Design, synthesis and antitumor activity of novel pyrazolo[3,4-d]pyrimidine derivatives as EGFR-TK inhibitors

Article abstract of DOI:10.1016/j.bioorg.2016.03.011

A novel series of 2-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-N-(4-substitutedbenzylidene)acetohydrazide (12a-g) was prepared and their structures were confirmed by spectral and elemental analyses. The cytotoxic activity of the newly syn

Full text of DOI:10.1016/j.bioorg.2016.03.011

Accepted Manuscript
Design, synthesis and antitumor activity of novel pyrazolo[3,4-d]pyrimidine
derivatives as EGFR-TK inhibitors
Mohamed A. Abdelgawad, Rania B. Bakr, Olla A. Alkhoja, Wafaa R. Mohamed
PII:
S0045-2068(16)30030-X
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.03.011
YBIOO 1898
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
24 November 2015
22 March 2016
23 March 2016
Please cite this article as: M.A. Abdelgawad, R.B. Bakr, O.A. Alkhoja, W.R. Mohamed, Design, synthesis and
antitumor activity of novel pyrazolo[3,4-d]pyrimidine derivatives as EGFR-TK inhibitors, Bioorganic Chemistry
(
2016), doi: http://dx.doi.org/10.1016/j.bioorg.2016.03.011
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Design, synthesis and antitumor activity of novel pyrazolo[3,4-d]pyrimidine
derivatives as EGFR-TK inhibitors
a,b*
Mohamed A. Abdelgawad , Rania B. Bakr , Olla A. Alkhoja , Wafaa R. Mohamed
b
c
d
a
Department of Pharmaceutical Chemistry, College of Pharmacy, Al Jouf University, Sakaka, Al Jouf-
2
014, KSA.
b
Department of Pharmaceutical Organic Chemistry, Faulty of Pharmacy, Beni-Suef University, Beni-
Suef-62514, Egypt.
c
Sharjah Institute for Medical Research, College of Medicine, University of Sharjah, Sharjah, UAE.
d
Department of Pharmacology, Faulty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt.
*
To whom correspondence should be addressed.
Mohamed A. Abdelgawad, Ph.D.
Tel.: (002)-011-43406061, 00966595435214
Fax: (002)-082-2317958
E-mail address: mohamed.abdelgawad@pharm.bsu.edu.eg
Key words: Pyrazolo[3,4-d]pyrimidine; Schiff bases; Antitumor; EGFR tyrosine kinase.

Abstract
novel
A
series
of
2-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-N-(4-
substitutedbenzylidene)acetohydrazide (12a-g) was prepared and their structures were confirmed by
spectral and elemental analyses. The cytotoxic activity of the newly synthesized compounds was
evaluated against breast carcinoma (MCF-7), non-small cell lung cancer (A549) and human colorectal
adenocarcinoma (HT-29) cell lines using MTT and colony formation assays. The tested compounds
showed a marked anticancer activity against all the tested cell lines, especially compound 12g, which was
the most potent anticancer agent with half maximal inhibitory concentrations (IC50) between 5.36 and
9
.09 µM. Docking studies into ATP binding site of EGFR protein tyrosine kinase were performed to
predict their scores and mode of binding to amino acids, In addition, the inhibitory activity of the target
compounds against epidermal growth factor receptor tyrosine kinase (EGFR-TK) was evaluated. Results
indicated the ability of the target compounds to inhibit EGFR-TK with half maximal inhibitory
concentrations (IC50) in the range of 4.18 to 35.88 µM. Furthermore, The most active compounds 12g,
1
2c and 12d were assayed against Fibroblast Growth Factor Receptor (FGFR), Insulin Receptor (IR) and
Vascular Endothelial Growth Factor Receptor (VEGFR). The activity of the reported compounds warrants
further optimization as novel members in cancer treatment protocols.
2

1
. Introduction
Protein Tyrosine Kinases (PTKs) represent a diverse and rapidly expanding superfamily of proteins that
play an important role in cellular activities including growth, differentiation, metabolism, adhesion,
motility and death [1-4]. The aberrant catalytic activity of many PTKs via mutation or over-expression
plays an important role in numerous pathological conditions including cancer [5-7]. The epidermal growth
factor receptor (EGFR) belongs to a large family of trans-membrane growth factor receptor PTKs. The
EGFR family has four members: HER2 (human epidermal growth factor receptor-2; also known as
erbB2) and its relatives HER1 (epidermal growth factor receptor; EGFR), HER3 and HER4. EGFR
overexpression has been implicated in numerous tumors of epithelial origin (e.g. Colon, breast, ovarian
and NSC lung cancer) [8]. Therefore, EGFR inhibition represents a rational approach for the development
of novel anticancer therapies which would act by competing with ATP for binding at the catalytic domain
of their target enzyme [9]. A number of small molecule EGFR kinase inhibitors had been evaluated in
TM
cancer clinical trials. For example, anilinoquinazoline-containing compounds, erlotinib (Tarceva ) [10]
TM
1) and gefitinib (Iressa ) [11] (2) which had been approved for the chemotherapeutic treatment of
(
TM
patients with advanced non-small lung cancer. Also, lapatinib (Tykerb ) [12, 13] (3) was approved for
the treatment of breast cancer (Fig. 1).
Pyrazolo[3,4-d]pyrimidine derivatives have drawn a great attention, since they are considered as an
isostere to purine nucleus and so exhibits a promising antitumor activity [14-17]. Different mechanisms
account for the cytotoxic effect of this class of compounds. They had been reported to act as glycogen
synthase kinase (GSK) inhibitors [18], cyclin dependent kinase (CDK) inhibitors [19], dual src/Ab1
kinase inhibitors [20] and epidermal growth factor receptor (EGFR) inhibitors [21].
On the other hand, Schiff bases were revealed as promising scaffolds for new anticancer agents [22-24].
Compound 4 exhibited promising cytotoxic activity against ACP-03 and SKMEL-19 with IC50 values
lower than 5 µM [25]. Moreover, Jesmin et al prepared and evaluated a set of Schiff bases as anticancer
3

