Synthesis of chromonylthiazolidines and their cytotoxicity to human cancer cell lines

Article abstract of DOI:10.3390/molecules20011151

Nine new chromonylthiazolidine derivatives were successfully semi-synthesized from paeonol. All of the compounds, including starting materials, the intermediate compound and products, were evaluated for their cytotoxic effects toward eight human cancer ce

Full text of DOI:10.3390/molecules20011151

Molecules 2015, 20, 1151-1160; doi:10.3390/molecules20011151
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Chromonylthiazolidines and Their Cytotoxicity to
Human Cancer Cell Lines
Hoang Le Tuan Anh 1, Nguyen Thi Cuc 1, Bui Huu Tai 1,2, Pham Hai Yen 1, Nguyen Xuan Nhiem 1,
Do Thi Thao 3, Nguyen Hoai Nam 1, Chau Van Minh 1, Phan Van Kiem 1 and Young Ho Kim 2,*
1
Institute of Marine Biochemistry, Vietnam Academy of Science and Technology,
18 Hoang Quoc Viet, Caugiay, Hanoi 10000, Vietnam;
E-Mails: anh_792002@yahoo.com (H.L.T.A.); cuc_k51a@yahoo.com (N.T.C.);
bhtaiich@gmail.com (B.H.T.); yeninpc@yahoo.com (P.H.Y.); nxnhiem@yahoo.com (N.X.N.);
namnguyenhoai@imbc.vast.vn (N.H.N.); cvminh@vast.ac.vn (C.V.M.);
phankiem@yahoo.com (P.V.K.)
2
College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea
3
Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet,
Caugiay, Hanoi 10000, Vietnam; E-Mail: thaodo74@yahoo.com
* Author to whom correspondence should be addressed; E-Mail: yhk@cnu.ac.kr;
Tel.: +82-42-821-5933; Fax: +82-42-823-6566.
Academic Editor: Jean Jacques Vanden Eynde
Received: 5 December 2014 / Accepted: 6 January 2015 / Published: 12 January 2015
Abstract: Nine new chromonylthiazolidine derivatives were successfully semi-synthesized
from paeonol. All of the compounds, including starting materials, the intermediate
compound and products, were evaluated for their cytotoxic effects toward eight human
cancer cell lines. The synthesized chromonylthiazolidines displayed weak cytotoxic effects
against the tested cancer cell lines, but selective cytotoxic effects were observed.
Compounds 3a and 3b showed the most selective cytotoxic effects against human
epidermoid carcinoma (IC50 44.1 ± 3.6 μg/mL) and breast cancer (IC50 32.8 ± 1.4 μg/mL)
cell lines, respectively. The results suggest that chromoylthiazolidines are potential low-cost,
and selective anticancer agents.
Keywords: anti-cancer agent; chromonylthiazolidine; Paeonia lactiflora; paeonol; selective
cytotoxicity; semi-synthesis

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1. Introduction
Cancer is one of the most serious global health threats. It is characterized by explosive proliferation
of cells. To date, over 100 types of cancer have been classified on the basis of the initially affected cells.
Annually, more than 11 million people are diagnosed with cancer and it is estimated that up to
16 million new cases will occur annually by 2020 [1]. Cancer has various causes, including genetic
factors, lifestyle, chemicals, and radiation exposure. Cancer patients can be saved if the cancer is
detected at an early stage and effective treatment is provided. Cancer treatment depends on the type of
cancer and its stage, but it usually comprises a combination of therapies such as surgery, radiation,
chemotherapy, and gene therapy. Among them, chemotherapy remains an important option for treatment
of cancer [2]. Many chemotherapeutic agents, such as 5-fluorouracil, cisplatin, paclitaxel and docetaxel,
have been approved and are currently used to treat cancer [3]. The main goal of anti-cancer agents is to
induce apoptosis of cancer cells. However, in some cases, the dose of anticancer drug needed to kill
cancer cells is toxic to normal cells and hence leads to many undesired side effects. Moreover, decreased
drug activity or development of drug resistance during long-term treatment is believed to cause failure
in over 90% of patients with metastatic cancer [4,5]. Therefore, discovery of novel anticancer agents
with high efficacy, selectivity, low costs and minimal side effects, and new approaches to cancer
treatment, is an exciting prospect.
