Synthesis and biological evaluation of novel millepachine derivative containing aminophosphonate ester species as novel anti-tubulin agents

Article abstract of DOI:10.1016/j.bioorg.2019.103486

A new series of millepachine derivative containing aminophosphonate ester moieties were designed and synthesized, and evaluated for their anticancer activities using MTT assay. Among all the compounds, compound 9m exhibited the most potent cytotoxic activ

Full text of DOI:10.1016/j.bioorg.2019.103486

Bioorganic Chemistry xxx (xxxx) xxxx
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and biological evaluation of novel millepachine derivative
containing aminophosphonate ester species as novel anti-tubulin agents
a,⁎,1
a,1
c
a
a
d
Xiaochao Huang
, Meng Wang , Chungu Wang , Weiwei Hu , Qinghong You , Tianhua Ma ,
a
c,⁎ b,⁎
d
Qiang Jia , Chunhao Yu , Zhixin Liao , Hengshan Wang
a
Jiangsu Key Laboratory of Regional Resource Exploitation and Medicinal Research, School of Chemical Engineering, Huaiyin Institute of Technology, Huaian 223003,
China
b
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences of Guangxi Normal
University, Guilin 541004, China
c
d
Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University, Nanjing 211189, China
Seasons Biotechnology (Taizhou) Co., Ltd, 21 Jiutiao Road, Jiaojiang District, Taizhou City, Zhejiang Province, 318000, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new series of millepachine derivative containing aminophosphonate ester moieties were designed and syn-
thesized, and evaluated for their anticancer activities using MTT assay. Among all the compounds, compound
Millepachine
Anti-cancer activity
Tubulin
9
m exhibited the most potent cytotoxic activity against all tested human cancer cell lines including multidrug
resistant phenotype, which inhibited cancer cell growth with IC50 values ranging from 0.85 to 3.09 μM, re-
spectively. In addition, its low cytotoxicity toward human normal liver cells HL-7702 and sensitivity toward to
doxorubicin or cisplatin-resistant cells indicated the possibility for cancer therapy. Furthermore, 9m sig-
nificantly induced cell apoptosis and cell cycle arrest in G2/M phase and dramatically disrupted the microtubule
organization. Moreover, a decrease in MMP, an increase in reactive oxygen species (ROS) generation and Bax/
Bcl-2 ratio, accompanied by activated caspase-3 and -9, were observed in HepG-2 cells after incubation with 9m,
indicating that the mitochondrial pathway was involved in the 9m-mediated apoptosis.
Apoptosis
1
. Introduction
Microtubules, as major components of the cytoskeleton, play critical
attention for their pharmacological activities [8]. Chalcones have been
reported for a broad spectrum of biological activities including anti-
oxidant, anti-HIV, anti-inflammatory, anti-malarial and anti-cancer
properties [8,9–11]. In addition, recent studies have indicated that
most of chalcones and its analogues significantly induced apoptosis in a
number of cell types [9,12–15]. In particular, millepachine (1a, Fig. 1),
a novel chalcone with a 2, 2-dimethylbenzopyran motif, was first iso-
lated from the millettia pachycarpa by Lijuan Chen group [16]. 1a and its
analogues (1b) have been found to exhibit the most potent cytotoxic
activity against a variety of human cancer cell lines, which can inhibit
strongly tubulin polymerization by binding to the colchicine site of
tubulin, and effectively induce cells to arrest in the G2/M phase of the
cell cycle [16,17]. However, the poor bioavailability and solubility
render these substances suboptimum for clinical treatment of cancer. In
an effort to further enhance the anticancer activity of millepachine, in
our previous work, a series of 1a derivative containing anilines were
synthesized through the formation of amide bond in the presence of
condensing agents, and evaluated their anti-proliferative activities
roles in a series of fundamental cell functions, such as cell division and
replication, cell signaling, cellular transport and motility [1–3].
Therefore, tubulin is recognized as important targets for anticancer
drug development. Among antitumor agents, three major groups have
been well characterized, the taxane site (e.g., paclitaxel and docetaxel)
for microtubule-stabilizing agents, the vinca alkaloids site (e.g., vin-
cristine, vinblastine, and vinorelbine), and the colchicine site (e.g.,
colchicine and CA-4) for microtubule polymerization inhibitors [4–7].
Unfortunately, drug resistance is one of the most important factors to
restrict its chemotherapeutic efficacy in addition to the well-known
serious side effects and poor solubility of these compounds. Thus, these
haves encouraged scientists to design and synthesize novel of antic-
ancer agents for cancer therapy.
Chalcones, also known as ɑ-ß-unsaturated ketones, represent an
important class of natural products, which received significant
⁎
Corresponding authors.
E-mail addresses: viphuangxc@126.com (X. Huang), zxliao@seu.edu.cn (Z. Liao), whengshan@163.com (H. Wang).
1
Co-first author: These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2019.103486
Received 2 September 2019; Received in revised form 23 October 2019; Accepted 27 November 2019
0045-2068/ © 2019 Published by Elsevier Inc.
Please cite this article as: Xiaochao Huang, et al., Bioorganic Chemistry, https://doi.org/10.1016/j.bioorg.2019.103486

X. Huang, et al.
Bioorganic Chemistry xxx (xxxx) xxxx
Fig. 1. Structures of natural products and millepachine analogs as potent inhibitors of tubulin polymerization.
[
18]. However, the research results showed that most of the new de-
Schmidt condensation of 4 and 5 in the presence of KOH (50% w/v
rivatives exhibited equivalent or less potent cytotoxic activity against
tested human cancer cells compared to 1a. Recent studies have hown
that introduction of a phosphate ester moiety to chemotherapy agents
could obviously improve the solubility of drugs and increase transport
through cellular membrane [19–21]. More importantly, many studies
indicated that phosphate esters could be easily hydrolyzed under phy-
siological conditions, suggesting introduction of aminophosphonate
esters in drugs was an effective strategy to obtain targeted antitumor
drugs [22–24]. In addition, most natural or synthetic aminopho-
sphonate compounds have been found to exhibit moderate anticancer
activity against a wide variety of cell lines [25–28]. Therefore, in order
to obtain more effective compounds, we designed and synthesized a
series of millepachine derivative containing aminophosphonate ester
species at the ortho-position of methoxyl group on the B-ring.
In present study, a new series of millepachine derivative containing
aminophosphonate ester moieties were synthesized and prepared as
shown in Fig. 2. Furthermore, we characterized these target compounds
and evaluated their in vitro anticancer activities as well as their me-
chanism of action. Compound 9m exhibited the most potent anticancer
activity against tested human cancer cell lines, with IC50 values ranging
from 0.85 to 3.09 μM, and also showed promising activities in drug-
resistant tumor cells. The results of mechanistic experiments revealed
that 9m could significantly induce cell apoptosis, and strongly inhibit
tubulin polymerization, and effectively induce cells to arrest in the G2/
M phase of the cell cycle. Furthermore, our study further demonstrated
that mitochondrial death pathway was involved in 9m induced HepG-2
cells apoptosis through regulating the expression of Bcl-2 family pro-
teins.
aqueous solution) in methanol at 0 °C for overnight to obtain the key
intermediate 6, and then treating with Fe powder and NH Cl in
4
ethanol/water smoothly to obtain 7. The key intermediate compounds
8a–8o was prepared according to the reported procedures [20,23].
Finally, the final targets compounds 9a–9o was obtained by the for-
mation of amide bond between 7 and 8a–8o in the presence of (2-(7-
Azabenzotriazol-1-yl)-N, N, N′, N′-tetramethyluroniumhexafluoropho-
sphate) (HATU)/Et N, respectively. The structures of these target
3
1 13
compounds were confirmed by H NMR, C NMR and high resolution
mass spectra (HR-MS).
2.2. In vitro cytotoxicity
In order to searching for potential target compounds that displayed
better activity, the reactions of millepachine analogue with different
aminophosphonate ester species were performed to obtain title com-
pounds 9a–9o. The synthesized millepachine analogues were evaluated
for their anti-proliferative activity against HepG-2 (hepatocellular),
A375 (melanoma), K562 (leukemia) and NCI-H460 (lung) cancer cells
and human normal liver cells HL-7702 using MTT assay. The cyto-
toxicities were expressed as IC50 values presented in Table 1. From the
results of the MTT assay, it was noted that some of the synthesized new
compounds (such as 9d, 9g, 9h, 9j, 9m, 9n and 9o) were significantly
more potent against the four cancer cell lines than that the parent
compound 1a. Especially, compound 9m exhibited potent anticancer
efficacy against the HepG-2, A375, K562, and NCI-H460 cancer cell
lines, with IC50 values of 0.85, 2.03, 1.96, and 2.61 μM, respectively.
Among the tested compounds, with the exception of compounds 9e, 9f,
9
k and 9l, the introduction of different substituent groups into the
2
. Results and discussion
benzene ring of para-position or meta-position caused a significant in-
crease in anti-proliferative efficacy relative to 9a in the cancer cell
lines. These results proved that para-position was better than meta-po-
sition. Interestingly, relative to 9b, the introduction of eOH group to
2
.1. Synthesis
The general steps to synthesize the target compounds 9a–9o have
the eOCH group of ortho-position caused a 2.49- to 3.63-fold increase
3
been outlined in Fig. 2. Firstly, treatment of compound 1 with 2 in the
in the potency of compound 9m. In addition, compound 9n, the in-
presence of 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) and catalytic
troduction of eNO
2
group to the eOCH group of ortho-position, caused
3
amounts of CuCl
2
·2H
2
O proceeded easily to obtain intermediate 3, and
a significant increase in anti-proliferative efficacy relative to 9b in the
cancer cell lines, especially in K562 cells, where compound 9n was
3.26-fold more potent than that of compound 9b (8.21 μM), with an
the resulting compound 3 was cyclized by heating in pyridine at 120 °C
for overnight and we obtained the intermediate 4. Secondly, by Claisen-
2

