EGFR inhibitors and apoptotic inducers: Design, synthesis, anticancer activity and docking studies of novel xanthine derivatives carrying chalcone moiety as hybrid molecules

Article abstract of DOI:10.1016/j.bioorg.2019.102997

One of the helpful ways to improve the effectiveness of anticancer agents and weaken drug resistance is to use hybrid molecules. therefore, the current study intended to introduce 20 novel xanthine/chalcone hybrids 9–28 of promising anticancer activity. Compounds 10, 11, 13, 14, 16, 20 and 23 exhibited potent inhibition of cancer cells growth with IC₅₀ ranging from 1.0 ± 0.1 to 3.5 ± 0.4 μM compared to doxorubicin with IC₅₀ ranging from 0.90 ± 0.62 to 1.41 ± 0.58 μM and that compounds 11 and 16 were the best. To verify the mechanism of their anticancer activity, compounds 10, 11, 13, 14, 16, 20 and 23 were evaluated for their EGFR inhibitory effect. The study results revealed that compound 11 showed IC₅₀ = 0.3 μM on the target enzyme which is more potent than staurosporine reference drug (IC₅₀ = 0.4 μM). Accordingly, the apoptotic effect of the most potent compounds 11 was extensively investigated and showed a marked increase in Bax level up to 29 folds, and down-regulation in Bcl2 to 0.28 fold, in comparison to the control. Furthermore, the effect of compound 11 on Caspases 3 and 8 was evaluated and was found to increase their levels by 8 and 14 folds, respectively. Also, the effect of compound 11 on the cell cycle and its cytotoxic effect were examined. Moreover, a molecular docking study was adopted to confirm mechanism of action.

Full text of DOI:10.1016/j.bioorg.2019.102997

Accepted Manuscript
EGFR inhibitors and apoptotic inducers: Design, synthesis, anticancer activity
and docking studies of novel xanthine derivatives carrying chalcone moiety as
hybrid molecules
Hesham A. Abou-Zied, Bahaa G.M. Youssif, Mamdouh F.A. Mohamed, Alaa
M. Hayallah, Mohamed Abdel-Aziz
PII:
S0045-2068(18)31402-0
https://doi.org/10.1016/j.bioorg.2019.102997
102997
DOI:
Article Number:
Reference:
YBIOO 102997
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 December 2018
21 March 2019
17 May 2019
Please cite this article as: H.A. Abou-Zied, B.G.M. Youssif, M.F.A. Mohamed, A.M. Hayallah, M. Abdel-Aziz,
EGFR inhibitors and apoptotic inducers: Design, synthesis, anticancer activity and docking studies of novel xanthine
derivatives carrying chalcone moiety as hybrid molecules, Bioorganic Chemistry (2019), doi: https://doi.org/
10.1016/j.bioorg.2019.102997
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EGFR inhibitors and apoptotic inducers: Design, synthesis,
anticancer activity and docking studies of novel xanthine derivatives
carrying chalcone moiety as hybrid molecules
Hesham A. Abou-Zied1, Bahaa G.M. Youssif2,3*, Mamdouh F. A. Mohamed4, Alaa M.
Hayallah1,2*, Mohamed Abdel-Aziz5
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Deraya
University, Minia, Egypt; 2Pharmaceutical Organic Chemistry Department,
3
Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt; Department of
Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Aljouf, Sakaka
2014, Saudi Arabia; 4Department of Pharmaceutical Chemistry, Faculty of
5
Pharmacy, Sohag University, 82524 Sohag, Egypt; Department of Medicinal
Chemistry, Faculty of Pharmacy, Minia University, 61519-Minia, Egypt.
Short running title: Synthesis, anticancer, and docking study of new xanthine-
chalcone hybrids
*To whom correspondence should be addressed:
Bahaa G. M. Youssif. Ph.D. Pharmaceutical Organic Chemistry Department, Faculty
of Pharmacy, Assiut University, Assiut 71526, Egypt
Tel.: (00966)-537240294
E-mail address: bgyoussif@ju.edu.sa
Alaa M. Hayallah. Ph.D. Pharmaceutical Organic Chemistry Department, Faculty
of Pharmacy, Deraya University, Minia, Egypt
Tel.: (002)-01283410110
E-mail address: alaa_hayalah@yahoo.com

Abstract
One of the helpful ways to improve the effectiveness of anticancer agents and weaken
drug resistance is to use hybrid molecules. therefore, the current study intended to
introduce 20 novel xanthine/chalcone hybrids 9-28 of promising anticancer activity.
Compounds 10, 11, 13, 14, 16, 20 and 23 exhibited potent inhibition of cancer cells
growth with IC50 ranging from 1.0±0.1 to 3.5±0.4 μM compared to doxorubicin with
IC50 ranging from 0.90± 0.62 to 1.41 ± 0.58 μM and that compounds 11 and 16 were
the best. To verify the mechanism of their anticancer activity, compounds 10, 11, 13,
14, 16, 20 and 23 were evaluated for their EGFR inhibitory effect. The study results
revealed that compound 11 showed IC50 = 0.3 µM on the target enzyme which is more
potent than staurosporine reference drug (IC50 = 0.4 µM). Accordingly, the apoptotic
effect of the most potent compounds 11 was extensively investigated and showed a
marked increase in Bax level up to 29 folds, and down-regulation in Bcl2 to 0.28 fold,
in comparison to the control. Furthermore, the effect of compound 11 on Caspases 3
and 8 was evaluated and was found to increase their levels by 8 and 14 folds,
respectively. Also, the effect of compound 11 on the cell cycle and its cytotoxic effect
were examined. Moreover, a molecular docking study was adopted to confirm
mechanism of action.
Keywords: Xanthine; Chalcone; Hybrids; EGFR; Anti-proliferative; Apoptotic assay.

