Design, synthesis and QSAR study of novel isatin analogues inspired Michael acceptor as potential anticancer compounds

Article abstract of DOI:10.1016/j.ejmech.2017.12.043

Molecular hybridization is considered as an effective tactic to develop drugs for the treatment of cancer. A series of novel hybrid compounds of isatin and Michael acceptor were designed and synthesized on the basis of association principle. These hybrid compounds were tested for cytotoxic potential against human cancer cell lines namely, BGC-823, SGC-7901 and NCI-H460 by MTT assay. Most compounds showed good anti-growth activities in all tested human cancer cells. SAR and QSAR analysis may provide vital information for the future development of novel anti-cancer inhibitors. Notably, compound 6a showed potent growth inhibition on BGC-823, SGC-7901 and NCI-H460 with the IC₅₀ values of 3.6 ± 0.6, 5.7 ± 1.2, 3.2 ± 0.7 μM, respectively. Besides, colony formation assays, wound healing assays and flow cytometry analysis indicated 6a exhibited a potent anti-growth and anti-migration ability in a concentration-dependence manner through arrested cells in the G2/M phase of cell cycle. Moreover, 6a significantly repressed tumor growth in a NCI-H460 xenograft mouse model. Overall, our findings suggested isatin analogues inspired Michael acceptor may provide promising lead compounds for the development of cancer chemotherapeutics.

Full text of DOI:10.1016/j.ejmech.2017.12.043

Accepted Manuscript
Design, synthesis and QSAR study of novel isatin analogues inspired Michael
acceptor as potential anticancer compounds
Jiabing Wang, Di Yun, Jiali Yao, Weitao Fu, Fangyan Huang, Liping Chen, Tao Wei,
Cuijuan Yu, Haineng Xu, Xiaoou Zhou, Yanqing Huang, Jianzhang Wu, Peihong Qiu,
Wulan Li
PII:
S0223-5234(17)31069-3
10.1016/j.ejmech.2017.12.043
EJMECH 10021
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 August 2017
Revised Date: 30 November 2017
Accepted Date: 13 December 2017
Please cite this article as: J. Wang, D. Yun, J. Yao, W. Fu, F. Huang, L. Chen, T. Wei, C. Yu, H. Xu,
X. Zhou, Y. Huang, J. Wu, P. Qiu, W. Li, Design, synthesis and QSAR study of novel isatin analogues
inspired Michael acceptor as potential anticancer compounds, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2017.12.043.
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Graphical abstract:

ACCEPTED MANUSCRIPT
Design, synthesis and QSAR study of novel isatin analogues inspired
Michael acceptor as potential anticancer compounds
Jiabing Wangab1, Di Yunab1, Jiali Yaoa1, Weitao Fuad1, Fangyan Huanga, Liping Chena, Tao Weia,
Cuijuan Yuab, Haineng Xuab, Xiaoou Zhouab, Yanqing Huanga, Jianzhang Wuab*, Peihong Qiua1*,
Wulan Liabc
*
(aChemical Biology Research Center, College of Pharmaceutical Sciences, Wenzhou Medical
Universtiy, Wenzhou, Zhejiang 325035, China )
(bWenzhou Biomedical Innovation Center, Wenzhou University and Wenzhou Medical University,
Wenzhou, Zhejiang 325035, China )
(cCollege of Information Science and Computer Engineering, The First Clinical Medical College,
Wenzhou Medical University, Wenzhou, Zhejiang 325035, China)
(dCollege of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China)
1 These authors contribute to this work equally.
*Correspondence authors.
Address: Chemical Biology Research Center, College of Pharmaceutical Sciences, Wenzhou
Medical Universtiy, Wenzhou, Zhejiang 325035, China. Tel./Tex: +86 577 86689984:
(Jianzhang Wu).
wjzwzmu@163.com (Jianzhang Wu)
Address: Chemical Biology Research Center, College of Pharmaceutical Sciences, Wenzhou
Medical Universtiy, Wenzhou, Zhejiang 325035, China. Tel./Tex: +86 577 86689984: (Peihong
Qiu).
wjzwzmc@126.com (Peihong Qiu)
Address: Chemical Biology Research Center, College of Pharmaceutical Sciences, Wenzhou
Medical Universtiy, Wenzhou, Zhejiang 325035, China. Tel./Tex: +86 577 86689984: (Wulan
Li).
lwlwzmu@163.com (Wulan Li)
Abstract
Molecular hybridization is considered as an effective tactic to develop drugs for the
treatment of cancer. A series of novel hybrid compounds of isatin and Michael
acceptor were designed and synthesized on the basis of association principle. These

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hybrid compounds were tested for cytotoxic potential against human cancer cell lines
namely, BGC-823, SGC-7901 and NCI-H460 by MTT assay. Most compounds
showed good anti-growth activities in all tested human cancer cells. SAR and QSAR
analysis may provide vital information for the future development of novel
anti-cancer inhibitors. Notably, compound 6a showed potent growth inhibition on
BGC-823, SGC-7901 and NCI-H460 with the IC50 values of 3.6±0.6, 5.7±1.2, 3.2±0.7
ꢀM, respectively. Besides, colony formation assays, wound healing assays and flow
cytometry analysis indicated 6a exhibited a potent anti-growth and anti-migration
ability in a concentration-dependence manner through arrested cells in the G2/M
phase of cell cycle. Moreover, 6a significantly repressed tumor growth in a NCI-H460
xenograft mouse model. Overall, our findings suggested isatin analogues inspired
Michael acceptor may provide promising lead compounds for the development of
cancer chemotherapeutics.
Key words: Chemotherapeutics; Association principle; Quantitative structure-activity
relationship; Anticancer.
1. Introduction
Cancers, one of the leading causes of mortality across the world, bring great pressure
to more and more human lives[1]. However, most of patients have been in the
advanced stage of cancer when definitely diagnosed, which makes them miss the best
operation time of treatment, and chemotherapy is the primary treatment for them.
Despite researchers made extraordinary effort to develop new type drugs and novel
therapeutic strategies for treating all sorts of cancers, there was no radical cure for
cancer across the globe[2]. Chemotherapeutic drugs with side effects and poor
tolerance limited their use in patients. Therefore, it remained tremendous challenges
for scientists to search for effective and unexplored anticancer agents which have
valid potency against tumor cells.
Over the past decade, there has been growing interest in searching for novel
chemotherapeutic agents based biologically active substances found in nature[3-5].
For example, isatin (2,3-dioxindole), is an endogenous substance in human bodies,
which has been documented as a distinguished pharmacological agent that shows a
broad range of biological actions, such as anti-bacterial[6], anti-inflammatory[7],
anti-oxidant[8] and anti-cancer[9]activities. It has been reported that compounds
bearing the isatin chemical scaffold possess diverse activities for the treatment of
cancers, like FDA approved drugs sunitinib maleate and toceranib phosphate. In

