Design, synthesis, and evaluation of chalcone analogues incorporate α,β-Unsaturated ketone functionality as anti-lung cancer agents via evoking ROS to induce pyroptosis

Article abstract of DOI:10.1016/j.ejmech.2018.08.072

Chalcone, a natural structure, demonstrates many pharmacological activities including anticancer, and one promising mechanism is to modulate the generation of ROS. It has been known that pyroptosis is associated with anticancer effects, whereas there is fewer researches about ROS-mediated pyroptosis triggered by chemotherapy drugs. Moreover, incorporation of a α,β-unsaturated ketone unit into chalcone may be an effective strategy for development of chemotherapy drugs. Hence, a number of chalcone analogues bearing a α,β-unsaturated ketone were synthesized from chalcone analogues 1 with modest anticancer activities as the lead compound. Structure-activity relationship (SAR) studies confirmed the function of α,β-unsaturated ketone to improve anticancer activity. Notably, compound 8, bearing a α,β-unsaturated ketone, is the most potent inhibitor of cancer, with IC₅₀ values on NCI-H460, A549 and H1975 cells of 2.3 ± 0.3, 3.2 ± 0.0 and 5.7 ± 1.4 μM, respectively. Besides, 8 showed antiproliferative ability against NCI-H460 cells in a time- and concentration-dependent manner through modulating ROS to induce caspase-3-mediated pyroptosis, and displayed a better safety profile in vivo. Overall, these results demonstrated that compound 8 is a candidate agent and a potential lead compound for development of chemotherapy drugs, and can be used as a probe to further examine the mechanism of ROS-dependent pyroptosis.

Full text of DOI:10.1016/j.ejmech.2018.08.072

Accepted Manuscript
Design, synthesis, and evaluation of chalcone analogues incorporate α,β-Unsaturated
ketone functionality as anti-lung cancer agents via evoking ROS to induce pyroptosis
Min Zhu, Jiabing Wang, Jingwen Xie, Liping Chen, Xiaoyan Wei, Xing Jiang, Miao
Bao, Yanyi Qiu, Qian Chen, Wulan Li, Chengxi Jiang, Xiaoou Zhou, Liping Jiang,
Peihong Qiu, Jianzhang Wu
PII:
S0223-5234(18)30744-X
10.1016/j.ejmech.2018.08.072
EJMECH 10683
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 February 2018
Revised Date: 24 August 2018
Accepted Date: 25 August 2018
Please cite this article as: M. Zhu, J. Wang, J. Xie, L. Chen, X. Wei, X. Jiang, M. Bao, Y. Qiu,
Q. Chen, W. Li, C. Jiang, X. Zhou, L. Jiang, P. Qiu, J. Wu, Design, synthesis, and evaluation of
chalcone analogues incorporate α,β-Unsaturated ketone functionality as anti-lung cancer agents via
evoking ROS to induce pyroptosis, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.08.072.
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Graphical abstract
NCI-H460
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Xn
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A549
H1975
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pyroptosis
Colony formation assay in NCI-H460

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Design, Synthesis, and Evaluation of Chalcone Analogs Incorporate
α,β-Unsaturated Ketone Functionality as Anti-lung Cancer Agents via
Evoking ROS to Induce Pyroptosis
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Min Zhua1, Jiabing Wangab1, Jingwen Xiea, Liping Chena, Xiaoyan Weica, Xing Jianga, Miao Baoad,
Yanyi Qiua, Qian Chena, Wulan Liae, Chengxi Jianga, Xiaoou Zhoua, Liping Jiangf, Peihong Qiua*,
Jianzhang Wua**
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(aChemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical
University, Wenzhou, Zhejiang 325035, China)
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(bMunicipal Hospital Affiliated to Medical School of Taizhou University, Taizhou, Zhejiang,
318000, China)
(cDepartment of Pharmacy, Zhejiang Cancer Hospital, Hangzhou, Zhejiang 310022, China)
(dDepartment of Pediatrics, The First Affiliated Hospital of Wenzhou Medical University,
Wenzhou, Zhejiang 325035, China)
(eCollege of Information Science and Computer Engineering, Wenzhou Medical University,
Wenzhou, Zhejiang 325035, China)
(fDepartment of Parasitology, Xiangya School of Medicine, Central South University, Changsha,
Hunan 410013, China)
(1 These authors contribute to this work equally)
*Correspondence author: Peihong Qiu
Address: Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou
Medical University, Wenzhou, Zhejiang 325035, China
E-mail: wjzwzmc@126.com
**Correspondence author: Jianzhang Wu
Address: Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou
Medical University, Wenzhou, Zhejiang 325035, China
E-mail: wjzwzmu@163.com
Disclosures/Conflicts of Interest: None declared
Abstract
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Chalcone, a natural structure, demonstrates many pharmacological activities
including anticancer, and one promising mechanism is to modulate the generation of
ROS. It has been known that pyroptosis is associated with anticancer effects, whereas
there is fewer researches about ROS-mediated pyroptosis triggered by chemotherapy
drugs. Moreover, incorporation of a α,β-unsaturated ketone unit into chalcone may be
an effective strategy for development of chemotherapy drugs. Hence, a number of
chalcone analogues bearing a α,β-unsaturated ketone were synthesized from chalcone
analogues 1 with modest anticancer activities as the lead compound. Structure-activity
relationship (SAR) studies confirmed the function of α,β-unsaturated ketone to
improve anticancer activity. Notably, compound 8, bearing a α,β-unsaturated ketone,
is the most potent inhibitor of cancer, with IC50 values on NCI-H460, A549 and
H1975 cells of 2.3±0.3, 3.2±0.0 and 5.7±1.4 µM, respectively. Besides, 8 showed
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antiproliferative ability against NCI-H460 cells in
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time- and
concentration-dependent manner through modulating ROS to induce
caspase-3-mediated pyroptosis, and displayed a better safety profile in vivo. Overall,
these results demonstrated that compound 8 is a candidate agent and a potential lead
compound for development of chemotherapy drugs, and can be used as a probe to
further examine the mechanism of ROS-dependent pyroptosis.
Keywords
Chalcone analogues; α,β-unsaturated ketone; Anticancer effect; ROS; Pyroptosis
1. Introduction
Lung cancer, one of the most leading reasons of morbidity and mortality across
the world [1], brings increasing pressure toward human health. Most patients are
diagnosed at an advanced stage, which makes them unable to be treated by surgical
removal, and chemotherapy is the most primary clinical treatment for them. Currently,
there are two main classes of small molecule chemotherapeutic drugs including
targeted drugs and cytotoxic drugs [2]. Targeted therapy is widely used because of the
strong efficacy for patients with specific genomic aberrations and the fewer side
effects. However, mutations frequently happened in lung cancer and thus the
successful targeted therapeutic strategy far eluded us [1-3]. What’s more, there is not a
potentially actionable molecular target for some patients [3], which results in targeted
drugs inactive. Hence, it is the first choice to apply the classical cytotoxic drugs for
patients ineffective in targeted therapies. However, the cytotoxic agents have more
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adverse side effects such as gastrointestinal reaction, liver dysfunction, kidney failure,
cardiovascular complications and others [4]. Thus, development of new
chemotherapeutic agents with great efficiency and reduced side effects is still very
urgent.
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At present, the diversity of natural products still provides a critical source of
bioactive lead compounds for development of new drugs, which is attributed to their
good activity and low toxicity [5-7]. For instance, chalcone, one of the numerous
natural compounds, has an extensive distribution in fruits, tea, vegetables and other
plants [8-10]. It’s also well-known that chalcones demonstrate wide biological
activities including anticancer [11], anti-inflammatory [12], analgesic [9], and
antioxidant [13], whereas the pharmacological activity ordinarily results in minor
effectiveness. It has been reported that the activities of synthetic chalcones were
obviously better than natural compounds [14-16]. In our previous study, chalcone
derivative 1 was found to possess a modest efficiency on inhibiting proliferation of
cancer cells [11], which may be further improved through ulteriorly optimization.
Most recently, Rana et al. has found that the α,β-unsaturated ketone functionality
(a Michael acceptor) in the α-methylene-γ-butyrolactone analogues is pivotal for the
inhibition of cancer cell growth [17]. And Heller et al. reported that combination of a
α,β-unsaturated ketone unit can significantly increase the anticancer activity of
triterpenoic acids compounds [18]. These examples showed that incorporating a
α,β-unsaturated ketone into a certain molecular structure is able to augment the
anticancer properties of secondary natural products [17-22], and α,β-unsaturated
ketone can be regarded as functionality structure to drugs design. Therefore, based on
remaining the scaffold of compound 1, we focused on molecular hybridization
strategy in order to design and synthesis a series of chalcone analogues bearing
α,β-unsaturated ketone scaffold, and evaluated their anti-lung cancer activities, and
determined whether α,β-unsaturated ketone group is able to further improve the
cytotoxicity.