Thiazolidine rings, notably 1,4-thiazolidinedione (TZD), have been used as a scaffold to develop
novel anticancer agents with a broad spectrum of cytotoxicity toward various human cancer cells [6,7].
Recently, TZD derivatives were emphasized as a new dawn in cancer chemotherapy [8,9]. Various
preclinical and clinical studies strongly suggest that the anticancer activity of TZDs is mediated in part
by PPAR-γ-dependent or -independent pathways. Several new TZDs, such as efatutazone, netoglitazone,
rosiglitazone and troglitazone, are being explored in terms of the mechanism underlying their anticancer
activity [8]. Having researched anticancer agents containing a thiazolidine core motif, we herein report
the synthesis of nine new chromonylthiazolidines and evaluation of their cytotoxicity against several
human cancer cell lines.
2. Results and Discussion
In the past decade, thiazolidines were emphasized as a new class of compounds with a wide range of
pharmacological activities, such as antihyperglycemic, euglycemic, anti-inflammatory, antimalarial,
antioxidant, antitumor, cytotoxic, antimicrobial, and antiproliferative effects [10–12]. The literature also
indicates that thiazolidines act as PPAR agonists and inhibit various important enzymes, such as
cyclin-dependent kinases (CDKs), glycogen synthase kinase (GSK), aldose reductase and bacterial
arylamine N-acetyltransferase (NAT) [8,10]. Due to their broad biological spectrum, thiazolidine
molecules have attracted the attention of medicinal chemists and a number of synthesis strategies have
been developed.
Chromone derivatives are abundant in all parts of plants and vegetables, such as flowers, fruits, seeds
and bark [13,14]. They show low mammalian toxicity and are present in large amounts in
food [15]. Many naturally occurring molecules contain chromone in their structures, especially
flavonoids, which exhibit a wide range of biological activities [14,16]. Therefore, we hypothesized that

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chromonylthiazolidines might have a broad spectrum of pharmacological activities as well as selective
cytotoxicity toward cancer cell lines. We thus aimed to design and optimize chromonylthiazolidines as
a potential anti-cancer candidate by anti-proliferative effects against several human cancer cell lines.
In our strategy, chromonylthiazolidines 3a–i were synthesized by a Knoevenagel condensation
reaction between 3-formyl-7-methoxychromone (2) and different thiazolidine derivatives (Scheme 1).
First, 3-formyl-7-methoxychromone (2) was semi-synthesized from the starting material paeonol (1),
which was isolated in abundance from Paeonia lactiflora (see Experimental Section). Through the
Vilsmeier–Haack reaction, compound 2 was easily obtained by treatment of paeonol with
dimethylformamide and phosphorous oxychloride [17]. Next, compound 2 was reacted with appropriate
thiazolidine derivatives in the presence of piperidine as a base catalyst to give compounds 3a–i.
However, the reaction yields of compounds 3g–i were low, so anhydrous sodium acetate and glacial
acetic acid were used as the catalyst and reaction medium, respectively, to improve reaction yields.
