Synthesis and biological evaluation of novel xanthine derivatives as potential apoptotic antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2019.05.015

A series of novel xanthine/NO donor hybrids containing 1,3,8-trisubstituted or 1,8-disubstituted xanthine derivatives were designed and synthesized. The synthesized compounds were tested in a cell viability assay using human mammary gland epithelial cell line (MCF-10A) where all the compounds exhibited no cytotoxic effects and more than 90% cell viability at a concentration of 50 μM. The oxime containing compounds 7a-b and 17-24 were more active as antiproliferative agents than their non-oxime congeners 6a-b and 9-16. Hydroxyimino-phenethyl scaffold compounds 17-24 were more active than the hydroxyimino-ethyl phenyl acetamide 7a-b derivatives. Compounds 18–20 and 22-24 exhibited inhibition of EGFR with IC₅₀ ranging from 0.32 to 2.88 μM. Compounds 18-20 and 22-24 increased the level of active caspase 3 by 4–8 folds, compared to the control cells in Panc-1 cell lines compared to doxorubicin as a reference drug. Compounds 18, 22 and 23 were the most caspase-3 inducers. Compounds 22 and 23 increased the levels of caspase-8 and 9 indicating activation of both intrinsic and extrinsic pathways and showed potent induction of Bax, down-regulation of Bcl-2 protein levels and over-expression of cytochrome c levels in Panc-1 human pancreas cancer cells. Compound 23 exhibited mainly cell cycle arrest at the Pre-G1 and G2/M phases in the cell cycle analysis of Panc-1 cell line. The drug likeness profiles of compounds 18-20 and 22-24 were predicted to have good to excellent drug likeness profiles specially compounds 18-20 and 23. Finally molecular docking study was performed at the EGFR active site to suggest thier possible binding mode. The hydroxyimino-phenethyl scaffold compounds 17-24 represent an interesting starting point to optimize their pharmacokinetics and pharmacodynamics profiles.

Full text of DOI:10.1016/j.ejmech.2019.05.015

Accepted Manuscript
Synthesis and biological evaluation of novel xanthine derivatives as potential
apoptotic antitumor agents
Mohamed Hisham, Bahaa G.M. Youssif, Essam Eldin A. Osman, Alaa M. Hayallah,
Mohamed Abdel-Aziz
PII:
S0223-5234(19)30424-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.015
EJMECH 11325
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 February 2019
Revised Date: 24 April 2019
Accepted Date: 6 May 2019
Please cite this article as: M. Hisham, B.G.M. Youssif, E.E.A. Osman, A.M. Hayallah, M. Abdel-Aziz,
Synthesis and biological evaluation of novel xanthine derivatives as potential apoptotic antitumor agents,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.05.015.
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ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel xanthine derivatives as
potential apoptotic antitumor agents
Mohamed Hisham 1, Bahaa G.M. Youssif2,3*, Essam Eldin A. Osman4, 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, Cairo University, 11562 Cairo, Egypt; Department of Medicinal
Chemistry, Faculty of Pharmacy, Minia University, 61519-Minia, Egypt.
Short running title: Synthesis and anticancer activity of novel xanthine derivatives
*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
Mohamed Abdel-Aziz. Ph.D. Medicinal Chemistry Department, Faculty of
Pharmacy, Minia University, Minia, Egypt
Tel.: (002)-01003311327
E-mail address: abulnil@hotmail.com

ACCEPTED MANUSCRIPT
Abstract:
A series of novel xanthine/NO donor hybrids containing 1,3,8-trisubstituted or 1,8-
disubstituted xanthine derivatives were designed and synthesized. The synthesized
compounds were tested in a cell viability assay using human mammary gland
epithelial cell line (MCF-10A) where all the compounds exhibited no cytotoxic
effects and more than 90% cell viability at a concentration of 50 µM. The oxime
containing compounds 7a-b and 17-24 were more active as antiproliferative agents
than their non-oxime congeners 6a-b and 9-16. Hydroxyimino-phenethyl scaffold
compounds 17-24 were more active than the hydroxyimino-ethyl phenyl acetamide
7a-b derivatives. Compounds 18-20 and 22-24 exhibited inhibition of EGFR with
IC50 ranging from 0.32 to 2.88 µM. Compounds 18-20 and 22-24 increased the level
of active caspase 3 by 4-8 folds, compared to the control cells in Panc-1 cell lines
compared to doxorubicin as a reference drug. Compounds 18, 22 and 23 were the
most caspase-3 inducers. Compounds 22 and 23 increased the levels of caspase-8 and
9 indicating activation of both intrinsic and extrinsic pathways and showed potent
induction of Bax, down-regulation of Bcl-2 protein levels and over-expression of
cytochrome c levels in Panc-1 human pancreas cancer cells. Compound 23 exhibited
mainly cell cycle arrest at the Pre-G1 and G2/M phases in the cell cycle analysis of
Panc-1 cell line. The drug likeness profiles of compounds 18-20 and 22-24 were
predicted to have good to excellent drug likeness profiles specially compounds 18-20
and 23. Finally molecular docking study was performed at the EGFR active site to
suggest thier possible binding mode. The hydroxyimino-phenethyl scaffold
compounds 17-24 represent an interesting starting point to optimize their
pharmacokinetics and pharmacodynamics profiles.
Keywords: Xanthine; Oxime; Hybrids; EGFR; Anti-proliferative; Apoptotic assay.

