Synthesis and selective cytotoxic activities on rhabdomyosarcoma and noncancerous cells of some heterocyclic chalcones

Article abstract of DOI:10.3390/molecules21030329

Chemically diverse heterocyclic chalcones were prepared and evaluated for cytotoxicity, aiming to push forward potency and selectivity. They were tested against rhabdomyosarcoma (RMS) and noncancerous cell line (LLC-PK1). The influence of heteroaryl patte

Full text of DOI:10.3390/molecules21030329

molecules
Article
Synthesis and Selective Cytotoxic Activities on
Rhabdomyosarcoma and Noncancerous Cells of Some
Heterocyclic Chalcones
Tuong-Ha Do 1, Dai-Minh Nguyen 2, Van-Dat Truong 2, Thi-Hong-Tuoi Do 2, Minh-Tri Le 2,
Thanh-Quan Pham 3, Khac-Minh Thai 2,* and Thanh-Dao Tran 2,
*
1
Faculty of Applied Sciences, Ton Duc Thang University, 19 Nguyen Huu Tho St., Tan Phong Ward, Dist. 7,
Ho Chi Minh City 700000, Vietnam; dotuongha@tdt.edu.vn
2
3
Faculty of Pharmacy, University of Medicine and Pharmacy, Ho Chi Minh City, 41 Dinh Tien Hoang St.,
Dist. 1, Ho Chi Minh City 700000, Vietnam; nguyendaiminh.medchem@gmail.com (D.-M.N.);
dattv.vn@gmail.com (V.-D.T.); hongtuoid99@gmail.com (T.-H.-T.D.); leminhtri1099@gmail.com (M.-T.L.)
Faculty of Chemical Engineering, Ho Chi Minh City University of Technology, Vietnam National University,
Ho Chi Minh City, 268 Ly Thuong Kiet St, Dist. 10, Ho Chi Minh City 700000, Vietnam;
ptquan@hcmut.edu.vn
*
Correspondence: thaikhacminh@uphcm.edu.vn or thaikhacminh@gmail.com (K.-M.T.);
tranthanhdao@uphcm.edu.vn or thanhdaot@yahoo.com (T.-D.T.); Tel.: +84-909-680-385 (K.-M.T.);
+84-903-716-482 (T.-D.T.); Fax: +84-8-3822-5435 (K.-M.T. & T.-D.T.)
Academic Editor: Julio A. Seijas Vázquez
Received: 20 January 2016; Accepted: 3 March 2016; Published: 9 March 2016
Abstract: Chemically diverse heterocyclic chalcones were prepared and evaluated for cytotoxicity,
aiming to push forward potency and selectivity. They were tested against rhabdomyosarcoma (RMS)
and noncancerous cell line (LLC-PK1). The influence of heteroaryl patterns on rings A and B was
studied. Heterocycle functionalities on both rings, such as phenothiazine, thiophene, furan and
pyridine were evaluated. Notably, the introduction of three methoxy groups at positions 3, 4, 5 on
ring B appears to be critical for cytotoxicity. The best compound, with potent and selective cytotoxicity
(IC50 = 12.51 µM in comparison with the value 10.84 µM of paclitaxel), contains a phenothiazine
moiety on ring A and a thiophene heterocycle on ring B. Most of the potential compounds only show
weak cytoxicity on the noncancerous cell line LLC-PK1.
Keywords: heterocyclic chalcones; cytotoxicity; rhabdomyosarcoma; LLC-PK1 cell; MTT
1. Introduction
Statistics indicate that cancer is the second most frequent cause of death in the U.S. only after
cardiovascular disease and the leading cause of death in the UK [1,2]. Some of the dominant drawbacks
of present anticancer drug therapy involve their lack of significant greater toxicity towards cancer
cells in comparison with normal tissue and the rise of multi-drug resistance. Tumor-selective cytotoxic
agents whose structures are sufficiently different from anticancer medication currently on the market
are therefore highly sought.
Soft tissue sarcomas constitute a heterogeneous group of neoplasms which accounts for
approximately 7% of all cancer cases in patients under the age of 20 [3–5]. According to pediatric
oncologists, these tumors are arbitrarily divided into two groups: rhabdomyosarcoma (RMS), the most
common soft tissue sarcomas in children less than 10 years of age, and non-RMS soft tissue sarcomas
(NRSTS), predominating in the older age groups [3].
Chemotherapy using well-known anticancer medicines including vinscristine, dactinomycin and
cyclophosphamide is mostly indicated for all patients with RMS [
3
,6
]. Other agents such as melphalan,
Molecules 2016, 21, 329; doi:10.3390/molecules21030329
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Molecules 2016, 21, 329
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methotrexate, doxorubicin, cisplatin, ifosfamide, toptecan and irenotican are active against RMS, but
in combination chemotherapy. To date, none of them have been shown to improve outcome [3].
