Novel pyrazolo[3,4-d]pyrimidines: design, synthesis, anticancer activity, dual EGFR/ErbB2 receptor tyrosine kinases inhibitory activity, effects on cell cycle profile and caspase-3-mediated apoptosis

Article abstract of DOI:10.1080/14756366.2018.1564046

A series of novel pyrazolo[3,4-d]pyrimidines was synthesised. Twelve synthesised compounds were evaluated for their anticancer activity against 60 human tumour cell lines by NCI (USA). Compound 7d proved prominent anticancer activity. It showed 1.6-fold more potent anti-proliferative activity against OVCAR-4 cell line with IC₅₀ = 1.74 μM. It also exhibited promising potent anticancer activity against ACHN cell line with IC₅₀ value 5.53 μM, representing 2.2-fold more potency than Erlotinib. Regarding NCI-H460 cell line, compound 7d (IC₅₀ = 4.44 μM) was 1.9-fold more potent than Erlotinib. It inhibited EGFR and ErbB2 kinases at sub-micromolar level (IC₅₀ = 0.18 and 0.25 μM, respectively). Dual inhibition of EGFR and ErbB2 caused induction of apoptosis which was confirmed by a significant increase in the level of active caspase-3 (11-fold). It showed accumulation of cells in pre-G1 phase and cell cycle arrest at G2/M phase.

Full text of DOI:10.1080/14756366.2018.1564046

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Novel pyrazolo[3,4-d]pyrimidines: design,
synthesis, anticancer activity, dual EGFR/ErbB2
receptor tyrosine kinases inhibitory activity,
effects on cell cycle profile and caspase-3-
mediated apoptosis
Mai Maher, Asmaa E. Kassab, Ashraf F. Zaher & Zeinab Mahmoud
To cite this article: Mai Maher, Asmaa E. Kassab, Ashraf F. Zaher & Zeinab Mahmoud
(2019) Novel pyrazolo[3,4-d]pyrimidines: design, synthesis, anticancer activity, dual EGFR/
ErbB2 receptor tyrosine kinases inhibitory activity, effects on cell cycle profile and caspase-3-
mediated apoptosis, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 532-546, DOI:
10.1080/14756366.2018.1564046
To link to this article: https://doi.org/10.1080/14756366.2018.1564046
©
2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 27 Jan 2019.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
019, VOL. 34, NO. 1, 532–546
2
https://doi.org/10.1080/14756366.2018.1564046
RESEARCH PAPER
Novel pyrazolo[3,4-d]pyrimidines: design, synthesis, anticancer activity, dual EGFR/
ErbB2 receptor tyrosine kinases inhibitory activity, effects on cell cycle profile and
caspase-3-mediated apoptosis
Mai Maher, Asmaa E. Kassab, Ashraf F. Zaher and Zeinab Mahmoud
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
ABSTRACT
ARTICLE HISTORY
A series of novel pyrazolo[3,4-d]pyrimidines was synthesised. Twelve synthesised compounds were eval-
uated for their anticancer activity against 60 human tumour cell lines by NCI (USA). Compound 7d proved
prominent anticancer activity. It showed 1.6-fold more potent anti-proliferative activity against OVCAR-4
cell line with IC50 ¼ 1.74 lM. It also exhibited promising potent anticancer activity against ACHN cell line
with IC50 value 5.53 lM, representing 2.2-fold more potency than Erlotinib. Regarding NCI-H460 cell line,
compound 7d (IC50 ¼ 4.44 lM) was 1.9-fold more potent than Erlotinib. It inhibited EGFR and ErbB2 kin-
ases at sub-micromolar level (IC50 ¼ 0.18 and 0.25 mM, respectively). Dual inhibition of EGFR and ErbB2
caused induction of apoptosis which was confirmed by a significant increase in the level of active cas-
pase-3 (11-fold). It showed accumulation of cells in pre-G1 phase and cell cycle arrest at G2/M phase.
Received 29 November 2018
Revised 12 December 2018
Accepted 14 December 2018
KEYWORDS
Pyrazolo[3,4-d]pyrimidines;
design; synthesis; anticancer
activity; EGFR; ErbB2;
caspase-3; cell cycle arrest
profile; apoptosis
1
. Introduction
adenosine triphosphate (ATP) to block the catalytic domain of
these receptors. They had been approved for the chemothera-
peutic treatment of cancer patients. The main structural features
of these drugs are quinazoline scaffold containing a substituted
phenyl amino pyrimidine moiety. Purine is a heterocyclic nucleus
which exists in the chemical architecture of various bioactive
compounds. It is an important pharmacophore, which is widely
used in the development of protein kinase inhibitors via introduc-
The EGFR family comprises four distinct membrane tyrosine kin-
ase receptors: (EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3 and HER4/
ErbB4) located in the plasma membrane and activated in
response to ligand binding . The interaction of EGFRs with the
ligands causes receptor homo- and hetero-dimerisation, this inter-
action initiates a cascade of events that control diverse biological
processes such as proliferation, differentiation, migration and
apoptosis. Thus, deregulation of this transduction pathway has
been implicated in many types of human cancers and is associ-
ated with poor clinical prognosis . Moreover, members of EGFR
family have been shown to be oncogenic. High expression levels
of EGFR and ErbB2 has been implicated in the development of
various types of cancers including breast, lung, colorectal, ovar-
ian, prostate, head and neck . EGFR and ErbB2 pathways are
interconnected since both receptors have the highest homology
among the EGFR family members in their kinase catalytic
domains and share many similar biochemical and kinetic proper-
1
,2
1
5
tion of substituents on the 2, 6 and 9 positions . Pyrazolo[3,4-
d]pyrimidine as a bioisostere of purine has drawn a considerable
attention as a privileged scaffold for the design and discovery of
2
–5
1
6,17
novel anticancer agents
. In addition, pyrazolo[3,4-d]pyrimi-
dine or its bioisostere pyrrolo[2,3-d]pyrimidine are common struc-
tural motifs of potent dual EGFR/ErbB2 inhibitors such as
1
8,
19
20
21
6
–8
PKI166 I (AEE788) , II and III (Figure 1). Motivated by all
these findings, we have designed and synthesised a series of pyr-
azolo[3,4-d]pyrimidines as novel small molecules targeting both
EGFR and ErbB2 tyrosine kinases to be useful for treatment of
cancer via inhibition of cell growth and induction of apoptosis.
Our strategy was directed towards carrying out the chemical
modifications on the general features of anilinoquinazoline scaf-
fold to substantiate the effect of such modifications on the anti-
9
ties . Additionally, the use of multiple ErbB receptors for cell pro-
liferation and survival in tumour cells and the synergistic
transforming effects of EGFR and ErbB2 lead to the hypothesis
that targeting both the EGFR and ErbB2 catalytic domains simul-
taneously through dual EGFR/ErbB2 inhibition would have super- cancer activity and to identify potent anticancer agents (Figure
ior therapeutic effects relative to single-agent treatment for 2). Initially, we aimed to replace the benzene moiety in the qui-
1
0
cancer . Furthermore, a multi-targeted approach may improve nazoline skeleton by an isostere (pyrazole one). This respected
the outcome of anti-EGFR therapies since the blockade of EGFR nucleus is always bearing a 4-fluorophenyl group at N1 since flu-
by EGFR tyrosine kinase inhibitors is insufficient to eradicate orinated compounds are one of the research hotspots in modern
2
2
established tumors because of independently activated survival medicinal chemistry . Moreover, incorporation of a fluorine atom
1
1
12
13
pathways . Erlotinib (TarcevaTM) , Gefitinib (IressaTM)
and provides compounds with enhanced both pharmacokinetic and
TM 14
Lapatinib (Tykerb
)
(Figure 1) are low-molecular-weight dual physicochemical properties as compared to their non-fluorinated
2
3,24
inhibitors of EGFR/ErbB2 tyrosine kinases that compete with analogs
. The second modification focused on introducing
CONTACT Asmaa E. Kassab
1562, Egypt
asmaa.kassab@pharma.cu.edu.eg
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Cairo University, Cairo
1
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
533
SO CH
2
3
CH
C
O
HN
F
O
HN
N
Cl
HN
O
N
O
O
N
O
N
Erlotinib (Tarceva TM
Lapatinib (Tykerb TM
)
)
F
O
NH
Cl
NH
N
N
O
N
HO
O
N
N
N
H
Gefitinib (Iressa TM
PKI 166
)
N
NH
N
N
N
N
H
N
I (AEE788)
HO
Cl
NH
O
O
Cl
N
F
NH2
NH
N
O
N
N
N
N
N
N
N
H
II
III
Structural analysis
N
Quinazoline scaffold or its bioisosteres pyrrolo[2,3-d]pyrimidine
or pyrazolo[3,4-d]pyrimidine carrying substituted phenylamino
moiety or amino group at at position 4 of pyimidine
N
Figure 1. Examples of dual EGFR/ErbB2 inhibitors.
various phenyl amino groups on the pyrimidine moiety. We have introduced at C-6 position of pyrazolopyrimidine core. Twelve of
introduced unsubstituted phenyl amino group, phenyl amino the newly synthesised pyrazolopyrimidines were subjected to in
group substituted with electron donating groups or phenyl vitro anticancer screening by the National Cancer Institute (USA)
amino group substituted with electron withdrawing groups. The against 60 different human cell lines. The most potent compound
third modification included incorporating the phenyl amino was selected to be further studied through determination of its
group to the pyrimidine nucleus through a spacer such as azo- half maximal inhibitory concentration (IC ) values against ovarian
5
0
methine group or piperazinyl linker. In the fourth modification, cancer OVCAR-4, lung cancer NCI-H460, NCI-H226 and renal can-
we have focused on replacement of the phenyl amino group by cer ACHN cell lines. In order to explore the mechanistic pathways
small pharmacophoric moieties as carbonyl, amino, morpholine, of the anticancer activity of 7d, it was evaluated in EGFR, ErbB2
4
-methylpiperazine or hydrazinyl groups. These groups at such and active caspase-3 assays. Moreover, we also investigated its
position are well acknowledged for the anticancer activity of the effect on the normal cell cycle profile and induction of apoptosis
25,26
fused pyrimidine rings
. Finally, additional amino group was in the OVCAR-4 cell line.

5
34
M. MAHER ET AL.
SO CH
CH
C
2
3
O
HN
F
HN
N
O
HN
N
Cl
O
O
O
O
N
N
Erlotinib (Tarceva TM
)
Lapatinib (Tykerb TM
)
Isosteric replacemant of benzene
ring with pyrazole
R
R
R
N
NH
N
NH
N
N
N
N
N
N
N
N
N
N
N
N
N
F
7
F
10
F
8
Keeping the phenylamino-
pyrimidine moiety
Incorporation of the phenylamino-
pyrimidine moiety with azomethine spacer
Incorporation of the phenylamino-
pyrimidine moiety with piperazinyl spacer
O
NH2
NH2
N
HN
NH2
NH
N
N
N
N
N
N
N
N
N
N
N
N
N
N
NH2
F
3
F
4
F
F
9
5
Replacing the phenylamino
moiety with carbonyl group
Replacing the phenylamino
moiety with amino group
Replacing the phenylamino
moiety with hydrazinyl group
Replacing the phenylamino
moiety with amino group and introducing
additional amino group at C-6
Figure 2. Design strategy for the target pyrazolo[3,4-d]pyrimidines.
