p-nitrobenzoyl
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Fig. 2 Rational design of the target compounds (series 1–5).
chloride was obtained from the corresponding acid using
thionyl chloride under reflux conditions as reported,30 which
was then reacted with different amines in DCM in the pres-
ence of triethylamine in a typical amide formation to give the
amide derivatives.31 The amide derivatives were reduced
using stannous chloride dihydrate to produce amino deriva-
tives 10a–d.32 Compounds 11a–f were obtained by the reac-
tion of the amino derivatives 10a–d with compounds 1a and
b in ethanol under reflux followed by purification of the
resulting precipitate. Finally, compounds 13a–d were
obtained by reacting commercially available benzo-
thiazolamines 12a–c and the chloro derivatives 1a and b
under the same conditions (Scheme 5).
2.2. Biological evaluation
2.2.1. VEGFR-2 enzyme inhibition assay. The aim of this
study was to identify whether any of the compounds can
affect the activity of VEGFR-2 tyrosine kinase. The percent
change of the enzymatic activity was evaluated against a ref-
erence kinase inhibitor control at 10 μM. The results
observed as % activity change compared to that of the control
are presented in Table S1 (c.f. ESI‡), and the intra-assay vari-
ability was determined to be less than 10%. Inhibition of tar-
get activity by the compounds gives negative (−) values while
activation of target activity gives positive (+) values. Unfortu-
nately, the profiling data for the compounds tested against
VEGFR-2 showed insignificant enzyme inhibition. In the light
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Scheme 1 Synthesis of 3a–e and 4a and b.
Scheme 2 Synthesis of 7a–e.
of this information, further investigation was conducted to
evaluate the antiproliferative activities of the synthesized
compounds as demonstrated next.
2.2.2. Antiproliferative activity
2.2.2.1. Anticancer screening at NIH, Bethesda, Maryland,
USA. All the prepared final compounds were submitted to the
National Cancer Institute “NCI” (www.dtp.nci.nih.gov); five
compounds (7a, 9a, 9c, 11a, and 13a) were selected under the
Developmental Therapeutic Program (DTP). The operation of
this screening utilizes 60 different human tumor cell lines,
representing leukemia, melanoma, and cancers of the lung,
colon, brain, ovary, breast, prostate, and kidney. Selection for
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Scheme 3 Synthesis of 9a–d.
Scheme 4 Synthesis of 11a–f.
Scheme 5 Synthesis of 13a–d.
screening is based on the ability of the submitted
compounds to add diversity to the NCI small molecule
compound collection. All selected compounds with NCI
codes NSC 775949/1, NSC 775947/1, NSC 775950/1, NSC
775951/1, and NSC 775948/1 were tested at a single dose
(10 μM) in the full NCI 60-cell panel. Among the five selected
compounds, compound 11a (NSC 775951/1) was further scre-
ened for the 5-log dose molar range as it has shown
prominent cell growth inhibition at 10 μM against a variety
of cell lines.
2.2.2.1.1. Primary in vitro single dose screening of
anticancer activity in full NCI 60-cell line panel. Primary
in vitro one dose (10 μM) anticancer activity screening was
performed in a full NCI 60-cell panel. The results for each
compound were reported as a mean graph of the percent
growth of the treated cells compared to that of the untreated
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Table 1 Percentage of cell growth of NCI 60 cancer cell lines displayed
by the final compounds (7a, 9a, 9c, 11a, and 13a)
control cells. The mean graph of 11a produced from the NCI
60-cell line screening program is illustrated in Fig. S1 (c.f.
ESI‡).34 This primary screening indicated that compound 11a
possesses higher selectivity against non-small cell lung can-
cer and CNS cancer cells with mean growth inhibition per-
centages of 73.56 and 62.91%, respectively. Compound 11a
showed remarkable cell growth inhibition at 10 μM against a
variety of cell lines as highlighted in Table 1, which
prompted the 5-dose study to be carried out.
The cell growth percentages of the target compounds (7a,
9a, 9c, 11a, and 13a) against the full 60-cell line panel are
presented in Table 1.
In light of the NCI 60-cell line screening results, the fol-
lowing observations could be made:
Panel/cell line
Percentage cell growth
7a
9a
9c
11a
13a
87.87
103.14
102.24
102.43
101.97
44.82b
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
63.56
66.90
49.56b
58.59b
63.51
80.64
80.79 78.92
99.77 86.89
90.33 87.28
93.07 89.56
96.50 86.36
91.95 83.27
64.93
106.24
73.17
89.52
89.53
67.48
Non-small cell lung cancer
A549/ATCC
HOP-62
HOP-92
78.01
93.96
64.36
71.44
84.73
75.65
83.52
104.13 88.76
103.51 81.58
98.79 97.82
16.57b
101.75
−66.54b 110.16
37.74b
97.54
100.78
106.46
45.67b
98.37
NCI-H226
NCI-H23
NCI-H322M
NCI-460
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
103.08 101.76 19.13b
108.51 92.70
84.12 75.01
93.68 93.09
72.04
63.97
42.17b
• Compound 7a showed good antiproliferative activity
against renal A498 and UO-31 cancer cell lines with cell
growth inhibition percentages of 41.05% and 49.27%, respec-
tively. Cell growth inhibition was also high for the prostate
PC-3 cancer cell line (44.4%) and leukemic K-562 cell line
(50.44%).
• Compound 13a showed good antiproliferative activity
against the leukemic SR cell line with a cell growth inhibition
percentage of 55.18%, as well as the non-small cell lung cancer
NCI-H322M cell line with an inhibition percentage of 54.33%.
• Compound 11a exhibited a prominent cell growth inhib-
itory effect in most of the tested cell lines as demonstrated in
Table 1.
2.2.2.1.2. In vitro five dose full NCI 60-cell panel assay.
Compound 11a (NSC 775951/1) was selected for further
screening at five different concentrations (0.01, 0.1, 1, 10,
and 100 μM) as it showed the most prominent cell growth
inhibition at 10 μM concentration against a variety of cell
lines. The assay results were used to create log concentration
vs. % growth curves and three response parameters namely
GI50 (median growth inhibition), TGI (total growth inhibition
= cytostatic activity), and LC50 (median lethal growth inhibi-
tion = cytotoxic activity) were calculated in μM for each cell
line.33 The results are shown in Fig. S2 and S3 (c.f. ESI‡) and
Table 2. Compound 11a exhibited remarkable anticancer
activity against most of the tested cell lines representing nine
different subpanels. GI50 values were between 1.71–23.9 μM,
except for five cell lines of different subpanels namely leuke-
mic cell line MOLT-4, colon cancer cell lines HCT-15 and
KM12, melanoma cell line MDA-MB-435, and breast cancer
cell line MCF7 showing GI50 values up to 75.6, 99.3, 57.8,
67.8 and 74.1 μM, respectively (Table 2).
