Synthesis and antioxidant activity of conjugates of hydroxytyrosol and coumarin

Article abstract of DOI:10.1016/j.bioorg.2020.104427

Antioxidants have been the subject of intense research interest due to their numerous health benefits. In this work, a series of new conjugates of hydroxytyrosol and coumarin were synthesized and evaluated for their free radical scavenging, toxicity and antioxidant mechanism in vitro. The all target compounds 14a–t exhibited better radical scavenging activity than BHT, hydroxytyrosol, and coumarin in both DPPH radical and ABTS⁺ radical cation scavenging assays. The structure-activity relationships study indicated that the number and position of hydroxyl groups on the coumarin ring were vital to a good antioxidant capacity. Furthermore, the most promising compound 14q showed less toxicity in hemolysis assay and weaker antiproliferative effects than BHT against normal WI-38 and GES cells, and enhanced viability of H₂O₂-induced HepG2 cells. Additionally, 14q decreased the apoptotic percentage of HepG2 cells, reduced the ROS produce and LDH release, and improved GSH and SOD levels in H₂O₂-treated HepG2 cells. Lastly, 14q exhibited more stability than hydroxytyrosol in methanol solution. These results revealed that conjugations of hydroxytyrosol and coumarin show better antioxidant capacity, and are the efficacious approach to finding novel potential antioxidant.

Full text of DOI:10.1016/j.bioorg.2020.104427

Journal Pre-proofs
Synthesis and antioxidant activity of conjugates of hydroxytyrosol and cou-
marin
Wen-Bo Li, Xue-Peng Qiao, Zi-Xiao Wang, Shuai Wang, Shi-Wu Chen
PII:
S0045-2068(20)31725-9
DOI:
https://doi.org/10.1016/j.bioorg.2020.104427
YBIOO 104427
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
25 September 2020
20 October 2020
21 October 2020
Please cite this article as: W-B. Li, X-P. Qiao, Z-X. Wang, S. Wang, S-W. Chen, Synthesis and antioxidant
activity of conjugates of hydroxytyrosol and coumarin, Bioorganic Chemistry (2020), doi: https://doi.org/
10.1016/j.bioorg.2020.104427
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Elsevier Inc. All rights reserved.

Synthesis and antioxidant activity of conjugates of
hydroxytyrosol and coumarin
Wen-Bo Li, Xue-Peng Qiao, Zi-Xiao Wang, Shuai Wang, Shi-Wu Chen *
School of Pharmacy, Lanzhou University, Lanzhou 730000, China
__________________
* Corresponding author.
E-mail
addresses:
chenshw@lzu.edu.cn
(S.-W.
Chen).
1

Abstract
Antioxidants have been the subject of intense research interest due to their numerous
health benefits. In this work, a series of new conjugates of hydroxytyrosol and coumarin
were synthesized and evaluated for their free radical scavenging, toxicity and
antioxidant mechanism in vitro. The all target compounds 14a‒t exhibited better radical
scavenging activity than BHT, hydroxytyrosol, and coumarin in both DPPH radical and
ABTS+ radical cation scavenging assays. The structure-activity relationships study
indicated that the number and position of hydroxyl groups on the coumarin ring were
vital to a good antioxidant capacity. Furthermore, the most promising compound 14q
showed less toxicity in hemolysis assay and weaker antiproliferative effects than BHT
against normal WI-38 and GES cells, and enhanced viability of H2O2-induced HepG2
cells. Additionally, 14q decreased the apoptotic percentage of HepG2 cells, reduced the
ROS produce and LDH release, and improved GSH and SOD levels in H2O2-treated
HepG2 cells. Lastly, 14q exhibited more stability than hydroxytyrosol in methanol
solution. These results revealed that conjugations of hydroxytyrosol and coumarin
show better antioxidant capacity, and are the efficacious approach to finding novel
potential antioxidant.
Keywords: Antioxidant; Free radical scavenging; Conjugates; Coumarin;
Hydroxytyrosol
2

1. Introduction
Oxidation is the most common and ubiquitous chemical reaction that affects both
living and non-living forms. Oxygen is vital for the survival of an aerobic organism,
however, it leads to the production of free radicals, especially reactive oxygen species
(ROS) and reactive nitrogen species (RNS) during biochemical processes. ROS/RNS
and initiated chains of biochemical reactions can cause peroxidation of DNA, protein,
and lipid, destroy their normal cellular functions, and lead to cell death and apoptosis
[1]. To maintain delicate oxidative homeostasis, ROS produced in living cells is
continuously neutralized by the antioxidant defense system. Antioxidant defense
mechanisms include both enzymatic and non-enzymatic mechanisms. Enzymatic
antioxidants comprise of antioxidant enzymes, such as superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidase (GSH-Px) and reductase, etc., they are highly
specific and detoxify specific free radicals. Non-enzymatic antioxidants, for example,
ascorbic acid, Vitamin E, carotenoids, glutathione, etc., are non-specific yet constitute
the first line of defense mechanism against free radicals/ROS [2,3].
Antioxidants molecules found in cells are used to prevent free radicals from taking
electrons from another molecule. Over the years, significant advancement has been
made relating to free radicals and the development of antioxidants. Various kinds of
natural, semi-synthetic, as well as synthetic free radical scavengers, have gained much
attention as chain-breaking antioxidants that protect aerobic organisms from oxidative
damage [4]. Especially, polyphenolic compounds of both natural and synthetic source
have drawn greater attention as free radical scavengers [5].
3

Hydroxytyrosol (1, Fig. 1) is the content of Olives, also the hydrolysis product and
antioxidant active ingredient of glycoside oleuropein (2) [6], which possesses many
biological activities including anti-oxidation and scavenging free radicals [7],
anticancer [8], cardiovascular activity [9], anti-inflammatory [10], antiviral [11], and
neural activity [12]. However, the poor stability and deterioration in air or light made it
difficult to store [13]. Therefore, it is meaningful to improve its activity and stability
by chemical modification, such as esterification [14].
The coumarins are heterocyclic compounds consisting of benzene and 2-pyrone
rings enriched in various plants like tonka beans. Coumarin and their derivatives exert
a vast array of bioactive properties such as antioxidant, anticoagulant, antibacterial,
anti-inflammatory, antitumor, antiviral, and enzyme inhibition [15]. Although higher
doses of coumarin are found to be hepatotoxic, they exhibit beneficial effects by
reducing the risk of cancer and other neuronal and cardiovascular ailments [16]. Most
of these effects can be attributed to their free radical scavenging effects. For example,
compound 4, a hybrids of coumarin and chalcone, presents good scavenging capacity,
low toxicity, and high cytoprotection [17]. Despite significant advancements in
antioxidant searches, coumarins remain one of the most versatile classes of compounds
for anti-oxidant drug design and discovery [18].
HO
HO
O
O
COOMe
OH
O
HO
O
O
O
OH
O
O
OH
OGlc
1
2
3
4
Fig. 1. The chemical structures of hydroxytyrosol (1), oleuropein (2), coumarin (3) and
4

4.
The conjugation principle involves the rational design of new chemical entities
based on the recognition of pharmacophoric subunits with the molecular structure of
two or more known bioactive derivatives [19]. The successful fusion of these subunits
produces a new conjugate that maintains the desired characteristics of the original
subunits. Hence, the conjugation principle in drug discovery is meaningful for the
optimization of natural products [20]. As our continuing efforts to find new compounds
with potent activities and lower toxicity based on a natural product [21‒23], considering
the excellent antioxidant activity of the orthodiphenolic moiety and good bio-
pharmacological activities of coumarin scaffold, we presented a series of conjugates of
coumarin and hydroxytyrosol, in which maintaining the orthodiphenolic moiety of
hydroxytyrosol to improve antioxidant activity (Fig. 2). Furthermore, their
antioxidative activities and mechanism were also studied.
OH
OH
OH
OH
O
O
O
O
Conjugation
SAR
O
O
R
1
3
+
O
O
Fig. 2. Design of target compounds.
2. Material and methods
2.1. Reagents and instruments
All materials were obtained from commercial suppliers (Bide Pharmatech Ltd.,
Shanghai, China and Energy Chemical, Shanghai, China) and were used without further
purification, unless stated otherwise. Reaction time was monitored by TLC (silica gel
5

