Molecular modeling of cytotoxic activity of a new terpenoid-like bischalcone

Article abstract of DOI:10.1039/c9nj03452h

This study describes the synthesis and structure of (1E,4E)-1-(3-chlorophenyl)-5-(2,6,6-trimethylcyclohex-1-en-1-yl)penta-1,4-dien-3-one (BC I). X-ray single-crystal diffraction and Hirshfeld surface analysis describe supramolecular arrangement and topological analysis. Theoretical calculations, such as QTAIM, frontier molecular orbital, MEP and infrared spectra assignments, were performed at the B3LYP/6-311++G(d,p) level of theory. Also, this work evaluates molecular docking against DNA (PDB ID: 1BNA) and cytotoxic activity against two tumor cell lines. The BC I molecule has a half chair conformation of the cyclohexene ring, and the supramolecular arrangements are stabilized by C₆-H₆?O₁ and C₇-H₇?O₁ interactions. MEP and docking analyses indicate an electrophilic attack that is likely to occur on the carbonyl group.

Full text of DOI:10.1039/c9nj03452h

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Molecular modeling of cytotoxic activity of a new
terpenoid-like bischalcone†
Cite this: New J. Chem., 2019,
43, 18451
Loide O. Sallum, *a Vera L. Siqueira,b Jean M. F. Custodio,
Nadia M. Borges,a
´
a
´
Aliny P. Lima,bc Davi C. Abreu,c Elisangela de P. S. Lacerda,
ˆ
Rosa S. Lima,b
c
Alisson M. de Oliveira,b Ademir J. Camargoa and Hamilton B. Napolitano
*
ad
This study describes the synthesis and structure of (1E,4E)-1-(3-chlorophenyl)-5-(2,6,6-trimethylcyclohex-1-
en-1-yl)penta-1,4-dien-3-one (BC I). X-ray single-crystal diffraction and Hirshfeld surface analysis
describe supramolecular arrangement and topological analysis. Theoretical calculations, such as QTAIM,
frontier molecular orbital, MEP and infrared spectra assignments, were performed at the B3LYP/6-
311++G(d,p) level of theory. Also, this work evaluates molecular docking against DNA (PDB ID: 1BNA)
and cytotoxic activity against two tumor cell lines. The BC I molecule has a half chair conformation
of the cyclohexene ring, and the supramolecular arrangements are stabilized by C6–H6ꢀ ꢀ ꢀO1 and
C7–H7ꢀ ꢀ ꢀO1 interactions. MEP and docking analyses indicate an electrophilic attack that is likely to occur
on the carbonyl group.
Received 3rd July 2019,
Accepted 16th October 2019
DOI: 10.1039/c9nj03452h
rsc.li/njc
pro-retinoid carotenoids (b-carotene) of carrots, roasted almonds,
Introduction
herbs and fruits.21,22 Naturally originating from carotenoids and
Structural modifications in organic compounds with known synthesized from citral and acetone, b-ionones are composed of
biological potential might increase their potential activity.1–5 a trimethylcyclohexene, an olefin portion and a carbonyl group.
Among these important compounds, chalcones have been They have been studied not only because of their application
widely used because their pharmacophore site is similar to in the fragrance industry, but also as anti-inflammatory23,24
those found in flavones and isoflavones (potent inhibitors and anticancer25 agents. Although the biological potential of
of DNA topoisomerase II).6 Added to this, chalcones have chalcones and b-ionones is well known, their fusion into a
a delocalized p-system composed of a carbonyl group, olefin terpenoid-like bischalcone has not been extensively studied so
portion and aromatic rings, which enables their interaction far. Fundik and coworkers synthesized some of these hybrid
with biological receptors7–12 and is responsible for some activities compounds by reacting a- and b-ionones with substituted
such as antitumor,1,2 anti-inflammatory,3 antiallergic4 and anti- benzaldehydes, furfural, and thiophene-2-carbaldehyde and
microbial.5 In this sense, several chalcone derivatives have been evaluated their antibacterial and antifungal activity.25 Also,
reported to exhibit significant DNA binding interactions acting Lima and coworkers25 prepared eight new chalcones with both
as potent anticancer agents.13–17 Usually, these compounds are these structural frameworks and tested them against three
synthesized from the Claisen–Schmidt condensation of an aceto- cancer cell lines: SF-295, HCT-116 and OVCAR-8.
