Correlating experimental electrochemistry and theoretical calculations in 2′-hydroxy chalcones: The role of the intramolecular hydrogen bond

Article abstract of DOI:10.1039/c5ra10140a

In this work we present a study on the molecular structure and electrochemical behavior of a series of methoxylated 2′-hydroxychalcones, whose antitumor activity has been previously described. Cyclic voltammetry was used to quantitatively characterize the

Full text of DOI:10.1039/c5ra10140a

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Cifuentes, R. Salazar, C. A. Escobar, B. Weiss-Lopéz, L. Santos and R. Araya-Maturana, RSC Adv., 2015,
DOI: 10.1039/C5RA10140A.
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Correlating Experimental Electrochemistry and Theoretical Calculations
in 2´-Hydroxy Chalcones: The Role of Intramolecular Hydrogen Bond
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Maximiliano Martínez-Cifuentes[a]*, Ricardo Salazar[b], Carlos A. Escobar[c], Boris E.
Weiss-López[d], Leonardo S. Santos[a], Ramiro Araya-Maturana[e,f]*
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a Laboratorio de Síntesis Asimétrica, Instituto de Química de los Recursos Naturales,
Universidad de Talca, Talca, casilla 747, Chile.
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b Departamento de Ciencias del Ambiente, Facultad de Química y Biología, Universidad
de Santiago de Chile, USACh, Casilla 40, Correo 33, Santiago, Chile
c Departamento de Ciencias Químicas Universidad Andres Bello, Av. República 275,
Santiago, Chile.
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d Departamento de Química, Facultad de Ciencias, Universidad de Chile, Casilla 653,
Santiago, Chile.
e Departamento de Química Orgánica y Fisicoquímica, Facultad de Ciencias Químicas Y
Farmacéuticas, Universidad de Chile, Casilla 233, Santiago 1, Chile.
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fInstituto de Química de los Recursos Naturales, Universidad de Talca, Talca, casilla 747,
Chile.
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Abstract: In this work we present a study about the molecular structure and
electrochemical behavior of a series of methoxylated 2´-hydroxychalcones, whose
antitumor activity has been previously described. Cyclic voltammetry was used to
quantitatively characterize the formation and stability of the anion radicals. The molecular
structure of the neutral compounds and their anion radicals, particularly the
intramolecular hydrogen bonds (IHB), were investigated through density functional theory
(DFT) calculations. Geometrical and frontier orbitals changes in the anion, relative to the
neutral species, were examined and the adiabatic and vertical electron affinities (AEA,
VEA) as well as vertical detachment energy (VDE) were calculated. Natural bond orbital
(NBO) analysis was used to get insights about the electronic characteristics of the IHB and
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the results were correlated with H-NMR chemical shifts. A direct relation among the
substitution pattern on rings A and B, the strength of the IHBs and the reduction potentials
was found. NBO energies (∆Eij(2)) show that the main contributions to the stabilization of
the IHBs arises from LP  σ* interactions. The strength of IHBs, given by ∆Eij(2), exhibit a
notable quantitative correlation with the experimental reduction potential, which, at least
to the best of our knowledge, has not been described before for any type of molecules. The
results show the importance of the methoxy substitution pattern on the IHB and redox
properties of these compounds. Our findings have potential implications in the design of
antitumor chalcones
Keywords
Chalcone; Hydrogen Bonding; Electrochemistry; DFT; NBO
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1. Introduction
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Studies concerning the chemistry of molecular radical ions has been an issue of great
interest in physical organic chemistry for many years.1,2,3 It is of fundamental importance to
understand their formation, properties and behavior, because radical species participate in
several important biochemical process, such as redox enzymatic reactions4,5 and DNA
strand break 4, 6-8, among others. At the same time, chemical reactions mediated by radical
species have received great attention for decades and remains as a very active field of
research.9-11
In this work the center of attention is a series of chalcone derivatives. These molecules are
known to have beneficial effects, such as antioxidant and tumor cell growth inhibitory
activities, as well as apoptosis inductors in several cancer cell lines. These activities explain
the great attention received by chalcones as chemo-preventive agents.12 Particularly
interesting is their ability to modify the mitochondrial membrane potential. On the above, it
was found that chalcones with fewer hydroxyl groups on the aromatic rings were more
effective as mitochondrial uncouplers.13 More recently, the role of the methoxylation in the
inhibition potency and cytotoxicity of chalcones towards breast cancer cells has been
critically studied.14 Furthermore, it has been demonstrated that 2´-hydroxychalcones induce
apoptosis and inhibit invasion of human breast cancer cells.15
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It has been suggested that the radical anion of 2′-hydroxychalcones play a central role in
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the catalyzed and uncatalyzed reactions with prenylflavonoid dienes,16,
to give
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biologically active prenylflavonoid derivatives.18,
The above has prompted the