Experimental part
with UV fluorescent silica gel (Merck 60 F 254) and visualised using
UV lamp. Solvent system used was ethyl acetate, hexane with dif-
ferent ratios.
General
All melting points were determined with Stuart SMP10 apparatus
ꢀ
1
and the values given are uncorrected. IR spectra (KBr, cm ) were
determined on Shimadzu IR 8400 s spectrophotometer (Faculty of
Pharmacy, Cairo University, Egypt).1H NMR and 13 C NMR spectra
were recorded on Bruker 400-BB 400 MHz spectrometers
1
–(4-Fluorophenyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-
one (3)
A well-stirred solution of compound 2 (1.08 g, 0.005 mol) in formic
(Microanalytical Unit, Faculty of Pharmacy, Cairo University, Egypt) acid (20 ml) was heated under reflux for 12 h. After cooling down,
using tetramethylsilane (TMS) as an internal standard. Chemical the formed precipitate was filtered, washed twice with water,
shift values are recorded in ppm on d scale. Mass spectra were dried and then recrystallised from absolute ethanol to afford 3.
ꢁ
ꢀ1
recorded on ISQLT single quadrapole mass spectrometer (at the Yield: 41%; mp: >300 C; IR (KBr) cm : 3122 (NH str), 3055, 3010
Regional Center for Mycology and Biotechnology, Microanalytical (CH aromatic str), 2978, 2920, 2872 (CH aliphatic str), 1697 (C¼O
1
Center, Al-Azhar University, Egypt). Elemental analyses were car- str), 1598, 1521 (C¼N, C¼C aromatic str); H NMR (DMSO-d ppm)
6
ried out at the Regional center for Mycology and Biotechnology, d: 2.52 (s, 3H, CH ), 7.36–7.40 (dd, 2H, J ¼ 8.84 Hz), 8.03–8.05 (dd,
3
Faculty of Pharmacy, Al-Azhar University, Egypt. The reactions pro- 2H, J ¼ 4.88 Hz, J ¼ 9 Hz, ArH), 8.14 (s, 1H, C6-H), 12.36 (s, 1H, NH,
gression was monitored by TLC using aluminum sheets precoated
2 6 3
D O exchangeable); 13 C NMR (DMSO-d ppm) d: 13.7 (CH ), 106.0,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
535
1
1
1
16.27, 116.50 (d, J ¼ 23 Hz), 123.81, 123.89 (d, J ¼ 8 Hz) 124.0, ArH), 8.91 (s, 1H, C6-H); 13 C NMR (DMSO-d
6 3
ppm) d: 14.3 (CH ),
35.11, 135.13 (d, J ¼ 2 Hz) 146.4, 149.5, (d, J ¼ 308 Hz, C-F), 152.6, 113.5, 116.57, 116.80 (d, J ¼ 23 Hz), 123.7, 134.5, 140.3, 143.7,
.
þ
58.4, 159.5 (Ar Cs), 161.9 (C¼O); MS (m/z, %): 244 [M , 27.56], 95 153.60, 156.01 (d, J ¼ 241 Hz, C-F), 156.4, 162.8 (Ar Cs). Anal. Calcd
eþ
[100.00](C H F ). Anal. Calcd for C H FN O (244.22): C, 59.01; H, for C H ClFN (262.67): C, 54.87; H, 3.07; N, 21.33. Found: C,
6
4
12
9
4
12
8
4
3
.71; N, 22.94. Found: C, 59.17; H, 3.78; N, 23.12.
55.12; H, 3.14; N, 21.59.
1
–(4-Fluorophenyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-
General procedure for the preparation of compounds (7a–d)
A mixture of the chloro derivative 6 (0.3 g, 0.001 mol) and the
amine (4)
A solution of compound 2 (0.3 g, 0.001 mol) in formamide (10 ml) selected aromatic amine (0.001 mol) was dispersed in isopropanol
was stirred while heated under reflux for 4 h. The reaction mixture (2 ml). The reaction mixture was heated under reflux for 6 h. The
was cooled, the formed solid was filtered, washed with aqueous reaction mixture was cooled. The obtained solid was filtered, dried
ethanol, dried and recrystallised from ethanol to give 4. Yield:
and recrystallised from ethanol to afford 7a–d.
ꢁ
ꢀ1
6
8%; mp: >300 C; IR (KBr) cm : 3498, 3394 (NH
2
str), 3124, 3055
(
CH aromatic str), 2924 (CH aliphatic str), 1597, 1450 (C¼N, C¼C
1
aromatic str); H NMR (DMSO-d6 ppm) d: 2.62 (s, 3H, CH ), 1–(4-Fluorophenyl)-3-methyl-N-phenyl-1H-pyrazolo[3,4-d]pyrimi
3
7
D
(
1
1
1
(
2
.33–7.38 (dd, 2H, J ¼ 8.80 Hz, J ¼ 8.92 Hz, ArH), 7.47 (br s, 2H, NH , din-4-amine (7a)
2
2
O exchangeable), 8.16–8.21 (dd, 2H, J ¼ 5 Hz, J ¼ 9 Hz, ArH), 8.26
ꢁ
ꢀ1
Yield: 75%; mp: 164–166 C; IR (KBr) cm : 3444 (NH str), 3082
s, 1H, C6-H); 13 C NMR (DMSO-d
6
ppm) d: 14.8 (CH
3
), 100.6,
(
(
CH aromatic str), 2920, 2850 (CH aliphatic str), 1570, 1515, 1496
C¼N, C¼C aromatic str); H NMR (DMSO-d ppm) d: 2.78 (s, 3H,
16.11, 116.34 (d, J ¼ 23 Hz), 122.55, 122.63 (d, J ¼ 8 Hz), 135.79,
1
6
35.81 (d, J ¼ 2 Hz), 143.3, 154.50, 157.06 (d, J ¼ 265 Hz), 158.8,
3
CH ), 7.16–7.20 (t, 1H, ArH), 7.37–7.43 (m, 4H, ArH), 7.70 (d, 2H,
.
þ
59.0, 161.2 (Ar Cs); MS (m/z, %): 243 [M , 61.00], 95 [100.00]
J ¼ 8 Hz, ArH), 8.18–8.21 (dd, 2H, J ¼ 4.9 Hz, J ¼ 8 Hz, ArH), 8.43 (s,
eþ
6
C H
4
F
). Anal. Calcd for C12H10FN
5
(243.24): C, 59.25; H, 4.14; N,
1
H, C6-H), 8.83 (s, 1H, NH, D O exchangeable); 13 C NMR (DMSO-
2
8.79. Found: C, 59.48; H, 4.16; N, 29.05.
d ppm) d: 15.1 (CH ), 101.8, 116.21, 116.43 (d, J ¼ 22 Hz), 122.89,
6
3
1
1
%
22.97 (d, J ¼ 8 Hz), 123.8, 124.7, 128.9, 135.5, 138.9, 142.9, 154.4,
56.0, 156.44, 159.07 (d, J ¼ 263 Hz, C-F), 161.4 (Ar Cs); MS (m/z,
1
–(4-Fluorophenyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidine-4,6-
.þ
): 319 [M , 8.22], 71 [100.00]. Anal. Calcd for C H FN (319.34):
diamine (5)
18 14 5
C, 67.70; H, 4.42; N, 21.93. Found: C, 67.96; H, 4.39; N, 22.19.
Guanidine HCl (2.1 g, 0.022 mol) was added to a solution of
sodium (2.6 g, 0.113 mol) in methanol (95 ml), the precipitated
sodium chloride was filtered off. Compound 2 (3.24 g, 0.015 mol) N,1-Bis(4-fluorophenyl)-3-methyl-1H-pyrazolo[3,4-d]-pyrimidin-4-
was added to the filtrate. The reaction mixture was heated under amine (7b)
reflux for 8 h. The mixture was cooled to room temperature. The
ꢁ
ꢀ1
Yield: 75%; mp: 206–208 C; IR (KBr) cm : 3444 (NH str), 3097,
formed solid was filtered, washed with aqueous ethanol and
ꢁ
3059 (CH aromatic str), 2916, 2850 (CH aliphatic str), 1593, 1566,
recrystallised from ethanol to yield 5.Yield: 83%; mp: >300 C; IR
1
ꢀ
1
1512 (C¼N, C¼C aromatic str); H NMR (DMSO-d
6
ppm) d: 2.77
(KBr) cm : 3392–3215 (2 NH
2
str), 3001 (CH aromatic str), 2962,
(
s, 3H, CH ), 7.21–7.26 (dd, 2H, J ¼ 8.8 Hz, J ¼ 8.8 Hz, ArH),
3
2
927, 2854 (CH aliphatic str), 1593, 1560, 1508 (C¼N, C¼C aro-
1
7.35–7.39 (dd, 2H, J ¼ 8.8 Hz, J ¼ 8.9 Hz, ArH), 7.67–7.71 (dd, 2H,
J ¼ 5 Hz, J ¼ 8.9 Hz, ArH), 8.16–8.20 (dd, 2H, J ¼ 4.9 Hz, J ¼ 7 Hz,
matic str); H NMR (DMSO-d ppm) d: 2.37 (s, 3H, CH ), 7.29–7.38
6
3
(
dd, 2H, J ¼ 8.8 Hz, J ¼ 8.9 Hz, ArH), 7.55 (s, 2H, NH , D O exchange-
2
2
2
ArH), 8.39 (s, 1H, C6-H), 8.85 (s, 1H, NH, D O exchangeable); 13 C
able), 8.05–8.08 (dd, 1H, J ¼ 4.9 Hz, J ¼ 8.9 Hz, ArH), 8.31–8.36 (dd,
NMR (DMSO-d
6 3
ppm) d: 15.2 (CH ), 101.7, 115.38 115.60 (d,
1
1
H, J ¼ 4.9 Hz, J ¼ 8.9 Hz, ArH), 8.34 (s, 2H, NH
2 2
, D O exchangeable);
J ¼ 22 Hz), 116.2, 116.46 (d, J ¼ 22 Hz), 122.91, 122.99 (d, J ¼ 8 Hz),
3 C NMR (DMSO-d ppm) d: 13.8 (CH ), 104.0, 116.02, 116.25 (d,
6
3
1
23.0, 126.09, 126.17, (d, J ¼ 8 Hz) 135.2, 135.5, 143.0, 154.4, 156.1,
56.4, 158.20, 160.5 (d, J ¼ 239 Hz, C-F), 159.08, 161.49 (d,
J ¼ 23 Hz), 116.55, 116.77 (d, J ¼ 22 Hz), 122.9, 127.01, 127.10 (d,
J ¼ 8 Hz), 135.8, 145.8, 159.2, 160.4, 167.4, 176.5 (Ar Cs); MS (m/z,
1
.þ
.þ
eþ
J ¼ 241 Hz, C-F) (Ar Cs); MS (m/z, %): 337 [M , 96.15], 336 [100.00]
%
): 258 [M , 99.78], 43 [100.00] (CH N ). Anal. Calcd for
3
eþ
(
M-H ). Anal. Calcd for C18
0.76. Found: C, 64.18; H, 3.94; N, 20.98.
13
H F
N
2 5
(337.33): C, 64.09; H, 3.88; N,
12
C H
11FN
6
(258.25): C, 55.81; H, 4.29; N, 32.54. Found: C, 56.04; H,
2
4
.35; N, 32.79.
