Structural characterization, DFT calculations, ADMET studies, antibiotic potentiating activity, evaluation of efflux pump inhibition and molecular docking of chalcone (E)-1-(2-hydroxy-3,4,6-trimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one

Article abstract of DOI:10.1016/j.molstruc.2020.129692

Chalcones are open-chain flavonoids characterized by two aromatic rings joined by a three-carbon α,β-unsaturated carbonyl system. Over the last several years, chalcones have instigated the interest of chemical and pharmacological researchers due to their

Full text of DOI:10.1016/j.molstruc.2020.129692

Journal Pre-proof
Structural characterization, DFT calculations, ADMET studies,
antibiotic potentiating activity, evaluation of efflux pump inhibition and
molecular docking of chalcone (E)-1-(2-hydroxy-3,4,6-
trimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one
Jayze da Cunha Xavier Investigation. Data curation. Formal analysis ,
Francisco Wagner Queiroz Almeida-Neto ,
Priscila Teixeira da Silva , Amanda Pereira de Sousa ,
Emmanuel Silva Marinho , M a´ rcia Machado Marinho ,
Janaina Esmeraldo Rocha , Priscila Ramos Freitas ,
Ana Carolina Justino de Ara u´ jo , Thiago Santiago Freitas ,
Carlos Em ´ı dio Sampaio Nogueira , Pedro de Lima-Neto ,
Paulo Nogueira Bandeira , Alexandre Magno Rodrigues Teixeira ,
Henrique Douglas Melo Coutinho , H e´ lcio Silva dos Santos
PII:
S0022-2860(20)32005-6
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.129692
MOLSTR 129692
To appear in:
Journal of Molecular Structure
Received date:
Revised date:
Accepted date:
2 August 2020
12 November 2020
26 November 2020
Please cite this article as: Jayze da Cunha Xavier Investigation. Data curation. Formal analysis ,
Francisco Wagner Queiroz Almeida-Neto , Priscila Teixeira da Silva , Amanda Pereira de Sousa ,
Emmanuel Silva Marinho ,
Priscila Ramos Freitas ,
Carlos Em ´ı dio Sampaio Nogueira ,
M a´ rcia Machado Marinho ,
Ana Carolina Justino de Ara u´ jo ,
Pedro de Lima-Neto ,
Janaina Esmeraldo Rocha ,
Thiago Santiago Freitas ,
Paulo Nogueira Bandeira ,
Alexandre Magno Rodrigues Teixeira , Henrique Douglas Melo Coutinho , H e´ lcio Silva dos Santos ,
Structural characterization, DFT calculations, ADMET studies, antibiotic potentiating activity,
evaluation of efflux pump inhibition and molecular docking of chalcone (E)-1-(2-hydroxy-3,4,6-
trimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one, Journal of Molecular Structure (2020), doi:
https://doi.org/10.1016/j.molstruc.2020.129692
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
©
2020 Published by Elsevier B.V.

Structural characterization, DFT calculations, ADMET studies, antibiotic
potentiating activity, evaluation of efflux pump inhibition and molecular
docking of chalcone (E)-1-(2-hydroxy-3,4,6-trimethoxyphenyl)-3-(4-
methoxyphenyl)prop-2-en-1-one
a
b
Jayze da Cunha Xavier , Francisco Wagner Queiroz Almeida-Neto , Priscila Teixeira da
a
f
c
g
Silva , Amanda Pereira de Sousa , Emmanuel Silva Marinho , Márcia Machado Marinho ,
a
a
Janaina Esmeraldo Rocha , Priscila Ramos Freitas, Ana Carolina Justino de Araújo ,
a
a,e
b
Thiago Santiago Freitas , Carlos Emídio Sampaio Nogueira , Pedro de Lima-Neto , Paulo
f
a
Nogueira Bandeira , Alexandre Magno Rodrigues Teixeira , Henrique Douglas Melo
a
a,d,f*
Coutinho , Hélcio Silva dos Santos
a
Department of Biological Chemistry, Regional University of Cariri, Crato, CE, Brazil,
Corresponding author. Postgraduate Program in Biological Chemistry, Department of
*
Biological Chemistry, Regional University of Cariri, Campus Pimenta II, CEP: 63.100-000,
Crato, CE, Brazil. E-mail addresses: helciodossantos@gmail.com
HIGHLIGHTS





A chalcone was synthesized by Claisen-Schmidt condensation reaction.
Structural, electronic and reactivity properties were obtained using DFT.
ADMET studies were made.
Antimicrobial activities assays were done.
Molecular docking studies were carried out.

b
Group of Theoretical Chemistry, Department of Analytical Chemistry and Physical
Chemistry, Federal University of Ceará, Fortaleza, CE, Brazil
c
Group of Theoretical Chemistry and Electrochemistry, State University of Ceará, Campus
FAFIDAM, Limoeiro do Norte, CE, Brazil.
d
Science and Technology Center, Postgraduate Program in Natural Sciences, State
University of Ceará, Fortaleza, CE, Brazil
e
Department of Physics, Regional University of Cariri, Juazeiro do Norte, CE, Brazil.
f
Center for Exact Sciences and Technology - Chemistry Course, Vale do Acaraú
University, Sobral, CE, Brazil
g
Iguatu Faculty of Education, Science and Letters (Fecli), State University of Ceará, Iguatu,
Ceará, Brazil
Graphical abstrcats

Abstract
Chalcones are open-chain flavonoids characterized by two aromatic rings joined by a
three-carbon α,β-unsaturated carbonyl system. Over the last several years, chalcones have
instigated the interest of chemical and pharmacological researchers due to their simple
chemical structure and varied biological activities. Here, we performed the electronic
properties,
of
the
chalcone,
(E)-1-(2-hydroxy-3,4,6-trimethoxyphenyl)-3-(4-
methoxyphenyl) prop-2-en-1-one synthesized by Claisen-Schmidt condensation reaction.
The density functional theory method was used with the B3LYP/6-311++G(d,p) level of
theory to compute the structural, electronic, and reactivity properties of the chalcone. In
addition, microbiological tests were performed to investigate the modulator potential and
efflux pump inhibition on Staphylococcus aureus multi-resistant strains. The spectroscopic
data analyses allowed drawing the molecular structure of the chalcone synthetized with
subsequent confirmation using the quantum chemical calculations. The addition of
chalcone to the growth medium caused a synergic effect by reduction of the minimum
inhibitory concentration (MIC) values for ciprofloxacin strain. Docking results showed that
the chalcone docks in almost the same way as the antibiotic against a MepA model.
Druglikeness criteria based on the rules of Lipinski and Veber evaluated that the chalcone
has the ideal physicochemical and pharmacokinetic properties to be a good candidate for
drug orally. The results confirm the structure of the synthesized chalcone and revealing that
the compound can be used as a possible inhibitor of the Mep A efflux pump.
Keywords:
Chalcone,
ATR-FTIR;
UV-Vis;
Fukui,
efflux
pump

1. Introduction
The search for new drugs is increasing due to the rapid growth of bacterial
resistance that has become a medical concern today [1-3]. The bacterial resistance comes
from factors such as selectivity (low plasma membrane permeability), adherence of the
drug to the cell wall, its expulsion by efflux pumps, and the action of enzymes minimizing
or inhibiting the antibiotic action [4, 5]. Among bacterial pathogens, S. aureus, despite
being present in the skin and nasal mucosa, is capable of causing many infections and
diseases both in animals and in humans since skin infections [6], toxic shock syndrome [7],
to sepsis [8]. These opportunists reach debilitated people in hospitals and are difficult to
treat. Fighting these infections has become a difficult problem for health worldwide due to
the growth of bacterial resistance to antibiotics [9]. In this sense, there are current interests
in finding new antimicrobial agents [10].
Understanding the mechanism of action and metabolism of the biologically active
molecules depends, to a large extent, on the analysis of its reactivity, which is determined
by intermolecular forces, that is, lipophilic, polar, electrostatic, and stereo interactions,
which depend on the characteristics of each molecule (electronic, Steric or conformational,
energetic and thermodynamic [11, 12]. It is precisely at this point that molecular modeling
techniques can make their contribution to the study and development of active molecules
because through it you can have a detailed description of the structure, intermolecular
interactions, and, if applicable, chemical reactions between the ligand and
biomacromolecules with chalcones [13-16].
Also, the electronic properties with respect to the frontier molecular orbitals, the
quantum reactivity descriptors, the Fukui functions (electronic and condensed), and the
molecular electrostatic potential were applied to characterize the reactive electrophilic and
nucleophilic sites, which is extremely important because in previous studies the higher
reactivity and electrophilicity of a synthetic chalcone, can be responsible for intermediated
interactions with the bacterial cell wall and when combined with the gentamicin drug
proved to be more efficient [17].
Previous studies showed that synthetic chalcones associated with antibiotics had
obtained increasingly satisfactory results in containing resistant pathogen [18-24].

