Synthesis and anti-tumor activity of EF24 analogues as IKKβ inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.11.077

EF24 is an IKKβ inhibitor (IC₅₀: 72 μM) containing various anti-tumor activities. In this study, a series of EF24 analogs targeting IKKβ were designed and synthesized. Several IKKβ inhibitors with better activities than EF24 were screened out and B3 showed best IKKβ inhibitory (IC₅₀: 6.6 μM). Molecular docking and dynamic simulation experiments further confirmed this inhibitory effect. B3 obviously suppressed the viability of Hela229, A549, SGC-7901 and MGC-803 cells. Then, in SGC-7901 and MGC-803 cells, B3 blocked the NF-κB signal pathway by inhibiting IKKβ phosphorylation, and followed arrested the cell cycle at G2/M phase by suppressing the Cyclin B1 and Cdc2 p34 expression, induced the cell apoptosis by down-regulating Bcl-2 protein and up-regulating cleaved-caspase3. Moreover, B3 significantly reduced tumor growth and suppressed the IKKβ-NF-κB signal pathway in SGC-7901 xenograft model. In total, this study present a potential IKKβ inhibitor as anti-tumor precursor.

Full text of DOI:10.1016/j.ejmech.2017.11.077

Accepted Manuscript
The synthesis and anti-tumor activity of EF24 analogues as IKKβ inhibitors
Rong Jin, Qiuxiang Chen, Song Yao, Encheng Bai, Weitao Fu, Ledan Wang, Jiabing
Wang, Xiaojing Du, Tao Wei, Haineng Xu, Chengxi Jiang, Peihong Qiu, Jianzhang
Wu, Wulan Li, Guang Liang
PII:
S0223-5234(17)30980-7
10.1016/j.ejmech.2017.11.077
EJMECH 9952
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 September 2017
Revised Date: 26 November 2017
Accepted Date: 27 November 2017
Please cite this article as: R. Jin, Q. Chen, S. Yao, E. Bai, W. Fu, L. Wang, J. Wang, X. Du, T. Wei, H.
Xu, C. Jiang, P. Qiu, J. Wu, W. Li, G. Liang, The synthesis and anti-tumor activity of EF24 analogues as
IKKβ inhibitors, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.11.077.
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Graphical abstract:
B
Anti-tumor activity

ACCEPTED MANUSCRIPT
The synthesis and anti-tumor activity of EF24 analogues as
IKKβ inhibitors
Rong Jinab1, Qiuxiang Chencb1, Song Yaob1, Encheng Baiab1, Weitao Fub1, Ledan
Wangd, Jiabing Wangb, Xiaojing Duba, Tao Weib*, Haineng Xub, Chengxi Jiangbe,
Peihong Qiub, Jianzhang Wube*, Wulan Libf*, Guang Liangbe
(aDepartment of Digestive Diseases, the First Affiliated Hospital of Wenzhou Medical University,
Wenzhou, Zhejiang 325000, China)
(bChemical Biology Research Center, College of Pharmaceutical Sciences, Wenzhou Medical
Universtiy, Wenzhou, Zhejiang 325035, China)
(cdepartment of ultrasound，the First Affiliated Hospital of Wenzhou Medical University, Wenzhou,
Zhejiang 325000, China)
(dDepartment of Gynecology and Obstetrics, The 2nd Affiliated Hospital of the Wenzhou Medical
University, Wenzhou, Zhejiang 325000, China)
(eWenzhou Biomedical Innovation Center, Wenzhou University and Wenzhou Medical University,
Wenzhou, Zhejiang 325000, China)
(fCollege of Information Science and Computer Engineering, The First Clinical Medical College,
Wenzhou Medical University, Wenzhou, Zhejiang 325035, China)
1 These authors contribute to this work equally
* Corresponding author: Tao Wei
Address: Chemical Biology Research Center, College of Pharmaceutical Sciences, Wenzhou
Medical University, Wenzhou, Zhejiang 325035, China
E-mail: weitao@wzmu.edu.cn
* Corresponding author: Jianzhang Wu
Address: Chemical Biology Research Center, College of Pharmaceutical Sciences, Wenzhou
Medical University, Wenzhou, Zhejiang 325035, China
E-mail: wjzwzmu@163.com
* Corresponding author: Wulan Li
Address: Chemical Biology Research Center, College of Pharmaceutical Sciences, Wenzhou
Medical University, Wenzhou, Zhejiang 325035, China
E-mail: lwlwzmu@163.com

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Abstract:
EF24 is an IKKβ inhibitor (IC50: 72 µM) containing various anti-tumor activities. In
this study, a series of EF24 analogs targeting IKKβ were designed and synthesized.
Several IKKβ inhibitors with better activities than EF24 were screened out and B3
showed best IKKβ inhibitory (IC50: 6.6 µM). Molecular docking and dynamic
simulation experiments further confirmed this inhibitory effect. B3 obviously
suppressed the viability of Hela229, A549, SGC-7901 and MGC-803 cells. Then, in
SGC-7901 and MGC-803 cells, B3 blocked the NF-κB signal pathway by inhibiting
IKKβ phosphorylation, and followed arrested the cell cycle at G2/M phase by
suppressing the Cyclin B1 and Cdc2 p34 expression, induced the cell apoptosis by
down-regulating Bcl-2 protein and up-regulating cleaved-caspase3. Moreover, B3
significantly reduced tumor growth and suppressed the IKKβ-NF-κB signal pathway
in SGC-7901 xenograft model. In total, this study present a potential IKKβ inhibitor
as anti-tumor precursor.
Key words: IKKβ inhibitor, synthesis, molecular docking, anti-tumor activity
1. Introduction
IκB kinase beta (IKKβ) is one of the key roles in IKKβ-NF-κB pathway that
regulates cell proliferation and survival [1, 2]. Under normal physiological condition,
NF-κB is located in the cytosol complexed with the inhibitory protein IκBα. Various
stimulus such as cytokines, free radicals, and bacterial or viral antigens lead to the
activation of IKKβ, which in turn, phosphorylated the IκBα protein, and separated
NF-κB from IκBα [3-5]. The activated NF-κB then nuclear translocated to form
DNA/NF-κB complex and regulated the expression of related genes, which can be
associated with cancer, inflammatory, autoimmune diseases and other
pathophysiological processes [6-9].
Notably, IKKβ-NF-κB pathway is constitutively active in many different types of
human tumors [10]. Silencing or inhibiting the expression of IKKβ or suppressing its
activation can present anti-tumor effect. Up to now, IKKβ has been regarded as an
important target for cancer therapy [11], and developing IKKβ inhibitors is considered
as one of the most promising strategies in the related tumors treatment. IKKβ
inhibitors are currently focused on small molecule inhibitor drugs such as SC-514,
PS1145, ML120B, and BMS345541. Among them, PS1145 could enhance apoptosis
in DMBA-induced tumor in male wistar rats, and overcome imatinib resistance
through suppressing the phosphorylation of IKKβ [12-14]. BMS345541 is a kind of
non-ATP competitive IKK inhibitor, and a couple of literature indicated that it can
induce apoptosis of tumor cells including melanoma, HTLV-1 and T-cell acute
lymphoblastic leukemia [15-17]. Although anti-tumor agents like those IKKβ
inhibitors were constantly reported [18, 19], rare of them succeeded in clinical
practice. So, it’s urgent to develop novel IKK inhibitors for anti-tumor therapy.
EF24 (3,5-bis((E)-2-fluorobenzylidene)piperidin-4-one) potently suppresses the

