Design, synthesis and anticancer evaluation of thieno[2,3-d]pyrimidine derivatives as dual EGFR/HER2 inhibitors and apoptosis inducers

Article abstract of DOI:10.1016/j.bioorg.2019.102944

Deregulation of many kinases is directly linked to cancer development and the tyrosine kinase family is one of the most important targets in current cancer therapy regimens. In this study, we have designed and synthesized a series of thieno[2,3-d]pyrimidine derivatives as an EGFR and HER2 tyrosine kinase inhibitors. All the synthesized compounds were evaluated in vitro for their inhibitory activities against EGFR^WT; and the most active compounds that showed promising IC₅₀ values against EGFR^WT were tested in vitro for their inhibitory activities against mutant EGFR^T790M and HER2 kinases. Moreover, the antitumor activities of these compounds were tested against four cancer cell lines (HepG2, HCT-116, MCF-7 and A431). Compounds 13g, 13h and 13k exhibited the highest activities against the examined cell lines with IC₅₀ values ranging from 7.592 ± 0.32 to 16.006 ± 0.58 μM comparable to that of erlotinib (IC₅₀ ranging from 4.99 ± 0.09 to 13.914 ± 0.36 μM). Furthermore, the most potent antitumor agent (13k) was selected for further studies to determine its effect on the cell cycle progression and apoptosis in MCF-7 cell line. The results indicated that this compound arrests G₂/M phase of the cell cycle and it is a good apoptotic agent. Finally, molecular docking studies showed a good binding pattern of the synthesized compounds with the prospective target, EGFR^WT and EGFR^T790M.

Full text of DOI:10.1016/j.bioorg.2019.102944

Accepted Manuscript
Design, synthesis and anticancer evaluation of thieno[2,3-d]pyrimidine deriva-
tives as dual EGFR/HER2 inhibitors and apoptosis inducers
Souad A. Elmetwally, Khaled F. Saied, Ibrahim H. Eissa, Eslam B. Elkaeed
PII:
S0045-2068(18)31098-8
https://doi.org/10.1016/j.bioorg.2019.102944
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Bioorganic Chemistry
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20 November 2018
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Please cite this article as: S.A. Elmetwally, K.F. Saied, I.H. Eissa, E.B. Elkaeed, Design, synthesis and anticancer
evaluation of thieno[2,3-d]pyrimidine derivatives as dual EGFR/HER2 inhibitors and apoptosis inducers,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.102944
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Design, synthesis and anticancer evaluation of thieno[2,3-d]pyrimidine derivatives as
dual EGFR/HER2 inhibitors and apoptosis inducers
Souad A. Elmetwallya, Khaled F. Saiedb*, Ibrahim H. Eissac, Eslam B. Elkaeedd*
a
Department of Basic Science, Higher Technological Institute, 10th of Ramadan City, 228, Egypt;
soad.abdelaziz@hti.edu.eg
b
Department of Basic Science, Oral and Dental Medicine, Nahda University, East Beni-Suef, 62511,
Beni-Suef, Egypt; fathykhaled905_7@yahoo.com
c
Department of Pharmaceutical Chemistry, Faculty of Pharmacy (Boys), Al-Azhar University, Nasr
City, 11884, Cairo, Egypt; Ibrahimeissa@azhar.edu.eg
d Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Boys), Al-Azhar University,
Nasr City, 11884, Cairo, Egypt; eslamkaeed@azhar.edu.eg
* Corresponding authors:
Eslam B. Elkaeed
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Boys), Al-Azhar University,
Nasr City 11884, Cairo, Egypt
Email:
eslamkaeed@azhar.edu.eg
1

Abstract
Deregulation of many kinases is directly linked to cancer development and the tyrosine kinase
family is one of the most important targets in current cancer therapy regimens. In this study, we have
designed and synthesized a series of thieno[2,3-d]pyrimidine derivatives as an EGFR and HER2 tyrosine
kinase inhibitors. All the synthesized compounds were evaluated in vitro for their inhibitory activities
against EGFRWT; and the most active compounds that showed promising IC50 values against EGFRWT
were tested in vitro for their inhibitory activities against mutant EGFRT790M and HER2 kinases.
Moreover, the antitumor activities of these compounds were tested against four cancer cell lines (HepG2,
HCT-116, MCF-7 and A431 ). Compounds 13g, 13h and 13k exhibited the highest activities against the
examined cell lines with IC50 values ranging from 7.592 ± 0.32 to 16.006 ± 0.58 µM comparable to that
of erlotinib (IC50 ranging from 4.99 ± 0.09 to 13.914 ± 0.36 µM). Furthermore, the most potent
antitumor agent (13k) was selected for further studies to determine its effect on the cell cycle progression
and apoptosis in MCF-7 cell line. The results indicated that this compound arrests G2/M phase of the cell
cycle and it is a good apoptotic agent. Finally, molecular docking studies showed a good binding pattern
of the synthesized compounds with the prospective target, EGFRWT and EGFRT790M
.
Key words: Anticancer; Apoptosis; EGFR-TK; EGFRWT; EGFRT790M; HER2; Docking; Thieno[2,3-
d]pyrimidine
1. Introduction
Cancer is a highly aggressive and lethal disease represents one of the greatest medical challenges
in both more and less economic countries. According to the WHO face sheet, published in September
2018 [1], cancer is classified as the second leading cause of death worldwide: it accounted for 9.6
million deaths in 2018. Mortality from cancer are projected to continue rising worldwide, with an
estimated 13 million deaths in 2030. Cancer is caused by gene mutations or interfering with normal cell
differentiation which initiated by drugs, viruses, smoking or diet [2].
Receptor tyrosine kinases (RTKs) are classes of signaling proteins which appear to be targeted
more frequently by oncogenic mutations [3]. Theses enzymes catalyze the transfer of the γ-phosphate
from ATP to tyrosine residues of its intracellular substrate and regulate the majority of cellular pathways
[4], especially those involved in cell growth, proliferation, survival, metabolism, differentiation and
apoptosis. Disruption of cell signaling cascades through kinase alterations (specially mutations, hyper-
2

