Design, synthesis, and primary activity assays of baicalein derivatives as cyclin-dependent kinase 1 inhibitors

Article abstract of DOI:10.1111/cbdd.13917

Malignant tumor is a disease with high mortality. Traditional treatment methods have many disadvantages, such as side-effects, drug resistance. Because cyclin-dependent kinase 1 (CDK1) plays an indispensable role in cell cycle regulation, it became an att

Full text of DOI:10.1111/cbdd.13917

Received: 28 October 2020
DOI: 10.1111/cbdd.13917
Revised: 27 April 2021
Accepted: 3 July 2021
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R E S E A R C H A R T I C L E
Design, synthesis, and primary activity assays of baicalein
derivatives as cyclin-dependent kinase 1 inhibitors
Jiajia Mou1
Shuang Qiu1
Danghui Chen1
Yanru Deng1
Teka Tekleab2
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1Department of Medicinal Chemistry,
School of Chinese Materia Medica, Tianjin
University of Traditional Chinese Medicine,
Tianjin, China
2Tianjin State Key Laboratory of Modern
Chinese Medicine, Tianjin Key Laboratory
of TCM Chemistry and Analysis, Tianjin
University of Traditional Chinese Medicine,
Tianjin, China
Abstract
Malignant tumor is a disease with high mortality. Traditional treatment methods have
many disadvantages, such as side-effects, drug resistance. Because cyclin-dependent
kinase 1 (CDK1) plays an indispensable role in cell cycle regulation, it became an
attractive target in rational anti-cancer drug discovery. Herein, we reported a se-
ries of baicalein derivatives, which remarkably repressed the proliferation of MCF-7
tumor cells and the activity of CDK1/cyclin B kinase. Among them, compound 4a
displayed better inhibition rate than flavopiridol against MCF-7 proliferation at the
concentration of 50 μg/ml, comparable to compound CGP74514A, while compound
3o possessed the best activity against CDK1/cyclin B kinase (IC50 = 1.26 μM). The
inhibitory activities toward the kinase well correlated with anti-proliferative activi-
ties. Molecular docking results suggested that compound 3o can interact with the key
amino acid residues, E81, L83, and D146, of CDK1 through hydrogen bond just like
flavopiridol does. And it can also form an extra hydrogen bond with D146 by its in-
troduced 7-acrylate group, which flavopiridol does not have. These findings proved
that baicalein derivatives can be used as CDK1 inhibitors fighting against cancer.
Correspondence
Jiajia Mou, Department of Medicinal
Chemistry, School of Chinese Materia
Medica, Tianjin University of Traditional
Chinese Medicine, Tianjin, China.
Email: moujiajia66@163.com
Funding information
National Natural Science Foundation of
China, Grant/Award Number: 21502142;
Tianjin Natural Science Foundation, Grant/
Award Number: 18JCYBJC28900
K E Y W O R D S
baicalein derivatives, cyclin-dependent kinase 1, inhibitor
1
INTRODUCTION
(Sánchez-Martínez et al., 2015). The cell cycle is a series
of events that lead to cell division and duplication (Lapenna
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Malignant tumor is a kind of disease caused by multiple
gene mutations under the concurrent action of genetic and
environmental factors, which leads to uncontrolled cell
cycle regulation and unlimited cell proliferation (Lapenna &
Giordano, 2009). In recent years, the incidence of tumor is in-
creasing, which always posts a threat to human health (Fouad
& Aanei, 2017; Hanahan & Weinberg, 2011). Traditional
chemotherapy has several disadvantages, side-effects,
drug resistance, and so on (Bradner et al., 2017; Solaki &
Ewald, 2018). Therefore, there is an urgent need to seek new
targets and anti-cancer drugs with high efficiency, selectivity,
and low toxicity.
& Giordano, 2009), which can be divided into four phases:
G1 (presynthetic growth), S (DNA synthesis), G2 (premi-
totic growth), and M (mitotic) phases. This process is regu-
lated by cyclin-dependent kinases (CDKs) and their cognate
cyclins, along with their endogenous inhibitors (CDKIs;
Malumbre & Barbacid, 2005, 2009). Deregulation of the
cell cycle occurs in the pathogenesis of various human dis-
eases, especially cancers, where CDKs, cyclins, and endog-
enous CDKIs are deeply involved. CDKs play key roles in
the regulation of the cell cycle (Denicourt & Dowdy, 2004;
Deshpande et al., 2005). Particularly, CDKs were found
to play an important part in tumorigenesis directly or
through signaling cascades indirectly (Uziel et al., 2006).
Hence, CDKs have become promising anti-cancer tar-
gets and it is believed that the inhibition of CDKs could
Since unraveling the mechanism of cell cycle regula-
tion in the early 1970s, cell cycle checkpoint has become
an important target for anti-cancer drug development
Chem Biol Drug Des. 2021;00:1–16.
wileyonlinelibrary.com/journal/cbdd
© 2021 John Wiley & Sons Ltd.
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MOU et al.
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effectively suppress tumor growth (Asghar et al., 2015;
Jorda et al., 2018; Roskoski, 2016, 2019). All CDKs have
two-lobed structures: N-terminal lobe rich in β sheets and
C-terminal lobe rich in α helices. These two lobes are joined
together by a hinge polypeptide strand. The cleft between
the two lobes is the binding site of ATP. Most inhibitors
developed nowadays belong to ATP-competitive inhib-
itors (Pavletich, 1999). Because of the similarity of ATP-
binding sites between different CDKs (Węsierska-Gądek
et al., 2009), most potent compounds entered clinical trials
in the early stage; for example, alvocidib (flavopiridol), mil-
ciclib (PHA-848125), roniciclib (BAY-1000394), AT7519,
and TG02 (SB-1317; Figure 1) are pan-CDK inhibitors
(Mou et al., 2020). Owing to the complex mechanisms of
pan-CDK inhibitors, they usually caused side-effects in-
cluding hepatic dysfunction, nausea, vomiting, and fatigue.
So development of selective small molecular CDK inhibi-
tors is necessary and urgent (Huwe et al., 2003).
CDK1 controls the entry from G2 phase to M phase in
mammalian cells (Shapiro, 2006). It is also reported that
CDK1 can drive all the events that are required in cell cycle
in the absence of interphase CDKs (CDK2, 3, 4 and 6;
Santamaría et al., 2007; Vassilev et al., 2006). Hence, CDK1
became a novel target for exploitation of selective CDK in-
hibitors as targeted anti-cancer drugs.
The co-crystal structures of CDK1/cyclin B-Cks2 with
several CDK inhibitors were resolved by Noble and Martin
et al. (Wood et al., 2019). In the co-crystal complex of CDK1/
cyclin B-Cks2 with flavopiridol (PDB: 6GU2), the chromone
core of flavopiridol is sandwiched between A31 and L135,
forms two hydrogen bonds with the main-chain amide of L83
and carbonyl of E81 in the hinge region, and forms hydropho-
bic interactions with the gatekeeper residue F80 of CDK1,
while the piperidinol moiety forms network of interactions
with K33 and D146. Finally, the chlorophenyl group of flavo-
piridol forms hydrophobic interactions with V18 and I10
(Wood et al., 2019). The binding mode of flavopiridol with
CDK1/cyclin B-Cks2 provides the fundament for the design
of CDK1 inhibitors with flavonoid scaffold.
Baicalein (Figure 1) is a natural flavonoid isolated from
the root of Scutellaria baicalensis Georgi or from baicalin
by hydrolysis (Shen et al., 2003). It can induce cell apoptosis
and cell cycle arrest by downregulating CDK1, CDK2, cy-
clin D2, and cyclin A and upregulating CDKIs in G1 and G2
phases, and also by downregulating the expression of CDK4/
cyclins B and D (Eichhorn & Efferth, 2012). Two compounds
with flavonoid scaffold, flavopiridol and P276-00 (Figure 1),
derived from the natural product rohitukine (Mahajan
et al., 2015; Figure 1) showed very strong CDK inhibitory
activities (Blachly et al., 2016; Cassaday et al., 2015) and
are currently used in the clinical trials for tumor treatment.
So we selected baicalein as lead compound and docked it to
the ATP-binding site of CDK1/cyclin B-Cks2 (PDB: 6GU2;
Figure 2) to determine its structural modification protocol
as a CDK1 inhibitor. The results showed that the chromone
core of baicalein can be accommodated to the hinge region
of CDK1 with the help of two hydrogen bonds with E81 and
L83. And it also forms hydrophobic interaction with V18,
A31 and L135. The phenyl group forms hydrophobic inter-
action with I10. But it does not form any interaction with the
FIGURE 1 FIGURE Selected
chemical structures of CDK inhibitors

MOU et al.
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FIGURE 2 (a) Binding mode of baicalein with CDK1 (baicalein in yellow, while flavopiridol in pale). (b) Interactions between baicalein and
CDK1's binding site
important D146 of CDK1 compared with flavopiridol. The
main reason is that there are no other hydrogen bond donors
substitute with various of aminomethyl
groups to interact with D146 and other
or acceptors with large volume on baicalein's A ring except
amino acid residues of CDK1
for hydroxyl groups.
B
Accordingly, in our subsequent structural modification
8
1
O
C
HO
HO
7
process, the key chromone core and phenyl group of baicalein
were kept, and hydrophobic groups to position-6 or position-7
and amine methylenes to position-8 were introduced to baica-
lein's A ring (Figure 3). It is expected that after modification,
the introduced substituent groups of baicalein could form hy-
drogen bonds with D146 and hydrophobic interactions with
other amino acid residues of CDK1, so as to improve baica-
lein derivatives' inhibitory activities toward CDK1.
2
3
A
6
substitute with hydrophobic groups
on 6- or 7-OH to interact with the
binding site of CDK1
4
5
OH
O
keep
FIGURE 3 Modification strategy of baicalein
In this paper, we choose CDK1 as the target and baica-
lein as the lead compound to modify the A ring of baicalein
based on the docking results (Figure 2). And then, we found
a new kind of CDK1 inhibitors with flavonoid as scaffold
through biological activity evaluations and structure–activity
relationship (SAR) assessment.
Technology Co., Ltd, Wuhan, China. Molecular docking was
conducted on Discovery Studio, version 5.
2.1.2
Synthesis procedures
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Synthesis of compounds 1
2
EXPERIMENTAL SECTION
To a solution of baicalein (4 mmol) in 30 ml methanol was
added 37% formaldehyde solution (6 mmol), one kind of sec-
ondary amine (4.8 mmol) in turn (Zhang et al., 2008). After
that, the mixture was stirred at 30–70°C. The progress of the
reaction was monitored by TLC until the reaction is com-
pleted and a large amount of yellow precipitate appeared.
The yellow precipitate was filtered under vacuum, washed
with a small amount of methanol, and dried in a vacuum oven
to obtain the product.
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2.1
Chemistry
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2.1.1
Reagents and instruments
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All solvents were of analytical reagents, commercially
available, and used without further purification. Thin-layer
chromatography (TLC) with silica gel precoated glass and
1
fluorescent indicator was used to monitor the reactions. H-
5,6,7-Trihydroxy-8-(morpholinomethyl)-2-phenyl-4H-
chromen-4-one (1a). Yellow powder, yield 93.5%. 1H-NMR
(DMSO-d6, 600 MHz) δ: 8.08–8.10 (m, 2H, H-2ʹ,6ʹ), 7.58–
7.63 (m, 3H, H-3ʹ,4ʹ,5ʹ), 6.96 (s, 1H, H-3), 3.95 (s, 2H, H-9),
3.62 (brs, 4H, H-11,12), 2.61 (brs, 4H, H-10,13). 13C-NMR
(DMSO-d6, 150 MHz) δ: 182.7 (C-4), 163.0 (C-2), 154.7 (C-
7), 148.5 (C-8a), 146.4 (C-5), 132.3 (C-4ʹ), 131.6 (C-1ʹ), 129.7
(C-3ʹ,5ʹ), 129.4 (C-6), 126.7 (C-2ʹ,6ʹ), 104.9 (C-4a), 104.1 (C-
3), 100.6 (C-8), 66.5 (C-11,12), 53.1 (C-10,13), 52.0 (C-9).
and 13C-NMR spectra were recorded on a Bruker AV-III-600
instrument. High-resolution mass spectral (MS) data were
determined on an Agilent 6540 UHD accurate mass Q-TOF/
MS instrument in a low-resonance electrospray mode (ESI).
CCK-8waspurchasedfromDojindoMolecularTechnologies,
Inc., Kumamoto, Japan. MCF-7 cells were purchased from
Lanmeng Biomedical Technology Co., Ltd, Hebei, China.
CDK1/cyclin B kinase was provided by Chundu Biomedical

