New hybrids based on curcumin and resveratrol: Synthesis, cytotoxicity and antiproliferative activity against colorectal cancer cells

Article abstract of DOI:10.3390/molecules26092661

We synthesized twelve hybrids based on curcumin and resveratrol, and their structures were elucidated by spectroscopic analysis. The chemopreventive potential of these compounds was evaluated against SW480 human colon adenocarcinoma cells, its metastatic derivative SW620, along with the non-malignant CHO-K1 cell line. Among the tested compounds, hybrids 3e and 3i (for SW480) and 3a, 3e and 3k (for SW620) displayed the best cytotoxic activity with IC₅₀ values ranging from 11.52 ± 2.78 to 29.33 ± 4.73 μM for both cell lines, with selectivity indices (SI) higher than 1, after 48 h of treatment. Selectivity indices were even higher than those reported for the reference drug, 5-fluorouracil (SI = 0.96), the starting compound resveratrol (SI = 0.45) and the equimolar mixture of curcumin plus resveratrol (SI = 0.77). The previous hybrids showed good antiproliferative activity.

Full text of DOI:10.3390/molecules26092661

molecules
Article
New Hybrids Based on Curcumin and Resveratrol: Synthesis,
Cytotoxicity and Antiproliferative Activity against Colorectal
Cancer Cells
Cristian Hernández , Gustavo Moreno † , Angie Herrera-R and Wilson Cardona-G *
†
Química de Plantas Colombianas, Faculty of Exact and Natural Sciences, Institute of Chemistry,
University of Antioquia (UdeA), Calle 70 No. 52–21, Medellín AA 1226, Colombia;
cristian.hernandezs@udea.edu.co (C.H.); gustavo.morenoq@gmail.com (G.M.);
angie.herrerar@udea.edu.co (A.H.-R.)
*
Correspondence: wilson.cardona1@udea.edu.co
†
Both authors contributed equally to this work and should be considered co-first authors.
Abstract: We synthesized twelve hybrids based on curcumin and resveratrol, and their structures
were elucidated by spectroscopic analysis. The chemopreventive potential of these compounds was
evaluated against SW480 human colon adenocarcinoma cells, its metastatic derivative SW620, along
with the non-malignant CHO-K1 cell line. Among the tested compounds, hybrids 3e and 3i (for
SW480) and 3a
from 11.52 2.78 to 29.33
after 48 h of treatment. Selectivity indices were even higher than those reported for the reference drug,
-fluorouracil (SI = 0.96), the starting compound resveratrol (SI = 0.45) and the equimolar mixture of
,
3e and 3k (for SW620) displayed the best cytotoxic activity with IC50 values ranging
±
±
4.73 M for both cell lines, with selectivity indices (SI) higher than 1,
µ
5
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
curcumin plus resveratrol (SI = 0.77). The previous hybrids showed good antiproliferative activity.
Citation: Hernández, C.; Moreno, G.;
Herrera-R, A.; Cardona-G, W. New
Hybrids Based on Curcumin and
Resveratrol: Synthesis, Cytotoxicity
and Antiproliferative Activity against
Colorectal Cancer Cells. Molecules
Keywords: curcumin; resveratrol; hybrid molecules; colorectal cancer; cytotoxicity; antiprolifera-
tive activity
2
021, 26, 2661. https://doi.org/
1. Introduction
1
0.3390/molecules26092661
Colorectal cancer (CRC) is the third most diagnosed malignancy and the fourth leading
cause of cancer-related death worldwide. Despite the progress in diagnosis and treatment,
incidence and mortality of CRC cancer is unacceptably high [1]. Current chemotherapy
Academic Editor: Brullo Chiara
is based on 5-fluorouracil, oxaliplatin and irinotecan, which, although effective, induce
severe side effects to the patients; thus, novel approaches for the treatment of colorectal
cancer are needed [2].
Received: 23 March 2021
Accepted: 26 April 2021
Published: 1 May 2021
Curcumin is one of the bioactive components of Curcuma longa Linn. Several studies
with this compound have revealed important functions in cancer control [
antiproliferative activity against different cancer cell lines and inhibition at different stages
of cancer cell progression [ ]. Various growth factor receptors and cell adhesion molecules
involved in tumor growth, metastasis, apoptosis and multidrug resistance are affected by
curcumin [ ]. Resveratrol has also received great interest in drug discovery mainly due
to its various biological activities including antitumor [ ], affecting all three stages of
3], including
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with regard to jurisdictional claims in
published maps and institutional affil-
iations.
4
5–7
8,9
carcinogenesis (initiation, promotion and progression) by modulating signal transduction
pathways that control cell division and growth, apoptosis, inflammation, angiogenesis and
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
metastasis [10
biological activity; however, resveratrol analogs containing methoxy groups have increased
the activity [ 17]. Both curcumin and resveratrol alone have shown restricted clinical
efficacy due to different factors, including low bioavailability and stability stands out [18 19].
–16]. Different studies have shown the importance of hydroxyl group for the
8,9,
,
The synthesis of curcumin and resveratrol hybrids has emerged as a promising strategy
in recent drug discovery research to develop highly active anticancer therapies. The hybrid
anticancer drug approach is an innovative synthetic strategy that combines two or more
4
.0/).
Molecules 2021, 26, 2661. https://doi.org/10.3390/molecules26092661
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2661
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bioactive scaffolds with relevant pharmacological action [20
,21], where the presence of
different pharmacophores in a single unit could improve the ability to inhibit more than
one biological target, displaying a synergistic effect in biological activity. The goal of the
molecular hybridization approach is to improve anticancer activity and selectivity with a
subsequent reduction in side effects regarding conventional chemotherapy [18].
Curcumin–monastrol hybrid (Figure 1A) showed maximum activity with a mean
growth percentage (GP) of 88.90; besides, it was found to be the most sensitive on MDA-
MB-231/ATCC (breast cancer), PC-3 (prostate cancer), SNB-75 (CNS cancer), RPMI-8226
(leukemia), MOLT-4 (leukemia), CCRF-CEM (leukemia) and HS 578T (breast cancer) cell
lines with GP values of 58.50, 59.60, 60.07, 64.11, 72.84 and 73.39, respectively [22]. On the
other hand, curcumin–quinolone hybrid (Figure 1B) was evaluated against two human
lung cancer cells (A549 and H-460), with IC50 values of 23.9 and 21.75
the MCF-7 human breast cancer cell line (IC50 values of 36.2 M); and SKOV-3 ovarian
carcinoma cells (IC50 value of 12.8 M) with selectivity against SKOV-3 cell line, induc-
ing changes in normal cell morphology and apoptosis [23]. Hybrid curcum–imidazol
Figure 1C) showed antiproliferative activity against LNCaP (human androgen-dependent
prostate cancer) cell line with an IC50 value of 1.0 M [24]. 1H-1,2,3-triazole tethered
curcumin–isatin hybrid (Figure 1D) was tested against a panel of cancer cell lines ex-
hibiting potent activity with IC50 values ranging from 1.12 to 5.67 M. Moreover, this
compound showed inhibition of tubulin polymerization at 1.2 M when evaluated in HCT-
16 cells [25]. Hybrid stilbene–chalcone (Figure 1E) showed high selectivity towards certain
ovarian cancer, non-small cell lung cancer and breast cancer cell lines with GI50 values
in the range of 1.28–34.1 M [26]. Resveratrol–oxadiazole hybrid F showed great activity
against SiHa, MDA-MB-231 and PANC-1 human cancer cells (GI50 < 0.1 M), being supe-
rior to the reference compound resveratrol [27]. Hybrids based on stilbene–coumarin
Figure 1G,H) exhibited selective activity against SW480 cells (IC50/48 h = 6.92 and
SI/48 h 400 for compound Figure 1G; IC50/48 h = 1.01 M and SI/48 h = 67.8 for
µM, respectively;
µ
µ
(
µ
µ
µ
1
µ
µ
(
≥
µ
compound Figure 1H). In addition, these compounds were able to preserve their activities
over time. The results achieved by these hybrids are even better than the lead compounds
(
coumarin and resveratrol) and the standard drug (5-FU) [28]. In addition, compound
Figure 1I, a resveratrol–chalcone hybrid, exhibited potent in vitro activity, causing growth
inhibition of HepG2, B16-F10 and A549 cell lines, with IC50 values of 0.2, 0.1 and 1.4 g/mL,
µ
respectively. This hybrid also exhibited significant tubulin polymerization inhibitory activ-
ity at 2.6 µg/mL [29] (Figure 1).
Figure 1. Hybrid molecules derived from curcumin and resveratrol with anticancer activity.

