Design, synthesis, and biological activities of 1-aryl-(3-(2-styryl)phenyl)prop-2-en-1-ones

Article abstract of DOI:10.1016/j.bioorg.2018.11.005

A moderate elevation in reactive oxygen species (ROS) levels can generally be controlled in normal cells, but may lead to death of cancer cells as the ROS level in cancer cells is already elevated. Therefore, a ROS-generating compound can act as a selecti

Full text of DOI:10.1016/j.bioorg.2018.11.005

Accepted Manuscript
Design, synthesis, and biological activities of 1-aryl-(3-(2-styryl)phe-
nyl)prop-2-en-1-ones
Soon Young Shin, Junho Lee, Jihyun Park, Youngshim Lee, Seunghyun Ahn,
Ji Hye Lee, Dongsoo Koh, Young Han Lee, Yoongho Lim
PII:
S0045-2068(18)30884-8
DOI:
https://doi.org/10.1016/j.bioorg.2018.11.005
YBIOO 2613
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 August 2018
12 September 2018
5 November 2018
Please cite this article as: S. Young Shin, J. Lee, J. Park, Y. Lee, S. Ahn, J. Hye Lee, D. Koh, Y. Han Lee, Y. Lim,
Design, synthesis, and biological activities of 1-aryl-(3-(2-styryl)phenyl)prop-2-en-1-ones, Bioorganic Chemistry
(2018), doi: https://doi.org/10.1016/j.bioorg.2018.11.005
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Design, synthesis, and biological activities of 1-aryl-(3-(2-styryl)phenyl)prop-2-en-1-ones
Soon Young Shina, Junho Leeb, Jihyun Parkb, Youngshim Leeb, Seunghyun Ahnb, Ji Hye Leec,
Dongsoo Kohc, Young Han Leea,*, Yoongho Limb,*
aDepartment of Biological Sciences, Konkuk University, Seoul 05029, Korea
bDivision of Bioscience and Biotechnology, BBRC, Konkuk University, Seoul 05029, Korea
cDepartment of Applied Chemistry, Dongduk Women’s University, Seoul 02748, Korea
*Corresponding authors:
Prof. Yoongho Lim; Division of Bioscience and Biotechnology, Konkuk University
Hwayang-Dong 1, Kwangjin-Ku, Seoul, Korea 05029
Tel.: 82-2-450-3760; Fax : 82-2-3437-6106; Email: yoongho@konkuk.ac.kr
Prof. Young Han Lee; Department of Biological Sciences, Konkuk University,
Tel.: 82-2-2049-6115; Email: yhlee58@konkuk.ac.kr
Abbreviations: CoMFA, comparative molecular field analysis; CoMSIA, comparative
molecular similarity index analysis; GI50, half-maximal cell growth inhibitory concentration;
SPP, 1-aryl-(3-(2-styryl)phenyl)prop-2-en-1-one; COSY, correlated spectroscopy; HMBC,
heteronuclear multiple bonded connectivities; NOESY, nuclear Overhauser enhanced
spectroscopy
1

Abstract
A moderate elevation in reactive oxygen species (ROS) levels can generally be controlled in
normal cells, but may lead to death of cancer cells as the ROS level in cancer cells is already
elevated. Therefore, a ROS-generating compound can act as a selective chemotherapeutic agent
for cancer cells that does not affect normal cells. In our previous study, a compound containing a
Michael acceptor was selectively cytotoxic to cancer cells without affecting normal cells;
therefore, we designed and synthesized 26 compounds containing a Michael acceptor. Their
cytotoxicities against HCT116 human colon cancer cell lines were measured by using a
clonogenic long-term survival assay. To derive the structural conditions required to obtain
stronger cytotoxicity against cancer cells, the relationships between the half-maximal cell growth
inhibitory concentration values of the synthesized compounds and their physicochemical
properties were evaluated by Comparative Molecular Field Analysis and Comparative Molecular
Similarity Indices Analysis. It was confirmed that the compound with the best half-maximal cell
growth inhibitory concentration triggered apoptosis through ROS generation, which then led to
stimulation of the caspase pathway.
key words: 1-aryl-(3-(2-styryl)phenyl)prop-2-en-1-one; CoMFA; CoMSIA; ROS generation;
poly(ADP-ribose) polymerase; apoptosis
2

1. Introduction
There are more than 100,000 publications containing “reactive oxygen species” (ROS) as a key
word [1]. Triplet molecular oxygen in the ground state contains two valence electrons with
parallel spins; conversion to ROS may occur by energy transfer or electron transfer reactions
with organic molecules [2]. Molecular oxygen can be reduced to superoxide; dismutation of
superoxide can produce hydrogen peroxide, which can then be reduced to hydroxyl radical.
Superoxide, hydrogen peroxide, and hydroxyl radical are types of ROS. Triplet molecular
oxygen may be converted to singlet oxygen by radiation, which is also a ROS [3]. ROS can be
produced via radiolytic processes in exogenous conditions and via the defense mechanisms of
cells and tissues in endogenous conditions [4]. The roles of ROS are known to be related to the
defense system against microbial invasion; in addition, ROS are related to cell signal
transduction and the cell cycle, including apoptosis, gene expression, and cell signaling cascades
[5]. Although the moderate production of ROS can elevate cell proliferation and differentiation,
excessive production can cause oxidative damage to biomolecules [6,7]. Consequently, the
homeostasis of ROS is crucial for normal cell growth and survival [8]. To prevent cellular
damage in normal conditions, the level of ROS is controlled; however, the level in cancer cells is
higher than in normal cells [9]. This phenomenon induces cell death in cancer cells. Although a
moderate elevation of ROS level can be controlled in normal cells, it may exceed the
antioxidative ability of cancer cells, which results in the cancer cell death [9]. Therefore, ROS-
generating compounds can act as selective chemotherapeutic agents against cancer cells that do
3

not affect normal cells [10]. Three such compounds, 2-(p-tolyl)-4H-benzo[h]chromen-4-one, 2-
allyl-1-((3,4-dimethoxyphenethyl)amino)-3-methylbenzo[4,5]imidazo[1,2-a]pyridine-4-
carbonitrile,
and
3-(1-acetyl-5-(furan-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)-6-chloro-4-
phenylquinolin-2(1H)-one, have been reported as ROS enhancers [11]. Piperlongumine is known
to be selectively toxic to cancer cells [12]. Based on the study of its analogs, it was suggested
that the Michael acceptor moiety was important for ROS generation [13]. A Michael acceptor
including α,β-unsaturated carbonyl group forms a covalent bond with glutathione (GSH) by a
Michael addition. For GSH to quench ROS, it should be remained as a reduced form [14].
However, S-alkylation of GSH by a Michael addition with a Michael acceptor leads to loss of the
possibility which GSH can be converted to its oxidized form [15]. This phenomenon prevents the
elimination of ROS, which causes accumulation of ROS. In the authors’ previous study, one
compound, (E)-3-(3,5-dimethoxyphenyl)-1-(2-methoxyphenyl)prop-2-en-1-one, containing a
Michael acceptor (Figure 1A) showed selective toxicity to cancer cells without affecting normal
cells [16]. Therefore, we designed and synthesized 26 compounds containing Michael acceptors
to find compounds able to generate ROS (Figure 1B). The design of the current compounds was
based on the combination of chalcone (Figure 1C) and resveratrol (Figure 1D). Chalcone and
resveratrol are secondary metabolites of plants. It has been reported that chalcone and resveratrol
derivatives exert anticancer activities in in vitro and in vivo experiments [16-21]. Their structures
were identified by using NMR spectroscopy and high-resolution mass spectrometry (HR/MS). It
has been previously explained why ROS-generating compounds should be studied for the
possible discovery of a chemotherapeutic agent. Therefore, the cytotoxicities of the designed
compounds against HCT116 human colon cancer cell lines were measured by using a clonogenic
4

