4-Substituted-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine derivatives: Design, synthesis, antitumor and EGFR tyrosine kinase inhibitory activity

Article abstract of DOI:10.1111/cbdd.12451

Four series of some 4-substituted-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine derivatives 5a-f, 6a-f, 8a-f, and 9a-f were designed to be screened for their antitumor activity. All compounds were evaluated against breast (MCF-7) and lung (A-549) cell lines. Six compounds 5a, 5b, 6b, 6e, 9e, and 9f displaying activity against both cell lines were further estimated for their EGFR-TK inhibitory activity where they revealed 41-91% inhibition and compound 6b elicited the highest activity (91%). A docking study of these compounds into the ATP-binding site of EGFR-TK demonstrated their binding mode where H-bonding interaction with Met793 through N¹ of pyrimidine or N² of pyrazole was observed. Four series of compounds comprising the pyrazolo[3,4-d]pyrimidine core substituted at position 4 with various heterocyclic substitutions were synthesized. Antitumor activity and EGFR-TK inhibition were evaluated.

Full text of DOI:10.1111/cbdd.12451

Chem Biol Drug Des 2014
Research Article
4-Substituted-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine
Derivatives: Design, Synthesis, Antitumor and EGFR
Tyrosine Kinase Inhibitory Activity
cancers such as non-small cell lung cancer (NSCLC),
breast, ovarian, colon, and prostate cancer and correlates
with a poor prognosis in many cancer patients (5). There-
fore, blocking of the kinase activity and its signal transduc-
Safinaz E.-S. Abbas, Enayat I. Aly, Fadi
M. Awadallah* and Walaa R. Mahmoud
Pharmaceutical Chemistry Department, Faculty of
Pharmacy, Cairo University, Kasr El-Eini Street, 11562
Cairo, Egypt
*Corresponding author: Fadi M. Awadallah,
fadi_mae@hotmail.com
tion pathway in cancer cells represents
a rational
approach to cancer therapy, and several drug discovery
efforts have targeted this aberrant kinase activity in cancer
(6,7). Inhibition of EGFR has been achieved through two
main approaches: by blocking ligand binding to the extra-
cellular domain with monoclonal antibodies or by using
small-molecule inhibitors that interact at the ATP-binding
site (8).
Four series of some 4-substituted-1-phenyl-1H-pyraz-
olo[3,4-d]pyrimidine derivatives 5a–f, 6a–f, 8a–f, and
9a–f were designed to be screened for their antitumor
activity. All compounds were evaluated against breast
(MCF-7) and lung (A-549) cell lines. Six compounds 5a,
5b, 6b, 6e, 9e, and 9f displaying activity against both
cell lines were further estimated for their EGFR-TK
inhibitory activity where they revealed 41–91% inhibi-
tion and compound 6b elicited the highest activity
(91%). A docking study of these compounds into the
ATP-binding site of EGFR-TK demonstrated their bind-
ing mode where H-bonding interaction with Met793
through N1 of pyrimidine or N2 of pyrazole was
observed.
Pyrazolopyrimidine is an interesting bioactive core used for
developing molecules of biological interest. Herein, through
our ongoing efforts to develop highly potent antitumor
agents, we focused on the pyrazolo[3,4-d]pyrimidines as a
privileged lead molecule for scheming bioactive agents
with promising antitumor activity. Screening the literature
declared the antitumor activity of many 4-aralkylamino/ani-
lino-pyrazolo[3,4-d]pyrimidines (9–18). Some of these
derivatives exerted their antitumor activity via inhibition of
EGFR tyrosine kinase as demonstrated by compounds
I–III (19–21). Moreover, many derivatives with potent antitu-
mor activity were found to possess 4-(un)substitutedphenyl-
piperidinyl IV (22) and 4-(un)substitutedphenyl-piperazinyl
moieties V (23) at position 4 of the pyrazolopyrimidine
nucleus (Chart 1). Accordingly, the 1-phenyl-pyrazolo[3,4-
d]pyrimidine was selected as a pharmacophoric core for
our target compounds. The first group of compounds 5a–f
was designed by incorporating phenylpiperidin-1-yl or (un)
substitutedphenylpiperazin-1-yl moieties at position 4 of
the pyrazolo[3,4-d]pyrimidine core.
Key words: antitumor, EGFR-TK inhibition, molecular model-
ing, pyrazolo[3,4-d]pyrimidines, synthesis
Received 2 August 2014, revised 25 September 2014 and
accepted for publication 4 October 2014
Protein tyrosine kinases (PTKs) are enzymes involved in
many cellular processes such as cell proliferation, metabo-
lism, and apoptosis. Several protein tyrosine kinases are
activated in cancer cells resulting in tumor growth and pro-
gression (1). Inappropriate or uncontrolled activation of
many of these kinases, by over-expression, constitutive
activation or mutation, has shown to result in uncontrolled
cell growth. PTKs can be broadly classified as receptor
(RTK) or non-receptor kinases (2). The epidermal growth
factor receptor (EGFR) protein belongs to the ErbB family
of receptor tyrosine kinases (RTKs), which plays an impor-
tant role in the regulation of cell growth, differentiation,
and survival (3). EGFR is a glycoprotein that contains an
Furthermore, regarding the pyrimidine template as an
important component of nucleic acids and essential building
block in many antitumor agents, several reports outlined the
biological interest of compounds bearing the 2-mercapto-
6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitrile moiety
as shown by compound VI which expressed antitumor
activity against leukemia, lung and ovarian carcinoma
(Chart 2) (24). Consequently, the design of the second and
third groups of target compounds involved hybridization
of the 1-phenylpyrazolopyrimidine nucleus to the 4-(un)sub-
stitutedphenyl-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
extracellular ligand-binding domain,
a transmembrane
region, and an intracellular domain with kinase activity (4).
Over-expression of these receptors was found in many
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12451
1

Abbas et al.
F
F
HO
NH
Cl
HO
N
N
N
HN
Cl
HN
N
HN
N
H3C
NH
O
S
N
N
N
N
N
N
H
N
N
N
H
I
II
III
R
H
N
Cl
N
R'
O
N
N
N
Br
N
N
N
N
N
N
N
N
H
Chart 1: Examples of some pyrazolo[3,4-d]
pyrimidines with antitumor activity.
IV
V
group either through a sulfanyl or hydrazinyl spacer to pro-
duce compounds with the general formulae 6a–f and 8a–
f, respectively.
activity against breast (MCF-7) and lung (A-549) cell lines.
Compounds exhibiting activity against both cell lines were
further subjected to in vitro EGFR tyrosine kinase activity
to validate the mechanism of action of the compounds. In
addition, flexible molecular docking study was performed
to explore the binding mode of these compounds at the
active site of the ATP-binding site of EGFR-TK.
In the same vein, and searching for another bioactive moi-
ety of interest in the field of chemotherapy, many citations
described the potent cytotoxic activity of imidazolone-
based compounds as exemplified by the derivative VII
(25). Also, it is worth mentioning that the imidazolone ring
could be regarded as the ring contracted non-classical
isostere of the pyrimidinone template used in compounds
6a–f and 8a–f. Motivated by these findings, it was thought
worthwhile to carry out structural modification of com-
pounds 6a–f via substituting the pyrimidinone template by
the ring contracted 2-phenyl-4-benzylidene-5-imidazolone
ring along with isosteric substitution of the sulfanyl linker
with the amino one to get the fourth group of target com-
pounds of general formula 9a–f (Chart 3). The substitution
patterns of the designed compounds were selected to tar-
get additional regions in the ATP-binding site of the protein
kinase domain to create differentially selective molecules.
The designed compounds were tested for their antitumor
Chemistry
The target compounds were synthesized as illustrated in
Schemes 1 and 2. The key intermediate 4-chloro-1H-pyr-
azolo[3,4-d]pyrimidine 4 was prepared starting from ethyl
2-cyano-3-ethoxyacrylate 1 as depicted in Scheme 1. The
latter was prepared according to the method of Guvigny
and Normant from ethyl cyanoacetate and triethyl orthofor-
mate in acetic anhydride (26). Reaction of the acrylate
ester 1 with phenyl hydrazine in ethanol yielded the pyraz-
ole derivative 2 (27) which upon condensation with form-
amide furnished 1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4
(5H)-one 3 (28). Chlorination of the pyrazolopyrimidinone 3
with phosphorous oxychloride gave the 4-chloro derivative
4 (29).
Cl
The first group of target compounds, 4-[(4-(un)substituted-
phenyl)piperidin/piperazin-1-yl]-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidines 5a–f, was synthesized from the corresponding
4-chloro intermediate 4 via reaction with 1-phenylpiperi-
dine or the appropriate phenylpiperazine in dry dimethylfor-
mamide (DMF) in the presence of triethylamine
(Scheme 2). The 1H NMR spectrum of 5a showed three
multiplet signals at 1.62–2.21, 2.73–3.21, and 3.47–
3.63 ppm assigned to the nine piperidine protons, in addi-
tion to multiplet signals in the aromatic region integrated
N
HN
N
S
CN
N
HN
N
H
O
O
VII
VI
Chart 2: Pyrimidin-6-one and imidazol-5-one derivatives with
potent cytotoxic activity.
2
Chem Biol Drug Des 2014