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We synthesized several hybrids based on curcumin and resveratrol (Figure 2), and the
cytotoxicity and antiproliferative effect were determined to identify possible therapeutic
approaches for the treatment of colorectal cancer.
Figure 2. Design of hybrids based on curcumin and resveratrol as anti-cancer agents.
2. Results and Discussion
2.1. Chemistry
In this study, the synthesis of the hybrids began with obtained chalcone 1 by mean the Claisen–
Schmidt condensation and ultrasonic irradiation assisted between 4-bromoacetotophenone and
vanillin (yield 63%) [30]. This compound has already been reported [31], and it was sub-
jected to cross-coupling reaction with various styrenes (
leading to the formation of hybrids 3a 3l with 35–55% yields. To improve these results,
other methodologies were tested such as (Pd(Ph ) , AgOAc, DMF) [33], Pd(AcO) , PPh ,
2) under palladium catalysis [32],
–
3
4
2
3
CH CO Na [34], Pd(AcO) and triethanolamine [28], but these results were not as ex-
3
2
2
pected. Styrenes (
2
) were synthesized via microwave assisted Wittig reaction between
substituted aldehydes and methyltriphenylphosphonium bromide [28
,35] and Knoeve-
nagel condensation reactions between 4-hydroxybenzaldehydes with malonic acid [28
,36
]
(
Scheme 1).
The structures of all compounds were established by a combined study of IR, ESI-MS,
H-NMR, C-NMR and COSY spectra. IR spectra exhibited characteristic absorption
1
13
peaks corresponding to C = O, C = C, C = CAr and C-HAr. ESI-MS spectra showed
+
characteristic [M+H] peaks corresponding to the molecular weights. The signals of H or
1
C atoms were performed using typical
δ
-values and coupling constants (J). The H-NMR
spectra of hybrids dissolved in CDCl showed signals of Ar-CH (~8.00 ppm, d, 2H), Ar-CH
3
(
~7.60 ppm, d, 2H), H-C = C-Htrans (~7.77 and 7.40 ppm) and -OCH (3.80–3.90 ppm).
3
13
C-NMR spectra of hybrids showed signals around 56.28, 128, 129, 145 and 189 ppm,
corresponding to -OCH , -C = C-, Ar-CH (x2), -C = C- and C = O, respectively.
3
2.2. Biological Activity
2
.2.1. Cytotoxic Effect of Hybrids Based on Curcumin and Resveratrol on SW480, SW620
and CHO-K1 Cell Lines
To determine the cytotoxic effect of the synthesized hybrids, they were evaluated
against SW480 human colon adenocarcinoma cells, its metastatic derivative SW620 and
the non-malignant CHO-K1 cell line, through the sulforhodamine B assay. In this exper-
iment, 5-FU was also included as the reference drug, as well as the parental subunits
(curcumin and resveratrol) and the equimolar mixture of these. Cytotoxicity was reported
as 50% inhibitory concentration (IC50 values). All results regarding the cytotoxic effect are
summarized in Table 1.

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Scheme 1. Synthesis of hybrids based on curcumin and resveratrol.

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Table 1. Cytotoxic effect of the hybrids against SW480, SW620 and CHO-K1 cell lines at 24 and 48 h.
2
4 h
48 h
SI
Hybrid
Compound
Structure
CHO-K1
IC50 (µM)
SW480
IC50 (µM)
SW620
IC50 (µM)
CHO-K1
IC50 (µM)
SW480
IC50 (µM)
SW620
IC50 (µM)
SI
SI
SI
3
a
122.43 ± 15.5
95.96 ± 2.76
1.28
165.7 ± 11.78
0.74
51.58 ± 10.46
95.08 ± 7.02
0.54
20.15±1.21
2.56
3
b
N.E.
N.E.
N.A.
N.E.
N.A.
N.E.
N.E.
N.A.
N.E.
N.A.
3
c
>3000
27.49 ± 2.00
34.15 ± 3.92
54.37 ± 6.93
>100
0.91
1.85
>3000
1.00
0.44
1.81
>3000
>200
>15
0.49
1.48
>3000
1.00
0.48
2.12
3
d
31 ± 2.93
100.6 ± 1.00
70.45 ± 3.8
55.46 ± 8.0
12.4 ± 3.41
43.27 ± 6.84
25.22 ± 3.54
29.18 ± 4.36
26.05 ± 4.24
20.44 ± 3.51
3
e
3
f
112.07 ± 15.8
84.37 ± 5.45
1.33
47.44 ± 10.53
2.36
23.89 ± 4.01
88.64 ± 1.84
0.27
27.62 ± 4.99
0.86