With regard to the sensitivity against some individual cell
lines (Table 2), compound 11a showed remarkable activity
against the CNS SNB-75 cancer cell line with a GI50 value of
1.71 and a TGI value of 5.40 and the melanoma MALME-3M
cell line with a GI50 value of 1.97 and a TGI value of 4.94.
The collective dose response curve and individual dose
response curves of all cell lines tested are shown in Fig. S2
and S3 (c.f. ESI‡), respectively.
92.69
NTa
80.29
86.45
107.01 91.21
108.06 98.03
106.36 94.82
72.41 87.15
65.37
NTa
109.89
105.79
99.36
97.82
103.33
98.87
37.65b
66.58
70.23
88.49
HT29
106.36 115.50 84.65
KM12
SW-620
71.63
84.39
98.25 80.16
98.71 100.85 72.91
100.20
CNS cancer
SF-268
SF-295
SF-539
SNB-19
85.57
87.16
79.19
85.63
88.34
96.64
108.02 99.67
107.12 97.83
99.64 92.44
95.97 NTa
102.49 86.37
105.58 94.99
36.92b
73.36
114.14
99.85
101.04
98.25
43.72b
48.73b
SNB-75
U251
−20.35b 106.68
40.19b
102.73
Melanoma
MALME-3M
M14
86.42
82.85
84.19
87.97
90.10
87.25
92.57
58.26b
92.76 85.12
101.33 93.61
90.10 83.23
115.23 NTa
95.95 90.43
94.27 94.40
2.93b
69.10
81.16
79.10
62.24
75.43
87.04
106.94
91.92
108.80
95.29
92.06
111.92
94.68
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
120.63 101.77 56.48b
93.28 104.81 63.86
81.82
86.60
86.98
75.11
87.92
84.79
100.04 101.96 71.12
112.54 101.52 87.44
91.33 80.57
98.93 92.84
113.12 97.95
107.36 94.55
99.59
108.46
103.78
96.04
107.04
105.64
111.66
30.27b
80.27
21.63b
74.18
100.62 107.57 99.73
54.02b
93.69
58.95b
74.61
71.58
79.54
75.01
100.45 102.91 72.06
103.25
66.37
106.72
102.89
98.96
97.55
117.68
91.72
A498
ACHN
CAKI-1
RXF-393
SN12C
77.87 85.22
103.91 98.61
99.06 93.66
101.80 97.85
97.38 NTa
50.40b
54.27b
62.29
62.16
18.96b
50.98b
57.72b
TK-10
UO-31
104.89 111.11 92.14
50.73b
79.60 83.61
Prostate cancer
PC-3
DU-145
55.60b
94.54
88.50 86.08
53.30b
98.26
103.47
103.66 100.20 54.57b
Breast cancer
MCF7
MDA-MB-231/ATCC 57.73
HS 578T
BT-549
T-47D
85.26
100.50 95.89
101.04 NTa
109.14 96.61
82.83
8.76b
0.22b
97.21
102.21
120.22
107.58
107.55
81.13
100.94 106.45 106.74 59.80b
The criterion for the selectivity of a compound depends
on the ratio obtained by dividing the full panel MID (the
62.31
100.20 82.52
76.80
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Table 1 (continued)
treatment of A549 cells with compound 11a significantly
boosted the protein expression of the cell cycle inhibitor
p27kip1 by about 3 folds (Fig. 6). To sum up, compound 11a
induced cell cycle arrest at G0/G1 phase and this could be at
least partly due to its ability to downregulate cyclin D1 and
upregulate p27kip1 levels.
Panel/cell line
Percentage cell growth
65.93 93.06 91.50
MDA-MB-468
76.06
80.31
a
b
NT: not tested. Italicized values represent those below 60.00%
growth.
2.2.2.2. Antiproliferative activity against MCF-7 human
breast cancer cell line. The rest of the synthesized compounds
(3a–e, 4a and b, 7b–e, 9b and d, 11b–f, and 13b–d) not cho-
sen by NCI were screened for their cytotoxic activity against
MCF-7 (breast) human tumor cell line utilizing the in vitro
sulforhodamine-B (SRB) standard method.33 Doxorubicin,
which is one of the most effective anticancer agents, was
used as the reference drug in this study. The relationship
between the surviving fraction and drug concentration was
plotted to obtain the survival curve of the breast cancer cell
line (MCF-7). The response parameter calculated was the IC50
value, which corresponds to the concentration required for
50% inhibition of cell viability. Table 3 shows the in vitro
cytotoxic activity of the synthesized compounds; it is appar-
ent that most of the synthesized analogs exhibit good anti-
proliferative properties against the tested human tumor cell
line. However, compound 11b exhibited more potent cyto-
toxic effects compared to the reference compound doxorubi-
cin with IC50 values of 1.12 and 1.172 μM, respectively. More-
over, there are six compounds that showed significant
antitumor activity.
average sensitivity of all cell lines toward the test agent) by
their individual subpanel MID (the average sensitivity of all
cell lines of a particular subpanel toward the test agent).
Ratios between 3 and 6 refer to moderate selectivity, and
ratios greater than 6 indicate high selectivity toward the cor-
responding cell line, while compounds not meeting either of
these criteria are rated as non-selective.35 As per this crite-
rion, compound 11a in this study was found to be highly
selective toward CNS cancer with a selectivity ratio of 8.25
and moderately selective toward non-small cell lung, renal
and ovarian cancer subpanels only with selectivity ratios of
4.51, 3.16 and 3.11, respectively, whereas it was found to be
non-selective against the remaining cell panels (Table 2).
2.2.2.1.3. Effects of compound 11a on the cell cycle.
Compound 11a was further investigated for its possible
mechanisms of anticancer activity using non-small cell lung
cancer cells based on the selectivity ratio drawn from the NCI
results. The cytotoxicity assay at 5 different concentrations
(0.1, 1, 10, 100, and 1000 μM) was repeated under our labora-
tory conditions using sulforhodamine-B (SRB) and the IC50 in
the A549 cell line was 66.6 μM (Fig. 3). Exposure of A549 lung
cancer cells to compound 11a at a concentration equal to its
IC50 for 48 h induced a significant (P < 0.001) increase in the
percentage of cells at G0/G1 phase by about 11.9% compared
to the untreated cells (Fig. 4). This could partly explain the
antiproliferative activity of compound 11a.