Finally, chromonylthiazolidines 3a–i were obtained with reaction yields ranging from 38% to 72% (see
Experimental Section, Table 1). The structures of the synthesized compounds were elucidated on the
1
13
basis of H-NMR, C-NMR and ESI-MS spectra. All spectral data were in accordance with assumed
structures (see Experimental Section). Chromonylthiazolidines 3a–i, paeonol (1), and the intermediate
compound 2 were evaluated for their effects on the viability of various human cancer cell lines: HepG2
(hepatocellular carcinoma), HL-60 (acute promyeloid leukemia), KB (epidermoid carcinoma), LLC
(Lewis lung carcinoma), LNCaP (hormone dependent prostate cancer), LU-1 (human lung cancer),
MCF7 (breast cancer) and SW480 (colon adenocarcinoma). Briefly, cancer cells were cultured with
various concentrations of synthetic compounds (0.8–100 μg/mL) for 3 days. Then, the cells were
incubated with MTT solution for 4 h. Formation of a violet precipitate, formazan, was monitored in
DMSO at a wavelength of 540 nm using a microplate reader. The cytotoxicity of compounds is expressed
as IC50 values. Ellipticine is an efficient anticancer drug. It functions by number of mechanisms such as
participating in cell cycle arrest, initiation of apoptosis, DNA-damaging [18,19]. Thus, in our
experiments, ellipticine was used as a positive control. Its IC50 values for HepG2, HL-60, KB, LLC,
LNCaP, LU-1, MCF-7 and SW480 cells were 1.45 ± 0.08, 0.56 ± 0.04, 0.43 ± 0.05, 0.98 ± 0.04,
0.86 ± 0.06, 1.29 ± 0.11, 0.49 ± 0.04 and 0.62 ± 0.05 μg/mL, respectively. Our results indicate that
the synthesized chromonylthiazolidines exhibit weak cytotoxic effects toward the eight tested human
cancer cell lines (Table 2). However, the results also suggest selective cytotoxic effects of
chromonylthiazolidines toward cancer cells. Notably, compound 3a showed weak cytotoxic effects
against KB and LU-1 cells (IC50 44.1 ± 3.6 and 52.9 ± 3.4 μg/mL, respectively), but it did not show
potent cytotoxic effects against the other cancer cell lines. Similarly, compound 3b exhibited stronger
cytotoxicity only against MCF-7 cells (IC50 32.8 ± 1.4) among the eight selected cancer cell lines. In
addition, the chromonylthiazolidine-2,4-dione derivatives 3a–c displayed cytotoxic effects stronger than
those of the chromonylthiazolidine-2-thione-4-one derivatives 3d–i. Compound 3c exhibited modest
cytotoxic effects toward all tested cancer cell lines. Meanwhile, compounds 3e and 3g showed significant
selective cytotoxic effects against half of the tested cancer cell lines (HepG2, HL-60, LLC and SW480).
All of the synthesized chromonylthiazolidines showed weak cytotoxicity against HL-60 and SW480
cells: IC50 values were 74.6 ± 4.6 to 94.8 ± 6.8 μg/mL and 71.4 ± 3.6 to 92.5 ± 4.12 μg/mL, respectively.

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Scheme 1. Synthesis of the chromonylthiazolidines.
Table 1. Synthesized chromonylthiazolidines 3a–i.
Compounds
X
O
O
R
H
Yield (%)
3a
3b
72
47
CH3
3c
O
38
3d
3e
3f
S
S
S
S
S
NH2
CH3
66
60
72
55
50
H2C-CH=CH2
H
3g
3h
H2C-COOH
3i
S
66
Table 2. Cytotoxicity of compounds 1, 2, and 3a–i toward various human cancer cell lines.
IC50 (μg/mL)
Comp.