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Table 1. Cont.
2
4 h
48 h
SI
Hybrid
Compound
Structure
CHO-K1
IC50 (µM)
SW480
IC50 (µM)
SW620
IC50 (µM)
CHO-K1
IC50 (µM)
SW480
IC50 (µM)
SW620
IC50 (µM)
SI
SI
SI
3
g
94.95 ± 1.67
97.41 ± 2.62
8.55 ± 2.47
11.1
2.06
33.72 ± 4.20
61.03 ± 12.76
2.82
32.38 ± 5.16
23.17 ± 4.07
39.09 ± 5.68
24.92 ± 1.65
0.83
0.93
27.6 ± 5.21
1.17
1.08
3
h
47.21 ± 8.39
1.60
21.36 ± 3.29
3
i
103.96 ± 5.03
42.14 ± 4.85
49.7 ± 4.56
7.98 ± 1.53
2.09
5.28
78.89 ± 6.20
28.11 ± 4.27
1.32
1.50
28.29 ± 9.30
24.28 ± 5.21
11.52 ± 2.78
41.14 ± 7.80
2.46
0.59
29.33 ± 4.73
28.8 ± 4.57
0.96
0.84
3
j
3
k
74.56 ± 4.20
88.5 ± 4.58
0.84
20.42 ± 2.43
34.19 ± 5.09
3.65
29.35 ± 2.29
85.8 ± 1.27
0.34
11.81 ± 1.20
2.49
3
l
89.96 ± 8.55
63.47 ± 8.16
1.42
2.63
24.84 ± 5.53
51.83 ± 5.85
0.48
29.75 ± 3.68
0.83
Curcumin
20.18 ± 2.41
118.4 ± 8.54
42.68 ± 4.03
543.5 ± 52.94
84.64 ± 6.60
153.6 ± 10.64
35.05 ± 2.71
1544 ± 127.9
0.24
0.77
1.22
0.35
163.6 ± 5.88
549 ± 38.5
0.12
0.22
0.38
0.59
36.29 ± 4.17
63.98 ± 8.92
19.93 ± 3.62
173.2 ± 14.61
90.44 ± 1.00
123 ± 4.48
0.40
0.52
0.69
0.99
22.21 ± 2.37
143.1 ± 4.03
25.81 ± 2.76
180.9 ± 18.8
1.63
0.45
0.77
0.96
Resveratrol
Mixture (Curcumin+Resveratrol)
111.2 ± 3.38
28.58 ± 2.67
5-FU
898.8 ± 60.74
174.3 ± 19.1
IC50 values were obtained from dose response curves for each compound. Selectivity index (SI) was calculated by the ratio of IC50 values in non-malignant CHO-K1 cells to IC50 of SW480 or SW620 cells. Data are
presented as the mean ± SE of at least three independent experiments. N.E., Not Evaluated; N.A., Not Applicable.

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Among the tested compounds, it was observed that hybrids 3e and 3i (for SW480),
together with 3a
values ranging from 11.52
,
3e and 3k (for SW620), displayed the best cytotoxic activity with IC50
2.78 to 29.18 4.36 M, after 48 h of treatment. These
±
±
µ
compounds demonstrate the objective of molecular hybridization, improving the biological
activity, since they were more active than parental compounds. They were also more
selective than the equimolar mixture and the reference drug. These results are in accordance
with those previously reported by Castrillon and colleagues (2019) [37], who also reported
cytotoxic and selective activity of different S-allyl cysteine hybrids, using the same model.
Moreover, similar results were reported by Herrera-R et al. (2018) [28], who synthesized
another class of hybrid compounds using different scaffolds as coumarin and stilbene
(
styrylcoumarin), finding better cytotoxic activity and selectivity when tested using an
in vitro model of colorectal cancer (CRC). Furthermore, compounds 3c 3g and 3j decreased
activity after 48 h of treatment, as evidenced by the increase of the IC50 value compared
to 24 h, for both cell lines. On the other hand, although hybrids 3d 3h and 3l improved
,
,
the activity over malignant cells (SW480 and SW620) after 48 h of treatment, a decrease in
selectivity was also observed due to the high toxicity on non-malignant cells. Compound
3b was not successfully evaluated due to solubility problems.
In the structure–activity relationship (SAR) study at 48 h, we noticed a synergistic
action of the parent subunits when they are linked to form a single structure in the hybrid.
The same result was displayed over SW480 cell line by other hybrids [28 37]. When we
compared compounds 3a and 3b with 3c, it was observed that electron donor groups
favored activity unlike electron withdrawing groups. Except for compounds 3d and 3i
all di-oxygenated compounds (3d 3j) showed better activity over SW620 cell line than
SW480 cells. These hybrids exhibited similar activity (20–29 M) on SW620 cell line.
,
,
–
µ
Hybrid 3i, which has oxygenated positions 3 and 4, showed the best activity on SW480.
The 3k hybrid, a combination of compounds 3b and 3h, exhibited a synergistic effect on
SW620 cells reflected in increased activity when compared to initial hybrids. In both mono-
oxygenated and di-oxygenated compounds, it was observed that those with hydroxyl
groups exhibited less activity than methoxylated compounds. The importance of hydroxyl
groups in compounds such as chalcones, coumarins, caffeic acid esters and allylcysteine–
caffeic acid hybrids [38–41] is related with better molecular recognition ability towards
target bioreceptors upon hydrogen bond formation [42], oxidation processes across radical
formation and/or the ability of metal complexation [43]. However, considering that our
compounds with hydroxyl groups were less active, this suggests they could probably have
different mechanism of action. The results of these compounds are in agreement with other
reports for several methoxy derivatives of resveratrol which were evaluated against SW480
or KB lines [44–46].
2
.2.2. Antiproliferative Effect of Hybrids Based on Curcumin and Resveratrol on SW480
and SW620 Cells
With the aim of testing the antiproliferative effect of the most cytotoxic and selective
hybrids (3e and 3i for SW480 and 3a 3e and 3k for SW620), they were analyzed through a
,
longer period. According to the results in comparison with the control, it was observed
that the activity was time- and concentration-dependent, as evidenced by the decrease in
cell viability in the highest concentrations (Table 2). Among the results, it was observed
that all compounds evaluated displayed significant antiproliferative activity and, thus,
viability percentages were reduced (p values < 0.01) in both SW480 and SW620 cells after
four days of treatment. Besides, in SW620 cells, hybrids 3e and 3k displayed signifi-
cant reduction in viability, after just two days of treatment with concentrations higher
than 10 µM (Figure 3A,B). In addition, when treated cells were observed with optical
microscope, the cellular morphology was severely perturbed, exhibiting changes in size
and shape after treatment. Similar results were presented in a previous investigation by
Herrera-R et al. (2018) [28], reporting the antiproliferative effect and morphological changes
in SW480 cells after treatment with different hybrids using a stilbene moiety, which high-