We can conclude from these results (Table 3) the struc-
ture–activity correlation of the synthesized compounds with
the following observations:
▶ The pyrazoloij3,4-d]pyrimidine ring is a suitable back-
bone for cytotoxic activity.
▶ Both the amide and thiazole series displayed consider-
able potency.
2.2.2.1.4. Effects of compound 11a on cell cycle regulatory
molecules. The significant cell cycle arrest induced by
compound 11a at G0/G1 phase was further substantiated
through assessing the expression levels of two cell cycle
regulatory molecules cyclin D1 and p27kip1 that are known to
regulate the transition at this phase of the cell cycle. Hall
and Peters previously demonstrated that cyclin D1 levels are
unregulated in several types of tumors including NSCLC.36
Treatment of A549 cells with compound 11a triggered a
significant reduction in the protein abundance of the cell
cycle inducer cyclin D1 by about 51% compared to control
cells as shown by immunocytochemistry (Fig. 5). Cyclin D1
expression is known to be maintained through G1 phase as it
is required for the transition of cells to S phase where its
levels decline again to allow for DNA synthesis.37 Therefore,
the evidenced reduction in cyclin D1 abundance after
treatment with compound 11a can justify the cell cycle arrest
at G0/G1 phase. p27/Kip1 is a member of the Cip/Kip family
of cyclin-dependent kinase inhibitors. They function via
forming heterotrimeric complexes with cyclins and CDKs,
with subsequent inhibition of kinase activity and blockage of
progression through G1/S phase.38 In the current study,
▶ The cytotoxicity of the alkylated thiazolone and urea
series depends on different substituents.
▶ The introduction of p-chloro in the phenyl moiety at the
N-1 position of pyrazolopyrimidine affects the activity dra-
matically; it may increase or decrease the cytotoxic activity
which might be due its effect on the length of the conforma-
tion adopted by the compound.
2.3. Enzyme inhibition assay
This assay was performed to determine whether any of the
compounds can modulate the activity of the protein kinase
targets identified from comparative analysis and target fish-
ing. The percent change of the enzymatic activity produced
by the synthesized compounds against EGFR, Aurora-A, SRC,
P38α, C-MET, PDGFR and HER2 kinases was evaluated
against a reference kinase inhibitor control at 10 μM (Table
S2, c.f. ESI‡).
The profiling data for the compounds tested against
EGFR, Aurora-A, SRC, P38α, C-MET, PDGFR and HER2
showed weak inhibition of some protein kinase targets by a
few compounds. The results of EGFR activity showed weak
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Table 2 NCI in vitro testing results for compound 11a (NSC 775951/1) at five-dose levels in μM
Panel/cell line
GI50IJμM)
SubpanelMIDb
71.8
Selectivity ratio(MIDa : MIDb)
0.41
TGIIJμM)
LC50IJμM)
Leukemia
CCRF-CEM
MOLT-4
RPMI-8226
SR
>100
75.6
>100
11.6
>100
>100
>100
49.1
>100
>100
>100
>100
Non-small cell lung cancer
A549/ATCC
HOP-62
6.63
4.51
4.38
2.62
2.53
3.35
10.9
15.8
7.09
6.40
51.3
8.78
8.29
21.5
>100
37.3
86.6
>100
>100
>100
>100
>100
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-460
NCI-H522
Colon cancer
COLO 205
HCT-2998
HCT-116
HCT-15
89.9
>100
>100
>100
62.26
0.48
12.5
>100
4.01
99.3
57.8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
KM12
SW-620
CNS cancer
SF-268
SF-295
SF-539
SNB-19
3.63
8.25
1.73
5.85
2.98
3.81
3.82
1.71
>100
13.3
17.1
18.9
5.40
>100
43.1
49.0
>100
35.9
SNB-75
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
A498
ACHN
CAKI-1
RXF-393
SN12C
TK-10
UO-31
17.27
4.49
1.97
18.3
67.8
13.6
3.79
21.7
6.42
17.4
22.3
4.94
>100
21.0
>100
>100
96.3
>100
>100
>100
>100
>100
>100
36.2
>100
>100
>100
>100
9.63
9.46
3.11
3.16
14.4
14.3
15.2
3.87
7.06
2.95
65.9
>100
95.2
19.3
>100
8.70
>100
>100
>100
>100
>100
51.1
3.47
3.83
3.32
14.0
2.43
8.26
16.5
23.9
18.3
39.4
11.0
>100
9.33
>100
49.5
>100
65.5
>100
37.0
>100
56.9
>100
>100
>100
Prostate cancer
PC-3
DU-145
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
11.2
2.67
1.38
11.8
10.6
44.8
48.1
>100
>100
21.69
74.1
5.09
3.37
5.69
20.2
21.7
>100
21.9
34.2
26.4
>100
>100
>100
80.6
>100
91.9
>100
>100
29.96
a
b
Italicized values represent those GI50 values which are less than 10 μM. MID = Average sensitivity of all cell lines in μM. MID = Average
sensitivity of all cell lines of a particular subpanel in μM.
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Fig. 3 Concentration–response curve of compound 11a (N) in A549
non-small cell lung cancer cells. Cells were exposed to 5 different
concentrations (0.1, 1, 10, 100, and 1000 μM) of the tested agent for 72
h and then cell viability was determined using SRB assay. Data are
mean SD (n = 3). Experiments were carried out in triplicate.
Fig. 5 The effect of compound 11a (N) on the protein abundance of
cyclin D1 in A549 cells assessed through immunocytochemistry. Data
are mean SD (n = 3). Experiments were carried out in triplicate.
calculated using Accord for Excel include atom based log P98
(A log P98),39 ADMET 2D polar surface area (ADMET 2D PSA),
aqueous solubility (AQ SOl), aqueous solubility level (AQ SOl
LEV), blood brain barrier value (BBB), blood brain barrier
level (BBB LEV), cytochrome P450 2D6 (CYP2D6), cytochrome
P450 2D6 probability (CYP PROB), hepatotoxicity level
(HEPATOX LEV), hepatotoxicity probability (HEPATOX
PROB), plasma protein binding logarithmic value (PPB LOG)
and plasma protein binding logarithmic level (PPB LEV).
