Synthesis and anti-tumor activity of novel aminomethylated derivatives of isoliquiritigenin

Article abstract of DOI:10.3390/molecules191117715

A series of new aminomethylated derivatives of isoliquiritigenin was synthesized. The structures of the compounds were confirmed by IR, MS, NMR, ¹³C-NMR and elemental analyses. Cytotoxic activities of these derivatives towards the human prostatic cell line PC-3, human mammary cancer cell line MCF-7 and human oophoroma cell line HO-8910 in vitro were tested. The IC50 values showed cytotoxic activities of some of these new derivatives were relatively strong. Furthermore, tumor growth inhibition in vivo of aminomethylated derivatives of isoliquiritigenin 15 was superior to that of isoliquritigenin and reached inhibition rates of 71.68%. The detailed synthesis, spectroscopic data, biological and pharmacologicalactivities of the synthesized compounds were provided.

Full text of DOI:10.3390/molecules191117715

Molecules 2014, 19, 17715-17726; doi:10.3390/molecules191117715
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Anti-Tumor Activity of Novel Aminomethylated
Derivatives of Isoliquiritigenin
1
,†
2,†
2
2
2
3
Haoran Fu , Yuhang Zhang , Xiqing Wang , Yingzhi Han , Xiao Peng , Thomas Efferth
2
,
and Yujie Fu *
1
School of Chemistry and Environment, Beihang University, Beijing 100191, China;
E-Mail: haoran_fu2014@163.com
2
Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University,
Harbin 150040, China; E-Mails: narutohang1991128@163.com (Y.Z.);
wangxiqing0622@163.com (X.W.); yingzhi_han@163.com (Y.H.);
pengxiao1986@aliyun.com (X.P.)
3
Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry,
University of Mainz, 55128 Mainz, Germany; E-Mail: efferth@uni-mainz.de
†
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed; E-Mail: yujie_fu2002@yahoo.com;
Tel./Fax: +86-451-8219-0535.
External Editor: Jean Jacques Vanden Eynde
Received: 25 August 2014; in revised form: 21 October 2014 / Accepted: 27 October 2014 /
Published: 31 October 2014
Abstract: A series of new aminomethylated derivatives of isoliquiritigenin was
1
3
synthesized. The structures of the compounds were confirmed by IR, MS, NMR, C-NMR
and elemental analyses. Cytotoxic activities of these derivatives towards the human
prostatic cell line PC-3, human mammary cancer cell line MCF-7 and human oophoroma
cell line HO-8910 in vitro were tested. The IC50 values showed cytotoxic activities of some
of these new derivatives were relatively strong. Furthermore, tumor growth inhibition in vivo
of aminomethylated derivatives of isoliquiritigenin 15 was superior to that of isoliquritigenin
and reached inhibition rates of 71.68%. The detailed synthesis, spectroscopic data,
biological and pharmacologicalactivities of the synthesized compounds were provided.

Molecules 2014, 19
17716
Keywords: aminomethylated derivatives of isoliquiritigenin; organic synthesis; Mannich
reaction; anti-tumor activity
1
. Introduction
Natural products play a prominent role in oncology because of their potent anticancer activity and
good tolerability in normal tissues. Therefore, it is very significant for scientists to screen active
compounds from plants and synthesized new derivatives with similar molecular structures as potential
anti-tumor drugs or pro-drugs [1–3]. As a common active ingredient of many Chinese herbal
medicines [4,5], flavonoids reveal little side effects in clinical research [6,7]. Among numerous
flavonoids, isoliquiritigenin (ISL) attracted the attention of many scientists. ISL is one of the most
important chalcone compound from licorice (Figure 1) and it has a variety of biological activities, such
as anti-tumor, anti-virus, anti-free radical, anti-lipid peroxidation and anti-HIV activity, among which
the anti-tumor effect came into the focus in recent years [8–12].
Figure 1. Structure of isoliquiritigenin.
The promising anti-tumor effect of ISL stimulates research to modify the chalcone structures, in
order to obtain novel derivatives with even stronger anti-tumor activities. So far, ISL and its
derivatives have been shown to exert anti-tumor activities by the following mechanism: inhibition of
cell proliferation [13,14]; induction of apoptosis [15,16], inhibition of angiogenesis [17], anti-oxidative
effects leading to enhanced expression of tumor suppressor genes and decreased expression of
oncogenes [18].