long-term survival assay [22]. The half-maximal cell growth inhibitory concentration (GI50) was
determined. The compound showing the best GI50 value, (E)-3-(2-((E)-4-methoxystyryl)-4,6-
dimethoxyphenyl)-1-(pyridin-2-yl)prop-2-en-1-one, was subjected to further study to determine
whether it stimulated ROS production, which was measured by using 2′,7′-dichlorofluorescin
diacetate dye. Further biological activities of the title compound were evaluated for the induction
of apoptosis using annexin V staining. The aims of this study were to find compounds
stimulating ROS generation and to determine the structural conditions that result in stronger
cytotoxicity against cancer cells, as determined from quantitative structure-activity relationship
(QSAR) calculations.
2. Materials and Methods
2.1. Preparation of 1-aryl-(3-(2-styryl)phenyl)prop-2-en-1-ones
We designed 26 derivatives of 1-aryl-(3-(2-styryl)phenyl)prop-2-en-1-one (named as SPP), as
listed in Table 1. Their synthetic procedures are summarized in Scheme 1. In brief, 2 mL POCl3
was added dropwise to a solution of trans-trimethoxystilbene I (2.7 g, 10 mmol) in 20 mL DMF
in an ice-water bath and stirred overnight at room temperature. The resulting reaction mixture
was poured into 400 mL ice-water and stirred for 1–2 h to yield a precipitate. The resulting solid
was filtered, washed with water, and air-dried. For purification, recrystallization from hot
methanol was performed to produce aldehyde compound II (yield: 30 %) and methyl aldehyde
compound III (yield: 15 %) respectively. The aldehyde II or III was treated with methyl
5

aromatic ketone IV (1.2–1.5 eq.) in ethanol. An excess of 50% KOH solution was added to the
reaction mixture and stirred at overnight at room temperature overnight. Precipitation was
obtained by the addition of 3 N HCl until the pH reached 3–4. The resulting solids were filtered
and washed with ethanol to yield the corresponding chalcone compounds 1–19 or 20–26, which
were purified by recrystallization. The synthesis of derivative 13 was performed as follows:
Compound II (284 mg, 0.95 mmol) and 2-acetylpyridine (180 mg, 1.48 mmol) were dissolved in
20 mL ethanol. To this reaction mixture, 2 mL aqueous (aq.) KOH (50 w/v %) was added and
stirred at room temperature for 13 h. After completion of the reaction, the reaction mixture was
acidified with 3 N HCl (pH = 3–4) to cause precipitation. The resulting solid was filtered and
washed with ethanol. Color: lemon; m.p.: 157–158 °C; yield: 80%. Similarly, the synthesis of
derivative 20 was performed as follows: Compound III (304 mg, 0.96 mmol) and 2-
methoxyacetophenone (192 mg, 1.26 mmol) were dissolved in 20 mL ethanol. To the reaction
mixture, 2 mL aq. KOH (50 w/v %) was added and stirred at room temperature for 28 h. After
completion, the reaction mixture was acidified by the addition of 3 N HCl (pH = 3–4) to yield
precipitation. The resulting solid was filtered and washed with ethanol. Recrystallization from
hot methanol was performed, yielding analytically pure compound 20. Color: yellow; m.p.: 94–
100 °C; yield: 82%.
Synthetic SPP derivatives were identified by using nuclear magnetic resonance (NMR)
spectroscopy, performed on an Avance 400 spectrometer system (9.4 T; Bruker, Karlsruhe,
Germany) at 25 °C. The experimental methods followed a previous report [23]. To confirm the
structural determination obtained from NMR spectroscopy, high-resolution mass spectrometry
(HR/MS) was performed by using an ultraperformance liquid chromatography (UPLC)-hybrid
6

quadrupole time-of-flight mass spectrometer with a Waters Acquity UPLC system (Waters Corp.,
Milford, MA); assistance was provided by Prof. Choong Hwan Lee at Konkuk University, Korea
[24]. All HR/MS data were collected in [M+H]+ mode. The spectroscopic data of the 26 SPP
derivatives synthesized for these studies are presented below.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(2-methoxyphenyl)prop-2-en-1-one
(1)
Color: orange; m.p. 104–106 °C; yield 28%; 1H NMR δ (in DMSO-d6) 3.64(s, 3H), 3.80(s, 3H),
3.83(s, 3H), 3.84(s, 3H), 6.38(d, J= 2.3 Hz, 1H), 6.69(d, J= 2.3 Hz, 1H), 6.86(d, J= 8.8 Hz, 1H),
6.88(d, J= 8.8 Hz, 1H), 6.89(d, J= 16.0 Hz, 1H), 6.98(ddd, J= 0.8, 7.4, 7.6 Hz, 1H), 7.24(d, J=
16.0 Hz, 1H), 7.38(d, J= 8.8 Hz, 1H), 7.38(d, J= 16.0 Hz, 1H), 7.38(ddd, J= 1.7, 7.4, 8.8 Hz, 1H),
7.61 (dd, J= 1.7, 7.6 Hz, 1H), and 7.99(d, J= 16.0 Hz, 1H); 13C NMR δ (in DMSO-d6) 55.4, 55.5,
55.7, 97.6, 103.5, 111.6, 114.2, 116.1, 120.6, 125.5, 128.1, 129.7, 130.0, 130.2, 130.3, 131.4,
132.4, 138.3, 141.4, 158.0, 159.6, 160.8, 161.6, and 194.0; HR/MS calcd. for C27H26O5 (M+H)+
431.1858, found 431.1865.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(3-methoxyphenyl)prop-2-en-1-one
(2)
1
Color: yellow; m.p. 66–70 °C; yield 90%; H NMR δ (in DMSO-d6) 3.75(s, 3H), 3.78(s, 3H),
3.87(s, 3H), 3.91(s, 3H), 6.60(d, J= 2.1 Hz, 1H), 6.81(d, J= 2.0 Hz, 1H), 6.95(d, J= 8.7 Hz, 1H),
7.07(d, J= 16.1 Hz, 1H), 7.18(dd, J= 2.6, 8.0 Hz, 1H), 7.32(dd, J= 7.7, 8.0 Hz, 1H), 7.34(dd, J=
1.6, 2.6 Hz, 1H), 7.39(d, J= 16.1 Hz, 1H), 7.44(dd, J= 1.6, 7.7 Hz, 1H), 7.55(d, J= 8.7 Hz, 1H),
7

7.70(d, J= 15.7 Hz, 1H), and 8.02(d, J= 15.7 Hz, 1H); 13C NMR δ (in DMSO-d6) 55.2, 55.6,
55.8, 56.0, 97.8, 104.3, 112.5, 114.6, 118.9, 120.5, 122.3, 124.7, 128.1, 129.5, 130.0, 130.9,
132.0, 138.2, 139.6, 141.5, 159.4, 159.5, 160.8, 161.7, and 189.6; HR/MS calcd. for C27H26O5
(M+H)+ 431.1858, found 431.1887 [25].
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one
(3)
1
Color: pale yellow; m.p. 118–124 °C; yield 92%; H NMR δ (in DMSO-d6) 3.77(s, 3H), 3.82(s,
3H), 3.87(s, 3H), 3.90(s, 3H), 6.59(d, J= 2.1 Hz, 1H), 6.80(d, J= 2.1 Hz, 1H), 6.95(d, J= 8.6 Hz,
1H), 6.99(d, J= 8.8 Hz, 1H), 7.06(d, J= 16.5 Hz, 1H), 7.34(d, J= 16.5 Hz, 1H), 7.54(d, J= 8.6 Hz,
13
1H), 7.56(d, J= 15.8 Hz, 1H), 7.93(d, J= 8.8 Hz, 1H), and 7.98(d, J= 15.8 Hz, 1H); C NMR δ
(in DMSO-d6) 55.2, 55.5, 55.9, 97.8, 104.1, 114.0, 114.3, 114.8, 124.1, 124.8, 129.5, 130.4,
130.9, 131.0, 131.8, 137.2, 141.2, 159.3, 160.6, 161.5, 162.9, and 187.9; HR/MS calcd. for
C27H26O5 (M+H)+ 431.1858, found 431.1867. [25]
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(2-hydroxy-5-methoxyphenyl)prop-2-
en-1-one (4)
1
Color: red orange; m.p. 138–144 °C; yield 25%; H NMR δ (in DMSO-d6) 3.66(s, 3H), 3.87(s,
3H), 3.99(s, 3H), 4.04(s, 3H), 6.71(d, J= 1.5 Hz, 1H), 6.93(d, J= 1.5 Hz, 1H), 6.99(d, J= 8.3 Hz,
1H), 7.02(d, J= 8.5 Hz, 1H), 7.19(d, J= 16.1 Hz, 1H), 7.21(dd, J= 2.9, 8.3 Hz, 1H), 7.37(d, J=
2.9 Hz, 1H), 7.52(d, J= 16.1 Hz, 1H), 7.67(d, J= 8.5 Hz, 1H), 7.82(d, J= 15.5 Hz, 1H), and
13
8.34(d, J= 15.5 Hz, 1H); C NMR δ (in DMSO-d6) 55.3, 55.4, 55.6, 56.0, 97.8, 104.9, 112.1,
8