Chalcones are precursors to flavonoids and isoflavonoids [25, 26], but they can also be
obtained by synthesis [23, 27]. This class of compound has aroused much interest due to
the broad spectrum of pharmacological activities that they present, including antimicrobial
[
23, 28], antioxidant [29], antinociceptive [30, 31], antiparasitic [32], antitumor [33], and
antiproliferative activities [34].
In this work, the chalcone (E)-1-(2-hydroxy-3,4,6-trimethoxyphenyl)-3-(4-
methoxyphenyl) prop-2-en-1-one (C H O ) was synthesized by Claisen-Schmidt
1
9
20
6
condensation reaction. The molecular structure was characterized by spectroscopy methods,
and the Density Functional Theory was used to determine the electronic and reactivity
properties. In addition, microbiological tests were performed to investigate the modulator
potential and efflux pump inhibition on S. aureus multi-resistant strains.
2. Material and methods
2.1 Spectroscopic methods: NMR, FTIR, UV-vis
1
13
The chemical reagents were purchased from Sigma-Aldrich. H and C NMR
spectra were obtained using a Bruker Spectrometer, model Avance DPX-500, operating at a
frequency of 500 MHz for hydrogen and 125 MHz for carbon, respectively. The spectra
were measured in CDCl , and chemical shifts are reported as δ values in parts per million
3
(
ppm) relative to CDCl . The attenuated total reflectance Fourier transform infrared
3
spectroscopy (ATR-FTIR) was performed using a Bruker vacuum infrared spectrometer
VERTEX 70V. The measurements were recorded at room temperature in the range 400 to
1
1
4
000 cm⁻, with a resolution of 2 cm⁻ and accumulating 60 scans per spectrum. The UV-
Visible absorption spectrum of the compound was obtained using a GENESYS 10S UV-
Vis spectrophotometer (Thermo Scientific, Waltham, MA, USA) in the wavelength range
of
210 to 400 nm with spectral resolution (1.8 nm). The samples were prepared at a
1
concentration of 0.1 mmol L⁻ using ethanol as a solvent to perform the absorption
measurements in the UV-Vis region. All absorption measurements were carried out at room
temperature in quartz cells with 1 cm optical path.

2.2 Synthesis of the chalcone
The compounds 2-hydroxy-3,4,6 trimethoxyacetophenone (2 mmol) and
Anisaldehyde (2 mmol) were placed in a volumetric flask (25 mL). Then 5 mL of ethanolic
NaOH (50%) solution was added and mixed with stirring for 48 h at room temperature. The
progress of the reaction was checked by TLC (n-hexane: ethyl acetate, 2:1). After 48 h, the
reaction mixture was neutralized with dilute HCl (10%) and ice water added. The product
was obtained as a yellow solid filtered under reduced pressure, washed with cold water, and
recrystallized from ethanol [27, 35] (Scheme 1).
2.3 Quantum chemical computational calculations
The quantum chemical calculations were used extensively to support the
experimental data during the structural characterization of a newly synthesized molecule
using the density functional theory (DFT) method. From a previous work from our group,
three DFT methods were compared for the characterization of a chalcone acetamide
derivative (PAAPFBA) [17], and all the methods used were accurate to describe the
structural and electronic properties of a chalcone derivative molecule. Hence, due to the
low computational cost, the title chalcone was geometrically optimized using the exchange-
correlation functional B3LYP at the 6-311++G(d,p) basis set [36, 37], which was the same
method used in other work of our group to describe the structural and electronic properties
of the chalcone derivative (2E,4E)-1-(2-hydroxy-3,4,6-trimethoxyphenyl)-5-phenylpenta-
2,4-dien-1-one (cinnamaldehyde) [38]. The quantum chemical calculations were carried out
using the Gaussian 09 [39] software and the Gauss View 5 [40] to draw the input molecule.
The optimization was done with chloroform as an implicit solvent using the IEF-PCM
solvation model [41-43] available in Gaussian 09. Conformational analysis was carried
from the optimized structure to obtain the potential energy surface (PES). Next, the
fundamental vibrational modes were computed at the same level of theory together with the
thermodynamic properties at 298.15 K and 1 atm of pressure. The absence of negative

frequencies implies that the simulated molecule can be used to describe the structural and
electronic properties of the newly synthesized chalcone. The infrared spectrum was
obtained from the optimized molecular geometry, and the theoretical wavenumbers were
1
1
scaled by 0.983 for below 1700 cm⁻ and by 0.958 for above 1700 cm⁻ [44]. The
theoretical assignments of the highlighted band from the experimental infrared spectrum
were done using the potential energy distribution (PED) with the VEDA [45] software, and
1
13
only the PED 10% were considered. Then, the H and C NMR spectra were calculated
using the GIAO method [46-48] available in Gaussian 09 to obtain the theoretical isotropic
shielding for the carbon (ꢀ) and hydrogen (ꢀ ) atoms. Tetramethylsilane (TMS) was used
ꢁ
ꢂ
as a reference compound to calculate the theoretical chemical shifts as follows: ꢃ ꢄ
ꢁ
ꢅꢀꢁ(ꢆꢇꢈ)ꢅꢉꢅꢀ and ꢃ ꢄꢅꢀ
ꢉꢀ . The frontier molecular orbitals (FMO) were
ꢂ
ꢁ
ꢂ
ꢂ(ꢆꢇꢈ)
calculated from the optimization calculation to understand the electronic density
distribution over the entire molecule and the energy values of those molecular orbitals were
used to compute the global quantum reactivity descriptors: the HOMO-LUMO energy gap
(
ꢊꢋꢌꢍꢎ, equation 1) [49]; From the Koopmans’ theorem [50], the ionization potential (I,
equation 2) and the electron affinity (A, equation 3); According to the works of Chermette
51] and Iczkowski and Margrave [52], the electronegativity (χ, equation 4); From the
[
Pearson’s Hard and Soft Acid and Base (HSAB) theory [53] and according to the Janak
theorem [54], and the work of Szentpály [55], the global hardness (η, equation 5) and the
global softness (S, equation 6) introduced in the work of Yang and Parr [56]; the
electrophilicity index (ω, equation 7) according to the work of Parr and Szentpály [57]; the
nucleophilicity index (ε, equation 8) from the work of Chattaraj [58]. The trial version of
the ChemCraft [59] software was used to render the frontier molecular orbitals.
ꢊꢋꢌꢍꢎ ꢄꢅꢋꢏꢐꢇꢑꢉꢋꢂꢑꢇꢑ
ꢒꢄꢅꢉꢅꢋꢂꢑꢇꢑ
(1)
(2)
(3)
ꢓꢄꢅꢉꢅꢋꢏꢐꢇꢑ
ꢒꢕꢓ
(4)
ꢔꢄꢅ
ꢖ

ꢒꢉꢓ
(
5)
6)
ꢗꢄꢅ
ꢖ
ꢙ
(
ꢘꢄꢅ
ꢗ
ꢔꢛ
ꢖꢗ
(7)
8)
ꢚꢄꢅ
ꢜꢄꢅ
ꢙ
(
ꢚ
To complete the understanding of the reactivity of chalcone, the following Fukui
functions are applied: Electronic [60] (equations 9 – 12) and condensed [61] (equations 13
ꢞ
ꢞ
ꢠ
ꢠ
ꢠ
ꢡ
–
15) to the nucleophilic (ꢝ and ꢝ ), electrophilic (ꢝ and ꢝ ), and radical (ꢝ and ꢝ )
ꢟ ꢟ ꢟ
attack; and the local quantum descriptors: the dual descriptor [62] (∆f, equation 16) and the
multiphilic index [63] (∆ω, equation 17) to characterize the nucleophilic and electrophilic
sites of the molecule. The electronic Fukui functions were obtained using the Multiwfn [64]
software, and the isosurfaces were rendered using the VESTA [65] software. The molecular
electrostatic potential (MEP) was computed at B3LYP/6-311++G(d,p) computational level
to complete the local analysis of the chemical reactivity of the chalcone. The Gabedit
program [66] was used to compute the MEP.
ꢣꢤ(ꢥ)
(9)
ꢝꢄꢅꢢ
ꢧ
ꢣꢦ
ꢨꢅ(ꢩ)
ꢝꢞꢪꢅꢫꢏꢐꢇꢑ
ꢝꢠꢪꢅꢫꢂꢑꢇꢑ
(10)
(11)
(12)
ꢝꢡ ꢪꢅꢫꢏꢐꢇꢑ
ꢕꢅꢫꢂꢑꢇꢑ
ꢖ
ꢞ
ꢝ ꢄꢬ (ꢦꢕꢙ)ꢉꢬ (ꢦ)
ꢟ
ꢟ
ꢟ
(13)
(14)
(15)
ꢠ
ꢝ ꢄꢬ (ꢦ)ꢉꢬ (ꢦꢉꢙ)
ꢟ
ꢟ
ꢟ
ꢬ (ꢦꢕꢙ)ꢉꢬ (ꢦꢉꢙ)
ꢟ
ꢟ
ꢡ
ꢝ ꢄꢅ
ꢟ
ꢖ
ꢞ
ꢠ
ꢊꢝꢄꢅꢝ ꢉꢝ
(16)
(17)
ꢟ
ꢟ
ꢊꢚꢄꢅꢚꢭꢊꢝ