In order to enhance water-solubility, bioavailability, and tumor inhibitory activities of ISL, we used
ISL as lead compound while remaining its hydroxyl and modifying side chains in A ring of chalcone
with nitrogen heterocyclic ring compounds and open-chain amino through Mannich reaction [19–21].
We chose the Mannich reaction in our study, because the reaction products Mannich alkaline
compounds have a quite wide range of biological activity, such as antibacterial, anticonvulsive,
anti-inflammatory, anti-tumor activities [22–25]. For this reason, Mannich reactions are widely used in
medicinal chemistry [26]. To the best of our knowledge, the synthesis of ISL derivatives with different
side chains in the A ring containing nitrogen atoms has not been reported yet.
In our present study, chalcone was first synthesized by a hydroxyl-protection method and a
consequent condensation reaction was performed in a base environment [27,28]. Then, different
nitrogen atoms side chains (heterocyclic azo and open chain amino) were attached to the A ring by

Molecules 2014, 19
17717
Mannich reactions. To test the cytotoxic activities of these derivatives in vitro, human prostatic PC-3,
human mammary cancer MCF-7 and human oophoroma HO-4980 cells were used. Additionally, the
in vivo anti-tumor activity of the compound with the lowest IC50 value was tested in the mice
transplanted with murine S180 sarcoma cells. Our results provide potential ISL derivatives for
anti-tumor therapy in the future.
2
. Results and Discussion
2
.1. Chemistry
ISL has several active spots, and the two hydroxyl groups in ring A partly attributed to its anti-tumor
activity [29]. ISL reveals poor water-solubility. It may be possible to improve its water-solubility
by attaching an amino-group to ring A. Meanwhile, by structure-activity-relationship analyses of a
series of anti-cancer drugs, we found that many functional groups in drugs contained nitrogen. As a
result, heterocyclic azo-groups and open chain amino-groups were chosen to modify ring A by the
Mannich reaction.
Scheme 1. The route of synthetic isoliquiritigenin derivatives.
Reagents and conditions: (a) pyridine-PPTS, 2,3-dihydropyran, CH
,3-dihydropyran, NaHCO , CH Cl , RT, 4.5 h; (c) Ba(OH) , CH OH 45 °C, 12 h; (d) PTSA, CH
e) amine, CH CHO, CH COOH, 16–18 °C.
2
Cl
2
, RT, 4 h; (b) pyridine-PPTS,
2
3
2
2
2
3
,
3
OH, RT, 6 h;
(
3
3

Molecules 2014, 19
17718
According to this idea, we achieved the synthesis of novel derivatives 7–16 (Scheme 1). In our
experiment, Substrate 1 (p-hydroxybenzaldehyde) and 2 (2,4-dihydroxyacetophenone) were first
separately protected by 2,3-dihydropyrane in the presence of pyridine-PPTS, and compounds 3 and 4
were obtained. Then, a condensation reaction of compounds 3 and 4 was conducted with Ba(OH)
2
as
catalyst and CH OH as solvent at 40 °C. Intermediate product 5 was obtained, which was converted to
3
chalcone 6 (ISL) in good yield by deprotection using PTSA. This method using hydroxyl-protection
before condensation reaction led to obviously higher yields compared with classical synthesis ways.
Next, compounds 7–16 were all synthesized by Mannich reactions using chalcone 6, formaldehyde and
amine in one-pot syntheses.
Interestingly, in the Mannich reaction, extending reaction times or enlarging reactant amounts did
not effect the final yields. However, byproducts considerably increased. When Mannich reaction was
stopped immediately after adding reagents, the highest yields were achieved. Mannich reactions are
often accompanied by many side reactions [30], and with several reaction loci, not only one but several
aminomethyl groups attached to the benzene ring of ISL during the reaction process. Besides, we
found that the addition of a little bit excessive aldehyde and amine also contributed to higher yields.