Graphical abstract
EGFR inhibitors and apoptotic inducers: Design, synthesis, anticancer activity and
docking studies of novel xanthine derivatives carrying chalcone moiety as hybrid
molecules
Hesham A. Abou-Zied1, Bahaa G.M. Youssif2,3, Mamdouh F. A. Mohamed4, Alaa M. Hayallah1,2*,
Mohamed Abdel-Aziz5
O
H
H3C
O
N
N
S
O
O
N
N
HN
CH3
11
Cl

Highlights
 A series of 20 novel compounds contain xanthine-chalcone hybrid was
designed and synthesized.
 The synthesized hybrids were evaluated for their in vitro antiproliferative
activity against different human cancer cell lines.
 Compounds 10, 11, 13, 14, 16, 20 and 23 exhibited potent inhibition of cancer
cells.
 Compound 11 was selected for cell cycle analysis and apoptosis assay
 Results revealed that these compounds showed programmed cell death and
cell cycle arrest
 Molecular modeling for compounds 10, 11, 20 and 21 were done against 3D
crystal structures of EGFR.

1. INTRODUCTION
Cancer is one of the primary causes of death universal [1]. Most patients who have
been diagnosed with cancer suffer from poor quality of life due to adverse
proceedings associated with cancer [2]. One of the most effective methods of
suppressing tumor growth and tumor eradication is chemotherapy [3]. However, many
patients undergoing chemotherapy have associated side effects such as
thrombocytopenia, anemia, nausea and vomiting [4]. Although, many research
studies have reported potential chemotherapeutic effects of new compounds, research
is still underway in the hope of finding new anticancer agents with improved efficacy
and high degree of safety toward normal host cells [5, 6]. Methylxanthine derivatives
such as caffeine (1) and theophylline (2) were found to induce apoptosis, and promote
cytotoxicity induced by doxorubicin [7]. Theophylline was found to induce
programmed cell death in various human cancer cell line and in a malignantly
transformed granulosa cell line when synergizing with gemcitabine or cisplatin [8].
The mechanism of the apoptogenic effect of theophylline has been found to involve
reduction of intracellular levels of the antiapoptotic mediator Bcl2. Therefore,
theophylline-based moieties comprise an attractive scaffold for structural modification
in search of novel antiproliferative agents with diverse pharmacodynamics [9].
Besides theophylline and its congeners other methylxanthines have been also
evaluated for anticancer and chemosensitivity-modulating agents. Pentoxifylline (3)
was found to increase the effectiveness of both radiotherapy and chemotherapy [10,
11]. A combination schedule of pentoxifylline with doxorubicin, exhibited synergistic
action and inhibited cell proliferation to a superior extent with regard to each drug
used alone [12].

Recent studies demonstrated that molecular hybridization of chalcone units with
biologically active pharmacophore produced new hybrids with synergistic biological
activity [13]. For example, Compound 4 showed remarkable cytotoxic activity against
human lung adenocarcinoma A549 cells with IC50 of 4.4 M compared to cisplatin.
The further mechanistic study of compound 4 demonstrated that 1, 2, 4-triazole-
chalcone hybrids induced apoptosis via increased level of proapoptotic protein Bax,
release of Cytochrome C from mitochondria and activation of caspase-3/8/9 proteins
[13]. The presence of chalcone derivativesas a key element, or a substituent or as a
side-chain in different biologically active compounds has encouraged the synthetic
chemists to synthesize new compounds bearing this moiety and hence, different newly
synthesized chalcones with their anti-cancer activities are discussed [14-17].
Encouraged by all these facts, the present work aimed at gathering two bioactive
entities chalcone and xanthine derivative in only one compact hybrid structure for the
purpose of synergism and/or decreasing the expected adverse effects. Synthesis of
novel hybrid compounds based on xanthine and chalcone pharmacophores through S-
alkylation of 1,3,8-trisubstituted and 1,8-disubstituted xanthine derivatives with
different acetylated chalcones.

O
N
O
N
O
N
O
H
N
N
N
H
N
N
N
N
O
N
O
N
O
Caffeine (1)
Theophylline (2)
Pentoxifylline (3)
O
H
H3CO
R1
O
N
N
S
O
N
O
O
S
N
N
R2
N
N
HN
O
HN
R3
OCH3
Compounds 9-28
Compound (4)
H3CO
OCH3
Figure 1: Structure of compounds 1-4 and rational compounds 9-28.
the newly synthesized compounds were identified using different spectroscopic
techniques including: IR, 1H NMR, 13C NMR as well as elemental microanalysis. All
target compounds were evaluated for their anti-proliferative activity in vitro on
selected human cancer cell lines. In order to investigate the mechanistic pathways of
the anticancer activity of the target compounds, the most potent hybrids were selected
to perform further investigations such as EGFR assay, cell cycle analysis, and
apoptosis markers.

2. RESULTS AND DISCUSSION
2.1. Chemistry
The synthesis of purine-2,6-diones 4a-b was described in Scheme 1. The intermediate
6-Amino-1,3-dimethyluracil 1a was prepared by condensation of N,N'-dimethylurea
with cyanoacetic acid, followed by stirring in 70% (w/v) sodium hydroxide solution
to afford the desired compound 1a [18-20]. 6-Amino-3-methyluracil 1b was prepared
by refluxing 6-aminouracil in hexamethyldisilazane (HMDS) in presence of a
catalytic amount of ammonium sulfate, followed by addition of methyl iodide to
afford the desired N3-substituted 6-aminouracil [21, 22]. Nitrosation of compounds
1a-b using sodium nitrite in 50% aqueous acetic acid yielded 2a-b. Sodium dithionite
reduction of 2a-b in aqueous ammonia solution yielded compounds 3a-b. Direct
cyclization of 5,6-diamino-1,3-dimethyluracil 3a using carbon disulfide in DMF
under reflux for 5 h afforded compound 4a while, Compound 4b was prepared by
direct cyclization of 5,6-diamino-1,3-dimethyluracil 3b using carbon disulfide in
ethanolic solution of KOH under reflux for 4 h.

