The discovery of novel sanjuanolide derivatives as chemotherapeutic agents targeting castration-resistant prostate cancer

Article abstract of DOI:10.1016/j.bioorg.2021.104880

There remains a critical need for more effective therapies for the treatment of castration-resistant prostate cancer (CRPC), which is the leading cause of death in patients with prostate cancer. In this study, a series of sanjuanolide derivatives were des

Full text of DOI:10.1016/j.bioorg.2021.104880

Bioorganic Chemistry 111 (2021) 104880
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
The discovery of novel sanjuanolide derivatives as chemotherapeutic
agents targeting castration-resistant prostate cancer
,
,
,
,
Guangbao Wanga 1, Xiaojing Chena 1, Nan Wanga 1, Yunbei Xiaob 1, Sheng Shua,
Ali Mohammed Mohammed Alsayeda, Lu Liua, Yue Maa, Peng Liua, Qianwen Zhanga,
Xiangjuan Chenc *, Zhiguo Liua *, Xiaohui Zhenga
,
,
,*
a Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical University, 1210 University Town, Wenzhou, Zhejiang 325035, China
b Department of Urology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang, China
c Department of Obstetrics and Gynecology, Shenzhen University General Hospital, Shenzhen University, Shenzhen, Guangdong 518000, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
There remains a critical need for more effective therapies for the treatment of castration-resistant prostate cancer
(CRPC), which is the leading cause of death in patients with prostate cancer. In this study, a series of sanjuanolide
derivatives were designed, synthesized and evaluated as potential anti-CRPC agents. Most of the compounds had
excellent selectivity for CRPC cells with IC50 values < 20 µM. Moreover, minimal side effects on human normal
hepatic MIHA cells and normal prostatic stromal myofibroblast WPMY-1 cells were observed, with IC50 > 100
µM. The representative compound S07 slowed down the proliferative rate of CRPC cells, promoted cell apoptosis
and caused G2/M phase accumulation, as well as G1/G0 phase reduction. Further mechanistic studies showed
that S07 treatment triggered intense DNA damage and provoked strong DNA damage response in a dose-
dependent manner. These findings suggested that sanjuanolide derivatives, especially S07, selectively induced
CRPC cell death by triggering intense DNA damage and DNA damage response.
Sanjuanolide
Castration-resistant prostate cancer
Anticancer activity
DNA damage
DNA damage response
1. Introduction
potential advantages as CRPC therapeutics.
Sanjuanolide (1, Fig. 1) is an isoprenylated chalcone first isolated
Castration-resistant prostate cancer (CRPC) is defined by disease
progression despite androgen-deprivation therapy (ADT) and is mainly
developed from prostate cancer with ADT treatment [1]. It is the leading
cause of death in prostate cancer patients [2–5]. At present, surgery,
chemotherapy, radiotherapy and immunotherapy are the primary
treatment options for CRPC [5–7]. Among them, chemotherapy is the
first choice for oncologists against metastatic CRPC [8–10]. However, it
has been reported that cancer cells can become drug resistant, which can
limit the effectiveness of expensive agents used to treat cancer and/or
lead to chemotherapy failure [4,5,11–13]. With these concerns, the
design and discovery of more effective drug candidates that selectively
target CRPC cells, with minimal side effects and low cost, could have
from Dalea frutescens A. Gray (Leguminosae) in 2016. In in vitro studies,
it exhibited promising anti-proliferative activity against PC3 and DU145
prostate cancer cells [14]. To further study the pharmacological action
of sanjuanolide, we have recently completed the first total synthesis of
(R)-, (S)- and (±)-sanjuanolide by a new and efficient synthetic strategy.
Additional biological studies have shown that the (R)-sanjuanolide is
important for anti-inflammatory activity [15]. Additionally, our previ-
ous studies indicated that chalcones, with an allyl or isoprenyl moiety at
position 5 in the A ring, showed effective inhibitory activity against
cancer and related diseases [16]. Thus, we proposed that replacement of
the isoprenyl group with an allyl moiety in sanjuanolide may provide
equivalent or better biological activity.
Abbreviations: CRPC, Castration-resistant prostate cancer; ADT, Androgen-deprivation therapy; MOM, Methoxymethyl; TBS, t-Butyldimethyl; DMF, Dimethyl
Formamide; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PARP, Poly ADP-ribose polymerase; Bcl2, B-cell lymphoma-2; FCM, Flow
cytometry; DSBs, DNA double-strand breaks; DDR, DNA damage response; IF, Immunofluorescence.
* Corresponding authors at: Department of Obstetrics and Gynecology, Shenzhen University General Hospital, Shenzhen University, Shenzhen, Guangdong
518000, China. (X. Chen). Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical University. 1210 University Town, Wenzhou,
Zhejiang 325035, China. (Z. Liu, X. Zheng).
E-mail addresses: 472346758@qq.com (X. Chen), lzgcnu@163.com (Z. Liu), zhengxh@wmu.edu.cn (X. Zheng).
1
These authors contribute equally to this work.
https://doi.org/10.1016/j.bioorg.2021.104880
Received 5 October 2020; Received in revised form 26 March 2021; Accepted 26 March 2021
Available online 29 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

G. Wang et al.
Bioorganic Chemistry 111 (2021) 104880
Table 1
Chemical structures and IC50 (μ
M) values determined by the MTT assay. a
Deletion
Compound
R
IC50 (µM)
PC3 b
DU145 b
MIHA b
WPMY-
1b
S01
S02
S03
–
21.04
± 0.24
19.6 ±
0.39
46.16 ±
1.16
>100
>100
>100
4′-Br
4′–NO2
58.05 ±
2.81
>100
Fig. 1. Chemical structures of sanjuanolide and the structural design of new
sanjuanolide derivatives.
12.56
± 0.02
>100
19.37
± 0.51
>100
12.04 ±
0.08
26.1 ±
0.10
32.81 ±
0.10
S04
S05
2′-CF3
3′-CF3
>100
37.02 ±
6.01
>100
35.23
± 4.27
>100
>100
37.8 ±
0.22
Inspired by these results, we aimed to discover novel CRPC thera-
peutic agents by replacing the substituted 2-methyl-3-hydroxyisoprenyl
group in sanjuanolide with a simple 2-methyl-3-hydroxyallyl group.
Moreover, optimization of the phenyl substituents on the B ring may
provide new lead compounds with enhanced CRPC activity. To our
knowledge, these types of sanjuanolide derivatives have not yet been
previously synthesized. Importantly, active derivative S07 showed
improved anti-tumor activities by triggering intense DNA damage and
DNA damage response against castration-resistant prostate cancer
(CRPC) in vitro and in vivo, suggesting the potential for sanjuanolide
derivatives to be developed as new anti-cancer agents for the treatment
of CRPC.
S06
S07
S08
S09
S10
S11
S12
S13
4′-CF3
4′-
40.78 ±
0.43
>100
15.55
± 0.51
29.80
± 1.95
26.47
± 0.96
26.46
± 3.36
50.01
± 2.66
28.07
± 0.18
87.94
± 4.36
>100
6.77 ±
0.17
>100
>100
>100
>100
methylpiperizine
4′- morpholine
>100
2′-OMe
53.12 ±
0.35
63.17
40.93 ±
1.05
± 1.21
83.11
3′-OMe
46.52 ±
0.55
60.92 ±
4.31
± 2.73
68.26
4′-OMe
88.74 ±
4.36
51.43 ±
1.20
± 1.41
59.71
2′,3′-OMe
3′,4′-OMe
>100
83.62 ±
1.63
± 2.557
>100
2. Results and discussion
>100
22.44 ±
0.57
2.1. Chemistry
S14
S15
S16
2′,4′-OMe
>100
>100
85.16 ±
4.80
>100
>100
>100
>100
2′,5′-Me
>100
>100
3′-OMe, 4′–OH
>100
>100
The synthesis of sanjuanolide derivatives (S01-S18) was accom-
plished as portrayed in Scheme 1, and their structures are shown in
Table 1. MOM protection of commercially available 2,4-dihydroxyaceto-
phenone (2), followed by NaHB4 reduction, provided 1-[2,4-bis(methox-
ymethoxy)phenyl]ethanol (3). The secondary alcohol 3 was protected
by TBS to give (1-(2,4-bis(methoxymethoxy)phenyl)ethoxy)(tert-butyl)
dimethylsilane (4). Treatment of 4 with n-BuLi and DMF afforded the
aldehyde 5, which was reacted efficiently with isopropenylmagnesium
bromide to give the corresponding allylic alcohol 6 in good yield. Sub-
sequently, acetylation of the alcoholic hydroxyl group with acetyl
chloride yielded the corresponding1-(3-(1-((tert-butyldimethylsilyl)oxy)
ethyl)-2,6-bis(methoxymethoxy)phenyl)-2-methylallyl acetate (7), ac-
cording to the procedure reported by Fang et al [15]. Furthermore,
preparation of acetophenone 9 was achieved through the TBS-
deprotection of 7 with TBAF, followed by oxidative workup of acetyl
chloride 8 with Dess-Martin periodinane in EtOH. Finally, aldol
condensation between the acetophenone 9 and various benzaldehydes
S17
2′,4′,6′-OMe
3′,4′,5′-OMe
10.93
± 0.55
18.43
± 0.37
7.78 ±
0.26
>100
>100
>100
S18
15.67 ±
0.45
29.55
± 2.03
>100
22.44 ±
0.57
Sanjuanolide
Cisplatin
20.3 ±
0.38
>100
6.21 ±
0.54
25.68 ±
1.79
36.62
51.6 ±
± 0.61
1.19
a
IC50 values were drug concentrations necessary for 50% inhibition of cell
viability. Data are average ± standard deviations of at least three independent
experiments. The drug treatment period was 48 h. bHuman castration-resistant
prostate cancer (CRPC) cells, PC3 and DU145; human normal hepatic cells
(MIHA) and human normal prostatic stromal myofibroblast cells (WPMY-1).
Scheme 1. General synthetic routes for S01-S18. Reagents and conditions: (a) i) NaH, MOMCl, THF, 0 ◦C, 4 h, ii) NaBH4, EtOH, 0 ◦C, 3 h, 81%; (b) TBSCl,
imidazole, CH2Cl2, rt, 6 h, 100%; (c) n-BuLi, DMF, THF, ꢀ 78 ◦C to 0 ◦C, 3 h, 58%; (d) isopropenylmagnesium bromide, THF, 0 ◦C, 2 h, 63%; (e) DMAP, Et3N, Ac2O,
CH2Cl2, 2 h, 88%; (f) TBAF, THF, rt, 5 h, 92%; (g) Dess-Martin periodinane, CH2Cl2, 4 h, 63%; (h) i) NaOH, EtOH, rt, overnight, ii) 4 M HCl, MeOH, 70 ◦C, 1.5 h,
35%–50%.
2

