Structural and Spectroscopic Properties of Isoconazole and Bifonazole—Experimental and Theoretical Studies

The paper compares the experimental FT-IR, UV-vis, and 1H NMR spectra of isoconazole and bifonazole with the density functional theory (DFT) calculations using different functionals. The results were compared with previously reported data related to their analogue, posaconazole. The analysis of calculated IR spectra with use of CAM-B3LYP (isoconazole) or B3LYP (bifonazole) functionals shows good accordance with the experimental IR spectrum. The best compatibility between the experimental and theoretical UV spectra was observed with the use of B3LYP or wB97XD functionals for isoconazole or bifonazole, respectively. The reason for the difference in the UV-vis spectra of isoconazole and bifonazole was discussed based on linear response time-dependent DFT and natural bond orbital methods. The calculated 1H NMR spectrum shows that the DFT formalism, particularly the B3LYP functional, give an accurate description of the isoconazole and bifonazole chemical shifts.


Introduction
Azoles are the most widely used antifungal drugs, acting by inhibiting the lanosterol 14-α-demethylase, the fungal cytochrome P450 enzyme (CYP51A1), which is responsible for conversion of lanosterol to the ergosterol, major sterol agent in the fungal cell wall [1]. The structure of the wall is disrupted by changing permeability of membrane and leading to the cell death. Two classes of antifungal azoles are currently in clinical use. The first class is the imidazole group, where compounds consist of two-nitrogen azole ring, and the second is the triazole group, which includes compounds with three nitrogens in the azole ring. Nitrogen atoms in imidazole and triazole moiety are the key to binding via coordination with the heme iron of the enzyme. Moreover, changes in the lipophilic side chain of azoles can cause the differences in affinity of binding with enzyme due interaction with CYP51 apoprotein [2]. These molecular aspects lead to the search for new azole analogues for a more effective treatment of mycoses.
Isoconazole belongs to the N-substituted imidazole class of azoles with four chlorine atoms and is closely similar in structure to miconazole. It is used in pharmaceutical preparations as nitrate salts and is the active pharmaceutical ingredient in the topical drugs Int. J. Mol. Sci. 2023, 24, 520 3 of 22
Regarding the analysis of MEP (Figure 2) for the charge distribution in the 1 and 2 optimized rotamers, we employed the keywords: "pop=full", as well as the natural bond orbital (NBO, key-words: "pop=nbo") as implemented in Gaussian software, and CHelpG (charges from electrostatic potential) procedure (key-word "pop=chelpg"). In the latter scheme, atomic charges are fitted to reproduce the molecular electrostatic potential at several points around the molecule.  (1) and bifonazole (2) and their optimized geometry at the CAM-B3LYP/6-31G(d,p) (1, left) or B3LYP/6-31G(d,p) levels of theory (2, right).
Regarding the analysis of MEP ( Figure 2) for the charge distribution in the 1 and 2 optimized rotamers, we employed the keywords: "pop=full", as well as the natural bond orbital (NBO, key-words: "pop=nbo") as implemented in Gaussian software, and CHelpG (charges from electrostatic potential) procedure (key-word "pop=chelpg"). In the latter scheme, atomic charges are fitted to reproduce the molecular electrostatic potential at several points around the molecule.
Of all the nitrogen atoms within the structure of 1 and 2 (Tables 1 and 2, respectively) the pyrrolic nitrogen's atoms N1 (2) and N1 (1) are characterized by more positive charge, however the application of the NBO charges methodology indicated these atoms as the ones having the negative charge value. The N1 (1) or N2 (2) atoms were found to have the most negative charge value when the CAM-B3LYP functional and the CHelpG charges methodology were applied. In the case of the CHelpG charges methodology, all chloride atoms within the structure of isoconazole 1 are characterized by negative values of charges. (1) or B3LYP/6-311++G(2d,3p)//B3LYP/6-31G(d,p) (2) levels of theory; gaseous phase; isovalue = 0.0004 a.u.; scale: red-blue from −6.243 ×10 −2 to +6.243 × 10 −2 .