O
O
O
O
N
N
b
a
N
NC
H
+
N
N
OH
H
H
O
O
N
NH2
CN
N,N-dimethyl urea
Cyanoacetic acid
1-(2-Cyano-acetyl)-1,3-dimethyl-urea
1a
O
O
H3C
c
N
HN
N
NH2
O
N
NH2
O
H
H
6-amino uracil
1b
O
N
O
N
O
R1
R1
R1
N
NH2
NH2
NO
f
d
N
e
O
N
O
NH2
O
NH2
N
R2
R2
R2
3a-b
1a-b
O
O
H
R1
N
H
N
N
R1
O
N
SH
S
N
N
O
N
H
N
R2
R2
4a-b
R1 = CH3; R2 = CH3 or H
Scheme 1: Synthesis of Xanthine derivatives 4a-b.
Reagents and reaction condition: a) acetic anhydride; b) NaOH 70 %; c) 1. HMDS,
2. CH3I, 3. Na2S2O3, NaHCO3; d) NaNO2/CH3COOH; e) 12.5% aq. NH3/ Na2S2O4; f)
CS2/ (DMF or ethanol)/ reflux.
The target hybrid molecules 9-28 were prepared according to Scheme 2. Chalcone
derivatives 7a-j were synthesized by a base catalyzed Claisen–Schmidt condensation
of 4-aminoacetophenone with substituted benzaldehyde derivatives [23]. Stirring
chalcones 7a-j with bromoacetylbromide in biphasic layer from potassium carbonate
solution in dichloromethane forming the corresponding acetylated chalcones 8a-j.
Alkylation of compounds 4a-b with acylated chalcones 8a-j afford the target

compounds 9-28 in good yields. The chemical structures of 9-28 were elucidated by
13
1
1H NMR and C NMR spectroscopy. The H NMR spectrum of compound 11 as a
representative example of 1,3-dimethyl xanthine derivatives 9-18 revealed the
appearance of asinglet signal with three protons integration at δ 3.22 ppm represented
(N1-CH3), while (N3-CH3) appeared at δ 3.39 ppm as singlet signal with three protons
integration, appearance of a singlet signal with two protons integration at δ 4.26 ppm
assigned for methylene protons (S-CH2-CO) of the linker. Furthermore, the two
chalcone protons appears at aromatic region as doublet signal at δ 7.73 and δ7.93 ppm
with coupling constant J = 15.00 Hz, two doublet of doublet signals related to
aromatic protons and also, appearance of a singlet signal at 10.71 ppm assigned to
amide proton NH, characteristic broad singlet signal at δ13.63 ppm represents (N7-H)
of xanthine nucleus. The 13C NMR spectrum of 11 showed the appearance of two
peaks at δ 28.18 and δ 30.20 ppm assigned to N1-CH3 and N3-CH3 of xanthine
moiety, while methylene carbon of the linker peak at δ 37.09 ppm, all other carbons
1
appear at their expected chemical shifts. H NMR spectrum of compound 19 as a
representative example of 3-methyl xanthine derivatives 19-28 showed asinglet signal
at δ 3.18 ppm represented (N1-CH3), a singlet signal at δ 4.24 ppm of methylene
protons (S-CH2-CO) of the linker. Moreover, the two chalcone protons appears at
aromatic region as doublet signal at δ 7.75 and δ 7.97 ppm with coupling constant J =
15.08 Hz and also, appearance of a broad singlet signal at δ11.89 ppm represents (N1-
H) of xanthine nucleus. The 13C NMR spectrum of 19 showed the appearance of one
peak at δ 27.43 ppm related to N1-CH3 of xanthine moiety, while methylene carbon of
the linker peak at δ36.96 ppm.

O
O
O
O
b
a
O
+
Br
N
H2N
R3
H2N
R3
R3
H
5
7a-g
6a-g
8a-g
O
O
H
N
R1
H
N
N
R1
c
S
O
N
8a-g
SH +
O
N
O
N
R2
N
HN
N
O
R2
4a-b
9-28
R3
Comp
9
R1
R2
R3
H
Comp
19
R1
R2
H
H
H
H
H
H
H
H
R3
H
Methyl Methyl
Methyl Methyl
Methyl Methyl
Methyl Methyl
Methyl Methyl
Methyl Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
10
2-Cl
4-Cl
4-Br
20
2-Cl
4-Cl
4-Br
11
21
12
22
13
4-OCH3
23
4-OCH3
3,4-di-OCH3
14
3,4-di-OCH3
24
15
Methyl Methyl 3,4,5-tri-OCH3
25
3,4,5-tri-OCH3
3-NO2
16
Methyl Methyl
3-NO2
26
17
18
Methyl Methyl
Methyl Methyl
4-CH3
27
28
Methyl
Methyl
H
H
4-CH3
2,4-di-CH3
2,4-di-CH3
Scheme 2: Synthesis of target compounds 9-28.
Reagents and reaction conditions: a) NaOH 60%; b) BrCH2COBr/ CH2Cl2; c) TEA/
acetonitrile

2.2. Evaluation of biological Activity
2.2.1. In vitro anticancer activity
2.2.1.1. Cell viability assay
Cell viability assay was carried out using human mammary gland epithelial cell line
(MCF-10A). All synthesized compounds 9-28 were treated with MCF-10A cells for 4
days and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
was used to determine the viability of cells. All compounds were exhibited non-toxic
with the majority of them reveling more than 90% cell viability at the concentration of
50 μM.
2.2.1.2. Antiproliferative activity
All final hybrids 9-28 were evaluated for their antiproliferative activity against four
cancer cell lines, human pancreas cancer cell line (Panc-1), breast cancer cell line
(MCF-7), colon cancer cell line (HT-29) and epithelial cancer cell line (A-549), using
MTT assay and Doxorubicin was used as the reference compound. GraphPad Prism
software (GraphPad Software, San Diego, CA, USA) was used to calculate the
median inhibition concentration (IC50) for all compounds.
As shown in Table 1, seven most active compounds 10, 11, 13, 14, 16, 20 and 23
among both 1,3-dimethyl substituted xanthine and 1-methyl substituted xanthine
derivatives exhibited potent inhibition of cancer cells growth with IC50 ranging from
1.0±0.1 to 3.5±0.4 μM compared to doxorubicin with IC50 ranging from 0.90± 0.62 to
1.41 ± 0.58 μM. Compound 16 possessed highest anti cancer activity among all new
hybrids against cancer cell growth with IC501.0±0.1 μM for the MCF-7 breast cancer
cell line. All other compounds showed moderate activity against growth of cancer cell
lines. Compound 16 displayed the peak anticancer potential and it was bearing 1,3-

