Theoretical and Experimental Spectroscopic Study on 2-Chloro-3-(substituted-phenylamino)-1,4-naphthoquinone Derivatives

The 1,4-naphthoquinones are an important group of compounds intensively studied because of their wide range of biological activities. Four 2-chloro-3-(substituted-phenylamino)-1,4 naphthoquinone derivatives were synthesized, and the vibrational modes of these molecules were assigned using Raman and Fourier transform infrared spectroscopy (FTIR) techniques. In addition, X-ray studies were performed for one of these derivatives. Density functional theory (DFT) calculations were also developed for these compounds and presented. In summary, the results obtained from these studies can assess chemical changes in the structures of functionalized quinones and the discovery of candidate biologically active compounds.


Introduction
][11][12] In the field of chemotherapy, several quinone derivatives, such as anthracyclines, are clinically important drugs used in cancer treatment. 13][16][17][18] Quinones exert their biological effects via quinonehydroquinone redox cycling, leading to reactive oxygen species (ROS) generation, such as superoxide anion radical (O 2 •− ) and hydroxyl radical (•OH). 19ROS production leads to an oxidant-antioxidant imbalance or oxidative stress and can lead to irreversible biomolecules damage such as lipids, proteins, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA), followed by cell death. 20,21Other DNA damage mechanisms associated with quinone derivatives include DNA alkylating reaction and intercalation in the DNA double helix. 22he quinone redox cycle may be influenced by adding electron-attracting or donating substituents to the quinoid system.4][5] Aminoquinone derivatives have also been used as a potential building block for the synthesis of bioactive modified quinones. 14,15,19everal computational and structural X-ray diffraction studies, 18,[23][24][25] in search for quantitative three-dimensional correlation structure-property (3D-QSAR) 26 have aided in the comprehension of these biological applications.The evaluation of the importance of intramolecular electronic properties, such as parameters highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energies, hardness, atomic charges, dipole moment, polarizability, molecular volume and Gibbs free energy, can provide indications of potential target molecules.Moreover, the crystalline package containing intermolecular interactions of the classic and non-classical hydrogen bond type can justify the importance of the biophoric sites of these substances. 27owever, theoretical-experimental studies of vibrational and electronic spectroscopy containing a quinonoidic ring have placed great emphasis on p-benzoquinone and its derivatives, 28,29 with a reduced approach to studies of complete aminoquinone assignment.Such techniques are routinely used in the quality control of pharmaceutics and may allow the identification and distinction of crystal forms. 30,31n this work, we described the synthesis, evaluated a new crystal structure, and investigated how electrondonating substituents by inductive effect and by resonance effect affect the electronic structure, molecular topology and vibrational properties of 2-chloro-3-(substitutedphenylamino)-1,4-naphthoquinone derivatives (Figure 1).

Experimental
All reagents and solvents used in the preparation of substances 1a-1d were used without purification.The progress of the reactions was routinely monitored by thin layer chromatography (TLC) on silica gel plates (60F-F254) aluminum sheets 20 × 20 cm (Merck KGaA, Darmstadt, Germany) and visualized using ultraviolet light (UV-254 and 366 nm).The eluent mixture used was prepared volume by volume (v/v).Compounds 1a-1d had their respective melting points (mp) determined with a Fisher Johns instrument (Niterói, Brazil), which are uncorrected.Purification of compounds was carried out by silica gel flash column chromatography from Merck (Darmstadt, Germany).Hydrogen nuclear magnetic resonance spectra ( 1 H NMR) were performed using CDCl 3 as solvent (Sigma-Aldrich, São Paulo, Brazil) using a Varian Unity Plus 500 MHz spectrometer and tetramethylsilane (TMS) as an internal standard.The respective chemical shifts (d) were expressed in parts per million (ppm) and the coupling constant (J) in hertz.
In a 50 mL flask bottom, 0.5 mmol of 2,3-dichloronaphthoquinone (2) in 10 mL of water and 0.5 mmol of aniline 3a or the analogues substituted 3b-3d were added.The mixture was kept under stirring at room temperature for 20 h.Upon completion of the reaction, they were filtered through a vacuum, and the precipitate was washed with water.Then, the substituted 2-amino-1,4-naphthoquinones 1a-1d were purified by chromatography column on a silica gel using a blend of hexane/ethyl acetate (7:3) as eluent.The solid obtained had a yield ranging from 84 to 90%. 21