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lights the potential of this scaffold in the approach to design molecules with potential
activity against CRC.
Table 2. Data of viability (%) at the lowest and the highest concentrations evaluated.
Time After Plating (Days)
Cell Line
Compound
Concentration (µM)
2
4
6
8
Viability (%) ± SE
0
53.9 ± 5.3
32.9 ± 1.3
2.3 ± 0.3
3.8 ± 2.7
5.2 ± 0.3
0.1 ± 0.1
Hybrid 3e
Hybrid 3i
Hybrid 3a
Hybrid 3e
Hybrid 3k
0.9 ± 0.2
0.5 ± 0.2
SW480
0
54.4 ± 7.4
2.0 ± 0.2
1.6 ± 0.2
0.1 ± 0.1
1.6 ± 0.3
30.3 ± 8.5
1.7 ± 0.1
0.2 ± 0.1
71.9 ± 1.9
16.1 ± 0.0
10.7 ± 4.7
2.5 ± 0.5
0.2 ± 0.3
0
0
0
0
80.6 ± 4
11.1 ± 4.2
4.1 ± 2.2
0.6 ± 0.5
SW620
5.0 ± 1
4.1 ± 0.0
0
0
0
56.1 ± 4.0
2.4 ± 0.7
12.1 ± 0.0
1.9 ± 0.8
0.1 ± 0.1
0
0
0
Figure 3. Antiproliferative effect of hybrids based on curcumin and resveratrol against: SW480 cell line (
metastatic derivative SW620 cell line ( ). Data are presented as the mean
* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). Vehicle control was assumed as 100% of viability.
A
); and its
B
±
SE of at least three independent experiments
(

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3. Conclusions
We showed for the first time that the synthesized hybrids based on curcumin and
resveratrol have chemopreventive potential in an in vitro model of CRC (using two dif-
ferent adenocarcinoma cells), involving selective growth inhibition and antiproliferative
activity, with changes in size and shape after treatment, better results than parental com-
pounds and exhibiting higher selectivity than the conventional chemotherapeutic (5-FU)
and the equimolar mixture, highlighting the potential of the strategy based on molecular
hybridization. Our findings suggest that these hybrid molecules could be promising chemo-
preventive agents against colorectal cancer, thus it is necessary to carry out further studies.
4
4
4
. Experimental Section
.1. Chemical Synthesis
.1.1. General Remarks
Microwave reactions were carried out in a CEM Discover microwave reactor in sealed
vessels (monowave, maximum power 300 W, temperature control by IR sensor and fixed
temperature). IR spectra were recorded on a Spectrum RX I FT-IR system (Perkin-Elmer,
1
13
Waltham, MA, USA) in KBr disks. H- and C-NMR spectra were recorded on a Varian
instrument operating at 300 and 75 MHz, respectively. The signals of the deuterated solvent
(
CDCl or DMSO-D ) were used as reference. Chemical shifts (
δ
) are expressed in ppm
with the solvent peak as reference and TMS as an internal standard; coupling constants (J)
are given in Hertz (Hz). Carbon atom types (C, CH, CH and CH ) were determined using
3
6
2
3
the DEPT pulse sequence. HRMS was obtained using a Bruker Impact II UHR-Q-TOF
mass spectrometry (Bruker Daltonik GmbH, Bremen Germany) in positive mode. Silica
gel 60 (0.063–0.200 mesh, Merck, Whitehouse Station, NJ, USA) was used for column
chromatography and precoated silica gel plates (Merck 60 F254 0.2 mm) were used for thin
layer chromatography (TLC). Monitoring of the reaction progress and product purification
was carried out by TLC.
4
.1.2. General Procedure for the Synthesis of
(
E)-1-(4-Bromophenyl)-3-(4-Hydroxy-3-Methoxyphenyl)prop-2-en-1-one (1)
This compound was prepared by the condensation of 4-bromoacetophenone 1 (10 mmoL,
2
.0 g) and vanillin 2 (10 mmoL, 1.52 g) in a solution of 20% KOH in ethanol (20 mL). The
reaction mixture was sonicated for 1 h and neutralized with a solution of 10% HCl. The
solid was filtered, sequentially washed with water, dried and recrystallized from ethanol
to obtain the corresponding chalcone in 63% yield (6.3 mmoL, 2.1 g).
4.1.3. Styrene Synthesis
Method 1: Wittig Reaction under microwave conditions.
Potassium carbonate (0.10 mol) and (ethyl)triphenylphosphonium iodide (0.016 mol)
were mixed and crushed in a mortar to a fine powder and transferred into a 250 mL Erlen-
meyer flask. Aldehyde (0.016 mol) was added to the powdered mixture and thoroughly
mixed. The Erlenmeyer flask was covered with a watch glass and placed into a microwave
along with a 1 L beaker of ice (to ensure that the reaction mixture did not overheat). The
microwave was run at full power for 12 min, making sure to stop the heating and replace
the ice when it had melted. A tiny sample of the crude reaction product was removed,
diluted in a small amount of hexanes and analyzed by TLC to check the reaction progress.
Upon completion, the crude solids were extracted with hexanes (3
×
20 mL). The com-
bined hexane extracts were placed in a round-bottomed flask and concentrated by rotary
evaporation to obtain a yellow oil which was purified by flash chromatography on silica
gel using hexanes or a mixture of hexanes/ethyl acetate as eluent.
Method 2: Knoevenagel condensation reactions:
Piperidine (0.75 mL, 7.6 mmoL) was added to a solution of aldehyde (5 mmoL) and
malonic acid (20 mmoL) in pyridine (21 mL). The mixture was heated at reflux for a
period of 30 min under microwave irradiation. Then, toluene (40 mL) was added to