X. Huang, et al.
Bioorganic Chemistry xxx (xxxx) xxxx
Fig. 2. Synthetic pathway to target compounds 9a–9o. Reagents and conditions: (i) CuCl
2
·2H
2
O, DBU, CH
3
CN, 0 °C, 5 h; (ii) Pyridine, 120 °C, overnight; (iii) KOH
(
50% w/v aqueous solution), CH
3
OH, 0 °C, overnight; (iv) Fe, NH
4
Cl, EtOH/H
2
O, 85 °C, 2 h; (v) HATU, Et
3
N, Dimethyl formamide (DMF), 50 °C, overnight.
IC50 value of 2.52 μM, respectively. Moreover, compounds 9d and 9j
were significantly more potent against the HepG-2, A375, K562, and
NCI-H460 cancer cell lines than that of compound 9a, but compounds
(such as eOCH and eCH group) or strong electron withdrawing
3
3
groups (such as eF and eNO group) into the benzene ring of para-
2
position or meta-position might be responsible for the enhanced anti-
9
e, 9f, 9k and 9l exhibited lower toxicity compared to 9a. In shorts, the
proliferative efficacy.
above results indicated that introduction of electron-donating groups
Table 1
Anti-proliferative activities of 9a–9o against human cancer cell lines.
Compd.
IC50 (μM)a
HepG-2
A375
K562
NCI-H460
HL-7702
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
1
a
b
c
d
e
f
6.26 ± 1.21
3.09 ± 1.17
5.22 ± 1.31
2.23 ± 1.09
18.39 ± 1.64
> 20
7.51 ± 1.24
5.06 ± 1.39
6.27 ± 1.42
3.26 ± 1.18
> 20
10.36 ± 1.18
8.21 ± 1.23
6.23 ± 1.35
3.12 ± 1.33
> 20
12.36 ± 1.63
7.25 ± 1.16
8.18 ± 1.51
4.66 ± 1.15
19.31 ± 1.81
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
18.21 ± 1.66
3.38 ± 0.95
6.28 ± 1.47
10.96 ± 1.69
5.74 ± 1.22
> 20
> 20
g
h
i
2.51 ± 1.22
5.33 ± 1.09
9.58 ± 1.73
4.87 ± 1.01
15.91 ± 1.76
> 20
4.35 ± 1.01
10.72 ± 1.55
7.36 ± 1.83
6.71 ± 1.80
> 20
5.27 ± 1.06
9.87 ± 1.83
9.82 ± 1.92
7.28 ± 1.07
> 20
j
k
l
> 20
> 20
> 20
m
n
o
0.85 ± 0.83
1.65 ± 0.94
4.84 ± 1.14
5.35 ± 0.43
2.03 ± 0.91
3.07 ± 0.81
6.09 ± 1.29
7.05 ± 0.72
1.96 ± 1.02
2.52 ± 1.06
5.30 ± 1.06
8.58 ± 1.01
2.61 ± 1.13
3.02 ± 1.07
7.58 ± 1.44
11.85 ± 1.08
b
a
a
b
Each data represents mean ± S.D. from three different experiments performed in triplicate.
Used as positive controls.
3

X. Huang, et al.
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Table 2
In vitro cell growth inhibitory effects of compound 9m on drug resistant cells.
Compd.
IC50 (μM)a
MCF-7
MCF-7/DOX
RFb
A549
A549/CDDP
RFb
9
m
1.75 ± 1.03
1.09 ± 0.91
9.05 ± 1.67
2.12 ± 1.15
43.5 ± 2.31
15.21 ± 2.07
1.24
39.9
1.68
3.09 ± 1.03
2.27 ± 1.15
6.05 ± 1.46
2.83 ± 1.64
2.95 ± 1.33
27.36 ± 2.03
0.92
1.3
c
DOX
d
CDDP
4.52
a
b
c
Each data represents mean ± S.D. from three different experiments performed in triplicate.
RF: Resistant factor = IC50 of drug resistant cancer cell/ IC50 of drug sensitive cancer cell.
Doxorubicin.
Cisplatin.
d
2
.3. Effect of compound 9m on multidrug resistant cells
while the wounds of cells showed 53.6% closure after the cells exposure
to 1a (10 μM) for 24 h, respectively. Notably, the wounds of HepG-2
cells exhibited 51.5% and 38.6% after incubation of 9m (5 and 10 μM)
for 24 h, respectively (Fig. 4). Overall, these results suggested that 9m
significantly attenuated the migration of HepG-2 cells in a dose-de-
pendent manner.
Drug resistance has become a major problem for the first-line che-
motherapy drugs [28,29]. For example, doxorubicin (DOX) resistant
human breast carcinoma cells MCF-7/DOX, cisplatin (CDDP) resistant
human lung cancer cells A549/CDDP. Therefore, to further investigate
the selectivity of compound 9m against a panel of parental and drug-
resistant cancer cells, commercially available human breast carcinoma
cells MCF-7, and the cell line MCF-7/DOX (doxorubicin resistant cells)
and A549/CDDP (cisplatin resistant cell lines) were selected. The cy-
totoxicities were expressed as IC50 values presented in Table 2. As
shown in Table 2, although a high activity in parental cancer cell lines
was exhibited, but the IC50 value of DOX against MCF-7/DOX resistant
cells was increased to 43.5 μM, respectively. It was noted that 9m was
not obviously changed for the DOX resistant cancer cells compared with
2.6. Compound 9m induced apoptosis in HepG-2 cells
In recently, many studies revealed that most of anti-mitotic cancer
agents exert their cytotoxic effects through apoptosis, thus we in-
vestigated features related to this pathway [9,16]. So, we investigated
the occurrence of apoptosis in HepG-2 cells treated for 24 h with
compound 9m using a dual Annexin V staining/PI assay detected by
flow cytometry. As shown in Fig. 5, after incubation with 9m at the
indicated concentrations (5 and 10 μM) for 24 h, the percentage of the
early and late stage apoptosis cells were increased from 21.59 to
DOX sensitive cells, with IC50 values of 1.75
±
1.03 μM and
2
.12 ± 1.15 μM toward this pair of cancer cells, respectively. It was
much significant to found that 9m had a low resistance factor (1.24)
compared to DOX (39.9). Interestingly, the similar trend was also ob-
served in cisplatin resistant human lung cancer cells A549/CDDP. In
shorts, these results suggested that 9m displayed practically equally
potent activities in the sensitive cells and the drug-resistant cells, in-
dicating that 9m might be useful in the treatment of drug refractory
tumors.
3
8.46%, but that of control group cells was only 3.85%. Moreover, the
early and late stage apoptosis of HepG-2 cells treatment with 9m in-
creased gradually in a concentration-dependent manner. Notably, the
3
8.46% induction of HepG-2 cell apoptosis with incubation with 10 μM
of 9m was much higher than that of 1a (17.18% apoptotic cells at the
same concentration). Overall, these results indicated that 9m effec-
tively induced apoptosis in HepG-2 cancer cells.
2.4. Immunofluorescence staining of tubulin
2.7. Cell cycle analysis
In order to investigate the microtubule disrupting effects of the
It is well established that G2/M cell cycle arrest is strongly asso-
ciated with inhibition of tubulin polymerization [31,32], thus the effect
of 9m on cell cycle were investigated in HepG-2 cells using PI-staining
by flow cytometry analysis, and 1a was served as a positive control. As
illustrated in Fig. 6, untreated group cells as negative control exhibited
13.88% accumulation in G2/M phase, when HepG-2 cells were in-
cubated with 1a for 24 h, approximately 24.49% of the cells were ar-
rested in G2/M phase as expected. Notably, the percentage of HepG-2
cells in the G2/M phase was 28.45% and 42.34% when the cells were
treated with 9m for 24 h at concentrations of 5 μM and 10 μM, re-
spectively (Fig. 6). In shorts, the above results demonstrated that 9m
induce cancer cell cycle arrest at the G2/M phase in a concentration-
dependent manner.
millepachine derivatives, we selected 9m as a representative compound
in a cell-based phenotypic screening. In present study, we determined
the effect on the cellular microtubule network treated with 5 and 10 μM
of 9m for 24 h, and stained for α-tubulin (green) and DNA (blue) using
confocal microscopy analysis. As shown in Fig. 3, confocal analysis of
HepG-2 cells demonstrated a well-organized microtubule network in
control cells in the absence of drug treatment, while cells treatment
with 5 μM of 1a displayed dramatically disrupted microtubule organi-
zation as expected. Notable, cells exposure to 9m (5 and 10 μM) for
2
4 h significantly disrupted microtubule formation, respectively
(
Fig. 3). These morphological microtubules changes demonstrated that
that 9m exerted similar effects to millepachine (1a) on the microtubule
network, indicating that 9m was most likely targeting tubulin.
2.8. Compound 9m triggered mitochondrial pathway dependent apoptosis
2
.5. Compound 9m inhibited the migration of HepG-2 cells in vitro
It is well known that mitochondria plays a crucial role in regulating
Recent studies have shown that microtubule targeting anticancer
cellular functions, and mitochondrial dysfunction has been proposed to
be involved in triggering apoptosis [33,34]. Therefore, to further in-
vestigate if 9m induced HepG-2 cells apoptosis was involved in a dis-
ruption of mitochondrial membrane integrity, the fluorescent probe JC-
1 was used to detect the mitochondrial membrane potential (MMP) by
flow cytometry analysis. As illustrated in Fig. 7, the MMP level in HepG-
2 cells was decreased to 81.99% compared with the control group cells
(94.76%) after exposure to 10 μM of 1a for 24 h. Notably, incubation of
agents have been shown to be active against the cancer vasculature
through inhibiting cell invasion and capillary tube formation [30].
Thus, in present study, the effect of 9m on cell migration, which was a
major mechanism involved in tumor invasion, was investigated through
scratching a HepG-2 cells monolayer and monitoring the percentage of
wound closure. As illustrated in Fig. 4, the wounds of HepG-2 cells
displayed 63.5% closure in the absence of drug treatment after 24 h,
4

