Design and synthesis of thienopyrimidine urea derivatives with potential cytotoxic and pro-apoptotic activity against breast cancer cell line MCF-7

Article abstract of DOI:10.1016/j.ejmech.2017.10.075

A series of novel tetrahydrobenzothieno[2,3-d]pyrimidine urea derivatives was synthesized according to fragment-based design strategy. They were evaluated for their anticancer activity against MCF-7 cell line. Three compounds 9c, 9d and 11b showed 1.5–1.03 folds more potent anticancer activity than doxorubicin. In this study, a promising multi-sited enzyme small molecule inhibitor 9c, which showed the most potent anti-proliferative activity, was identified. The anti-proliferative activity of this compound appears to correlate well with its ability to inhibit topoisomerase II (IC₅₀ = 9.29 μM). Moreover, compound 9c showed excellent VEGFR-2 inhibitory activity, at the sub-micromolar level with IC₅₀ value 0.2 μM, which is 2.1 folds more potent than sorafenib. Moreover, activation of damage response pathway of the DNA leads to cell cycle arrest at G2/M phase, accumulation of cells in pre-G1 phase and annexin-V and propidium iodide staining, indicating that cell death proceeds through an apoptotic mechanism. Compound 9c showed potent pro-apoptotic effect through induction of the intrinsic mitochondrial pathway of apoptosis. This mechanistic pathway was confirmed by a significant increase in the expression of the tumor suppressor gene p53, elevation in Bax/BCL-2 ratio and a significant increase in the level of active caspase-3. Quantitative structure-activity relationship (QSAR) studies delivered equations of five 3D descriptors with R² = 0.814. This QSAR model provides an effective technique for understanding the observed antitumor properties and thus could be adopted for developing effective lead structures.

Full text of DOI:10.1016/j.ejmech.2017.10.075

Accepted Manuscript
Design and synthesis of thienopyrimidine urea derivatives with potential cytotoxic and
pro-apoptotic activity against breast cancer cell line MCF-7
Eman F. Abdelhaleem, Mohammed K. Abdelhameid, Asmaa E. Kassab, Manal M.
Kandeel
PII:
S0223-5234(17)30877-2
10.1016/j.ejmech.2017.10.075
EJMECH 9867
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 July 2017
Revised Date: 28 September 2017
Accepted Date: 28 October 2017
Please cite this article as: E.F. Abdelhaleem, M.K. Abdelhameid, A.E. Kassab, M.M. Kandeel, Design
and synthesis of thienopyrimidine urea derivatives with potential cytotoxic and pro-apoptotic activity
against breast cancer cell line MCF-7, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.10.075.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design and Synthesis of Thienopyrimidine Urea Derivatives
with Potential Cytotoxic and Pro-apoptotic Activity against
Breast Cancer Cell Line MCF-7
a
a
Eman F. Abdelhaleem , Mohammed K. Abdelhameid , Asmaa E. Kassab
a
b
,
Manal M. Kandeel .
a
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy,
Cairo University, Cairo 11562, Egypt.
b
Pharmaceutical Chemistry Department, Faculty of Pharmaceutical
Sciences and Pharmaceutical Industries, Future University, Cairo 12311,
Egypt.
Address: 33 Kasr El-Aini Street, Cairo, Egypt.
1

ACCEPTED MANUSCRIPT
Design and Synthesis of Thienopyrimidine Urea Derivatives
with Potential Cytotoxic and Pro-apoptotic Activity against
Breast Cancer Cell Line MCF-7
Correspondence to: Asmaa E. Kassab, Pharmaceutical Organic
Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo
1
1562, Egypt.
Tel: 002023639307.
Fax: 002023635140.
E-mail: asmaa.kassab@pharma.cu.edu.eg
2

ACCEPTED MANUSCRIPT
Abstract
A series of novel tetrahydrobenzothieno[2,3-d]pyrimidine urea
derivatives was synthesized according to fragment-based design strategy.
They were evaluated for their anticancer activity against MCF-7 cell line.
Three compounds 9c, 9d and 11b showed 1.5-1.03 folds more potent
anticancer activity than doxorubicin. In this study, a promising multi-
sited enzyme small molecule inhibitor 9c, which showed the most potent
anti-proliferative activity, was identified. The anti-proliferative activity of
this compound appears to correlate well with its ability to inhibit
topoisomerase II (IC = 9.29 µM). Moreover, compound 9c showed
5
0
excellent VEGFR-2 inhibitory activity, at the sub-micromolar level with
IC value 0.2 µM, which is 2.1 folds more potent than sorafenib.
5
0
Moreover, activation of damage response pathway of the DNA leads to
cell cycle arrest at G2/M phase, accumulation of cells in pre-G1 phase
and annexin-V and propidium iodide staining, indicating that cell death
proceeds through an apoptotic mechanism. Compound 9c showed potent
pro-apoptotic effect through induction of the intrinsic mitochondrial
pathway of apoptosis. This mechanistic pathway was confirmed by a
significant increase in the expression of the tumor suppressor gene p53,
elevation in Bax/ BCL-2 ratio and a significant increase in the level of
active caspase-3. Quantitative structure-activity relationship (QSAR)
2
studies delivered equations of five 3D descriptors with R = 0.814. This
QSAR model provides an effective technique for understanding the
observed antitumor properties and thus could be adopted for developing
effective lead structures.
3

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Key words: Thieno[2,3-d]pyrimidines; Aryl urea; Fragment-based
Design; Synthesis; Anticancer activity; Topoisomerase II; VEGFR-2;
Cell cycle arrest profile; Apoptosis; QSAR.
1
. Introduction
Cancer is a major health problem worldwide. Among the various
types of malignant tumors, breast cancer is the second leading cause
of death in women [1]. In advanced solid tumor including breast cancer,
tumor growth and metastasis has been linked to overexpression of DNA
topoisomerase (topo) enzymes and pathological angiogenesis via vascular
endothelial growth factor (VEGFR) pathway [2]. Topoisomerases are a
family of enzymes that control DNA synthesis, modulators of topo are
now widely used as cytotoxic agents [3], this class of drugs acts either by
topo poisoning via interchelation with DNA as doxorubicin and
amsacrine (figure 1) or acts as catalytic inhibitors as TSC24 (figure 1)
[4]. The common structural basis for the action of these drugs is linear or
curved polycyclic core bearing a flexible side chain that anchors DNA by
hydrogen bond formation [5]. As a catalytic topo inhibitor, TSC24 acts
by blocking the ATP-binding site of the enzyme leading to inactivation of
enzyme [6]. Topo modulators are efficient; however, they produce
detrimental secondary effects [5]. Therefore, it would be useful to design
newer products, such as aroylthiourea derivative I (figure 1), which will
be more potent and better tolerated, being able to interfere with both topo
and tyrosine kinases [7, 8]. This dual activity increases cytotoxic
properties of the compound and decreases its side effects. Activation of
the DNA damage response pathway leads, in most cases, to drug-induced
apoptosis and/or to cell cycle arrest in G1, S, and G2 phases [9]. Vascular
Endothelial Growth Factor Receptor-2 (VEGFR-2), also known as Kinase
4