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One of the characterized anticancer mechanisms of chalcones is up-regulating
the generation of intracellular reactive oxygen species (ROS) [8, 23-27]. The
elevating production of ROS is responsible for the inflammasome-dependent
pyroptosis [28-30]. In addition, inflammasomes are found to exhibit anticancer effects
through inducing cell pyroptosis [30], a programmed cell death that can also be
triggered by chemotherapy drugs in various cancer cell lines [31-33]. Nevertheless,
there is no report about the relationship between ROS evoked by chemotherapeutics
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and pyroptosis in cancer cells. Thus, in the present study, we started a new anticancer
mechanism study of the active compounds and found that increased ROS can result in
pyroptosis in lung cancer cells.
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2. Results and discussion
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2.1 Design and synthesis of chalcone analogues
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To increase the anticancer effects produced by a change in the structure of
chalcone-based compound 1, the first series of analogues were obtained by acylation
reaction of the amine group on “A” ring of 1. To our surprise, compared with
compound 1, except compound 8 containing an acrylamide motif, others showed
lower cytotoxic against all the tested human lung cancer cell lines, which suggested
that α,β-unsaturated carbonyl may strengthen the anticancer activity. Compound 11
was synthesized to investigate the function of α,β-unsaturated ketone located in
chalcone skeleton, while retaining the α,β-unsaturated ketone of “A” ring.
Furthermore, in order to develop anticancer compounds with higher efficiency,
another series of chalcone analogues were designed, and different groups were
introduced on “B” ring while “A” ring retained the acrylamide substituents.
The synthesis of two series of chalcone derivatives 2-9 (Table 1) and 12-29
(Table 2) was performed according to the synthetic pathway shown in Scheme 1.
Initially, compounds of the first series (2-9) were prepared from the lead compound 1
with various acyl chlorides, in the presence of triethylamine as the acid binding agent.
Afterwards, another series of chalcone analogues 12-29 were synthesized.
4-aminoacetophenone (10) reacted with acrylyl chloride to give the intermediate 11.
Aldol condensation reaction was performed between 11 and a variety of substituted
benzaldehyde by using 40% NaOH as the base to give the chalcone derivatives 12-29.
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All the chalcone analogues were characterized by LC-MS, HPLC, H-NMR and
13C-NMR (Supplementary information). The characteristic data of all products
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including color, yield, melting points, LC-MS, HPLC, H-NMR and 13C-NMR
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spectrum of compounds were presented in chemistry synthetic section.
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Scheme 1. The general route to produce the chalcone analogues 2-9 and 12-29. Reagents and
conditions: (a): acyl chloride, THF, 0 °C; (b): benzaldehyde, 40%NaOH, EtOH, room temperature.
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Table 1. The chalcone analogues 2-9
Comp.
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R1
Comp.
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-CH=CH2
-CH2CH3
-CH3
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Table 2. The chalcone analogues 12-29
Comp.
R2
Comp.
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2,5-OCH3
2-Br
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2,4-Cl
4-N(CH3)2
4-OCH3
2-F
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4-Cl
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3,4-Cl
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3,4,5-OCH3
3,4-OCH3
2,3-Cl
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/
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2,3-OCH3
2-CF3
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2-F 5-OCH3
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a “/” represents none.
2.2 In vitro screening of chalcone derivatives against lung cancer cells
The first series of the synthesized chalcone analogues (2-9) were evaluated for
their in vitro antiproliferative activity against three human lung cancer cell lines,
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namely, NCI-H460, A549 and H1975, using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenylte-trazolium bromide (MTT) assay. BMS-345541 (BMS) and Xn
(xanthohumol) were used as positive controls. As shown in Figure 1A-C, it is
noticeable that most compounds showed lower activity with no clear structural
activity relationships (SARs). Among three cancer cell lines, these compounds
exhibited relatively larger cytotoxic against NCI-H460 cells. Interestingly, introducing
a α,β-unsaturated ketone in compound 8 displayed potent growth inhibition against
the three lung cancer cells, which indicated that the Michael acceptor is a favorable
feature to increase anticancer potential. Additionally, these results showed that
anticancer activity of compound 8 was more pronounced than that of BMS and Xn.
Among compounds 1, 8 and 11, compared to compounds 1 and 11 with only one
α,β-unsaturated ketone, it was found that compound 8 bearing two α,β-unsaturated
ketones displayed higher growth inhibition toward cancer cells, which suggested that
compound containing two α,β-unsaturated ketones may show more potent anticancer
activity.
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Based on these results above, in order to obtain more effective anticancer
compounds, a series of compound 8 analogues (12-29) by varying the substitutions on
“B” ring were further developed (Table 2). According to the in vitro cytotoxic
activities, the structural activity relationships (SARs) of the chalcone analogues 12-29
have been proposed. Close observation of results from Figure 1D-F indicated that
almost all compounds selectively displayed higher anticancer activity to the
NCI-H460 and H1975 cells, whether electron donating groups or electron
withdrawing groups on ring “B”. In comparison with compound 18 without
substitution on phenyl ring, analogues bearing substitution on phenyl ring showed
different levels of anticancer activities, especially compound 13 (4-dimethylamino)
and compound 20 (2-trifluoromethyl) showed lower effects on these three cancer cells.
Compound 20 with 2-trifluoromethyl substitution on phenyl ring was selectively
active on NCI-H460 cells and moderately active on both A549 and H1975 cells.
Moreover, results exhibited that analogues 13 (4-dimethylamino), 14 (4-methoxy), 23
(4-chloro), 24 (3,4-dichloro), 25 (3,4-difluoro), and 27 (3,4-dimethoxy) showed potent
inhibition on both NCI-H460 and H1975 cells and lower effects on A549 cells.
Furthermore, it should be noted that different substitutions on the “B” ring didn’t play
any significant role in activity to NCI-H460 cancer cells.
Lastly, in recent years, the PAINS (pan-assay interference compounds) present a
major problem for medicinal chemistry. PAINS are small molecules that are reactive
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under assay conditions and produce false-positive signals [34]. However, several
studies have suggested that some PAINS might not be that promiscuous or
problematic and could even be true quality probes [35-36]. And it has been
emphasized by Gomes et al. that chalcones, as perspective drugs against cancer,
bacteria, etc., were mightily underestimated due to the PAINS filtering [10]. In this
study, the anticancer efficiencies of these chalcone compounds have been determined
by MTT assay, it is possible to exclude that the studied derivatives act in a
non-specific way as PAINS. Based on these results, the most active compounds 8, 22
and 26 from these derivatives were taken-up for further detailed studies.
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Figure 1. The growth inhibition rate of compounds against NCI-H460 cells (A, D), A549 cells (B,
E), and H1975 cells (C, F). The cells were treated with chalcone analogues, BMS and Xn
(xanthohumol) at 5 µM for 72 h, finally determined by the MTT assay.
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2.3 Active compounds inhibited growth of lung cancer cell lines
On the basis of their promising cytotoxicity, compounds 8, 22 and 26 were
selected for subsequent evaluation of their IC50 on three lung cancer cell lines. As
listed in Table 3, each compound showed potent growth inhibition on NCI-H460,
A549 and H1975 cells with the IC50 values in the range of 2.3±0.3-5.7±1.4,
2.2±0.9-5.2±0.7 and 2.0±1.1-5.4±1.2 µM, respectively, which are approximately 2.2-
to 3.8-fold more potent than Xn. These results ulteriorly validated that incorporating a
α,β-unsaturated ketone unit into chalcone scaffold can significantly enhance their
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anticancer effects. Moreover, as shown in Figure 2A, when NCI-H460 cells were
treated with compounds 8, 22 and 26 for a series of times, the population of viable
cells decreased in a time-dependent manner. Furthermore, the colony formation assay
exhibited that these compounds can obviously restrain the colony formation in a
concentration-dependent pattern compared to control group (Figure 2B). More
interestingly, all compounds exhibited a greater suppression on colony formation at
2.5 µM than Xn at 5 µM (Figure 2B). Collectively, these data suggested that
compounds 8, 22 and 26 can efficaciously inhibit the growth of three lung cancer cell
lines.
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Table 3. The IC50 (µM) of selected compounds against lung cancer cell lines.
Compound
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Figure 2. Three active compounds exhibited inhibitory effects on NCI-H460 cells. (A) NCI-H460
cells were treated with compounds 8, 22 and 26 and Xn at 5 µM for 12, 24, 48 and 72 h, and the
cell viability was measured by MTT assay. (B) NCI-H460 cells were exposed to compounds 8, 22
and 26 at 1.25, 2.5 and 5 µM or Xn at 5 µM. Then medium was changed to fresh medium and
allowed to form colonies.