HRGF254). Column chromatography was performed on silica gel (200–300 mesh)
from Yantai Xinnuo Chemicals (Yantai, Shandong, China). All melting points were
measured with a Büchi Melting Point X-4 apparatus (Beijing Taike, Beijing, China)
and were uncorrected. High-resolution mass spectra (HRMS) were taken in ESI mode
on Bruker Apextm Ⅱ 4.7T LC-MS (Bruker, GER). 1H NMR and 13C NMR spectrum
were recorded on Agilent-NMR-INOVA600 (Agilent, Palo Alto, CA, USA) with TMS
as an internal standard, all chemical shift values were reported as ppm. The purity was
measured by HPLC (Waters 1525, US).
2.2. Preparation of the target compounds
2-(3,4-Dihydroxyphenyl)acetic acid 6 as starting material was esterified to give
intermediate 7, which was further protected with benzyl chloride (BnCl), and reduced
to yield key intermediate 9 as shown in Scheme 1, with 58% yield for overall 3 steps.
O
O
OH
OH
i
ii
iii
O
O
O
HO
BnO
BnO
HO
OH
OBn
OBn
OH
9
6
8
7
Scheme 1. Reagents and conditions: i) H2SO4, MeOH, 75℃; ii) K2CO3, BnCl, MeCN,
reflux; iii) LiAlH4, THF, 80℃.
The target compounds 14a‒t were prepared from m-hydroxylbenzaldehydes 10a‒t
as outlined in Scheme 2. Firstly, ethyl 2-oxo-2H-chromene-3-carboxylates (11a‒t)
were yielded by Perkin reaction of 10a‒t with diethyl carbonate under piperidine
catalysis. Then 11a‒t were changed into 12a‒t by hydrolysis directly in the presence
of 10% NaOH, or after benzyl protection of hydroxyl group with benzyl chloride
6

(BnCl). And carboxylic acids 12a‒t were esterified with 9 using 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N,N-
dimethylaminopyridine (DMAP) catalysis to give 13a‒t. Finally, the targets 14a‒t were
obtained by deprotection of 13a‒t with BBr3.
O
O
O
OH
H
OEt
ii
i
R1
R
R
or iii,ii
O
O
OH
10a-t
O
O
11a-t
12a-t
O
O
OH
O
O
OBn
OH
OBn
O
O
iv
v
R
R1
O
O
14a-t
13a-t
d
h
l
R= 6-OH, R1= 6-OBn
R=R1= 7-Cl
c
g
k
o
s
R=R1= 6-Cl
b
f
R=R1= 6-CH3
R=R1= 6-NO2
R=R1= 7-OCH3
R=R1= 8-OCH3
R=R1= 7,8-OCH3
a
e
i
R=R1= H
R=R1= 6-OCH3
R=R1= 7-CH3
R=R1= 8-CH3
R=R1= 5,7-OCH3
R=R1= 6,8-Cl
R=R1= 8-Cl
j
R= 7-OH, R1= 7-OBn
R= 8-OH, R1= 8-OBn
p
t
R=R1= 6,8-C(CH3)3
R=R1= 5,6-phenyl ring
n
r
m
q
R= 7,8-OH, R1= 7,8-OBn
Scheme 2. Reagents and conditions: i) diethyl carbonate, piperidine, CH3COOH, 75℃;
ii) 10% NaOH, EtOH, heat; iii) Cs2CO3, BnCl, DMF, 80℃; iv) 9, EDCI, DMAP,
CH2Cl2, rt; v) BBr3 (1 M in CH2Cl2), CH2Cl2, ‒40℃.
2.2.1. Synthetic procedure of compound 7
To a solution of 3,4-dihydroxyphenylacetic acid 6 (5.65 g, 33.6 mmol) in MeOH
(100 mL), H2SO4 (98%, 10 drops) was slowly added in atmosphere of argon, followed
by further refluxing for 6 h. After the reaction was completed, the solution was
concentrated under reduced pressure. The residue was dissolved in ethyl acetate (30
mL), and the solution was washed with saturated NaHCO3 solution. The aqueous phase
was extracted with ethyl acetate (20 mL×3), washed with saturated NaCl solution (20
mL), dried over anhydrous Na2SO4, and evaporated in vacuo to give the crude product
7, which was directly used in the next step without further purification.
7

2.2.2. Synthetic procedure of compound 8
A solution of benzyl chloride (BnCl, 11.5 mL, 100 mmol), potassium carbonate
(K2CO3, 13.8 g, 100 mmol) and 7 in MeCN (80 mL) was stirred for 24 h at 80 ℃ in
atmosphere of argon. Next, the solvent was evaporated under vacuum, and the residue
was dissolved in ethyl acetate (30 mL) and washed with H2O (20 mL). Then aqueous
phase was extracted with ethyl acetate (20 ml×3), washed with saturated NaCl (20 ml),
dried over Na2SO4, and filtered. The solvent was evaporated in vacuo, and purified by
column chromatography (PE: EA = 20:1) to give 8 as a colorless liquid. Yield: 64%
1
(two steps); H NMR (600 MHz, CDCl3): δ 7.46‒7.25 (m, 10H), 6.90‒6.77 (m, 3H),
5.15 (s, 2H), 5.14 (s, 2H), 3.65 (s, 3H), 3.51 (s, 2H).
2.2.3. Synthetic procedure of compound 9
To a solution of 8 (11.3 g, 31.2 mmol) in dry THF (80 mL), lithium
tetrahydroaluminum (LiAlH4, 0.59 g, 15.6 mmol) was slowly added in atmosphere of
argon. The mixture was warmed to 80℃ and stirred for 4 h. Then, the resulting mixture
was cooled to room temperature, and water/ether (1:1) was added to quench the reaction.
The organic extracts were filtered, and evaporated in vacuo. The crude product was
purified by column chromatography (PE: EA = 5:1) to provide 2-(3,4-
Bis(benzyloxy)phenyl)ethan-1-ol (9) (9.48 g, 28.4 mmol) as white solid; Yield: 91%;
1H NMR (600 MHz, CDCl3): δ 7.46‒7.25 (m, 10H), 6.90‒6.77 (m, 3H), 5.15 (s, 2H),
5.14 (s, 2H), 3.65 (s, 3H), 3.51 (s, 2H).
2.2.4. General synthetic procedure of compounds 11a‒t
8

Compounds 10 (10 mmol) and diethyl malonate (20 mmol) in EtOH (20 mL) were
added piperidine (2.5 mmol) and AcOH (2 drops). Following the reaction mixture was
warmed to 75℃ and stirred for 1 h. The mixture was poured into the cold water to yield
a large amount of solid. The solid product was filtered off and recrystallized with
H2O/EtOH to afford compounds 11a‒t in good yields, while 11g, 11j, and 11p were
used directly in the next reaction without purification.
1
2.2.4.1. Ethyl 2-oxo-2H-chromene-3-carboxylate (11a). Yield: 82%; white solid; H
NMR (600 MHz, DMSO-d6): δ 8.74 (s, 1H), 7.92‒7.39 (m, 3H), 4.29 (q, 2H, J = 7.2
Hz), 1.30 (t, 3H, J = 7.2 Hz).
2.2.4.2. Ethyl 6-methyl-2-oxo-2H-chromene-3-carboxylate (11b). Yield: 88%; yellow
solid; 1H NMR (600 MHz, DMSO-d6): δ 8.68 (s, 1H), 7.70‒7.34 (m, 3H), 4.30 (q, 2H,
J = 7.2Hz), 2.38 (s, 3H), 1.31 (t, 3H, J = 7.2 Hz).
2.2.4.3. Ethyl 6-chloro-2-oxo-2H-chromene-3-carboxylate (11c). Yield: 92%; white
solid; 1H NMR (600 MHz, DMSO-d6): δ 8.72 (s, 1H), 8.06‒7.48 (m, 3H), 4.31 (q, 2H,
J = 7.2 Hz), 1.32 (t, 3H, J = 7.2 Hz).
2.2.4.4. Ethyl 6-hydroxy-2-oxo-2H-chromene-3-carboxylate (11d). Yield: 86%; yellow
solid; 1H NMR (600 MHz, DMSO-d6): δ 9.91 (s, 1H), 8.67 (s, 1H), 7.30‒7.15 (m, 3H),
4.29 (q, 2H, J = 6.6 Hz), 1.31 (t, 3H, J = 6.6 Hz).
2.2.4.5. Ethyl 6-methoxy-2-oxo-2H-chromene-3-carboxylate (11e). Yield: 83%; red
solid; 1H NMR (600 MHz, DMSO-d6) δ 8.71 (s, 1H), 7.50‒7.33 (m, 3H), 4.30 (q, 2H,
J = 6.6 Hz), 3.82 (s, 3H), 1.31 (t, 3H, J = 6.6 Hz).
4.1.4.6. Ethyl 6-nitro-2-oxo-2H-chromene-3-carboxylate (11f). Yield: 86%; brown
9