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Insert Domain Receptor (KDR), is a Receptor Tyrosine Kinase and is one
of the major regulators of both physiologic and pathologic angiogenesis.
Therapies based on targeted antiangiogenic agents, such as sorafenib
(
figure 2), are supposed to be less toxic than conventional chemotherapy
10] and, when used in combination with doxorubicin, increases potency
[
of chemotherapy [11]. Sorafenib is structurally a member of diaryl urea
family that act as (Receptor Tyrosine Kinase) RTK inhibitors, especially
as VEGFR-2 inhibitors, and induces apoptosis of cancer cells [12]. Other
members of this family are, a p38 inhibitor (doramapimod), compounds
II and III are VEFGR-2 inhibitors (figure 2). Compound II is with
substituted thienopyrimidine heterocyclic core, while compound III is a
quinoline derivative. The main structural features of this urea family are
head group formed from orthogonal polycyclic ring that bind to adenine
region of ATP and phenyl urea tail that bind to allosteric site of kinase
enzyme [13]. The therapeutic chemotherapy protocol must be balanced in
terms of therapeutic effectiveness and minimization of treatment-
associated side effects. Multi-sited enzyme small molecule inhibitors are
now one of the potential fields in cancer drug discovery to reach the same
therapeutic goal. One of the used approaches to obtain such multi-sited
enzyme inhibitors is fragment-based lead generation. Fragments are
scaffolds that usually form part of drugs that are responsible of biological
activities. These fragments are then combined to generate lead
compounds [14]. Based on a structural analysis of the topo and VEGFR-2
inhibitors, we have constructed a ‘hybrid-design’ model (figure 3) with
topo and VEGFR-2 inhibitors structural features. We have used
polycyclic fused tetrahydrobenzothienopyrimidine, as large coplanar
core, or orthogonal ring with aniline moiety, as head part that satisfies
ATP domain of both enzymes, attached to aryl urea as tail part that can
anchor DNA or interacts with the binding site of both enzymes, as
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antagonists with some structural modification in the head or tail group
(figure 3). In the current study, all target compounds were evaluated for
their anti-proliferative activity in vitro against MCF-7 cell line. In order
to explore the mechanistic pathways of the anticancer activity of the
synthesized compounds, we chose the most potent compound 9c to
perform extra investigations such as cell cycle analysis, topoisomerase II,
and VEGFR-2 assays and apoptosis markers. In this work, quantitative
structure-activity relationship (QSAR) studies were also performed, for
validating the observed antitumor properties.
2
. Results and discussion
2
. 1. Chemistry
The synthesis of the target compounds is outlined in Schemes 1 -3. Our
primary starting material 4-chloro-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (1) [15] reacted with
hydrazine hydrate to afford 2 [15]. Reacting with the appropriate
isocyanates in methylene chloride, the 4-hydrazinyl-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine
(2)
gave
the
1
thienopyrimidine urea derivatives 3a-e. The H NMR spectra of 3a-e
displayed the presence of signals at δ 7.12-8.13 ppm, corresponding to
different NH-Ar groups which were not present in 2. On the other hand,
1
3
C NMR spectra of compounds 3b & 3d revealed the appearance of
C=O) carbon at δ 166.07 and 155.98 ppm, respectively. In the present
(
study, hoping to synthesize the triazolothienopyrimidine 5, the
chlorothienopyrimidine 1 was allowed to react with thiosemicarbazide in
ethanol. Actually, unexpected hydrazinecarbothioamide derivative 4 was
obtained after heating under reflux for 8 h. The IR spectrum of compound
4
indicated the presence of three absorption bands at the range 3417-3147
-1
cm , due to two NH and NH groups, in addition to the C=S group that
2
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-1
appeared as an absorption band at 1292 cm . Further evidence was
1
obtained from the H NMR spectrum that showed four exchangeable
signals at δ 7.67, 7.77, 8.63 and 9.29 ppm assigned to NH and two NH
2
13
protons. Further structural evidence stemmed from the C NMR
spectrum of compound 4 that revealed the presence of (C=S) carbon at δ
1
82.23 ppm. In the present study, compound 5 was prepared by reacting
the 4-chlorothienopyrimidine derivative 1 with thiosemicarbazide in
ethanol and extending the reflux time to 20 h. The IR spectrum of 5
lacked the presence of C=S group, which confirmed the success of
1
cyclization. The H NMR spectrum showed only one exchangeable signal
at δ 6.45 ppm due to NH protons. Reacting the triazolothienopyrimidine
2
1
derivative 5 with the appropriate isocyanates afforded 6a-c. H NMR
spectra of these compounds displayed the characteristic signals
1
3
corresponding to different NH-Ar groups. The C NMR spectra of
compounds 6a & 6b revealed the appearance of signals due to (C=O)
carbon at δ 167.10 and 167.08 ppm, respectively. The
triazolothienopyrimidine derivatives 7a,b were achieved by the reaction
1
of compound 5 with the appropriate isothiocyanates. The H NMR
spectra of 7a,b displayed the presence of the characteristic signals of
1
3
aromatic protons at δ 7.13-7.65 ppm. In addition, the C NMR spectrum
of compound 7a showed the appearance of signal at δ 174.60 ppm
corresponding to (C=S) carbon. The thienopyrimidine diarylurea
derivatives 9a-e were obtained by reacting the thienopyrimidine 8 [16]
1
with the appropriate isocyanates in methylene chloride. The H NMR
spectra of 9a-e showed the appearance of three exchangeable singlet
signals at δ 7.78-9.52 ppm corresponding to three NH protons, in addition
to the expected signals corresponding to different NH-Ar protons. The
1
3
C NMR spectra of compounds 9b & 9e revealed the appearance of
(C=O) carbon at δ 165.96 & 165.49 ppm, sequentially. Reacting
7

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compound 9b or 9d and 4-bromophenacyl bromide in ethanol afforded
1
compounds 10a,b. The H NMR spectra of these derivatives revealed the
1
3
disappearance of the signals due to two NH protons. Moreover, C NMR
spectra showed the disappearance of the signal corresponding to (C=O)
carbons. The synthesis of compounds 11a-g was accomplished through
reacting the thienopyrimidine derivative 8 with the appropriate
1
isothiocyanates. The H NMR spectra of 11a-g showed the presence of
three exchangeable singlet signals at δ 7.96-10.74 ppm, corresponding to
13
three NH protons. The C NMR spectra of compounds 11a & 11b
showed the appearance of signals at δ 172.47 & 172.46 ppm,
respectively, corresponding to (C=S) carbon. Cyclization of compounds
1
1a or 11e with 4-bromophenacyl bromide resulted in the formation of
1
compounds 12a,b. The H NMR spectra of these derivatives lacked the
presence of the two exchangeable singlet signals due to the two NH
protons. Compound 14 was obtained in a good yield through reacting the
2
-choromethyl derivative 13 [17] with p-phenylene diamine in ethanol in
1
the presence of catalytic amount of triethylamine. The H NMR spectrum
of 14 showed additional two exchangeable signals at δ 8.28 and 5.75
ppm, due to NH and NH protons of 2-aryl amino methyl group, which
2
were not present in the starting chloromethyl derivative 13, in addition to
the signals at δ 6.61-7.36 ppm, corresponding to the aromatic protons.
Finally, compound 14 was reacted with the appropriate isothiocyanates to
1
give 15a-e. The H NMR spectra of 15a-e displayed four exchangeable
signals at δ 7.67-12.18 ppm, assigned to four NH protons. On the other
1
3
hand, the C NMR spectra of compounds 15d & 15e showed the
appearance of signals at δ 180.08 & 180.66 ppm, respectively
corresponding to (C=S) carbons.
2
. 2. In vitro anticancer activity
8

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The anticancer activity of the newly synthesized compounds was
examined against MCF-7 cell line compared to doxorubicin as reference
anticancer drug. The results of the mean values of experiments performed
in triplicate, expressed as half maximal inhibitory concentration (IC )
5
0
values, were summarized in Tables 1-3 and presented graphically in
figures 4 and 5. The in vitro results showed that most of the test
compounds showed moderate to significant antitumor activity against
MCF-7 cell line. Three compounds 9c, 9d and 11b showed 1.5-1.03 folds
more potent anticancer activity than doxorubicin. Three compounds 3d,
3
e and 6a showed anticancer activity comparable to doxorubicin with
IC values 28.02, 22.65 and 29.06 µM, respectively. Compounds 3c, 9b,
5
0
9
e, 11a, 11c-f and 12a showed moderate anticancer activity. The
antitumor activity correlation of the newly synthesized compounds
showed that among the diaryl urea and thiourea derivatives compounds
9
c, 9d and 11b, bearing electron withdrawing groups, showed more
potent anticancer activity than the other unsubstituted and substituted
derivatives with electron donating groups and were found to be more
potent than doxorubicin. Moreover, compounds 9c and 11b bearing
electron withdrawing group at p-position were found to be more potent
than compound 9d. Another interesting phenomenon is that introducing
two groups into the 2 and 6 positions of the terminal phenyl ring in
compounds 9a, 9b and 11c resulted in lower anticancer activity.
Replacement of the orthogonal 4-anilinothienopyrimidine head with
pseudo tetracyclic core with movement of the diaryl urea tail to position 2
in 15a-e caused a dramatic loss in the anticancer activity. Replacement of
NH-phenyl linker in 9a-e with NH only in 3a-e resulted in lower
anticancer activity. Further analysis of compounds 3a-e clearly revealed
that the groups on the phenyl ring had a notable influence on activity.
Compounds 3c-e with CF group at m-position showed comparable
3
9

