Discovery of 4H-thieno[3,2-b]pyrrole derivatives as potential anticancer agents

Article abstract of DOI:10.1002/jhet.4285

In this study, we describe the discovery of the 4H-thieno[3,2-b]pyrrole derivatives as an useful scaffold to obtain potent lead compounds for the treatment of colon cancer. We first started with the 4H-thieno[3,2-b]pyrrole derivatives which come from comp

Full text of DOI:10.1002/jhet.4285

Discovery of 4H-thieno[3,2-b]pyrrole derivatives as potential
anticancer agents
Bo Fanga,b, Chunsheng Hua,b, Yong Dinga, Hongxia Qinb, Yafei Luoa, Zhigang Xua,b,
Jiangping Menga,b, Zhongzhu Chena,b
a
National & Local Joint Engineering Research Center of Targeted and Innovative Therapeutics, College of Pharmacy &
International Academy of Targeted Therapeutics and Innovation, Chongqing University of Arts and Sciences,
Chongqing, China.
b
Chongqing Engineering Laboratory of Targeted and Innovative Therapeutics, Chongqing Key Laboratory of Kinase
Modulators as Innovative Medicine, Chongqing Collaborative Innovation Center of Targeted and Innovative
Therapeutics, Chongqing University of Arts and Sciences, Chongqing, China.
Correspondence
Jiangping Meng, National & Local Joint Engineering Research Center of Targeted and Innovative Therapeutics, College
of Pharmacy & International Academy of Targeted Therapeutics and Innovation, Chongqing University of Arts and
Sciences, Chongqing, 402160, China. E-mail: mengjp2006@163.com
Zhongzhu Chen, National & Local Joint Engineering Research Center of Targeted and Innovative Therapeutics, College of
Pharmacy & International Academy of Targeted Therapeutics and Innovation, Chongqing University of Arts and
Sciences, Chongqing, 402160, China. E-mail: 18883138277@163.com
Bo Fang and Chunsheng Hu contributed equally to this work.
Received: ((will be filled in by the editorial staff))
((Additional Supporting Information may be found in the online version of this article.)).((Please delete if not appropriate))
Abstract. In this study, we describe the discovery of the 4H- validated by molecular docking and biological activity
thieno[3,2-b]pyrrole derivatives as a useful scaffold to obtain analysis. The results of apoptosis and flow cytometry
potent lead compounds for the treatment of colon cancer. We demonstrated that compound 8i induce cell apoptosis
first started with the 4H-thieno[3,2-b]pyrrole derivatives probably by inhibiting activity of methionine
which come from compound libraries screening, and then aminopeptidase 2, therefore compound 8i may be a potent
optimized their structures based on the cellular activities and inhibitor to methionine aminopeptidase 2.
pharmacophore models. The inhibition rate of cell growth
assay demonstrated that this series compounds showed better
Keywords: Thieno[3,2-b]pyrrole derivatives; Colon
cancer; Structure optimization; Target prediction;
Methionine aminopeptidase 2
inhibitory activities against colon cancer cells than other
tested tumor cells. Moreover, the target of the most active
compound 8i was explored by target fishing strategy and
identifying hit molecules as starting points in drug
research and development.[5-8] Therefore, to address
the increasingly severe situation of colorectal cancer,
we try to find suitable lead compounds by using the
method of compound library screening which will be
introduced in detail below, and optimize the structure
of lead compounds to obtain the potential compounds
which deserve further research and development. The
screening results suggest that the inhibition rates of
compounds I-IV are more than 50% on typical
colorectal cancer cells (HT29 and/or HCT116)
(Figure 1), moreover, the shared feature of these
compounds is 3-phenyl-4H-thieno[3,2-b]pyrrole
skeleton.
1. Introduction
Colon cancer is the third most commonly diagnosed
cancer globally, and ranks among the three most
important causes of cancer-related mortality
worldwide.[1] Current colon treatment options include
surgery, radiation therapy, and chemotherapy. Due to
the lack of an early diagnosis, most patients are not
eligible for surgical resection.[2,3] In addition, a major
drawback of the use of cancer chemotherapy results
from the lack of tumor specificity shown by most
anticancer drugs.[4] Therefore, new and effective
anticancer drugs are required for the management of
colon carcinoma.
Although some literatures reported that several 4H-
thieno[3,2-b]pyrrole derivatives showed antitumor,
antidiabetic and antiallergic effects by targeting
As an effective means of discovering novel lead
compounds, screening compound libraries for
specific targets or cells is a powerful technique for
LSD1,[9,10]
CHK1,[11]
CENP-E,[12]
glycogen
This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process
which may lead to differences between this version and the Version of Record. Please
cite this article as doi: 10.1002/jhet.4285
This article is protected by copyright. All rights reserved.

Figure 1. Four potent lead compounds from preliminary inhibition rates screening experiment.
Phosphorylase,[13,14] Human Histamine H4[15] etc., to
2. Results and Discussion
date there is no clinic drugs containing 4H-
thieno[3,2-b]pyrrole scaffold which fused by pyrrole
2.1. Discovery of Lead Compounds
and thiophene groups. Therefore, the 4H-thieno[3,2-
It is generally known that computer-aided drug
design (CADD) can find lead compounds through
virtual screening at low cost with the fast pace of the
development of computer technology in the field of
drug research and development. However, due to the
intricate mechanisms between drug molecules and
biological cells, there is still a large gap between the
measured and predicted activities of most of the lead
compounds found by CADD. Therefore, it is still an
effective way to find lead compounds with new
structural characteristics by measuring the activities
of compounds library molecules. Herein, we tested
the inhibition rate of no less than 400 drug-like
molecules on common tumor cells by screening
typical commercial drug-like compounds library, the
related tumor cells include Hela (cervical cancer cell),
b]pyrrole
derivatives
may
have
special
pharmacological properties due to their chemical
structure obvious difference from current drugs.
Given the necessity of developing new anti-colon
cancer drugs and the possible biological activities of
4H-thieno[3,2-b]pyrrole derivatives based on the
results of the compound libraries screening, we
designed and synthesized a series of 4H-thieno[3,2-
b]pyrrole derivatives and studied their anti-tumor
properties. Furthermore, in view of their potential in
anti-colon cancer, we also explored the possible
targets of these compounds through reverse targeting
strategy and verified the result by molecular docking
and biological experiments. These studies will
provide valuable choices for molecular biologists and
the pharmaceutical industry.
Table 1. The inhibition rate of cell growth assay of first series of target conmounds (20μM) on common tumor cellsa.
Compd.
Hela
23.89
0.62
U87
H1975
60.08
13.04
44.85
14.78
54.91
56.86
MCF-7
54.25
56.38
62.97
2.37
Hep3B
35.05
16.75
46.18
7.88
HT29
70.22
14.15
66.78
65.58
24.27
69.03
HCT116
72.27
52.63
69.25
2.90
I
25.09
62.30
59.39
-0.76
42.56
69.82
II
III
57.89
1.66
IV
Erlotinib
Paclitaxel
34.32
53.25
38.75
46.59
26.67
38.93
41.66
53.35
a) The data were presented from three independent experiments.
This article is protected by copyright. All rights reserved.

