Anti-angiogenic and anticancer effects of baicalein derivatives based on transgenic zebrafish model

Article abstract of DOI:10.1016/j.bmc.2018.07.037

Angiogenesis leads to tumor neovascularization by promoting tumor growth and metastatic spread, therefore, angiogenesis is considered as an attractive target for potential small molecule anticancer drug discovery. Herein, we report the structural modification and biological evaluation of baicalein derivatives, among which compound 42 had potent in vivo anti-angiogenic activity and wide security treatment window in transgenic zebrafish model. Further, 42 exhibited the most potent inhibitory activity on HUVEC proliferation, migration and tube formation in vitro. Moreover, 42 significantly inhibited growth of human lung cancer A549 cells and weak influence on human normal fibroblast L929 cells. The present research demonstrated that the significant anti-angiogenic and anticancer effects, which provided the supportive evidence for 42 could be used as a potential compound of cancer therapy.

Full text of DOI:10.1016/j.bmc.2018.07.037

Accepted Manuscript
Anti-angiogenic and anticancer effects of baicalein derivatives based on trans-
genic zebrafish model
Xueyang Jiang, Junting Zhou, Qinghua Lin, Guiyi Gong, Haopeng Sun,
Wenyuan Liu, Qinglong Guo, Feng Feng, Wei Qu
PII:
S0968-0896(18)30850-2
https://doi.org/10.1016/j.bmc.2018.07.037
BMC 14474
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 May 2018
19 July 2018
22 July 2018
Please cite this article as: Jiang, X., Zhou, J., Lin, Q., Gong, G., Sun, H., Liu, W., Guo, Q., Feng, F., Qu, W., Anti-
angiogenic and anticancer effects of baicalein derivatives based on transgenic zebrafish model, Bioorganic &
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.07.037
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Anti-angiogenic and anticancer effects of baicalein derivatives based
on transgenic zebrafish model
Xueyang Jiang,a Junting Zhou,a Qinghua Lin,a Guiyi Gong,a Haopeng
Sun,c Wenyuan Liu,d Qinglong Guo,e Feng Feng, a,b,f* Wei Qua,b*
a Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009,
China;
b Key Laboratory of Biomedical Functional Materials, China Pharmaceutical University, Nanjing
210009, China;
c Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing, 210009, China;
d Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University),
Ministry of Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China;
e
Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University,
24 Tongjiaxiang, Nanjing 210009, People’s Republic of China
f Jiangsu Food & Pharmaceutical Science College, Huaian 223003, China
Abstract: Angiogenesis leads to tumor neovascularization by promoting tumor
growth and metastatic spread, therefore, angiogenesis is considered as an attractive
target for potential small molecule anticancer drug discovery. Herein, we report the
structural modification and biological evaluation of baicalein derivatives, among
which compound 42 had potent in vivo anti-angiogenic activity and wide security
treatment window in transgenic zebrafish model. Further, 42 exhibited the most potent
inhibitory activity on HUVEC proliferation, migration and tube formation in vitro.
Moreover, 42 significantly inhibited growth of human lung cancer A549 cells and
weak influence on human normal fibroblast L929 cells. The present research
demonstrated that the significant anti-angiogenic and anticancer effects, which
provided the supportive evidence for 42 could be used as a potential compound of
cancer therapy.
Key words: baicalein derivatives; anti-angiogenesis; transgenic zebrafish model;
anticancer
1. Introduction
Cancer is a common illness which endangers human health seriously and is one of the
major causes of death around world.1 In the past decades, the rate of morbidity of
cancer has sharply increased for various reasons such as environmental pollution,

unhealthy lifestyles, and high stress levels. Previous studies have indicated that the
occurrence and development of tumor is closely related to angiogenesis.2,3
Angiogenesis is a process of capillaries sprouting from preexisting blood vessels
which is related to various physiological and pathological processes and has the
capability to maintain the normal growth and balance of human body.4 Once the
process of angiogenesis is out of balance, various diseases can be induced such as
inflammation, ischaemic and cancer.5 The free diffusion of oxygen which is needed
for the tumor growth happens only in capillary terminal. Therefore, the induction of
new blood vessels is a necessary condition for tumors to recruit oxygen, nutrients and
disseminate to distant sites,6 and inhibiting abnormal angiogenesis can prevent tumor
from growing. Simultaneously, previous research have shown that the inhibition of
abnormal angiogenesis can prevent tumor cells from proliferation, migration and
invasion through vascular tissue.7 Therefore, the inhibition of angiogenesis has been
the focus of new therapeutic strategies against cancer in recent years.
Baicalein, a main flavonoid extracted from the dried root of S. baicalensis Georgi
(also known as Chinese Huang Qin), has been demonstrated to exert multiple
pharmacological effects including anti-cancer,8 antiviral,9 antioxidant,10 and
anti-inflammatory11 properties. Emerging researches have shown that baicalein
inhibited tumor angiogenesis through disrupting the development of tumor
vasculature. Miocinovic et al. reported that the average number and length of sprouts
formed by the endothelial cell aggregates of the baicalein-treated group were notably
decreased.12 Simultaneously, Park, YG et al. found that baicalein could decreased the
proliferation and migration in B16F10 cells, and significantly inhibited tube formation
and cell migration in human umbilical vein endothelial cells (HUVECs).13 In addition,
baicalein could inhibit the phosphorylation of VEGFR2 and ERK in HUVECs, which
inhibited new blood vessel formation.14 Therefore, baicalein may represent a
promising angiogenesis inhibitor for the treatment of cancer. Unfortunately, some
drawbacks of baicalein have limited its clinical application such as poor oral
bioavailability and low aqueous solubility. In order to overcome these problems,
recent studies have focused on the synthesis of baicalein derivatives as novel
anticancer agents, which showed multiple biological effects, such as inducing cell
apoptosis,15 inhibiting cell proliferation,16 reducing inflammation17 and so on.
However, few of them have aimed to improve the effect on inhibiting angiogenesis.
Based on these former findings, in this paper we hypothesized that introduction of
hydrophilic groups and N-containing heterocycles at the 6-position of baicalein was
aimed to obtain novel anticancer agents.
Phenotype-driven drug discovery is a novel approach which utilizes the phenotypic

changes of animals to find effective compounds. Compared to the traditional
target-driven drug discovery strategy, the phenotype-driven one has many advantages
as follows: i) the pharmacokinetic and physicochemical properties of the compounds
can be directly reflected in the screening;18 ii) researchers do not have to identify the
exact target of the disease in advance; iii) the animals after treatment reflect not only
the effectiveness, but also the toxicity, side effects and the process of transport of the
drug intuitively. In recent years, phenotype-driven chemical genetic screens in
zebrafish have become a popular strategy for both dissection of developmental
mechanisms and discovery of effective treatment approach.19,20 Zebrafish has
emerged as an excellent model to carry out the angiogenesis studies associated with
cancer.21 There is a rapid translation from screen results to humans due to the high
level of gene conservation between zebrafish and mammals22: 71.4% of human genes
and about 87% of known human disease-related genes can be found orthologous gene
in the zebrafish genome, respectively23.
Transgenic zebrafish [Tg(fli1:EGFP)], exhibiting vasculature-specific expression of
enhanced green fluorescent protein (EGFP) under the control of the fli-1 promoter
during embryonic and larval development, where the endothelial cells can be directly
observed using fluorescence microscope, is an ideal model in vivo for studying
antiangiogenic agents.24 The major advantages include their high degree of genetic,
physiologic, and pharmacologic similarity with human, as well as the small size,
optical transparency, rapid development, and large numbers of their embryos and
larvae. Compared to the wildtype zebrafish, the Tg (fli1:EGFP) zebrafish can provide
much higher resolution of vasculature, and the processing procedure is easy and fast
to facilitate observation, which allowed the rapid analysis of live embryos in response
to drugs.25 Moreover, the toxicity of drugs could also be determined by the influence
on the embryo development and body integrity, by observing whether the
anti-angiogenic activity of drugs is through a direct vasculature targeting effect or
through a secondary effect caused by development blocking.26 In this paper, we
adopted the phenotype-driven drug discovery strategy as guidance and used the
zebrafish [Tg(flia:EGFP)] as an in vivo screening model to evaluate the
anti-angiogenic effects of the synthesized baicalein derivatives, in addition to a
HUVECs in vitro model.
2. Results and discussion
2.1 Chemistry
The general method used for the synthesis of 6-O-alkyl alcohol baicalein

derivatives from baicalein was described in Scheme 1. Benzylation of baicalein was
conducted by reaction with benzyl bromide to provide 7-O-benzyl baicalein (1). Alkyl
chains contained terminal hydroxyl group were selectively introduced at 6-OH of
intermediate 1 with various bromoalkyl alcohols in the presence of K2CO3 to afford
the corresponding derivatives 2-6. Besides, three or five-carbon linkers were
introduced to the 6-OH of 1 to form intermediates 14 and 15, which were
subsequently reacted with ethanolamine to provide compounds 16 and 17.
Debenzylation of compounds 2-6, 12, 16 and 17 gave rise to compounds 7-11, 13, 18
and 19. Acylation of 2 was conducted by reaction with succinic anhydride to afford
intermediate 22. Then the debenzylation of compound 22 gave rise to compound 23.
Scheme 1. Synthesis of 7-OH-6-O substituted baicaleinsa
aReagents and conditions: (a) benzyl bromides, NaHCO3, acetone, reflux, 9 h, 69%; (b)
bromoalkyl alcohol, K2CO3, KI, acetone, 75 ℃, 8 h, 49-72%; (c) 10% Pd/C, H2, MeOH, AcOH, rt,
overnight, 59-71%; (d) dibromoalkanes, K2CO3, DMF, 70 ℃, 6 h, 37-64%; (e) ethanolamines,
TEA, MeCN, reflux, 8 h, 59-73%; (f) i: CH3I, MeCN, rt, 5 h; ii: 10% Pd/C, H2, MeOH, rt, 9 h,
29-51%; (g) bromoalkanes, KHCO3, DMF, 55 ℃, 6 h, 49-72%; (h) succinyloxide, DMAP, DCM,
rt, 7 h, 69%.
The synthesis of amido-baicalein derivatives was described in Scheme 2.
Alkylation of 1 was conducted by reaction with methyl chloroacetate to afford 24.
Next, debenzylation of compound 24 was conducted with anhydrous AlCl3 to
quantitatively afford compound 25, which was subsequently reacted with different
amines in N,N-dimethylformamide (DMF) to afford compounds 26-34. Compound 24