agents [26]. From this series, compound 5 showed 70.81% reduction of tumor weight comparing with that
of reference compound bleomycin (Fig. 2).
All those experimental evidences and in continuation of our ongoing program related to the synthesis of
antitumor compounds [27, 28], we now describe the synthesis of a new series of pyrazolo[3,4-
d]pyrimidine derivatives to be tested as possible inhibitors of EGFR and, consequently, as antitumor
agents.
In this work, we have carried out the chemical modifications on the general features of
anilinoquinazoline-containing compounds (Fig. 1). These modifications comprise a replacement of the
benzene moiety in the quinazoline skeleton by a pyrazole moiety in an attempt to enhance the cytotoxic
activity. Another modification was done by introducing Schiff bases at C-4 of pyrazolopyrimidine core,
with the goal to develop more effective target molecules utilizing a fragment-based drug design approach.
2
. Results and discussion
2
.1. Chemistry
The starting compound 3,6-dimethyl-1-phenyl-1,5-dihydropyrazolo[3,4-d]pyrimidin-4-one (9) was
prepared from basic hydrolysis of ethyl 5-amino-3-methyl-1-phenyl-1H-pyrazole-4-carboxylate (6) to
afford carboxylic acid derivative 7 which was heated with acetic anhydride to give a cyclized product,
3
,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-d][1,3]oxazin-4-one (8) followed by reflux with formamide
(
Scheme 1) [27].
The synthesis of the target 2-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-N-(4-
substitutedbenzylidene)acetohydrazide derivatives 12a-g was accomplished in accordance with the
sequence of reactions depicted in Scheme 1. Reaction of 3,6-dimethyl-1-phenyl-1,5-dihydropyrazolo[3,4-
d]pyrimidin-4-one (9) with ethyl chloroacetate in dry acetone afforded ethyl 2-(3,6-dimethyl-1-phenyl-
1
H-pyrazolo[3,4-d]pyrimidin-4-yloxy) acetate (10) in good yield (79%). Hydrazinolysis of the ester 10
with hydrazine hydrate afforded the corresponding 2-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-
4

d]pyrimidin-4-yloxy)acetohydrazide (11) which was condensed with the appropriate aromatic aldehydes
in acetic acid to afford the target products (12a-g) in good yields (54-65%). All the prepared compounds
1 13
have been characterized by IR, H NMR, C NMR, mass spectra and elemental analyses.
2
.2. Pharmacological screening
The newly synthesized compounds were screened in vitro for their anti-proliferative activity against three
cancer cell lines; breast carcinoma (MCF-7), Non-Small Cell Lung Cancer (A549) and Human Colorectal
Adenocarcinoma (HT-29) cell lines. The results were expressed in terms of IC50 values (the concentration
that resulted in a 50% inhibition) where the well-known anticancer agents doxorubicin and erlotinib were
used as positive controls (Table 1).
Concerning the activity against breast carcinoma cell line (MCF-7), compound 12g was the most active
one, its IC50 = 6.14 µM, followed by compounds 12c and 12d with IC50 values of 7.22 and 8.74 µM
respectively .The rest of the tested compounds are arranged in the following order 12e > 12a > 12b > 12f
according to their IC50 values (Table 1).
Activity against Non-Small Cell Lung Cancer (A549) revealed that; compound 12g showed higher
activity than erlotinib (IC50 = 11.68 µM) with an IC50 value of 9.09 µM followed by compounds 12e and
1
2c with IC50 values of 12.92 and 13.59 µM respectively. The other tested compounds are arranged in the
following order 12d > 12b > 12a > 12f according to their IC50 values. These results were confirmed by
colony formation assay (Fig. 3). All the tested compounds showed potent activity against Human
Colorectal Adenocarcinoma (HT-29) cell lines, their order of antitumor activity is as following 12g > 12d
>
12e > 12c > 12b > 12a > 12f with IC50 range (5.36-13.34 µM). Compound 12g with trimethoxy
benzylidene moiety represented a promising candidate molecule, as it has been the most potent cytotoxic
compound against all of the tested cancer cell lines, with IC50 values between 5.36 and 9.09 µM.
Further exploration for the mode of action of the target compounds 12a-f was performed through docking
these compounds into EGFR-TK. Moreover, the inhibitory properties against these receptors were also
evaluated. Compound 12g was the most potent inhibitor (IC50 = 4.18 µM) followed by compound 12c
5

(
IC50 = 4.72 µM) and the other compounds showed inhibitory effect that had IC50 range of 5.01-35.88
µM. In addition, the most active compounds 12g, 12c and 12d were assayed against Fibroblast Growth
Factor Receptor (FGFR), Insulin Receptor (IR) and Vascular Endothelial Growth Factor Receptor
(
VEGFR) in order to investigate the selectivity of these compounds. Activity against Fibroblast Growth
Factor Receptor (FGFR) revealed that compounds 12g, 12c and 12d had IC50 = 12.01, 12.86, 9.59 µM,
respectively, while Activity against Insulin Receptor (IR) showed that compounds 12g, 12c and 12d had
IC50 = 8.20, 12.58, 17.62 µM, respectively. In addition, results against Vascular Endothelial Growth
Factor Receptor (VEGFR) produced IC50 = 8.37, 10.14, 12.92 µM corresponding to 12g, 12c and 12d,
respectively (Table 2).
2
.3. Molecular docking study
To know the plausible mode of interactions of the target compounds 12a-g within the EGFR tyrosine
kinases, molecular docking experiments were performed using X-ray crystal structure data for EGFR
obtained from the protein data bank. In this work all the newly synthesized compounds were docked into
ATP binding site of EGFR-TK to explore the ability of the novel pyrazolo[3,4-d]pyrimidines 12a-g to act
as EGFR-TK inhibitors. Erlotinib was redocked into EGFR with a root mean standard deviation (rmsd) =
1
.1524, erlotinib revealed score energy (S) = -27.35 kcal/mol. The main interactions of erlotinib with
EGFR resulted in two main residues Thr-766 and Met-769 amino acids, sequentially forming two
hydrogen bonding interactions with N-1 and N-3 of quinazoline ring at a distance equal to 2.70 and 2.78
°
A , respectively (Fig. 4).
The docking results including the energy associated with intermolecular interactions (affinity in kcal/mol)
obtained upon computational docking for all compounds (12a-g and erlotinib) within EGFR active site
and hydrogen bonding interactions between the amino acid residues and functional groups of compounds
are summarized in Table 3. Compounds 12g and 12c showed better docking scores than erlotinib, the best
docking score was assigned for compound 12g (-28.89 Kcal/mol), it showed three hydrogen bonding
°
with Thr766 (3.21 A ), ii) OCH
°
with Lys721 (3.42 A ), iii) OCH
interactions; i) OCH
3
3
3
with Lys721
6

