Discovery of new apoptosis-inducing agents for breast cancer based on ethyl 2-amino-4,5,6,7-tetra hydrobenzo[b]thiophene-3-carboxylate: Synthesis, in vitro, and in vivo activity evaluation

Article abstract of DOI:10.3390/molecules25112523

A multicomponent synthesis was empolyed for the synthesis of ethyl 2-amino-4,5,6,7- tetrahydrobenzo[b]thiophene-3-carboxylate 1. An interesting cyclization was obtained when the amino-ester 1 reacted with ethyl isothiocyanate to give the benzo[4,5]thieno[

Full text of DOI:10.3390/molecules25112523

molecules
Article
Discovery of New Apoptosis-Inducing Agents for
Breast Cancer Based on Ethyl 2-Amino-4,5,6,7-Tetra
Hydrobenzo[b]Thiophene-3-Carboxylate: Synthesis,
In Vitro, and In Vivo Activity Evaluation
Emad M. Gad 1,*, Mohamed S. Nafie 1 , Elsayed H. Eltamany 1, Magdy S. A. G. Hammad 1,
Assem Barakat 2,3 and Ahmed T. A. Boraei 1,
*
1
Chemistry Department, Faculty of Science, Suez Canal University, Ismailia 41522, Egypt;
Mohamed_nafie@science.suez.edu.eg (M.S.N.); s.eltamany51@yahoo.com (E.H.E.);
magdy_hammad83@yahoo.com (M.S.A.G.H.)
Chemistry Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia;
ambarakat@ksu.edu.sa
2
3
Chemistry Department, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia,
Alexandria 21321, Egypt
*
Correspondence: emad_gad@science.suez.edu.eg (E.M.G); ahmed_tawfeek83@yahoo.com (A.T.A.B.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 26 April 2020; Accepted: 25 May 2020; Published: 28 May 2020
Abstract: A multicomponent synthesis was empolyed for the synthesis of ethyl 2-amino-4,5,6,7-
tetrahydrobenzo[b]thiophene-3-carboxylate
amino-ester reacted with ethyl isothiocyanate to give the benzo[4,5]thieno[2,3-d][1,3]thiazin-4-one
Acylation of the amino-ester with chloroacetyl chloride in DCM and Et3N afforded the acylated ester
. The amino-ester was cyclized to benzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one 8, which was reacted
with some alkylating agents leading to alkylation at nitrogen 9–13. Hydrazide 14 was utilized as a
synthon for the synthesis of the derivatives 15 19. Chloro-thieno[2,3-d]pyrimidine 20 was synthesized
and reacted with the hydrazine hydrate to afford the hydrazino derivative 21, which was used as a
scaffold for getting the derivatives 22 28. Nucleophilic substitution reactions were used for getting
the compounds 29 35 from chloro-thieno[2,3-d]pyrimidine 20. In the way of anticancer therapeutics
1. An interesting cyclization was obtained when the
1
3.
1
4
1
–
–
–
development, the requisite compounds were assessed for their cytotoxicity in vitro against MCF-7
and HepG-2 cancer cell lines. Twelve compounds showed an interesting antiproliferative potential
with IC50 from 23.2 to 95.9
apoptosis in MCF-7 cells with a significant 26.86% reduction in cell viability. The in vivo study
revealed a significant decrease in the solid tumor mass (26.6%) upon treatment with compound
µM. The flow cytometric analysis results showed that hit 4 induces the
4.
Moreover, in silico study as an agonist for inhibitors of JAK2 and prediction study determined their
binding energies and predicted their physicochemical properties and drug-likeness scores.
Keywords: benzo[b]thiophene; pyrimidinone; alkylation; MCF-7; HepG-2; JAK2 inhibitor
1. Introduction
The development and discovery of high efficacy anticancer agents by many molecular oriented
strategies and techniques have increased in the last decades [1]. Globcan in 2018 provided a statistical
report worldwide on the global burden of cancer, which shows 18.1 million new cancer incidences and
9.6 million mortalities. The female breast cancer is the second type of cancer, leading to death with
11.6%. The statics show that in every 8–10 women, one gets developed with breast cancer. Moreover,
the fourth fatal cancer mortality is liver cancer with 8.2% [2].
Molecules 2020, 25, 2523; doi:10.3390/molecules25112523
www.mdpi.com/journal/molecules

Molecules 2020, 25, 2523
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There are several reasons for the tremendous growth of cancer mortality and new cases worldwide
due to an increasing number of the population, high rate of aging, as well as prevalence changes and
distribution of the risk factor for cancer linked with socioeconomic development. More specifically,
the risk factors responsible for the development of breast cancer are reproductive factors (e.g., late
menopause, multiparty, premature menarche, do not breastfeed), genetic factors (if her mother
has breast cancer), lifestyle and dietary related factors (e.g., obesity, drinking, and smoking), and
environmental factors (exogenous estrogen exposure for a long time). There are several types of breast
cancer treatments including chemotherapy, radiotherapy, surgery, immune, and hormone therapy
but still these treatments have diverse side effects. The urgent need to discover and develop a high
efficacy anticancer therapy is highly challenged. Several therapeutic potentials have been discovered
for cancer treatment with different targets; one of the most promising targets for cancer therapies is the
JAK family [3]. JAK family members (JAK1, JAK2, JAK3, and TYK2) play an important role in the
pathogenesis of many immunological disorders and cellular malignancies. The activity of JAK kinase
was described in a series of abnormal cell proliferation of the hematologic neoplasias, including B-cell
non-Hodgkin’s and Hodgkin’s lymphomas, myeloid leukemias, and lymphoid [4]. One of the most
molecular targets and techniques for antiproliferative and pro-apoptotic effects are to design potential
JAK inhibitors.
Fused heterocycles, based on pyrimidine scaffolds, have gained significant attention due to a wide
range of pharmacological applications [
anticancer agents [ ], EGFR kinase inhibitors (compound I, IC50 = 2.6 [
and compound III, IC50 = 0.007 M, respectively) [ ], compound IV selective against HepG-2
IC50 = 14.9 µM) [10], compound selective against the MCF-7 breast cancer line (IC50 = 0.10 M) [11],
5]. Especially, thienopyrimidines have been discovered as
6,
7
8
]; compound II IC50 = 0.008
,
,
µ
9
(
V
µ
and tyrosine kinase inhibitors (compound VI) [12]. TargeGen, Inc. et al. have been designed as an
JAK2 inhibitor based on the core structure of anilinopyrimidine, and the cell proliferation assay has
shown the EC50 = 3 nM in a JAK2 V617F (compound VI) [13]. The benzo[b]-thiophene scaffold plays a
crucial rule for many STAT3 inhibitors [14,15]. Additionally, the substituted cycloalkyl[b]thiophene
core structure possessed a cytotoxic activity [16], also exhibited biological activities such as the
AChE inhibitor [17], I3-AG85 inhibitor (compound VIII) [18], as well as the HCV replication
inhibitor (compound IX, Figure 1) [19], antiviral [20], anti-inflammatory [21,22], antibacterial [23,24],
and antitumor activities [25–29]. In the proceeding text, we have synthesized a set of compounds
based on the cycloalkyl[b]thiophene with different hybrids tethered pharmacophoric/functional groups
diversity (Figure 1). The target hybrids were examined against two cancer cell lines (breast cancer
MCF-7 and liver cancer HepG-2), followed by an in vivo study along with the flow cytometric analysis
for the most active member were also carried out. Finally, an in silico molecular docking study such as
the JAK2 inhibitor determined their binding energies and predicted their physicochemical properties
and drug-likeness scores.

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Figure 1. Biologically active compounds based on benzo[b]-thiophene and cycloalkyl[b]thiophene and
pharmacophoric/functional groups relationship.

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2. Results and Discussion
2.1. Chemistry
2.1.1. Synthesis
The benzo[b]thiophene analogue
1
was synthesized in high yield using the multicomponent
strategy from ethyl cyanoacetate, cyclohexanone, sulfur and triethylamine in ethanol. The amino-ester
was reacted with phenyl isothiocyanate in ethanol containing Et3N to afford the N,N-disubstituted
thiourea derivative . Whereas, when it was reacted with ethyl isothiocyanate under the same
1
2
conditions, surprisingly it afforded the 2-(ethylamino)-5,6,7,8-tetrahydro-4H-benzo[4,5]thieno[2,3-
d][1,3]thiazin-4-one 3 (Scheme 1).
Scheme 1. Multicomponent synthesis of 1 and its reaction with isothiocyanates.
Acylation of the amino-ester
. Compound was used for the alkylation of cyclohexylamine to afford the alkylated product
The benzoylation of the benzo[b]thiophene analogue was done by a reaction with benzoyl chloride
. The reaction of with benzene sulfonyl
1 with chloroacetyl chloride in DCM and Et3N afforded the acylated
ester
4
4
5.
1
in benzene containing triethylamine to afford the amide
chloride in ethanol yielded the sulfonamide 7 (Scheme 2).
6
1
Scheme 2. Synthesis of 4–7 starting from compound 1.
Cyclization of the amino-ester
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one
acrylonitrile as the Michael acceptor in ethanol containing Et3N led to an addition at nitrogen to give
the Michael adduct . Alkylation of with benzyl bromide, phenacyl bromide, 4-methoxyphenacyl
1
was done by reflux with formamide for 3 h to give
8
. Michael additions of pyrimidinone to
8
9
8
bromide, and ethyl chloroacetate in DMF and K2CO3, afforded the N-alkylated products 10–13,
respectively (Scheme 3).

