Novel 3′,5′-diprenylated chalcones inhibited the proliferation of cancer cells in?vitro by inducing cell apoptosis and arresting cell cycle phase

Article abstract of DOI:10.1016/j.ejmech.2017.03.077

A double Claisen rearrangements synthetic strategy was established for the total synthesis of 4,4′-dimethyl medicagenin (compound 6c). A series of its analogs also were prepared, including two novel 3′,5′-diprenylated chalcones, in which ring B was replaced by azaheterocycle. The structures of the twenty-two newly synthesized compounds were confirmed by ¹H NMR, ¹³C NMR and ESI-MS. In?vitro, the cytotoxicity of the target compounds was evaluated using cancer cells. Noticeably, compound 10 exhibited broad-spectrum cytotoxicity on PC3 prostate cancer cells, MDA-MB-231 breast cancer cells (MDA), HEL and K562 erythroleukemia cells with IC₅₀ values of 2.92, 3.14, 1.85 and 2.64?μM, respectively. Further studies indicated that compound 10 induced apoptosis and arrested the cell cycle phase of the above mentioned four cancer cell lines. By contrast, compound 6g selectively displayed potent inhibitory activity against the proliferation of HEL cells with an IC₅₀ value of 4.35?μM. Compound 6g slightly induced apoptosis and arrested cell cycle phase of HEL cells. Preliminary structure-activity relationship studies indicated that, in all cancer cell lines evaluated, the 3-pyridinyl group was essential for cytotoxicity.

Full text of DOI:10.1016/j.ejmech.2017.03.077

Accepted Manuscript
Novel 3′,5′-diprenylated chalcones inhibited the proliferation of cancer cells in vitro by
inducing cell apoptosis and arresting cell cycle phase
Zhonghang Wen, Yongqiang Zhang, Xinghui Wang, Xiaoping Zeng, Zhanxing Hu, Yi
Liu, Yuxin Xie, Guangyi Liang, Jianguo Zhu, Heng Luo, Bixue Xu
PII:
S0223-5234(17)30245-3
10.1016/j.ejmech.2017.03.077
EJMECH 9337
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 December 2016
Revised Date: 3 March 2017
Accepted Date: 30 March 2017
Please cite this article as: Z. Wen, Y. Zhang, X. Wang, X. Zeng, Z. Hu, Y. Liu, Y. Xie, G. Liang, J. Zhu,
H. Luo, B. Xu, Novel 3′,5′-diprenylated chalcones inhibited the proliferation of cancer cells in vitro by
inducing cell apoptosis and arresting cell cycle phase, European Journal of Medicinal Chemistry (2017),
doi: 10.1016/j.ejmech.2017.03.077.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel 3',5'-diprenylated chalcones inhibited the proliferation of
cancer cells in vitro by inducing cell apoptosis and arresting cell cycle
phase
Zhonghang Wen a,b,c#, Yongqiang Zhang a,b,c#, Xinghui Wang a,b, Xiaoping Zeng a,b, Zhanxing Hu a,b,
Yi Liu a,b,c, Yuxin Xie d, Guangyi Liang a,b, Jianguo Zhu e*, Heng Luo a,b*, Bixue Xu a,b*
a State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang
550014, P.R. China;
b Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences,
Guiyang 550014, P.R. China;
c School of Pharmaceutical Sciences, Guizhou University, Guiyang 550025, P.R. China;
d School of Pharmaceutical Sciences, Guizhou Medical University, Guiyang 550025, P.R. China;
e Guizhou Provincial People’s Hospital, Guiyang 550002, P.R. China.
Abstract
A double Claisen rearrangements synthetic strategy was established for the total synthesis of
4,4'-dimethyl medicagenin (compound 6c). A series of its analogs also were prepared, including two
novel 3',5'-diprenylated chalcones, in which ring B was replaced by azaheterocycle. The structures
13
of the twenty-two newly synthesized compounds were confirmed by 1H NMR, C NMR and ESI-
MS. In vitro, the cytotoxicity of the target compounds was evaluated using cancer cells. Noticeably,
compound 10 exhibited broad-spectrum cytotoxicity on PC3 prostate cancer cells, MDA-MB-231
breast cancer cells (MDA), HEL and K562 erythroleukemia cells with IC50 values of 2.92, 3.14,
1.85 and 2.64 µM, respectively. Further studies indicated that compound 10 induced apoptosis and
arrested the cell cycle phase of the above mentioned four cancer cell lines. By contrast, compound
6g selectively displayed potent inhibitory activity against the proliferation of HEL cells with an IC50
value of 4.35 µM. Compound 6g slightly induced apoptosis and arrested cell cycle phase of HEL
cells. Preliminary structure-activity relationship studies indicated that, in all cancer cell lines
evaluated, the 3-pyridinyl group was essential for cytotoxicity.
1 Introduction
Cancer is the second leading cause of morbidity and mortality worldwide [1]. The use of
conventional chemotherapy is the primary treatment approach for curing cancer. Despite enormous
efforts in developing better leads and novel chemotherapeutic strategies, the lack of selectivity and
development of drug-resistance diminishes the efficacy of cancer chemotherapy [2]. Therefore, the
search and development for improved treatment strategies and more effective anti-cancer agents
Corresponding authors. Tel.: +86 851-83807713; E-mail: bixue_xu@126.com (B.X. Xu); Tel.: +86 851-83876210; E-mail:
luohengzz@sina.com (H. Luo); Tel.: +86 851-83876458; E-mail: doctorzhujianguo@163.com (J.G. Zhu)
# Both authors contributed equally to this work.
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remains an essential task.
Prenylated chalcones have drawn a great deal of attention owing to their unique structure and
extensive bioactivities. Various prenylated chalcones have been investigated as anti-malarial [3],
anti-inflammatory [4], anti-bacterial [5], anti-diabetic [6] and anti-cancer [7] agents. Natural
prenylated chalcones under the current study have xanthohumol [8], broussochalcone A [9],
spinochalcone A [10], 4,4'-dimethyl medicagenin [11], etc. Xanthohumol and its analogs have been
hot topics in the field of anti-cancer research [12-20], broussochalcone A enhanced cytotoxicity of
mitomycin C in treatment of MCF-7 cells may be associated with the quinone reductase-inducing
potential of broussochalcone A [21], spinochalcone A showed good anti-proliferative activity
against DU-145 cells, and high selectivity compared with normal cells [22]. However, few reports
have been published on the biological activities of 4,4'-dimethyl medicagenin (6c), and there is a
lack of diversity about the structural modification of its ring B. Therefore it seems to be important
to develop synthetic routes of 4,4'-dimethyl medicagenin and its analogs for elucidation of the
relation between structures and promising activities. In this paper, we report on the design and
synthesis of a series of prenylated chalcones as analogs of 4,4'-dimethyl medicagenin, comprising
of two novel 3',5'-diprenylated chalcones, in which ring B was replaced by azaheterocycle. The
inhibitory activity was assessed in vitro using four cancer cell lines, including PC3, MDA-MB-231
(MDA), HEL and K562 cells.
Figure 1. Chemical structure of four natural prenylated chalcones. Xanthohumol was isolated from Humulus
lupulus L., broussochalcone A was isolated from Broussonetia papyrifera, spinochalcone A was isolated from
Tephrosia spinosa, and 4,4'-dimethyl medicagenin was isolated from sophora subprostrata Chun et T. Chen.
2 Results and discussion
2.1 Chemistry
Several methods have been reported for the preparation of 3',5'-diprenylated chalcones, all
showing as a synthetic challenge for the formation of the double C-prenyl side-chains. Sugamoto et
al [23]. carried out the double prenylation of the C-3 and C-5 of 2'-hydroxy-4'-
methoxyacetophenone
by
using
Montmorillonite
K10
catalyzed
4'-methoxy-2'-
prenoxyacetophenone via [1,3]-sigmatropic rearrangement, only two steps it taken, however, the
yield was only 7%. Narender et al [3]. accomplished the total synthesis of Medicagenin, 2',4'-
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dihydroxy-3',5'-C-diprenylacetophenone as the key intermediate was obtained by reacting 2',4'-
dihydroxyacetophenone with 2-methyl-but-3-en-2-ol in the presence of BF3·OEt2 in low yield
(21%), and this synthetic condition was not easy to control. Wang et al [7]. also finished the total
synthesis of Medicagenin in 8 steps and 8.5% overall yield, the formation of the double C-prenyl
side-chains of chalcone by using Florisil® catalyzed prenyl phenyl ethers, however, the yield was
very low (2%). Therefore, the challenging synthetic route and accompanying poor yield limited this
approach for further use.
To optimize the synthetic route as well as to improve the yield of the total synthesis of 3',5'-
diprenylated chalcones, we developed an efficient method as showed in Scheme 1, Lindlar catalytic
hydrogenation [24], double Claisen rearrangements and Aldol condensation are the key steps in this
route, the most important intermediate 2'-hydroxy-4'-methoxy-3',5'-diprenylacetophenone (5) was
achieved in 5 steps and 24% overall yield. Currently, this approach has not been described in other
studies.
