Synthesis of flavonoids nitrogen mustard derivatives and study on their antitumor activity in vitro

Article abstract of DOI:10.1016/j.bioorg.2020.103613

Several novel flavonoids nitrogen mustard derivatives were synthesized and evaluated for antiproliferative activity against seven human cancer cell lines (HeLa, A549, HepG2, MCF7, SH-SY5Y, PC-3, DU145) by the MTT assay in vitro. The resulting IC₅₀

Full text of DOI:10.1016/j.bioorg.2020.103613

Journal Pre-proofs
Synthesis of flavonoids nitrogen mustard derivatives and study on their antitu-
mor activity in vitro
Xi Yan, Jinglei Song, Meixuan Yu, Hao-Ling Sun, Haijun Hao
PII:
S0045-2068(19)31538-X
DOI:
https://doi.org/10.1016/j.bioorg.2020.103613
YBIOO 103613
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
19 September 2019
25 December 2019
21 January 2020
Please cite this article as: X. Yan, J. Song, M. Yu, H-L. Sun, H. Hao, Synthesis of flavonoids nitrogen mustard
derivatives and study on their antitumor activity in vitro, Bioorganic Chemistry (2020), doi: https://doi.org/
10.1016/j.bioorg.2020.103613
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Research paper
Synthesis of flavonoids nitrogen mustard derivatives and study on their antitumor activity in
vitro
a*
a
a
Xi Yan , Jinglei Song , Meixuan Yu , Hao-Ling Sun a *, Haijun Hao b *
aCollege of Chemistry, Beijing Normal University, Beijing 100875, PR China
bDepartment of Organic Chemistry, College of Science, Beijing University of Chemical Technology, Beijing 100029, PR China
Key words: Flavonoids, Nitrogen mustard derivatives, Anticancer, Cell apoptosis, Cell cycle
ABSTRACT
Several novel flavonoids nitrogen mustard derivatives were synthesized and evaluated for antiproliferative activity against seven human cancer cell lines (HeLa, A549, HepG2, MCF7,
SH-SY5Y, PC-3, DU145) by the MTT assay in vitro. The resulting IC50 showed that most compounds exhibited better inhibitory activity against seven cell lines. IC50 values of some
compounds were lower than well-known melphalan. In particular, compound 8b was the most promising compound which inhibited HeLa cells with IC50 value of 1.43μM. It showed
excellent antitumor activity against these seven cell lines. Besides, it could arrest cell cycle of HeLa in G2/M phase and induce cell apoptosis. The loss of mitochondrial membrane potential
may be an apoptotic mediating factor.
1.
Introduction
limiting factors in clinical therapy. To solve these problems, medicinal
chemists have made a lot of effort in the past. Kun et al.19 reported several dual-
targeting (IDO1 and DNA) anticancer agents, evaluated their antitumor
activities and studied the anti-tumor mechanism. Xiang et al. 20synthesized and
evaluated a series of enmein-type diterpenoid hybrids coupled with nitrogen
mustards, and some of them exhibited both cytotoxicity and selectivity.
Wenbing et al.21 described novel aromatic nitrogen mustards with various
aromatic substituents and boronic esters, which could cross-link DNA
effectively and enhanced the solubility. Rajesh K. Singh et al.22designed
substituted 1, 4-dihydropyridine derivatives with nitrogen mustard
pharmacophore. Novel derivatives of brefeldin A containing nitrogen mustards
showed antiproliferative activities, and the most promising one could induce
apoptosis, arrest cell cycle at the G1 phase and induce the intrinsic apoptotic
mitochondrial pathway in Bel-7402 cells23.
As one of leading causes of death in the world, cancer has led to enormous
socio-economic losses to human. Although there are considerable progresses
in cancer therapy, it’s still necessary to design novel anticancer agents for drug
resistance of tumors1. Flavonoids are a kind of polyphenolic secondary
metabolites, which exit widely in leguminous plants2. Flavonoids have aroused
great interest of medicinal chemists for their broad-spectrum biological
activities, such as anticancer, antioxidant, antibacterial, anti-osteoporosis
effect, antidiabetic, antiangiogenic, anti-inflammatory effects and so on3-9
.
Dihydroxyflavones, especially those containing ortho-dihydroxy group, are
strong antioxidants.10 As the most abundant soy isoflavones, genistein could
prevent estrogen-dependent cancer for its structural similarity to 17β-
oestradiol11. Daniel et al. 12found that genistein could downregulate lipid
synthesis to inhibit cell proliferation of human lung cancer cell line. Hideki et
al. 13discovered that genistein could inhibit cell growth of prostate cancer by
suppressing the activity of telomerase. Chrysin is also a potent anticancer agent
as a kind of flavone. Chrysin can inhibit cell proliferation and induce cell
apoptosis according to the literature14. Beibei et al. 15found that chrysin could
suppress the expression of hypoxia-inducible factor-1α in tumor cells. In order
to search for new flavonoid anticancer drugs containing nitrogen mustard
group, we chose genistein and chrysin as parent compounds for modification.
