Design, molecular modeling and anticancer evaluation of thieno[2,3-d]pyrimidine derivatives as inhibitors of topoisomerase II

Article abstract of DOI:10.1016/j.bioorg.2019.103492

Synthesis of 4-(3,5-dimethyl-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine 1 and its functionalized reactions as nucleophile with various electrophilic reagents were performed through facile methods to yield different cyclic and acyclic derivatives (2–17). The structures of the newly synthesized compounds were established by their elemental analysis and spectral data. Derivatives 4, 14, 16, and 17 in addition to the parent compound 1 had IC₅₀ at ~4–10 μM against HepG2 and MCF7 and were selected for further investigations. All derivatives had high IC₅₀ values (>60 μM) against normal fibroblasts WI38 indicating selectivity against cancer cell lines. Derivatives 4, 14, and 17 up-regulated the expression of p53 by ~3–4 folds. All derivatives caused a significant ~3-fold increase in the expression of executive caspase 3 and significant elevation in cleaved caspase 3 activity. The elevation in the expression of caspase 3 by compound 1 and derivative 16 was not accompanied by an increase in p53 expression or cleaved caspase 3 activity. These two thienopyrimidines may act directly on caspase 3. Derivative 17 was unique in reducing the expression of Topo II by ~60%. The molecular docking showed that derivatives 4 and 17 with high binding energies could bind and inhibit Topoisomerase II (Topo II). In accordance with the docking modelling, derivatives 4 and 17 reduced the Topo II concentration by 82 and 90%, respectively, compared to the untreated cells. However, the parent compound 1 also caused a significant 34% reduction in the enzyme concentration although it was not predicted as a ligand for the enzyme in the docking study. Taken together, derivatives 4, 14 and 17 showed selective cytotoxicity, could arrest cell cycle, and induce apoptosis. Furthermore, they could serve as cytostatic agents by inhibiting/reducing Topo II.

Full text of DOI:10.1016/j.bioorg.2019.103492

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Design, Molecular Modeling and Anticancer Evaluation of Thieno[2,3-d]pyri-
midine Derivatives as Inhibitors of Topoisomerase II
Souad A. El-Metwally, Ali K. Khalil, Wael M. El-Sayed
PII:
S0045-2068(19)30750-3
DOI:
https://doi.org/10.1016/j.bioorg.2019.103492
YBIOO 103492
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Design, Molecular Modeling and Anticancer Evaluation of Thieno[2,3-d]pyrimidine
Derivatives as Inhibitors of Topoisomerase II
Souad A. El-Metwallya, Ali K. Khalilb, Wael M. El-Sayedc,*
a Higher Technological Institute, 10th Ramadan city, Egypt
b University of Ain Shams, Faculty of Science, Department of Chemistry, Abbassia 11566,
Cairo, Egypt
c University of Ain Shams, Faculty of Science, Department of Zoology, Abbassia 11566, Cairo,
Egypt
* Corresponding author;
Wael M. El-Sayed,
Tel: +202/2482-1633
Fax: +202/2684-2123
Email; waelelhalawany@hotmail.com, wael_farag@sci.asu.edu.eg
Running title: Thienopyrimidine Derivatives as Topoisomerase Inhibitors
1

Abstract
Synthesis of 4-(3,5-Dimethyl-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidine 1 and its functionalized reactions as nucleophile with various electrophilic reagents
were performed through facile methods to yield different cyclic and acyclic derivatives (2-17).
The structures of the newly synthesized compounds were established by their elemental analysis
and spectral data. Derivatives 4, 14, 16, and 17 in addition to the parent compound 1 had IC50 at
~ 4-10 µM against HepG2 and MCF7 and were selected for further investigations. All
derivatives had high IC50 values (> 60 µM) against normal fibroblasts WI38 indicating selectivity
against cancer cell lines. Derivatives 4, 14, and 17 up-regulated the expression of p53 by ~ 3-4
folds. All derivatives caused a significant ~ 3-fold increase in the expression of executive
caspase 3 and significant elevation in cleaved caspase 3 activity. The elevation in the expression
of caspase 3 by compound 1 and derivative 16 was not accompanied by an increase in p53
expression or cleaved caspase 3 activity. These two thienopyrimidines may act directly on
caspase 3. Derivative 17 was unique in reducing the expression of topo II by ~60%. The
molecular docking showed that derivatives 4 and 17 with high binding energies could bind and
inhibit Topoisomerase II (Topo II). In accordance with the docking modelling, derivatives 4 and
17 reduced the Topo II concentration by 82 and 90%, respectively, compared to the untreated
cells. However, the parent compound 1 also caused a significant 34% reduction in the enzyme
concentration although it was not predicted as a ligand for the enzyme in the docking study.
Taken together, derivatives 4, 14 and 17 showed selective cytotoxicity, could arrest cell cycle,
and induce apoptosis. Furthermore, they could serve as cytostatic agents by inhibiting/reducing
Topo II.
2

Key words: Thienopyrimidine; Semicarbazide derivative; Cytotoxic; Topoisomerase II;
Apoptosis; p53.
3

