Design, synthesis and anticancer evaluation of 1H-pyrazolo[3,4-d]pyrimidine derivatives as potent EGFRWT and EGFRT790M inhibitors and apoptosis inducers

Article abstract of DOI:10.1016/j.bioorg.2018.06.017

In our attempt to develop effective EGFR-TKIs, two series of 1H-pyrazolo[3,4-d]pyrimidine derivatives were designed and synthesized. All the newly synthesized compounds were evaluated in vitro for their inhibitory activities against EGFR^WT. Compounds 15_b, 15_j, and 18_d potently inhibited EGFR^WT at sub-micro molar IC₅₀ values comparable to that of erlotinib. Moreover, thirteen compounds that showed promising IC₅₀ values against EGFR^WT were tested in vitro for their inhibitory activities against mutant EGFR^T790M. Compounds 17_d and 17_f exhibited potent inhibitory activities towards EGFR^T790M comparable to osimertinib. Compounds that showed promising IC₅₀ values against EGFR^WT were further tested for their anti-proliferative activities against three cancer cell lines bearing EGFR^WT (MCF-7, HepG2, A549), and two cancer cell lines bearing EGFR^T790M (H1975 and HCC827). Compounds 15_g, 15_j, 15_n, 18_d and 18_e were the most potent anticancer agents against the EGFR^WT containing cells, while compounds 15_e, 17_d and 17_f showed promising anti-proliferative activities against EGFR^T790M containing cells. Furthermore, the most active compound 18_d was selected for further studies regarding to its effects on cell cycle progression and induction of apoptosis in the HepG2 cell line. The results indicated that this compound is good apoptotic agent and arrests G₀/G₁and G₂/M phases of cell cycle. Finally, molecular docking studies were performed to investigate binding pattern of the synthesized compounds with the prospective targets, EGFR^WT (PDB: 4HJO) and EGFR^T790M (PDB: 3W2O).

Full text of DOI:10.1016/j.bioorg.2018.06.017

Accepted Manuscript
Design, synthesis and anticancer evaluation of 1H-pyrazolo[3,4-d]pyrimidine
WT
T790M
derivatives as potent EGFR and EGFR
inhibitors and apoptosis inducers
Ahmed A. Gaber, Ashraf H. Bayoumi, Ahmed M. El-morsy, Farag F. Sherbiny,
Ahmed B.M. Mehany, Ibrahim H. Eissa
PII:
S0045-2068(18)30301-8
DOI:
https://doi.org/10.1016/j.bioorg.2018.06.017
YBIOO 2397
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
28 March 2018
7 June 2018
11 June 2018
Please cite this article as: A.A. Gaber, A.H. Bayoumi, A.M. El-morsy, F.F. Sherbiny, A.B.M. Mehany, I.H. Eissa,
WT
Design, synthesis and anticancer evaluation of 1H-pyrazolo[3,4-d]pyrimidine derivatives as potent EGFR and
T790M
EGFR
inhibitors and apoptosis inducers, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.
2
018.06.017
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Design, synthesis and anticancer evaluation of 1H-pyrazolo[3,4-d]pyrimidine derivatives as
WT
T790M
potent EGFR and EGFR
inhibitors and apoptosis inducers
a
a
a*
a
Ahmed A. Gaber , Ashraf H. Bayoumi , Ahmed M. El-morsy , Farag F. Sherbiny , Ahmed. B. M.
b
c
Mehany Ibrahim H. Eissa *
a
Pharmaceutical organic chemistry department, Faculty of Pharmacy (Boys), Al-Azhar University,
Cairo, 11884, Egypt
b
Zoology department, Faculty of Science, Al-Azhar University, Cairo 11884, Egypt.
c
Medicinal chemistry department, Faculty of Pharmacy (Boys), Al-Azhar University, Cairo,
1
*
1884, Egypt.
Corresponding authors:
Ahmed M. El-morsy
Pharmaceutical organic chemistry department, Faculty of Pharmacy (Boys), Al-Azhar
University, Nasr City 11884, Egypt
Email: Ahmedelmorsy232@yahoo.com
Ibrahim H. Eissa
Medicinal chemistry department, Faculty of Pharmacy (Boys), Al-Azhar University, Cairo,
1
1884, Egypt
Email:
Ibrahimeissa@azhar.edu.eg

Abstract
In our attempt to develop effective EGFR-TKIs, two series of 1H-pyrazolo[3,4-
d]pyrimidine derivatives were designed and synthesized. All the newly synthesized
WT
compounds were evaluated in vitro for their inhibitory activities against EGFR .
WT
Compounds 15
b
, 15
j
, and 18 potently inhibited EGFR at sub-micro molar IC50 values
d
comparable to that of erlotinib. Moreover, thirteen compounds that showed promising
WT
IC50 values against EGFR were tested in vitro for their inhibitory activities against
T790M
mutant EGFR
. Compounds 17
d
and 17 exhibited potent inhibitory activities towards
f
T790M
EGFR
comparable to osimertinib. Compounds that showed promising IC50 values
WT
against EGFR
were further tested for their anti-proliferative activities against three
WT
cancer cell lines bearing EGFR (MCF-7, HepG2, A549), and two cancer cell lines
T790M
bearing EGFR
(H1975 and HCC827). Compounds 15
g
, 15
j
, 15
n
, 18
d
and 18 were
e
WT
the most potent anticancer agents against the EGFR
containing cells, while
compounds15
e
, 17
d
and 17
f
showed promising anti-proliferative activities against
was selected for
T790M
EGFR
containing cells. Furthermore, the most active compound 18
d
further studies regarding to its effects on cell cycle progression and induction of
apoptosis in the HepG2 cell line. The results indicated that this compound is good
apoptotic agent and arrests G
0
/ G
1
and G /M phases of cell cycle. Finally, molecular
2
docking studies were performed to investigate binding pattern of the synthesized
WT
T790M
compounds with the prospective targets, EGFR (PDB: 4HJO) and EGFR
W2O).
(PDB:
3
Key words: Anticancer; EGFR-TKIs; Docking; NSCLC; 1H-Pyrazolo[3,4-
WT
T790M
d]pyrimidine; EGFR ; EGFR