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Graphical Abstract

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Highlights
1. A series of 20 novel compounds including xanthine/oxime hybrid was
designed and synthesized.
2. The synthesized compounds were evaluated for their in vitro antiproliferative
activity against different human cancer cell lines.
3. The oxime containing compounds 7a-b and 17-24 were more active as
antiproliferative agents than their non-oxime congeners 6a-b and 9-16.
4. Hydroxyimino-phenethyl scaffold compounds 17-24 were more active than
the hydroxyimino-ethyl phenyl acetamide 7a-b derivatives.
5. Compounds 18-20 and 22-24 exhibited inhibition of EGFR with IC50 ranging
from 0.32 to 2.88 µM and molecular docking for compounds 17-24 were
performed at the active site of EGFR kinase domain.
6. Compounds 18-20 and 22-24 increased the level of active caspase-3 by 4-8
folds.
7. Results revealed that these compounds resulted in programmed cell death and
cell cycle arrest in Panc-1 cells.

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1. Introduction
Xanthine derivatives have been widely investigated for their biological effects. They
represent an important class of therapeutic agents with diverse biological activities
such as antiasthmatic [1], antimicrobial [2], CNS stimulant [3], anti-inflammatory [4],
adenosine receptor antagonism [5], phosphodiesterase (PDE) inhibition [6],
phosphoenol pyruvate carboxykinase (PEPCK) inhibition [7] and antitumor activity
[8]. Methylxanthine derivatives such as theophylline (1) and caffeine (2) (Figure 1)
were found to induce apoptosis, and promote cytotoxicity induced by doxorubicin [9].
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 [10]. The mechanism of the apoptogenic effect of
theophylline has been found to involve reduction of intracellular levels of the
antiapoptotic mediator Bcl2 [11]. Therefore, theophylline-based moieties comprise an
attractive scaffold for structural modification in search of novel antiproliferative
agents with diverse pharmacodynamics. It was found that 1-methyl-3-propyl-7-
butylxanthine (MPBX) (3) showed strong inhibitory activity compared to caffeine
[12, 13]. Moreover, methylxanthines (4) and (5) bearing 8-alkylthio substitution
showed potent VEGFR-2 and BRAF inhibitory activities respectively [14, 15].
Nitric oxide-releasing oximes have various potential activities that depend on
releasing NO from their structures. These activities include antihypertensive [16],
anti-inflammatory, gastroprotective [17] and antiproliferative activities [18]. Several
mechanisms can explain the antiproliferative activity of oximes, but the most accepted
one is through the release of nitric oxide and/or induction of apoptosis [19]. NO may
be converted inside the cell into reactive nitrogen species (RNS) such as NO2 and
N2O, which target P53 causing cytochrome c release and caspases activation [20].

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caspases are a group of vital mediators for apoptosis, but the most critical one is
caspase-3, which can activate death protease leading to cleavage of many key cellular
proteins [21]. Also, oxime moiety is characterized by the ease of coordination with
metal ions and can participate in hydrogen bonding with amino acid residues in the
active site of different enzymes. Consequently, oxime moiety can improve the binding
of the whole molecule with its binding site [22].
Enlightened by the aforementioned information and in continuation of our efforts in
probing for novel effective anticancer agents [23-27] we designed and synthesized a
series of novel 1-methyl or 1,3-dimethyl xanthine derivatives substituted at C8,
similar to the kinase inhibitors (4) and (5), with either 2-(4-acetylphenylamino)-2-
oxoethylthio in compounds 6a-b and their oxime derivatives 7a-b or with 2-oxo-2-
(un)substituted phenethylthio in compounds 9-16 and their oxime derivatives 17-24 to
examine the structure activity relationship and the impact of changing the spacer on
the anticancer activity. The oxime derivatives 7a-b and 17-24 are viewed as hybrids
containing methyl xanthine, an important pharmacophore that exhibits potential role
as anticancer agent; and a NO releasing oxime as the second pharmacophore which
have the ability to overcome the resistance to chemotherapy and refractoriness to
conventional therapeutics which is one of the major challenges in the treatment of
cancer. The cytotoxic activities of these hybrids on the proliferation of 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) were determined by MTT assay,
respectively. To investigate the possible anticancer mechanism of the synthesized
hybrids, EGFR enzymatic assay was performed. Molecular docking study of the most
active compounds inside the active site of EGFR enzyme was done. Cell cycle

ACCEPTED MANUSCRIPT
analysis and apoptosis through caspase-3, 8 and caspase-9 expression levels were
screened, in addition to expression level of Bax and Bcl-2.
O
N
O
N
O
N
H
N
N
N
N
N
N
N
N
N
O
O
O
Caffeine (2)
Theophylline (1)
MPBX (3)
O
O
N
N
N
N
HN
S
S
O
O
N
O
N
O
N
OH
(5)
(4)
O
O
H
H
R1
O
R1
O
HO
N
N
N
N
S
N
S
O
HO
N
N
N
N
N
HN
R2
R2
7a-b
17-24
R3
Figure 1: Reported antitumor xanthine derivatives and the new designed compounds
7a-b and 17-24
2. RESULTS AND DISCUSSION
2.1.Chemistry
Compounds 4a-b were synthesized as described in Scheme 1. Condensation of N,N'-
dimethylurea with cyanoacetic acid, followed by stirring in 70% (w/v) sodium
hydroxide solution yielded the desired compound 1a [28, 29]. 6-Amino-3-
methyluracil 1b was prepared by refluxing 6-aminouracil in hexamethyldisilazane
(HMDS) followed by addition of methyl iodide to afford the desired N3-substituted 6-

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aminouracil [30, 31]. Nitrosation of compounds 1a-b using sodium nitrite in 50%
aqueous acetic acid yielded 2a-b [30]. Sodium dithionite reduction of 2a-b in aqueous
ammonia solution yielded compounds 3a-b [32]. Direct cyclization of 5,6-diamino-
1,3-dimethyluracil 3a using carbon disulfide in DMF under reflux for 5 h afforded
compound 4a [33] 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 [34].
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-Cyanoacetyl)-1,3-dimethylurea
1a
O
O
c
N
HN
O
N
NH2
O
N
NH2
H
H
6-amino uracil
1b
O
N
O
N
O
R1
R1
R1
NH2
NH2
NO
d
f
N
e
N
O
N
R2
O
NH2
O
NH2
N
R2
R2
1a-b
3a-b
2a-b
O
H
O
H
R1
N
R1
O
N
N
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.