1
–(4-Fluorophenyl)-3-methyl-N-(p-tolyl)-1H-pyrazolo[3,4-d]-
4
-Chloro-1–(4-fluorophenyl)-3-methyl-1H-pyrazolo[3,4-
pyrimidin-4-amine (7c)
d]pyrimidine (6)
ꢁ
ꢀ1
Yield: 70%; mp: 203–205 C; IR (KBr) cm : 3444 (NH str), 3012 (CH
Compound 3 (0.55 g, 0.0023 mol) was mixed with phosphorus oxy-
chloride (80 ml). Ten drops of DMF was then added dropwise to
this mixture. The resulting mixture was heated under reflux with
aromatic str), 2920, 2850 (CH aliphatic str), 1566, 1512, 1450 (C¼N,
1
C¼C aromatic str); H NMR (DMSO-d
6
ppm) d: 2.32 (s, 3H, benzylic
stirring for 15 h. The reaction mixture was cooled, poured on ice- CH
), 2.76 (s, 3H, CH
3
), 7.22 (d, 2H, J ¼ 8 Hz, ArH), 7.36–7.40 (dd,
3
cold water (50 ml) with continuous stirring and sprinkling of 2H, J ¼ 8.8 Hz, J ¼ 8.9 Hz, ArH), 7.56 (d, 2H, J ¼ 8 Hz, ArH), 8.17 –
sodium carbonate till neutralisation. The precipitated product was 8.21 (dd, 2H, J ¼ 4.9 Hz, J ¼ 8.9 Hz, ArH), 8.39 (s, 1H, C6-H), 8.75
filtered, washed several times with aqueous ethanol, dried and (s, 1H, NH, D
O exchangeable); 13 C NMR (DMSO-d
ppm) d: 15.2
), 101.6, 116.21, 116.43 (d, J ¼ 22 Hz),
78–180 C; IR (KBr) cm : 3067 (CH aromatic str), 2950, 2916 (CH 122.86, 122.94 (d, J ¼ 8 Hz), 124.0, 129.3, 129.8, 133.9, 135.56,
2
6
3 3
recrystallised from absolute ethanol to afford 6. Yield: 71%; mp: (CH ), 21.0 (benzylic CH
ꢁ
ꢀ1
1
1
aliphatic str), 1577, 1550 (C¼N, C¼C aromatic str); H NMR 135.59 (d, J ¼ 3 Hz), 136.3, 142.9, 154.4, 156.1, 156.4, 159.05, 161.46
.
þ
(
DMSO-d
6
ppm) d: 2.74 (s, 3H, CH
3
), 7.45 (d, 2H, ArH), 8.14 (d, 2H, (d, J ¼ 241 Hz, C-F) (Ar Cs); MS (m/z, %): 333 [M , 70.98], 71

5
36
M. MAHER ET AL.
[
2
100.00]. Anal. Calcd for C19
1.01. Found: C, 68.70; H, 4.91; N, 21.37.
H
16FN
5
(333.36): C, 68.46; H, 4.84; N, 4–(4-(4-Chlorophenyl)piperazin-1-yl)-1–(4-fluorophenyl)-3-
methyl-1H-pyrazolo[3,4-d]pyrimidine (8c)
ꢁ
ꢀ1
Yield: 80%; mp: 167–169 C; IR (KBr) cm : 3043 (CH aromatic str),
920, 2850 (CH aliphatic str), 1600, 1566, 1512, 1458 (C¼N, C¼C
2
N-(3-Chloro-4-fluorophenyl)-1–(4-fluorophenyl)-3-methyl-1H-
pyrazolo[3,4-d] pyrimidin-4-amine (7d)
1
aromatic str); H NMR (DMSO-d ppm) d: 2.67 (s, 3H, CH ),
6
3
3
7
8
2
.20–3.35 (m, 8H, 4CH of piperazine), 6.99–7.03 (m, 2H, ArH),
ꢁ
ꢀ1
Yield: 80%; mp: 234–236 C; IR (KBr) cm : 3444 (NH str), 3100,
.07–7.11 (dd, 2H, J ¼ 5 Hz, J ¼ 9 Hz, ArH), 7.36–7.42 (m, 2H, ArH),
3
1
2
7
7
016 (CH aromatic str), 2954, 2920, 2850 (CH aliphatic str), 1570,
.13–8.18 (dd, 2H, J ¼ 5 Hz, J ¼ 9 Hz, ArH), 8.42 (s, 1H, C6-H); 13 C
1
512, 1465 (C¼N, C¼C aromatic str); H NMR (DMSO-d
6
ppm) d:
6 3 2
NMR (DMSO-d ppm) d: 17.5 (CH ), 45.6, 46.6, 48.1, 49.4 (4CH of
.78 (s, 3H, CH
3
), 7.36–7.40 (dd, 2H, J ¼ 8.80 Hz, J ¼ 8.84 Hz, ArH),
piperazine), 102.6, 116.2 (d, C-F), 118.4, 123.3, 123.8, 135.1, 142.1,
43.2, 146.4, 148.0, 149.5, 152.6, 155.3, 156.6, 158.3, 159.9,164.5
.42–7.47 (dd, 1H, J ¼ 9 Hz, J ¼ 9.1 Hz, ArH), 7.70–7.74 (m, 1H, ArH),
.99–8.01 (dd, 1H, J ¼ 6.8 Hz, J ¼ 2.4 Hz, ArH), 8.16–8.19 (dd, 2H,
1
.þ
(
Ar Cs); MS (m/z, %): 422 [M , 0.95], 256 [100.00]. Anal. Calcd for
20ClFN (422.89): C, 62.48; H, 4.77; N, 19.87. Found: C, 62.75;
H, 4.84; N, 19.65.
J ¼ 4.9 Hz, J ¼ 8.8 Hz, ArH), 8.46 (s, 1H, C6-H), 8.90 (s, 1H, NH, D O
2
C
22
H
6
exchangeable); 13 C NMR (DMSO-d6 ppm) d: 15.2 (CH ), 101.8,
3
1
1
1
1
06.0, 116.26, 116.48 (d, J ¼ 22 Hz), 116.82, 117.04 (d, J ¼ 22 Hz),
19.13, 119.31 (d, J ¼ 18 Hz), 122.97, 123.05 (d, J ¼ 8 Hz), 123.82,
23.91 (d, J ¼ 9 Hz), 124.19, 124.26 (d, J ¼ 7 Hz), 125.3, 135.4, 136.2,
42.9, 153.07, 155.72 (d, J ¼ 265 Hz, C-F), 154.37, 156.34 (d,
1
–(4-Fluorophenyl)-4–(4-(4-methoxyphenyl)piperazin-1-yl)-3-
.
þ
methyl-1H-pyrazolo[3,4-d]pyrimidine (8d)
J ¼ 197 Hz) (Ar Cs); MS (m/z, %): 371 [M , 0.87], 258 [100.00]. Anal.
ꢁ
ꢀ1
Calcd for C18H12ClF N
2 5
(371.77): C, 58.15; H, 3.25; N, 18.84. Found: Yield: 96%; mp: 188–190 C; IR (KBr) cm : 3082, 3059 (CH aro-
C, 58.43; H, 3.30; N, 19.12.
matic str), 2954, 2920, 2850 (CH aliphatic str), 1558, 1508, 1473
1
(
C¼N, C¼C aromatic str); H NMR (DMSO-d
CH ), 3.20 (t, 4H, 2CH of piperazine), 3.70 (s, 3H, OCH
H, 2CH2 of piperazine), 6.86 (d, 2H, J ¼ 9 Hz, ArH), 6.97 (d, 2H,
6
ppm) d: 2.68 (s, 3H,
3
2
3
), 3.90 (t,
General procedure for the preparation of compounds (8a–d)
4
Compound 6 (0.262 g, 0.001 mol) was added to the selected sub- J ¼ 9 Hz, ArH), 7.37 – 7.41 (t, 2H, J ¼ 8.8 Hz, ArH), 8.15–8.18 (dd, 2H,
stituted amine (0.001 mol) and triethyl amine (0.1 g, 0.001 mol) in J ¼ 4.9 Hz, J ¼ 9 Hz, ArH), 8.42 (s, 1H, C6-H); 13 C NMR (DMSO-d
absolute ethanol (18 ml). The reaction mixture was heated under ppm) d: 17.5 (CH ), 48.3 (OCH ), 50.2 (2CH of piperazine), 55.5 (2
6
3
3
2
reflux for 15 h. After cooling down to room temperature, the pre- CH2 of piperazine), 102.6, 108.0, 114.8, 116.18, 116.40 (d,
cipitated product was filtered, dried and recrystallised from etha- J ¼ 22 Hz), 118.3, 123.23, 123.31 (d, J ¼ 8 Hz), 135.3, 138.2, 142.1,
nol to obtain compounds 8a–d.
145.5, 150.3, 153.75, 155.23 (d, J ¼ 248 Hz, C-F), 155.3, 159.9 (Ar
.þ
Cs); MS (m/z, %): 418 [M , 20.75], 256 [100.00]. Anal. Calcd for
C H FN O (418.47): C, 66.01; H, 5.54; N, 20.08. Found: C, 66.23; H,
2
3
23
6
1
–(4-Fluorophenyl)-3-methyl-4-morpholino-1H-pyrazolo[3,4-
5
.60; N, 20.37.
d]pyrimidine (8a)
ꢁ
ꢀ1
Yield: 75%; mp: 160–162 C; IR (KBr) cm : 3041, 3014 (CH aro-
1
–(4-Fluorophenyl)-4-hydrazinyl-3-methyl-1H-pyrazolo[3,4-d]-
matic str), 2978, 2963, 2948, 2926 (CH aliphatic str), 1560, 1512,
1
pyrimidine (9)
1
3
2
479 (C¼N, C¼C aromatic str); H NMR (DMSO-d ppm) d: 2.62 (s,
6
H, CH ), 3.74–3.78 (m, 8H, 4CH of morpholine), 7.35–7.40 (dd,
3
2
A mixture of compound 6 (0.289 g, 0.0011 mol) and hydrazine
hydrate (99%, 0.6 g, 0.012 mol) in absolute ethanol (20 ml) was
heated under reflux with stirring for 8 h. The obtained solution
was left to cool down. The produced precipitate was filtered, dried
H, J ¼ 8.8 Hz, J ¼ 8.9 Hz, ArH), 8.13–8.17 (dd, 2H, J ¼ 4.9 Hz,
J ¼ 8.9 Hz, ArH), 8.40 (s, 1H, C6-H); 13 C NMR (DMSO-d ppm) d:
6
1
7.4 (CH ), 48.7, 66.4 (4CH of morpholine), 102.4, 116.08, 116.31
3 2
(
d, J ¼ 23 Hz), 123.06, 123.14 (d, J ¼ 8 Hz), 135.36, 135.39 (d,
and recrystallised from 96% ethanol to yield 9. Yield: 69%; mp:
J ¼ 3 Hz), 141.9, 155.0, 155.2, 159.09, 159.9, 161.51 (d, J ¼ 242 Hz,
ꢀ1
ꢁ
2
08–210 C; IR (KBr) cm : 3309–3120 (NH, NH
2
str), 3047, 3008
.