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addition, varieties of inhibitors with isatin moiety proved outstanding anti-cancer
effects in clinical trials, including the tyrosine kinase inhibitors SU5416 and SU6668.
SU5416 was found to exhibit extreme potency for multiple NCI cell lines, and
SU6668 had significant anti-tumor activity in xenografts[10, 11]. Therefore, it’s
believed that anti-tumor drugs containing isatin scaffold could possess broader
spectrum of cytotoxicity to cancer cells.
The α,β-unsaturated ketone (Michael acceptor) pharmacophore has been considered
as a common skeleton that many naturally occurring agents possess [12-14]. Such as
curcumin, a nature chemopreventive agent, displays strong anti-cancer activity by
inhibiting cancer cell growth and inducing cancer cell apoptosis[15]. Moreover,
chalcones, another typical edible natural products, also have a wide range of
pharmacological activities, like xanthohumol[16]. Chemically, the biological activities
of curcumin and chalcones are largely attributed to the presence of α,β-unsaturated
ketone, as removal of this group makes them inactive[17-20]. Furthermore, many
studies have shown that the incorporation of a Michael acceptor into molecules often
resulted in drugs for improved cytotoxicity[21,22]. Hence, α,β-unsaturated ketone can
be regarded as functionality structure to drugs design.
To date, molecular hybrids is the one of most popular strategies to develop new
anti-cancer agents based on combination of structural features of two different active
fragments, which not only reduce the risk of drug-drug interactions but also improve
the pharmacological activities[23-27]. In this study, inspired by the structural features
of isatin and α,β-unsaturated ketone, we envisioned that the merging of these two
bioactive scaffolds in a single chemical entity may lead to the development of
promising new leads with potential anticancer activity. Therefore, owing to our
interest in the synthesis and biological evaluation, we focused on molecular
hybridization strategy to design and synthesis a novel series of isatin analogues
bearing α,β-unsaturated ketone scaffold, evaluated their anti-tumor activities, and
established the QSAR model to obtain a deeper insight into the molecular description
of compounds activity.

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Figure 1. Design of novel isatin analogues bearing α,β-unsaturated ketone as anti-cancer agents
2. Results and discussion
2.1. Chemistry
The synthetic pathway adopted to synthesize the compounds were as described as in
Scheme 1. 22 compounds were synthesized and purified (Table 1). All compounds
were purified by column chromatography or re-crystallization. The compounds were
obtained in good yields (70%-80%) after purification. The products were
characterized by analysis and comparison of their spectral data of liquid
chromatography-mass spectrometry (LC-MS) and 1H-NMR. These characteristic data
1
including color, yield, melting points, LC-MS and H-NMR, spectrum of compounds
were presented in chemistry synthetic section.
A
O
B
R2
N
NH2
e
O
O
H
O
O
N
N
H
H
5a
6a-6d
Scheme 1. The general route to produce the isatin analogues bearing α,β-unsaturated ketone.
Reagents and conditions: (a): CCl3CH(OH)2, NH2OH HCl, Na2SO4; (b): concentrated H2SO4; (c):
acetophenones, diethylamine, EtOH; (d): EtOH, HCl, reflux; (e): acyl chloride, THF, 0 °C.

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Table 1. The isatin analogues bearing α,β-unsaturated ketone
Comp.
5a
R
3-NH2
4-OCH3
4-Cl
Comp.
5l
R
4-Br
5b
5c
5m
5n
5o
4-NO2
3,4,5-OCH3
3,5-F
5d
5e
2,4-OCH3
2-F
5p
5q
5r
2-OCH3
a
5f
2-Cl
/
5g
4-F
4-NH2, 3,5-Cl
-C2H3
5h
5i
2,5-Cl
2-Br
6a
6b
6c
-CH(CH3)2
-C2H5
5j
3-Cl
5k
3,4-Cl
6d
-CH3
a “/” represents none.
2.2. Evaluation of the cytotoxic activities in vitro
All the synthesized compounds were evaluated for the inhibitory activity in vitro
against the growth of three tumor cell lines namely, BGC-823 (human gastric cancer
cell line), SGC-7901 (human gastric cancer cell line), NCI-H460 (human large cell
lung cancer cell line) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay[28], and curcumin (cur) and xanthohumol (xn) were used as
positive controls. As shown in Table 2, it is noticeable from the initial screening that
most compounds showed significant improvement in the anticancer activity in all
tested human cancer cells compared with positive controls. Furthermore, in order to
get insight in to the SARs, we varied the substitutions on aryl ring of these analogues.
Based on the in vitro cytotoxic activities, the structural activity relationships (SARs)
of the isatin analogues 5a-5r and 6a-6d have been proposed (Figure 2).Interestingly, it
is noted that most analogues were more active on BGC-823 and NCI-H460 cells,
respectively. In comparison with 5q without substitution on phenyl ring, analogues
bearing substitution on phenyl ring showed different levels of anti-cancer activities.
Especially for compounds 5c and 6a displayed remarkable activity against three
cancer cells with IC50 values ranging from 3.2±0.7 µM to 8.5±0.9 µM, while 5h
(2,5-dichloro) was found to be inactive on all tested cancer cells. In addition, the
generally N-acylated compounds (6a-6d) exhibited better activity against three cancer
cells compared with N-unsubstituted compound 5a. Among compounds 6a-6d, 6a
containing Michael acceptor was found to be more potent anticancer activity
compared to others. Compound 5b with 2-methoxy substitution on phenyl ring was

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selectively active on NCI-H460 cells and moderately active on both BGC-823 and
SGC-7901 cells. Moreover, results showed that analogues 5d (2,4-dimethoxy) with
increasing number of methoxy substitutions on phenyl ring showed enhanced
cytotoxicity against BGC-823 and SGC-7901 cells exception of NCI-H460 cells
compared with 5b (4-methoxy) and 5p (2-methoxy). But 3,4,5-trimethoxy (5n)
substitutions on the phenyl ring were lower active on all the cell lines studied, which
suggested the position of electron-donating substitutions on phenyl ring could affect
the activity. Furthermore, there are opposite phenomenon in the benzene ring with
electron-withdrawing substitution such as chloro substitution, in other words,
compound 5k (3,4-dichloro) and 5h (2,5-dichloro) with increasing number of chloro
substitutions exhibited lower anti-cancer activity than 5c (4-chloro), 5e (2-chloro) and
5j (3-chloro). Additionally, the similar phenomenon was occurred in benzene ring
containing fluoro substitution. Taken together, the results demonstrated electron-
withdrawing substitutions to the disadvantaged of the anticancer activity and Michael
acceptor on phenyl ring were favorable for anti-cancer activity, respectively.
Table 2 IC50 (µM) of analogues against human cancer cell lines
Compound
BGC-823
SGC-7901
NCI-H460
5a
5b
5c
5d
5e
5f
29.9±2.7
19.8 ±1.2
6.9±0.4
14.4±1.5
23.2±2.0
8.5±0.9
20.4±1.3
5.5±0.3
6.5±0.7
5.4±0.3
8.5±0.3
7.7±0.6
7.5±1.4
61.6±1.6
11.0±0.9
9.8±0.7
13.7±1.3
11.5±3.7
18.4±1.4
10.9±0.3
13.3±1.7
10.2±2.0
10.4±1.0
6.2±0.1
3.2±0.7
9.7±0.7
9.2±0.8
11.5±0.3
17.4±0.0
12.8±0.4
12.4±1.2
56.3±6.1
13.1±0.1
8.4±0.6
11.8±0.7
9.1±1.4
5g
5h
5i
11.1±2.3
53.7 ±1.7
9.5±0.1
5j
7.7±0.3
5k
5l
13.2±1.3
9.0 ±0.9
14.3±1.2
13.9±0.9
15.7±1.9
10.2±1.3
13.2±1.1
8.5±2.1
17.8± 1.2
10.2±1.0
18.2±1.3
18.3±0.6
17.6±0.3
16.3±0.7
9.3±0.5
5m
5n
5o
5p
5q
5r
11.9±0.8
5.7±1.2
6a
6b
3.6 ±0.6
6.2±1.0
8.6±0.6