IR Analysis
The theoretical analysis of isoconazole (1) and bifonazole (2) IR spectrum was limited to the DFT formalism without correction term. We carried out the computations of 1 and 2 vibrational frequencies using the same level of theory as was used for the SCF optimization procedure and Grimme's D3 empirical (GD3) dispersion model [20] (for rotamers optimized in gaseous phase using B3LYP, CAM-B3LYP, PB0, M06L, M062X, wB97XD functionals; Tables 3 and 4), and the Petersson-Frisch dispersion model from the APFD functional (for rotamer optimized using APF functional) [21]. The resultant IR spectra are shown in Figure 3. Small differences between the experimental and calculated vibrational modes can be observed because the experimental results were obtained in solid phase whereas the theoretical calculations were carried out in gaseous phase.    From the spectra given in Figure 3, we can conclude that the use of the B3LYP/6-31G(d,p), CAM-B3LYP/6-31G(d,p), or even wB97XD/6-31G(d,p) and PBE1PBE/6-31G(d,p) approaches for the rotamers optimization gives the highest conformity of the theoretical IR bands with the experimental spectrum, particularly in the ca. 3100-2800 cm −1 and ca. 1750-500 cm −1 range. The utilization of the Grimme's D3 empirical dispersion model leads to better consistency between the theoretical and experimental IR spectral values. Moreover, including the diffuse functions in B3LYP/6-311++G(d,p) approach did not lead to the theoretical IR spectrum being comparable with the experimental one (as is relatively more time-consuming in comparison with the B3LYP/6-31G(d,p) functional). In the computed spectra of 1 and 2, the estimated υ and δ C-Cl, N=N, C-N (within the azole moiety), and γ C-H (related with benzene and azole rings) absorptions were in excellent accordance with the experimental and literature data [22]. To the best of our knowledge, in the literature, there are no data regarding IR spec- From the spectra given in Figure 3, we can conclude that the use of the B3LYP/6-31G(d,p), CAM-B3LYP/6-31G(d,p), or even wB97XD/6-31G(d,p) and PBE1PBE/6-31G(d,p) approaches for the rotamers optimization gives the highest conformity of the theoretical IR bands with the experimental spectrum, particularly in the ca. 3100-2800 cm −1 and ca. 1750-500 cm −1 range. The utilization of the Grimme's D3 empirical dispersion model leads to better consistency between the theoretical and experimental IR spectral values. Moreover, including the diffuse functions in B3LYP/6-311++G(d,p) approach did not lead to the theoretical IR spectrum being comparable with the experimental one (as is relatively more time-consuming in comparison with the B3LYP/6-31G(d,p) functional). In the computed spectra of 1 and 2, the estimated υ and δ C-Cl, N=N, C-N (within the azole moiety), and γ C-H (related with benzene and azole rings) absorptions were in excellent accordance with the experimental and literature data [22]. To the best of our knowledge, in the literature, there are no data regarding IR spectrum simulation using DFT formalism for isoconazole 1. It turned out that the use of the B3LYP, CAM-B3LYP, wB97XD, or PBE1PBE functional was comparatively more effective because these approaches generally afford results without significant errors. A similar conclusion cannot be drawn from the method involving other functionals used in our investigations.

UV-Vis Analysis
The UV spectra regarding the absorption bands of 1 and 2 are in accordance with the literature data [11]. The spectra (Figures 4 and 5) display an absorption band at 273 (for 1) or 254 nm (for 2) nm, which did not change with the concentration used. However, other absorption bands of analytes 1 or 2 are observed at 219-229 (1) or 201-213 (2) nm and 281-283 (1) nm, which migrated as a function of concentration. trum simulation using DFT formalism for isoconazole 1. It turned out that the use of the B3LYP, CAM-B3LYP, wB97XD, or PBE1PBE functional was comparatively more effective because these approaches generally afford results without significant errors. A similar conclusion cannot be drawn from the method involving other functionals used in our investigations.

UV-Vis Analysis
The UV spectra regarding the absorption bands of 1 and 2 are in accordance with the literature data [11]. The spectra (Figures 4 and 5) display an absorption band at 273 (for 1) or 254 nm (for 2) nm, which did not change with the concentration used. However, other absorption bands of analytes 1 or 2 are observed at 219-229 (1) or 201-213 (2) nm and 281-283 (1) nm, which migrated as a function of concentration.   trum simulation using DFT formalism for isoconazole 1. It turned out that the use of the B3LYP, CAM-B3LYP, wB97XD, or PBE1PBE functional was comparatively more effective because these approaches generally afford results without significant errors. A similar conclusion cannot be drawn from the method involving other functionals used in our investigations.