Where the ꢬ (ꢦꢕꢙ), ꢬ (ꢦ), and ꢬ (ꢦꢉꢙ)are the Hirshfeld charge population
ꢟ
ꢟ
ꢟ
[
67] on the atom k of the anionic, neutral, and cationic species.
Finally, the UV-Vis spectrum was calculated using the Time-Dependent Density
Functional Theory (TD-DFT) using the CAM-B3LYP [51] at 6-311++G(d,p) level of
theory in ethanol as an implicit solvent and the electronic transition was assignment using
the GaussSum 3.0.1 [68, 69].
2.4 Assays for modulation of the antibiotic resistance and evaluation of efflux pump
inhibition by MIC reduction
Antimicrobial activity of the chalcone was evaluated against S. aureus K2068,
which are resistant to ciprofloxacin due to overexpression of the MepA genes encoding the
MepA efflux pumps. The SA-K2068 strains were provided by Dr. Glenn W. Kaatz, John
Dingell VA Medical Center, Detroit, MI, USA. Evaluation of the modulatory effect on the
resistance to antibiotics was performed according to previously described [70]. Briefly, 150
µL of each suspended bacterial inoculum in saline solution, corresponding to 0.5 of the
McFarland scale, were added to microtubes (2mL) together with 1350 µL of Brain heart
infusion (BHI) as a control. In tests, 150 µL of each suspended bacterial inoculum in saline
solution, corresponding to 0.5 of the McFarland scale, were added together with EPIs
(MIC/8) and completed with brain heart infusion. These were then transferred to 96-well
microtiter plates, and 100 µL of the antimicrobial drug and ethidium bromide serial
dilutions were performed (1:1). The plates were incubated at 37 °C for 24 h, and bacterial
growth was assessed by using resazurin. MIC was defined with antibiotics and ethidium
bromide concentrations ranging from 0.5 to 512 µg/mL. The MIC of controls was assessed
using the antibiotics and ethidium bromide alone [71].
2.5 Docking procedure

The MepA model was generated by retrieving the protein sequence for the NCTC 8325
strain from the Uniprot database. Then, the SWISS-MODEL [72] service was used to build
the homology model. Of all the 50 templates generated, the one with the best Global Model
Quality Estimation score was based on the structure of the multidrug and toxic compound
extrusion transporter of the Bacillus halodurans (PDB-ID: 5C6N). For the docking
procedure, which was carried out using the Autodock Vina [73] software, the grid box was
defined as a 126Åx126Åx126Å box around the geometrical center of the model, with
spacing = 0.431 e exhaustiveness = 8. Partial Gasteiger charges were added to ligand
atoms, non-polar hydrogen atoms were mixed while all other parameters were kept at their
default values. The best results were chosen based on the binding score.
2.6 Evaluation of physicochemical properties and drug-likeness criteria
Calculations of physical and chemical properties, which include acid dissociation
constant (pKa), molecular weight (MW), consensus partition coefficient (Clog P), hydrogen
bond donor (HBD) and acceptor (HBA) count, distribution coefficient at pH 7.4 (log
DpH7.4), solubility coefficient at pH 7.4 (log SpH7.4), topological polar surface area (TPSA),
number of rotatable bonds (Nrot) were performed using the plugins provided in the
MarvinSketch®
code [74]. To validate the ―drug-likeness‖ criteria [75], the
bioavailability radar was used, generated on the SwissADME web-server [76], which uses
an algorithm based on a filter that combines the descriptors of the ―rule of five‖ of Lipinski
et al. [75] (MW≤500, log P≤5, H-bond donors≤5 e H-bond acceptors≤10) and the rule by
7
Veber et al., (2002) (TPSA≤140 e rotatable bonds≤10), establishing the parameters of
lipophilicity
(-0.7<log
P<5.0),
size
(150g/mol<MW<500g/mol),
polarity
(20Ų<TPSA<130Ų), insolubility (-6<log S<0), unsaturation (0.25<Fraction Csp3<1) and
flexibility (0<Num. Rotatable bonds<9) as a determining spatial spectrum for good oral
bioavailability.
2.7Virtual screening of pharmacokinetics properties
Molecular descriptors of structure-activity relationship (QSAR) of the properties of
absorption, distribution, metabolism, and excretion (ADME) were used to evaluate the
pharmacokinetic behavior of chalcone. Through the MW, log P, and TPSA descriptors,

statistical evaluation was made for the models of human intestinal absorption (HIA) [77],
human adenocarcinoma colon cells permeability (Caco-2) [78], blood-brain barrier
penetration (BBB) [79] and P-glycoprotein substrate (Pgp) [80].
2.8 Evaluation of the multi-target bioactivity
To
evaluate
bioactivity,
the
code
SMILES
[
COc2ccc(C=CC(=O)c1c(O)c(OC)c(OC)cc1OC)cc2] was generated, using the plug-in
JSME molecular editor [81], deployed on the web-server Molinspiration
(https://molinspiration.com/) and submitted to interaction simulation with main human
biological targets: G-protein coupled receptor (GPCR), ion channel modulator, a kinase
inhibitor, nuclear receptor ligand, protease inhibitor, and enzyme inhibitor. The simulation
is based on the test of similarity of the ―drug-likeness‖ properties of known compounds
deposited in the server database, and the results are available in scores, where values above
0.5 indicate strong interaction with the specified target, between 0.2 and 0.5 moderate
interactions and for values less than 0.2 low interaction potential.
2.9 Acute oral toxicity prediction model
For the prediction of Acute oral toxicity, the LabMol web-server LabMol
(http://stoptox.labmol.com.br) was used, which has its algorithm based on the structure-
activity relationship (QSAR) of the acute oral toxicity model in bees, meeting the
guidelines of the Organization for Economic Cooperation and Development (OECD) and
the Registration, Evaluation, Authorization, and restriction of chemical substances
(REACH). For this test, results with a confidence index of 70% have closer proximity to
the acute toxicity indexes in bees and are expressed in a contribution map by the toxic
fragments, where the green-colored region is the positive contribution and the region
colored red the negative contributions to this class of toxicity.
2.10 Statistical Analysis
All bacteriological tests were performed in triplicates. Data were analyzed using a two-
way ANOVA followed by Bonferroni’s post-hoc test (where p<0.05 was considered

significant). The geometric mean of the triplicates was used as the central data ± standard
error of the mean. The GraphPad Prism 5.0 statistical program was used for the analysis.
3. Results and discussion
3.1 Conformational analysis
Chalcones may exist in two isomeric forms, E- (trans) or Z- (cis). The E-isomer is
the thermodynamically most stable form in most cases [82]. The title chalcone possesses
the trans form. It is clearly known that there are differences in the structural conformations
of chalcones, especially between some of their dihedral angles [83].
The relaxed PES scan of the title chalcone calculated at the B3LYP/6-311++G (d,p)
level for the dihedral angle  (C6-C1-C-C) with step variation of 15° is shown in Figure
1. The dashed red line in the plot in this figure corresponds the zero energy. The potential
energy curve shows three minimum energy structures, two global minima around -180 °
and + 180 °, and a local minimum at 0°. The conformations at -180 ° and + 180 are
equivalent, and correspond the configurations I and III, while the conformation at 0°
corresponds the configuration II. According to our calculation, the configuration I is more
stable than configurations II and III by 1.23 kJ/mol. It is also noted that the conformers I, II
and III presented the isomeric form E- (trans). Figures. 2 shows the lower energy
conformers of the title chalcones.
3.1 NMR data
1
The H NMR spectrum of this chalcone showed signals at δ 3.78 - 3.91 ppm relative
H
to the hydrogens of methoxy groups. These signals can also be observed in the
1
3
C NMR spectrum at δ 55.5 to 60.9 ppm. The signals at δ 7.24 and 7.75 ppm (J = 14.6
C
H
Hz) were attributed to doublets referring to α,β-unsaturated hydrogens, whose coupling
1
3
constant (J) confirms the stereochemistry E, in the C NMR, these signals are observed at
δC 142.9 (Cβ) and 128.4 (Cα) being related to olefinic carbons α and β, respectively. The
singlet observed at δ 5.93 ppm refers to hydrogen attached to the carbon 5' whose signal is
H
1
3
observed at δ 87.3ppm in the C NMR spectrum. The signal at δ 193.3 ppm concerns to
C
C