As a kind of DNA bifunctional alkylating agents, nitrogen mustards are
still widely used in clinic, such as melphalan, chlorambucil and
cyclophosphamide. They exert cytotoxicity through binding to DNA and
cross-linking the two chains to prevent DNA replication16. However, nitrogen
mustards are so toxic to normal cells that they bring many side effects to
patients17. These side effects and the drug resistance18 have become major
Natural flavonoids have low toxicity and many of them are food and
medicine homologous. We hope to find a new kind of nitrogen mustard
derivatives with antitumor activity by using flavonoids as carriers. According
to the principle of combination and hybridization, we designed and synthesized
a series of genistein and chrysin nitrogen mustard derivatives, which have
never been reported in the literature. The cytotoxic activities of genistein and
chrysin nitrogen mustard derivatives were evaluated and the possible
mechanisms of cell proliferation inhibition were studied.
2. Results and Discussion
2.1 Chemistry
The synthetic routes for genistein and chrysin nitrogen mustard derivatives
were shown in Scheme 1. Genistein or chrysin reacted with bromide to obtain
No color should be used for any figures in print.
*Corresponding author. College of Chemistry, Beijing Normal University, Beijing 100875,
PR China.

E-mail address: yanxi@bnu.edu.cn (Xi Yan).
HO
O
O
O
O
O
Br
n
a
b
OH
OH
OH
OH
O
1
2a-c
HO
O
O
Cl
O
O
N
N
n
n
c
OH
OH
O
3a-c
Cl
OH
OH
OH
4a-c
2a,3a,4a: n=1
2b,3b,4b: n=2
2c,3c,4c: n=3
O
O
HO
O
O
Br
n
d
e
OH
O
6a-c
OH
N
5
Cl
O
O
O
HO
O
O
N
f
n
n
Cl
OH
OH
OH
O
8a-c
7a-c
6a,7a,8a: n=1
6b,7b,8b: n=2
6c,7c,8c: n=3
Scheme 1. Synthesis of genistein and chrysin nitrogen mustard derivatives (4a-c, 8a-c). Reagents and conditions: (a) K2CO3, acetone, 1,2-dibromoethane, 1,3-
dibromopropane, or 1,4-dibromobutane, reflux, 8h, 69%-75%(for 2a-c); (b) NH(CH2CH2OH)2, CH3CN, reflux, 48h, 85%-88%(for 3a-c); (c) SOCl2, CH2Cl2,
reflux, 48h, 87%-92%(for 4a-c); (d) K2CO3, acetone, 1,2-dibromoethane, 1,3-dibromopropane, or 1,4-dibromobutane, reflux, 8h, 67%-89%(for 6a-c); (e)
NH(CH2CH2OH)2, CH3CN, reflux, 48h, 89%-96%(for 7a-c); (f) SOCl2, CH2Cl2, reflux, 48h, 79%-84%(for 8a-c).
intermediates (2a-c, 6a-c) in the presence of potassium carbonate. The
intermediates 2a-c, 6a-c and diethanolamine refluxed in acetonitrile to give
compounds 3a-c, 7a-c. Then the target compounds 4a-c, 8a-c were obtained by
the chlorination reaction of 3a-c, 7a-c using SOCl2 in CH2Cl2.
7.86μM, 11.31μM, 7.34μM, respectively, which were lower than that of
melphalan. More interestingly, we found that the carbon linker length between
flavonoid scaffold and the nitrogen mustard group influenced antiproliferative
activity regularly. Target compounds of three-carbon linker were more active
than those of two-carbon linker. And those of four-carbon linker had the worst
anticancer activity. Valeriy A. Bacherikov et al.24 and Dong-Jun Fu et al.25 have
also found the similar pattern. These suggested that the linker played a
significant role in their activities. Toxicity of these compounds towards normal
cells (human embryonic lung fibroblasts) was also tested, but they do not show
selectivity for tumor cells.
