Melatonin derivatives combat with inflammation-related cancer by targeting the Main Culprit STAT3

Article abstract of DOI:10.1016/j.ejmech.2020.113027

The combination between two well-studied bioactive compounds melatonin and salicylic acid with proper modifications unexpectedly creates a sharp pair of “scissors” cutting off the vicious connection between inflammation and cancer by targeting a key contributor Signal Transducers and Activators of Transcription 3 (STAT3) in the two pathological processes. A representative compound P-3 with IC₅₀ values on each tested cell line ranging from 7.37 to 18.62 μM among the designed melatonin derivatives is equipped with the ability of curbing inflammation-promoting cancer by down-regulating the expression, activation and nuclear translocation of STAT3, breaking the feedforward loop of STAT3 activation by decreasing the expression of pro-tumorigenic cytokines, and inducing cell apoptosis through ROS triggered Cyto-c/Caspase-3 pathway. This study suggests that the melatonin derivative P-3 is likely to become a promising chemical structure for developing the novel anti-cancer agents taking effect through hindering the mutual-promoting processes between inflammation and cancer.

Full text of DOI:10.1016/j.ejmech.2020.113027

Journal Pre-proof
Melatonin Derivatives Combat with Inflammation-related Cancer by Targeting the
Main Culprit STAT3
Shumeng Ma, Longqing Zhu, Xiaohong Fan, Tian Luo, Dan Liu, Ziyi Liang, Xiaoling
Hu, Tao Shi, Wen Tan, Zhen Wang
PII:
S0223-5234(20)30999-5
DOI:
https://doi.org/10.1016/j.ejmech.2020.113027
EJMECH 113027
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 August 2020
Revised Date: 22 October 2020
Accepted Date: 12 November 2020
Please cite this article as: S. Ma, L. Zhu, X. Fan, T. Luo, D. Liu, Z. Liang, X. Hu, T. Shi, W. Tan, Z.
Wang, Melatonin Derivatives Combat with Inflammation-related Cancer by Targeting the Main Culprit
STAT3, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.113027.
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IL-6
TNF-
Inflammation
STAT3
Cancer
α
Compound P-3
O
Combat
Expression
N
H
HN
inflammation-
related cancer
Nuclear translocation
N
H
Cyto-c/Caspase-3
pathway
Cl
IC50=8.42 ±0.34 μM
on HCT-116 cells
ROS
Apoptosis
IC50=9.96 ±0.66 μM
on MGC-803 cells

Melatonin Derivatives Combat with Inflammation-related
Cancer by Targeting the Main Culprit STAT3
Shumeng Ma1, §, Longqing Zhu1, §, Xiaohong Fan1, §, Tian Luo1, Dan Liu1, Ziyi Liang1,
Xiaoling Hu1, Tao Shi1, *, Wen Tan1, *, Zhen Wang 1, 2, *
1 School of Pharmacy, Lanzhou University, Lanzhou, 730000, China
2
State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou
University, Lanzhou, 730000, China
§ These authors made equal contributions to this work.
*Corresponding authors: zhenw@lzu.edu.cn; tanwen@lzu.edu.cn; shit18@lzu.edu.cn
Abstract
The combination between two well-studied bioactive compounds melatonin and salicylic
acid with proper modifications unexpectedly creates a sharp pair of “scissors” cutting off the
vicious connection between inflammation and cancer by targeting a key contributor Signal
Transducers and Activators of Transcription 3 (STAT3) in the two pathological processes. A
representative compound P-3 with IC50 values on each tested cell line ranging from 7.37 to 18.62
μM among the designed melatonin derivatives is equipped with the ability of curbing
inflammation-promoting cancer by down-regulating the expression, activation and nuclear
translocation of STAT3, breaking the feedforward loop of STAT3 activation by decreasing the
expression of pro-tumorigenic cytokines, and inducing cell apoptosis through ROS triggered
Cyto-c/Caspase-3 pathway. This study suggests that the melatonin derivative P-3 is likely to
become a promising chemical structure for developing the novel anti-cancer agents taking
effect through hindering the mutual-promoting processes between inflammation and cancer.
Keywords: melatonin; N-salicylic acid; inflammation-associated cancer; STAT3
1. Introduction
In human body as an organic whole, any pathological process can hardly be regarded as an
isolated event. In the past few decades, an increasing number of evidences revealed the intricate
and intertwined relationships between inflammation and cancer [1-4]. Chronic inflammation
induced by external factors such as bacterial infection [5], smoking [6], and high calorie diet [7],
are likely to contribute to gene instability or damage as well as proto-oncogene mutation,

further leading to tumorigenesis. It can also accelerate tumor development by provoking
immunosuppression and impairing anti-cancer immunity [5]. After tumor initiation, the
necrosis of cancer cells caused by a scarcity of blood supply or microbism as the result of
barrier deterioration may also elicit inflammation [8]. Such concomitant inflammation
contributes to the formation of tumor microenvironment constituted by diverse immune cells,
cancer cells and cytokines and chemokines they produced, which in turn drives the progression
of malignancy in most solid tumor [8-9]. Cancer cells may further amplify the tumor
microenvironment by recruiting more immune cells with chemokines, aggravating and
maintaining the inflammatory state [10]. Consequently, a chronic feedforward loop of
tumorigenesis and metastasis is established [11] (Figure 1).
A linchpin between the chronic inflammation and cancer is the Signal Transducers and
Activators of Transcription 3 (STAT3). To be specific, STAT3 as a nuclear transcription factor
persistently being activated in cancer cells and inflammatory cells in tumor microenvironment
is responsible for transducing cytoplasmic signals of extracellular stimuli and regulating genes
involved in tumor proliferation, survival, angiogenesis, invasion and additionally genes of
cancer-promoting inflammatory mediators [12-14]. The activation of STAT3 and its subsequent
consequences have been well-studied in many classic inflammation-dependent cancers such as
colorectal cancer and gastric cancer [15-18]. It has been demonstrated that during the process of
chronic gastrointestinal inflammation, the chance of tumorigenesis largely ascends due to the
activation of STAT3 as well as the production of pro-tumorigenic cytokines interleukin-6 (IL-6),
tumor necrosis factor (TNF-α), etc. [17-18]. Therefore, impeding the mutual-promoting
processes between chronic inflammation and cancer by suppressing the biological functions of
STAT3 or the expression of pro-tumorigenic cytokines in tumor microenvironment could be a
promising strategy to combat inflammation-related colorectal and gastric carcinoma [19-20].
Melatonin as a well-known hormone produced by pineal gland has diverse physiological
functions including affecting circadian rhythms [21], changing cellular redox status [22], and
anti-tumor [23]. Notably, it has been reported that melatonin exerts its multiple bioactivities,
especially its anti-tumor effect, partially via STAT3 pathway [24-27]. Another “old friend”
salicylic acid, which is famous for its derivative acetylsalicylic acid, a classical
anti-inflammation agent also known as Aspirin, once again amazed us with its novel derivatives
represented by S3I-201.1066 entitled with great anti-cancer activity by inhibiting STAT3
aberrant activation and functions in human cancer cells. [28-30] In our previous research, the
designed compounds following the combination strategy between melatonin and N-salicyloyl
derivatives had been proven that it exerted satisfying anti-neuroinflammatory activity by

inhibiting cyclooxygenase (COX), scavenging radicals, and inhibiting activation of glial cells
[31] (Figure 2). The modification also granted a lower acidity to these compounds compared to
the majority of non-steroidal anti-inflammatory drugs (NSAIDs) by shielding the acid group. In
consideration of a “reversed” pH gradient (intracellular pH is higher than the extracellular pH)
in most cancer cells compared to normal cells [32], and most anticancer drugs are ionizable, an
appropriate pKa trait of anti-cancer drugs, which determines the portion of drugs in a neutral
form (accessible to cancer focus and permeable into cancer cytomembrane), could enhance the
drugs’ efficiency [33]. Enlightened by the studies above, a series of modifications had been
done on the structure of melatonin and N-salicylic acid following the similar modification
strategy with the aim of making a combination of anti-cancer and anti-inflammation properties
from the two fragments (Scheme 1).
In short, based on firstly the potential anti-tumor and anti-inflammation activity of melatonin
N-salicyloyl derivatives, secondly the proper acidity of these compounds compared to classic
NSAIDs and thirdly the close connection between inflammation and cancer, the anti-tumor
effect of these compounds on inflammation-dependent cancers, especially on coloectal and
gastric carcinoma, was explored and a brief structure and activity relationship (SAR) was
concluded. Subsequently, the primary anti-tumor mechanisms of the representative compound
P-3 were studied with 5-FU set as a positive control. In addition, the toxicity and
pharmacokinetic profile of compound P-3 were evaluated, which may provide a guidance for
further structure optimization and hit-to-lead transformation. More significantly, this research
may provide us with an approach of thinking out of the box to discover novel
“anti-inflammatory anti-tumor agents”.
Figure 1. Relationships between inflammation and cancer.

2. Results and discussion
2.1. Chemical synthesis
The derivatives of melatonin were synthesized by employing a well-established
methodology displayed as follows [31] (Scheme 1). To be specific, a condensation reaction
was happened between N-salicylic acid derivatives and melatonin derivatives under the
presence of EDCI, HOBt and triethylamine. All compounds were identified by NMR and mass
spectrometry with 70%-90% overall yields and purity no less than 95.0% monitored by HPLC.
Compounds P-1 [34], P-2 [31], P-3 [34], P-4 [31], P-5 [35], P-6 [36], P-7 [37], P-8 [31] have
been reported previously, while compounds Q-1 to Q-7 have not been reported.