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anticancer activity to doxorubicin. While the introduction of two groups
at 2 and 6 positions in compounds 3a and 3b resulted in a marked
decrease in the activity, suggesting that substitution at 2 and 6 positions
of the phenyl ring was not tolerated for this region. Replacement of the
orthogonal 4-anilinothienopyrimidine head in 9a-e and 11a-g with a rigid
coplanar tetracyclic head in 6a-c and 7a, b decreased the anticancer
activity. It was found that urea derivatives 6 a-c bearing electron
withdrawing group on the phenyl ring were more potent than thiourea
derivatives 7a,b with unsubstituted phenyl ring or phenyl carrying
electron donating group. Rigidification of urea or thiourea tail to oxazole
or thiazole ring in compounds 10a,b and 12a,b resulted in much less
anticancer activity. According to these findings, we concluded that a
flexible diaryl urea scaffold at 4-position of thieno[2,3-d]pyrimdine core
with the terminal phenyl ring bearing electron withdrawing group at p-
position had a positive effect on antitumor activity.
2
. 3. Cell cycle analysis and detection of apoptosis
The most active compound 9c was selected to further study its effects on
cell cycle progression and induction of apoptosis in the MCF-7 cell line.
Exposure of MCF-7 cells to compound 9c at its IC value for 24h and its
50
effect on the normal cell cycle profile and induction of apoptosis were
analyzed. Exposure of MCF-7 cells to compound 9c resulted in an
interference with the normal cell cycle distribution of this cell line.
Compound 9c induced a significant increase in the percentage of cells at
pre-G1 and G2/M phases by 15.1 and 2.2 folds, respectively (figure 6)
compared to control. Accumulation of cells in pre-G1 phase, confirmed
by the presence of a sub-G1 peak in the cell cycle profile analysis, may
have resulted from degradation or fragmentation of the genetic materials,
indicating a possible role of apoptosis in compound 9c-induced cancer
1
0

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cell death and cytotoxicity. On the other hand, the accumulation of the
cells in G2/M phase may have resulted from G2 arrest.
2
. 4. Apoptosis determination by Annexin-V assay
To ensure the ability of compound 9c to induce apoptosis, a
biparametric cytofluorimetric analysis was performed, using Propidium
iodide (PI), which stains DNA and enters only dead cells, and
fluorescent immunolabeling of the protein annexin-V, which binds to
phosphatidylserine (PS) expressed on the surface of the apoptotic cells
and fluoresces green, after interacting with the labeled annexin-V [18].
Dual staining of annexin-V with PI permits discrimination between live
cells, early apoptotic cells, late apoptotic cells and necrotic cells. As
shown in figure 7, after 24 h of treatment with compound 9c at its IC50
concentration (7.10 µM) a decrease in the percentage of the survived
cells was observed. Moreover, a significant increase in the percentage of
annexin-V positive cells (30 folds more than control) occurred,
indicating an early apoptosis (lower right quadrant).
2
. 5. Effect of compound 9c on the level of p53/ Bax/ BCL-2:
Tumor cells can acquire resistance to apoptosis by the expression of the
anti-apoptotic proteins such as BCL-2 or by the down-regulation or
mutation of the pro-apoptotic proteins, such as Bax. The expression of
both BCL-2 and Bax is regulated by the p53 tumor suppressor gene [19].
As compound 9c showed significant apoptosis inducing ability, further
investigation was carried out to measure the apoptotic activity, especially
the intrinsic pathway through the evaluation of p53, Bax, BCL-2 and
caspase-3. Exposure of MCF-7 cells to compound 9c at IC for 24h
50
resulted in significant increase in the expression of the tumor suppressor
gene p53 and the pro-apoptotic protein Bax, with consequent reduction of
the expression levels of the anti-apoptotic protein BCL-2 compared to the
control (Table 4 and figure 8). As a result, the test compound 9c showed
1
1

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significant elevation in Bax/ BCL-2 ratio, which supports its ability to
promote the therapeutic response in MCF-7 cells.
2
. 6. Effect of compound 9c on the level of active caspase-3 (key
executer of apoptosis):
Two principle apoptotic pathways were identified, the death receptor (or
extrinsic) and the mitochondrial (or intrinsic) pathways [20]. The two
pathways converge on the same terminal, which was initiated by the
activation of caspase-3 and results in DNA fragmentation [21]. In the
current study, compound 9c significantly elevated the Bax / BCL-2
ratio. Therefore, the subsequent step was to assess the level of active
caspase-3, which is the key executer of apoptosis. Treatment of MCF-7
cells with compound 9c at concentration 7.10 µM produced a significant
increase in the level of active caspase-3 (Table 5 and figure 9). In
conclusion, compound 9c showed potent pro-apoptotic effect by
induction of the intrinsic mitochondrial pathway of apoptosis.
2
. 7. In vitro topoisomerase II assay:
As a result of the effect of compound 9c on S phase of the cell cycle in
which DNA replicated and the cell cycle arrested at G2/M phase, the
inhibition of topoisomerase II enzyme was suspected as expected
mechanism of action of this compound. So, the assay was performed to
evaluate the topoisomerase II inhibitory activity. Compound 9c showed
promising activity (IC = 9.29 µM), which was less than 10 µM and
5
0
comparable to that of doxorubicin, a known potent topoisomerase II
inhibitor (IC = 3.50 µM), so it proved that compound 9c exerted its anti-
5
0
proliferative activity through inhibition of topoisomerase II.
. 8. Molecular docking of compound 9c in the active site of
topoisomerase II
2
1
2

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Compound 9c showed promising activity in the topoisomerase II
inhibition assay with IC value 9.29 µM. At this stage, molecular
5
0
docking study was carried out to investigate its plausible binding pattern
and its interaction with the key amino acids in the active site of the
topoisomerase II. The ability of the synthesized compound 9c to interact
with the key amino acids in the active site rationalized its good activity.
We used The X-ray crystallographic structure of Topoisomerase II co-
crystallized with DNA (PDB ID: 1ZXM). As can be seen in figure 10,
compound 9c interacted by its NH of diaryl urea moiety as an H-bond
donor with Thr 744. As H-bond acceptor, it interacted with its C=O
group of urea scaffold with Tyr 734. Its docking energy score was -10.92
kcal/mol.
2
. 9. In vitro VEGFR-2 assay
In order to prove the success of fragment based drug design approach,
we extended the biological enzyme assay inhibitory activity to VEGFR-2.
The IC value of compound 9c was evaluated compared to reference
5
0
VEGFR-2 inhibitor (sorafenib). Compound 9c showed excellent VEGFR-
inhibitory activity, at the sub-micromolar level with IC value 0.2 µM,
2
5
0
which was 2.1 folds more potent than sorafenib (IC = 0.42 µM).
5
0
2
. 10. Molecular docking of compound 9c in the active site of
VEGFR-2
Docking study was performed for compound 9c, which showed excellent
activity in the VEGFR-2 enzyme inhibition assay, which was 2.1 folds
more potent than sorafenib, in order to reveal the binding mode of this
compound in the active site of the VEGFR-2. To that end, we used (PDB
ID: 4ASD), which has VEGFR-2 co-crystallized with sorafenib as
inhibitor. The ability of the synthesized compound 9c to interact with the
same key amino acids in the active site rationalized its excellent activity,
1
3

