Discovery of potent dual egfr/her2 inhibitors based on thiophene scaffold targeting h1299 lung cancer cell line

Article abstract of DOI:10.3390/ph14010009

Dual targeting of epidermal growth factor receptor (EGFR) and human EGFR-related receptor 2 (HER2) is a proven approach for the treatment of lung cancer. With the aim of discov-ering effective dual EGFR/HER2 inhibitors targeting non-small cell lung cancer cell line H1299, three series of thieno[2,3-d][1,2,3]triazine and acetamide derivatives were designed, synthesized, and biologically evaluated. The synthesized compounds displayed IC₅₀ values ranging from 12 to 54 nM against H1299, which were superior to that of gefitinib (2) at 40 μM. Of the synthesized com-pounds, 2-(1H-pyrazolo[3,4-b]pyridin-3-ylamino)-N-(3-cyano4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)acetamide (21a) achieved the highest in vitro cytotoxic activity against H1299, with an IC₅₀ value of 12.5 nM in situ, and 0.47 and 0.14 nM against EGFR and HER2, respectively, values comparable to the IC₅₀ of the approved drug imatinib (1). Our synthesized compounds were promising, demonstrating high selectivity and affinity for EGFR/HER2, especially the hinge region forming a hydrophobic pocket, which was mediated by hydrogen bonding as well as hydrophobic and electrostatic inter-actions, as indicated by molecular modeling. Moreover, the designed compounds showed good affinity for T790M EGFR, one of the main mutants resulting in acquired drug resistance. Furthermore, both pharmacokinetic and physicochemical properties of the designed compounds were within the appropriate range for human usage as predicted by the in Silico ADME study. The designed compound (21a) might serve as an encouraging lead compound for the discovery of promising anti-lung cancer agents targeting EGFR/HER2.

Full text of DOI:10.3390/ph14010009

pharmaceuticals
Article
Discovery of Potent Dual EGFR/HER2 Inhibitors Based on
Thiophene Scaffold Targeting H1299 Lung Cancer Cell Line
Ranza Elrayess 1,† , Yasmine M. Abdel Aziz 1,
*
, Mohamed Saleh Elgawish 2,† , Marwa Elewa 1,
Asmaa S. A. Yassen 1,
*
, Sameh S. Elhady 3 , Hosam A. Elshihawy 1 and Mohamed M. Said 1
1
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Suez Canal University,
Ismailia 41522, Egypt; ranza.el-rayes@pharm.suez.edu.eg (R.E.); Marwa_elewa@pharm.suez.edu.eg (M.E.);
hosamelshihawy74@hotmail.com (H.A.E.); msaid123eg@yahoo.com (M.M.S.)
Medicinal Chemistry Department, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt;
2
mohamed_elgawish@pharm.suez.edu.eg
Department of Natural Products, Faculty of Pharmacy, King Abdulaziz University,
3
Jeddah 21589, Saudi Arabia; ssahmed@kau.edu.sa
*
Correspondence: yassmin_morsy@pharm.suez.edu.eg (Y.M.A.A.);
asmaa_yaseeen@pharm.suez.edu.eg (A.S.A.Y.); Tel.: +20-1277718225 (Y.M.A.A.); +20-1096206738 (A.S.A.Y.);
Fax: +20-064-3230741 (Y.M.A.A. & A.S.A.Y.)
†
The authors contributed equally.
Abstract: Dual targeting of epidermal growth factor receptor (EGFR) and human EGFR-related
receptor 2 (HER2) is a proven approach for the treatment of lung cancer. With the aim of discov-
ering effective dual EGFR/HER2 inhibitors targeting non-small cell lung cancer cell line H1299,
three series of thieno[2,3-d][1,2,3]triazine and acetamide derivatives were designed, synthesized,
and biologically evaluated. The synthesized compounds displayed IC50 values ranging from 12 to
54 nM against H1299, which were superior to that of gefitinib (2) at 40 µM. Of the synthesized com-
pounds, 2-(1H-pyrazolo[3,4-b]pyridin-3-ylamino)-N-(3-cyano4,5,6,7-tetrahydrobenzo[b]thiophen-2-
yl)acetamide (21a) achieved the highest in vitro cytotoxic activity against H1299, with an IC50 value of
12.5 nM in situ, and 0.47 and 0.14 nM against EGFR and HER2, respectively, values comparable to the
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Elrayess, R.; Abdel Aziz, Y.M.;
Elgawish, M.S.; Elewa, M.; Yassen, A.S.A.;
Elhady, S.S.; Elshihawy, H.A.; Said,
M.M. Discovery of Potent Dual EGFR/
HER2 Inhibitors Based on Thiophene
Scaffold Targeting H1299 Lung Cancer
Cell Line. Pharmaceuticals 2021, 14, 9.
https://dx.doi.org/ 10.3390/ph14010009
IC50 of the approved drug imatinib (1). Our synthesized compounds were promising, demonstrating
high selectivity and affinity for EGFR/HER2, especially the hinge region forming a hydrophobic
pocket, which was mediated by hydrogen bonding as well as hydrophobic and electrostatic inter-
actions, as indicated by molecular modeling. Moreover, the designed compounds showed good
affinity for T790M EGFR, one of the main mutants resulting in acquired drug resistance. Furthermore,
both pharmacokinetic and physicochemical properties of the designed compounds were within
the appropriate range for human usage as predicted by the in Silico ADME study. The designed
compound (21a) might serve as an encouraging lead compound for the discovery of promising
anti-lung cancer agents targeting EGFR/HER2.
Received: 24 November 2020
Accepted: 21 December 2020
Published: 24 December 2020
Keywords: thieno[2,3-d][1,2,3]triazine; acetamide; H1299; HER2; EGFR
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional claims
in published maps and institutional
affiliations.
1. Introduction
Lung cancer continues to be one of the major causes of cancer-related mortality, and
non-small cell lung cancer (NSCLC) cases account for approximately 90% of all lung can-
cer [
1
]. Treatment options for patients with advanced lung cancer, including chemotherapy,
radiation, or surgery, are still insufficient, and around half of limited-stage lung cancer
patients relapse [ ]. Receptor tyrosine kinases (RTKs) play a significant role in cellular
Copyright: © 2020 by the authors. Li-
censee MDPI, Basel, Switzerland. This
article is an open access article distributed
under the terms and conditions of the
Creative Commons Attribution (CC BY)
license (https://creativecommons.org/
licenses/by/4.0/).
2,3
signaling pathways and regulate the majority of cellular processes, such as cell metabolism,
differentiation, proliferation, and apoptosis. Among the well-known RTKs, members of the
ERbB receptor tyrosine kinase family, including epidermal growth factor receptor (EGFR;
HER1/erbB-1), human EGFR-related receptor 2 (HER2; erbB-2/neu), HER3 (erbB-3), and
Pharmaceuticals 2021, 14, 9. https://dx.doi.org/10.3390/ph14010009
https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021, 14, 9
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HER4 (erbB-4), represent a promising strategy for targeted therapy in patients with NSCLC
due to observed patterns of oncogenic mutations of EGFR and HER2 [4]. Therefore, tar-
geting the ERbB receptor could be a promising strategy in NSCLC eradication. HER2
overexpression currently serves as a biomarker of poor prognosis in many forms of human
cancer and is considered to be responsible for around 11–32% of NSCLC tumors, with
increased gene copy numbers recognized in 2–23% of cases. As HER2 overexpression
is found to potentiate EGFR signaling, dual inhibition of these two pathways is of great
clinical interest [5].
First-generation EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib (
2), imatinib
(
1
), lapatinib ( ), and erlotinib ( ) have been approved by the US Food and Drug Ad-
4
3
ministration (FDA) for treatment of EGFR-activating mutation-positive NSCLC patients
(Figure 1). Acquired drug resistance, however, often occurs after approximately 12 months
of treatment with gefitinib (2) or imatinib (1) [6,7]. Lapatinib (4), the first dual oral inhibitor
of EGFR and HER2, was approved by the FDA in 2007 for patients with advanced or
metastatic breast cancer (Figure 1). These drugs bind competitively and reversibly to the
adenosine triphosphate (ATP) binding site of EGFR/HER2, achieving target selectivity by
identifying unique characteristics of specific ATP-binding features [5–7]. Unfortunately, the
ability of these EGFR inhibitors to successfully treat NSCLC is short-lived in subsequent
therapies due to acquired T790M missense secondary mutations in EGFR, contributing
to drug resistance in around 50% of patients. The mutation of EGFR T790M restores ATP
affinity to levels close to those of wild type EGFR, preventing the first generation of TKIs
from binding ATP at higher levels. To overcome this resistance, second-generation TKIs,
represented by afatinib (5) (Vizimpro™, Pfizer Inc., New York, NY, USA) and neratinib (6)
(Nerlynx , Puma Biotechnology Inc.), were developed. They have been reported to be
™
superior to first-generation EGFR TKIs in cases of EGFR mutation [6,7].
The majority of the second generation has been developed by structure-guided incor-
poration of an electrophilic moiety into inhibitors possessing binding affinity to the target
of interest. This electrophilic moiety is designed to be attacked by the highly nucleophilic
sulfhydryl of cysteine residues, forming a covalent bond [8]. Initially, covalent inhibitors
will bind noncovalently and, then, if the reactive moiety trajectory is suitable, covalent
bond formation will take place, permanently deactivating TK activity. High selectivity is
one big benefit of covalent kinase inhibitors. Additionally, the covalency could allow for
extension of the pharmacodynamic period without for the need for elevated drug concen-
trations [9,10]. Despite the therapeutic success of first- and second-generation EGFR TKIs,
EGFR-TK mutation-related resistance remains a significant clinical problem. Great effort
has been made to develop alternative strategies, resulting in third-generation TKIs. Third-
generation irreversible EGFR inhibitors containing a Michael acceptor functional group
have been developed in order to resolve the problem of second-generation irreversible
inhibitors with limited wild type (WT) EGFR activity. These inhibitors have acquired in-
creased ATP binding through irreversible alkylation of a cysteine residue. However, these
irreversible inhibitors have so far demonstrated only partial clinical efficacy, except in a few
cases [11]. Recently, however, interest in irreversible TK inhibitors has had a resurgence
from a risk–benefit perspective. The reactivity of electrophilic moieties must be tuned to
prevent rapid in vivo quenching and the formation of protein adducts, ultimately leading
to hepatotoxicity and mutagenicity [
kinase are the causes of limited clinical efficacy of third-generation TKIs such as avitinib
). (Figure 1) C797S mutations have been reported in cell-free plasma DNA samples from
9,10]. High toxicity and weak binding to mutant
(
7
patients who acquired resistance [12]. These data indicate that the development of drugs
that do not depend on covalent reaction with Cys797 is important for potency or selectivity.