U87 (glioma cell), H1975 (lung carcinoma cell), 2.2. Chemistry
MCF-7 (breast cancer cell), Hep3B (hepatoma cell),
HT29 and HCT116 (colon cancer cell). Since the
target of this series of compounds is not yet clear, the
typical small molecule targeted drug Erlotinib and
natural broad-spectrum anticancer drug Paclitaxel
were chosen as positive control drugs.
The synthetic route of the target compounds 8a-8n
is outlined in Schemes 1. Methyl 2-chloroacetate 1
reacted with sodium azide in acetone and water at
60 °C to give methyl 2-azidoacetate 2 in yield of 85%,
the following condensation of compound 2 and 4-
bromothiophene-2-carbaldehyde 3 at the present of
sodium methanolate in mehanol to produce methyl-2-
azido-3-(4-bromothiophen-2-yl)acrylate 4 in yield of
65%, then methyl 3-bromo-4H-thieno[3,2-b]pyrrole-
5-carboxylate 5 was obtained in yield of 82% by
intramolecular cyclization of compound 4 in xylene
under reflux. After this, compound 5 reacted with
phenyboronic under conventional coupling reaction
condition to give methyl 3-phenyl-4H-thieno[3,2-
b]pyrrole-5-carboxylate 6 in yield of 60%, then
hydrolyzed by sodium hydroxide aqueous solution
and acidized by hydrochloric acid to give the key
The results showed that most of compounds library
molecules showed weak or no activity against these
tumor cells. However, some compounds containing
4H-thieno[3,2-b]pyrrole skeleton (I-IV, Table 1)
showed strong inhibitory activity on colon cancer
cells. In particular, the inhibitory rates of compounds
I, III and IV on HT29 at 20 μM were 70.22%,
66.78% and 65.58%, while the inhibitory rates of
compounds I, II and III on HCT116 at 20 μM also
reached 72.27%, 52.63% and 69.25%, respectively.
These results showed that this kind of compounds has
the potential to be lead compounds, especially
compounds I and III showed strong inhibitory
activities against both colon cancer cells, which were
superior to or equivalent to the control drugs
Erlotinib and Paclitaxel. Therefore, based on these
compounds with 4H-thieno[3,2-b]pyrrole skeleton
structure, we plan to carry out structural optimization
on this kind of compounds in order to discover more
active anti-tumor compounds and study their
preliminarily mechanism of action.
intermediate
3-phenyl-4H-thieno[3,2-b]pyrrole-5-
carboxylic acid 7 in yield of 96%. Finally, target
compounds 8a-8n were prepared in yields ranging
from 52% to 88% by condensation reaction of
compound 7 with various amine at the present of
dimethylaminopyridine, 3-(3-dimethylaminopropyl)-
1-ethylcarbodiimide hydrochloride and 1-hydroxy-
benzotriazole in dimethyl formamide.
As shown in scheme 1, we first synthesized four
compounds for the preliminary biological activities
Scheme 1. Reagents and conditions: (a) acetone, H2O; (b) NaOMe, MeOH; (c) xylene, reflux; (d) Pd(PPh3)4,
Na2CO3, toulene, EtOH, H2O, 100 °C; (e) NaOH, H2O, then HCl; (f) Opportune amine, DMAP, EDCI, HOBT,
DMF.
This article is protected by copyright. All rights reserved.

evaluation, which was used to compare with
compounds I-IV and optimize the structure of the
target molecules. The amines which were chosen for
coupling with intermediate 7 are based on the
following considerations (together with compounds I-
IV): (a) To investigate the influence of various
amines to the biological activities, the species of
amines including aromatic amines, benzylamines and
aliphatic amines; (b) To explore the effect of the aryl
groups in aromatic amines and benzylamines, the aryl
group could be (halogenated)phenyl, pyridyl,
thiazolyl and so on. Therefore, based on the
structures of compounds I-IV, four amines were used
to synthesize the target compounds, and all these
compounds were investigated the cell viabilities to
typical tumor cells by MTT assay.
(HT29 and HCT116) than other tumor cells, but also
revealed that the biological activities of these
compounds was strongly associated with the kinds of
their amino groups, and more specifically, aromatic
amine groups of target compounds (I and III) are
superior moieties than corresponding benzylamine
and alkyl amine groups (IV, 8c and 8d) based on
these biological data. In additon, the different aryl in
aromatic amine groups make the compounds
exhibiting different biological activities, for instance,
six-membered
aromatic
ring
such
as
(halogenated)phenyl and pyridyl (I-IV and 8a) are
more beneficial to improve the biological activities of
target compounds than five-membered aromatic ring
(8b). Moreover, the inhibitory activities of compound
8a on cells HT29 and HCT116 were significantly
better than the control drug Erlotinib, and were
comparable to the control drug Paclitaxel.
The cell viability data was shown in Table 2, these
results not only showed that these compounds may
exhibit more potential in against colon cancer cells
Table 2. The inhibition rate of cell growth assay of first series of target conmounds (20μM) on common tumor cellsa.
Compd.
Hela
44.64
17.00
8.44
U87
H1975
9.32
MCF-7
-1.81
39.39
28.70
21.49
8.30
Hep3B
1.32
HT29
47.64
63.53
32.29
59.19
7.24
HCT116
37.43
59.03
15.02
39.13
10.84
41.66
53.35
40.53
36.66
0.40
6
38.12
31.03
16.25
5.62
8.04
8a
2.14
8b
3.40
38.72
4.68
1.38
8c
2.04
-0.96
26.67
38.93
8d
34.32
53.25
42.56
69.82
54.91
56.86
38.75
46.59
24.27
69.03
Erlotinib
Paclitaxel
a) The data were presented from three independent experiments.
Figure 2. HipHop pharmacophore model for training set. (A) The best HipHop pharmacophore model. (B) The
configuration of the most active compound I. (C) The best HipHop model mapped with the most active
compound I in the training set. The features are color coded with green (hb_acceptor); magenta (hb_donor);
yellow (ring_aromatic feature); light blue (hydrophobic feature).
This article is protected by copyright. All rights reserved.

Furthermore, it’s well known that pharmacophore
model is widely applied to explore common chemical
characteristics among a considerable number of
structures with great diversity and used as a query for
searching chemical databases and finding new
chemical entities.[16,17] Therefore, the pharmacophore
model of these compounds was generated based on
their cytotoxic activities in order to better understand
corresponding structure-activity relationships and
effectively optimize the structure of target
compounds furtherly using the HipHop module from
Discovery Studio 2018 package. The Feature
Mapping tool was used firstly to generate all
pharmacophore feature of these compounds, the
‘principal’ and ‘MaxOmit-Feat’ values were 2 and 0
for high active compounds (I and III), 1and 1 for
moderate activite compounds (8a), 0 and 2 for low
active compounds (6, II, IV and 8b-8d) respectively,
these compounds form the training set. The
pharmacophore feature includes acceptor (HBA),
donor (HBD), hydrophobe (H), ionizable positive and
ring aromatic which will be used for generating
HipHop pharmacophore model by Common feature
Pharmacophore generation. The parameter settings
are as follows: conformation generation (best),
maximum conformations (200), energy threshold (10),
features (hb_acceptor, hb_donor, hydrophobic,
pos_ionizable and ring aromatic), and the rest
HipHop parameters were kept at their default values.
The top ten hypotheses were generated based on the
training set molecules using Common Feature
Pharmacophore Generation to identify the common
features necessary to responsible for a desired
biological activity. Figure 2A shows the best HipHop
pharmacophore hypothesis, which contains five
features: two hydrogen-bond acceptors, one
hydrogen-bond donor, one aromatic moiety and one
hydrophobic feature, and figure 2B displays the steric
configuration of the most active compound I. In
addition, figure 2C presents the mapping of the
pharmacophore model onto the most active
compound I in the training set. The HipHop
pharmacophore hypothesis explicitly demonstrated
the importance of hb_donor, hb_acceptor,
ring_aromatic moiety and hydrophobic feature.
Therefore, more kinds of amines will be used to
synthesize the following target compounds for
investigating their effect on biological activities,
these newly synthesized compounds will be served as
a test set to validate the reliability of the HipHop
pharmacophore model.
Table 3. The inhibition rate of cell growth assay of second series of target conmounds (20μM) on common tumor cellsa.
Compd.
Hela
U87
H1975
51.60
6.09
MCF-7
52.95
8.20
Hep3B
26.89
11.24
31.98
33.28
70.44
1.49
HT29
68.95
69.07
67.69
74.42
76.23
50.30
66.17
39.39
34.74
5.85
HCT116
78.52
32.69
72.20
73.86
86.41
5.18
26.58
6.87
32.54
30.52
31.79
21.91
48.28
42.03
3.45
8e
8f
32.69
32.33
67.46
1.20
57.58
56.16
78.87
2.21
54.31
55.87
66.97
4.55
8g
8h
8i
8j
-2.86
1.70
12.75
12.29
22.15
8.19
21.93
3.55
10.53
2.31
34.83
8.64
8k
1.73
8l
43.83
6.18
22.55
1.25
31.18
1.08
12.69
3.58
7.03
8m
19.41
58.64
41.66
53.35
8n
19.56
34.32
53.25
43.47
42.56
69.82
65.43
54.91
56.86
35.91
38.75
46.59
15.52
26.67
38.93
75.97
24.27
69.03
8o
Erlotinib
Paclitaxel
a) The data were presented from three independent experiments.
This article is protected by copyright. All rights reserved.