HepG2
>100
HL-60
>100
KB
>100
LLC
>100
LNCaP
>100
LU-1
>100
MCF7
>100
SW480
1
>100
2
>100
>100
>100
>100
>100
>100
>100
>100
3a
3b
3c
3d
3e
3f
3g
3h
3i
>100
82.2 ± 4.5
75.3 ± 3.9
92.3 ± 5.2
74.6 ± 4.6
94.8 ± 6.8
87.6 ± 6.7
84.6 ± 5.4
94.2 ± 7.1
84.3 ± 4.2
0.56 ± 0.04
44.1 ± 3.6
84.6 ± 4.2
74.1 ± 5.1
90.2 ± 2.4
>100
87.4 ± 6.3
>100
77.4 ± 5.8
81.6 ± 6.3
84.2 ± 4.1
92.4 ± 5.7
>100
52.9 ± 3.4
>100
66.0 ± 2.7
32.8 ± 1.4
52.7 ± 3.6
88.4 ± 7.4
>100
71.4 ± 3.6
90.1 ± 4.8
85.4 ± 7.4
84.3 ± 6.2
87.6 ± 4.8
81.3 ± 5.6
90.8 ± 6.4
85.7 ± 3.2
92.5 ± 4.1
0.62 ± 0.05
86.3 ± 6.4
78.4 ± 5.8
94.5 ± 6.3
81.4 ± 7.4
78.4 ± 3.4
88.4 ± 6.7
91.3 ± 8.2
>100
90.1 ± 7.7
>100
65.5 ± 4.1
59.0 ± 4.3
>100
90.2 ± 4.9
>100
>100
91.6 ± 7.6
>100
>100
65.9 ± 4.7
>100
>100
90.8 ± 5.2
87.4 ± 6.4
92.8 ± 6.4
0.98 ± 0.04
>100
88.4 ± 3.5
90.2 ± 2.7
0.43 ± 0.05
81.9 ± 4.3
85.1 ± 6.1
85.6 ± 5.6
>100
>100
80.9 ± 6.9
Ellipticine 1.45 ± 0.08
0.86 ± 0.06 1.29 ± 0.11 0.49 ± 0.04

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3. Experimental Section
3.1. General Experimental Procedures
Melting points were determined on an Electrothermal 9100 melting point apparatus. NMR spectroscopy
was recorded on AVANCE 500 MHz (Bruker, Karlsruhe, Germany) instrument. Electrospray ionization
mass spectrometry (ESI-MS) was measured by Agilent 1100 Series LC/MSD Trap SL (Agilent
Technologies, Waldbronn, Germany). HR-ESI-MS spectra were recorded on MicroQ-TOP III mass
spectrometer (Bruker Daltonics, Bremen, Germany). 3-Methyl-2,4-thiazolidinedione (used for the
synthesis of 3b) and N-(3-methoxybenzyliden)-3-amino-2-thione-4-thiazolidinone (using for the
synthesis of 3i) were synthetic compounds. They were easily obtained from reactions between
2,4-thiazolidinedione/3-amino-2-thione-4-thiazolidinone and iodomethane/3-methoxybenzaldehyde,
respectively [20,21]. Other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
3.2. Isolation of Paeonol (1)
Dried and powdered of Paeonia lactiflora leaves (3 kg) were extracted with chloroform
(20 L × 3 times) at room temperature for 3 times. After removal of solvent, a chloroform extract (130 g)
was obtained. This crude extract was separated on a silica gel chromatography column eluted with a
gradient solvent system of n-hexane/acetone (1/0, 50/1, 10/1, 5/1, and 1/1, each 3 L stepwise) to give
five fractions C1‒C5. Fraction C3 (67 g) was recrystallized several times in methanol to obtain paeonol
(1, 11.4 g, 0.38% w/w of dried material) [22]. ESI-MS: m/z 167.0 [M+H]+; 1H-NMR (500 MHz, CDCl3)
δ (ppm): 12.73 (1H, s, OH), 7.63 (1H, d, J = 8.0 Hz, H-Ar), 6.44 (1H, dd, J = 8.0, 2.0 Hz, H-Ar), 6.42
13
(1H, d, J = 2.0 Hz, H-Ar), 3.83 (3H, s, OCH3), 2.55 (3H, s, COCH3). C-NMR (125 MHz, CDCl3) δ
(ppm): 202.5, 166.1, 165.3, 132.3, 113.9, 107.6, 100.9, 55.5, 26.2.
