Novel thienopyrimidine derivatives as dual EGFR and VEGFR-2 inhibitors: design, synthesis, anticancer activity and effect on cell cycle profile

Article abstract of DOI:10.1080/14756366.2019.1593160

Aim: Design and synthesis of thienopyrimidine derivatives as dual EGFR and VEGFR-2 inhibitors.Material and methods: A series of novel 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidine derivatives with different substituents on C-4 position was synthesized and evaluated for their anticancer activity against MCF-7 cell line. EGFR, VEGFR-2 inhibitory assay, the cell cycle analysis and apoptosis induction ability of the most potent compound 5f were evaluated.Results: Most of the compounds showed moderate to significant anticancer activity. Compound 5f exhibited the most potent anticancer activity being 1.73- and 4.64-folds more potent than erlotinib and doxorubicin, respectively. Compound 5f showed potent EGFR inhibitory activity being 1.18-folds more potent than reference standard erlotinib and it also showed good VEGFR-2 inhibitory activity at the micromolar level with IC₅₀ value 1.23?μM. Compound 5f caused induction of cell cycle arrest at G2/M phase and accumulation of cells in pre-G1 phase. Compound 5f induced cellular apoptosis.

Full text of DOI:10.1080/14756366.2019.1593160

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Novel thienopyrimidine derivatives as dual
EGFR and VEGFR-2 inhibitors: design, synthesis,
anticancer activity and effect on cell cycle profile
Aml E.-S. Mghwary, Ehab M. Gedawy, Aliaa M. Kamal & Suzan M. Abuel-
Maaty
To cite this article: Aml E.-S. Mghwary, Ehab M. Gedawy, Aliaa M. Kamal & Suzan M. Abuel-
Maaty (2019) Novel thienopyrimidine derivatives as dual EGFR and VEGFR-2 inhibitors: design,
synthesis, anticancer activity and effect on cell cycle profile, Journal of Enzyme Inhibition and
Medicinal Chemistry, 34:1, 838-852, DOI: 10.1080/14756366.2019.1593160
To link to this article: https://doi.org/10.1080/14756366.2019.1593160
©
2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
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Published online: 28 Mar 2019.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
019, VOL. 34, NO. 1, 838–852
2
https://doi.org/10.1080/14756366.2019.1593160
RESEARCH PAPER
Novel thienopyrimidine derivatives as dual EGFR and VEGFR-2 inhibitors: design,
synthesis, anticancer activity and effect on cell cycle profile
a
a,b
a,c
a
Aml E.-S. Mghwary , Ehab M. Gedawy , Aliaa M. Kamal and Suzan M. Abuel-Maaty
a
b
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt; Department of Pharmaceutical
c
Chemistry, Faculty of Pharmacy and Pharmaceutical Industries, Badr University in Cairo BUC, Cairo, Egypt; Department of Organic Chemistry,
Faculty of Pharmacy, October University for Modern Science and Arts (MSA), Cairo, Egypt
ABSTRACT
ARTICLE HISTORY
Aim: Design and synthesis of thienopyrimidine derivatives as dual EGFR and VEGFR-2 inhibitors.
Material and methods: A series of novel 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidine
derivatives with different substituents on C-4 position was synthesized and evaluated for their anticancer
activity against MCF-7 cell line. EGFR, VEGFR-2 inhibitory assay, the cell cycle analysis and apoptosis induc-
tion ability of the most potent compound 5f were evaluated.
Results: Most of the compounds showed moderate to significant anticancer activity. Compound 5f exhib-
ited the most potent anticancer activity being 1.73- and 4.64-folds more potent than erlotinib and doxo-
rubicin, respectively. Compound 5f showed potent EGFR inhibitory activity being 1.18-folds more potent
than reference standard erlotinib and it also showed good VEGFR-2 inhibitory activity at the micromolar
level with IC50 value 1.23 mM. Compound 5f caused induction of cell cycle arrest at G2/M phase and accu-
mulation of cells in pre-G1 phase. Compound 5f induced cellular apoptosis.
Received 10 January 2019
Revised 26 February 2019
Accepted 27 February 2019
KEYWORDS
Thieno[2,3-d]pyrimidines;
design; synthesis; anticancer
activity; EGFR; VEGFR-2;
vandetanib; apoptosis
Introduction
and VEGFR-2 represents a promising cancer treatment proto-
1
5,16
col
been discovered
Figure 1) that exhibited potent inhibitory activity against both
. Recently, several EGFR/VEGFR-2 dual inhibitors have
Protein kinases have a crucial role in signal transduction pathways
that mediate several cellular functions . Protein kinase inhibitors
has received great attention in the last decades due to their
ongoing role in fighting cancer . The epidermal growth factor
R
1
4,17–19
V
1
, such as vandetanib (ZD6474, Caprelsa ,
2
0
2
EGFR and VEGFR-2 (with IC50¼0.50, 0.04 mM, respectively) .
Vandetanib had FDA approval in 2011 for treatment of thyroid
receptor (EGFR) is a tyrosine kinase transmembrane receptor that
mediates several signal transduction cascades [Ras/MAPK, PI3K/
Akt, Jak/STAT] which regulate cell proliferation, growth and apop-
2
1
cancer . In addition, such 4-anilinoquinazoline derivative substi-
tuted with halogen atoms in position 2 and 4 of the anilino group
showed significant anticancer activity against breast, colorectal
3
tosis . EGFR over-expression is implicated in various types of can-
2
2–26
and lung cancers
.
cer such as breast, colon, ovarian and prostate through enhancing
the cancer cell proliferation, invasiveness, metastasis and angio-
1
8
In 2010, Garofalo et al. designed several 4-anilinoquinazoline
derivatives with urea or carbamate ester moieties (I–III) (Figure 1).
It was reported that presence of urea entity enhanced the VEGFR-
4–7
genesis . Moreover, EGFR-signaling pathways stimulate vascular
8
endothelial growth factor (VEGF) which is considered as the key
2
inhibitory activity and abolished the activity against EGFR. On
inducer of tumor angiogenesis. Angiogenesis is the process of
blood vessel formation that starts with dilatation and increase in
the vascular permeability of the existing capillaries and venules.
Followed by activation, migration and proliferation of the endo-
thelial cells, of the blood vessels to form the new capillaries .
Binding of VEGF with VEGFR-2 stimulates signaling pathways
the other hand, derivatives with carbamate ester group (com-
pound II) showed promising dual EGFR/VEGFR-2 inhibitory activ-
1
8
ity . Furthermore, the presence H-donor/acceptor group at the
para position of the 4-anilino moiety such as sulfonamide or
amide group (compound IV–VI) improved the binding to both
EGFR and VEGFR-2 receptors and as a result enhanced the dual
9
(
p38MAPK, PI3K/Akt) that mediate several cellular functions such
1
9,27
as proliferation, migration, survival and vascular permeability for enzyme inhibitory activity
. Finally, presence of halide in the
10
the tumor cell and hence promotes angiogenesis . Furthermore, phenyl ring of 4-anilino or aryloxy moiety enhance the dual inhibi-
it has been demonstrated that VEGFR-2 are highly expressed in tory activity (compound III, VII) (Figure 1)
cancer cells especially endothelial cells . In addition, both glyco-
1
4,28
.
11
EGFR inhibitors differ in their structure but have common phar-
proteins, EGFR and VEGFR-2 are closely related; inhibition of EGFR macophoric features, the first pharmacophore is a flat aromatic
decreasing the expression of VEGF, whereas targeting of VEGFR-2 heterocyclic fused system that acts as hydrogen bond acceptor
1
2–14
potentiate the anticancer activity of EGFR inhibitors
.
(HBA) to reside in adenine binding pocket (hinge segment).
Therefore, recent therapies based on dual inhibition of both EGFR Moreover, EGFR inhibitors should have a hydrophobic moiety
CONTACT Ehab M. Gedawy
ehab.gedawy@pharma.cu.edu.eg
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Cairo University,
Cairo, Egypt
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
839
Figure 1. Structures of potent dual EGFR and VEGFR-2 inhibitors.
such as phenyl ring (hydrophobic head) linked to aromatic hetero- another hydrogen bond acceptor group as well as a hydrogen
cyclic fused system with NH spacer to occupy the hydrophobic bond donor group (such as ureido or amide moiety) to interact
3
0
region I of the enzyme. Finally, another hydrophobic moiety that with NH of Asp1044 and C¼O of Glu883, respectively .
is attached or fused to the aromatic flat, (hydrophobic tail) to
occupy hydrophobic region I .