dimethyl xanthine backbone while on the other side compound 26 was bearing same
substitution pattern as compared to 16 and difference was 1-methyl xanthine moiety
but it showed almost six folds less activity. Compound 11 of 1,3-dimethyl xanthine
(R3 = 4-Cl) class showed highly potent anticancer activity (IC50 = 1.3µM) against
breast cancer cell line (MCF-7). Interestingly, compound 21, with R3 = 4-Cl at
position of 1-methyl xanthine core showed weak cytotoxicity against breast cancer
cell line (IC50 = 9.4µM), which is 7 fold less activity than 11. From the above-
mentioned results, it can be concluded that most of 1,3-dimethyl xanthine hybrids 9-
18 were better antiproliferative agents of cancer cells as compared to other derivatives
19-28 bearing 1-methyl xanthine core.
Table 1: Antiproliferative activity of the target compounds 9-28.
Compound
number
Antiproliferative activity IC50 ± SEM (μM)
Panc-1
MCF-7
HT-29
A-549
6.7±1.5
9
4.9±2.4
1.9±1.1
1.7±1.6
2.91±0.2
2.1±1.1
2..61±0.2
3.3±2.4
1.3±0.9
9.7±2.1
5.6±2.4
6.9±1.5
5.1±2.4
1.8±0.7
1.3±0.9
2.8±0.5
1.1±0.7
1.5±0.5
4.5±2.4
1.0±0.1
11.1±1.7
6.4±2.4
6.8±2.7
5.9±1.7
2.3±1.6
1.7±0.9
3.7±0.4
1.9±1.6
3.4±0.4
4.3±1.7
1.2±0.7
11.6±1.5
6.1±1.7
6.2±2.4
10
11
12
13
14
15
16
17
18
19
1.7±0.7
1.8±0.7
3.5±0.2
2.2±0.7
3.2±0.2
5.1±1.5
1.5±0.5
10.2±1.9
7.1±1.5
7.7±1.6

20
3.3±0.9
7.8±1.7
7.8±1.7
2.7±1.6
6.2±1.5
4.6±1.2
6.2±1.5
5.5±2.5
6.8±1.5
3.0±0.1
9.4±2.1
9.4±2.1
2.3±0.9
7.4±1.4
5.3±1.2
6.4±2.7
5.9±3.0
7.2±1.7
3.2±0.7
6.5±2.4
6.5±2.4
2.7±0.9
8.9±2.7
3.1±1.4
5.3±2.4
6.7±2.4
8.2±1.5
1.01 ± 0.82
3.5±0.5
7.3±2.1
7.3±2.1
2.8±0.7
7.4±2.1
6.6±1.6
6.7±1.6
6.1±1.1
8.1±2.1
1.21 ± 0.80
21
22
23
24
25
26
27
28
Doxorubicin
1.41 ± 0.58 0.90± 0.62
Moreover, the substitution variations ( R3) at position 4 of chalcone phenyl ring can
be correlated with the activity of compounds. Interestingly, in 1,3-dimethyl xanthine
series three compounds 10, 11 and 16 with an electron withdrawing substitution
groups on chalcone phenyl ring showed better inhibitory activity against almost all the
cell lines tested compared to compounds 17 and 18 which were bearing electron
donating substitution groups and that NO2, Cl and OMe were the best.
2.2.2. Inhibition of Epidermal Growth Factor Receptor Activity (EGFR-TK)
EGFR-TK assay was performed to assess the EGFR inhibitory potency of novel
compounds and the results are included in Table 2. The results from this assay
complement the findings of cancer cell-based assay. All investigated compounds 10,
11, 13, 14, 16 and 23 exhibited inhibition of EGFR with IC50 ranging from 0.3 to 1.6
μM. According to data presented, three derivatives 10, 11 and 14 selected from
xanthine-chalcone hybrids were found to be most potent and their EGFR inhibitory
activities were close to the positive reference staurosporine (IC50 = 0.4 μM). This

experiment shows that these compounds are potent EGFR inhibitors and can possibly
be used as anticancer agents.
Table 2: Effects of compounds 10, 11, 13, 14, 16, 23, and staurosporine on EGFR.
Compd. No.
EGFRIC50(μM)
10
11
13
14
16
0.6
0.3
1.5
0.9
1.6
23
1.2
0.4
staurosporine
2.2.3. Apoptotic markers activation assay
Apoptosis, also called programmed cell death, involves several morphological and
biochemical events [24]. There are proapoptotic proteins such as Bad, Bax, Bid, BcL-
Xs and Bim, as well as antiapoptotic members such as Bcl-2, Bc-LXl and Bcl-W [25].
Anti-apoptotic proteins act as inhibitors of apoptosis by blocking the release of
cytochrome-c while proapoptotic members function as activators of its release. When
the ratio of proapoptotic proteins exceeds antiapoptotic ones, the outer mitochondrial
membrane becomes permeable leading to a cascade of events. Cytochrome C is
released which activates caspase-9 which then activates caspase-3 which in turn
stimulates apoptosis by attacking many valuable proteins required by the cell [26].