þ
eþ
3 5
H O ).
C-F) (Ar Cs); MS (m/z, %): 313 [M , 7.01], 57 [100.00] (C
Anal. Calcd for C16 O (313.33): C, 61.33; H, 5.15; N, 22.35.
Found: C, 61.71; H, 5.19; N, 22.52.
(
CH aromatic str), 2962, 2850 (CH aliphatic str), 1593, 1512 (C¼N,
H
16FN
5
1
C¼C aromatic str); H NMR (DMSO-d ppm) d: 2.62 (s, 3H, CH ),
6
3
4
.80 (s, 2H, NH , D O exchangeable), 7.32–7.38 (dd, 2H, J ¼ 7 Hz,
2
2
J ¼ 9 Hz, ArH), 8.14–8.19 (dd, 2H, J ¼ 5 Hz, J ¼ 9 Hz, ArH), 8.35 (s,
1H, C6-H), 8.90 (s, 1H, NH, D O exchangeable); 13 C NMR (DMSO-
ppm) d: 15.4 (CH
1
–(4-Fluorophenyl)-3-methyl-4–(4-methylpiperazin-1-yl)-1H-
2
pyrazolo[3,4-d]pyrimidine (8b)
d
6
3
), 99.7, 116.11, 116.33 (d, J ¼ 22 Hz), 122.76,
1
1
22.84 (d, J ¼ 8 Hz), 135.70, 135.72 (d, J ¼ 2 Hz), 142.8, 153.8,
56.49, 158.2, 158.94 (d, J ¼ 245 Hz, C-F), 161.3 (Ar Cs); MS (m/z,
ꢁ
ꢀ1
Yield: 88%; mp: 253–255 C; IR (KBr) cm : 3089, 3043 (CH aro-
matic str), 2951, 2920, 2850 (CH aliphatic str), 1558, 1516, 1458
.
þ
1
%): 258 [M , 100.00]. Anal. Calcd for C12H11FN (258.25): C, 55.81;
6
(
C¼N, C¼C aromatic str); H NMR (DMSO-d
6
ppm) d: 2.26 (s, 3H,
of piperazine)
.76–3.78 (m, 4H, 2CH of piperazine), 7.35–7.39 (dd, 2H, J ¼ 8.8 Hz,
H, 4.29; N, 32.54. Found: C, 55.98; H, 4.37; N, 32.82.
3 3 2
N-CH ), 2.62 (s, 3H, CH ), 3.30–3.34 (m, 4H, 2CH
3
2
J ¼ 8.7 Hz, ArH), 8.13–8.16 (dd, 2H, J ¼ 4.9 Hz, J ¼ 8.7 Hz, ArH), 8.38
General procedure for the preparation of compounds (10a–d)
(
s, 1H, C6-H); 13 C NMR (DMSO-d ppm) d: 17.3 (CH ), 45.7 (N-CH ),
6 3 3
4
1
1
5
6
7.9 (2CH of piperazine), 54.5 (2CH of piperazine), 102.6, 116.10, Five drops of glacial acetic acid were added to a solution of com-
2
2
6.34 (d, J ¼ 24 Hz)), 123.1, 135.3, 142.0, 149.7, 155.1, 155.2, 159.09, pound 9 (0.774 g, 0.003 mol) and the selected aromatic aldehyde
.
þ
59.9, 161.52 (d, J ¼ 243 Hz, C-F) (Ar Cs); MS (m/z, %): 326 [M , (0.003 mol) in absolute ethanol (20 ml). The reaction mixture was
eþ
.27], 256 [100.00] (M-70 ). Anal. Calcd for C17
H
19FN
6
(326.37): C, heated under reflux for 6 h. After cooling the formed precipitate
2.56; H, 5.87; N, 25.75. Found: C, 62.80; H, 5.94; N, 26.01.
was filtered, dried and recrystallised from ethanol to give 10a–d.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
537
1
–(4-Fluorophenyl)-4–(2-(methoxybenzylidene)hydrazinyl)-3-
J ¼ 273 Hz, C-F), 149.8, 150.1, 150.4, 150.8, 151.3, 155.5, 159.5 (Ar
.þ
methyl-1H-pyrazolo[3,4-d]pyrimidine (10a)
Cs and N¼CH); MS (m/z, %): 347 [M , 100.00]. Anal. Calcd for
(347.35): C, 62.24; H, 4.06; N, 28.23. Found: C, 62.52; H,
.17; N, 28.61.
ꢀ1
18 7
C H14FN
4
ꢁ
Yield: 43%; mp: 266–268 C; IR (KBr) cm : 3394 (NH str), 3062,
001 (CH aromatic str), 2916, 2846 (CH aliphatic str), 1508, 1462
3
1
(
C¼N, C¼C aromatic str); H NMR (DMSO-d
6
ppm) d: 2.67 (s, 3H,
CH
3
), 3.86 (s, 3H, OCH
3
), 7.05 (d, 2H, J ¼ 8 Hz, ArH), 7.38–7.42 (t,
Anticancer activity
2
8
1
1
1
1
H, J ¼ 8.8 Hz, ArH), 7.87–7.90 (m, 2H, ArH), 8.02–8.07 (m, 2H, ArH),
Measurement of anticancer activity against a panel of 60
cell lines
.19 (s, 1H, N¼CH), 8.21 (s, 1H, NH, D O exchangeable), 8.47 (s,
2
H, C6-H); 13 C NMR (DMSO-d6 ppm) d: 14.8 (CH ), 55.8 (OCH ),
3
3
06.1, 111.6, 114.6, 116.23, 116.46 (d, J ¼ 23 Hz), 121.3, 123.73,
23.80 (d, J ¼ 7 Hz), 127.6, 128.0, 129.9, 132.1, 135.1, 138.9, 145.0,
49.5, 155.3, 159.40, 161.42 (d, J ¼ 202 Hz, C-F) (Ar Cs and N¼CH);
Anticancer activity screening of the newly synthesised compounds
was measured in vitro utilising 60 different human tumour cell
lines provided by US National Cancer Institute according to previ-
.þ
eþ
MS (m/z, %): 376 [M , 64.38], 243 [100.00] (M-133 ). Anal. Calcd
27–29
ously reported standard procedure
as follows: Cells are inocu-
for C H FN O (376.39): C, 63.82; H, 4.55; N, 22.33. Found: C,
2
0
17
6
lated into 96-well microtitre plates in 100 ml. After cell inoculation,
6
4.07; H, 4.59; N, 22.17.
ꢁ
the microtitre plates are incubated at 37 C, 5% CO
2
, 95% air and
1
00% relative humidity for 24 h prior to addition of experimental
compounds. After 24 h, two plates of each cell line are fixed in
situ with TCA, to represent a measurement of the cell population
for each cell line at the time of drug addition (Tz). Experimental
1
–(4-Fluorophenyl)-4–(2-(4-hydroxy-3-methoxybenzylidene)-
hydrazinyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidine (10b)
ꢁ
ꢀ1
Yield: 75%; mp: 221–223 C; IR (KBr) cm : 3510 (OH str), 3444 (NH compounds are solubilised in dimethyl sulphoxide at 400-fold the
str), 3055, 3005 (CH aromatic str), 2920, 2850 (CH aliphatic str), desired final maximum test concentration and stored frozen prior
1
1
600, 1573, 1516 (C¼N, C¼C aromatic str); H NMR (DMSO-d6 to use. At the time of compound addition, an aliquot of frozen
ppm) d: 2.86 (s, 3H, CH ), 3.83 (s, 3H, OCH ), 6.84–6.89 (dd, 2H, concentrate is thawed and diluted to twice the desired final max-
3
3
J ¼ 4.7 Hz, J ¼ 8 Hz, ArH), 7.29–7.34 (dd, 2H, J ¼ 4.7 Hz, J ¼ 8 Hz, imum test concentration with complete medium containing
ArH), 7.42 (s, 1H, ArH), 7.92–7.95 (m, 2H, ArH), 8.25 (s, 1H, N¼CH), 50 mg/mL gentamicin. Aliquots of 100 ml of the compounds dilu-
8
1
2
.52 (s, 1H, C6-H), 9.55 (br s, 1H, NH, D O exchangeable), 11.84 (s, tions are added to the appropriate microtitre wells already con-
H, OH, D O exchangeable); 13 C NMR (DMSO-d6 ppm) d: 14.5 taining 100 ml of medium, resulting in the required final
2
(
1
1
CH ), 55.9 (OCH ), 110.5, 111.3, 115.79, 115.97 (d, J ¼ 18 Hz), 116.2, compound concentration. Following compound addition, the
3
3
ꢁ
23.1, 123.80, 123.92 (d, J ¼ 12 Hz), 124.5, 125.9, 127.1, 135.2, plates are incubated for an additional 48 h at 37 C, 5% CO , 95%
2
38.8, 145.3, 148.44, 149.4, 150.44 (d, J ¼ 200 Hz, C-F), 161.0 (Ar Cs air, and 100% relative humidity. For adherent cells, the assay is
.
þ
and N¼CH); MS (m/z, %): 392 [M , 22.72], 300 [100.00]. Anal. terminated by the addition of cold trichloroacetic acid (TCA). Cells
Calcd for C H FN O (392.39): C, 61.22; H, 4.37; N, 21.42. Found: are fixed in situ by the gentle addition of 50 ml of cold 50% (w/v)
2
0
17
6 2
C, 61.43; H, 4.45; N, 21.80.
TCA (final concentration, 10% TCA) and incubated for 60 min at
ꢁ
4
C. The supernatant is discarded, and the plates are washed five
times with tap water and air-dried. Sulphorhodamine B (SRB) solu-
tion (100 ml) at 0.4% (w/v) in 1% acetic acid is added to each well,
and plates are incubated for 10 min at room temperature. After
staining, unbound dye is removed by washing five times with 1%
acetic acid and the plates are air-dried. Bound stain is subse-
quently solubilised with 10 mM trizma base, and the absorbance is
read on an automated plate reader at a wavelength of 515 nm.