Single crystal X-ray
Single crystal X-ray data for 1d were collected on a Bruker D8 Venture diffractometer (Niterói, Brazil) using graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) at 298 K. Data collection, cell refinement and data reduction were performed with Bruker Instrument Service v4.2.2, APEX3 and SAINT, 32,33 respectively.The absorption correction using equivalent reflections was performed with the SADABS program. 34The structure solutions and full-matrix least-squares refinements based on F 2 were performed with the SHELX package. 35,36All H atoms were refined with fixed individual displacement parameters [Uiso\(H) = 1.2 Ueq (Csp 2 and C ar ) or 1.5 U eq (Csp 3 )] using a riding model.All non-hydrogen atoms were refined anisotropically.Structure illustrations were generated using ORTEP-3 for Mercury and the crystallographic tables were constructed using Olex2. 37,38he equipment is located at the Multiuser X-Ray Diffraction Laboratory, LDRX-UFF of the Instituto de Física of the Universidade Federal Fluminense, Brazil.

Vibrational and electronic spectroscopy
The Fourier transform infrared (FTIR) spectra were measured on Nicolet TM iS50 (Niterói, Brazil) spectrometer equipment with an attenuated total reflectance accessory and LaTGS detector for the ranges of 4000-400 cm -1 and 600-50 cm -1 with an average of 128 scans at room temperature with 4 cm -1 resolution.Data analysis was performed on the Omnic software.
The FT-Raman spectra were obtained using a Bruker MultiRAM spectrometer (Niterói, Brazil) in the region between 3500-70 cm -1 with 2 cm -1 resolution.The exciting laser wavelength radiation (Nd:YAG laser line as the excitation source) presented a wavelength of 1064 nm, and the spectra were recorded at room temperature with a germanium detector kept in liquid nitrogen.The samples were measured in the hemispheric opening of an aluminum sample holder.Data analysis was performed using the OPUS software.
The UV-visible measurements were done in the solid state diluted in MgO and in an acetonitrile solution using a Cary 5000, UV-Vis NIR spectrometer (Niterói, Brazil) and a Cary 50, UV-Vis spectrometer (Niterói, Brazil), respectively.Both measurements were performed from 190 to 800 nm with 0.1 s nm -1 resolution.Data analysis was performed using the Cary WinUV software.
The equipment are located at the Multiuser Spectroscopy Laboratory (LAME-UFF) of the Instituto de Química of the Universidade Federal Fluminense, Brazil.

Calculations
The molecular properties were evaluated using the program Gaussian 09 for Linux, 39 with the CAM-B3LYP functional for the development of the density functional theory (DFT) calculation.This functional was selected because it describes the high accuracy systems, including corrections for a better description of long-range interactions. 40,41A theoretical polar environment was simulated by means of the integral equation formalism in the implicit solvation method of the polarizable continuous model (IEFPCM) implicit solvation method, 42 using acetonitrile, dichloromethane and dimethyl sulfoxide (DMSO) as solvents to emulate possible intermolecular interactions around the analyzed molecules.The basis set used for developing of all the calculations was the double zeta 6-311G(d,p) for all atoms. 43The partial atomic charges and the electrostatic potential surfaces (ESP) were calculated according to the Merz-Singh-Kollman scheme. 44,45The evaluation of the molecular orbitals was carried out using a Mulliken population analysis, and the percentage of atomic orbitals and the graphical population analysis software GaussSum were employed. 46Every visualization and check of the calculated data were done through the ChemCraft 1.8 program. 47sing as a starting point different conformations for the molecular units containing different dihedral angles between the quinone and the phenyl group, interconnected by the amino group, the most stable structural variations of the molecular units of the compounds were analyzed together with their vibrational spectra for acetonitrile data without the presence of imaginary harmonic vibrational modes, indicating that the analysis of isolated units alone, such as neglecting the intermolecular interactions, still allowed good agreement between the calculated and experimental data (crystallographic).The vibrational wavenumber, Raman scattering, and infrared adsorption intensities were visualized through the potential energy distribution (PED) analysis using the VEDA 4 software. 48][51][52][53] Transition energies and oscillator strengths in the UV-Vis spectra of the optimized structures were obtained from timedependent density functional theory (TD-DFT) calculations.The evaluation of the theoretical methods was accomplished using the first 50 lowest energy states in the solution.The analysis of the TD-CAM-B3LYP states and the spectra simulation were carried out with the GaussSum software, using Gaussian functions with half-widths of 3000 cm -1 . 46he cluster computers and programs used are located at the Multiuser Computational Chemistry Laboratory, LMQC-UFF, of the Instituto de Química of the Universidade Federal Fluminense, Brazil.

