1Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 29 | Number 03 | Article 347295
The luminescent Chalcones. Its potential use as a luminescent and antitumoral agents
JOURNAL VITAE
School of Pharmaceutical and
Food Sciences
ISSN 0121-4004 | ISSNe 2145-2660
University of Antioquia
Medellin, Colombia
Filliations
1Dirección de Investigación y
Postgrado, Universidad de Aconcagua,
Vitacura, Chile
2Departamento de Ciencias Biológicas
y Químicas, Facultad de Medicina y
Ciencia, Universidad San Sebastián,
Campus Los Leones, Lota 2465,
Providencia, Santiago, Chile.
3Facultad de Medicina, Universidad
de Atacama, Copayapu 485, 1531772
Copiapó, Chile.
*Corresponding
Rodrigo Ramirez-Tagle
rodrigoramireztagle@gmail.com
Received: 23 August 2021
Accepted: 13 August 2022
Published: 31 August 2022
The luminescent Chalcones. Its potential
use as a luminescent and antitumoral
agents
Chalconas luminiscentes. Su uso potencial como agentes
luminiscentes y antitumorales
Rodrigo Ramirez-Tagle1 , Leonor Alvarado-Soto1, Carlos A. Escobar2 ,
Cesar Echeverria-Echeverria 3
ABSTACT
Background: Hepatocellular carcinoma (HCC) is one of the most diagnosed cancers
worldwide. Chemoprevention of HCC can be achieved using natural or synthetic compounds
that reverse, suppress, detect, or prevent cancer progression. Objectives: In this study,
both the antiproliferative effects and luminescent properties of 2’-hydroxychalcones were
evaluated. Methods: Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) colorimetric assay, spectroscopy assays, and density
functional theory (DFT) calculations were used to determine the luminescent properties of
2´-hydroxychalcones. Results: Cytotoxic effects of 2´-hydroxychalcones were observed over
the HepG2 and EA.hy926 cells. Since the chalcone moiety could be used as a fluorescent
probe, these compounds may be helpful in cancer diagnosis and tumor localization. They
may enable tumor observation and regression through the fluorescence during treatment;
therefore, the compounds are a potential candidate as novel anticancer agents acting on human
hepatomas. Conclusions: This report describes the chalcones’ use as a specific luminescent
biomarker in tumor cells. We also report the cellular uptake of 2’-hydroxychalcones, their
cellular distribution, and the mechanisms that may be responsible for their cytotoxic effects.
Keywords: 2’-hydroxychalcones; anticancer agent; luminescent biomarker; Cytotoxic effects
ORIGINAL RESEARCH
Published 31 August 2022
Doi: https://doi.org/10.17533/udea.vitae.v29n3a347295
The luminescent Chalcones. Its potential use as a luminescent and antitumoral agents
JOURNAL VITAE
School of Pharmaceutical and
Food Sciences
ISSN 0121-4004 | ISSNe 2145-2660
University of Antioquia
Medellin, Colombia
Filliations
1Dirección de Investigación y
Postgrado, Universidad de Aconcagua,
Vitacura, Chile
2Departamento de Ciencias Biológicas
y Químicas, Facultad de Medicina y
Ciencia, Universidad San Sebastián,
Campus Los Leones, Lota 2465,
Providencia, Santiago, Chile.
3Facultad de Medicina, Universidad
de Atacama, Copayapu 485, 1531772
Copiapó, Chile.
*Corresponding
Rodrigo Ramirez-Tagle
rodrigoramireztagle@gmail.com
Received: 23 August 2021
Accepted: 13 August 2022
Published: 31 August 2022
The luminescent Chalcones. Its potential
use as a luminescent and antitumoral
agents
Chalconas luminiscentes. Su uso potencial como agentes
luminiscentes y antitumorales
Rodrigo Ramirez-Tagle1 , Leonor Alvarado-Soto1, Carlos A. Escobar2 ,
Cesar Echeverria-Echeverria 3
ABSTACT
Background: Hepatocellular carcinoma (HCC) is one of the most diagnosed cancers
worldwide. Chemoprevention of HCC can be achieved using natural or synthetic compounds
that reverse, suppress, detect, or prevent cancer progression. Objectives: In this study,
both the antiproliferative effects and luminescent properties of 2’-hydroxychalcones were
evaluated. Methods: Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) colorimetric assay, spectroscopy assays, and density
functional theory (DFT) calculations were used to determine the luminescent properties of
2´-hydroxychalcones. Results: Cytotoxic effects of 2´-hydroxychalcones were observed over
the HepG2 and EA.hy926 cells. Since the chalcone moiety could be used as a fluorescent
probe, these compounds may be helpful in cancer diagnosis and tumor localization. They
may enable tumor observation and regression through the fluorescence during treatment;
therefore, the compounds are a potential candidate as novel anticancer agents acting on human
hepatomas. Conclusions: This report describes the chalcones’ use as a specific luminescent
biomarker in tumor cells. We also report the cellular uptake of 2’-hydroxychalcones, their
cellular distribution, and the mechanisms that may be responsible for their cytotoxic effects.
Keywords: 2’-hydroxychalcones; anticancer agent; luminescent biomarker; Cytotoxic effects
ORIGINAL RESEARCH
Published 31 August 2022
Doi: https://doi.org/10.17533/udea.vitae.v29n3a347295
2Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 29 | Number 03 | Article 347295Rodrigo Ramirez-Tagle, Leonor Alvarado-Soto, Carlos A. Escobar, Cesar Echeverria-Echeverria
INTRODUCTION
Chalcones are precursors of flavonoids and
isoflavonoids present in edible plants. Their
derivatives have attracted increasing attention
due to their numerous potential pharmacological
applications. They have displayed a broad spectrum
of pharmacological activities that derive from the
changes introduced in their structure by adding
different substituents in their aromatic rings. These
variations proved to be helpful for developing new
medicinal agents, having improved potency and
lesser toxicity (1–3).
Chalcones are important structural units, most of
which exhibit significant biological activity. The wide
range of biological activity associated with many
Chalcone derivatives isolated from natural products
has stimulated interest in developing synthetic
strategies to synthesize heterocyclic systems
based on the chalcone core. Along with efficient
methodologies implemented for their preparation,
investigations have also focused on studying their
reactivity and assessing their possible biological
activities (4–6). Particularly in the pharmaceutical
field, chalcones and their derivatives have been used
in different applications for treating cancer (6–12).