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development of biomimetic total synthesis, mostly via catalysis, of these natural
products.17,20, 21
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Few studies that directly relate electrochemical properties with theoretical calculations have
been carried out in chalcone type structures. We found a computational study about the
electron affinities of substituted chalcones and their comparison with experimental
reduction potentials that showed a highly linear correlation, however none of these
molecules presented IHB.22 On the other hand, oxidation potential of hydroxychalcones
were studied by cyclic voltammetry and some thermodynamic and kinetic parameters
compared with theoretical calculations.23 Among their results, the authors claim that the
IHB, present in some of the studied structures, does not modify the redox properties, but it
does affect the molecular conformation, maintaining planar both, the neutral and the radical
species.23
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In this work, we study the influence of molecular structure, particularly the role of IHB, on
the reduction potential, formation and stability of the radical anion of 2`-hydroxychalcones
of pharmacological interest. In a previous study, cyclic voltammetry was employed to
quantitatively characterize the formation and stability of the anion radical from three 2’-
hydroxychalcones.24
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In this work, the molecular structure and electrochemical behaviour of six methoxylated 2´-
hydroxychalcones (Figure 1), which have been previously reported as antitumoral agents,
were examined in detail.25,14 First, cyclic voltammetry is used to quantitatively characterize
the formation and stability of the anion radical of the series Ch4, Ch5 and Ch6 and the
results compared with three previously studied molecules (Ch1-Ch3).24 Then, the

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molecular structures of all six compounds, particularly their intramolecular hydrogen bond
(IHB) O2´-H2´…O7´ in the neutral species and in their radical anions, were investigated
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through DFT calculations. The strength of IHB O2´-H2´ O7´ was experimentally assessed
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through H-NMR chemical shift of H2. A comparison of geometrical parameters and
frontier orbitals between the neutral and the anion radical is presented, in particular,
adiabatic and vertical electron affinities (AEA, VEA) as well as vertical detachment
energies (VDE) were calculated. Finally, natural bond orbital (NBO) analysis was used to
get insight about the electronic characteristics of the IHB present in these molecules.
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2. Experimental Section
2.1. Synthesis
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Chalcones were prepared following a previously described methodology,24 by adding , in a
dropwise manner, a solution of the corresponding substituted benzaldehyde, (7.34 mmol in
ethanol, 20 mL) to a stirred mixture of 4-methoxy-2-hydroxyacetophenone solution (7.34
mmol, in ethanol, 20 mL) and potassium hydroxide solution (2g in 10 mL distilled water).
The mixture was allowed to react overnight at room temperature. Then, it was diluted with
distilled water (200 mL), neutralized with hydrochloric acid, and extracted four times with
ethyl acetate (50 mL). The compounds were crystallized from ethanol. All these
compounds have been described before.24,26 1H-NMR spectra were obtained for all
compounds in DMSO and along with melting points were used to ensure the identity of the
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products. In addition, H-NMR spectra from Ch3 and Ch4 in CDCl3 were also obtained.
1H-NMR chemical shifts of H2 were used to experimentally assess the strength of IHB O2´-
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H2´ O7´.