Twenty-one 3',5'-diprenylated chalcones were synthesized through the route described in
Scheme 1. Treating the starting material 1 with 3-chloro-3-methylbutyne at -5 ºC in the presence of
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and CuCl2 provided compound 2 in 87% yield [25].
Reduction of 2 by Lindlar catalytic hydrogenation and subsequently Claisen rearrangement at 120
ºC in N,N-diethylaniline afforded compound 3 in 71% yield over two steps [24]. The compound 5
was synthesized by prenylation of 3 and followed by Claisen rearrangement at 170 ºC in N,N-
diethylaniline. Aldol coupling [26] compound 5 with different derivatives of benzaldehyde afforded
the corresponding 3',5'-diprenylated chalcones (6a-6k) in low to good yields (12-60%). The
deprotection of compounds 6a and 6b were performed by using two methods [27,28], treatment of
6a with diluted hydrochloric acid (3 M) in methanol at 60 ºC to give 7a in 58% yield, the reaction
of 6b with p-toluene sulfonic acid monohydrate (p-TsOH.H2O) in methanol at room temperature
provided 7b in better yield (84%) than former. Different substituents were introduced to the 4-
hydroxyl of 7a or 7b to afford the corresponding compounds (8a-8h) in low to good yields (14-
94%) by using Kim’s method [29]. As shown in Scheme 2, two novel 3',5'-diprenylated chalcones
(9, 10), in which ring B was replaced by azaheterocycle were synthesized by Aldol coupling
between compound 5 and the N-methylpyrrole-2-carboxaldehyde or 3-pyridinecarboxaldehyde,
respectively. In addition, we have tried to replace ring B of 4,4'-dimethyl medicagenin with a
variety of nitrogen heterocycles by treating compound 5 with a variety of aromatic aldehydes,
which contain nitrogen atoms, such as, 2-pyrrolecarbaldehyde, imidazole-4-carboxaldehyde, 5-
methoxyindole-3-carboxaldehyde, 2-pyridinecarboxaldehyde, 4-pyridinecarboxaldehyde, 4-
quinolinecarboxaldehyde, etc. Unfortunately, we failed to get the desired compounds.
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Scheme 1. Synthetic route of 3',5'-diprenylated chalcones. Reagents and conditions: (i) CuCl2, DBU, acetonitrile,
3-chloro-3-methylbutyne, -5 ºC, 11 h; (ii) Lindlar catalyst(Pd/CaCO3), H2, ethyl acetate, RT, 12 h; (iii) N,N-
diethylaniline, 120 ºC, 45 h; (iv) K2CO3, DMF, prenyl bromine, 50 ºC, 30 h; (v) N,N-diethylaniline, 170 ºC, 24 h;
(vi) benzaldehyde derivatives, NaOH, ethanol, RT; (vii) HCl (3M), methanol, 60 ºC, 1 h (7a); TsOH.H2O,
methanol, RT, 15 h (7b); (viii) R4OH, DMAP, EDCI, CH2Cl2, 0 ºC-RT (8a-8h).
Scheme 2. Synthesis of target compound 9 and 10. Reagents and conditions: (i) NaOH, ethanol, RT, 37 h; (ii)
NaOH, ethanol, RT, 35 h.
2.2 In vitro cytotoxicity assay
The cytotoxic activities of the synthesized 3', 5'-diprenylated chalcones were evaluated using
four cell lines, including PC3, MDA, HEL and K562 cells. We used the MTT (3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyltetrazolium bromide) assay to determine the cell viability after treatment
with 5 µmol/L of compound (Figure 2 and Table 1). The result indicated that compound 6g only
displayed a potent inhibitory effect on the proliferation of HEL cells at a relatively low
concentration. By contrast, compound 10 exhibited broad-spectrum cytotoxic effects on all cancer
cells evaluated.
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Figure 2. Inhibition of 3',5'-diprenylated chalcones on the proliferation of cancer cells in vitro. Cancer cells lines
PC3, MDA, HEL and K562 were incubated with chalcones at a concentration of 5 µmol/L and the effect on cell
proliferation was investigated. Data are presented as mean ± SD of three independent experiments.
Table 1
The structures and inhibition after treating cancer cells with 5 µmol/L of target compounds respectively. Data are
presented as the mean ± SD of three independent experiments.
Inhibition (%)
Compd
Ar
PC3
MDA
HEL
PC3
17.3 ± 1.7
14.0 ± 0.4
NAa
3.9 ± 0.3
6a
6b
6c
6d
13.1 ± 2.1
24.6 ± 1.3
10.6 ± 0.9
29.9 ± 4.1
14.5 ± 1.1
19.0 ± 1.7
6.3 ± 0.7
29.5 ± 3.2
NA
NA
32.0 ± 0.7
7.5 ± 0.6
15.0 ± 1.8
13.4 ± 2.2
26.2 ± 8.3
27.7 ± 1.6
23.6 ± 5.7
NA
NA
NA
6e
6f
21.2 ± 3.6
11.6 ± 2.8
NA
23.4 ± 2.1
21.5 ± 5.3
12.1 ± 2.9
26.5 ± 1.9
85.4 ± 4.4
NA
23.0 ± 5.8
NA
6g
6h
6i
12.3 ± 1.7
11.7 ± 4.4
19.3 ± 3.6
31.9 ± 2.0
31.8 ± 2.0
6j
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Inhibition (%)
Compd
Ar
PC3
MDA
HEL
PC3
20.9 ± 0.4
30.2 ± 1.4
NA
18.9 ± 5.4
6k
7a
7b
26.0 ± 2.2
25.6 ± 1.9
19.3 ± 1.6
33.5 ± 5.7
22.2 ± 3.9
12.7 ± 5.4
NA
10.2 ± 3.6
23.0 ± 1.2
36.5 ± 1.0
19.2 ± 6.9
29.9 ± 4.1
12.7 ± 2.3
21.4 ± 5.3
34.3 ± 1.0
13.5 ± 1.8
NA
38.5 ± 2.4
19.6 ± 1.8
28.3 ± 7.5
NA
5.7 ± 2.0
NA
NA
8.1 ± 1.0
6.3 ± 1.4
NA
8a
8b
8c
8d
8e
8f
NA
OMe
N
NA
O
O
NA
NA
7.4 ± 2.7
NA
4.0 ± 1.2
NA
NA
NA
8g
8h
NA
NA
NA
11.7 ± 1.3
90.4 ± 3.2
89.7 ± 5.4
33.8 ± 1.3
83.4 ± 5.4
87.1 ± 4.2
NA
9.9 ± 1.8
86.2 ± 5.6
90.6 ± 3.8
9
91.7 ± 2.7
89.2 ± 4.7
10
doxorubicinb
a No activity; b Used as a positive contro1.
The relation between concentration and inhibition was analyzed to calculate the IC50 values of
selected compounds 6g and 10 (Figure 3A). The results indicated a dose-dependent trend of the
inhibitory response of compound 6g on HEL cells and compound 10 on PC3, MDA, HEL and K562
cells. The IC50 values are summarized in Table 2 and showed that the cytotoxicity of compound 10
on all cancer cells was close to that of the positive control (doxorubicin). In contrast, the
cytotoxicity of compound 6g on HEL cells was lower than that of doxorubicin (P < 0.05).
Microscopical observation indicated morphological changes of the cancer cells (Figure 3B-E), we
found the numbers of HEL cells (Figure 3B) were significantly reduced after the treatment of 5
µmol/L of compounds 6g or 10. In addition, a reduced number of apoptotic bodies were found when
compared to the control. Compared to the control, an increase in apoptotic bodies, rounder cells and
an increased intercellular space were found in MDA cells (Figure 3C), PC3 cells (Figure 3D) and
K562 cells (Figure 3E) after treatment with compound 10. In addition, spindle-like untreated MDA
cells and PC3 cells easily adhered, and demonstrated a clear cell membrane, which indicated that
MDA cells and PC3 cells underwent apoptosis after treatment with compound 10.
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Figure 3. (A) Inhibition on growth of cancer cells in vitro after treatment with compounds. (B-E) Morphological
changes of cancer cell lines HEL (B), MDA (C), PC3 (D) and K562 (E) Induced by treatment with 5 µmol/L of
tested compound for 48 h, morphological changes were observed by an inverted microscope.
Table 2
Cytotoxicity screening of selected compounds in vitro.
IC50/ µM
Compd
PC3
MDA
ND
HEL
4.35 ± 0.84*
PC3
ND
NDa
6g
10
2.92 ± 0.47
2.63 ± 0.38
3.14 ± 0.35
2.25 ± 0.42
1.85 ± 0.28
2.13 ± 0.18
2.64 ± 0.18
2.41 ± 0.39
doxorubicinb
a Not determined; b Used as a positive contro1; *P < 0.05, versus the control group.
Cells were treated with 5 µmol/L of compound 10 for 48 h at 37 ºC and then cells were
harvested and stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI).
Cells were analyzed by flow cytometry to confirm that cancer cells underwent apoptosis. Four
cancer cell lines that were treated with compound 10 showed a significant increase in apoptosis
when compared to untreated cells (P < 0.01, Figure 4A and B). This was especially true for PC3
cells and MDA cells. At 5 µmol/L, compound 6g displayed a lower apoptosis rate, whereas at 10
µmol/L, a higher apoptosis rate was observed in HEL cells (P < 0.01, Figure 5A and B).