Chalcones can be used as a fluorescent probe
because of their pi-conjugated propenone core
(13–15). Therefore, the potential use of luminescent
chalcones in biological and medical fields depends
on the knowledge of their mode of action.
Up to date, luminescent biomarker properties
of chalcones were almost unknown. Therefore,
to promote the use of chalcones as biomarkers
in biological and medical fields, more studies
concerning their cellular uptake, subcellular
localization, and cytotoxicity (3,6–8,10,11,14) should
be undertaken.
In this study, we evaluated both the antitumoral
activity and the luminescent properties of a set of
synthetic chalcone derivatives (Figure 1) to assess
their potential as biomarker agents.
Chalcones 2´ 4´ 5´ 2 3 4 5
1 OH OCH3 H OCH3 OCH3 H H
2 OH H H OCH3 H H OCH3
3 OH H H OCH3 H OCH3 H
4 H H Br H OCH3 OCH3 H
Figure 1: Chemical structures of the studied chalcones
MATERIALS AND METHODS
Synthesis of Chalcones
The synthesis of the compounds 1, 2, 3, 4 has been
previously described as follows:
Chalcones were prepared following a previously
described methodology (8,11,16) by adding, in a
dropwise manner, a solution of the corresponding
substituted benzaldehyde, (7.34 mmol in 98 ethanol,
20 mL) to a stirred mixture of 2´-hydroxyacetophenone
solution (7.34 99 mmol, in ethanol, 20 mL) and
potassium hydroxide solution (2g in 10 mL distilled
water). The mixture was allowed to react overnight at
room temperature. Then, it was diluted with distilled
RESUMEN
ANTECEDENTES: El carcinoma hepatocelular (CHC) es uno de los cánceres más diagnosticados en todo el mundo. La quimio
prevención del CHC se puede lograr utilizando compuestos naturales o sintéticos que reviertan, supriman, detecten o prevengan
la progresión del cáncer. OBJETIVOS: En este estudio, se investigó tanto los efectos antiproliferativos como las propiedades
luminiscentes de las 2’-hidroxicalconas. MÉTODOS: La viabilidad celular se evaluó usando el ensayo colorimétrico (MTT), los
ensayos de espectroscopia y los cálculos DFT se usaron para determinar las propiedades luminiscentes de las 2´-hidroxichalconas.
RESULTADOS: Se observaron efectos citotóxicos sobre las líneas celulares del tipo HepG2 y EA.hy926. Dado que la estructura
de la 2´-hidroxichalcona puede ser usada como sonda fluorescente, estos compuestos pueden ser útiles en el diagnóstico del
cáncer y la localización del tumor, ya que pueden permitir la observación a través de la fluorescencia y la regresión del tumor
durante el tratamiento, por lo que son candidatas potenciales como nuevos agentes anticancerígenos que podrían actuar
sobre hepatomas humanos. CONCLUSIONES: Este trabajo describe el uso de las 2´-hidroxichalconas como un biomarcador
luminiscente específico para células tumorales. También informamos la captación celular de 2›-hidroxicalconas, su distribución
celular y los mecanismos que pueden ser responsables de sus efectos citotóxicos.
Palabras Claves: 2’-hidroxichalconas; agente anticancerígeno; biomarcador luminiscente; efectos citotóxicos.
INTRODUCTION
Chalcones are precursors of flavonoids and
isoflavonoids present in edible plants. Their
derivatives have attracted increasing attention
due to their numerous potential pharmacological
applications. They have displayed a broad spectrum
of pharmacological activities that derive from the
changes introduced in their structure by adding
different substituents in their aromatic rings. These
variations proved to be helpful for developing new
medicinal agents, having improved potency and
lesser toxicity (1–3).
Chalcones are important structural units, most of
which exhibit significant biological activity. The wide
range of biological activity associated with many
Chalcone derivatives isolated from natural products
has stimulated interest in developing synthetic
strategies to synthesize heterocyclic systems
based on the chalcone core. Along with efficient
methodologies implemented for their preparation,
investigations have also focused on studying their
reactivity and assessing their possible biological
activities (4–6). Particularly in the pharmaceutical
field, chalcones and their derivatives have been used
in different applications for treating cancer (6–12).
Chalcones can be used as a fluorescent probe
because of their pi-conjugated propenone core
(13–15). Therefore, the potential use of luminescent
chalcones in biological and medical fields depends
on the knowledge of their mode of action.
Up to date, luminescent biomarker properties
of chalcones were almost unknown. Therefore,
to promote the use of chalcones as biomarkers
in biological and medical fields, more studies
concerning their cellular uptake, subcellular
localization, and cytotoxicity (3,6–8,10,11,14) should
be undertaken.
In this study, we evaluated both the antitumoral
activity and the luminescent properties of a set of
synthetic chalcone derivatives (Figure 1) to assess
their potential as biomarker agents.
Chalcones 2´ 4´ 5´ 2 3 4 5
1 OH OCH3 H OCH3 OCH3 H H
2 OH H H OCH3 H H OCH3
3 OH H H OCH3 H OCH3 H
4 H H Br H OCH3 OCH3 H
Figure 1: Chemical structures of the studied chalcones
MATERIALS AND METHODS
Synthesis of Chalcones
The synthesis of the compounds 1, 2, 3, 4 has been
previously described as follows:
Chalcones were prepared following a previously
described methodology (8,11,16) by adding, in a
dropwise manner, a solution of the corresponding
substituted benzaldehyde, (7.34 mmol in 98 ethanol,
20 mL) to a stirred mixture of 2´-hydroxyacetophenone
solution (7.34 99 mmol, in ethanol, 20 mL) and
potassium hydroxide solution (2g in 10 mL distilled
water). The mixture was allowed to react overnight at
room temperature. Then, it was diluted with distilled
RESUMEN
ANTECEDENTES: El carcinoma hepatocelular (CHC) es uno de los cánceres más diagnosticados en todo el mundo. La quimio
prevención del CHC se puede lograr utilizando compuestos naturales o sintéticos que reviertan, supriman, detecten o prevengan
la progresión del cáncer. OBJETIVOS: En este estudio, se investigó tanto los efectos antiproliferativos como las propiedades
luminiscentes de las 2’-hidroxicalconas. MÉTODOS: La viabilidad celular se evaluó usando el ensayo colorimétrico (MTT), los
ensayos de espectroscopia y los cálculos DFT se usaron para determinar las propiedades luminiscentes de las 2´-hidroxichalconas.