Due to hydrophobic feature of compounds plays a
colon cancer cells suggested that these compounds
very important role in HipHop pharmacophore model, may be potential lead compounds in the study of new
introducing more hydrophobic groups such as various
halogen and alkyl groups into target compounds may
be an effective strategy to improve their potency for
tumor inhibition. Therefore, based on above HipHop
pharmacophore model, ten target compounds were
synthesized and their biological activities have been
evaluated, the data of cell viability assay was shown
in table 3, the result demonstrated that this series
compounds also showed better inhibitory activities
against colon cancer cells than other tumor cells. The
inhibitory activities of compounds 8e, 8g-8i against
drugs against colon cancer. Especially, compound 8i
is a most active compound against colon cancer cells
and almost have high inhibitory activities to all tested
tumor cells, not only that, the inhibitory activities of
compound 8i on all tested tumor cells have
completely surpassed control drugs Erlotinib and
Paclitaxel except the inhibitory activity of compound
8i on cell U87 is slightly lower than control drug
Paclitaxel. Therefore, compound 8i was chosen for
further studying to reveal its anti-tumor mechanism.
Figure 3. HipHop pharmacophore model for test set. (A) The best HipHop pharmacophore model Hypo10. (B) The
Hypo10 mapped with the most active compound 8i. (C) The Hypo10 mapped with compound 8h. (D) The Hypo10
mapped with compound I. (E) The overlapped conformation of three most active compounds (8i, 8h and I). (F) The
Hypo10 mapped with three active compounds (8i, 8h and I). The features are color coded with green (hb_acceptor);
magenta (hb_donor); yellow (ring_aromatic feature); light blue (hydrophobic feature).
Figure 4. (A) Compound 8g of the test set mapped to Hypo3. (B) The most active compound 8i of the test set
mapped to Hypo3. The features are color coded with green (hb_acceptor); magenta (hb_donor); yellow
(ring_aromatic feature); light blue (hydrophobic feature).
This article is protected by copyright. All rights reserved.

Because the structural optimization strategy for the
second series of compounds is based on the HipHop
pharmacophore model established by the first series
of compounds (training set), so the second series of
compounds will be serve as the test set to validate the
reliability of HipHop model by using Ligand Profiler
tool, the parameter settings are as follows: input file
pharmacophores (above ten pharmacophores),
is one other pharmacophore model (Hypo3) in
HipHop may explain why compound 8g exhibits high
activities. As shown in Figure 4, compound 8g fit all
features of Hypo3 very well, in particular, the
aromatic feature may be overlapped by 4-
bromophenyl ring very well. Additionally, the most
active compound 8i was still the best matched
compound in Hypo3. These evidences illustrated that
the strategy of optimizing the structure of compounds
through the HipHop pharmacophore model was
feasible.
In order to study the possible target of this kind of
compounds, the most active compound 8i was used as
ligand to search the potential target by reverse virtual
screening, also known as in silico or computational
target fishing.[18] Furthermore, the result of the
reverse virtual screening will be verified by
molecular docking.
conformation
generation
(best),
maximum
conformations (200), energy threshold (10), save
conformations (true), maximum omitted features (-1),
scale fit values (false), aligned ligands (true) and the
rest parameters were kept at their default values.
Surprisingly, the results of Ligand Profiler are in
good agreement with the experimental values, that is
to say, most of the active compounds can match the
model very well, these properties can also be seen
from Figure 3 which depicted the conformations of
compounds 8i, 8h and I mapped onto Hypo10.
Especially, compound 8i, as the most active
compound, successfully fit all chemical features in
the Hypo10 model, and is the most matched
compound in all ten models. Additionally, the
trifluoromethyl of compound 8i, as the hydrophobic
group, have significant effect to improve the
inhibitory activities, we can also see that one
hydrophobic feature was overlapped with the
trifluoromethyl moiety. However, we must recognize
that just relying on single pharmacophore model
(Hypo10) may not match all compounds perfectly,
such as highly active compound 8g couldn’t match
well with pharmacophore of Hypo10. Even so, there
Table 4. The pharmacophores and corresponding fitvalues
of the target prediction screeninga.
Compound Pharmacophore
1KQ0-01
Fitvalue
4.45067
4.42972
4.40169
4.26285
4.26164
4.21522
4.18110
3D15-03
3D7Z-03
3D2K-02
3DKO-06
1WBT-05
3DJ7-03
8i
a) The best five pharmacophores were presented.
Figure 5. 3D conformations of compound 8i docked into methionine aminopeptidase 2 (PDB code: 1KQ0).
This article is protected by copyright. All rights reserved.

Herein, the Ligand Profiler module, which was
based on the reverse pharmacophore searching
method, will be used to predict the potential target of
compound 8i by reverse virtual screening. The
parameter settings are as follows: Input Ligands
(compound 8i), Input PharmaDB Pharmacophores
(All (noshape and shape)), Model Selection (All
compounds (8i and 8h) were docked into the crystal
structure of METAP2 enzyme and top ten poses were
examined
on
the
basis
of
CDOCKER
(protein–ligand
INTERACTION
ENERGY
interaction energy) to evaluate the nature and type of
interactions. It was observed that compound 8i
docked very well with a CDOCKER INTERACTION
ENERGY of -51.57. As shown in Figure 5 and 6, we
may see that there were two NH groups and one
fluorine atom in compound 8i showed three hydrogen
bonds interaction with His339, His231 and His382
with the distance of 3.14 Å, 3.24 Å and 3.45 Å
respectively. Additionally, the oxygen atom of
carbonyl group in compound 8i showed metal-
acceptor interaction with two zinc ion. Six amino
acids namely Ala414, Phe219, His339, His231,
His382 and Leu328 made the hydrophobic
environment around compound 8i (Figure 6).
Furthermore, compound 8h also showed prominent
interaction (Figure 7) with a good CDOCKER
INTERACTION of -36.76. Similar to compound 8i,
there were two hydrogen bonds (2.94 Å to His-339
and 3.51 Å to Asp-262), two metal-acceptors
interaction (2.17 Å and 2.10 Å) and hydrophobic
environment between compound 8h with METAP2.
However, compound 8i showed better docking
capacity with METAP2 than compound 8h based on
the data of CDOCKER INTERACTION ENERGY
and hydrogen bond. These results theoretically
explain why compound 8i has better inhibitory
activities in colon cancer cells than compound 8h,
and have been confirmed in further biological
experiments.
Models),
Conformation
Generation
(FAST),
Maximum Omitted features (-1), Save Aligned
Ligands (true) and the rest parameters were kept at
their default values. The calculation result (Table 4)
indicated that there are two pharmacophore models
(1kq0-1 and 3d15-3) related to cancer with the
highest Fitvalues, which indicated that methionine
aminopeptidase 2 (METAP2) or auroral kinase A
might be the target of compound 8i. The results of
biological experiments suggested to us that
compound 8i could exert inhibitory effects on tumor
cells probably relating to cell apoptosis but not cell
cycle. Therefore, METAP2 will be used in molecular
docking to study its possible mechanisms of action.
The CDOCKER module in DS2018 was utilized to
perform the probable interaction between compound
8i and methionine aminopeptidase 2. A high
resolution crystal structure of METAP2 bound with
an inhibitory molecule, A849519 (PDB code 1KQ0)
was selected for molecular docking studies. All water
molecules were removed from 1KQ0 before docking
calculations. In the protein structure hydrogen atoms
were added and CHARMm forcefield was applied for
energy minimization. Binding site present in the
protein was defined within the radius of 7.0 Å. For
convenience of comparison, two most active
Figure 6. 2D conformations of compound 8i docked into methionine aminopeptidase 2 (PDB code: 1KQ0).
This article is protected by copyright. All rights reserved.

Figure 7. 3D conformations of compound 8h docked into methionine aminopeptidase 2 (PDB code: 1KQ0).
Figure 8. Cytotoxic activities of compound 8i against human colon cancer HCT116 and HT29 cells. Dose- and
time- dependent anti-proliferative activities of compound 8i against HCT116 and HT29 cells. The values
represent the means of three separate experiments.
The anti-proliferative effect of compound 8i on the
viability of HCT116 and HT29 cells were assessed
using the MTT method. As shown in Figure 8, we
treated compound 8i at different times and doses and
it inhibited the growth of HCT116 and HT29 cells in
a time- and dose-dependent manner. As a result,
compound 8i was found to have anticancer activities
after 48h treatment in HCT116 and HT29 cells at an
IC50 of 10.93 and 6.25μM, respectively. However,
compound 8i did not show obvious cytotoxicity at
100μM against normal colon FHC cell. Next, the role
of compound 8i in the regulation of colony formation
ability of HCT116 and HT29 cells were investigated.
As shown in Figure 9, compound 8i significantly
inhibited the colony formation of HCC116 and HT29
cells in dose-dependent manner.
Compound 8i was docked into human MetAP2
according to the result of target prediction and
molecular docking. Previously researches reported
that MetAP2 has been found to be overexpressed in
colon cancer cell lines and in samples from colon
cancer patients when compared to normal tissue.[19-21]
Moreover, various reports suggested that MetAP2
plays a critical role in cell proliferation and tumor
growth. Downregulation of MetAP-2 expression
significantly induces apoptosis in rat hepatoma
cells.[22] To determine whether the growth inhibitory
effect of compound 8i is associated with the
induction of apoptotic cell death by inhibition of
MetAP2
activity,
flow
cytometry
was
This article is protected by copyright. All rights reserved.

