Synthesis, crystal structure, and biological evaluation of a series of phloretin derivatives

Article abstract of DOI:10.3390/molecules191016447

A one-step synthesis of phloretin derivatives 2-11 from phloretin in good to excellent yields is reported. Their structures were characterized by ¹H-NMR, ¹³C-NMR and MS, and the structures of 8 and 11 were determined by X-ray diffrac

Full text of DOI:10.3390/molecules191016447

Molecules 2014, 19, 16447-16457; doi:10.3390/molecules191016447
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Crystal Structure, and Biological Evaluation of a
Series of Phloretin Derivatives
Li Wang 1,†,*, Zheng-Wei Li 2,†, Wei Zhang 3,†, Rui Xu 1, Fei Gao 3, Yang-Feng Liu 4 and
Ya-Jun Li 3,*
1
Department of Scientific Research, The Affiliated Hospital of Xi’an Medical University,
Xi’an 710077, China
2
Department of Anesthesia, The Affiliated Hospital of Xi’an Medical University, Xi’an 710077, China
3
Department of Neurology, The Affiliated Hospital of Xi’an Medical University, Xi’an 710077, China
4
Department of Neurology, The People’s Liberation Army No. 451 Hospital, Xi’an 710032, China
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: piaolanger7828@163.com (L.W.);
liyajun9@hotmail.com (Y.-J.L.); Tel./Fax: +86-29-8427-7893 (Y.-J.L.).
External Editor: Derek J. McPhee
Received: 12 August 2014; in revised form: 10 September 2014 / Accepted: 23 September 2014 /
Published: 13 October 2014
Abstract: A one-step synthesis of phloretin derivatives 2–11 from phloretin in good to
excellent yields is reported. Their structures were characterized by 1H-NMR, 13C-NMR and
MS, and the structures of 8 and 11 were determined by X-ray diffraction analysis. A
mechanism for the formation of 9–11 is proposed. Compared with the anticancer drug
docetaxel, phloretin, phloretin derivatives and phlorizin exhibited moderate cytotoxicity
toward the MDA-MB-231, SPC-A1, A549, MCF-7 and EC109 cell lines. Among all of the
tested compounds, 7 exhibited the strongest cytotoxicity toward the five cell lines and was
more active than docetaxel in MDA-MB-231 cells. Our findings suggest that these
derivatives hold great promise for further development as anticancer agents.
Keywords: phloretin derivatives; X-ray diffraction analysis; mechanism; anticancer activity

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1. Introduction
Phloretin, a naturally occurring flavonoid, belongs to the dihydrochalcone chemical class and consists
of a C6-C3-C6 skeleton structure (two aromatic rings connected by a C3 chain, Figure 1) [1–3]. Phloretin
is primarily found in apples, pears, and other succulent fruits. It exhibits various biological and
pharmacological activities, such as anti-inflammatory, anticancer [4], antimutagenic and
anticarcinogenic activities, and has been used as a cosmeceutical ingredient [5–7]. Recently, its
antitumor properties have also attracted much attention from pharmacologists. Many studies indicate
that phloretin inhibits the growth of human leukemia, bladder cancer, and rat mammary adenocarcinoma
cells in vivo and induces apoptosis of B16 murine melanoma and HL60 human leukemia cells [8,9].
Phloretin also exerts a strong inhibitory effect on the proliferation of SMMC-7721, HT-29 and
human liver cancer cell lines [10–12]. Phloretin can be used for the treatment of hepatomas and
other tumors; it is therefore currently being evaluated as a potential chemotherapeutic agent.
Phlorizin (phloretin-2'-O-glucoside, Figure 2) is the product of phloretin glycosylation. Since its
discovery more than 170 years ago, phlorizin has been widely investigated in biofilm research and the
field of human medicine [13–18].
Figure 1. The structure of phloretin.
5'
A
3
HO
3'
OH
4 OH
2
B
OH O
Figure 2. The structure of phlorizin.
