Synthesis of novel pyrazolo[3,4-d]pyrimidine derivatives as potential anti-breast cancer agents

Article abstract of DOI:10.1016/j.ejmech.2012.09.031

A series of new 1-aryl-4-benzylidenehydrazinyl-3-methylsulphanyl- pyrazolo[3,4-d]pyrimidines 6a-p was synthesized. The cytotoxic activity of the newly synthesized compounds against human breast cancer cell line, MCF7 was investigated. Most of the test compounds showed potent antitumor activity comparable to that of doxorubicin. The 1-phenyl series (6a-i) exhibited better antitumor activity than 1-(4-methoxyphenyl) series (6j-p). 4-[2-(4- Fluorobenzylidene)hydrazinyl]-3-(methylsulphanyl)-1-phenyl-1H-pyrazolo[3,4-d] pyrimidine (6d) was the most active compound in this study with IC₅₀ equal to 7.5 nM.

Full text of DOI:10.1016/j.ejmech.2012.09.031

European Journal of Medicinal Chemistry 57 (2012) 323e328
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of novel pyrazolo[3,4-d]pyrimidine derivatives as potential anti-breast
cancer agents
,
Mohammed K. Abd El Hamid a, Marko D. Mihovilovic b, Hala B. El-Nassan a
*
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, 33 Kasr El-Aini Street, Cairo 11562, Egypt
b Institute of Applied Synthetic Chemistry, Vienna University of Technology, Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new 1-aryl-4-benzylidenehydrazinyl-3-methylsulphanyl-pyrazolo[3,4-d]pyrimidines 6aep
was synthesized. The cytotoxic activity of the newly synthesized compounds against human breast
cancer cell line, MCF7 was investigated. Most of the test compounds showed potent antitumor activity
comparable to that of doxorubicin. The 1-phenyl series (6aei) exhibited better antitumor activity than 1-
(4-methoxyphenyl) series (6jep). 4-[2-(4-Fluorobenzylidene)hydrazinyl]-3-(methylsulphanyl)-1-
phenyl-1H-pyrazolo[3,4-d]pyrimidine (6d) was the most active compound in this study with IC50
equal to 7.5 nM.
Received 29 June 2012
Received in revised form
21 September 2012
Accepted 24 September 2012
Available online 29 September 2012
Keywords:
Ó 2012 Elsevier Masson SAS. All rights reserved.
Pyrazolo[3,4-d]pyrimidine
Antitumor activity
MCF7
1. Introduction
[13e15]. Although, the SAR of these derivatives as GSK inhibitors
was fully investigated, none of these publications described the
Cancer is considered as the most serious health problem all over
the world. Although, the development of novel chemotherapeutic
agents has significantly progressed within the last 60 years, success
in developing non-cytotoxic “targeted” drugs with fewer side
effects has occurred only in the last decade. Despite these advances,
the treatment of most types of solid tumors (e.g. breast and
ovarian) is still a problem and survival rates remain significantly
low [1]. Therefore, the discovery of new potent, safe and selective
antitumor agents is strongly needed.
The pyrazolo[3,4-d]pyrimidine nucleus is considered as an iso-
stere to the purine nucleus and hence exhibits promising antitumor
activity by acting as ATP competitive inhibitor for many kinase
enzymes. Indeed, many pyrazolo[3,4-d]pyrimidines were reported
to exhibit potent anti-tumor activity [2e6]. Their cytotoxic activi-
ties might be attributed to inhibition of several enzymes such as Src
kinase [5], tyrosine kinase [7,8], mammalian target of rapamycin
(mTOR) [9], cyclin dependent kinase (CDK) [10e12] and glycogen
synthase kinase (GSK) [13e15].
antitumor activity of such a nucleus.
On the other hand, the presence of a methylsulphanyl group at
position 3 of the pyrazolo[3,4-d]pyrimidine nucleus was reported
to enhance the antitumor activity of the nucleus [4,11,21].
Prompted by these claims, we assumed that incorporating these
potent pharmacophores together may result in strong anticancer
agents that act on breast cancer as an example of the solid tumors.
In the present work, new 4-benzylidenehydrazinyl-pyrazolo[3,4-d]
pyrimidine derivatives 6aep were synthesized, incorporating the
3-methylsulphanyl group and varying the substitution on the
phenyl ring at position 1 [H or 4-CH3O] and the phenyl ring at
benzylidenehydrazinyl group in order to study the effect of the
substitution at these two positions on the antitumor activity of the
nucleus against MCF7 cell line.
2. Results and discussion
2.1. Chemistry
Hydrazinyl derivatives have been claimed to exhibit antitumor
effect especially against breast cancer cell lines [16e20]. Recently,
a numberof publications had emerged describing the GSK inhibitory
activity of 4-benzylidenehydrazinylpyrazolo[3,4-d]pyrimidines
Schemes 1 and 2 outline the synthesis of the target compounds.
The synthesis of the starting pyrazole derivatives 2a and 2b was
accomplished via reacting bis(methylsulphanyl)methylenemalono-
nitrile (1) with phenylhydrazine in absolute ethanol [22] or with 4-
methoxyphenylhydrazine HCl in ethanol and triethylamine. The
formation of compound 2b was confirmed by IR that showed CN band
* Corresponding author. Tel.: þ20 223632245; fax: þ20 223635140.
E-mail address: hala_bakr@hotmail.com (H.B. El-Nassan).
at 2202 cmꢀ1 as well as NH2 bands at 3410, 3325 cmꢀ1 1H NMR
.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.031

324
M.K. Abd El Hamid et al. / European Journal of Medicinal Chemistry 57 (2012) 323e328
NC
S
NC
S
NC
S
N
b
a
N
H2N
H2N
N
N
NC
S
1
O
2b
2a
Reagents: a) C6H5NHNH2, C2H5OH; b) 4-CH3OC6H4NHNH2.HCl, (C2H5)3N, C2H5OH
Scheme 1. Preparation of the starting pyrazole derivatives.
spectrum of compound 2b showed an exchangeable singlet signal at
6.91ppm corresponding totheNH2 protons. Also, themassspectrum
of compound 2b showed a molecular ion peak at m/z 260.
Cyclization of pyrazole derivatives 2a,b with formic acid affor-
ded pyrazolo[3,4-d]pyrimidin-4-ones 3a [22] and 3b in high yields.