°
3.28 A ) (Fig. 5). In addition, compound 12c showed docking score (-27.66 Kcal/mol) and revealed three
(
°
°
3
hydrogen bonding interactions; i) OCH with Thr766 (1.39 A ), ii) C=O with Lys721 (2.50 A ), iii) CH=N
°
with Lys721 (1.69 A ) (Fig. 6). Compound 12a revealed docking score (-20.12 Kcal/mol) forming two
hydrogen bonding interactions with Met 769 and Glu 738 amino acids (Table 3). Compounds 12d and 12e
also showed good docking scores (affinity in kcal/mol = -22.66 and -20.32, respectively) with only one
hydrogen bonding interaction with Met 769. In addition, compounds 12b and 12f displayed docking
scores (affinity in kcal/mol = -25.07 and -26.92, respectively) with only one hydrogen bonding interaction
with Asp831 or Cys773. All the synthesized compounds 12a-g were evaluated for their inhibitory activity
against EGFR-TK and the obtained results supported the molecular docking studies.
3
. Conclusion
In conclusion, a series of new 2-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-N-(4-
substitutedbenzylidene) acetohydrazide derivatives (12a-g) was synthesized. The cytotoxic activity of the
newly synthesized compounds was evaluated against breast carcinoma (MCF-7), non-small cell lung
cancer (A549) and human colorectal adenocarcinoma (HT-29) cell lines. All the tested compounds
revealed a potent antitumor activity, particularly N-(3,4,5-trimethoxybenzylidene)-2-(3,6-dimethyl-1-
phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)acetohydrazide (12g), which exhibited the highest
antitumor activity among the tested compounds, with IC50 range equal 5.36-9.09 µM. Docking studies
revealed the ability of the target compounds to fit into the active site of EGFR-TK and can act as
promising inhibitors for these receptors, compound 12g displayed the best score energy (-28.89
Kcal/mol). In addition, compound 12g was the most potent inhibitor for EGFR-TK followed by
compound 12c and these results confirmed the molecular docking studies. Furthermore, the most active
compounds 12g, 12c and 12d were assayed against Fibroblast Growth Factor Receptor (FGFR), Insulin
Receptor (IR) and Vascular Endothelial Growth Factor Receptor (VEGFR). The obtained results showed
that these compounds exhibited more potent EGFR inhibitory activity rather than the other tested kinases.
7

4
. Experimental
4
.1. Chemistry
Melting points were determined on a Thomas-Hoover capillary apparatus and are uncorrected. Infrared
IR) spectra were recorded as films on NaCl plates using a Nicolet 550 Series II Magna FT-IR
(
1
spectrometer. H NMR and C NMR spectra were measured on a BrukerAvance III 400 MHz for H and
13
1
1
00 MHz for C (Bruker AG, Switzerland) with BBFO Smart Probe and Bruker 400 AEON Nitrogen-
3
1
Free Magnet, Faculty of Pharmacy, Beni-Suef University, Egypt in DMSO-d with TMS as the internal
6
standard, where J (coupling constant) values are estimated in Hertz (Hz) and chemical shifts were
recorded in ppm on δ scale. Mass spectra (MS) were recorded on Hewlett Packard 5988 spectrometer.
Microanalyses for C, H and N were carried out on Perkin-Elmer 2400 analyzer (Perkin-Elmer, Norwalk,
CT, USA) at the Micro analytical unit of Cairo University, Egypt and all compounds were within + 0.4%
of the theoretical values. All other reagents, purchased from the Aldrich Chemical Company (Milwaukee,
WI), were used without further purification. 3,6-dimethyl-1-phenyl-1,5-dihydropyrazolo[3,4-d]pyrimidin-
4
-one (9) was prepared according to a literature procedure [27].
4
.1.1. Ethyl 2-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)acetate (10).
A mixture of 3,6-dimethyl-1-phenyl-1,5-dihydropyrazolo[3,4-d]pyrimidin-4-one (9, 10 mmol, 2.40 g),
ethyl chloroacetate (1.22g, 10 mmol) in dry acetone (20 mL) and anhydrous potassium carbonate (10
mmol, 1.38 g) was heated under reflux for 6 h. After cooling, the reaction mixture was poured into ice-
cold water. The separated solid was filtered, and crystallized from ethanol to give pure compound 10 (1.5
g, 46%) as a buff powder: mp 120-122°C; IR (film) 3074 (CH aromatic), 2931 (CH aliphatic), 1739
-
1 1
(
C=O), 1559 (C=N) cm ; H NMR (CDCl
CH ), 2.66 (s, 3H, pyrimidine CH ), 4.27 (q, 2H, J = 7.6 Hz, CH
H, phenyl H-4), 7.46-7.50 (m, 2H, phenyl H-3, H-5), 8.04 (d, J = 7.5 Hz, 2H, phenyl H-2, H-6); C
3
) δ 1.36 (t, 3H, J = 7.6 Hz, CH
3
CH
2
), 2.56 (s, 3H, pyrazole
3
3
2
CH ), 4.86 (s, 2H, CH
3
2
O), 7.30-7.37 (m,
1
3
1
NMR (CDCl
3
3 3 3 2
) δ 13.67 (pyrazole CH ), 14.47 (ester CH ), 23.98 (pyrimidine CH ), 45.32 (OCH ), 61.94
/
), 104.18 (C-3 ), 121.75 (phenyl C-2, C-6), 126.90 (phenyl C-4), 129.56 (phenyl C-3, C-5),
(
ester CH
2
8

/
38.61 (phenyl C-1), 146.46 (C-7 ), 151.23 (C-3), 159.85 (C-6), 160.21 (C-4), 168.53 (C=O); EIMS (m/z)
1
3
6
4
+
26 (M , 11.32%), 59 (100%). Anal.Calcd for C17
.
18 4 3
H N O : C, 65.59; H, 5.85; N, 18.05. Found: C,
5.30; H, 5.80; N, 17.90.
.1.2. 2-(3,6-Dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)acetohydrazide (11).
A mixture of compound 10 (10 mmol, 3.26 g), hydrazine hydrate (99.9%) (1 mL, 20 mmol) in ethanol
(
20 mL) was heated under reflux for 4 h. After cooling, the separated solid was filtered, dried and
crystallized from ethanol to give pure compound 11 (2.00 g, 64%) as a white powder: mp 260-263 °C; IR
(
film) 3432-3284 (br, NH and NH
2
), 3069 (CH aromatic), 2942 (CH aliphatic), 1661 (C=O), 1560 (C=N)
) δ 2.39 (s, 3H, pyrazole CH ), 2.54 (s, 3H, pyrimidine CH ), 4.73 (s, 2H, NH2,
O exchangeable), 4.77 (s, 2H, CH O), 7.35-7.39 (m, 1H, phenyl H-4), 7.52-7.57 (m, 2H, phenyl H-3,
H-5), 8.04 (d, J = 7.5 Hz, 2H, phenyl H-2, H-6), 9.95 (s, 1H, NH, D
-1 1
cm ; H NMR (DMSO-d
6
3
3
D
2
2
1
3
2
O exchangeable); C NMR (DMSO-
/
), 104.15 (C-3 ), 121.79 (phenyl C-2, C-
d
6
1
6
) δ 13.78 (pyrazole CH
3
), 21.94 (pyrimidine CH
3
), 49.06 (OCH
2
/
), 127.16 (phenyl C-4), 129.57 (phenyl C-3, C-5), 138.82 (phenyl C-1), 146.18 (C-7 ), 153.34 (C-3),
+
.
59.10 (C-6), 159.15 (C-4), 160.22 (C=O); EIMS (m/z) 312 (M , 0.63%), 77 (100%). Anal.Calcd for
: C, 57.68; H, 5.16; N, 26.91. Found: C, 57.40; H, 5.20; N, 27.20.
.1.3. General procedure for the preparation of 12a-g.
15 16 6 2
C H N O
4
A mixture of 11 (10 mmol. 3.12 g) and benzaldehyde derivatives (10 mmol) in acetic acid (10 mL) was
refluxed for 10 h, and the reaction mixture left to cool. The obtained solid was filtered off, and
crystallized from ethanol/acetic acid (1:1). Physical and spectral data are listed below.
4
.1.3.1.
N-(4-Hydroxybenzylidene)-2-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
yloxy)acetohydrazide (12a).
White powder (1.5 g, 36%); mp 290-292°C; IR (film) 3429-3180 (br, OH and NH), 3078 (CH aromatic),
-
1 1
2
2
7
954 (CH aliphatic), 1684 (C=O), 1563 (C=N) cm ; H NMR (DMSO-d
6
) δ 2.50 (s, 3H, pyrazole CH
3
),
.55 (s, 3H, pyrimidine CH ), 5.28 (s, 2H, CH O), 6.84 (d, J = 8.4 Hz, 2H, hydroxyphenyl H-3, H-5),
3
2
.36-7.39 (m, 1H, phenyl H-4), 7.53-7.59 (m, 4H, phenyl H-3, H-5, hydroxyphenyl H-2, H-6), 8.06 (d, J
9