Structures
Structural analysis of compounds listed in this study, such as 1a and 1b, are reported in the literature. 54,55The main characteristic confirmed is that the phenyl and naphthoquinone rings are not in the same plane.The compounds 1a and 1b present crystalline packing with P21/c and Pna21 group spaces, respectively.This class of compounds present intermolecular interaction, with detached for hydrogen bonds connected by systems with N…O in the range from 2.70 to 3.05 Å, which can be treated as resonance assisted hydrogen bonding.Thus, NH…O(2) interaction in 1a and 1b is observed between adjacent units involving the amine and carbonyl group of naphthoquinone which is vicinal to the halogen group.
Finally, even though we did not perform an X-ray diffraction structural analysis for the 1d compound, we evaluated the skeleton centered on the secondary amine between the naphthoquinone and phenyl groups for the 2-chloro-3-((3,5-dimethoxyphenyl)amino)-1,4-naphthoquinone. 56This compound presents a P-1 space group with the smallest resonance assisted hydrogen bonding effect, presenting an N…O distance of 3.227(3) Å, as observed for the 1c structure.
5][56] The other studied parameters from the theoretical calculations agree with the experimental results, as observed in Table 1 and Tables S2 and S3 found in SI section.
The analysis by root-mean-square deviation (RMSD) was performed to facilitate the comparison of crystallographic data with theoretical data and the results are shown in Figure S1 of the SI section.Note that, in this analysis, only the compounds 1a, 1b, and 1c were evaluated because their molecular structures present higher similarity.The observed results indicate a reasonable approximation between the IEFPCM-optimized data and the crystallographic data, with RMSD < 0.5.The theoretical data resulted in divergence only in the methoxy group in 1c, with RMSD = 0.40.Therefore,  these results indicate that bond length and bending in the 2-chloro-3-phenylamino-1,4-napthoquinones skeleton are close to those determined experimentally, demonstrating that the theoretical approach can describe the system with great accuracy and contribute to understanding additional topics, such as vibrational and electronic spectra.Even though 1d has not been analyzed in isolation without considering the crystalline structure, it is clear that it follows the same trend as the other compounds in this study.
Finally, by evaluating the partial atomic charges calculated through the Merz-Singh-Kollman (MSK) and natural charge scheme, as available in Tables S4 and S5 in the SI section, it was possible to identify the influence of the more electronegative atoms that result in the stronger intermolecular interactions.For example, in all molecules 1a-1d, oxygen (O 1 and O 2 ) and nitrogen (N 1 ) atoms have the more negative charge values.These atoms are then involved in the main interactions detected in the crystal packing.The amino group has a hydrogen atom with a strong positive charge.It indicates an electron deficiency that justifies the formation of hydrogen bonds.The other oxygen atoms of the methoxy groups through MSK charge for the compounds 1c and 1d also have a negative charge, but due to the position of the amino group in the benzene ring, an inductive effect that removes electron density is perceived.This observation was not corroborated by the natural charge obtained by the NBO calculation, since the partial atomic charges of the oxygen atoms show similar values, as observed in Table S5.
Interestingly, even though 1c has a methoxy group that is an electron density donor by resonance effect in the para position, the charge concentration on the nitrogen atom of the amino group is higher than the previous cases in 1d; however, it is still lower than the compound without a substituent (Figure 5).The evaluation of the charges of the naphthoquinone groups showed certain regularity regardless of the compounds evaluated.The oxygen (O 2 ) that is adjacent to the chlorine group linked to carbon (C 3 ) has a charge concentration higher than the oxygen (O 1 ) adjacent to the amino group.
A recent study by Rajalakshmi et al. 54 using several substituents in the phenyl group indicated the effect on the amino group when the nature of the inductive and resonance effects could affect NH group's acidity and the action itself in the hydrogen bonds between adjacent units.For example, the compound 3-chloro-2-(4-chlorophenylamino)-1,4-naphthoquinone presents in its crystalline packaging with an N…O distance of 2.966 Å and NH…O of 131.4º with N−C phenyl of 1.407 Å.In this case, the chloro substituent on the phenyl group has a weak deactivating action.This implies an action of removing the electron density of the amine group, which makes it more acidic.In addition, the quinone ring has an O=C−C=C−NHR π-conjugated system that allows evaluating this effect on the C=O bond distance (1.233 Å), which is longer than the reference molecule 1a unsubstituted (1.227 Å).Specifically for molecule 1c of our work, containing a methoxy substituent on the phenyl group, which is a strong activating substituent (Figure 5), an increase in the N(1)−C(11) bond distance to 1.415 Å is observed, according to the X-ray diffraction.This distance was greater than the N−C phenyl distance for compounds 1a (1.410 Å) and 1b (1.412 Å) in X-ray diffraction, 54,55 where the first is unsubstituted and the second has a methyl substituent which is a weak activator group.Associated with the C=O bond distance vicinal to the chloro group of quinone, with values of 1.229 and 1.230 Å, respectively for compounds 1b and 1c, the effect of resonance assisted hydrogen bonding confirms the influence of the substituents in the phenyl group (R).The crystalline packaging for 1a, 1b and 1c present N…O distance from 3.049 to 2.983 Å. 54 These crystallographic observations were corroborated by the theoretical data since the RMSD analysis indicated great similarity between the experimental and theoretical structures.But to support this observation, we performed an analysis by natural resonance theory (NRT) that indicates the percentage contribution of different resonance structures in relation to a reference and prediction of NBO delocalization, available in Tables S6-S9 in the SI section.The highest weight of structures with a concentration of partial positive atomic charge on the nitrogen atom of the amine was found for the structure of compound 1a (26.15%), followed by structures of compound 1b (18.69%) and finally for compounds 1c and 1d (6.87 and 6.48%).This indicates the effect of the methoxy substituent with an activating action on the phenyl group, increasing the charge density on the amino group, which makes it more basic.
Figure 6 shows the electrostatic potential surfaces (ESP) of the molecules.These surfaces permit interpreting the overall charge distribution of the molecules.The red color describes the higher concentration of negative charges, while the blue color describes the most positive sites.The hydrogen atom of the amino group is the most positively charged.Contrarily, the oxygen (O) in the carbonyl group of naphthoquinones is the most negatively charged atom, corroborating information described in the literature for these functional groups in naphthoquinone systems. 20In terms of the electrostatic aspect, the benzenoid ring has a neutral characteristic even in the presence of more electronegative atoms while the naphthoquinone group is positively charged.