G. Wang et al.
Bioorganic Chemistry 111 (2021) 104880
in ethanolic KOH solution resulted in protected target molecules. The
target molecules were deprotected with concentrated HCl in methanol,
generating target compounds S01–S18 over two steps. Both analytical
and spectral data of all synthesized compounds are in full agreement
with the proposed structures.
and DU145 cells, relative to compounds S01. However, compound S04,
with a trifluoromethyl moiety at the ortho-position of the phenyl ring,
resulted in a dramatic loss of anti-proliferative activity, indicating that
the introduction of an electron-withdrawing group at the ortho-position
is not beneficial to the activity. Interestingly, compound S07, which has
a 4′-methylpiperizine at the para-position of the phenyl ring, displayed
the greatest activity against PC3 and DU145 cells. Replacement of 4-
methylpiperizine of S07 with a morphine motif (compound S08)
resulted in a substantial reduction or loss of anti-proliferative effects.
The introduction of 2′-methoxy (compound S09) or 3′-methoxy
(compound S10) substitution showed an almost equivalent activity in
comparison with the compounds S01 on PC3 and DU145 cells. However,
the introduction of methoxy (compound S11) to the C-4′ position, as
oppose to phenyl ring, resulted in a substantial loss of anti-proliferative
effects. Finally, incorporation of different disubstituted or trisubstituted
electron-donating groups onto the phenyl ring provided compounds
S12–S18, and these compounds displayed varying degrees of inhibition
toward PC3 cells. However, almost no activity toward DU145 cells was
observed, except for compound S18 with an IC50 value of 15.67 µM.
Taken together, the structure–activity relationships in this series are
complex, but the data provide valuable information for the design of
novel anti-cancer agents.
2.2. The cytotoxicity and antiproliferative activities of synthetic
compounds toward castration-resistant prostate cancer
The anticancer activity of the synthesized compounds was evaluated
for potential anti-castration-resistant prostate cancer (CRPC) activity
using MTT assay, while the well-known anticancer drug cisplatin and
sanjuanolide were used as the positive controls. Human CRPC cells, PC3
and DU145, were cultured in the presence or absence of test compounds
for 48 h, and the cell viabilities were determined. The results are sum-
marized in Table 1. The initial screening showed that compared to
human normal hepatic cells (MIHA) and human normal prostatic stro-
mal myofibroblast cells (WPMY-1), a majority of compounds preferred
to exhibit higher cytotoxicity to both CRPC PC3 and DU145 cells. This
suggests that compounds S01–S18 could potentially be developed as
selective antitumor agents against CRPC. Among the 15 tested com-
́
pounds studied, compound S07, with 4-methylpiperizine at the R posi-
tion, proved to be the most active against PC3 and DU145 cells, with IC50
values of 15.55 µM and 6.77 µM, respectively. In addition, compound
S07, possessed comparable anticancer activity to cisplatin, but S07 had
fewer side effects. Thus, it was selected as a candidate in this study.
To investigate the influence of the B ring substituents on the anti-
cancer activity, we introduced various electron-withdrawing groups,
such as bromine, nitro group and trifluoromethyl groups at the phenyl
ring in compound S01, resulting in compounds S02–S06. Compounds
S02, S03 and S05 demonstrated equal or greater potency against PC3
2.3. Active compound S07 inhibits cell proliferation and disrupts tubulin
polymerization
In order to further explore the antiproliferative activity of active
compound S07 in CRPC cells, the long-term proliferation assay was
carried out under the 0.5IC50 and IC50 values at the indicated concen-
trations 4.0 µM, 8.0 µM or 16.0 µM. The results clearly showed that even
in a relatively low dose, S07 could significantly slow down the
A)
B)
DU145
10
8
PC3
8
6
4
2
0
Control
8.0 µM
16.0 µM
Control
4.0 µM
8.0 µM
*
*
*
6
*
*
*
*
*
*
*
*
*
4
2
0
2
4
6
8
2
4
6
8
Time.d
Time.d
C)
D)
PC3
DU145
DAPI
Merge
DAPI
α-Tublin
Merge
α-Tublin
Control
Control
8.0 µM
4.0 µM
8.0 µM
16.0 µM
Fig. 2. S07 inhibits the proliferation and disrupts tubulin polymerization of castration-resistant prostate cancer (CRPC) cells. The proliferation curves of
PC3 (A) and DU145 (B) in the control and S07-treated cells at the indicated concentrations of 0.5IC50 and IC50 concentrations of S07 upon PC3 (8.0 µM and 16.0 µM)
and DU145 (4.0 µM and 8.0 µM), respectively. Values are the average ± SD of three independent experiments. p values were calculated using Two-way ANOVA (*p <
0.05, **p < 0.01, ***p < 0.001). Immunofluorescence assay analysis of the effect of S07 on the organizations of cellular microtubule network. PC3 (C) and DU145 (D)
cells were treated with 0.5IC50 and IC50 concentrations of S07 upon PC3 (8.0 µM and 16.0 µM) and DU145 (4.0 µM and 8.0 µM), respectively. Then, microtubules
were stained with anti-α-tubulin-FITC antibody (green), and nucleuses were stained with DAPI (blue). Images were performed by using Nikon microscope (Nikon,
Japan). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3

G. Wang et al.
Bioorganic Chemistry 111 (2021) 104880
proliferation rate of CRPC PC3 and DU145 cells (Fig. 2A and 2B).
Considering the results from MTT assay and long-term proliferation
assay together, the 0.5IC50 and IC50 concentrations of S07 were chosen
in the following assay. Furthermore, we observed that compared with
untreated group, S07 treatment would disrupt the tubulin polymeriza-
tion and organization of CRPC PC3 and DU145 cells (Fig. 2C and 2D) in a
concentration-dependent manner. Since the tubulin polymerization and
functional organization played a key role of cell mitosis [17], S07 might
finally lead to cell cycle arrest due to the inhibition of tubulin
polymerization.
that S07 treatment increased the percentage of G2/M phase and S phase
cells while reducing the percentage of cells in the G0/G1 phase relative
to the control. Since cells transverse the cell cycle in several well-
controlled phases: cells commit to enter the cell cycle in G1/G0 phase
and prepare to duplicate their DNA in S phase, then chromatids and
daughter cells separate in M phase [18]. Furthermore, it has been re-
ported that G1/G0 phase arrest might enhance proliferation, and G2/M
phase arrest might result in cell apoptosis [19]. S07 arrested CRPC cells
at the G2/M and S phases and reduced the G0/G1 phase, demonstrating
that the slower CRPC cell proliferation rate in S07-treated cells might
also induce CRPC cell apoptosis.
2.4. Active compound S07 arrests cell cycle of PC3 and DU145 cells in
G2/M phase
2.5. Active compound S07 induces cell apoptosis by regulating the
expression of apoptosis-related protein
In order to explore the in vivo evidence that active compound S07
slowed down the proliferation rate of CRPC cells by cell cycle arrest. The
cell cycle distribution of CRPC cells PC3 and DU145 treated and/or
untreated with active compound S07 for 48 h was analyzed by flow
cytometry assay. As shown in Fig. 3, S07 arrested both PC3 and DU145
cells at the G2/M phase in a dose-dependent manner. It is worth noting
Apoptosis is the common anticancer mechanism of natural products,
and the cell cycle arrest data indicated that S07 would induce cell
apoptosis. Therefore, the well-known apoptosis-related proteins,
cleaved-PARP [20], caspase-3, cleaved-caspase-3 [21] and Bcl2, which
are widely regarded as markers of apoptosis, were chosen in the western
A)
Control
8.0 µM
16.0 µM
Control
8.0 µM
4.0 µM
C)
B)
D)
120
120
PC3
DU145
Dip G1 (%)
Dip S (%)
Dip G1 (%)
Dip S (%)
100
80
60
40
20
0
100
80
60
40
20
0
Dip G2 (%)
Dip G2 (%)
Control 8.0 µM 16.0 µM
Control 4.0 µM 8.0 µM
Fig. 3. G2/M phase arrest in S07-treated castration-resistant prostate cancer (CRPC) cells. 0.5IC50 and IC50 concentrations of S07-treated PC3 (8.0 µM and
16.0 µM) and DU145 (4.0 µM and 8.0 µM), or untreated cells (Control) were subjected to cell cycle FACS analysis. The representative images of S07-treated PC3 (A)
and DU145 (C) cells. B and D, Quantification of A and C. Values are average ± SD of three independent experiments.
4