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Fig. 3. Effects of 9m on the microtubule network of cells. Untreated HepG-2 cells were served as negative control, and cells treated with 9m (5 and 10 μM) for 24 h
were fixed in methanol and stained with α-tubulin and counterstained with 4, 6-diamidino-2-phenylindole (DAPI). Millepachine (1a) was used as positive drug.
Microtubules and unassembled tubulin are shown in green. DNA, stained with DAPI, was shown in blue. Data are expressed as the mean ± SEM of three independent
experiments.
Fig. 4. Migration inhibition (wound-healing assay) of HepG-2 cells treated without or with the tested 9m (5, 10 μM) and 1a (10 μM) for 24 h at the indicated
concentrations. Typical images were taken at 0 and 24 h. The widths of wounds are indicated with the lines (mm). Widths are statistically significant with P < 0.05.
5

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Bioorganic Chemistry xxx (xxxx) xxxx
Fig. 5. Representative flow cytometric histograms
of apoptotic HepG-2 cells after 24 h treatment with
9
m (5 and 10 μM) and 1a (10 μM) as positive
control. The cells were harvested and labeled with
annexin-V-FITC and PI, and analyzed by flow cy-
tometry. Q1, Q2, Q3, and Q4 represent four dif-
ferent cell states: necrotic cells, late apoptotic or
necrotic cells, living cells and apoptotic cells, re-
spectively. Data are expressed as the mean ± SEM
for three independent experiments.
HepG-2 cells with 9m (5 and 10 μM) induced the dissipation of MMP in
a dose-dependent manner, the MMP level in HepG-2 cells was de-
creased to 80.86% and 61.59%, respectively (Fig. 7). In shorts, these
results indicated that incubation of HepG-2 cells with 9m cause a de-
crease the MMP level in a dose-dependent manner (Fig. 7), which de-
monstrated the activation of mitochondria mediated apoptosis.
Previously, many studies have indicated that the ratio of Bcl-2/Bax is
decreased, resulting to the release of apoptogenic factors, such as cy-
tochrome c [37]. Subsequently, cytochrome c further inducted activa-
tion of caspase-3, and -9 [38].Therefore, in the present study, to further
investigate whether such a mechanism was involved in apoptosis in-
duced by 9m, the expression of cytotochrome c, caspase-3 and -9 were
determined by Western blot analysis. As shown in Fig. 9, the expression
of cytotochrome c, caspase-3 and -9 began to increase after incubation
with 9m for 24 h. These results indicated that mitochondrial death
pathway was involved in 9m induced HepG-2 cells apoptosis through
regulating the expression of Bcl-2 family proteins.
2.9. Compound 9m triggered ROS generation
Recent studies have shown that ROS generation was considered to
play an important role in mitochondrial depolarization and apoptosis
induction [35,36]. Therefore, we used the fluorescent probe 2′, 7′-di-
chlorofluoresceindiacetate (DCFH-DA) to evaluate intracellular ROS
levels in the presence or absence of 9m. As illustrated in Fig. 8, after the
HepG-2 cells exposure to 10 μM of 1a for 24 h, the production of ROS
level was enhanced to 16.76% compared with control group cells
3
. Conclusions
In this study, a series of millepachine derivative containing ami-
nophosphonate ester moieties were synthesized and evaluated their
anticancer activities. Most of these synthesized compounds were sig-
nificantly more potent than the parent molecule 1a against both drug-
sensitive and drug-resistant cancer cells. Especially, the most potent
compound, 9m, exhibited potent anticancer efficacy against the HepG-
(
1.06%). Notably, incubation of HepG-2 cells with 9m (5 and 10 μM)
induced the production of ROS level in a dose-dependent manner, the
production of ROS level in HepG-2 cells was increased to 21.26% and
3
1
9.77%, respectively (Fig. 8). More importantly, when cells exposure to
0 μM of 9m, the production of ROS level was more than two times that
2
, A375, K562, and NCI-H460 cancer cell lines, with IC50 values of 0.85,
of 1a (39.77% vs 16.76%), respectively. In short, these results indicated
that ROS mediated apoptosis in HepG-2 cells incubated with 9m.
2
.03, 1.96, and 2.61 μM, respectively. Notably, 9m showed practically
equally potent activities in the DOX or CDDP sensitive cells and re-
sistant cells, and displayed low toxicity toward to human normal liver
cells HL-7702. Moreover, flow cytometry analysis and immuno-
fluorenscence staining assay suggested that 9m induced cell apoptosis
and cell cycle arrest in G2/M phase through significantly inhibited
microtubule polymerization. More importantly, the mitochondrial
dysfunction and ROS overproduction up-regulated the expression of
Bcl-2 family proteins, and then induced the mitochondrial release of
certain cellular components, such as cytochrome c and triggered cas-
pases family proteases such as caspase-3 and -9, finally leading to cell
2.10. Compound 9m regulates the expression of apoptosis-related proteins
The above results demonstrated that 9m induced HepG-2 cells
apoptosis was closely related to the mitochondrial pathway. The Bcl-2
family proteins have been described as key regulators of MMP [37].as
illustrated in Fig. 9, western blot analysis displayed that 9m up-regu-
lated the expression of Bax (pro-apoptosis Bcl-2 family protein), and
suppressed the expression of Bcl-2 (anti-apoptosis Bcl-2 family protein).
6