Antitumor and Kinase Inhibition of Pyrazolopyrimidines
R
CN
R
O
R
O
N
O
X
N
CN
HN
N
HN
N
N
N
NH
HN
S
HN
N
N
N
R
N
N
N
N
N
N
N
N
N
N
N
N
Chart 3: General structures of the target
compounds.
6a-f
5a-f
8a-f
9a-f
O
O
O
O
N
NH
a
b
N
NH2
O
N
O
N
C
N
N
3
1
2
c
Cl
N
N
N
N
Scheme 1: Preparation of compound 4.
Reagents and reaction conditions. (a)
PhNHNH2, ethanol, reflux 15 h; (b)
HCONH2, 200–210 °C, 8 h; (c) POCl3, reflux
1 h.
4
for the additional phenyl ring of the phenylpiperidine moi-
ety. As for the piperazine derivatives 5b–e, H NMR spec-
reported procedure by refluxing 4-chloropyrazolopyrimidine
4 with hydrazine hydrate in ethanol [29]. Reacting the
4-hydrazinyl derivative 7 with the appropriate pyrimidine-5-
carbonitrile i in refluxing absolute ethanol afforded the third
group of compounds 8a–f (Scheme 2). The structure of
compounds 8a–f was confirmed by IR, 1H NMR, and
Mass spectroscopic analyses. 1H NMR spectra of 8a–f
showed similar signals to those revealed with compounds
6a–f, in addition to increase in the integration of broad
exchangeable signals appearing in the range of 11.27–
12.42 ppm assigned to 3 NH protons in all derivatives,
except 8c whose hydrazinyl protons appeared separately
as a broad exchangeable signal at 6.60 ppm.
1
tra revealed the characteristic two triplets of the piperazine
ring in the range of 3.20–3.49 and 3.75–4.23 ppm
assigned for the 4 protons of the 2 methylene groups at
N4 and N1 of the piperazine ring, respectively. In addition,
other characteristic protons such as the methoxy protons
of 5d that appeared as singlet signal at 3.94 ppm were
revealed.
The second group of compounds 6a–f was synthesized
by reacting the 4-chloro-pyrazolopyrimidine 4 and the
appropriate 2-mercapto-6-oxo-4-phenyl-1,6-dihydropyrimi-
dine-5-carbonitrile derivative i (30) in absolute ethanol in
the presence of potassium carbonate. The target com-
pounds 6a–f were characterized by IR spectra that
showed the cyano and carbonyl stretching vibrations at
2229–2206 and 1714–1654 cmꢀ1, respectively. 1H NMR
spectra of 6a–f revealed increase in the integration of aro-
matic protons attributed to the phenyl ring of the substi-
tuted pyrimidine moiety, in addition to the singlet signal of
the NH proton appearing in the range of 10.20–
12.42 ppm which disappeared upon deuteration. The
4-hydrazinyl derivative 7 was obtained according to the
The fourth group including 4-(un)substitutedbenzylidene-2-
phenyl-1[(1-phenyl-1H-pyrazolo[3,4-d]pyrimidine-4-yl)amino]-
1H-imidazol-5(4H)-ones 9a–f was obtained via reaction of
the 4-hydrazinyl derivative 7 with the appropriate oxazol-
ones ii (31,32) in glacial acetic acid in the presence of
freshly fused sodium acetate (Scheme 2). The structures
assigned to compounds 9a–f were confirmed from IR
spectra that showed NH bands at 3422–3332 cmꢀ1, in
addition to the carbonyl bands vibrating at 1705–
1637 cmꢀ1 (c.f. carbonyl bands of oxazolones ii appeared
Chem Biol Drug Des 2014
3

Abbas et al.
Cl
N
N
N
N
a
4
O
R''
CN
HN
HS
c
b
N
R
i
R'
O
X
N
CN
HN
NHNH2
S
N
N
R
N
N
N
N
N
N
N
N
N
N
N
6a-f
5a-f
7
O
R
X
CH
N
R'
H
R''
N
O
a
b
c
d
e
f
H
H
CF3
H
H
Cl
F
d
e
N
H
O
R
CN
ii
N
OCH3
H
CN
HN
HS
N
O
R
N
H
N
O
R
HN
HN
N
i
Scheme 2: Synthesis of compounds 5a–f,
6a–f, 7, 8a–f, and 9a–f. Reagents and
reaction conditions. (a) Phenylpiperidine/
appropriate phenylpiperazine, dry DMF,
triethylamine, reflux 15 h; (b) appropriate
derivative i, absolute ethanol, anh. K2CO3,
reflux 20 h; (c) NH2NH2.H2O, ethanol, reflux,
2.5 h; (d) appropriate derivative i, dry DMF,
TEA, reflux, 20 h; (e) appropriate derivative
ii, gl. acetic acid, fused CH3COONa, boiling
water bath, 3 h.
N
NH
N
N
HN
N
N
N
N
N
N
N
9a-f
8a-f
R, a = H, b = F, c = Cl, d = OCH3, e = N(CH3)2, f = OH
at higher frequency, 1793–1762 cmꢀ1). 1H NMR spectra
revealed an increase in the integration of aromatic protons
attributed to the 2 phenyl rings of the imidazolone moiety,
in addition to the methine (-CH=) proton that appeared
within the aromatic region and the singlet signal of the NH
proton in the range of 9.90–13.40 ppm which disappeared
upon deuteration. Other characteristic protons of some
compounds appeared as singlet signals, namely the meth-
oxy protons of compound 9d at 3.86 ppm, the six protons
of dimethylamino group of 9e at 2.55 ppm, and the OH
proton of 9f at 5.80 ppm.
noted, 1H NMR spectra were recorded in CDCl3 or
DMSO-d6 on Varian mercury 300BB at 300 MHz or Bruker
Avance III 400 MHz FT-NMR at 400 MHz. 13C NMR spec-
tra were run at 75.46 or 100 MHz. Chemical Shifts were
given in d as parts per million (ppm) downfield from tetra-
methylsilane (TMS) as internal standard. The electron
impact (EI) mass spectra were recorded on Finnigan Mat
SSQ 7000 (70 ev) mass spectrometer. Elemental micro-
analysis was performed at the Regional Center for Mycol-
ogy and Biotechnology, Azhar University. TLC were
monitored on Macherey-Nagel Alugram Sil G/UV254 silica
gel plates (0.2 mm thickness) using chloroform/methanol
(9.5:0.5) as eluent. The spots were visualized using Vilber
Lourmet ultraviolet lamp at k = 254 and 266 nm. All
reagents and solvents were purified and dried by standard
techniques.
Experimental Section
General methods
Melting points were determined with Gallenkamp digital
melting point Griffin apparatus 1901 and were uncor-
rected. FT-IR spectra were recorded on Bruker FT-IR
8400S spectrophotometer using KBr cell. Unless otherwise
Compounds 1 (26), 2 (27), 3 (28), 4 (29), i (30), ii (31,32),
5f (23), and 7 (29) were prepared according to the
reported procedures.
4
Chem Biol Drug Des 2014