The structures of the compounds were drawn using DS
ViewerPro Suite software and appended to Accord for Excel
software, then the parameters were calculated.
2.4.1. Results of ADMET studies. All the parameters calcu-
lated are tabulated in Table 4. The ADMET plot is a 2D
plot generated using calculated PSA_2D and A log P98
properties.
Fig. 4 Effect of compound 11a (N) on DNA-ploidy flow cytometric
analysis of A549 cells. The cells were treated with compound 11a (25
mM) for 48 h. Data are mean SD (n = 3). Experiments were carried
out in triplicate.
Blood Brain Barrier (BBB) and Human Intestinal Absorp-
tion (HIA) plots (Fig. 7) were drawn using all the compounds.
In the BBB plot, 19 compounds fall outside the 99%
ellipse (undefined). Hence, these compounds may not be able
to penetrate the blood brain barrier, and the chances of CNS
side effects are low or absent. On the contrary, seven com-
pounds from the benzyloxy and benzothiazole series, and the
cyclohexylamide (11a) compound lie inside the 99% ellipse,
thus they have a medium to high degree of penetration.
In the HIA plot, seven compounds fall outside the 99%
confidence ellipse (undefined), whereas the remaining com-
pounds fall inside the 99% ellipse. Hence, compounds 4a,
9a, 11a and b, and 13a–c are expected to possess good
human intestinal absorption. Compound 7e shows very low
absorption as it contains three chloro groups. The ADME
aqueous solubility logarithmic level of most of the com-
pounds was found to be 2, 1 or 0 which indicates very low
inhibition by two compounds (11b and 13a) while those for
Aurora-A showed weak inhibition by one compound (3b).
Weak inhibition by 7a was also shown for the protein kinase
target PDGFR.
2.4. ADMET studies
Computer-aided ADMET studies were performed by using the
software Accord for Excel (Accelrys Discovery Studio 2.5 soft-
ware). These studies are solely based on the chemical struc-
ture of the molecule. Some of the parameters that are
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Fig. 6 The effect of compound 11a (N) on the protein expression of p27kip1 in A549 cells assessed through Western blotting. Data are mean SD
(n = 3). Experiments were carried out in triplicate.
aqueous solubility. The hepatotoxicity score predicts the hep-
atotoxic nature of the chemical compounds. The ADME.
HEPATOTOX.PROB values of most of the compounds lie in
the range of 0.93–0.98. Hence, the compounds are likely to
possess hepatotoxicity, except for two compounds both
containing a cyclohexyl ring instead of a phenyl one. Further
studies are necessary to determine the hepatotoxic dose
levels. Score predicts the hepatotoxic nature of the studied
chemical compound through its effect on CYP2D6 score.
More than half of the compounds are predicted as non-
inhibitors of CYP2D6, so the results are reliable. Hence, the
side effects (i.e. liver dysfunction) are not expected upon
administration of these compounds. The plasma protein
binding model predicts whether a compound is likely to be
highly bound to carrier proteins in the blood. There is a high
probability that these compounds can reach the desired tar-
gets as all of the compounds showed more than 95% plasma
protein binding.
On the other hand, compounds 3b, 7a, and 11b showed
weak inhibition against Aurora-A, EGFR, and PDGFR kinases,
respectively.
The structures of the weak inhibitors will be optimized to
achieve optimum binding through SAR studies and hence,
the activity will be enhanced. Such modification may involve
insertion of a methylene group between the aryl moiety and
N-1 of pyrazolopyrimidine to render the molecule more flexi-
ble to accommodate better fitting with the target enzyme.
This suggested modification was based on the fact that the
cyclohexylamide derivative displayed the highest activity.
However, the exact inhibitory mechanism of these com-
pounds could not be fully described but is still a subject for
future investigation.
3 Experimental
3.1. Chemistry
The starting materials and reagents were purchased from
Sigma-Aldrich and Alfa-Aesar Organics and used without fur-
ther purification. Melting points were determined on a Stuart
Scientific apparatus and uncorrected reactions were moni-
tored using thin layer chromatography (TLC) performed on
0.255 mm silica gel plates with visualization under UV light
(254 nm). FT-IR spectra were recorded on a Perkin-Elmer
spectrophotometer using KBr discs. 1H-NMR spectra were
recorded on a Perkin-Elmer 300 MHz spectrometer in δ scale
(ppm) and J (Hz) using TMS as the standard signal at
Professor Dr. Ahmed Farag's Laboratory at Cairo University.
13C-NMR spectra were recorded on a Perkin-Elmer 400 MHz
spectrometer in δ scale (ppm) at the NMR Center of the Fac-
ulty of Pharmacy, Cairo University. EI-MS spectra were
recorded on a Finnigan Mat SSQ 7000 (70 eV) mass spectro-
meter at the Microanalytical Center at Cairo University. Ele-
mental analysis was performed at the Microanalytical Center
at Al-azhar University.
PSA is a key property that has been linked to drug bio-
availability. Thus, passively absorbed molecules with PSA
>140 are thought to have low bioavailability. All the synthe-
sized compounds have PSA ranging from 60.87–94.86, thus
they theoretically should present good passive oral
absorption.
2.5. Final conclusion
In this study, we have designed and synthesized a novel
series of pyrazoloij3,4-d]pyrimidine derivatives and tested
their activity against VEGFR-2.
Biological evaluation revealed that compound 11a
displayed significant activity on an NCI 60-cell panel. It
showed high specificity against CNS cancer, and moderate
specificity against NSCLC, ovarian and renal cancer cell lines.
Further investigation showed that compound 11a induced
cell cycle arrest at G0/G1 phase and this could be at least
partly due to its ability to downregulate cyclin D1 and
upregulate p27kip1 levels.
The reported intermediates 1a and b as well as 2a and b
were synthesized according to the reported experimental
procedures.
Also, compounds 3b, d and e, 7d, 9b, 11b, and 11f showed
high antiproliferative activity against the MCF-7 breast cancer
cell line.