α,β-unsaturated carbonyl group. The ketone absorbs 203.8 ppm, however, the presence of
α, β unsaturation, causes a displacement to the high field, and the probable cause is the
delocalization of charge by the benzene ring or by the double bond that makes carbonyl
carbon less electron deficient. In addition, the signs at δ 159.5 (C-4´), 158.7 (C-2´), 158.6
C
(C-6´), 130.3 (C-3´), and 107.1 (C-1´) refer to the non-hydrogenated carbons present in ring
A of the chalcone. The signals at δ 7.53 (d, J = 8.4 Hz, H-2/6) and 7.89 ppm (d, J = 8.4
H
13
Hz,
H-3/5) are related to the signs at 125.2 (C-2/6) and 130.3 (C-3/5) in the C
NMR spectrum, and the signs at δ 131.0 (C-4) and 130.7 ppm (C-1) refer to the non-
C
hydrogenated carbons present in ring B (Table 1). This chalcone has two aromatic rings
linked by a three-carbon α,β-unsaturated carbonyl system. The ring A is formed by the
atoms C1ʹ, C2ʹ, C3ʹ, C4ʹ, C5ʹ and C6ʹ, and the ring B is formed by the atoms C1, C2, C3,
C4, C5, and C6. The molecule was drawn according to this experimental result, and the
geometrical optimization calculation carried out using the B3LYP/6-311++G(d,p)
computational level in chloroform as an implicit solvent. The optimized structure for the
1
13
chalcone is shown in Figure 2. The H and NMR were theoretically calculated at the same
level of theory as the optimization. The theoretical results for the calculated chemical shift
were displaced in Table 1. To understand the correlation between the experimental and
theoretical data, the linear fitting was made using the experimental and calculated chemical
1
13
shifts for the H and C NMR (Figure 3). The linear correlation was computed with the
coefficient of determination (R²) of 0.98669 with the linear equation of y = 0.94812x +
0.77857. This result shows an excellent agreement between the experimental and
theoretical data; hence the chalcone had its molecular structure confirmed to be the same as
in Figure 2.
The molecular structure of the chalcone was described to the in the global minimum of
energy due to the absence of any imaginary frequency in the vibrational spectrum; hence
the simulated molecule describes the real molecule of the chalcone. The molecule is not
planar: the ring A is about 54.161° out of the plane delimited by the α,β-unsaturated
carbonyl. The O18, C8, Cα, and Cβ structure (α,β-unsaturated carbonyl) is almost planar
with a little deviation of -1.081°. The ring B is in the same plane as the structure previously
describes with an angle of only 0.112°. The four methoxy groups showed bond length for
the carbon (ring)-oxygen sigma bond of 1.38151 Å (C3′-O27), 1.35811 Å (C4′-O28),

1.36371 Å (C6′-O24), and 1.35837 Å (C4-O41). The hydroxyl group showed a bond length
of 1.35693 Å (C2′-O23), and the carbonyl group value of 1.22753 Å (C8-O10). The π-bond
between the carbon atoms Cα and Cβ demonstrate the bond length of 1.34848 Å, and the
bonds Cα-H12 and Cβ-H14 the values respectively of 1.08240 Å and 1.08747 Å. The
bonds C8-C1′, C8-Cα, and Cβ-C1 have a bond length of 1.50254 Å, 1.47647 Å, and
1
.45611 Å, respectively. For the methyl groups, the bond lengths are 1.44312 Å (O27-
C29), 1.09379 Å (C29-H31), 1.08916 Å (C29-H30), and 1.09052 Å (C29-H32); 1.42931 Å
O28-C33), 1.08798 Å (C33-H34), 1.09370 Å (C33-H35), and 1.09349 Å (C33-H36);
.42683 Å (O24-C37), 1.08816 Å (C37-H38), 1.09408 Å (C33-H39), and 1.09397 Å (C37-
H40); 1.42929 Å (O41-C42), 1.08794 Å (C42-H43), 1.09377 Å (C42-H44), and 1.09375 Å
C42-H45). For the ring A, the unique C-H bond showed a bond length of 1.07783 Å. For
the ring B, the four C-H bonds have the bond length of 1.08345 Å (C2-H21), 1.08352 Å
C3-H22), 1.08129 Å (C5-H25), and 1.08486 Å (C6-H20). Finally, the carbon-carbon
bonds in the rings A and B are described by the following bond lengths: for the 1.40420 Å
C1′-C2′), 1.39896 Å (C2′-C3′), 1.39864 Å (C3′-C4′), 1.39976 Å (C4′-C5), 1.39723 Å (C5′-
C6′), and 1.40792 Å (C6′-C1′) respectively in the ring A; for the 1.41173 Å (C1-C2),
(
1
(
(
(
1
1
.38091 Å (C2-C3), 1.40589 Å (C3-C4), 1.39800 Å (C4-C5), 1.39287 Å (C5-C6), and
.40295 Å (C6-C1) respectively for ring B.
Finally, the thermodynamics properties were computed at 298.15 K and 1 atm of
pressure to complete the structural description of the chalcone using B3LYP/6-
11++G(d,p) computational level in chloroform as an implicit solvent. The calculated
3
values of the thermodynamic properties Internal Energy (U), the Heat Capacity at constant
volume (C ), Enthalpy (H), Entropy (S), and Gibbs Free Energy (G) were respectively
v
-
1
-1 -1
-1
-1 -1
2
6
39.639 kcal.mol , 92.406 cal.mol K 703.771 kcal.mol , 175.04 cal.mol .K , and
-1
51.583 kcal.mol . The Zero-Point Energy (ZPE) and the Electronic Energy (E ) were
0
-1
respectively 223.900 kcal.mol and -1,187.683697 Hartree. For comparison, the
thermodynamics properties Internal Energy, Enthalpy, Entropy, and Gibbs Free Energy for
-1
the chalcones previously studied [17, 38] are respectively 181.231 kcal.mol , 532.4667
-1
-1
-1
-1
kcal.mol , 148.9 cal.K mol , and 488.072 kcal.mol for the PAAPFBA chalcone [17];
-1
-1
-1
-1
-1
2
39.837 kcal.mol , 704.866 kcal.mol , 173.234 cal.K mol , and 653.217 kcal.mol for
the cinnamaldehyde chalcone [38]. The molecular structure of the chalcone is more similar

to the cinnamaldehyde chalcone; hence the values for the thermodynamics properties are
similar. However, the PAAPFBA chalcone has a fluorine atom bonded in ring B and an
acetamide group bonded in ring A and the values for the same properties are different from
the values obtained to the HPHENYL and cinnamaldehyde chalcones, which shows how
the ligands in both phenyls ring A and B can change the properties of the entire molecule.
3.2 Infrared analyses
The ATR-IR absorbance spectrum of the chalcone is presented in Figure 4. We
highlighted 18 bands in the infrared spectrum, assigned with the help of the DFT
calculations, and listed in Table 2. The linear fitting was made using those highlighted
infrared bands. The result is shown in Figure 5. The coefficient of determination (R²) has a
value of 0.99812, with the linear equation y = 0.9945x + 5.9665. This result shows an
excellent agreement of the experimental values and the theoretical wavenumbers.
Therefore, the theoretical model for the liquid phase using chloroform as solvent is reliable
and complement the understanding of the ATR-FTIR spectrum of the title chalcone.
Infrared spectra of 2-bromo-4-chlorobenzaldehyde in solid phase and in n-hexane,
cyclohexane, chloroform, benzene, toluene as non-polar solvents and ethanol, 2-propanol,
methanol as polar solvents were investigated by Parlak and Ramasami [84]. They showed
that the theoretical infrared spectra in solutions show excellent agreement with the
experimental data in solid phase, mainly for the carbonyl stretching mode.
-1
The bands of wavenumbers at 3119, 2933, and 2832 cm in the experimental infrared
spectrum of the title chalcone are associated with stretching modes of the groups, CβH14
(97%), asymmetrical stretching in the methyl group C37H39 (48%) and C37H40 (51%),
and symmetrical stretching of the methyl group C37H39 (46%) and C37H40 (44%),
-1
respectively, whereas the infrared band at 1619 cm is associated with the mixture of
carbonyl stretching mode (C8=O18) with Cα-Cβ stretching mode. The C-C stretching
-1
modes of the ring B are observed in the infrared spectrum at 1547 cm with the movement
of the atoms C4-C5 mainly (PED = 21%) and the CαCβ stretching (PED = 12%).
−
1
The ATR-IR absorbance bands observed at 1507 cm corresponds to a combination of
the bending modes of the H21C2C3 (14%), H20C6C5 (14%), H22C3C4 (14%), and
-1
H26C5C6 (15%), whereas the infrared band at 1468 cm corresponds to the bending modes