Vibrational analyses
Theoretical-experimental vibrational studies involving compounds containing the 1,4-naphtoquinone group have been reported in recent years using density functional theory assistance through the B3LYP hybrid method with different double or triple zeta basis functions with or without pseudopotential, in addition to the use of an implicit solvent method for the study of solvation effects. 57,58The use of techniques such as potential energy distribution (PED) analysis of theoretical vibrational spectra is also reported.However, as they are the simplest target molecules and have non-functionalized amino groups or only the functionalized 1,4-naphtoquinone molecule, the observed spectra are simpler and have fewer bands and fewer couplings than the molecules in this study. 59,60hus, solid-state experimental spectra for compounds show characteristic bands of the most important chemical groups in each compound between 4000 and 600 cm -1 in Figure 7 and from 600 to 150 cm -1 in Figure S2 (SI section).The broad band of the amino group in the infrared and Raman spectra and multiple bands between 1675 and 1500 cm -1 due to C=C and C=O stretching modes are the most evident bands.The complete assignment of all spectra is given below, with five regions considered: 3500-2800, 1800-1300, 1300-1200, 1000-600 and 600-150 cm -1 , where the first four regions belong to the mid-infrared spectrum and the latter to the far-infrared spectrum.This division was also accompanied by the Raman spectrum.
All vibrational modes of the four compounds 1a-1d were classified as part of either the benzene ring or the naphthoquinone ring.The substituents were identified as methoxy or methyl groups according to the components of each derivative, as illustrated in Figure 7.
The molecules presented between 84 and 96 normal vibrational modes in the theoretical spectra.Tables 2 complete tables with all the theoretical vibrations are available in the SI section (Tables S10-S13).
Assignment of the normal modes was done using Veda 4.1 software, 48 which performs potential energy distribution analyses to identify the most significant components in each vibrational mode and describes them using internal coordinates.The percentage contributions of each coupled mode at the same frequency were indicated in parentheses.The sum of all possible representations for a given vibrational mode was indicated by the sum of multiple assignments.
The theoretical and experimental wavenumbers showed high correlation for the regions between 3100 and 150 cm -1 .
The coefficient of determination (R 2 ) for the correlation between experimental and theoretical wavenumbers was between 0.998 and 0.999.These results are available in the SI section (Figure S3).Thus, by simulating the theoretical spectra according to the values of the calculated scale factors between 0.953 and 0.960, there was a good theoretical and experimental correlation, wherein the intensities of the spectra were comparable and allowed adequate assignment.Scaling factors were applied to the calculated wavenumbers in the SI section (Tables S10-S13).
in Figure 7 was classified as the region associated with the N−H stretching mode.The Raman spectrum showed a single band of low intensity between 3250 and 3220 cm -1 .The infrared spectra of the solid phase have a medium intensity broadband indicating the influence of intermolecular interactions, particularly hydrogen bonding.This profile confirms the presence of the secondary aromatic amino group.Due to the presence of the hydrogen bonds in the crystal packing, this band was not included in the calculation of the scale factor mentioned in the previous item.Aromatic C−H stretching modes were observed for all compounds in the region between 3040 and 3100 cm -1 .The asymmetric and symmetric aromatic ν CH modes (benzenoid and naphthoquinone groups) were assigned.The methoxylated compounds 1c and 1d showed the lower definition of these bands in the infrared spectra.Stretching modes of the aliphatic C−H bonds were observed in the region between 2850 and 3000 cm -1 .The theoretical information and the differences between the vibrational modes were essential in the assignment of these bands, due to the spectra complexity in this region.Bands in 2958, 2921 and 2852 cm -1 were identified in 1b and 1c as asymmetric and symmetric ν CH modes.In the spectrum of derivative 1d, a larger number of bands is noticed, reflecting the increase in the number of methoxy groups.