Introduction
Traditional chemotherapeutic agents are cytotoxic to both cancer and normal cells. They
usually cause oxidative stress that evokes apoptosis without selectivity leading to sever and
sometimes life-threatening effects. On top of that, they may result in secondary cancers or lose
their effectiveness due to the emerging of cancer cell resistance. Therefore, the research is focused
on targeted therapy and cytostatic agents. Thienopyrimidine derivatives have attracted much
attention since they are well known to have a wide range of biological activities. Versatile natural
products such as purines, pyrrolopyrimidines, pyridopyrimidines, pteridines, agrochemicals, and
veterinary products possess fused pyrimidines [1-3]. Pyrimidine derivatives and heterocyclic
pyrimidines showed immense biological activities such as anticancer [4], antiviral [5], antitumor
[6], anti-inflammatory [7], and antimicrobial [8, 9] agents. Aromatic and heteroaromatic
compounds bearing an o-amino nitrile or o-amino ester group represent a remarkable class of
substrates utilized as precursors for the synthesis of various condensed pyrimidine heterocyclic
systems [10]. Thieno[2,3-d]pyrimidine derivatives bearing a phenylamino substituent at the 4-
position have shown remarkable anticancer activity as tyrosine kinase inhibitors [11, 12].
Structure-activity relationships studies revealed that various substituted thienopyrimidines,
particularly at 4-position with various substituents such as anilino or heterocycles result in
enhanced anticancer activities [12-16], Figure 1.
To test the hypothesis and fulfill the aim of the study, 17 thienopyrimidines derivatives
were synthesized from the parent compound; 4-hyrazino compound (1) [17] by different
substitution at the 4-position. Compound 1, as a strong nucleophile, was allowed to react with
diverse electrophilic centers such as diketonic compounds, anhydrides, acid halides, esters, alkyl
halides, isocyanate, isothiocyanate derivatives, carboxylic acids, nitrous acid, triethyl
4

orthoformate, carbon disulphide, acrylonitrile, and aldehydes together with thioglycolic acid. The
antiproliferative activity of these new derivatives and the parent compound was tested against
epithelial human liver (HepG2) and breast (MCF-7) cell lines in addition to the normal human
fibroblasts (WI-38) and compared to the activity of doxorubicin. The most active derivatives were
selected for further investigations on the key genes involved in the cell cycle and/or apoptosis in
liver cells. Finally, the active derivatives were examined by molecular modeling as inhibitors for
topoisomerase II. In addition, the concentration of the later enzyme was also estimated.
NH2
N
NH
N
N
O
N
N
S
N
N
N
N
N
O
N
S
S
HO
O
O
GDC-0941
GNE-490
O
N
NH2
N
N
N
N
N
N
HN
O
HO
S
S
N
R2
R1
O
Known IKK inhibtor

thienopyrimidine-base analoges
GNE-493
Figure 1: Thienopyrimidines as anticancer agents
Topoisomerases are enzymes that participate in the control of supercoiling and prevention of DNA
tangling, therefore, it is essential for the growth of cancer cells. Consequently, topoisomerases
5

have become popular targets for cancer chemotherapeutic agents [18]. Several synthetic chemical
analogs have been designed and used as topoisomerase inhibitors that block the action of
topoisomerases (I and II) or decrease their expression and/or protein content [19, 20]. This
reduction in activity or protein level will affect the topology and stability of DNA leading to single
and double stranded breaks that harm the integrity of the genome leading to apoptosis [21]. Drugs
poison or inhibit Topo II by two mechanisms. Etoposide, teniposide, doxorubicin, daunorubicin,
and amsacrine inhibit the religation of DNA, whereas the ellipticines, azatoxins, and genistein
enhance the formation of Topo II cleavage complex. The DNA intercalator doxorubicin poisons
Topo II at low concentration and suppresses the enzyme concentration at higher concentration [18-
21].
2. Results and discussion
2.1 Chemistry
Due to all of the aforementioned facts and as a continuation of our previous work [11, 22-
25], the scope of our investigation has been broadened to evaluate the potency of some synthetic
analogues of thieno[2,3-d]pyrimidine derivatives as plausible topoisomerase inhibitors.
Therefore, a profound assessment of those derivatives as anticancer agents capable of targeting
topoisomerase II has been performed. Initially, a series of thieno-pyrimidine with diverse moieties
at position 4 has been constructed. The synthetic strategy for constructing them has emanated from
allowing the starting compound 4-hyrazino derivative (1) [17] as a strong nucleophile to react with
diverse electrophilic centers. As electrophilic scafflods, diketonic compounds, anhydrides, acid
halides, esters, alkyl halides, isocyanate, isothiocyanate derivatives, carboxylic acids, nitrous acid,
6

triethyl orthoformate, carbon disulphide, acrylonitrile were utilized. Also aldehydes together with
thioglycolic acid have been used as one pot reaction in order to build different thienopyrimidine
derivatives.
When the starting compound 1 was subjected to react with diketonic compounds such as
acetyl acetone, it gave the expected product 2 (Scheme 1). 1H NMR of 2 revealed the presence of
two quartet peaks at  2.9 & 2.2 ppm assuring the presence of the two methyl groups. In addition,
13
its C NMR showed two peaks at  11.8 and 13.6 ppm which confirmed the existence of the
methyl groups coinciding with the literature [17].
As anticipated, when compound 1 was treated with acid anhydride namely phthalic
anhydride, it gave derivative 3. The structure of derivative 3 was confirmed by the appearance of
two carbonyl peaks at 1790 and 1733 cm-1 in IR and in addition to two peaks at 166.47, 166.75
ppm in 13C NMR. As an additional proof, 1H NMR revealed the presence of two doublets at 8.0
and 8.4 ppm corresponding to four aromatic hydrogens of the phenyl group. On the other hand,
the reaction of compound 1 with maleic anhydride proceeded to completion to afford derivative 4.
In both reactions with the anhydrides, the reaction seemed to commence with the amino group
attack on one of the two carbonyl groups followed by a simple condensation reaction.
Unambiguously, the 13C NMR spectrum proved the structure by exhibiting peaks at 168.5, 168.5
1
ppm for the two carbonyl groups whereas the H NMR showed a doublet peak at  6.4 ppm
representing the two methine hydrogens of the pyrrrolidinone moiety. Meanwhile, IR spectrum
showed characteristic peaks for two carbonyl groups at 1725, 1765 cm-1 (Scheme 1).
7