RESULTADOS: Se observaron efectos citotóxicos sobre las líneas celulares del tipo HepG2 y EA.hy926. Dado que la estructura
de la 2´-hidroxichalcona puede ser usada como sonda fluorescente, estos compuestos pueden ser útiles en el diagnóstico del
cáncer y la localización del tumor, ya que pueden permitir la observación a través de la fluorescencia y la regresión del tumor
durante el tratamiento, por lo que son candidatas potenciales como nuevos agentes anticancerígenos que podrían actuar
sobre hepatomas humanos. CONCLUSIONES: Este trabajo describe el uso de las 2´-hidroxichalconas como un biomarcador
luminiscente específico para células tumorales. También informamos la captación celular de 2›-hidroxicalconas, su distribución
celular y los mecanismos que pueden ser responsables de sus efectos citotóxicos.
Palabras Claves: 2’-hidroxichalconas; agente anticancerígeno; biomarcador luminiscente; efectos citotóxicos.
3Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 29 | Number 03 | Article 347295
The luminescent Chalcones. Its potential use as a luminescent and antitumoral agents
water (200 mL), neutralized with hydrochloric acid,
and extracted four times with ethyl acetate (50 mL).
The compounds were crystallized from ethanol.
All these compounds have been described before
(8,11,16).
Cell culture
The HepG2 hepatocellular carcinoma cell line (HB
8065; American Type Culture Collection, ATCC),
derived from a human hepatoblastoma (17), was
maintained in Dulbecco´s modified Eagle´s (DMEM)-
high glucose (GIBCO) with 10% heat-inactivated
fetal bovine serum (FBS), and 50 U mL-1 penicillin-
streptomycin (Sigma). HUVEC-derived endothelial
cell line (EA.hy926) was kindly provided by C-J
Edgell. It was grown in DMEM-low glucose (GIBCO)
supplemented with 10% heat-inactivated FBS, 2
mmol L-1
, and 50 U mL-1 penicillin-streptomycin
(Sigma). All cell cultures were grown at 37 ºC in a
5%: 95% CO2 : air atmosphere.
Cell viability assay
C e l l v i a b i l i t y w a s e v a l u a t e d u s i n g t h e
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) colorimetric assay (Invitrogen,
Eugene, Oregon, USA). Cell viability was quantified
by the amount of MTT reduction assay (18) HepG2 and
EA.hy926 were exposed to different concentrations
of chalcones for 48 hours. Af ter treatment
cells were co-incubated with MTT (0.5 mg/mL)
for 4 hours, and then solubilized with an acidified
(0.04 N HCl) isopropanol/dimethyl sulfoxide (DMSO)
solution in equal proportions. Optical density
was measured at 540 nm. All experiments were
performed as triplicates. Cell survival (%) data were
plotted and adjusted to a sigmoidal best-fit curve,
where: EC50 is the chalcone concentration to reach
the half-maximal cell survival.
Epifluorescence microscope in live cells
HepG2 and EA.hy926 cells cultured in 6-well
plates were exposed to different concentrations of
chalcones for 24 hours. The cells were washed twice
using phosphate-buffered saline (PBS) and visualized
using a FLOID ® Cell Imaging Station (N = 3-5).
UV-Vis absorption measurements
Electronic spectroscopic data were collected at
room temperature on a MeOH solution using a Jasco
V-630 UV-vis spectrophotometer. Measurements
were recorded between 1000-190 nm with a data
interval of 0.1 nm.
Fluorescence measurements
Emission and excitation spectra were recorded
on a JASCO spectrofluorometer (FP-6200) using
a 1 cm path length JASCO spectrofluorometer
(FP6200) using a quartz cuvette of 1 cm path length.
Excitation wavelengths for all complexes were set
to 347, 405, 460, and 362 nm. Those wavelengths
correspond to the maximum absorption in the
UV-Vis spectra. The fluorometric characterization
was carried out at room temperature. All emission
spectra measurements were recorded between 250
and 750 nm.
Computational Details
The geometry optimizations and computational
properties of the Chalcone family were performed
using density functional theory (DFT) implemented
in the Amsterdam density functional package
(ADF2021) (19).
The computations of the ground and excited states
were carried out using the ZORA Hamiltonian
incorporating scalar relativistic corrections (20).
Triple-ӡ slater basis set plus two polarization
functions (TZ2P) (21) and the nonlocal correction
for the exchange and correlation (XC) incorporated
via the general gradient approximation (GGA)
within the functional BLYP were employed. The
molecular structures were fully optimized without
symmetry constriction. The excitation energies
were estimated by Time-Dependent Density
Functional Theory (TDDFT) (22). This methodology
is based on the linear response formalism within
the iterative Davison procedure implemented in the
ADF code. The calculations performed by the first-
principles method let to obtain accurate excitation
energies and oscillator strengths for the calculated
complexes. Solvation effects were simulated by a
‘‘Conductor-like Screening Model’’ (COSMO) (23)
using methanol as solvent.
RESULTS AND DISCUSSIONS
A luminescent biomarker is a possible indicator of
the biological state of the disease. It is characteristic
of a specific state and, therefore, can be used as a
marker for a target disease. These biomarkers are
usually appropriated to study cellular processes and
monitor or recognize disruption or alterations in
the cellular processes of cancer cells. Luminescent
biomarkers, specifically cancer biomarkers, indicate
cancer presence, and detecting them can verify the
existence of that specific disease (24,25).
The luminescent Chalcones. Its potential use as a luminescent and antitumoral agents
water (200 mL), neutralized with hydrochloric acid,
and extracted four times with ethyl acetate (50 mL).
The compounds were crystallized from ethanol.
All these compounds have been described before
(8,11,16).
Cell culture
The HepG2 hepatocellular carcinoma cell line (HB
8065; American Type Culture Collection, ATCC),
derived from a human hepatoblastoma (17), was
maintained in Dulbecco´s modified Eagle´s (DMEM)-
high glucose (GIBCO) with 10% heat-inactivated
fetal bovine serum (FBS), and 50 U mL-1 penicillin-
streptomycin (Sigma). HUVEC-derived endothelial
cell line (EA.hy926) was kindly provided by C-J
Edgell. It was grown in DMEM-low glucose (GIBCO)
supplemented with 10% heat-inactivated FBS, 2
mmol L-1
, and 50 U mL-1 penicillin-streptomycin
(Sigma). All cell cultures were grown at 37 ºC in a
5%: 95% CO2 : air atmosphere.