Region between 1800-1300 cm -1
Bands of high intensity due to ν C=C , ν C=O and ν CN modes and coupled stretching modes were observed in the experimental spectra between 1670 and 1450 cm -1 , as shown in Figure 7.In infrared spectra, ν C=O modes in the naphthoquinone group were identified in two bands at 1675/1670 and 1635/1633 cm -1 .The ν C=C modes of the aromatic groups were observed in the 1593 cm -1 as a high intensity band.The band at 1564/1558 cm -1 was assigned as a coupled mode of ν C=C and ν CN , followed by bands at 1520 and 1500 cm -1 that have been associated with HCC and HNC angle strain coupling modes.In the Raman spectra, these bands are observed, but especially on the band at 1636/1635 cm -1 as ν C=O modes.These data reinforce the similarity of the four compounds with their benzenoid and naphthoquinone groups.
Region between 1300-1000 cm -1 The region between 1285 and 1130 cm -1 shows greater complexity for the spectrum in Figure 7 containing the two methoxy groups compared to the other systems studied.The compound 1d presents an envelope of bands where the most intense band is found at 1236 cm -1 associated with the coupling ν C-O-CH3 , ν CC and ν HCC Benz .This compound still presents intense bands at 1284 and 1130 cm -1 that are associated with ν CC and ν CCH Naphtq.modes.These two bands are the most prominent among the other compounds.Even though the 1c substance contains the methoxy group, it has been identified as participating in the coupled mode at 1284 cm -1 .In the Raman spectra, the band at 1255 cm -1 was highlighted for the compounds 1a and 1b, assigned as ν CC + ν CN mode and presents in both aromatic groups.For the compounds 1c and 1d, this intense band is observed at 1268 and 1293 cm -1 .
Region between 1000-600 cm -1 The infrared spectrum in Figure 7 for the compound 1d reinforces the distinction between the compounds according to the highest number of substituents present.The characteristic region of aromatic rings respiration and deformation outside the plane was observed with bands at 854, 831 802, 748, 719, 685 and 654 cm -1 .Furthermore, the first highlighted band is assigned by coupling the breathing mode of the aromatic rings with the ν CCl .For the other compounds 1a-1c, this mode stands out as a band of medium intensity found in the other compounds at 848/818 cm -1 , in addition to the out-of-plane deformation of the aromatic rings at 717/715 cm -1 .In the Raman spectrum, this region stands out for a band of weak intensity between 686 and 653 cm -1 related to the d CCC angular deformation of the aromatic rings.