114.5, 115.1, 119.2, 120.6, 123.0, 124.4, 125.2, 128.5, 130.1, 132.7, 139.5, 142.6, 152.2, 157.3,
160.1, 161.7, 162.9, and 194.0; HR/MS calcd. for C27H26O6 (M+H)+ 447.1808, found 447.1770.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-
en-1-one (5)
1
Color: brown; m.p. 148–152 °C; yield 30%; H NMR δ (in DMSO-d6) 3.78(s, 3H), 3.81(s, 3H),
3.88(s, 3H), 3.93(s, 3H), 6.43(dd, J= 9.1, 2.5 Hz, 1H), 6.49(d, J= 2.5 Hz, 1H), 6.60(d, J= 2.3 Hz,
1H), 6.81(d, J= 2.3 Hz, 1H), 6.96(d, J= 8.7 Hz, 1H), 7.07(d, J= 16.0 Hz, 1H), 7.37(d, J= 16.0 Hz,
1H), 7.56(d, J= 8.7 Hz, 1H), 7.69(d, J= 15.4 Hz, 1H), 7.80(d, J= 9.1 Hz, 1H), and 8.10(d, J=
15.4 Hz, 1H); 13C NMR δ (in DMSO-d6) 55.2, 55.6, 55.7, 56.0, 97.8, 101.0, 104.3, 107.4, 114.0,
114.3, 114.5, 122.5, 124.5, 128.2, 129.5, 131.6, 132.3, 137.9, 141.8, 159.4, 161.0, 161.9, 165.6,
165.7, and 191.9; HR/MS calcd. for C27H26O6 (M+H)+ 447.1808, found 447.1801.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (6)
Color: yellow; m.p. 136–140 °C; yield 48%; 1H NMR δ (in DMSO-d6) 3.78(s, 3H), 3.89(s, 3H),
3.94(s, 3H), 6.62(d, J= 2.2 Hz, 1H), 6.83(d, J= 2.2 Hz, 1H), 6.90(dd, J= 7.2, 8.0 Hz, 1H), 6.96(d,
J= 8.7 Hz, 1H), 6.98(d, J= 8.8 Hz, 1H), 7.08(d, J= 16.1 Hz, 1H), 7.38(d, J= 16.1 Hz, 1H),
7.52(ddd, J= 1.2, 7.2, 8.8 Hz, 1H), 7.56(d, J= 8.7 Hz, 1H), 7.73(d, J= 15.5 Hz, 1H), 7.84(dd, J=
1.2, 8.0 Hz, 1H), and 8.12(d, J= 15.5 Hz, 1H); 13C NMR δ (in DMSO-d6) 55.2, 55.6, 56.1, 97.8,
104.3, 114.3, 114.4, 117.8, 119.2, 121.1, 123.0, 124.4, 128.2, 129.4, 129.9, 132.4, 135.9, 138.8,
142.0, 159.4, 161.1, 161.6, 162.1, and 193.9; HR/MS calcd. for C26H24O5 (M+H)+ 417.1702,
found 417.1692.
9

(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (7)
Color: orange; m.p. 162–166 °C; yield 26%; 1H NMR δ (in DMSO-d6) 3.77(s, 3H), 3.86(s, 3H),
3.90(s, 3H), 6.58(d, J= 1.7 Hz, 1H), 6.79(d, J= 1.7 Hz, 1H), 6.83(d, J= 8.5 Hz, 1H), 6.95(d, J=
8.4 Hz, 1H), 7.05(d, J= 16.1 Hz, 1H), 7.33(d, J= 16.1 Hz, 1H), 7.53(d, J= 15.6 Hz, 1H), 7.54(d,
13
J= 8.4 Hz, 1H), 7.85(d, J= 8.5 Hz, 1H), 7.95(d, J= 15.6 Hz, 1H), and 10.39(s, 1H); C NMR δ
(in DMSO-d6) 55.3, 55.5, 56.0, 97.8, 104.1, 114.3, 115.0, 115.5, 124.3, 125.0, 128.1, 129.6,
130.8, 131.8, 137.4, 141.2, 159.4, 160.6, 161.5, 162.0, and 187.8; HR/MS calcd. for C26H24O5
(M+H)+ 417.1702, found 417.1672 [26].
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(2-hydroxy-5-nitrophenyl)prop-2-en-
1-one (8)
Color: orange; m.p. 168–172 °C; yield 48%; 1H NMR δ (in DMSO-d6) 3.85(s, 3H), 3.93(s, 3H),
4.00(s, 3H), 6.47(d, J= 1.6 Hz, 1H), 6.74(d, J= 1.6 Hz, 1H), 6.92(d, J= 8.5 Hz, 1H), 6.95(d, J=
16.0 Hz, 1H), 7.08(d, J= 9.2 Hz, 1H), 7.35(d, J= 16.0 Hz, 1H), 7.50(d, J= 8.5 Hz, 1H), 7.89(d, J=
15.2 Hz, 1H), 8.31(dd, J= 2.4, 9.2 Hz, 1H), 8.39(d, J= 15.2 Hz, 1H), and 8.80(d, J= 2.4 Hz, 1H);
13C NMR δ (in DMSO-d6) 55.5, 55.7, 56.1, 97.9, 104.6, 114.3, 114.5, 115.1, 119.5, 120.5, 124.6,
126.3, 128.3, 129.7, 130.5, 133.5, 139.6, 141.9, 143.8, 160.1, 162.0, 163.0, 168.6, and 193.8;
HR/MS calcd. for C26H23NO7 (M+H)+ 462.1553, found 462.1559.
(E)-3-(2-((E)-4-<ethoxystyryl)-4,6-dimethoxyphenyl)-1-(5-bromo-2-hydroxyphenyl)prop-2-en-
1-one (9)
10

Color: orange; m.p. 156–160 °C; yield 27%; 1H NMR δ (in DMSO-d6) 3.86(s, 3H), 3.99(s, 3H),
4.04(s, 3H), 6.70(d, J= 2.2 Hz, 1H), 6.95(d, J= 2.2 Hz, 1H), 7.02(d, J= 8.7 Hz, 1H), 7.04(d, J=
8.7 Hz, 1H), 7.19(d, J= 16.3 Hz, 1H), 7.54(d, J= 16.3 Hz, 1H), 7.67(d, J= 8.7 Hz, 1H), 7.70(dd,
J= 2.3, 8.7 Hz, 1H), 7.81(d, J= 15.5 Hz, 1H), 8.05(d, J= 2.3 Hz, 1H), 8.31(d, J= 15.5 Hz, 1H),
13
and 11.97(s, 1H); C NMR δ (in DMSO-d6) 55.4, 55.8, 56.3, 98.1, 105.0, 110.5, 114.8, 115.3,
120.8, 123.5, 123.6, 125.1, 128.7, 130.3, 132.5, 133.1, 138.5, 140.2, 143.0, 160.3, 161.8, 162.0,
163.2, and 193.5; HR/MS calcd. for C26H23BrO5 (M+H)+ 495.0807, found 495.0826.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(4-fluoro-2-hydroxyphenyl)prop-2-en-
1-one (10)
Color: orange; m.p. 195–200 °C; yield 89%; 1H NMR δ (in DMSO-d6) 3.75(s, 3H), 3.85(s, 3H),
3.86(s, 3H), 6.28(dd, J= 2.5, 8.5 Hz, 1H), 6.44(dd, J= 2.5, 12.4 Hz, 1H), 6.56(d, J= 2.3 Hz, 1H),
6.82(d, J= 2.3 Hz, 1H), 6.92(d, J= 8.8 Hz, 1H), 7.07(d, J= 16.1 Hz, 1H), 7.38(d, J= 16.1 Hz, 1H),
7.54(d, J= 8.8 Hz, 1H), 7.69(dd, J= 8.0, 8.8 Hz, 1H), 7.87(d, J= 15.8 Hz, 1H), and 7.92(d, J=
15.8 Hz, 1H); 13C NMR δ (in DMSO-d6) 55.1, 55.4, 55.8, 97.7, 104.8, 103.6, 105.5, 114.2, 115.7,
122.0, 124.6, 125.2, 128.2, 129.6, 131.6, 132.7, 137.1, 140.6, 159.2, 160.4, 161.1, 165.1, 169.3,
and 191.0; HR/MS calcd. for C26H23FO5 (M+H)+ 435.1596, found 435.1577.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(5-fluoro-2-hydroxyphenyl)prop-2-en-
1-one (11)
Color: orange; m.p. 154–156 °C; yield 80%; 1H NMR δ (in DMSO-d6) 3.85(s, 3H), 3.98(s, 3H),
4.02(s, 3H), 6.68(d, J= 2.2 Hz, 1H), 6.93(d, J= 2.2 Hz, 1H), 7.00(d, J= 8.7 Hz, 1H), 7.06(dd, J=
11

