Synthesis, characterization and preliminary biological evaluation of chrysin amino acid derivatives that induce apoptosis and EGFR downregulation

Article abstract of DOI:10.1080/10286020.2019.1702028

Chrysin amino acid derivatives were synthesized to evaluate for their antiproliferative activities. Among them, N-(7-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)valeryl)-L-leucine (8c) displayed the most remarkable inhibitory activities against MCF-7 cells with IC₅₀ values of 16.6 μM. Preliminary mechanistic studies showed that 8c could inhibit the colony formation and migration of MCF-7 cells. Flow cytometry analysis demonstrated that 8c mediated cell apoptosis and the prolongation of cell cycle progression in G1/S-phase against MCF-7 cells. Besides, 8c displayed the moderate inhibition against EGFR. Western blot assay suggested that 8c significantly inhibited EGFR phosphorylation. Molecular docking showed that 8c can bind the EGFR kinase well.

Full text of DOI:10.1080/10286020.2019.1702028

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Synthesis, characterization and preliminary
biological evaluation of chrysin amino acid
derivatives that induce apoptosis and EGFR
downregulation
Yun-Mei Liu, Yang Li, Rong-Fang Liu, Jie Xiao, Bin-Ning Zhou, Qi-Zhi Zhang &
Jian-Xin Song
To cite this article: Yun-Mei Liu, Yang Li, Rong-Fang Liu, Jie Xiao, Bin-Ning Zhou, Qi-Zhi Zhang
& Jian-Xin Song (2019): Synthesis, characterization and preliminary biological evaluation of chrysin
amino acid derivatives that induce apoptosis and EGFR downregulation, Journal of Asian Natural
Products Research, DOI: 10.1080/10286020.2019.1702028
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2019.1702028
Synthesis, characterization and preliminary biological
evaluation of chrysin amino acid derivatives that induce
apoptosis and EGFR downregulation
Yun-Mei Liua,b, Yang Lib , Rong-Fang Liub, Jie Xiaob, Bin-Ning Zhoub, Qi-Zhi
Zhangb and Jian-Xin Songa
aKey Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of
Education of China), Key Laboratory of the Assembly and Application of Organic Functional
b
molecules of Hunan Province, Hunan Normal University, Changsha 410081, China; Institute of
Pharmacy & Pharmacology, Hunan Province Cooperative Innovation Center for Molecular Target
New Drug Study, University of South China, Hengyang 421001, China
ABSTRACT
ARTICLE HISTORY
Received 5 April 2019
Accepted 3 December 2019
Chrysin amino acid derivatives were synthesized to evaluate for
their antiproliferative activities. Among them, N-(7-((5-hydroxy-4-
oxo-2-phenyl-4H-chromen-7-yl)oxy)valeryl)-L-leucine (8c) displayed
the most remarkable inhibitory activities against MCF-7 cells with
IC50 values of 16.6 lM. Preliminary mechanistic studies showed
that 8c could inhibit the colony formation and migration of
MCF-7 cells. Flow cytometry analysis demonstrated that 8c medi-
ated cell apoptosis and the prolongation of cell cycle progression
in G1/S-phase against MCF-7 cells. Besides, 8c displayed the mod-
erate inhibition against EGFR. Western blot assay suggested that
8c significantly inhibited EGFR phosphorylation. Molecular dock-
ing showed that 8c can bind the EGFR kinase well.
KEYWORDS
Chrysin derivatives; EGFR;
amino acid; antitumor
CONTACT Yun-Mei Liu
28 Changsheng west Rd, Hengyang 421001, PR China; Jian-Xin Song
lymhsm@126.com
Institute of Pharmacy & Pharmacology, University of South China,
jxsong@hunnu.edu.cn Key Laboratory
of the Assembly and Application of Organic Functional Molecules of Hunan Province, Hunan Normal University,
Changsha 410081, China
Supplemental data for this article is available online at https://doi.org/10.1080/10286020.2019.1702028.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

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Y.-M. LIU ET AL.
1. Introduction
It was reported that chryisn exhibits many biological activities and possesses a wide
range of pharmacological effects, such as anti-inflammatory [1], anti-estrogenic [2]
and anticancer [3]. Recently, several researchers have attempted to modify the profile
of chrysin and found that chrysin derivatives have diverse activities including anti-
cancer effects [4]. Noteworthy, part of chrysin derivatives showed potent epidermal
growth factor receptor (EGFR) inhibitory activity [5]. EGFR, as a transmembrane, is
an important class of protein tyrosine kinases [6]. Studies have shown that EGFR
overexpression is associated with different types of cancers, including renal carcin-
oma, lung, breast, esophageal, colon, pancreatic and prostate cancer. Accordingly, the
EGFR family is a major target of antitumor targets and EGFR inhibitors may be
effective in the treatment of cancer [7].
Considering that amino acids are the basic substance in biological system and have
special physiological functions, for example good biocompatibility and cell affinity
[8]. Through modification with amino acid, the parent structure can either increase
the interaction and selectivity toward tumor cells or reduce the side effect and
improve the bio-availability [9].
Overexpression of EGFR contributes to cell proliferation, survival and motility as
well as angiogenesis, which are responsible for all important aspects of tumorigenesis
[10]. For this reason, many companies are actively developing inhibitors targeting
EGFR. Several studies have been approved for the use of quercetin as inhibitors of
EGFR [11, 12]. However, there are few studies about the research on the chrysin
derivatives targeted EGFR. Previously, two series of amino acid derivatives containing
chrysin were prepared from 7-O-carboxyethyl chrysin (4a) and 7-O-carboxybutyl
chrysin (4b) and amino acid methyl easters by treatment with amidated reagent [13,
14]. Currently, we investigated whether the EGFR phosphorylation is involved in the
antiproliferative activity of potent compound 8c against MCF-7 cells. Specifically,
compound 8c was further studies to explore its mechanism on the apoptosis signal
pathway by flow cytometry (FCM), clonogenicity assay, cell migration, enzyme-linked
immunosorbent assay (ELISA) and western blot assay. Docking simulations were per-
formed using the X-ray crystallographic structure of the target protein EGFR in com-
plex with inhibitors to explore the binding modes of 8c at the active site.
2. Results and discussion
2.1. Chemistry
Here, we smoothly synthesized two series of chrysin amino acid analogues. All com-
pounds were identified by spectroscopic data. The synthesis routes are outlined in
Scheme 1 [13, 15].
2.2. Biological activity
2.2.1. Antiproliferative activity
All the final products were screened for the antiproliferative activities against the
human breast cancer MCF-7 cells and human liver cancer HepG-2 cells by the MTT