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Figure 4. (A) Flow cytometric histograms of apoptosis induced by compound 10 in four cancer cell lines. After
treatment for 48 h with 5 µmol/L of compound 10, cells were harvested, stained with annexin-V-FITC and PI and
analyzed by flow cytometry. (B) Compound 10-induced apoptosis in the investigated cell lines compared to
DMSO-treated control cells. Data show the percentages of early and late apoptotic cells (right quadrants) and
present the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01, versus the control group.
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Figure 5. (A) Flow cytometric histograms of apoptotic HEL cells, 48 h after treatment with 5 µmol/L or 10
µmol/L of compound 6g. Cells were harvested, stained with annexin-V-FITC and PI and analyzed by flow
cytometry. (B) Compound 6g-induced apoptosis in HEL cells compared to DMSO-treated control cells. Data
show the percentages of early and late apoptotic cells (right quadrants) and present the mean ± SD of three
independent experiments. *P < 0.05; **P < 0.01, versus the control group.
2.3 Effects of active compounds on cell cycle phase arrest of cancer cells
The effects of two selected compounds 6g and 10 on cell cycle phase arrest in cancer cells
were evaluated by flow cytometry. After treatment for 48 h with 5 µmol/L of compound 6g or 10,
cells were harvested and analyzed by flow cytometry. Results showed that in PC3 cells, compound
10 can prolong the G1 phase (P < 0.01), shorten the S phase (P < 0.01) and G2 phase (P < 0.05)
compared to the DMSO-treated control cells (Figure 6A1 and A2). Cell cycle phase arrest of
compound 10 in MDA cells differed from that in PC3 cells, compound 10 only shortened the S
phase (P < 0.01) and prolonged the G2 phase (P < 0.01) of MDA cells (Figure 6B1 and B2). By
contrast, compound 10 can significantly reduce G1 (P < 0.01) and slightly shorten G2 phases (P <
0.05) of K562 cells (Figure 6C1 and C2), and noticeably prolong the G1 phase (P < 0.01) and
considerably shorten the S phase (P < 0.01) of HEL cells (Figure 6D1 and D2). Compound 6g
slightly prolonged the G1 phase (P < 0.05) and shortened the G2 phase (P < 0.05) of HEL cells
(Figure 7A1 and A2). The above results showed that compound 10 caused cell cycle phase arrest in
several cancer cells, indicating that the anti-cancer activity of compound 10 may be involved in the
regulation of cell cycle-associated genes.
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Figure 6. Effects of compound 10 on cell cycle phase arrest in PC3 (A1), MDA (B1), K562 (C1) and HEL (D1)
cells. Cells were treated for 48 h with 5 µmol/L of compound 10. Cells were then fixed and stained with PI to
analyze the DNA content by flow cytometry. Percentages of PC3 (A2), MDA (B2), K562 (C2) and HEL (D2)
cells in different phases of the cell cycle. Data are presented as the mean ± SD from three independent
experiments. *P < 0.05; **P < 0.01, versus the control group.
Figure 7. (A1) Effects of compound 6g on cell cycle phase arrest in HEL cells. Cells were treated for 48 h with 5
µmol/L of compound 6g. Cells were fixed and stained with PI to analyze the DNA content by flow cytometry.
(A2) Percentages of cells in the different phases of the cell cycle. Data are presented as the mean ± SD from three
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independent experiments. *P < 0.05; **P < 0.01, versus the control group.
2.4 Structure-activity relationship studies
To obtain a series of analogs, the aromatic ring B of 4,4'-dimethyl medicagenin (6c) was
modified. Based on the cytotoxicity results (Table 1-2), preliminary structure-activity relationship
could be drawn. Replacement of the ring B of compound 6c with 3-pyridinyl resulted in greatly
improved cytotoxicity in all cancer cell lines evaluated, the improvement was approximately three-
four fold compared to the standard compound 6c. While the replacement of ring B of compound 6c
with N-methyl-2-pyrrolyl only slightly improved cytotoxicity in MDA cells. Changing the
substituents on ring B of compound 6c was detrimental to activity versus erythroleukemia cells
(HEL and K562), only addition of a nitro group to position 3 of compound 6c could increase
cytotoxic activity in HEL cells. Molecules bearing electron-donating groups, such as in compounds
6b, 6f and 7b, show a better inhibitory effect in MDA cells, whereas the introduction of electron-
withdrawing groups to position 4 of compound 7b (compounds 8e-8h, respectively) resulted in
decreased cytotoxicity in MDA cells. Addition of a group to position 3 of compound 6c slightly
improved cytotoxicity in MDA cells, such as in compounds 6d, 6g, 6j and 6k. However, this
structural change was not associated with improved cytotoxic activity in PC3 cells. Cumulatively,
the evaluation of the compounds mentioned above is indicative of a greater preference for the
structural modification of 4,4'-dimethyl medicagenin (6c) in relation to cytotoxicity. Therefore, the
best-performing new compound 10 may be selected as a lead compound that further explored in
future.
3 Conclusion
In this study, a library of novel 3′,5′-diprenylated chalcones as analogs of 4,4'-dimethyl
medicagenin (6c) were synthesized. Cytotoxicity of these target compounds was evaluated by MTT
assays, which indicated that compound 10 possessed broad-spectrum cytotoxicity in PC3, MDA,
HEL and K562 cells with IC50 values of 2.92, 3.14, 1.85 and 2.64 µM, respectively. Further studies
indicated that compound 10 induced apoptosis and arrested cell cycle phase of PC3, MDA, HEL
and K562 cells. By contrast, compound 6g selectively displayed potent inhibitory activity against
proliferation of HEL cells with an IC50 value of 4.35 µM. Compound 6g slightly induced apoptosis
and arrested cell cycle phase of HEL cells. Preliminary structure-activity relationship studies
indicated that, in all cancer cell lines evaluated, the 3-pyridinyl group was essential for cytotoxicity.
4 Experimental section
4.1 General
Mass spectra (MS) were obtained on an electrospray ionization (ESI) mode on HP-5793 mass
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spectrometer (Hewlett-Packard, USA). Nuclear magnetic resonance (NMR) spectra were recorded
13
on INOVA-400 (1H NMR, 400 MHz; C NMR, 100 MHz, Varian Unit, USA) or WNMR-I (1H
13
NMR, 500 MHz; C NMR, 125 MHz, WIPM, China) with TMS as an internal standard. Melting
points (mp) were determined on an X-4 microscope melting point apparatus. Column
chromatography was performed on silica gel (300-400 mesh) and thin-layer chromatography (TLC)
analysis was carried out on silica gel plates GF254 purchased from Qingdao Ocean Chemical
Reagent Co. (Qingdao, China). Other reagents were analytical grade or guaranteed reagent
commercial product and used without further purification, unless otherwise noted.
4.2 Chemical synthesis
4.2.1 Synthesis of 4'-methoxy-2'-((2-methylbut-3-yn-2-yl)oxy)acetophenone (2)
To a solution of 2-hydroxy-4-methoxyacetylbenzene 1 (10.11 g, 59.62 mmol) in 65 mL
CH3CN, CuCl2 (43.0 mg, 0.06 mmol) was added, after stirring at -5 ºC for 20 min, the 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU, 13.65 mL, 89.43 mmol) followed by 3-chloro-3-
methylbutyne (10.60 mL, 89.43 mmol) were added dropwise. Then the mixture was stirred further
11 h at -5 ºC, ice concentrated hydrochloric acid was added to adjust pH = 2. After removal of
solvent, the residue was poured into 100 mL water, and the light gray precipitate was collected by
suction filtration, washed with petroleum ether and dried to obtain 2 (12.05 g, 87%). ESI-MS m/z:
1
255.1 [M + Na]+; H NMR (400 MHz, CDCl3) δ (ppm): 7.75 (d, J = 8.8 Hz, 1H), 7.18 (d, J = 2.4
Hz, 1H), 6.59 (dd, J = 8.8, 2.4 Hz, 1H), 3.83 (s, 3H), 2.68 (s, 1H), 2.57 (s, 3H), 1.74 (s, 6H).
4.2.2 Synthesis of 2'-hydroxy-4'-methoxy-3'-prenylacetophenone (3)
To a solution of 2 (11.59 g, 49.90 mmol) in 20 mL ethyl acetate was added 5% Pd/CaCO3
(1.16 g, 10% wt. of 2). Three purges of vacuum/argon followed by three purges of vacuum/H2 were
performed. After stirring for 12 h at room temperature under hydrogen, the reaction mixture was
filtered through diatomite and the filtrate was concentrated, the obtained product was used in the
next reaction without further purification. The previous crude product (6.19 g) was dissolved in 30
mL N, N-diethylaniline and heated at 120 ºC for 45 h under argon. The reaction mixture was cooled
to room temperature, 100 mL 1 M dilute hydrochloric acid was added and the product was extracted
with ethyl acetate, and the organic phase was washed with brine, dried over MgSO4. Then the
solvent was evaporated under reduced pressure, the obtained residue was purified by column
chromatography on silica gel (1:35 ethyl acetate–petroleum ether) to afford 3 (4.8 g, 71% yield,
over two steps) as yellow oil. ESI-MS m/z: 257.0 [M + Na]+; 1H NMR (500 MHz, CDCl3) δ (ppm):
12.72 (s, 1H), 7.60 (d, J = 8.9 Hz, 1H), 6.46 (d, J = 8.9 Hz, 1H), 5.22 – 5.17 (m, 1H), 3.90 (s, 3H),
3.36 (s, 2H), 2.58 (s, 3H), 1.79 (s, 3H), 1.68 (s, 3H).