phenone and a benzaldehyde, but these reactants can be altered
to increase their biological activities.18
Considering the known biological properties of such hybrid
compounds, BC I was synthesized from b-ionone and 3-chloro-
Another important aromatic class of compounds is b-ionones, benzaldehyde, and its molecular and supramolecular architec-
which occur in many essential oils, such as those from roses, tures were described using single crystal X-ray diffraction
violets, boronias and petunias.19,20 In food, they are present in (XRD) and Hirshfeld surface (HS) analysis. In order to achieve
better insights into the molecular structure, frontier molecular
orbital (FMO), molecular electrostatic potential (MEP) chemical
a
´
´
´
´
Grupo de Quımica Teorica e Estrutural, Universidade Estadual de Goias, Anapolis,
GO, Brazil. E-mail: loide.sallum@gmail.com, hbnapolitano@gmail.com;
Tel: +55-(62)-3328-1192
reactivity, vibrational assignment and quantum theory of atoms
in molecules (QTAIM) analyses at the B3LYP/6-311++G(d,p)
level of theory were undertaken. In addition, the mouse
tumor lines sarcoma 180 (S-180) (ATCCs TIB-66) and Ehrlich
tumor (ATCCs CCL-77) were used in biological assays, which
were also investigated at the molecular level via docking
simulations.
b
c
´
Faculdade do Instituto Brasil, Anapolis, GO, Brazil
ˆ
´
´
ˆ
Instituto de Ciencias Biologicas, Universidade Federal de Goias, Goiania, GO, Brazil
d
´
´
´
´
Laboratorio de Novos Materiais, Centro Universitario de Anapolis, Anapolis, GO,
Brazil
† CCDC 1936534. For crystallographic data in CIF or other electronic format see
DOI: 10.1039/c9nj03452h
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Experimental and computational
procedures
interaction by using this tool, which is helpful to study the
crystal packing behavior. In a HS, de is the distance from the
nearest nucleus of an outside molecule to the surface, which
provides the close intermolecular contacts, while di is the
Synthesis and crystallization
A bischalcone like-terpenoid was synthesized using a volumetric distance from inside to the surface, which provides studies of
flask reactor of 50 mL size, to which was added 10 mL of methanol, the molecule itself. The normalized contact distance (dnorm),
b-ionone (1.5 mM) and 3-chlorobenzaldehyde (2.5 mM) with a high which combines the normalized de and di with the van der
purity rate. Then, five drops of 30% aqueous potassium hydroxide Waals radius for each atom involved in this close contact with
solution was added into the reactional medium. The reaction the surface, is used to analyze intermolecular interactions.33
mixture was shaken for 90 minutes at room temperature. At the Fingerprint34 plots provide quantitative information on the
end of the reaction time, the mixture was treated with HCl solution type of intermolecular contacts as well as their frequency. The
(10%), washed with water and then dichloromethane was added to software Crystal Explorer 3.135 was used to generate HS inter-
the separation funnel to drag out the organic phase. Dichloro- molecular interactions and calculated 2D fingerprint plots.
methane was evaporated at room temperature for 24 hours
Theoretical calculations
and then isopropyl alcohol was added for compound recrystal-
lization, as shown in Scheme 1. The synthesis process was
accompanied by thin-layer chromatography. Claisen-Schmidt
methodology was used in the experimental protocol. Infrared
spectra (IR) were recorded on a PerkinElmer Frontier in the
range of 4000–700 cmꢁ1 using the attenuated total reflection
(ATR) technique. ATR-FTIR (cmꢁ1): 3020, 2955, 2930, 2867,
1647, 1617, 1583, 1475, 1431, 1357, 1324, 1267, 1204, 1180,
1134, 1093, 1078, 998, 906, 871, 784, 715, 703.
Electronic structure calculations were carried out using the
Gaussian 0936 program package for BC I. The full geometry
optimization was carried out using density functional theory
(DFT) with the exchange–correlation functional B3LYP37 and
the basis set 6-311++G(d,p).38,39 This functional is commonly
used in quantum chemistry for geometry optimization. The
studied electronic properties included frontier molecular orbital
energies: highest occupied molecular orbitals (HOMOs) and
lowest unoccupied molecular orbitals (LUMOs) and MEP. The
QTAIM analysis was performed using the initial geometry generated
from X-ray refinement data and assignments of infrared frequencies
were also performed.