UV-Vis Analysis
The UV spectra regarding the absorption bands of 1 and 2 are in accordance with the literature data [11]. The spectra (Figures 4 and 5) display an absorption band at 273 (for 1) or 254 nm (for 2) nm, which did not change with the concentration used. However, other absorption bands of analytes 1 or 2 are observed at 219-229 (1) or 201-213 (2) nm and 281-283 (1) nm, which migrated as a function of concentration.   The vertical excited states were calculated for each optimized rotamer of compounds 1 and 2 at the functional/6-311++G(2d,3p) level of theory in gas phase, as well as in methanol, chloroform, dichloromethane, and acetonitrile (CPCM solvation model).
In the case of isoconazole 1 (Figure 6), the highest correspondence to the experimental data, especially with reference to the 273 nm band, was obtained using the M06L functional (absolute value of ∆ = 17.37 nm), B3LYP functional (absolute value of ∆ = 20.18 nm), and the B3LYP/6311++G(d,p) approximation (absolute value of ∆ = 20.94 nm). Whereas in the case of bifonazole 2 (Figure 7), with a reference to the experimental band 254 nm, the highest agreement was possible using the M06L, M062X, PBE1PBE, and wB97XD functionals (absolute value of ∆ = 33.60 nm). It was also noted that in the case of bifonazole 2 for all functionals, the first band of absorption also corresponded to the maximum absorption in the theoretical UV spectrum (except for the use of the M06L functional, where the maximum absorption corresponded to the second band of absorption). It can also be concluded that the implementation of the wB97XD functional for 2 resulted in the formation of two explicit absorption bands. Therefore, the use of the functional wB97XD seems to be a favorable approach for the correct prediction of UV-vis spectra of the investigated bifonazole. The results of calculations involving the first excited states of 1 and 2 and using different functionals are collected in Tables 5 and 6 maximum absorption in the theoretical UV spectrum (except for the use of the M06L functional, where the maximum absorption corresponded to the second band of absorption). It can also be concluded that the implementation of the wB97XD functional for 2 resulted in the formation of two explicit absorption bands. Therefore, the use of the functional wB97XD seems to be a favorable approach for the correct prediction of UV-vis spectra of the investigated bifonazole. The results of calculations involving the first excited states of 1 and 2 and using different functionals are collected in Tables 5 and 6, Figures 6 and 7, as well as in Tables S1 and S2 (Supplementary Material).      The contours of LUMO and HOMO orbitals for 1 and 2 (visualized based on the checkpoint file (.chk) generated during the TD-DFT computations) are presented in Figures 8 and 9, respectively. The highest occupied molecular orbital (HOMO) is located mainly over all structure of analytes 1 and 2, except for the azole moieties. The lowest unoccupied molecular orbital (LUMO) covers only the azole ring of 1 or all structure of the bifonazole 2. It turned out that the isoconazole and bifonazole HOMO orbitals are not similar to the HOMO orbitals of posaconazole, itraconazole, voriconazole, and fluconazole. Dissimilarity between them is also related with the lowest occupied molecular orbital (LUMO) of 1, which covers the only the diazole residue without dihalogenophenyl ring [6,7,23].  The HOMO-LUMO gap calculated for isoconazole 1 at the B3LYP/6-311++G(2d,3p) level is 5.2004 eV, corresponding to an electron transition from spinorbital 106 to spinorbital 107. It can be assigned to the calculated first excitation state at 263.64 nm (the HOMO−LUMO contribution relatively to the first excited state, calculated as duplicated coefficient square, is 99%, oscillator strength f = 0.0102, coefficient 0.70499, calculated energy is 4.4164 eV; data taken from the output file) and is slightly higher than for bifonazole 2 where that gap was estimated at 5.0181 eV [B3LYP/6-311++G(2d,3p)//B3LYP/6-31G(d,p) approach]. On the other hand, the HOMO-LUMO gap calculated for 2 at the wB97XD/6-311++G(2d,3p) level is 8.8147 eV is related to an electron transition from spinorbital 82 to spinorbital 83 and the first excitation state at  The HOMO-LUMO gap calculated for isoconazole 1 at the B3LYP/6-311++G(2d,3p) level is 5.2004 eV, corresponding to an electron transition from spinorbital 106 to spinorbital 107. It can be assigned to the calculated first excitation state at 263.64 nm (the HOMO−LUMO contribution relatively to the first excited state, calculated as duplicated coefficient square, is 99%, oscillator strength f = 0.0102, coefficient 0.70499, calculated energy is 4.4164 eV; data taken from the output file) and is slightly higher than for bifonazole 2 where that gap was estimated at 5.0181 eV [B3LYP/6-311++G(2d,3p)//B3LYP/6-31G(d,p) approach]. On the other hand, the HOMO-LUMO gap calculated for 2 at the wB97XD/6-311++G(2d,3p) level is 8.8147 eV is related to an electron transition from spinorbital 82 to spinorbital 83 and the first excitation state at The HOMO-LUMO gap calculated for isoconazole 1 at the B3LYP/6-311++G(2d,3p) level is 5.2004 eV, corresponding to an electron transition from spinorbital 106 to spinorbital 107. It can be assigned to the calculated first excitation state at 263.64 nm (the HOMO−LUMO contribution relatively to the first excited state, calculated as duplicated coefficient square, is 99%, oscillator strength f = 0.0102, coefficient 0.70499, calculated energy is 4.4164 eV; data taken from the output file) and is slightly higher than for bifonazole 2 where that gap was estimated at 5.0181 eV [B3LYP/6-311++G(2d,3p)//B3LYP/6-31G(d,p) approach]. On the other hand, the HOMO-LUMO gap calculated for 2 at the wB97XD/6-311++G(2d,3p) level is 8.8147 eV is related to an electron transition from spinorbital 82 to spinorbital 83 and the first excitation state at 244.40 nm and is lower than for isoconazole 1 where that gap was estimated at 9.1657 eV (wB97XD/6-311++G(2d,3p)//wB97XD/6-31G(d,p) approach).
Next, for 1 and 2, we computed several descriptors related to HOMO-LUMO electron transition, i.e., electronegativity (χ), chemical hardness (η) and electronic potential using the orbital energy of the HOMO and the orbital energy of the LUMO based on the DFT formalism, as well as the chemical potential (µ) of the molecule using Koopman's theorem [24] With regard to other derivatives of 1 and 2 containing azole moiety with antifungial activity, i.e., voriconazole and fluconazole, and the above descriptors, we used in previ-ous studies B3LYP/6-311++G(2d,3p)//B3LYP/6-31G(d,p) approach [23] or additionally computed at the wB97XD/6-311++G(2d,3p)//wB97XD/6-31G(d,p) level of theory. Regarding these approximations, these descriptors are as follows On the basis of the solvatochromism phenomenon, we decided to plot experimental UV-vis spectra for 1 and 2 including, in addition to methanol, the following solvents: chloroform and acetonitrile (Figures 10 and 11). In the case of isoconazole 1, the absorption band migrated at ca. 276 nm and was more shifted towards longer wavelengths in the case of chloroform (reaching the highest intensity), while it did not change in the methanol and acetonitrile medium. A similar phenomenon was observed for bifonazole 2 at ca. 254 nm, with the absorption band reaching a relatively lower intensity in the chloroform environment. Subsequently, using the TF-DFT method, theoretical UV-vis spectra were determined for 1 and 2 (Tables 7 and 8). Analysis of the first excited state (λ 1 ) and the λ max bands of the theoretical UV-vis spectra of 1 (  The discussion presented above provides important data relating to, e.g., the effect of reaction field and solvent polarity on the values of λmax and first excited state (λ1) of theoretical UV-vis spectra of the azoles studied, depending on the functionals used for calculations. To the best of our knowledge, regarding this phenomenon, a wealth of experimental and theoretical approaches has not yet been studied in terms of azoles 1 and 2.