For suspension cells, the methodology is the same except that the
assay is terminated by fixing settled cells at the bottom of the
wells by gently adding 50 ml of 80% TCA (final concentration,
1
–(4-Fluorophenyl)-3-methyl-4–(2-(3,4,5-trimethoxybenzylidene)-
hydrazinyl)-1H-pyrazolo[3,4-d]pyrimidine (10c)
ꢁ
ꢀ1
Yield: 69%; mp: 222–224 C; IR (KBr) cm : 3421 (NH str), 3062 (CH
aromatic str), 2920, 2850 (CH aliphatic str), 1581, 1504, 1454 (C¼N,
1
C¼C aromatic str); H NMR (DMSO-d
6 3
ppm) d: 2.67 (s, 3H, CH ),
3
.85 (s, 6H, 2 OCH ), 3.86 (s, 3H, CH O), 7.22 (s, 2H, ArH), 7.36–7.41
3 3
(
dd, 2H, J ¼ 5.2 Hz, J ¼ 8.9 Hz, ArH), 8.08–8.10 (m, 3H, ArH þ NH),
8
1
1
1
1
.31 (s, 1H, N¼CH), 8.66 (s, 1H, C6-H); 13 C NMR (DMSO-d
6
ppm) d:
4.7 (CH ), 56.4 (2 OCH ), 60.63 (OCH ), 106.0, 110.9, 116.18,
3
3
3
1
6% TCA). Using the absorbance measurements [time zero, (Tz),
control growth, (C), and test growth in the presence of compound
Ti)], the percentage growth is calculated for each compound.
Percentage growth inhibition is calculated as:
16.40 (d, J ¼ 22 Hz), 121.20, 123.63 (d, J ¼ 243 Hz, C-F), 124.0,
28.3, 129.7, 132.3, 133.4, 135.2, 136.4, 138.3, 140.6, 141.6, 150.1,
.þ
(
53.6, 161.6 (Ar Cs and N¼CH); MS (m/z, %): 436 [M , 3.40], 269
eþ
[100.00] (M-167 ). Anal. Calcd for C22
H
21FN
O
6 3
(436.44): C, 60.54;
Â
Ã
H, 4.85; N, 19.26. Found: C, 60.82; H, 4.91; N, 19.38.
ðTi – TzÞ=ðC – TzÞ x 100 for concentrations for which Ti >=¼ Tz:
Â
Ã
ðTi – TzÞ=Tz x 100 for concentrations for which Ti < Tz:
1
–(4-Fluorophenyl)-3-methyl-4–(2-(pyridin-4-yl-methylidene)
hydrazinyl)-1H-pyrazolo[3,4-d]pyrimidine (10d)
ꢁ
ꢀ1
Yield: 51%; mp: 262–264 C; IR (KBr) cm : 3433 (NH str), 3070, Measurement of IC50 against lung cancer NCI-H460, NCI-
3
1
3
039 (CH aromatic str), 2920, 2850 (CH aliphatic str), 1562, 1516, H226, ovarian cancer OVCAR-4 and renal cancer ACHN
1
473 (C¼N, C¼C aromatic str); H NMR (DMSO-d ppm) d: 2.58 (s, cell lines
6
H, CH
3
), 7.36–7.42 (dd, 2H, J ¼ 4.9 Hz, J ¼ 9.2 Hz, ArH), 7.92 (d, 2H,
Cell culture
.19 (s. 1H, N¼CH), 8.44 (s, 1H, C6-H), 8.65 (d, 2H, J ¼ 5.8 Hz, pyr- Cell Line cells were obtained from American Type Culture
idyl H), 12.15 (s, 1H, NH, D O exchangeable); 13 C NMR (DMSO-d Collection, cells were cultured using Dulbecco’s Modified Eagle’s
ppm) d: 14.5 (CH ), 102.5, 116.23, 116.46 (d, J ¼ 23 Hz), 121.4, medium (DMEM) (Invitrogen/Life Technologies) supplemented
J ¼ 5.8 Hz, pyridyl H), 8.01–8.05 (dd, 2H, J ¼ 4.9 Hz, J ¼ 9.2 Hz, ArH),
8
2
6
3
1
22.0, 123.79, 123.88 (d, J ¼ 9 Hz), 135.1, 142.7, 145.68, 148.41 (d, with 10% foetal bovine serum (FBS) (Hyclone), 10 lg/mL of insulin

5
38
M. MAHER ET AL.
(Sigma), and 1% penicillin-streptomycin. All of the other chemicals Measurement of inhibitory activity against ErbB2
and reagents were from Sigma, or Invitrogen. Plate cells (cells
density 1.2 – 1.8 ꢂ 10,000 cells/well) in a volume of 100 mL com-
plete growth medium and 100 lL of the tested compound per
well in a 96-well plate for 24 h before the MTT assay.
Compound 7d was evaluated against ErbB2 enzyme using
HTScanVR HER2/ErbB2 Kinase Assay Kit according to manufacturer’s
instructions. In brief, add 10 lL of 10 mM ATP to 1.25 ml of 6 lM
substrate peptide. Dilute the mixture with dH 0 to 2.5 ml. Transfer
2
ꢁ
enzyme from ꢀ80 C to ice. Allow enzyme to thaw on ice. Micro-
0
centrifuge briefly at 4 C. Return immediately to ice. Add 10 lL of
Cell culture protocol
V
R
dithiothreitol (DTT) (1.25 M) to 2.5 ml of HTScan tyrosine kinase
Remove culture medium to a centrifuge tube. Briefly rinse the cell buffer. Transfer 1.2 ml of DTT/Kinase buffer to each enzyme.
layer with 0.25% (w/v) Trypsin 0.53 lM ethylenediaminetetraacetic Incubate 12.5 lL of the reaction cocktail with 12.5 lL/well of
acid (EDTA) solution to remove all traces of serum which contains pre-diluted compound of interest (10 lM) for 5 min at room tem-
Trypsin inhibitor. Add 2.0 to 3.0 ml of Trypsin EDTA solution to perature. Add 25 lL of ATP/substrate cocktail to 25 lL/well pre-
flask and observe cells under an inverted microscope until cell incubated reaction cocktail/compound. Incubate reaction plate at
layer is dispersed. Add 6 to 8 ml of complete growth medium and room temperature for 30 min. Add 50 lL/well stop buffer to stop
aspirate cells by gently pipetting. Transfer the cell suspension to the reaction. Transfer 25 lL of each reaction and 75 lL dH
the centrifuge tube with the medium and centrifuge for 5 to to a 96-well streptavidin coated, plate and incubate at room
0 min. Discard the supernatant. Resuspend the cell pellet in fresh temperature for 60 min. Wash three times with 200 lL/well phos-
2
O/well
1
growth medium. Add appropriate aliquots of the cell suspension phate-buffered saline (PBS). Dilute primary antibody, phospho-
ꢁ
to new culture vessels. Incubate cultures at 37 C for 24 h. After tyrosine mAb (P-Tyr-100), 1:1000 in PBS with 1% bovine serum
treatment of cells with the serial concentrations of the compound albumin (BSA). Add 100 lL/well primary antibody. Incubate at
ꢁ
to be tested incubation is carried out for 48 h at 37 C, then the room temperature for 60 min and wash three times. Prepare
appropriate dilution of horseradish peroxidase (HRP) labelled sec-
ondary antibody in PBS with 1% BSA. Add 100 lL/well secondary
antibody solution. Incubate at room temperature for 30 min and
plates are to be examined under the inverted microscope and
proceed for the MTT assay.
0
0
wash five times. Add 100 lL/well 3,3 ,5,5 -tetramethylbenzidine
MTT assay protocol
(TMB) substrate. Incubate at room temperature for 15 min. Add
1
4
00 lL/well of stop solution. Mix well and read the absorbance at
50 nm with a microtitre plate reader.
The 3–(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
3
0
(MTT) method of monitoring in vitro cytotoxicity is well suited
for use with multiwell plates. The assessment of cell population
growth is based on the capability of living cells to reduce the yel- Measurement of the effect of compound 7d on the level of
low product MTT to a blue product, formazan, by a reduction caspase-3 protein (Marker of apoptosis):
reaction occurring in the mitochondria. The five cell lines were
The level of human active caspase-3 protein was evaluated using
incubated for 24 h in 96-microwell plates. The number of living
Invitrogen (Catalog KHO1091) ELISA kit. The manufacturer’s
cells in the presence or absence (control) of the various test com-
instructions were followed in the following procedures. Add
pounds is directly proportional to the intensity of the blue colour,
1
00 lL of the standard diluent buffer to the zero standard wells.
measured by spectrophotometry using (ROBONIK P2000
Spectrophotometer) at a wavelength of 570 nm. Measure the
background absorbance of multiwell plates at 690 nm and sub-
tract from the 570 nm measurement. Five concentrations ranging
from 0.01 lM to 100 lM (with semi-log decrease in concentration)
were tested for each of the compounds under study. Each experi-
ment was carried out in triplicate. The IC50 values [the concentra-
tion required for 50% inhibition of cell viability] were calculated
using sigmoidal dose response curve-fitting models.
Add 100 lL of standards and controls or diluted samples to the
appropriate microtitre wells. Cover wells with and incubate for 2 h
at room temperature. Thoroughly aspirate or decant solution from
wells and discard the liquid. Pipette 100 lL of caspase-3 (active)
detection antibody solution into each well. Cover plate and incu-
bate for 1 h at room temperature. Add 100 lL Anti-Rabbit IgG HRP
working solution to each well. Cover wells with the plate cover
and incubate for 30 min at room temperature. Add 100 lL of stabi-
lised chromogen to each well. The liquid in the wells will begin to
turn blue. Incubate for 30 min at room temperature. Stop solution
has been added to each well. The solution in the wells should
change from blue to yellow. Read the plate within 2 h after add-
Measurement of inhibitory activity against EGFR
Compound 7d was selected to be evaluated against EGFR enzyme ing the stop solution. Use a curve fitting software to generate the
TM
using EnzyChrom Kinase Assay Kit (EKIN-400) according to man- standard curve.
ufacturer’s instructions. In brief, set up 20 lL reaction mixture con-
taining the EGFR kinase, ATP and substrate in the provided assay
buffer. Set up a blank control that contains ATP and substrate but
Cell cycle analysis of compound 7d
no enzyme. Incubate at desired temperature for 30 min. Prepare The OVCAR-4 cells were treated with compound 7d at its IC50
9
3
00 lL 10 lM (adenosine diphosphate) ADP premix by mixing 3 lL concentration for 24 h. After treatment, the cells were washed
mM standard and 897 lL distilled water. Transfer 20 lL standards twice with ice-cold PBS, collected by centrifugation, and fixed in
into separate wells of the plate. Prepare enough working reagent ice-cold 70% (v/v) ethanol, washed with PBS, re-suspended with
for each well. Add 40 lL working reagent to each assay well. Tap 0.1 mg/mL RNase, stained with 40 mg/mL propidium iodide (PI),
plate to mix. Incubate at room temperature for 10 min. Read fluor- and analysed by flow cytometry using FACS Calibur (Becton
3
1
escence intensity at kexc ¼ 530 nm and kem ¼ 590 nm.Calculate Dickinson) . The cell cycle distributions were calculated using
kinase activity. Cell-Quest software (Becton Dickinson). Exposure of OVCAR-4 cells

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
539
H3C
CN
H3C
CN
CN
N
a
NH2
N
C2H5O
F
2
1
b
c
d
O
NH2
H3C
NH2
H3C
H3C
N
NH
N
N
N
N
N
N
N
N
N
N
NH2
F
F
4
F
3
5
Reagents and conditions:
a) 4-F-Phenyl hydrazine; ethanol; reflux 5 h.
b) Formic acid; reflux 9 h.
c) Formamide; reflux 4 h.
d) Guanidine HCl; reflux 6 h.