2.2 Cytotoxic activity assay
The antiproliferative activity of genistein and chrysin nitrogen mustard
derivatives against seven human cancer cell lines (Human Cervical Cancer Cell
Line HeLa, Human Lung Tumor Cell Line A549, Human Hepatoma Cell Line
HepG2, Human Breast Carcinoma Cell Line MCF7, Human Neuroblastoma
Cell Line SH-SY5Y, Human Prostatic Carcinoma Cell Line PC-3, Human
Prostatic Carcinoma Cell Line DU145) in vitro were evaluated by the MTT
essay using melphalan as the positive control. The resulting IC50 values were
summarized in Table 1. As we can see, compound 4a-c and 8a-c all showed
antiproliferative activity against these seven human cancer cell lines, whose
cytotoxic activities improved a lot compared with raw materials. Among these
flavonoid mustard derivatives, as the most promising target compound,
compound 8b inhibited HeLa, PC-3, DU145, MCF-7, SH-SY5Y, HepG2,
A549 cell lines with IC50 values of 1.43μM, 2.32μM, 2.91μM, 4.90μM,
2.3 The morphological analysis
One important sign of cell apoptotic is the aberrant morphological changes
of chromatin. After being treated with compound 8b (0μM, 1μM, 2μM, 4μM)
for 48h, HeLa cells were stained with DAPI and observed under the Laser
Scanning Confocal Microscopy. The results were shown in Fig.1. The drug-
treated cells displayed chromatin condensation and nuclear fragmentation and
the formation apoptotic body compared with the control group, which were the

typical morphological characteristics of apoptosis cells.
of compound 8b (0μM, 0.3μM, 0.8μM, 1.5μM) for 48h and then measured by
flow cytometry. As shown in Fig.2, the apoptosis rate of HeLa cells at different
concentrations increased from 3.63% of the control group to 5.70%, 7.79% and
9.29%, respectively. Hence, compound 8b may induce apoptosis of HeLa cells
in a dose-dependent manner.
2.4 Cell apoptosis assay
To explore the mechanism of cytotoxic activity of compound 8b further, cell
apoptosis assay on HeLa cells was performed using an Annexin V-FITC&PI
Apoptosis Detection Kit. HeLa cells were treated with different concentrations
Fig. 1. Morphological change of apoptosis in HeLa cells which were treated with compound 8b (0μM, 1μM, 2μM, 4μM) for 48h. The treated cells were stained
with DAPI and observed by a Laser Scanning Confocal Microscopy.
Fig. 2. Effects of compound 8b on cell apoptosis of HeLa cells. The cells were treated with different concentrations of compound 8b (0μM, 0.3μM, 0.8μM,
1.5μM) for 48h and then measured by flow cytometry.
Table 1 The cytotoxicity of formononetin nitrogen mustard derivatives.
IC50 (μmol/L)
compound
HeLa
20.14
16.37
36.52
4.28
HepG2
36.91
36.73
>100
16.86
11.31
23.91
>100
>100
13.36
A549
21.36
16.64
58.90
10.34
7.34
SH-SY5Y
25.33
12.77
25.36
11.04
7.86
DU145
6.07
MCF-7
18.02
14.90
28.14
7.67
PC-3
15.25
14.53
18.00
8.47
CCC-HPF-1
18.69
21.93
51.09
5.50
4a
4b
4.23
4c
18.32
5.19
8a
8b
1.43
2.91
4.90
2.32
1.53
8c
6.06
11.43
>100
>100
23.39
23.66
>100
11.88
>100
>100
15.66
12.68
70.88
95.31
20.21
9.54
7.97
genistein
chrysin
melphalan
77.26
>100
10.72
>100
>100
19.81
>100
49.18
20.31
>100
24.33

Fig. 3. Effects of compound 8b on cell cycle of HeLa cells. HeLa cells were treated with compounds 8b (0μM, 0.5μM, 0.8μM) for 48h and then measured by flow
cytometry.
Fig. 4. Mitochondrial membrane potential change of HeLa cells which were treated with compound 8b (0μM, 1μM, 2μM) for 48h. The treated cells were stained
with JC-1 and observed by a Laser Scanning Confocal Microscopy.
2.5 Cell cycle assay
results were shown in Fig.4. With the increase of the concentration of 8b, the
proportion of red and green fluorescence decreased from 3.35 to 2.57 and 2.05
by the analysis of Image J software. Hence, the loss of mitochondrial
membrane potential may be an apoptotic mediating factor.
In order to investigate the effect of our synthesized flavonoid nitrogen
mustard derivatives on cell cycle of humor cells, the most promising compound
8b against HeLa was chosen to study by flow cytometry according to the
results of the MTT assay. HeLa cells were treated with different concentrations
of compound 8b (0μM, 0.5μM, 0.8μM) for 48h and then measured by flow
cytometry. As shown in Fig.3, the percentages of cells in G2/M phase at
different concentrations increased from 12.23% of the control group to 34.21%
and 52.37%. It suggests that the compound 8b can arrest cell cycle of HeLa in
G2/M phase.
3.
Conclusions
A series of genistein and chrysin nitrogen mustard derivatives have been
synthesized and evaluated for cytotoxic activity against seven human cancer
cell lines. The results showed that most compounds inhibited seven human
cancer cell lines well better than raw materials and melphalan. It's remarkable
that the most effective compound 8b inhibited HeLa, PC-3, DU145, MCF-7
cell lines with IC50 values of 1.43μM, 2.32μM, 2.91μM, 4.90μM, respectively,
which were more than 4 times higher than melphalan. We also found that
compound 8b could induce cell apoptosis of HeLa cells, which may attribute
to cell cycle arrest in G2/M phase. The loss of mitochondrial membrane
potential may be an apoptotic mediating factor. Hence, compound 8b was a
potential anticancer agent.