The disappearance of the CN and NH2 bands in the IR spectrum of
compound 3b, together with the appearance of a single NH band at
3437 cmꢀ1 and C]O band at 1681 cmꢀ1 provided proof for the
formation of 3b. 1H NMR spectrum of compound 3b revealed the
disappearance of the NH2 characteristic signal and the appearance
corresponding to the H-6 proton of the pyrimidine ring. The mass
spectrum of compound 3b showed a molecular ion peak at m/z 288.
Chlorination of pyrazolo[3,4-d]pyrimidin-4-one derivatives 3a,b
with POCl3 gave 4-chloro derivatives 4a,b. The latter compounds
were reacted with hydrazine hydrate to give the corresponding 4-
hydrazinyl derivatives 5a,b.
The target compounds were obtained by reacting 4-hydrazinyl
derivatives 5a,b with the appropriate aromatic aldehyde in
ethanol and glacial acetic acid. The formation of compounds 6aep
was confirmed by 1H NMR spectra that demonstrated the appear-
d
of an exchangeable singlet signal at
d
12.39 ppm corresponding to
ance of a singlet signal at d 8.2e8.6 ppm corresponding to the N]CH
the NH proton. Besides, a singlet signal appeared at
d
8.13 ppm
proton as well as an exchangeable singlet signal at d 11e12 ppm
NC
S
O
S
Cl
S
H
N
N
H2N
N
b
N
N
a
N
N
N
N
N
3a,b
2a,b
4a,b
X
X
X
R
c
N
HN
S
NHNH
2
S
N
N
N
d
N
N
N
N
N
5a,b
6a-p
X
X
For 6a-i: X=H, R= 4-NH , 4-Br, 4-Cl, 4-F, 2-OH, 3-OH, 4-OH, 3-CH O, 2-Cl-4-NO
2
3
2
For 6j-p: X=CH O, R= 4-Br, 4-Cl, 4-F, 2-OH, 3-OH, 4-OH, 4-CH O
3
3
Reagents: a) HCOOH; b) POCl ; c) N H , C H OH; d) RC H CHO, C H OH
3
2
4
2
5
6
4
2 5
Scheme 2. Synthesis of the target compounds 6aep.

M.K. Abd El Hamid et al. / European Journal of Medicinal Chemistry 57 (2012) 323e328
325
corresponding to the NH proton. 13C NMR spectra of compounds
6a,b,e,h,i,j and 6p showed N]CH carbon signal at 99e104 ppm.
Besides, the mass spectra of 6aep showed the corresponding
molecular ion peaks and peaks due to loss of [RC6H4CH]N] and/or
loss of [RC6H4C]N].
40
d
35
30
25
20
15
10
5
2.2. In vitro anticancer screening
The newly synthesized compounds were evaluated for their
in vitro cytotoxic activity against human breast cancer cell line
(MCF7) using Doxorubicin as the reference drug.
0
Dox 6a 6b 6c 6d 6e 6f 6g 6h 6i
6j 6k 6l 6m 6n 6o 6p
Fig. 1. IC50 in nM of the synthesized compounds and doxorubicin against human
breast adenocarcinoma cell line (MCF7).
The relation between surviving fraction and drug concentra-
tions was plotted to obtain the survival curve of MCF7 tumor cell
line after addition of the specified compound. The parameter used
here is IC50, which corresponds to the concentration required for
50% inhibition of cell viability.
The IC50 of the synthesized compounds compared to the refer-
ence drug are shown in Table 1 and the results are represented
graphically in Fig. 1.
From the results in Table 1, it was found that most of the test
compounds showed potent antitumor activity comparable to that
of doxorubicin.
Structurally, the test compounds belong to two series: 1-phenyl
series and 1-(4-methoxyphenyl) series. In general, the 1-phenyl
series (6aei) exhibited better antitumor activity than 1-(4-
methoxyphenyl) series (6jep).
Careful examination of the influence of the substituents on the
benzylidenehydrazinyl group (R) on the antitumor activity showed
the following:
-
-
In both series, the influence of 4-halide substituents on the
antitumor activity was in the order F > Br > Cl
In case of 1-phenyl series, no difference was observed with the
hydroxy group at different position of the ring (ortho, meta and
para). Whilst, in 1-(4-methoxyphenyl) series, the order of
antitumor activity was 3-OH > 2-OH > 4-OH.
Compound 6d (X ¼ H, R ¼ 4-F) was the most active compound in
this study with IC50 equal to 7.5 nM.
Table 1
3. Conclusion
Results of in vitro cytotoxic activity of the synthesized compounds on human breast
adenocarcinoma cell line (MCF7).
In summary, a series of new 1-aryl-4-benzylidenehydrazinyl-3-
methylsulphanyl-pyrazolo[3,4-d]pyrimidines 6aep was synthe-
sized. The cytotoxic activity of the newly synthesized compounds
against human breast cancer cell line (MCF7) was investigated. The
1-phenyl series (6aei) exhibited betterantitumoractivity than 1-(4-
methoxyphenyl) series (6jep). Most of the test compounds showed
potent antitumor activity comparable to that of doxorubicin, espe-
cially, compound (6d) which displayed the highest activity among
the test compounds with IC50 equal to 7.5 nM. Further studies are
still needed to determine the exact mechanism of the antitumor
action as well as to explore the SAR of other positions of the nucleus.
R
N
HN
S
N
N
N
N
4. Experimental part
6a-p
4.1. General
X
Melting points were determined using a Griffin apparatus and
were uncorrected. IR spectra were recorded on Shimadzu IR 435
spectrophotometer and values were represented in cmꢀ1. 1H NMR
were carried out on Varian Gemini 300 MHz spectrophotometer,
Main Defense Chemical Laboratory, Cairo, Egypt and Bruker AC 200
(200 MHz) spectrometer, Institute for synthetic chemistry, Vienna
University of Technology, Vienna, Austria. TMS was used as an
internal standard and chemical shifts were recorded in ppm on
Compound no.
X
R
IC50 in nMa
Doxorubicin
e
e
5.4
12
10.9
12.1
7.5
10.7
10.7
10.3
11.1
15.7
23
31.1
12.1
13.3
9.9
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
H
H
H
H
H
H
H
H
4-NH2
4-Br
4-Cl
4-F
2-OH
3-OH
4-OH
3-CH3O
2-Cle4-NO2
4-Br
4-Cl
4-F
2-OH
3-OH
4-OH
4-CH3O
d
scale and coupling constants (J) are given in Hz. 13C NMR were
carried out on Bruker AC 200 (200 MHz) spectrometer, Institute for
synthetic chemistry, Vienna University of Technology, Vienna,
Austria. The electron impact (EI) mass spectra were recorded on
Hewlett Packard 5988 spectrometer, Microanalytical center, Cairo
University, Cairo, Egypt. Analytical thin layer chromatography (TLC)
on silica gel plates containing UV indicator was employed routinely
to follow the course of reactions and to check the purity of prod-
ucts. All reagents and solvents were purified and dried by standard
techniques.
