Theoretical Investigation of the Structural, Spectroscopic, Electronic, and Pharmacological Properties of 4-Nerolidylcathecol, an Important Bioactive Molecule

4-Nerolidylcatechol (4NRC), a secondary metabolite described as a potent antioxidant that presents anti-inflammatory, antimalarial, analgesic, and cytotoxic properties, has been receiving prominence in the catechol class. In this work, a theoretical DFTstudy of the vibrational, structural, and quantum properties of 4-nerolidylcatechol (4NRC) using the B3LYP/6-311G (2d,p) level is presented.'e theoretical molecular geometry data were compared with the X-ray data of a similar molecule in the associated literature and a conformational study is presented, with the aim of providing a good comprehension of the 4NRC structural arrangement and stability. Also, HOMO-LUMO energy gap and natural bond orbitals (NBOs) were performed and discussed. 'e calculated UV spectrum showed similarity to the experimentally obtained data, with transitions assigned. 'e comparative IR studies revealed that intermolecular hydrogen bonds that stabilize dimeric forms are plausible and also allowed the assignment of several characteristic vibrations. Molecular docking calculations with DNA topoisomerase I-DNA complex (TOPO-I), glyceraldehyde 3-phospate dehydrogenase (GAPDH), and Plasmodium falciparum lactate dehydrogenase (PfLDH) showed binding free energies of −6.3, −6.5, and −7.6 kcal/mol, respectively, which indicates that 4NRC is a good competitive inhibitor for these enzymes.


Introduction
Catechols are a group of small molecules with physiological importance, acting as effective structural units in many adrenergic, Parkinson's disease [1], attention-deficit hyperactivity disorder (ADHD) [2], bronchodilator, antidepressants, antihypertensive drugs, perfumes, and agrochemical ingrediants (for the production of pesticides) [3].In organic chemistry, catechol (1,2-dihydroxybenzene) and its derivatives have widely attracted scientists since decades principally for the design of mussel-inspired synthetic adhesives and coatings [4].With respect to their biological importance, catechols can occur as metabolites [5,6] or as endogenous compounds, such as neurotransmitter and their precursors as adrenaline, noradrenaline, dopamine, and L-DOPA [1,5,6].
Catechols could act as antioxidants, preventing lipid peroxidation, and as pro-oxidant-damaging macromolecules such as DNA [7].
is work discusses 4NRC, isolated from Piper peltatum L., through a theoretical DFT approach (geometry, HOMO-LUMO orbitals, natural bond orbitals, and conformational analysis) based on experimental data (X-ray, UV, and FTIR), providing a more detailed description of the 4NRC structure and properties.Docking calculations were also performed with the objective of evaluating the interaction of this molecule with enzymes that justify its antimalarial and cytotoxic potential.As far as we know, no theoretical molecular modeling study that discusses the spectroscopic behavior of 4NRC with the quantum chemical DFT approach has been presented.It is important to mention that theoretical quantum models, such as DFT, and advanced software constitute a powerful tool for the study of the properties of several compounds and ultimately for the study of properties of secondary metabolites of plants [23][24][25][26].However, it is important to mention that a theoretical study of the interaction of 4NRC with 2hydroxypropyl-β-cyclodextrin (HP-b-CD) was performed previously but through a mechanical molecular approach [27].