Antitumor and Kinase Inhibition of Pyrazolopyrimidines
General procedure for the synthesis of compounds
(5a–f)
(C3 of pyrazolopyrimidine); 139.1 (C1 of 1-phenyl ring);
147.8 (C7a of pyrazolopyrimidine), 153.0 (C1 of phenyl ring
on piperazine); 156.5 (C6 of pyrazolopyrimidine); 160.4 (C4
of pyrazolopyrimidine). MS m/z: 356.10 [M]+. Anal. Calcd.
for C21H20N6 (356.42): C, 70.77; H, 5.66; N, 23.58.
Found: C, 70.82; H, 5.64; N, 23.73.
To a solution of the 4-chloro derivative 4 (0.46 g, 2 mmol)
in dry DMF (10 mL), the appropriate phenyl piperazine or
4-phenylpiperidine (2.1 mmol) and triethylamine (5 mL)
were added and heated under reflux for 15 h. The solution
was poured onto ice/water. The crude product was filtered
off, dried, and crystallized from ethanol to give compounds
5a–f.
4-[4-(2-Methoxyphenyl)piperazin-1-yl]-1-phenyl-
1H-pyrazolo[3,4-d]pyrimidine (5d)
Creamy powder; m.p. 273–274 °C, yield, 0.60 g; 78%. IR
1-Phenyl-4-[(4-phenylpiperidin)-1-yl]-1H-pyrazolo
[3,4-d]pyrimidine (5a)
υ
max/cmꢀ1: 3059 (CH aromatic); 2947, 2831 (CH ali-
phatic). 1H NMR (CDCl3, 300 MHz): d 3.26 (t, 4H, J = 5,
CH2-2 and CH2-6 piperazine); 3.94 (s, 3H, OCH3); 4.23
(t, 4H, J = 5, CH2-3 and CH2-5 piperazine); 6.94-8.14
(m, 9H, Ar-Hs); 8.19 (s, 1H, pyrazole CH); 8.49 (s, 1H,
pyrimidine CH). 13CNMR (CDCl3, 75 MHz): d 43.8 (piper-
azine Cs); 55.4 (OCH3); 121.1, 122.1, 124.0, 125.9,
126.0, 127.2, 128.3, 129.8 (aromatic Cs); 136.0 (C3 of
pyrazolopyrimidine); 138.1 (C1 of 1-phenyl ring); 144.5 (C1
of phenyl piperazine); 146.5 (C2 of phenyl on piperazine);
150.5 (C7a of pyrazolopyrimidine), 157.8 (C6 of pyra-
zolopyrimidne); 159.4 (C4 of pyrazolopyrimidine). MS m/z:
386.15 [M]+. Anal. Calcd. for C22H22N6O (386.45): C,
68.38; H, 5.74; N, 21.75. Found: C, 68.59; H, 5.40; N,
21.95.
Creamy powder; m.p. 260–261 °C, yield, 0.46 g; 65%. IR
υ
max/cmꢀ1: 3043 (CH aromatic); 2920, 2850 (CH aliphatic).
1H NMR (CDCl3, 300 MHz): d 1.62–2.21 (m, 4H, piperi-
dine); 2.73–3.21 (m, 1H, piperidine); 3.47–3.63 (m, 4H,
piperidine); 7.18–8.27 (m, 11H, Ar-Hs and pyrazole CH);
8.55 (s, 1H, pyrimidine CH). 13C NMR (CDCl3, 100 MHz):
d 31.1 (C3 + C5 of piperidine); 42.0 (C4 of piperidine); 46.3
(C2 + C6 of piperidine); 108.0, 122.2, 127.0, 127.6, 128.9,
129.6 (aromatic Cs); 136.4 (C3 of pyrazolopyrimidine);
138.7 (C1 of 1-phenyl ring); 149.3 (C7a of pyrazolopyrimi-
dine), 152.3 (C6 of pyrazolopyrimidine); 157.7 (C4 of pyraz-
olopyrimidine). MS m/z: 355.35 [M]+. Anal. Calcd. for
C22H21N5 (355.44): C, 74.34; H, 5.96; N, 19.70. Found:
C, 74.42 H, 6.02; N, 19.83.
4-[4-(4-Chlorophenyl)piperazin-1-yl]-1-phenyl-1H-
pyrazolo[3,4-d]pyrimidine (5e)
1-Phenyl-4-[4-(4-trifluoromethylphenyl)piperazin-
1-yl]-1H-pyrazolo[3,4-d]pyrimidine (5b)
Creamy powder; m.p. 285–287 °C, yield, 0.60 g; 70%. IR
Creamy powder; m.p. 284–285 °C, yield, 0.50 g; 65%. IR
υ
max/cmꢀ1: 3116 (CH aromatic); 2974, 2885 (CH aliphatic);
υ
max/cmꢀ1: 3109 (CH aromatic); 2958, 2843 (CH ali-
779 (C-Cl). 1H NMR (CDCl3, 300 MHz): d 3.39 (t, 4H,
J = 5, CH2-2 and CH2-6 piperazine), 4.21 (t, 4H, J = 5,
CH2-3 and CH2-5 piperazine), 6.87–8.06 (m, 9H, Ar-Hs),
8.11 (s, 1H, pyrazole CH), 8.30 (s, 1H, pyrimidine CH). 13C
NMR (DMSO-d6, 100 MHz): d 45.3 (piperazine Cs); 121.2,
122.5, 122.9, 125.0, 126.4, 128.3, 129.6, 129.8 (aromatic
Cs); 135.6 (C3 of pyrazolopyrimidine); 137.5 (C1 of 1-phe-
nyl ring); 148.7 (C7a of pyrazolopyrimidine), 152.7 (C1 of
phenyl ring on piperazine); 160.0 (C6 of pyrazolopyrimi-
dine); 162.1 (C4 of pyrazolopyrimidine). Anal. Calcd. for
C21H19ClN6 (390.87): C, 64.53; H, 4.90; N, 21.50. Found:
C, 64.27; H, 4.79; N, 21.67.
phatic). 1H NMR (CDCl3, 300 MHz): d 3.49 (t, 4H, J = 6,
CH2-2 and CH2-6 piperazine); 4.23 (t, 4H, J = 6, CH2-3
and CH2-5 piperazine); 7.09–8.14 (m, 9H, Ar-Hs); 8.18 (s,
1H, pyrazole CH); 8.49 (s, 1H, pyrimidine CH). 13C NMR
(DMSO-d6, 100 MHz):
d 47.2 (piperazine Cs); 121.6,
122.6, 124.1, 126.3, 128.1, 129.6, 130.5 (aromatic
Cs + CF3); 135.5 (C3 of pyrazolopyrimidine); 139.1 (C1 of
1-phenyl ring); 149.0 (C7a of pyrazolopyrimidine), 155.8
(C1 of phenyl ring on piperazine); 156.9 (C6 of pyrazolo-
pyrimidine); 158.6 (C4 of pyrazolopyrimidine). MS m/z:
425.35 [M + 1]+.Anal. Calcd. for C22H19F3N6 (424.42): C,
62.26; H, 4.51; N, 19.80. Found: C, 62.49 H, 4.80; N,
19.93.
4-[4-(4-Fluorophenyl)piperazin-1-yl]-1-phenyl-1H-
pyrazolo[3,4-d]pyrimidine (5f)
1-Phenyl-4-[(4-phenylpiperazin)-1-yl]-1H-pyrazolo
[3,4-d]pyrimidine (5c)
Brown powder; m.p. 288–291 °C, yield, 0.42 g; 60%. IR
Light creamy powder; m.p. 275–277 °C, yield, 0.52 g;
70%. IR υmax/cmꢀ1: 3109 (CH aromatic); 2989 (CH ali-
phatic) (23). 1H NMR (CDCl3, 300 MHz): d 3.31 (t, 4H,
J = 6, CH2-2 and CH2-6 piperazine), 4.20 (t, 4H, J = 6,
CH2-3 and CH2-5 piperazine), 6.92-8.17 (m, 10H, Ar-Hs
and pyrazole CH), 8.48 (s, 1H, pyrimidine CH). 13C NMR
υ
max/cmꢀ1: 3107 (CH aromatic); 2972, 2883 (CH aliphatic).
1H NMR (CDCl3, 300 MHz): d 3.20 (t, 4H, J = 6, CH2-2
and CH2-6 piperazine); 3.75 (t, 4H, J = 6, CH2-3 and
CH2-5 piperazine); 7.16-8.06 (m, 10H, Ar-Hs), 8.14 (s, 1H,
pyrazole CH); 8.29 (s, 1H, pyrimidine CH). 13C NMR
(DMSO-d6, 100 MHz):
d 44.2 (piperazine Cs); 122.3,
122.6, 124.8, 126.3, 127.3, 129.3, 129.7, 131.2 (aro-
matic Cs); 135.6 (C3 of pyrazolopyrimidine); 137.5 (C1 of
1-phenyl ring); 148.7 (C7a of pyrazolopyrimidine), 152.7
(DMSO-d6, 100 MHz):
d 48.2 (piperazine Cs); 121.8,
123.5, 124.2, 127.5, 127.9, 129.8 (aromatic Cs); 136.1
Chem Biol Drug Des 2014
5