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MOU et al.
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5,6,7-Trihydroxy-2-phenyl-8-(thiomorpholinomethyl)-4H-
chromen-4-one (1b). Yellow powder, yield 88.7%. 1H-NMR
(DMSO-d6, 600 MHz) δ: 12.78 (s, 1H, 5-OH), 8.08–8.09 (m,
2H, H-2ʹ,6ʹ), 7.59–7.63 (m, 3H, H-3ʹ,4ʹ,5ʹ), 6.95 (s, 1H, H-3),
3.97 (s, 2H, H-9), 2.87 (t, J = 5.0 Hz, 4H, H-10,13), 2.67 (t,
J = 5.0 Hz, 4H, H-11,12). 13C-NMR (DMSO-d6, 150 MHz)
δ: 182.7 (C-4), 163.0 (C-2), 154.9 (C-7), 148.5 (C-8a), 146.4
(C-5), 132.3 (C-4ʹ), 131.6 (C-1ʹ), 129.7 (C-6), 129.4 (C-3ʹ,5ʹ),
126.8 (C-2ʹ,6ʹ), 104.9 (C-4a), 104.1 (C-3), 100.7 (C-8), 54.4
(C-10,13), 52.6 (C-9), 27.6 (C-11,12).
8-((Dipropylamino) methyl)-5,6,7-trihydroxy-2-phenyl-4H-
chromen-4-one (1g). Yellow powder, yield 92.4%. 1H-
NMR (DMSO-d6, 600 MHz) δ: 12.57 (s, 1H, 5-OH), 8.04–
8.06 (m, 2H, H-2ʹ,6ʹ), 7.56–7.61 (m, 3H, H-3ʹ,4ʹ,5ʹ), 6.85 (s,
1H, H-3), 4.27 (s, 2H, H-9), 2.77 (t, J = 6.0 Hz, 4H, H-10,13),
1.64 (m, 4H, H-11,14), 0.88 (t, J = 6.0 Hz, 6H, H-12,15).
13C-NMR (DMSO-d6, 150 MHz) δ: 181.7 (C-4), 161.8 (C-2),
160.7 (C-7), 148.8 (C-8a), 144.9 (C-5), 132.0 (C-4ʹ), 131.8
(C-1ʹ), 130.3 (C-6), 129.6 (C-3ʹ,5), 126.6 (C-2ʹ,6ʹ), 105.0 (C-
4a), 102.3 (C-3), 97.7 (C-8), 54.8 (C-10,13), 50.4 (C-9), 18.7
(C-11,14), 11.8 (C-12,15).
5,6,7-Trihydroxy-8-((4-methylpiperazin-1-yl)methyl)-2-
phenyl-4H-chromen-4-one (1c). Yellow powder, yield
Synthesis of compound 2
1
89.6%. H-NMR (DMSO-d6, 600 MHz) δ: 8.07–8.08 (m,
To a solution of baicalein (4 mmol) in 30 ml dimethylforma-
mide (DMF) were added bromobenzyl (5.2 mmol, 0.62 ml),
potassium carbonate (12 mmol), and potassium iodide
(12 mmol) successively (Gao et al., 2015). The mixture was
stirred for 7 hr under nitrogen atmosphere at 60–70°C. After
that, the mixture was filtered under vacuum and a few drops
of formic acid were added. The filtrate was then evaporated
under vacuum and dispersed in cold water to form a suspen-
sion. The suspension was neutralized and filtered to obtain
the crude product. The crude product was purified by silica
gel column chromatography and recrystallization.
2H, H-2ʹ,6ʹ), 7.57–7.61 (m, 3H, H-3ʹ,4ʹ,5ʹ), 6.93 (s, 1H, H-3),
4.02 (s, 2H, H-9), 2.69 (brs, 4H, H-10,13), 2.51 (brs, 4H, H-
11,12), 2.20 (s, 3H, H-14). 13C-NMR (DMSO-d6, 150 MHz)
δ: 182.4 (C-4), 162.6 (C-2), 156.2 (C-7), 148.4 (C-8a), 146.0
(C-5), 132.2 (C-4ʹ), 131.6 (C-1ʹ), 131.4 (C-6), 129.6 (C-3ʹ,5ʹ),
126.7 (C-2ʹ,6ʹ), 104.9 (C-4a), 103.7 (C-3), 100.0 (C-8), 54.7
(C-10,13), 52.2 (C-11,12), 45.9 (C-9), 40.0 (C-14).
2-Phenyl-8-((pyrrolidin-1-yl) methyl)-4H-chromene-5,6,7-
triol (1d). Yellow powder, yield 92.5%. 1H-NMR (DMSO-
d6, 600 MHz) δ: 8.05–8.06 (m, 2H, H-2ʹ,6ʹ), 7.57–7.58 (m,
3H, H-3ʹ,4ʹ,5ʹ), 6.81 (s, 1H, H-3), 4.35 (s, 2H, H-9), 3.12
(brs, 4H, H-10,13), 1.90 (brs, 4H, H-11,12). 13C-NMR
(DMSO-d6, 150 MHz) δ: 180.9 (C-4), 163.8 (C-2), 161.0
(C-7), 150.1 (C-8a), 143.7 (C-5), 132.0 (C-4ʹ), 131.7 (C-1ʹ),
130.7 (C-6), 129.6 (C-3ʹ,5ʹ), 126.5 (C-2ʹ,6ʹ), 104.9 (C-4a),
101.0 (C-3), 97.2 (C-8), 53.2 (C-10,13), 49.7 (C-9), 23.4
(C-11,12).
7-(Benzyloxy)-5,6-dihydroxy-2-phenyl-4H-chromen-4-one
(2). Yellow crystal, yield 65%. 1H-NMR (DMSO-d6,
600 MHz) δ: 12.57 (s, 1H, 5-OH), 8.82 (s, 1H, 6-OH), 8.05–
8.07 (m, 2H, H-2ʹ,6ʹ), 7.54–7.62 (m, 5H, H-3ʹ,4ʹ,5ʹ,3ʺ,5ʺ),
7.42–7.45 (m, 2H, H-2ʺ,6ʺ), 7.35–7.38 (m, 1H, H-4ʺ), 7.04
(s, 1H, H-8), 6.97 (s, 1H, H-3), 5.30 (s, 2H, H-9). 13C-NMR
(DMSO-d6, 150 MHz) δ: 182.2 (C-4), 163.1 (C-2), 153.5
(C-7), 149.6 (C-8a), 146.4 (C-5), 136.2 (C-1ʺ), 131.8 (C-4ʹ),
130.8 (C-1ʹ), 130.4 (C-6), 129.0 (C-3ʹ,5ʹ), 128.4 (C-3ʺ,5ʺ),
127.9 (C-4ʺ), 127.7 (C-2ʺ,6ʺ), 126.2 (C-2ʹ,6ʹ), 105.3 (C-
4a), 104.6 (C-3), 92.4 (C-8), 70.2 (C-9). HRMS (ESI) m/z
[M + H]+ Calcd for C22H17O5 361.1076 found 361.1077.
8-((2H-Pyrrol-1(5H)-yl) methyl)-5,6,7-trihydroxy-2-phenyl-
1
4H-chromen-4-one (1e). Yellow powder, yield 91.8%. H-
NMR (DMSO-d6, 600 MHz) δ: 8.05–8.07 (m, 2H, H-2ʹ,6ʹ),
7.56–7.61 (m, 3H, H-3ʹ,4ʹ,5ʹ), 6.88 (s, 1H, H-3), 5.89 (brs,
2H, H-11,12), 4.38 (s, 2H, H-9), 3.80 (brs, 4H, H-10,13).
13C-NMR (DMSO-d6, 150 MHz) δ: 181.8 (C-4), 161.9 (C-2),
159.7 (C-7), 149.0 (C-8a), 145.1 (C-5), 132.0 (C-4ʹ), 131.8
(C-1ʹ), 130.2 (C-6), 129.6 (C-3ʹ,5ʹ), 127.0 (C-2ʹ,6ʹ), 126.6 (C-
11,12), 104.9 (C-4a), 102.5 (C-3), 99.1 (C-8), 59.4 (C-10,13),
50.3 (C-9).
Synthesis of compounds 3
To a solution of different carboxylic acid (2.2 mmol) in 30 ml
anhydrous THF was added HOBt (2.6 mmol) under −5°C
(Mou et al., 2009). DCC (2.6 mmol) dissolved in 20 ml of an-
hydrous THF was dropped into the above reaction solution.
The mixture was stirred at −5°C for 12 hr to get the active
ester and then filtered under vacuum. The filtrate would be
used in the next step.
8-((Dimethylamino) methyl)-5,6,7-trihydroxy-2-phenyl-4H-
chromen-4-one (1f). Yellow powder, yield 89.8%. 1H-NMR
(DMSO-d6, 600 MHz) δ: 8.05–8.07 (m, 2H, H-2ʹ,6ʹ), 7.56–
7.59 (m, 3H, H-3ʹ,4ʹ,5ʹ), 6.82 (s, 1H, H-3), 4.21 (s, 2H, H-9),
2.61 (s, 6H, H-10,11). 13C-NMR (DMSO-d6, 150 MHz) δ:
181.2 (C-4), 162.6 (C-2), 161.3 (C-7), 149.8 (C-8a), 144.2
(C-5), 131.9 (C-4ʹ), 131.8 (C-1ʹ), 130.5 (C-6), 129.6 (C-3ʹ,5ʹ),
126.5 (C-2ʹ,6ʹ), 104.9 (C-4a), 101.5 (C-3), 97.0 (C-8), 53.5
(C-9), 43.2 (C-10,11).
To a solution of one of compounds 1 (2.2 mmol) in 50 ml
anhydrous THF were added dimethylaminopyridine (DMAP,
0.43 mmol) and triethylamine (0.8 ml) in turn under the
stirring condition. Then, the above active ester filtrate was
dropped into the reaction solution slowly. After completion
(monitored by TLC), the organic solvent was removed and
the residue was purified by silica gel column chromatography