Crystallographic characterization
Single crystal XRD data collection was performed on a Bruker
APEX II CCD diffractometer using graphite monochromated
MoKa radiation (0.71073 Å) at 296 K. The structure was solved
using Olex2,26 with the ShelXS27 structure solution program
using direct methods and refined with the ShelXL28 refinement
package using least squares minimization. Olex2 was also used
to prepare the crystallographic information file (CIF). The non-
hydrogen atoms were refined anisotropically. The hydrogen
bonded to carbon was placed geometrically and refined using
a riding model with a distance of methyl C–H = 0.96 Å,
methylene C–H = 0.97 Å and aromatic C–H = 0.93 Å. In the
case of hydrogens, their Uiso was set to 1.2 Ueq of the bonded
carbon, except for the methyl group, whose Uiso(H) = 1.5 Ueq of
the corresponding carbon. The programs Ortep29 and Mercury
(version 3.10)30 were used to prepare the artwork representa-
tions for publication. The intermolecular interactions were
checked by Platon software.31 The BC I molecule was deposited
in the Cambridge Structural Database under code 1936534.†
The HS32 is a spatial map used to visualize the surface of
Molecular docking
Molecular docking studies were carried out using the AutoDock-
Vina software.40 Receptor and ligand were prepared using the
dock prep tool of AutoDock tools implemented in the Chimera
software.41 The receptor X-ray crystallographic structure was
retrieved from the Protein Data Bank (PDB)42 and in its prepara-
tion the solvent and ligand were deleted, any hydrogens were
added and the energy was minimized by the minimized structure
of Chimera. The ligand assigned charge was managed with the
general Amber ff12SB.43 The receptor search volume option was
enclosed in a box centered at the geometric point (13.00, 17.97,
and 13.00 Å) within the grid size (43.94 ꢂ 53.77 ꢂ 47.11 Å). Chains
of non-standard residues and all non-standard residues have been
set to false. All other parameters were kept as the default setting.
Cell culture and cytotoxicity assay using MTT
molecules that compares the electron density of a molecule The murine sarcoma-180 tumor cells (S180) (ATCCs# TIB-66),
with that of the entire crystal and measures the distribution of and Ehrlich-ascites tumor (ATCCs CCL-77t) and non-tumor
close contact interactions. We can explain the intermolecular cells (Vero) (ATCCs CCL-81t) were cultured in suspension in
Scheme 1 Chemical scheme of the synthesized BC I.
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RPMI 1640 and DMEM media (Sigma Chemical Co., MO), respec-
tively, supplemented with 10% fetal calf serum, 100 mg mLꢁ1
penicillin, and 100 mg mLꢁ1 streptomycin. The cultures were
incubated in a humidified incubator (Thermo Scientific) at
37 1C with 5% CO2.
The cytotoxic effect of BC I was evaluated using MTT assay with
S180, Ehrlich-ascites tumor cells and Vero non-tumor cells as
described previously by Mosman, 1983.44 Briefly, 1.0 ꢂ 105 S180
cells and 2.0 ꢂ 104 Vero cells were plated in 96-well tissue culture
plates and treated with different concentrations of BC I and
cisplatin (0.2–200 mM) for 48 h. After treatment, 10 mL of MTT
(1 mg mLꢁ1) was added to each well, and the plates were incubated
at 37 1C for an additional 3 h. The purple formation crystals were
dissolved in 50 mL of SDS, and the absorbance was determined at
545 nm using a Stat Fax 2100 microplate reader. The cell viability
was calculated and the IC50 was obtained from dose–response
curves using GraphPad Prism 4.02 for Windows. The selectivity of
BC I to tumor cells was assessed in terms of the selectivity index.
Fig. 1 Ortep diagram of ellipsoids at 50% probability level with the atomic
numbering scheme for BC I. Hydrogen atoms are in arbitrary radii.
respectively. The molecular conformation from the b-ionone
mean plane calculated through cyclohexene and core carbonyl
atoms forms an angle of 49.591 observed in terpenoid-like chalcones,
as shown in Fig. 2. This molecular conformation is a common
structural framework present in many anticancer terpenoid-like
chalcones.45,46 The C10–C11–C12–C13 and C10–C11–C12–C17
dihedral angles are 134.01 and ꢁ47.71. The C2–C1–C7–C8 and
C6–C1–C7–C8 dihedral angles are 3.71 and ꢁ178.41, respectively,
which indicates coplanarity in this region.