of the H39C37H38 (16%), H34C33H36 (22%), H35C33H34 (29%), H38C37H40 (30%).
-1
The more intense infrared band located at 1415 cm is associated with the stretching modes
-1,
of the C3′C2′ (16%), C2’C1’ (16%), O23C2′ (18%) in ring A. At 1333 and 1290 cm there
are mixed vibrational modes for the bending and stretching of the following group of atoms
respectively: H21C2C3 (10%), H12CαCβ (11%), H14CβCα (15%), and CβCα (11%) at
-
1
-1
1
1
333 cm ; H21C2C3 (11%), H14CβCα (18%), and CβC1′ (14%) at 1290 cm . The band at
-
1
210 cm is associated with the bending and stretching modes of the groups H7C5′C4′
(14%) and O27C3′ (26%), respectively. Stretching modes of the carbon-oxygen bonds
-1
O28C33 (11%) and O28C4′ (17%) are observed in the band at 1151 cm . A strong infrared
band, which is associated with the stretching of the methoxy group O24C37 (16%), O23C2′
-1
(
20%), is observed at 1117 cm . Stretching and bending modes of the O27C29 (23%),
−
1
C4C5C6 (12%), and C2C3C4 (21%) are observed at 992 cm .
-1
At 828 cm it was observed mixed modes between the out of plane deformations and
two torsions respectively in the ring B for the group of atoms O41C3C5C4 (10%),
H22C3C4C5 (10%), and H21C2C3C4 (13%). Out of plane deformation and torsion of the
ring A respectively are observed in the wavenumbers 678 (O28C5′C3′C4′ with PED = 13%
-1
and O24C5′C1′C6′ with PED = 14%) and 588 cm (H25O23C2’C3’ with PED = 82%),
-1
whereas the mode at 425 cm has low intensity and corresponds to bending modes of the
C33O28C4′ (13%) and C6C1Cβ (15%). Therefore, the vibrational analysis allowed us to
obtain information on the main infrared bands of the chalcone.
3.3 Electronic Properties analysis
The Frontier Molecular Orbitals (FMO) for the title chalcone were calculated using
the B3LYP/6-311++G(d,p) computational level, and they are shown in Figure 6. The
Highest Occupied Molecular Orbital (HOMO) is related to the molecule’s ability to donate
electronic density (nucleophilic character), while the Lowest Unoccupied Molecular Orbital
(
LUMO) is related to the molecule’s ability to accept electronic density (electrophilic
character). The HOMO is mainly spread over the phenyl group of the ring A due to the π-
bonds, also is spread over the oxygen atoms of the methoxy groups (O24, O27, and O28),
in the π-bond between the carbon atoms CαCβ, in the oxygen atom (O18) of the carbonyl

group, in the π-bonds of the phenyl group in the ring B, and in the oxygen atom (O41) of
the methoxy group. Besides, the LUMO is mainly spread over the π* antibonding positions
of the phenyl group in the ring B, the bond between the CαCβ, and the oxygen atom (O18)
of the carbonyl group. There is a little contribution of the π* antibonding for the ring A.
Hence, the region of the molecule responsible for donating electronic density is the ring A,
the ring B, and the olefin group (CαCβ), while the region responsible for accepting
electronic density is the ring B and the α,β-unsaturated carbonyl group. The electronic
transition between the HOMO and the LUMO is π → π*.
To understand the chemical behavior of the chalcone, the quantum reactivity
descriptors were computed at the same level of theory from the energies values of the
HOMO and the LUMO, and they are shown in Table 3. The values of the quantum
descriptors show that the title chalcone has an electrophilic character due to the higher
values of the electronegativity and the electrophilicity index. The LUMO as a spread over
the entire molecule, the extra negative charge. Besides, the molecule has a high ionization
potential, which means it is difficult to donate electronic density. This result is confirmed
by the low value of the nucleophilicity index. The molecule can be classified as a hard
molecule due to the high value of the global hardness in comparison with the global
softness and the high value of the energy gap, which agrees with the difficulty of
electronic-donation of this molecule. Therefore, the chalcone should interact better with
substrates that have a good electronic donation character. Also, in Table 3 is shown the
comparison of the quantum reactivity descriptors computed for the chalcone and the
PAAPFBA [17] and cinnamaldehyde [38] chalcones. The chalcone should be more reactive
than the PAAPFBA and should be less reactive than the cinnamaldehyde chalcone due to
the values of the HOMO-LUMO energy gap. The chalcone has a lower value of
electronegativity; hence this molecule also has the lower electrophilic character,
consequently the higher nucleophilic character, which is proved by the higher value of the
energy value for the HOMO when compared to the others chalcones.
3.4 Fukui functions and local reactivity descriptors analysis
The Fukui functions (Electronic and Condensed) are used to understand how each
atom behaves during a chemical reaction. The quantum reactivity descriptors give an

overview of the molecule’s reactivity; however, the Fukui functions together with the dual
descriptor (∆f) and the multiphilic index (∆ω), a more selective analysis can be made on
each atom.
ꢞ
The Electronic Fukui functions for the nucleophilic attack (ꢝ ), electrophilic attack
ꢠ
ꢡ
(
ꢝ ), and radical attack (ꢝ ) are shown in Figure 7. The green and the blue colored
represent the positive and the negative signs of the Fukui functions, respectively. The
higher the value of the Fukui function, the higher the propensity for the determined attack
(nucleophilic, electrophilic, or radical). For the nucleophilic attack, the most susceptible
atoms are Cα, Cβ, O18, C2, C4, C6, and O41. In this case, the molecule behaves like an
ꢞ
electrophilic site; hence the ꢝ function is related to the LUMO. These atoms have mainly
the LUMO spread over them; hence they are most prone to accept electronic density. For
the electrophilic attack, the most susceptible atoms are C1, C2, C3, C4, C5, C6, C8, O41,
ꢠ
Cα, Cβ, O18, C1′, C3′, O24, and O28. The ꢝ function is related to the HOMO because the
molecule behaves as a nucleophilic site. Despite a larger number of atoms that are
susceptible to donate electronic density, this molecule has a low energy level for the
HOMO and higher ionization potential due to the more delocalization of the electronic
ꢡ
density within the molecule. For the radical attack (ꢝ ), the most susceptible atoms are C1,
C2, C3, C4, C5, C6, O41, Cα, Cβ, O18, C1′, C2′, C3′, and the hydrogen atoms. The carbon
and oxygen atoms can stabilize the radical due to the resonance effect, and the hydrogen
atoms are lost during the reaction between a radical species.
The results from the calculations of the Condensed Fukui functions, dual descriptor,
and multiphilic index are shown in Table 4. The Condensed Fukui functions were
computed from the Hirshfeld charge analysis [67] for the anionic (N+1), neutral (N), and
cationic (N-1) species of the title chalcone. According to the results, for a nucleophilic
attack, the most susceptible atoms are C2′, C5′, C1, C2, C3, C4, C5, C6, O24, O27, C33,
and C37. For the electrophilic attack, the most propensity atoms are C1′, C2′, C4′, C5′, C6′,
C8, C2, O27, C29, C37, and C42. For the radical attack, the most susceptible atoms are C1′,
C2′, C4′, C5′, C6′, C1, C3, C5, O24, O27, O28, C29, C33, C37, and C42. The relation
between the Condensed Fukui functions and the local reactivity descriptors (the dual
descriptor and the multiphilic index) lies in the following fact: if both ∆f and ∆ω are
positive, the reactive site has an electrophilic character, and if ∆f and ∆ω are negative, the

reactive site has nucleophilic character. The values in Table 4 show that the electrophilic
sites are in atoms C1′, C2′, C3′, C4′, C5′, C6′, C1, C3, C5, O24, O27, O28, C29, C33, C37,
O41, and C42. The nucleophilic sites are in the atoms C8, Cα, Cβ, O18, C2, C4, and C6.
The negative values for the condescended Fukui functions confirm the analysis made by the
Electronic Fukui Functions that the HOMO is not too available to donate electronic density
since the electronic density is spread over the entire molecule due to the resonance effect,
hence due to the higher nucleophilic character of the chalcone when compared to the
PAAPFBA [17] and cinnamaldehyde [38], the title chalcone can use the inner molecular
orbitals (HOMO-1, HOMO-2, etc.) to donate electronic density during a nucleophilic attack
[
85, 86].
3.5 Molecule Electrostatic Potential (MEP)
The Molecular Electrostatic Potential (MEP) was computed at B3LYP/6-
11++G(d,p) level of theory (Figure 8). The MEP has the following color scheme: the red-
3
colored is related to a negatively charged region, the yellow-colored to a partially negative
charge region, the green-colored to a neutral region, the light blue color to a partially
positive region, and the blue-colored to a positively charged region. The nucleophilic sites
of the molecule are displayed as red to yellow colored regions due to higher concentration
of electronic density. The electrophilic sites are displayed as light blue to blue colored
regions due to the electron deficient. The oxygen atoms from the carbonyl, methoxy, and
hydroxyl groups, the π electrons from the phenyl rings, and the π electrons from the CαCβ
bond are most susceptible to interact with positive regions of a substrate. The hydrogen
atoms with a partially positive charge are most susceptible to interact with negative regions
from other molecules. These results are directly related to the Fukui functions and the FMO
analysis.
3.6 UV-Vis spectrum analysis
In addition, the spectrum of UV-Vis absorption of chalcone is presented in Figure 9.
Analyzing this spectrum, it is possible to observe the maximum wavelength, λmax, at 375
nm, which corresponds to an energy value of 3.31 eV. For the theoretical characterization,

