Design, synthesis and antitumour and anti-angiogenesis evaluation of 22 moscatilin derivatives

Article abstract of DOI:10.1016/j.bmc.2019.04.027

Two series of moscatilin derivatives were designed, synthesized and evaluated as anti-tumor and anti-angiogenesis agents. Most of these compounds showed moderate-to-obvious cytotoxicity against five cancer cell lines (A549, HepG2, MDA-MB-231, MKN-45, HCT116). Among these cell lines, compounds had obvious effects on HCT116. Especially for 8Ae, the IC₅₀ was low to 0.25 μM. 8Ae can inhibit the viability and induce the apoptosis of HCT116 cells but exhibit no cytotoxic activity in noncancerous NCM460 colon cells. 8Ae can also arrest the G2/M cell cycle in HCT116 cells by inhibiting the α-tubulin expression. Zebrafish bioassay-guided screen showed the 22 moscatilin derivatives had potent anti-angiogenic activities and compound 8Ae had better activities than positive compound. Molecular docking indicated 8Ae interacted with tubulin at the affinity of ?7.2 Kcal/mol. In conclusion, compound 8Ae was a potential antitumor and anti-angiogenesis candidate for further development.

Full text of DOI:10.1016/j.bmc.2019.04.027

Accepted Manuscript
Design, synthesis and antitumour and anti-angiogenesis evaluation of 22 mo-
scatilin derivatives
Li Guan, Junting Zhou, Qinghua Lin, Huilin Zhu, Wenyuan Liu, Baolin Liu,
Yanbo Zhang, Jie Zhang, Jing Gao, Feng Feng, Wei Qu
PII:
S0968-0896(19)30498-5
https://doi.org/10.1016/j.bmc.2019.04.027
BMC 14881
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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27 March 2019
12 April 2019
16 April 2019
Please cite this article as: Guan, L., Zhou, J., Lin, Q., Zhu, H., Liu, W., Liu, B., Zhang, Y., Zhang, J., Gao, J., Feng,
F., Qu, W., Design, synthesis and antitumour and anti-angiogenesis evaluation of 22 moscatilin derivatives,
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.04.027
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Design, synthesis and antitumour and anti-angiogenesis
evaluation of 22 moscatilin derivatives
Li Guana,b, Junting Zhoua, Qinghua Lina, Huilin Zhua, Wenyuan Liuc, Baolin Liud, Yanbo Zhange,
Jie Zhanga, Jing Gaof, Feng Fenga,g,h* and Wei Qua,g*
a Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009,
China
b Institute of Medicine, Xi’an Medical University, Xi’an 710021, China
c
Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical
University), Ministry of Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing
210009, China
d
Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University,
Nanjing 210009, China
e School of Chinese Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong,
Hong Kong, China
f The Joint Laboratory of Chinese Pharmaceutical University and Taian City Centrol Hopspital,
Taian City Centrol Hospitol, Taian 271000, China
g Key Laboratory of Biomedical Functional Materials, China Pharmaceutical University, Nanjing
211198, China
h Jiang Su Food and Pharmaceutical Science College, huai'an 223003,China
* Corresponding authors
E-mail addresses: fengfeng@cpu.edu.cn (F. Feng), popoqzh@126.com (W. Qu)
Abstract:
Two series of moscatilin derivatives were designed, synthesized and evaluated as
anti-tumor and anti-angiogenesis agents. Most of these compounds showed
moderate-to-obvious cytotoxicity against five cancer cell lines (A549, HepG2,
MDA-MB-231, MKN-45, HCT116). Among these cell lines, compounds had obvious
effects on HCT116. Especially for 8Ae, the IC50 was low to 0.25 μM. 8Ae can inhibit

the viability and induce the apoptosis of HCT116 cells but exhibit no cytotoxic
activity in noncancerous NCM460 colon cells. 8Ae can also arrest the G2/M cell
cycle in HCT116 cells by inhibiting the α-tubulin expression. Zebrafish
bioassay-guided screen showed the 22 moscatilin derivatives had potent
anti-angiogenic activities and compound 8Ae had better activities than positive
compound. Molecular docking indicated 8Ae interacted with tubulin at the affinity of
-7.2 Kcal/mol. In conclusion, compound 8Ae was a potential antitumor and
anti-angiogenesis candidate for further development.
Keywords:
Antitumor; Anti-angiogenesis; Bibenzyl; Moscatilin derivatives, tubulin
1. Introduction
Cancer has become a disease seriously impact on human health. Anticancer drugs kill
cancer cells through several ways such as inhibiting sustaining proliferation,
interfering cell cycle, anti-angiogenesis, etc.1
Microtubules, composed of α- and β-tubulin that form heterodimers, are the
2
components of cytoskeleton, . Microtubules participate in many cellular processes,
including intracellular transport, maintenance of cell shape, polarity, cell signaling,
mitosis and cytokinesis3. The role of microtubules in mitosis makes them as important
cellular targets for anticancer drug developments. Because of their key role in cell
proliferation, microtubules have been recognized as one of the successful and
efficacious drug targets for the discovery of novel anticancer chemotherapeutics4-8.
Microtubule-inhibiting agents currently in clinic therapies work through suppressing
the microtubule dynamics by misdirecting the formation of functional mitotic spindle
in fast-dividing tumor cells that arrests the cells in G2–M phase, thereby leading to
apoptosis of the tumor cells.
Angiogenesis is the process of new blood vessel formation which is required for the
sustained growth and proliferation of solid tumors.9-10 Accordingly, the search for
angiogenesis inhibitors has become a leading line of investigation in anticancer
research11-12. Angiogenesis inhibitors translated into several drugs in the market have
clearly improved outcomes in patients with different tumor types and metastatic

disease.13Tumour cells could influence angiogenesis by releasing proangiogenic
molecules such as vascular endothelial growth factor (VEGF) and basic fibroblast
growth factor (bFGF) 14-15. In recent years, in vivo chemical screening in zebrafish
(TG(flia:EGFP))16-20 has emerged as an ideal method to study anti-angiogenic
substances and identify promising lead compounds.
Moscatilin [4-(4-hydroxy-3-methoxyphenethyl)-2,6-dimethoxyphenol, MST] is a
bibenzyl compound extracted from orchid Dendrobium loddigesii or D. nobile, both
of which have been used as Chinese traditional medicines for reducing fever and
replenishing body fluid 21-22(Figure 1). Moscatilin has various pharmacological
21
22
properties such as anti-inflammatory 23, antioxidant , antiplatelet aggregation and
anticancer activities against several cancer cell lines by targeting c-Jun NH2-terminal
24-25
protein kinase, inducing cell cycle G2-M arrest
and cell apoptosis 21, 25. Recently
studies indicated that MST could inhibit tumor migration and metastasis26-27
.
To discover more potent antitumor and anti-angiogenesis agents, we designed two
series of bibenzyls derived from moscatilin. We assayed their anti-proliferation
activities against 5 cancer cell lines (MKN-45, A549, HepG2, HCT116 and
MDA-MB-231) and discovered their angiogenic activities by zebrafish-guided model.
22 compounds displayed moderate to good activities, especially for compound 8Ae.
8Ae could inhibit cell viability and induce apoptosis on HCT116 effectively. It could
also arrest G2/M cell cycle by inhibit α-tubulin protein expression. Molecular docking
indicated that the affinity of 8Ae-tubulin complex was -7.2 Kcal/mol. In conclusion,
compound 8Ae was a potential antitumor and anti-angiogenic candidate for further
development.
2. Results and discussion
2.1 Chemistry
Moscatilin (MST) and its two series (A and B) derivatives were synthesized with
Wittig reaction as a key step, which was used widely for the synthesis of bibenzyl
skeleton.28 The hydroxyl group of 1 should be first acetylated to gain
4-acetoxy-3,5-dimethoxylbenzaldehyde (1a). 4-Acetoxy-3,5-dimethoxyl benz-
aldehyde(1a) or 3,4-dimethoxybenzaldehyde (1b) were used as the starting material