O
N
N
HN
O
Phithalic anhydride
EtOH/reflux
S
N
H2N
S
N
N
O
O
NH
(3)
O
N
N
H3C
CH3
N
N
EtOH / Ref. 2 hr
H3C
S
N
HN
N
O
(1)
(2)
Maleic anhydride
EtOH/reflux
N
CH3
S
(4)
(Scheme 1)
Utilizing another type of electrophilic reagents such as benzoyl chloride, chloro acetyl
chloride, and acetyl chloride was investigated by subjecting compound 1 to react with them. The
mechanistic pathway for the synthesis of the expected products 5, 6 and 7 commenced with the
loss of hydrogen halide followed by the attack of N-3 of the pyrimidine ring on the carbonyl group
to give the expected cyclic products. The structure of derivative 5 was confirmed by the appearance
of the five aromatic protons as two doublet peaks at  8.5 ppm for 2H and one triplet peak at  7.5
ppm for 3H in the 1H NMR spectra. In case of chloro methyl derivative 6, a singlet peak at  5.0
ppm appeared for the methylene group while in case of the methyl derivative 7, a singlet peak for
1
the methyl group at  2.7 ppm was found. IR and H NMR spectra of all the three revealed the
13
absence of the NH and NH2 peaks. However, in case of the methyl derivative 7, its C NMR
showed a peak at 14.7 ppm for the methyl group. Furthermore, the structure of derivative 7 was
authentically proved from the reaction of compound 1 with glacial acetic acid and/or acetic
anhydride which showed coincidental accordance with the proposed structure of 7 (Scheme 2).
8

H
N
H
N
O
OH
H
N
N
N
N
N
N
S
S
(8)
N
O
N
EtOH / 3 h
Cl
Ph
Cl
N
N
N
S
N
HCOOH or
PhCOCl
N
(5)
triethyl orthoformate
EtOH / 3 h
S
N
H2N
NH
(9)
N
S
N
(1)
N
N
Cl
CH3COCl
EtOH / 3 h
N
N
N
NaNO2
ClCH2COCl
THF / 3h
N
S
N
CH3COOH
N
(6)
S
N
N
N
(10)
CH3
N
S
N
(7)
(Scheme 2)
Surprisingly, when the reaction with chloroacetyl chloride was performed in ethanol as a
solvent, the reaction proceeded via a different route which was initiated with the removal of two
moles of HCl to afford the product 8. The structure of this compound was proved by the appearance
of both the NH and C=O of lactam ring peaks in the IR spectrum at 3241 and 1661cm-1,
respectively. Compound 8 is believed to be existing in a tautomeric equilibrium (Scheme 2), since
1
it has two singlet peaks in its H NMR spectrum at  8.4 and 9.2 ppm corresponding to N-C=H
9

proton pyrimidine hydrogen, respectively accompanied with the appearance of two weak peaks at
 1.9 and 3.2 ppm for both the OH and NH protons.
When the hydrazino compound 1 was refluxed with formic acid, the reaction resulted in
the formation of triazolo derivative 9 where a singlet peak appeared at  8.5 for the one proton of
the thriazole ring and lacked any peaks for NH or NH2 functional group in both IR and 1H NMR
spectra. As an additional proof, compound 9 was also authentically prepared by refluxing the
starting compound 1 with triethyl orthoformate in a neat reaction (Scheme 2).
To achieve the target of constructing the desired tetrazine derivative 10, a freshly prepared
ice cold solution of nitrous acid was added to compound 1. Structural elucidation of derivative 10
was ascertained by the absence of the NH and NH2 peaks in either the IR or 1H NMR spectrum.
Meanwhile, a singlet peak at  9.1 ppm corresponding to the pyridine aromatic proton has been
shown in the 1H NMR spectrum (Scheme 2).
Fusion of compound 1 with ethyl benzoyl acetate at 180οC afforded the pyrazolone
derivative 11. The structure of derivative 11 was confirmed by the absence of both NH and NH2
absorption peaks in IR and the presence of a carbonyl absorption band at 1665 cm-1 while in the
1H NMR spectrum a broad peak for 5H phenyl protons at  7.0-7.5 ppm and a peak at  7.9 ppm
for the pyrazolone proton, a CH proton peak at  8.4 ppm for pyrimidine proton, and one OH
proton at  10.4 ppm. The appearance of the OH and NH peaks could be interpreted by the
existence of the pyrazolone derivative 11 in different tautomeric forms as shown in (Scheme 3)
The nucleophilic attitude of hydrazine compound 1 was further evaluated when it was allowed to
react with benzyl chloride as an alkylating reagent. The reaction proceeded to afford the N-dialkyl
product 12 where two moles of the alkylating agent were consumed (Scheme 3).
10

Ph
N
Ph
N
H
O
O
HO
O
N
N
N
N
Ph
OEt
N
N
fusion
H2N
S
S
NH
(11)
N
Ph
S
N
Ph
N
NH
(1)
Ph-CH2Cl/EtOH
N
tetrahydrofuran
ref. 4h
S
N
(12)
(Scheme 3)
The structure of the alkylated product 12 was confirmed by the absence of the NH2
1
absorption band from the IR spectrum. H NMR revealed the existence of doublet peak at  2.1
ppm corresponding to one methylene group and another peak for the other methylene group at 
2.1 ppm besides absorption peaks at 7.2-10.0 ppm corresponding to the twelve protons; 10
aromatic hydrogen appear as (m, 1H) at  7.43 ppm, (m, 5H) at  7.8 ppm, (d, 3H) at  8.4 ppm
(d, 1H) at  8.9 ppm, pyrimidine one hydrogen at  8.54 ppm, and one NH proton as a singlet peak
at  9.6 ppm.
As the last electrophilic reagent of those which have been utilized, acrylonitrile has been
exploited. It is worth mention that the nature of the product was found to be dependent on the
reaction conditions under which the reaction was performed. For example, under refluxing an
ethanolic solution of compound 1 with acrylonitrile, a simple nucleophilic attack on the highly
deficient acrylonitrile carbon (C-3) took place to give product 13 IR spectrum of such compound
11