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Fig. 6. Cell cycle arrest effect of 9m. HepG-2 cells treated with 9m (5 and 10 μM) and 1a (10 μM) for 24 h. Then, the cells were trypsinized, harvested and washed
three times with ice-PBS for PI-stained DNA content detected by flow cytometry.
apoptosis. In shorts, our results demonstrated that the newly developed
millepachine analogue 9m exhibited significant antitumor efficacy in
vitro, and has the potential to be further developed into a promising
antitumor compound for human cancers.
(yield: 6.3 g, 75.9%) which was used directly without further pur-
1
ification. H NMR (600 MHz, CDCl
3
) δ 7.75 (d, J = 8.8 Hz, 1H), 7.18
(d, J = 2.3 Hz, 1H), 6.60–6.58 (m, 1H), 3.83 (s, 3H), 2.67 (s, 1H), 2.57
+
(s, 3H), 1.74 (s, 6H). HR-MS (m/z) (ESI): calcd for C14
H
16
O
3
[M+H]
:
2
3
33.1177; found: 233.1179.
4
. Experimental section
Synthesis of compound 4. A solution of compound 3 (6.96 g,
0.0 mmol) in dry pyridine (120 mL) was stirred at 120 °C for overnight
All chemicals and solvents were analytical reagent grade and com-
and monitored by TLC. After removing the solvent under reduced
pressure, and then 2 N HCl (aq) was added to adjust pH = 2. The
mixture was added dichloromethane (300 mL), washed with water, and
mercially available, and used without further purification unless noted
specifically. Column chromatography was performed using silica gel
(
200–300 mesh). Mass spectra were measured on an Agilent 6224 TOF
the organic layer was dried over anhydrous Na
2
SO and concentrated
4
1
13
LC/MS instrument. H NMR and C NMR spectra were recorded in
CDCl with a Bruker 400 or 600 MHz NMR spectrometer.
under reduced pressure. The resulting crude product was purified by
chromatography on silica gel eluted with petroleum ether/ethyl acetate
3
1
to obtain compound 3 as a yellow oil (yield: 5.0 g, 71.8%). H NMR
4.1. General procedure for the preparation of compounds 9a–9o
(600 MHz, CDCl ) δ 7.74 (d, J = 8.9 Hz, 1H), 6.66 (d, J = 10.0 Hz,
3
1
H), 6.47 (d, J = 8.9 Hz, 1H), 5.61 (d, J = 10.0 Hz, 1H), 3.86 (s, 3H),
Synthesis of compound 3. To a solution of compound 1 (8.3 g,
2.61 (s, 3H), 1.48 (s, 6H). HR-MS (m/z) (ESI): calcd for C14
H
16
O [M
3
+
5
0.0 mmol) in dry CH CN (100 mL), CuCl ·2H O (25 mg, 1.5 mmol),
3
2
2
+Na] : 255.0997; found: 255.1001.
DBU (7.5 mL, 75.0 mmol) and 3-chloro-3-methyl-1-butyne (7.7 g,
Synthesis of compound 6. A solution of 50% KOH (25 mL, aq) was
added dropwise to a stirred solution of 4 (4.6 g, 12.92 mmol) and 5
(3.6 g, 12.92 mmol) in methanol (50 mL) at 0 °C and stirred at the same
temperature for overnight. After completion of reaction, the mixture
was poured into ice-water (300 mL), and adjusted to pH = 2–3 with
con.HCl. The precipitated was filtered, washed with water and dried to
offer the crude product which was purified by chromatography on silica
gel eluted with petroleum ether/ethyl acetate to obtain compound 6 as
7
5.0 mmol) were added at 0 °C and then the mixture reaction was
stirred at the same temperature for 5 h, which was monitored by TLC.
After completion of reaction for 5 h, the mixture was cooled at room
temperature, then 2 N HCl (aq) was added to adjust pH = 2. After
removing the solvent under reduced pressure, the residue was poured
into water (500 mL) to yield yellow precipitate that was collected by
filtration and washed with petroleum ether to obtain 3 as a yellow solid
7

X. Huang, et al.
Bioorganic Chemistry xxx (xxxx) xxxx
Fig. 7. Compound 9m decreased the MMP of
HepG-2 cells. The HepG-2 cells were treated with
9
m and 1a at the indicated concentrations for 24 h
followed by incubation with the fluorescence probe
JC-1 for 30 min. Then, the cells were analyzed by
flow cytometry. The experiments were performed
three times, and the results of the representative
experiments were shown.
1
a yellow solid (yield: 3.8 g, 74.5%). H NMR (600 MHz, CDCl
3
) δ 8.15
washed with water (3 × 200 mL), dried over anhydrous Na
2
SO and
4
(
(
d, J = 2.1 Hz, 1H), 7.75–7.73 (m, 2H), 7.71 (d, J = 3.3 Hz, 1H), 7.60
concentrated under pressure. The crude product which was purified by
d, J = 15.7 Hz, 1H), 7.12 (d, J = 8.7 Hz, 1H), 6.69 (d, J = 10.0 Hz,
chromatography on silica gel eluted with CH
2
Cl
2
/CH
3
OH to give the
1
1
3
H), 6.53 (d, J = 8.9 Hz, 1H), 5.64 (d, J = 10.0 Hz, 1H), 4.00 (s, 3H),
target compound 9a as a yellow solid (yield: 145 mg, 73.2%). H NMR
.89 (s, 3H), 1.51 (s, 6H). HR-MS (m/z) (ESI): calcd for C22
H
21NO
6
[M
(600 MHz, CDCl ) δ 8.72 (s, 1H), 7.77 (s, 1H), 7.70 (d, J = 8.8 Hz, 1H),
3
+
+
H] : 396.1447; found: 396.1441.
7.63 (s, 2H), 7.49 (s, 2H), 7.35 (t, J = 7.6 Hz, 2H), 7.29 (d, J = 7.6 Hz,
1H), 7.22–7.20 (m, 1H), 7.09 (d, J = 8.4 Hz, 2H), 6.77 (d, J = 8.5 Hz,
1H), 6.69 (d, J = 10.0 Hz, 1H), 6.65 (d, J = 8.4 Hz, 2H), 6.51 (d,
J = 8.9 Hz, 1H), 5.64 (d, J = 10.0 Hz, 1H), 4.79 (d, J = 24.4 Hz, 1H),
4.17–4.11 (m, 2H), 3.97–3.89 (m, 1H), 3.89 (s, 3H), 3.70–3.66 (m, 1H),
3.61 (s, 3H), 3.60 (s, 2H), 1.54 (t, J = 5.3 Hz, 6H), 1.31 (t, J = 7.1 Hz,
Synthesis of compound 7. To a solution of compound 6 (4.2 g,
0.46 mmol) in ethanol (60 mL)/water (5 mL) was added iron powder
1
(
3.6 g, 63.8 mmol) and NH Cl (342 mg, 6.4 mmol), and then the
4
mixture stirred at 85 °C for 4 h. After completion of reaction, the
mixture was cooled to room temperature and filtered through celite.
The filter cake was washed with dichloromethane (100 mL), and con-
1
3
3H), 1.13 (t, J = 7.1 Hz, 3H). C NMR (150 MHz, CDCl ) δ 190.15,
3
centrated under pressure. The residue was dissolved in CH
2
Cl
2
169.52, 158.24, 153.69, 149.28, 145.67, 145.57, 141.64, 135.77,
131.55, 130.44, 128.98, 128.72, 128.67, 128.10, 128.05, 127.86,
127.82, 125.85, 125.24, 124.00, 121.80, 117.78, 116.58, 114.29,
110.49, 109.87, 103.34, 63.46, 63.33, 56.46, 55.77, 55.73, 44.27,
(
200 mL), washed with water. The organic layer dried over anhydrous
Na
2
SO , filtered, and concentrated under reduced pressure. The crude
4
product was purified by chromatography on silica gel eluted with
1
CH
2
Cl
2
/CH
3
OH to obtain 7 as a yellow solid (yield: 3.45 g, 90.8%). H
27.95, 16.48, 16.23. HR-MS (m/z) (ESI): calcd for C41
H
45
N
2
O P [M
8
+
NMR (600 MHz, CDCl
3
) δ 7.68 (d, J = 8.8 Hz, 1H), 7.58 (d,
+Na] : 747.2811; found: 747.2818.
J = 15.7 Hz, 1H), 7.52 (d, J = 15.7 Hz, 1H), 7.02–7.70 (m, 2H), 6.98
(
d, J = 2.0 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H), 6.69 (d, J = 10.0 Hz,
4.1.2. Synthesis of target compound 9b
1
3
H), 6.50 (d, J = 8.8 Hz, 1H), 5.62 (d, J = 10.0 Hz, 1H), 3.88 (s, 3H),
The compound 9b was prepared from compound 8b and compound
.87 (s, 3H), 1.50 (s, 6H). HR-MS (m/z) (ESI): calcd for C22
H
23NO
4
[M
7 according to describe for 9a method: 150 mg, 72.8% yield as a yellow
+
1
+
H] : 366.1705; found: 366.1722.
solid; H NMR (400 MHz, CDCl
3
) δ 8.69 (s, 1H), 7.75 (s, 1H), 7.67 (d,
J = 9.7 Hz, 1H), 7.61 (s, 2H), 7.38 (d, J = 8.3 Hz, 2H), 7.19 (d,
J = 8.4 Hz, 1H), 7.06 (d, J = 7.6 Hz, 2H), 6.86 (d, J = 7.9 Hz, 2H),
6.75 (d, J = 8.0 Hz, 1H), 6.67 (d, J = 10.0 Hz, 1H), 6.62 (d,
J = 7.6 Hz, 2H), 6.48 (d, J = 8.1 Hz, 1H), 5.61 (d, J = 10.0, 1.0 Hz,
1H), 4.71 (d, J = 23.9 Hz, 1H), 4.14–4.07 (m, 2H), 3.97–3.91 (m, 1H),
3.86 (s, 3H), 3.76 (s, 3H), 3.72–3.66 (m, 1H), 3.61 (s, 3H), 3.57 (s, 2H),
4
.1.1. Synthesis of target compound 9a
To a solution of compound 8a (124 mg, 0.329mmol) in dry DMF
N (57 μL,
(
3 mL) was added HATU (156 mg, 0.411 mmol), Et
3
0
.411 mmol) and 7 (100 mg, 0.274 mmol), and stirred at 50 °C for
overnight and monitored by TLC. The resulting mixture was stirred at
room temperature for overnight and monitored by TLC. After comple-
1
3
1.52 (s, 6H), 1.28 (t, J = 7.0 Hz, 3H), 1.13 (t, J = 7.0 Hz, 3H). C NMR
tion of reaction, the mixture was diluted with CH
2
Cl
2
(100 mL) and
(100 MHz, CDCl ) δ 190.14, 169.54, 159.41, 158.26, 153.70, 149.33,
3
8

