New bis-isoxazole with monoterpenic skeleton: regioselective synthesis, spectroscopic investigation, electrochemical, and density functional theory (DFT) studies

A novel bis-isoxazole was synthesized from (R)-Carvone and p-methylbenzaldoxime, via two successive [3+2] cycloaddition reactions (32CA). The newly obtained bis-isoxazole has been fully characterized by HRMS and NMR spectroscopy. The HMBC experiment was performed to determine the stereo and the regioselectivity of the reaction. The electrochemical behavior of the studied compound, in oxidation and reduction processes, was examined using the cyclic voltammetry technique. In addition, the regioselectivity of the [3+2] cycloaddition reaction and the molecular structure of the title compound was performed by density functional theory (DFT). The HOMO and LUMO orbitals were investigated to determine the electronic properties of the synthesized compound. Besides, the global reactivity indexes were used to explain the regioselectivity for the formation of the bis-isoxazole, the theoretical results are in good agreement with experimental findings.

often resulting in the oxidation or reduction of a metal complex [28].Among the electrochemical techniques, cyclic voltammetry is the most popular and commonly employed technique to investigate the reduction and oxidation processes of molecular species.
These bibliographic findings prompt us to expand our research work aiming at the preparation and theoretical study of heterocyclic systems with monoterpenic skeleton [29][30][31][32].The target compound was designed by the incorporation of two isoxazole coresintoa monoterpenic skeleton by reacting (R)-carvone with an arylonitrile oxide via two sequential 1,3-dipolar cycloaddition reactions (Figure 1).Through the obtained results, we discussed the electronic and structural effects in order to carry out a comparative study of its electrochemical properties.
We report also an experimental NMR investigation of the formation of the uniquely obtained bis-isoxazole regioisomer 3, followed by a study on the plausible mechanism for this regioselective synthesis of the stoichiometric compound 3 via the local reactivity indexes.HOMO, LUMO, and NBO analyses were performed using density-functional theory (DFT).The electrochemical activity of the studied compounds (2 and 3) was carried out using the cyclic voltammetry technique.

Experimental section 2.1. Instruments and reagents
All chemicals were used as obtained from commercial sources (Aldrich and Acros).Melting points (m.p.) were determined using a capillary apparatus and are inaccurate.Analytical thin-layer chromatography (TLC) was performed on plates precoated with E. Merck silica gel 60 F254 to a thickness of 0.25 mm.HRMS were obtained on a Q-TOF micromass spectrometer. 1H and 13 C NMR spectra were recorded in CDCl 3 with a 500 MHz Bruker Avance III spectrometer with a BBFO + probe.Chemical shifts (δ) are expressed in parts per million (ppm).They were recorded relative to solvent CDCl 3 signal (7.26 ppm and 77.16 ppm).The (R)-carvone-isoxazole 2 was prepared according to the reported method [33].

Cyclic voltammetry
All the measurements were carried out using a potentiostat equipped with a three-electrode cell, consisting of two Pt electrodes (diameter = 0.1 cm) as working and counter-electrode as well as a saturated calomel reference electrode (SCE) (3 M KCl), all connected to a Voltalab 10 system (PGZ 100 radiometer) controlled by the Volta master 4 software.The potential was scanned between -2000mV and +2000mV at a scan rate ranging from 50 to 1000 mV/S.As the electrolyte, an anhydrous acetonitrile (CH 3 CN) solution containing the synthesized compound (2 or 3) and tetrabutylammonium perchlorate (Et 4 ClNO 4 ) in concentrations of 10-3 and 0.1 M, respectively was used at room temperature.UV-Vis spectra were recorded using a UV-6300 PC double-beam spectrophotometer (200-800 nm) operating at 1 nm resolution with a scanning speed of 100 nm min-1 at room temperature.All the measurements were carried out in acetonitrile containing a concentration of 10 -6 M for two compounds studied.
After stirring for 10 min (the reaction was monitored by TLC) at room temperature, the layers were separated, and the aqueous layer was extracted with CH 2 Cl 2 (3 × 10 mL).The combined organic extracts were dried over anhydrous Na 2 SO 4 and the solvent was evaporated in vacuo.The residue was purified by column chromatography using hexane/ Ethylacetate mixture (82:18) as eluent.The structures, spectroscopic analysis (HRMS), and the systematic name (according to IUPAC) of two products 2 and 3 are mentioned in Table 2.