Region between 600-150 cm -1
As previously discussed, this region presents a great complexity, as it presents several active bands for all compounds in Figure S2 in the SI section.However, this region allows for greater differentiation between the compounds through the profile and intensity of the bands, also featuring a fingerprint region to distinguish these compounds.However, the intense band between 390 and 382 cm -1 stands out in all compounds for describing the out-of-plane deformation of aromatic rings with low intensity bands between 360 and 331 cm -1 which is assigned by the coupling of ν CC + ν CCl + d CC=O .In the Raman spectrum, the band between 296 and 283 cm -1 is an indication for all compounds as τ CCC Naphtq.+ τ CCN Naphtq.+ τ CCCl Naphtq.

Electronic spectra
The compounds 1a-1d listed in this study show a color that ranges from purple to black in solid state, which in solution with the indicated solvents shows a red-orange color, typical of dyes containing anthraquinone groups. 61he experimental UV-Vis spectra for different compounds and in different solvents show high similarity, as observed in other studies containing aminoquinones. 23All the spectra in the solution presented a lower intensity band in the visible region between 470 and 500 nm, which was responsible for a bluish-green to blue-green absorption that justifies the colors observed in solution 62,63 presented in Figure 8.This spectral region is similar to the study observed by Rajalakshmi et al. 54 with an aqueous N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid (HEPES) buffer:N,Ndimethylformamide (DMF) (2:8 v/v) solution.In the spectra in the ultraviolet region, two bands are observed: the first, a low-intensity band between 320 and 335 nm, and the second, a high-absorptivity band ranging from 275 to 283 nm.Using acetonitrile as a solvent, the highlighted spectrum obtained presents a medium intensity band ranging from 228 to 240 nm, which is not defined for the other solvents due to their transparency regions.When evaluating the solid-state spectra in Figure S4 in the SI section, a very broad and intense band is noticeable between 400 and 700 nm in the visible region and a more defined band in the ultraviolet region between 277 and 292 nm.The characteristics of the first band may explain the reason for the color variation observed in the solid state, since there is an overlap of multiple absorption bands in this region influenced by crystal packing with multiple intermolecular interactions.
The deconvolution spectra were obtained using different Gaussian numbers; five for acetonitrile, four for dichloromethane, and three for DMSO.Some bands were estimated at below 200 nm to aid in the deconvolution process.The overall-fit parameters and the molar absorptivities are presented in Table S14 of the SI section.Notably, all compounds 1a-1d have molar absorptivity values at around 10 4 in the UV region and 10 3 in the visible.When assessing the visible band with the different solvents, a bathochromic effect accompanied by a hypochromic effect is observed when comparing these bands in acetonitrile/CH 2 Cl 2 to DMSO.This can be explained by the difference in polarity between these solvents and the aromatic groups.Another relevant effect related to this band was the solvent-independent bathochrome shift used in ascending order for the compounds 1a < 1b < 1c < 1d.This demonstrates an effect of the different substituents as highlighted in the charge analysis, as it should also be observed in the HOMO-LUMO boundary orbitals that will be evaluated in the next section.When evaluating the values of molar absorptivity, we can infer a strong participation of π →π * transitions to the intense band in the ultraviolet region and n→π * in the visible region with the effect of a donor substituent such as methoxy containing isolated electron pairs that generate a known bathochromatic effect on the transitions.There is also the participation of auxochromic groups present in naphthoquinone that has a profile resembling p-benzoquinone 28,64 but is difficult to predict without an analysis of orbitals and electronic excitations as reported by Pereira-da-Silva et al.

