Vibrational characterization of active drug to the treatment of chagas disease, benznidazole by using force fields and internal coordinates

: Two experimental structures of benznidazole active drug used to the treatment of Chagas disease have been structurally characterized and its vibrational spectra completely assigned combining B3LYP/6-311++G** calculations with the experimental FT-IR and FT-Raman spectra and the SQMFF methodology. The most stable conformer of benznidazole found in the study of the potential energy surface is in agreement with that experimentally observed by X-ray diffraction at room temperature while the other one was observed with the heating up to 195 ºC. Both differs in the positions of CH 2 groups of acetamide fragment. Their structural properties in gas phase and ethanol solution were computed by using natural bond orbital (NBO), atoms in molecules (AIM), Merz-Kollman (MK) charges, molecular electrostatic potentials (MEP) and frontier orbitals calculations by using the hybrid B3LYP method and the 6-31G* and 6-311++G** basis sets. Additional WB97XD/6-311++G** calculations show that the energy values optimized for the most stable species in both media present lower values than the obtained with the B3LYP/6-31G* method. The vibrational assignments for those two conformers in both media were obtained from their corresponding harmonic force fields together with the scaled force constants.


Figure 1. Conformations s-cis and s-trans of Benznidazole
The physicochemical characterization of benznidazole by using the infrared and Raman spectra and the studies of thermal stability and compatibility were also performed by Soares-Sobrinho et al. but in this study the vibrational assignments of both spectra for benznidazole were no reported (Soares-Sobrinho, et al., 2010).
Obviously, the complete characterization of this important drug by using the vibrational spectroscopy is very important to identify with this technique their presence in different media.
Hence, the aims of this work are to perform the complete assignments of the vibrational spectra for those two experimental structures observed of benznidazole combining DFT calculations with the experimental infrared and Raman spectra by using their harmonic force fields, normal internal coordinates, transferable scaling factors and the SQMFF methodology (Pulay, et al., 1983;Rauhut, & Pulay, 1995;Sundius, 2002).
For these purposes, the potential energy surfaces for conformations cis and trans of benznidazole were studied in gas phase and in ethanol solution by using the B3LYP/6-31G(d) and 6-311++G(d,p) and WB97XD methods (Becke, 1988;Lee, Yang, & Parr, 1988;Chai, & Head-Gordon, 2018) while the solvent effects in solution were considered with the polarized continuum (PCM) and solvation models (Tomasi, & Persico, 1994;Miertus, Scrocco, & Tomasi, 1981;Marenich, Cramer, & Truhlar, 2009).Here, our results suggest two stable cis conformers for benznidazole, named C2 and C4, where the assignments of both forms s-Cis were considered in the vibrational analyses because only these structures were experimentally observed by X-ray diffraction although C4 was observed not at room temperature as C2 (Soares-Sobrinho, et al., 2008;Honorato, et al., 2014).At this time, the structural, topological and vibrational properties were studied in both media for the most stable C2 structure of benznidazole observed at room temperature.
Then, the harmonic scaled force constants for those structures of benznidazole in both media were reported for both methods.Additionally, the frontier orbitals were used to predict their reactivities and behaviours in both media by using different global descriptors (Parr, & Pearson, 1983;Karrouchi, et al., 2021;Laurella, 66 et al., 2022;Karrouchi, et al., 2021;Mortada, et al., 2022).
Between both structures only changes in the positions of two CH2 groups linked to benzyl and acetamide fragment are observed with the heating, as mentioned by Honorato et al. (Honorato, et al., 2014).However, the optimizations of C4 in both media show that this structure is the same than C2 but with opposite dihedral angles.Thus, for the C2 conformation experimentally observed, atomic natural population (NPA) and Merz-Kollman (MK) charges were studied in the two media (Besler, Merz, & Kollman, 1990).Moreover, the molecular electrostatic potentials were calculated by using those latter charges while the donoracceptor interactions and bond orders were computed by using NBO calculations (Glendening, et al., 1996).Intra-molecular interactions were evaluated taking into account the atoms in molecules theory by using the topological properties calculated with the AIM2000 program (Bader, 1990;Biegler-Köning, Schönbohm, & Bayles, 2001).In this work, the frontier orbitals were first calculated and, then, the gap values and, after that, with these values the chemical potential (μ), electronegativity (χ), global hardness (η), global softness (S) and global electrophilicity index (ω) descriptors were predicted by means of know equations (Parr, & Pearson, 1983;Karrouchi, et al., 2021;Laurella, et al., 2022;Karrouchi, et al., 2021;Mortada, et al., 2022).Harmonic force 67 fields and the scaled force constants were determined with the SQMFF methodology and the Molvib program using normal internal coordinates and transferable scaling factors (Pulay, et al., 1983;Rauhut, & Pulay, 1995;Sundius, 2002) while in the vibrational assignments of IR and Raman spectra we considered potential energy distribution (PED) contribution ≥ 10%.Further, the 1 H and 13 C chemical shifts were computed applying the Gauge-Independent Atomic Orbital (GIAO) method using tetramethylsilane (TMS) as reference (Ditchfield, 1974).Here, Timedependent DFT calculations (TD-DFT) at the B3LYP/6-311++G** levels of theory were applied to predict the ultraviolet-visible spectrum of BENZ in ethanol solution using the Gaussian 16 program (Frisch, et al., 2019).In addition, the TDDFT calculations were also employed to predict the rotatory strengths by ECD.