X. Huang, et al.
Bioorganic Chemistry xxx (xxxx) xxxx
Fig. 8. Intracellular production of ROS by 9m (5 and 10 μM) and 1a (10 μM) following a 24 h incubation using 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-
DA) staining analysis by flow cytometry. The experiments were performed three times, and the results of the representative experiments were shown.
1
1
1
4
45.81, 145.66, 141.64, 131.55, 130.39, 129.00, 128.97, 128.16,
27.58, 125.93, 125.19, 123.99, 121.83, 117.89, 116.59, 114.37,
14.11, 110.52, 109.95, 103.36, 63.39, 63.27, 56.11, 55.76, 55.25,
4.1.3. Synthesis of target compound 9c
The compound 9c was prepared from compound 8c and compound
7 according to describe for 9a method: 163 mg, 80.7% yield as a yellow
1
4.27, 38.62, 27.95, 16.48, 16.29. HR-MS (m/z) (ESI): calcd for
solid; H NMR (400 MHz, CDCl
3
) δ 8.70 (s, 1H), 7.74 (s, 1H), 7.68 (d,
+
C
42
H
47
N
2
O
9
P [M+Na] : 777.2917; found: 777.2909.
J = 8.8 Hz, 1H), 7.61 (s, 2H), 7.36 (d, J = 2.1 Hz, 1H), 7.34 (d,
Fig. 9. HepG-2 cells were incubated with 9m (5 and 10 μM) and 1a (10 μM) for 24 h. (A) Representative Western blots of the effects of 9m on the expression of
apoptosis-related proteins. GAPDH was used as internal control. (B) Percentage expression levels of cytotochrome c (Cyto c), Bcl-2, Bax, Apaf-1, caspase-3 and
caspase-9. The percentage values are those relative to the GAPDH.
9