H
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
34.4
35
a
The values given are means of three experiments.

326
M.K. Abd El Hamid et al. / European Journal of Medicinal Chemistry 57 (2012) 323e328
5-Amino-3-methylsulphanyl-1-phenyl-1H-pyrazole-4-
4.1.4.2. 4-Hydrazinyl-1-(4-methoxyphenyl)-3-(methylsulfanyl)-pyr-
azolo[3,4-d]pyrimidine (5b). Yield: 80%; mp: 175e176 ꢁC; IR
(cmꢀ1): 3300, 3200 (NH/NH2), 2924, 2854 (CH-aliphatic); 1H NMR
carbonitrile (2a) [22] and 3-methylsulphanyl-1-phenyl-1H-pyr-
azolo[3,4-d]pyrimidin-4(5H)-one (3a) [22] were prepared accord-
ing to the published method.
(300 MHz, DMSO-d6) d ppm d 2.59 (s, 3H, SCH3), 3.82 (s, 3H, OCH3),
4.83 (br s, 2H, NH2, D2O exchangeable), 7.07 (d, 2H, J ¼ 9.0 Hz, Are
H), 7.96 (d, 2H, J ¼ 9.0 Hz, AreH), 8.01 (s, 1H, AreH), 8.28 (s, 1H, NH,
D2O exchangeable), MS m/z: 302 [Mþ, 37.5%], 287 [(M ꢀ CH3)þ,
87.5%], 60 [100%].
4.1.1. 5-Amino-3-methylsulphanyl-1-(4-methoxyphenyl)-1H-
pyrazole-4-carbonitrile (2b)
A solution of bis(methylsulphanyl)methylenemalononitrile (1)
(1.70 g, 10 mmol), 4-methoxyphenylhydrazine HCl (1.75 g,
10 mmol) and triethylamine (2 mL) in absolute ethanol (20 mL)
was heated under reflux for 5 h. The reaction mixture was
cooled and the precipitate formed was filtered, dried and crys-
tallized from ethanol. Yield: 57%; mp: 128e129 ꢁC; IR (cmꢀ1):
3410, 3325 (NH2), 2202 (CN), 2927, 2850 (CH-aliphatic); 1H NMR
4.1.5. General procedure for the synthesis of 1-aryl-4-[-2-
(substituted benzylidene)hydrazinyl]-3-(methylsulphanyl)-1H-
pyrazolo[3,4-d]pyrimidines 6aep
A
mixture of 4-hydrazinylpyrazolo[3,4-d]pyrimidines 5a,b
(1 mmol) and the appropriate aromatic aldehyde (1 mmol) in
absolute ethanol (25 mL) and glacial acetic acid (2 mL) was heated
under reflux for 4 h. The solvent was concentrated under reduced
pressure, and the solid formed was filtered, dried and crystallized
from acetic acid.
(300 MHz, DMSO-d6)
d ppm 2.50 (s, 3H, SCH3), 3.78 (s, 3H,
OCH3), 6.91 (s, 2H, NH2, D2O exchangeable), 7.03 (d, 2H,
J ¼ 8.7 Hz, AreH), 7.36 (d, 2H, J ¼ 8.7 Hz, AreH); MS m/z: 260
[Mþ, 100%].
4.1.2. 3-Methylsulphanyl-1-(4-methoxyphenyl)-1H-pyrazolo[3,4-d]
pyrimidin-4(5H)-one (3b)
4.1.5.1. 4-[2-(4-Aminobenzylidene)hydrazinyl]-3-(methylsulphanyl)-
1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (6a). Yield: 97%; mp: 140e
141 ꢁC; IR (cmꢀ1): 3320, 3200 (NH/NH2), 2924, 2854 (CH-
A mixture of pyrazole derivative 2b (2.78 g, 10 mmol) and formic
acid (85%, 40 mL) was heated under reflux for 8 h. The reaction was
cooled, and the separated solid was filtered, dried and crystallized
from formic acid. Yield: 92%; mp: 268e269 ꢁC; IR (cmꢀ1): 3437 (NH),
2920, 2850 (CH-aliphatic), 1681 (CO); 1H NMR (300 MHz, DMSO-d6)
aliphatic); 1H NMR (200 MHz, CDCl3)
d
ppm 2.54 (s, 3H, SCH3), 5.16
(s, 2H, NH2, D2O exchangeable), 6.86e7.74 (m, 12H, AreH þ NH);
13C NMR (DMSO-d6)
ppm 14.1 (SCH3), 103.2 (N]CH), 114.5,
d
116.4, 120.6, 120.9, 122.6, 122.9, 126.2, 129.0, 129.2, 129.3, 129.9,
d
ppm 2.59 (s, 3H, SCH3), 3.80 (s, 3H, OCH3), 7.08 (d, 2H, J ¼ 8.7 Hz, Are
138.3, 144.1 (aromatic carbons); MS m/z: 375 [Mþ, 6.5%], 374
H), 7.84 (d, 2H, J ¼ 8.7 Hz, AreH), 8.13 (s, 1H, AreH), 12.39 (s, 1H, NH,
[(M
ꢀ
1)þ, 4.3%], 257 [(M
ꢀ
H2NC6H4C]N)þ, 40.2%], 256
D2O exchangeable); MS m/z: 288 [Mþ, 100%], 287 [(M ꢀ 1)þ, 63.6%].
[(M ꢀ H2NC6H4CH]N)þ, 27.2%], 224 [17.8%], 92 [H2NC6Hþ4 , 25.6%],
77 [C6Hþ5 , 100%], 76 [C6Hþ4 , 12.8%].