Abbas et al.
(C1 of phenyl ring on piperazine); 160.0 (C6 of pyrazolo-
pyrimidine); 162.1 (C4 of pyrazolopyrimidine). MS m/z:
374.40 [M]+. Anal. Calcd. for C21H19FN6 (374.42): C,
67.37; H, 5.12; N, 22.45. Found: C, 67.08; H, 5.27; N,
22.27.
4-(4-Chlorophenyl)-6-oxo-2-[(1-phenyl-1H-pyrazolo
[3,4-d]pyrimidin-4-yl)thio]-1,6-dihydropyrimidine-5-
carbonitrile (6c)
Brown powder; m.p. 288–291 °C, yield, 3.38 g; 74%. IR
υ
max/cmꢀ1: 3421 (NH); 3118 (CH aromatic); 2198 (CꢁN);
1651 (C=O). 1H NMR (DMSO-d6, 300 MHz): d 7.21–8.13
(m, 9H, Ar-Hs); 8.18 (s, 1H, pyrazole CH); 8.53 (s, 1H,
pyrimidine CH); 10.20 (s, 1H, NH exchanged with D2O).
13C NMR (DMSO-d6, 100 MHz): d 108.1 (C5 of pyrimidi-
none); 122.2, 125.8, 126.6, 127.6, 128.2, 128.9, 129.6,
129.7 (aromatic Cs + CN group); 136.5 (C3 of pyrazolo-
pyrimidine); 138.7 (C1 of 1-phenyl ring); 149.3 (C7a of pyr-
azolopyrimidine); 152.3 (C6 of pyrazolopyrimidine); 157.7
(C4 of phenyl on pyrimidinone C-Cl); 161.4 (C2 of pyrimidi-
none); 163.6 (C=O); 172.7 (C4 of pyrimidinone); 176.3 (C4
of pyrazolopyrimidine). Anal. Calcd. for C22H12ClN7OS
(457.89): C, 57.71; H, 2.64; N, 21.41. Found: C, 57.60; H,
2.90; N, 21.31.
General procedure for the synthesis of compounds
(6a–f)
A mixture of the 4-chloro derivative 4 (2.30 g, 10 mmol),
the appropriate 2-mercapto-dihydropyrimidine-5-carbonitri-
le derivative i (10 mmol) and anhydrous potassium carbon-
ate (1.37 g, 10 mmol) was heated under reflux in absolute
ethanol (20 mL) for 20 h. The reaction mixture was filtered
while hot, and the residue was washed with hot ethanol.
The filtrate and washings were concentrated, and the sep-
arated crude product was filtered off and crystallized from
acetonitrile.
6-Oxo-4-phenyl-2-[(1-phenyl-1H-pyrazolo[3,4-d]
pyrimidin-4-yl)thio]-1,6-dihydropyrimidine-5-
carbonitrile (6a)
4-(4-Methoxyphenyl)-6-oxo-2-[(1-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-4-yl)thio]-1,6-
dihydropyrimidine-5-carbonitrile (6d)
Brown powder; m.p. 264–265 °C, yield, 3.20 g; 76%. IR
υ
Creamy powder; m.p. 296–297 °C, yield, 3.67 g; 81%. IR
max/cmꢀ1: 3394 (NH); 3113 (CH aromatic); 2214 (CꢁN);
υ
max/cmꢀ1: 3340 (NH); 3116 (CH aromatic); 2927 (CH ali-
1678 (C=O). 1H NMR (CDCl3, 300 MHz): d 7.27–8.06 (m,
10H, Ar-Hs); 8.06 (s, 1H, pyrazole CH); 8.30 (s, 1H,
pyrimidine CH); 11.00 (s, 1H, NH exchanged with D2O).
MS m/z: 423.10 [M]+. 13C NMR (CDCl3, 100 MHz): d
108.0 (C5 of pyrimidinone); 122.2, 122.3, 127.6, 127.9,
128.7, 129.1, 129.6, 129.8, 132.5 (aromatic Cs + CN
group); 136.4 (C3 of pyrazolopyrimidine); 138.6 (C1 of 1-
phenyl ring); 147.9 (C7a of pyrazolopyrimidine); 152.3 (C6
of pyrazolopyrimidine); 160.7 (C2 of pyrimidinone); 163.2
(C=O); 174.2 (C4 of pyrimidinone); 179.1 (C4 of pyrazolo-
pyrimidine). Anal. Calcd. for C22H13N7OS (423.45): C,
62.40; H, 3.09; N, 23.15. Found: C, 62.10; H, 3.30; N,
23.47.
phatic); 2214 (CꢁN); 1658 (C=O). 1H NMR (DMSO-d6,
300 MHz) d 3.85 (s, 3H, OCH3); 7.06–8.20 (m, 9H, Ar-Hs);
8.33 (s, 1H, pyrazole CH); 8.51 (s, 1H, pyrimidine CH);
12.40 (s, 1H, NH exchanged with D2O). 13C NMR (DMSO-
d6, 75 MHz): d 55.5 (OCH3); 107.5 (C5 of pyrimidinone);
120.9, 121.6, 126.9, 127.2, 129.0, 129.1, 130.8 (aromatic
Cs + CN group); 135.9 (C3 of pyrazolopyrimidine); 138.2
(C1 of 1-phenyl ring); 148.6 (C7a of pyrazolopyrimidine);
157.1 (C6 of pyrazolopyrimidine); 158.5 (C4 of phenyl on
pyrimidinone); 160.3 (C2 of pyrimidinone); 162.3 (C=O);
176.1 (C4 of pyrimidinone); 178.6 (C4 of pyrazolopyrimi-
dine). Anal. Calcd. for C23H15N7O2S (453.48): C, 60.92; H,
3.33; N, 21.62. Found: C, 61.23; H, 3.61; N, 21.95.
4-(4-Fluorophenyl)-6-oxo-2-[(1-phenyl-1H-pyrazolo
[3,4-d]pyrimidin-4-yl)thio]-1,6-dihydropyrimidine-5-
carbonitrile (6b)
4-[4-(N,N0-Dimethylamino)phenyl]-6-oxo-2-[(1-
phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)thio]-1,6-
dihydropyrimidine-5-carbonitrile (6e)
Dark creamy powder; m.p. 249–251 °C, yield, 3.50 g;
80%. IR υmax/cmꢀ1: 3398 (NH); 3113 (CH aromatic); 2218
(CꢁN); 1654 (C=O). 1H NMR (CDCl3, 300 MHz): d 7.44–
8.16 (m, 9H, Ar-Hs); 8.28 (s, 1H, pyrazole CH); 8.47 (s,
1H, pyrimidine CH); 11.50 (s, 1H, NH exchanged with
D2O). 13C NMR (CDCl3, 100 MHz): d 108.0 (C5 of pyrimidi-
none); 122.1, 122.4, 127.7, 128.2, 128.5, 128.7, 129.1,
129.3, 129.7, 130.0, 131.3 (aromatic Cs + CN group);
135.5 (C3 of pyrazolopyrimidine); 138.7 (C1 of 1-phenyl
ring); 148.3 (C7a of pyrazolopyrimidine); 150.9 (C6 of pyraz-
olopyrimidine); 162.7 (C2 of pyrimidinone); 165.2 (C=O);
173.8 (C4 of pyrimidinone); 179.2 (C4 of pyrazolopyrimi-
dine). MS m/z: 442.20 [M + 1]+. Anal. Calcd. for
C22H12FN7OS (441.44): C, 59.86; H, 2.74; N, 22.21.
Found: C, 59.93; H, 2.73; N, 22.00.
Yellow powder; m.p. 220–222 °C, yield, 3.90 g; 85%. IR
υ
max/cmꢀ1: 3298 (NH); 3110 (CH aromatic); 2920 (CH ali-
phatic); 2206 (CꢁN); 1697 (C=O). 1H NMR (DMSO-d6,
300 MHz): d 3.08 (s, 6H, 2CH3); 6.82-8.51 (m, 9H, Ar-Hs);
8.32 (s, 1H, pyrazole CH); 8.51 (s, 1H, pyrimidine CH); 12.40
(s, 1H, NH exchanged with D2O). 13C NMR (DMSO-d6,
100 MHz): d 62.3 (CH3), 107.8 (C5 of pyrimidinone); 116.1,
116.8, 122.3, 122.6, 127.1, 129.2, 129.6 (aromatic Cs + CN
group), 134.4 (C3 of pyrazolopyrimidine); 138.1 (C1 of 1-phe-
nyl ring); 150.1 (C7a of pyrazolopyrimidine); 151.2 (C6 of pyr-
azolopyrimidine); 157.5 (C4 of phenyl on pyrimidinone);
158.6 (C2 of pyrimidinone); 167.2 (C=O); 176.0 (C4 of pyri-
midinone); 178.8 (C4 of pyrazolopyrimidine). MS m/z: 466.20
[M]+. Anal. Calcd. for C24H18N8OS (466.52): C, 61.79; H,
3.89; N, 24.02. Found: C, 62.01; H, 4.09; N, 24.67.
6
Chem Biol Drug Des 2014