solid; 1H NMR (600 MHz, DMSO-d6): δ 8.94 (s, 1H), 8.93‒87.66 (m, 3H), 4.33 (q, 2H,
J = 6.6 Hz), 1.33 (t, 3H, J = 6.6 Hz).
2.2.4.7. Ethyl 7-chloro-2-oxo-2H-chromene-3-carboxylate (11h). Yield: 67%; yellow
solid; 1H NMR (600 MHz, DMSO-d6): δ 8.78 (s, 1H), 7.96‒7.49 (m, 3H), 4.30 (q, 2H,
J = 7.2 Hz), 1.31 (t, 3H, J = 7.2 Hz).
2.2.4.8. Ethyl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate (11i). Yield: 75%; white
solid; 1H NMR (600 MHz, DMSO-d6): δ 10.68 (s, 1H), 8.53 (s, 1H), 7.53‒6.77 (m, 3H),
4.35 (q, 2H, J = 7.2 Hz), 1.39 (t, 3H, J = 7.2 Hz).
2.2.4.9. Ethyl 8-methyl-2-oxo-2H-chromene-3-carboxylate (11k). Yield: 82%; white
solid; 1H NMR (600 MHz, DMSO-d6): δ 8.73 (s, 1H), 7.75‒7.30 (m, 3H), 4.30 (q, 2H,
J = 6.6 Hz), 2.37 (s, 3H), 1.32 (t, 3H, J = 6.6 Hz).
2.2.4.10. Ethyl 8-chloro-2-oxo-2H-chromene-3-carboxylate (11l). Yield: 62%; yellow
solid; 1H NMR (600 MHz, DMSO-d6): δ 8.80 (s, 1H), 7.92‒7.41 (m, 3H), 4.31 (q, 2H,
J = 7.2 Hz), 1.32 (t, 3H, J = 7.2 Hz).
2.2.4.11. Ethyl 8-hydroxy-2-oxo-2H-chromene-3-carboxylate (11m). Yield: 90%;
white solid; 1H NMR (600 MHz, DMSO-d6): δ 10.39 (s, 1H), 8.68 (s, 1H), 7.32‒7.19
(m, 3H), 4.27 (q, 2H, J = 7.2 Hz), 1.29 (t, 3H, J = 7.2 Hz).
2.2.4.12. Ethyl 8-methoxy-2-oxo-2H-chromene-3-carboxylate (11n). Yield: 78%; red
solid; 1H NMR (600 MHz, DMSO-d6): δ 8.72 (s, 1H), 7.45‒7.32 (m, 3H), 4.28 (q, 2H,
J = 6.6 Hz), 3.90 (s, 3H), 1.29 (t, 3H, J = 6.6 Hz).
2.2.4.13. Ethyl 5,7-dimethoxy-2-oxo-2H-chromene-3-carboxylate (11o). Yield: 87%;
green solid; 1H NMR (600 MHz, DMSO-d6): δ 8.61 (s, 1H), 6.64‒6.55 (m, 2H), 4.26
10

(q, 2H, J = 6.6 Hz), 3.95 (s, 3H), 3.90 (s, 3H), 1.29 (t, 3H, J = 6.6 Hz).
2.2.4.14. Ethyl 7,8-dihydroxy-2-oxo-2H-chromene-3-carboxylate (11q). Yield: 80%;
green solid; 1H NMR (600 MHz, DMSO-d6): δ 8.62 (s, 1H), 7.26‒6.84 (m, 2H), 4.24
(q, 2H, J = 7.2 Hz), 1.28 (t, 3H, J = 7.2 Hz).
2.2.4.15. Ethyl 7,8-dimethoxy-2-oxo-2H-chromene-3-carboxylate (11r). Yield: 97%;
white solid; 1H NMR (600 MHz, DMSO-d6): δ 8.71 (s, 1H), 7.68‒7.18 (m, 2H), 4.27
(q, 2H, J = 7.2 Hz), 3.95 (s, 3H), 3.83 (s, 3H), 1.30 (t, 3H, J =7.2 Hz).
2.2.4.16. Ethyl 6,8-dichloro-2-oxo-2H-chromene-3-carboxylate (11s). Yield: 93%;
white solid; 1H NMR (600 MHz, DMSO-d6): δ 8.73 (s, 1H), 8.08‒8.05 (m, 2H), 4.32
(q, 2H, J = 7.2 Hz), 1.32 (t, 3H, J = 7.2 Hz).
2.2.4.17. Ethyl 3-oxo-3H-benzo[f]chromene-2-carboxylate (11t). Yield: 89%; yellow
1
solid; H NMR (600 MHz, DMSO-d6): δ 9.34 (s, 1H), 8.57‒8.06 (m, 3H), 7.79‒7.57
(m, 3H), 4.35 (q, 2H, J = 7.2 Hz), 1.37 (t, 3H, J = 7.2 Hz).
2.2.5. General synthetic procedure of compounds 12a‒t
Compound 11 (5 mmol) in EtOH (5 mL) were added 10% NaOH (10 mL). Next,
the mixture was warmed to 80℃ and stirred for 15 min. The resulting mixture poured
off in cold water to precipitate a large amount of solid product. The solid product was
filtered off and recrystallized by EtOH to afford acid compounds 12a‒t (except 12d,
12i, 12m, and 12q) in good yields.
For compounds 12d, 12i, 12m, and 12q, compounds 11d, 11i, 11m, and 11q (2.46
mmol) were firstly protected with BnCl (7.37 mmol) in the presence of K2CO3 (7.37
mmol) in DMF (15mL). Then a similar procedure of 11a was performed to provide
11

desired compounds 12d, 12i, 12m, and 12q.
1
2.2.5.1. 2-Oxo-2H-chromene-3-carboxylic acid (12a). Yield: 82%; white solid; H
NMR (600 MHz, DMSO-d6): δ 8.74 (s, 1H), 7.91‒7.39 (m, 3H).
2.2.5.2. 6-Methyl-2-oxo-2H-chromene-3-carboxylic acid (12b). Yield: 92%; green
solid; 1H NMR (600 MHz, DMSO-d6): δ 8.70 (s, 1H), 8.04‒7.48 (m, 3H), 2.51 (s, 3H).
2.2.5.3. 6-Chloro-2-oxo-2H-chromene-3-carboxylic acid (12c). Yield: 86%; yellow
solid; 1H NMR (600 MHz, DMSO-d6): δ 8.66 (s, 1H), 7.68‒7.33 (m, 3H), 2.38 (s, 3H).
2.2.5.4. 6-(Benzyloxy)-2-oxo-2H-chromene-3-carboxylic acid (12d). Yield: 94%;
yellow solid; 1H NMR (600 MHz, DMSO-d6): δ 8.69 (s, 1H), 7.57‒7.34 (m, 8H), 5.16
(s, 1H).
2.2.5.5. 6-Methoxy-2-oxo-2H-chromene-3-carboxylic acid (12e). Yield: 82%; yellow
solid; 1H NMR (600 MHz, DMSO-d6): δ 8.70 (s, 1H), 7.48‒7.32 (m, 3H), 3.82 (s, 3H).
2.2.5.6. 6-Nitro-2-oxo-2H-chromene-3-carboxylic acid (12f). Yield: 84%; yellow solid;
1H NMR (600 MHz, DMSO-d6): δ 8.91-8.89 (m, 2H), 7.64‒8.50 (m, 2H).
2.2.5.7. 7-Methyl-2-oxo-2H-chromene-3-carboxylic acid (12g). Yield: 91%; white
solid; 1H NMR (600 MHz, DMSO-d6): δ 8.72 (s, 1H), 7.79‒7.24 (m, 3H).
2.2.5.8. 7-Chloro-2-oxo-2H-chromene-3-carboxylic acid (12h). Yield: 86%; white
solid; 1H NMR (600 MHz, DMSO-d6): δ 8.65 (s, 1H), 7.92‒7.46 (m, 3H).
2.2.5.9. 7-(Benzyloxy)-2-oxo-2H-chromene-3-carboxylic acid (12i). Yield: 95%;
yellow solid; 1H NMR (600 MHz, DMSO-d6): δ 8.70 (s, 1H), 7.83‒7.05 (m, 8H), 5.24
(s, 2H).
2.2.5.10. 7-Methoxy-2-oxo-2H-chromene-3-carboxylic acid (12j). Yield: 90%; red
12