=
7.5 Hz, 2H, phenyl H-2, H-6), 8.12 (s, 1H, N=CH), 9.96 (s, 1H, NH, D
2
O exchangeable), 11.66 (s, 1H,
1
O exchangeable); C NMR (DMSO-d
3
OH, D
2
6
) δ 13.68 (pyrazole CH
3
), 24.03 (pyrimidine CH ), 45.44
3
/
), 103.33 (C-3 ), 116.17 (hydroxyphenyl C-3, C-5), 121.76 (phenyl C-2, C-6), 125.33
(
(
OCH
2
hydroxyphenyl C-1), 127.08 (phenyl C-4), 129.20 ((hydroxyphenyl C-2, C-6), 129.67 (phenyl C-3, C-5),
/
38.68 (phenyl C-1), 145.24 (N=CH), 146.43 (C-7 ), 151.38 (C-3), 158.31 (hydroxyphenyl C-4), 159.87
1
+
.
C-6), 160.46 (C-4), 168.24 (C=O); EIMS (m/z) 416 (M , 0.46%) , 80 (100%). Anal.Calcd for
(
C
22
H
20
N
6
O
3
: C, 63.45; H, 4.84; N, 20.18. Found: C, 63.20; H, 4.60; N, 20.10.
4
.1.3.2.
N-(4-Fluorobenzylidene)-2-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
yloxy)acetohydrazide (12b).
White crystals in 59 % yield. mp 280-282 °C (from dioxane); IR (film): 3436 (NH), 1654 (C=O), 1598
-
1 1
(
C=N) cm ; H NMR (DMSO-d
6
) δ 2.51 (s, 3H, pyrazole CH
O), 7.28-7.36 (m, 3H, phenyl H-4, fluorophenyl H-3, H-5), 7.54-7.57 (m, 2H, phenyl H-3, H-5),
.80 (d, J = 8.2 Hz, 2H, fluorophenyl H-2, H-6), 8.05 (d, J = 7.5 Hz, 2H, phenyl H-2, H-6), 8.23 (s, 1H,
3 3
), 2.55 (s, 3H, pyrimidine CH ), 5.31 (s,
2
H, CH
2
7
1
O exchangeable); C NMR (DMSO-d
3
N=CH), 11.93 (s, 1H, NH, D
2
6
3
) δ 13.65 (pyrazole CH ), 23.99
/
), 103.30 (C-3 ), 116.26 (fluorophenyl C-3, C-5), 116.48 (fluorophenyl C-
(
pyrimidine CH
3
), 45.05 (OCH
2
2
, C-6), 121.77 (phenyl C-2, C-6), 127.10 (phenyl C-4), 129.66 (phenyl C-3, C-5), 130.90 (fluorophenyl
/
C-1), 138.63 (phenyl C-1), 143.89 (N=CH), 146 (C-7 ), 151.34 (C-3), 158.31 (C-6), 160.38 (C-4), 162.34
+
fluorophenyl C-4), 168.67 (C=O); EIMS (m/z) 418 (M , 25%), 240 (100%). Anal.Calcd for
.
(
C
22
H19FN
6
O
2
: C, 63.15; H, 4.58; N, 20.08. Found: C, 63.20; H, 4.85; N, 20.10.
4
.1.3.3.
N-(4-Methoxybenzylidene)-2-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
yloxy)acetohydrazide (12c).
Yellow crystals in 65 % yield. mp 279-282 °C (from acetic acid); IR (film): 3435 (NH), 3058 (CH
-
1
1
aromatic), 2917 (CH aliphatic), 1678 (C=O), 1596 (C=N) cm ; H NMR (DMSO-d
6
) δ 2.52 (s, 3H,
pyrazole CH ), 2.59 (s, 3H, pyrimidine CH ), 4.87 (s, 2H, CH O), 3.80 (s, 3H, CH O), 7.01 (d, J = 7.6 Hz,
2
3
3
3
1
0

2
H, methoxyphenyl H-3, H-5), 7.35-7.39 (m, 1H, phenyl H-4), 7.53-7.59 (m, 2H, phenyl H-3, H-5), 7.68
(
d, J = 7.6 Hz, 2H, methoxyphenyl H-2, H-6), 8.06 (d, J = 7.5 Hz, 2H, phenyl H-2, H-6), 8.18 (s, 1H,
1
O exchangeable); C NMR (DMSO-d
3
N=CH), 11.75 (s, 1H, NH, D
2
6
3
) δ 13.68 (pyrazole CH ), 24.04
/
), 103.34 (C-3 ), 114.77 (methoxyphenyl C-3, C-5), 121.69
(
(
(
pyrimidine CH
3
), 45.02 (OCH
2
), 55.76 (OCH
3
phenyl C-2, C-6), 126.89 (phenyl C-4), 126.98 (methoxyphenyl C-1), 127.01 (phenyl C-4), 129.02
methoxyphenyl C-2, C-6), 129.64 (phenyl C-3, C-5), 138.73 (phenyl C-1), 144.80 (N=CH), 146.38 (C-
/
7
4
4
), 151.33 (C-3), 158.27 (methoxyphenyl C-4), 160.40 (C-6), 161.28 (C-4), 168.35 (C=O); EIMS (m/z)
+
.
30 (M , 9%), 55 (100%). Anal.Calcd for C23
22 6 3
H N O : C, 64.17; H, 5.15; N, 19.52. Found: C, 63.80; H,
.80; N, 19.35.
4
.1.3.4.
N-(4-Chlorobenzylidene)-2-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
yloxy)acetohydrazide (12d).
White crystals in 61 % yield. mp 299-300 °C (from acetic acid); IR (film): 3420 (NH), 3056 (CH
-
1
1
aromatic), 2923 (CH aliphatic), 1676 (C=O), 1595 (C=N) cm ; H NMR (DMSO-d
pyrazole CH ), 2.50 (s, 3H, pyrimidine CH ), 4.83 (s, 2H, CH O), 7.35-7.37 (m, 1H, phenyl H-4), 7.50-
.59 (m, 4H, phenyl H-3, H-5, chlorophenyl H-2, H-6), 7.90 (d, J = 7.4 Hz, 2H, chlorophenyl H-3, H-5),
6
) δ 2.38 (s, 3H,
3
3
2
7
8
.02 (d, J = 7.5 Hz, 2H, phenyl H-2, H-6), 8.72 (s, 1H, N=CH), 11.89 (s, 1H, NH, D
2
O exchangeable);
1
3
/
), 103.16 (C-3 ),
C NMR (DMSO-d
6
) δ 13.47 (pyrazole CH
3
), 24.84 (pyrimidine CH
3
), 45.72 (OCH
2
1
2
15.44 (chlorophenyl C-3, C-5), 121.56 (phenyl C-2, C-6), 126.76 (phenyl C-4), 129.23 (chlorophenyl C-
/
, C-6), 129.95 (phenyl C-3, C-5), 138.63 (phenyl C-1), 145.80 (N=CH), 146.22 (C-7 ), 147.96
(
chlorophenyl C-1), 151.22 (C-3), 158.85 (chlorophenyl C-4), 160.50 (C-6), 161.33 (C-4), 168.44 (C=O);
.
+.
6 2
EIMS (m/z) 436 (M+2 , 5%), 434 (M , 15%), 73 (100%). Anal.Calcd for C22H19ClN O : C, 60.76; H,
4
.40; N, 19.32. Found: C, 60.70; H, 4.75; N, 19.60.
.1.3.5. 2-(3,6-Dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-N-(4-nitrobenzylidene)
4
acetohydrazide (12e).
1
1