2.2.3.1. Caspase-3 activation assay
Xanthine-chalcone hybrids 10, 11, 13, 14, 16 and 23 were evaluated as caspase-3
activators against Panc-1 human pancreas cancer cell line and the results are listed in
Table 3. The results revealed that compounds 11, 14 and 23 possessed remarkable
over expression of caspase-3 protein level (532.2 ±5.13, 373.2 ±3.92 and 387.4 ±12.2
pg/mL, respectively) compared to the reference staurosporine (503.2 ± 4.22 pg/mL).
The most active compound which was bearing 4-Cl on the phenyl ring of chalcone
moiety 11 caused over-expression of caspase-3 protein level (532.2 ±5.13 pg/mL) in
Panc-1human pancreas cancer cell line about 8 folds higher than control, and higher
than that of staurosporine (Figure 2). So, the above results may be considered as a
suggestion that apoptosis may be attributed to over-expression of caspase-3 which
induced by the tested compounds.
Table 3: Caspase-3 level for compounds 10, 11, 13, 14, 16, 23, and staurosporine in
human pancreas cancer cell line (Panc-1).
Compound Caspase-3
Number
Conc (Pg/ml)
315.20 ± 9.27
532.20 ± 5.13
254.70 ± 3.14
373.20 ± 3.92
339.80 ± 2.19
387.40 ± 12.2
Fold change
4.80
10
11
13
14
16
23
8.10
3.87
5.68
5.17
5.90
Staurosporine 503.20 ± 4.22
Control 65.64
7.66
1

Figure 2: Caspase-3 level for compounds 10, 11, 13, 14, 16, 23, and staurosporine in
human pancreas cancer cell line (Panc-1).
2.2.3.2. Caspase-8, Bax and Bcl-2 levels assay
The most three active caspase-3 activator xanthine-chalcone hybrids 11, 14 and 16
were further studied for their effect on caspase-8, Bax and Bacl-2 levels against Panc-
1human pancreas cancer cell line using staurosporine as a reference, which are
mentioned in Table 4. The results showed that all of the tested compounds revealed
remarkable increase in both caspase-8 and Bax levels compared to staurosporine.
compound 14 which was bearing 3,4-dimethoxy groups on the phenyl ring of
chalcone moiety possessed the maximum over-expression of caspapse-8 level (1.43
ng/mL) followed by compound 11 (1.21 ng/mL) and that of the reference doxorubicin
(1.755 ng/mL) (Figure 3). Moreover, 1,3-dimethyl xanthine-chalcone hybrid 11
showed a comparable induction of Bax (235 pg/mL) compared to doxorubicin (276
pg/mL) with 25 fold higher than control untreated Panc-1 cancer cells. Finally,
compound 16 which was bearing 3-NO2 group on the phenyl ring of chalcone moiety

caused equipotent down-regulation of Bcl-2 protein level (1.22 ng/mL) followed by
compound 11 (1.46 ng/mL) in Panc-1cell line compared to staurosporine (0.98
ng/mL).
Table 4: Caspase-8, Bax and Bcl-2 levels for compounds 11, 14, 16, and
staurosporine on human pancreas cancer cells (Panc-1).
Compound
Number
Caspase-8
Bax
Bcl-2
Fold
(ng/ml) change
Conc
Fold
change
14.33
16.92
12.77
20.76
1
Conc
(Pg/ml)
235.20
208.90
188.50
276.19
8.26
Fold
Conc
(ng/ml)
1.21
change
28.46
25.27
22.80
33.42
1
11
14
1.46
1.87
1.22
0.98
5.08
3.48
2.71
4.16
5.18
1
1.43
16
1.08
staurosporine
Control
1.75
0.084
2.0
1.5
1.0
0.5
0.0
11
14
16
Staturosporine
Cont.
e
11
14
16
Cont.
Staturosporin
Figure 3 : Caspase-8 level for compounds 11, 14, 16, and staurosporine in human
pancreas cancer cells (Panc-1).

2.2.4. Flow cytometric Cell cycle analysis
Cell cycle analysis was performed for the most active compound 11 as standard drug
against Panc-1 human pancreas cancer cell line.the percentages of cells of Panc-1cell
in G0/G1 phase of the cell cycle in the control was 53.64% which recorded a
noteworthy decrease to 26.67% upon treatment with compound 11 (Figure 4 and 5).
These percentages were markedly increased at the G2/M phase to 48.56% for
compound 11 due to accumulation of cells at this phase. Furthermore, it is obvious
that the apoptotic cell percentage was increased from 1.79% for control untreated
Panc-1cancer cell to 27.15% and 33.57% in treated cells with compound 11 and
doxorubicin, respectively, (Figure 5 and 6). The results indicated that the percentage
of the late apoptosis is more than that of early apoptosis which a good evidence for
irreversible apoptosis caused by compounds 11 (Figure 5 and 6). According to the
above results, it is clear that the compound 11 exhibited pre G1 apoptosis and cell
cycle arrest at G2/M phase. Moreover, it is obvious that the tested compounds not
cytotoxic but antiproliferative causing programmed cell death and cell cycle arrest.
60
%G0-G1
%S
40
20
0
%G2-M
%Pre-G1
11
Control
Figure 4: Cell cycle analysis on Panc-1cell line treated with compound 11

30
20
10
0
11
Control
s
i
s
o
% Late
% Total
% Early
% Necr
Figure 5: Percentage of apoptosis and necrosis for compound 11 onPanc-1cell line.
Figure 6: Cell cycle analysis and Apoptosis induction analysis using Annexin V/PI of
compound 11 and control untreated RPMI8226 cell.

2.3. Docking study
In order to achieve some structural insights into EGFR inhibitory activities of the
newly synthesized target compounds, a molecular docking simulation was done using
CDOCKER embedded in the Discovery Studio software (Accelrys® software
corporation, San Diego, USA). The 3D crystal structure of EGFR (PDB ID: 1M17) in
complex with AQ4999 was used for this docking study [27, 28]. The most active
disubstituted compounds 10 and 11 and their corresponding monosubstituted
derivatives 20 and 21 were selected to be docked into EGFR binding site. The
superimposition of the active docked poses inside protein binding pocket were shown
in Figure 7. As a first step, validation of the docking protocol settings was done
through the re-docking of the extracted co-crystallized ligand AQ4999 from the 3D
structure 1M17 using the same protocol for the docked compounds. The used docking
protocol closely reproduced the bound structure with RMSD value of 0.73 Å
confirming the confidence in our docking study. Interestingly, the docking studies
were in consistence with the results of EGFR inhibitory assay. The inspection of
docking results revealed that the two docked ligands 10 and 11 adopted a nearly
similar disposition inside the ATP binding pocket of the EGFR. The docking results
of the most potent compound 10 and 11 against EGFR indicated that it fits well inside
the ATP-active site. Also, it was found that chalcone moiety aligns near the DFG
motif which plays a pivotal role in the kinase activity regulation forming a network of
hydrogen bonds with Gly767, Met769 residues in compound 10 and with Arg817 and
Asp831 residues in compound 11, in addition to many hydrophobic interactions as
shown in Figure 8. The xanthine moiety was oriented towards the hinge region and
interestingly making two additional hydrogen bonds with Pro770 and His871 residue
in both compounds, in addition to many hydrophobic interactions with Phe771 and