Motivated by these facts, herein, we designed and synthesized
novel cyclohepta[4,5]thieno[2,3-d]pyrimidine derivatives as dual
29
On the other hand, the structural requirements for VEGFR-2 EGFR and VEGFR-2 inhibitors (Figure 2). The structural modifica-
kinase inhibitory activity is a flat aromatic ring system with a tions involved replacement of 4-anilinoquinazoline scaffold with is
hydrogen bond acceptor group to interact with NH of Cys917. In their bioisostere thieno[2,3-d]pyrimidine core fused to large lipo-
addition to the presence of another hydrophobic group with philic cycloalkyl group guided by their reported potent anticancer

8
40
A. E.-S. MGHWARY ET AL.
N
O
O
O
O
S
S
NH2
N
N
CH3
H
HN
N
HN
N
N
N
S
S
4
a
4b,c
p-substituted with sulphonamide group as H-bond doner & H-bond acceptor
with additional pyrimidine ring to improve binding with ATP-binding site of both enzymes
O
NH2
O
N
CH3
O
O
N
O
N
O
N
N
N
N
S
S
S
5
b
5j
5
k
p-substituted with amino (H-bond doner)/ methoxy or nitro group (H-bond acceptor)
X
O
N
S
N
5
c-i
aryloxy moiety substituted with haogen atoms and/or methyl group
Figure 2. Design strategy of the targeted thieno[2,3-d]pyrimidines derivatives as dual EGFR and VEGFR-2 inhibitors.
3
1,32
activity
. In addition, we explored the substitution of the phe- a Bruker Advance 400 MHz NMR (Bruker Corp., Billerica, MA) spec-
nyl ring of 4-anilino or 4-aryloxy moieties with, halogen, hydrogen trometer, Microanalytical Unit, Faculty of Pharmacy, Cairo
bond donor (such as amino group), hydrogen bond acceptor University, Egypt. Chemical shift values were recorded in ppm on
(
nitro, methoxy groups) or hydrogen bond donor/acceptor (sul- d scale using tetramethylsilane (TMS) as internal standard and
13
coupling constants (J) were given in Hz. C NMR spectra were
carried out using a Bruker Advance 100 MHz spectrometer,
Microanalytical Unit, Faculty of Pharmacy, Cairo University, Egypt.
Microanalyses for C, H and N were carried out at the Regional
Center for Mycology and Biotechnology, Faculty of Pharmacy, Al-
Azhar University. Mass spectra were recorded on a GCMP-QP1000
EX Mass spectrometer. Progress of the reactions was monitored
by TLC using aluminum sheets precoated with UV fluorescent sil-
ica gel (Merck 60 F254, Kenilworth, NJ) and spots were visualized
by UV lamp. The solvent system used was chloroform:benzene:me-
thanol [4.5:5:0.5] and few drops of TEA. Chemicals were purchased
from Acros Organics, Geel, Belgium and Sigma-Aldrich, St. Louis,
phonylamino group) to substantiate the effect of such variations
on the dual EGFR and VEGFR-2 inhibitory activity as well as their
anticancer activity against MCF-7 breast cancer cell line. The cyto-
toxicity of the most potent compounds was estimated on MCF-
1
0A (the human mammary epithelial cell line) to determine their
safety to normal cells.
Material and methods
Melting points were determined on Stuart SMP10 apparatus and
the values were uncorrected. IR spectra were carried out using KBr
discs on Shimadzu IR spectrophotometer (FTIR, 8400S, Kyoto, MO. The cytotoxic activity, the cell cycle, in vitro EGFR and VEGFR-
Japan), Faculty of Pharmacy, Cairo University, Egypt and values 2 inhibitory activities assays were evaluated at the Confirmatory
ꢀ
1 1
were represented in cm . H NMR spectra were carried out using Unit, Vaccines and Sera Co. (VACSERA), Egypt.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
841
Chemistry
The reaction mixture was allowed to cool to room temperature,
the solid formed was filtered, dried and crystallized from absolute
ethanol to afford compounds 5a–k.
2
(
-Amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carbonitrile
1), 6,7,8,9-tetrahydro-3H-cyclohepta[4,5]thieno[2,3-d]pyrimidin-4(5H)-
one (2) and 4-chloro-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-
3
-((6,7,8,9-Tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimi-
ꢁ
din-4-yl)oxy)aniline (5a). Yield: 61%; m.p.: 179–180 C; IR (KBr)
3
3
d]pyrimidine
reported procedures.
(3)
were
prepared
according
to
v
max: 3452, 3325 (NH
2
), 3109, 3055 (C–H aromatic), 2916, 2846
ꢀ1
1
(
C–H aliphatic) cm
m, 4H, 2CH
.24–3.26 (m, 2H, CH
dd, 1H, J ¼ 8 Hz, 2 Hz, ArH), 6.39 (t, 1H, J ¼ 2 Hz, ArH), 6.48 (dd, 1H,
J ¼ 8 Hz, 2 Hz, ArH), 7.06 (t, 1H, J ¼ 8 Hz, ArH), 8.47 (s, 1H, C2-H)
; H NMR (400 MHz, DMSO-d ): d 1.68–1.72
6
General procedure for the preparation of N-substituted-4-
(6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidin-4-
(
3
(
2
), 1.87–1.88 (m, 2H, CH
), 5.28 (s, 2H, NH
2
), 2.94–2.97 (m, 2H, CH
2
),
[
2
2
, D O exchangeable), 6.34
2
yl)amino]benzenesulfonamides (4a–c). A mixture of 4-chloro-
,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidine (3)
1.50 g, 0.0063 mol) and the appropriate substituted benzenesulfo-
6
(
1
3
ppm; C NMR (100 MHz, DMSO-d
6
): d 26.9, 27.3, 28.4, 29.6, 31.9,
namide (0.0063 mol) in isopropanol (15 mL) was heated under
reflux for 10 h. The reaction mixture was allowed to cool, the solid
formed was filtered, dried and crystallized from absolute ethanol
to give compounds 4a–c.
1
1
1
1
07.4, 109.1, 111.6, 119.8, 130.1, 132.6, 140.2, 150.7, 152.1, 153.5,
þ
þ
63.2, 166.7 ppm; EIMS [m/z, %]: 312 [M þ 1· , 37.96], 311 [M· ,
00.00]; Anal. Calcd for C17 OS (311.40): C, 65.57; H, 5.50; N,
3.49. Found: C, 65.31; H, 5.73; N, 13.68.
17 3
H N
4-((6,7,8,9-Tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimi-
4
-((6,7,8,9-Tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimi-
din-4-yl)amino)benzenesulfonamide (4a). Yield: 86%; m.p.:
ꢁ
ꢁ
din-4-yl)oxy)aniline (5b). Yield: 30%; m.p.>300 C; IR (KBr) vmax
:
2
71–272 C; IR (KBr) vmax: 3414, 3360, 3290 (NH, NH ), 3082 (C–H
2
ꢀ
1
1
2
3440–3400 (NH ), 3030 (C–H aromatic), 2920, 2850 (C–H aliphatic),
aromatic), 2916, 2846 (C–H aliphatic) cm
; H NMR (400 MHz,
ꢀ
1 1
1
4
3
612 (C¼N) cm
;
H NMR (400 MHz, DMSO-d ): d 1.69–1.70 (m,
6
6
DMSO-d ): d 1.70–1.74 (m, 4H, 2CH
2 2
), 1.86–1.87 (m, 2H, CH ),
H, 2CH ), 1.87–1.88 (m, 2H, CH ), 2.94–2.97 (m, 2H, CH ),
.25–3.28 (m, 2H, CH
2
2
2
2
.93–2.96 (m, 2H, CH ), 3.16–3.18 (m, 2H, CH ), 7.22 (br.s, 2H, NH ,
2
2
2
2
), 5.12 (s, 2H, NH
2
, D
2
O exchangeable), 6.61
D O exchangeable), 7.72 (d, 2H, J ¼ 8.8 Hz, ArH), 7.78 (d, 2H,
2
(
d, 2H, J ¼ 8.8 Hz, ArH), 6.89 (d, 2H, J ¼ 8.8 Hz, ArH), 8.43 (s, 1H, C2-
J ¼ 8.8 Hz, ArH), 8.47 (s, H, C
2
–H), 9.05 (s, 1H, NH, D
able) ppm; C NMR (100 MHz, DMSO-d ): d 27.0, 27.1, 29.2, 29.3,
1.1, 118.9, 119.4, 121.6, 126.8, 132.6, 138.4, 143.2, 151.3, 154.9,
2
O exchange-
13
1
3
H) ppm; C NMR (100 MHz, DMSO-d6): d 27.0, 27.3, 28.5, 29.6,
6
3
1.9, 114.7, 122.7, 132.7, 136.9, 139.9, 142.4, 146.9, 152.1, 163.9,
3
1
þ
þ
166.4 ppm; EIMS [m/z, %]: 311 [M· , 9.79], 298 [100.00]; Anal.