was also reacted with corresponding amines to provide compounds 35-44 using the
same method.
Scheme 2. Synthesis of 7-OBn-6-O substituted baicaleinsa
aReagents and conditions: (a) methyl bromoacetate, K2CO3, DMF, 60 ℃, 5 h, 91%; (b) anhydrous
AlCl3, N,N-dimethylaniline, DCM, rt, 9 h, 86%; (c) N-containing heterocycles or chains, DMF,
80 ℃, 64-82%; (for 34) 1.5N LiOH, THF/MeOH, rt, 4 h, 83%.
2.2 Biological activity
2.1.1 In vivo assessment of anti-angiogenic activity and toxicity of baicalein
derivatives
Transgenic zebrafish with expression of enhanced green fluorescent protein (EGFP)
was applied for the primary investigation of antiangiogenic effects of the target
molecules. The anti-angiogenic activities and toxicities of baicalein derivatives at
various concentrations were evaluated on transgenic zebrafish from 24 hours of
fertilization (hpf) to 72 hpf (Table 1). Baicalein showed strong toxicity to zebrafish
embryos, indicating that it has affected other developmental key genes or pathways in
zebrafish embryos before demonstrating neovascularization. The baicalein derivatives,
with alkyl alcohol side chains, displayed visible anti-angiogenic effects. Compounds 8,
10 and 11 showed the ability to inhibit angiogenesis at low concentrations (1-2.5 μM),
and had satisfactory AI values (4-6). However, these three compounds also produced
toxicities to zebrafish embryos while exerting anti-angiogenic activities, with a TI
value of 1. It was noteworthy that 7 and 9, with an ethanol or n-butanol substituents,
displayed potent anti-angiogenic activities with LOEC of 5 μM. Importantly,
increasing the concentration of the compounds to 10 μM caused toxicity to the
development of zebrafish embryos. In particular, compound 7 has the highest AI value
(10) in this series, indicating shorter link chain with lower toxicity. These findings
suggested that substituents with a terminal polar group, such as hydroxyl group, might
have a drastic effect on the anti-angiogenic activity and toxicity to zebrafish.
Encouraged by the above results, branched hydroxyl, amino and carboxyl were

introduced in the 6-position of baicalein by alkyl chains, resulting in compounds 18,
19 and 23. However, these compounds manifested neither toxicity to the larvae, nor
potent anti-angiogenic effect. Only 19, with ethanolamine group, showing
anti-angiogenic activity on zebrafish at a moderate concentration of 25 μM, and had
satisfactory AI values (4).
Table 1. In vivo assessments of alkoxy-substituted baicalein derivatives in zebrafish
Ra
LOEC (μM)b
LOTC (μM)c
LC100 (μM)d TIe
AIf
10
Compd.
7
5
10
50
15
2
1
2.5
5
2.5
10
1
6
6
5
4
2
2
4
8
30
5
2
1
1
1
1
2
9
1
10
11
13
18
19
2.5
50
100
25
2.5
50
100
50
10
100
>200
100
H
H
50
50
100
1
1
2
2
20
21
100
100
>200
50
>75
0.5
75
1
1.5
1
1.5
2
23
H
0.5
Bai.

1
1.5
5
1.5
5
SU5416
aThe 7-positions of compounds 20 and 21 are prenyl and 3-hydropropyl groups, respectively.
bLOEC: lowest observed effect concentration, it was the treatment concentration that resulted
c
in antiangiogenic effects on most of the zebrafish larval population; LOTC: lowest observed
d
toxity concentration; LC100: totally lethal concentration, it was a concentration that resulted
in 100% mortality of the embryos during the incubation period; eTI: toxity index,
LOTC/LOEC, which was proportional to the specificity of antiangiogenic activity of the
f
compounds. AI: antiangiogenic index, LC100/LOEC, was considered as the concentration
range between LOEC and LC100, it revealed the sub-lethal effects of the compounds with
respect to its efficacious concentrations. Results from at least 20 embryos per condition.
Considering the prevalence of the hydroxyl group in anti-angiogenic activity, we
next introduced different acylamine substituted groups to 6-position of baicalein.In
addition, we also discussed the effect of substitutions at 7-position of baicalein.
Unlike the previous alkyl alcohol series, the antiangiogenic activities of derivatives of
the 7-hydroxy group were generally lower than 7-benzyl group in the series (Table 2).
Derivatives bearing aliphatic amines at 6-position (26-29, 35 and 36) manifested no
anti-angiogenic effect and little toxicity to the larvae, no matter whether the 7-position
was phenolic or benzyl-substituted. Compound 31, with piperazine terminal
substitution, possessed anti-angiogenic activity, with an LOEC value of 10 μM. In
contrast, compounds 30 and 32, with N-methylpiperazine and N-ethylpiperazine
substituents, respectively, lost their anti-angiogenic activities. Interestingly, compound
42, with the piperazine acetamide group at 6-position, exhibited potent
anti-angiogenic activity, with an LOEC value of 5 μM and the lowest toxicity to
zebrafish embryos, with an AI/TI value of 20 and 5, respectively. After studying the
individual effects of substitutions at 6- and 7-position, we summarized several
important structure-activity information. Introducing an alkyl alcohol substituents in
the 6-position could improve anti-angiogenic activities, and shortened chain length
contributed to decreased toxicities. Additionally, with regard to the 6-O-acetamide
substituted scaffold, replacing phenolic hydroxyl group with benzyl group did not
significantly enhance antiangiogenic activities, whereas compound 42, with a
piperazine group, displayed the most potent antiangiogenic activities. Importantly, it
showed good safety with an AI value of 20.
Table 2. In vivo assessments of amido-substituted baicalein derivatives in zebrafish

R1
H
R2
LOEC (μM)
200
LOTC (μM)
200
100
>200
200
50
LC100 (μM)
200
TI
AI
Compd.
26
1
1
1
2
H
100
>200
>200
200
27
H
>200
200
N/Aa
1
N/A
28
H
1
2
29
H
50
100
1
30
H
10
15
50
1.5
1
5
31
H
100
100
50
>200
100
2
32
H
50
1
2
33
H
>50
100
>50
100
100
N.d.
10
50
N/A
1
N/A
2
34
Bn
Bn
Bn
Bn
Bn
200
35
100
200
1
2
36
N.d.b
10
N.d.
50
N/A
1
N/A
5
37
38
200
200
200
1
1
39

Bn
Bn
Bn
Bn
Bn
25
75
5
25
75
100
100
1
1
5
1
1
4
1.3
20
2
40
41
42
43
44
25
100
100
50
100
50
>200
100
2
aN/A, not applicable; bN.d., not determined due to precipitation.
2.2.2 Identification of a new baicalein derivative 42 as a potent angiogenic
inhibitor
Next, we selected the most potent molecule 42 for additional studies. The
intersegmental vessels (ISVs) and dorsal long axis blood vessels (DLAV) are the most
easily observed angiogenic vessels in zebrafish embryos at 48 hpf. Normally, ISVs
appears as a smooth fence structure at 48 hpf (Fig. 1A). The inhibitory effect of
different doses of 42 on ISVs formation in zebrafish embryos was shown in Fig. 1B-E.
Zebrafish embryos at 48 hpf treated with 42 showed a dose-dependent inhibition of
ISVs formation compared to vehicle control. 42 affected the formation of ISVs with
mild anti-angiogenic effect at 10 μM, and almost completely inhibited ISVs and
DLAV formation at 50 μM. Importantly, the overall morphology and structure of
treated embryos were generally normal by 48 hpf. At late stages of embryos when
ISVs were already established, 42 treatment did not cause visible changes in blood
vessel morphology. These results indicated that 42 causes anti-angiogenesis by
blocking new blood vessel formation and not by destruction of existing vasculature.
Due to the significant therapeutic potential of 42 in terms of its largest AI value in
vivo, we subsequently investigated its anti-angiogenic activity in mammalian model.

Fig. 1 Compound 42 inhibited ISVs and DLAV angiogenesis in Tg (fli1: EGFP) zebrafish. (A)
Vehicle-treated control. Arrow indicated EGFP expressing ISVs; (B–E) 42 treated embryos at
concentrations of 5 μM, 10 μM, 25 μM and 50 μM. (F) SU5416 treated embryos at
concentration of 2.5 μM. Defects in ISVs formation were marked with asterisks. The
magnified views of A–F (40 magnification) were shown in on the right of the corresponding
images (90 magnification), respectively.
2.2.3 Effects of compound 42 on HUVEC proliferation, migration and tube
formation in vitro
Angiogenesis is a complex process, typically involving endothelial cell proliferation
and alignment to form tubular structures. In order to determine the potential
anti-angiogenic effect of 42, we further explored cytotoxicity on HUVECs in vitro
using cell counting kit-8 (CCK-8) method.27 We treated HUVECs with increasing
concentrations of 42 ranging from 5 to 50 μM for 48 h. As shown in Fig. 2, 42
dose-dependently inhibited HUVEC proliferation, with significant inhibition at 25 and
50 μM, and an IC50 value of 57.1 ± 2.4 μM. These results suggested that compound 42
suppressed the proliferation of HUVECs and the process of angiogenesis.