Cell cycle analysis and detection of apoptosis by flow cytometry
XRD data are shown in Table 1. BC I crystallized into the
P21/c space group with unit cell parameters: a = 11.4168(13) Å,
b = 14.2639(18) Å, c = 11.1744(14) Å and b = 106.469(3)1. The
selected bond lengths and angles and conformationally important
dihedral angles are listed in Table 2. Table 2 shows theoretical
and experimental geometric parameters in good agreement with
one another. The major difference of about 4.59% is in contrac-
tion strengthening of the C7–C8 (1.285 Å) experimental bond, with
a C7–C8 (1.344 Å) theoretical length. The theoretical calculation
model suggests geometric parameters very close to experimental
ones, regardless of supramolecular arrangements.
BC I crystal packing is stabilized by bifurcated hydrogen
bonds through C6–H6ꢀꢀꢀO1 and C7–H7ꢀꢀꢀO1 in an R12(6)33 ring
motif, involving a carbonyl group along the c axis (Fig. 3), wherein
C–H groups are hydrogen bonding donors pertained to the
a,b-unsaturated ketone and aromatic ring, respectively. Also, this
The S180 cells were treated with BC I (20 mM) for 24 h and 48 h.
Briefly, 3.0 ꢂ 105 cells were harvested by centrifugation, washed
with phosphate-buffered saline (PBS), fixed with 70% (v/v) cold
ethanol and stored overnight at ꢁ20 1C. The fixed cells were washed
with PBS and incubated with propidium iodide (PI; Sigma-Aldrich)
containing 0.05% RNase. The samples were incubated at 4 1C in the
dark and analyzed by flow cytometry (FACSCalibur, BD Biosciences).
The percentage of cells in the G0/G1, S, G2/M and sub-G1 phases
was analyzed using ModFit software.
The cell death of S180 tumor cells was examined using the
Annexin V-FITC apoptosis detection kit. S180 cells were treated
with BC I (20 mM) for 48 h. Briefly, 3.0 ꢂ 105 cells were harvested
and washed with PBS. The cells were re-suspended in 400 mL of
binding buffer. Next, 5 mL of Annexin V-FITC and 1 mL of PI were
added. Flow cytometric analysis was performed immediately after
supravital staining. Data acquisition and analysis were performed
on a flow cytometer (FACSCalibur, BD Biosciences) using Cell
Quest software. The criteria for positivity in cells in early stages
of apoptosis were Annexin V-positive and PI-negative and
criteria for cells in the late stages of apoptosis were both
Annexin V-positive and PI-positive. Cells positive for Annexin V
were stained fluorescent green and those positive for PI were
stained fluorescent red.
Results and discussion
Solid state characterization
The asymmetric unit of BC I is shown in the Ortep diagram
in Fig. 1. The stereochemistry of C7QC8 and C10QC11 is
(E)-configuration. This molecule has a cyclohexene ring in the
half chair conformation with C13 deviated by 0.205 Å from the
mean plane defined by C12–C17–C16–C15–C14. This conforma-
tion also reveals the equatorial position on C11 and C13.
In the C15-flap half-chair conformation, the dihedral angles
C17–C12–C13–C14 and C16–C17–C12–C13 are 11.21 and ꢁ0.31,
Fig. 2 Representation of angles formed by planes of core carbonyl atoms
and cyclohexene for BC I. y = 49.591.
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Table 1 Crystal data and structure refinement for BC I
Chemical formula
MW
Crystal system, space group
Temperature
a, b, c
C20H23O1Cl1
314.83 g molꢁ1
Monoclinic, P21/c
296(2) K
11.4168(13) Å, 14.2639(18) Å,
11,1744(14) Å
a, b, g
V
901, 106.469 (3)1, 901
1745.1(4) Å3
Z
4
Radiation type
Mo Ka
0.219
0.0823
0.2431
m (mmꢁ1
)
R[F2 4 2s(F2)]
wR(F2)
S
1.030
3603
213
No. of reflections
No. of parameters
H-Atom treatment
H atoms treated by a mixture
of independent and constrained
refinement
Fig. 3 Molecular arrangement of BC
C6–H6ꢀ ꢀ ꢀO1 and C7–H7ꢀ ꢀ ꢀO1.
I
depicting hydrogen bonds
crystal packing is stabilized by two intermolecular hydrogen
bonding C6–H6ꢀꢀꢀO1 and C7–H7ꢀꢀꢀO1 interactions, which caused
two molecules to bind in a unit cell (Table 3).
Table 3 Hydrogen-bond geometry (Å, 1) for BC I
The dnorm HS (ranging from ꢁ0.511 to 1.470 Å) is visualized
in Fig. 4. This HS analysis is helpful in the identification of the
most dominant intermolecular interactions among neighboring
packs.32 These interactions observed on de and di surfaces of BC I
appear as two identical red spots. The red spot represents the
HꢀꢀꢀO (4.1%) and OꢀꢀꢀH (4.7%) interactions.