Methodologies
4-Nerolidylcatechol was isolated of inflorescences from P. peltatum L. collected in March 2017 at the Federal University of Amazonas Campus, northern sector, Manaus City, Amazonas, Brazil (SISBIO n. 55863-1).e sample was dried, ground, and subjected to successive extraction in an ultrasonic bath, obtaining hexane and ethyl acetate extracts.A portion of the ethyl acetate extract was subjected to Sephadex LH20 column with hexane, dichloromethane, and methanol (2 : 5 :1).After TLC analysis, part of catechol-rich fraction was submitted to preparative TLC plate and eluted with hexane and ethyl acetate (7 : 3, two times) for isolation of 4NRC.e substance was confirmed by comparison with a 4NRC sample previously isolated by our group.e substance shows oil aspect and was solubilized with chloroform and then mixed with KBr salt until the solvent was evaporated for obtention of the FT-IR spectrum.e pastille was analyzed by a ermo Scientific Nicolet iS10 infrared spectrometer in the spectral region from 4000 to 400 cm −1 .
e sample was solubilized with ethanol and analyzed by an ultraviolet visible spectrometer (Global Trade, UV-5100) in the range between 190 and 300 nm. e quantum-chemical calculations were performed on the Gaussian 09 D.01 Program [28] on Debian LINUX (8.0 version) platform using an INTEL Quadcore ™ PC 16 GB.e DFT approach was selected for the calculations using the 6-311G (2d,p) basis sets and the B3LYP functional.To evaluate conformers' stability and energy barriers, scan calculations have been performed which consisted in 30 steps varying by 12 °(totalizing a 360 °turn) the dihedral angles C11-C12-C13-C14, C13-C14-C16-C17, and C16-C17-C18-C19.e most stable conformations (potential energy wells) were resubmitted to the same scan calculations procedure in order to find the structures with the lowest potential energy (minima geometry).All the minima structures were optimized by the force gradient method using Berny's algorithm and a standard analytical harmonic vibrational analysis (no imaginary frequencies or negative eigenvalues were found).e obtained theoretical IR spectra from the calculated DFT vibrational wavenumbers were uniformly scaled by a factor of 0.98.e UV spectra were calculated using the time-dependent density functional theory (TD-DFT) with B3LYP/6-311G (2d,p) and 6-311G++ (2d,p) basis set in methanol (polarizable continuum model).NBO values were obtained with the program NBO 3.1, implemented in the GAUSSIAN 09 package.e assignments of the calculated IR wavenumbers were aided by the animation option present in GAUSSVIEW 5.0, which promotes a visual presentation of the vibrational modes [29].e potential energy distribution (PED) was calculated using the VEDA 4 software package [30].e basis set superposition error (BSSE) of the interaction energies of the dimer was corrected by the counterpoise (CP) method [31].e methodology consisted in the single point (a posteriori) CP corrections on the conventionally optimized structures (which are not minima on the CPcorrected surfaces).

HOMO and LUMO Analysis.
e molecular orbital (MO) is a very important concept in quantum chemistry, being extensively employed to describe the chemical behavior.
e highest occupied molecular orbital (HOMO) and lower unoccupied molecular orbital (LUMO) are the two most important molecular orbitals in a molecule as both are used to describe various chemical properties such as reactivity and kinetics [33][34][35].Also, these orbitals are an indispensable tool for the description of other phenomena involving molecular electronic structures, such as charge transfer, photoexcitation, and molecular electronics.According to the Janak theorem [36] and Perdew et al. [37] MO theory approaches, the HOMO energy (ε HOMO ) is related to the IP, and the LUMO energy (ε LUMO ) has been used to estimate the electron affinity (EA), making it possible to calculate the important reactivity indices hardness (η), chemical potential (μ), electronegativity (χ), and electrophilicity index (ω) which are defined as follows [36][37][38]: e hardness (η), defined as the second derivative of the total energy, together with the concept of electronegativity and the principle of equalization of electronegativities, has been used to develop the principle of hard and soft acids and bases.Electrophilicity index (ω) [39,40] is the measure of the extent of the electrophilicity of a molecule.e chemical potential (μ) provides a global reactivity index, being interpreted as a charge transfer from a system of higher chemical potential to the one of lower chemical potential.Electronegativity (χ) represents the power to attract electrons and is equal to the negative of the chemical potential.All these properties were calculated for 4NRC at B3LYP/6-311G (2d,p) basis set using the above equations, and their values are shown in Table 1.As seen in Figure 4, the calculated HOMO and LUMO for 4NRC are located in the aromatic moiety (catechol moiety), indicating that this region is more susceptible to accepting or donating electrons, implying that the catechol moiety is the portion of higher reactivity and activity of the molecule.e HOMO-LUMO gap energy value (5.67 eV) indicates a good reactivity of 4NRC.e small hardness (η) value (2.83 eV) shows its high polarizability, leading to infer that 4NRC presents soft molecule behavior, with the electrons further from the nucleus (in the case of the phenolic moiety).e electronegativity (χ) and electrophilicity index (ω) values reveals that 4NRC has good attractive electron power and acts as a moderate electrophile.

