Discovery of 7-bromo-1,4-dihydrothieno[3’,2’:5,6]thiopyrano[4,3-c]pyrazole-3-carboxamide derivatives as the potential epidermal growth factor receptors for tyrosine kinase inhibitors

Article abstract of DOI:10.1007/s00044-019-02354-0

Small molecule tyrosine kinase inhibitors (TKIs) targeting at epidermal growth factor receptor (EGFR) in recent years have made great progress in the treatment of advanced non-small cell cancer (NSCLC). Although as the first-line treatment for sensitizing EGFR mutation-positive metastatic NSCLC, gefitinib has also behaved quite a lot of side effect and EGFR tolerance. Herein, a novel series of 7-bromo-1,4-dihydrothieno[3’,2’:5,6]thiopyrano[4,3-c]pyrazole-3-carboxamide derivatives were designed and synthesized, and screened for their inhibitory activity on the EGFR high-expressing human lung adenocarcinoma cell line A549 and human large cell lung cancer cell line NCI-H460 by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric assay. The calculated IC₅₀ values were reported. Compound 8h demonstrated the most potent inhibitory activity (IC₅₀ = 9.57 ± 2.20 μmol L^–1 for A549 and IC₅₀ = 13.04 ± 1.21 μmol L^–1 for NCI-H460), comparable to the positive-control gefitinib (IC₅₀ = 8.58 ± 1.65 μmol L^–1 for A549 and IC₅₀ = 18.66 ± 5.01 μmol L^–1 for NCI-H460). Conclusively, 7-bromo- 1,4-dihydrothieno[3’,2’:5,6]thiopyrano[4,3-c]pyrazole-3-carboxamide derivatives as the EGFR–TKIs were discovered, and could be used as potential leading compounds for further research.

Full text of DOI:10.1007/s00044-019-02354-0

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02354-0
ORIGINAL RESEARCH
Discovery of 7-bromo-1,4-dihydrothieno[3’,2’:5,6]thiopyrano[4,3-c]
pyrazole-3-carboxamide derivatives as the potential epidermal
growth factor receptors for tyrosine kinase inhibitors
Qian Xie1 Jingjing Wang1 Miao Liu1 Xin Wang1 Di Jiao1 Qingqing Ma1 Zhe Jin1 Qingguo Meng1,2
Chun Hu
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Received: 21 February 2019 / Accepted: 30 April 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Small molecule tyrosine kinase inhibitors (TKIs) targeting at epidermal growth factor receptor (EGFR) in recent years have
made great progress in the treatment of advanced non-small cell cancer (NSCLC). Although as the first-line treatment for
sensitizing EGFR mutation-positive metastatic NSCLC, gefitinib has also behaved quite a lot of side effect and EGFR
tolerance. Herein, a novel series of 7-bromo-1,4-dihydrothieno[3’,2’:5,6]thiopyrano[4,3-c]pyrazole-3-carboxamide deriva-
tives were designed and synthesized, and screened for their inhibitory activity on the EGFR high-expressing human lung
adenocarcinoma cell line A549 and human large cell lung cancer cell line NCI-H460 by using 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide colorimetric assay. The calculated IC50 values were reported. Compound 8h demonstrated
the most potent inhibitory activity (IC50 = 9.57 2.20 μmol L–1 for A549 and IC50 = 13.04 1.21 μmol L–1 for NCI-H460),
comparable to the positive-control gefitinib (IC50 = 8.58 1.65 μmol L–1 for A549 and IC50 = 18.66 5.01 μmol L–1 for
NCI-H460). Conclusively, 7-bromo- 1,4-dihydrothieno[3’,2’:5,6]thiopyrano[4,3-c]pyrazole-3-carboxamide derivatives as
the EGFR–TKIs were discovered, and could be used as potential leading compounds for further research.
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Keywords EGFR Inhibitors Heterocycle Synthesis Anticancer activities
Introduction
of lung cancer, small molecule tyrosine kinase inhibitors
(TKIs) targeting at epidermal growth factor receptor
(EGFR) in recent years have made great progress in the
treatment of advanced NSCLC (Sharma et al. 2007). Stu-
dies have found that most NSCLC cancer tissues express
ligands such as EGF and TGFα, and activating EGFR
mutations are present in 5–15% of NSCLC in the United
States and Europe and in 44–55% of NSCLC in east Asia
(Kohno et al. 2015).
Lung cancer is the leading cause of cancer death worldwide
(Parkin et al. 2005). Eighty percent of lung cancers belong
to non-small cell lung cancer (NSCLC), and 70% of
NSCLC is diagnosed at an advanced stage, which is diffi-
cult to be cured by surgery or radiotherapy (Schiller et al.
2002). With the deeper study of the molecular mechanism
Targeting activating EGFR mutations with TKIs showed
a promising response and prolonged patients’ lives in phase
III trials. Gefitinib showed superiority to paclitaxel/carbo-
platin in the Iressa Pan-Asia Study (IPASS) on advanced
EGFR-mutant lung adenocarcinoma (Mok et al. 2009;
Fukuoka et al. 2011). The National Comprehensive Cancer
Network (NCCN) guideline for NSCLC has suggested
using gefitinib for first-line treatment of sensitizing EGFR
mutation-positive metastatic NSCLC after 2016 (NCCN,
2016). Although occurring at low frequency, progressive
respiratory dysfunction, including acute interstitial pneu-
monia, is the most-severe adverse effect of gefitinib
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02354-0) contains supplementary
material, which is available to authorized users.
* Chun Hu
chunhu@syphu.edu.cn
1
Key Laboratory of Structure-based Drug Design & Discovery,
Ministry of Education, Shenyang Pharmaceutical University,
Shenyang 110016, China
2
Department of Pharmacy, Yantai Univeristy, Yantai 264005,
China

Medicinal Chemistry Research
Fig. 1 The structure of some
EGFR–TKIs
(Inoue et al. 2003), which has limited the therapeutic benefit
of gefitinib. Moreover, ~50% of patients have acquired
EGFR-resistant mutations after treatment with gefitinib,
which also occurred in the treatment with other
EGFR–TKIs (Pao et al. 2005). Consequently, discovering
more-effective EGFR–TKIs with lower side effects is still
the current hot spot in the research and development
of drugs.