Comp.
R1
R2
R3
Comp
.
X
R1
R2
R3
P-1
P-2
OMe
H
H
Ph
Ph
Q-1
N
N
H
H
H
H
Me
Q-2
Q-3
P-3
H
H
3-Cl-2-Me-Ph
N
H
H
P-4
P-5
OMe
H
H
H
3-Cl-2-Me-Ph
Me
Q-4
Q-5
N
N
H
H
H
H
benzyl
3,4-di-OMe-phenethyl
P-6
P-7
P-8
H
H
Me
H
Me
Q-6
Q-7
N
N
H
H
H
H
4-hydroxybutyl
2,3-dimethylphenyl
2,3-dimethylphenyl
4-Me-benzenesulfonyl
OH
H
Scheme 1. Synthesis of melatonin derivatives.
2.2. Biological studies
2.2.1. Anti-proliferation activity and SARs
The anti-proliferation activity of compounds P-1 to P-8 and Q-1 to Q-7 were evaluated on
several inflammation-related cancer cell lines, which were gastric cancer cell lines (MGC-803,
SGC-7901, HGC-27, AGS), colon cancer cell line (HCT-116), breast cancer cell line (MCF-7),
hepatoma carcinoma cell line (HepG2), and lung cancer cell line (A549) using MTT assay with
5-FU as a positive control, and melatonin as a reference. The results indicated that most
compounds showed improved anti-proliferation activity towards tested cancer cell lines
compared to the parent compound melatonin especially on colon and gastric cancer cells (Table
1). Besides, the structure activity relationships (SARs) of the compounds with different
substituent groups on the nitrogen of N-salicylic acid fragment were that firstly compounds
with aliphatic substituents no matter a chain or a ring had a narrow anti-tumor spectrum than
those substituted by an aromatic ring; secondly, regarding the number of carbon atoms between
aromatic ring and the nitrogen atom, naught is better than one or two carbon atoms; thirdly, for
those whose substituents on aromatic ring with diverse electronic effects, a substituent with
electron-withdrawing effect in meta position (compound P-3) was better than a donating one
(compound P-7). Notably, compound P-3 exhibited the best anti-proliferation activity in
general towards the tested cell lines possibly because the fragment connected to melatonin-like
fragment is a tolfenamic acid, which has also been reported with anti-tumor activity [38-39].

However, retaining the methoxy group of melatonin (compound P-4) showed weaker
anti-proliferation activity than removing it (compound P-3). Thus, compound P-3 was selected
for further anti-cancer study on colon cancer cell HCT-116 and gastric cancer cell MGC-803.
Table 1. Anti-proliferation activity of compounds on different cancer cell lines.
IC50 ± SDa (μM)
Comp.
HCT-116
MCF-7
HePG-2
A549
MGC-803
SGC-7901
HGC-27
AGS
24.67±1.74
29.04±1.64
>30
>30
12.75±1.06
>30
>30
>30
P-1
17.23±0.72
8.42±0.34
20.40±0.72
>30
19.57±0.30
12.82±0.56
25.26±3.65
>30
21.27±0.28
7.37±0.90
>30
11.06±1.20
9.96±0.66
9.80±0.57
>30
>30
15.46±0.61
27.95±0.78
>30
>30
15.49±1.32
19.93±2.35
>30
14.7±1.98
9.12±0.42
16.02±1.19
>30
P-2
P-3
P-4
P-5
P-6
P-7
P-8
Q-1
Q-2
Q-3
Q-4
Q-5
Q-6
Q-7
18.62±1.44
21.93±0.98 17.78±0.76
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
16.60±3.93
14.42±2.92
22.24±0.96
>30
>30
>30
>30
>30
22.55±0.98
16.60±1.97
8.97±0.61
>30
>30
21.69±1.48
16.96±0.86
28.43±0.09
28.75±1.89
28.60±1.76
>30
>30
>30
>30
25.28±0.51
>30
16.46±0.47
>30
>30
26.96±0.32
28.28±1.18
19.26±4.61
26.74±0.28
19.93±0.69
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
6.10±2.17
>30
13.16±2.89
>30
>30
>30
>30
>30
29.15±2.01
>30
>30
>30
>30
>30
11.04±1.44
>30
5-FU
Melatonin
aMTT method; cells were treated with compounds at different concentrations for 72 h; the data were mean ± SD
(n=3).
To determine the appropriate dose of compound P-3 in subsequent assays, CCK8 test was
employed to collect comparatively accurate cell viability of HCT-116 and MGC-803 cells
treated by compound P-3 at different concentrations for 24 h and 48 h. In the 24 h viability
study (indicated by orange lines in Figure 3 ), the HCT-116 cells had high viability (cell
viability no less than 80%) at low compound P-3 concentration until the concentration was
above 36 μM, whereas MGC-803 cell could maintain high viability only when the
concentration was less than 20 μM. When exposed to compound P-3 for 48 h (indicated by blue
lines in Figure 3), the tested concentrations less or around 20 μM and 10 μM could keep
HCT-116 cells and MGC-803 cells respectively in a high viability state.

A
B
Figure 3. Effects of compound P-3 on cell viability of HCT-116 and MGC-803 cells. (A) Cell viability of
HCT-116 cells exposed to compound P-3 at different concentrations for 24 h or 48 h (B) Cell viability of
MGC-803 cells exposed to compound P-3 at different concentrations for 24 h or 48 h.
2.2.2. Compound P-3 inhibited the proliferation of HCT-116 and MGC-803 cells and arrested the
cell cycle of HCT-116 at G0/G1 phase
Human tumor Clonogenic Assays (HTCA) is a common method utilized to predict the
anti-proliferation activity of drugs in in vitro drug screening. Within a short time period, a single
cancer cell could rapidly proliferate to form a colony of daughter cells, which is large enough to be
observed with bare eyes after being stained by crystal violet dye. Thus, the proliferation ability of
cancer cells can be evaluated by counting the number of colonies larger than a specific threshold.
The outcomes showed that compound P-3 exerted inhibitory effects on the clonogenicity process of
both HCT-116 and MGC-803 cells in a concentration-dependent manner (Figure 4A and 4B).
EdU(5-ethynyl-2’-deoxyuridine), a terminal alkyne-containing nucleoside analog, can
incorporate into DNA during the progress of DNA synthesis due to its similarity to thymidine. The
alkyne of EdU can react with an azide-containing fluorochrome in a Cu (I)-catalyzed reaction,
forming a stable covalent bond. The detection of fluorescent signals of incorporated EdU in DNA
provides the information on cell proliferation and the proportion of cells in S-phase [40]. The results
indicated that compound P-3 could decrease the percentage of cells at S-phase, which indicated its
inhibitory effect on cell proliferation of HCT-116 (Figure 4C) and MGC-803 (Figure 4D).

A
B
C
D
Figure 4. Effect of compound P-3 on the proliferation of HCT-116 and MGC-803 cells. (A). Effect of
compound P-3 on clonogenicity of HCT-116 cells at different concentrations after 9-days treatment. (B).
Effect of compound P-3 on clonogenicity of MGC-803 cells at different concentrations after 6-days
treatment. (C) EdU proliferation assay of HCT-116 cells (magnification 20 ×, scale bar = 100 μm) treated
with compound P-3 at different concentrations for 24 h. (D) EdU proliferation assay of MGC-803 cells
(magnification 10 ×, scale bar = 200 μm) treated with compound P-3 at different concentrations for 24 h.
Data are presented as mean ± SD from three independent experiments. ** P<0.01, *** P<0.001 versus
the control group.
Next, HCT-116 and MGC-803 cells treated with compound P-3 were subjected to cell cycle assay
via flow cytometry (FCM) based on propidium iodide (PI) staining method. As depicted in Figure
5A, the cell cycle of HCT-116 cells was arrested at G0/G1 phase by compound P-3 with the portion
of this phase growing from 70.45% to 84.74% as concentrations of P-3 rose from 0 to 20 μM.
Nevertheless, no obvious effect on cell cycle was detected on MGC-803 cells as can be seen in the
Supporting Information.

A
Figure 5. (A) Cell cycle distribution of HCT-116 cells treated with compound P-3 at different
concentrations for 48 h and the quantitative analysis of the cell cycle distribution at each phase. Data are
presented as mean ± SD from three independent experiments. * P < 0.05, ** P<0.01, *** P<0.001 versus
the control group.
2.2.3. Compound P-3 induced cell apoptosis on HCT-116 cells
To determine the apoptosis-inducing effect of compound P-3, firstly flow cytometry (FCM) was
applied with two fluorescent dyes PI and Annexin-V. A concentration-dependent (2.5 μM, 5 μM, 10
μM, 20 μM) increase (from 2.14% to 42.50%) appeared on the portion of early stage apoptosis of
HCT-116 (Figure 6A). In comparison, the apoptosis-inducing effect on MGC-803 cells was quite
weak (Supporting Information), which means the cytotoxicity effect of compound P-3 on
MGC-803 cells might not exert by inducing apoptosis only.
Afterwards, Hoechst staining and Giemsa staining assays were performed on HCT-116 cells by
treating the cells with compound P-3 at different concentrations (1.25 μM, 2.5 μM, 5 μM, 10 μM
and 2.5 μM, 5 μM, 10 μM, 15 μM respectively). The Hoechst staining assay further verified that
compound P-3 was able to induce the apoptosis of HCT-116 cells indicated by the increased
percentage of apoptotic cells with twinkling cell nuclei stained by Hoechst 33342 (Figure 6B).
Consistently, HCT-116 cells in Giemas staining assay showed that HCT-116 cells appeared classic
morphological changes of apoptosis such as apoptosis body formation, chromatin pyknosis and
plasmatorrhexis compared to the control group, in which the chromatin distributed evenly in cell
nuclei and the cytomembranes were integrated (Figure 6C).