3.3. Synthesis of 3-Formyl-7-methoxychromone (2)
A solution of paeonol (1, 3.32 g, 0.02 mol) in N,N'-dimethylformamide (DMF, 10 mL) was added to
a 100 mL round-bottomed flask and stirred in an ice bath (0–5 °C). Next, phosphorus oxychloride
(POCl3, 4 mL) was slowly added. After that, the reaction mixture was warmed and kept at room
temperature for 30 min. The reaction solution was poured into iced water. The reaction product was
filtered, washed with distilled water, and re-crystalized in a solvent system of n-hexane/ethyl acetate to
yield 3-formyl-7-methoxychromone (2, 3.08 g, 76%) [23]. ESI-MS: m/z 205.2 [M+H]+, 227.0 [M+Na]+;
1H-NMR (500 MHz, CDCl3) δH (ppm): 10.38 (1H, d, J = 4.0 Hz, CHO), 8.48 (1H, brs, H-2), 8.21 (1H,
d, J = 9.0 Hz, H-5), 7.06 (1H, d, J = 9.0 Hz, H-6), 6.92 (1H, s, H-8), 3.93 (3H, s, OCH3).
3.4. Synthesis of Chromonylthiazolidines 3a‒i
3.4.1. Procedure 1
Compound 2 (100 mg, 0.5 mmol) and 2,4-thiazolidinedione or 2-thione-4-thiazolidinone derivatives
(0.5 mmol) were dissolved in ethanol (5 mL). Piperidine (100 μL) was added as a catalyst and the
reaction mixture was stirred at room temperature for 5 h. Then, the reaction mixture was poured into

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iced water to yield a precipitate. The product was filtered, washed with distilled water and recrystallized
from the indicated solvents to yield compounds 3a‒f.
5-((7-Methoxy-4-oxo-4H-chromen-3-yl)methylene)thiazolidine-2,4-dione (3a): From EtOH/DMF (1:1);
Mp. 183–184 °C; HR-ESI-MS m/z: 304.0261 [M+H]+ (calcd [C14H10O5NS]+ for 304.0274), 326.0085
1
[M+Na]+ (calcd [C14H9O5NSNa]+ for 326.0094); ESI-MS m/z: 304.0 [M+H]+; H-NMR (500 MHz,
DMSO-d6) δH ppm: 12.38 (1H, s, NH), 8.72 (1H, s, H-2), 8.02 (1H, d, J = 8.5 Hz, H-5); 7.57 (1H, s, H-1'),
7.19 (1H, s, H-8), 7.12 (1H, d, J = 8.5 Hz, H-6), 3.91 (3H, s, 7-OCH3); 13C-NMR (125 MHz, DMSO-d6)
δC ppm: 173.8, 169.0, 167.3, 164.3, 160.2, 126.9, 124.8, 124.4, 117.7, 116.6, 115.4, 100.9, 56.2.
5-((7-Methoxy-4-oxo-4H-chromen-3-yl)methylene)-3-methyl-thiazolidine-2,4-dione
(3b):
From
EtOH/DMF (1:1); Mp. 186–187 °C; HR-ESI-MS m/z: 340.0246 [M+Na]+ (calcd [C15H11O5NSNa]+ for
340.0250); ESI-MS: m/z 318.1 [M+H]+; 1H-NMR (500 MHz, DMSO-d6) δH (ppm): 8.83 (1H, s, H-2),
8.03 (1H, d, J = 9.0 Hz, H-5), 7.69 (1H, s, H-1'), 7.23 (1H, d, J = 2.0 Hz, H-8), 7.14 (1H, dd, J = 9.0,
2.0, H-6), 3.92 (3H, s, 7-OCH3), 3.08 (3H, s, NCH3).