D–Hꢀ ꢀ ꢀA
D–H Hꢀ ꢀ ꢀA Dꢀ ꢀ ꢀA D–Hꢀ ꢀ ꢀA Symmetry code
BC I C6–H6ꢀ ꢀ ꢀO1 1.04 2.59 3.563 156.00
x, 1.5 ꢁ y, ꢁ1/2 + z
C7–H7ꢀ ꢀ ꢀO1 1.04 2.37 3.354 158.00
x, 1.5 ꢁ y, ꢁ1/2 + z
The HOMO and LUMO energies are good approximations
for ionization and electron affinity energies, respectively.47
The HOMO orbital is localized on the b-ionone. The b-ionone
appears to have p bonding, which is characteristic of the nucleo-
Fig. 5 displays 2D fingerprint plots of BC I, where most of the
contact is due to HꢀꢀꢀH contacts, which adds up to 55.3% of the HS
of BC I (Fig. 5a) because it is an organic compound. The contribu-
tions of OꢀꢀꢀH/HꢀꢀꢀO contacts, characterized by the spikes at the
bottom of the fingerprint plot, represent 8.9%, of the HS in BC I
(Fig. 5b). As for CꢀꢀꢀH/HꢀꢀꢀC and ClꢀꢀꢀH/HꢀꢀꢀCl contacts of BC I,
20.8% and 14.2% of the HS are observed (Fig. 5c and d); however,
the QTAIM analysis did not find these interactions.
philic region. The HOMO energy is negative (ꢁ147.05 kcal molꢁ1
)
and appears as a p bonding orbital. The LUMO orbital is a p
antibonding orbital localized on the CQO bond of the carbonyl
group. The LUMO energy is negative (ꢁ59.56 kcal molꢁ1). These
results show that BC I is an electrophilic species. This orbital
analysis gives information about interactions in both occupied
and unoccupied orbital spaces that could enhance the analysis of
Molecular modeling analysis
The root mean square (RMS) value of the compared calculated intermolecular interactions.
and experimental geometries obtained by structural overlapping
MEP is an important physicochemical tool that gives informa-
was 0.0247, as shown in Fig. 6. The overlay shows a good tion about molecular interactions and is very useful to predict the
agreement of geometric parameters. The cyclohexene ring exhibits reactive sites to be attacked in a chemical reaction. The electrostatic
dihedral angles of C14–C15–C16–C17 with the value of ꢁ45.91 potential can be calculated through eqn (1), as follows:
(Table 2).
ð
rðr0Þ
X
Za
The FMO obtained from Kohn–Sham analysis of BC
was carried out at the B3LYP/6-311++G(d,p) level of theory (Fig. 7).
I
VðrÞ ¼
ꢁ
dr0
(1)
jr ꢁ Raj
jr ꢁ r0j
a
Table 2 Relevant experimental and theoretical geometric parameters for BC I. Bond distance (Å), angles (1) and dihedral angles (1). The calculation was
carried out at the B3LYP/6-311++G(d,p) level of theory, in the gas phase
Experimental
Theoretical
Experimental
Theoretical
C7–C8
C10–C11
C12–C17
C17–C12–C11
C17–C12–C13
C2–C1–C7
C8–C7–C1
1.285(6)
1.332(5)
1.341(5)
123.3(3)
122.8(3)
123.2(4)
125.7(4)
1.3445
1.3445
1.3547
123.23
122.82
122.78
127.02
C7–C8–C9–O1
C6–C1–C7–C8
C10–C11–C12–C17
C12–C13–C14–C15
C13–C14–C15–C16
C14–C15–C16–C17
C15–C16–C17–C12
180.0(4)
ꢁ178.4(4)
ꢁ47.7(6)
ꢁ41.6(5)
60.0(6)
177.32
ꢁ179.75
ꢁ48.09
ꢁ46.63
60.23
ꢁ45.9(6)
ꢁ42.89
17.3(6)
15.18
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Fig. 6 Overlapping of B3LYP/6-311++G(d,p) level of theory (black) and
X-ray (yellow) structures of BC I.
with a value of about ꢁ34.19 kcal molꢁ1. On the other hand,
the positive region (blue) is around the hydrogen atom of the
a,b-unsaturated ketone, with an isovalue potential energy of
about 21.65 kcal molꢁ1. In this region, the chlorine atom at
the meta position with the aromatic ring is responsible for the
electron withdrawing effect. From these results, we can conclude
that the electrophilic attack is likely to occur on the carbonyl
group. The IR absorption bands are provided in Table 4, and the
experimental and calculated FTIR spectra are shown in Fig. 9.