Cell viability assay
C e l l v i a b i l i t y w a s e v a l u a t e d u s i n g t h e
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) colorimetric assay (Invitrogen,
Eugene, Oregon, USA). Cell viability was quantified
by the amount of MTT reduction assay (18) HepG2 and
EA.hy926 were exposed to different concentrations
of chalcones for 48 hours. Af ter treatment
cells were co-incubated with MTT (0.5 mg/mL)
for 4 hours, and then solubilized with an acidified
(0.04 N HCl) isopropanol/dimethyl sulfoxide (DMSO)
solution in equal proportions. Optical density
was measured at 540 nm. All experiments were
performed as triplicates. Cell survival (%) data were
plotted and adjusted to a sigmoidal best-fit curve,
where: EC50 is the chalcone concentration to reach
the half-maximal cell survival.
Epifluorescence microscope in live cells
HepG2 and EA.hy926 cells cultured in 6-well
plates were exposed to different concentrations of
chalcones for 24 hours. The cells were washed twice
using phosphate-buffered saline (PBS) and visualized
using a FLOID ® Cell Imaging Station (N = 3-5).
UV-Vis absorption measurements
Electronic spectroscopic data were collected at
room temperature on a MeOH solution using a Jasco
V-630 UV-vis spectrophotometer. Measurements
were recorded between 1000-190 nm with a data
interval of 0.1 nm.
Fluorescence measurements
Emission and excitation spectra were recorded
on a JASCO spectrofluorometer (FP-6200) using
a 1 cm path length JASCO spectrofluorometer
(FP6200) using a quartz cuvette of 1 cm path length.
Excitation wavelengths for all complexes were set
to 347, 405, 460, and 362 nm. Those wavelengths
correspond to the maximum absorption in the
UV-Vis spectra. The fluorometric characterization
was carried out at room temperature. All emission
spectra measurements were recorded between 250
and 750 nm.
Computational Details
The geometry optimizations and computational
properties of the Chalcone family were performed
using density functional theory (DFT) implemented
in the Amsterdam density functional package
(ADF2021) (19).
The computations of the ground and excited states
were carried out using the ZORA Hamiltonian
incorporating scalar relativistic corrections (20).
Triple-ӡ slater basis set plus two polarization
functions (TZ2P) (21) and the nonlocal correction
for the exchange and correlation (XC) incorporated
via the general gradient approximation (GGA)
within the functional BLYP were employed. The
molecular structures were fully optimized without
symmetry constriction. The excitation energies
were estimated by Time-Dependent Density
Functional Theory (TDDFT) (22). This methodology
is based on the linear response formalism within
the iterative Davison procedure implemented in the
ADF code. The calculations performed by the first-
principles method let to obtain accurate excitation
energies and oscillator strengths for the calculated
complexes. Solvation effects were simulated by a
‘‘Conductor-like Screening Model’’ (COSMO) (23)
using methanol as solvent.
RESULTS AND DISCUSSIONS
A luminescent biomarker is a possible indicator of
the biological state of the disease. It is characteristic
of a specific state and, therefore, can be used as a
marker for a target disease. These biomarkers are
usually appropriated to study cellular processes and
monitor or recognize disruption or alterations in
the cellular processes of cancer cells. Luminescent
biomarkers, specifically cancer biomarkers, indicate
cancer presence, and detecting them can verify the
existence of that specific disease (24,25).
4Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 29 | Number 03 | Article 347295Rodrigo Ramirez-Tagle, Leonor Alvarado-Soto, Carlos A. Escobar, Cesar Echeverria-Echeverria
The optical properties of the compounds indicate
that all the structures allow σ → σ* or π → π* transitions
in the UV-Vis region with high extinction coefficients
(Figures 2 and 3). The frontier orbitals are the highest
occupied MO (HOMO) and the lowest unoccupied
MO (LUMO). We focused on these two orbitals
because they are the closest in energy. These
orbitals are intimately involved in chemical reactivity
because they are the most available to electrophiles
and nucleophiles, respectively. Another key change
has to do with the frontier orbitals, the π (HOMO)
and π* (LUMO) orbitals.
Figure 2. Experimental UV-Vis absorption spectra of the
compounds 1 to 4.
Figure 3. Experimental emission spectra of compounds 1, 2, 3
and 4.
The HOMO represents the ability to donate an
electron, and LUMO as an electron acceptor
represents the ability to get an electron. This
electronic absorption corresponds to the transition
from the ground to the first excited state. This is
mainly described by one electron excitation from
the highest occupied molecular or orbital (LUMO)
the highest occupied molecular orbital (HOMO) and
lowest unoccupied molecular orbital (LUMO) are the
main orbitals taking part in chemical stability. Also, 3D
plots of HOMOs and LUMOs for the title compounds
are shown in Figure 4 and considered all occupied
and unoccupied energy levels in a diagram that shows
how the energy changed among the levels.
Figure 4 shows the orbital energy levels of the
frontier Kohn-Sham molecular orbitals (MOs) and
the isosurfaces plots of the lowest unoccupied
molecular orbital (LUMO+1 and LUMO) and the
highest occupied molecular orbitals (HOMO and
HOMO-1). In the case of the LUMO energy of
the studied chalcones, these lie at approximately
-3.1 eV. In chalcones 3 and 4, the HOMO energy
is approximately -5.2 eV. Chalcone 1 displays the
most stable HOMO showing the largest HOMO-
LUMO gap, and chalcone 2 displays the less stable
HOMO showing the shortest HOMO-LUMO gap.
In all compounds, the HOMOs are confined to
the p-orbitals of the same ring, but 3 and 4 show
significance of the bridge orbitals. HOMO-1 orbitals
are confined to the p-orbitals over the other ring.
On the contrary, the LUMO orbitals are extended
over the whole molecules with few compositions of
the substituents.
The optical properties of the compounds indicate
that all the structures allow σ → σ* or π → π* transitions
in the UV-Vis region with high extinction coefficients
(Figures 2 and 3). The frontier orbitals are the highest
occupied MO (HOMO) and the lowest unoccupied
MO (LUMO). We focused on these two orbitals
because they are the closest in energy. These
orbitals are intimately involved in chemical reactivity
because they are the most available to electrophiles
and nucleophiles, respectively. Another key change
has to do with the frontier orbitals, the π (HOMO)
and π* (LUMO) orbitals.
Figure 2. Experimental UV-Vis absorption spectra of the
compounds 1 to 4.
Figure 3. Experimental emission spectra of compounds 1, 2, 3
and 4.
