Experimental and Computational Investigation of the Oxime Bond Stereochemistry in c-Jun N-terminal Kinase 3 Inhibitors 11H-Indeno[1,2-b]quinoxalin-11-one Oxime and Tryptanthrin-6-oxime

11H-Indeno[1,2-b]quinoxalin-11-one oxime (IQ-1) and tryptanthrin-6-oxime are potent c-Jun N-terminal kinase 3 (JNK-3) inhibitors demonstrating neuroprotective, anti-inflammatory and anti-arthritic activity. However, the stereochemical configuration of the oxime carbon–nitrogen double bond (E- or Z-) in these compounds was so far unknown. In this contribution, we report the results of the determination of the double bond configuration in the solid state by single crystal X-ray diffraction and in solution by 1D and 2D NMR techniques and DFT calculations. It was found that both in the solid state and in solution, IQ-1 adopts the E-configuration stabilized by intermolecular hydrogen bonds, in contrast to previously assumed Z-configuration that could be stabilized only by an intramolecular hydrogen bond.


Introduction
Every year, the number of diseases associated with inflammatory processes in the body, heart dysfunction and the regulation of metabolism is increasing [1]. Enzymes of the c-Jun N-terminal kinase (JNK) family are known to play an important role in human body functioning [2]. JNKs are involved in the regulation of inflammation [3], participate in signaling pathways leading to apoptosis and necrosis [4,5], and regulate some transcriptional and non-transcriptional processes that damage the brain neurons and cardiomyocytes during ischemia/reperfusion [6,7]. JNKs are also involved in the embryonic development of the heart, the regulation of metabolism [8], and the normal functioning of the myocardium. JNK activation is an important link in the pathogenesis of cancer [9], obesity [10], diabetes mellitus [11], insulin resistance [10], and metabolic syndrome [8].
The natural alkaloid tryptanthrin can be considered as a structural analogue of 11Hindeno-[1,2-b]-quinoxalin-11-one. Tryptanthrin contains a quinazoline fragment annulated to indole with two carbonyl groups at positions 6 and 12. A large number of natural and synthetic tryptanthrin derivatives containing various substituents at six positions of the indolo [2,1-b]quinazoline core are known [27][28][29][30]. Tryptanthrin-6-oxime (Trp-Ox, Scheme 1) was shown to express JNK3 inhibitory activity comparable to that of IQ-1 [31]. Trp-Ox significantly attenuated the development of collagen-induced arthritis and collagenantibody-induced arthritis in mice, indicating it as a promising agent against the rheumatoid arthritis [32]. An O-acyl derivative of Trp-Ox demonstrated strong binding with JNK1-3 isoforms with nanomolar values of dissociation constants and inhibited the pro-Pharmaceutics 2023, 15, 1802 3 of 20 duction of proinflammatory cytokines, indicating the potential of Trp-Ox derivatives as anti-inflammatory drugs [33].
Despite a wide range of potential therapeutic activity, the stereochemistry of the oxime C=N double bond in IQ-1 and Trp-Ox is still unknown; in some works, they were assigned as individual Zor E-isomers, or considered as a mixture of dynamically interconverting isomers [34]. However, it is well known that the stereochemical configuration of drugs plays a crucial role in their activity, including the active pharmaceutical ingredients among the class of oximes [35,36]. Thus, fluovoxamine expresses the antidepressant only in the form of E-isomer [37]. For better understanding of the mechanisms of action of the oximes IQ-1 and Trp-Ox, it is therefore important to establish the correct stereochemical configurations of these compounds. In this work, we report the experimental, i.e., X-ray diffraction in the solid state and NMR in solution and theoretical investigation of E/Z-isomerism or IQ-1 and Trp-Ox that allowed to unambiguously assign them as E-isomers.

NMR and X-ray Diffraction Equipment
NMR spectra were recorded on a Bruker Advance 500 instrument (Billerica, MA, USA). The solvent residual peaks were used as internal standards. The operating frequencies were 500. 13  Diffraction data for single crystals of compounds IQ-1 and Trp-Ox·Py were collected with a Bruker D8 Venture diffractometer (Bruker Corporation, Billerica, MA, USA) with a CMOS PHOTON III detector and IµS 3.0 source (mirror optics, λ(CuKα) = 1.54178 Å). The ϕand ω-scanning techniques were employed to measure the intensities.
The structures were solved by the dual-space algorithm (SHELXT [42]) and refined by the full-matrix least-squares technique (SHELXL [43]) in the anisotropic approximation (except hydrogen atoms). Positions of the hydrogen atoms were calculated geometrically and refined in the riding model.