Chemistry
The first step in the synthesis of the desired hybrid bis-isoxazole 3 from (R)-carvone is the preparation of mono-adduct 2 according to the procedure reported in our previous work [33].The second step was achieved through [3+2] cycloaddition reaction by treating the mono-adduct 2, with a stoichiometric amount of p-methyl arylonitrile (generated in situ from the corresponding oxime using 5% bleach solution).The reaction was performed at 0 °C, in dichloromethane as a solvent to yield the corresponding bis-isoxazole 3 in a high stereo and regioselective manner with low yield (11%) (Scheme1).
The low yield (11%) of the bis-isoxazole 3 has prompted us to carry out an optimization study of the reaction yield by increasing the number of equivalents of p-methylbenzonitrile oxide.The obtained results are shown in Table 3.
As revealed in Table 3, the yield of the reaction increases with the increase of the number of equivalents of p-methyl arylonitrile, such as the use of three equivalents of dipole gave the desired compound with an improved yield of 48 %.On the other hand, under the same operating conditions, there is a drop in yield from 48% to 41% in the case of 3.5 and 4 equivalents.This drop-in yield is likely due to the effect of the excess dipole on the formed bis-isoxazole 3.

H NMR and 13 C NMR spectrum analysis of compound 3
The mono-adduct 2 was synthesized and identified according to the reported work [33].Similarly, the newly synthesized bis-isoxazole 3 was fully characterized from its spectral NMR (1D & 2D) and HRMS data.In HRMS spectrometry, bisisoxazole 3 reveals its pseudo molecular ion at m/z = 417.2127[M+H] + , which is consistent with the molecular formula C 26 H 28 N 2 O 3 .In NMR spectra of the hybrid compound 3, and in comparison with those of starting mono-adduct 2, we can easily state that the attack of the 1,3-dipole occurs on the internal double bond of the terpenic skeleton.Indeed, the first noteworthy data ARE the disappearance of resonances (δ 1 H 6.76 ppm; δ 13 C 144.50 ppm), characterizing the methane group (HC=) of the internal double bond of the precursor 2. These letters are replaced in δ 1 H NMR spectra by one hydrogen doublet of doublet δ 1 H 3.75 ppm J = 1.1 & 6.8 Hz attributed to the proton at C3a position, while in 13 C NMR spectra, we noted two shielded signal at 55.60 and 87.79 ppm assigned respectively to C3a and C7a of isoxazole.
The 1,3-dipolar cycloaddition reaction of the 4-methylbenzonitrile oxide on the C=C dipolarophile of compound 2 can theoretically take place at both or one of its two faces according to two opposite directions leading to the formation of two possible regioisomers (A & B) each in the form of two possible diastereomers (A: A1 and/or A2) and (B: B1 and/ or B2) (Figure 2).
To resolve this structural problem, 2D (two-dimensional) NMR spectroscopy was carried out, and HMBC (heteronuclear multiple bond correlation) experiments (Figure 3) allowed us to rule out B1 and B2 and confirm the A1 or A2 stereoisomers.