Huang and Xing48 scaled by 0.961 the values of DFT vibrational
frequencies for the results obtained at the B3LYP/6-311++G(d,p)
level of theory. In chalcones, we must consider the resonance
effects of a,b-unsaturated ketones, which result in a lowering of
the frequency absorptions for carbonyl groups and alkenes. The
good correlation suggests that BC I in crystalline arrangement is
very close to the computed data obtained in the gas phase.
The QTAIM analysis was carried out within the quantum
chemical calculations required using Gaussian 09. This is a
powerful tool to analyze the inter-atomic interactions based on
electron density distribution.49 Nevertheless, the QTAIM affects
the supramolecular arrangement of molecules in the crystalline
state. The Multiwfn program50 was used within the QTAIM
approach. These interactions can be described and visualized
with the help of molecular graphs, and they show a crucial
bond path linking the proton-acceptor atoms, as shown in
Fig. 10 and Table 5.
Fig. 4 Hirshfeld surfaces plotted for the BC I dnorm surface. Dotted lines
are used to represent hydrogen bonds.
BC I is formed by two hydrogen bonds (C6–H6ꢀ ꢀ ꢀO1 and
C7–H7ꢀ ꢀ ꢀO1). The bond critical point (BCP) describes the
stationary point between these hydrogen donor atoms and
the acceptor atoms as a confirmation of the existence of
hydrogen bonding interaction; for the electron density at the
BCP of the proton-acceptor, r(r), a value of 0.022 a.u. was
calculated for C6–H6ꢀꢀꢀO1 and 0.017 a.u. for C7–H7ꢀꢀꢀO1 (in the
range of 0.002–0.040 a.u. typical of hydrogen bonds); the
second Hessian eigenvalue l2 is negative (so sign l2) and
identifies the bonding interaction; the positive Laplacian,
between 0.028 and 0.032 for C6–H6ꢀ ꢀ ꢀO1 and C7–H7ꢀ ꢀ ꢀO1,
Fig. 5 Fingerprint plots for BC I quantifying the different types of contacts
(a) Hꢀ ꢀ ꢀH, (b) Oꢀ ꢀ ꢀH, (c) Cꢀ ꢀ ꢀH and (d) Clꢀ ꢀ ꢀH. The full fingerprint is shown in
gray. The internal distance (di) from a given point on the Hirshfeld surface
and the closest external contact is (de).
where V(r) is the potential energy with a positive unit charge at r; Za respectively, is observed for noncovalent hydrogen bond inter-
is the nuclear charge a located at Ra, and r(r0) is the electron density actions. The total energy density value E(r) is small and negative
at point r0.
To understand the MEP for BC I, we used the tridimensional
for C6–H6ꢀ ꢀ ꢀO1, and C7–H7ꢀ ꢀ ꢀO1.
The hydrogen bond energy (EHB) is the energy obtained
MEP representation (Fig. 8), in which the most negative region from the interaction energy. According to Espinosa–Molins–
(red) is localized on the oxygen atom of the carbonyl group, Lecomte,51 the electron density at the BCP permits the
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Fig. 7 Frontier molecular orbitals for BC I derived from Kohn–Sham analysis at the B3LYP/6-311++G(d,p) level of theory with the isovalue of 0.02 atomic
units.
Fig. 8 Molecular electrostatic potential surface mapped for BC I. (a) The red-coloured carbonyl region is rich in electrons, and (b) the blue-coloured
region is electron-depleted. The density isovalue of 4.0 ꢂ 10ꢁ4 electrons per Bohr3 was used to generate the molecular electrostatic potential surfaces.
correlation of EHB to the potential energy of the electrons
V(rbcp), as shown in eqn (2):
Table 4 Theoretical and experimental vibrational assignments for BC I.
The calculation was carried out at the B3LYP/6-311++G(d,p) level of
1
EHB ¼ Vðrbcp
Þ
(2)
2
theory, in the gas phase
High values of EHB and the modulus of V(rbcp) are indicative
Vibrational mode
Exp. freq.a Scaled freq.a,b
2
of covalence if |V(rbcp)| 4 2|G(rbcp)| and r r(r) o 0. According
c
d
n
n
sym/asym(C–H)Ar ,Alk
3020–3000 3010–3000
2955–2867 2974–2857
to Rozas,52 these interactions have a hydrogen bond energy
EHB below 12 kcal molꢁ1, which is classified as weak. The
a,b-unsaturated group is stabilized by hydrogen bonds of
C–Hꢀ ꢀ ꢀO linking them with oxygen at the carbonyl group.