It can be found that the structure of most small molecules
with EGFR-inhibitory activity reported in the literatures
possess heterocyclic scaffold, such as quinazoline, quino-
line, thienopyrimidine, furopyrimidine, and tetra-
hydropyridothiophenepyrimidine, and the side chain with
substituted amine group (Fig. 1) (Coumar et al. 2010;
Wissner & Mansour, 2008; Wood et al. 2008; Wu et al.
2010; Zhou et al. 2011). Previous studies in our laboratory
have shown that 4,5-dihydro-1H-thieno[2’,3’:2,3]thiepino
[4,5-c]pyrazole-3-carboxamide derivatives might be potent
EGFR inhibitors and showed remarked antitumor activity
(Ke et al. 2018). Based on the principle of scaffold hopping
(Guo, 2008), hybridization, and bioisosterism, a novel
heterocyclic scaffold, thieno[3’,2’:5,6]thiopyrano[4,3-c]
pyrazole, was designed as the target scaffold. The molecular
docking has showed that the interaction of the target thieno
[3’,2’:5,6]thiopyrano[4,3-c]pyrazole derivatives with EGFR
was similar to the interation of gefitinib with EGFR. Bro-
mination is a frequent modification in medicinal chemistry
and can play a significant improvement on the biological
activity (Neumann et al. 2008), and introduction of the
bromine atom at the parent ring was considered pre-
ferentially. As known, the piperazine groups have often
appeared in the structures of EGFR inhibitors (Adly et al.
2018; Ke et al. 2018; Sun et al. 2015; Zhang et al. 2016),
and the molecular docking results also showed that the
target compounds with piperazine side chain have stronger
affinity and selectivity for EGFR than that of gefitinib.
Fig. 2 The structure of gefitinib and designed compounds
Therefore, a novel series of 7-bromo-1,4-dihydrothieno
[3’,2’:5,6]thiopyrano[4,3-c]pyrazole-3-carboxamide deriva-
tives were synthesized in satisfactory yields (Fig. 2). All the
target compounds were screened for their antitumor activity
on the EGFR high-expressing human lung adenocarcinoma
cell line A549 and human large cell lung cancer cell line
NCI-H460. The screening results showed that several target
compounds exhibited moderate antitumor activity and
compound 8h demonstrated the most potent inhibitory
activity.
Results and discussion
Molecular docking studies
A docking study aiming at the characterization of the
interaction between the target molecule and EGFR at
molecular level was carried out and more-effective
EGFR–TKIs were expected to be discovered.
In this study, docking model was selected from Protein
Data Bank of Research Collaborator for Structural Bioin-
formatics (RCSB PDB), and the docking study was pre-
pared by Molegro Virtual Docker (MVD) 2008 (v3.0)
(Thomsen & Christensen, 2006). Initially, the docking

Medicinal Chemistry Research
studies were carried out on the EGFR–erlotinib complex
(PDB code 4HJO) (Park et al. 2012), using the Discovery
Studio 2016 client to remove the small-molecule ligand of
erlotinib in 4HJO to obtain the three-dimensional crystal
structure of EGFR, and then the designed compounds and
the positive-control gefitinib were docked to the crystal
structure EGFR under MVD software. The detailed docking
data will be described below, using compound 8h with two
trifluoromrthyl groups at the 3 and 5 positions of the phenyl
ring as an example.
From Fig. 3 and Fig. 4, it can be seen that compound 8h
was inserted inside the protein and the N atom of its pyr-
azole ring can form a hydrogen bond with the amino acid
residue THR830 of EGFR, and similarly the N atom at the
N1 position on the quinoline ring of gefitinib also has a
hydrogen bond with the amino acid residue of EGFR. At
the same time, the amino-acid residue LYS721of EGFR
exhibited hydrogen bond interaction with the S atom on the
dihydrothiophene ring of 8h and the N atom at the N1
position on the quinoline ring of gefitinib. The amino-acid
residue THR766 of EGFR also has conventional hydrogen
bond interaction with the S atom on the dihydrothiophene
ring of 8h. Hence, the importance of dihydrothienopyrazole
ring for the binding of small ligand molecules to EGFR is
determined. In addition, there is a hydrogen bond interac-
tion with the amino-acid residue SER696 of EGFR on the F
atom of the trifluoromethyl group of 8h. Hence, we can
conclude that the type and position of the substituent on the
aromatic ring of the aryl piperazine attached to the parent
ring will have an effect on the activity of the target com-
pounds. From Fig. 4 and Fig. 5, it can be seen that the
amino-acid residues LEU694, LYS721, THR830, ALA719,
Fig. 5 Interactions of 8h (blue) and gefinitib (red) with EGFR in a
three-dimensional diagram
Fig. 3 Interactions of 8h and the active site of EGFR in H-bonds level
Fig. 4 Interactions of 8h (left) and gefinitib (right) with EGFR in a two-dimensional diagram

Medicinal Chemistry Research
Scheme 1 Reagents and
conditions: a NaOC2H5/
C2H5OH, reflux, N2; then HCl
(aq). b oxalyl chloride, r.t, 1 h,
N2; then SnCl4, 0 °C, 12 h, N2.
c NaOCH3, dimethyl oxalate, r.t,
24 h. d) 80% hydrazine hydrate,
reflux, 4 h. e Br2, CH3COOH/
H2O, 0 °C, 24 h. f NaOH/H2O,
reflux; 18% HCl(aq). g Amines
or 1arylpiperazines, EDCI/
HOBt, Et3N, r.t. 24 h
and VAL702 formed some weak interactions between the
compound 8h and gefitinib.
presence of EDCI and HOBt and were purified by column
chromatography on silica gel with eluent of ethyl acetate/
petroleum ether (1:1).
The molecular docking results revealed that the target
compound 8h and its analogs were potential inhibitors of
EGFR, and might exhibit similar effect to gefinitib.
Therefore, we synthesized a novel series of 7-bromo- 1,4-
dihydrothieno[3’,2’:5,6]thiopyrano[4,3-c]pyrazole-3-car-
boxamide derivatives, aiming to assay their inhibitory
activity to EGFR.