NMR Chemical Shift Calculations
The calculations were performed using Gaussian 09 package [44]. The structures of IQ-1 and Trp-Ox were fully optimized at the DFT level of theory employing the three-parameter hybrid B3LYP functional [45][46][47][48] and 6-31+G(d,p) or 6-311+G(2d,p) basis set [49][50][51][52]. The energy minimum character of the found stationary points was confirmed by the absence of imaginary frequencies calculated in a harmonic approximation. Grimme's empirical dispersion correction with Becke-Johnson damping (GD3BJ) [53] was applied for all B3LYP calculations. Solvation effects were taken into account using the IEFPCM dielectric continuum model [54] and dimethyl sulfoxide as a solvent.

Thermodynamic Calculations for IQ-1 and Trp-Ox
The calculations were carried out using the ORCA 5.0 software package [65]. The search for energy minima and the calculation of the thermodynamic characteristics of the Eand Z-isomers of the studied oximes were carried out for the "in" and "out" O-H bond orientations. The D3BJ dispersion correction [53] was taken into account in all the cases of the DFT method application. Model chemistries B3LYP[G] [45][46][47][48]53]/6-31+G(d) [49][50][51][52]/ IEFPCM(DMSO) [54] and RI-B2PLYP [66,67]/def2-SVP [61,62]/SMD(DMSO) [68] were used for the geometry optimization of Eand Z-isomers and transition states for E/Zisomerization. Gibbs free energies were calculated using the Quasi-RRHO approach [69] which provides the most correct account for low-frequency normal vibrations, especially important in calculations using the microsolvation model. Nudged Elastic Band with Climbing Image (NEB-CI) methodology [70] was used for finding the transition states of E/Z-isomerization reactions. Powder of oxime IQ-1 was prepared as previously reported [31]. A sample of 10 mg of IQ-1 was placed in a mixture of 400 µL of acetone and 20 µL of water; the mixture was refluxed on a hot plate until complete dissolution of the solid phase and allowed to cool to room temperature while remaining on a powered-off hot plate.

Tryptanthrin-6-oxime (Trp-Ox)
Powder of oxime Trp-Ox was prepared as previously reported [31]. A sample of 5 mg Trp-Ox was placed in a mixture of 600 µL of dimethylformamide and 420 µL of water; the mixture was refluxed on a hot plate until complete dissolution of the solid phase and then placed in a temperature-controlled oven, in which it was allowed to cool from 100 • C to 30 • C at a rate of 1.4 • C/h (during 50 h).

Tryptanthrin-6-oxime Pyridine Solvate (Trp-Ox·Py)
A sample of 18 mg Trp-Ox was dissolved in 200 µL of pyridine at 80 • C and allowed to naturally cool to room temperature. Single crystals of IQ-1 were obtained by recrystallization from the acetone-water mixture with slow cooling of the solution. The oxime IQ-1 crystallizes in a monoclinic crystal system, space group Ia ( Table 1). The asymmetric unit consists of one formula unit ( Figure 1) and the unit cell contains four formula units.  The molecules are linked into supramolecular chains via hydrogen bonds between  Based on the X-ray crystal structure, the configuration of the oxime C=N double bond was unambiguously determined as an E-isomer ( Crystallization of tryptanthrin-6-oxime from pyridine provided single crystals of the solvate Trp-Ox·Py. This compound crystallizes in a monoclinic crystal system, space group P21/n ( Table 1). The asymmetric unit is represented by one Trp-Ox molecule and The aromatic tetracyclic systems of IQ-1 are involved in π-π stacking interactions which join them into perpendicular chains. The distance between the planes of the cycles is 3.345 Å (Figure 2b).

Crystal Structure of Tryptanthrin-6-oxime-pyridine Solvate (Trp-Ox·Py)
Crystallization of tryptanthrin-6-oxime from pyridine provided single crystals of the solvate Trp-Ox·Py. This compound crystallizes in a monoclinic crystal system, space group P2 1 /n ( Table 1). The asymmetric unit is represented by one Trp-Ox molecule and one pyridine molecule ( Figure 3a); the unit cell contains four formula units. The solvate pyridine molecule is bound to the oxime group of Trp-Ox through a hydrogen bond, d(O2-H2···N4) = 2.633(2) Å, ∠(O2-H2···N4) = 176 • . The oxime and pyridine molecules are placed almost perpendicular to each other, and the angle between the planes of two molecules is 84.5 • ( Figure S1). The geometrical configuration of the oxime C=N double bond is Ewith C2-N3 and N3-O2 bond lengths of 1.293(2) and 1.376(2) Å, and a bond angle Figure 2. Crystal packing features in the structure of IQ-1: (a) Supramolecular chains of IQ-1 molecules formed by hydrogen bonds, view along the crystallographic axis a; (b) Supramolecular chains of IQ-1 molecules formed by π-π stacking interactions, view along the crystallographic axis c.