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Scheme 3. Alkylation of pyrimidinone 8 with different alkylating agents.
Hydrazinolysis of the ester 13 to hydrazide 14 was done by a reaction with hydrazine hydrate
in ethanol. The heating hydrazide 14 with phenyl isothiocyanate in dioxane for 6 h gives the phenyl
thiosemicarbazide product 15. The hydrazide 14 was refluxed with benzaldehyde and acetophenone
in ethanol to give the hydrazones 16 and 17, respectively. Linking the 4-amino-1,2,4-triazolethione
moiety to the benzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one system separated by a methylene spacer
was achieved by a reaction with CS2/aq. KOH/EtOH, to give potassium dithiocarbazate 18, which was
cyclized by a reaction with hydrazine hydrate and acidification to give 19 (Scheme 4).
Scheme 4. Formation of hydrazones, thiosemicarbazide, and linking new 1,2,4-triazole nucleus to the
system via methylene spacer 19.
The chlorination of
8 was achieved by reflux in POCl3 to yield the chloro derivative 20. A reaction
of 20 with NH2NH2 in ethanol formed the hydrazino compound 21. The benzoylation of 21 with

Molecules 2020, 25, 2523
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benzoyl chloride provided the N-benzoyl derivative 22, while the condensation of 21 with benzaldehyde
and acetophenone afforded compounds 23 and 24, respectively (Scheme 5).
Scheme 5. Synthesis and condensation of the hydrazino derivative 21.
Cyclization of the hydrazino derivative 21 to triazolo-pyrimidines 25 and 26 was done by reflux
in acetic and formic acids, respectively. The condensation of 21 with acetyl acetone in ethanol yielded
the pyrazolo derivative 27. A reaction of 21 with carbon disulfide or phenyl isothiocyanate led to
cyclization to the triazolo-pyrimidine-thione 28 (Scheme 6).
Scheme 6. Cyclization of the hydrazino derivative 21.
Oxygen nucleophiles such as sodium methoxide and sodium ethoxide were used to substitute
the chloride of 20 to afford the ethers 29 and 30, respectively. A reaction of the chloro derivative 20
with nitrogen nucleophiles such as p-toluidine, benzyl amine, 4-aminobenzoic acid, and 4-acetylaniline
afforded the substituted products 31–34, respectively. The reaction of the chloro derivative 20 with
2-aminobenzoic acid afforded the pyrimido-quinazolinone 35 (Scheme 7).
2.1.2. Structural Characterization
The 1H NMR of
group protons (CH3CH2O) appeared at
carbons at 165.53 (C=O), 59.03 (OCH2), and 14.79 ppm for CH3. The structure of
exchangeable signals at 11.78 and 10.88 ppm. The thiocarbonyl and carbonyl groups were detected at
176.36 and 166.02 ppm, respectively. Compound missed the ester group signals and showed only
1
displayed the amino group as an exchangeable signal at
4.15 and 1.25 ppm. 13C NMR showed the ester group
showed the two
δ 7.15 ppm. The ethoxy
δ
δ
2
δ
δ
3

Molecules 2020, 25, 2523
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the NH signal at
δ
13.43 ppm in addition to the ethyl group signals at 4.40 and 1.20 ppm. The ethyl
group carbon signals were found at
structure of . The acetamido group signals in
for CH2Cl. The two carbonyl groups were identified at
and sulfonamide NMR showed the NH signals at
cyclization, the structure of showed the NH as an exchangeable signal at 12.20 ppm and pyrimidine
CH at δ 7.96 ppm. The carbonyl group was identified at δ 162.89 ppm. The aza-Michael addition and
nitrogen alkylation of to produce 13 were deduced from the missing NH signal and illustrated
the methylene carbon signals directly attached to the ring nitrogen (NCH2) at 41.84, 48.83, 52.05,
51.64, and 47.25 ppm, respectively. The NMR of hydride 14 showed the -CO-NHNH2 group as
two exchangeable signals at 9.36 and 4.25 ppm, whereas the carbonyl carbon was identified at
166.55 ppm. The thiosemicarbazide 15 displayed three NH signals at 10.56, 9.77, and 9.33 ppm.
The thiocarbonyl and carbonyl carbons were detected at 181.06, 167.10, and 162.50 ppm. Hydrazones
16 and 17 were confirmed from the disappearance of the NH2 signal and the detection of benzylidene
CH signals around 8.30 ppm. Compound 19 displayed the NH of the ring at 13.54 ppm and the NH2
protons at 5.61 ppm. The C=S was found at 167.32 ppm. The structure of 20 missed the NH signal,
while 21 illustrated the NHNH2 exchangeable signals at 7.81 and 4.55 ppm. The benzoylated product
22 showed two NH signals at 9.64 and 8.29 ppm. The hydrazones 23 and 24 displayed the NH signal
at 11.79 and 11.49 ppm, respectively, while the benzylidene CH was observed at 7.80 ppm in the
δ
41.08 and 12.26 ppm, which strongly recommend the cyclic
were found at 11.66 ppm for NH and 4.57 ppm
165.45 and 164.23 ppm. The benzamide
12.03 and 10.36 ppm, respectively. Due to
3
4
δ
6
7
δ
8
8
9–
δ
δ
δ
δ
δ
δ
δ
δ
δ
δ
case of 23. The cyclic structures 25 and 26 did not show any NH signal; only CH signals appeared in
the aromatic region at 9.48 ppm in the case of 25 and two of the CH signals at 9.55 and 8.60 ppm in
the case of 26. The pyrazole ring structure 27 revealed two CH protons at
pyrimidine and pyrazole CH. The triazole-thione moiety construction in the case of 28 was identified
from the detection of an NH at 14.62 ppm and the thiocarbonyl carbon at 160.56 ppm. The ethers 29
and 30 showed the CH signal of the pyrimidine ring at 8.55 and 8.52 ppm in addition to the alkoxy
group indicating signals. The NH signals appeared in the spectra of 31 34 at 8.34, 8.13, 5.09, and 8.49
9.34 ppm, and the
δ 8.97 and 6.17 ppm for the
δ
δ
–
δ
ppm, respectively. The five-ring fused system 35 displayed the pyrimidine CH at
four phenyl protons appeared between δ 8.35 and 7.57 ppm.
δ
Scheme 7. Synthesis of compounds 29–35 from the chloro derivative 20.

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2.2. Biological Activity
2.2.1. Antitumor Activity Evaluation
The synthesized compounds were tested for their antiproliferative activity against MCF-7 and
HepG-2 cancer cell lines. Twelve compounds displayed a significant activity, which ranged from 23.2
to 95.9
49.9 M. Compounds
95.9 µM. The remaining derivatives showed a lower activity (Table 1).
µM. Compounds
4
,
24
,
29
,
30, and 31 were the most active with an IC50 range from 23.2 to
µ
5
,
15
,
21,
26
,
27, 28, and 33 revealed a moderate activity in the range from 52.9 to
Table 1. The in vitro inhibition % found using (100
µg/mL) of a single dose and IC50 in
µg/mL and µM
of the tested compounds on MCF-7 and HepG-2 cell lines.
MCF-7
HepG-2
Inhibition %
Entry
IC50
µg/mL
IC50
µM
IC50
IC50
Inhibition %
µg/mL
µM
2
27.12551
46.93572
80.625
ND
ND
7
ND
ND
23.2
52.9
ND
131.3
ND
ND
ND
ND
ND
ND
154.5
53.2
ND
ND
ND
ND
127.1
ND
ND
40.3
ND
71.2
76.3
59.1
49.9
31.6
33.2
ND
95.9
ND
42.85714
43.42105
78.63636
76.41221
45.17544
73.58779
52.41228
81.79825
78.18182
72.98246
73.07018
66.22807
69.34211
73.24561
77.09091
79.09091
77.17557
34.21053
69.73684
30.61224
3.508772
78.63636
29.82456
77.27273
77.72727
47.36842
76.36364
78.72727
80.45455
51.83673
81.36364
77.63636
ND
ND
11.7
19.3
ND
37
ND
ND
3
4
38.8
52.9
ND
5
75.91912
38.56502
74.77941
49.0284
19.3
ND
48
6
7
101.2
ND
8
ND
ND
ND
ND
ND
ND
43
ND
36.5
26.5
16.5
31
9
45.06726
67.70833
45.29148
38.93871
56.65172
61.13602
60.46338
66.66667
67.70833
68.41912
34.90284
60.38864
31.98381
1.00005
140.7
89.4
50.9
87.5
94.0
120.7
53.2
68.2
110.4
334.42
ND
10
11
12
13
14
15
16
17
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
27.5
33.6
22
22
ND
ND
ND
ND
28
25
42
38.5
ND
13.6
ND
ND
10
61.7
ND
ND
ND
13
ND
72.39583
38.7145
31.0
ND
ND
16.4
21.7
15.5
11
ND
29
70.67708
79.0625
125.9
114.6
ND
32.6
ND
14.5
8.8
70.40359
80.20833
72.91667
76.04167
47.36842
71.875
65.8
37.6
57.5
ND
7.4
9.8
17
ND
31.2
ND
ND
40
122.9
ND
57.8125
ND
ND: Not determined.