General procedure for the preparation of 1-IJsubstituted
phenyl)-3-IJ4-IJ(1-substituted phenyl-1H-pyrazoloij3,4-d]pyrimidin-4-
yl)amino)phenyl)urea (3a–e). To a solution of 1-IJ4-aminophenyl)-
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Table 3 Cytotoxic activity of the synthesized compounds (3a–e, 4a and b, 7b–e, 9b and d, 11b–f, and 13b–d)
Entry
1
Compoundcode
R
X
R′
IC50 (μM)on MCF-7a
3a
Ph
NH
55.07
2
3
3b
3c
Ph
Ph
NH
NH
4.20
36.33
4
5
3d
3e
p-Chlorophenyl
p-Chlorophenyl
NH
NH
3.15
9.05
6
4a
Ph
O
>100
7
8
4b
7b
p-Chlorophenyl
O
82.53
46.06
Ph
NH
9
7c
Ph
Ph
NH
NH
44.04
1.60
10
7d
11
7e
p-Chlorophenyl
NH
>100
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Table 3 (continued)
Entry
12
Compoundcode
R
X
R′
IC50 (μM)on MCF-7a
9b
Ph
NH
4.36
13
14
9d
p-Chlorophenyl
NH
NH
>100
11b
Ph
1.12
15
16
11c
11d
Ph
NH
NH
75.87
p-Chlorophenyl
>100
17
18
11e
11f
p-Chlorophenyl
p-Chlorophenyl
NH
NH
>100
4.92
19
20
13b
13c
Ph
Ph
NH
NH
>100
>100
21
13d
p-Chlorophenyl
NH
85.59
1.172
22
Doxorubicin
a
The values given are means of three experiments.
3-substituted phenylurea (3 mmol) in ethanol (20 ml), an equi-
molar amount of 4-chloro-1-IJ4-substituted phenyl)-1H-
pyrazoloij3,4-d]pyrimidine (1a and b) was added followed by
addition of 0.2 ml of triethylamine and the mixture was heated
under reflux for 18 h. The reaction mixture was monitored by
TLC till completion of the reaction. The reaction mixture was
then filtered while hot and the precipitate was crystallized from
an ethanol/water mixture to give the target compounds 3a–e.
1-IJ2-Chlorophenyl)-3-IJ4-IJ(1-phenyl-1H-pyrazoloij3,4-d]pyrimidin-
4-yl)amino)phenyl)urea (3a). Yield 54.31%; m.p. >280 °C; FT-IR
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Table 4 Computer-aided ADMET screening results of the synthesized compounds
CPD ID BBB_Leva Absorp_Levb AQ SOl LEVc Hepatoxd Hepatox Prob CYP2D6e PPB_Levf A log P98 Unk_A log P98 ADEM_PSA_2Dg
3a
3b
3c
3d
3e
4a
4b
7a
7b
7c
7d
7e
9a
9b
9c
9d
11a
11b
11c
11d
11e
11f
13a
13b
13c
13d
4
4
4
4
4
4
4
4
4
4
4
4
1
1
1
4
2
4
4
4
4
4
1
1
1
4
1
1
1
1
1
0
1
1
2
2
2
3
0
1
1
2
0
0
2
1
1
2
0
0
0
1
1
1
1
1
1
2
1
1
1
0
0
0
1
1
1
1
1
1
1
1
1
0
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
0
1
1
1
1
1
1
0.966
0.96
0.96
0.953
0.966
0.96
0
0
1
1
0
1
1
0
0
0
0
0
1
1
1
1
1
1
0
0
1
0
0
1
0
0
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
4.983
4.983
5.067
5.496
5.647
4.329
4.993
6.148
6.232
6.812
6.812
7.477
5.193
5.857
5.857
6.522
4.692
5.075
6.017
5.356
5.739
6.682
5.068
5.554
5.246
5.91
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
94.862
94.862
94.862
94.862
94.862
90.982
90.982
72.594
72.594
72.594
72.594
72.594
60.871
60.871
60.871
60.871
82.052
82.052
82.052
82.052
82.052
82.052
63.202
63.202
63.202
63.202
0.96
0.993
0.993
0.993
0.993
0.986
0.986
0.986
0.986
0.986
0.311
0.973
0.933
0.337
0.966
0.98
0.993
0.887
0.986
0.986
a
b
BBB_Level; 4 = undefined, 2 = medium penetration, 1 = high penetration. Absorp_Level; 3 = very low absorption, 2 = low absorption, 1 =
c
moderate absorption, 0 = good absorption. AQ SOl LEV; 2 = low solubility, 1 = very low but soluble, 0 = extremely low solubility.
d
e
f
Hepatotox_Level; 1 = toxic, 0 = non-toxic. CYP 2D6; 1 = likely to inhibit, 0 = non-inhibitor. PPB (plasma protein binding); 2 = more than
g
95%, 1 = more than 90%, 0 = less than 90%. PSA (polar surface area); cpds must have log P value not greater than 5.0 to attain a reasonable
probability of being well absorbed.
Fig. 7 Human intestinal absorption (HIA) and blood brain barrier plot for the newly synthesized compounds.
(ν max, cm−1): 3310 (NH), 1705 (CO amide), 1630 (CN);
1H-NMR (300 MHz, DMSO-d6): δ ppm 10.15 (s, 1H, NH, D2O
exchangeable), 9.44 (s, 1H, NH, D2O exchangeable), 8.50 (s,
2H, heterocyclic H), 8.30 (s, 1H, NH, D2O exchangeable),
8.23–8.17 (m, 3H, ArH), 7.75 (d, J = 7.3 Hz, 2H, ArH), 7.59–
7.44 (m, 5H, ArH), 7.39–7.33 (m, 2H, ArH), 7.03 (t, J = 7.5 Hz,
1H, ArH); MS (Mwt.: 455.13): m/z (% rel. int.) 457.00 (M+ + 2,
10.28), 456.00 (M+ + 1, 8.4), 455.00 (M+, 32.12), 328.05
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(74.97), 302.05 (100); Anal. calcd for C24H18ClN7O: C, 63.23; H,
3.98; N, 21.51; found: C, 63.41; H, 3.96; N, 21.63.
The analytical characterization data for compounds 3b–e
are provided in the ESI.‡
General procedure for the preparation of 3-substituted
benzyl-6-amino-2,3-dihydrobenzoijd]thiazol-2-one (6a–d). To a
solution of appropriate 3-substituted benzyl-6-amino-2,3-
dihydrobenzoijd]thiazol-2-one (5a–d, 5 mmol) in an ethanol/
THF mixture (3 : 1, 100 ml), 10% Pd/C (0.2 g) was added and
the mixture was hydrogenated in a bar-shaker hydrogenator
at 35 psi and room temperature for 6 h. The mixture was fil-
tered through celite and the filtrate was concentrated to give
crude amines which were then crystallized from diethyl ether
to yield pure crystals of compounds 6a–d. 6a: R2 = 4-Cl; yield
87.7%; m.p. 123–127 °C. 6b: R2 = 2,4-diCl; yield 99%; m.p.