Structural Study
First, B3LYP/6-31G* calculations were performed for BENZ, three s-CIS (C1, C2 and C4) and one s-TRANS named C3 and, then, hybrid B3LYP/6-311++G** calculations in order to analyse the populations of those conformers of BENZ.Hence, a comparison of theoretical C2 and C4 structures of BENZ with the corresponding experimental at room temperature determined by X-ray diffraction by Soares-Sobrinho et al (Soares-Sobrinho, et al., 2008) and by Honorato et al with the heating (Honorato, et al., 2014) are shown in Figure 3.  Soares-Sobrinho, et al., 2008;Honorato, et al., 2014 Then, total and relative energies, dipolar moment, volume and population values for those conformers of BENZ calculated in the two media with both B3LYP and WB97XD methods and the 6-311++G** basis set are shown in Table 1.Two dihedral C11-C9-N5-C10 and N4-C8-C10-N5 angles are also included in that table together with the populations.Regarding Table 1, we observed that the energy values of C1 and C2 with both methods are approximately similar between them but with differences of 0.52 kJ/mol in gas phase.The energy, V, µ and population values for both in solution with the B3LYP/6-311++G** method are the same and the only difference observed between them is the positive C11-C9-N5-C10 dihedral angle.Then, C1 structure is the same than C4 in gas phase but this latter structure is observed with the heating while C2 is observed at room temperature.Thus, the only structure experimentally observed at room temperature is C2.Besides, the C4 form in gas phase is different from the optimized in solution, as can be seen in Figure S1 (Appendix 1).Table 1 also shows that C3 structure presents the higher relative energy (18.88 kJ/mol) and dipole moment in gas phase by using the B3LY/6-311++G** level of theory and, consequently, null population is observed in both media.Hence, taking into account that C2 is the only conformer experimentally observed at room temperature, the properties for this form of BENZ were predicted by using the higher level of theory.Additional WB97XD/6-311++G** calculations have optimized C2 in gas phase with an E value of -909.0311Hartrees and µ= 2.38 D. An E value enough lower than the obtained with the B3LYP/6-31G* method in the same medium (-909.0831Hartrees), as reported for other species (Sundius, & Brandán, 2023;Romano, E., et al., 2023).When the dipole moment values for the C1, C2, C3 and C4 conformers in gas phase are graphically represented by using the B3LYP/6-311++G** method we observed that the magnitudes, directions and orientations of their vectors are different among them, as shown in Figure S2 (Appendix 1).In C1, C2 and C4 the vectors are oriented in direction to C=O bonds on to acetamide fragment while in C3 the vector is located within the CH2 group near to benzyl ring, as observed in Figure S2 (Appendix 1).Probably, the positions, directions and orientations of dipole moments vectors and, their magnitudes explain the presence of C2 in the solid phase, as was reported for other compounds (Romano, et al., 2011;Romano, et al., 2013;Romano, Ladetto, & Brandán, 2013;Guzzetti, et al., 2013).Apparently, these results could have impact on structural and electronic properties and, on its reactivities and behaviours in both media.
Theoretical geometrical parameters calculated for C2 in both media using two B3LYP methods with the 6-31G* and 6-311++G** basis sets are compared in