solid; 1H NMR (600 MHz, DMSO-d6): δ 8.71 (s, 1H), 7.83‒6.99 (m, 3H), 3.89(s, 3H).
2.2.5.11. 8-Methyl-2-oxo-2H-chromene-3-carboxylic acid (12k). Yield: 72%; white
solid; 1H NMR (600 MHz, DMSO-d6): δ 8.73 (s, 1H), 7.74‒7.29 (m, 3H), 2.37 (s, 3H).
2.2.5.12. 8-Chloro-2-oxo-2H-chromene-3-carboxylic acid (12l). Yield: 84%; white
solid; 1H NMR (600 MHz, DMSO-d6): δ 8.76(s, 1H), 7.88‒7.38 (m, 3H).
2.2.5.13. 8-(Benzyloxy)-2-oxo-2H-chromene-3-carboxylic acid (12m). Yield: 92%;
white solid; 1H NMR (600 MHz, DMSO-d6): δ 8.73 (s, 1H), 7.51‒7.31 (m, 8H), 5.27
(s, 2H).
2.2.5.14. 8-Methoxy-2-oxo-2H-chromene-3-carboxylic acid (12n). Yield: 94%; yellow
solid; 1H NMR (600 MHz, DMSO-d6): δ 8.70 (s, 1H), 7.43‒7.32 (m, 3H), 3.90 (s, 3H).
2.2.5.15. 5,7-Dimethoxy-2-oxo-2H-chromene-3-carboxylic acid (12o). Yield: 90%;
white solid; 1H NMR (600 MHz, DMSO-d6): δ 12.90 (s, 1H), 8.63 (s, 1H), 6.63‒6.54
(m, 2H), 3.94 (s, 3H), 3.90 (s, 3H).
2.2.5.16. 5,7-Di-tert-butyl-2-oxo-2H-chromene-3-carboxylic acid (12p). Yield: 79%;
yellow solid; 1H NMR (600 MHz, DMSO-d6): δ 8.74(s, 1H), 7.81‒7.65 (m, 2H), 1.56
(s, 9H), 1.32 (s, 9H).
2.2.5.17. 7,8-Bis(benzyloxy)-2-oxo-2H-chromene-3-carboxylic acid (12q). Yield: 83%;
1
yellow solid; H NMR (600 MHz, DMSO-d6): δ 8.70 (s, 1H), 7.66‒7.28 (m, 12H),
5.31(s, 2H), 5.10 (s, 2H).
2.2.5.18. 7,8-Dimethoxy-2-oxo-2H-chromene-3-carboxylic acid (12r). Yield: 64%;
white solid; 1H NMR (600 MHz, DMSO-d6): δ 8.70 (s, 1H), 7.67‒7.18 (m, 2H), 3.94
(s, 3H), 3.83 (s, 3H).
13

2.2.5.19. 6,8-Dichloro-2-oxo-2H-chromene-3-carboxylic acid (12s). Yield: 91%; white
solid; 1H NMR (600 MHz, DMSO-d6): δ 8.70 (s, 1H), 8.07‒8.03 (m, 2H).
2.2.5.20. 3-Oxo-3H-benzo[f]chromene-2-carboxylic acid (12t). Yield: 92%; green
1
solid; H NMR (600 MHz, DMSO-d6): δ 9.27 (s, 1H), 8.51‒8.02 (m, 3H), 7.74‒7.52
(m, 3H).
2.2.6. General synthetic procedure of compounds 13a‒t
To a solution of 9 (1.0 mmol) and 12 (1.2 mmol) in dry DCM (5 mL), DMAP (2.0
mmol) and EDCI (1.2 mmol) were added under an atmosphere of argon, and the mixture
was allowed to stir for 24 h. Next, the resulting mixture was washed with 1M HCl (5
mL). The organic phases were dried, filtered, and evaporated in vacuo. Compounds
13b‒t were obtained by chromatography column (PE: EA = 10:1), but 13q and 13r
were used directly in the next reaction without purification.
2.2.6.1. 3,4-Bis(benzyloxy)phenethyl-2-oxo-2H-chromene-3-carboxylate (13a). Yield:
1
71%; white solid; H NMR (600 MHz, CDCl3): δ 8.44 (s, 1H), 7.65‒7.28 (m, 13H),
6.94‒6.79 (m, 3H), 5.16 (s, 2H), 5.14 (s, 2H), 4.48 (t, 2H, J = 6.6 Hz), 2.99 (t, 2H, J =
6.6 Hz).
2.2.6.2.
3,4-Bis(benzyloxy)phenethyl-6-methyl-2-oxo-2H-chromene-3-carboxylate
(13b). Yield: 81%; yellow solid; 1H NMR (600 MHz, CDCl3): δ 8.39 (s, 1H), 7.45‒7.25
(m, 13H), 6.94‒6.81 (m, 3H), 5.16 (s, 2H), 5.14 (s, 2H), 4.48 (t, 2H, J = 6.6 Hz), 2.99
(t, 2H, J = 6.6 Hz), 2.40 (s, 3H).
2.2.6.3.
3,4-Bis(benzyloxy)phenethyl-6-chloro-2-oxo-2H-chromene-3-carboxylate
(13c). Yield: 72%; yellow solid; 1H NMR (600MHz, CDCl3): δ 8.33 (s, 1H), 7.60‒7.28
14

(m, 13H), 6.92‒6.79 (m, 3H), 5.16 (s, 2H), 5.14 (s, 2H), 4.48 (t, 2H, J = 6.6 Hz), 2.98
(t, 2H, J = 6.6 Hz).
2.2.6.4.
3,4-Bis(benzyloxy)phenethyl-6-hydroxy-2-oxo-2H-chromene-3-carboxylate
1
(13d). Yield: 81%; yellow solid; H NMR (600 MHz, DMSO-d6): δ 8.65 (d, 1H),
7.54‒7.26 (m, 18H), 7.29‒6.81 (m, 3H), 5.10 (s, 2H), 5.08 (s, 2H), 5.07 (s, 2H), 4.39
(t, 2H, J = 6.6 Hz), 2.91 (t, 2H, J = 6.6 Hz).
2.2.6.5. 3,4-Bis(benzyloxy)phenethyl-6-methoxy-2-oxo-2H-chromene-3-carboxylate
1
(13e). Yield: 81%; yellow solid; H NMR (600 MHz, DMSO-d6): δ 8.67 (s, 1H),
7.45‒7.28 (m, 13H), 7.10‒6.82 (m, 3H), 5.08 (s, 2H), 5.07 (s, 2H), 4.39 (t, 2H, J = 6.6
Hz), 3.76 (s, 3H), 2.92 (t, 2H, J = 6.6 Hz).
2.2.6.6. 3,4-Bis(benzyloxy)phenethyl-6-nitro-2-oxo-2H-chromene-3-carboxylate (13f).
Yield: 60%; white solid; 1H NMR (600 MHz, DMSO-d6): δ 8.94 (s, 1H), 8.93‒7.65 (m,
3H), 7.42‒7.28 (m, 10H), 7.12‒6.84 (m, 3H), 5.09 (s, 2H), 5.08 (s, 2H), 4.43 (t, 2H, J
= 6.6 Hz), 2.94 (t, 2H, J = 6.6 Hz).
2.2.6.7.
3,4-Bis(benzyloxy)phenethyl-7-methyl-2-oxo-2H-chromene-3-carboxylate
1
(13g). Yield: 40%; white solid; H NMR (600 MHz, DMSO-d6): δ 8.67 (s, 1H),
7.76‒7.22 (m, 13H), 7.09‒6.81 (m, 3H), 5.08 (s, 2H), 5.07 (s, 2H), 4.38 (t, 2H, J = 6.6
Hz), 2.91 (t, 2H, J= 6.6 Hz), 2.43 (s, 3H).
2.2.6.8.
3,4-Bis(benzyloxy)phenethyl-7-chloro-2-oxo-2H-chromene-3-carboxylate
1
(13h). Yield: 48%; White solid; H NMR (600 MHz, DMSO-d6): δ 8.73 (s, 1H),
7.92‒7.83 (m, 10H), 7.09‒6.81 (m, 3H), 5.08 (s, 2H), 5.07 (s, 2H), 4.39 (t, 2H, J = 6.6
Hz), 2.91 (t, 2H, J = 6.6 Hz).
15