for A and B series respectively. The aldehyde of 1a or 1b was first reducted by sodium
borohydride to get 2a or 2b. The hydroxyl of 2a or 2b was chlorinated to obtain 3a or
3b, respectively. 3a and 3b reacted with triphenylphosphine to prepare the two
phosphonium salt (4a and 4b), the key reagent for the Wittig reaction. The
phosphonium salt was dissolved completely with R-replaced benzaldehyde in THF
and deprotonated with NaH to yield a mixture of Z- and E-stilbenes, which were
directly reduced by treatment with catalytic amounts of 10% Pd-C over H2 (4 atm) to
afford bibenzyl skeleton. After deacetylation with sodium methylate, moscatilin and
its two series derivatives were finally obtained through a total 6-step procedure, which
were summarized inScheme 1.
Scheme 1 Synthesis of moscatilin and its A/B series analogs. a) Acetic anhydride,
DCM, DMAP, rt, 30 min; b) NaBH4, EtOH, 0 °C, rt, 30 min; c) SOCl2, Et3N, DCM,
0 °C, rt, 3 h; d) PPh3, toluene, reflux, 5 h; e) benzaldehyde analogs, NaH, THF, Ar,
0 °C, rt, overnight; f) H2, 10% Pd/C, EtOAc, 4 atm; g) MeONa / MeOH, rt, 30 min;
H+/H2O.
All compounds were purified by recrystallization or chromatography technology. The
22 compounds which contain 14 of A series (X ring: R1=OH, R2=OMe) and 8 of B
series(X ring: R1=OMe, R2=H) showed good drug property (ClogP<5). Their
structure were shown in Table 1. The analytical and spectroscopic data confirmed
their structures, which were shown as details in the experimental section.
Table 1. The structure of 22 moscatilin derivatives

Compound
Compound
ClogP
ClogP
R
H
H
R1
OH OMe 3.55
OMe H 4.25
R2
R
R1
OH OMe 4.42
OMe H 3.7
OH OMe 4.39
OMe H 2.06
R2
1Aa
2Ba
13Aj
14Bj
p-fluro-
p-fluro-
15Ak o-aza
16Bk o-aza
17Am p-aza
3Ab 3,4-dichloro-
4Ac p -NO2-
5Bc p -NO2-
OH OMe 4.86
OH OMe 3.3
OMe H
OH OMe 4.27
OMe H 4.96
OH OMe 4.27
3.99
OH OMe 2.75
OH OMe 2.06
OH OMe 3.7
p-chloro-
p-chloro-
o-fluro
m-fluro
6Ad
7Bd
18As
19At
8Ae o-chloro-
20Au 2',4'-dichloro- OH OMe 3.7
o-chloro-
OMe H
OMe H
OMe H
4.96
3.43
2.69
2',3'-dichloro- OH OMe 4.98
2'-methoxy OH OMe 4.86
moscatilin 3.47
9Be
21Av
3'-hydroxy-4'-methoxy
4'-hydroxy-3'-methoxy
p-bromo-
10Bf
11Bg
12Ai
22Aw
23MST
OH OMe 3.43
2.2 In Vitro Antiproliferative Activities
The newly synthesized MST and its two series of 22 derivatives were initially
evaluated for their antiproliferative activities against five human cancer cell lines,
A549 (human lung carcinoma), HepG2 (human liver carcinoma), MDA-MB-231
(human breast carcinoma), MKN-45 (human gastric carcinoma) and HCT116 (human
colon carcinoma) by the conventional MTT assay, using MST and gambogic acid(GA)
as reference agent. The results were shown in Table 2.
For 22 MST derivatives, Y rings were riched with substituted groups, like
hydrogen, halogen, methoxy, nitro and pyridine ring and different substituted
position. In total, A series were obviously with better activities than B series for four
cancer cell lines (A549 cells, HepG2 cells, MDA-MB-231 cells and MKN-45 cells),
which indicated that the 4-hydroxyl and 5-OMe in compounds is of great importance
for the activity. However, for HCT116 cells, A and B seires have no obvious
difference, both of which exhibited better activity than other four cell lines.
Compound 8Ae (Fig2A), as a star molecular, aroused our attention. For its Y ring,
an ortho-chloro group was substituted, which exhibited the most potent activity than
others. We then synthesized 3 compounds (20Au, 21Av and 22Aw) with ortho
substitution and all of them showed good activities similar to 8Ae, which confirmed
that ortho substituted compounds may improve the activity to a large extent.
Replacement ofR=o-chloro (8Ae) by R= o-OMe (22Aw) killed less tumor cells, but
still have better activity than others, which revealed that ortho-chloro had positive