NPA and MK Charges, MEP and Bond Orders
The interesting pharmacological properties observed in BENZ could probably be associated to the hydrophobic and hydrophilic sites and, for these reasons, the identifications of these possible reaction sites are necessaries requirements.Besides, the bond orders are related to forces of the different bonds and to those reactivity sites because when a bond is weak, the break of that bond is expected, so, for this reason, the bond order (BO) values is other very important property that must be studied.Hence, the atomic natural population (NPA), Merz-Kollman (MK) charges, molecular electrostatic potential (MEP) and bond orders (BO) values are investigate for C2 in both media (Besler, Merz, & Kollman, 1990).These three properties were analysed only for the acceptors (N and O atoms) and donors of H bonds (N-H) groups of BEZ in both media by using the B3LYP/6-311++G** method.Table 3 summarize those properties while Figure 4 shows the behaviours of two atomic charges studied on the atoms involved in acceptors and donor's groups of H bonds of C2.The exhaustive analysis of charges shows different curves indicating that the behaviours of MK charges are different from NPA ones.However, few changes are observed in both charges in ethanol solution.From the figure 5 it is observed that the range colours decrease from ±0.056 a.u. with the B3LYP/6-31G* method to ±0.056 a.u. with the higher level of theory while the colorations observed in the different regions are approximately the same.Thus, the intense red colours are observed on the N6 of imidazole ring and on the O atoms of NO2 and on C=O groups.These sites correspond to nucleophilic regions while the blue colours are observed on the N-H and CH2 groups of acetamide fragment and correspond to electrophilic sites.In general, the green colours are observed on benzyl ring and correspond to inert sites.
In relation to the BO values, Table 3 show that the three O atoms and N6, N7 and H24 have lower values in solution while N4 and N5 present higher BO values in this medium.These variations in the BOs are related to their hydrations because they are clear sites of formation of H bonds.

Stability Studies
The stability study of BENZ is important due to its observed biological properties as a consequence of acceptors and donor's groups and, also because we need to know why C2 is experimentally observed when the other stable C1 and C4 conformers have approximately the same energy values than C2 (Caryn, 2015;Rassi, Rassi, & Marin-Neto, 2010;Bern, 2015;Figueroa, et al., 2018;Tonin, et al., 2009).Hence, to examine the stabilities in both media NBO and AIM calculations were performed (Glendening, et al., 1996;Bader, 1990;Biegler-Köning, Schönbohm, & Bayles, 2001).Thus, the donor-acceptor energy interactions and their topological properties were predicted for C2 in both media and these results are presented in Tables S1 and S2 (Appendix 2), respectively (Glendening, et al., 1996).Regarding first the donor-acceptor energy interactions computed by using the two levels of theory from Table S1 ( and LP(1)N6→ π*C13-C16 interactions are only predicted in solution with the B3LYP/6-311++G** method.Thus, the evaluation of total energy with both methods show that C2 is most stable in solution (4683.20 kJ/mol) while in gas phase the higher stability is observed with the B3LYP/6-31G* level of theory (2900.57kJ/mol).