Chalcones represent an important group of the flavonoid family, which includes a
large number of naturally occurring and synthetic molecules. The chemical structure of
chalcones consists of two aromatic rings joined by a three carbon,
system (1,3-diphenylprop-2-en-1-one) [ ]. They have been documented with diverse biological
activities including antibacterial [ 14], anti-inflammatory [15 17], antioxidant [18 21], anti-tumor
α,β-unsaturated carbonyl
7
8
–
–
–
effects [22–27]. Recent studies have demonstrated that chalcones are absorbed in the daily diet
and appear to be promising as potential chemopreventive and chemotherapeutic compounds [28].
Although the availability of heterocyclic chalcones from natural sources is limited, they have been
reported to possess a wide range of bioactivities, especially cytotoxic activity [29]. Compared to the
current cancer drugs, chalcones have the advantages of being inexpensive, available and less toxic. In
addition, the ease of synthesis also makes them an attractive drug scaffold. Thus, the identification
of new heterocyclic chalcones with anticancer activity against cancerous cells including RMS is of
great interest.
Very recently, the structure-activity relationship of cytotoxic chalcone compounds were established
based on the systematic review of literature from 2007 to 2014 [30]. The general results are summarized
in Figure 1. Supported by these conclusions, we herein carry out the structural modification of both aryl
rings, including the replacement of aryl rings with heteroaryl moieties and introduction of methoxy
groups on rings A and B for enhancement of anticancer properties.
Figure 1. Structure- anticancer activity relationship of chalcone compounds [30].
All synthesized chalcones were evaluated for their cytotoxicity on RMS cells by a MTT
(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) cell proliferation assay. Additionally, a
similar assay was conducted on a noncancerous cell line (renal tubule cells, LLC-PK1) to identify the
most selective anticancer agents.
2. Results and Discussion
2.1. Chemistry
The synthesis of all the target compounds was accomplished by classical Claisen-Schmidt
condensation between an acetophenone derivative and a corresponding aryl aldehyde in
methanolic/KOH at room temperature, as depicted in Scheme 1 [31
a one-step reaction produced five new substituted chalcones
moieties and fifteen known derivatives 10
–
33]. This aldol condensation via
1,
2,
3
,
7,
8
and with phenothiazine
9
4–6
,
–
20, as listed in Tables 1 and 2. To our knowledge, the
chemical data of the new compounds and some others have not been described in the literature until
now, so their spectral data is presented herein for the first time.

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Scheme 1. General key step for the synthesis of heterocyclic chalcones.
Table 1. Group of chalcone analogues with ring A as phenothazine moiety.
Ring B
Structure of Phenothiazinyl Chalcones
No.
Ref.
R1
R2
R3
R4
<N> 1
<N> 1
<N> 1
[31]
1
2
3
4
5
6
7
8
9
H
H
H
H
H
H
Cl
OCH3
H
H
Br
H
H
H
H
H
N(CH3)2
OCH2C6H5
H
Cl
H
OCH3
H
H
H
H
H
H
H
H
H
[32]
[31]
<N> 1
<N> 1
<N> 1
OCH3
2-thiophenyl
1
<N>: new compound.
Table 2. Group of chalcone analogues with ring A as thiophene/furan/pyridine/phenyl moieties.
Ring A
X
Ring B
No.
Chalcone Structure
Ref.
R1
R2
R3
R4
10
11
12
13
14
15
S
S
S
S
O
O
H
OH
H
H
H
H
H
H
OCH3
OCH3
H
Cl
H
N(CH3)2
OCH3
OCH3
N(CH3)2
H
H
H
[32]
[33]
[33]
OCH3 [33]
OCH3 [33]
H
H
[33]
16
R1: H; X: N
H
OCH3
OCH3
OCH3 [33]
17 *
18 *
19 *
20 *
R1: H; X: C-OH
R1: H; X: C-OH
R1:OCH3; X: C-OH
R1:OCH3;X: C-OH
3-pyridyl
[34]
[34]
[34]
[34]
2-pyridyl
3-pyridyl
2-pyridyl
*: known compounds but their spectra data was not available.
2.2. Evaluation of Cytotoxic Activities
The MTT assay for determination of cell viability was adapted from previously described
procedures [35]. The cytotoxicity assay results are shown in Table 3 and the cytotoxic profile of
the potent synthesized heterocyclic chalcones on noncancerous cells (LLC-PK1) is displayed in Table 4.
Overall, the studied heterocyclic chalcone compounds demonstrate low to strong cytotoxic
properties on RMS cells, in which compounds
values below 20 M. These results are in accordance with reported intitial structure-activity relationship
of anticancer chalcones, i.e., heteroaryl moieties either on ring A or ring B of chalcones provide
7, 8, 9 and 16 exhibit promising potentials with IC50
µ
cytotoxicity on cancer cells [30]. More interestingly, compounds
absence of toxicity on the noncancerous LLC-PK1 cell line.