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2.2.2. Flow Cytometric Analysis
To demonstrate the apoptotic mechanistic mode of action, the MCF-7 cell line was treated with
compound (IC50 = 23.2 M, 48 h incubation) compared to untreated cells as the control, which
4
µ
include flow cytometric analyses including an FITC/Annexin-V-FITC/PI differential apoptosis/necrosis
assessment, DNA content-flow cytometry aided cell cycle analysis, and acridine orange quantitative
autophagy assessment. All flow cytometric methodologies were performed according to the standard
protocols, as previously described by Kattan et al., 2020 [30].
FITC/Annexin-V-FITC/PI Differential Apoptosis/Necrosis Assessment
Double-staining with annexin-FITC and propidium iodide (PI) on MCF-7 cells treated with
compound 4 (IC50 = 23.2 µM), and nontreated cells served as the negative control for 48 h to investigate
whether it has an induction of apoptosis and/or cell cycle progression. As displayed in Figure 2,
results show that the treatment caused apoptosis in MCF-7 cells with a substantial cell viability
reduction of 26.86%. The percentage of cells sub-populating in early apoptosis (AV+/PI ) was 8.73,
−
which is 2.3 times more relative to the untreated regulation. In addition, the average late apoptotic
sub-population (AV+/PI+) count was 18.13%, which is 6.6 times higher than the untreated control.
Moreover, the tested compound 4 induced an increase with 1.89-fold (6.29%, compared to 3.33% for
control) in cell death via necrosis, as shown in Figure 2. The results proved that the tested compound
4
effectively induced apoptosis and necrosis in MCF-7 cells.
Figure 2. Induction of apoptosis in MCF-7 cells by compound
annexin-V/propidium iodide stained MCF-7 cells: ( ) MCF-7 cells nontreated with the negative control
and treated with compound ; ( ) bar chart representation of the apoptosis analysis results, quadrant
4 (23.2 µM, 48 h). Cytograms showing
A
4
B
charts show Q2-1 (necrotic cells, AV–/PI+), Q2-2 (late apoptotic cells, AV+/PI+), Q2-3 (normal cells,
AV–/PI–), Q2-4 (early apoptotic cells, AV+/PI–). * p < 0.05 unpaired t-test compared to negative control.
DNA Content-Flow Cytometry Aided Cell Cycle Analysis
Cell cycle analysis is an important test that demonstrates the cell proliferation percentage during
cell growth in every phase following cytotoxic compound treatment. Therefore, to analyze the cell cycle
kinetics of MCF-7 cells treated with compound 4 (IC50 = 23.2 µM, 48h incubation), DNA flow cytometry
was carried out to indicate phase interference with the cell cycle of the compound’s. As shown in
(Figure 3), results showed an induction an increase at G2/M-phase cell-cycle arrest with 1.48-fold
(25.56%, compared to 17.23% for control), and at S-phase cell-cycle arrest with 1.39-fold (23.38%,
compared to 16.76% for control) in cell population distribution, this may have resulted in genetic
material degradation due to the apoptosis induction indicating compound 4 antiproliferative.

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Figure 3. Cytogram reflecting the cell cycle distribution of MCF-7 cells (A): MCF-7 cells untreated
and treated with compound (23.2 M, 48 h); ( ) bar chart representation of the percentage of cell
4
µ
B
population in different MCF-7 cell cycle phases. * p < 0.05 unpaired t-test compared to negative control.
Acridine Orange Quantitative Autophagy Assessment
Herein, we further investigated the effect of compound
process within MCF-7 using the acridine orange lysosomal stain coupled with the flow cytometric
analysis. The tested compound did not induce any significant cell death by autophagy, it inhibited
4 (IC50 = 23.2 µM) on the autophagy
4
autophagic cell death (9.59%, compared to 11.24% for control) and this proves the antiproliferative
activity of compound 4 through apoptosis and necrosis as a dual activity (Figure 4).
Figure 4. Autophagic cell death assessment in MCF-7 treated with compound
4 (IC50 = 23.2 µM) using
the acridine orange lysosomal stain coupled with the flow cytometric analysis. The green curve for the
negative control (untreated), and the red one for the compound 4-treated cells.
2.2.3. In Silico Molecular Docking Studies
A molecular docking study was made to investigate the binding interactions of hit 4 towards three
proteins as JAK2 inhibitors; 3ZMM, 4C62, and 5AEP whose crystal structures complexed with their
co-crystallized ligands were easily accessible from the Protein Data Bank. The co-crystallized ligands
of the studied proteins form hydrogen bonds with Leu 932 as the key amino acid for interaction.
As seen in (Table 2) with 3D images, compound
4 was docked inside the protein active site of the
studied proteins and formed one hydrogen bond with bond length (Aº) through the carbonyl group
oxygen as a hydrogen bond acceptor with the key amino acid Leu 932 as their co-crystallized ligand

Molecules 2020, 25, 2523
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with binding energy
−
14.32,
−
13.39, and 11.38 Kcal/mol, respectively. Additionally, compound 4
−
formed lipophilic interactions with the nonpolar amino acids (Leu 155, Val 24, Ile 140, Leu 90, and Leu
141) inside the receptor pocket. Different models obtained from the docking studies indicated that the
designed target
4 showed promising binding activity as JAK2 inhibitors, and this may be the proposed
mode of action for the anti-breast cancer activity.
Table 2. Analysis of ligand-receptor interactions with binding energies of docked compound 4 inside
three proteins 3ZMM, 4C62, and 5AEP as Jak2 inhibitors.
Binding
Affinity
(Kcal/mol)
Bond
Length
(Aº)
Protein
(PDB Code)
Type of
Interaction
Interaction Moiety
Involved
Amino Acid
Leu 932
3ZMM
–14.32
H-acceptor
1.97
C=O
4C62
–13.39
H-acceptor
1.84
C=O
Leu 932

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Table 2. Cont.
Binding
Affinity
(Kcal/mol)
Bond
Length
(Aº)
Protein
(PDB Code)
Type of
Interaction
Interaction Moiety
Involved
Amino Acid
5AEP
–11.38
H-acceptor
1.67
C=O
Leu 932
Superimposed compound
5AEP proteins.
4
(orange), and the co-crystalized ligand (green) of the three studied 3ZMM
,
4C62, and
2.2.4. Bioinformatics Study
Compounds with high binding affinity through ligand-receptor interactions and binding energy
towards the investigated target (Jak2/STAT3) were subjected to bioinformatics study to predict the
ADME pharmacokinetics properties. Drugs consistent with Lipinski’s "five rule" (Ro5) are considered
prospective in future [31
–
34]. For drug absorption through the intestine, TPSA surface topological
31] The
polar area values should be 140, and the barrier to blood brain should be as low as 90 Å2 [30
,
investigated compounds had good well-permeability and absorption. As shown in (Table 3) and
(Figure 5), compounds had 0-1 Hydrogen-bond donor and 3-4 Hydrogen-bond acceptors. In addition,
the tested compounds had log P range from 2.82 to 4.80, so they had strong toleration by cell membranes.
For managing conformational changes and for oral bioavailability, the number of rotabile bond (nrotb)
should be less than 10 [31,35,36]. All the investigated compounds had 1–5 nrotb. Regarding to
drug-likeness scores, compounds with positive values should be considered like drugs; all the tested
compounds showed 0.15–0.73 (positive values), so they seem to be drug-like.
Table 3. In silico ADME pharmacokinetics properties.
Molinspiration 2018.10
PSA
MolSoft
#
MWt
(D)
MV
(A3)
Solubility Drug-Likeness
Log P
nrotb
nviolations HBA
HBD
(A2)
55.40
37.81
50.17
35.02
(mg/L)
187.99
2.51
Score
0.48
0.48
0.73
0.15
4
301.79
295.40
322.43
234.31
254.18
267.17
290.43
209.15
2.82
4.80
4.33
3.03
5
2
3
2
0
0
0
0
4
3
4
4
1
1
1
0
31
24
30
2.13
14.58
“Mwt: Molecular Weight, MV: Molecular Volume, PAS: Polar Surface Area, Log P: Octanol-water partition coefficient,
nrotb: number of rotatable bond, nviolations: number of violations, HBA: Hydrogen Bond Acceptor, HBD: Hydrogen
Bond Donor” [37].

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Figure 5. Plotting of Drug-likeness score of compound
4 using MolSoft. Non-drug like behavior
(green-colored curve) and drug-like behavior (blue-colored curve). Compounds with zero or negative
value should not seem to be as drug. Compound
score. It should be regarded as drug-like.
4 has a value of 0.48 (positive value) as a drug-likeness
2.2.5. In Vivo Study
Solid Ehrlich Carcinoma (SEC) was treated daily with compound 4 to evaluate the in vivo effect
on tumor cell growth, beginning on day 7 following inoculation of tumor cells. The compound had
a total of seven doses, starting on day 7. At the end of the procedure, the weight of the solid tumor
mass was measured. There was an increase in the solid tumor mass in SEC-bearing mice. During
the experimental duration, an increase in the solid tumor weight of around 155 mg was observed via
the tumor development. The antitumor effect of compound
significant decrease in the solid tumor mass by 54% (70 mg) and 67% (50 mg), respectively (Figure 6).
Accordingly, treatments with compound significantly inhibited the percentage of tumor
4 and 5-FU was elucidated; there was a
4
by 26.6% (tumor volume = 22 mm3) compared to the 5-FU treatment 33.3% (tumor volume =
20 mm3). The achieved results indicate that compound
antitumor agent.
4 has a promising therapeutic potential as an
At the end of the experiment, animals from different groups were anesthetized, and blood samples
were collected for the determination of liver enzymes ALT, AST levels, and CBC parameters, including
Hb content, RBCs and WBCs counts. As seen in Table 4, in SEC-bearing mice, liver enzymes ALT and
AST were found to be significantly increased to 84.65 and 97.64 (U/L), respectively, as compared to
normal mice, this could be contributed to the hepatocellular damage following tumor inoculation.
The treatment with compound 4, such as 5-FU, substantially reduced liver enzyme, where elevated
transaminases ALT were restored to 64.28, AST to 69.89 close to the normal values measured in normal
mice (43.53 and 45.75, respectively), indicating a noticeable improvement in the hepatocellular toxicity
induced by SEC proliferation.