Molecules 2021, 26, 2661
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◦
the cooled reaction mixture and the solvent volume reduced in vacuum at 30–40 C.
Additional toluene (20 mL) was then added and the solvent again removed in vacuum to
eliminate all traces of pyridine. This procedure afforded a residue which was purified by
column chromatography over silica gel. Elution with 9:1 hexane/ethyl acetate afforded the
corresponding vinylphenol.
4.1.4. General Procedure for the Mizoroki-Heck Reactions
Styrene (0.3 mmoL), bromochalcone (0.3 mmoL, 100 mg), pyrrolidine (0.6 mmoL,
5
0 µL), Pd(OAc) (6 mg, 0.027 mmoL), LiCl (12 mg, 0.28 mmoL) and DMF (1 mL) were
2
placed in a 10 mL flat-bottomed flask equipped with a magnetic stirring bar. The mixture
◦
was stirred and heated to 170 C at 200 W for a period of 4 min, under microwave irradiation.
The mixture of reaction was allowed to cool, and then 100 mL of HCl 5% were added. Then,
this mixture was extracted with dichloromethane, concentrated and the crude residue was
purified by column chromatography (silica gel, hexane-ethyl acetate mixture) affording the
coupling products in 35–55% yield.
(
E)-3-(4-Hydroxy-3-methoxyphenyl)-1-(4-((E)-4-hydroxystyryl)phenyl)prop-2-en-1-one (3a)
◦
−1
Yield 20%; m.p. 119–120 C; IR (cm ): νmax 3271 (OH), 1645 (C = O), 1593 (C = C),
1
1
512 (C = C ), 1267 (C-O-C). H-NMR (300 MHz, DMSO-d6)
δ
8.13 (d, J = 8.1 Hz, 2H),
Ar
7
7
.80–7.62 (m, 4H), 7.53–7.45 (m, 3H), 7.35 (d, J = 16.4 Hz, 1H), 7.27 (dd, J = 8.1, 2.0 Hz, 1H),
.13 (d, J = 16.4 Hz, 1H), 6.80 (dd, J = 8.1, 2.0 Hz, 3H), 3.87 (s, 3H). 1 C-NMR (75 MHz,
188.32 (C = O), 158.51 (Ar-O), 151.50 (Ar-O),148.72 (Ar-O), 145.26, 142.52
C = C), 136.69 (Ar), 131.72 (Ar), 129.44 (2C Ar), 128.87 (2C Ar), 128.09 (Ar), 126.57 (2C Ar),
3
DMSO-d6)
(
δ
1
5
26.01 (Ar), 124.97 (Ar), 124.51 (C = C), 118.40 (Ar), 116.23 (Ar), 116.16 (2C Ar), 112.03 (Ar),
+
+
6.23 (OCH ). HRMS-ESI (m/z): 373,1435 [M+H] calcd. for C H O [M + H] 373,1361
3
24 21
4
(
E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-((E)-4-methoxystyryl)phenyl)prop-2-en-1-one (3b)
◦
−1
Yield 50%; m.p. 187–190 C; IR (cm ): νmax 3248 (OH), 1641 (C = O), 1568 (C = C),
1
1
452 (C = CAr), 1263 (C-O-C); H-NMR (300 MHz, DMSO-d6)
δ
8.15 (d, J = 8.2 Hz, 2H), 7.78
(
2
d, J = 15.4 Hz, 1H), 7.74 (d, J = 8.2 Hz, 2H), 7.69 (d, J = 15.4 Hz, 1H), 7.61 (d, J = 8.6 Hz,
H), 7.52 (d, J = 1.4 Hz, 1H), 7.41 (d, J = 16.5 Hz, 1H), 7.30 (dd, J = 8.2, 1.4 Hz, 1H), 7.22 (d,
J = 16.5 Hz, 1H), 6.98 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.2 Hz, 1H), 3.88 (s, 3H), 3.80 (s, 3H).
1
3
C-NMR (75 MHz, DMSO)
δ 188.42 (C = O), 159.90 (Ar-O), 150.45 (Ar-O), 148.50 (Ar-O),
145.16 (C = C), 142.35 (Ar), 136.78 (Ar), 131.31 (C = C), 129.76 (C = C), 129.47 (2C Ar), 129.05
(Ar), 128.75 (2C Ar), 126.73 (Ar), 126.62 (Ar), 125.58 (Ar), 124.70 (C = C), 118.90 (Ar), 116.07
(
3
Ar), 114.73 (2C Ar), 114.36 (Ar), 112.12 (Ar), 56.28 (OCH ), 55.66 (OCH ). HRMS-ESI (m/z):
3
3
+
+
87,1589 [M+H] calcd. for C H O [M + H] 387.1518
25 23 4
(
E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-((E)-4-nitrostyryl)phenyl)prop-2-en-1-one (3c)
◦
−1
Yield 35%; m.p. 227–230 C; IR (cm ): νmax 3232 (OH), 1643 (C = O), 1556 (C = C),
1
1
452 (C = CAr), 1303 (NO ), 1276 (C-O-C); H-NMR (300 MHz, DMSO-d6)
δ
8.13 (d, J = 8.4
2
Hz, 2H), 7.81 (d, J = 15.5 Hz, 1H), 7.73–7.63 (m, 3H), 7.53 (d, J = 1.5 Hz, 1H), 7.49 (d, J = 8.8
Hz, 2H), 7.34 (d, J = 16.6 Hz, 1H), 7.30 (dd, J = 8.1, 1.5 Hz, 1H), 7.08 (d, J = 16.6 Hz, 1H),
.85 (d, J = 8.1 Hz, 1H), 6.73 (d, J = 8.8 Hz, 2H), 3.88 (s, 3H). 1 C-NMR (75 MHz, DMSO)
3
δ
6
1
88.30 (C = O), 150.81 (Ar-O), 150.13 (Ar-O), 148.45 (Ar-NO ), 144.93 (C = C), 145.02 (Ar),
2
142.99 (Ar), 136.17 (Ar), 132.13 (Ar), 129.50 (C = C), 128.55 (2C Ar), 126.83 (C = C), 126.42
(
Ar), 126.31 (Ar), 124.84 (Ar), 124.58 (C = C), 122.81 (Ar), 119.07 (Ar), 116.04 (Ar), 112.58 (2C
+
Ar), 112.11 (Ar), 56.29 (OCH ). HRMS-ESI (m/z): 402,1977 [M+H] calcd. for C H NO
[
3
24 20
5
+
M + H] 402.1263
E)-1-(4-((E)-2,3-Dimethoxystyryl)phenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-
-one (3d)
(
1
◦
−1
Yield 55%; m.p. 63–65 C; IR (cm ): νmax 3359 (OH), 1651 (C = O), 1575 (C = C), 1510
1
(
C = C ), 1267 (C-O-C); H-NMR (600 MHz, CDCl )
δ
8.03 (d, J = 8.1 Hz, 2H), 7.77 (d,
Ar
3
J = 15.5 Hz, 1H), 7.66 (d, J = 8.1 Hz, 2H), 7.59 (d, J = 16.4 Hz, 1H), 7.40 (d, J = 15.5 Hz, 1H),
.27 (d, J = 8.3 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 7.18 (d, J = 16.4 Hz, 1H), 7.14 (s, 1H), 7.08 (t,
J = 8.0 Hz), 6.96 (d, J = 8.3 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 3.97 (OCH ), 3.90 (OCH ), 3.88
7
3
3
(
OCH3). 13C-NMR (75 MHz, Chloroform-d)
δ 189.79 (C = O), 153.17 (Ar-O), 148.36 (Ar-O),