7, 8, 9 and 16 show an almost complete

Molecules 2016, 21, 329
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Table 3. Cytotoxic profile of the potent synthesized heterocyclic chalcones on RMS cells.
Compounds
IC50 (µM)
Compounds
IC50 (µM)
1
2
3
4
5
6
7
8
9
Nd
Nd
Nd
Nd
Nd
11
12
13
14
15
16
17
18
19
20
48.77 ˘ 2.42
Nd
26.64 ˘ 1.42
29.06 ˘ 1.82
77.34 ˘ 4.69
19.57 ˘ 1.04
92.85 ˘ 6.14
91.12 ˘ 2.45
26.62 ˘ 1.72
20.77 ˘ 1.33
10.84 ˘ 0.47
Nd
18.04 ˘ 1.63
19.65 ˘ 1.63
12.51 ˘ 1.74
49.55 ˘ 3.20
10
Paclitaxel was used as the reference
Table 4. Cytotoxic properties of the potent synthesized heterocyclic chalcones on noncancerous
LLC-PK1 cells.
% Cell Viability on Noncancer LLC-PK1 cells
Compounds
100 µM
50 µM
10 µM
7
8
9
94.8 ˘ 7.6
105.2 ˘ 5.4
97.1 ˘ 5.0
104.3 ˘ 6.5
109.4 ˘ 10.9
103.2 ˘ 6.4
98.2 ˘ 7.3
100.2 ˘ 6.5
97.3 ˘ 10.2
98.8 ˘ 10.6
99.1 ˘ 3.1
16
101.9 ˘ 12.1
20 µM
10 µM
108.1 ˘ 8.2
2 µM
Paclitaxel (reference)
75.0 ˘ 3.6
103.4 ˘ 6.0
For chalcones with a phenothiazine moiety, substitution on the B-ring by halogen gave variable
results: a single Cl at position 2 was found to increase cytotoxicity greatly ( ), whereas a single
Cl at position 4 or Br atom at position 3 ( and ) brought limited effect as compared to their
phenyl counterpart ( ). Moreover, phenothiazinyl chalcones with other functional groups displayed
insignificant cytotoxicity. Notably, the presence of a thiophene ring led to the most potent chalcone in
our series ( ). From this observation, it is apparent that an electronegative center at the position 2 of
7
3
4
5
9
ring B enhance cytotoxicities greatly. Besides, the presence of two methoxy groups at positions 2 and 4
on ring B followed the same trend as that of electronegative groups.
In terms of chalcones with ring B replaced with pyridine moieties (typically chalcones 19 and 20
in comparison with 17 and 18), a methoxy group at position 41 on ring A also enhances the cytotoxic
properties. This has emphasized the role of alkoxy group at position 41 of 21-hydroxychalcones in
exhibiting the cytotoxic profile.
The activities of 3,4,5-trimethoxy-containing chalcone analogues (13, 14 and 16) are consistent
with what has been hypothesized. Additionally, the order of heterocycles’ contribution to cytotoxicity
is as followed: pyridine > thiophene > furan.
More interestingly, chalcone 14 shows its cytotoxicity ascending on proliferation inhibition 15.1%,
26.5% and 42.9% when decreasing concentrations of 100 µM, 50 µM and 25 µM, respectively. This led
us to speculate that the action mode of these heterocyclic chalcones was similar to that of colchicine
and combrestatin A-4, mostly by the similarity in their structure. The planar structure of heterocycles
pyridine, furan and thiophene may be responsible for the cytotoxic properties.
3. Experimental Section
3.1. General Information
All reagents and chemicals were obtained from commercial sources, and used without further
purification. Melting points were determined on open capillary tubes and are uncorrected (using

Molecules 2016, 21, 329
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a Gallenkamp apparatus). UV spectra were measured on a Hitachi U-2010 instrument (Hitachi
1
High-Technologies, Tokyo, Japan). H- and 13C-NMR spectra were recorded on a Bruker Avance II
instrument at 500 MHz and 125 MHz, respectively (Bruker Corporation, Billerica MA, USA). Chemical
shifts are reported in parts per million (ppm) downfield relative to tetramethylsilane as an internal
standard. Peak splitting patterns are abbreviated as m (multiplet), s (singlet), brs (broad singlet), d
(doublet), bd (broad doublet), t (triplet) and dd (doublet of doublets). MS spectra were acquired
on an Agilent LC-MS 1200 Series instrument (Agilent Technologies, Santa Clara, CA USA) with MS
detector of micrOTOF-QII Bruker. Thin layer chromatography (TLC) used Merck silicagel F-254 plates
(thickness = 0.25 mm). Column chromatopraphy used silica gel 60, 320–400 mesh.