2.6 Mitochondrial membrane potential
The mitochondria play an important role in activating cell apoptosis. The
change of mitochondrial membrane potential is one of the earliest events in
apoptosis. Hence, HeLa cells were treated with different concentrations of
compound 8b (0μM, 1μM, 2μM) for 48h. After being stained with JC-1, HeLa
cells were observed under the Laser Scanning Confocal Microscopy. The

4. Materials and Methods
nyl)-4H-chromen-4-one (3a)
4.1 General
Yield 88%, light yellow solid. mp 139-1410C; 1H NMR (400 MHz,
METHANOL-D4) δ 8.08 (s, 1H), 7.37 (d, J = 8.5 Hz, 2H), 6.84 (d, J = 8.5 Hz,
2H), 6.51 (d, J = 1.9 Hz, 1H), 6.35 (d, J = 2.0 Hz, 1H), 4.15 (t, J = 5.6 Hz, 2H),
3.63 (t, J = 5.7 Hz, 4H), 3.01 (t, J = 5.6 Hz, 2H), 2.77 (t, J = 5.7 Hz, 4H). MS
(ESI): Calcd.C21H23NO7, [M+H+] m/z: 402.15, found: 402.1180.
4.2.2.2 7-[3-[bis(2-hydroxyethyl)amino]propoxy]-5-hydroxy-3-(4-hydroxyph
enyl)-4H-chromen-4-one (3b)
All reagents and solvents were purchased in a commercial way and used
without further treatment. The 98.5% pure genistein and 98% pure chrysin
were bought from Ciyuan Biotechnology Co., Ltd of Shanxi. Thin layer
chromatography (TLC) was adopted to monitor reactions on silica gel plates.
Silica gel for purification of the compounds: 100~200 mesh, Qingdao Marine
Chemical Factory, Qingdao, PR China. Melting points were determined on an
1
X-4 micro melting point apparatus and uncorrected. H NMR and 13C NMR
Yield 85%, light yellow solid. mp 157-1590C; 1H NMR (600 MHz,
METHANOL-D4) δ 8.09 (s, 1H), 7.38 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.5 Hz,
2H), 6.52 (s, 1H), 6.35 (s, 1H), 4.14 (t, J = 6.0 Hz, 2H), 3.62 (t, J = 5.7 Hz,
4H), 2.74 (t, J = 7.0 Hz, 2H), 2.68 (t, J = 5.7 Hz, 4H), 2.02 – 1.90 (m, 2H). MS
(ESI): Calcd.C22H25NO7, [M+H+] m/z: 416.16, found: 416.0802.
4.2.2.3 7-[4-[bis(2-hydroxyethyl)amino]butoxy]-5-hydroxy-3-(4-hydroxyphe
nyl)-4H-chromen-4-one (3c)
spectra were recorded on Japan Electron Nuclear Magnetic Resonance
Hydrogen Spectrometer. MS spectra were recorded on the AB SCIEX TRIPLE
TOF 5600+ MS Spectrum.
4.2 Synthesis
4.2.1 General procedure for the synthesis of compounds 2a-c
To a solution of genistein (1mmol, 1eq) in 50mL anhydrous acetone was
added potassium carbonate (2mmol, 2eq) and the mixture was stirred for 15min
at room temperature. Then 1, 2-dibromoethane, 1, 3-dibromopropane, or 1, 4-
dibromobutane (10mmol, 10eq) was slowly added. The reaction was heated to
reflux for 8h. The solvent was removed under vacuum. The resulting residue
was washed with petroleum ether and distilled water successively, and filtered
under reduced pressure. Then the residue was dried and purified by silica gel
column chromatography (MeOH/CH2Cl2, 1:60) to give compounds 2a-c.
4.2.1.1 7-(2-bromoethoxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-o
ne (2a)
Yield 86%, light yellow solid. mp 141-1420C; 1H NMR (400 MHz,
METHANOL-D4) δ 8.08 (s, 1H), 7.40 – 7.35 (m, 2H), 6.87 – 6.82 (m, 2H),
6.50 (d, J = 2.4 Hz, 1H), 6.34 (d, J = 2.0 Hz, 1H), 4.07 (t, J = 6.4 Hz, 2H), 3.62
(t, J = 6.0 Hz, 4H), 2.70 – 2.60 (m, 6H), 1.86 – 1.77 (m, 2H), 1.71 – 1.62 (m,
2H). MS (ESI): Calcd.C23H27NO7, [M+H+] m/z: 430.18, found: 430.3159.