effect on anticancer treatment. Compound 8Ae might be a new novel compound than
MST for the deep research.
Table 2. The antiproliferative activity of 22 MST derivatives against 5 cancer
cells.
IC50(μM)a
Com.
A549
10.94±1.21 7.83±0.97 >25
>25 2.08±0.12 >25
HepG2
MDA-MB-231 MKN-45
HCT116
1.04±0.03 0.53±0.02
21.94±2.06 2.56±0.29
20.64±1.73 1.86±0.02
15.43±2.90 3.16±0.83
13.72±1.40 5.97±0.31
1Aa
2Ba
13.43±1.19 16.08±1.33 2.52±0.19
9.13±0.27 11.02±0.38 8.05±0.26
3Ab
4Ac
>25
22.92±2.12 8.78±
>25
2.13±0.38 2.19±0.22 21.07±1.41
>25 4.93±0.54 >25
1.6±0.10
>25
5Bc
>25
>25
0.33±0.01
6Ad
0.74±0.12 1.25±0.21
1.52±0.17 0.52±0.02
24.53±1.98 0.25±0.01
2.04±0.11 6.16±0.31
7Bd
8Ae
9Be
4.79±0.71 4.05±0.71 0.081±0.02
3.64±0.28 2.27±0.23 1.64±0.21
>25
5.26±0.28
10Bf
11Bg
12Ai
13Aj
14Bj
15Ak
16Bk
17Am
18As
19At
20Au
21Av
22Aw
23MST
GA
2.06±0.21 2.69±0.04
1.49±0.05 15.85±1.24
10.7±1.04 1.19±0.02
3.98±0.06 7.89±0.43
0.66±0.12 3.06±0.38
11.07±1.49 0.93±0.02
3.81±0.21 3.74±0.22
>25
8.49±1.09 >25
4.15±0.65 5.47±0.33 >25
>25
>25
>25
>25
>25
>25
0.92±0.10
4.84±0.72 >25
13.75±1.42 10.49±2.11
2.17±0.40 6.3±0.74
1.03±0.15 9.22±0.88
>25
0.88±0.08
2.24±0.12 12.39±1.39 >25
3.84±0.33 6.74±0.70 >25
3.99±0.41 4.77±0.48 6.87±0.92
1.74±0.12 2.48±0.34 >25
2.97±0.08 7.57±0.43
4.46±0.13 2.98±0.05
0.011±0.21 8.78±0.39
2.48±0.17 14.53±1.01
3.51±0.31 2.29±0.02
4.27±0.65 3.11±0.43
2.1±0.15
3.76±0.74 0.082±0.02
1.47±0.11 2.36±0.42 1.33±0.12
2.3 Effect of 8Ae on noncancerous NCM460 colon cells
We used 8 different concentrations of 8Ae (0.1μM, 1μM, 2μM, 5μM, 10 μM,
20μM, 30μM, 40μM and 50 μM) to detect its toxic effects on NCM460 colon cells.
The cell viability of noncancerous NCM460 colon cells after different concentration
of 8Ae treatments was shown in Fig 1. It was observed that compound 8Ae had little
toxic effect, the cell viability all exceed 84.4% (50 μM). Therefore, we concluded
that 8Ae was toxic to human colon carcinoma HCT116 cells and had little effect on
normal NCM460 colon cells, which suggested that 8Ae could be a good candidate

for colon carcinoma therapy.
Fig 1 Cell viability of NCM460 after 8Ae treatments
2.3 Effect of 8Ae on cell viability and apoptosis
The structure of compound 8Ae was shown in Fig 2A. The morphological
assessment in HCT116 cells treated with 1, 5, and 10 μM of compound 8Ae for 48 h
were shown in Fig2B. With the increased concentration of 8Ae, HCT116 cells were
distorted significantly, grew slowly, and some cells appeared tensile deformation,
while the untreated cells displayed normal and healthy shapes. To evaluate whether
8Ae could induce apoptotic cell death, DAPI staining was used. As shown in Fig2C,
untreated HCT116 cells were stained equably with blue fluorescence under the
fluorescent microscope, demonstrating the steady chromatinic distribution in
nucleolus. 8Ae-treated HCT116 cells emitted bright fluorescence, owing to the
chromatin congregation and the nucleolus pyknosis, which are considered as the early
phenomena of apoptosis. In further, Annexin V/PI staining assay was used to confirm
the apoptosis induced by 8Ae. After cells were treated with 1, 5 and 10 µM 8Ae or
gambogic acid(GA) for 48 h, the early apoptotic rates were 24.8%, 34.5%, 36.9% and
33.0%, respectively, and their late apoptotic rates were 18.1%, 22.4%, 37.2% and
31.8%, respectively (Fig 2D). Quantitative analysis of Annexin V/PI double-staining
was shown in Fig 2E. Taken together, these results indicated that compound 8Ae have
the ability to inhibit cell growth and induce typical apoptosis in human colon

carcinoma HCT116 cells.
Fig 2.Compound 8Ae inhibits the viability and induces apoptosis of HCT116 Cells. A)
The structure of compound 8Ae B). Morphological changes of HCT116 cells were
observed under an inverted light microscope (400×) C) Nucleolus morphologic
changes observed by fluorescent microscope (400×).Apoptotic cells were observed
for apoptotic bodies and nuclei pyknosis. D) Annexin V/PI double-staining assay of
the toxicity of 8Ae (1, 5, and 10 μM) and GA (1 μM) was analyzed by flow cytometry.
(E) Quantitative analysis of Annexin V/PI double-staining.
2.4 Effect of 8Ae on cell-cycle in HCT116 cells.
To evaluate whether the inhibition of growth due to compound 8Ae is associated
with cell-cycle perturbation, the distribution of cells in different phases of the cell
cycle was determined by flow cytometry, analyzed using FlowJo7.6 analysis software.
The control group of HCT116 cell population profile indicates that most of the cells
were in G1- and S-phases, for G1/G0 53.97%, G2/M 11.88% and S 34.15%. The
compound Ae caused a statistical significant increase in the G2–M cell population
from 11.88% to 85.22% in 5 μM and to 82.47% in 10 μM, respectively for 48 hours
and concomitant decrease in G1 and S cell population was observed. The distribution
of cells in different phases of the cell cycle is summarized in Fig 3A and quantitative
analysis in Fig 3B. Western blot analysis showed that compound 8Ae could down
regulate the α-tubulin expression, which could influence the cell cycle (Fig3C). The

data above showed that compound 8Ae could induce apoptosis by arresting G2/M via
reducing α-tubulin expression.
Fig 3 Effects of compound 8Ae on HCT116 cell cycle progress. (A) Cells were
treated with different concentrations of 8Ae (5μM and 10μM) or control group for 48
h and then analyzed by flow cytometry. (B) Quantitative analysis of cell cycle
progress. (C) The expression of α-tubulin in HCT116 cells.
2.5 In vivo zebrafish-guided angiogenesis assay.
In order to evaluate the anti-angiogenic activities of the synthesized compounds in
an efficient way, we firstly screened all the compounds at 50 μM in zebrafish-guided
assay model. Compounds with significant effect were assayed the minimum effective
dose. After anesthetized with 0.016% tricaine (Sigma-Aldrich), the inter-segmental
blood vessels (ISVs) of embryos were observed and imaged at 72 hpf under a
fluorescence microscope (IX71, Olympus, Japan). SU5416 was served as a positive
control. The minimum effective doses were showed in Table 3. Compound 8Ae still
exhibited the most potent activity in all the 24 compounds (Fig 4).
Table 3. Antiangiogenic activity of moscatilin analogues.

Com.
Effective dose(μM) 20-25 >50 20-25 NT
Com. 9Be
Effective dose(μM) 20-25 40-50 >50 2.5-5.0 >50 >50
Comp.
Effective dose(μM) >50
1Aa 2Ba 3Ab 4Ac
5Bc 6Ad
7Bd
8Ae
>50 >50
>50
0.62-1.25
10Bf 11Bg 12Ai 13Aj 14Bj
15Ak 16Bk
25-50 >50
17Am 18As 19At 20Au 21Av 22Aw MST SU5416
10-15 15-20 5-10 5-10 1.25-2.5 2.5-5.0 1.25-2.5
Fig 4 Compound 8Ae inhibited ISVs angiogenesis in zebrafish. Lateral view of
Tg(fli1:EGFP) zebrafish embryos at 72 hpf immersed in control (0.1% DMSO), 8Ae
and positive SU5416 (40 × magnification) .
2.6 Molecular docking
The theoretical binding mode between 8Ae and tubulin is shown in Fig 5. Compound
8Ae adopted an “V-shaped” conformation in the pocket of tubulin. The bibenz group
of 8Ae stretched into the hydrophobic pocket that consisted of Leu-255, Ala-316,
Ala-317 and Thr-353, forming stable hydrophobic bonds. Detailed analysis showed
that the 2’-chlorophenyl group of 8Ae formed CH–π interactions with the residues of
Leu-255, Ala-316 and Ala-317. It was shown that the residues of Glu-200 (bond
length: 1.8 Å), Tyr-202 (length: 3.1 Å) formed two hydrogen bonds with 8Ae, which
were the main interactions between 8Ae and tubulin. In addition, the estimated
binding energies was −7.4 kcal mol−1 for 8Ae.