3.2. General Procedure for the Synthesis of Chalcones 1–20
A solution of heteroaryl methyl ketone (5 mM) and aryl/heteroaryl aldehyde (5 mM) in 15 mL
of so˝lvent (DMSO for chalcones
1–9; methanol for the other compounds 10–20) was cooled to
5–10 C in an ice bath. The cooled solution was treated with a small portion of pulverized KOH
(10 mM). The reaction mixture was magnetically stirred for 60 min and then left overnight or longer
and monitored by thin layer chromatography using hexane–acetone (5:1) as developing solvent.
The resulting dark solution was diluted with ice water and carefully acidified using dilute hydrochloric
acid. The chalcones, which separated as a yellow solid, were collected by filtration after washing with
water and further purified by crystallization from appropriate solvents. The crude product was further
purified by column chromatography if necessary. Coupling constants (J) from the proton nuclear
magnetic resonance (1H-NMR) spectra clearly indicated that all synthesized chalcone analogues were
geometrically pure and were exclusively trans (E) isomers with JC2=C3 calculated in the range of
15–16 Hz.
3.3. Product Characterization
(E)-3-(4-(˝N,N-Dimethylamino)phenyl)-1-(10H-phenothiazin-2-yl)prop-2-en-1-one (
1). Yield 45%. Mp:
232–233 C. EIMS m/z: 371.1286 [M
´
H]´. UV (
λ
max
nm, MeOH): 204, 248, 292, 425. IR (KBr)
1
cm´1: 3314, 1650, 1600. H-NMR (DMSO)
δ
ppm: 8.75 (s, 1H, NH), 7.67–7.64 (d, 1H, J3´2 = 15.5 Hz
,
H3), 7.65–7.63 (d, 2H, J22´32 = J62´52 = 9Hz, H2”, H6”), 7.55 (d, J31´41 = 7.5 Hz, 1H, H31), 7.47 (d,
J
2´3 = 15.5 Hz, 1H, H2), 7.30 (s, 1H, H11), 7.05 (d, J41´31 = 7.5 Hz, 1H, H41), 7.00 (t, J81´71 = J81´91
=
7.5 Hz), 6.92 (d, J61´71 = 7.5 Hz, 1H, H61), 6.76 (t, J71´61 = 7.5 Hz, 1H, H71), 6.74 (d, J32´22 = 9 Hz,
J52´62 = 9Hz, 2H, H3”, H5”), 6.67 (d, J81´91 = 7.5 Hz, 1H, H9’), 3.00 (s, 6H, NMe2). 13C-NMR (DMSO)
δ
ppm: 187.4 (C1=O), 151.9 (C4”), 144.9 (C3), 142.0 (C13’), 141.2 (C11”), 137.7 (C2’), 130.6 (C2”, C6”),
127.9 (C81), 126.2 (C61), 126.01(C41), 122.5 (C1”), 122.0 (C71), 122.0 (C2), 121.9 (C3’), 115.8 (C11), 115.3
1
(C12 ), 114.5 (C14’), 112.9 (C9 ), 111.7 (C3”, C5”), 39.63 (NMe2).
˝
(E)-3-(4-Benzyloxyphenyl)-1-(10H-phenothiazin-2-yl)prop-2-en-1-one (2). Yield 55%. Mp: 215–216 C. EIMS
m/z: 436.2321 [M
´
H]´. UV ( max nm, MeOH): 204, 247, 303. IR (KBr) cm´1: 3366, 1653, 1571, 1257.
λ
ppm: 8.76 (s, 1H, NH), 7.81 (d, J22´32 = 8.5 Hz, J52´62 = 8.5 Hz, 2H, H2”, H6”),
1H-NMR (DMSO)
δ
7.69 (d, J3´2 = 15.5 Hz, 1H, H3), 7.64 (d, J2´3 = 15.5 Hz, 1H, H2), 7.59 (dd, J31´41 = 8 Hz, J31´11 = 1.5 Hz,
1
3´33
3´53
3´23
3´43
1H, H3 ), 7.47 (d, J2
= 7.5 Hz, J6
= 7.5 Hz, 2H, H3”’, H5”’), 7.34 (t, J4
= 7.5 Hz, 2H, H2”’, H6”’), 7.41 (dd, J3
= 7.5 Hz, J3
=
3´53
3´63
3´33
3´53
J3
= 7.5 Hz, J5
= J4
= 7.5 Hz, 1H, H4”’), 7.30 (d,
J11´31 = 1.5 Hz, 1H, H11), 7.1 (d, J32´22 = 8.5 Hz, J52´62 = 71.5 Hz, 2H, H3”, H5”), 7.07 (d, J31´41 = 8 Hz
,
1
1
1
1H, H4 ), 7.01 (m, 1H, H8 ), 6.92 (d, J61´71 = 7.5 Hz, 1H, H6 ), 6.77 (m, 1H, H7 ), 6.66 (d, J91´81 = 7.5 Hz,
1H, H91), 5.18 (s, 2H, CH2O). 13C-NMR (DMSO)
δ ppm 187.8 (C1=O), 160.4 (C4”), 143.7 (C3), 142.1
(C131), 141.1 (C111), 137.11 (C21), 136.7 (C1”’), 130.6 (C2”, C6”), 128.4 1(C3”’, C5”’), 127.9 (C811), 127.9
1
1
(C2”’, C6”’), 127.7 (C4”’), 127.5 (C1”), 126.2 (C6 ), 126.0 (C4 ), 123.2 (C7 ), 122.3 (C2), 122.0 (C1 ), 119.4
1
1
1
1
(C3 ), 115.2 (C12 , C14 ), 114.5 (C9 ), 112.9 (C3”, C5”), 69.4 (CH2O).