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2.2 Electrochemical experiments.
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Cyclic Voltammetry (CV) experiments were carried out in DMSO with
tetrabutylammonium hexafluorophosphate (TBFP) as supporting electrolyte using a totally
automated BAS-100 voltammetric analyzer attached to a PC computer with the BAS 100-
W software (v. 2.3), for total control of the experiments, data acquisition and treatment. All
CV experiments were carried out with 1.0 mM solution of each chalcone. A hanging
mercury drop electrode (HMDE) was used as working electrode (area 2.27 mm2). Platinum
wire was used as auxiliary electrode and all potentials were measured against a non
aqueous Ag/Ag+ CH Instrument 112 reference electrode. All the electrochemical
experiments were obtained after purging the cell with N2 for ten minutes before each run,
and the temperature was maintained at 25  1 ºC. Resistance was compensated before each
experiment. To obtain comparable results within the complete series we choose this
methodology, because it is the same previously used with Ch1-Ch3.23
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2.3 Computational details
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The calculations were carried out using the Gaussian 0927 program package, running in a
Microsystem cluster of blades. Geometries were optimized at DFT B3P86/6-31+G(d,p)
level, using the conductor-like polarizable continuum model (C-PCM) approach to model
the role of the solvent, DMSO. This methodology has been proved to be reliable in the
study of redox properties of chalcones.28-30 No imaginary vibrational frequencies were
found at the optimized geometries, which indicate that they are true minima of the potential
energy surface. In solution, DMSO molecules can also act as hydrogen bond acceptors31, 32
and thus they compete with the intramolecular hydrogen bond discussed here. It has been

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shown in previous works that even in this type of proton acceptor solvent, the
intramolecular hydrogen bond remains. 33,23
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From frequency calculations, zero-point vibrational energies (ZPVE) were obtained and
used to correct the calculated enthalpies. The IHB analysis were obtained using
hyperconjugation stabilization energies within the framework of NBO analysis.34 The NBO
method involves a population analysis, which distributes computed electron density into
orbitals in the way chemist think, in terms of physical organic chemistry. The interaction
between filled orbitals and empty antibonding orbitals represents the deviation of the
molecule from the Lewis structure and can be used as a measurement of delocalization due
to the presence of hydrogen bond interaction. These hyperconjugative interactions play an
important role in hydrogen bonding. Donor-acceptor interaction energies (stabilization
energy, ΔEij(2)) can be calculated using second-order perturbation theory analysis.34 Natural
resonance theory (NRT) calculations35,36,37, available from the NBOPro6 software,38 have
been used to determine the bond order and the fraction of covalency and ionicity of the O-
H…O IHB.
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Electron affinities and bond dissociation energies were calculated as follow (where H
corresponds to enthalpies):39
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Adiabatic electron affinity (AEA) = H(optimized neutral) – H(optimized anion)
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Vertical electron affinity (VEA) = H(optimized neutral) – H(anion at optimized neutral
geometry)

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Vertical detachment energy (VDE) = H(neutral at optimized anion geometry) –
H(optimized anion)
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3. Results and Discussion
3.1. Electrochemistry
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Chalcones 4-6 were electrochemically reduced on Hg electrode in a non-aqueous medium
containing DMSO as solvent and tetrabutylammonium hexafluorophosphate (TBFP) as
supporting electrolyte, in the same way as it was previously reported for Ch1-Ch3.24 The
cyclic voltammograms were obtained for all three compounds, showing a behavior for the
redox process similar to that observed in Ch1-Ch3.24 (Figure 2).
When the reduction potentials are compared (Table 1), it can be noticed that Ch5 needs
more energy for reducing than Ch4 and Ch6. Similar behavior was reported24 for Ch1-
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Ch3, where Ch2 needs more energy than Ch1 and Ch3 to be reduced. A systematic
decrease in the reduction potential is observed for Ch4-Ch6 when compared with their
respective analogues Ch1-Ch3, indicating that the presence of the -OCH3 substituent, in
para position with respect to the carbonyl on ring A, increases the energy of the molecular
electronic state hindering the electron capture.
Under the mentioned experimental conditions, the one electron reduction process produces
very well resolved cyclic voltammograms due to the formation of the anion radicals. When
the concentration of the species in solution increased, an increase in the magnitude of the
current for the couple was observed (Figure 3A). The stability of the anion radical depends