Fig 5 The docking of 8Ae binding to the active pocket of tubulin (4YJ2).
3. Conclusion
In summary, two series of moscatilin derivatives were designed, synthesized and
evaluated as antitumor and anti-angiogenesis agents. Most of these compounds
exhibited good anti-proliferative activities against five cancer cell lines. Compound
8Ae, as a star molecular, could inhibit the viability and apoptosis in HCT116 cells but
showed little toxic effect on normal NCM460 colon cells. The early apoptotic rates
were 24.8%-36.9% higher than the late apoptotic rates (18.1%, 22.4%-37.2%).
Meanwhile, 8Ae effected the cell cycle progress and arrests G2/M cell cycle by
decreasing the α-tubulin expression. Molecular docking indicated that 8Ae-tubulin
complex had a good affinity at -7.4 kcal mol-1. In vivo zebrafish-guided
anti-angiogenesis assay showed that 22 moscatilin derivatives had different effective
doses. 8Ae as the best molecular whose minimum effective dose low to 0.62-1.25 μM.
Therefore, our studies indicated that 8Ae is a promising candidate as an anticancer
and anti-angiogenic agent.
4. Experimental section
4.1 Chemistry
All chemicals (reagent grade) used were purchased from Aladdin Shanghai
Biochemical Technology Co., Ltd. or Sinopharm Chemical Reagent Co., Ltd. (China).
Reaction progress was monitored using analytical thin layer chromatography (TLC)

on precoated silica gel GF254 (Qingdao Haiyang Chemical Plant, Qingdao, China)
plates and the spots were detected under UV light (λ=254 nm).
13
1H and C NMR spectra were measured on a Bruker 300 MHz spectrometer at
298T. Chemical shifts were reported in ppm using the residual solvent line as internal
standard. Splitting patterns were designed as s (singlet), d (doublet), t (triplet), m
(multiplet).
Mass spectra were obtained on an MS Agilent 1100 Series LC/MSD Trap mass
spectrometer (ESI-MS). Column chromatography was performed on silica gel
(200-300 mesh; Qingdao Marine Chemical Inc.)
4.1.1. Synthetic intermediates
4.1.1.1. 4-formyl-2,6-dimethoxyphenyl acetate (1a)
Syringaldazine (8.33 g, 0.046 mol) and catalyst DMAP were dissolved in DCM (10
mL), then acetic anhydride (4.75 mL, 0.051mol) was slowly added. After the mixture
was stirred at room temperature for 1 h, the solution was washed with diluted water
(10 mL × 3). Organic layer was dried over anhydrous Na2SO4 and concentrated to get
white crystals 10.19 g, 99.8 % yield; mp 118-119 ºC; IR vmax (KBr, cm-1): 3099, 2940,
1
1759, 1691, 1603, 1499; H-NMR (300 MHz, CDCl3) δ 9.93(1H, s, -CHO), 7.17(2H,
s, H-3, H-5), 3.92(6H, s, 2×-OCH3), 2.38(3H, s, -COCH3); 13C-NMR (75 MHz,
CDCl3) δ 191.1, 168.1, 152.8, 134.3, 115.0, 106.0, 56.3, 20.4; ESI-MS m/z: 225.1
[M+H]+ .
4.1.1.2. 4-(hydroxymethyl)-2,6-dimethoxyphenyl acetate (2a)
To a solution of 1a (8.6 g, 0.038 mol) in EtOH (10 mL), NaBH4 (0.53g, 0.014mol)
was slowly added under ice bath. After stirred at room temperature for 30 min, 5%
HCl was added to adjust pH to 7. Concentrated under vacuum, residue was extracted
with DCM (10 mL × 3), then organic layer was dried over anhydrous Na2SO4 and
concentrated to get white solid 8.34 g, 96.5 % yield; mp 98-99 ºC; IR vmax (KBr, cm-1):
3530, 3126, 2944, 1748, 1605, 1508; 1H-NMR (300 MHz, CDCl3) δ 6.66 (1H, s, H-3,
H-5), 4.69 (2H, s, Ar-CH2-), 3.84 (6H, s, 2×-OCH3), 2.36 (3H, s, -COCH3); 13C-NMR
(75 MHz, CDCl3) δ 168.8, 152.1, 139.6, 115.3, 103.1, 65.2, 56.1, 20.5; ESI-MS m/z:
227.1 [M+H]+.

4.1.1.3. 4-(chloromethyl)-2,6-dimethoxyphenyl acetate (3a)
To a solution of 2a (8.3 g, 0.037 mol) in DCM (10 mL), Et3N (6.2 mL, 0.043mol),
SOCl2 (3.0 mL, 0.043mol) was slowly dropped under ice salt bath. After stirred at
room temperature for 3 h, the solution was washed with diluted water (10 mL × 3).
Organic layer was dried over anhydrous Na2SO4 and concentrated to get light yellow
solid 7.96 g, 88.7 % yield; mp 75-76 ºC; IR vmax (KBr, cm-1): 3162, 2971, 1759, 1606,
1
1508; H-NMR (300MHz, CDCl3) δ 6.85 (1H, s, H-3, H-5), 4.73 (2H, s, Ar-CH2-),
13
3.76 (6H, s, 2×-OCH3), 2.24 (3H, s, -COCH3); C-NMR (75 MHz, CDCl3) δ 168.3,
152.0, 139.4, 115.0, 103.2, 60.7, 56.1, 20.4; ESI-MS m/z: 227.1 [M+H]+.
4.1.1.4. (4-acetoxy-3,5-dimethoxybenzyl) triphenylphosphonium chloride (4a)
3a (7.8 g, 0.032 mol) and PhP3 (8.4g, 0.032 mol) were dissolved in toluene (5 mL)
under reflux for 6 h. After cooled to temperature, the mixture was filtrated and washed
with toluene under vacuum. Dried under infrared light and 14.63 g white powder was
1
obtained, 90.2 % yield; H-NMR (300 MHz, DMSO-d6) δ 7.92 (3H, m, ArH), 7.76
(6H, dt, J = 3.6Hz, 8.1Hz, ArH), 7.69 (6H, m, ArH), 6.30 (2H, d, J = 2.4Hz, H-3, H-5),
5.09 (2H, J = 15Hz, Ar-CH2-), 3.37 (6H, s, 2×-OCH3 ), 2.21 (3H, s, ×-OCH3);
13C-NMR (75 MHz, DMSO-d6) δ 168.4, 152.0, 151.9, 135.5, 134.8, 134.6, 130.6,
130.4, 126.9, 126.7, 118.8, 117.6, 108.5, 108.4, 56.2, 29.4, 28.8, 20.6; ESI-MS m/z:
541.2 [M+Cl]-.
4.1.2 General procedure for the preparation of 1Aa~23MST
4a (500 mg, 0.99 mmol) and corresponding benzaldehyde analogs were dissolved
in anhydrous THF (3 mL) under Ar protection. NaH (28.4 mg, 1.18 mmol) was added
slowly under ice salt bath. After overnight reaction in room temperature, the solution
was concentrated. Then the mixture of trans- and cis- stilbenes was recrystallized in
methanol. To the mixture of trans- and cis- stilbenes in 15 mL MeOH, 30 mg 10%
Pd/C was added, violently stirring under a hydrogen atmosphere. After completion of
the reaction, Pd/C was filtered and washed with little MeOH. Then CH3ONa was
added to the reaction mixture to remove the acetyl group. The residue was purified by
silica gel column chromatography (petroleum ether- ethyl acetate = 20:1~5:1 as the
eluent) to afford products.