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The topological properties of C2 in the two media were also evaluated with the AIM 2000 program to predict their stabilities (Bader, 1990;Biegler-Köning, Schönbohm, & Bayles, 2001).Hence, the Bader's theory was used to analyse the intra-molecular interactions (Bader, 1990) calculating the electron density distribution, ρ(r), the values of the Laplacian, ∇ 2 ρ(r), the eigenvalues (λ1, λ2, λ3) of the Hessian matrix and the λ1/λ3 ratio in the bond critical points (BCPs) and ring critical points (RCPs).Thus, with these parameters it is possible to identify the characteristics and type of interaction.Thus, when an interaction present high values of ρ(r) and ∇ 2 ρ(r), the ratio λ1/λ3> 1 and ∇ 2 ρ(r) < 0 the interaction is covalent (shared interaction) while the interaction is ionic or highly polar covalent when λ1/λ3< 1 and ∇ 2 ρ(r) > 0 (closedshell interaction).Hence, in Table S2 (Appendix 2) are summarized those parameters for C2 in both media while in Figure 6 can be seen details of the molecular models for the C2 conformer of BENZ in gas phase by using the B3LYP/6-31G* and B3LYP/6-311++G** levels of theory, respectively.The figure shows three H bonds interactions with the B3LYP/6-31G* method which are, N5-H24•••O3, C8-H21•••O3 and C10-O1•••H26 interactions while with the higher level of theory only are predicted the two first interactions.Thus, Table S2 (Appendix 2) shows that the number of H bonds interactions and their properties are dependent on the used method.Analysing the interactions in ethanol solution with both methods from Figure 7 we observed for each method one same interaction but with different characteristics, as can be seen from Table S2 (Appendix 2).This way, Figure 7 shows the only N7-O3•••C8 interaction predicted for both methods in solution by using the B3LYP/6-31G* method.A same graphic is observed with the other method and, for this reason, the corresponding figure is not presented here.Higher densities are predicted for the H bonds interactions in gas phase with the B3LYP/6-31G* method than the other one while on the contrary it is predicted for the RCPAs of rings and of new RCPNs.
The presence of three and two H bonds in C2 with both basis sets could justify its experimental presence in the solid phase.Note that RCPA1and RCPA2 belong to the benzyl and imidazole rings, respectively while the new ring critical points are identified as RCPN.Both NBO and AIM studies support the high stability of C2 conformer in the gas phase due to the presence of three different N-H H interactions in gas phase and two in ethanol solution, as was observed in the crystal packing of BENZ in the solid phase (Soares-Sobrinho, et al., 2008).Besides, the high total energy value predicted by NBO analysis for C2 in ethanol solution using the B3LYP/6-311++G** method, as compared with the other one, reveal the influence of level of theory and the medium on the total stabilization energy.

Frontier orbitals and Descriptors
Reactivities and behaviours of C2 in gas phase and ethanol solution have been predicted by using the gap energy values calculated from the frontier orbitals, as suggested by Parr and Pearson (Parr, & Pearson, 1983).Then, with the gap values the chemical potential (μ), electronegativity (χ), global hardness (η), global softness (S) and global electrophilicity index (ω) descriptors were estimated using known equations (Karrouchi, et al., 2021;Laurella, et al., 2022;Karrouchi, et al., 2021;Mortada, et al., 2022;Sundius, & Brandán, 2023;Castillo, et al., 2018;Romano, E., et al., 2023).Thus, in Table S3 (Appendix 2) are summarized the gap values for C2 in the two media together with the calculated descriptors by using both levels of theory.The equations used in the calculations of descriptors are given in the same table .The results for C2 are compared with reported for the thiol and thione forms of 1,3-benzothiazole tautomers, a compound with potential antimicrobial activity because these species also present NO2 groups in the five member's rings and have benzyl rings, as shown in Figure S3 (Appendix 1) (Romani, & Brandán, 2015).
Regarding first the gap values for C2 with both methods, we observed that lower gap values are observed in solution, for which, C2 is most reactive in solution.From the comparisons with the two tautomers of 1,3-benzothiazole, the thione form in gas phase is most reactive than C2 and thiol in the two media.Thus, the fused benzyl and thiazole rings increase the reactivity of compound.When the descriptors are evaluated we observed for the most reactive thiazole species < chemical potential (μ), < electronegativity (χ) and < global hardness (η).Thus, this study show that the different methods have few influence on the gap values and descriptors.Hence, the low gap values and the high reactivities of these species could explain their biological activities.