(E)-3-(3-Bromophenyl)-1-(10H-phenothiazin-2-yl)prop-2-en-1-one (
). Yield 43%. Mp: 230–232 ˝C. EIMS
m/z: 405.9936 [M
H]´. UV ( max nm, MeOH): 245, 307, 450. IR (KBr) cm´1: 3349, 1651, 1587, 582.
3
´
λ

Molecules 2016, 21, 329
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1H-NMR (DMSO)
δ
ppm 8.78 (s, 1H, NH), 8.17 (s, 1H, H2”), 7.87–7.85 (d, J3´2 = 15.5 Hz, 1H, H3), 7.83
(d, J62´52 = 7.5 Hz, 1H, H6”), 7.67–7.64 (d, 1H, J2´3 = 15.5 Hz, H2), 7.66 (d, J42´52 = 8Hz, 1H, H4”), 7.63
(d, J31´41 = 8 Hz, 1H, H31), 7.41 (t, 1H, H5”), 7.30 (s, 1H, H11), 7.08 (d, J41´31 = 8 Hz, 1H, H41), 7.01 (m,
1
1
1
1
1H, H8 ), 6.92 (d, J61´71 = 7.5 Hz, 1H, H6 ), 6.77 (m, 1H, H7 ), 6.66 (d, J91´81 = 8 Hz, 1H, H9 ). 13C-NMR
(DMSO)
δ
ppm: 187.8 (C1=O), 142.1 (C3), 141.9 (C131), 141.0 (C11”), 137.2 (C1”), 136.6 (C21), 133.0
1
1
1
1
(C2”), 130.9 (C4”), 130.7 (C5”), 128.1 (C8 ), 128.0 (C6”), 126.2 (C6 ), 126.1 (C4 ), 123.9 (C3”), 123.4 (C7 ),
1
1
1
1
1
122.7 (C3 ), 122.3 (C1 ), 122.1 (C2), 115.1 (C12 ), 114.6 (C14 ), 112.9 (C9 ).
(E)-3-(2-Chlorophenyl)-1-(10H-phenothiazin-2-yl)prop-2-en-1-one (
). Yield 57%. Mp: 202–203 ˝C. EIMS
m/z: 386.0331 [M + Na]+. UV ( max nm, MeOH): 204, 248, 309, 449. IR (KBr) cm´1: 3354, 1654, 1590, 754.
1H-NMR (DMSO)
7
λ
δ
ppm: 8.79 (s, 1H, NH), 8.16 (d, J32´42 = 7.5 Hz, 1H, H3”), 8.00 (d, J3´2 = 15.5 Hz
,
1H, H3), 7.84 (d, J2´3 = 15.5 Hz, 1H, H2), 7.64 (d, J31´41 = 8 Hz, 1H, H31), 7.57 (d, J62´52 = 7.5 Hz, 1H,
H6”), 7.46 (m, 2H, H4”, H5”), 7.30 (s, 1H, H11), 7.09 (d, J41´31 = 8 Hz, 1H, H41), 7.01 (m, 1H, H81), 6.92
(d, J61´71 = 7.5 Hz, 1H, H61), 6.66 (m, 1H, H71), 6.66 (d, J91´81 = 8 Hz, 1H, H91). 13C-NMR (DMSO)
δ
ppm: 187.7 (C1=O), 142.1 (C3), 141.0 (C131), 138.2 (C111), 136.5 (C21), 134.2 (C2”), 132.2 (C1”), 131.9
(C3”), 130 (C4”), 128.4 (C81), 127.91(C6”), 127.6 (C51), 126.2 (C5”), 126.1 (C41), 124.5 (C71), 124.4 (C2),
1
1
1
122.7 (C3 ), 115.1 (C12 ), 114.5 (C14 ), 112.8 (C9 ).
(E)-3-(2,4-Dimethoxyphenyl)-1-(10H-phenothiazin-2-yl)prop-2-en-1-one (8
). Yield 49%. Mp: 178–179 ˝C.