4.1.3. 1-Aryl-4-chloro-3-methylsulphanyl-pyrazolo[3,4-d]
pyrimidines (4a,b)
4.1.5.2. 4-[2-(4-Bromobenzylidene)hydrazinyl]-3-(methylsulphanyl)-
1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (6b). Yield: 71%; mp: 192e
193 ꢁC; IR (cmꢀ1): 3205 (NH), 2924, 2854 (CH-aliphatic); 1H NMR
A
suspension of the pyrazolo[3,4-d]pyrimidin-4-ones 3a,b
(10 mmol) in phosphorus oxychloride (80 mL) was heated under
reflux for 8 h. The reaction was cooled, poured onto ice-cold water
(200 mL) and the precipitate was filtered, dried and crystallized
from ethanol.
(200 MHz, DMSO-d6/CF3COOD)
(m, 10H, AreH), 8.67 (s, 1H, N]CH), 11.99 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6)
ppm 13.0 (SCH3), 101.7 (N]
d ppm 2.66 (s, 3H, SCH3), 7.29e8.44
d
CH), 120.7, 121.1, 123.1, 126.5, 129.6, 130.1, 131.4, 134.5, 138.1, 144.4,
4.1.3.1. 4-Chloro-3-methylsulphanyl-1-phenyl-pyrazolo[3,4-d]pyrim-
147.2, 148.4, 152.2 (aromatic carbons); MS m/z: 440 [(M þ 2)þ,
idine (4a). Yield: 60%; mp: 90e91 ꢁC; IR (cmꢀ1): 2924, 2854 (CH-
15.6%], 438 [Mþ, 20.7%], 283 [(M
ꢀ
BrC6H4)þ, 23.1%], 257
aliphatic); 1H NMR (200 MHz, DMSO-d6)
d
ppm 2.60 (s, 3H, SCH3),
[(M ꢀ BrC6H4C]N)þ, 99.6%], 256 [(M ꢀ BrC6H4CH]N)þ, 51.8%], 224
7.30e8.90 (m, 6H, AreH), MS m/z: 278 [(M þ 2)þ, 0.1%], 276 [Mþ,
[24.7%], 77 [C6Hþ5 , 100%], 76 [C6Hþ4 , 31.9%].
0.3%], 80 [100%], 77 [C6Hþ5 , 22.3%].
4.1.5.3. 4-[2-(4-Chlorobenzylidene)hydrazinyl]-3-(methylsulphanyl)-
1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (6c). Yield: 73%; mp: 208e
209 ꢁC; IR (cmꢀ1): 3421 (NH), 2924, 2850 (CH-aliphatic); 1H NMR
4.1.3.2. 4-Chloro-1-(4-methoxyphenyl)-3-methylsulphanyl-pyrazolo
[3,4-d]pyrimidine (4b). Yield: 72%; mp: 157e158 ꢁC; IR (cmꢀ1):
2900, 2800 (CH-aliphatic); 1H NMR (300 MHz, DMSO-d6)
d
ppm
(200 MHz, DMSO-d6) d ppm 2.50 (s, 3H, SCH3), 7.30e8.38 (m, 10H,
2.62 (s, 3H, SCH3), 3.81 (s, 3H, OCH3), 7.06e8.12 (m, 5H, AreH); MS
m/z: 308 [(M þ 2)þ, 39.4%], 306 [Mþ, 100%], 77 [C6Hþ5 , 25.4%], 76
[C6Hþ4 , 19.8%].
AreH), 8.69 (s, 1H, N]CH),12.04 (s,1H, NH, D2O exchangeable); MS
m/z: 396 [(M þ 2)þ, 5.5%], 394 [Mþ, 22.0%], 257 [(M ꢀ ClC6H4C]
N)þ, 42.5%], 256 [(M ꢀ ClC6H4CH]N)þ, 26.8%], 224 [25.2%], 77
[C6Hþ5 , 100%], 76 [C6Hþ4 , 25.2%].
4.1.4. 1-Aryl-4-hydrazinyl-3-methylsulphanyl-pyrazolo[3,4-d]
pyrimidines (5a,b)
4.1.5.4. 4-[2-(4-Fluorobenzylidene)hydrazinyl]-3-(methylsulphanyl)-
1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (6d). Yield: 71%; mp: 237e
238 ꢁC; IR (cmꢀ1): 3421 (NH), 2924, 2854 (CH-aliphatic); 1H NMR
A mixture of 4-chloropyrazolo[3,4-d]pyrimidine derivative 4a,b
(10 mmol) and hydrazine hydrate (99%, 2 mL, 40 mmol) in absolute
ethanol (35 mL) was heated under reflux for 3 h. The reaction was
cooled, and the separated solid was filtered, dried and crystallized
from ethanol.
(200 MHz, CDCl3) d ppm 2.64 (s, 3H, SCH3), 7.31e8.09 (m,10H, AreH),
8.30 (s,1H, N]CH),11.50 (s,1H, NH, D2O exchangeable); MS m/z: 378
[Mþ, 30.0%], 377 [(M ꢀ 1)þ, 13.4%], 283 [(M ꢀ FC6H4)þ, 13.4%], 257
[(M ꢀ FC6H4C]N)þ, 91.2%], 256 [(M ꢀ FC6H4CH]N)þ, 39.5%], 224
[30.1%],121[(FC6H4C]N)þ, 38.4%], 95[FC6H4þ,19.9%],77[C6Hþ5 ,100%].
4.1.4.1. 4-Hydrazinyl-3-(methylsulfanyl)-1-phenyl-pyrazolo[3,4-d]
pyrimidine (5a). Yield: 84%; mp: 191e92 ꢁC; IR (cmꢀ1): 3325, 3201
(NH/NH2), 2924, 2854 (CH-aliphatic); 1H NMR (200 MHz, DMSO-
4.1.5.5. 4-[2-(2-Hydroxybenzylidene)hydrazinyl]-3-(methyl-
sulphanyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (6e). Yield: 96%;
mp: 203e204 ꢁC; IR (cmꢀ1): 3614 (OH), 3394 (NH), 2924, 2854 (CH-
d6) d ppm 2.61 (s, 3H, SCH3), 4.95 (br s, 2H, NH2, D2O exchangeable),
7.42e8.36 (m, 6H, AreH), 8.60 (br s, 1H, NH, D2O exchangeable), MS
m/z: 272 [Mþ, 24.5%], 77 [C6H5þ, 47.4%], 76 [C6Hþ4 , 19.8%], 64 [100%].