In addition, the hydrophobic interaction contributes to this
structural stability.
sym/asym(CH3) and nsym/asym(CH2)
n(CQO)
1647
1636
c
n(CQC)Ar
1617; 1475 1615; 1566; 1402
e
n(CQC)Heex
1583
1324
1431
1267
1180
1134
1566
1321
1441
1285
1168
1114
1089; 1109
n(C–C)Hex
d(CH3) in plane
d
d(C–H)Alk in plane
d(C–H)Hex out of plane
d(C–H)Hex and d(C–H)Alk in plane
n(C–C)Hex /Alk and d(C–H)Hex in plane 1093
d(C–H)Ar in plane
d(C–H)Alk out of plane
e
e
d
e
d
e
DNA docking analysis
c
1078; 1204 1051; 1144
DNA represents a traditional target for a wide range of anti-
cancer drugs, especially in regions of DNA involved in vital
processes.53,54 Drug–DNA interaction could change the DNA
structure as well as the cell metabolism and even terminate cell
growth. Along these lines, the development of new drugs that have
improved toxicity and pharmacokinetic profiles is critical. In order
to explore the interaction mechanisms and progress with the
design of a specific DNA target drug, the determination of binding
d
998;
906; 1357
871
966; 1001
1345
880
d(CH3) out of plane
c
d
d(C–H)Ar and d(C–H)Alk out of plane
c
d(C–H)Ar out of plane
784
715
766
713
e
d(C–CO) and d(C–H)Hex in plane
n(C–Cl)
703
705
n = stretching; d = bending; sym = symmetric; asym = asymmetric;
a
b
c
d
e
cmꢁ1
.
Scale factor 0.961. Ar = aromatic ring. Alk = alkene. Hex =
cyclohexene ring.
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mode is crucial. Because of the problems and monetary costs according to the AutoDock scoring function was selected. The
of experimental strategies for determining the crystallographic study revealed a good binding affinity with DNA and the highest
structure of the complex, in silico methods are sought, to predict affinity of ꢁ6.9 kcal molꢁ1 docking energy. Generally, all poses
binding modes.54
have shown high capability to bind DNA in a groove binding
The interaction of BC I with DNA (PDB ID: 1BNA) was mode. Fig. 11 shows that the bischalcone was completely
investigated by molecular docking,55 and the lowest energy enfolded in the minor groove of the targeted DNA and stabilized
by van der Waals interaction and hydrophobic contacts in the
GC-rich region. In particular, minor groove binders are an impor-
tant series of derivatives in anticancer treatment.54 In this model,
the compound BC I formed two hydrogen bonds between the
oxygen atom of the carbonyl group and hydrogen of two guanines,
which are DG16 and DG10, with the bond length of 2.42 Å and
1.88 Å, respectively, as shown in Fig. 12. In this manner, an
accessible acceptor atom in the compound, which is involved in
hydrogen bonding with hydrogen of guanine, assumes an essential
role in the DNA binding studies of bischalcone. Consequently, a
good correlation between previous theoretical outcomes and
molecular docking provides valuable information on the inter-
action of BC I with DNA, since there is no co-crystallization with
DNA information.
Cytotoxic activity
The cytotoxic activity of BC I for the tumor cell line S180,
Ehrlich-ascites and non-tumor Vero cells was assayed by
observing cell viability after 48 h of exposure. Fig. 13 shows
the dose–response curves for S180 (a), Ehrlich-ascites (b) and
Vero cells (c) analysed by MTT assay.
Fig. 9 Experimental (red) and theoretical (black) overlapped FT-IR spectra
of BC I.
As shown in Table 6, BC I inhibited the viability of the
S180 and Ehrlich-ascites tumor cell lines, with IC50 values of
19.83 mM and 38.35 mM, respectively. The results in Table 6 and
the dose–response curves in Fig. 13a and b show that BC I is
more active against S180 cells compared with the Ehrlich-ascites
cells. Under the same conditions, cisplatin showed IC50 values
of 29.05 mM against S180 tumor cells and 62.42 mM against
Ehrlich-ascites tumor cells (Table 6). In particular, BC I
has a higher SIa of 1.33 for S180 cells and SIb of 0.68 for
Ehrlich-ascites tumor cells compared with cisplatin, with the
SIa of 0.3 (S180) and SIb of 0.15 (Ehrlich-ascites). These results
suggest that BC I is generally more selective for S180 cancer
cells compared with Vero non-tumor cells.