NBO Analysis
Considering the conclusions drawn from the UV-vis analysis, we carried out natural bond orbitals (NBO) studies (CPCM solvation model and methanol used as solvent). The NBO analysis was performed at the wB97XD/6-311++G(2d,3p) level of theory using the NBO 3.0 approach as implemented in Gaussian G16 A.03 software for rotamers previously optimized in the wB97XD/6-31G(d,p) approximation. Our attention was focused on the oxygen and nitrogen atoms, as well as aromatic rings whose electrons were important for the distribution of HOMO and LUMO orbitals (Figure 1). The second order perturbation theory, which involves Fock matrix in the NBO basis, shows intramolecular hyper-conjugative interactions.
The fundamental structural differences between isoconazole 1 and bifonazole 2 are due to the presence of the two 1,3-chlororophenyl systems and oxygen atom bridge in the structure 1 compared to the other azole tested.
The O1-C12 bond in compound 1 (Figure 1) can be characterized by an almost completely filled (1.98838e) 2-centre hybrid bonding orbital (polarization coefficient 0.8222) formed by the overlap of s (28.27% s) and p (71.66% p2.53) orbitals. The oxygen atom has a greater contribution (67.60%) to the formation of this σO-N bonding orbital. This bond is also an NBO density donor to the following bonds formed by the antibonding orbital BD*: C4-C5, and C13-C14, as well as antibonding Rydberg orbitals RY* centered on atoms: C5, and C13. The analogous O-C bond in posaconazole, itraconazole voriconazole, and fluconazole [6,7,23] can be characterized similarly.