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Figure 6. Solid Ehrlich Carcinoma (SEC)-bearing mice model. Different solid tumor mass, volume,
tumor inhibition ratio (TIR %) values are attributed to the antitumor activity. Mean SEM values
of mice in each group (n = 8). * Mean SEM values with a significant difference in tumor volume
±
±
and tumor weight values compared to the SEC control using an unpaired t-test (p
GraphPad prism.
≤
0.05) using
Table 4. Evaluation of liver enzymes and hematological parameters in the studied groups.
Treatments
ALT *# (I/U)
AST *# (I/U)
Positive Control
43.53 ± 1.06
45.75 ± 0.97
7.9 ± 0.86
SEC Control
84.65 ± 2.1
97.64 ± 1.75
3.99 ± 0.21
3.28 ± 0.73
4.89 ± 0.49
SEC + Comp 4
64.28 ± 1.64
59.89 ± 1.85
5.99 ± 0.49
SEC + 5-FU
51.31 ± 1.41
50.64 ± 0.97
7.02 ± 0.83
4.98 ± 021
Hb *# (g/dL)
RBCs count *# (× 106/µL)
5.14 ± 0.65
4.43 ± 0.47
WBCs count *# (× 103/µL)
3.69 ± 0.53
4.21 ± 0.43
3.98 ± 0.32
#
* Mean
using GraphPad prism.
±
SEM values of mice in each group (n = 8). Values are significantly different (p
≤
0.05), un-paired test
Regarding hematological parameters in SEC-bearing mice, all CBC parameters were changed
upon treatment with compound where the Hb content and RBCs were significantly decreased to
be 3.99 (g/dL) and 3.28 (106/
L), respectively, while the WBCs count was significantly increased to
be 4.89 (103/
L) compared to the normal control levels. Myelosuppression and anemia are the main
4
µ
µ
issues of cancer chemotherapy. Anemia in a tumor-bearing mouse is primarily due to reductions in
the RBC count and hemoglobin, which are either hemolytic or myelopathic [30]. CBC parameters
for hemoglobin, RBC, and WBC levels have been greatly improved by compound
4 treatments.
CBC parameters improved to 5.99, 4.43, and 4.21, respectively, relative to normal control values 7.9,
5.14, and 3.69, hence demonstrating the ability of the tested compound to cure the change of the
hematological parameters.
3. Materials and Methods
All general information about the equipment used in this text, biological activities assays (in vitro
MTT assay, flow cytometric analysis, in silico molecular docking, bioinformatics study, and in vivo
SEC model) of full protocols have been amended in the Supplementary Materials.

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3.1. Synthesis
Synthesis of (
1
): Sulpher (0.02 mol) was added to a mixture of ethyl cyanoacetate (0.03 mol)
and cyclohexanone (0.03 mol) in 30 mL of ethanol followed by the addition of Et3N (4.3 mmol), the
mixture kept on stirring at an ambient temperature for 2 h, then refluxed for further 2 h. The reaction
progress was monitored by TLC then, cooled, added to ice water, and kept overnight to complete the
precipitation. The ppt was filtered, washed with water, dried, and recrystallized from ethanol to give
yellow needle crystals.
Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (1)
Yield: 86%, m.p. 110–111 ◦C; 1H NMR (400 MHZ, DMSO-d6):
NH2), 4.15 (q, 2H, OCH2CH3), 2.42, 2.61 (2 m, 4H, 2 CH2cyhex), 1.67–1.69 (m, 4H, 2 CH2cyhex), 1.25 (t,
3H, J 6.0 Hz, CH3); 13C NMR (100 MHZ, DMSO-d6):
165.53, 163.33, 131.80, 115.97, 103.25 (4 Cthiophene
δ 7.15 (s, 2H, D2O exchangeable,
δ
,
C=O), 59.03 (OCH2), 26.98, 24.41, 23.31, 22.19 (4 CH2cyhex), 14.79 (CH3); CHN calcd. for: C11H15NO2S
[225.08]: C, 58.64; H, 6.71; N, 6.22; S, 14.23; found: C, 58.54; H, 6.69; N, 6.26; S, 14.12.
Synthesis of (2) and (3): A mixture of amino-ester 1 (2.2 mmol) and the appropriate isothiocyanate
(2.2 mmol) and Et3N 0.5 mL was refluxed in 30 mL of absolute ethanol for 3 h then left to cool. Acidified
with concentrated HCl, the formed ppt was collected by filtrations, washed with water, dried, and
recrystallized from ethanol.
Ethyl 2-(3-phenylthioureido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (2)
Yield: 73%, m.p. 173–174 ◦C; 1H NMR (400 MHz, DMSO-d6):
δ 11.78 (s, 1H, D2O exchangeable,
NH), 10.88 (s, 1H, D2O exchangeable, NHPh), 7.49 (d, 2H, Ph), 7.41 (t, 2H, Ph), 7.24 (t, 1H, Ph), 4.23
(q, 2H, OCH2CH3), 2.72 (s, 2H, CH2cyhex), 2.60 (s, 2H, 2 CH2cyhex), 1.73 (s, 4H, 2 CH2cyhex), 1.28 (t,
3H, OCH2CH3); 13C NMR (100 MHz, DMSO-d6):
δ 176.36 (C=S), 166.02 (C=O), 150.00, 138.69, 130.49,
129.35, 126.34, 126.14, 124.78, 112.42, (4 Cthiophene, 6 Cphenyl), 60.72 (OCH2CH3), 26.34, 24.12, 23.01,
22.91, (4 CH2cyhex), 14.52 (CH2CH3); CHN calcd. for: C18H20N2O2S2 [360.49]: C, 59.97; H, 5.59; N, 7.77;
S, 17.79; found: C, 59.95; H, 5.56; N, 7.73; S, 17.77.
2-(Ethylamino)-5,6,7,8-tetrahydro-4H-benzo[4,5]thieno[2,3-d][1,3]thiazin-4-one (3)
Yield: 70%, m.p. 265–267 ◦C; 1H NMR (400 MHz, DMSO-d6):
NH), 4.40 (q, 2H, NHCH2CH3), 2.78, 2.64 (2brs, 4H, 2 CH2cyhex), 1.75 (m, 4H, 2 CH2cyhex), 1.20 (t, 3H,
CH2CH3 ); 13C NMR (100 MHz, DMSO-d6):
173.56, 156.64 (C=O, Cthiazin), 149.02, 131.38, 128.77,
δ 13.43 (s, 1H, D2O exchangeable,
δ
116.17 (4 Cthiophene), 41.08 (NHCH2CH3), 25.32, 24.40, 22.91, 22.01 (4 CH2cyhex), 12.26 (CH2CH3); CHN
calcd. for: C12H14N2OS2 [266.38]: C, 54.11; H, 5.30; N, 10.52; S, 24.07; found: C, 54.22; H, 5.32; N, 10.50;
S, 24.04.
Acylation with Chloroacetyl Chloride: A mixture of
1 (4.5 mmol) and chloroacetyl chloride
(13.5 mmol) in 30 mL of methylene chloride containing Et3N (0.5 mL) was stirred for 3 h in an ice bath.
The formed solid product was collected by filtration, dried, and recrystallized from ethanol.
Ethyl 2-(2-chloroacetamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (4)
Yield: 75%, m.p. 110–112 ◦C; 1H NMR (400 MHz, DMSO-d6):
NH), 4.57 (s, 2H, CH2Cl), 4.32 (q, 2H, OCH2CH3), 2.71 (s, 4H, 2 CH2cyhex), 2.61 (s, 2H, 2 CH2cyhex),
1.73 (s, 4H, 2 CH2cyhex), 1.32 (t, 3H, CH2CH3); 13C NMR (100 MHz, DMSO-d6):
165.45, 164.23
δ 11.66 (s, 1H, D2O exchangeable,
δ
(C=Oamide+ester), 145.55, 131.18, 127.30, 112.78 (4 Cthiophene), 61.00 (OCH2CH3), 43.01 (CH2Cl), 26.24,
24.20, 22.89, 22.70 (4 CH2cyhex), 14.52 (CH2CH3); CHN calcd. for: C13H16ClNO3S [301.79]: C, 51.74; H,
5.34; N, 4.64; S, 10.62; found: C, 51.76; H, 5.30; N, 4.62; S, 10.63.
Synthesis of (5): A mixture of 4 (1.6 mmol) and cyclohexylamine (1.6 mmol) was refluxed in 30 mL
of absolute ethanol for 3 h and cooled to room temperature. The formed solid was filtered, dried, and
recrystallized from ethanol.
Ethyl 2-(2-(cyclohexylamino)acetamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (5)