1
. Introduction
Although there have been great advances in the detection and treatment of cancer, it
remains one of the greatest medical challenges. Cancer is a huge global health burden,
touching every region and socio-economic level. It is the second major cause of death
worldwide, exceeded only by heart diseases [1]. When normal cells lose their regulatory
mechanisms that control the growth and multiplication, cancer cells are produced. Cancer
is caused by gene mutations or interfering with normal cell differentiation which initiated
by drugs, viruses, smoking or diet [2].
It has been known that protein kinases (PKs) play a vital role in regulation of
almost all major cellular functions, such as cell proliferation, growth, metabolism,
survival, differentiation and apoptosis [3]. These enzymes catalyze the transfer of the
gamma phosphate group from ATP to specific serine, threonine or tyrosine hydroxyl
groups on target protein substrates involved in a number of cell signaling pathways [4].
The disruption of cell signaling cascades through kinase alterations (especially hyper-
activation, hyper-production, or mutations) leads to several diseases, including cancer [5],
inflammation [6], neurological disorders [7], autoimmune, cardiovascular disorders [8]
and diabetes [9].
Epidermal growth factor receptor (EGFR) is one of the protein kinases family. It
is a key mediator having a crucial role in cell growth, proliferation, survival, and
migration [10, 11]. Overexpression of EGFR-tyrosine kinase (EGFR-TK) is a common
feature in many human solid tumors as [12]. breast cancer [3] and hepatocellular
carcinoma (HCC) [13]. Therefore, EGFR-TK is considered as a rational target for the
design of new anticancer agents [14-16].
The discovery and development of novel therapeutic anti-EGFR-TK drugs for
treatment of malignancy are some of the most important goals in modern medicinal
chemistry. First-generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs), erlotinib 1
[
17] and gefitinib 2 [18, 19], provide significant clinical benefits in cases of non-small-
cell lung cancer (NSCLC). However, disease progression develops ultimately after 9-14
months of treatment in most patients who respond to therapy. In addition, resistance due
to EGFR-TK mutations affects about 50% of NSCLC patients [20, 21]. Second
generation EGFR-TKIs as afatinib 3[22] and canertinib 4 [23] have been developed to

overcome EGFR-TK mutation-related resistance and used clinically in treatment of
NSCLC patients with actively mutated EGFR-TK. Unfortunately, these agents showed a
relatively low maximal-tolerated-dose (MTD) due to the nonselective inhibition against
WT
wild-type EGFR-TK (EGFR ) and/or other kinases, leading to poor clinical patient
outcomes [24, 25]. Third-generation EGFR-TKIs, rociletinib 5 [26] and avitinib 6 [27]
showed advantageous properties in terms of residence times and toxicity profiles. Both
rociletinib and avitinib were designated to treat mutant NSCLC. Rociletinib in phase III
clinical trials exhibited serious hyperglycemia problem in NSCLC patients [28] (Fig. 1).
1
H-Pyrazolo[3,4-d]pyrimidine nucleus is an important pharmacophore, presented
in a number of anticancer agents [29-31], including EGFR-TKIs [32]. In 1997, compound
was generated by Novartis co-workers, using a pharmacophore model for ATP-
7
competitive inhibitors interacting with the active site of the EGFR-TK. This compound
showed low nanomolar efficacy for inhibiting the targeted enzyme, EGFR-TK [33].
Compound 8 is another example of 1H-pyrazolo [3,4-d]pyrimidines with anti EGFR-TK
activity, having IC50 value of 5 nM against the BT474C cell line [34] (Fig. 1).
For several years, our research group has designed and synthesized many new
anticancer derivatives [35-39]. Based on our previous researches and attractiveness of
tyrosine kinases as promising targets for the design of new cancer agents, it was decided
to introduce new 1H-pyrazolo[3,4-d]pyrimidine derivatives having inhibitory activities
WT)
T790M
against wild-type EGFR-TK (EGFR and mutant EGFR-TK (EGFR
). Besides, the
most active members were examined for their anti-proliferative activities against a
number of cancer cell lines in which these types of enzyme exist. Moreover, the most
active compound 18 was investigated for its apoptosis induction potential in HepG2 cell
d
line. Cell cycle assay was performed to determine the cell phase which may be arrested
by 1H-pyrazolo[3,4-d]pyrimidine derivatives. Finally, a molecular docking of the tested
compounds was carried out to investigate their binding patterns with the prospective
target.

Fig. 1: Basic pharmacophoric features of EGFR-TK inhibitors.