2
.2. Evaluation of Biological Activity
2.2.1. Cytotoxicity Assay
We examined the cytotoxicity of ISL nitrogenous derivatives against three cancer cell lines (PC-3,
MCF-7 and HO-8910) and normal human THP-1 cells using MTT growth inhibition assays [31]. ISL
was used as a positive control. For THP-1, all derivatives and ISL showed almost no cytotoxicity
against normal cells. The IC50 values were all above 100 µM. The IC50 values for the three cancer cell
lines were listed in Table 1. Although not all of these compounds exhibited considerable cytotoxicities
against these cell lines, some still showed good cytotoxicities in vitro experiment.
Table 1. Cytotoxicity of isoliquiritigenin (ISL) derivatives.
Cytotoxicity (IC50, μM)
Compound
R
1
R
2
mp, °C
PC-3 a MCF-7 HO-4980 THP-1
7
8
9
Me
CH
H
Me
OCH CH
>100
48.64
28.32
>100
66.02
57.05
>100
87.72
35.14
>100
36.90
>100
>100
73.58
84.56
>100
84.78
>100
66.01
42.94
81.84
44.11
>100
96.43
>100
98.14
97.32
>100
>100
89.01
37.85
74.87
58.43
>100
>100
>100
>100
>100
>100
>100
>100
>100
>176
>180
>168
>180
>176
>175
>170
>180
106.1–8
2
CH
2
2
2
C
6
H
11
1
1
1
1
1
1
1
0
1
2
3
4
5
6
i-Pr
Et
H
i-Pr
Et
CHCH
3
C
2
H
5
H
t-Bu
H
C
2
H
4
OH
OH
C
2
4
H OH
C
2
H
4
CH
2
CH
2
CH
2
CH
2
>100 116.5–7.4
>100
ISL
-
-
a
PC-3, human prostate cancer cells; MCF-7, human breast cancer cells; HO-4980, human ovarian cancer cells.

Molecules 2014, 19
17719
For PC-3, compounds 9 and 15 had stronger growth inhibition effect, and the IC50 were 28.32 µM
and 35.14 µM for 72 h, respectively. These two compounds showed stronger cytotoxicities than ISL.
For HO-8910, 15 showed the strongest cytotoxicity with the IC50 values of 37.85 µM. For MCF-7,
only 15 resulted in strong growth inhibition. Compound 15 was more potent than ISL against HO-8910
and MCF-7. Besides, the monoethanolamine substituted compound 14 showed much improved activity
compared with the other derivatives, while compound 15 containing two hydroxyl attached with the
nitrogen atom showed the most potent cytotoxicity. We conclude that amines containing hydroxyl
located in 3′-position of the A ring could be highly beneficial for the activity of the compounds. In
addition, compound 15 attached with amines containing two hydroxyl groups resulted in more potent
cytotoxicities than compound 14 modified with amines containing only one hydroxy. Therefore, ISL
derivative 15 was used to evaluate anti-tumor activity in S180 tumor-bearing mice for further studies.
Table 2. Effect of ISL and ISL derivative 15 (40 mg/kg, 80 mg/kg) on tumor weight and
inhibitory ratio in S180 tumor-bearing mice.
Dose
Mice Number
a
b
Group
Treatment
TW (g) x ± SD BWC (%) TIR (%)
(
mg/kg) Begin End
I
normal saline
ISL
-
10
10
10
10
10
10
10
10
1.23 ± 0.534 **
0.85 ± 0.369 **
0.74 ± 0.618 *
0.35 ± 0.442 **
+4.4
−3.0
-
II
40
40
80
32.44
39.72
71.68
III
IV
ISL derivative 15
ISL derivative 15
+12.8
+7.8
Student’s t-test was used to compare tumor volumes of treated mice. * p values < 0.05 were considered
significant, ** p values < 0.01 were considered very significant. a Percentage of mice body-weight change
(
BWC) after drug treatment: BWC% = (mean BW final day/mean BW first day × 100) − 100; ‘‘+” means
b
bodyweight increase; ‘‘−” means body-weight decrease. Tumor inhibitory rate: TIR% = (1 − average tumor
weight of experimental group/the average tumor weight of model control group).