the UV-Vis absorption spectrum was computed using the Time-Dependent Density
Functional Theory (TD-DFT) at CAM-B3LYP/6-311++G(d,p) level of theory in ethanol
media. The calculated UV-Vis spectrum is shown in Figure 10. The maximum value for the
absorption wavelength was found at 333.10 nm, which correspondent energy of 3.7221 eV
with an oscillator strength of 0.5846. The electronic transitions which are responsible for
this transition are the HOMO-2→LUMO (6%), the HOMO-4→LUMO (29%), and the
HOMO→LUMO (56%). Hence, the absorption band in the experimental UV-Vis spectrum
of the chalcone is described mainly for the π → π* transition between the HOMO and the
LUMO, and the difference between the experimental and theoretical maximum wavelength
is less than 50 nm.
3.8 Evaluation of the major microspecies
By analyzing the calculated pKa value of chalcone in the order of 7.76, it is possible to
observe the chemical balance between the microspecies of the molecular system of the
substance. The graph expressed in Figure 11 shows the distribution of chalcone
microspecies as a function of pH variation, where it is worth noting that the loaded species
is formed close to the physiological pH levels (approximately 7.4), since the blue curve
shows a trend decreasing the concentration of the neutral species of the compound, while
the concentration of the ionic species shown by the orange curve tends to 100%.
Considering the relations established by the Henderson–Hasselbalch equation and the
calculated value compatible with experimental data from other phenolic hydroxyls, at the
time when pH> pKa, the chemical equilibrium shifts towards the formation of the ionic
species as the most abundant in the system, since such behavior occurs only at high basic
pH levels. Thus, it was possible to identify a concentration of 69.49% associated with
neutral species as the major microspecies at pH 7.4 (Figure 12).
3.9 Evaluation of the physicochemical properties
Chalcones are often mentioned in studies that relate to structure-activity (QSAR). The
predicted and observed antimycobacterial, antibacterial, antituberculosis, and anticancer

biological activities show a linear trend of fidelity between theoretical molecular
descriptors and the bioactivity performed by these small molecules [75, 87-89]. Directly
associated with lipophilicity and permeability, important descriptors of rational drug
planning such as partition coefficient between n-octanol and water (log P ) e topological
o/w
polar surface area (TPSA) and topological polar surface area (TPSA) can provide
information on the pharmacokinetic behavior of a given substance, as well as its oral
bioavailability. As a rule, most of the experimental findings involving these two properties
in theoretical screens suggest that compounds with positive values of log P are associated
with compounds with hydrophobic behavior and TPSA≤140Ų values with compounds
capable of penetrating diverse biological membranes [76, 88, 89]. Thus, the calculated
value of Clog P in the order of 3.61 indicates that chalcone is predominantly lipophilic,
while its TPSA value of 74.22Ų indicates a good capacity for cell permeability (Table 5).
In the filter that combines the drug-likeness criteria of Lipinski's ―rule of five‖
(MW≤500, log P≤5, H-bond donors≤5 and H-bond acceptors≤10) [75] and Veber's
parameters (TPSA≤140 e rotatable bonds≤10) [89], the bioavailability radar shown in
Figure 12 shows the ideal physicochemical properties that refine chalcone as a good
candidate for an oral drug [76]. It is verifiable that, despite the violation in the unsaturation
score (INSATU) of type Fsp3 <0.25, the compound does not violate any criteria of the rules
of Lipinski and Veber, satisfying the conditions within the ideal spectrum that combines
lipophilicity properties (LIPO), size molecular (SIZE), polarity (POLAR), flexibility
(FLEX) and insolubility (INSOLU) (Figure 12).
3.10 Virtual screening of pharmacokinetics properties
In a systematic statistical analysis that relates structure to permeability, some
descriptors make it possible to predict intestinal absorption of small molecules, such as the
distribution coefficient (log D), which, directly related to lipophilicity (log P), indicates the
absorption potential in specific pH. In these conditions, the lipophilicity indexes
incorporated into the log D values between 0 and 3 are associated with compounds with
good intestinal absorption. In addition, the model is used to identify anticancer compounds
by permeability in Caco-2 model cells, associated with compounds with log P between 1

and 10 [77, 90]. The calculated log value DpH7.4 3.45 indicates that chalcone has a high
intestinal absorption (Figure 13A), while the model indicates that the compound has
moderate permeability potential in Caco-2 cells, which can vary from 20 -70%. In addition,
it is possible to observe that the decrease in the values of log D as a function of the increase
in pH implies in the reduction of the potential for absorption of the compound, behavior
related to ionic species resulting from the chemical balance between the microspecies in
physiological pH, at the same time in there is an increase in the solubility coefficient
evaluated in log S -3.74 at pH 7.4 (Figure 13B) with a moderate concentration of
approximately 0.06 mg / mL, reaching high concentrations that can reach up to 344.36 mg /
mL at high pH levels (Figure 13C ).
As activity in the drug distribution stage, the transport of substances in the central
nervous system (CNS) is an analysis of great importance in pharmacokinetic studies, since
only 15% of compounds that penetrate the blood-brain barrier (BBB) perform efflux by
plasma proteins, such as P-glycoprotein (Pgp) [91]. In experimental observations made by
Kelder et al. (1999) [78], compounds with TPSA values TPSA<60-70Ų are more likely to
be active in the CNS by penetrating the BBB in phase II clinical tests, while the statistical
observations refine compounds as substrates of Pgp those with log P> 0 and MW between
200 and 400 g / mol [79]. Through the filter that combines the two observations to describe
the behavior of chalcone in the CNS, it can be observed that, despite the ellipse formed
between the values of MW and Clog P showing chalcone as a substrate for Pgp, the
substance has a high probability of being inactive in the CNS because it does not permeate
from the BBB (Table 5).
3.11 Evaluation of the multi-target bioactivity
The prediction of toxicological effects is gaining more and more prominence in
pharmacokinetic studies, especially in the identification of toxic fragments that intercalate
between the cyclic structures of DNA molecules in an intracellular environment, resulting
in tumorigenicity. The so-called mutagens are precursors of mutagenicity that involves the
DNA structures of a host in the development of tumorigenic activity in human cells. In this
context, the virtual screening by the interaction with the different human biological targets

allows to identify the carcinogenic activity of a certain chemical species [79, 92-95]. Thus,
the score of interaction with the target of G-protein coupled receptor (GPCR) in the order
of -0.10 associated with a low index of chalcone interaction with this target, located in the
membranes of human cells, indicating minimal toxicological risk due to modification of
intracellular gene pool (Table 5).
Considered one of the main ion channel modulators, the human Ether-to-go-go-Related
Gene (hERG) ion transporter is associated with the electrical conduction of the system that
surrounds the human heart and in the modulation of cells in the nervous system, and its
inhibition results in complications such as cardiac arrhythmia and cancer events such as
leukemia [96-100], where the inhibition score of -0.08 for chalcone indicates low
cardiotoxic risk (Table 5).
In the stage of phase I metabolism, the cytochrome P450 (CYP450) isoenzymes, in
particular the CYP3A4 and CYP2C9 enzymes, play a fundamental role in the
biotransformation of drugs through O- type oxidation reactions [101]. Physico-chemical
properties such as log DpH7.4 and log P are related to CYP450 substrates since the sites of
interaction with these enzymes are predominantly lipophilic [102]. The positive values of
log DpH7.4 and log P suggest that chalcone interacts as a substrate of the main CYP450
isoenzymes, while the inhibition score of them evaluated at 0.02 indicates the low
probability of inhibitory actions of these isoenzymes, indicating that the compound is
subject to drug modifications by oxidation of the phenolic hydroxyl (Table 5).
Figure 13D shows the contributions of the structure-activity relationship (QSAR) to the
chalcone acute oral toxicity model. In this model, the green-colored area represents the
positive contributions to the toxicity model, showing the non-toxic molecular fragments,
while the red-colored region indicates the negative contributions. Thus, the toxicity model
of the test indicates, with a 70% reliability index, that the minimum oral dose administered
does not result in toxicity.
3.12 Potential Antibacterial Activity and Evaluation of Efflux Pump Inhibition
The modulating effect of the chalcone on the resistance of the SA-K2068 strain to
ciprofloxacin can be seen in Figure 14. It can be seen that the addition of chalcone in

subinhibitory concentrations to the growth medium caused a reduction of the MIC values
for ciprofloxacin strain. The synergic effect presented by the chalcone was the same as the
modulating effect observed for CCCP, which is cited in the literature as an inhibitor of the
MepA efflux pump [71]. This effect against the MepA efflux pump is due to the inhibition
of the efflux pump mechanism, which can be proven by similar behavior observed
examining the association of this chalcone with ethidium bromide (Figure 14). Previous
studies showed a similar effect when the DB Thiphene chalcone was tested for the SA-
K2068 strain. DB Thiophene showed a better inhibition potential for CCCP-sensitive efflux
pumps than the standard inhibitor, also acting as a strong inhibitor of efflux pumps in the
Ciprofloxacin test [71].
3.13 Docking Results
In order to better understand the MepA inhibition mechanism displayed by the
chalcone, a docking essay was carried out. Both the chalcone and ciprofloxacin were
individually docked against a MepA model. Figure 15 shows the best poses of both
molecules docked on the binding site of the MepA mode; as can be clearly seen from the
figure, the chalcone docks to the model in almost the same way as the antibiotic. It also
interacts with essentially the same residues. Figure 16 shows the two-dimensional map of
the interactions between the title chalcone and the MepA model. Despite of the HOMO is
mainly spread over the ring A, the ring B makes a π interaction with the amino acids
methionine (MET 142) and phenylalanine (PHE 62) since this chalcone has electronic
density to donate, and the LUMO is mainly spread over the ring B; hence this chalcone can
also receive electronic density from the amino acids.
The binding energy of the best pose of chalcone was -6.8kcal/mol. Although the
binding energy is essentially a ranking system for the poses of the ligand, one could use it
to compare affinities to the model, provided the ligands being compared have similar
molecular weights. With that caveat in mind, the binding energy of the ciprofloxacin
antibiotic was -7.6kcal/mol. The slightly higher affinity of ciprofloxacin could be attributed
to the hydrogen bond between the antibiotic and the Asn205 residue, which is shorted than
the hydrogen bond between chalcone and Gln284. Also, these binding energies are not only