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Scheme 1. Synthesis of amino acid derivatives containing chrysin.
assay. 5-Fluorouracil (5-FU) was used as reference compound. The cells were propa-
gated for 48 hours in the test substance, and the antiproliferative results were sum-
marized in Table 1. Against MCF-7 cells, compound 8c showed the most potent
activity with IC50 values of 16.6 lm, which is comparable to the positive control 5-FU
(IC50¼57.1 lm). Against HepG-2 cells, compounds 4b and 6d were the most potent
agents compared with other compounds, and the IC50 values were 39.4 and 47.1 lm,
respectively. They showed inferior antiproliferative effects than 5-FU (IC50
¼
29.2 lm). Furthermore, the toxicity against normal human umbilical vein endothelial
cell line HUVEC was also examined. Two series of amino acid derivatives containing
chrysin manifested apparent un-toxic effect on HUVEC.
Based on our previous studies [15, 16], the introduction of the amino acid
group at the C-7 position of chrysin largely improves inhibitory activities against
cancer cell lines. Initially, all chrysin derivatives were evaluated in terms of their
inhibitory activities in MCF-7 and HepG-2 cells. As shown in Table 1, all of com-
pounds generally showed various degrees of inhibitory effects. In MCF-7 cells,
compared with amino acid ester derivatives 5a–5d and 6a–6d, amino acid deriva-
tives 7a–7d and 8a–8d exhibited better inhibitory effects. The most obvious reason
for this phenomenon is the formation of potential hydrogen bonding between the
carboxyl group and target protein. Additionally, compared with compounds 7a–7d
and 8a–8d, the results showed that chrysin coupling with long-chain can achieve
good inhibitory activity. This is consistent with the conclusions reported in previ-
ous research. Nevertheless, as shown in Figure 1, L-leucine analogue 8c could
maintain two hydrogen bonds with the hinge region, which should be responsible
for the raised inhibitory potency of 8c. Specifically, isobutyl group (8c) reaches
deep into a pocket and forms hydrophobic interactions with various aromatic resi-
dues. However, the bulky phenyl group (8a) and the methyl group (8d) led to loss
of inhibitory activity. Unfortunately, in HepG-2 cells, it should be noted that there
are no clear structure-activity relationship for chrysin amino acid derivatives,
underscoring the importance of the flavonoid scaffold and the types of amino
acids for the activity.

4
Y.-M. LIU ET AL.
Table 1. Inhibitory activity of compounds 3a–8d against the tested cell lines.
IC50 (lm)
Compounds
HepG-2
MCF-7
HUVEC
3a
3b
4a
4b
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
Chrysin
5-FU
102 4.9
110 1.3
259 4.3
39.4 3.9
>150
>150
>150
>150
>150
>150
>150
47.1 1.3
>150
58.8 0.6
>150
72.2 1.0
58.7 1.3
>150
106 2.7
131 5.2
>150
49.2 1.0
26.1 3.2
74.4 4.3
>150
>150
>150
>150
>150
>150
N.D.
>150
N.D.
>150
>150
98.2 5.1
109 4.3
91.6 0.5
62.8 4.6
44.5 0.3
>150
>150
63.6 3.1
>150
139 3.9
>150
N.D.
>150
>150
>150
>150
N.D.
N.D.
>150
N.D.
>150
N.D.
>150
>150
>150
31.2 1.1
16.6 0.8
>150
143 1.9
57.1 3.2
29.2 3.2
2.2.2. Clonogenicity assay and cell migration
The excellent antiproliferative inhibition of potential compound 8c toward MCF-7
cells encouraged us to examine the effect on colony and migration of those cells. The
colony formation of cancer cells indicates an indirect method of neoplastic transform-
ation [17]. As shown in Figure 2A, treatment of MCF-7 cells with 8c for 10 days led
to obvious fewer colonies in a concentration-dependent manner compared with the
control, and the colony formation was almost inhibited completely at 20 lmol/L. This
study offered new evidence for the anticancer activity of 8c. On the side, we assessed
the role of 8c on cell migration, a key determinant of malignant progression and
metastases [18]. As show in Figure 2B, treatment of MCF-7 cells with 8c for 48 h at
referential concentrations obviously inhibited cell migrations in a concentration-
dependent manner. This demonstrated that 8c could block the MCF-7 cell migration
significantly.
2.2.3. Cell apoptosis and G1/S- phase cell cycle arrest induced by compound 8c
In order to identify the effect of 8c on tumor cell apoptosis and cycle progression,
FCM were performed on MCF-7 cells treated with different concentrations of 8c for
48 h. As shown in Figure 3A and B, 8c increased the proportion of cells in G1/S
phase and decreased the fraction in G2-phase in a dose-dependent manner, indicating
that 8c arrested MCF-7 cells at G1/S phase. Moreover, 8c induced cellular apoptosis
in a concentration-dependent manner (Figure 3C), and the percentage of apoptotic
cells induced by 8c at 5, 10 and 20 lmol/L were 6.9%, 7.1% and 8.2%, respectively.
As shown in Figure 3D, the apoptotic cells were also remarkably (P ＜0.05 or P＜
0.01) increased in a dose-dependent manner. These results indicated that 8c can
mediate cell apoptosis and a prolongation of cell cycle progression in G1/S phase
against MCF-7.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Figure 1. (A) Representative images of MCF-7 cell colonies after treatment with compound 8c at
different concentrations for 10 days. (B) Wound healing assay. Effect of compound 8c on migration
of MCF-7 cells. The images were captured using phase contrast microscopy before (0 h), and after
48 h of treatment with compound 8c.
2.2.4. Inhibitory activity against EGFR tyrosine kinase
Over-expression of certain growth factor receptor kinases such as epidermal growth
factor receptor (EGFR) has been implicated as being important in cancer [19]. EGFR
is over-expressed in plenty tumors including those derived from the brain, lung, blad-
der, head and neck [20]. In order to test whether compound 8c modulates the expres-
sion of EGFR, we performed ELISA assay and western blot.
As shown in Figure 4A, the assay was used to manufacture standard curve
and rapidly examine the EGFR concentration of sample. The protein levels of
EGFR were measured and the results were analyzed (Figure 4B). The protein
levels of EGFR in MCF-7 cells were significantly decreased in a dose-dependent
manner, after treated with 8c for 48 h. It is demonstrated that 8c was moderately
active inhibitors of EGFR kinase. As shown in Figure 5, western blot assay was
employed to evaluate the inhibition of EGFR autophosphorylation of compound
8c in MCF-7 cell lines. Importantly, 8c did not affect the overall EGFR content
in MCF-7 cells at any concentration tested, indicating that p-EGFR suppression
was not an artifact of compound cytotoxicity. The results suggested that
compound 8c can block EGFR phosphorylation in a dose-dependent manner in
MCF-7 cell line.

