Design, synthesis, anti-lung cancer activity, and chemosensitization of tumor-selective MCACs based on ROS-mediated JNK pathway activation and NF-κB pathway inhibition

Article abstract of DOI:10.1016/j.ejmech.2018.03.051

EF24 and F35 both were effective monocarbonyl curcumin analogues (MCACs) with excellent anti-tumor activity, however, drug defect such as toxicity may limit their further development. To get anti-lung cancer drugs with high efficiency, low toxicity and chemosensitization, a series of analogues based on EF24 and F35 were designed and synthesized. A number of compounds were found to exhibit cytotoxic activities selectively towards lung cancer cells compared to normal cells. Among these compounds, 5B was considered as an optimal anti-tumor agent for lung cancer cells with IC₅₀ values ranging from 1.0 to 1.7 μM, selectivity index (SI, as a logarithm of a ratio of IC₅₀ value for normal and cancer cells) were all above 1.1, while the SI of EF24 and F35 were less than 0.8. Consistent with selectivity in vitro, 5B was observed to show lower toxicity in acute toxicity experiment than EF24 and F35 respectively. Further, 5B was found to exert anti-tumor effects through ROS-mediated activation of JNK pathway and inhibition of NF-κB pathway. 5B could significantly enhance the sensitivity of A549 cells to cisplatin or 5-Fu. These findings suggested that 5B was an effective and less toxic MCAC and provided a promising candidate for anti-tumor drugs.

Full text of DOI:10.1016/j.ejmech.2018.03.051

Accepted Manuscript
Design, synthesis, anti-lung cancer activity, and chemosensitization of tumor-selective
MCACs based on ROS-mediated JNK pathway activation and NF-κB pathway
inhibition
Liping Chen, Qian Li, Bixia Weng, Jiabing Wang, Yangyang Zhou, Dezhi Cheng,
Thanchanok Sirirak, Peihong Qiu, Jianzhang Wu
PII:
S0223-5234(18)30296-4
10.1016/j.ejmech.2018.03.051
EJMECH 10316
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 December 2017
Revised Date: 28 February 2018
Accepted Date: 18 March 2018
Please cite this article as: L. Chen, Q. Li, B. Weng, J. Wang, Y. Zhou, D. Cheng, T. Sirirak, P. Qiu, J.
Wu, Design, synthesis, anti-lung cancer activity, and chemosensitization of tumor-selective MCACs
based on ROS-mediated JNK pathway activation and NF-κB pathway inhibition, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.051.
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Design, Synthesis, anti-lung Cancer Activity, and Chemosensitization
of Tumor-selective MCACs Based on ROS-mediated JNK Pathway
Activation and NF-κB Pathway Inhibition
Liping Chena, Qian Lia, Bixia Wenga, Jiabing Wanga, Yangyang Zhouab, Dezhi Chengc,
Thanchanok Sirirakd, Peihong Qiua, Jianzhang Wua*
(aChemical Biology Research Center, College of Pharmaceutical Sciences, Wenzhou Medical
Universtiy, Wenzhou, Zhejiang 325035, China)
(
bDepartment of Gastroenterology, The First Affiliated Hospital of Wenzhou Medical University,
Wenzhou, Zhejiang 325035, China)
(cDepartment of Thoracic Surgery, The First Affiliated Hospital of Wenzhou Medical University,
Wenzhou, Zhejiang 325035, China)
(
dFaculty of Pharmaceutical Sciences, Burapha University, Bangsaen, Chonburi 20131, Thailand
)
*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)
Abstract
EF24 and F35 both were effective monocarbonyl curcumin analogs (MCACs) with
excellent anti-tumor activity, however, drug defect such as toxicity may limit their
further development. To get anti-lung cancer drugs with high efficiency, low toxicity
and chemosensitization, a series of analogues based on EF24 and F35 were designed
and synthesized. A number of compounds were found to exhibit cytotoxic activities
selectively towards lung cancer cells compared to normal cells. Among these
compounds, 5B was considered as an optimal anti-tumor agent for lung cancer cells
with IC50 values ranging from 1.0 to 1.7 µM, selectivity index (SI, as a logarithm of a
ratio of IC50 value for normal and cancer cells) were all above 1.1, while the SI of
EF24 and F35 were less than 0.8. Consistent with selectivity in vitro, 5B was
observed to show lower toxicity in acute toxicity experiment than EF24 and F35
respectively. Further, 5B was found to exert anti-tumor effects through ROS-mediated
activation of JNK pathway and inhibition of NF-κB pathway. 5B could significantly

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enhance the sensitivity of A549 cells to cisplatin or 5-Fu. These findings suggested
that 5B was an effective and less toxic MCAC and provided a promising candidate for
anti-tumor drugs.
Keywords: MCACs; Synthesis; Toxicity; Anti-tumor activities; Chemosensitization
1. Introduction
To date, lung cancer is one of the most threatening malignant tumors against human
health[1,2]. There are 1800,000 new cases of lung cancer worldwide every year[3].
Due to the lack of valid methods for early diagnosis and unobvious symptoms of early
lung cancer, most patients are found at an advanced stage and can not be treated by
surgical removal, thus the main efficacious therapy is chemotherapy[4,5]. With the
discovery of a variety of cancer-specific molecular biomarkers, personalized therapy
for the tumor specific targets has gradually become a popular issue in the
development of anti-lung cancer drugs[1,3]. Unfortunately, it is not just the frequent
appearance of resistance concomitant with targeted therapies, but the higher budgets
for targeted drugs that account for the limited use of targeted preparations[6,7]. In
addition, not all driver mutations have been identified in lung cancer cells and
matched with effective targeted drugs[8,9], leading to the classical cytotoxic drugs
remain the first choice for patients ineffective in targeted therapies[10]. Whereas, the
use of cytotoxic drugs such as cisplatin, paclitaxel are accompanied by side effects
and chemoresistance[11-13], which may restrict drug dosage, impose the burden of
disease and impair the level of patients’ health-related quality of life. To change the
current status of lung cancer treatment, less toxic and effective compounds as well as
chemosensitizers therefore have received increasing attention.
Natural products without obvious toxicity have been an important source of new drug
discovery and inspired the drug development having novel scaffolds[14]. Curcumin,
extracted from the Curcuma longa with outstanding pharmacological activities[15,16],
has attracted a lot of attention. Unfortunately, no successful progress has been made in
the curcumin-related clinical trials, which could partly be explained by its instability
as well as pharmacokinetic deficiencies caused by unstable β-diketone
structure[16,17]. As a consequence, many scholars have focused on the study of
altering curcumin-unstable β-diketone to monocarbonyl curcumin analogues
(MCACs), which have been reported to retain or raise the biological activities with
improvement of the pharmacokinetics[17-20]. Among the derivatives, EF24 is a
hot-studied MCAC, found to have superior anti-tumor activities[21]. Nevertheless,