Yellow crystals in 72 % yield. mp > 300 °C (from acetic acid); IR (film): 3415 (NH), 3078 (CH aromatic),
-
1 1
2
2
963 (CH aliphatic), 1676 (C=O), 1560 (C=N) cm ; H NMR (DMSO-d
6
3
) δ 2.51 (s, 3H, pyrazole CH ),
.57 (s, 3H, pyrimidine CH ), 5.37 (s, 2H, CH O), 7.37-7.39 (m, 1H, phenyl H-4), 7.53-7.57 (m, 2H,
2
3
phenyl H-3, H-5), 8.00-8.04 (m, 4H, phenyl H-2, H-6, nitrophenyl H-2, H-6), 8.06 (s, 1H, N=CH), 8.29
1
3
(
d, J = 8.4 Hz, 2H, nitrophenyl H-3, H-5), 12.17 (s, 1H, NH, D
2
O exchangeable); C NMR (DMSO-d ) δ
6
/
), 103.31 (C-3 ), 121.73 (phenyl C-2, C-6),
1
1
3.67 (pyrazole CH
3
), 24.21 (pyrimidine CH
3
), 45.11 (OCH
2
24.49 (nitrophenyl C-3, C-5), 127.06 (phenyl C-4), 128.38 (nitrophenyl C-2, C-6), 129.65 (phenyl C-3,
/
C-5), 138.68 (phenyl C-1), 140.57 (nitrophenyl C-4), 142.63 (N=CH), 146.40 (C-7 ), 148.31 (nitrophenyl
+
.
C-4), 151.34 (C-3), 158.26 (C-6), 160.36 (C-4), 169.14 (C=O); EIMS (m/z) 445 (M , 2.74%), 281
100%). Anal.Calcd for C22 : C, 59.32; H, 4.30; N, 22.01. Found: C, 59.05; H, 4.60; N, 21.85.
.1.3.6. N-(4-Trifluoromethylbenzylidene)-2-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
(
19 7 4
H N O
4
yloxy)acetohydrazide (12f).
Buff powder in 55 % yield. mp 297-298 °C (from acetic acid); IR (film): 3423 (NH), 3043 (CH aromatic),
-
1 1
2
2
924 (CH aliphatic), 1673 (C=O), 1554 (C=N) cm ; H NMR (DMSO-d
6
3
) δ 2.52 (s, 3H, pyrazole CH ),
.57 (s, 3H, pyrimidine CH ), 5.53 (s, 2H, CH O), 7.35-7.40 (m, 1H, phenyl H-4), 7.53-7.57 (m, 2H,
2
3
phenyl H-3, H-5), 7.81 (d, J = 8 Hz, 2H, trifluoromethylphenyl H-3, H-5), 7.96 (d, J = 8 Hz, 2H,
trifluoromethylphenyl H-2, H-6), 8.03 (d, J = 7.5 Hz, 2H, phenyl H-2, H-6), 8.15 (s, 1H, N=CH), 12.07 (s,
1
O exchangeable); C NMR (DMSO-d
3
1
4
H, NH, D
2
6
3 3
) δ 13.65 (pyrazole CH ), 24.01 (pyrimidine CH ),
/
), 103.30 (C-3 ) 121.72 (phenyl C-2, C-6), 123.17 (trifluoromethylphenyl C-4), 125.87
5.08 (OCH
2
(
(
trifluoromethylphenyl C-3, C-5), 127.05 (phenyl C-4), 128.00 (trifluoromethylphenyl C-2, C-6), 129.65
/
), 138.22 (phenyl C-1), 143.33 (N=CH), 146.40 (C-7 ), 151.32 (C-3),
phenyl C-3, C-5), 130.03 (CF
3
+
58.28 (trifluoromethylphenyl C-1), 160.34 (C-6), 164.30 (C-4), 168.98 (C=O); EIMS (m/z) 468 (M ,
.
1
1
19 3 6 2
8%), 265 (100%). Anal.Calcd for C23H F N O : C, 58.97; H, 4.09; N, 17.94. Found: C, 59.15; H, 4.20;
N, 17.70.
1
2