The cytotoxic activity of chalcone and chalcone derivatives
can result from cycle cell arrest, induction of death cell,
or a combination of these two mechanisms.56,57 Flow cyto-
metric analysis was performed to determine the possible
cytotoxicity mechanisms of BC I in S180 tumor cells. As shown
in the cell cycle analysis (Fig. 14), exposure of the S180 cells
to BC I led to a significant increase in the percentage of
cells in the G0/G1 and S phases, which was accompanied
Fig. 10 Molecular graphs showing the BCP yellow points.
2
Table 5 Topological parameters of BC I describing intermolecular interactions (electron density at BCP (r(r))), Laplacian (r r(r)), the potential electron
energy density (V(r)), the kinetic electron energy density (G(r)), the total electron energy density (E(r)), |V(r)|/|G(r)| and bond energy (EHB
)
2
b
BCP
Interaction
r(r)a
r r(r)a
V(r)a
G(r)a
E(r)a
EHb
Kind of interaction
1
2
C6–H6ꢀ ꢀ ꢀO1
C7–H7ꢀ ꢀ ꢀO1
0.02211
0.01756
0.02819
0.03243
ꢁ0.00932
ꢁ0.01175
0.00818
0.00993
ꢁ0.00113
ꢁ0.00182
ꢁ2.9241
ꢁ3.6866
Weak
Weak
a
b
Atomic units. kcal molꢁ1
.
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by a corresponding reduction in the percentage of cells in the
G2/M phase. The fraction of cells increased from 11.28%
(phase G0/G1) and 38% (phase S) in the control to 19.26%
(phase G0/G1) and 63.30% (phase S) after exposure to 20 mM
BC I. In relation to the G2/M phase, there was a decrease in the
fraction of cells from 50.70% in the control to 17.43% in cells
after exposure to BC I.
To evaluate the mechanism of S180 cell death induced by
BC I, the FITC-Annexin V/PI assay was used. As shown in
Fig. 15a and b, after exposure of the S180 cells to 20 mM BC I
for 48 h, the cells in initial apoptosis (Annexin V+ and PIꢁ)
represented 1.88%, cells in late apoptosis (Annexin V+ and PI+)
represented 12.97% and cells in necrosis (Annexin Vꢁ and PI+)
represented 39.04% (p o 0.001) of the total cells. The distribu-
tion of cells is shown in the dot plot in Fig. 15b. The results
showed that the mechanism of S180 cell death induced by BC I
may involve a necrosis and apoptosis process. Thus, the two
Fig. 11 Molecular docking for BC I with DNA (PDB ID: 1BNA).
Table 6 IC50 (mM) and selectivity indexes (SIs) of BC I and cisplatin in S180,
Ehrlich-ascites tumor cells and Vero non-tumor cells
IC50 (mM)
S180
Ehrlich
Vero
SIa
SIb
BC I
19.83 ꢃ 1.22 38.35 ꢃ 1.39 26.43 ꢃ 1.49 1.33 0.68
Cisplatin 29.05 ꢃ 1.88 62.42 ꢃ 1.85 8.75 ꢃ 0.25 0.3 0.14
SIa(IC50 Vero/IC50 S180). SIb(IC50 Vero/IC50 Ehrlich).
Fig. 12 Molecular docking for BC I showing two hydrogen-bonding
interactions with DNA (PDB ID: 1BNA).
Fig. 13 Dose–response curves for (a) S180, (b) Ehrlich-ascites and
(c) Vero cells analysed by MTT assay. The data are the means ꢃ SD of
three experiments. Significant differences from the untreated control are
indicated by ***p o 0.001.
Fig. 14 Effect of BC I on the cell cycle distribution of S180 cells after 48 h
of exposure. The data are the means ꢃ SD of three experiments. Significant
differences from the untreated control are indicated by ***p o 0.001.
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`
´
Amparo a Pesquisa do Estado de Goias (FAPEG) for financial
support. Also, the authors are grateful to the High Performance
´
Computing Center of the Universidade Estadual de Goias (UEG).
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The BC I compound was synthesized by Claisen–Schmidt metho-
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˜
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´
˜
de Pessoal de Nıvel Superior (CAPES) and the Fundaçao de
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