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mainly on the reaction media, and can be monitored by following the decay of the
corresponding oxidation current, Ip,a, subsequent to the electrochemical generation (return
sweep). In effect, a suitable measurement of the stability of the radical anion is expressed
by the current ratio parameter, Ipa / Ipc, which reveals the tendency of an electrochemically
generated species, i.e. the anion radical, to undergo subsequent chemical reactions.40 The
current ratio equals to one in the absence of further reactions but decreases if the radical
subsequently reacts.
The calculation of the ratios Ipa / Ipc for the three species show that when the scan rate
increases, it takes values close to 1 (Figure 3B), but when the concentration increases, the
ratio of currents decreases (Figure 3C), indicative of a non-reversible process.
3.2. DFT calculations
3.2.1. Geometries, electro affinities and molecular orbital analysis
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Some key selected geometrical parameters and calculated AEAs, VEAs and VDEs for
Ch1-Ch6 and their respective anon radicals are presented in Figure 4 and Table 2
respectively. It has been previously reported that for the most stable trans configuration of
chalcones, two conformers can exist, namely s-cis and s-trans. It has been found that the
difference in energy between the two conformers is in favor of the s-cis isomer by 5.6
kcal/mol, with a barrier of 8.3 kcal. In addition, it has been demonstrated that properties
such as bond dissociation energy and many energetic properties will not be modified from
the s-cis to the s-trans conformers.28 Thus, to carry out our calculations only the s-cis
conformers were taken into account.

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As can be observed from the table attached to Figure 4, most geometrical parameters do
not show significant differences between the neutral and the anion radical species, for all
chalcones. Nevertheless, the C=C - bond increases its length by 0.03-0.04 Å, while the
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C-C7´ bond shortens by 0.04-0.07 Å in the anion radicals. Carbonyl C7´=O7´ also present a
bond lengthening of 0.06-0.09 Å. The only notable geometrical differences between the
neutral species and the anion radicals was found for parameters corresponding to the IHB,
for instance, the distance O2´-H2´ lengthens by 0.06-0.08 Å for Ch1-Ch4 and notably by
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0.37-0.36 Å for Ch5 and Ch6, while oppositely, the distance H2´ O7´, shortens by 0.20-
0.21 Å for Ch1-Ch4 and 0.47 Å for both Ch5 and Ch6. In addition, the angle O2´-H2´-O7´
increased from 150-152° to 159-160° for all chalcones. These results indicate that the IHB
and the double bond are mostly affected upon the electron attachment.
Particularly interesting is that the presence of a para-methoxy substituent on position 4´ of
ring A, along with the presence of a methoxy substituent on ring B at C4 or C5 in the anion
radical, shortens the H2´´…O7 distance (lengthening the O2´-H2´ distance) to a point in which
H1´…O7 could be consider a covalent bond, in particular for Ch5 and Ch6. However, the
latter do not occur for Ch4, which exhibit the methoxy group in C3. Even though both,
Ch4 and Ch6 present a methoxy substituent in ortho- and meta- position with regard to the
double bond, only the ortho-methoxy group can deliver electron density to the C7´=O7´
bond through conjugation with the C-Cdouble bond. In the case of Ch4, both methoxy
groups are neighbors, which can affect the ability of the methoxy in C2 to remain in the
ring plane and deliver electron density to the C7´=O7´ bond, reducing the effect exhibited in
Ch5 and Ch6.Futher bond order analysis (vide infra) helps to rationalizes these results.

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Table 2 shows the values of AEA, VEA and VDE calculated for Ch1-Ch6. If the VEA is
positive, the attachment of the electron to the molecule is energetically favorable, even
before geometrical rearrangements occur, i.e. the molecule can attract the excess of charge
density. On the other hand, a positive AEA indicates that the formed anion is stable in its
final geometry, i.e. that once the electron is trapped by the molecule, it stays there long
enough to rearrange and possibly to play a role in chemical reactions. When VDE is
positive the energy of the neutral molecule is higher than their anion, and it is stable with
respect to the vertical electron auto-detachment. VDE can be interpreted as the vertical
ionization potential of the anion. If the nuclear configuration of the anion and the neutral
molecule are similar, the values of VEA and VDE can be understood as lower and upper
bounds of the AEA, as can be seen in Table 2. Only in the case that electron attachment
generates a significant change in geometry, VDE could be lower than AEA.41
We found that all AEAs are positive, indicating a favorable tendency to form stable anion
radicals in this series. For Ch1-Ch3 calculated AEAs correlate well with observed
reduction potentials, with Ch2 showing the lowest potential and the lower AEA. The same
tendency is observed for Ch4-Ch6, where Ch5 also possesses the lowest potential and the
lowest AEA. These results indicate that the presence of a methoxy substituent in C4 of ring
B (Ch2 and Ch5) decreases the ability of chalcones to form a stable anion radical. When
Ch1-Ch3 are compared with Ch4-Ch5, they present higher AEA and higher reduction
potential. These results indicate that the presence of a methoxy group at C4´ of ring A
decreases the ability of the molecule to form a stable anion radical. On the other hand, all
VEAs are positive, which indicate that all molecules have favorable tendency to take an
electron. The effects of substituents have a similar impact for VEAs than for AEAs, which