close to each other, but also on par with those reported on the literature for MepA and
NorA models. Rezende-Jꢀnior et al. [103] have reported that the binding energies of
several chalcones to a NorA model are approximately 7 kcal/mol.
4. Conclusion
The analyses allowed drawing the molecular structure of the chalcone synthetized with
subsequent confirmation using the quantum chemical calculations with an excellent linear
correlation between the experimental and theoretical chemical shifts. The spectroscopic
analysis allowed obtaining information of the main infrared bands of chalcone, and together
with the theoretical assignments of the vibrational modes of those band, the Infrared
spectroscopic analysis contributed to confirm the molecular structure of chalcone, and the
electronic characterization identified the regions of the molecule that are responsible to
donate (nucleophilic) and to accept (electrophilic) electronic density. The drug-likeness
criteria based on the rules of Lipinski and Veber evaluated that the compound has the ideal
physicochemical properties for the realization of its active ingredient as an oral drug, and
the low interaction with plasma proteins indicates a molecular fraction of the bioavailable
substance for interactions with specific biological targets in docking tests and molecular
dynamics. The molecular size and lipophilicity models suggest that the substance has a high
probability of being an inactive drug in the CNS, and its transport by Pgp indicates that
there is no risk of toxicity due to residual drug accumulation in the blood. The structure-
activity relationship (QSAR) of chalcone and the low rates of interaction with the targets
GPCR and Ion CM suggest that the minimum oral dose administered does not present a
toxic risk to the nervous system and the respiratory system, constituting a good candidate
for drug orally. In addition, the microbiological tests show that the chalcone can be used as
a possible inhibitor of the Mep A efflux pump, also revealing that chalcone can be used as a
base for the design of substances with antibiotic modifying activity.
CRediT authorship contribution statement
Jayze da Cunha Xavier: Investigation. Data curation. Formal analysis, Writing - original
draft. Francisco W. Q. Almeida-Neto: Software, Validation, Formal analysis. Priscila T.

Amanda P. de Sousa:
da Silva: Data curation.
Data curation. Emmanuel S. Marinho: Formal
analysis, Writing—review and editing, Márcia M. Marinho: Formal analysis, Writing—
review and editing. Janaina E. Rocha: Methodology. Priscila R. Freitas: Methodology.
Ana C. J. de Araújo: Methodology. Thiago S. Freitas: Methodology. Carlos E. S.
Nogueira: Software, Formal analysis, Writing—review and editing. Pedro de Lima-Neto:
Conceptualization. Paulo N. Bandeira: Conceptualization, Resources. Alexandre M. R.
Teixeira: Formal analysis, Funding acquisition. Henrique D. M. Coutinho: Methodology,
Resources. Hélcio Silva dos Santos: Supervision, Project administration, Formal analysis,
Writing—review and editing.
Declaration of competing interests
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Acknowledgments: The authors thank FUNCAP, CAPES, and CNPq for financial support
and scholarship. A.M.R. Teixeira acknowledges the financial support from the CNPq
(Grant#: 305719/2018-1). The authors also thank Centro Nacional de Processamento de
Alto Desempenho (CENAPAD) of the Federal University of Ceará (UFC) for providing
computational resources. Thanks to the India government program "Research Training
Fellowship for Developing Country Scientist" (RTF-DCS/2019/000192).
References
[
1] D.E. Payne, B.R. Boles, Emerging interactions between matrix components during biofilm
development, Current Genetics, 62 (2016) 137-141. https://doi.org/10.1007/s00294-015-0527-5.
[
2] M. Jamal, W. Ahmad, S. Andleeb, F. Jalil, M. Imran, M.A. Nawaz, T. Hussain, M. Ali, M.
Rafiq, M.A. Kamil, Bacterial biofilm and associated infections, Journal of the Chinese Medical
Association, 81 (2018) 7-11. https://doi.org/10.1016/j.jcma.2017.07.012.

[
3] S.J. Dancer, The effect of antibiotics on methicillin-resistant Staphylococcus aureus, Journal of
Antimicrobial Chemotherapy, 61 (2008) 246-253. https://doi.org/10.1093/jac/dkm465.
[
(
4] F. Aqil, I. Ahmad, M. Owais, Evaluation of anti-methicillin-resistant Staphylococcus aureus
MRSA) activity and synergy of some bioactive plant extracts, Biotechnology Journal, 1 (2006)
1093-1102. https://doi.org/10.1002/biot.200600130.
[
5] H.D.M. Coutinho, J.G.M. Costa, E.O. Lima, V.S. Falcao-Silva, J.P. Siqueira, Jr., Enhancement
of the antibiotic activity against a multiresistant Escherichia coli by Mentha arvensis L. and
chlorpromazine, Chemotherapy, 54 (2008) 328-330. https://doi.org/10.1159/000151267.
[
6] S. Krishna, L.S. Miller, Host-pathogen interactions between the skin and Staphylococcus aureus,
Current Opinion in Microbiology, 15 (2012) 28-35. http://dx.doi.org/10.1016/j.mib.2011.11.003.
[
7] P.M. Dugourd, A. Dupont, T. Hubiche, C. Chiaverini, A. Alkhalifa, L. Roudiere, A. Tristan,
C.A. Gustave, P. Del Giudice, Staphylococcal toxic shock syndrome should be considered in the
event of diffuse erythema with fever and shock, Annales de Dermatologie et de Venereologie, 146
(
2019) 287-291. https://doi.org/10.1016/j.annder.2018.12.002.
[
8] S.P. Bergin, T.L. Holland, V.G. Fowler, S.Y.C. Tong, Bacteremia, Sepsis, and Infective
Endocarditis Associated with Staphylococcus aureus, in: F. Bagnoli, R. Rappuoli, G. Grandi (Eds.)
Staphylococcus aureus: Microbiology, Pathology, Immunology, Therapy and Prophylaxis, Springer
International Publishing, Cham, 2017, pp. 263-296.
[
9] P.M. Wright, I.B. Seiple, A.G. Myers, The Evolving Role of Chemical Synthesis in
Antibacterial Drug Discovery, Angewandte Chemie-International Edition, 53 (2014) 8840-8869.
https://doi.org/10.1002/anie.201310843.
[
10] M.T.A. Oliveira, A.M.R. Teixeira, H.D.M. Coutinho, I.R.A. Menezes, D.M. Sena, H.S. Santos,
B.M. de Mesquita, M.R.J.R. Albuquerque, P.N. Bandeira, R. Braz-Filho, Identification and
Modulatory Activity Assessment of 2-Hydroxy-3,4,6-trimethoxyacetophenone Isolated from Croton
anisodontus Mull. Arg.(Euphorbiaceae), Natural Product Communications, 9 (2014)
1934578X1400900520. http://dx.doi.org/10.1177/1934578X1400900520.
[
11] P.H.M. Torres, A.C.R. Sodero, P. Jofily, F.P. Silva-Jr, Key Topics in Molecular Docking for
Drug Design, International Journal of Molecular Sciences, 20 (2019).
https://doi.org/10.3390/ijms20184574.
[
12] S. Saikia, M. Bordoloi, Molecular Docking: Challenges, Advances and its Use in Drug
Discovery Perspective, Current Drug Targets, 20 (2019) 501-521.
https://doi.org/10.2174/1389450119666181022153016.
[
13] D.G. do Rego, B.G. de Oliveira, Um Estudo Químico-quântico da Covalência Intermolecular
em Sistemas Estabilizados por Ligações de Hidrogênio π∙∙∙ H e N∙∙∙ H: Cálculos DFT, ChelpG,
NBO e QTAIM, Orbital: The Electronic Journal of Chemistry, 1 (2016) 1-11.
[
14] S. Burmaoglu, S. Ozcan, S. Balcioglu, M. Gencel, S.A.A. Noma, S. Essiz, B. Ates, O. Algul,
Synthesis, biological evaluation and molecular docking studies of bis-chalcone derivatives as
xanthine oxidase inhibitors and anticancer agents, Bioorganic Chemistry, 91 (2019) 103149.
https://doi.org/10.1016/j.bioorg.2019.103149.