MOU et al.
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to get the corresponding compound 3. (The eluant was di-
chloromethane: methanol; sometimes, further recrystalliza-
tion was needed.)
6.92 (d, J = 16.1 Hz, 1H, H-15), 4.16 (s, 2H, H-9), 3.69 (brs,
4H, H-12,13), 2.80 (brs, 4H, H-11,14). 13C-NMR (DMSO-d6,
150 MHz) δ: 182.4 (C-4), 164.4 (C-10), 163.3 (C-2), 160.2
(C-7), 152.7 (C-8a), 152.1 (C-5), 146.8 (C-16), 134.4 (C-
1ʺ), 132.5 (C-4ʹ), 131.3 (C-1ʹ), 131.0 (C-4ʺ), 129.7 (C-3ʹ,5ʹ),
129.5 (C-3ʺ,5ʺ), 129.1 (C-2ʺ,6ʺ), 126.9 (C-2ʹ,6ʹ), 122.9 (C-6),
117.4 (C-15), 105.2 (C-4a), 103.0 (C-3), 99.5 (C-8), 65.9 (C-
12,13), 52.7 (C-9), 52.4 (C-11,14). HRMS (ESI) m/z [(M-
acid) + H]+ Calcd for C20H20NO6 370.1291 found 370.1286.
5,6-Dihydroxy-8-(morpholinomethyl)-4-oxo-2-phenyl-4H-
chromen-7-yl benzoate (3a). Yellow powder, yield 21.2%.
1H-NMR (DMSO-d6, 600 MHz) δ: 13.17 (s, 1H, 5-OH),
8.12–8.13 (m, 4H, H-2ʹ,6ʹ,2ʺ,6ʺ), 7.72–7.79 (m, 1H, H-4ʺ),
7.57–7.65 (m, 5H, H-3ʹ,4ʹ,5ʹ,3ʺ,5ʺ), 7.01 (s, 1H, H-3), 4.17
(s, 2H, H-9), 3.68 (brs, 4H, H-12,13), 2.81 (brs, 4H, H-
11,14). 13C-NMR (DMSO-d6, 150 MHz) δ: 182.3 (C-4),
164.1 (C-10), 163.2 (C-2), 160.7 (C-7), 152.8 (C-8a), 152.1
(C-5), 134.4 (C-4ʺ), 132.5 (C-4ʹ), 131.4 (C-1ʹ), 130.3 (C-
2ʺ,6ʺ), 129.7 (C-3ʹ,5ʹ), 129.4 (C-3ʺ,5ʺ), 129.2 (C-1ʺ), 126.9
(C-2ʹ,6ʹ), 123.1 (C-6), 105.2 (C-4a), 102.7 (C-3), 99.5 (C-8),
65.8 (C-12,13), 52.7 (C-9), 52.4 (C-11,14). HRMS (ESI) m/z
[M + H]+ Calcd for C27H24NO7 474.1553 found 474.1549.
5,6-Dihydroxy-8-(morpholinomethyl)-4-oxo-2-phenyl-4H-
chromen-7-yl methacrylate (3e). Yellow powder, yield
19.5%. 1H-NMR (DMSO-d6, 600 MHz) δ: 8.10–8.11 (m, 2H,
H-2ʹ,6ʹ), 7.59–7.64 (m, 3H, H-3ʹ,4ʹ,5ʹ), 6.99 (s, 1H, H-3), 6.29
(s, 1H, H-16E), 5.90 (s, 1H, H-16Z), 4.14 (s, 2H, H-9), 3.69
(brs, 4H, H-12,13), 2.79 (brs, 4H, H-11,14), 2.01 (s, 3H, H-
17). 13C-NMR (DMSO-d6, 150 MHz) δ: 182.3 (C-4), 164.7
(C-10), 163.2 (C-2), 160.2 (C-7), 152.6 (C-8a), 152.0 (C-
5), 135.4 (C-15), 132.5 (C-4ʹ), 131.3 (C-1ʹ), 129.7 (C-3ʹ,5ʹ),
128.2 (C-6), 126.9 (C-2ʹ,6ʹ), 122.9 (C-16), 105.2 (C-4a),
102.9 (C-3), 99.5 (C-8), 65.9 (C-12,13), 52.7 (C-9), 52.4 (C-
11,14), 18.6 (C-17). HRMS (ESI) m/z [(M-acid) + H]+ Calcd
for C20H20NO6 370.1291 found 370.1294.
5,6-Dihydroxy-8-(morpholinomethyl)-4-oxo-2-phenyl-4H-
chromen-7-yl 2-bromobenzoate (3b). Yellow crystal, yield
1
59.4%. H-NMR (DMSO-d6, 600 MHz) δ: 13.20 (s, 1H, 5-
OH), 8.12–8.15 (m, 3H, H-2ʹ,6ʹ,6ʺ), 7.85–7.86 (m, 1H, H-3ʺ),
7.57–7.65 (m, 5H, H-3ʹ,4ʹ,5ʹ,4ʺ,5ʺ), 7.01 (s, 1H, H-3), 4.21 (s,
2H, H-9), 3.70 (brs, 4H, H-12,13), 2.87 (brs, 4H, H-11,14).
13C-NMR (DMSO-d6, 150 MHz) δ: 182.2 (C-4), 163.1 (C-
2,10), 161.1 (C-7), 153.1 (C-8a), 152.2 (C-5), 135.1 (C-4ʺ),
134.6 (C-1ʺ), 132.6 (C-3ʺ), 132.4 (C-6ʺ), 131.4 (C-4ʹ), 130.7
(C-1ʹ), 129.7 (C-3ʹ,5ʹ), 128.4 (C-6), 126.9 (C-2ʹ,6ʹ), 123.0 (C-
5ʺ), 122.0 (C-2ʺ), 105.2 (C-4a), 102.5 (C-3), 99.3 (C-8), 65.7
(C-12,13), 52.6 (C-9), 52.3 (C-11,14). HRMS (ESI) m/z [(M-
acid) + H]+ Calcd for C20H20NO6 370.1291 found 370.1284.
5,6-Dihydroxy-4-oxo-2-phenyl-8-(thiomorpholinomethyl)-
4H-chromen-7-yl benzoate (3f). Yellow crystal, yield
1
70.1%. H-NMR (DMSO-d6, 600 MHz) δ: 13.14 (s, 1H, 5-
OH), 8.12–8.14 (m, 4H, H-2ʺ,6ʺ,2ʹ,6ʹ), 7.75–7.77 (m, 1H,
H-4ʺ), 7.60–7.64 (m, 5H, H-3ʹ,4ʹ,5ʹ,3ʺ,5ʺ), 7.01 (s, 1H, H-3),
4.20 (s, 2H, H-9), 3.06 (t, J = 5.0 Hz, 4H, H-11,14), 2.75 (t,
J = 5.0 Hz, 4H, H-12,13). 13C-NMR (DMSO-d6, 150 MHz)
δ: 181.7 (C-4), 163.5 (C-10), 162.6 (C-2), 160.6 (C-7), 152.3
(C-8a), 151.5 (C-5), 133.8 (C-4ʺ), 131.9 (C-4ʹ), 130.8 (C-1ʹ),
129.7 (C-2ʺ,6ʺ), 129.1 (C-3ʹ,5ʹ), 128.8 (C-3ʺ,5ʺ), 128.6 (C-1ʺ),
126.4 (C-2ʹ,6ʹ), 122.5 (C-6), 104.7 (C-4a), 102.0 (C-3), 98.8
(C-8), 54.8 (C-11,14), 53.3 (C-9), 26.2 (C-12,13). HRMS
(ESI) m/z [M + H]+ Calcd for C27H24NO6S 490.1324 found
490.1346.
5,6-Dihydroxy-8-(morpholinomethyl)-4-oxo-2-phenyl-4H-
chromen-7-yl 4-methoxybenzoate (3c). Yellow crystal,
yield 70.3%. 1H-NMR (DMSO-d6, 600 MHz) δ: 13.07 (s, 1H,
5-OH), 8.12–8.13 (m, 2H, H-2ʹ,6ʹ), 8.06 (d, J = 12.0 Hz, 2H,
H-2ʺ,6ʺ), 7.60–7.65 (m, 3H, H-3ʹ,4ʹ,5ʹ), 7.09 (d, J = 12.0 Hz,
2H, H-3ʺ,5ʺ), 7.02 (s, 1H, H-3), 4.17 (s, 2H, H-9), 3.86 (s, 3H,
H-15), 3.67 (brs, 4H, H-12,13), 2.81 (brs, 4H, H-11,14). 13C-
NMR (DMSO-d6, 150 MHz) δ: 182.3 (C-4), 164.1 (C-10),
163.7 (C-4ʺ), 163.2 (C-2), 160.7 (C-7), 152.7 (C-8a), 152.2
(C-5), 132.5 (C-1ʺ,2ʺ,6ʺ), 131.4 (C-6), 129.7 (C-3ʹ,5ʹ), 126.9
(C-2ʹ,6ʹ), 123.2 (C-4ʹ), 121.3 (C-1ʹ), 114.6 (C-3ʺ,5ʺ), 105.2
(C-4a), 102.8 (C-3), 99.5 (C-8), 65.8 (C-15), 56.1 (C-12,13),
52.7 (C-9), 52.4 (C-11,14). HRMS (ESI) m/z [M + H]+ Calcd
for C28H26NO8 504.1658 found 504.1692.
5,6-Dihydroxy-4-oxo-2-phenyl-8-(thiomorpholinomethyl)-
4H-chromen-7-yl 2-bromobenzoate (3g). Light yellow
1
powder, yield 66.5%. H-NMR (DMSO-d6, 600 MHz) δ:
13.18 (s, 1H, 5-OH), 8.12–8.15 (m, 3H, H-2ʹ,6ʹ,6ʺ), 7.83–
7.86 (m, 1H, H-3ʺ), 7.57–7.64 (m, 5H, H-3ʹ,4ʹ,5ʹ,4ʺ,5ʺ), 7.00
(s, 1H, H-3), 4.23 (s, 2H, H-9), 3.11 (brs, 4H, H-11,14), 2.78
(brs, 4H, H-12,13). 13C-NMR (DMSO-d6, 150 MHz) δ: 182.1
(C-4), 163.1 (C-10), 163.0 (C-2), 161.6 (C-7), 153.1 (C-8a),
152.2 (C-5), 135.0 (C-4ʺ), 134.5 (C-1ʺ), 132.6 (C-3ʺ), 132.4
(C-6ʺ), 131.4 (C-4ʹ), 130.7 (C-1ʹ), 129.7 (C-3ʹ,5ʹ), 128.4 (C-
6), 126.9 (C-2ʹ,6ʹ), 123.1 (C-5ʺ), 121.9 (C-2ʺ), 105.2 (C-4a),
102.3 (C-3), 99.1 (C-8), 53.8 (C-11,14), 53.2 (C-9), 26.6 (C-
12,13). HRMS (ESI) m/z [M + H]+ Calcd for C27H23BrNO6S
568.0429 found 568.0315.
(2E)-5,6-Dihydroxy-8-(morpholinomethyl)-4-oxo-2-phenyl-
4H-chromen-7-yl cinnamate (3d). Yellow powder, yield
1
50.2%. H-NMR (DMSO-d6, 600 MHz) δ: 13.15 (s, 1H, 5-
OH), 8.11–8.12 (m, 2H, H-2ʹ,6ʹ), 7.86 (d, J = 16.1 Hz, 1H,
H-16), 7.80–7.82 (m, 2H, H-2ʺ,6ʺ), 7.59–7.64 (m, 3H, H-
3ʹ,4ʹ,5ʹ), 7.44–7.47 (m, 3H, H-3ʺ,4ʺ,5ʺ), 7.01 (s, 1H, H-3),