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2.4 Compound 8 exhibited anticancer effect by regulating the generation of ROS
ROS are recognized as a pivotal factor in numerous cellular signaling pathways
including inflammation, proliferation, metabolism, autophagy, apoptosis, etc [37-39].
The regulation of intracellular ROS levels could be exploited for selective therapeutic
approach of cancer, and ROS-based therapeutic strategies continued to be an
important direction for research of anticancer agents nowadays. Studies early have
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demonstrated that many anticancer agents [40-43] including chalcones are able to
up-regulate the production of intracellular ROS. Consequently, based on its better
activity, we selected compound 8 in the interest of determining whether the anticancer
effect of active compounds are involved in ROS-mediated therapy. Results were
showed in Figure 3A, NCI-H460 cells were incubated with 10 µM compound 8 for 3,
6, 12 and 24 h, the levels of intracellular ROS were augmented in comparison to the
control group. In addition, the promoted intracellular ROS accumulation induced by 8
was distinctly suppressed by N-acetyl cysteine (NAC), which is one of the ROS
scavengers (Figure 3B). Generally, these data suggested that compound 8 can
modulate the generation of intracellular ROS.
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Afterwards, MTT and colony formation assay were performed to ulteriorly
determine if the enhancement of intracellular ROS are responsible for the anticancer
activity of compound 8. As shown in Figure 3C-D, there was almost no cell death
after incubation NCI-H460 cells with NAC alone, whereas the growth of cancer cells
was significantly suppressed after treating with 8 alone. Moreover, when NAC and
compound 8 were applied together, the inhibitory efficacy of compound 8 was
evidently attenuated. This indicated that NAC can not only restrain the generation of
ROS but also suppress the cell death. Additionally, there was a significant difference
between only NAC treated group and compound 8 with NAC treated group, which
indicated that NAC can obviously suppress the anticancer effect of 8 (Figure 3C). On
the basis of these results, we confirmed that promotion of intracellular ROS levels
have a significant function in inducing cell death, and at least partly, explain the
anticancer activity of compound 8.
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Figure 3. Compound 8 exhibited cytotoxicity against NCI-H460 cells through increasing ROS. (A)
ROS levels were measured after treating with 8 at 10 µM for indicated times. (B) Pretreatment
with 5 mM NAC for 1 h and then cells were incubated with 10 µM 8 for 3 h. ROS generation were
assessed by flow cytometry. (C-D) Cells were pre-incubated with 5 mM NAC for 1 h before
exposing to compound 8 (5 µM). (C) Cell viability was measured via MTT assay after treating
with 8 for 48 h. *** P<0.001 vs only compound 8 treated group. # P<0.05 vs NAC treated group.
(D) The medium was changed after treating with compound 8 for 12 h and cells were growing in
fresh medium for approximately 8 days.
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2.5 Compound 8 displayed cytotoxicity against NCI-H460 cells via ROS-based
pyroptosis
Pyroptosis is an inflammatory form of lytic programmed cell death. It has been
reported that chemotherapy drugs, such as 5-fluorouracil (5-FU) [32] and cisplatin
[31], have the ability to trigger pyroptosis in NCI-H460 cells. Therefore, we have
sought to investigate whether compound 8 could lead to pyroptosis. As seen in Figure
4A, the morphologic signs of pyroptosis were observed under optical microscope after
treatment with 8, characterized by the loss of osmotic potential, cytoplasmic swelling
and cellular content releasing. Likewise, caspase-3 is known to play a key role in
NCI-H460 cells pyroptosis triggered by chemotherapy drugs [31]. Then western blot
analysis was utilized for studying the expression of this protein. Results showed that
compound 8 decreased pro-caspase-3 expression in a time-dependent manner (Figure
4C), which suggested that compound 8 may trigger pyroptosis upon degradation of
pro-caspase-3.
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What’s more, it has been known that the production of ROS is involved in the
inflammation-dependent pyroptosis. But there is still fewer researches on the
interrelation between pyroptosis and ROS both induced by chemotherapy drugs in
cancer cells. Hence, we sought to preliminarily investigate if the increased ROS act on
the pyroptosis triggered by compound 8. NCI-H460 cells were pretreated with NAC
for 4 h and then treated with 8. As shown in Figure 4B, it was found that pyroptosis is
suppressed by NAC, and we can draw a conclusion that increased ROS by compound
8 can lead to pyroptosis in lung cancer cells. In summary, these results indicated that
compound 8 probably exhibits anticancer efficacy by targeting ROS to trigger
caspase-3-mediated pyroptosis in NCI-H460 cells. However, in the previous
investigation, V Derange`re [33] discovered that ROS do not affect the occurrence of
pyroptosis in human colorectal carcinoma HCT116 cells. Thus it suggested that not all
cancer cells’ pyroptosis are associated with the increase of ROS. These findings
suggested that ROS-dependent pyroptosis may be a new target with promising
therapeutic application, and could be explored as an approach for therapy of cancers.
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Figure 4. Compound 8 triggered pyroptosis in NCI-H460 cells. (A-B) Static bright field cell
images of pyroptosis were obtained with microscopic imaging. (A) Cells were stimulated with 20
µM compound 8 and 20 µg/mL cisplatin for indicated times. (B) NCI-H460 cells were pretreated
with NAC (20 mM) for 4 h and then treated with 8 (20 µM) for 24 h. (C) NCI-H460 cells were
treated with 8 (5 µM) for indicated times and western blot was performed for detection of
pro-caspase-3.
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2.6 Toxicity examination of 8 in animal models
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In present study, the toxicity exhibited in vivo of compound 8 was investigated to
aid in future application of active compounds. Therefore, acute toxicity experiment
was further carried out to determine the toxicity of compound 8. Results were showed
in Figure 5, no mortality was observed for any of 8-treated mice while the BALB/c
mice treated with EF24, which is a candidate compound with two Michael acceptors
[44, 45] and excellent anticancer activities [46, 47], appeared a 33.3% mortality rate.
This data suggested that 8 has lower toxicity than EF24. Generally, based on these
results, it was confirmed that compound 8 showed a better safety profile in animal
models compared with EF24.
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Figure 5. Survival chart of BALB/c mice after treatment with compound 8 in an in vivo acute
toxicity experiment. All compounds were administrated with a single dose of 500 mg/kg via ip
injection at the first day only.
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3. Conclusion
In present work, a structural optimization on the lead compound 1 was conducted
with the aim to improve the anticancer efficacy. Two series of new chalcone
derivatives were designed and synthesized via incorporation of functional groups, and
their antiproliferative effects against three lung cancer cells were evaluated. It was
found that, in comparison to compound 1, BMS and Xn, incorporation of a
α,β-unsaturated ketone functional group help to markedly increase the
antiproliferative efficacy of most compounds. Particularly, compound 8, bearing a
α,β-unsaturated ketone unit, displayed the most potent anticancer effects with the IC50
values of 2.3±0.3, 3.2±0.0 and 5.7±1.4 µM, respectively, against NCI-H460, A549
and H1975 cells. Likewise, 8 demonstrated excellent cytotoxicity against NCI-H460
cells in time- and concentration-dependent manner, and was a potent intracellular
ROS inducer. Preliminary mechanism studies exhibited that compound 8 significantly
induce death of NCI-H460 cells, at least partially, by promoting the levels of
intracellular ROS to trigger caspase-3-mediated pyroptosis. More importantly, in
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healthy BALB/c mice, ip administration of 8 at 500 mg/kg was found to have a better
safety profile than EF24. Taken together, these results indicated that α,β-unsaturated
ketone functionality has crucial significance for anti-lung cancer effects of chalcone
analogues, and derivative 8 has superior activity in vitro, and the mechanism of
ROS-mediated pyroptosis deserves further investigating using 8 as a probe.