Crystal Structure of Tryptanthrin-6-oxime-pyridine Solvate (Trp-Ox·Py)
Crystallization of tryptanthrin-6-oxime from pyridine provided single crystals of the solvate Trp-Ox·Py. This compound crystallizes in a monoclinic crystal system, space group P21/n ( Table 1). The asymmetric unit is represented by one Trp-Ox molecule and one pyridine molecule ( Figure 3a); the unit cell contains four formula units. The solvate pyridine molecule is bound to the oxime group of Trp-Ox through a hydrogen bond, d(O2-H2···N4) = 2.633(2) Å, ∠(O2-H2···N4) = 176°. The oxime and pyridine molecules are placed almost perpendicular to each other, and the angle between the planes of two molecules is 84.5° ( Figure S1). The geometrical configuration of the oxime C=N double bond is E-with C2-N3 and N3-O2 bond lengths of 1.293(2) and 1.376(2) Å, and a bond angle  Several types of intermolecular interactions were identified in crystal packing of Trp-Ox·Py. Thus, pyridine molecules and the neighboring Trp-Ox molecules are involved in C-H···O short contacts via the hydrogen atoms of pyridine rings at position 4 and the carbonyl groups at position 12 of the tryptanthrin tetracycles, d(C18-H18···O1) = 3.347(2) Å, ∠(C18-H18···O1) = 167.7 • . These contacts join the molecules into supramolecular chains (Figure 3b). In turn, Trp-Ox molecules are involved in π-π stacking which packs the molecules into perpendicular chains (Figure 3c). The distance between the planes of tryptanthrin tetracycles is 3.326 Å.

Crystal Structure of Tryptanthrin-6-oxime (Trp-Ox)
In order to avoid the formation of solvate with pyridine, which can make one of the geometrical oxime isomers more favorable and thus influence the geometry of the C=N oxime bond, a series of other solvents (DMSO, DMF, acetone, acetonitrile, their mixtures with water) and crystallization conditions were evaluated to obtain single crystals suitable for X-ray structure determination. It was found that almost in all the tested crystallization conditions, Trp-Ox formed amorphous precipitates or microcrystalline conglomerates unsuitable for structural analysis. Only slow cooling of the Trp-Ox solution in DMF water afforded a microcrystalline product, the crystal structure of which was determined using synchrotron X-ray radiation.
Tryptanthrin-6-oxime (Trp-Ox) crystallizes in a monoclinic crystal system, space group P2 1 ( Table 1). The asymmetric unit consists of two formula units of the oxime. One of the Trp-Ox molecules in the asymmetric unit is disordered over two equally populated positions differing in the relative orientation of two crystallographically independent molecules ( Figure S2). Similarly to the structure of Trp-Ox·Py, the oxime C=N bond adopts E-configuration ( Figure 4). Therefore, formation of pyridine solvate described above is not the main factor determining the C=N bond configuration. The geometrical parameters of the oxime group in Trp-Ox are close to those in Trp-OX·Py, only the oxime C=N double bond is somewhat longer, d(C11-N3) = 1.319(8) Å, and the N3-O2 bond is slightly shorter (1.347 (7)  In order to avoid the formation of solvate with pyridine, which can make one of the geometrical oxime isomers more favorable and thus influence the geometry of the C=N oxime bond, a series of other solvents (DMSO, DMF, acetone, acetonitrile, their mixtures with water) and crystallization conditions were evaluated to obtain single crystals suitable for X-ray structure determination. It was found that almost in all the tested crystallization conditions, Trp-Ox formed amorphous precipitates or microcrystalline conglomerates unsuitable for structural analysis. Only slow cooling of the Trp-Ox solution in DMF water afforded a microcrystalline product, the crystal structure of which was determined using synchrotron X-ray radiation.
Tryptanthrin-6-oxime (Trp-Ox) crystallizes in a monoclinic crystal system, space group P21 (Table 1). The asymmetric unit consists of two formula units of the oxime. One of the Trp-Ox molecules in the asymmetric unit is disordered over two equally populated positions differing in the relative orientation of two crystallographically independent molecules ( Figure S2). Similarly to the structure of Trp-Ox·Py, the oxime C=N bond adopts Econfiguration ( Figure 4). Therefore, formation of pyridine solvate described above is not the main factor determining the C=N bond configuration. The geometrical parameters of the oxime group in Trp-Ox are close to those in Trp-OX·Py, only the oxime C=N double bond is somewhat longer, d(C11-N3) = 1.319(8) Å, and the N3-O2 bond is slightly shorter (1.347 (7)    Summarizing the results obtained from X-ray diffraction analysis, one can conclude that intermolecular hydrogen bonds involving the oxime group can form only for the E-isomer. For the Z-isomer, such bonding is sterically hindered and only intramolecular interactions are possible. Apparently, multiple intermolecular interactions involving E-oxime molecules are more energetically favorable than single intramolecular hydrogen bonds in the Z-isomer, and only the E-isomer crystallizes in the solid phase.