UV-Vis Analysis.
e electronic spectrum of 4NRC in ethanol was compared to the calculated spectrum at timedependent density functional using B3LYP/6-311 G (2d,p) and B3LYP/6-311++G (2d,p) basis set in ethanol by the PCM model as shown in Figure 5. e experimental spectrum of the structure presented bands at 200 and 280 nm that were assigned to the sum of the σ ⟶ π * and π ⟶ π * transitions characteristic of catechols with conjugated system [22].e theoretical spectrum calculated at B3LYP/6-311++G (2d,p) presented an intense electronic transition of 6.03 eV at 205.46 nm (oscillator strength f � 0.2438) with major contributions from H − 3 ⟶ L (24.8%), H − 3 ⟶ L + 3 (13.7%),and H − 3 ⟶ L + 2 (12.23%), being equivalent to the experimental band at 200 nm.
e calculation also predicted intense electronic transition at 257.51 nm (4.81 eV) that are equivalent to the experimental bands at 280 nm, corresponding to the contribution of the electronic transition from H ⟶ L (83.46%).In B3LYP/6-311 (2d,p) calculation, the theoretical spectrum presented an intense electronic transition of 6.07 eV at 204.31 nm (oscillator strength f � 0.2505), with major contributions from H ⟶ L + 2 (82.57%) and a transition of 4.90 eV at 252.87 nm (oscillator strength f � 0.1357) with major contributions from H ⟶ L (82.88%).Concerning the employed basis set, B3LYP 6-311++G (2d,p) presented a better performance; however, B3LYP 6-311 G (2d,p) showed close values to the experimental ones too, revealing a good applicability of this base for the prediction of absorption spectra.

IR Analysis.
e assignment of the experimental bands to the vibration modes was made using the lowest energy structure, conformation B, by the calculated potential energy distribution (PED).A total of 153 normal vibration modes were obtained; however, only the modes between 400 and 4000 cm −1 were compared with the experimental IR data (Table 2).Figure 6 shows the superimposition of the experimental and theoretical IV spectra of 4NRC.e bands between 3200 and 2500 cm −1 were assigned to OH (band at 3468 cm −1 ), aromatic C-H (band at 2968 cm −1 ), and CH 2 stretching modes (bands at 2932 and 2923 cm −1 ).Bands     in the dimer, 3595.62 cm −1 , is closer to the experimental ones, 3468 cm −1 , while the monomer showed discrepant wavenumber values, 3767.29 and 3712.40 cm −1 .Regarding the stability of the proposed dimer, the analysis was given through ∆E (interaction energy value), defined as follows: where the interaction energy ΔE is −8.26 kcal/mol, which reveals that the dimer is more stable in relation to the separated monomers.Applying the counterpoise correction, the corrected ΔE value is −4.58 kcal/mol.It is important to note that each monomeric unit of the proposed dimer presents slight distortions in the geometry compared to the most stable calculated conformer (conformer B), which makes the value of ΔE questionable, as slight changes in the geometry imply differences in the energy value.e counterpoise correction methodology considers only the energies of the monomers that constitute the dimer, but does not consider the fact that the monomers can change their geometry when they form a dimer.In view of this, other ΔE calculation can be taken into account in this case, considering the energy of monomer B and the corrected energy of the CP dimer (counterpoise corrected energy): which results in a corrected ΔE of −3.51 kcal/mol and an incorrect ΔE of −7.15 kcal/mol.

NBO Study.
Natural bonding orbitals (NBOs) are localized orbitals of few centers (typically 1 or 2) and of maximum occupancy that are composed of natural hybrid orbitals (NHOs) from the optimized linear combination of NAOs of a given set of atoms.e NBO analysis provides very important information, especially for organic chemists, of a molecule because it presents a localized representation of the natural Lewis structure (NLS) of the wave function, such as delocalisation degree of the electron structure, orbital interactions charge balance, and binding order.e orbital interactions (hyperconjugation interactions), which constitute a notorious stabilizing factor of a molecule, are given by the second-order perturbation energies E (2) [donor (i) ⟶ acceptor (j)] in the NBOs calculations, which is defined as follows [41][42][43]: us, NBOs constitute an important theoretical tool for analysis, because they provide a description of valence bond type of the wave function closely linked to the classical concepts of Lewis structure, which makes the NBOs useful in understanding the delocalisation of the electron density [44][45][46][47].e NBO analysis (Table S2) for the title molecule revealed strong hyperconjugative intramolecular interactions of π ⟶ π * , σ ⟶ π * , and LP ⟶ π * transitions that lead to an intramolecular electronic density transfer, causing stabilization of the molecular system in 4NRC.e second-order perturbation energy analysis shows greater conjugations in the benzene ring principally by π ⟶ π * and LP ⟶ π * interactions as C5-C5 ⟶ C1-C2 (19.46 kcal/ mol), C1-C2 ⟶ C3-C4 (20.03 kcal/mol), C3-C4 ⟶ C5-C6(18.77kcal/mol), C3-C4 ⟶ C1-C3 (21.53 kcal/mol), O1 ⟶ C3-C4 (21.98 kcal/mol), and O2 ⟶ C3-C4 (21.98 kcal/ mol).σ ⟶ σ * hyperconjugative intramolecular interactions also occur and contributes to the stabilization of the system deserving prominence in the interactions C13-H13 ⟶ C14-C15 (7.89 kcal/mol), C18-H18 ⟶ C19-C21 (7.88 kcal/mol), C9-H9 ⟶ C7-C8 (7.77 kcal/mol), and C9-H9 ⟶ C8-H8 (5.15 kcal/mol)).For the dimer, NBO calculations revealed LP ⟶ σ * hyperconjugative intermolecular interactions in order of 4 kcal/mol between the OH groups that form the O-H-O hydrogen bonds.[48] is a very useful online tool that estimates the pharmacological activities of a molecule based on SAR (structure activity relationship) analysis from MDDR database (produced by Accelrys and Prous Science) [49,50].It works on the principle that the biological activity of a compound equates to its structure, thus comparing the molecular structure with more than 200,000 active compounds, allowing to estimate more than 3000 kinds of biological activities.e PASS prediction gives Pa (probable activity) and Pi (probable inactivity) values, which can vary from 0.000 to 1.000; in general, Pa + Pi ≠ 1, since these probabilities are calculated independently [50].e PASS prediction results for the studied molecule are listed in Table 3.