6
Y.-M. LIU ET AL.
Figure 2. Effects of compound 8c on cell cycle and apoptosis of MCF-7 cells. (A) Flow cytometry
analysis of cell cycle distribution for MCF-7 cells treated with compound 8c (0, 5, 10, 20 lm) for
48 h. (B) Compound 8c blocked cell cycle G1/S arrest of MCF-7 cells and also had effects in the G2
phase. (C) Apoptotic effect of compound 8c on MCF-7 cell line after treatment for 48 h. Q1, Q2, Q3
and Q4 respectively represent cells damaged during the procedure, the earlier apoptotic cells, the
late apoptotic cells and live cells. (D) Compound 8c increased cell apoptotic numbers. Error bar
ꢀ
ꢀꢀ
show the SD, “ ” p＜0.05, “ ” p＜0.01, compared with the control cells at 48 h.
2.2.5. Binding mode of 8c into EGFR
To get deeper insights into the possible binding mode of the designed chrysin deriva-
tives in target kinase, the most potent inhibitor 8c was docked into the ATP binding
site of this model of EGFR complex structure (PDB ID: 1M17). This model was able
to give an explanation and understanding of good activity observed. As shown in
Figure 1, in the binding model of compound 8c and EGFR, there are three hydrogen
bonds. The carboxyl group on the L-leucine formed a hydrogen bond with ASP-831,
and carbonyl group of L-leucine also formed H-bond with LYS-721. Moreover, the
oxygen on the 7-position formed a H-bond with the backbone of THR-830. The

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
Figure 3. (A) The double antibody sandwich-ELISA was used to manufacture standard curve and
rapidly examine the EGFR concentration of sample. (B) EGFR kinase inhibitory activity of 8c on
ꢀ
MCF-7 cells after treatment for 48 h. Error bar show the SD, “ ” p＜0.05, compared with the con-
trol cells at 48 h.
molecular docking results, along with the kinase assay data, illustrated that compound
8c may have a potential inhibitory effect on EGFR.
3. Experimental
3.1. Chemistry
The reagents (chemical) and solvents were bought from Aladdin Chemical (Shanghai,
China), and used without further purification. The tetramethylsilane (TMS) and solv-
1
ent signals were used as internal standards at 25 ꢁC, and H-NMR was recorded by
using a Bruker AVANCE-500 spectrometer (Sweden, Germany). Melting points were
determined on a Thermo Scientific electrothermal digital melting point apparatus
(California, USA). Mass spectra were acquired on APCI-MS spectrometer (Thermo,
California, USA) and elemental analysis was measured on Perkin Elmer 2400 CHN
(Waltham, USA).
1
The H NMR and 13C NMR spectra data of compounds 3a–8d were supplied as
supplementary material.
3.2. Ethyl 2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy) propanoate (3a)
The mixture of chrysin (2.54 g, 0.01 mol) and K2CO3 (1.59 g, 0.0115 mol) was added
in acetone (150 ml), keeping the reaction solution below 60 ꢁC. The resulting mixture
was stirred at 60 ꢁC for 1 h, then ethyl 2-bromopropionate (1.81 g, 0.011 mol) was
added dropwise into the solution. The mixture was kept stirring at 60 ꢁC for 12 h.
After TLC detection to show no starting materials, the mixture was cooled to room
temperature and then the precipitate was removed by filtration. The filtrate was con-
centrated and the residue was purified by column chromatography on silica gel, elut-
ing with dichloromethane/acetone (50:1) to give a light yellow solid 3a (2.55 g, yield
1
60%) [14]. m.p.100–103 ꢁC, H NMR (500 MHz, DMSO-d6) d 12.81 (s, 1 H), 8.09 -
7.95 (m, 2 H), 7.69 – 7.65 (m, 3 H), 6.77 (d, J ¼ 2.0 Hz, 2 H), 6.38 (d, J ¼ 2.0 Hz, 1 H),
5.21 - 5.18 (m, 1 H), 4.19 - 4.12 (m, 2 H), 1.56 (d, J ¼ 6.7 Hz, 3 H), 1.21 (s, 3 H).; 13C
NMR (126 MHz, DMSO-d6) d 182.5 (s), 171.1 (s), 164.1 (s), 163.7 (s), 161.7 (s), 157.7

8
Y.-M. LIU ET AL.
Figure 4. Inhibition of EGFR autophosphorylation of compound 8c in MCF-7 cell line.
Figure 5. The crystal structure of EGFR (PDB ID:1M17, download from the PDB) in complex with
potent compound 8c. The protein and ligand are colored in red and green, respectively. Hydrogen
bonds are represented with dotted yellow lines. The image was generated with Pymol.
(s), 132.7 (s), 131.0 (s), 129.6 (s), 129.6 (s), 127.0 (s), 127.0 (s), 105.9 (s), 105.7 (s),
99.2 (s), 94.3(s), 72.6 (s), 61.6 (s), 18.4 (s), 14.5 (s). ESI-MS: m/z 354.11 [M þ H]þ.
Elemental analysis: Found: C, 67.82%; H, 5.09%; calcd for C20H18O6: C, 67.79%;
H, 5.12%.
3.3. Ethyl 5-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)pentanoate (3b)
A mixture of chrysin (2.54 g, 0.01 mol) and anhydrous potassium carbonate (1.59 g,
0.0115 mol) was dissolved in acetone (150 ml) and stirred at 60 ꢁC for 1 h. Ethyl 5-
bromovalerate (2.09 g, 0.011 mol) was added to the reaction mixture and then stirred
for 12 h at 60 ꢁC. When TLC showed the reaction was completed, evaporation of the
solvent under reduced pressure gave a residue, which was purified by column