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Yield: 69%, m.p. 140–141 ◦C; 1H NMR (400 MHz, DMSO-d6):
(100 MHz, DMSO-d6):
δ
1.29–4.69 (m, 28H); 13C NMR
δ
171.22, 164.75 (2 C=Oamide+ester), 146.07, 131.04, 126.12, 111.77 (4 Cthiophene,),
60.46 (OCH2CH3), 57.35, 49.99, 33.25, 26.33, 26.17, 24.89, 24.26, 23.01, 22.85 (9 CH2cyhex, CHcyhex), 14.67
(CH2CH3); CHN calcd. for: C19H28N2O3S [364.50]: C, 62.61; H, 7.74; N, 7.69; S, 8.80; found: C, 62.60;
H, 7.70; N, 7.66; S, 8.81.
Synthesis of (6): A mixture of 1 (3.8 mmol), benzoyl chloride (4.3 mmol), and few drops of
triethylamine was refluxed for 4 h in dry benzene. The reaction mixture was concentrated and
cooled at room temperature. The formed ppt was collected and recrystallized from ethanol to obtain
yellow crystals.
Ethyl 2-benzamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (6)
Yield: 8%, m.p. 170–172 ◦C; 1H NMR (400 MHz, DMSO-d6):
NH), 7.64–7.93 (m, 5H, phenyl), 4.35 (q, 2H, OCH2CH3), 2.66, 2.76 (2brs, 4H, 2 CH2cyhex), 1.76 (brs, 4H,
2 CH2cyhex), 1.35 (t, 3H, CH2CH3); 13C NMR (100 MHz, DMSO-d6):
166.23, 163.06 (2 C=Oester+amide
δ 12.03 (s, 1H, D2O exchangeable,
δ
)
,
147.01, 133.21, 132.54, 131.21, 129.64, 127.46, 127.03 (4 Cthiophene, 6 Cphenyl), 61.04 (OCH2CH3), 26.29,
24.27, 22.97, 22.75 (4 CH2cyhex), 14.52 (OCH2CH3); CHN calcd. for: C18H19NO3S [329.41]: C, 65.63; H,
5.81; N, 4.25; S, 9.73; found: C, 65.60; H, 5.84; N, 4.15; S, 9.5.
Synthesis of (7): A mixture of compound 1 (2.2 mmol) and benzenesulfonyl chloride (2.2 mmol)
was refluxed in 30 mL of absolute ethanol for 4 h then cooled to room temperature. The ppt was
collected by filtration, washed with ethanol, and recrystallized from ethanol.
Ethyl 2-(phenylsulfonamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (7)
Yield: 73%, m.p. 107–108 ◦C; 1H NMR (400 MHz, DMSO-d6):
7.68 (t, 1H, Ph), 7.60 (t, 2H, Ph), 4.14 (q, 2H, OCH2CH3), 2.56–2.59 (m, 4H, 2 CH2cyhex), 1.68–1.69 (m,
4H, 2 CH2cyhex), 1.21 (t, 3H, OCH2CH3); 13C NMR (100 MHz, DMSO-d6):
163.84 (C=Oester), 142.70,
δ 10.36 (s, 1H, NH), 7.81 (d, 2H, Ph),
δ
139.46, 133.87, 132.91, 130.13, 129.82, 127.29, 120.47 (4 Cthiophene+, 6 Cphenyl), 60.76 (OCH2CH3), 25.92,
24.47, 22.81, 22.42 (4 CH2cyhex), 14.38 (OCH2CH3); CHN calcd. for: C17H19NO4S2 [365.46]: C, 55.87; H,
5.24; N, 3.83; S, 17.54; found: C, 55.88; H, 5.26; N, 3.80; S, 17.56.
Synthesis of (8): A mixture of 1 (4.5 mmol) and formamide (0.44 mol) was refluxed for 3 h. Cooled,
poured onto cold water, and left at room temperature overnight, the formed ppt was filtered off and
recrystallized from methanol to give brown needle crystals.
4,5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (8)
Yield: 90%, m.p. 250–251 ◦C; 1H NMR (400 MHz, DMSO-d6):
NH), 7.96 (s, 1H, CHpyrimid.), 2.72, 2.86 (2brs, 4H, 2 CH2cyhex), 1.74–1.80 (m, 4H, 2 CH2cyhex); 13C NMR
(100 MHz, DMSO-d6): 162.89, 158.1, 145.2, 132.57, 131.3, 123.2 (4 Cthiophene, 2 Cpyrimidone), 25.79, 24.90,
δ 12.20 (s, 1H, D2O exchangeable,
δ
22.94, 22.24 (4 CH2cyhex); CHN calcd. for: C10H10N2OS [206.26]: C, 58.23; H, 4.89; N, 13.58; S, 15.54;
found: C, 58.25; H, 4.90; N, 13.57; S, 15.50.
Michael Addition Procedures: A mixture of pyrimidinone
8 (3.4 mmol), acrylonitrile (4.6 mmol),
and Et3N (0.3 mL) in 25 mL of ethanol was refluxed for 6 h, left to cool, a solid product formed, filtered
off, and recrystallized from ethanol to give colorless crystals.
3-(4-Oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-yl)propanenitrile (9)
Yield: 73%, m.p. 173–175 ◦C; 1H NMR (400 MHz, DMSO-d6):
(m, 2H, NCH2), 3.02–3.04 (m, 2H, CH2CH2CN), 2.89 (brs, 2H, CH2cyhex), 2.75 (brs, 2H, CH2cyhex),
1.79 (brs, 4H, 2 CH2cyhex); 13C NMR (100 MHz, DMSO-d6):
162.10 (C=O pyrimidone), 157.20, 147.55,
δ 8.37 (s, 1H, CHpyrimidine), 4.23–4.25
δ
133.79, 131.35, 122.15, 118.56 (6 C(1pyrimidone+4thiophene+CN)), 41.84 (NCH2), 25.77, 25.02, 22.85, 22.20
(4 CH2cyhex), 17.49 (CH2CN); CHN calcd. for: C13H13N3OS [259.33]: C, 60.21; H, 5.05; N, 16.20; S, 12.36;
found: C, 60.23; H, 5.10; N, 16.23; S, 12.34.
General Procedure for Preparation of Compounds (10–13): To a mixture of pyrimidinone 8
(3.0 mmol), the appropriate alkyl halide (3.3 mmol) and anhydrous potassium carbonate (3.6 mmol)

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was refluxed in 20 mL of DMF for 4 h. Then cooled, poured in cold water, the ppt was filtered off and
recrystallized from ethanol to afford the alkylated products 10–13.
3-Benzyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (10)
Yield: 73%, m.p. 190 ◦C; 1H NMR (400 MHz, DMSO-d6):
Hphenyl), 5.18 (s, 2H, NCH2), 2.90–2.89 (m, 2H, CH2cyhex), 2.75–2.74 (m, 2H, CH2cyhex), 1.81–1.75 (m, 4H,
2 CH2cyhex); 13C NMR (100 MHz, DMSO-d6):
162.01 (C=Opyrimidone), 157.32, 147.92, 137.36, 133.62,
δ 8.47 (s, 1H, CHpyrimidine), 7.34 (s, 5
δ
131.38, 129.07, 128.09, 122.42 (11 C(1pyrimidone+4thiophene+6phenyl)), 48.83 (NCH2), 25.81, 25.01, 22.84, 22.21
(4 CH2cyhex); CHN calcd. for: C17H16N2OS [296.39]: C, 68.89; H, 5.44; N, 9.45; S, 10.82; found: C, 68.88;
H, 5.46; N, 9.43; S, 10.80.
3-(2-Oxo-2-phenylethyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (11)
Yield: 85% pale brown crystals, m.p. 190–192 ◦C; 1H NMR (400 MHz, DMSO-d6 ):
CHpyrimidine), 8.10 (d, 2 Hphenyl), 7.75 (t, 2 Hphenyl), 7.62 (t, 1 Hphenyl), 5.60 (s, 2H, NCH2), 2.86 (brs, 2H,
CH2cyhex), 2.78 (brs, 2H, CH2cyhex), 1.78–1.83 (m, 4H, 2CH2cyhex); 13C NMR (100 MHz, DMSO-d6 ):
δ 8.29 (s, 1H,
δ
193.25 (C=Ophenacyl), 162.22 (C=Opyrimidine), 157.26, 148.28, 134.91, 134.59, 133.67, 131.33, 129.47, 128.50,
122.20 (11 C(4thiophene+ 1pyrimidine+6phenyl)), 52.05 (NCH2), 25.76, 25.03, 22.88, 22.22 (4 CH2cyhex); CHN
calcd. for: C18H16N2O2S [324.40]: C, 66.64; H, 4.97; N, 8.64; S, 9.88; found: C, 66.60; H, 4.95; N, 8.66;
S, 9.90.
3-(2-(4-methoxyphenyl)-2-oxoethyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (12)
Yield: 90% as yellow crystals, m.p. 145–146 ◦C; 1H NMR (400 MHz, DMSO-d6 ):
δ 8.27 (s, 1H,
CHpyrimidine), 8.08 (d, 2 Hphenyl), 7.14 (d, 2 Hphenyl), 5.53 (s, 2H, NCH2), 3.90 (s, 3H, OCH3 ), 2.86 (s,
2H, CH2cyhex), 2.78 (s, 2H, CH2cyhex), 1.77–1.81 (m, 4H, 2 CH2cyhex); 13C NMR (100 MHz, DMSO-d6):
δ
191.48 (C=Ophenacyl), 164.35 (C=Opyrimidine), 162.24, 157.29, 148.36, 133.58, 131.32, 130.88, 127.81,
122.20, 114.72 (11 C(4thiophene+ 1pyrimidine+6phenyl)), 56.14 (OCH3), 51.64 (NCH2), 25.76, 25.02, 22.88, 22.22
(4 CH2cyhex); CHN calcd. for: C19H18N2O3S [354.42]: C, 64.39; H, 5.12; N, 7.90; S, 9.05; found: C, 64.41;
H, 5.14; N, 7.93; S, 9.00.
Ethyl 2-(4-oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-yl)acetate (13)
Yield: 82% pale brown needles, m.p. 113–114 ◦C; 1H NMR (400 MHz, DMSO-d6):
CHpyrimdine), 4.79 (s, 2H, NCH2), 4.18 (q, J=8.0 Hz, 2H, OCH2CH3), 2.87 (brs, 2H, CH2cyhex), 2.65 (brs,
2H, CH2cyhex), 1.79 (m, 4H, 2CH2cyhex), 1.23 (t, 3H, CH3); 13C NMR (100 MHz, DMSO-d6):
168.29
δ 8.30 (s, 1H,
δ
(C=Oester), 162.17 (C=Opyrimidine), 157.21, 147.95, 133.85, 131.29, 122.08 (4 Cthiophene, 1 Cpyrimidine), 61.71
(OCH2CH3), 47.25 (NCH2), 25.72, 25.00, 22.85, 22.19 (4 CH2cyhex), 14.45 (CH2CH3); CHN calcd. for:
C14H16N2O3S [292.35]: C, 57.52; H, 5.52; N, 9.58; S, 10.97; found: C, 57.48; H, 5.50; N, 9.61; S, 10.99.
Synthesis of (14): A mixture of ester 13 (3.0 mmol) and hydrazine hydrate (6.0 mmol) in 30 mL of
ethanol was refluxed for 3 h, the reaction mixture was cooled, the solid product was filtered off, dried,
and recrystallized from EtOH/DMF.
2-(4-Oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-yl)acetohydrazide (14)
◦
1
Yield: 91% as white crystals, m.p. 246–248 C; H NMR (400 MHz, DMSO-d6):
δ 9.36 (s, 1H,
D2O exchangeable, NH), 8.22 (s, 1H, CHpyrimdine), 4.58 (s, 2H, NCH2), 4.27 (s, 2H, D2O exchangeable,
NH2), 2.86 (brs, 2H, CH2cyhex), 2.76 (brs, 2H, CH2cyhex), 1.77–1.80 (m, 4H, 2 CH2cyhex); 13C NMR
(100 MHz, DMSO-d6):
δ 166.55, 162.13 (2 C=O(amide+pyrimidone)), 157.32, 148.55, 133.36, 131.28, 122.13
(5 C(1pyrimidine+4thiophene)), 46.85 (NCH2), 25.77, 25.00, 22.88, 22.24 (4 CH2cyhex); CHN calcd. for:
C12H14N4O2S [278.33]: C, 51.78; H, 5.07; N, 20.13; S, 11.52; found: C, 51.80; H, 5.17; N, 20.11; S, 11.55.
Synthesis of (15): To a mixture of hydrazide 14 (3.0 mmol) in 30 mL of dioxane, phenyl
isothiocyanate (3.7 mmol) was added, the reaction mixture was kept under reflux for 6 h, concentrated,
cooled, and left overnight. The formed ppt was collected and recrystallized from ethanol to give 15 as
white crystals.