Figure 9. Compound 8i inhibited HCT116 and HT29 cancer cells colony formation. (A) The image of HCT116 and HT29
cancer cells colony formation with compound 8i at concentration of 0, 5, 10 and 20 μM. (B and C) The number of colonies
was counted in per plate. data presented are representative of those obtained in at least three separated experiments. **P＜
0.01 vs group with 0 μM compound 8i.
performed. HCT116 and HT29 cells were exposed to
various concentrations (0, 5, 10 and 20 μM) of
compound 8i for 48 h and analyzed using propidium
iodide (PI)/Annexin V double staining followed by
flow cytometry.
As shown in Figure 11, the levels of pro-apoptotic
proteins Bax and PARP were markedly increased in
HCT116 and HT29 cells by compound 8i treatment,
whereas levels of anti-apoptotic proteins Bcl-2 and p-
Bad were reduced compared with the DMSO control.
The Bax/Bcl-2 ratio is an important factor for
regulating mitochondrial cytochrome C release and is
usually used as an indicator of cells apoptosis. The
dysregulation of the balance between proliferation
and cell death represents a pro-tumorigenic principle
in human colon cancer.[26] Apoptosis plays an
important role in regulating the development,
homeostasis, and elimination of unwanted and
malignant cells. It is a valuable therapeutic method in
clinical practice that inducing apoptosis in human
colon cancer cells via inhibition of the anti-apoptotic
factor, Bcl-2, or the activation of a pro-apoptotic
factor, Bax, which induces activation of
mitochondrial apoptotic pathways.[27-29] In addition,
Bax is localized in the cytoplasm and mitochondrial
membrane, inducing release cytochrome C into the
cytosol followed by recruitment and activation of
caspases to lead to cell apoptosis.[30,31] Our results
consistent with the previous reports indicated that
Bax/Bcl-2 ratio was increased by compound 8i
treatment, which regulated HCT116 and HT29 cells
apoptosis by mitochondrial cytochrome C pathway.
As shown in Figure 10, we observed a dose-
dependent increase in apoptotic cells in the presence
of compound 8i in HCT116 and HT29 cells. The
results showed that the HCT116 cell was more
sensitive to compound 8i than HT29. The apoptotic
cell death of HCT116 was upto 32.96%, 30.52% and
67.64% after incubation with compound 8i at 5, 10
and 20 μM, respectively. The apoptotic cell death of
HT29 was upto 13.03%, 15.82% and 26.89% after
incubation with compound 8i at 5, 10 and 20 μM,
respectively. As a result, these data indicated that the
primary mode of cell death is through apoptosis,
which is induced by inhibition of MetAP2 activity
with the compound 8i.
The mitochondrial or intrinsic apoptosis pathway
was mediated by pro-apoptotic proteins (such as Bax
and PARP) and anti-apoptotic proteins (such as Bcl-2
and p-Bad). MetAP2 also affects the pathway of
apoptosis by regulation of Bcl-2 protein
expression.[23] Inhibition of MetAP2 in mesothelioma
cells depresses both mRNA and protein expression of
the Bcl-2.[21] Previously studies suggested that
MetAP2 plays a major role in proliferative and
apoptosis pathways.[21,24,25]
This article is protected by copyright. All rights reserved.

Figure 10. Apoptosis driven effects of compound 8i against HCT116 and HT29 cells. (A) HCT116 and HT29
cells were collected for flow cytometry analysis after incubated with compound 8i at different concentration for
48h. (B and C) The graph showed cell apoptosis rates at 0, 5, 10 and 20 μM compound compound 8i. *P＜0.05,
**P＜0.01 vs group with 0μM compound 8i.
Figure 11. Effects of compound 8i on protein expression in HCT116 and HT29 cells. Cancer cells were incubated with
compound 8i at concentration of 0, 5, 10 and 20 μM for 48 h. β-tubulin was used for normalization. Western blot data
presented are representative of those obtained in at least three separated experiments.
This article is protected by copyright. All rights reserved.

Figure 12. compound 8i induced caspase3 and 8 activation in HCT116 and HT29 cells. Cancer cells were incubated with
compound 8i at concentration of 0, 5, 10 and 20 μM for 48 h. β-Tubulin was used for normalization. Western blot data
presented are representative of those obtained in at least three separated experiments.
Phosphorylation of H2AX, called γH2AX, is
recognized as an excellent marker of DNA DSBs.[32]
γH2AX was reported to be a useful tool to monitor
the response of patients to cancer therapy by
genotoxic chemicals. Because γH2AX is also
generated by apoptosis, a γH2AX-assay might assess
genotoxic risk incorrectly [33]. Therefore, we
investigated capacity of compound 8i to induce
apoptosis on measurements of γH2AX by western
blot. The data showed that the expression of γH2AX
was increased with dose-dependent increase of
compound 8i (Figure 11).
The MetAP2 is overexpression in Colon
adenocarcinoma HCT116 and HT29 cell lines, which
is responsible for the reported reduction in protein
levels of caspase cascades.[21,24] A hand of studies
reported that caspase cascades are crucial effectors
and mediators of apoptosis induction and caspase3
and 8 have been linked to mitochondrial death
pathway,[34-36] therefore the expression of cleaved-
caspase3 and 8 in HCT116 and HT29 cells were
analyzed. The data revealed that treatment with
compound 8i increased the activities of caspase3 and
8 in both colon cancer cell lines to a significantly
greater extent than untreated cells (Figure 12).
Apoptosis disorders are an additional cause for the
occurrence of colon cancer. The initiation of
apoptosis is typically triggered via caspase3, which is
being used to treat cancer. Therefore, compound 8i
which increase caspase3 and 8 activities or γH2AX
expression by inhibition of MetAP2 activity, may be
effective tools to improve colon cancer prognosis.
compound libraries screening, molecular docking and
target fishing strategies. The results showed that
compound 8i, which bearing
a
hydrophobic
trifluoromethyl group, was the most active compound
against all tested tumor cell lines, especially in
HCT116 (IC50 10.93 μM) and HT29 (IC50 6.25 μM)
cells. The molecular biological target of compound 8i
was explored by target fishing strategy, the result
demonstrated that methionine aminopeptidase 2
probably be the target of compound 8i, which was
further validated by molecular docking. The
biological results suggested that compound 8i
induced the apoptosis of HCT116 and HT29 colon
cancer
cells
probably through
methionine
aminopeptidase 2, and in accordance with the result
of target fishing and molecular docking. This study
provides valuable information for the development of
new anti-cancer drugs and offer more options for
biologists and pharmaceutical industries.
4. Experimental Section
4.1. Chemistry
1H and 13C NMR were recorded on a Bruker 400
spectrometer. The high-resolution mass spectra (HRMS)
were recorded on an Thermo UItiMate 3000-Q Exactive
mass spectrometer with ESI resource. LC-MS analyses
were performed on a Shimadzu-2020 LC-MS instrument
using the following conditions: Shim-pack VPODS C18
column (reverse phase, 150 x 2.0mm); 80% acetonitrile
and 20% water over 6.0 min; flow rate of 0.4 mL/min; UV
photodiode array detection from 200 to 300nm. The
products were purified by Biotage Isolera™ Spektra
Systems
and
n-Hexane/EtOAc
or
Dichloromethane/Methanol solvent systems. Microwave
irradiation experiments were carried out in a Biotage®
Initiator Classic microwave apparatus with continuous
irradiation power from 0 to 400W with utilization of the
standard absorbance level of 250W maximum power. The
reactions were carried out in 5 or 10 mL glass tubes, sealed
with microwave cavity. The reaction was irradiated at a
required ceiling temperature using maximum power for the
stipulated time. Then it was cooled to 50oC with gas jet
3. Conclusion
In summary, this paper presents researches of the
discovery of 4H-thieno[3,2-b]pyrrole scaffold, the
optimization of structure, the exploration of structure-
activity relationship and the mechanism of action of
the 4H-thieno[3,2-b]pyrrole derivatives based on the
This article is protected by copyright. All rights reserved.