A
B
C
Figure 6. Apoptosis analysis of HCT-116 cells induced by compound P-3. (A) Percentage of apoptotic
HCT-116 cells treated with compound P-3 at different concentrations for 48 h detected by flow cytometry.
(B) Percentages of apoptotic HCT-116 cells treated with P-3 at different concentrations for 48 h
visualized by Hoechst 33342 staining (magnification 20 ×, scale bar = 100 μm). The cell nuclei of
apoptotic HCT-116 cells in Hoechst staining assay were stained with light blue fluorescence. (C)
Apopotic morphological changes of HCT-116 cells induced by the treatment of compound P-3 at
different concentrations for 48 h (magnification 40 ×, scale bar = 100 μm). Red arrow denotes apoptotic
cells characterized by cell shrinkage, condensed chromatin, apoptotic body formation and cytoskeleton
disintegration. Data are presented as mean ± SD from three independent experiments. * P < 0.05, **
P<0.01, *** P<0.001 versus the control group.
2.2.4. Compound P-3 weakened the invasion and migration ability of HCT-116 and MGC-803 cells
As cancer cells undergo metastasis (invasion and migration of a new tissue), they penetrate and
attach to the basal matrix of target tissue, allowing the cancer cells to form a focus in deep tissues or
transfer to another tissues to form a new tumor focuses, which is a crucial trick played by most
cancer cells against cancer therapies [41]. In order to test the effect of compound P-3 on the invasion
and migration of cancer cells, transwell chambers were used. Seduced by high FBS-containing
medium in the lower chambers, the vast majority of serum-starved HCT-116 cells and MGC-803
cells moved towards the lower chamber through polycarbonate membrane in the migration assay or

through polycarbonate membrane covered with matrigel in the invasion assay. In comparison, after
being treated with compound P-3, both cell lines demonstrated an impaired mobility in two assays
in a concentration-dependent manner as shown in Figure 7A and Figure 7B.
A
A
B
Figure 7. Effect of compound P-3 on the metastasis of HCT-116 and MGC-803 cells. (A) Inhibitory
effect on the invasion and migration ability of HCT-116 cells treated with compound P-3 at different
concentrations for 36 h. (magnification 10 ×, scale bar = 100 μm) (B) Inhibitory effect on invasion and
migration ability of MGC-803 cells treated with compound P-3 at different concentrations for 24 h.
(magnification 10 × and scale bar = 200 μm for invasion; magnification 20 × and scale bar = 100 μm for
migration). Data are presented as mean ± SD from three independent experiments. * P < 0.05, ** P<0.01,
*** P<0.001 versus the control group.
2.2.5. Compound P-3 down-regulated the expression of pro-tumorigenic cytokines, STAT3, Bcl-2
and inhibited the nuclear translocation of STAT3
In tumor microenvironment, various pro-inflammatory cytokines such as IL-6, IL-10, IL-21,
IL-23, produced by immune cells can bind to and activate the cell surface receptors, recruiting
members of the Janus kinase (JAK) family to act as intermediaries for activation of STAT3.
JAK-mediated phosphorylation of an invariant tyrosine residue on the receptor-bound STAT3
monomer induces the phosphorylated STAT3 (p-STAT3) monomers to associate with each other.
The activated dimers then translocate into the nucleus and bind to specific DNA-response elements
in the promoters of target genes and thereby regulate the expression of certain genes such as IL-6,
IL-17, B-cell lymphoma-x (Bcl-x), B-cell lymphoma-2 (Bcl-2), and vascular endothelial growth
factor (VEGF), which are related to tumor proliferation, survival, angiogenesis, and invasion [42].

In this process, IL-6 as both the activator and the targeted gene of STAT3 contributes to the form of
a feedforward loop of STAT3 activation in tumor-elicited inflammation [43]. In addition, TNF-α is
also an important pro-tumorigenic inflammatory cytokine that have been shown to participate in
both the initiation and progression of cancer [17]. Taking the significant role of IL-6 and TNF-α in
tumor-elicited inflammation into consideration, the effect of compound P-3 on the two cytokines
was evaluated with ELISA kits. After treated with compound P-3 for 24 h at the concentrations of 5,
10, 15 and 20 μM, the cell supernatant of mouse mononuclear macrophage RAW 264.7 was
collected for ELISA. According to the result, compound P-3 decreased the release of IL-6 and
TNF-α in RAW 264.7 in a concentration-dependent manner (Figure 8A), which might mitigate the
overactivation of STAT3 and hinder the process of tumorgenesis.
To further study the direct influence of compound P-3 on STAT3, western blot assay was
performed, which displayed an obvious decrease on the expression of STAT3, phosphorylated
STAT3 and STAT3 regulated gene Bcl-2 in MGC-803 cells and HCT-116 cells (Figure 8B). In
addition, nuclear translocation of STAT3 is an indispensable process in order to regulate the
expression of tumorgenesis-related genes. Therefore, the effect of compound P-3 on the nuclear
translocation of STAT3 was detected by Hoechst dye and fluorochrome-conjugated antibody. From
the photographed pictures of HCT-116 and MGC-803 cells exposed to compound P-3 for 3 h, an
obvious inhibitory effect on the nuclear translocation of STAT3 was observed in both cell lines.
Moreover, in consistent with western blot assay, a decline in gross amount of STAT3 in compound
P-3-treated group was also observed from the comparatively sparse STAT3 indicated by red
fluorescence compared to the control group (Figure 8C).

A
B
C
Figure 8. (A) Compound P-3 down-regulated the levels of IL-6 and TNF-α in RAW 264.7. The cell
viability of RAW 264.7 is higher than 80% under the treatment of compound P-3 at indicated
concentrations. (B) Compound P-3 suppressed the expression of STAT3, phosphorylated STAT3, and
Bcl-2 in both HCT-116 and MGC-803 cells. (C) Compound P-3 inhibited the nuclear translocation and
the total amount of STAT3 in both HCT-116 and MGC-803 cells. Data are presented as mean ± SD from
three independent experiments. * P < 0.05, ** P<0.01, *** P<0.001 versus the control group.
2.2.6. Docking study revealing the possible interaction model between compound P-3 and STAT3
To predict the interaction model between compound P-3 and STAT3 in Src Homology 2 (SH2)
domain, which plays an essential role in the process of STAT3 phosphorylation and following
dimerization, a docking study was carried out based on the crystal structure of STAT3 homo dimer
(PDB code: 1BG1) . As can be seen in Figure 9, bending from the linker amide bond, the

melatonin-like part could reach to a hydrophilic sub-pocket of the SH2 domain (composed of
ARG609, SER61l, SER613 and LYS591), while the other tolfenamic acid part could access to a
relative hydrophobic sub-pocket (woven by ILE597, ILE634, LYS592, ARG595). In this process,
two kinds of interaction forces played important roles, which were hydrogen bond interaction and
p-π interaction. To be specific, amide carbonyl formed one strong hydrogen bond (2.45 Å) between
the CO and LYS591 and the other strong hydrogen bond (2.27 Å) between the NH and SER636,
locating the center of P-3 in between the two adjacent sub-pockets. Further, a p-π interaction (3.52 Å)
between the indole ring of the melatonin-like part and LYS591 contributed to the binding process. In
general, the amide bond between melatonin-like part and salicylic derivatives is crucial for
anchoring the compound into the pocket of SH2 domain and all these interactions rationalized the
effect of the compound P-3 on STAT3.
Figure 9. 3D diagram of compound P-3 inserting into the SH2 domain of STAT3. (A) Residues in the
active site were displayed in line style. Compound P-3 was represented with sticks (turquoise). The dash
lines showed the potential hydrogen bond (yellow), p-π conjugated bond (green) with bond length
indicated by purple numbers. (B) 3D structure of the protein was presented in surface and several
residues were displayed. P-3 was represented with sticks (Pink). Arrows denoted potential binding
sub-pockets accessed by P-3.
2.2.7. Compound P-3 induced HCT-116 cells apoptosis via ROS-triggered Cyto-c/Caspase-3
pathway
Reactive oxygen species (ROS) is a double-edged sword to cancer cells. In lower content, it
functions as signal molecules driving the proliferation and other events required for tumor
progression. That is the reason why transformed cells appear to generate more ROS than do normal
cells. Nevertheless, evidence also showed that diverse cancer chemotherapeutic agents may be
selectively toxic to tumor cells by augmenting oxidant stress and pushing these already stressed
cells beyond their limit [44]. Hence, triggering ROS explosion could be a valuable strategy to
initiate the apoptosis of cancer cells. Marked by fluorescent probe DCFH-DA, the cells undergoing
ROS explosion can be observed under microscope. The results indicated that the degree and extent

of ROS explosion drastically increased with the rise of the dosage of compound P-3 in HCT-116
cells as displayed in Figure 10A.
Bcl-2 residues in the permeability transition pore (PT pore) of mitochondria while
Bcl-2-associated X (Bax) scatters at the mitochondria matrix. When mitochondria subjected to
stimuli, such as overwhelming ROS, Bax could promote the opening of PT pore, allowing
cytochrome c, an apoptosis signal transducer, to release from the mitochondria into cytoplasm to
activate the caspase cascade, inducing cell apoptosis. On the contrary, Bcl-2 could impede the
opening of PT pore to antagonize the apoptosis effect of Bax [45]. Western blot assay revealed that
compound P-3 was capable of down-regulating the expression of Bcl-2, Cyto-c and cleaved
caspase-3 and up-regulated the expression of Bax (Figure 10B). Based on all the findings above,
another anti-tumor mechanism of compound P-3 on HCT-116 cells could be through ROS triggered
Cyto-c/Caspase-3 pathway.
A
B
Figure 10. (A) Compound P-3 triggered the explosion of ROS in HCT-116 cells. (magnification 20 ×,
scale bar = 100 μm) (B) P-3 regulated the expression of proteins involved in Cyto-c/Caspase3 pathway.
Data are presented as mean ± SD from three independent experiments. * P < 0.05, ** P<0.01, ***
P<0.001 versus the control group.
2.2.8. Acute toxicity study of compound P-3 on mice
Acute toxicity evaluation was performed with compound P-3 on three female and three male
Kunming mice weighing form 19 to 22 g. Compound P-3 at the dose of 1000 mg/kg was given by
intragastric administration in consideration of the focused gastrointestinal tumor in this study. In the
following 14 days, all the mice survived and in a good condition that the hairs, feeding frequency
and amount, activity were as normal. Therefore, compound P-3 can be regarded as a comparatively