G. Wang et al.
Bioorganic Chemistry 111 (2021) 104880
(µM)
(µM)
8.0
Control
8.0
16.0
Control
4.0
A)
C)
Cleaved-PARP
Caspase-3
Cleaved-PARP
Caspase-3
Cleaved-Caspase-3
Bcl2
Cleaved-Caspase-3
Bcl2
GAPDH
GAPDH
1.0
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.8
0.6
0.4
0.2
0.0
B)
**
**
0.8
**
*
*
*
0.6
0.4
0.2
0.0
Control 8.0 µM 16.0 µM
Control 8.0 µM 16.0 µM
Control 8.0 µM 16.0 µM
0.6
0.4
0.2
0.0
0.6
0.4
0.2
0.0
0.4
0.3
0.2
0.1
0.0
D)
*
*
*
*
*
*
Control 4.0 µM 8.0 µM
Control 4.0 µM 8.0 µM
Control 4.0 µM 8.0 µM
Fig. 4. S07 regulates the expression of apoptosis-relative proteins cleaved-PARP, caspase-3, cleaved-caspase-3 and Bcl2 in castration-resistant prostate
cancer (CRPC) cells. Western blot determination of the abundance of cleaved-PARP, caspase-3, cleaved-caspase-3 and Bcl2 in the control and S07-treated PC3 (A)
and DU145 (C) cells. Cells were treated with 0.5IC50 and IC50 concentrations of S07-treated PC3 (8.0 µM and 16.0 µM) and DU145 (4.0 µM and 8.0 µM) for 48 h and
subjected to western blot using antibodies against cleaved-PARP, caspase-3, cleaved- caspase-3 and Bcl2. GAPDH was used as a loading control. B and D, Quan-
tification of A and C. Values are average ± SD of three independent experiments. Unpaired Student’s two-tailed t test was used to determine the statistical significance
(*p < 0.05, **p < 0.01).
blot assay. The results show that S07 decreased the expression of
cleavage-PARP and increased the expression of caspase-3 and Bcl2
proteins in a dose-dependent manner (Fig. 4). The results further indi-
cate that S07 induces cell apoptosis by regulating the expression of
apoptosis-related proteins, PARP, caspase-3 and Bcl2.
cancer cells by introducing mass DNA damage [22]. This is based on the
widely accepted concept that nonproliferating cells are more resistant to
DNA damage and/or DNA damage response than proliferating cells
[23]. To investigate DNA damage in S07-treated human CRPC cells, PC3
and DU145 cells were exposed to 4.0, 8.0 or 16.0 μM of S07 for 48 h, and
To test this, flow cytometry (FCM) assay was performed to analyze
the cell apoptosis in control and S07 treated CRPC cells. Consequently,
S07 induced CRPC cells apoptosis at a dose- and time-dependent
manner, especially in time-dependent manner (Fig. 5 and Figure S1).
To exclude the possibility that S07 might induce CRPC cell senescence, a
beta-galactosidase (SA-β gal) staining assay was performed in the pre-
sent or absent of S07 treated CPRC cells. We observed that there is no
significant difference in the percentage of senescent cells between un-
treated group and S07-treated group (Figure S2), demonstrating that
S07 did not induce cell senescence. Taken together, these results indi-
cate that S07 could induce apoptosis in CRPC cells, presumably as a
result of its effects on the expression of apoptosis-related protein PARP,
caspase-3 and Bcl2. This is consistent with other reports showing that
the cleaved-PARP, caspase-3 and Bcl2 levels correlate with cell cycle
arrest and apoptosis [20,21].
the level of DNA damage was assessed by comet assay. As expected, S07
induced a significant amount of DNA damage in PC3 and DU145 cells,
leading to DNA fragments that leave the genome and form “tails” during
the neutral and alkaline comet assay (Fig. 6A and 6C, Figure S3A and
S3C). A percentage of tail DNA was used to indicate the abundance of
fragments induced by DNA lesions. The results show that the level of
DNA damage in S07-treated CRPC cells significantly increased in a
concentration-dependent manner (Fig. 6B and Figure S3B). Accordingly,
>20% of cells displayed higher DSB levels than the control (5% tail DNA
signal) (Fig. 6D and Figure S3D). Collectively, these results demonstrate
that active compound S07 achieved excellent anticancer activity by
triggering intense DNA damage.
2.7. Active compound S07 treatment provoked strong DNA damage
response
2.6. Active compound S07 treatment triggered intense DNA damage
The ability to repair DNA lesion is critical for cell viability [24,25],
and previous reports indicated that cancer cells are endowed with that
same innate, or even stronger, DNA repair capacity as normal cells [24].
Chemotherapy agents, such as some natural products, are used to kill
5

G. Wang et al.
Bioorganic Chemistry 111 (2021) 104880
2days
**
A)
B)
15.0
10.0
5.0
Control
8.0 µM
4.0 µM
ns
0.0
Control 4.0 µM 8.0 µM
Annexin V-FLITC staining
4.0 µM
7days
**
C)
D)
Control
8.0 µM
100.0
80.0
60.0
40.0
20.0
0.0
***
Annexin V-FLITC staining
Control 4.0 µM 8.0 µM
Fig. 5. S07 treatment induces apoptosis of castration-resistant prostate cancer (CRPC) cells. FACS analysis was performed to detect the apoptotic DU145 cells
treated with 0.5IC50 and IC50 concentrations of S07-treated PC3 (8.0 µM and 16.0 µM) or DU145 (4.0 µM and 8.0 µM), for 2 days (A) and/or 7 days (C). B and D,
Quantification of A and C. Values are average ± SD of three independent experiments. Unpaired Student’s two-tailed t test was used to determine the statistical
significance (*p < 0.05, **p < 0.01, ***p < 0.001).
alkaline
4.0 µM
neutral
4.0 µM
A)
C)
Control
8.0 µM
Control
8.0 µM
B)
D)
120
100
80
60
40
20
0
80
60
40
20
0
350
300
250
200
150
100
50
500
400
300
200
100
0
***
***
***
***
***
***
***
***
0
Control
4.0 µM
8.0 µM
Control
4.0 µM
8.0 µM
Control 4.0 µM
8.0 µM
Control
4.0 µM
8.0 µM
Fig. 6. S07 treatment triggers intense DNA damage in castration-resistant prostate cancer (CRPC) DU145 cells in a concentration-dependent manner. A
and C, Representative images of comet assay in neutral and alkaline of 0.5IC50 and IC50 concentrations of S07-treated DU145 (4.0 µM and 8.0 µM), respectively. B and
D, The percentages of DNA in the tail for S07-treated DU145 and the percentage of S07-treated DU145 with over 10% tail DNA. Cells were treated with indicated
concentration of S07 for 48 h, and ≥ 500 cells were examined in each group. Values are average ± SD of three independent experiments. Unpaired Student’s two-
tailed t test was used to determine the statistical significance (***p < 0.001).
Persistent DNA double-strand breaks (DSBs) induce a potent DNA
damage response (DDR), leading to cell cycle arrest, cell senescence or
apoptosis that ultimately results in lethality at the cellular level [26]. To
explore the fate of S07-induced DNA lesions, immunofluorescence (IF)
assay was performed, using antibody to 53BP1 (a widely used marker for
DSBs) [27]. In Fig. 7, the number of 53BP1 foci per nucleus was calcu-
lated in both the control and S07-treated PC3 and/or DU145 cells. As
expected, the number of 53BP1 foci in PC3 cells increased to an average
of ~ 13 foci per nucleus 48 h after 10 µM S07 treatment, and the number
of 53BP1 foci per cell was found to be dose-dependent. Interestingly, the
6