1
.1. Rational drug design
The ATP binding pocket of EGFR-TK consists of five main parts; adenine
pocket, hydrophilic ribose pocket, hydrophobic region I, hydrophobic region II and
phosphate binding region. (Fig. 2) [40-42]. The majority of EGFR-TKIs do not exploit
the ribose and phosphate binding site [42]. Accordingly, structure-activity relationships
(
SAR) of EGFR-TKIs revealed that they have a Y shape [43] and share four common
pharmacophoric features as shown in Fig. 1 and Fig. 3 [44].
i) The core structure of most inhibitors consists of a flat hetero aromatic ring
system that contains at least one H-bond acceptor. It occupies the adenine binding pocket
(
hinge segment) and can form hydrogen bonding interactions with the key amino acid
residues; Met793, Thr790 and Thr854 [45]. ii) Terminal hydrophobic head interacting
with the hydrophobic region I. This hydrophobic head is a phenyl ring with different
extra hydrophobic substitutions [44]. iii) Imino group (NH, spacer), occupying the linker
region between the adenine binding region and the hydrophobic region I [46]. iv)
Hydrophobic tail directly attached to the flat hetero aromatic ring system which occupies
the hydrophobic region II [40, 47].
The main target of this work was synthesis of new 1H-pyrazolo[3,4-d]pyrimidines
having the same essential pharmacophoric features of the reported and clinically used
EGFR-TKIs as erlotinib 1 . The core of our molecular design rational comprised
bioisosteric modification strategies of EGFR-TKIs at four different positions (Fig. 4).
The first position was the flat hetero aromatic ring system, where 1H-
pyrazolo[3,4-d]pyrimidine nucleus was used as a bioisostere for quinazoline moiety of
erlotinib 1 . The choice of this moiety was based on an important bio-isoeteric
considerations. Frist of all, the bicyclic structure of 1H-pyrazolo[3,4-d]pyrimidine core is
convenient to the large size space of the adenine binding region [43, 48]. Moreover, the
heterocyclic nitrogen atoms serve as hydrogen-bond acceptors conferring excellent
EGFR-TK potency [48]. The second position was the terminal hydrophobic head.
Different hydrophobic moieties including (substituted) phenyl, aromatic heterocyclic,
fused aromatic or aliphatic structures were selected. The third position was the linker
(
spacer) region, where the linker length as well as number of its hydrogen acceptor and/or
hydrogen acceptor groups were modified. The different linkers may be one atom (e.g.

imino group as compound 19), two atoms (e.g. hydrazono group as compound 16), three
atoms (e.g. ketohydrazinyl group as compounds 18a-e, methylenehydrazinyl group as
compounds 15a-g,i,j and acetohydrazonoyl group as compounds 15k-o), four atoms (e.g.
thiosemicarbazide moiety as compounds 17
d,f), five atoms (e.g. allylidenehydrazinyl moiety as compound 15
e.g. pyrazole ring as compound 20). The fourth position was the hydrophobic tail.
a
,
c,e and semicarbazide moiety as compounds
1
7
b
,
h
) or cyclic structure
(
Phenyl ring was incorporated at position-6 of 1H-pyrazolo[3,4-d]pyrimidine nucleus to
occupy the hydrophobic region II of ATP binding site.
The wide variety of modifications enabled us to study the SAR of these
compounds as effective anti-cancer agents with potential EGFR-TK inhibitory activity
which is considered as a crucial objective of our work. All modification pathways and
molecular design rationale were illustrated and summarized in fig. 4 & 5.
Fig. 2: ATP binding site of EGFR-TK cavity composed of five main parts [41].

Fig. 3: The basic structural requirements for erlotinib as reported EGFR-TK inhibitor
Fig. 4: Summary for the possible modifications of EGFR-TK inhibitors.

Fig. 5: Rational of molecular design of the new proposed EGFR-TK inhibitors
2
2
. Results and discussion
.1.Chemistry
The designed compounds were synthesized as outlined in Schemes 1 & 2.
Ethoxymethylene malononitrile 9 [49] was allowed to react with phenyl hydrazine to
produce 5-amino-1-phenyl-1H-pyrazole-4-carbonitrile 10 [29]. Compound 10 was
underwent a partial hydrolysis using alcoholic NaOH to produce carboxamide derivative
1
1 [50]. 1,6-Diphenyl pyrazolo[3,4-d]pyrimidine core 12 [51] was formed from the

reaction of 11 with methyl benzoate with subsequent chlorination using phosphoryl
trichloride to afford compound 13 [52]. The obtained compound 13 was heated with
hydrazine hydrate to afford 4-hydrazinyl-1,6-diphenyl-1H-pyrazolo[3,4-d]pyrimidine 14.
1
The IR spectrum of 14 demonstrated stretching bands at 3444, 3352 and 3190 cm-
1
corresponding to NH
2
and NH, respectively. Moreover, H NMR of this compound
showed two exchangeable signals at δ 4.73 and 9.89 ppm corresponding to NH
NH, respectively. Refluxing of 14 with commercially available aromatic aldehydes or
acetophenones in the presence of glacial acetic acid afforded the target compounds 15a-o
2
and
.
In addition, refluxing of 14 with commercially available isatin in the presence of glacial
acetic acid afforded the target compound 16 (Scheme 1).
Reaction of compound 14 with appropriate isocyanates and/or isothiocyanates
namely, ethyl isothiocyanates, ethyl isocyanates, cyclohexyl isothiocyanates, cyclohexyl
isocyanates, phenyl isothiocyanates, and phenyl isocyanates, afforded the target
thiosemicarbazide and semicarbazide derivatives 17a-f, respectively, depending on the
1
reported procedure of thiosemicarbazide and semicarbazide syntheses [53-57]
spectra of these compounds showed characteristic three D
corresponding to three NH groups. The IR spectra of these compounds demonstrated
.
H NMR
2
O exchangeable signals
-1
stretching bands around 3320 cm corresponding to the three NH groups, and other
-1
bands around 1666 cm corresponding to C=O groups in compounds 17
b
, 17
d
and 17 .
f
On the other hand, compound 14 was allowed to react with commercially available
benzoyl chloride derivatives namely, benzoyl chloride, 4-methoxybenzoyl chloride, 4-
methyl benzoyl chloride, 4-fluorobenzoyl chloride and 4-chlorobenzoyl chloride in
presence of catalytic amount of TEA to produce the target benzohydrazide derivatives
1
1
8
a-e, respectively. H NMR spectra of compounds 18a-e exhibited two D
2
O exchangeable
signals corresponding to two NH groups. The IR spectra of these compounds
-1
demonstrated stretching bands ranging from 3320 to 3341 cm corresponding to the two
-1
NH groups, and other bands ranging from 1672 to 1688 cm corresponding to C=O
groups Additionally, phthalimide derivative 19 was prepared by the reaction of
compound 14 with commercially available phthalic anhydride, depending on the reported
procedure [58]. The produced compound showed a characteristic D
2
O exchangeable
signal corresponding to the NH group. The IR spectrum of this compound demonstrated