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further clinical development into an applicable drug candidate may be impeded by its
toxicity[22,23]. Similarly, a series of MCACs with great pharmacological activities
have been designed and synthesized in our previous study[24-27]. For example, F35,
F36, as EF24 analogues with structure of multiple methoxys on benzene ring, found
to exhibit preferable anti-inflammatory activities by inhibiting the NF-κB pathway.
With the research deep-going, F35 was also identified as a good anti-tumor compound.
However, like EF24, F35 exhibited toxicity to a certain degree in vitro and vivo.
Given that multi-methoxy phenyl was a key functional group in many natural
products with excellent anti-tumor activities, including piperlongumine,
combretastatin-A4 and terameprocol[28-30]. It has been reported that N-substituted
curcumin analogues on the piperidone moiety showed limited toxicity towards normal
cells[31,32]. Thus, for the purpose of finding efficient and low-toxicity anti-lung
cancer compounds, a series of piperid-4-one-containing MCACs bearing the structure
of multi-methoxy phenyl (3,4,5-OCH3 or 3,4-OCH3) were designed and synthesized
based on the lead compounds of EF24, F35 and F36. Among these compounds, 5B, as
an optimal compound with higher efficiency and lower toxicity superior to EF24 and
F35, was selected for the study of anti-tumor mechanism and found to exert
anti-tumor effects via ROS-mediated JNK and NF-κB pathways preliminaryly.
2. Results and discussion
2.1 Design and synthesis
In the present study, we synthetized a series of MCACs. Piperidone embedded in two
benzene rings conjugated with flanking C=C bonds was used to displace the central
keto-enol curcuminoid moiety. MCACs with 3,4,5-trimethoxyl or 3,4-dimethoxyl
substituents on both benzene rings were designed and synthesized (Figure 1A).
Generally, MCACs (F35, F36) can be easily achieved by aldol condensation reaction
between 4-piperidinone and aldehydes, and MCACs (5B-13B) with N-substitution
with different groups (methyl, ethyl, n-propyl, propionyl, formyl propionyloxy,
2-fluorobenzoyl, isobutyryl) on the piperidone moiety can be synthesized by F35, F36
and various alkyl halide or acyl chloride, respectively (Figure 1B). The structures of
MCACs were shown in Figure 1C, and confirmed by nuclear magnetic resonance
(1H-NMR) spectroscopy and Liquid Chromatography-Mass Spectrometry (LC-MS)

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1
analysis. The color, melting point, LC-MS and H-NMR spectrum of compounds
were presented in the chemical section.
Figure 1. Design, synthesis and structures of MCACs. (A) Design of MCACs. (B) Synthetic
pathway to MCACs. Reagents and conditions: (Ι) H2O/CH3CH2OH, 40%NaOH, room
tempreature; (ΙΙ) NaH, THF, 0 °C. (C) Chemical structures of synthesized MCACs.
2.2 The inhibition effects to cell viability
All compounds were evaluated by measuring the anti-viability activities to human
lung cancer cells H460, A549, H1650 and H1975 using MTT assay[33]. The half
inhibitory concentration (IC50) of these compounds were measured and listed in Table
1. Except that compound 11B showed low activity, other compounds induced a
significant loss of cell viability on the four types of cancer cells, whose IC50 reached
approximately 5.0 µM. In recent years, our research group has been engaged in
MCACs medicinal chemistry research and reviewed on the pharmacological activities
of 607 MCACs reported in the literature. Among them, many MCACs had good
anti-tumor effects, namely, the IC50 upon cancer cells generally reached the level of
1-5 µM except for some of them less than 1 µM. The results showed that the tested
compounds demonstrated a significant anti-viability effects against H460 cells and
most of them with the IC50 were less than 5 µM, especially 5B, 12B and 13B, whose
IC50 were within 2.0 µM; The same results could be observed in A549 cells, especially
compounds 10B, 12B and 13B, whose IC50 had reached 0.8 µM. Similarly, in the
other two cell lines, the majority of tested substances also showed excellent inhibitory
efficacy, which were just as effective as EF24, F35, and better than curcumin.
Therefore, the anti-tumor activities of these compounds designed in this essay had