64.3 ppm; EIMS [m/z, %]: 374 [M· , 45.91], 129 [100.00]; Anal.
(374.48): C, 54.52; H, 4.84; N, 14.96. Found:
Calcd for C H N OS (311.40): C, 65.57; H, 5.50; N, 13.49. Found:
1
7 17 3
18 4 2 2
Calcd for C17H N O S
C, 65.83; H, 5.65; N, 13.72.
C, 54.89; H, 4.97; N, 15.31.
N-(Pyrimidin-2-yl)-4-((6,7,8,9-tetrahydro-5H-cyclohepta[4,5]-
thieno[2,3-d]pyrimidin-4-yl)amino)benzenesulfonamide
4
-(4-Bromophenoxy)-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]-
ꢁ
thieno[2,3-d]pyrimidine (5c). Yield: 73%; m.p.: 135–136 C; IR (KBr)
vmax: 3101, 3066 (C–H aromatic), 2916, 2854 (C–H aliphatic) cm ;
H NMR (400 MHz, DMSO-d ): d 1.70–1.72 (m, 4H, 2CH ), 1.88–1.90
6 2
(4b).
ꢀ
1
ꢁ
Yield: 77%; m.p.: 243–245 C; IR (KBr) vmax: 3456, 3371 (2 NH),
1
ꢀ
1
3
101, 3078, 3035 (C–H aromatic), 2927, 2850 (C–H aliphatic) cm ;
1
2 2 2
(m, 2H, CH ), 2.97–3.00 (m, 2H, CH ), 3.25–3.28 (m, 2H, CH ), 7.29
H NMR (400 MHz, DMSO-d ): d 1.64–1.72 (m, 4H, 2CH ), 1.82–1.84
6
2
(
d, 2H, J ¼ 8.8 Hz, ArH), 7.66 (d, 2H, J ¼ 8.8 Hz, ArH), 8.48 (s, 1H, C2-
(
(
m, 2H, CH ), 2.92–2.94 (m, 2H, CH ), 3.11–3.14 (m, 2H, CH ), 6.00
s, 1H, NH, D
2
2
2
13
H) ppm; C NMR (100 MHz, DMSO-d
3
1
6
): d 26.9, 27.3, 28.5, 29.6,
1.8, 118.3, 119.8, 124.9, 132.5, 133.0, 140.7, 151.7, 151.8, 162.8,
66.8 ppm; Anal. Calcd for C H BrN OS (375.28): C, 54.41; H, 4.03;
2
O exchangeable), 6.57 (d, 2H, J ¼ 8.8 Hz, ArH), 7.01 (t,
1
8
H, J ¼ 4.8 Hz, ArH), 7.62 (d, 2H, J ¼ 8.8 Hz, ArH), 8.47 (s, 1H, C2-H),
.52 (d, 2H, J ¼ 4.8 Hz, ArH), 9.07 (s, 1H, NH, D O exchangeable);
17 15
2
2
1
3
N, 7.46. Found: C, 54.68; H, 4.22; N, 7.68.
C NMR (100 MHz, DMSO-d6): d 27.0, 27.1, 29.1, 29.4, 31.1, 112.6,
4
-(2-Chlorophenoxy)-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]-
1
1
15.9, 120.4, 125.3, 129.0, 130.2, 132.5, 138.5, 151.8, 153.4, 157.4,
57.6, 158.7 ppm; EIMS [m/z, %]: 452 [M· , 8.09], 133 [100.00];
ꢁ
þ
thieno[2,3-d]pyrimidine (5d). Yield: 21%; m.p.>300 C; IR (KBr)
vmax: 3040 (C–H aromatic), 2920, 2850 (C–H aliphatic), 1620 (C¼N)
Anal. Calcd for C H N O S (452.55): C, 55.73; H, 4.45; N, 18.57.
Found: C, 55.61; H, 4.62; N, 18.74.
N-(4-Methylpyrimidin-2-yl)-4-((6,7,8,9-tetrahydro-5H-cyclohep-
ta[4,5]thieno[2,3-d]pyrimidin-4-yl)amino)benzenesulfonamide
2
1 20 6 2 2
ꢀ
1
1
cm
1
;
H NMR (400 MHz, DMSO-d
6
): d 1.71–1.73 (m, 4H, 2CH
2
),
), 3.33–3.34 (m, 2H,
CH ), 7.35–7.40 (m, 1H, ArH), 7.45–7.52 (m, 2H, ArH), 7.64 (d, 1H,
2 2
.90–1.91 (m, 2H, CH ), 2.99–3.02 (m, 2H, CH
2
1
3
ꢁ
J ¼ 8 Hz, ArH), 8.48 (s, 1H, C2–H) ppm; C NMR (100 MHz, DMSO-
): d 26.9, 27.3, 28.5, 29.6, 31.8, 119.5, 125.3, 126.5, 127.9, 129.0,
30.8, 132.5, 140.9, 148.3, 151.9, 152.3, 167.0 ppm; Anal. Calcd for
15ClN OS (330.83): C, 61.72; H, 4.57; N, 8.47. Found: C, 61.89;
m, 2H, CH ), 3.11–3.14 (m, 2H, CH ), 4.77 (s, 1H, NH, D O H, 4.66; N, 8.63.
(
4c). Yield: 77%; m.p. 260–262 C; IR (KBr) vmax: 3437, 3228 (2 NH),
ꢀ
1
d
6
3
070, 3035 (C–H aromatic), 2920, 2850 (C–H aliphatic) cm ;
1
1
H NMR (400 MHz, DMSO-d
6
): d 1.64–1.70 (m, 2H, CH
2
),1.72–1.77
(m, 2H, CH
2
), 1.82–1.83 (m, 2H, CH
2
), 2.33 (s, 3H, CH ), 2.91–2.93
3
C
17
H
2
(
2
2
2
exchangeable), 6.91 (d, 1H, J ¼ 8.8 Hz, ArH), 7.72 (d, 2H, J ¼ 8.8 Hz,
4-(4-Chlorophenoxy)-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]-
ꢁ
ArH), 7.95 (d, 2H, J ¼ 8.8 Hz, ArH), 8.34 (d, 1H, J ¼ 8.8 Hz, ArH), 8.47 thieno[2,3-d]pyrimidine (5e). Yield: 53%; m.p. 115–116 C; IR (KBr)
1
3
ꢀ1
(
s, 1H, C2-H), 9.11(s, 1H, NH, D O exchangeable);
C NMR
vmax: 3105 (C–H aromatic), 2912, 2854 (C–H aliphatic) cm ;
2
1
(100 MHz, DMSO-d6): d 23.7, 27.0, 27.1, 29.1, 29.3, 31.1, 115.3,
H NMR (400 MHz, DMSO-d
6
): d 1.70–1.72 (m, 4H, 2CH
2
), 1.88–1.89
), 7.34
1
1
19.7, 120.5, 129.2, 132.5, 133.9, 138.5, 144.4, 151.5, 154.5, 157.1, (m, 2H, CH
2
), 2.96–2.99 (m, 2H, CH ), 3.25–3.28 (m, 2H, CH
2
2
þ
58.0, 165.1, 168.7 ppm; EIMS [m/z, %]: 466 [M· , 27.71], 70 (d, 2H, J ¼ 8.8 Hz, ArH), 7.53 (d, 2H, J ¼ 8.8 Hz, ArH), 8.48 (s, 1H, C2-
1
3
[
100.00]; Anal. Calcd for C H N O S (466.58): C, 56.63; H, 4.75; H) ppm; C NMR (100 MHz, DMSO-d
6
): d 26.9, 27.3, 28.5, 29.6,
31.8, 119.8, 124.5, 130.0, 130.2, 132.5, 140.5, 151.3, 151.9, 162.9,
General procedure for the preparation of 4-(aryloxy)-6,7,8,9- 166.9 ppm; Anal. Calcd for C17 15ClN OS (330.83): C, 61.72; H, 4.57;
tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidines (5a-k). A N, 8.47. Found: C, 61.56; H, 4.71; N, 8.79.
solution of 4-chloro-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]-
4-(4-Chloro-3-methylphenoxy)-6,7,8,9-tetrahydro-5H-cyclo-
22 22 6 2 2
N, 18.01. Found: C, 56.87; H, 4.89; N, 18.25.