Molecules 2021, 26, 2661
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147.27 (Ar-O), 146.87 (Ar-O), 145.07 (C = C), 142.00 (Ar), 137.32 (Ar), 131.03 (Ar), 129.06 (2C
Ar), 128.84 (C = C), 127.59 (Ar), 126.75 (2C Ar), 125.61 (C = C), 124.28 (C = C), 123.45 (Ar),
1
5
19.71 (Ar), 117.99 (Ar), 114.95 (Ar), 111.99 (Ar), 110.07 (Ar), 61.29 (OCH ), 56.07 (OCH ),
3 3
+
+
5.89 (OCH ). HRMS-ESI (m/z): 417,1696 [M+H] calcd. for C H O [M + H] 417.1623
3
26 25
5
(
E)-1-(4-((E)-2,4-Dimethoxystyryl)phenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-
1
-one (3e)
◦
−1
Yield 40%; m.p. 78–82 C; IR (cm ): νmax 3349 (OH), 1649 (C = O), 1575 (C = C), 1508
1
(C = C ), 1259 (C-O-C); H-NMR (600 MHz, CDCl )
δ
7.99 (d, J = 7.9 Hz, 2H), 7.76 (d,
Ar
3
J = 15.5 Hz, 1H), 7.60 (d, J = 7.9 Hz, 2H), 7,53 (d,1H, J = 15.5 Hz), 7,53 (d, 1H, J = 8.5 Hz),
.40 (d, J = 15.5 Hz, 1H), 7.22 (d, J = 7.8 Hz, 1H), 7.13 (s, 1H), 7.06 (d, J = 15.5 Hz, 1H), 6.95
d, J = 7.9 Hz, 1H), 6.53 (d, J = 8.5 Hz, 1H), 6.48 (s, 1H), 4.60 (OH), 3.95 (OCH ), 3.88 (OCH ),
7
(
3
3
3
.84 (OCH3). 13C-NMR (75 MHz, Chloroform-d)
δ 189.77 (C = O), 161.13 (Ar-O), 158.42
(
Ar-O), 148.31 (Ar-O), 146.88 (Ar-O), 144.85 (C = C), 142.85 (Ar), 136.63 (Ar), 129.04 (2C
Ar), 127.69 (C = C), 127.64 (Ar), 126.31 (2C Ar), 126.09 (C = C), 125.80 (C = C), 123.37 (Ar),
19.74 (Ar), 118.98 (Ar), 114.96 (Ar), 110.12 (Ar), 105.17 (Ar), 98.51 (Ar), 56.06 (OCH ), 55.59
1
3
+
+
(OCH ), 55.50 (OCH ). HRMS-ESI (m/z): 417,1696 [M+H] calcd. for C H O [M + H]
3
3
26 25
5
417.1623
(
E)-1-(4-((E)-2,5-Dimethoxystyryl)phenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-
1
1
-one (3f)
◦
−1
Yield 38%; m.p. 47–51 C; IR (cm ): νmax 3379 (OH), 1651 (C = O), 1577 (C = C),
508 (C = C ), 1265 (C-O-C); H-NMR (300 MHz, CDCl )
1
δ
8.01 (d, J = 8.3 Hz, 2H), 7.77
Ar
3
(d, J = 15.6 Hz, 1H), 7.63 (d, J = 8.3 Hz, 2H), 7.56 (d, J = 15.6 Hz, 1H), 7.40 (d, J = 15.5 Hz,
1
H), 7.22 (d, J = 7.2 Hz, 1H), 7.18–7.08 (m, 3H), 6.96 (d, J = 8.2 Hz, 1H), 6.84 (s, 2H), 6.18 (s,
13
OH), 3.94 (OCH ), 3.85 (OCH ), 3.82 (OCH ). C-NMR (75 MHz, Chloroform-d)
δ 189.78
3
3
3
(
(
C = O), 153.74 (Ar-O), 151.73 (Ar-O), 148.38 (Ar-O), 146.91 (Ar-O), 145.05 (C = C), 142.18
Ar), 137.10 (Ar), 129.03 (2C Ar), 128.19 (C = C), 127.57 (Ar), 126.67 (2C Ar), 126.58 (C = C),
1
1
25.99 (C = C), 123.41 (Ar), 119.62 (Ar), 114.98 (Ar), 114.54 (Ar), 112.31 (Ar), 111.81 (Ar),
+
10.16 (Ar), 56.25 (OCH ), 56.04 (OCH ), 55.85 (OCH ). HRMS-ESI (m/z): 417,1696 [M+H]
3
3
3
+
calcd. for C H O [M + H] 417.1623
26
25
5
(
E)-1-(4-((E)-2,6-Dimethoxystyryl)phenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-
1
1
-one (3g)
◦
−1
Yield 35%; m.p. 87–90 C; IR (cm ): νmax 3365 (OH), 1649 (C = O), 1573 (C = C),
510 (C = C ), 1249 (C-O-C); H-NMR (300 MHz, CDCl )
1
δ
8.01 (d, J = 8.0 Hz, 2H), 7.76
Ar
3
(d, J = 15.6 Hz, 1H), 7.60–7.66 (m, 4H), 7.41 (d, J = 15.6 Hz, 1H), 7.25–7.17 (m, 2H), 7.14 (s,
1
H), 6.96 (d, J = 8.2 Hz, 1H), 6.60 (d, J = 8.3 Hz, 2H), 6.12 (s, OH), 3.95 (s, 3H), 3.91 (s, 6H).
13
C-NMR (75 MHz, Chloroform-d)
Ar-O), 144.73 (C = C), 143.92 (Ar), 136.64 (Ar), 131.13 (C = C), 128.94 (2 Ar), 128.06 (Ar),
27.69 (Ar), 126.44 (2 Ar), 123.33 (C = C), 122.74 (Ar), 119.87 (C = C), 114.89 (Ar), 114.28
Ar), 110.06 (Ar), 103.97 (2 Ar), 56.06 (OCH ), 55.88 (2 OCH ). HRMS-ESI (m/z): 417,1696
δ 189.86 (C = O), 158.91 (2 Ar-O), 148.20 (Ar-O), 146.82
(
1
(
[
3
3
+
+
M+H] calcd. for C H O [M+H] 417.1623
26
25
5
(
E)-1-(4-((E)-3,5-Dimethoxystyryl)phenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-
1
1
-one (3h)
◦
−1
Yield 35%; m.p. 49–51 C; IR (cm ): νmax 3388 (OH), 1651 (C = O), 1577 (C = C),
508 (C = C ), 1267 (C-O-C); H-NMR (300 MHz, CDCl )
1
δ
8.00 (d, J = 8.4 Hz, 2H), 7.76
Ar
3
(d, J = 15.5 Hz, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 15.5 Hz, 1H), 7.21 (dd, J = 8.2,
2
.0 Hz, 1H), 7.16–7.08 (m, 3H), 6.96 (d, J = 8.2 Hz, 1H), 6.69 (d, J = 2.2 Hz, 3H), 6.42 (tapp
,
13
J = 2.2 Hz, 2H), 3.93 (OCH ), 3.82 (2 OCH ). C-NMR (75 MHz, Chloroform-d) δ 189.69
3
3
(C = O), 161.04 (2 Ar-O), 148.49 (Ar-O), 146.97 (Ar-O), 145.18 (C = C), 141.43 (Ar), 138.77
(Ar), 137.34 (Ar), 131.26, 129.07 (2C Ar), 128.09 (C = C), 127.49 (C = C), 126.66 (2C Ar), 123.43
(
5
C = C), 119.46 (Ar), 115.04 (Ar), 110.24 (Ar), 104.91 (2C Ar), 100.59 (Ar), 56.04 (OCH ),
3
+
+
5.44 (2 OCH ). HRMS-ESI (m/z): 417,1696 [M+H] calcd. for C H O [M + H] 417.1623
3 26 25 5
(
E)-1-(4-((E)-3,4-dimethoxystyryl)phenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-
1-one (3i)