NMR Study
The predicted 1 H and 13 C NMR chemical shifts for C2 in ethanol solution were calculated for both levels of theory by using the GIAO method (Ditchfield, 1974) and are respectively compared in Tables 4 and 5 with the corresponding experimental ones for Benz in CHCl3 taken from Ref (SpectraBase, n.a.) by using the RMSD values.In general, an overestimation in the theoretical values is observed with both methods as compared with the experimental ones and a better concordance for the 1 H nucleus (0.7 ppm) than for the 13 C nucleus (6.7-8.1 ppm) was observed.An uninspected resulted is obtained because usually better correlations are observed with the 6-311++G** basis set than the other one due to that generate better-quality results for the 1 H nucleus than the C atoms.However, here similar RMSD values are predicted for the 1 H nucleus with the 6-31G* basis set and a lower value for the C atoms.Here, the similarity in the RMSD values for both H and C nucleuses support the presence of C2 in the liquid phase and probably also will be present in the solid state.Besides, these reasonable correlations suggest that the structures of C2 are optima to perform the vibrational studies with both methods.

Vibrational Study
In this analysis, we have considered the two C2 and C4 structures s-Cis experimentally observed for BENZ by X-ray diffraction at room temperature with the heating, respectively (Soares-Sobrinho, et al., 2008;Honorato, et al., 2014).Figures 8 and 9 show comparisons of predicted IR and Raman spectra of C2 and C4 conformers of BENZ in ethanol and gas phase, respectively by using the B3LYP/6-311++G** method with the corresponding experimental Attenuated Total Reflectance Infrared (ATR-IR) and Raman spectra in the solid phase (Soares-Sobrinho, et al., 2010;SpectraBase, n.a.).We see reasonable concordances between the IR spectra of both forms in solution because the calculations in gas phase were performed for the isolated molecule where the forces packing were not considered while in solution the used method consider solute-solvent interactions and, hence, a higher number of bands are observed, as in the experimental spectrum.Comparisons of both predicted Raman spectra with the corresponding experimental one after the conversion from scattering activities to Raman intensities by using known equations show very good correlations (Keresztury, et al., 1993).Optimized C2 and C4 conformers show C1 symmetries and due to the 31 atoms present in both structures 87 normal vibration modes are expected for these species and, where all the vibration modes present activities in both spectra.Complete vibrational assignments have been performed using the SQMFF methodology and the Molvib program with potential energy distribution (PED) contributions ≥ 10%.In that procedure, normal internal coordinates and scaling factors were used (Pulay, et al., 1983;Rauhut, & Pulay, 1995;Sundius, 2002).Table 6 shows observed and calculated wavenumbers and assignments for C2 and C4 conformations of BENZ in gas phase using the B3LYP/6-311++G** level of theory.Here, the increasing in the intensities of IR and Raman bands between 1745-1503 cm -1 (Figure 9) are due to changes conformational, as mentioned by Honorate et al (2014).Hence, differences in the vibrational assignments and in the intensities of some IR and Raman bands for both IR spectra of C2 and C4 can be seen from Figs 8 and 9. Note: Abbreviations: ν, stretching; β, deformation in the plane; γ, deformation out of plane; wag, wagging; τ, torsion; ρ, rocking; τw, twisting; δ, deformation; a, antisymmetric; s, symmetric; A1, Benzyl; A2, imidazole rings; a This work, b From SQMFF/B3LYP/6-311++G** method, c From SpectraBase, n.a.
Discussions on assignments of the most important groups are given below.