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4. Experimental section
8
4.1 Chemistry
9
All chemical reagents and solvents were available from Sigma-Aldrich Aladdin
(Beijing, China) and used without further purification. Thin-layer chromatography
(TLC) uses silica gel GF25 monitoring reaction, observed by UV light. Mass
spectrometry (MS) was performed through an Agilent 1100 LC-MS (Agilent, Palo
Alto, CA, USA). Melting point was measured in an opened capillary that under the
Fisher-Johns melting apparatus, and uncorrected. Using 600 MHz (1H) and 400 MHz
(13C) spectra (Bruker Corporation, Switzerland) record nuclear magnetic resonance
spectroscopy (NMR) with TMS as an internal standard. Chemical shifts were
performed with CDCl3 and DMSO-d6, 1H NMR coupling constants (J) was displayed
by hertz (Hz), and multiplicity was expressed as follows: s = singlet, d = doublet, t =
triplet, dd = doublet of doublets, m = multiplet. The analysis and purification of HPLC
were done an on Agilent 1100 series instrument, using a Agilent ZORBAX SB-C18
column of 4.6 mm i.d.. The purity of all compounds was determined by HPLC
analysis to be ≥95%. Data of novel chemical were pressed as follows:
4.1.1 (E)-N-(4-(3-(2-chlorophenyl)acryloyl)phenyl)benzamide (2)
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1
Pale yellow powder, 50.32% yield, mp 214.1-216.1 °C. H-NMR (600 MHz,
DMSO-d6), δ:10.613 (s, 1H, NH), 8.233 (d, J=8.4 Hz, 1H, Ar-H3’), 8.224 (d, J=8.4
Hz, 2H, Ar-H2, Ar-H6), 8.057 (d, J=15.6 Hz, 1H, β-H), 8.021 (d, J=15.6 Hz, 1H, α-H),
8.024 (d, J=9.0 Hz, 2H, Ar-H2’’, Ar-H6’’), 7.991 (d, J=8.4 Hz, 2H, Ar-H3, Ar-H5),
7.628 (t, J=8.4 Hz, 1H, Ar-H4’’), 7.580 (d, J=9.0 Hz, 2H, Ar-H3’’, Ar-H5’’), 7.499-7.462
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(m, 2H, Ar-H4’, Ar-H5’), 7.069 (d, J=7.8 Hz, 1H, Ar-H6’). C-NMR (400 MHz,
DMSO), δ: 187.439, 166.014, 143.925, 137.957, 134.556, 134.264, 132.441, 132.334,
131.876, 131.815, 129.983, 129.827×2, 128.544, 128.420×2, 127.782×2, 127.639,
124.853, 119.589×2. HPLC: purity 95.5%. LC-MS m/z: 362.28[M+1]+, calcd for
C22H16ClNO2: 361.09.
4.1.2 (E)-4-chloro-N-(4-(3-(2-chlorophenyl)acryloyl)phenyl)benzamide (3)
1
Pale yellow powder, 58.85% yield, mp 182.2-182.5 °C. H-NMR (600 MHz,
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DMSO-d6), δ: 10.668 (s, 1H, NH), 8.010 (d, J=9.0 Hz, 2H, Ar-H2’’, Ar-H6’’), 7.992 (d,
J=9.0 Hz, 2H, Ar-H3’’, Ar-H5’’), 7.902 (d, J=16.2 Hz, 1H, β-H), 7.887 (d, J=16.2 Hz,
1H, α-H), 7.581-7.555 (m, 1H, Ar-H6’), 7.497-7.439 (m, 2H, Ar-H4’, Ar-H5’), 7.214 (d,
J=8.4 Hz, 2H, Ar-H2, Ar-H6), 7.189 (d, J=8.4 Hz, 2H, Ar-H3, Ar-H5), 7.031 (d, J=8.4
Hz, 1H, Ar-H3’). 13C-NMR (400 MHz, DMSO), δ: 187.439, 166.014, 143.925,
137.957, 134.556, 134.264, 132.441, 132.334, 131.876, 131.815, 129.983, 129.827×2,
128.544, 128.420×2, 127.782×2, 127.639, 124.853, 119.589×2. HPLC: purity 96.4%.
LC-MS m/z: 396.16[M+1]+, calcd for C22H15Cl2NO2: 395.05.
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4.1.3 (E)-N-(4-(3-(2-chlorophenyl)acryloyl)phenyl)-2-phenylacetamide (4)
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Pale yellow powder, 65.54% yield, mp 171.1-171.9 °C. H-NMR (600 MHz,
CDCl3), δ: 8.182 (d, J=15.6 Hz, 1H, β-H), 8.005 (d, J=8.4 Hz, 2H, Ar-H2, Ar-H6),
7.765 (dd, J=1.8, 5.4 Hz, 1H, Ar-H3’), 7.607 (d, J=8.4 Hz, 2H, Ar-H3, Ar-H5), 7.459
(dd, J=3.6, 7.2 Hz, 2H, Ar-H5’, Ar-H6’), 7.435 (s, 1H, α-H), 7.399 (s, 1H, Ar-H4’’),
7.384 (d, J=4.2 Hz, 2H, Ar-H2’’, Ar-H6’’), 7.361-7.358 (m, 2H, Ar-H3’’, Ar-H5’’), 7.340
(d, J=6.0 Hz, 1H, Ar-H4’), 3.809 (s, 2H, CH2). 13C-NMR (400 MHz, DMSO), δ:
187.737, 170.281, 144.337, 138.454, 136.086, 134.717, 132.855, 132.441, 132.257,
130.565×2, 130.422, 129.118, 129.665×2, 128.817, 128.216, 127.115×2, 125.143,
118.962×2, 43.889. HPLC: purity 96.4%. LC-MS m/z: 376.14[M+1]+, calcd for
C23H18ClNO2: 375.10.
4.1.4 (E)-N-(4-(3-(2-chlorophenyl)acryloyl)phenyl)acetamide (5)
1
Pale yellow powder, 50.32% yield, mp 157.6-158.7 °C. H-NMR (600 MHz,
DMSO-d6), δ: 10.340 (s, 1H, NH), 8.202 (d, J=8.4 Hz, 1H, Ar-H3’), 8.153 (d, J=8.4
Hz, 2H, Ar-H2, Ar-H6), 8.019 (d, J=8.4 Hz, 1H, β-H), 7.977 (d, J=15.6 Hz, 1H, α-H),
7.772 (d, J=8.4 Hz, 2H, Ar-H3, Ar-H5), 7.569 (d, J=7.8 Hz, 1H, Ar-H6’), 7.562-7.075
(m, 2H, Ar-H4’, Ar-H5’), 2.101 (s, 3H, CH3). 13C-NMR (400 MHz, DMSO), δ:
187.733, 169.493, 144.451, 138.309, 134.663, 132.282, 132.231, 130.522×2, 129.076,
127.908, 125.284, 118.762×2, 24.659. HPLC: purity 95.6%. LC-MS m/z:
300.04[M+1]+, calcd for C17H14ClNO2: 299.07.
4.1.5 (E)-2-((4-(3-(2-chlorophenyl)acryloyl)phenyl)carbamoyl)benzoic acid (6)
1
Pale yellow powder, 58.32% yield, mp 165.1-166.5 °C. H-NMR (600 MHz,
DMSO-d6), δ: 8.224 (d, J=15.6 Hz, 1H, β-H), 8.170 (d, J=9.0 Hz, 2H, Ar-H2, Ar-H6),
7.989 (d, J=9.0 Hz, 2H, Ar-H2’’, Ar-H6’’), 7.825 (d, J=9.0 Hz, 2H, Ar-H3’’, Ar-H5’’),
7.770 (dd, J=1.8, 7.2 Hz, 1H, Ar-H3’), 7.687 (d, J=9.0 Hz, 2H, Ar-H3, Ar-H5), 7.514 (d,
J=15.6 Hz, 1H, α-H), 7.459 (dd, J=1.8, 7.2 Hz, 1H, Ar-H6), 7.320-7.368 (m, 2H,
Ar-H4, Ar-H5). 13C-NMR (400 MHz, DMSO), δ: 187.330, 168.620, 163.609, 143.543,
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143.434, 141.000, 132.812, 132.355, 132.218, 131.532, 129.944×2, 127.762, 126.642,
126.600, 123.325, 118.766×2, 118.475, 117.969, 117.831, 116.971, 116.831. HPLC:
purity 97.0%. LC-MS m/z: 404.15 [M-1]+, calcd for C23H16ClNO4: 405.08.
4.1.6 (E)-N-(4-(3-(2-chlorophenyl)acryloyl)phenyl)-2-fluorobenzamide (7)
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Pale yellow powder, 50.32% yield, mp 163.6-164.3 °C. H-NMR (600 MHz,
6
DMSO-d6), δ: 8.200 (d, J=15.6 Hz, 1H, β-H), 8.088 (d, J=8.4 Hz, 2H, Ar-H2, Ar-H6),
7.841 (d, J=8.4 Hz, 2H, Ar-H3, Ar-H5), 7.769 (d, J=7.2 Hz, 1H, Ar-H3’), 7.546-7.582
(m, 1H, Ar-H6’’), 7.516 (d, J=15.6 Hz, 1H, α-H), 7.451 (d, J=8.4 Hz, 1H, Ar-H6’),
7.360-7.315 (m, 4H, Ar-H4’, Ar-H5’, Ar-H4’’, Ar-H5’’), 7.261-7.198 (m, 1H, Ar-H3’’).