G. Wang et al.
Bioorganic Chemistry 111 (2021) 104880
Fig. 7. S07 treatment provokes intense DNA
damage response in castration-resistant prostate
A)
B)
Merge
DAPI
53BP1
16
12
8
cancer (CRPC) cells in
a
concentration-
***
dependent manner. A and C, Representative im-
ages of 53BP1 foci in PC3 and DU145 cells at the
0.5IC50 and IC50 concentrations of S07-treated PC3
(8.0 µM and 16.0 µM) and DU145 (4.0 µM and 8.0
µM), respectively. B and D, Quantifying 53BP1 foci
per cell of A and C, respectively. Cells were treated
with S07 for 48 h, and ≥ 200 cells were examined in
each group. Values are average ± SD of three in-
dependent experiments. Unpaired Student’s two-
tailed t test was used to determine the statistical
significance (**p < 0.01, ***p < 0.001).
Control
***
8.0 µM
4
16.0 µM
0
Control 8.0 µM 16.0 µM
Merge
DAPI
53BP1
C)
D)
16
12
8
***
**
Control
4.0 µM
8.0 µM
4
0
Control 4.0 µM 8.0 µM
relative intensity of 53BP1 foci also increased accordingly (Fig. 7A). We
obtained similar results when DU145 cells were exposed to various
concentrations of S07 (Fig. 7B). The results are consistent with the re-
sults from the western blot assay, showing S07 treatment significantly
increased the expression of γ-H2AX and Rad 51 (Fig. 8) in a dose-
dependent manner. These results, combined the data from comet
assay, further supported the conclusion that S07 induced the death of
CRPC cells by triggering strong DNA damage and DNA damage response.
S07 was exposed to water outside of DNA, as well mainly stabilized in a
large hydrophobic cavity formed on the basis of a number hydrophobic
nucleotides, including DC-15 (cytosine), DG-16 (guanine) and DA-17
(adenine), explaining why even as light change could not be tolerated
at this position.
3. Conclusion
In conclusion, a series of novel sanjuanolide derivatives were
designed and synthesized. The evaluation of their inhibitor activities
against PC3 and DU145 cell lines showed the majority of synthetic
compounds exhibited higher cytotoxicity to both PC3 and DU145 cells,
compared to human normal MIHA and WPMY-1 cells. In general, de-
rivative S07 showed the best anticancer activity on PC3 cells, with
comparable cytotoxicity to the lead compounds sanjuanolide and
cisplatin. Further mechanism studies showed that S07 inhibited cell
viability, arrested the cell cycle and induced cell death of castration-
resistant prostate cancer (CRPC) cells by triggering intense DNA dam-
age and provoking stronger DNA damage response. These results reveal
the potential of sanjuanolide derivatives as a novel chemotherapeutic
agent for targeting CRPC cells.
2.8. Molecular docking
Binding of small organic ligands to DNA is among the widely used
strategies for the development of novel antitumor therapeutics [28,29].
To investigate the structural mechanism underlying S07 binding to
DNA, we docked the S07 molecule into the crystal structure of DNA by
using the software Auto Dock 4.0. As shown in Fig. 9, the entire S07
molecule lodges inside the major groove of double-stranded DNA. From
the in-silico modeling analysis, several hydrogen bonds were predicted
between compound S07 and the surrounding nucleotides in the drug
binding pocket. DA-18 (adenine) was involved in two hydrogen bonding
interactions by one of the interacting with the carbonyl oxygen atom of
α
, β-unsaturated carbonyl groups of S07, and the other amino hydrogen
4. Materials and methods
atom interacting with one of the phenolic group in S07. In addition, thee
carbonyl oxygen atom in DT-7 (thymine) also interacted, by hydrogen
bonding, with the secondary alcohol and the phenolic group of S07,
respectively. Similarly, the hydrogen atom in the N1 atom of the imid-
azole in DA-6 (adenine) was engaged in a hydrogen bond with the
phenolic group of S07. Moreover, The 4′-methylpiperizine side chain of
4.1. Chemistry
4.1.1. General
All reagents and solvents were purchased from Alfa Aesar or Sigma
7

G. Wang et al.
Bioorganic Chemistry 111 (2021) 104880
A)
C)
D)
Control
4.0
8.0 (µM)
Control
8.0
16.0 (µM)
γ-H2AX
γ-H2AX
Rad 51
Rad 51
GAPDH
GAPDH
B)
0.8
0.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.4
**
*
*
*
0.6
0.4
0.2
0.0
0.3
***
*
0.4
0.2
0.0
*
*
0.2
0.1
0.0
Control 4.0 µM 8.0 µM
Control 4.0 µM 8.0 µM
Control 8.0 µM 16.0 µM
Control 8.0 µM 16.0 µM
Fig. 8. S07 regulates the expression of DNA damage response-relative protein γ-H2AX and Rad51 in castration-resistant prostate cancer (CRPC) cells.
Western blot determination of the abundance of γ-H2AX and Rad51 in the control and S07-treated PC3 (A) and DU145 (C) cells. Cells were treated with 0.5IC50 and
IC50 concentrations of S07 (PC3, 8.0 µM and 16.0 µM; DU145, 4.0 µM and 8.0 µM), for 48 h and subjected to western blot analysis using antibodies against γ-H2AX
and Rad51. GAPDH was used as a loading control. B and D, Quantification of A and B. Values represent the average ± SD of three independent experiments. Unpaired
Student’s two-tailed t test was used to determine the statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001).
DA-6
DA-7
DA-18
Fig. 9. The binding modes of S07 with the major groove of double-stranded DNA.
Aldrich and were used as received, unless noted otherwise. Thin layer
chromatography was carried out t using Kiesel-gel 60 F254 glass-backed
plates. Flash column chromatography was performed with silica gel 60
as the stationary phase. Melting points were determined on a Fisher-
Johns melting apparatus and were uncorrected. Nuclear magnetic
resonance (NMR) spectra were recorded on a Bruker Avance DRX 500
instrument (1H, 500 MHz, 13C,126 MHz). The chemical shifts are
expressed in parts per million relative to internal TMS, and coupling
constants (J) are in Hz. Analytical liquid chromatography/mass spec-
trometry (LC/MS) was recorded on a Bruker Esquire 3000tSpectrometer.
8