Pharmaceuticals 2021, 14, 9
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F
O
HN
4
Cl
HN
4
6
6
7
N
O
O
O
O
3
3
N
N
O
O
2
2
7
N
1
N
1
Erlotinib 3
Gefitinib 2
First Generation
First Generation
2
3
0
0
0
0
4
2
O
F
N
3
N
H
H
HN
4
Cl
2
N
N
N
N
6
O
3
2
N
N
1
NH
O
S
O
N
1
2001
O
2007
FDA-Approval
Imatinib 1
First Generation
Lapatinib 4
First Generation
Cl
F
HN
O
O
N
HN
4
6
HN
O
O
4
3
N
O
N
2
3
2
N
N
1
7
HN
4
Cl
N
H
N
1
NH
H
N
6
3 CN
N
O
O
2
O
N
1
7
F
Afatinib 5
N
N
Second Generation
Neratinib 6
Second Generation
central aromatic /hetero
aromatic system
NH spacer
CN
Avitinib 7
Third Generation
Hydrophobic tail
Hydrophobic head
Amide linker
Figure 1. Representation of chemical structures of first-, second-, and third-generation tyrosine kinase inhibitors (TKIs).
Recently, it has been proposed that HER2/EGFR heterodimers or HER2/HER2 ho-
modimers trigger a cascade of signal transduction pathways that are responsible for tumor
invasion, progression, and metastasis. Therefore, dual EGFR/HER2 inhibition could be
more effective than individual EGFR or HER2 inhibition [13]. In view of this, and in
continuing the ongoing strategy aimed at developing new scaffolds for the discovery of
potent TKIs, in the current study, a new series of thienotriazine and N-(3-cyanothiophen-2-
yl)acetamide derivatives was designed and chemically synthesized (Figure 2).

Pharmaceuticals 2021, 14, 9
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OH
NH2
HN
4
HN
N
N
3
2
4
S
N
N
3
2
N
1
Compound 8
O
S
S
N
1
NH
O
Compound 13b
HN
NC
N
N
2
N
3
H
O
S
1
Compound 9
Reported compounds
Designed TK inhibitors
Target compounds
Series A
Series C
Series B
CN
2
Ar
O
HN
3
R
N
NH
S
R
N
1
CH
CN
O
HN
3
R
2
3
NH
N
4
2
N
HN
S
1
R= 3-NO2, 4-OCH3
N
1
3
N
4
2
N
HN
S
1
N
S
R= H, 2-OH, 3-NO2, 4-OCH3
central aromatic /hetero
aromatic system
CN
Hydrophobic head
NH spacer
Hydrophobic tail
Amide linker
Figure 2. Reported and designed derivatives as dual epidermal growth factor receptor (EGFR)/human EGFR-related
receptor 2 (HER2) inhibitors targeting H1299.
Rationale and Design
It was found that most of the quinazoline-based anticancer agents that selectively in-
hibit EGFR/HER2 overexpression develop resistance over a long course of treatment [14,15]
.
Thus, there is an urgent need to create novel core structures that could replace this quina-
zoline core, retaining the advantage of the two nitrogen atoms necessary for binding
with key amino acids in kinase active sites. In 2014, a study published by our group

Pharmaceuticals 2021, 14, 9
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reported the synthesis of a promising thieno[2,3-d][1,2,3]triazine-based structure as an
EGFR inhibitor (
and synthesized another thiophene-based derivative (
8
) with an IC50 of about 38 nM [15]. Moreover, our group designed
) with an IC50 of about 30.8 nM
9
against EGFR (Figure 2) [15]. In 2020, our research group succeeded in designing a novel
m-phenylenediamine thieno [1–3] triazine derivative (13b) as a promising EGFR inhibitor
with an IC50 of about 0.33 nM. Compound (13b) was targeted to inhibit the growth of the
challenging H1299 lung cancer cell line with an IC50 of about 25 nM [16]. This success
prompted us to develop a novel series of EGFR/HER2 inhibitors targeting H1299.
Moreover, it was observed that the structure of the majority of clinically approved
TKIs is characterized by a hydrophobic head and hydrophobic tail linked to a central
heteroaromatic system and NH spacer, as shown in Figure 1. A literature survey based on
the leading dual EGFR/HER2 targeting compounds lapatinib (4) and neratinib (6) shows
that the upper right warhead, or the hydrophobic tail, is essential for potency of kinase
inhibition, while the lower left warhead, the hydrophobic head, is crucial for the dual
EGFR/HER2 inhibitory activity. Therefore, to design dual TKIs, we kept the 4-aminoaniline
with modified substitution at the meta position as the upper right warhead. On the other
hand, the lower right warhead serves as an electrophilic moiety for covalent bond formation
in the majority of second- and third-generation agents, and owing to the limited clinical
benefit and toxicity, the left warhead was modified in the current study to cyclopentyl
and/or cyclohexyl (series A and B) in an attempt to abolish the cytotoxicity. In addition,
the hinge region of TKIs, which is occupied by the adenine ring of ATP, was modified to be
occupied by thienotriazine in series A and B, and tetrahydrobenzothiophene in series C.
Modification of the core scaffold was intended to diminish the cellular resistance common
with quinazoline and improve the potency based on our previous publication. Another
new modification in this study is a change in the position of the upper warhead from
position 4 of quinoline and quinazoline in the majority of approved TKIs to position 2 of
tetrahydrobenzothiophene, keeping the hydrophobic tail to mimic the dual EGFR/HER2
inhibitors. The cyano group, which is characteristic of neratinib (6), was kept at position 3
to mimic N3 of quinazoline-based TKIs. In this way, various regions of ATP-binding sites
could be targeted by such substitution patterns on the mutant EGFR protein kinase domain
to design molecules that are differentially selective. The design strategies are displayed in
Figures 1 and 2.
2. Results and Discussion
2.1. Chemistry
The synthetic methods for series compounds A/B and C were illustrated in Schemes
1 and 2. Compounds (11
Series A and B compounds were obtained by condensation of compounds (13a
–
13) were synthesized as reported in our earlier research [16].
) with
,b
different aromatic aldehydes (Scheme 1) [17]. Scheme 2, involving the synthesis of series C,
started with the reaction of 2-amino-5,6-dihydro-4H-cyclohexa[b]thiophene-3-carbonitrile
(
18) with chloroacetyl chloride to yield compound (19). Then, coupling of compound (19
with heteroaryl amines (20a and 22) [18] and hydrazine hydrate gave compounds (21a ),
23), and (24) respectively [19]. The hydrazide derivative (24) was condensed with different
aromatic aldehydes, yielding compounds (25a,b) [17].
)
,b
,b
(

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Scheme 1. Synthesis of dihydrocyclopentathienotriazine and tetrahydrobenzothienotriazine derivatives (series A, B).
Scheme 2. Synthesis of tetrahydrobenzothieno-acetamide derivatives (series C).