4.6, 9.1 Hz, 1H), 7.18(d, J= 16.1 Hz, 1H), 7.40(ddd, J= 3.2, 8.0, 9.1 Hz, 1H), 7.53(d, J= 16.1 Hz,
1H), 7.66(d, J= 8.7 Hz, 1H), 7.71(dd, J= 3.2, 9.2 Hz, 1H), 7.86(d, J= 15.5 Hz, 1H), and 8.29(d,
13
J= 15.4 Hz, 1H); C NMR δ (in DMSO-d6) 55.6, 56.0, 56.3, 98.7, 105.1, 114.8, 115.5, 115.7,
120.2, 122.3, 123.5, 123.7, 125.0, 128.8, 130.7, 133.2, 140.0, 143.5, 157.0, 160.0, 160.9, 162.7,
163.6, and 194.3; HR/MS calcd. for C26H23FO5 (M+H)+ 435.1232, found 435.1219.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(5-chloro-2-hydroxyphenyl)prop-2-en-
1-one (12)
Color: yellow; m.p. 156–160 °C; yield 76%; 1H NMR δ (in DMSO-d6) 3.84(s, 3H), 3.89(s, 3H),
3.93(s, 3H), 6.41(d, J= 2.2 Hz, 1H), 6.69(d, J= 2.2 Hz, 1H), 6.92(d, J= 8.7 Hz, 1H), 6.93(d, J=
16.0 Hz, 1H), 6.95(d, J= 8.9 Hz, 1H), 7.30(d, J= 16.0 Hz, 1H), 7.37(dd, J= 2.5, 8.9 Hz, 1H),
7.48(d, J= 8.7 Hz, 1H), 7.62(d, J= 15.4 Hz, 1H), 7.71(d, J= 2.5 Hz, 1H), 8.30(d, J= 15.4 Hz,
13
1H); C NMR δ (in DMSO-d6) 55.5, 55.6, 55.9, 97.7, 104.3, 114.5, 115.4, 120.1, 121.2, 121.7,
123.4, 125.1, 128.2, 128.9, 129.7, 132.6, 135.7, 140.5, 142.9, 159.9, 161.4, 162.1, 162.4, and
193.6; HR/MS calcd. for C26H23ClO5 (M+H)+ 451.1312, found 451.1246.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(2-hydroxynaphthalen-1-yl)prop-2-en-
1-one (13)
1
Color: red orange; m.p. 146–150 °C; yield 48%; H NMR δ (in DMSO-d6) 3.80(s, 3H), 3.84(s,
3H), 3.87(s, 3H), 6.57(d, J= 2.2 Hz, 1H), 6.79(d, J= 2.2 Hz, 1H), 6.85(d, J= 16.1 Hz, 1H), 6.91(d,
J= 8.7 Hz, 1H), 6.96(d, J= 16.1 Hz, 1H), 7.10(d, J= 16.1 Hz, 1H), 7.14(d, J= 8.7 Hz, 1H), 7.25(d,
J= 8.9 Hz, 1H), 7.35(ddd, J= 1.4, 6.9, 8.2 Hz, 1H), 7.44(ddd, J= 1.4, 6.9, 8.2 Hz, 1H), 7.58(d, J=
12

8.1 Hz, 1H), 7.61(d, J= 16.1 Hz, 1H), 7.90(d, J= 8.2 Hz, 1H), 7.90(d, J= 8.9 Hz, 1H), and
10.21(s, 1H); 13C NMR δ (in DMSO-d6) 55.2, 55.5, 55.9, 97.9, 103.6, 113.7, 114.2, 118.3, 120.2,
123.0, 123.1, 123.4, 127.1, 127.6, 127.9, 128.2, 129.1, 130.7, 130.8, 131.6, 132.0, 139.7, 140.8,
152.4, 159.3, 160.7, 161.8, and 197.7; HR/MS calcd. for C30H26O5 (M+H)+ 467.1858, found
467.1822.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(thiophen-2-yl)prop-2-en-1-one (14)
Color: yellow; m.p. 140–144 °C; yield 71%; 1H NMR δ (in DMSO-d6) 3.78(s, 3H), 3.88(s, 3H),
3.93(s, 3H), 6.61(d, J= 2.0 Hz, 1H), 6.82(d, J= 2.0 Hz, 1H), 6.96(d, J= 8.5 Hz, 1H), 7.08(d, J=
16.1 Hz, 1H), 7.23(ddd, J= 0.8, 3.8, 5.0 Hz, 1H), 7.36(d, J= 16.1 Hz, 1H), 7.54(d, J= 15.2 Hz,
1H), 7.56(d, J= 8.5 Hz, 1H), 7.90(dd, J= 0.8, 5.0 Hz, 1H), 8.00(dd, J= 0.8, 3.8 Hz, 1H), and
13
8.01(d, J= 15.2 Hz, 1H); C NMR δ (in DMSO-d6) 55.2, 55.5, 56.0, 97.8, 104.2, 114.3, 114.4,
123.6, 124.4, 128.1, 128.9, 129.5, 132.2, 134.9, 137.2, 141.6, 145.6, 159.4, 160.8, 161.8, and
181.8; HR/MS calcd. for C24H22O4S (M+H)+ 407.1283, found 407.1280.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(thiophen-3-yl)prop-2-en-1-one (15)
Color: yellow; m.p. 122–125 °C; yield 83%; 1H NMR δ (in DMSO-d6) 3.83(s, 3H), 3.88(s, 3H),
3.89(s, 3H), 6.42(d, J= 2.2 Hz, 1H), 6.72(d, J= 2.2 Hz, 1H), 6.89(d, J= 8.3 Hz, 1H), 6.93(d, J=
16.1 Hz, 1H), 7.43(d, J= 15.7 Hz, 1H), 7.46(d, J= 8.3 Hz, 1H), 7.53(dd, J= 0.9, 4.9 Hz, 1H),
7.73(dd, J= 2.6, 4.9 Hz, 1H), 7.76(d, J= 16.1 Hz, 1H), 8.03(dd, J= 0.9, 2.6 Hz, 1H), and 8.18(d,
13
J= 15.7 Hz, 1H); C NMR δ (in DMSO-d6) 55.5, 55.6, 55.8, 97.7, 103.8, 114.4, 116.1, 125.1,
13