safe compound with oral LD50 value higher than 1000 mg/kg.
2.2.9. Pharmacokinetic study of compound P-3 on rats
The pharmacokinetic property of compound P-3 was evaluated on five male Sprague-Dawley
(SD) rats weighing 300 ± 10g by intragastric administration at the dosage of 100mg/kg. The
pharmacokinetic parameters were shown in Table 2 and the pharmacokinetic curve can be seen in
Figure 11, which indicated that compound P-3 quickly reached its peak concentration at 0.1495
μg/mL one hour later after oral administration. The concentration of compound P-3 declined by half
7.58 hours later after peaking, which displayed a comparatively long time-span of action for
compound P-3.
Table 2. Pharmacokinetic parameters of compound P-3 obtained from serum samples of five SD rats
subjected to intragastric administration of compound P-3 at the dosage of 100mg/kg.
Parametera t1/2 Tmax
Cmax
0.1495
μg/mL
AUC0-t AUC0-inf_obs AUC0-t/o-inf_obs
Cl/F_obs
51.75
P-3
7.58
h
1.00
h
1.67
1.93
0.87
-
Unit
μg/mL*h
μg/mL*h
(mg/kg)/(μg/mL)/h
a
The analyzing method was Linear Trapezoidal in a non-compartmental model using the
PKsolver2.0 computer program.
Figure 11. Pharmacokinetic curve of compound P-3 obtained from serum samples of five SD rats
subjected to intragastric administration of compound P-3 at the dosage of 100 mg/kg. Data for
compound P-3 was shown as the mean ± SD, n = 5.
3. Conclusion
To conclude, inspired by the vital role of inflammation in tumor initiation and progression,

potential anti-cancer and anti-inflammation effects of melatonin derivatives, and the merit of proper
acidity of the compounds achieved by combination strategy, a series of melatonin derivatives were
designed and synthesized to explore their anti-tumor effect on inflammation-dependent cancers.
The results displayed the great anti-proliferation effect of these compounds especially for
compound P-3 with IC50 values on each tested cell line ranging from 7.37 to 18.62 μM. In
subsequent research, favorable effects of compound P-3 on decreasing the release of
pro-tumorigenic cytokines IL-6 and TNF-α in immune cells RAW 264.7, down-regulating the
expression of STAT3 and inhibiting the activation of STAT3 in HCT-116 and MGC-803 cells,
blocking the nuclear translocation of STAT3 in HCT-116 and MGC-803 cells to curb
inflammation-induced cancer were also disclosed. Additionally, another ROS-dependent
mechanism of compound P-3 to induce the apoptosis of HCT-116 cells was also explored. All in all,
the strategy of obstructing the progression from chronic inflammation to cancer by crushing the
pivotal participators in the mutual-promotion processes between the two diseases has been proven
to be feasible and is well worth further practicing and developing.
4. Experiment
4.1. Chemistry
All chemicals used were of reagent grade. Proton (1H) and carbon (13C) NMR spectra (400 or 300
1
MHz for H-NMR; 101 MHz for 13C-NMR) were recorded on a Bruker spectrometer (Bruker
Company, Germany). NMR spectra used DMSO-d6, CDCl3 or acetone-d6 as solvent (TMS as the
internal standard). Proton chemical shifts are reported relative to a residual solvent peak (CDCl3 at
7.26 ppm, DMSO-d6 at 2.50 ppm, acetone-d6 at 2.05 ppm). Carbon chemical shifts are reported
relative to a residual solvent peak (CDCl3 at 77.00 ppm, DMSO-d6 at 39.96 ppm, acetone-d6 at
29.70 ppm). The values of the chemical shifts are expressed in ppm and the coupling constants (J) in
hertz. The following abbreviations were used to designate multiplicities: s = singlet, d = doublet, t =
triplet, q = quartet, quint = quintet, m = multiplet, br = broad. High-resolution mass spectrometry
(HRMS) analysis was performed using a Bruker Daltonics Apex II 47e Specification (Bruker
Company, Germany).
4.2. HPLC analysis
The purity was determined by high performance liquid chromatography (HPLC). Purity of all
final compounds was more than 95% (Agilent 1260 Infinity II; USA). The column was Eclipse Plus
C18 (4.6 × 150 mm, 4 µm). Chromatographic conditions for all compounds: Mobile phase: 0-7 min,
MeOH:H2O = 20:80; 7-16 min MeOH:H2O = 95:5, 16-25 min MeOH:H2O = 20:80; Wavelength:

254 nm; Column temperature: 25 °C; Flow rate of 0.5 mL/min.
4.3. General procedure for the preparation of compounds
A mixture of ortho-iodo benzoic acid (1.0 mmol, 1.0 eq), amine (1.2 mmol, 1.2 eq), K2CO3 (1.0
mmol, 1.0 eq), copper powder (10 mol%) and DMF (2.0 mL) were stirred at 100 °C in the schlenk
tube for 12 h under argon condition, and then cooled to room temperature. The dark green solid was
diluted with water and acidified with HCl until pH of 2 was reached. A large amount of black solid
was precipitated. The suspension was filtered, washed with an excess of water to remove amine and
then filter cake was dissolved in dichloromethane. The solvent was removed by rotary evaporation
under reduced pressure and dried over sodium sulfate and concentrated. The crude compounds,
o-aminobenzoic acid derivatives, were obtained without further purification. Then o-aminobenzoic
acid derivatives (1 mmol, 1.0 eq), tryptamine (1 mmol, 1.0 eq), EDCI·HCl (1.2mmol, 1.2 eq),
HOBT (1.2mmol, 1.2 eq) were dissolved in dichloromethane (5 mL) at 0 °C, then Et3N (2.5 eq) was
added. The reaction system was then increased to room temperature and stirred for 8 h. After
completion of the reaction detected by TLC, the reaction liquid was removed by rotary evaporation
under reduced pressure. Then the resulting residue was purified by silica gel flash column
chromatography to afford the desired product as a solid and the total yield in two steps was 50-90%.
4.3.1. N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-2-(phenylamino)benzamide (P-1)
white solid (75% yield), mp 121–122 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.67 (s, 1H, CNHC), 9.77
(s, 1H, CNHCH), 8.69 (t, J = 5.4 Hz, 1H, NHCO), 7.63 (d, J = 8.0 Hz, 1H, arom.), 7.37 – 7.26 (m, 4H,
arom.), 7.23 (d, J = 8.7 Hz, 1H, arom.), 7.16 (s, 1H, NHCHC), 7.14 (s, 2H, arom.), 7.07 (d, J = 2.0 Hz,
arom.), 6.97 (t, J = 7.3 Hz, 1H, arom.), 6.85 – 6.77 (m, 1H, arom.), 6.71 (dd, J = 8.7, 2.2 Hz, 1H,
3.73 (s, 3H, OCH3), 3.53 (dd, J = 13.2, 6.8 Hz, 2H, CH2CH2NH), 2.92 (t, J = 7.3 Hz, 2H, CCH2CH2).
NMR (101 MHz, DMSO-d6) δ 169.21 (NHCO), 153.44 (arom.), 144.71 (arom.), 141.99 (arom.),
(arom.), 131.83 (arom.), 129.87 (arom.), 129.25 (arom.), 128.07 (arom.), 123.77 (arom.), 122.17
119.83 (arom.), 119.36 (arom.), 118.49 (arom.), 115.36 (arom.), 112.48 (arom.), 112.15 (arom.), 111.55
(arom.), 100.59 (arom.), 55.71 (CH3N), 40.51(CH2CH2NH), 25.49 (CCH2CH2). HRMS (ESI-TOF) m/z:
[M + H]+ Calcd for C24H24N3O2 386.1863; found 386.1867.
4.3.2. N-(2-(1H-indol-3-yl)ethyl)-2-(methyl(phenyl)amino)benzamide (P-2)
white solid (50% yield), mp 103–105 °C. 1H NMR (400 MHz, CDCl3) δ 8.33 – 8.25 (m, 1H, CNHCH),
8.21 (dd, J = 7.8, 1.7 Hz, 1H, NHCO ), 7.63 (d, J = 8.2 Hz, 1H, arom.), 7.54 (d, J = 7.9 Hz, 1H, arom.),
7.37 (td, J = 7.6, 1.8 Hz, 1H, arom.), 7.33 – 7.24 (m, 2H, arom.), 7.23 – 7.13 (m, 3H, arom., NHCHC),