Molecular orbitals analysis
The ground state for all compounds 1a-1d consists of the frontier region of occupied molecular orbitals distributed between 7 and 12 eV for CAM-B3LYP.Table 6 briefly presents these orbitals in an IEFPCM (solvent MeCN) with the associated Koopmans' energy and Mulliken analysis of the virtual and occupied orbitals.Five occupied orbitals and five virtual ones with lower energy were analyzed for 1c and 1d, while for the other compounds five occupied orbitals and eleven virtual ones with lower energy are highlighted.HOMO, LUMO, and other molecular orbitals of compounds are shown in Figure 9.The same analysis performed for the data obtained with the IEFPCM model with CH 2 Cl 2 and DMSO is found in the SI section in Tables S15 and S16.
According to Borges et al., 65 through DFT calculations it is feasible to predict the cytotoxic or cytoprotective effect of quinones by redox mechanism.For this, it is possible to use as a parameter the energy of the molecular orbitals HOMO and LUMO of compounds 1a-1d, whose values are shown in Table 6.It is generally observed that quinones that exhibit higher εHOMO have the ability to donate electrons more easily, being carriers of cytoprotective properties.On the other hand, substances with high εLUMO imply electron acceptor molecules, which have cytotoxic properties.Thus, it can be noted that the quinones 1a-1d studied in this work, especially the compounds 1b and 1c-1d, which contain electron-donating substituents by the electron-donating inductive effect and by the resonance effect, respectively, have high values of εHOMO and low values of εLUMO.These results indicate a greater capacity to donate electrons and, therefore, having probable cytoprotective effects. 66ased on Koopman's theorem, it is worth mentioning that the HOMO-LUMO energy difference for the compounds fluctuates from 5.48 to 5.23 eV in solvent  acetonitrile and is very close to the other results obtained for the other two-solvent systems.A relevant observation is that the highest barriers are found for compounds 1a, without a para substituent on the benzene ring, 1b with a methyl substituent (5.39 eV) and the lowest for compounds 1c and 1d with methoxy substituents.Such values are compatible with the inverse order of the activation strength of the substituents on phenyl group, where more activating groups tend to make the species more reactive to the detriment of less activating groups.
Orbitals close to the HOMO-LUMO border of H-3, H-2 through L+1 are characterized by the participation of orbitals associated with πC=C Benz.and πC=C Naphtq and isolated pairs of oxygen (carbonyl), nitrogen (amino) and chlorine (halogen) atoms.Specifically, for methoxy groups, orbitals where the most relevant participation exists for these groups are found in regions farther away from the HOMO-LUMO boundary orbitals.The electronic density of states (DOS) revealed that variations in the presence of substituent groups are found in the upper and lower energy ranges, different from those observed for the orbitals near the HOMO-LUMO border (Figure 10).This analysis confirms the similarity of the boundary orbitals, indicating the dominance of the benzenoid group orbital in HOMO and the naphthoquinonic group in LUMO.These trends are also reproduced in data with IEFPCM in CH 2 Cl 2 and DMSO, with the results shown in Figures S5 and S6 of the SI section.This analysis confirms the observation highlighted by Rajalakshmi et al. 54 that intramolecular charge transfer (ICT) occurs from the benzenoid group to the quinone ring.