Bioactivity in cell line
Using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-
zolium bromide (MTT) colorimetric assay, and using gefi-
tinib as a positive-control drug, 15 target compounds were
evaluated on the EGFR, high-expressing human lung ade-
nocarcinoma cell line A549, and human large cell lung
cancer cell line NCI-H460, which were commonly used in
EGFR–TKI-related studies for the effect of cell proliferation
levels (Mosmann, 1983; Sun et al. 2016; Zhang et al. 2017;
Zhou et al. 2016). The antitumor activity of these 15 target
compounds was preliminary screened (final concentration
of drug is 8 μM). The calculated IC50 value, the concentra-
tion (mu-gram/mL) of a compound that was able to cause
50% growth inhibition with respect to the control culture,
was summarized in Table 1.
In general, the screening results showed that most of the
target compounds exhibit remarked antiproliferative activ-
ities against A549 cells, and most of the target compounds
hardly exhibit any antiproliferative activities against NCI-
H460 cells except 7e, 7f, 8b, 8h, and 8i.
In A549 cells, the IC50 values of compounds 7e, 7f, 8c,
8f, and 8h were close to the positive-control drug gefitinib,
indicating that these compounds exhibit significant pro-
liferation inhibitions on A549 lung cancer cells; in NCI-
H460 lung cancer cells, the IC50 values of the compound 8h
were smaller than that of the positive-control drug gefitinib,
suggesting that these drugs have a stronger inhibitory effect
on proliferation of NCI-H460 lung cancer cells. As shown
Synthesis
The synthesis of 7-bromo- 1,4-dihydrothieno[3’,2’:5,6]
thiopyrano[4,3-c]pyrazole-3-carboxamide derivatives 7a–7f
and 8a–8i is described in Scheme 1.
4-(Thiophen-2-ylthio)butanoic acid (1), obtained by
treating 2-mercaptothiophene with γ-butyrolactone under
the nitrogen gas, was reacted with oxalyl chloride and tin
tetrachloride under nitrogen gas to give 2 (Ponticello et al.
1988) in the yield of 25–47.5%. The reaction of 2 with
dimethyl oxalate in dry toluene at room temperature for 24 h
in the presence of sodium hydride yielded the compound 3
(Wang et al. 1990) in the field of 60%. A mixture of 3 and
hydrazine hydrate in acetic acid was then refluxed for 4 h to
yield 4 (Zhao et al. 2002). Then bromine was added to a
mixed solution of 95% glacial acetic acid and compound 4
while stirring in an ice bath, and allowed to react at room
temperature for 24 h after the completion of the dropwise
addition to give 5. Compound 5 was refluxed in aqueous
sodium hydroxide for 2 h, and then 18% hydrochloric acid
was used to yield compound 6 in the field of 75–90%. The
target compounds 7a–7f and 8a–8i were obtained by
reacting 6 with amines or piperazine derivatives in the

Medicinal Chemistry Research
Table 1 IC50 values of the target compounds 7a–7f and 8a–8i against A549 and NCI-H460
Substituents
No.
IC50(μM)
R1
C2H5
H
R2
R3
A549
NCI-H460
-67.29 101.53
-11.14 5.72
-27.73 15.66
-14.65 6.31
28.31 23.13
23.70 15.45
-93.76 53.15
42.12 34.69
-43.32 13.66
-16.32 9.08
-16.12 7.74
-24.61 17.68
-19.69 3.91
13.04 1.21
C2H5
25.13 6.91
14.31 1.94
4.71 28.95
17.66 8.45
11.77 1.10
12.73 4.68
19.12 1.98
3.39 27.14
12.06 2.29
7.67 38.51
25.43 51.63
10.57 2.20
20.30 6.57
9.57 2.20
7a
7b
H
7c
H
C6H5
7d
H
7e
H
7f
8a
8b
8c
8d
8e
8f
8g
8h
-72.53 126.56
8.58 1.65
9.46 72.03
8i
18.66 5.01
gefitinib
in Table 1, the compound 8h displayed the most-potent
inhibitory activity (IC50 = 9.57 2.20 μmol L–1 for A549
and IC50 = 13.04 1.21 μmol L–1 for NCI-H460), compar-
able to the positive-control gifitinib (IC50 = 8.58
1.65 μmol L–1 for A549 and IC50 = 18.66 5.01 μmol L–1
for NCI-H460).
By analyzing the effect of side-chain changes on activ-
ity, it was found that the target compounds with substituted
piperazine side chains (8a–8i) are significantly more active
than the compounds with other amine side chains (7a–7f).
The compound which introduced a methyl group or a
methoxy group (8f and 8g) at the 4-position of the phenyl
ring in the substituted aryl piperazine side chain has high
biological activity, but when an electron-withdrawing
group (8e) at this position was introduced, the biological
activity is reduced. It can be seen that the introduction of a
smaller electron-donating group at this position is advan-
tageous for enhancing the biological activity. When two
trifluoromethyl groups were introduced at the 3 and 5
positions of the phenyl ring, this compound (8h) showed
strong EGFR-inhibitory activity in human lung adeno-
carcinoma cell line A549, whereas the EGFR-inhibitory
activity of compound 8i is significantly reduced after
removing one trifluoromethyl group. It can be seen that
there is a close relationship between the position of the
substituent and the biological activity. When one or two
electron-withdrawing groups are introduced at different
positions of the phenyl ring (such as 8a–8e), the biological
activity of the compound varies greatly. These results
suggest that there is an interesting correlation between the
position and size of the substituents and the biological
activity.

Medicinal Chemistry Research
Conclusion
performed on a Waters spectrometer (Massachusetts, USA).
High resolution mass spectrometry (HRMS) analyses were
performed on an Agilent Technologies 6530 Accurate-Mass
Q-TOF Mass Spectrometer (Santa Clara, CA, USA). The
purities were determined by high-performance liquid chro-
matography (HPLC) using an Agilent 1100 series HPLC
(Santa Clara, USA).