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
chromatography on silica gel, eluting with dichloromethane/acetone (50:1) to give a
light yellow solid 3b [14]. m.p.101.0–102.1 ꢁC. 1H NMR (500 MHz, DMSO-d6) d
12.77 (s, 1 H), 8.07 - 8.00 (m, 2 H), 7.68 - 7.65 (m, 3 H), 6.99 (s, 1 H), 6.76 (s, 1 H),
6.34 (s, 1 H), 4.14 - 4.06 (m, 4 H), 2.38 (t, J ¼ 6.9 Hz, 2 H), 1.74- 1.63 (m, 4 H), 1.19
(s, 3 H).13C NMR (126 MHz, DMSO-d6) d 182.5 (s), 173.2 (s), 165.1 (s), 161.6 (s),
157.8 (s), 132.6 (s), 131.1 (s), 129.6 (s), 129.6 (s), 126.9 (s), 126.9 (s), 105.8 (s), 105.3
(s), 98.9 (s), 93.6 (s), 68.6 (s), 60.2 (s), 33.5 (s), 28.2 (s), 21.5 (s), 14.6 (s). ESI-MS: m/
z 382.14 [M þ H]þ. Elemental analysis: Found: C, 69.05%; H, 5.82%; calcd for
C22H22O6: C, 69.10%; H, 5.80%.
3.4. General synthetic procedure of the compounds 4a–4b
The mixture of compound 3a or 3b (0.005 mol) in CH3OH (50 ml) was treated with
1 N NaOH (pH ¼ 10–11). After 6 h at 60 ꢁC, the solvent was treated with 1 N HCl
(pH¼ 2–3). After standing 6 h, the residue was filtered and washed with saturated
salt water, 3.8% HCl and water, and dried in vacuo to give light yellow solid 4a or
4b [14].
3.4.1. 2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)propanoic acid (4a)
Yield: 90%, m.p.241.6–243.5 ꢁC.1H NMR (500 MHz, DMSO-d6) d 12.80 (s, 1 H), 8.08
- 8.00 (m, 2 H), 7.65 – 7.63 (m, 3 H), 7.02 (s, 1 H), 6.73 (d, J ¼ 1.9 Hz, 1 H), 6.35 (d,
J ¼ 1.9 Hz, 1 H), 5.13 - 5.07 (m, 1 H), 1.56 (d, J ¼ 6.7 Hz, 3 H); 13C NMR (126 MHz,
DMSO-d6) d 182.50 (s), 172.70 (s), 164.0 (d, J ¼ 7.0 Hz), 161.7 (s), 157.7 (s), 132.6 (s),
131.0 (s), 129.6 (s), 129.6 (s), 126.9 (s), 126.9 (s), 105.8 (s), 105.6 (s), 99.2 (s), 94.1
(s), 72.5 (s), 18.5 (s). ESI-MS: m/z 326.08 [M þ H]þ. Elemental analysis: Found: C,
66.26%; H, 4.32%; calcd for C18H14O6: C, 66.30%; H, 4.29%.
3.4.2. 7-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)valeric acid (4b)
1
Yield: 90%, m.p. 174.2–175.5 ꢁC. H NMR (500 MHz, DMSO-d6) d 12.79 (s, 1 H),
12.07 (s, 1 H), 8.10 - 8.09 (m, 2 H), 7.72 – 7.46 (m, 3 H), 7.02 (s, 1 H), 6.79 (s, 1 H),
6.37 (s, 1 H), 4.12 - 4.09 (m, 2 H), 2.30 - 2.33 (m, 2 H), 1.81 – 1.72 (m, 2 H), 1.72 –
1.59 (m, 2 H).13C NMR (126 MHz, DMSO-d6) d 182.51 (s), 174.8 (d, J ¼ 1.0 Hz),
165.2 (s), 163.9 (s), 161.6 (s), 157.8 (s), 132.6 (s), 131.1 (s), 129.6 (s), 129.6 (s), 126.9
(s), 126.9 (s), 105.8 (s), 105.3 (s), 98.9 (s), 93.7 (s), 68.7 (s), 33.7 (s), 28.3 (s), 21.5 (s).
ESI-MS: m/z 354.11 [M þ H]þ. Elemental analysis: Found: C, 67.81%; H, 5.09%; calcd
for C20H18O6: C, 67.79%; H, 5.12%.
3.5. General synthetic procedure of the compounds 5a–6d
Compound 4a or 4b (0.5 mmol) was dissolved in DMF (10 ml) and treated with
HOBt (270.2 mg, 2 mmol) and EDCl (383.4 mg, 2 mmol). The mixture was stirred for
1 h at 0–4 ꢁC. Then, DIPEA (349.3 ll, 2 mmol) and amino acid methyl ester hydro-
chloride (2 mmol) were added drop by drop. After reaction at room temperature for
24 h, the cold water (50 ml) was added to the mixture. The residue was filtered and