7.09 – 7.04 (m, 1H, arom.), 6.98 (dd, J = 7.8, 1.1 Hz, 1H, arom.), 6.91 (t, J = 7.3 Hz, 1H, arom.), 6.63 –
6.50 (m, 3H, arom.), 3.77 (q, J = 6.5 Hz, 2H, CH2CH2NH), 2.92 (t, J = 6.6 Hz, 2H, CCH2CH2), 2.69 (s,
3H, CH3). 13C NMR (101 MHz, CDCl3) δ 165.71 (NHCO), 149.12 (arom.), 148.13 (arom.), 136.45
(arom.), 132.44 (arom.), 131.45 (arom.), 130.94 (arom.), 128.95 (arom.), 127.72 (arom.), 126.95
(arom.), 126.53 (arom.), 122.37 (arom.), 122.01 (arom.), 119.74 (arom.), 119.27 (arom.), 118.74
(arom.), 116.27 (arom.), 112.44 (arom.), 111.23 (arom.), 40.24 (CH3N), 39.32 (CH2CH2NH), 24.87
(CCH2CH2). HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C24H24N3O 370.1914; found 370.1910.
4.3.3. N-(2-(1H-indol-3-yl)ethyl)-2-((3-chloro-2-methylphenyl)amino)benzamide (P-3)
1
white solid (90% yield), mp 164–165 °C. H NMR (400 MHz, DMSO-d6) δ 10.84 (s, 1H, CNHCH),
9.79 (s, 1H, CNHC), 8.75 (t, J = 5.4 Hz, 1H, NHCO), 7.66 (d, J = 7.8 Hz, 1H, arom.), 7.58 (d, J = 7.8
Hz, 1H, arom.), 7.37 – 7.30 (m, 2H, arom.), 7.29 (d, J = 5.7 Hz, 1H, arom.), 7.21 (s, 1H, arom.), 7.19 (s,
1H, NHCHC), 7.15 (t, J = 8.0 Hz, 1H, arom.), 7.07 (t, J = 7.5 Hz, 1H, arom.), 7.04 – 6.95 (m, 2H,
arom.), 6.83 (t, J = 7.5 Hz, 1H, arom.), 3.56 (dd, J = 13.3, 7.0 Hz, 2H, CH2CH2NH), 2.98 (t, J = 7.4 Hz,
2H, CCH2CH2), 2.29 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 169.27 (NHCO), 145.15 (arom.),
142.00 (arom.), 136.69 (arom.), 134.80 (arom.), 132.29 (arom.), 129.15 (arom.), 127.92 (arom.),
127.82 (arom.), 127.73 (arom.), 123.58 (arom.), 123.09 (arom.), 121.36 (arom.), 119.28 (arom.),
118.97 (arom.), 118.71 (arom.), 118.67 (arom.), 118.48 (arom.), 115.32 (arom.), 112.28 (arom.),
111.82 (arom.), 40.52 (CH2CH2NH), 25.42 (CCH2CH2), 14.99 (CH3). HRMS (ESI-TOF) m/z: [M +
Na]+ Calcd for C24H22ClNaN3O 426.1524; found 426.1363.
4.3.4. 2-((3-chloro-2-methylphenyl)amino)-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-ben-zamide (P-4)
white solid (85% yield), mp 177–178 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.65 (s, 1H, CNHCH),
9.80 (s, 1H, CNHC), 8.72 (t, J = 5.7 Hz, 1H, NHCO), 7.65 (d, J = 7.3 Hz, 1H, arom.), 7.31 (t, J = 7.5
Hz, 1H, arom.), 7.25 (d, J = 7.7 Hz, 1H, arom.), 7.21 (t, J = 6.0 Hz, 1H, arom.), 7.18 (s, 1H, NHCHC),
7.13 (dd, J = 9.4, 4.6 Hz, 2H, arom.), 7.07 (d, J = 1.7 Hz, 1H, arom.), 7.01 (d, J = 8.2 Hz, 1H, arom.),
6.82 (t, J = 7.3 Hz, 1H, arom.), 6.70 (dd, J = 8.7, 2.1 Hz, 1H, arom.), 3.73 (s, 3H, OCH3), 3.53 (dd, J =
13.6, 6.8 Hz, 2H, CH2CH2NH), 2.92 (t, J = 7.4 Hz, 2H, CCH2CH2), 2.27 (s, 3H, CH3). 13C NMR (101
MHz, DMSO-d6) δ 169.23 (NHCO), 153.40 (arom.), 145.14 (arom.), 141.97 (arom.), 134.76 (arom.),
132.30 (arom.), 131.79 (arom.), 129.13 (arom.), 128.03 (arom.), 127.82 (arom.), 123.73 (arom.),
123.54 (arom.), 119.21 (arom.), 118.86 (arom.), 118.47 (arom.), 115.27 (arom.), 112.42 (arom.),
112.08 (arom.), 111.52 (arom.), 100.56 (arom.), 55.67 (OCH3), 40.45 (CH2CH2NH), 25.44 (CCH2CH2),
14.92 (CH3). HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C25H25ClN3O2 434.1630; found 434.1627.
4.3.5. N-(2-(1H-indol-3-yl)ethyl)-2-(methylamino)benzamide (P-5)
1
white solid (75% yield), mp 114–115 °C. H NMR (400 MHz, DMSO-d6) δ 10.83 (s, 1H, CNHCH),
8.43 (t, J = 5.3 Hz, 1H, NHCO), 7.70 (q, J = 5.1 Hz, 1H, arom.), 7.59 (d, J = 7.8 Hz, 1H, arom.), 7.52
(d, J = 7.8 Hz, 1H, arom.), 7.35 (d, J = 8.0 Hz, 1H, arom.), 7.28 (t, J = 7.7 Hz, 1H, arom.), 7.18 (s, 1H,
NHCHC), 7.07 (t, J = 7.4 Hz, 1H, arom.), 6.99 (t, J = 7.3 Hz, 1H, arom.), 6.62 (d, J = 8.3 Hz, 1H,
CNHCH3), 6.55 (t, J = 7.4 Hz, 1H, arom.), 3.50 (q, J = 7.0 Hz, 2H, CH2CH2NH), 2.94 (t, J = 7.5 Hz,
2H, CCH2CH2), 2.77 (d, J = 5.0 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 169.52 (NHCO),

150.54 (arom.), 136.71 (arom.), 132.69 (arom.), 128.59 (arom.), 127.74 (arom.), 123.05 (arom.),
121.38 (arom.), 118.78 (arom.), 118.68 (arom.), 115.72 (arom.), 114.33 (arom.), 112.40 (arom.), 111.84
(arom.), 110.91 (arom.), 40.32 (CH2CH2NH), 29.74 (CH3), 25.65 (CCH2CH2). HRMS (ESI-TOF) m/z:
[M + H]+ Calcd for C18H20N3O 294.1601; found 294.1603.
4.3.6. N-(2-(1H-indol-3-yl)ethyl)-2-(dimethylamino)benzamide (P-6)
1
white solid (50% yield), mp 167–169 °C. H NMR (400 MHz, DMSO-d6) δ 10.88 (s, 1H, CNHCH),
9.13 (t, J = 5.2 Hz, 1H, NHCO), 7.66 (dd, J = 7.7, 1.7 Hz, 1H, arom.), 7.60 (d, J = 7.8 Hz, 1H, arom.),
7.39 – 7.33 (m, 2H, arom.), 7.23 (d, J = 2.2 Hz, 1H, arom.), 7.13 – 6.96 (m, 4H, arom., NHCHC), 3.62
(q, J = 7.0 Hz, 2H, CH2CH2NH), 2.97 (t, J = 7.1 Hz, 2H, CCH2CH2), 2.45 (s, 6H, N(CH3)2). 13C NMR
(101 MHz, DMSO-d6) δ 167.07 (NHCO), 151.94 (arom.), 136.81 (arom.), 131.53 (arom.), 130.43
(arom.), 128.07 (arom.), 127.63 (arom.), 123.37 (arom.), 122.40 (arom.), 121.43 (arom.), 119.22
(arom.), 118.81 (arom.), 118.70 (arom.), 112.12 (arom.), 111.83 (arom.), 44.30 (N(CH3)2), 40.01
(CH2CH2NH), 25.19 (CCH2CH2). HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C19H22N3O 308.1757;
found 308.1759.
4.3.7. N-(2-(1H-indol-3-yl)ethyl)-2-((2,3-dimethylphenyl)amino)benzamide (P-7)
1
white solid (85% yield), mp 143–145 °C. H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H, CNHCH),
9.66 (s, 1H, CNHC), 8.69 (t, J = 5.6 Hz, 1H, NHCO), 7.66 – 7.57 (m, 2H, arom.), 7.35 (d, J = 8.1 Hz,
1H, arom.), 7.26 – 7.23 (m, 1H, arom.), 7.21 (d, J = 2.2 Hz, 1H, arom.), 7.13 – 7.04 (m, 3H, arom.,
NHCHC), 7.01 – 6.96 (m, 1H, arom), 6.92 (d, J = 7.0 Hz, 1H, arom), 6.87 – 6.83 (m, 1H, arom), 6.75 –
6.70 (m, 1H, arom), 3.57 (dd, J = 13.4, 7.1 Hz, 2H, CH2CH2NH ), 2.99 (t, J = 7.4 Hz, 2H, CCH2CH2),
2.27 (s, 3H, CH3), 2.12 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 169.52 (NHCO), 146.63
(arom.), 139.80 (arom.), 138.13 (arom.), 136.72 (arom.), 132.28 (arom.), 129.83 (arom.), 129.07
(arom.), 127.77 (arom.), 126.31 (arom.), 125.49 (arom.), 123.11 (arom.), 121.40 (arom.), 120.10
(arom.), 118.77 (arom.), 118.71 (arom.), 117.75 (arom.), 117.21 (arom.), 114.38 (arom.), 112.35
(arom.), 111.86, 40.51, 25.52, 20.78, 14.06. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C25H26N3O
384.2070; found 384.2074.
4.3.8. 2-((2,3-dimethylphenyl)amino)-N-(2-(5-hydroxy-1H-indol-3-yl)ethyl)ben-zamide (P-8)
1
white solid (75% yield), mp 139–140 °C. H NMR (400 MHz, DMSO-d6) δ 10.49 (d, J = 1.6 Hz, 1H,
CNHCH), 9.62 (s, 1H, CNHC), 8.64 (t, J = 5.6 Hz, 1H, NHCO), 8.60 (s, 1H, OH), 7.64 (dd, J = 7.9, 1.3
Hz, 1H, arom.), 7.26 – 7.21 (m, 1H, arom.), 7.13 (d, J = 8.6 Hz, 1H, NHCHC), 7.09 (dt, J = 11.0, 5.3
Hz, 3H, arom.), 6.92 (dd, J = 9.3, 4.5 Hz, 2H, arom.), 6.85 – 6.81 (m, 1H, arom.), 6.75 – 6.70 (m, 1H,
arom.), 6.61 (dd, J = 8.6, 2.3 Hz, 1H, arom.), 3.52 (dd, J = 14.4, 6.4 Hz, 2H, CH2CH2NH), 2.92 – 2.85
(m, 2H, CCH2CH2), 2.27 (s, 3H, CH3), 2.12 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 169.45
(NHCO), 150.65 (arom.), 146.67 (arom.), 139.79 (arom.), 138.12 (arom.), 132.25 (arom.), 131.29
(arom.), 129.92 (arom.), 129.03 (arom.), 128.38 (arom.), 126.29 (arom.), 125.52 (arom.), 123.52
(arom.), 120.24 (arom.), 117.71 (arom.), 117.19 (arom.), 114.33 (arom.), 112.12 (arom.), 111.76
(arom.), 111.31 (arom.), 102.74 (arom.), 40.28 (CH2CH2NH), 25.67 (CCH2CH2), 20.73 (CH3), 14.04
(CH3). HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C25H26N3O2 300.2020; found 400.2017.