[
15] S. Burmaoglu, E.A. Kazancioglu, R. Kaya, M. Kazancioglu, M. Karaman, O. Algul, I. Gulcin,
Synthesis of novel organohalogen chalcone derivatives and screening of their molecular docking
study and some enzymes inhibition effects, Journal of Molecular Structure, 1208 (2020) 127868.
https://doi.org/10.1016/j.molstruc.2020.127868.
[
16] P. Yadav, J.K. Yadav, A. Agarwal, S.K. Awasthi, Insights into the interaction of potent
antimicrobial chalcone triazole analogs with human serum albumin: spectroscopy and molecular
docking approaches, RSC Advances, 9 (2019) 31969-31978. https://doi.org/10.1039/C9RA04192C.
[
17] F.W.Q. Almeida-Neto, L.P. da Silva, M.K.A. Ferreira, F.R.S. Mendes, K.K.A. de Castro, P.N.
Bandeira, J.E.S.A. de Menezes, H.S. dos Santos, N.K.V. Monteiro, E.S. Marinho, P. de Lima-Neto,
Characterization of the structural, spectroscopic, nonlinear optical, electronic properties and
antioxidant activity of the N-{4’-[(E)-3-(Fluorophenyl)-1-(phenyl)-prop-2-en-1-one]}-acetamide,
Journal of Molecular Structure, 1220 (2020) 128765.
https://doi.org/10.1016/j.molstruc.2020.128765.
[
18] P.T. Silva, L.L.M. A., J.C. Xavier, M.C.S. Carvalho , M.O. Moraes, C. Pessoa, F.W.A. Barros,
P.N. Bandeira, C.S.P. Cavalcante, A.M.R. Teixeira, R.O.S. Fontenelle, H.S. Santos, Atividade
Citotóxica e Antifúngica de Chalconas Sintetizadas a partir de uma Acetofenona Natural Isolada de
Croton anisodontus, Revista Virtual de Química, 12 (2020) 712-723. https://doi.org/10.21577/1984-
6835.20200057.
[
19] P.T. Silva, T.S. Freitas, D.M. Sena, Jr.; , P.N. Bandeira, M.S.S. Julião, E.S. Marinho, A.A.C.
Alcanfor, E.M. Marinho, P. Lima-Neto, C.E.S. Nogueira, H.D.M. Coutinho, A.L.A.B. Leal, H.M.
Barreto, N. Martins, Rodrigues Teixeira, A. M.;, H.S. Santos, Structural, Vibrational and
Electrochemical Analysis and Antibacterial Potential of Isomeric Chalcones Derived from Natural
Acetophenone, Applied Sciences, 10 (2020) 4713. http://dx.doi.org/10.3390/app10144713.
[
20] T.S.d. Freitas, J.d.C. Xavier, R.L.S. Pereira, J.E. Rocha, D.F. Muniz, P.T. da Silva, J.P.
da Hora, H.S. dos Santos, P.N. Bandeira, C.E.S. Nogueira, A.M.R. Teixeira, H.D.M. Coutinho,
Direct antibacterial and antibiotic resistance modulatory activity of chalcones synthesized from the
natural product 2-hydroxy-3,4,6-trimethoxyacetophenone, FEMS Microbiology Letters, 367 (2020).
http://dx.doi.org/10.1093/femsle/fnaa124.
[
21] C.A.N. Ferraz, S.R. Tintino, A.M.R. Teixeira, P.N. Bandeira, H.S. Santos, B.G. Cruz, C.E.S.
Nogueira, T.F. Moura, R.L.S. Pereira, D.M. Sena, T.S. Freitas, J.E. Rocha, H.D.M. Coutinho,
Potentiation of antibiotic activity by chalcone (E)-1-(4′-aminophenyl)-3-(furan-2-yl)-prop-2-en-1-
one against gram-positive and gram-negative MDR strains, Microbial Pathogenesis, 148 (2020)
104453. https://doi.org/10.1016/j.micpath.2020.104453.
[
22] P.T. da Silva, J.d.C. Xavier, T.S. Freitas, M.M. Oliveira, H.D.M. Coutinho, A.L.A.B. Leal,
H.M. Barreto, P.N. Bandeira, C.E.S. Nogueira, D.M. Sena, F.W.Q. Almeida-Neto, E.S. Marinho,
H.S. Santos, A.M.R. Teixeira, Synthesis, spectroscopic characterization and antibacterial evaluation
by chalcones derived of acetophenone isolated from Croton anisodontus Müll.Arg, Journal of
Molecular Structure, DOI https://doi.org/10.1016/j.molstruc.2020.129403(2021) 129403.
https://doi.org/10.1016/j.molstruc.2020.129403.
[
23] A.M.R. Teixeira, H.S. Santos, P.N. Bandeira, M.S.S. Juliao, P.T.C. Freire, V.N. Lima, B.G.
Cruz, P.T. da Silva, H.D.M. Coutinho, D.M. Sena, Structural, spectroscopic and microbiological
characterization of the chalcone 2E-1-(2 '-hydroxy-3 ',4 ',6 '-trimethoxyphenyl)-3-(phenyl)-prop-2-

en-1-one derived from the natural product 2-hydroxy-3,4,6-trimethoxyacetophenone, Journal of
Molecular Structure, 1179 (2019) 739-748. https://doi.org/10.1016/j.molstruc.2018.11.075.
[
24] P.N. Bandeira, R.O.S. Fontenelle, P.S. Costa, H.S. Santos, T.L.G. Lemos, Atividade
Antifúngica In Vitro Contra Trychophyton Rubrum de P-Aminochalcones e 3'-Metoxi-4'-Hidroxi
Chalcona, Revista Virtual de Química, 12 (2020) 703-711. https://doi.org/10.21577/1984-
6835.20200056.
[
25] Z. Rozmer, P. Perjési, Naturally occurring chalcones and their biological activities,
Phytochemistry Reviews, 15 (2016) 87-120. https://doi.org/10.1007/s11101-014-9387-8.
[
26] C.L. Zhuang, W. Zhang, C.Q. Sheng, W.N. Zhang, C.G. Xing, Z.Y. Miao, Chalcone: A
Privileged Structure in Medicinal Chemistry, Chemical Reviews, 117 (2017) 7762-7810.
http://dx.doi.org/10.1021/acs.chemrev.7b00020.
[
27] P.N. Bandeira, T.L.G. Lemos, H. S. Santos, M.C.S. de Carvalho, D.P. Pinheiro, M.O. de
Moraes Filho, C. Pessoa, F.W.A. Barros-Nepomuceno, T.H.S. Rodrigues, P.R.V. Ribeiro, H.S.
Magalhães, A.M.R. Teixeira, Synthesis, structural characterization, and cytotoxic evaluation of
chalcone derivatives, Medicinal Chemistry Research, DOI https://doi.org/10.1007/s00044-019-
02434-1(2019). https://doi.org/10.1007/s00044-019-02434-1.
[
28] T.R. Garcia, T.S. de Freitas, H.S. dos Santos, P.N. Bandeira, M.S.S. Julião, J.E. Rocha, C.E.S.
Nogueira, R.L.S. Pereira, A.C.H. Barreto, P.T.C. Freire, H.D.M. Coutinho, A.M.R. Teixeira,
Structural, vibrational and electrochemical analysis and antibiotic activity study of chalcone (2E)-1-
(
1
3ʹ,-methoxy-4ʹ,-hydroxyphenyl)-3-(3-nitrophenyl)prop-2-en-1-one, Journal of Molecular Structure,
216 (2020) 128358. https://doi.org/10.1016/j.molstruc.2020.128358.
[
29] T.O. Ajiboye, M.T. Yakubu, A.T. Oladiji, Electrophilic and Reactive Oxygen Species
Detoxification Potentials of Chalcone Dimers is Mediated by Redox Transcription Factor Nrf-2,
Journal of Biochemical and Molecular Toxicology, 28 (2014) 11-22.
https://doi.org/10.1002/jbt.21517.
[
30] M.K.A. Ferreira, A.W. da Silva, F.C.O. Silva, C.L.A. Holanda, S.M. Barroso, J.d.R. Lima,
A.E. Vieira Neto, A.R. Campos, P.N. Bandeira, H.S. dos Santos, T.L.G. de Lemos, S.M.C.
Siqueira, F.E.A. Magalhães, J.E.S.A. de Menezes, Anxiolytic-like effect of chalcone N-{(4′-[(E)-3-
(
4-fluorophenyl)-1-(phenyl) prop-2-en-1-one]} acetamide on adult zebrafish (Danio rerio):
Involvement of the GABAergic system, Behavioural Brain Research, 374 (2019) 111871.
https://doi.org/10.1016/j.bbr.2019.03.040.
[
31] M.K.A. Ferreira, A.W. da Silva, F.C.O. Silva, A.E. Vieira Neto, A.R. Campos, S.A. Alves
Rodrigues Santos, A.M. Rodrigues Teixeira, J. da Cunha Xavier, P.N. Bandeira, C.E. Sampaio
Nogueira, D.H.A. de Brito, E.L. Rebouças, F.E.A. Magalhães, J. de Menezes, H.S. Dos Santos,
Anxiolytic-like effect of chalcone N-{4'[(2E)-3-(3-nitrophenyl)-1-(phenyl)prop-2-en-1-one]}
acetamide on adult zebrafish (Danio rerio): Involvement of the 5-HT system, Biochemical and
Biophysical Research Communications, 526 (2020) 505-511.
https://doi.org/10.1016/j.bbrc.2020.03.129.
[
32] E.O. Ajaiyeoba, O.O. Ogbole, O.O. Abiodun, J.S. Ashidi, P.J. Houghton, C.W. Wright,
Cajachalcone: An antimalarial compound from Cajanus cajan leaf extract, Journal of Parasitology
Research 2013 (2013). https://doi.org/10.1155/2013/703781.