Pharmaceuticals 2021, 14, 9
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The structures of the synthesized compounds were confirmed by microanalyses and
spectral data (IR, 1H NMR, and EI-MS). Mass spectra showed the expected molecular ion
peaks. IR spectra of the synthesized compounds (21a,b) collectively showed secondary
amine signals at around 3300–3500 cm−1, which indicates coupling between 2-chloro-N-
(3-cyano-4,5,6,7-tetrahydrobenzo[b] thiophen-2-yl)acetamide (19) and heteroaryl amines
1
(
20a
compounds (14
8.70 (ppm) and a single peak for CH proton in the range of 7.4 to 8.4
of compound (15a) showed a single peak corresponding to OH proton at 10.15
the spectrum of compound (16a) showed a single sharp peak at 4.06 (ppm) corresponding
to the OCH3 group of anisaldehyde. A single peak for CH proton appeared at 7.85 (ppm)
and 7.64
(ppm) for compounds (15b) and (17b), respectively. The 1H NMR spectrum of
compound (15b) showed a single peak corresponding to OH proton at 8.25
(ppm). The 1H
NMR spectrum of compound (21a) showed the appearance of single peak at 11.88 (ppm)
and multiple peaks at 6.95 (ppm) corresponding to NH proton and three aromatic protons
of heteroarylamine, respectively. The NH proton of the coupling reaction appeared as a
single peak at 3.65 (ppm). Another single peak belonging to proton of NH of thiophene
appeared at 5.49 (ppm). Finally, a single sharp peak accounting for CH2 appeared at
3.08
(ppm). The 1H NMR spectrum of compound (21b) showed the appearance of single
peaks at 11.69 and 6.58 (ppm), corresponding to NH proton and single aromatic proton
of the heteroaryl amine, respectively. A single peak for NH proton of thiophene appeared
at 5.01 (ppm) and a sharp peak of CH2 appeared at 3.65 (ppm). The spectrum also
,
b
and 22) with the disappearance of signal corresponding to NH2. H NMR spectra of
–
17a) showed multiple peaks of aromatic protons in the range of 7.80 to
(ppm). The spectrum
(ppm), and
δ
δ
δ
δ
δ
δ
δ
δ
δ
δ
δ
δ
δ
δ
δ
showed two single sharp peaks for the two methyl groups of the heteroarylamine at 2.42
and 2.54 δ (ppm).
2.2. Biological Evaluation
2.2.1. In Vitro Cytotoxic Activity against H1299 Cell Line
The in vitro cytotoxic activities of the synthesized compounds (13
sured using sulfo-rhodamine B (SRB) assay [20] against the NSCLC cell line H1299 and
gefitinib ( ) as a reference drug. This cancer cell line has high expression of EGFR and
HER2 enzymes [ ]. As seen in Table 1, the results revealed that the synthesized compounds
showed versatile antitumor activity against the tested cell line. The synthesized compounds
13 25a ) showed IC50 values in the range of 12 to 54 nM, superior to that of gefitinib ( ) at
40 M. Compound (21a) was the most potent, with IC50 = 12.5 nM, followed by compound
21b), with IC50 = 13.68 nM. The viability curve of compound (21a), with the most potent
–25a,b) were mea-
2
3
(
–
,
b
2
µ
(
anticancer activity, is displayed in Figure 3.
Table 1. In vitro cytotoxic activity of compounds (13
–
25a,b) against non-small cell lung cancer
(NSCLC) cell line H1299.
Compound No.
IC50 against H1299
13a
14a
15a
16a
17a
28.79 nM
25.68 nM
32.81 nM
34.91 nM
54.80 nM
25 nM
13b
15b
17b
21a
21b
23
25a
25b
34.00 nM
38.13 nM
12.50 nM
13.68 nM
18.41 nM
18.53 nM
18.47 nM
40 µM
Gefitinib (2)

Pharmaceuticals 2021, 14, 9
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Figure 3. Viability curve of compound (21a), with cytotoxic activity against H1299 NSCLC cell line.
2.2.2. EGFR and HER2 Kinase Inhibitory Assay
The most active compound (21a) was chosen for further evaluation of in vitro EGFR
and HER2 kinase inhibitory activity. The results showed that the IC50 values of compound
(21a) were 0.47 and 0.14 nM against EGFR and HER2, respectively. This is superior to the
value of gefitinib ( ) against EGFR (1.9 nM). It is comparable to IC50 values for imatinib (1)
2
0.11 nM against EGFR and 0.06 nM against HER2 (Table 2).
Table 2. In vitro inhibitory activity of compound 21a against EGFR and HER2 enzymes.
Compound No.
IC50 against EGFR (nM)
IC50 against HER2 (nM)
(21a)
0.47
1.9
0.11
0.14
–
0.06
59 [21]
Gefitinib (2)
Imatinib (1)
Neratinib (6)
92 [21]
2.3. Molecular Modeling
2.3.1. Molecular Docking
Computer-aided simulation of drug design saves effort, time, and energy compared
to conventional methods of drug production. Molecular-based drug design was proposed
in order to predict the possible interactions of the target compounds with EGFR/HER2.
Using Glide in the Schrodinger program for docking, the modulation activity of erlotinib (
3)
and afatinib ( ) derivatives against the EGFR/HER2 receptor was determined, as shown in
5
Figure 4. The designed compounds were docked into the ATP-binding pocket as a thienotri-
azine and tetrahydrobenzothiophene scaffold, mimicking the ATP adenine ring occupying
the hinge region motif of TKs. The positions of thienotriazine (14a), tetrahydrobenzothio-
phene (21a), erlotinib (3), and afatinib (5) were similar, which implies that they might share
the same biological activity. The docking results show, in detail, that the designed TKI
formed hydrogen bonds, hydrophobic contacts, and hydrophobic–hydrophilic interactions
with the target TKs. N1 of the triazine motif in series A and B could act as a hydrogen bond
acceptor and form hydrogen bonds with MET769 (distance 2.4 Å, angle 143.6) in the hinge
region, mimicking the N1 of quinazoline in the approved drug erlotinib (3) (distance 2.2 A,
angle 135.7). In series C, a tetrahydrobenzothiophene scaffold occupied the hinge region in
the same position of quinazoline, forming a hydrogen bond with crystal water, mimicking
the N3 of erlotinib’s quinazoline. The phenyleneimine and pyrazolopyridine moieties
deeply penetrated the hydrophobic pocket, forming hydrophobic–hydrophobic, Van der
Waals, and
LEU753, and LEU764, mimicking the phenylacetylene of erlotinib (
moieties, such as by adding hydroxy, nitro, or methoxy groups, could enhance affinity
π
–
π
interactions with key amino acids such as ALA719, ILE720, MET742,
3). Modifying these

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through the formation of hydrogen bonds with GLU738 and LYS721. The incorporation
of CN at position 3 of tetrahydrobenzothiophene could confer series C with a certain su-
periority by forming hydrophobic interactions with LEU694 and VAL702. Extra precision
glide docking of compounds (14a) and (21a) with the active domain of EGFR showed
reasonable docking scores of
−
6.78 and
−
7.69 and glide E-model values of
−
64.6 and
−71.2 kcal mol−1, respectively.
(A)
(B)
(C)
(D)
Figure 4.
(
A
) Three-dimensional binding modes and overlay of erlotinib (
3
), compound (14a), and compound (21a) in
) in the EGFR catalytic domain. ( ) 2D
D) 2D representation of compound (21a) in the EGFR
the catalytic domain of EGFR (PDB 1M17). (
representation of compound (14a) in the EGFR catalytic domain. (
B) 2D representation of erlotinib (
3
C
catalytic domain.
On the other hand, the docking study of the designed compounds in human HER2
showed high affinity of these compounds toward this receptor. Thienotriazine and tetrahy-
drobenzothiophene occupied the hinge region, forming hydrogen bonds with the MET803
and water molecules there, as shown in Figure 5. Additionally, the hydrophobic heads in
the upper right position of series A and B and at position 2 of series C deeply penetrated the
hydrophobic pocket in HER2, forming hydrogen bond, hydrophobic, Van der Waals, and
π–
π
interactions. The inclusion of large substitutions was intended to improve the affinity
of the designed compounds toward HER2, generating dual-acting compounds mimicking
lapatinib ( ) and neratinib ( ). The extra precision docking score and glide energy showed
the highest affinity of the design compounds compared to lapatinib ( ) and neratinib ( ).
For (15a) and (21a), the docking scores were 10.5 and 8.8, and the glide energy values
were 81.4 and
77.03 kcal mol−1, respectively. These values are higher than those of
lapatinib ( ) ( 7.8 and ) ( 4.3 and
79.8 kcal mol−1) and neratinib ( 52.6 kcal mol−1).
These results are in agreement with those of in situ and in vitro screening studies.
4
6
4
6
−
−
−
−
4
−
−
6
−
−