Evaluation of the Oxime Group Configuration in IQ-1 and Tpr-Ox in Solution by NMR Methods
As shown in the previous section, in the solid state, both IQ-1 and Trp-Ox oximes have the double bond E-configuration. At the same time, for biomedical applications, it is important to know the spatial structure of the drug in solution.
The solubility of IQ-1 and Trp-Ox in water (about 0.54 mg/L, 2 µM) is insufficient for their study by NMR in water; therefore, dimethyl sulfoxide-d 6 was used as a solvent.
The 1 H NMR spectrum of the oxime IQ-1 contains two signals at 13.36 and 13.26 ppm in the region characteristic of the hydroxyl protons of the oxime group ( Figure S3). The ratio of the integral signal intensities is 1.00:0.11. In the resonance region of aromatic protons (7.6-8.6 ppm), the signals of the two forms overlap, but the integral intensity of the signals corresponds to the ratio indicated above ( Figure S3). It is obvious that two sets of signals arise from the Eand Z-isomers of the oxime, with one of them noticeably predominating. In the 13 C NMR spectrum of compound IQ-1, there is a set of 15 signals related to the main isomer, but there are also weak signals that can be attributed to the second isomer ( Figure S4). Summarizing the results obtained from X-ray diffraction analysis, one can conclude that intermolecular hydrogen bonds involving the oxime group can form only for the Eisomer. For the Z-isomer, such bonding is sterically hindered and only intramolecular interactions are possible. Apparently, multiple intermolecular interactions involving E-oxime molecules are more energetically favorable than single intramolecular hydrogen bonds in the Z-isomer, and only the E-isomer crystallizes in the solid phase.

Evaluation of the Oxime Group Configuration in IQ-1 and Tpr-Ox in Solution by NMR Methods
As shown in the previous section, in the solid state, both IQ-1 and Trp-Ox oximes have the double bond E-configuration. At the same time, for biomedical applications, it is important to know the spatial structure of the drug in solution.
The solubility of IQ-1 and Trp-Ox in water (about 0.54 mg/L, 2 µM) is insufficient for their study by NMR in water; therefore, dimethyl sulfoxide-d6 was used as a solvent.
The 1 H NMR spectrum of the oxime IQ-1 contains two signals at 13.36 and 13.26 ppm in the region characteristic of the hydroxyl protons of the oxime group ( Figure S3). The ratio of the integral signal intensities is 1.00:0.11. In the resonance region of aromatic protons (7.6-8.6 ppm), the signals of the two forms overlap, but the integral intensity of the signals corresponds to the ratio indicated above ( Figure S3). It is obvious that two sets of signals arise from the E-and Z-isomers of the oxime, with one of them noticeably predominating. In the 13 C NMR spectrum of compound IQ-1, there is a set of 15 signals related to the main isomer, but there are also weak signals that can be a ributed to the second isomer ( Figure S4). In the 1 H and 13 C NMR spectra of Trp-Ox, only one set of signals is observed ( Figures S5 and S6); therefore, in a solution, this compound is almost completely represented by one isomer. The signal of the oxime proton is located at 13.63 ppm ( Figure S5).
The assignment of signals in the 1 H and 13 C NMR spectra of IQ-1 and Trp-Ox was performed using 2D NMR spectroscopy methods HSQC ( 1 H/ 13 C), HMBC ( 1 H/ 13 C), HMBC ( 1 H/ 15 N). The 2D NMR spectra plots are shown in Figures S7-S12 and the assignment of the signals is given in Table 2.
Nuclear Overhauser effect 2D NMR spectroscopy (NOESY) was used to identify IQ-1 and Trp-Ox isomers. Two possible correlations for the isomeric forms of both oximes are shown in Scheme 2. In the experimental NOESY spectra of both IQ-1 ( Figure S13) and Trp-Ox ( Figure S14), only one cross-peak involving the oxime proton is observed, and in both cases it corresponds to the interactions only possible for E-isomers (Scheme 2). NOESY interaction of OH protons with H8 (IQ-1) or H4 (Trp-Ox) in E-isomers should not take place because the distance between these atoms (~7 Å) surpasses the upper limit for NOE cross-peak observation (4-5 Å) [71]. In Z-isomers, the internuclear distance for OH and H4/H8 pair is in the suitable range (3.3-3.4 Å), but the corresponding cross-peaks are not detected in the NOESY spectra probably due to low concentration of this minor isomer. Table 2. Assignment of the signals in NMR 1 H and 13 C of IQ-1 and Trp-Ox in DMSO-d 6 .