Indeed, in the HMBC spectrum (Figure 3), we note a 2 J CH correlation between the C3a-H proton (δ 1 H 3.75) and the C3=N imine carbon (δ 13 C 158.66) of the isoxazole ring, while no correlation was observed between this latter and the protons of C7a-CH 3 methyl group (δ 1 H 1.42).This deep examination of the HMBC spectrum informs us about the regioselective formation of isomer A (A1 or A2) (Figure 4-A).In the same spectrum, we can also note a 3 J CH correlation between the C3a-H proton (δ 1 H 3.75) and the methyl carbon at the C7a position.This three bond correlation would be present in both A1 and A2 diastereoisomers (Figure 4-B).Thus, the 1,3-dipolar cycloaddition reaction of 4-methylbenzonitrile oxide on isoxazoline 2 was highly regioselective.This result corroborates the one obtained in our recently published study, which showed that the C3 carbon (in beta position  of the carbonyl group) is more nucleophilic than the C2 carbon (in alpha position of the carbonyl group) [45].Therefore, the bis-isoxazoline 3 is obtained in a unique manner where the oxygen atom of the 1,3-dipole is linked to the more hindered carbon of the cyclohexenic double bond of the isoxazoline 2. Furthermore, the 1,3-dipolar cycloaddition reaction of 4-methylbenzonitrile oxide on isoxazoline 2 was revealed to be also highly diastereoselective.This could be ascribed to the fact that the Si face of the cyclohexenic double bond of 2 is much more sterically hindered than the Re face.Therefore, the approach of the 1,3-dipole on the isoxazoline 2 should be favoured on the Re face thus producing the obtained A1 diastereoisomer (Figure 5).
If the regioselectivity of the reaction was unambiguously established, its diastereoselectivity and, so, the absolute configuration of the two newly formed asymmetric carbons C3a and C7a remained unknown.In an attempt to resolve  this problem, we have carried out a DFT.Theoretical study where 1 H and 13 C NMR chemical shifts of compound 3 were calculated at the B3LYP/6-31G(d,p) using GIAO approach in the solvent phase.The calculations were performed in CDCl 3 solvent by using C-PCM formalism, and the selected shifts were compared with the experimental values as illustrated in Table 4.
According to Table 4, we can see that the highest chemical shift value was that of the carbonyl group (C7 atom), which was observed experimentally at 205.83 ppm, while the corresponding calculated values were 214.64 ppm for A1 and 219.97 ppm for A2.Concerning the C8 and C9 carbon atoms of the two methyl groups, which were revealed with the lowest chemical shift values, they were observed experimentally at 20.06 and 24.37 ppm, while, theoretically, they were noted at 19.52 and 25.27 ppm for diastereomer A1 and 17.61 and 23.01 ppm for diastereomer A2.A slight difference was observed between the C5' and C7a isoxazolic carbons; their experimental chemical shift values were recorded respectively at 87.69 and 87.79 ppm, while their theoretical ones were recorded at 94.51 and 92.30 ppm for A1, and at 95.96 and 96.33 ppm for A2, respectively.
Among the two Csp 3 methine groups, the one at C3a position ( 1 H 3.76, 13 C 55.79) is more deshielded than the one at C5 position ( 1 H 2.35, 13 C 39.10) because of the inductive withdrawing effect of the adjacent imine group.The calculated chemical shift values of C3a-H methine group were ( 1 H 3.59, 13 C 59.71) for A1 and ( 1 H 3.41, 13 C 61.66) for A2.On the other hand, those of C5-H methine group were ( 1 H 2.24, 13 C 39.53) for the diastereomer A1 and ( 1 H 1.99, 13 C 43.41) for the diastereomer A2.
The conclusion to be drawn concerning the comparability between the experimental and the theoretical findings is that the experimental NMR data are consistent with the computed values from the optimized structure of diastereomer A1 (Figure 6).According to this NMR analysis, which allowed an approximate assignment of each carbon and its protons, we can relatively conclude that the absolute configuration of both new stereogenic centers (C3a & C7a) is 3aS and 7aR.