cooling. All reagents and solvents were obtained from
commercial sources and used without further purification.
column chromatography using ethylacetate/hexane (1:19-
1:5) to yield corresponding product 8a-8n.
N,3-diphenyl-4H-thieno[3,2-b]pyrrole-5-carboxamide (8a)
1
Yellow solid, 112 mg, yield 88%. H NMR (400 MHz,
4.2. Synthesis
Methyl 2-azidoacetate (2)
DMSO-d6) δ 11.85 (s, 1H), 10.01 (s, 1H), 7.83 – 7.76 (m,
7.4 Hz, 4H), 7.69 (s, 1H), 7.49 (s, 2H), 7.40 – 7.36 (m, 4H),
7.09 (d, J = 6.5 Hz,, 1H). 13C NMR (100 MHz, DMSO-d6)
δ 159.50, 139.59, 138.83, 134.08, 132.35, 129.28, 129.18,
127.85, 127.42, 127.28, 124.75, 124.59, 123.79, 120.45,
105.61. HRMS (E) calcd for C19H15N2OS+ [M+H]+,
319.08996; found, 319.09079.
To a solution of methyl 2-chloroacetate (1.09 g, 10.0
mmol) in acetone/water (20 mL, 2/1, v/v) was added NaN3
(1.30 g, 20.0 mmol) at room temperature, after addition the
mixture was heated to 60 ºC for 3 hours, then the mixture
was evaporated and the residue was extracted by ethyl
acetate and wash by saturated NaHCO3 solution and brine,
dried and concentrated to give crude compound 2 as a light
yellow oil (0.98 g, yield: 85%), which was used for next
step without further purification.
3-Phenyl-N-(thiazol-2-yl)-4H-thieno[3,2-b]pyrrole-5-
carboxamide (8b)
1
Yellow solid, 84 mg, yield 64%. H NMR (400 MHz,
DMSO-d6) δ 12.44 (s, 1H), 12.03 (s, 1H), 7.82 (d, J = 7.4
Hz, 2H), 7.78 (s, 1H), 7.58 (s, 1H), 7.55 (d, J = 3.5 Hz,
1H), 7.51 (t, J = 7.7 Hz, 2H), 7.39 (t, J = 7.3 Hz, 1H), 7.26
(d, J = 3.5 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ
158.91, 158.51, 139.79, 138.22, 133.87, 130.00, 129.34,
Methyl (E)-2-azido-3-(4-bromothiophen-2-yl)acrylate (4)
To a solution of NaOMe (1.35 g, 25.0 mmol) in MeOH (10
mL) was added a solution of 4-bromothiophene-2-
carbaldehyde (3, 1.91 g, 10.0 mmol,) and methyl 2-
azidoacetate (2.42 mL, 25.0 mmol) in MeOH (10 mL)
dropwise at -10 °C over 30 minutes and stirred for 2 hours
at this temperature The mixture was allowed to stir for an
additional 2.5 h at 0o C and then poured into ice-water,
extracted with ethyl acetate, washed with water. The
organic layer was dried over Na2SO4 and concentrated
under reduced pressure. The residue was purified by
column chromatography using ethylacetate/hexane (1:19)
to yield pure product 4 (1.87 g, 65%).
127.96, 127.30, 127.10, 125.92, 125.15, 114.06, 107.71.
+
HRMS (E) calcd for C16H12N3OS2 [M+H]+, 326.04163;
found, 326.04178.
(R)-3-phenyl-N-(1-phenylethyl)-4H-thieno[3,2-b]pyrrole-
5-carboxamide (8c)
1
Yellow solid, 101 mg, yield 73%. H NMR (400 MHz,
DMSO-d6) δ 11.60 (s, 1H), 8.55 (d, J = 8.0 Hz, 1H), 7.78
(d, J = 7.4, 2H), 7.62 (s, 1H), 7.47 (t, J = 7.6 Hz, 2H), 7.41
– 7.31 (m, 5H), 7.25 – 7.22 (m, 2H), 5.22 – 5.15 (m, 1H),
1.50 (d, J = 7.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ
160.12, 145.27, 138.20, 134.22, 132.54, 129.31, 128.72,
127.79, 127.31, 127.21, 127.11, 126.55, 124.52, 123.85,
104.94, 48.43, 22.73. HRMS (E) calcd for C21H19N2OS+
[M+H]+, 347.12126; found, 347.12115.
Methyl
(5)
3-bromo-4H-thieno[3,2-b]pyrrole-5-carboxylate
A solution of compound 4 (2.88 g, 10 mmol) in dry toluene
(100 mL) was slowly added into boiling xylene (100 mL)
over 1 h, then the reaction mixture was refluxed for an
additional 3 h. After the reaction completed the mixture
was cooled and the solvent was removed under reduced
pressure. The residue was purified by column
chromatography using ethylacetate/hexane (1:4) to yield
pure compound 5 (2.13 g, 82%) .1H NMR (400 MHz,
DMSO-d6) δ 12.53 (s, 1H), 7.63 (s, 1H), 7.19 (s, 1H), 3.83
(s, 3H). 13C NMR (101 MHz, DMSO) δ 161.41, 140.25,
127.95, 126.89, 123.47, 108.78, 94.55, 52.00. HRMS (E)
calcd for C8H7BrNO2S+ [M+H]+, 259.93754; found,
259.93787.
morpholino(3-phenyl-4H-thieno[3,2-b]pyrrol-5-
yl)methanone (8d)
Light yellow solid, 70 mg, yield 56%. 1H NMR (400 MHz,
DMSO-d6) δ 11.77 (s, 1H), 7.79 (d, J = 7.8 Hz, 2H), 7.63
(s, 1H), 7.45 (t, J = 7.5 Hz, 2H), 7.34 (t, J = 7.3 Hz, 1H),
6.84 (s, 1H), 3.71 (d, J = 3.3 Hz, 4H), 3.64 (d, J = 3.8 Hz,
4H). 13C NMR (100 MHz, DMSO-d6) δ 162.37, 137.57,
134.08, 130.40, 129.20, 127.72, 127.24, 127.09, 124.33,
123.23, 104.75, 66.74, 45.60. HRMS (E) calcd for
C17H17N2O2S+ [M+H]+, 313.10052; found, 313.10086.
N-(3-fluorophenyl)-3-phenyl-4H-thieno[3,2-b]pyrrole-5-
Methyl 3-phenyl-4H-thieno[3,2-b]pyrrole-5-carboxylate
(6)
carboxamide (8e)
A
mixture of compound 5 (2.08 g, 8.0 mmol),
1
Yellow solid, 105 mg, yield 78%. H NMR (400 MHz,
phenylboronic acid (1.22 g, 10.0 mmol), Na2CO3 (1.06 g,
10.0 mmol) and Pd(PPh3)4 (925 mg, 0.8 mmol) in
toulene/EtOH (20 mL, 3/1, v/v) was degassed and
protected by N2, then the mixture was stirred at 100 ºC for
40 minutes in microwave apparatus. After the reaction
completed the mixture was dried and purified by column
DMSO-d6) δ 11.90 (s, 1H), 10.18 (s, 1H), 7.83 – 7.76 (m,
3H), 7.71 (s, 1H), 7.53 – 7.47 (m, 3H), 7.42 – 7.36 (m, 3H),
6.91 (t, J = 7.9 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ
162.60 (d, J = 241.4 Hz), 159.70, 141.44 (d, J = 11.1 Hz),
139.10, 134.02, 131.96, 130.78 (d, J = 10.1 Hz), 129.27,
127.87, 127.44, 127.28, 124.88 (d, J = 15.2 Hz), 116.04,
110.16 (d, J = 21.2 Hz), 107.17, 106.91 105.90. HRMS (E)
calcd for C19H14FN2OS+ [M+H]+, 337.08054; found,
337.08066.
chromatography (ethylacetate/hexane
= 1/5) 1to give
compound 6 (1.23 g, 60%) as a yellow solid. H NMR
(400 MHz, DMSO-d6) δ 12.15 (s, 1H), 7.79 (d, 7.7 Hz,
2H), 7.74 (s, 1H), 7.45 (t, J = 7.2 Hz, 2H), 7.37 (d, J = 6.8
Hz, 1H), 7.22 (s, 1H), 3.83 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 161.56, 139.95, 133.79, 129.13, 127.85,
127.79, 127.57, 127.36, 126.05, 125.08, 108.62, 51.88.
HRMS (E) calcd for C14H12NO2S+ [M+H]+, 258.05833;
found, 258.05856.
N-(furan-2-ylmethyl)-3-phenyl-4H-thieno[3,2-b]pyrrole-5-
carboxamide (8f)
Light yellow solid, 87 mg, yield 67%. 1H NMR (400 MHz,
DMSO-d6) δ 11.63 (s, 1H), 8.71 (t, J = 5.6 Hz, 1H), 7.78 (d,
J = 7.5 Hz, 2H), 7.64 (s, 1H), 7.60 (s, 1H), 7.47 (t, J = 7.6
Hz, 2H), 7.36 (t, J = 7.3 Hz, 1H), 7.18 (d, J = 1.1 Hz, 1H),
6.42 (t, J = 2.2 Hz, 1H), 6.32 (d, J = 3.0 Hz, 1H), 4.50 (d, J
= 5.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 160.71,
152.87, 142.62, 138.28, 134.13, 132.24, 129.27, 127.79,
127.29, 127.17, 124.57, 123.96, 110.98, 107.54, 104.96,
35.96. HRMS (E) calcd for C18H15N2O2S+ [M+H]+,
323.08487; found, 323.08463.
3-Phenyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid (7)
To a solution of compound 6 (1.54 g, 6.0 mmol) in
MeOH/H2O (60 mL, 3/1, v/v) was added LiOH (432 mg,
18.0 mmol) at room temperature, then the mixture was
stirred and heated at 60 ºC for 2 hours. After the reaction
completed the mixture was evaporated to remove MeOH,
then ice-water (20 mL) was added and the pH was adjusted
to 3-4, then the suspension was filtered and the cake was
washed by water and dried to give compound 7 (1.40 g,
yield: 96%) as a white powder.
N-(4-bromophenyl)-3-phenyl-4H-thieno[3,2-b]pyrrole-5-
carboxamide (8g)
Light brown solid, 129 mg, yield 81%. 1H NMR (400 MHz,
DMSO-d6) δ 11.88 (s, 1H), 10.13 (s, 1H), 7.82 (d, J = 7.4
Hz, 2H), 7.76 (d, J = 8.8 Hz, 2H), 7.70 (s, 1H), 7.55 (d, J =
8.8 Hz, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.41 – 7.36 (m, 2H).
13C NMR (100 MHz, DMSO-d6) δ 159.55, 139.03, 139.01,
134.03, 132.07, 131.99, 129.27, 127.86, 127.43, 127.28,
124.84, 124.80, 122.29, 115.37, 105.84. HRMS (E) calcd
for C19H14BrN2OS+ [M+H]+, 397.00047; found, 397.00107.
N-(3-bromophenyl)-3-phenyl-4H-thieno[3,2-b]pyrrole-5-
carboxamide (8h)
General Procedures for the Preparation of 8a-8n
A suspension of compound 7 (97 mg, 0.4 mmol), EDCI
(96 mg, 0.5 mmol), HOBT (68 mg, 0.5 mmol) and DMAP
(61 mg, 0.5 mmol) in DCM (3 mL) was stirred for 0.5 hour
at room temperature, then a solution of amine (0.5 mmol)
was added dropwise at room temperature and stirred
overnight. After the reaction completed the mixture was
extracted with dichloromethane, washed with brine. The
organic layer was dried over Na2SO4 and concentrated
under reduced pressure. The residue was purified by
This article is protected by copyright. All rights reserved.