G. Wang et al.
Bioorganic Chemistry 111 (2021) 104880
4.1.2. Synthesis of 1-[2,4-bis(methoxymethoxy)phenyl]ethanol (3)
To a solution of 1-(2,4-dihydroxyphenyl)ethanone (1) (12.0 g, 78.9
mmol) in anhydrous THF (100 mL) was added sodium hydride in an ice-
bath. After 30 min, the mixture was added chloromethyl methyl ether
(19.1 g, 236.6 mmol) slowly. After stirring for 4 h at room temperature,
the resulting mixture waspoured into water (100 mL), extracted with
EtOAc (3 × 50 mL). The combined organic layers were washed with-
water (100 mL), brine (100 mL), dried over anhydrous MgSO4, filtered,
and concentrated in vacuo.
to room temperature and stirring was continued for 2 h. The resulting
solution was poured into water (20 mL) and extracted with EtOAc (3 ×
20 mL). The combined organic layers were washed with water (50 mL),
brine (50 mL), dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. The residue was purified by chromatography on silica gel gave
compound 6 as a yellow oil (1.46 g, 63%). 1H NMR (500 MHz, CDCl3) δ
(ppm): 7.40 (d, J = 10.0 Hz, 1H), 6.93 (d, J = 10.0 Hz, 1H), 5.42 (s, 1H),
5.17–5.13 (m, 3H), 4.99–4.95 (m, 1H), 4.90–4.87 (m, 3H), 3.57 (s, 3H),
3.44 (s, 3H), 1.69 (s, 3H), 1.36 (d, J = 10.0 Hz, 3H),0.85 (m, 9H), 0.04 (s,
3H), ꢀ 0.04 (s, 3H). ESI-MS m/z: 450 (M + Na)+.
Thecrude liquid (10.5 g, 43.7 mmol) was dissolved in EtOH (50 mL)
at ice-bathtemperature, followed by sodium borohydride (4.96 g, 43.7
mmol) and the mixture was then allowed to warmup to room temper-
ature and stirring was continued for 3 h. The resulting mixture was
quenched withsaturated aqueous NH4Cl (50 mL), and extracted with
EtOAc (3 × 50 mL). The combined organic layers were washed with-
water (100 mL), brine (100 mL), dried over anhydrous MgSO4,filtered,
and concentrated in vacuo. The residue was purified by chromatography
on silica gel to give compound 3 as a colorless oil (9.80 g, 81%).1H NMR
(500 MHz, CDCl3) δ (ppm): 7.28 (d, J = 10.0 Hz, 1H), 6.79 (s,1H), 6.71
(d, J = 10.0 Hz, 1H), 5.21 (d, J = 5.0 Hz, 2H), 5.15 (s, 1H), 5.11 (d, J =
5.0 Hz, 2H), 5.09–5.08 (m, 1H),3.49 (s, 3H), 3.47 (s, 3H), 1.19 (d, J =
5.0 Hz, 3H. ESI-MS m/z: 265.1 (M + Na)+.
4.1.6. Synthesis of 1-{3-{1-[(tert-butyldimethylsilyl)oxy]ethyl}-2,6-bis
(methoxymethoxy) phenyl}-2-methylallyl acetate (7)
A solution of 1-{3-{1-[(tert-butyldimethylsilyl)oxy]ethyl}-2,6-bis
(methoxymethoxy)phenyl} ꢀ 2-methylprop-2-en-1-ol (6) (1.41 g, 3.3
mmol) in dry CH2Cl2 (20 mL) was added Et3N(1.5 mL, 9.9 mmol) por-
tionwise, followed by acetic anhydride (0.72 mL,6.6 mmol) and 4-dime-
thylaminopyridine (DMAP) (38 mg, 0.31 mmol) at room temperature.
After 2 h, the reaction mixture was quenched with saturated aqueous
NH4Cl (20 mL) and extracted with EtOAc (3 × 20 mL). The combined
organic layers were washed with a solution of aqueous citric acid (50
mL), brine (50 mL), dried over anhydrous MgSO4, filtered, and
concentrated in vacuo. The residue was purified by chromatography on
silica gel gave compound 7 as a yellow oil (1.10 g, 88%). 1H NMR (500
MHz, CDCl3) δ (ppm): 7.44 (d, J = 10.0 Hz, 1H), 6.90 (d, J = 10.0 Hz,
1H), 6.76 (s, 1H), 5.24 (d, J = 5.0 Hz, 1H), 5.12–5.10 (m, 2H), 4.98 (d, J
= 5.0 Hz, 1H), 4.93–4.86 (m, 2H), 4.78 (s, 1H), 3.55 (s, 3H), 3.44 (s, 3H),
2.07 (s, 3H), 1.61 (s, 3H), 1.35–1.33 (m, 3H), 0.84 (s, 9H), ꢀ 0.01 (s, 3H),
ꢀ 0.10 (s, 3H). ESI-MS m/z: 491.3 (M + Na)+.
4.1.3. Synthesis of {1-[2,4-bis(methoxymethoxy)phenyl]ethoxy}(tert-
butyl)dimethylsilane (4)
To a solution of 1-[2,4-bis(methoxymethoxy)phenyl]ethanol (3)
(9.70 g, 40.0 mmol) inanhydrous CH2Cl2 (20 mL) was added imidazole
(8.18 g, 120.1 mmol) at room temperature followed by tert-butyldime-
thylsilyl chloride (TBSCl) (15.1 g, 100.1 mmol) and the mixture was
stirred at this temperature for 6 h. The reaction mixture was quenched
with saturated aqueous NH4Cl (20 mL) and extracted with EtOAc (3 ×
20 mL). The combined organic layers were washed with water (50 mL),
brine (50 mL), dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. The residue was purified by chromatography on silica gel
provided compound 4 as a colorless oil (11.5 g, 100%). 1H NMR (500
MHz, CDCl3) δ (ppm): 7.42 (d, J = 10.0 Hz, 1H), 6.74 (s, 1H), 6.70 (d, J
= 10.0 Hz, 1H), 5.18 (s, 2H), 5.17–5.167 (m, 1H), 5.15 (s, 1H), 3.49
(S,3H), 3.48 (s, 3H), 1.35 (d, J = 5.0 Hz, 3H), 0.91 (s, 9H), 0.05 (s, 3H),
ꢀ 0.02 (s, 3H). ESI-MS m/z: 379.2 (M + Na)+.
4.1.7. Synthesis of 1-[3-(1-hydroxyethyl)-2,6-bis(methoxymethoxy)
phenyl]-2-methylallyl acetate (8)
A solution of 1-{3-{1-[(tert-butyldimethylsilyl)oxy]ethyl}-2,6-bis
(methoxymethoxy) phenyl}-2-methylallyl acetate (7) (1.0 g, 2.13
mmol) in anhydrous THF (15 mL) was treated with tetra-n-buty-
lammonium fluoride (16.5 mg, 6.3 mmol) at room temperature. After
stirring for 5 h, the resulting mixture was poured into cool water (20 mL)
and extracted with EtOAc (3 × 20 mL). The combined organic layers
were washed with water (50 mL), brine (50 mL), dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. The residue was purified by
chromatography on silica gel gave compound 8 as a colorless liquid
(0.83 g, 92%). 1H NMR (500 MHz, CDCl3) δ (ppm): 7.43 (d, J = 10.0,
1H), 6.95 (s, 1H), 5.18–5.15 (m, 3H), 5.07 (d, J = 10.0 Hz, 1H),
5.02–4.92 (m, 2H), 4.83 (s, 1H), 3.63 (s, 3H), 3.46 (s, 3H), 2.10 (s, 3H),
1.68 (s, 3H), 1.51 (d, J = 5.0 Hz, 3H). ESI-MS m/z: 377.1 (M + Na)+.
4.1.4. Synthesis of 3-{1-[(tert-butyldimethylsilyl)oxy]ethyl}-2,6-bis
(methoxymethoxy) benzaldehyde (5)
A 2.5 M solution of n-BuLi in THF(9.4 mL, 22.8 mmol) was added
dropwise to a solution of {1-[2,4-bis(methoxymethoxy)phenyl]ethoxy}
(tert-butyl)dimethylsilane (4) (5.80 g, 16.3 mmol) in anhydrous THF
(30 mL) at ꢀ 78 ◦C under a nitrogen atmosphere. After 1 h, dry DMF (4.0
mL, 26.0 mmol) was added dropwise. The reaction solution was then
allowed to warmup to room temperature and stirring was continued for
2 h. The resulting solution was quenched with saturated aqueous NH4Cl
(20 mL) and extracted with EtOAc (3 × 20 mL). The combined organic
layers were washed with water (100 mL), brine (100 mL), dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was
purified by chromatography on silica gel gave compound 5 as a yellow
oil (3.60 g, 58.1%).1H NMR (500 MHz, CDCl3) δ (ppm): 10.46 (s, 1H),
7.73 (d, J = 10.0 Hz, 1H), 7.01 (d, J = 10.0 Hz, 1H), 5.28–5.25 (m, 3H),
5.11 (d, J = 5.0 Hz, 1H), 5.00 (d, J = 5.0 Hz, 1H), 3.58 (s, 3H), 3.52 (s,
3H), 1.37 (d, J = 5.0 Hz, 3H), 0.90 (s, 9H), 0.06 (s, 3H), ꢀ 0.03 (s, 3H).
ESI-MS m/z: 407.2 (M + Na)+.
4.1.8. Synthesis of 1-[3-acetyl-2,6-bis(methoxymethoxy)phenyl]-2-meth-
ylallyl acetate (9)
To a stirring solution of 1-[3-(1-hydroxyethyl)-2,6-bis(methoxyme-
thoxy)phenyl]-2-methylallyl acetate (8) (0.8 g, 2.34 mmol) in dry
CH2Cl2 (10 mL) at 0 ◦C was added Dess-Martin periodinane (3.0 g, 7.1
mmol) slowly. After 4 h, the mixture was taken up in saturated aqueous
NaHCO3 (10 mL) and the aqueous layer was extracted with EtOAc (3 ×
10 mL). The combined organic layers were washed with water (30 mL),
brine (30 mL), dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. The residue was purified by chromatography on silica gel gave
1
compound 9 as a colorless liquid (0.54 g, 63%). H NMR (500 MHz,
CDCl3) δ (ppm):7.55 (d, J = 10.0, 1H), 6.93 (d, J = 5.0 Hz, 1H), 6.85 (s,
1H), 5.21–5.18 (m, 2H), 5.00–4.92 (m, 3H), 4.84 (s, 1H), 3.50 (s, 3H),
3.46 (s, 3H), 2.57 (s, 3H), 2.09 (s, 3H), 1.71 (s, 3H). ESI-MS m/z: 375.1
(M + Na)+.
4.1.5. Synthesis of 1-{3-{1-[(tert-butyldimethylsilyl)oxy]ethyl}-2,6-bis
(methoxymethoxy)phenyl}-2-methylprop-2-en-1-ol (6)
To a stirred solution of 3-{1-[(tert-butyldimethylsilyl)oxy]ethyl}-2,6-
bis(methoxymethoxy) benzaldehyde (5) (2.30 g, 5.90 mmol) in anhy-
drous THF (20 mL) was added a 0.5 M solution of isopropenyl magne-
sium bromide in THF(13.0 mL,8.9 mmol) slowly at ꢀ 30 ◦C under a
nitrogen atmosphere. The reaction solution was then allowed to warmup
4.1.9. General procedure for synthesis of sanjuanolide derivatives S01-S18
To
a stirring solution of 1-[3-acetyl-2,6-bis(methoxymethoxy)
phenyl]-2-methylallyl acetate (9) (145 mg, 0.41 mmol) and various
substituted benzaldehydes (0.41 mmol) in EtOH (8 mL), KOH (69 mg,
9