-1
-1
stretching band at 3218 cm corresponding to the NH group, and other band at 1713 cm
corresponding to the two C=O groups. Finally, treatment of compound 14 with
acetylacetone yielded a pyrazolo derivative 20 (cyclized product). Compound 20 was
1
characterized by H NMR spectrum with absence of the D
2
O exchangeable signal of NH
proton. The IR spectrum of this compound demonstrated disappearance of stretching
band of NH group due to the cyclization of the final compound (Scheme 2).
Scheme 1. Reagents and conditions of reaction; (a) phenyl hydrazine, absolute ethanol,
reflux, 2 h; (b) sodium hydroxide, absolute ethanol, reflux, 5 h; (c) methyl benzoate,

sodium ethoxide, absolute ethanol, HCl, reflux, 14 h; (d) phosphoryl trichloride, reflux, 6
h; (e) hydrazine hydrate 99%, reflux, 8 h; (f) app. aromatic aldehydes or acetophenones,
absolute ethanol, glacial acetic acid, reflux.; (g) isatin, glacial acetic acid, reflux, 24 h.
Scheme 2. Reagents and conditions of reaction: (a) app. isothiocyanates or isocyanates,
butanol, RT; (b) app. benzoyl chlorides, DMF, RT.; (c) phthalic anhydride, glacial acetic
acid, reflux 16 h; (d) acetyl acetone, glacial acetic acid, reflux 48 h.

2
2
.2. Biological evaluation
WT
.2.1. EGFR kinase inhibitory assay
WT
According to the rational drug design, a series of novel EGFR inhibitors bearing
1
H-pyrazolo[3,4-d]pyrimidine scaffold were designed and synthesized. The kinase
inhibitory activities of the synthesized compounds were evaluated according to
WT
Homogeneous time resolved fluorescence (HTRF) assay [59] against EGFR . Erlotinib
WT
as one of the most potent EGFR inhibitors was used as a positive control (Table 1).
WT
In general, most of the synthesized compounds could interfere with the EGFR
activity exhibiting stronger activities than erlotinib as compounds 15
, 15 , 17 , 17 , 17 , 17 , 18 and 18 , with IC50 values ranging from 0.09 ± 0.11 to 0.4ꢀ
0.21 µM. While compounds 15 , 15 , 15 , 15 , 17 and 17 showed comparable
inhibitory activities to erlotinib with IC50 values ranging from 0.44 ± 0.24 to 0.73 ± 0.31
µM. . On the other hand, compounds 14, 15 , 15 , 15 , 15 , 16, 18 , 18 , 18 and 19
b
, 15
e
, 15
g
, 15
h
, 15
j
,
1
5
n
o
a
b
d
f
d
e
±
c
f
i
l
c
e
a
d
k
m
a
b
c
showed weaker activities than erlotinib with IC50 values ranging from 0.92 ± 0.43 to 4.91
WT
±
0.61 µM. Compound 20 was inactive against EGFR .
The most active inhibitor was 18
d
(IC50 = 0.09 ± 0.11 µM), bearing a
ketohydrazinyl linker between the phenyl group of the side chain and the 1H-
pyrazolo[3,4-d]pyrimidine moiety. This compound was 4.5 times more active than
erlotinib (IC50 = 0.42 ± 1.80 µM). However, replacing the chlorine atom with a fluorine
one at the para position of phenyl moiety, the produced compound 18
decreased inhibitory activity (IC50 = 0.4ꢀ ± 0.21 µM). Moreover, 4-methoxy and 4-methy
substituted derivatives as compounds 18 (IC50 = ꢀ.01± 0.28 µM), and 18 (IC50 = ꢀ.ꢁ4 ±
.19 µM), respectively, showed lower activities than the 4-flouro and 4-chloro analogs
e
showed a
b
c
0
indicating that grafting a large electron withdrawing substituents, as fluorine and
chlorine, at the 4-positions is beneficial for the activity. With regard to the un-substituted
derivative 18 (IC50 = ꢀ.ꢂꢃ± 0.32 µM), had a lower activity than the substituted ones.
a

When the ketohydrazinyl linker was replaced by a methylenehydrazinyl or
acetohydrazonoyl ones, the yielded compounds 15a-g,i,j and 15k-o exhibited variable
activities depending on the type of hydrophobic head. Compounds 15
b
( 4-hydroxy
phenyl derivative) and 15 (naphthyl derivative) were found to be the most potent
j
counterparts in this two groups, as they were 2.2 and 2.6 times more active than erlotinib
with IC50 values of 0.19 ± 0.13 and 0.16 ± 0.11 µM, respectively. Besides, compounds
1
5
e
(4-nitro phenyl derivative), 15
derivative), 15 (4-flouro phenyl derivative) and 15
possessed excellent activities (IC50 = 0.23 ± 0.31, 0.28 ± 0.22, 0.51 ± 0.23, 0.34 ± 0.25
and 0.39 ± 0.17 µM, respectively). On the other hand, compounds 15 (4-methoxy phenyl
derivative), 15 (4-chloro phenyl derivative), and 15 (4-aminophenyl derivative), showed
almost equipotent activities with that of erlotinib with IC50 values of 0.49 ± 0.10, 0.44 ±
g
(2,6-dichloro phenyl derivative), 15
i
(thiophenyl
n
o
(4-bromo phenyl derivative),
c
f
l
0
.29 and 0.73 ± 0.31 µM, respectively. In addition, compounds 15
a
(phenyl derivative),
(4-methyl phenyl
1
5
d
(4-methyl phenyl derivative), 15 (phenyl derivative) and 15
k
m
WT
derivative), exhibited weaker anti EGFR activities with IC50 of 4.91 ± 0.61, 1.84 ±
0
.19, 1.02 ± 0.41 and 0.92 ± 0.43 µM, respectively.
The addition of a long linker of four carbon atoms (thiosemicarbazide and
semicarbazide moieties) between the hydrophobic head and the pyrazolo [3,4-
d]pyrimidine moiety was beneficial. The majority of the produced compounds 17 , 17
had inhibitory activities higher than that of erlotinib with IC50 values of 0.35
0.21, 0.29 ± 0.14, 0.26 ± 0.14 and 0.32 ± 0.20 µM, respectively. While the other
and 17 showed comparable inhibitory activities to erlotinib with IC50
a
b
,
1
7
d
, and 17
f
±
compounds 17
c
e
values of 0.44 ± 0.24 and 0.56 ± 0.19 µM, respectively.
When linker extended to be five carbon atoms (allylidenehydrazinyl moiety), the
yielded compound 15 exhibited strong activity with IC50 value of 0.32 ± 0.18 µM.
h
Finally, compound 20 incorporating a cyclic structure linker (pyrazole ring), was
WT
found to be inactive against EGFR . The absence of suitable size of the hydrophobic
head (e.g. phenyl ring) as a pharmacophoric feature in designing compound 20 may be
WT
the cause of its inactivity against EGFR .
T790M
2
.2.2. EGFR
kinase inhibitory assay