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2-(2-(4-Oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-yl)acetyl)-N-phenylhydrazine-1-
carbothioamide (15)
Yield: 85%, m.p. 155–156 ◦C; 1H NMR (400 MHz, DMSO-d6):
δ 10.56, 9.77, 9.33 (3s, 3H, D2O
exchangeable, 3NH), 8.30 (s, 1H, CHpyrimidone), 7.59 (d, 2H, J 8 Hz, 2HAr), 7.35 (t, 2H, 2HAr), 7.17–7.20
(m, 1H, 1HAr ), 4.74 (s, 2H, NCH2), 2.81 (brs, 2H, CH2cyhex), 2.77 (brs, 2H, CH2cyhex), 1.74–1.80 (m,
4H, 2 CH2cyhex); 13C NMR (100 MHz, DMSO-d6):
δ 181.06 (C=S), 167.10, 162.5 (2 C=Opyrimidone+amide),
157.88, 148.23, 139.38, 133.91, 131.16, 128.57, 125.55, 125.21, 122.12 (11 C(1pyrimidone+4thiophene+6phenyl)),
47.98 (NCH2), 25.76, 25.01, 22.84, 22.18 (4 CH2cyhex); CHN calcd. for: C19H19N5O2S2 [413.51]: C, 55.19;
H, 4.63; N, 16.94; S, 15.51; found: C, 55.23; H, 4.60; N, 16.92; S, 15.50.
General Procedures for Synthesis of Compounds (16) and (17): To a mixture of hydrazide (3.0
mmol) in 30 mL of ethanol, the selected aldehyde or ketone (3.3 mmol) was added and refluxed for 6
h. Then, concentrated and left overnight, the formed ppt was recrystallized from ethanol to give 16
and 17.
(E)-N’-Benzylidene-2-(4-oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-yl)acetohydrazide (16
)
Yield: 52%, m.p. 230 ◦C; 1H NMR (400 MHz, DMSO-d6):
δ
11.65 (s, 1H, D2O exchangeable, NH),
8.29 (s, 1H, N=CH), 8.08 (s, 1H, CHpyrimidone), 7.73–7.74 (m, 2H, Ph), 7.46 (brs, 3H, Ph), 5.20 (s, 2H,
NCH2), 2.89 (brs, 2H, CH2cyhex), 2.78 (brs, 2H, CH2cyhex), 1.79–1.84 (m, 4H, 2CH2cyhex); 13C NMR
(100 MHz, DMSO-d6):
δ 168.69, 162.16 (2 C=Oamide+pyrimidone), 157.46, 148.61, 144.78, 134.37, 133.52,
131.30, 130.55, 129.33, 127.38, 122.16 (12 C(1pyrimidone+4thiophene+ 6phenyl+N=CH)), 46.94 (NCH2), 25.78,
25.01, 22.87, 22.23 (4 CH2cyhex); CHN calcd. for: C19H18N4O2S [366.44]: C, 62.28; H, 4.95; N, 15.29; S,
8.75; found: C, 62.30; H, 4.92; N, 15.31; S, 8.73.
(E)-2-(4-Oxo-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-yl)-N’-(1-phenylethylidene)
acetohydrazide (17)
Yield: 50%, m.p. 230 ◦C; 1H NMR (400 MHz, DMSO-d6):
δ 11.00 (s, 1H, D2O exchangeable, NH),
8.30 (s, 1H, CHpyrimidone), 7.80–7.86 (m, 2H, 2HAr), 7.43 (brs, 3H, 3HAr), 5.23 (s, 2H, NCH2), 2.88 (brs,
2H, CH2cyhex), 2.78 (brs, 2H, CH2cyhex), 2.31 (s, 3H, CH3), 1.79–1.81 (m, 4H, 2 CH2cyhex); 13C NMR
(100 MHz, DMSO-d6):
δ 169.55, 162.19 (C=Oamide+pyrimidone), 157.50, 149.44, 148.59, 138.32, 133.56,
131.29, 129.77, 128.90, 126.83, 126.58, 122.17 (12 C(N=C+1pyrimidone+4thiophene+6phenyl)), 47.37 (NCH2),
25.77, 25.00, 22.85, 22.21 (4 CH2cyhex), 14.09 (CH3); CHN calcd. for: C20H20N4O2S [380.46]: C, 63.14; H,
5.30; N, 14.73; S, 8.43; found: C, 63.12; H, 5.33; N, 14.75; S, 8.42.
Synthesis of (19): To hydrazide 14 (3.0 mmol) in 30 mL of absolute ethanol containing aq. KOH
(6.0 mmol), carbon disulphide (6.0 mmol) and the mixture was stirred at room temperature for 3 h,
then the solvent was removed on a water bath to get the yellow ppt of 18, which was directly used
for the next step without further purification. To potassium salt 18 (3.0 mmol) in 10 mL of water,
hydrazine hydrate (6.0 mmol) was added and the mixture was refluxed for 5 h, then cooled, diluted
with water, and acidified by concentrated HCl. The formed ppt was collected, dried, and recrystallized
from ethanol to give white plates crystals from 19.
3-((4-Amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidin-4(3H)-one (19)
◦
1
Yield: 77%, m.p. 269–271 C; H NMR (400 MHz, DMSO-d6 ):
δ 13.54 (s, 1H, exchangeable,
NH), 8.38 (s, 1H, CHpyrimidine), 5.61 (s, 2H, D2O exchangeable, NH2), 5.25 (s, 2H, NCH2), 2.87 (t, 2H,
CH2cyhex), 2.77 (t, 2H, CH2cyhex), 1.75–1.83 (m, 4H, 2 CH2cyhex); 13C NMR (100 MHz, DMSO-d6):
δ
167.32 (C=Sthioamide), 162.06 (C=Opyrimidone), 157.03, 148.62, 148.14, 133.80, 131.35, 122.17 (6
C(triazole+pyrimidine+4thiophene), 40.2 (NCH2), 25.75, 25.01, 22.84, 22.19 (4 CH2cyhex); CHN calcd. for:
C13H14N6OS2 [334.42]: C, 46.69; H, 4.22; N, 25.13; S, 19.18; found: C, 46.66; H, 4.20; N, 25.15; S, 19.21.