IQ-1
one isomer. The signal of the oxime proton is located at 13.63 ppm ( Figure S5).
The assignment of signals in the 1 H and 13 C NMR spectra of IQ-1 and Trp-Ox was performed using 2D NMR spectroscopy methods HSQC ( 1 H/ 13 C), HMBC ( 1 H/ 13 C), HMBC ( 1 H/ 15 N). The 2D NMR spectra plots are shown in Figures S7-S12 and the assignment of the signals is given in Table 2. Trp-Ox one isomer. The signal of the oxime proton is located at 13.63 ppm ( Figure S5). The assignment of signals in the 1 H and 13 C NMR spectra of IQ-1 and Trp-Ox was performed using 2D NMR spectroscopy methods HSQC ( 1 H/ 13 C), HMBC ( 1 H/ 13 C), HMBC ( 1 H/ 15 N). The 2D NMR spectra plots are shown in Figures S7-S12 and the assignment of the signals is given in Table 2. To obtain additional confirmation of the oxime double bond geometry, DFT calculations of NMR chemical shifts for E-and Z-isomers of IQ-1 and Trp-Ox were carried out. Development of computational resources rendered quantum-chemical calculations of To obtain additional confirmation of the oxime double bond geometry, DFT calculations of NMR chemical shifts for Eand Z-isomers of IQ-1 and Trp-Ox were carried out. Development of computational resources rendered quantum-chemical calculations of NMR spectral parameters a powerful tool for establishing the structures of organic compounds, including complex natural products [72][73][74][75] and even stereoisomers [76,77].
Five computational protocols known for their good accuracy in predicting 1 H and 13 C chemical shifts for organic compounds were chosen for the calculations; these protocols are further referred to as Methods A-E. The model chemistries used together with their benchmark root mean square deviations (RSMD) for 1 H and 13 C chemical shifts are given in Table 3. Using the indicated model chemistries, isotropic magnetic shielding constants for Eand Z-isomers of IQ-1 and Trp-Ox were calculated, and the obtained values were converted to NMR chemical shifts in DMSO using the corresponding slope and intercept coefficients ( Table 3). The complete list of the calculated chemical shifts is given in Tables S1-S2 and the observed values of RMSD and linear correlation coefficients between the calculated and the experimental chemical shifts (r 2 ) are given in Table 4. The obtained prediction accuracy parameters indicate that Method A showed the best performance in terms of RMSD, while Method B provided the best linear correlation between the calculated and the experimental values, which is consistent with the benchmark performance of these computational protocols (Table 3). Representative examples of the correlation plots (obtained for 1 H chemical shifts using Method A) are shown in Figure 6, and the complete set of the plots is shown in Figures S15-S24.  Figure 6, and the complete set of the plots is shown in Figures S15-S24. As one can see from Figure 6 and Table 4, much be er calculation-experiment correlations were observed for E-isomers of IQ-1 and Trp-Ox for all computational models used. Therefore, a dominating isomer in solution of IQ-1 as well as the only detected isomer of Trp-Ox should be identified as E-isomers.   As one can see from Figure 6 and Table 4, much better calculation-experiment correlations were observed for E-isomers of IQ-1 and Trp-Ox for all computational models used. Therefore, a dominating isomer in solution of IQ-1 as well as the only detected isomer of Trp-Ox should be identified as E-isomers.