13C-NMR (400 MHz, DMSO), δ: 187.439, 166.014, 143.925, 137.957, 134.556,
134.264, 132.441, 132.334, 131.876, 131.815, 129.983, 129.827×2, 128.544,
128.420×2, 127.782×2, 127.639, 124.853, 119.589×2. HPLC: purity 95.1%. LC-MS
m/z: 380.13[M+1]+, calcd for C22H15ClFNO2: 379.08.
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4.1.7 (E)-N-(4-(3-(2-chlorophenyl)acryloyl)phenyl)acrylamide (8)
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Pale yellow powder, 54.6% yield, mp 176.3-177.3 °C. H-NMR (600 MHz,
DMSO-d6), δ: 10.548 (s, 1H, NH), 8.236 (dd, J=1.8, 9.0 Hz, 1H, β-H), 8.204 (d,
J=9.0 Hz, 2H, Ar-H2, Ar-H5), 8.027 (d, J=3.6 Hz, 2H, Ar-H3, Ar-H6), 7.878 (d, J=9.0
Hz, 2H, Ar-H3’, α-H), 7.583 (dd, J=1.8, 6.0 Hz, 1H, Ar-H6’), 7.503-7.464 (m, 2H,
Ar-H5’, Ar-H4’), 6.509-6.464 (m, 1H, CO-CH), 6.333 (dd, J=1.8, 15.6 Hz, 1H, CH),
5.841 (dd, J=1.2, 8.4 Hz, 1H, CH). 13C-NMR (400 MHz, DMSO), δ: 187.346,
163.757, 163.630, 140.676, 134.107, 133.255, 132.232, 132.005, 131.526, 130.029×2,
128.704, 128.191, 127.814, 125.232, 124.965, 118.809×2. HPLC: purity 96.7%.
LC-MS m/z: 312.10[M+1]+, calcd for C18H14ClNO2: 311.07.
4.1.8 (E)-N-(4-(3-(2-chlorophenyl)acryloyl)phenyl)propionamide (9)
1
Pale yellow powder, 62.78% yield, mp 164.6-167.7 °C. H-NMR (600 MHz,
CDCl3), δ: 8.135 (d, J=15.6 Hz, 1H, β-H), 7.864 (d, J=8.4 Hz, 2H, Ar-H2, Ar-H6),
7.756 (d, J=8.4 Hz, 1H, Ar-H3’), 7.582 (d, J=8.4 Hz, 2H, Ar-H3, Ar-H5), 7.441 (d,
J=15.6 Hz, 1H, α-H), 7.361-7.333 (m, 2H, Ar-H5’, Ar-H6’), 7.329-7.308 (m, 1H,
Ar-H4’), 3.781 (s, 3H, CH3), 1.216-1.497 (m, 2H,CH2). 13C-NMR (400 MHz, DMSO),
δ: 187.696, 173.141, 144.527, 138.284, 134.735, 132.829, 132.396, 132.143,
130.535×2, 130.487, 129.107, 128.135, 125.130, 118.779×2, 30.179, 9.864. HPLC:
purity 99.2%. LC-MS m/z: 314.12[M+1]+, calcd for C18H16ClNO2: 313.09.
4.1.9 N-(4-acetylphenyl)acrylamide (11)
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Pale yellow powder, 61.32% yield, mp 142.1-144.9 °C. H-NMR (600 MHz,
DMSO-d6), δ: 10.456 (s, 1H, NH), 7.952 (d, J=9.6 Hz, 2H, Ar-H2, Ar-H6), 7.841 (d,
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J=9.6 Hz, 2H, Ar-H3, Ar-H5), 6.497-6.443 (m, 1H, CO-CH), 6.315 (d, J=20.4 Hz, 1H,
CH), 5.821 (d, J=12.0 Hz, 1H, CH), 2.537 (s, 3H, CH3). 13C-NMR (400 MHz,
DMSO), δ: 197.723, 164.274, 142.743, 132.792, 131.038, 129.795×2, 128.935,
119.405, 113.753. HPLC: purity 98.2%. LC-MS m/z: 189.96[M+1]+, calcd for
C11H11NO2: 189.08.
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4.1.10 (E)-N-(4-(3-(2,4-dichlorophenyl)acryloyl)phenyl)acrylamide (12)
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Pale yellow powder, 50.32% yield, mp 214.1-216.1 °C. H-NMR (600 MHz,
8
DMSO-d6), δ: 10.717 (s, 1H, NH), 8.279 (d, J=8.5 Hz, 1H, β-H), 8.200 (d, J=8.6 Hz,
2H, Ar-H2, Ar-H6), 7.892 (t, J=8.4 Hz, 2H, Ar-H3, Ar-H5), 7.817-7.772 (m, 2H, Ar-H3’,
Ar-H6’), 7.644-7.564 (m, 2H, Ar-H5’, α-H), 6.512 (t, J=16.8 Hz, 1H, CO-CH), 6.328 (t,
J=16.8 Hz, 1H, CH), 5.825 (t, J=20.4 Hz, 1H, CH). 13C-NMR (400 MHz, DMSO), δ:
187.564, 164.095, 144.211, 137.143, 135.972, 135.565, 132.547×2, 131.900×2,
130.614, 130.322, 130.005, 128.437, 128.392, 125.780, 119.216×2. HPLC: purity
98.6%. LC-MS m/z: 346.09[M+1]+, calcd for C18H13Cl2NO2: 345.03.
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10
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12
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4.1.11 (E)-N-(4-(3-(4-(dimethylamino)phenyl)acryloyl)phenyl)acrylamide (13)
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Pale yellow powder, 53.8% yield, mp 201.4-202.5 °C. H-NMR (600 MHz,
DMSO-d6), δ: 8.048 (d, J=8.0 Hz, 2H, Ar-H2, Ar-H6), 7.815 (d, J=15.0 Hz, 1H, β-H),
7.765 (d, J=6.0 Hz, 2H, Ar-H3, Ar-H5), 7.565 (d, J=6.0 Hz, 2H, Ar-H2’, Ar-H6’), 7.369
(d, J=15.0 Hz, 1H, α-H), 6.710 (d, J=8.4 Hz, 2H, Ar-H3’, Ar-H5’), 6.500 (d, J=18.0 Hz,
1H, CO-CH), 6.370-6.325 (m, 1H, CH), 5.827 (d, J=12.0 Hz, 1H, CH), 3.063 (s, 6H,
NCH3×2). 13C-NMR (400 MHz, DMSO), δ: 189.230, 163.651, 152.105, 145.677,
141.486, 134.930, 130.983, 130.405×2, 129.684×2, 128.397, 122.769, 119.262,
116.662, 111.882×2, 40.083×2. HPLC: purity 98.6%. LC-MS m/z: 320.87[M+1]+,
calcd for C20H20N2O2: 320.15.
4.1.12 (E)-N-(4-(3-(4-methoxyphenyl)acryloyl)phenyl)acrylamide (14)
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Pale yellow powder, 52.5% yield, mp 155.1-157.9 °C. H-NMR (600 MHz,
CDCl3), δ: 8.051 (d, J=8.4 Hz, 2H, Ar-H2, Ar-H5), 7.823 (s, 1H, β-H), 7.773 (d, J=8.4
Hz, 2H, Ar-H3, Ar-H6), 7.623 (d, J=8.4 Hz, 2H, Ar-H2’, Ar-H6’), 7.446 (d, J=15.6 Hz,
1H, α-H), 6.960 (d, J=8.4 Hz, 2H, Ar-H5’, Ar-H4’), 6.510 (d, J=16.8 Hz, 1H, CO-CH),
6.356-6.311 (m, 1H, CH), 5.845 (d, J=10.2 Hz, 1H, CH), 3.879 (s, 3H, CH3).
13C-NMR (400 MHz, DMSO), δ: 189.076, 163.634, 161.725, 144.505, 141.800,
134.377, 130.894, 130.210×2, 129.839×2, 128.571, 127.706, 119.542, 119.303,
114.460×2, 55.400. HPLC: purity 99.4%. LC-MS m/z: 308.14[M+1]+, calcd for
C19H17NO3: 307.12.