revealed the presence of CN absorption peak at 2252 cm-1 and two absorption peaks for two NH at
3382, 3289 cm-1 whereas 13C NMR spectrum showed peaks for the two methylene groups CH2-
CN, CH2-N at  14.7, 22.1 ppm, respectively and an absorption peak for the cyano group at
 120.11 ppm. Meanwhile, 1H NMR showed peaks at  1.7, 2.7, 1.9, 3.3 ppm corresponding to the
two methylene groups CH2-CN, CH2-N, respectively. However, under fusion conditions, the same
reaction was initially preceded by attacking of the amino group as a nucleophile on the β carbon
of one mole of acrylonitrile following Michael reaction to give derivative 13 as an intermediate
which in turn its NH as a nucleophile further attacked the other cyano group to give the cyclic
product 14. The structure of derivative 14 was verified by the presence of absorption peaks at 2246
and 3378 cm-1 in the IR spectrum corresponding to CN and NH2 groups, respectively. In addition,
1
13C NMR spectrum showed a peak at  119.3 ppm corresponding to the cyano group. H NMR
revealed a singlet peak at  3.6 ppm (CH2, pyrazole) (Scheme 4).
H
N
HN
N
CN
HO
O
CN
Dioxane
N
S
H2N
N
reflux
S
NH
NH
(13)
Salicylaldehyde
N
N
HSCH2COOH
CH3COOH/ reflux
S
N
S
N
N
N
H2N
N
N
CN
CN
(1)
(15)
fusion
S
(14)
(Scheme 4)
Condensation of compound 1 with salicyaldehyde in the presence of thioglycolic acid in
acetic acid under reflux condition gave the isothiazolidine derivative 15 (Scheme 4). Its structure
12

was confirmed by the presence of absorption peaks in IR spectrum at 3470, 1650 cm-1
corresponding to the hydroxyl group and carbonyl group, respectively and lacking of absorption
peaks for the NH2 group. Also, the mass spectrum of derivative 15 gave a molecular ion peak at
m/z 398.9 (4.93 %).
The reaction of compound 1 with carbon disulphide in pyridine gave the expected triazolo-
thione derivative 16 (Scheme 5) with no absorption band for the NH2 group in the IR spectrum
and a singlet peak at  14.6 corresponding to SH proton in the 1H NMR spectrum. Also, its mass
spectrum assured the structure by the presence of M+ peak at m/z 262.0 (100%). When compound
1 was treated with carbon disulphide or α- naphthyl isothiocyanate or phenyl isothiocyanate gave
the same product 16. The plausible mechanism of formation of derivative 16 is illustrated in
(Scheme 6). This was confirmed by the absence of the aromatic hydrogens and appearance of a
1
singlet broad absorption peak for the SH proton at  14.6 ppm in the H NMR spectra of both
compounds which means the release of both the α-naphthyl amine and aniline fragments during
the attack upon the C=S bond followed by cyclization and releasing of the amine moiety. However,
this was not the behavior in the case of phenyl isocyanate where the reaction was halted at the step
of forming the Semicarbazide derivative 17 with no further cyclization (Scheme 5). This can be
attributed due to the lower reactivity of isocyanates derivatives compared with their corresponding
isothiocyanates. The structure of 17 was proved by the presence of the carbonyl absorption peak
at 1653 cm-1 in the IR spectrum and the appearance of five protons of the phenyl group at  7.1-
7.5 ppm in its 1H NMR spectrum.
13

H
O
N
Ph-N=C=S /EtOH/reflux
CS2/Pyridine
NH
H2N
HN
NH
N
N
SH
PhN=C=O
N
N
N
EtOH, reflux
S
N
S
N
S
N
(17)
(16)
(1)
-naphthyl isothiocyanate
EtOH/reflux
(Scheme 5)
H
N
N
S
S
R
R
..
NH
N
H2N
NH2
NH
HN
N
HN
N
N
N
~ H
R- N=C=S
S
N
S
S
(1)
- R-NH2
N
N
SH
N
R =
or
S
N
(16)
Scheme 6: Suggested mechanistic pathway for the release of amino moiety.
2.2. Biology
Doxorubicin was cytotoxic to both liver and breast cell lines in addition to normal
fibroblasts with IC50 at ~ 4-5 µM (Table 1). Thienopyrimidine derivatives having IC50 equal to
or less than ~ 10 µM were considered active. Derivatives 4, 14, 16, and 17 in addition to the
parent compound 1 had IC50 at ~ 4-10 µM against HepG2 and MCF7 and were selected for
further investigations. All derivatives had high IC50 values (> 60 µM) against normal fibroblasts
14

WI38 indicating selectivity against cancer cell lines. All new derivatives were less cytotoxic to
the normal fibroblasts than the parent compound 1 which had an IC50 at ~ 40 µM in normal cells.
Table 1: Influence of the thienopyrimidine derivatives on the viability of HepG2, MCF7 and
WI-38 cells.
IC50 (µM)
Cpd
HepG2
MCF7
WI-38
DOX
1
4.50±0.22
7.84±0.81
49.47±3.13
30.02±2.25
6.71±0.77
38.13±2.67
53.23±3.37
73.16±4.01
65.48±3.80
76.85±4.21
22.90±1.83
84.37±4.83
16.96±1.47
>100
4.17±0.21
10.32±1.10
56.62±3.40
17.28±1.53
5.71±0.67
32.09±2.31
45.38±3.10
81.57±4.70
69.20±3.91
92.73±5.20
27.16±1.90
>100
5.23±0.87
39.91±7.67
81.81±9.71
68.67±4.13
60.67±3.18
68.31±14.35
59.01±4.57
>100
2
3
4
5
6
7
8
66.33±9.33
>100
9
10
11
12
13
14
15
32.54±5.33
>100
14.27±1.21
>100
64.00±5.37
>100
4.38±0.51
41.85±2.90
3.96±0.31
37.45±2.83
74.71±11.23
45.25±3.17
15