Based on the structure of known EGFR–TKI inhibitors, and
the principle of scaffold hopping, hybridization, and bioi-
sosterism, combining structural optimization, a novel series
of 7-bromo-1,4-dihydrothieno[3’,2’:5,6]thiopyrano[4,3-c]
pyrazole-3-carboxamide derivatives, was designed as the
target compounds. The binding mode of 8h, a representative
of the target compounds, to EGFR was simulated by
Molegro Virtual Docker software. It was found that the N
atom of pyrazole ring and the S atom of dihydrothiophene
ring can both form a hydrogen bond with the amino-acid
residue in the EGFR protein. As the target compounds,
these novel structures lay a foundation for the discovery of
novel EGFR–TKI inhibitors through synthesis and phar-
macological screening studies.
A novel series of 7-bromo-1,4-dihydrothieno[3’,2’:5,6]
thiopyrano[4,3-c]pyrazole-3-carboxamide derivatives were
successfully synthesized and screened for their inhibitory
activity on EGFR high-expressing human lung adenocarci-
noma cell line A549 and human large cell lung cancer cell
line NCI-H460 using the MTT colorimetric assay. As shown,
a structure–activity relationship was clearly discernible, and
several of the compounds exhibited moderate antitumor
activity. Particularly, compound 8h demonstrated the most-
potent inhibitory activity (IC50 = 9.57 2.20 μmol L–1 for
A549 and IC50 = 13.04 1.21 μmol L–1 for NCI-H460),
comparable to the positive-control gefitinib (IC50 = 8.58
1.65 μmol L–1 for A549 and IC50 = 18.66 5.01 μmol L–1 for
NCI-H460). It is hoped that these novel compounds, as
EGFR–TKIs, could be used as potential leading compounds
for further research.
Synthesis of 4-(thiophen-2-ylthio)butanoic acid (1)
The sodium ethoxide was prepared with sodium (3.34 g,
145 mmol) and ethanol (120 mL). 2-mercaptothiophene
(9.80 g, 84 mmol) was added dropwise to this solvent and
stirred 30 min at room temperature. γ-Butyrolactone
(11.5 mL, 128 mmol) was added and the mixture was
refluxed for 19 h under N2. The mixture was concentrated,
and the residue was solved in water. The solution was
acidified with 18% hydrochloric acid to pH 1–2, and then
extracted three times with EtOAc. The combined organic
layer was dried, filtered, and concentrated. The residue was
recrystallized from petroleum ether to give 1 (8.08 g,
47.4%) as a white solid. mp. 40–42 °C.
Synthesis of 6,7-dihydrothieno[2,3-b]thiepin-4(5H)-
one (2)
To a solution of 1 (4.04 g, 20 mmol) add 2 drops of DMF in
CH2Cl2 (20 mL), oxalyl chloride (2.81 g, 22 mmol) was
added dropwise at room temperature under N2. After the
mixture was stirred for 1 h, it was cooled to 0 °C and a
solution of SnCl4 (2.57 g, 10 mmol) in CH2Cl2 (10 mL) was
added dropwise. The solution was stirred at 0 °C for 12 h,
and then H2O was added. The organic layer was separated
and washed with saturated Na2CO3 and brine, respectively.
After drying and filtration, the resulting organic solution
was concentrated to dryness under vacuum. The residue was
recrystallized from petroleum ether to yield 2 (0.92 g,
25.0%) as a white solid. mp: 59.0–60.5 °C (lit. 65 °C
Experimental section
Materials and methods
All the starting materials were commercially available and
directly used without further purification. All the reaction
progresses were monitored by thin layer chromatography
with the silica gel plates (Qingdao Jiyida silica reagent
factory, Qingdao, China). The melting points were deter-
mined on a melting-point apparatus with microscope
(Zhengzhou Mingyi Instrument Equipment Co., Ltd.,
Zhengzhou, China), and were uncorrected. The infrared
(IR) spectra were recorded on a Bruker IFS55 spectrometer
(Faellanden, Switzerland) utilizing KBr pellets. 1H and 13C-
NMR spectra were recorded on a Bruker Avance spectro-
meter (Karlsruhe, Germany) and with TMS as an internal
standard and DMSO-d6 or CDCl3 as the solvent, chemical
shifts (d values), and coupling constants (J values) were,
respectively, given in ppm and Hz. ESI mass spectra were
(Cagniant &
Cagniant 1966)). 1H-NMR (600 MHz,
DMSOd6): δ 2.77(2H, t, J = 5.1 Hz, S-CH2), 3.47(2H, t,
J = 5.1 Hz, CH2C=O), 7.35(1H, d, J = 5.4 Hz, H-7), 7.38
(1H, d, J = 5.4 Hz, H-8).
Synthesis of methyl oxo(4-oxo-4,5,6,7-
tetrahydrothieno[2,3-b]thiepin-5-yl)acetate (3)
The sodium methoxide was prepared with sodium (0.46 g,
20 mmol) and methanol (20 mL), which were removed by
distillation when sodium was dissolved completely. A
solution of 2 (1.84 g, 10 mmol) and dimethyl oxalate
(2.36 g, 20 mmol) in dry toluene (20 mL) was added to the
sodium methoxide. The mixture was stirred for 24 h at room
temperature. Then the mixture was poured into 100 mL

Medicinal Chemistry Research
water. The aqueous layer was separated, and the organic
layer was extracted three times with 10% NaOH (each
15 mL). The combined aqueous layer was acidified with
18% hydrochloric acid to yield 3 (1.62 g, 60.0%) as a
red oil.
(0.10 g, 0.7 mmol), 1 mL Et3N and absolutely dry CH2Cl2
(20 mL) was stirred for 24 h at room temperature. The mixture
was filtered and the filtrate was washed with 20 mL of 1N
HCl, saturated Na2CO3 and brine, respectively. Then the
organic layer was dried, filtered, and concentrated. The resi-
due was purified by column chromatography on silica with
EtOAc-petroleum (3:1, v/v) as the eluent to yield product as a
white solid.