150–155 °C. 6c: R2 = 3,4-diCl; yield 95%; m.p. 157–158 °C. 6d:
R2 = 3-Br; yield 82%; m.p. 135–137 °C.
General procedure for the preparation of 6-IJ(1-IJ4-substituted
phenyl)-1H-pyrazoloij3,4-d]pyrimidin-4-yl)amino)-3-IJsubstituted
benzyl)benzoijd]thiazol-2IJ3H)-one (7a–e). To a solution of 6-
amino-3-IJsubstituted benzyl)benzoijd]thiazol-2IJ3H)-one (6a–d, 3
mmol) in ethanol (20 ml), an equimolar amount of 4-chloro-1-
IJ4-substituted phenyl)-1H-pyrazoloij3,4-d]pyrimidine (1a and b)
was added after which 0.2 ml of triethylamine was added and
the mixture was stirred under reflux for 25 h. The reaction mix-
ture was then filtered while hot and the precipitate was crystal-
lized from an ethanol/water mixture to give the target com-
pounds 7a–e.
3-IJ4-Chlorobenzyl)-6-IJ(1-phenyl-1H-pyrazoloij3,4-d]pyrimidin-
4-yl)amino)benzoijd]thiazol-2IJ3H)-one (7a). Yield 55.4%; m.p.
200 °C; FT-IR (ν max, cm−1): 3300 (NH), 1690 (CO), 1640
(CN); 1H-NMR (300 MHz, DMSO-d6): δ ppm 10.29 (s, 1H,
NH, D2O exchangeable), 8.51 (s, 2H, heterocyclic H), 8.27 (s,
1H, ArH), 8.19 (d, J = 7.4 Hz, 2H, ArH), 7.59–7.54 (m, 3H,
ArH), 7.45–7.35 (m, 5H, ArH), 7.32 (d, J = 7.3 Hz, 1H, ArH),
5.20 (s, 2H, aliphatic CH2); MS (Mwt.: 484.96): m/z (% rel.
int.) 486.05 (M+ + 2, 27.08), 485.05 (M+ + 1, 21.97), 484.05
(M+, 66.38), 359.05 (78.12), 125 (100); Anal. calcd for
C25H17ClN6OS: C, 61.92; H, 3.53; N, 17.33; found: C, 62.07; H,
3.59; N, 17.48.
General procedure for the preparation of 1-substituted
phenyl-3-IJ4-IJ(1-phenyl-1H-pyrazoloij3,4-d]pyrimidin-4-
yl)oxy)phenyl)urea (4a and b). To a solution of 1-IJ4-hydroxy-
phenyl)-3-phenylurea (3 mmol) in acetonitrile (20 ml), an equi-
molar amount of 4-chloro-1-IJ4-substituted phenyl)-1H-
pyrazoloij3,4-d]pyrimidine was added. A catalytic amount of
cesium carbonate was added to the mixture which was then
heated under reflux overnight. The reaction mixture was then
filtered while hot and the precipitate was crystallized from an
ethanol/water mixture to give the target compounds 4a and b.
1-Phenyl-3-IJ4-IJ(1-phenyl-1H-pyrazoloij3,4-d]pyrimidin-4-
yl)oxy)phenyl)urea (4a). Yield 23%; m.p. >300 °C; FT-IR
1
(ν max, cm−1): 3315 (NH), 1675 (CO), 1630 (CN); H-NMR
(300 MHz, DMSO-d6): δ ppm 8.80 (s, 1H, NH, D2O exchange-
able), 8.70 (s, 1H, NH, D2O exchangeable), 8.66 (s, 1H, hetero-
cyclic H), 8.41 (s, 1H, heterocyclic H), 8.18 (d, J = 7.4 Hz, 2H,
ArH), 7.63–7.56 (m, 4H, ArH), 7.48–7.42 (m, 3H, ArH), 7.31–
7.27 (m, 4H, ArH), 6.99 (t, J = 6.0 Hz, 1H, ArH); 13C-NMR (400
MHz, DMSO-d6): 164.10, 156.41, 155.18, 153.07, 146.68,
140.12, 138.73, 138.27, 133.69, 129.86, 129.27, 127.49, 122.68,
122.36, 121.65, 119.88, 118.73, 104.09; MS (Mwt.: 422.44): m/z
(% rel. int.) 423.20 (M+ + 1, 4.14), 422.20 (M+, 14.05), 329.10
(45.17), 303.15 (78.97), 93.10 (100); Anal. calcd for
C24H18N6O2: C, 68.24; H, 4.29; N, 19.89; found: C, 68.27; H,
4.34; N, 20.14.
1-IJ4-IJ(1-IJ4-Chlorophenyl)-1H-pyrazoloij3,4-d]pyrimidin-4-
yl)oxy)phenyl)-3-phenylurea (4b). Yield 16%; m.p. charring
at 230 °C; FT-IR (ν max, cm−1): 3300 (NH), 1675 (CO), 1625
(CN); 1H-NMR (300 MHz, DMSO-d6): δ ppm 10.37 (s, 1H,
NH, D2O exchangeable), 9.30 (s, 1H, NH, D2O exchangeable),
8.69 (s, 1H, heterocyclic H), 8.36 (s, 1H, heterocyclic H), 8.25
(d, J = 7.4 Hz, 2H, ArH), 7.69–7.64 (m, 4H, ArH), 7.55 (d, J =
6.0 Hz, 2H, ArH), 7.27–7.19 (m, 4H, ArH), 6.91 (t, J = 6.0 Hz,
1H, ArH); MS (Mwt.: 456.11): m/z (% rel. int.) 458.10 (M+ + 1,
10.64), 456.10 (M+, 32.15), 366.10 (41.23), 93.10 (100); Anal.
calcd for C24H17ClN6O2: C, 63.09; H, 3.75; N, 18.39; found: C,
63.17; H, 3.79; N, 18.51.
