Toxicity assessments of Chalcone and some Synthetic Chalcone Analogues in a Zebrafish model

Article abstract of DOI:10.3390/molecules19010641

The aim of this study was to investigate the in vivo toxicities of some novel synthetic chalcones. Chalcone and four chalcone analogues 1a-d were evaluated using zebrafish embryos following antibody staining to visualize their morphological changes and muscle fiber alignment. Results showed that embryos treated with 3'-hydroxychalcone (compound 1b) displayed a high percentage of muscle defects (96.6%), especially myofibril misalignment. Ultrastructural analysis revealed that compound 1b-treated embryos displayed many muscle defect phenotypes, including breakage and collapse of myofibrils, reduced cell numbers, and disorganized thick (myosin) and thin (actin) filaments. Taken together, our results provide in vivo evidence of the myotoxic effects of the synthesized chalcone analogues on developing zebrafish embryos.

Full text of DOI:10.3390/molecules19010641

Molecules 2014, 19, 641-650; doi:10.3390/molecules19010641
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Toxicity Assessments of Chalcone and Some Synthetic Chalcone
Analogues in a Zebrafish Model
Ya-Ting Lee 1, Tsorng-Harn Fong 2, Hui-Min Chen 2, Chao-Yuan Chang 1, Yun-Hsin Wang 1,
Ching-Yuh Chern 3,* and Yau-Hung Chen 1,*
1
Department of Chemistry, Tamkang University, 151, Yingzhuan Road, Danshui Dist.,
New Taipei City 25137, Taiwan; E-Mails: joanna20520@gmail.com (Y.-T.L.);
frank00634@hotmail.com (C.-Y.C.); 129180@mail.tku.edu.tw (Y.-H.W.)
2
Department of Anatomy, School of Medicine, College of Medicine, Taipei Medical University,
Taipei 11031, Taiwan; E-Mails: thfong@tmu.edu.tw (T.-H.F.);
chm7805@tmu.edu.tw (H.-M.C.)
3
Department of Applied Chemistry, National Chia-Yi University, Chia-Yi 60004, Taiwan
* Authors to whom correspondence should be addressed;
E-Mails: cychern@mail.ncyu.edu.tw (C.-Y.C.); yauhung@mail.tku.edu.tw (Y.-H.C.);
Tel.: +886-2-2621-5656 (ext. 3009) (Y.-H.C.); Fax: +886-2-2620-9924 (Y.-H.C.).
Received: 31 October 2013; in revised form: 4 December 2013 / Accepted: 17 December 2013 /
Published: 7 January 2014
Abstract: The aim of this study was to investigate the in vivo toxicities of some novel
synthetic chalcones. Chalcone and four chalcone analogues 1a–d were evaluated using
zebrafish embryos following antibody staining to visualize their morphological changes
and muscle fiber alignment. Results showed that embryos treated with 3'-hydroxychalcone
(compound 1b) displayed a high percentage of muscle defects (96.6%), especially myofibril
misalignment. Ultrastructural analysis revealed that compound 1b-treated embryos
displayed many muscle defect phenotypes, including breakage and collapse of myofibrils,
reduced cell numbers, and disorganized thick (myosin) and thin (actin) filaments. Taken
together, our results provide in vivo evidence of the myotoxic effects of the synthesized
chalcone analogues on developing zebrafish embryos.
Keywords: chalcone; embryogenesis; muscle; toxicity; zebrafish