Fig. 2 Compound 42 inhibited cell proliferation in HUVECs in a dose-dependent manner
under normal conditions. Attached cells were treated by various concentrations of 42 for 48 h.
Cell growth was quantified by CCK-8 assay. Data were presented as mean ± SEM (n=3).
Since Metastasis is a property of malignant cancer cells and cells migration is a
crucial step of both angiogenesis and tumor progression, we also investigated the
effects of 42 on the ability of endothelial cells to form tubular structures using Boyden
Chamber migration assay. As shown in Fig. 3, HUVECs migration was inhibited in
various concentrations after 72 h treatment of 42. We observed that 42 was able to
decrease HUVEC migration compared with control. Quantitative determination of the
migrated cells showed 25 μM of 42 inhibited migration of endothelial cells by up to
50% compared with control.
Fig. 3 Compound 42 inhibited HUVEC migration by a Boyden chamber migration assay and
a scratch-wound assay. (F) After 8 hours, the HUVECs that migrated through the membrane
were stained and quantified. For each treatment condition, four measurements were taken
randomly in each well of three replicates. Percentage of inhibition was expressed using
control wells at 100%. Data were presented as mean ± SEM (n=3).
Tube formation is also indispensable for angiogenesis. When cultured on a
membrane-basement matrix layer, untreated HUVECs form an extensive and
complete network of capillary-like structures within 6 h in vitro, mimicking the events

occurring during vessel network formation in vivo. 42 was confirmed to
dose-dependently inhibited tube formation in vitro HUVEC morphogenesis evaluation
at different concentrations (Fig. 4B–E). Indeed, compared with control, 42 led to a
24.9%, 54.2% and 72.6% reduction of the formation of capillary-like structures at 25,
50 and 100 μM, respectively. The reduction was evidenced by fragments of
unconnected tubes with fewer branch points and shorter tube length (Fig. 4C, D and
E), indicating 42 also inhibits endothelial cell differentiation on Matrigel.
Fig. 4 Compound 42 inhibited HUVEC tube formation. Images showing the morphological
features of 42-treated HUVECs on Matrigel. The vessel length decreased after 12 h
incubation with 42. (F) The vessel length in each treatment conditions was quantified. Data
were presented as mean ± SEM (n=3).
2.2.4. Effect of compound 42 on cells viability and apoptosis induction of lung
cancer cells
Angiogenesis plays a critical role in the proliferation of various cancers cells. In
this regard, in order to investigate the potential anticancer potency of these
Anti-angiogenic compounds, 42 was selected to investigate its anti-proliferative
activity against human lung cancer cell (A549). Highly consistent with the
anti-angiogenic activity, 42 significantly inhibited growth of A549 cells, with IC50
value 4.73 μM (see Fig. s1). We further evaluated the cytotoxicity of 42 in human
normal fibroblast L929 cell lines and compound 42 displayed weak cytotoxicity with
IC50 value of 19.1 μM, which suggested 42 may be an ideal anticancer candidate with
weak toxicity on normal tissues.
Apoptosis is a key mechanism for most cancer therapies.28 In order to investigate
whether the observed cytotoxic activity of 42 on A549 cells are mediated via
apoptosis, it (including Q3, the early apoptotic cells and Q2, late apoptotic cells) was
measured with Annexin V-FITC/PI double-labeled flow cytometry and populations of
apoptotic cells were quantified. 42 significantly increased late apoptosis of A549 cells.

As shown in Fig. 5, the late apoptosis rate was 23%, 36% and 62% after treatment
with 1, 4 and 8 μM of 42 for 48 h, respectively. Further, the activation of caspase-3
was detected by activity assay in which the activities of caspase-3 was determined by
the amount of p-nitroaniline (pNA) produced. According to Fig. 5F, we found that
caspase-3 was activated following 42, compared to the control group, which further
confirmed that apoptosis was activated.
Fig. 5 Effect of 42 on apoptosis of A549 cells. (A-E) Cell apoptosis was measured by
Annexin V‑ FITC/PI flow cytometry. (F) The activity of caspase-3 was measured using
caspase-3 Activity Assay kits. Data were presented as mean ± SEM (n=3).
3. Conclusions
A new series of baicalein derivatives have been prepared and their effects on
anti-aniogenic and anticancer activities have been evaluated. In vivo phenotype-based
screening of all the derivatives in zebrafish, compound 42 was identified as an
effective and low toxicity angiogenesis inhibitor. The anti-angiogenic activity of 42
was confirmed by testing on HUVECs proliferation, migration and tube formation in
vitro mammalian assays. The further evaluation of anticancer activity showed that 42
achieved an IC50 value of 4.73 μM in inhibition of A549 cell growth via inducing late
apoptosis, and a weaker cytotoxicity in human normal fibroblast L929 cells. Overall,
these preliminary results highlight that the antiangiogenic properties of baicalein

derivatives may represent new insights for their applications in the treatment of
cancer.
4. Experimental protocols
4.1 Chemistry
1H-NMR and 13C-NMR spectra were recorded with a Bruker Avance 300 MHz
spectrometer at 300 K, using TMS as an internal standard. MS spectra data were
obtained on an Agilent LC−MS 6120 instrument with an ESI mass selective detector
in positive ion mode. HRMS were performed with LTQ Orbitrap XL (Thermo Fisher
Scientific) using electrospray ionization (ESI). IR spectra were recorded on a
Perkin-Elmer spectrophotometer. Melting points were determined on a RY-1 micro
melting point apparatus (Tianjin Analytical Instrument Corp., Tianjin, China).
Analytical and preparative thin-layer chromatography (TLC) were performed on silica
gel GF/UV 254, and the chromatograms were developed on silica gel (200–300 mesh,
Merck) and visualized under UV light at 254 and 365 nm. Dichloromethane, acetone
and tetrahydrofuran were dried by standard procedures and stored over molecular
sieves. All other solvents and chemicals were reagent grade and used without further
purification.
4.1.3. General procedure for the preparation of 7-11, 13 and 23.
To a solution of benzyl substituted compounds (0.3 mmol) in 15 mL MeOH, 30 mg
10% Pd/C and a drop acetic acid was added, violently stirring under a hydrogen
atmosphere. After completion of the reaction, Pd/C was filtered and washed with little
MeOH. Then the reaction mixture was concentrated under vacuum. The residue was
purified by silica gel column chromatography (CH2Cl2-MeOH = 40:1 as the eluent) to
afford debenzylation products as a yellow powder.
4.1.3.1. 5,7-dihydroxy-6-(2-hydroxyethoxy)-2-phenyl-4H-chromen-4-one (7). Yield
1
89%; yellowish powder; mp 115-116 °C; H-NMR (DMSO-d6, 300 MHz, δ ppm):
12.96 (s, 1H, 5-OH), 8.07 (d, J = 7.5 Hz, 2H, H-2′, H-6′), 7.58 (m, 3H, H-3′, H-4′,
H-5′), 6.97 (s, 1H, H-8), 6.66 (s, 1H, H-3), 4.02 (t, J = 6.1 Hz, 2H, -OCH2-), 3.70 (t, J
= 6.1 Hz, 2H, -OCH2-); 13C-NMR (DMSO-d6, 75 MHz, δ ppm): 182.25, 163.22,
157.48, 152.64, 152.55, 131.98, 130.70, 130.43, 129.09, 126.36, 104.63, 104.38,
94.40, 74.33, 60.04; MS (ESI) m/z = 313.2 [M - H]-, 337.1 [M + Na]+; HRMS (ESI):
m/z calcd for C18H17O6: 315.0863; found: 315.0874 [M + H]+.
4.1.3.2. 5,7-dihydroxy-6-(3-hydroxypropoxy)-2-phenyl-4H-chromen-4-one (8). Yield

1
86%; yellowish powder; mp 118-119 °C; H-NMR (DMSO-d6, 300 MHz, δ ppm):
12.92 (s, 1H, 5-OH), 8.07 (d, J = 7.5 Hz, 2H, H-2′, H-6′), 7.58 (m, 3H, H-3′, H-4′,
H-5′), 6.97 (s, 1H, H-8), 6.65 (s, 1H, H-3), 4.05 (t, J = 6.1 Hz, 2H, -OCH2-), 3.61 (t, J
= 6.1 Hz, 2H, -OCH2-) , 1.83 (m , 2H, -CH2-); 13C-NMR (DMSO-d6, 75 MHz, δ ppm):
182.24, 163.18, 157.74, 152.79, 152.49, 131.97 130.74, 130.42, 129.11, 126.37,
104.64, 104.34, 94.32, 70.29, 58.16, 32.56; MS (ESI) m/z = 329.1 [M + H]+ , 351.1
[M + Na]+; HRMS (ESI): m/z calcd for C18H17O6: 329.1020; found: 329.1050 [M +
H]+.
4.1.3.3. 5,7-dihydroxy-6-(4-hydroxybutoxy)-2-phenyl-4H-chromen-4-one (9). Yield
1
79%; yellowish powder; mp 125-127 °C; H-NMR (DMSO-d6, 300 MHz, δ ppm):
12.90 (s, 1H, 5-OH), 10.73 (s, 1H, 7-OH), 8.05 (dd, J = 8.1 Hz, 2.1 Hz, 2H, H-2′,
H-6′), 7.57 (m, 3H, H-3′, H-4′, H-5′), 6.95 (s, 1H, H-8), 6.62 (s, 1H, H-3), 4.43 (m, 1H,
-CH2-OH), 3.94 (t, J = 6.1 Hz, 2H, -OCH2-), 3.42 (m, 2H, -OCH2-), 1.70 (m , 2H,
CH2-), 1.57 (m , 2H, -CH2-); 13C-NMR (DMSO-d6, 75 MHz, δ ppm): 182.37, 164.91,
157.37, 152.94, 149.68, 141.60, 130.82, 129.36, 127.92, 129.57, 110.59, 107.61,
98.27, 69.94, 63.18, 28.17, 24.59; HRMS (ESI): m/z calcd for C19H19O6: 343.176;
found: 343.1182 [M + H]+.
4.1.3.4. 5,7-dihydroxy-6-((5-hydroxypentyl)oxy)-2-phenyl-4H-chromen-4-one (10).
1
Yield 51%; yellowish powder; mp 152-153 °C; H-NMR (DMSO-d6, 300 MHz, δ
ppm): 12.92 (s, 1H, 5-OH), 10.73 (s, 1H, 7-OH), 8.08 (d, J = 7.5 Hz, 2H, H-2′, H-6′),
7.60 (m, 3H, H-3′, H-4′, H-5′), 6.98 (s, 1H, H-8), 6.65 (s, 1H, H-3), 4.39 (s, 1H,
-CH2OH), 3.96 (t, J = 6.6 Hz, 2H, -OCH2-), 3.40 (m, 2H, -OCH2-), 1.71 (m, 2H,
-CH2-), 1.45-1.47 (m , 4H, 2×-CH2-); 13C-NMR (DMSO-d6, 75 MHz, δ ppm): 183.19,
163.82, 160.10, 153.37, 150.29, 143.53, 131.39, 129.44, 128.43, 128.64, 110.34,
109.07, 94.93, 70.06, 62.99, 33.57, 29.43, 26.18; HRMS (ESI): m/z calcd for
C20H21O6: 357.1333; found: 357.1327 [M + H]+.
4.1.3.5. 5,7-dihydroxy-6-((6-hydroxyhexyl)oxy)-2-phenyl-4H-chromen-4-one (11).
1
Yield 49%; yellowish powder; mp 131-132 °C; H-NMR (CDCl3, 300 MHz, δ ppm):
12.95 (s, 1H, 5-OH), 7.98 (m, 2H, H-2′, H-6′), 7.56 (m, 3H, H-3′, H-4′, H-5′), 6.82 (s,
1H, H-8), 6.73 (s, 1H, H-3), 4.27 (t, J = 6.6 Hz, 2H, -OCH2-), 4.06 (m, 2H, -OCH2-),
13
1.81 (m, 2H, -CH2-), 1.68 (m, 2H, -CH2-), 1.44-1.57 (m , 4H, 2×-CH2-); C-NMR
(CDCl3, 75 MHz, δ ppm): 181.99, 163.27, 159.67, 153.49, 152.92, 132.18, 130.92,
130.31, 129.42 125.73, 104.01, 102.10, 94.83, 75.1, 61.94, 31.29, 28.67, 25.27, 25.19;
HRMS (ESI): m/z calcd for C21H23O6: 371.1489; found: 371.1491 [M + H]+
4.1.3.6. 6-(2,3-dihydroxypropoxy)-5,7-dihydroxy-2-phenyl-4H-chromen-4-one (13).
Yield 56%; yellowish powder; mp 165-167 °C; 1H-NMR (MeOD, 300 MHz, δ ppm):