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indicate that the presence of a methoxy groups at C4´ of ring A and C4 of ring B decrease
the ability of the molecule to take an electron and form a stable anion radical. Besides, all
VDEs are positive, which indicates that electron auto-detachment from the anion radical is
unlikely. In this case it is observed that the presence of a methoxy substituent at C4 of ring
B (Ch2 and Ch5) increases the possibility of an electron auto-detachment from the anion
radical, but does not affect in the same way to Ch4 and Ch6. Ch4 present a lower VDE
than their analog Ch1, but Ch5 and Ch6 present a higher VDE than Ch2 and Ch3, which
indicates that a methoxy substituent at C4 of ring B decreases the capability of suffering an
electron auto-detachment in Ch4, but increases in Ch5 and Ch6.
The attachment of an extra electron to a neutral molecule leads to one of two different types
of anions, valence-bound or dipole-bound anion.42 In the first case, the attached electron is
strongly bound occupying a valence molecular orbital. In the second case, the attached
electron is weakly bound, mainly by electrostatic charge-dipole interactions.43 Therefore, a
valence-bound anion suffer a much notable effect on their structural parameters than a
dipole-bound anion.44,45 In our cases, we found that the differences between AEAs and
VEAs is in all cases 0.2-0.3 eV. These differences indicate that the radical anions formed in
all molecules should be valence-bound anions, since a difference energy of ~0.3 eV is close
to the expected amount for relaxation energy of a valence-bound anion.39, 46 With the
purpose to study the distribution of the excess electron density of the anion radicals, the
singly occupied molecular orbitals (SOMOs) of the stable and vertically attached electron
were obtained (see Figure 1 and Figure 2 of electronic supplementary information, ESI).
This approach also helps to distinguish between valence-bound and dipole-bound anions,
given that SOMO of a dipolo-bound anion is centered in a limited part of the molecules,

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while SOMO of a valence-bound anion is distributed throughout the whole molecule.6, 39, 46
All anion radicals display valence-bound character, confirming the above statement about
the difference between AEAs and VEAs.
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Correlations between the experimental reduction potentials (-Ep,c) and calculated AEAs and
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VEAs are presented in Figure 5A and Figure 5B respectively. This kind of correlations
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have been made for different kind of compounds
and an interpretation of the results
can be carried out from the work of Parker.50 We found that the correlation between VEAs
and experimental reduction potentials (R2 = 0.98) was better than the correlation with
AEAs (R2=0.88). The above result suggests that the reduction potential depends more on
the ability of taking an extra electron than on the ability to form a stable anion radical. The
slopes in both cases are close to one, which indicate the absence of secondary effects such
as differential solvation and relaxation of the resulting anions. The results suggest that the
different methoxy substitution patterns of these chalcones do not modify such secondary
effects which remain fairly constant, regardless the electron affinities.
These results support the reliability of our calculations and are in agreement with
previously reported works, which states that the energetics of the reduction process should
be controlled by the first single-electron uptake.48 -51
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3.2.2. NBO analysis
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With the aim of studying the electronic characteristics and quantifying the strength of the
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(2)
O7´ H2´-O2´ IHB present in these chalcones, we carried out a detailed NBO analysis. Eij
is an appropriate tool to analyze hydrogen bonding, and allow us to quantify the strength of