activation or hyper-production) leads to several diseases includes cancer [5-7], neurological disorders
[8], inflammation [9], diabetes [10], autoimmune and cardiovascular disorders [11].
Epidermal growth factor receptor (EGFR) is a principal subfamily of the protein kinases, also
called ErbB or HER receptors, that consists of four members: EGFR (ErbB1, HER1), ErbB2 (HER2),
ErbB3 (HER3) and ErbB4 (HER4). EGFR is a key mediator playing a very important role in the
regulation of fundamental cellular processes including: proliferation, survival as well as migration [12,
13]. EGFR overexpression is one of the most extensively studied for its role in development of many
human solid tumors as non-small cell lung cancer (NSCLC) [14], hepatocellular carcinoma (HCC) [15]
and breast cancer [16]. Novel molecular strategies designed to target specific molecules affecting
regulatory mechanisms involved in the control of cancer cell proliferation enable to improve cancer
therapy efficiency compared to conventional chemotherapy and/or radiotherapy protocols. The aim of
targeted therapies is inhibition and down-regulation or inactivation of overactive proteins responsible for
triggering of aberrant cellular pathways [17-20].
HER2 is another member of the human epidermal growth factor receptor family which has a
similar structure to EGFR [21]. Overexpression of this oncogene plays an important role in the
progression of certain aggressive types of cancer [22].
Due to the vital role of overactive EGFR and HER2 tyrosine kinases as an important hallmarks of
different types of cancer, such as lung cancer, colorectal cancer, pancreatic cancer, and head and neck
cancer; inhibition of EGFR and HER2 pathway is a well-known target for effective anticancer therapies
[22-27].
First generation of EGFR inhibitors are small molecules tyrosine kinase inhibitors (TKIs) such as
erlotinib [28] 1 and gefitinib [29] 2; they bind to the adenosine triphosphate (ATP)-binding site of the
receptor, inhibiting intracellular tyrosine kinase domain (TKD) of the receptor. Both compounds have
been used in the clinic to some success. However, only stabilizing effects of up to 12 months could be
achieved. Although the overall response to EGFR-TKIs is high for patients with EGFR mutations,
almost all patients subsequently develop acquired resistance to gefitinib and erlotinib within 10-16
months [30]. Due to the sobering performance of first generation EGFR inhibitors in the clinic,
considerable effort has been put in the development of alternative approaches, resulting in second
generation EGFR inhibitors; afatinib [31] 3 and canertinib [32] 4 were shown to effectively overcome
EGFR-TK mutation-related resistance in vivo and xenograft models [33] and subsequently used
successfully in clinical trials [34]. Unfortunately, they failed to meet endpoints against EGFR-mutant
lung adenocarcinoma in clinical trials due to the limited therapeutic window offered by these agents [35,
3

36]. As such, third generation EGFR-TKIs were developed: osimertinib 5 and avitinib 6 are irreversible
EGFR-TKIs with a puckered ring structure that can bind the TKD active site in the presence of mutant
EGFR [37-39]. These agents have been proven to be active in patients [40, 41], and one of them,
osimertinib, is FDA approved in 2015 to treat NSCLC. However, resistance to osimertinib is emerging
[42], suggesting that alternative strategies and/or EGFR-TKIs are needed [43] (Fig. 1).
Figure 1. Basic pharmacophoric features of EGFR-TK inhibitors.
Thieno[2,3-d]pyrimidine nucleus is an important pharmacophore, presented in a number of
anticancer agents [44-59], including EGFR and HER2-TKIs [44-46, 59, 60]. Compound 7 was designed
and synthesized by Chia-Hsien Wu et al, using knowledge-based design strategy for ATP-competitive
inhibitors interacting with the active site of the EGFR-TK. This compound showed low nanomolar
efficacy for inhibiting the targeted enzyme, EGFR-TK [61]. Recently, another group, Milik et al,
reported a new compound, 8, as another example of thieno[2, 3-d]pyrimidines with anti EGFR and
HER2-TK activity, having IC50 value of 13.9 μM against the NCI-H1975 cell line [59] (Fig. 2).
4

Figure 2. Reported thieno[2, 3-d]pyrimidine derivatives as anti EGFR compounds
Based on the previous researches and attractiveness of tyrosine kinases as promising targets for the
design of new cancer agents, it was decided to introduce new thieno[2,3-d]pyrimidine derivatives having
inhibitory activities against wild-type EGFR-TK (EGFRWT). All the compounds were tested against
EGFRWT enzyme and also screened for their anti-proliferative activities against a number of cancer cell
lines. The most active compounds were further tested against the mutated EGFRT790M and HER2 kinase.
Moreover, the most active compound 13k was investigated for its apoptosis induction potential in MCF-
7 cell line. The cell phase which may be arrested by compound 13k was determined by cell cycle assay.
Finally, inhibitor molecular docking study, utilizing the crystal structure of EGFR kinase domain, of the
active compounds was performed to investigate their binding patterns with the potential EGFRWT and
EGFRT790M target.
Rational drug design
EGFR-TK domains consist of two lobes (or subdomains): The N-terminal and the C-terminal
lobes. The lobes are connected by a short polypeptide chain, which is known as the linker, or the hinge
region [62]. The ATP-EGFR binding site is divided into several distinct regions, although all these
together form one space suitable for inhibitor binding; Adenine binding site/linker region, ribose binding
pocket, phosphate binding pocket, back hydrophobic region and front hydrophobic region (Fig. 3) [62,
63].
5

Figure 3. Pharmacophore model of the ATP-binding site of EGFR-TK
The linker region lies in the cleft at the junction of the N- and C-terminal lobes and it is a key
section of the ATP nucleotide binding site – the adenosine moiety of ATP interacts via hydrogen bonds
with the main chain of the linker region, and the rest interacts with glycine-rich, phosphate binding loop.
In principle, many properly substituted (heterocyclic) moieties that can interact with the ATP-
binding site of an enzyme can serve as cores of kinase inhibitor. Among a number of different 5,6-
heterocyclic systems, the thieno[2,3-d]pyrimidine core proved particularly suitable for the development
of kinase inhibitors. Therefore, our goal was to synthesize several thieno[2,3-d]pyrimidine sub-series
having the same essential pharmacophoric features of the reported and clinically used EGFR-TKIs as
erlotinib 1 (Fig. 4). The core of our molecular design rational comprised bio-isosteric modification
strategies of EGFR-TKIs at four different positions (Fig. 5).
The first position was the hetero aromatic ring system, where the thieno[2,3-d]pyrimidine nucleus
was used as a bio-isostere for quinazoline moiety of erlotinib 1. The choice of this moiety was based on
an important bio-isosteric considerations. First of all, the bicyclic structure of thieno[2,3-d]pyrimidine
core is convenient to the large size space of the adenine binding region [64]. Moreover, the heterocyclic
nitrogen atoms serve as hydrogen-bond acceptors conferring excellent EGFR-TK potency [63]. The
second position was the terminal hydrophobic head. Different hydrophobic moieties including
(substituted) phenyl, aromatic, and fused aromatic heterocyclic structures were selected. The third
position was the linker (spacer) region, where the linker length as well as number of its hydrogen
acceptor and/or hydrogen acceptor groups were modified. The different linkers may be NH- as
compounds 13a-i, cyclic structure (e.g. NHPhNH) as compound 13j and NHNH- as compound 13k. The
6

fourth position was the hydrophobic tail. Fused cyclohexyl group was incorporated at position-2,3-d of
thienopyrimidine nucleus to occupy the front hydrophobic region of ATP binding site. All modification
pathways and molecular design rationale were illustrated and summarized in Fig. 5.
Figure 4. The basic structural requirements for erlotinib as reported EGFR-TK inhibitor.
Figure 5. Rational of molecular design of the new proposed EGFR-TK inhibitors.
2. Results and discussion
2.1. Chemistry
According to the rational drug design, a series of thieno[2,3-d]pyrimidine scaffold was synthesized
as outlined in Schemes 1-5. 2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (9), is the key
intermediate for the synthesis of thieno[2,3-d]pyrimidines, which can be synthesized according to the
7

reported methodology [65], by the condensation of commercially available cyclohexanone with
malononitrile and sulphur powder using morpholine as catalyst. Fusion of compound 9 with formic acid
afforded 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-one (10). Chlorination of 10 with POC13
yielded 4-chloro derivative 11 (Scheme 1) which served as a facile intermediate for nucleophilic
substitution reactions with amines, hydrazine hydrate, sodium azide and N-phenylthiourea to afford our
target compounds.
Scheme 1. Synthesis of compound 11
A possible mechanism is speculated and described in Scheme 2. The first step is a Knoevenagel
condensation of methylene-active nitrile with a α-methylene carbonyl component of cyclohexanone to
produce an α-β-unsaturated nitrile intermediate mediated by morpholine, which is then thiolated at the γ-
methylene group with sulfur powder. The sulfurated compound undergoes cyclization to form the
thiophene product, followed by an aromatization rearrangement through proton transfer (P.T).
Cyclization of compound 9 was achieved by fusion with formic acid in the presence of water molecule.
The last rearrangement of the cyclized product regenerates the aromaticity and allows the formation of
the desired product 10.
8