5'
A
3
HO
3'
OH
4 OH
2
B
O
O
O
HO
HO
OH
OH
Phloretin and its derivatives have a broad and important role in various fields of human health and
have become a focus of natural products research. Although numerous studies show that phloretin has
anticancer effects, its solubility in lipids and water is poor, the interaction target of phloretin is unclear,
and its pharmacodynamic effects are not optimal. In addition, little research has been performed
regarding the antitumor activities of phloretin derivatives, except for phlorizin and its derivatives.
Therefore, to improve the bioavailability of phloretin and investigate the antitumor activities of phloretin
derivatives compared with docetaxel, the synthesis of a series of phloretin derivatives was accomplished
and their antitumor activity has been evaluated against various cancer cell lines, including A549 (human
lung cancer cell line), SPC-A1 (human non-small-cell lung cancer cell line), EC109 (human esophageal
cancer cell line), MCF-7 (human breast adenocarcinoma cell line) and MDA-MB-231 (human breast
cancer cell line) cells.

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2. Results and Discussion
The synthesis of a series of phloretin derivatives is outlined in Scheme 1, starting from phloretin (1),
which is commercially available. By varying the reaction temperature, 2–4 were synthesized by
methylation of the phenolic hydroxyl groups of phloretin. A mixture of phloretin (1, 10 mmol) and acetic
anhydride (50 mmol) was refluxed for 5 h to give 5 in 85% yield. Under different reaction conditions, a
mixture of phloretin 1 (10 mmol) and acetic anhydride (50 mmol) was stirred under reflux overnight in
the presence of triethylamine (50 mmol). The progress of the reaction was monitored by thin-layer
chromatography (TLC). During the reaction, three new products whose structures differed completely
from that of 5 were detected by TLC. The three products (compounds 8, 9 and 11) were isolated in 19%,
5.1% and 66% yields, respectively. The carbonyl of phloretin had isomerized to the enol form, which
was acetylated to afford 8. Compound 10 was synthesized using a similar method. Phloretin was treated
with PhCH2Br to give 6. Compound 7 was readily prepared by acylation of phloretin with
2,2,2-trichloroethyl chloroformate. The new compounds 7 and 10 were characterized by MS, 1H-NMR
and 13C-NMR. Compounds 8 and 11 were also characterized by X-ray diffraction analysis; their
structures are shown in Figures 3 and 4.
Scheme 1. Synthesis of phloretin derivatives 2–11.
2
3
R1=H, R2=R3=R4=CH3
R1=R4=H, R2=R3=CH3
OR1
O
4
5
6
7
R1=R2=R4=H, R3=CH3
R1=R2=R3=R4=COCH3
R1=H, R2=R3=R4=CH2Ph
R1=R2=R3=R4=COOCH2CCl3
R2O
RO
OR3
OR4
OR
OH
O
RO
8
9
R=COCH3
HO
OH
OH
1
OR OR
R1=H, R2=CH3
R1O
O
OR1
R2
10 R1=H, R2=CF3
11 R1=COCH3, R2=CH3
O
OH
Reagents and conditions: 2: (CH3)2SO4, K2CO3, (CH3)2CO, r.t., 3 h, 80%; 3 and 4: (CH3)2SO4, K2CO3,
(CH3)2CO, 0 °C, 3 h, 65% for 3 and 21% for 4; 5: (CH3CO)2O, reflux, 5 h, 85%; 6: PhCH2Br, K2CO3,
(CH3)2CO, 0 °C to r.t., 5 h, 50%; 7: Cl3CCH2OCOCl, K2CO3, (CH3)2CO, 0 °C to r.t., 80%. 8, 9 and
11: (CH3CO)2O, (CH3CH2)3N, reflux, overnight, 19% for 8, 5.1% for 9 and 66% for 11. 10:
(CF3CO)2O, (CH3CH2)3N, reflux, overnight, 78%.
The 1H-NMR spectra of 2–4 show chemical shifts at 14.028, 14.094 and 12.321 ppm, respectively;
these shifts are assigned to the 2-OH and are farther downfield than the usual value of 9.91 ppm because
of intramolecular hydrogen bonding. The same explanation is applicable to the chemical shifts of 14.037,
12.950, 12.200 and 12.852 ppm observed in 9–11, respectively.