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the donor-acceptor orbital interaction. H-NMR chemical shifts of H2 were used as an
experimental measurement of the strength of the IHB. Variation of solvent (DMSO and
CDCl3) do not affect this parameter, showing that the IHBs are strong enough to be
independent of the medium. Previously, we have reported a direct correlation between
(2)
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NBO stabilization energy Eij and H-NMR chemical shift of the hydrogen engaged in
the IHB in a series of hydroquinones.52 We found a similar behaviour for these chalcone
(2)
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derivatives. The second order stabilization energy Eij for the IHB and the H-NMR
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chemical shift ofH2 are presented in Table 3. For all molecules, we found that the *O2´-H2´
antibonding orbitals act as electron acceptors and oxygen 7´ lone pair (LPO7´) act as an
electron donor in the IHBs. The total stabilization energy for the IHB (LP1+2O7´  *O2´-
H2´) in Ch1, Ch2 and Ch3 are 43.9, 47.7 and 45.0 kcal/mol, respectively. A similar
behavior is observed for the analogue series Ch4, Ch5 and Ch6, with stabilization energies
of 49.6, 53.0 and 50.4 kcal/mol respectively. The series Ch1-Ch3 has lower reduction
potentials when compared with their analogs in the series Ch4-Ch6. The stabilization
energies for the IHBs are pointing in the same direction that the reduction potential, which
indicates that the strength of IHB has a strong influence in the capability of these chalcones
to capture an extra electron. Hyperconjugation is favored by a lower difference between
orbital energies ɛj–ɛI and a higher overlap between them (Fij). For all LP O7´  * O2´-H2´
interactions, we found that the difference among ∆Eij(2) can be attributed to the difference in
the overlap ability between orbitals, instead of their energy differences.
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Few quantitative relationships of NBO second-order stabilization energy with other
characteristic parameters of the hydrogen bond have been reported. Senthilkumar et al.53
showed a quantitative correlation between NBO energies and calculated hydrogen bond

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distances at B3LYP and MP2 level, in formic acid dimers. Also, recently we found good
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correlations among the experimental H-NMR chemical shift of O-H,52 electrochemical
oxidation potentials54 and NBO stabilization energy for the interaction O-H…O-C, in a
series of hydroquinones possessing IHB. As far as we know, there are no other reports in
the literature about a quantitative correlation between experimental IHB parameters and
1
calculated NBO energies. Figure 6a shows a good correlation between the H-NMR
chemical shift of H2´ and the Eij(2) for IHB. Having shown that this stabilization energy is
a good parameter for the IHB strength in these chalcones, we study their correlation with
the experimental reduction potentials (-Ep,c) and the results are displayed in Figure 6b. As
observed from this figure, there exist a good correlation between the strength of the IHB
and the experimental reduction potentials (R2 = 0.90). This correlation allow us to establish
that, in this series of chalcone derivatives, the IHB strength is a very important factor to
determine their capability to attach an extra electron, a remarkable observation.
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NBO method, besides determining the second-order stabilization energies, also allow us to
carry out a bond order analysis and to establish the covalency and ionicity of the bonds,
based in the natural resonance theory (NRT, for details see references).35,36,37,55 We focused
on calculating the bond order and the degree of covalency and ionicity for the bonds
involved in the O2´-H2´...O7´ IHB, both, in the neutral and the anion radical species. There
were three resonance structures considered by the NBO programme, third resonance
structures were neglected from all the analysis, since they represented less than 2% in all
cases. Figure 7 presents the resonance structures 1 and 2, along with the main data from
NRT calculations. Resonance structure 1 was always the major contributor to the neutral
species (~95%) and has been consistently major for Ch1 to Ch3 than Ch4 to Ch6. The

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latter observation is supported by the small but consistent bond order differences for O-H in
Ch1 to Ch3 when compared with Ch4 to Ch6 (see table attached to Figure 7). The O-H
bond for all chalcones has a low covalent character, around 40%. On the other hand, as can
...
be expected, the bond orders for H2´ O7´ is lower than for O2´-H2´, being higher for Ch4 to
Ch6 than for Ch1 to Ch3. The ionic character dominates this bond in all cases (96-97%).
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When the results for the anion radicals are examined, a very different behaviour is observed
between Ch1-Ch3 and Ch4-Ch6. Weights of resonance structures for Ch1 to Ch3 are
very similar to those that have the neutral species, ~92% for resonance structure 1 and ~6%
for resonance structure 2. Natural bond orders for O2´-H2´ of Ch1-Ch3 are ~0.46, with a
...
30% of covalency and 70% of ionicity, while H2' O7´ present a natural bond order around
0.03 with ~91% of ionicity for all three cases. On the other hand, for Ch4 the resonance
structure 2 takes a much greater weight than for Ch1 to Ch3, reaching ~34%, while for
Ch4 and Ch5 the resonance structure 2 becomes the dominant one, with a 93% for both.
Natural bond order and covalency of O2´-H2´ for Ch4 decreases slightly, compared with
Ch1 to Ch3, to values of 0.3214 and 28% respectively. However, both natural bond order
and covalency notably decrease to ~0.028 and 8% respectively for Ch5 and Ch6.
..
Moreover, H2'. O2' bond in Ch4 presents a notable increases in their natural bond order and
even more for Ch5 and Ch6. The above was accompanied by a decrease in ionic character
and an increase in covalent character, being more important for Ch5 and Ch6. Taking
together, these results evidence that the presence of the methoxy substituents on ring B do
not significantly affect the IHB in the radical anion when compared with the neutral
molecule. Besides, the presence of methoxy substituent in C4´ of ring A has a high impact,
modifying the IHB of the anion radical compared with the neutral molecule, being more