Synthesis of methyl 4,5-dihydro-1H-thieno[2’,3’:2,3]
thiepino[4,5-c]pyrazole-3-carboxylate (4)
N,N-Diethyl-7-bromo-1,4-dihydrothieno[3’,2’:5,6]
thiopyrano[4,3-c]pyrazole-3-carboxamide (7a): white
solid, yield 50%. mp. 147–148 °C; IR (KBr, cm−1): 3212,
2958, 2917, 2849, 1735, 1583, 1535, 1508, 1485, 1461,
A solution of 3 (0.54 g, 2 mmol) and 80% hydrazine hydrate
(2 mL, 32 mmol) in acetic acid (10 mL) was refluxed for
4 h. The reaction mixture was poured into ice–water mixture
(50 g) and allowed to stand overnight. A precipitate was
collected to yield 4 (0.40 g, 75.0%) as a white solid. mp:
1
1375, 1215, 1159, 967, 849; H-NMR (600 MHz, CDCl3):
δ 1.25(m, 6H, 2CH3), 3.54(m, 4H, 2N-CH2), 4.12(s, 2H, S-
CH2), 7.72(s, 1H, H-10); 13C-NMR (101 MHz, DMSO-d6)
δ 162.83 (C=O), 143.03 (C-1), 135.57 (C-2), 131.65 (C-6),
127.55, 126.07, 110.25, 108.89 (Ar-C), 43.30, 43.10 (H2C-
N-CH2), 26.19 (C-3), 15.17, 13.30(2CH3); HRMS (ESI)
m/z (neg): 369.969569, C13H13BrN3OS2 [M-H]-(calcd.
369.968890); purity (HPLC): 98.53%.
N-tert-Butyl-7-bromo-1,4-dihydrothieno[3’,2’:5,6]
thiopyrano[4,3-c]pyrazole-3-amide (7b): white solid,
yield 75%. mp. 205–207 °C; IR (KBr, cm−1): 3364, 3201,
2919, 1638, 1553, 1526, 1492, 1450, 1385, 1296, 1220,
1167, 969, 863; 1H-NMR (600 MHz, CDCl3): δ 1.49(s, 9H,
C(CH3)3), 4.49(s, 2H, S-CH2), 7,21(s, 1H, H-10), 7.40(s,
1H, NHC=O); HRMS (ESI) m/z (neg): 369.969569,
C13H13BrN3OS2 [M-H]-(calcd. 369.968890); purity
(HPLC): 98.02%.
1
94.0–95.7 °C (lit. 94–96 °C (Sun et al. 2011)). H-NMR
(600 MHz, CDCl3): δ 3.97(3H, s, O-CH3), 4.33(2H, s, S-
CH2), 7.18(1H, d, J = 5.3 Hz, H-7), 7.40 (1H, d, J =
5.3 Hz, H-8).
Synthesis of 7-bromo-1,4-dihydrothieno[3’,2’:5,6]
thiopyrano[4,3-c]pyrazole-3-carboxylic acid methyl
ester (5)
A mixture of 4 (2.52 g, 0.01 mol), 50 mL glacial acetic acid,
and 5 mL water was placed in a round-bottom flask, and
bromine (2.08 g, 0.013 mol) was added dropwise with stir-
ring in an ice bath. Then the mixture was reacted at room
temperature for 24 h. The reaction liquid was poured into
200 mL ice–water, and a white solid was precipitated, and
1
filtered, to give a white solid (2.52 g). H-NMR (400 MHz,
N-Isopropyl-7-bromo-1,4-dihydrothieno[3’,2’:5,6]
thiopyrano[4,3-c]pyrazole-3-amide (7c): white solid, yield
69%. mp. 189–190 °C; IR (KBr, cm−1): 3400.5, 3207.5,
2974.5, 2921.3, 1637.4, 1556.3, 1528.3, 1452.1, 1384.6,
DMSOd6) δ 3.86 (s, 3H, -O-CH3), 4.38 (s, 2H, S-CH2),7.47
(s, 1H, Ar-H). ESI-MS: m/z (pos) 330.9 C10H7BrN2O2S2
[M+H]+ (calcd 329.9).
1
1175.4, 967.5, 836.5; H-NMR (600 MHz, DMSO-d6): δ
Synthesis of 7-bromo-1,4-dihydrothieno[3’,2’:5,6]
thiopyrano[4,3-c]pyrazole-3-carboxylic acid (6)
1.14(d, 6H, J = 6.6 Hz, 2CH3), 4.48–4.52(m, 1H, N-CH),
4.39(s, 2H, S-CH2), 7.50(s, 1H, H-10), 8.08(s, 1H,
NHC=O); 13C-NMR (101 MHz, DMSO-d6) δ 161.38
(C=O), 141.96 (C-1), 136.54 (C-2), 131.99 (C-6), 129.10,
126.34, 109.28, 108.80 (Ar-H), 67.84 (N-CH), 30.28 (C-3),
14.35, 11.26 (2CH3); HRMS (ESI) m/z (neg): 355.953471,
C12H11BrN3OS2 [M-H]-(calcd. 355.953240); purity
(HPLC): 97.80%.
A 100 mL round bottom flask was charged with 5 (0.62 g,
2 mmol), 20 mL water, and 0.4 g sodium hydroxide, and
heated to reflux for 2 h. The reaction mixture was adjusted
to pH 2 with 18% hydrochloric acid, and a white solid
1
(0.57 g) was precipitated in the field of 90.0%. H-NMR
(400 MHz, DMSO-d6) δ 4.37 (s, 2H, S-CH2), 7.39 (s, 1H,
Ar-H), 12.92 (s, 1H, -OH). ESI-MS: m/z (pos) 316.9
C9H5BrN2O2S2 [M+H]+ (calcd 315.9).
N-Phenyl-7-bromo-1,4-dihydrothieno[3’,2’:5,6]thio-
pyrano[4,3-c]pyrazole-3-amide (7d): white solid, yield
30%. mp. 190–191 °C; IR (KBr, cm−1): 3443.8, 2919.8,
2850.5, 1730.5, 1612.4, 1555.1, 1441.0, 1384.2, 1296.2,
1
General procedure for the synthesis of 7-bromo- 1,4-
dihydrothieno[3’,2’:5,6]thiopyrano[4,3-c]pyrazole-3-
carboxamids (7a–7f and 8a–8i)
1114.0, 1038.4, 778.5; H-NMR (600 MHz, DMSO-d6): δ
4.42 (s, 2H, S-CH2), 7.36–7.43 (m, 5H, Ar-H), 7.42 (s, 1H,
H-10), 8.82 (s, 1H, NHC=O), 13.64 (s, 1H, NH); MS (ESI)
m/z (neg): 390.1, C15H8BrN3OS2 [M-H]-(calcd. 389.9).