126.0, 126.3, 127.6, 128.1, 128.5, 129.9, 131.7, 138.2, 141.8, 143.7, 159.8, 160.9, 161.7, and
184.7; HR/MS calcd. for C24H22O4S (M+H)+ 407.1283, found 407.1281.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(1H-pyrrol-2-yl)prop-2-en-1-one (16)
Color: yellow; m.p. 182–184 °C; yield 20%; 1H NMR δ (in DMSO-d6) 3.77(s, 3H), 3.87(s, 3H),
3.90(s, 3H), 6.19(dd, J= 2.1, 3.6 Hz, 1H), 6.59(d, J= 2.0 Hz, 1H), 6.81(d, J= 2.0 Hz, 1H), 6.90(m,
1H), 6.94(d, J= 8.7 Hz, 1H), 7.08(d, J= 16.1 Hz, 1H), 7.13(m, 1H), 7.33(d, J= 16.1 Hz, 1H),
13
7.35(d, J= 15.7 Hz, 1H), 7.54(d, J= 8.7 Hz, 1H), 7.93(d, J= 15.7 Hz, 1H), and 11.93(s, 1H); C
NMR δ (in DMSO-d6) 55.2, 55.5, 55.9, 97.8, 103.9, 110.0, 114.2, 115.0, 116.0, 124.9, 125.4,
126.0, 128.0, 129.5, 131.5, 133.1, 134.6, 140.6, 159.3, 160.3, 161.1, and 178.3; HR/MS calcd.
for C24H23NO4 (M+H)+ 390.1705, found 390.1738.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(thiazol-2-yl)prop-2-en-1-one (17)
Color: yellow; m.p. 160–162 °C; yield 60%; 1H NMR δ (in DMSO-d6) 3.84(s, 3H), 3.90(s, 3H),
3.94(s, 3H), 6.44(d, J= 2.3 Hz, 1H), 6.74(d, J= 2.3 Hz, 1H), 6.91(d, J= 8.7 Hz, 1H), 6.96(d, J=
16.0 Hz, 1H), 7.42(d, J= 16.0 Hz, 1H), 7.52(d, J= 8.7 Hz, 1H), 7.64(d, J= 3.0 Hz, 1H), 8.00(d, J=
13
3.0 Hz, 1H), 8.00(d, J= 16.0 Hz, 1H), and 8.45(d, J= 16.0 Hz, 1H); C NMR δ (in DMSO-d6)
55.5, 55.6, 55.9, 97.7, 103.9, 114.3, 115.9, 123.4, 125.2, 125.9, 128.4, 130.1, 132.4, 140.3, 142.8,
144.8, 159.8, 161.6, 162.3, 169.6, and 182.8; HR/MS calcd. for C23H21NO4S (M+H)+ 408.1270,
found 408.1283.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(furan-2-yl)prop-2-en-1-one (18)
14

Color: yellow; m.p. 182–184 °C; yield 20%; 1H NMR δ (in DMSO-d6) 3.78(s, 3H), 3.88(s, 3H),
3.92(s, 3H), 6.60(d, J= 2.2 Hz, 1H), 6.73(dd, J= 1.5, 3.4 Hz, 1H), 6.82(d, J= 2.2 Hz, 1H), 6.96(d,
J= 8.7 Hz, 1H), 7.07(d, J= 16.1 Hz, 1H), 7.35(d, J= 16.1 Hz, 1H), 7.42(dd, J= 0.9, 3.4 Hz, 1H),
7.45(d, J= 15.8 Hz, 1H), 7.55(d, J= 8.7 Hz, 1H), 7.99(dd, J= 0.9, 1.5 Hz, 1H), and 8.01(d, J=
15.8 Hz, 1H); 13C NMR δ (in DMSO-d6) 55.2, 55.5, 56.0, 97.8, 104.2, 112.8, 114.3, 114.4, 118.0,
123.5, 124.5, 128.1, 129.5, 132.2, 136.8, 141.6, 147.8, 155.3, 159.4, 160.8, 161.7, and 177.5;
HR/MS calcd. for C24H22O5 (M+H)+ 391.1545, found 391.1548.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxyphenyl)-1-(pyridin-2-yl)prop-2-en-1-one (19)
Color: yellow; m.p. 157–158 °C; yield 80%; 1H NMR δ (in DMSO-d6) 3.80(s, 3H), 3.86(s, 3H),
3.88(s, 3H), 6.40(d, J= 2.3 Hz, 1H), 6.71(d, J= 2.3 Hz, 1H), 6.87(d, J= 8.7 Hz, 1H), 6.94(d, J=
16.2 Hz, 1H), 7.39(ddd, J= 0.8, 4.8, 7.8 Hz, 1H), 7.42(d, J= 16.2 Hz, 1H), 7.52(d, J= 8.7 Hz, 1H),
7.80(ddd, J= 1.6, 7.8, 7.8 Hz, 1H), 8.14(dd, J= 0.8, 7.8 Hz, 1H), 8.22(d, J= 16.1 Hz, 1H), 8.32(d,
13
J= 16.1 Hz, 1H), and 8.62(dd, J= 1.6, 4.8 Hz, 1H); C NMR δ (in DMSO-d6) 55.5, 55.6, 55.9,
97.6, 103.7, 114.2, 116.5, 122.9, 124.4, 125.9, 126.6, 128.4, 130.2, 131.7, 136.2, 138.9, 142.1,
149.0, 155.0, 159.7, 161.2, 161.8, and 190.5; HR/MS calcd. for C25H23NO4 (M+H)+ 402.1705,
found 402.1667.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxy-3-methylphenyl)-1-(2-methoxyphenyl)prop-2-
en-1-one (20)
1
Color: pale yellow; m.p. 96–100 °C; yield 82%; H NMR δ (in DMSO-d6) 2.11(s, 3H), 3.67(s,
3H), 3.82(s, 3H), 3.86(s, 3H), 3.90(s, 3H), 6.34(d, J= 16.6 Hz, 1H), 6.41(s, 1H), 6.75(dd, J= 0.8,
15

7.9 Hz, 1H), 6.76(ddd, J= 0.8, 7.6, 7.7 Hz, 1H), 6.84(d, J= 16.6 Hz, 1H), 6.86(dd, J= 1.8, 8.7 Hz,
1H), 7.22(ddd, J= 1.8, 7.6, 7.9 Hz, 1H), 7.29(dd, J= 1.8, 8.7 Hz, 1H), 7.36(dd, J= 1.8, 7.7 Hz,
1H), 7.44(d, J= 16.1 Hz, 1H), and 7.78(d, J= 16.1 Hz, 1H); 13C NMR δ (in DMSO-d6) 12.7, 55.5,
55.6, 55.7, 93.8, 111.5, 114.1, 115.1, 117.5, 120.4, 124.4, 127.9, 129.2, 129.6, 129.8, 130.0,
131.5, 136.2, 142.2, 142.5, 157.4, 159.3, 159.6, 159.8, and 196.3; HR/MS calcd. for C28H28O5
(M+H)+ 445.2015, found 445.2058.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxy-3-methylphenyl)-1-(3-methoxyphenyl)prop-2-
en-1-one (21)
Color: yellow; m.p. 116–120 °C; yield 14%; 1H NMR δ (in DMSO-d6) 2.09(s, 3H), 3.75(s, 3H),
3.78(s, 3H), 3.91(s, 3H), 3.99(s, 3H), 6.41(d, J= 16.6 Hz, 1H), 6.70(s, 1H), 6.95(d, J= 8.8 Hz,
1H), 7.13(d, J= 16.6 Hz, 1H), 7.14(dd, J= 2.6, 8.0 Hz, 1H), 7.32(dd, J= 7.7, 8.0 Hz, 1H), 7.34(dd,
J= 1.6, 2.6 Hz, 1H), 7.45(dd, J= 1.6, 7.7 Hz, 1H), 7.53(d, J= 8.8 Hz, 1H), 7.71(d, J= 15.7 Hz,
13
1H), and 8.00(d, J= 15.7 Hz, 1H); C NMR δ (in DMSO-d6) 12.5, 55.1, 55.2, 55.8, 55.9, 94.5,
112.3, 113.9, 114.2, 116.3, 118.5, 120.4, 122.3, 124.2, 127.8, 129.3, 129.8, 135.5, 139.8, 140.4,
141.9, 159.3, 159.4, 159.8, and 189.7; HR/MS calcd. for C28H28O5 (M+H)+ 445.2015, found
445.2017.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxy-3-methylphenyl)-1-(4-methoxyphenyl)prop-2-
en-1-one (22)
Color: yellow; m.p. 138–144 °C; yield 45%; 1H NMR δ (in DMSO-d6) 2.16(s, 3H), 3.79(s, 3H),
3.81(s, 3H), 3.88(s, 3H), 3.93(s, 3H), 6.44(s, 1H), 6.46(d, J= 16.5 Hz, 1H), 6.79(d, J= 8.8 Hz,
16