4.3.9. N-(2-(1H-indol-3-yl)ethyl)-2-(cyclopropylamino)benzamide (Q-1)
yellow solid (63% yield), mp 128.2-130.1 °C. 1H NMR (400 MHz, CDCl3) δ 8.14 (s, 1H, CNHCH),
7.70 (s, 1H, NHCO), 7.63 (d, J = 7.8 Hz, 1H, arom.), 7.36 (d, J = 8.1 Hz, 1H, arom.), 7.29 (t, J = 7.7
Hz, 1H, arom.), 7.21 (t, J = 7.5 Hz, 1H, arom. ), 7.12 (q, J = 6.0, 5.6 Hz, 3H, NHCHC, arom.), 7.02 (s,
1H, arom.), 6.53 (t, J = 7.5 Hz, 1H, arom.), 6.11 (t, J = 5.6 Hz, 1H, NHCH(CH2)2), 3.72 (q, J = 6.3 Hz,
2H, NHCH2CH2), 3.05 (t, J = 6.6 Hz, 2H, CCH2CH2), 2.44 – 2.36 (m, 1H, NHCH(CH2)2), 0.74 (q, J =
6.2 Hz, 2H, (CH(CH2)2) 0.56 – 0.48 (m, 2H, (CH(CH2)2). 13C NMR (101 MHz, CDCl3) δ 169.55
(NHCO), 150.05 (arom.), 136.26 (arom.), 132.32 (arom.), 127.08 (arom.), 126.85 (arom.), 122.12
(arom.), 121.99 (arom.), 119.38 (arom.), 118.62 (arom.), 115.15 (arom.), 115.05 (arom.), 112.97
(arom.), 112.83 (arom.), 111.17 (arom.), 39.67 (NHCH2CH2) 25.13 (NHCH(CH2)2, 24.22 (CCH2CH2),
7.29 (CH(CH2)2). HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C20H22N3O 320.1757; found 320.1750.
4.3.10. N-(2-(1H-indol-3-yl)ethyl)-2-(cyclopentylamino)benzamide (Q-2)
yellow solid (68% yield), mp 152.4-152.6 °C. 1H NMR (400 MHz, CDCl3) δ 8.20 (s, 1H, CNHCH),
7.66 – 7.59 (m, 2H, NHCO, arom.), 7.35 (d, J = 8.1 Hz, 1H, arom.), 7.26 – 7.17 (m, 2H, arom,
NHCHC), 7.15 – 7.07 (m, 2H, arom.), 7.00 (s, 1H, arom.), 6.69 (d, J = 8.4 Hz, 1H, arom.), 6.44 (t, J =
7.5 Hz, 1H, arom.), 6.12 (t, J = 5.7 Hz, 1H, NHCH(CH2CH2)2), 3.84 – 3.75 (m, 1H, NHCH(CH2CH2)2 ),
3.71 (q, J = 6.3 Hz, 2H, CH2CH2NH), 3.05 (t, J = 6.6 Hz, 2H, CCH2CH2), 2.04 – 1.94 (m, 2H,
CH(CH2CH2)2), 1.78 – 1.68 (m, 2H, CH(CH2CH2)2), 1.64 – 1.50 (m, 4H, CH(CH2CH2)2). 13C NMR
(101 MHz, CDCl3) δ 169.84 (NHCO), 149.26 (arom.), 136.34 (arom.), 132.51 (arom.), 127.26 (arom.),
127.15 (arom.), 122.14 (arom.), 122.10 (arom.), 119.41 (arom.), 118.67 (arom.), 114.80 (arom.), 114.03
(arom.), 112.86 (arom.), 112.37 (arom.), 111.26 (arom.), 53.64 (NHCH(CH2)2), 39.72 (NHCH2CH2),
33.34 (CH(CH2CH2)2), 25.19 (CCH2CH2), 24.08 (CH(CH2CH2)2). HRMS (ESI-TOF) m/z: [M + H]+
Calcd for C22H26N3O 348.2070; found 348.2069.
4.3.11. N-(2-(1H-indol-3-yl)ethyl)-2-(cyclohexylamino)benzamide (Q-3)
yellow solid (75% yield), mp 124.1-125.1 °C 1H NMR (400 MHz, CDCl3) δ 8.29 (s, 1H, CNHCH),
7.66 (d, J = 7.7 Hz, 2H, NHCO, arom.), 7.39 (d, J = 8.1 Hz, 1H, arom.), 7.29 – 7.21 (m, 2H, arom.),
7.18 – 7.12 (m, 2H, arom., NHCHC), 7.03 (s, 1H, arom.), 6.72 (d, J = 8.4 Hz, 1H, arom.), 6.46 (t, J =
7.4 Hz, 1H, arom.), 6.18 (t, J = 5.7 Hz, 1H, NHCH(CH2CH2)2CH2), 3.76 (q, J = 6.4 Hz, 2H,
NHCH2CH2), 3.35 (s, 1H, NHCH(CH2CH2)2CH2), 3.09 (t, J = 6.6 Hz, 2H, CCH2CH2), 2.10 – 2.00 (m,
2H, NHCH(CH2CH2)2CH2), 1.84 – 1.75 (m, 2H, NHCH(CH2CH2)2CH2), 1.70 – 1.59 (m, 1H,
NHCH(CH2CH2)2CH2), 1.42 – 1.28 (m, 5H, NHCH(CH2CH2)2CH2). 13C NMR (101 MHz, CDCl3) δ
169.72 (NHCO), 148.60 (arom.), 136.19 (arom.), 132.37 (arom.), 127.34 (arom.), 127.00 (arom.),
121.98 (arom.), 121.94 (arom.), 119.21 (arom.), 118.50 (arom.), 114.52 (arom.), 113.74 (arom.), 112.65
(arom.), 111.86 (arom.), 111.13 (arom.), 50.41 (NHCH(CH2CH2)2CH2), 39.58 (NHCH2CH2), 32.61
(NHCH(CH2CH2)2CH2)),
25.69
(CCH2CH2),
25.03
(NHCH(CH2CH2)2CH2),
24.59
(NHCH(CH2CH2)2CH2). HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C23H28N3O 362.2227; found
362.2216.