6
MOU et al.
|
5,6-Dihydroxy-4-oxo-2-phenyl-8-(thiomorpholinomethyl)-
12,13), 2.23 (s, 3H, H-15). 13C-NMR (DMSO-d6, 150 MHz)
δ: 181.7 (C-4), 164.1 (C-10), 163.5 (C-2), 162.6 (C-7), 153.1
(C-8a), 152.1 (C-5), 134.3 (C-4ʺ), 132.3 (C-4ʹ), 131.5 (C-1ʹ),
130.2 (C-2ʺ,6ʺ), 129.6 (C-3ʹ,5ʹ), 129.4 (C-3ʺ,5ʺ), 129.4 (C-1ʺ),
126.8 (C-2ʹ,6ʹ), 123.7 (C-6), 106.5 (C-4a), 105.1 (C-3), 101.4
(C-8), 53.5 (C-11,14), 51.4 (C-9), 50.2 (C-12,13), 45.4 (C-
15). HRMS (ESI) m/z [(M-acid) + H]+ Calcd for C21H23N2O5
383.1607 found 383.1622.
4H-chromen-7-yl 4-methoxybenzoate (3h). Light yellow
1
powder, yield 68.3%. H-NMR (DMSO-d6, 600 MHz) δ:
13.09 (s, 1H, 5-OH), 8.13 (d, J = 12.0 Hz, 2H, H-2ʺ,6ʺ), 8.03–
8.04 (m, 2H, H-2ʹ,6ʹ), 7.60–7.65 (m, 3H, H-3ʹ,4ʹ,5ʹ), 7.06 (d,
J = 12.0 Hz, 2H, H-3ʺ,5ʺ), 7.02 (s, 1H, H-3), 4.19 (s, 2H, H-
9), 3.84 (s, 3H, H-15), 3.08 (brs, 4H, H-11,14), 2.74 (brs, 4H,
H-12,13). 13C-NMR (DMSO-d6, 150 MHz) δ: 182.2 (C-4),
164.0 (C-10), 163.7 (C-4ʺ), 163.0 (C-2), 161.4 (C-7), 152.8
(C-8a), 152.2 (C-5), 132.4 (C-2ʺ,6ʺ,4ʹ), 131.4 (C-1ʹ), 129.7
(C-3ʹ,5ʹ), 126.9 (C-2ʹ,6ʹ), 123.3 (C-6), 121.3 (C-1ʺ), 114.6
(C-3ʺ,5ʺ), 105.2 (C-4a), 102.6 (C-3), 99.4 (C-8), 56.1 (C-15),
53.9 (C-11,14), 53.3 (C-9), 26.7 (C-12,13). HRMS (ESI) m/z
[M + H]+ Calcd for C28H26NO7S 520.1430 found 520.1440.
5,6-Dihydroxy-8-((4-methylpiperazin-1-yl)methyl)-4-oxo-
2-phenyl-4H-chromen-7-yl 2-bromobenzoate (3l). Light
yellow powder, yield 32.8%. 1H-NMR (DMSO-d6, 600 MHz)
δ: 13.23 (s, 1H, 5-OH), 8.08–8.11 (m, 3H, H-2ʹ,6ʹ,6ʺ), 7.83–
7.84 (m, 1H, H-3ʺ), 7.54–7.63 (m, 5H, H-3ʹ,4ʹ,5ʹ,4ʺ,5ʺ), 6.94
(s, 1H, H-3), 4.25 (s, 2H, H-9), 2.97 (brs, 4H, H-11,14), 2.51
(brs, 4H, H-12,13), 2.24 (s, 3H, H-15). 13C-NMR (DMSO-d6,
150 MHz) δ: 181.6 (C-4), 164.2 (C-10), 163.2 (C-2), 162.4
(C-7), 153.4 (C-8a), 152.1 (C-5), 135.0 (C-4ʺ), 134.4 (C-1ʺ),
132.5 (C-3ʺ), 132.2 (C-6ʺ), 131.5 (C-4ʹ), 131.1 (C-1ʹ), 129.6
(C-3ʹ,5ʹ), 128.4 (C-6), 126.8 (C-2ʹ,6ʹ), 123.7 (C-5ʺ), 121.8 (C-
2ʺ), 105.1 (C-4a), 101.0 (C-3), 98.6 (C-8), 53.3 (C-11,14),
52.1 (C-9), 51.4 (C-12,13), 45.4 (C-15). HRMS (ESI) m/z
[(M-acid) + H]+ Calcd for C21H23N2O5 383.1607 found
383.1694.
( 2 E ) - 5 , 6 - D i h y d r o x y - 4 - o x o - 2 - p h e n y l - 8 -
(thiomorpholinomethyl)-4H-chromen-7-yl
cinnamate
1
(3i). Light yellow crystal, yield 69.5%. H-NMR (DMSO-
d6, 600 MHz) δ: 13.11 (s, 1H, 5-OH), 8.11–8.13 (m, 2H, H-
2ʹ,6ʹ), 7.86 (d, J = 16.1 Hz, 1H, H-16), 7.80–7.82 (m, 2H,
H-2ʺ,6ʺ), 7.59–7.65 (m, 3H, H-3ʹ,4ʹ,5ʹ), 7.45–7.48 (m, 3H,
H-3ʺ,4ʺ,5ʺ), 7.01 (s, 1H, H-3), 6.91 (d, J = 16.1 Hz, 1H, H-
15), 4.18 (s, 2H, H-9), 3.04 (brs, 4H, H-11,14), 2.76 (brs, 4H,
H-12,13). 13C-NMR (DMSO-d6, 150 MHz) δ: 182.3 (C-4),
164.4 (C-10), 163.2 (C-2), 160.6 (C-7), 152.7 (C-8a), 152.1
(C-5), 146.8 (C-16), 134.4 (C-1ʺ), 132.5 (C-4ʹ), 131.3 (C-1ʹ),
131.3 (C-4ʺ), 129.7 (C-3ʹ,5ʹ), 129.5 (C-3ʺ,5ʺ), 129.1 (C-2ʺ,6ʺ),
126.9 (C-2ʹ,6ʹ), 123.0 (C-6), 117.4 (C-15), 105.2 (C-4a),
102.8 (C-3), 99.4 (C-8), 53.9 (C-11,14), 53.3 (C-9), 26.8 (C-
12,13). HRMS (ESI) m/z [M + H]+ Calcd for C29H26NO6S
516.1481 found 516.1505.
5,6-Dihydroxy-8-((4-methylpiperazin-1-yl)methyl)-4-oxo-2-
phenyl-4H-chromen-7-yl 4-methoxybenzoate (3m). Yellow
crystal, yield 50.9%. 1H-NMR (DMSO-d6, 600 MHz)
δ:13.08 (s, 1H, 5-OH), 8.11–8.12 (m, 2H, H-2ʹ,6ʹ), 8.02 (d,
J = 12.0 Hz, 2H, H-2ʺ,6ʺ), 7.58–7.64 (m, 3H, H-3ʹ,4ʹ,5ʹ), 7.04
(d, J = 12.0 Hz, 2H, H-3ʺ,5ʺ), 6.97 (s, 1H, H-3), 4.22 (s, 2H,
H-9), 3.83 (s, 3H, H-16), 2.92 (brs, 4H, H-12,13), 2.51 (brs,
4H, H-11,14), 2.20 (s, 3H, H-15). 13C-NMR (DMSO-d6,
150 MHz) δ: 181.7 (C-4), 163.9 (C-10), 163.7 (C-4ʺ), 163.6
(C-2), 162.5 (C-7), 153.0 (C-8a), 152.2 (C-5), 132.3 (C-
1ʺ,2ʺ,6ʺ), 131.5 (C-6), 129.7 (C-3ʹ,5ʹ), 126.8 (C-2ʹ,6ʹ), 123.8
(C-4ʹ), 121.6 (C-1ʹ), 114.6 (C-3ʺ,5ʺ), 105.1 (C-4a), 101.5 (C-
3), 98.8 (C-8), 56.0 (C-16), 53.5 (C-11,14), 52.3 (C-9), 51.6
(C-12,13), 45.6 (C-15). HRMS (ESI) m/z [M + H]+ Calcd for
C29H29N2O7 517.1975 found 517.1980.
5,6-Dihydroxy-4-oxo-2-phenyl-8-(thiomorpholinomethyl)-
4H-chromen-7-yl methacrylate (3j). Light yellow crystal,
1
yield 59.8%. H-NMR (DMSO-d6, 600 MHz) δ: 13.11 (s,
1H, 5-OH), 8.10–8.11 (m, 2H, H-2ʹ,6ʹ), 7.58–7.64 (m, 3H,
H-3ʹ,4ʹ,5ʹ), 6.99 (s, 1H, H-3), 6.23 (s, 1H, H-16E), 5.89 (s,
1H, H-16Z), 4.16 (s, 2H, H-9), 3.03 (brs, 4H, H-11,14), 2.76
(brs, 4H, H-12,13), 2.01 (s, 3H, H-17). 13C-NMR (DMSO-d6,
150 MHz) δ: 182.3 (C-4), 164.7 (C-10), 163.2 (C-2), 160.6
(C-7), 152.7 (C-8a), 152.0 (C-5), 135.4 (C-15), 132.5 (C-4ʹ),
131.3 (C-1ʹ), 129.7 (C-3ʹ,5ʹ), 128.2 (C-6), 126.9 (C-2ʹ,6ʹ),
123.0 (C-16), 105.2 (C-4a), 102.7 (C-3), 99.4 (C-8), 53.9 (C-
11,14), 53.3 (C-9), 26.8 (C-12,13), 18.6 (C-17). HRMS (ESI)
m/z [(M-acid) + H]+ Calcd for C20H20NO5S 386.1062 found
386.1068.
(2E)-5,6-Dihydroxy-8-((4-methylpiperazin-1-yl)methyl)-4-
oxo-2-phenyl-4H-chromen-7-yl cinnamate (3n). Yellow
1
powder, yield 52.9%. H-NMR (DMSO-d6, 600 MHz) δ:
13.13 (s, 1H, 5-OH), 8.09–8.11 (m, 2H, H-2ʹ,6ʹ), 7.83 (d,
J = 16.1 Hz, 1H, H-17), 7.78–7.80 (m, 2H, H-2ʺ,6ʺ), 7.58–
7.64 (m, 3H, H-3ʹ,4ʹ,5ʹ), 7.46–7.47 (m, 3H, H-3ʺ,4ʺ,5ʺ), 6.95
(s, 1H, H-3), 6.88 (d, J = 16.1 Hz, 1H, H-16), 4.20 (s, 2H, H-
9), 2.90 (brs, 4H, H-11,14), 2.51 (brs, 4H, H-12,13), 2.24 (s,
3H, H-15). 13C-NMR (DMSO-d6, 150 MHz) δ: 181.9 (C-4),
164.4 (C-10), 162.7 (C-2), 152.9 (C-7), 152.1 (C-8a), 146.5
(C-5), 134.4 (C-17), 132.3 (C-1ʺ), 131.5 (C-4ʹ), 131.2 (C-1ʹ),
129.7 (C-3ʹ,5ʹ), 129.5 (C-3ʺ,4ʺ,5ʺ), 129.0 (C-2ʺ,6ʺ), 126.8 (C-
5,6-Dihydroxy-8-((4-methylpiperazin-1-yl)methyl)-4-oxo-2-
phenyl-4H-chromen-7-yl benzoate (3k). Yellow powder,
yield 18.9%. 1H-NMR (DMSO-d6, 600 MHz) δ: 13.16 (s, 1H,
5-OH), 8.11–8.13 (m, 4H, H-2ʹ,6ʹ,2ʺ,6ʺ), 7.71–7.77 (m, 1H,
H-4ʺ), 7.58–7.62 (m, 5H, H-3ʹ,4ʹ,5ʹ,3ʺ,5ʺ), 6.95 (s, 1H, H-3),
4.22 (s, 2H, H-9), 2.92 (brs, 4H, H-11,13), 2.51 (brs, 4H, H-