4.1.13 (E)-N-(4-(3-(2-fluorophenyl)acryloyl)phenyl)acrylamide (15)
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Pale yellow powder, 57.9% yield, mp 177.9-179.2 °C. H-NMR (600 MHz,
DMSO-d6), δ: 10.545 (s, 1H, NH), 8.178 (d, J=9.0 Hz, 2H, Ar-H2, Ar-H6), 8.133 (t,
J=15.0 Hz, 1H, β-H), 8.005 (d, J=15.6 Hz, 1H, α-H), 7.878 (d, J=9.0 Hz, 2H, Ar-H3,
Ar-H5), 7.833 (d, J=12.0 Hz, 1H, Ar-H6’), 7.526 (d, J=7.2 Hz, 1H, Ar-H3’),
7.350-7.318 (m, 2H, Ar-H5’, Ar-H4’), 6.509-6.404 (m, 1H, CO-CH), 6.333 (d, J=15.6
Hz, 1H, CH), 5.838 (d, J=11.4 Hz, 1H, CH). 13C-NMR (400 MHz, DMSO), δ:
188.994, 163.686, 162.775, 160.749, 142.146, 137.278, 131.806, 131.736, 130.863,
130.031×2, 129.761, 128.642, 124.478, 124.413, 119.350, 116.371, 116.196. HPLC:
purity 99.5%. LC-MS m/z: 296.10[M+1]+, calcd for C18H14FNO2: 295.10.
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4.1.14 (E)-N-(4-(3-(2,6-difluorophenyl)acryloyl)phenyl)acrylamide (16)
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Pale yellow powder, 58.2% yield, mp 203.9-205.1 °C. H-NMR (600 MHz,
DMSO-d6), δ: 10.535 (s, 1H, NH), 8.067 (d, J=8.4 Hz, 2H, Ar-H2, Ar-H6), 7.876 (t,
J=15.3 Hz, 3H, Ar-H3, Ar-H5, β-H), 7.682 (d, J=16.2 Hz, 1H, α-H), 7.582-7.533 (m,
1H, Ar-H4’), 7.258 (t, J=17.4 Hz, 2H, Ar-H1’, Ar-H5’), 6.498-6.453 (m, 1H, CO-CH),
6.321 (dd, J=1.8, 15.0 Hz, 1H, CH), 5.826 (dd, J=1.8, 8.4 Hz, 1H, CH). 13C-NMR
(400 MHz, DMSO), δ: 184.750, 164.376, 144.183, 132.985, 132.763, 132.709×2,
132.423, 130.195×2, 129.142, 128.933, 127.744, 119.842×2, 112.890×2, 112.732.
HPLC: purity 95.8%. LC-MS m/z: 314.06[M+1]+, calcd for C18H13F2NO2: 313.09 .
4.1.15 (E)-N-(4-(3-(3-fluorophenyl)acryloyl)phenyl)acrylamide (17)
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Pale yellow powder, 61.4% yield, mp 143.3-145.1 °C. H-NMR (600 MHz,
DMSO-d6), δ: 10.540 (s, 1H, NH), 8.206 (d, J=9.0 Hz, 2H, Ar-H2, Ar-H5), 8.030 (d,
J=15.6 Hz, 1H, β-H), 7.880 (s, 2H, Ar-H3, Ar-H4), 7.861 (d, J=5.4 Hz, 1H, α-H),
7.715 (t, J=8.4 Hz, 2H, Ar-H6’, Ar-H5’), 7.526-7.490 (m, 1H, Ar-H4’), 7.294 (td, J=1.8,
6.0 Hz, 1H, Ar-H2’), 6.509-6.464 (m, 1H, CO-CH), 6.333 (dd, J=1.8, 15.0 Hz, 1H,
13
CH), 5.840 (dd, J=1.2, 8.4 Hz, 1H, CH). C-NMR (400 MHz, DMSO), δ: 188.737,
163.616, 142.887, 142.266, 131.257, 130.491, 130.440, 129.949×2, 127.918, 124.310,
123.748, 119.654×2, 117.190, 117.051, 114.614, 114.464. HPLC: purity 95.1%.
LC-MS m/z: 296.16[M+1]+, calcd for C18H14FNO2: 295.10.
4.1.16 N-(4-cinnamoylphenyl)acrylamide (18)
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Pale yellow powder, 51.3% yield, mp 191.8-192.4 °C. H-NMR (600 MHz,
DMSO-d6), δ: 10.516 (s, 1H, NH), 8.178 (d, J=8.4 Hz, 2H, Ar-H2, Ar-H6), 7.949 (d,
J=15.6 Hz, 1H, β-H), 7.893-7.855 (m, 4H, Ar-H3, Ar-H5, Ar-H2’, Ar-H6’), 7.729 (d,
J=15.6 Hz, 1H, α-H), 7.461 (t, J=6.6 Hz, 3H, Ar-H3’, Ar-H4’, Ar-H5’), 6.502-6.457 (m,
1H, CO-CH), 6.323 (dd, J=1.8, 15.0 Hz, 1H, CH), 5.828 (dd, J=1.8, 8.4 Hz, 1H, CH).
13C-NMR (400 MHz, DMSO), δ: 188.995, 163.584, 144.629, 141.931, 134.979,
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134.114, 130.831, 130.491, 129.969×2, 128.954×2, 128.685, 128.432×2, 121.865,
119.302. HPLC: purity 97.8%. LC-MS m/z: 278.06[M+1]+, calcd for C18H15NO2:
277.11.
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4.1.17 (E)-N-(4-(3-(2,3-dimethoxyphenyl)acryloyl)phenyl)acrylamide (19)
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Pale yellow powder, 62.4% yield, mp 166.1-167.7 °C. H-NMR (600 MHz,
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DMSO-d6), δ: 10.517 (s, 1H, NH), 8.156 (d, J=9.0 Hz, 2H, Ar-H2, Ar-H6), 7.972 (d,
J=7.8 Hz, 1H, β-H), 7.910-7.852 (m, 3H, Ar-H3, Ar-H5, α-H), 7.618 (t, J=9.0 Hz, 1H,
Ar-H5), 7.156 (d, J=4.8 Hz, 2H, Ar-H4’, Ar-H6’), 6.502-6.456 (m, 1H, CO-CH), 6.323
(d, J=17.4 Hz, 1H, CH), 5.828 (d, J=10.2 Hz, 1H, CH), 3.843 (s, 3H, OCH3), 3.800 (s,
3H, OCH3). 13C-NMR (400 MHz, DMSO), δ: 189.352, 163.587, 153.268, 146.049,
141.888, 139.484, 134.216, 130.866, 129.995×2, 129.194, 128.608, 127.508, 124.171,
123.435, 119.751, 119.271, 114.318. HPLC: purity 98.3%. LC-MS m/z:
338.16[M+1]+, calcd for C20H19NO4: 337.13.
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8
9
10
11
12
13
14
15
16
17
18
19
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22
23
24
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29
30
31
32
33
34
35
4.1.18 (E)-N-(4-(3-(2-(trifluoromethyl)phenyl)acryloyl)phenyl)acrylamide (20)
1
Pale yellow powder, 65.1% yield, mp 180.9-184.0 °C. H-NMR (600 MHz,
DMSO-d6), δ: 10.717 (s, 1H, NH), 8.280 (d, J=9.0 Hz, 1H, β-H), 8.200 (d, J=9.0 Hz,
2H, Ar-H2, Ar-H6), 8.059 (d, J=15.6 Hz, 1H, Ar-H3) 7.959 (d, J=15.6 Hz, 1H, Ar-H5),
7.907-7.886 (m, 3H, α-H, Ar-H2’, Ar-H6’), 7.817 (t, J=16.2 Hz, 1H, Ar-H6’), 7.573 (d,
J=6.6 Hz, 1H, Ar-H4’), 6.526-6.490 (m, 1H, CO-CH), 6.328 (t, J=16.8 Hz, 1H, CH),
13
5.834 (d, J=10.2 Hz, 1H, CH). C-NMR (400 MHz, DMSO), δ: 187.210, 163.639,
143.747, 137.240, 133.000, 132.894, 132.044, 130.330, 130.085×2, 128.717, 127.802,
126.260, 126.140, 126.094, 125.265, 118.812×2. HPLC: purity 97.7%. LC-MS m/z:
346.09[M+1]+, calcd for C19H14F3NO2: 345.10.
4.1.19 (E)-N-(4-(3-(2,5-dimethoxyphenyl)acryloyl)phenyl)acrylamide (21)
1
Pale yellow powder, 60.32% yield, mp 173.3-176.5 °C. H-NMR (600 MHz,
DMSO-d6), δ: 10.527 (s, 1H, NH), 8.178 (d, J=9.0 Hz, 2H, Ar-H2, Ar-H6), 8.028 (d,
J=15.6 Hz, 1H, β-H), 7.924 (d, J=15.6 Hz, 1H, α-H), 7.868 (d, J=8.4 Hz, 2H, Ar-H3,
Ar-H5), 7.561 (d, J=2.4 Hz, 1H, Ar-H4’), 7.050 (t, J=4.2 Hz, 2H, Ar-H3’, Ar-H6’),
6.508-6.463 (m, 1H, CO-CH), 6.330 (dd, J=1.8, 15.0 Hz, 1H, CH), 5.836 (dd, J=1.8,
13
16.8 Hz, 1H, CH), 3.852 (s, 3H, OCH3), 3.811 (s, 3H, OCH3). C-NMR (400 MHz,
DMSO), δ: 189.698, 163.841, 154.277, 153.773, 141.875, 139.936, 139.911, 131.358,
129.938 ×2, 127.711, 123.721, 119.551×2, 117.472, 114.435, 113.296, 56.482, 56.053.