Tyr77, all these interactions may greatly contribute to the high in potency of
compounds 10 and 11.
On the hand, the docking results of the monosubstituted derivatives 20 and 21 against
EGFR indicated that they adopted a similar binding pattern to that of compound 10
and 11, however, there was adecrease in the number of engaged hydrogen bonds.
Compound 20 forms one hydrogen bond with His781 residue, while compound 21
forms two hydrogen bonds with Met769 and his781 residue Figure 9. These results
confirm our rationalized design that the disubstituted derivatives are more potent than
the mono substituted ones.
Figure 7: Superimposition of the active docked poses 10, 11, 20 and 21 inside the
EGFR active site (PDB code: 1M17) where EGFR protein is represented as a
secondary structure (flatribbon display style). The binding pockets are depicted
according to atom charges. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)

Figure 8: (A) Docking and binding pattern of compound 10 (pink) into ATP-active
site of EGFR kinase (PDB code: 1M17); (B) Docking and binding pattern of
compound 11 (red) into ATP-active site of EGFR kinase. Hydrogen bonds were
represented as dashed green lines. All hydrogens were removed for the purposes of
clarity.
Figure 9: (A) Docking and binding pattern of compound 20 (cyan) into ATP-active
site of EGFR kinase (PDB code: 1M17); (B) Docking and binding pattern of
compound 21 (yellow) into ATP-active site of EGFR kinase. Hydrogen bonds were
represented as dashed green lines.

4. EXPERIMENTAL
4.1. Chemistry
All reagents for synthesis were purchased from Sigma-Aldrich (St. Louis Mo., USA)
and MERCK (Schuchardt, Germany). All solvents used in this work weren’t further
distilled before to be used. The Reactions were monitored by TLC (Thin-layer
Chromatography) Pre-coated aluminum sheets kieselgel 60 F254 with fluorescent
indicator (MERCK). Melting points (m.p.) were determined on Stuart electrothermal
melting point apparatus and were uncorrected. 1H NMR spectra were recorded using a
Bruker Advance 400 MHz NMR spectrometer, Faculty of Pharmacy, Beni Suef
University; chemical shift (δ) in ppm relative to TMS (δ=0 ppm) as internal standard
and DMSO-d6as solvent. Coupling constant (J) in Hz and the signal are designed as
13
follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. C NMR spectra
were carried out on Bruker Advance 101 MHZ spectrometer, Faculty of Pharmacy,
Beni Suef University; chemical shift (δ) in ppm relative to TMS (δ=0 ppm) as internal
standard and DMSO-d6as solvent. LC-MS was carried out using an Agilent 6420
triple Quad LC Mass Spectrometer, Faculty of Pharmacy, Minia University.
Elemental analyses were recorded on Shimadzu GC/ MS-QP5050A, Regional center
for Mycology and Biotechnology, Al-Azhar University.
Compounds 1a-b [29-30], 2a-b [31], 3a-b [31], 4a [20], 4b [21], 7a-j [16, 17] and 8
a-j [17] were prepared according to the previous reported studies.

4.1.1. General procedure for synthesis of Xanthine-Chalcone hybrids (9-28)
To a stirred solution of 4a-b (0.9 mmol) and the appropriate acetylated chalcone
derivative 8a-j (0.9 mmol) in acetonitrile, TEA (0.18 mL/mol) was added, the
reaction mixture was stirred at room temperature until a precipitate formed. The
obtained precipitate was filtrated off and recrystallized from acetonitrile to afford the
target compounds 9-28.
4.1.1.1. 2-((1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)thio)-N-(4-
((E)-cinnamoylphenyl)acetamide (9)
1
Straw yellow crystals (0.35 g, 83% yield); m.p > 300 ºC; H NMR (400 MHz,
DMSO-d6) δ (ppm): 13.63 (1H, s, N7-H), 10.73 (1H, s, O=C-NH), 8.18 (2H, d, J =
8.6 Hz, Ar-H), 7.95 (1H, d, J = 15.6 Hz, CH=CH), 7.87-7.91 (2H, m, Ar-H), 7.78
(2H, d, J = 8.6 Hz,Ar-H), 7.73 (1H, d, J = 15.6 Hz, CH=CH), 7.47-7.49 (3H, m, Ar-
13
H), 4.26 (2H, s, SCH2), 3.38 (3H, s, N3-CH3), 3.18 (3H, s, N1-CH3); C NMR (101
MHz, DMSO-d6) δ (ppm): 187.99, 166.74, 154.56, 151.45, 148.82, 147.83, 143.91,
143.63, 135.22, 133.01, 130.97, 130.44, 129.37, 129.27, 122.41, 118.99, 108.27,
36.96, 30.20, 28.16; Anal. Calcd. For C24H21N5O4S (475.13): C, 60.62; H, 4.45; N,
14.73. Found: C, 60.51; H, 4.58; N, 14.63.
4.1.1.2. 2-((1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)thio)-N-(4-
((E)-3-(2-chlorophenyl)acryloyl)phenyl)acetamide (10)
1
Orange powder (0.37g, 80.6% yield); m.p > 300 ºC; H NMR (400 MHz, DMSO-d6)
δ (ppm): 13.48 (1H, s, N7-H), 10.73 (1H, s, O=C-NH), 8.20-8.23 (3H, m, CH=CH +
Ar-H), 7.93-8.09 (2H, m, Ar-H), 7.78 (2H, d, J = 8.5 Hz, Ar-H), 7.54-7.60 (1H, m,
Ar-H), 7.43-7.51 (2H, m, Ar-H + CH=CH), 4.24 (2H, s, SCH2), 3.39 (3H, s, N3-