Band Assignments
NH modes.Generally, the NH stretching modes in compounds containing these groups are assigned between 3400 and 3300 cm -1 and, in the previous assignment reported by Soares-Sobrinho et al for BENZ this mode was assigned to the IR band at 3330 cm -1 (Soares- Sobrinho, et al., 2010;Karrouchi, et al., 2021;Karrouchi, et al., 2021;Mortada, et al., 2022).Hence, the IR band of medium intensity at 3330 cm -1 is without difficulty assigned to those vibration modes of C2 and C4, as observed in Table 6.The in-plane deformation or rocking mode are predicted by calculations for both conformers between 1517 and 1480 cm -1 (Soares- Sobrinho, et al., 2010;Karrouchi, et al., 2021;Karrouchi, et al., 2021;Mortada, et al., 2022).Hence, those modes are assigned accordingly.The SQM calculations predicted the out-of-plane deformation modes of C2 and C4 conformers coupled with other modes and between 652 and 573 cm -1 , hence; these modes are assigned to the shoulders at 683 and 572 cm -1 , as indicated in Table 6.
CH modes.In both conformers of BENZ there are seven aromatic C-H stretching modes (five of benzyl rings and two of imidazole rings).The SQM calculations predicted the stretching modes of imidazole rings between 3135 and 3112 cm -1 , hence; the IR bands between 3180 and 3160 cm -1 are assigned to those vibration modes, as expressed in Table 6.The C-H stretching modes of benzyl rings are predicted between 3058 and 3022 cm -1 , therefore, they are assigned in that mentioned region.The rocking modes for C2 and C4 conformers are predicted between 1480 and 1064 cm -1 , hence, they are assigned in these regions, as detailed in Table 6 (Karrouchi, et al., 2021;Laurella, et al., 2022;Karrouchi, et al., 2021;Mortada, et al., 2022).
CH2 modes.The positions of these groups in C2 is different from C4, as was observed by infrared spectroscopy by Honorate et al (2014).Thus, changes in the intensities of some bands between 1510 and 1400 cm -1 region are related to deformation modes of these groups predicted between 1415 and 1412 cm -1 .The SQM calculations predict the waging, rocking and twisting modes for these two conformers in approximately the same regions.Thus, the group of bands at 1367/1349, 1297/1181 and 974/793 cm -1 are assigned to those vibration modes.
NO2 modes.Generally, the two NO2 antisymmetric and symmetric stretching modes are assigned between 1584 and 1335 cm -1 (Romani, & Brandán, 2015;Castillo, et al., 2017).These N=O bonds in C2 and C4 conformers have double bond characters and, hence, the strong and medium intensity IR bands respectively at 1535 and 1287 cm -1 can easily be assigned to those modes for both conformers of BENZ.Normally, the NO2 deformation modes are assigned between 841 and 821 cm -1 while the wagging modes of both forms are assigned at 775-756 cm -1 .In C2 and C4, the NO2 deformation and wagging modes are predicted in the same region, hence, they were assigned to the weak band at 835 cm -1 and to the strong band at 744 cm -1 , respectively (Romani, & Brandán, 2015;Castillo, et al., 2017).The rocking modes are clearly predicted in the same region and, for this reason, those modes are assigned to the medium intensity IR band at 558 cm -1 , in accordance to those two tautomers which were assigned at 545 cm -1 (Romani, & Brandán, 2015).
In those two thiol and thione tautomers, the twisting modes are predicted by SQM calculations between 66 and 63 cm -1 while for C2 and C4 these modes are predicted at 98 and 105 cm -1 , respectively (Romani, & Brandán, 2015;Castillo, et al., 2017).Obviously, due to the low wavenumbers values those modes were not assigned.This analysis shows clearly that the vibration modes related to these groups for C2 and C4 are predicted in the same region.

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Skeletal modes.The C=O stretching modes of the two conformers together with the C=C of benzyl rings are predicted between 1680 and 1571 cm -1 , thus, the very strong IR band at 1685 and 1565 cm -1 are assigned to those stretching modes of both conformers.Besides, some N=C stretching modes of imidazole rings and acetamide fragment are predicted by the SQM/B3LYP/6-311++G** calculations with double bond character while other N-C and C-C stretching modes with partial double bond character, thus, the bands between 1478 and 1287 cm -1 can be assigned to those modes, as observed in similar species (Karrouchi, et al., 2021;Karrouchi, et al., 2021;Mortada, et al., 2022).However, the N-C and C-C stretching modes with simple bond character are assigned between 1248 and 694 cm -1 , as predicted by the SQM calculations and, as observed in Table 6.