EIMS m/z: 388.1154 [M
1570, 1272, 1150. H-NMR (DMSO)
´
H]´. UV (
λ
δ
max nm, MeOH): 204, 248, 304, 365. IR (KBr) cm´1: 3310, 1643,
ppm: 8.76 (s, 1H, NH), 7.95 (d, J3´2 = 15.5 Hz, 1H, H3), 7.86
1
(d, J62´52 = 8.5 Hz, 1H, H6”), 7.61 (d, J2´3 = 15.5 Hz, 1H, H2), 7.52 (dd, J31´41 = 8 Hz, J31´11 = 1.5 Hz,
1H, H31), 7.28 (d, J11´31 = 1.5 Hz, 1H, H11), 7.06 (d, J41´31 = 8 Hz, 1H, H41), 7.01 (m, 1H, H81), 6.92
(d, J61´71 = 7 Hz, 1H, H61), 6.80 (m, 1H, H71), 6.64 (m, 3H, H91, H3”, H5”), 3.90 (s, 3H, 2”-OMe), 3.84
(s, 3H, 41-OMe). 13C-NMR (DMSO)
δ
ppm: 187.9 (C1=O), 163.1 (C4”), 160 (C2”), 142.1 (C131), 141.2
(C111), 138.6 (C3), 137.3 (C21), 130.1 (C6”), 127.9 (C81), 126.2 (C61), 126.1 (C41), 123.0 (C71), 122.1 (C2),
122.0 (C31), 118.9 (C1”), 115.9 (C11), 115.2 (C121), 114.5 (C141), 112.9 (C91), 106.3 (C5”), 98.3 (C3”), 55.8
1
1
(2 -MeO), 55.5 (4 -CH3O).
(E)-3-(Thiophen-2-yl)-1-(10H-phenothiazin-2-yl)prop-2-en-1-one (
9
). Yield 60%. Mp: 229–230 ˝C. EIMS
´´
1
m/z: 334.0042 [M
´
H] . UV (
λ
max nm, CH2Cl2): 249, 340. IR (KBr) cm´1: 3342, 1645, 1601. H-NMR
(DMSO) δ ppm: 8.78 (s, 1H, NH), 7.89 (d, J3´2 = 15 Hz, 1H, H3), 7.79 (d, J42´52 = 5 Hz, 1H, H5”), 7.67
(d, J32´42 = 3.5 Hz, 1H, H3”), 7.50 (dd, J31´41 = 8 Hz, J31´11 = 1.5 Hz, 1H, H31), 7.40 (d, J2´3 = 15 Hz,
1H, H2), 7.27 (d, J11´31 = 1.5 Hz, 1H, H11), 7.19 (dd, J42´32 = 3.5 Hz, J42´52 = 5 Hz, 1H, H4”), 7.07 (d,
J41´31 = 8 Hz, 1H, H41), 7.00 (m, 1H, H81), 6.92 (dd, J61´71 = 8 Hz, 1H, H61), 6.77 (m, 1H, H71), 6.66 (dd,
J91´71 = 1.5 Hz, J91´81 = 1.5 Hz, 1H, H91). 13C-NMR (DMSO)
δ
ppm: 187.4 (C1=O), 142.1 (C131), 141.1
(C111), 139.7 (C2”), 136.8 (C21), 136.6 (C3), 132.9 (C5”), 130.4 (C3”), 1281.7 (C4”), 128.0 (C81), 126.2 (C2),
1
1
1
1
1
1
126.2 (C6 ), 123.4 (C4 ), 122.2 (C7 ), 120.0 (C1 ), 115.2 (C12 ), 114.6 (C14 ), 112.9 (C9 ).
˝
(E)-3-(Pyridin-2-yl)-1-(2-hydroxyphenyl)prop-2-en-1-one (17). Yield 52%. Mp: 136–138 C. EIMS m/z:
1
226.0861 [M
´
H]+. IR (KBr) cm´1: 1641, 1577. H-NMR (CDCl3),
δ
ppm: 12.72 (s, 1H, OH); 8.8 (s, 1H,
H2-pyridine), 8.24–8.27 (d, 1H, J3´2 = 15 Hz, H3), 8.03-8.05 (dd, 1H, J62´42 = 1.5 Hz, J62´52 = 8 Hz, H6”),
7.83–7.86 1(d, 1H, J2´3 = 15 Hz, H2), 7.74–7.77 (t, 1H, H4”), 7.47–7.51 (dd, 1H, H31-pyridine), 7.30–7.32
(t, 1H, H4 -pyridine), 7.02–7.04 (d, 1H, H3”), 6.93–6.96 (t, 1H, H5 -pyridine). 13C-NMR (CDCl3),
δ ppm:
1
193.1 (C1=O), 163.7 (C2”), 151.41(C21, C61), 150.1 (C21), 141 (C3), 136.7 (C4”), 134.7 (C41), 130.4 (C31),
129.7 (C6”), 123.8 (C2), 122.1 (C5 ), 119.8 (C5”), 119 (C1”), 118.8 (C3”).