3-Benzyl-5-((7-methoxy-4-oxo-4H-chromen-3-yl)methylene)thiazolidine-2,4-dione
(3c):
From
EtOH/toluene (1:1); Mp. 211–212 °C; HR-ESI-MS m/z: 416.0551 [M+Na]+ (calcd [C21H15O5NSNa]+
for 416.0563); ESI-MS: m/z 394.0 [M+H]+; 1H-NMR (500 MHz, DMSO-d6) δH (ppm): 8.85 (1H, s, H-2),
8.03 (1H, d, J = 9.0 Hz, H-5), 7.72 (1H, s, H-1'), 7.33–7.28 (5H, m, H-Phenyl), 7.23 (1H, d, J = 2.0 Hz,
H-8), 7.14 (1H, dd, J = 9.0, 2.0 Hz, H-6), 4.81 (2H, s, NCH2), 3.92 (3H, s, 7-OCH3).
3-Amino-5-((7-methoxy-4-oxo-4H-chromen-3-yl)methylene)-2-thioxothiazolidin-4-one (3d): From
EtOH; Mp. 219–220 °C; HR-ESI-MS m/z: 335.0140 [M+H]+ (calcd [C14H11O4N2S2]+ for 335.0155),
1
356.9967 [M+Na]+ (calcd [C14H10O4N2S2Na]+ for 356.9974); ESI-MS: m/z 335.0 [M+H]+; H-NMR
(500 MHz, DMSO-d6) δH (ppm): 8.95 (1H, s, H-2), 8.03 (1H, d, J = 8.5 Hz, H-5), 7.57 (1H, s, H-1'),
7.23 (1H, s, H-8), 7.14 (1H, d, J = 8.5 Hz, H-6), 3.92 (3H, s, 7-OCH3); 13C-NMR (125 MHz, DMSO-d6)
δC ppm: 191.1, 174.0, 164.5, 163.9, 163.1, 157.2, 127.1, 126.9, 121.5, 117.9, 116.5, 115.6, 101.3, 56.2.
5-((7-Methoxy-4-oxo-4H-chromen-3-yl)methylene)-3-methyl-2-thioxothiazolidin-4-one (3e): From
EtOAc; Mp. 197–198 °C; HR-ESI-MS m/z: 356.0014 [M+Na]+ (calcd [C15H11O4NS2Na]+ for 356.0022);
ESI-MS: m/z 334.0 [M+H]+; 1H-NMR (500 MHz, DMSO-d6) δH (ppm): 8.95 (1H, s, H-2), 8.04 (1H, d,
J = 9.0 Hz, H-5), 7.55 (1H, s, H-1'), 7.24 (1H, s, H-8), 7.15 (1H, d, J = 9.0 Hz, H-6), 3.92 (1H, s,
7-OCH3), 3.38 (3H, s, NCH3).
3-Allyl-5-((7-methoxy-4-oxo-4H-chromen-3-yl)methylene)-2-thioxothiazolidin-4-one (3f): From EtOAc;
Mp. 202–203 °C; HR-ESI-MS m/z: 382.0169 [M+Na]+ (calcd [C17H13O4NS2Na]+ for 382.0178);
ESI-MS: m/z 360.0 [M+H]+; 1H-NMR (500 MHz, DMSO-d6) δH (ppm): 8.95 (1H, s, H-2), 8.03 (1H, d,
J = 9.0 Hz, H-5), 7.54 (1H, s, H-1'), 7.23 (1H, d, J = 2.5 Hz, H-8), 7.14 (1H, dd, J = 9.0; 2.5 Hz, H-6),
5.85 (1H, m, H-Vinyl), 5.17 (1H, dd, J = 10.5; 1.0 Hz, H-Vinyl), 5.10 (1H, dd, J = 17.5; 1.0 Hz,
H-Vinyl), 4.62 (2H, d, J = 5.0 Hz, NCH2), 3.91 (3H, s, 7-OCH3); 13C-NMR (125 MHz, DMSO-d6) δC
(ppm): 196.4, 174.10, 166.8, 164.5, 163.0, 157.2, 130.3, 127.0, 126.8, 123.5, 117.8, 117.5, 116.5, 115.6,
101.1, 56.2, 45.8.