NBO Analysis
Considering the conclusions drawn from the UV-vis analysis, we carried out natural bond orbitals (NBO) studies (CPCM solvation model and methanol used as solvent). The NBO analysis was performed at the wB97XD/6-311++G(2d,3p) level of theory using the NBO 3.0 approach as implemented in Gaussian G16 A.03 software for rotamers previously optimized in the wB97XD/6-31G(d,p) approximation. Our attention was focused on the oxygen and nitrogen atoms, as well as aromatic rings whose electrons were important for the distribution of HOMO and LUMO orbitals (Figure 1). The second order perturbation theory, which involves Fock matrix in the NBO basis, shows intramolecular hyper-conjugative interactions.
The fundamental structural differences between isoconazole 1 and bifonazole 2 are due to the presence of the two 1,3-chlororophenyl systems and oxygen atom bridge in the structure 1 compared to the other azole tested.
The O1-C12 bond in compound 1 (Figure 1) can be characterized by an almost completely filled (1.98838e) 2-centre hybrid bonding orbital (polarization coefficient 0.8222) formed by the overlap of s (28.27% s) and p (71.66% p2.53) orbitals. The oxygen atom has a greater contribution (67.60%) to the formation of this σ O-N bonding orbital. This bond is also an NBO density donor to the following bonds formed by the antibonding orbital BD*: C4-C5, and C13-C14, as well as antibonding Rydberg orbitals RY* centered on atoms: C5, and C13. The analogous O-C bond in posaconazole, itraconazole voriconazole, and fluconazole [6,7,23] can be characterized similarly.
The N2-C1 bond within the azole ring in compound 1 (Figure 1) can be characterized by two almost completely filled (1.98607e) 2-centre bonding hybrid BD orbitals (polarization coefficients 0.8030 and 0.5960) formed by the overlap of: s (33.56% s) and p (66.41% p1.98) orbitals (in this bond the nitrogen atom has a greater contribution (64.48%) to the formation of this bonding orbital). This bond is also an NBO density donor to the following bonds formed by the antibonding orbitals BD*: N1, C2, C4, N2-C2, N2-C4, and C2-H2.
The N1-C20 bond in the bifonazole 2, analogous to the N2-C1 bond in isoconazole 1, can be characterized by an almost filled (1.98539e) 2-centre hybrid BD (polarization coefficient 0.8027) formed by the overlap of s (33.39% s) and p (66.58% p1.99) orbitals. The nitrogen atom has a greater contribution (64.44%) to the formation of the σ N-C bonding orbital. This bond is also an NBO density donor to the following bonds formed by the antibonding orbitals BD*: N1-C7, N1-C22, and C22-H18, as well as the antibonding Rydberg orbitals RY* of atoms: N2, C7, and C22.
Considering the above data, we can conclude that the distribution of the NBOs for rotamers of isoconazole and bifonazole almost identically covers especially the azole nitrogen atoms. The sole difference, discussed above, is connected with the NBO donoracceptor interaction, including the hyper-conjugate interaction energy ( Figure 1). The differences come down to the fact that a dichlorophenyl ring and -CH 2 -O-CH-bridge are present in the isoconazole structure as opposed to the bifonazole structure.