CH3), 3.22 (3H, s, N1-CH3); 13C NMR (101 MHz, DMSO-d6) δ(ppm): 187.77,
166.79, 154.57, 151.45, 148.79, 147.83,143.85, 138.47, 134.76, 132.82, 132.70,
132.37, 130.60, 130.48, 129.01, 128.15, 125.16, 119.02, 108.30, 36.95, 30.20, 28.18;
Anal. Calcd. For C24H20ClN5O4S (509.96): C, 56.52; H, 3.95; N, 13.73. Found: C,
56.79; H, 4.11; N, 13.94.
4.1.1.3. 2-((1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)thio)-N-(4-
((E)-3-(4-chlorophenyl)acryloyl)phenyl)acetamide (11)
Pale yellow powder (0.38g, 82% yield); m.p > 300 ºC; 1H NMR (400 MHz, DMSO-
d6) δ (ppm): 13.63 (1H, s, N7-H), 10.71 (1H, s, O=C-NH), 8.18 (2H, d, J = 8.8 Hz,
Ar-H), 7.97 (1H, d, J = 15.6 Hz, CH=CH), 7.93 (2H, d, J = 8.6 Hz, Ar-H), 7.77 (2H,
d, J = 8.8 Hz, Ar-H), 7.71 (1H, d, J = 15.6 Hz, CH=CH),7.53 (2H, d, J = 8.6 Hz, Ar-
13
H), 4.26 (2H, s, SCH2), 3.39 (3H, s, N3-CH3), 3.22 (3H, s, N1-CH3); C NMR (101
MHz, DMSO) δ (ppm): 187.86, 166.91, 153.79, 151.39, 148.68, 143.73, 142.42,
135.44, 134.22, 132.91, 130.98, 130.51, 129.40, 123.16, 118.98, 108.32, 99.99, 37.45,
30.20, 28.18; Anal. Calcd. For C24H20ClN5O4S (509.96): C, 56.52; H, 3.95; N, 13.73.
Found: C, 56.09; H, 4.12; N, 14.01.
4.1.1.4. 2-((1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)thio)-N-(4-
((E)-3-(4-bromophenyl)acryloyl)phenyl)acetamide (12)
1
White crystal (0.43g, 86% yield); m.p> 300 ºC; H NMR (400 MHz, DMSO-d6) δ
(ppm): 13.63 (1H, s, N7-H), 10.72 (1H, s, O=C-N H), 8.18 (2H, d, J = 8.8 Hz, Ar-H),
8.00 (1H, d, J = 15.7 Hz, CH=CH), 7.87 (2H, d, J = 8.8 Hz, Ar-H), 7.78 (2H, d, J =
8.5 Hz, Ar-H), 7.76 (1H, d, J = 15.7 Hz, CH=CH),7.68 (2H, d, J = 8.5 Hz, Ar-H),
13
4.26 (2H, s, SCH2), 3.39 (3H, s, N3-CH3), 3.22 (3H, s, N1-CH3); C NMR (101
MHz, DMSO) δ (ppm): 187.89, 166.94, 153.80, 151.40, 148.73, 143.74, 142.52,

134.55, 132.91, 132.34, 131.19, 130.51, 124.32, 123.21, 119.00, 108.33, 99.98, 37.09,
30.19, 28.18; Anal. Calcd. For C24H20BrN5O4S (554.42): C, 51.99; H, 3.64; N, 12.63.
Found: C, 52.23; H, 3.81; N, 12.89.
4.1.1.5. 2-((1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)thio)-N-(4-
((E)-3-(4-methoxyphenyl)acryloyl)phenyl)acetamide (13)
White powder (0.36 g, 79.5% yield); m.p > 300 ºC; 1H NMR (400 MHz, DMSO-d6) δ
(ppm): 13.63 (1H, s, N7-H), 10.69 (1H, s, O=C-NH), 8.15 (2H, d, J = 8.8 Hz,Ar-H),
7.84 (2H, d, J = 8.8 Hz, Ar-H), 7.79 – 7.73 (3H, m, CH=CH + Ar-H), 7.70 (1H, d, J =
15.5 Hz, CH=CH), 7.02 (2H, d, J = 8.8 Hz, Ar-H), 4.26 (2H, s, SCH2), 3.83 (3H, s,
OCH3), 3.39 (3H, s, N3-CH3), 3.22 (3H, s, N1-CH3); 13C NMR (101 MHz, DMSO) δ
(ppm): 187.85, 166.85, 161.74, 153.79, 151.38, 148.71, 148.68, 143.88, 143.44,
133.29, 131.15, 130.28, 127.86, 119.86, 118.96, 114.85, 108.32, 55.84, 37.10, 30.20,
28.17; Anal. Calcd. For C25H23N5O5S (505.55): C, 59.39; H, 4.59; N, 13.85. Found:
C, 59.21; H, 4.80; N, 14.12.
4.1.1.6. 2-((1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)thio)-N-(4-
((E)-3-(3,4-dimethoxyphenyl)acryloyl)phenyl)acetamide (14)
White powder (0.39 g, 81% yield); m.p > 300 ºC; 1H NMR (400 MHz, DMSO-d6) δ
(ppm):13.63 (1H, s, N7-H),10.73 (1H, s, O=C-NH), 8.17 (2H, d, J = 8.7 Hz, Ar-H),
7.83 (1H, d, J = 15.5 Hz, CH=CH), 7.77 (2H, d, J = 8.7 Hz,Ar-H), 7.69 (1H, d, J =
15.5 Hz, CH=CH), 7.54 (1H, s,Ar-H), 7.38 (1H, d, J = 8.3 Hz, Ar-H), 7.03 (1H, d, J =
8.3 Hz, Ar-H), 4.26 (2H, s, SCH2), 3.87 (3H, s, OCH3), 3.82 (3H, s, OCH3), 3.39 (3H,
13
s, N3-CH3), 3.22 (3H, s, N1-CH3); C NMR (101 MHz, DMSO) δ (ppm): 187.84,
166.89, 153.78, 151.66, 151.39, 149.47, 148.71, 148.63, 144.44, 143.47, 133.29,
130.32, 128.04, 124.35, 119.89, 118.94, 112.00, 111.12, 108.40, 56.21, 56.06, 37.10,