˝
(E)-3-(Pyridin-3-yl)-1-(2-hydroxyphenyl)prop-2-en-1-one (18). Yield 65%. Mp: 120–122 C. EIMS m/z:
1
1
H]+. H-NMR (CDCl3),
δ ppm: 12.62 (s, 1H, OH); 8.9 (s, 1H, H2 -pyridine); 8.66–8.67 (d,
226.0852 [M
´
1
1
1H, H6 -pyridine); 7.98–7.99 (d, 1H, J = 7.5 Hz, H4 -pyridine); 7.89–7.92 (d, 1H, 7.5 Hz, H6”); 7.88–7.91
(d, 1H, J3´2 = 16 Hz, H3); 7.72–7.75 (d, 1H, J2´3 = 16 Hz, H2); 7.51–7.54 (t, 1H, J = 1.5 Hz, 8.5 Hz,
H4”); 7.39–7.42 (t, 1H, J = 5 Hz, 8 Hz, H5-pyridine); 7.04-7.06 (d, 1H, J = 8.5 Hz, H3”); 6.59–6.50 (t,
1
δ ppm: 193.1 (C1=O); 163.7 (C2”); 151.4 (C4 -pyridine); 150.1
1H, J = 7.5 Hz, H5”). 13C-NMR (CDCl3),

Molecules 2016, 21, 329
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(C21-pyridine); 141.5 (C3); 136.7 (C4”); 134.7 (C61-pyridine); 130.4 (C11-pyridine); 129.6 (C6”); 123.8
1
(C2); 122.1 (C5 -pyridine); 119.8 (C5”); 119 (C1”); 118.7 (C3”)
(E)-3-(Pyridin-2-yl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-en-1-one (19). Yield 65% over two steps. Mp
˝
1
130–131 C. EIMS m/z: 256.0861 [M + H]+. H-NMR (CDCl3),
δ ppm: 13,36 (s, 1H, OH), 8.68–8.69 (m,
1H, H31), 8.17 (d, J3´2 = 15 Hz, 1H, H3), 7.94 (d, J52´62 = 8.5 Hz, 1H, H6”), 7.81 (d, J2´3 = 15 Hz, 1H,
H2), 7.75 (dt, J5´6 = J5´4 = 7.5 Hz, J5´3 = 1.5 Hz, 1H, H51), 7.46 (d, J6´5 = 7.5 Hz, 1H, H61), 7.27–7.31
(m, 1H, H4 ), 6.47–6.50 (m, 2H, H3”, H5”), 3.86 (s, 3H, OCH3). 13C-NMR (CDCl3),
δ ppm: 192 (C1=O),
1
1
1
1
1
166.7 (C4”), 166.4 (C2”), 153 (C1 ), 150.2 (C3 ), 142.2 (C3), 136.9 (C5 ), 131.8 (C6”), 125.6 (C2), 124.5 (C6 ),
1
124.4 (C4 ), 114.2 (C1”), 107.7 (C5”), 101 (C3”), 55.61 (OCH3).
(E)-3-(Pyridin-3-yl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-en-1-one (20). Yield 60% over two steps. Mp
˝
128–129 C. EIMS m/z: 256.0962 [M
´
H]+. 1H-NMR (DMSO),
δ ppm: 13.32 (s, 1H, OH), 9.03
(s, 1H, H21-pyridine), 8.61–8.62 (m, 1H, H41-pyridine), 8,36 (d, J62´52 = 8 Hz, 1H, H6”), 8.28 (d,
1
J61´51 = 8 Hz, 1H, H6 -pyridine), 8.13 (d, J3-2 = 15.5 Hz, 1H, H3), 7.83 (d, J2´3 = 15,5 Hz, 1H, H2), 7.49
1
(dd, J51´61 = 8 Hz, 1H, H5 ), 6.57 (dd, J52´62 = 8 Hz, J52´32 = 2,5 Hz, 1H, H5”), 6.52 (d, J32´52 = 2.5 Hz,
1H, H3”), 3,85 (s, 1H, OCH3). 13C-NMR (DMSO),
δ
ppm: 191.4 (C1=O), 166.1 (C4”), 165.7 (C2”), 151
1
1
1
1
1
(C4 ), 150 (C2 ), 140.5 (C3), 135.2 (C6 ), 132.7 (C6”), 130.6 (C1 ), 123.8 (C2), 123.1 (C5 ), 113.7 (C1”), 107.5
(C5”), 100.9 (C2”), 55.7 (OCH3).