16
17
5.51±0.4
9.19±0.9
73.33±8.11
89.13±9.93
6.17±0.93
5.89±0.87
After 48h of incubation, the viability of cells was measured with 5 different concentrations (three wells per every
concentration) of the investigated compounds by MTT assay. The IC50 was determined from the dose-response curves
as the mean of two parallel experiments. Doxorubicin (Dox) was used as a positive control.
To explore the molecular mechanism(s) through which these active derivatives exerted
their antiproliferative activity, the effect of the most active derivatives (4, 14, 16 and 17) and the
parent compound 1 on the expression of key genes involved in cell cycle and apoptosis (cdk1,
p53, caspase 3, and topo II) was investigated in HepG2 cells and compared to that of doxorubicin
(Table 2). Doxorubicin was the only treatment that affected the expression of cdk1 causing ~
70% reduction compared to the untreated cells in agreement with previous reports [26, 27]. The
ability of the cell to progress to S phase depends on the cdk1 [28]. The downregulation of cdk1
by doxorubicin along with the elevation in p53 expression suggest cell cycle arrest. p53, the
guardian of the genome, is a tumor suppressor gene. It is mutated in more than 50% of human
cancer types [29]. It plays a central role in the regulation of cell cycle progression, DNA
stability, and apoptosis. DNA damage induces the elevation in p53 expression which arrests the
cell cycle at the G1/S checkpoint, stimulates the DNA repair mechanisms, and if the damage is
sever, p53 initiates apoptosis [30]. Derivatives 4, 14, and 17 upregulated the expression of p53
by ~ 3-4 folds compared to ~ 2-folds elevation caused by doxorubicin. Similar effects were
previously reported for doxorubicin [31]. The elevations in p53 caused by the parent compound
and derivative 16 did not achieve a statistical significance.
16

All derivatives investigated as well as doxorubicin caused a significant ~ 3-fold increase
in the expression of executive caspase 3 (Table 2). The elevation of p53 in the current study
could be responsible for the parallel elevation in the expression of caspase 3. Caspase 3 is the
main execution member responsible for apoptosis induced by both extrinsic and intrinsic
pathways [32]. Procaspase 3 is cleaved and activated by other initiator caspases in a well-
controlled cascade. However, the elevation in the expression level of caspase 3 by compound 1
and derivative 16 was not accompanied by an increase in p53 expression. These two
Thienopyrimidines may act directly on caspase 3. Currently, there is no clinically approved drug
that specifically activates effector caspase 3 directly without going through the regular activation
by initiator caspases and p53. Therefore, these derivatives could be chemically modified to
maximize their specific caspase targeting potential. Since many tumor cells overexpress
procaspase 3 [33], having specific targeted drugs that bypass all upstream apoptotic events and
directly hit effector caspase 3 will be a substantial advance and this will significantly reduce the
resistance of tumor cells to chemotherapeutic agents. In this study, we did not measure the
protein level of enzyme activity of the cleaved caspase 3. Since a linear correlation between gene
expression and enzyme level or activity is not always present due to post-transcriptional and
translational events, therefore, these derivatives deserve further investigations. Many FDA-
approved chemotherapeutic agents were found to elevate p53 expression [34], and induce
caspases [35] in tumor cells
Derivative 17 was the only treatment that significantly affected the expression of topo II
resulting in ~60% reduction compared to the untreated cells. Doxorubicin also significantly
reduced the expression of topo II but to a lesser extent; ~40%. Topo II prevents the DNA tangling
17

by double stranded breaks and is an essential enzyme for DNA replication and cell proliferation
[36]. Inhibitors of Topo II are extensively examined as antineoplastic agents [37].
Table 2: Effect of thienopyrimidine derivatives on the expression of p53, cdk1, caspase 3
and topo II in HepG2 after 8 h incubation.
Treatment
p53
Cdk1
Caspase 3
Topo II
(fold induction from untreated cells)
DOX
1
2.37 ± 0.31a
1.27 ± 0.24
3.01 ± 0.42a
2.67 ± 0.10a
1.43 ± 0.09
3.83 ± 0.11a
0.31 ± 0.02a
1.24 ± 0.17
0.88 ± 0.21
1.15 ± 0.18
1.00 ± 0.35
0.92 ± 0.24
3.37 ± 0.57a
2.97 ± 0.21a
2.78 ± 0.05a
2.51 ± 0.06a
2.97 ± 0.12a
3.10 ± 0.21a
0.57 ± 0.09a
0.90 ± 0.11
0.92 ± 0.16
1.11 ± 0.04
0.93 ± 0.09
0.37 ± 0.04a
4
14
16
17
18

a Significant difference (p< 0.05) as compared to untreated cells. The data are expressed as mean ± SEM
for two independent experiments.
Molecular docking
A molecular docking study was performed using MOE 2008.10 software for the most active
synthesized compounds reported in Table 1 in addition to doxorubicin as a reference DNA
intercalator, into the DNA binding site of topoisomerase II (ID: 3qx3). According to the interaction
results presented in Table 3, the most active compounds 4 and 17 showed high binding energies
with DNA-Topo II target, with energies of -27.78 and -36.74 kcal/mol, respectively, compared to
doxorubicin (-34.80 kcal/mol). All compounds formed a network of molecular interactions (H-
bonds, vdw, and π-aromatic) with the active site residues of Topo II when analyzed in 2D plots as
shown in Figure 2.
The proposed binding mode of derivative 4 is the formation of various interactions in the
binding pocket of the enzyme. A hydrogen bond between the NH and DTB9 was formed with a
distance of 2.77 A° and the planar aromatic system was involved in a hydrophobic interaction with
LysA739, ThrA783 and TyrA773 residues (Figure 2A). The same behaviour was exhibited by
derivative 17 which showed two hydrogen bonds interaction between the two amidic NH groups
and the C=O group of AspA479 with a distance of 2.95 and 2.43 A°. In addition, the planar
aromatic system (thieno-pyrimidine) showed hydrophobic interactions with LysA739, ThrA783
and TyrA773 residues. Moreover, the benzene ring moiety was oriented to form aromatic stacking
interaction with LysA456 and ArgA503 (Figure 2B). Camptothecin (CPT) binds to topoisomerase
and DNA by hydrogen bonds forming in a ternary complex and thereby stabilizing it. This prevents
the re-ligation of DNA and therefore causes DNA damage and apoptosis [38]. CPT showed
19

promising clinical results, and two analogues (topotecan and irinotecan) of CPT were approved by
FDA as effective cancer therapeutic agents [39]. Another analogue of CPT; a lurtotecan with a
pyrimidine moiety was clinically used in treating resistant ovarian cancer [40].
Table 3. The docking binding free energies of thienopyrimidine derivatives against
topoisomerase II
Binding free
Compound
energy
(kcal/mol)
-19.01
-27.78
-18.11
-16.18
-36.74
-34.80
1
4
14
16
17
Doxorubicin
(A)
20

