Synthesis, Crystal Structure, DFT, and Anticancer Activity of Some Imine-Type Compounds via Routine Schiff Base Reaction: An Example of Unexpected Cyclization to Oxazine Derivative

The synthesis, characterization, and anticancer properties of three imine-type compounds 1–3 and an unexpected oxazine derivative 4 are presented. The reaction of p-dimethylaminobenzaldehyde or m-nitrobenzaldehyde with hydroxylamine hydrochloride afforded the corresponding oximes 1–2 in good yields. Additionally, the treatment of benzil with 4-aminoantipyrine or o-aminophenol was investigated. Routinely, the Schiff base (4E)-4-(2-oxo-1,2-diphenylethylideneamino)-1,2-dihydro-1,5-dimethyl-2-phenylpyrazol-3-one 3 was obtained in the case of 4-aminoantipyrine. Unexpectedly, the reaction of benzil with o-aminophenol proceeded with cyclization to produce the 2,3-diphenyl-2H-benzo[b][1,4]oxazin-2-ol 4. The structures of compounds 3 and 4 were unambiguously determined by single crystal X-ray diffraction. Hirshfeld analysis of molecular packing revealed the importance of the O…H (11.1%), N…H (3.4%), C…H (29.4%), and C…C (1.6) interactions in the crystal stability of 3. In the case of 4, the O…H (8.8%), N…H (5.7%), and C…H (30.3%) interactions are the most important. DFT calculations predicted that both compounds have a polar nature, and 3 (3.4489 Debye) has higher polarity than 4 (2.1554 Debye). Different reactivity descriptors were calculated for both systems based on the HOMO and LUMO energies. The NMR chemical shifts were calculated and were found well correlated with the experimental data. HepG2 growth was suppressed by the four compounds more than MCF-7. The IC50 values of 1 against HepG2 and MCF-7 cell lines were the lowest, and it is considered the most promising candidate as an anticancer agent.


Introduction
The transformation of carbonyl functional groups into imine derivatives is a wellknown conversion in organic synthesis. Oximes are imine compounds having the general formula HO-N=CR 1 R 2 and are described as a significant class of compounds with diverse applications, not only for the protection of carbonyl functionality but also for the purification and/or characterization of compounds bearing a carbonyl group [1,2]. Oxime derivatives have several applications in medicine and can be employed as antidotes for nerve agents [3]. They might function as cholinesterase inhibitors as well. Drugs that suppress cholinesterase are used to treat dementia and Alzheimer's symptoms [4,5]. Oximes are employed as intermediates in the preparation of caprolactam, which is a precursor of nylon 6 [6]. The transformations to nitriles [7], nitro products [8,9], nitrones [10], amines [11], and aza heterocycles [12] are some of the synthetic uses of oximes. It has been reported that oximes are also useful reagents for selective activation [13] and widely employed as intermediates for the synthesis of amides by Beckmann rearrangement [14,15], fungicides, and Compounds 1 and 2 were characterized by FT-IR, 1 H, and 13 C{ 1 H} NMR spectroscopy and elemental analyses. The FT-IR spectra of p-dimethylaminobenzaldoxime 1 and m-nitrobenzaldoxime 2 showed the characteristic bands at wavenumbers 3246 and 3298 cm −1 (O−H) and 1606 and 1617 cm −1 (C=N), respectively, which confirm the formation of the aryl aldoximes 1 and 2. In the 1 H NMR spectra of 1 and 2, we detected the absence of the signal of the aldehyde functional group at ca. 10 ppm, and new signals at 7.98 and Scheme 1. Synthesis of aryl aldoximes 1 and 2.
Compounds 1 and 2 were characterized by FT-IR, 1 H, and 13 C{ 1 H} NMR spectroscopy and elemental analyses. The FT-IR spectra of p-dimethylaminobenzaldoxime 1 and m-nitrobenzaldoxime 2 showed the characteristic bands at wavenumbers 3246 and 3298 cm −1 (O−H) and 1606 and 1617 cm −1 (C=N), respectively, which confirm the formation of the aryl aldoximes 1 and 2. In the 1 H NMR spectra of 1 and 2, we detected the absence of the signal of the aldehyde functional group at ca. 10 ppm, and new signals at 7.98 and 8.36 ppm due to the imine protons CH=N of 1 and 2, respectively, were observed. Moreover, in the 13 C{ 1 H} NMR spectra of 1 and 2, the signal of the aldehyde at ca. 190 ppm was not detected, and new signals at 150.3 and 147.2 ppm due to the oxime carbons C=NOH of 1 and 2, respectively, were observed, confirming the formation of the aryl aldoximes 1 and 2 (see Section 3).

