Design, Synthesis and Anticancer Evaluation of New 7-O-Alkylation Genistein Derivatives

Article abstract of DOI:10.1007/s11094-020-02298-5

Genistein is a phytoestrogen compound which possesses multiple biological activities such as anti-cancer, while its application is limited by poor pharmacokinetic properties. Structural modification is an effective approach to get genistein derivatives wi

Full text of DOI:10.1007/s11094-020-02298-5

DOI 10.1007/s11094-020-02298-5
Pharmaceutical Chemistry Journal, Vol. 54, No. 9, December, 2020 (Russian Original Vol. 54, No. 9, September, 2020)
DESIGN, SYNTHESIS AND ANTICANCER EVALUATION
OF NEW 7-O-ALKYLATION GENISTEIN DERIVATIVES
Yuyan Ren,1,2,3,** Hongfei Chen,1,2,3,** Xu Yao,1,2,3 Zehua Yang,1,2,3
Tingjuan Wu,1,2,3 Yu Guo,1,2 Junhui Xiao,1,2 and Xing Zheng1,2,3,*
Original article submitted October 27, 2019.
Genistein is a phytoestrogen compound which possesses multiple biological activities such as anti-cancer,
while its application is limited by poor pharmacokinetic properties. Structural modification is an effective ap-
proach to get genistein derivatives with better activities and approved pharmacokinetic properties. Based on
previous research work, we synthesized two series of genistein derivatives bearing halogen substituents, and
evaluated their inhibitory effects on the cancer cell lines MCF-7, MDA-MB-231, and MDA-MB-435. Among
these derivatives, compound XI displayed the best inhibitory activity against MCF-7, MDA-MB-231 and
MDA-MB-435 cell lines in vitro, which is worth further studies.
Keywords: genistein derivatives; isoflavone; anticancer activity; MTT assay; synthesis.
1. INTRODUCTION
vivo instability, and poor solubility [13]. Up to now, numer-
ous research works have studied the structure activity
relationship of genistein and its derivatives, in the hope of
getting potent anti-cancer agents through structural modifi-
cation [14 – 16].
Breast cancer is the fifth leading cause of cancer-related
death worldwide and the second leading cause of cancer
death among women, accounting for approximately 626679
deaths in 2018 [1]. It is noteworthy that the breast cancer
morbidity in some Asian countries such as China and Japan
is on a relatively lower level [2]. This is probably related
with Asian peoples having high consumption of beans that
are rich in flavonoids. As a member of plant polyphenols, the
flavonoid phenol groups in their structures may contribute to
their anticancer activity [3].
Our group had previously designed and synthesized sev-
eral series of flavone derivatives and carried out their activity
evaluation in vitro and in vivo, which showed that many of
them possessed potent anticancer activity [17 – 19]. Inspired
by the previous research work, we have designed and synthe-
sized two series of genistein derivatives and evaluated their
anticancer activity in vitro against MCF-7, MDA-MB-231,
and MDA-MB-435 cell lines.
Genistein (4¢,5,7-trihydroxyisoflavone) is an isoflavo-
noid compound widely distributed in plants [4, 5], which had
been emphasized by more than 3600 papers in the past de-
cade because of its multiple biological effects such as the
anticancer effect [6 – 9] and especially anti-breast cancer ac-
tivity [19, 11]. Genistein is also a phytoestrogen due to its
structural features similar to those of estradiol-17b estrogen
[12]. However, the application of genistein is limited because
of its structural characteristics, susceptibility to oxidation, in
2. RESULTS AND DISCUSSION
2.1. Chemistry
All the new genistein derivatives were synthesized via a
route presented in Fig. 1 under the indicated optimum condi-
tions. Phloroglucinol and two kinds of benzacetonitrile (Ia
and Ib) were used as starting compounds for the preparation
of target genistein derivatives. The yield of IIb was signifi-
cantly higher than that of IIa, mainly in the hydrolysis pro-
cess of recrystallization. In step 2, we used icy water to
quench the reaction, after which a pale yellow precipitate
was obtained. In the presence of KI in step 3, the reaction
1
Department of Pharmacy, University of South China, Hengyang, 421001,
China.
2
Hunan Province Cooperative Innovation Center for Molecular Target New
Drug Study, Hengyang, 421001, China.
3
Group of Lead Compound, University of South China, Hengyang,
421001, China.
*
e-mail: zhengxing5018@yahoo.com; zhengxing9166@sohu.com
**
These two authors contributed equally.
924
0091-150X/20/5409-0924 © 2020 Springer Science+Business Media, LLC

Design, Synthesis and Anticancer Evaluation of New 7-O-Alkylation Genistein Derivatives
925
Fig. 1. Synthetic route for compounds II – XXI. Reagents and conditions: (i) ZnCl , HCl (g), Et O, 0°C; (ii) PCl , BF .Et O, DMF; (iii)
2
2
5
3
2
K CO , KI, RBr, acetone, reflux.
2
3
time could be greatly shortened, and the reflux temperature
could also be lowered [20].
that compounds show decreased total score when the alkyl
side chains become too long (³ 9 atoms) as can be explained
by their steric hindrances.
All halogenated hydrocarbons used in the alkylation re-
action were bromo-alkanes. Due to the effect of hydrogen
bonding between 7-OH group and 4-carbonyl, the alkylation
of genistein only occurred at the 7-OH group. All products
were accurately analyzed by spectroscopic techniques, and
the results were in full accordance with the proposed struc-
tures (Fig. 2).
From the binding mode of these compounds with estro-
gen receptor (Fig. 3), it can be seen that the alkyl side chain
is located in the hydrophobic pocket, forming hydrophobic
interaction with the receptor.
2.3. Biological Activity
Totally 18 genistein derivatives were evaluated for their
antiproliferative activity against MCF-7, MDA-MB-231, and
MDA-MB-435 cell lines. The results are summarized in Ta-
ble 2. From these results, we preliminarily concluded that
some of the synthesized compounds displayed cellular activ-
ity comparable with the positive control drugs cisplatin
(DDP) and Tamoxifen (TAM). Among all the drugs tested,
compounds IX, XI, XIV and XV showed moderate
antiproliferative activity. Compound XI showed the best ac-
tivity, while other compounds did not exhibit evident inhibi-
tory effect.
2.2. Virtual Screening
Based on the structure of estrogen receptor and the previ-
ous structure activity relationship study, we applied CADD
(computer-aided drug design) to the rational design of the
genistein derivatives, and used the SYBYL-Surflex docking
to test the compounds’ in silico affinity for the estrogen re-
ceptor.
According to the virtual screening results (Table 1), com-
pounds with longer alkyl side chains were expected to be
stronger estrogen blockers, the probable reason being that the
long lipophilic side chains are preferred for binding to the
hydrophobic pocket of estrogen receptor. However, it seems
As was mentioned above, the virtual screening results
showed that compounds with longer alkyl side chains were

926
Yuyan Ren et al.
Fig. 2. Two series of genistein derivatives.
likely to be better anti-breast cancer agents. However, the
cellular evaluation results were quite another story, in which
compounds VIII and XIX with the longest alkyl side chains
did not show any activity. The possible reason for this dis-
crepancy is that the activity of compounds is dramatically af-
fected by their hydrophilicity/lipophilicity balance.
As can be seen from Table 2, in series 1, compound IX
with C H side chain is more powerful than compounds
2
5
(such as IV, V, and VI) with longer side chains. This is prob-
ably due to decreased hydrophilicity of compounds IV, V,
and VI caused by their longer alkyl side chains. Compound
XI with a longer C H Br chain exhibits the best activity,
4
8

Design, Synthesis and Anticancer Evaluation of New 7-O-Alkylation Genistein Derivatives
927
which can be explained by its increased hydrophilicity re-
lated to a more stable O- ion form. In series 2 (because of the
lower lipophilicity of -Br group compared to -CF group)
3
compounds need relatively more lipophilic groups to get
better activity. Accordingly, compounds XIV and XV
showed the best potential activity in this series. These results
indicate that the 7-O-alkylation of genistein is an effective
approach to obtaining compounds with improved antiproli-
ferative activity. Further pharmacodynamic and pharmaco-
kinetic evaluation as well as toxicological study of these lead
compounds is underway, in the hope of producing drug can-
didates for drug development.
3. EXPERIMENTAL CHEMICAL PART
3.1. Instrumentation and Chemicals
Fig. 3. The binding mode of a designed genistein derivative with
Melting points were measured on a Kofler apparatus, un-
corrected. The 1H NMR and 13C NMR spectra were recorded
estrogen receptor.
on a Bruker AM 400(400-MHz) spectrometer in CDCl with
3
Me Si as internal standard. All chemical shifts (d ) are ex-
4
5-Hydroxy-7-propoxy-3-(3-(trifluoromethyl)phenyl)-
pressed as parts per million (ppm) and coupling constants (J)
are given in Hertz. Mass spectra were recorded on Waters
Micromass GCT Premier and Thermo Fisher Scientific LTO
FT Ultra using EI ionization at 70 eV and DART positive op-
eration mode. All reagents were commercial materials used
without further purification.
4H-chromen-4-one (IV): white needles, 0.24 g, yield:
1
65.6%, m.p. 125 – 128°C, H NMR (400 MHz, CDCl ) d
12.63 (s, 1H), 7.93 (s, 1H), 7.80 – 7.73 (m, 2H), 7.66 (d,
3
3.2. General Procedures for Preparation of Compounds
TABLE 1. Results Virtual Screening of the Synthesized Com-
IX – XXI
pounds
A mixture of compound I (10 mmol, Ia 1.85 g, or Ib
B ring substituent
Compound
Side chain
Total score
1.96 g), phloroglucin (12 mmol, 1.52 g), ZnCl (10 mmol,
2
1.36 g), and 80 mL of anhydrous diethyl ether were stirred
on ice bath for 4 – 8 h under addition of dry HCl gas, and
then the mixture was put into fridge for two days. Then, the
addition of dry HCl gas was continued for another 4 – 8 h on
ice bath, after which the mixture was placed in fridge for
IX
IV
C2H5
C3H7
4.7837
6.2982
7.9467
7.2333
7.8648
8.4146
9.1928
9.7859
8.3277
5.2892
6.3325
6.7209
6.9042
7.1115
8.2815
9.2914
8.4162
5.9875
3¢-CF3
3¢-CF3
3¢-CF3
3¢-CF3
3¢-CF3
3¢-CF3
3¢-CF3
3¢-CF3
3¢-CF3
4¢-Br
V
C4H9
VI
C5H11
C6H13
C7H15
C8H17
C9H19
C4H8Br
C2H5
three days. The obtained solid was recrystallized in H O to
VII
X
2
give light yellow solid II (yield 54%, IIa 3.12 g, or IIb
3.22 g). Intermediate II (5 mmol, IIa 1.56 g, or IIb 1.62 g)
was dissolved in 30 mL DMF, after which a mixture of boron
trifluoride etherate (40 mmol, 5.68 g) and DMF (40 mmol,
3.1 mL) was added dropwise (on ice bath) and 20 – 30 min
later the mixture was transferred onto 80°C oil bath, and a
XII
VIII
XI
XX
XIII
XV
XIV
XVI
XVII
XVIII
XIX
XXI
mixture of PCl (15 mmol, 3.12 g) and DMF (15 mmol,
5
C3H7
4¢-Br
1.17 mL) was added dropwise. Then, the reaction mixture
was refluxed for 3 – 5 h, ice water was added, and the mix-
ture was filtered to get light yellow solid III (yield 85%, IIIa
1.37 g, or IIIb 1.41 g). A mixture of III (1 mmol, IIIa
0.32 g, or IIIb 0.33 g), K CO (3 mmol, 0.41 g), KI (1mmol,
C4H9
4¢-Br
C5H11
C6H13
C7H15
C8H17
C9H19
C4H8Br
4¢-Br
4¢-Br
2
3
4¢-Br
0.17 g), RBr (3 mmol), and 30 mL of acetone was stirred at
60 – 80°C with reflux for 2 – 5 h. Then, the mixture was con-
centrated and purified by flash column chromatography to
obtain the desired product as white solid or pale yellow nee-
dles.
4¢-Br
4¢-Br
4¢-Br

928
Yuyan Ren et al.
J = 7.9 Hz, 1H), 7.58 (t, J = 7.8 Hz, 1H), 6.43 (d, J = 2.3 Hz,
1H), 6.40 (d, J = 2.3 Hz, 1H), 4.00 (t, J = 6.6 Hz, 2H),
1.90 – 1.80 (m, 2H), 1.06 (t, J = 7.4 Hz, 3H). 13C NMR
5-Hydroxy-7-(pentyloxy)-3-(3-(trifluoromethyl)phenyl)-
4H-chromen-4-one (VI: white needles, 0.25 g, yield:
1
63.3%, m.p. 110 – 112°C, H NMR (400 MHz, CDCl ) d
3
(101 MHz, CDCl ) d 180.0, 165.4, 162.6, 157.9, 153.5,
12.62 (s, 1H), 7.92 (s, 1H), 7.80 – 7.72 (m, 2H), 7.65 (d,
J = 7.9 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H), 6.41 (d, J = 2.2 Hz,
1H), 6.39 (d, J = 2.2 Hz, 1H), 4.02 (t, J = 6.6 Hz, 2H),
1.85 – 1.77 (m, 2H), 1.47 – 1.36 (m, 4H), 0.94 (t, J = 7.1 Hz,
3
132.3(d, J = 1.0 Hz), 131.7, 131.0 (q, J = 32.4 Hz), 129.0,
125.6 (q, J = 3.9 Hz), 125.1 (q, J = 3.7 Hz), 128.09 – 119.79
(m), 122.8, 106.0, 98.9, 93.1, 70.2, 22.3, 10.4. MS (EI, 70
eV): 364.0923 (C H O F ; calc. 364.0922).
3H). 13C NMR (101 MHz, CDCl ) d 180.0, 165.4, 162.6,
19 15
4 3
3
7-Butoxy-5-hydroxy-3-(3-(trifluoromethyl)phenyl)-4H-
157.8, 153.5, 132.3 (d, J = 0.9 Hz), 131.7, 131.0 (q,
J = 32.4 Hz), 129.0, 125.6 (q, J = 3.8 Hz), 125.1 (q,
J = 3.6 Hz), 128.04 – 119.83 (m), 122.7, 105.9, 98.9, 93.1,
68.8, 28.6, 28.0, 22.4, 14.0. MS (EI, 70 eV): 392.1236
(C H O F , calc. 392.1235).
chromen-4-one (V): white needles, 0.26 g, yield: 69.2%,
m.p. 135 – 137°C, 1H NMR (400 MHz, CDCl ) d 12.63 (s,
3
1H), 7.93 (s, 1H), 7.80 – 7.73 (m, 2H), 7.66 (d, J = 7.9 Hz,
1H), 7.58 (t, J = 7.7 Hz, 1H), 6.42 (d, J = 2.2 Hz, 1H), 6.40
(d, J = 2.3 Hz, 1H), 4.04 (t, J = 6.5 Hz, 2H), 1.81 (dt,
J = 14.4, 6.5 Hz, 2H), 1.50 (dt, J = 14.8, 7.4 Hz, 2H), 0.99 (t,
21 19
4 3
7-(Hexyloxy)-5-hydroxy-3-(3-(trifluoromethyl)phenyl)-
4H-chromen-4-one (VII): white needles, 0.23 g, yield:
57.7%, m.p. 97 – 99°C, 1H NMR (400 MHz, CDCl ) d 12.63
J = 7.4 Hz, 3H). 13C NMR (101 MHz, CDCl ) d 180.0,
3
3
165.4, 162.6, 157.9, 153.5, 132.3 (d, J = 1.0 Hz), 131.7,
131.0 (q, J = 32.4 Hz), 129.0, 125.6 (q, J = 3.8 Hz), 125.1 (q,
J = 3.7 Hz), 128.05 – 119.79 (m), 122.8, 106.0, 98.9, 93.1,
68.5, 30.9, 19.1, 13.7. MS (EI, 70 eV): 378.1086
(C H O F ; calc. 378.1079).
(s, 1H), 7.93 (s, 1H), 7.79 – 7.74 (m, 2H), 7.66 (d,
J = 7.8 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H), 6.42 (d, J = 2.2 Hz,
1H), 6.39 (d, J = 2.3 Hz, 1H), 4.03 (t, J = 6.6 Hz, 2H),
1.85 – 1.78 (m, 2H), 1.51 – 1.43 (m, 2H), 1.37 – 1.33 (m,
4H), 0.92 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl ) d
20 17
4 3
3
TABLE 2. Anti-Breast Cancer Cell Activity of Synthesized Compounds
MCF-7
MBA-MD-231
MBA-MD-435
Compound
IC50, m M
(n = 5)
IC50, m M
(n = 5)
IC50, m M
(n = 5)
aEmax, %
aEmax, %
aEmax, %
DDP
TAM
IV
2.3 ± 0.1
11.1 ± 1.9
>100
96.8 ± 3.1
94.4 ± 5.6
<50
2.7 ± 0.3
13.4 ± 1.5
>100
95.3 ± 5.8
84.8 ± 4.6
<50
6.37 ± 0.4
13.1 ± 2.1
>100
92.1 ± 4.3
90.5 ± 10.5
<50
V
>100
<50
>100
<50
>100
<50
VI
>100
<50
>100
<50
>100
<50
VII
VIII
IX
>100
<50
>100
<50
>100
<50
>100
<50
>100
<50
>100
<50
25.1 ± 3.4
>100
85.5 ± 5.5
<50
32.7 ± 2.8
>100
84.0 ± 6.6
<50
30.7 ± 2.9
>100
77.5 ± 8.2
<50
X
XI
14.6 ± 2.2
>100
93.9 ± 8.4
<50
18.6 ± 2.8
>100
85.6 ± 9.4
<50
22.3 ± 1.8
>100
83.9 ± 4.2
<50
XII
XIII
XIV
XV
>100
<50
>100
<50
>100
<50
18.2 ± 2.8
25.9 ± 4.7
>100
89.7 ± 9.1
87.6 ± 7.8
<50
29.0 ± 4.4
46.8 ± 5.2
>100
78.3 ± 3.8
75.3 ± 6.0
<50
39.3 ± 6.9
52.0 ± 4.4
>100
72.3 ± 10.2
64.5 ± 2.9
<50
XVI
XVII
XVIII
XIX
XX
>100
<50
>100
<50
>100
<50
>100
<50
>100
<50
>100
<50
>100
<50
>100
<50
>100
<50
>100
<50
>100
<50
>100
<50
XXI
>100
<50
>100
<50
>100
<50
a E
is the maximum effect at high drug concentration.
max

Design, Synthesis and Anticancer Evaluation of New 7-O-Alkylation Genistein Derivatives
929
122.8, 106.1, 98.8, 93.1, 67.6, 33.1, 29.2, 27.6. ESI-MS:
457.0254 (C H O BrF , (M+H(+).
180.0, 165.4, 162.6, 157.7, 153.5, 132.3 (d, J = 0.9 Hz),
131.7, 131.0 (q, J = 32.4 Hz), 129.0, 125.6 (q, J = 3.9 Hz),
125.1 (q, J = 3.7 Hz), 128.05 – 119.82 (m), 122.8, 106.0,
98.9, 93.1, 68.8, 31.5, 28.9, 25.6, 22.6, 14.0. MS (EI, 70 eV):
406.1389 (C H O F ; calc. 406.1392).
+
20 17
4
3
5-Hydroxy-7-(octyloxy)-3-(3-(trifluoromethyl)phenyl)-
4H-chromen-4-one (XII): white needles, 0.21 g, yield:
48.5%, m.p. 90 – 92°C, 1H NMR (400 MHz, CDCl ) d 12.65
3
22 21
4 3
(s, 1H), 7.95 (s, 1H), 7.82 – 7.75 (m, 2H), 7.68 (d,
J = 7.8 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 6.44 (d, J = 2.2 Hz,
1H), 6.41 (d, J = 2.2 Hz, 1H), 4.05 (t, J = 6.6 Hz, 2H),
1.88 – 1.79 (m, 2H), 1.48 (dd, J = 15.0, 7.0 Hz, 2H),
1.41 – 1.29 (m, 8H), 0.92 (t, J = 6.9 Hz, 3H). 13C NMR
5-Hydroxy-7-(nonyloxy)-3-(3-(trifluoromethyl)phenyl)-
4H-chromen-4-one (VIII): white needles, 0.23 g, yield:
52.4%, m.p. 85 – 88°C, 1H NMR (400 MHz, CDCl ) d 12.61
3
(s, 1H), 7.90 (d, J = 2.3 Hz, 1H), 7.78 – 7.70 (m, 2H), 7.63
(d, J = 7.7 Hz, 1H), 7.55 (t, J = 7.7 Hz, 1H), 6.39 (t,
J = 2.2 Hz, 1H), 6.36 (d, J = 2.3 Hz, 1H), 4.00 (td, J = 6.4,
1.5 Hz, 2H), 1.83 – 1.74 (m, 2H), 1.48 – 1.39 (m, 2H),
1.37 – 1.23 (m, 10H), 0.87 (t, J = 6.7 Hz, 3H). 13C NMR
(101 MHz, CDCl ) d 180.0, 165.4, 162.6, 157.9, 153.5,
3
132.3 (d, J = 1.0 Hz), 131.7, 131.0(q, J = 32.4 Hz), 129.0,
125.6 (q, J = 3.8 Hz), 125.1 (q, J = 3.7 Hz), 128.09 – 119.74
(m), 122.8, 106.0, 98.9, 93.1, 68.8, 31.8, 29.3, 29.2, 28.9,
(101 MHz, CDCl ) d 180.0, 165.5, 162.7, 157.9, 153.5,
25.9, 22.6, 14.1. ESI-MS: 435.1776 (C H O F , (M+H(+).
+
3
24 26
4 3
132.3 (d, J = 0.8 Hz), 131.7, 131.0 (q, J = 32.5 Hz), 129.0,
125.6 (q, J = 3.8 Hz), 125.1 (q, J = 3.7 Hz), 128.06 – 119.82
(m), 122.8, 106.0, 98.9, 93.1, 68.8, 31.9, 29.5, 29.3, 29.2,
28.9, 25.9, 22.7, 14.1. MS (EI, 70 eV): 448.1872
(C H O F ; calc. 448.1861).
3-(4-Bromophenyl)-5-hydroxy-7-propoxy-4H-chromen-
4-one (XIII): white needles, 0.27 g, yield: 72.0%, m.p.
1
140 – 142°C, H NMR (400 MHz, CDCl ) d 12.69 (s, 1H),
3
7.87 (s, 1H), 7.57 (d, J = 8.3 Hz,2H), 7.41 (d, J = 8.3 Hz,
2H), 6.39 (d, J = 7.2 Hz, 2H), 3.98 (t, J = 6.5 Hz, 2H),
1.89 – 1.78 (m, 2H), 1.05 (t, J = 7.4 Hz, 3H). 13C NMR
25 27
4 3
7-Ethoxy-5-hydroxy-3-(3-(trifluoromethyl)phenyl)-
4H-chromen-4-one (IX): white needles, 0.26 g, yield:
1
(101 MHz, CDCl ) d 180.2, 165.3, 162.6, 157.8, 153.1,
3
75.2%, m.p. 180 – 182°C, H NMR (400 MHz, CDCl ) d
3
131.7, 130.4, 129.7, 122.9, 122.6, 106.0, 98.8, 93.0, 70.2,
22.3, 10.4. ESI-MS: 375.0226 (C H O Br+, (M+H(+).
12.66 (s, 1H), 7.95 (s, 1H), 7.82 – 7.76 (m, 2H), 7.69 (d,
J = 7.8 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 6.44 (d, J = 2.2 Hz,
1H), 6.42 (d, J = 2.2 Hz, 1H), 4.14 (q, J = 7.0 Hz, 2H), 1.48
18 16
4
3-(4-Bromophenyl)-5-hydroxy-7-(pentyloxy)-4H-chro-
men-4-one (XIV): white needles, 0.27 g, yield: 67.4%, m.p.
1
(t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl ) d 180.1,
3
117 – 119°C, H NMR (400 MHz, CDCl ) d 12.66 (s, 1H),
3
165.2, 162.7, 157.9, 153.5, 132.3 (d, J = 1.0 Hz), 131.7,
131.0 (q, J = 32.4 Hz), 129.0, 125.6 (q, J = 3.8 Hz), 125.1 (q,
J = 3.7 Hz), 128.12 – 119.77 (m), 122.8, 106.0, 98.9, 93.1,
7.85 (s, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.3 Hz,
2H), 6.37 (d, J = 2.0 Hz, 1H), 6.35 (s, 1H), 3.99 (t,
J = 6.5 Hz, 2H), 1.83 – 1.74 (m, 2H), 1.40 (qd, J = 13.8,
6.7 Hz, 4H), 0.92 (t, J = 6.8 Hz, 3H). 13C NMR (101 MHz,
64.3, 14.5. ESI-MS: 351.0837 (C H O F , (M+H(+).
+
18 14
4 3
7-(Heptyloxy)-5-hydroxy-3-(3-(trifluoromethyl)phenyl)-
CDCl ) d 180.2, 165.3, 162.6, 157.8, 153.1, 131.7, 130.4,
3
4H-chromen-4-one (X): white needles, 0.21 g, yield: 49.2%,
m.p. 90 – 92°C, 1H NMR (400 MHz, CDCl ) d 12.63 (s, 1H),
129.7, 122.9, 122.6, 106.0, 98.8, 93.0, 68.7, 28.6, 28.0, 22.4,
14.0. ESI-MS: 403.0539 (C H O Br+, (M+H(+).
3
20 20
4
7.93 (s, 1H), 7.80 – 7.73 (m, 2H), 7.66 (d, J = 7.8 Hz, 1H),
7.57 (t, J = 7.8 Hz, 1H), 6.42 (d, J = 2.2 Hz, 1H), 6.39 (d,
J = 2.2 Hz, 1H), 4.03 (t, J = 6.6 Hz, 2H), 1.86 – 1.77 (m,
2H), 1.51 – 1.42 (m, 2H), 1.39 – 1.29 (m, 6H), 0.90 (t,
3-(4-Bromophenyl)-7-butoxy-5-hydroxy-4H-chromen-
4-one (XV): white needles, 0.28 g, yield: 71.3%, m.p.
1
123 – 126°C, H NMR (400 MHz, CDCl ) d 12.69 (s, 1H),
3
7.88 (s, 1H), 7.57 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.3 Hz,
2H), 6.40 (d, J = 2.2 Hz, 1H), 6.38 (d, J = 2.1 Hz, 1H), 4.03
(t, J = 6.5 Hz, 2H), 1.84 – 1.75 (m, 2H), 1.49 (dt, J = 14.7,
7.4 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz,
J = 6.8 Hz, 3H). 13C NMR (101 MHz, CDCl ) d 180.1, 165.,
3
162.74 (s), 157.97 (s), 153.59 (s), 132.42 (d, J = 1.0 Hz),
131.8, 131.14 (q, J = 32.6 Hz), 129.1, 125.7 (q, J = 3.8 Hz),
125.2 (q, J = 3.8 Hz), 128.23 – 119.85 (m), 122.9, 106.1,
99.0, 93.2, 68.9, 31.8, 29.1, 29.0, 26.0, 22.7, 14.2. ESI-MS:
CDCl ) d 180.2, 165.3, 162.6, 157.8, 153.1, 131.7, 130.4,
129.7, 122.9, 122.6, 106.0, 98.8, 93.0, 68.4, 30.9, 19.1, 13.8.
ESI-MS: 389.0384 (C H O Br+, (M+H(+).
3
421.1620 (C H O F , (M+H(+).
+
23 24
4 3
19 18
4
7-(4-Bromobutoxy)-5-hydroxy-3-(3-(trifluoromethyl)-
phenyl)-4H-chromen-4-one (XI): white needles, 0.24 g,
yield: 53.4%, m.p. 113 – 116°C, 1H NMR (400 MHz,
3-(4-Bromophenyl)-7-(hexyloxy)-5-hydroxy-4H-chro-
men-4-one (XVI): white needles, 0.25 g, yield: 60.4%, m.p.
1
122 – 124°C, H NMR (400 MHz, CDCl ) d 12.69 (s, 1H),
3
CDCl ) d 12.64 (s, 1H), 7.94 (s, 1H), 7.81 – 7.73 (m, 2H),
7.88 (s, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.3 Hz,
2H), 6.40 (d, J = 2.2 Hz, 1H), 6.38 (d, J = 2.2 Hz, 1H), 4.02
(t, J = 6.6 Hz, 2H), 1.86 – 1.75 (m, 2H), 1.47 (dt, J = 14.4,
7.0 Hz, 2H), 1.39 – 1.31 (m, 4H), 0.92 (t, J = 6.8 Hz, 3H).
3
7.66 (d, J = 7.8 Hz, 1H), 7.58 (t, J = 7.8 Hz, 1H), 6.42 (d,
J = 2.0 Hz, 1H), 6.39 (d, J = 2.0 Hz, 1H), 4.08 (t, J = 5.7 Hz,
2H), 3.50 (t, J = 6.4 Hz, 2H), 2.07 (dd, J = 14.4, 7.6 Hz, 2H),
2.00 (dd, J = 13.0, 6.3 Hz, 2H). 13C NMR (101 MHz,
13C NMR (101 MHz, CDCl ) d 180.2, 165.3, 162.6, 157.8,
3
CDCl ) d 180.1, 165.0, 162.7, 157.8, 153.5, 132.3 (d,
J = 0.9 Hz), 131.6, 130.9 (d, J = 1.6 Hz), 129.0, 125.6 (q,
J = 3.9 Hz), 125.2 (dd, J = 7.4, 3.7 Hz), 127.42 – 120.05 (m),
153.1, 131.7, 130.4, 129.7, 122.9, 122.7, 106.0, 98.8, 93.0,
68.8, 31.5, 28.9, 25.6, 22.6, 14.0. ESI-MS: 417.0694
(C H O Br+, (M+H(+).
3
21 22
4

930
Yuyan Ren et al.
3-(4-Bromophenyl)-7-(heptyloxy)-5-hydroxy-4H-chro-
4. EXPERIMENTAL BIOLOGICAL PART
men-4-one (XVII): white needles, 0.24 g, yield: 56.8%, m.p.
1
Setting cisplatin (DDP) and tamoxifen as the positive
control drugs, all synthesized compounds were tested for
their antiproliferative effect on three cancer cell lines:
MCF-7, MDA-MB-231 and MDA-MB-435 (all purchased
from The Cell Bank of Type Culture Collection of Chinese
Academy of Sciences). The cancer cell lines were cultured in
high glucose DMEM medium (from HyClone) supplemented
with 10% fetal bovine serum (from ScienCell) and permitted
166 – 169°C, H NMR (400 MHz, CDCl ) d 12.69 (s, 1H),
3
7.88 (s, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz,
2H), 6.40 (d, J = 2.1 Hz, 1H), 6.37 (d, J = 2.1 Hz, 1H), 4.02
(t, J = 6.6 Hz, 2H), 1.85 – 1.76 (m, 2H), 1.50 – 1.41 (m, 2H),
1.36 (dd, J = 14.4, 11.1 Hz, 6H), 0.90 (t, J = 6.7 Hz, 3H). 13
C
NMR (101 MHz, CDCl ) d 180.2, 165.3, 162.6, 157.9,
3
153.1, 131.7, 130.4, 129.7, 122.9, 122.7, 106.0, 98.8, 93.0,
68.8, 31.7, 29.0, 28.9, 25.9, 22.6, 14.1. ESI-MS: 431.0852
(C H O Br+, (M+H(+).
in a humidified incubator at 37°C under 5% CO atmo-
2
22 24
4
sphere.
3-(4-Bromophenyl)-5-hydroxy-7-(octyloxy)-4H-chro-
MTT assay. The MCF-7, MDA-MB-231 and
MDA-MB-435 cells were seeded in a 96-well plate (at den-
sity of 2 ´ 103, 3 ´ 103 and 5 ´ 103 per well, respectively)
and allowed to grow to the desired confluence. The cells
were treated with various concentrations of genistein deriva-
tives for 72 h (genistein derivatives were dissolved in DMSO
to final concentrations of 100, 30, 10, 3, 1, 0.3 m M, and the
DMSO content never exceeded 0.05%). After 72 h exposure,
20 m L MTT solution (5 mg·mL-1 in DMSO) was added to
each well and then incubated for 4 h. Control cells were in-
cubated with a media containing an equivalent solvent
amount with DDP or TAM instead of genistein derivatives.
The supernatant was removed carefully by pipetting from
wells without disturbing the attached cells, and formazan
crystals were solubilized by adding 200 m L of DMSO to each
well and shaking for 15 min. The absorbance at 490 nm was
men-4-one (XVIII): white needles, 0.22 g, yield: 48.8%,
m.p. 92 – 93°C, 1H NMR (400 MHz, CDCl ) d 12.69 (s, 1H),
3
7.88 (s, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz,
2H), 6.40 (d, J = 2.2 Hz, 1H), 6.38 (d, J = 2.1 Hz, 1H), 4.02
(t, J = 6.5 Hz, 2H), 1.85 – 1.76 (m, 2H), 1.46 (dt, J = 15.0,
7.7 Hz, 2H), 1.32 (dd, J = 14.7, 7.6 Hz, 8H), 0.89 (t,
J = 6.7 Hz, 3H). 13C NMR (101 MHz, CDCl ) d 180.2,
3
165.3, 162.6, 157.9, 153.1, 131.7, 130.4, 129.7, 122.9, 122.7,
106.0, 98.8, 93.0, 68.8, 31.8, 29.3, 29.2, 28.9, 25.9, 22.6,
14.1. ESI-MS: 445.1006 (C H O Br+, (M+H(+).
23 26
4
3-(4-Bromophenyl)-5-hydroxy-7-(nonyloxy)-4H-chro-
men-4-one (XIX): white needles, 0.20 g, yield: 45.2%, m.p.
1
108 – 110°C, H NMR (400 MHz, CDCl ä 12.69 (s, 1H),
3
7.88 (s, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz,
2H), 6.40 (d, J = 2.1 Hz, 1H), 6.38 (d, J = 1.9 Hz, 1H), 4.02
(t, J = 6.6 Hz, 2H), 1.85 – 1.76 (m, 2H), 1.46 (dt, J = 14.4,
7.1 Hz, 2H), 1.32 (dt, J = 26.5, 10.8 Hz, 10H), 0.89 (t,
measured with a microplate reader, and the IC value was
50
defined as the concentration at which 50% survival of cells
was observed.
J = 6.7 Hz, 3H). 13C NMR (101 MHz, CDCl ) d 180.2,
3
165.4, 162.7, 157.9, 153.1, 131.8, 130.5, 129.7, 123.0, 122.7,
106.0, 98.8, 93.0, 68.8, 31.9, 29.5, 29.3, 29.2, 28.9, 25.9,
22.7, 14.1. ESI-MS: 459.1169 (C H O Br+, (M+H(+).
ACKNOWLEDGEMENTS
24 28
4
This research was supported by the National Natural Sci-
ence Foundation of China (No. 81273537); the Key Project
of Hunan Province Science and Technology Department
(No.2016DK2001), the Key Project of Hengyang Science
and Technology Department (No. 2017KJ166), the Key Dis-
ciplines of Hunan Province and Zhengxiang Scholar Pro-
gram of the University of South China, Joint Funds of Hunan
Province and Hengyang City (2017JJ4050), Hunan Provin-
cial Natural Science Foundation of China(2018JJ3456), and
the Project of Education Department of Hunan Province
(17C1398).
3-(4-Bromophenyl)-7-ethoxy-5-hydroxy-4H-chromen-
4-one (XX): white needles, 0.24 g, yield: 67.3%, m.p.
1
161 – 163°C, H NMR (400 MHz, CDCl ) d 12.69 (s, 1H),
3
7.87 (s, 1H), 7.57 (d, J = 8.5 Hz, 2H), 7.40 (d, J = 8.6 Hz,
2H), 6.39 (d, J = 2.2 Hz, 1H), 6.37 (d, J = 2.2 Hz, 1H), 4.10
(q, J = 7.0 Hz, 2H), 1.45 (t, J = 7.0 Hz, 3H). 13C NMR
(101 MHz, CDCl ) d 180.2, 165.1, 162.6, 157.8, 153.1,
131.7, 130.4, 129.7, 122.9, 122.7, 106.0, 98.7, 93.0, 64.3,
14.5. ESI-MS: 361.0070 (C H O Br+, (M+H(+).
3
17 13
4
7-(4-Bromobutoxy)-3-(4-bromophenyl)-5-hydroxy-4H-
chromen-4-one (XXI): pale yellow needles, 0.25 g, yield:
1
54.0%, m.p. 116 – 118°C, H NMR (400 MHz, CDCl ) d
3
CONFLICTS OF INTEREST
12.70 (s, 1H), 7.89 (s, 1H), 7.57 (d, J = 8.5 Hz, 2H), 7.41 (d,
J = 8.5 Hz, 2H), 6.40 (d, J = 2.1 Hz, 1H), 6.37 (d, J = 2.1 Hz,
1H), 4.06 (dd, J = 10.5, 5.8 Hz, 2H), 3.50 (t, J = 6.4 Hz, 1H),
3.27 (t, J = 6.7 Hz, 1H), 2.12 – 1.89 (m, 4H). 13C NMR
The authors declare that they have no conflicts of inter-
est.
(101 MHz, CDCl ) d 180.2, 165.0, 162.7, 157.8, 153.1,
131.8, 130.4, 129.6, 123.0, 122.7, 106.2, 98.7, 93.0, 67.6,
3
REFERENCES
+
33.1, 29.2, 27.6. ESI-MS: 466.9487 (C H O Br ,
(M+H(+).
1. F. Bray, J. Ferlay, I. Soerjomataram, et al., CA Cancer J. Clin.,
68(6), 394 – 424 (2018).
19 17
4
2

Design, Synthesis and Anticancer Evaluation of New 7-O-Alkylation Genistein Derivatives
931
2. K. Sak, Asian Pac. J. Cancer Prev., 18(9), 2309 – 2328 (2017).
3. S. Thota, D. A. Rodrigues, and E. J. Barreiro, Mini Rev. Med.
Chem., 18(15), 1265 – 1269 (2018).
12. M. Ono, K. Ejima, T. Higuchi, et al., Nutr. Cancer, 69(8),
1300 – 1307 (2017).
13. E. M. Shehata, Y. S. Elnaggar, S. Galal, et al., Int. J. Pharm.,
511(2), 745 – 56 (2016).
4. S. R. Lepri, R. C. Luiz, L. C. Zanelatto, et al., Cytotechnology,
65(2), 213 – 22 (2013).
14. Y. Ning, M. Xu, X. Cao, et al., Oncol. Rep., 38(2), 949 – 958
(2017).
5. A. Antosiak, K. Milowska, K. Maczynska, et al., Med. Chem.
Res., 26(1), 64 – 73 (2017).
15. P. Zhou, C. Wang, Z. Hu, et al., BMC Cancer, 17(1), 813 (2017).
16. F. Ardito, M. R. Pellegrino, D. Perrone, et al., Oncol. Targets
Ther., 10, 5405 – 5415 (2017).
6. V. Mukund, D. Mukund, V. Sharma, et al., Crit. Rev. Oncol.
Hematol., 119(13 – 22 (2017).
7. C. Spagnuolo, G. L. Russo, I. E. Orhan, et al., Adv. Nutr., 6(4),
408 – 19 (2015).
17. Z. Wang, X. Deng, S. Xiong, et al., Nat. Prod. Res., 32(24),
2900 – 2909 (2018).
8. Y. Mizushina, K. Shiomi, I. Kuriyama, et al., Int. J. Oncol.,
43(4), 1117 – 24 (2013).
18. Y. Guo, L. Shen, X. Yao, et al., Luminescence, 32(8),
1368 – 1384 (2017).
9. C. K. Taylor, R. M. Levy, J. C. Elliott, et al., Nutr. Rev., 67(7),
398 – 415 (2009).
19. Y. Wei, Q. Zheng, G. Tang, et al., Med. Chem., 12(5), 441 – 7
(2016).
10. S. Poschner, A. Maier-Salamon, M. Zehl, et al., Front.
Pharmacol., 8, 699 (2017).
20. X. Qiang, Z. Sang, W. Yuan, et al., Eur. J. Med. Chem., 76,
314 – 331 (2014).
11. D. G. Pons, J. Vilanova-Llompart, A. Gaya-Bover, et al., Int. J.
Food Sci. Nutr., No. 2, 1 – 9 (2019).