(B)
(C)
Figure 2: Calculated binding mode of derivatives 4 (A), 17 (B), and doxorubicin (C) (grey sticks) within
the binding pocket of topo II receptor. The active pocket has been represented as yellow surface. Other
residues have been hidden for sake of clarity.
From the gene expression profile, it was clear that only derivative 17 inhibited the
expression of topo II but the docking study showed that derivatives 4 and 17 are projected to bind
and inhibit the enzyme activity. The molecular modeling is directed to the protein and not to the
genome level. Therefore, the topo II protein levels were measured in HepG2 after treatment with
21

the most active antiproliferative derivatives at their IC50 values (Figure 3). The results were
somewhat parallel to the docking results. Derivatives 14 and 16 were devoid of any significant
effect (15 and 17% decrease, respectively) compared to the untreated cells. In accordance with the
docking modelling, derivatives 4 and 16 reduced the enzyme concentration by 82 and 90%,
respectively, compared to the untreated cells. However, the parent compound 1 also caused a
significant 34% reduction in the enzyme concentration although it was not shown to bind the
enzyme in the docking study. This could be due to its effect on the post-transcriptional processing
of the mRNA or translation of the protein; a hypothesis that needs further investigation.
Doxorubicin caused ~ 87% decrease in the enzyme concentration as compared with the untreated
cells. The antitumor activity of doxorubicin is mainly due to intercalation of DNA and reduction
in the concentration and activity of Topo II [41, 42].
1400
1200
1000
*,+
800
600
400
*
200
0
*
*
Figure 3: The inhibitory activity of the novel thienopyrimidine derivatives against topoisomerase II (Topo
II) in HepG2 cells. Each compound has been assigned three wells and the experiment was performed twice.
+
Data are expressed as mean ± SEM. * Significant difference compared to untreated cells. Significant
difference compared to doxorubicin-treated cells
22

Effect of the active compounds (1, 4, 14, 16 and 17) on the cleaved caspase 3 activity in HepG2
Since a linear correlation between gene expression and protein expression does not always
exists and since caspase 3 exists as inactive procaspase 3, measuring the cleaved caspase 3 activity
is considered the most accurate way to examine the effect of the most active compounds from the
previous investigations (1, 4, 14, 16 and 17) on caspase 3 and link it to apoptosis. Compounds 4,
14, and 17 caused a significant elevation in the enzyme activity comparable to that caused by
doxorubicin (Figure 4). These results do not correlate well with those reported for genes in table
2, where all compounds significantly elevated the gene expression. Therefore, we believe that
measuring qPCR alone is not a sensitive measure due to the complexity of translation and post-
translational processing. This clearly indicates that compounds 4, 14, and 17 induce apoptosis
through caspase 3 while compounds 1 and 17 work through different pathway(s).
700
600
500
*
*
*
*
400
300
200
100
0
Figure 4: The effect of derivatives 1, 4, 14, 16 and 17 on the cleaved caspase 3 activity in HepG2 cells.
Data are expressed as mean ± SEM, n=3. * significant inhibition compared to untreated cells.
Conclusions
Previous studies revealed that various substituted thienopyrimidines at 4-position with various
substituents enhanced their anticancer activities. Consequently, 17 thienopyrimidines derivatives
were synthesized from the parent compound; 4-hyrazino compound (1) by different substitution at
23

the 4-position. These new derivatives were examined for their antiproliferative activity, their
effects on key genes involved in cell cycle and apoptosis. Finally they were evaluated as inhibitors
for TopoII by molecular docking and by measuring the enzyme concentration in liver cells.
Derivatives 4, 14, 16, and 17 in addition to the parent compound 1 had IC50 at ~ 4-10 µM against
HepG2 and MCF7 and were selected for further investigations. All derivatives had high IC50 values
(> 60 µM) against normal fibroblasts WI38 indicating selectivity against cancer cell lines.
Derivatives 4, 14, and 17 up-regulated the expression of p53 by ~ 3-4 folds which functions to
arrest cell cycle, induce DNA repair, or induce apoptosis. These same derivatives significantly
elevated the cleaved caspase 3 activity indicating the occurrence of apoptosis. All derivatives
caused a significant ~ 3-fold increase in the expression of caspase 3, the final and most important
member of execution phase of apoptosis. Compound 1 and derivative 16 induced caspase 3
expression without affecting p53 expression but did not affect the cleaved caspase 3 activity.
Derivative 16 was unique in reducing the expression of topo II by ~60%. The molecular docking
showed that derivatives 4 and 17 with high binding energies could bind and inhibit Topo II. In
accordance with the docking modelling, derivatives 4 and 17 reduced the Topo II concentration
by 82 and 90%, respectively, compared to the untreated cells. Taken together, derivatives 4, 14,
and 17 showed selective cytotoxic, could arrest cell cycle, and induce apoptosis. Furthermore, they
could serve as cytostatic agents by inhibiting/reducing Topo II and therefore, they deserve further
investigations as small molecules targeted therapies.
3. Experimental
3.1 Chemistry
24

