Discovery of 3,3a,4,5-tetrahydro-2H-benzo[g]indazole containing quinoxaline derivatives as novel EGFR/HER-2 dual inhibitors

Article abstract of DOI:10.1039/c5ra02576a

In the present study, twenty-five pyrazole-quinoxaline derivatives (4a-4y) were designed and synthesized, and their biological activity as potential EGFR or HER-2 kinase inhibitors was evaluated. Among them, compound 4l displayed better antiproliferative

Full text of DOI:10.1039/c5ra02576a

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Discovery of 3,3a,4,5-tetrahydro-2H-benzo[g]
indazole containing quinoxaline derivatives as
novel EGFR/HER-2 dual inhibitors
Cite this: RSC Adv., 2015, 5, 24814
a
a
a
a
b
bc
*
Xi Zong, Jin Cai, Junqing Chen, Chunlong Sun, Lushen Li and Min Ji
In the present study, twenty-five pyrazole–quinoxaline derivatives (4a–4y) were designed and synthesized, and
their biological activity as potential EGFR or HER-2 kinase inhibitors was evaluated. Among them, compound 4l
displayed better antiproliferative activity against A549 and MCF-7 cell lines than Eroltinib. Further kinase
inhibitory activity assay results indicated that compound 4l demonstrated the most potent enzyme inhibitory
activity. Docking simulations were then performed to position compounds 4l and 4x into the active binding
site of EGFR to determine the probable binding model. 3D-QSAR models were built to aid in the effective
design of the presently studied and future EGFR inhibitors. These discoveries suggested that the title
compounds are potential EGFR/HER-2 dual inhibitors and compound 4l may be a promising lead
compound for the development of novel antitumor agents, potentially via inhibiting EGFR/HER-2.
Received 10th February 2015
Accepted 12th February 2015
DOI: 10.1039/c5ra02576a
www.rsc.org/advances
epidermal growth factor receptor 4 (HER-4/Erb-4).11 Among these,
as the targets in current cancer research, EGFR and HER-2 have
1. Introduction
been implicated in the development and malignancy of many
human cancers,7 because their overexpression or abnormal acti-
vation oen causes the malignant transformation of cells, such as
non-small cell lung cancer (NSCLC), and prostate, breast,
stomach, colon, and ovarian cancers.12–15
Cancer is a major problem in human health and remains the
second highest cause of mortality worldwide, with millions of
cases every year.1 The use of molecularly targeted therapeutics,
which are highly selective and do not have the serious toxicity
associated with conventional cytotoxic drugs, has become an
attractive approach for cancer chemotherapy.2 Protein kinases
catalyze the phosphorylation of tyrosine and serine/threonine
residues in various proteins,3 thus, protein kinases are exten-
sively targeted in order to discover inhibitors suitable as
potential cancer-treatment drugs.4–7 In recent years, a variety of
novel anticancer lead compounds have been developed that
target mutant or aberrantly expressed oncogenic growth factor
receptor and non-receptor tyrosine kinases in the mitogenic or
proliferative signal transduction pathways of cancer cells.8
As a transmembrane protein tyrosine kinase (PTK), the
epidermal growth factor receptor (EGFR) family plays a crucial
role in regulating cell proliferation, survival, adhesion, migration,
differentiation and apoptosis, and has been identied to play a
critical role in cancer.9,10 The EGFR family consists of the human
epidermal growth factor receptor (EGFR/ErbB-1), human
epidermal growth factor receptor 2 (HER-2/ErbB-2), human
epidermal growth factor receptor 3 (HER-3/Erb-3), and human
Recently, thousands of small organic molecules with
different structures have been developed as multi-target inhib-
itors.16–18 Compounds that inhibit the kinase activity of EGFR
and/or HER-2 aer binding to their ATP binding sites are of
potential interest as new therapeutic antitumor agents.19
A
number of tyrosine kinase inhibitors (TKIs) targeting EGFR and
HER-2 have been approved for the clinical treatment of cancer
by the FDA. As shown in Fig. 1, the EGFR inhibitors Getinib,
Erlotinib, Lapatinib, Vandetanib, Icotinib and Afatinib have
been marketed and launched successfully to treat various kinds
of cancer in clinical practice. There are also several EGFR kinase
inhibitors, such as Dacomitinib, that are already showing
promise in phase III clinical trials.
In previous studies, several pyrazole derivatives have been
reported to possess bioactivity in a wide range of applications.
The pyrazole moiety makes up the core structure of numerous
biologically active compounds, such as antiviral/antitumor,20–22
antibacterial,23,24 anti-inammatory,25 analgesic,26 fungistatic,27
and antihyperglycemic28 agents. Recently, special attention has
been paid to the usage of pyrazole to develop anticancer agents.
Lv et al. reported a series of novel pyrazole derivatives (PD,
Fig. 1) which showed modest to potent EGFR TK inhibitory
activity.29 In addition, a series of novel EGFR and HER-2
inhibitors (N1, N2, Fig. 1) with a thiazolyl-pyrazoline scaffold
have been discovered.30,31 Moreover, it was demonstrated that
aSchool of Chemistry & Chemical Engineering, Southeast University, Nanjing 210096,
China
bSchool of Biological Science & Medical Engineering, Southeast University, Nanjing
210096, China. E-mail: jimin@seu.edu.cn; Fax: +86-0512-62729923; Tel: +86-0512-
62729923
cSuzhou Key Laboratory of Biomaterials and Technologies & Collaborative Innovation
Center of Suzhou Nano Science and Technology, Suzhou, 215123, China
24814 | RSC Adv., 2015, 5, 24814–24823
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
Fig. 1 Chemical structures of EGFR and HER-2 inhibitors.
quinoxaline derivatives display a broad spectrum of biological summarized in Scheme 1. To explore the inuence of the
activity; some of these compounds have been described as substituted group at the 4-position of the phenyl and 8-position
potential candidates for the treatment of cancer.32–34
of the tricyclic heterocycles on the compounds’ activity,
However, to our knowledge, few reports have been dedicated compounds 4a–4o were synthesized. We also designed
to the design and synthesis of a type of antitumor compound compounds 4p–4y to expand the structural diversity of the
that contains pyrazole and quinoxaline moieties simulta- target compounds. Firstly, the diverse substituted chalcones
neously. A perusal of the literature revealed that the B ring of (2a–2y) were afforded by treating tetralone derivatives with the
pyrazole derivatives (PD, N1 and N2) has a great inuence on appropriate aldehyde in the presence of a solution of sodium
their activity. In our continued search for potential EGFR and hydroxide in methanol. Secondly, cyclization of the different
HER-2 inhibitory agents with novel scaffolds, we constrained chalcones with thiosemicarbazide in the presence of excess
the spatial location of the B ring by replacing the 3,5-diphenyl- sodium hydroxide gave tricyclic heterocycles containing a
4,5-dihydro-1H-pyrazole moiety with a 3-phenyl-3,3a,4,5-tetra- thiourea group (3a–3y). Finally, the target compounds 4a–4y
hydro-2H-benzo[g]indazole moiety, then combined the novel were obtained by reacting 3a–3y with 2,3-dichloroquinoxaline in
tricyclic pyrazole derivative with a thiazolo quinoxaline ring. ethanol. All of the target compounds gave satisfactory analytical
The two combined substructures were expected to exhibit a and spectroscopic data.
synergistic anticancer effect (Fig. 2).
2.2. Biological activity
2.2.1. Antiproliferation assay. Newly synthesized deriva-
tives 4a–4y were assessed for their antiproliferative activity by
employing the MTT-based assay, using Erlotinib and Getinib
2. Results and discussion
2.1. Chemistry
The protocol for the synthesis of the novel tricyclic heterocycles as positive controls against four cultured cancer cell lines
containing thiazolo[4,5-b]quinoxaline derivatives (4a–4y) is including A549, MCF-7, Hela and HepG2 (Table 1). Most target
Fig. 2 Design strategy and modification of novel EGFR/HER-2 inhibitors.
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 24814–24823 | 24815

View Article Online
RSC Advances
Paper
Scheme 1 Synthetic route for the preparation of the target compounds 4a-4y. Reagents and conditions: (a) ArCHO, 8 N NaOH, ethanol, 2 h, r.t.