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3.4.2. Procedure 2
A solution of compound 2 (100 mg, 0.5 mmol), 2-thione-4-thiazolidinone derivatives (0.5 mmol),
and sodium acetate (50 mg) in glacial acetic acid (5 mL) was added to a 50 mL round-bottomed flask.
The reaction mixture was refluxed for 2 h and then poured into ice water to yield a precipitate. The
product was filtered, washed with distilled water and recrystallized from ethanol (unless otherwise
indicated) to yield compounds 3g‒i.
5-((7-Methoxy-4-oxo-4H-chromen-3-yl)methylene)-2-thioxothiazolidin-4-one (3g): From EtOH; Mp.
192–193 °C; HR-ESI-MS m/z: 320.0031 [M+H]+ (calcd [C14H10O4NS2]+ for 320.0046), 341.9857
[M+Na]+ (calcd [C14H9O4NS2Na]+ for 341.9865); ESI-MS: m/z 320.0 [M+H]+, 318.0 [M−H]−; 1H-NMR
(500 MHz, DMSO-d6) δH (ppm): 8.86 (1H, s, H-2), 8.02 (1H, d, J = 9.0 Hz, H-5), 7.37 (1H, s, H-1'), 7.22
(1H, d, J = 2.5 Hz, H-8), 7.13 (1H, dd, J = 9.0, 2.5 Hz, H-6), 3.91 (3H, s, 7-OCH3). 13C-NMR (125 MHz,
DMSO-d6) δC (ppm): 198.6, 174.1, 164.5, 163.9, 162.2, 157.2, 127.0, 117.7, 116.6, 115.6, 101.0, 56.3.
2-(5-((7-Methoxy-4-oxo-4H-chromen-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (3h):
From EtOH; Mp. 223–224 °C; HR-ESI-MS m/z: 378.0101 [M+H]+ (calcd [C16H12O6NS2]+ for 378.0101),
399.9912 [M+Na]+ (calcd [C16H11O6NS2Na]+ for 399.9920); ESI-MS: m/z 378.0 [M+H]+, 375.9 [M−H]−;
1H-NMR (500 MHz, DMSO-d6) δH ppm: 8.97 (1H, s, H-2), 8.04 (1H, d, J = 8.5 Hz, H-5), 7.58 (1H, s,
H-1'), 7.24 (1H, d, J = 2.5 Hz, H-8), 7.14 (1H, dd, J = 8.5, 2.5 Hz, H-6), 4.17 (2H, s, NCH2), 3.92 (3H,
s, 7-OCH3); 13C-NMR (125 MHz, DMSO-d6) δC ppm: 196.7, 174.2, 167.3, 166.7, 164.6, 163.4, 157.3,
127.0, 127.0, 123.2, 117.7, 116.5, 115.7, 101.0, 56.3, 44.9.
5-((7-Methoxy-4-oxo-4H-chromen-3-yl)methylene)-3-((3-methoxybenzylidene)amino)-2-thioxothiazol-
idin-4-one (3i): From EtOH/toluene (1:1); Mp. 234–235 °C; HR-ESI-MS m/z: 475.0381 [M+Na]+ (calcd
1
[C22H16O5N2S2Na]+ for 475.0393); ESI-MS: m/z 453.0 [M+H]+; H-NMR (500 MHz, DMSO-d6) δH
ppm: 9.02 (1H, s, N=CH), 8.88 (1H, s, H-2), 8.07 (1H, d, J = 8.5 Hz, H-5), 7.61 (1H, s, H-1'), 7.49 (3H,
overlapped, H-Ar), 7.27 (1H, d, J = 2.5 Hz, H-8), 7.24 (1H, m, H-Ar), 7.17 (1H, dd, J = 8.5, 2.5 Hz,
H-6), 3.93 (3H, s, 7-OCH3), 3.84 (3H, s, Ar-OCH3).