1H), 6.88(d, J= 8.6 Hz, 1H), 6.99(d, J= 16.5 Hz, 1H), 7.43(d, J= 8.6 Hz, 1H), 7.69(d, J= 15.8 Hz,
1H), 7.88(d, J= 8.8 Hz, 1H), and 8.08(d, J= 15.8 Hz, 1H); 13C NMR δ (in DMSO-d6) 12.8, 55.5,
55.7, 55.9, 93.9, 113.7, 114.3, 115.6, 117.6, 124.3, 124.9, 127.9, 130.1, 130.8, 131.9, 136.0,
140.4, 142.0, 159.1, 159.7, 159.8, 162.9, and 190.5; HR/MS calcd. for C28H28O5 (M+H)+
445.2015, found 445.2028.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxy-3-methylphenyl)-1-(2-hydroxy-5-
methoxyphenyl) prop-2-en-1-one (23)
Color: orange brown; m.p. 182–186 °C; yield 80%; 1H NMR δ (in DMSO-d6) 2.20(s, 3H), 3.62(s,
3H), 3.84(s, 3H), 3.93(s, 3H), 3.98(s, 3H), 6.48(s, 1H), 6.50(d, J= 16.5 Hz, 1H), 6.92(d, J= 9.0
Hz, 1H), 6.92(ddd, J= 2.4, 3.0, 8.8 Hz, 1H), 7.05(d, J= 16.5 Hz, 1H), 7.07(dd, J= 3.0, 9.0 Hz,
1H), 7.24(d, J= 3.0 Hz, 1H), 7.48(ddd, J= 2.4, 3.0, 8.8 Hz, 1H), 7.84(d, J= 15.5 Hz, 1H), 8.30(d,
J= 15.5 Hz, 1H), and 12.70(s, 1H); 13C NMR δ (in DMSO-d6) 12.8, 55.5, 55.8, 56.0, 93.8, 112.6,
114.4, 115.3, 118.0, 119.2, 120.2, 121.5, 123.6, 124.5, 128.1, 129.9, 136.4, 141.8, 142.4, 151.7,
158.0, 159.6, 159.9, 160.5, and 194.4; HR/MS calcd. for C28H28O6 (M+H)+ 461.1600, found
461.1611.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxy-3-methylphenyl)-1-(3,4-dimethoxyphenyl)prop-
2-en-1-one (24)
Color: yellow; m.p. 146–149 °C; yield 25%; 1H NMR δ (in DMSO-d6) 2.16(s, 3H), 3.80(s, 3H),
3.84(s, 3H), 3.88(s, 3H), 3.90(s, 3H), 3.93(s, 3H), 6.44(s, 1H), 6.46(d, J= 16.5 Hz, 1H), 6.68(d,
J= 8.3 Hz, 1H), 6.87(dd, J= 1.9, 8.7 Hz, 1H), 6.99(d, J= 16.5 Hz, 1H), 7.42(dd, J= 1.9, 8.7 Hz,
17

1H), 7.49(dd, J= 1.9, 8.3 Hz, 1H), 7.51(d, J= 1.9 Hz, 1H), 7.70(d, J= 15.8 Hz, 1H), and 8.09(d,
J= 15.8 Hz, 1H); 13C NMR δ (in DMSO-d6) 12.8, 55.5, 55.7, 55.9, 56.0, 56.1, 93.9, 110.0, 110.9,
114.3, 115.6, 117.6, 123.0, 124.0, 124.9, 127.9, 130.1, 132.1, 136.0, 140.3, 142.0, 149.1, 152.7,
159.1, 159.7, 159.8, and 190.2; HR/MS calcd. for C29H30O6 (M+H)+ 475.2121, found 475.2127.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxy-3-methylphenyl)-1-(3-hydroxyphenyl)prop-2-
en-1-one (25)
Color: yellow; m.p. 150–154 °C; yield 48%; 1H NMR δ (in DMSO-d6) 2.09(s, 3H), 3.78(s, 3H),
3.91(s, 3H), 3.99(s, 3H), 6.41(d, J= 16.5 Hz, 1H), 6.71(s, 1H), 6.95(d, J= 8.8 Hz, 1H), 6.97(d, J=
8.1 Hz, 1H), 7.14(d, J= 16.5 Hz, 1H), 7.22(dd, J= 8.1, 8.1 Hz, 1H), 7.28(s, 1H), 7.29(d, J= 8.1
Hz, 1H), 7.54(d, J= 8.8 Hz, 1H), 7.70(d, J= 15.8 Hz, 1H), 7.95(d, J= 15.8 Hz, 1H), and 9.85(s,
13
1H); C NMR δ (in DMSO-d6) 12.5, 55.3, 55.9, 56.0, 94.6, 113.9, 114.2, 114.3, 116.3, 118.9,
119.7, 122.4, 124.2, 127.9, 129.3, 129.8, 135.6, 139.8, 140.2, 142.0, 157.7, 159.3, 159.4, 159.7,
and 190.0; HR/MS calcd. for C27H26O5 (M+H)+ 431.1858, found 431.1846.
(E)-3-(2-((E)-4-Methoxystyryl)-4,6-dimethoxy-3-methylphenyl)-1-(thiophen-2-yl)prop-2-en-1-
one (26)
Color: yellow; m.p. 174–176 °C; yield 20%; 1H NMR δ (in DMSO-d6) 2.18(s, 3H), 3.87(s, 3H),
4.01(s, 3H), 4.11(s, 3H), 6.55(d, J= 16.4 Hz, 1H), 6.84(s, 1H), 7.02(d, J= 8.6 Hz, 1H), 7.23(dd,
J= 3.8, 4.9 Hz, 1H), 7.26(d, J= 16.4 Hz, 1H), 7.64(d, J= 8.6 Hz, 1H), 7.84(d, J= 15.6 Hz, 1H),
13
7.94(dd, J= 1.0, 3.8 Hz, 1H), 7.99(dd, J= 1.0, 4.9 Hz, 1H), and 8.16(d, J= 15.6 Hz, 1H); C
NMR δ (in DMSO-d6) 12.4, 55.3, 55.9, 94.8, 114.5, 117.0, 122.6, 124.8, 128.2, 128.9, 130.1,
18

132.0, 134.3, 136.4, 139.9, 142.7, 146.8, 160.2, 160.5, and 182.8; HR/MS calcd. for C25H24O4S
(M+H)+ 421.1440, found 421.1425.
2.2. Clonogenic assay
The effect of SPP derivatives on the clonogenicity of HCT116 cells was determined as described
previously [22]. Briefly, HCT116 colon cancer cells were seeded onto 24-well tissue culture
plates (BD FalconTM; Becton Dickinson Immunocytometry Systems, San Jose, CA) at a density
of 2 × 103 cells per well. After 24 h, the cells were treated with different concentrations of the
compounds for 7 days and stained with 0.1% (w/v) crystal violet [27].
2.3. Cell viability assay
The effect of the compound SPP19 on cell viability was determined by using a Cell Counting
Kit-8 (CCK-8; Dojindo Molecular Technologies, Gaithersburg, MD) in accordance with the
manufacturer’s instructions. Briefly, exponentially growing HCT116 cells (3 × 103 cells/sample)
were treated with different concentrations of SPP19 (0, 10, 20, 50, and 100 μM). After 24 h,
CCK-8 solution containing the water-soluble tetrazolium salt WST-8 (2-(2-methoxy-4-
nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) was
added and incubated for 1 h. The absorbance at 450 nm was measured by using an Emax
Endpoint ELISA Microplate Reader (Molecular Devices, Sunnyvale, CA) [28].
2.4. Apoptosis assay by annexin V staining
19