Figure 2. Effect of ISL and ISL derivative 15 on S180 tumor growth. ISL (40 mg/kg) was
used as control drug. (A): Untreated negative control; (B): Treatment with low dose ISL
derivative 15 (40 mg/kg); (C): Treatment with high dose ISL derivative 15 (80 mg/kg);
(
D): Treatment with ISL (40 mg/kg) as positive control.
2
.2.2. In Vivo Toxicity and Antitumor Studies
S180 tumor-bearing mice were treated with 80 or 40 mg/kg ISL derivative 15 and tumor inhibition
rates of 71.68% and 39.72%, respectively, were obtained (Table 2). For comparison, ISL (40 mg/kg)

Molecules 2014, 19
17720
was analyzed as positive control. Changes in body weight were used as parameter for toxicity. Loss of
body weight was not found after exposure of animals to 80 or 40 mg/kg ISL derivative 15, whereas
ISL caused a slight weight loss in animals. Tumors were surgically removed after treatment with ISL
and derivative 15, and the results showed significant inhibition of tumor growth (Figure 2). The
activity of ISL derivative 15 in S180 tumor-bearing mice indicated that ISL derivative 15 might be an
attractive candidate for clinical use in oncology.
3. Experimental Section
3
.1. Chemistry
1
13
H (500 MHz) and C (125 MHz) NMR spectra were recorded with a Bruker instrument, and
reported with TMS as internal standard and DMSO. Chemical shifts (δ values) and coupling constants
J values) are given in ppm and Hz. MS analysis was carried out on an API-3000 LC-MS-MS
instrument. The melting points were determined on a WRS-1B digital melting point apparatus
Shanghai Precision & Scientific Instruments Co., Shanghai, China). Elemental analyses were carried
(
(
out in a EA3000 CHNS-O analyzer. TLC analysis was carried out on silica gel plates GF254. Flash
chromatography (Lisure Science (Suzhou) Co., Ltd., Suzhou, China) was performed with silica gel
3
00–400 mesh using a glass column. Unless otherwise indicated, reagents and solvents were obtained
from commercial suppliers.
Procedure for chalcone 6: p-hydroxybenzaldehyde (219.8 mg, 1.8 mmol) and pyridine-PPTS
(45.2 mg, 0.18 mmol) were dissolved in dichloromethane (5 mL),2,3-dihydropyran (0.25 mL, 2.7 mmol)
was dropwise added. The reaction was stirred at room temperature for 4 h, the mixture was treated
with saturated NaCl and extracted with ether. Solvent was removed under reduced pressure to obtain
oily crude product (Yield: 94.4%). Similarly, 2,4-dihydroxyacetophenone (152.2 mg, 1.0 mmol),
pyridine-PPTS (25.1 mg, 0.1 mmol), and 2,3-dihydropyran (0.14 mL, 1.5 mmol) were added into a
round bottom flask and stirred for 2 h, NaHCO
.5 h, the mixture was filtered. Oily crude 4 product was obtained after solvent was removed under
reduced pressure (Yield: 91.2%). These two hydroxyl-proctected products were dissolved in CH OH
5 mL), Ba(OH) (428.4 mg, 2.5 mmol) was added as catalyst. The reaction was stirred at 45 °C (TLC
detected) and after 12 h it was diluted with CH Cl . Then, HCl was added to adjust the system to weak
acid. The mixture was extracted with CH Cl . The condensation product 5 was obtained after solvent
was removed in vacuo and was following dissolved in CH OH (Yield: 77.9%). PTSA (4.8 mg,
.028 mmol) was added and the mixture was stirred at room temperature for 6 h, the reaction mixture
3
(80.0 mg, 1 mmol) was added, continued for another
2
3
(
2
2
2
2
2
3
0
was poured into ice water and yellow crystals were filtered, washed and then dried. Finally, target
compound 6 was obtained (Yield: 95.8%).