30.21, 28.17; Anal. Calcd. For C26H25N5O6S (535.57): C, 58.31; H, 4.70; N, 13.08.
Found: C, 58.43; H, 4.63; N, 13.31.
4.1.1.7. 2-((1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)thio)-N-(4-
((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)acetamide (15)
1
White crystals (0.39 g, 81% yield); m.p> 300 ºC; H NMR (400 MHz, DMSO-d6) δ
(ppm): 13.63 (1H, s, N7-H), 10.72 (1H, s, O=C-NH), 8.18 (2H, d, J = 8.8 Hz, Ar-H),
7.89 (1H, d, J = 15.5 Hz, CH=CH), 7.78 (2H, d, J = 8.8 Hz, Ar-H), 7.68 (1H, d, J =
15.5 Hz, CH=CH), 7.23 (2H, s, Ar-H), 4.27 (2H, s, SCH2), 3.87 (6H, s, 2OCH3), 3.72
13
(3H, s, OCH3), 3.39 (3H, s, N3-CH3), 3.22 (3H, s, N1-CH3); C NMR (101 MHz,
DMSO) δ (ppm): 187.89, 166.89, 153.80, 153.56, 151.39, 148.73, 148.70, 144.42,
143.60, 140.13, 133.11, 130.76, 130.43, 121.52, 118.95, 108.34, 106.95, 60.60, 56.61,
37.10, 30.20, 28.17; Anal. Calcd. For C27H27N5O7S (565.60): C, 57.34; H, 4.81; N,
12.38. Found: C, 57.59; H, 4.89; N, 12.54.
4.1.1.8. 2-((1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)thio)-N-(4-
((E)-3-(3-nitrophenyl)acryloyl)phenyl)acetamide (16)
1
Yellowish white powder (0.41g, 87% yield); m.p> 300 ºC; H NMR (400 MHz,
DMSO-d6) δ (ppm): 13.63 (1H, s, N7-H), 10.74 (1H, s, O=C-NH), 8.78 (1H, s, Ar-H),
8.34 (1H, d, J = 7.8 Hz, Ar-H), 8.29 – 8.25 (1H, m, Ar-H), 8.23 (2H, d, J = 8.8 Hz,
Ar-H), 8.17 (1H, d, J = 15.7 Hz, CH=CH), 7.84 (1H, d, J = 15.7 Hz, CH=CH), 7.80 –
7.72 (3H, m, Ar-H), 4.26 (2H, s, SCH2), 3.39 (3H, s, N3-CH3), 3.22 (3H, s, N1-CH3);
13C NMR (101 MHz, DMSO) δ (ppm): 187.82, 166.95, 153.80, 151.38, 148.90,
148.72, 148.69, 143.90, 141.36, 137.15, 135.55, 132.70, 130.81, 130.69, 125.18,
125.03, 123.37, 119.03, 108.35, 37.10, 30.20, 28.17; Anal. Calcd. For C24H20N6O6S
(520.52): C, 55.38; H, 3.87; N, 16.15. Found: C, 55.57; H, 4.01; N, 16.42.

4.1.1.9. 2-((1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)thio)-N-(4-
((E)-3-(p-tolyl)acryloyl)phenyl)acetamide (17)
1
Straw yellow powder (0.38 g, 85.5% yield); m.p> 300 ºC; H NMR (400 MHz,
DMSO-d6) δ (ppm): 13.63 (1H, s, N7-H), 10.72 (1H, s, O=C-NH), 8.16 (2H, d, J =
8.7 Hz, Ar-H), 7.89 (1H, d, J = 15.6 Hz, CH=CH), 7.77 (4H, dd, J = 8.0, 8.0 Hz, Ar-
H), 7.70 (1H, d, J = 15.6 Hz, CH=CH), 7.28 (2H, d, J= 8.7 Hz, Ar-H), 4.24 (2H, s,
13
SCH2), 3.39 (3H, s, N3-CH3), 3.22 (3H, s, N1-CH3), 2.36 (3H, s, CH3); C NMR
(101 MHz, DMSO) δ (ppm): 187.95, 166.89, 153.82, 151.39, 148.71, 148.70, 143.97,
143.57, 141.03, 133.12, 132.51, 130.37, 129.99, 129.31, 121.33, 118.98, 108.35,
37.09, 30.20, 28.17, 21.56; Anal. Calcd. For C25H23N5O4S (489.55): C, 61.34; H,
4.74; N, 14.31. Found: C, 61.58; H, 4.90; N, 14.52.
4.1.1.10. 2-((1,3-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)thio)-N-(4-
((E)-3-(2,4-dimethylphenyl)acryloyl)phenyl)acetamide (18)
Straw yellow crystals (0.35g, 78% yield); m.p> 300 ºC; 1H NMR (400 MHz, DMSO-
d6) δ (ppm): 13.63 (1H, s, N7-H), 10.71 (1H, s, O=C-NH), 8.16 (2H, d, J = 8.8 Hz,
Ar-H), 7.95 (1H, d, J = 15.4 Hz, CH=CH), 7.90 (1H, d, J = 8.5 Hz, Ar-H), 7.82 –
7.73 (3H, m, Ar-H + CH=CH), 7.10 (2H, d, J = 5.2 Hz, Ar-H), 4.26 (2H, s, SCH2),
3.39 (3H, s, N3-CH3), 3.22 (3H, s, N1-CH3), 2.41 (3H, s, CH3), 2.31 (3H, s, CH3); 13C
NMR (101 MHz, DMSO) δ (ppm): 187.97, 166.88, 153.80, 151.38, 148.71, 143.56,
140.92, 140.68, 138.40, 133.13, 131.90, 130.99, 130.37, 127.55, 127.28, 122.09,
118.99, 108.34, 99.98, 37.09, 30.20, 28.17, 21.41, 19.73; Anal. Calcd.
ForC26H25N5O4S (503.57): C, 62.01; H, 5.00; N, 13.91. Found: C, 61.89; H, 4.88; N,
14.17.