2.2.6.9.
3,4-Bis(benzyloxy)phenethyl-7-hydroxy-2-oxo-2H-chromene-3-carboxylate
1
(13i). Yield: 83%; white solid; H NMR (600 MHz, DMSO-d6): δ 8.67 (s, 1H),
7.81‒6.81 (m, 18H), 5.24 (s, 2H), 5.08 (s, 2H), 5.07 (s, 2H), 4.36 (t, 2H, J = 6.6 Hz),
2.90 (t, 2H, J = 6.6 Hz).
2.2.6.10. 3,4-Bis(benzyloxy)phenethyl-7-methoxy-2-oxo-2H-chromene-3-carboxylate
(13j). Yield: 65%; yellow solid; 1H NMR (600 MHz, CDCl3): δ 8.40(s, 1H), 7.45‒7.27
(m, 13H), 6.94‒6.79 (m, 3H), 5.15 (s, 2H), 5.12 (s, 2H), 4.46 (t, 2H, J = 6.6 Hz), 3.90
(s, 3H), 2.98 (t, 2H, J = 6.6 Hz).
2.2.6.11.
3,4-Bis(benzyloxy)phenethyl-8-methyl-2-oxo-2H-chromene-3-carboxylate
1
(13k). Yield: 44%; white solid; H NMR (600 MHz, DMSO-d6): δ 8.70 (s, 1H),
7.72‒7.10 (m, 13H), 6.99‒6.83 (m, 3H), 5.09 (s, 2H), 5.08 (s, 2H), 4.30 (t, 2H, J = 6.6
Hz), 2.93 (t, 2H, J = 6.6 Hz), 2.37 (s, 3H).
2.2.6.12.
3,4-Bis(benzyloxy)phenethyl-8-chloro-2-oxo-2H-chromene-3-carboxylate
1
(13l). Yield: 59%; white solid; H NMR (600 MHz, DMSO-d6): δ 8.74 (s, 1H),
7.89‒7.28 (m, 13H), 7.27‒6.82 (m, 3H), 5.08 (s, 2H), 5.07 (s, 2H), 4.40 (t, 2H, J = 6.6
Hz), 2.90 (t, 2H, J = 6.6 Hz).
2.2.6.13. 3,4-Bis(benzyloxy)phenethyl-8-hydroxy-2-oxo-2H-chromene-3-carboxylate
1
(13m). Yield: 68%; yellow solid; H NMR (600MHz, DMSO-d6): δ 8.40 (s, 1H),
7.47‒7.11 (m, 18H), 6.94‒6.72 (m, 3H), 5.25 (s, 2H), 5.16 (s, 2H), 5.13 (s, 2H), 4.47
(t, 2H, J = 6.6 Hz), 2.98 (t, 2H, J = 6.6 Hz).
2.2.6.14. 3,4-Bis(benzyloxy)phenethyl-8-methoxy-2-oxo-2H-chromene-3-carboxylate
1
(13n). Yield: 84%; yellow solid; H NMR (600 MHz, DMSO-d6): δ 8.69 (s, 1H),
16

7.43‒7.11 (m, 13H), 6.99‒6.83 (m, 3H), 5.10 (s, 1H), 5.09 (s, 1H), 4.40 (t, 2H, J = 6.6
Hz), 3.92 (s, 3H), 2.93 (t, 2H, J = 6.6 Hz).
2.2.6.15.
3,4-Bis(benzyloxy)phenethyl-5,7-dimethoxy-2-oxo-2H-chromene-3-
1
carboxylate (13o). Yield: 75%; white solid; H NMR (600 MHz, CDCl3): δ 8.78 (s,
1H), 8.64 (s, 1H), 7.44‒7.28 (m, 10H), 6.94‒6.81 (m, 3H), 6.41-6.26 (m, 2H), 5.15 (s,
1H), 5.13 (s, 1H), 4.45 (t, 2H, J = 7.2 Hz), 3.88 (s,6H) , 2.82 (t, 2H, J = 7.2 Hz).
2.2.6.16.
3,4-Bis(benzyloxy)phenethyl-6,8-di-tert-butyl-2-oxo-2H-chromene-3-
1
carboxylate (13p). Yield: 94%; white solid; H NMR (600 MHz, CDCl3): δ 8.45 (s,
1H), 7.68‒7.26 (m, 12H), 7.27‒6.81 (m, 3H), 5.17 (s, 2H), 5.14 (s, 2H), 4.48 (t, 2H, J
= 7.2 Hz), 2.99 (t, 2H, J = 7.2 Hz), 1.53 (s, 9H), 1.35(s, 9H).
2.2.6.17.
3,4-Bis(benzyloxy)phenethyl-6,8-dichloro-2-oxo-2H-chromene-3-
1
carboxylate (13s). Yield: 78%; yellow solid; H NMR (600 MHz, CDCl3): δ 8.29 (s,
1H), 7.68‒7.26 (m, 12H), 6.92‒6.79 (m, 3H), 5.16 (s, 2H), 5.14 (s, 2H), 4.49 (t, 2H, J
=7.2 Hz), 2.98 (t, 2H, J = 7.2 Hz).
2.2.6.18.
3,4-Bis(benzyloxy)phenethyl-3-oxo-3H-benzo[f]chromene-2-carboxylate
1
(13t). Yield: 77%; white solid; H NMR (600 MHz, DMSO-d6): δ 9.27 (s, 1H),
8.50‒8.08 (m, 3H), 7.76‒7.34 (m, 3H), 7.32-7.26 (m, 10H), 6.88‒7.15 (m, 3H), 5.09 (s,
2H), 5.08 (s, 2H), 4.46 (t, 2H, J = 6.6 Hz), 2.99 (t, 2H, J = 6.6 Hz).
2.2.7. General synthetic procedure of compounds 14a‒t
A solution of 13 (0.40 mmol) in dry DCM (2 mL) added 1M BBr3 in DCM (2 mL)
at -40℃ followed by stirring for 15 min. Next, the resulting mixture was added water
to quench and extracted with ethyl acetate (10 mL×3). The combined organic extracts
17

were washed with saturated NaCl (10 mL), dried over Na2SO4, filtered, and evaporated
in vacuo. The crude product was purified by column chromatography (DCM: MeOH=
100:1 to 50:1) to give target compounds 14a‒t.
2.2.7.1. 3,4-Dihydroxyphenethyl-2-oxo-2H-chromene-3-carboxylate (14a). Yield: 50%;
1
yellow powder; m.p.: 115‒117 ℃; H NMR (600 MHz, DMSO-d6) δ 8.77 (brs, 2H),
8.68 (s, 1H), 7.90‒7.40 (m, 3H), 6.69‒6.55 (m, 3H), 4.35 (t, 2H, J = 6.6 Hz), 2.84 (t,
2H, J = 6.6 Hz); 13C NMR (150 MHz, DMSO-d6) δ 162.4, 155.9, 154.5, 148.6, 145.1,
143.8, 134.5, 130.2, 128.3, 124.8, 119.6, 117.7, 116.3, 116.1, 115.5, 66.0, 33.7; HRMS
(ESI) m/z: Calcd for C18H14O6Na [M+Na]+ 349.0688; found, 349.0697; HPLC purity =
96.08% (MeOH: H2O = 85:15, 1 mL/min, tR = 3.960 min).
2.2.7.2. 3,4-Dihydroxyphenethyl-6-methyl-2-oxo-2H-chromene-3-carboxylate (14b).
Yield: 78%; yellow powder; m.p.: 140‒142 ℃; 1H NMR (600 MHz, DMSO-d6): δ 8.81
(d, 1H), 8.74 (s, 1H), 8.60 (s, 1H), 7.68‒7.34 (m, 3H), 6.68‒6.55 (m, 3H), 4.35 (t, 2H,
J = 6.6 Hz), 2.82 (t, 2H, J = 6.6 Hz), 2.38 (s, 3H); 13C NMR (150 MHz, DMSO-d6) δ
162.4, 156.0, 152.7, 148.5, 145.1, 143.8, 135.4, 134.2, 129.6, 128.3, 119.6, 115.9, 115.6,
66.0, 33.7, 20.1; HRMS (ESI) m/z Calcd for C19H16O6Na [M+Na]+ 363.0845; found,
363.0854; HPLC purity = 96.51% (MeOH: H2O = 85:15, 1 mL/min, tR = 4.280 min).
2.2.7.3. 3,4-Dihydroxyphenethyl-6-chloro-2-oxo-2H-chromene-3-carboxylate (14c).
Yield: 47%; yellow powder; m.p.: 178‒180 ℃; 1H NMR (600 MHz, DMSO-d6): δ 8.80
(d, 1H), 8.74 (s, 1H), 8.66 (s, 1H), 8.05‒7.48 (m, 3H), 6.68‒6.55 (m, 3H), 4.36 (t, 2H,
J = 6.6 Hz), 2.83 (t, 2H, J = 6.6 Hz); 13C NMR (150 MHz, DMSO-d6): δ 162.1, 155.4,
153.1, 147.3, 145.1, 143.8, 133.8, 129.0, 128.4, 128.2, 119.6, 119.1, 118.7, 118.2, 116.3,
18