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NF-κB signaling pathway and induces death of lung, breast, ovarian, and cervical
cancer cells through directly inhibiting the catalytic activity of IKKβ [20-22].
However, according to known literature [23] (IC50: 72 µM) and our early work
(34.5% inhibitory ratio at 20 µM), its inhibition to IKKβ was not remarkable enough.
Therefore, designing newly IKKβ inhibitors based on EF24 is a convenient, effective
and necessary strategy to develop novel anti-tumor drugs. In this study, taking EF24
as a precursor, we synthesized a series of EF24 analogs and screened out better IKKβ
inhibitors than EF24.
2. Results and discussion
2.1 Chemistry
The basic structure of EF24 can be described as terminal phenyl chain like
conjugated alkone, which could be abstracted as the combination of ketone bridges
and substituted phenyl (Scheme 1A). In order to modify its activity and find better
IKK inhibitors, combination of different ketone bridges (containing five/six
membered cycle ketones and acetone) and substituted phenyl furnished four kinds of
EF24 analogs which differed from each other. And the synthesis of these novel EF24
analogs can be easily achieved by aldol condensation reaction between (cyclic)
ketones and aldehydes under diluted acids or bases catalyzation (Scheme 1B).
Generally, the synthesis of A, B, and D series can be catalyzed by NaOH/EtOH/H2O
system, but this system didn’t work on the C series which contained phenolic
hydroxyl group, so we turn to AcOH/HCl/EtOH system, and it worked though only
low yields were got. The structures of A-D series compounds were shown in Scheme
1C, and these compounds were confirmed by nuclear magnetic resonance (1H NMR)
spectroscopy and mass spectrometry (MS) analysis. Several novel compounds
(Scheme 1C: A4, A6, C5 and D1-D4) was firstly reported in this study.
Scheme 1. The optimization design (A), synthetic routes (B) and structure (C) of EF24 analogs.
Synthetic procedures: ketone (1.0 mmol) and aldehyde (2.5 mmol) was dissolved into 10 mL
o
EtOH, catalyzed by 40% NaOH or AcOH/HCl, and refluxed at 78 C for hours until the reaction
was accomplished.

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2.2 The screening of inhibitory activity to IKKβ kinase
In order to find better IKKβ inhibitors than EF24, considering the structural feature
of EF24, four series of EF24 analogues were designed (Scheme 1A, 1C) and their
inhibitory activity to IKKβ kinase was tested (Figure 1A). Firstly, different ketone
bridges and aldehydes with different substitution positions and numbers of fluorine
atom were matched to furnish analogues of series A. As shown in Scheme 1A, 1C
and Figure 1A, the substitution of the ketone bridge decreased the inhibitory activity,
while increased fluorine atom number or fluorine positional change promoted the
activity. Then, replacing the electron-withdraw group fluorine to approximations such
as bromine, chlorine and trifluoromethyl, and the matches provided analogues of
series B. It’s basically found other halogens promoted the activity, while the
substitution of the ketone bridge draw it back. What’s more, electron-donating group
phenolic hydroxyl was also investigated in the combination of kinds of ketone bridges,
referred to analogues of series C. The results indicated that phenolic hydroxyl did no
favor to IKKβ inhibitory in the EF24 analogues, even changing the kind of ketone
bridge. Finally, another kind of electron-donating group N-alkyl was explored in this
combination to provide analogues of series D. The results further confirmed that
electron-donating groups on benzene ring decreased the IKKβ inhibitory, as well as
replacing the ketone bridge.
Then some analogues with relatively better inhibitory than EF24 at 20 µM level
were measured IC50 values. As shown in Figure 1B, analogue B3 possessed the best
activity and the IC50 value was as low as 6.6 µM.
A
B
Comp.
A2
IC50 (µM)
14.0±1.4
A3
A4
A7
B3
7.3±2.1
10.7±0.8
19.6±2.8
6.6±1.5
Figure 1. The inhibitory activity against IKKβ kinase of EF24 analogues. (A) The inhibitory rates
of compounds at 20 µM. (B) The IC50 values of active compounds. Results shown are the
means±S.E.M.s of three independent tests.
2.3. Molecular docking and molecular dynamics simulation
Until the crystal structure of IκB kinase β inhibitor was revealed [24], rare
interactions mechanism between IKKβ kinase and its inhibitors were reported. In
order to explore the inhibitory mechanism of the EF24 analogs, molecular docking
and dynamics simulation experiment were carried out. We investigated interactions

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between the active compound B3 and the kinase domain of IKKβ (The
crystallographic co-ordinates for human IKKβ (PDB ID: 4KIK) was obtain from the
Protein Data Bank [25]) using the software Autodock (version 4.2.6) [26]. From an
overall determination of root mean square deviations (RMSD) and estimated free
energy, the only pose of docking results were chosen as initial structures for molecular
dynamics (MD) simulation. The RMSD of the receptor in IKKβ/B3 complex was
converged after 10-20 ns in 50 ns MD simulations, and the RMSD of backbone atoms
was ≈ 3-4 Å (Figure 2A). Binding energies for B3 were calculated by MM-GBSA
using 500 snapshots from 40 to 50 ns stable trajectories. The only one pose of B3
were carried out for MD simulation. The result showed that B3 interacted with ten
key residues: Leu-21, Val-29, Ile-165, Gly-22, Val-152, Glu-149 Thr-23, Met-96,
Ala-42 and Gly-24 (Figure 2B). Interestingly, except Glu-149 and Thr-23, all these
residues were hydrophobic amino acids, indicating that B3 likely interact primarily
with non-polar residues in IKKβ to exert their inhibitory activity. Kinases can exist as
active and/or inactive states in cells. The hydrophobic residues in kinases interact to
form “hydrophobic clusters” that stabilize the inactive conformation and interfere
with ATP binding. The whole molecule of B3 was sandwiched in a hydrophobic cleft
between the residues Leu-21, Val-29, Ile-165, Gly-22, Val-152, Glu-149 Thr-23,
Met-96, Ala-42 and Gly-24 (Figure 2C). The phenyl ring in B3 created p-δ stacking
with the isopropyl chains of Leu-21 and Val-152 to stabilize the binding conformation,
and another phenyl ring occupied the main hydrophobic pocket by making
hydrophobic contacts with residues Gly-22, Val-29, and Ile-165. In summary, a
critical hydrophobic interaction between B3 with IKKβ kinase domain was predicted
by a rational method combined with molecular docking and molecular dynamics
simulation. These methods clarified the interaction mechanisms of B3 with IKKβ. The
further verification of co-crystallization of IKKβ/B3 is in progress.
Figure 2. Molecular docking and dynamics analysis of B3. (A) Time evolution of the RMSD of
IKKβ and B3 are shown with green lines and orange lines, respectively. (B) Per-residue of top 10
contribution to the binding effective energy of B3. Per-residue contributions were calculated by
the MM-GBSA decomposition method. (C) Last snapshot of IKKβ/B3 in 50 ns MD simulations.
B3 (green), top 10 of residues contribution to the binding effective energy (cyan).