3.4. MTT Assay of Cytotoxic Activity
Human muscle rhabdomyosarcoma (RMS) cell line (ATCC® CCL-136
™) was provided by World
Health Organization (WHO) for The Institute of Pasteur in Ho Chi Minh City, Vietnam according to
the Poliovirus Monitoring Project protocols.
Pig Kidney Epithelial (LLC-PK1) cell line (ATCC® CL-101
™) was a generous gift of Prof. Jeong
Hill Park, Research Institute of Pharmaceutical Science (College of Pharmacy, Seoul National University,
Seoul 151-172, Korea).
RMS or LLC-PK1 cells were seeded and cultured in EMEM medium (Gibco, Waltham, MA, USA)
supplemented with 10% fetal calf serum (FCS) (Gibco), 2 mM L-glutamine, 100 IU/mL penicillin and
100
at 37 C.
The samples were dissolved in dimethyl sulfoxide (DMSO) at 10 mM and then diluted with
culture medium to get three concentrations of 100; 50; 10 M or 25; 12.5; 2.5 M for cell proliferation
µg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) in a humidified atmosphere of 5% CO2
˝
µ
µ
assays. The final concentration of DMSO in culture medium was 1%; 0.5% or 0.1% (v/v), which did not
affect cell viability [35]. DMSO at different concentrations or was used as a blank control. Paclitaxel
(Anzatax®, Mayne Pharma, Australia) was used as the reference compound at the concentration of 1, 5
and 10 µM (directly dissolved in culture medium).
The cells were cells were seeded in 96-well plates at 4.2
at 37 C, 5% CO2, 100
control (DMSO) was added and incubated for 24 hours at 37 C, 5% CO2.
ˆ
104 cell/cm2. After 24 h of incubation
µL of chalcone samples at tested concentrations, reference compound, and blank
˝
˝
Cell viability was evaluated as mitochondrial succinate dehydrogenase (SDH) activity, a marker
of viable cells, using MTT test as described by Denizot and Lang [36]. Briefly, SDH activity was
detected after 3 hours incubation in culture medium without FCS containing 0.05 mg/mL MTT
(3-4,5-dimethyl-thiazol-2-yl-2,5 diphenyl-tetrazolium bromide, Sigma-Aldrich), which is converted
into formazan dissolved in acidified isopropanol (Merck, Darmstadt, Germany) by agitation for 10 min
at room temperature. The produced purple solution was spectrophotometrically measured at 570 nm
on Multiskan™ FC microplate Photometer (Thermo Scientific, Waltham, MA, USA).
Each assay was done in triplicate and the standard deviation of absorbance was less than 10% of
the mean. The results were expressed as Mean + SD of six cultures of one representative experiment
and statistically analyzed by Mann-Whitney test with SPSS 22.0 for Windows software. Threshold of
significance was set at p < 0.05.

Molecules 2016, 21, 329
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The percentage of proliferation inhibition was calcutated as follows:
% Proliferation inhibition “ 100 ´ rpAsample{Ablank controlq ˆ 100s
(1)
The IC50 values were calculated based on the linear regression between the concentrations of
testing compound and the inhibition percentages of cells. The chalcone compounds with significant
potential against RMS cells (IC50 < 20 µM) were then tested for their toxicity on LLC-PK1 cells.
4. Conclusions
Several lines of investigation suggest that by replacing the original phenyl ring of chalcones by
heteroaryl moieties, resulting heterocyclic chalcone compounds may be effective anticancer agents.
The study presented herein demonstrate1d that some representative heterocyclic chalcones, namely,
phenothiazinyl chalcones and pyridinyl 2 -hydroxychalcones exhibit promising anticancer properties.
Thus, these classes of chalcones, whose structures are far different from anticancer drugs currently
on the market, exhibit potential cytotoxicity. This study will provide further assistance to the design
of more potent and selective cytotoxic chalcones and will allow us to correlate the structural features
with the biological chemistry of chalcone compounds.
Acknowledgments: This work was supported by the Vietnam’s National Foundation for Science and Technology
Development—NAFOSTED (Grant #104.01.2012.78 to Thanh-Dao Tran).
Author Contributions: This study was designed, directed and coordinated by T.-D. Tran, K.-M. Thai and M.-T. Le
.
The chemical synthesis and product characterization was performed and analyzed the data by T.-H. Do, D.-M.
Nguyen, V.-D. Truong, T.-Q. Pham, K.-M. Thai and T.-D. Tran. The MTT assay of cytotoxic activity was planned
and performed by T.-H. Do, D.-M. Nguyen, K.-M. Thai, T.-D. Tran, T.-H.-T. Do. The manuscript was written by
T.-H. Do, D.-M. Nguyen, T.-H.-T. Do, K.-M. Thai and T.-D. Tran. All authors give final approval of the version to
be submitted and final version.
Conflicts of Interest: The authors declare no conflict of interest.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).