MOU et al.
7
|
2ʹ,6ʹ), 123.4 (C-6), 117.6 (C-16), 105.1 (C-4a), 101.8 (C-3),
98.9 (C-8), 53.7 (C-11,14), 52.3 (C-9), 51.5 (C-12,13), 45.5
(C-15). HRMS (ESI) m/z [M + H]+ Calcd for C30H29N2O6
513.2026 found 513.2063.
13.16 (s, 1H, 5-OH), 8.10–8.13 (m, 2H, H-2ʺ,6ʺ), 8.06–
8.07 (m, 2H, H-2ʹ,6ʹ), 7.72–7.74 (m, 1H, H-4ʺ), 7.57–7.61
(m, 5H, H-3ʹ,4ʹ,5ʹ,3ʺ,5ʺ), 6.85 (s, 1H, H-3), 5.91 (brs, 2H,
H-12,13), 4.48 (s, 2H, H-9), 4.00 (brs, 4H, H-11,14). 13C-
NMR (DMSO-d6, 150 MHz) δ: 180.9 (C-4), 167.4 (C-10),
164.3 (C-2), 161.6 (C-7), 153.9 (C-8a), 152.3 (C-5), 134.0
(C-4ʺ), 132.0 (C-4ʹ), 131.8 (C-1ʹ), 130.1 (C-2ʺ,6ʺ), 129.9 (C-
1ʺ), 129.6 (C-3ʹ,5ʹ), 129.3 (C-3ʺ,5ʺ), 126.7 (C-2ʹ,6ʹ), 125.9
(C-12,13), 124.8 (C-6), 105.0 (C-4a), 99.3 (C-3), 98.1 (C-8),
59.4 (C-11,14), 51.0 (C-9). HRMS (ESI) m/z [M + H]+ Calcd
for C27H22NO6 456.1447 found 456.1469.
5,6-Dihydroxy-8-((4-methylpiperazin-1-yl)methyl)-4-oxo-2-
phenyl-4H-chromen-7-yl methacrylate (3o). Light yellow
1
powder, yield 40.3%. H-NMR (DMSO-d6, 600 MHz) δ:
13.06 (s, 1H, 5-OH), 7.08–7.10 (m, 2H, H-2ʹ,6ʹ), 7.57–7.63
(m, 3H, H-3ʹ,4ʹ,5ʹ), 6.93 (s, 1H, H-3), 6.26 (s, 1H, H-17E),
5.87 (s, 1H, H-17Z), 4.20 (s, 2H, H-9), 2.89 (brs, 4H, H-
11,14), 2.51 (brs, 4H, H-12,13), 2.23 (s, 3H, H-15), 2.00 (s,
H, H-18). 13C-NMR (DMSO-d6, 150 MHz) δ: 181.8 (C-4),
164.8 (C-10), 163.0 (C-2), 162.6 (C-7), 152.9 (C-8a), 152.0
(C-5), 135.6 (C-16), 132.3 (C-4ʹ), 131.5 (C-1ʹ), 129.6 (C-
3ʹ,5ʹ), 127.9 (C-6), 126.8 (C-2ʹ,6ʹ), 123.5 (C-17), 105.1 (C-
4a), 101.6 (C-3), 98.7 (C-8), 53.7 (C-11,14), 52.4 (C-9), 51.5
(C-12,13), 45.6 (C-15), 18.7 (C-18). HRMS (ESI) m/z [(M-
acid) + H]+ Calcd for C21H23N2O5 383.1607 found 383.1607.
8-((2H-Pyrrol-1(5H)-yl)methyl)-5,6-dihydroxy-4-oxo-2-
phenyl-4H-chromen-7-yl 4-methoxybenzoate (3s). Light
yellow powder, yield 52.6%. 1H-NMR (DMSO-
d6,600 MHz) δ: 13.13 (s, 1H, 5-OH), 8.04–8.07 (m, 4H,
H-2ʺ,6ʺ,2ʹ,6ʹ), 7.56–7.60 (m, 3H, H-3ʹ,4ʹ,5ʹ), 7.10–7.11 (m,
2H, H-3ʺ,5ʺ), 6.86 (s, 1H, H-3), 5.97 (brs, 2H, H-12,13),
4.47 (s, 2H, H-9), 3.98 (brs, 4H, H-11,14), 3.87 (s, 3H, H-
15). 13C-NMR (DMSO-d6, 150 MHz) δ: 180.9 (C-4), 167.1
(C-10), 163.9 (C-2), 163.8 (C-4ʺ), 161.7 (C-7), 153.7 (C-
8a), 152.3 (C-5), 132.3 (C-2ʺ,6ʺ), 132.0 (C-4ʹ), 131.7 (C-
1ʹ), 129.6 (C-2ʹ,6ʹ), 126.7 (C-3ʹ,5ʹ), 126.0 (C-12,13), 124.7
(C-6), 122.0 (C-1ʺ), 114.5 (C-3ʺ,5ʺ), 105.0 (C-4a), 99.6
(C-3), 98.1 (C-8), 59.4 (C-15), 56.0 (C-11,14), 51.1 (C-9).
HRMS (ESI) m/z [M + H]+ Calcd for C28H24NO7 486.1553
found 486.1619.
5,6-Dihydroxy-4-oxo-2-phenyl-8-((pyrrolidin-1-yl)methyl)-
4H-chromen-7-yl benzoate (3p). Yellow crystal, yield
1
24.8%. H-NMR (DMSO-d6, 600 MHz) δ: 13.20 (s, 1H,
5-OH), 8.08–8.13 (m, 4H, H-2ʺ,6ʺ,2ʹ,6ʹ), 7.69–7.75 (m,
1H, H-4ʺ), 7.58 (m, 5H, H-3ʹ,4ʹ,5ʹ,3ʺ,5ʺ), 6.81 (s, 1H, H-3),
4.38 (s, 2H, H-9), 3.24 (brs, 4H, H-11,14), 1.93 (brs, 4H,
H-12,13). 13C-NMR (DMSO-d6, 150 MHz) δ: 180.4 (C-4),
169.0 (C-10), 164.3 (C-2), 161.1 (C-7), 154.3 (C-8a), 152.3
(C-5), 133.9 (C-4ʺ), 131.9 (C-4ʹ), 131.8 (C-1ʹ), 130.1 (C-
1ʺ), 130.1 (C-2ʺ,6ʺ), 129.6 (C-3ʹ,5ʹ), 129.2 (C-3ʺ,5ʺ), 126.6
(C-2ʹ,6ʹ), 125.3 (C-6), 104.9 (C-4a), 98.3 (C-3), 98.0 (C-8),
53.2 (C-11,14), 40.4 (C-9), 23.3 (C-12,13). HRMS (ESI)
m/z [(M-acid) + H]+ Calcd for C20H20NO5 354.1341 found
354.1341.
8-((2H-Pyrrol-1(5H)-yl)methyl)-5,6-dihydroxy-4-oxo-2-
phenyl-4H-chromen-7-yl methacrylate (3t). Light yellow
1
powder, yield 63.7%. H-NMR (DMSO-d6, 600 MHz) δ:
13.10 (s, 1H, 5-OH), 8.04–8.06 (m, 2H, H-2ʹ,6ʹ), 7.56–7.60
(m, 3H, H-3ʹ,4ʹ,5ʹ), 6.84 (s, 1H, H-3), 6.23 (s, 1H, H-16E),
5.92 (brs, 2H, H-12,13), 5.83 (s, 1H, H-16Z), 4.45 (s, 2H,
H-9), 3.97 (brs, 4H, H-11,14), 1.99 (s, 3H, H-17). 13C-NMR
(DMSO-d6, 150 MHz) δ: 180.9 (C-4), 167.0 (C-10), 164.9
(C-2), 161.7 (C-7), 153.6 (C-8a), 152.1 (C-5), 135.9 (C-15),
132.0 (C-4ʹ), 131.7 (C-1ʹ), 129.6 (C-3ʹ,5ʹ), 127.3 (C-6), 126.7
(C-2ʹ,6ʹ), 126.0 (C-12,13), 124.6 (C-16), 105.0 (C-4a), 99.5
(C-3), 98.0 (C-8), 59.4 (C-11,14), 51.1 (C-9), 18.7 (C-17).
HRMS (ESI) m/z [M + H]+ Calcd for C24H22NO6 420.1447
found 420.1554.
5,6-Dihydroxy-4-oxo-2-phenyl-8-((pyrrolidin-1-yl)methyl)-
4H-chromen-7-yl 4-methoxybenzoate (3q). Yellow powder,
1
yield 17.9%. H-NMR (CDCl3, 600 MHz) δ: 12.89 (s, 1H,
5-OH), 8.24 (d, J = 12.0 Hz, 2H, H-2ʺ,6ʺ), 7.82–7.83 (m, 2H,
H-2ʹ,6ʹ), 7.53–7.54 (m, 3H, H-3ʹ,4ʹ,5ʹ), 6.97 (d, J = 12.0 Hz,
2H, H-3ʺ,5ʺ), 6.63 (s, 1H, H-3), 4.28 (s, 2H, H-9), 3.88 (s,
3H, H-15), 2.87 (brs, 4H, H-11,14), 1.92 (brs, 4H, H-12,13).
13C-NMR (CDCl3, 150 MHz) δ: 182.4 (C-4), 164.2 (C-10),
163.8 (C-4ʺ), 162.9 (C-2), 160.5 (C-7), 152.8 (C-8a), 151.5
(C-5), 132.7 (C-2ʺ,6ʺ), 131.7 (C-4ʹ), 131.6 (C-1ʹ), 129.2 (C-
3ʹ,5ʹ), 126.1 (C-2ʹ,6ʹ), 123.1 (C-6), 121.4 (C-1ʺ), 113.7 (C-
3ʺ,5ʺ), 105.6 (C-4a), 103.7 (C-3), 98.8 (C-8), 55.5 (C-15),
53.6 (C-11,14), 51.9 (C-9), 23.7 (C-12,13). HRMS (ESI) m/z
[M + H]+ Calcd for C28H26NO7 488.1709 found 488.1741.
8-((Dimethylamino)methyl)-5,6-dihydroxy-4-oxo-2-phenyl-
4H-chromen-7-yl benzoate (3u). Yellow crystal, yield
43.8%. 1H-NMR (DMSO-d6, 600MHz) δ: 8.28–8.29 (m, 2H,
H-2ʺ,6ʺ), 7.83–7.84 (m, 2H, H-2ʹ,6ʹ), 7.49–7.63 (m, 6H, H-
3ʹ,4ʹ,5ʹ,3ʺ,4ʺ,5ʺ), 6.65 (s, 1H, H-3), 4.09 (s, 2H, H-9), 2.50
(s, 6H, H-11,12). 13C-NMR (DMSO-d6, 150 MHz) δ: 182.5
(C-4), 164.4 (C-10), 163.2 (C-2), 159.7 (C-7), 152.9 (C-8a),
151.9 (C-5), 133.5 (C-4ʺ), 131.8 (C-4ʹ), 131.6 (C-1ʹ), 130.5
(C-2ʺ,6ʺ), 129.2 (C-3ʹ,5ʹ), 129.0 (C-1ʺ), 128.5 (C-3ʺ,5ʺ), 126.1
(C-2ʹ,6ʹ), 123.0 (C-6), 105.7 (C-4a), 104.1 (C-3), 98.5 (C-8),
8-((2H-Pyrrol-1(5H)-yl)methyl)-5,6-dihydroxy-4-oxo-2-
phenyl-4H-chromen-7-yl benzoate (3r). Light yellow
1
powder, yield 29.7%. H-NMR (DMSO-d6, 600 MHz) δ:

8
MOU et al.
|
55.5 (C-9), 44.3 (C-11,12). HRMS (ESI) m/z [(M-acid) + H]+
Calcd for C18H18NO5 328.1185 found 328.1200.
13C-NMR (DMSO-d6, 150 MHz) δ: 180.9 (C-4), 167.9 (C-
10), 163.4 (C-2), 161.7 (C-7), 153.7 (C-8a), 152.3 (C-5),
134.9 (C-4ʺ), 134.2 (C-1ʺ), 132.4 (C-3ʺ), 132.0 (C-6ʺ), 131.7
(C-4ʹ), 131.6 (C-1ʹ), 129.6 (C-3ʹ,5ʹ), 128.4 (C-6), 126.7 (C-
2ʹ,6ʹ), 124.5 (C-5ʺ), 121.6 (C-2ʺ), 105.0 (C-4a), 99.3 (C-3),
97.2 (C-8), 54.3 (C-11,14), 50.0 (C-9), 17.8 (C-12,15), 11.5
(C-13,16). HRMS (ESI) m/z [(M-acid) + H]+ Calcd for
C18H18NO5 384.1811 found 384.1815.
8-((Dimethylamino)methyl)-5,6-dihydroxy-4-oxo-2-phenyl-
4H-chromen-7-yl 2-bromobenzoate (3v). Yellow powder,
yield 16.0%. 1H-NMR (DMSO-d6, 600MHz) δ: 8.24 (d,
J = 7.4 Hz, 1H, H-6ʺ), 7.83–7.84 (m, 2H, H-2ʹ,6ʹ), 7.72 (d,
J = 7.7 Hz, 1H, H-3ʺ), 7.54–7.58 (m, 3H, H-3ʹ,4ʹ,5ʹ), 7.37–
7.44 (m, 2H, H-4ʺ,5ʺ), 6.65 (s, 1H, H-3), 4.11 (s, 2H, H-9),
2.52 (s, 6H, H-11,12). 13C-NMR (DMSO-d6, 150 MHz) δ:
182.4 (C-4), 163.3 (C-10), 163.2 (C-2), 159.9 (C-7), 153.0
(C-8a), 152.1 (C-5), 134.6 (C-4ʺ), 133.2 (C-1ʺ), 132.6 (C-
3ʺ), 131.8 (C-6ʺ), 131.5 (C-4ʹ), 130.6 (C-1ʹ), 129.2 (C-3ʹ,5ʹ),
127.2 (C-6), 126.1 (C-2ʹ,6ʹ), 122.8 (C-5ʺ), 122.8 (C-2ʺ), 105.7
(C-4a), 103.9 (C-3), 98.5 (C-8), 55.3 (C-9), 44.2 (C-11,12).
HRMS (ESI) m/z [(M-acid) + H]+ Calcd for C18H18NO5
328.1185 found 328.1170.
8-((Dipropylamino)methyl)-5,6-dihydroxy-4-oxo-2-phenyl-
4H-chromen-7-yl 4-methoxybenzoate (3z). Yellow powder,
yield 29.4%. 1H-NMR (DMSO-d6, 600 MHz) δ: 13.12 (s, 1H,
5-OH), 8.04–8.08 (m, 4H, H-2ʹ,6ʹ,2ʺ,6ʺ), 7.57–7.62 (m, 3H,
H-3ʹ,4ʹ,5ʹ), 7.10–7.12 (m, 2H, H-3ʺ,5ʺ), 6.85 (s, 1H, H-3),
4.37 (s, 2H, H-9), 3.87 (s, 3H, H-17), 2.92 (t, J = 6.0 Hz, 4H,
H-11,14), 1.65–1.71 (m, 4H, H-12,15), 0.86 (t, J = 6.0 Hz,
6H, H-13,16). 13C-NMR (DMSO-d6, 150 MHz) δ: 181.1 (C-
4), 167.5 (C-10), 163.8 (C-2,4ʺ), 161.8(C-7), 153.3 (C-8a),
152.2 (C-5), 132.3 (C-2ʺ,6ʺ), 132.0 (C-4ʹ), 131.7 (C-1ʹ), 129.6
(C-3ʹ,5ʹ), 126.7 (C-2ʹ,6ʹ), 124.5 (C-6), 122.0 (C-1ʺ), 114.6
(C-3ʺ,5ʺ), 105.0 (C-4a), 99.7 (C-3), 97.3 (C-8), 56.0 (C-17),
54.4 (C-11,14), 50.1 (C-9), 17.9 (C-12,15), 11.6 (C-13,16).
HRMS (ESI) m/z [M + H]+ Calcd for C30H32NO7 518.2179
found 518.2181.
8-((Dimethylamino)methyl)-5,6-dihydroxy-4-oxo-2-phenyl-
4H-chromen-7-yl methacrylate (3w). Yellow powder, yield
1
17.5%. H-NMR (CDCl3, 600 MHz) δ: 7.82–7.83 (m, 2H,
H-2ʹ,6ʹ), 7.54–7.57 (m, 3H, H-3ʹ,4ʹ,5ʹ), 6.64 (s, 1H, H-3),
6.47 (s, 1H, H-14E), 5.80 (s, 1H, H-14Z), 4.07 (s, 2H, H-9),
2.50 (s, 6H, H-11,12), 2.11 (s, 3H, H-15). 13C-NMR (CDCl3,
150 MHz) δ: 182.5 (C-4), 165.1 (C-10), 163.2 (C-2), 159.6
(C-7), 152.8 (C-8a), 151.8 (C-5), 135.2 (C-13), 131.8 (C-4ʹ),
131.6 (C-1ʹ), 129.2 (C-3ʹ,5ʹ), 127.7 (C-6), 126.1 (C-2ʹ,6ʹ),
123.0 (C-14), 105.7 (C-4a), 104.0 (C-3), 98.5 (C-8), 55.4
(C-9), 44.3 (C-11,12), 18.5 (C-15). HRMS (ESI) m/z [(M-
acid) + H]+ Calcd for C18H18NO5 328.1185 found 328.1194.
8-((Dipropylamino)methyl)-5,6-dihydroxy-4-oxo-2-phenyl-
4H-chromen-7-yl 2-(2-chlorophenyl)acetate (3A). Yellow
1
powder, yield 59.7%. H-NMR (DMSO-d6, 600 MHz) δ:
13.12 (s, 1H, 5-OH), 8.05–8.06 (m, 2H, H-2ʹ,6ʹ), 7.56–7.60
(m, 4H, H-3ʹ,4ʹ,5ʹ,6ʺ), 7.48–7.49 (m, 1H, H-3ʺ), 7.33–7.35
(m, 2H, H-4ʺ,5ʺ), 6.83 (s, 1H, H-3), 4.35 (s, 2H, H-9), 4.05 (s,
2H, H-17), 2.91 (t, J = 7.8 Hz, 4H, H-11,14), 1.65–1.71 (m,
4H, H-12,15), 0.87 (t, J = 6.0 Hz, 6H, H-13,16). 13C-NMR
(DMSO-d6, 150 MHz) δ: 181.1 (C-4), 168.4 (C-10), 167.3
(C-2), 161.8 (C-7), 153.3 (C-8a), 152.1 (C-5), 134.3 (C-4ʺ),
132.9 (C-1ʺ), 132.6 (C-3ʺ), 132.3 (C-6ʺ), 132.0 (C-4ʹ), 131.7
(C-1ʹ), 129.6 (C-3ʹ,5ʹ), 127.6 (C-6), 126.7 (C-2ʹ,6ʹ), 126.4 (C-
5ʺ), 124.4 (C-2ʺ), 105.0 (C-4a), 99.6 (C-3), 97.3 (C-8), 54.4
(C-11,14), 48.8 (C-9), 38.3 (C-17), 18.0 (C-12,15), 11.6 (C-
13,16). HRMS (ESI) m/z [M + H]+ Calcd for C30H31ClNO6
536.1840 found 536.1971.
8-((Dipropylamino)methyl)-5,6-dihydroxy-4-oxo-2-phenyl-
4H-chromen-7-yl benzoate (3x). Yellow powder, yield
29.8%. 1H-NMR (CDCl3, 600 MHz) δ: 13.18 (s, 1H, 5-OH),
8.07–8.13 (m, 4H, H-2ʺ,6ʺ,2ʹ,6ʹ), 7.72–7.77 (m, 1H, H-4ʺ),
7.58–7.61 (m, 5H, H-3ʹ,4ʹ,5ʹ,3ʺ,5ʺ), 6.85 (s, 1H, H-3), 4.38 (s,
2H, H-9), 2.94 (t, J = 6.0 Hz, 4H, H-11,14), 1.66–1.70 (m,
4H, H-12,15), 0.86 (t, J = 6.0 Hz, 6H, H-13,16). 13C-NMR
(CDCl3, 150 MHz) δ: 181.0 (C-4), 167.7 (C-10), 164.2 (C-2),
161.8 (C-7), 153.5 (C-8a), 152.2 (C-5), 134.1 (C-4ʺ), 132.0
(C-4ʹ), 131.7 (C-1ʹ), 130.2 (C-2ʺ,6ʺ), 129.8 (C-1ʺ), 129.6 (C-
3ʹ,5ʹ), 129.3 (C-3ʺ,5ʺ), 126.7 (C-2ʹ,6ʹ), 124.5 (C-6), 105.0 (C-
4a), 99.6 (C-3), 97.2 (C-8), 54.4 (C-11,14), 50.1 (C-9), 17.9
(C-12,15), 11.6 (C-11,16). HRMS (ESI) m/z [(M-acid) + H]+
Calcd for C18H18NO5 384.1811 found 384.1833.
(2E)-8-((Dipropylamino)methyl)-5,6-dihydroxy-4-oxo-2-
phenyl-4H-chromen-7-yl cinnamate (3B). Yellow powder,
yield 62.3%. 1H-NMR (DMSO-d6,600 MHz) δ: 13.13 (s, 1H,
5-OH), 8.07–8.08 (m, 2H, H-2ʹ,6ʹ), 7.80 (d, J = 16.1 Hz, 1H,
H-18), 7.79–7.82 (m, 2H, H-2ʺ,6ʺ), 7.57–7.62 (m, 3H, H-
3ʹ,4ʹ,5ʹ), 7.46–7.47 (m, 3H, H-3ʺ,4ʺ,5ʺ), 6.89 (d, J = 16.1 Hz,
1H, H-17), 6.85 (s, 1H, H-3), 4.37 (s, 2H, H-9), 2.92 (t,
J = 7.7 Hz, 4H, H-11,14), 1.65–1.69 (m, 4H, H-12,15),
0.87 (t, J = 7.4 Hz, 6H, H-13,16). 13C-NMR (DMSO-d6,
150 MHz) δ: 181.1 (C-4), 167.3 (C-10), 164.5 (C-2), 161.8
(C-7), 153.2 (C-8a), 152.2 (C-5), 146.0 (C-18), 134.5 (C-1ʺ),
8-((Dipropylamino)methyl)-5,6-dihydroxy-4-oxo-2-phenyl-
4H-chromen-7-yl 2-bromobenzoate (3y). Yellow powder,
1
yield 53.9%. H-NMR (DMSO-d6, 600 MHz) δ: 13.23 (s,
1H, 5-OH), 8.07–8.10 (m, 3H, H-2ʹ,6ʹ,6ʺ), 7.82–7.84 (m, 1H,
H-3ʺ), 7.55–7.61 (m, 5H, H-3ʹ,4ʹ,5ʹ,4ʺ,5ʺ), 6.85 (s, 1H, H-3),
4.40 (s, 2H, H-9), 2.96 (t, J = 7.8 Hz, 4H, H-11,14), 1.67–
1.70 (m, 4H, H-12,15), 0.87 (t, J = 7.4 Hz, 6H, H-13,16).