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2.4. B3 down-regulated p-IKKα,β and inhibited the degradation of IκBα leading to
IKK-NF-κB pathway suppression
IKK-NF-κB pathway can be triggered by a variety of stimulus including growth
factors, cytokines, lymphokines, UV, pharmacological agents, and stress. Then, IκBα
is phosphorylated and followed by occurrence of ubiquitination and degradation. This
results in the exposure of the nuclear localization signals (NLS) on NF-κB subunits
and the subsequent translocation of p65 to the nucleus. Our data indicated that B3
could dose-dependently inhibited TNF-α induced phosphorylation of IKKα,β and
degradation of IκBα (Figure 3A, 3B). We further investigated the effect of B3 on p65
translocation in SGC-7901 cell lines. Results demonstrated that B3 could decrease
nucleus translocation of p65 induced by TNF-α treatment (Figure 3C). Taken together,
B3 may suppress IKK-NF-κB signaling pathway via targeting IKKβ.
Figure 3. B3 inhibited TNF-α induced activation of IKK-NF-κB pathway. SGC-7901 (A) and
MGC-803 (B) cells were treated with B3 for 1 h and followed stimulated with TNF-α for 10 min.
Then, proteins were extracted and analyzed by western blot assay. IKKα,β and GAPDH were
control band for p-IKKα,β and GAPDH, respectively. **p < 0.01 and *p < 0.05 denote significant
changes compared with the TNF-α group. (C) B3 decreased TNF-α induced p65 nuclear
translocation. SGC-7901 cells were pre-treated with B3 (10 µM) or BMS345541 (10 µM) for 1h,
followed treatment with TNF-α (1 ng/mL) for 1h. After stained, translocation of p65 was observed
under a fluorescence microscope. The red color represents p65 staining, the blue color represents
nuclei staining, and the reddish blue color represents nuclear translocation.
2.5. B3 suppressed the viability of cancer cells
It’s well known that NF-κB is constitutively active in many kinds of tumors leading
to malignant proliferation and anti-apoptosis [27]. EF24 and BMS345541 were
reported as IKK targeted agents and had multi anti-cancer capabilities [28, 29]. Since
B3 was verified to be IKKβ inhibitor, its anti-tumor activity was investigated. At first,
its ability of anti-growth was studied in four kinds of different tumor cells including
Hela229, A549, SGC-7901 and MGC-803 cells. B3 obviously suppressed the viability
of these cancer cells and its potency was equal to EF24 and better than BMS345541
(BMS) by MTT assay (Table 1).

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Table 1. The compounds’ IC50 values to the viability of different kinds of cancer cells.
IC50 (µM)
Comp.
HeLa229 A549
SGC-7901 MGC-803
B3
1.8±0.7
1.9±0.5
9.4±4.3
2.1±0.9
1.2±0.6
1.0±0.1
8.3±4.5
1.8±0.3
2.0±0.9
14.2±2.6
EF24
2.8±2.1
9.3±1.7
BMS345541
2.6. B3 arrested the cell cycle at G2/M period in SGC-7901 and MGC-803 cells and
suppressed the cyclinB1 and Cdc2 p34 expression
IKK-NF-κB cascade can regulate the expression of cell cycle proteins and
influence cell cycle progression. Anti-tumor agents usually suppressed tumor cell
growth through regulating cyclin proteins, which resulted in cell cycle arrest [30, 31].
To further verify the anti-tumor activity of B3, the cell cycle and cyclin proteins of
SGC-7901 cells treated with B3 were detected by FMC and western blotting test. It
found that the cell cycle of SGC-7901 cells were arrested at G2/M period (Figure 4A,
4C) and crucial protein cyclin B1 and Cdc2 p34 were down-regulated, too (Figure
4D). Similar results could be also reproduced in MGC-803 cells (Figure 4B, 4C and
4E). Cyclin proteins like Chk1, Cdc25C, Cdc2 and Cyclin B1 are highly involved in
cell cycle, especially in G2/M transition, and high Cyclin B1 expression levels are
found in variety of cancers such as breast, cervical, gastric, and lung cancer [32]. Our
results showed that B3 decreased Cdc2 and Cyclin B1 expression in dose-effect
manner, which means selected cancer cells were actually sensitive to B3 treatment
and its growth activity was cut down partially due to the lack of crucial cyclin
proteins.
Figure 4. B3 arrested cell cycle at G2/M period and down-regulated cyclin B1 and Cdc2 p34

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protein expression. (A-C) Cell cycle components analysis by flow cytometry. SGC-7901 (A) and
MGC-803 (B) cells were treated with B3 for 12 h, than cells were collected, stained and analyzed
by flow cytometry. (D-E) Cyclin proteins expression detection. SGC-7901 (D) and MGC-803 (E)
cells were treated with B3 for 12 h and Cyclins were analyzed by western blot assay. *p < 0.05,
**p < 0.01 and ***p < 0.001 denote significant changes compared with the DMSO group.
2.7. B3 induced apoptosis in gastric cancer cells and down-regulated Bcl-2 protein
expression while up-regulated cleaved caspase-3
In tumor cells, IKK-NF-κB axis can upregulate survival-related genes and
downregulate apoptotic-related genes, which resulted in malignant proliferation,
anti-apoptosis and chemotherapy drug resistance [33]. Bcl-2 (B-cell lymphoma-2)
protein is one of the most important survival proteins and can activate tumor cells to
escape from procedural apoptosis. In contrast, caspases protein family played
essential roles in programmed cell death. And the activation of caspases is considered
as bio-index in procedural apoptosis [34]. The cell apoptosis involved executers
include caspase 3, 6 and 7. In our study, B3 dose-dependently induced cell apoptosis
in SGC-7901 and MGC-803 (Figure 5A, 5B) cells, and had better effect than
BMS345541. What’s more, after treatment with B3, Bcl-2 protein expression was
suppressed while cleaved-caspase 3 was up-regulated (Figure 5C, 5D). It indicated
that B3 induced apoptosis-related proteins regulation and led to cell apoptosis.
Figure 5. B3 induced SGC-7901 and MGC-803 cell apoptosis, and regulated apoptosis-related
protein expression. (A) Flow cytometry for cell apoptosis. SGC-7901 or MGC-803 cells were
treated with agents as indicated for 12 h. Then apoptosis was detected. (B) Statistics of cell
apoptosis. (C-D) Apoptosis-related proteins expression analysis. Cells were treated with B3 for 12
h and apoptins were analyzed by western blot assay. *p < 0.05, **p < 0.01 and ***p < 0.001
denote significant changes compared with the DMSO group.
2.8. B3 suppressed the SGC-7901 cell growth in nude mice xenograft model
Xenograft tumor model is a common method to evaluate the anti-tumor activity in
vivo. We further established a xenograft tumor model through subcutaneously injected
SGC-7901 cells to nude mice. Our data showed that B3 can significantly restrict
tumor growth (Figure 6A). Meanwhile, there was no significant decrease in mouse