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4.2.3 Synthesis of 4'-methoxy-2'-prenoxy-3'-prenylacetophenone (4)
To a mixture of 3 (5.74 g, 24.51 mmol) and anhydrous K2CO3 (7.10 g，51.0 mmol) in 18 mL
of anhydrous DMF, prenyl bromide (6.5 mL, 50.25 mmol) was added under argon. The contents
were stirred at 50 ºC for 30 h, after cooling to room temperature, the reaction mixture was poured
into water and extracted with ethyl acetate, and the organic phase was washed with brine, dried over
MgSO4. After removal of solvent, the obtained residue was purified by column chromatography on
silica gel (1:60 ethyl acetate–petroleum ether) to afford 4 (5.3 g, 72% yield) as light yellow oil. ESI-
MS m/z: 325.0 [M + Na]+; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.55 (d, J = 8.7 Hz, 1H), 6.68 (d, J
= 8.7 Hz, 1H), 5.54 – 5.47 (m, 1H), 5.19 – 5.12 (m, 1H), 4.30 (d, J = 7.1 Hz, 2H), 3.86 (s, 3H), 3.38
(d, J = 6.7 Hz, 2H), 2.59 (s, 3H), 1.76 (s, 6H), 1.67 (d, J = 1.1 Hz, 3H), 1.59 (s, 3H).
4.2.4 Synthesis of 2'-hydroxy-4'-methoxy-3',5'-diprenylacetophenone (5)
A solution of 4 (5.30 g, 17.53 mmol) in 30 mL N,N-diethylaniline was heated at 170 ºC for 24
h under argon. The reaction mixture was cooled to room temperature, 100 mL 1 M dilute
hydrochloric acid was added and the product was extracted with ethyl acetate, and the organic
phase was washed with brine, dried over MgSO4. After removal of solvent, the obtained residue
was purified by column chromatography on silica gel (1:60 ethyl acetate–petroleum ether) to give 5
1
(2.83 g, 53% yield) as yellow oil. ESI-MS m/z: 325.1 [M + Na]+; H NMR (400 MHz, CDCl3) δ
(ppm): 7.40 (s, 1H), 5.29 – 5.19 (m, 2H), 3.76 (s, 3H), 3.38 (d, J = 6.7 Hz, 2H), 3.30 (d, J = 7.2 Hz,
2H), 2.57 (s, 3H), 1.79 (s, 3H), 1.76 (s, 3H), 1.73 (s, 3H), 1.68 (d, J = 1.1 Hz, 3H).
4.2.5 Synthesis of compounds 6a-6k, 9 and 10
4.2.5.1 (E)-2'-Hydroxy-3,4'-dimethoxy-4-methoxymethoxy-3',5'-diprenylchalcone (6a)
To solution of 5 (1.20 g, 3.97 mmol) and 3-methoxy-4-(methoxymethoxy) benzaldehyde (1.17
g, 5.96 mmol) in 14 mL ethanol, NaOH (2.48 g, 37.57 mmol) was added and the reaction mixture
was stirred at room temperature for 41 h. After neutralized the excessive NaOH with 1 M dilute
hydrochloric acid, the resulting mixture was extracted with ethyl acetate, and the organic phase was
washed with brine, dried over MgSO4. After removal of solvent under reduced pressure, the
obtained residue was purified by column chromatography on silica gel (2:3 chloroform–petroleum
ether) to give 6a (714.7 mg, 37% yield) as yellow oil. ESI-MS m/z: 503.0 [M + Na]+; 1H NMR (500
MHz, CDCl3) δ (ppm): 13.27 (s, 1H), 7.84 (d, J = 15.4 Hz, 1H), 7.58 (s, 1H), 7.46 (d, J = 15.4 Hz,
1H), 7.24 (d, J = 8.7 Hz, 1H), 7.20 (d, J = 8.3 Hz, 1H), 7.17 (s, 1H), 5.33 – 5.23 (m, 4H), 3.97 (s,
3H), 3.78 (s, 3H), 3.53 (s, 3H), 3.42 (d, J = 6.5 Hz, 2H), 3.34 (d, J = 7.0 Hz, 2H), 1.81 (s, 3H), 1.79
(s, 3H), 1.76 (s, 3H), 1.70 (s, 3H).
4.2.5.2 (E)-3-Ethoxy-2'-hydroxy-4-methoxymethoxy-4'-methoxy-3',5'-diprenylchalcone (6b)
6b was obtained from 5 and 3-ethoxy-4-(methoxymethoxy) benzaldehyde as described for the
14

ACCEPTED MANUSCRIPT
1
synthesis of 6a: 53% yield as yellow oil. ESI-MS m/z: 517.3 [M + Na]+; H NMR (500 MHz,
CDCl3) δ (ppm): 13.27 (s, 1H), 7.82 (d, J = 15.4 Hz, 1H), 7.58 (s, 1H), 7.45 (d, J = 15.4 Hz, 1H),
7.22 (dd, J = 8.4, 1.6 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), 7.17 (d, J = 1.5 Hz, 1H), 5.33 – 5.23 (m,
4H), 4.18 (q, J = 7.0 Hz, 2H), 3.78 (s, 3H), 3.54 (s, 3H), 3.42 (d, J = 6.5 Hz, 2H), 3.35 (d, J = 7.1
Hz, 2H), 1.81 (s, 3H), 1.80 (s, 3H), 1.77 (s, 3H), 1.70 (s, 3H), 1.51 (t, J = 7.0 Hz, 3H).
4.2.5.3 (E)-2'-Hydroxy-4,4'-dimethoxy-3',5'-diprenylchalcone (6c)
6c was obtained from 5 and 4-methoxybenzaldehyde as described for the synthesis of 6a: 56%
1
yield as yellow oil. ESI-MS m/z: 443.2 [M + Na]+; H NMR (400 MHz, CDCl3) δ (ppm): 13.32 (s,
1H), 7.86 (d, J = 15.4 Hz, 1H), 7.62 (s, 1H), 7.60 (s, 1H), 7.59 (s, 1H), 7.47 (d, J = 15.4 Hz, 1H),
6.97 (s, 1H), 6.95 (d, J = 1.9 Hz, 1H), 5.34 – 5.23 (m, 2H), 3.87 (s, 3H), 3.78 (s, 3H), 3.42 (d, J =
6.6 Hz, 2H), 3.34 (d, J = 7.0 Hz, 2H), 1.81 (s, 3H), 1.79 (s, 3H), 1.77 (s, 3H), 1.70 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ (ppm): 192.94, 166.34, 162.96, 162.36, 161.93, 144.56, 133.00, 132.03,
130.50, 128.42, 127.65, 125.56, 123.77, 123.17, 122.63, 118.24, 116.74, 114.60, 61.41, 55.58,
28.36, 25.92, 25.88, 23.10, 18.13, 18.10.
4.2.5.4 (E)-2'-Hydroxy-3,4,4'-trimethoxy-3',5'-diprenylchalcone (6d)
6d was obtained from 5 and 3,4-dimethoxybenzaldehyde as described for the synthesis of 6a:
1
32% yield as yellow oil. ESI-MS m/z: 473.2 [M + Na]+; H NMR (500 MHz, CDCl3) δ (ppm):
13.30 (s, 1H), 7.84 (d, J = 15.3 Hz, 1H), 7.59 (s, 1H), 7.45 (d, J = 15.4 Hz, 1H), 7.26 – 7.24 (m,
1H), 7.16 (d, J = 1.7 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 5.31 (t, J = 7.1 Hz, 1H), 5.26 (t, J = 6.6 Hz,
1H), 3.97 (s, 3H), 3.95 (s, 3H), 3.78 (s, 3H), 3.42 (d, J = 6.5 Hz, 2H), 3.35 (d, J = 7.1 Hz, 2H), 1.81
(s, 3H), 1.79 (s, 3H), 1.76 (s, 3H), 1.70 (s, 3H). 13C NMR (100 MHz, CDCl3) δ (ppm): 192.81,
162.99, 162.39, 151.68, 149.35, 144.81, 133.10, 132.08, 128.32, 127.90, 125.47, 123.80, 123.50,
123.10, 122.59, 118.43, 116.70, 111.25, 110.20, 61.40, 56.18, 56.03, 28.23, 25.92, 25.90, 23.10,
18.12.