Molecules 2021, 26, 2661
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◦
−1
Yield 55%; m.p. 67–70 C; IR (cm ): νmax 3390 (OH), 1649 (C = O), 1577 (C = C), 1508
1
(
C = C ), 1265 (C-O-C); H-NMR (600 MHz, CDCl )
δ
8.01 (d, J = 8.3 Hz, 2H), 7.77 (d,
Ar
3
J = 15.5 Hz, 1H), 7.60 (d, J = 8.3 Hz, 2H), 7.40 (d, J = 15.5 Hz, 1H), 7.23 (dd, J = 8.2, 1.5 Hz,
1
H), 7.19 (d, J = 16.2 Hz, 1H), 7.13 (d, J = 1.5 Hz, 1H), 7.11–7.07 (m, 2H), 7.02 (d, J = 16.2 Hz,
H), 6.96 (d, J = 8.2 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 3.96 (OCH ), 3.96 (OCH ), 3.91 (OCH ).
1
3
3
3
13
C-NMR (75 MHz, Chloroform-d)
δ 189.68 (C = O), 149.51 (Ar-O), 149.22 (Ar-O), 148.32
(
Ar-O), 146.86 (Ar-O), 145.00 (C = C), 141.95 (Ar), 136.98 (Ar), 131.17 (Ar), 129.89 (C = C),
1
1
29.10 (2C Ar), 127.60 (C = C), 126.33 (2C Ar), 125.67 (Ar), 123.37 (C = C), 120.56 (Ar), 119.64,
14.94 (Ar), 111.22 (Ar), 110.16 (Ar), 108.85 (Ar), 56.07 (OCH ), 56.01 (OCH ), 55.95 (OCH ).
3
3
3
+
+
HRMS-ESI (m/z): 417,1696 [M+H] calcd. for C H O [M + H] 417.1623
26
25
5
(E)-3-(4-Hydroxy-3-methoxyphenyl)-1-(4-((E)-4-hydroxy-3-methoxystyryl)phenyl)prop-
2
-en-1-one (3j)
◦
−1
Yield 21%; m.p. 142–146 C; IR (cm ): νmax 3232 (OH), 1641 (C = O), 1589 (C = C),
1
1
508 (C = CAr), 1269 (C-O-C); H-NMR (300 MHz, CDCl )
δ
8.01 (d, J = 8.3 Hz, 2H), 7.77 (d,
3
J = 15.5 Hz, 1H), 7.58 (d, J = 8.3 Hz, 2H), 7.40 (d, J = 15.5 Hz, 1H), 7.22 (dd, J = 8.3, 1.9 Hz,
H), 7.15–7.10 (m, 1H), 7.08–7.03 (m, 2H), 6.99 (d, J = 9.8 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H),
.10 (s, OH), 5.88 (s, OH), 3.95 (s, 2 OCH3). 13C-NMR (75 MHz, Chloroform-d)
189.70
1
6
δ
(
(
C = O), 148.34 (Ar-O), 146.88 (Ar-O), 146.86 (Ar-O), 146.25 (Ar-O), 145.03 (C = C), 142.01
Ar), 136.87 (Ar), 131.32 (Ar), 129.45 (C = C), 129.10 (2C Ar), 127.58 (C = C), 126.27 (2C Ar),
1
1
25.32 (C = C), 123.36 (Ar), 121.08 (Ar), 119.57 (Ar), 114.96 (Ar), 114.75 (Ar), 110.19 (Ar),
+
08.48 (Ar), 56.06 (OCH ), 55.98 (OCH ). HRMS-ESI (m/z): 403,1540 [M+H] calcd. for
3
3
+
C H O [M + H] 403.1467
25
23
5
(E)-3-(4-Hydroxy-3-methoxyphenyl)-1-(4-((E)-3,4,5-trimethoxystyryl)phenyl)prop-2-en-
1
1
-one (3k)
◦
−1
Yield 55%; m.p. 74–77 C; IR (cm ): νmax 3390 (OH), 1651 (C = O), 1579 (C = C),
504 (C = C ), 1265 (C-O-C); H-NMR (600 MHz, CDCl )
1
δ
8.03 (d, J = 8.2 Hz, 2H), 7.77
Ar
3
(d, J = 15.6 Hz, 1H), 7.62 (d, J = 8.1 Hz, 2H), 7.41 (d, J = 15.6 Hz, 1H), 7.24 (dd, J = 8.2,
1
6
.9 Hz, 1H), 7.17 (d, J = 16.2 Hz, 1H), 7.14 (d, J = 1.9 Hz, 1H), 7.06 (d, J = 16.2 Hz, 1H),
1
3
.97 (d, J = 8.2 Hz, 1H), 6.78 (s, 2H), 3.97 (OCH ), 3.93 (2 OCH ), 3.89 (OCH ). C-NMR
3
3
3
(75 MHz, Chloroform-d)
δ
189.69 (C = O), 153.51 (2 Ar-O), 148.35 (Ar-O), 146.87 (Ar-O),
1
45.12 (C = C), 141.56 (Ar), 137.28 (Ar-O), 132.54 (Ar), 131.28 (Ar), 129.11 (2C Ar), 127.57
(
C = C), 127.09 (C = C), 126.51 (2C Ar), 123.37 (C = C), 119.61 (Ar), 114.95 (Ar), 110.18 (Ar),
1
03.90 (2C Ar), 61.06 (OCH ), 56.20 (OCH ), 56.08 (OCH ). HRMS-ESI (m/z): 447,1803
3
3
3
[
M+H]+ calcd. for C H O : 447.1729
27 27 6
(
E)-1-(4-((E)-4-Hydroxy-3,5-dimethoxystyryl)phenyl)-3-(4-hydroxy-3-methoxyphenyl)
prop-2-en-1-one (3l)
◦
−1
Yield 40%; m.p. 86–88 C; IR (cm ): νmax 3332 (OH), 1641 (C = O), 1589 (C = C), 1508
1
(
C = C ), 1269 (C-O-C); H-NMR (300 MHz, CDCl )
δ
8.02 (d, J = 8.3 Hz, 2H), 7.77 (d,
Ar
3
J = 15.6 Hz, 1H), 7.59 (d, J = 8.3 Hz, 2H), 7.40 (d, J = 15.6 Hz, 1H), 7.19–7.12 (dd, J = 8.3,
.8 Hz, 1H), 7.16 (d, J = 16.0 Hz, 1H), 7.14–7.11 (m, 1H), 7.00 (d, J = 16.0 Hz, 2H), 6.95
d, J = 8.3 Hz, 1H), 6.79 (s, 2H), 6.02 (s, OH), 5.69 (s, OH), 3.96 (OCH ), 3.95 (2 OCH ).
1
(
3
3
1
3
C-NMR (75 MHz, Chloroform-d)
δ 189.64 (C = O), 148.31 (Ar-O), 147.29 (2 Ar-O), 146.85
(Ar-O), 145.02 (C = C), 141.82 (Ar-O), 136.99 (Ar), 135.41 (Ar), 131.49 (Ar), 129.10 (2C Ar),
128.42 (C = C), 127.59 (C = C), 126.30 (2C Ar), 125.69 (Ar), 123.34 (C = C), 119.59 (Ar), 114.93
(
[
Ar), 110.17 (Ar), 103.69 (2C Ar), 56.38 (2 OCH ), 56.07 (OCH ). HRMS-ESI (m/z): 433,1646
3
3
+
M+H] calcd. for C H O : 433.1572.
26
25
6
4.2. Biological Activity Assays
4.2.1. Cell Lines and Culture Medium
Biological assays were performed using an adenocarcinoma colon cancer cell line
SW480), its metastatic derivative (SW620) and non-malignant cells (CHO-K1). These were
(
obtained from The European Collection of Authenticated Cell Cultures (ECACC, England)
and maintained in Dulbecco’s Modified Eagle Medium, supplemented with 10% heat-
◦
inactivated (56 C) horse serum, 1% penicillin/streptomycin and 1% non-essential amino