In the crystal structure of 8, the dihedral angle between the two phenyl rings is 87.91°. In the
intermolecular interaction C25-H25A…O3, the H25A…O3 distance is 2.694 Å, which is slightly shorter
than the sum of the van der Waals radii of 2.72 Å, whereas the C20-H20…O2 distance of 2.499 Å is

Molecules 2014, 19
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shorter than the C25-H25A…O3 distance. These results suggest that the C20-H20…O2 interaction is
quite strong and is the greatest contributor to the stability of the molecules.
Figure 3. The molecular structure of 8, showing the atom numbering scheme. For the sake of
clarity, H atoms have been omitted. Displacement ellipsoids are drawn at the 30%
probability level.
Figure 4. The molecular structure of 11, showing the atom numbering scheme. For the sake
of clarity, H atoms have been omitted. Displacement ellipsoids are drawn at the 30%
probability level.
In a stereoview of the crystal structure of 11, several weak C-H...O interactions of the types
3
Caryl-H…O and Csp -H…O are present, which causes the molecule to dimerize. The molecules are linked
by weak Caryl-H…O hydrogen bonds, and C18-H18 and C25-H25 interact with the carbonyl O atoms
(O4 and O11, respectively) of different neighboring molecules. The new formed ring
(O3\C11\C12\C13\C14\C19) is nearly planar, with a dihedral angle of 2.30°. The phenyl rings
C14-C19 (centroid Cg1) and C24-C29 (centroid Cg2) are involved in π–π interactions, with a Cg1…Cg2
centroid-centroid distance of 3.660 Å, which makes them nearly coplanar, with a dihedral angle of 4.77°.
These hydrogen bonds and other interactions appear to play an important role in controlling the
molecular conformation.
Much attention has been devoted to the structures of 9–11. A proposed mechanism of their formation,
from a theoretical perspective, is presented in Scheme 2. Phloretin (1) can react with the acid anhydride
to give intermediate a, in which the α-hydrogen of the carbonyl group can be deprotonated in the

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presence of triethylamine (acting as a base) to form enolate b. The α-carbon of the negatively charged
enolate then attacks the carbonyl carbon of the nearby acetyl group through an addition-elimination
reaction to afford intermediate d. Intermediate d is protonated to give e. Intermediate e, as a 1,3-dione,
readily interconverts to the enol form f. Finally, 9–11 are formed by an intramolecular dehydration
reaction of intermediate f under heating.
Scheme 2. A proposed mechanism of formation for 9–11.
R2
O
HO
HO
O
OH
R1O
O
O
OR1
(CH3CH2)3N
OH
OH
a
1
O
R2
O
R1O
O
OR1
R1O
R2
OR1
O
O
O
OH
OH
c
b
HO
O
R1O
R2
OR1
O
R1O
R2
O
O
OR1
H+
tautomerizm
O
OH
OH
e
d
HO
OH
R1O
R2
OR1
R1O
O
OR1
R2
9
R1=H, R2=CH3
-H2O
10 R1=H, R2=CF3
11 R1=COCH3, R2=CH3
O
OH
O
OH
f
Phloretin and its derivatives were screened in vitro for their antitumor activities against A549,
SPC-A1, EC109, MCF-7 and MDA-MB-231 cells using the standard MTT method, with the antitumor
drug docetaxel used as a positive control. The in vitro cytotoxicity screening assays were conducted at
different compound concentrations. All of the experiments were carried out in triplicate. The IC50 values
were calculated from the percentage of cytotoxicity by nonlinear curve fitting and are presented in Table 1.
Among all of the investigated compounds, 7 exhibits the most potent growth inhibition of
MDA-MB-231 cells, with an IC50 value of 0.839 μM, indicating that it is more potent than docetaxel,
which exhibited an IC50 value of 0.998 μM. Except for 7, all of the compounds exhibit systematic
cytotoxicity in MDA-MB-231 cells, and their cytotoxicities are weaker than that of docetaxel. Moreover,
the cytotoxicities of phloretin and phlorizin are lower than that of docetaxel in MDA-MB-231 cells.