Thirteen compounds (15
b
, 15
e
, 15
g
, 15
h
, 15
j
, 15
n
, 15
o
, 17
a
, 17
b
, 17
d
, 17f, 18
d
and 18 )
e
WT
that showed promising IC50 values against EGFR were further evaluated for their
T790M
inhibitory activities against mutant EGFR
. Osimertinib was tested as a positive
control. The results revealed that compounds 17
d
(IC50 = 2.72 ± 0.21 nM) and 17 (IC50 =
f
T790M
3
.90 ± 0.331nM) exhibited potent inhibitory activities towards EGFR
comparable to
osimertinib (IC50 = 2.81 ± 1.6 nM). While compounds 15 and 18
j
d
possessed moderate
activities with IC50 values of 4.81 ± 0.43 and 5.30 ± 0.52 nM, respectively. On the other
hand, compounds 15 , 15 , 15 , 15 , 15 , 17 , 17 and 18 showed weak activities when
compared with the reference drug, with IC50 values ranging from 6.54 ± 0.64 to 11.80 ±
b
g
h
n
o
a
b
e
1
.13 nM (Table 1).
2
.2.3. In vitro anti-proliferative activities
Thirteen compounds (15
b
, 15
e
, 15
g
, 15
h
, 15
j
, 15
n
, 15
o
, 17
a
, 17
b
, 17
d
, 17f, 18 and
d
WT
1
8
e
) that showed promising IC50 values against EGFR were further tested for their
anti-proliferative activities against five cancer cell lines (human breast adenocarcinoma
MCF-7), hepatocellular carcinoma (HepG2) and non-small cell lung cancer cells (A549,
(
H1975 and HCC827)) using on MTT assay [60]. MCF-7, HepG2 and A549 cells have the
WT
overexpressed EGFR [61-63], while H1975 and HCC827 cells harbor different EGFR
L858R/T790M
del
mutations (H1975 harboring EGFR
mutation, and HCC827 harboring EGFR
E746-A750
mutation) [64, 65]. Four commercially available drugs (lapatinib, sorafinib,
erlotinib and osimertinib) were used in this test as positive controls.
As shown in Table 2, the obtained results revealed that most of the synthesized
compounds showed excellent, moderate to weak anti-proliferative activities against the
tested cell lines. For the activity against human breast adenocarcinoma cell line (MCF-7),
compounds 15
lapatinib (IC50 = 6.60 ± 0.30 µM ) with IC50 values ranging from 0.50 ± 1.90 to 4.90 ±
.21 µM. While compounds 15 , 17 , 17 , 17 and 17 , were less potent than lapatinib
b
, 15
e
, 15
g
, 15
h
, 15
j
, 15
n
, 18
d
and 18
e
were found to be more potent than
0
o
a
b
d
f
with IC50 values ranging from 6.74 ± 1.92 to 12.07 ± 2.30 µM. For hepatocellular
carcinoma cell line (HepG2), all the tested compounds were more potent than sorafenib
(
IC50 = 4.00 ± 0.4 µM) with IC50 values ranging from 0.01 ± 0.71 to 1.53 ± 0.72 µM.