General procedure for the preparation of 3-substituted
benzyl-6-nitro-2,3-dihydrobenzoijd]thiazol-2-one (5a–d). To a
stirred solution of 6-nitro-3H-benzothiazol-2-one (2.943 g, 15
mmol) in acetone (22.5 ml), water (0.75 ml) and 85% KOH
(0.99 g, 30 mmol) were added followed by addition of the
appropriate substituted benzyl chloride (15 mmol) in one
portion. The reaction mixture was refluxed for 24 h, then
cooled to 5 °C after which 60 g of ice-cold water was added
and the mixture was stirred at 0–10 °C for 1 h. The obtained
solid was filtered, washed with cold water and diethyl ether,
dried at room temperature and crystallized from acetone/
water to give pure compounds 5a–d. 5a: R2 = 4-Cl; yield
95.5%; m.p. 110–112 °C. 5b: R2 = 2,4-diCl; yield 90%;
m.p. 136–137 °C. 5c: R2 = 3,4-diCl; yield 92%; m.p. 142–146 °C.
5d: R2 = 3-Br; yield 80%; m.p. 119–120 °C.
The analytical characterization data for compounds 7b–e
are provided in the ESI.‡
General procedure for the preparation of 4-IJ(4-substituted
benzyl)oxy)aniline (8a and b). A 4-nitrophenyl substituted
benzyl ether derivative (17.4 mmol) was dissolved in ethanol
(50 ml) before adding stannous chloride dihydrate (19.04 g,
84.4 mmol). The mixture was stirred under reflux for 4 h.
The reaction was monitored by TLC using dichloromethane
as an eluent. After completion of the reaction, the solvent
was evaporated under vacuum and the mixture was treated
with a concentrated aqueous solution of sodium bicarbonate
until the effervescence ceased. The reaction mixture was then
extracted with ethyl acetate (4 × 50 ml), and the organic layers
were collected, dried over anhydrous sodium sulfate, filtered
and evaporated to dryness. The obtained residue was crystal-
lized from methanol to give 8a and b. 8a: R3 = H; yield 97%;
m.p. 44–45 °C. 8b: R3 = Cl; yield 71%; m.p. 106–108 °C.
General procedure for the preparation of N-IJ4-IJ(4-
substituted benzyl)oxy)phenyl)-1-IJ4-substituted phenyl)-1H-
pyrazoloij3,4-d]pyrimidin-4-amine (9a–d). To a solution of
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4-IJ(4-substituted benzyl)oxy)aniline (8a and b, 3 mmol) in
ethanol (20 ml), an equimolar amount of 4-chloro-1-IJ4-
substituted phenyl)-1H-pyrazoloij3,4-d]pyrimidine (1a and b)
was added followed by the addition of 0.2 ml of triethylamine
and the mixture was stirred under reflux for 48 h. It was
followed by TLC till completion of the reaction. The reaction
mixture was then filtered while hot and the precipitate was
crystallized from an ethanol/water mixture to give the target
compounds 9a–d.
N-IJ4-IJBenzyloxy)phenyl)-1-phenyl-1H-pyrazoloij3,4-d]pyrimidin-
4-amine (9a). Yield 35%; m.p. 190–192 °C; FT-IR (ν max, cm−1):
3290 (NH), 1610 (CO), 1640 (CN); 1H-NMR (300 MHz,
DMSO-d6): δ ppm 10.09 (s, 1H, NH, D2O exchangeable), 8.46
(s, 2H, heterocyclic H), 8.20 (d, J = 6.0 Hz, 2H, ArH), 7.69 (d, J
= 7.6 Hz, 2H, ArH), 7.55 (t, J = 6.0 Hz, 2H, ArH), 7.48–7.33 (m,
6 H, ArH), 7.08 (d, J = 7.6 Hz, 2H, ArH), 5.13 (s, 2H, aliphatic
CH2); MS (Mwt.: 393.44): m/z (% rel. int.) 394.15 (M+ + 1,
38.16), 393.25 (M+, 50.03), 302.15 (100), 91.05 (69.89); Anal.
calcd for C24H19N5O: C, 73.27; H, 4.87; N, 17.80; found: C,
73.39; H, 4.84; N, 17.96.
d6): 165.29, 156.50, 154.76, 153.52, 141.92, 139.14, 134.20,
130.01, 129.69, 128.54, 126.91, 121.30, 120.43, 103.23, 48.76,
32.98, 25.77, 25.46; MS (Mwt.: 412.49): m/z (% rel. int.) 413.15
(M+ + 1, 14.30), 412.15 (M+, 49.23), 329.10 (100), 314.05
(72.52); Anal. calcd for C24H24N6O: C, 69.88; H, 5.86; N,
20.37; found: C, 70.03; H, 5.84; N, 20.45.
The analytical characterization data for compounds 11b–f
are provided in the ESI.‡
General procedure for the preparation of 5,6-disubsti-
tuted-N-IJ1-IJ4-substituted phenyl)-1H-pyrazoloij3,4-d]pyrimidin-
4-yl)benzoijd]thiazol-2-amine (13a–d). To a solution of 5,6-
disubstituted-benzoijd]thiazol-2-amine (12a–c, 5 mmol) in eth-
anol (20 ml), an equimolar amount of 4-chloro-1-IJ4-
substituted phenyl)-1H-pyrazoloij3,4-d]pyrimidine (1a and b)
was added after which 0.2 ml of triethylamine was added to
the mixture which was then stirred under reflux for 48 h. The
reaction was monitored by TLC till the reaction was finished.
Then, the reaction mixture was filtered while hot and the pre-
cipitate was crystallized from an ethanol/water mixture to
give the target compounds 13a–d.
The analytical characterization data for compounds 9b–d
are provided in the ESI.‡
6-Methyl-N-IJ1-phenyl-1H-pyrazoloij3,4-d]pyrimidin-4-yl)benzo-
ijd]thiazol-2-amine (13a). Yield 21.3%; m.p. >250 °C; FT-IR (ν
max, cm−1): 3310 (N–H), 2985 (aliphatic CH), 1680 (CN);
1H-NMR (300 MHz, DMSO-d6): δ ppm 12.82 (s, 1H, NH, D2O
exchangeable), 8.78 (s, 2H, heterocyclic H), 8.20 (d, J = 6.0
Hz, 2H, ArH), 7.78 (s, 1H, ArH), 7.69–7.55 (m, 3H, ArH), 7.40
(t, J = 6.0 Hz, 1H, ArH), 7.27 (d, J = 6.0 Hz, 1H, ArH), 2.37 (s,
3H, aliphatic CH3); 13C-NMR (400 MHz, DMSO-d6): 155.32,
153.45, 138.93, 134.49, 133.17, 132.68, 129.73, 127.89, 127.16,
121.59, 121.51, 103.40, 21.49; MS (Mwt.: 358.42): m/z (% rel.
int.) 360.10 (M+ + 2, 5.59), 359.05 (M+ + 1, 17.01), 358.15 (M+,
61.98), 77.05 (100); Anal. calcd for C19H14N6S: C, 63.67; H,
3.94; N, 23.45; found: C, 63.81; H, 3.87; N, 23.55.