Molecules 2021, 26, 2661
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acids (Gibco Invitrogen, Carlsbad, USA). For all experiments, horse serum was reduced to
3
%, and the medium was supplemented with 5 mg/mL transferrin, 5 ng/mL selenium
and 10 mg/mL insulin (ITS-defined medium; Gibco, Invitrogen, Carlsbad, CA, USA) [28].
4.2.2. Cytotoxic Activity
Cytotoxicity of the synthesized hybrids, lead and reference compounds was evaluated
through Sulforhodamine B (SRB) assay, a colorimetric test that is based on staining of total
cellular protein of adherent cells. Cells were seeded to a final density of 20,000 cells/well
◦
in 96-well tissue culture plates and incubated at 37 C in a humidified atmosphere at 5%
CO . All cultures were allowed to grow for 24 h, and afterward they were treated with
2
DMSO (vehicle control 1%) or increasing concentrations (0.01–200 µM) of the synthesized
hybrids, as well as curcumin and resveratrol (lead compounds), the equimolar mixture
of these and 5-fluorouracil (5-FU; the standard drug). After treatment, cells were fixed
◦
with trichloroacetic acid (50% v/v) (MERCK) for a period of 1 h at 4 C. Cell proteins were
determined by staining with 0.4% (w/v) SRB (Sigma-Aldrich, United States), and then they
were washed with 1% acetic acid for the removal of unbound SRB and left for air-drying.
Protein bound SRB was solubilized in 10 mM Tris-base and the absorbance was measured
at 492 nm in a microplate reader (Mindray MR-96A) [46]. All experiments were performed
at least in triplicate.
4.2.3. Antiproliferative Activity
Antiproliferative effect of the most active compounds was also tested through Sul-
forhodamine B (SRB) assay. Briefly, cells were seeded to a final density of 2500 cells/well
in 96-well tissue culture plates and incubated in the same conditions described for cyto-
toxicity. Cultures were allowed to grow for 24 h and then were treated with increasing
concentrations of the selected hybrids (10–160
or DMSO (vehicle control, 1%), for 0, 2, 4, 6 and 8 days. Culture media was replaced every
8 h. After each incubation time, cells were fixed, stained and read as previously described
for this technique [37].
µM, ranges dependent on the IC50 values)
4
4.2.4. Statistical Analysis
All experiments were performed at least three times. Data are reported as mean
standard error). Statistical differences between control group (non-treated) and treated
±
SE
(
cells were evaluated by one-way ANOVA followed by the Dunnett’s test. Values with
p
≤
0.05 were considered significant. Data were analyzed with GraphPad Prism version
7.04 for Windows (Graph Pad Software, San Diego, CA, USA).
Author Contributions: C.H.: Synthesis of hybrid molecules. G.M.: evaluation of biological activities,
formal analysis, investigation. A.H.-R.: Conceptualization, Methodology, Validation, evaluation of
biological activities, formal Analysis, Investigation, Writing—Original Draft, Writing—Review &
Editing. W.C.-G.: Conceptualization, Methodology, Resources, Writing—Original Draft, Writing—
Review & Editing, Supervision, Project Administration, Funding Acquisition. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was funded by University of Antioquia and MINCIENCIAS, through the Pro-
gram Ecosistema Científico (Alianza NanoBioCancer Cod. FP44842-211-2018 project number 58537).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments: The work was funded by University of Antioquia and MINCIENCIAS, through the
Program Ecosistema Científico (Alianza NanoBioCancer Cod. FP44842-211-2018 project number 58537).
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the hybrid compounds based on curcumin and resveratrol are
available from the authors.

Molecules 2021, 26, 2661
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References
1
2
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