H
2
ꢁ
thieno[2,3-d]pyrimidine (3) (1.90 g, 0.0080 mol) and phenolic com- hepta[4,5]thieno[2,3-d]pyrimidine (5f). Yield: 23%; m.p.>300 C;
pound (0.0096 mol) in alcoholic potassium hydroxide solution IR (KBr) vmax: 3059, 3032 (C–H aromatic), 2924, 2850 (C–H ali-
ꢀ
1 1
(
6
KOH 0.54 g in 20 mL ethanol) was heated under reflux for 20 h. phatic) cm ; H NMR (400 MHz, DMSO-d ): d 1.68–1.71 (m, 4H,

8
42
A. E.-S. MGHWARY ET AL.
2
CH
2
), 1.87–1.88 (m, 2H, CH
2
), 2.34 (s, 3H, CH
), 7.15 (dd, 1H, J ¼ 8.8 Hz, J ¼ 2.8 Hz,
3
), 2.95–2.98 (m, 2H, Biological assay
CH ), 3.24–3.26 (m, 2H, CH
2
2
Measurement of anticancer activity
ArH), 7.31 (d, 1H, J ¼ 2.8 Hz, ArH), 7.49 (d, 1H, J ¼ 8.8 Hz, ArH), 8.47
1
3
(s, 1H, C2-H) ppm; C NMR (100 MHz, DMSO-d
6
): d 20.0, 26.9, 27.3, Human breast cancer cell line (MCF-7) and the human mammary
2
1
8.5, 29.6, 31.8, 119.8, 121.8, 125.1, 130.2, 130.4, 132.5, 137.5, epithelial cell line (MCF-10A) were obtained from American Type
40.5, 151.1, 151.9, 162.9, 166.8 ppm; Anal. Calcd for C H ClN OS Culture Collection, they were cultured using Dulbecco’s Modified
18
17
2
(344.86): C, 62.69; H, 4.97; N, 8.12. Found: C, 62.83; H, 4.69; N, 8.35. Eagle’s medium (DMEM) (Invitrogen/Life Technologies, Carlsbad,
4
-(4-Fluorophenoxy)-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]-
CA) supplemented with 10% fetal bovine serum (FBS) (Hyclone,
ꢁ
thieno[2,3-d]pyrimidine (5g). Yield: 22%; m.p.>300 C; IR (KBr) USA), 10 mg/mL of insulin (Sigma-Aldrich, St. Louis, MO), and 1%
v
max: 3078, 3012 (C–H aromatic), 2920, 2850 (C–H aliphatic), 1620 penicillin–streptomycin. All of the other chemicals and reagents
ꢀ
1 1
(C¼N) cm
;
H NMR (400 MHz, DMSO-d
6
): d 1.72–1.75 (m, 4H, were purchased from Sigma-Aldrich, or Invitrogen.
2
CH ), 1.87–1.90 (m, 2H, CH ), 2.97–3.00 (m, 2H, CH ), 3.26–3.29
2
2
2
(
2
m, 2H, CH ), 7.28–7.34 (m, 4H, ArH), 8.47 (s, 1H, C2-H) ppm;
1
3
Cell culture protocol
C NMR (100 MHz, DMSO-d ): d 26.9, 27.3, 28.5, 29.6, 31.8,116.6,
6
1
1
16.8, 119.8, 124.4, 124.5, 132.6, 136.9, 140.5, 148.5, 151.9, 163.1,
66.7 ppm; EIMS [m/z, %]: 314 [M· , 5.12], 303 [100.00]; Anal.
The culture medium was removed to a centrifuge tube. The cell
layer was rinsed with 0.25% (w/v) trypsin 0.53 mM EDTA solution
by which all traces of serum containing trypsin inhibitor were
removed. Then, 2.0–3.0 mL of trypsin EDTA solution was added to
a flask and cells were monitored under an inverted microscope
until cell layer was dispersed (usually within 5–15 min). Complete
growth medium (6.0–8.0 mL) was added. The cell suspension was
then centrifuged for 5–10 min. The cell pellet was resuspended in
a fresh growth medium and suitable aliquots of the cell suspen-
sion were added to new culture vessels. Cultures were incubated
þ
Calcd for C H FN OS (314.38): C, 64.95; H, 4.81; N, 8.91. Found:
C, 65.11; H, 4.89; N, 9.14.
1
7
15
2
4
-(o-Tolyloxy)-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]-
ꢁ
thieno[2,3-d]pyrimidine (5h). Yield: 24%; m.p.>300 C; IR (KBr)
vmax: 3066, 3024 (C–H aromatic), 2924, 2846 (C–H aliphatic), 1589
ꢀ
1
1
(
2
C¼N) cm
CH ), 1.88–1.90 (m, 2H, CH
CH ), 3.30–3.33 (m, 2H, CH ), 7.22 (t, 2H, ArH), 7.28 (d, 1H,
;
H NMR (400 MHz, DMSO-d
6
): d 1.70–1.71 (m, 4H,
2
2
), 2.08 (s, 3H, CH
3
), 2.97–2.99 (m, 2H,
2
2
J ¼ 7.6 Hz, ArH), 7.35 (d, 1H, J ¼ 7.6 Hz, ArH), 8.44 (s, 1H, C2-H)
ꢁ
at 37 C for 24 h.
1
3
ppm; C NMR (100 MHz, DMSO-d ): d 16.3, 26.9, 27.3, 28.5, 29.6,
6
3
1
1.9, 119.5, 122.9, 126.3, 127.6, 130.4, 131.6, 132.7, 140.4, 150.9,
þ
52.0, 162.7, 166.7 ppm; EIMS [m/z, %]: 311 [M þ 1e· , 38.80], 310 MTT assay protocol
þ
[M· , 81.28], 56 [100.00]; Anal. Calcd for C H N OS (310.41): C,
18 18 2
Anticancer activity of the newly synthesized compounds was eval-
6
9.65; H, 5.84; N, 9.02. Found: C, 69.88; H, 6.07; N, 9.31.
uated in vitro against MCF-7 according to the 3-(4,5-dimethylthia-
4
-(p-Tolyloxy)-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]-
3
4
ꢁ
zol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT)
method .
thieno[2,3-d]pyrimidine (5i). Yield: 22%; m.p.>300 C; IR (KBr)
V
R
ꢀ
1
1
Doxorubicin (Adriamycin ) and erlotinib were used as reference
standards. Cells were plated (cells density 1.2–1.8 ꢂ 10,000 cells/
well) in a volume of 100 mL complete growth medium and 100 mL
of the tested compound per well in a 96-well plate for 24 h before
the MTT assay. Treatment of the cells with different concentrations
vmax: 3032 (C–H aromatic), 2920, 2850 (C–H aliphatic) cm
NMR (400 MHz, DMSO-d6): d 1.70–1.72 (m, 4H, 2CH ), 1.87–1.90
m, 2H, CH ), 2.35 (s, 3H, CH ), 2.97–2.99 (m, 2H, CH ), 3.27–3.30
; H
2
(
(
2
3
2
m, 2H, CH
2
), 7.15 (d, 2H, J ¼ 8.4 Hz, ArH), 7.27 (d, 2H, J ¼ 8.4 Hz,
13
ArH), 8.45 (s, 1H, C2–H) ppm; C NMR (100 MHz, DMSO-d
6
): d
(
0.39, 1.56, 6.25, 25 and 100 mM) of the test compounds, doxorubi-
2
1
5
6
0.9, 26.9, 27.3, 28.5, 29.6, 31.9, 119.8, 122.2, 130.5, 132.6, 135.2,
ꢁ
þ
cin and erlotinib followed by incubation for 48 h at 37 C. Then
cultures were removed from incubator into laminar flow hood or
other sterile work area. Each vial of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) [M-5655] was reconstituted
with 3 mL of medium or balanced salt solution without phenol
red and serum. Consequently, reconstituted MTT was added in an
amount equal to 10% of the culture medium volume. Cultures
were incubated for 2–4 h based on cell type and maximum cell
density. Cultures were removed from incubator and the afforded
formazan crystals were dissolved via addition of MTT solubilization
solution [M-8910]. Color intensity was measured spectrophotomet-
rically by ROBONIK P2000 spectrophotometer at a wavelength of
570 nm. The percentage of surviving cells was plotted against
drug concentration to get the survival curve of MCF-7 after each
compound. The IC50 value [the concentration required for 50%
inhibition of cell viability] for each test compound and reference
drug compound doxorubicin and erlotinib was calculated in mM.
40.3, 148.5, 150.3, 152.0, 163.3 ppm; EIMS [m/z, %]: 311 [M þ 1e· ,
þ
1.51], 310 [M· , 15.82]; Anal. Calcd for C18
H
18 2
N OS (310.41): C,
9.65; H, 5.84; N, 9.02. Found: C, 69.87; H, 5.98; N, 9.31.