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The target hybrid ketone intermediates 6a-b and 9-16 and their corresponding final
oximes 7a-b and 17-24 were prepared according to Scheme 2. N-(4-Acetylphenyl)-2-
bromoacetamide 5 was prepared in high yield through stirring of p-amino
acetophenone with bromoacetyl bromide [35]. Alkylation of compounds 4a-b with N-
(4-acetylphenyl)-2-bromoacetamide
5
afforded the corresponding ketones
intermediate 6a-b in good yields.
O
O
a
O
H2N
Br
N
H
p-Aminoacetophenone
5
O
H
O
H
O
R1
O
R1
N
N
N
O
N
b
Br
SH
+
S
O
N
N
N
O
H
N
O
N
HN
R2
R2
4a-b
6a-b
5
O
c
R3
O
d
H
N
Br
R1
O
N
HO
S
O
8a-d
N
N
N
O
HN
R2
H
N
R1
O
N
S
O
7a-b
R1 = CH3 ; R2 = H or CH3
N
N
R2
c
O
R3
9-16
H
N
R1
N
S
N OH
N
O
N
R2
17-24
R3
R3 = H, OCH3, Cl, Br
Scheme 2: Synthesis of compounds 6a-b, 7a-b and 9-24.
Reagents and reaction conditions: a) BrCOCH2Br, CH2Cl2, K2CO3, H2O; b)
CH3CN, TEA reflux 8 hr; c) NH2OH.HCl, CH3COONa, EtOH reflux 12 hr; d) 1%
NaOH.

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Comp R1
R2
CH3 CH3 ----
CH3 ----
CH3 CH3 ----
R3
Comp R1
R2
H
R3
CH3
CH3
Cl
Br
H
6a
6b
7a
7b
9
15
16
17
18
19
20
21
22
23
24
H
H
CH3 CH3
CH3
H
----
H
CH3 CH3 OCH3
CH3 CH3
CH3 CH3
CH3 CH3
Cl
Br
CH3 CH3 OCH3
10
11
12
13
14
CH3 CH3
CH3 CH3
Cl
Br
CH3
CH3
CH3
CH3
H
H
H
H
H
OCH3
Cl
CH3
CH3
H
H
H
OCH3
Br
Compounds 9-16 were prepared by the interaction of equimolar amounts of
compounds 4a-b and the appropriate p-(un)substituted phenacyl bromides 8a-e under
the conditions of Schotten-Baumann reaction. The conversion of the ketones
derivatives 6a-b and 9-16 into their corresponding oximes 7a-b and 17-24 has been
carried out in good yield upon heating with hydroxylamine HCl in absolute ethanol.
The chemical structures of hybrid ketone intermediates 6a-b and 9-16 and their
1
corresponding final oximes 7a-b and 17-24 were elucidated by H NMR and 13C
1
NMR spectroscopy. The H NMR spectrum of compound 15 as a representative
example of 1-methyl xanthine derivatives revealed the appearance of a singlet signal
with three protons integration at δ 3.17 ppm corresponding to (N1-CH3) and
appearance of a singlet signal at δ 4.97 ppm assigned for methylene protons (S-CH2-
CO) of the linker. Furthermore, The two p-substituted phenyl protons appear at
aromatic region as pair of doublets at δ 7.65 ppm and δ 8.05 ppm with coupling
constant J = 8.4 Hz. Also, appearance of a singlet signal at δ 11.79 ppm assigned to

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(N3-H) and characteristic broad singlet signal at δ13.40 ppm represents (N7-H) that
13
are both characterized to xanthine nucleus. The C NMR spectrum of compound 15
showed N3-CH3 appears at δ 27.30 ppm, the methylene group (SCH2) appears at δ
38.60 ppm, aromatic carbons appear at δ 128.92-139.03 ppm and one carbonyl group
of the ketone at δ192.12 ppm. The 1H NMR spectrum of oxime compound 23 showed
the appearance of a downfield singlet at δ 11.93 ppm, related to the hydroxyl group in
addition to the appearance of same characteristic features as its ketonic derivative
compound 15. The OH proton appeared to be highly downfield shifted due to its
13
intramolecular hydrogen bonding with the sulphur atom. The C NMR spectrum of
compound 23 showed the disappearance of the ketonic carbonyl due its conversion to
ketoxime group (C=NOH) which appears at δ 154.69 ppm and all other carbons
appear at their expected chemical shifts. A characteristic feature of the mass spectra of
the oximes 7a-b and 17-24 is the appearance of a weak abundance for the molecular
ion peaks (Supplementary data). Finally, data from the elemental analyses further
confirmed the assigned structures.
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 final compounds 6a-b, 7a-b and 9-24 were incubated
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 exhibited no cytotoxic effects and more than 90% cell viability
was reported for the majority of the tested compounds at 50 µM.