N-Benzyl-7-bromo-1,4-dihydrothieno[3’,2’:5,6]thio-
pyrano[4,3-c]pyrazole-3-amide (7e): white solid, yield
56%. mp 98–100 °C; IR (KBr, cm−1): 3421.9, 2920.9,
A mixture of 6 (1.01 g, 4 mmol), amines or piperazine deri-
vative (4 mmol), 1ethyl-3-(3dimethylaminopropyl)carbodii-
mide hydrochloride (1.00 g, 5 mmol), 1hydroxybenzotriazole

Medicinal Chemistry Research
2851.7, 1629.7, 1446.5, 1384.2, 1274.3, 1114.1, 850.9; 1H-
NMR (600 MHz, DMSO-d6): δ 4.40 (d, 2H, Ar-CH), 4.42
(s, 2H, S-CH2), 7. 36–7.43(m, 5H, Ar-H), 7.42 (s, 1H, H-
10), 8.82(s, 1H, NHC=O), 13.64(s, 1H, NH); 13C-NMR
(101 MHz, DMSO-d6) δ 162.34 (C=O), 140.19 (C-1),
136.50 (C-11), 133.96 (C-2), 131.72 (C-6), 129.12, 128.69,
127.81, 127.45, 127.17, 127.01, 126.27, 109.45, 108.88
(Ar-H), 42.34 (NH-CH2), 25.85 (C-3); HRMS (ESI) m/z
C19H15BrFN4OS2 [M-H]-(calcd. 476.986017); purity
(HPLC): 99.12%.
1-(7-Bromo-1,4-dihydrothieno[3’,2’:5,6]thiopyrano
[4,3-c]pyrazole-3-formyl)-4-(3-bromophenyl)piperazine
(8c): white solid, 0.82 g, yield 38%. mp. 150–151 °C; IR
(KBr, cm−1): 3418.7, 3217.5, 2920.0, 2850.7, 1590.4,
1480.6, 1442.9, 1384.6, 1224.6, 1157.9, 999.9, 938.2,
1
764.3; H-NMR (600 MHz, CDCl3): δ 3.20–3.27(m, 4H,
(neg):
403.953743,
C16H11BrN3OS2
[M-H]-(calcd.
Ar-N(CH2)2), 3.98–4.04(m, 4H, CON(CH2)2), 4.26(s, 2H,
S-CH2), 6.85(m, 1H, Ar-H), 7.00–7.03(m, 1H, Ar-H),
7.05–7.09(m, 1H, Ar-H), 7.11–7.14(m, 1H, Ar-H), 7.23 (s,
1H, H-10); 13C-NMR (101 MHz, DMSO-d6) δ 161.68
(C=O), 152.58 (C-14), 132.17 (C-1), 131.97 (C-2), 131.61
(C-18), 131.23 (C-6), 129.12, 126.28, 123.00, 121.87,
118.26, 114.87, 110.41, 108.96 (Ar-H), 48.74, 48.37 (C-11,
C-12), 46.52, 42.02 (C-10, C-13), 25.92 (C-3); HRMS
(ESI) m/z (neg): 536.906767, C19H15Br2N4OS2 [M-H]-
(calcd. 536.905951); purity (HPLC): 99.62%.
403.953240); purity (HPLC): 98.68%.
1-(7-Bromo-1,4-dihydrothieno[3’,2’:5,6]thiopyrano
[4,3-c]pyrazole-3-formyl)-4-(2-methylphenyl)piperazine
(7f): white solid, yield 48%. mp.167–170 °C; IR (KBr, cm−1):
3442.6, 2920.4, 2851.6, 1621.6, 1491.9, 1443.3, 1384.0,
1
1327.3, 1274.9, 1224.2, 1126.5, 1021.8, 1000.2, 762.3; H-
NMR (600 MHz, CDCl3): δ 4.22(s, 2H, S-CH2), 4.60(d, 2H,
N-CH), 6.29(d, 1H, J = 6 Hz, H-19), 6.33(d, 1H, J = 6 Hz, H-
21), 7.20(dd, 1H, J = 6 Hz, 6 Hz, H-20), 7.25(s, 1H, H-10);
13C-NMR (101 MHz, DMSO-d6) δ 162.24 (C=O), 153.01 (C-
1), 142.30 (C-11), 141.61 (C-14), 136.54 (C-2), 131.76 (C-6),
127.47, 126.19, 110.91, 109.42, 108.89, 107.19 (Ar-H), 35.65
(NH-CH2), 25.88 (C-3); HRMS (ESI) m/z (neg): 393.932823,
C14H9BrN3O2S2 [M-H]-(calcd. 393.932504); purity (HPLC):
99.41%.
1-(7-Bromo-1,4-dihydrothieno[3’,2’:5,6]thiopyrano
[4,3-c]pyrazole-3-formyl)-4-(3,4-difluorophenyl)piper-
azine (8d): white solid, yield 44%. mp. 190–192 °C; IR
(KBr, cm−1): 3444.2, 2919.8, 1656.7, 1560.4, 1417.7,
1384.3, 1276.6, 1116.2; 1H-NMR (600 MHz, DMSO-d6): δ
3.12–3.16 (m, 4H, Ar-N(CH2)2), 3.70–3.74(t, 4H, CON
(CH2)2), 4.27(s, 2H, S-CH2), 6.72–6.77(m, 1H, Ar-H),
7.00–7.04 (m, 1H, Ar-H), 7.25 (d, 1H, J = 9.6 Hz, Ar-H),
7.45(s, 1H, H-10); 13C-NMR (101 MHz, DMSO-d6) δ
160.06 (C=O), 151.31 (C-16), 149.02 (C-1), 148.71 (C-14),
148.63 (C-17), 144.71 (C-2), 142.22 (C-6), 126.26, 117.84,
117.67, 112.02, 108.94, 105.38, 105.17 (Ar-H), 49.46,
48.86 (C-11, C-12), 46.47, 41.97 (C-10, C-13), 26.01 (C-3);
HRMS (ESI) m/z (neg): 494.976868, C19H14BrF2N4OS2
[M-H]-(calcd. 494.976595); purity (HPLC): 99.44%.