3.5. Cell Culture and Cell Viability Assay
Eight human cancer cell lines including HepG2 (hepatocellular carcinoma), HL-60 (acute promyeloid
leukemia), KB (epidermoid carcinoma), LLC (Lewis lung carcinoma), LNCaP (hormone dependent
prostate cancer), LU-1 (human lung cancer), MCF7 (breast cancer), and SW480 (colon adenocarcinoma)
were used for cytotoxic evaluation. Cells were obtained from the American Type Culture Collection
(Manassas, VA, USA) and cultured in DMEM or RPMI-1640 containing 2 mM L-glutamine, 1.5 g/L
sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate and supplement with
fetal bovine serum (FBS) 10%. The MCF7 medium was further added with 0.01 mg/mL bovine insulin
while LNCaP medium was supplied with 10 nM of testosterone. The cells were incubated at 37 °C in a
humidified atmosphere (95% air and 5% CO2).
The inhibitory effects of compounds on the growth of human cancer cell lines were determined by
measuring metabolic activity using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

Molecules 2015, 20
1158
(MTT) assay as previous described [24]. Briefly, human cancer cell lines (1 × 105 cells/mL) were treated
for 3 days with series concentrations 0.8, 4.0, 20.0, 50.0, and 100.0 µg/mL of the compounds dissolved
in DMSO (100 mg/mL for stock solution, and a final concentration of DMSO in all experiments was
0.1%, v/v). After incubation, 0.1 mg (50 µL of a 2 mg/mL solution) MTT solution was added to each
well and the cells were then incubated at 37 °C for 4 h. The culture medium was then carefully aspirated
and dimethylsulfoxide (150 µL) was added to each well to dissolve the formazan crystals. The plates
were read immediately at 540 nm on a microplate reader (TECAN GENIOUS). All the experiments were
performed three times and the mean absorbance values were calculated. The results are expressed as the
percentage of inhibition that produced a reduction in the absorbance by the treatment of the compounds
compared to the untreated controls. A dose-response curve was generated and the inhibitory
concentration of 50% (IC50) was determined for each compound as well as each cell line. Ellipticine was
used as a positive control.
3.6. Statistical Analysis
All data were expressed as mean ± S.D. of at least three independent experiments performed in
triplicates. Statistical significance is indicated by one-way ANOVA followed by Dunnett’s multiple
comparison test using GraphPad Prism 6 program (GraphPad Software Inc., San Diego, CA, USA),
p < 0.05.
4. Conclusions
In summary, the semi-synthesis of chromonylthiazolidines from paeonol, which was isolated in
abundance from P. lactiflora, was inexpensive and gave good yields. The assumed structures of the
1
synthesized compounds agreed with their H-NMR, 13C-NMR and HR-ESI-MS spectra data. All
compounds were evaluated in term of their cytotoxic effects against eight human cancer cell lines:
HepG2, HL-60, KB, LLC, LNCaP, LU-1, MCF-7 and SW480 cell lines. The synthesized
chromonylthiazolidines displayed weak cytotoxic effects against the tested cancer cell lines, but
selective cytotoxic effects were observed, suggesting that chromonylthiazolidines could be low-cost,
selective anticancer agents.
Acknowledgments
This research is funded by Vietnam National Foundation for Science and Technology Development
(NAFOSTED) under grant number 104.01.2010.55.
Author Contributions
Anh H.L.T., Cuc N.T., Tai B.H., Yen P.H. and Nhiem N.X. performed synthesis and structure elucidation.
Thao D.T. contributed to evaluation of bioactivity. Anh H.L.T., Nam N.H., Minh C.V., Kiem P.V. and
Kim Y.H. prepared the manuscript and supervised whole research project.
Conflicts of Interest
The authors declare that there is no conflict of interest.

Molecules 2015, 20
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Sample Availability: Samples of the compounds 3a–i are available from the authors.
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