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different for Ch4 than for Ch5 and Ch6. The later suggests that the difference described
above in the values of VDEs between Ch4 and Ch5-Ch6 is closely related to their
difference in the radical anion IHB structure. There is a switching of the minimum in the
potential energy surface along the IHB coordinate.
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3.3 Implications with the biological activities of 2´-hydroxichalones
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The results of this work provide insights about the effect of methoxy substituents on the
IHB properties of Ch1-Ch6 and their ability to take an electron and generate a stable anion
radical. As we mentioned in the introduction, previous works have shown that this
25, 14,26
chalcones can inhibit the proliferation of different cancer cells lines.
where the
presence of methoxy substituents seems to play a critical role. It has been proposed that
some of the anti-proliferative activities of these compounds is related to their capability of
inducing mitochondrial mediated apoptosis.25 The ability of chalcones to generate radical
species could impact, for example, in their capability to modify the mitochondrial
membrane potential, inhibit the electron chain or affect their ability to act as uncoupler of
the oxidative phosphorylation.13,15
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Our results suggest that if it is necessary to increase the ability of the chalcone to form an
anion radical and their persistency in time, the presence of the methoxy substituent in C4 of
ring B should be avoided; their presence do not significantly affect the IHB in the radical
anion. On the other hand, the presence of a methoxy substituent in C4´ has opposite impact
in neutral and radical anion species. For the neutral specie, it decreases the ability to form a
radical anion, but once it is formed , it presents lower possibility to return back to the

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neutral form. The above can have a high impact on the IHB, making it weaker for the
anion radical than for the neutral species.
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Our results also suggest that in addition to the predictability of the AEAs and VEAs to
determine the reduction potential of these 2´-hydroxichalcone derivatives, the strength of
the IHB is also an acceptable parameter to predict this important experimental data, which
has been widely related to different biological activities. Nevertheless, this relationships
needs to be tested for a more extensive series of chalcones to establish their actual
reliability.
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4. Conclusions
Cyclic Voltammetry is a very useful technique to investigate the formation and stability of
anion radicals from chalcone derivatives. We found that the presence of a -OCH3
substituent in para position with respect to the carbonyl on ring A, decreases the energy of
the electronic state of the anion radical when compared with derivatives without this
substituent. This is closely related with the energy of the molecular orbital where the extra
electron is going to be placed. The calculation of the ratios Ipa / Ipc for Ch4-Ch6 shows that,
as expected, the formed radicals appears to be stable at low concentration, but when the
concentration increases they experience subsequent reactions.
…
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399
DFT results show that the IHB O2´-H2´ O7´ and the double bond C=C are the most
affected regions upon the electron attachment process. From the analysis of the AEAs,
VEAs and VDEs, we found that all chalcones can take an electron and form a stable
radical anion. Besides the auto-detachment process is very unlikely for all. The presence of
methoxy groups in C4 of ring B and in C4´ of ring A decreases the ability to take an