10
Y.-M. LIU ET AL.
purified with a silia gel column and was eluted with dichloromethane/acetone (12:1)
to afford a yellow solids 5a–6d.
3.5.1. N-(2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)isopropionyl)-L-glycine
methyl ester (5a)
Yield: 60%, m.p.176.0–177.8 ꢁC.1H NMR (500 MHz, DMSO-d6) d 12.79 (s, 1 H), 8.71
(t, J ¼ 5.9 Hz, 1 H), 8.11 - 8.13 (m, 2 H), 7.69 – 7.55 (m, 3 H), 7.06 (s, 1 H), 6.80 (d,
J ¼ 2.0 Hz, 1 H), 6.42 (d, J ¼ 2.0 Hz, 1 H), 5.00 - 4.98 (m, 1 H), 3.91 - 3.87 (m, 2 H),
3.63 (s, 3 H), 1.51 (d, J ¼ 6.6 Hz, 3 H).13C NMR (126 MHz, DMSO-d6) d 182.1 (s),
171.0 (s), 164.5 (s), 163.1 (s), 161.1 (s), 157.2 (s), 132.2 (s), 130.6 (s), 129.1 (d), 126.5
(d), 105.4 (s), 105.2 (s), 99.2 (s), 93.2 (s), 73.8 (s), 51.7 (s), 40.4 (s), 18.5 (s). ESI-MS:
m/z 397.12 [M þ H]þ. Elemental analysis: Found: C, 63.48%; H, 4.81%; N, 3.50%;
calcd for C21H19O7: C, 63.47%; H, 4.82%; N, 3.52%.
3.5.2. N-(2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)isopropionyl)-L-alanine
methyl ester (5b)
Yield: 55%, m.p.178.2–180.0 ꢁC.1H NMR (500 MHz, DMSO-d6) d 12.79 (s, 1 H), 8.75
- 8.69 (m, 1 H), 8.11 - 8.09 (m, 2 H), 7.67 – 7.54 (m, 3 H), 7.07 (s, 1 H), 6.75 (s, 1 H),
6.40 (d, J ¼ 6.3 Hz, 1 H), 4.96 - 4.89 (m, 1 H), 4.37 - 4.36 (m, 1 H), 3.67 – 3.56 (m,
3 H), 1.51 (d, J ¼ 6.2 Hz, 3 H), 1.34 (d, J ¼ 7.2 Hz, 3 H). 13C NMR (126 MHz, CDCl3)
d 182.4 (s), 172.9 (s), 170.7 (s), 164.2 (s), 162.6 (s), 162.4 (s), 157.8 (s), 132.0 (s),
131.1 (s), 129.1 (s), 126.3 (s), 106.5 (s), 105.9 (s), 99.6 (s), 99.4 (s), 93.9 (s), 93.6 (s),
75.2 (s), 52.6 (s), 47.8 (s), 18.6 (s), 18.1 (s). ESI-MS: m/z 411.13 [M þ H]þ. Elemental
analysis: Found: C, 64.25%; H, 5.14%; N, 3.39%; calcd for C22H21O7: C, 64.23%; H,
5.15%; N, 3.40%.
3.5.3. N-(2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)isopropionyl)-L-leucine
methyl ester (5c)
Yield: 58%, m.p. .84.9–86.4 ꢁC.1H NMR (500 MHz, DMSO-d6) d 12.78 (s, 1 H), 8.68 -
8.67 (m, 1 H), 8.14 - 8.08 (m, 2 H), 7.67 – 7.57 (m, 3 H), 7.07 (s, 1 H), 6.72 - 6.71 (m,
1 H), 6.39 (s, 1 H), 5.01 – 4.88 (m, 1 H), 4.42 – 4.29 (m, 1 H), 3.61 - 3.59 (m, 3 H),
1.51 (t, J ¼ 6.2 Hz, 2 H), 0.91 (s, 3 H), 0.87 (s, 3 H).13C NMR (126 MHz, CDCl3) d
182.3 (s), 172.9 (s), 172.7 (s), 171.0 (s), 162.7 (s), 132.0 (s), 131.0 (s), 129.1 (s), 126.2
(s), 105.8 (s), 99.7 (s), 99.5 (s), 93.9 (s), 93.5 (s), 75.3 (s), 52.3 (s), 50.3 (s), 41.1 (s),
25.0 (s), 24.8 (s), 22.7 (s), 21.9 (s), 21.5 (s), 18.50 (s), 1.0 (s). ESI-MS: m/z 453.18
[M þ H]þ. Elemental analysis: Found: C, 66.18%; H, 6.02%; N, 3.10%; calcd for
C25H27O7: C, 66.21%; H, 6.00%; N, 3.09%.
3.5.4. N-(2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)isopropionyl)-L-phenyl-
alanine methyl ester (5d)
Yield: 59%, m.p 162.3–164.0 ꢁC.1H NMR (500 MHz, DMSO-d6) d 12.79 (s, 1 H), 8.84
- 8.83 (m, 1 H), 8.10 - 8.09 (m, 2 H), 7.67 – 7.55 (m, 3 H), 7.20 - 7.12 (m, 5 H), 7.05
(d, J ¼ 3.7 Hz, 1 H), 6.65 - 6.63 (m, 1 H), 6.33 - 6.31 (m, 1 H), 4.94 - 4.87 (m, 1 H),
4.55 - 4.54 (m, 1 H), 3.66 (s, 3 H), 3.15 – 2.90 (m, 2 H), 1.36 (d, 6.6 Hz, 3 H). 13C
NMR (126 MHz, DMSO-d6) d 182.5 (s), 172.2 (s), 172.0 (s), 171.1 (s), 170.8 (s), 163.6

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
11
(s), 161.7 (s), 161.5 (s), 157.6 (s), 137.8 (d), 132.7 (d), 131.1 (s), 129.7 (s), 129.5 (s),
128.6 (s), 128.4 (s), 126.9 (s), 105.3 (d), 99.9(s), 94.6(s), 74.3(s), 53.9 (s), 52.4 (s), 18.9
(s). ESI-MS: m/z 487.16 [M þ H]þ. Elemental analysis: Found: C, 69.03%; H, 5.17%;
N, 2.89%; calcd for C28H25O7: C, 68.99%; H, 5.17%; N, 2.87%.
3.5.5. N-(7-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)valeryl)-L-glycine
methyl ester (6a)
Yield: 55%, m.p. 164.3–166.0 ꢁC.1H NMR (500 MHz, CDCl3) d 12.67 (s, 1 H), 7.86 -
7.84 (m, 2 H), 7.57 – 7.45 (m, 3 H), 7.29 (s, 1 H), 6.64 (s, 1 H), 6.47 – 6.25 (m, 3 H),
4.10 - 4.07 (m, 3 H), 3.76 - 3.74 (m, 3 H), 2.40 - 2.37 (m, 2 H), 1.87 (s, 3 H).13C NMR
(126 MHz, CDCl3) d 182.4 (s), 172.8 (s), 170.6 (s), 165.0 (s), 163.9 (s), 162.0 (s), 157.7
(s), 131.8 (s), 131.2 (s), 129.1 (s), 129.1 (s), 126.3 (s), 126.3 (s), 105.7 (d), 98.6 (s),
93.0 (s), 68.2 (s), 52.4 (s), 41.2 (s), 35.7 (s), 28.4 (s), 22.1 (s). ESI-MS: m/z 425.15
[M þ H]þ. Elemental analysis: Found: 64.91%; H, 5.47%; N, 3.29%; calcd for
C23H23O7: C, 64.93%; H, 5.45%; N, 3.29%.
3.5.6. N-(7-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)valeryl)-L-alanine
methyl ester (6b)
Yield: 60%, m.p. 151.8–153.2 ꢁC.1H NMR (500 MHz, CDCl3) d 12.67 (s, 1 H), 7.85 -
7.83 (m, 2 H), 7.40 - 7.35 (m, 3 H), 6.65 (s, 1 H), 6.50 – 5.89 (m, 4 H), 4.62 (d,
J ¼ 5.8 Hz, 1 H), 3.76 (s, 3 H), 2.08 - 2.05 (m, 2 H), 1.86 - 1.65 (m, 4 H), 1.42 (s,
3 H).13C NMR (126 MHz, CDCl3) d 182.4 (s), 173.7 (s), 172.2 (s), 165.0 (s), 163.9 (s),
162.0 (s), 157.7 (s), 131.8 (s), 131.2 (s), 129.1 (s), 126.3 (s), 105.7 (s), 98.6 (s), 93.0
(s), 68.2 (s), 52.5 (s), 48.0 (s), 35.8 (s), 28.4 (s), 22.1 (s), 18.4 (s). ESI-MS: m/z 439.16
[M þ H]þ. Elemental analysis: Found: C, 65.60%; H, 5.71%; N, 3.20%; calcd for
C24H25O7: C, 65.59%; H, 5.73%; N, 3.19%.
3.5.7. N-(7-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)valeryl)-L-leucine
methyl ester (6c)
1
Yield: 49%, m.p. 139.1–140.5 ꢁC. H NMR (500 MHz, DMSO-d6) d 12.79 (s, 1 H),
8.28 - 8.27 (m, 1 H), 8.10 - 8.09 (m, 2 H), 7.60 - 7.57 (m, 3 H), 7.02 (s, 1 H), 6.79 (s,
1 H), 6.37 (s, 1 H), 4.38 – 4.21 (m, 1 H), 4.09 - 4.07 (m, 2 H), 2.51 (s, 1 H), 2.22 - 2.20
(m, 2 H), 1.77 – 1.70 (m, 2 H), 1.69 – 1.63 (m, 2 H), 1.59 – 1.49 (m, 2 H), 0.88 (d,
J ¼ 6.5 Hz, 3 H), 0.83 (d, J ¼ 6.5 Hz, 3 H).13C NMR (126 MHz, DMSO-d6) d 182.5 (s),
173.7 (s), 172.7 (s), 165.2 (s), 163.9 (s), 161.6 (s), 157.8 (s), 132.6 (s), 131.1 (s), 129.6
(s), 126.9 (s), 105.8 (s), 105.3 (s), 99.0 (s), 93.6 (s), 68.6 (s), 52.2 (s), 50.6 (s), 34.9 (s),
28.2 (s), 24.7 (s), 23.2 (s), 22.2 (s), 21.6 (s). ESI-MS: m/z 481.21 [M þ H]þ. Elemental
analysis: Found: C, 67.31%; H, 6.51%; N, 2.93%; calcd for C27H31O7: C, 67.35%; H,
6.49%; N, 2.91%.
3.5.8. N-(7-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)valeryl)-L-phenylalan-
ine methyl ester (6d)
Yield: 52%, m.p. 151.2–153.0 ꢁC.1H NMR (500 MHz, DMSO-d6) d 12.78 (s, 1 H), 8.46
- 8.45 (m, 1 H), 8.07 - 8.05 (m, 2 H), 7.59 - 7.56 (m, 3 H), 7.32 – 7.15 (m, 5 H), 6.99
(s, 1 H), 6.74 (s, 1 H), 6.33 (s, 1 H), 4.50 (d, J ¼ 4.2 Hz, 1 H), 4.01 (s, 2 H), 3.60 (s,