HCT116 cells (1 × 106 cells/sample) were treated with different concentrations of compound
SPP19 (0, 25, and 50 μM) for 24 h, fixed with 4% paraformaldehyde, and incubated with
fluorescein isothiocyanate (FITC)-conjugated annexin V in accordance with the manufacturer’s
instructions (ChemoMetec). Propidium iodide (PI) staining was used to identify dead cells. The
fluorescence intensities of FITC-annexin V-positive cells and PI-positive cells were analyzed by
using a NucleoCounter NC-3000 instrument, as described previously [29].
2.5. Detection of intracellular ROS
HCT116 cells were incubated with 10 μM DCF-DA (Sigma-Aldrich) for 30 min and then
exposed to 25 μM SPP19. After 1 and 12 h, the cells were collected and the fluorescence
intensity was measured by using a FACSCalibur flow cytometer (Becton Dickinson
Immunocytometry Systems, Jose, CA, USA), as described previously [17].
2.6. Immunoblot analysis
HCT116 cells were extracted in 20 mM pH 7.2 HEPES buffer containing 1% Triton X-100, 10%
glycerol, 150 mM NaCl, 10 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride in an ice
bath. The lysates were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred
to nitrocellulose membranes. The membranes were incubated with the corresponding primary
antibodies and developed by using the enhanced chemiluminescence detection system
(Amersham Pharmacia Biotech, Piscataway, NJ, USA) [30].
2.7. QSAR calculations
20

Three-dimensional quantitative structure-activity relationships (3D-QSARs) were performed by
using an Intel Core 2 Quad Q6600 (2.4 GHz) Linux PC with Sybyl 7.3 software (Tripos, St.
Louis, MO, USA). The experiment details are contained in a previously reported method [31].
2.8. Statistical Analysis
All statistical analyses were performed by using one‑ way analysis of variance (ANOVA)
followed by a Kruskal-Wallis test using GraphPad Instat software (La Jolla, CA). P values of
less than 0.05 indicated statistical significance [29].
3. Results and Discussion
Among the 26 SPP derivatives synthesized, 23 compounds are novel [25,26]—all except SPP 2,
3, and 7. Therefore, one compound, (E)-3-(2-((E)-4-methoxystyryl)-4,6-dimethoxyphenyl)-1-(2-
hydroxynaphthalen-1-yl)prop-2-en-1-one (SPP 13) was selected for structural determination. In
1
the H NMR spectrum of SPP 13, a total of 17 peaks were observed; 14 were found in the
aromatic region. Of these, four peaks at 6.85, 6.96, 7.10, and 7.61 ppm were a doublet with a
coupling constant of 16.1 Hz, indicating that they are from trans conformations of vicinal protons
of alkene groups (CH=CH). The positions of the peaks were determined by using COSY and
HMBC NMR experiments. For example, the peak at 7.61 ppm must be H-β, because of the
correlation with both the carbonyl carbon (C=O, 197.7 ppm) and the quaternary carbon (C-1',
113.7 ppm) in the HMBC spectrum. In addition, a cross peak between the peaks at 7.10 ppm and
7.61 ppm was present in the COSY spectrum. Therefore, the peaks at 7.10 and 7.61 ppm were
21

assigned as H-α and H-β. The other two peaks at 6.85 and 6.96 ppm were H-7' and H-8',
1
respectively, and assigned in a similar way. Two H peaks at 6.57 and 6.79 ppm were a doublet
4
with a coupling constant of 2.2 Hz, which is a typical JHH coupling constant. Therefore, they
were assigned as H-3' and H-5', respectively. Two peaks at 7.14 and 6.91 ppm had 2H of
integrated intensity, suggesting H-2"/6" and H-3"/5", respectively. The remaining peaks in the
aromatic region were from the naphthalene moiety. The three peaks at approximately 3.80 ppm
were methoxy protons. The positions of the methoxy protons were also determined by 2D NMR
experiments, such as HMBC and NOESY. All methoxy protons showed long-range coupling
1
with an adjacent quaternary carbon in the HMBC spectrum. For example, the H peak at 3.87
ppm showing a singlet and 3H of integrated intensity was correlated with 160.7 ppm (C-2'),
which indicated that the peak must be 2'-OCH3. In addition, NOE cross peaks between the
methoxy protons and the adjacent protons supported the positional determinations.
There are many methods to screen the anticancer effects of the compounds. The 26 SPP
derivatives contain different substituents at two positions, but they have a common feature.
Therefore, the clonogenic long-term survival assay was adapted for use here. Although this
method requires a long experimental period, such as a minimum of 7 days, it is good for the
discrimination of the cytotoxicity of compounds with similar structures. The inhibitory effects of
the growth of HCT116 human colon cancer cells caused by different concentrations of the
compounds synthesized are shown in Figure 2. The first clonogenic assay was performed at 0, 1,
5, 10, and 20 μM, and the second assay was at lower concentrations, such as 0, 0.1, 0.5, 1, and 5
μM. The clonogenic survival of the cancer cells was evaluated by using densitometry [32] and
the half-maximal cancer cell growth inhibitory concentrations (GI50) were determined by using
22

SigmaPlot software (SYSTAT, Chicago, IL). These values were between 3.41 and 91.42 μM, as
listed in Table 1. The GI50 values with error bars are graphed as shown in Suppl. Figure 1. The
biological data for 3D-QSAR calculations were the negative logarithmic scales of the GI50 values
(pGI50). To determine the 3D structures of the SPP derivatives tested here, the crystal structure of
(E)-4,6-dimethoxy-2-(4-methoxystyryl)-3-methylbenzaldehyde was used as a template [33]. The
3D structures of all derivatives were determined based on the modification of this template
structure. Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular
Similarity Indices Analysis (CoMSIA) were used for the 3D-QSAR calculations. The 26
derivatives were separated into the training set, used to build the QSAR model, and the test set,
used to validate the model. Five derivatives (SPP 9, 10, 14, 17, and 24) were selected arbitrarily
and hierarchical clustering analysis was performed to confirm whether they belonged to the same
structural group. As shown in Suppl. Figure 2, the five derivatives belonged to the separate
groups. The derivatives contained in the training set were aligned to confirm whether they
interacted with each other. As shown in Suppl. Figure 3, they were well aligned.
CoMFA calculations were iterated to find the best model able to elucidate the
relationships between the structural properties of the SPP derivatives and their cytotoxicities
obtained from the clonogenic long-term survival assay in HCT116 human colon cancer cells. Of
the many CoMFA models, one model was chosen, which showed a good cross-validation
correlation coefficient (0.503). In this CoMFA model, the non-cross-validated coefficient,
optimal number of components, standard error estimation, and F values were 0.958, 5, 0.078, and
68.287, respectively. CoMFA provides information about steric and electrostatic field descriptors.
In this CoMFA model, these descriptors contributed 64.4% and 35.6%, respectively. Using this
23

CoMFA model, the pGI50 values of the derivatives contained in the training set were calculated.
As listed in Suppl. Table 1, the residuals between the experimental values of pGI50 and the
values calculated using this CoMFA model were between 0.12% and 13.59%. Similarly, the
residuals in the test set were between 7.18% and 28.79%. The experimental values of pGI50 were
plotted against the pGI50 values predicted by this CoMFA model (Suppl. Figure 4). The plot
indicated that this CoMFA model was reliable. To visualize the steric and electrostatic field
descriptors calculated by this CoMFA model, contour maps were generated by using Sybyl
software. SPP 19 is illustrated in the contour maps of CoMFA model as a template. In the steric
field maps of CoMFA, yellow (14% contribution) and green (86% contribution) contours were
observed in the second and fifth position of the pyridine structure, respectively (Figure 3A). The
yellow contour indicates that the bulky group may decrease the biological activity. For example,
the structures of SPP 5 and SPP 3 are identical, except for the substituents at C-2: SPP 5 has
hydroxyl group instead of the H in SPP 3, but the GI50 values were poorer. In contrast, green
contours were observed mainly at the C-5 position, where the bulky group is thought to increase
the biological activity. SPP 4, with a bulky methoxy group at C-5, had a GI50 of 25.85 µM,
which is more active than the values of SPP 5 (91.42 µM) and SPP 6 (27.72 µM). In addition,
SPP 8 and SPP 9 have bulky groups of -NO2 and -Br at the C-5 position, respectively, which
showed good GI50 values. The electrostatic field maps were displayed in Figure 3B. The blue
(17% contribution) and red (83% contribution) contours indicate that electropositive and
electronegative groups enhance the activity, respectively. The electropositive group at the second
position increases the activity, such as the nitrogen atom of SPP 19. Red contours were observed
near C-3 and C-5, indicating that electronegative groups may increase the activity. SPP 18, with
24