Antitumor and Kinase Inhibition of Pyrazolopyrimidines
4-(4-Hydroxyphenyl)-6-oxo-2-[(1-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-4-yl)thio]-1,6-
dihydropyrimidine-5-carbonitrile (6f)
exchanged with D2O). 13C NMR (DMSO-d6, 100 MHz) d
108.0 (C5 of pyrimidinone); 114.6, 115.5, 117.2, 122.5,
125.7, 127.1, 129.5, 129.7, 130.0 (aromatic Cs + CN
group); 134.1 (C3 of pyrazolopyrimidine); 138.6 (C1 of
1-phenyl); 147.0 (C7a of pyrazolopyrimidine); 154.2 (C6 of
pyrazolopyrimidine); 159.0 (C2 pyrimidinone); 161.7 (C=O);
165.5 (C4 of pyrazolopyrimidine); 171.3 (C4 of pyrimidi-
none). MS m/z: 439.10 [M]+. Anal. Calcd. for C22H14FN9O
(439.40): C, 60.13; H, 3.21; N, 28.69. Found: C, 60.21; H,
3.28; N, 28.76.
Dark creamy powder; m.p. 262–263 °C, yield, 3.45 g; 79%.
IR υmax/cmꢀ1: 3288 (OH); 3161 (NH); 3082 (CH aromatic);
1
2229 (CꢁN); 1714 (C=O). H NMR (DMSO-d6, 300 MHz): d
7.14–8.10 (m, 9H, Ar-Hs); 8.19 (s, 1H, pyrazole CH); 8.32 (s,
1H, pyrimidine CH); 11.60 (s, 1H, OH, exchanged with D2O),
12.42 (s, 1H, NH exchanged with D2O). 13C NMR (DMSO-
d6, 100 MHz): d 108.1 (C5 of pyrimidinone); 116.2, 116.9,
122.2, 122.8, 127.5, 129.6 (aromatic Cs + CN group),
134.5 (C3 of pyrazolopyrimidine); 138.6 (C1 of 1-phenyl ring);
149.2 (C7a of pyrazolopyrimidine); 152.3 (C6 of pyrazolopyr-
imidine); 155.1 (C4 of phenyl on pyrimidinone); 157.7 (C2 of
pyrimidinone); 163.1 (C=O); 176.0 (C4 of pyrimidinone);
179.0 (C4 of pyrazolopyrimidine). MS m/z: 439.10 [M]+. Anal.
Calcd. for C22H13N7O2S (439.45): C, 60.13; H, 2.98; N,
22.31. Found: C, 60.00; H, 3.21; N, 22.54.
4-(4-Chlorophenyl)-6-oxo-2-[2-(1-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-4-yl)hydrazinyl]-1,6-
dihydropyrimidine-5-carbonitrile (8c)
Creamy powder; m.p. > 300 °C, yield, 0.75 g; 85%. IR
υ
max/cmꢀ1: 3394 (NHs); 3050 (CH aromatic); 2209 (CꢁN);
1663 (C=O). 1H NMR (DMSO-d6, 300 MHz): d 6.60 (s,
2NHs exchanged with D2O); 7.27–7.75 (m, 9H, Ar-Hs);
8.00 (s, 1H, pyrazole CH); 8.18 (s, 1H, pyrimidine CH);
11.27 (s, 1H, NH exchanged with D2O). 13C NMR (DMSO-
d6, 100 MHz) d 108.0 (C5 of pyrimidinone); 115.1, 116.5,
118.0, 123.3, 124.2, 126.6, 127.1, 129.5, 131.8 (aromatic
Cs + CN group); 135.2 (C3 of pyrazolopyrimidine); 140.6
(C1 of 1-phenyl); 146.8 (C7a of pyrazolopyrimidine); 155.2
(C6 of pyrazolopyrimidine); 157.8 (C2 pyrimidinone); 159.7
(C=O); 162.5 (C4 of pyrazolopyrimidine); 173.9 (C4 of pyri-
midinone). MS m/z: 457.00, 455.00 [M + 2, M]+. Anal.
Calcd. for C22H14ClN9O (455.86): C, 57.96; H, 3.10; N,
27.65. Found: C, 57.94; H, 3.14; N, 27.78.
General procedure for the synthesis of compounds
(8a–f)
A mixture of the 4-hydrazinyl derivative 7 (0.45 g, 2 mmol),
the appropriate 2-mercapto-dihydropyrimidine-5-carbonitri-
le derivative i (2 mmol) and triethylamine (3 mL) in DMF
(5 mL) was heated under reflux for 20 h. The mixture was
cooled and poured onto ice/water. The obtained precipi-
tate was filtered off, washed with water, dried, and crystal-
lized from DMF/water.
6-Oxo-4-phenyl-2-[2-(1-phenyl-1H-pyrazolo[3,4-d]
pyrimidin-4-yl)hydrazinyl]-1,6-dihydropyrimidine-5-
carbonitrile (8a)
4-(4-Methoxyphenyl)-6-oxo-2-[2-(1-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-4-yl)hydrazinyl]-1,6-
dihydropyrimidine-5-carbonitrile (8d)
Yellow powder; m.p. 260–262 °C, yield, 0.63 g; 75%. IR
υ
max/cmꢀ1: 3435, 3415 (NHs); 3026 (CH aromatic); 2220
Creamy powder; m.p. 282–283 °C, yield, 0.74 g; 82%. IR
(CꢁN); 1718 (C=O). 1H NMR (DMSO-d6, 300 MHz): d 7.38–
8.32 (m, 10H, Ar-Hs); 8.33 (s, 1H, pyrazole CH); 8.50 (s, 1H,
pyrimidine CH); 12.42 (s, 3H, NHs exchanged with D2O).
13C NMR (DMSO-d6, 100 MHz) d 108.1 (C5 of pyrimidinone);
114.8, 115.0, 115.4, 116.7, 121.3, 122.2, 124.4, 127.5,
129.1, 129.6, 130.4 (aromatic Cs + CN group); 133.9 (C3 of
pyrazolopyrimidine); 138.7 (C1 of 1-phenyl); 149.2 (C7a of
pyrazolopyrimidine); 152.3 (C6 of pyrazolopyrimidine); 157.6
υ
max/cmꢀ1: 3448, 3298 (NHs); 3113 (CH aromatic); 2920
(CH aliphatic); 2206 (CꢁN); 1697 (C=O). 1H NMR (DMSO-
d6, 300 MHz): d 3.87 (s, 3H, OCH3); 7.13-8.20 (m, 9H,
Ar-Hs); 8.31 (s, 1H, pyrazole CH); 8.33 (s, 1H, pyrimidine
CH); 12.40 (s, 3H, NHs exchanged with D2O). 13C NMR
(DMSO-d6, 100 MHz) d 55.3 (OCH3); 108.1 (C5 of pyrimid-
inone); 115.2, 115.6, 116.3, 118.2, 121.4, 122.6, 126.7,
128.5, 129.3 (aromatic Cs + CN group); 133.1 (C3 of pyr-
azolopyrimidine); 137.9 (C1 of 1-phenyl); 145.7 (C7a of pyr-
azolopyrimidine); 153.8 (C6 of pyrazolopyrimidine); 158.8
(C2 pyrimidinone); 161.1 (C=O); 163.6 (C4 of pyrazolopyr-
imidine); 174.0 (C4 of pyrimidinone). MS m/z: 451.10 [M]+.
Anal. Calcd. for C23H17N9O2 (451.44): C, 61.19; H, 3.80;
N, 27.92. Found: C, 61.22; H, 3.77; N, 28.04.
(C2 pyrimidinone); 162.8 (C=O); 164.0 (C4 of pyrazolopyrimi-
+
dine); 174.3 (C4 of pyrimidinone). MS m/z: 421.30 [M]
.
Anal. Calcd. for C22H15N9O (421.41): C, 62.70; H, 3.59; N,
29.91. Found: C, 62.78; H, 3.62; N, 30.11.
4-(4-Fluorophenyl)-6-oxo-2-[2-(1-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-4-yl)hydrazinyl]-1,6-
dihydropyrimidine-5-carbonitrile (8b)
Dark creamy powder; m.p. 218–219 °C, yield, 0.70 g;
80%. IR υmax/cmꢀ1: 3414-3294 (NHs); 3055 (CH aro-
matic); 2220 (CꢁN); 1674 (C=O). 1H NMR (DMSO-d6,
300 MHz): d 7.00–7.95 (m, 9H, Ar-Hs); 8.23 (s, 1H, pyraz-
ole CH); 8.45 (s, 1H, pyrimidine CH); 12.10 (s, 3H, NHs
4-[4-(N,N0-Dimethylamino)phenyl]-6-oxo-2-[2-(1-
phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)
hydrazinyl]-1,6-dihydropyrimidine-5-carbonitrile
(8e)
Yellow powder; m.p. 286–288 °C, yield, 0.74 g; 80%. IR
υ
max/cmꢀ1: 3400 (NHs); 3045 (CH aromatic); 2951 (CH
Chem Biol Drug Des 2014
7