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body weight, which indicated the controllable toxicity of B3 treatment in mice
(Figure 6B). Further detection of tumor tissues mainly focused on the anti-tumor
mechanism of B3 in vivo. Results indicated that B3 can significantly block
IKK-NF-κB pathway in vivo (Figure 6C, 6D). As stated above, Bcl-2 as a crucial
apoptin could be signally down-regulated by B3 in vivo. Our aforementioned results
favored that B3 displayed outstanding anti-tumor activity in vitro and in vivo as a
novel IKKβ inhibitor.
Figure 6. Anti-tumor activity of B3 in vivo. (A) Tumor volume was monitored.
Statistical analysis of TGI (tumor growth inhibition) was conducted using a Student t
test. TGI = (RTVVehicle - RTVtreatment)/RTVVehicle × 100. RTV = relative tumor
volume on the given day versus at the start of treatment. ip means intraperitoneal
injection. (B) Mice body weight. (C-D) Western blot assay on the level of p-IKKα,β,
IκBα and Bcl-2 from respective tumor tissue lysates. *p < 0.05 denote significant
changes compared with the DMSO group.
3. Conclusion
In this study, a series of EF24 analogs targeting to IKKβ were designed and
synthesized. Several analogs showed better activity against IKKβ kinase than that of
EF24, and B3 showed best IKKβ inhibitory (IC50: 6.6 µM). Due to recently revelation
of the crystal structure of IKKβ, rare interactions mechanism between IKKβ kinase
and its inhibitors were reported. Through molecular docking and molecular dynamics
simulation, we found that B3 occupied the IKKβ catalytic pocket and was stabilized
by hydrophobic interaction with Leu-21, Val-152, Gly-22, etc. At cells level, B3 could
inhibit the phosphorylation of IKKβ and IKK-NF-κB axis, and suppressed the
expression of Cyclin B1 and Cdc2 p34, which leaded to the G2/M phase arrest and
tumor cell growth inhibition. Also, B3 could down-regulated the expression of Bcl-2
and up-regulated the expression of cleaved-caspase 3, which resulted in the apoptosis
of tumor cells. In vivo, based on targeting to IKKβ, B3 also exhibited good anti-tumor
activity in SGC-7901 xenograft model.

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In summary, we modified and obtained several EF24 analogs with better IKKβ
inhibitory activity, and presented a potential IKKβ inhibitor B3 with excellent
anti-gastric cancer activity. But the activity of these inhibitors need to be further
enhanced and the interaction between the inhibitors and IKKβ should be further
investigated by co-crystallization.
4. Materials and methods
4.1. Instrumentation and chemicals
All reactions were monitored by TLC and the products were purified by column
chromatography or recrystallization. Melting points were tested in open capillary
tubes on a Fisher–Johns melting apparatus. Low-resolution mass spectra were
performed on a Bruker Esquire HCT spectrometer and High-resolution mass spectra
1
were recorded on a Bruker micrOTOF-QII spectrometer, respectively. The H-NMR
spectra were obtained on a 600 MHz apparatus (Bruker Corporation, Ettlingen,
Germany) using tetramethylsilane (TMS) as an internal standard. All chemical
reagents were commercial products purchased from Sigma–Aldrich, Fluka, and
Aladdin and used without further purification. Reference compound EF24 (A1) was
synthesized in our laboratory. And BMS345541 was commercial bought from
Sigma-Aldrich chemical company.
4.2. General procedure for the preparation of compounds A1-D7
For the reactions, 4-piperidinone or corresponding ketones (1 mmol) and
2-bromobenzaldehyde or corresponding aldehydes (2.5 mmol) were dissolved in
ethanol (10 mL). The mixture was refluxed at 78 °C and added with 0.5 mL 40%
NaOH or AcOH/HCl. Reactions were monitored by TLC, when the reactions were
accomplished, cooled on ice. The products could precipitate or be purified by
crystallization and column chromatography using PE/EA as the eluent. Among these
compounds, A4, A6, C5, D1, D2, D3 and D4 were unreported.
4.2.1. (3E,5E)-3,5-bis(2-Fluorobenzylidene)piperidin-4-one (A1). Light yellow
1
powder, 44% yield, mp 139.6-142.5 °C (Lit, 189-190 °C) [35]. H-NMR (CDCl3), δ:
9.86 (s, 1H, -NH), 8.05 (s, 2H, Ar-CH=C×2), 7.92 (d, J = 7.8 Hz, 2H, Ar-H4×2), 7.84
(t, J = 15.0 Hz, 2H, Ar-H6×2), 7.71 (t, J = 15.6 Hz, 2H, Ar-H3×2), 7.57 (d, J = 7.8 Hz,
2H, Ar-H5×2), 4.29 (s, 4H, CH2-N-CH2). ESI-MS m/z: 312.12 (M+H)+, calcd for
C19H15F2NO: 311.11.
4.2.2. (3E,5E)-3,5-bis(2,6-Difluorobenzylidene)piperidin-4-one (A2). Light yellow
1
powder, 33% yield, mp 164.1-166.9 °C [36]. H-NMR (CDCl3), δ: 7.60 (s, 2H,
Ar-CH=C×2), 7.27-7.37 (m, 2H, Ar-H4×2), 6.86-6.96 (m, 4H, Ar-H3×2, Ar-H5×2),
3.80 (s, 4H, CH2-N-CH2), 1.90 (brs, 1H, N-H). ESI-MS m/z: 348.10 (M+H)+, calcd
for C19H13F4NO: 347.09.