Scheme 2. Proposed reaction mechanism for compound 10 formation
Reaction of compound 11 with appropriate amines namely, 2-aminobenzoic acid, 2-chloroaniline,
4-nitroaniline, 4-methoxyaniline, 4-aminobenzoic acid, 1-(4-aminophenyl)ethan-1-one, 2-amino-5-
iodobenzoic
acid,
4-aminophenol,
2-amino-4,5,6,7-tetra-hydrobenzo[b]thiophene-3-carbonitrile,
benzene-1,2-diamine, and 4-hydrazinyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (12),
afforded the target thieno[2,3-d]pyrimidine derivatives 13a-k respectively in 60-90 % yield, as shown in
Scheme 3. The IR spectra of these compounds demonstrated stretching bands around 3400 cm-1
corresponding to the NH group, and other bands ranging from 1655 to 1693 cm-1 corresponding to C=O
groups in compounds 13a, 13e, 13f and 13g.
The 4-hydrazino derivative 12 was prepared by reaction of compound 11 with 1.1 equivalent of
hydrazine hydrate at room temperature [66]; when half equivalent amount of hydrazine hydrate was
used, pyrimidine 13k product was formed (Scheme 4). The MS spectrum of this compound exhibited
correct molecular ion peak at m/z = 408.
9

Scheme 3. Synthesis of compound 13a-k
Scheme 4. Reaction of 11 with hydrazine hydrate
The 4-chloro derivative 11 was further cyclized into the higher polycyclic compounds, tetrazolo
14 and triazolo 15 derivatives by reaction with sodium azide and thiosemicarbazide respectively in acetic
10

acid [66], while the reaction with N-phenylthiourea yielded the unexpected 4-mercapto derivative 16
[67] instead of N-phenylthiourea derivative 17 (Scheme 5). Proposed mechanism for compound 16
formation is described in Scheme 6.
Scheme 5. Synthesis of compound 14, 15 and 16
Scheme 6. Proposed reaction mechanism for compound 16 formation
2.2. Biological evaluation
2.2.1.EGFRWT,EGFRT790M and HER2 kinase inhibitory assay
Thirteen compounds were tested for their EGFR kinase inhibitory activities using EGFR Kinase
Assay Kit (BPS biosciences). Erlotinib as one of the most potent EGFRWT inhibitors was used as a
positive control. Most of the test compounds showed comparable inhibitory activities to erlotinib (0.387
11

µM) as shown in Table 1. The high reactivity of compound 13g indicating that grafting a bulky iodo
electron withdrawing substituent at the 4-position and carboxylic group at the 2-position of the phenyl
ring may be beneficial for the activity; In addition, when the NH linker is extended by one more NH
group as in compound 13k, the yielded compound exhibited potent activity comparable to erlotinib.
Further testing against the mutated EGFRT790M was done for compounds 13g, 13h, 13i and 13k.
Compound 13k was found to be the most active one against EGFRT790M comparable to erlotinib.
In addition to the inhibitory effect against EGFR kinase, the most active compounds were further
evaluated for their selectivity against HER2 kinase using HER2 Kinase Assay Kit (BPS biosciences).
Fortunately, compound 13k have the strongest HER2 inhibitory effect with IC50 value of 0.415 µM.
Table 1 In vitro enzymatic inhibitory activities of thieno[2,3-d]pyrimidines against EGFRWT, EGFRT790M and HER2
EGFRWT EGFRT790M
IC50 (µM) IC50 (µM) IC50 (µM)
HER2
EGFRWT EGFRT790M
IC50 (µM) IC50 (µM) IC50 (µM)
HER2
Comp.
Comp.
NTa
NTa
NTa
NTa
NTa
NTa
NTa
NTa
NTa
0.608
NTa
NTa
NTa
NTa
NTa
NTa
NTa
NTa
0.711
0.630
0.443
0.956
0.908
NTa
0.858
0.722
NTa
11
12
13h
13i
0.560
0.865
1.440
13a
13j
NTa
0.352
NTa
0.415
NTa
13b
13c
13k
14
0.278
0.459
NTa
NTa
NTa
0.605
0.488
16.352
0.174
0.550
0.475
0.387
13d
13e
13f
13g
15
16
NTa
NTa
Erlotinib
0.241
0.117
a NT: Compounds not tested.
Bold font indicates superior potency than erlotinib
2.2.2.In vitro anti-proliferative activities
The anti-proliferative activities of the synthesized compounds were evaluated against four cancer cell
lines; hepatocellular carcinoma (HepG2), colon carcinoma (HCT-116), human breast adenocarcinoma
(MCF-7) and human epithelial carcinoma (A431) using MTT assay [68]. These cancer cell lines have a
high expression of EGFR [69-72]. The commercially available erlotinib was used in this test as a
reference compound. As shown in Table 2, the obtained results revealed that the examined compounds
showed versatile anti-proliferative activities against the tested cell lines. Compound 13g showed IC50
12

near to the reference compound (erlotinib) in HCT-116 and A431 cells, while compound 13h evidently
reduced the viability of HCT-116 cells with IC50 of 10.14 ± 1.1 µM which was comparable to that of
erlotinib (13.91 ± 0.36 µM). Also, in alignment with the inhibitory effects on HCT-116, compound 13h
exhibited s strong cytotoxic effect against HepG2 with IC50 of 13.02 ± 1.00. Compound 13i have a
strong cytotoxic effect against A431 cells with IC50 of 13.77 ± 0.71. The most potent cytotoxic agent,
compound 13k, exhibit a significant activity against HepG2, MCF-7 and A431 cells with IC50 values of
7.975 ± 0.37, 7.592 ± 0.32 and 9.46 ± 0.44 µM, respectively. However, it showed a moderate activity
against HCT-116. The rest of compounds (11, 13a-f, 13j and 16) didn't show promising cytotoxic
activities as the mentioned compounds.
Table 2. In vitro anti-proliferative activities towards HepG2, HCT-116, MCF-7 and A431 cell lines
IC50 (µM)a
Compound
HepG2
HCT-116
MCF-7
A431
NTb
54.89 ± 3.10
58.05 ± 2.70
67.48 ± 3.20
11
NTb
NTb
NTb
NTb
12
NTb
87.63 ± 4.30
71.82 ± 3.60
81.60 ± 3.90
13a
NTb
36.10 ± 2.20
85.32 ± 4.10
42.52 ± 2.50
67.72 ± 3.70
35.08 ± 1.90
26.446 ± 1.33
13.02 ± 1.00
27.29 ± 1.60
70.92 ± 3.90
7.975 ± 0.37
40.03 ± 2.40
64.06 ± 3.40
26.26 ± 1.70
62.57 ± 3.30
50.73 ± 2.60
16.006 ± 0.58
10.14 ± 1.10
30.18 ± 1.90
90.76 ± 4.30
50.29 ± 2.71
46.45 ± 2.50
77.9 ± 3.60
13b
13c
13d
13e
13f
13g
13h
13i
NTb
NTb
25.77 ± 1.80
75.75 ± 3.40
53.71 ± 3.10
23.928 ±1.44
12.68 ± 1.30
37.08 ± 2.00
95.34 ± 4.60
7.592 ± 0.32
NTb
NTb
16.14 ± 0.62
47.05 ± 1.29
13.77 ± 0.71
NTb
13j
13k
9.46 ± 0.44
NTb
NTb
NTb
NTb
NTb
NTb
NTb
14
15
NTb
20.39 ± 1.50
6.733 ± 0.33
17.72 ± 1.40
13.914 ± 0.36
17.39 ± 1.60
3.107 ± 0.14
NTb
16
Erlotinib
4.99 ± 0.09
13