4.2.3 General procedure for the synthesis of compounds 4a-c
To a solution of 3a-c (1mmol, 1eq) in 50mL CH2Cl2 was added SOCl2 (8mmol,
8eq). The reaction was heated to reflux for 48h. The solvent was removed
under vacuum. Then the residue was dried and purified by silica gel column
chromatography (MeOH/CH2Cl2, 1:60) to give compounds 4a-c.
4.2.3.1 7-[2-[bis(2-chloroethyl)amino]ethoxy]-5-hydroxy-3-(4-hydroxypheny
l)-4H-chromen-4-one (4a)
Yield 70%, yellow solid. mp 181-1820C; 1H NMR (400 MHz, DMSO-D6) δ
12.96 (s, 1H), 9.58 (s, 1H), 8.41 (s, 1H), 7.38 (d, J = 8.6 Hz, 2H), 6.82 (d, J =
8.7 Hz, 2H), 6.69 (d, J = 2.3 Hz, 1H), 6.43 (d, J = 2.3 Hz, 1H), 4.47 – 4.43
(m, 2H), 3.86 – 3.78 (m, 2H).
Yield 92%, light yellow solid. mp 148-1500C; 1H NMR (600 MHz,
METHANOL-D4) δ 8.20 (s, 1H), 7.44 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.7 Hz,
2H), 6.68 (d, J = 2.3 Hz, 1H), 6.51 (d, J = 2.3 Hz, 1H), 4.60 – 4.54 (m, 2H),
4.11 (t, J = 6.2 Hz, 4H), 3.94 – 3.89 (m, 2H), 3.86 (t, J = 6.2 Hz, 4H). MS
(ESI): Calcd.C21H21Cl2NO5, [M+H+] m/z: 438.08, found: 438.0252.13C NMR
(101 MHz, METHANOL-D4) δ 180.45, 163.19, 161.76, 157.63, 157.36,
154.50, 130.11, 122.61, 120.89, 115.16, 105.84, 98.56, 93.12, 63.38, 54.12,
51.43, 37.60.
4.2.1.2 7-(3-bromopropoxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-
one (2b)
Yield 69%, yellow solid. mp 130-1320C; 1H NMR (600 MHz, DMSO-D6) δ
9.59 (s, 1H), 8.41 (s, 1H), 7.38 (d, J = 8.6 Hz, 2H), 6.81 (d, J = 8.6 Hz, 2H),
6.68 (d, J = 2.2 Hz, 1H), 6.42 (d, J = 2.2 Hz, 1H), 4.20 (t, J = 6.0 Hz, 2H),
3.66 (t, J = 6.6 Hz, 2H), 2.26 (p, J = 6.3 Hz, 2H).
4.2.1.3 7-(4-bromobutoxy)-5-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-o
ne (2c)
4.2.3.2 7-[3-[bis(2-chloroethyl)amino]propoxy]-5-hydroxy-3-(4-hydroxyphe
nyl)-4H-chromen-4-one (4b)
Yield 75%, yellow solid. mp 138-1400C; 1H NMR (400 MHz, DMSO-D6) δ
12.95 (s, 1H), 9.59 (s, 1H), 8.41 (s, 1H), 7.39 (d, J = 8.7 Hz, 2H), 6.82 (d, J =
8.7 Hz, 2H), 6.66 (d, J = 2.3 Hz, 1H), 6.41 (d, J = 2.3 Hz, 1H), 4.14 (t, J =
6.3 Hz, 2H), 3.62 (t, J = 6.6 Hz, 2H), 2.01 – 1.92 (m, 2H), 1.90 – 1.81 (m,
2H).
1
Yield 91%, light gray solid. mp 282-2890C; H NMR (600 MHz, MET
HANOL-D4) δ 8.15 (s, 1H), 7.41 (d, J = 8.6 Hz, 2H), 6.88 (d, J = 8.6
Hz, 2H), 6.58 (d, J = 2.1 Hz, 1H), 6.41 (d, J = 2.2 Hz, 1H), 4.24 (
t, J = 5.6 Hz, 2H), 4.07 (t, J = 6.1 Hz, 4H), 3.78 (t, J = 6.1 Hz, 4H),
3.65 – 3.55 (m, 2H), 2.36 – 2.30 (m, 2H). MS (ESI): Calcd.C22H23Cl
2NO5, [M+H+] m/z: 452.10, found: 452.0548.13C NMR (101 MHz, DM
SO-D6) δ 180.92, 164.62, 162.27, 158.10, 157.97, 154.93, 130.64, 123.
07, 121.46, 115.65, 106.05, 98.93, 93.38, 66.27, 53.76, 50.44, 37.82, 2
3.25.
4.2.2 General procedure for the synthesis of compounds 3a-c
To a solution of 2a-c (1mmol, 1eq) in 50mL acetonitrile was added
diethanolamine (10mmol, 10eq). The reaction was heated to reflux for 48h.