Molecular Docking Studies. PASS (prediction of activity spectra)
Among the various predicted properties, the following activities deserve attention: antioxidant, free radical scavenger, antiprotozoal, antieczematic, hypolipemic, antineoplastic,    hepatoprotectant, antihelmintic, lipid metabolism regulator, cholesterol inhibitor, MMP9 expression inhibitor, and topoisomerase I inhibitor.In fact, some of these activities have been confirmed in vitro [9, 11-13, 15-18, 20].Guided by the antineoplastic activity of 4NRC, molecular docking calculations were performed on AutoDock-Vina [51] with DNA Topoisomerase I complex with DNA (topo I-DNA) due to the fact that a large number of structures used for the treatment of human (antineoplastic) cancers, such as brain, ovary, colon, and lung cancers are eukaryotic topo I inhibitors.e second enzyme chosen for docking calculations was Plasmodium falciparum lactate dehydrogenase (PfLDH) due to the parasite's dependence on glycolysis for energy production.Because the lactate dehydrogenase (LDH) enzymes found in P. vivax, P. malariae, and P. ovale (pLDH) exhibit great similarity to PfLDH (about 90%), this enzyme is an effective drug target against malaria [52].Human glyceraldehyde 3-phospate dehydrogenase (GAPDH) was the third target enzyme chosen because it catalyzes the sixth step of glycolysis and thus serves to break down glucose for energy and carbon molecules.e glycolytic and antiapoptotic functions of this enzyme promote tumorigenesis because they contribute to the proliferation and protection of tumor cells.GAPDH is overexpressed in multiple human cancers, such as cutaneous melanoma, being positively correlated with tumor progression [53,54].
e docking calculations in AutoDock Vina (ADTV) consist in a number of sequential steps, where each step involves a random perturbation of the conformation followed by a local optimization that uses the Broyden-Fletcher-Goldfarb-Shanno algorithm [55] (an efficient quasi-Newton method).Each local optimization involves many "evaluations" of the scoring function as well as its derivatives in the position-orientation-torsion coordinates, resulting in structures that are accepted or not [51,55].e X-ray crystal structure of human topoisomerase I (PDB ID: 1k4t) [56], Plasmodium falciparum lactate dehydrogenase (PDB ID: 1cet) [57], and GAPDH (PDB ID: 1u8F) [58] were obtained from the Protein Data Bank website (http://www.rcsb.org/pdb/).For topoisomerase I, water molecules and topotecan were removed; Gasteiger charges were assigned to the PDBQT file format generated by ADTV.A grid box size of 10 × 16 × 10 Å was centered at the site of DNA cleavage of topo I-DNA complex (x � 21.260, y � −3.683, and z � 28.289).For Plasmodium falciparum lactate dehydrogenase, a grid box size of 16 × 16 × 10 Å was centered at the site of chloroquine interaction (x � 35.726, y � 10.811, and z � 19.711).For GAPDH, the same procedure was performed with a grid box size of 18 x 20 x 20 Å centered at the site of NAD interaction (Figure S1(c)).e validation of each grid box was done by removing the ligands (inhibitor) and   Journal of Chemistry redocking them at the same site using the proposed grid box.en, a comparison between the docked structure and the X-ray structure was performed through the RMSD calculation.For all the performed calculations, the obtained RMSD values were lower than 2 (Figures S1(a)-S1(c)).
Free energy of binding (ΔG) analysis demonstrated that 4NRC molecule docked with ΔG values of −6.3, −6.5, and −7.6 kcal/mol with PfLDH, TOPO I, and GAPDH, respectively, while the known inhibitors (or ligands) docked with ΔG values of −7.0, −12.6, and −10.6 kcal/mol, respectively (Figure 8).Binding modes analysis demonstrated that the title molecule docked at the PfLDH site similar to chloroquine (Figure 8(a)); however, studies confirm that chloroquine acts as a weak inhibitor of PfLDH, with mild selectivity for the parasite enzyme [56], but low levels of inhibition may contribute to the biological efficacy of the drug, a factor that seems to apply to 4NRC molecule too.4NRC binds at the site of the macromolecule by noncovalent alkyl-alkyl and alkyl-π interactions, where the catechol moiety interacts with Ala 98 and Ile54 (alkyl-π) and the side chain interacts with Phe 100, Ile 119, Leu 115, and Lys 118 residues (alkyl-alkyl, Figure 9(a)).A hydrogen bond between the hydroxyl group of the catechol moiety and Tyr 85 residue was also confirmed.
With TOPO I, the binding modes analysis showed that 4NRC docked at the site of DNA cleavage, between the base pairing, similar to topotecan (Figure 8(b)); however, for having four conjugated aromatic rings and a planar structure, topotecan molecule presents a lower ΔG and a more effective docking than 4NRC.On the other hand, the calculations for 4NRC revealed that catechol moiety binds to DC 112, DA 113, and TGP11 base pairs by π-π interactions, besides forming a hydrogen bond (with the DC 112 base).e side chain interacts by weak noncovalent interaction with Glu 356, preventing the DNA cleavage (see Figure 9(b)).For GAPDH, the binding modes revealed that 4NRC binds at the site similar to NAD molecule (Figure 8(c)).Despite the high value of ΔG in relation to ligand, the catechol moiety of 4NRC forms an intermolecular hydrogen bond with ASN9, which makes 4NRC a competitive inhibitor for this essential glycolytic enzyme.Weak π-alkyl and alkyl-alkyl type interactions with Asp35, Gly10, r99, Ile14, and Arg13 residues were also registered in the active site, thus contributing to the inhibition of GAPDH (Figure 9(c)).