1
Brown solid, 121 mg, yield 76%. H NMR (400 MHz,
CDCl3) δ 159.24, 139.82, 139.09, 133.84, 132.99, 132.66,
132.32, 131.99, 129.28, 129.11, 127.93, 127.12, 126.84,
126.52, 125.43, 125.24, 124.41, 121.69, 119.53, 117.54,
103.40. HRMS (E) calcd for C21H13F6N2OS+ [M+H]+,
455.06473; found, 455.06476.
DMSO-d6) δ 11.89 (s, 1H), 10.14 (s, 1H), 8.09 (s, 1H),
7.82 (d, J = 7.4 Hz, 2H), 7.75 (d, J = 7.8 Hz, 1H), 7.71 (s,
1H), 7.49 (t, J = 7.3 Hz, 2H), 7.42 – 7.27 (m, 4H). 13C
NMR (100 MHz, DMSO-d6) δ 159.63, 141.29, 139.11,
134.02, 131.90, 131.20, 129.28, 127.87, 127.43, 127.27,
126.29, 125.00, 124.84, 122.60, 121.96, 119.04, 105.95.
HRMS (E) calcd for C19H14BrN2OS+ [M+H]+, 397.00047;
found, 397.00104.
4.3. Pharmacophore models, target prediction and
molecular docking
The pharmacophore models, target prediction and
molecular docking were all performed using Discovery
3-Phenyl-N-(3-(trifluoromethyl)phenyl)-4H-thieno[3,2-
b]pyrrole-5-carboxamide (8i)
Studio
2018R2
software
package.
Specifically,
1
Yellow solid, 107 mg, yield 69%. H NMR (400 MHz,
pharmacophore models were carried out by using
pharmacophores module, which includes Edit and Cluster
Features, Create Pharmacophore Automatically and Search,
Screen and Profile tools. And target prediction was
performed using Ligand Profiler protocol which contains a
new pharmacophore database called “PharmaDB”.
Moreover, molecular docking was carried out by using
pharmacophores module called “CDOCKER” protocol,
which is an accurate molecular docking strategy based on
CHARMm forcefield. The X-ray crystal structure of
METAP2 (PDB code 1KQ0) complexed with ligand D-
methionine was used as the reference receptor structure in
the docking calculations, which was downloaded from the
Protein Data Bank of RSCB.
DMSO-d6) δ 11.92 (s, 1H), 10.32 (s, 1H), 8.24 (s, 1H),
8.05 (d, J = 7.3 Hz, 1H), 7.83 (d, J = 6.6 Hz, 2H), 7.73 (s,
1H), 7.62 (d, J = 7.3 Hz, 1H), 7.51 – 7.38 (m, 5H). 13C
NMR (100 MHz, DMSO-d6) δ 159.82, 140.48, 139.19,
134.00, 131.80, 130.44, 130.05, 129.73, 129.28, 127.88,
127.42, 127.26, 126.02, 125.10, 124.87, 123.78, 123.31,
122.39, 120.00, 119.96, 116.40, 116.35, 106.00. HRMS
(E) calcd for C20H14F3N2OS+ [M+H]+, 387.07734; found,
387.07776.
(S)-3-phenyl-N-(1-phenylethyl)-4H-thieno[3,2-b]pyrrole-5-
carboxamide (8j)
1
Yellow solid, 104 mg, yield 75%. H NMR (400 MHz,
DMSO-d6) δ 11.60 (s, 1H), 8.55 (d, J = 7.9 Hz, 1H), 7.78
(d, J = 7.7 Hz, 2H), 7.62 (s, 1H), 7.47 (t, J = 7.6 Hz, 2H),
7.41 – 7.31 (m, 6H), 7.21 (s, 1H), 5.20 – 5.17 (m, 1H),
1.50 (d, J = 7.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 160.12, 145.28, 138.20, 134.22, 132.53, 129.31, 128.73,
127.79, 127.31, 127.21, 127.11, 126.55, 124.52, 123.86,
104.94, 48.43, 22.73. HRMS (E) calcd for C21H19N2OS+
[M+H]+, 347.12126; found, 347.12115.
4.4. Cell culture
Human colon cancer cells (HCT116, HT29) and normal
colon cell (FHC) were obtained from the Nanjing Cobioer
Co., Ltd (Jiangsu, China). Cells were grown in RPMI 1640
medium (Gibco, Beijing, China) containing 10% fetal
bovine serum (Gibico, New York, NY, USA), penicillin
(100U/mL, Gibco, Beijing, China), and streptomycin
(100μg/mL, Gibco, Beijing, China). FHC was cultured in
DMEM:F12 medium containing 10% FBS, 10ng/mL
cholera toxin, 5μg/mL insulin, 5μg/mL transferrin and
100ng/mL hydrocortisone (Cobioer, Jiangsu, China). The
cells were incubated at 37°C in a humidified air with 5%
CO2.
3-Phenyl-N-(thiophen-2-ylmethyl)-4H-thieno[3,2-
b]pyrrole-5-carboxamide (8k)
1
Yellow solid, 90 mg, yield 66%. H NMR (400 MHz,
DMSO-d6) δ 11.67 (s, 1H), 8.88 (s, 1H), 7.79 (d, J = 7.1
Hz, 2H), 7.64 (s, 1H), 7.48 – 7.36 (m, 4H), 7.18 (s, 1H),
7.05 (s, 1H), 6.97 (s, 1H), 4.67 (s, 2H). 13C NMR (100
MHz, DMSO-d6) δ 160.72, 143.19, 138.30, 134.10, 132.23,
129.25, 127.78, 127.31, 127.19, 127.14, 125.99, 125.59,
124.60, 123.98, 104.82, 37.72. HRMS (E) calcd for
C18H15N2OS2+ [M+H]+, 339.06203; found, 339.06219.
N-cyclopropyl-3-phenyl-4H-thieno[3,2-b]pyrrole-5-
carboxamide (8l)
4.5. In vitro cytotoxicity assay
Cells were seeded in 96-well dish (1×104 cells/well). After
12 h of culture, the cells were treated with compouds at 20
μM or a series of concentration for 24, 48 and 72h,
respectively. Upon completion of the incubation, stock 3-
Light yellow solid, 59 mg, yield 52%. 1H NMR (400 MHz,
DMSO-d6) δδ 11.52 (s, 1H), 8.24 (d, J = 3.5 Hz, 1H), 7.78
(d, J = 7.3 Hz, 2H), 7.61 (s, 1H), 7.47 (t, J = 7.5 Hz, 2H),
7.37 (d, J = 7.3 Hz, 1H), 7.10 (s, 1H), 2.84 – 2.80 (m, 1H),
0.73 – 0.71 (m, 2H), 0.57 – 0.55 (m, 2H). 13C NMR (100
(4,5-dimethylthiazole)-2,5-diphenyltetrazolium
bromide
(MTT) dye solution (20μL) was added to each well and
incubated for additional 4 h at 37°C. Then, 100μL
MHz, DMSO-d6) δ 162.07, 138.07, 134.17, 132.59, 129.26, dimethylformamide was added to solublilze the MTT
128.80, 127.77, 127.31, 124.50, 123.70, 104.45, 23.00,
6.33. HRMS (E) calcd for C16H15N2OS+ [M+H]+,
283.08996; found, 283.08966.
formazan. The cell viability was obtained by measuring the
optical density at 490 nm using a microplate reader
(Cytation 5; BioTek, Winooski, VT, USA). The IC50
values were determined by plotting the percentage viability
versus concentration on a logarithmic graph and reading
off the concentration at which 50% of cells remain viable
relative to the DMSO control.
N-(tert-butyl)-3-phenyl-4H-thieno[3,2-b]pyrrole-5-
carboxamide (8m)
Light yellow solid, 77 mg, yield 64%. 1H NMR (400 MHz,
DMSO-d6) δ 11.52 (s, 1H), 7.77 (d, J = 7.6 Hz, 2H), 7.59
(s, 2H), 7.48 (t, J = 7.5 Hz, 2H), 7.36 (t, J = 7.3 Hz, 1H),
7.13 (s, 1H), 1.40 (s, 9H). 13C NMR (100 MHz, DMSO-d6)
δ 160.54, 137.87, 134.32, 133.52, 129.35, 127.78, 127.30,
127.21, 124.34, 123.48, 104.80, 51.25, 29.29. HRMS (E)
calcd for C17H19N2OS+ [M+H]+, 299.12126; found,
299.12106.
4.6. Colony formation assay
HCT116 and HT29 cells were trypsinized, counted, and
seeded into 6-well dish at a density of xx cells/well. Cells
were treated with compound 8i at 5, 10, 20μM or DMSO
for 12-14 day to allow for colony formation. After
incubation, colonies were fixed and stained with 1%
methylene blue in 50% ethanol at room temperature, and
colonies consisting of more than 100 cells per well were
counted by an inverted microscope (Olympus IX53;
Olympus, Tokyo, Japan). Three independent experiments
were carried out.
Tert-butyl
4-(3-phenyl-4H-thieno[3,2-b]pyrrole-5-
carbonyl)piperazine-1-carboxylate (8n)
Light yellow solid, 92 mg, yield 56%. 1H NMR (400 MHz,
DMSO-d6) δ 11.77 (s, 1H), 7.79 (d, J = 7.5 Hz, 2H), 7.63
(s, 1H), 7.45 (t, J = 7.6 Hz, 2H), 7.34 (d, J = 7.4 Hz, 1H),
6.86 (s, 1H), 3.70 (s, 4H), 3.42 (s, 4H), 1.42 (s, 9H). 13
C
NMR (100 MHz, DMSO-d6) δ 162.46, 154.30, 137.62,
134.53, 134.06, 130.46, 129.20, 127.72, 127.25, 127.10,
124.34, 123.30, 104.86, 79.66, 43.57, 28.52. HRMS (E)
calcd for C22H26N3O3S+ [M+H]+, 412.16894; found,
412.16565.
4.7. Annexin V-FITC apoptosis assay
HCT116 and HT29 cells were treated with compound 8i at
0, 5, 10 and 20μM for 24h. Cells were stained with annexin
V-FITC and PI (Beyotime Biotechnology Co. Ltd., Jiangsu,
China) for 15min at room temperature in annexin V
binding buffer, then analyzed using flow cytometry BD C6
(BD Biosciences, San Jose, CA). For each sample, data
form 10,000 cells were recorded and the results were
N-(3,5-bis(trifluoromethyl)phenyl)-3-phenyl-4H-
thieno[3,2-b]pyrrole-5-carboxamide (8o)
1
Light yellow solid, 133 mg, yield 73%. H NMR (400
MHz, CDCl3) δ 9.64 (s, 1H), 8.14 (s, 2H), 7.92 (s, 1H),
7.63 – 7.62 (m, 3H), 7.47 (t, J = 7.6 Hz, 2H), 7.40 – 7.35
(m, 2H), 7.05 (d, J = 1.9 Hz, 1H). 13C NMR (100 MHz,
This article is protected by copyright. All rights reserved.