Molecules 2020, 25, 2523
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Synthesis of (20): The benzothieno[2,3-d]pyrimidine
8
(4.5 mmol) was refluxed in POCl3 (20 mL)
for 3 h. The mixture was cooled and added into ice/water. The formed ppt was filtered off, washed
several times with water, dried, and recrystallized from EtOH to give colorless needle crystals.
4-Chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (20)
Yield: 64%, m.p. 110–112 ◦C; 1H NMR (400 MHz, DMSO-d6):
2H, CH2cyhex), 2.88 (s, 2H, CH2cyhex), 1.85 (s, 4H, 2 CH2cyhex); 13C NMR (100 MHz, DMSO-d6):
δ
8.78 (s, 1H, CHpyrimdine), 2.98 (s,
158.00,
δ
145.25, 132.65, 131.31, 123.22 (5 CCHpyrimidine+4Cthiophene)), 25.76, 24.90, 22.92, 22.22 (4 CH2cyhex); CHN
calcd. for: C10H9ClN2S [224.70]: C, 53.45; H, 4.04; N, 12.47; S, 14.27; found: C, 53.47; H, 4.03; N, 12.49;
S, 14.25.
Synthesis of (21): A mixture 20 (3.6 mmol) and hydrazine hydrate (0.03 mmol) in 35 mL of ethanol
was refluxed for 5 h, then concentrated to half, the formed solid product was filtered off, dried, and
recrystallized from ethanol.
4-Hydrazineyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (21)
Yield: 85% as pale brown crystals, m.p. 175–176 ◦C; 1H NMR (400 MHz, DMSO-d6):
CHpyrimidine), 7.81 (s, 1H, D2O exchangeable, NH), 4.55 (s, 2H, D2O exchangeable, NH2), 2.93 (brs, 2H,
CH2cyhex), 2.76 (brs, 2H, CH2cyhex), 1.80 (brs, 4H, 2 CH2cyhex); 13C NMR (100 MHz, DMSO-d6):
164.42,
δ 8.33 (s, 2H,
δ
158.76 (2 Cpyrimidine), 152.93, 131.97, 127.26, 115.32 (4 Cthiophene), 25.89, 25.35, 22.63, 22.50 (4 CH2cyhex);
CHN calcd. for: C10H12N4S [220.29]: C, 54.52; H, 5.49; N, 25.43; S, 14.55; found: C, 54.49; H, 5.51; N,
25.44; S, 14.57.
Synthesis of (22): To hydrazide 21 (1.8 mmol) in 20 mL of absolute ethanol, benzoyl chloride
(2 mmol) was added, the reaction mixture was refluxed for 6 h, then cooled. The formed solid product
was filtered off and recrystallized from acetic acid.
N’-(5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)benzohydrazide (22)
Yield: 68% brown crystals, m.p. 198–200 ◦C; 1H NMR (400 MHz, DMSO-d6):
8.29 (s, 2H, CHpyrimdine+NH), 7.59–7.60 (m, 5H, Ar), 3.22 (brs, 2H, CH2cyhex), 2.97 (brs, 2H, CH2cyhex),
1.91–1.95 (m, 4H, 2 CH2cyhex); 13C NMR (100 MHz, DMSO-d6):
164.38 (C=O), 153.33, 149.51, 138.69,
δ 9.64 (s, 1H, NH),
δ
137.41, 131.13, 130.43, 129.44, 129.18, 127.66, 120.05 (12 C(2pyrimdine+ thiophene+phenyl)), 25.40, 23.00, 22.17,
21.44 (4 CH2cyhex); CHN calcd. for: C17H16N4OS [324.40]: C, 62.94; H, 4.97; N, 17.27; S, 9.88; found: C,
62.90; H, 4.96; N, 17.24; S, 9.85.
The General Procedure for Synthesis of Compounds (23) and (24): A mixture of 21 (1.6 mmol)
and the selected aldehyde or ketone (2.0 mmol) in 20 mL of ethanol, was refluxed for 4 h, on cooling,
the solid product was filtered off, dried, and recrystallized from ethanol to give compounds 23 and 24
.
(E)-4-(2-benzylidenehydrazineyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (23)
◦
1
Yield: 80% pale brown needles, m.p. 108–110 C; H NMR (400 MHz, DMSO-d6):
δ
11.79 (s,
1H, D2O exchangeable, NH), 8.40 (s, 1H, CHpyrimidine), 7.94 (brs, 2 Hphenyl), 7.80 (s, 1H, N=CH-Ph),
7.39–7.46 (m, 3 Hphenyl), 3.01 (brs, 2H, CH2cyhex), 2.76 (brs, 2H, CH2cyhex), 1.80 (brs, 4H, 2 CH2cyhex); 13
NMR (100 MHz, DMSO-d6): 157.81, 153.32 (2 Cpyrimidine), 149.51, 144.31, 135.91, 132.78, 131.35, 130.33,
C
δ
129.80, 128.55, 119.37 (11 C(4thiophene+7N=CH-C6H5)), 26.99, 25.20, 22.86, 22.52 (4 CH2cyhex); CHN calcd.
for: C17H16N4S [308.40]: C, 66.21; H, 5.23; N, 18.17; S, 10.40; found: C, 66.20; H, 5.26; N, 18.20; S, 10.20.
(E)-4-(2-(1-phenylethylidene)hydrazineyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (24)
Yield: 85% red crystals, m.p. 178–180 ◦C; 1H NMR (400 MHz, DMSO-d6):
δ 11.49 (s, 1H, D2O
exchangeable, NH ), 8.02 (brs, 2 Hphenyl), 7.78 (s, 1H, CHpyrimidine), 7.41–7.43 (m, 3 Hphenyl), 3.04 (brs,
2H, CH2cyhex), 2.75 (brs, 2H, CH2cyhex), 2.45 (s, 3H, CH3), 1.81 (brs, 4H, 2 CH2cyhex); 13C NMR (100 MHz,
DMSO-d6):
δ 157.97, 157.32, 147.94, 144.42, 139.20, 132.73, 131.34, 129.36, 128.53, 127.01, 120.14 (13 C
(4thiophene+6phenyl+2pyrimidine+N=C), 26.87, 25.18, 22.88, 22.65 (4 CH2cyhex), 14.68 (CH3); CHN calcd. for:
C18H18N4S [322.43]: C, 67.05; H, 5.63; N, 17.38; S, 9.94; found: C, 67.04; H, 5.66; N, 17.34; S, 9.99.

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Synthesis of (25) and (26): Hydrazino derivative 21 (2.7 mmol) was refluxed in a glacial acetic acid
or formic acid (3.5 mmol) for 4 h, then concentrated, cooled, and poured into cold water. The formed
ppt was filtered off, washed with water, dried, and recrystallized from ethanol.
3-Methyl-8,9,10,11-tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine (25)
Yield: 80%, m.p. 180 ◦C; 1H NMR (400 MHz, DMSO-d6):
CH2cychex), 2.91 (brs, 2H, CH2cychex), 2.56 (s, 3H, CH3), 1.90 (brs, 4H, 2 CH2cyhex); 13C NMR (100 MHz,
DMSO-d6): 164.68, 153.05, 148.96 (3 C(2pyrimidine+1triazol)), 138.35, 136.97, 128.92, 119.96 (4 Cthiophene),
δ 9.48 (s, 1H, CHpyrimidine), 3.05 (brs, 2H,
δ
25.33, 22.95, 22.15 (4 CH2cyhex), 14.64 (CH3); CHN calcd. for: C12H12N4S [344.32]: C, 58.99; H, 4.95; N,
22.93; S, 13.12; found: C, 58.98; H, 4.97; N, 22.90; S, 13.05.
8,9,10,11-Tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine (26)
◦
1
Yield: 65%, m.p. 136 C; H NMR (400 MHz, DMSO-d6):
(s, 1H, CHtriazole), 3.02–3.04 (m, 2H, CH2cyhex), 2.89–2.90 (m, 2H, CH2cyhex), 1.89–1.93 (m, 4H, 2
CH2cyhex); 13C NM (100 MHz, DMSO-d6):
155.09, 152.86, 148.26, 138.69, 137.48, 128.86, 120.14 (7
δ 9.55 (s, 1H, CHpyrimdine), 8.60
δ
C(1triazole+2pyrimdine+4thiophene)), 25.28, 25.22, 22.89, 22.10 (4 CH2cyhex); CHN calcd. for: C11H10N4S
[230.29]: C, 57.37; H, 4.38; N, 24.33; S, 13.92; found: C, 57.39; H, 4.40; N, 24.31; S, 13.90.
Synthesis of (27): A mixture of hydrazino derivative 21 (2.7 mmol) and acetyl acetone (2.8 mmol) in
20 mL abs. ethanol was refluxed for 6 h on cooling, a ppt is formed which was filtered and recrystallized
from ethanol.
4-(3,5-Dimethyl-1H-pyrazol-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (27)
◦
1
Yield: 67% yellow needles, m.p. 151–153 C; H NMR (400 MHz, DMSO-d6):
CHpyrimidine), 6.17 (s, 1H, CHpyrazolyl), 2.92 (brs, 2H, CH2cyhex), 2.21–2.30 (m, 8H, 2 CH3+CH2cyhex),
1.83–1.84 (m, 2H, CH2cyhex), 1.66 (brs, 2H, CH2cyhex); 13C NMR (100 MHz, DMSO-d6):
170.45, 152.00
δ 8.97 (s, 1H,
δ
(2 Cpyrimidine), 151.77, 149.42, 141.85, 139.55, 127.50, 126.14, 107.85 (7 C(3pyrazole+4thiophene)), 25.95, 24.91,
22.58, 22.13 (4 CH2cyhex), 13.70, 11.80 (2CH3); CHN calcd. for: C15H16N4S [284.38]: C, 63.35; H, 5.67; N,
19.70; S, 11.28; found: C, 63.38; H, 5.65; N, 19.71; S, 11.26.
Synthesis of (28)
Method A: To the hydrazino derivative 21 (2.7 mmol) in 20 mL of ethanol containing aq. KOH
(6.0 mmol) carbon disulfide (3.0 mmol) was added, the reaction mixture was refluxed for 6 h, then
cooled, diluted with water, and acidified with concentrated HCl. A yellow ppt is formed, filtered off,
washed with water, dried, and recrystallized from ethanol to give brown crystals.
Method B: A mixture of hydrazino derivative 21 (1.8 mmol) and phenyl isothiocyanate (2.0 mmol)
in 10 mL of ethanol was refluxed for 6 h. After that cooling, the solid product is formed, filtered off,
and recrystallized from ethanol.
8,9,10,11-Tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine-3(2H)-thione (28)
◦
1
Yield: 85A%, 65B%, m.p. 283–285 C; H NMR (400 MHz, DMSO-d6):
exchangeable, NH), 8.87 (s, 1H, CHpyrimidine), 2.90 (brs, 4H, 2 CH2cyhex), 1.88 (brs, 4H, 2 CH2cyhex); 13
NMR (100 MHz, DMSO-d6): 160.56, 151.28, 143.19 (3 C(pyrimidine+C=S thioamide)), 138.78, 135.69, 129.59,
δ 14.62 (s, 1H, D2O
C
δ
117.23 (4 Cthiophene ), 25.14, 22.88, 22.05 (4 CH2cyhex); CHN calcd. for: C11H10N4S2 [262.35]: C, 50.36; H,
3.84; N, 21.36; S, 24.4; found: C, 50.32; H, 3.86; N, 21.32; S, 24.3.
The General Procedure for Synthesis of Compounds (29) and (30): The chloro derivative 20
(1.8 mmol) was refluxed in sodium methoxide or sodium ethoxide (prepared from 0.23 g of sodium in
30 mL of ethanol or methanol) for 6 h after finishing and the solvent was concentrated. The formed
solid product was filtered off and recrystallized from the water-ethanol mixture.
4-Methoxy-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (29)
Yield: 71.8% colorless needle crystals, m.p. 98–100 ◦C; 1H NMR (400 MHz, DMSO-d6):
1H, CHpyrimidine), 4.04 (s, 3H, OCH3), 2.86 (brs, 2H, CH2cyhex), 2.80 (brs, 2H, CH2cyhex), 1.82–1.81 (m,
δ 8.55 (s,