The mixture was cooled to room temperature to crystallize, filtered under
reduced pressure and washed with water. Then the residue was dried and
purified by silica gel column chromatography (MeOH/CH2Cl2, 1:40) to give
compounds 3a-c.
4.2.3.3 7-[4-[bis(2-chloroethyl)amino]butoxy]-5-hydroxy-3-(4-hydroxyphen
yl)-4H-chromen-4-one (4c)
4.2.2.1 7-[2-[bis(2-hydroxyethyl)amino]ethoxy]-5-hydroxy-3-(4-hydroxyphe

Yield 87%, light gray solid. mp 235-2500C; 1H NMR (600 MHz,
METHANOL-D4) δ 8.12 (s, 1H), 7.46 – 7.27 (m, 2H), 6.91 – 6.76 (m, 2H),
6.55 (d, J = 2.2 Hz, 1H), 6.38 (d, J = 2.2 Hz, 1H), 4.16 (t, J = 5.8 Hz, 2H),
4.01 (t, J = 6.1 Hz, 4H), 3.72 (t, J = 6.0 Hz, 4H), 3.53 – 3.34 (m, 2H), 2.02 –
1.95 (m, 2H), 1.95 – 1.89 (m, 2H). MS (ESI): Calcd.C23H25 Cl2NO5, [M+H+]
m/z: 466.11, found: 466.25.13C NMR (101 MHz, DMSO-D6) δ 180.90,
164.91, 162.26, 158.09, 157.98, 154.89, 130.64, 123.02, 121.48, 115.64,
105.92, 98.89, 93.36, 68.36, 53.59, 52.80, 37.80, 25.97, 20.24.
romen-4-one (7b)
Yield 89%, light yellow solid. mp 100-1020C; 1H NMR (600 MHz,
CHLOROFORM-D) δ 12.71 (s, 1H), 7.88 (dd, J = 8.1, 1.4 Hz, 2H), 7.58 – 7.48
(m, 3H), 6.66 (s, 1H), 6.51 (d, J = 2.2 Hz, 1H), 6.37 (d, J = 2.2 Hz, 1H), 4.14
(t, J = 5.9 Hz, 2H), 3.66 (t, J = 5.2 Hz, 4H), 2.78 (t, J = 6.8 Hz, 2H), 2.71 (t, J
= 5.2 Hz, 4H), 2.07 – 1.88 (m, 2H). MS (ESI): Calcd.C22H25NO6, [M+H+] m/z:
400.17, found: 400.1818.
4.2.5.3 7-[4-[bis(2-hydroxyethyl)amino]butoxy]-5-hydroxy-2-phenyl-4H-chr
omen-4-one (7c)
4.2.4 General procedure for the synthesis of compounds 6a-c
To a solution of chrysin (1mmol, 1eq) in 50mL anhydrous acetone was added
potassium carbonate (4mmol, 4eq) and the mixture was stirred for 15min at
room temperature. Then 1,2-dibromoethane, 1,3-dibromopropane, or 1,4-
dibromobutane (10mmol, 10eq) was slowly added. The reaction was heated to
reflux for 8h. The solvent was removed under vacuum. The resulting residue
was washed with petroleum ether and distilled water successively, and filtered
under reduced pressure. Then the residue was dried and purified by silica gel
column chromatography (CH2Cl2) to give compounds 6a-c.
Yield 96%, light yellow solid. mp 83-850C; 1H NMR (600 MHz,
METHANOL-D4) δ 7.95 (d, J = 7.3 Hz, 2H), 7.58 – 7.49 (m, 3H), 6.71 (s,
1H), 6.61 (s, 1H), 6.30 (d, J = 1.3 Hz, 1H), 4.06 (t, J = 5.9 Hz, 2H), 3.60 (t, J
= 5.9 Hz, 4H), 2.65 (t, J = 5.9 Hz, 4H), 2.63 – 2.59 (m, 2H), 1.83 – 1.77 (m,
2H), 1.69 – 1.62 (m, 2H). MS (ESI): Calcd.C23H27NO6, [M+H+] m/z: 414.18,
found: 414.1972.
4.2.6 General procedure for the synthesis of compounds 8a-c
To a solution of 7a-c (1mmol, 1eq) in 50mL CH2Cl2 was added SOCl2 (8mmol,
8eq). The reaction was heated to reflux for 48h. The solvent was removed
under vacuum. Then the residue was dried and purified by silica gel column
chromatography (MeOH/CH2Cl2, 1:100) to give compounds 8a-c.