HPLC: purity 96.7%. LC-MS m/z: 338.16[M+1]+, calcd for C20H19NO4: 337.13.
4.1.20 (E)-N-(4-(3-(2-bromophenyl)acryloyl)phenyl)acrylamide (22)
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Pale yellow powder, 67.56% yield, mp 191.8-192.4 °C. H-NMR (600 MHz,
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DMSO-d6), δ: 10.537 (s, 1H, NH), 8.190 (d, J=8.4 Hz, 3H, Ar-H2, Ar-H6, β-H), 7.974
(d, J=4.8 Hz, 2H, Ar-H3, Ar-H5), 7.870 (d, J=8.4 Hz, 2H, Ar-H3’, α-H), 7.743 (d, J=7.8
Hz, 1H, Ar-H6’), 7.496 (t, J=15.0 Hz, 1H, Ar-H5), 7.401-7.376 (m, 1H, Ar-H4’),
6.503-6.457 (m, 1H, CO-CH), 6.325 (dd, J=1.8, 15.0 Hz, 1H, CH), 5.831 (dd, J=1.2,
9.0 Hz, 1H, CH). 13C-NMR (400 MHz, DMSO), δ: 187.346, 163.757, 163.630,
140.676, 134.107, 133.255, 132.232, 132.005, 131.526, 130.029×2, 128.704, 128.191,
127.814, 125.232, 124.965, 118.809×2. HPLC: purity 96.2%. LC-MS m/z:
355.93[M+1]+, calcd for C18H14BrNO2: 355.02.
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4.1.21 (E)-N-(4-(3-(4-chlorophenyl)acryloyl)phenyl)acrylamide (23)
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10
11
12
13
14
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Pale yellow powder, 62.54% yield, mp 168.0-173.8 °C. H-NMR (600 MHz,
CDCl3), δ: 8.057 (d, J=8.4 Hz, 2H, Ar-H2, Ar-H6), 7.790-7.765 (m, 3H, β-H, Ar-H3,
Ar-H5), 7.560 (d, J=7.8 Hz, 2H, Ar-H2’, Ar-H6’), 7.534 (d, J=15.6 Hz, 1H, α-H), 7.417
(d, J=7.8 Hz, 2H, Ar-H3’, Ar-H5’), 6.515 (d, J=16.8 Hz, 1H, CO-CH), 6.349-6.304 (m,
13
1H, CH), 5.858 (d, J=10.2 Hz, 1H, CH). C-NMR (400 MHz, DMSO), δ: 188.995,
163.584, 144.629, 141.931, 134.979, 134.114, 130.837, 130.491, 129.969×2,
128.954×2, 128.685, 128.432×2, 121.865, 119.302. HPLC: purity 98.9%. LC-MS m/z:
312.10[M+1]+, calcd for C18H14ClNO2: 311.07.
4.1.22 (E)-N-(4-(3-(3,4-dichlorophenyl)acryloyl)phenyl)acrylamide (24)
1
Pale yellow powder, 65.79% yield, mp 168.9-171.3 °C. H-NMR (600 MHz,
CDCl3), δ: 8.057 (d, J=7.8 Hz, 2H, Ar-H2, Ar-H6), 7.966 (t, J=7.8 Hz, 1H, β-H), 7.786
(d, J=7.8 Hz, 2H, Ar-H3, Ar-H5), 7.735 (d, J=12.6 Hz, 3H, α-H, Ar-H2’, Ar-H3’), 7.284
(s, 1H, Ar-H6’), 6.517 (d, J=16.8 Hz, 1H, CO-CH), 6.348-6.303 (m, 1H, CH), 5.864 (d,
J=10.2 Hz, 1H, CH). 13C-NMR (400 MHz, DMSO), δ: 187.239, 163.641, 143.742,
137.815, 135.059, 131.812, 131.506, 130.087×2, 129.830, 128.394, 127.840, 127.165,
123.211, 118.815×2, 118.725. HPLC: purity 95.4%. LC-MS m/z: 346.03[M+1]+,
calcd for C18H13Cl2NO2: 345.03.
4.1.23 (E)-N-(4-(3-(3,4-difluorophenyl)acryloyl)phenyl)acrylamide (25)
1
Pale yellow powder, 62.54% yield, mp 180.1-182.5 °C. H-NMR (600 MHz,
CDCl3), δ: 8.057 (d, J=7.8 Hz, 2H, Ar-H2, Ar-H6), 7.966 (t, J=7.8 Hz, 1H, β-H), 7.786
(d, J=7.8 Hz, 2H, Ar-H3, Ar-H5), 7.735 (d, J=12.6 Hz, 4H, α-H, Ar-H2’, Ar-H2’,
Ar-H3’), 7.284 (s, 1H, Ar-H6’), 6.517 (d, J=16.8 Hz, 1H, CO-CH), 6.348-6.303 (m, 1H,
CH), 5.864 (d, J=10.2 Hz, 1H, CH). 13C-NMR (400 MHz, DMSO), δ: 187.564,
164.095, 144.211, 137.143, 135.972, 135.565, 132.547×2, 131.900×2, 130.614,
130.322, 130.005, 128.437, 128.392, 125.780, 119.216×2. HPLC: purity 95.5%.
LC-MS m/z: 314.02[M+1]+, calcd for C18H13F2NO2: 313.09.
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4.1.24 (E)-N-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)acrylamide (26)
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Pale yellow powder, 67.19% yield, mp 186.5-188.1 °C. H-NMR (600 MHz,
3
DMSO-d6), δ: 10.515 (s, 1H, NH), 8.188 (d, J=9.0 Hz, 2H, Ar-H2, Ar-H6),
7.910-7.865 (m, 3H, Ar-H3, Ar-H5, β-H), 7.683 (d, J=15.6 Hz, 1H, α-H), 7.229 (s, 2H,
Ar-H2’, Ar-H6’), 6.502-6.456 (m, 1H, CO-CH), 6.323 (dd, J=1.8, 15.0Hz, 1H, CH),
5.829 (dd, J=1.2, 7.8 Hz, 1H, CH), 3.868 (s, 6H, OCH3×2), 3.716 (s, 3H, OCH3).
13C-NMR (400 MHz, DMSO), δ: 189.050, 163.629, 153.543×2, 144.827×2, 141.922,
134.150, 130.842, 130.416, 129.931×2, 128.651, 121.243, 119.326×2, 105.588×2,
60.973, 56.297×2. HPLC: purity 96.9%. LC-MS m/z: 368.11[M+1]+, calcd for
C21H21NO5: 367.14.
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12
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4.1.25 (E)-N-(4-(3-(3,4-dimethoxyphenyl)acryloyl)phenyl)acrylamide (27)
1
Pale yellow powder, 61.22% yield, mp 110.3-112.9 °C. H-NMR (600 MHz,
CDCl3), δ: 8.059 (d, J=9.0 Hz, 2H, Ar-H2, Ar-H6), 7.782-7.755 (m, 3H, Ar-H3, Ar-H5,
β-H), 7.421 (d, J=15.6 Hz, 1H, α-H), 7.261 (d, J=7.8 Hz, 1H, Ar-H2’), 7.183 (d, J=1.2
Hz, 1H, Ar-H5’), 6.926 (d, J=8.4 Hz, 1H, Ar-H6’), 6.514 (d, J=16.8 Hz, 1H, CO-CH),
6.349-6.304 (m, 1H, CH), 5.853 (d, J=10.2 Hz, 1H, CH), 3.979 (s, 3H, OCH3), 3.956
(s, 3H, OCH3). 13C-NMR (400 MHz, DMSO), δ: 189.681, 166.340, 151.531, 149.491,
148.728, 144.841×2, 143.228, 142.310, 129.869×2, 128.604, 127.827, 123.104,
111.294, 110.879, 110.394, 56.012×2. HPLC: purity 99.0%. LC-MS m/z:
338.16[M+1]+, calcd for C20H19NO4: 337.13.