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4.2.3. (3E,5E)-3,5-bis(4-Fluorobenzylidene)piperidin-4-one (A3). Light yellow
powder, 69% yield, mp 184.9-186.0 °C [37, 38]. 1H-NMR (DMSO-d6), δ: 7.57 (s, 2H,
Ar-CH=C×2), 7.53-7.56 (m, 4H, Ar-H2×2, Ar-H6×2), 7.29 (t, J = 9.0 Hz, 4H, Ar-H3×2,
Ar-H5×2), 3.96 (s, 4H, CH2-N-CH2). ESI-MS m/z: 312.21 (M+H)+, calcd for
C19H15F2NO: 311.11.
4.2.4. (3E,5E)-3,5-bis(3-fluorobenzylidene)piperidin-4-one (A4). Light yellow powder,
1
47% yield, mp 119.5-121.1 °C . H-NMR (CDCl3), δ: 7.74 (s, 2H, Ar-CH=C×2),
7.37-7.40 (m, 2H, Ar-H2×2), 7.16 (d, J = 7.8 Hz, 2H, Ar-H6×2), 7.06-7.08 (m, 4H,
Ar-H4×2, Ar-H5×2), 4.13 (s, 4H, CH2-N-CH2). ESI-MS m/z: 312.21 (M+H)+, calcd
for C19H15F2NO: 311.11
4.2.5. 2,5-bis((E)-2-fluorobenzylidene)cyclopentan-1-one (A5). Yellow powder, 91%
yield, mp 207-209 oC [36]. 1H-NMR (CDCl3), δ: 7.83 (s, 2H, Ar-CH=C×2), 7.60 (t, J
= 13.8 Hz, 2H, Ar-H4×2), 7.40-7.37 (m, 2H, Ar-H6×2), 7.22 (t, J = 15.0 Hz, 2H,
Ar-H3×2), 7.15 (t, J = 18.6 Hz, 2H, Ar-H5×2), 3.07 (s, 4H, CH2CH2). ESI-MS m/z:
297.19 (M+H)＋, calcd for C19H14F2O: 296.10.
4.2.6. (3E,5E)-3,5-bis(2-fluorobenzylidene)-1-propylpiperidin-4-one (A6). Yellow
powder, 96% yield, mp 87.0-89.9 °C. 1H-NMR (CDCl3), δ: 7.70 (s, 2H, Ar-CH=C×2),
7.51-7.48 (m, 4H, Ar-H4×2, Ar-H6×2), 7.34 (d, J = 11.4 Hz, 2H, Ar-H3×2), 7.31 (d, J
= 7.8 Hz, 2H, Ar-H5×2), 3.72 (s, 4H, CH2-N-CH2), 2.43 (t, J = 14.4 Hz, 2H, CH2),
1.34-1.30 (m, 2H, CH2), 0.75 (t, J = 13.2 Hz, 3H, CH3). ESI-MS m/z: 354.16 (M+H)+,
calcd for C22H21F2NO: 353.15.
4.2.7. (3E,5E)-3,5-bis(2-fluorobenzylidene)-tetrahydropyran-4-one (A7). Yellow
1
powder, 72% yield, mp 143.8-146.6°C (Lit, 157-158 °C) [39]. H-NMR (CDCl3), δ:
7.91 (s, 2H, Ar-CH=C×2), 7.36-7.39 (m, 1H, Ar-H6), 7.17-7.20 (m, 2H, Ar-H3, Ar-H4),
7.13-7.16 (m, 1H, Ar-H5), 4.78 (s, 4H, CH2-O-CH2). ESI-MS m/z: 313.10 (M+H)+,
calcd for C19H14F2O2: 312.09.
4.2.8. (3Z,5Z)-3,5-bis(2-fluorobenzylidene)-tetrahydrothiopyran-4-one (A8). Yellow
1
powder, 15% yield, mp 121.1-130.4 °C [40]. H-NMR (CDCl3), δ: 7.77 (s, 2H,
Ar-CH=C×2), 7.34-7.37 (m, 2H, Ar-H6×2), 7.30-7.33 (m, 2H, Ar-H4×2), 7.18 (t, J =
7.8 Hz, 2H, Ar-H5×2), 7.13 (t, J = 9.0 Hz, 2H, Ar-H3×2), 3.78 (s, 4H, CH2-S-CH2).
ESI-MS m/z: 313.08 (M+H)+, calcd for C19H14F2S: 312.07.
4.2.9. (2E,5E)-2,5-bis(2-bromobenzylidene)cyclopentanone (B1). Yellow powder,
1
87% yield, mp 163.1-164.5 °C [36]. H-NMR (CDCl3), δ: 7.88 (s, 2H, Ar-CH=C×2),
7.68 (dd, J = 1.2 Hz, J = 0.6 Hz, 2H, Ar-H4×2), 7.54 (dd, J = 1.2 Hz, J = 1.2 Hz, 2H,
Ar-H6×2), 7.38 (t, J = 14.4 Hz, 2H, Ar-H3×2), 7.24 (td, J = 1.2 Hz, J = 1.2 Hz, J = 1.8
Hz, 2H, Ar-H5×2), 2.99 (s, 4H, CH2CH2). ESI-MS m/z: 419.07 (M+H)+, calcd for
C19H14Br2O: 417.94.

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4.2.10 (3E,5E)-3,5-bis(2-bromobenzylidene)-1-methylpiperidin-4-one (B2). Yellow
1
powder, 85% yield, mp 144.2-146.0 °C [41]. H-NMR (CDCl3), δ: 7.98 (s, 2H,
Ar-CH=C×2), 7.68 (d, J = 7.8 Hz, 2H, Ar-H3×2), 7.37 (t, J = 7.8 Hz, 2H, Ar-H5×2),
7.23-7.28 (m, 4H, Ar-H4×2, Ar-H6×2), 3.65 (s, 4H, CH2-N-CH2), 2.40 (s, 3H, N-CH3).
ESI-MS m/z: 447.97 (M+H)+, calcd for C20H17Br2NO: 446.96.
4.2.11. (3E,5E)-3,5-bis(2-bromobenzylidene)piperidin-4-one (B3). Light yellow
1
powder, 13% yield, mp 150.3-152.7 °C (Lit, 220-222 °C) [35]. H-NMR (CDCl3), δ:
7.90 (s, 2H, Ar-CH=C×2), 7.65 (d, J = 7.8 Hz, 2H, Ar-H3×2), 7.33 (t, J = 7.8 Hz, 2H,
Ar-H5×2), 7.16-7.23 (m, 4H, Ar-H4×2, Ar-H6×2), 3.97 (s, 4H, N-CH2×2), 1.79 (brs,
1H, N-H). ESI-MS m/z: 433.95 (M+H)+, calcd for C19H15Br2NO: 432.95.
4.2.12. (3E,5E)-3,5-bis(2-(trifluoromethyl)benzylidene)piperidin-4-one (B4). Yellow
1
powder, 82% yield, mp 153.5-155.7 °C [42]. H-NMR (CDCl3), δ: 7.65 (s, 2H,
Ar-CH=C×2), 7.51-7.45 (m, 4H, Ar-H4×2, Ar-H6×2), 7.34-7.31 (m, 4H, Ar-H5×2),
7.30 (t, J = 9.6 Hz, 2H, Ar-H3×2), 3.90 (s, 4H, CH2-N-CH2). ESI-MS m/z: 412.21
(M+H)+, calcd for C21H15F6NO: 411.10.
4.2.13. 2,5-bis((E)-2-chlorobenzylidene)cyclopentan-1-one (B5). Light yellow powder,
1
78% yield, mp 150.8-152.4 °C [43]. H-NMR (DMSO-d6), δ: 7.77-7.75 (m, 2H,
Ar-H2×2), 7.71 (s, 2H, Ar-CH=C×2), 7.61-7.60 (m, 2H, Ar-H4×2), 7.45 (d, J = 1.8 Hz,
2H, Ar-H5×2), 7.46 (t, J = 6.0 Hz, 2H, Ar-H3×2), 3.05 (s, 4H, CH2-CH2). ESI-MS m/z:
329.10 (M+H)+, calcd for C19H14Cl2O: 328.04.
4.2.14. (3E,5E)-3,5-bis(2-chlorobenzylidene)-1-methylpiperidin-4-one (B6). Light
Kelly powder, 78% yield, mp 143.9-145.2 °C [44]. 1H-NMR (CDCl3), δ: 8.01 (s, 2H,
Ar-CH=C×2), 7.45 (d, J = 9.0 Hz, 2H, Ar-H6×2), 7.28-7.32 (m, 4H, Ar-H3×2,
Ar-H4×2), 7.23-7.26 (m, 2H, Ar-H5×2), 3.65 (s, 4H, CH2-N-CH2), 2.37 (s, 3H,
N-CH3). ESI-MS m/z: 358.07 (M+H)+, calcd for C20H17Cl2NO: 357.06.
4.2.15. (3E,5E)-3,5-bis(2-chlorobenzylidene)piperidin-4-one (B7). Light yellow
1
powder, 22% yield, mp 214.3-216.7 °C (Lit, 215-217°C) [35]. H-NMR (CDCl3), δ:
7.96 (s, 2H, Ar-CH=C×2), 7.45 (d, J = 9.0 Hz, 2H, Ar-H3×2), 7.40 (d, J = 8.4 Hz, 2H,
Ar-H6×2), 7.29 (m, 2H, Ar-H4×2), 7.20 (m, 2H, Ar-H5×2), 3.99 (d, J = 1.2 Hz, 4H,
CH2-N-CH2). ESI-MS m/z: 345.15 (M+H)+, calcd for C19H15Cl2NO: 343.05.
4.2.16. (3E,5E)-3,5-bis(3-hydroxybenzylidene)piperidin-4-one (C1). Brown-yellow
1
powder, 10% yield, mp 295.2-297.6 °C [44]. H-NMR (DMSO-d6), δ: 7.77 (s, 2H,
-OH×2), 7.32 (s, 2H, Ar-CH=C×2), 6.88-6.94 (m, 6H, Ar-H), 4.45 (s, 4H,
CH2-N-CH2). ESI-MS m/z: 308.14 (M+H)+, calcd for C19H16O4: 307.12.
4.2.17. (3E,5E)-3,5-bis(4-hydroxybenzylidene)piperidin-4-one (C2). Yellow powder,
1
8% yield, mp 295.5-296.4 °C (carbonization) (Lit, >300 °C) [45]. H-NMR