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1. Introduction
Chalcone (1,3-diphenyl-2-propen-1-one), is an important compound for the biosynthesis of
flavonoids. Chalcones have a general structure consisting of two phenyl groups, both with hydroxyl
group, connected by a C3 bridge. Natural occurring chalcones, as well as synthetic chalcone analogues,
have been demonstrated to possess many pharmaceutical effects including anti-inflammatory,
anti-oxidant, anti-nociceptive, anti-parasites, and anti-proliferative activities [1–7]. Moreover,
some chalcones can be conjugated or hybridized with other compounds and thus become potential
anti-cancer therapeutic agents [8–10]. These observations highlight the importance and multiple
applications of chalcones. However, current knowledge regarding the toxic effects of chalcones in
vertebrates during embryogenesis is still limited.
To date, many kinds of chalcones have been reported, including synthetic and natural ones. For
their toxicity assessment, most previous studies used cancer cell lines [11–13], some used rat liver
epithelial cells [14], and only few reports used rat (or dog) as a model to test the embryotoxicity and
teratogenicity of some specific chalcones [15]. To exand our knowledge on chalcone toxicity
(especially embryotoxicity), development of an alternative model is essential. The optical transparency
and clear developmental stages of zebrafish (Danio rerio) embryos allows noninvasive and
dynamic evaluation of embryotoxicity in vivo. In this study, we used embryonic zebrafish as a model
to assess the toxic effects of chalcones (chalcone and some chalcone analogues) on muscle development
in vivo. We generated a series of time- and dose-dependent chalcone exposure experiments. Subtle
changes in the muscle fiber alignment can be easily observed by staining with specific monoclonal
antibodies. This strategy is excellent for studying chalcone-induced myotoxicity during early
embryonic development.
2. Results and Discussion
2.1. Chemistry
For this study, we synthesized four chalcones 1a–d (Figure 1A). These aldol compounds were
obtained using a procedure similar to one described previously [16–19]. Unfortunately, the reaction
yields using this method was very low for the chalcones 1a and 1d (~15%). It is possible that
intramolecular hydrogen bonding such as those observed in 1a or 1d prevents the aldol reaction.
Therefore, O-isoproxylacetophenones 3a, 3b, and 3c were used as the starting material for our
synthesis. The preparations of O-isoproxylacetophenones 3a–c were straightforward. Acetophenones
2a–c were protected with isopropyl bromide and potassium carbonate in DMF in excellent yield.
(89%, 91% and 90%, respectively). The isolated products 3a–c were then reacted with appropriate
benzaldehydes and 5 N KOH to provide intermediates 4a–c and 6 in 85%, 81%, 90% and 87% yields.
The O-isopropyl ether group was removed quantitatively with BCl3 to afford the target chalcones 1a,
1b, 1c and 1d (Figure 1B).

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Figure 1. (A) Structures of chalcone and compounds 1a–d; (B) Synthesis of compounds 1a–d.
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2.2. Titration and Survival Rates Analysis
In order to access the toxic effects of chalcone and the synthetic chalcone analogues 1a–d on
zebrafish larvae, first of all, we treated zebrafish embryos with low dosages of compounds (0.1, 0.5
and 1 ppm) via exposure methods I–V (12–24, 12–36, 12–48, 12–60, 12–72 hpf; Figure 2A) to
calculate the survival rates. As shown in Figure 2B–E, 99.2%–100% of the no treatment control
(mock, 0 ppm) embryos and 96.4%–100% of chalcone and compounds 1a–d-treated embryos were
alive after exposure via exposure protocols I–V. No significant differences in the survival rates were
observed between no treatment control and low-dosage-treated groups (0.1, 0.5 and 1 ppm). Next, we
treated zebrafish embryos with high dosages of chalcone and compounds 1a–d (3 and 5 ppm) via
exposure methods I–V to calculate the survival rates. Results showed that the survival rates decreased
as the time of exposure and the concentration (3 and 5 ppm) of chalcone and compounds 1a–d
increased. At the end of the examination (72 h postfertilization, hpf), almost no embryos survived
treatment with 5 ppm of chalcone and compounds 1a–d. On the other hand, we noticed that 3 ppm of
compound 1b-exposed embryos (via method II: 12–36 hpf) displayed high survival rates (99.3%) and
high percentage of malformed phenotypes (96.6%). Therefore, we used compound 1b-exposed
embryos via exposure method II (12–36 hpf) as material for subsequent analysis.

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Figure 2. (A) Exposure protocols used in this study; (B–E) Survival analysis of zebrafish
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embryos after chalcone, compounds 1a–d treatment.
2.3. Phenotypic Changes after Chalcone Treatment
To explore the toxicities of the chalcones (chalcone and the synthetic chalcone analogues 1a–d)
during zebrafish embryogenesis, we exposed zebrafish embryos to different chalcones (3 ppm) via
exposure methods II (12–36 hpf), and recorded their malformation phenotypes. Results showed that

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embryos derived from the compounds 1a–d (3 ppm)-treated groups displayed shorter body length,
curved trunks and malformed somite boundary than those with either no treatment control or the
chalcone-treated group (Figure 3A vs. 3B–F). For further investigation, the monoclonal antibody F59
was employed to visualize the alignments of muscle fibers in no treatment control and chalcones
(including chalcone and compounds 1a–d)-treated zebrafish embryos. In both no treatment (mock)
control and chalcone-treated embryos, muscle fibers aligned well in the chevron-shaped somatic
hemi-segment (Figure 3A’,B’). In compounds 1a–d-treated embryos, muscle fibers were both split
and short by 36 hpf, and lost their integrity and aligned disorderly (Figure 3C’–F’). These
observations suggested that synthetic chalcone analogues (compounds 1a–d) are myotoxic and can
impair myofibril alignment.
Figure 3. Chalcones exposure affects myofibril alignment. (A–F) Visible and defective
phenotypes of zebrafish embryos after chalcones treatment. Embryos were exposed to
water (no treatment control; A) or water containing 3 ppm of chalcones (including
chalcone and compounds 1a–d; (A’–F’) F59 monoclonal antibody staining of zebrafish
embryo derived from each groups. All the photos were taken from the lateral view and
were of developmental stages at 36 hpf. The exposure duration ranges from 12 to 36 hpf.
2.4. Chalcones Affect Myofibril Ultrastructures and Alignment
We have shown that chalcone-treated embryos have a shorter body length and curved trunk. Is it
possible such malformations are due to the disorganization of myofibrils? To address this question, we
carried out hematoxylin and eosin Y (H&E) staining and electron microscopy (EM) experiments. After
H&E staining, boundaries of the segments of myofibrils in the embryos derived from the no treatment
control group are compact with normal morphology (Figure 4A). In contrast, myofibrils’ organization
are fractured in the compound 1b-treated embryos (Figure 4B). Ultrastructural analysis indicated that
in compound 1b-treated embryos many myofibrils are broken and collapsed. The numbers of
myofibrils is greatly decreased. The remainder are thinner and with fragmentary cytosolic
components. Although the Z-line is still visible, the thick (myosin) and thin (actin) filaments are
disorganized (Figure 5A vs. 5B). Notably, cytosolic electron density in the compound 1b-treated
embryos is more greatly decreased than in the no treatment control group (Figure 5B vs. 5A).
However, mitochondria electron density in the compound 1b-treated embryos is increased, indicating
that some substances are being produced or accumulated in the mitochondria. Taken together, we
concluded that compound 1b affects myofibril alignment and organization.

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Figure 4. Compound 1b affects myofibril alignment. Representative images of
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hemetoxylin (H) & eosin Y (E) staining of zebrafish embryo after exposure to water (A) or
water containing 3 ppm of compound 1b. (B) Dashed lines mark the boundary of somites.
Figure 5. Compound 1b affects ultrastructure of myofibril. Representative images of TEM
of zebrafish embryo after exposure to water (A) or water containing 3 ppm of compound 1b.
(B) Arrows indicate the positions of Z-line. M: mitochondria; N: nucleus.
3. Experimental
3.1. General Information
Proton (300 MHz) and carbon (75 MHz) NMR spectra were recorded on a Varian Mercury-300
NMR spectrometer (Agilent, Santa Clara, CA, USA). Cchemical shifts are reported on the δ scale as
parts per million (ppm) downfield from tetramethylsilane (TMS) used as internal reference. Mass
spectra were measured with a VG Analytical Model 70–250 s Mass Spectrometer (Varian, Palo Alto,
CA, USA). All reagents were used as obtained commercially.
3.2. Synthesis of Chalcone Analogues
A mixture of the corresponding acetophenone 2a–c (1 equiv), isopropyl bromide (1.1 equiv), and
potassium carbonate (1.1 equiv) in DMF (0.3 M) was stirred at 70 °C for 3 h. Then, the reaction

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mixture was added to water and extracted with CH2Cl2. The purified O-isopropyl product 3a–c was
obtained in excellent yield by column chromatography (silicagel, 70–230 mesh) using CH2Cl2 as
eluent. The isolated product 3a–c (1 equiv) was then reacted with the appropriate benzaldehyde
(1 equiv) and 5N KOH at room temperature until the aldehyde was consumed. After that, HCl (10%)
was added until neutrality. In the cases where the chalcones precipitated, they were filtered and
crystallized from MeOH. In the other cases, the product was purified using column chromatography with
EtOAc/hexane as eluent. The O-isopropyl ether groups of 4a–c, 6 (1 equiv) were removed quantitatively
with BCl3 (1.1 equiv) in CH2Cl2 (0.3 M) at 0 °C for 2 h to afford the target chalcones 1a–d.
1
1-(2-Hydroxyphenyl)-3-phenylpropenone (1a). Mp 89–90 (lit. 89 °C) [20]; H-NMR (CDCl3): 12.80
(1H, s, -OH), 7.93 (1H, d, J = 15.6 Hz), 7.93 (1H, d, J = 8.0 Hz), 7.66 (1H, d, J = 15.6 Hz),
7.68–7.65 (1H, m), 7.55–7.42 (5H, m), 7.03 (1H, dd, J = 8.0 Hz, 1.0 Hz), 6.95 (1H, td, J = 7.2 Hz, 1.0 Hz);
13C-NMR (CDCl3): 193.8, 163.5, 145.4, 136.4, 134.5, 130.9, 129.6, 129.0, 128.6, 120.0, 119.9, 118.8,
118.6; EI-MS m/z (rel.int.%): 224 [M+,15], 175 (18), 131 (18), 119 (100), 91 (94), 77 (28), 65 (33).
1
1-(3-Hydroxyphenyl)-3-phenylpropenone (1b). Mp 120–121 °C (lit. 120.2–120.6 °C) [17]; H-NMR
(CDCl3): 7.83 (1H, d, J = 15.6 Hz), 7.66–7.58 (3H, m), 7.51 (1H, d, J = 15.6 Hz), 7.45–7.34 (5H, m),
13
7.14 (1H, ddd, J = 8.0 Hz, 2.4 Hz, 1.2 Hz), 4.8–4.2 (1H, br s, -OH); C-NMR (CDCl3):190.9, 156.5,
145.5, 139.4, 134.7, 130.7, 129.9, 129.0, 128.6, 121.9, 120.9, 120.5, 115.2; EI-MS m/z (rel.int.%): 224
[M+,100], 223 (79), 195 (14), 121 (40), 103 (20), 93 (19), 65 (19).
1
1-(4-Hydroxyphenyl)-3-phenylpropenone (1c). Mp 179–180 °C (lit. 175–179 °C) [19]; H-NMR
(CDCl3): 8.00 (2H, d, J = 8.8 Hz), 7.80 (1H, d, J = 15.6 Hz), 7.63 (2H, dd, J = 2.6 Hz, 2.0 Hz), 7.53 (1H, d,
13
J = 15.6 Hz), 7.42–7.40 (3H, m), 6.93 (2H, d, J = 8.8 Hz), 2.10–1.90 (1H, br s, -OH); C-NMR
(CDCl3):188.4, 162.9, 143.8, 136.4, 132.0, 131.2, 131.0, 129.8, 129.4, 123.0, 116.2; EI-MS m/z
(rel.int.%): 224 [M+,100], 223 (79), 103 (15), 65 (20).
1-(2-Hydroxyphenyl)-3-(3-hydroxyphenyl)propenone (1d). Mp 180–181 °C (lit. 180–181 °C) [21];
1H-NMR (acetone-d6): 12.87 (1H, br s, -OH), 8.60 (1H, br s, -OH), 8.26 (1H, dd, J = 8.4 Hz, 1.6 Hz),
7.98 (1H, d, J = 15.6 Hz), 7.85 (1H, d, J = 15.6 Hz), 7.56 (1H, ddd, J = 8.6 Hz, 8.6 Hz, 1.6 Hz),
7.38–7.25 (3H, m), 7.03–6.94 (3H, m); 13C-NMR (acetone-d6):195.0, 164.5, 158.7, 146.3, 137.4,
137.1, 131.3, 130.9, 121.4, 121.3, 120.8, 119.8, 119.0, 118.9, 116.2; EI-MS m/z (rel.int.%):
240 [M+,100], 239 (79), 191 (15), 147 (18), 107 (81), 65 (33).
3.3. Fish Care and Chemicals Treatment
Mature zebrafish (AB strain) was supplied by the Zebrafish Core at Academia Sinica
(ZCAS, Taipei, Taiwan). Embryos were produced using standard procedures [22–24] and were staged
according to standard criteria: hours postfertilization, hpf; or days postfertilization (dpf) [25].
3.4. Histology, Antibody Labeling and Images
The procedures for H&E staining, antibody labeling and cryosection have been described
previously [26–29], except that F59 (Hybridoma Bank, Iowa City, IA, USA) was used as primary

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antibody. The procedures of embedding and cryosectioning described by Chen and Tsai [30] were
followed, except that embryos developed at 36 hpf were used and sections of 10 μm were obtained.
All embryos were observed under a microscope (DM 2500, Leica, Wetzlar, Germany) equipped with
Nomarski differential interference contrast optics and a fluorescent module having GFP and
DsRed filter cubes (Leica). Images of embryos were captured at specific stages with a digital carema
(Sony, Tokyo, Japan), or were examined by a Leica SP2 confocal microscope.
3.5. Electron Microscopy
The zebrafish embryos after exposure to water or water containing compound 1b were fixed with
2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) overnight at 4 °C.
Subsequently, the embryos were washed in buffer twice for 15 min each and then postfixed using
1% osmium tetroxide in 0.1 M cacodylate buffer for 1 h at room temperature. Samples were
dehydrated in an ethanol series and embedded in resin using standard procedures. Ultrathin sections
were cut and double-stained with uranyl acetate and lead citrate, and then examined in a Hitachi H-600
electron microscope (Hitachi, Tokyo, Japan).
4. Conclusions
This study provide a myotoxic assessment of chalcone and some synthetic chalcone analogues in a
zebrafish model. This toxicity information should prove useful for further structure-activity
relationship analysis.
Acknowledgments
This project was supported by the National Science Council, Republic of China, under grant
numbers NSC 97-2313-B-032-001-MY3 and NSC 101-2313-B-032-001-MY3. We are also grateful to
Taiwan Zebrafish Core Facility at Academia Sinica (TZCAS: NSC 102-2321-B-001-038) for
providing AB strain zebrafish.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Mohamad, A.S.; Akhtar, M.N.; Zakaria, Z.A.; Perimal, E.K.; Khalid, S.; Mohd, P.A.; Khalid, M.H.;
Israf, D.A.; Lajis, N.H.; Sulaiman, M.R. Antinociceptive activity of a synthetic chalcone, flavokawin
B on chemical and thermal models of nociception in mice. Eur. J. Pharmacol. 2010, 647, 103–109.
2. Wu, J.; Lee, J.; Cai, Y.; Pan, Y.; Ye, F.; Zhang, Y.; Zhao, Y.; Yang, S.; Li, X.; Liang, G.
Evaluation and discovery of novel synthetic chalcone derivatives as anti-inflammatory agents.
J. Med. Chem. 2011, 54, 8110–8123.
3. Ajaiyeoba, E.O.; Ogbole, O.O.; Abiodun, O.O.; Ashidi, J.S.; Houghton, P.J.; Wright, C.W.
Cajachalcone: An antimalarial compound from Cajanus cajan leaf extract. J. Parasitol. Res. 2013,
2013, 703781.

Molecules 2014, 19
649
4. Ajiboye, T.O.; Yakubu, M.T.; Oladiji, A.T. Electrophilic and reactive oxygen species
detoxification potentials of chalcone dimers is mediated by redox transcription factor Nrf-2.
J. Biochem. Mol. Toxicol. 2013, doi:10.1002/jbt.21517.
5. Chen, Y.H.; Wang, W.H.; Wang, Y.H.; Lin, Z.Y.; Wen, C.C.; Chern, C.Y. Evaluation of
anti-inflammatory effect of chalcone and chalcone analogues in a zebrafish model. Molecules
2013, 18, 2052–2060.
6. Pan, Y.; Chen, Y.; Li, Q.; Yu, X.; Wang, J.; Zheng, J. The synthesis and evaluation of novel
hydroxyl substituted chalcone analogs with in vitro anti-free radicals pharmacological activity and
in vivo anti-oxidation activity in a free radical-injury Alzheimer’s model. Molecules 2013, 18,
1693–1703.
7. Wei, H.; Zhang, X.; Wu, G.; Yang, X.; Pan, S.; Wang, Y.; Ruan, J. Chalcone derivatives from the
fern Cyclosorus parasiticus and their anti-proliferative activity. Food Chem. Toxicol. 2013, 60,
147–152.
8. Sashidhara, K.V.; Kumar, A.; Kumar, M.; Sarkar, J.; Sinha, S. Synthesis and in vitro evaluation of
novel coumarin-chalcone hybrids as potential anticancer agents. Bioorg. Med. Chem. Lett. 2010,
20, 7205–7211.
9. Fang, X.; Yang, B.; Cheng, Z.; Yang, M.; Su, N.; Zhou, L.; Zhou, J. Synthesis and antitumor
activity of novel mustard-linked chalcones. Arch. Pharm. 2013, 346, 292–299.
10. Kamal, A.; Kashi Reddy, M.; Viswanath, A. The design and development of imidazothiazole-chalcone
derivatives as potential anticancer drugs. Expert Opin. Drug Discov. 2013, 8, 289–304.
11. Neves, M.P.; Lima, R.T.; Choosang, K.; Pakkong, P.; de São José Nascimento, M.;
Vasconcelos, M.H.; Pinto, M.; Silva, A.M.; Cidade, H. Synthesis of a natural chalcone and its
prenyl analogs—Evaluation of tumor cell growth-inhibitory activities, and effects on cell cycle
and apoptosis. Chem. Biodivers. 2012, 9, 1133–1143.
12. Shin, S.Y.; Yoon, H.; Ahn, S.; Kim, D.W.; Kim, S.H.; Koh, D.; Lee, Y.H.; Lim, Y.
Chromenylchalcones showing cytotoxicity on human colon cancer cell lines and in siloco docking
with aurora kinases. Bioorg. Med. Chem. 2013, 21, 4250–4258.
13. De Vasconcelos, A.; Campos, V.F.; Nedel, F.; Seixas, F.K.; Dellagostin, O.A.; Smith, K.R.;
de Pereira, C.M.; Stefanello, F.M.; Collares, T.; Barschak, A.G. Cytotoxic and apoptotic effects of
chalcone derivatives of 2-acetylthiophene on human colon adenocarcinoma cells. Cell Biochem.
Funct. 2013, 31, 289–297.
14. Forejtníková, H.; Lunerová, K.; Kubínová, R.; Jankovská, D.; Marek, R.; Suchŷ, V.;
Vondrácek, J. Chemoprotective and toxic potentials of synthetic and natural chalcones and
dihydrochalcones in vitro. Toxicology 2005, 208, 81–93.
15. Waalkens-Berendsen, D.H.; Kuilman-Wahls, M.E.; Bär, A. Embryotoxicity and teratogenicity
study with neohesperidin dihydrochalcone in rats. Regul. Toxicol. Pharmacol. 2004, 40, 74–79.
16. Ohkatsu, Y.; Satoh, T. Antioxidant and photo-antioxidant activities of chalcone derivatives.
J. Jpn. Pet. Inst. 2008, 51, 298–308.
17. Karki, R.; Thapa, P.; Kang, M.J.; Jeong, T.C.; Nam, J.M.; Kim, H.L.; Na, Y.; Cho, W.J.;
Kwon, Y.; Lee, E.S. Synthesis, topoisomerase I and II inhibitory activity, cytotoxicity, and
structure-activity relationship study of hydroxylated 2,4-diphenyl-6-aryl pyridines. Bioorg. Med.
Chem. 2010, 18, 3066–3077.

Molecules 2014, 19
650
18. Kurniadewi, F.; Juliawaty, L.D.; Syah, Y.M.; Achmad, S.A.; Hakim, E.H.; Koyama, K.;
Kinoshita, K.; Takahashi, K. Phenolic compounds from Cryptocarya konishii: Their cytotoxic and
tyrosine kinase inhibitory properties. J. Nat. Med. 2010, 64, 121–125.
19. Qian, Y.P.; Shang, Y.J.; Teng, Q.F.; Chang, J.; Fan, G.J.; Wei, X.; Li, R.R.; Li, H.P.; Yao, X.J.;
Dai, F.; et al. Hydroxychalcones as potent antioxidants: Structure-activity relationship analysis
and mechanism considerations. Food Chem. 2011, 126, 241–248.
20. Moorthy, N.S.H.N.; Singh, R.J.; Singh, H.P.; Gupta, S.D. Synthesis, biological evaluation and in
silico metabolic and toxicity prediction of some flavanone derivatives. Chem. Pharm. Bull. 2006,
54, 1384–1390.
21. Karki, R.; Thapa, P.; Yoo, H.Y.; Kadayat, T.M.; Park, P.H.; Na, Y.; Lee, E.; Jeon, K.H.;
Cho, W.J.; Choi, H.; et al. Dihydroxylated 2,4,6-triphenyl pyridines: Synthesis, topoisomerase I
and II inhibitory activity, cytotoxicity, and structure-activity relationship study. Eur. J. Med.
Chem. 2012, 49, 219–228.
22. Westerfield, M. The Zebrafish Book, 3rd ed.; University of Oregon Press: Eugene, OR, USA,
1995.
23. Chen, Y.H.; Wang, Y.H.; Yu, T.H.; Wu, H.J.; Pai, C.W. Transgenic zebrafish line with
over-expression of Hedgehog on the skin: A useful tool to screen Hedgehog-inhibiting
compounds. Transgenic Res. 2009, 18, 855–864.
24. Wang, Y.H.; Cheng, C.C.; Lee, W.J.; Chiou, M.L.; Pai, C.W.; Wen, C.C.; Chen, W.L.;
Chen, Y.H. A novel phenotype-based approach for systematically screening antiproliferation
metallodrugs. Chem. Biol. Interact. 2009, 182, 84–91.
25. Kimmel, C.; Ballard, W.W.; Kimmel, S.R.; Ullmann, B.; Schilling, T.F. Stages of embryonic
development in the zebrafish. Dev. Dyn. 1995, 203, 253–310.
26. Chen, Y.H.; Lin, Y.T.; Lee, G.H. Novel and unexpected functions of zebrafish CCAAT box
binding transcription factor (NF-Y) B subunit during cartilages development. Bone 2009, 44,
777–784.
27. Lee, G.H.; Chang, M.Y.; Hsu, C.H.; Chen, Y.H. Essential roles of basic helix-loop-helix
transcription factors, Capsulin and Musculin, during craniofacial myogenesis of zebrafish.
Cell Mol. Life Sci. 2011, 68, 4065–4078.
28. Chen, Y.H.; Chang, C.Y.; Wang, Y.H.; Wen, C.C.; Chen, Y.C.; Hu, S.C.; Yu, D.S.; Chen, Y.H.
Embryonic exposure to diclofenac disturbs actin organization and leads to myofibril misalignment.
Birth Defects Res. B Dev. Reprod. Toxicol. 2011, 92, 139–147.
29. Ding, Y.J.; Chen, Y.H. Developmental nephrotoxicity of aristolochic acid in a zebrafish model.
Toxicol. Appl. Pharmacol. 2012, 261, 59–65.
30. Chen, Y.H.; Tsai, H.J. Treatment with myf5-morpholino results in somite patterning and brain
formation defects in zebrafish. Differentiation 2002, 70, 447–456.
Sample Availability: Samples of the all compounds in this study are available from Prof. Ching-Yuh Chern.
© 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/3.0/).