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as indicated by its docking pattern and docking score, compared to that of
sorafenib (figure 11). As can be seen, compound 9c interacted by its NH
of diayl urea moiety as H-bond donor with the key amino acid Glu 885.
As H-bond acceptor, it interacted with its C=O group of urea scaffold
with the key amino acid Asp 1046. The N1 of pyrimidine ring interacted
as an H-bond acceptor with the Cys 919. Moreover, the superior activity
of compound 9c against VEGFR-2 (IC = 0.2 µM) was supported by its
50
marked docking score (-16.83 kcal/mol.), which was higher than that of
sorafenib (-13.39 kcal/mol).
2
. 11. QSAR study
The QSAR study was performed using Discovery Studio 2.5 software
Accelrys Inc., San Diego, CA, USA). A set of 21 compounds (3a-d, 6a-
(
c, 7a,b, 9a,b, 9d,e , 11a-g and 12a) was used as a training set for a QSAR
modeling. Compounds 9c, 10a and 12b were adopted as an external test
subset for validating the QSAR models.
2
. 11. 1. QSAR modeling
Many molecular descriptors were calculated for each compound
employing a calculated molecular properties module. The 3D structures
of the training set analogs were imported into the Discovery Studio to
calculate various molecular descriptors for each antitumor active agent.
Moreover, energies of highest occupied and lowest unoccupied molecular
orbitals (HOMO and LUMO) [22, 23] of each of the training set
compounds were determined using this software and imported as
additional descriptors. Firstly, Genetic function approximation (GFA)
was employed to search for the best possible QSAR regression equation,
then Multiple linear regression (MLR) analysis was employed to optimize
QSAR models that combine high quality binding pharmacophores with
other molecular descriptors and being capable of correlating bioactivity
variation across the used training set collection [22, 23]. Equation 1
1
4

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shows our best performing QSAR models. Figure 12 exhibits the
corresponding scatter plots of experimental versus estimated bioactivity
values for the training set compounds against MCF-7 breast human tumor
cell line. Potency (IC ) (the concentration required to produce 50%
5
0
inhibition of cell growth compared to the control experiment) against
2
2
2
MCF-7 tumor cell line (N = 21, r = 0.814, r
= 0.752, r
=
adjusted
prediction
0
.716).
Equation 1
IC = 32468 – 1010 Jurs-RPCS – 18675 Rad of Gyration + 286.31 PMI-
5
0
mag + 156.36 PMI-X − 383.46 PMI-Z.
Abbreviations used: Jurs-RPCS, is the Relative Positive Charge Surface
area. Jurs descriptors combines shape and electronic information to
characterize molecules; Rad of Gyration, is the radius of gyration, it is a
molecular property, defined as the mass weighted. Mathematically, it is
the root mean square average distance of all atoms in the molecular
system from their centre of mass; PMI-mag, Principal Moment of Inertia,
calculates the principal moments of inertia about the principal axes of a
molecule; PMI-X, Principal moment of inertia at X axis; PMI-Z,
Principal moment of inertia at Z axis. The goodness of the model was
2
validated by squared correlation coefficient (R ) and residuals between
the predicted and experimental activity of the test set and training set
2
(
Table 6). R values for MCF-7 cell line QSAR models are 0.814.
2
. 11. 2. Validation of QSAR
QSAR models were validated employing leave one-out cross-validation,
2
2
r (squared correlation coefficient value) and r prediction (predictive
squared
correlation coefficient value) [23]. In addition to external validation of
the determined QSAR equations was performed utilizing three of our
synthesized analogues exhibiting promising (9c), mild (10a) and inactive
1
5

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(
12b) anticancer properties. The observed activities and those provides by
QSAR study, are presented in Table 7.
3
. Conclusion
In summary, a series of novel tetrahydrobenzothieno[2,3-d]pyrimidine
urea derivatives was synthesized according to fragment-based design.
They were evaluated for their anticancer activity against MCF-7 cell line.
Three compounds 9c, 9d and 11b showed 1.5-1.03 folds more potent
anticancer activity than doxorubicin. The most potent compound 9c
showed promising inhibitory activity against topoisomerase II (IC = 9.29
5
0
µM). At the same time, it showed excellent VEGFR-2 inhibitory activity
at the sub-micromolar level with IC value 0.2 µM, which was 2.1 folds
50
more potent than sorafenib. Moreover, it induced a significant increase in
the percentage of cells at pre-G1 and G2/M phases by 15.1 and 2.2 folds
respectively, compared to control, indicating a possible role of apoptosis
in compound 9c-induced cancer cell death and cytotoxicity. Compound
9
c showed potent pro-apoptotic effect through induction of the intrinsic
mitochondrial pathway of apoptosis. This mechanistic pathway was
confirmed by a significant increase in the expression of the tumor
suppressor gene p53, elevation in Bax/ BCL-2 ratio and a significant
increase in the level of active caspase-3, compared to the control.
Compound 9c is a promising multi-targeted lead for the design and
synthesis of potent anticancer agents. QSAR studies delivered equations
2
of five 3D descriptors with R = 0.814.The most important and
controlling descriptors in anticancer properties were 3D descriptors
including Jurs-RPCS, Rad of Gyration, PMI-mag, PMI-X and PMI-Z.
External validation of the established QSAR models utilizing three of our
synthesized analogues exhibiting promising (9c), mild (10a) and inactive
(
12b) anticancer properties, revealed good agreement between the
experimental and the calculated data. It can be concluded that this QSAR
1
6

ACCEPTED MANUSCRIPT
model provides an effective technique for understanding the observed
anticancer properties and thus could be adopted for developing effective
lead structures.
4
4
. Experimental
. 1. Chemistry
4
. 1. 1. General
Melting points were obtained on a Griffin apparatus and were
uncorrected. Microanalyses for C, H and N were carried out at the
Regional Center for Mycology and Biotechnology, Faculty of Pharmacy,
Al-Azhar University. IR spectra were recorded on Shimadzu IR 435
spectrophotometer (Shimadzu Corp., Kyoto, Japan) Faculty of Pharmacy,
-1 1
Cairo University, Cairo, Egypt and values were represented in cm . H
NMR spectra were carried out on Bruker 400MHz (Bruker Corp.,
Billerica, MA, USA) spectrophotometer, Faculty of Pharmacy, Cairo
University, Cairo, Egypt. Tetramethylsilane (TMS) was used as an
internal standard and chemical shifts were recorded in ppm on δ scale and
1
3
coupling constants (J) were given in Hz. C NMR spectra were carried
out on Bruker 100MHz spectrophotometer, Faculty of Pharmacy, Cairo
University, Cairo, Egypt. Mass spectra were recorded on a GCMP-
QP1000 EX Mass spectrometer. Progress of the reactions were monitored
by TLC using precoated aluminum sheet silica gel MERCK 60F 254 and
was visualized by UV lamp.
4
. 1. 2. General procedure for the preparation of N-aryl-2-(5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)hydrazine
carboxamide (3a-e)
A solution of 4-hydrazinyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidine (2) (0.22 g, 0.001 mol) in methylene chloride (4 mL) at 0
⁰
C was treated with the selected isocyanate (0.0011 mol) and stirred
1
7