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2.2.1.2. Antiproliferative activity
All final hybrids 6a-b, 7a-b and 9-24 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.
Graph Pad Prism software (Graph Pad Software, San Diego, CA, USA) was used to
calculate the median inhibition concentration (IC50) for all compounds. Results were
illustrated in Table 1. Compounds 6a-b and 9-16, non-oxime derivatives, showed
weak antiproliferative activities against all cancer cell lines while, compounds 7a-b
and 17-24, containing NO releasing oxime moiety, were more active than 6a-b and 9-
16 against all the assayed cancer cell lines, which reflects the importance of the oxime
moiety on the anticancer activity of the title scaffold. Results revealed that
hydroxyimino-phenethyl scaffold compounds 17-24 were more active than the
hydroxyimino-ethyl phenyl acetamide derivatives 7a-b suggesting the importance of a
specific location for this moiety.
Concerning the antiproliferative effect of compounds 17-24, the substitution pattern
of the xanthine core (R1 and R2) and the p-substitution (R3) in the pendant phenyl
ring appears important. The 1-methyl xanthine-oxime hybrid 21 (IC50 = 2.1±1.4 to
5.6±1.6 µM) was more active than its 1,3-dimethyl congener 17 (IC50 = 5.5±2.50 to
6.7±2.4 µM). Contrarily, grafting p-substitution in 1,3-dimethyl xanthines 18-20 was
generally correlated with good to excellent antiproliferative activities against all
cancer cell lines compared to the 1-methyl derivatives 22-24 (Table 1). The nature of
the p-substitution was having marginally variable effects on the antiproliferative
activity of these compounds in the tested cell lines. It was suggested that the

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differences could mainly be attributed to the pharmacokinetics profile of these
compounds though the pharmacodynamic effects could not be rolled out completely.
Table 1: Antiproliferative activity of the target compounds 6a-b, 7a-b and 9-24
Compound
No.
Antiproliferative activity IC50 ± SEM (µM)
Panc-1
5.1±1.7
MCF-7
6.1±1.5
HT-29
6.4±1.5
6.9±1.7
3.1±1.4
6.2±2.4
5.9±1.7
8.1±1.5
9.9±2.7
6.5±2.4
11.1±1.5
6.7±2.4
7.2±1.7
11.5±1.5
6.7±2.4
1.7±0.4
1.2±0.7
1.7±0.9
2.1±1.4
1.7±0.40
1.80±0.73
2.0±0.90
1.01 ± 0.82
A-549
6.9±1.5
7.7±1.5
6.6±1.6
7.3±2.1
6.7±1.5
7.9±1.9
8.4±2.1
7.9±1.6
11.9±1.9
6.1±1.1
8.1±1.5
11.9±1.9
6.1±1.1
1.5±0.2
1.5±0.5
1.8±0.7
5.6±1.6
1.5±0.20
1.89±0.45
2.1±0.70
1.21 ± 0.80
6a
5.9±2.4
6.1±2.4
6b
4.6±1.2
5.3±1.2
7a
7.7±1.5
9.1±1.7
7b
4.9±2.4
5.1±2.4
9
6.4±2.1
7.6±1.7
10
7.2±1.5
8.4±1.4
11
7.4±1.5
7.6±2.7
12
9.4±2.1
11.6±1.7
5.9±3.0
13
5.5±2.5
14
6.1±2.4
6.3±2.4
15
9.7±2.1
11.2±1.7
5.9±3.00
0.8±0.50
1.0±0.10
1.3±0.90
4.3±.92
16
5.5±2.50
0.91±0.20
1.3±0.90
1.7±1.60
3.6±.80
17
18
19
20
21
1.91±0.20
1.70±0.86
2.1±1.60
1.41 ± 0.58
1.8±0.50
1.40±0.22
1.7±0.90
0.90± 0.62
22
23
24
Doxorubicin

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2.2.2. Inhibition of Epidermal Growth Factor Receptor Activity (EGFR-TK)
EGFR-TK assay was performed to assess the EGFR inhibitory potency of the 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 18,
19, 20, 22, 23 and 24 exhibited inhibition of EGFR with IC50 ranging from 0.32 to
2.88 µM. According to data presented three derivatives 18, 19 and 20 selected from
xanthine hybrids were found to be the most potent and their EGFR inhibitory
activities were close to the positive reference erlotinib (IC50 = 0.08 µM). EGFR assay
results demonstrated that these compounds may potentially function via EGFR
inhibition.
Table 2: Effects of compounds (18, 19, 20, 22, 23, 24 and erlotinib) on EGFR.
Compound No.
EGFR IC50 (µM)
18
0.32
0.40
0.45
0.71
0.82
2.88
0.08
19
20
22
23
24
Erlotinib

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2.2.3. Apoptosis assay
Apoptosis defects in cancer cells are the key impediment that limits the therapeutic
efficacy of anticancer agents, thus the development of novel agents targeting
programmed cell death has become a vital task for clinical application [36].
Therefore, to disclose the proapoptotic potential of our goal compounds, compounds
18-20 and 22-24 were tested for their potential ability to induce the apoptosis cascade.
2.2.3.1. Activation of Proteolytic Caspases Cascade
Activation of caspases plays a key role in the instigation and completing of the
apoptotic process [37]. Among the caspases, the executioner caspase-3 is a vital
player that cleaves multiple proteins in the cells, leading to apoptotic cell death [38].
The effect of compounds 18-20 and 22-24 on caspase-3 were evaluated and
compared to Doxorubicin as a reference drug. The results revealed that the tested
compounds 18-20 and 22-24 showed an increase in the level of active caspase-3 by
4-8 folds, compared to the control cells, and that 18, 22 and 23 were the most active
compounds that possessed remarkable over expression of caspase-3 protein level
(460.1 ± 2.33, 502.4 ± 4.48, and 529.7 ± 9.45 pg/mL, respectively) compared to the
reference doxorubicin (503.2 ± 4.22 pg/mL). The most active compound 23 showed
an increase in the level of active caspase-3 by 8 folds, compared to the control cells,
and induced caspase-3 higher than that of doxorubicin as a reference drug (Figure 2).