a IC50 values are the mean ± S.D. of three separate experiments; IC50 (µM): 1- 10 (very strong), 11-20 (strong), 21-50
(moderate), 51-100 (weak), more than 100 (non-cytotoxic)
b NT: Compounds not tested for their anti-proliferative activities.
Bold font indicates a high potency relative to erlotinib
2.2.3.In-vitro DNA-flow cytometric (cell cycle) analysis.
The most active compound 13k was selected for further studies regarding to its effect on cell cycle
progression in the MCF-7 cell line. DMSO was used as a negative control. Cell cycle stages were being
recognized through flow cytometry after propidium iodide (PI) staining followed by RNAse treatment.
The MCF-7 cells were incubated with 1 µM of compound 13k for 24 h. The flow cytometry was
employed to quantify cell populations in different cell cycle phases (pre-G1, G1, S and G2/M phases).
The results indicated that treating MCF-7 cells with compound 13k resulted in an interference with
the normal cell cycle distribution of this cell line. This compound induced a significant increase in the
percentage of cells at phases of pre-G1, which could be indicative of apoptosis, and G2/M by 16.3 and 4.7
folds respectively, comparing to the control. Such increase was accompanied by a significant decrease in
the percentage of cells at the G1 and S-phase of the cell cycle. This result clearly indicated that
compound 13k arrests G2/M phase of cell cycle (Figs. 6, 7 & Table 3).
Figure 6. Cell cycle analysis of MCF-7 cells treated with DMSO.
14

Figure 7. Cell cycle analysis of MCF-7 cells treated with compound 13k at 1 µM concentration
Table 3. Effect of compound 13k and DMSO on cell cycle of MCF-7 cell line.
Sample data
Sample/cell line
13k/MCF-7
Results
Conc
(µM)
% G1
22.67
49.82
% S
% G2/M % Pre-G1
Comment
cell growth arrest
at G2/M phase
1
-
27.51
39.52
49.82
10.66
23.47
1.44
DMSO/MCF-7
2.2.4. Determination and assay of apoptosis using Annexin V-FITC staining.
The mode of cell death induced by compound 13k was further investigated to determine whether
death is due to apoptosis or necrosis. This was elucidated using Annexin V/PI assay. Annexin V
conjugated with FITC is used to stain cells in combination with PI. Cells that stained positive for
Annexin V/PI represents the cells in the late apoptotic stage that have lost membrane integrity [73].
As EGFR-TK inhibitors can induce cancer cell apoptosis [74], the apoptotic nature of compound
13k against MCF-7 cells was evaluated via flow cytometry detection using AnnexinV-FITC and PI
double staining. The representative dot plots of flow cytometric analyses of MCF-7 cells demonstrated
15

four different distributions (Fig. 8): healthy cells (Lower Left; Annexin V and PI negative), cells in early
apoptosis (Lower Right; Annexin V positive and PI negative), cells in late apoptosis (Upper Right;
Annexin V and PI positive) and dead or necrotic cells (Upper Left; Annexin V negative and PI positive).
The results shown in Fig. 8 and Table 4 revealed that application of compound 13k on MCF-7 cells for
48 h with increasing its concentration from 0 μM to 1 μM, increases the early apoptosis ratio (lower right
quadrant of the cytogram) from 0.72% to 7.92%, and increases the late apoptosis ratio (upper right
quadrant of the cytogram) from 0.27% to 12.47 %. This means that treating MCF-7 cells with compound
13k resulted in a significant increase in the percentage of apoptotic cells compared to non-treated
control. Additionally, a slight increase in number of necrotic cells was seen. The total number of early
and late apoptotic cells is higher compared to the number of necrotic cells, indicating that apoptosis is
the main mechanism by which 13k causes cell death.
Figure 8. Flow cytometry analysis of MCF-7 cell line treated with DMSO and compound 13k.
Table 4. Effect of compound 13k and DMSO on the percentage of annexin V-FITC-positive staining in MCF-7 cells.
Sample data
Sample/cell line
13k/MCF-7
Apoptosis (%)
Early
Necrosis (%)
-------
Conc
(µM)
Total
23.47
1.44
Late
12.47
0.27
1
-
7.92
3.08
DMSO/MCF-7
0.72
0.45
16

2.3. Docking studies
Additionally, molecular modeling studies were performed in order to rationalize the anticancer
activities of the proposed compounds. All the synthesized thieno[2,3-d]pyrimidine derivatives were
subjected to docking study together with the internal ligand, erlotinib, as a reference molecule to explore
their calculated binding modes with the EGFR wild type receptor (EGFRWT, PDB: 4HJO)[75]. The ATP
binding pocket of EGFRWT consist of five main parts; sugar pocket, two hydrophobic region, adenine
binding pocket, and phosphate binding region. The sugar pocket contains Cys773 residue, which is
unique to the EGFR-TK and provides both potency and selectivity. The back hydrophobic region
comprises Thr766, Lys721 and Thr830 residues, playing a crucial role in inhibitor selectivity. The front
hydrophobic region is formed by Gly772 and Leu694 residues. The adenine region contains electrostatic
interaction between the amino group of the adenine ring and Met769 residue. The phosphate binding
region offers little opportunity in terms of inhibitor binding affinity due to high solvent exposure [63,
76]. (Fig. 9).
Figure 9. Superposition of erlotinib (grey sticks) in the binding pocket of EGFRWT receptor (PDB ID code: 4HJO). The active pocket was
represented as a yellow surface. The surrounding residues have been depicted as green sticks.
The results of docking studies against the EGFRWT revealed that the synthesized compounds have
similar orientations inside the ATP binding site. The designed compounds gave good binding energies
ranging from -13.78 to -25.27 kcal/mol (Table 5). The binding mode of the co-crystallized ligand,
17