7.93 (d, J = 8.1 Hz, 2H, H-2′, H-6′), 7.54 (m, 3H, H-3′, H-4′, H-5′), 6.63 (s, 1H, H-8),
6.44 (s, 1H, H-3), 4.20 (m, 1H, -CH2CH-), 4.02 (m, 1H, -CHCH2-), 3.96 (m, 1H,
-CHCH2-), 3.65 (m, 2H, -CH2-); 13C-NMR (DMSO-d6, 75 MHz, δ ppm): 182.16,
164.38, 161.27, 152.68, 149.91, 144.18, 132.91, 129.34, 128.17, 128.38, 110.62,
107.92, 95.63, 72.94, 69.35, 62.73; HRMS (ESI): m/z calcd for C18H17O7: 345.0969;
found: 345.0971 [M + H]+.
4.1.3.7.
4-(2-((5,7-dihydroxy-4-oxo-2-phenyl-4H-chromen-6-yl)oxy)ethoxy)-4-
1
oxobutanoic acid (23). Yield 66%; white solid; mp 198-199 °C; H-NMR (MeOD,
300 MHz, δ ppm): 7.96 (m, 2H, H-2′, H-6′), 7.57 (m, 3H, H-3′, H-4′, H-5′), 6.70 (d, J
= 8.2 Hz, 2H, H-2′′, H-6′′), 6.57 (d, J = 8.2 Hz, 2H, H-3′′, H-4′′), 4.40 (m, 2H,
-OCH2-), 4.32 (m, 2H, -OCH2-), 2.62 (m, 2H, -CH2-), 2.52 (m, 2H, -CH2-); 13C-NMR
(MeOD, 75 MHz, δ ppm): 183.37, 172.15, 170.32, 161.13, 158.60, 153.51, 153.12,
132.81, 131.91, 131.38, 129.17, 128.78, 105.31, 99.81, 94.81, 72.38, 65.19, 29.61,
29.36; HRMS (ESI): m/z calcd for C21H19O9: 415.1024; found: 415.1020 [M + H]+.
4.1.5 Synthesis of 16, 17
A mixture of intermediates (compound 14 (508 mg, 1 mmol) for 16, compound 15
(450 mg, 1 mmol) for 17), 2-aminoethanol (122 mg, 2 mmol), and TEA (3 mmol) in
30 mL MeCN was refluxed for 3 h. After the mixture was cooled to room temperature,
the solvent was removed, and the crude product obtained was purified by column
chromatography (CH2Cl2-MeOH = 30:1 as the eluent) on silica gel.
4.1.5.1.
7-hydroxy-6-(3-((2-hydroxyethyl)(methyl)amino)propoxy)-5-methoxy-2-
phenyl-4H-chromen-4-one (16). Yield 41%; yellowish powder; mp 214-216 °C;
1H-NMR (MeOD, 300 MHz, δ ppm): 7.92 (m, 2H, H-2′, H-6′), 7.52 (m, 3H, H-3′,
H-4′, H-5′), 7.56 (s, 2H, H-8, H-3), 4.13 (t, J = 4.1 Hz, 2H, -OCH2-), 3.95 (t, J = 4.1
Hz, 2H, -OCH2-), 3.90 (s, 3H, -OCH3-), 3.34 (t, J = 5.1 Hz, 2H, -NCH2-), 3.31 (t, J =
13
5.1 Hz, 2H, -NCH2-), 2.94 (s, 3H, -NCH3-), 1.93 (m, 2H, -CH2-); C-NMR (MeOD,
75 MHz, δ ppm): 178.20, 167.41, 161.05, 156.64, 151.74, 141.49, 131.58, 130.93,
128.70, 125.58, 106.40, 106.02, 100.98, 72.31, 61.11, 57.04, 56.25, 56.09, 40.40,
24.51; HRMS (ESI): m/z calcd for C22H26O6N: 400.1755; found: 400.1753 [M + H]+.
4.1.5.2. 7-(benzyloxy)-5-hydroxy-6-((5-((2-hydroxyethyl)amino)pentyl)oxy)-2-phenyl
1
-4H-chromen-4-one (17). Yield 64%; yellowish powder; mp 135-136 °C; H-NMR
(DMSO-d6, 300 MHz, δ ppm): 12.79 (s, 1H, 5-OH), 8.10 (d, J = 7.5, 8.2 Hz, 2H, H-2′,
H-6′), 7.59 (m, 3H, H-3′, H-4′, H-5′), 7.44 (m, 5H, Ar-H), 7.12 (s, 1H, H-8), 7.06 (s,
1H, H-3), 5.28 (s, 2H, -OCH2-), 3.94 (t, J = 6.0 Hz, 2H, -OCH2-), 3.62 (m, 2H,
-OCH2-), 3.57 (m, 1H, -CH2NH-), 2.95 (m, 2H, -NHCH2-), 2.83 (m, 2H, -NHCH2-),

1.62 (m, 4H, 2×-CH2-), 1.44 (m, 2H, -CH2-); MS (ESI) m/z = 488.2 [M - H]-.
4.1.6 Synthesis of 18 and 19
To a solution of intermediates (compound 17(196 mg, 0.4 mmol) for 19, compound
16 (151 mg, 0.3 mmol) for 18) in 15 mL THF, 35 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 THF. Then the reaction mixture was concentrated under
vacuum, and the crude product obtained was purified by column chromatography
(CH2Cl2-MeOH = 15:1 as the eluent) on silica gel.
4.1.6.1.
7-hydroxy-6-(3-((2-hydroxyethyl)(methyl)amino)propoxy)-5-methoxy-2-
1
phenyl-4H-chromen-4-one (18). Yield 51%; yellow soild; mp 191-163 °C; H-NMR
(MeOD, 300 MHz, δ ppm): 7.91 (d, J = 7.5 Hz, 2H, H-2′, H-6′), 7.51 (m, 3H, H-3′,
H-4′, H-5′), 6.6 (s, 2H, H-8, H-9), 4.13 (t, J = 6.1 Hz, 2H, -OCH2-4), 3.95 (t, J = 6.1
Hz, 2H, -OCH2-), 3.90 (s, 3H, -OCH3), 3.33-3.36 (m, 2H, -CH2-), 3.31 (m, 2H, -CH2-),
2.94 (s, 3H, -NCH3), 1.93 (m, 2H, -CH2-); 13C-NMR (MeOD, 75 MHz, δ ppm):
178.20, 167.41, 161.05, 156.64, 151.74, 141.49, 131.58, 130.93, 128.70, 125.58,
106.40, 106.02, 100.98, 72.31, 61.11, 57.04, 56.25, 56.09, 48.19, 46.77, 40.40, 24.51;
HRMS (ESI): m/z calcd for C22H26O6N: 400.1755; found: 400.1753 [M + H]+.
4.1.6.2.
5,7-dihydroxy-6-((5-((2-hydroxyethyl)amino)pentyl)oxy)-2-phenyl-4H-
1
chromen-4-one (19). Yield 46%; yellow oil; H-NMR (MeOD, 300 MHz, δ ppm):
7.90 (d, J = 7.6 Hz, 2H, H-2′, H-6′), 7.52 (m, 3H, H-3′, H-4′, H-5′), 6.64 (s, 1H, H-8),
6.49 (s, 1H, H-3), 4.06 (t, J = 6.1 Hz, 2H, -OCH2-), 3.83 (t, J = 6.1 Hz, 2H, -OCH2-),
3.31 (m, 2H, -CH2-), 3.13 (m, 2H, -CH2-), 1.82 (m, 4H, 2×-CH2-), 1.64 (m, 2H,
-CH2-); 13C-NMR (MeOD, 75 MHz, δ ppm): 182.34, 163.74, 160.47, 153.45, 152.39,
131.45, 131.17, 131.01, 128.71, 128.41, 125.81, 103.87, 103.63, 71.94, 56.54, 49.21,
48.13, 28.74, 25.34, 22.54; HRMS (ESI): m/z calcd for C22H26O6N: 400.1755; found:
400.1745 [M + H]+.
4.1.7 Synthesis of 20, 21
Baicalein (360 mg, 1 mmol) and anhydrous potassium bicarbonate (150 mg, 1.5
mmol) was dissolved in 5 mL N, N-dimethylformamide, then corresponding
isoprenylbromide and 3-bromopropyl alcohol (1.5 mmol) was added. The mixture
was stirred at 55 °C for 4 h. Then the reaction mixture was poured into water (45 mL)
and extracted with ethyl acetate (3×25 mL). The organic layer was separated, washed
with water (2×10 mL) and dried over anhydrous sodium sulfate. Organic solvent was
evaporated under reduced pressure and the residue was purified by column
chromatography (CH2Cl2-MeOH = 60:1 as the eluent) on silica.