4.2.5.5 (E)-2'-Hydroxy-3,4,4',5-tetramethoxy-3',5'-diprenylchalcone (6e)
6e was obtained from 5 and 3,4,5-trimethoxybenzaldehyde as described for the synthesis of
6a: 43% yield as yellow oil. ESI-MS m/z: 503.3 [M + Na]+; 1H NMR (500 MHz, CDCl3) δ (ppm):
13.23 (s, 1H), 7.80 (d, J = 15.4 Hz, 1H), 7.58 (s, 1H), 7.46 (d, J = 15.4 Hz, 1H), 6.87 (s, 2H), 5.33 –
5.28 (m, 1H), 5.28 – 5.24 (m, 1H), 3.94 (s, 6H), 3.91 (s, 3H), 3.79 (s, 3H), 3.42 (d, J = 6.6 Hz, 2H),
3.35 (d, J = 7.1 Hz, 2H), 1.81 (s, 3H), 1.79 (s, 3H), 1.76 (s, 3H), 1.70 (s, 3H). 13C NMR (125 MHz,
CDCl3) δ 192.66, 163.14, 162.44, 153.59, 144.74, 140.63, 133.17, 132.10, 130.39, 128.30, 125.51,
123.84, 123.01, 122.52, 119.90, 116.60, 105.79, 61.37, 61.16, 56.28, 28.14, 25.88, 23.06, 18.08.
4.2.5.6 (E)-4-Allyloxy-3-ethoxy-2'-hydroxy-4'-methoxy-3',5'-diprenylchalcone (6f)
6f was obtained from 5 and 4-allyloxy-3-ethoxybenzaldehyde as described for the synthesis of
15

ACCEPTED MANUSCRIPT
1
6a: 60% yield as yellow powder, m.p. 43-45 ºC. ESI-MS m/z: 513.2 [M + Na]+; H NMR (500
MHz, CDCl3) δ (ppm): 13.30 (s, 1H), 7.82 (d, J = 15.3 Hz, 1H), 7.58 (s, 1H), 7.43 (d, J = 15.4 Hz,
1H), 7.21 (dd, J = 8.3, 1.8 Hz, 1H), 7.18 (d, J = 1.8 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 6.09 (ddt, J =
17.1, 10.5, 5.2 Hz, 1H), 5.44 (dd, J = 17.3, 1.4 Hz, 1H), 5.34 – 5.28 (m, 2H), 5.28 – 5.24 (m, 1H),
4.68 (dd, J = 3.9, 1.4 Hz, 2H), 4.17 (q, J = 7.0 Hz, 2H), 3.78 (s, 3H), 3.42 (d, J = 6.6 Hz, 2H), 3.35
(d, J = 7.1 Hz, 2H), 1.81 (s, 3H), 1.80 (s, 3H), 1.77 (s, 3H), 1.71 (s, 3H), 1.51 (t, J = 7.0 Hz, 3H).
13C NMR (125 MHz, CDCl3) δ 192.83, 162.97, 162.38, 151.09, 149.06, 144.85, 132.98, 132.05,
128.33, 128.10, 125.46, 123.78, 123.23, 123.10, 122.60, 118.42, 118.18, 116.71, 113.49, 112.52,
69.87, 64.78, 61.38, 28.22, 25.88, 23.08, 18.10, 14.94.
4.2.5.7 (E)-2'-Hydroxy-4,4'-dimethoxy-3-nitro-3',5'-diprenylchalcone (6g)
6g was obtained from 5 and 4-methoxy-3-nitrobenzaldehyde as described for the synthesis of
1
6a: 16% yield as yellow oil. ESI-MS m/z: 464.2 [M - H]-; H NMR (500 MHz, CDCl3) δ (ppm):
13.10 (s, 1H), 8.18 (d, J = 2.1 Hz, 1H), 7.80 (d, J = 15.7 Hz, 1H), 7.77 (dd, J = 9.2, 2.6 Hz, 1H),
7.57 (s, 1H), 7.53 (d, J = 15.5 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H), 5.33 – 5.22 (m, 2H), 4.03 (s, 3H),
3.79 (s, 3H), 3.42 (d, J = 6.7 Hz, 2H), 3.35 (d, J = 7.0 Hz, 2H), 1.81 (s, 3H), 1.79 (s, 3H), 1.77 (s,
3H), 1.70 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 192.21, 163.44, 162.54, 154.43, 141.54, 134.90,
133.25, 132.21, 128.47, 127.60, 125.99, 124.96, 123.95, 122.98, 122.43, 121.09, 116.47, 114.01,
61.46, 56.95, 32.07, 29.85, 29.52, 28.38, 25.91, 23.08, 22.85, 18.16, 18.12, 14.30.
4.2.5.8 (E)-2'-Hydroxy-4-dimethylamino-4'-methoxy-3',5'-diprenylchalcone (6h)
6h was obtained from 5 and 4-dimethylaminobenzaldehyde as described for the synthesis of
1
6a: 12% yield as yellow powder, m.p. 118-120 ºC. ESI-MS m/z: 456.1 [M + Na]+; H NMR (500
MHz, CDCl3) δ (ppm): 13.52 (s, 1H), 7.87 (d, J = 15.2 Hz, 1H), 7.59 (s, 1H), 7.56 (d, J = 8.8 Hz,
2H), 7.38 (d, J = 15.2 Hz, 1H), 6.72 (d, J = 8.8 Hz, 2H), 5.34 – 5.25 (m, 2H), 3.78 (s, 3H), 3.42 (d,
J = 6.5 Hz, 2H), 3.35 (d, J = 7.0 Hz, 2H), 3.07 (s, 6H), 1.81 (s, 3H), 1.80 (s, 3H), 1.78 (s, 3H), 1.71
(s, 3H). 13C NMR (125 MHz, CDCl3) δ (ppm): 192.89, 162.54, 162.29, 162.23, 152.26, 145.70,
132.90, 131.93, 130.76, 128.27, 125.24, 123.62, 123.30, 122.79, 116.98, 115.07, 111.97, 61.39,
40.31, 28.35, 25.94, 25.89, 23.11, 18.12.
4.2.5.9 (E)-3-Fluoro-2'-hydroxy-4,4'-dimethoxy-3',5'-diprenylchalcone (6i)
6i was obtained from 5 and 3-fluoro-4-methoxybenzaldehyde as described for the synthesis of
1
6a: 40% yield as yellow powder, m.p. 62-64 ºC. ESI-MS m/z: 461.2 [M + Na]+; H NMR (400
MHz, CDCl3) δ (ppm): 7.79 (d, J = 15.4 Hz, 1H), 7.57 (s, 1H), 7.47 – 7.39 (m, 2H), 7.36 (d, J = 8.6
Hz, 1H), 7.00 (t, J = 8.5 Hz, 1H), 5.28 (mf, J = 13.4, 4.7 Hz, 2H), 3.95 (s, 3H), 3.78 (s, 3H), 3.41 (d,
J = 6.5 Hz, 2H), 3.34 (d, J = 6.9 Hz, 2H), 1.80 (s, 3H), 1.79 (s, 3H), 1.77 (s, 3H), 1.70 (s, 3H). 13C
NMR (125 MHz, CDCl3) δ (ppm): 192.63, 163.16, 162.43, 153.51, 151.54, 149.95, 143.38, 133.09,
16

ACCEPTED MANUSCRIPT
132.12, 128.43, 128.13, 126.53, 125.76, 123.85, 123.09, 122.54, 119.52, 116.62, 114.97, 114.82,
113.26, 61.43, 56.39, 28.37, 25.91, 25.88, 23.08, 18.14, 18.10.
4.2.5.10 (E)-3-Chloro-2'-hydroxy-4,4'-dimethoxy-3',5'-diprenylchalcone (6j)
6j was obtained from 5 and 3-chloro-4-methoxybenzaldehyde as described for the synthesis of
1
6a: 47% yield as yellow powder, m.p. 101-103 ºC. ESI-MS m/z: 477.0 [M + Na]+; H NMR (500
MHz, CDCl3) δ (ppm): 13.20 (s, 1H), 7.77 (d, J = 15.4 Hz, 1H), 7.71 (d, J = 1.8 Hz, 1H), 7.58 (s,
1H), 7.49 (dd, J = 8.5, 1.7 Hz, 1H), 7.46 (d, J = 15.4 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 5.32 – 5.28
(m, 1H), 5.28 – 5.23 (m, 1H), 3.97 (s, 3H), 3.79 (s, 3H), 3.42 (d, J = 6.6 Hz, 2H), 3.35 (d, J = 6.9
Hz, 2H), 1.81 (s, 3H), 1.80 (s, 3H), 1.78 (s, 3H), 1.70 (s, 3H). 13C NMR (125 MHz, CDCl3) δ
(ppm): 192.61, 163.20, 162.44, 156.99, 142.98, 133.11, 132.09, 129.63, 129.44, 128.49, 128.47,
125.73, 123.85, 123.42, 123.11, 122.56, 119.55, 116.62, 112.16, 61.41, 56.45, 29.83, 28.38, 25.88,
23.08, 18.14, 18.09, 18.04.