General procedure for ISL derivatives 7–16: ISL 128 mg (0.5 mmol) were put into a round bottom
flask, and substituted amines (0.6 mmol) were added. Temperature was kept at 16–18 °C. The mixture
of 36% formaldehyde solution (0.046 ml, 0.6 mmol) and acetic acid (2 ml) was quickly and dropwise
added under electromagnetic stirring. The reaction immediately stopped, when the agents has been
dropwise added. The mixture was treated with water and extracted with EtOAc. The organic phase was

Molecules 2014, 19
17721
dried over Na
2
SO . The solvent was removed under reduced pressure, and the crude mixture was purified
4
by column chromatography with EtOAc/MeOH (5:1 to 1:2 v/v) as eluent to afford the target compounds.
(
E)-1-(3-(dimethylaminomethyl)-2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one
(7).
According to the general procedure for ISL derivatives using dimethylamine as amine, the lead
compound was obtained as yellow powder. Weight: 43.5 mg. Yield: 27.8%. Melting point: >176 °C;
1H-NMR (500 MHz, DMSO): 2.41 (6H, s, (CH
.82~6.84 (2H, m, H5, H6), 7.67~7.75 (4H, m, H2′,3′, 5′, 6′), 8.03 (1H, d, J = 9 Hz, CH
)
N-), 3.84 (2H, s, -CH
N), 6.22 (1H, d, J = 9 Hz, H3),
);
3
2
2
6
2
13C-NMR (125 MHz, DMSO): 190.65, 172.59, 170.20, 164.47, 160.58, 143.63, 132.05, 131.46,
+
1
26.36, 118.04, 116.29, 111.01, 110.15, 106.89, 54.17, 43.76. ESI-MS, m/z [M+H] 314.3. For
C
18
H19NO
4
: C, 68.99; H, 6.11; N, 4.47. Found: C, 68.90; H, 6.08; N, 4.42.
(E)-1-(2,4-dihydroxy-3-(morpholinomethyl)phenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one (8). According
to the general procedure for ISL derivatives using morpholine as amine, the lead compound was
1
obtained as yellow powder. Weight: 62.84 mg. Yield: 35.4%. Melting point: >180 °C; H-NMR
(
500 MHz, DMSO): 2.51 (4H, s, -(CH2)
2
N), 3.61 (4H, s, -(CH
2
)
2
O), 3.74 (2H, s, -CH
2
N), 6.38 (1H, d,
J = 9 Hz, H3), 6.83~6.85 (2H, m, H5, H6), 7.75~7.77 (4H, m, H2′, 3′, 5′, 6′), 8.13 (1H, d, J = 9 Hz,
1
3
H2); C-NMR (125 MHz, DMSO): 191.68, 165.27, 163.75, 160.27, 44.30, 131.47, 131.24, 125.75,
+
1
17.31, 115.82, 112.35, 107.99, 107.75, 66.00, 52.54, 51.99. ESI-MS, m/z [M+H] 356.3. Anal. Calcd.
For C20
H21NO
5
: C, 67.59; H, 5.96; N, 3.94. Found: C, 67.54; H, 5.89; N, 3.87.
(
E)-1-(3-(cyclohexylaminomethyl)-2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one
(9).
According to the general procedure for ISL derivatives using cyclohexylamine as amine, the lead
compound was obtained as yellow powder. Weight: 60.9 mg. Yield: 33.2%. Melting point: >168 °C;
1H-NMR (500 MHz, DMSO): 1.14~1.99 (10H, m, -CH
), 2.74 (1H, s, -CHN), 4.01 (2H, s, -CH N),
2
2
5
.99 (1H, d, J = 9 Hz, H3), 6.80~6.82 (2H, m, H5, H6), 7.59~7.70 (4H, m, H2′, 3′, 5′, 6′), 7.88 (1H, d,
13
J = 9 Hz, H2); C-NMR (125 MHz, DMSO): 188.20, 175.04, 164.28, 159.67, 141.63, 131.33, 130.63,
1
3
26.14, 117.91, 115.74, 111.99, 108.27, 105.14, 54.78, 29.88, 25.02, 23.93. ESI-MS, m/z [M+H]+
68.4. Anal. Calcd. For C22 : C, 71.91; H, 6.86; N, 3.81. Found: C, 71.89; H, 6.87; N, 3.83.
H25NO
4
(
E)-1-(3-(diisopropylamino)-methyl)-2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one (10).