4.1.7.1.
5,6-dihydroxy-7-((3-methylbut-2-en-1-yl)oxy)-2-phenyl-4H-chromen-4-one
1
(20). Yield 81%; yellowish powder; mp 165-166 °C; H-NMR (CDCl3, 300 MHz, δ
ppm): 12.49 (s, 1H, 5-OH), 7.89 (m, 2H, H-2′, H-6′), 7.53 (m, 3H, H-3′, H-4′, H-5′),
6.68 (s, 1H, H-8), 6.62 (s, 1H, H-3), 5.56 (m, 1H, -CH-), 4.71 (d, J = 4.6 Hz, 2H,
-OCH2-), 1.84 (s, 3H, -CH3), 1.81 (s, 3H, -CH3); 13C-NMR (CDCl3, 75 MHz, δ ppm):
182.14, 163.48, 151.79, 150.08, 145.14, 139.15, 131.23, 130.90, 129.39, 128.54,
125.71, 117.87, 105.45, 104.78, 90.88, 65.78, 25.33, 17.82; MS (ESI) m/z = 361.1 [M
+ Na]+; HRMS (ESI): m/z calcd for C20H19O5: 339.1227; found: 339.1219 [M + H]+.
4.1.7.2. 5,6-dihydroxy-7-(3-hydroxypropoxy)-2-phenyl-4H-chromen-4-one (21). Yield
1
85%; yellowish powder; mp 168-169 °C; H-NMR (DMSO-d6, 300 MHz, δ ppm):
12.51 (s, 1H, 5-OH), 8.69 (s, 1H, 6-OH), 8.10 (m, 2H, H-2′, H-6′), 7.59 (m, 3H, H-3′,
H-4′, H-5′), 7.01 (s, 1H, H-8), 6.98 (s, 1H, H-3), 4.60 (t, J = 4.6 Hz, 1H, -CH2OH),
4.20 (t, J = 4.6 Hz, 2H, -OCH2-), 3.61 (m, 2H, -OCH2-), 1.94 (m, 2H, -CH2-);
13C-NMR (DMSO-d6, 75 MHz, δ ppm): 182.28, 163.07, 153.96, 149.75, 146.05,
131.93, 130.84, 130.13, 129.09, 126.31, 105.16, 104.62, 91.88, 66.08, 57.17, 31.79;
MS (ESI) m/z = 327.1 [M - H]-, 329.1 [M + H]+; HRMS (ESI): m/z calcd for
C18H17O6: 329.1020; found: 329.1015 [M + H]+.
4.1.7.3. 4-(2-((7-(benzyloxy)-5-hydroxy-4-oxo-2-phenyl-4H-chromen-6-yl)oxy)ethoxy)
-4-oxobutanoic acid (22). To a solution of compound 2 (400 mg, 0.98 mmol) in
CH2Cl2 (10 mL) were added succinic anhydride (200 mg, 2 mmol) and DMAP (24.5
mg, 0.2 mmol), and the mixture was reacted at room temperature for 8 h. The solution
was concentrated under vacuum, and the residue was purified by column
chromatography (CH2Cl2-MeOH = 30:1 as the eluent) on silica gel. Yield 59%;
yellowish powder; mp 119-120 °C; 1H-NMR (DMSO-d6, 300 MHz, δ ppm): 7.93 (m,
2H, H-2′, H-6′), 7.85 (m, 5H, Ar-H), 7.61 (m, 3H, H-3′, H-4′, H-5′), 6.86 (s, 1H, 8-H),
6.79 (s, 1H, 3-H), 5.22 (s, 2H, -OCH2-), 4.37 (t, J = 6.1 Hz, 2H, -OCH2-), 4.30 (t, J =
6.1 Hz, 2H, -OCH2-), 2.58 (t, J = 6.0 Hz, 2H, -CH2-), 2.49 (t, J = 6.0 Hz, 2H, -CH2-);
MS (ESI) m/z = 503.1 [M - H]-.
4.1.7.4. Methyl-2-((7-(benzyloxy)-5-hydroxy-4-oxo-2-phenyl-4H-chromen-6-yl)oxy)
acetate (24). Baicalein (3.6 g, 10 mmol) and anhydrous potassium carbonate (3.45 g,
25 mmol) was dissolved in 10 mL DMF, then methyl chloroacetate (2.17 g, 20 mmol)
was added. The mixture was stirred at 60 °C under a nitrogen atmosphere for 9 h.
Then the reaction mixture was poured into water (45 mL). The precipitate was filtered
off, washed with water and recrystallized from ethanol. Yield 78%; yellowish powder;
mp 157-159 °C; 1H-NMR (CDCl3, 300 MHz, δ ppm): 12.85 (s, 1H, 5-OH), 7.85 (dd,
J = 1.8, 6.5 Hz, 2H, H-2′, H-6′), 7.54 (m, 5H, Ar-H), 7.39 (m, 3H, H-3′, H-4′, H-5′),
6.66 (s, 1H, 8-H), 6.61 (s, 1H, 3-H), 5.25 (s, 2H, -OCH2-), 4.78 (s, 2H, -OCH2-), 3.76

(s, 3H, -OCH3); MS (ESI) m/z = 433.1 [M + H]+.
4.1.7.5. Methyl-2-((5,7-dihydroxy-4-oxo-2-phenyl-4H-chromen-6-yl)oxy)acetate (25).
To a solution of compound 24 (6.5 g, 15 mmol) in dry CH2Cl2 (20 mL) were added
anhydrous AlCl3 (6 g, 45 mmol) and N, N-dimethylaniline (364 mg, 3 mmol) and the
mixture was reacted at room temperature for 3 h. Thereafter, the reaction mixture was
slowly poured into excess 1 M HCl and the reaction mixture was stirred for 10 min.
The precipitate was filtered off, washed with water and recrystallized from methanol.
1
Yield 83%; white solid; mp 162-163 °C; H-NMR (DMSO-d6, 300 MHz, δ ppm):
13.01 (s, 1H, 5-OH), 10.74 (s, 1H, 7-OH), 8.03 (m, 2H, H-2′, H-6′), 7.55 (m, 3H, H-3′,
H-4′, H-5′), 6.92 (s, 1H, H-8), 6.62 (s, 1H, H-3), 4.67 (s, 2H, -OCH2-), 3.70 (s, 3H,
-CH3); 13C-NMR (DMSO-d6, 75 MHz, δ ppm): 182.69, 170.04, 163.74, 157.56,
153.11, 152.53, 132.45, 131.11, 129.54, 128.78, 126.82, 105.09, 104.70, 94.89, 68.85,
52.14; HRMS (ESI): m/z calcd for C18H15O7: 343.0812; found: 343.0844 [M + H]+.
4.1.8 Synthesis of 26-44.
4.1.8.1. 2-((5,7-dihydroxy-4-oxo-2-phenyl-4H-chromen-6-yl)oxy)-N-(2-hydroxyethyl)
acetamide (26). A mixture of compound 25 (150 mg, 0.44 mmol) and 2-aminoethanol
(61 mg, 1 mmol) in 3 ml DMF, and the mixture was stirred at 80 °C for 5 h. Then the
reaction mixture was poured into water (30 mL). The precipitate was filtered off,
washed with water and recrystallized from ethanol. Yield 78%; white solid; mp
151-152 °C; 1H-NMR (DMSO-d6, 300 MHz, δ ppm): 13.05 (s, 1H, 5-OH), 8.38 (t, J =
4.2 Hz, 1H, CONH), 8.06 (d, J = 6.2 Hz, 2H, H-2′, H-6′), 7.57 (m, 3H, H-3′, H-4′,
H-5′), 6.97 (s, 1H, H-8), 6.66 (s, 1H, H-3), 4.50 (s, 2H, -OCH2-), 3.47 (t, J = 4.5 Hz,
13
2H, -OCH2-), 3.26 (t, J = 4.5 Hz, 2H, -NHCH2-); C-NMR (DMSO-d6, 75 MHz, δ
ppm): 182.22, 169.96, 163.34, 157.37, 152.95, 152.37, 132.03, 130.61, 129.84,
129.09, 126.37, 104.60, 104.26, 94.72, 71.49, 29.44, 41.39; MS (ESI) m/z = 370.1 [M
- H]-; HRMS (ESI): m/z calcd for C19H18O7N: 372.1078; found: 372.1113 [M + H]+;
calcd for C19H17O7NNa: 394.0897; found: 394.0928 [M + Na]+.
4.1.8.2.
2-((5,7-dihydroxy-4-oxo-2-phenyl-4H-chromen-6-yl)oxy)-N-(2-
(dimethylamino)ethyl)acetamide (27). According to the procedure for the preparation
of 26, 25 (150 mg, 0.44 mmol) and N, N-dimethyl-1, 2-ethanediamine (88 mg, 1
1
mmol) yielded 27 (79%) as a white solid; mp 138-139 °C; H-NMR (CDCl3, 300
MHz, δ ppm): 8.46 (m, 1H, CONH), 7.68 (m, 2H, H-2′, H-6′), 7.42 (m, 3H, H-3′, H-4′,
H-5′), 6.34 (s, 2H, H-8, H-3), 4.62 (s, 2H, -OCH2), 3.56 (m, 2H, NHCH2), 2.77 (m,
2H, NCH2), 2.48 (s, 6H, 2×CH3); 13C-NMR (CDCl3, 75 MHz, δ ppm): 181.79, 170.83,
163.04, 159.39, 153.28, 151.95, 131.60, 131.18, 130.75, 128.45, 125.65, 104.22,
103.67, 95.28, 71.33, 57.21, 43.98, 35.31; HRMS (ESI): m/z calcd for C21H23O6N2:

399.1551; found: 399.1579 [M + H]+.
4.1.8.3.
2-((5,7-dihydroxy-4-oxo-2-phenyl-4H-chromen-6-yl)oxy)-N,N-bis(2-
hydroxyethyl)acetamide (28). According to the procedure for the preparation of 26, 25
(150 mg, 0.44 mmol) and diethanolamine (105 mg, 1 mmol) yielded 28 (79%) as a
yellowish powder; mp 189-190 °C; 1H-NMR (DMSO-d6, 300 MHz, δ ppm): 13.01 (s,
1H, 5-OH), 11.98 (s, 1H, 7-OH), 7.98 (d, J = 7.2 Hz, 2H, H-2′, H-6′), 7.52 (m, 3H,
H-3′, H-4′, H-5′), 6.87 (s, 1H, H-8), 6.57 (s, 1H, H-3), 5.00 (s, 2H, OCH2), 3.57 (m,
13
4H, 2×OCH2), 3.45 (m, 2H, NCH2), 3.37 (m, 2H, NCH2); C-NMR (DMSO-d6, 75
MHz, δ ppm): 182.64, 172.61, 163.74, 158.50, 153.69, 153.09, 132.37, 131.28,
131.04, 129.45, 126.72, 104.95, 104.74, 95.15, 71.65, 58.67, 49.96, 48.98; HRMS
(ESI): m/z calcd for C24H20O5N2: 416.1367; found: 416.1372 [M + H]+.
4.1.8.4.
2-((5,7-dihydroxy-4-oxo-2-phenyl-4H-chromen-6-yl)oxy)-N,N-
diethylacetamide (29). According to the procedure for the preparation of 26, 25 (150
mg, 0.44 mmol) and diaethylamin (73 mg, 1 mmol) yielded 29 (75%) as a white
1
solid; mp 151-152 °C; H-NMR (CDCl3, 300 MHz, δ ppm): 13.00 (s, 1H, 5-OH),
11.53 (s, 1H, 7-OH), 7.89 (d, J = 6.5 Hz, 2H, H-2′, H-6′), 7.54 (m, 3H, H-3′, H-4′,
H-5′), 6.64 (s, 1H, H-8), 6.60 (s, 1H, H-3), 4.88 (s, 2H, OCH2), 3.47 (q, J = 6.1 Hz,
2H, NCH2), 3.26 (q, J = 6.1 Hz, 2H, NCH2), 1.26 (t, J = 6.1 Hz, 3H, CH2CH3), 1.20 (t,
J = 6.1 Hz, 3H, CH2CH3); 13C-NMR (CDCl3, 75 MHz, δ ppm): 182.32, 169.85,
163.55, 157.98, 153.55, 152.51, 131.31, 131.20, 130.82, 128.56, 125.80, 104.49,
94.77, 70.34, 40.54, 40.26, 13.35, 12.30; MS (ESI) m/z = 382.1 [M-H]-; 406.1 [M +
Na]+; HRMS (ESI): m/z calcd for C21H22O6N: 384.1442; found: 384.1436 [M + H]+.
4.1.8.5.
5,7-dihydroxy-6-(2-oxo-2-(piperazin-1-yl)ethoxy)-2-phenyl-4H-chromen
-4-one (30). According to the procedure for the preparation of 26, 25 (150 mg, 0.44
mmol) and 1, 4-diazacyclohexane (86 mg, 1 mmol) yielded 30 (65%) as a yellow oil;
1
mp 172-173 °C; H-NMR (CDCl3, 300 MHz, δ ppm): 7.86 (m, 2H, H-2′, H-6′), 7.52
(m, 3H, H-3′, H-4′, H-5′), 6.59 (s, 1H, H-8), 6.55 (s, 1H, H-3), 4.86 (s, 2H, OCH2),
3.66 (m, 4H, N(CH2)2), 2.91 (m, 4H, NH(CH2)2); 13C-NMR (CDCl3, 75 MHz, δ ppm):
182.79, 169.60, 164.06, 158.46, 154.09, 152.94, 131.76, 129.03, 128.70, 128.38,
126.28, 105.10, 105.03, 95.26, 70.67, 45.84, 45.59, 45.25, 43.35; HRMS (ESI): m/z
calcd for C21H21O6N2: 397.1394; found: 397.1426 [M + H]+.
4.1.8.6.
5,7-dihydroxy-6-(2-(4-methylpiperazin-1-yl)-2-oxoethoxy)-2-phenyl-4H-
chromen-4-one (31). According to the procedure for the preparation of 26, 25 (150 mg,
0.44 mmol) and 4-methylpiperazine (100 mg, 1 mmol) yielded 31 (83%) as a
yellowish powder; mp 164-165 °C; 1H-NMR (CDCl3, 300 MHz, δ ppm): 12.99 (s, 1H,
5-OH), 7.88 (m, 2H, H-2′, H-6′), 7.54 (m, 3H, H-3′, H-4′, H-5′), 6.63 (s, 1H, H-8),

6.59 (s, 1H, H-3), 4.88 (s, 2H, OCH2), 3.71 (t, J = 6.0 Hz, 2H, NCH2), 3.38 (t, J = 6.0
13
Hz, 2H, NCH2), 2.45 (m, 4H, N(CH2)2), 2.32 (s, 3H, NCH3); C-NMR (CDCl3, 75
MHz, δ ppm): 182.32, 169.13, 163.61, 157.73, 153.56, 152.46, 131.31, 130.98,
130.85, 128.56, 125.81, 104.62, 104.54, 94.72, 70.21, 54.00, 53.83, 45.46, 43.46,
41.81; HRMS (ESI): m/z calcd for C22H23O6N2: 411.1551; found: 411.1581 [M + H]+.
4.1.8.7.
5,7-dihydroxy-6-(2-(4-(2-hydroxyethyl)piperazin-1-yl)-2-oxoethoxy)-2-
phenyl-4H-chromen-4-one (32). According to the procedure for the preparation of 26,
25 (150 mg, 0.44 mmol) and 1-piperazineethanol (130 mg, 1 mmol) yielded 32 (57%)
1
as a yellowish powder; mp 182-183 °C; H-NMR (DMSO-d6, 300 MHz, δ ppm):
13.03 (s, 1H, 5-OH), 11.57 (s, 1H, 7-OH), 8.06 (d, J = 6.2 Hz, 2H, H-2′, H-6′), 7.59
(m, 3H, H-3′, H-4′, H-5′), 6.98 (s, 1H, H-8), 6.65 (s, 1H, H-3), 4.88 (s, 2H, OCH2),
4.42 (m, 1H, CH2OH), 3.49 (m, 2H, OCH2), 3.35 (m, 4H, CON(CH2)2), 2.38 (m, 6H,
13
N(CH2)3); C-NMR (DMSO-d6, 75 MHz, δ ppm): 182.26, 169.30, 163.33, 157.98,
153.22, 152.65, 132.06, 131.04, 130.61, 129.11, 126.38, 104.57, 104.29, 94.69, 70.85,
59.91, 58.44, 52.69, 52.47, 43.79, 41.76; MS (ESI) m/z = 441.2 [M+H]+; 463.2 [M +
Na]+; HRMS (ESI): m/z calcd for C23H25O7N2: 441.1656; found: 441.1666 [M + H]+.
4.1.8.8.
5,7-dihydroxy-6-(2-(4-hydroxypiperidin-1-yl)-2-oxoethoxy)-2-phenyl-4H-
chromen-4-one (33). According to the procedure for the preparation of 26, 25 (150 mg,
0.44 mmol) and 4-hydroxypiperidine (101 mg, 1 mmol) yielded 33 (69%) as a
yellowish powder; mp 173-174 °C; 1H-NMR (DMSO-d6, 300 MHz, δ ppm): 13.02 (s,
1H, 5-OH), 11.85 (s, 1H, 7-OH), 8.04 (d, J = 7.5 Hz, 2H, H-2′, H-6′), 7.56 (m, 3H,
H-3′, H-4′, H-5′), 6.95 (s, 1H, H-8), 6.62 (s, 1H, H-3), 4.89 (s, 2H, OCH2), 3.85 (m,
1H, OCH), 3.71 (m, 2H, NCH2), 3.16 (m, 2H, NCH2), 1.72 (m, 2H, CHCH2), 1.40 (m,
2H, CHCH2); 13C-NMR (DMSO-d6, 75 MHz, δ ppm): 182.25, 169.17, 163.32, 157.98,
153.21, 152.65, 132.04, 131.12, 130.59, 129.08, 126.35, 104.54, 104.30, 94.67, 70.88,
64.86, 41.24, 40.28, 33.80, 33.41; HRMS (ESI): m/z calcd for C22H22O7N: 412.1391;
found: 412.1401 [M + H]+.
4.1.8.9. 2-((5,7-dihydroxy-4-oxo-2-phenyl-4H-chromen-6-yl)oxy)acetic acid (34). A
mixture of compound 25 (150 mg, 0.44 mmol) and 1.5 N lithium hydroxide (84 mg, 2
mmol) in 5 ml THF/MeOH (v/v 3:1), and the mixture was stirred at room temperature
for 5 h. Then adjust the pH of the solution to 3.0 with 2 N HCl. The precipitate was
filtered off, and washed with water. Yield 83%; white solid; mp 151-152 °C; Yield
1
93%; white solid; mp 155-156 °C; H-NMR (DMSO-d6, 300 MHz, δ ppm): 13.04 (s,
1H, 5-OH), 8.06 (d, J = 7.1 Hz, 2H, H-2′, H-6′), 7.59 (m, 3H, H-3′, H-4′, H-5′), 6.96
(s, 1H, H-8), 6.67 (s, 1H, H-3), 4.64 (s, 2H, OCH2); 13C-NMR (DMSO-d6, 75 MHz, δ
ppm): 182.71, 172.06, 163.76, 157.56, 153.15, 152.53, 132.47, 131.10, 130.23,
129.55, 126.82, 105.07, 104.78, 69.05; HRMS (ESI): m/z calcd for C17H13O7:

329.0656; found: 329.0248 [M + H]+.
4.1.8.10.
2-((7-(benzyloxy)-5-hydroxy-4-oxo-2-phenyl-4H-chromen-6-yl)oxy)-N-(2-
hydroxyethyl) acetamide (35). According to the procedure for the preparation of 26,
24 (216 mg, 0.5 mmol) and 2-aminoethanol (61 mg, 1 mmol) yielded 35 (73%) as a
1
light yellow powder; mp 209-211 °C; H NMR (300 MHz, CDCl3, δ ppm): 12.86 (s,
1H, Ar-OH), 8.04 (m, 1H, -NHCO-), 7.79-7.81 (m, 2H, Ar-H), 7.38-7.54 (m, 8H,
Ar-H), 6.61 (s, 1H, -COCH-), 6.59 (s, 1H, Ar-H), 5.15 (s, 2H, -OCH2-), 4.56 (s, 2H,
-COCH2O-), 3.61 (t, 2H, -CH2CH2OH), 3.30 (t, 2H, -CONHCH2-); 13C NMR (75
MHz, CDCl3, δ ppm): 182.43, 170.78, 164.21, 156.88, 153.20, 152.04, 134.91, 131.99,
131.55, 130.75, 129.02, 128.87, 128.70, 127.64, 126.15, 106.01, 105.30, 91.83, 72.65,
71.20, 62.05; HRMS (ESI): m/z calcd for C26H24NO7: 462.1547; found: 462.1552 [M
+ H]+.
4.1.8.11. 2-((7-(benzyloxy)-5-hydroxy-4-oxo-2-phenyl-4H-chromen-6-yl)oxy)-N,N-bis
(2-hydroxyethyl)acetamide (36). According to the procedure for the preparation of 26,
24 (216 mg, 0.5 mmol) and diethanolamine (105 mg, 1 mmol) yielded 36 (68%) as a
1
light yellow powder; mp 249-252 °C; H NMR (300 MHz, CDCl3, δ ppm): 7.75 (m,
2H, Ar-H), 7.34-7.45 (m, 8H, Ar-H), 6.54 (s, 1H, -COCH-), 6.51 (s, 1H, Ar-H), 5.14
(s, 2H, -OCH2-), 4.77 (s, 2H, -COCH2O-), 3.78 (m, 2H, -CONCH2-), 3.66 (m, 2H,
13
-CONCH2-), 3.55 (m, 2H, -CH2CH2OH), 3.48 (m, 2H, -CH2CH2OH); C NMR (75
MHz, CDCl3, δ ppm): 182.38, 169.81, 163.91, 157.59, 153.27, 152.70, 135.31, 131.84,
130.79, 130.71, 128.95, 128.70, 128.40, 127.48, 126.10, 106.01, 105.24, 91.99, 71.43,
70.98, 60.61, 60.35, 51.47, 50.38; HRMS (ESI): m/z calcd for C28H28NO8: 506.1809;
found: 506.1810 [M + H]+.
4.1.8.12. 7-(benzyloxy)-6-(2-(4-ethylpiperazin-1-yl)-2-oxoethoxy)-5-hydroxy-2-phenyl
-4H-chromen-4-one (37). According to the procedure for the preparation of 26, 24
(216 mg, 0.5 mmol) and 1-ethylpiperazine (114 mg, 1 mmol) yielded 37 (63%) as a
1
light yellow powder; mp 229-231 °C; H NMR (300 MHz, CDCl3, δ ppm): 12.74 (s,
1H, Ar-OH), 7.82 (m, 2H, Ar-H), 7.43-7.59 (m, 8H, Ar-H), 6.61 (s, 1H, -COCH-),
6.58 (s, 1H, Ar-H), 5.17 (s, 2H, -OCH2-), 4.68 (s, 2H, -COCH2O-), 3.71 (m, 2H,
-CONCH2-), 3.61 (m, 2H, -CONCH2-), 2.38 (m, 2H, -CH2CH2NCH2-), 2.36 (m, 2H,
-CH2CH2NCH2-), 2.30 (m, 2H, -CH2CH3), 1.06 (t, 3H, J = 5.79Hz, -CH2CH3); 13C
NMR (75 MHz, CDCl3, δ ppm): 182.50, 166.20, 163.96, 157.70, 153.41, 153.16,
135.33, 131.82, 130.99, 130.85, 128.98, 128.72, 128.41, 127.54, 126.15, 106.20,
105.45, 91.90, 71.75, 71.03, 52.72, 52.25, 52.10, 45.38, 41.74, 11.81; HRMS (ESI):
m/z calcd for C30H31N2O6: 515.2177; found: 515.2179 [M + H]+.
4.1.8.13.
7-(benzyloxy)-5-hydroxy-6-(2-(4-methylpiperazin-1-yl)-2-oxoethoxy)-2-

phenyl-4H-chromen-4-one (38). According to the procedure for the preparation of 26,
24 (216 mg, 0.5 mmol) and 4-methylpiperazine (100 mg, 1 mmol) yielded 38 (79%)
as a yellowish powder; mp 238-239 °C; 1H-NMR (CDCl3, 300 MHz, δ ppm): 12.89 (s,
1H, 5-OH), 7.68 (m, 2H, H-2′, H-6′), 7.58 (m, 3H, H-3′, H-4′, H-5′), 7.25-7.31 (m, 5H,
Ar-H), 6.61 (s, 1H, H-8), 6.60 (s, 1H, H-3), 5.19 (s, 2H, -OCH2-), 4.79 (s, 2H,
-OCH2-), 3.81 (t, J = 6.0 Hz, 2H, -NCH2-), 3.37 (t, J = 6.0 Hz, 2H, -NCH2-), 2.58 (m,
13
4H, -N(CH2)2-), 2.49 (s, 3H, -NCH3); C-NMR (CDCl3, 75 MHz, δ ppm): 182.31,
168.39, 163.54, 157.67, 153.38, 152.93, 131.9, 130.78, 130.96, 128.73, 128.59,
127.94, 127.30, 125.89, 104.67, 104.29, 94.91, 7132, 70.29, 54.38, 53.17, 45.81,
43.31, 41.29; HRMS (ESI): m/z calcd for C29H29O6N2: 501.2020; found: 501.2018 [M
+ H]+.
4.1.8.14.
7-(benzyloxy)-5-hydroxy-6-(2-(4-(2-hydroxyethyl)piperazin-1-yl)-2-
oxoethoxy)-2-phenyl-4H-chromen-4-one (39). According to the procedure for the
preparation of 26, 24 (216 mg, 0.5 mmol) and 1-(2-hydroxyethyl) piperazine (130 mg,
1
1 mmol) yielded 39 (68%) as a yellowish powder; mp 278-279 °C; H NMR (300
MHz, DMSO-d6, δ ppm): 12.86 (s, 1H, Ar-OH), 8.08 (m, 2H, Ar-H), 7.43-7.59 (m,
8H, Ar-H), 7.08 (s, 1H, -COCH-), 7.04 (s, 1H, Ar-H), 5.25 (s, 2H, -OCH2-), 4.56 (s,
2H, -COCH2O-), 4.45 (s, 1H, -CH2OH), 3.47 (m, 2H, -CH2OH), 3.41 (m, 4H,
13
-CON(CH2-)2), 2.33 (m, 4H, -CH2NCH2CH2OH), 2.18 (m, 2H, -CH2OH); C NMR
(75 MHz, DMSO-d6, δ ppm): 182.59, 165.75, 163.81, 158.01, 153.30, 152.52, 136.14,
132.49, 130.87, 129.47, 128.92, 128.50, 126.72, 105.76, 105.36, 93.04, 71.32, 71.03,
60.39, 58.73, 53.52, 53.09, 45.15, 41.54; HRMS (ESI): m/z calcd for C30H31O7N2:
531.2126; found: 531.2120 [M + H]+.
4.1.8.15.
7-(benzyloxy)-5-hydroxy-6-(2-(4-hydroxypiperazin-1-yl)-2-oxoethoxy)-2-
phenyl-4H-chromen-4-one (40). According to the procedure for the preparation of 26,
24 (216 mg, 0.5 mmol) and 4-hydroxypiperidinehydrochloride (137 mg, 1 mmol)
yielded 40 (73%) as a yellowish powder; mp 235-237 °C; 1H NMR (300 MHz, CDCl3,
δ ppm): 12.70 (s, 1H, Ar-OH), 7.78 (m, 2H, Ar-H), 7.32-7.49 (m, 8H, Ar-H), 6.58 (s,
1H, -COCH-), 6.54 (s, 1H, Ar-H), 5.15 (s, 2H, -OCH2-), 4.67 (s, 2H, -COCH2O-),
3.98 (m, 2H, -NCH2-), 3.81 (m, 1H, -CHOH), 3.25 (m, 1H, -NCH2-), 3.09 (m, 1H,
-NCH2-), 2.92 (s, 1H, -CHOH), 1.79 (m, 2H, -CH2CH2CH-), 1.46 (m, 2H,
13
-CH2CH2CH-); C NMR (75 MHz, CDCl3, δ ppm): 182.46, 166.38, 163.95, 157.69,
153.31, 152.94, 135.35, 131.83, 130.92, 130.86, 128.95, 128.70, 128.38, 127.49,
126.12, 106.09, 105.30, 91.93, 71.74, 70.97, 66.82, 42.78, 39.38, 34.38, 33.59;
HRMS (ESI): m/z calcd for C28H27O7N2: 503.1813; found: 503.1819 [M + H]+.
4.1.8.16.
7-(benzyloxy)-5-hydroxy-6-(2-oxo-2-(4-(pyrimidin-2-yl)piperazin-1-yl)
ethoxy)-2-phenyl-4H-chromen-4-one (41). According to the procedure for the