a furan ring that contained a negatively charged oxygen at the C-3 position, showed better
activity than SPP 14 and SPP 16, which contained the less negatively charged -NH and -S groups,
respectively.
Similar to CoMFA calculations, CoMSIA calculations were performed using the same
training set and the test set. Whereas CoMFA provides information about the steric and
electrostatic field descriptors, CoMSIA provides information about the steric and electrostatic
field descriptors, as well as hydrophobic interactions, hydrogen bond (H-bond) donors, and H-
bond acceptor field descriptors. After many iterations of CoMSIA calculations, the model with a
cross-validated coefficient of 0.626 was chosen. This model included three field descriptors,
such as the steric, electrostatic, and H-bond donor descriptors, which contributed 7.4%, 55.8%,
and 36.8%, respectively. Here, the non-cross-validated coefficient, the optimal number of
components, standard error estimation, and F values were 0.954, 6, 0.085, and 48.154,
respectively. The pGI50 values of the derivatives contained in the training set predicted by using
this CoMSIA model are listed in Suppl. Table 1. The residuals between the experimental values
of pGI50 and the predicted values were between 0.24% and 8.66%. Similarly, the residuals for
the test set were between 16.49% and 30.70%. The experimental values of pGI50 were plotted
versus the pGI50 values predicted using this CoMSIA model (Suppl. Figure 5), and the plot
indicated that this CoMSIA model was considered reliable. The contour maps for CoMSIA were
generated to visualize the results obtained from the CoMSIA model. The steric and electrostatic
contours of the CoMSIA model (Suppl. Figure 6A, 6B) were similar to those of the CoMFA
model. In the steric contour maps, two yellow contours at the C-4 and C-3' positions were newly
observed; this may provide an explanation for why compounds with the methoxy group at C-4,
25

such as SPP 3, SPP 5, and SPP 22, had poor activity. In contrast, a bulky group at C-3' may have
a weaker effect on the activity. Although some of the SPP derivatives without a CH3 group at the
C-3' position showed better activities than the derivatives with a CH3 group, the differences in
GI50 values between the derivatives were small. The contour maps for the H-bond donor of the
CoMSIA model, illustrated with cyan and purple colors, indicate the H-bond donor-favored and -
disfavored regions, respectively (Suppl. Figure 6C). Based on the H-bond donor contour maps of
the CoMSIA model, the H-bond donor groups were favored near the C-4 and C-5 region. SPP 7,
with a hydroxyl group at the C-4 position, and SPP 25, with a hydroxyl group at the C-5 position,
showed good GI50 values of 7.51 µM and 7.08 µM, respectively.
Based on the CoMFA and CoMSIA models, the pharmacophores of the SPP structures
were elucidated, as shown in Figure 4. A bulky group was disfavored in the C-2 and C-4
positions, but favored at the C-5 position. In addition, electropositive groups, such as nitrogen or
methoxy groups, are favored at the C-2 position and electronegative groups are favored at the C-
3 and C-5 position. The presence of an H-bond donor group at C-4 and C-5 enhances the activity.
We selected SPP 19 for the evaluation of its cytotoxic effect. HCT116 cells in the
exponential growth phase were treated with different concentrations of SPP 19 (0, 5, 10, 25, and
50 μM) for 24 h and the cell viability was measured. Treatment with >10 μM SPP 19
significantly reduced the viability of HCT116 cells (Figure 5). It has been reported that
phosphatidylserine (PS) in the inner plasma membrane translocates to the outer membrane
during apoptosis. As annexin V preferentially binds to PS [34], apoptotic cells can be identified
by using the annexin V binding assay. To investigate whether SPP 19 triggers apoptosis, an
annexin V staining assay was performed. HCT116 cells treated with SPP 19 (25 or 50 μM) for
26

24 h were incubated with fluorescein isothiocyanate (FITC)-conjugated annexin V (AV) and
with propidium iodide (PI) to stain dead cells. The PI/AV double-negative populations (top
panels, lower left quadrant in each cytogram) represent viable cells, whereas PI negative/AV
positive populations (top panels, lower right quadrant) represent early apoptotic cells. PI/AV
double-positive populations (top panels, upper right quadrant) are late apoptotic or dead cells. As
shown in Figure 6A, the AV-positive populations increased from 8% (0 μM) to 84% (25 μM)
and 90% (50 μM) after SPP 19 treatment, which suggested that SPP 19 triggered apoptotic cell
death in HCT116 cells. Caspases (cysteine-dependent aspartate-directed proteases) function as
mediators of apoptosis [35]. To determine whether SPP 19 induced caspase activation, caspase
activity was determined through the measurement of the cleavage of poly(ADP-ribose)
polymerase (PARP), a well-known substrate for executioner caspases (e.g., caspase-3 and
caspase-7), by using immunoblotting analysis. SPP 19 treatment increased the abundance of
cleaved PARP in a time-dependent manner (Figure 6B). These data suggested that SPP 19
triggered apoptosis through a caspase-mediated pathway.
Many types of cancer cells are more sensitive to ROS than normal cells [36,37]; thus,
ROS production is predicted to offer a valuable therapeutic approach to targeting cancer cells
[38,39]. Given that ROS activates caspase in a variety of mammalian cells [40], we determined
whether SPP 19 stimulated intracellular ROS production by using a H2-2′,7′-dichlorofluorescin
diacetate (DCF-DA) probe. We found that SPP 19 treatment led to detectable ROS accumulation
after 1 h, with this level maintained until 12 h (Figure 7A). To further evaluate the effect of SPP
19 on ROS production, intracellular ROS was visualized by using fluorescence microscopy
(Figure 7B). To determine whether SPP 19-induced ROS production was functionally associated
27

with the induction of apoptosis, HCT116 cells were pretreated with 2 mM N-acetyl cysteine
(NAC), a thiol-containing ROS scavenger. SPP 19-induced PARP cleavage was substantially
reduced by pretreatment with NAC (Figure 7C). These results suggested that SPP 19-induced
apoptosis occurred through a ROS-dependent caspase-mediated pathway.
4. Conclusion
To find compounds generating ROS, 26 SPP derivatives designed to contain a Michael acceptor,
were synthesized. Of these 26 derivatives, 23 were novel. The derivatives were identified by
using NMR spectroscopy and HR/MS. Their cytotoxicity to HCT116 colon cancer cells were
measured by using a clonogenic long-term survival assay. Their half-maximal cell growth
inhibitory effect was between 3.41 and 91.42 μM. The compound with the strongest cytotoxicity
was examined by using a DCF-DA probe to confirm whether it generated ROS. As expected, the
title compound, (E)-3-(2-((E)-4-methoxystyryl)-4,6-dimethoxyphenyl)-1-(pyridin-2-yl)prop-2-
en-1-one, caused an accumulation of ROS at 1 h. In addition, intracellular ROS determined by
the title compound was confirmed by using fluorescence microscopy. ROS generation by the title
compound was linked to the induction of apoptosis, which was confirmed by pretreatment with
N-acetyl cysteine. The search for ROS-generating compounds is linked to the discovery of new
chemotherapeutic agents. Therefore, the structural conditions that show better cytotoxicity were
derived based on CoMFA and CoMSIA. Although the presence of a bulky group at the C-2 and
C-4 positions is not favored, it is favored at the C-5 position. In addition, electropositive groups,
such as nitrogen or methoxy groups, are favored at the C-2 position and electronegative groups
28

are favored at the C-3 and C-5 positions. The presence of the H-bond donor group at C-4 and C-
5 enhances cytotoxicity. The design of the 26 compounds containing a Michael acceptor
synthesized in this study were based on the combination of chalcone and resveratrol. Their
cytotoxicities were not improved greatly compared with those caused by known chalcone and
resveratrol derivatives; however, 23 compounds synthesized here were novel and one was
confirmed to result in ROS generation. These findings can assist in the design of novel
compounds with a combination of the structures of chalcone and resveratrol for use as
chemotherapeutic agents.
Acknowledgments
This study was supported by the Basic Science Research Program (NRF-
2016R1D1A1A09919045). SY Shin was supported by the KU Research Professor Program of
Konkuk University.
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