Synthesis and Characterization of Compounds 3 and 4
The corresponding (4E)-4-(2-oxo-1,2-diphenylethylideneamino)-1,2-dihydro-1,5-dimethyl-2-phenylpyrazol-3-one 3 and 2,3-diphenyl-2H-benzo[b] [1,4]oxazin-2-ol 4 were synthesized, in ca. 88% yield, by treatment of benzil with 4-aminoantipyrine or o-aminophenol in refluxing EtOH (Scheme 2). Compounds 3 and 4 were characterized by FT-IR, 1 H, and 13 C{ 1 H} NMR s copy, elemental analyses, and single crystal X-ray diffraction. The FT-IR spec compound 3 showed the characteristic band at 1663 cm −1 (C=N), which confi presence of the imine moiety. In the 1 H NMR spectrum of 3, the new signals at 2.94 ppm due to the methyl protons were observed, and the signals in the range 7.8 ppm due to the phenyl protons were also detected. Moreover, in the 13 C{ 1 H spectrum of 3, the new signals at 158.7 and 162.1 ppm confirm the presence of th (C(N)=O) and imino (C=N) moieties, respectively. The presence of the carbon signal at 194.5 ppm indicates that the condensation occurs to only one carbonyl benzil (see Section 3).
The FT-IR spectrum of compound 4 showed the characteristic bands at 32 1667 (C=N), 1589 (C=C), and 1092 (C-O), which confirms the formation of cycloa In the 1 H NMR spectrum of 4, a broad signal at 3.82 ppm due to the OH proton served, and the signals in the range of 6.5 to 7.9 ppm due to the phenyl protons w detected. Moreover, in the 13 C{ 1 H} NMR spectrum of 4, the new signal at 15 confirms the presence of the imino (C=N) moiety. The presence of the signal at 9 due to the hemiacetal carbon (OCO) confirms the nucleophilic attack of the h group of o-aminophenol on the carbonyl group of benzil, followed by a ring cl produce the cycloadduct 2,3-diphenyl-2H-benzo[b] [1,4]oxazin-2-ol 4 (see Section

X-ray Structure Description
In the structure of compound 3 (Figure 1 Compounds 3 and 4 were characterized by FT-IR, 1 H, and 13 C{ 1 H} NMR spectroscopy, elemental analyses, and single crystal X-ray diffraction. The FT-IR spectrum of compound 3 showed the characteristic band at 1663 cm −1 (C=N), which confirms the presence of the imine moiety. In the 1 H NMR spectrum of 3, the new signals at 2.32 and 2.94 ppm due to the methyl protons were observed, and the signals in the range of 7.1 to 7.8 ppm due to the phenyl protons were also detected. Moreover, in the 13 C{ 1 H} NMR spectrum of 3, the new signals at 158.7 and 162.1 ppm confirm the presence of the amido (C(N)=O) and imino (C=N) moieties, respectively. The presence of the carbonyl (C=O) signal at 194.5 ppm indicates that the condensation occurs to only one carbonyl group of benzil (see Section 3).
The FT-IR spectrum of compound 4 showed the characteristic bands at 3240 (OH), 1667 (C=N), 1589 (C=C), and 1092 (C-O), which confirms the formation of cycloadduct 4. In the 1 H NMR spectrum of 4, a broad signal at 3.82 ppm due to the OH proton was observed, and the signals in the range of 6.5 to 7.9 ppm due to the phenyl protons were also detected. Moreover, in the 13 C{ 1 H} NMR spectrum of 4, the new signal at 159.9 ppm confirms the presence of the imino (C=N) moiety. The presence of the signal at 94.7 ppm due to the hemiacetal carbon (OCO) confirms the nucleophilic attack of the hydroxyl group of o-aminophenol on the carbonyl group of benzil, followed by a ring closure to produce the cycloadduct 2,3-diphenyl-2H-benzo[b] [1,4]oxazin-2-ol 4 (see Section 3).