The HOMO represents the ability to donate an
electron, and LUMO as an electron acceptor
represents the ability to get an electron. This
electronic absorption corresponds to the transition
from the ground to the first excited state. This is
mainly described by one electron excitation from
the highest occupied molecular or orbital (LUMO)
the highest occupied molecular orbital (HOMO) and
lowest unoccupied molecular orbital (LUMO) are the
main orbitals taking part in chemical stability. Also, 3D
plots of HOMOs and LUMOs for the title compounds
are shown in Figure 4 and considered all occupied
and unoccupied energy levels in a diagram that shows
how the energy changed among the levels.
Figure 4 shows the orbital energy levels of the
frontier Kohn-Sham molecular orbitals (MOs) and
the isosurfaces plots of the lowest unoccupied
molecular orbital (LUMO+1 and LUMO) and the
highest occupied molecular orbitals (HOMO and
HOMO-1). In the case of the LUMO energy of
the studied chalcones, these lie at approximately
-3.1 eV. In chalcones 3 and 4, the HOMO energy
is approximately -5.2 eV. Chalcone 1 displays the
most stable HOMO showing the largest HOMO-
LUMO gap, and chalcone 2 displays the less stable
HOMO showing the shortest HOMO-LUMO gap.
In all compounds, the HOMOs are confined to
the p-orbitals of the same ring, but 3 and 4 show
significance of the bridge orbitals. HOMO-1 orbitals
are confined to the p-orbitals over the other ring.
On the contrary, the LUMO orbitals are extended
over the whole molecules with few compositions of
the substituents.
5Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 29 | Number 03 | Article 347295
The luminescent Chalcones. Its potential use as a luminescent and antitumoral agents
The biological properties of chalcones and the
potential use of luminescent chalcones depends on
the investigation of their cellular uptake, subcellular
localization, and cytotoxicity.
Considering that EA.hy926 and HepG2 cells showed
a cytotoxic response to the chalcones, we exposed
these cells to a value close to IC50 for 24 hours and
observed by a FLOID ® Cell Imaging Station the
intrinsic fluorescence. Cell membrane penetration of
the EA.hy926 and HepG2 cells for 24 hours showed
chalcones inside the cells, localized mainly in the
cytoplasm. The HepG2 and EA.hy926 cells showed
similar intracellular accumulation of chalcones,
whereas cells treated with the vehicle did not exhibit
green fluorescence (Figures 5a and 6a). Cells treated
with chalcones for 24 hours showed intracellular
green marks that were attributed to the presence
of the chalcones (Figures 5b,c,d,e, and 6b,c,d,e ).
These results demonstrated that chalcones entered
to the cells.
Figure 4. DFT calculated Isosurfaces of the frontier molecular orbitals and energy levels diagram.
Figure 5. Incorporation and morphologic changes in EA.hy926 cells exposed to chalcones. EA.hy926 cells were grown por 24 h, then
treated with chalcones for 24 h. Figures show representative phase-contrast images from at least three separated experiments of EA
exposed to vehicle (control) (A), 10 μM of compound1(B), 10 μM of compound 2(C), 10 μM of compound 3(D), 5 μM of compound 4(E).
Figure 6. Incorporation and morphologic changes in HepG2 cells exposed to chalcones. HepG2 cells were grown for 24 h, then
treated with chalcones for 24 h. Figures show representative phase-contrast images from at least three separates experiments of
HepG2 exposed to vehicle (control) (A), 15 μM of compound 1(B), 30 μM of compound 2(C), 30 μM of compound 3(D), 10 μM of
compound 4(E).
The luminescent Chalcones. Its potential use as a luminescent and antitumoral agents
The biological properties of chalcones and the
potential use of luminescent chalcones depends on
the investigation of their cellular uptake, subcellular
localization, and cytotoxicity.
Considering that EA.hy926 and HepG2 cells showed
a cytotoxic response to the chalcones, we exposed
these cells to a value close to IC50 for 24 hours and
observed by a FLOID ® Cell Imaging Station the
intrinsic fluorescence. Cell membrane penetration of
the EA.hy926 and HepG2 cells for 24 hours showed
chalcones inside the cells, localized mainly in the
cytoplasm. The HepG2 and EA.hy926 cells showed
similar intracellular accumulation of chalcones,
whereas cells treated with the vehicle did not exhibit
green fluorescence (Figures 5a and 6a). Cells treated
with chalcones for 24 hours showed intracellular
green marks that were attributed to the presence
of the chalcones (Figures 5b,c,d,e, and 6b,c,d,e ).
These results demonstrated that chalcones entered
to the cells.
Figure 4. DFT calculated Isosurfaces of the frontier molecular orbitals and energy levels diagram.
Figure 5. Incorporation and morphologic changes in EA.hy926 cells exposed to chalcones. EA.hy926 cells were grown por 24 h, then
treated with chalcones for 24 h. Figures show representative phase-contrast images from at least three separated experiments of EA
exposed to vehicle (control) (A), 10 μM of compound1(B), 10 μM of compound 2(C), 10 μM of compound 3(D), 5 μM of compound 4(E).
Figure 6. Incorporation and morphologic changes in HepG2 cells exposed to chalcones. HepG2 cells were grown for 24 h, then
treated with chalcones for 24 h. Figures show representative phase-contrast images from at least three separates experiments of
HepG2 exposed to vehicle (control) (A), 15 μM of compound 1(B), 30 μM of compound 2(C), 30 μM of compound 3(D), 10 μM of
compound 4(E).
6Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 29 | Number 03 | Article 347295Rodrigo Ramirez-Tagle, Leonor Alvarado-Soto, Carlos A. Escobar, Cesar Echeverria-Echeverria
The current report describes for the first time
the use of chalcones as a specific luminescent
biomarker agent against tumor cells. We studied
the cytotoxicity of chalcones in different cell cultures
from tumoral cell lines (table 1 and figure 7) to
assess its antitumor activities. We also report the
cellular uptake of luminescent chalcones and their
subcellular distribution (Figures 5 and 6).
Table 1. The IC 50 value on EA.hy926 and HepG2 incubated with
different chalcones for 48 hours. Cell viability was measured using
the MTT assay. Data area expressed as mean +/- SEM from the
three independent experiment, each performed in triplicate.
Chalcone EA.hy926 IC 50/μM HepG2 IC50/μM
1 7.66 ± 0.46 10.65 ± 0.98
2 6.76 ± 1.89 27.61 ± 2.51
3 10.73 ± 0.11 31.26 ± 2.42
4 3.92 ± 1.34 8.27 ± 0.19
Figure 7. Effect of Chalcones on growth of (a) HepG2 and (b)
EA.hy926 cells, which were treated with various concentrations
of chalcones (0.5-2 mg/mL) for 48h.