erlotinib, exhibited an energy binding of -25.17 kcal/mol. Quinazoline nucleus was oriented in the
adenine pocket of the receptor, forming a hydrogen bond (3.01 A°) between the N-1 atom of pyrimidine
ring and Met769, while the phenyl ring formed pi-sigma interaction with Lue694. The bis (2-
methoxyethoxy) groups occupied the front hydrophobic region forming hydrophobic interaction with
Gly772, Pro770 and Leu694 residues. Besides, the ethynyl phenyl moiety occupied the back
hydrophobic pocket forming pi-cation interaction with Lys721, and hydrophobic interaction with
Thr830, Lue764, Ala719 and Thr766 residues (Fig. 9). Further docking studies have been done for the
most active compounds 13g-i and 13k against the mutated EGFR (EGFRT790M, PDB: 3W2O); the docked
compounds 13g-i have almost similar binding free energy while the most active one, 13k, has a binding
free energy of -28.56 which is higher than that of erlotinib ligand (Table 5).
Table 5. The docking binding free energies of the synthesized compounds against EGFRWT and EGFRT790M
Binding free energy (kcal/mol)
Compound
EGFRWT
-14.04
-16.30
-18.51
-18.07
-21.96
-21.38
-22.99
-24.84
-19.10
-23.18
-20.38
-25.27
-22.11
-14.64
-14.48
-13.78
-24.87
EGFRT790M
-
11
12
-
-
13a
13b
13c
13d
13e
13f
-
-
-
-
-
-21.78
13g
13h
13i
-21.69
-21.92
-
13j
-28.56
13k
14
-
-
-
15
16
-26.66
Erlotinib
18

The binding mode of compound 13j was higher than that of erlotinib, with affinity value of -25.27
kcal/mol. The thieno[2,3-d]pyrimidine moiety occupied the adenine binding site forming a hydrogen
bond with Lys704 with a distance of 3.17 A°. The cyclohexyl moiety occupied the front hydrophobic
pocket forming hydrophobic interactions with Phe771 and Pro770 residues. In addition, the central
phenyl moiety of NH-Ph-NH linker forms hydrophobic interaction with Leu694, gly772 and Met769.
Moreover, this linker length provides the other thieno[2,3-d]pyrimidine moiety higher flexibility to get
closer to Val702, Arg817, Leu820 and Cys773 forming hydrophobic interaction in the back hydrophobic
region (Fig. 10).
The binding mode of compound 13f was as like as that of erlotinib, with affinity value of −24.84
kcal/mol. General hydrophobic interactions seen between the adenine ring, cyclohexyl and phenyl
moieties with Val702, Leu694, cys773, Thr830, Lys721 and Thr766 of the adenine and two hydrophobic
pockets (Fig. 11).
Similar results were obtained for synthetic inhibitor 13h. This compound showed an energy
binding of −23.18 kcal/mol. An electrostatic interaction involving H-bond noted between the adenine N
atom and water molecule binding to Thr766 and Thr830. The adenine and two hydrophobic binding
pockets also showed varying interactions between the adenine ring with Ala 719 / Leu820, cyclohexyl
ring with Val702, and 4-OH phenyl with Leu719 / Thr 830 (Fig. 12).
Compound 13k has a binding energy nearly similar to that of erlotinib. Pyrimidine nucleus
positioned closely towards Met-769 forming two hydrogen bonds with the N3 and the linker NH with a
distance of 2.95 and 2.92 A°, respectively. On the other side of 13k, a third hydrogen bond formed
between the N1 and Lys704 with a shorter distance of 2.78 A° (Fig. 13). The binding mode of 13k with
the mutated EGFRT790M, showed a hydrogen bond formed with Met793 with a distance of 3.21 A˚
(Fig.14).
In general, most of the synthetic inhibitors have moderate to strong potency relative to the internal
ligand, erlotinib.
19

Figure 10. Calculated binding mode of compound 13j (grey sticks) within the binding pocket of EGFRwt receptor. The active pocket has
been represented as yellow surface. Important binding sites residues have been depicted as green sticks; other residues have been hidden for
sake of clarity.
Figure 11. Calculated binding mode of compound 13f (grey sticks) within the binding pocket of EGFRwt receptor. The active pocket has
been represented as yellow surface. Important binding sites residues have been depicted as green sticks.
20

Figure 12. Calculated binding mode of compound 13h (grey sticks) within the binding pocket of EGFRwt receptor. The active pocket has
been represented as yellow surface. Important binding sites residues have been depicted as green sticks.
Figure 13. Calculated binding mode of compound 13k (grey sticks) within the binding pocket of EGFRwt receptor. The active pocket has
been represented as yellow surface. Important binding sites residues have been depicted as green sticks.
21

Figure 14. Calculated binding mode of compound 13k (grey sticks) within the binding pocket of EGFRT790M receptor. The active pocket has
been represented as yellow surface. Important binding sites residues have been depicted as green sticks.
3. Structure-activity relationships (SAR)
Observing the results of biological tests in Table 2, valuable data about the structure-activity
relationships of thieno[2,3-d]pyrimidine derivatives as potential EGFR-TK inhibitors could be deduced
in comparison to the tested standard inhibitor.
Initially, the effect of different linkers on the activity against EGFRWT was investigated. It was
found that replacement of the NH linker with cyclic (NHPhNH-) linker in compound 13j provide very
weak inhibition activity (IC50 range 0. 2 .34 µM), while compound 13k with one more NH group
in the linker was found to be strong cytotoxic inhibitor (IC50 range = 7.59 9.46 µM) in HepG2, MCF-7
and A431 cell lines. In absence of the linker and the hydrophobic head and replacement them by SH
group in compound 16, the growth inhibition of the cancer cell lines showed moderate activity (IC50
range = 1 .3 20.39 µM).
The impact of the substitution on the phenyl group of terminal hydrophobic head was explored.
The high activity of compound 13h (IC50 range 10.14 13.02 µM) may be attributed to the presence of
4-OH substituent as an electron donating group on the phenyl ring, however, replacement of 4-OH in
compound 13h by bulkier electron donating group (4-OCH3) decreased the growth inhibition, like in the
case of compound 13d. The presence of electron withdrawing group such as 2-COOH in compounds
13a,g, 2-Cl in 13b and 4-NO2 in 13c decreased the growth inhibition activity.
22

Finally, more bulky hydrophobic head and the longer NHNH linker in 13k dimer are capable of
extending into the back pocket of the receptor forming three H-bonds, resulting in an effective HepG2,
MCF-7 and A431 cell line growth inhibition.
4. Conclusion
In summary, sixteen thieno[2,3-d]pyrimidine derivatives (11, 12, 13a-k, 14, 15 and 16) were
designed, synthesized and evaluated for their inhibitory activities against EGFRWT ,EGFRT790M and
HER2 kinases. Compounds 13g and 13k showed excellent inhibitory activities. The tested compounds
were further evaluated in vitro for their inhibitory action against four cancer cell lines (HepG2, HCT-
116, MCF-7 and A431) and compared with erlotinib as a positive control. In this study, three of the
synthesized compounds (13g, 13h and 13k) showed growth cell line inhibitory effect similar to standard
control with IC50 values ranging from 7.592 ± 0.32 to 16.006 ± 0.58 µM. The SAR studies pointed that,
the less bulky electron donating group substitution on the terminal hydrophobic head seems to be
preferred. Also, it was found that a longer linker and bulky hydrophobic head in 13k is advantageous.
Further, compound 13k was tested for its effect on cell cycle progression and induction of apoptosis in
the MCF-7 cell line. It was found that it has apoptotic effect and can arrest G2/M phase of cell cycle.
Also, molecular docking studies was carried out to recognize the binding pattern of the synthesized
compounds against EGFRWT and EGFRT790M receptor. Most of the synthesized compounds showed good
binding modes with higher binding energies. Extension of this work could provide more information
towards the optimization of other thieno-pyrmidine derivatives that can be considered as interesting
candidates for further development of more potent anticancer agents. The results of this extended work
will be reported in due course.
5. Experimental
5.1. Chemistry
5.1.1.General
All Melting points were measured on a Gallen-kamp melting point apparatus and were
uncorrected. The IR spectra were recorded on Perkin Elmer 1600 FT IR spectrophotometer using KBr
1
discs (λ max in cm−1). H NMR spectra were measured on Bruker spectrometer, TMS was used as
internal standard and DMSO-d6 as solvent, otherwise stated. Chemical shifts (δ) are reported in ppm
downfield from TMS and coupling constant (J) values were given in Hertz (Hz). Signal multiplicities
were represented by s (singlet), d (doublet), t (triplet), q (quadruplet), and m (multiplet). Mass spectra
23