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(DMSO-d6), δ: 10.25 (s, 2H, 4-OH×2), 7.77 (s, 2H, Ar-CH=C×2), 7.39 (d, J = 8.4 Hz,
4H, Ar-H2×2, Ar-H6×2), 6.91 (d, J = 7.8 Hz, 4H, Ar-H3×2, Ar-H5×2), 4.43 (s, 4H,
CH2-N-CH2). ESI-MS m/z: 308.14 (M+H)+, calcd for C19H17NO3: 307.12.
4.2.18. 2,5-Bis(4-hydroxybenzylidene)cyclopentanone (C3). Yellow crystal, 89% yield,
o
1
mp > 300 C [46]. H-NMR (DMSO-d6), δ: 10.05 (s, 2H, 4-OH×2), 7.54 (d, J = 8.4
Hz, 4H, Ar-H2×2, Ar-H6×2), 7.34 (s, 2H, Ar-CH=CO×2), 6.88 (d, J = 8.4 Hz, 4H,
Ar-H3×2, Ar-H5×2), 3.02 (s, 4H, CH2CH2). ESI-MS m/z: 293.28 (M+H)+, calcd for
C19H16O3: 292.11.
4.2.19. 2,5-bis((E)-4-hydroxybenzylidene)cyclopentan-1-one (C4). Light green
1
powder, 41% yield, mp 295.2-297.6 °C [46]. H-NMR (DMSO-d6), δ: 9.64 (s, 2H,
3-OH×2), 7.34 (s, 2H, Ar-CH=C×2), 7.29 (m, 2H, Ar-H5×2), 7.11 (d, J = 7.8 Hz, 2H,
Ar-H6×2), 7.09 (s, 2H, Ar-H2×2), 6.85 (dd, J = 2.4 Hz, J = 2.4 Hz, 2H, Ar-H4×2), 3.07
(s, 4H, CH2-CH2). ESI-MS m/z: 293.10 (M+H)+, calcd for C19H16O3: 292.10.
4.2.20. (3E,5E)-3,5-bis(3-hydroxybenzylidene)-tetrahydropyran-4-one (C5). Kelly
powder, 40% yield, mp 197.2-198.6 °C (carbonization). 1H-NMR (CDCl3), δ: 8.61 (s,
2H, Ar-CH=C×2), 7.65 (s, 2H, Ar-H2×2), 7.31 (t, 2H, Ar-H6×2), 6.89-6.91 (m, 4H,
Ar-H4×2, Ar-H5×2), 4.94 (s, 4H, CH2-O-CH2). ESI-MS m/z: 309.10 (M+H)+, calcd
for C19H16O4: 308.10.
4.2.21. (3E,5E)-3,5-bis(4-hydroxybenzylidene)-tetrahydropyran-4-one (C6). Kelly
1
power, 44% yield, mp > 200 °C [47]. H-NMR (Acetone-d6), δ: 8.93 (s, 2H,
Ar-CH=C×2), 7.652 (s, 2H, 4-OH×2), 7.31 (d, J = 8.4 Hz, 4H, Ar-H2×2, Ar-H6×2),
6.94 (d, J = 8.4 Hz, 4H, Ar-H3×2, Ar-H5×2), 4.92 (s, 4H, CH2-O-CH2). ESI-MS m/z:
309.10 (M+H)+, calcd for C19H16O4: 308.10.
4.2.22. 1,5-Bis(4-hydroxyphenyl)penta-1,4-dien-3-one (C7). Brown powder, 45%
o
1
yield, mp 246.3-248.6 °C (Lit. 243-245 C) [48]. H-NMR (DMSO-d6), δ: 10.04 (s,
2H, 4-OH×2), 7.66 (d, J = 15.6 Hz, 2H, Ar-CH=C×2), 7.62 (d, J = 8.4 Hz, 4H,
Ar-H2×2, Ar-H6×2), 7.10 (d, J = 16.2 Hz, 2H, CH=CO×2), 6.84 (d, J = 8.4 Hz, 4H,
Ar-H3×2, Ar-H5×2). ESI-MS m/z: 267.04 (M+H)+, calcd for C17H14O3: 266.09.
4.2.23. 1-methyl-3,5-bis((E)-4-(piperidin-1-yl)benzylidene)piperidin-4-one (D1).
1
Yellow powder, 35% yield, mp 214.1-214.8 °C. H-NMR (CDCl3), δ: 7.76 (s, 2H,
Ar-CH=C×2), 7.35 (d, J = 9.0 Hz, 4H, Ar-H2×2, Ar-H6×2), 6.93 (d, J = 9.0 Hz, 4H,
Ar-H3×2, Ar-H5×2), 3.80 (s, 4H, CH2-N-CH2), 3.31 (t, J = 10.8 Hz, 8H, N-CH2×4),
2.51 (s, 3H, -CH3), 1.73-1.69 (m, 8H, -CH2×4), 1.65 (t, J = 10.8 Hz, 4H, -CH2×2).
ESI-MS m/z: 456.34 (M+H)+, calcd for C30H37N3O: 455.29.
4.2.24. 3,5-bis((E)-4-(piperidin-1-yl)benzylidene)tetrahydro-4H-pyran-4-one (D2).
1
Yellow powder, 50% yield, mp 239.3-241.6 °C. H-NMR (CDCl3), δ: 7.77 (s, 2H,
Ar-CH=C×2), 7.26 (d, J = 8.4 Hz, 4H, Ar-H2×2, Ar-H6×2), 6.92 (d, J = 9.6 Hz, 4H,