115.5, 66.1, 33.6; HRMS (ESI) m/z: Calcd for C18H13ClO6Na [M+Na]+ 383.0298;
found, 383.0312; HPLC purity = 99.64% (MeOH: H2O = 85:15, 1 mL/min, tR = 4.389
min).
2.2.7.4. 3,4-Dihydroxyphenethyl-6-hydroxy-2-oxo-2H-chromene-3-carboxylate (14d).
Yield: 54%; yellow powder; m.p.: 213‒215 ℃; 1H NMR (600 MHz, DMSO-d6): δ 9.91
(s, 1H), 8.79 (s, 1H), 8.72 (s, 1H), 8.58 (s, 1H), 7.29‒7.15 (m, 3H), 6.66‒6.53 (m, 3H),
4.32 (t, 2H, J = 6.6 Hz), 2.81 (t, 2H, J = 6.6 Hz); 13C NMR (150 MHz, DMSO-d6): δ
162.6, 156.2, 154.0, 148.5, 147.9, 145.1, 143.8, 128.3, 122.7, 119.6, 118.2, 117.6, 117.1,
116.3, 115.6, 113.8, 67.0, 33.7; HRMS (ESI) m/z: Calcd for C18H14O7Na [M+Na]+
365.0637; found, 365.0650; HPLC purity = 95.72% (MeOH: H2O = 85:15, 1 mL/min,
tR = 3.863 min).
2.2.7.5. 3,4-Dihydroxyphenethyl-6-methoxy-2-oxo-2H-chromene-3-carboxylate (14e).
Yield: 54%; yellow powder; m.p.: 146‒147 ℃; 1H NMR (600 MHz, DMSO-d6): δ 8.79
(d, 1H), 8.73 (s, 1H), 8.62 (s, 1H), 7.45‒7.32 (m, 3H), 6.66‒6.53 (m, 3H), 4.33 (t, 2H,
J = 6.6 Hz), 3.81 (s, 3H), 2.81 (t, 2H, J = 6.6 Hz); 13C NMR (150 MHz, DMSO-d6): δ
162.6, 156.2, 155.8, 149.1, 148.6, 145.2, 143.9, 128.5, 122.5, 119.7, 118.2, 117.8, 117.4,
116.3, 115.7, 111.1, 66.1, 55.9, 33.8; HRMS (ESI) m/z: Calcd for C19H16O7Na
[M+Na]+ 379.0794; found, 379.0807; HPLC purity = 99.02% (MeOH: H2O = 85:15, 1
mL/min, tR = 4.177 min).
2.2.7.6. 3,4-Dihydroxyphenethyl-6-nitro-2-oxo-2H-chromene-3-carboxylate (14f).
Yield: 45%; yellow powder; m.p.: 214‒216 ℃; 1H NMR (600 MHz, DMSO-d6): δ 8.93
(d, 1H), 8.89 (s, 1H), 8.81 (s, 1H), 8.75 (s, 1H), 8.52‒8.50 (m, 1H), 7.65 (d, 1H),
19

13
6.56‒6.68 (m, 3H), 4.38 (t, 2H, J = 6.6 Hz), 2.84 (t, 2H, J = 6.6Hz); C NMR (150
MHz, DMSO-d6): δ 162.0, 158.1, 155.0, 147.7, 145.1, 143.9, 143.7, 128.6, 128.6, 126.1,
119.6, 119.4, 118.2, 117.8, 116.3, 115.6, 66.3, 33.7; HRMS (ESI) m/z: Calcd for
C18H13NO8Na [M+Na]+ 394.0539; found, 394.0552; HPLC purity = 95.51% (MeOH:
H2O = 85:15, 1 mL/min, tR = 3.979 min).
2.2.7.7. 3,4-Dihydroxyphenethyl-7-methyl-2-oxo-2H-chromene-3-carboxylate (14g).
Yield: 40%; yellow powder; m.p.: 165‒168 ℃; 1H NMR (600 MHz, DMSO-d6): δ 8.79
(s, 1H), 8.72 (s, 1H), 8.63 (s, 1H), 7.77‒7.23 (m, 3H), 6.66‒6.53 (m, 3H), 4.32 (t, 2H,
J = 6.6 Hz), 2.81 (t, 2H, J = 6.6 Hz), 2.43 (s, 3H); 13C NMR (150 MHz, DMSO-d6): δ
162.5, 156.0, 154.7, 148.7, 146.1, 145.1, 143.8, 130.0, 129.9, 128.3, 126.0, 119.5, 116.3,
116.3, 116.2, 115.6, 115.4, 66.9, 33.7, 21.5; HRMS (ESI) m/z: Calcd for C18H14O6Na
[M+Na]+ 363.0845; found, 363.0859; HPLC purity = 98.08% (MeOH: H2O = 85:15, 1
mL/min, tR = 4.266 min).
2.2.7.8. 3,4-Dihydroxyphenethyl-7-chloro-2-oxo-2H-chromene-3-carboxylate (14h).
Yield: 60%; yellow powder; m.p.: 170‒172 ℃; 1H NMR (600 MHz, DMSO-d6): δ 8.78
(s, 1H), 8.72 (s, 1H), 8.69 (s, 1H), 7.93‒7.48 (m, 3H), 6.66‒6.53 (m, 3H), 4.34 (t, 2H,
J = 6.6Hz), 2.81 (t, 2H, J = 6.6 Hz); 13C NMR (150 MHz, DMSO-d6): δ 162.2, 155.3,
154.9, 148.0, 145.1, 143.8, 138.8, 131.6, 131.5, 128.3, 125.2, 119.6, 117.5, 116.8, 116.3,
115.6, 66.1, 33.7; HRMS (ESI) m/z: Calcd for C18H13ClO6Na [M+Na]+ 383.0298;
found, 383.0311; HPLC purity = 99.26% (MeOH: H2O = 85:15, 1 mL/min, tR = 4.504
min).
2.2.7.9. 3,4-Dihydroxyphenethyl-7-hydroxy-2-oxo-2H-chromene-3-carboxylate (14i).
20

1
Yield: 42%; yellow powder; m.p.: 202‒204 ℃; H NMR (600 MHz, DMSO-d6): δ
11.12 (s, 1H), 8.81 (s, 1H), 8.74 (s, 1H), 8.60 (s, 1H), 6.74‒7.55 (m, 6H), 4.32 (t, 2H, J
= 7.2 Hz), 2.82 (t, 2H, J = 7.2 Hz); 13C NMR (150 MHz, DMSO-d6): δ 164.1, 162.7,
157.1, 156.3, 149.4, 145.1, 143.8, 132.1, 128.4, 119.6, 116.3, 115.5, 114.0, 111.9, 110.4,
101.8, 65.7, 33.8; HRMS (ESI) m/z: Calcd for C18H14O7Na [M+Na]+ 365.0637; found,
365.0650; HPLC purity = 97.22% (MeOH: H2O = 85:15, 1 mL/min, tR = 3.530 min).
2.2.7.10. 3,4-Dihydroxyphenethyl-7-methoxy-2-oxo-2H-chromene-3-carboxylate (14j).
Yield: 48%; yellow powder; m.p.: 185‒188 ℃; 1H NMR (600 MHz, DMSO-d6): δ 8.80
(s, 1H), 8.73 (s, 1H), 8.66 (s, 1H), 7.84‒7.01 (m, 3H), 6.68‒6.55 (m, 3H), 4.33 (t, 2H,
J = 7.2 Hz), 3.90 (s, 3H), 2.82 (t, 2H, J = 7.2 Hz); 13C NMR (150 MHz, DMSO-d6): δ
164.8, 162.6, 157.0, 156.1, 149.1, 145.1, 143.8, 131.6, 128.3, 119.5, 116.3, 115.5, 113.3,
111.3, 110.3, 65.709, 56.2, 33.7; HRMS (ESI) m/z: Calcd for C19H16O7Na [M+Na]+
379.0794; found, 379.0807; HPLC purity = 96.96% (MeOH: H2O = 85:15, 1 mL/min,
tR = 4.168 min).
2.2.7.11. 3,4-Dihydroxyphenethyl-8-methyl-2-oxo-2H-chromene-3-carboxylate (14k).
Yield: 67%; yellow powder; m.p.: 149‒150 ℃; 1H NMR (600 MHz, DMSO-d6): δ 8.83
(s, 1H), 8.76 (s, 1H), 8.67 (s, 1H), 7.74‒7.32 (m, 3H), 6.69‒6.56 (m, 3H), 4.36 (t, 2H,
J = 7.2 Hz), 2.84 (t, 2H, J = 7.2 Hz), 2.39 (s, 3H); 13C NMR (150 MHz, DMSO-d6): δ
162.5, 156.0, 152.8, 149. 0, 145.1, 143.8, 135.6, 128.4, 128.0, 125.1, 119.6, 117.5,
117.2, 116.4, 115.6, 66.0, 33.7, 14.8; HRMS (ESI) m/z: Calcd for C19H16O6Na
[M+Na]+ 363.0843; found, 363.0857; HPLC purity = 98.18% (MeOH: H2O = 85:15, 1
mL/min, tR = 4.374 min).
21