Abbas et al.
aliphatic); 2208 (CꢁN); 1705 (C=O). 1H NMR (DMSO-d6,
300 MHz): d 3.08 (s, 6H, 2CH3); 6.82-8.18 (m, 9H, Ar-Hs);
8.20 (s, 1H, pyrazole CH); 8.32 (s, 1H, pyrimidine CH);
12.40 (s, 3H, NHs exchanged with D2O). 13C NMR
(DMSO-d6, 75 MHz) d 61.3 (CH3); 107.6 (C5 of pyrimidi-
none); 111.6–129.1 (aromatic Cs + CN group); 135.9 (C3
of pyrazolopyrimidine); 138.2 (C1 of 1-phenyl); 148.7 (C7a
of pyrazolopyrimidine); 148.9 (C4 of phenyl on pyrimidi-
none); 153.6 (C6 of pyrazolopyrimidine); 157.1 (C2 pyrimidi-
none); 163.4 (C=O); 163.6 (C4 of pyrazolopyrimidine);
176.2 (C4 of pyrimidinone). MS m/z: 464.10 [M]+. Anal.
Calcd. for C24H20N10O (464.48): C, 62.06; H, 4.34; N,
30.16. Found: C, 62.15; H, 4.41; N, 30.31.
129.3 (aromatic Cs); 136.4 (C3 of pyrazolopyrimidine);
139.1 (C1 of 1-phenyl ring); 149.3 (C7a of pyrazolopyrimi-
dine), 152.6 (C2 of pyrazolone); 158.4 (C6 of pyrazolopyr-
imidine); 165.0 (C=O); 170.7 (C4 of pyrazolopyrimidine).
MS m/z: 457.00 [M] +. Anal. Calcd. for C27H19N7O
(457.50): C, 70.89; H, 4.19; N, 21.43. Found: C, 70.96; H,
4.22; N, 21.59.
4-(4-Fluorobenzylidene)-2-phenyl-3-[(1-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-4-yl)amino]-1H-imidazol-5
(4H)-one (9b)
White powder; m.p. 218–220 °C, yield, 0.38 g; 80%. IR
υ
max/cmꢀ1: 3414 (NH); 3062 (CH aromatic); 2950 (CH ali-
phatic); 1693 (C=O). 1H NMR (DMSO-d6, 400 MHz): d
7.38-8.19 (m, 15H, Ar-Hs + methine H); 8.32 (s, 1H, pyr-
azole CH); 8.38 (s, 1H, pyrimidine CH); 12.45 (s, 1H, NH
exchanged with D2O). 13C NMR (DMSO-d6, 100 MHz): d
106.1 (methine C); 116.5, 116.8, 122.2, 125.5, 127.6,
128.4, 129.6, 129.8, 130.6 (aromatic Cs); 135.2 (C3 of
pyrazolopyrimidine); 138.7 (C1 of 1-phenyl ring); 149.2 (C7a
of pyrazolopyrimidine); 152.3 (C2 of pyrazolone); 157.7 (C6
of pyrazolopyrimidine); 162.7 (C4 of phenyl C-F); 163.6
(C=O); 175.3 (C4 of pyrazolopyrimidine). MS m/z: 475.00
[M]+. Anal. Calcd. for C28H18FN7O (475.49): C, 68.20; H,
3.82; N, 20.62. Found: C, 68.29; H, 3.81; N, 20.71.
4-(4-Hydroxyphenyl)-6-oxo-2-[2-(1-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-4-yl)hydrazinyl]-1,6-
dihydropyrimidine-5-carbonitrile (8f)
Creamy powder; m.p. 256–258 °C, yield, 0.70 g; 79%. IR
υ
max/cmꢀ1: 3286 (OH); 3159, 3113 (NHs); 3024 (CH aro-
matic); 2229 (CꢁN); 1716 (C=O). 1H NMR (DMSO-d6,
300 MHz): d 6.93–8.23 (m, 9H, Ar-Hs); 8.32 (s, 1H, pyraz-
ole CH); 8.40 (s, 1H, pyrimidine CH); 10.79 (s, 1H, OH
exchanged with D2O); 12.42 (s, 3H, NHs exchanged with
D2O). 13C NMR (DMSO-d6, 100 MHz) d 108.1 (C5 of pyri-
midinone); 116.9, 117.0, 122.2, 122.9, 127.6, 129.7 (aro-
matic Cs + CN group); 134.4 (C3 of pyrazolopyrimidine);
138.7 (C1 of 1-phenyl); 149.2 (C7a of pyrazolopyrimidine);
152.3 (C6 of pyrazolopyrimidine); 155.1 (C4 of phenyl on
pyrimidinone); 157.6 (C2 pyrimidinone); 163.1 (C=O); 163.4
(C4 of pyrazolopyrimidine); 173.2 (C4 of pyrimidinone). MS
m/z: 437.10 [M]+. Anal. Calcd. for C22H15N9O2 (437.13):
C, 60.41; H, 3.46; N, 28.82. Found: C, 60.47; H, 3.49; N,
28.98.
4-(4-Chlorobenzylidene)-2-phenyl-3-[(1-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-4-yl)amino]-1H-imidazol-5
(4H)-one (9c)
Yellow powder; m.p. > 300 °C, yield, 0.40 g; 85%. IR
υ
max/cmꢀ1: 3338 (NH); 3057 (CH aromatic); 2941 (CH ali-
phatic); 1697 (C=O). 1H NMR (CDCl3, 300 MHz): d 7.21-
8.22 (m, 15H, Ar-Hs + methine H); 8.24 (s, 1H, pyrazole
CH); 8.51 (s, 1H, pyrimidine CH); 11.80 (s, 1H, NH
exchanged with D2O). 13C NMR (CDCl3, 100 MHz): d
108.1 (methine C); 115.2, 122.2, 125.8, 126.7, 127.6,
128.2, 128.9, 129.7, 129.8, 131.8, 133.8 (aromatic Cs);
135.0 (C3 of pyrazolopyrimidine); 138.7 (C1 of 1-phenyl
ring); 149.2 (C7a of pyrazolopyrimidine), 152.3 (C2 of py-
razolone); 157.7 (C6 of pyrazolopyrimidine); 162.4 (C4 of
phenyl C-Cl); 167.6 (C=O); 172.4 (C4 of pyrazolopyrimi-
dine). Anal. Calcd. for C27H18ClN7O (491.94): C, 65.92; H,
3.69; N, 19.93. Found: C, 65.98; H, 3.73; N, 20.04.
General procedure for the synthesis of compounds
(9a–f)
An equimolar mixture of the appropriate oxazolones ii
(1 mmol) and the 4-hydrazinopyrazolo[3,4-d]pyrimidine
derivative 7 (0.23 g, 1 mmol) in glacial acetic acid (5 mL)
containing freshly fused sodium acetate (0.03 g,
0.36 mmol) was heated in a boiling water bath with con-
stant stirring for 3 h. The solid product separated on cool-
ing was filtered off, washed with water, and crystallized
from ethanol.
4-(4-Methoxybenzylidene)-2-phenyl-3-[(1-phenyl-
1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino]-1H-
imidazol-5(4H)-one (9d)
4-Benzylidene-2-phenyl-3-[(1-phenyl-1H-pyrazolo
[3,4-d]pyrimidin-4-yl)amino]-1H-imidazol-5(4H)-one
(9a)
Creamy powder; m.p. 282–283 °C, yield, 0.40 g; 82%. IR
Creamy powder; m.p. 260–262 °C, yield, 0.34 g; 75%. IR
υ
max/cmꢀ1: 3342 (NH); 3100 (CH aromatic); 2924 (CH ali-
υ
max/cmꢀ1: 3421 (NH); 3058 (CH aromatic); 2976 (CH ali-
phatic); 1637 (C=O). 1H NMR (DMSO-d6, 300 MHz): d
3.86 (s, 3H, OCH3); 7.09–8.29 (m, 15H, Ar-Hs + methine
H); 8.32 (s, 1H, pyrazole CH); 8.33 (s, 1H, pyrimidine CH);
12.40 (s, 1H, NH exchanged with D2O). 13C NMR (CDCl3,
100 MHz): d 55.5 (OCH3); 107.5 (methine C); 114.2,
122.3, 125.7, 127.4, 128.0, 128.9, 130.1, 131.8 (aromatic
phatic); 1661 (C=O). 1H NMR (CDCl3, 300 MHz): d 7.25–
8.20 (m, 16H, Ar-Hs + methine H); 8.22 (s, 1H, pyrazole
CH); 8.26 (s, 1H, pyrimidine CH); 10.20 (s, 1H, NH
exchanged with D2O). 13C NMR (DMSO-d6, 100 MHz): d
108.1 (methine C); 122.2, 124.1, 126.3, 127.4, 128.3,
8
Chem Biol Drug Des 2014