MOU et al.
9
|
132.1 (C-4ʹ), 131.7 (C-1ʹ), 131.1 (C-4ʺ), 129.6 (C-3ʹ,5ʹ), 129.4
(C-3ʺ,5ʺ), 129.0 (C-2ʺ,6ʺ), 126.7 (C-2ʹ,6ʹ), 124.3 (C-6), 118.1
(C-17), 105.0 (C-4a), 99.8 (C-3), 97.3 (C-8), 54.5 (C-11,14),
50.3 (C-9), 18.1 (C-12,15), 11.6 (C-13,16). HRMS (ESI) m/z
[M + H]+ Calcd for C31H32NO6 514.2230 found 514.2253.
131.1 (C-1ʹ), 129.7 (C-3ʹ,5ʹ), 126.9 (C-2ʹ,6ʹ), 107.2 (C-4a),
106.1 (C-3), 105.0 (C-8), 73.5 (C-10), 72.2 (C-13), 54.8 (C-
14,17), 50.6 (C-9), 28.7 (C-11), 27.7 (C-15,16), 24.7 (C-12).
HRMS (ESI) m/z [M + H]+ Calcd for C24H26NO5S 440.1532
found 440.1603.
8-((Dipropylamino)methyl)-5,6-dihydroxy-4-oxo-2-phenyl-
2,3,4,5-Tetrahydro-7-((3,4,5,11-tetrahydro-12-hydroxy-
11-oxo-9-phenyl-2H-[1,4]dioxocino[2,3-g]chromen-7-
yl)methyl)-12-hydroxy-9-phenyl-[1,4]dioxocino[2,3-g]
chromen-11-one (5). Yellow powder, yield 10.7%. 1H-
NMR (CDCl3, 600 MHz) δ: 12.85 (s, 2H, 2 × 5-OH), 7.92–
7.93 (m, 4H, 2 × H-2ʹ,6ʹ), 7.50–7.55 (m, 6H, 2 × H-3ʹ,4ʹ,5ʹ),
6.69 (s, 2H, 2 × H-3), 4.43 (s, 2H, H-9), 4.28 (brs, 4H,
2 × H-13), 4.19 (brs, 4H, 2 × H-10), 1.65 (brs, 8H, 2 × H-
11,12). 13C-NMR (CDCl3, 150 MHz) δ: 183.1 (2 × C-4),
163.9 (2 × C-2), 157.3 (2 × C-7), 153.0 (2 × C-8a), 150.3
(2 × C-5), 131.9 (2 × C-6), 131.6 (2 × C-4ʹ), 130.6 (2 × C-
1ʹ), 129.2 (2 × C-3ʹ,5ʹ), 126.2 (2 × C-2ʹ,6ʹ), 108.6 (2 × C-
4a), 105.6 (2 × C-3), 104.8 (2 × C-8), 73.5 (2 × C-13), 70.3
(2 × C-10), 28.9 (2 × C-12), 23.9 (2 × C-1), 18.3 (C-9).
HRMS (ESI) m/z [M + H]+ Calcd for C39H33O10 661.2074
found 661.2093.
4H-chromen-7-yl methacrylate (3C). Light yellow powder,
1
yield 52.5%. H-NMR (DMSO-d6, 600 MHz) δ: 13.10 (s,
1H, 5-OH), 8.05–8.07 (m, 2H, H-2ʹ,6ʹ), 7.56–7.62 (m, 3H, H-
3ʹ,4ʹ,5ʹ), 6.83 (s, 1H, H-3), 6.23 (s, 1H, H-18E), 5.84 (s, 1H, H-
18Z), 4.35 (s, 2H, H-9), 2.92 (t, J = 6.0 Hz, 4H, H-11,14), 1.99
(s, 3H, H-19), 1.66–1.71 (m, 4H, H-12,15), 0.87 (t, J = 6.0 Hz,
6H, H-13,16). 13C-NMR (DMSO-d6, 150 MHz) δ: 181.0 (C-
4), 167.4 (C-10), 164.8 (C-2), 161.8 (C-7), 153.3 (C-8a), 152.1
(C-5), 135.9 (C-17), 132.0 (C-4ʹ), 131.7 (C-1ʹ), 129.6 (C-3ʹ,5ʹ),
127.4 (C-6), 126.7 (C-2ʹ,6ʹ), 124.3 (C-18), 105.0 (C-4a), 99.6
(C-3), 97.2 (C-8), 54.4 (C-11,14), 50.1 (C-9), 18.7 (C-19), 17.9
(C-12,15), 11.6 (C-13,16). HRMS (ESI) m/z [(M-acid) + H]+
Calcd for C18H18NO5 384.1811 found 384.1790.
Synthesis of compounds 4 and 5
Compounds 4 and 5 were prepared by the method mentioned
above for the preparation of compound 2, employing com-
pounds 1a, b as starting materials and 1, 4-dibromobutane
instead of bromobenzyl.
2.2
Activity assays
|
2.2.1
Anti-MCF-7 tumor cell
|
2,3,4,5-Tetrahydro-12-hydroxy-7-(morpholinomethyl)-
proliferative experiment
9-phenyl-[1,4]dioxocino
[2,3-g]
chromen-11-one
(4a). Yellow crystal, yield 9.7%. 1H-NMR (DMSO-d6,
600 MHz) δ: 12.94 (s, 1H, 5-OH), 8.10–8.11 (m, 2H, H-
2ʹ,6ʹ), 7.59–7.64 (m, 3H, H-3ʹ,4ʹ,5ʹ), 7.04 (s, 1H, H-3), 4.64
(t, J = 5.6 Hz, 2H, H-10), 4.15 (t, J = 5.1 Hz, 2H, H-13), 3.71
(s, 2H, H-9), 3.53 (brs, 4H, H-15,16), 2.46–2.51 (m, 4H, H-
14,17), 1.95–1.97 (m, 2H, H-11), 1.73–1.74 (m, 2H, H-12).
13C-NMR (DMSO-d6, 150 MHz) δ: 183.1 (C-4), 163.9 (C-2),
158.0 (C-7), 153.6 (C-8a), 151.2 (C-5), 132.6 (C-6), 131.4
(C-4ʹ), 131.1 (C-1ʹ), 129.7 (C-3ʹ,5ʹ), 126.9 (C-2ʹ,6ʹ), 106.9
(C-4a), 106.1 (C-3), 105.0 (C-8), 73.5 (C-10), 72.2 (C-13),
66.8 (C-15,16), 53.6 (C-14,17), 50.2 (C-9), 28.7 (C-11), 24.7
(C-12). HRMS (ESI) m/z [M + H]+ Calcd for C24H26NO6
424.1760 found 424.1820.
All the target compounds and controls were dissolved in
dimethyl sulfoxide (DMSO) to prepare 5 mg/ml stock solu-
tions (Meegan et al., 2001). The stock solution was filtered
by 0.2 μM filter and diluted into 5 concentrations (50, 10, 2,
0.4, and 0.08 μg/ml) with starving medium.
MCF-7 cells were diluted to 100,000/ml. 0.1 ml of cell
suspension was inoculated into 96-well plate. After 24 hr of
culture, the primary medium was discarded and replaced with
starving medium (1640 medium +2% fetal bovine serum) for
12 hr. Then, the medium was discarded and replaced with
compounds with different concentrations in eight multiple
wells. After 72 hr of culture, 10 μl of CCK-8 reagent was
added into each well and the 96-well plates were incubated for
1 hr at 37°C. The OD value was measured at the wavelength of
450 nm. The inhibition rates on 50 μg/ml were recorded. The
IC50 values were also obtained from the plot of activity versus
inhibitor concentration by using GraphPad Prism 5 software.
2 , 3 , 4 , 5 -Te t ra h y d ro - 1 2 - h y d ro x y - 9 - p h e n y l - 7 -
(thiomorpholinomethyl)-[1,4]dioxocino[2,3-g]chromen-11-
1
one (4b). Yellow crystal, yield 12.0%. H-NMR (DMSO-
d6, 600 MHz) δ: 12.93 (s, 1H, 5-OH), 8.09–8.10 (m, 2H,
H-2ʹ,6ʹ), 7.59–7.64 (m, 3H, H-3ʹ,4ʹ,5ʹ), 7.02 (s, 1H, H-3),4.62
(t, J = 5.6 Hz, 2H, H-10), 4.15 (t, J = 5.6 Hz, 2H, H-13), 3.71
(s, 2H, H-9), 2.73 (brs, 4H, H-14,17), 2.51–2.57 (m, 4H, H-
15,16), 1.95 (m, 2H, H-11), 1.74 (m, 2H, H-12). 13C-NMR
(DMSO-d6, 150 MHz) δ: 183.1 (C-4), 163.9 (C-2), 158.0
(C-7), 153.6 (C-8a), 151.2 (C-5), 132.6 (C-6), 131.4 (C-4ʹ),
2.2.2
Anti-CDK1/cyclin B kinase
|
activity experiment
In vitro CDK1/cyclin B assay was performed as described by
the manufacturer (Ha et al., 2016). All the test compounds

10
MOU et al.
|
and positive controls were dissolved in DMSO to prepare
10 mmol/L stock solutions. The stock solutions were diluted
20 times by double distilled water and then 10 times by buffer
to 50 μmol/L working concentration (two times of the test
concentration) and stored in refrigerator at −20°C. CDK1/
cyclin B kinase was aliquoted into the buffer and incubated
with test compounds at various concentrations (25, 5, 1, 0.2,
and 0.04 μmol/L) at 20°C for 30 min. The 10 μl mixture
was incubated with buffer containing Reagent C (130 μl),
Reagent D (20 μl), Reagent E (20 μl), and Reagent F (20 μl)
at room temperature for 3 min, and then, the final mixture
was immediately put into 30°C spectrophotometer (340-nm
wavelength) for detection. The value of every minute was
recorded until 90 min. The D-value between the 0-min value
and the 90-min value is used to calculate the IC50 value.
According to the above conditions, baicalein and compound
3o were docked to the flavopiridol binding site of CDK1.
Other docking parameters in the program were kept default.
The docking simulation using CDOCKER was prepared ac-
cording to the user guidance. The binding pose figure was
prepared by PyMOL.
3
RESULTS AND DISCUSSION
Chemistry
|
3.1
|
The synthetic route of all target compounds is outlined in
Scheme 1 with baicalein as the starting material. Compounds
1a–g were synthesized by Mannich reaction of baicalein
with formaldehyde and various secondary amines in metha-
nol. Compound 2 was synthesized by etherification of bai-
calein with bromobenzyl under the catalyst of K2CO3 and
KI in N, N-DMF. Compounds 3a-z and 3A-C were obtained
by esterification of compounds 1a-g with various carbox-
ylic acids in the presence of N, N′-dicyclohexylcarbodiimide
(DCC) and 1-hydroxybenzotriazole (HOBt) as catalysts in
tetrahydrofuran (THF). Compounds 4a, b and compound 5
were produced by etherification of compounds 1a, b with
1,4-dibromobutane in a method similar to the synthesis of
compound 2.
2.2.3
Molecular docking study
|
Molecular docking study was finished by CDOCKER pro-
gram of Discovery Studio to explore the predicted bind-
ing mode of baicalein and compound 3o with CDK1 (Yan
et al., 2020). The co-crystal structure of CDK1/cyclin B-
Cks2 with flavopiridol (PDB: 6GU2) was obtained from the
PDB database. The edge water molecules were removed,
and hydrogen atoms were added to the protein by clean pro-
tein module. And then, the corresponding amino acids were
protonated and energy minimization was performed. The
structures of baicalein and compound 3o were introduced.
Hydrogen atoms and CHARMm field were added. According
to the binding position of flavopiridol in CDK1, a sphere
with a radius of 9.0 × 10–10 m was set, and then, flavopiridol
was extracted from the complex. Flavopiridol was re-docked
to the original protein by CDOCKER program. The RMSD
value of the docking conformation and crystal conformation
was calculated to determine the credibility of docking results.
1
Compounds 1 (Table 1) were characterized by H, 13C-
1
NMR techniques. Compounds 3 were characterized by H,
13C-NMR, and ESI-MS techniques and chemical method
(Table 2). Compounds 2, 4 (Table 3), and 5 were character-
ized by 1H, 13C-NMR, and ESI-MS techniques.
From 1H-NMR, 13C-NMR, and ESI-MS data, it can be con-
firmed that compounds 3 were generated by esterification of
only one equivalent carboxylic acid with compounds 1, which
were single-substituted products. The single peak at δ12.9 ppm
1
or so in the H-NMR spectrum is the signal of 5-phenolic
R1
N
R1
N
R2
HO
R2
O
O
HO
HO
O
O
O
O
O
O
a
c
R3
HO
HO
OH
OH
OH
OH
baicalein
1
3
b
d
O
O
R1
O
O
O
N
R2
O
O
O
O
O
SCHEME 1 Synthesis of baicalein
derivatives. Reagents and conditions: (a)
HCHO, NHR1R2/MeOH; (b) BnBr, K2CO3,
KI/DMF; (c) carboxylic acid, DCC, HOBt/
anhydrous THF; (d) BrCH2CH2CH2CH2Br,
K2CO3, KI/DMF
O
CH2
HO
+
OH
O
2
OH
O
O
OH
O
4
5