NMR Analysis
The signals in the 1 H NMR spectra of isoconazole 1 and bifonazole 2 were registered in DMSO-d6 (Tables 9 and 10 Figure 1). The theoretical 1 H NMR spectra of 1 and 2 using the B3LYP functional (MAE = 0.25 or 0.24 for 1 or 2, respectively; DMSO as solvent) show the highest conformity of the chemical shifts with the experimental data (Table 9). In the case of 1, the largest values of percentage error (∆δ) more than 12% were found for the H5 and H10 methylene protons. These errors are due to steric reasons, namely proximity of the oxygen atom (closest distance CH 2 . . . O1 is 2.58 or 2.08 Å for the H5 or H10 atoms, respectively) or H1 atoms within the azole ring (distance CH 2 . . . H1 is 2.56 Å) and dichlorophenyl ring (distance CH 2 . . . Cl3 is 2.65 Å). Fundamentally, it should be emphasized that in the case of isoconazole 1, the calculated values of chemical shifts in the 1 H NMR spectrum presented significant correspondence with experimental data (MAE error range 0.25-0.64). Table 10. Experimental (δ exp ) and calculated chemical shifts (I) for compound 2; errors (∆), relative percentage errors (∆δ); calculated NMR shielding (B3LYP/631G(d,p)//B3LYP/6-31G(d,p)/DMSO) for proton H ref = 31.7468 ppm for TMS; MAD = 0.24 (atoms numbering as is in Figure 1).

Atoms
For bifonazole 2, the compliance of the estimated values of chemical shifts with the experimental data of 1 H NMR spectrum was expressed in the following ranges of MAE error values: 0.25−0.78. In the case of 2, the theoretical 1 H NMR spectra using the B3LYP functional (MAE = 0.25 for DMSO as solvent) show the highest conformity of the chemical shifts with the experimental data ( Table 10). The highest values of percentage error (∆δ) exceeding ca. 8% were related to the H9 proton of the phenyl ring, which was caused by steric reasons. We noticed the proximity of the second phenyl rings (closest distance H9 ... H11 is ca. 2.33 Å). Moreover, the closest distance H6 ... H18 (within the azole ring) equaled ca. 2.58 Å and resulted in 6% percentage error (∆δ). The closest distance H6 ... H10 (within the phenyl ring) equaled ca. 2.38 Å and presented the error 6% too. The percentage error (∆δ) 7% was found for the H16 within the azole ring is due to proximity of the rotating phenyl ring (closest distance H16 . . . C8 is 2.72 Å).
In consideration of the data presented, it should be emphasized that these data show that the DFT formalism, particularly the B3LYP functional, results in a correct description of the isoconazole and bifonazole 1 H NMR chemical shifts, with the worst results being obtained using the M062X functional.

Spectroscopy
The IR spectra were recorded in KBr (1.00 mg of compound 1 or 2 per 300 mg of KBr) on a Shimadzu IRAffinity-1 spectrometer.
The NMR spectra were recorded at 298 K on a NMR 700 MHz (16.44 T) AVANCE III Bruker spectrometer operating at 500 or NMR 500 MHz (11.74 T) AVANCE III Bruker spectrometer operating at 700 MHz ( 1 H) and 126 MHz ( 13 C). Bifonazole (10 mg) or isoconazole (10 mg) were dissolved in 500 µL of d 6 -DMSO (Aldrich). TMS was used as an internal standard.
Isoconazole (1) High resolution mass spectrometry analysis was performed using Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with TriVersa NanoMate ESI ion source (Advion BioSciences ltd., Ithaca, NY, USA) working in direct infusion mode. 5 µL sample aliquots were infused directly into mass spectrometer, after ion current stabilization, spectra were acquired for 5 min. TriVersa source was operating at 1.25 psi nitrogen pressure and ionization voltage was set to 1.05 kV. HRMS data were collected in positive ion mode within the range of m/z 100-1500 at the resolution of 140,000 (at m/z 200, full width at half maximum, FWHM). All analyses were performed using automatic gain control (AGC) set to target value of 3 × 106 and ion injection time (IT) was set to 100 ms.
The 1 H, 13 C, H-H COSY, H-C HSQC, and HR MS spectra of 1 or 2 are given in the supplementary material (Figures S5a-S8).