Scheme 1. The synthetic path and reagents for the preparation of compounds 1–5.
3
2
33
to compound 7d resulted in an interference with the normal cell as previously reported . Compound 2
was prepared by reflux-
cycle distribution as indicated.
ing 2–(1-ethoxyethylidene) malononitrile (1) with 4-fluorophenyl-
hydrazine hydrochloride in absolute ethanol in the presence of
triethylamine. Heating under reflux the amino cyano derivative 2
in formic acid afforded compound 3. The IR spectrum of com-
Annexin V-FITC assay
pound 3 was useful in tracing the disappearance of CꢃN and NH
2
Annexin V fluorescein isothiocyanate (FITC)/PI apoptosis detection
kit (BD Biosciences) was used according to the manufacturer’s
instructions to measure the apoptotic activity of compound 7d.
OVCAR-4 cells were incubated with Annexin V-FITC. Apoptosis was
absorption bands, while it showed the absorption band at
ꢀ
1
1
1
697 cm due to C¼O group. The H NMR spectrum displayed
the presence of a singlet signal at d 8.14 ppm due to C6-H. In add-
ition to the appearance of an exchangeable singlet signal at d
6
induced by addition of compound 7d at its IC50 to 4 ꢂ 10 cell/T
1
2.36 ppm corresponding to NH proton. The 13C NMR spectrum
7
5 flask and incubation for 24 h. The cells then were collected by
6
pointed the formation of compound 3 by the appearance of C¼O
carbon at d 161.9 ppm. Compound 4 was prepared in a good yield
through the condensation of the amino cyano derivative 2 with
trypsinisation and 0.5 ꢂ 10 cells were washed twice with PBS and
stained with 5 lL Annexin V-FITC and 5 lL PI in 1 ꢂ binding buffer
for 15 min at room temperature in the dark. Analyses were per-
formed using FACS Calibur flow cytometer (BD Biosciences, San
Jose, CA).
formamide. The IR spectrum confirmed the disappearance of the
1
CꢃN group of the respective compound 2. Furthermore, the
H
NMR spectrum showed an exchangeable singlet signal at d
.47 ppm corresponding to the NH protons, in addition to a
7
2
Results and discussion
singlet signal at d 8.26 ppm indicating the C6-H proton of the pyr-
imidine ring. Reacting 5-amino-1–(4-fluorophenyl)-3-methyl-1H-
pyrazole-4-carbonitrile (2) with guanidine in methanol gave the
Chemistry
The synthesis of the new compounds is illustrated in Schemes 1 target compound 5. The IR spectrum showed the appearance of
ꢀ
1
and 2. The preparation of the starting material 1 was achieved via bands at 3392 – 3215 cm
attributed to two NH
2
groups. In
1
heating triethyl orthoacetate and malononitrile in acetic anhydride accordance, the H NMR spectrum of compound 5 displayed two

5
40
M. MAHER ET AL.
Scheme 2. The synthetic path and reagents for the preparation of compounds 6–10.
ꢀ
1
ꢀ1
1
D
2
2
O exchangeable signals at d 7.55 and 8.34 ppm attributed to groups at 3122 cm
and 1697 cm , respectively. Further
-amino and 4-amino protons with the appropriate integration. structural evidence stemmed from the H NMR spectrum was the
The chloro derivative 6 was prepared by the reaction of the pyra- disappearance of the signal of NH proton. The reaction of the
zolopyrimidone 3 with phosphorus oxychloride. The IR spectrum 4-chloropyrazolo[3,4-d]pyrimidine 6 with the appropriate aromatic
lacked the absorption bands corresponding to NH and C¼O amine in isopropyl alcohol afforded compounds 7a–d. The IR

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
541
ꢀ5
ꢀ5
Table 1. Growth inhibition percentages obtained from the single dose (10 M) Table 2. Growth inhibition percentages obtained from the single dose (10 M)
test for compounds 3–6, 7c, d.
test for compounds 8a, c, d, 9 and 10b, c.
Compound
Compound
8d
3
4
5
6
7c
7d
Panel/cell line
8a
8c
9
10b
10c
Panel/cell line
Leukemia
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
HOP-62
HOP-92
ꢀ1.13 ꢀ12.67 ꢀ16.80
1.47 ꢀ8.18 0.70
2.31 ꢀ8.74
0.86 30.91 38.04
4.81
6.74
1.69
3.30
7.87
8.85 13.97 16.02 14.97
22.48 25.51 11.84 11.79
1.69 ꢀ4.11
1.98 ꢀ11.90 15.44
0.26 1.78
1.83 4.27
ꢀ3.68 11.36 ꢀ4.00 ꢀ3.06 24.18 ꢀ3.02
0.58 ꢀ0.48 13.75
5.79
7.19
3.49 22.66 38.29
6.45 ꢀ6.21 7.52
7.03
1.29
ꢀ10.17 ꢀ17.49 ꢀ3.69
2.21 ꢀ4.47 9.77
2.00 ꢀ8.37
ꢀ1.58ꢀ
3.35 ꢀ1.68 14.42
6.02 25.37
1.18 10.96
8.97 20.25 22.95 13.68
ꢀ2.93
36.18
ꢀ6.53
10.32
ꢀ9.27
4.09
ꢀ4.88 ꢀ1.96 11.58
3.08 40.51
3.39 14.46
ꢀ0.09 ꢀ3.31 ꢀ4.08 ꢀ5.98
19.50 20.55 7.21 17.03 12.38 10.04
1.51 0.38
4.27 ꢀ6.39
4.73 13.39 39.54 40.41
5.22 15.47 ꢀ2.30 5.82
9.95 19.05 9.39 14.25
1.50 23.88 14.39 13.49
ꢀ8.23 ꢀ4.35 ꢀ4.87 ꢀ11.72 11.95 ꢀ3.23
8.03 8.23 3.59 10.35 27.06 7.26
2.36 ꢀ0.67
19.10 6.25 9.08
ꢀ8.24 ꢀ8.35 15.44 ꢀ14.49 38.70
ꢀ1.54 1.64 7.91 3.79 22.78
ꢀ7.40 ꢀ8.08 15.05 ꢀ10.21 62.91
4.73 11.79 7.86 4.63 7.53
7.26 8.14 5.32 10.04 12.02
ꢀ9.30 ꢀ18.78 ꢀ0.07 ꢀ9.33 58.46
6.49 2.08 58.95 10.98 10.44
ꢀ13.58
4.05
9.27 15.93
NCI-H226
NCI-H23
ꢀ8.48
7.59
0.28 20.68
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS Cancer
5.19
5.51
3.90
ꢀ9.62
4.92
ꢀ14.86 ꢀ18.39 ꢀ13.53 ꢀ14.88 ꢀ13.30 ꢀ3.96
ꢀ7.19 ꢀ1.97 ꢀ7.56 ꢀ11.74 ꢀ4.39 ꢀ6.78
ꢀ2.16
ꢀ1.64
ꢀ6.88
3.83
ꢀ2.52
3.79 ꢀ5.66
1.34 1.79
3.20 19.23
2.41
ꢀ7.69
3.74
1.69
3.59
7.42
5.10
3.99 ꢀ4.51
0.27
2.74
3.15
ꢀ8.66 ꢀ2.75
ꢀ4.62 ꢀ3.69
3.34 ꢀ6.50
7.98
1.32 ꢀ0.49 2.75
9.55 11.70 14.38
0.99 ꢀ1.11 1.96
ꢀ2.04 ꢀ8.49 ꢀ5.55 ꢀ5.82 18.91 ꢀ5.30
HT29
KM12
SW-620
CNS Cancer
7.56
0.04
5.97 ꢀ1.26
1.62
3.79 28.14
7.70
8.83
5.82
1.48
3.50
1.24
2.24 0.40
ꢀ12.29 ꢀ18.66 ꢀ16.79 ꢀ2.88 ꢀ8.11 11.14
ꢀ4.76 ꢀ3.16 ꢀ0.63 ꢀ8.79 ꢀ30.86 ꢀ11.68
SF-268
SF-295
SF-593
SNB-19
SNB-75
U251
ꢀ1.71
ꢀ4.14
ꢀ0.99
ꢀ3.69
0.29
3.71 ꢀ2.30 ꢀ1.16
ꢀ3.53 ꢀ1.89 ꢀ4.11 ꢀ3.83 39.96
ꢀ1.06 2.81 ꢀ7.81 ꢀ1.94 22.58
ꢀ9.19 ꢀ7.94 7.79 7.21 20.45
8.18 5.33
6.36 ꢀ5.20 25.04
ꢀ7.56 ꢀ6.63 5.15 2.65 24.02
1.06 3.94
SF-268
SF-295
SF-593
SNB-19
SNB-75
U251
ꢀ0.79 4.51 ꢀ2.90 25.55 29.01 6.35
0.61 ꢀ0.93 ꢀ2.40 ꢀ3.92 ꢀ5.51 ꢀ2.66
ꢀ8.46
5.14 ꢀ4.77
0.92 0.02
ꢀ1.70 ꢀ0.06 ꢀ2.50
3.89
8.43 ꢀ0.70
3.85 13.15
6.67 ꢀ4.87
2.02 11.34
5.93 ꢀ0.56 17.50 19.53
ꢀ1.70
9.85
5.64
0.92
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-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
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-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
2.19
ꢀ8.22
ꢀ7.65
ꢀ7.05
ꢀ5.10
ꢀ14.06
0.31
1.75
2.19
6.64 ꢀ0.99 8.68
ꢀ2.33
4.93
4.22
0.28 13.28 18.99
9.33
5.86 ꢀ7.80 ꢀ4.30
1.70 1.56
0.75 1.01
ꢀ10.46
5.14 19.00 ꢀ12.36 12.58
ꢀ3.62 ꢀ2.93 5.89
ꢀ4.47 ꢀ0.94
3.95 ꢀ8.00 ꢀ2.10
2.73
ꢀ5.15 ꢀ7.73 ꢀ8.83 ꢀ6.94 0.10
1.74 3.86 10.97 10.95 2.61
ꢀ3.55 ꢀ4.89 ꢀ4.92 ꢀ11.09 ꢀ4.63
2.00 0.86
5.94 ꢀ3.74 ꢀ0.44
ꢀ2.24 ꢀ14.50 ꢀ6.10 ꢀ9.22
1.49
ꢀ12.61 ꢀ3.74 ꢀ8.74
ꢀ0.67 5.22 ꢀ1.01
ꢀ5.90 ꢀ0.44 ꢀ6.35 ꢀ3.21 ꢀ14.11
N.T. N.T. N.T. N.T. N.T.
4.56 ꢀ2.02
9.37 ꢀ1.69 18.31 10.15 ꢀ1.74
2.10 ꢀ7.04
1.30
3.85 43.36 15.34
ꢀ8.15 ꢀ14.19 ꢀ18.68 17.45 ꢀ7.62 ꢀ1.41
0.35
N.T.
N.T.
N.T.
N.T.
12.63
N.T.
N.T.