4
-(4-Methoxyphenoxy)-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]-
ꢁ
thieno[2,3-d]pyrimidine (5j). Yield: 26%; m.p.>300 C; IR (KBr)
ꢀ
1
v
max: 3055, 3032 (C–H aromatic), 2920, 2850 (C–H aliphatic) cm ;
1
H NMR (400 MHz, DMSO-d ): d 1.69–1.71 (m, 4H, 2CH ), 1.88–1.89
6
2
(m, 2H, CH
2
), 2.96–2.98 (m, 2H, CH
2 2
), 3.26–3.29 (m, 2H, CH ), 3.79
(
s, 3H, OCH ), 7.00 (d, 2H, J ¼ 9.2, ArH), 7.19 (d, 2H, J ¼ 9.2, ArH),
3
13
8
2
1
6
.45 (s, 1H, C2-H) ppm; C NMR (100 MHz, DMSO-d ): d 26.9, 27.3,
8.5, 29.6, 31.8, 55.9, 115.1, 119.7, 123.4, 132.7, 140.2, 145.7, 152.0,
þ
57.2, 163.5, 166.6 ppm; EIMS [m/z, %]: 327 [M þ 1e· , 25.15], 326
þ
[
M· , 100.00]; Anal. Calcd for C18
18
H N
2 2
O S (326.41): C, 66.23; H,
5
.56; N, 8.58. Found: C, 66.44; H, 5.72; N, 8.80.
4
-(4-Nitrophenoxy)-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]-
ꢁ
thieno[2,3-d]pyrimidine (5k). Yield: 78%; m.p. 166–169 C; IR (KBr)
ꢀ
1
v
max: 3101, 3059 (C–H aromatic), 2920, 2843 (C–H aliphatic) cm ;
1
H NMR (400 MHz, DMSO-d ): d 1.70–1.72 (m, 4H, CH ), 1.88–1.89
6
2
(
(
m, 2H, CH
2
), 2.98–3.00 (m, 2H, CH
2
2
), 3.24–3.27 (m, 2H, CH ), 7.61
In vitro measurement of inhibitory activity of compound 5a, 5b,
d, 2H, J ¼ 9.2 Hz, ArH), 8.35 (d, 2H, J ¼ 9.2 Hz, ArH), 8.52 (s, 1H, C2-
5d–g and 5k against EGFR
1
3
H) ppm; C NMR (100 MHz, DMSO-d ): d 26.9, 27.3, 28.5, 29.6,
6
3
1
1.8, 120.0, 123.7, 125.9, 132.4, 141.1, 145.2, 151.8, 157.7, 162.2, Compounds 5a, 5b, 5d, 5e, 5f, 5g and 5k were further submitted
þ
þ
67.3 ppm; EIMS [m/z, %]: 342 [M þ 1e· , 29.82], 341 [M· , 100.00]; for evaluation against EGFR (Cat. # 40321) using EGFR assay kit:
Anal. Calcd for C H N O S (341.38): C, 59.81; H, 4.43; N, 12.31. cloud clone SEA757Hu 96 tests according to manufacturer’s
1
7 15 3 3
Found: C, 59.72; H, 4.61; N, 12.49.
instructions. The assay kit exits as 96 well form with purified

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
843
recombinant EGFR enzyme, EGFR substrate, ATP and kinase assay for 30 min then washed five times with 200 mL/well PBS/T followed
0
0
buffer for 100 enzyme reactions. All samples and controls were by addition of 100 mL/well 3,3 ,5,5 -tetramethylbenzidine (TMB)
tested in triplicate. 5ꢂ Kinase assay buffer, ATP and 50ꢂ PTK sub- substrate and incubation at room temperature for 15 min and
strate were thawed. The master mixture was constructed (25 mL finally addition of 100 mL/well of stop solution and mix well. The
per well), consequently 25 mL was added to every well. About 5 mL absorbance was read at 450 nm with a microtiter plate reader. The
inhibitor solution was added to each well labeled as “Test values of % activity versus a series of compound concentrations
Inhibitor” while for the “Positive control” and “Blank”, 5 mL of the (0.01 mM–0.1 mM–1 mM–10 mM) was then plotted using non-linear
buffer solution without inhibitor (Inhibitor buffer) was added.
regression analysis of sigmoidal dose-response curve. The IC50 val-
About 3 mL of 1ꢂ kinase assay buffer was developed through ues for compounds 5a, 5b and 5d–f against VEGFR-2 were deter-
mixing 600 mL of 5ꢂ kinase assay buffer with 2400mL water. About mined by the concentration causing a half-maximal percent
2
0mL of 1ꢂ kinase assay buffer was added to the wells designated activity and the data were compared with vandetanib as standard
as “Blank”. EGFR enzyme was thawed on ice. Consequent to the VEGFR-2 inhibitor.
first thaw, the tube containing enzyme was shortly spun so as to
recover full content of the tube. The amount of EGFR required for
the assay was calculated then the enzyme was diluted to 1 ng/mL Molecular docking of the compound 5b and 5f
with 1ꢂ kinase assay buffer. Reaction was initiated via addition of
The molecular docking of the compounds 5b and 5f were performed
using Molecular Operating Environment (MOE, 10.2008; Montreal,
2
0mL of diluted EGFR enzyme to the wells designated “Positive
ꢁ
Control” and “Test Inhibitor Control” then incubation at 30 C for
Quebec, Canada) software. Compounds were minimized with MOE
4
0min. Kinase-Glo Max reagent was thawed. After the 40 min reac-
ꢀ1 ꢀ1
till an RMSD gradient of 0.05 kcal mol
Å
using MMFF94ꢂ force
tion, 50mL of Kinase-Glo Max reagent was added to each well.
Plates were covered with aluminum foil and incubated at room
temperature for 15min. Finally, luminescence was measured using
the microplate Robonik P2000 ELISA Reader (India). Erlotinib, lapati-
nib and gefitinib were selected as reference drugs due to their
potent inhibitory activity of EGFR.
field and the partial charges were automatically calculated. The X-ray
crystallographic structures of EGFR co-crystallized with erlotinib (PDB
ID: 1m17) and VEGFR-2 co-crystallized with tivozanib as inhibitor
(PDB ID: 4ASE) were downloaded from the protein data bank.
Enzyme structure was inspected at first for missing bonds, atoms and
contacts. The receptor was prepared for docking study using
Protonate 3D protocol in MOE with default options and water for
crystallization was kept as it’s important for binding with the active
site. The cocrystallized ligand was used to determine the active site
for docking. Triangle matcher placement method and London dG
scoring function were used for docking. Docking setup was first vali-
dated by re-docking of the cocrystallized ligand (erlotinib and tivoza-
nib) in the pocket of the active site of both receptors with energy
score (S)¼ ꢀ10.97 and ꢀ17.04 kcal/mol, respectively. The validated
setup was subsequently utilized in order to predict the ligands recep-
tor interactions at the active site for compounds 5b and 5f. Poses
displaying the best superimposition mode on the ligand and binding
energy score for each compound were analyzed to identify potential
interactions with amino acids in the active site of each enzyme.
In vitro measurement of inhibitory activity of compounds 5a,
5b and 5d–f against VEGFR-2
Compounds 5a, 5b and 5d–f was submitted for evaluation against
VEGFR-2 [#7788] using recombinant human VEGFR-2/KDR ELISA kit
according to manufacturer’s instructions. About 10 mL of 10 mM
ATP was added to 1.25 mL 6 mM substrate peptide followed by
dilution of the mixture with distilled water to 2.50 mL to make 2ꢂ
ATP/substrate cocktail ([ATP] ¼ 40 mM, [substrate] ¼ 3 mm). The
ꢁ
enzyme was immediately transferred from –80 C to ice and was
allowed to thaw on ice. Microcentrifuge was performed briefly at
ꢁ
4
(
C and returned immediately to ice. About 10 mL of dithiothreitol
V
R
DTT) (1.25 M) was added to 2.5 mL of 4ꢂ HTScan Tyrosine
Kinase Buffer [240 mM 4-(2-hydroxyethyl)-1-piperazineethanesul-
fonic acid (HEPES) pH 7.5, 20 mM MgCI , 20 mM MnCI , 12 mM Cell cycle analysis of compound 5f
Na VO ] to make DTT/Kinase buffer. About 0.6 mL of DTT/Kinase
2
2
3
4
Cell cycle were carried out using propidium iodide flow cytometry kit
Abcam, ab139418) according to the manufacturer’s instructions. The
buffer was transferred to each enzyme tube to make 4ꢂ reaction
mixture. Exactly 12.50 mL of the 4ꢂ reaction mixture was incu-
bated with 12.50 mL/well of prediluted tested compounds 5a, 5b
and 5d–f (around 0.01, 0.1, 1 and 10 mM) for 5 min at room tem-
perature. About 25 mL of 2ꢂ ATP/substrate cocktail was added to
(
MCF-7 cells were treated with 0.66 mM of compound 5f for 24 h.