4.2.5.11 (E)-3-Bromo-2'-hydroxy-4,4'-dimethoxy-3',5'-diprenylchalcone (6k)
6k was obtained from 5 and 3-bromo-4-methoxybenzaldehyde as described for the synthesis of
1
6a: 32% yield as yellow powder, m.p. 120-122 ºC. ESI-MS m/z: 522.0 [M + Na]+; H NMR (400
MHz, CDCl3) δ (ppm): 7.91 – 7.88 (m, 1H), 7.77 (d, J = 15.4 Hz, 1H), 7.58 (s, 1H), 7.54 (dd, J =
8.5, 1.8 Hz, 1H), 7.46 (d, J = 15.4 Hz, 1H), 6.94 (d, J = 8.5 Hz, 1H), 5.33 – 5.21 (m, 1H), 3.96 (s,
3H), 3.78 (s, 3H), 3.41 (d, J = 6.5 Hz, 2H), 3.35 (d, J = 6.9 Hz, 2H), 1.80 (s, 6H), 1.77 (s, 3H), 1.70
(s, 3H). 13C NMR (125 MHz, CDCl3) δ (ppm): 192.59, 163.18, 162.44, 157.84, 142.84, 133.14,
132.77, 132.12, 130.20, 128.97, 128.47, 125.73, 123.84, 123.10, 122.54, 119.50, 116.61, 112.57,
111.97, 61.42, 56.56, 28.37, 25.91, 25.89, 23.08, 18.15, 18.11.
4.2.5.12 (E)-1-(2-Hydroxy-4-methoxy-3,5-diprenyl)phenyl-3-(N-methyl-2-pyrrolyl)-pro- pene-1-one
(9)
9 was obtained from 5 and N-methyl-pyrrole-2-carbaldehyde as described for the synthesis of
6a: 58% yield as yellow oil. ESI-MS m/z: 416.1 [M + Na]+; 1H NMR (400 MHz, CDCl3) δ (ppm):
13.49 (s, 1H), 7.86 (d, J = 15.0 Hz, 1H), 7.55 (s, 1H), 7.32 (d, J = 15.0 Hz, 1H), 6.87 (dd, J = 4.0,
1.2 Hz, 1H), 6.85 (d, J = 2.2 Hz, 1H), 6.25 (dd, J = 3.5, 2.7 Hz, 1H), 5.33 – 5.24 (m, 2H), 3.79 (s,
3H), 3.78 (s, 3H), 3.41 (d, J = 6.6 Hz, 2H), 3.34 (d, J = 7.1 Hz, 2H), 1.81 (s, 3H), 1.79 (s, 3H), 1.76
(s, 3H), 1.70 (d, J = 0.8 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 192.43, 162.67, 162.21, 133.05,
132.09, 131.95, 130.44, 128.35, 128.09, 125.34, 123.65, 123.11, 122.72, 116.79, 115.31, 112.91,
110.12, 61.37, 34.56, 28.23, 25.91, 25.88, 23.08, 18.10.
4.2.5.13 (E)-1-(2-Hydroxy-4-methoxy-3,5-diprenyl)phenyl-3-(3-pyridinyl)-propene-1-one (10)
10 was obtained from 5 and nicotinaldehyde as described for the synthesis of 6a: 59% yield as
yellow oil. ESI-MS m/z: 414.0 [M + Na]+; 1H NMR (500 MHz, CDCl3) δ (ppm): 12.77 (s, 1H), 8.56
17

ACCEPTED MANUSCRIPT
(d, J = 1.3 Hz, 1H), 8.35 – 8.31 (m, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.54 (d, J = 15.6 Hz, 1H), 7.34
(d, J = 15.6 Hz, 1H), 7.07 (dd, J = 7.8, 4.8 Hz, 1H), 5.00 – 4.92 (m, 2H), 3.48 (s, 3H), 3.11 (d, J =
6.6 Hz, 2H), 3.04 (d, J = 7.0 Hz, 2H), 1.51 (s, 3H), 1.48 (s, 3H), 1.46 (s, 3H), 1.40 (s, 3H). 13C
NMR (125 MHz, CDCl3) δ (ppm):192.19, 163.48, 162.54, 151.24, 150.01, 140.74, 134.86, 133.16,
132.11, 130.67, 128.44, 125.94, 123.91, 122.89, 122.62, 122.40, 116.39, 61.38, 28.28, 25.87, 25.83,
23.03, 18.08, 18.04.
4.2.6 Synthesis of (E)-2',4-Dihydroxy-3,4'-dimethoxy-3',5'-diprenylchalcone (7a)
6a (714.7 mg, 1.49 mmol ) was dissolved in 25 mL methanol. To this solution, 7.2 mL of 3 M
diluted hydrochloric acid was added. After the contents were stirred at 60 ºC for 1 h, the reaction
mixture was poured into water and extracted with ethyl acetate, and the organic phase was washed
with brine, dried over MgSO4. After removal of solvent under reduced pressure, the obtained
residue was purified by column chromatography on silica gel (2:1 chloroform–petroleum ether) to
1
give 7a (373.4.0 mg, 57.5% yield) as yellow oil. ESI-MS m/z: 459.1 [M + Na]+; H NMR (500
MHz, CDCl3) δ (ppm): 13.31 (s, 1H), 7.83 (d, J = 15.3 Hz, 1H), 7.58 (s, 1H), 7.43 (d, J = 15.3 Hz,
1H), 7.24 (dd, J = 8.2, 1.6 Hz, 1H), 7.13 (d, J = 1.6 Hz, 1H), 6.98 (d, J = 8.2 Hz, 1H), 5.97 (s, 1H),
5.33 – 5.24 (m, 2H), 3.98 (s, 3H), 3.79 (s, 3H), 3.42 (d, J = 6.6 Hz, 2H), 3.35 (d, J = 7.0 Hz, 2H),
1.82 (s, 3H), 1.79 (s, 3H), 1.77 (s, 3H), 1.71 (s, 3H). 13C NMR (100 MHz, CDCl3) δ (ppm): 192.83,
162.97, 162.37, 148.55, 146.91, 145.00, 133.04, 132.06, 128.34, 127.53, 125.48, 123.79, 123.56,
123.15, 122.60, 118.17, 116.70, 115.07, 110.30, 61.40, 56.08, 28.28, 25.91, 25.89, 23.09, 18.12,
18.11.
4.2.7 Synthesis of (E)-3-Ethoxy-2',4-dihydroxy-4'-methoxy-3',5'-diprenylchalcone (7b)
6b (1.07 g, 2.16 mmol) was dissolved in 20 mL methanol. To this solution, p-toluenesulfonic
acid monohydrate (2.39 g, 12.56 mmol) was added. After the contents were stirred at room
temperature for 15 h, the reaction mixture was poured into water and extracted with ethyl acetate,
and the organic phase was washed with brine, dried over MgSO4. After removal of solvent under
reduced pressure, the obtained residue was purified by column chromatography on silica gel (2:1
chloroform–petroleum ether) to give 7b (800.6 mg, 83.7% yield) as yellow powder, m.p. 86-88 ºC.
1
ESI-MS m/z: 473.1 [M + Na]+; H NMR (400 MHz, CDCl3) δ (ppm): 7.82 (d, J = 15.4 Hz, 1H),
7.58 (s, 1H), 7.42 (d, J = 15.4 Hz, 1H), 7.23 (dd, J = 8.3, 1.2 Hz, 1H), 7.11 (d, J = 1.3 Hz, 1H), 6.97
(d, J = 8.2 Hz, 1H), 6.03 (s, 1H), 5.34 – 5.22 (m, 2H), 4.19 (q, J = 7.0 Hz, 2H), 3.78 (s, 3H), 3.41
(d, J = 6.5 Hz, 2H), 3.34 (d, J = 7.0 Hz, 2H), 1.81 (s, 3H), 1.79 (s, 3H), 1.76 (s, 3H), 1.70 (s, 3H),
1.50 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ(ppm): 192.86, 162.98, 162.38, 148.68,
146.22, 145.07, 133.03, 132.03, 128.34, 127.49, 125.47, 123.79, 123.39, 123.18, 122.63, 118.13,
116.72, 115.03, 111.24, 64.78, 61.38, 28.28, 25.88, 23.09, 18.10, 14.95.