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Ar-H3×2, Ar-H5×2), 4.96 (s, 4H, CH2-O-CH2), 3.32 (t, J = 10.8 Hz, 8H, N-CH2×4),
1.72-1.69 (m, 8H, -CH2×4), 1.65 (t, J = 10.2 Hz, 4H, -CH2×2). ESI-MS m/z: 443.26
(M+H)+, calcd for C29H34N2O2: 442.26.
4.2.25.
3,5-bis((E)-4-(diethylamino)benzylidene)-1-methylpiperidin-4-one
(D3).
1
Orange powder, 21% yield, mp 195.7-197.1 °C. H-NMR (CDCl3), δ: 7.76 (s, 2H,
Ar-CH=C×2), 7.35 (d, J = 8.4 Hz, 4H, Ar-H2×2, Ar-H6×2), 6.70 (d, J = 7.8 Hz, 4H,
Ar-H3×2, Ar-H5×2), 3.82 (s, 4H, CH2×2), 3.43 (t, J = 13.2 Hz, 8H, CH2-N-CH2×2),
2.52 (s, 3H, N-CH3), 1.22 (t, J = 13.8 Hz, 12H, CH3×4). EI-MS m/z: 432.25(M+1)+,
calcd for C28H37N3O: 431.29.
4.2.26. 3,5-bis((E)-4-(diethylamino)benzylidene)-1-propylpiperidin-4-one (D4). Red
1
powder, 33% yield, mp 167.8-169.1 °C. H-NMR (CDCl3), δ: 7.76 (s, 2H,
Ar-CH=C×2), 7.35 (d, J = 3.0 Hz, 4H, Ar-H2×2, Ar-H6×2), 6.70 (d, J = 9.0 Hz, 4H,
Ar-H3×2, Ar-H5×2), 3.88 (s, 4H, CH2×2), 3.44-3.40 (m, 8H, CH2-N-CH2×2), 2.55 (t, J
= 15 Hz, 2H, N-CH2), 1.57-1.53 (m, 2H, CH2), 1.22 (t, J = 13.8 Hz, 12H, CH3×4),
0.90 (t, J = 10.8 Hz, 3H, CH3). EI-MS m/z: 460.38(M+1)+, calcd for C30H41N3O:
459.33.
4.3. IKK inhibitory activity assay
The kinase activity of IKKβ was determined by Mobility Shift Assay on EZ Reader
(Caliper Life Sciences, MA). Due to our limited experimental condition, the kinase
activity screening was authorized to ChemPartner (Shanghai, China). The specific
protocol is described briefly as follows: The enzyme solution was prepared in 1.25×
kinase base buffer (62.5 mM HEPES, 0.001875% Brij-35, 12.5 mM MgCl2, 2.5 mM
DTT), stop buffer (100 mM HEPES, 0.015% Brij-35, 0.2% coating reagent No. 3, 50
mM EDTA) The compound was dissolved in DMSO, and diluted to the specific
concentration with water in 384-well plate. 5 mL of compound solution was diluted
with 10 mL of 2.5× enzyme solution. 10 mM EDTA was used as the blank control.
The mixture was incubated at room temperature for 10 min. 10 mL of peptide solution
(2.5×, FAM-P22, add FAM-labeled peptide) was added to each well of the 384-well
assay plate. After incubation at 28 oC for 1 h, 25 mL of stop buffer was added into the
mixture to stop the reaction. Conversion data were collected on Caliper EZ reader
(Hopkinton, MA), the percent inhibition of kinase activity was calculated based on the
formula below:
Inhibition % = (max - conversion)/(max - min) × 100%
“Max” stands for DMSO control; “Min” stands for low control.
To determine the half-maximal inhibitory concentration of the compounds to IKKβ
activity, 10 gradient concentrations of compounds (100, 33.33, 11.11, 3.70, 1.23, 0.41,
0.14, 0.046, 0.015, and 0.005 mM) were set up. The inhibition ratios of different
concentrations relative to kinase were calculated and the concentration inhibition rate
curve was fitted by GraphPad Prism.

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4.4. Molecular docking
As one of the most widely used computational approaches for structure-based drug
design, molecular docking study was used to predict the binding pose of B3 in IKKβ’s
binding site by using the software Autodock (version 4.2.6) [26]. The crystallographic
co-ordinates for human IKKβ (PDB ID: 4KIK) was obtained from the Protein Data
Bank (PDB). Prior to docking, protein structure was prepared by removing water
molecules and other ligands using PyMol software. A grid box size of 60 × 60 × 60
dimensions with a spacing of 0.375 Å between the grid points was implemented and
covered almost the entire IKKβ binding site. The grid parameter files are created with
setting up of map files directly. The Lamarckian genetic algorithm (LGA) was applied
to deal with the protein and inhibitors interactions. Trials of 100 dockings, the number
of individuals in population was set to 300, 25 million of maximum number of energy
evaluations and other settings were set default. AutoDockTools version 1.5.6 and
PyMol was used to analyze the docking results.
4.4. Molecular dynamics (MD) simulations
Molecular dynamics (MD) simulations were applied to verify the binding mode of
inhibitors to IKKβ predicted by molecular docking. The charge of B3 was first
obtained by the restrained electrostatic potential (RESP) fitting technique based on the
electrostatic potential computed by Gaussian 09 at Hartree-Fock (HF) SCF/6-31G*
level of theory [49]. Afterwards, the topology and parameter files of B3 were
generated by using the antechamber module in AMBER11 simulation package [50].
Molecular mechanics parameters from the ff99SB and GAFF force fields were
assigned to the protein and the ligands, respectively [51, 52]. TIP3P water molecules
were used to solvate the complex, extending at least 10 Å from the protein. In
addition, an appropriate number of sodium ions was used to keep the systems neutral.
MD simulations were carried out with AMBER 11 software packages [53, 54].
Prior to the MD productive simulation, we performed an equilibration protocol
consisting of an initial minimization of the water box of 5000 steps, 2500 steps for the
steepest descent and 2500 steps in the conjugate gradient. Then, the TIP3P water box
was heated at constant volume until 300 K using a time constant for the heat bath with
a coupling time of 100 ps. Equilibration was at 300 K and constant pressure of 100 ps.
Before the production of MD, the whole system was equilibrated at 100 ps at constant
pressure of 1 bar with Langevin temperature scaling with a collision frequency of 2 ps.
The last step used 50 ns of MD simulation without any restraints. Furthermore,
nonbonded interactions were cut off at 8.0 Å. Periodic Boundary Conditions were
turned on in every step of the whole process [55]. Particle mesh Ewald (PME) was
also performed to deal with the long-range electrostatic interactions under periodic
boundary conditions [56]. The SHAKE method was used to constrain hydrogen atoms
and the time step was set to 2 fs [57]. The coordinates were saved every 10 ps for the
subsequent analysis.

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4.5. MM/GBSA binding free energy calculation
The binding free energy (△Gbind) between the receptor and ligand in each system
was calculated by the Molecular Mechanics/Generalized Born Surface Area
(MM/GBSA) method based on the 500 snapshots evenly extracted from 40 to 50 ns
according to the following equations [14, 25, 58, 59].
(1)
(2)
(3)
(4)
In equation (2), ∆EMM and ∆Gsol represent the molecular mechanical energy and
solvation energy. T∆S, at temperature T, represents the change of the conformational
entropy upon ligand binding. In equation (3), ∆EMM can be decomposed into three
terms: the intramolecular energy (∆Einternal, including bond, angle, and dihedral
energies), van der Waals energy (∆EVDW) and electrostatic energy (∆Eelec). In equation
(4), ∆GGB and ∆GSA are the polar and nonpolar components of the solvation free
energy, respectively. ∆GGB was calculated using the the “OBC” model (igb = 5) [60];
∆GSA was computed based on the solvent-accessible surface area (SASA) estimated
with a fast linear combination of the pairwise overlap (LCPO) algorithm using a
probe radius of 1.4 Å [49]: ∆GSA= 0.0072 × ∆SASA; −T∆S was not considered here
due to expensive computational cost and low prediction accuracy [59, 61]. In the
∆GGB calculations, the solvent and solute dielectric constants were set to 80 and 1,
respectively.
4.6. Cell culture and viability Assay
Human gastric adenocarcinoma cell line SGC-7901 (GCD150), human lung cancer
cell line A549 (GCD063) were purchased from the Cell Bank of the Chinese
Academy of Sciences (Wuhan, China). Human gastric adenocarcinoma cell line
MGC-803 (TCHu 86) and Human cervical carcinoma cell line Hela229 (TCHu 20)
were obtained from the Shanghai Institute of Biosciences and Cell Resources Center
(Shanghai, China). SGC-7901, MGC-803 and A549 were cultured in Roswell Park
Memorial Institute (RPMI) 1640 medium (11875093, Gibco, Eggenstein, Germany)
with 10% fetal bovine serum (FBS; 10099141, Gibco) and 1%
penicillin–streptomycin (15140122, Gibco). Hela229 was maintained in Dulbecco’s
modified Eagle’s medium (11995040, Gibco) containing 10% FBS and 1%
penicillin–streptomycin. All cancer cells were cultured in a humidified Thermo Forma
incubator (Thermo Fisher Scientific, Massachusetts, USA) containing 5% CO2 at
37°C; they were cultured for a maximum of 6 months.
MTT assay was employed to assess cell viability. Cells were seeded in 96-well