Approximately 40% of the compounds inhibit cell growth in A549 cells, and the cytotoxicities of 3
(IC50 = 19 μM) and 4 (IC50 = 23 μM) are 3.7- and 3-fold greater than that of docetaxel (IC50 = 70 μM),
respectively. The cytotoxicity of 7 (IC50 = 51 μM) is stronger than that of docetaxel, whereas the
cytotoxicities of phloretin and phlorizin are weaker than that of docetaxel.
Compared with docetaxel, all of the compounds exhibit better growth inhibition of SPC-A1 cells. For
example, the anti-proliferative effects of 3 (IC50 = 33 μM) and 7 (IC50 = 47 μM) are 5- and 3.2-fold,
respectively, greater than that of docetaxel (IC50 = 149 μM), and the cytotoxicities of phloretin and
phlorizin are similar to that of docetaxel. With respect to EC109 cells, 3, 4 and 7 exhibit better

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cytotoxicities than docetaxel. The cytotoxicities of the remaining compounds are generally weaker than
that of docetaxel. In contrast, most of the compounds exhibit are relatively active toward MCF-7 cells,
but are less active than docetaxel. With an IC50 value of 10 μM, 7 exhibits significantly weaker activity
compared to docetaxel (IC50 = 1.46 μM) in MCF-7 cells.
Table 1. In vitro anti-proliferative activity of 1–11 against human cancer cell lines.
IC50 (μM)
Compound
MDA-MB-231
30.54 ± 1.95
14.73 ± 1.28
14.43 ± 0.99
6.93 ± 0.78
A549
SPC-A1
147 ˃ 100
EC109
MCF-7
1
133 ˃ 100
132 ˃ 100
18.79 ± 0.54
23.15 ± 0.92
103 ˃ 100
105 ˃ 100
50.81 ± 0.66
95.43 ± 0.83
62.80 ± 0.96
113 ˃ 100
110 ˃ 100
120 ˃ 100
70.30 ± 1.06
148 ˃ 100
78.64 ± 0.86
117 ˃ 100
2
131 ˃ 100
138 ˃ 100
3
32.90 ± 1.13
39.29 ± 0.78
94.35 ± 1.25
96.40 ± 0.38
47.25 ± 0.93
101 ˃ 100
30.35 ± 0.76
31.79 ± 1.09
109 ˃ 100
8.42 ± 0.74
137 ˃ 100
4
5
40.886 ± 1.94
37.615 ± 1.15
0.839 ± 0.83
33.593 ± 0.67
31.69 ± 1.64
21.27 ± 1.36
92.168 ± 0.86
3.649 ± 1.19
0.998 ± 1.04
104 ˃ 100
6
82.48 ± 1.18
42.14 ± 0.88
95.41 ± 1.39
87.26 ± 0.91
103 ˃ 100
66.21 ± 1.22
10.16 ± 1.04
12.49 ± 0.89
88.73 ± 1.62
111 ˃ 100
7
8
9
10
91.5 ± 1.63
94.50 ± 1.36
97.20 ± 1.45
133 ˃ 100
11
107 ˃ 100
108 ˃ 100
Phlorizin
Docetaxel
116 ˃ 100
15.99 ± 0.67
1.46 ± 1.52
149 ˃ 100
56.44 ± 1.62
In summary, 7 exhibits the most potent growth inhibition in most cell lines, except for MCF-7,
compared to docetaxel. Interestingly, 7 contains 12 chlorine atoms, which suggests that chloro substituents
may play a role in increasing its cytotoxicity. Holla et al. synthesized a series of 2-chloro-1,4-bis-(5-
substituted-1,3,4-oxadiazol-2-ylmethyleneoxy)phenylene derivatives and evaluated their anticancer
activities [19]. Their research showed that compounds that contain two or more chloro substituents
exhibit better anticancer activities than compounds with less than two chloro substituents, which is a
result similar to ours. Furthermore, our results also show that 3–8 exhibit better anti-proliferative effects
than phloretin in the five cell lines tested. The molecular structure of phloretin includes four hydroxyl
groups. With an increase in hydroxyl groups, the cytotoxicity increases. We conclude that 7 exhibits
broad-spectrum antitumor activity worthy of further pharmacological investigation.