For the activity against the wild-type non-small cell lung cancer cell line (A549),
compounds 15 , 15 , 15 , 15 , 17 , 17 , 18 and 18 were more potent than erlotinib (IC50
1.80 ± 0.91 µM ) with IC50 values ranging from 0.62 ± 1.70 to 1.70 ± 0.21 µM. While
compounds 15 and 17 were less potent than erlotinib with IC50 values
g
j
n
o
d
f
d
e
=
b
, 15e, 15h, 17
a
b
ranging from 1.95 ± 1.01 to 4.16 ± 0.71 µM.
For the activity against the mutant-type non-small cell lung cancer cell line
(
H1975), compounds 15
e
, 17
d
and 17
f
were more potent than osimertinib (IC50 = 0.03 ±
0
.08 µM ) with IC50 values ranging from 0.02 ± 0.08 to 0.03 ± 0.07 µM. While
compounds 15b, 15j, 15n, 17
b
and 18
d
showed moderate anti-proliferative activities with
IC50 values ranging from 0.04 ± 0.01 to 0.06 ± 0.01µM. Moreover, compounds 15 , 15h,
g
1
5
o, 17a, and 18 exhibited weak activities with IC50 values ranging from 0.07 ± 0.10 to
e
0
.09 ± 0.27µM.
For the activity against the mutant-type non-small cell lung cancer cell line
HCC827), only compound 15 (IC50 = 0.04 ± 0.03 µM) was more potent than osimertinib
IC50 = 0.05 ± 0.01µM). While compounds 17 and 17 showed moderate anti-
(
(
e
d
f
proliferative activities with IC50 values of 0.06 ± 0.24 and 0.09 ± 0.08 µM, respectively.
Moreover, compounds 15 and 18 exhibited weak
activities with IC50 values ranging from 0.11 ± 0.01 to 0.27 ± 0.49 µM.
Table 1: In vitro enzymatic inhibitory activities of the target of 1H-pyrazolo[3,4-
b
, 15g, 15h, 15j, 15n, 15o, 17a, 17b, 18
d
e
WT
790M
d]pyrimidines against EGFR and EGFR
.
WT
T790M
WT
T790M
a
EGFR
EGFR
EGFR
EGFR
Comp.
Comp.
16
a
a
a
IC50 (µM)
IC50 (nM)
IC50 (µM)
IC50 (nM)
b
b
1
4
3.59 ± 0.30
4.91 ± 0.61
NT
3.85 ± 0.38
NT
b
1
5
a
NT
17
17
17
17
17
17
a
0.35 ± 0.21 8.52 ± 0.80
1
5
b
0.19 ± 0.13 7.81 ± 0.71
b
0.29 ± 0.14 6.54 ± 0.64
b
b
1
5
c
0.49 ± 0.10
1.84 ± 0.19
0.23 ± 0.31
0.44 ± 0.29
NT
NT
c
0.44 ± 0.24
NT
b
1
1
1
5
d
d
0.26 ± 0.14 2.72 ± 0.21
b
3
.20 ± 0.25
0.56 ± 0.19
NT
5
e
e
b
5
f
NT
f
0.32 ± 0.20 3.90 ± 0.33

b
b
b
1
0.32 ± 0.91
NT
NT
NT
1
1
5
g
0.28 ± 0.22
18
a
ꢀ.ꢂꢃ ± 0.32
9
.63 ± 0.90
5
h
0.32 ± 0.18
0.51 ± 0.23
0.16 ± 0.11
1.02 ± 0.41
0.73 ± 0.31
0.92 ± 0.43
0.34 ± 0.25
0.39 ± 0.17
18
18
18
b
ꢀ.01 ± 0.28
ꢀ.ꢁ4 ± 0.19
0.09 ± 0.11
b
1
1
5
i
NT
c
4
.81 ± 0.43
5.30 ± 0.52
5
j
d
b
1
5
k
NT
18
e
0.14 ± 0.21 8.41 ± 0.80
b
b
NT
NT
1
5
l
49
20
2.78 ± 0.31
b
c
b
b
1
1
1
5
m
NT
NA
NT
NT
6
.80 ± 0.62
5
n
Erlotinib
Osimertinib
0.42 ± 1.80
b
1
1.80 ± 1.13
2.81 ± 1.6
5
o
NT
a
b
c
d
Data were expressed as Mean ± Standard error (S.E.) of three independent experiments.
NT: Compounds not tested.
NA: Compounds having IC50 value >50 µM.
Bold figures indicate superior potency than erlotinib
Table 2: In vitro anti-proliferative activities towards MCF7, A549, HepG2, H1975 and
HCC827 cell lines
a
IC50 ( µM )
Comp.
MCF7
HepG2
A549
H1975
HCC827
1
5
b
2.36 ± 1.80
3.90 ± 0.80
1.89 ± 1.71
4.65 ± 0.30
0.81 ± 1.00
0.35 ± 1.61 1.95 ± 1.01
0.01 ± 1.01 2.90 ± 1.61
0.06 ± 1.71 1.07 ± 1.70
0.44 ± 1.00 4.16 ± 0.71
0.0ꢀ ± 0.21 1.70 ± 0.21
0.ꢁꢂ ± 0.30 0.81 ± 1.01
0.74 ± 1.62 1.22 ± 0.40
0.06 ± 0.01
0.03 ± 0.02
0.08 ± 0.09
0.08 ± 0.03
0.04 ± 0.01
0.18 ± 0.01
0.04 ± 0.03
0.23 ± 0.09
0.22 ± 0.02
0.11 ± 0.01
1
1
5
e
5
g
1
5
h
1
5
j
1
1
1
5
n
4.90 ± 0.21
0.05 ± 0.47
0.09 ± 0.27
0.15 ± 0.10
5
o
a
6.74 ± 1.92
0.27 ± 0.49
0.19 ± 0.13
0
0
.07 ± 0.10
.05 ± 0.13
7
12.07 ± 2.30 1.53 ± 0.72 2.64 ± 0.40
11.57 ± 2.51 1.12 ± 1.71 3.34 ± 1.61
0.15 ± 0.08
1
7
b