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plate (NEST Biotechnology Co. LTD., Wuxi, China). After cultured overnight, cells
were exposed to B3, EF24 or BMS345541 for 72 h. Then 20 µL
methylthiazoletetrazolium (MTT; 0.5 mg/mL; M5655; Sigma, St Louis, Missouri,
USA) was added to the culture medium and incubated at 37 °C for 4 h. At the end of
incubation, produced crystals were dissolved in 150 µL dimethyl sulfoxide (D2650,
Sigma) and than optical density (OD) values were quantified at a wavelength of 490
nm. Inhibition rate of cell growth was measured using the formula: inhibition rate (%)
= [1-OD490 (experiment group)/OD490 (control group)] × 100%. The 50% inhibition
of cell proliferation (IC50) values were calculated from log dose response curves.
4.7. Cell cycle assay
SGC-7901 and MGC-803 cells (3x105 per well) were seeded in six-well plates and
cultured overnight. After treated with B3 for 12 h, cells were collected, fixed by 75%
ice alcohol and stained with propidium iodide (PI) containing RNase (550825, BD
Biosciences Clontech, San Jose, CA, USA). Then cell cycle was detected by flow
cytometry and analyzed using the Flow Jo analysis program (version 7.6.1).
4.8. Cell apoptosis assay
The cell apoptosis assay was performed using an annexin V-FITC apoptosis
detection kit (556547, BD Biosciences Clontech). SGC-7901 and MGC-803 cells
(5×105 cells/well) were seeded in six-well plates. After cells adhered to the wells
overnight, cells were treated with different concentrations of B3 or BMS345541 for
12 h. Then the cells were processed according to the manufacturer’s instructions and
followed subjected to a FACSCalibur instrument (BD Biosciences Clontech). Results
were analyzed using the Flow Jo analysis program.
4.9. Western blotting
The treated cells were collected and lysated. Then an amount of 80 µg of the whole
cell lysates was separated by 10% sodium dodecyl sulfate−polyacrylamide gel
electrophoresis and electro-transferred to a PVDF membrane (Millipore,
Massachusetts, USA). Each membrane was pre-incubated for 1 h at room temperature
in Tris-buffered saline, pH 7.6, containing 0.05% Tween 20 and 5% non-fat milk and
then probed with specific antibodies against p-IKKα,β, IKKα,β, IκB, Cyclin B1, Cdc2
p34, Bcl-2, Cleaved-Caspase3, or GAPDH (Santa Cruz Biotech Co. Ltd., CA).
Immuno-reactive bands were then detected by incubating with secondary antibody
conjugated with horseradish peroxidase (Santa Cruz Biotech Co. Ltd.) and visualized
using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA). Quantity One
v4.62 software (Bio-Rad) was employed to quantify the optical density of the bands.
5.0. Immunofluorescence staining

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SGC-7901 cell suspension (1×105 cells per well) was inoculated on coverslips that
were partitioned previously into a 6-well plate (NEST Biotechnology Co. LTD.). After
24 h, cells were treated with 10 µM B3 or BMS345541 for 1 h and followed cultured
with TNFα for 1 h. Cells were fixed with 4% formaldehyde in PBS for 15 min and
washed with PBS for three times. Washed cells were blocked in 1% bovine serum
albumin (BSA; A7906, Sigma) in PBS at room temperature for 1 h. Thereafter, cells
were washed three times with PBS and incubated with the antibody NF-κB p65
(1:100, sc-8008, Santa Cruze) with 1% BSA in PBS at 4 ℃ overnight. After three
washes, fluorescein PE-labeled polyclonal mouse anti-rabbit IgG antibody (1:100,
sc-3753, Santa Cruze) was used to stain the cells. The nucleus was counterstained by
DAPI (C1006, Beyotime Biotech, Nantong, China). The stained cells were
photographed by an fluorescence microscope (Nikon, Tokyo, Japan) to assess nuclear
translation of NF-κB p65.
5.1. In vivo experiments
All animal experiments were followed according with the Wenzhou Medical
University’s Policy on the Care and Use of Laboratory Animals. Operations for
animal experiments were approved by the Wenzhou Medical University Animal
Policy and Welfare Committee. For analysis of tumorigenicity, SGC7901 cells (5×106
cells/mice) were mixed with 100 µL PBS, and were injected subcutaneously into the
flank of 6-week-old, athymic nude mice (18-22g). Animals were housed under barrier
conditions and fed with a standard rodent diet and water. After 1 week, tumor volume
reached 50 mm3 and mice were randomly divided into two treatment groups (n=6): (I)
solvent containing 10% castor oil and 90% PBS as vehicle control and (II) 10mg/kg
of L50H25 (dissolved mentioned solvent). Vehicle and B3 were administered via
intraperitoneal injection every other day for 22 days. The tumor volumes were
determined by measuring length (L) and width (W) with a caliper. And tumor volume
was calculated according to the following formula: volume = (π/6)*L*W2. At the end
of treatment, tumors were removed from euthanized mice and, snap-frozen in liquid
nitrogen, and stored at −80°C. Tumors tissue homogenates were lysed and separated
by SDS-PAGE to examine the expression of p-IKKα,β, IκB and Bcl-2 expressions
were determined by Western blot assay.
5.2. Statistics
All experiments were repeated more than three times. The results are showed as
means±S.E.M.s. Statistics were analyzed using student’s t-test mode in GraphPad Pro
(GraphPad, San Diego, CA). P values less than 0.05 (P <0.05) were considered as
significance.
Acknowledgement
This work was supported by the National Natural Science Foundation of China

ACCEPTED MANUSCRIPT
(Grant Nos. 81703766, 81402839, 81272462) the ZheJiang Province Natural Science
Fund of China (Grant Nos. LY17H160059, LY15H280014), Project of Wenzhou
Sci-Tech Bureau (Y20170161) and granted by the Opening Project of Zhejiang
Provincial Top Key Discipline of Pharmaceutical Sciences.
Supplementary data
1
Electronic supplementary information including H NMR, and MS spectra for all
compounds is available.
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Highlights:
1. Newly IKKβ inhibitors based on EF24 were designed and synthesized, and
several compounds had better activity than that of EF24.
2. The inhibitory effect of active compound B3 against IKKβ kinase was confirmed
by molecular docking.
3. By inhibiting IKKβ-NF-κB axis, compound B3 exhibited excellent anti-tumor
activity in vitro and in vivo.