Molecules 2020, 25, 2523
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4H, 2 CH2cyhex); 13C NMR (100 MHz, DMSO-d6):
δ
166.88, 163.91 (2 Cpyrimidine), 152.67, 135.01, 127.49,
118.56 (4 Cthiophene), 54.28 (OCH3), 25.91, 25.32, 22.83, 22.26 (4 CH2cyhex); CHN calcd. for: C11H12N2OS
[220.29]: C, 59.97; H, 6.35; N, 12.60; S, 14.42; found: C, 59.99; H, 6.34; N, 12.61; S, 14.38.
4-Ethoxy-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (30)
Yield: 67% white solid, m.p. 109 ◦C; 1H NMR (400 MHz, DMSO-d6):
4.52 (q, 2H, CH2CH3), 2.89–2.91 (m, 2H, CH2cyhex), 2.79–2.81 (m, 2H, CH2cyhex), 1.82–185 (m, 4H, 2
CH2cyhex), 1.40 (t, 3H, CH2CH3); 13C NMR (100MHz, DMSO-d6):
167, 163.58 (2 Cpyrimidine), 152.70,
δ 8.52 (s, 1H, CHpyrimidine),
δ
134.88, 127.59, 118.57 (4 Cthiophene), 62.69 (OCH2), 25.86, 25.35, 22.86, 22.32 (4 Ccyhex), 14.66 (CH2CH3);
CHN calcd. for: C12H14N2OS [234.31]: C, 61.51; H, 6.02; N, 11.96; S, 13.68; found: C, 61.53; H, 6.05; N,
11.99; S, 13.66.
The General Procedure for Synthesis of Compounds (31–35): A mixture of chloro derivative
20 (2.0 mmol) and the appropriate amine (2.1 mmol) in 20 mL of ethanol was refluxed for 6 h.
The solvent was concentrated and on cooling a solid product is formed which was filtered off, dried,
and recrystallized from ethanol to afford the corresponding compound 31–35.
N-(p-Tolyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (31)
Yield: 71%, brown plates crystals, m.p. 140–142 ◦C; 1H NMR (400 MHz, DMSO-d6):
δ 8.34 (s, 1H,
D2O exchangeable, NH), 7.98 (s, 1H, CHpyrimidine), 7.55 (d, J 8.0 Hz, 2H, 2HAr), 7.17 (d, J 8 Hz, 2H, 2CH,
2HAr), 3.12 (brs, 2H, CH2cyhex), 2.82 (brs, 2H, CH2cyhex), 2.30 (s, 3H, CH3), 1.86 (brs, 4H, 2 CH2cyhex);
13C NMR (100 MHz, DMSO-d6):
δ 166.06, 155.51 (2 Cpyrimidine), 152.55, 137.02, 133.20, 132.96, 129.29,
127.01, 122.83, 117.02 (10 C(4thiophene+6phenyl)), 25.89, 25.52, 22.61, 22.48 (4 CH2cyhex), 20.93 (CH3); CHN
calcd. for: C17H17N3S [295.40]: C, 69.12; H, 5.80; N, 14.22; S, 10.85; found: C, 69.10; H, 5.81; N, 14.25;
S, 10.79.
N-Benzyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (32)
Yield: 65.5%, m.p. 162–164 ◦C; 1H NMR (400 MHz, DMSO-d6):
δ 8.54 (d, 1H, J 8.0 Hz, phenyl),
8.43 (s, 1H, CHpyrimidine), 8.13 (s, 1H, D2O exchangeable, NH), 6.98–7.11 (m, 4H, phenyl), 3.92 (s, 2H,
NHCH2Ph), 3.07–3.09 (m, 2H, CH2cyhex), 2.82–2.84 (m, 2H, CH2cyhex), 1.87–1.94 (m, 4H, 2 CH2cyhex);
13C NMR (100 MHz, DMSO-d6):
δ 165.60, 154.84 (2 Cpyrimidine), 152.71, 149.38, 134.05, 128.79, 126.24,
123.66, 121.03, 117.15, 111.42 (10 C(6phenyl+4thiophene)), 56.77 (CH2-NHBn), 25.94, 25.49, 22.58, 22.40 (4
CH2cyhex); CHN calcd. for: C17H17N3S [295.40]: C, 69.12; H, 5.80; N, 14.22; S, 10.85; found: C, 69.10; H,
5.81; N, 14.25; S, 10.79.
4-((5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)benzoic acid (33)
◦
1
Yield: 88% yellow crystals, m.p. 269–271 C; H NMR (400 MHz, DMSO-d6): 8.50 (s, 2H,
CHpyrimidine+COOH), 7.93 (d, 2H, J 8.0 HZ, 2HAr), 7.82 (d, 2H, J 8.0 Hz, 2HAr), 5.09 (s, 1H, D2O
exchangeable, NH), 3.16 (brs, 2H, CH2cyhex), 2.86 (brs, 2H, CH2cyhex), 1.92 (brs, 4H, 2 CH2cyhex); 13
C
NMR (100 MHz, DMSO-d6):
δ 167.43 (C=O), 165.69, 154.72 (2 Cpyrimidine), 151.97, 143.99, 134.49, 130.50,
127.13, 125.30, 120.89, 118.14 (10 C(4thiophene+ 6phenyl)), 25.71, 25.59, 22.60, 22.40 (4 CH2cyhex); CHN calcd.
for: C17H15N3O2S [325.39]: C, 62.75; H, 4.65; N, 12.91; S, 9.85; found: C, 62.80; H, 4.60; N, 12.93; S, 9.87.
1-(4-((5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)phenyl)ethan-1-one (34)
Yield: 67% brown crystals, m.p. 173–175 ◦C; 1H NMR (400 MHz, DMSO-d6):
exchangeable, NH), 8.38 (s, 1H, CHpyrimidine), 7.94 (d, 2H, J 8.0 Hz, 2HAr), 7.83 (d, 2H, J 8.0 Hz, 2HAr),
3.14 (brs, 2H, CH2cyhex), 2.84 (brs, 2H, CH2cyhex), 2.54 (s, 3H, OCH3), 1.87 (brs, 4H, 2 CH2cyhex); 13
NMR (100 MHz, DMSO-d6): 196.81 (C=O), 166.86, 154.51 (2 Cpyrimidine), 152.27, 144.48, 134.42, 131.65,
δ 8.49 (s, 1H, D2O
C
δ
129.55, 126.94, 120.42, 118.17 (10 C(6phenyl+4thiophene)), 26.81 (CH3), 25.7, 25.59, 22.60, 22.42 (4 CH2cyhex);
CHN calcd. for: C18H17N3OS [323.41]: C, 66.85; H, 5.30; N, 12.99; S, 9.91; found: C, 66.82; H, 5.31; N,
12.96; S, 9.90.

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1,2,3,4-Tetrahydro-9H-benzo[4’,5’]thieno[2’,3’:4,5]pyrimido[6,1-b]quinazolin-9-one (35)
Yield: 70% yellow crystals, m.p. 285 ◦C; 1H NMR (400 MHz, DMSO-d6):
δ 9.34 (s, 1H, CHpyrimdine),
8.35 (d, 1 Hphenyl), 7.96 (t, 1 Hphenyl), 7.78 (d, 1 Hphenyl), 7.57 (t, 1 Hphenyl) 2.91, 1.90 (4 CH2cyhex); CHN
calcd. for: C17H13N3OS [307.37]: C, 66.43; H, 4.26; N, 13.67; S, 10.43; found: C, 66.40; H, 4.24; N, 13.69;
S, 10.40.
3.2. Biological Experimental Assays (Full Protocols, see Supplementary Materials)
3.2.1. In Vitro
In vitro work regarding cell culturing, cytotoxic screening using the MTT assay, and the IC50
calculations were made according to Mosmann 1983 (see Supplementary Materials) [38].
3.2.2. Flow Cytometry
All flow cytometry including FITC/Annexin-V-FITC/PI differential apoptosis/necrosis assessment,
DNA content-flow cytometry aided cell cycle analysis, and acridine orange quantitative autophagy
assessment were made according to Nafie et al., 2020 [39].
3.2.3. In Silico Molecular Docking
All in silico studies including ligand optimization, protein preparation, and molecular docking
calculation were investigated followed by the reported [40].
3.2.4. Bioinformatics
Bioinformatics study (in silico and bioactivity prediction) of the most active compounds were
calculated using a set of software’s including MolSoft, Molinspiration [41], and SwissADME [42
]
websites as previously described by Youssef et al., 2020 [37].
3.2.5. In Vivo (SEC) Model
Experiment design and methodology including tumor volume and percentage of tumor inhibition
were summarized in Figure 7.
Figure 7. Summarized methodology of Solid Ehrlich Carcinoma (SEC)-bearing mice in vivo.

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3.2.6. Statistical Analysis
Data were represented by the value of the mean for three different replicates, with the standard
error of the mean (mean SEM). All statistical analysis was done as using GraphPad Prism software
±
version 7.0. The statistical difference between two groups was examined using the unpaired t-test.
The significance level was set at p < 0.05.
4. Conclusions
In conclusion, approximately 33 compounds were synthesized and characterized. The target
compounds were evaluated in vitro against HepG-2 and MCF-7 cancer cell lines. Hit
4 was found
to be the most active, and the cell cycle analysis showed that this lead induces apoptosis. Moreover,
the in vivo study demonstrates that our target significantly reduces the tumor mass. The in silico
molecular docking shows the binding of 4 as an agonist for JAK2 inhibitors.
Supplementary Materials: The following are available online, Full protocols for the biological assays; Figure
S1–S87 copies of the HNMR and CNMR spectrum of the synthesized compounds.
Author Contributions: Conceptualization, E.H.E., A.T.A.B., and E.M.G.; synthesis and characterization,
M.S.A.G.H., E.H.E., A.T.A.B., and E.M.G.; in vivo, in silico, bioinformatics experiments and flow cytometric
analyses discussion, M.S.N.; writing original manuscript, A.T.A.B., M.S.N., and A.B.; revision and editing, A.T.A.B.,
M.S.N., and A.B. All authors have read and agree to the published version of the manuscript.
Funding: Researchers Supporting Project, King Saud University.
Acknowledgments: The authors would like to extend their sincere appreciation to the Researchers Supporting
Project (RSP-2019/64), King Saud University, Riyadh, Saudi Arabia.
Conflicts of Interest: The authors declare no conflict of interest.
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