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Figure 2: Caspase-3 level for compounds 18, 19, 20, 22, 23, 24 and doxorubicin in
human pancreas cancer cell line (Panc-1).
To outline compounds 22 and 23 apoptotic mechanism whether it is through the
induction of the intrinsic or the extrinsic pathway or both, their effect on caspase-8
and caspase-9 was also evaluated. Result revealed that compound 22 increases the
levels of caspase-8 and 9 by 2.58 and 14.49 folds, respectively while compound 23
showed 3.47 and 14.63 fold increasing in the level of caspase-8 and 9, respectively
compared to the control cells, which indicates activation of both intrinsic and extrinsic
pathways with more prominent effect on the intrinsic pathway, since caspase-9
levels were higher (Table 3).

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Table 3: Effects of compounds (18, 19, 20, 22, 23, 24 and doxorubicin) on active
caspases-3, 8, 9 and cytochrome c in Panc-1human pancreas cancer cell line.
Compound
Number
Caspase-3
Caspase-8
Caspase-9 Cytochrome C
Conc (pg/ml) Fold change Conc Fold Conc Fold Conc Fold
(ng/ml) change (ng/ml) change (ng/ml) change
460.1 ±2.33
270.2 ±6.05
388 ±6.94
7.00
4.11
5.91
7.65
8.06
4.99
7.66
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
18
--
19
--
20
502.4 ±4.48
529.7 ±9.45
328 ±7.24
0.44
0.59
--
2.58 13.52 14.49 0.5262 11.43
3.47 14.63 15.68 0.4696 10.20
22
23
24
--
--
--
--
--
503.2 ±4.22
1.75 10.07 16.23 17.40 0.6042 13.134
0.17 0.93 0.046
Doxorubicin
65.64
1
1
1
1
Control
2.2.3.2. Bax and Bcl-2 levels assay
The most active caspase-3 activator hybrids 22 and 23 were further studied for their
effect on Bax and Bacl-2 levels against Panc-1 human pancreas cancer cell line using
doxorubicin as a reference, which are mentioned in Table 4. The results showed that
22 and 23 elicited remarkable increase in Bax level compared to doxorubicin.
Compound 23 showed a comparable induction of Bax (351 pg/mL) compared to
doxorubicin (276 pg/mL) with 12.7 fold higher than control untreated Panc-1 cancer
cells followed by compound 22 (246.9 pg/mL and 8.9 fold change). Finally,
compound 23 caused a down-regulation of Bcl-2 protein level (1.97 ng/mL) followed
by compound 22 (2.79 ng/mL) in Panc-1cell line compared to doxorubicin (0.98
ng/mL),

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Table 4: Bax and Bcl-2 levels for compounds 22, 23, and doxorubicin
on human pancreas cancer cells (Panc-1).
Bax
Bcl-2
Fold
(pg/ml) change (ng/ml) change
Compound Number
Conc
Fold
Conc
22
23
246.9
351
8.9
12.7
33.42
2.795
1.978
0.983
0.3378
0.2390
Doxorubicin
276.19
0.193276
8.264
1
5.086
1
Control
2.2.3.3. Cytochrome C assay
Cytochrome c concentration in the cell has a critical role for activation of caspases
and initiating intrinsic apoptosis pathway [39]. Xanthine hybrids 22 and 23 were
evaluated as cytochrome c against Panc-1 human pancreas cancer cell line and the
results are listed in Table 3. Compounds 22 and 23 cause over-expression of
Cytochrome C level in Panc-1 human pancreas cancer cell about 11 and 10 fold,
respectively higher than control. So, the above results may be considered as a
suggestion that apoptosis may be attributed to over-expression of cytochrome c and
activation of the intrinsic apoptotic pathway induced by the tested compounds.
2.2.3.4. Flow cytometric cell cycle analysis
Cell cycle analysis was performed for the most active compound 23 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 35.08% upon treatment with compound 23 while the percentage of cells in
the S phase were marginally reduced with compound 23 (35.56%) compared to the
control (36.41%) (Figure 3 and Figure 4). The percentage of Panc-1 human pancreas

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cancer cell line at the G2/M phase increase to 34.36% upon treatment with compound
23. Furthermore, it is obvious that the apoptotic cell percentage in the Pre-G1 phase
was increased from 1.79% for control untreated Panc-1 human pancreas cancer cell to
17.34% and 22.17% in treated cells with 23 and doxorubicin, respectively, (Figure 4
and Figure 5). According to the above results, it is clear that the compound 23
exhibited mainly cell cycle arrest at the Pre-G1 and G2/M phases. Moreover, it is
obvious that the tested compound is not cytotoxic but antiproliferative causing
programmed cell death and cell cycle arrest.
Figure 3: Cell cycle analysis on Panc-1cell line treated with compound 23

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Figure 4. Percentage of apoptosis and necrosis for compounds 23 on Panc-1cell line.
Figure 5: Cell cycle analysis and Apoptosis induction analysis using annexin V/PI of
compound 23 and control untreated Panc-1cell line.
2.4. Drug Likeness Profile
Since the pharmackokinetics profile of the tested compounds was of intrest, the drug
likeness profiles of compounds 18-20 and 22-24 were predicted using Swiss ADME
server [40]. The results of the drug likeness profile of these compounds are shown in
Table 5 (Appendix A). All the compounds were predicted to have high oral
absorption except for the mono methylated derivatives 22 and 24 and all the
compounds were predicted to have no brain penetration potential propably due to thier
high TPSA (Total Polar Surface Area) and low logP values. Similar to erlotinib, all
the compounds showed no violation to Lipinski and Ghosefilters while compounds