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(A)
(B)
(D)
(C)
Figure 5.
(
A
) Three-dimensional binding modes and overlay of lapatinib (
4
), compound (15a), and compound (21a) in
) in the catalytic domain of HER2. ( ) 2D
D) 2D representation of compound (21a) in the catalytic
the catalytic domain of HER2 (PDB 3PP0). (
representation of compound (15a) in the catalytic domain of HER2. (
B
) 2D representation of lapatinib (
4
C
domain of HER2.
Acquired drug resistance normally develops after about 12 months of treatment with
gefitinib ( ) or erlotinib ( ), which is attributed to secondary T790M mutation. To confirm
2
3
the benefits of the designed compounds in terms of their affinity toward mutated TKs, the
molecular docking study was extended to include mutated T790M protein (PDB 4G5P),
as shown in Figure 6. The designed compounds occupied the same position as afatinib

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(
5). The amide link at position 2 of the thiophene ring and the cyano group at position
3 of the highest potent compound (21a) formed a hydrogen bond (2.01 A, angle 108.9)
with MET793 in the hinge region, mimicking the hydrogen bond of N1 of the quinazoline
ring in afatinib (5) (2.19 Å, angle 116.8). Pyrazolopyridine participated in hydrophobic
interactions, enforcing the binding affinity. The electrophilic moiety and hydrophobic tail
at the left side of quinazoline formed extra hydrogen bonds, hydrophobic interactions,
and covalent bonds with CYS797. Extra precision glide docking of compounds (14a) and
(
21a) with the active domain of T790M EGFR showed reasonable docking scores of
and 7.5 and glide E-model values of 72.12 and
66.88 kcal mol−1, respectively. These
) ( 10.5 and
103.4 kcal mol−1); however,
−
7.7
−
−
−
values were not as high as to those of afatinib (
5
−
−
they might better than those of lapatinib (4) (−6.2 and −87.2 kcal mol−1).
(A)
(B)
(C)
) Three-dimensional binding modes and overlay of afatinib (
(D)
Figure 6.
(
A
5
) and compounds (14a) and (21a) in the catalytic
domain of T790M EGFR (PDB 4G5P). ( ) 2D representation of afatinib (
B
5
) in the catalytic domain of T790M EGFR. (
C) 2D
representation of compound (14a) in the catalytic domain of T790M EGFR. (D) 2D representation of compound (21a) in the
catalytic domain of T790M EGFR.

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2.3.2. Physicochemical Properties and In Silico ADME Prediction
The pharmacokinetic properties of drug-like molecules are primarily determined by
the physicochemical properties of the designed molecules, such as solubility in aqueous
solution, lipophilicity, hydrogen bonding capability, polar surface area, chemical stability,
etc. Significantly, the physiochemical properties of a compound are the key to turning
a biologically active compound into a therapeutically effective drug [16]. Therefore, our
promising novel compound (21a) and moderately active compound (15a) were subjected
to prediction of physicochemical properties using ADME predictions made by the QikProp
v4.3 function of Schrodinger 10-1 prior to in vivo studies, with the licensed drug lapatinib
(4) used as a reference compound. According to the QikProp suite, polar surface area (PSA),
partition coefficient (log PO/W), and number of rotatable bonds were predicted in order
to test the possible drug-like properties of compounds. As seen in Table 3, compounds
(
(
15a) and (21a) exhibited appropriate physicochemical properties compared to lapatinib
). Caco-2 cell permeability (QPPCaco) was used as a marker for intestinal absorption
4
or permeation. Compound (15a) showed a high partition coefficient and, consequently,
the highest cell permeability. The most active compound (21a) and compound (15a) might
be considered as promising lead molecules for designing potent EGFR inhibitors with
excellent membrane permeability and oral bioavailability.
Table 3. In Silico ADME prediction parameters of designed and reference molecules.
ADME Prediction
Parameters
Compound
(15a)
Compound
(21a)
Lapatinib
(4)
mol MW a
Donor-HB b
Accept-HB c
QPlogPo/w d
PSA e
371.4
1
5
352.1
2.25
5.7
581.06
1
8.25
6.3
95.35
–8.2
187.5
0.8
376.5
1.19
10
4.3
2.4
62.2
–5.6
1143.7
–0.6
1080
0.55
5
116.8
–6.2
96.8
–1.9
59.5
0.26
5
QPlogS f
QPPCaco g
QPlogBB h
QPPMDCK i
QPlogKhsa j
#rotor k
% Human Oral
100
76.9
78.8
Absorption l
Acceptable ranges: a: mol MW (molecular weight) < 500 amu; b: Donor-HB (hydrogen bond
donor) < 5;
c: Accept-HB (hydrogen bond acceptor) < 10; d: QPlogPo/w (octanol/water parti-
tion coefficient) < 5; e: PSA (Van der Waals surface area of polar nitrogen and oxygen atoms) = 7–200;
f: QPlogS (Conformation-independent predicted aqueous solubility) < 0.5; g: QPPCaco (Caco-2 cell
permeability)
≤
5; h: QPlogBB (Predicted brain/blood partition coefficient) <25 poor, h > 500 great;
i: QPPMDCK (Predicted apparent MDCK cell permeability in nm/s. MDCK cells are considered to be
a good mimic for the blood–brain barrier) < 25 poor, i > 500 great; j: QPlogKhsa (Prediction of binding
to human serum albumin) =
(Predicted qualitative human oral absorption) > 80% is high, l < 25% is poor [16].
l
−
1.5 to 1.5; k: rotatable bond = 0–15, : % Human Oral Absorption
2.3.3. Molecular Orbital Energy Study and Molecular Electrostatic Potential (MESP)
A molecular electrostatic potential (MESP) map is a useful tool for understanding
the sites for nucleophilic reactions, electrophilic attacks, and intermolecular interactions.
Additionally, it widely used for the detection of iso-surface values, showing the locations
of positive and negative electrostatic potential of designed compounds [16]. The MESP
values of compounds (21a) and lapatinib (4) are shown in Figure 7. Red indicates the most
electronegative region, blue the most electropositive region, and green the region of zero
potential. The nitrogen atoms of quinazoline and aniline and the oxygen group of the furan
ring of lapatinib (
with greater positive charge. Thus, electrophilic attack and nucleophilic interaction with
the receptor are more favorable for these parts of lapatinib ( ). The remaining parts of
4) have a greater negative charge surrounded by some carbon atoms
4

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the structure are shown in green, which means they can bind through hydrophobic and
Van der Waals interactions. In the same manner, the electronegative and electropositive
regions of the designed compounds are located around 3-cyanotetrahydrothiophen and
pyrazolopyridine, making these regions better able to interact with charged parts of the
EGFR/HER2 receptors. The electronegative region around pyrazolopyridine distinguished
it somewhat from lapatinib (
4), which lacks this. The docking study showed that the
designed compounds have higher affinity toward EGFR/HER2 compared to lapatinib (
4).
Therefore, we attributed the high affinity and biological activity observed in the in vitro
study to the inclusion of a heterocyclic rather than aromatic system in this region that is
common in TKIs.
(B)
(A)
Figure 7. Molecular electrostatic potential mapping for (A) the most potent compound (21a) and (B) lapatinib (4) from
density functional theory (DFT) calculations. Green indicates zero potential regions, blue indicates electropositive regions,
and red indicates electronegative regions.
Moreover, the Mulliken atomic charges (Figure 8) are in agreement with the MESP
results. The negative charge is mainly located on nitrogen atoms of the quinazoline of
lapatinib (4) and on the cyano group and nitrogen of the amide link and nitrogen atoms of
pyrazolopyridine in the most potent compound (21a). The highest negative charge in both
lapatinib (4) and the representative compound give this atom the highest reactivity in the
interaction with EGFR and HER2 receptors.
(A)
Figure 8. Cont.