Then, the cells were washed twice with ice-cold PBS, centrifuged and
fixed using ice-cold 66% (v/v) ethanol, washed with PBS, re-suspended
with 0.1 mg/mL RNase and stained with 200 mL propidium iodide (PI).
Finally, cells were analyzed by flow cytometry. The cell cycle distribu-
tions were calculated using Cell-Quest software (Becton Dickinson). An
interference with the normal cell cycle distribution resulted upon
exposure of MCF-7 cells to this compound as indicated.
2
5 mL/well pre-incubated reaction mixture/compound. Reaction
plate was incubated at room temperature for 30 min. Then, 50 mL/
well stop buffer (50 mM EDTA, pH 8) was added to stop the reac-
tion. About 25 mL of each reaction and 75 mL distilled water/well
was transferred to a 96-well streptavidin coated plate and incu-
bate at room temperature for 60 min and subsequently washed
three times with 200 mL/well PBS (phosphate-buffered saline)/T.
Primary antibody, Phospho-Tyrosine mAb (P-Tyr-100) were diluted
Measurement of apoptosis using Annexin-V-FITC apoptosis
detection kit
1
1
:1000 in PBS/T with 1% BSA (bovine serum albumin). About
00 mL/well primary antibody was added and incubated at room Apoptosis was evaluated utilizing Annexin V-FITC/PI apoptosis
temperature for 60 min followed by washing three times with detection kit (Biovision, Mountain View, CA #K101-25) according
00 mL/well PBS/T.
to the manufacturer’s instructions after staining the cells with
Appropriate dilution of HRP labeled secondary antibody in Annexin V fluorescein isothiocyanate (FITC) and counterstaining
PBS/T was prepared with 1% BSA (1:500 dilution for anti-mouse with propidium iodide (PI).
2
5
IgG or 1:1000 for anti-rabbit IgG). About 100 mL/well secondary
About 1–5 ꢂ 10 cells were exposed to compound 5f at its IC50
antibody solution was added and incubated at room temperature concentration (0.66 mM) for 24 h. The cells then were centrifuged

8
44
A. E.-S. MGHWARY ET AL.
and in case of adherent cells, cells were gently trypsinized and formic acid gave 6,7,8,9-tetrahydro-3H-cyclohepta[4,5]thieno[2,3-
washed once with serum-containing media followed by resuspen- d]pyrimidin-4(5H)-one (2) which was subsequently underwent
sion in 500 mL of binding buffer. About 5 mL of Annexin V-FITC chlorination upon heating under reflux with phosphorus oxychlor-
and 5 mL of (PI 50 mg/mL) were added and incubated at room ide to afford 4-chloro-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]-
3
3
temperature for 5 min in the dark. Analyses were carried out using thieno[2,3-d]pyrimidine (3) as reported . Reacting compound 3
flow cytometer (Ex ¼ 488 nm; Em ¼ 530 nm) using FITC signal with the appropriate substituted benzenesulphonamide in isopro-
detector (usually FL1) and PI staining by the phycoerythrin emis- panol resulted in 4-arylaminothienopyrimidine derivatives 4a–c. IR
spectra showed the appearance of two absorption bands at
sion signal detector (usually FL2).
ꢀ
1
3
456–3228 cm
2
corresponding to NH, NH groups. Moreover,
1
H NMR spectrum of compound 4a elucidated two exchangeable
Results and discussion
singlet peaks at d 7.22 and 9.05 ppm relative to NH and NH pro-
2
tons. Additionally, compounds 4b and 4c showed two exchange-
Chemistry
able singlet peaks at
d
6.00, 9.07 and 4.77, 9.11 ppm
The target compounds were synthesized as outlined in Figure 3. corresponding to two NH protons. It also displayed the expected
Our primary starting material 2-amino-5,6,7,8-tetrahydro-4H-cyclo- signals corresponding to the aromatic protons of 4a–c, compound
hepta[b]thiophene-3-carbonitrile (1) was synthesized utilizing one 4c further revealed the appearance of CH group as singlet signal
3
3
5–37
13
step Gewald reaction
through the reaction of cycloheptanone at d 2.33 ppm. Additionally, C NMR spectra of compounds 4a–c
with malononitrile and sulfur in presence of catalytic amount of was as a further evidence and it revealed an increase in absorbance
33
diethylamine as reported by Arya . Reacting compound 1 with peaks in the aromatic region corresponding to the aromatic carbons.
Figure 3. The synthetic pathway and reagents for the preparation of the target compounds 4a–c and 5a–k.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
845
Table 1. Results of in vitro anticancer activity of the newly synthesized compounds 4a–c and 5a–k against MCF-7 cell line.
SO NHR
2
R1
O
N
HN
N
N
N
S
S
5
a-k
4a-c
1
a
ꢃ
b
ꢃ
Compound No.
R
R
IC50 , mM ±SD
IC50 , mM ±SD
Selectivity index
4
4
a
b
H
–
39.75 ± 4.11
7.12 ± 0.55
–
–
–
–
–
–
N
N
N
4
c
N
10.98 ± 1.24
–
–
CH3
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
–
–
–
–
–
–
–
–
–
–
–
–
–
3-NH
4-NH
4-Br
2-Cl
4-Cl
4-Cl-3-CH
4-F
2
2
1.98 ± 0.21
1.47 ± 0.11
6.65 ± 0.41
1.34 ± 0.07
2.76 ± 0.33
0.66 ± 0.05
1.23 ± 0.04
87.43 ± 5.4
21.07 ± 2.7
5.01 ± 0.31
2.76 ± 0.19
3.06 ± 0.19
1.14 ± 0.05
–
–
49.5
–
–
72.74 ± 3.98
–
–
–
–
3
153.16 ± 7.57
232.1
–
–
–
–
–
–
–
–
–
–
–
2-CH
3
4-CH3
4-OCH
4-NO
j
k
3
2
–
Doxorubicin
Erlotinib
–
–
27.58 ± 1.55
69.41 ± 4.24
a
IC50 on MCF-7 cell line.
IC50 on normal mammary epithelial cell line (MCF-10A).
The results given are means of three experiments.
b
ꢃ
Furthermore, compound 4c showed absorbance peak at d 23.7 ppm results expressed as half maximal inhibitory concentration (IC50
)
corresponding to CH . The nucleophilic substitution of 4-chlorothieno- values, were summarized in Table 1 and graphically illustrated in
3
pyrimidine 3 with different substituted phenols was achieved via dis- Figure 4. According to the in vitro results, it was revealed that all
solving various substituted phenols in potassium hydroxide and of the test compounds except 5h exhibited moderate to signifi-
ethanol in order to form potassium phenoxide that subsequently cant anticancer activity against MCF-7 cell line. Seven compounds
heated under reflux with 4-chlorothienopyrimidine 3 to afford com- 5a, 5b, 5d–g and 5k showed 4.64–1.11-folds more potent anti-
pounds 5a–k. Compounds 5a–k were verified by spectral data and cancer activity than doxorubicin, six of them 5a, 5b, 5d, 5e, 5g
elemental analysis. IR spectra of compound 5a and 5b showed two
and 5k were comparable to erlotinib while compound 5f was
more potent than erlotinib. Four compounds 4b, 4c, 5c and 5j
displayed anticancer activity comparable to both doxorubicin and
erlotinib with IC50 values 7.12, 10.98, 6.65 and 5.01 mM, respect-
ively. Compounds 4a and 5i showed moderate anticancer activity.
From the activity results, the relation between the test com-
pounds and the activity can be concluded as follows:
ꢀ
1
absorption bands at 3452–3325 and 3440–3400 cm , respectively
1
2
which corresponding to NH groups. Furthermore, H NMR revealed
the expected signals of the aromatic protons corresponding to the
substituted phenoxy rings which appear at range of d 6.34–8.52 ppm,
also compounds 5a and 5b confirmed the presence of NH group as
2
singlet exchangeable signal at d 5.28 and 5.12 ppm, respectively. In
addition, compound 5f, 5h and 5i showed singlet signal at d 2.34,
2
.08 and 2.35 ppm, respectively which is an evidence for the presence
1
.
The introduction of different substituents at C-4 position has
pronounced influence on the antitumor activity.
a. Presence of benzenesulphonamide moieties compounds
1
of CH
O-CH
3
group while H NMR spectrum of compound 5j elucidated the
3
group as singlet signal at d 3.79 ppm. On the other hand,
13
C NMR revealed the peaks corresponding to the carbon atoms in
4a–c exhibited moderate activity, Furthermore, com-
compounds 5a–k, additionally, compound 5f, 5h and 5i showed
absorbance peak at 20.0, 16.3 and 20.9 ppm, respectively which are
corresponding to CH
ance peak at
O–CH group.
pounds 4b and 4c bearing pyrimidine ring in benzene-
sulphonamide moiety displayed more potent anticancer
activity than 4a. On the other hand, in compound 4c,
introduction of electron donating group (–CH ) at pos-
3
ition-4 of the pyrimidine ring showed slight decrease in
the anticancer activity compared with 4b.