CONCLUSION
Based on their fluorescent properties, the chalcones
may be helpful for cancer diagnostics, tumor
localization, and may enable the observation
through the fluorescence of tumor regression during
treatment. The findings reported here represent
the first attempts to use chalcones as biomarkers
in cancer disease. We are currently planning
new experiments to shed light on the complex
mechanisms involved.
REFERENCES
1. Rahman MA, Chalcone: A Valuable Insight into the Recent
Advances and Potential. Chem. Sci. 2011, 2011, 1–16, DOI: https://
doi.org/10.4172/2150-3494.1000021
2. Kumar V, Kumar S, Hassan M, Wu H, Thimmulappa RK, Kumar A,
Sharma SK, Parmar VS, Biswal S, Malhotra S V Novel chalcone
derivatives as potent Nrf2 activators in mice and human lung
epithelial cells. J. Med. Chem. 2011, 54, 4147–59, DOI: https://
doi.org/10.1021/jm2002348.
3. Marquina S, Maldonado-Santiago M, Sánchez-Carranza J N,
Antúnez-Mojica M, González-Maya L, Razo-Hernández RS,
Alvarez L, Design, synthesis and QSAR study of 2′-hydroxy-4′-
alkoxy chalcone derivatives that exert cytotoxic activity by the
mitochondrial apoptotic pathway. Bioorganic Med. Chem. 2019,
27, 43–54, DOI: https://doi.org/10.1016/J.BMC.2018.10.045.
4. Tadigoppula N, Korthikunta V, Gupta S, Kancharla P, Khaliq T,
Soni, A, Srivastava RK, Srivastava K, Puri S K, Raju K S R, et al.
Synthesis and insight into the structure-activity relationships of
chalcones as antimalarial agents. J. Med. Chem. 2013, 56, 31–45,
DOI: https://doi.org/10.1021/jm300588j.
5. Shenvi S, Kumar K, Hatti K S Rijesh K, Diwakar L, Reddy G C,
Synthesis, anticancer and antioxidant activities of 2,4,5-trimethoxy
chalcones and analogues from asaronaldehyde: structure-activity
relationship. Eur. J. Med. Chem. 2013, 62, 435–42, DOI: https://
doi.org/10.1016/j.ejmech.2013.01.018.
6. Zarate X, Schott E, Escobar C A, Lopez-Castro R, Echeverria
C, Alvarado-Soto L, Interaction of Chalcones with CT-DNA by
Spectrophotometric Analysis and Theoretical Simulations. Quim.
Nova 2016, 39, 914–918.
7. Dickson J, Flores L, Stewart M, LeBlanc R, Pati H N, Lee M,
Holt H, Synthesis and Cytotoxic Properties of Chalcones: An
Interactive and Investigative Undergraduate Laboratory Project
at the Interface of Chemistry and Biology. J. Chem. Educ. 2006,
83, 934, DOI: https://doi.org/10.1021/ed083p934.
8. Echeverria C, Santibañez J F, Donoso-Tauda O, Escobar C ,
Ramirez-Tagle R , Structural antitumoral activity relationships
of synthetic chalcones. Int. J. Mol. Sci. 2009, 10, 221–31, DOI:
https://doi.org/10.3390/ijms10010221.
9. Detsi A, Majdalani M, Kontogiorgis C, Hadjipavlou-Litina D,
Kefalas P, Natural and synthetic 2’-hydroxy-chalcones and
aurones: synthesis, characterization and evaluation of the
antioxidant and soybean lipoxygenase inhibitory activity. Bioorg.
Med. Chem. 2009, 17, 8073–85, DOI: https://doi.org/10.1016/j.
bmc.2009.10.002.
10. Boumendjel A, Boccard J, Carrupt P A, Nicolle E, Blanc M, Geze
A, Choisnard L, Wouessidjewe D, Matera E L, Dumontet C,
Antimitotic and antiproliferative activities of chalcones: forward
structure-activity relationship. J. Med. Chem. 2008, 51, 2307–10,
DOI: https://doi.org/10.1021/jm0708331.
11. Ramirez-Tagle R, Escobar C, Romero V, Montorfano I, Armisén R,
Borgna V, Jeldes E, Pizarro L, Simon F, Echeverria C, Chalcone-
Induced Apoptosis through Caspase-Dependent Intrinsic
Pathways in Human Hepatocellular Carcinoma Cells. Int. J. Mol.
Sci. 2016, 17, 260, DOI: https://doi.org/10.3390/ijms17020260.
12. Suwito H, Jumina M, Pudjiastuti P, Fanani M Z, Kimata-Ariga
Y, Katahira R, Kawakami T, Fujiwara T, Hase T, et al. Design
The current report describes for the first time
the use of chalcones as a specific luminescent
biomarker agent against tumor cells. We studied
the cytotoxicity of chalcones in different cell cultures
from tumoral cell lines (table 1 and figure 7) to
assess its antitumor activities. We also report the
cellular uptake of luminescent chalcones and their
subcellular distribution (Figures 5 and 6).
Table 1. The IC 50 value on EA.hy926 and HepG2 incubated with
different chalcones for 48 hours. Cell viability was measured using
the MTT assay. Data area expressed as mean +/- SEM from the
three independent experiment, each performed in triplicate.
Chalcone EA.hy926 IC 50/μM HepG2 IC50/μM
1 7.66 ± 0.46 10.65 ± 0.98
2 6.76 ± 1.89 27.61 ± 2.51
3 10.73 ± 0.11 31.26 ± 2.42
4 3.92 ± 1.34 8.27 ± 0.19
Figure 7. Effect of Chalcones on growth of (a) HepG2 and (b)
EA.hy926 cells, which were treated with various concentrations
of chalcones (0.5-2 mg/mL) for 48h.
CONCLUSION
Based on their fluorescent properties, the chalcones
may be helpful for cancer diagnostics, tumor
localization, and may enable the observation
through the fluorescence of tumor regression during
treatment. The findings reported here represent
the first attempts to use chalcones as biomarkers
in cancer disease. We are currently planning
new experiments to shed light on the complex
mechanisms involved.