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(B)
Figure 8. Mulliken atomic charges for (A) the most potent compound (21a) and (B) lapatinib (4).
The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular
orbital (LUMO) maps of the representative compound (21a) and lapatinib ( ) are shown
4
in Figure 9. On the one hand, it was observed that the electrons were delocalized on
quinazoline, aniline, furan, and amino moieties in the HOMO map, whereas on the LUMO
map, the electrons are mainly delocalized on quinazoline and aniline groups. On the other
hand, the HOMO and LUMO maps of the target compounds are delocalized around the
3-cyanothiophene and amide linkage. The frontier molecular orbitals are mainly delo-
calized on the moieties or groups that mainly participate in drug–receptor interaction.
Therefore, we can conclude that the biological activity might be facilitated via the quinazo-
line moiety of lapatinib (
through hydrophobic, hydrophilic, Van der Waals, and
bond formation with key amino acids in the catalytic domain. The LUMO map and part
of the HOMO map of the designed compounds and lapatinib ( ) are concentrated on
4) and the 3-cyanothiophene moiety of the designed compound
π–π
interactions or via hydrogen
4
3-cyanothiophene and quinazoline, making these moieties superior in drug–receptor in-
teraction. Besides this, the amide linkage also participated in this interaction. The results
of density functional theory (DFT) study are in accordance with those of the docking
and in vitro cytotoxicity studies. Gaussian 09 software (Gauss View 5.0, Gaussian, Inc.,
Wallingford, CT, USA) was utilized for all computational calculation.
TK proteins are ATP-dependent, and the ATP-binding domain is conserved in TKs and
serves as the receptor for anticancer agents. The discovery of novel inhibitors is focused
mainly on searching for compounds containing the proper chemical moieties that can fulfill
the requirements for interaction with the adenine-binding site (hinge region); 4-anilino-
quinoline and/or quinazoline are features common in several approved TKIs. However, it
was found that for most of the quinazoline-based anticancer agents that selectively inhibit
EGFR/HER2 overexpression, resistance is developed over long courses of treatment. The
discovery of a novel core structure with various substitutions at different positions is thus
of great importance.

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HOMO (Compound (21a))
LUMO (Compound (21a))
HOMO (Lapatinib) (4))
LUMO (Lapatinib (4))
Figure 9. Highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) maps for
compound (21a) and lapatinib (4) from DFT calculations. Green indicates positive molecular orbital and red indicates
negative molecular orbital.
In this study, and based on our previous publication, thienotriazine and
3-cyanotetrahydrobenzothiophene were selected as the core scaffold to design novel dual-
acting EGFR/HER2 drug-like molecules. The designed compounds displayed promising
in situ cytotoxicity against H1299 NSCLC and in vitro activity against EGFR and HER2,
comparable to the approved TKIs imatinib (1) and gefitinib (2). The molecular modeling
along with the computational studies revealed the significance of the two core moieties—
thienotriazine and 3-cyanotetrahydrobenzothiophene, which functioned as the adenine of
ATP in the hinge region, forming hydrogen bonding interactions. Modifying the upper
right hydrophobic head through the addition of some heterocyclic systems (pyrazolopy-
ridine and isoxazolopyridine) resulted in superior activity of the designed compound
compared with the approved dual-acting drug lapatinib (4). Heterocyclic systems suc-
ceeded in forming extra hydrogen bonding in addition to the hydrophobic interaction
in the hydrophobic region. The red electronic map of MESP and the highly negative
charge localized in these systems, shown by Mulliken charge distribution, potentiated the
merit of these moieties. Furthermore, alteration in the position of the substitution from
position 4 on quinazoline in the common approved TKI drugs to position 2 in our series
C enforced the potency and conferred to the designed compounds a certain benefit in
strongly interacting with TKs at different sites. Molecular modeling also revealed that
these modifications increased the interaction of the designed compounds with the protein
kinase harboring the T790M mutation, which is responsible for acquired drug resistance.
Figure 10 can summarize the cytotoxic activity of the designed molecules based on the
observed structure–activity relationships (SAR).

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Cyno group involved in hydrogen bond formation
mimic nitrogens in quinazoline
Cyclohexyl ring is hydrophobic
head important for hydrophobic
interaction in
CN
Bulky group is essential
to occupy hydrophobic
pocket.
Heteroatoms are
involved in hydrogen
bonding and
O
H
N
N
NH
N
N
H
S
hydrophobic/hydrophilic
interactions
Thiophene
(
central heteroaromatic
system) is essential
for activity mimic
quinazoline in the
approved drug
Amide group
is important for
hydrogen
bonding
Figure 10. The observed structure–activity relationships of the designed molecules as promising dual EGFR/HER2 inhibitors.
3. Materials and Methods
3.1. Instrument
The Varian Mercury Vx 300 NMR spectrometer or JEOL LA (300 MHz for 1H NMR)
1
were used to measure H NMR spectra. Electron impact mass spectra (EI-MS) were
recorded on a Shimadzu GCMS-QP 5050A gas chromatograph mass spectrometer (70 eV).
Melting points were calculated in open capillaries using a Gallenkemp melting point
apparatus and are uncorrected. Infrared (IR) spectra were recorded on a Shimadzu FT-IR
8101 PC IR spectrophotometer (KBr pellets).
3.2. Chemicals and Reagents
Commercial chemicals and solvents of reagent grade were used directly in synthesis
without further purification.
3.3. Experimental
Chemistry
General procedure for synthesis of compounds (14a–17a)
A mixture of 3-(5,6-dihydro-7H-cyclopenta[4:5]thieno[2,3-d][1,2,3]triazin-4-ylamino)
benzene-1,3-diamine (13a) [16] (0.01 mol) and suitable aromatic aldehyde (0.01 mol) in
methanol (20 mL) in the presence of a catalytic amount of glacial acetic acid was refluxed
for 5 h, with the progression of reaction monitored using TLC. The solvent was removed
under reduced pressure, and the product recrystallized from chloroform [17].
3-(5,6-Dihydro-7H-cyclopenta[4:5]thieno[2,3-d][1,2,3]triazin-4-ylamino)-N-
benzylidenebenzene-1,3-diamine (14a)
Mass spectrum: m/z (%): 371 (M+, 1.50%), 361 (1.33%), 353 (1.21%), 343 (7.82%), 316
(11.55%), 108 (100%). IR (cm−1): 752 (C–S), 1261 (C–N), 1624 (C=N), 3429 (N–H). 1H NMR
(DMSO, 300 MHz):
δ (ppm) 1.29 (m, 2H), 3.08 (t, 2H), 3.27 (t, 2H), 7.08 (m, 8H), 7.71 (s, 1H),
7.80 (s, 1H), 11.81 (s, 1H). Elemental analysis calculated for C21H17N5S: C, 67.90; H, 4.61; N,
18.85. Found: C, 68.09; H, 4.66; N, 19.08.
3-(5,6-Dihydro-7H-cyclopenta[4:5]thieno[2,3-d][1,2,3]triazin-4-ylamino)-N-(2-
hydroxybenzylidene)benzene-1,3-diamine (15a)
Mass spectrum: m/z (%): 387 (M+, 0.76%), 375 (2.45%), 347 (6.59%), 345 (9.14%), 212
(85.96%), 119 (100%). IR (cm−1): 756 (C–S), 1149 (C–O), 1276 (C–N), 1616 (C=N), 3221