3
group while compound 5j displayed absorb-
55.9 ppm confirming the presence of the
d
3
b. Replacement of the benzenesulphonamide substituents
with phenoxy moieties 5a–k showed marked increase in
the anticancer activity. Additional analysis of compounds
5a–k elucidated that the presence of H-bond donor
group such as amino group in compounds 5a and 5b
interestingly enhanced the anticancer activity. Moreover,
Biological assay
In vitro anticancer activity
The newly synthesized compounds were evaluated for their anti-
cancer activity against MCF-7 cell line compared to doxorubicin
and erlotinib as reference anticancer drugs using MTT assay. The

8
46
A. E.-S. MGHWARY ET AL.
100
90
80
70
60
50
40
30
20
10
0
Comound
Figure 4. Graphical representation of IC50 expressed in mM of test compounds against MCF-7 cell line compared to doxorubicin and erlotinib.
introduction of H-bond acceptor groups such as Table 2. Results of in vitro EGFR kinase activity of compounds 5a, 5b, 5d–g
methoxy or nitro groups (compounds 5j and 5k, respect- and 5k.
IC , mM
ꢃ ± SD
ively) resulted in potent anticancer activity.
Compound No.
5
0
The potency was also favored by substitution with halo-
5
a
0.07 ± 0.003
0.042 ± 0.002
0.07 ± 0.004
0.069 ± 0.003
0.028 ± 0.003
0.20 ± 0.011
0.46 ± 0.018
0.03 ± 0.002
0.05 ± 0.003
0.03 ± 0.002
gens, compounds 5c–g (with IC50 ¼ 0.66–6.65 mM). 5b
5
5
5
d
e
f
Moreover, compound 5f that showed presence of
methyl group at position-3 in addition to 4-chloro atoms
revealed the most potent anticancer activity with IC50 in
micromolar range (IC50 ¼ 0.66 mM).
5g
5k
2
.
.
Furthermore, our study also demonstrated that 4-substituted Erlotinib
Lapatinib
Gefitinib
aryloxy derivatives were more potent anticancer activity than 2-
and 3-substituted ones, such as compound compare 5a with
ꢃThe results given are means of three experiments.
14
5
b, compound 5f with 5d and compound 5i with 5h position .
3
Finally, compounds 5b and 5f (the most potent compounds in
the present work) were tested for their possible anticancer
activity on the human mammary epithelial cell line (MCF-10A).
The results revealed that the compounds showed IC50 of 72.74
and 153.16 mM, respectively. They exhibited approximately
ꢄ
Changing p-amino substituent by H-acceptor group (p-nitro)
dramatically decreased the enzyme inhibition activity (compare
compound 5b and 5k).
4
9.5–232 times more selectivity against the tumor cell line.
Introduction of chloro substituent in the aryloxy ring slightly
increased the EGFR inhibitory activity (compound 5d and 5e)
while replacing p-chloro with p-fluoro greatly decreased the
enzyme inhibition (compare compound 5e and 5g). On the other
Measurement of in vitro EGFR and VEGFR-2 inhibition
The target compounds 5a, 5b, 5d–g and 5k that showed potent
anticancer activity against MCF-7 breast cancer cell line were
tested for their inhibitory activity against EGFR. The IC50 value of
the tested compounds were evaluated compared to reference
EGFR inhibitors (erlotinib, gefitinib and lapatinib). Compounds 5a,
hand, compound 5f that possessed (3-CH ) group in addition to
3
p-chloro substituent revealed potent EGFR inhibitory activity.
Furthermore, the compounds 5a, 5b and 5d–f that exhibited
potent EGFR inhibitory activity were further submitted for VEGFR-
2
biological enzyme assay to confirm the dual inhibitory activity
5d and 5e exhibited comparable inhibitory activity on EGFR to
of these compounds.
that of the reference EGFR inhibitor lapatinib but compounds 5b
and 5f displayed excellent EGFR inhibitory activity, at the sub-
micromolar level with IC50 value 0.042 and 0.028 mM, respectively,
compound 5b was 1.24-folds more potent than lapatinib but com-
parable with both erlotinib and gefitinib, while compound 5f was
The IC50 value of compounds 5a, 5b and 5d–f were compared
to reference VEGFR-2 inhibitor (vandetanib). Compounds 5b, 5d
and 5f showed good VEGFR-2 inhibitory activity, at the sub-micro-
molar level with IC50 value 0.51, 0.74 and 1.23 mM, respectively,
which were comparable to that of vandetanib (IC50 ¼ 0.43 mM),
1
.83–1.18-folds more potent than the three reference drugs erloti-
nib, gefitinib and lapatinib (IC50 ¼ 0.033, 0.038 and 0.052 mM, moreover, compound 5e exhibited the most potent VEGFR-2
respectively) (Table 2 and Figure 5).
inhibitory activity with IC50 value 0.34 mM while compound 5a dis-
Examining the structures and EGFR inhibitory activity of the played the least VEGFR-2 inhibitory activity with IC50 value
selected compounds demonstrated the following structure–activity 1.71 mM compared to vandetanib (Table 3 and Figure 6).
relationship:
Examining the structures of the selected compounds demon-
strated the following structure–activity relationship as dual EGFR
and VEGFR-2 inhibitors:
ꢄ
Presence of p-amino substituent (in compound 5b) interest-
ingly enhanced the EGFR inhibitory activity while changing the
position of amino group from p- to m-position decreased the
enzyme inhibition activity (compare compound 5a and 5b).
ꢄ
Presence of H-bond donor group (NH
aryloxy moiety (compound 5b) showed potent dual EGFR and
) in p-position of the
2

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
847
0
0
0
0
0
0
.6
.5
.4
.3
.2
.1
0
Compound
Figure 5. Graphical representation for IC50 of EGFR assay in mM of compounds 5a, 5b, 5d–g and 5k.
0
.028 mM) was supported by their good docking score (ꢀ10.13
Table 3. Results of in vitro VEGFR-2 kinase activity of compounds 5a, 5b
and 5d–f.
and ꢀ10.51 kcal/mol, respectively), which was comparable to that
ꢃ
Compound No.
IC50, mM ±SD of erlotinib (ꢀ10.97 kcal/mol), additionally, their excellent superim-
position on the ligand (erlotinib) in the active site.
5
5
5
5
5
a
b
d
e
f
1.71 ± 0.062
0.51 ± 0.016
0.74 ± 0.025
0.34 ± 0.013
1.23 ± 0.028
0.43 ± 0.012
Molecular docking of compound 5b and 5f in the active site of
VEGFR-2
Vandetanib
ꢃThe results given are means of three experiments.
Docking study was performed for compound 5b and 5f which dis-
played good activity in the VEGFR-2 enzyme inhibition assay,
which were comparable to that of vandetanib, in order to illus-
trate the binding mode of these compounds in the active site of
the VEGFR-2. Accordingly, we used (PDB ID: 4ASE), which has
VEGFR-2 co-crystallized with tivozanib as inhibitor. Also, vandeta-
nib which was chosen as a reference compound depending on
similarity in its structure with those of compounds 5b and 5f was
docked to ensure the validation of VEGFR-2 inhibitory activity
results of both 5b and 5f.
According to results elucidated in (Figure 8), the reference com-
pound vandetanib and the novel synthesized 4-aryloxythieno[2,3-
d]pyrimidine compounds 5b and 5f interact in VEGFR-2 ATP-binding
site through N1 of the pyrimidine ring as H-bond acceptor with the
key amino acid Cys919, following an orientation similar to the co-
crystallized tivozanib, as shown in Figure 8(A–C). The good activity
of compound 5b and its superior activity when compared to 5f
VEGFR-2 inhibitory activity with IC50 ¼ 0.042 and 0.51 mM,
respectively.
ꢄ
Replacement of p-amino group with halogen and methyl
group (compound 5f) showed potent EGFR inhibitory activity
but moderate VEGFR-2 inhibitory activity with IC50 ¼ 0.028 and
1
.23 mM, respectively.