The growth inhibitory effects of all compounds on MDA-MB 231, SPC-A1, A549, MCF-7 and
EC109 cell lines were measured by the MTT assay after 3 days of incubation. The IC50 values, defined
as the drug concentration at which 50% of the cells are viable, were calculated from the respective
logarithmic cytotoxicity curves of the different cancer cells. The data are shown as the means ± SD from
three independent experiments.
3. Experimental Section
3.1. General Information
1
The H-NMR and 13C-NMR spectra were recorded on a Bruker 300 MHz Fourier-transform
(FT)-NMR spectrometer, and chemical shift values are reported as δ ppm relative to tetramethylsilane

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(TMS) in CDCl3 or DMSO-d6. The mass spectra were recorded on a Waters ACQUITY TQD LC-MS/MS
mass spectrometer. Crystal structures were determined using a Bruker SMART APEX CCD X-ray
single-crystal diffractometer. The MTT assay was performed using an American Thermo FC microplate.
Chemicals and solvents were obtained from commercial sources. Thin-layer chromatography (TLC)
was performed on pre-coated silica gel aluminum-backed plates (Merck F254, Darmstadt, Germany).
Chromatograms were visualized under UV light at 254 and 365 nm.
3.2. MTT Assay
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay was performed as
described by Ho et al. [19]. Cells were seeded in a 96-well plate at a concentration of 1.0 × 104 cells/well
and allowed to adhere overnight. Five replicates were prepared for each treatment and cultured for 48 or
72 h. After 20 μL of MTT (5 mg/mL) was added to each well, the cells were cultured for another 4 h.
The supernatant was discarded. After 150 μL of DMSO was added to each well, the samples were
incubated for 30 min and then swirled for 10 min. The absorbance A at 570 nm was measured using a
microplate reader. Experiments were repeated three times.
3.3. Synthesis
3.3.1. Carbonic Acid 3,5-bis-(2,2,2-Trichloroethoxycarbonyloxy)-2-{3-[4-(2,2,2-trichloroethoxy-
carbonyloxy) phenyl] propionyl}phenyl Ester 2,2,2-Trichloroethyl Ester (7)
2,2,2-Trichloroethoxycarbonyl chloride (10 mL) was slowly added to a solution of phloretin
(1, 2.74 g, 10 mmol) in acetone (30 mL) in an ice bath and then heated at reflux for 3 h. After phloretin
was observed to have disappeared by TLC, the residue obtained by evaporation of the solvent was
purified by column chromatography using a binary solvent mixture of petroleum ether-ethyl acetate (5:1)
to obtain 7 as a white solid (6.4 g, 66%). MS m/z: 993.48 [M+Na]+; 1H-NMR (CDCl3, 300 MHz) δ: 7.27
(t, 4H, Ph), 7.12 (d, 2H, J = 5.5 Hz, Ph), 4.86 (s, 8H, CH2), 3.18 (t, 2H, CH2), 2.99 (t, 2H, CH2).
13C-NMR (DMSO-d6, 75 MHz): 197.6, 152.0, 151.4, 151.0, 150.9, 148.8, 147.6, 138.7, 129.5, 124.9,
120.8, 120.7, 115.1, 94.5, 94.1, 76.9, 76.6, 76.5, 75.3, 44.9, 28.3.
3.3.2. 5,7-Dihydroxy-3-(4-hydroxybenzyl)-2-trifluoromethylchromen-4-one (10)
Trifluoroacetic anhydride (10 mL) was added to a mixture of phloretin (1, 2.74 g, 10 mmol) and
triethylamine (10 mL) in acetone (50 mL) and heated at reflux for 5 h. After a new compound was
observed by TLC, the reaction mixture was diluted with water (50 mL) and extracted with
dichloromethane (3 × 50 mL). The organic phases were collected and dried over anhydrous magnesium
sulfate. The solution was filtered under vacuum and purified by column chromatography using a binary
solvent mixture of petroleum ether-ethyl acetate (5:1-3:1) to afford 10 (2.74 g, 78%). MS m/z: 352.98
1
[M+H]+; H-NMR (DMSO-d6, 300 MHz) δ: 12.20 (s, 1H, OH), 11.22 (s, 1H, OH), 9.24 (s, 1H, OH),
7.00 (d, 2H, J = 2.0 Hz, Ph), 6.68 (d, 2H, J = 2.5 Hz, Ph), 6.42 (s, 1H, Ph), 6.29 (s, 1H, Ph), 3.82 (t, 2H,
13
CH2). C-NMR (DMSO-d6, 75 MHz): 180.4, 165.5, 161.3, 156.3, 155.8, 147.6, 147.3, 128.8, 127.7,
121.8, 115.1, 103.7, 99.7, 93.9, 26.6.