X. Huang, et al.
Bioorganic Chemistry xxx (xxxx) xxxx
J = 2.2 Hz, 1H), 7.20–7.17 (m, 1H), 7.13 (d, J = 8.0 Hz, 2H), 7.06 (d,
J = 8.4 Hz, 2H), 6.75 (d, J = 8.5 Hz, 1H), 6.67 (d, J = 10.0 Hz, 1H),
55.74, 44.25, 27.96, 16.47, 16.28. HR-MS (m/z) (ESI): calcd for
C
41
H44BrN
2
O
8
P [M+Na]+: 827.1896; found: 827.1880.
6
.62 (d, J = 8.5 Hz, 2H), 6.48 (d, J = 8.9 Hz, 1H), 5.61 (d,
J = 10.0 Hz, 1H), 4.73 (d, J = 24.1 Hz, 1H), 4.14–4.09 (m, 2H),
.97–3.90 (m, 1H), 3.86 (s, 3H), 3.71–3.67 (m, 1H), 3.60 (s, 3H), 3.57
s, 2H), 2.30 (s, 3H), 1.52 (s, 6H), 1.29 (t, J = 7.1 Hz, 3H), 1.12 (t,
4.1.7. Synthesis of target compound 9g
3
The compound 9g was prepared from compound 8g and compound
(
7 according to describe for 9a method: 143 mg, 68.1% yield as a yellow
1
3
1
J = 7.1 Hz, 3H). C NMR (100 MHz, CDCl
3
) δ 190.12, 169.51, 158.25,
solid; H NMR (600 MHz, CDCl
3
) δ 8.68 (s, 1H), 8.19 (d, J = 8.5 Hz,
1
1
1
1
1
7
53.70, 149.31, 145.84, 145.69, 141.62, 137.73, 137.69, 132.66,
2H), 7.73 (s, 1H), 7.67–7.65 (m, 3H), 7.60 (s, 2H), 7.20–7.19 (m, 1H),
7.08 (d, J = 8.3 Hz, 2H), 6.77 (d, J = 8.5 Hz, 1H), 6.66 (d,
J = 10.0 Hz, 1H), 6.56 (d, J = 8.4 Hz, 2H), 6.48 (d, J = 8.9 Hz, 1H),
5.60 (d, J = 10.0 Hz, 1H), 4.87 (d, J = 25.2 Hz, 1H), 4.17–4.11 (m,
2H), 4.05–4.02 (m, 1H), 4.01–3.88 (m, 1H), 3.85 (s, 3H), 3.68 (s, 3H),
3.57 (s, 2H), 1.51 (s, 6H), 1.29 (t, J = 7.1 Hz, 3H), 1.18 (t, J = 7.1 Hz,
31.55, 130.40, 129.37, 128.97, 128.80, 128.17, 127.76, 127.71,
25.93, 125.18, 123.96, 121.84, 117.85, 116.59, 114.33, 110.51,
09.92, 103.35, 63.39, 63.26, 56.49, 55.76, 55.69, 44.28, 27.95, 21.12,
6.48, 16.26. HR-MS (m/z) (ESI): calcd for C42
H
47
N
2
O
8
P [M+Na]+
:
61.2968; found: 761.2977.
1
3
3
H). C NMR (150 MHz, CDCl ) δ 190.15, 169.30, 158.26, 153.66,
3
4
.1.4. Synthesis of target compound 9d
149.21, 147.63, 145.50, 144.91, 143.98, 141.60, 131.54, 130.51,
128.93, 128.75, 128.68, 128.03, 125.90, 125.11, 124.85, 123.79,
121.76, 118.07, 116.60, 114.23, 110.48, 109.96, 103.36, 63.85, 63.61,
56.43, 55.78, 55.73, 44.17, 27.94, 16.48, 16.30. HR-MS (m/z) (ESI):
The compound 9d was prepared from compound 8d and compound
7
according to describe for 9a method: 149mg, 73.4% yield as a yellow
1
solid; H NMR (400 MHz, CDCl
3
) δ 8.70 (s, 1H), 7.74 (s, 1H), 7.67 (d,
J = 8.8 Hz, 1H), 7.61 (s, 2H), 7.47–7.43 (m, 2H), 7.20–7.18 (m, 2H),
calcd for C41
H
44
3
N O
10P [M+Na]+: 792.2662; found: 792.2664.
7
1
.07 (d, J = 8.4 Hz, 2H), 7.02 (t, J = 8.6 Hz, 2H), 6.76 (d, J = 8.5 Hz,
H), 6.67 (d, J = 10.0 Hz, 1H), 6.59 (d, J = 8.4 Hz, 2H), 6.48 (d,
4.1.8. Synthesis of target compound 9h
J = 8.9 Hz, 1H), 5.61 (d, J = 10.0 Hz, 1H), 4.75 (d, J = 24.1 Hz, 1H),
The compound 9h was prepared from compound 8h and compound
4
3
.15–4.09 (m, 2H), 3.99–3.95 (m, 1H), 3.86 (s, 3H), 3.79–3.71 (m, 1H),
7 according to describe for 9a method: 149mg, 72.3% yield as a yellow
1
.64 (s, 3H), 3.58 (s, 2H), 1.52 (s, 6H), 1.29 (t, J = 7.1 Hz, 3H), 1.14 (t,
solid; H NMR (600 MHz, CDCl
3
) δ 8.73 (s, 1H), 7.78 (s, 1H), 7.71 (d,
1
3
J = 7.1 Hz, 3H). C NMR (100 MHz, CDCl
3
) δ 190.12, 169.41, 163.69,
J = 8.8 Hz, 1H), 7.65 (s, 2H), 7.29–7.27 (m, 1H), 7.23–7.21 (m, 1H),
7.10 (d, J = 8.2 Hz, 2H), 7.07 (d, J = 16.4 Hz, 2H), 6.83 (d,
J = 8.2 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H), 6.70 (d, J = 10.0 Hz, 1H),
6.66 (d, J = 8.4 Hz, 2H), 6.52 (d, J = 8.8 Hz, 1H), 5.65 (d,
J = 10.0 Hz, 1H), 4.77 (d, J = 24.3 Hz, 1H), 4.19–4.14 (m, 2H),
4.00–3.96 (m, 1H), 3.90 (s, 3H), 3.80 (s, 3H), 3.74–3.70 (m, 1H), 3.64
(s, 3H), 3.62 (s, 2H), 1.56 (s, 6H), 1.31 (t, J = 7.1 Hz, 3H), 1.16 (t,
1
1
1
1
1
7
61.27, 158.26, 153.69, 149.29, 145.53, 145.39, 141.60, 131.55,
30.43, 129.52, 129.38, 128.95, 128.82, 128.14, 125.96, 125.15,
24.31, 121.83, 117.96, 116.60, 115.73, 115.49, 114.30, 110.51,
09.96, 103.36, 63.41, 63.34, 56.13, 55.76, 55.73, 44.24, 27.95, 16.47,
6.26. HR-MS (m/z) (ESI): calcd for
C
41
H44FN
2
O
8
P
[M+Na]+
:
65.2717; found: 765.2728.
1
3
J = 7.1 Hz, 3H). C NMR (150 MHz, CDCl ) δ 190.15, 169.53, 159.85,
3
4
.1.5. Synthesis of target compound 9e
158.24, 153.69, 149.29, 145.70, 145.60, 141.64, 137.40, 131.55,
130.44, 129.65, 128.98, 128.72, 128.11, 125.85, 125.24, 124.04,
121.80, 120.25, 117.77, 116.57, 114.31, 113.47, 113.42, 113.39,
110.49, 109.87, 103.33, 63.53, 63.36, 56.52, 55.77, 55.72, 55.24,
The compound 9e was prepared from compound 8e and compound
7
according to describe for 9a method: 167 mg, 81.2% yield as a yellow
1
solid; H NMR (400 MHz, CDCl
3
) δ 8.70 (s, 1H), 7.74 (s, 1H), 7.68 (d,
J = 8.8 Hz, 1H), 7.61 (s, 2H), 7.47 (s, 1H), 7.37 (d, J = 8.4 Hz, 1H),
44.28, 27.95, 16.49, 16.26. HR-MS (m/z) (ESI): calcd for C42
H
47
N
2
O P
9
7
6
.29 (d, J = 7.9 Hz, 2H), 7.21–7.18 (m, 1H), 7.08 (d, J = 8.3 Hz, 2H),
.76 (d, J = 8.5 Hz, 1H), 6.67 (d, J = 10.0 Hz, 1H), 6.60 (d,
[M+Na]+: 777.2917; found: 792.2907.
J = 8.4 Hz, 2H), 6.49 (d, J = 8.9 Hz, 1H), 5.61 (d, J = 10.0 Hz, 1H),
4.1.9. Synthesis of target compound 9i
4
4
.96–4.84 (m, 1H), 4.73 (d, J = 24.6 Hz, 1H), 4.18–4.11 (m, 2H),
The compound 9i was prepared from compound 8i and compound 7
.01–3.95 (m, 1H), 3.87 (s, 3H), 3.81–3.74 (m, 1H), 3.64 (s, 3H), 3.59
according to describe for 9a method: 138 mg, 68.8% yield as a yellow
1
(
s, 2H), 1.52 (s, 6H), 1.30 (t, J = 7.1 Hz, 3H), 1.15 (t, J = 7.1 Hz, 3H).
solid; H NMR (600 MHz, CDCl
3
) δ 8.69 (s, 1H), 7.74 (s, 1H), 7.68 (d,
1
3
C NMR (100 MHz, CDCl
3
) δ 190.14, 169.39, 158.25, 153.69, 149.29,
J = 8.8 Hz, 1H), 7.61 (s, 2H), 7.35 (d, J = 1.9 Hz, 1H), 7.34 (d,
J = 1.9 Hz, 1H), 7.20–7.18 (m, 1H), 7.13 (d, J = 7.9 Hz, 2H), 7.05 (d,
J = 8.3 Hz, 2H), 6.75 (d, J = 8.5 Hz, 1H), 6.67 (d, J = 10.0 Hz, 1H),
6.62 (d, J = 8.4 Hz, 2H), 6.49 (d, J = 8.8 Hz, 1H), 5.61 (d,
J = 10.0 Hz, 1H), 4.73 (d, J = 24.2 Hz, 1H), 4.15–4.08 (m, 2H),
3.98–3.91 (m, 1H), 3.86 (s, 3H), 3.69–3.64 (m, 1H), 3.60 (s, 3H), 3.57
(s, 2H), 2.29 (s, 3H), 1.52 (s, 6H), 1.29 (t, J = 7.0 Hz, 3H), 1.12 (t,
1
1
1
6
45.44, 145.29, 141.60, 138.29, 134.63, 131.55, 130.52, 129.92,
28.97, 128.83, 128.25, 128.14, 127.79, 126.06, 125.96, 125.18,
24.41, 121.84, 117.89, 116.60, 114.24, 110.51, 109.93, 103.35,
3.55, 63.48, 56.48, 55.76, 55.69, 44.25, 27.96, 16.45, 16.23. HR-MS
+
(
m/z) (ESI): calcd for C41
H44ClN
2
O
8
P [M+Na] : 781.2422; found:
7
81.2412.
1
3
J = 7.1 Hz, 3H). C NMR (150 MHz, CDCl ) δ 190.18 169.57, 158.26,
3
4
.1.6. Synthesis of target compound 9f
153.70, 149.30, 145.78, 145.68, 141.65, 137.75, 132.60, 131.56,
130.42, 129.38, 128.99, 128.76, 128.13, 127.75, 125.89, 125.24,
123.92, 121.82, 117.82, 116.59, 114.33, 110.50, 109.90, 103.35,
The compound 9f was prepared from compound 8f and compound 7
according to describe for 9a method: 146 mg, 66.4% yield as a yellow
1
solid; H NMR (400 MHz, CDCl
3
) δ 8.70 (s, 1H), 7.73 (s, 1H), 7.68 (d,
63.41, 63.28, 56.19, 55.77, 55.69, 44.28, 27.95, 21.15, 16.48, 16.26.
+
J = 8.8 Hz, 1H), 7.61 (s, 2H), 7.46 (d, J = 8.3 Hz, 2H), 7.36 (d,
J = 2.1 Hz, 1H), 7.34 (d, J = 2.1 Hz, 1H), 7.21–7.19 (m, 1H), 7.07 (d,
J = 8.3 Hz, 2H), 6.77 (d, J = 8.5 Hz, 1H), 6.67 (d, J = 10.0 Hz, 1H),
HR-MS (m/z) (ESI): calcd for C42
H
47
N
2
O
8
P [M+Na] : 761.2968;
found: 761.2973.
6
.58 (d, J = 8.4 Hz, 2H), 6.49 (d, J = 8.9 Hz, 1H), 5.61 (d,
4.1.10. Synthesis of target compound 9j
J = 10.0 Hz, 1H), 4.72 (d, J = 24.5 Hz, 1H), 4.15–4.08 (m, 2H),
.01–3.95 (m, 1H), 3.87 (s, 3H), 3.81–3.75 (m, 1H), 3.63 (s, 3H), 3.58
s, 2H), 1.52 (s, 6H), 1.29 (t, J = 7.1 Hz, 3H), 1.16 (t, J = 7.1 Hz, 3H).
The compound 9j was prepared from compound 8j and compound 7
4
according to describe for 9a method: 129mg, 63.5% yield as a yellow
1
(
solid; H NMR (400 MHz, CDCl
3
) δ 8.70 (s, 1H), 7.74 (s, 1H), 7.68 (d,
1
3
C NMR (100 MHz, CDCl
3
) δ 190.15, 169.38, 158.26, 153.69, 149.29,
J = 8.8 Hz, 1H), 7.61 (s, 2H), 7.34–7.28 (m, 2H), 7.19 (d, J = 8.4 Hz,
2H), 7.08 (d, J = 8.3 Hz, 2H), 6.96 (t, J = 7.8 Hz, 1H), 6.76 (d,
J = 8.4 Hz, 1H), 6.67 (d, J = 10.0 Hz, 1H), 6.60 (d, J = 8.3 Hz, 2H),
6.48 (d, J = 8.9 Hz, 1H), 4.76 (d, J = 24.6 Hz, 1H), 4.18–4.08 (m, 2H),
1
1
1
45.41, 145.26, 141.61, 135.11, 131.80, 131.55, 130.47, 129.55,
28.96, 128.83, 128.12, 125.96, 125.17, 124.41, 121.98, 121.84,
16.61, 114.33, 110.52, 109.96, 103.36, 63.54, 63.47, 56.34, 55.77,
10