4.2.6.1 7-[2-[bis(2-chloroethyl)amino]ethoxy]-5-hydroxy-2-phenyl-4H-chro
men-4-one (8a)
4.2.4.1 7-(2-bromoethoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (6a)
Yield 67%, yellow solid. mp 157-1590C; 1H NMR (600 MHz,
CHLOROFORM-D) δ 12.74 (s, 1H), 7.91 – 7.86 (m, 2H), 7.63 – 7.45 (m, 3H),
6.68 (s, 1H), 6.53 (d, J = 2.2 Hz, 1H), 6.38 (d, J = 2.2 Hz, 1H), 4.37 (t, J = 6.3
Hz, 2H), 3.67 (t, J = 6.3 Hz, 2H).
4.2.4.2 7-(3-bromopropoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (6b)
Yield 77%, yellow solid. mp 163-1670C; 1H NMR (400 MHz,
CHLOROFORM-D) δ 12.72 (s, 1H), 7.94 – 7.84 (m, 2H), 7.60 – 7.47 (m, 3H),
6.67 (s, 1H), 6.52 (d, J = 2.3 Hz, 1H), 6.38 (d, J = 2.2 Hz, 1H), 4.20 (t, J = 5.8
Hz, 2H), 3.61 (t, J = 6.4 Hz, 2H), 2.41 – 2.29 (m, 2H).
Yield 84%, light yellow solid. mp 138-1400C; 1H NMR (400 MHz,
METHANOL-D4) δ 8.02 (d, J = 6.6 Hz, 2H), 7.66 – 7.52 (m, 3H), 6.86 – 6.79
(m, 2H), 6.49 (d, J = 1.7 Hz, 1H), 4.61 – 4.53 (m, 2H), 4.08 (t, J = 6.0 Hz, 4H),
3.95 – 3.79 (m, 6H). MS (ESI): Calcd.C21H21Cl2NO4, [M+H+] m/z: 422.08,
found: 422.0545.13C NMR (101 MHz, DMSO-D6) δ 182.66, 164.12, 164.00,
161.71, 157.79, 132.76, 131.06, 129.71, 127.00, 105.97, 105.83, 99.18, 94.07,
55.47, 54.83, 52.01.
4.2.4.3 7-(4-bromobutoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (6c)
Yield 89%, yellow solid. mp 147-1480C; 1H NMR (400 MHz,
CHLOROFORM-D) δ 12.69 (s, 1H), 7.89 – 7.85 (m, 2H), 7.57 – 7.48 (m, 3H),
6.65 (s, 1H), 6.48 (d, J = 2.2 Hz, 1H), 6.35 (d, J = 2.2 Hz, 1H), 4.07 (t, J = 6.0
Hz, 2H), 3.49 (t, J = 6.4 Hz, 2H), 2.12 – 2.03 (m, 2H), 2.03 – 1.94 (m, 2H).
4.2.5 General procedure for the synthesis of compounds 7a-c
To a solution of 6a-c (1mmol, 1eq) in 50mL acetonitrile was added
diethanolamine (10mmol, 10eq). The reaction was heated to reflux for 48h.
The mixture was cooled to room temperature to crystallize, filtered under
reduced pressure and washed with water. Then the residue was dried and
purified by silica gel column chromatography (MeOH/CH2Cl2, 1:60) to give
compounds 7a-c.
4.2.6.2 7-[3-[bis(2-chloroethyl)amino]propoxy]-5-hydroxy-2-phenyl-4H-chr
omen-4-one (8b)
Yield 80%, light yellow solid. mp 119-1210C; 1H NMR (600 MHz,
METHANOL-D4) δ 8.02 (d, J = 7.1 Hz, 2H), 7.66 – 7.53 (m, 3H), 6.81 (s,
1H), 6.74 (d, J = 1.9 Hz, 1H), 6.42 (d, J = 2.1 Hz, 1H), 4.28 (t, J = 5.6 Hz, 2H),
4.08 (t, J = 6.1 Hz, 4H), 3.80 (t, J = 6.1 Hz, 4H), 3.67 – 3.58 (m, 2H), 2.40 –
2.33 (m, 2H). MS (ESI): Calcd.C22H23 Cl2NO4, [M+H+] m/z: 436.10, found:
436.33.13C NMR (101 MHz, DMSO-D6) δ 182.55, 164.74, 163.98, 161.69,
157.80, 132.68, 131.06, 129.66, 126.94, 105.87, 105.54, 99.02, 93.81, 66.26,
53.75, 50.41, 37.91, 23.35.