aliphatic); 1H NMR (200 MHz, DMSO-d6)
d ppm 2.60 (s, 3H, SCH3),

M.K. Abd El Hamid et al. / European Journal of Medicinal Chemistry 57 (2012) 323e328
327
6.90e8.40 (m, 10H, AreH), 8.60 (s, 1H, N]CH), 10.20 (s, 1H, OH, D2O
133.6, 134.0, 145.9, 147.1, 148.8, 153.7, 156.0, 160.2 (aromatic carbons);
exchangeable), 12.00 (s, 1H, NH, D2O exchangeable); 13C NMR
MS m/z: 470 [(M
þ
2)þ, 19.3%], 468 [(M)þ, 19.3%], 287
(DMSO-d6)
d
ppm 14.9 (SCH3), 101.7 (N]CH), 116.0, 119.2, 120.6,
[(M ꢀ BrC6H4C]N)þ, 28.1.6%], 286 [(M ꢀ BrC6H4CH]N)þ, 33.3%],183
[(81BrC6H4C]N)þ, 52.6%], 181 [(79BrC6H4C]N)þ, 54.4%], 107
[CH3OC6Hþ4 , 36.8%], 102 [C6H4CNþ, 100%], 76 [C6Hþ4 , 63.2%].
121.0, 126.4, 129.4, 131.2, 138.1, 138.5, 144.3, 145.5, 148.5, 150.2,
153.7, 156.9 (aromatic carbons); MS m/z: 376 [Mþ, 9.4%], 375
[(M
ꢀ
1)þ, 3.7%], 257 [(M
ꢀ
HOC6H4C]N)þ, 15.2%], 256
[(M ꢀ HOC6H4CH]N)þ, 6.2%], 240 [100%], 224 [19.4%], 121
[(HOC6H4CH]NH)þ, 67.1%], 120 [(HOC6H4CH]N)þ, 46.5%], 77
[C6Hþ5 , 46.8%].
4.1.5.11. 4-[2-(4-Chlorobenzylidene)hydrazinyl]-1-(4-methoxyphenyl)-
3-(methylsulphanyl)-1H-pyrazolo[3,4-d]pyrimidine (6k). Yield: 70%;
mp: 238e239 ꢁC; IR (cmꢀ1): 3209 (NH), 2924, 2835 (CH-aliphatic);
1H NMR (200 MHz, DMSO-d6/CF3COOD)
d ppm 2.62 (s, 3H, SCH3),
4.1.5.6. 4-[2-(3-Hydroxybenzylidene)hydrazinyl]-3-(methyl-
sulphanyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (6f). Yield: 39%;
mp: 223e224 ꢁC; IR (cmꢀ1): 3286 (NH/OH), 2924, 2850 (CH-
3.80 (s, 3H, OCH3), 7.02 (d, 2H, J ¼ 8.7 Hz, AreH), 7.50 (d, 2H,
J ¼ 8.8 Hz, AreH), 7.65 (d, 2H, J ¼ 8.7 Hz, AreH), 7.95 (d, 2H, J ¼ 8.8 Hz,
AreH), 8.20 (s,1H, AreH), 8.35 (s, 1H, N]CH),11.87 (br s,1H, NH, D2O
exchangeable); MS m/z: 287 [(M ꢀ ClC6H4C]N)þ, 17.2%], 286
[(M ꢀ ClC6H4CH]N)þ, 34.4%], 102 [C6H4CNþ, 42.2%], 76 [C6Hþ4 ,
42.2%], 64 [100%].
aliphatic); 1H NMR (200 MHz, DMSO-d6)
d ppm 2.60 (s, 3H, SCH3),
7.20e8.40 (m, 10H, AreH), 8.50 (s, 1H, N]CH), 10.00 (s, 1H, OH, D2O
exchangeable),12.00 (s,1H, NH, D2O exchangeable); MS m/z: 376 [Mþ,
28.5%], 375 [(M ꢀ 1)þ, 21.6%], 283 [(M ꢀ HOC6H4)þ, 25.9%], 257
[(Mꢀ HOC6H4C]N)þ, 65.5%], 256[(Mꢀ HOC6H4CH]N)þ, 44.8%], 224
[25.9%], 120 [(HOC6H4CH]N)þ, 21.1%], 77 [C6H5þ, 100%], 76 [C6Hþ4 ,
17.0%].
4.1.5.12. 4-[2-(4-Flourobenzylidene)hydrazinyl]-1-(4-methoxyphenyl)-
3-(methylsulphanyl)-1H-pyrazolo[3,4-d]pyrimidine (6l). Yield: 91%;
mp: 258e259 ꢁC; IR (cmꢀ1): 3209 (NH), 2924, 2850 (CH-aliphatic);
1H NMR (300 MHz, DMSO-d6)
d ppm 2.55 (s, 3H, SCH3), 3.80 (s, 3H,
4.1.5.7. 4-[2-(4-Hydroxybenzylidene)hydrazinyl]-3-(methylsulphanyl)-
1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (6g). Yield: 55%; mp: 262e
263 ꢁC; IR (cmꢀ1): 3371 (NH/OH), 2924, 2854 (CH-aliphatic); 1H
OCH3), 6.94e8.39 (m, 9H, AreH), 8.41 (s, 1H, N]CH), 11.98 (br s, 1H,
NH, D2O exchangeable); MS m/z: 408 [Mþ, 16.7%], 407 [(M ꢀ 1)þ,
45.2%], 287 [(M ꢀ FC6H4C]N)þ, 16.7%], 286 [(M ꢀ FC6H4CH]N)þ,
66.7%], 122 [(FC6H4CHN)þ, 50.0%], 121 [(FC6H4C]N)þ, 100%], 107
[(CH3OC6H4)þ, 47.6%], 95 [FC6H4þ, 45.2%], 76 [C6Hþ4 , 28.6%].