4.1.2.1 4-(4-hydroxy-3-methoxyphenethyl)-2,6-dimethoxyphenol (23MST). Yield 51%;
white crystal; 1H-NMR (300 MHz, CDCl3) δ6.85 (1H, d, J = 8.1 Hz, H-5’), 6.78 (1H,
d, J = 8.1 Hz, H-6’), 6.63 (1H, s, H-2’), 6.38 (2H, s, H-3, H-5), 5.52, 5.41 (2H, s,
ArOH), 3.86 (9H, s, 3×-OCH3), 2.83 (4H, s, -CH2CH2-); 13C-NMR (75MHz, CDCl3) δ
160.2, 155.8, 132.4, 128.5, 127.8, 127.3, 121.1, 115.0, 111.8, 105.1, 56.2, 56.1, 38.1,
37.6; ESI-MS m/z: 327.1 [M+Na]+.
1
4.1.2.2 2,6-dimethoxy-4-phenethylphenol (1Aa). Yield 45 %; yellow oil; H-NMR
(300 MHz, CDCl3) δ 7.33, 7.24, 7.20 (5H, m, ArH), 6.37 (2H, s, H-2, H-6), 5.40 (1H,
13
brs, -OH), 3.86 (6H, s, 2×-OCH3), 2.89 (4H, m, ArCH2CH2Ar); C-NMR (75 MHz,
CDCl3) δ 150.1, 144.8, 143.1, 141.8, 131.9, 130.5, 130.2, 128.1, 105.1, 56.3, 38.7,
37.3; ESI-MS m/z: 281.1 [M+Na]+, 267.1 [M-H]-.
1
4.1.2.3 1, 2-dimethoxy-4-phenethylbenzene (2Ba). Yield 62 %; yellow oil; H-NMR
(300 MHz, CDCl3) δ 7.34-7.19 (5H, m, ArH), 6.82 (1H, d, J = 8.1 Hz, H-6), 6.75 (1H,
dd, J = 2.1, 8.1 Hz, H-5), 6.66 (1H, d, J = 1.8 Hz, H-3), 3.89 (3H, s, -OCH3), 3.85 (3H,
13
s, -OCH3), 2.91 (4H, brs, -CH2CH2-); C-NMR (75 MHz, CDCl3) δ 149.2, 144.8,
143.1, 141.8, 131.9, 130.5, 130.2, 128.1, 105.1, 56.3, 37.7, 37.1; ESI-MS m/z:
243.1[M+H]+.
4.1.2.4 4-(3,4-dichlorophenethyl)-2,6-dimethoxyphenol (3Ab). Yield 51 %; yellow
1
crystal; H-NMR (300 MHz, CDCl3) δ 7.34 (1H, d, J = 8.1Hz, H-5’), 7.27 (1H, brs,
H-2’), 6.98 (1H, dd, J = 1.8, 8.1Hz, H-6’), 6.35 (1H, s, H-2, H-6), 5.41 (1H, brs,
ArOH), 3.86 (6H, s, 2×-OCH3), 2.84 (4H, m, -CH2CH2-); 13C-NMR (75 MHz, CDCl3)
δ 148.0, 146.8, 142.2, 141.8, 131.9, 130.5, 130.2, 128.1, 105.1, 56.3, 37.7, 37.3;
ESI-MS m/z: 349.0 [M+Na]+, 325.1 [M-H]-.
4.1.2.5 2,6-dimethoxy-4-(4-nitrophenethyl) phenol (4Ac). Yield 39 %; yellow oil;
1H-NMR (300 MHz, CDCl3) δ 8.15 (2H, d, J = 8.7 Hz, H-3’, H-5’), 7.31 (2H, d, J =
8.7 Hz, H-2’, H-6’), 6.34 (2H, s, H-2, H-6), 5.41 (1H, s, ArOH), 3.0 (4H, m,
13
-CH2CH2-); C-NMR (75MHz, CDCl3) δ 160.2, 155.8, 132.4, 128.5, 127.8, 127.3,
121.1, 115.0, 111.8, 105.1, 56.2, 56.1, 38.1, 37.6; ESI-MS m/z: 304.1 [M+H]+.
4.1.2.6 1,2-dimethoxy-4-(4-nitrophenethyl) benzene (5Bc). Yield 57 %; yellow solid;
1H-NMR (300 MHz, CDCl3) δ 7.34-7.19 (5H, m, ArH), 6.82 (1H, d, J = 8.1 Hz, H-6),