Molecular docking of compounds 5b and 5f in the active site
of EGFR
Docking study was carried out for compounds 5b and 5f which
showed excellent activity in the EGFR enzyme inhibition assay to
illustrate the molecular reasons for the observed inhibition activity
profile, such as the amino acids in the EGFR active site that sup-
posed to be involved in the expected hydrogen bonding interac-
tions. Based on the obtained results with the semi-rigid docking
approach, the novel synthesized 4-aryloxythieno[2,3-d]pyrimidine
compounds interacted in the EGFR ATP-binding site where the N1
of pyrimidine ring interacted as an H-bond acceptor with the
amino acid Met 769 and N3 of pyrimidine ring also as an H-bond
acceptor interacted through water molecule with the amino acid
Thr 766 in a similar orientation to that of the co-crystallized drug
erlotinib in the reference crystallographic structure (PDB ID:
(
IC50 ¼ 0.51 mM) probably correlated with the ability of the amino
group to form two hydrogen bonds with the side chain of the
amino acid Glu885 and the C¼O from the Asp1046 backbone
(
Figure 8(E,F)). These findings support results previously described in
38–40
the literature
which indicate the correlation of hydrogen bond
interactions with these amino acids for VEGFR-2 potent inhibition.
Additionally, the good activity of compounds 5b and 5f against
VEGFR-2 (IC50 ¼ 0.51 and 1.23 mM, respectively) were validated by
their good docking score (ꢀ12.02 and ꢀ12.90 kcal/mol, respectively).
1
m17), as shown in Figure 7(A,B). As previously mentioned, the
increased potency of compound 5b of almost 1.24-folds more
potent than lapatinib but comparable with both erlotinib and
gefitinib against EGFR (IC50 ¼ 0.042 mM) could be related to the
Cell cycle analysis and detection of apoptosis
hydrogen bond donating ability of the amino group, which pro- The most potent compound 5f was chosen for further investiga-
vides an additional interaction with the side chain of amino acid tion of its effects on cell cycle progression and induction of apop-
Glu738 (Figure 7(C)). For compound 5f, which displayed the most tosis in the MCF-7 cell line. Exposure of MCF-7 cells to compound
significant inhibitory activity being 1.83–1.18-folds more potent 5f at its IC50 value for 24 h and its effect on the normal cell cycle
than erlotinib, gefitinib and lapatinib could be attributed to the profile and induction of apoptosis were analyzed. Exposure
presence of arene cation interaction between the phenyl ring and of MCF-7 cells to compound 5f caused an interference with the
the amino acid Lys721 (Figure 7(D). Furthermore, the significant normal distribution of cell cycle of this cell line. Compound 5f
activity of compound 5b and 5f against EGFR (IC50 ¼ 0.042 and induced a pronounced increase in the percentage of cells at

8
48
A. E.-S. MGHWARY ET AL.
2
1
1
1
1
.8
.6
.4
.2
1
0
0
0
0
.8
.6
.4
.2
0
5a
5b
5d
5e
5f
vandetanib
Compound
Figure 6. Graphical representation for IC50 of VEGFR-2 assay in mM of compounds 5a, 5b and 5d–f.
Figure 7. (A) Superposition of 5b (green) with erlotinib (magenta) inside the EGFR tyrosine kinase ATP-binding site. (B) Superposition of 5f (pink) with erlotinib
magenta) inside the EGFR tyrosine kinase ATP-binding site. (C) Binding interactions of 5b (green) with EGFR showing good binding energy score value (score:
10.13). (D) Binding interactions of 5f (pink) with EGFR showing the best binding energy score value (score: ꢀ10.51).
(
ꢀ
pre-G1 and G2/M phases by 11.80 and 4.75-folds, respectively degradation or fragmentation of the genetic material, indicating a
Table 4 and Figure 9) compared to control. Accumulation of cells possible role of apoptosis in compound 5f-induced cancer cell
(
in pre-G1 phase, confirmed by the presence of a sub-G1 peak in death and cytotoxicity. Moreover, G2 arrest may have resulted in
the cell cycle profile analysis, which may have resulted from the accumulation of the cells in G2/M phase.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
849
Figure 8. (A) Superposition of vandetanib (orange) with tivozanib (blue) inside the VEGFR-2 tyrosine kinase ATP-binding site. (B) Superposition of 5b (green) with tivo-
zanib (blue) inside the VEGFR-2 tyrosine kinase ATP-binding site. (C) Superposition of 5f (pink) with tivozanib (blue) inside the VEGFR-2 tyrosine kinase ATP-binding
site. (D) 2D binding interactions of vandetanib (orange) with EGFR showing the best binding energy score value (score: ꢀ18.48). (E) 2D binding interactions of 5b
(
green) with VEGFR-2 showing good binding energy score value (score: ꢀ12.02). (F) 2D binding interactions of 5f (pink) with EGFR showing the best binding energy
score value (score: ꢀ12.90).
Apoptosis detection by annexin V-FITC assay
Table 4. Results of cell cycle inhibition activity of compound 5f.
Compound No.
f/MCF-7
Control/MCF-7
%G0–G1
%S
%G2/M
%Pre-G1
The ability of compound 5f to induce apoptosis was investigated
through a biparametric cytofluorimetric analysis, using propidium
iodide (PI), which stains DNA and is allowed to enter only the
5
34.19
55.93
33.16
37.2
32.65
6.87
17.58
1.49

8
50
A. E.-S. MGHWARY ET AL.
Figure 9. Effect of compound 5f (0.66 lM) on DNA-ploidy flow cytometric analysis of MCF-7 cells after 24 h.
more than control) occurred, indicating an early apoptosis (lower
Table 5. Results of apoptotic activity of compound 5f.
right quadrant). Furthermore, the study showed significant
increase in the percentage of the late apoptotic and necrotic cells
Apoptosis
Compound No.
f/MCF-7
Control/MCF-7
%Total
%Early
%Late
Necrosis
(annexin-V positive, PI positive cells) about 24.66-folds (upper right
5
17.58
1.49
5.34
0.88
9.37
0.38
2.87
0.23
quadrant) and 12.48-folds (upper left quadrant) more than control.
dead cells, and fluorescent conjugate of the protein annexin V Conclusion
with fluorescein isothiocyante (FITC), that binds to phosphatidyl-
A
series of novel 4-substituted-6,7,8,9-tetrahydro-5H-cyclohep-
serine (PS) expressed on the surface of the apoptotic cells and flu-
oresces green, after interacting with the labeled annexin-V. Dual
staining of annexin-V with PI grants differentiation between viable
cells, early apoptotic cells, late apoptotic cells and necrotic cells.
As shown in Table 5 and Figure 10, after 24 h of treatment with
compound 5f at its IC50 concentration (0.66 mM) a decrease in the
percentage of the survived cells was observed. Compound 5f
caused a pronounced elevation in the percentage of the total
ta[4,5]thieno[2,3-d]pyrimidine derivatives was synthesized and was
evaluated for their anticancer activity against MCF-7 cell line.
Seven compounds 5a, 5b, 5d–g and 5k showed 4.64–1.11-folds
more potent anticancer activity than doxorubicin, six of them 5a,
5
b, 5d, 5e, 5g and 5k were comparable to erlotinib while com-
pound 5f showed the most significant anticancer activity being
.64 and 1.73-folds more potent than both doxorubicin and erloti-
4
apoptosis 17.58% compared to control which showed 1.49% and nib, respectively. Moreover, Compounds 5b revealed potent dual
the percentage of the necrosis 2.87% which was higher than con- EGFR and VEGFR-2 inhibitory activity with IC50 ¼ 0.042 and
trol that demonstrated only 0.23%. Moreover,
a
significant 0.51 mM, respectively. Molecular docking attributed its potent dual
increase in the percentage of annexin-V positive cells (6.07-folds activity to the presence of additional hydrogen bonding

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
851
Figure 10. Representative dot plots of MCF-7 cells treated with 5f (0.66 lM) for 24 h and analyzed by flow cytometry after double staining of the cells with annexin-V
FITC and PI.
interaction of the p-amino group with Glu738 amino acid of EGFR
enzyme and with Asp1046 and Glu88 of VEGFR-2. On the other
hand, compound 5f that displayed potent EGFR inhibitory activity
with moderate VEGFR-2 inhibitory activity with IC50 ¼ 0.028 and
4. Masuda H, Zhang D, Bartholomeusz C, et al. Role of epider-
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1.23 mM, respectively. The increased potency of compound 5f against
EGFR compared to its potency against VEGFR-2 enzyme could be
related to its ability to form extra arene cation interaction between
the phenyl ring and the amino acid Lys721 of EGFR enzyme.
Also, compound 5f caused a pronounced increase in the per-
centage of cells at pre-G1 and G2/M phases by 11.80 and 4.75-
folds, respectively, compared to control, which confirm a possible
role of apoptosis in compound 5f-induced cancer cell death and
cytotoxicity. Compound 5f displayed potent apoptotic activity.
6
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