were recorded on a Shimadzu GC-MS-QP 1000X spectrometer operating at 70 e.V. All the new
compounds were analyzed for C, H and N and agreed with the proposed structures within ± 0.4% of the
theoretical values by the automated CHN analyzer. Reactions progression was monitored by TLC Merck
Kieselgel 60 F254 aluminum packed plates. Solvents were purchased from ADWIC Company and used
without further purification, hydrazine hydrate (Merck grade), and the other chemicals from Sigma-
Aldrich.
5.1.2. 4-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine 11
Step 1: Equimolar amount (0.01 mol) of malononitrile, sulphur and cyclohexanone were taken in a
round bottom flask containing 20 ml of ethanol. The mixture was stirred for 5 min then morpholine
(0.012 mol) was added drop wise to the reaction mixture at 60 °C with constant stirring over 30 min.
Later, the reaction mixture was allowed to stir for 5 h at room temperature and left in refrigerator
overnight. The crystals thus formed were collected by filtration and washed with cold ethanol. Further
purification by recrystallization from ethanol afford compound 9 in 56 % yield, m.p. 145 ºC.
Step 2: To a mixture of 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophen-carbonitrile (9, 0.9 g) and
formic acid (10 ml) was added 0.1 ml HCl. After fusion for 3 h, the reaction mixture was cooled and
water (20 ml) was added. The precipitated was filtered and washed thoroughly with water and hexanes to
give 5,6,7,8 tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (10, 0.8 g, 77%); m.p. 250 ºC; IR:
1
3520, 2934, 2874, 1660, 1543. H NMR (400 MHz, DMSO-d6) δ 12.27 (br. s., 1H), 7.94 (s, 1H), 2.89
(br. s., 2H), 2.76 (br. s., 2H), 1.76 (br. s., 4H).
Step 3: To a mixture of compound 10 (0.8 g) and POCl3 (10 ml), triethyl amine (1 ml) was added
dropwise over 30 min. Then, the reaction mixture was heated at 65°C for 4 h. Water was then added
followed by sodium bicarbonate. The resulting mixture was extracted with ethyl acetate. The organic
layer was concentrated and the crude compound was crystallized using a mixture of hexanes: ethyl
acetate (20:1), to afford 4-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (11, 0.52 g, 60%);
1
m.p. 118 ºC; IR: 2937, 2861, 1660, 1128; H NMR (400 MHz, DMSO-d6) δ 8.72 (s, 1H), 2.96 (br. s.,
2H), 2.82 (br. s., 2H), 1.80 (br. s., 4H).
5.1.3.4-hydrazinyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine 12
A solution of absolute ethanol (30 ml) containing pyrimidine 11 (0.5 g, 2.23 mmol) and hydrazine
hydrate (0.12 ml, 2.25 mmol) was stirred overnight. The produced solid was filtered and recrystallized
from ethanol to give the target compound 12 (95%).
24

White crystals (95%); m.p. 184 186 (Lit. [66] m.p. 184 oC); IR: 3307, 2939, 2833, 1560; 1H NMR
13
(400 MHz, CDCl3) δ 8.5 (s, 1H), 6.5 (s, 1H), 4.1 (br. s., 2H), 2.8 (t, 4H), 1.98 (m, 4H); C-NMR (101
MHz, CDCl3) δ 16 .1, 1 8. , 1 2.6, 134.1, 12 .0, 11 .1, 26.3, 2 .4, 22. , 22.4.
5.1.4.General procedure for preparation of compounds 13a-k
A mixture of 4-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]-pyrimidine (11, 0.5 g, 2.23
mmol) and appropriate amine (2.23 mmol) was heated under reflux in absolute ethanol for 4 h. After
completion, the solid product was filtered off and crystallized from the appropriate solvent to give 13a-k
respectively.
5.1.4.1. 2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)benzoic acid 13a
Synthesized according to general procedure 5.1.4 using 2-aminobenzoic acid. The residue was
crystallized from Ethanol/DMF to give 13a (80%) as buff crystals; m.p. 285-287 °C; IR: 3448, 2928,
1
1689, 1596, 752; H NMR (400 MHz, DMSO-d6)  10.98 (s, 1H, OH, exchanged with D2O), 8.73 (d, J =
9.67 Hz, 1H), 8.49 (s, 1H), 8.01 (dd, J = 1.76, 7.91 Hz, 1H), 7.61 (t, J = 7.03 Hz, 1H), 7.10 - 7.15 (m,
1H), 3.17 (br. s., 2H), 2.85 (br. s., 2H), 1.87 (br. s., 4H); ESI-MS m/z: 297 (M+ -H,-HCN; 7%), 292
(100%), 280 (72%); Anal. calcd. for C17H15N3O2S: C, 62.75; H, 4.65; N, 12.91 S, 9.85. Found: C, 62.77;
H, 4.63; N, 12.87; S, 9.81.
5.1.4.2. N-(2-chlorophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine 13b
Synthesized according to general procedure 5.1.4 using 2-chloroaniline. The residue was
crystallized from Ethanol/DMF to give 13b (60%) as white crystals; m.p. 167-170 °C; IR: 3417, 2932,
2855, 1601, 742; 1H NMR (400 MHz, DMSO-d6) δ 8.34 (s, 1H), 8.11 (br. s., 1H), 7.52 (br. s., 1H), 7.36
(br. s., 1H), 7.19 (br. s., 1H), 3.11 (s, 2H), 2.82 (s, 2H), 1.83 (m, 4H); Anal. calcd. for C16H14ClN3S: C,
60.85; H, 4.47; N, 13.31; S, 10.15. Found: C, 60.90; H, 4.53; N, 13.34; S, 10.19.
5.1.4.3. N-(4-nitrophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine 13c
Synthesized according to general procedure 5.1.4 using 4-nitrobenzenamine. The residue was
crystallized from Ethanol/DMF to give 13c (77%) as yellow crystals; m.p. 202-204 ºC (Lit. [77] m.p. 200
oC); IR: 3435, 3111, 2924, 2862, 853; 1H NMR (400 MHz, DMSO-d6)  8.98 (br. s., 1H), 8.56 (s, 1H),
8.16 - 8.21 (m, J = 9.23 Hz, 2H), 7.88 - 7.93 (m, J = 9.23 Hz, 2H), 3.13 (br. s., 2H), 2.82 (br. s., 2H),
1.83 (br. s., 4H); Anal. calcd. for C16H14N4O2S: C, 58.88; H, 4.32; N, 17.17; S, 9.82. Found: C, 58.84;
H, 4.35; N, 17.22; S, 9.85.
5.1.4.4. N-(4-methoxyphenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine 13d
25