6.75 (1H, dd, J = 2.1, 8.1 Hz, H-5), 6.66 (1H, d, J = 1.8 Hz, H-3), 3.89 (3H, s, -OCH3),
13
3.85 (3H, s, -OCH3), 2.91 (4H, brs, -CH2CH2-); C-NMR (75MHz, CDCl3) δ 157.2,
155.8, 132.4, 128.5, 127.8, 127.3, 121.1, 115.0, 111.8, 105.1, 56.2, 56.1, 38.1, 37.6;
ESI-MS m/z: 380.3 [M+Na]+, 286.1 [M-H]-.
4.1.2.7 4-(4-chlorophenethyl)-2,6-dimethoxyphenol (6Ad). Yield 36 %; yellow solid;
1H-NMR (300 MHz, CDCl3) δ 7.25 (2H, d, J = 8.4 Hz, H-3’, H-5’), 7.09 (2H, d, J =
8.1 Hz, H-2’, H-6’), 6.34 (2H, s, H-2, H-6), 5.43 (1H, s, ArOH), 3.85(6H, s,
2×-OCH3), 2.86 (4H, m, -CH2CH2-); 3C-NMR (75 MHz, CDCl3) δ 148.0, 146.8, 142.2,
141.8, 131.9, 130.5, 130.2, 128.1, 105.1, 56.3, 37.7, 37.3; ESI-MS m/z: 315.1
[M+Na]+.
4.1.2.8 4-(4-chlorophenethyl)-1,2-dimethoxybenzene (7Bd). Yield 49 %; yellow oil;
1H-NMR (300 MHz, CDCl3) δ 7.25 (2H, d, J = 8.4 Hz, H-3’, H-5’), 7.09 (2H, d, J =
8.4 Hz, H-2’, H-6’), 6.80 (1H, d, J = 8.4 Hz, H-5), 6.70 (1H, dd, J = 1.8, 8.4 Hz, H-5),
6.70 (1H, d, J = 1.8 Hz, H-3), 3.88 (3H, s, -OCH3), 3.85 (3H, s, -OCH3), 2.86 (4H, m,
13
-CH2CH2-); C-NMR (75MHz, CDCl3) δ 159.3, 156.3, 131.4, 128.7, 127.5, 127.3,
121.1, 115.0, 111.8, 105.1, 56.2, 56.1, 38.1, 37.6; ESI-MS m/z: 314.7 [M+K]+.
4.1.2.9 4-(2-chlorophenethyl)-2, 6-dimethoxyphenol (8Ae). Yield 47 %; white solid;
1H-NMR (300 MHz, CDCl3) δ7.38 (1H, m, H-3’), 7.18-7.11 (3H, m, H-4’, 5’, 6’),
6.39 (2H, s, H-3, H-5), 5.41 (1H, s, ArOH), 3.87 (6H, s, 2×-OCH3), 3.05-2.84 (4H, m,
13
-CH2CH2-); C-NMR (75MHz, CDCl3) δ 160.2, 155.8, 132.4, 128.5, 127.8, 127.3,
121.1, 115.0, 111.8, 105.1, 56.2, 56.1, 38.1, 37.6; ESI-MS m/z: 315.1 [M+Na]+.
4.1.2.10 4-(2-chlorophenethyl)-1,2-dimethoxybenzene (9Be). Yield 69 %; yellow oil;
1H-NMR (300 MHz, CDCl3) δ 7.38 (1H, m, H-3’), 7.19-7.16 (3H, m, ArH), 6.82 (1H,
d, J = 8.1 Hz, H-6), 6.77 (1H, dd, J = 1.8, 8.1 Hz, H-5), 6.70 (1H, brs, H-3), 3.89 (3H,
3
s, -OCH3), 3.86 (3H, s, -OCH3), 3.07-2.87 (4H, m, -CH2CH2-); C-NMR (75 MHz,
CDCl3) δ 148.0, 146.8, 142.2, 141.8, 131.9, 130.5, 130.2, 128.1, 105.1, 56.3, 37.7,
37.3; ESI-MS m/z: 299.1 [M+Na]+.
4.1.2.11 5-(3,4-dimethoxyphenethyl)-2-methoxyphenol (10Bf). Yield 54 %; white solid;
1H-NMR (300 MHz, CDCl3) δ 6.82-6.63 (6H, ArH), 5.62 (1H, brs, ArOH), 3.87 (6H,
13
s, 2×-OCH3), 3.86 (3H, s, -OCH3), 2.83 (4H, brs, -CH2CH2-); C-NMR (75MHz,

CDCl3) δ 158.5, 146.4, 145.1, 144.8, 142.9, 134.4, 120.2, 119.8, 115.0, 111.9, 111.2,
110.6, 56.0, 55.9, 55.8, 37.6, 37.5; ESI-MS m/z: 311.1 [M+Na]+.
4.1.2.12 4-(3,4-dimethoxyphenethyl)-2-methoxyphenol (11Bg). Yield 39 %; white
solid; 1H-NMR (300 MHz, CDCl3) δ 6.67-6.64 (6H, ArH), 5.50 (1H, brs, ArOH), 3.88
(3H, s, -OCH3), 3.86 (6H, s, 2×-OCH3), 2.85 (4H, brs, -CH2CH2-); 13C-NMR (75MHz,
CDCl3) δ 160.2, 155.8, 132.4, 128.5, 127.8, 127.3, 121.1, 115.0, 111.8, 105.1, 56.2,
56.1, 38.1, 37.6; ESI-MS m/z: 311.1 [M+Na]+.
4.1.2.13 4-(4-bromophenethyl)-2, 6-dimethoxyphenol (12Ai). Yield 32 %; yellow oil;
1H-NMR (300 MHz, CDCl3) δ 7.42-7.02 (4H, m, ArH), 6.37 (2H, s, H-3, H-5), 5.41
(1H, s, ArOH), 3.86 (6H, s, 2×-OCH3), 2.95-2.85 (4H, m, -CH2CH2-); 13C-NMR
(75MHz, CDCl3) δ 152.2, 155.8, 132.4, 127.5, 127.8, 127.3, 121.1, 115.0, 111.8,
105.1, 56.2, 56.1, 38.1, 37.6; ESI-MS m/z: 359.0 [M+Na]+, 335.1 [M-H]-.
4.1.2.14 4-(4-fluorophenethyl)-2, 6-dimethoxyphenol (13Aj). Yield 35 %; yellow solid;
1H-NMR (300 MHz, CDCl3) δ 7.10 (2H, m, H-3’, H-5’), 6.97 (2H, m, H-2’, H-6’),
6.34 (2H, s, H-3, H-5), 5.41 (1H, s, ArOH), 3.85 (6H, s, 2×-OCH3), 2.85 (4H, m,
13
-CH2CH2-); C-NMR (75MHz, CDCl3) δ 160.2, 155.8, 132.4, 128.5, 127.8, 127.3,
121.1, 115.0, 111.8, 105.1, 56.2, 56.1, 38.1, 37.6; ESI-MS m/z: 299.1 [M+Na]+, 275.1
[M-H]-.
4.1.2.15 4-(4-fluorophenethyl)-1,2-dimethoxybenzene (14Bj). Yield 45 %; yellow
1
solid; H-NMR (300 MHz, CDCl3) δ 7.12 (2H, m, H-3’, H-5’), 6.97 (2H, m, H-2’,
H-6’), 6.81 (1H, d, J = 8.1 Hz, H-6), 6.71 (1H, dd, J = 1.8, 8.1 Hz, H-5), 6.64 (1H, d,
J = 1.8 Hz, H-3), 3.88 (3H, s, -OCH3), 3.85 (3H, s, -OCH3), 2.87 (4H, m, -CH2CH2-);
13C-NMR (75MHz, CDCl3) δ 157.2, 154.8, 132.4, 128.5, 127.8, 127.3, 121.1, 115.0,
111.8, 105.1, 56.2, 56.1, 38.1, 37.6; ESI-MS m/z: 282.7 [M+Na]+, 260.6[M+H]+.
4.1.2.16 2, 6-dimethoxy-4-(2-(pyridin-2-yl)ethyl)phenol (15Ak). Yield 51 %; yellow
solid; 1H-NMR (300 MHz, CDCl3) δ 8.57 (1H, d, J = 3.9 Hz, H-3’), 7.57 (1H, dt, J =
1.8, 7.8 Hz, H-4’), 7.12 (1H, m, H-5’), 7.08 (1H, d, J = 7.8 Hz, H-6’), 6.39 (2H, s,
H-3, H-5), 5.89 (1H, brs, ArOH), 3.81 (6H, s, 2×-OCH3), 3.10-2.94 (4H, m,
13
-CH2CH2-); C-NMR (75MHz, CDCl3) δ 161.1, 160.6, 149.2, 147.0, 136.4, 132.5,
128.8, 123.2, 121.2, 115.0, 105.1, 56.1, 40.6, 36.4; ESI-MS m/z: 299.1 [M+Na]+,