4.1.26 (E)-N-(4-(3-(2,3-dichlorophenyl)acryloyl)phenyl)acrylamide (28)
1
Pale yellow powder, 67.88% yield, mp 205.3-208.1 °C. H-NMR (600 MHz,
DMSO-d6), δ: 10.565 (s, 1H, NH), 8.200 (dd, J=1.2, 8.4 Hz, 3H, β-H, Ar-H2, Ar-H6),
8.024 (d, J=3.6 Hz, 2H, Ar-H3, Ar-H5), 7.879 (d, J=9.0 Hz, 2H, α-H, Ar-H3’), 7.749 (t,
J=7.8 Hz, 1H, Ar-H5’), 7.490 (t, J=15.6 Hz, 1H, Ar-H6’), 6.510-6.465 (m, 1H, CO-CH),
6.332 (dd, J=1.8, 8.4 Hz, 1H, CH), 5.841 (dd, J=1.8, 8.4 Hz, 1H, CH). 13C-NMR (400
MHz, DMSO), δ: 187.239, 163.641, 143.742, 137.815, 135.059, 131.812, 131.506,
130.087×2, 129.830, 128.394, 127.821, 127.165, 126.211, 118.815×2, 118.725. HPLC:
purity 98.6%. LC-MS m/z: 346.03[M+1]+, calcd for C18H13Cl2NO2: 345.03.
4.1.27 (E)-N-(4-(3-(2-fluoro-5-methoxyphenyl)acryloyl)phenyl)acrylamide (29)
1
Pale yellow powder, 67.88% yield, mp 151.2-154.7 °C. H-NMR (600 MHz,
CDCl3), δ: 8.054 (d, J=8.4 Hz, 2H, Ar-H2, Ar-H6), 7.884 (s, 1H, β-H), 7.785 (d, J=8.4
Hz, 2H, Ar-H3, Ar-H5), 7.634 (d, J=16.2 Hz, 1H, α-H), 7.121 (s, 1H, Ar-H3’), 7.070 (t,
J=18.6 Hz, 1H, Ar-H4’), 6.939-6.919 (m, 1H, Ar-H6’), 6.510 (d, J=16.8 Hz, 1H,
CO-CH), 6.361-6.216 (m, 1H, CH), 5.848 (d, J=10.2 Hz, 1H, CH), 3.852 (s, 3H,
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OCH3). 13C-NMR (400 MHz, DMSO), δ: 188.997, 163.683, 155.856, 142.152,
137.367, 133.831, 130.381×2, 130.852, 130.041, 124.609, 124.553, 119.345, 117.403,
117.336, 116.969, 116.776, 113.673, 55.896. HPLC: purity 95.6%. LC-MS m/z:
326.19[M+1]+, calcd for C19H16FNO3: 325.11.
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5
4.2 Synthetic procedures
6
4.2.1 General procedure for synthesis of chalcone derivatives 2-9 and 11
A mixture of compound 1 or 10 (1 mmol), various acyl chloride (2 mmol), and
anhydrous THF (10 mL) at 0 °C condition with triethylamine as a catalyst and stirred
for 30 min. Then the resulting mixture was then allowed slowly to warm to room
temperature. When TLC monitoring showed complete consumption of the starting
material, the reaction mixture was evaporated under reduced pressure. Then, the
mixture was extracted with CH2Cl2 and water. Subsequently, the resulting mixture
was washed with brine, dried by Na2SO4, and concentrated in vacuo to provide
residue. Finally, the residue was purified by silica gel column to obtain desired
products.
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4.2.2 General procedure for synthesis of chalcone derivatives 12-29
A mixture of compound 11 (1 mmol), a variety of substituted benzaldehyde, and
40% NaOH in EtOH (10 mL), stirred at room temperature for 12 h. Then, the reaction
mixture concentrated in vacuo to provide residue. Finally, the residue was purified by
silica gel column to obtain desired products.
4.3 Cells culture
The human lung cancer cell line NCI-H460 was obtained directly from the
Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Human
lung cancer cell line A549 was purchased from the Cell Bank of the Chinese Academy
of Sciences (Wuhan, China). And human lung cancer cell line H1975 was purchased
from the Shanghai Institute of Biosciences and Cell Resources Center (Shanghai,
China). Three cell lines were grown in RPMI-1640 medium (Gibco) supplemented
with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin and 100 mg/mL
streptomycin (Gibco). All cell lines were cultured at 37 °C in a humidified atmosphere
containing 5% CO2. Furthermore, cells were used for the appropriate experiments
until growing to the logarithmic phase. BMS-345541, xanthohumol, dimethyl
sulfoxide (DMSO) and MTT were purchased from Sigma-Aldrich (St. Louis, MO).
4.4 MTT assay
Lung cancer cell lines NCI-H460, A549 and H1975 were seeded in 96-well plate
with 3000 per well, and exposed to compounds for indicated times after overnight
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culturing.
Then
20
µL
solution
of
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) (5 mg/mL) was added to every well. After 4 h
incubation, 150 µL of DMSO was added to dissolve the formazan crystals of
intracellular and the absorbance (A value) was measured at 490 nm using an
Enzyme-labeled meter (MD, USA). Therefore, the percentage of inhibitory effect was
calculated as [1－value of compound-treated group (A)/control group (A)]×100%,
and the values of IC50 were determined through Prism 5 (GraphPad Software).
4.5 Colony formation assay
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9
The colony formation assay was performed to evaluate the effect of analogs on
proliferation. NCI-H460 cells were seeded in 6-well plate with 1000 per well and
incubated with compounds for 12 h after overnight growth. Next, medium was
changed and cells were cultured for approximately 8 days with normal medium. At
last, cells were combined with crystal violet and images were obtained with camera.
4.6 Intracellular ROS determined by flow cytometry
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Influences of compound 8 on ROS generation were assessed by flow cytometry.
NCI-H460 cells were cultured in 6-well plate for 24 h with 300, 000/well and then
treated with compounds for a certain time in the absence or presence of NAC. Then
medium was changed to serum-free medium and cells were incubated with DCFH-DA
(10 µM) (Beyotime Institute of Biotechnology, China) for 30 min at 37 °C in the dark.
Finally, the cells were collected and the samples were analyzed by flow cytometry
(BD, USA).
4.7 Western blot analysis
NCI-H460 cells were growing in the 6-well plate for 24 h and then exposed to
compound 8 for indicated times. Next, cells were lysed with lysis solution on the ice.
The concentrations of protein were detected by the Bradford colorimetric method.
After that, samples were separated through sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) (10% acrylamide gels) and transferred to PVDF. 5%
skim milk was used to block the PVDF membranes before staining with the primary
antibodies against caspase-3 and β-actin (Cell Signaling Technology, USA) at 4 °C.
After overnight, membranes were incubated with secondary antibody for 1 h at room
temperature and the immune-reactive complexes were tested with ECL (Enhanced
chemiluminescence kit) (Bio-Rad, Hercules, CA). Determination analysis was
performed using the Image Lab Software 5.1 (Bio-Rad).
4.8 Determination of pyroptosis in NCI-H460 cells
To detect the morphology of pyroptosis cells, NCI-H460 cell lines were seeded
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into 6-well plate with 300, 000/well 24 h before stimulation. Cells were exposed to 20
µg/mL cisplatin (24 h) and 20 µM compound 8 (19 and 24 h) in the presence or
absence of NAC (20 mmol/L). Static bright field cell images were obtained with
microscopic imaging (Nikon, Japan). Cells subjected to DMSO were used as control
group.
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4.9 In vivo toxicity examination
7
Wild-type BALB/c mice (male) were purchased from Shanghai Slaccas Lab
Animal Co. Ltd. The 18 mice (26-33 g) were randomly divided into 3 groups (n=6),
including vehicle group, EF24 group and compound 8 group. Toxicity examination
was performed with compounds (500 mg/Kg) via intraperitoneal (ip) injection at the
first day only. All the mice were housed under 12 h light-dark cycles at 25 °C and free
for water and diet. In addition, the mortality of the animals within 14 days was kept a
record. Then these mice were euthanasia together.
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4.10 Statistical analysis
These data were presented as mean±standard error (SEM) for three independent
experiments. Statistical comparisons among results were performed using one-way
analysis of variance (ANOVA). P<0.05 was set as the criterion of statistical
significance.
Acknowledgment
The work was supported by Zhejiang Province Natural Science Funding of China
(LY15H280014, LY17H160059, LQ18H280008), the Technology Foundation for
Medical Science of Zhejiang Province (2015KYA1506), the National Natural Science
Foundation of China (81803580, 81272462), Project of Wenzhou Sci-Tech Bureau
(Y20170158) and Granted by the Opening Project of Zhejiang Provincial Top Key
Discipline of Pharmaceutical Sciences.
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Highlights
1. Incorporation of a α,β-unsaturated ketone functionality into chalcone can
significantly improve the cytotoxicity.
2. Compound 8 showed more excellent inhibitory activity than positive controls
against several kinds of lung cancer cells.
3. Compound 8 was able to trigger ROS-mediated pyroptosis in NCI-H460 cells.