1
7
d
9.10 ± 1.22
7.72 ± 1.21
0.50 ± 1.90
1.61 ± 0.40
6.60 ± 0.30
0.01 ± 1.01 1.10 ± 0.21
0.36 ± 0.33 0.96 ± 1.01
0.01 ± 0.71 0.62 ± 1.70
4.ꢃ1 ± 1.04 0.85 ± 0.21
0.02 ± 0.08
.03 ± 0.07
0.06 ± 0.24
0.09 ± 0.08
0
1
7
f
1
8
d
0.04 ± 0.33
0.12 ± 0.07
0.19 ± 0.05
0
.07 ± 0.04
1
8
e
b
b
b
b
Lapatinib
Sorafenib
Erlotinib
NT
NT
NT
NT
NT
b
b
b
b
NT
NT
NT
4.00 ± 0.42
b
b
b
b
NT
NT
NT
NT
1.80 ± 0.91
b
b
b
0
.03 ± 0.08
0.05 ± 0.01
Osimertinib
NT
NT
NT
a
IC50 values are the mean ± S.D. of three separate experiments.
b
c
NT: Compounds not tested for their anti-proliferative activities.
Bold figures indicate superior potency than positive controls.
2
.2.4. In-vitro DNA-flow cytometric (cell cycle) analysis.
The most active compound 18 was selected for further studies regarding to its
d
effect on cell cycle progression and induction of apoptosis in the HepG2 cell line. DMSO
was used as a negative control. Cell cycle stages were being recognized through flow
cytometry after PI staining followed by RNAse treatment. The HepG2 cells were
incubated with 1 µM of this compound for 24 h, then its effect on the cell cycle profile
was analyzed.
The results revealed that exposure of HepG2 cells to compound 18
interference with the normal cell cycle distribution of this cell line. This compound
induced a significant increase in the percentage of cells at phases of pre-G , which could
be indicative of apoptosis, G /G and G /M by 12, 1.7 and 1.2 folds respectively,
d
leaded to an
1
0
1
2
comparing to the control. Such increase was accompanied by a significant decrease in the
percentage of cells at the S-phase of the cell cycle. These result clearly indicated that
compound 18
d
arrests G
0
/ G
1
and G /M phases of cell cycle (Fig. 6. & Fig. 7 & Table 3).
2

Fig. 6: Cell cycle analysis of HepG2 cells treated with DMSO.

Fig. 7: Cell cycle analysis of HepG2 cells treated with compound 18 at 1 µM
d
concentration
Table 3: Effect of compound 18 and DMSO on cell cycle of HepG2 cell line
d
Conc.
pre-G
(%)
1
G
0
/G
1
S
G /M
2
Sample
(
μM)
(%)
(%)
15.94
51.80
(%)
1
8
d
/ HepG2
1
-
12.42
0.59
46.38
27.34
25.26
20.27
DMSO / HepG2
2
.2.5. Annexin V-FITC apoptosis assay
In order to confirm the apoptosis induction, Annexin V binding studies by flow
cytometer was carried out. To determine early and late apoptosis, Annexin V conjugated
with FITC is used to stain cells in combination with Propidium Iodide (PI). As
phosphatidyl serine externalization is the hallmark of apoptotic cells, Annexin V binds to
the membrane and gives fluorescence. Thus, Annexin V stained cells represent cells with
intact membranes and externalized phosphatidyl serine. Cells that stained positive for
Annexin V/PI represents the cells in the late apoptotic stage that have lost membrane
integrity [66].

As EGFR-TK inhibitors can induce cancer cell apoptosis [67], the apoptotic
nature of compound 18 against HepG2 cells was evaluated via flow cytometry detection
using AnnexinV-FITC and propidium iodide (PI) double staining. The results revealed
that application of compound 18 on HepG2 cells for 48 h with increasing its
d
d
concentration from 0 μM to 1 μM, increases the early apoptosis ratio (lower right
quadrant of the cytogram) from 0.21% to 15.61%, and increases the late apoptosis ratio
(
higher right quadrant of the cytogram) from 0.12 % to 45.45%. This means that
compound 18 induced almost up to 185 folds both early and late cellular apoptosis when
compared with the control. These data suggest that compound 18 is good apoptotic agent
Fig. 8 & Table 4).
d
d
(
Fig. 8: Effect of compound 18
d
and DMSO on the percentage of annexin V-FITC-
and DMSO on the percentage of annexin V-FITC-
positive staining in HepG2 cells
Table 4: Effect of compound 18
d
positive staining in HepG2 cells.
Concentration Early apoptosis
Late apoptosis
(%)
Sample/ cell line
(
μM)
(%)

1
8
d
/HepG2
1
-
15.61
0.21
45.48
0.12
DMSO/HepG2
2
.3.Docking studies
Additionally, the synthesized compounds were docked into the ATP binding
WT
pockets of Wild-type EGFR-TK (EGFR , PDB: 4HJO) [68] and mutant EGFR-TK
T790M
(
EGFR
, PDB: 3W2O)[69] to investigate the putative interaction mechanism with the
EGFR-TK target.
The cavities of EGFR and EGFR
WT
T790M
consist of five main parts; adenine binding
pocket, hydrophilic ribose pocket, hydrophobic region I, hydrophobic region II and
phosphate binding region. The adenine binding pocket is of mostly hydrophobic
character. The N1 and N6 nitrogen atoms of the adenine ring of ATP are involved in a
hydrogen bonding interaction with two amino acids, Gln767 and Met769, of the hinge
region. The hydrophilic ribose pocket contains Cys773 residue, which is unique to the
EGFR-TK and provides both potency and selectivity. The hydrophobic region I
comprises Thr766 and Thr860 residues, playing a crucial role in inhibitor selectivity. The
hydrophobic region II is formed by Leu694 and Gly772 residues. The phosphate binding
region has high solvent exposure and is not of primary importance with respect to binding
affinity [41, 42]. (Fig. 1).
T790M
To date, several EGFR
inhibitors have been developed to target Cys797
residue located at the lip of the adenosine triphosphate (ATP)-binding cleft and Met793
of the hinge region. These compounds were rationally designed to make a covalent bond
T790M
with C797, which provides the potential to inhibit EGFR
mutation [69].
WT
The results of docking studies against the EGFR revealed that the synthesized
compounds have similar orientations inside the ATP binding site. The designed
compounds gave good binding energies ranging from - 16.90 to - 26.96 kcal/mol (Table
5
).
The binding mode of the co-crystallized ligand, erlotinib, exhibited an energy
binding of - 25.17 kcal/mol. Quinazoline nucleus was oriented in the adenine pocket of
the receptor, The N1 atom of pyrimidine ring formed a hydrogen bond with Met769 with
a distance of 7.ꢀꢃA˚. The phenyl ring formed pi-sigma interaction with Lue694. The
ethynylphenyl moiety occupied the hydrophobic pocket I forming pi-cation interaction