NMR (200 MHz, DMSO-d6)
d ppm 2.58 (s, 3H, SCH3), 6.81e8.09 (m,
10H, AreH), 8.27 (s, 1H, N]CH), 9.92 (s, 1H, OH, D2O exchangeable),
11.84 (br s, 1H, NH, D2O exchangeable); MS m/z: 376 [Mþ, 10.4%], 375
[(M
ꢀ
1)þ, 6.1%], 257 [(M
ꢀ
HOC6H4C]N)þ, 30.7%], 256
4.1.5.13. 4-[2-(2-Hydroxybenzylidene)hydrazinyl]-1-(4-methoxy-
[(M ꢀ HOC6H4CH]N)þ, 15.3%], 224 [17.8%], 121 [(HOC6H4CH]NH)þ,
14.7%], 120 [(HOC6H4CH]N)þ, 16.6%], 77 [C6Hþ5 , 54.0%], 55 [100%].
phenyl)-3-(methylsulphanyl)-1H-pyrazolo[3,4-d]pyrimidine
Yield: 48%; mp: 158e159 ꢁC; IR (cmꢀ1): 3410, 3275 (NH/OH), 2924,
2835 (CH-aliphatic); 1H NMR (200 MHz, DMSO-d6)
ppm 2.89 (s,
(6m).
d
4.1.5.8. 4-[2-(3-Methoxybenzylidene)hydrazinyl]-3-(methyl-
sulphanyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (6h). Yield: 54%;
mp: 238e239 ꢁC; IR (cmꢀ1): 3205 (NH), 2924, 2835 (CH-aliphatic);
3H, SCH3), 3.80 (s, 3H, OCH3), 6.84e8.17 (m, 9H, AreH), 8.50 (s, 1H,
N]CH), 10.10 (br s, 1H, OH, D2O exchangeable), 12.17 (br s, 1H, NH,
D2O exchangeable); MS m/z: 406 [Mþ, 21.4%], 405 [(M ꢀ 1)þ, 28.6%],
286 [(M ꢀ HOC6H4CH]N)þ, 28.6%], 121 [(HOC6H4CHNH)þ, 100%],
120 [(HOC6H4CH]N)þ, 71.4%], 119 [(HOC6H4C]N)þ, 75.0%], 93
[(HOC6H4)þ, 60.7%], 76 [C6H4þ, 39.3%].
1H NMR (200 MHz, DMSO-d6/CF3COOD)
d ppm 2.62 (s, 3H, SCH3),
3.78 (s, 3H, OCH3), 6.97e8.45 (m, 10H, AreH), 8.58 (s, 1H, N]CH),
11.02 (br s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6/
CF3COOD)
d ppm 14.0 (SCH3), 55.1 (OCH3), 99.8 (N]CH), 112.1,
114.2, 117.8, 121.2, 126.2, 128.9, 129.9, 137.7, 144.3, 157.1, 157.9, 158.7,
4.1.5.14. 4-[2-(3-Hydroxybenzylidene)hydrazinyl]-1-(4-methoxyphenyl)-
3-(methylsulphanyl)-1H-pyrazolo[3,4-d]pyrimidine (6n). Yield: 69%;
mp: 179e180 ꢁC; IR (cmꢀ1): 3271, 3213 (NH/OH), 2931, 2835 (CH-
159.4,161.2,161.6 (aromatic carbons); MS m/z: 390 [Mþ, 22.2%], 389
[(M
ꢀ
1)þ, 8.3%], 257 [(M
ꢀ
CH3OC6H4C]N)þ, 100%], 256
[(M ꢀ CH3OC6H4CH]N)þ, 39.8%], 224 [23.1%], 77 [C6Hþ5 , 83.0%], 76
aliphatic); 1H NMR (200 MHz, DMSO-d6)
d ppm 2.62 (s, 3H, SCH3),
[C6Hþ4 , 14.4%].
3.80 (s, 3H, OCH3), 6.82e8.17 (m, 9H, AreH), 8.50 (s, 1H, N]CH),
9.88 (br s, 1H, OH, D2O exchangeable), 12.17 (br s, 1H, NH, D2O
exchangeable); MS m/z: 120 [(HOC6H4CH]N)þ, 21.2%], 107
[(CH3OC6H4)þ, 18.2%], 106 [(HOC6H4CH)þ, 21.2%], 55 [100%].
4.1.5.9. 4-[2-(2-Chloro-4-nitrobenzylidene)hydrazinyl]-3-(methyl-
sulphanyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (6i). Yield: 45%;
mp: 234e235 ꢁC; IR (cmꢀ1): 3201 (NH), 2924, 2835 (CH-aliphatic),
1531, 1342 (NO2); 1H NMR (200 MHz, DMSO-d6)
d
ppm 2.60 (s, 3H,
SCH3), 7.31e8.17 (m, 9H, AreH), 8.50 (s, 1H, N]CH), 12.17 (br s, 1H,
NH, D2O exchangeable); 13C NMR (DMSO-d6)
ppm 12.9 (SCH3),
4.1.5.15. 4-[2-(4-Hydroxybenzylidene)hydrazinyl]-1-(4-methoxy-
phenyl)-3-(methylsulphanyl)-1H-pyrazolo[3,4-d]pyrimidine
Yield: 30%; mp: 113e114 ꢁC; IR (cmꢀ1): 3205, 3170 (NH/OH), 2966,
2835 (CH-aliphatic); 1H NMR (200 MHz, DMSO-d6)
ppm 2.63 (s, 3H,
(6o).
d
101.7 (N]CH), 121.0, 123.2, 124.9, 126.5, 128.9, 131.7, 132.4, 135.9,
138.0, 144.5, 148.0, 148.1, 148.2, 149.9, 150.4 (aromatic carbons); MS
m/z: 441 [(M þ 2)þ, 10.0%], 439 [Mþ, 28.4%], 283 [(M ꢀ Cl(NO2)
d
SCH3), 3.80 (s, 3H, OCH3), 6.89 (d, 2H, J ¼ 8.7 Hz, AreH), 7.40 (d, 2H,
J ¼ 9.0 Hz, AreH), 7.65 (d, 2H, J ¼ 8.6 Hz, AreH), 7.85 (d, 2H, J ¼ 9.0 Hz,
AreH), 8.17 (s,1H, AreH), 8.50 (s,1H, N]CH), 10.15 (br s,1H, OH, D2O
exchangeable), 12.17 (br s, 1H, NH, D2O exchangeable); MS m/z: 404
C6H3)þ, 34.0%], 257 [(M
ꢀ
Cl(NO2)C6H3C]N)þ, 56.0%], 256
[(M ꢀ Cl(NO2)C6H3CH]N)þ, 40.2%], 224 [24.5%], 185 [(37Cl(NO2)
C6H3CH]N)þ, 4.2%], 183 [(35Cl(NO2)C6H3CH]N)þ, 12.0%], 77
[C6Hþ5 , 100%].
[(M
ꢀ
2)þ, 12.0%], 287 [(M
ꢀ
HOC6H4C]N)þ, 23.2%], 286
[(M ꢀ HOC6H4CH]N)þ, 24.0%], 120 [(HOC6H4CH]N)þ, 41.6%], 119
[(HOC6H4C]N)þ, 65.6%], 107 [(CH3OC6H4)þ, 52.8%], 106
[(HOC6H4CH)þ, 27.2%], 93 [(HOC6H4)þ, 28.0%], 76 [C6Hþ4 , 20.8%], 55
[100%].