12
Y.-M. LIU ET AL.
3 H), 3.04 - 2.97 (m, 2 H), 2.16 - 1.58 (m, 6 H).13C NMR (126 MHz, DMSO-d6) d
182.5 (s), 172.7 (s), 172.6 (s), 165.2 (s), 163.8 (s), 161.6 (s), 157.8 (s), 137.8 (s), 132.6
(s), 131.0 (s), 129.6 (s), 129.5, 128.6 (s), 128.6 (s), 127.0 (s), 126.9, 126.9 (s), 105.7 (s),
105.3 (s), 98.9 (s), 93.6 (s), 68.6 (s), 53.9 (s), 52.3 (s), 37.1 (s), 34.9 (s), 28.1 (s), 22.1
(s). ESI-MS: m/z 515.19 [M þ H]þ. Elemental analysis: Found: C, 67.86%; H, 5.69%;
N, 2.73%; calcd for C30H29O7: C, 69.89%; H, 5.67%; N, 2.72%.
3.6. General method of synthesis of compounds 7a–8d
0.1 N KOH (10 ml) was added to a stirred mixture of 5a–6d (0.2 mmol) in absolute
ethyl alcohol (10 ml), followed by stirring for 24 h at room temperature.The pH of
mixture was adjusted to 2–3 with 1 N H2SO4 solution.The residue was filtered off,
which was subsequently washed with saturated NaCl solution and water, and dried
under vacuum to afford 7a–8d.
3.6.1. N-(2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)isopropionyl)-L-gly-
cine (7a)
Yield: 60%, m.p. 250.0–251.5 ꢁC.1H NMR (500 MHz, DMSO-d6) d 12.77 (s, 1 H), 8.57
(s, 1 H), 8.05 - 8.04 (m, 2 H), 7.59 - 7.56 (m, 3 H), 7.00 (s, 1 H), 6.75 (s, 1 H), 6.39 (s,
1 H), 4.96 (d, J ¼ 6.1 Hz, 1 H), 3.88 - 3.79 (m, 2 H), 1.51 (d, J ¼ 5.7 Hz, 3 H).13C NMR
(126 MHz, DMSO-d6) d 182.5 (s), 171.4 (d), 164.0 (s), 163.6 (s), 161.6 (s), 157.6 (s),
132.6 (s), 131.0 (s), 129.6 (s), 129.6 (s), 126.9 (s), 126.9 (s), 105.8 (d), 99.7 (s), 94.3
(s), 74.3 (s), 41.0 (s), 19.0 (s). ESI-MS: m/z 383.10 [M þ H]þ. Elemental analysis:
Found: C, 62.67%; H, 4.48%; N, 3.63%; calcd for C20H17O7: C, 62.66%; H, 4.47%;
N, 3.65%.
3.6.2. N-(2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)isopropionyl)-L-alanine
(7b)
Yield: 55%, m.p.163.1–164.8 ꢁC. 1H NMR (500 MHz, DMSO-d6) d 12.79 (s, 1 H),
12.77 (s, 1 H), 8.57 - 8.56 (m, 1 H), 8.07 - 8.06 (m, 2 H), 7.60 - 7.54 (m, 3 H), 7.02 (s,
1 H), 6.73 (s, 1 H), 5.02 – 4.89 (m, 1 H), 4.31 - 4.27 (m, 1 H), 1.50 (d, J ¼ 6.0 Hz, 3 H),
1.39 – 1.31 (m, 2 H). 13C NMR (126 MHz, DMSO-d6) d 182.5 (s), 174.1 (s), 170.7 (s),
170.5 (s), 164.1 (s), 163.7 (s), 161.6 (s), 157.6 (s), 132.6 (s), 131.1 (s), 129.6 (s), 126.9
(s), 105.9 (s), 105.6 (s), 99.5 (s), 94.2 (s), 74.1 (s), 47.9 (s), 19.1 (s), 18.8 (s), 17.5 (s).
ESI-MS: m/z 397.12 [M þ H]þ. Elemental analysis: Found: C, 63.49%; H, 4.78%; N,
3.54%; calcd for C21H19O7: C, 63.47%; H, 4.82%; N, 3.52%.
3.6.3. N-(2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)isopropionyl)-L-leu-
cine (7c)
1
Yield: 58%, m.p. 164.5–166.4 ꢁC. H NMR (500 MHz, DMSO-d6) d 12.71 (s, 1 H),
8.54 - 8.53 (m, 1 H), 8.22 – 7.95 (m, 2 H), 7.60 - 7.57 (m, 3 H), 7.02 (s, 1 H), 6.68 -
6.67 (m, 1 H), 6.35 - 6.34 (m, 1 H), 5.08 – 4.78 (m, 1 H), 4.32 - 4.31 (m, 1 H), 3.39 (s,
1 H), 1.50 - 1.49 (m, 5 H), 0.81 (s, 3 H), 0.76 (s, 3 H). 13C NMR (126 MHz, CDCl3) d
182.4 (s), 171.6 (s), 164.3 (s), 162.7 (s), 157.7 (s), 157.6 (s), 132.1 (s), 130.8 (s), 130.7
(s), 129.1 (s), 129.0 (s), 126.3 (s), 126.2 (s), 106.2 (s), 105.6 (s), 105.5 (s), 99.6 (s),