G. Wang et al.
Bioorganic Chemistry 111 (2021) 104880
1.2 mmol) was added slowly. The reaction mixture was stirred at room
temperature for 16 h, then quenched with saturated aqueous NH4Cl
solution (10 mL) and extracted with EtOAc (3 × 10 mL). The combined
organic layers were concentrated under reduced pressure. The residue
was then dissolved in MeOH (6 mL), followed by 4 mol/L HCl dropwise,
MS m/z: 377.1 (M - H)-.
4.1.9.7. (E)-1-[2,4-Dihydroxy-3-(1-hydroxy-2-methylallyl)phenyl]-3-[4-
(4-methylpiperazin-1-yl)phenyl]prop-2-en-1-one (S07). Yellow powder,
1
◦
48.7% yield, m.p: 68.6–70.1 C. H NMR (500 MHz, CDCl3) δ (ppm):
7.54–7.64 (m, 1H), 7.23 (d, J = 8.5 Hz, 1H), 7.14 (d, J = 8.7 Hz, 1H),
6.86 (dd, J = 14.2, 5.7 Hz, 2H), 6.53 (s, 1H), 6.32 (d, J = 8.7 Hz, 1H),
5.52 (s, 1H), 4.76 (s, 3H), 3.20–3.30 (m, 6H), 2.62 (dd, J = 18.3, 12.8
Hz, 7H), 1.83 (s, 3H). 13C NMR (126 MHz, CDCl3) δ (ppm): 190.3, 163.2,
160.1, 146.5, 145.4, 131.8, 130.5, 130.2, 127.3, 125.1, 116.0, 114.8,
113.7, 109.3, 105.1, 70.1, 54.6, 46.9, 45.9, 19.6. ESI-MS m/z: 409.0 (M
+ H)+.
◦
and the reaction mixture was stirred for 1.5 h at 70 C. The resulting
mixture was quenched with saturated aqueous NH4Cl solution (10 mL)
and extracted with EtOAc (3 × 10 mL). The combined organic layers
were washed with water (30 mL), brine (30 mL), dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. The residue was purified by
chromatography on silica gel to give target compounds S01-S18.
4.1.9.1. (E)-1-[2,4-Dihydroxy-3-(1-hydroxy-2-methylallyl)phenyl]-3-phe-
nylprop-2-en-1-one (S01). Yellow powder, 46% yield, m.p:
147.3–150.1 ◦C. 1H NMR (500 MHz, CDCl3) δ (ppm): 13.6 (s, 1H), 7.86
(d, J = 15.0 Hz, 1H), 7.67–7.63 (m, 4H), 7.55 (d, J = 15.0 Hz, 1H),
7.42–7.41 (m, 3H), 6.56 (s, 1H), 6.36 (d, J = 5.0 Hz, 1H), 4.80 (s, 2H),
3.58–3.56 (m, 1H), 1.81 (s, 3H). 13C NMR (126 MHz, CDCl3) δ (ppm):
192.1, 160.1, 159.3, 144.3, 134.9, 130.6, 129.8, 128.9, 128.5, 120.4,
114.5, 112.8, 110.7, 107.4, 69.7, 29.3, 18.9. ESI-MS m/z: 309.3 (M - H)-.
4.1.9.8. (E)-1-[2,4-Dihydroxy-3-(1-hydroxy-2-methylallyl)phenyl]-3-(4-
morpholinophenyl) 1prop-2-en-1-one (S08). Yellow powder, 36.3% yield,
m.p:87.8–91.2 ◦C. H NMR (500 MHz, CDCl3) δ (ppm):14.2 (s, 1H), 7.87
(d, J = 15.4 Hz, 1H), 7.84 (d, J = 11.6 Hz, 1H), 7.58 (d, J = 8.6 Hz, 1H),
7.43 (d, J = 15.4 Hz, 1H), 6.91 (d, J = 8.6 Hz, 2H), 6.73 (d, J = 9.0 Hz,
1H), 5.63 (s, 1H), 5.30–5.25 (m, 2H), 5.01 (s, 1H), 4.91 (s, 1H), 3.87 (s,
4H), 3.48 (s, 3H), 3.29 (s, 4H), 1.79 (s, 3H). 13C NMR (500 MHz, CDCl3)
δ (ppm): 192.6, 163.3, 160.2, 152.9, 146.5, 145.3, 130.4, 125.6, 118.2,
116.3, 115.4, 114.6, 109.3, 105.1, 94.2, 77.3, 77.0, 76.8, 66.6, 56.5,
47.9, 19.6. ESI-MS m/z: 418.4 (M + Na)+.
4.1.9.2. (E)-1(4-Bromophenyl)-1-[2,4-dihydroxy-3-(1-hydroxy-2-methyl-
allyl)phenyl]prop-2-en-1-one (S02). Yellow powder, 43.2% yield, m.p:
195.6–198.3 ◦C. 1H NMR (500 MHz, CDCl3) δ (ppm): 13.50 (s, 1H), 7.79
(d, J = 15.0 Hz, 1H), 7.65 (d, J = 9.0 Hz, 1H), 7.56–7.49 (m, 5H), 6.56 (s,
1H), 6.37 (d, J = 9.0 Hz, 1H), 4.78 (s, 2H), 1.82 (s, 3H). 13C NMR (126
MHz, CDCl3) δ (ppm): 191.7, 160.1, 159.4, 142.8, 133.8, 132.2, 129.8,
128.6, 124.9, 121.0, 114.4, 112.7, 110.8, 107.5, 69.7, 28.5, 18.9. ESI-
MS m/z: 387.2 (M - H)-.
4.1.9.9. (E)-1-[2,4-Dihydroxy-3-(1-hydroxy-2-methylallyl)phenyl]-3-(2-
methoxyphenyl)prop-2-en-1-one (S09). Yellow liquid, 38.2% 1H NMR
(400 MHz, CDCl3) δ (ppm): 13.73 (s, 1H), 8.19 (d, J = 15.6 Hz, 1H), 7.81
(d, J = 8.82 Hz, 1H), 7.71 (d, J = 15.6 Hz, 1H), 7.63 (d, J = 6.08 Hz, 1H),
7.39 (t, J = 7.48 Hz, 1H), 7.00 (t, J = 7.48 Hz, 1H), 6.95 (d, J = 8.32 Hz,
1H), 6.54 (d, J = 8.82 Hz, 1H), 6.29 (s, 1H), 4.07 (s, 2H), 3.93 (s, 3H),
1.67 (s, 3H). 13C NMR (101 MHz, CDCl3) δ (ppm): 192.7, 163.8, 159.3,
158.9, 141.9, 140.1, 131.9, 130.6, 129.5, 123.8, 121.0, 120.8, 115.9,
113.9, 111.9, 111.3, 107.0, 58.3, 55.6, 15.9. ESI-MS m/z: 338.4 (M - H)-.
4.1.9.3. (E)-1-[2,4-Dihydroxy-3-(1-hydroxy-2-methylallyl)phenyl]-3-(4-
nitrophenyl)prop-2-en-1-one (S03). Yellow powder, 38.4% yield, m.p:
151.3–153.7 ◦C. 1H NMR (500 MHz, CDCl3) δ (ppm): 13.69 (s, 1H), 9.25
(s, 1H), 8.29 (d, J = 10.0 Hz, 2H), 7.87 (d, J = 15.0 Hz, 1H), 7.80–7.78
(m, 3H), 7.70 (d, J = 15.0 Hz, 1H), 6.51 (d, J = 5.0 Hz, 1H), 5.92 (s, 1H),
5.16 (s, 1H), 5.00 (s, 1H), 1.82 (s, 3H). 13C NMR (126 MHz, CDCl3) δ
(ppm): 191.1, 164.3, 163.6, 144.3, 141.0, 140.9, 137.2, 130.9, 128.9,
124.3, 113.2, 113.1, 112.6, 109.7, 104.3, 72.7, 18.4. ESI-MS m/z: 354.1
(M - H)-.
4.1.9.10. (E)-1-[2,4-Dihydroxy-3-(1-hydroxy-2-methylallyl)phenyl]-3-(3-
methoxyphenyl)prop-2-en-1-one (S10). Yellow liquid, 36.9% 1H NMR
(400 MHz, CDCl3) δ (ppm): 13.73 (s, 1H), 7.87 (d, J = 15.4 Hz, 1H), 7.70
(d, J = 8.88 Hz, 1H), 7.63 (d, J = 8.68 Hz, 2H), 7.47(d, J = 15.4 Hz, 1H),
6.97 (d, J = 8.68 Hz, 2H), 6.60 (s, 1H), 6.39 (d, J = 8.88 Hz, 1H), 4.80 (s,
2H), 4.11 (s, 3H), 1.85 (s, 3H). 13C NMR (101 MHz, CDCl3) δ (ppm):
192.1, 163.9, 163.3, 160.0, 144.9, 144.4, 130.5, 130.0, 121.2, 120.7,
120.4, 120.3, 116.6, 116.4, 115.8, 1137, 107.2, 103.1, 102.8, 55.4,
29.7, 16.6. ESI-MS m/z: 338.4 (M - H)-.
4.1.9.4. (E)-1-[2,4-Dihydroxy-3-(1-hydroxy-2-methylallyl)phenyl)-3-[2-
(trifluoromethyl)phenyl]prop-2-en-1-one (S04). Yellow powder, 35.3%
yield, m.p: 123.7–126.5 ◦C. 1H NMR (500 MHz, CDCl3) δ (ppm): 13.43
(s, 1H), 7.87 (d, J = 20.0 Hz, 2H), 7.80 (s, 1H), 7.68 (d, J = 20.0 Hz, 2H),
7.59–7.54 (m, 2H), 6.57 (s, 1H), 6.39 (d, J = 10.0 Hz, 1H), 4.79 (s, 2H),
1.83 (s, 3H). 13C NMR (126 MHz, CDCl3) δ (ppm): 199.1, 158.8, 155.4,
146.7, 143.4, 132.1, 131.5, 130.6, 127.9, 127.4, 125.3, 110.1, 109.3,
105.3, 105.0, 101.8, 94.4, 69.9, 19.9. ESI-MS m/z: 377.1 (M - H)-.
4.1.9.11. (E)-1-[2,4-Dihydroxy-3-(1-hydroxy-2-methylallyl)phenyl]-3-(4-
methoxyphenyl)prop-2-en-1-one (S11). Yellow liquid, 39.4%. 1H NMR
(400 MHz, CDCl3) δ (ppm): 13.89 (s, 1H), 7.90 (d, J = 15.4 Hz, 1H), 7.85
(d, J = 8.96 Hz, 1H), 7.64 (d, J = 8.56 Hz, 2H), 7.52 (d, J = 15.4 Hz, 1H),
6.98 (d, J = 8.56 Hz, 2H), 6.58 (d, J = 8.96 Hz, 1H), 6.32 (s, 1H), 4.10 (s,
2H), 3.89 (s, 3H), 1.71 (s, 3H). 13C NMR (101 MHz, CDCl3) δ (ppm):
192.1, 163.8, 161.8, 159.3, 144.35, 141.9, 130.4, 127.6, 117.8, 115.9,
114.5, 113.9, 111.9, 107.1, 58.4, 55.5, 15.9. ESI-MS m/z: 338.3 (M - H)-.
4.1.9.5. (E)-1-[2,4-Dihydroxy-3-(1-hydroxy-2-methylallyl)phenyl]-3-[3-
(trifluoromethyl) phenyl]prop-2-en-1-one (S05). Yellow powder, 42.6%
yield, m.p: 127.6–131.3 ◦C. 1H NMR (500 MHz, CDCl3) δ (ppm): 13.43
(s, 1H), 7.86–7.82 (m, 3H), 7.65 (d, J = 5.0 Hz, 1H), 7.59–7.54 (m, 3H),
6.54 (s, 1H), 6.36 (d, J = 5.0 Hz, 1H), 4.76 (s, 2H), 3.56 (s, 1H), 1.80 (s,
3H). 13C NMR (126 MHz, CDCl3) δ (ppm): 191.4, 160.1, 159.5, 142.2,
135.7, 131.7, 129.9, 129.5, 128.7, 126.8, 124.7, 124.6, 122.2, 114.4,
112.6, 110.7, 107.6, 107.4, 69.7, 46.4, 18.9. ESI-MS m/z: 377.1 (M - H)-.
4.1.9.12. (E)-1-[2,4-Dihydroxy-3-(1-hydroxy-2-methylallyl)phenyl]-3-
(2,3-dimethoxyphenyl) prop-2-en-1-one (S12). Yellow powder, 43.6%
yield, m.p: 135.2–138.9 ◦C. 1H NMR (500 MHz, CDCl3) δ (ppm): 13.59
(s, 1H), 8.15 (d, J = 20.0 Hz, 1H), 7.66–7.62 (m, 2H), 7.09 (d, J = 10.0
Hz, 1H), 6.98 (d, J = 5.0 Hz, 1H), 6.57 (s, 1H), 6.35 (d, J = 10.0 Hz, 1H),
4.76 (s, 2H), 3.90 (s, 6H), 1.81 (s, 3H). 13C NMR (126 MHz, CDCl3) δ
(ppm): 192.4, 160.1, 159.2, 153.3, 149.2, 139.2, 129.8, 129.0, 124.2,
121.9, 119.9, 114.4, 112.8, 110.7, 107.4, 69.7, 61.3, 55.9, 18.9. ESI-MS
m/z: 371.2 (M + H)+.
4.1.9.6. (E)-1-[2,4-Dihydroxy-3-(1-hydroxy-2-methylallyl)phenyl]-3-(4-
(trifluoromethyl) phenyl)prop-2-en-1-one (S06). Yellow powder, 44.1%
yield, m.p: 178.8–181.6 ◦C. 1H NMR (500 MHz, CDCl3) δ (ppm): 13.42
(s, 1H), 7.86 (d, J = 15.0 Hz, 1H), 7.74 (d, J = 10.0 Hz, 2H), 7.69–7.63
(m, 4H), 6.57 (s, 1H), 6.38 (s, 1H), 4.79 (s, 2H), 1.83 (s, 3H). 13C NMR
(126 MHz, CDCl3) δ (ppm): 191.2, 160.2, 159.6, 142.1, 138.3, 136.7,
129.8, 128.5, 125.9, 125.9, 114.4, 112.7, 110.8, 107.6, 69.8, 18.9. ESI-
4.1.9.13. (E)-1-[2,4-Dihydroxy-3-(1-hydroxy-2-methylallyl)phenyl]-3-
(3,4-dimethoxyphenyl) prop-2-en-1-one (S13). Yellow powder, 42.5%
10