Calculation of Thermodynamic Parameters of the Isomeric IQ-1 and Trp-Ox
Having established the dominating forms of IQ-1 and Trp-Ox in the solid state and in solution, it was necessary to estimate the relative thermodynamic stability of Eand Z-isomers as well as evaluate the possibility of their interconversion. For this purpose, DFT calculations were carried out for Eand Z-isomers of IQ-1 and Trp-Ox and the transition state for E/Z-isomerization.
Geometry optimization of IQ-1 and Trp-Ox isomers in DMSO using the B3LYP functional, the 6-31+G(d) basis set, and the CPCM solvation model leads to the results presented in Table 5. For both isomers, two possible conformers designated as "in" and "out" with respect to the position of the oxime proton were considered in the calculations.
For both oximes, the E-isomer has a thermodynamically more stable "out" conformer, which is obviously explained by a steric repulsion between the hydrogen atoms of the oxime group and the heterocycle in the "in" conformers. Z-isomers of IQ-1 and Trp-Ox are more stable in the form of "in"-conformers, probably due to the formation of an intramolecular hydrogen bond OH···N. Table 5. Calculated enthalpies and Gibbs free energies for Eand Z-isomers and conformers of IQ-1 and Trp-Ox in DMSO. The search for transition states (TS) of oxime isomerization was carried out using the Nudged Elastic Band with Climbing Image (NEB-CI) methodology [70] followed by refinement of the TS structures. The analysis of normal vibrations shows the presence of a single imaginary frequency, which unambiguously indicates the attainment of the first-order saddle points. The imaginary frequency for both IQ-1_TS and Trp-Ox_TS corresponds to the in-plane inversion of the oxime nitrogen atom, i.e., the isomerization occurs without rotation around the C=N bond and is not accompanied by breaking of the π-bond. This is consistent with the experimental and theoretical results obtained for other compounds containing the C=N bond [78,79]. In this regard, the geometric structure of the found transition states is characterized by an approximately linear arrangement of the C=N-O atomic group. The calculated barriers for the E/Z-isomerization are about 200 kJ/mol (Table 5). Hence, at room temperature in DMSO, the interconversion of the isomers is extremely unlikely.