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6c
6d
7.5±0.9
9.1±1.5
19.5±0.8
11.5±0.7
14.1 ±0.3
26.2±0.7
10.3±0.6
12.3±1.1
20.3±0.3
Curcumin
Xanthohumol
7.8±0.3
10.0±0.5
6.9±0.4
Figure 2. SAR analysis of isatin inspired α,β-unsaturated ketone analogues
2.3 Quantitative evaluation of structure-activity relationship(QSAR)
Quantitative structure-activity relationship (QSAR) has been widely applied to predict
the biological effects of chemicals[29]. Hence, a QSAR model was constructed to
further investigate the SAR of the isatin inspired α,β-unsaturated ketone analogues. In
this study, the missing value of the molecular descriptors were removed and random
forest algorithm was used to find the best feature subset[30,31]. The descriptors of
small molecules predicted in this study could correctly predict compounds’
categorical biological activities. As shown in Figure 3A, for BGC-823 cells, it was
noted that the predicted accuracy could reach 87.4%. Furthermore, the results also
showed the most five relative important molecular descriptors are bcutm11, bcutp5, J,
ATSp1, GATSv3 (Figure 3A). Meanwhile, the developed QSAR models also showed
good predictability for SGC-7901 and NCI-H460 cell lines, and the predicted
accuracy could reach 74.2% and 73.6%, respectively (Figure 3B and 3C). Therefore,
the presented models indicated different descriptors exert distinct influence on the
anti-cancer activity of compounds for different cancer cells. In summary, the results of
SAR and QSAR may provide vital clues for the development of new inhibitors to
combat different cancers.

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Figure 3. Quantitative evaluation of structure-activity relationship (QSAR). The predicted
accuracy by random forest algorithm based 500 iterations (Internal: four fifths of the data set,
External: one fifth of data set) and the most five relative important molecular descriptors
calculated by random forest algorithm for (A) BGC-823 cells, (B) SGC-7901 cells and (C)
NCI-H460 cells.
For BGC-823 cells line: GATSv3: Geary autocorrelation-lag3/weighted by atomic van der Waals
volumes. ATSp1: Broto-Moreau autocorrelation of a topological structure-lag1/weighted by
atomic polarizabilities. J: Balaban’s J index. bcutp5: Highest eigenvaluen.5 of Burden
matrix/weighted by atomic polarizabilities. bcutm11: Burden descriptors based on atomic mass.
For SGC-7901 cells line: J: Balaban’s J index. S35: Sum of E-State of atom type: dO. Shev:
EState indices over all non-hydrogen atoms. Hy: Hydrophilic index. Scar: The sum of the EState
indices over all C atoms.
For NCI-H460 cells line: GATSv8: Geary autocorrelation descriptors based on atomic van der

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Waals volume. Qmin: Most negative charge in
a
molecule. MATSe6: Moran
autocorrelation-lag6/weighted by atomic Sanderson electronegativities. MATSm6: Moran
autocorrelation-lag6/weighted by atomic masses. IC2: Information content with order 2 proposed
by Basak.
2.4 The anti-growth activity of 6a
Based on the promising cytotoxic activity, the most active compound 6a bearing
α,β-unsaturated skeleton was screened from this series and taken-up for further
detailed studies. 6a appraised as more potent by comparing their IC50 values with that
of curcumin and xanthohumol. As shown in table 2, 6a displayed potent growth
inhibition on three cancer cells with IC50 values in the range of 3.2±0.7-5.7±1.2 ꢀM,
which are better than curcumin and xanthohumol, respectively. Moreover, for better
understanding of the potency of compound 6a, we further evaluated the anti-cancer
activity of 6a (5 and 10 ꢀM) in NCI-H460 cells. As shown in Figure 4A, B, when
cells were treated with 6a for 72 h, the population of viable cells decreased in a
concentration-dependent manner and time-dependent manner, respectively.
Furthermore, the effect of 6a was more pronounced than that of xanthohumol and
curcumin. The results suggested that 6a had good anti-growth activity.
2.5 Colony formation assay of 6a in NCI-H460 cells
To confirm the anti-growth activity of 6a, the colony formation assay was performed
to measure the effect of 6a in NCI-H460 cells. The cells were treated with 6a at
various concentrations (10 and 20 ꢀM), xanthohumol and curcumin. As shown in
Figure 4C, it is found that treatment of cells with 6a remarkably reduced the
population of viable cells in a dose-dependent manner compared with DMSO-treated
group. Notably, 6a at 10 ꢀM exhibited even more stronger anti-growth effect than
xanthohumol (10 ꢀM) and curcumin (20 ꢀM). These findings demonstrated that 6a
can significantly inhibit NCI-H460 cells growth.
2.6 6a can inhibit the migration of NCI-H460 cells
In generally, the mortality and prognosis in cancer patients associate with metastatic
potential of tumors, and cancer cells have a strong ability of migration[32-34], so we
further investigated the effect of 6a on cells migration capability using wound healing
assay[35]. In this study, NCI-H460 cells were treated with 6a, xanthohumol and
curcumin alone after creating the scratch in the confluent monolayers of cultured cells.
After the compound treatment of 0 h and 48 h, we captured the images of the injured
area. As shown in Figure 4D, the wound area was almost completely healed in

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DMSO-treated control after 48 h, while compound 6a can inhibit the healing of
wounded area in a concentration-dependent manner. What’s more, 6a displayed more
stronger cellular migration inhibition ability than curcumin and xanthohumol. The
results showed 6a had the ability to inhibit the migration capability of NCI-H460
cells.
Figure 4. 6a inhibited growth and migration in NCI-H460 cells. (A) (B) The anti-growth activity
of 6a. The NCI-H460 cells were treated with 6a, curcumin and xanthohumol, finally determined
by the MTT assay. (C) Representative images of NCI-H460 cells colonies a fter treatment 6a with
various concentrations. The NCI-H460 cells were treated with 6a, xanthohumol and curcumin,
then cells were transferred to normal medium and allowed to form colonies, and finally colonies
were stained with crystal violet staining solution and photographed. (D) Effect of 6a on migration
of NCI-H460 lung cancer cells. The images were captured using phase contrast microscopy before
(0 h), and after 48 h of treatmemt with 6a, xanthohumol and curcumin.
2.7 Treatment of NCI-H460 cells with compound 6a resulted in G2/M phase arrest
Usually, anticancer agents have the ability of preventing the growth of cancer cells by
altering the regulation of cell cycle at a specific checkpoint[36]. To understand
whether the anti-growth activity of 6a in NCI-H460 cells is on account of cell cycle
arrest, cell cycle distribution of NCI-H460 cells was determined by flow-cytometry
analysis. Cells were treated with 6a at concentrations of 5 ꢀM, 10 ꢀM, 20 ꢀM for 48 h,
and stained with propidium iodide and further analyzed by flow cytometry. Treatment
of NCI-H460 cells with 6a results in an increase in the DNA content in G2/M phase