4.3.12. N-(2-(1H-indol-3-yl)ethyl)-2-(benzylamino)benzamide (Q-4)
yellow solid (83% yield), mp 118.6-120.0 °C. 1H NMR (400 MHz, CDCl3) δ 8.14 – 8.02 (m, 2H,
CNHCH, NHCO), 7.66 (d, J = 7.9 Hz, 1H, arom.), 7.42 – 7.35 (m, 3H, arom.), 7.35 – 7.30 (m, 2H,
arom.), 7.25 – 7.12 (m, 5H, arom., NHCHC), 7.09 (d, J = 2.2 Hz, 1H, arom.), 6.59 (d, J = 8.3 Hz, 1H,
arom.), 6.50 (t, J = 7.5 Hz, 1H, CNHCH2), 6.19 – 6.11 (m, 1H, arom.), 4.41 (d, J = 5.6 Hz, 2H,
CCH2NH), 3.76 (q, J = 6.4 Hz, 2H, CH2CH2NH), 3.09 (t, J = 6.6 Hz, 2H, CCH2CH2). 13C NMR (101
MHz, CDCl3) δ 169.73 (NHCO), 149.37 (arom.), 139.16 (arom.), 136.35 (arom.), 132.61 (arom.),
128.57 (arom.), 127.19 (arom.), 127.15 (arom.), 127.04 (arom.), 126.94 (arom.), 122.29 (arom.),
122.08 (arom.), 119.55 (arom.), 118.76 (arom.), 115.36 (arom.), 114.83 (arom.), 113.03 (arom.), 112.02
(arom.), 111.26 (arom.), 47.05 (CH2CH2NH), 39.74 (CCH2NH), 25.27 (CCH2CH2). HRMS (ESI-TOF)
m/z: [M + H]+ Calcd for C24H24N3O 370.1914; found 370.1905.
4.3.13. N-(2-(1H-indol-3-yl)ethyl)-2-((3,4-dimethoxyphenethyl)amino)benzamide (Q-5)
yellow solid (85% yield), mp 60.0-61.0 °C. 1H NMR (400 MHz, CDCl3) δ 8.26 (s, 1H, CNHCH),
7.70 – 7.60 (m, 2H, NHCO, arom.), 7.35 (d, J = 8.1 Hz, 1H, arom.), 7.26 – 7.23 (m, 1H, arom.), 7.22 –
7.17 (m, 1H, NHCHC), 7.16 – 7.08 (m, 2H, arom.), 6.97 (s, 1H, arom.), 6.83 – 6.76 (m, 3H, arom.),
6.67 (d, J = 8.4 Hz, 1H, arom.), 6.48 (t, J = 7.4 Hz, 1H, CNHCH2 ), 6.14 (d, J = 5.9 Hz, 1H, arom. ),
3.87 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.71 (q, J = 6.3 Hz, 2H, NHCH2CH2), 3.35 (q, J = 6.6, 6.1 Hz,
2H, NHCH2CH2), 3.04 (t, J = 6.5 Hz, 2H, CCH2CH2), 2.89 (t, J = 7.2 Hz, 2H, CCH2CH2). 13C NMR
(101 MHz, CDCl3) δ 169.67 (NHCO), 149.27 (arom.), 148.69 (arom.), 147.36 (arom.), 136.33 (arom.),
132.60 (arom.), 132.05 (arom.), 127.24 (arom.), 127.14 (arom.), 122.10 (arom.), 120.53 (arom.),
119.37 (arom.), 118.63 (arom.), 115.20 (arom.), 114.54 (arom.), 112.77 (arom.), 111.98 (arom.), 111.43
(arom.), 111.26 (arom.), 111.14 (arom.), 55.78 (OCH3), 55.71 (OCH3), 44.90 (CH2CH2NH), 39.74
(CH2CH2NH), 35.16 (CCH2CH2), 25.20 (CCH2CH2). HRMS (ESI-TOF) m/z: [M + H]+ Calcd for
C27H30N3O3 444.2282; found 444.2271.
4.3.14. N-(2-(1H-indol-3-yl)ethyl)-2-((4-hydroxybutyl)amino)benzamide (Q-6)
pale yellow solid (76% yield), mp 109.8-110.6 °C. 1H NMR (400 MHz, acetone-d6) δ 10.07 (s, 1H,
CNHCH), 8.00 (t, J = 5.3 Hz, 1H, NHCO ), 7.73 (t, J = 5.8 Hz, 1H, arom.), 7.65 (d, J = 7.9 Hz, 1H,
arom.), 7.51 (dd, J = 7.9, 1.4 Hz, 1H, arom.), 7.41 – 7.35 (m, 1H, arom.), 7.27 – 7.20 (m, 2H, arom.),
7.13 – 7.07 (m, 1H, CCHNH), 7.05 – 6.99 (m, 1H, CNHCH2), 6.69 (d, J = 8.2 Hz, 1H, arom.), 6.52 –
6.44 (m, 1H, arom.), 3.71 – 3.65 (m, 3H, CH2CH2NH, OH), 3.62 (q, J = 5.9 Hz, 2H, CH2CH2OH), 3.18
(q, J = 6.6 Hz, 2H, NHCH2CH2), 3.06 (t, J = 7.3 Hz, 2H, CCH2CH2), 1.78 – 1.62 (m, 4H,
NHCH2CH2CH2CH2OH). 13C NMR (101 MHz, acetone-d6) δ 170.98 (NHCO), 151.38 (arom.), 138.2 0
(arom.), 133.61 (arom.), 129.23 (arom.), 129.11 (arom.), 123.83 (arom.), 122.60 (arom.), 119.90
(arom.), 119.87 (arom.), 116.61 (arom.), 115.26 (arom.), 114.02 (arom.), 112.66 (arom.), 112.43
(arom.), 62.72 (NHCHCH2CH2CH2OH), 43.85 (NHCHCH2CH2CH2OH), 41.46 (NHCH2CH2), 31.84
(NHCHCH2CH2CH2OH), 27.03 (CCH2CH2), 26.77 (NHCHCH2CH2CH2OH). HRMS (ESI-TOF) m/z:
[M + H]+ Calcd for C21H26N3O2 352.2020; found 352.2014.
4.3.15. N-(2-(1H-indol-3-yl)ethyl)-2-(4-methylphenylsulfonamido)benzamide (Q-7)
1
yellow solid (79% yield), mp 156.5-157.2 °C. H NMR (400 MHz, CDCl3) δ 10.90 (s, 1H, CNHSO2),
8.12 (s, 1H, CNHCH), 7.70 (d, J = 8.1 Hz, 2H, NHCO, arom.), 7.64 (d, J = 8.3 Hz, 1H, arom.), 7.58 (d,

J = 7.9 Hz, 1H, arom.), 7.40 (d, J = 8.1 Hz, 1H, arom.), 7.34 (t, J = 7.8 Hz, 1H, arom.), 7.23 (t, J = 7.6
Hz, 1H, arom.), 7.17 (s, 1H, CNHCH), 7.15 (s, 1H, arom), 7.11 (dd, J = 7.5, 4.2 Hz, 2H, arom.), 7.04 (s,
1H, arom.), 6.93 (t, J = 7.6 Hz, 1H, arom.), 6.11 (s, 1H, arom.), 3.67 (q, J = 6.3 Hz, 2H, CH2CH2NH),
3.02 (t, J = 6.6 Hz, 2H, CCH2CH2), 2.31 (s, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ 168.16 (NHCO),
143.51 (arom.), 138.79 (arom.), 136.58 (arom.), 136.37 (arom.), 132.46 (arom.), 129.49 (arom.),
127.23 (arom.), 127.08 (arom.), 126.48 (arom.), 123.27 (arom.), 122.39 (arom.), 122.10 (arom.),
121.34 (arom.), 121.00 (arom.), 119.65 (arom.), 118.56 (arom.), 112.53 (arom.), 111.38 (arom.), 40.08
(CH2CH2NH), 25.03 (CCH2CH2), 21.49 (CH3). HRMS (ESI-TOF) m/z: [M + H]+ Calcd for
C24H24N3O3S 434.1533; found 434.1537.

4.4. Cells culture
All cell lines were purchased from the American Type Culture Collection (ATCC, USA). Among
them, HCT-116, SGC-7901, MGC-803, HGC-27, MCF-7 cell lines were incubated in RPMI 1640
media supplemented with 10% FBS, 100 U/mL penicillin and streptomycin at 37 °C with 5% CO2,
while A549, HePG2, and RAW 264.7 cell lines were incubated in DMEM media supplemented with
10% FBS, 100 U/mL penicillin and streptomycin. Compounds dissolved in DMSO solution were
added into cell cultural medium with the final concentrations of DMSO less than 0.1%.
4.5. Proliferation evaluation
MTT method was utilized to evaluate the anti-proliferation effect of the compounds on various
kinds of cancer cell lines for primary screening. In details, cells in logarithmic phase were seeded
onto 96-well plates at the density of 5×103 per well with media added in periphery wells. After
adherence, compounds at gradient concentrations were added in triplicate, making the final
concentrations of each compound 6.25, 12.5, 25, 50 and 100 μM. After incubation for 72 h, 10 μL of
MTT solution was added to incubate with cells for 4h. Then, the MTT solution was disposed and the
formed formazan crystals were fully dissolved by 150 μL of DMSO for absorbance measurement by
microplate reader (Bio-Rad Laboratories, CA, USA) at 570 nm. IC50 values were calculated with
IBM SPASS statistics 25.0 software following the formula listed below. Data were given as mean ±
SD from three independent experiments conducted in triplicate. Inhibitory rate=1-(OD570 value of
tested well - OD570 value of background)/(OD570 value of control - OD570 value of background)
100%.
×
Cell viability was monitored using a Cell Counting Kit-8 (CCK8) (Meilunbio,
MA0218-5-Apr-01F) according to the manufacturer’s instructions. To be specific, MGC-803 and
HCT-116 cells were seeded onto 96-well plates (5×103 cells/well). Cell viability was documented
every 24 h for 2 days and detected by adding 10 μL of CCK-8 solution into each well and incubation
for 1h under 5% CO2 at 37 °C. The number of viable cells was assessed by absorbance measurement
at 450 nm using a microplate reader (Bio-Rad Laboratories, CA, USA). The percentage of survival
was determined using the following equation. Data were given as mean±SD from three independent
experiments conducted in triplicate. Cell viability=(OD450 value of tested well - OD450 value of
background)/(OD450 value of control - OD450 value of background)
×100%.
4.6. EdU incorporation assay
HCT-116 and MGC-803 cells were seeded onto 96-well plates at the density of 5×103 per well
and incubated overnight. After adherence, compound P-3 at gradient concentrations was added into
each well with the final concentrations of 1.25, 2.5, 5, 10 μM for HCT-116 cells and 2, 4, 8, 16 μM

for MGC-803 cells. 5-FU of the highest concentration in each concentration group as a positive
control and DMSO as vehicle were added. After incubation for 24 h, EdU staining assay was
performed according to the manufacturer’s instructions of Cell-Light EdU Apollo 567 In Vitro Kit
(Ribobio, Cat.#: C10310-1). Briefly, 100 μL of 50 μM EdU diluted in RPMI 1640 medium was
added into each well and incubated for 2 h. The medium was then disposed and cells were washed
with PBS twice. Afterwards, 50 μL of 4% paraformaldehyde was added to fix cells for 30 min at
room temperature followed by adding 50 μL of 2mg/mL glycine and incubating on shaker for 5 min.
Next, 100 μL of 0.5% TritonX-100 solution was added into each well and washed away 10 min later
with PBS before Apollo staining for 30 min in the dark (100 μL per well). Finally, 100 μL of
Hoechst 33342 (Solarbio, Cat.#:B8030) was added and incubated for 30 min on shaker. The stained
cells can be observed under a fluorescence microscope (Moticam Pro 282B, China).
4.7. Clonogenicity assay
HCT-116 and MGC-803 cells were seeded onto 6-well plates with each well containing 500-1000
cells. After adherence, compound P-3 at gradient concentrations was added into each well of
MGC-803, making the final concentrations 2, 4, 8, 16 μM and into each well of HCT-116, making
the final concentrations of 1.25, 2.5, 5, 10 μM. 5-FU of the highest concentrations in each
concentration group was added as a positive control. After incubation for 6 days for MGC-803 and 9
days for HCT-116, medium was removed and cells were fixed with methanol for 15 min before
stained with 1% crystal violet dye. Afterwards, excess dye was rinsed before colony-counting
according to the rule that the accumulation of more than 50 cells can be counted as one colony. The
relative colony ratio was determined using the following equation. The results were calculated from
at least three independent experiments. Relative colony ratio = (colonies of drug treated
group/colonies of control group) × 100%.
4.8. Cell cycle and apoptosis analysis
HCT-116 cells and MGC-803 cells were seeded onto 6-well plates and co-cultured with
compound P-3 at indicated concentrations (2.5, 5, 10, and 20 μM for 48 h). Afterwards, cells were
collected by trypsinisation, washed with PBS three times and fixed with 75% ethanol at 4 °C
overnight. In the following day, fixed cells were washed with PBS again, treated with Rnase, and
stained with propidium iodide (PI) provided in cell cycle staining kit (MULTI SCIENCES, Cat.#:
CCS012) for 30 min in the dark. The stained cells were analyzed by flow cytometry (BD
LSRFortessa, USA) within 1 hour. The cell debris were gated out and at least 20,000 cells in each
sample were analyzed to determine the distribution of fixed cells in each stage of cell cycle (G0/G1,
S, and G2/M phases) with modfitLT software. The apoptosis analysis was performed according to