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3.4. X-ray Crystallographic Data
CCDC 1026553 and 1026554 contain the supplementary crystallographic data for this paper. These
data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: deposit@ccdc.cam.ac.uk).
3.5. Characterization of Compounds 2–6, 8, 9 and 11
1-(2-Hydroxy-4,6-dimethoxy-phenyl)-3-(4-methoxy-phenyl)-propan-1-one (2): ESI-MS m/z: 317.11
[M+H]+, 340 [M+Na]+; 1H-NMR (CDCl3, 300 MHz) δ 2.900 (t, 2H, CH2), 3.252 (t, 2H, CH2), 3.705 (s,
9H, OCH3), 5.919 (s, 1H, Ph), 6.070 (s, 1H, Ph), 6.852 (d, 2H, Ph), 7.140 (d, 2H, Ph), 14.028 (s, 1H,
OH). 13C-NMR (DMSO-d6,75 MHz): 204.45, 165.79, 165.60, 162.40, 155.39, 131.30, 129.11, 115.06,
105.49, 93.79, 90.87, 56.06, 55.64, 45.67, 29.31.
1-(2-Hydroxy-4,6-dimethoxy-phenyl)-3-(4-hydroxy-phenyl)-propan-1-one (3): ESI-MS m/z: 303.62
[M+H]+; 1H-NMR (DMSO-d6, 300 MHz) δ 2.936 (t, 2H, CH2), 3.243 (t, 2H, CH2), 3.801 (s, 3H, OCH3),
3.821 (s, 3H, OCH3), 5.338 (s, 1H, OH), 5.919 (s, 1H, Ph), 6.071 (s, 1H, Ph), 6.779 (d, 2H, Ph), 7.100
(d, 2H, Ph), 14.094 (s, 1H, OH).
1-(2,4-Dihydroxy-6-methoxy-phenyl)-3-(4-hydroxy-phenyl)-propan-1-one (4): ESI-MS m/z: 289.04
[M+H]+; 1H-NMR (DMSO-d6, 300 MHz) δ 2.778(t, 2H, CH2), 3.220 (t, 2H, CH2), 3.741 (s, 3H, OCH3),
5.956 (s, 2H, Ph), 6.661 (d, 2H, Ph), 7.009 (d, 2H, Ph), 9.141 (s, 1H, OH),12.321 (s, 2H, OH). 13C-NMR
(DMSO-d6, 75 MHz): 169.22, 165.43, 163.96, 155.38, 131.48, 129.12, 115.04, 104.55, 93.20, 55.31,
45.58, 29.29.
Acetic acid 4-[3-oxo-3-(2,4,6-triacetoxy-phenyl)-propyl]-phenyl ester (5): ESI-MS m/z: 443.28 [M+H]+;
1H-NMR (CDCl3, 300 MHz) δ 2.120 (s, 6H, CH3), 2.270 (s, 6H, CH3), 2.974 (t, 2H, CH2), 3.070 (t, 2H,
CH2), 6.917 (s, 2H, Ph), 7.003 (d, 2H, Ph), 7.212 (d, 2H, Ph).
3-(4-Benzyloxy-phenyl)-1-(2,4-bis-benzyloxy-6-hydroxy-phenyl)-propan-1-one (6): ESI-MS m/z: 545.35
[M+H]+; 1H-NMR (CDCl3, 300 MHz) δ 2.846 (t, 2H, CH2), 3.242 (t, 2H, CH2), 5.036 (s, 6H), 6.112 (m,
2H, Ph), 6.836 (m, 4H, Ph), 7.206–7.428 (m, 15H, Ph), 14.037 (s, 1H, OH). 13C-NMR (DMSO-d6,
75 MHz): 204.32, 165.74, 164.51, 162.77, 162.23, 161.43, 160.84, 156.37, 141.02, 137.32, 133.04,
128.96, 128.55, 128.47, 128.39, 128.26, 128.16, 127.96, 127.85, 127.51, 114.51, 106.70, 105.80, 105.20,
94.84, 92.59, 89.76, 71.03, 69.75, 69.10, 45.48, 28.87.