IR spectral analysis
Subsequently, an FT-IR spectral study of the title compound was carried out.The structure of compound 3 was confirmed according to its IR spectral data, the existence of the band at υ max = 1720 cm -1 corresponds to the carbonyl group (C=O).The characteristic band of the aromatic nucleus (C=C) has been revealed at 1515 cm -1 .However, the scaled harmonic vibrational frequencies of compound 3 were calculated from the optimized structure with DFT/B3LYP using a 6-31G(d,p) basis set at room temperature in the region between 400 and 4000 cm -1 .The experimental and theoretical spectrums plotted on the transmittance (%) against the wavenumber (cm -1 ) are shown in Figure 6a,6b.
The calculated frequencies of the diastereoisomer A1 and A2 were compared with the observed values, and the results found are grouped in Table 5.
The cycloaddition reaction of compound 2 in the presence of the 1,3-dipole is illustrated in Scheme 2. To get a closer understanding of the regioselectivity experimentally, DFT calculations were performed at the B3LYP/6-311G(d,p) level of both reagents.The optimized geometries obtained and the numbering of the atoms of the most stable conformation of both reactants are shown in Figure 8.
The global reactivity indexes are potent tools that can explain the reaction mechanism and are measured through the global electron density transfer (GEDT) value [46][47].In this regard, the global electrophilicity and nucleophilicity indexes are calculated and summarized in Table 6.The electronic chemical potential value of dipole, μ = -3.94eV, is closer to that of the compound 2, μ = -3.88eV.These results confirm that this 32CA reaction will have a nonpolar character.The electrophilicity ω indices (1.63 and 1.54 eV) and the nucleophilicity N indices (2.93 and 2.65 eV) of the compound 2 and dipole illustrate that both reagents act as moderate electrophiles and moderate nucleophiles within the electrophilicity and nucleophilicity scales, confirming the nonpolar character of this cycloaddition reaction and suggests high activation energy for the formation of the corresponding products [48].
Recently, Domingo et al. have proposed the Parr functions as a helpful tool to explain the regioselectivity that is experimentally observed in 32CA reactions [49].The authors confirmed that the ethylene group could participate in 32CA reactions by forming the first new single bond through their most electrophilic center.Therefore, the local reactivity of compound 2 was analyzed by the electrophilic P k + Parr functions (Figure 9).The P k + Parr function analysis indicates that the carbon C19 atom with a P k + value of 0.24 is more electrophilic center than the carbon atoms C20 and C21, with P k + values of 0.12 and 0.01, respectively.These results indicate that the first single bond formation will involve the most electrophilic C19 carbon explaining the experimentally observed results.
In order to illuminate the experimental results, two reaction paths associated with regioisomeric approach modes to the regio-and diastereofacial attacks for this 32CA reaction between product 2 and dipole were considered (Figure 9).The results indicate that the above-mentioned reaction paths proceed via a one-step mechanism, and the relative energies are donated in Figure 10.The activation energies associated with the reaction paths conduct to the formation of regioisomeric A are 19.09 and 20.33 kcal/mol for A1 and A2, respectively.In the case of the regioisomeric B, the results were found to be 20.30and 21.91 kcal/mol for the compounds B1 and B2, respectively.These results confirm that the formation of the compounds A1 and A2 are more exothermic than the formation of B1 and B2.Also, the activation energy associated with TS-A1 (1.321 kcal/mol) is lower than that associated with TS-B1.The theoretical results are in good agreement with the experimental observations (Figure 10).

Electrochemistry study 3.4.1. Cyclic voltammetry measurements
The electrochemical properties of compounds 2 and 3 were performed by cyclic voltammetry in acetonitrile solution at a scan rate of 200 mV/s in the potential range from 2 to -2 V at room temperature are shown in Figure 11, and their electrochemical data are given in Table 5.The analysis of cyclic voltammetry spectra of the 2, 3, and their comparison with the blank spectrum show that new anodic and cathodic peaks appear as indicated in Figure 11.Indeed, the anodic current peaks observed around -1.22 and -1.48 V for compounds 2 and 3, respectively, are located in a negative range and precede the cathodic current peaks, which appear around -1 and -1.25 V for compounds 2 and 3, respectively; this indicates that it is not a Red/Ox system of the same grouping in the molecules, which can be explained by the irreversibility of the examined compounds [50][51].On the other hand, these oxidation peaks observed in Figure 11 can be explained by oxidation at the α-position of the carbonyl group or/and the α-position of the double bonds, knowing that the hydrogens at these positions are labile.Then, the reduction peaks may be due to protonation of nitrogen atoms or/and reduction of nonaromatic double bonds or the carbonyl group to alcohol. Figure 12 represents the effect of scan rate on the simulated voltammograms for 10 -3 M of compounds 2 and 3 in a 0.1 M Et 4 NClO 4 /acetonitrile solution at different scan rates (50-1000 mV/s) at room temperature.The peak current intensities of both compounds increase with increasing scan rate.However, the oxidation and reduction peaks of compound 2 have the same appearance as that of compound 3, but they move in the negative direction with a higher height of current intensities than compound 2.This phenomenon can be attributed to the steric effect of the 5-methyl-3-p-tolyl-4,5-dihydroisoxazole group, which influences the electrochemical properties at the electrode surface.
On the other side, the HOMO, LUMO energy levels, and corresponding band gaps E g CV of the two compounds were also determined by using the cyclic voltammetry method by measuring the first oxidation ( !" !#$%& ) ( '%( !#$%& ) and the first reduction ( '%( !#$%& ) potentials of two studied compounds (as shown in Table 7) according to equations ( 1) and ( 2) [52][53][54]:    The band gaps E g CV were calculated from the difference between the onset of oxidation and reduction, according to the following equation [52][53][54]: The band gaps (E g CV ) of compound 3 are higher than those of compound 2; this can be attributed to the increased size of molecule 3. Compared to compound 2, compound 3 has higher stability due to the lower HOMO levels.However, the LUMO energy level of compound 2 is higher than that of compound 3, which allows for electron transfer after excitation.