Force Field
To characterize the force of different bonds, the scaled force constants are useful parameters associated to the positions of bands observed in the vibrational spectra.Hence, these factors for C2 and C4 of BENZ were computed in gas phase and ethanol solution at the B3LYP/6-311++G** level of theory by using the SQMFF methodology and the Molvib program (Pulay, et al., 1983;Rauhut, & Pulay, 1995;Sundius, 2002).These parameters for the two conformers in both media are shown in Note: Units are mdyn Å -1 for stretching and mdyn Å rad -2 for angle deformations.a This work.
A very important observation is detected in the scaled f(C-C-N) force constants related to the acetamide fragments because the values are different for both conformers in the two media.These variations are justified by the different f(νC-N) and f(νC-C) force constants values, as observed in Table 7.

Ultraviolet-Visible Spectrum
The electronic spectra for the C2 and C4 conformers of BENZ were predicted in ethanol solution at the B3LYP/6-311++G** and B3LYP/6-311G** levels of theory and both are compared in Figure 10.Different level of theory was used for C4 due to the difficulty to obtain the spectrum with the other method.The corresponding experimental obtained for BENZ in aqueous solution by Nothenberg et al (1991) showed two bands, one intense absorption at 203 nm and other of lower intensity at 314 nm while in the predicted spectra for C2 conformer the maximum appeared at 177 nm while the band of lower intensity at 307 nm.On the contrary, C4 shows a shoulder at 234 nm and a maximum at 285 nm.Thus, the spectrum of C2 shows a very good concordance with the experimental obtained in the solvent of higher polarity (water, 1.85 D) because both bands are located at higher wavelengths than the corresponding predicted in ethanol (1.66 D), for which, the observed hypsochromic shift is in agreement with the change in solvent polarity.Due to the presence of C=C, C=O and C=N double bonds these absorptions can be quickly assigned to π→π* and n→π* transitions, as reported for similar compounds and as suggested by NBO calculations (Nogueira Silva, et al., 2008;Honorato, et al., 2014;Nothenberg, Funayama, & Najjar, 1991).

Conclusions
In this work, two C2 and C4 stable cis conformers of benznidazole active drug used to the treatment of Chagas disease have been theoretically characterized and its vibrational spectra completely assigned combining B3LYP/6-311++G** calculations with the 83 experimental FT-IR and FT-Raman spectra and the SQMFF methodology.Both forms were experimentally determined by X-ray diffraction and they differ in the positions of the CH2 groups linked to benzyl and imidazole rings.C2 was observed at room temperature while C4 with the heating.Changes in the positions, directions and orientations of dipole moments vectors and in the magnitudes could explain the presence of C2 in the solid phase.The mapped MEP surfaces show that the nucleophilic regions are located on N6 of imidazole ring and on the O atoms of NO2 and on C=O groups while the electrophilic sites are observed on the N-H and CH2 groups of acetamide fragment.
The NBO studies show that C2 is most stable in solution and that the E values are strongly dependent on method and basis set.The AIM analyses reveal the high stability of C2 in the gas phase and in ethanol solution, as supported by the different N-H The low gap value and the high reactivity of C2 could explain their biological activities.
Reasonable concordances were found among the experimental FTIR, FT-Raman, UV-Visible and 1 H and 13 C-NMR spectra and their corresponding theoretical ones.Complete vibrational assignments of the 87 normal vibration modes for both conformers of BENZ were reported for first time together with the scaled force constants for C2 and C4 conformers. 89

Figure 3 .
Figure 3. Comparisons Between the Theoretical C2 and C4 molecular structures of benznidazole with the corresponding experimental determined respectively at roomtemperature and with the heating Source:Soares-Sobrinho, et al., 2008;Honorato, et al., 2014

Figure 4 .
Figure 4. Variations Observed on Calculated MK and NPA Charges on N, O and H Atoms Corresponding to C2 of BENZ in Both Media by Using the B3LYP/6-311++G** Method.N and O Atoms Belong to NO2 Group, to Imidazole and to Acetamide Fragment While the H Atom to NH Group.