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
13
93.7 (s), 75.1 (s), 40.9 (s), 24.9 (s), 22.8 (s), 21.5 (s), 18.5 (s). ESI-MS: m/z 437.18
[M þ H]þ. Elemental analysis: Found: C, 65.60%; H, 5.72%; N, 3.17%; calcd for
C24H25O7: C, 65.59%; H, 5.73%; N, 3.19%.
3.6.4. N-(2-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)isopropionyl)-L-phenyl-
alanine (7d)
Yield: 59%, m.p. 197.6–199.5 ꢁC.1H NMR (500 MHz, DMSO-d6) d 12.78 (s, 1 H), 8.47
- 8.46 (m, 1 H), 8.05 - 8.03 (m, 2 H), 7.58 - 7.55 (m, 3 H), 7.18 - 7.13 (m, 5 H), 7.06 –
6.94 (m, 1 H), 6.59 - 6.58 (m, 1 H), 6.32 - 6.31 (m, 1 H), 4.87 - 4.86 (m, 1 H), 4.53 (s,
1 H), 3.56 – 2.78 (m, 2 H), 1.33 (d, 6.6 Hz, 3 H), 0.82 (s, 1 H) . 13C NMR (126 MHz,
DMSO-d6) d 182.5 (s), 173.2 (s), 173.0 (s), 171.0 (s), 170.6 (s), 164.0 (s), 163.6 (s),
161.6 (s), 161.5 (s), 157.6 (s), 138.0 (s), 132.6 (s), 131.1 (s), 129.6 (s), 129.4 (s), 128.5
(s), 128.3 (s), 126.9 (s), 105.9 (s), 99.6 (s), 94.3 (s), 93.9 (s), 74.3 (s), 53.5 (s), 19.0 (s),
18.9 (s). ESI-MS: m/z 473.15 [M þ H]þ. Elemental analysis: Found: C, 68.53%; H,
4.92%; N, 2.98%; calcd for C27H23O7: C, 68.49%; H, 4.90%; N, 2.96%.
3.6.5. N-(7-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)valeryl)-L-glycine (8a)
Yield: 55%, m.p. 201.8–202.5 ꢁC.1H NMR (500 MHz, DMSO-d6) d 12.79 (s, 1 H), 8.16
- 8.15 (m, 1 H), 8.09 - 8.07 (m, 2 H), 7.60 - 7.57 (m, 3 H), 7.02 (s, 1 H), 6.79 (s, 1 H),
6.37 (s, 1 H), 4.10 (s, 2 H), 3.74 (d, J ¼ 5.4 Hz, 2 H), 2.22 (t, J ¼ 6.8 Hz, 2 H), 1.75 (s,
2 H), 1.68 (d, J ¼ 6.8 Hz, 2 H).13C NMR (126 MHz, DMSO-d6) d 182.5 (s), 172.8 (s),
171.9 (s), 165.2 (s), 161.6 (s), 157.8 (s), 132.6 (s), 131.1 (s), 129.6 (s), 129.6 (s), 126.9
(s), 126.9 (s), 105.8 (s), 105.3 (s), 99.0 (s), 93.6 (s), 68.7 (s), 41.2 (s), 35.0 (s), 28.3 (s),
22.2 (s). ESI-MS: m/z 411.13 [M þ H]þ. Elemental analysis: Found: C, 64.25%; H,
5.14%; N, 3.39%; calcd for C22H21O7: C, 64.23%; H, 5.15%; N, 3.40%.
3.6.6. N-(7-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)valeryl)-L-alanine (8b)
1
Yield: 60%, m.p. 202.4–203.5 ꢁC. H NMR (500 MHz, DMSO-d6) d 12.72 (s, 1 H),
8.16 - 8.15 (m, 1 H), 7.99 - 7.97 (m, 2 H), 7.55 - 7.52 (m, 3 H), 6.89 (s, 1 H), 6.63 (s,
1 H), 6.25 (s, 1 H), 4.28 – 4.18 (m, 1 H), 4.01 (s, 2 H), 3.18 (s, 1 H), 2.21 (s, 2 H), 1.68
- 1.64 (m, 4 H), 1.25 (d, 7.0 Hz, 3 H). 13C NMR (126 MHz, DMSO-d6) d 182.3 (s),
174.8 (s), 172.3 (s), 165.1 (s), 163.7 (s), 161.6 (s), 157.7 (s), 132.4 (s), 131.0 (s), 129.5
(s), 129.5 (s), 126.7 (s), 126.7 (s), 105.6 (s), 105.2 (s), 98.8 (s), 93.4 (s), 68.6 (s), 47.9
(s), 35.0 (s), 28.3 (s), 22.2 (s), 17.7 (s). ESI-MS: m/z 425.15 [M þ H]þ. Elemental ana-
lysis: Found: C, 64.93%; H, 5.44%; N, 3.30%; calcd for C23H23O7: C, 64.93%; H,
5.45%; N, 3.29%.
3.6.7. N-(7-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)valeryl)-L-leucine (8c)
1
Yield: 49%, m.p. 152.5–154.0 ꢁC. H NMR (500 MHz, DMSO-d6) d 12.79 (s, 1 H),
12.47 (s, 1 H), 8.09 - 8.07 (m, 2 H), 7.74 – 7.49 (m, 3 H), 7.02 (s, 1 H), 6.79 (s, 1 H),
6.37 (s, 1 H), 4.31 – 4.21 (m, 1 H), 4.10 (t, J ¼ 6.1 Hz, 2 H), 2.21 (d, J ¼ 2.3 Hz, 2 H),
1.73 - 1.43 (m, 8 H), 0.85 (s, 3 H), 0.86 (s, 3 H).13C NMR (126 MHz, DMSO-d6) d
182.5 (s), 174.8 (s), 172.5 (s), 165.2 (s), 163.9 (s), 161.6 (s), 157.8 (s), 132.6 (s), 131.1
(s), 129.6 (s), 129.6 (s), 126.9 (s), 126.9 (s), 105.8 (s), 105.3 (s), 99.0 (s), 93.6 (s), 68.7
(s), 50.6 (s), 35.0 (s), 28.3 (s), 24.8 (s), 23.3 (s), 22.2 (s), 21.7 (s). ESI-MS: m/z 467.19