General procedure for the preparation of 4-amino-N-
substituted benzamide (10a–d). Stannous chloride dihydrate
(0.39 g, 1.76 mmol) was added to a solution of N-substituted-
4-nitrobenzamide (9a–d, 0.59 mmol) in ethanol (8 ml) and
the reaction mixture was stirred under reflux for 3 h. The
resulting mixture was partitioned between ethyl acetate (50
ml) and an aqueous solution of sodium carbonate (5%, 30
ml); the organic layer was separated, washed with brine (30
ml), dried over anhydrous sodium sulfate, filtered and evapo-
rated under reduced pressure. The crude residue was purified
by crystallization using hexane/EtOAc to yield pure title com-
pounds 10a–d. 10a: R4 = cyclohexyl; yield 82%; m.p. 183–184
°C. 10b: R4 = 3-chlorophenyl; yield 86%; m.p. 142–144 °C.
10c: R4 = 2-chlorophenyl; yield 88%; m.p. 159–160 °C. 10d: R4
= 4-chloro-3-CF3phenyl; yield 44%; m.p. 178–180 °C.
The analytical characterization data for compounds 13b–d
are provided in the ESI.‡
3.2. Biological evaluation
General procedure for the preparation of N-substituted-
4-IJ(1-IJ4-substituted phenyl)-1H-pyrazoloij3,4-d]pyrimidin-4-
yl)amino)benzamide (11a–f). To a solution of 4-amino-N-
chlorobenzamide (10a–d, 3 mmol) in ethanol (20 ml), an
equimolar amount of 4-chloro-1-IJ4-substituted phenyl)-1H-
pyrazoloij3,4-d]pyrimidine (1a and b) was added after which
0.2 ml of triethylamine was added and the mixture was then
stirred under reflux for 72 h. The reaction mixture was fil-
tered while hot and the precipitate was crystallized from an
ethanol/water mixture to give the target compounds 11a–f.
N-Cyclohexyl-4-IJ(1-phenyl-1H-pyrazoloij3,4-d]pyrimidin-
4-yl)amino)benzamide (11a). Yield 67.3%; m.p. 272–273 °C; FT-IR
(ν max, cm−1): 2975 (aliphatic CH), 3310 (NH), 1705 (CO),
1670 (CN); 1H-NMR (300 MHz, DMSO-d6): δ ppm 10.38
(s, 1H, NH, D2O exchangeable), 8.61 (s, 2H, heterocyclic H),
8.21 (d, J = 7.6 Hz, 2H, ArH), 7.93 (dd, J = 6.0, 1.8 Hz, 4H,
ArH), 7.58 (t, J = 6.0 Hz, 2H, ArH), 7.37 (t, J = 6.0 Hz, 1H,
ArH), 3.78 (p, 1H, NH–CH–IJCH2)2), 1.82–1.59 (m, 6H, cyclo-
hexyl), 1.31 (p, 4H, cyclohexyl); 13C-NMR (400 MHz, DMSO-
3.2.1. Enzyme inhibition assay. The determination of
in vitro enzyme inhibition for the synthesized compounds
was carried out in KINEXUS Corporation, Vancouver, British
Columbia, Canada. Kinexus uses a radioactive assay format
for profiling evaluation of protein kinase targets and all
assays were performed in a designated radioactive working
area. Protein kinase assays were carried out at ambient tem-
perature for 30 minutes (depending on the target) in a final
volume of 25 μL according to the following assay reaction
receipt: 5 μL of diluted active protein kinase target (~10–50
nM final protein concentration in the assay), 5 μL of peptide
substrate, 5 μL of kinase assay buffer, 5 μL of compound (var-
ious concentrations) and 5 μL of 28P-ATP (250 μM stock
solution).
The assay was initiated by the addition of 28P-ATP and the
reaction mixture was incubated at 30 °C for 20–40 minutes
depending on the protein kinase target. After the incubation
period, the assay was terminated by spotting 10 μL of the
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reaction mixture onto a multiscreen phosphocellulose P81
plate. The multiscreen phosphocellulose P81 plate was
washed 3 times for approximately 15 minutes each in 1%
phosphoric acid solution. The radioactivity on the P81 plate
protein expression of this marker in the tested cells
according to the reported procedure43 and the experimental
part is provided in the ESI.‡
was counted in the presence of scintillation fluid in a TriLux Author contribution
scintillation counter. A blank control was set up for each pro-
MFT designed and performed cell culture studies, flow
tein kinase target, which included all the assay components
except the appropriate substrate (replaced with an equal vol-
ume of kinase assay buffer). The corrected activity for the
protein kinase target was determined by removing the blank
control value.40
cytometry, western blotting and immunocytochemistry exper-
iments as well as data analysis and writing. AE performed
SRB–MCF-7 cytotoxicity screening.
Acknowledgements
3.2.2. Antitumor activity screening
3.2.2.1. Screening at NIH, Bethesda, Maryland, USA. The
cytotoxicity of the synthesized compounds was assayed using
the standardized assay procedure of the National Cancer
Institute (NCI Bethesda, Maryland, USA).41
3.2.2.2. Cell culture. The experimental procedure of cell
culture assay is provided in the ESI.‡
3.2.2.3. SRB cytotoxicity assay. SRB assay was performed
according to the reported procedure42 and the experimental
part is provided in the ESI.‡
The authors would like to thank the Science and Technology
Development Fund (STDF) for funding the center for drug dis-
covery and development research at the Faculty of Pharmacy
Ain Shams University (Project No. 5251) and for partial
funding of this study. The authors are also grateful for the
National Cancer Institute (NCI), Bethesda, Maryland, USA, for
performing the in vitro anticancer testing for the selected
compounds over their panel of cell lines.
3.2.2.4. DNA-flow cytometry analysis. A549 cells at a density
of 2 × 105 cells were exposed to the tested compounds at
their IC50 concentration for 48 h. The cells were collected by
trypsinization, washed in PBS and then fixed in ice-cold abso-
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