X. Huang, et al.
Bioorganic Chemistry xxx (xxxx) xxxx
4
.02–3.96 (m, 1H), 3.86 (s, 3H), 3.81–3.72 (m, 1H), 3.64 (s, 3H), 3.59
4.1.14. Synthesis of target compound 9n
(
s, 2H), 1.52 (s, 6H), 1.29 (t, J = 7.1 Hz, 3H), 1.15 (t, J = 7.1 Hz, 3H).
The compound 9n was prepared from compound 8n and compound
1
3
C NMR (100 MHz, CDCl
3
) δ 190.13, 169.41, 164.25, 161.83, 158.26,
7 according to describe for 9a method: 148 mg, 67.5% yield as a yellow
1
1
1
1
1
1
7
53.69, 149.30, 145.48, 145.33, 141.61, 138.83, 131.55, 130.49,
30.20, 128.96, 128.81, 128.15, 125.95, 125.18, 124.38, 123.61,
21.84, 117.89, 116.60, 115.11, 114.87, 114.58, 114.27, 110.52,
09.94, 103.36, 63.51, 63.44, 56.51, 55.76, 55.75, 44.25, 27.95, 16.46,
solid; H NMR (600 MHz, CDCl
3
) δ 8.69 (s, 1H), 7.96 (s, 1H), 7.75 (s,
1H), 7.67 (d, J = 8.7 Hz, 2H), 7.60 (s, 2H), 7.20 (d, J = 8.3 Hz, 1H),
7.09 (d, J = 8.0 Hz, 2H), 7.07 (d, J = 8.8 Hz, 1H), 6.77 (d, J = 8.4 Hz,
1H), 6.67 (d, J = 10.0 Hz, 1H), 6.59 (d, J = 8.0 Hz, 2H), 6.49 (d,
J = 8.8 Hz, 1H), 5.61 (d, J = 10.0 Hz, 1H), 4.75 (d, J = 24.2 Hz, 1H),
4.17–4.11 (m, 2H), 4.06–4.02 (m, 1H), 3.92 (s, 3H), 3.91–3.88 (m, 1H),
6.24. HR-MS (m/z) (ESI): calcd for
C
41
H44FN
2
O
8
P
[M+Na]+
:
65.2717; found: 765.2712.
3
3
1
1
1
1
5
.86 (s, 3H), 3.69 (s, 3H), 3.59 (s, 2H), 1.52 (s, 6H), 1.30 (t, J = 7.1 Hz,
13
H), 1.20 (t, J = 7.1 Hz, 3H). C NMR (150 MHz, CDCl ) δ 190.15,
3
4.1.11. Synthesis of target compound 9k
69.35, 158.25, 153.67, 152.66, 149.27, 145.11, 145.01, 141.62,
39.53, 133.34, 131.55, 130.53, 128.96, 128.73, 128.46, 128.05,
25.88, 125.17, 124.93, 124.71, 121.78, 117.99, 116.59, 114.28,
13.85, 110.48, 109.95, 103.35, 63.78, 63.58, 56.63, 55.81, 55.78,
5.34, 44.22, 27.95, 16.49, 16.34. HR-MS (m/z) (ESI): calcd for
The compound 9k was prepared from compound 8k and compound
7
according to describe for 9a method: 134 mg, 64.7% yield as a yellow
1
solid; H NMR (400 MHz, CDCl
3
) δ 8.69 (s, 1H), 7.73 (s, 1H), 7.67 (d,
J = 8.8 Hz, 1H), 7.61 (s, 2H), 7.42 (d, J = 2.2 Hz, 1H), 7.40 (d,
J = 2.3 Hz, 1H), 7.30 (d, J = 8.3 Hz, 2H), 7.21–7.18 (m, 1H), 7.07 (d,
J = 8.4 Hz, 2H), 6.76 (d, J = 8.5 Hz, 1H), 6.67 (d, J = 10.0 Hz, 1H),
11P [M+Na]+: 822.2768; found: 822.2764.
N O
C
42
H
46
2
6
.58 (d, J = 8.5 Hz, 2H), 6.48 (d, J = 8.9 Hz, 1H), 5.61 (d,
4
.1.15. Synthesis of target compound 9o
J = 10.0 Hz, 1H), 4.74 (d, J = 24.5 Hz, 1H), 4.15–4.10 (m, 2H),
.01–3.95 (m, 1H), 3.86 (s, 3H), 3.80–3.74 (m, 1H), 3.63 (s, 3H), 3.58
s, 2H), 1.52 (s, 6H), 1.29 (t, J = 7.1 Hz, 3H), 1.15 (t, J = 7.1 Hz, 3H).
The compound 9o was prepared from compound 8o and compound
4
7
according to describe for 9a method: 159mg, 71.3% yield as a yellow
1
(
solid; H NMR (400 MHz, CDCl
J = 8.8 Hz, 1H), 7.60 (s, 2H), 7.20 –7.18 (m, 1H), 7.09 (d, J = 8.3 Hz,
H), 6.76 (d, J = 8.5 Hz, 1H), 6.70 (d, J = 2.2 Hz, 2H), 6.65 (t,
J = 9.5 Hz, 3H), 6.48 (d, J = 8.9 Hz, 1H), 5.61 (d, J = 10.0 Hz, 1H),
3
) δ 8.69 (s, 1H), 7.76 (s, 1H), 7.66 (d,
1
3
C NMR (100 MHz, CDCl ) δ 190.14, 169.39, 158.26, 153.69, 149.29,
3
1
1
1
5
45.44, 145.29, 141.61, 134.53, 133.86, 131.55, 130.46, 129.20,
29.15, 128.95, 128.82, 128.12, 125.96, 125.16, 124.38, 121.83,
17.96, 116.60, 114.32, 110.51, 109.96, 103.36, 63.52, 63.45, 56.26,
2
4
.67 (d, J = 24.0 Hz, 1H), 4.15 –4.09 (m, 2H), 4.00–3.91 (m, 1H), 3.86
5.76, 55.73, 44.24, 38.61, 27.95, 16.47, 16.28. HR-MS (m/z) (ESI):
(
(
s, 3H), 3.83 (s, 6H), 3.80 (s, 3H), 3.76–3.70 (m, 1H), 3.64 (s, 3H), 3.59
+
calcd for C41
H44ClN
2
O
8
P [M+Na] : 781.2422; found: 781.2421.
s, 2H), 1.51 (s, 6H), 1.29 (t, J = 7.0 Hz, 3H), 1.15 (t, J = 7.1 Hz, 3H).
1
3
C NMR (100 MHz, CDCl ) δ 190.16, 169.47, 158.26, 153.67, 153.42,
3
1
1
1
49.29, 145.85, 145.71, 141.61, 137.74, 131.53, 131.44, 130.43,
28.94, 128.82, 128.14, 125.96, 125.12, 124.24, 121.82, 117.98,
16.60, 114.28, 110.51, 109.99, 104.88, 103.36, 63.45, 63.33, 60.84
4.1.12. Synthesis of target compound 9l
The compound 9l was prepared from compound 8l and compound 7
according to describe for 9a method: 151 mg, 68.6% yield as a yellow
1
(s), 57.11, 56.20, 55.76, 55.73, 44.26, 27.95, 16.50, 16.32. HR-MS (m/
solid; H NMR (400 MHz, CDCl
3
) δ 8.70 (s, 1H), 7.75 (s, 1H), 7.68 (d,
z) (ESI): calcd for
C
44
H
51
N
2
O
11
P
[M+Na]+
: 837.3128; found:
J = 8.8 Hz, 1H), 7.63 (d, J = 1.7 Hz, 1H), 7.62 (s, 2H), 7.41 (t,
J = 6.7 Hz, 2H), 7.22 (d, J = 7.9 Hz, 1H), 7.20–7.18 (m, 1H), 7.09 (d,
J = 8.3 Hz, 2H), 6.76 (d, J = 8.4 Hz, 1H), 6.67 (d, J = 10.0 Hz, 1H),
8
37.3120.
.2. In vitro cytotoxicity
In this study, all human cancer cell lines including HepG-2, A375,
4
6
.61 (d, J = 8.4 Hz, 2H), 6.49 (d, J = 8.8 Hz, 1H), 5.62 (d,
J = 10.0 Hz, 1H), 4.72 (d, J = 24.5 Hz, 1H), 4.18–4.09 (m, 2H),
.02–3.94 (m, 1H), 3.87 (s, 3H), 3.81–3.73 (m, 1H), 3.64 (s, 3H), 3.59
s, 2H), 1.52 (s, 6H), 1.30 (t, J = 7.0 Hz, 3H), 1.16 (t, J = 7.1 Hz, 3H).
4
K562, NCI-H460, MCF-7, MCF-7/DOX, A549, A549/CDDP cancer cells
and human normal liver cells HL-7702 using MTT assay were purchased
from the Shanghai Cell Bank of the Chinese Academy of Sciences.
Culture medium Roswell Park Memorial Institute (RPMI-1640), phos-
phate buffered saline (PBS, pH = 7.2), fetal bovine serum (FBS), and
Antibiotice-Antimycotic came from KeyGen Biotech Company (China).
All cancer cells were cultivate in the supplemented with 10% FBS, and
human normal liver HL-7702 cells were cultivate in the supplemented
with 20% FBS, 100 units/ml of penicillin and 100 g/ml of streptomycin
(
1
3
C NMR (100 MHz, CDCl ) δ 190.12, 169.39, 158.26, 153.69, 149.31,
3
1
1
1
6
45.43, 145.29, 141.61, 138.60, 131.55, 131.17, 130.76, 130.51,
30.20, 128.96, 128.80, 128.14, 126.50, 125.94, 125.18, 124.42,
22.80, 121.83, 117.89, 116.60, 114.24, 110.52, 109.94, 103.36,
3.56, 63.49, 56.44, 55.79, 55.76, 44.24, 27.96, 16.45, 16.23. HR-MS
+
(
m/z) (ESI): calcd for C41
H44BrN
2
O
8
P [M+Na] : 827.1896; found:
8
27.1887.
in a humidified atmosphere of 5% CO at 37 °C. Tested compounds
2
4.1.13. Synthesis of target compound 9m
were dissolved to stock concentrations of 2 mM with DMSO (Sigma);
the lead compound 1a used as a positive control, and the cytotoxicity of
all target compounds against the tested cancer cells and human normal
cells was investigated using MTT assay. All data were independently
tested repeated in triplicate.
The compound 9m was prepared from compound 8 m and com-
pound 7 according to describe for 9a method: 127 mg, 60.4% yield as a
1
yellow solid; H NMR (400 MHz, CDCl
3
) δ 8.69 (s, 1H), 7.75 (s, 1H),
7
.67 (d, J = 8.8 Hz, 1H), 7.61 (s, 2H), 7.18–7.16 (m, 1H), 7.05 (d,
J = 7.5 Hz, 3H), 6.95 (d, J = 8.2 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H),
.74 (d, J = 8.4 Hz, 1H), 6.66 (d, J = 10.0 Hz, 1H), 6.62 (d,
J = 8.2 Hz, 2H), 6.48 (d, J = 8.9 Hz, 1H), 5.61 (d, J = 10.0 Hz, 1H),
6
4.3. Immunofluorescence assay
4
.66 (d, J = 24.0 Hz, 1H), 4.16–4.08 (m, 2H), 3.98–3.92 (m, 1H), 3.86
s, 3H), 3.83 (s, 3H), 3.75–3.67 (m, 1H), 3.62 (s, 3H), 3.57 (s, 2H), 1.52
s, 6H), 1.29 (t, J = 7.1 Hz, 3H), 1.15 (t, J = 7.1 Hz, 3H). C NMR
100 MHz, CDCl ) δ 190.19, 169.62, 158.27, 153.71, 149.38, 146.55,
HepG-2 cells were seeded in each well of six-well plates at the
5
(
(
(
density of 0.5 × 10 cells/mL of the RPMI-1640 medium with 10% FBS
1
3
to the final volume of 2 mL. Cells treated with 9m (5.0 or 10 μM) and
3
1a (10 μM) in a humidified atmosphere of 5% CO at 37 °C for 24. After
2
1
1
1
5
45.94, 145.85, 145.70, 141.69, 131.55, 130.39, 128.98, 128.74,
24 h treatment, cells were fixed with 4% paraformaldehyde at 37 °C for
30 minuntes, and then permeabilized with 0.5% Triton X-100/PBS for
15 minuntes. After blocking for 30 minuntes in 5% BSA/PBS, cells were
washed with PBS and incubated with a-tubulin for 2 h, and then tubulin
was immunostained with monoclonal antibody to a-tubulin followed by
28.65, 128.15, 125.89, 125.24, 123.93, 121.82, 119.59, 117.84,
16.58, 114.38, 114.13, 110.82, 110.52, 109.93, 103.37, 63.48, 63.33,
6.24, 55.93, 55.76, 55.75, 44.26, 27.95, 16.47, 16.28. HR-MS (m/z)
+
(
ESI): calcd for C42
H
47
N
2
O
10P [M+Na] : 793.2866; found: 793.2858.
11