Optical properties
The UV/Vis experimental spectra are shown in Figure 13.The maxima for compounds 2 and 3 are located in the UV-Vis at λ max = 218.81;265.19 nm (Abs = 0.2263 and 0.3427, respectively) and at λ max = 220.09;268.40 nm (Abs = 0.2402 and 0.4538, respectively), respectively.Comparing these two experimental spectra, we can note that the spectrum of compound 3 shows a slight shift and has a higher absorbance value than compound 2 due to its stronger conjugate effect [55].This shift corresponds to an optical band gap (E g Opt ) from 5.35 eV (compound 2) to 5.37 eV (compound 3) (Table 8).The excellent  absorption properties of compound 3 are associated with the large delocalization of the most occupied molecular orbital (HOMO) and the least occupied molecular orbital (LUMO) [56].These transitions can be measured using the energy of the long-wave edge of the exciton absorption band [57].The optical band gap (E g opt ) values have been calculated according to equation (4) [58] and are presented in Table 6.However, the difference between E g CV and E g Opt is due to the progression of reduction, oxidation, and the difference in energy level between the LUMO of the electron transport units and the HOMO of hole transport units, respectively.This result has been confirmed by DFT calculations.

Conclusion
In this study, we have described a synthesis of new chiral bis-isoxazole having monoterpenic skeleton, via a 1,3-dipolar cycloaddition reaction of (R)-Carvone-isoxazole with the nitrile oxide.The cycloaddition reaction was revealed to be highly regioselective.This regioselectivity was identified by a spectroscopic study of type HMBC and computational study (DFT).Cyclic voltammetry indicates that the oxidation potentials of compounds 2 and 3 vary depending upon the cycle count of the isoxazole.Moreover, DFT calculations were used to explain the regioselectivity for the formation of the bisisoxazole, and the results are in good agreement with experimental findings.

Acknowledgments
We are grateful to Cadi Ayyad University and Chouaïb Doukkali University for providing financial support.

Figure 1 .
Figure 1.The design strategy of the new bis-isoxazole hybrid.

Figure 4 -
Figure 4-A, B. Main HMBC cross-peaks correlations were observed in the 2D-NMR spectra of compound 3.

Figure 5 .
Figure 5.The proposed mechanism of the formation of the stereoisomer A1 of bis-isoxazoline 3.

Figure 6 .
Figure 6.Correlation curves between the predicted and experimental 1 H NMR. (a) and 13 C NMR, (b) chemical shifts for product 3.

Figure 9 .
Figure 9. Three-dimensional representation of the Mulliken atomic spin densities of 2 together with the P k +

Figure 10 .
Figure 10.Relative energie for the studied reactions paths of the 32CA of benzonitrile oxide with compound 2.

Figure 12 .
Figure 12.Cyclic voltammograms of 10 -3 M of 2, and 3 compounds in a solution of 0.1 M Et 4 NClO 4 / acetonitrile at different scanning speeds at room temperature.

Table 1 .
The quantity of the volume of NaOCl added to the number of equivalents of dipole.

Table 2 .
IUPAC name, molecular structure, molecular formula, melting point, and analytical data of the studied isoxazole derivatives.

Table 3 .
Effect of the 1,3-dipole amounts on the reaction yield.

Table 5 .
Comparison of the experimental and theoretical vibrational spectra analysis of the compound.3.

Table 6 .
Electronics properties and global reactivity indexes of compound 2 and dipole.

Table 7 .
Cyclic voltammetry parameters for the two molecules tested in a 0.1 M Et 4 NClO 4 /acetonitrile solution at room temperature.

Table 8 .
Absorption edge and band gap energy of compounds 2 and 3.