MOU et al.
11
|
TABLE 1 Substituent groups of compounds 1
4a exhibited the best inhibition rate (96.7%), comparable to
CGP74514A (96.7%; Figure 1), which is a selective CDK1
inhibitor (Imbach et al., 1999), and better than flavopiridol
(90.0%), while compounds 3C, 3o, and 3c exhibited compa-
rable activity with flavopiridol (87.8%, 86.9%, and 86.0%,
respectively).
R
HO
HO
O
O
OH
The substituent groups at position-7 and position-8
determined the activities of the target compounds.
Cpds.
1a
R
Cpds.
1e
R
For position-8, the 8-aminomethylene substitution is very
important to inhibit the growth of tumor cells. Compound 2,
with a benzyloxy group at position-7 and no substituent group
at position-8, showed no inhibitory activity toward MCF-7
tumor cell proliferation, suggesting the importance of the
nitrogen-containing functional group in position-8. The rea-
son may be that the nitrogen atom in the nitrogen-containing
functional group can form hydrogen bond with the target,
thus enhancing the activity of the compound. The results also
demonstrated that compounds with position-8 linked with
N-methylpiperazine methylene or thiomorpholine methylene
have better activities. For example, the inhibition rate of 3o is
up to 86.9%, and 1b is up to 84.8%. The reason may be due to
the extra hydrogen bond interaction with target by the second
nitrogen atom in N-methylpiperazine and the sulfur atom in
thiomorpholine.
O
N
N
1b
1c
1d
1f
N
S
N
1g
N
H3C N
N
N
hydroxyl. So esterification took place on 6- or 7-phenolic hy-
droxyl group. As there is no hydrogen atom on the ester bond,
the 2D-NMR spectrum cannot determine whether the acyla-
tion is on the 6- or 7-phenolic hydroxyl group.
In our research, the position of acylation was confirmed
by the chemical method. Because the flavonoids with O-
diphenol hydroxyl groups in structure can form green to
brown or even black precipitate (Scheme 2) with strontium
chloride (SrCl2) in ammonia methanol solution. By this
method, it can be determined whether there are O-diphenol
hydroxyl groups in compounds 3, and then determined the
position of substitution.
For position-7, cinnamoyloxy- and α-methacryloyloxy-
substituted compounds have excellent activities. The inhibi-
tion rate of compound 3d is 79.4%, and 3C is 87.8%. The
reason may be that compared with the aryl acyloxy group,
cinnamoyloxy and α-methacryloyloxy groups have longer
chains and better flexibility, can stretch into the active site of
the target to form hydrophobic interactions.
In addition, 6, 7-bietherification products have better ac-
tivities than those of 7-esterification products. For example,
4a is the most active compound, with inhibition rate up to
96.7%. The reason may be that the hydrocarbon part of the
octad dioxane can form hydrophobic interaction with the tar-
get. On the other hand, the oxygen atom of the ether bond is
hydrophilic, while the hydrocarbon part is lipophilic, so it is
easy to pass the membrane of tumor cells.
From the combination of 1H, 13C-NMR and ESI-MS tech-
niques and chemical method, it can be confirmed that the ac-
curate structures of compounds 3 were as in Scheme 1.
3.2
Results of activity assays
|
3.2.1
Anti-proliferative activity toward MCF-
|
7 tumor cells
3.2.2
Anti-CDK1/cyclin B kinase activity
|
Cell counting kit-8 (CCK-8) assay was carried out for evalu-
ating the anti-proliferative activities of the target compounds
coupled with three positive controls. The IC50 value of fla-
vopiridol is 0.08 μM, a little higher than the IC 50 of 0.03 μM
reported in the literature (Ahn et al., 2007). At the concentra-
tion of 50 μg/ml, the inhibition rates of the test compounds on
MCF-7 tumor cells are shown in Table 4.
The results of inhibition rates at the concentration of
50 μg/ml showed that 20 target compounds had higher activ-
ities than that of baicalein (63.9%). Among them, compound
In anti-MCF-7 tumor cell proliferative assay, a half of the
target compounds showed good activities. In order to eluci-
date the CDK1/cyclin B kinase inhibition activities of the tar-
get compounds, the compounds with desirable anti-MCF-7
activities were chosen to conduct the anti-CDK1/cyclin B
kinase activity assay. CGP74514A was used as positive con-
trol. The IC50 value of CGP74514A is 0.20 μM, compara-
ble to the IC50 of 0.11 μM reported in the literature (Imbach
et al., 1999). The results are shown in Table 5.

12
MOU et al.
|
TABLE 2 Substituent groups of compounds 3
R
O
O
O
O
R3
HO
OH
Cpds.
3a
R
R3
Cpds.
3p
R
R3
O
N
N
N
N
3b
3q
O
H3C
O
O
Br
3c
3r
3s
O
O
N
N
N
N
N
H3C
O
3d
H3C
3e
3f
3t
O
N
3u
N
S
N
3g
3h
3v
N
S
S
N
N
Br
Br
3w
N
H3C
O
3i
3j
3x
3y
N
N
S
S
N
N
Br
3k
3l
3z
N
N
H3C N
H3C N
N
N
H3C
O
3A
N
Br
3m
3B
N
H3C N
N
H3C
O
(Continues)

MOU et al.
13
|
TABLE 2 (Continued)
R
O
O
O
O
R3
HO
OH
Cpds.
3n
R
R3
Cpds.
3C
R
R3
N
H3C N
N
N
3o
H3C N
which can be attributed to the ether groups in their structures.
The better histocompatibility of ether bond makes it more
permeable through the membrane of tumor cells, but it did
not show any advantage in kinase activity assay. Compounds
1b and 3f with 8-thiomorpholine methylene groups showed
desirable activities in anti-CDK1/cyclin B kinase activity
assay. That coincides with their anti-MCF-7 tumor cell pro-
liferative activity assay results.
In order to analyze the correlation between cell activity
and kinase inhibition activity, the Pearson correlation coeffi-
cient analysis was performed using SPSS 19. From the anal-
ysis results, it can be seen that p value equals to .006 (<.01),
indicating that there is a linear correlation between the two
variables, and r value equals to .714, indicating that there is
a strong correlation between the two variables. They were
positively correlated with each other (Figure 4).
TABLE 3 Substituent groups of compounds 4
R
O
O
O
OH O
Cpds.
4a
R
Cpds.
4b
R
O
N
S
N
The results showed that most of the test compounds pos-
sessed good inhibitory activities against CDK1/cyclin B
with the IC50 value distributing between 1.26 and 30.17 μM,
which is, however, weaker than that of the positive control
(IC50 = 0.20 μM). Compound 3o showed the best anti-CDK1/
cyclin B activity (IC50 = 1.26 μM), and compounds 3f, 1b,
and 3C had considerable activities (2.36, 2.43, and 2.72 μM,
respectively).
Compounds 3o and 3C with 7-α-methacryloyloxy groups
possessed excellent inhibitory activities against both MCF-7
tumor cells and CDK1/cyclin B kinase, which fully verified
the importance of the 7-α-methacryloyloxy group in the tar-
get compounds, while compounds 4a and 3c only performed
well in anti-MCF-7 tumor cell proliferative activity assay,
3.3
Docking study of compound 3o with
|
CDK1/cyclin B
Compound 3o showed the best activity in anti-CDK1/cyclin
B kinase activity assay. With the aim of exploring the inter-
action mode, molecular simulation work of compound 3o
binding with CDK1/cyclin B-Cks2-flavopiridol co-crystal
complex (PDB: 6GU2) was carried out. Random confor-
mation search was employed to identify predicted ligand–
protein binding conformation that are closer to the crystal
ones. The RMSD value of flavopiridol docking conformation
R1 R2
R1 R2
N
O
N
O
R3
O
O
O
OH
R3
O
O
O
Sr2+
+
HO
O
SCHEME 2 Reaction of O-diphenol
hydroxyl with strontium chloride
OH
O
Sr

14
MOU et al.
|
TABLE 4 Inhibition rates of the test compounds (50 μg/ml) on
MCF-7 tumor cells
TABLE 5 Activities of chosen target compounds
CDK1/
MCF-7
cyclin B
Inhibition
rate (%)
Inhibition
rate (%)
Cpds.
Cpds.
IC50
1a
1b
1c
1d
1e
1f
77.4
84.8
81.5
52.9
58.2
64.2
74.2
0.0
3o
3p
3q
3r
3s
3t
3u
3v
3w
3x
3y
3z
3A
3B
3C
4a
86.9
16.3
53.5
62.2
7.7
54.8
30.1
0.0
65.8
74.1
19.9
34.0
47.4
64.8
87.8
96.7
ND
65.9
63.9
90.0
96.7
Cpds.
IC50 (μM)
(μM)
CGP74514A
7.74
24.33
24.29
27.34
28.48
25.41
30.14
28.23
29.46
24.10
26.16
25.86
32.11
15.39
20.18
32.48
0.20
2.43
16.72
17.36
18.21
2.36
NA
1b
3c
3d
3e
3f
1g
2
3i
3j
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
0.0
NA
11.9
86.0
79.4
78.4
76.8
56.7
45.2
79.6
79.1
62.0
30.1
14.8
78.9
3n
3o
3w
3x
3B
3C
4a
5
19.27
1.26
5.16
22.79
30.17
2.72
NA
4b
5
30.11
Note: NA means “no activity.”
Baicalein
Flavopiridol
CGP74514A
Note: ND means “not determined.”
compared with its crystal conformation was 1.3209. So, the
binding results are creditable. In the compound 3o-CDK1
binding results, it can be seen that compound 3o binds to
CDK1's active site in a resembling stretching conforma-
tion with flavopiridol (Figure 5a). The chromone core of
compound 3o can occupy hinge region of CDK1 and forms
hydrogen bonds with E81 and L83 and hydrophobic inter-
actions with F80, V18, and V64. The phenyl group forms
hydrophobic interactions with I10 and V18. The intro-
duced 8-nitromethyl piperazine methylene moiety interacts
with D146 just as flavopiridol does, while the introduced
7-acrylate group also interacts with D146 through hydrogen
bond and V64 through hydrophobic interaction (Figure 5b).
The fact that compound 3o can form an extra interaction
with D146 through its 7-acrylate group may be the reason
for its best activity. It could be concluded that position-7
substitution is crucial to CDK1 binding. It is worth to men-
tion that molecular docking has certain guiding significance
for the structural modification, but it is not completely con-
sistent with the actual situation.
FIGURE 4 Bar chart of the IC50 of test compounds against MCF-
7 tumor cells and CDK1/cyclin B
4
CONCLUSIONS
|
A series of baicalein derivatives were designed and synthesized
with CDK1 as the target. Anti-MCF-7 tumor cell proliferative
assay results showed that compound 4a possessed higher inhibi-
tion rate than flavopiridol and was comparable to CGP74514A
at the concentration of 50 μg/ml. Half of the target compounds

MOU et al.
15
|
FIGURE 5 (a) Compound 3o and flavopiridol bind to the hinge region of CDK1 with a similar stretching conformation (compound 3o in
green, while flavopiridol in pale). (b) Binding mode of compound 3o with CDK1
exhibited better activities against MCF-7 proliferation than the
lead compound baicalein. The nitrogen atom in position-8 can
easily form hydrogen bond with the target, so 8-aminomethylene
substitution, especially 8-N-methylpiperazine methylene or
thiomorpholine methylene substitution, shows excellent activ-
ity toward tumor cells. Position-8 substitution is very important
to baicalein derivatives' inhibition activity. Flexible substitu-
ent groups in position-7, for example, cinnamoyloxy and α-
methacryloyloxy groups, could adapt to the active site of the
target through hydrogen bond and hydrophobic interactions,
the corresponding compounds showed considerable activities.
6, 7-Bietherification products have better activities than those
of 7-esterification products. Anti-CDK1/ cyclin B kinase results
of the test target compounds showed that compound 3o has
the best IC50. The molecular docking results showed that com-
pound 3o can interact with the important amino acid residues
E81, L83, and D146 of CDK1 through hydrogen bond just like
flavopiridol does. And it can also form an extra hydrogen bond
with D146 by its introduced 7-acrylate group, which flavopiri-
dol does not have. So, position-7 substitution is also beneficial
to CDK1 binding. Correlation analysis results demonstrated that
there is a correlation between the inhibition of MCF-7 tumor
cell proliferation and the inhibition of kinase activity.
CONFLICT OF INTEREST
There are no conflicts of interest to declare.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available
from the corresponding author upon reasonable request.
ORCID
Jiajia Mou
https://orcid.org/0000-0002-4344-1514
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