Structure B3LYP[G]/6-31+G(d)/CPCM(DMSO) RI-B2PLYP/def2-SVP/SMD(DMSO
According to the results presented in Table 5, the E-isomers of the studied oximes are thermodynamically more stable than the corresponding Z-isomers. Thus, the difference in the Gibbs free energies of two isomers (in the form of the most stable conformers) is 2.05 kJ/mol for IQ-1 and 0.92 kJ/mol for Trp-Ox. However, the use of the double hybrid B2PLYP functional that accounts for the electron correlation in the framework of the secondorder perturbation theory, along with the SMD solvation model, more accurate for the solvation energy estimation [68], leads to a greater thermodynamic stability of the Z-isomer for both oximes ( Table 5). The RI-B2PLYP calculations were carried out by refining the previously optimized structures. In this case, the transition states were optimized using the OptTS option of ORCA 5.0 program. The nature of the refined saddle points remained the same, i.e., the single imaginary frequency corresponded to a planar inversion of the oxime nitrogen atom through an approximately linear configuration of the C=N-O atomic group (Figure 7a,b).
The use of a more accurate approximation does not significantly change the calculated isomerization barriers compared to the data obtained with the B3LYP functional and the CPCM solvation model. However, the calculated stability of IQ-1 and Trp-Ox Z-isomers becomes higher than that of the corresponding E-isomers by 6-8 kJ/mol (Table 5). It should be noted that the additional thermochemistry calculations for the E (out) and Z (in) conformers of IQ-1 at other levels of theory using the SMD model led to similar results. Thus, the values of ∆G • 298 (Z/E) and barrier heights (in kJ/mol) are 3.1 and 222 (RI-B2PLYP/def2-TZVP), 3.5 and 217 (B3LYP/6-311++G(2d,2p)), 4.0 and 226 (RI-MP2/def2-SVPD). In all of these cases, the sign of ∆G • 298 (Z/E) does not agree with the results of NMR experiments (Section 3.2) indicating a higher stability of E-isomers of oximes IQ-1 and Trp-Ox. This discrepancy can be explained by a specific solvation of the oximes by a DMSO molecule which can form an intermolecular hydrogen bond with the oxime OH group in the "out" orientation. To evaluate the effect of such interactions on the relative stability of geometric isomers, DFT calculations of IQ-1 and Trp-Ox were performed using the microsolvation model with one explicit DMSO molecule.
in the Gibbs free energies of two isomers (in the form of the most stable conformers) is 2.05 kJ/mol for IQ-1 and 0.92 kJ/mol for Trp-Ox. However, the use of the double hybrid B2PLYP functional that accounts for the electron correlation in the framework of the second-order perturbation theory, along with the SMD solvation model, more accurate for the solvation energy estimation [68], leads to a greater thermodynamic stability of the Zisomer for both oximes ( Table 5). The RI-B2PLYP calculations were carried out by refining the previously optimized structures. In this case, the transition states were optimized using the OptTS option of ORCA 5.0 program. The nature of the refined saddle points remained the same, i.e., the single imaginary frequency corresponded to a planar inversion of the oxime nitrogen atom through an approximately linear configuration of the C=N-O atomic group (Figure 7a,b). A preliminary search for optimal orientations of one DMSO molecule relative to E (out) or Z (out) conformers of two studied oximes, as well as the transition states of Z/Eisomerization, was carried out using the PBEh-3c composite method [80] that provides high speed and acceptable accuracy of calculations. The effect of bulk solvent was taken into account within the CPCM model. Geometry optimizations of the "oxime·DMSO" or "TS·DMSO" microsolvates led to an almost linear arrangement of O-H···O atom group (Figure 7c,d) with an internuclear distance H···O near 1.5 Å indicative of a very strong binding between the highly polar DMSO molecule and the oxime group. The angles H···O=S are about 124-125 • . It should be noted that according to the Cambridge Structural Database, similar intermolecular interactions between oximes and DMSO solvate molecules are observed in 22 solid-state structures [81][82][83][84][85][86][87][88][89][90][91][92][93][94][95] with the mean O-H···O distance 1.816 Å and the shortest distances of 1.65-1.69 Å [88,89].
Based on the optimization results for several structures of each microsolvate differing in the angles of the DMSO molecule rotation about the O-H···O axis and the direction of pyramidal sulfur atom inversion, the structures with the lowest energy were selected for further optimization using a double hybrid functional at the RI-B2PLYP/def2-SVP/SMD(DMSO) level of theory. For the Eand Z-isomers of oximes IQ-1 and Trp-Ox (conformers E (out) or Z (out)), such structures are characterized by the DMSO methyl group orientation towards the oxime nitrogen atom, whereas in a transition state microsolvate, the sulfur atom of DMSO is directed towards the nitrogen atom of the heterocycle and to the nearest proton of the benzene ring (Figure 7c,d). The vibrational analysis of microsolvates of Eand Z-isomers confirms the attainment of energy minima on the potential energy surface, while the "TS·DMSO" microsolvates have a single imaginary frequency, which again corresponds to the planar inversion of the oxime nitrogen atom. Table 6 shows the Gibbs free energies and some geometric characteristics of the calculated microsolvates. In microsolvates, the distance r(OH···O) for transition states is somewhat shorter than that for the Eand Z-isomers of compounds IQ-1 and Trp-Ox, in accordance with the larger s-character of the oxime nitrogen atom at the top of the inversion barrier than in the minima on the PES. The corresponding increase in the nitrogen electronegativity can be responsible for the transition state stabilization by the DMSO molecule and for the decrease in the calculated isomerization barriers by about 15 kJ/mol compared with the results obtained using purely continuum solvation models (Table 5; Table 6).
Accounting for the specific solvation with dimethyl sulfoxide leads to higher calculated stabilities of E-isomers relative to Z-isomers (Table 6), in accordance with the experimental results. Thus, the calculated ∆G • 298 values for the isomerization process E out ·DMSO → Z out ·DMSO are approximately 11.0 and 14.1 kJ/mol for IQ-1 and Trp-Ox, respectively. The reason for a lower thermodynamic stability of the Z-isomer may be its transition from "in" to "out" conformation due to the formation of an intermolecular hydrogen bond with the DMSO molecule and the resulting break of the intramolecular hydrogen bond between the OH group and the nitrogen atom of heterocycle. This is confirmed, for example, by Gibbs energies of the solvate formation in processes (1) and (2) calculated in the RI-B2PLYP/def2-SVP/SMD(DMSO) approximation: Z in + DMSO → Z out ·DMSO; ∆G • 298 = −6.5 kJ/mol (IQ-1), −3.0 kJ/mol (Trp-Ox).
On the other hand, the E-isomer, along with the effect of specific solvation, remains stabilized due to the weak intramolecular hydrogen bond between the oxime oxygen atom and the nearest hydrogen atom in the benzene ring: the corresponding distances O···H are 2.42 and 2.47 Å in the microsolvates of IQ-1 and Trp-Ox.
We also performed DFT calculations of the microsolvates, each containing one explicit water molecule as a hydrogen bond acceptor attached to the oxime OH group of the transition states and of the Eand Z-isomer "out" conformers in an aqueous solution. The calculations made in the RI-B2PLYP/def2-SVP/SMD (Water) approximation led to the results very similar to those obtained for the DMSO microsolvates.  Figure 8). However, water solubilities of the investigated parent oximes are very low, and efforts were undertaken to synthesize more soluble derivatives like oximates of alkali metals [13,17] and compounds containing amine or carboxylic substituents in the benzene rings [19] of IQ-1 and Trp-Ox.
Zin + DMSO → Zout·DMSO; ΔG°298 = −6.5 kJ/mol (IQ-1), −3.0 kJ/mol (Trp-Ox). ( On the other hand, the E-isomer, along with the effect of specific solvation, remains stabilized due to the weak intramolecular hydrogen bond between the oxime oxygen atom and the nearest hydrogen atom in the benzene ring: the corresponding distances O···H are 2.42 and 2.47 Å in the microsolvates of IQ-1 and Trp-Ox. We also performed DFT calculations of the microsolvates, each containing one explicit water molecule as a hydrogen bond acceptor attached to the oxime OH group of the transition states and of the E-and Z-isomer "out" conformers in an aqueous solution. The calculations made in the RI-B2PLYP/def2-SVP/SMD (Water) approximation led to the results very similar to those obtained for the DMSO microsolvates. The calculated ΔG°298 values for the Eout·H2O → Zout·H2O interconversion equal 6.1 and 12.1 kJ/mol for IQ-1 and Trp-Ox, while the TS·H2O microsolvates display Gibbs energies 194.5 and 190.4 kJ/mol above the corresponding Eout·H2O species formed by IQ-1 and Trp-Ox, respectively ( Figure 8). However, water solubilities of the investigated parent oximes are very low, and efforts were undertaken to synthesize more soluble derivatives like oximates of alkali metals [13,17] and compounds containing amine or carboxylic substituents in the benzene rings [19] of IQ-1 and Trp-Ox.