14
Y.-M. LIU ET AL.
[M þ H]þ. Elemental analysis: Found: C, 66.81%; H, 6.25%; N, 2.99%; calcd for
C26H29O7: C, 66.80%; H, 6.25%; N, 3.00%.
3.6.8. N-(7-((5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)valeryl)-L-phenylalan-
ine (8d)
1
Yield: 52%, m.p. 204.5–206.8 ꢁC. H NMR (500 MHz, DMSO-d6) d 12.79 (s, 1 H),
12.72 (s, 1 H), 8.21 - 8.20 (m, 1 H), 8.07 - 8.05 (m, 2 H), 7.58 - 7.55 (m, 3 H), 7.22 -
7.17 (m, 5 H), 7.00 (s, 1 H), 6.73 (s, 1 H), 6.33 (s, 1 H), 4.48 - 4.47 (m, 1 H), 4.00 (s,
2 H), 3.08 - 2.76 (m, 2 H), 2.15 - 1.58 (m, 6 H). 13C NMR (126 MHz, DMSO-d6) d
182.5 (s), 173.0 (s), 170.0 (s), 164.1 (s), 164.0 (s), 161.6 (s), 157.6 (s), 138.0 (s), 132.7
(s), 131.1 (s), 129.6 (s), 129.4 (s), 129.4 (s), 128.4 (s), 128.3 (s), 127.0 (s), 126.7 (s),
106.0 (s), 105.7 (s), 99.6 (s), 94.4 (s), 94.0 (s), 78.0 (s), 53.3 (s), 36.7 (s), 28.7 (d,
J ¼ 7.0 Hz), 24.6 (s), 22.4 (s). ESI-MS: m/z 501.18 [M þ H]þ. Elemental analysis:
Found: C, 69.44%; H, 5.44%; N, 2.79%; calcd for C29H27NO7: C, 69.45%; H, 5.43%;
N, 2.79%.
3.7. Biology
3.7.1. MTT cytotoxicity assay
The antiproliferative activities of those novel compounds were determined by the 3-
[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) method. Two
cancer cells were seeded in a density in 96-well plates (5 ꢂ 103 cells per well). After
24 h of incubation, cells were treated with different concentrations of compounds (16,
32, 64, 128 lmol/L) for 48 h. Then, 0.5% MTT solution was added to each well. After
4 h of incubation, formazan formed from MTT was extracted by adding 200 ll
DMSO. Cell viability was assessed at 570 nm by a microplate reader (Biotech,
Shanghai, China). The concentration causing 50% inhibition of cells growth (IC50)
was determined by the Logit method.
3.7.2. Clonogenicity assay
To determine the survival of the MCF-7 cells treated with compound 8c, 1000 tumor
cells were planted in six-well plate and incubated for 24 h. Then, fresh medium con-
taining different concentrations (0, 5, 10, 20 lmol/L) of test compound was added to
the plate. After incubation for 10 day at 37 ꢁC, the cells were fixed with 4% parafor-
maldehyde and stained with 0.5% crystal violet. The cells were imaged.
3.7.3. Wound healing assay
Cells were seeded in 6-well plate and allowed to grow to nearly 100% confluence in
culture medium. Subsequently, a cell-free line was manually formed by scratching the
confluent cell monolayers with a 200 ll pipette tip. Then the cells were treated with
different concentrations of potent compound and photographed on an
inverted microsope.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
15
3.7.4. Cell cycle assay
Cell cycle assay was determined by Cell Cycle and Apoptosis Analysis Kit (Beyotime
Biotechnology, Jiangsu, China), according to the manufacturer’s instructions and refer-
ence methods [21].
3.7.5. Cell apoptosis assay
Cells (2 ꢂ 105 cells per well) were treated with different concentrations of potent com-
pound for 48 h. Then, cells were collected after incubation, washed twice in cold
phosphate buffer saline (PBS), and treated with the Annexin-V-FITC/PI apoptosis kit
(MultiSciences Biotech., Hangzhou, China). The stained cells were analyzed by flow
cytometry [22].
3.7.6. The assay for EGFR inhibition
Human EGFR ELISA kit was purchased from Boster Biological Technology (Wuhan,
China. Catalog #EK0327). Human EGFR were expressed and purified as described
[23]. The experiments were performed according to the manufacturer’s instructions.
The inhibition rate (%) was calculated using the following equation: [1-(A490/
A490control)] ꢂ 100%.
3.7.7. Western blotting
Western blot analysis was carried out as described previously [13]. Cells were treated
with compound 8c or DMSO for a specified period time. Then, the cells were washed
twice with cold PBS and lysed in cold radioimmunoprecipitation assay (RIPA) buffer.
Lysates were cleared by centrifugation. Antibodies against the following were used:
phospho-EGFR and EGFR, GAPDH (All from Cell Signaling Technology, Beverly,
MA, USA).
3.8. Molecule docking simulations
To explore the interaction mechanism between the newly synthesized amino acid
derivatives containing chrysin and target protein, molecular docking for compound
8c into three-dimensional EGFR complex structure (PDB ID:1M17, download from
the PDB) was carried out using the SYBYL-XL 2.0 [24]. The image was generated
with Pymol [25].
Disclosure statement
The authors declare that there are no conflicts of interest.
Funding
We are grateful to the Hunan Provincial Department of Education Key Project (18A244), the
University of South China Students Research Learning and Innovative Experimental Project
(2018XJXZ200),Graduate student science foundation of University of South China (2018496)
and the University of South China Students Research Learning and Innovative Experimental
Project (2018XJXZ359) for financial support.

16
Y.-M. LIU ET AL.
ORCID
Yang Li
http://orcid.org/0000-0003-3855-7720
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