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(N–H), 3352 (OH). 1H NMR (DMSO, 300 MHz):
δ
(ppm) 1.14 (m, 2H), 2.12 (t, 2H), 2.73 (t,
2H), 4.54 (s, 1H), 7.84 (m, 7H), 7.90 (s, 1H), 8.52 (s, 1H), 10.15 (s, 1H). Elemental analysis
calculated for C21H17N5OS: C, 65.10; H, 4.42; N, 18.08. Found: C, 65.34; H, 4.47; N, 18.39.
3-(5,6-Dihydro-7H-cyclopenta[4:5]thieno[2,3-d][1,2,3]triazin-4-ylamino)-N-(3-
nitrobenzylidene)benzene-1,3-diamine (16a)
Mass spectrum: m/z (%): 401 (M+, 3.10%), 389 (79.4%), 362 (1.80%), 322 (1.20%), 293
(2.40%), 261 (1.50%), 249 (100%). IR (cm−1): 690 (C–S), 1246 (C–O–C), 1261 (C–N), 1678
(C=N), 3444 (N–H). 1H NMR (DMSO, 300 MHz):
δ (ppm): 1.15 (m, 2H), 3.65 (t, 2H), 3.70 (t,
2H), 4.06 (s, 3H), 7.30 (m, 7H), 7.85 (s, 1H), 8.70 (s, 1H), 10.20 (s, 1H). Elemental analysis
calculated for C22H19N5OS: C, 65.81; H, 4.77; N, 17.44. Found: C, 66.04; H, 4.84; N, 17.68.
3-(5,6-Dihydro-7H-cyclopenta[4:5]thieno[2,3-d][1,2,3]triazin-4-ylamino)-N-(4-
methoxybenzylidene)benzene-1,3- diamine (17a)
Mass spectrum: m/z (%): 416 (M+, 0.88%), 407 (2.47%), 403 (2.56%), 376 (4.56%), 222
(12.24%), 108 (100%). IR (cm−1): 732 (C–S), 1350 (NO2), 1527 (NO2), 1612 (C=N), 3363
(N–H). 1H NMR (DMSO, 300 MHz):
δ (ppm): 1.80 (m, 2H), 2.62 (t, 2H), 2.64 (t, 2H), 4.80 (s,
1H), 7.28 (m, 6H), 7.31 (s, 1H), 7.34 (s, 1H), 8.50 (s, 1H). Elemental analysis calculated for
C21H16N6O2S: C, 60.56; H, 3.87; N, 20.18. Found: C, 60.73; H, 3.91; N, 20.43.
General procedure for synthesis of compounds (15b) and (17b)
A mixture of 3-(5,6,7,8-Tetrahydro-7H-cyclohexa[4:5]thieno[2,3-d][1,2,3]triazin-
4ylamino)benzene-1,3-diamine (13b) [16] (0.01 mol) and suitable aromatic aldehyde (0.01 mol)
in methanol (20 mL) in the presence of a catalytic amount of glacial acetic acid was refluxed
for 5 h, with the progression of reaction monitored using TLC. The solvent was removed
under reduced pressure, and the product recrystallized from chloroform [17].
3-(5,6,7,8-Tetrahydro-7H-cyclohexa[4:5]thieno[2,3-d][1,2,3]triazin-4-ylamino)-N-(2-
hydroxybenzylidene)benzene-1,3-diamine (15b)
Mass spectrum: m/z (%): 401 (M+, 7.20%), 390 (3.80%), 375 (2.10%), 361 (6.49%), 346
(100%), 303 (5.30%). IR (cm−1): 756 (C–S), 1678 (C=N), 3228 (N–H), 3363 (OH). 1H NMR
(DMSO, 300 MHz):
δ (ppm): 1.13 (m, 4H), 3.05 (t, 2H), 3.10 (t, 2H), 3.60 (s, 1H), 7.13 (m,
7H), 7.81 (s, 1H), 7.85 (s, 1H), 8.25 (s, 1H). Elemental analysis calculated for C22H19N5OS:
C, 65.81; H, 4.77; N, 17.44. Found C, 65.58; H, 4.39; N, 17.76.
3-(5,6,7,8-Tetrahydro-7H-cyclohexa[4:5]thieno[2,3-d][1,2,3]triazin-4-ylamino)-N-(3-
nitrobenzylidene)benzene-1,3- diamine (17b)
Mass spectrum: m/z (%): 430 (M+, 5.10%), 384 (8.60%), 354 (10.40%), 296 (6.30%), 103
(100%). IR (cm−1): 732 (C–S), 1350 (NO2), 1527 (NO2), 3360 (N–H). 1H NMR (DMSO, 300
MHz):
δ (ppm): 1.14 (m, 4H), 3.04 (t, 2H), 3.11 (t, 2H), 3.20 (s, 1H), 7.31 (m, 6H), 7.37 (s, 1H),
7.45 (s, 1H), 7.64 (s, 1H).
2-Chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)acetamide (19)
To a stirred solution of 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carb◦onitrile
11) [22] (0.10 mol) in acetone (20 mL), chloroacetyl chloride (0.05 mol) at 0–5 C was
(
added dropwise and maintained in an ice bath. The reaction mixture was stirred at room
temperature for an additional 4 h. Then, 40 mL of 10% HCl was added to the mixture.
The formed precipitate was filtered and washed with HCl (10%) and water. Melting point:
175–177 ◦C [23].
General procedure for synthesis of compounds (21a,b)
The amine derivatives (20a,b) (0.009 mol) [24] were dissolved in dry dichloromethane
(DCM) (25.0 mL) and triethylamine (TEA) (1.5 mL) under nitrogen atmosphere. Compound
(19) (0.0045 mol) was slowly added to the stirred solution, and the solution was then
refluxed for 6–9 h. The mixture was then cooled and poured onto crushed ice. The
formed precipitate was filtered, washed with water, dried and recrystallized from absolute
ethanol [4].
2-(1H-Pyrazolo[3,4-b]pyridin-3-ylamino)-N-(3-cyano4,5,6,7-tetrahydrobenzo[b]
thiophen-2-yl)acetamide (21a)
Mass spectrum: m/z (%): 354 (M+2, 4.29%), 353 (4.83%), 352 (6.96%), 320 (57.61%), 291
(38.64%), 178 (100%). IR (cm−1): 789 (C–S), 1689 (C=O), 2224 (C
N), 3214 (N–H), 3338
≡

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(N–H), 3443 (N–H). 1H NMR (DMSO, 300 MHz):
δ
(ppm): 1.20 (m, 4H), 3.06 (m, 4H), 3.08
(s, 2H), 3.65 (s, 1H), 5.49 (s, 1H), 6.95 (m, 3H), 11.88 (s, 1H). Elemental analysis calculated
for C17H16N6OS: C, 57.94; H, 4.58; N, 23.85. Found: C, 58.21; H, 4.42; N, 24.18.
N-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(4,6-dimethyl-1H-pyrazolo
[3,4-b]pyridin-3-ylamino)acetamide (21b)
Mass spectrum: m/z (%) 382 (M+2, 1.22%), 381 (1.08%), 380 (3.65%), 348 (1.44%), 320
(13.61%), 143 (100%). IR (cm−1): 778 (C–S), 1689 (C=O), 2224 (C
≡
N), 3285 (N–H), 3388
(N–H), 3444 (N–H). 1H NMR (DMSO, 300 MHz):
δ (ppm): 1.18 (m, 4H), 1.75 (s, 1H), 2.42 (s,
3H), 2.54 (s, 3H), 3.02 (m, 4H), 3.65 (s, 2H), 5.01 (s, 1H), 6.58 (s, 1H), 11.69 (s, 1H). Elemental
analysis calculated for C19H20N6OS: C, 59.98; H, 5.30; N, 22.09. Found: C, 60.29; H, 5.43;
N, 22.41.
N-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(isoxazolo[3,4-b]pyridin-
3-ylamino)acetamide (23)
Compound (22) [24] was dissolved in dry DCM (25.0 mL) and TEA (1.5 mL) under
nitrogen atmosphere. Compound (19) (0.0045 mol) was added slowly to the stirred solution,
then heated under reflux for 6–9 h. The mixture was then cooled and poured onto crushed
ice. The formed precipitate was filtered and washed with water, dried and recrystallized
from absolute ethanol [4].
Mass spectrum: m/z (%): 355 (M+2, 0.39%), 354 (1.95%), 353 (0.60%), 343 (4.50%), 313
(6.29%), 144 (100%). IR (cm−1): 692 (C–S), 1689 (C=O), 2224 (C
≡
N), 3220 (N–H), 3446
(N–H). 1H NMR (DMSO, 300 MHz):
δ (ppm): 1.79 (m, 4H), 3.17 (m, 4H), 3.82 (s, 2H), 4.09
(s, 1H), 7.09 (m, 3H), 9.78 (s, 1H). Elemental analysis calculated for C17H15N5O2S: C, 57.78;
H, 4.28; N, 19.82. Found: C, 58.01; H, 4.37; N, 20.09.
N-(3-Cyano-4, 5, 6, 7-tetrahydrobenzo[b]thiophen-2-yl)-2-hydrazinyl acetamide (24)
A mixture of hydrazine hydrate 80% (0.05 mol, 0.25 mL) and 2-chloro-N-(3-cyano-
4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)acetamide (19) (0.0015 mol) in absolute ethanol
(20 mL) was refluxed for 4 h, with the progression of reaction monitored using TLC. The
reaction mixture was cooled and poured into water. The formed precipitate was filtered and
recrystallized from ethanol to give the desired hydrazide. Melting point: 140–142 ◦C [19].
General procedure for synthesis of compounds (25a,b)
A mixture of compound (24) (0.01 mol) and the suitable aromatic aldehyde (0.01 mol)
dissolved in methanol (20 mL) in the presence of a catalytic amount of glacial acetic acid
was refluxed for 5 h, with the progression of reaction monitored using TLC. The solvent
was removed under reduced pressure, and the product recrystallized from chloroform [17].
N-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(2-(3-nitrobenzylidene)
hydrazinyl)acetamide (25a)
Mass spectrum: m/z (%) 385 (M+2, 0.66%), 384 (1.26%), 383 (2.27%), 369 (16.60%), 339
(5.59%), 311 (100%). IR (cm−1): 736 (C–S), 1265 (NO2), 1526 (NO2), 1689 (C=O), 2221 (C
≡
N),
3088 (N–H), 3443 (N–H). 1H NMR (DMSO, 300 MHz):
δ (ppm): 1.78 (m, 4H), 2.15 (s, 1H),
2.45 (m, 4H), 3.62 (s, 2H), 7.80 (m, 3H), 8.71 (s, 1H), 8.88 (s, 1H), 11.63 (s, 1H). Elemental
analysis calculated for C18H17N5O3S: C, 56.38; H, 4.47; N, 18.27. Found: C, 56.70; H, 4.58;
N, 18.49.
N-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(2-(4-methoxybenzylidene)
hydrazinyl)acetamide (25b)
Mass spectrum: m/z (%) 370 (M+2, 2.36%), 369 (6.14%), 368 (21.44%), 354 (100%), 338
(5.20%), 296 (39.14%). IR (cm−1): 695 (C–S), 1256 (OCH3), 1689 (C=O), 2225 (C
≡
N), 3220
(N–H), 3446 (N–H). 1H NMR (DMSO, 300 MHz):
δ (ppm): 1.74 (m, 4H), 2.16 (s, 1H), 2.56
(m, 4H), 3.63 (s, 2H), 3.82 (s, 3H), 7.04 (m, 4H), 8.62 (s, 1H), 11.64 (s, 1H). Elemental analysis
calculated for C19H20N4O2S: C, 61.94; H, 5.47; N, 15.21. Found: C, 62.23; H, 5.60; N, 15.49.
3.4. Biological Assays
3.4.1. Cytotoxic Activity against H1299
The potential cytotoxic activity of the target compounds on H1299 lung cancer cell line
was tested using sulfo-rhodamine B (SRB) assay according to Skehan method [20]. Single