Conclusions
In summary, using solid-state X-ray diffraction analysis, 1D and 2D NMR spectroscopy in DMSO solution and DFT calculations it was confirmed that the preferable stereochemistry of the oxime double bond in JNK3 inhibitors IQ-1 and Trp-Ox is E-configuration. In a solid state, E-isomers are stabilized by intermolecular hydrogen bonds involving the oxime OH group and nitrogen atoms of the heterocyclic rings. In a DMSO solution, according to DFT calculations, the oxime OH group forms a strong hydrogen with the oxygen atom of the solvent molecule, resulting in the stabilization of the E-isomer, while for the Z-isomer, formation of the indicated hydrogen bond is sterically hindered. The energy barrier between Zand E-isomers in DMSO is about 200 kJ/mol at 298 K, making their interconversion almost impossible at room temperature. Therefore, in biomedical studies involving IQ-1 and Trp-Ox inhibitors, they should be considered as E-isomers.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics15071802/s1, Figure S1: Fragment of the crystal structure of Trp-Ox·Py showing the relative orientation of pyridine and tryptanthrin rings; Figure S2: Crystal packing diagram of Trp-Ox showing two disordered positions of molecules (orange and green), view along axis b; Figure S3: 1 H NMR spectrum of IQ-1 in DMSO-d 6 at 500 MHz; Figure S4 Figure S15: Correlation plots between the experimental and the calculated NMR chemical shifts (Method A) for IQ-1; Figure S16: Correlation plots between the experimental and the calculated NMR chemical shifts (Method B) for IQ-1; Figure S17: Correlation plots between the experimental and the calculated NMR chemical shifts (Method C) for IQ-1; Figure S18: Correlation plots between the experimental and the calculated NMR chemical shifts (Method D) for IQ-1; Figure S19: Correlation plots between the experimental and the calculated NMR chemical shifts (Method E) for IQ-1; Figure S20: Correlation plots between the experimental and the calculated NMR chemical shifts (Method A) for Trp-Ox; Figure S21: Correlation plots between the experimental and the calculated NMR chemical shifts (Method B) for Trp-Ox; Figure S22: Correlation plots between the experimental and the calculated NMR chemical shifts (Method C) for Trp-Ox; Figure S23: Correlation plots between the experimental and the calculated NMR chemical shifts (Method D) for Trp-Ox; Figure S24: Correlation plots between the experimental and the calculated NMR chemical shifts (Method E) for Trp-Ox; Table S1: Calculated chemical shifts in NMR 1 H and 13 C spectra of Eand Z-isomers of IQ-1 and experimental chemical shifts in DMSO-d 6 ; Table S2: Calculated chemical shifts in NMR 1 H and 13 C spectra of Eand Z-isomers of Trp-Ox and experimental chemical shifts in DMSO-d 6