4
.1.3.7.
N-(3,4,5-Trimethoxybenzylidene)-2-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-
yloxy)acetohydrazide (12g).
Yellow crystals in 67 % yield. mp > 300 °C (from ethanol); IR (film): 3416 (NH), 3046 (CH aromatic),
-
1 1
2
2
862 (CH aliphatic), 1664 (C=O), 1601 (C=N) cm ; H NMR (DMSO-d
6
) δ 2.52 (s, 3H, pyrazole CH
3
),
.57 (s, 3H, pyrimidine CH ), 3.71 (s, 3H, CH O), 3.90 (s, 6H, 2CH O), 5.33 (s, 2H, CH
3
3
3
2
O), 7.07 (s, 2H,
trimethoxyphenyl H-2, H-6),7.36-7.39 (m, 1H, phenyl H-4), 7.54-7.58 (m, 2H, phenyl H-3, H-5), 7.99 (s,
1
O exchangeable); C NMR
3
1
H, N=CH), 8.06 (d, J = 8 Hz, 2H, phenyl H-2, H-6), 11.90 (s, 1H, NH, D
2
(
(
DMSO-d
OCH
6
) δ 13.69 (pyrazole CH
3
), 24.09 (pyrimidine CH
3
), 45.15 (OCH ), 56.41 (2OCH ), 60.60
3
2
/
), 103.33 (C-3 ), 104.71 (trimethoxyphenyl C-2, C-6), 121.72 (phenyl C-2, C-6), 127.06 (phenyl C-
3
4
), 129.82 (phenyl C-3, C-5), 129.97 (trimethoxyphenyl C-1), 138.71 (phenyl C-1), 139.64
/
trimethoxyphenyl C-4), 144.81 (N=CH), 146.41 (C-7 ), 151.38 (C-3), 153 (trimethoxyphenyl C-3, C-5),
(
+
58.23 (C-6), 160.46 (C-4), 168.59 (C=O); EIMS (m/z) 490 (M , 3%), 241 (100%). Anal.Calcd for
.
1
25 26 6 5
C H N O
: C, 61.22; H, 5.34; N, 17.13. Found: C, 61.50; H, 5.05; N, 17.10.
4
4
.2. Pharmacological studies
.2.1. Cell Viability Analysis:
Cell viability was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
tetrazolium reduction assay (Sigma-Aldrich, St. Louis, MO, USA) in triplicate as described previously
[
29]. In brief, cells were seeded into 96-well tissue culture plates in DMEM containing 10% FBS to a
final volume of 0.2 ml. The cells were subjected to different treatments after 24 h of seeding. The cells
were then incubated for 48 h with doxorubicin or Erlotinib (positive controls), test drugs or vehicle
(
DMSO). The media were then removed, replaced by 200 µL DMEM containing 0.5 mg/mL of MTT and
cells were incubated for 2 h. Next, the supernatants were removed and the precipitated formazan was
1
3

dissolved by adding 200 µl of DMSO. Absorbance at 570 nm was determined using a microplate reader
Model 450 Mioroplate Reader; Bio-Rad). Results were calculated by subtracting blank readings.
(
4
.2.2. Colony formation Assay
To assess the ability of test compounds to inhibit the clonogenicity of cancer cells (an indirect measure of
tumorigenicity), colony formation assay was performed essentially as mentioned before [30]. In a brief,
A549 cells were seeded in six-well, flat-bottomed plates and incubated overnight. The cells were then
treated with erlotinib or test compounds at 10 µM for 48h. The culture media containing the different
agents were then replaced with fresh media and the cells were allowed to grow at 37 °C for 10 days. The
grown colonies were then fixed by 95% ethanol and the number of colonies was determined by staining
with 2% crystal violet in 20% ethanol and was expressed as a percentage survival of the untreated control.
4
.2.3. Kinases inhibitory activity
Kinase activity was determined using Kinase-Glo Plus luminescence kinase assay kit according to the
manufacturer instructions. The assays depend on the quantitation of the amount of ATP remaining in the
solution of kinase reaction [31].
4
.2.4. Molecular docking
The crystal structures of erlotinib bound at EGFR (PDB:ID 1M17) [32] (obtained from protein data bank
at research collaboration for structural Bioinformatics (RSCB) protein database [PDB]. Docking of the
co-crystallized ligand should be carried out to study the scoring energy (s), root mean standard deviation
(
rmsd) and amino acid interactions. The rmsd which is measure of superposing was 1.1524 Aº for the lead
compound. Docking was performed using London dG force and refinement of the results was done using
force field energy. Preparation of the synthesized compounds for docking was achieved via their 3D
structure built by Molecular Operating Environment (MOE, Version 2005.06, Chemical Computing
1
4

Group Inc., Montreal, Quebec, Canada). Certain procedures were taken before docking which include:3D
protonation of the structures, running conformational analysis using systemic search, selecting the least
energetic conformer and applying the same docking protocol used with ligands. Docking for the
synthesized compounds was applied. Amino acid interactions and the hydrogen bond lengths were
summarized in (Table 3).
4
.2.5. Statistical analysis
The presented data are mean ± SD, and the statistical analysis was performed using one way ANOVA
followed by Dunnett multiple comparisons test. Differences were considered significant at p < 0.05.
Statistical analysis was performed using SPSS for Windows (SPSS, Inc., Chicago, IL).
References
[
1] A. Detsi, M. Majdalani, C.A. Kontogiorgis, D. Hadjipavlou-Litina, P. Kefalas, Natural and synthetic
'-hydroxy-chalcones and aurones: synthesis, characterization and evaluation of the antioxidant and
2
soybean lipoxygenase inhibitory activity, Bioorganic & medicinal chemistry, 17 (2009) 8073-8085.
2] P. Traxler, P. Furet, Strategies toward the design of novel and selective protein tyrosine kinase
inhibitors, Pharmacology & therapeutics, 82 (1999) 195-206.
[
[
[
[
3] P.S. Sharma, R. Sharma, T. Tyagi, Receptor tyrosine kinase inhibitors as potent weapons in war
against cancers, Current pharmaceutical design, 15 (2009) 758-776.
4] A. Ocana, R. Serrano, R. Calero, A. Pandiella, Novel tyrosine kinase inhibitors in the treatment of
cancer, Current drug targets, 10 (2009) 575-576.
5] S. Vogel, M. Barbic, G. Jurgenliemk, J. Heilmann, Synthesis, cytotoxicity, anti-oxidative and anti-
inflammatory activity of chalcones and influence of A-ring modifications on the pharmacological
effect, European journal of medicinal chemistry, 45 (2010) 2206-2213.
[
6] D.G. Piotrowska, M. Cieslak, K. Krolewska, A.E. Wroblewski, Design, synthesis and cytotoxicity of a
new series of isoxazolidines derived from substituted chalcones, European journal of medicinal
chemistry, 46 (2011) 1382-1389.
1
5