Figure 5 .
Figure 5. Calculated Electrostatic Potential Surfaces on the Molecular Surface of C2 of BENZ in Gas Phase by Using the Different Levels of Theory.Isodensity Value of 0.005.
Appendix 2), we observed five different types of interactions: ∆ETπ→π*, ∆ETπ→LP, ∆ETπ*→π*, ∆ETLP→π* and ∆ETLP→σ* which are related to the double bonds of the benzyl ring (C=C), to the NH and NO2 groups and to C=O bond of acetamide fragment.Besides, the lone pairs of the O and N atoms are clearly involved in the ∆ETπ→LP, ∆ETLP→π* and ∆ETLP→σ* interactions and present higher energy values in solution.In particular, the ∆ETπ→LP interactions are predicted by the two methods only in ethanol solution while the E values are strongly dependent on method and basis set because the LP(1)N5→ σ*O1-C10, LP(1)N6→ π*N4-C12

Figure
Figure 8. Experimental ATR-IR Spectrum in the Solid State of BENZ Compared with the Corresponding Predicted for C2 and C4 Conformers in Ethanol Solution by Using B3LYP/6-311++G** Level of Theory Source: SpectraBase, n.a.

Figure
Figure 9. Experimental Raman Spectrum in the Solid State of BENZ Compared with the Corresponding Predicted for C2 and C4 Conformers in Ethanol Solution by Using B3LYP/6-311++G** Level of Theory Source: Soares-Sobrinho, et al., 2010

Figure 10 .
Figure 10.Predicted Electronic Spectra for C2 and C4 of BENZ in Ethanol Solution at the B3LYP/6-311++G** Level of Theory

Table 2
(Soares-Sobrinho et al, 2008)s in ethanol solution.These calculations show changes in the different dihedral angles related to the imidazole and benzyl rings and to acetamide fragment in gas phase and in ethanol solution but a very important observation for C2 in the two media and with both methods is the similarity in the RMSDs values of dihedral angles.Thus, the values decrease from 209.4/208.4° in gas phase to 179.5/179.3 in solution.Hence, due to the presence of donor N-H group and of acceptors N and O atoms in the structure of BENZ could also be expected H bonds interactions in solution, as the strong intermolecular N-H•••O hydrogen bonds observed in the crystal packing of BENZ in the solid phase(Soares-Sobrinho et al, 2008).

Table 2 . Comparison of Calculated Geometrical Parameters for C2 of Benz in Gas Phase and Ethanol Solution by Using Different Levels of Theory with the Corresponding Experimental Ones
Soares-Sobrinho, et al., 2008brinho, et al., 2008Source:Soares-Sobrinho, et al., 2008

Table 3 . Atomic Natural Population (NPA), Merz-Kollman (MK) Charges, Molecular Electrostatic Potential (MEP) and Bond Orders (BO) Acceptors and Donor Groups of Most Stable C2 Conformer of BENZ in Gas Phase and Ethanol Solution by Using the B3LYP/6- 311++G** Levels of Theory
Note: Atomic charges and MEP values in a.u.

Table 5 . Observed and Calculated 13 C Chemical Shifts (δ in ppm) for BENZ in Ethanol Solution at Two Levels of Theory
Note: a This work, b From SpectraBase, (n.a.) for benz in CHCl3

Table 7 . Comparison of Scaled Internal Force Constants for C2 and C4 Conformers of BENZ in Gas Phase and Ethanol Solution by Using the B3LYP/6311++G** Level of Theory
•••O, C-H•••O and C-O•••H interactions of H bonds.Besides, both studies reveal the influence of level of theory and the medium on the total stabilization energy.