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22-24 showed no more than one violation for some other pharmacokinetics filters as
Veber, Egan or Muegge filters. Compounds 18-20 and 22-24 were free from alerts for
Pan Assay Interfering substances (PAINS) and predicted not to be substrates for Pgp
(P-glycoprotein). While erlotinib was having the potential to be an inhibitor of
CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4, compounds 18-20 and 22-24
were predicted to have no CYP (Cytochrome P450) inhibitory effects except for the
bromo compound 20 towards CYP2C9 and CYP3A4 and 24 towards CYP3A4 only.
Thus, these compounds may be considered as having good to excellent Drug likeness
profiles specially compounds 18-20 and 23.
2.5. Molecular Docking Study
Docking of compounds 18-20 and 22-24 was performed at the active site of the
crystal structure of the EGFR tyrosine kinase domain in complex with erlotinib to
explore their binding mode as EGFR inhibitors using MOE software package. Two
PDB files for erlotinib in complex with EGFR (4JHO and 1M17) are available at the
RSCB protein data bank [41, 42]. erlotinib binds to the ATP binding site of the EGFR
kinase in its active conformation (DFG-Asp in, αC-helix in) as seen in 1M17 complex
in its inactive conformation (DFG-Asp in, αC-helix out) as seen in 4JHO and based
on this it may be classified as type I or type I½ kinase inhibitor respectively.
Since both files show the “DFG-Asp in” conformation in the activation loop where
Asp831 was pointed towards the ATP binding site, the PDB file 4JHO was selected
based on its better quality as expressed by R-free, clash score, Ramchandran and side
chain outliers despite its slightly lower resolution (2.75A for 4JHO compared to 2.6 A
for 1M17). The docking results are shown in Table 6 (Appendix A). The N1 in the
quinazoline core of erlotinib was engaged in a key hydrogen bond with Met769 in the
hinge region of the EGFR kinase and N3 was forming a water mediated hydrogen

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bond with the gatekeeper Thr766 while its substituted aniline ring at C4 was oriented
towards a hydrophobic pocket close to the gate keeper Thr766 in the EGFR kinase
domain (Figure 6, Appendix A).
In the top ranked docking poses, compounds 18-20 and 22-24 were generally oriented
within the active site of the EGFR kinase in such a way where the 1-methyl or the 1,3-
dimethyl xanthine moiety were oriented towards the solvent accessible area and the
phenyl ring from the side chain at C8 was buried deep toward another hydrophobic
pocket lined by Met742, Leu753 and Leu764 which is not occupied by the 4-anilino
ring in erlotinib (Figure 7).
Figure 7: The top docking pose of compounds 18 (yellow), 19 (cyan) and 20
(magenta) overlaid on erlotinib (green) in the EGFR active kinase domain (PDB:
4JHO).

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Similar to erlotinib, the xanthine core in the docked compounds was engaged in
hydrogen bonds with the backbone NH of the Met769 via the C6 oxygen and
sometimes the N8 was forming a water mediated hydrogen bond to THr766.
Interestingly, the oxime moiety was placed in the vicinity of the Asp831 in the
activation loop (DFG motif) and Lys721 in β3 strand of the EGFR kinase domain and
in most of the top scored docking poses, the oxime was forming one or two additional
hydrogen bonds with these amino acids (Figure 8). The lack of these additional
hydrogen bonds may justify the weaker activity in compounds lacking the oxime
moiety or where it was appended to the acetyl group on the phenyl ring 7a and 7b.
The methoxy substituent in compounds 18 and 22 appears superior to the chloro or
bromo derivatives by virtue of occupying the hydrophobic cavity more nicely in terms
of steric and electronic properties thus being more deeply buried.
Figure 8: The top docking pose of compound 19 (magenta) overlaid on erlotinib
(green) in the EGFR active kinase domain (PDB: 4JHO), Met769, Thr766, Lys721
and Asp831 amino acids forming hydrogen bonds are colored orange

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Figure 9: The top docking pose of compound 18 (magenta) overlaid on Erlotinib
(green) in the EGFR active kinase domain (PDB: 4JHO), Met769, Thr766, Lys721
and Asp831 amino acids forming hydrogen bonds are colored orange
3. EXPERIMENTAL
3.1. Chemistry
General Details: See Appendix A
Compounds 1a [28-29], 1b [30, 31], 2a-b [30], 3a-b [32], 4a [33], 4b [34] and 5 [35]
were prepared according to the previous reported studies.
3.1.1. General procedure for synthesis of Xanthine/Unsubstituted P-amino
acetophenone Hybrids (6a-b)
An equimolar mixture of 4a or 4b, compound 5 (1 mmol, 0.256 g) and TEA (1.2
mmol, 0.121 g) in acetonitrile (50 mL) was heated at reflux for 4-8 hr. The reaction