4.2.5.1 7-[2-[bis(2-hydroxyethyl)amino]ethoxy]-5-hydroxy-2-phenyl-4H-chr
omen-4-one (7a)
4.2.6.3 7-[4-[bis(2-chloroethyl)amino]butoxy]-5-hydroxy-2-phenyl-4H-chro
men-4-one (8c)
Yield 91%, light yellow solid. mp 165-1670C; 1H NMR (400 MHz,
CHLOROFORM-D) δ 12.71 (s, 1H), 7.87 (dd, J = 8.0, 1.6 Hz, 2H), 7.58 – 7.47
(m, 3H), 6.65 (s, 1H), 6.51 (d, J = 2.2 Hz, 1H), 6.36 (d, J = 2.2 Hz, 1H), 4.12
(t, J = 5.3 Hz, 2H), 3.66 (t, J = 5.2 Hz, 4H), 3.04 (t, J = 5.3 Hz, 2H), 2.82 (t, J
= 5.2 Hz, 4H). MS (ESI): Calcd.C21H23NO6, [M+H+] m/z: 386.15, found:
386.0789.
Yield 79%, light gray solid. mp 150-1510C; 1H NMR (600 MHz,
METHANOL-D4) δ 8.02 (s, 2H), 7.80 – 7.29 (m, 3H), 6.79 (s, 2H), 6.39 (s,
1H), 4.22 (s, 2H), 4.04 (s, 4H), 3.73 (s, 4H), 3.45 (s, 2H), 2.29 – 1.78 (m,
4H).MS (ESI): Calcd.C23H25Cl2NO4, [M+H+] m/z: 450.12, found: 450.22.13C
NMR (101 MHz, DMSO-D6) δ 182.55, 165.08, 163.96, 161.69, 157.84,
132.71, 131.10, 129.71, 126.98, 105.89, 105.43, 99.03, 93.80, 68.46, 53.57,
52.93, 37.66, 26.08, 20.26.
4.2.5.2 7-[3-[bis(2-hydroxyethyl)amino]propoxy]-5-hydroxy-2-phenyl-4H-ch
4.3 MTT assay
The MTT assay was used to evaluate antiproliferative activities of compounds

4a-c and 8a-c against seven human cancer cell lines (HeLa, A549, HepG2,
MCF7, SH-SY5Y, PC-3, DU145) in vitro, with melphalan used as the positive
control. Tumor Cells were seeded in 96-well plates and incubated at 370C in
an atmosphere containing 5% CO2 overnight. After being treated with various
concentrations of the synthesized compounds and the positive control drug for
48h, every well was added 20μL MTT (5mg/mL) and 100μL DMEM and
incubated at 370C for 4 h. Then the upper liquid was removed and 150μL
DMSO was added. Being incubated at 370C for 20min, the absorbance (OD)
values were measured at 490nm by a microplate reader (Epoch, BioTek
Instruments). The inhibitory concentration 50% (IC50) was calculated by SPSS
20.
cytometry.
4.6 Cell cycle assay
HeLa cells were seeded in 6-well plates and treated with different
concentrations of compound 8b (0μM, 0.5μM, 0.8μM) for 48h after being
incubated overnight. The cells were collected, washed with PBS, and fixed
with 70% ethanol at 40C for 24h. Then the cells were washed with PBS, treated
with 100μL RNase A at 370C for 30min and stained with PI at 40C for 30min
in the darkness. At last, the treated cells were analyzed by flow cytometry to
quantify cell cycle distribution.
4.7 Mitochondrial membrane potential
HeLa cells were seeded in glass bottom of cell culture dishes and treated with
compound 8b (0μM, 1μM, 2μM) for 48h after being incubated overnight. Then
the cells were washed with PBS and stained with JC-1 at 370C for 20min in
darkness. After being washed with JC-1 binding buffer (1 ×), the cells were
observed with the Laser Scanning Confocal Microscopy.
Author contributions
4.4 The morphological analysis
HeLa cells were seeded in glass bottom of cell culture dishes and treated with
compound 8b (0μM, 1μM, 2μM, 4μM) for 48h after being incubated overnight.
Then the cells were washed with PBS and stained with DAPI at 370C for 15min
(10μg/mL in PBS) in darkness. After being washed with PBS, the cells were
observed with the Laser Scanning Confocal Microscopy.
The manuscript was written through contributions of all authors. All authors
have given approval to the final version of the manuscript.
Acknowledgments
4.5 Cell apoptosis assay
HeLa cells were seeded in 6-well plates and treated with different
concentrations of compound 8b (0μM, 0.3μM, 0.8μM, 1.5μM) for 48h after
being incubated overnight. The cells were collected and washed with cold PBS
This work was supported by the National Science and Techonology Major
Project (2014zx09507007-003).
×
Declarations of interest
and Binding Buffer (1 ). Then the cells were resuspended with 100μL
×
None.
Binding Buffer(1 ), stained with 5μL Annexin V-FITC for 10min and 5μL
PI for 5min at room temperature in the darkness and analyzed by flow
Appendix A . Supplementary data
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Graphical abstract

Highlights
Six new flavonoids nitrogen mustard derivatives were synthesized. 8b, the most promising compound, could
arrest cell cycle of HeLa in G2/M phase, induce cell apoptosis and cause the loss of mitochondrial membrane
potential.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential
competing interests: