Synthesis of chalcones with antiproliferative activity on the SH-SY5Y neuroblastoma cell line: Quantitative Structure–Activity Relationship Models

Article abstract of DOI:10.1007/s00044-018-2245-2

Chalcones are a group of molecules with a broad spectrum of biological activities, being especially appealing for their antiproliferative effects on several cancer cell lines. For this reason, we synthesized 23 chalcones with good to excellent yields and assessed their effect on the viability of the SH-SY5Y neuroblastoma cell line and on primary human fibroblasts. The results indicated that 18 of these compounds were more active than 5-fluorouracil in the cancer cell line and one of them was more selective than this reference drug. To identify structural features related to the antiproliferative activity of these compounds, as well as, the selectivity on the cancer cell line, a 2D-QSAR analysis was performed. The QSAR model (q² = 0.803; r² = 0.836) showed that lipophilicity (CLogP) is the most important factor to increase their cytotoxicity on the cancer cell line. On the other hand, the selectivity QSAR model (q² = 0.917; r² = 0.916) showed that changes in the Mulliken’s charge of the carbonyl group and at the C4’ position in the chalcone core can increase the selectivity for SH-SY5Y cell line compared to normal fibroblasts.

Full text of DOI:10.1007/s00044-018-2245-2

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2245-2
ORIGINAL RESEARCH
Synthesis of chalcones with antiproliferative activity on the SH-SY5Y
neuroblastoma cell line: Quantitative Structure–Activity Relationship
Models
1
Alejandro Madrid2 Mauricio Reyna3 Caroline Weinstein-Oppenheimer3,4 Jaime Mella4,5
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Marco Mellado
Cristian O. Salas6 Elizabeth Sánchez7 Mauricio Cuellar3,4
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Received: 8 May 2018 / Accepted: 7 September 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Chalcones are a group of molecules with a broad spectrum of biological activities, being especially appealing for their
antiproliferative effects on several cancer cell lines. For this reason, we synthesized 23 chalcones with good to excellent
yields and assessed their effect on the viability of the SH-SY5Y neuroblastoma cell line and on primary human fibroblasts.
The results indicated that 18 of these compounds were more active than 5-fluorouracil in the cancer cell line and one of them
was more selective than this reference drug. To identify structural features related to the antiproliferative activity of these
compounds, as well as, the selectivity on the cancer cell line, a 2D-QSAR analysis was performed. The QSAR model (q2 =
0.803; r2 = 0.836) showed that lipophilicity (CLogP) is the most important factor to increase their cytotoxicity on the cancer
cell line. On the other hand, the selectivity QSAR model (q2 = 0.917; r2 = 0.916) showed that changes in the Mulliken’s
charge of the carbonyl group and at the C4’ position in the chalcone core can increase the selectivity for SH-SY5Y cell line
compared to normal fibroblasts.
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Keywords Chalcones Antiproliferative activity 2D-QSAR SH-SY5Y Cancer Neuroblastoma
Introduction
number is projected to be 11 million (WHO 2015).
Worldwide cancer of the lungs, liver, colon, stomach, and
breasts cause the majority of cancer deaths each year (WHO
2015). Most of cancer chemotherapies are cytotoxic with
levels of toxicity that limit their usefulness (Ramalho et al.
2013). In recent years, therapies directed at certain mole-
cular targets have been developed for cancer treatments to
reduce their toxic effects and damage to by stander normal
tissues. Nevertheless, there is still a need for novel antic-
ancer drugs (Ramalho et al. 2013).
Cancer is one of the principal causes of death in the world.
According to the World Health Organization, in 2008, 7.6
million deaths were caused by cancer, and in 2030 this
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-018-2245-2) contains supplementary
material, which is available to authorized users.
* Marco Mellado
Valparaíso, Chile
marco.mellado@pucv.cl
4
Centro de Investigación Farmacopea Chilena, Universidad de
Valparaíso, Santa Marta 183, Valparaíso, Chile
* Mauricio Cuellar
mauricio.cuellar@uv.cl
5
Instituto de Química y Bioquímica, Facultad de Ciencias,
Universidad de Valparaíso, Avenida Gran Bretaña 1111, Playa
Ancha, Casilla 5030, Valparaíso, Chile
1
Instituto de Química, Pontificia Universidad Católica de
Valparaíso, Avenida Universidad 330, Valparaíso, Chile
6
Departamento de Química Orgánica, Facultad de Química,
Pontificia Universidad Católica de Chile, Avda. Av. Vicuña
2
Departamento de Química, Facultad de Ciencias Naturales y
Exactas, Universidad de Playa Ancha, Avenida Leopoldo Carvallo
270, Playa Ancha, Valparaíso, Chile
Mackenna 4860, Macul, Santiago, Chile
7
Centro de Biotecnología, Universidad Técnica Federico Santa
3
Escuela de Química y Farmacia, Facultad de Farmacia,
María, Avenida España 1680, Valparaíso, Chile
Universidad de Valparaíso, Avenida Gran Bretaña 1093,

Medicinal Chemistry Research
Neuroblastoma is a neoplasia derived from neuron cells
from the sympathetic nervous system. It is considered one
of the most common extracranial tumors and represents
15% of all childhood cancer deaths (Dodurga et al. 2014).
Treatment for this neoplasia is diverse according to the age
and the state of development of the disease. The Interna-
tional Neuroblastoma Staging System (INSS) states that
tumors above stage II are those that require mandatory
chemotherapeutic intervention due to the poor efficiency of
surgery for tumor remission (Navarro et al. 2006). For this
reason, it is relevant to find new highly effective and
selective molecules that offer new therapeutic choices to
control this devastating disease (Ҫitişh et al. 2015).
In this sense, natural products have been a wide source of
therapeutic agents. The most studied family of natural
products is polyphenols, mainly flavonoids and related
compounds due to their multiple pharmacological applica-
tions on cancer and other diseases (Agrawal 2011; Pandey
and Rizvi 2009). Chalcones are open chain flavonoids,
which exhibit several biological properties such as anti-
inflammation, anti-mutagenesis, and cytotoxicity (Alegaon
et al. 2014; Anto et al. 1994; Avila et al. 2008; Kamal et al.
2011; Luo et al. 2012; Pilatova et al. 2010; Yadav et al.
2011). Examples of natural chalcones (1a–g) and flavonoids
(2a–c) with antiproliferative activity on cancer cell lines are
shown in Fig. 1. Flavokawain A (1a) and B (1b) have
antiproliferative and pro-apoptotic effects on bladder cancer
cells (Zi and Simoneau 2005), while xanthoangelol (1c)
shows the same effects on liver cancer cell lines (Moon
et al. 2010); xanthohumol (1d) exhibits anticancer effects in
breast and prostate cancer cell lines (Vanhoecke et al. 2005;
Vene et al. 2012); butein (1e) has shown antiproliferative
and pro-apoptotic effects on the neuroblastoma IMR-23 cell
line (Tabata et al. 2005). Other compounds such as apigenin
(2a), apigenin-7-O-β-D-glucopyranoside (2b), and brac-
flavone (2c), isoliquiritigenin (1f) and their derivative 2’-O-
methyl-isoliquiritigenin (1g) have shown antiproliferative
effects on the SH-SY5Y cancer cell line, however, with a
half inhibitory concentration (IC50) above 30 µM, similar
performance was shown by luteolin 7–O–β–D –glucopyr-
anosiduronicacid- (1→2)-β-D-glucopyranoside (2d), and
chrysoeriol 7-O-β- D-glucopyranosiduronicacid- (1→2)-β-D-
glucopyranoside (2e)with IC50 around 30 µM on the same
cancer cell line (Conrad et al. 2009; Chan et al. 2007; Hu
et al. 2014; Tabata et al. 2005; Yu et al. 2007).
The structure–activity relationship analysis has shown
that the stereo-electronic properties play an important role
on the antiproliferative activity (Lessa et al. 2010; Montana
and Batalla 2009; Singh et al. 2011), for instance the
cytotoxicity on primary skin melanoma (WM-115), human
leukemia promyelocytic cell line (HL-60), and lympho-
blastic lines (NALM-6) have been linked to the electronic
properties of the molecules (Kupcewicz et al. 2014).
Despite the above, there are no reports of quantitative
structure–activity analysis for chalcone or flavonoids
nucleus on the cytotoxicity for the neuroblastoma SH-
SY5Y cancer cell line.
The antiproliferative activity of chalcones on diverse
cancer cell lines, encouraged us to pursue the synthesis and
characterization of 23 chalcones, as well as, the assessment
of their effect on the SH-SY5Y cancer cell line (neuro-
blastoma) and a non-neoplastic human primary culture
(fibroblasts) viability. The Hanch’s analysis technique (2D-
QSAR) was utilized to distinguish the structural require-
ments responsible for biological activity and accordingly
propose new derivatives with improved activity and
selectivity.
Experimental procedure
Material and methods
Melting point was measured in a Fischer Scientific appa-
ratus. Infrared spectra were recorded in Buck Scientific
M500. The recorded range was 600 to 4000 cm−1, all
samples were registered on an ATR system (Attenuated
1
Total Reflectance). H and 13C-NMR, 2D-HSQC, and 2D-
OH
O
O
R1
O
R2
R2
R3
R6
R5
R1O
R4
OH
1a R1=OH, R2=R6= H, R3=R4=R5= OMe
1b R1= OH, R2=R5=R6= H, R3=R4= OMe
1c R1=R5= OH, R2= Geranyl, R3= OMe, R4=R6= H
1d R1=R5= OH, R2=R6=H, R3=R4= OMe
1e R1=R5=R6= OH, R2=R4= H, R3= OMe
1f R1=R5= OH, R2=R4=R6= H, R3= OMe
1g R1=OMe, R2=R4=R6= H, R3=R5= OH
2a R1=R2=R3= H
R3
2b R1=Glu, R2=R3= H
2c R1=H, R2= O-iso-butenyl, R3= OH
2d R1=Glucoside-(1-2)-Glucuronic acid, R2= H, R3= OH
2e R1= Glucoside-(1-2)-Glucuronic acid, R2= H, R3= OMe
Fig. 1 Natural chalcones and flavonoids with antiproliferative activity on several cancer cell lines, including neuroblastoma SH-SY5Y

Medicinal Chemistry Research
HMBC spectra were recorded on a Bruker Advance 400
NMR spectrometer, operating at 400.13 MHz for H and
(2E)-3-(2-methylphenyl)-1-phenylprop-2-en-1-one (5b)
1
100.6 MHz for 13C, respectively. Chemical shifts were
reported in δ (ppm downfield from the TMS resonance) and
coupling constants (J) are given in Hz. GC-MS was carried
out using Agilent Technologies 6890 model with automatic
ALS, mass detector HP MD 5973 in splitless mode (5 min).
All reagents and reactants were purchased from commercial
suppliers and were either of analytical reagent grade or
chemically pure: Acetophenone (3) Benzaldehyde (4a), 2-
methylbenzaldehyde (4b), 2-methoxybenzaldehyde (4c), 3-
methybenzaldehyde (4d), 3-methoxybenzaldehyde (4e), 4-
methylbenzaldehyde (4f), 4-methoxybenzaldehyde (4g), 4-
N,N-dimethylaminebenzaldehyde (4h), 2,5-dimethox-
ybenzaldehyde (4i), 2,4,5-trimethoxybenzaldehyde (4j),
3,4-dimethoxybenzaldehyde (4k), 3,4-dioxomethyl-benzal-
dehyde (4l), 2-hydroxy-3-methoxybenzaldehyde (4m), 4-
hydroxybenzaldehyde (4n), 2,5-dihydroxybenzaldehyde
(4o), 4-hydroxy-3-methoxybenzaldehyde (4p) were pur-
chased from Aldrich and Merck. The compounds 2-
hydroxy-3-methoxy-5-nitro-benzaldehyde (4q), 2-hydroxy-
3-nitro-benzaldehyde (4r) were obtained by nitration (see
Electronic Supplementary Material); 2-O-hexyloxy-3-
methoxybenzaldehyde (4s), 2-O-hexyloxy-benzaldehyde
(4t), bis 2,5-O-hexyloxy-benzaldehyde (4u), 3-methoxy-4-
O-hexyloxy-benzaldehyde (4v) were obtained by alkylation
as described in a previous a report (see Electronic Supple-
mentary Material); 2,5-dimethoxy-4-bromobenzaldehyde
(4w) was synthesized according to a previous report (Tapia
et al. 2014).
Orange oil (88% yield). IR (νmax): 3069, 2970, 1682, 1606,
1518, 1420 cm−1. All spectroscopic data (1H-NMR, 13C-
NMR, and EI-MS) are consistent with previous report
(Zhang et al. 2015) (see Electronic Supplementary
Material).
(2E)-3-(2-methoxyphenyl)-1-phenylprop-2-en-1-one (5c)
Orange solid (97% yield). MP: 58–60 °C. IR (νmax): 3068,
2961, 2934, 1662, 1618, 1544, 1483, 1224 cm−1. All
spectroscopic data (1H-NMR, 13C-NMR, and EI-MS) are
consistent with previous report (Suwito et al. 2014) (see
Electronic Supplementary Material).
(2E)-3-(3-methylphenyl)-1-phenylprop-2-en-1-one (5d)
Yellow solid (69% yield). MP: 63–65 °C. IR (νmax): 3086,
2988, 2926, 1663, 1572, 1500, 1449 cm−1. All spectro-
scopic data (1H-NMR, 13C-NMR, and EI-MS) are con-
sistent with previous report (Zhang et al. 2015) (see
Electronic Supplementary Material).
(2E)-3-(3-methoxyphenyl)-1-phenylprop-2-en-1-one (5e)
Yellow oil (55% yield). IR (νmax): 3112, 2918, 2859, 1666,
1584, 1539, 1502, 1453, 1222 cm−1. All spectroscopic data
(1H-NMR, 13C-NMR, and EI-MS) are consistent with pre-
vious report (Zhang et al. 2015) (see Electronic Supple-
mentary Material).
General procedure for synthesizing chalcones (5a–
w)
(2E)-3-(4-methylphenyl)-1-phenylprop-2-en-1-one (5f)
Saturated ethanolic NaOH (20 mL) was added under stirring
to a solution of acetophenone (3; 250 mg, 2.08 mmol) and
appropriate benzaldehydes (4a–w; 2.70 mmol) in ethanol
(2.5 mL). The mixture was stirred for 48 h at room tem-
perature. After, the reaction mixture was neutralized with
HCl 5% until pH ≈ 7 and extracted with ethyl acetate (3 × 50
mL). Then, the organic layer was dried, concentrated, and
purified by flash column chromatography using hexane:
EtOAc (1:0–1:1) as eluent, to obtain the corresponding
chalcones (5a–w). The compounds 6o–r were obtained
using acidic media (see Electronic Supplementary Material).
Yellow solid (43% yield). MP: 87–91 °C. IR (νmax): 3000,
2936, 2899, 1662, 1592, 1542, 1512, 1455 cm−1. All
spectroscopic data (1H-NMR, 13C-NMR, and EI-MS) are
consistent with previous report (Zhang et al. 2015) (see
Electronic Supplementary Material).
(2E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (5g)
Yellow solid (99% yield). MP: 70–72 °C. IR (νmax): 3066,
2929, 1662, 1596, 1546, 1511, 1466, 1239, 1214 cm−1. All
spectroscopic data (1H-NMR, 13C-NMR, and EI-MS) are
consistent with previous report (Zhang et al. 2015) (see
Electronic Supplementary Material).
(2E)-1,3-diphenylprop-2-en-1-one (5a)
Yellow solid (99% yield). MP: 65-69 °C. IR (νmax): 3134,
1669, 1592, 1548, 1514 cm−1. All spectroscopic data (1H-
NMR, 13C-NMR, and EI-MS) are consistent with previous
report (Zhang et al. 2015) (see Electronic Supplementary
Material).
(2E)-3-[4-(dimethylamino)phenyl]-1-phenylprop-2-en-1-one
(5h)
Orange solid (82% yield). MP: 109–111 °C. IR (νmax): 3062,
2966, 1644, 1564, 1532, 1486, 1460, 1228, 1167 cm−1. All

Medicinal Chemistry Research
spectroscopic data (1H-NMR, 13C-NMR, and EI-MS) are
consistent with previous report (Batovska et al. 2007) (see
Electronic Supplementary Material).
(2E)-(3-oxo-3-phenylprop-1-en-1yl)1,4-phenylene diacetate
(5o)
White solid (17% yield). MP: 121–125 °C. IR (νmax): 3089,
(2E)-3-(2,5-dimethoxyphenyl)-1-phenylprop-2-en-1-one (5i)
2932, 1765, 1678, 1608, 1512, 1488, 1235, 1211, 1170 cm
1
−1. H-NMR (CDCl3, 400 MHz): δ = 7.99 (2H, d, J = 8.4
Orange oil (68% yield). IR (νmax): 3042, 2958, 1653, 1601,
1502, 1441, 1233 cm−1. All spectroscopic data (1H-NMR,
13C-NMR, and EI-MS) are consistent with previous report
(Suwito et al. 2014) (see Electronic Supplementary
Material).
Hz, H-2+H-6), 7.81 (1H, d, J = 15.8 Hz, H-β), 7.57 (1H,
dd, J = 7.3, 7.3 Hz, H-4), 7.49 (1H, d, J = 7.8 Hz, H-3’),
7.47 (2H, d, J = 8.4 Hz, H-3+H-5), 7.49 (1H, d, J = 15.8
Hz, H-α), 7.15 (2H, d, J = 1.2 Hz, H-4+H-6,), 2.35 (3H, s,
CH3CO2-C-2’), 2.30 (CH3CO2-C-5’). 13C-NMR (CDCl3,
100 MHZ): δ = 189.8 (C, CO), 169.0 (C, CH3CO2-C-2’),
168.9 (C, CH3CO2-C-5’), 148.3 (C, C-5’), 147.0 (C, C-2’),
137.7 (C, C-1), 136.9 (CH, C-β), 132.9 (CH, C-4), 128.6
(4xCH, C-2 + C-3 + C-5 + C-6), 128.4 (C, C-1’), 124.5
(CH, C-3’), 124.2 (CH, C-4’), 124.0 (CH, C-6’), 120.0 (CH,
C-α), 20.9 (CH3, CH3CO2-C-5’), 20.8 (CH3, CH3CO2-C-
2’). EI-MS m/z 324 [M+](2), 240 (100). M+ calc. for
C19H16O5.
(2E)-3-(2,4,5-trimethoxyphenyl)1-phenyl-prop-2-en-1-one
(5j)
Yellow solid (74% yield). MP: 85–89 °C. IR (νmax): 3016,
2916, 2846, 1661, 1593, 1494, 1214 cm−1. All spectro-
scopic data (1H-NMR, 13C-NMR, and EI-MS) are con-
sistent with previous report (Shenvi et al. 2013) (see
Electronic Supplementary Material).
(2E)-3-(4-hydroxy-3-methoxyphenyl)-1-phenylprop-2-en-1-
one (5p)
(2E)-3-(3,4-dimethoxyphenyl)-1-phenylprop-2-en-1-one (5k)
Orange oil (79% yield). IR (cm−1): 3042, 2964, 1658, 1590,
1508, 1470, 1242 cm−1. All spectroscopic data (1H-NMR,
13C-NMR, and EI-MS) are consistent with previous
report (Tran et al. 2012) (see Electronic Supplementary
Material).
Pale orange solid (81% yield). MP: 81–83 °C. IR (νmax):
3220, 2932, 2897, 1687, 1617, 1511, 1474, 1242 cm−1. All
spectroscopic data (1H-NMR, 13C-NMR, and EI-MS) are
consistent with previous report (Batovska et al. 2007) (see
Electronic Supplementary Material).
(2E)-3-(1,3-benzodioxol-5-yl)-1-phenylprop-2-en-1-one (5l)
(2E)-3-(2-hydroxy-3-methoxy-5-nitrophenyl)-1-phenylprop-
2-en-1-one (5q)
Pale yellow solid (95% yield). MP: 48–50 °C. IR (νmax):
3085, 2958, 2920, 1659, 1607, 1578, 1468, 1225 cm−1. All
spectroscopic data (1H-NMR, 13C-NMR, and EI-MS) are
consistent with previous report (Zhang et al. 2015) (see
Electronic Supplementary Material).
Yellow solid (46% yield). MP: < 260 °C. IR (νmax): 3062,
2928, 2894, 1678, 1609, 1566, 1546, 1479, 1367, 1236,
1182 cm−1 1H-NMR (Acetone d6, 400 MHz): δ = 8.41
.
(1H, d, J = 2.5 Hz, H-4’), 8.17 (2H, d, J = 7.4 Hz, H-2+H-
6), 8.17 (1H, d, J = 15.8 Hz, H-β), 8.08 (1H, d, J = 15.8 Hz,
H-α), 7.83 (1H, d, J = 2.5 Hz, H-6’), 7.65 (1H, dd, J = 7.3,
7.4 Hz, C-4), 7.57 (2H, d, J = 7.4 Hz, H-3+H-5), 4.05 (3H,
s, CH3O-C-3’). 13C-NMR (Acetone d6, 100 MHZ): δ =
190.0 (C, CO), 152.9 (C, C-2’), 148.8 (C, C-3’), 139.0 (C,
C-1), 137.6 (C, C-5’), 133.8 (CH, C-4), 129.6 (2xCH, C-2
+ C-6), 129.4 (2xCH, C-3 + C-5), 125.0 (CH, C-β), 122.2
(CH, C-α), 117.2 (CH, C-4’), 107.9 (CH, C-6’), 57.1 (CH3,
CH3O-C-3’). EI-MS m/z 299 [M+] (100). M+ calc. for
C16H13NO5.
(2E)-3-(2-hydroxy-3-methoxyphenyl)-1-phenylprop-2-en-1-
one (5m)
Orange solid (68% yield). MP: 107–109 °C. IR (νmax):
3383, 2914, 1665, 1598, 1479, 1249, 1222 cm−1. All
spectroscopic data (1H-NMR, 13C-NMR, and EI-MS) are
consistent with previous report (Batovska et al. 2007) (see
Electronic Supplementary Material).
(2E)-3-(4-hydroxyphenyl)-1-phenylprop-2-en-1-one (5n)
(2E)-3-(2-hydroxy-3-nitrophenyl)-1-phenylprop-2-en-1-one
(5r)
Orange solid (85% yield). MP: 183–187 °C. IR (νmax):
3421, 3024, 1647, 1594, 1566, 1513, 1180 cm−1. All
spectroscopic data (1H-NMR, 13C-NMR, and EI-MS) are
consistent with previous report (Batovska et al. 2007) (see
Electronic Supplementary Material).
Yellow solid (66% yield). MP: 134–138 °C. IR (νmax):
3211, 1687, 1607, 1534, 1322, 1234 cm−1 1H-NMR
.
(Acetone d6, 400 MHz): δ = 8.33 (1H, d, J = 7.6 Hz, H-

Medicinal Chemistry Research
4’), 8.22 (1H, d, J = 7.6 Hz, H-6’), 8.15 (2H, d, J = 6.8 Hz,
H-2+H-6), 8.14 (1H, d, J = 15.9 Hz, H-β), 8.00 (1H, d, J =
15.9 Hz, H-α), 7.65 (1H, dd, J = 7.2, 7.2 Hz, H-4), 7.56
(2H, dd, J = 7.2, 7.2 Hz, H-3+H-5), 7.18 (1H, dd, J = 7.2,
7.2 Hz, H-5’). 13C-NMR (Acetone d6, 100 MHZ): δ =
189.8 (C, CO), 154.2 (C, C-2’), 138.8 (C, C-1), 136.9
(2xCH, C-β + C-4), 135.5 (C, C-3’), 133.8 (CH, C-6’),
129.6 (2xCH, C-2 + C-6), 129.3 (2xCH, C-3 + C-5), 127.6
(CH, C-4’), 127.1 (C, C-1’), 125.2 (CH, C-α), 120.8 (CH,
C-5’). EI-MS m/z 269 [M+] (100). M+ calc. for C15H11NO4.
4), 128.4 (2xCH, C-2 + C-6), 128.4 (2xCH, C-3 + C-5),
123.8 (C, C-1’), 122.9 (CH, C-α), 122.5 (CH, C-5’), 112.0
(CH, C-3’), 68.4 (CH2, OCH2CH2CH2CH2CH2CH3), 31.5
(CH2, OCH2CH2CH2CH2CH2CH3), 29.2 (CH2, OCH2
CH2CH2CH2CH2CH3), 25.9 (CH2, OCH2CH2CH2CH2
CH2CH3), 22.5 (CH2, OCH2CH2CH2CH2CH2CH3), 13.9
(CH3, OCH2CH2CH2CH2CH2CH3). EI-MS m/z 308 [M+] (3),
207 (100). M+ calc. for C21H24O2.
(2E)-3-[2,5-bis(hexyloxy)phenyl]-1-phenylprop-2-en-1-one
(5u)
(2E)-3-[2-(hexyloxy)-3-methoxyphenyl]-1-phenylprop-2-en-
1-one (5s)
Orange oil (82% yield). IR (νmax): 3102, 3078, 2929, 2861,
1
1661, 1601, 1575, 1497, 1470, 1220, 1175 cm−1. H-NMR
Yellow oil (62% yield). IR (νmax): 3076, 2937, 2903, 1663,
(CDCl3, 400 MHz): δ = 8.05 (1H, d, J = 15.9 Hz, H-β),
8,01 (2H, d, J = 7.3 Hz, H-2+H-6), 7.66 (1H, d, J = 15.9
Hz, H-α), 7.57 (1H, dd, J = 7.3,7.3 Hz, H-4), 7.49 (2H, dd,
J = 7.3, 7.3 Hz, H-3+H-5), 7.15 (1H, d, J = 3.0 Hz, H-6’),
6.92 (1H, dd, J = 9.0, 3.0 Hz, H-4’), 6.85 (1H, d, J = 9.0
Hz, H-3’), 4.00 (2H, t, J = 6.4 Hz, OCH2CH2CH2
CH2CH2CH3), 3.95 (2H, t, J = 6.4 Hz, OCH2CH2CH2
CH2CH2CH3), 1.85 (2H, q, J = 6.4 Hz, OCH2CH2CH2CH2
CH2CH3), 1.78 (2H, q, J = 6.4 Hz, OCH2CH2CH2CH2
CH2CH3), 1.49 (4H, m, 2xOCH2CH2CH2CH2CH2CH3),
1.36 (8H, m, 2xOCH2CH2CH2CH2CH2CH3), 0.87 (6H, m,
2xOCH2CH2CH2CH2CH2CH3). 13C-NMR (CDCl3, 100
MHZ): δ = 191.2 (C, CO), 152.9 (2xC, C-2’ + C-5’), 140.7
(CH, C-β), 138.5 (C, C-1), 132.5 (C, C-4), 128.5 (4xCH, C-
2 + C-3 + C-5 + C-6), 124.5 (C, C-1’), 123.2 (CH, C-α),
118.0 (CH, C-4’), 115.0 (CH, C-6’), 113.5 (CH, C-3’), 69.2
(CH2, OCH2CH2CH2CH2CH2CH3), 68.7 (CH2, OCH2
CH2CH2CH2CH2CH3), 31.6 (2xCH2, OCH2CH2CH2CH2
CH2CH3), 29.4 (OCH2CH2CH2CH2CH2CH3), 29.3 (CH2,
OCH2CH2CH2CH2CH2CH3), 25.9 (CH2, OCH2CH2CH2
CH2CH2CH3), 25.7 (CH2, OCH2CH2CH2CH2CH2CH3),
22.6 (2xCH2, OCH2CH2CH2CH2CH2CH3), 14.0 (2xCH3,
OCH2CH2CH2CH2CH2CH3). EI-MS m/z 408 [M+] (100).
M+ calc. for C27H36O3.
1
1601, 1580, 1548, 1474, 1224 cm−1. H-NMR (CDCl3, 400
MHz): δ = 8.11 (1H, d, J = 16.0 Hz, H-β), 7.95 (2H, d, J =
7.1 Hz, H-2+H-6), 7.51 (1H, d, J = 16.0 Hz, H-α), 7.46 (1H,
dd, J = 8.6, 8.6 Hz, H-4), 7.39 (2H, dd, J = 7.1, 7.1 Hz, H-3
+H-5), 7.20 (1H, d, J = 8.0 Hz, H-6’), 6.97 (1H, dd, J = 8.0,
8.0 Hz, H-5’), 6.84 (1H, d, J = 8.0 Hz, H-4’), 3.93 (2H, t, J =
6.7 Hz, OCH2CH2CH2CH2CH2CH3), 3.75 (3H, s, CH3O-3’),
1.72 (2H, q, J = 6.7 Hz, OCH2CH2CH2CH2CH2CH3), 1.40
(2H, m, OCH2CH2CH2CH2CH2CH3), 1.26 (4H, m,
OCH2CH2CH2CH2CH2CH3), 0.83 (3H, t, J = 6.7 Hz,
OCH2CH2CH2CH2CH2CH3). 13C-NMR (CDCl3, 100 MHZ):
δ = 190.1 (C, CO), 152.8 (C, C-2’), 147.8 (C, C-3’), 139.6
(CH, C-β), 137.9 (C, C-1), 132.1 (CH, C-4), 128.7 (C, C-1’),
128.1 (2xCH, C-2 + C-6), 128.0 (2xCH, C-3 + C-5), 123.5
(CH, C-6’), 122.8 (C, C-4’), 118.9 (CH, C-α), 113.8 (CH, C-
5’), 73.6 (CH2, OCH2CH2CH2CH2CH2CH3), 55.3 (CH3,
CH3O), 31.2 (CH2, OCH2CH2CH2CH2CH2CH3), 29.8 (CH2,
OCH2CH2CH2CH2CH2CH3), 25.2 (CH2, OCH2CH2CH2CH2
CH2CH3), 22.2 (CH2, OCH2CH2CH2CH2CH2CH3), 13.6
(CH3, OCH2CH2CH2CH2CH2CH3). EI-MS m/z 324 [M+-14]
(5), 240 (100). M+ calc. for C22H26O3.
(2E)-3-[2-(hexyloxy)phenyl]-1-phenylprop-2-en-1-one (5t)
Orange oil (77% yield). IR (νmax): 3115, 2949, 1655, 1587,
(2E)-3-[4-(hexyloxy)-3-methoxyphenyl]-1-phenylprop-2-en-
1-one (5v)
1
1542, 1491, 1239 cm−1. H-NMR (CDCl3, 400 MHz): δ =
8.11 (1H, d, J = 15.8 Hz, H-β), 8.03 (2H, d, J = 7.6 Hz, H-2
+H-6), 7.73 (1H, d, J = 15.8 Hz, H-α), 7.61 (1H, d, J = 7.6
Hz, H-4), 7.56 (1H, d, J = 7.1 Hz, H-6’), 7.49 (2H, dd, J =
7.6, 7.6 Hz, H-3+H-5), 7.34 (1H, dd, J = 7.6, 7.6 Hz, H-4’),
6.98 (1H, dd, J = 7.6, 7.6 Hz, H-5’), 6.92 (1H, d, J = 7.6 Hz,
H-3’), 4.05 (2H, t, J = 6.4 Hz, OCH2CH2CH2CH2CH2CH3),
1.88 (2H, q, J = 6.4 Hz, OCH2CH2CH2CH2CH2CH3), 1.52
(2H, m, OCH2CH2CH2CH2CH2CH3), 1.34 (4H, m,
OCH2CH2CH2CH2CH2CH3), 0.91 (3H, t, J = 6.4 Hz,
OCH2CH2CH2CH2CH2CH3). 13C-NMR (CDCl3, 100 MHZ):
δ = 191.0 (C, CO), 158.4 (C, C-2’), 140.8 (CH, C-β), 138.5
(C, C-1), 132.4 (CH, C-6’), 131.6 (CH, C-4’), 130.0 (CH, C-
Yellow solid (80% yield). MP: 55–59 °C. IR (νmax): 3014,
2937, 2864, 1662, 1585, 1546, 1509, 1470, 1247 cm−1. 1H-
NMR (CDCl3, 400 MHz): δ = 8.00 (2H, d, J = 8.0 Hz, H-2
+H-6), 7.76 (1H, d, J = 15.6 Hz, H-β), 7.57 (1H, ddd, J =
8.0, 8.0, 1.4 Hz, H-4), 7.49 (2H, ddd, J = 8.0, 8.0, 1.4 Hz,
H-3+H-5), 7.38 (1H, d, J = 15.6 Hz, H-α), 7.20 (1H, dd, J
= 8.3, 1.9 Hz, H-6’), 7.16 (1H, d, J = 1.9 Hz, H-2’), 6.88
(1H, d, J = 8.3 Hz, H-5’), 4.05 (2H, t, J = 6.9 Hz,
OCH2CH2CH2CH2CH2CH3), 3.92 (3H, s, CH3O-C-3’),
1.86 (2H, q, J = 6.9 Hz, OCH2CH2CH2CH2CH2CH3), 1.46
(2H, m, OCH2CH2CH2CH2CH2CH3), 1.34 (4H, m,

Medicinal Chemistry Research
OCH2CH2CH2CH2CH2CH3), 0.90 (3H, t, J = 6.9 Hz,
OCH2CH2CH2CH2CH2CH3). 13C-NMR (CDCl3, 100
MHZ): δ = 190.8 (C, CO), 151.1 (C, C-4’), 149.5 (C, C-3’),
145.1 (CH, C-β), 138.5 (C, C-1), 132.5 (C, C-4), 128.5
(2xCH, C-2 + C-6), 128.4 (2xCH, C-3 + C-5), 127.6 (C, C-
1’), 123.1 (C, C-6’), 119.8 (CH, C-α), 112.3 (CH, C-5’),
110.6 (CH, C-2’), 69.0 (CH2, OCH2CH2CH2CH2CH2CH3),
56.0 (CH3, CH3O-C-3’), 31.5 (CH2, OCH2CH2CH2
CH2CH2CH3), 28.9 (CH2, OCH2CH2CH2CH2CH2CH3),
25.5 (CH2, OCH2CH2CH2CH2CH2CH3), 22.5 (CH2,
OCH2CH2CH2CH2CH2CH3), 14.0 (CH3, OCH2CH2CH2
CH2CH2CH3). EI-MS m/z 338 [M+] (100). M+ calc. for
C22H26O3.
at a density of 15,000 cells/cm2 and incubated for 24 h.
Then, both cell types were exposed to the synthetic chal-
cones at concentrations from 200 to 1.0 μM, using 0.1%
dimethylsulfoxide (DMSO) in DMEM as the solvent. Each
condition was tested in triplicate. Control cells were incu-
bated with 1% DMSO in DMEM. In addition, cells were
cultured in the presence of 5-fluorouracil (5-FU), an anti-
neoplastic drug. After 48 h, cell viability was assessed by
adding a 4 mg/L resazurin® (Sigma-Aldrich, St. Louis,
USA) solution in cell culture media and measuring fluor-
escence after 4 h at an excitation and emission wavelength
of 544 and 590 nm, respectively.
Computational details
(2E)-3-(4-bromo-2,5-dimethoxyphenyl)-1-phenylprop-2-en-
1-one (5w)
All structures shown in Scheme 1 were optimized using the
Gaussian 03 program (Frisch et al. 2004). To obtain the
different chemical properties, a DFT-B3LYP-6-311G + +
(d) optimization was performed. In addition, every opti-
mized geometry was verified by frequency calculations (no
imaginary frequency) in their potential energy surface. The
quantum reactivity descriptors as dipolar moment (DM),
Mülliken’s charges, highest occupied molecular orbital
(HOMO), lowest unoccupied molecular orbital (LUMO),
LUMO + 1 were obtained directly from output file, while
chemical potential (µ), hardness (η), and electrophilic global
index (ω) were calculated with the following equations
(Barua et al. 2012).
Yellow solid (55% yield). MP: 137–139 °C. IR (νmax):
3052, 2986, 2940, 1654, 1577, 1490, 1394, 1212 cm−1. 1H-
NMR (CDCl3, 400 MHz): δ = 7.97 (1H, d, J = 15.8 Hz, H-
β), 7.99 (2H, d, J = 8.7 Hz, H-2+H-6), 7.58 (1H, d, J =
15.8 Hz, H-α), 7.57 (1H, dd, J = 8.7,8.7 Hz, H-4), 7.47 (2H,
dd, J = 8.7, 8.7 Hz, H-3+H-5), 7.10 (1H, s, H-3’), 7.09
(1H, s, H-6’), 3.88 (CH3O-C-5’), 3.83 (CH3O-C-2’). 13C-
NMR (CDCl3, 100 MHZ): δ = 190.7 (C, CO), 153.1 (C, C-
2’), 150.0 (C, C-5’), 139.3 (CH, C-β), 138.1 (C, C-1), 132.6
(C, C-1’), 128.4 (4xCH, C-2 + C-3 + C-5 + C-6), 123.4
(CH, C-4), 123.1 (CH, C-α), 116.7 (CH, C-3’), 114.7 (C, C-
4’), 111.9 (CH, C-6’), 56.7 (CH3, CH3O-C-2’), 56.2 (CH3,
CH3O-C-5’). EI-MS m/z 347 [M+] (100), 349 [M+ + 2]
(100). M+ calc. for C17H15O3.
ðELUMO þ EHOMO
Þ
Þ
ð1Þ
ð2Þ
μ ¼
η ¼
2
ðELUMO À EHOMO
Bioactivity
2
Cell culture
μ2
ð3Þ
ω ¼
2η
Human neuroblastoma cell line SH-SY5Y (ATTC CRL-
2266) and primary culture of human fibroblasts were
maintained in cell culture medium (CM) containing Dul-
becco’s Modified Eagle Medium (DMEM; Corning, Man-
assas, USA) supplemented with 10% fetal calf serum (FCS;
Biological Industries, Belt Haemek, Israel), 2 mM gluta-
mine (Gibco, Carlsbad, USA) and 1% antibiotic solution of
penicillin/streptomycin (Gibco, Carlsbad, USA). Cells were
grown in T-75 culture flasks in an incubator with a humi-
dified atmosphere at 37 °C and 5.0% CO2.
In addition, steric descriptors as molecular surface (MS),
molecular volume (MV), lipophilicity index (CLogP), and
molar refractivity (MR), were obtained after molecular
mechanic (MM) optimization using the ChemDraw
software.
QSAR Study
Cytotoxicity assay
The Quantitative Structure–Activity Relationship (QSAR)
Study was carried out using multiple linear regressions
according to a previous report of our research group with
small modifications (Mellado et al. 2018). We developed
several regression models between pIC50 (-log10(IC50)) or
pSI (-log10(SI)) values and the descriptors previously
The cytotoxic effect of synthetic compounds was evaluated
according to Ahmed et al. (1994), based on the reduction of
resazurin to resorufin by viable cells. Initially, fibroblasts
and SH-SY5Y cells were seeded on wells of 96-wells plates

Medicinal Chemistry Research
Scheme 1 Chemical structures of chalcone synthesized through the Claisen-Schmidt condensation
mentioned above: molecular surface (MS) and substituent
molecular surface (SMS), molecular volume (MV) and
substituent molecular volume (SMV), molar refractivity
(MR) and substituent molecular refractivity (SMR), lipo-
philicity index (CLogP), squared lipophilicity index
(CLogP2), dipole moments (DM), HOMO and LUMO
energies, LUMO+1 energy, energy difference between
LUMO and HOMO (L-H), chemical potential (µ), hardness
(η), electrophilic global index (ω), and Mulliken’s charges
at C1, C2, C3, C4, C5, C6, C1’, C2’, C3’, C4’, C5’, C6’,
Cα, Cβ, CO and CO.
different reaction conditions were studied (5o–r, see Elec-
tronic Supplementary Material). Hence, the compound 5o
was obtained in acidic medium using concentrated sulfuric
acid, as usual reaction conditions reported by several
authors, to catalyze the synthesis of chalcone derivatives,
(Gharib et al. 2013; Kumar Akanksha 2007; Petrov et al.
2008; Rahmani et al. 2014; Rocchi et al. 2014) reaching
only a yield of 17%. Furthermore, the compounds 5q and 5r
were synthesized using p-toluenesulfonic acid (TsOH) in
dichloromethane with modest results (up to a yield of 66%).
Finally, 5p was synthesized by Lewis acid catalysis (AlCl3),
which provided the proposed product with a yield of 81%
(see Scheme 1).
Cross-validation
To confirm the structural assignation of the chalcones,
different spectroscopic assays were performed (IR, NMR,
and MS). In the infrared spectra, all the compounds pre-
sented an absorption peak at the zone of υ ≈ 1665 cm−1
corresponding to the stretching of the conjugated carbonyl
group (C=O), (Silverstein et al. 2005) in addition to the
To avoid a random correlation between pIC50 or pSI values
and all descriptors, the cross-validation of the QSAR was
carried out by the Golbraikh methodology using the Eq. 4.
(Golbraikh and Tropsha 2002) A value of q2 equal or higher
than 0.5 is considered acceptable.
1
signals corresponding to other functional groups. The H-
P
2
ðyobs À byÞ
NMR spectra showed two protons coupled (δ ≈ 7.5 and 7.9
ppm) corresponding to an unsaturated system with trans
geometry (J ≈ 15 Hz), a typical feature of chalcone com-
pounds. In addition, other NMR experiments (13C, 2D-
HSQC, and 2D-HMBC) allowed to determine connectivity
between the double bond, the carbonyl group, and two
aromatic rings. Furthermore, by mass spectrometry analysis,
characteristic signals of the molecular ion for each com-
pound were observed (5a–w), followed by their respective
fragmentation pattern. Finally, it is important to highlight
that eight of the chalcones reported here are new com-
pounds (5o, 5q–w).
q2 ¼ 1 À
ð4Þ
P
2
ðyobs À yÞ
Where yobs is the experimental pIC50 or pSI; by is the pIC50 or
pSI calculated by the QSAR model, and y is the average
pIC50 or pSI of all the compounds used in QSAR model.
Results and discussion
Chemistry
The synthesis of chalcone derivatives (5a–w) was per-
formed starting from acetophenone (3) and the corre-
sponding benzaldehyde through the Claisen-Schmidt
reaction in alkaline conditions (5a–n and 5s–w). In addi-
tion, due to poor yields obtained in compounds 5o–r,
Antiproliferative activity
All synthesized compounds (5a–w) were evaluated as
antiproliferative agents against neuroblastoma SH-SY5Y

Medicinal Chemistry Research
Table 1 IC50 v of compounds
5a–w on SH-SY5Y cell line and
fibroblast primary culture, and
selectivity index values
Compounds Substituent
IC50 SH-SY5Y (µM) IC50 fibroblast (µM) Selective index (SI)a
5a
5b
5c
H
38.58 1.73
19.20 0.69
28.28 1.07
34.97 0.50
37.57 0.89
44.10 0.61
46.05 1.22
34.04 0.70
17.64 0.27
33.56 0.15
22.69 0.63
33.17 0.80
98.50 3.33
50.03 1.48
13.76 0.01
33.37 1.02
41.84 1.20
37.28 1.11
36.19 1.07
18.26 0.29
35.66 0.16
41.31 1.60
69.26 0.12
44.66 1.92
109.5 1.06
18.61 0.43
18.67 0.22
21.64 0.40
37.82 0.53
40.56 0.77
16.47 0.41
35.82 2.27
85.00 1.40
26.46 1.52
39.55 0.18
117.4 0.97
> 200
1.1
1.9
1.3
0.5
0.9
0.9
1.5
1.3
6.2
0.6
0.8
0.7
0.4
0.8
1.2
1.1
1.3
0.7
0.9
1.8
-
2’-CH3
2’OCH3
5d
5e
3’-CH3
3’-OCH3
5f
4’-CH3
5g
5h
5i
4’-OH
4’-OCH3
4’-N(CH3)2
2’-OH-3’-OCH3
2’,5’-(OCH3)2
3’,4’-(OCH3)
3’-OCH2O-4’
2’,4’,5’-(OCH3)3
2’,5’-(OAc)2
3’-OCH3-4’-OH
5j
5k
5l
5m
5n
5o
5p
5q
5r
2’-OH-3’-OCH3-5’-NO2 67.12 1.08
2’-OH-3’-NO2
37.08 0.49
44.30 0.13
64.65 3.38
> 200
5s
2’-OC6H13-3’-OCH3
2’-OC6H13
5t
5u
5v
5w
5-FU
2’,5’-(OC6H13)2
3’-OCH3-4’-OC6H13
2,5-(OCH3)2-4-Br
47.42 1.96
47.5 0.90
15.95 0.73
207 11.3
20.47 1.00
15.25 0.57
4.4
0.4
1.0
aSI = IC50values on fibroblast /IC50 values on SH-SY5Y
cancer cell line, as well as, on non-cancer primary culture of
fibroblasts using the Ahmed method. 5-FU was used as
positive control. From this assay, IC50 values on both cell
cultures were calculated and summarized in Table 1.
As shown in Table 1, a wide-range of compounds are
more active on the SH-SY5Y cancer cell line than the
activity of these chalcones could be related with another
molecular feature (see QSAR model of SH-SY5Y activity).
Altogether with the high antiproliferative activity elicited
by the most promising compounds, the selectivity is an
important feature for anticancer drug-development, due to
the undesirable side effect of chemotherapeutic agents in
normal cells (Ramalho et al. 2013). Subsequently, com-
pounds 5g, 5i, 5q, and 5t showed the lowest anti-
proliferative activity on the non-cancer cells (IC50 > 60 µM,
see Table 1). Interestingly, compound 5i is the only com-
pound with high activity on SH-SY5Y cell line. This
compound showed a 1.4-folds better selectivity index than
5-FU. To understand the influence of substituents pattern in
the cytotoxicity and selectivity see the next section (QSAR
model of selectivity index).
positive control 5-FU (17 of 23 compounds have an IC50
<
47.42 µM), while only one compound (5u) is inactive. The
four most active compounds on the SH-SY5Y cancer cell
line (5b, 5i, 5o, and 5w; IC50 < 20 µM, see Table 1) have a
C2’ and/or C4’ electron-donor substituent in resonance with
the carbonyl group of the chalcone scaffold, therefore, the
antiproliferative activity of these compounds could be
related with their electronic properties, which is consistent
with a previous report (Kupcewicz et al. 2014).
Moreover, the compounds 5m, 5n, 5q, 5t, and 5v
showed lower antiproliferative activity than 5-FU. These
compounds present oxygenated functions (OH, OMe, and
others) on B ring, similar to compounds 1e and 2c–e (see
Fig. 1) that have good antiproliferative activity on neuro-
blastoma IMR-23 and SH-SY5Y cells (Moon et al. 2010;
Tabata et al. 2005; Vanhoecke et al. 2005; Vene et al. 2012;
Zi and Simoneau 2005). Therefore, the antiproliferative
QSAR model of SH-SY5Y activity
To determine the structural features that allow modulating
the antiproliferative activity on the SH-SY5Y cancer cell
line,
a
Quantitative Structure–Activity Relationship
(QSAR) Study was performed. Several multivariable cor-
relations allowed to find that CLogP and (CLogP)2 were the

Medicinal Chemistry Research
more important features for antiproliferative activity on the
SH-SY5Y cell line (see Electronic Supplementary Mate-
rial). The Eq. 5 shows the result of the QSAR model for the
synthesized chalcones.
the charges, and molecular size (Chan et al. 2007; Stouch
and Gudmundsson 2002), while the LUMO descriptor is
related with the capacity to accept electrons (Lopez et al.
2013). Both descriptors have been related with anti-
proliferative activity on cancer and normal cell lines (Boiani
et al. 2004; Itokawa et al. 1989; Sabet et al. 2010).
pIC50 SH À SY5Y ¼ À0:806ð 0:745Þ
ð5Þ
þ 2:43ð 0:337ÞCLogP À 0:263ð 0:036ÞCLogP2
QSAR model of selectivity index
n = 14; r = 0.914; r2 = 0.836; SD = 0.086; F = 28.0; q2 =
0.803.
A Quantitative Structure–Activity Relationship Model was
developed to determine the structural features that modify
the selectivity index. A multivariable correlation analysis
between pSI (-log(SI)) and several descriptors (mentioned
in the experimental section) showed that the Mülliken
charge on C4’ and CO explain the selectivity of the syn-
thesized compounds. The Eq. 7 shows the result of the
QSAR model using selectivity index as the dependent
variable.
The Eq. 5 was developed using fourteen of twenty-three
compounds. Nine compounds were excluded as outliers.
The correlation of the partition octanol-water coefficient
calculated (ClogP) with the antiproliferative effect on SH-
SY5Y cancer cell line is concordant with reports in the
literature, which indicate that the substituents that increase
lipophilia are necessary to pass through the cell membranes
and exert their antiproliferative effects. (Kadam et al. 2007;
Kubinyi 1993) Moreover, the lipophilicity descriptor
(CLogP) allowed to choose the most appropriate sub-
stituents to improve the pharmacokinetics properties of the
molecules. (Karcher and Devillers 1990). Nonetheless, the
maximum lipophilia value is CLogP = 4.63, because higher
values produce a decrease of the antiproliferative activity.
The excluded compounds of the first QSAR model
(compounds 5d, 5f, 5g, 5m, 5o, 5p, 5r, and 5v), despite
having the same structural nucleus were outliers. The
structural modifications in these compounds could be rela-
ted to a change on their pharmacological target. These
compounds might activate other cell death mechanisms,
which is coherent with a previous report in PC-3, H460, and
HeLa cell lines (Verma and Hansch 2005). Thus, using the
same procedure of the first QSAR study, after several
multivariable correlations we found that dipolar moment
(DM), LUMO + 1, and CLogP showed correlation with the
antiproliferative activity (see Electronic Supplementary
Material). The Eq. 6 shows the results of the QSAR model
for outlier compounds and the antiproliferative activity on
the SH-SY5Y cancer cell line.
pSI ¼ 5:81ð 0:512Þþ7:47ð 0:886ÞC4′ þ 1:61ð 0:290ÞCO
ð7Þ
N = 15; r = 0.957; r2 = 0.916; SD = 0.081; F = 65.7; q2 =
0.917.
Only fifteen compounds were used for this analysis.
From the physical point of view, Mülliken’s charge is a
descriptor related to electronegativity and charge of the
linked atom (Bultinck et al. 2004). It was found that elec-
tronegative groups attached directly to C4’ and carbonyl
group (CO) increase the selectivity index. Remarkably, the
use of the Mülliken charge as descriptor in QSAR studies
about chalcones and antiproliferative effect have been pre-
viously reported (Liu and Go 2007; Ng et al. 2016), as well
as, for other pharmacophore nucleus (Matysiak 2007;
Matysiak 2008; Nikolic and Agababa 2009; Nikolic 2008).
The summary of QSAR studies with proposal mod-
ifications to change the antiproliferative activity, as well as,
the selectivity on SH-SY5Y cell lines is shown in Fig. 2.
ADME-Tox evaluation
pIC50 outlier ¼ 3:57ð 0:263Þ þ 0:251ð 0:115ÞCLogP
À 0:042ð 0:013ÞCLogP2þ0:056ð 0:011ÞDM
À 7:72ð 1:85ÞLUMOþ1
In order to evaluate the pharmacokinetics and potential
toxicity of the most active chalcone derivatives, we per-
formed a calculation of several pharmacokinetic parameters
ð6Þ
n = 8; r = 0.994; r2 = 0.989; SD = 0.038; F = 66.5; q2 =
0.983.
O
Electron
Lipophilic
substituent
max
R2
R1
Withdrawing
substituent
A comparison between Eq. 5 and 6 shows that in the
latter, the contribution of CLogP is significantly decreased
and other variables take relevance (DM and LUMO + 1),
confirming the potential allosteric effect of outlier com-
pounds (Verma and Hansch 2005). On the other hand, DM
is related to the molecular conformation, heterogeneity of
R3
increase
CLogP= 4.63
Selectivity index
R2
Fig. 2 Two-dimensional (2D) pharmacophore for the antiproliferative
activity and selectivity index of chalcone derivatives on the neuro-
blastoma SH-SY5Y cell line

Medicinal Chemistry Research
Table 2 Calculated
Propertya
Compounds
5b
pharmacokinetic parameters for
the most active chalcone
derivatives
5i
5o
5w
5t
5g
BBB
1.55
1.03
0.01
0.38
6.69
1.15
Buffer solubility (mg/L)
Caco2
17.31
54.60
Inhibitor
Inhibitor
Non
30.22
56.57
Non
18.47
28.33
Inhibitor
Inhibitor
Non
4.27
1.43
93.30
28.52
Inhibitor
Inhibitor
Non
54.03
Inhibitor
Inhibitor
Non
51.85
Inhibitor
Inhibitor
Non
CYP2C19 inhibition
CYP2C9 inhibition
CYP2D6 inhibition
CYP2D6 substrate
CYP3A4 inhibition
CYP3A4 substrate
HIA
Inhibitor
Non
Non
Non
Non
Non
Non
Non
Non
Non
Inhibitor
Non
Non
Inhibitor
Non
Non
Non
Weakly
100
Weakly
98.78
0.04
Non
100
98.78
1.14
100
95.82
40.15
Inhibitor
91.88
28.86
MDCK
117.28
Inhibitor
4.27
20.80
Inhibitor
98.96
0.15
Pgp inhibition
Inhibitor
92.63
7.29
Inhibitor
88.12
4.55
Inhibitor
95.65
0.71
Plasma protein binding (%) 93.32
Pure water solubility (mg/ 5.76
L)
Skin permeability (cm/h) -1.50
-2.00
3.91
-2.30
3.12
-2.22
4.51
-1.25
5.94
-1.93
3.32
SKlogP
4.21
hERG inhibition
medium
risk
medium
risk
medium
risk
medium
risk
medium
risk
medium
risk
aBBB in vivo blood–brain barrier penetration (C.brain/C.blood), Caco2 in-vitro Caco2 cell permeability (nm/
s), HIA human intestinal absorption (%), MDCK in-vitro MDCK cell permeability (nm/s), Pgp inhibition in-
vitro P-glycoprotein inhibition, SKlogP Calculated logP by SK atomic types, hERG inhibition in-vitro
Human ether-a-go-go related gene channel inhibition
using PreAdmet online platform (https://preadmet.bmdrc.
kr/adme/). As shown in Table 2, these selected compounds
5i, 5b, 5t, and 5g elicited a high penetration through the
blood–brain barrier. This is a very significant finding for the
in-vivo pharmacokinetic properties.
In addition, intestinal absorption (HIA, Caco2) is high,
which would allow oral administration. Moreover, these
molecules are not inhibitors of 2D6 cytochromes, nor are
they significant substrates of cytochrome 3A4. A probably
disadvantage of these compounds, related to this parameters
is their low water solubility according to high SKlogP
values. However, the solubility improves considerably in a
buffer medium especially for compound 5g, which has a
high value of solubility, due to hydroxyl group on C-5’.
Finally, they do not present a high risk of inhibition of the
hERG anti-target, so there is no increased risk of
cardiotoxicity.
cells. These results indicated that 18 compounds were more
potent than a widely used anticancer agent (5-FU), and one
molecule exhibited higher selectivity index than this drug.
In addition, two 2D-QSAR models were developed, estab-
lishing the structural features that could be used to increase
their cytotoxicity on the SH-SY5Y neuroblastoma cell line,
as well as, to improve their selectivity. So, electron-
withdrawing groups in resonance with the carbonyl group
and / or C4’ should increase selectivity, while a lipophilic
group (a saturated chain with three carbons or a mono-
unsaturated chain with four carbons), should increase the
antiproliferative effect of chalcones on the SH-SY5Y cancer
cell line. Finally, the most active compounds elicited pro-
mising ADME-Tox properties, determined by some calcu-
lated parameters: high penetration through the blood–brain
barrier, good oral absorption, and low probability of cardio-
toxic effects.
Acknowledgements We thank the Dirección de Investigación y
Postgrado (DGIP) of Universidad Técnica Federico Santa María, sci-
entific initiation project 2014 (PIIC-MM) and CONICYT Programa
Formación de Capital Humano Avanzado 21130456 and Fondecyt
grants 1141264; 11130701 and Fondecyt Postdoctorado grant
3180408. Miss Ursula Martínez and Dr. Guillermo Diaz Fleming of
Laboratorio de Espectroscopía Atómica y Molecular (CESPAM),
Universidad de Playa Ancha for technical support in infrared
spectroscopy.
Conclusions
Twenty-three chalcones were synthesized with different
substitutions in the B ring, changing their stereo-electronic
properties. All the synthetic chalcones were tested for
antiproliferative activity on the SH-SY5Y cancer cell line
and a primary culture of human fibroblasts as non-cancer

Medicinal Chemistry Research
Compliance with ethical standards
relationship for antitumor-activity of long-chain phenols from
Ginkgo-Biloba L. Chem Pharm Bull 37:1619–1621
Kadam SS, Mahandik KR, Bothara KG (2007) Principles of medicinal
chemistry. Nirali Prakashan, Pune, India
Conflict of interest The authors declare no conflict of interest.
Kamal A, Prabhakar S, Janaki Ramaiah M, Venkat Reddy P, Ratna
Reddy C, Mallareddy A, Shankaraiah N, Lakshmi Narayan
Reddy T, Pushpavalli SN, Pal-Bhadra M (2011) Synthesis and
anticancer activity of chalcone-pyrrolobenzodiazepine conjugates
linked via 1,2,3-triazole ring side-armed with alkane spacers. Eur
J Med Chem 46:3820–3831
Karcher W, Devillers J (1990) Practical applications of quantitative
structure – activity relationships (QSAR) on environmental
chemistry and toxicology. Kluwer Academic Publisher, Italy
Kubinyi H (1993) QSAR: Hansch analysis and related approaches.
Weinheim, Alemania, Editorial VCH
Kumar A, Akanksha (2007) Zirconium chloride catalyzed efficient
synthesis of 1,3-diaryl-2-propenones in solvent free conditions
via aldol condensation*. J Mol Catal A-Chem 274:212–216
Kupcewicz B, Jarzecki AA, Malecka M, Krajewska U, Rozalski M
(2014) Cytotoxic activity of substituted chalcones in terms of
molecular electronic properties. Bioorg Med Chem Lett 24:4260–
4265
Lessa JA, Mendes IC, da Silva PRO, Soares MA, dos Santos RG,
Speziali NL, Romeiro NC, Barreiro EJ, Beraldo H (2010) 2-
Acetylpyridine thiosemicarbazones: Cytotoxic activity in nano-
molar doses against malignant gliomas. Eur J Med Chem
45:5671–5677
References
Agrawal A (2011) Pharmacological activities of flavonoids: a review.
Int J Pharm Sci Nanotech 4:1394–1398
Ahmed SA, Gogal Jr. RM, Walsh JE (1994) A new rapid and simple
non-radioactive assay to monitor and determine the proliferation
of lymphocytes: an alternative to [3H]thymidine incorporation
assay. J Immunol Methods 170:211–224
Alegaon SG, Algawadi KR, Vinod D, Unger B, Khatib NA (2014)
Synthesis, pharmacophore modeling, and cytotoxic activity of 2-
thioxothiazolidin-4-one derivatives. Med Chem Res 23:5160–
5173
Anto RJ, Kutaan G, Kuttan R, Sathyanarayana K, Rao MNA (1994)
Tumor-reducing and antioxidant activities of sydnone-substituted
chalcones. J Clin Biochem Nutr 17:73–80
Avila HP, Smania EDA, Delle Monache F, Junior AS (2008)
Structure-activity relationship of antibacterial chalcones. Bioor-
gan Med Chem 16:9790–9794
Barua N, Sarmah P, Hussain I, Deka RC, Buragohain AK (2012) DFT-
based QSAR models to predict the antimycobacterial activity of
chalcones. Chem Biol Drug Des 79:553–559
Batovska D, Parushev S, Slavova A, Bankova V, Tsvetkova I, Ninova
M, Najdenski H (2007) Study on the substituents’ effects of a
series of synthetic chalcones against the yeast Candida albicans.
Eur J Med Chem 42:87–92
Boiani M, Cerecetto H, Gonzalez M (2004) Cytotoxicity of furoxans:
quantitative structure-activity relationships study. Farmaco
59:405–412
Liu X, Go ML (2007) Antiproliferative activity of chalcones with
basic functionalities. Bioorg Med Chem 15:7021–7034
Lopez JM, Ensuncho AE, Robles JR (2013) Global and local reactivity
descriptors for the design of new anticancer drugs based on cis-
platinum(Ii). Quim Nova 36:1308–U1278
Luo Y, Song R, Li Y, Zhang S, Liu ZJ, Fu J, Zhu HL (2012) Design,
synthesis, and biological evaluation of chalcone oxime deriva-
tives as potential immunosuppressive agents. Bioorg Med Chem
Lett 22:3039–3043
Bultinck P, De Winter H, Langenaeker W, Tollenaere JP (2004)
Computational. Medicinal Chemistry for Drug Discovery USA.
Marcel Dekker, Marcel Dekker, pp 295–322
Matysiak J (2007) Evaluation of electronic, lipophilic and membrane
affinity effects on antiproliferative activity of 5-substituted-2-
(2,4-dihydroxyphenyl)-1,3,4-thiadiazoles against various human
cancer cells. Eur J Med Chem 42:940–947
Matysiak J (2008) QSAR of antiproliferative activitv of N-substituted
2-amino-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazoles in various
human cancer cells. Qsar Comb Sci 27:607–617
Chan K, Jensen NS, Silber PM, O’Brien PJ (2007) Structure-activity
relationships for halobenzene induced cytotoxicity in rat and
human hepatocytes. Chem Biol Interact 165:165–174
Ҫitişh V, Dodurga Y, Eroğlu C, Seҫme M, Avci CB, Şatiroğlu-Tufan
NL (2015) Temozolomide may induce cell cycle arrest by
interacting with URG4/URGCP in SH-SY5Y neuroblastoma
cells. Tumor Biol. 36:6765–6772
Mellado M, Madrid A, Martinez U, Mella J, Salas CO, Cuellar M
(2018) Hansch’s analysis application to chalcone synthesis by
Claisen-Schmidt reaction based in DFT methodology. Chem Pap
72:703–709
Montana AM, Batalla C (2009) The rational design of anticancer
platinum complexes: The importance of the structure-activity
relationship. Curr Med Chem 16:2235–2260
Moon DO, Kim MO, Choi YH, Hyun JW, Chang WY, Kim GY
(2010) Butein induces G(2)/M phase arrest and apoptosis in
human hepatoma cancer cells through ROS generation. Cancer
Lett 288:204–213
Navarro S, Amann G, Beiske K, Cullinane CJ, d’Amore ESG, Gam-
bini C, Mosseri R, De Bernardi B, Michon J, Peuchmaur M
(2006) Prognostic value of International Neuroblastoma Pathol-
ogy Classification in localized resectable peripheral neuroblastic
tumors: A histopathologic study of Localized Neuroblastoma
European Study Group 94.01 trial and protocol. J Clin Oncol
24:695–699
Conrad J, Forster-Fromme B, Constantin MA, Ondrus V, Mika S,
Mert-Balci F, Klaiber I, Pfannstiel J, Moller W, Rosner H,
Forster-Fromme KBeifuss U (2009) Flavonoid glucuronides and
a chromone from the aquatic macrophyte stratiotes aloides. J Nat
Prod 72:835–840
Dodurga Y, Gundogdu G, Tekin V, Koc T, Satiroglu-Tufan NL, Bagci G,
Kucukatay V (2014) Valproic acid inhibits the proliferation of
SHSY5Y neuroblastoma cancer cells by downregulating URG4/
URGCP and CCND1 gene expression. Mol Biol Rep 41:4595–4599
Gharib A, Pesyan NN, Jahangir M, Roshani M, Scheeren JW (2013) A
catalytic crossed-aldol condensation of ketones with aromatic and
non-aromatic aldehydes by silica supported Preyssler hetero-
pOlyacids catalyst. Bulg Chem Com 45:314–325
Golbraikh A, Tropsha A (2002) Beware of q2! J Mol Graph Model
20:269–276
Frisch MJ, Trucks GW, Schlegel HB, Gonzalez C, Pople JA (2004)
Gaussian 03, Revision C.02. Gaussian, Inc, Wallingford CT
Hu QF, Niu DY, Wang SJ, Qin YH, Yang ZY, Zhao GL, Yang ZX,
Gao XM, Chen ZY (2014) New flavones from garcinia bracteata
and their biological activities. Chem Nat Com 50:985–988
Itokawa H, Totsuka N, Nakahara K, Maezuru M, Takeya K, Kondo M,
Inamatsu M, Morita H (1989) A quantitative structure activity
Ng HL, Chen SY, Chew EH, Chui WK (2016) Applying the designed
multiple ligands approach to inhibit dihydrofolate reductase and
thioredoxin reductase for antiproliferative activity. Eur J Med
Chem 115:63–74

Medicinal Chemistry Research
Nikolic K, Agababa D (2009) Design and QSAR study of analogs of
gamma-tocotrienol with enhanced antiproliferative activity
against human breast cancer cells. J Mol Graph Model 27:777–
783
derivatives as inhibitors of the ferredoxin - ferredoxin-NADP
+reductase interaction of Plasmodium falciparum: pursuing new
antimalarial agents. Molecules 19:21473–21488
Tabata K, Motani K, Takayanagi N, Nishimura R, Asami S, Kimura
Y, Ukiya M, Hasegawa D, Akihisa T, Suzuki T (2005) Xan-
thoangelol, a major chalcone constituent of Angelica keiskei,
induces apoptosis in neuroblastoma and leukemia cells. Biol
Pharm Bull 28:1404–1407
Tapia RA, Salas CO, Vazquez K, Espinosa-Bustos C, Soto-Delgado J,
Varela J, Birriel E, Cerecetto H, Gonzalez M, Paulino M (2014)
Synthesis and biological characterization of new aryloxyindole-
4,9-diones as potent trypanosomicidal agents. Bioorg Med Chem
Lett 24:3919–3922
Tran TD, Do TH, Tran NC, Ngo TD, Huynh TN, Tran CD, Thai KM
(2012) Synthesis and anti Methicillin resistant Staphylococcus
aureus activity of substituted chalcones alone and in combination
with non-beta-lactam antibiotics. Bioorg Med Chem Lett
22:4555–4560
Vanhoecke B, Derycke L, Van Marck V, Depypere H, De Keukeleire
D, Bracke M (2005) Antiinvasive effect of xanthohumol, a pre-
nylated chalcone present in hops (Humulus lupulus L.) and beer.
Int J Cancer 117:889–895
Vene R, Benelli R, Minghelli S, Astigiano S, Tosetti F, Ferrari N
(2012) Xanthohumol impairs human prostate cancer cell growth
and invasion and diminishes the incidence and progression of
advanced tumors in TRAMP mice. Mol Med 18:1292–1302
Verma RP, Hansch C (2005) An approach toward the problem of
outliers in QSAR. Bioorgan Med Chem 13:4597–4621
WHO. 2015. World Health Organization: Media Centre: Cancer
Journal. Available from: http://www.who.int/mediacentre/fa
ctsheets/fs297/en/
Yadav VR, Prasad S, Sung B, Aggarwal BB (2011) The role of
chalcones in suppression of NF-kappaB-mediated inflammation
and cancer. Int Immunopharmacol 11:295–309
Yu XL, Wang W, Yang M (2007) Antioxidant activities of compounds
isolated from Dalbergia odorifera T. Chen and their inhibition
effects on the decrease of glutathione level of rat lens induced by
UV irradiation. Food Chem 104:715–720
Nikolic KM (2008) Design and QSAR study of analogs of alpha-
tocopherol with enhanced antiproliferative activity against human
breast adenocarcinoma cells. J Mol Graph Model 26:868–873
Pandey KB, Rizvi SI (2009) Plant polyphenols as dietary antioxidants
in human health and disease. Oxid Med Cell Longev 2:270–278
Petrov O, Ivanova Y, Gerova M (2008) SOCl2/EtOH: Catalytic sys-
tem for synthesis of chalcones. Catal Comm 9:315–316
Pilatova M, Varinska L, Perjesi P, Sarissky M, Mirossay L, Solar P,
Ostro A, Mojzis J (2010) In vitro antiproliferative and anti-
angiogenic effects of synthetic chalcone analogues. Toxicol Vitr
24:1347–1355
Rahmani S, Amoozadeh A, Kolvari E (2014) Nano titania-supported
sulfonic acid: An efficient and reusable catalyst for a range of
organic reactions under solvent free conditions. Catal Comm
56:184–188
Ramalho SD, Bernades A, Demetrius G, Noda-Perez C, Vieira PC,
Dos Santos CY, da Silva JA, de Moraes MO, Mousinho KC
(2013) Synthetic chalcone derivatives as inhibitors of cathepsins
K and B, and their cytotoxic evaluation. Chem Biodivers
10:1999–2006
Rocchi D, Gonzalez JF, Menendez JC (2014) Montmorillonite clay-
promoted, solvent-free cross-aldol condensations under focused
microwave irradiation. Molecules 19:7317–7326
Sabet R, Mohammadpour M, Sadeghi A, Fassihi A (2010) QSAR
study of isatin analogues as in vitro anti-cancer agents. Eur J Med
Chem 45:1113–1118
Shenvi S, Kumar K, Hatti KS, Rijesh K, Diwakar L, Reddy GC (2013)
Synthesis, anticancer and antioxidant activities of 2,4,5-tri-
methoxy chalcones and analogues from asaronaldehyde:
structure-activity relationship. Eur J Med Chem 62:435–442
Silverstein RM, Webster FX, Kiemle DJ (2005) Spectrometric Iden-
tification of Organic Compounds, 7th edn. Wiley & Sons Inc,
USA
Singh RK, Lange TS, Kim KK, Brard L (2011) A coumarin derivative
(RKS262) inhibits cell-cycle progression, causes pro-apoptotic
signaling and cytotoxicity in ovarian cancer cells. Invest New
Drugs 29:63–72
Zhang L, Wang A, Wang W, Huang Y, Liu X, Miao S, Liu J, Zhang T
(2015) Co-N-C catalyst for C-C coupling reactions: On the cat-
alytic performance and active sites. ACS Catal 5:6563–6572
Zi X, Simoneau AR (2005) Flavokawain A, a novel chalcone from
kava extract, induces apoptosis in bladder cancer cells by invol-
vement of Bax protein-dependent and mitochondria-dependent
apoptotic pathway and suppresses tumor growth in mice. Cancer
Res 65:3479–3486
Stouch TR, Gudmundsson A (2002) Progress in understanding the
structure-activity relationships of P-glycoprotein. Adv Drug Deliv
Rev 54:315–328
Suwito H, Jumina M, Pudjiastuti P, Fanani MZ, Kimata-Ariga Y,
Katahira R, Kawakami T, Fujiwara T, Hase T, Sirat HM, Pus-
paningsih NN (2014) Design and synthesis of chalcone