[
[
7] S. Shenvi, K. Kumar, K.S. Hatti, K. Rijesh, L. Diwakar, G.C. Reddy, Synthesis, anticancer and
antioxidant activities of 2,4,5-trimethoxy chalcones and analogues from asaronaldehyde: structure-
activity relationship, European journal of medicinal chemistry, 62 (2013) 435-442.
8] J.H. Cheng, C.F. Hung, S.C. Yang, J.P. Wang, S.J. Won, C.N. Lin, Synthesis and cytotoxic, anti-
inflammatory, and anti-oxidant activities of 2',5'-dialkoxylchalcones as cancer chemopreventive
agents, Bioorganic & medicinal chemistry, 16 (2008) 7270-7276.
[
[
[
9] M. Cherry, D.H. Williams, Recent kinase and kinase inhibitor X-ray structures: mechanisms of
inhibition and selectivity insights, Current medicinal chemistry, 11 (2004) 663-673.
10] J. Smith, Erlotinib: small-molecule targeted therapy in the treatment of non-small-cell lung cancer,
Clinical therapeutics, 27 (2005) 1513-1534.
11] G.R. Simon, J.C. Ruckdeschel, C. Williams, A. Cantor, A. Chiappori, C.M. Rocha Lima, S. Antonia,
E. Haura, H. Wagner, L. Robinson, E. Sommers, M. Alberts, G. Bepler, Gefitinib (ZD1839) in
previously treated advanced non-small-cell lung cancer: experience from a single institution, Cancer
control : journal of the Moffitt Cancer Center, 10 (2003) 388-395.
[
[
12] A.J. Tevaarwerk, J.M. Kolesar, Lapatinib: a small-molecule inhibitor of epidermal growth factor
receptor and human epidermal growth factor receptor-2 tyrosine kinases used in the treatment of
breast cancer, Clinical therapeutics, 31 Pt 2 (2009) 2332-2348.
13] K. Bouchalova, M. Cizkova, K. Cwiertka, R. Trojanec, D. Friedecky, M. Hajduch, Lapatinib in
breast cancer - the predictive significance of HER1 (EGFR), HER2, PTEN and PIK3CA genes and
lapatinib plasma level assessment, Biomedical papers of the Medical Faculty of the University
Palacky, Olomouc, Czechoslovakia, 154 (2010) 281-288.
[
[
14] C.C. Cheng, R.K. Robins, Potential Purine Antagonists. VI. Synthesis of 1-Alkyl- and 1-Aryl-4-
substituted Pyrazolo[3,4-d]pyrimidines, The Journal of Organic Chemistry, 21 (1956) 1240-1256.
15] Z.H. Ismail, S.M. Abdel-Gawad, A. Abdel-Aziem, M.M. Ghorab, Synthesis of Some New
Biologically Active Sulfur Compounds Containing Pyrazolo[3,4-d] pyrimidine Moiety, Phosphorus,
Sulfur, and Silicon and the Related Elements, 178 (2003) 1795-1805.
[
16] F. Carraro, A. Naldini, A. Pucci, G.A. Locatelli, G. Maga, S. Schenone, O. Bruno, A. Ranise, F.
Bondavalli, C. Brullo, P. Fossa, G. Menozzi, L. Mosti, M. Modugno, C. Tintori, F. Manetti, M.
Botta, Pyrazolo[3,4-d]pyrimidines as Potent Antiproliferative and Proapoptotic Agents toward A431
and 8701-BC Cells in Culture via Inhibition of c-Src Phosphorylation, Journal of medicinal
chemistry, 49 (2006) 1549-1561.
1
6

[
[
[
[
[
17] M.M. el-Enany, M.M. Kamel, O.M. Khalil, H.B. el-Nassan, Synthesis and antitumor activity of
novel 6-aryl and 6-alkylpyrazolo[3,4-d]pyrimidin-4-one derivatives, Eur J Med Chem, 45 (2010)
5
286-5291.
18] 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, Bioorganic & medicinal
chemistry letters, 14 (2004) 2127-2130.
19] D.C. Kim, Y.R. Lee, B.S. 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) 525-532.
20] S. Schenone, C. Brullo, O. Bruno, F. Bondavalli, L. Mosti, G. Maga, E. Crespan, F. Carraro, F.
Manetti, C. Tintori, M. Botta, Synthesis, biological evaluation and docking studies of 4-amino
substituted 1H-pyrazolo[3,4-d]pyrimidines, Eur J Med Chem, 43 (2008) 2665-2676.
21] S. Schenone, O. Bruno, F. Bondavalli, A. Ranise, L. Mosti, G. Menozzi, P. Fossa, F. Manetti, L.
Morbidelli, L. Trincavelli, C. Martini, A. Lucacchini, Synthesis of 1-(2-chloro-2-phenylethyl)-6-
methylthio-1H-pyrazolo[3,4-d]pyrimidines 4-amino substituted and their biological evaluation,
European Journal of Medicinal Chemistry, 39 (2004) 153-160.
[
[
22] S. Ren, R. Wang, K. Komatsu, P. Bonaz-Krause, Y. Zyrianov, C.E. McKenna, C. Csipke, Z.A.
Tokes, E.J. Lien, Synthesis, biological evaluation, and quantitative structure-activity relationship
analysis of new Schiff bases of hydroxysemicarbazide as potential antitumor agents, Journal of
medicinal chemistry, 45 (2002) 410-419.
23] R. Pignatello, A. Panico, P. Mazzone, M. Pinizzotto, A. Garozzo, P. Fumeri, Schiff bases of N-
hydroxy-N′-aminoguanidines as antiviral, antibacterial and anticancer agents, European journal of
medicinal chemistry, 29 (1994) 781-785.
[
[
24] M. Shingare, D. Ingle, SYNTHESIS OF PYRIMIDINE SCHIFF-BASES AS ANTICANCER
AGENTS, JOURNAL OF THE INDIAN CHEMICAL SOCIETY, 53 (1976) 1036-1037.
25] E.d.L. Chazin, P.d.S. Sanches, E.B. Lindgren, W.T. Vellasco Júnior, L.C. Pinto, R.M.R. Burbano,
J.D. Yoneda, K.Z. Leal, C.R.B. Gomes, J.L. Wardell, Synthesis and Biological Evaluation of Novel
6
2
-Hydroxy-benzo [d][1, 3] oxathiol-2-one Schiff Bases as Potential Anticancer Agents, Molecules,
0 (2015) 1968-1983.
[
26] M. Jesmin, M. Ali, J. Khanam, Antitumor activities of some schiff bases derived from benzoin,
salicylaldehyde, aminophenol and 2, 4-di nitrophenyl hydrazine, Thai J Pharm Sci, 34 (2010) 20-31.
1
7

[
[
[
[
27] K.R. Abdellatif, E.K. Abdelall, M.A. Abdelgawad, R.R. Ahmed, R.B. Bakr, Synthesis, docking study
and antitumor evaluation of certain newly synthesized pyrazolo [3, 4-d] pyrimidine derivatives,
Organic Chemistry:An Indian Journal, 10(4) (2014).
28] K.R. Abdellatif, E.K. Abdelall, M.A. Abdelgawad, R.R. Ahmed, R.B. Bakr, Synthesis and
Anticancer Activity of Some New Pyrazolo [3, 4-d] pyrimidin-4-one Derivatives, Molecules, 19
(
2014) 3297-3309.
29] H.A. Omar, E.-S.A. Arafa, S.A. Salama, H.H. Arab, C.-H. Wu, J.-R. Weng, OSU-A9 inhibits
angiogenesis in human umbilical vein endothelial cells via disrupting Akt–NF-κB and MAPK
signaling pathways, Toxicology and applied pharmacology, 272 (2013) 616-624.
30] K.H. Lee, W.Y. Ho, S.J. Wu, H.A. Omar, P.J. Huang, C.C. Wang, J.H. Hung, Modulation of cyclins,
p53 and mitogen-activated protein kinases signaling in breast cancer cell lines by 4-(3,4,5-
trimethoxyphenoxy)benzoic acid, International journal of molecular sciences, 15 (2014) 743-757.
31] D. Balzano, S. Santaguida, A. Musacchio, F. Villa, A general framework for inhibitor resistance in
protein kinases, Chemistry & biology, 18 (2011) 966-975.
[
[
32] J. Stamos, M.X. Sliwkowski, C. Eigenbrot, Structure of the epidermal growth factor receptor kinase
domain alone and in complex with a 4-anilinoquinazoline inhibitor, Journal of Biological Chemistry,
2
77 (2002) 46265-46272.
1
8