interpreted with FlowJo software (Tree Star, FlowJo LLC,
Ashland, OR).
[9] G. Badr, H. I. Gul, C. Yamali, A. A. M. Mohamed, B.
M. Badr, M. Gul, A. Abo Markeb, N. Abo El-Maali,
Bioorg. Chem. 2018, 78, 46-57.
4.8. Immunoblot analysis
[10] G. Dewson, T. Kratina, H. W. Sim, H. Puthalakath, J.
M. Adams, P. M. Colman, R. M. Kluck, Mol. Cell 2008,
30, 369-380.
HCT116 and HT29 cells were solubilized in RIPA lysis
buffer containing protease and phosphatase inhibitor
cocktails. Following centrifugation at 12,000g for 15min at
4°C, the concentration of protein in the supernatant was
[11] H. Chen, M. Kanai, A. Inoue-Yamauchi, H. Tu, Y.
Huang, D. Ren, H. Kim, S. Takeda, D. E. Reyna, P. M.
Chan, Y. T. Ganesan, C. Liao, E. Gavathiotis, J. J.
Hsieh, E. H. Cheng, Nat. Cell Biol. 2015, 17, 1270-
1281.
determined using
a
protein assay kit (Beyotime
Biotechnology Co. Ltd., Jiangsu, China). Protein samples
(20μg) were loaded into a SDS-polyscrylamide gel,
separated by SDS-PAGE, transferred onto polyvinylidene
fluoride (PVDF) membrane, and probed with specific
antibodies against the following proteins: cleaved-caspase3,
cleaved-caspase8, cleaved-caspase9, γH2AX, H2AX, Bax,
Bcl2, PARP, p-Bad (all form CST, Danvers, MA). The
blots were thoroughly rinsed and then incubated with an
HRP-labeled species-matched secondary antibody for 1h at
room temperature. Protein bands were detected by
enhanced chemiluminescence (ECL, Thermo Fisher
Scientific (China) Co., Ltd., Shanghai, China) and the
signals were recorded using Tanon 5200 Luminescent
Imaging Workstation (Tanon Science & Technology Co.,
Ltd., Shanghai, China) and software that helps to quantify
the protein band intensities.
[12] H. Chen, Y. Lee, S. Y. Huang, P. Shi, P. Hsu, C.
Chang, Oncotarget 2018, 9, 13167-13180.
[13] I. Çoruh, Ö. Çevik, K. Yelekçi, T. Djikic, Ş. G.
Küçükgüzel, Arch. Pharm. 2018, 351, 1700195.
[14] I. Endo, M. Gonen, A. C. Yopp, K. M. Dalal, Q.
Zhou, D. Klimstra, M. DʼAngelica, R. P. DeMatteo, Y.
Fong, L. Schwartz, N. Kemeny, E. OʼReilly, G. K.
Abou-Alfa, H. Shimada, L. H. Blumgart, W. R.
Jarnagin, Ann. Surg. 2008, 248, 84-96.
Acknowledgements
[15] J. Boström, D. G. Brown, R. J. Young, G. M. Keserü,
Nat. Rev. Drug Discov. 2018, 17, 922.
We acknowledge the financial support from the National Natural
Science Foundation of China (Grant Nos. 21901028 and
81902357), Natural Science Foundation of Chongqing (Grant
Nos. cstc2019jcyj-msxmX0202 and cstc2019jcyj-msxmX0012),
Science and Technology Research Program of Chongqing
Municipal Education Commission (Grant Nos. KJQN201801305,
KJQN202001327 and KJQN201801321) and Chongqing
University of Arts and Sciences (Grant Nos. 2017RBX08,
2017ZBX07 and R2016BX09).
[16] J. D. Venable, H. Cai, W. Chai, C. A. Dvorak, C. A.
Grice, J. A. Jablonowski, C. R. Shah, A. K. Kwok, K. S.
Ly, B. Pio, J. Wei, P. J. Desai, W. Jiang, S. Nguyen, P.
Ling, S. J. Wilson, P. J. Dunford, R. L. Thurmond, T.
W. Lovenberg, L. Karlsson, N. I. Carruthers, J. P.
Edwards, J. Med. Chem. 2005, 48, 8289-8298.
[17] J. L. Mauriz, J. Martín-Renedo, A. García-Palomo, M.
J. Tuñón, J. González-Gallego, Curr. Drug Targets
2010, 11, 1439-1457.
References
[18] J. Parmentier, B. Portevin, R. M. Golsteyn, A. Pierré,
J. Hickman, P. Gloanec, G. De Nanteuil, Bioorg. Med.
Chem. Lett. 2009, 19, 841-844.
[1] A. Catalano, M. Romano, I. Robuffo, L. Strizzi, A.
Procopio, Am. J. Pathol. 2001, 159, 721-731.
[2] A. Harada, K. Matsuzaki, A. Takeiri, M. Mishima,
Mutat. Res. Genet. Toxicol. Environ. Mutagen. 2014,
771, 23-29.
[19] J. Shen, L. Zeng, L. Pan, S. Yuan, M. Wu, X. Kong,
Oncol. Lett. 2018, 15, 4557-4563.
[20] J. Sun, M. Li, S. Qian, F. Guo, X. Dang, X. Wang, Y.
Xue, H. Zhu, Bioorg. Med. Chem. Lett. 2013, 23, 2876-
2879.
[3]A. Iida, A. Yamaguchi, K. Hirose, J. Surg. Oncol. 2000,
73, 219-23.
[4] A. Ivashkevich, C. E. Redon, A. J. Nakamura, R. F.
[21] L. Sartori, C. Mercurio, F. Amigoni, A. Cappa, G.
Fagá, R. Fattori, E. Legnaghi, G. Ciossani, A. Mattevi,
G. Meroni, L. Moretti, V. Cecatiello, S. Pasqualato, A.
Romussi, F. Thaler, P. Trifiró, M. Villa, S. Vultaggio,
O. A. Botrugno, P. Dessanti, S. Minucci, E. Zagarrí, D.
Carettoni, L. Iuzzolino, M. Varasi, P. Vianello, J. Med.
Chem. 2017, 60, 1673-1692.
Martin, O. A. Martin, Cancer Lett. 2012, 327, 123-133.
[5] A. K. Bhattacharjee, J. W. Pomponio, S. A. Evans, D.
Pervitsky, R. K. Gordon, Bioorgan. Med. Chem. 2013,
21, 2651-2662.
[6] A. M. Birch, P. W. Kenny, N. G. Oikonomakos, L.
Otterbein, P. Schofield, P. R. O. Whittamore, D. P.
Whalley, Bioorg. Med. Chem. Lett. 2007, 17, 394-399.
[22] M. A. Abu-Helalah, H. A. Alshraideh, M. D. Da Na,
M. T. Al-Hanaqtah, A. Abuseif, K. Arqoob, A. Ajaj, J.
Gastrointest. Canc. 2016, 47, 36-46.
[7] C. Günther, E. Martini, N. Wittkopf, K. Amann, B.
Weigmann, H. Neumann, M. J. Waldner, S. M. Hedrick,
S. Tenzer, M. F. Neurath, C. Becker, Nature 2011, 477,
335-339.
[23] P. R. Adiyala, V. Tekumalla, I. B. Sayeed, V. L.
Nayak, A. Nagarajan, M. A. Shareef, B. Nagaraju, A.
Kamal, Bioorg. Chem. 2018, 76, 288-293.
[8] C. J. Gerry, S. L. Schreiber, Nat. Rev. Drug Discov.
2018, 17, 333-352.
[24] P. Selvakumar, A. Lakshmikuttyamma, U. Das, H. N.
Pati, J. R. Dimmock, R. K. Sharma, Mol. Cancer 2009,
8, 65.
This article is protected by copyright. All rights reserved.