X. Huang, et al.
Bioorganic Chemistry xxx (xxxx) xxxx
fluorescence antibody. Cells were visualized by fluorescence micro-
scope after the nuclei of cells were labeled with DAPI.
4.9. Western blot assay
Western blot analysis was performed as described previously [35].
HepG-2 cells were seeded in each well of six-well plates at the density of
4.4. Cell wound-healing assay
5
0
.5 × 10 cells/mL of the RPMI-1640 medium with 10% FBS to the
final volume of 2 mL, and cells incubated for overnight in a humidified
atmosphere of 5% CO at 37 °C. Cells treated with 9m (5.0 or 10 μM)
HepG-2 cells were seeded in 6-well plates and allowed to grow
to ≥ 95% confluent. After being washed with thrice in PBS, and then
wounds were created perpendicular to the lines by 20 μL tips, and
unattached cells were removed by washing with thrice in PBS. Cells
treated with 9m (5.0 or 10 μM) and 1a (10 μM) in a humidified at-
2
and 1a (10 μM) in a humidified atmosphere of 5% CO at 37 °C for 24.
2
After incubation, cells were collected, centrifuged, and washed thrice in
PBS. The pellet was then re-suspended in lysis buffer containing
1
1
50 mM NaCl, 50 mM Tris (pH 7.4), 1% (w/v) sodium deoxycholate,
% (v/v) Triton X-100, 0.1% (w/v) SDS, and 1 mM EDTA (Beyotime,
mosphere of 5% CO at 37 °C for 24. After 24 h treatment, the cells were
2
washed with thrice in PBS, and then photographed to mark the final
scratched tracks. The migration rates analyzed by Equation 1:
Migration rate (%) = (d1 − d2)/d1. The d1 and d2 represented the
width of wound at 0 and 24 h, respectively.
China). The lysates were incubated at 37 °C for 30 min, and centrifuged
at 20000g at 4 °C for 10 min. The protein concentration in the super-
natant was analyzed by the BCA protein assay reagents. Equal amounts
of protein per line were was separated on 12% SDS polyacrylamide gel
electrophoresis and transferred to PVDF Hybond-P membrane (GE
Healthcare). Membranes were incubated with 5% skim milk in Tris-
buffered saline with Tween 20 (TBST) buffer for 1 h and then the
membranes being gently rotated overnight at 4 °C. Membranes were
then incubated with primary antibodies against Cyto c, Bcl-2, Bax,
Apaf-1, caspase-3, caspase-9 and PARP or GAPDH for overnight at 4 °C.
After three washes in TBST, the membranes were next incubated with
peroxidase labeled secondary antibodies for 2 h. Then all membranes
were washed with TBST four times for 20 min and the protein blots
were detected by chemiluminescence reagent (Thermo Fischer Scien-
tifics Ltd.). The X-ray films were developed with developer and fixed
with fixer solution.
4.5. Cell apoptosis assay
HepG-2 cells were seeded in each well of six-well plates at the
5
density of 0.5 × 10 cells/mL of the RPMI-1640 medium with 10% FBS
to the final volume of 2 mL, and cells incubated for overnight in a
humidified atmosphere of 5% CO at 37 °C. Cells treated with 9m (5.0
2
or 10 μM) and 1a (10 μM) in a humidified atmosphere of 5% CO at
2
3
7 °C for 24. After 24 h incubation, cells were collected, washed thrice
in PBS, and re-suspended in 120 μL of binding buffer at a final con-
6
centration of 0.5 × 10 cells/mL, and then cells were treated with 5 μL
of annexin V-FITC and 5 μL of PI in the dark at 4 °C for 30 min, and
examined by system software (Cell Quest; BD Biosciences).
Declaration of Competing Interest
4.6. Cell cycle assay
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
HepG-2 cells were seeded in each well of six-well plates at the
5
density of 0.5 × 10 cells/mL of the RPMI-1640 medium with 10% FBS
to the final volume of 2 mL, and cells incubated for overnight in a
humidified atmosphere of 5% CO at 37 °C. Cells treated with 9m (5.0
2
Acknowledgements
or 10 μM) and 1a (10 μM) in a humidified atmosphere of 5% CO at
2
3
7 °C for 24. After 24 h incubation, cells were collected, washed thrice
We are grateful to the National Natural Science Foundation of China
in ice-PBS, fixed with ice-cold 70% ethanol at −20 °C for overnight.
The cells were treated with 100 μg /mL RNase A for 30 min at 37 °C
after washed thrice in ice-cold PBS, and finally stained with PI at 1 mg/
ml in the dark at 4 °C for 30 min detected by flow cytometry.
(
Grant Nos. 21977021 and 81760626), and the Ministry of Education
Innovation Team Fund (IRT_16R15, 2016GXNSFGA380005). The au-
thors would also like to thank the Natural Science Foundation of
Guangxi Province (AB17292075) and Guangxi Funds for Distinguished
Experts, and the Key University Science Research Project of Jiangsu
Province (18KJA360001) and the Postgraduate Research & Practice
Innovation Program of Jiangsu Province (SJCX18-0899).
4
.7. Mitochondrial membrane potential (MMP) assay
HepG-2 cells were seeded in each well of six-well plates at the
5
density of 0.5 × 10 cells/mL of the RPMI-1640 medium with 10% FBS
to the final volume of 2 mL, and cells incubated for overnight in a
Appendix A. Supplementary material
humidified atmosphere of 5% CO at 37 °C. Cells treated with 9m (5.0
2
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103486.
or 10 μM) and 1a (10 μM) in a humidified atmosphere of 5% CO
2
at
3
1
7 °C for 24. After 24 h treatment, cells were then stained with 2 μM JC-
in the dark at room temperature for 30 min. After 30 min of in-
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