1-(7-Bromo-1,4-dihydrothieno[3’,2’:5,6]thiopyrano
[4,3-c]pyrazole-3-formyl)-4-(4-fluorophenyl) piperazine
(8e): white solid. mp. 185–186 °C; IR (KBr, cm−1): 3443,
2922, 2852, 1605, 1507, 1443, 1384, 1225, 1116; 1H-NMR
(600 MHz, CDCl3): δ 3.17–3.23 (m, 4H, Ar-N(CH2)2),
4.02–4.05 (m, 4H, CON(CH2)2), 4.24 (s, 2H, S-CH2),
7.0–7.04 (m, 4H, Ar-H), 7.24 (s, 1H, H-10); 13C-MR
(101 MHz, DMSO-d6) δ 162.01 (C=O), 157.93 (C-17),
148.18 (C-1), 142.34 (C-14), 135.82 (C-2), 131.87 (C-6),
127.47, 126.15, 118.22, 118.14, 115.91, 115.70, 110.66,
108.96 (Ar-H), 50.31, 49.71 (C-11, C-12), 46.72, 42.14 (C-
10, C-13), 26.09 (C-3); HRMS (ESI) m/z (neg):
476.986845, C19H15BrFN4OS2 [M-H]-(calcd. 476.986017);
purity (HPLC): 98.58%.
1-(7-Bromo-1,4-dihydrothieno[3’,2’:5,6]thiopyrano
[4,3-c]pyrazole-3-formyl)-4-(3-chloro-4-fluorophenyl)
piperazine (8a): white solid, yield 36%. mp. 120 °C; IR
(KBr, cm−1): 3233, 2924, 2853, 1745, 1609, 1586, 1502,
1464, 1382, 1223, 1154, 998; 1H-NMR (600 MHz, CDCl3):
δ 3.16(t, 4H, J = 6.0 Hz, Ar-N(CH2)2), 3.95(t, 4H, J =
6.0 Hz, CON(CH2)2), 4.26(s, 2H, S-CH2), 6.76–6.80(m, 1H,
H-23), 6.95(dd, 1H, J = 6.3 Hz, J = 2.4 Hz, H-27), 7.05
(dd, 1H, J = 8.7 Hz, J = 9 Hz, H-26), 7.25 (s, 1H, H-10);
13C-NMR (101 MHz, DMSO-d6) δ 162.00 (C=O), 152.65
(C-17), 150.28 (C-1), 148.72 (C-14), 131.84 (C-2), 130.09
(C-6), 126.26, 120.14, 119.96, 117.62, 117.41, 117.20,
116.50, 108.96 (Ar-H), 49.55, 48.97 (C-11, C-12), 46.52,
41.97 (C-10, C-13), 29.49 (C-3); HRMS (ESI) m/z (neg):
510.948158,
C19H14BrClFN4OS2
[M-H]-(calcd.
510.947045); purity (HPLC): 99.74%.
1-(7-Bromo-1,4-dihydrothieno[3’,2’:5,6]thiopyrano
[4,3-c]pyrazole-3-formyl)-4-(2-fluorophenyl)piperazine
(8b): white solid, yield 39%. mp. 78–80 °C; IR (KBr, cm−1):
3221, 2921, 2851, 1720, 1641, 1500, 1446, 1394, 1385,
1287, 1240, 1151, 999; 1H-NMR (600 MHz, CDCl3): δ 3.11
(t, 4H, J = 10.2 Hz, Ar-N(CH2)2), 4.02(t, 4H, J = 10.2 Hz,
CON(CH2)2), 4.24(s, 2H, S-CH2), 7.01–7.04(m, 3H, Ar-H),
7.23(s, 1H, H-10), 7.38(d, 1H, J = 7.8 Hz, Ar-H); 13C-NMR
(101 MHz, DMSO-d6) δ 161.62 (C=O), 156.68 (C-15),
140.01 (C-1), 139.92 (C-2), 126.27, 125.34, 125.31, 123.34,
123.26, 120.12, 120.09, 116.56, 116.36, 108.97 (Ar-H),
51.18, 50.73 (C-11, C-12), 46.99, 42.32 (C-10, C-13), 25.90
1-(7-Bromo-1,4-dihydrothieno[3’,2’:5,6]thiopyrano
[4,3-c]pyrazole-3-formyl)-4-(4-trimethoxyphenyl) piper-
azine (8f): white solid, yield 44%. mp. 90 °C; IR (KBr, cm−1):
2922, 1730, 1618, 1511, 1446, 1379, 1255, 1224, 1186, 1137,
1096, 1034, 992, 816; 1H-NMR (600 MHz, CDCl3)):
δ3.12–3.15(m, 4H, Ar-N(CH2)2), 3.96–4.03(m, 7H, CON
(C-3);
HRMS
(ESI)
m/z
(neg):
476.986542,

Medicinal Chemistry Research
(CH2)2, O-CH3), 4.28(s, 2H, S-CH2), 6.85(dd, 2H, J = 7.2 Hz,
2.4 Hz, Ar-H), 6.91(dd, 2H, J = 7.2 Hz, 2.4 Hz, Ar-H), 7.24 (s,
1H, H-10); 13C-NMR (101 MHz, DMSO-d6) δ 162.00 (C=O),
153.78 (C-17), 145.61 (C-1), 142.39 (C-14), 135.80 (C-2),
131.86 (C-6), 129.11, 127.48, 126.15, 118.49, 114.75, 110.61,
108.96, 99.98 (Ar-H), 55.65 (O-CH3), 51.01, 50.43 (C-11, C-
12), 46.87, 42.25 (C-10, C-13), 26.08 (C-3); HRMS (ESI) m/z
48.74, 48.09 (C-11, C-12), 46.47, 42.00 (C-10, C-13),
26.09 (C-3); HRMS (ESI) m/z (neg): 526.983766,
C20H15BrF3N4OS2 [M-H]-(calcd. 526.982823); purity
(HPLC): 99.72%.
Molecular docking
(neg):
489.006763,
C20H18BrN4O2S2
[M-H]-(calcd.
The complexes for the enzyme were those deposited in the
RCSB Protein Data Bank for the EGFR (PDB code: 4HJO)
after eliminating the inhibitor (erlotinib) and water mole-
cules. The missing residues were built and the polar
hydrogen atoms acid residues were added. Molecular
docking was carried out using MVD. The docking was set
at 1500 maximum iterations, with a simplex evolution
population size of 50 and a minimum of 10 runs. The
schematic diagrams of interactions between 4HJO and the
small molecules were analyzed by PyMOL 1.7.