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cells were plated in 96-multiwell plates for 24 h before treatment with the compounds. A
single concentration of the tested compounds was added to the cell monolayer. Monolayer
cell was incubated with the compounds for 48 h at 37 ◦C and in atmosphere of 5% CO2.
After 48 h, cell was fixed, washed and stained with Sulforhodamine-B stain. Acetic acid
was then used to wash excess stain and attached stain was recovered with Tris ethylenedi-
aminetetraacetic acid (EDTA) buffer. Color intensity was measured in an enzyme-linked
immunosorbent assay (ELISA). Percentage of the surviving and inhibition were tabulated.
3.4.2. Measurement of Potential EGFR/HER2 Inhibitory Activity (IC50)
Measurement of potential EGFR/ HER2 inhibitory activities was performed using
Kinase-Glo Plus luminescence kinase assay kit (Promega Corporation, Madison, WI,
USA) [25]. Mix kinases, ATP, substrates, and compounds in the reaction buffer of 25 mM
HEPES (pH 7.4), 10 mM MgCl2, 0.01% Triton X-100, 100
µg/mL BSA, 2.5 mM DTT in a
384-well plate. Total reaction volume was 10
µ
L. The assay plate was incubated at 30 ◦C
for 1 h, and the reaction was stopped by the addition of equal volume of kinase glo plus
reagent. The fluorescence was measured. The signal was correlated with the remaining
amount of ATP in the reaction and was inversely correlated with the kinase activity.
3.5. Molecular Modeling Study
3.5.1. Protein Preparation for Docking Study
The X-ray crystal structure of the catalytic domain of the EGFR enzyme complex with
erlotinib (3) (PDB 1M17 resolution 2.6 A), the human HER2 kinase domain with ligand
03Q (PDB 3PP0 resolution 2.25 A), and EGFR kinase T790M in complex with BIBW2992
(afatinib (5)) (PDB 4G5P resolution 3.17A) were obtained from Protein Data Bank and
further prepared by the protein preparation wizards available in the Glide program of
Schrodinger 10.1. The preparation portion adds hydrogen after ensuring chemical accuracy
and neutralizes side chains that are neither close to the binding cavity nor involved in
the formation of salt bridges. For this reason, the OPLS-2005 force field was used, and
then the active site of the protein was established. Water molecules were extracted in the
next step, and H atoms were added to the crystal structure, most likely hydroxyl and thiol
hydrogen atom positions, protonation states, and tautomers of the His residue and chi
“flip” assignment were selected by the protein assignment script provided by Schrodinger
for Asn, Gln, and His residues. Using the OPLS2005 force field, minimization was carried
out to alleviate steric clashes until the average root mean square deviation (RMSD) of the
non-hydrogen atoms reached a maximum value of 0.3 A [16,26,27].
3.5.2. Ligand Preparation
All compounds were constructed using the Maestro 10.1 fragment library and pre-
pared using LigPrep 2.1, which can generate several structures with different ionization
states, tautomers, stereochemistry, and ring conformations from each input structure. For
optimization, the OPLS-2005 force field was used, which generates the ligand’s low-energy
conformer. Partial atomic charges were allocated, and potential ionization states were
issued at a pH of 7.0. For each ligand, energy minimization was carried out until it met the
RMSD cutoff of 0.010 A. The resulting structures were then adopted to carry out modeling
studies [27,28].
3.5.3. Molecular Docking
The designed compounds were docked to the EGFR (PDB 1M17), HER2 (PDB 3PP0),
and EGFR kinase T790M in complex with BIBW2992 (afatinib (5)) (PDB 4G5P resolution
3.17A) binding sites using Glide, grid-based ligand docking with energetics software from
Schrodinger 10.1, to test the docking parameters. We scaled the Van der Waals radii of
receptor atoms by 0.8 with a partial atomic charge of 0.15 to soften the potential for nonpolar
parts of the receptor. At the center of the active site, a grid box with coordinates X = 10
,
Y = 10, and Z = 10 was created. The ligands were docked with the active site using “extra-

Pharmaceuticals 2021, 14, 9
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precision” glide docking (Glide XP), which flexibly docks ligands. Glide internally creates
conformations and passes them through a series of filters. The XP docking technique has
been clarified elsewhere. Using a Glide score feature, the final best docked structure was
selected. In most comparable docking conformations, the lowest energy docking complex
was found. Finally, for further analysis, the lowest energy docked complex was chosen [16].
3.5.4. In Silico ADME Prediction
Drug-like properties of the synthesized compounds were evaluated in accordance
with Lipinski’s rule of five ADME. This was used to evaluate whether these compounds
have properties that would allow them to be orally active drugs for humans. The drug-
like action of our compounds was predicted using module QikProp (v4.2; Schrodinger
2015-1) [16,26].
4. Conclusions
New chemical compounds belonging to tetrahydrobenzothieno[2,3-d][1,2,3]triazine,
dihydrocyclopentathieno[2,3-d][1,2,3]triazine, and 3-cyanotetrahydrobenzothiophene bear-
ing various heterocyclic systems at positions 4 and 2 were designed, synthesized, struc-
turally elucidated, and biologically evaluated as anti-lung cancer agents. The synthesized
compounds exhibited potent in situ cytotoxic activity toward human lung carcinoma cell
line H1299 compared to gefitinib (2), with IC50 values ranging from 12.5 to 54.8 nM. The
designed compounds showed antiproliferative activity through competitive inhibition of
EGFR and/or HER2 receptors. The designed compound (21a) has shown comparable po-
tency to imatinib (1) but more potent than neratinib (6) in in vitro kinase inhibition against
EGFR and HER2. Results of molecular modeling further supported the potent inhibitory
activity of the proposed compounds, which helps in understanding the various interac-
tions between ligands and receptor sites. The tetrahydrobenzothieno[2,3-d][1,2,3]triazine
scaffold and 3-cyanotetrahydrobenzothiophene occupied the ATP-binding site, showing
the same drug–receptor interactions as two approved drugs: lapatinib (4) and neratinib (6).
Additional binding sites, especially in hydrophobic pockets, were provided by our struc-
tural modification. The introduction of new functional groups with positions different to
those commonly found in approved drugs could allow the designed compounds to interact
with alternative binding sites, which could solve the problem of acquired drug resistance.
Our new scaffolds might also serve as promising lead compounds for the discovery of new
drugs to overcome acquired resistance in NSCLC patients.
Author Contributions: Conceptualization, R.E., Y.M.A.A., M.E., A.S.A.Y., H.A.E., and M.M.S.;
methodology, R.E., Y.M.A.A., M.E., A.S.A.Y., and H.A.E.; software, M.S.E.; writing—original draft
preparation, R.E., Y.M.A.A., M.E., A.S.A.Y., and H.A.E.; writing—review and editing, R.E., Y.M.A.A.,
M.E., A.S.A.Y., and H.A.E.; resources, S.S.E.; funding acquisition, S.S.E.; R.E and M.S.E. contributed
equally. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz
University, Jeddah, Saudi Arabia, under grant no. FP-127-42.
Acknowledgments: The Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah,
Saudi Arabia, funded this project under grant no. FP-127-42. The authors, therefore, acknowledge
and thank DSR for technical and financial support.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Zhao, J.; Xia, Y. Targeting Her2 Alterations in Non–Small-Cell Lung Cancer: A Comprehensive Review. JCO Precis. Oncol. 2020, 4,
411–425. [CrossRef]
2.
Li, X.; Wu, J.; Cao, H.; Ma, R.; Wu, J.; Zhong, Y.; Feng, J. Blockade of DNA Methylation Enhances the Therapeutic Effect of
Gefitinib in Non-Small Cell Lung Cancer Cells. Oncol. Rep. 2013, 29, 1975–1982. [CrossRef]

Pharmaceuticals 2021, 14, 9
21 of 21
3.
4.
Kang, H.N.; Kim, S.; Yun, M.R.; Kim, H.R.; Lim, S.M.; Kim, M.; Hong, K.; Kim, S.; Kim, H.; Pyo, K.; et al. Er2, a Novel Human
Anti-Egfr Monoclonal Antibody Inhibit Tumor Activity in Non-Small Cell Lung Cancer Models. Lung Cancer 2016, 95, 57–64.
[CrossRef] [PubMed]
Yassen, A.S.A.; Elshihawy, H.E.A.E.A.; Said, M.M.A.; Abouzid, K.A.M. Molecular Modelling and Synthesis of Quinazoline-Based
Compounds as Potential Antiproliferative Agents. Chem. Pharm. Bull. 2014, 62, 454–466. [CrossRef] [PubMed]
Charles, S.; Futreal, A.; Eisen, T. Her2-Targeted Therapies in Non–Small Cell Lung Cancer. Clin. Cancer Res. 2006, 12, 4377s–4383s.
Jianming, Z.; Yang, P.L.; Gray, N.S. Targeting Cancer with Small Molecule Kinase Inhibitors. Nat. Rev. Cancer 2009, 9, 28–39.
Elmetwally, S.A.; Saied, K.F.; Eissa, I.H.; Elkaeed, E.B. Design, Synthesis and Anticancer Evaluation of Thieno [2, 3-D] Pyrimidine
Derivatives as Dual Egfr/Her2 Inhibitors and Apoptosis Inducers. Bioorgan. Chem. 2019, 88, 102944. [CrossRef]
Emeline, L.; Barluenga, S.; Moras, D.; Wurtz, J.; Winssinger, N. Cysteine Mapping in Conformationally Distinct Kinase Nucleotide
Binding Sites: Application to the Design of Selective Covalent Inhibitors. J. Med. Chem. 2011, 54, 1347–1355.
Smith, A.J.; Zhang, X.; Leach, A.G.; Houk, K.N. Beyond Picomolar Affinities: Quantitative Aspects of Noncovalent and Covalent
Binding of Drugs to Proteins. J. Med. Chem. 2009, 52, 225–233. [CrossRef]
5.
6.
7.
8.
9.
10. Lanning, B.R.; Whitby, L.R.; Dix, M.M.; Douhan, J.; Gilbert, A.M.; Hett, E.C.; Johnson, T.O.; Joslyn, C.; Kath, J.C.; Niessen, S. A
Road Map to Evaluate the Proteome-Wide Selectivity of Covalent Kinase Inhibitors. Nat. Chem. Biol. 2014, 10, 760–767. [CrossRef]
11. Shuhang, W.; Cang, S.; Liu, D. Third-Generation Inhibitors Targeting Egfr T790 m Mutation in Advanced Non-Small Cell Lung
Cancer. J. Hematol. Oncol. 2016, 9, 34.
12. Yu, H.A.; Tian, S.K.; Drilon, A.E.; Borsu, L.; Riely, G.J.; Arcila, M.E.; Ladanyi, M. Acquired Resistance of Egfr-Mutant Lung Cancer
to a T790m-Specific Egfr Inhibitor: Emergence of a Third Mutation (C797s) in the Egfr Tyrosine Kinase Domain. JAMA Oncol.
2015, 1, 982–984. [CrossRef] [PubMed]
13. Zhuo, L.; Wang, L.; Feng, M.; Yi, Y.; Zhang, W.; Liu, W.; Li, L.; Liu, Z.; Li, Y.; Ma, X. New Acrylamide-Substituted Quinazoline
Derivatives with Enhanced Potency for the Treatment of Egfr T790m-Mutant Non-Small-Cell Lung Cancers. Bioorgan. Chem.
2018, 77, 593–599.
14. Siyuan, Y.; Tang, C.; Wang, B.; Zhang, Y.; Zhou, L.; Xue, L.; Zhang, C. Design, Synthesis and Biological Evaluation of Novel
Egfr/Her2 Dual Inhibitors Bearing a Oxazolo [4, 5-G] Quinazolin-2 (1h)-One Scaffold. Eur. J. Med. Chem. 2016, 120, 26–36.
15. Mohamed, S.; Elshihawy, H. Synthesis, Anticancer Activity and Structure-Activity Relationship of Some Anticancer Agents Based
on Cyclopenta (B) Thiophene Scaffold. Pak. J. Pharm. Sci. 2014, 27.
16. Ranza, E.; Aziz, Y.M.A.; Elgawish, M.S.; Elewa, M.; Elshihawy, H.A.; Said, M.M. Pharmacophore Modeling, 3d-Qsar, Synthesis,
and Anti-Lung Cancer Evaluation of Novel Thieno [2, 3-D] [1, 2, 3] Triazines Targeting Egfr. Arch. Pharm. 2020, 353, 1900108.
17. Adesh, D.; Srivastava, S.K.; Srivastava, S.D. Conventional and Microwave Assisted Synthesis of 2-Oxo-4-Substituted Aryl-
Azetidine Derivatives of Benzotriazole: A New Class of Biological Compounds. Bioorgan. Med. Chem. Lett. 2011, 21, 569–573.
18. Al-Obaid, A.M.; Abdel-Hamide, S.G.; El-Kashef, H.A.; Alaa, A.M.; El-Azab, A.S.; Al-Khamees, H.A.; El-Subbagh, H.I. Sub-
stituted Quinazolines, Part 3. Synthesis, in Vitro Antitumor Activity and Molecular Modeling Study of Certain 2-Thieno-4
(3h)-Quinazolinone Analogs. Eur. J. Med. Chem. 2009, 44, 2379–2391. [CrossRef]
19. Canan, K.; Ayhan-Kılcıgil, G.; Özbey, S.; Kaynak, F.B.; Kaya, M.; Çoban, T.; Can-Eke, B. Synthesis and Antioxidant Properties of
Novel N-Methyl-1, 3, 4-Thiadiazol-2-Amine and 4-Methyl-2h-1, 2, 4-Triazole-3 (4h)-Thione Derivatives of Benzimidazole Class.
Bioorgan. Med. Chem. 2008, 16, 4294–4303.
20. Philip, S.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J.T.; Bokesch, H.; Kenney, S.; Boyd, M.R. New
Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening. JNCI J. Nat. Cancer Inst. 1990, 28, 1107–1112.
21. 1 Product Results for Neratinib. Available online: https://www.selleckchem.com/search.html?searchDTO.searchParam=
Neratinib&sp=Neratinib (accessed on 20 November 2020).
22. Gewald, K. Heterocyclen Aus Ch-Aciden Nitrilen, Vii. 2-Amino-Thiophene Aus A-Oxo-Mercaptanen Und Methylenaktiven
Nitrilen. Chem. Berichte 1965, 98, 3571–3577. [CrossRef]
23. Lou, Z.; Li, Z.; Chunfen, X.; Yong, Y.; Fanbo, Z.; Kaixun, H. Anticancer Activities of Some Arylcarbamoylalkyltriphenylphospho-
nium Chlorides. Med. Chem. Res. 2007, 16, 380–391.
24. Kundariya, D.S.; Bheshdadia, B.M.; Joshi, N.K.; Patel, P.K. Synthesis, Characterization and Pharmacological Evaluation of Some
Novel Schiff Bases Containing 1h-Pyrazolo [3, 4-B] Pyridine Moiety. Int. J. ChemTech. Res. 2011, 44, 4385–4392.
25. Available online: http://www.bpsbioscience.com/biochemical-based-assays (accessed on 21 November 2020).
26. Elrayess, R.; Elgawish, M.S.; Elewa, M.; Nafie, M.S.; Elhady, S.S.; Yassen, A.S.A. Synthesis, 3d-Qsar, and Molecular Modeling
Studies of Triazole Bearing Compounds as a Promising Scaffold for Cyclooxygenase-2 Inhibition. Pharmaceuticals 2020, 13, 370.
[CrossRef] [PubMed]
27. Ghareb, N.; El-Sayed, N.M.; Abdelhameed, R.; Yamad, K.; Elgawish, M.S. Toward a treatment of diabesity: Rational design,
synthesis and biological evaluation of benzene-sulfonamide derivatives as a new class of PTP-1B inhibitors. Bioorgan. Chem. 2019
86, 322–338. [CrossRef] [PubMed]
,
28. Elgawish, M.S.; Kishikawa, N.; Helal, M.A.; Ohyama, K.; Kuroda, N. Molecular modeling and spectroscopic study of quinone–
protein adducts: Insight into toxicity, selectivity, and reversibility. Toxicol. Res. 2015, 4, 843–847. [CrossRef]