3.1.1 General
Melting points were determined in an open capillary in a Gallenkamp melting point
apparatus (Sanyo Gallenkamp, UK) and are uncorrected. Pre-coated silica gel plates (Kieselgel
0.25 mm, 60 F254, Merck, Germany) were used for thin layer chromatography. IR spectra (KBr
1
disc) were recorded using an FT-IR spectrophotometer (Perkin Elmer, USA). H NMR spectra
were scanned on a NMR spectrophotometer (Bruker AXS Inc., Switzerland), operating at 400
MHz for 1H and 13C NMR. Chemical shifts are expressed in  values (ppm) relative to TMS as an
internal standard, using DMSO-d6 or CDCl3 as a solvent. Elemental analyses were done on a model
2400 CHNSO analyzer (Perkin Elmer, USA). All values were within ±0.4 % of the theoretical
values. All reagents used were obtained from Sigma (St. Louise, USA) and was directly used for
the preparation of the target compounds.
3.1.2 Synthesis
Formation of4-( 3,5-dimethyl-1H-pyrazolyl) and 3,4-dimethyl-triazin-4-ol.
thienopyrimidine derivatives (2):
An amount of 0.005 moles of some diketonic compound ; acetyl acetone was added to a
solution of compound 1 (1.1 g, 0.005 mole) in 30 mL ethanol and refluxed for 2 h. The formed
precipitate was filtered off and crystalized from the suitable solvent.
4-(3,5-Dimethyl-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (2)
C15H16N4S: Pale buff crystals (ethanol); m.p. (260οC); yield (80 %); IR (KBr νcm-1) 2939,
2861, 1566; 1H NMR (CDCl3) δ ppm: 1.60 (m, 2H, CH2cyclohexane), 1.76 (m, 2H, CH2cyclohexane), 2.1
(s, 3H, CH3), 2.2 (s, 3H, CH3), 2.8 (m, 4H, 2CH2cyclohexane), 6.1 (s, 1H, =CH), 8.9 (s, 1H pyrimidine);
25

13C NMR (CDCl3): 11.8, 13.3, 22.3, 22.5, 24.9, 26.1, 77.3, 107.5, 126.4, 127.1, 139.6, 141.5,
150.0, 151.4; Anal. Calcd: C, 63.35; H, 5.67; N, 19.70; Anal. Found: C, 63.26; H, 5.55; N, 19.63.
Formation of isoindoline-1,3-dione&furan-2(5H)-one derivatives 3 and 4:
A quantity of 0.005 moles of some acid anhydrides; phthalic or maleic anhydride was
added to a solution of compound 1 (1.1 g 0.005 mole) in 30 mL ethanol and refluxed for 3h where
a precipitate was formed during the reaction. After completion of the reaction, the precipitate was
separated by filtration and crystalized from the appropriate solvent.
2-((5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)isoindoline-1,3-dione
(3)
C18H14N4O2S: White crystals (Ethanol / DMF); m.p. (268οC); yield (80 %); IR (KBr νcm-
1) 3402, 3354, 2939, 1790, 1733; 1H NMR (CDCl3) δ ppm: 1.7 (m, 4H, 2CH2cyclohexane), 2.7-2.9 (m,
4H, 2CH2cyclohexane), 7.7 (d, 2Haromatic), 8.0 (d, 2Haromatic), 8.1(s, 1HPyrimidine), 8.4 ( s,1H ,NH, D2O
exchangeable ); 13CNMR (CDCl3): 20.8, 22.2, 25.4, 25.8, 116.3, 124.2 ,125.1, 130.0, 134.8, 136.0,
151.9, 155.1, 166.4, 166.7, 176.3; Anal. Calcd: C, 61.35; H, 4.00; N, 15.99; Anal. Found: C, 61.25;
H, 3.95; N, 15.88.
5-(2-(5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)hydrazono)furan -2(5H)-one
(4)
C14H12N4O2S: Yellow crystals (DMF); m.p. (205οC); yield (60 %); IR (KBr ν/ cm-1) 3337,
3049, 2949, 1645;1H NMR(DMSO-d6) δ ppm; 1.8 (m, 4H, 2CH2cyclohexane), 2.7 (d, 2 H,
CH2cyclohexane), 3.0 (d ,2H, CH2cyclohexane), 6.4 (d, 2Holefinic), 8.3 (s, 1Hpyrimidine), 8.7 (s ,1H ,NH, D2O
26

exchangeable); 13C NMR (DMSO-d6): 22.07, 22.47, 24.89, 25.63, 115.22, 126.69, 129.12, 129.97
,133.17, 133.67, 152.01, 163.11 ,166.75, 168.57; MS m/z: 300 [M+1, 71.76%], 204.01 (100%),
81.92 (62.46 %), 53.84 (61.06 %); Anal. Calcd: C, 55.99; H, 4.03; N, 18.65; Anal. Found: C,
55.79; H, 3.936; N, 18.46.
Formation of 3-alkyl-8,9,10,11-tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-
c]pyrimidine derivatives 5, 6 and 7:
A 0.005 mole of different acid halides; benzoyl chloride, chloro acetyl chloride and acetyl
chloride was added to a solution of compound 1 (1.1 g, 0.005 mole) in 30 mL ethanol for 6 and 8
(tetrahydrofuran for 7) and refluxed for 3 h. After completion, the precipitated solid was filtered
off and crystallize from the appropriate solvent.
3-Phenyl-8,9,10,11-tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine(5):
C17H14N4S: Creamy crystals (ethanol/ DMF); m.p. (211οC); yield (70%); IR (KBr νcm-1),
1
3054, 2976, 2934, 1622; H NMR (CDCl3) δ ppm: 2.1 (m, 4H, 2CH2 cyclohexane), 3.2 (m, 2H,
13
CH2cyclohexane), 3.3 (m, 2H, CH2cyclohexane), 7.5 (t, 3 Haromatic), 8.5 (d, 2 Haromatic), 9.2 (s, 1 Hpyrimidine); C
NMR (CDCl3): 22.2, 23.0, 25.3, 25.5, 120.4, 127.6, 128.5, 129.3, 130.3,130.5, 135.3, 138.8, 149.6,
153.4, 165.0; Anal. Calcd.: C, 66.64; H, 4.61; N, 18.29; Anal. Found: C, 66.54; H, 4.51; N, 18.40.
3-(Chloromethyl)-8,9,10,11-tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-
c]pyrimidine (6)
C12H11ClN4S, White crystals (ethanol /DMF); m.p. (170οC); yield (80 %); IR (KBr ν cm-1)
1
3060, 3015, 2935, 1612; H NMR (DMSO- d6) δ ppm: 1.9 (m, 4Hcyclohexane), 2.9 (d, 2H,
CH2cyclohexane), 3.0 (d, 2H, CH2cyclohexane), 5.0 (s, 2H, CH2), 9.6 (s ,1Hpyrimidine); 13C NMR (CDCl3):
27