Antitumor and Kinase Inhibition of Pyrazolopyrimidines
Cs); 134.1 (C3 of pyrazolopyrimidine); 137.9 (C1 of 1-
phenyl ring); 147.1 (C7a of pyrazolopyrimidine); 150.3 (C2
of pyrazolone); 156.2 (C6 of pyrazolopyrimidine); 164.4 (C4
of phenyl C-Cl); 166.9 (C=O); 171.9 (C4 of pyrazolopyrimi-
dine). MS m/z: 487.00 [M]+. Anal. Calcd. for C28H21N7O2
(487.52): C, 68.98; H, 4.34; N, 20.11. Found: C, 68.95; H,
4.42; N, 20.19.
removed from liquid nitrogen storage and was immediately
placed into a water bath; then, it was cultured in a flask
containing complete media and incubated in an incubator
at 37 °C. The cells were seeded in a plate, 55 000 per
well for A-549 cell line and 35 000 per well for MCF-7 for
24 h at 37 °C. The tested compounds were added at dif-
ferent concentrations (0, 0.125, 0.25, 0.50, and 1 lmol/
mL) in each well. Triplicate wells were prepared for each
individual dose. The same procedure was applied for neg-
ative control and positive control drug (doxorubicin); then,
all were incubated for 24 h. 100 lL of neutral red was
added per well then incubated for 3 h. Excess unbound
dye was removed using 150 lL of destain solution per
well. The color intensity was measured in an ELISA reader
at a wavelength of 490 nm. The percentage inhibition of
growth of tumor cells was measured for all the tested
compounds against breast MCF-7 and lung A-549 and is
presented in Table 1.
4-[4-(N,N0-Dimethylamino)benzylidene]-2-phenyl-3-
[(1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)
amino]-1H-imidazol-5(4H)-one (9e)
Brown powder; m.p. 286–288 °C, yield, 0.40 g; 80%. IR
υ
max/cmꢀ1: 3332 (NH); 3039 (CH aromatic); 2897 (CH ali-
phatic); 1693 (C=O). 1H NMR (DMSO-d6, 300 MHz): d
2.55 (s, 6H, 2CH3); 7.38–8.19 (m, 15H, Ar-Hs + methine
H); 8.30 (s, 1H, pyrazole); 8.33 (s, 1H, pyrimidine CH);
12.40 (s, 1H, NH exchanged with D2O). 13C NMR (CDCl3,
100 MHz): d 62.5 (CH3), 107.9 (methine C); 115.5, 117.9,
122.2, 124.8, 127.6, 128.7, 129.5, 130.4 (aromatic Cs);
133.6 (C3 of pyrazolopyrimidine); 138.8 (C1 of 1-phenyl
ring); 145.2 (C7a of pyrazolopyrimidine); 149.3 (C2 of py-
razolone); 157.0 (C6 of pyrazolopyrimidine); 165.4 (C4 of
phenyl C-Cl); 168.3 (C=O); 174.5 (C4 of pyrazolopyrimi-
dine). Anal. Calcd. for C29H24N8O (500.57): C, 69.59; H,
4.83; N, 22.39. Found: C, 69.62; H, 4.81; N, 22.48.
In vitro EGFR tyrosine kinase inhibition
The kinase inhibitor compound profiling (KICP) was carried
out at Kinexus Bioinformatics Corporation, Kinexus labora-
tory, Vancouver, B.C. Canada. The method was based on
Table 1: Percentage inhibition of breast MCF-7 and lung A-549
tumor cell lines caused by the synthesized compounds at a single
dose of 1 lmol/mL and IC50 (lmol/mL) of compounds showing
more than 50% inhibition
4-(4-Hydroxybenzylidene)-2-phenyl-3-[(1-phenyl-
1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino]-1H-
imidazol-5(4H)-one (9f)
Breast MCF-7
% inhibition
Lung A-549
% inhibition
Brown powder; m.p. 256–257 °C, yield, 0.37 g; 79%. IR
IC50 in
lmol/
IC50 in
lmol/
υ
max/cmꢀ1: 3410 (OH); 3230 (NH); 3062 (CH aromatic);
2929 (CH aliphatic); 1705 (C=O). 1H NMR (DMSO-d6,
300 MHz): d 5.80 (s, 1H, OH exchanged with D2O), 7.28–
8.22 (m, 15H, Ar-Hs + methine H), 8.30 (s, 1H, pyrazole
CH), 8.38 (s, 1H, pyrimidine CH), 12.40 (s, 1H, NH
exchanged with D2O). 13C NMR (CDCl3, 100 MHz): d
108.2 (methine C); 114.4, 116.3, 122.2, 124.4, 126.3,
127.2, 128.5, 131.3 (aromatic Cs); 134.3 (C3 of pyrazolo-
pyrimidine); 138.4 (C1 of 1-phenyl ring); 145.2 (C7a of pyr-
azolopyrimidine); 148.8 (C2 of pyrazolone); 155.1 (C6 of
pyrazolopyrimidine); 165.4 (C4 of phenyl C-Cl); 167.6
(C=O); 173.4 (C4 of pyrazolopyrimidine). MS m/z: 473.10
[M]+. Anal. Calcd. for C27H19N7O2 (473.50): C, 68.49; H,
4.04; N, 20.71. Found: C, 68.53; H, 4.08; N, 20.82.
Compound at 1 lmol/mL mL
at 1 lmol/mL mL
5a
5b
5c
5d
5e
5f
83.36
71.27
NA
0.72
0.70
–
–
–
0.85
–
0.08
–
–
0.12
–
76.33
61.17
46.18
67.05
68.23
NA
0.28
0.88
–
0.16
0.16
–
30.14
17.35
59.68
17.60
84.20
44.20
35.80
63.60
21.60
30.20
NA
34.60
46.95
17.60
88.39
77.70
84.06
10.00
NA
6a
6b
6c
6d
6e
6f
48.09
62.35
45.29
NA
–
0.24
–
–
66.76
67.64
42.94
45.58
61.17
70.00
NA
0.15
0.94
–
8a
8b
8c
8d
8e
8f
–
–
–
–
–
0.18
0.28
–
In vitro antitumor activity
The cytotoxic activity was carried out at the Biotechnology
Laboratory, Cairo University Research Park (CURP), Fac-
ulty of Agriculture, Cairo University (33,34). The cytotoxic
activity of the tested compounds was measured in vitro
using the neutral red uptake assay method. It is based on
the ability of viable cells to incorporate and bind the supra-
vital dye neutral red in the lysosomes. The amount of dye
extracted from the cells is directly proportional to the cell
mass. The cryovial containing the frozen cells was
–
0.10
0.20
0.10
–
–
0.10
0.84
29.41
22.35
47.64
29.41
26.33
52.67
67.94
–
–
–
–
–
0.32
0.28
9a
9b
9c
9d
9e
9f
78.30
63.60
NA, no activity.
Chem Biol Drug Des 2014
9

Abbas et al.
radioisotope assay in which direct quantification of radio-
labeled phosphate from labeled 33P-ATP onto a peptide or
protein substrate of the target protein kinase was carried
out. This assay provided a direct measure of the effect of
a compound on the enzyme activity rather than a measure
of the ability of a compound to bind near the active site of
the kinase, Herein, profiling of the six compounds against
EGFR (T790M/L858R mutant) at single concentration of
50 lM in duplicate was performed. Gefitinib was used as a
positive control at dose of 10 lM.
face and subjected to energy minimization using the
MMFF94X force field. The produced model was subjected
to Systematic Conformational Search where all items were
set as default with RMS gradient of 0.01 kcal/mol and
ꢀ
RMS distance of 0.1 A. The X-ray crystallographic
structure of EGRF-TK co-crystallized with its ligand was
downloaded from the protein data bank (PDB) code
3W2O. The enzyme was prepared for docking as followed:
ꢂ
The ligand molecule was removed from the active site.
Tested solutions were prepared to give final volume of
25 lL according to the following component ratios:
Component 1: 5 lL of diluted active EGFR; Component
2: 5 lL of stock solution of peptide substrate; Compo-
nent 3: 5 lL of kinase assay buffer; Component 4: 5 lL
of compound (50 lM) or 10% DMSO (negative control)
or 1 lM staurosporine (positive control), and Component
5: 5 lL of 33P-ATP (250 lM stock, 0.8 lCi). The assay
was initiated by the addition of 33P-ATP, and the
reaction mixture was incubated at room temperature for
20–30 min.
ꢂ
H atoms were added to the structure with their standard
geometry.
ꢂ
MOE Alpha Site Finder was used for the active sites
search in the enzyme structure, and dummy atoms were
created from the obtained alpha spheres.
ꢂ
Docking of the least energetic conformer for each com-
pound in the active site of the ATP-binding site of EGFR-
TK was performed.
After the incubation period, the assay was terminated by
spotting 10 lL of the reaction mixture onto multiscreen
phosphocellulose P81 plate. The multiscreen phosphocel-
lulose P81 plate was washed three times for approximately
15 min each in a 1% phosphoric acid solution. The radio-
activity in the captured 33P-labeled protein substrate on
the P81 plate is quantified using a Trilux scintillation coun-
ter. Blank control was performed with equal volume of
assay dilution buffer instead of the substrate.
ꢂ
Docking protocol was verified by redocking of the
co-crystallized ligand in the vicinity of the active site of the
enzyme with energy score = 26.2922 kCal/mol and
RMSD = 1.78.
Results and Discussion
In vitro antitumor activity
The synthesized compounds were screened for their anti-
tumor activity against breast (MCF-7) and lung (A-549) cell
lines at a single dose of 1 lmol/mL. Compounds eliciting
more than 50% inhibition in the single-dose screening
were screened at different dose levels to calculate their
IC50 values (lmol/mL) (Table 1).
The results were observed as % activity change compared
to control (Table 2). Values of the results are significant if
they cause >25% change in activity compared to the
control.
Docking studies
Concerning breast (MCF-7) cell lines: The results of the sin-
gle-dose screening of the synthesized compounds against
breast (MCF-7) cell line showed ten compounds achieving
more than 50% inhibition. These compounds were further
screened at different dose levels to calculate their IC50 val-
ues. Regarding the activity of the series of compounds 5a–
f, the 4-trifluoromethylphenylpiperazin-1-yl compound 5b
was the most active (IC50 = 0.70 lmol/mL) followed by the
4-phenylpiperidin-1-yl 5a derivative (IC50 = 0.72 lmol/mL)
then the 4-fluorophenyl piperazin-1-yl analogue 5f
(IC50 = 0.85 lmol/mL). On the other hand, out the deriva-
tives bearing the 5-cyano-4-(4-substitutedphenyl)-6-oxo-
pyrimidine template through a sulfanyl linker emerged two
promising compounds, the 4-(4-fluorophenyl) derivative 6b
and its 4-(4-N,N’-dimethylamino)phenyl analogue 6e which
possessed IC50 = 0.08 and 0.12 lmol/mL, respectively. It
is worth mentioning that compound 6b was the most
active among all synthesized compounds. Also, it was
The molecular docking study was carried out on Molecular
Operating Environment (MOE, 10.2008) software provided
by chemical computing group, Canada. The program
operated on Windows XP operating system installed on
Intel Pentium IV 2.80 MHz processor, 512 MB RAM. The
target compounds were built using the MOE builder inter-
Table 2: The EGFR tyrosine kinase assays of over EGFR tyrosine
kinase at a single dose concentration of 50 lM
Compound
% Inhibition
5a
5b
6b
6e
9e
9f
41
51
91
71
59
77
10
Chem Biol Drug Des 2014