Conclusions
Our computations proved that the use of the CAM-B3LYP or B3LYP functional seems to be comparatively more effective in the IR spectra predictions of isoconazole 1 and bifonazole 2 because these approaches generally afford results without significant errors. The best conformity with the experimental UV spectra was obtained with the use of B3LYP/6-31G(d,p) (for isoconazole 1) or wB97XD/6-31G(d,p (for bifonazole 2) methods. The HOMO-LUMO gap calculated for isoconazole 1 at the B3LYP/6-311++G(2d,3p) level is 5.2004 eV, corresponding to an electron transition from spinorbital 106 to spinorbital 107. It can be assigned to the calculated first excitation state at 263.64 nm (the HOMO−LUMO contribution relative to the first excited state, calculated as duplicated coefficient square, is 99%, oscillator strength f = 0.0102, coefficient 0.70499, calculated energy is 4.4164 eV; data taken from the output file) and is slightly higher than for bifonazole 2, where that gap was estimated at 5.0181 eV (B3LYP/6-311++G(2d,3p)//B3LYP/6-31G(d,p) approach). On the other hand, the HOMO-LUMO gap calculated for 2 at the wB97XD/6-311++G(2d,3p) level is 8.8147 eV is related to an electron transition from spinorbital 82 to spinorbital 83 and the first excitation state at 244.40 nm and is lower than for isoconazole 1 where that gap was estimated at 9.1657 eV (wB97XD/6-311++G(2d,3p)//wB97XD/6-31G(d,p) approach). For 1 and 2, we computed several descriptors related to HOMO-LUMO electron transition, i.e., electronegativity (χ), chemical hardness (η), and electronic potential, using the orbital energy of the HOMO and the orbital energy of the LUMO based on the DFT formalism, as well as the chemical potential (µ) of the molecule using Koopman's theorem. For 1, first ionization potential (I) reached higher values compared to 2 (except for the use of CAM-B3LYP, APF and functionals). In addition, the use of wB97XD functional resulted in the largest value of this descriptor relative to both azoles, as well as the fact that chemical potential (µ) took negative values (for the other functionals, µ values were positive). Moreover, analysis of the first excited state (λ 1 ) and the λ max bands of the theoretical UV-vis spectra of 1 or 2 indicates that the highest absorption values were observed for the use of the M06L functional, and the lowest for the CAM-B3LYP functional. Besides, for 2, the first excited state (λ 1 ) and the λ max bands reach the same values for the functional: CAM-B3LYP, PBE1PBE, M062X and APF (in methanol and acetonitrile medium). In our work we compared 1 H NMR experimental and theoretical spectra of 1 and 2. The calculated data show that the DFT formalism, particularly B3LYP functionals, result in a correct description of the isoconazole and bifonazole 1 H NMR chemical shifts. In the case of 1 the largest values of percentage error (∆δ) to be more than 12% were found for the H5 and H10 methylene protons. These errors are due to steric reasons, namely proximity of the oxygen atom (closest distance CH 2 . . . O1 is 2.58 or 2.08 Å for the H5 or H10 atoms, respectively) and H1 atoms within the azole ring (distance CH 2 . . . H1 is 2.56 Å) and dichlorophenyl ring (distance CH 2 . . . Cl3 is 2.65 Å). In the case of isoconazole 1, the calculated values of chemical shifts in the 1 H NMR spectrum gave significant correspondence with experimental data (MAE error ranged from 0.25−0.64). For bifonazole 2, the compliance of the estimated values of chemical shifts with the experimental data of the 1 H NMR spectrum was expressed in the following ranges of MAE error values: 0.25−0.78. In the case of 2, the theoretical 1 H NMR spectra using the B3LYP functional (MAE = 0.25 for DMSO as solvent) show the highest conformity of the chemical shifts with the experimental data. The highest values of percentage error (∆δ) exceeding ca. 8% were related to the H9 proton of the phenyl ring, which was caused by steric reasons. The above conclusions show that our proposed methodology seems to be a potentially useful tool for the prediction of IR and UV-vis properties of biologically active conazoles. We wish to investigate this standpoint further in the near future, additionally involving the tuned range-separated functionals [43][44][45][46][47] (especially regarding the TD-DFT computations and all investigated so far azoles [6][7][8]23]).

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/ijms24010520/s1. The supplement contains Cartesian coordinates of the rotamers and complete results of the UV-vis, and NMR spectra and calculations.