2.2.7.12. 3,4-Dihydroxyphenethyl-8-chloro-2-oxo-2H-chromene-3-carboxylate (14l).
1
Yield: 46%; yellow powder; m.p.: 57‒60 ℃; H NMR (600 MHz, DMSO-d6): δ 8.79
(s, 1H), 8.72 (s, 1H), 8.70 (s, 1H), 7.89‒7.39 (m, 3H), 6.66‒6.53 (m, 3H), 4.35 (t, 2H,
J = 6.6 Hz), 2.82 (t, 2H, J = 6.6 Hz); 13C NMR (150 MHz, DMSO-d6): δ 162.1, 155.0,
149.9, 148.4, 145.1, 143.8, 134.2, 129.2, 128.3, 125.3, 119.6, 119.5, 119.4, 118.3, 116.3,
115.6, 66.2, 33.7; HRMS (ESI) m/z: Calcd for C18H13ClO6Na [M+Na]+ 383.0298;
found, 383.0311; HPLC purity = 97.18% (MeOH: H2O = 85:15, 1 mL/min, tR = 4.264
min).
2.2.7.13.
3,4-Dihydroxyphenethyl-8-hydroxy-2-oxo-2H-chromene-3-carboxylate
(14m). Yield: 33%; yellow powder; m.p.: 178‒181 ℃; 1H NMR (600MHz, DMSO-d6):
δ 10.40 (s, 1H), 8.81 (s, 1H), 8.74 (s, 1H), 8.61 (s, 1H), 7.32‒7.22 (m, 3H), 6.68‒6.55
(m, 3H), 4.35 (t, 2H, J= 7.2 Hz), 2.84 (t, 2H, J = 7.2 Hz); 13C NMR (150 MHz, DMSO-
d6): δ 162.5, 155.8, 149.1, 145.1, 144.4, 143.8, 143.2, 128.3, 124.8, 120.6, 120.1, 119.6,
118.6, 117.4, 116.3, 115.5, 66.0, 33.7. HRMS (ESI) m/z: Calcd for C18H14O7Na
[M+Na]+ 365.0637; found, 365.0649; HPLC purity = 95.42% (MeOH: H2O = 85:15, 1
mL/min, tR = 3.948 min).
2.2.7.14.
3,4-Dihydroxyphenethyl-8-methoxy-2-oxo-2H-chromene-3-carboxylate
(14n). Yield: 40%; yellow powder; m.p.: 90‒92 ℃; 1H NMR (600 MHz, DMSO-d6): δ
8.82 (s, 1H), 8.75 (s, 1H), 8.65 (s, 1H), 7.44‒7.34 (m, 3H), 6.69‒6.56 (m, 3H), 4.36 (t,
2H, J = 7.2 Hz), 3.93 (s, 3H), 2.84 (t, 2H, J = 7.2 Hz); 13C NMR (150 MHz, DMSO-
d6): δ 162.4, 155.6, 148.9, 146.2, 145.1, 143.8, 128.3, 124.8, 121.1, 119.6, 118.235,
117.7, 116.5, 116.3 115.6, 79.1, 66.0, 56.4, 33.7; HRMS (ESI) m/z: Calcd for
22

C19H16O7Na [M+Na]+ 379.0794; found, 379.0806; HPLC purity = 97.71% (MeOH:
H2O = 85:15, 1 mL/min, tR = 4.001 min).
2.2.7.15. 3,4-Dihydroxyphenethyl-5,7-dimethoxy-2-oxo-2H-chromene-3-carboxylate
(14o). Yield: 54%; yellow powder; m.p.: 192‒195 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 8.78 (s, 1H), 8.73 (s, 1H), 8.57 (s, 1H), 6.67‒6.64 (m, 3H), 6.55‒6.54 (m, 2H), 4.31
(t, 2H, J = 7.2 Hz), 3.95 (s, 3H), 3.90 (s, 3H), 2.80 (t, 2H, J = 7.2 Hz); 13C NMR (150
MHz, DMSO-d6): δ 166.4, 162.4, 158.2, 157.6, 156.1, 145.1, 143.8, 134.5, 128.4, 119.6,
116.2, 115.5, 95.2, 93.0, 65.5, 56.6, 56.3, 33.7; HRMS (ESI) m/z: Calcd for
C20H18O8Na [M+Na]+ 409.0899; found, 409.0911; HPLC purity = 95.53% (MeOH:
H2O = 85:15, 1 mL/min, tR = 4.351 min).
2.2.7.16. 3,4-Dihydroxyphenethyl-6,8-di-tert-butyl-2-oxo-2H-chromene-3-carboxylate
(14p). Yield: 48%; yellow powder; m.p.: 70‒73 ℃; 1H NMR (600 MHz, DMSO-d6): δ
8.80 (s, 1H), 8.73 (s, 1H), 8.70 (s, 1H), 7.80‒7.66 (m, 2H), 6.70‒6.57 (m, 3H), 4.35 (t,
13
2H, J = 7.2 Hz), 2.84 (t, 2H, J = 7.2 Hz), 1.46 (s, 9H), 1.34 (s, 9H); C NMR (150
MHz, CDCl3): δ 163.4, 157.4, 152.0, 150.9, 147.9, 143.9, 142.9, 137.5, 130.1, 124.2,
121.1, 117.9, 116.2, 115.3, 66.6, 35.1, 34.7, 34.4, 31.2, 29.7; HRMS (ESI) m/z: Calcd
for C26H30O6 [M+Na]+ 461.1952; found, 461.1940; HPLC purity = 95.74% (MeOH:
H2O = 85:15, 1 mL/min, tR = 8.871 min).
2.2.7.17. 3,4-Dihydroxyphenethyl-7,8-dihydroxy-2-oxo-2H-chromene-3-carboxylate
(14q). Yield: 52%; yellow powder; m.p.: 228‒230 ℃; 1H NMR (600 MHz, DMSO-d6):
δ 8.54 (s, 1H), 6.54‒6.24 (m, 2H), 4.31 (t, 2H, J = 6.6 Hz), 2.82 (t, 2H, J = 6.6 Hz); 13C
NMR (150 MHz, DMSO-d6): δ 162.8, 156.3, 152.9, 149.9, 145.1, 144.9, 143.8, 131.8,
23

128.4, 121.6, 119.6, 116.3, 115.5, 13.3, 111.5, 111.0, 65.6, 33.7; HRMS (ESI) m/z:
Calcd for C18H14O8Na [M+Na]+ 381.0586; found, 381.0601; HPLC purity = 98.07%
(MeOH: H2O = 85:15, 1 mL/min, tR = 3.542 min).
2.2.7.18. 3,4-Dihydroxyphenethyl-7,8-dimethoxy-2-oxo-2H-chromene-3-carboxylate
(14r). Yield: 43%; yellow powder; m.p.: 141‒144 ℃; 1H NMR (600 MHz,DMSO-d6):
δ 8.80 (s, 1H), 8.73 (s, 1H), 8.64 (s, 1H), 7.67‒7.19 (m, 2H), 6.68‒6.55 (m, 3H), 4.33
(t, 2H, J =7.2 Hz), 3.95 (s, 3H), 3.83 (s, 3H), 2.82 (t, 2H, J = 7.2 Hz); 13C NMR (150
MHz, DMSO-d6): δ 162.7, 157.6, 155.9, 149.4, 148.5, 145.1, 143.8, 134.8, 128.5, 126.2,
119.7, 116.4, 115.6, 113.7, 112.5, 110.0, 65.9, 60.9, 65.7, 33.8; HRMS (ESI) m/z: Calcd
for C20H18O8Na [M+Na]+ 409.0899; found, 409.0911; HPLC purity = 95.84% (MeOH:
H2O = 85:15, 1 mL/min, tR = 3.917 min).
2.2.7.19.
3,4-Dihydroxyphenethyl-6,8-dichloro-2-oxo-2H-chromene-3-carboxylate
(14s). Yield: 67%; yellow powder; m.p.: 218‒222 ℃; 1H-NMR (600 MHz, DMSO-d6):
δ 8.77 (s, 1H), 8.71 (s, 1H), 8.65 (s, 1H), 8.06‒8.02 (m, 2H), 6.67‒6.54 (m, 3H), 4.36
(t, 2H, J = 7.2 Hz), 2.82 (t, 2H, J = 7.2 Hz); 13C NMR (150 MHz, DMSO-d6): δ 162.5,
155.9, 155.1, 145.2, 143.9, 136.1, 129.7, 129.1, 128.990, 128.9, 128.5, 126.4, 121.9,
119.6, 116.4, 116.3, 115.5, 111.7, 65.9, 33.7; HRMS (ESI) m/z: Calcd for
C18H12Cl2O6Na [M+Na]+ 416.9909; found, 416.9922; HPLC purity = 96.78% (MeOH:
H2O = 85:15, 1 mL/min, tR = 4.852 min).
2.2.7.20. 3,4-Dihydroxyphenethyl-3-oxo-3H-benzo[f]chromene-2-carboxylate (14t).
Yield: 52%; yellow powder; m.p.: 187‒190℃; 1H NMR (600 MHz, DMSO-d6): δ 9.25
(s, 1H), 8.82 (s, 1H), 8.77 (s, 1H), 8.48‒8.08 (m, 3H), 7.84‒7.58 (m, 3H), 6.60‒6.74
24