4.1.5.10. 4-[2-(4-Bromobenzylidene)hydrazinyl]-1-(4-methoxyphenyl)-
3-(methylsulphanyl)-1H-pyrazolo[3,4-d]pyrimidine (6j). Yield: 52%;
mp: 254e255 ꢁC; IR (cmꢀ1): 3394 (NH), 2924, 2835 (CH-aliphatic);
1H NMR (300 MHz, DMSO-d6)
d
ppm 2.50 (s, 3H, SCH3), 3.80 (s, 3H,
OCH3), 6.94e8.35 (m, 9H, AreH), 8.42 (s, 1H, N]CH), 12.00 (br s, 1H,
NH, D2O exchangeable); 13C NMR (DMSO-d6/CF3COOD)
ppm 14.3
(SCH3), 55.8 (OCH3), 103.5 (N]CH), 113.4, 115.9, 126.8, 131.8, 132.8,
4.1.5.16. 4-[2-(4-Methoxybenzylidene)hydrazinyl]-1-(4-methoxy-
phenyl)-3-(methylsulphanyl)-1H-pyrazolo[3,4-d]pyrimidine
Yield: 61%; mp: 228e229 ꢁC; IR (cmꢀ1): 3205 (NH), 2927, 2835 (CH-
aliphatic); 1H NMR (300 MHz, DMSO-d6)
ppm 2.50 (s, 3H, SCH3),
(6p).
d
d

328
M.K. Abd El Hamid et al. / European Journal of Medicinal Chemistry 57 (2012) 323e328
[4] M.M. El-Enany, M.M. Kamel, O.M. Khalil, H.B. El-Nassan, Synthesis and anti-
tumor activity of novel 6-aryl and 6-alkylpyrazolo[3,4-d]pyrimidin-4-one
derivatives, Eur. J. Med. Chem. 45 (2010) 5286e5291.
[5] A. Kumar, I. Ahmad, B.S. Chhikara, R. Tiwari, D. Mandal, K. Parang, Synthesis of
3-phenylpyrazolopyrimidine-1,2,3-triazole conjugates and evaluation of their
Src kinase inhibitory and anticancer activities, Bioorg. Med. Chem. Lett. 21
(2011) 1342e1346.
[6] G.S. Hassan, H.H. Kadry, S.M. Abou-Seri, M.M. Ali, A.E. Mahmoud, Synthesis
and in vitro cytotoxic activity of novel pyrazolo[3,4-d]pyrimidines and related
pyrazole hydrazones toward breast adenocarcinoma MCF-7 cell line, Bioorg.
Med. Chem. 19 (2011) 6808e6817.
[7] S. Schenone, O. Bruno, F. Bondavalli, A. Ranise, L. Mosti, G. Menozzi, P. Fossa,
S. Donnini, A. Santoro, M. Ziche, F. Manetti, M. Botta, Antiproliferative activity
of new 1-aryl-4-amino-1H-pyrazolo[3,4-d]pyrimidine derivatives toward the
human epidermoid carcinoma A431 cell line, Eur. J. Med. Chem. 39 (2004)
939e946.
[8] R. Ducray, P. Ballard, B.C. Barlaam, M.D. Hickinson, J.G. Kettle, D.J. Ogilvieb,
C.B. Trigwell, Novel 3-alkoxy-1H-pyrazolo[3,4-d]pyrimidines as EGFR and
erbB2 receptor tyrosine kinase inhibitors, Bioorg. Med. Chem. Lett. 18 (2008)
959e962.
[9] K.J. Curran, J.C. Verheijen, J. Kaplan, D.J. Richard, L. Toral-Barza, I. Hollander,
J. Lucas, S. Ayral-Kaloustian, K. Yu, A. Zask, Pyrazolopyrimidines as highly
potent and selective, ATP-competitive inhibitors of the mammalian target of
rapamycin (mTOR): optimization of the 1-substituent, Bioorg. Med. Chem.
Lett. 20 (2010) 1440e1444.
[10] D.C. Kim, Y.R. Lee, B. Yang, K.J. Shin, D.J. Kim, B.Y. Chung, K.H. Yoo, Synthesis
and biological evaluations of pyrazolo[3,4-d]pyrimidines as cyclin-dependent
kinase 2 inhibitors, Eur. J. Med. Chem. 38 (2003) 525e532.
[11] J.A. Markwalder, M.R. Arnone, P.A. Benfield, M. Boisclair, C.R. Burton, C. Chang,
S.S. Cox, P.M. Czerniak, C.L. Dean, D. Doleniak, R. Grafstrom, B.A. Harrison,
R.F. Kaltenbach, D.A. Nugiel, K.A. Rossi, S.R. Sherk, L.M. Sisk, P. Stouten,
G.L. Trainor, P. Worland, S.P. Seitz, Synthesis and biological evaluation of 1-
Aryl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one inhibitors of cyclin-
dependent kinases, J. Med. Chem. 47 (2004) 5894e5911.
3.78(s, 3H, OCH3), 3.80 (s, 3H, OCH3), 6.83e8.38 (m,10H, AreHþ N]
CH),11.89 (br s,1H, NH, D2O exchangeable); 13C NMR (CDCl3)
ppm
d
16.2 (SCH3), 49.9, 51.2 (OCH3), 104.3 (N]CH), 116.8, 126.8, 126.8,
126.9, 126.9, 127.5, 128.1, 128.4, 128.8, 128.9, 134.0, 134.8, 144.2
(aromatic carbons); MS m/z: 420 [Mþ, 5.3%], 419 [(M ꢀ 1)þ, 11.7%],
287 [(M ꢀ CH3OC6H4C]N)þ, 14.6%], 286 [(M ꢀ CH3OC6H4CH]N)þ,
28.7%], 134 [(CH3OC6H4CH]N)þ, 83.0%], 133 [(CH3OC6H4C]N)þ,
100%], 120 [(CH3OC6H4CH)þ, 25.1%], 107 [(CH3OC6H4)þ, 21.1%], 76
[C6Hþ4 , 33.3%].
4.2. Biological testing
4.2.1. Materials and methods
The human breast adenocarcinoma cell line (MCF7) was ob-
tained as a gift from NCI, MD, USA.
All chemicals and solvents were purchased from SigmaeAldrich.