preparation of 26, 24 (216 mg, 0.5 mmol) and 2-(1-piperazinyl)pyrimidine (164 mg, 1
mmol) yielded 41 (62%) as a yellowish powder; mp 286-289 °C; 1H NMR (300 MHz,
CDCl3, δ ppm): 12.79 (s, 1H, Ar-OH), 8.31 (d, 2H, J = 6.31 Hz, Ar-OH), 7.80 (m, 2H,
Ar-H), 7.39-7.49 (m, 8H, Ar-H), 6.61 (s, 1H, Ar-H), 6.58 (s, 1H, Ar-H), 6.51 (m, 1H,
Ar-H), 5.16 (s, 2H, -OCH2-), 4.73 (s, 2H, -COCH2O-), 3.60-3.77 (m, 8H,
-N(CH2CH2)2N-); 13C NMR (75 MHz, CDCl3, δ ppm): 182.47, 166.68, 163.94,
161.36, 157.62, 153.41, 153.12, 135.25, 131.80, 130.98, 128.96, 128.72, 128.43,
127.53, 126.13, 110.52, 110.14, 106.17, 105.42, 91.86, 71.97, 71.05, 53.39, 45.39,
43.80, 43.16, 41.69; HRMS (ESI): m/z calcd for C32H29O6N4: 565.2082; found:
565.2079 [M + H]+.
4.1.8.17. 7-(benzyloxy)-5-hydroxy-6-(2-oxo-2-(piperazin-1-yl)ethoxy)-2-phenyl-4H-
chromen-4-one (42). According to the procedure for the preparation of 26, 24 (216 mg,
0.5 mmol) and piperazine (86 mg, 1 mmol) yielded 42 (83%) as a yellowish powder;
mp 247-249 °C; 1H NMR (300 MHz, CDCl3, δ ppm): 7.75 (m, 2H, Ar-OH), 7.31-7.45
(m, 8H, Ar-H), 6.55 (s, 1H, -COCH-), 6.53 (s, 1H, Ar-H), 5.12 (s, 2H, -OCH2-), 4.64
(s, 2H, -COCH2O-), 3.61 (m, 2H, -CONCH2-), 3.54 (m, 2H, -CONCH2-), 2.79 (m, 2H,
-CH2CH2NH-), 2.74 (m, 2H, -CH2CH2NH-); 13C NMR (75 MHz, CDCl3, δ ppm):
182.50, 166.47, 163.98, 157.72, 153.44, 153.10, 135.38, 131.93, 130.94, 130.82,
129.05, 128.80, 128.54, 127.68, 126.19, 106.19, 105.42, 91.97, 71.72, 71.11, 46.21,
45.81, 45.28, 42.37; HRMS (ESI): m/z calcd for C28H27O6N2: 487.1864; found:
487.1859 [M + H]+.
4.1.8.18.
7-(benzyloxy)-5-hydroxy-6-(2-oxo-2-(piperidin-1-yl)ethoxy)-2-phenyl-4H-
chromen-4-one (43). According to the procedure for the preparation of 26, 24 (216 mg,
0.5 mmol) and piperidine (85 mg, 1 mmol) yielded 43 (82%) as a yellowish powder;
mp 213-216 °C; 1H NMR (300 MHz, CDCl3, δ ppm): 12.76 (s, 1H, Ar-OH), 7.86 (m,
2H, Ar-H), 7.35-7.53 (m, 8H, Ar-H), 6.66 (s, 1H, -COCH-), 6.61 (s, 1H, Ar-H), 5.22
(s, 2H, -OCH2-), 4.70 (s, 2H, -COCH2O-), 3.61 (t, 2H, J = 8.31 Hz, -NCH2-), 3.46 (t,
2H, J = 8.31 Hz, -NCH2-), 1.83 (m, 2H, -CH2CH2CH2-), 1.80 (m, 2H, -CH2CH2CH2-);
13C NMR (75 MHz, CDCl3, δ ppm): 182.58, 166.58, 163.95, 157.72, 153.31, 152.97,
135.50, 131.78, 131.11, 128.99, 128.66, 128.28, 127.42, 126.18, 125.96, 106.27,
105.51, 92.00, 73.46, 72.04, 71.02, 60.08, 46.07, 45.92, 26.02, 23.81; HRMS (ESI):
m/z calcd for C29H28O6N: 486.1911; found: 486.1917 [M + H]+.
4.1.8.19.
7-(benzyloxy)-5-hydroxy-6-(2-morpholino-2-oxoethoxy)-2-phenyl-4H-
chromen-4-one (44). According to the procedure for the preparation of 26, 24 (216 mg,
0.5 mmol) and morpholine (87 mg, 1 mmol) yielded 43 (78%) as a yellowish powder;
mp 219-221 °C; 1H NMR (300 MHz, CDCl3, δ ppm): 12.76 (s, 1H, Ar-OH), 7.78 (m,
2H, Ar-H), 7.40-7.43 (m, 8H, Ar-H), 6.58 (s, 1H, -COCH-), 6.56 (s, 1H, Ar-H), 5.13

(s, 2H, -OCH2-), 4.65 (s, 2H, -COCH2O-), 3.49-3.65 (m, 8H, -N(CH2CH2)2O); 13C
NMR (75 MHz, CDCl3, δ ppm): 182.45, 166.45, 163.95, 157.66, 153.40, 153.10,
135.22, 131.85, 130.89, 128.97, 128.74, 128.53, 127.69, 126.12, 106.12, 105.36,
71.72, 71.06, 66.69, 66.53, 46.03, 42.11; HRMS (ESI): m/z calcd for C28H26O7N:
488.1704; found: 488.1709 [M + H]+.
4.2. Biological assays
4.2.1 Anti-angiogenic screening of phytochemicals in zebrafish model
Adult zebrafish were kept and raised according to the zebrafish book guidelines. We
used 20 dechorinated 24 hpf-embryos per experimental group in a 24-well plate and
incubated with 1 mL of embryo water containing various concentrations of drug at
28.5 °C for additional 48 h.29 In order to evaluate the compounds’ activities in a fast
and efficient way, we tested all the compounds at doses of 1, 5, 10, 25, 50, 75, 100,
150, 200 μM. 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.
4.2.2 In vitro proliferation assay
Proliferation assay was performed as described.27 HUVEC (3000 per well) were
seeded in 96-well plate in the EGM-2 medium for 12 h for attachment. Then cells
were treated for 48 h with EGM-2 medium containing vehicle 0.1% dimethyl
sulfoxide (DMSO) and various concentrations of 42. Cell growth was assessed using
a cell-counting kit-8 (CCK-8, Dojindo, Japan) according to the protocol provided. The
spectrophotometric absorbance of each well was measured by a multi-detection
microplate reader (Synergy HT, BioTeks, USA) at a wavelength of 450 nm. Each
treatment was performed in triplicate. The IC50 was calculated by GraphPad Prism 6
statistical software (San Diego, CA, USA).
4.2.3 In vitro migration assay
Boyden chamber migration assay: HUVEC (8000 per well) were seeded on 48-well
chamber (8 μm pore-size, AP48, Neuro Probe). The top chamber contained vehicle or
various concentrations of 42. Cells were allowed to migrate for 8 hours. Non-migrated
cells were scraped with a cotton swab, and migrated cells were fixed with 100%
methanol and stained with 0.05% crystal violet. Images were taken at 0 h and 12 h
independently through an inverted microscope (Nikon, Japan). The migrated cells

were counted manually. The values were observed from four randomly selected fields.
The percentage of inhibition was expressed using control wells at 100%.
4.2.4 In vitro tube formation assay
Matrigel (growth factor reduced; BD Biosciences, USA) was thawed at 4 °C
overnight. Each well of pre-chilled 96-well plates was coated with 100 μl of Matrigel,
incubated and solidified at 37 °C for 120 min. HUVECs at the density of 2 × 104 per
well in EGM-2 containing the indicated concentrations of 42 or 2 μM SU5416were
placed onto the Matrigel layer and incubated for 12 h. The network formation was
visualized and imaged under an inverted microscope (Nikon, Japan) at 100 ×
magnification. The tube length was quantified by Image Pro Plus 6.0 software. The
values were observed from four randomly selected fields.
4.2.5 Cell toxicity against A549 and L929 cells
DMEM medium and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) were purchased from Sigma. Newborn calf serum was purchased from
Hangzhou Sijiqing Biological Engineering Materials. All other chemicals were of
analytical grade or the highest chromatographic purity available. Human lung cancer
cell (A549) and fibroblast cell (L929) were seeded into 96-well plates at a density of
4×104 cells per mL in DMEM medium supplemented with 10% fetal bovine serum,
5% horse serum, 100U/mL penicillin and 100U/mL streptomycin in a humidified
atmosphere of 5% CO2 at 37 °C, respectively. After 24h incubation, different
concentrations of compound 42 was added and incubated with the cells for 48h. Then,
the cell viability was measured with MTT assay. The absorbance was determined at
570 nm using a microplate reader. Compared with the normal cells, the viability of
cells treated with drugs is calculated by the following formula: OD (drug-treated)/OD
(normal cells) × 100%. Data are expressed as mean ± SD.
4.2.6 Apotosis assay and caspase‑ 3 activity assay
Apoptosis was assessed by staining with Annexin V-FITC and propidium iodide (PI)
using the apoptosis detection kit (BD Biosciences, Brazil), according to the
manufacturer’s protocol. A549 cells (2 × 105/well) were placed in 12-well culture
plates at 37 °C and 5% CO2. After 24 h cells were incubated with medium in absence
(control cells) or presence of Bothropoidin (10 and 40 g/mL). After 24 h cells were
incubated with 5 L of annexin V-FITC and 2 g/mL PI for 30 min at 25 °C. After
incubation the cells were examined by flow cytometry (BD Accure C6, USA) and
analyzed via the software FlowJo (FlowJo v.7.6.5; Tree Star Inc., Ashland, OR, USA).

The activities of caspase-3 was determined by caspase-3 Activity Assay kit (Beyotime
institute of biotechnology, Jiangsu, China) as the manufacturer's instructions. The
cells were collected and lysed by cell lysis buffer followed by centrifuging at 15,000
rpm for 15 min at 4 ˚C. After assaying protein concentration, 110 µg of protein was
added in 50 µL cell lysis buffer. Next, the reaction buffer and 10 µL of
Ac-DEVD-pNA substrate (2 mM) was added to the lysis buffer. The reaction mixtures
were incubated at 37 ˚C for 3 h followed by detecting the activity of caspase-3 at 405
nm in a microtiter plate reader.
4.2.7 Statistical analysis
Data are expressed as mean ± SEM based on at least three independent experiments.
Statistical analysis was performed using one-way ANOVA with a Tukey’s test by
SPSS 10.0 for Windows (SPSS, Inc, Chicago, IL, USA). P<0.05 was considered
significant.
Acknowledgments
This research work was financially supported by the Fundamental Research Funds for
the Central Universities (JKZY2013001) and the Priority Academic Program
Development of Jiangsu Higher Education Institutions.
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