5.29
ꢀ5.82
ꢀ8.19
ꢀ10.42
ꢀ0.16
ꢀ1.22
ꢀ2.82
11.14
6.71 24.43
1.54 27.56
ꢀ3.29 25.56
ꢀ5.65 1.21 ꢀ8.47 32.23 24.19 ꢀ0.30
ꢀ10.71 ꢀ1.90 ꢀ1.77 17.82 9.65 ꢀ8.66
9.45 23.49 20.55 26.65
ꢀ2.81 ꢀ12.89 ꢀ8.39 ꢀ6.58 9.57
ꢀ4.38 1.28 ꢀ0.80 ꢀ3.91 53.12
ꢀ4.61 ꢀ1.61 ꢀ12.31 ꢀ0.50 4.92
6.34 2.02 16.63
ꢀ3.95 ꢀ2.91 ꢀ7.96 ꢀ7.64 ꢀ30.85 ꢀ11.41
ꢀ4.64 ꢀ7.15
ꢀ5.87
ꢀ6.35
3.66
6.62 ꢀ3.70
3.69
1.33
1.29
3.79
7.98
3.64 ꢀ1.23
4.30 ꢀ2.25
0.66 ꢀ3.45 ꢀ8.11 ꢀ6.46 5.13
ꢀ5.01 ꢀ6.26 13.68 ꢀ16.74 13.87
ꢀ7.07 12.56
0.81
7.06
Renal Cancer
Renal Cancer
7
86-0
1.16
0.02
9.41
2.79 ꢀ0.09
0.56 21.33
5.97 ꢀ5.43
786-0
A498
ACHN
CAKI-1
10.64 ꢀ2.04 ꢀ4.57 ꢀ3.72 11.35
1.85
A498
ACHN
CAKI-1
0.68 ꢀ3.11
4.71
4.93
ꢀ10.31
N.T.
9.66 ꢀ0.85
2.13 ꢀ0.34
1.48 ꢀ3.34 22.01
ꢀ4.68
0.98 ꢀ3.79
4.11 ꢀ12.22 60.41
2.07
N.T.
7.34
N.T.
4.01
N.T.
N.T.
N.T.
N.T.
1.02
N.T.
N.T.
N.T.
N.T.
RXF 393
SN12C
TK-10
UO-31
ꢀ11.41 ꢀ17.32 ꢀ13.02 ꢀ3.61 ꢀ13.69 ꢀ0.39
RXF 393
SN12C
TK-10
UO-31
ꢀ15.68 ꢀ16.53 ꢀ9.45
5.47
8.30 14.90
4.90
3.39
4.99
ꢀ2.50
ꢀ3.17
11.67
ꢀ1.40 ꢀ2.74
1.02 48.38 21.52
1.12 13.35 6.31
ꢀ4.14 ꢀ12.97 ꢀ23.0
9.04 ꢀ6.68
9.75 ꢀ30.90 ꢀ47.95 ꢀ51.2
11.27 ꢀ36.27
11.34 14.20 11.83
8.66 25.50
5.57 34.89 21.31 28.67 35.57 31.11
Prostate Cancer
PC-3
Prostate Cancer
PC-3
1.46
1.03
3.59 14.76
7.23 17.21
5.06 11.35 12.47 13.76 27.02 16.52
DU-145
Breast Cancer
ꢀ6.51
ꢀ7.26 ꢀ9.51
3.05 ꢀ8.97 ꢀ3.13
DU-145
Breast Cancer
ꢀ7.70 ꢀ5.88 ꢀ11.21 ꢀ0.84 12.39 ꢀ4.94
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
2.70
9.82
0.73 11.58
6.19 15.69
MCF7
9.07
ꢀ10.39
ꢀ4.78
3.10
7.92
3.74
1.60 11.52 14.72
2.36 23.08 15.55 16.46
1.96
ꢀ9.78 ꢀ10.78 ꢀ9.11 10.81 ꢀ2.67 14.16
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
0.13
ꢀ3.59
0.23
ꢀ4.94 ꢀ3.27
4.54
0.70 21.46
ꢀ8.83 ꢀ4.84
5.99 ꢀ4.22
2.13 ꢀ10.8
0.89 13.94
9.79 ꢀ17.43 ꢀ11.5
3.96 ꢀ4.23
ꢀ22.24 10.32
ꢀ7.80 ꢀ3.83
9.63 ꢀ1.06
4.18 ꢀ2.65
0.28 14.64 27.35
8.70 ꢀ14.97 ꢀ1.69
3.85 32.00 20.15 16.75
MDA-MB-468
ꢀ11.51 ꢀ13.19 ꢀ15.49
0.37 18.48
8.05 ꢀ4.81
ꢄ
ꢄ
NT: not tested.
NT: not tested.
Bold values signifies the growth inhibition parentage is higher than 50%.

5
42
M. MAHER ET AL.
spectra of 7a–d showed the presence of an absorption band at Cancer Institute (USA) against 60 different human cell lines includ-
ꢀ
1
1
3
444 cm corresponding to NH group. The H NMR spectra sup- ing (leukemia, non-small cell lung cancer, colon cancer, CNS can-
ported the formation of 7a–d through the appearance of the D O cer, melanoma, ovarian cancer, renal cancer, prostate cancer and
2
exchangeable singlet signal at d 8.75 – 8.90 ppm. The appearance breast cancer). The selected compounds were evaluated at single
ꢀ
5
of extra aromatic protons corresponding to substitution on the dose (1 0 M). The growth inhibition percentages obtained from
4
-amino moiety was another significant proof of the success of the single dose test for the selected compounds are shown in
Tables 1 and 2. The analysis of the obtained in vitro data revealed
that two lung cell lines NCI-H226 and NCI-H460 were sensitive to
compound 7d with growth inhibition percentages 62.91 and
the reaction. The synthesis of the target compounds 8a–d was
accomplished through the reaction of the 4-chloropyrazolo[3,4-
d]pyrimidine derivative 6 with the selected secondary amine in
ethanol in the presence of a catalytic amount of triethylamine.
5
8.46, sequentially. Compound 7d also displayed potent cytotoxic
1
activity against renal cell line ACHN with growth inhibition per-
centage 60.41 and against ovarian cancer cell line OVCAR-4 with
growth inhibition percentage 53.12. Compound 6 showed promis-
ing selectivity against lung cancer cell line NCI-H522 with growth
inhibition percentage 58.95. Other test compounds exhibited no
activity against most investigated cell lines.
The H NMR spectra of these compounds showed the presence of
the 4 CH
.20 – 3.78 ppm, while the 13C NMR spectra indicated the
presence of 4 CH carbons at d 45.6 – 66.4 ppm. The hydrazinyl
2
protons of the morpholinyl or piperazinyl moiety at d
3
2
derivative 9 was obtained in a good yield via reacting the 4-chlor-
opyrazolo[3,4-d]pyrimidine 6 with hydrazine hydrate in ethanol.
The IR spectrum of compound 9 showed two absorption bands at
ꢀ
1
Table 4. IC50 values for the inhibitory activity of compound 7d against EGFR
and ErbB2 enzymes.
the range 3309 – 3120 cm indicating the presence of both NH
1
and NH
2
groups. The H NMR spectrum revealed the presence of
ꢄ
IC50 (mM ±SD)
two D O exchangeable singlet signals at d 4.80 and 8.90 ppm with
2
proper integration, corresponding to NH2 and NH protons, Enzyme
sequentially. Condensation of the 4-hydrazinyl derivative 9 with EGFR
7d
Erlotinib
0.186 ± 0.730
0.254 ± 0.640
0.030 ± 0.002
0.115 ± 0.720
ErbB2
the selected aromatic aldehyde in ethanol in the presence of gla-
1
ꢄThe values given are means of three experiments.
cial acetic acid yielded compounds 10a–d in good yields. The H
NMR of the target compounds revealed the appearance of a sing-
let signal at the range of d 8.19 – 8.31 ppm corresponding to
N¼CH proton, in addition to additional characteristic aromatic
protons denoting the success of the condensation reaction.
1
00
EGFR
90
Erb B2
8
7
6
0
0
0
Growth inhibition against a panel of 60 human tumor cell lines
In this study, 12 of the newly synthesised pyrazolopyrimidines
were subjected to in vitro anticancer screening by the National
50
4
3
0
0
Table 3. Concentrations required for 50% inhibition of cell viability (IC50) of
compound 7d compared to Erlotinib.
ꢄ
20
IC50 (lM ±SD)
1
0
0
Panel/ cell line
7d
Erlotinib
Lung cancer
Lung cancer
Lung cancer
Renal cancer
Ovarian cancer
NCI-H460
NCI-H460 (EGFR-WT)
NCI-H226
ACHN
4.44 ± 0.26
18.73 ± 0.71
17.36 ± 2.13
5.53 ± 0.31
1.74 ± 0.06
8.36 ± 0.69
64.54 ± 2.59
11.36 ± 0.97
12.41 ± 1.88
0
1
2
3
4
5
Log Conc (nM)
OVCAR-4
2.79 ± 0.41 Figure 4. Line representation of the effect of compound 7d on EGFR and ErbB2
ꢄThe results given are means of three experiments.
tyrosine kinases.
25
20
15
10
5
0
7
d
Erloꢀnib
NCI-H460
NCI-H226
Cell line
Figure 3. Graphical representation for concentrations required for 50% inhibition of cell viability (IC50) of compound 7d compared to Erlotinib.
ACHN
OVCAR-4

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
543
Detection of IC50 of compound 7d against lung cancer NCI-
potent than Erlotinib. It also showed promising potent anticancer
H460, NCI-H226, renal cancer ACHN and ovarian cancer OVCAR- activity against renal (ACHN) cell line with IC50 value 5.53 lM rep-
4
cell lines
resenting 2.2 folds more potency than Erlotinib. Regarding lung
cancer NCI-H460 cell line, compound 7d (IC50 ¼ 4.44 lM) was 1.9
folds more potent than Erlotinib. It is worthy mention that com-
pound 7d (IC50 ¼ 18.73 lM) was almost 3.5 folds more potent
than Elrlotinib against NCI-H460 EGFR-WT cell line. It possessed
Compounds 7d was selected to be further studied through deter-
mination of its half maximal inhibitory concentration (IC50) values
against the most sensitive cancer cell lines compared to Erlotinib
as a reference anticancer drug. The results of the mean values of
experiments performed in triplicate were summarised in Table 3
and represented graphically in Figure 3. The in vitro results
moderate anticancer activity against NCI-N226 cell line with IC50
¼
1
7.36 lM. Structural activity relationship analysis revealed that
showed that the most sensitive cell line for compound 7d was anti-proliferative activity of the newly synthesised pyrazolo[3,4-
ovarian (OVCAR-4) with IC50 ¼ 1.74 lM, which was 1.6 times more d]pyrimidines correlates well with substitution pattern on position
4
. It is worth noting that compound 7d, a highly potent
anticancer agent was among pyrazolopyrimidines 7a–d having a
phenyl amino group directly attached to the pyrimidine nucleus.