18

ACCEPTED MANUSCRIPT
4.2.8 Synthesis of compounds 8a-8h
4.2.8.1 (E)-2'-Hydroxy-3,4'-dimethoxy-4-nicotinoyloxy-3',5'-diprenylchalcone (8a)
To an ice solution of 7a (50.0 mg, 0.115 mmol) and nicotinic acid (14.3 mg, 0.116 mmol) in 2
mL dry CH2Cl2, 4-dimethylaminopyridine (DMAP, 21.1 mg, 0.174 mmol) followed by 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 54.9 mg, 0.288 mmol) was added
under argon. After the contents were stirred at 0 ºC-RT for 16 h, the reaction mixture was poured
into saturated ammonium chloride solution and extracted with ethyl acetate, and the organic phase
was washed with brine, dried over MgSO4. After removal of solvent, the obtained residue was
purified by preparation plate on silica gel (1:1 ethyl acetate–petroleum ether) to give 8a (58.5 mg,
1
94% yield) as yellow oil. ESI-MS m/z: 564.3 [M + Na]+; H NMR (400 MHz, CDCl3) δ (ppm):
13.19 (s, 1H), 9.42 (d, J = 1.6 Hz, 1H), 8.87 (dd, J = 4.8, 1.6 Hz, 1H), 8.48 (dt, J = 8.0, 1.9 Hz, 1H),
7.87 (d, J = 15.4 Hz, 1H), 7.59 (s, 1H), 7.55 (d, J = 15.5 Hz, 1H), 7.49 (dd, J = 7.9, 4.9 Hz, 1H),
7.33 (dd, J = 8.3, 1.7 Hz, 1H), 7.26 (d, J = 3.8 Hz, 1H), 7.25 (d, J = 8.2 Hz, 2H), 5.33 – 5.24 (m,
2H), 3.90 (s, 3H), 3.79 (s, 3H), 3.43 (d, J = 6.6 Hz, 2H), 3.35 (d, J = 7.0 Hz, 2H), 1.81 (s, 3H), 1.78
(s, 3H), 1.76 (s, 3H), 1.71 (s, 3H). 13C NMR (100 MHz, CDCl3) δ (ppm): 192.67, 163.34, 163.31,
162.50, 154.19, 151.66, 151.63, 143.81, 141.54, 137.89, 134.33, 133.17, 132.12, 128.44, 125.73,
125.32, 123.91, 123.62, 123.52, 123.01, 122.52, 121.53, 121.25, 116.60, 112.20, 61.41, 56.11,
28.26, 25.90, 25.88, 23.09, 18.11, 18.10.
4.2.8.2 (E)-2'-Hydroxy-3,4'-dimethoxy-4-(4-dimethylaminobenzoyloxy)-3',5'-diprenyl chalcone (8b)
8b was obtained from 7a and 4-(dimethylamino)benzoic acid as described for the synthesis of
8a: 43% yield as yellow oil. ESI-MS m/z: 606.2 [M + Na]+; 1H NMR (500 MHz, CDCl3) δ (ppm):
13.22 (s, 1H), 8.09 (s, 1H), 8.07 (s, 1H), 7.87 (d, J = 15.4 Hz, 1H), 7.59 (s, 1H), 7.53 (d, J = 15.4
Hz, 1H), 7.30 (dd, J = 8.2, 1.7 Hz, 1H), 7.23 (s, 1H), 7.22 (d, J = 8.1 Hz, 2H), 6.72 (s, 1H), 6.70 (s,
1H), 5.33 – 5.29 (m, 1H), 5.28 – 5.24 (m, 1H), 3.89 (s, 3H), 3.79 (s, 3H), 3.42 (d, J = 6.6 Hz, 2H),
3.35 (d, J = 7.0 Hz, 2H), 3.09 (s, 6H), 1.81 (s, 3H), 1.78 (s, 3H), 1.76 (s, 3H), 1.71 (s, 3H). 13C
NMR (125 MHz, CDCl3) δ (ppm): 192.80, 164.97, 163.17, 162.44, 153.87, 152.10, 144.31, 142.74,
133.45, 133.20, 132.33, 132.11, 128.46, 125.65, 123.99, 123.83, 122.99, 122.55, 121.66, 120.61,
116.64, 115.46, 112.14, 110.90, 61.41, 56.13, 40.24, 28.22, 25.91, 23.08, 18.11.
4.2.8.3 (E)-2'-Hydroxy-3,4'-dimethoxy-4-(5-methylthiophene-2-carbonyloxy)-3',5'-diprenylchalcone
(8c)
8c was obtained from 7a and 5-methylthiophene-2-carboxylic acid as described for the
1
synthesis of 8a: 53% yield as yellow oil. ESI-MS m/z: 583.3 [M + Na]+; H NMR (500 MHz,
CDCl3) δ (ppm): 13.19 (s, 1H), 7.86 (d, J = 15.4 Hz, 1H), 7.82 (d, J = 3.7 Hz, 1H), 7.58 (s, 1H),
7.53 (d, J = 15.5 Hz, 1H), 7.30 (dd, J = 8.2, 1.7 Hz, 1H), 7.23 – 7.21 (m, 2H), 6.86 (d, J = 3.7 Hz,
19

ACCEPTED MANUSCRIPT
1H), 5.33 – 5.23 (m, 2H), 3.90 (s, 3H), 3.79 (s, 3H), 3.42 (d, J = 6.6 Hz, 2H), 3.35 (d, J = 7.1 Hz,
2H), 2.58 (s, 3H), 1.81 (s, 3H), 1.78 (s, 3H), 1.76 (s, 3H), 1.71 (s, 3H). 13C NMR (125 MHz, CDCl3)
δ (ppm): 192.72, 163.22, 162.46, 159.97, 151.89, 149.72, 144.03, 141.76, 135.67, 133.94, 133.19,
132.14, 129.54, 128.45, 126.89, 125.69, 123.86, 123.75, 122.98, 122.51, 121.53, 120.92, 116.60,
112.18, 61.41, 56.14, 28.23, 25.90, 23.07, 18.11, 16.06.
4.2.8.4 (E)-2'-Hydroxy-3,4’-dimethoxy-4-(2-indolylacetyloxy)-3',5'-diprenylchalcone (8d)
8d was obtained from 7a and 2-indolylacetic acid as described for the synthesis of 8a: 54%
1
yield as yellow powder, m.p. 143-145 ºC. ESI-MS m/z: 616.2 [M + Na]+; H NMR (500 MHz,
CDCl3) δ (ppm): 13.11 (s, 1H), 8.15 (s, 1H), 7.81 (d, J = 15.2 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H),
7.56 (s, 1H), 7.49 (d, J = 15.3 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.29 – 7.27 (m, 1H), 7.25 – 7.21
(m, 2H), 7.20 – 7.15 (m, 2H), 7.08 (d, J = 8.1 Hz, 1H), 5.34 – 5.30 (m, 1H), 5.30 – 5.26 (m, 1H),
4.12 (s, 2H), 3.85 (s, 3H), 3.83 (s, 3H), 3.47 (d, J = 6.6 Hz, 2H), 3.39 (d, J = 7.0 Hz, 2H), 1.88 (s,
3H), 1.85 (s, 3H), 1.82 (s, 3H), 1.77 (s, 3H). 13C NMR (125 MHz, CDCl3) δ (ppm): 192.73, 169.94,
163.24, 162.46, 151.62, 144.00, 142.07, 136.24, 133.85, 133.19, 132.14, 128.44, 127.32, 125.68,
123.87, 123.49, 123.46, 122.98, 122.52, 122.43, 121.48, 120.90, 119.84, 119.08, 116.60, 112.08,
111.35, 107.96, 61.40, 55.93, 31.24, 28.23, 25.90, 25.89, 23.08, 18.10.
4.2.8.5 (E)-3-Ethoxy-4-(furan-2-carbonyloxy)-2'-hydroxy-4'-methoxy-3',5'-diprenylchalcone (8e)
8e was obtained from 7b and furan-2-carboxylic acid as described for the synthesis of 8a: 14%
1
yield as yellow oil. ESI-MS m/z: 567.0 [M + Na]+; H NMR (500 MHz, CDCl3) δ (ppm): 13.18 (s,
1H), 7.84 (d, J = 15.4 Hz, 1H), 7.69 (d, J = 0.8 Hz, 1H), 7.57 (s, 1H), 7.52 (d, J = 15.5 Hz, 1H),
7.42 (d, J = 3.1 Hz, 1H), 7.29 (dd, J = 8.3, 1.6 Hz, 1H), 7.24 – 7.21 (m, 2H), 6.61 (dd, J = 3.5, 1.7
Hz, 1H), 5.33 – 5.28 (m, 1H), 5.28 – 5.24 (m, 1H), 4.14 (q, J = 7.0 Hz, 2H), 3.79 (s, 3H), 3.42 (d, J
= 6.6 Hz, 2H), 3.34 (d, J = 7.0 Hz, 2H), 1.81 (s, 3H), 1.78 (s, 3H), 1.76 (s, 3H), 1.70 (s, 3H), 1.38 (t,
13
J = 7.0 Hz, 3H). C NMR (125 MHz, CDCl3) δ (ppm): 192.73, 163.26, 162.49, 156.30, 151.12,
147.39, 143.99, 143.80, 141.54, 134.10, 133.21, 132.14, 128.44, 125.70, 123.90, 123.61, 123.00,
122.53, 121.46, 121.03, 119.80, 116.62, 113.40, 112.36, 64.81, 61.42, 28.25, 25.90, 23.09, 18.12,
14.78.