ACCEPTED MANUSCRIPT
overnight as the temperature slowly rose to room temperature. To the
resulting suspension, hexane was added to yield more precipitate. The
solid was filtered, dried and recrystallized from the suitable solvent to
give 3a-e.
4
.
1.
2.1.
N-(2,6-Dichlorophenyl)-2-(5,6,7,8-
tetrahydrobenzo[4,5]thieno-
[2,3-d]pyrimidin-4-
o
yl)hydrazinecarboxamide (3a). mp 184-186 C (toluene); yield 86%; IR
-1
1
(
KBr) v : 3360, 3236, 3217 (3NH), 1670 (C=O) cm ; H NMR
max
(
DMSO-d ): δ 1.80-1.85 (m, 4H, 2CH ), 2.75-2.85 (m, 2H, CH ), 3.05-
6
2
2
3
.10 (m, 2H, CH ), 7.25-7.30 (m, 2H, ArH), 7.48 (t, 1H, J = 7.24 Hz,
2
ArH), 8.33 (s, 1H, NH, D O exchangeable), 8.41 (s, 1H, C2-H), 8.55 (s,
2
1
H, NH, D O exchangeable) and 8.62 (s, 1H, NH, D O exchangeable)
2
2
+
+
+
ppm; MS [m/z, %]: 409 [M+2˥· , 0.52], 4.8 [M+1˥· , 0.64] and 407 [M· ,
0
1
.42]. Anal. Calcd for C H Cl N OS (408.30): C, 50.01; H, 3.70; N,
1
7
15
2
5
7.15. Found: C, 50.17; H, 3.68; N, 17.43.
4
. 1. 2. 2. N-(2-Chloro-6-methylphenyl)-2-(5,6,7,8-tetrahydrobenzo[4,5]-
o
thieno[2,3-d]pyrimidin-4-yl)hydrazinecarboxamide (3b). mp 178-180
C
(
ethanol); yield 61 % ; IR (KBr) v : 3441, 3321, 3271 (3NH),
max
-1 1
1
662(C=O) cm ; H NMR (DMSO-d ): δ 1.80-1.85 (m, 4H, 2CH ), 2.23
6
2
(
s, 3H, CH ), 2.80-2.85 (m, 2H, CH ), 3.00-3.10 (m, 2H, CH ), 7.12-7.20
3
2
2
(
m, 2H, ArH), 7.31 (t, 1H, ArH), 8.17, 8.18 (2s, 1H, NH, D O
2
exchangeable), 8.26 (s, 1H, NH, D O exchangeable), 8.40 (s, 1H, C2-H),
2
1
3
and 8.42, 8.54 (2s, 1H, NH, D O exchangeable) ppm; C NMR (DMSO-
2
d6): δ 18.5, 22.2, 22.4, 25.1, 25.3 (5 aliphatic Cs), 112.6, 127.2, 128.2,
1
29.5, 132.6, 133.5, 134.0, 138.4, 139.3, 142.7, 151.8 157.3 (aromatic
Cs) and 166.0 (C=O) ppm. Anal. Calcd for C H ClN OS (387.89): C,
18
18
5
5
5.74; H, 4.68; N, 18.06. Found: C, 55.89; H, 4.74; N, 18.31.
1
8

ACCEPTED MANUSCRIPT
4
. 1. 2. 3. N-(4-Chloro-3-(trifluoromethyl)phenyl)-2-(5,6,7,8-tetrahydro-
benzo[4,5]thieno[2,3-d]pyrimidin-4-yl)hydrazinecarboxamide (3c). mp
o
1
88-190 C (toluene); yield 73 % ; IR (KBr) v : 3309, 3255, 3190
max
-1
1
(
3NH), 1651 (C=O) cm ; H NMR (DMSO-d ): δ 1.80-1.90 (m, 4H,
6
2
8
1
CH ), 2.80-2.90 (m, 2H, CH ), 3.00-3.15 (m, 2H, CH ), 7.69 (d, 1H, J =
2
2
2
.0 Hz, ArH), 7.89 (d, 1H, J = 8.0 Hz, ArH), 8.13 (s, 1H, ArH), 8.44 (s,
H, C2-H), 8.59 (s, 1H, NH, D O exchangeable), 8.98, 9.40 (2s, 1H, NH,
2
D O exchangeable), and 10.71 (s, 1H, NH, D O exchangeable) ppm; MS
2
2
+
+
[
m/z, %]: 443 [M+2˥· , 0.52] and 441 [M· , 0.90]. Anal. Calcd for
C H ClF N OS (441.86): C, 48.93; H, 3.42; N, 15.85. Found: C, 49.08
1
8
15
3
5
;
H, 3.45; N, 16.02.
4
. 1. 2. 4. N-(2-Fluoro-5-(trifluoromethyl)phenyl)-2-(5,6,7,8-tetrahydro-
benzo[4,5]thieno[2,3-d]pyrimidin-4-yl)hydrazinecarboxamide (3d). mp
o
1
78-180 C (n-hexane); yield 94% ; IR (KBr) v : 3421, 3321, 3244
max
-1
1
(
3NH), 1697 (C=O) cm ; H NMR (DMSO-d ): δ 1.85-1.90 (m, 4H,
6
2
CH ), 2.80-2.90 (m, 2H, CH ), 3.00-3.10 (m, 2H, CH ), 7.41 (s, 1H,
2
2
2
ArH), 7.49 (t, 1H, J = 10.4 Hz, ArH), 7.61 (t, 1H, J = 10.4 Hz, ArH), 8.62
s, 1H, C2-H), 8.90 (s, 1H, NH, D O exchangeable), 9.05 (s, 1H, NH,
(
2
D O exchangeable) and 9.19, 10.93 (2s, 1H, NH, D O exchangeable)
2
2
1
3
ppm; C NMR (DMSO-d ): δ 22.2, 22.4, 25.2, 25.9 (4 aliphatic Cs),
6
1
1
12.8, 113.5, 115.9, 116.1, 116.5, 116.7 (aromatic Cs), 122.7 (CF3),
25.5, 125.9, 127.0, 128.2, 134.2, 137.2 (aromatic Cs) and 155.9 (C=O)
ppm. Anal. Calcd for C H F N OS (425.4): C, 50.82; H, 3.55; N, 16.46.
1
8
15
4
5
Found: C, 50.93; H, 3.51; N, 16.58.
. 1. 2. 5. 2-(5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)-
4
N-(3-(trifluoromethyl)phenyl)hydrazinecarboxamide (3e). mp 168-170
o
C (methylene chloride); yield 89% ; IR (KBr) v : 3290, 3267, 3217
max
-1
1
(
3NH), 1670 (C=O) cm ; H NMR (DMSO-d ): δ 1.80-1.90 (m, 4H,
6
1
9

ACCEPTED MANUSCRIPT
2
1
CH ), 2.80-2.90 (m, 2H, CH ), 3.00-3.10 (m, 2H, CH ), 7.27-7.29 (m,
2
2
2
H, ArH), 7.47-7.49 (m, 1H, ArH), 7.72-7.74 (m, 1H, ArH), 7.97 (s, 1H,
ArH), 8.00 (s, 1H, NH, D O exchangeable), 8.32 (s, 1H, NH, D O
2
2
exchangeable), 8.45 (s, 1H, C2-H) and 8.78, 9.46 (2s, 1H, NH, D O
2
exchangeable) ppm;. Anal. Calcd for C H F N OS (407.41): C, 53.06;
1
8
16
3
5
H, 3.96; N, 17.19. Found: C, 53.49; H, 4.01; N, 17.32.
4
. 1. 3. Procedure for the preparation of 2-(5,6,7,8-tetrahydrobenzo-
[4,5]thieno[2,3-d]pyrimidin-4-yl)hydrazinecarbothioamide (4)
A
mixture of 4-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidine (1) (0.23 g, 0.001 mol) and thiosemicarbazide (0.1 g, 0.001
mol) in absolute ethanol (30 mL) was heated under reflux for 8 h. The
separated solid was filtered while hot, dried and recrystallized from
o
ethanol to give 4. mp 212-214 C; yield 71% ; IR (KBr) v : 3417,
max
-
1 1
3
1
7
1
302, 3147 (NH , 2NH), 1292 (C=S) cm ; H NMR (DMSO-d ): δ 1.75-
2
6
.85 (m, 4H, 2CH ), 2.75-2.80 (m, 2H, CH ), 3.00-3.05 (m, 2H, CH ),
2
2
2
.67, 7.77 (2s, 2H, NH , D O exchangeable), 8.39 (s, 1H, C2-H), 8.63 (s,
2
2
H, NH, D O exchangeable) and 9.29 (s, 1H, NH, D O exchangeable)
2
2
1
3
ppm; C NMR (DMSO-d ): δ 22.3, 22.5, 25.3, 25.6 (4 aliphatic Cs),
6
1
16.2, 127.0, 133.7, 152.5, 157.1, 165.4 (aromatic Cs) and 182.2 (C=S)
ppm. Anal. Calcd for C H N S (279.38): C, 47.29; H, 4.69; N, 25.07.
11
13
5 2
Found: C, 47.54; H, 4.76; N, 25.34.
4
. 1. 4. Procedure for the preparation of 8,9,10,11-tetrahydrobenzo-
[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidin-3-amine (5)
A
mixture of 4-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidine (1) (0.23 g, 0.001 mol) and thiosemicarbazide (0.1 g, 0.001
mol) in absolute ethanol (30 mL) was heated under reflux for 20 h. The
reaction mixture was cooled, then the separated solid was filtered, dried
2
0