275.1 [M-H]-.
4.1.2.17 2-(3,4-dimethoxyphenethyl)pyridine (16Bk). Yield 57 %; yellow oil;
1H-NMR (300 MHz, CDCl3) δ 8.58 (1H, qt, J = 0.9, 4.8 Hz, H-3’), 7.58 (1H, dt, J =
1.8, 7.8 Hz, H-4), 7.13 (1H, m, H-5), 7.09 (1H, d, J = 7.5 Hz, H-6), 3.86 (3H, s,
-OCH3), 3.83 (3H, s, -OCH3), 3.05 (4H, m, -CH2CH2-); 13C-NMR (75MHz, CDCl3) δ
151.1, 150.6, 149.2, 147.0, 136.4, 132.5, 128.8, 123.2, 121.2, 115.0, 105.1, 56.1, 40.6,
36.4; ESI-MS m/z: 266.1 [M+Na]+, 244.1[M+H]+.
4.1.2.18 2,6-dimethoxy-4-(2-(pyridin-4-yl)ethyl)phenol (17Am). Yield 46 %; yellow
solid; 1H-NMR (300 MHz, CDCl3) δ 8.50 (2H, d, J = 5.7 Hz, H-3’, 5’), 7.09 (2H, d, J
= 4.0 Hz, H-2’, 6’), 6.33 (2H, s, H-3, H-5), 5.31 (1H, brs, ArOH), 3.84 (6H, s,
2×-OCH3), 2.89 (4H, m, -CH2CH2-); 13C-NMR (75MHz, CDCl3) δ 160.2, 155.8,
132.4, 128.5, 127.8, 127.3, 121.1, 115.0, 111.8, 105.1, 56.2, 56.1, 38.1, 37.6; ESI-MS
m/z: 282.1 [M+Na]+.
4.1.2.19 4-(2-fluorophenethyl)-2,6-dimethoxyphenol (18As). Yield 51 %; yellow solid;
1H-NMR (300 MHz, CDCl3) δ 7.21-7.01 (4H, m, ArH), 6.38 (2H, s, H-3, H-5), 5.40
(1H, brs, ArOH), 3.86 (6H, s, 2×-OCH3), 2.86 (4H, m, -CH2CH2-); 13C-NMR (75MHz,
CDCl3) δ 157.2, 154.8, 132.4, 128.5, 127.8, 127.3, 121.1, 115.0, 111.8, 105.1, 56.2,
56.1, 38.1, 37.6; ESI-MS m/z: 299.1 [M+Na]+.
4.1.2.20 4-(3-fluorophenethyl)-2,6-dimethoxyphenol (19At). Yield 37 %; yellow oil;
1H-NMR (300 MHz, CDCl3) δ 7.23 (1H, m, H-2’), 6.95-6.88 (3H, m, ArH), 6.36 (2H,
s, H-3, H-5), 5.43 (1H, brs, ArOH), 3.86 (6H, s, 2×-OCH3), 2.87 (4H, m, -CH2CH2-);
13C-NMR (75MHz, CDCl3) δ 161.1, 160.6, 149.2, 147.0, 136.4, 132.5, 128.8, 123.2,
121.2, 115.0, 105.1, 56.1, 40.6, 36.4; ESI-MS m/z: 299.1 [M+Na]+.
4.1.2.21 4-(2,4-dichlorophenethyl)-2,6-dimethoxyphenol (20Au). Yield 40 %; yellow
1
oil; H-NMR (300 MHz, CDCl3) δ 7.39 (1H, d, J = 2.1 Hz, H-3’), 7.14 (1H, dd, J =
1.8, 8.1 Hz, H-5’), 7.03 (1H, d, J = 8.4 Hz, H-6’), 6.36 (2H, s, H-3, H-5), 5.43 (1H,
brs, ArOH), 3.86 (6H, s, 2×-OCH3), 3.01-2.80 (4H, m, -CH2CH2-); 13C-NMR (75MHz,
CDCl3) δ 156.1, 153.2, 132.4, 128.5, 127.8, 127.3, 121.1, 115.0, 111.8, 105.1, 56.2,
56.1, 38.1, 37.6; ESI-MS m/z: 349.0 [M+Na]+.
4.1.2.22 4-(2,3-dichlorophenethyl)-2,6-dimethoxyphenol (21Av). Yield 46 %; yellow

oil; 1H-NMR (300 MHz, CDCl3) δ 7.34 (1H, dd, J = 1.5, 7.8 Hz, H-4’), 7.10 (1H, t, J
= 7.8 Hz, H-5’), 7.01 (1H, dd, J = 1.5, 7.5 Hz, H-6’), 5.43 (1H, brs, ArOH), 3.87 (6H,
s, 2×-OCH3), 3.08-2.83 (4H, m, -CH2CH2-); 13C-NMR (75MHz, CDCl3) δ 151.1,
150.6, 149.2, 147.0, 136.4, 132.5, 128.8, 123.2, 121.2, 115.0, 105.1, 56.1, 40.6, 36.4;
ESI-MS m/z: 349.0 [M+Na]+, 325.2 [M-H]-.
4.1.2.23 2, 6-dimethoxy-4-(2-methoxyphenethyl) phenol (22Aw). Yield 42 %; yellow
1
solid; H-NMR (300 MHz, CDCl3) δ 7.24 (1H, m, H-3’), 7.09 (1H, m, H-4’), 6.89
(2H, m, H-5’, 6’), 6.40 (2H, s, H-3, H-5), 5.40 (1H, brs, ArOH), 3.86 (9H, s,
3×-OCH3), 2.90-2.85 (4H, m, -CH2CH2-) ; 13C-NMR (75MHz, CDCl3) δ161.1, 160.6,
149.7, 148.3, 137.1, 133.4, 129.8, 123.6, 120.9, 114.0, 105.5, 55.9, 56.7, 40.9, 36.7;
ESI-MS m/z: 311.1 [M+Na]+.
4.2 Cell culture, animals and chemicals.
Human gastric adenocarcinoma cell line(MKN-45), human non-small-cell lung
carcinoma (A549), human hepatoma (HepG2), human colon carcinoma (HCT116),
human breast adenocarcinoma (MDA-MB-231) and noncancerous NCM460 colon
cells were purchased from the Institute of Biochemistry and Cell Biology (Shanghai,
29
China) and grown in DMEM medium (Gibco). The media was supplemented with
115 units/mL of penicillin G (Gibco, Milano, Italy), 115 μg/mL of streptomycin
(Invitrogen), and 10% fetal bovine serum (Invitrogen). Transgenic zebrafish Tg
(fli-1:EGFP) were obtained from Model Animal Research Center of Nanjing
University. All cells were incubated at 37 °C in 5% CO2 (v/v). SU5416, and dimethyl
sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Stock
solutions (25 mM) of synthesized compounds were obtained by dissolving them in
dimethyl sulfoxide (DMSO).
4.3 Cell Morphology
HCT116 cells were seeded in 12-well plates at a density of 1×105 cells per well.
After overnight adherence, they were exposed to increasing concentrations of
compound 8Ae for 48 h. Cells were imaged using fluorescence microscope. In order
to visualize nuclear morphological changes, cells were stained with Hoechst 33342
(Sigma, USA).