According to the general procedure for ISL derivatives using diisopropylamine as amine, the lead
compound was obtained as yellow powder. Weight: 57.56 mg. Yield: 31.2%. Melting point: >180 °C;
1H-NMR (500 MHz, DMSO): 1.12 (12H, s, -CH
d, J = 9 Hz, H3), 6.82~6.84 (2H, m, H5, H6), 7.70~7.74 (4H, m, H2′, 3′, 5′, 6′), 8.00 (1H, d, J = 9 Hz,
), 3.23 (2h, s, -CHN), 3.96 (2H, s, -CH N), 6.16 (1H,
3
2
1
3
H2); C-NMR (125 MHz, DMSO): 190.52, 170.27, 163.17, 160.02, 143.27, 131.01, 125.89, 117.50,
+
1
15.78, 110.58, 109.59, 106.60, 49.11, 30.66, 18.27. ESI-MS, m/z [M+H] 370.3. Anal. Calcd. For
C
22
H27NO
4
: C, 71.52; H, 7.37; N, 3.79. Found: C, 71.51; H, 7.35; N, 3.75.
(
E)-1-(3-((diethylamino)methyl)-2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one
(11).
According to the general procedure for ISL derivatives using diaethylamin as amine, the lead compound
1
was obtained as yellow powder. Weight: 72.97 mg. Yield: 42.8%. Melting point: >176 °C; H-NMR
(
500 MHz, DMSO): 1.09 (6H, t, J = 7 Hz, CH
3
), 2.71~2.75 (4H, q, J = 7 Hz, (CH
2
2
) N-), 3.92 (2H, s,

Molecules 2014, 19
17722
-
CH
2
N), 6.19 (1H, d, J = 9 Hz, H3), 6.82~6.84 (2H, m, H5, H6), 7.67~7.75 (4H, m, H2′, 3′, 5′, 6′), 8.02
13
(1H, d, J = 9 Hz, H2); C-NMR (125 MHz, DMSO): 190.80, 170.48, 164.19, 160.52, 143.69, 131.85,
1
3
31.48, 126.38, 118.03, 116.27, 111.08, 110.13, 106.68, 49.28, 46.45, 10.79. ESI-MS, m/z [M+H]+
42.1. Anal. Calcd. For C20 : C, 70.36; H, 6.79; N, 4.10. Found: C, 70.43; H, 6.74; N, 4.20.
H23NO
4
(
E)-1-(3-((sec-butylamino)methyl)-2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one
(12).
According to the general procedure for ISL derivatives using sec-butylamine as amine, the compound
1
was obtained as yellow powder. Weight: 46.71 mg. Yield: 27.4%. Melting point: >175 °C; H-NMR
(
500 MHz, DMSO): 0,92~0.93 (6H, d, J = 7 Hz, CH3), 1.82~1.88 (1H, m, -CHN), 2.56 (2H, d,
J = 7 Hz, -CH -), 3.99 (2H, s, -CH N), 6.04 (1H, d, J = 9 Hz, H3), 6.81~6.83 (2H, m, H5, H6),
.61~7.71 (4H, m, H2′, 3′, 5′, 6′), 7.91 (1H, d, J = 9 Hz, H2); C-NMR (125 MHz, DMSO): 189.17,
2
2
1
3
7
1
5
6
74.56, 164.68, 160.22, 142.45, 131.84, 131.19, 126.59, 118.34, 116.24, 111.95, 109.23, 105.78,
+
4.73, 44.17, 26.79, 20.58. ESI-MS, m/z [M+H] 342.3. Anal. Calcd. For C20
H
23NO : C, 70.36; H,
4
.79; N, 4.10. Found: C, 70.33; H, 6.84; N, 4.07.
(
E)-1-(3-((tert-butylamino)methyl)-2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one
(13).