4.2.1.1. Measurement of potential cytotoxicity. The cytotoxic activity
of the newly synthesized compounds was measured in vitro on
human breast adenocarcinoma cell line (MCF7) using
Sulforhodamine-B stain (SRB) assay applying the method of Skehan
et al. [23].
Cells were plated in 96-multiwell plate (104 cells/well) for 24 h
before treatment with the test compounds to allow attachment of
the cells to the wall of the plate. Test compounds were dissolved in
DMSO and diluted with saline to the appropriate volume. Different
[12] A.J. Peat, J.A. Boucheron, S.H. Dickerson, D. Garrido, W. Mills, J. Peckham,
F. Preugschat, T. Smalley, S.L. Schweiker, J.R. Wilson, T.Y. Wang, H.Q. Zhou,
S.A. Thomson, Novel pyrazolopyrimidine derivatives as GSK-3 inhibitors,
Bioorg. Med. Chem. Lett. 14 (2004) 2121e2125.
[13] A.J. Peat, D. Garrido, J.A. Boucheron, S.L. Schweiker, S.H. Dickerson, J.R. Wilson,
T.Y. Wang, S.A. Thomson, Novel GSK-3 inhibitors with improved cellular
activity, Bioorg. Med. Chem. Lett. 14 (2004) 2127e2130.
[14] T.L. Smalley, A.J. Peat, J.A. Boucheron, S. Dickerson, D. Garrido, F. Preugschat,
S.L. Schweiker, S.A. Thomson, T.Y. Wang, Synthesis and evaluation of novel
heterocyclic inhibitors of GSK-3, Bioorg. Med. Chem. Lett. 16 (2006) 2091e
2094.
[15] C. Luma, J. Kahl, L. Kessler, J. Kucharski, J. Lundstrm, S. Miller, H. Nakanishi,
Y. Pei, K. Pryor, E. Roberts, L. Sebo, R. Sullivan, J. Urban, Z. Wang, 2,5-
Diaminopyrimidines and 3,5-disubstituted azapurines as inhibitors of
glycogen synthase kinase-3 (GSK-3), Bioorg. Med. Chem. Lett. 18 (2008)
3578e3581.
[16] J. Pandey, R. Pal, A. Dwivedi, K. Hajela, Synthesis of some new diaryl and
triaryl hydrazine derivatives as possible estrogen receptor modulators, Arz-
neim. Forsch., Drug Res. 52 (2002) 39e44.
[17] H. Zhang, J. Drewe, B. Tseng, S. Kasibhatla, S.X. Cai, Discovery and SAR of
indole-2-carboxylic acid benzylidenehydrazides as a new series of potent
apoptosis inducers using a cell based HTS assay, Bioorg. Med. Chem. 12 (2004)
3649e3655.
[18] N. Demirbas, S. Karaoglu, A. Demirbas, K. Sancak, Synthesis and antimicrobial
activities of some new 1-(5-phenylamino-[1,3,4]thiadiazol-2-yl)methyl-5-
oxo-[1,2,4]triazole and 1-(4-phenyl-5-thioxo-[1,2,4]triazol-3-yl)methyl-5-
oxo-[1,2,4]triazole derivatives, Eur. J. Med. Chem. 39 (2004) 793e804.
[19] M.T. Cocco, C. Congiu, V. Lilliu, V. Onnis, Synthesis and in vitro antitumoral
activity of new hydrazinopyrimidine-5-carbonitrile derivatives, Bioorg. Med.
Chem. 14 (2005) 366e372.
concentrations of the test compound (0, 5, 12.5, 25 and 50 mg/mL)
were added to the cell monolayer. Triplicate wells were prepared
for each individual dose. Monolayer cells were incubated with the
test compound for 48 h at 37 ꢁC in atmosphere of 5% CO2. After 48 h,
cells were fixed with trichloroacetic acid, washed with water and
stained for 30 min with 0.4% (wt/vol) Sulforhodamine-B stain dis-
solved with 1% acetic acid. Excess stain was removed by four
washes with 1% acetic acid and attached stain was recovered with
Tris EDTA buffer. Color intensity was measured in ELISA reader. The
relation between surviving fraction and compound concentration
was plotted and IC50 [the concentration required for 50% inhibition
of cell viability] was calculated for each compound and results are
given in Table 1 and represented graphically in Fig. 1.
Acknowledgments
We are grateful to all members of the Department of Cancer
Biology, National Cancer Institute, Cairo, Egypt, for carrying out the
cytotoxicity testing. The authors thank Dr. Michael Schnürch,
Institute of Applied Synthetic Chemistry, Vienna University of
Technology, Vienna, Austria for the help in performing the NMR
spectra. Mohammed K. Abd El Hameid would like to thank OeAD,
Austria for providing travel grant to support this work.
[20] S. Rollas, S¸ .G. . Küçükgüzel, Biological activities of hydrazone derivatives,
Molecules 12 (2007) 1910e1939.
[21] O.A. Fathalla, N.A. Mohamed, E.M. Abbas, Sh. I. Abd-Elmoez, A.M. Soliman,
Synthesis and evaluation of some new pyrazolopyrimidine and thiazolidin-4-
one derivatives as antimicrobial and anticancer, World J. Chem. 4 (2009)
141e148.
[22] Y. Tominaga, Y. Honkawa, M. Hara, A. Hosomi, Synthesis of pyrazolo[3,4-d]
pyrimidine derivatives using ketene dithioacetals, J. Heterocycl. Chem. 27
(1990) 775e785.
[23] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica,
J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyd, New colorimetric assay for
anticancer-drug screening, J. Natl. Cancer Inst. 82 (1990) 1107e1112.
References
[1] D.E. Thurston, Chemistry and Pharmacology of Anticancer Drugs, 1st ed.,
(2007). p.1.
[2] A.E. Rashad, A.E. Mahmoud, M.M. Ali, Synthesis and anticancer effects of some
novel pyrazolo[3,4-d]pyrimidine derivatives by generating reactive oxygen
species in human breast adenocarcinoma cells, Eur. J. Med. Chem. 46 (2011)
1019e1026.
[3] M.M. Ghorab, F.A. Ragab, S.I. Alqasoumi, A.M. Alafeefy, S.A. Aboulmagd,
Synthesis of some new pyrazolo[3,4-d]pyrimidine derivatives of expected
anticancer and radioprotective activity, Eur. J. Med. Chem. 45 (2010) 171e178.