X-ray Structure Description
In the structure of compound 3 (Figure 1  result showed that the phenyl rings A, B, and C are out of the plane of pyrazole ring D. Analysis of bond lengths revealed that C16-N1 is 1.403(5) Å and C7=N1 is 1.283(5) Å, with the C-N and C=N bond lengths indicating single and double bonds, respectively, which is similar to Schiff base compounds [35,36]. The torsional angles of C16/N1/C7/C6 and C16/N1/C7/C8, which are connected to the phenyl rings A (  In the structure of compound 4 (Figure 2), the dihedral angles between the three phenyl rings identified by A (C1-C6), B (C8-C13), and C (C15-C20) are A/B = 85.08(10) • , A/C = 53.86(12) • , and B/C = 64.86(12) • , respectively. This result shows that the phenyl rings B and C are out of the plane of phenyl ring A. Analysis of bond lengths revealed that C1-N1 is 1.422(3) Å and C14=N1 is 1.280(2) Å, with the C-N and C=N bond lengths indicating single and double bonds, respectively, which is similar to Schiff base compounds [35,36]. In the molecular structure, the crystal packing is stabilized by intramolecular C-H...O, intermolecular O-H...N hydrogen bonds, and C-H. . . π short interactions.   Table 1. The majority of these short contacts appeared as sharp spikes in the fingerprint plots ( Figure 4), which sheds light on the importance of these intermolecular contacts in the molecular packing of 3. lecular packing. The Hirshfeld surfaces are shown in Figure 3. All red spots in the dnorm map represent regions where there are short distance contacts, and those are considered significant for molecular packing. The most important contacts are O…H, N…H, C…H, and C…C interactions. Their percentages are 11.1, 3.4, 29.4, and 1.6%, respectively. The contact distances of these short contacts are depicted in Table 1. The majority of these short contacts appeared as sharp spikes in the fingerprint plots ( Figure 4), which sheds light on the importance of these intermolecular contacts in the molecular packing of 3.     As clearly seen from Figure 5, there are many different contacts controlling the molecular packing of this compound. The most abundant interaction in the crystal structure of 3 is the H…H contacts, which contribute to more than half the intermolecular interactions. The H…H contact percentage is 53.1%. Similarly, the contribution of this interaction in the molecular packing is 51.9% for compound 4.    As clearly seen from Figure 5, there are many different contacts controlling the mo lecular packing of this compound. The most abundant interaction in the crystal structur of 3 is the H…H contacts, which contribute to more than half the intermolecular interac tions. The H…H contact percentage is 53.1%. Similarly, the contribution of this interac tion in the molecular packing is 51.9% for compound 4.   Figure 7). Although there is a higher percentage of the C. . . C contacts in this compound (3.6%) compared to 3, there are no significant C. . . C interactions detected in 4, where all appeared as the blue region in the d norm . Additionally, the importance of the π-π stacking interactions in the former is further revealed from the shape index with red/blue triangles and curvedness, with a flat green area where these features are totally absent in the latter. The only contacts that appeared as red spots in d norm and sharp spikes in the fingerprint plots are the O. . . H, N. . . H, and C. . . H interactions ( Figure 6). The interaction distances are summarized in Table 1. Similarly, the Hirshfeld surfaces of 4 shown in Figure 6 revealed the importance of the O…H, N…H and C…H interactions in the molecular packing of this compound. These interactions contributed by 8.8, 5.7, and 30.0%, respectively, from the whole contacts detected in 4 ( Figure 7). Although there is a higher percentage of the C…C contacts in this compound (3.6%) compared to 3, there are no significant C…C interactions detected in 4, where all appeared as the blue region in the dnorm. Additionally, the importance of the π-π stacking interactions in the former is further revealed from the shape index with red/blue triangles and curvedness, with a flat green area where these features are totally absent in the latter. The only contacts that appeared as red spots in dnorm and sharp spikes in the fingerprint plots are the O…H, N…H, and C…H interactions ( Figure  6). The interaction distances are summarized in Table 1. A clear difference between the two compounds is the shorter O…H and N…H contacts in 4 compared to 3. In the former, the hydrogen-acceptor distance of the C-H…O interaction is 2.284 Å (O2…H20), while the corresponding values range from 2.420 Å (O1…H2) to 2.557 Å (O1…H25B) in the latter. In addition, the N1…H1 (2.028 Å) contact in 4 occurred at a shorter distance than the N1…H23 (2.482 Å) in 3. In contrast, the Molecules 2023, 28, x FOR PEER REVIEW 9 of 1 C6…H3 contact (2.718 Å) in 4 is longer than those found in 3. In the latter, the shorte C…H interaction is C7…H23 (2.608 Å).