REFERENCES
1. Rahman MA, Chalcone: A Valuable Insight into the Recent
Advances and Potential. Chem. Sci. 2011, 2011, 1–16, DOI: https://
doi.org/10.4172/2150-3494.1000021
2. Kumar V, Kumar S, Hassan M, Wu H, Thimmulappa RK, Kumar A,
Sharma SK, Parmar VS, Biswal S, Malhotra S V Novel chalcone
derivatives as potent Nrf2 activators in mice and human lung
epithelial cells. J. Med. Chem. 2011, 54, 4147–59, DOI: https://
doi.org/10.1021/jm2002348.
3. Marquina S, Maldonado-Santiago M, Sánchez-Carranza J N,
Antúnez-Mojica M, González-Maya L, Razo-Hernández RS,
Alvarez L, Design, synthesis and QSAR study of 2′-hydroxy-4′-
alkoxy chalcone derivatives that exert cytotoxic activity by the
mitochondrial apoptotic pathway. Bioorganic Med. Chem. 2019,
27, 43–54, DOI: https://doi.org/10.1016/J.BMC.2018.10.045.
4. Tadigoppula N, Korthikunta V, Gupta S, Kancharla P, Khaliq T,
Soni, A, Srivastava RK, Srivastava K, Puri S K, Raju K S R, et al.
Synthesis and insight into the structure-activity relationships of
chalcones as antimalarial agents. J. Med. Chem. 2013, 56, 31–45,
DOI: https://doi.org/10.1021/jm300588j.
5. Shenvi S, Kumar K, Hatti K S Rijesh K, Diwakar L, Reddy G C,
Synthesis, anticancer and antioxidant activities of 2,4,5-trimethoxy
chalcones and analogues from asaronaldehyde: structure-activity
relationship. Eur. J. Med. Chem. 2013, 62, 435–42, DOI: https://
doi.org/10.1016/j.ejmech.2013.01.018.
6. Zarate X, Schott E, Escobar C A, Lopez-Castro R, Echeverria
C, Alvarado-Soto L, Interaction of Chalcones with CT-DNA by
Spectrophotometric Analysis and Theoretical Simulations. Quim.
Nova 2016, 39, 914–918.
7. Dickson J, Flores L, Stewart M, LeBlanc R, Pati H N, Lee M,
Holt H, Synthesis and Cytotoxic Properties of Chalcones: An
Interactive and Investigative Undergraduate Laboratory Project
at the Interface of Chemistry and Biology. J. Chem. Educ. 2006,
83, 934, DOI: https://doi.org/10.1021/ed083p934.
8. Echeverria C, Santibañez J F, Donoso-Tauda O, Escobar C ,
Ramirez-Tagle R , Structural antitumoral activity relationships
of synthetic chalcones. Int. J. Mol. Sci. 2009, 10, 221–31, DOI:
https://doi.org/10.3390/ijms10010221.
9. Detsi A, Majdalani M, Kontogiorgis C, Hadjipavlou-Litina D,
Kefalas P, Natural and synthetic 2’-hydroxy-chalcones and
aurones: synthesis, characterization and evaluation of the
antioxidant and soybean lipoxygenase inhibitory activity. Bioorg.
Med. Chem. 2009, 17, 8073–85, DOI: https://doi.org/10.1016/j.
bmc.2009.10.002.
10. Boumendjel A, Boccard J, Carrupt P A, Nicolle E, Blanc M, Geze
A, Choisnard L, Wouessidjewe D, Matera E L, Dumontet C,
Antimitotic and antiproliferative activities of chalcones: forward
structure-activity relationship. J. Med. Chem. 2008, 51, 2307–10,
DOI: https://doi.org/10.1021/jm0708331.
11. Ramirez-Tagle R, Escobar C, Romero V, Montorfano I, Armisén R,
Borgna V, Jeldes E, Pizarro L, Simon F, Echeverria C, Chalcone-
Induced Apoptosis through Caspase-Dependent Intrinsic
Pathways in Human Hepatocellular Carcinoma Cells. Int. J. Mol.
Sci. 2016, 17, 260, DOI: https://doi.org/10.3390/ijms17020260.
12. Suwito H, Jumina M, Pudjiastuti P, Fanani M Z, Kimata-Ariga
Y, Katahira R, Kawakami T, Fujiwara T, Hase T, et al. Design
7Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 29 | Number 03 | Article 347295
The luminescent Chalcones. Its potential use as a luminescent and antitumoral agents
and synthesis of chalcone derivatives as inhibitors of the
ferredoxin - Ferredoxin-NADP+ reductase interaction of
Plasmodium falciparum: Pursuing new antimalarial agents.
Molecules 2014, 19, 21473–21488, DOI: https://doi.org/10.3390/
MOLECULES191221473.
13. Jagtap AR, Satam VS, Rajule RN, Kanetkar VR, Synthesis of
highly fluorescent coumarinyl chalcones derived from 8-acetyl-
1,4-diethyl-1,2,3,4-tetrahydro-7H-pyrano(2,3-g)quinoxalin-7-one
and their spectral characteristics. Dye. Pigment. 2011, 91, 20–25,
DOI: https://doi.org/10.1016/j.dyepig.2011.01.011.
14. Asiri A, Mar wani H, Green Synthesis, Charac terization,
Photophysical and Electrochemical Properties of Bis-chalcones.
Int. J. Electrochem. 2014, 9, 799–809.
15. Xue Y, Mou J, Liu Y, Gong X, Yang Y, An L, An ab initio simulation
of the UV/Visible spectra of substituted chalcones. Cent. Eur. J.
Chem. 2010, 8, 928–936, DOI: https://doi.org/10.2478/s11532-
010-0058-3.
16. Zarate X, Schott E, Escobar CA, Lopez-Castro R, Echeverria
C, A l v ar a d o -S oto L , Ra mir ez-Tagle, R . I nte r ac t io n of
chalcones with CT-DNA by spectrophotometric analysis and
theoretical simulations. Quim. Nova 2016, 39, DOI: https://doi.
org/10.5935/0100-4042.20160114.
17. Javitt B, Hep G2 as a resource cholesterol, for metabolic and bile
studies: lipoprotein , acids. FASEB 1990, 4, 161–168, DOI: https://
doi.org/10.1096/fasebj.4.2.2153592
18. Mosmann T, Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J.
Immunol. Methods 1983, 65, 55–63.
19. te Velde G , Bickelhaupt F M, Baerends E J, Fonseca Guerra C,
van Gisbergen S J A , Snijders J G , Ziegler T, Chemistry with
ADF, Journal of Comp Chem , 2001, 22, 931–967, DOI: https://
doi.org/10.1002/jcc.1056.