Synthesized according to general procedure 5.1.4 using 4-methoxyaniline. The residue was
crystallized from Ethanol to give 13d (85%) as brown crystals; m.p. 235-237 °C; IR: 3394, 2925, 2858,
1616, 1582, 1247, 832 cm-1; 1H NMR (400 MHz, DMSO-d6)  8.70 (br. s., 1H), 8.45 (s, 1H), 7.45 - 7.50
(m, J = 8.79 Hz, 2H), 6.95 - 7.01 (m, J = 8.79 Hz, 2H), 3.78 (s, 3H), 3.13 (br. s., 2H), 2.84 (br. s., 2H),
13
1.85 (br. s., 4H); C NMR (101 MHz, DMSO-d6)  22.2, 22.4, 25.4, 25.7, 55.7, 114.3, 116.8, 126.2,
127.5, 131.1, 134.1, 150.1, 155.4, 157.2, 161.9. Anal. calcd. for C17H17N3OS: C, 65.57; H, 5.50; N,
13.49; S, 10.30. Found: C, 65.59; H, 5.45; N, 13.41; S, 10.1.
5.1.4.5. 4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)benzoic acid 13e
Synthesized according to general procedure 5.1.4 using 4-aminobenzoic acid. The residue was
o
crystallized from Ethanol/DMF to give 13e (88%) as yellow crystals; m.p. 280-282 C; IR: 3414, 3053,
2930, 1803, 1655; 1H NMR (400 MHz, DMSO-d6)  8.58 (s, 1H), 8.52 (s, 1H), 7.90 - 7.94 (m, J = 8.79
Hz, 2H), 7.79 - 7.82 (m, J = 8.79 Hz, 2H), 3.15 (br. s., 2H), 2.85 (br. s., 2H), 1.85 (br. s., 4H); ESI-MS
m/z: 325 (M+, 98%), 53.56 (100%); Anal. calcd. for C17H15N3O2S: C, 62.75; H, 4.65; N 12.9; S, 9.85.
Found: C, 62.72; H, 4.70; N; 12.95; S, 9.82
5.1.4.6. 1-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)ethan-1-one 13f
Synthesized according to general procedure 5.1.4 using 4-aminoacetophenone. The residue was
crystallized from Ethanol to give 13f (67%) as brown crystals; m.p. 181-183 °C; IR: 3457, 3051, 2925,
2859, 1666, 1600, 811 cm-1; 1H NMR (400 MHz, DMSO-d6)  8.49 (s, 1H), 7.92 - 7.96 (m, J = 9.23 Hz,
2H), 7.80 - 7.84 (m, J = 8.79 Hz, 2H), 3.14 (br. s., 2H), 2.84 (br. s., 2H), 2.54 (s, 3H), 1.85 (br. s., 4H);
ESI-MS m/z: 323 (M+, 100%), 308 (23%), 80 (27%), 69 (88%); Anal. calcd. for C18H17N3OS: C, 66.85;
H, 5.30; N, 12.99; S, 9.91. Found: C, 66.86; H, 5.34; N, 12.96; S, 9.87.
5.1.4.7. 5-iodo-2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)benzoic acid 13g
Synthesized according to general procedure 5.1.4 using 2-amino-5-iodobenzoic acid. The residue
was crystallized from DMF to give 13g (80%) as buff crystals; m.p. 250-252 °C; IR: 3211, 3155, 3075,
2925, 2853, 1693, 1587, 579 ; 1H NMR (400 MHz, DMSO-d6)  10.92 (s, 1H), 8.59 (d, J = 8.79 Hz, 1H),
8.49 (s, 1H), 8.22 (d, J = 2.20 Hz, 1H), 7.89 (dd, J = 2.20, 9.23 Hz, 1H), 3.12 (br. s., 2H), 2.84 (br. s.,
2H), 1.85 (br. s., 4H); ESI-MS m/z: 451 (M+, 10%), 433 (100%); Anal. calcd. for C17H14IN3O2S: C,
45.24; H, 3.13; N, 9.31; S, 7.11. Found: C, 45.23; H, 3.15; N, 9.33; S, 7.13.
5.1.4.8. 4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)phenol 13h
Synthesized according to general procedure 5.1.4 using 4-aminophenol. The residue was
crystallized from Ethanol/DMF to give 13h (80%) as brown crystals; m.p. 230-333 °C; IR: 3409, 3201,
26

2948, 2853, 1619, 1585. 1220 cm-1; 1H NMR (400 MHz, DMSO-d6)  8.88 (br. s., 1H), 8.46 (s, 1H), 7.28 -
7.36 (m, J = 8.79 Hz, 2H), 6.79 - 6.88 (m, J = 8.79 Hz, 2H), 3.11 (br. s., 2H), 2.84 (br. s., 2H), 1.84 (br.
s., 4H); 13C NMR (101 MHz, DMSO-d6)  162.2, 155.0, 150.0, 148.0, 133.3, 129.1, 127.1, 125.9, 116.2,
115.2, 25.4, 25.0, 22.0, 21.9; Anal. calcd. for C16H15N3OS: C, 64.62; H, 5.08; N, 14.13; S, 10.87. Found:
C, 64.66; H, 5.10; N, 14.15; S, 10.83.
5.1.4.9. 2-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)-4,5,6,7-
tetrahydrobenzo[b]thiophene-3-carbonitrile 13i
Synthesized
according
to
general
procedure
5.1.4
using
2-amino-4,5,6,7-
tetrahydrobenzo[b]thiophene-3-carbonitrile. The residue was crystallized from Ethanol/DMF to give 13i
1
(90%) as yellow crystals; m.p. 220-222 ºC; IR: 3435, 2928, 2850, 2233, 1628, 1586, 1502; H NMR
(400 MHz, DMSO-d6)  9.26 (s, 1H), 2.99 (br. s., 2H), 2.93 (br. s., 2H), 2.90 (br. s., 2H), 2.78 (br. s.,
2H), 1.87 (br. s., 4H), 1.79 (br. s., 4H); ESI-MS m/z: 367 (M+1, 6.9%), 72 (81%), 44 (100%); Anal.
calcd. for C19H18N4S2: C, 62.27; H, 4.95; N, 15.29; S, 17.50. Found: C, 62.30; H, 5.99; N, 15.30; S,
17.42.
5.1.4.10.N1,N2-bis(5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)benzene-1,2-diamine 13j
Synthesized according to general procedure 5.1.4 using 2-benzene-1,2-diamine. The residue was
washed with hot DMSO to give 13j (85%) as white crystal; m.p. 269-271 °C; IR: 3370, 3318, 3122,
2928, 2908, 1648, 1570; 1H NMR (400 MHz, DMSO-d6)  8.17 (s, 2H), 6.77 (br. s., 4H), 2.90 (br. s.,
4H), 2.74 (br. s., 4H), 1.80 (br. s., 8H); ESI-MS m/z: 484 (M+, 5.68%), 323 (100%), 279 (54.0%); Anal.
calcd. for C26H24N6S2: C, 64.44; H, 4.99; N, 17.34; S, 13.23. Found: C, 64.45; H, 5.08; N, 17.37; S,
13.22.
5.1.4.11.1,2-bis(5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)hydrazine 13k
Synthesized
according
to
general
procedure
5.1.4
using
4-hydrazinyl-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (12). The residue was crystallized from Ethanol/DMF to
give 13k (90%) as grey powder; m.p. > 300 ºC, IR: 3238, 2929, 2835, 1621, 1582; ESI-MS m/z: 409
(M+1, 2.03%), 408 (M+, 1.7%), 303 (33%), 264 (67%), 77 (100%). Anal. calcd. for C20H20N6S2: C,
58.80; H, 4.93; N, 20.57. Found: C, 58.85; H, 4.97; N, 20.60.
Alternative method for preparation of 13k: A mixture of compound 11 (0.5 g, 2.23 mmol) and half
equivalent of hydrazine hydrate (0.060 ml, 1.30 mmol) was refluxed for 4 h in absolute ethanol (30 ml).
The formed solid product was filtered off and washed with hot DMSO to give 13k.
5.1.5. General procedure for preparation of compounds 14-16
27