ACCEPTED MANUSCRIPT
and recrystallized from ethanol to give 5. mp 212-214 C; yield 77% ; IR
o
-1 1
(
KBr) v : 3282, 3221 (NH ) cm ; H NMR (DMSO-d ): δ 1.80-1.90 (m,
max
2
6
4
H, 2CH ), 2.80-2.85 (m, 2H, CH ), 2.90-2.95 (m, 2H, CH ), 6.45 (s, 2H,
2
2
2
NH , D O exchangeable) and 9.14 (s, 1H, C2-H) ppm. Anal. Calcd for
2
2
C H N S (245.30): C, 53.86; H, 4.52; N, 28.55. Found: C, 54.12; H,
1
1
11
5
4
.60; N, 28.81.
4
. 1. 5. General procedure for the preparation of 1-aryl-3-(8,9,10,11-
tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidin-3-
yl)urea (6a-c)
A solution of 8,9,10,11-tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]-
triazolo[4,3-c]pyrimidin-3-amine (5) (0.23 g, 0.001 mol) in methylene
chloride (4 mL) at 0 ⁰ C was treated with the selected isocyanate (0.0011
mol) and stirred overnight as the temperature slowly rose to room
temperature. To the resulting suspension, hexane was added to yield more
precipitate. The solid was filtered, dried and recrystallized from the
suitable solvent to give 6a-c.
4
. 1. 5. 1. 1-(2-Chloro-6-methylphenyl)-3-(8,9,10,11-tetrahydrobenzo-
[4,5] thieno [3,2-e][1,2,4]triazolo[4,3-c]pyrimidin-3-yl)urea (6a) mp
o
1
1
62-164 C (ethanol); yield 44% ; IR (KBr) v : 3255, 3217 (2NH),
max
-1 1
650 (C=O) cm ; H NMR (DMSO-d ): δ 1.80-1.90 (m, 4H, 2CH ), 2.29
6
2
(
s, 3H, CH ), 2.80-2.90 (m, 2H, CH ), 2.95-3.05 (m, 2H, CH ), 7.05 (s,
3
2
2
1
H, NH, D O exchangeable), 7.17-7.38 (m, 3H, ArH), 9.16 (s, 1H, C2-H)
2
1
3
and 9.89 (s, 1H, NH, D O exchangeable) ppm; C NMR (DMSO-d ): δ
2
6
1
1
8.9, 22.1, 22.9, 25.2, 25.4 (5 aliphatic Cs), 118.0, 127.4, 127.6, 128.4,
29.4, 129.6, 132.4, 135.8, 136.8, 138.9, 139.2, 148.6, 153.3 (aromatic
Cs) and 167.1 (C=O) ppm. Anal. Calcd for C H ClN OS (412.90): C,
19
17
6
5
5.27; H, 4.15; N, 20.35. Found: C, 55.45; H, 4.23; N, 20.59.
2
1

ACCEPTED MANUSCRIPT
4
. 1. 5. 2. 1-(2-Fluoro-5-(trifluoromethyl)phenyl)-3-(8,9,10,11-
tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidin-3-
o
yl)urea (6b) mp 138-140 C (ethanol); yield 36% ; IR (KBr) v : 3379,
max
-
1 1
3
4
224 (2NH), 1666 (C=O) cm ; H NMR (DMSO-d ): δ 1.75-1.95 (m,
6
H, 2CH ), 2.80-2.90 (m, 2H, CH ), 2.95-3.00 (m, 2H, CH ), 7.11 (s, 1H,
2
2
2
NH, D O exchangeable), 7.37-7.53 (m, 2H, ArH), 9.12 (s, 1H, ArH),
2
1
3
9
.15 (s, 1H, C2-H) and 9.59 (s, 1H, NH, D O exchangeable) ppm; C
2
NMR (DMSO-d ): δ 22.1, 22.9, 25.2, 25.4 (4 aliphatic Cs), 118.0, 120.2,
6
1
1
22.9 (CF ), 125.6, 128.3, 128.5, 128.6, 135.7, 136.7, 145.2, 148.6,
3
52.4, 154.2, 164.2 (aromatic Cs) and 167.0 (C=O) ppm. Anal. Calcd for
C H F N OS (450.41): C, 50.67; H, 3.13; N, 18.66. Found: C, 50.84; H,
1
9
14
4
6
3
.16; N, 18.92.
1. 5.
4
.
3.
1-(8,9,10,11-Tetrahydrobenzo[4,5]thieno[3,2-
e][1,2,4]triazolo
[4,3-c]pyrimidin-3-yl)-3-(3-
o
(
trifluoromethyl)phenyl)urea (6c) mp 148-150 C (isopropanol); yield
-1
1
3
8% ; IR (KBr) v : 3255, 3151 (2NH), 1670 (C=O) cm ; H NMR
max
(
DMSO-d ): δ 1.85-1.95 (m, 4H, 2CH ), 2.85-2.90 (m, 2H, CH ), 2.95-
6
2
2
3
.00 (m, 2H, CH ), 7.17 (s, 1H, NH, D O exchangeable), 7.30 (s, 1H,
2
2
ArH), 7.43 (s, 1H, C2-H), 7.52-7.56 (m, 1H, ArH), 7.67-7.71 (m, 1H,
ArH), 7.95-8.01 (m, 1H, ArH) and 9.15, 9.87 (2s, 1H, NH, D O
2
+
exchangeable) ppm; MS [m/z, %]: 432 [M· , 0.91]. Anal. Calcd for
C H F N OS (432.42): C, 52.77; H, 3.50; N, 19.43. Found: C, 53.02; H,
1
9
15
3
6
3
.54; N, 19.60.
4
. 1. 6. General procedure for the preparation of 1-aryl-3-(8,9,10,11-
tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidin-3-
yl)thiourea (7a,b)
2
2

ACCEPTED MANUSCRIPT
An equimolar mixture of 8,9,10,11-tetrahydrobenzo[4,5]thieno[3,2-
e][1,2,4]triazolo[4,3-c]pyrimidin-3-amine (5) (1.23 g, 0.005 mol), the
selected isothiocyanate (0.005 mol) and anhydrous potassium carbonate
(0.70 g, 0.005 mol) in dioxane (25 mL) was heated under reflux for 25 h.
The reaction mixture was cooled, poured into ice cold water (50 mL) and
the separated solid was filtered, dried and recrystallized from ethanol to
give 7a,b.
4
. 1. 6. 1. 1-Phenyl-3-(8,9,10,11-tetrahydrobenzo[4,5]thieno[3,2-e]-
o
[
1,2,4]triazolo[4,3-c]pyrimidin-3-yl)thiourea (7a) mp 108-110 C; yield
-1
1
7
6% ; IR (KBr) v : 3421, 3344 (2NH), 1219 (C=S) cm ; H NMR
max
(
DMSO-d ): δ 1.75-1.90 (m, 4H, 2CH ), 2.65-2.80 (m, 2H, CH ), 2.85-
6
2
2
3
5
.00 (m, 2H, CH ), 6.46 (s, 1H, NH, D O exchangeable), 7.15-7.65 (m,
2
2
H, ArH), 8.54 (s, 1H, C2-H) and 9.15 (s, 1H, NH, D O exchangeable)
2
1
3
ppm; C NMR (DMSO-d ): δ 22.7, 22.9, 25.2, 25.4 (4 aliphatic Cs),
6
1
1
18.0, 118.5, 123.8, 128.6, 129.0, 129.3, 132.2, 135.8, 136.8, 148.6,
49.1, 153.4, 167.0 (aromatic Cs) and 174.6 (C=S) ppm. Anal. Calcd for
C H N S (380.49): C, 56.82; H, 4.24; N, 22.09. Found: C, 57.06; H,
1
8
16
6 2
4
.31; N, 22.34.
4
. 1. 6. 2. 1-(8,9,10,11-Tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo-
o
[
4,3-c]pyrimidin-3-yl)-3-(m-tolyl)thiourea (7b) mp 102-104 C; yield
-1
1
4
2% ; IR (KBr) v : 3429, 3313 (2NH), 1249 (C=S) cm ; H NMR
max
(
DMSO-d ): δ 1.80-1.90 (m, 4H, 2CH ), 2.30 (s, 3H, CH ), 2.70-2.95 (m,
6
2
3
4
H, 2CH ), 6.45 (s, 1H, NH, D O exchangeable), 7.13-7.22 (m, 1H,
2
2
ArH), 7.25-7.33 (m, 1H, ArH), 7.46 (s, 1H, ArH), 7.55-7.59 (m, 1H,
ArH), 9.16 (s, 1H, C2-H) and 10.01, 10.28 (2s, 1H, NH, D O
2
exchangeable) ppm. Anal. Calcd for C H N S (394.52): C, 57.84; H,
1
9
18
6 2
4
.60; N, 21.30. Found: C, 58.11; H, 4.67; N, 21.57.
2
3