G. Wang et al.
Bioorganic Chemistry 111 (2021) 104880
yield, m.p: 169.3–173.6 ◦C. 1H NMR (500 MHz, CDCl3) δ (ppm): 13.68
(s, 1H), 7.83 (d, J = 15.0 Hz, 1H), 7.69 (d, J = 10.0 Hz, 1H), 7.42 (d, J =
15.0 Hz, 1H), 7.24 (d, J = 10.0 Hz, 1H), 7.15 (s, 1H), 6.90 (d, J = 10.0
Hz, 1H), 6.57 (s, 1H), 6.37 (d, J = 5.08 Hz, 1H), 4.77 (s, 2H), 3.96 (s,
3H), 3.94 (s, 3H), 1.82 (s, 3H). 13C NMR (126 MHz, CDCl3) δ (ppm):
191.9, 160.1, 159.1, 151.6, 149.4, 144.4, 129.7, 128.5, 127.9, 123.3,
118.1, 114.6, 112.83, 111.2, 110.3, 107.2, 69.7, 56.0 (d), 19.0. ESI-MS
m/z: 371.0 (M + H)+.
Center of Peking Union Medical College and were cultured at 37 ◦C
under 5% CO2 in DMEM (Dulbecco’s Modified Eagle Medium) supple-
mented with 10% fetal calf serum (PPA) and 100 U/mL penicillin and
streptomycin (HyClone™).
4.3. Cell viability assay
The cell viability was assessed with the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay as described previously
[30]. Cells (4 × 103 cells/well in 96-well plates) were incubated at 37 ◦C
with or without S07 treatment for 48 h, after which MTT (0.5 mg/mL)
4.1.9.14. (E)-1-[2,4-Dihydroxy-3-(1-hydroxy-2-methylallyl)phenyl]-3-
(2,4-dimethoxyphenyl) prop-2-en-1-one (S14). Yellow powder, 45.7%
yield, m.p:1776.7–180.5 ◦C. 1H NMR (500 MHz, CDCl3) δ (ppm): 8.12
(d, J = 15.0 Hz, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.58 (d, J = 15.0 Hz, 1H),
7.57 (d,J = 10.0 Hz, 1H), 6.58–6.52 (m, 2H), 6.48 (s, 1H), 6.36 (d, J =
was added at 20
μL/well for another 4 h. The reaction product, for-
mazan, was dissolved in 100
μ
L of DMSO after discarding the culture
medium. Cell viability was determined by reading the absorbance at
560 nm by a spectrophotometer (DTX880, Beckman Coulter, CA, USA).
10.0 Hz, 1H), 4.76 (s, 2H), 3.92 (s, 3H), 3.87 (s, 3H), 1.82 (s, 3H). 13
C
NMR (126 MHz, CDCl3) δ (ppm): 192.7, 163.6, 163.2, 160.6, 142.5,
140.0, 131.2, 130.8, 118.5, 117.2, 113.5, 113.3, 1113, 108.6, 105.5,
98.6, 79.9, 77.3, 77.0, 76.8, 65.4, 55.6, 55.5, 18.5. ESI-MS m/z: 371.2
(M + H)+.
4.4. Long-term proliferation studies
PC3 and DU145 cells (5.0 × 105) were seeded in a 10 cm2 dish. After
6 h, S07 was added to a final concentration of 4.0, 8.0 or 16.0 µM. The
medium was changed every three days until confluency was reached
[31].
4.1.9.15. (E)-1-[2,4-Dihydroxy-3-(1-hydroxy-2-methylallyl)phenyl]-3-
(2,5-dimethylphenyl) prop-2-en-1-one (S15). Yellow powder, 49.6%
yield, m.p: 127.6–130.3 ◦C. 1H NMR (500 MHz, CDCl3) δ (ppm): 13.61
(s, 1H), 8.16 (d, J = 15.0 Hz, 1H), 7.70 (d, J = 10.0 Hz, 1H), 7.50–7.46
(m, 2H), 7.13 (s, 2H), 6.58 (s, 1H), 6.38 (d, J = 5.0 Hz, 1H), 5.35–5.30
(m, 1H), 4.78 (s, 2H), 2.45 (s, 3H), 2.37 (s, 3H), 1.83(s, 3H). 13C NMR
(126 MHz, CDCl3) δ (ppm): 191.1, 159.1, 158.2, 141.1, 134.8, 134.5,
132.6, 130.3, 129.9, 128.9, 127.4, 125.9, 120.1, 113.5, 111.8, 109.7,
106.4, 68.7, 19.9, 18.3), 17.9. ESI-MS m/z: 338.3 (M - H)-.
4.5. Western blot
Cells were lysed and boiled for 10 min. Proteins were separated by
SDS-PAGE, transferred to a PVDF membrane and detected with relevant
antibodies against cleaved-PARP (CST), caspase-3 (CST), cleaved-
caspase-3 (CST), γ-H2AX (CST) or GAPDH (CST).
4.6. Cell cycle distribution analysis
4.1.9.16. (E)-1-[2,4-Dihydroxy-3-(1-hydroxy-2-methylallyl)phenyl]-3-(4-
hydroxy-3-methoxy phenyl)prop-2-en-1-one (S16). Yellow powder,
32.4% yield, m.p: 143.6–147.5 ◦C. 1H NMR (500 MHz, CDCl3) δ (ppm):
14.02 (s, 1H), 7.87–7.84 (s, 2H), 7.45 (d, J = 15.0 Hz, 1H), 7.25–7.13
(m, 3H), 6.75 (d, J = 10.0 Hz, 2H), 5.64 (s, 1H), 5.50 (s, 1H), 5.28 (s,
2H), 5.01 (s, 1H), 4.92 (s, 1H), 3.94 (s, 3H), 1.79 (s, 3H). 13C NMR (126
MHz, CDCl3) δ (ppm): 192.6, 163.3, 160.3, 150.3, 149.2, 146.4, 145.3,
130.4, 128.8, 122.9, 118.2, 117.0, 115.3, 112.0, 109.4, 105.2, 97.2,
94.2, 62.2, 56.6, 19.6. ESI-MS m/z: 357.2 (M + H)+.
Cells were cultured in the absence or presence of 0, 4.0, 8.0 or 16.0
μ
M of S07 for 2 days or 7 days. Cells were trypsinized, washed and
stained with propidium iodide before the cell cycle distribution was
assessed on a flow cytometer (BD (Becton Dickinson) FACS Calibur, BD
Biosciences).
4.7. Annexin V/PI apoptosis assay
Cells were seeded in 6 cm2 dishes at a density of 3.0 × 105 cells per
dish and incubated at 37 ◦C for 6 h until cells attached to the dish. S07
was added to the medium at a final concentration of 0, 4.0, 8.0 or 16.0
4.1.9.17. (E)-1-[2,4-Dihydroxy-3-(1-hydroxy-2-methylallyl)phenyl]-3-
(2,4,6-trimethoxyphenyl) prop-2-en-1-one (S17). Yellow powder, 44.2%
yield, m.p: 116.4–118.3 ◦C. 1H NMR (500 MHz, CDCl3) δ (ppm):14.04 (s,
1H), 8.32 (d, J = 15.0 Hz, 1H), 7.91 (d, J = 15.0 Hz, 1H), 7.65 (d, J = 5.0
Hz, 1H), 6.58 (s, 1H), 6.34 (d, J = 10.0 Hz, 1H), 6.13 (s, 2H), 4.75 (s,
2H), 3.92 (s, 6H), 3.86 (s, 3H), 1.81 (s, 3H). 13C NMR (126 MHz, CDCl3)
δ (ppm): 193.8, 163.4, 161.9 (d), 160.00, 158.5, 135.6, 129.7, 128.2,
119.8, 115.0, 113.1, 110.6, 106.9, 106.7, 90.6 (d), 69.6, 55.9 (d), 55.4,
18.9. ESI-MS m/z: 401.3 (M + H)+.
μ
M. After 48 h or 7 days, cells were harvested for Annexin V/PI
apoptosis assay. The assay was performed following the protocol pro-
vided by the Annexin V/PI apoptosis Kit (Sigma). The cells were assessed
on a flow cytometer (BD FACSCalibur, BD Biosciences).
4.8. Senescence-associated beta-galactosidase (SA-β gal) staining asssay
A beta-galactosidase (SA-β gal) staining kit was obtained from Sigma.
The assay was performed following the manufacturer’s instructions. In
brief, cells were washed once with PBS and fixed with stationary liquid
provided in the kit at room temperature for 15 min. Next, the cells were
incubated overnight at 37 ◦C in the dark with 1 mL of a working solution
containing 0.05 mg/ml of 5-bromo-4-chloro-3-indolyl-b-d-galactopyr-
anoside (X-gal). Subsequently, the cells were observed under a normal
light microscope (Nikon, Japan).
4.1.9.18. (E)-1-[2,4-Dihydroxy-3-(1-hydroxy-2-methylallyl)phenyl]-3-
(3,4,5-trimethoxyphenyl) prop-2-en-1-one (S18). Yellow powder, 47.5%
yield, m.p: 142.8–145.1 ◦C. 1H NMR (500 MHz, CDCl3) δ (ppm): 13.60
(s, 1H), 7.78 (d, J = 15.0 Hz, 1H), 7.67 (d, J = 10.0 Hz, 1H), 7.45–7.43
(m, 1H), 6.86 (s, 2H), 6.57 (s, 1H), 6.41 (d, J = 10.0 Hz, 1H), 4.77 (s,
2H), 3.93 (s, 6H), 3.90 (s, 3H), 1.82 (s, 3H). 13C NMR (126 MHz, CDCl3)
δ (ppm): 191.9, 160.1, 159.2, 153.5 (d), 144.4, 140.7, 130.3, 129.7,
128.5, 119.6, 114.5, 112.8, 110.8, 107.3, 105.9 (d), 69.7, 61.0, 56.3 (d),
18.9. ESI-MS m/z: 399.1 (M - H)-.
4.9. Immunofluorescence (IF) assay of DNA damage
4.2. Cell culture
Immunofluorescence (IF) assays were performed as previously
described [31]. Briefly, cells on a coverslip were fixed with 4% para-
formaldehyde and permeabilized in 0.5% Triton X-100 (in 1 × PBS).
Cells were incubated overnight at 4 ◦C with primary antibodies against
53BP1 (CST), washed three times and incubated with secondary
Castration-resistant Prostate Cancer (CRPC) PC3 and DU145 cells,
human normal hepatic cells (MIHA) and human normal prostatic stro-
mal myofibroblast cells (WPMY-1) were obtained from the Cell Resource
11

G. Wang et al.
Bioorganic Chemistry 111 (2021) 104880
antibodies (DyLight 488-conjugated anti-mouse). The coverslip was
washed with PBS about three times. The cells were washed and mounted
with DAPI. Fluorescence was detected and imaged using a Nikon Ti
microscope. To quantify the degree of DNA damage, > 200 cells from
each group were randomly chosen from three independent experiments.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
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