with Lys721, and hydrophobic interaction with Thr766, Thr830, Ala719, Lue764, Ile720
and Ile765 residues. Besides, the bis (2-methoxyethoxy) groups occupied the
hydrophobic region II forming hydrophobic interaction with Leu694 and Gly772 residues
(
Fig 9).
WT
Fig. 9: Erlotinib docked into the active site of EGFR
green) with Met769 residue. The hydrophobic interactions are represented in orange
lines.
formed one hydrogen bond
(
Compound 15 as a representative example showed a binding mode as like as that
b
of erlotinib, with affinity value of - 24.78 kcal/mol. The 1H-pyrazolo[3,4-d]pyrimidine
moiety was oriented in the adenine pocket of the receptor. The two nitrogen atoms of
hydrazone moiety formed two hydrogen bonds with Met769 with a distance of 1.58 and
2
.19 A˚. The hydroxyphenyl moiety occupied the hydrophobic pocket I forming
hydrophobic interactions with Ala719, Lue820 and Val 702 residues, while the hydroxyl
group formed two hydrogen bonds with Thr766 and Thr830 with a distance of 2.32 and
2
.04A˚, respectively. Moreover, the phenyl group at position-6 occupied the hydrophobic

region II forming pi-cation interaction with Lys704, and hydrophobic interaction with
Leu694 Gly772 and Pro770 residues (Fig 10 & 11).
WT
Fig. 10: Compound 15 docked into the active site of EGFR formed four hydrogen
b
bonds (green) with Met769, Thr766, Thr766 and Thr830, residues. The pi interactions are
represented in orange lines.

Fig. 11: Mapping surface showing compound 15 occupying the active pocket of
b
WT
EGFR .
The binding mode of compound 18 was as like as that of erlotinib, with affinity
d
value of - 24.93 kcal/mol. The 1H-pyrazolo[3,4-d]pyrimidine moiety occupied the
adenine binding site forming a hydrogen bond with Lys721 with a distance of 1.64 A˚.
The two nitrogen atoms of ketohydrazinyl group moiety formed two hydrogen bonds
with Asp831 and Thr830 with a distance of 1.56 and 1.87A˚, respectively. The fluoro
phenyl moiety occupied the hydrophobic pocket I forming hydrophobic interactions with
Phe832, Lue843, Lue764, Lue753 and Thr766 residues. In addition, the phenyl group at
position-6 occupied the hydrophobic region II (Fig 12 & 13).

WT
Fig. 12: Compound 18 docked into the active site of EGFR formed three hydrogen
d
bonds (green) with Lys721, Asp831 and Thr830 residues. The pi interactions are
represented in orange lines.

Fig. 13: Mapping surface showing compound 18
d
occupying the active pocket of
WT
EGFR .
T790M
The results of docking studies against the mutant EGFR-TK (EGFR
)
revealed that the synthesized compounds have similar orientations inside the ATP
binding site. Osimertinib and TAK-285(co-crystallized ligand) were used as reference
ligands. The designed compounds gave good binding energies ranging from - 19.27 to -
2
7.46 kcal/mol (Table 5).
The binding mode of the co-crystallized ligand, TAK-285, showed an energy
binding of - 27.35 kcal/mol. Pyrrolo[3,2-d]pyrimidine moiety was oriented in the adenine
pocket of the receptor forming pi-sigma interaction with Leu844 and Ala743. The N1
atom of pyrimidine ring formed a hydrogen bond with Met793 with a distance of 2.23 A˚.
The 3-(trifluoromethyl)phenoxy group occupied the hydrophobic region I forming
hydrophobic interaction with Ile759 and Lys745. One fluoro atom of trifluoromethane
moiety formed one hydrogen bond with Lys745 with a distance of 1.41 A˚. The N-ethyl-
3
-hydroxy-3-methylbutanamide moiety occupied the hydrophobic pocket II. The
hydroxyl group formed a hydrogen bond with Ser720 with a distance of 1.79 A˚. The
central phenyl moiety formed pi-sigma interaction with Lys745, Val726 and Met790 (Fig
1
4).

T790M
Fig. 14: Co-crystallized ligand (TAK-285) docked into the active site of EGFR
, the
hydrogen bonds are represented in green dashed lines and the pi interactions are
represented in orange dashed lines
Compound 17 as a representative example showed a binding mode as like as that
d
of co-crystalized ligand, TAK-285, with affinity value of - 26.50 kcal/mol. The 1H-
pyrazolo [3,4-d]pyrimidine moiety was oriented in the adenine binding pocket forming
pi-sigma interactions with Leu844, Met793, Leu718 and Ala743. Also, N2 atom of
pyrazole moiety formed one hydrogen bond with Met793 with a distance of 1.95 A˚. The
cyclohexyl moiety occupied the hydrophobic pocket I forming hydrophobic interactions
with Lys745. Moreover, the phenyl group at position-6 occupied the hydrophobic region
II forming pi-sigma interactions with Cys797 and Leu844 (Fig 15).

T790M
Fig. 15: Binding of compound 17
d
with EGFR
, the hydrogen bonds are represented
in green dashed lines and the pi interactions are represented in orange dashed lines.
The binding mode of compound 18 was as like as that of TAK-285, with affinity
d
value of - 27.13 kcal/mol. The 1H-pyrazolo[3,4-d]pyrimidine moiety occupied the
adenine binding site forming pi-sigma interactions with Leu844, Leu718, Met793 and
Ala743. N2 atom of pyrazole moiety formed one hydrogen bond with Met793 with a
distance of 2.21 A˚. The 4-fluorophenyl moiety occupied the hydrophobic pocket I
forming hydrophobic interactions with Lys745. Moreover, the phenyl group at position-6
occupied the hydrophobic region II forming pi-sigma interaction with Cys797 (Fig 16).