65–83%; (b) thiosemicarbazide, NaOH, ethanol, 8 h, reflux, 62–75%; (c) 2,3-dichloroquinoxaline, ethanol, 8 h, reflux, 31–57%.
compounds were found to possess broad antiproliferative (IC50 ¼ 3.04 mM) also showed antiproliferative activity against
activity with IC50 values ranging from 2.02 to 31.54 mM. Among the A549 cell line that was higher than Erlotinib (IC50 ¼ 4.28
them, compounds 4k and 4v displayed the most broad- mM) and comparable to Getinib (IC50 ¼ 2.96 mM).
spectrum antiproliferative activity; their corresponding IC50
2.2.2. EGFR and HER-2 inhibitory activity. The aforemen-
values were all below 7.00 mM against the four cancer cell lines. tioned compounds were designed to assess the EGFR/HER-2
It is noticeable that several compounds exerted antiproliferative inhibitory activity of novel tricyclic heterocycle-containing
activity against the A549 and MCF-7 cell lines comparable to thiazolo[4,5-b]quinoxaline derivatives. Therefore, all of the
that of the positive control drugs, such as 4b, 4k, 4l, 4o, 4q, 4t, 4v compounds were assayed for their inhibitory activity against
and 4y. In particular, the most signicant inhibition was ach- EGFR and HER-2 tyrosine kinases, using Erlotinib as a positive
ieved by compound 4l with IC50 ¼ 1.91 mM toward the MCF-7 control.
cell line, which was better than that of Erlotinib (IC50 ¼ 2.32
As shown in Table 2, the pyrazole–quinoxaline derivatives
mM) and Getinib (IC50 ¼ 6.26 mM). In addition, compound 4l (4a–4y) have demonstrated fairly good EGFR and HER-2 inhib-
itory activity. Subsequent structure–activity relationship (SAR)
analysis indicated that the substituted group on ring A (R2)
played important roles in inhibiting kinase activity; compounds
with an electron-donating substituent at the 4-position of ring A
Table 1 In vitro antiproliferative activity (IC50, mM) of compounds 4a–
4y against four human tumor cell lines
exhibited better potency than those with an electron-
IC50a (mM)
withdrawing group. For example, 4b (IC50 ¼ 2.26 mM) > 4e
(IC50 ¼ 8.26 mM) > 4a (IC50 ¼ 9.82 mM) > 4d (IC50 ¼ 15.67 mM) >
Compounds R1
R2
X
A549 MCF-7 Hela
HepG2
4c (IC50 ¼ 21.24 mM), which was illustrated by the potency order:
OCH3 > CH3 > H > Cl > F. In addition, compounds (4d and 4e)
with methyl or chlorine at the 4-position of ring A displayed
more potent activity than compounds (4g–4j) with methyl or
chlorine at the 2-position and 3-position of ring A. Compounds
(4k–4o) with an electron-donating methoxy group at the 4-
position of ring B (R1) displayed higher inhibitory activity than
those (4p–4y) with a methyl or hydrogen substituent. Therefore,
the biological data demonstrated that an electron-donating
substituent at the R1 and/or R2 position might increase the
inhibitory activity against EGFR/HER-2. Replacing the 3,3a,4,5-
tetrahydro-2H-benz[g]indazole ring with a 2,3,3a,4-tetrahydro[1]
4a
H
H
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
O
O
O
O
O
O
O
O
O
O
8.32
4.38
28.26 15.26
15.26 11.33
4.62
15.34 28.26
15.26 18.26
8.88
15.36 10.16
13.22 14.87
5.38
3.04
14.19 12.28
24.73
9.59
4.12
4.26
10.92 28.22
24.96 13.36
6.82
4.47 12.8
6.38
18.65 21.34
28.26 16.26
6.88
3.28
2.98
8.25
5.38 10.08
15.24 17.18
8.89
12.25 19.56
9.28 10.08
10.33
16.28
18.27 19.23
6.28
7.38
10.25 12.61
7.46 15.25
16.23 14.57
5.88 12.56
4.38
2.58
4b
H
4-OCH3
4-F
4-Cl
4-CH3
2,4-Cl
3-Cl
4c
H
4d
H
4e
H
5.86
5.58
4f
H
4g
H
4h
4i
H
H
2-Cl
8.23
5.36
8.99
3-CH3
2-CH3
H
4j
H
4k
OCH3
3.48
1.91
4.18
5.76
4l
OCH3 4-OCH3
OCH3 4-F
OCH3 4-Cl
OCH3 4-CH3
4m
4n
4o
8.23
2.53
6.83
3.23
benzopyrano[4,3-c]pyrazole ring only resulted in
a slight
decrease in activity (4a–4e vs. 4p–4t). For the given compounds,
it was observed that the IC50 values of the inhibitors against
EGFR were consistent with those against HER-2, which is
possibly attributable to the high sequence homology in the
catalytic domains of these two kinases. Among these twenty-ve
pyrazole–quinoxaline derivatives, 4l seemed to be the most
potent EGFR and HER-2 inhibitor with IC50 values of 0.28 mM
and 1.26 mM, respectively, compared with the positive control
Erlotinib’s IC50 values of 0.08 mM and 0.23 mM.
Taking the biological data as a whole, we could reach the
preliminary conclusion that some of the target compounds are
potent EGFR/HER-2 inhibitors, and are helpful for the further
research of EGFR inhibitors and expanding the types of
compounds that act as EGFR inhibitors.
4p
H
H
4q
H
4-OCH3
4 F
4-Cl
4-CH3
H
4-OCH3
4 F
7.63
15.66 18.59
6.12
4r
H
4s
H
31.54
6.81
2.02
5.38
26.98 24.45
17.64 17.05
7.46 11.38
9.88
7.26
8.29
5.66
4t
H
3.99
4u
4v
4w
4x
4y
CH3
CH3
CH3
CH3
CH3
2.02
4-Cl
4-CH3
13.67
4.28
2.96
3.28
2.32
6.26
Erlotinib
Getinib
1.93
4.12
3.02
3.18
a
Antiproliferative activity was measured using the MTT assay. The
values are the average of three independent experiments run in
triplicate. The variation was generally 5–10%.
24816 | RSC Adv., 2015, 5, 24814–24823
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
Table 2 Enzyme inhibition activities of compounds 4a–4y against Table
3
The median cytotoxic concentration (CC50) data of all
EGFR and HER-2
compounds
a
IC50a (mM)
Compounds
CC50 (mmol)
Compounds
EGFRb
HER-2b
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
4w
4x
51.28
55.43
46.78
50.36
53.28
55.33
46.72
49.38
52.68
51.47
61.33
78.44
55.56
51.42
68.45
48.28
56.12
41.56
48.33
52.67
48.16
59.26
51.38
48.34
56.25
68.03
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
4w
4x
9.82
2.26
21.24
15.67
8.26
15.83
14.63
15.82
9.26
15.28
4.68
0.28
8.16
18.23
0.96
3.32
11.43
3.68
18.43
26.58
12.38
17.46
11.06
3.04
13.98
20.88
5.34
1.26
15.24
25.46
3.23
8.68
5.25
30.46
28.4
5.67
11.34
2.35
33.56
12.8
1.23
18.26
15.34
2.36
5.23
0.86
15.28
6.8
1.23
4y
Erlotinib
4y
4.66
0.23
a
The cytotoxicity of each compound was expressed as the concentration
of compound that reduced cell viability to 50% (CC50).
Erlotinib
0.08
a
Errors were in the range of 5–10% of the reported values, from three
different assays. b Human recombinant enzymes, by the esterase assay
(4-nitrophenylacetate as substrate).
of compound 4l forms a p–p interaction with Phe699. As shown
in Fig. 3C and D, a similar p–p interaction also occurs between
the terminal phenyl moiety of compound 4x and Phe699.
Furthermore, the oxygen of the tricyclic moiety forms a
hydrogen bond with Met769 and the nitrogen of the quinoxa-
line moiety forms hydrogen bonds with Lys721 and Asp831.
Comparing these models (Fig. 3E and F), it is obvious that
compounds 4l and 4x are able to nicely occupy the active
binding pocket of EGFR, as well as Erlotinib. Interestingly, the
similar hydrogen bonding interactions with Met769 all
occurred in the binding pocket of 4l, 4x and Erlotinib in
complex with EGFR.
2.2.3. Cytotoxicity test. As shown in Table 3, all of the target
compounds (4a–4y) were evaluated for their toxicity against the
human kidney epithelial 293T cell line using the MTT assay;
these compounds were tested at multiple doses to study the
viability of 293T cell. The median cytotoxic concentration (CC50
)
showed that all of the tested compounds displayed almost no
cytotoxicity in vitro against 293T cells.
2.3. Molecular docking
In summary, the docking analysis strongly suggests that the
substituents on the two different rings and the quinoxaline
moiety form hydrogen bonding interactions and p–p interac-
tions with the protein residues in the ATP binding site, thus
resulting in dramatic EGFR inhibition activity.