Figure, scheme and table captions
Fig. 1. Chemical structures of erlotinib (1), gefitinib (2), lapatinib (3) and the designed pyrazolo[3,4-
d]pyrimidine derivatives (12a-g).
Fig. 2. Chemical structure of some reported Schiff bases 4, 5.
Fig. 3. Colony formation in A549 cells untreated and treated with erlotinib or test compounds at 10 µM
for 48h. Colonies were determined after allowed to grow in fresh media for 10 days post treatment.
Columns, mean; bars, S.D. (n=3). *Significant difference from control at p < 0.01.
Fig. 4. A) The interactions of erlotinib with EGFR (using MOE site finder program), the red dotted line
represents H-bonding interaction between N-3 and Thr-766 through a water bridge (red sphere), and the
violet dotted line represents H-bonding interaction between N-1 and Met-769. B) 2D interactions of
erlotinib with Thr-766 and Met-769 acid residues.
Fig. 5. Binding of the most active compound 12g inside EGFR active site. A) The interactions of 12g with
EGFR (using MOE site finder program), the red dotted line represents H-bonding interaction between
3
OCH and Thr-766 through a water bridge (red sphere), and the violet dotted lines represent H-bonding
3
interaction between 2 OCH and Lys 721. B) 2D interactions of 12g with Thr-766 and Lys 721 acid
residues.
Fig. 6. A) The interactions of 12c with EGFR (using MOE site finder program), the violet dotted line
represents H-bonding interactions between OCH and Thr-766, C= O and Lys 721 and =N and Lys 721.
3
B) 2D interactions of 12c with Thr-766 and Lys 721 acid residues.
Scheme 1. Synthesis of 2-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-N-(4-
substitutedbenzylidene)acetohydrazide derivatives 12a-g. Reagents and conditions: a) NaOH, CH
3
OH,
reflux, 5h; b) (CH
3
CO)
2
O, reflux, 5h; c) HCONH
2
, reflux, 6h.; d) ClCH
2
COOC
2
H
5
, CH
3
COCH
3
, K CO
2
3
,
reflux, 6h; e) NH NH
2
2
, C
2
H
5
OH, reflux, 4h; f) ArCHO, CH
3
COOH, 10h.
Table 1. IC50 values for the newly synthesized compounds on three cancerous cell lines; MCF-7, A549
and HT-29 using the MTT assay.
1
9

Table 2. IC50 values of the test compounds on Epidermal Growth Factor Receptor (EGFR), Fibroblast
Growth Factor Receptor (FGFR), Insulin Receptor (IR) and Vascular Endothelial Growth Factor Receptor
(
VEGFR).
Table 3: Molecular modeling data for compounds 12a-g and erlotinib during docking in the active site of
EGFR (PDB:ID 1M17).
2
0

Table 1
IC50 ± SD (µM)
Compounds
2a
2b
2c
2d
2e
2f
2g
MCF-7
A549
HT-29
1
12.31 ± 2.68
17.23 ± 2.01
11.25 ± 3.07
1
13.23 ± 2.91
7.22 ± 1.42
8.74 ± 1.51
10.19 ± 2.14
15.27 ± 2.17
6.14 ± 2.13
2.01 ± 0.87
ND
16.11 ± 2.74
13.59 ± 3.11
14.42 ± 1.97
12.92 ± 1.24
18.97 ± 2.78
9.09 ± 2.36
1.17 ± 0.35
11.68 ± 2.18
9.32 ± 2.74
8.20 ± 2.36
7.35 ± 3.04
8.08 ± 2.84
13.34 ± 1.96
5.36 ± 1.98
ND
1
1
1
1
1
Doxorubicin
Erlotinib
ND
Antiproliferative activity was measured using the MTT assay. Values are the average of three
independent experiments run in triplicate. ND, not determined
2
1

Compounds IC50 (µM)
EGFR
VEGFR
-
IR
-
FGFR
-
1
1
1
1
1
1
1
2a
2b
2c
2d
2e
2f
10.50
30.02
4.72
-
-
-
10.14
12.92
-
12.58
17.62
-
12.86
9.59
-
5.01
11.49
35.88
4.18
-
8.37
-
8.20
-
12.01
2g
Table 2
2
2

Table 3
o
Distance (A ) from Functional
Compound
No.
Affinity
No. of
hydrogen
bonds
2
Kcal/mol
-20.12
main residue
3.17 Met769
group
-CO
1
2a
1
.27
Glu738
Asp831
Thr766
-OH
-NH
1
2b
2c
-25.07
-27.66
1
3
1.39
1.39
1
-OCH
3
2
1
.50
.69
Lys721
-CO
Lys721
Met769
Met769
Cysp773
Thr766
-N=CH
-CO
1
2d
2e
2f
2g
-22.66
-20.32
-26.92
-28.89
1
1
1
3
2.79
1.89
2.58
3.21
1
-CO
1
-CO
1
OCH
3
3
3
3
3
.42
.28
Lys721
OCH
Lys721
Met769
OCH
-N1
Erlotinib
-27.35
2
2.70
2
.78
Thr766
-N3
2
3

2
4

Highlights
Novel pyrazolo[3,4-d]pyrimidine derivatives were designed and synthesized
-
-
-
All compounds were evaluated against a panel of cancer cell lines
The tested compounds showed a marked anticancer activity against all the cell
lines
-
-
Compound 12g was the most potent agent with IC between 5.36 and 9.09 µM
50
Docking and kinase assays revealed the ability of these compounds to inhibit
EGFR
2
5

Graphical abstract
H
N
1
R
O
N
N
O
2
N
R
N
3
R
N
1
2
3
12a, R = H, R = OH, R = H
1
2
3
1
1
2b, R = H, R = F, R = H
1
2
3
2c, R = H, R = OCH , R = H
1
3
2
3
1
1
2d, R = H, R = Cl, R = H
1
2
3
2e, R = H, R = NO , R = H
3
2
1
2
1
2f, R = H, R = CF , R = H
3
1
2
3
1
2g, R = OCH , R = OCH , R = OCH
3
3
3
A series of pyrazolo[3,4-d]pyrimidine derivatives 12a-g was prepared and screened against breast
carcinoma (MCF-7), non-small cell lung cancer (A549) and human colorectal adenocarcinoma (HT-29)
cell lines. All compounds revealed antitumor activity especially 12g.
2
6