4.4 Antiproliferative Assays
Individual wells of 96-well plate were inoculated with 100 μL of complete medium
containing 5 × 103 cancer cells (MKN-45, A549, HepG2, HCT116 and
MDA-MB-231). The plates were incubated at 37 °C in a humidified 5% CO2
incubator for 12 h prior to the experiments. After medium removal, 100 μL of fresh
medium containing the test compound at different concentrations was added to each
well and incubated at 37 °C for 72 h. The percentage of DMSO in the medium has
never exceeded 0.1%, which was also the maximum DMSO concentration in all
cell-based assays. Cell viability was test by the (3-(4,5-dimethylthiazol-2-yl)
-2,5-diphenyltetra-zolium bromide (MTT) assay. MTT (0.5 mg/mL, final
concentration) was added and incubated for 2 hours at 37 °C. Formazan crystals were
dissolved by DMSO, and absorbance was measured at a wavelength of 570 nm by a
1500 microplate reader (Thermo Fisher Scientific Co.). Cell viability was expressed
as a percentage of untreated group.
4.5 Annexin V/PI Double Staining Assay
HCT116 cells were cultured in 6-well plates at density of 2 × 105 cells per well, and
after overnight adherence, they were exposed to increasing concentrations of
compound 8Ae. After 48 h, apoptosis-mediated cell death was examined by a double
staining method with Annexin V-FITC Apoptosis Detection Kit (Beyotime, China)
according to the manufacturer’s instructions. Flow cytometric data acquisition and
analysis were performed in a Becton Dickinson FACS Calibur flow cytometer using
CellQuest software (BD Biosciences, Franklin Lakes, NJ). The lower left section of
fluorocytogram (Annexin V-, PI-) represents the normal cells, lower right section of
fluorocytogram (Annexin V+, PI-) represents early apoptosis cells, and upper right
section of fluorocytogram (Annexin V+, PI+) represents late apoptosis cells.
4.6 Cell Cycle Analysis
HCT116 cells were seeded in 6-well plates at density of 2 × 105 cells per well, and
after overnight adherence, they were exposed to increasing concentrations of
compound 8Ae for 48h. Then cells were harvested, fixed in cold 70% ethanol
overnight, treated with RNase A at 37 °C for 30 min, and incubated with PI solution

(Solarbio, China) at 4 °C for 15 min. Cell cycle distribution was analyzed using a
FACS flow cytometer.
4.7 Western Blot
Cells were treated with indicated concentration of 8Ae for 48 h, and then cells were
collected and lysed in lyses buffer (100 mM Tris-Cl, pH 6.8, 4% (m/v) sodium
dodecylsulfonate, 20 % (v/v) glycerol, 200 mM β-mercaptoethanol, 1 mM
phenylmethylsulfonyl fluoride, and 1 g/mL aprotinin). Lysates were centrifuged at
12000g for 30min at 4 °C. The concentration of total proteins was measured using the
BCA assay method with Thermo scientific varioskan flash (Thermo scientific, USA)
at 562 nm. Protein samples were separated with 10% SDS–PAGE gel and transferred
onto PVDF membrane (0.45 μm, Millipore Co., Ltd.) by semi-dry electrophoretic
transfer. Subsequently, the membranes were probed with primary antibodies overnight
at 4 °C, and then were incubated with HRP-conjugated secondary antibody at room
temperature for 2 h. Antibodies used were: α-tubulin (1:1000), anti-GADPH(1:1000).
The values of band intensities were detected enhanced chemiluminescence (ECL) and
quantized by Image-Pro Plus 6.0 software.
4.8 Anti-angiogenic screening in zebrafish-guided angiogenesis model
Adult zebrafish were kept and raised according to the Zebrafish book guidelines. 20
dechorinated 24 hpf-embryos were used per experimental group in a 24-well plate and
incubated with 1ml of embryo water containing various concentrations of drug at
28.5 °C for additional 48 h. In order to evaluate the anti-angiogenesis activities of
synthesized compounds in a fast and efficient way, we screened all the compounds at
dose 50 μM. Compounds with obvious effect were assayed the minimum effective
dose. After anesthetized with 0.016% tricaine (Sigma-Aldrich), the inter-segmental
blood vessels (ISVs) of embryos were observed and imaged at 72 hpf under a
fluorescence microscope (IX71, Olympus, Japan). SU5416 was served as a positive
control. All zebrafish studies were approved by the Institutional Animal Care and Use
Committee at Nanjing Tech University.
4.9 Molecular docking
Molecular docking studies were performed to investigate the binding mode

30
between 8Ae and tubulin using AutoDock Vina 1.1.2. The tubulin protein crysral
31
(4YJ2) was downloaded from prontein data bank(PDB). Perotein and ligand were
separated by pymol software and then the formula of protein was transferred from pdb
to pdbqt. The 3D structures of 8Ae was obtained using ChemBioDraw Ultra14.0 and
ChemBio3D Ultra 14.0 software programs. The AutoDockTools 1.5.6 package was
employed to generate the docking input files. The search grid of tubulin was identified
as center_x = -16.516, center_y = 64.771, and center_z = -37.543 with dimensions
size_x: 15, size_y: 15, and size_z: 15. After all of the preparation, we use autodock
vina to dock. For Vina docking, the default parameters were used if not mentioned.
The best-scoring poses as judged by the Vina docking score were chosen and visually
analyzed using PyMOL 1.7.6 software (http://www.pymol.org/).
4.10 Statistical analysis
All experiments were repeated at least three times. Values are given as means±SEM.
Data were analyzed using GraphPad Prism 6.0 software (GraphPad Software Inc., La
Jolla, CA). Statistical significance was assessed by One-Way analysis of variance
(ANOVA) or Student's t test. P values less than 0.05 were considered statistically
significant.
Acknowledgements
This research work was financially supported by the National Natural Science
Foundation of China (No. 81373956, No.81274064, No.81502950 and No.81573557),
Seed Funding Program for Basic Research from HKU (201111159043), the Priority
Academic Program Development of Jiangsu Higher Education Institutions, the
Fundamental Research Funds for the Central Universities (2015ZD010), Huahai
Graduate Innovation Fund (NO. CX14B-007HH),College Students Innovation Project
for the R&D of Novel Drugs (J1030830) and Xi'an Medical University Ph.D.
Research Startup Fund Project(2017DOC06). Besides, this work was appreciated for
the support from the joint laboratory of China pharmaceutical university and Taian
city central hospital.
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Highlights:

Two series of moscatilin derivatives were designed, synthesized and evaluated as
anti-tumor and anti-angiogenesis agents.



8Ae can inhibit the viability and induce the apoptosis of HCT116 cells.
8Ae could arrest G2–M cell-cycle by inhibiting α-tubulin expression.
Zebrafish bioassay-guided screen showed compound 8Ae had better
anti-angiogenic activities than positive compound.