Antitumor and Kinase Inhibition of Pyrazolopyrimidines
Chart 6: Docking of compound 5b in the ATP-binding site of the
EGFR tyrosine kinase enzyme.
Chart 4: Co-crystallized ligand interaction with ATP-binding site
of the EGFR tyrosine kinase enzyme (PDB code 3W2O).
Chart 7: Docking of compound 6b in the ATP-binding site of the
Chart 5: Docking of compound 5a in the ATP-binding site of the
EGFR tyrosine kinase enzyme.
EGFR tyrosine kinase enzyme.
sulfanyl linker (6a–f) or the 5-oxoimidazoline via an amino
spacer (9a–f) at position 4 of the pyrazolopyrimidine core
were more active than those bearing either 6-oxopyrimidine
moiety through a hydrazinyl bridge (8a–f) or hetero-alicyclic
moiety (piperidine or piperazine) directly attached to the
pyrazolopyrimidine core (5a–f).
observed that the series bearing the 6-oxopyrimidine moi-
ety via a hydrazinyl spacer was generally of lower potency
than that possessing the sulfanyl linker 6a–f except for the
4-(4-hydroxyphenyl) derivative 8f (IC50 = 0.10 lmol/mL).
Finally, the compounds bearing the ring contracted 5-oxo-
imidazoline moiety 9a–f were potentially more active than
their 6-oxopyrimidine congeners. The 4-(4-fluorobenzylid-
ene) derivative 9b and the 4-(4- N,N’-dimethylaminobenzy-
lidene) derivative 9e were equipotent, and they were the
most active in this series (IC50 = 0.10 lmol/mL). The 4-
benzylidene compound 9a possessed half the potency of
9b and 9e (IC50 = 0.20 lmol/mL). On the other hand, the
4-(4-hydroxybenzylidene) analogue 9f was the least potent
in this series (IC50 = 0.84 lmol/mL). Based on the antitu-
mor activity of the tested compounds and their IC50 values,
it could be concluded that derivatives bearing the substi-
Regarding lung (A-549) cell line: The single-dose screening
of the synthesized compounds against lung A-549 cell line
revealed that eleven compounds exhibited more than 50%
inhibition, and accordingly, IC50 values for these com-
pounds were calculated (Table 1). The results showed that
compounds bearing the 4-phenylpiperidine/4-phenylpiper-
azine moieties were more effective against lung cell lines
than breast cell lines as demonstrated by 5a, 5b, 5d, and
5e (IC50 = 0.28, 0.88, 0.16, and 0.16 lmol/mL, respec-
tively). The 4-(2-methoxyphenyl)piperazin-1-yl derivative 5d
and its 4-(4-chlorophenyl)piperazin-1-yl analogue 5e were
tuted heterocyclic moieties 6-oxopyrimidine through
a
Chem Biol Drug Des 2014
11

Abbas et al.
In general, there was no consistent relation between the
lipophilicity and/or electronic nature of the substituents (R)
and the activity in any of the series.
Finally, it could be observed that some derivatives
explored activity against both cell lines such as com-
pounds 5a, 5b, 6b, 6e, 9e, and 9f. These compounds
represent candidates for future optimization.
In vitro EGFR tyrosine kinase inhibition
Compounds exhibiting activity against both tested cell lines
were subjected to EGFR tyrosine kinase inhibition testing.
Gefitinib was used as positive control at dose of 10 lM
where it exhibited 100% inhibition. The profiling data of the
tested compounds 5a, 5b, 6b, 6e, 9e, and 9f against
EGFR showed modest inhibition of EGFR target ranging
from 41% to 91% as presented in Table 2. All compounds
except 5a showed inhibition percent more than 50%, and
two compounds demonstrated more than 75% inhibition,
namely compounds 6b and 9f. The 4-(4-phenyl)piperidin-
1-yl derivative 5a, the 4-(4-trifluoromethyl)phenyl-piperazin-
1-yl derivative 5b, and the 3-substituted-4-(4-N,N’-dimeth-
Chart 8: Docking of compound 6e in the ATP-binding site of the
EGFR tyrosine kinase enzyme.
ylamino)benzylidene-2-phenyl-1H-imidazol-5(4H)-one
9e
showed moderate to good activity (41%, 51%, and 59%
inhibition, respectively). Remarkable inhibitory activity was
observed with compounds 6e and 9f (71% and 77%
inhibition, respectively). The most potent compound was
the 2-substituted-4-(4-fluorophenyl)-6-oxo-1,6-dihydropyr-
imidine-5-carbonitrile derivative 6b (91% inhibition).
Docking studies
Compounds 5a, 5b, 6b, 6e, and 9e eliciting activity
against both tested cell lines and showing good to potent
EGFR tyrosine kinase inhibition (41–91%) were docked into
the active site of the ATP-binding site of EGRF-TK to visu-
alize the orientation and binding mode of the active com-
pounds. Molecular Operating Environment (MOE 10.2008)
software provided by chemical computing group, Canada
was used. The co-ordinates of the EGFR-TK structure
were obtained from the crystal structure of EGFR with its
co-crystallized inhibitor: N-(2-(4-[(3-chloro-4-(3-(trifluorom-
ethyl)phenoxy)phenyl)amino]-5H-pyrrolo[3,2-d]pyrimidin-5-
yl)ethyl)-3-hydroxy-3-methylbutanamide (PDB code 3W2O)
which was chosen because of the structure similarity
between the inhibitor and our target compounds. The
energy minimization for the compounds was performed
with MOE with MMFF94X force field, and the partial
charges were automatically calculated. The root mean
square difference (RMSD) between the top docking pose
and original crystallographic geometry was 1.78. The main
interaction between the co-crystallized ligand and the
enzyme-active site is a H-bond involving N1-pyrimidine
Chart 9: Docking of compound 9e in the ATP-binding site of the
EGFR tyrosine kinase enzyme.
equipotent, and they were the most active in this series.
Compounds bearing the 6-oxopyrimidine moiety via the
sulfanyl linker 6a–f showed considerable activity where the
4-(4-N,N’-dimethylamino)phenyl
analogue
6e
(IC50 = 0.15 lmol/mL) was the most active among this
series followed by compounds 6b and 6f (IC50 = 0.24 and
0.94 lmol/mL, respectively). Alternatively, replacing the
sulfanyl linker by a hydrazinyl one emerged two potentially
active compounds, namely 8c and 8d with IC50 = 0.18
and 0.28 lmol/mL, respectively. On the other hand, the
ring contracted 5-oxoimidazoline derivatives 9e and 9f
exhibited good potencies (IC50 = 0.32 and 0.28 lmol/mL),
respectively. By analyzing the results of the lung A-549 cell
line, it could be claimed that the series eliciting consider-
able activity were the piperidin-/piperazin-1-yl derivatives
5a–f and the 6-oxopyrimidine derivatives linked through
the sulfanyl spacer 6a–f.
ꢀ
and the amino acid Met793 with a bond length of 3.05 A
in addition to some hydrophobic interactions at different
positions of the structure (Chart 4). Compound 5a under-
went
H-bond
interaction
with
Met793
(bond
12
Chem Biol Drug Des 2014

Antitumor and Kinase Inhibition of Pyrazolopyrimidines
length = 3.20 A) through N2-pyrazole, in addition to
5(4H)-one moiety 9a–f was synthesized by reacting the
4-hydrazinyl intermediate 7 with the appropriate oxazol-
ones ii. All compounds were screened against breast
(MCF-7) and lung (A-549) cell lines, where series 5a–f,
6a–f, and 9a–f presented compounds that were the
most effective against either or both cell lines. Six com-
pounds displayed activity against both cell lines. EGRF-
TK inhibitory activity of these compounds showed mod-
erate activity. The most active compound was 6b show-
ing (91% enzyme inhibition). Docking of these
compounds into the ATP-binding site of EGRF-TK dem-
onstrated that the binding mode of these compounds
involved H-bond interaction with Met793 through the N1
of pyrimidine ring or the N2 of pyrazole ring with a bond
ꢀ
arene–cation interaction between Lys745 and the 4-phen-
ylpiperidine ring (Chart 5). Compound 5b showed similar
H-bond interaction with Met793 through N1-pyrimidine
with a bond length 3.19 A (Chart 6). Derivatives 6b and
6e revealed a H-bond interaction involving their N2-pyraz-
ꢀ
ꢀ
ole and Met793 with bond length = 3.16 and 3.18 A,
respectively. Arene–cation interaction with the phenyl ring
on the 6-oxopyrimidine moiety and Lys745 was also
observed for both compounds (Charts 7 and 8). The same
binding mode was observed for compound 9e that inter-
acted with Met793 via a H-bond through its N2-pyrazole
ꢀ
(bond length = 3.17 A) and arene–cation interaction
between Lys745 and the benzylidene ring (Chart 9). The
docking results suggested that the target compounds
were successfully docked into the active site of the ATP-
binding site of EGFR-TK and that the binding mode was
similar to that of the docked ligand as demonstrated by
the interactions with Met793. Most compounds showed
additional interactions with Lys745 through the phenyl
rings located on the substituents at position 4 of the pyr-
azolopyrimidine nucleus. Good correlation could be
observed between the binding mode of the compounds in
the ATP-binding site of EGFR-TK and their biological
screening results. For instance, the closer interaction of
ꢀ
length ranging from 3.16 to 3.20 A. Additional arene–
cation interaction with Lys745 was revealed with some
compounds. Good correlation between the docking
study and EGFR-TK inhibition supported targeting of the
EGFR-TK and presented compound 6b as a promising
and attractive antitumor agent that might be a promising
lead for further studies.
Acknowledgments
ꢀ
compound 6b with Met793 (H-bond = 3.16 A) was coin-
The authors would like to thank Dr. Yassin M. Nissan,
Molecular Modeling Laboratory of the Microanalytical Unit,
Faculty of Pharmacy, Cairo University for his kind help in
performing the molecular docking study.
ciding with its high potency against breast cells
(IC50 = 0.08 lmol/mL) and high in vitro inhibition percent
of EGFR tyrosine inhibition (91%). On the other hand,
ꢀ
compound 5a which formed a H-bond of 3.20 A with
Met793 showed a lower potency (0.72 lmol/mL) against
breast cells and a lower in vitro inhibition of EGFR tyrosine
kinase (41%).
Conflict of Interest
The authors have declared no conflict of interest.
Conclusion
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