Acetic acid 4-[3-acetoxy-3-(2,4,6-triacetoxy-phenyl)-allyl]-phenyl ester (8): ESI-MS m/z: 485.5
1
[M+H]+; H-NMR (CDCl3, 300 MHz) δ 2.151 (s, 3H, CH3), 2.261 (s, 3H, CH3), 2.322 (s, 6H, CH3),
2.427 (s, 3H, CH3), 3.836 (s, 2H, CH2), 5.799 (t, 1H, CH), 6.979 (m, 2H, Ph), 7.166-7.260 (m, 4H, Ph).
13C-NMR (CDCl3, 75 MHz): 169.22, 153.57, 148.67, 129.35, 128.77, 121.81, 121.59, 113.86, 109.22,
20.86, 20.79, 20.42, 18.18.

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5,7-Dihydroxy-3-(4-hydroxy-benzyl)-2-methyl-chromen-4-one (9): ESI-MS m/z: 299.09 [M+H]+;
1H-NMR (DMSO-d6, 300 MHz) δ 2.384(s, 3H, CH3), 3.670 (s, 2H, CH2), 6.172 (s, 1H, Ph), 6.303 (s, 1H,
Ph), 6.660 (d, 2H, Ph), 7.030 (d, 2H, Ph), 9.162 (s, 1H, OH), 10.767 (s, 1H, OH), 12.950 (s, 1H, OH).
Acetic acid 4-(7-acetoxy-5-hydroxy-2-methyl-4-oxo-4H -chromen-3-ylmethyl)-phenyl ester (11): ESI-MS
m/z: 383 [M+H]+; 1H-NMR (CDCl3, 300 MHz) δ 2.273 (s, 3H, CH3), 2.316 (s, 3H, CH3), 2.401 (s, 3H,
CH3), 3.863 (s, 2H, CH2), 6.524 (s, 1H, Ph), 6.670 (s, 1H, Ph), 6.976 (d, 2H, Ph), 7.244 (d, 2H, Ph),
12.852 (s, 1H, OH). 13C-NMR (DMSO-d6, 75 MHz): 181.35, 169.02, 168.36, 166.36, 160.70, 156.06,
155.62, 148.78, 136.44, 128.94, 121.66, 118.47, 107.33, 104.91, 101.02, 27.96, 20.89, 20.78, 18.48.
4. Conclusions
In conclusion, we synthesized a series of phloretin derivatives and tested their antitumor activities
toward the MDA-MB-231, SPC-A1, A549, MCF-7 and EC109 cell lines. Most of the tested compounds
exhibited moderately strong cytotoxicity toward the five cell lines. Notably, 7 exhibited greater
cytotoxicity compared to docetaxel toward the five cell lines tested. Compounds 3 and 4 also
demonstrated excellent anticancer activity. Therefore, these derivatives may hold great promise as
therapeutic agents for the treatment of human cancers.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/10/16447/s1.
Acknowledgments
This study was partially supported by the Chinese National Science & Technology Major Project
(Grants 2011ZX09401-308-23), the Scientific Research Program Funded by the Shaanxi Provincial
Education Department (No.14JK1628), the Medical Project of the Bureau of Science and Technology
Xi’an City and the Scientific Research Project of the Affiliated Hospital of Xi’an Medical University
(No. XYFY2013-02).
Author Contributions
Y.-J.L. and L.W. conceived and designed the study. L.W. and Z.-W.L. performed the syntheses. R.X.
and Y.-F.L. performed the NMR and analyses. W.Z. and F.G. performed the anticancer activity
evaluations. L.W. and W.Z. wrote the paper. Z.-W.L. and Y.-J.L. reviewed and edited the manuscript.
All authors read and approved the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.
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