To further investigate the interactions between these
compounds and EGFR, molecular docking of the 3,3a,4,5-tet-
rahydro-2H-benz[g]indazole derivative 4l and 2,3,3a,4-tetrahy-
dro[1]benzopyrano[4,3-c]pyrazole derivative 4x into the ATP
binding site of EGFR kinase were performed on the binding
model based on the EGFR complex structure (1M17.pdb).35
The docking pose of 4l in its complex with EGFR is shown in
Fig. 3A and B. In the binding mode, the oxygen of the methoxy
2.4. 3D-QSAR model
moiety forms hydrogen bonding interactions with Met769 and To study the bioactivity of the pyrazole–quinoxaline derivatives
Gly697. This may explain why compound 4l, with a methoxy in greater depth, a 3D-QSAR model was built according to the
substituent at R1 and R2, could display increased inhibitory compounds synthesized and their corresponding capabilities.36
activity against EGFR kinase. In addition, a hydrogen bonding In doing so, we intended to explain the mechanism of the SAR,
interaction is observed between the nitrogen of the quinoxaline and aid in the discovery of more potent novel antagonists of
moiety and Asp831. Simultaneously, the terminal phenyl moiety EGFR. This model was executed by the built-in QSAR soware of
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 24814–24823 | 24817

View Article Online
RSC Advances
Paper
Fig. 5 (a) 3D-QSAR model coefficients on electrostatic potential grids.
Blue represents positive coefficients and red represents negative
coefficients. (b) 3D-QSAR model coefficients on van der Waals grids.
Green represents positive coefficients and yellow represents negative
coefficients.
Table 4 Experimental and predicted inhibitory activity of compounds
4a-4y against EGFR protein using the 3D-QSAR model
EGFR
Compounds
Actual-pIC50
Predicted-pIC50
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
5.01
5.65
4.67
4.80
5.08
4.97
5.33
5.74
5.03
4.82
5.25
6.55
5.09
4.74
6.02
5.48
5.91
4.74
4.81
5.63
5.28
6.07
4.82
5.17
5.91
5.08
5.70
4.56
4.64
5.14
5.12
5.24
5.61
5.26
5.00
5.31
6.54
4.99
4.92
6.01
5.41
5.83
4.71
4.75
5.58
5.47
6.04
4.76
5.10
5.811
Fig. 3 Docking modes of compounds in the binding pockets of EGFR
(PDB ID: 1M17). Interactions between the protein and the ligand are
shown as yellow dotted lines, the residues are shown as orange sticks,
and the ligands are shown as stick models in green (4l), cyan (4x) and
magenta (Erlotinib). (A) The predicted binding mode of 4l with EGFR;
(B) the surface of the binding site of 4l with EGFR; (C) the predicted
binding mode of 4x with EGFR; (D) the surface of the binding site of 4x
with EGFR; (E) the predicted binding mode of Erlotinib with EGFR; (F)
the docking modes of compounds 4l, 4x and Erlotinib in the active site
of EGFR.
4s
4t
4u
4v
4w
4x
4y
medicinal chemistry lab (http://www.sanjeevslab.org/tools-
IC50.html). By a random selection, these compounds were
divided into a test set containing 5 compounds and a training
set comprising the rest of the 20 compounds.
Fig. 4 Plot of the experimental vs. predicted values for the EGFR
inhibitory activity of the training set and test set.
By default, the alignment conformation of each molecule
possessed the lowest CDOCKER_INTERACTION_ENERGY of all
of the docked poses. The critical regions (steric or electrostatic)
affecting the binding affinity were identied by this 3D-QSAR
model. Using a CHARMM force eld and PLS regression, the
model was set up with a conventional R2 of 0.95, indicating this
DS 3.5 (Discovery Studio 3.5, Accelrys, Co. Ltd), with all of the
molecules converted to their active conformation and the cor-
responding IC50 (mM) values transformed from an Indian
24818 | RSC Adv., 2015, 5, 24814–24823
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
model possesses a good predictive capability. The relationship
between the observed and predicted values has been shown
graphically in Fig. 4.
4.2. Synthesis
4.2.1. General synthetic procedure of chalcones (2a–2y). To
a solution of tetralone derivatives 1a–1d (50 mmol) and appro-
priate aldehyde (50 mmol) in ethanol (20 mL) was slowly added
an aqueous solution of NaOH (0.012 mol, 8 N) at room
temperature. The mixture was stirred for 2 h. The solid
precipitate was collected by ltration and then washed with cold
ethanol (30 mL) three times. The solid was dried in vacuo to give
each chalcone (2a–2y).
4.2.2. General synthetic procedure of pyrazole derivatives
(3a–3y). A mixture of chalcones 2a–2y (10 mmol), thio-
semicarbazide (10 mmol) and NaOH (25 mmol) in ethanol (25
mL) was reuxed for 8 h. The solution was poured into ice water
and then ltered. The residue was washed with a small amount
of methanol and dried in vacuo to give each pyrazole derivative
(3a–3y).
4.2.3. General synthetic procedure of dihydropyridin con-
taining thiazolinone derivatives (4a–4y). A mixture of pyrazole
derivatives 3a–3y (10 mmol) and 2,3-dichloroquinoxaline (10
mmol) in absolute ethanol (15 mL) was reuxed for 8 h. The
progress of the reaction was monitored using TLC. Aer the
completion of the reaction, the solvent was removed under
reduced pressure and the residue was recrystallized from
methanol to give 4a–4y.
4.2.3.1. 2-(3-Phenyl-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-
yl)thiazolo[4,5-b]quinoxaline (4a). White solid. Yield 51%; m.p.
201–203 ꢀC; 1H NMR (DMSO-d6, 300 MHz); d: 7.94 (d, J ¼ 6.4 Hz,
1H, Ar-H); 7.90 (d, J ¼ 7.8 Hz, 1H, Ar-H); 7.84–7.81 (m, 2H, Ar-H);
7.63–7.53 (m, 2H, Ar-H); 7.49–7.32 (m, 2H, Ar-H); 7.30–7.28 (m,
1H, Ar-H); 7.03–6.89 (m, 4H, Ar-H); 6.08 (d, J ¼ 10.7 Hz, 1H, CH);
4.10–4.03 (m, 1H, CH); 3.01–2.82 (m, 2H, CH2); 1.74–1.60 (m,
1H, CH2); 0.96–0.81 (m, 1H, CH2). ESI-MS m/z: 434.2 [M + H]+.
Anal. calcd for C26H19N5S (%): C, 72.03; H, 4.42; N, 16.15.
Found: C, 72.43; H, 4.33; N, 16.28.
Also, the molecules aligned with the iso-surfaces of the 3D-
QSAR model coefficients on electrostatic potential grids
(Fig. 5a) and van der Waals grids (Fig. 5b) were listed. The
electrostatic map displayed the favorable (in blue) or unfavor-
able (in red) electrostatic eld regions in a contour plot, while
the energy grids corresponding to the favorable (in green) or
unfavorable (in yellow) steric effects were also marked out. For
compounds based on the 3D-QSAR model, possessing strong
van der Waals attraction forces in the green areas and a polar
group in the blue electrostatic potential areas means achieving
potent bioactivity. The results yielded by the model were
accordant with the actual ndings, in terms of identifying the
potent compounds. Thus, this promising model could provide
guidelines for the design and optimization of more effective
tubulin inhibitors and pave the way for further study.
3. Conclusions
In this work, a series of pyrazole–quinoxaline derivatives that
potentially function as inhibitors of EGFR and HER-2 kinases
have been synthesized, and most of them exhibited potent
affinity for EGFR or HER-2 kinase as well as excellent anti-
proliferative activity. Compound 4l showed excellent EGFR/
HER-2 inhibitory activities and better antiproliferative activity
against A549 and MCF-7 cell lines than Erlotinib. Aer analysis
of the binding models of compounds 4l and 4x with EGFR, it
was found that the models of compounds 4l or 4x in complex
with the ATP binding site were similar to that of Erlotinib. The
quinoxaline moiety might play a crucial role in the compounds’
EGFR inhibition activity by forming p–p interactions and
hydrogen bonding interactions with the residues in the binding
pocket. Finally, QSAR models were built with the activity data
and binding conformations to begin our work in this paper, as
well as to provide a reliable tool for the reasonable design and
synthesis of potent tyrosine kinase inhibitors. In conclusion,
the preliminary evaluation results demonstrated that the newly
developed tricyclic heterocycle-containing pyrazoles combined
with a thiazolo quinoxaline ring, acting as potent dual EGFR
and HER-2 inhibitors, may possess therapeutic potential for
cancer treatment.
4.2.3.2. 2-(3-(4-Methoxyphenyl)-3,3a,4,5-tetrahydro-2H-benzo[g]-
indazol-2-yl)thiazolo[4,5-b]quinoxaline (4b). White solid. Yield 47%;
m.p. 186–187 ꢀC; 1H NMR (DMSO-d6, 300 MHz); d: 8.04 (d, J ¼ 7.4
Hz, 1H, Ar-H); 7.98 (d, J ¼ 7.7 Hz, 1H, Ar-H); 7.86–7.84 (m, 1H, Ar-
H); 7.73–7.62 (m, 2H, Ar-H); 7.48–7.36 (m, 2H, Ar-H); 7.31–7.29 (m,
1H, Ar-H); 7.12 (d, J ¼ 8.2 Hz, 2H, Ar-H); 6.91 (d, J ¼ 8.5 Hz, 2H, Ar-
H); 6.10 (d, J ¼ 10.6 Hz, 1H, CH); 4.14-4.02 (m, 1H, CH); 3.70 (s, 3H,
CH3); 3.02–2.86 (m, 2H, CH2); 1.84–1.81 (m, 1H, CH2); 1.06–0.96
(m, 1H, CH2). ESI-MS m/z: 464.3 [M + H]+. Anal. calcd for
C27H21N5OS (%): C, 69.96; H, 4.57; N, 15.11. Found: C, 69.85; H,
4.49; N, 15.28.
4. Experiments
4.2.3.3. 2-(3-(4-Fluorophenyl)-3,3a,4,5-tetrahydro-2H-benzo[g]-
indazol-2-yl)thiazolo[4,5-b]quinoxaline (4c). White solid. Yield
48%; m.p. 202–204 ꢀC; 1H NMR (DMSO-d6, 300 MHz); d: 8.08 (d,
J ¼ 7.5 Hz, 1H, Ar-H); 7.99–7.95 (m, 1H, Ar-H); 7.82–7.79 (m, 1H,
Ar-H); 7.40–7.28 (m, 3H, Ar-H); 7.23–7.20 (m, 1H, Ar-H); 7.15–
7.09 (m, 3H, Ar-H); 7.05–7.00 (m, 2H, Ar-H); 6.03 (d, J ¼ 10.6 Hz,
1H, CH); 3.87–3.37 (m, 1H, CH); 2.92–2.76 (m, 2H, CH2); 1.78–
1.74 (m, 1H, CH2); 0.80–0.66 (m, 1H, CH2). ESI-MS m/z: 452.2
[M + H]+. Anal. calcd for C26H18FN5S (%): C, 69.16; H, 4.02; N,
15.51. Found: C, 69.33; H, 4.09; N, 15.41.
4.1. Materials and measurements
All chemicals and reagents used in this study were analytical
grade. Thin-layer chromatography (TLC) was performed on
silica gel plates with a uorescent indicator. All of the analyzed
samples were homogeneous when assessed using TLC in at
1
least two different solvent systems. All of the H NMR spectra
were recorded on a Bruker DPX 300 model spectrometer in
DMSO-d6 and the chemical shis (d) were reported in parts per
million (ppm). ESI-MS spectra were recorded on a Mariner
System 5304 mass spectrometer. Elementary analyses were
performed on an Elementar Vario EL III instrument.
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 24814–24823 | 24819

View Article Online
RSC Advances
Paper
2H, Ar-H); 7.08–7.01 (m, 1H, Ar-H); 6.95–6.80 (m, 2H, Ar-H); 6.13
(d, J ¼ 10.2 Hz, 1H, CH); 3.58–3.47 (m, 1H, CH); 2.85–2.76 (m,
2H, CH2); 2.48 (s, 3H, CH3); 1.80–1.72 (m, 1H, CH2); 0.86–0.75
(m, 1H, CH2). ESI-MS m/z: 448.2 [M + H]+. Anal. calcd for
4.2.3.4. 2-(3-(4-Chlorophenyl)-3,3a,4,5-tetrahydro-2H-benzo[g]-
indazol-2-yl)thiazolo[4,5-b]quinoxaline (4d). Yellow solid. Yield
1
ꢀ
37%; m.p. 211–213 C; H NMR (DMSO-d6, 300 MHz); d: 8.05–
7.97 (m, 2H, Ar-H); 7.89–7.84 (m, 1H, Ar-H); 7.72–7.63 (m, 2H,
Ar-H); 7.58–7.48 (m, 2H, Ar-H); 7.44–7.35 (m, 3H, Ar-H); 7.31–
7.23 (m, 2H, Ar-H); 6.17 (d, J ¼ 10.7 Hz, 1H, CH); 4.06–4.00 (m,
1H, CH); 2.98–2.87 (m, 2H, CH2); 1.72–1.70 (m, 1H, CH2); 0.97–
0.93 (m, 1H, CH2). ESI-MS m/z: 468.2 [M + H]+. Anal. calcd for
C
27H21N5S (%): C, 72.46; H, 4.73; N, 15.65. Found: C, 72.61; H,
4.69; N, 15.58.
4.2.3.10. 2-(3-(M-tolyl)-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-
2-yl)thiazolo[4,5-b]quinoxaline (4j). White solid. Yield 42%; m.p.
188–190 ꢀC; 1H NMR (DMSO-d6, 300 MHz); d: 8.12–7.96 (m, 2H,
Ar–H); 7.80 (d, J ¼ 7.7 Hz, 1H, Ar–H); 7.35 (d, J ¼ 7.6 Hz, 2H, Ar-
H); 7.30–7.22 (m, 2H, Ar-H); 7.13–7.10 (m, 2H, Ar-H); 7.08–6.92
(m, 3H, Ar-H); 6.03 (d, J ¼ 10.7 Hz, 1H, CH); 3.56–3.41 (m, 1H,
CH); 2.82–2.69 (m, 2H, CH2); 2.39 (s, 3H, CH3); 1.81–1.75 (m,
1H, CH2); 0.85–0.73 (m, 1H, CH2). ESI-MS m/z: 448.2 [M + H]+.
Anal. calcd for C27H21N5S (%): C, 72.46; H, 4.73; N, 15.65.
Found: C, 72.61; H, 4.66; N, 15.68.
4.2.3.11. 2-(8-Methoxy-3-phenyl-3,3a,4,5-tetrahydro-2H-benzo
[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4k). White solid. Yield
43%; m.p. 196–198 ꢀC; 1H NMR (DMSO-d6, 300 MHz); d: 7.98 (d,
J ¼ 6.8 Hz, 1H, Ar-H); 7.84 (d, J ¼ 7.3 Hz, 1H, Ar-H); 7.71–7.64
(m, 2H, Ar-H); 7.50–7.42 (m, 1H, Ar-H); 7.34–7.29 (m, 3H, Ar-H);
7.21–7.20 (m, 3H, Ar-H); 7.07–7.05 (m, 1H, Ar-H); 6.16 (d, J ¼
10.7 Hz, 1H, CH); 4.16–4.08 (m, 1H, CH); 3.85 (s, 3H, CH3); 2.89–
2.81 (m, 2H, CH2); 1.85–1.82 (m, 1H, CH2); 0.86–0.79 (m, 1H,
CH2). ESI-MS m/z: 464.2 [M + H]+. Anal. calcd for C27H21N5OS
(%): C, 69.96; H, 4.57; N, 15.11. Found: C, 69.69; H, 4.49; N,
15.28.
4.2.3.12. 2-(8-Methoxy-3-(4-methoxyphenyl)-3,3a,4,5-tetrahy-
dro-2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4l). White
solid. Yield 34%; m.p. 184–186 ꢀC; 1H NMR (DMSO-d6, 300
MHz); d: 8.01 (d, J ¼ 7.2 Hz, 1H, Ar-H); 7.96 (d, J ¼ 7.7 Hz, 1H, Ar-
H); 7.86–7.83 (m, 1H, Ar-H); 7.73–7.66 (m, 2H, Ar-H); 7.45–7.34
(m, 2H, Ar-H); 7.31–7.28 (m, 1H, Ar-H); 7.12 (d, J ¼ 8.2 Hz, 2H,
Ar-H); 6.90 (d, J ¼ 8.5 Hz, 1H, Ar-H); 6.11 (d, J ¼ 10.6 Hz, 1H,
CH); 4.14–4.08 (m, 1H, CH); 3.70 (s, 3H, CH3); 3.59 (s, 3H, CH3);
3.04–2.83 (m, 2H, CH2); 1.84–1.80 (m, 1H, CH2); 1.02–0.94 (m,
1H, CH2). ESI-MS m/z: 494.2 [M + H]+. Anal. calcd for
C
26H18ClN5S (%): C, 66.73; H, 3.88; N, 14.97. Found: C, 66.63; H,
3.69; N, 14.78.
4.2.3.5. 2-(3-(P-tolyl)-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-
2-yl)thiazolo[4,5-b]quinoxaline (4e). Yellow solid. Yield 40%; m.p.
191–193 ꢀC; 1H NMR (DMSO-d6, 300 MHz); d: 8.12–8.03 (m, 1H,
Ar-H); 7.97 (d, J ¼ 7.7 Hz, 1H, Ar-H); 7.82–7.78 (m, 1H, Ar-H);
7.42–7.30 (m, 2H, Ar-H); 7.33–7.21 (m, 2H, Ar-H); 7.15–7.10
(m, 2H, Ar-H); 7.08–7.01 (m, 1H, Ar-H); 6.95–6.80 (m, 2H, Ar-H);
6.13 (d, J ¼ 10.3 Hz, 1H, CH); 3.57–3.37 (m, 1H, CH); 2.82–2.66
(m, 2H, CH2); 2.41 (s, 3H, CH3); 1.80–1.73 (m, 1H, CH2); 0.85–
0.79 (m, 1H, CH2). ESI-MS m/z: 448.2 [M + H]+. Anal. calcd for
C
27H21N5S (%): C, 72.46; H, 4.73; N, 15.65. Found: C, 72.63; H,
4.69; N, 15.58.
4.2.3.6. 2-(3-(2,4-Dichlorophenyl)-3,3a,4,5-tetrahydro-2H-benzo
[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4f). White solid. Yield
1
ꢀ
57%; m.p. 196–198 C; H NMR (DMSO-d6, 300 MHz); d: 8.04–
7.99 (m, 2H, Ar-H); 7.89–7.81 (m, 2H, Ar-H); 7.74–7.67 (m, 2H,
Ar-H); 7.47–7.30 (m, 4H, Ar-H); 7.00 (d, J ¼ 8.3Hz, 1H, CH); 6.41
(d, J ¼ 11.3 Hz, 1H, CH); 4.36–4.30 (m, 1H, CH); 3.04–2.87 (m,
2H, CH2); 1.94–1.90 (m, 1H, CH2); 1.10–1.07 (m, 1H, CH2). ESI-
MS m/z: 502.2 [M + H]+. Anal. calcd for C26H17Cl2N5S (%): C,
62.16; H, 3.41; N, 13.94. Found: C, 62.32; H, 3.35; N, 14.04.
4.2.3.7. 2-(3-(3-Chlorophenyl)-3,3a,4,5-tetrahydro-2H-benzo[g]-
indazol-2-yl)thiazolo[4,5-b]quinoxaline (4g). Yellow solid. Yield
1
ꢀ
32%; m.p. 186–187 C; H NMR (DMSO-d6, 300 MHz); d: 8.01–
7.94 (m, 2H, Ar-H); 7.88–7.82 (m, 1H, Ar-H); 7.70–7.53 (m, 3H,
Ar-H); 7.48–7.43 (m, 1H, Ar-H); 7.40–7.32 (m, 3H, Ar-H); 7.30–
7.24 (m, 2H, Ar-H); 6.13 (d, J ¼ 10.6 Hz, 1H, CH); 4.03–3.98 (m,
1H, CH); 2.95–2.82 (m, 2H, CH2); 1.70–1.65 (m, 1H, CH2); 0.96–
0.90 (m, 1H, CH2). ESI-MS m/z: 468.3 [M + H]+. Anal. calcd for
C
28H23N5O2S (%): C, 68.13; H, 4.70; N, 14.19. Found: C, 68.28; H,
4.59; N, 14.38.
C
26H18ClN5S (%): C, 66.73; H, 3.88; N, 14.97. Found: C, 66.63; H,
4.2.3.13. 2-(3-(4-Fluorophenyl)-8-methoxy-3,3a,4,5-tetrahy-
3.96; N, 14.86.
dro-2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline
(4m).
4.2.3.8. 2-(3-(2-Chlorophenyl)-3,3a,4,5-tetrahydro-2H-benzo[g]-
indazol-2-yl)thiazolo[4,5-b]quinoxaline (4h). Yellow solid. Yield
42%; m.p. 187–189 ꢀC; 1H NMR (DMSO-d6, 300 MHz); d: 8.11 (d,
J ¼ 8.1 Hz, 1H, Ar-H); 8.07–7.95 (m, 2H, Ar–H); 7.72–7.69 (m, 1H,
Ar–H); 7.66–7.59 (m, 1H, Ar-H); 7.51 (d, J ¼ 7.9 Hz, 1H, Ar-H);
7.43 (d, J ¼ 9.1 Hz, 1H, Ar-H); 7.34–7.29 (m, 3H, Ar-H); 7.21–
7.13 (m, 2H, Ar-H); 6.20 (d, J ¼ 10.5 Hz, 1H, CH); 4.11–4.05 (m,
1H, CH); 2.99–2.83 (m, 2H, CH2); 1.85–1.71 (m, 1H, CH2); 1.07–
1.03 (m, 1H, CH2). ESI-MS m/z: 468.2 [M + H]+. Anal. calcd for
1
ꢀ
White solid. Yield 47%; m.p. 181–183 C; H NMR (DMSO-d6,
300 MHz); d: 8.10–8.01 (m, 1H, Ar-H); 7.97–7.92 (m, 1H, Ar-H);
7.65 (d, J ¼ 2.6 Hz, 1H, Ar-H); 7.55–7.51 (m, 1H, Ar-H); 7.28–
7.20 (m, 1H, Ar-H); 7.16–7.09 (m, 3H, Ar-H); 7.04–6.94 (m, 3H,
Ar-H); 6.01 (d, J ¼ 10.6 Hz, 1H, CH); 3.80 (s, 3H, CH3); 3.80–3.67
(m, 1H, CH); 2.81–2.73 (m, 2H, CH2); 1.77–1.73 (m, 1H, CH2);
0.76–0.61 (m, 1H, CH2). ESI-MS m/z: 482.2 [M + H]+. Anal. calcd
for C27H20FN5OS (%): C, 67.34; H, 4.19; N, 14.54. Found: C,
67.63; H, 4.29; N, 14.48.
C
26H18ClN5S (%): C, 66.73; H, 3.88; N, 14.97. Found: C, 66.62; H,
4.2.3.14. 2-(3-(4-Chlorophenyl)-8-methoxy-3,3a,4,5-tetrahydro-
2H-benzo[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4n). Yellow
solid. Yield 33%; m.p. 203–205 ꢀC; 1H NMR (DMSO-d6,
300 MHz); d: 8.03–7.91 (m, 2H, Ar-H); 7.88–7.67 (m, 3H, Ar-H);
7.58–7.48 (m, 2H, Ar-H); 7.44–7.38 (m, 1H, Ar-H); 7.35–7.32
(m, 1H, Ar-H); 7.31–7.22 (m, 2H, Ar-H); 6.13 (d, J ¼ 10.7 Hz, 1H,
CH); 4.12–4.07 (m, 1H, CH); 3.81 (s, 3H, CH3); 2.87–2.81 (m, 2H,
3.86; N, 16.89.
4.2.3.9. 2-(3-(M-tolyl)-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-
2-yl)thiazolo[4,5-b]quinoxaline (4i). Yellow solid. Yield 48%; m.p.
178–180 ꢀC; 1H NMR (DMSO-d6, 300 MHz); d: 8.08 (d, J ¼ 7.6 Hz,
1H, Ar-H); 7.99 (d, J ¼ 8.3 Hz, 1H, Ar-H); 7.82–7.77 (m, 1H, Ar-H);
7.42–7.31 (m, 2H, Ar-H); 7.30–7.21 (m, 2H, Ar-H); 7.15–7.10 (m,
24820 | RSC Adv., 2015, 5, 24814–24823
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
CH2); 1.66–1.61 (m, 1H, CH2); 0.95–0.88 (m, 1H, CH2). ESI-MS 7.7 Hz, 1H, Ar-H); 7.97 (d, J ¼ 4.4 Hz, 1H, Ar-H); 7.82–7.78 (m,
m/z: 498.2 [M + H]+. Anal. calcd for C27H20ClN5OS (%): C, 1H, Ar-H); 7.42–7.22 (m, 4H, Ar-H); 7.13–7.09 (m, 2H, Ar-H);
65.12; H, 4.05; N, 14.06. Found: C, 65.33; H, 4.09; N, 14.28.
7.03–6.93 (m, 3H, Ar-H); 6.33 (d, J ¼ 10.6 Hz, 1H, CH); 4.42–
4.2.3.15. 2-(8-Methoxy-3-(p-tolyl)-3,3a,4,5-tetrahydro-2H-benzo- 4.37 (m, 1H, CH2); 3.88–3.78 (m, 1H, CH2); 3.47–3.37 (m, 1H,
[g]indazol-2-yl)thiazolo[4,5-b]quinoxaline (4o). Yellow solid. Yield CH); 2.41 (s, 3H, CH3). ESI-MS m/z: 450.2 [M + H]+. Anal. calcd
1
ꢀ
32%; m.p. 202–204 C; H NMR (DMSO-d6, 300 MHz); d: 7.92– for C26H19N5OS (%): C, 69.47; H, 4.26; N, 15.58. Found: C, 69.63;
7.89 (m, 1H, Ar-H); 7.97 (d, J ¼ 7.7 Hz, 1H, Ar-H); 7.80 (d, J ¼ 8.7 H, 4.39; N, 15.48.
Hz, 1H, Ar-H); 7.35 (d, J ¼ 5.4 Hz, 1H, Ar-H); 7.30–7.21 (m, 2H,
4.2.3.21. 8-Methyl-3-phenyl-2-(thiazolo[4,5-b]quinoxalin-2-yl)-
Ar-H); 7.13–7.07 (m, 2H, Ar-H); 7.05–7.01 (m, 1H, Ar-H); 6.93– 2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole (4u). White solid.
1
ꢀ
6.77 (m, 2H, Ar-H); 6.10 (d, J ¼ 10.9 Hz, 1H, CH); 3.81 (s, 3H, Yield 42%; m.p. 187–189 C; H NMR (DMSO-d6, 300 MHz); d:
CH3); 3.57–3.35 (m, 1H, CH); 2.85–2.62 (m, 2H, CH2); 2.38 (s, 7.99 (d, J ¼ 8.7 Hz, 2H, Ar-H); 7.82–7.77 (d, J ¼ 6.7 Hz, 1H, Ar-H);
3H, CH3); 1.82–1.71 (m, 1H, CH2); 0.88–0.82 (m, 1H, CH2). ESI- 7.42–7.21 (m, 4H, Ar-H); 7.16–7.11 (m, 2H, Ar-H); 7.08–7.03 (m,
MS m/z: 478.2 [M + H]+. Anal. calcd for C28H23N5OS (%): C, 1H, Ar-H); 6.93–6.83 (m, 2H, Ar-H); 6.03 (d, J ¼ 10.1 Hz, 1H, CH);
70.42; H, 4.85; N, 14.66. Found: C, 70.63; H, 4.59; N, 14.48.
4.2.3.16. 3-Phenyl-2-(thiazolo[4,5-b]quinoxalin-2-yl)-2,3,3a,4-
4.51–4.42 (m, 1H, CH2); 3.80–3.77 (m, 1H, CH2); 3.55–3.43 (m,
1H, CH); 2.48 (s, 3H, CH3). ESI-MS m/z: 450.2 [M + H]+. Anal.
tetrahydrochromeno[4,3-c]pyrazole (4p). White solid. Yield 49%; calcd for C26H19N5OS (%): C, 69.47; H, 4.26; N, 15.58. Found: C,
1
ꢀ
m.p. 193–195 C; H NMR (DMSO-d6, 300 MHz); d: 8.04 (d, J ¼ 69.63; H, 4.33; N, 15.48.
6.3 Hz, 1H, Ar-H); 7.97 (d, J ¼ 7.8 Hz, 1H, Ar-H); 7.88–7.81 (m,
4.2.3.22. 3-(4-Methoxyphenyl)-8-methyl-2-(thiazolo[4,5-b]qui-
2H, Ar-H); 7.66–7.55 (m, 2H, Ar-H); 7.51–7.37 (m, 3H, Ar-H); noxalin-2-yl)-2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole
(4v).
1
ꢀ
7.30–7.23 (m, 2H, Ar-H); 7.10–6.95 (m, 2H, Ar-H); 5.98 (d, J ¼ Yellow solid. Yield 36%; m.p. 192–194 C; H NMR (DMSO-d6,
10.7 Hz, 1H, CH); 4.63–4.55 (m, 1H, CH); 4.14–4.12 (m, 1H, CH); 300 MHz); d: 8.02–7.95 (m, 1H, Ar–H); 7.88–7.82 (m, 1H, Ar-H);
4.07–4.03 (m, 1H, CH). ESI-MS m/z: 436.2 [M + H]+. Anal. calcd 7.77 (d, J ¼ 8.7 Hz, 1H, Ar-H); 7.45–7.30 (m, 3H, Ar-H); 7.22–7.17
for C25H17N5OS (%): C, 68.95; H, 3.93; N, 16.08. Found: C, 68.73; (m, 2H, Ar-H); 7.10–6.88 (m, 3H, Ar-H); 6.25 (d, J ¼ 10.4 Hz, 1H,
H, 3.79; N, 16.28.
CH); 4.44–4.33 (dd, J ¼ 10.3 and 12.8 Hz, 1H, CH2); 3.81 (s, 3H,
CH3); 3.65–3.59 (dd, J ¼ 6.0 and 10.2 Hz, 1H, CH2); 3.55–3.42 (m,
4.2.3.17. 3-(4-Methoxyphenyl)-2-(thiazolo[4,5-b]quinoxalin-2-
yl)-2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole (4q). White solid. 1H, CH); 2.33 (s, 3H, CH3). ESI-MS m/z: 480.2 [M + H]+. Anal.
1
ꢀ
Yield 43%; m.p. 207–209 C; H NMR (DMSO-d6, 300 MHz); d: calcd for C27H21N5O2S (%): C, 67.62; H, 4.41; N, 14.60. Found: C,
8.03 (d, J ¼ 7.3 Hz, 1H, Ar-H); 7.97 (d, J ¼ 7.5 Hz, 1H, Ar-H); 7.86– 67.38; H, 4.49; N, 14.48.
7.74 (m, 3H, Ar-H); 7.46–7.34 (m, 2H, Ar-H); 7.32–7.25 (m, 1H,
4.2.3.23. 3-(4-Fluorophenyl)-8-methyl-2-(thiazolo[4,5-b]quinox-
Ar-H); 7.12–6.99 (m, 4H, Ar-H); 6.20 (d, J ¼ 10.4 Hz, 1H, CH); alin-2-yl)-2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole (4w). White
4.44–4.41 (m, 1H, CH2); 4.24–4.12 (m, 1H, CH); 3.86–3.76 (m, solid. Yield 42%; m.p. 199–201 ꢀC; 1H NMR (DMSO-d6, 300
1H, CH2); 3.78 (s, 3H, CH3); . ESI-MS m/z: 466.2 [M + H]+. Anal. MHz); d: 8.12–8.01 (m, 1H, Ar-H); 7.96–7.92 (m, 1H, Ar-H); 7.62
calcd for C26H19N5O2S (%): C, 67.08; H, 4.11; N, 15.04. Found: C, (d, J ¼ 3.6 Hz, 1H, Ar-H); 7.54–7.50 (m, 1H, Ar-H); 7.28–7.21 (m,
67.32; H, 4.22; N, 15.15.
1H, Ar-H); 7.16–7.09 (m, 3H, Ar-H); 7.04–6.94 (m, 3H, Ar-H); 6.06
4.2.3.18. 3-(4-Fluorophenyl)-2-(thiazolo[4,5-b]quinoxalin-2-yl)- (d, J ¼ 10.9 Hz, 1H, CH); 4.55–4.49 (dd, J ¼ 11.1 and 13.2 Hz, 1H,
2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole (4r). White solid. CH2); 3.80–3.69 (m, 1H, CH); 3.55–3.49 (dd, J ¼ 5.1 and 10.2 Hz,
1
Yield 37%; m.p. 211–213 C; H NMR (DMSO-d6, 300 MHz); d: 1H, CH2); 2.34 (s, 3H, CH3). ESI-MS m/z: 468.2 [M + H]+. Anal.
ꢀ
8.06 (d, J ¼ 7.3 Hz, 1H, Ar-H); 7.99–7.97 (m, 1H, Ar-H); 7.82–7.76 calcd for C26H18FN5OS (%): C, 66.79; H, 3.88; N, 14.98. Found:
(m, 1H, Ar-H); 7.40–7.33 (m, 1H, Ar-H); 7.30–7.28 (m, 2H, Ar-H); C, 66.63; H, 3.79; N, 14.73.
7.23–7.20 (m, 1H, Ar-H); 7.15–7.09 (m, 3H, Ar-H); 7.05–7.00 (m,
4.2.3.24. 3-(4-Chlorophenyl)-8-methyl-2-(thiazolo[4,5-b]quinox-
2H, Ar-H); 6.02 (d, J ¼ 10.9 Hz, 1H, CH); 4.66–4.58 (m, 1H, CH2), alin-2-yl)-2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole (4x). White
4.11–4.08 (m, 1H, CH2); 3.87–3.37 (m, 1H, CH). ESI-MS m/z: solid. Yield 51%; m.p. 205–207 ꢀC; 1H NMR (DMSO-d6,
454.2 [M + H]+. Anal. calcd for C25H16FN5OS (%): C, 66.21; H, 300 MHz); d: 8.04–7.96 (m, 2H, Ar-H); 7.85–7.80 (m, 2H, Ar-H);
3.56; N, 15.44. Found: C, 66.53; H, 3.49; N, 15.53.
7.75–7.63 (m, 2H, Ar-H); 7.47–7.30 (m, 4H, Ar-H); 6.94 (d, J ¼
4.2.3.19. 3-(4-Chlorophenyl)-2-(thiazolo[4,5-b]quinoxalin-2-yl)- 8.3 Hz, 1H, CH); 6.31 (d, J ¼ 10.2 Hz, 1H, CH); 4.60–4.51 (m, 1H,
2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole (4s). Yellow solid. CH2); 4.26–4.20 (m, 1H, CH); 3.88–3.79 (m, 1H, CH2); 2.41 (s, 3H,
1
Yield 31%; m.p. 174–176 C; H NMR (DMSO-d6, 300 MHz); d: CH3). ESI-MS m/z: 484.1 [M + H]+. Anal. calcd for C26H18ClN5OS
ꢀ
8.01–7.95 (m, 2H, Ar-H); 7.88–7.82 (m, 1H, Ar-H); 7.720–7.66 (m, (%): C, 64.52; H, 3.75; N, 14.47. Found: C, 64.63; H, 3.59; N, 14.38.
2H, Ar-H); 7.54–7.49 (m, 2H, Ar-H); 7.43–7.37 (m, 3H, Ar-H);
4.2.3.25. 8-Methyl-2-(thiazolo[4,5-b]quinoxalin-2-yl)-3-(p-tolyl)-
7.31–7.25 (m, 2H, Ar-H); 6.07 (d, J ¼ 10.1 Hz, 1H, CH); 4.41– 2,3,3a,4-tetrahydrochromeno[4,3-c]pyrazole (4y). Yellow solid.
1
ꢀ
4.33 (dd, J ¼ 10.1 and 12.2 Hz, 1H, CH2); 4.06–3.97 (m, 1H, CH); Yield 43%; m.p. 196–198 C; H NMR (DMSO-d6, 300 MHz); d:
3.89–3.77 (dd, J ¼ 6.1 and 10.2 Hz, 1H, CH2). ESI-MS m/z: 470.1 8.03 (d, J ¼ 7.2 Hz, 1H, Ar-H); 7.92 (d, J ¼ 7.3 Hz, 1H, Ar-H); 7.82–
[M + H]+. Anal. calcd for C25H16ClN5OS (%): C, 63.89; H, 3.43; N, 7.72 (m, 1H, Ar-H); 7.42–7.21 (m, 4H, Ar-H); 7.13–7.02 (m, 2H,
14.90. Found: C, 63.63; H, 3.49; N, 14.78.
Ar-H); 6.92–6.80 (m, 2H, Ar-H); 6.19 (d, J ¼ 10.8 Hz, 1H, CH);
4.2.3.20. 2-(Thiazolo[4,5-b]quinoxalin-2-yl)-3-(p-tolyl)-2,3,3a,4- 4.49–4.39 (dd, J ¼ 10.3 and 12.8 Hz, 1H, CH2); 3.77–3.69 (dd, J ¼
tetrahydrochromeno[4,3-c]pyrazole (4t). Yellow solid. Yield 32%; 5.1 and 10.2 Hz, 1H, CH2); 3.53–3.44 (m, 1H, CH); 2.48 (s, 3H,
1
m.p. 197–199 C; H NMR (DMSO-d6, 300 MHz); d: 8.10 (d, J ¼ CH3); 2.44 (s, 3H, CH3); ESI-MS m/z: 464.2 [M + H]+. Anal. calcd
ꢀ
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 24814–24823 | 24821

View Article Online
RSC Advances
Paper
for C27H21N5OS (%): C, 69.96; H, 4.57; N, 15.11. Found: C, 69.63; some modications. Cell lines were grown to log phase in
H, 4.49; N, 15.28.
DMEM supplemented with 10% fetal bovine serum, under a
humidied atmosphere of 5% CO2 at 37 C. Cell suspensions
ꢀ
were prepared and 100 mL per well was dispensed into 96-well
plates giving 105 cells per well. The plates were returned to the
incubator for 24 h to allow the cells to reattach. Subsequently,
cells were treated with the target compounds at increasing
concentrations in the presence of 10% FBS for 48 h. Then, cell
viability was assessed using the conventional 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
reduction assay and carried out strictly according to the
4.3. Preparation and purication of EGFR and HER-2 and
the inhibitory assay
A 1.6 kb cDNA encoding the EGFR cytoplasmic domain (EGFR-
CD, amino acids 645–1186) and 1.7 Kb cDNA encoding the
human HER-2 cytoplasmic domain (HER-2-CD, amino acids
676–1245) were cloned into the baculoviral expression vectors
pBlueBacHis2B and pFASTBac HTc (Huakang Company,
China), separately. A sequence that encodes (His)6 was located
at the 50 upstream of the EGFR and HER-2 sequences. Sf-9 cells
were infected for 3 days for protein expression. Sf-9 cell pellets
were solubilized at 0 ꢀC in a buffer at pH 7.4 containing 50 mM
HEPES, 10 mM NaCl, 1% Triton, 10 mM ammonium molybdate,
100 lM sodium vanadate, 10 mg mLꢁ1 aprotinin, 10 mg mLꢁ1
manufacturer’s instructions (Sigma). The absorbance (OD570
)
was read on an ELISA reader (Tecan, Austria).
4.5. Experimental protocol of the docking study
leupeptin, 10 mg mLꢁ1 pepstatin, and 16 mg mLꢁ1 benzamidine Molecular docking of compounds 4l and 4x into the three
HCl for 20 min followed by 20 min centrifugation. The crude dimensional X-ray structure of EGFR (PDB code: 1M17) was
extract supernatant was passed through an equilibrated Ni-NTA carried out using the Discovery Studio (version 3.5) as imple-
superow packed column and washed with 10 mM and then mented through the graphical user interface DS-CDOCKER
100 mM imidazole to remove nonspecically bound material. protocol. The three-dimensional structures of the aforemen-
Histidine tagged proteins were eluted with 250 and 500 mM tioned compounds were constructed using Chem. 3D ultra 12.0
imidazole and dialyzed against 50 mM NaCl, 20 mM HEPES, soware [Chemical Structure Drawing Standard; Cambridge
10% glycerol, and 1 mg mLꢁ1 each of aprotinin, leupeptin, and So corporation, USA (2013)], then they were energetically
pepstatin for 2 h. The entire purication procedure was per- minimized by using MMFF94 with 5000 iterations and a
formed at 4 ꢀC or on ice.
minimum RMS gradient of 0.10. The crystal structures of the
The EGFR and HER-2 kinase assays were both set up to assess protein complex were retrieved from the RCSB Protein Data
the level of autophosphorylation based on DELFIA/time-resolved Bank (http://www.rcsb.org/pdb/home/home.do). All bound
uorometry. Compounds 4a–4y were dissolved in 100% DMSO waters and ligands were eliminated from the protein. The
and diluted to the appropriate concentrations with 25 mM HEPES molecular docking was performed by inserting compounds 4l
at pH 7.4. In each well, 10 mL of each compound was incubated and 4x into the binding pocket of EGFR based on the binding
with 10 mL (12.5 ng for HER-2 or 5 ng for EGFR) recombinant mode. The types of interactions of the docked protein with the
enzyme (1 : 80 dilution in 100 mM HEPES) for 10 min at room ligand-based pharmacophore model were analyzed aer the
temperature. Then, 10 mL of 5 mM buffer (containing 20 mM end of molecular docking.
HEPES, 2 mM MnCl2, 100 mM Na3VO4, and 1 mM DTT) and 20 mL
of 0.1 mM ATP–50 mM MgCl2 were added for 1 h. Positive and
negative controls were included in each plate by incubation of the
enzyme with or without ATP–MgCl2. At the end of incubation, the
liquid was aspirated, and plates were washed three times with
wash buffer. A 75 mL (400 ng) sample of europium labeled anti-
phosphotyrosine antibody was added to each well for another 1
h of incubation. Aer washing, an enhancement solution was
added and the signal was detected using a Victor multilabel
reader (Wallac Inc.) with excitation at 340 nm and emission at 615
nm. The percentage of auto-phosphorylation inhibition by the
compounds was calculated using the following formula: 100% ꢁ
[(negative control)/(positive control ꢁ negative control)]. The IC50
was obtained from curves of the percentage inhibition with eight
concentrations of the compound. As the amount of contamina-
tion in the enzyme preparation is fairly low, the majority of the
signal detected by the anti-phosphotyrosine antibody is from
EGFR or HER-2.
4.6. 3D-QSAR
The ligand-based 3D-QSAR approach was performed using the
QSAR soware of DS 3.5 (Discovery Studio 3.5, Accelrys, Co.
Ltd). The training sets were composed of inhibitors with the
corresponding pIC50 values, which were converted from the
obtained IC50 (mM) values, and test sets comprised compounds
of data sets, as listed in Table 4.
All of the denition of the descriptors can be seen in the
“Help” of the DS 3.5 soware and they were calculated using the
QSAR protocol of DS 3.5. The alignment conformation of each
molecule was the one with the lowest interaction energy in the
docking results of CDOCKER. The predictive ability of 3D-QSAR
modeling can be evaluated based on the cross-validated corre-
lation coefficient, which qualies the predictive ability of the
models. A Y scrambling procedure was performed to investigate
the risk of chance correlations. The inhibitory potencies of
compounds were randomly reordered 30 times and subjected to
leave-one-out validation tests. The models were also validated
4.4. Antiproliferative activity
The antiproliferative activities of the prepared compounds were by test sets, in which the compounds were not included in the
evaluated using a standard (MTT)-based colorimetric assay with training sets.
24822 | RSC Adv., 2015, 5, 24814–24823
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
21 S. Manfredini, R. Bazzanini, P. G. Baraldi, M. Bonora,
M. Marangoni, D. Simoni, A. Pani, F. Scintu, E. Pinna,
L. Pisano and P. La Colla, Anti-Cancer Drug Des., 1996, 11,
193–204.
22 H. J. Park, K. Lee, S. J. Park, B. Ahn, J. C. Lee, H. Cho and
K. I. Lee, Bioorg. Med. Chem. Lett., 2005, 15, 3307–3312.
23 A. Tanitame, Y. Oyamada, K. Ofuji, M. Fujimoto, N. Iwai,
Y. Hiyama, K. Suzuki, H. Ito, H. Terauchi, M. Kawasaki,
K. Nagai, M. Wachi and J. Yamagishi, J. Med. Chem., 2004,
47, 3693–3696.
Acknowledgements
This work is supported by The Fundamental Research Funds for
the Central Universities (2242014R30019), Technology Sup-
porting Program of Jiangsu province (BE2012657), and National
Basin Research Program of China (no. 2011CB933503). We
thank Pro. Hai-liang Zhu (State Key Laboratory of Pharmaceu-
tical Biotechnology, Nanjing University) for a license to use their
soware.
24 S. Guniz Kucukguzel, S. Rollas, H. Erdeniz, M. Kiraz,
A. Cevdet Ekinci and A. Vidin, Eur. J. Med. Chem., 2000, 35,
761–771.
References and notes
1 A. S. El-Azab, M. A. Al-Omar, A. A. Abdel-Aziz, N. I. Abdel-Aziz,
M. A. el-Sayed, A. M. Aleisa, M. M. Sayed-Ahmed and
S. G. Abdel-Hamide, Eur. J. Med. Chem., 2010, 45, 4188–4198.
2 L. Seymour, Cancer Treat. Rev., 1999, 25, 301–312.
3 J. D. Jordan, E. M. Landau and R. Iyengar, Cell, 2000, 103,
193–200.
4 S. K. Rabindran, D. D. Ross, L. A. Doyle, W. Yang and
L. M. Greenberger, Cancer Res., 2000, 60, 47–50.
5 D. M. Shin, F. R. Khuri, B. S. Glisson, L. Ginsberg,
V. M. Papadimitrakopoulou, G. Clayman, J. J. Lee,
K. K. Ang, S. M. Lippman and W. K. Hong, Cancer, 2001,
91, 1316–1323.
25 T. D. Penning, J. J. Talley, S. R. Bertenshaw, J. S. Carter,
P. W. Collins, S. Docter, M. J. Graneto, L. F. Lee,
J. W. Malecha, J. M. Miyashiro, R. S. Rogers, D. J. Rogier,
S. S. Yu, G. d. Anderson, E. G. Burton, J. N. Cogburn,
S. A. Gregory, C. M. Koboldt, W. E. Perkins, K. Seibert,
A. W. Veenhuizen, Y. Y. Zhang and P. C. Isakson, J. Med.
Chem., 1997, 40, 1347–1365.
26 G. Menozzi, L. Mosti, P. Fossa, F. Mattioli and M. Ghia, J.
Heterocycl. Chem., 1997, 34, 963–968.
27 R. Sridhar, P. T. Perumal, S. Etti, G. Shanmugam,
M. N. Ponnuswamy, V. R. Prabavathy and N. Mathivanan,
Bioorg. Med. Chem. Lett., 2004, 14, 6035–6040.
6 P. Cohen, Nat. Rev. Drug Discovery, 2002, 1, 309–315.
7 A. Levitzki, Acc. Chem. Res., 2003, 36, 462–469.
8 A. A. Adjei, J. Natl. Cancer Inst., 2001, 93, 1062–1074.
9 X. W. Zhang, J. Gureasko, K. Shen, P. A. Cole and J. Kuriyan,
Cell, 2006, 125, 1137–1149.
10 S. Guo, L. S. Colbert, M. Fuller, Y. Zhang and R. R. Gonzalez-
Perez, Biochim. Biophys. Acta, 2010, 1806, 108–121.
11 K. S. Kolibaba and B. J. Druker, Biochim. Biophys. Acta, 1997,
1333, F217–F248.
12 P. Traxler, Expert Opin. Ther. Targets, 2003, 7, 215–234.
13 S. S. Sridhar, L. Seymour and F. A. Shepherd, Lancet Oncol.,
2003, 4, 397–406.
14 J. Vansteenkiste, Expert Rev. Anticancer Ther., 2004, 4, 5–17.
15 S. Madhusudan and T. S. Ganesan, Clin. Biochem., 2004, 37,
618–635.
16 C. Y. Hsieh, P. C. Tsai, C. H. Tseng, Y. L. Chen, L. S. Chang
and S. R. Lin, Toxicol. In Vitro, 2013, 27, 1–10.
17 Y. Y. Xu, Y. Cao, H. Ma, H. Q. Li and G. Z. Ao, Bioorg. Med.
Chem., 2013, 21, 388–394.
18 N. C. Valerie, B. Dziegielewska, A. S. Hosing, E. Augustin,
L. S. Gray, D. L. Brautigan, J. M. Larner and
J. Dziegielewski, Biochem. Pharmacol., 2013, 85, 888–897.
19 A. J. Bridges, Curr. Med. Chem., 1999, 6, 825–843.
20 S. Manfredini, R. Bazzanini, P. G. Baraldi, M. Guarneri,
D. Simoni, M. E. Marongiu, A. Pani, E. Tramontano and
P. La Colla, J. Med. Chem., 1992, 35, 917–924.
28 G. R. Bebernitz, G. Argentieri, B. Battle, C. Brennan,
B. Balkan, B. F. Burkey, M. Eckhardt, J. Gao, P. Kapa,
R. J. Strohschein, H. F. Schuster, M. Wilson and D. D. Xu,
J. Med. Chem., 2001, 44, 2601–2611.
29 P. C. Lv, H. Q. Li, J. Sun, Y. Zhou and H. L. Zhu, Bioorg. Med.
Chem., 2010, 18, 4606–4614.
30 P. C. Lv, D. D. Li, Q. S. Li, X. Lu, Z. P. Xiao and H. L. Zhu,
Bioorg. Med. Chem. Lett., 2011, 21, 5374–5377.
31 K. M. Qiu, H. H. Wang, L. M. Wang, Y. Luo, X. H. Yang,
X. M. Wang and H. L. Zhu, Bioorg. Med. Chem., 2012, 20,
2010–2018.
32 P. Corona, A. Carta, M. Loriga, G. Vitale and G. Paglietti, Eur.
J. Med. Chem., 2009, 44, 1579–1591.
33 F. A. Rodrigues, S. Bomm Ida, B. C. Cavalcanti, O. Pessoa
Cdo, J. L. Wardell, S. M. Wardell, A. C. Pinheiro,
C. R. Kaiser, T. C. Nogueira, J. N. Low, L. R. Gomes and
M. V. de Souza, Bioorg. Med. Chem. Lett., 2014, 24, 934–939.
34 T. R. Mielcke, A. Mascarello, E. Filippi-Chiela, R. F. Zanin,
G. Lenz, P. C. Leal, L. D. Chirardia, R. A. Yunes,
R. J. Nunes, A. M. O. Battastini, F. B. Morrone and
M. M. Campos, Eur. J. Med. Chem., 2012, 48, 255–264.
35 J. Stamos, M. X. Sliwkowski and C. Eigenbrot, J. Biol. Chem.,
2002, 277, 46265–46272.
36 W. J. Egan, K. M. Merz Jr and J. J. Baldwin, J. Med. Chem.,
2000, 43, 3867–3877.
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 24814–24823 | 24823