22.1, 23.0, 25.4, 25.6, 35.1, 127.1, 129.0, 136.8, 139.9, 144.7, 149.8, 152.6; Anal. Calcd: C, 51.70;
H, 3.98; N, 20.10; Anal. Found: C, 51.66 H, 3.78; N, 19.95.
3-Methyl-8,9,10,11-tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimi-dine (7):
C12H12N4S: White crystals (Ethanol-DMF); m.p. (255οC); yield (80 %); IR (KBr ν cm-1)
3055, 2974, 2931, 1790, 1618; 1H NMR (CDCl3) δ ppm, 2.0 (m, 4H, 2CH2cyclohexane) ,2.7 (s, 3H,
13
CH3) 3.0 (m, 2H, CH2cyclohexane), 3.2 (m, 2H, CH2cyclohexane), 9.1 (s, 1HPyrimidine), C NMR: 14.7,
22.1, 23.0, 24.4, 25.5, 120.1, 129.0, 135.1, 138.8, 149.3, 153.6, 165.0; Anal. Calcd: C, 58.99; H,
4.59; N, 22.93; Anal. Found: C, 58.87; H, 4.53; N, 22.89.
Formation of 9,10,11,12-tetrahydro-2H-benzo[4',5']thieno[2',3':4,5]pyrimido[6,1-
c][1,2,4]triazin-3-ol (8):
A 0.005 mole of chloroacetyl chloride (7 mL) was added to a solution of compound 1 in
30 mL ethanol and refluxed for 3 h. After the reaction being completed the precipitated solid was
filtered off and crystallize from the appropriate solvent. C12H12N4OS: White crystals (ethyl
1
acetate); m.p. (137οC); yield (75%); IR KBr ν cm-1) 3241 (NH), 3027, 2921, 1661 (C=O); H
NMR (CDCl3)  ppm: 1.9 (broad, 1H, NH, D2O exchangeable), 2.0 (m, 4H, 2CH2cyclohexane), 3.0
(d, 2H, CH2cyclohexane and 1H, OH, D2O exchangeable), 3.2 (d, 2H, CH2cyclohexane), 8.4 (s, 1H, N-
13
C=H), 9.3 (s, 1Hpyrimidine); C NMR (CDCl3): 22.1, 22.9, 25.2, 25.6, 76.2, 120.6, 129.0, 135.5,
139.5, 148.6, 153.5, 154.6; Anal. Calcd.: C, 55.37; H, 4.65; N, 21.52; Anal. Found: C, 55.35; H,
4.55; N, 21.42.
Formation of 8,9,10,11-tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine
derivatives (9)
28

A 0.005 mole of formic or neat triethyl orthoformate was added to a solution of compound
1 (1.1g; 0.005 mole) in 30 mL ethanol and refluxed for 4 h. The precipitated solid was filtered off
and crystalize from the suitable solvent. C11H10N4S: white crystals (Ethanol); m.p. (130-132 οC );
yield ( 55 % ); IR (KBr ν cm-1 ) 3060, 3018, 2935, 1613; 1H NMR (CDCl3) δ ppm: 2.0 ( m, 4 H,
13
2CH2cyclohexane), 3.0- 3.1 (m, 4 H, 2CH2cyclohexane), 8.4 (s, 1Htriazolo), 9.1 (s, 1Hpyrimidine); C NMR
(CDCl3): 22.1, 22.9, 25.4, 26.5, 120.4, 129.0, 135.4, 139.4, 140.3, 153.4, 154.5; Anal. Calcd.: C,
57.37; H, 4.38; N, 24.33; Anal. Found: C, 57.27; H, 4.30; N, 24.20.
Formation of 8,9,10,11-tetrahydrobenzo [4,5] thieno[3,2-e]tetrazolo[1,5-c]pyrimidine (10)
To a solution of compound 1 (1.1 g, 0.005 mole) in 20 mL acetic acid in an ice bath, a
solution of sodium nitrite (0.4g /2 mL H2O) was added drop wise with stirring for 30 minutes.
After complete addition, the mixture was left under stirring for another 30 minutes. The formed
solid was collected by filtration, dried and crystallized from ethanol. C10H9N5S: Pale buff crystals
(ethanol); m.p. (129οC); yield (70 %); IR (KBr ν cm-1) 3099, 3068, 2937, 1605; 1H NMR (CDCl3)
δ ppm: 2.0 (m, 4H, 2CH2cyclohexane), 3.0 (m, 2H, CH2cyclohexane), 3.2 (m, 2H, CH2cyclohexane), 9.5 (s,
1Hpyrimidine); 13C NMR (CDCl3): 22.4, 22.5, 25.4, 26.2, 125.4, 128.4, 134.0, 142.2,, 152.6, 165.0;
Anal. Calcd: C, 51.93; H, 3.92; N, 30.28; Anal. Found: C, 51.73; H, 3.88; N, 30.22.
Formation of 5-phenyl-2-(5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-2,4-
dihydro-3H-pyrazol-3-one (11):
A mixture of compound 1 (1.1 g; 0.005 mole) and ethyl benzoyl acetate (1 mL; 0.005 mole)
was fused at 180οC for 5 h. When the reaction was completed, the formed solid was triturated by
petroleum ether (60-80), filtered and crystallize from the suitable solvent. C19H16N4OS: Brown
crystals (ethanol); m.p. (209οC); yield (50%); IR (KBr ν cm-1) 3231, 2917, 2848, 1665; 1H NMR
29