According to the general procedure for ISL derivatives using tert-butylamine as amine, the lead
compound was obtained as yellow powder. Weight: 65.64 mg. Yield: 38.5%. Melting point: >170 °C;
1H-NMR (500 MHz, DMSO): 1.27 (9H, s, CH3), 3.98 (2H, s, -CH
N), 5.99 (1H, d, J = 9 Hz, H3),
2
1
3
6.81~6.83 (2H, m, H5, H6), 7.59~7.70 (4H, m, H2′, 3′, 5′, 6′), 7.88 (1H, d, J = 9 Hz, H2); C-NMR
(
125 MHz, DMSO): 188.59, 164.80, 160.18, 142.09, 131.89, 126.63, 118.39, 116.24, 112.66, 108.63,
+
1
05.64, 54.03, 37.54, 26.47. ESI-MS, m/z [M+H] 342.2. Anal. Calcd. For C20
H
23NO : C, 70.36; H,
4
6
.79; N, 4.10. Found: C, 70.39; H, 6.83; N, 4.06.
1
-(2,4-dihydroxy-3-((2-hydroxyethylamino)methyl)phenyl)-3-(4-hydroxyphenyl)propen-1-one
(14).
According to the general procedure for ISL derivatives using monoethanolamine as amine, the lead
compound was obtained as yellow powder. Weight: 41.62 mg. Yield: 25.3%. Melting point: >180 °C;
1H-NMR (500 MHz, DMSO): 2.82 (2H, s, -CH
OH), 3.58 (2H, s, -CH
N), 4.01 (2H, s, -CH N), 6.03
2
2
2
(
1H, d, J = 8 Hz, H3), 6.82~6.83 (2H, m, H5, H6), 7.63~7.69 (4H, m, H2′, 3′, 5′, 6′), 7.90 (1H, d,
13
J = 8 Hz, H2); C-NMR (125 MHz, DMSO): 165.03, 160.16, 142.20, 131.89, 131.14, 126.64, 118.40,
+
1
16.23, 112.23, 108.95, 105.79, 58.14, 49.26, 43.30. ESI-MS, m/z [M+H] 330.5. Anal. Calcd. For
C
18
H
19NO : C, 65.64; H, 5.81; N, 4.25. Found: C, 65.66; H, 5.85; N, 4.21.
5
(E)-1-(3-((bis(2-hydroxyethyl)amino)methyl)-2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one
(15). According to the general procedure for ISL derivatives using diethanolamine as amine, the lead
compound was obtained as yellow powder. Weight: 69.94 mg. Yield: 37.5%. Melting point:
1
1
06.1–106.8 °C; H-NMR (500 MHz, DMSO): 2.74 (4H, s, -(CH
2
2
) N), 3.58~3.60 (4H, t, J = 6 Hz,
-
CH
2
OH), 3.98 (2H, s, -CH2N), 6.28 (1H, d, J = 9 Hz, H3), 6.83~6.85 (2H, m, H5, H6), 7.74~7.76
1
3
(4H, m, H2′, 3′, 5′, 6′), 8.08 (1H, d, J = 9 Hz, H2); C-NMR (125 MHz, DMSO): 191.61, 172.47,
1
5
6
68.05, 164.05, 160.66, 144.30, 131.88, 131.6, 212.63, 1117.93, 116.30, 112.05, 109.29, 108.12,
+
8.07, 55.86, 50.29, 21.53. ESI-MS, m/z [M+H] 374.1. Anal. Calcd. For C20
H
23NO : C, 64.33; H,
6
.21; N, 3.75. Found: C, 64.32; H, 6.27; N, 3.69.

Molecules 2014, 19
E)-1-(2,4-dihydroxy-3-(pyrrolidin-1-ylmethyl)phenyl-3-(4-hydroxyphenyl)prop-2-en-1-one
17723
(
(16).
According to the general procedure for ISL derivatives using pyrrolidine as amine, the lead compound
was obtained as yellow powder. Weight: 60.0 mg. Yield: 35.4%. Melting point: 116.5–117.1 °C;
1H-NMR (500 MHz, DMSO): 1.84 (4H, s, -CH2-CH2-), 2.82~2.84 (4H, t, J = 6 Hz, (CH
)
2
2
N-), 3.99
(
2H, s, -CH
2
N), 6.18 (1H, d, J = 9 Hz, H3), 6.82~6.84 (2H, m, H5, H6), 7.65~7.73 (4H, m, H2′, 3′, 5′,
1
3
6
1
′), 8.00 (1H, d, J = 9 Hz, H2); C-NMR (125 MHz, DMSO): 190.20, 172.54, 171.03, 164.57, 160.48,
43.31, 132.02, 131.39, 126.43, 118.10, 116.28, 110.64, 110.51, 107.105, 334 , 50.31, 23.54.