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(Figure 5). The results showed that 6a-induced G2/M phase of cell cycle arrest may
be one of the possible mechanisms of cell growth inhibition.
A
DMSO
6a(5µM)
6a(10µM)
6a(20µM)
Xn(10µM)
Cur(20µM)
B
120
80
40
0
G2/M
S
G1
5
10
6a
20
10
Xn
(µM)
20
Cur
DMSO
Figure 5. Effect of 6a on cell cycle progression in NCI-H460 cells. (A) Cells were treatment with
6a for 48 h, then the cell cycle distribution was analysed by using propidium iodide staining
method and analysed by flow cytometry. (B) Data of 8000 cells were collected for each data file.
The percentage of the cell population in G1, S and G2/M phase were calculated by using the (BD
Biosciences, CA).
2.8 6a inhibited NCI-H460 xenograft tumor growth in vivo
To determine whether the anti-tumor potency of compound 6a in vivo, a subcutaneous
xenograft model of NCI-H460 cells in nude mice was used. Intraperitoneal
administration of 6a and curcumin at a dose of 15 mg/kg every day for 15 days
significantly reduced NCI-H460 tumor volume and weight versus vehicle control
(Figure 6A-B, and Figure S1 in the Supporting information). Furthermore, 6a had
more remarkable reduction of tumor growth than positive drug curcumin. Importantly,
as shown in Figure 6C, 6a treatment for 15 days was well tolerated, without no serious
side effects such as weight loss and abnormal behavior were observed. Moreover,
based on the histology analysis, no obvious severe histopathological changes in liver
and lung tissues were observed in 6a-operated and curcumin-treated rats, when
compared with DMSO-treated rats (Figure 6E). Additionally, Bcl-2 is the first
apoptosis-related gene acknowledged to play a important role in tumerogenesis and is
overexpressed in a range of cancers[37], so west blotting assay was performed to
further measure the effect of 6a. As shown in Figure 6D, 6a can effectively decrease
the expression of Bcl-2, and the efficacy was more pronounced than curcumin. Taken

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together, these results demonstrated that 6a had a potent effect on inhibiting tumor
growth without obvious toxicity in vivo.
Figure 6. 6a inhibited NCI-H460 xenograft tumor growth in vivo. 6a significantly inhibited (A)
tumor volume and (B) tumor weight versus vehicle control and positive drug curcumin. (C)Body
weight of the mice in each group at the end of the observation period. (D) Reducing the expression
level of protein Bcl-2 from tumor tissue by 6a. (E) The histopathological changes in liver and lung
tissues by 6a. The liver and lung tissues of nude mice were stained with hematoxylin and eosin
then were examined under light microscope. Data are expressed as the mean ± SD, n ≥ 6. **P <
0.01, *P < 0.05 vs vehicle-treated group. one way ANOVA, followed by Tukey’s multiple
comparison test.
3. Conclusion

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In summary, a series of isatin inspired α,β-unsaturated ketone analogues were
designed, synthesized based combination principles, and evaluated for their
anti-cancer potential against three human cancer cell lines. In the initial screening,
interestingly, compound 6a displayed a broad of spectrum of growth inhibition on all
the tested cancer cells and showed the most potent anticancer activity on the
NCI-H460 cell lines( IC50 value 3.2±0.7 ꢀM). The SAR and QSAR analysis
confirmed that the inhibition potency was correlated with the electron-donating
substituents on benzyl ring and the Michael acceptor in the synthetic compounds.
Additionally, compound 6a effectively inhibited the growth of NCI-H460 cells both in
a concentration dependent manner and time dependent manner, respectively. Colony
formation and wound healing assays indicated that 6a possessed potent anti-growth
and anti-migration ability. Further biological assessment of 6a revealed that it can
trigger cell cycle arrest in G2/M phase. Moreover, 6a significantly inhibited the
growth of NCI-H460 xenograft tumor in vivo. Collectively, our findings suggest isatin
inspired α,β-unsaturated ketone analogues offer promising leads for the further
development of anticancer agents.
4. Experimental
4.1. Chemistry
Unless otherwise noted, all chemical reagents were obtained commercially from
Sigma-Aldrich, Aladdin (Beijing, China) and used without further purification.
Reactions were monitored by thin-layer chromatography (TLC) using silica gel
GF254, and the chromatograms were conducted on silica gel (200−300 mesh) and
visualized under UV light at 254 and 365 nm. Melting points (mp) were determined
on open capillary tubes on a Fisher-Johns melting apparatus, and were uncorrected.
Mass spectra (MS) were determined on an Agilent 1100 LC-MS (Agilent, Palo Alto,
1
CA, USA). H spectral data were obtained from 600 MHz spectrometer (Bruker
Corporation, Switzerland) with TMS as an internal standard. Chemical shifts (d) are
reported in parts per million (ppm) relative to the residual solvent signal. Coupling
constant values (J) are given in Hertz (Hz) and refer to apparent multiplicities.
Splitting patterns were described as follows: s = singlet, d = doublet, t = triplet, dd =
doublet of doublets, m = multiplet. The chemical data of novel compounds are
presented as follows:

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(E)-3-(2-(3-aminophenyl)-2-oxoethylidene)indolin-2-one(5a) red-brown powder,
70.82% yield，mp166.2-168.7°C. 1H-NMR (DMSO), δ: 10.780 (s, 1H, NH), 7.926 (d,
J=7.8Hz, 1H, Ar-H5), 7.626 (s, 1H, Ar-CO-CH=C), 7.382 (t, J=15.0Hz, 1H, Ar-H5’),
7.260-7.213 (m, 2H, Ar-H3, Ar-H6’), 7.187 (d, J=7.2Hz, 1H, Ar-H4’), 6.941 (t,
J=15.0Hz, 1H, Ar-H2’), 6.880 (d, J=7.8Hz, 2H, Ar-H4, Ar-H2), 5.480 (s, 2H, 3’-NH2).
EI-MS m/z:265.10[M+1]+, calcd for C16H12N2O2: 264.09.
(E)-3-(2-(4-methoxyphenyl)-2-oxoethylidene)indolin-2-one(5b) red powder 74.73%
1
yield, mp195.5-197.1°C (Lit.[38] 192-194°C). H-NMR (DMSO), δ: 8.052 (d,
J=9.0Hz, 2H, Ar-H2’ Ar-H6’), 7.926 (d, J=7.8Hz, 1H, Ar-H5), 7.681 (s, 1H,
Ar-CO-CH=C), 7.319 (t, J=15.0Hz, 1H, Ar-H3), 7.116 (d, J=8.4Hz, 2H, Ar-H3’,
Ar-H5’), 6.928 (t, J=15.0Hz, 1H, Ar-H4), 6.885 (d, J=7.8Hz, 1H, Ar-H2), 3.868 (s, 1H,
4’-OCH3). EI-MS m/z: 280.07[M+1]+, calcd for C17H13NO3：279.09.
(E)-3-(2-(4-chlorophenyl)-2-oxoethylidene)indolin-2-one(5c) red-brown powder,
74.19% yield, mp191.4-192.6°C (Lit.[38] 182-185°C). 1H-NMR (DMSO), δ: 10.808
(s, 1H, NH), 8.080 (d, J=9.0Hz, 2H, Ar-H2’, Ar-H6’), 8.038 (d, J=7.8Hz, 2H, Ar-H5),
7.683 (s, 1H, Ar-CO-CH=C), 7.661 (d, J=8.4Hz, 2H, Ar-H3’, Ar-H5’), 7.350 (t,
J=15.6Hz, 1H, Ar-H3), 6.957 (t, J=15.0Hz, 1H, Ar-H4), 6.884 (d, J=9.0Hz, 1H,
Ar-H2). EI-MS m/z: 284.24, 285.80, 287.11[M+1]+, calcd for C16H10ClNO2: 283.04.
(E)-3-(2-(2,4-dimethoxyphenyl)-2-oxoethylidene)indolin-2-one(5d)
saffron
1
powder, 71.32% yield, mp163.8-165.4°C. H-NMR (DMSO), δ: 10.698 (s, 1H, NH),
8.049 (d, J=7.8Hz, 1H, Ar-H5), 7.792 (d, J=9.0Hz, 1H, Ar-H6), 7.611 (s, 1H,
Ar-CO-CH=C), 7.312 (t, J=7.8Hz, 1H, Ar-H3), 6.940 (t, J=7.8Hz, 1H, Ar-H4), 6.862
(d, J=7.8Hz, 1H, Ar-H2), 6.697-6.711 (m, 2H, Ar-H5’, Ar-H3’), 3.887 (s, 3H, 2’-OCH3),
3.877 (s, 3H, 4’-OCH3). EI-MS m/z: 310.09 [M+1]+, calcd for C18H15NO4: 309.10.
(E)-3-(2-(2-fluorophenyl)-2-oxoethylidene)indolin-2-one(5e) saffron powder, 83.60%
1
yield, mp181.6-183.1°C. H-NMR (DMSO), δ: 10.817 (s, 1H, NH), 8.074 (d,
J=7.8Hz, 1H, Ar-H5), 7.929 (td, J=1.8Hz, J=7.8Hz, 1H, Ar-H6’), 7.724-7.762(m, 1H,
Ar-H3’), 7.539 (d, J=3.6Hz, 1H, Ar-H4’), 7.404-7.436 (m, 2H, Ar-H3, Ar-H5’), 7.381 (s,
1H, Ar-CO-CH=C), 6.994 (t, J=7.8Hz, 1H, Ar-H4), 6.893 (d, J=7.8Hz, 1H, Ar-H2).
EI-MS m/z: 268.09, 269.00[M+1]+, calcd for C16H10FNO2: 267.07.
(E)-3-(2-(2-chlorophenyl)-2-oxoethylidene)indolin-2-one(5f) yellow powder, 77.97%
1
yield, mp167.9-170.1°C. H-NMR (DMSO), δ: 10.836 (s, 1H, NH), 8.297 (d,
J=7.8Hz, 1H, Ar-H5), 7.776 (d, J=7.8Hz, 1H,Ar-H6’), 7.625 (d, J=0.6Hz, 1H, Ar-H3’),
7.617 (d, J=1.2Hz, 1H, Ar-H4’), 7.540-7.513 (m, 1H, Ar-H5’), 7.400 (td, J=1.2Hz,
7.8Hz, 1H, Ar-H3), 7.367 (s, 1H, Ar-CO-CH=C), 7.013 (td, J=1.2Hz, 7.8Hz, 1H,
Ar-H4), 6.902 (d, J=6.6Hz, 1H, Ar-H2). EI-MS m/z: 284.05, 286.13[M+1]+, calcd for
C16H10ClNO2: 283.04.
(E)-3-(2-(4-fluorophenyl)-2-oxoethylidene)indolin-2-one(5g) saffron powder, 77.97%
1
yield, mp190.9-192.3°C (Lit.[39] 186 °C) H-NMR (DMSO), δ: 10.801 (s, 1H, NH),

ACCEPTED MANUSCRIPT
8.154(td, J=1.2Hz, 6.6Hz, 2H, Ar-H2’, Ar-H6’), 8.000 (d, J=7.8Hz, 1H, Ar-H5), 7.691
(s, 1H, Ar-CO-CH=C), 7.433-7.403 (m, 2H, Ar-H3’, Ar-H5’), 7.343 (td, J=1.2Hz,
7.8Hz, 1H, Ar-H3), 6.950 (td, J=1.2Hz, 7.8Hz, 1H, Ar-H4), 6.884 (d, J=7.8Hz, 1H,
Ar-H2). EI-MS m/z: 268.22, 269.00[M+1]+, calcd for C16H10FNO2: 267.07.
(E)-3-(2-(2,5-dichlorophenyl)-2-oxoethylidene)indolin-2-one(5h)
red powder,
77.67% yield, mp230.8-232.3°C. 1H-NMR (DMSO), δ: 10.851 (s, 1H, NH), 8.343 (d,
J=7.8Hz, 1H, Ar-H5), 7.837 (d, J=2.4Hz, 1H, Ar-H4’), 7.682 (dd, J=2.4Hz, 8.4Hz, 1H,
Ar-H6’), 7.648 (d, J=8.4Hz, 1H, Ar-H3’), 7.412 (td, J=1.2Hz, 7.8Hz, 1H, Ar-H3),
7.323 (s, 1H, Ar-CO-CH=C), 7.024 (td, J=0.6Hz, 8.4Hz, 1H, Ar-H4), 6.904 (d,
J=7.8Hz, 1H, Ar-H2). EI-MS m/z: 317.90, 319.92[M+1]+, calcd for C16H9Cl2NO2:
317.00.
(E)-3-(2-(2-bromophenyl)-2-oxoethylidene)indolin-2-one(5i) red powder, 70.32%
yield, mp152.9-155.2°C List[40]. 1H-NMR (DMSO), δ: 10.835 (s, 1H, NH), 8.340 (d,
J=7.8Hz, 1H, Ar-H5), 7.790 (d, J=7.8Hz, 1H, Ar-H6’), 7.741 (dd, J=1.8Hz, 7.8Hz, 1H,
Ar-H3’), 7.567 (td, J=1.2Hz, 7.8Hz, 1H, Ar-H4’), 7.522 (td, J=1.2Hz, 7.8Hz, 1H,
Ar-H5’), 7.408 (td, J=1.2Hz, 7.8Hz, 1H, Ar-H3), 7.333 (s, 1H, Ar-CO-CH=C), 7.022 (t,
J=15.6Hz, 1H, Ar-H4), 6.903 (d, J=7.8Hz, 1H, Ar-H2). EI-MS m/z: 328.00, 330.02,
331.12[M+1]+, calcd for C16H10BrNO2: 326.99.
(E)-3-(2-(3-chlorophenyl)-2-oxoethylidene)indolin-2-one(5j) red powder, 79.19%
1
yield, mp185.9-186.3°C. H-NMR (DMSO), δ: 10.810 (s, 1H, NH), 8.077 (d,
J=7.8Hz, 1H, Ar-H5), 8.031 (t, J=8.4Hz, 2H, Ar-H2’, Ar-H4’), 7.785 (dt, J=1.2Hz,
7.8Hz, 1H, Ar-H6’), 7.681 (s, 1H, Ar-CO-CH=C), 7.634 (t, J=15.6Hz, 1H, Ar-H5’),
7.360 (td, J=1.2Hz, 7.8Hz, 1H, Ar-H3), 6.966 (t, J=15.0Hz, 1H, Ar-H4), 6.895 (d,
J=7.8Hz, 1H, Ar-H2). EI-MS m/z: 284.11, 286,13[M+1]+, calcd for C16H10ClNO2:
283.04.
(E)-3-(2-(3,4-dichlorophenyl)-2-oxoethylidene)indolin-2-one(5k) red powder,
1
74.92% yield, mp230.2-231.1°C List[41]. H-NMR (DMSO), δ: 10.814 (s, 1H, NH),
8.226 (d, J=1.8Hz, 1H, Ar-H5), 8.113 (d, J=7.8Hz, 1H, Ar-H2’), 8.050 (dd, J=2.4Hz,
8.4Hz, 1H, Ar-H5’), 7.854 (d, J=8.4Hz, 1H, Ar-H6’), 7.668 (s, 1H, Ar-CO-CH=C),
7.364 (td, J=1.2Hz, 7.8Hz, 1H, Ar-H3), 6.969 (td, J=1.2Hz, 7.8Hz, 1H, Ar-H4), 6.883
(d, J=7.8Hz, 1H, Ar-H2). EI-MS m/z: 317.90, 320.18[M+1]+, calcd for C16H9Cl2NO2:
317.00.
(E)-3-(2-(4-bromophenyl)-2-oxoethylidene)indolin-2-one(5l) saffron powder, 75.43%
yield, mp203.8-205.2°C List[40]. 1H-NMR (DMSO), δ: 10.805 (s, 1H, NH), 8.045 (d,
J=7.8Hz, 1H, Ar-H5), 8.000 (dd, J=1.8Hz, 8.4Hz, 2H, Ar-H2’, Ar-H6’), 7.808 (dd,
J=1.8Hz, 2.4Hz, 2H, Ar-H3’, Ar-H5’), 7.677 (s, 1H, Ar-CO-CH=C), 7.353 (td, J=0.6Hz,
7.2Hz, 1H, Ar-H3), 6.958 (td, J=1.2Hz, 7.8Hz, 1H, Ar-H4), 6.884 (d, J=7.8Hz, 1H,
Ar-H2). EI-MS m/z: 327.93, 329.95[M+1]+, calcd for C16H10BrNO2: 326.99.
(E)-3-(2-(4-nitrophenyl)-2-oxoethylidene)indolin-2-one(5m) red powder, 77.24%

ACCEPTED MANUSCRIPT
1
yield, mp249.5-250.3°C (List[41] 246-248 °C). H-NMR (DMSO), δ: 10.838 (s, 1H,
NH), 8.387 (d, J=7.8Hz, 1H, Ar-H5), 8.289 (d, J=9.0Hz, 2H, Ar-H2’, Ar-H6), 8.165 (d,
J=7.8Hz, 2H, Ar-H3’, Ar-H5), 7.715 (s, 1H, Ar-CO-CH=C), 7.375 (t, J=15.0Hz, 1H,
Ar-H3), 6.979 (t, J=15.6Hz, 1H, Ar-H4), 6.890 (d, J=7.8Hz, 1H, Ar-H2). EI-MS m/z:
294.98[M+1]+, calcd for C16H10N2O4: 294.06.
(E)-3-(2-oxo-2-(3,4,5-trimethoxyphenyl)ethylidene)indolin-2-one(5n) red powder,
1
80.67% yield, mp169.5-170.4°C List[42]. H-NMR (DMSO), δ: 10.792 (s, 1H, NH),
8.005 (d, J=7.8Hz, 1H, Ar-H5), 7.728 (s, 1H, Ar-CO-CH=C), 7.353 (t, J=15.6Hz, 3H,
Ar-H2, Ar-H3, Ar-H4), 6.951 (t, J=15.0Hz, 1H, Ar-H2’), 6.880 (d, J=7.8Hz, 1H, Ar-H6’),
3.877 (s, 6H, 3’, 5’-OCH3), 3.777 (s, 3H, 4’-OCH3). EI-MS m/z: 340.17[M+1]+, calcd
for C19H17NO3: 339.11.
(E)-3-(2-(3,5-difluorophenyl)-2-oxoethylidene)indolin-2-one(5o) red powder, 78.90%
1
yield, mp197.3-200.7°C. H-NMR (DMSO), δ: 10.823 (s, 1H, NH), 8.127 (d,
J=7.8Hz, 1H, Ar-H5), 7.757 (d, J=6.6Hz, 1H, Ar-H2’), 7.736 (d, J=6.6Hz, 1H, Ar-H6’),
7.643 (s, 1H, Ar-H4’), 7.633 (s, 1H, Ar-CO-CH=C), 7.372 (td, J=1.2Hz, 7.8Hz, 1H,
Ar-H3), 6.976 (td, J=1.2Hz, 7.8Hz, 1H, Ar-H4), 6.888 (d, J=7.8Hz, 1H, Ar-H2). EI-MS
m/z: 286.26, 286.91[M+1]+, calcd for C16H9F2NO2: 285.06.
(E)-3-(2-(2-methoxyphenyl)-2-oxoethylidene)indolin-2-one(5p) yellow powder,
1
73.60% yield, mp160.8-161.8°C List[40]. H-NMR (DMSO), δ: 10.746 (s, 1H, NH),
8.206 (d, J=7.8Hz, 1H, Ar-H5), 7.709 (d, J=7.8Hz, 1H, Ar-H6’), 7.640 (t, J=15.6Hz,
1H, Ar-H4’), 7.573 (s, 1H, Ar-CO-CH=C), 7.349 (t, J=15.0Hz, 1H, Ar-H5’), 7.237 (d,
J=8.4Hz, 1H, Ar-H3’), 7.113 (t, J=15.0Hz, 1H, Ar-H3), 6.974 (t, J=15.0Hz, 1H,
Ar-H4), 6.878 (d, J=7,8Hz, 1H, Ar-H2), 3.890 (s, 3H, 2’-OCH3). EI-MS m/z:
280.07[M+1]+, calcd for C17H13NO2: 279.09.
(E)-3-(2-oxo-2-phenylethylidene)indolin-2-one(5q) yellow powder, 75.90% yield,
1
mp191.5-192.3°C (Lit.[38] 194-196°C). H-NMR (DMSO), δ: 10.797 (s, 1H, NH),
8.069 (d, J=7.8Hz, 2H, Ar-H2’, Ar-H6’), 8.007 (d, J=7.8Hz, 1H, Ar-H5), 7.733 (s, 1H,
Ar-CO-CH=C), 7.713 (d, J=6.6Hz, 1H, Ar-H4’), 7.605 (t, J=15.6Hz, 2H, Ar-H3’,
Ar-H5’), 7.343 (t, J=14.4Hz, 1H, Ar-H3), 6.850 (t, J=15.0Hz, 1H, Ar-H4), 6.885 (d,
J=7.8Hz, 1H, Ar-H2). EI-MS m/z: 250.12[M+1]+, calcd for C16H11NO2: 249.09.
(E)-3-(2-(4-amino-3,5-dichlorophenyl)-2-oxoethylidene)indolin-2-one(5r) yellow
1
powder, 73.60% yield, mp244.2-245.7°C. H-NMR (DMSO), δ: 10.750 (s, 1H, NH),
7.973 (d, J=7.8Hz, 1H, Ar-H5), 7.908 (d, J=7.8Hz, 2H, Ar-H2’, Ar-H6’), 7.607 (s, 1H,
Ar-CO-CH=C), 7.320 (t, J=15.6Hz, 1H, Ar-H3), 6.937 (t, J=14.4Hz, 1H, Ar-H4),
6.886 (d, J=7.8Hz, 1H, Ar-H2), 6.710 (s, 2H, 4’-NH2). EI-MS m/z: 332.75, 333.14,
334.96, 336.92[M+1]+, calcd for C16H11Cl2NO2: 332.01.
(E)-N-(3-(2-(2-oxoindolin-3-ylidene)acetyl)phenyl)acrylamide(6a)
powder, 70.53% yield, mp222.8-224.1°C List[43]. H-NMR (DMSO), δ: 10.780 (s,
red-brown
1

ACCEPTED MANUSCRIPT
1H, NH), 10.583 (s, 1H, CO-NH-Ar), 8.075(d, J=9.0Hz, 2H, Ar-H5, Ar-H2’), 7.976 (d,
J=7.2Hz, 1H, Ar-H4’), 7.833(d, J=8.4Hz, 2H, Ar-H5’, Ar-H6’), 7.707 (s, 1H,
Ar-CO-CH=C), 7.330 (td, J=1.2Hz, 7.8Hz, 1H, Ar-H3), 6.941 (td, J=1.2Hz, 7.8Hz, 1H,
Ar-H4), 6.880 (d, J=7.8Hz, 1H, Ar-H2), 6.495-6.450 (m, 1H, N-CO-CH=C), 6.321 (dd,
J=1.2Hz, 7.8Hz, 1H, CO-CH=C), 5.831 (dd, J=1.8Hz, 1.8Hz, 1H, N-CO-CH=C).
ESI-MS m/z: 319.08 [M+1]+, calcd for C19H14N2O3: 318.10.
(E)-N-(3-(2-(2-oxoindolin-3-ylidene)acetyl)phenyl)isobutyramide(6b)
saffron
1
powder, 75.33% yield, mp192.8-193.8°C. H-NMR (DMSO), 10.811 (s, 1H, NH),
10.124 (s, 1H, CO-NH-Ar), 8.348 (t, J=3.6Hz, 1H, Ar-H5), 8.007 (d, J=7.2Hz, 1H,
Ar-H2’), 7.971 (dd, J=1.2Hz, 7.8Hz, 1H, Ar-H4’), 7.729 (d, J=7.8Hz, 1H, Ar-H5’),
7.682 (s, 1H, Ar-CO-CH=C), 7.524 (t, J=16.2Hz, 1H, Ar-H6’), 7.349 (td, J=1.2Hz,
7.8Hz, 1H, Ar-H3), 6.960 (td, J=1.2Hz, 7.8Hz, 1H, Ar-H4), 6.890 (d, J=7.8Hz, 1H,
Ar-H2), 2.629-2.584(m, 1H, CH), 1.125 (s, 3H, CH3), 1.114 (s, 3H, CH3). ESI-MS
m/z: 335.16[M+1]+, calcd for C20H18N2O3: 334.13.
(E)-N-(3-(2-(2-oxoindolin-3-ylidene)acetyl)phenyl)propionamide(6c)
red-brown
1
powder, 78.72% yield, mp191.1-192.5°C. H-NMR (DMSO), δ: 10.808 (s, 1H, NH),
10.156 (s, 1H, CO-NH-Ar), 8.321 (t, J=3.0Hz, 1H, Ar-H5), 8.002 (d, J=7.8Hz, 1H,
Ar-H2’), 7.953 (dd, J=1.2Hz, 7.2Hz, 1H, Ar-H4’), 7.724 (dt, J=1.2Hz, 6.6Hz, 1H,
Ar-H5’), 7.677 (s, 1H, Ar-CO-CH=C), 7.523 (t, J=15.6Hz, 1H, Ar-H6’), 7.347 (td,
J=1.2Hz, 7.8Hz, 1H, Ar-H3), 6.957 (td, J=1.2Hz, 7.8Hz, 1H, Ar-H4), 6.889 (d,
J=7.8Hz, 1H, Ar-H2), 2.370-2.332 (m, 2H, N-CO-CH2), 1.098 (t, J=7.2Hz, 3H, CH3).
ESI-MS m/z: 321.10[M+1]+, calcd for C19H16N2O3: 320.12.
(E)-N-(3-(2-(2-oxoindolin-3-ylidene)acetyl)phenyl)acetamide (6d) yellow powder
70.71% yield, mp 236.1-238.5°C. 1H-NMR (DMSO) δ: 10.809 (s, 1H, NH), 10.232 (s,
1H, CO-NH-Ar), 8.286 (t, J=1.2Hz, 1H, Ar-H5), 7.999 (d, J=7.2Hz, 1H, Ar-H2’),
7.937 (dd, J=1.2Hz, 7.2Hz, 1H, Ar-H4’), 7.731 (d, J=7.8Hz, 1H, Ar-H5’), 7.672 (s, 1H,
Ar-CO-CH=C), 7.538-7.512 (m, 1H, Ar-H6’), 7.348 (td, J=1.2Hz, 7.8Hz, 1H, Ar-H3),
6.957 (td, J=1.2Hz, 7.8Hz, 1H, Ar-H4), 6.889 (d, J=7.8Hz, 1H, Ar-H2), 2.073 (s, 3H,
CH3). ESI-MS m/z: 307.10[M+1]+, calcd for C18H14N2O3: 306.10.
4.2. Synthetic procedures
4.2.1. General synthetic procedure for the synthesis of 2
To a stirred solution of chloral hydrate (2 g, 16.5 mmol) in water (30 mL), anhydrous
sodium sulfate (13 g, 91 mmol) was added. The reaction was stirred at 35 °C, then the
aqueous solution of aniline (10 mmol), concentrated hydrochloric acid(1.2 mL), and
hydroxylamine hydrochloride (2.5 g, 37 mmol) were sequentially added. The mixture
was stirred at 80 °C for 2 hours. After 1 hour, the mixure was cooled to 60 °C, during
which a precipitate was formed. After filtration and washing with water, the crude
product was uesd next step without purification.

ACCEPTED MANUSCRIPT
4.2.2. General synthetic procedure for the synthesis of 3
To a stirred solution of compound 2 (1 g, 6.1 mmol) in concentrated sulfuric acid (30
mL), the mixture was stirred at 60-65 °C for 30 minutes. Then the reaction stirred at
80 °C for 15 minutes. After 30 minutes, the mixture was cooled to 70 °C, and ice
water (100 mL) was added. The mixture was kept at 0-5 °C for 1 h, during which a
precipitate was formed, After filtration and washing with water, the crude product
was dissolved in NaOH solution at 60 °C, and added acetic acid to neutralize. Then
the mixture cooled to room temperature, the precipitate was formed. After filtration
and washing with water, 10% cold ethanol, the crude product was purified by silica
gel chromatography.
4.2.3. General synthetic procedure for the synthesis of compound 5a-5r
To a stirred solution of compound 3 (0.3 g, 2.04 mmol) in ethanol (2 mL), equivalent
substituted acetophenone was added. The reaction stirred at room temperature for
2-24 h, the precipitate was formed. After filtration and washing with water, the crude
product was uesd next step without purification. Then the crude product was
dissolved in ethanol solution (10 mL), concentrated hydrochloric acid (1 mL) was
added. The mixture was stirred at 60 °C for 2 h. Then the mixture was poured into ice
water, the precipitate was formed. After filtration and washing with water, the crude
product was purified by silica gel chromatography using hexane and ethyl acetate
gradient to obtain desired products 5a-5r.
4.2.4. General synthetic procedure for the synthesis of compound 6a-6d
To a stirred solution of compound 5a (0.2 g, 0.75 mmoL) in anhydrous
tetrahydrofuran (5 mL), and cooled to 0 °C, 1.5 equivalent acid chloride and 3 drops
triethylamine were added. The reaction stirred at room temperature for 2 h. Then the
mixture was poured into ice water, the precipitate was formed. After filtration and
washing with water, the crude product was purified by silica gel chromatography
using hexane and ethyl acetate gradient to obtain desired products 6a-6d.
4.3 Biological evaluation
4.3.1 Cell culture and reagents
NSCLC cell line NCI-H460, human gastric cancer cell lines SGC-7901 and BGC-823
were purchased from the Institute of Biochemistry and Cell Biology, Chinese

ACCEPTED MANUSCRIPT
Academy of Sciences. NCI-H460, SGC-7901 and BGC-823 cells were cultured in
RPMI 1640 medium (Gibco Technologies, Eggenstein, Germany) containing 10%
fetal bovine serum (FBS) and 1% streptomycin, and all cells were cultured in
incubator containing 5% CO2 at 37 °C with RPMI 1640 medium. Curcumin,
xanthohumol, dimethylsulfoxide (DMSO) and methyl thiazolyl tetrazolium (MTT)
were obtained from Sigma-Aldrich (St.Louis,MO). Antibodies including anti-Bcl-2,
anti-GAPDH and goat anti-rabbit IgG-HRP were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Propidium Iodide (PI) were purchased from BD
Pharmingen (Franklin Lakes, NJ).
4.3.2 MTT assay
NCI-H460, SGC-7901 and BGC-823 cells were seeded in 96-well plates at 4000 cells
per well for 24 h. The tumor cells were incubated with compounds (60, 20, 20/3, 20/9,
20/27 µM) for 72 h. After treatment, a solution of MTT (5 mg/mL) prepared in
phosphate buffer solution (PBS) was added to the cells in each well for 4 h at 37 °C
.
Then the MTT was aspirated, added 150 µL DMSO per well, shook 10 mins,
quantified with a microplate reader (SpectraMax M2/M2e, Molecular Devices,
Sunnyvale, USA) at a wavelength of 490 nm. The percentage of growth inhibition
rate was calculated from the comparison with the vehicle control. The IC50 values of
each compound were calculated by Graph Pad Pro 5.0 (San Diego, CA).
4.3.3 Quantitative structure-activity relationships analysis
All compounds were drawn and saved as SDF format. The descriptors of compounds
were obtained by upload the SDF format structures to the ChemoPy descriptor web
server (http://www.scbdd.com/chemopy_desc/index/). In the present study, the
descriptors include the Geometric descriptors, CPSA descriptors, WHIM descriptors,
RDF descriptors, MoRSE descriptors, etc. Then, the missing value of the molecular
descriptors were removed and random forest algorithm was used to find the best
feature subset. In this algorithm, 5-fold cross validation was repeated 5 times to assess
the results. The number of iterations was set to 500 to acquire the best molecular
descriptor subset.
4.3.4 Colony formation assay
NCI-H460 cells were seeded into 6-well plates at a density of 1000 cells per well for
24 h, and treated with 6a, curcumin, xanthohumol and vehicle (DMSO) for 18 h. The
culture medium was replaced by the fresh RPMI 1640 medium, aspirated the medium,

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washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, washed with
PBS three times, and finally stained with crystal violet 15 min, PBS washed twice.
4.3.5 Cell migration assay
NCI-H460 cells (8×106 cells/well) were seeded on 6-well tissue culture plates and
grown to 100% confluence. We scratched the cells in the same with using a sterile
pipette tip and washed with PBS to remove the floating cells, then treated with 6a,
curcumin, xanthohumol and vehicle (DMSO) for 48 h. Then the images were captured
with a microscopic camera system (Nikon, Tokyo, Japan).
4.3.6 Cell cycle analysis
NCI-H460 cells (3×105 cells/well) were seeded in six well plates. cells were treated
with 6a, curcumin , xanthohumol and vehicle (DMSO) for 48 h, washed with PBS,
fixed in 75% ice cold ethanol for 4 h. Then cells were stained with 500 µL of
propidium iodide (PI) containing RNase (550825, BD Biosciences 35 Clontech, San
Jose, CA, USA) for 10 mins at 4ꢁ°C away from light and filtered with a 200-mesh
gauze. Cell cycle analysis was performed in an FACS Calibur
Biosciences, CA)
flow cytometer (BD
4.3.7 Anti-tumor study in vivo
The antitumor effect of 6a was evaluated by xenograft tumor assay in nude mice.
NCI-H460 cells were collected and subcutaneously injected into the right flank of
6-week-old BALB/C-nu nude mice. Seven days after injection of NCI-H460 cells,
mice were intraperitoneally injected with 6a solution (36 mg 6a dissolved in 12 mL of
solvent (solvent = PBS: castor oil: polyethylene glycol = 7:2:1) at a dose of 15
mg/kg/Day, mice were injected intraperitoneally with curcumin solution (36 mg of
curcumin dissolved in 12 mL of solvent) intraperitoneally and injected with solvent (n
= 5 per group) in the intraperitoneal group of mice in the blank group for 16 day. The
tumor volume was measured by measuring their length (l) and width (w) to determine
the tumor and using the formula:V = (3.14/6) * ((w * w) * l). The weight of nude
mice was recorded every two days. The weight of tumors were recorded on the day of
killing the nude mice.
4.3.8 Western blotting analysis
The homogenized tumor tissue was lysed. The protein concentration of all samples
was determined using the Bradford Protein Assay Kit (Bio-Rad, Hercules, CA). The
lysates were separated by SDS-PAGE electrophoresis and then transferred to PVDF

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membranes. After blocking for 1.5 h at room temperature with TBST containing 5%
skim milk, the membrane was incubated with primary antibody Bcl-2 overnight at
4
°C. After washing three times with TBST, the membrane was incubated for 1.5 h
with the corresponding secondary antibody. The depth of the bands was detected with
the ECL kit (Bio-Rad, Hercules, CA), the results were analyzed with Quantity One
software (BioRad).
4.3.9 Hematoxylin and eosin (H&E) staining
Liver and the lungs were taken from the nude mice, and then fixed with
paraformaldehyde. Tissue was embedded in paraffin and cut into 5 µm paraffin
sections. H&E staining on paraffin sections was performed according to the
instructions for the staining kit (C0105; Beyotime Biotech).
4.3.10 Statistical analysis
The results were expressed as mean ± standard error (SEM). And all experiments
were repeated at least three times. We used Student's t in GraphPad Pro (GraphPad,
San Diego, CA, USA) to examine the statistics. If P values were less than 0.05 (p&lt;
0.05) then they were considered statistically significant.
Acknowledgement
This work was supported by the ZheJiang Province Natural Science Fund of China
(Grant Nos. LY17H160059), the National Natural Science Foundation of China
(Grant Nos. 81703766, 81402839), The Opening Project of Zhejiang Provincial Top
Key Discipline of Pharmaceutical Sciences, National Undergraduate Training
Programs for Innovation and Entrepreneurship (201710343004), Wenzhou Medical
Universtiy Undergraduate Training Programs for Innovation and Entrepreneurship
(wyx2017101038).
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Highlights
◇ A series of novel isatin analogues inspired Michael acceptor were designed and
synthesized on the basis of association principle, and most analogues showed good
anti-growth activity towards cancer cells.
◇ A quantitative structure-activity relationship analysis (QSAR) model was
constructed to investigate these analogues.
◇ 6a significantly inhibited NCI-H460 cells growth in vitro and had more remarkable
reduction of tumor growth than positive drug curcumin in vivo.