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generally reached a good level. Given the above, the existence of these structures
including EF24, F35 basic skeleton and multi-methoxy phenyl (3,4,5-OCH3 or
3,4-OCH3) that may contributed to good activities.
Table 1. Cytotoxicity and selectivity index (SI) of compounds on various human lung cancer cells
and normal cells
IC50a (µM) for cell lines/SIb
Compound
H460
A549
H1650
H1975
HL7702
F35 (3A)
F36 (4A)
5B
1.5±0.6/0.70
6.4±1.8/0.37
1.0±0.3/1.42
3.1±1.9/0.77
4.9±6.0/0.42
3.6±1.8/0.43
4.1±3.8/0.71
2.7±1.8/0.19
>60c/ncd
1.2±0.7/0.80
2.9±3.1/0.72
1.7±0.2/1.19
1.3±0.9/1.15
4.2±2.8/0.48
3.6±4.2/0.43
4.2±1.8/0.70
0.8±0.9/0.72
17.9±15.5/ncd
0.7±0.8/0.80
0.8±0.9/1.18
15.9±3.1/ncd
8.6±0.8/0.25
4.2±3.1/0.26
4.0±3.5/0.28
7.6±0.9
15.1±7.2
26.4±3.3
18.2±0.9
12.8±2.8
9.8±0.1
21.0±0.9
4.2±0.1
>60c
16.7±7.4/-0.04 10.3±5.9/0.17
1.5±0.1/1.25
9.0±2.9/0.31
8.2±2.3/0.19
1.0±0.6/1.42
6.6±6.8/0.44
6.5±0.1/0.29
6B
7B
8B
12.0±3.6/-0.09 8.5±2.6/0.06
16.0±12.2/0.12 11.2±5.9/0.27
9B
10B
4.4±1.2/-0.02
>60c/ncd
1.7±0.5/0.39
>60c/ncd
11B
12B
1.5±0.7/0.47
2.0±0.2/0.79
16.8±6.9/ncd
2.8±2.6/0.74
1.9±1.0/0.36
10.8±0.9/0.05
53.2±5.9/ncd
9.3±1.7/0.22
1.4±0.2/0.50
1.9±0.2/0.81
32.0±16.9/ncd
8.0±2.5/0.28
4.4±1.6
12.2±6.7
>60c
13B
BAY
BMS345541
15.3±4.8
Curcumin
EF24
8.5±1.1/-0.07
1.2±0.9/0.63
6.8±0.2/0.02
2.4±2.6/0.33
20.5±5.7/-0.45 1.4±0.3/0.71
1.7±0.4/0.48 1.3±0.1/0.59
7.2±0.6
5.1±1.6
a IC50 was the concentration of a drug that reduced cell viability by 50% relative to the untreated control.
b
SI has been calculated as a logarithm of a ratio of IC50 value for normal cells (HL7702) and the IC50 value for
cancer cells (H460, A549, H1650 and H1975).
c The maximum use of the compound concentration.
d nc (not calculated) – the SI could not be calculated because of lack of the IC50 value for normal/cancer cells.
2.3 In vitro and vivo toxicity
For the purpose to select efficient and hypotoxic anti-tumor compounds, the
cytotoxicity of tested substances were investigated further on normal human liver

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cells HL7702. The results were showed in Table 1. It is found that most compounds
showed higher IC50 of HL7702 than the cancer cells, indicating selectivity of HL7702
towards cancer cells at different extent, while the lead compound curcumin induced
no significant selectivity. In particular, compound 5B was more cytotoxic in four
types of cancer cells with selectivity index (SI, as a logarithm of a ratio of IC50 value
for normal and cancer cells) values in the range of 1.1-1.5. More delightfully, 5B had
the advantage of being more selective than EF24 and F35, whose SI were within 0.8.
Thus, it was speculated that the raised selectivity of 5B, 13B and others, even higher
than EF24, F35 and F36, may be related to N-substitutions.
As highlighted above, the advantage of 5B was its high activity and low
hepatotoxicity in vitro. Further, acute toxicity experiment was carried out to determine
whether 5B was safer than EF24 and F35 in vivo[34]. The study was performed on
mice by recording the changes in body weight. It was shown in Figure 2 that the
effect of 5B on body weight was lower than EF24 and F35 respectively, regardless of
whether it was administered intraperitoneally or intragastrically, which were in
agreement with in vitro findings, although there was no denying that 5B showed a
certain degree of toxicity in vivo. These findings suggested that as EF24 and F35
derivatives, 5B performed better selectivity in vitro and lower toxicity in vivo.
Figure 2. 5B showed less toxicity than EF24 and F35 respectively in vivo. (A) Changes in body
weight of C57BL/6 mice administered by intraperitoneal injection with vehicle (6% castor oil), 5B,
EF24, F35. The weight of the mice were observed for 15 days. (B) Changes in body weight of
C57BL/6 mice treated by lavage with vehicle (0.5% CMC-Na), 5B, EF24 were monitored for 14
days.

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2.4 5B induced cell cycle arrest and apoptosis in A549 cells
To study the potential mechanistic pathways responsible for the inhibitory effects, the
changes in cell cycle distribution and apoptosis in response to 5B were analyzed. As
shown in Figure 3, A549 cells treated with 5B clearly induced a dose-dependent arrest
of G2/M phase, while the cell proportion in G2/M phase induced by positive control
BMS345541 was lower than 5B with the concentration of 5 µM. The results in Figure
4A-C indicated that 5B induced obvious cell apoptosis in a dose-dependent manner.
Compared to 5B (10 µM) treated cells, BMS345541 (20 µM) induced less percentage
of apoptosis. To determine the role of proapoptotic effects in 5B-mediated cells,
apoptosis-related proteins were also studied. As depicted in Figure 4D-E, the cleavage
of poly (ADP-ribose) polymerase (PARP), an apoptosis marker, by activated caspase
3 was observed in a dose-dependent increase after 24 h treatment. Similarly,
5B-treated groups showed a significant increase in the level of Bax, a Bcl-2 family
protein. All results indicated that 5B had the capacity of anti-tumor activities by
arresting cell cycle and inducing apoptosis.
Figure 3. 5B induced cell cycle arrest of G2/M phase in A549 cells. (A) Flow cytometry was
performed to study the effects of 5B in cell cycle. A549 cells were treated with 5B (1 µM, 5 µM
and 10 µM) for 10 h, BMS345541 (20 µM) was used as positive control. (B) The frequency
distribution bar chart of G1/G0, S, G2/M. (C) Histograms displayed DNA content of G2/M. *, p <

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0.05; **, p < 0.01; ***, p < 0.001, vs DMSO.
Figure 4. 5B induced apoptosis in A549 cells. (A-B) 5B induced increased apoptotic morphology
and apoptotic ratio respectively. A549 cells were treated with 5B (1 µM, 5 µM and 10 µM) or
BMS345541 (20 µM) for 24 h. (C) The proportion of apoptotic cells from (B) was calculated after
Annexin V/PI double staining. (D-E) Western blot was performed to assess the expression of
apoptosis-related proteins Cle-PARP and Bax after 5B or BMS345541 treatment for 24 h. *, p <
0.05, vs DMSO.
2.5 5B induced the generation of ROS
Reactive Oxygen Species (ROS) was recognized as a key factor in many cellular
signaling events, including cell apoptosis and necrosis[35-37]. In cancer cells,
apoptosis was induced at a higher level of ROS, therefore, in search of ROS-inducing
compounds to target cancer has been considered as a potential strategy[35-37].
Curcumin derivatives including EF24 were reported to exhibit anti-tumor activities by
generating ROS[38-40]. Consequently, the level of intracellular ROS was monitored
using a ROS-sensitive fluorogenic dye (dichlorodihydrofluorescein diacetate,
DCF-DA) by flow cytometry to investigate whether 5B could generate ROS in A549
cells. Imaging analysis revealed that 5B strongly generated ROS in the range of 0 to 9
h, but slightly decreased at the time of 12 h (Figure 5A). In addition, ROS generation

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was observed dose-dependently after treating the cells with 5B (1 µM, 5 µM and 10
µM) for 9 h (Figure 5B). Further, ROS inhibitor N-acetyl cysteine (NAC) was used to
identify the role of ROS in mediating 5B’s anti-tumor effects. As shown in Figure 5C,
the generation of ROS was greatly attenuated when cells were pre-treated with NAC
for 1 h. And as expected, apoptosis induced by 5B was almost completely reversed by
NAC pre-treatment (Figure 5D). These results suggested that ROS production
mediated 5B-activated apoptosis.
Figure 5. 5B induced apoptosis in A549 cells was dependent on the generation of ROS. (A-B)
ROS generation was measured in cells following exposure to 5B (10 µM) for various time periods
and various concentrations for 9 h. (C) A549 cells were pre-treated with or without NAC (5 mM)
for 1 h before exposure to 5B (10 µM) for 9 h. ROS production was measured by flow cytometry.
(D) Effects of ROS decrease on cell apoptosis. A549 cells were pre-treated with or without NAC
(5 mM) for 1 h before exposure to 5B (10 µM) for 24 h. Percentage of cell apoptosis was
determined by flow cytometry. *, p < 0.05; **, p < 0.01; ***, p < 0.001, vs DMSO. #, p < 0.05; ##,
p < 0.01, vs 5B.
2.6 The activation effects of 5B on JNK pathway
It has been reported that in response to ROS, JNK signaling pathway was often

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activated, ultimately leading to apoptosis, which was also found in curcumin analogs
including EF24[41,42]. Thus, the effects of 5B on the JNK pathway and the
relationship between ROS and JNK were examined. Western blot analysis revealed
that treatment with 5B at a set four time points increased the phosphorylation of
JNK1/2, and treatment for 6 h multiplied the phosphorylation of JNK1/2 in a
dose-dependent manner (Figure 6A-B). Subsequently, to make sure if 5B induced
apoptosis through activating JNK pathway, its specific JNK inhibitor namely
SP600125 was applied to this study. It was not difficult to find that 5B-induced JNK
phosphorylation and apoptosis were effectively abolished by SP600125 (Figure 6C,
6E and 6F). Furthermore, pre-treatment with NAC for 1 h downregulated the
phosphorylation of JNK1/2 and attenuated 5B-induced apoptosis (Figure 6D and 5D).
These results suggested that the activated JNK pathway induced by 5B was at least
partly exerted in A549 cells and promoted apoptosis, and the multiplied ROS levels
mediated the enhanced JNK pathway.
Figure 6. 5B induced apoptosis in A549 cells via ROS-mediated JNK pathway. (A-B) The
expression of p-JNK was detected by western blot. A549 cells were exposed to 5B (10 µM) for
various time periods and various concentrations for 6 h. GAPDH was used as internal control.
(C-D) A549 cells were pre-treated with or without SP600125 (10 µM) and NAC (5 mM) for 1 h
before exposure to 5B (10 µM) for 6 h respectively. Western blot analysis was performed to assess

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the expression of p-JNK. (E) Blocking of JNK pathway abolished the cell apoptosis induced by
5B. A549 cells were pre-treated with or without SP600125 (10 µM) for 1 h before exposure to 5B
(10 µM) for 24 h. Induction of apoptosis was determined by flow cytometry. (F) The percentage of
apoptotic cells from (E) was calculated. *, p < 0.05; **, p < 0.01; ***, p < 0.001, vs DMSO. #, p <
0.05; ##, p < 0.01, vs 5B.
2.7 The inhibition effects of 5B on NF-κB pathway
The abnormal NF-κB activation has been reported to correlate with the occurrence
and development of lung cancer[43,44]. Namely, under normal circumstances, NF-κB
is connected with its inhibitor IκBα in a non-active state. In certain cases of lung
cancer, IKK (inhibitor kappa B kinase) is being continuously phosphorylated, that is,
in a high activity state. Activated IKK introduces phosphorylation of its substrate
IκBα. Then phosphorylated IκBα dissociates from the NF-κB. Soon afterwards NF-κB
enters the nucleus and starts regulating anti-tumor apoptotic protein Bcl-2 expression,
promoting tumor formation. It was known from previous studies that F35 and EF24
inhibited NF-κB signaling pathway playing anti-inflammatory effects[24,45]. Herein,
we performed western blot analysis to determine the effects of TNFα-induced IκBα
degradation on A549 via compound 5B (1 µM, 5 µM and 10 µM), BMS345541 was
selected as control. As shown in Figure 7A, TNFα-induced IκBα degradation was
inhibited when stimulated by TNFα for 15 min after 1 h of 5B action. Additionally,
the inhibitory effect induced by 5B was restrained in plasmid IKKβ transfected cells
relative to mock-vehicle group (Figure 7C), all of which suggested that 5B-induced
apoptosis was mediated by NF-κB pathway. Western blot was used to confirm the
success of plasmid IKKβ transfection (Figure 7B).
Reports showed that NF-κB inhibition was associated with ROS generation, which
played the important role in anti-tumor[46]. Therefore, we further confirmed whether
ROS production stimulated 5B-induced apoptosis by inhibiting NF-κB pathway.
Interestingly, pre-treatment with NAC for 1 h reduced the expression of IκBα (Figure
7D), indicating 5B-induced NF-κB inhibition was activated by ROS inhibitor, ROS
generation had created a significant inhibitory activity on the NF-κB pathway. Given
the above, ROS production triggered 5B-induced apoptosis via activating JNK
pathway and inhibiting NF-κB pathway, more importantly, there were still few reports
on the anti-tumor effects of curcumin analogues via ROS-mediated JNK and NF-κB
pathways.

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Figure 7. 5B induced apoptosis in A549 cells via ROS-mediated NF-κB pathway. (A) A549 cells
were pre-incubated with 5B of different concentrations or BMS345541 (20 µM) for 1 h before the
addition of TNFα (1 ng/mL) for 15 min. The expression of IκBα was detected by western blot.
GAPDH was used as internal control. (B) A549 cells were infected with plasmid IKKβ (2 ng/mL)
or empty vector. The transfection effect was confirmed by western blot. (C) A549 cells transfected
with plasmid IKKβ or empty vector were treated with 5B (60, 20, 6.67 µM) for 24 h. The cell
vitality underlying Ad10’s action was determined using MTT assay (D) The effects of ROS
inhibitor on IκBα degradation. A549 cells were pretreated with or without NAC (5 mM) for 1 h
before exposure to 5B (10 µM) for 1 h. IκBα level was analyzed by western blot. GAPDH was
used as internal control. **, p < 0.01, vs DMSO. #, p < 0.05, vs 5B.
2.8 The chemosensitization effects of 5B
Cisplatin is a first-line anti-lung cancer chemotherapeutic drug, whose use is limited
due to its dose-related side effects[47]. Searching for compounds combined with
cisplatin to reduce the amount of cisplatin and improve the efficacy has bright
prospects. It has been reported that NF-κB activation was a mechanism contributing to
tumor resistance to cisplatin[12,13], which prompted us to investigate that whether 5B,
as a NF-κB inhibitor, had sensitizing effects with cisplatin. As consistent with
previous reports, NF-κB could be activated by cisplatin (Figure 8A). In addition,
cisplatin-induced IKK phosphorylation could be inhibited via 5B in a dose-dependent
manner (Figure 8B). Ulteriorly, clonogenic assay verified that the combination of 5B
and cisplatin could significantly inhibit the formation of colony and increase cisplatin
sensitivity (Figure 8C). Similarly, the combination with anti-lung cancer drug 5-Fu
also existed unexpected combined effects (Figure 8C). Undeniably, high-efficient and
low-toxic compound 5B with capacity of combination with anti-lung cancer drugs
deserved to be further studied.

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Figure 8. The chemosensitization effects of 5B with cisplatin (Pt) and 5-Fu in A549 cells. (A)
IκBα level was measured in cells following exposure to Pt of various concentrations for 24 h and
TNFα (1 ng/mL) for 15 min. GAPDH was used as internal control of western blot analysis. (B)
A549 cells were pretreated with 5B (1 µM, 5 µM and 10 µM) or BMS345541 (20 µM) for 1 h
before exposure to Pt (20/3 µM) for 24 h. Western blot analysis was performed to assess the
expression of p-IKKβ. (C) Clonogenic assay of A549 cells treated with increasing concentrations
of 5B, Pt, or their combination as well as 5B, 5-Fu or their combination as indicated. (D) Percent
inhibition at each concentration of the drugs from (C) was calculated and represented as the
percent of control. *, p < 0.05; **, p < 0.01, vs DMSO. #, p < 0.05; ##, p < 0.01; ###, p < 0.001, vs
(DMSO + Pt).
3. Conclusion
Lung cancer remains the main cause of death worldwide and there still lacks anti-lung
cancer drugs without obvious side effects. In this study, a series of MCACs with
promising anti-tumor activities have been designed and synthesized based on EF24
and F35 motif. Notably, synthesized compounds generally exhibited better selectivity
than curcumin, several of whom proved to display more prominent selectivity in
relation to reference compounds, EF24, F35 and F36. Among them, 5B was
considered as a promising anti-tumor agent in selectively killing cancer and normal
cells with greatly enhanced inhibitory potential. Given that all compounds were

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designed and featured with mulit-methoxy group as well as nitrogen substituents,
which may be associated with changes in activity and toxicity respectively. More
importantly, this assumption needs to be further fully confirmed. Even more
gratifying is that, preferred compound 5B showed less toxicity in vivo than EF24 and
F35, indicating that 5B had a considerable potential in lung cancer research.
Thereupon, molecular mechanism of 5B was elaborated and found to induce cell
cycle arrest in G2/M phase. In addition, 5B subsequently induced apoptosis by
activating JNK and inhibiting NF-κB signaling pathways respectively, both of which
were associated with the generation of ROS. Nevertheless, there were few reports on
anti-tumor activities of MCACs via ROS-mediated JNK and NF-κB pathways.
Meanwhile, 5B was proved to have combined effects with cisplatin or 5-Fu, which, to
a certain extent, reducing the dose of cisplatin or 5-Fu for therapy. Conceivably, the
discovery of 5B with high efficiency, low toxicity and chemosensitization highlighted
its potential therapeutic applications in lung cancer treatment.
4. Experimental section
4.1 Synthesis
4.1.1 Chemistry
All reagents were obtained from commercial suppliers Sigma-Aldrich and Aladdin.
The reaction processes were monitored by thin-layer chromatography via using silica
gel GF254, and the chromatograms were performed with silica gel (200-300 mesh)
and visualized under UV light at 254 or 365 nm. Final compounds of melting points
were determined on open capillary tubes on a Fisher-Johns melting apparatus. Mass
spectrometry analyses were determined on an Agilent 1100 LC-MS (Agilent, Palo
1
Alto, CA, USA). H spectral data were recorded on 600 MHz spectrometer (Bruker
Corporation, Switzerland). The chemical data of compounds were presented as
follows:
3,5-bis((E)-3,4,5-trimethoxybenzylidene)piperidin-4-one (3A): yellow powder, 68.8% yield,
mp 191.7-194.1 °C (Lit[24] 193.7-194.0 °C). 1H-NMR (CDCl3) δ: 7.751 (s, 2H, H-α×2), 6.643 (s,
4H, H-2, H-6×2), 4.220 (d, J=1.2Hz, 4H, CH2×2), 3.912 (d, J=5.4Hz, 18H, 3-OCH3, 4-OCH3,
5-OCH3×2). LC-MS m/z: 456.20, calcd for C25H29NO7: 455.19.
3,5-bis((E)-3,4-dimethoxybenzylidene)piperidin-4-one (4A): yellow powder, 78.7% yield, mp
1
159.2-161.2 °C (Lit[24] 162.2-165.4 °C). H-NMR(CDCl3) δ: 7.758 (s, 2H, H-α×2), 7.008 (d,
J=8.4Hz, 2H, H-2×2), 6.908-6.933 (m, 4H, H-5, H-6×2), 4.191 (s, 4H, CH2×2), 3.895-3.947 (m,
12H, 4-OCH3, 3-OCH3×2). LC-MS m/z: 396.29, calcd for C23H25NO5: 395.17.
1-methyl-3,5-bis((E)-3,4,5-trimethoxybenzylidene)piperidin-4-one (5B): yellow powder, 45.9%

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1
yield, mp 143.7-146.0 °C Lit[48]. H-NMR (CDCl3) δ: 7.768 (s, 2H, H-α×2), 6.647 (s, 4H, H-2,
H-6×2), 3.917 (d, J=5.4Hz, 18H, 3-OCH3, 4-OCH3, 5-OCH3×2), 3.818 (s, 4H, CH2×2), 2.497 (s,
3H, CH3). LC-MS m/z: 470.29, calcd for C28H31NO7: 469.21.
3,5-bis((E)-3,4-dimethoxybenzylidene)-1-methylpiperidin-4-one (6B): yellow powder, 59.7%
1
yield, mp 158.8-161.8 °C (Lit[24] 157.3-159.4 °C). H-NMR (CDCl3) δ: 7.784 (s, 2H, H-α×2),
7.029 (d, J=8.4Hz, 2H, H-6×2), 6.949 (t, J=19.8Hz, 4H, H-2, H-5×2), 3.940 (d, J=9.0Hz, 12H,
3-OCH3, 4-OCH3×2), 3.808 (s, 4H, CH2×2), 2.506 (s, 3H, CH3). LC-MS m/z: 410.26, calcd for
C24H27NO5: 409.19.
1-ethyl-3,5-bis((E)-3,4,5-trimethoxybenzylidene)piperidin-4-one (7B): yellow powder, 65.9%
1
yield, mp 150.3-153.2 °C Lit[48]. H-NMR(CDCl3) δ: 7.762 (s, 2H, H-α×2), 6.642 (s, 4H, H-2,
H-6×2), 3.892 (t, J=17.4Hz, 22H, 3-OCH3, 4-OCH3, 5-OCH3×2, CH2×2), 2.634 (d, J=7.2Hz, 2H,
CH2), 1.089 (t, J=13.8Hz, 3H, CH3). LC-MS m/z: 484.45, calcd for C27H33NO7: 483.23.
3,5-bis((E)-3,4-dimethoxybenzylidene)-1-ethylpiperidin-4-one (8B): yellow powder, 57.3%
1
yield, mp 186.1-187.4 °C (Lit[24] 188.7-190.8 °C). H-NMR (CDCl3) δ: 7.799 (s, 2H, H-α×2),
7.035-7.052 (m, 2H, H-2×2), 6.977 (d, J=1.8Hz, 2H, H-6×2), 6.947 (d, J=8.4Hz, 2H, H-5×2),
3.944 (t, J=10.2Hz, 12H, 3-OCH3, 4-OCH3×2), 3.878 (s, 4H, CH2×2), 2.637-2.673 (m, 2H, CH2),
1.107 (t, J=14.4Hz, 3H, CH3). LC-MS m/z: 424.35, calcd for C25H29NO5: 423.20.
3,5-bis((E)-3,4-dimethoxybenzylidene)-4-methylene-1-propylpiperidine (9B): yellow powder,
1
58.6% yield, mp 166.5-167.6 °C (Lit[24] 162.7-165.2 °C). H-NMR(CDCl3) δ: 7.769 (s, 2H,
H-α×2), 7.022 (dd, J=1.8, 8.4Hz, 2H, H-6×2), 6.955 (d, J=1.2Hz, 2H, H-2×2), 6.926 (d, J=8.4Hz,
2H, H-5×2), 3.952 (s, 6H, 3-OCH3×2), 3.924 (s, 6H, 4-OCH3×2), 3.854 (s, 4H, CH2×2), 2.520 (t,
J=18.4Hz, 2H, CH2), 1.502-1.463 (m, 2H, CH2), 0.884 (t, J=7.2Hz, 3H, CH3). LC-MS m/z:
438.25, calcd for C26H31NO5: 437.22.
1-propionyl-3,5-bis((E)-3,4,5-trimethoxybenzylidene)piperidin-4-one (10B): yellow powder,
55.6% yield, mp 165.9-167.9 °C. 1H-NMR (CDCl3) δ: 7.800 (d, J=33.0Hz, 2H, H-α×2), 6.752 (s,
2H, H-2×2), 6.625 (s, 2H, H-6×2), 4.971 (s, 2H, CH2), 4.774 (s, 2H, CH2), 3.937 (d, J=13.2 Hz,
18H, 3-OCH3, 4-OCH3, 5-OCH3×2), 2.271-2.234 (m, 2H, CH2), 1.073 (t, J=15.0Hz, 3H, CH3).
LC-MS m/z: 512.25, calcd for C28H33NO8: 511.22.
4-oxo-4-(4-oxo-3,5-bis((E)-3,4,5-trimethoxybenzylidene)piperidin-1-yl)butanoic acid (11B):
1
yellow powder, 60.4% yield, mp 171.5-174.4 °C. H-NMR (CDCl3) δ: 7.689 (d, J=18.6Hz, 2H,
H-α×2), 6.871 (d, J=9.0Hz, 4H, H-2, H-6×2), 4.988 (d, J=22.8Hz, 4H, CH2×2), 3.909 (d, J=6.0Hz,
12H, 3-OCH3, 5-OCH3×2), 3.780 (d, J=16.2Hz, 6H, 4-OCH3×2), 2.579-2.600 (m, 2H, CH2),
2.480-2.527 (m, 2H, CH2). LC-MS m/z: 556.36, calcd for C29H33NO10: 555.21.
1-(2-fluorobenzoyl)-3,5-bis((E)-3,4,5-trimethoxybenzylidene)piperidin-4-one (12B): yellow
1
powder, 65.6% yield, mp 175.6-176.2 °C. H-NMR (CDCl3) δ: 7.762 (s, 1H, H-6’), 7.620 (s, 1H,
H-4’), 7.331-7.369 (m, 1H, H-α), 7.263-7.290 (m, 1H, H-α’), 7.077-7.104 (m, 1H, H-5’), 7.304 (t,
J=18.6Hz, 1H, H-3’), 6.951 (s, 2H, H-2×2), 6.582 (s, 2H, H-6×2), 5.162 (s, 2H, CH2), 4.790 (s,
2H, CH2), 3.935 (s, 6H, 4-OCH3×2), 3.781 (t, J=34.2Hz, 12H, 3-OCH3, 5-OCH3×2). LC-MS m/z:
578.28, calcd for C32H32FNO8: 577.21.
1-isobutyryl-3,5-bis((E)-3,4,5-trimethoxybenzylidene)piperidin-4-one (13B): yellow powder,
43.6% yield, mp 152.6-156.0 °C. 1H-NMR (CDCl3) δ: 7.809 (d, J=33.0Hz, 2H, H-α×2), 6.744 (s,
2H, H-2×2), 6.636 (s, 2H, H-6×2), 4.971 (s, 2H, CH2), 4.832 (s, 2H, CH2), 3.937 (d, J=13.8 Hz,
18H, 3-OCH3, 4-OCH3, 5-OCH3×2), 2.626-2.670 (m, 2H, CH2), 1.043 (d, J=7.2Hz, 6H, CH3×2).
LC-MS m/z: 526.34, calcd for C29H35NO8: 525.24.

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4.1.2 Synthetic procedures
4.1.2.1 General synthetic procedure for the synthesis of F35 and F36
4-piperidone hydrochloride hydrate 2 (5.0 mmol) and aryl aldehydes (10.0 mmol
3,4,5-trimethoxybenzaldehyde, 3,4-dimethoxybenzaldehyde) were dissolved in the
mixture solvent of ethanol and water (10:1) and 40% NaOH aqueous solution was
added into the solution to catalyze the reaction. The reaction stirred at room
temperature for 12 h, the precipitate was formed. After filtration and washing with
water and then recrystallized from ethanol to obtain desired products.
4.1.2.2. General synthetic procedure for the synthesis of 5B-13B
NaH (60% dispersion in mineral oil, 2.0 mmol) was added in portions to a stirred
solution of F35 or F36 (2.0 mmol) in anhydrous THF (10.0 mL) cooled in an ice bath.
The resulting mixture was then allowed slowly to warm to room temperature. After
stirring for 30 min, different alkyl halide or acyl chloride (3.0 mmol) in anhydrous
THF (3.0 mL) was added drop wise. When TLC monitoring showed complete
consumption of the starting material, the reaction mixture was evaporated under
reduced pressure. The resulting solid was purified by silica gel chromatography using
hexane and ethyl acetate gradient to obtain desired products.
4.2 Cells culture and MTT assay
Human lung cancer cell lines H460, A549, H1650, H1975 and normal human liver
cell line HL7702 were all obtained from Chinese academy of sciences, a typical cell
library culture preservation committee (China, shanghai). All cell lines were cultured
in 1640 medium (Gibco, Eggenstein, Germany), which contained 10%
heat-inactivated fetal bovine serum, 100 IU/mL penicillin and 100 µg/mL
streptomycin, growing in the humidifying cultivating environment of 5% CO2 at
37 °C.
The rate of cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. In short, H460, A549, H1650, H1975 and
HL7702 cells were seeded in 96-well plates with the cell density of 4000 per hole and
left adherenced overnight. The various concentrations of compounds were then
diluted in 1640 medium for three holes repeatability. The cell viability was detected
after 72 h incubation with tested substances, drugs were acted in transfected cells for
24 h besides, 25 µL MTT (5 mg/mL) was then added per hole for 4 h at incubator,

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DMSO dissolving crystallization, and the absorbance was detected at 490 nm by
ELIASA.
4.3 Acute toxicity experiment
To assess the toxicity of the preferred compound, acute toxicity test was used. The
C57BL/6 mice were purchased from SHANGHAI SLAC and observed for one week
in a controlled environment (air-conditioning, 50 ± 10% relative humidity, T= 22 ±
2 °C) prior to the experiments. The mice were then randomly assigned to different
groups, respectively administered by intraperitoneal injection and lavage two ways to
assess subjects compounds at a volume of 1 g/kg body weight. Solution of the
compounds of intraperitoneal injection was prepared in 6% castor oil and gavage
solvent was 0.5% CMC-Na. After more than ten days of continuous observation, the
state and weight of the mice were recorded.
4.4 Cell cycle
2 mL A549 cell suspension (6 x 105) was vaccinated in the diameter of 60 mm Petri
dishes and allowed to be cultured overnight. The cells were then incubated with
compounds with prescribed concentrations for 10 h. All cells were harvested by
digesting with trypsin and washed with PBS (Sigma-Aldrich, Germany) and then
fixed with ice-cold 70% ethanol in PBS for 30 min at -20 °C. Fixed cells were washed
with PBS, treated with RNase A (10 mg/mL) and resuspended in 50 µg/mL propidium
iodide (PI) for staining. Cell cycle distribution was performed with FACSCalibur flow
cytometer (BD Biosciences, CA).
4.5 Western blot
The cells were lysed in protein lysate buffer for 10 min. Total proteins were
centrifuged at 12,000 rpm for 10 min at 4 °C and quantified. Protein samples were
separated with 10% SDS-PAGE gel. After electrophoresis, the proteins in the
gel were transfered to PVDF membrane, incubated with 5% skim milk for 1.5 h. After
overnight incubation with the primary antibody, protein samples were incubated with
HRP-conjugated secondary antibodies for 1 h. The results were detected by Quantity
One software. The anti-p-IKKβ, anti-IKKβ, anti-IκBα, anti-GAPDH, anti-Bax,
anti-cleaved PARP, anti-p-JNK, anti-JNK, goat anti-rabbit IgG-HRP, goat anti-

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mouse IgG-HRP antibodies were obtained from Santa Cruz Biotechnology (Santa
Cruz, CA).
4.6 Apoptosis assay
For cell apoptosis analysis, after treatment for 24 h, cells were collected by the
pancreatin without EDTA. In some experiments, NAC (5 mM) was pre-incubated for
1 h before the exposure of tested substances. Cells were stained with 3 µL FITC
Annexin V and 1 µL PI for 15 min at room temperature avoiding light, and
immediately analyzed on FACSCalibur flow cytometer (BD Biosciences, CA). The
PI/RNase staining buffer was purchased from BD Bioscience (Franklin Lakes, NJ).
4.7 Hoechst 33258
A549 cells were inoculated in the Petri dishes of 3 cm diameter 1.2×106 cells per well
overnight, then replaced liquid, added compounds with the prescribed concentrations.
After 24 h drug action, the cells were fixed and stained by Hoechst 33258. The
nuclear morphometry was observed by the fluorescence microscope. Under the
magnification of 200, compounds could induce cell apoptosis obviously. Hoechst
33258 kit was obtained from Beyotime Institute of Biotechnology.
4.8 Determination of the level of ROS
The generation of ROS was detected by flow cytometry. A549 cells (6×105) were
seeded in the diameter of 60 mm Petri dishes and exposed to 10 µM of compound for
different times or various concentrations for 9 h. In some experiments, NAC (5 mM)
was pre-incubated for 1 h before the compounds exposure, and ROS levels were
determinated. Cells were incubated with DCF-DA (10 µM) in the dark at 37 °C for 30
min. Then the cells were collected, washed in PBS and analyzed by flow cytometer.
4.9 Cell transfection
Cells were seeded in 6-well dishes at a density of 3 × 105 cells per well and cultured
at 37 °C with 5% CO2 overnight. Before transfection, cells were washed by PBS and
added serum-free medium. Next, the mixture of high levels of plasmid and 2 µL of
lipofectamine 2000 as well as separate lipofectamine 2000 were added to the cell
culture in each well respectively. After 6 h, the complex medium was removed and
replaced by the 1640 medium contained 10% fetal bovine serum (FBS) and 1%

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penicillin-streptomycin solution. After further incubated for 12 h, cells were rinsed
twice with PBS, harvested by cell lysate and then detected by western blot.
4.10 Clonogenic assay
A549 cells were seeded in 6-well plates with the cell density of 1000 per hole and
exposed to the absence or presence of drugs with various concentrations for holes
respectively. Growth media with or without drugs was replaced after 2 days. And 8
days later, cells were fixed with methanol (1%) and formaldehyde (1%), stained with
0.5% crystal violet, then photographed by camera.
4.11 Statistical analysis
Data were expressed as means ± SEM of three independent experiments. Student’s
t-test and two-way ANOVA were employed to analyze the statistical comparisons
between sets of data. Significant differences were established at P < 0.05.
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.
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Highlights
• A series of EF24 analogues with cytotoxic activities selectively towards lung cancer cells were
designed and synthesized. 5B showed lower toxicity than EF24 both in vitro and vivo.
• 5B could significantly enhance the sensitivity of A549 cells to cisplatin or 5-Fu.
• 5B exerted anti-tumor activities by activating JNK and inhibiting NF-κB signaling pathways
respectively, both of which were associated with the generation of ROS.