+
ESI-MS, m/z [M+H] 340.3. Anal. Calcd. For C20
H
21NO : C, 70.78; H, 6.24; N, 4.13. Found: C, 70.85;
4
H, 6.20; N, 4.15.
3
.2. Biological Assays
3.2.1. Cytotoxicity Assay
Cytotoxicity was evaluated against four human cell lines (MCF-7 breast cancer, PC-3 prostatic
cancer, HO-8910 ovarian cancer, and THP1 normal cells). Briefly, MCF-7, PC-3 and HO-8910 cells
5
were separately plated in 96-well culture plates (1 × 10 cells/well). After 24 h incubation, cells were
treated with compounds 7–16 (0, 3.13, 6.25, 12.5, 25, 50 and 100 µM) for 72 h. MTT solution
(
5 mg/mL) was then added to each well. After 4 h incubation, the formazan precipitate was dissolved
in 100 mL dimethyl sulfoxide (DMSO), and then the absorbance was measured in an ELISA reader
Thermo Molecular Devices Co., Union City, CA, USA) at 570 nm. The cell viability ratio was
calculated by the following formula: Inhibitory ratio (%) = (1 − ODtreated/ODcontrol) × 100%.
(
3
.2.2. Animal Treatment
Female BALB/c mice (6–8 weeks old, 25 ± 2 g) were obtained from the Third Affiliated Hospital of
Harbin Medical University. S180 sarcoma cells were purchased from the Experimental Animal Center
of Peking University Health Science Center (Heilongjiang, China). The animals were housed in
polypropylene cages and maintained under controlled conditions of 12 h light/12 h dark photoperiod
and 55 ± 5% relative humidity at room temperature (28 ± 2 °C). After fed in our facility for 1 week,
4
0 mice were induced S180 tumor cells according to Wu et al. with some modification [32]. All mice
6
were subcutaneously implanted with 1 × 10 cells/mice on the right flank. After inoculation for 24 h,
all animals were randomly divided into one untreated control group, one treated positive control group
and two treated experimental groups (each group contained 10 mice).
Group I (untreated control) received orally the same volume of 0.9% normal saline once per day as
the treated groups. Group II (treated positive control) received orally ISL at a dosage of 40 mg/kg body
weight once per day. Group III (low dose of ISL derivative 15 treatment) received ISL derivative 15
orally (dissolved in 0.2 mL normal saline) at a dosage of 40 mg/kg body weight once per day. Group
IV (high-dose of ISL derivative 15 treatment) received ISL derivative 15 orally (dissolved in 0.2 mL
normal saline) at a dosage of 80 mg/kg body weight once per day.

Molecules 2014, 19
. Conclusions
A series of novel aminomethylated derivatives of ISL were synthesized and their anti-tumor
17724
4
activities in vitro were evaluated against three cancer cell lines. Through the results, we found that ISL
derivatives contained two or more aminemethyl and nitrogen connected with methylene substituent
exhibited considerable cytotoxicity. S180 tumor-bearing mice were treated with 80 or 40 mg/kg ISL
derivative 15 and tumor inhibition rates were 71.68% and 39.72%, respectively. Our results indicated
that ISL derivative 15 might be an attractive candidate for clinical use in oncology in the future and the
scheme we designed to synthesize ISL derivatives might become a useful method for chemical
synthesis and modification of flavone drugs.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/11/17715/s1.
Acknowledgments
The authors gratefully acknowledge the financial support by the fundamental research funds for the
Central University (2572014AA12).
Author Contributions
H.R. Fu and Y.H. Zhang together designed the research project, synthesized compounds 7–16,
interpreted the results, prepared the manuscript, analyzed IR and MS spectra and performed in vitro
experiment. X.Q Wang, Y.Z. Han analyzed NMR. X. Peng and T. Efferth performed in vivo
experiment. Y.J. Fu instructed the whole work.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
©
2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/4.0/).
(