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mixture was evaporated to dryness. The residue was crystallized from aqueous
ethanol affording the pure white products 6a-b.
3.1.1.1.
N-(4-Acetylphenyl)-2-((1,3-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-
purin-8-yl)thio)acetamide (6a)
White powder; yield: (0.225g , 75%); m.p: >300 oC; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 2.44 (s, 3H, COCH3), 3.21 (s, 3H, N1-CH3), 3.37 (s, 3H, N3-CH3), 4.24 (s,
2H, SCH2), 7.45 (2H, d, J = 8 Hz, Ar-H), 7.94 (2H, d, J = 8 Hz, Ar-H), 10.67 (s,1H,
CONH), 13.62 (s,1H, N7-H); 13C NMR (100 MHz, DMSO-d6) δ (ppm) : 26.88,
28.15, 30.20, 37.06, 118.84, 119.27, 129.99, 132.54, 139.59, 143.53, 147.36, 151.37,
153.79, 166.29, 196.79; Anal. Calcd. For C17H17N5O4S (387.41): C, 52.71; H, 4.42;
N, 18.08; S, 8.28. Found: C, 52.84; H, 4.58; N, 18.21; S, 8.35
3.1.1.2.N-(4-Acetylphenyl)-2-((1-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-
yl)thio)acetamide (6b)
White powder; yield: (0.216 g, 72%); m.p: >300 oC; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 2.53 (s, 3H, COCH3), 3.18 (s, N1-CH3), 4.22 (s, 2H, S-CH2), 7.71 (d, 2H, J
= 8.5 Hz, Ar-H), 7.94 (d, 2H, J = 8.5 Hz, Ar-H), 10.67 (s, 1H, CONH), 11.88 (s, 1H,
N3-H), 13.47 (s, 1H, N7-H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 26.91, 27.43,
36.94, 118.87, 122.23, 130.01, 132.42, 143.52, 151.46, 153.26, 155.07, 157.55,
166.71, 196.97; Anal. Calcd. For C16H15N5O4S (373.39): C, 51.47; H, 4.05; N, 18.76
; S, 8.59. Found: C, 51.32; H, 4.21; N, 18.98 ; S, 8.66.
3.1.2. General procedure for synthesis of Xanthine/p-Substituted Phenacyl
Hybrids (9-16)
To a stirred solution of compound 4a or 4b (4.7 mmol) in aqueous sodium hydroxide
1% w/v (20 mL), the appropriate p-(un) substituted phenacyl bromide 8a-f (4.7 mmol)

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dissolved in ethanol (5 mL) was added in portion wise manner. The reaction mixture
was stirred at ambient temperature for 4 hr, and then cooled in an ice bath for 3 hr.
The product was filtered off, washed with water, then diethyl ether, and dried,
recrystallized from ethanol to give compounds 9-16.
3.1.2.1. 1,3-Dimethyl-8-((2-oxo-2-phenylethyl)thio)-3,7-dihydro-1H-purine-2,6-
dione (9)
o
1
White powder; yield: (0.24 g, 80%); m.p: 226 C; H NMR (400 MHz, DMSO-d6) δ
(ppm): 3.21 (s, 3H, N1-CH3), 3.42 (s, 3H, N3-CH3), 4.52 (s, 2H, SCH2), 7.37-7.74 (m,
5H, Ar-H), 13.58 (s,1H, N7-H); 13C NMR (100 MHz, DMSO-d6) δ (ppm) : 25.50,
28.16, 30.21, 101.34, 126.44, 128.92, 129.53, 134.87, 147.25, 151.38, 152.91, 153.80,
193.47; Anal. Calcd. For C15H14N4O3S (330.36): C, 54.54; H, 4.27; N, 16.96; S, 9.70.
Found: C, 54.80; H, 4.39; N, 17.12; S, 9.64.
3.1.2.2. 8-((2-(4-Methoxyphenyl)-2-oxoethyl)thio)-1,3-dimethyl-3,7-dihydro-1H-
purine-2,6-dione (10)
White powder; yield: (0.24 g, 81%); m.p: 221 o C; 1H NMR (400 MHz, DMSO-d6) δ
(ppm) :3.21 (s, 3H, N1-CH3), 3.42 (s, 3H, N3-CH3), 3.8 (s, 3H, OCH3), 4.52 (s, 2H,
SCH2), 7.18 (d, 2H, J = 8 Hz, Ar-H), 7.61 (d, 2H, J = 8 Hz, Ar-H), 13.58 (s,1H, N7-
H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 25.42, 28.16, 30.15, 55.66, 108.48,
127.19, 128.73, 131.26, 148.62, 151.40, 152.47, 153.80, 164.04, 192.12;Anal. Calcd.
For C16H16N4O4S (360.39): C, 53.32; H, 4.48; N, 15.55; S, 8.90. Found C, 53.19; H,
4.55; N, 15.87; S, 8.79.

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3.1.2.3.
8-((2-(4-Chlorophenyl)-2-oxoethyl)thio)-1,3-dimethyl-3,7-dihydro-1H-
purine-2,6- dione (11)
White powder; yield: (0.246 g, 82%); m.p: 233 o C; 1H NMR (400 MHz, DMSO-d6) δ
(ppm): 3.21 (s, 3H, N1-CH3), 3.42 (s, 3H, N3-CH3), 4.52 (s, 2H, SCH2), 7.44 (d, 2H,
13
J = 8.4 Hz, Ar-H), 8.05 (d, 2H, J = 8.4 Hz, Ar-H), 13.59 (s, 1H, N7-H); C NMR
(100 MHz, DMSO-d6) δ (ppm) 25.44, 28.18, 30.23, 108.50, 128.54, 130.84, 131.82,
134.15, 146.71, 148.08, 151.38, 153.82, 192.12; Anal. Calcd. For C15H13ClN4O3S
(364.80): C, 49.39; H, 3.59; N, 15.36; S, 8.79. Found C, 49.54; H, 3.68; N, 15.59; S,
8.75.
3.1.2.4
8-((2-(4-Bromophenyl)-2-oxoethyl)thio)-1,3-dimethyl-3,7-dihydro-1H-
purine-2,6-dione (12)
White powder; yield: (0.246 g, 82%); m.p: 230 o C; 1H NMR (400 MHz, DMSO-d6) δ
3.21 (s, 3H, N1-CH3), 3.42 (s, 3H, N3-CH3), 4.55 (s, 2H, SCH2), 7.96 (d, 2H, J = 8.4
Hz, Ar-H), 8.23 (d, 2H, J = 8.4 Hz, Ar-H), 13.63 (s,1H, N7-H); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 25.44, 28.18, 30.23, 108.50, 128.54, 130.84, 131.82, 134.15,
146.71, 148.08, 151.38,153.82, 192.12; Anal. Calcd. For C15H13BrN4O3S (409.26):
C, 44.02; H, 3.20; N, 14.69; S, 8.01. Found: C, 44.04; H, 3.28; N, 15.06; S, 8.41.
3.1.2.5 1-Methyl-8-((2-oxo-2-phenylethyl)thio)-3,7-dihydro-1H-purine-2,6-dione
(13)
White powder; yield: (0.237 g, 79%); m.p: 290 o C; 1H NMR (400 MHz, DMSO-d6) δ
(ppm): 3.17 (s, 3H, N1-CH3), 4.98 (s, 2H, SCH2), 7.5-8.04 (m, 5H, Ar-H), 11.80(s,
1H, N3-H), 13.402 (s, 1H, N7-H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 27.42,
38.67, 119.61, 128.86, 129.33, 134.26, 135.75, 147.89, 149.00, 151.39, 154.50,