[25] P. Vianello, L. Sartori, F. Amigoni, A. Cappa, G.
Fagá, R. Fattori, E. Legnaghi, G. Ciossani, A. Mattevi,
G. Meroni, L. Moretti, V. Cecatiello, S. Pasqualato, A.
Romussi, F. Thaler, P. Trifiró, M. Villa, O. A.
Botrugno, P. Dessanti, S. Minucci, S. Vultaggio, E.
Zagarrí, M. Varasi, C. Mercurio, J. Med. Chem. 2017,
60, 1693-1715.
[26] R. A. Gonzalez-Polo, P. Boya, A. L. Pauleau, A. Jalil,
N. Larochette, S. Souquere, E. L. Eskelinen, G. Pierron,
P. Saftig, G. Kroemer, J. Cell Sci. 2005, 118, 3091-102.
[27] R. Kumar, R. Bavi, M. G. Jo, V. Arulalapperumal, A.
Baek, S. Rampogu, M. O. Kim, K. W. Lee, Sci. Rep.-
UK 2017, 7, 10827.
[28] S. Kuriyama, T. Tsuji, T. Sakuma, T. Yamamoto, M.
Tanaka, Cell Death Discov. 2018, 4, 11.
[29] S. N. L. Bennett, A. D. Campbell, A. Hancock, C.
Johnstone, P. W. Kenny, A. Pickup, A. T. Plowright, N.
Selmi, I. Simpson, A. Stocker, D. P. Whalley, P. R. O.
Whittamore, Bioorg. Med. Chem. Lett. 2010, 20, 3511-
3514.
[30] S. Tian, J. Wang, Y. Li, D. Li, L. Xu, T. Hou, Adv.
Drug Deliver. Rev. 2015, 86, 2-10.
[31] T. Hirayama, M. Okaniwa, T. Imada, A. Ohashi, M.
Ohori, K. Iwai, K. Mori, T. Kawamoto, A. Yokota, T.
Tanaka, T. Ishikawa, Bioorgan. Med. Chem. 2013, 21,
5488-5502.
[32] T. Kanno, H. Endo, K. Takeuchi, Y. Morishita, M.
Fukayama, S. Mori, Lab. Invest. 2002, 82, 893-901.
[33] T. Rodrigues, D. Reker, P. Schneider, G. Schneider,
Nat. Chem. 2016, 8, 531-541.
[34] T. Rodrigues, D. Reker, P. Schneider, G. Schneider,
Nat. Chem. 2016, 8, 531-541.
[35] Y. Zhang, Z. Li, Q. Min, A. Palida, Y. Zhang, R.
Tang, L. Chen, H. Li, Bioorg. Chem. 2018, 77, 478-484.
[36] Z. Jia, H. H. Yang, Y. Liu, X. Wang, Appl. Biochem.
Biotech. 2018, 186, 145-160.
This article is protected by copyright. All rights reserved.