ACCEPTED MANUSCRIPT
4
. 1. 7. General procedure for the preparation of 1-aryl-3-(4-((5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)urea
9a-e)
(
1
A solution of N -(5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)benzene-1,4-diamine (8) (0.90 g, 0.003 mol) in methylene chloride (4
mL) at 0 ⁰C was treated with the selected isocyanate (0.0033 mol) and
stirred overnight as the temperature slowly rose to room temperature. To
the resulting suspension, hexane was added to complete precipitation.
The solid was filtered, dried and recrystallized from the suitable solvent
to give 9a-e.
4
. 1. 7. 1. 1-(2,6-Dichlorophenyl)-3-(4-((5,6,7,8-tetrahydrobenzo[4,5]-
o
thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)urea (9a) mp 258-260 C
(
toluene); yield 55% ; IR (KBr) v : 3419, 3329, 3309 (3NH), 1635
max
-1
1
(
C=O) cm ; H NMR (DMSO-d ): δ 1.75-1.85 (m, 4H, 2CH ), -2.85
6
2
2
.75 (m, 2H, CH ), 3.05-3.10 (m, 2H, CH ), 6.58 (d, 2H, J = 8.2 Hz,
2
2
ArH), 7.20 (d, 2H, J = 8.2 Hz, ArH), 7.44 (d, 2H, J = 8.4 Hz, ArH), 7.55
t, 1H, J = 8.4 Hz, ArH), 7.78 (s, 1H, NH, D O exchangeable), 8.01 (s,
(
2
1
H, NH, D O exchangeable), 8.11, 8.96 (2s, 1H, NH, D O exchangeable)
2
2
+
and 8.23 (s, 1H, C2-H) ppm; MS [m/z, %]: 488 [M+5˥· , 1.96], 487
+
+
+
+
[
M+4˥· , 3.75], 486 [M+3˥· , 10.06], 485 [M+2˥· , 22.48], 484 [M+1˥· ,
+
6
5
2.23] and 483 [M· , 27.94]. Anal. Calcd for C H Cl N OS (484.40): C,
2
3
19
2
5
7.03; H, 3.95; N, 14.46. Found: C, 57.19; H, 3.98; N, 14.58.
4
. 1. 7. 2. 1-(2-Chloro-6-methylphenyl)-3-(4-((5,6,7,8-tetrahydrobenzo-
[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)urea (9b) mp 268-270
o
C (ethanol); yield 58% ; IR (KBr) v : 3419, 3365, 3275 (3NH), 1637
max
-1 1
(
C=O) cm ; H NMR (DMSO-d ): δ 1.80-1.90 (m, 4H, 2CH ), 2.26 (s,
6
2
3
H, CH ), 2.80-2.90 (m, 2H, CH ), 3.00-3.15 (m, 2H, CH ), 7.06-7.22 (m,
3
2
2
2
4

ACCEPTED MANUSCRIPT
3
7
H, ArH), 7.42 (d, 2H, J = 8.7 Hz, ArH), 7.52 (d, 2H, J = 8.7 Hz, ArH),
.97 (s, 1H, NH, D O exchangeable), 8.10 (s, 1H, NH, D O
2
2
exchangeable), 8.31 (s, 1H, C2-H) and 8.79, 8.91 (2s, 1H, NH, D O
2
1
3
exchangeable) ppm; C NMR (DMSO-d ): δ 18.9, 22.4, 22.6, 25.5, 25.9
6
(
5 aliphatic Cs), 118.5, 123.8, 127.0, 127.6, 129.1, 129.4, 132.2, 132.9,
33.5, 134.7, 136.4, 139.0, 139.2, 144.7, 152.6, 153.2, 153.6, 155.6
aromatic Cs) and 165.9 (C=O) ppm. Anal. Calcd for C H ClN OS
1
(
(
2
4
22
5
463.98): C, 62.13; H, 4.78; N, 15.09. Found: C, 62.32; H, 4.79; N, 15.18.
. 1. 7. 3. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-((5,6,7,8-
4
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)urea
o
(
9c) mp 218-220 C (ethanol); yield 74% ; IR (KBr) v : 3439, 3410,
max
-
1 1
3
4
321 (3NH), 1635 (C=O) cm ; H NMR (DMSO-d ): δ 1.85-1.90 (m,
6
H, 2CH ), 2.85-2.90 (m, 2H, CH ), 3.10-3.15 (m, 2H, CH ), 7.38 (s, 1H,
2
2
2
ArH), 7.45 (d, 2H, J = 6.0 Hz, ArH), 7.57 (d, 2H, J = 6.0 Hz, ArH), 7.61-
7
1
9
.66 (m, 1H, ArH), 8.10-8.14 (m, 1H, ArH), 8.35 (s, 1H, C2-H), 8.90 (s,
H, NH, D O exchangeable), 9.04 (s, 1H, NH, D O exchangeable), and
2
2
.35, 9.44 (2s, 1H, NH, D O exchangeable) ppm; MS [m/z, %]: 520
2
+
+
+
[
M+3˥· , 0.81], 519 [M+2˥· , 0.82] and 518 [M+1˥· , 1.04]. Anal. Calcd
for C H ClF N OS (517.95): C, 55.65; H, 3.70; N, 13.52. Found: C,
24
19
3
5
5
5.79 ; H, 3.68; N, 13.69.
4
. 1. 7. 4. 1-(2-Fluoro-5-(trifluoromethyl)phenyl)-3-(4-((5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)urea
o
(
9d) mp 188-190 C (ethanol); yield 71% ; IR (KBr) v : 3419, 3383,
max
-
1 1
3
4
334 (3NH), 1705 (C=O) cm ; H NMR (DMSO-d ): δ 1.80 -1.90 (m,
6
H, 2CH ), 2.80-2.90 (m, 2H, CH ), 3.10-3.20 (m, 2H, CH ), 7.39-7.62
2
2
2
(
m, 6H, ArH), 8.59 (s, 1H, ArH), 8.61 (s, 1H, C2-H), 9.48 (s, 1H, NH,
D O exchangeable), 9.50 (s, 1H, NH, D O exchangeable) and 9.52 (s, 1H,
2
2
2
5