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193.42; Anal. Calcd. For C14H12N4O3S (316.34): C, 53.16; H, 3.82; N, 17.71; S,
10.13. Found: C, 53.18; H, 3.98; N, 17.97; S, 10.28 .
3.1.2.6
8-((2-(4-Methoxyphenyl)-2-oxoethyl)thio)-1-methyl-3,7-dihydro-1H-
purine-2,6-dione (14)
White powder; yield: (0.240 g , 80%); m.p: 300 o C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 3.17 (s,3H, N1-CH3),3.86 (s, 3H, OCH3), 4.94 (s, 2H, SCH2), 7.08 (d, 2H, J
= 8 Hz, Ar-H), 8.00 (d, 2H, J = 8 Hz, Ar-H), 11.81 (s, 1H, N3-H), 13.402 (s, 1H, N7-
H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 27.41,32.57, 56.11, 108.22, 114.51,
128.57, 131.23, 147.85, 149.09, 151.40, 154.51, 164.06, 191.76.; Anal. Calcd. For
C15H14N4O4S (346.36): C, 52.02; H, 4.07; N, 16.18; S, 9.26. Found: C, 52.31; H,
3.95; N, 16.41; S, 9.34.
3.1.2.7 8-((2-(4-Chlorophenyl)-2-oxoethyl)thio)-1-methyl-3,7-dihydro-1H-purine-
2,6-dione (15)
o
1
White powder; yield: (0.246 g, 82 %); m.p:> 300 C; H NMR (400 MHz, DMSO-
d6) δ (ppm): 3.17 (s, 3H, N1-CH3), 4.97 (s, 2H, SCH2), 7.65 (d, 2H, J = 8.4 Hz, Ar-
H), 8.05 (d, 2H, J = 8.4 Hz, Ar-H), 11.79 (s, 1H, N3-H), 13.40 (s, 1H, N7-H); 13C
NMR (100 MHz, DMSO-d6) δ (ppm): 28.18, 30.23, 108.50, 122.90, 128.54, 130.84,
131.82, 148.57, 151.38, 152.36, 153.82, 192.12. Anal. Calcd. For C14H11ClN4O3S
(350.78): C, 47.94; H, 3.16; N, 15.97; S, 9.14. Found: C, 48.12; H, 3.50; N, 16.24; S,
9.03.
3.1.2.9 8-((2-(4-Bromophenyl)-2-oxoethyl)thio)-1-methyl-3,7-dihydro-1H-purine-
2,6-dione (16)
White powder; yield: (0.25 g, 83 %); m.p: > 300 o C; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 3.17 (s, 3H, N1-CH3), 4.97 (s, 2H, SCH2), 7.79 (d, 2H, J = 8 Hz, Ar-H),

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7.96 (d, 2H, J = 8 Hz, Ar-H), 11.79 (s, 1H, N3-H), 13.402 (s, 1H, N7-H); 13C NMR
(100 MHz, DMSO-d6) δ (ppm): 27.42, 38.43, 118.58, 126.07, 130.35, 131.72,
134.57, 144.90, 146.95, 153.85, 155.83, 193.17. Anal. Calcd. For C14H11BrN4O3S
(395.23): C, 47.40; H, 2.81; N, 15.18; S, 8.11. Found: C, 47.59; H, 2.88; N, 15.37;
S, 8.13.
3.1.3. General procedure for synthesis of oxime derivatives (7a-b and 17-24)
To a stirred mixture of the appropriate ketone, 6a-b or 9-16, (1 mmol) in absolute
ethanol (10 mL), (4 mmol, 0.277 g) hydroxylamine hydrochloride and anhydrous
sodium acetate (4 mmol, 0.328 g) were added. The mixture was heated at reflux for
12 hr. The formed precipitate was filtered while hot, washed with ethanol, dried and
recrystallized from acetonitrile to give oximes 7a-b and 17-24.
3.1.3.1. 2-((1,3-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)thio)-N-(4-
(1-(hydroxyimino)ethyl)phenyl)acetamide (7a)
White powder; yield: (0.225 g, 75%); m.p: >300 oC; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 2.44 (s, 3H, COCH3), 3.29 (s, 3H, N1-CH3), 3.37 (s, 3H, N3-CH3), 4.24 (s,
2H, SCH2), 7.71 (d, 2H, J = 8.8 Hz, Ar-H), 7.94 (d, 2H, J = 8.8 Hz, Ar-H), 10.67 (s,
1H, N=C-NH),11.10 (s,1H, C=NOH), 13.62 (s,1H, N7-H); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 26.88, 28.15, 30.20, 37.06, 108.31, 119.27, 126.53, 129.99,
132.40, 139.59, 143.53, 151.37, 153.79, 166.86, 196.93.; Anal. Calcd. For
C17H17N5O4S (387.41): C, 50.71; H, 4.42; N, 20.98; S, 7.97. Found: C, 50.93; H,
4.75; N, 21.15; S, 8.09.