Further analysis of these compounds clearly revealed the substitu-
tion pattern on this phenyl amino moiety had a notable effect on
the anticancer activity. Grafting 3-Cl and 4-F substituents to the
Table 5. Effect of compound 7d on active caspase-3.
ꢄ
Compound
Caspase3 conc. (Pg/mL )
7
d
568.50 ± 14.0
52.51 ± 2.46
Cont.OVCAR-4
ꢄ
The values given are means ± SD of three experiments.
4
-anilino moiety enhanced the anti-proliferative activity against
several tumor cell lines, otherwise 4-F and 4-CH3 substituents
resulted in inactive members. Furthermore, the incorporation of
the phenyl amino group through azomethine or piperazinyl linker
regardless of the substitution pattern on the phenyl group
abolished activity. Finally, replacement of the 4-anilino group
with pharmacophoric moieties as carbonyl, amino, morpholine,
7
6
5
4
3
2
1
00
00
00
00
00
00
00
0
7
d
cont.OVCAR-4
4
-methylpiperazine or hydrazinyl groups or introducing additional
60
50
40
30
20
10
0
7
d
Cont. Ovcar-4
Compound
Figure 5. Graphical representation for active caspase-3 assay of compounds 7d.
Table 6. Cell cycle distribution of compound 7d.
Cell cycle distribution
Compound
%G0/G1
%S
%G2/M
%Apoptosis
%
G0/G1
%S
Cell cycle stage
Figure 7. Graphical representation of the cell cycle analysis of compound 7d.
%G2/M
%Apoptosis
7
d
29.77
53.92
33.79
39.33
36.44
6.75
21.71
1.99
Cont. OVCAR-4
Figure 6. Effect of compound 7d (1.74 lM) on DNA-ploidy flow cytometric analysis of OVCAR-4 cells after 24 h.

5
44
M. MAHER ET AL.
Figure 8. Representative dot plots of OVCAR-4 cells treated with 7d (1.74 lM) for 24h and analyzed by flow cytometry after double staining of the cells with annexin-
V FITC and PI.
25
20
15
10
5
0
Table 7. Effect of compound 7d on apoptosis and necrosis.
7
d
%Apoptosis
Cont.Ovcar-4
Compound
Total
Early
Late
%Necrosis
7
d
21.71
1.99
7.52
0.88
11.31
0.72
2.88
0.39
Cont.OVCAR-4
3
4,35
enzyme activity
. To evaluate the effect of compound 7d on
the level of active caspase-3, OVCAR-4 cells were treated with com-
pound 7d at its IC50 value for 24 h, before the enzyme assay.
Compound 7d resulted in a significant increase (almost 11 folds) in
the level of active caspase-3 compared to control (Table 5 and
Figure 5).
Total apoptosis Early apoptosis Late apoptosis
Stage of apoptosis
Necrosis
Cell cycle analysis and detection of apoptosis
The effect of the most active compound 7d on the cell cycle pro-
gression and induction of apoptosis in OVCAR-4 cells was studied.
OVCAR-4 cells were exposed to compound 7d at its IC50 values
for 24 h and its effect on the normal cell cycle profile and induc-
tion of apoptosis was analysed. Exposure of OVCAR-4 cells to com-
pound 7d resulted in an interference with the normal cell cycle
distribution of this cell line. Interestingly, compound 7d resulted
in an increase in the cells accumulated in pre-G1 phase by almost
Figure 9. Graphical representation of effect of compound 7d on apoptosis
and necrosis.
amino group at C-6 position resulted in a dramatic loss in the
anti-proliferative activity.
Measurement of the effect of 7d on EGFR enzyme
1
1-folds compared to control, the apoptotic activity was affirmed
We have investigated the inhibitory effect of compound 7d on EGFR
enzyme in vitro. The anti-proliferative activity of this compound
appeared to correlate well with its ability to inhibit EGFR at sub-
micromolar range with IC50 value 0.18 mM (Table 4 and Figure 4).
by the presence of a sub-G1 peak which may result from degrad-
ation or fragmentation of the genetic materials. Also, it showed
significant increase in the percentage of cells at G2/M phases by 5
folds, compared to control (Table 6 and Figures 6, 7). These results
suggest that compound 7d exerted its cytotoxic activity by pro-
moting cycle arrest at G2/M phase and apoptotic induction.
Measurement of the effect of 7d on ErbB2 enzyme
Since EGFR and ErbB2 receptors have the highest homology among
the EGFR family members in their kinase catalytic domains and share
many similar biochemical and kinetic properties. We have measured
the inhibitory effect of compound 7d on ErbB2 tyrosine kinase in
vitro. Compound 7d showed excellent inhibitory activity, at the sub-
micromolar level with IC50 value 0.25 mM (Table 4 and Figure 4).
Apoptosis determination by Annexin-V assay
The apoptotic activity of compound 7d was further studied using
Annexin V-FITC assay, which includes dual staining using Annexin
2þ
V, a Ca -dependent protein, and Propidium iodide (PI). Annexin
V binds to phosphatidylserine (PS) expressed only on the surface
of the apoptotic cells and fluoresces green after interacting with
the fluorochrome labelled annexin-V. On the other hand, PI stains
DNA and enters only dead cells. This assay can give a differential
Measurement of the effect of compound 7d on caspase-3 level
EGFR and ErbB2 tyrosine kinase inhibitors are well known to analysis to the percentages of living, early apoptotic, late apop-
3
6
potentiate the intrinsic apoptotic pathway via increase in caspase-3 totic and necrotic cells . As shown in Figures 8, 9 and Table 7,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
545
after 24 h of treatment of OVCAR-4 cells with compound 7d at its
IC50 concentration a decrease in the percentage of the survived
cells was observed. Moreover, a significant increase in the percent-
age of Annexin-V positive cells (almost 9 folds more than control)
occurred indicating an early apoptosis (lower right quadrant). In
addition, an increase by 7 folds more than control in the percent-
age of PI positive cells (upper left quadrant) indicating necrotic
cells. Some treated cells were in a late apoptotic stage (upper
right quadrant), this was indicated by the significant increase in
the percentage of Annexin V positive, PI positive cells (16 folds
more than control).
3. Perry JE, Grossman ME, Tindall DJ. Epidermal growth factor
induces cyclin D1 in a human prostate cancer cell line.
Prostate 1998;35:117–24.
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Conclusion
A series of novel pyrazolo[3,4-d]pyrimidines was synthesised.
Twelve synthesised compounds were evaluated for their anti-
cancer activity by NCI (USA). Compound 7d exhibited potent anti-
cancer activity at low concentrations. Pyrazolopyrimidine 7d
proved marked anticancer activity higher than Erlotinib. It showed
1
103–13.
8
.
.
Slamon DJ, Godolphin W, Jones LA, et al. Studies of the
HER-2/neu proto-oncogene in human breast and ovarian
cancer. Science 1989;244:707–12.
Brignola PS, Lackey K, Kadwell SH, et al. Comparison of the
biochemical and kinetic properties of the type 1 receptor
tyrosine kinase intracellular domains. Demonstration of dif-
ferential sensitivity to kinase inhibitors. J Biol Chem 2002;
277:1576–85.
9
1
.6-fold more potent anti-proliferative activity against ovarian
(
OVCAR-4) cell line with IC50 ¼ 1.74 lM. It also exhibited promis-
ing potent anticancer activity against renal (ACHN) cell line with
IC50 value 5.53 lM representing 2.2-fold more potency than
Erlotinib. Regarding lung cancer NCI-H460 cell line, compound 7d
(
IC50 ¼ 4.44 lM) was 1.9 folds more potent than Erlotinib. It is 10. Moulder SL, Yakes FM, Muthuswamy SK, et al. Epidermal
worthy mention that compound 7d was almost 3.5-fold more
potent than Elrlotinib against NCI-H460 EGFR-WT cell line. It pos-
sessed moderate anticancer activity against NCI-N226 cell line
with IC50 ¼ 17.36 lM. The anti-proliferative activity of pyrazolopyr-
growth factor receptor (HER1) tyrosine kinase inhibitor
ZD1839 (Iressa) inhibits HER2/neu (erbB2)-overexpressing
breast cancer cells in vitro and in vivo. Cancer Res 2001;61:
8887–95.
imidine 7d appeared to correlate well with its ability to inhibit 11. Manstein V, Yang C, Richter D, et al. Resistance of cancer
both EGFR and ErbB2 tyrosine kinases at sub-micromolar level
with IC50 values 0.18 and 0.25 mM, sequentially. Dual inhibition of
EGFR and ErbB2 enzymes leads to induction of the intrinsic path-
cells to targeted therapies through the activation of com-
pensating signaling loops. Curr Signal Trans Ther 2013;8:
193–202.
way of apoptosis. This mechanistic pathway was confirmed by a 12. Smith JE. Small-molecule targeted therapy in the treatment
significant increase in the level of active caspase-3, which is the
of non-small-cell lung cancer. Clin Therapeut 2005;27:
key executer of apoptosis compared to the control (10.8 folds).
1513–34.
Moreover, compound 7d showed accumulation of cells in pre-G1 13. Simon GR, Ruckdeschel JC, Williams C, et al. Gefitinib
phase and annexin-V and propidium iodide staining in addition to
cell cycle arrest at G2/M phase. Pyrazolo[3,4-d]pyrimidine 7d is a
privileged multi-targeted scaffold for the design and discovery of
novel anticancer agents.
(ZD1839) in previously treated advanced non-small-cell lung
cancer: experience from a single institution, Cancer control.
J Moffitt Cancer Center 2003;10:388–95.
14. Burris HA. Dual kinase inhibition in the treatment of breast
cancer: initial experience with the EGFR/ErbB-2 inhibitor
lapatinib. Oncologist 2004;9:10–15.
5. Gray NS, Wodicka L, Thunnissen AMWH, et al. Exploiting
chemical libraries, structure, and genomics in the search for
kinase inhibitors. Science 1998;281:533–8.
Acknowledgements
1
1
1
The authors are grateful to all members of National Institute of
Health, Meryland, USA for carrying out the anticancer screening.
The authors thank E Rashwan, Head of the confirmatory diagnos-
tic unit VACSERA-EGYPT, for carrying out the cytotoxicity testing,
cell cycle analysis, active caspase-3 and EGFR and ErbB2 inhib-
ition assays.
6. Chauhan M, Kumar R. Medicinal attributes of
pyrazolo[3,4-d]pyrimidines: a review. Bioorg Med Chem
2
013;21:5657–68.
7. Petrie CR, Cottam HB, Mckernan PA, et al. Synthesis and bio-
logical activity of 6-azacadeguomycin and certain 3,4,6-tri-
substituted pyrazolo[3,4-d]pyrimidine ribonucleosides. J Med
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Disclosure statement
1
1
8. Traxler P, Bold G, Buchdunger E, et al. Tyrosine kinase inhibi-
tors: from rational design to clinical trials. Med Res Rev
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
2
001;21:499–512.
9. Traxler P, Allegrini PR, Brandt R, et al. AEE788: a dual family
epidermal growth factor receptor/ErbB2 and vascular endo-
thelial growth factor receptor tyrosine kinase inhibitor with
antitumor and antiangiogenic activity. Cancer Res 2004;64:
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