4.2.8.6
(E)-3-Ethoxy-2'-hydroxy-4'-methoxy-3',5'-diprenyl-4-(thiophene-2-carbonyloxy)chalcone
(8f)
8f was obtained from 7b and thiophene-2-carboxylic acid as described for the synthesis of 8a:
1
65% yield as yellow oil. ESI-MS m/z: 583.0 [M + Na]+; H NMR (500 MHz, CDCl3) δ (ppm):
12.87 (s, 1H), 7.68 (dd, J = 3.7, 1.2 Hz, 1H), 7.52 (d, J = 15.4 Hz, 1H), 7.36 (dd, J = 5.0, 1.2 Hz,
1H), 7.20 (d, J = 15.4 Hz, 1H), 6.97 (dd, J = 8.2, 1.6 Hz, 1H), 6.93 (s, 1H), 6.92 – 6.90 (m, 2H),
6.88 – 6.85 (m, 1H), 5.01 – 4.96 (m, 1H), 4.96 – 4.91 (m, 1H), 3.82 (q, J = 7.0 Hz, 2H), 3.47 (s,
20

ACCEPTED MANUSCRIPT
3H), 3.11 (d, J = 6.6 Hz, 2H), 3.03 (d, J = 7.0 Hz, 2H), 1.50 (s, 3H), 1.47 (s, 3H), 1.45 (s, 3H), 1.39
(s, 3H), 1.05 (t, J = 6.9 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ (ppm): 192.72, 163.23, 162.46,
160.01, 151.18, 144.02, 142.05, 134.96, 133.95, 133.70, 133.16, 132.46, 132.09, 128.45, 128.16,
125.67, 123.85, 123.60, 123.00, 122.53, 121.47, 120.92, 116.60, 113.46, 64.84, 61.39, 28.23, 25.87,
23.07, 18.09, 14.79.
4.2.8.7 (E)-4-(5-Chloro-thiophene-2-carbonyloxy)-3-ethoxy-2'-hydroxy-4'-methoxy-3',5'-diprenyl-
chalone (8g)
8g was obtained from 7b and 5-chloro-thiophene-2-carboxylic acid as described for the
1
synthesis of 8a: 58% yield as yellow oil. ESI-MS m/z: 617.0 [M + Na]+; H NMR (500 MHz,
CDCl3) δ (ppm): 13.19 (s, 1H), 7.84 (d, J = 15.4 Hz, 1H), 7.79 (d, J = 4.0 Hz, 1H), 7.58 (s, 1H),
7.52 (d, J = 15.5 Hz, 1H), 7.28 (dd, J = 8.3, 1.6 Hz, 1H), 7.23 – 7.20 (m, 2H), 7.02 (d, J = 4.0 Hz,
1H), 5.33 – 5.28 (m, 1H), 5.28 – 5.24 (m, 1H), 4.13 (q, J = 7.0 Hz, 2H), 3.79 (s, 3H), 3.42 (d, J =
6.6 Hz, 2H), 3.35 (d, J = 7.1 Hz, 2H), 1.81 (s, 3H), 1.79 (s, 3H), 1.76 (s, 3H), 1.71 (s, 3H), 1.37 (t, J
= 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ (ppm): 192.69, 163.26, 162.48, 159.01, 151.06,
143.92, 141.76, 138.78, 134.58, 134.12, 133.17, 132.11, 130.56, 128.44, 127.72, 125.69, 123.88,
123.49, 123.01, 122.53, 121.42, 121.05, 116.60, 113.39, 64.83, 61.40, 28.24, 25.88, 23.08, 18.10,
14.80.
4.2.8.8
chalcone (8h)
8h was obtained from 7b and (E)-3-(pyridin-4-yl) acrylic acid as described for the synthesis of
(E)-4-((E)-3-(4-Pyridinyl)acryloyloxy)-3-ethoxy-2'-hydroxy-4'-methoxy-3',5'-diprenyl-
8a: 47% yield as yellow oil. ESI-MS m/z: 604.2 [M + Na]+; 1H NMR (500 MHz, CDCl3) δ (ppm):
13.18 (s, 1H), 8.82 (d, J = 1.3 Hz, 1H), 8.65 (d, J = 3.6 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.88 (d, J
= 16.1 Hz, 1H), 7.84 (d, J = 15.4 Hz, 1H), 7.58 (s, 1H), 7.52 (d, J = 15.4 Hz, 1H), 7.39 (dd, J = 7.9,
4.9 Hz, 1H), 7.28 (dd, J = 8.2, 1.6 Hz, 1H), 7.23 (d, J = 1.6 Hz, 1H), 7.18 (d, J = 8.1 Hz, 1H), 6.75
(d, J = 16.1 Hz, 1H), 5.33 – 5.28 (m, 1H), 5.28 – 5.23 (m, 1H), 4.15 (q, J = 7.0 Hz, 2H), 3.79 (s,
3H), 3.42 (d, J = 6.6 Hz, 2H), 3.35 (d, J = 7.1 Hz, 2H), 1.81 (s, 3H), 1.79 (s, 3H), 1.77 (s, 3H), 1.70
(s, 3H), 1.42 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ (ppm): 192.69, 164.13, 163.25,
162.47, 151.45, 151.00, 150.01, 143.98, 143.13, 141.98, 134.61, 133.96, 133.16, 132.10, 130.08,
128.42, 125.67, 124.01, 123.87, 123.46, 123.00, 122.51, 121.46, 120.95, 119.08, 116.59, 113.25,
64.72, 61.39, 28.23, 25.87, 23.07, 18.09, 14.83.
4.3 Cytotoxicity assay
4.3.1 Cell culture
Cancer cell lines were obtained from the biology laboratory in the Key Laboratory of
21

ACCEPTED MANUSCRIPT
Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences (Guiyang,
China). Cells were maintained in DMEM (supplemented with 10% FBS, 100 µg/ mL penicillin, and
100 µg/mL streptomycin) and cultured at 37 ºC in a CO2 incubator (5% CO2 and 95% air, 95%
humidity). Freshly thawed cells were passaged at least 3 times before they were used in
experiments.
4.3.2 MTT assay
Cytotoxicity was evaluated by MTT assay. Briefly, PC3, MDA, K562 and HEL cells were
seeded in 96-well plates at density of 4×103 to 8×103 cells/well and allowed to adhere for 24 h.
After cells were exposed for 48 h to different concentrations of compounds, cells were evaluated
under an inverted microscope equipped with a CCD camera (Nikon, Japan). 20 µL of MTT solution
(5 mg/mL) was then added to each well and incubated at 37ºC for 4 h. The supernatant was
removed, and 150 µL DMSO solution was added. Plates were gently shaken to dissolve blue
formazan crystals and the absorbance was read at 490 nm using a microplate reader (Thermo
Scientific, Vario Skan Flash, USA).
4.4 Cell apoptosis assay
Apoptosis was evaluated by a microscope and flow cytometry based on the annexin V-
fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining kit (BD Pharmingen, San
Diego, USA). Apoptotic cells were defined as Annexin-V-positive. Compound-treated cells were
trypsinized, washed twice with PBS and transferred into microcentrifuge tubes for centrifugation at
1,000 rpm for 5 min at room temperature and resuspended in binding buffer. PI (Sigma, St. Louis,
USA) was then added to a final concentration of 20 µg/mL. The cells was transferred to a round-
bottom 96-well plate and analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, USA).
4.5 Cell cycle analysis
After treatment of the cells with compounds, cells were trypsinized, washed twice with PBS,
and transferred to microcentrifuge tubes for centrifugation at 1,000 rpm for 5 min at room
temperature. To avoid cell clumping, cells were fixed by addition of ice-cold ethanol and blocked
for 1 h at 4 °C in 5% BSA/PBS. After washing twice with PBS, the cells were then resuspended in
PBS containing 1 µg/mL RNase and incubated at 37ºC for 30 min. PI was added to a final
concentration of 20 µg/mL. Cells was transferred to round-bottom 96-well plates and analyzed by
flow cytometry using FACS Array (Becton Dickinson, Franklin Lakes, USA). Histograms of PI
signal intensity were generated and the percentages of cells in G0, G1, S and G2/M phases were
determined.
4.6 Statistical analysis
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Data were analyzed by SPSS 18.0 software and presented as mean ± SD of three independent
experiments. For all measurements, one-way ANOVA followed by Student’s test was used to assess
the statistical significance between groups. The LSD method was used to assess the statistical
significance between every two groups. A statistically significant difference was considered at the
level of P < 0.05.
Declaration of ethics
The authors declare that there is no ethical issue to declare.
Declaration of interest
The authors declare that there is no conflict of interest regarding the publication of this paper.
Acknowledgment
The authors are deeply thankful to the staff members of the analytical center for spectral
measurement and activity test centre for anti-cancer activity of the Key Laboratory of Chemistry for
Natural Products of Guizhou Province and Chinese Academy of Sciences (Guiyang, China). The
work was supported by the Specific Project of China Postdoctoral Science Foundation (No.
2015T80106), P.R. China, the Special Fund for Outstanding Youth in Science and Technology,
Guizhou Province (No. QKHRZ [2015]24), P.R. China, the Main Science Project of Guizhou
province (No. [2013]6012), P.R. China, the postdoctoral Science Foundation of Beijing (No. 2015-
ZZ-45), P.R. China and the Natural Science Foundation of Guizhou province (No. [2014]2099),
P.R. China.
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Highlights
1. A double Claisen rearrangements synthetic strategy was established for synthesis
of 3',5'-diprenylated chalcones.
2. Twenty-three novel 3',5'-diprenylated chalcones were prepared.
3. Preliminary SARs of these compounds against four cancer cell lines were
analyzed.
4. Compound 10 showed potent cytotoxicity versus all cancer cells tested.
5. Compound 10 inhibited the proliferation of cancer cells in vitro by inducing cell
apoptosis and arresting cell cycle phase.