20. van Lenthe E, Baerends E J, Snijders J G , Van Relativistic total
energy using regular approximations. J. Chem. Phys. 1994, 101,
DOI: https://doi.org/10.1063/1.467943
21. Snijders J G, Vernooijs P, Baerends E J , Roothaan-Hartree-Fock-
Slater atomic wave functions: Single-zeta, double-zeta, and
extended Slater-type basis sets for 87Fr-103Lr. Atomic Data and
Nuclear Data Tables 26, (6) 1981, 26, 483–509, DOI: https://doi.
org/10.1016/0092-640X(81)90004-8
22. Casida M E, Huix-Rotllant M, Progress in time-dependent density-
functional theory. Annu. Rev. Phys. Chem. 2012, 63, 287–323,
DOI: https://doi.org/10.1146/annurev-physchem-032511-143803.
23. Pye C C, Ziegler T, van Lenthe E, Louwen J N, An implementation
of the conductor-like screening model of solvation within the
Amsterdam density functional package — Part II. COSMO for
real solvents 1. Can. J. Chem. 2009, 87, 790–797, DOI: https://
doi.org/10.1139/V09-008.
24. Choi Y E, Kwak J W, Park J W, Nanotechnology for early cancer
detection. Sensors (Basel). 2010, 10, 428–55, DOI: https://doi.
org/10.3390/s100100428.
25. Sharma R, Kumar R, Kodwani R, Kapoor S, Khare A, Bansal
R, Khurana S, Singh S, Thomas J, Roy B, et al. A Review on
Mechanisms of Anti Tumor Activity of Chalcones. Anticancer.
Agents Med. Chem. 2015, 16, 200–211, DOI: https://doi.org/10
.2174/1871520615666150518093144.
26. Ramirez-Tagle R, Escobar C A, Romero V, Montorfano I, Armisén
R, Borgna V, Jeldes E, Pizarro L, Simon F, Echeverria C, Chalcone-
induced apoptosis through caspase-dependent intrinsic
pathways in human hepatocellular carcinoma cells. Int. J. Mol.
Sci. 2016, 17, DOI: https://doi.org/10.3390/ijms17020260.
The luminescent Chalcones. Its potential use as a luminescent and antitumoral agents
and synthesis of chalcone derivatives as inhibitors of the
ferredoxin - Ferredoxin-NADP+ reductase interaction of
Plasmodium falciparum: Pursuing new antimalarial agents.
Molecules 2014, 19, 21473–21488, DOI: https://doi.org/10.3390/
MOLECULES191221473.
13. Jagtap AR, Satam VS, Rajule RN, Kanetkar VR, Synthesis of
highly fluorescent coumarinyl chalcones derived from 8-acetyl-
1,4-diethyl-1,2,3,4-tetrahydro-7H-pyrano(2,3-g)quinoxalin-7-one
and their spectral characteristics. Dye. Pigment. 2011, 91, 20–25,
DOI: https://doi.org/10.1016/j.dyepig.2011.01.011.
14. Asiri A, Mar wani H, Green Synthesis, Charac terization,
Photophysical and Electrochemical Properties of Bis-chalcones.
Int. J. Electrochem. 2014, 9, 799–809.
15. Xue Y, Mou J, Liu Y, Gong X, Yang Y, An L, An ab initio simulation
of the UV/Visible spectra of substituted chalcones. Cent. Eur. J.
Chem. 2010, 8, 928–936, DOI: https://doi.org/10.2478/s11532-
010-0058-3.
16. Zarate X, Schott E, Escobar CA, Lopez-Castro R, Echeverria
C, A l v ar a d o -S oto L , Ra mir ez-Tagle, R . I nte r ac t io n of
chalcones with CT-DNA by spectrophotometric analysis and
theoretical simulations. Quim. Nova 2016, 39, DOI: https://doi.
org/10.5935/0100-4042.20160114.
17. Javitt B, Hep G2 as a resource cholesterol, for metabolic and bile
studies: lipoprotein , acids. FASEB 1990, 4, 161–168, DOI: https://
doi.org/10.1096/fasebj.4.2.2153592
18. Mosmann T, Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J.
Immunol. Methods 1983, 65, 55–63.
19. te Velde G , Bickelhaupt F M, Baerends E J, Fonseca Guerra C,
van Gisbergen S J A , Snijders J G , Ziegler T, Chemistry with
ADF, Journal of Comp Chem , 2001, 22, 931–967, DOI: https://
doi.org/10.1002/jcc.1056.
20. van Lenthe E, Baerends E J, Snijders J G , Van Relativistic total
energy using regular approximations. J. Chem. Phys. 1994, 101,
DOI: https://doi.org/10.1063/1.467943
21. Snijders J G, Vernooijs P, Baerends E J , Roothaan-Hartree-Fock-
Slater atomic wave functions: Single-zeta, double-zeta, and
extended Slater-type basis sets for 87Fr-103Lr. Atomic Data and
Nuclear Data Tables 26, (6) 1981, 26, 483–509, DOI: https://doi.
org/10.1016/0092-640X(81)90004-8
22. Casida M E, Huix-Rotllant M, Progress in time-dependent density-
functional theory. Annu. Rev. Phys. Chem. 2012, 63, 287–323,
DOI: https://doi.org/10.1146/annurev-physchem-032511-143803.
23. Pye C C, Ziegler T, van Lenthe E, Louwen J N, An implementation
of the conductor-like screening model of solvation within the
Amsterdam density functional package — Part II. COSMO for
real solvents 1. Can. J. Chem. 2009, 87, 790–797, DOI: https://
doi.org/10.1139/V09-008.
24. Choi Y E, Kwak J W, Park J W, Nanotechnology for early cancer
detection. Sensors (Basel). 2010, 10, 428–55, DOI: https://doi.
org/10.3390/s100100428.
25. Sharma R, Kumar R, Kodwani R, Kapoor S, Khare A, Bansal
R, Khurana S, Singh S, Thomas J, Roy B, et al. A Review on
Mechanisms of Anti Tumor Activity of Chalcones. Anticancer.
Agents Med. Chem. 2015, 16, 200–211, DOI: https://doi.org/10
.2174/1871520615666150518093144.
26. Ramirez-Tagle R, Escobar C A, Romero V, Montorfano I, Armisén
R, Borgna V, Jeldes E, Pizarro L, Simon F, Echeverria C, Chalcone-
induced apoptosis through caspase-dependent intrinsic
pathways in human hepatocellular carcinoma cells. Int. J. Mol.
Sci. 2016, 17, DOI: https://doi.org/10.3390/ijms17020260.