Conclusion
4-Nerolidylcatechol was comprehensively characterized with its spectral behavior and quantum properties described.
e interatomic distances and angles proved to be plausible compared to the X-ray data for a similar structure.e conformational analysis of 4NRC showed seven stable conformations in the gas phase, but, comparing the potential energies values, conformer B is the most stable rotamer by an average ∆E ≈ 0.1 kcal/mol.e small HOMO-LUMO energy gap calculated for both structures are directly related to their amount of chemical hardness (2.83 eV), which lead to classifying them as soft molecules when compared to other structures in the associated literature.e electronegativity (χ) and electrophilicity index (ω) values reveal that 4NRC has good attractive power and acts as a moderate electrophile, which can justify in part its known antioxidant potential.
e comparative IR studies showed that the intermolecular hydrogen bonds of the proposed dimer are plausible (making them closest to the experimental ones) and also revealed several characteristic vibrations of 4NRC, thus complementing the vibrational study for these type of   molecules.e molecular docking results revealed good interactions of the title molecule with the DNA-topoisomerase I complex, glyceraldehyde 3-phospate dehydrogenase (GAPDH), and Plasmodium falciparum lactate dehydrogenase (PfLDH), critical enzymes for antimalarial and antitumor activities.

Figure 1 :
Figure 1: Optimized structure of the most stable conformation in the gas phase of 4-nerolidylcatechol (4NRC) with scheme of atom numbering.

Figure 6 :
Figure 6: Comparison between experimental and calculated spectra of 4NRC.

Figure 8 :
Figure 8: Superimpositions of the docked 4NRC structures and cocrystallized ligands into the selected enzymes, PfLDH, TOPO I, and GAPDH.(a) Comparison of the docked 4NRC (magenta) and cocrystallized structure of chloroquine (green); (b) comparison of the docked 4NRC (blue) and cocrystallized structure of topotecan (pink); (c) comparison of the docked 4NRC (pink) and cocrystallized structure of NADH (green).

Table 3 :
PASS prediction for the activity spectrum of 4NRC with Pa > 0.4