the manufacturer’s instructions of apoptosis detection kit (Solarbio, Cat.#: CA1050). HCT-116 and
MGC-803 cells were seeded onto 6-well plates and co-cultured with compound P-3 at indicated
concentrations (2.5, 5, 10, 20μM) for 48 h. Afterwards, cells were collected by trypsinisation and
washed by PBS three times. Then, cells were resuspended in 1X binding buffer with 5 μl of FITC
Annexin-V and 10 μL PI added into cell suspension. After incubation for 15 min in the dark, another
400 μL of 1X binding buffer was added into each sample. The stained cells were analyzed by flow
cytometry (BD LSRFortessa, USA). The cell debris were gated out and at least 10,000 cells in each
sample were analyzed. Apoptotic cells were quantified with Flowjo software. The results were
calculated from at least three independent experiments.
4.9. Hoechst staining and Giemsa staining assays
HCT-116 cells at the density of 5×104 were seeded onto 6-well plates and incubated for 24 h.
After adherence, cells were treated with compound P-3 at the concentrations of 1.25, 2.5, 5, 10μM
for Hoechst staining assay or at 2.5, 5, 10, 15μM for Giemsa staining assay. After incubation for 48,
the supernatant was disposed and cells were washed with PBS twice before fixed with 4%
paraformaldehyde for 15 min. Subsequently, fixed cells were stained with 10μg/mL of Hoechst
33342 for 30 min in the dark or stained with Giemsa Staining solution (Solarbio, Cat.#: G1010) for
30 min. The redundant dye was washed away with PBS and the fluorescence or morphological
changes of cells were observed under a microscopy (Moticam Pro 282B, China).
4.10. Transwell assays
Migration and invasion of HCT-116 and MGC-803 cells were assessed using QCM™ 24-Well
Colorimetric Cell Migration Assay Kit (Costar) according to the manufacturer’s instructions.
Briefly, 8×104 of HCT-116 cells in 200 μL of serum-free medium were added into the upper
chambers. 600 μL of 20% FBS-containing medium as a chemoattractant was added into the lower
chambers. Cells were incubated with compound P-3 at different concentrations (2.5, 5, 10, 15 μM)
for 36 h (HCT-116 cells) or 24 h (MGC-803 cells) respectively at standard condition (5% CO2,
37 °C). Non-migrating cells on the upper chambers were then scraped off with cotton swabs. Cells
that migrated to the bottom of the membrane were slightly washed with PBS once and fixed with
4% paraformaldehyde, and then stained for 15 min with 1% crystal violet dye. After washing away
the excess dye, the stained cells were visualized under a microscope (Moticam Pro 282B, China).
For invasion assay, 50 μL of diluted matrigel (1:8) was pre-coated onto the polycarbonate
membrane and incubated at 37 °C, 5% CO2 for 4 h. After solidification, the supernatant was
disposed before 70 μL of serum-free medium was added into each well and incubated at standard
condition for 30 min to hydrate the matrigel. Next, the supernatant was disposed again and the cells

at the density of 16×104 was added into 200 μL of serum and anti-body-free medium in the upper
chambers and treated with compound P-3 at different concentrations (2.5, 5, 10, 15 μM). The
following steps were the same to the migration assay. To minimize the bias, at least three randomly
selected fields were quantified, and the average numbers of the cells were calculated. The results
were calculated from at least three independent experiments.
4.11. Western blot analysis
Cells in a 6-well pate were lysed using 100 μL of RIPA buffer (Solarbio, Cat.#: R0010, China)
supplemented with Protease and phosphatase inhibitor cocktail (50 ×, Beyotime, Cat.#: P-1045,
China) after treated with P-3 for 48 h. The protein concentration in lysates was measured using a
BCA Protein Assay Kit (Solarbio, Cat.#: PC0020). Next, 30 to 60 μg of proteins were resolved and
mixed with 4 ×loading buffer (Solarbio, Cat.#: P1016), electrophoresed in a 8%-12% SDS-PAGE
for separation, and transferred to polyvinylidene difluoride (PVDF) membranes of 0.45 μm
(Millipore Corporation, USA). The membranes were then blocked with 5% non-fat milk in Tris
Buffered Saline Tween (TBST) for 2 h at room temperature on shaker and then incubated with
diluted (1:500-1:1000) β-actin antibody (Affinity, Bioscience, USA, Cat.#: AF7018), STAT3
antibody (Affinity, Bioscience, USA, Cat.#: AF6294), p-STAT3 antibody (Affinity, Bioscience,
USA, Cat.#: AF3293), Bcl-2 antibody (Affinity, Bioscience, USA, Cat.#: AF6139), Bax antibody
(Affinity, Bioscience, USA, Cat.#: AF0120), Cyto-c antibody (Affinity, Bioscience, USA,
Cat.#:BF0714) and Cleaved-caspase-3 antibody (Affinity, Bioscience, USA, Cat.#: DF6879) at 4 °C
overnight. Then, uncombined primary antibodies were washed away with TBST before the bands
were incubated with goat anti-rabbit IgG H+L (Affinity, Bioscience, USA, Cat.#: HRP-S0001) or
goat anti-mouse IgG H+L (Affinity, Bioscience, USA, Cat.#: HRP-S0002) as secondary antibody at
room temperature on shaker for 2 h. Protein blots were visualized by Tanon imaging system (Tanon
Science & Technology Co., Ltd, China) after applying with ECL reagent (Millpore, Billerica, MA)
and the gray level of each protein bands was detected with ImageJ software. The relative
concentrations of each protein were presented as the relative signal intensity of loading control
β-actin. The results were calculated from at least three independent experiments.
4.12. ROS generation determination
HCT-116 cells were seeded onto 6-well plates at the density of 1×105/mL and incubated at
standard condition. After adherence, cells were exposed to compound P-3 at the concentrations of 2,
4, 8,16 μM for 36 h. Subsequently, 1 μg/mL of DCFH-DA was added into each well and incubated
with cells at 37 °C for 30 min in the dark. Afterwards, excess dye was washed away with PBS. ROS
production in cells of each group can be visualized by green fluorescence and compared after being

photographed under a fluorescence microscope (Moticam Pro 282B, China). To minimize the bias,
at least three randomly selected fields were photographed. The results were calculated from at
three independent experiments.
4.13. Nuclear translocation assay of STAT3
MGC-803 cells grown overnight on confocal glass slides were treated with compound P-3 of 10
μM in drug-treated group or equal volume of DMSO in control group for 3 h. Then, cells were
slightly rinsed with PBS and fixed with 50 μL of pre-cooled 4% paraformaldehyde for 15 min.
Afterwards, sealant (1% BSA and 0.3% Triton x-100 diluted in PBS) was added and incubated
with cells for 1 h at room temperature. After being washed three times with PBS, cells were
stained with primary STAT3 antibody (Affinity, Bioscience, USA, Lot. AF6294) diluted in sealant
at the rate of 1:100 overnight at 4 °C, followed by being incubated with Alexa Fluor® 488 Goat
Anti-Rabbit (IgG) secondary antibody (Invitrogen, Thermo Fisher Scientific,USA, Cat.#: A11011)
diluted in sealant at the rate of 1:200 for 2 h in the dark at 37 °C. Then, cells were washed with
PBS five times and incubated with 2 μg/mL of DAPI diluted in PBS for 15 min and washed with
PBS twice. Images were captured with a confocal laser microscope (Zeiss LSM700，Switzerland).
The results were calculated from at least three independent experiments.
4.14. ELISA of IL-6 and TNF-α
RAW 264.7 cells in 6-well plates were stimulated with 2 μg/mL of LPS (Solarbio, Lot.L8880,
China) except for vehicle group. Simultaneously, compound P-3 at different concentrations (5, 10,
15, 20 μM) was added into the drug-treated group. In the model group, equal volume of DMSO
were added. After 24 h, cell supernatant was collected and centrifuged to dispose cell debris. Then,
ELISA were performed according to the manufacturer's instructions of ELISA kits (TNF-α
:
Elabscience, E-EL-M0049c; IL-6: Elabscience, E-EL-M0044c). The optical values were detected
by micro reader (Bio-Rad Laboratories, CA, USA) at the wavelength of 450 nm. The
concentrations of IL-6 and TNF-α were determined by comparing the optical density value of each
sample to the standard curves using ELISACalc. The results were calculated from at least three
independent experiments.
4.15. Docking assay
Molecular docking study of compound P-3 with the SH2 domain of STAT3 was executed using
Schrodinger software package. Firstly, the X-ray crystal structure of STAT3 was obtained from
Protein Data Bank (PDB code: 1BG1) and prepared with the Protein Preparation Wizard model.
The specific process was including the removal of one monomer, the addition of missing hydrogen