Electronic transitions
The first 50 singlet electronic excited states were calculated through the TD-CAM-B3LYP method.The observation of spin-allowed π →π * transitions was the major concern in this work; therefore, the calculations were restricted to singlet states.All calculations were carried out without symmetry and with ground-state orbitals, to obtain the oscillator strength between the ground state and all excited states.The results of the energy and oscillator strength of the main calculated transitions are listed in Table 7, using the IEFPCM, with MeCN solvent.Tables S17 and S18 show the calculated excited states for IEFPCM with CH 2 Cl 2 and DMSO (SI section).All calculations presented the first transition with a monoreferential nature and a dominant configuration coefficient involving the HOMO-LUMO orbitals.The states obtained in the ultraviolet region presented a multiconfigurational nature according to the values of the configuration interaction coefficients.By comparing the experimental results with the calculated excitations with proportional oscillator strengths, it can be seen that variations of less than 0.5 eV were observed in the calculations for the first bands in the UV-Vis spectra for the CAM-B3LYP orbitals regardless of the solvent used.
Concerning the experimental observations and theoretical results, the final assignments of the UV-Vis spectra electronic transitions are summarized in Table 8.The first transitions of each case are associated with the following assignments πC=C Benz.+ πC=C Naphtq.+ nΟ + nCl + nN→πC=C Naphtq.+ nΟ.In other words, the influence of chromophore groups is confirmed in this band, without the participation of orbitals that indicate the presence of methyl or methoxy groups.However, single paired substituents are noted here.The other bands follow this trend, highlighting the nature of the transition π→π * with a multiconfigurational nature in some transitions.
These results are compatible with several studies already carried out with different methods, such as semiempirical, ab initio and DFT, for structures containing quinone groups with benzoquinone and its functionalized derivatives. 23,28

Conclusions
The synthesis and theoretical-experimental characterization of the four 2-chloro-3-(phenylamino)-1,4naphthoquinone derivatives presented in this work allowed corroborating and extending the observations highlighted in other studies for this compound class reported in the literature.Specifically, in this work, the combination of structural analysis by X-ray diffraction with the presentation of a new structure and associated with a broad theoretical discussion about the factors influencing the molecular characteristics enabled us to confirm the relevance of the intermolecular forces present in these derivatives.The analysis of the solid-phase vibrational and electronic spectra described in this article corroborated the analysis of the charge distribution, the presence of electronegative functional groups, and the study of the potential energy surface.Furthermore, the success in the spectroscopic assignment based on the isolated molecular units with the application of the implicit solvation model confirmed that such structures can show the maintenance of these conformations.Such information is relevant, since it can provide subsidies for the planning of new 1,4-naphthoquinone derivatives that can be explored for the development of compounds with potentiated biological activities.

Figure 9 .
Figure 9. Orbital representation at the CAM-B3LYP level of compounds 1a-1d.The contour values of the orbitals are all 0.03 a.u.

Table 5 .
Experimental and theoretical vibrational wavenumbers of 1d

Table 7 .
Main singlet transition energies and oscillator strength from the ground state for compounds 1a-1d, for TD-CAM-B3LYP/IEFPCM (solvent MeCN) a CI weights to the orbital list, shown in Table6; b oscillator strength.