(m, 3H), 4.41 (t, 2H, J = 6.6 Hz), 2.89 (t, 2H, J = 6.6 Hz); 13C NMR (150 MHz, DMSO-
d6): δ 162.5, 155.9, 155.2, 145.2, 143.9, 136.1, 129.7, 129.1, 129.0, 128.9, 128.5, 126.4,
121.9, 119.6, 116.4, 116.3, 115.5, 111.7, 65.9, 33.7; HRMS (ESI) m/z: Calcd for
C22H16O6Na [M+Na]+ 399.0845; found, 399.0856; HPLC purity = 96.20% (MeOH:
H2O = 85:15, 1 mL/min, tR = 4.513 min).
2.3. Antioxidant assays
2.3.1. Cell survival conditions
HepG2 WI-38 cells obtained from General Hospital of Lanzhou Military
Command were grown at 37℃ in DMEM supplemented with 10% fetal bovine serum,
and 1% penicillin/streptomycin in a humidified atmosphere containing 5% CO2. The
medium was replaced once every second day. All materials were obtained from
commercial suppliers (Solarbio and Beyotime Biotechnology). Each of the experiments
was done in triplicate and the mean value of each reading was reported within.
2.3.2. Free radical scavenging assay
2.3.2.1. DPPH radical scavenging assay
1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities of all the
synthesized compounds 14a‒t, 1, and 3 were examined by DPPH assay [24]. Butylated
hydroxytoluene (BHT) was used as the standard. DPPH was prepared into a 10 mM
stock solution with ethanol to produce DPPH free radicals, and then diluted with
ethanol. 100 μL of various concentration solutions of compounds 14a‒t, 1, and 3 in
methanol was mixed with 100 μL of 0.5 mM DPPH• solution in a 96-well plate, then
25

the mixture was incubated at 25 ℃ for 1 h in the dark. The absorbance was measured
at 517 nm using a spectrophotometer. The antiradical activity was calculated as IC50
according to % scavenged [DPPH•].
% scavenged [DPPH•] = [(Absorbance of control‒Absorbance of sample)/Absorbance
of control] × 100
2.3.2.2. ABTS radical cation scavenging assay
The 2,2'-azino-di(3-ethylbenzthiazoline sulphonate) (ABTS) radical cation
scavenging activities of all the synthesized compounds 14a‒t, 1, and 3 were examined
using ABTS assay [24]. Distilled water (10 mL) was prepared into a mixed solution of
ABTS salt (4 mM) and K2S2O8 (1.41 mM), and placed in the dark at 25℃ for 24h to
form ABTS+• free radicals, then diluted with 100mL of ethanol. 50 μL of various
concentration solutions of compounds 14a‒t, 1, and 3 in methanol were added to the
96-well plate, 150 μL of ABTS+• solution was added, and incubated at 25℃ for 10 min
in the dark. The absorbance was measured at 734 nm. The antiradical activity was
calculated as IC50 according to %scavenged [ABTS+•].
%
scavenged [ABTS+•]
=
[(Absorbance of control
‒
Absorbance of
sample)/Absorbance of control] × 100
2.3.3. Cytotoxic assay
2.3.3.1. Hemolysis assay
The toxicity was evaluated on 5% (v/v) suspension of rat RBCs in PBS (control).
After 2ꢀh of RBCs hemolysis incubated with different concentrations (200, 100, 50, and
26

25ꢀμM) of test compounds (14d, 14i, 14m, 14q, 3, 1, and BHT), incubation liquids were
centrifuged for 5ꢀmin (10000 r/min). Hemolysis was determined as hemoglobin content
in the supernatant at λ 540ꢀnm. The H2O-treated controls were assigned a hemolysis of
100%.
2.3.3.2. Cytotoxic assay in human normal cells line WI-38 and GES by MTT
Cells were plated in 96-well plates at a density of 5×103 cells per well in 100 μL
medium. After 24 h incubation, compounds 14d, 14i, 14m, 14q, 3, 1, and BHT (200,
100, 50, 25ꢀand 12.5 μM) were added to each well and incubated for 48 h. After attached
cells were incubated with 5 mg/mL (Sigma, USA) for another 4 h, the suspension was
solubilized in dimethyl sulfoxide (DMSO). The absorbance of the 490 nm was
measured using a multifunction microplate reader (Molecular Devices, Flex Station 3).
The DMSO-treated controls were assigned negative control.
2.3.4. Cell viability assay in H2O2-damaged WI-38 and HepG2 cells by MTT
HepG2 cells were plated in 96-well plates at a density of 5×103 cells per well in
100 μL medium. After 24 h incubation, compound 14q (50, 25ꢀand 12.5 μM) was added
to each well and incubated for 1 h. Subsequently, H2O2 (400 μM) was added and cells
were incubated for 12 h to induce cell injury. MTT assay was performed as mentioned
above. The no compound-treated controls were assigned a cell viability value of 100%.
Cell viability (%) = OD value (sample group) / OD value (blank control) × 100%.
2.3.5. Apoptosis assay in H2O2-damaged HepG2 cells
Cells apoptosis was assayed by the Annexin-V-FITC Apoptosis Detection Kit (BD
27

Biosciences, USA) according to the manufacturer's instructions. Briefly, HepG2 cells
were plated in 6-well plates at a density of 1×106 cells per well in 4 mL medium. After
24 h incubation, compounds 14q (50, 25, 5 and 0 μM) were added to well and incubated
for 1 h. Subsequently, H2O2 (400 μM) was added and cells were incubated for 12 h to
induce cell injury. Damaged HepG2 cells were harvested, washed twice with ice-cold
PBS, and resuspended in 1× binding buffer at a concentration of 1×106 cells/ mL.
Subsequently, the cells were stained with 5 μL Annexin-V-FITC and 5 μL PI (50
mg/mL) for 10 min in the dark at 25℃, and analyzed by flow cytometry. The no H2O2-
treated were assigned control.
2.3.6. ROS level assay
ROS level assay was measured at 488 excitation wavelength and 525 emission
wavelength using reactive oxygen species assay kit (Beyotime Biotechnology S0033)
by fluorescence spectrophotometer (RF-5301PC, Japan, Takatsu). HepG2 cells were
plated in 6 cm dishes at a density of 1.0×106 cells per dish in 4 mL medium. After 24h
incubation, compounds 14q (50, 25, 5 and 0 μM) were added to each dish and incubated
for 1 h. Subsequently, H2O2 (400 μM) was added and cells were incubated for 12 h to
induce cell injury. ROS level was measured according to the manufacturer's instructions.
The no H2O2-treated were assigned control.
2.3.7. LDH, SOD, and GSH level assay
HepG2 cells were plated in 6 cm dishes at a density of 1.0×106 cells per dish in 4
mL medium. After 24 h incubation, compounds 14q (50, 25, 5 and 0 μM) were added
28

to each dish and incubated for 1 h. Subsequently, H2O2 (400 μM) was added and cells
were incubated for 12 h to induce cell injury. LDH level was measured at 450 nm by
LDH assay kit (Beijing Solarbio BC0680), SOD level was measured at 560 nm by SOD
assay kit (Beijing Solarbio BC0170), and GSH level was measured at 405 nm based
on the rate of oxidation of reduced glutathione to oxidized glutathione by reduced
glutathione assay kit (Beijing Solarbio BC1175). Their levels were measured according
to the manufacturer's instructions. The no H2O2-treated were assigned control.
2.3.8. Stability assay
Compound 14q and hydroxytyrosol were completely dissolved in methanol, and
stored in 25℃or 50℃ for 0, 0.5, 1, 2, 4 and 6 days. The contents of compound 14q and
hydroxytyrosol contents were measured by HPLC (MeOH: H2O= 80:20, 1 mL /min,
LC-100HP, Wufeng, China), and expressed with their remaining percentage.
3. Results and discussion
Twenty new target compounds were synthesized as described in the Materials and
1
Methods, and their structures were elucidated by H NMR, 13C NMR (see
Supplementary data) and high resolution mass spectrometry, all spectral data were in
accordance with assumed structures. The purities of synthesized target compounds
were all above 95% by HPLC method.
The antioxidant capacity of the target conjugates 14a‒t were tested using two
different methods, namely, the DPPH radical and ABTS radical cation scavenging
assays [24]. BHT, hydroxytyrosol and coumarin were used as a positive control, the
29