489.006004); purity (HPLC): 98.96%.
1-(7-Bromo-1,4-dihydrothieno[3’,2’:5,6]thiopyrano
[4,3-c]pyrazole-3-formyl)-4-(4-methylphenyl)piperazine
(8g): white solid, yield 30%. mp. 185–186 °C; IR (KBr, cm−1):
3441, 2920, 2852, 1610, 1513, 1445, 1384, 1156, 1023, 813,
1
619; H-NMR (600 MHz, CDCl3): δ 3.11–3.16(m, 4H, Ar-N
(CH2)2), 3.95–4.03(m, 7H, CON(CH2)2, Ar-CH3), 4.28(s, 2H,
S-CH2), 6.85(dd, 2H, J = 7.2 Hz, 2.4 Hz, H-10), 6.91(dd, 2H,
J = 7.2 Hz, 2.4 Hz, H-10), 7.24 (s, 1H, H-10); 13C-NMR
(101 MHz, DMSO-d6) δ 162.01 (C=O), 149.21 (C-1), 142.37
(C-14), 135.81 (C-2), 131.86 (C-6), 129.88, 128.68, 127.48,
126.67, 126.15, 116.77, 116.64, 110.63, 108.94 (Ar-H), 50.06,
49.44 (C-11, C-12), 46.75, 42.16 (C-10, C-13), 26.10 (C-3),
20.54 (CH3); HRMS (ESI) m/z (neg): 473.011806,
C20H18BrN4OS2 [M-H]-(calcd. 473.011089); purity (HPLC):
97.21%.
Bioactivity in cell line
In order to determine the anticancer activity of our target
compounds in vitro, the MTT assay was used. A549 cells,
Hela cells, SW480 cells, HepG2 cells, and HL7702 cells
were harvested in the logarithmic growth phase and seeded
in 96-well plates at a density of 8000 cells per well, and
cultured at 37 °C in humidified atmosphere containing 5%
CO2 in Dulbecco’s modified Eagle’s medium (or RPMI-
1640) with 10% fetal bovine serum for 24 h before any
treatments. Tested compounds were dissolved in DMSO
and diluted in the culture fluid to get various concentrations.
The cells were treated with target compounds subsequently
and incubated overnight. Then 20 μL of MTT (5 mg/mL)
was added in each well and after 4 h incubation, the medium
was removed immediately and MTT formazan was solubi-
lized in 150 μL DMSO. The optical densities were mea-
sured with a microplate reader (Bio-Tek instruments, INC.
USA) at 490 nm. Inhibitory effects were determined as
IC50 value.
1-(7-Bromo-1,4-dihydrothieno[3’,2’:5,6]thiopyrano
[4,3-c]pyrazole-3-formyl)-4-(3,5-trifluoromethylphenyl)
piperazine (8h): white solid, yield 45%. mp. 220–221 °C;
IR (KBr, cm−1): 3421.2, 2918.2, 1610.2, 1581.2, 1485.5,
1
1400.4, 1275.0, 1175., 1131.4, 995.3, 963.6, 862.4; H-
NMR (600 MHz, DMSO-d6): δ 3.40–3.48(m, 4H, Ar-N
(CH2)2), 3.75–3.78(m, 2H, CONCH2), 4.11–4.14(m, 2H,
CONCH2), 4.31(s, 2H, S-CH2), 7.33(s, 1H, Ar-H), 7.45(s,
1H, H-10), 7.50(s, 2H, Ar-H), 13.64(s, 1H, NH); 13C-NMR
(101 MHz, DMSO-d6) δ 162.12 (C=O), 151.85 (C-14),
142.27 (C-1), 135.85 (C-2), 131.89, 131.74 (C-16, C-18),
131.42 (C-6), 131.10, 130.01 (2CF3), 127.45, 126.16,
125.40, 122.69, 114.88, 110.77, 108.95 (Ar-H), 48.03,
47.35 (C-11, C-12), 46.24, 41.87 (C-10, C-13), 26.10 (C-3);
HRMS (ESI) m/z (neg): 594.970282, C21H14BrF6N4OS2
[M-H]-(calcd. 594.970208); purity (HPLC): 98.63%.
Acknowledgements This work was supported by the National Science
Foundation of China (NSFC) for the grant No. 21342006 and the
Program for Innovative Research Team of the Ministry of Education of
China for the grant No. IRT_14R36. We thank Molegro ApS for
kindly providing a free evaluation copy of their software package and
Schrödinger, LLC for allowing the use of PyMOL Molecular Graphics
System.
1-(7-Bromo-1,4-dihydrothieno[3’,2’:5,6]thiopyrano
[4,3-c]pyrazole-3-formyl)-4-(3-trifluoromethylphenyl)
piperazine (8i): white solid, yield 45%. mp. 185–186 °C;
IR (KBr, cm−1): 3444.9, 3219.1, 2918.2, 2849.8, 1591.1,
1444.4, 1384.4, 1349.9, 1319.6, 1269.0, 1222.1, 1163.5,
1
1112.2, 1075.2, 999.6, 946.4, 864.1, H-NMR (600 MHz,
Compliance with ethical standards
DMSO-d6): δ 3.26–3.34(m, 8H, N(CH2CH2)2N), 4.27(s,
2H, S-CH2), 7.09 (d, 1H, J = 7.7 Hz, Ar-H), 7.24 (d, 1H,
J = 8.4 Hz, Ar-H), 7.43 (t, 1H, J = 8.0 Hz, Ar-H), 7.50 (s,
1H, H-10); 13C-NMR (101 MHz, DMSO-d6) δ162.08
(C=O), 151.47 (C-14), 142.32 (C-1), 135.83 (C-2), 131.89
(C-6), 130.54 (C-16), 130.48 (CF3), 127.46, 126.15,
123.52, 119.47, 115.42, 111.77, 110.71, 108.96 (Ar-H),
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with ani-
mals, performed by any of the authors. Informed consent was obtained
from all individual participants included in the study.

Medicinal Chemistry Research
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