ACCEPTED MANUSCRIPT
NH, D O exchangeable) ppm. Anal. Calcd for C H F N OS (501.5): C,
2
24 19
4
5
5
7.48; H, 3.82; N, 13.96. Found: C, 57.63; H, 3.87; N, 14.18.
4
. 1. 7. 5. 1-(4-((5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)amino)phenyl)-3-(3-(trifluoromethyl)phenyl)urea (9e) mp 238-240
o
C (ethanol); yield 92% ; IR (KBr) v : 3419, 3304, 3200 (3NH), 1631
max
-1
1
(
C=O) cm ; H NMR (DMSO-d ): δ 1.80-1.85 (m, 4H, 2CH ), -2.85
6
2
2
.80 (m, 2H, CH ), 3.10-3.15 (m, 2H, CH ), 7.30 (d, 2H, J = 6.2 Hz,
2
2
ArH), 7.39-7.61 (m, 3H, ArH), 8.02 (d, 2H, J = 6.2 Hz, ArH), 8.11 (s,
H, ArH), 8.34 (s, 1H, C2-H), 8.88 (s, 1H, NH, D O exchangeable), 9.02
1
2
(
s, 1H, NH, D O exchangeable) and 9.24, 9.32 (2s, 1H, NH, D O
2
2
exchangeable) ppm; δ 22.3, 22.4, 25.4, 25.8 (4 aliphatic Cs), 114.5,
1
1
1
1
16.8, 118.6, 119.9, 122.2, 123.2 (aromatic Cs), 123.8 (CF ), 125.9,
3
26.9, 130.4, 133.6, 133.7, 134.2, 135.4, 140.6, 140.7, 152.3, 153.0,
+
55.4 (aromatic Cs) and 165.4 (C=O) ppm; MS [m/z, %]: 486 [M+3˥· ,
+
+
+
.91], 485 [M+2˥· , 3.92], 484 [M+1˥· , 12.43] and 483 [M· , 5.11].
Anal. Calcd for C H F N OS (483.51): C, 59.62; H, 4.17; N, 14.48.
24
20
3
5
Found: C, 59.84; H, 4.21; N, 14.62.
1
4
. 1. 8. General procedure for the preparation of N -(3-aryl-4-(4-
4
bromophenyl)oxazol-2(3H)-ylidene)-N -(5,6,7,8-tetrahydrobenzo[4,5]-
thieno[2,3-d]pyrimidin-4-yl)benzene-1,4-diamine (10a,b)
A mixture of compound 9b or 9d (0.001 mol), 4-
bromophenacylbromide (0.001 mol) and anhydrous sodium acetate
(0.082 g, 0.001 mol) in absolute ethanol (10 mL) was heated under reflux
for 6 h. After cooling, the separated solid was filtered, dried and
recrystallized from ethanol to afford 10a,b.
2
6

ACCEPTED MANUSCRIPT
1
4
. 1. 8. 1. (Z)-N -(4-(4-Bromophenyl)-3-(2-chloro-6-methylphenyl)-
4
oxazol-2(3H)-ylidene)-N -(5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]-
o
pyrimidin-4-yl)benzene-1,4-diamine (10a) mp 208-210 C; yield 83% ;
-1 1
IR (KBr) v : 3417 (NH) cm ; H NMR (DMSO-d ): δ 1.80-1.90 (m,
max
6
4
H, 2CH ), 2.27 ( s, 3H, CH ), 2.80-2.85 (m, 2H, CH ), 3.10-3.15 (m, 2H,
2
3
2
CH ), 7.09 (d, 2H, J = 8.4 Hz, ArH), 7.34 (d, 2H, J = 8.0 Hz, ArH), 7.44
2
(d, 2H, J = 8.0 Hz, ArH), 7.50-7.61 (m, 2H, ArH), 7.71 (d, 2H, J = 8.4
Hz, ArH), 8.03 (s, 1H, NH, D O exchangeable ), 8.15 (s, 1H, oxazole H),
2
1
3
8
1
1
1
1
.23 (s, 1H, C2-H), and 8.33 (t, 1H, ArH) ppm; C NMR (DMSO-d ): δ
6
9.0, 22.4, 22.5, 25.5, 25.9 (5 aliphatic Cs), 116.8, 117.0, 118.4, 120.1,
23.9, 124.2, 127.1, 127.2, 127.4, 127.5, 129.4, 130.2, 131.7, 132.1,
32.2, 132.4, 133.0, 133.2, 133.3, 134.5, 136.6, 138.9, 152.5, 153.4,
55.5, 155.6 and 166.0 (aromatic Cs) ppm. Anal. Calcd for
C H BrClN OS (643.00): C, 59.77; H, 3.92; N, 10.89. Found: C, 60.04;
3
2
25
5
H, 3.97; N, 11.18.
1
4
. 1. 8. 2. (Z)-N -(4-(4-Bromophenyl)-3-(2-fluoro-5-(trifluoromethyl)-
4
phenyl)oxazol-2(3H)-ylidene)-N -(5,6,7,8-tetrahydrobenzo[4,5]thieno-
o
[
2,3-d]pyrimidin-4-yl)benzene-1,4-diamine (10b) mp 214-216 C; yield
-1 1
8
5% ; IR (KBr) v : 3417 (NH) cm ; H NMR (DMSO-d ): δ 1.80-1.90
max
6
(
m, 4H, 2CH ), 2.80-2.85 (m, 2H, CH ), 3.15-3.20 (m, 2H, CH ), 7.37 (d,
2
2
2
2
H, J = 8.8 Hz, ArH), 7.47 (d, 2H, J = 8.4 Hz, ArH), 7.62 (d, 2H, J = 8.4
Hz, ArH), 7.66 (d, 2H, J = 8.8 Hz, ArH), 7.81 (t, 2H, ArH), 8.23 (s, 1H,
C2-H), 8.45 (s, 1H, oxazole H), 8.46 (s, 1H, ArH), and 9.66 (s, 1H, NH,
1
3
D O exchangeable) ppm; C NMR (DMSO-d ): δ 22.4, 22.6, 25.5, 25.8
2
6
(4 aliphatic Cs), 114.1, 116.4, 116.5, 116.7, 116.9, 118.7 (aromatic Cs),
1
1
23.8 (CF ), 125.4, 125.6, 127.1, 128.5, 128.6, 128.7, 130.2, 131.7,
3
31.9, 132.4, 132.5, 133.0, 133.3, 134.1, 135.5, 152.6 (aromatic Cs),
2
7

ACCEPTED MANUSCRIPT
1
52.8 (C-F), 155.5 and 156.2 (aromatic Cs) ppm. Anal. Calcd for
C H BrF N OS (680.51): C, 56.48; H, 3.26; N, 10.29. Found: C, 56.71;
3
2
22
4
5
H, 3.31; N, 10.47.
. 1. 9. General procedure for the preparation of 1-aryl -3-(4-((5,6,7,8-
4
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)thio-
urea (11a-g)
1
A mixture of N -(5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)benzene-1,4-diamine (8) (1.50 g, 0.005 mol), the selected
isothiocyanate (0.005 mol) and anhydrous potassium carbonate (0.70 g,
0
.005 mol) in dioxane (25 mL) was heated under reflux for 12 h. The
reaction mixture was cooled, poured into ice cold water (50 mL) and the
separated solid was filtered, dried and recrystallized from isopropanol to
give 11a-g.
4
. 1. 9. 1. 1-Phenyl-3-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]-
o
pyrimidin-4-yl)amino)phenyl)thiourea (11a) mp 178-180 C; yield 91%
-1 1
;
IR (KBr) v : 3446, 3342, 3257 (3NH), 1222 (C=S) cm ; H NMR
max
(
DMSO-d ): δ 1.70-1.85 (m, 4H, 2CH ), 2.75-2.80 (m, 2H, CH ), 3.05-
6
2
2
3
1
1
2
1
.15 (m, 2H, CH ), 6.91-7.75 (m, 9H, ArH), 8.33 (s, 1H, C2-H), 9.40 (s,
2
H, NH, D O exchangeable), 10.63 (s, 1H, NH, D O exchangeable) and
2
2
1
3
0.74 (s, 1H, NH, D O exchangeable) ppm; C NMR (DMSO-d ): δ
2
6
2.4, 22.6, 25.5, 25.9 (4 aliphatic Cs), 118.6, 119.5, 122.2, 122.6, 123.1,
23.8, 124.0, 127.1, 129.2, 129.7, 140.2, 152.4, 153.0, 160.0 (aromatic
+
Cs) and 172.4 (C=S) ppm; MS [m/z, %]:433 [M+2˥· , 0.41] and 432
+
[
M+1˥· , 0.56]. Anal. Calcd for C H N S (431.58): C, 64.01; H, 4.90;
23
21
5 2
N, 16.23. Found: C, 64.18; H, 4.96; N, 16.41.
4
. 1. 9. 2. 1-(4-Bromophenyl)-3-(4-((5,6,7,8-tetrahydrobenzo[4,5]thieno-
o
[
2,3-d]pyrimidin-4-yl)amino)phenyl)thiourea (11b) mp 180-182 C;
2
8