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electron and form a stable radical anion. On the other hand, the presence of a methoxy
group in C4 of ring B, increases the ability to experience an electron auto-detachment by
the radical anion, in all cases. However, the presence of a methoxy group at C4´ of ring A
has a different affect on Ch4 that on Ch5 and Ch6. For Ch4 the presence of a methoxy
decreases the trend to experience an electron auto-detachment once the anion radical is
formed, but for Ch5 and Ch6 this trend increases.
406
407
408
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412
VEAs exhibit a better correlation than AEAs, with experimental reduction potentials which
suggests that it is more influenced by the capability of taking the extra electron than by
forming a stable anion radical. The slopes of the correlations, close to one, strongly
suggests the absence of secondary effects, such as differential solvation and relaxation of
the resulting anions. These indicate that the different methoxy substituents patterns of
these chalcones does not modify such secondary effects, which remain fairly constant,
regardless the electro affinity values.
413
414
415
416
417
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419
Analyses of the second order stabilization NBO energies (∆Eij(2)) for the IHBs, allows us to
quantify the strength of the donor-acceptor orbital interaction. A comparison with 1H-
NMR data shows that NBO energy is a good parameter to measure the strength of the
IHB. Stabilization energies for the IHBs are pointing in the same direction that reduction
potentials, and they present a good semi-quantitative correlation (R2 =0.90), which
indicates that the strength of IHB has a significant influence in the reduction potential of
these chalcones.
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421
Natural bond order analysis shows that the presence of a methoxy substituent on ring B
does not significantly affect the IHB in the radical anion, when compared with the neutral

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species. On the other hand, the presence of a methoxy substituent at C4´ of ring A has a
significant impact weakening the IHB of the radical anion when compared with the
neutral species.
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430
Our results highlight the possibility of establishing direct links between molecular structure
details, such as the position of substituents on the aromatic rings and the presence of
intramolecular hydrogen bonds, with the electrochemical behavior of methoxylated 2-
hydroxychalcones. We hope this work drives the search for similar correlations in a broader
spectrum of molecules engaged in IHB, and can be useful in the design of new chalcones
with potential pharmacological activity.
431
5. Acknowledgements
432
433
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437
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439
We are grateful to FONDECYT grants 1140753, FONDECYT/POSTDOCTORADO
3140286 (M.M-C) and ACT grant 1107.
Electronic Supporting Information available: Optimized geometries, SOMO´s and energies
for all molecules.
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Figure 1. Compounds studied in this work
535
Compound
R
-OCH3

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Ch1
H
H
3
4
5
3
4
5
Ch2
Ch3
Ch4
Ch5
Ch6
H
OCH3
OCH3
OCH3
536
537
538
Figure 2. Cyclic voltammogram of 1 mM 2’-hydroxy chalcone derivatives in DMSO + 0.1
M TBPF at 1.0 Vs-1.
539
540
541
542
543
544
Figure 3. A: Cyclic voltagramm for chalcone 4 at different concentrations, with a scan rate
of 5 Vs-1. B: Dependence of the current ratio with the sweep rate for the one-electron redox
couple of chalcone 4. C: Current ratio dependence of chalcone 4 at different concentrations
and different sweep rates. All experiments were performed in DMSO and 0.1 M TBPF.

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555
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Figure 4. Main geometrical parameters for neutral and radical anion chalcones

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Ch1
Ch2
Ch3
Ch4
Ch5
Ch6
C-C
C-C7´
C7´=O7´
1.35 (1.39) 1.36 (1.39) 1.35 (1.39) 1.35 (1.39) 1.36 (1.39) 1.35 (1.39)
1.46 (1.41) 1.45 (1.41) 1.46 (1.41) 1.46 (1.41) 1.47 (1.40) 1.46 (1.40)
1.26 (1.32) 1.26 (1.32) 1.26 (1.32) 1.26 (1.32) 1.26 (1.35) 1.26 (1.35)
1.00 (1.07) 1.01 (1.08) 1.01 (1.07) 1.01 (1.09) 1.02 (1.39) 1.01 (1.37)
1.57 (1.37) 1.55 (1.35) 1.56 (1.37) 1.54 (1.33) 1.53 (1.06) 1.54 (1.07)
O2´-H2´
..
H2´´
O
7´
O2´-H2´-O7´ 150 (159) 151 (160) 151 (160) 152 (160) 152 (160) 152 (160)
560
561
562
Figure 5. Correlation graphics of experimental reduction potentials vs. theoretical AEAs
563
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(5A) and VEAs (5B).

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Figure 6. Correlations between experimental chemical shift for H2´ in DMSO-d6 (A) and
reduction potentials (B) vs. NBO stabilization energies for LP  * interactions in IHB
O7´...H2-O2´.
574
575
576
577
578
Figure 7. Resonance structures and main data from NRT calculation