A mixture of 4-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]-pyrimidine 11(0.5 g, 2.23 mmol)
and (2.3 mmol) each of sodium azide (0.15 g), thiosemicarbazide hydrochloride (0.28 g) or N-
phenylthiourea (0.34gm) was refluxed for 4 h in glacial acetic acid (15 ml ). After cooling, the mixture
was poured over ice/water mixture. The formed solid was filtered, dried and crystallized from
(Ethanol/DMF) to give the target compounds 14, 15 and 16, respectively.
5.1.5.1. 8,9,10,11-tetrahydrobenzo[4,5]thieno[3,2-e]tetrazolo[1,5-c]pyrimidine 14
Yield: 70%; creamy crystals; m.p. 130 ºC (Lit. [66] m.p. 132 ºC). 1H NMR (400 MHz, CDCl3)  9.42 (s,
1H), 3.12 (br. s., 2H), 3.25 (br. s., 2H), 2.09 (br. s., 4H).
5.1.5.2. 8,9,10,11-tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine-3(2H)-thione 15
1
Yield: 60%; brown crystals; m.p. 272 ºC (Lit. [66] m.p. 275
277 ºC); H NMR (400 MHz,
DMSO-d6)  9.42 (s, 1H), 2.99 (br. s., 1H), 2.91 (br. s., 2H), 2.79 (br. s., 1H), 1.87 (br. s., 2H), 1.80 (br.
s., 2H).
5.1.5.3. 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine-4-thiol 16
Yield: 65%; white crystals; m.p. 243 ºC (Lit. [66] m.p. 241 243 ºC). 1H NMR (400 MHz, DMSO-d6) 
13.68 (br. s., 1H), 8.08 (d, J = 3.52 Hz, 1H), 3.17 (br. s., 2H), 2.76 (br. s., 2H), 1.77 (d, J = 5.71 Hz, 4H).
5.2. Biological evaluation
5.2.1.EGFRWT and HER2 kinase inhibitory assay
The in vitro inhibitory activities of the synthesized compounds against EGFRWT and HER2 were
carried out using EGFR and HER2 Kinase Assay Kit (BPS biosciences). At first, EGFRWT/HER2 and
their substrates were incubated with the synthesized compounds in enzymatic buffer for 40 min at 30 ºC
in order to start the enzymatic reaction. The reaction was stopped by addition of detection reagent
(Kinase-Glo Max reagent), followed by incubation at room temperature for 15 min. Then the IC50 values
were determined by ROBONIK P2000 ELISA reader. All samples and controls were tested in duplicate.
5.2.2.EGFRT790M kinase inhibitory assay
The in vitro inhibitory activities of the synthesized compounds against EGFRT790M were carried out
using EGFRT790M/ADP-GloTM Kinase Assay system (Promega). At first, EGFRT790M and their substrates
were incubated with the synthesized compounds in enzymatic buffer for 60 min at room temperature in
order to start the enzymatic reaction. The reaction was stopped by addition of detection reagent (ADP-
GloTM reagent), followed by incubation at room temperature for 40 min. Then, kinase detection reagent
was added, and the mixture was incubated for further 30 min. The IC50 values were determined after
luminescence record. All samples and controls were tested in duplicate.
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5.2.3.In vitro anti-proliferative activities
Anti-proliferative activities of the synthesized compounds were carried out based on MTT
assay[68]. The kit was used according to manufacturer’s protocol. Briefly, HepG2, HCT-116, MCF-7
and A431 cell lines were seeded at a density of 6000 cells/well in quadruplicates wells of a 96-well
tissue culture plates with 50 µl of complete media. Cells were allowed to attach and grow overnight at
37°C in a 5% CO2 humidified atmosphere. Then, for each well, the growth medium was exchanged with
0.1 ml of fresh medium containing graded concentrations of the test compounds to be or equal DMSO
and incubated for two days. Then 10 μl MTT solution ( μg/ml) was added to each well, and the cells
were incubated for additional 4 h. The crystals of MTT-formazan were dissolved in 100 μl of DMSO;
the absorbance of each well was measured at 490 nm using an automatic ELISA reader system (TECAN,
CHE). The IC50 values were calculated using the nonlinear regression fitting models (Graph Pad, Prism
Version 5). The means of three independent experiments gave the reported results. Statistical differences
were analyzed according to one-way ANOVA test wherein the differences were considered to be
significant at p < 0.05.
5.2.4.In-vitro DNA-Flow cytometric (cell cycle) analysis
To determine the distribution of cell lines in each phase of cell cycle, the PI was used to stain the
DNA content of each cell line. At a density of 1×106
3×106 cells/dish, MCF-7 cells were seeded in 30
mm tissue culture plates in 5 ml of complete medium. Cells were incubated and allowed to adhere in
CO2 atmosphere. After 24 h adherence, cells were incubated with compound 13k for 24 h. Then, the cell
pellets were collected by trypsinization and washed twice with PBS washing buffer and fixed with 70%
ice cold ethanol for a minimum of 24 h at -20 °C. The cells were stained with PI and RNase Staining
Solution according to the manufacturer's instructions. Cell-cycle distribution was evaluated using a BD
FACSCalibur flow cytometer. Data were collected from three individual experiments.
5.2.5.Annexin V-FITC apoptosis assay
Annexin V-FITC apoptosis detection kit (BD biosciences) was used to quantify the percentage of
cells undergoing apoptosis and to determine the mode of cell death whether by apoptosis or necrosis in
the presence or absence of the active compound 13k. The experiment was carried out according to the
manufacturer’s protocol. Briefly, cells were seeded (1×106 3×106) per dish and allowed to adhere
overnight in CO2 incubator. Following 24 h incubation, Compound 13k was added, and plates were
incubated for another 24 h in CO2 atmosphere. Both adherent and nonadherent cells were trypsinized,
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