DFT Studies
The B3LYP method is one of the most common and accurate methods for predictin the molecular structure and electronic and spectroscopic properties of molecular system [37][38][39]. The structures of 3 and 4 were optimized using the B3LYP/6-31G(d,p) method The calculated and experimental structures along with their overlay are shown in Figure  8 and 9, respectively, while the calculated geometric parameters are listed in Tables S and S2 (Supplementary Materials). There are some deviations between the calculated an experimental data that could be attributed to the well-known fact that the experimenta structure belongs to a molecule in the crystal that is affected by the intermolecular inte actions with the neighboring molecules, while the calculated structure is for a single iso lated molecule in the gas phase.

DFT Studies
The B3LYP method is one of the most common and accurate methods for predicting the molecular structure and electronic and spectroscopic properties of molecular systems [37][38][39]. The structures of 3 and 4 were optimized using the B3LYP/6-31G(d,p) method. The calculated and experimental structures along with their overlay are shown in Figures 8 and 9, respectively, while the calculated geometric parameters are listed in Tables S1 and S2 (Supplementary Materials). There are some deviations between the calculated and experimental data that could be attributed to the well-known fact that the experimental structure belongs to a molecule in the crystal that is affected by the intermolecular interactions with the neighboring molecules, while the calculated structure is for a single isolated molecule in the gas phase.
For 3, the molecular structure comprised different charged regions, as shown from the natural charge analysis and the map of electron density over electrostatic potential shown in Figure 8. The negatively charged regions are related to the oxygen atoms of the carbonyl groups. As a result, the molecule of 3 has a net dipole moment of 3.4489 Debye. Based on the natural charge analysis, the two oxygen atoms of the carbonyl groups have high negative charges, while the hydrogen atoms are the most positive atomic sites along with the carbonyl carbon. These regions are mapped with red and blue colors for the most negative and most positive regions, respectively. Another electronic feature that is related to molecular reactivity is the frontier molecular orbitals HOMO and LUMO. Their energies are calculated to be −5.4399 and −1.221 eV, respectively, leading to ionization potential (I = −E HOMO ), electron affinity (A = −E LUMO ), chemical potential (µ = −(I + A)/2), hardness (η = (I − A)/2), and electrophilicity index (ω = µ 2 /2η) [40][41][42][43][44][45] of 5.4399, 1.6221, −3.5310, 3.8178, and 1.6329 eV, respectively. The distribution of the HOMO and LUMO is delocalized over the conjugated π-system, indicating a π-π intramolecular charge transfer due to this lowest energy excitation, where the HOMO to LUMO excitation energy is 3.8178 eV.  For 3, the molecular structure comprised different charged regions, as shown from the natural charge analysis and the map of electron density over electrostatic potential shown in Figure 8. The negatively charged regions are related to the oxygen atoms of the  For 3, the molecular structure comprised different charged regions, as shown from the natural charge analysis and the map of electron density over electrostatic potential shown in Figure 8. The negatively charged regions are related to the oxygen atoms of the Similarly, the optimized structure and the different electronic parameters of 4 are presented in Figure 9. In addition, the most negative atomic sites are the O-atoms of hydroxyl and cyclic ether groups, respectively. Their natural charges are calculated to be −0.7505 and −0.5424 e, respectively. The carbon atoms bonded to these O-sites are the most positive. Their natural charges are 0.5517 and 0.3119, respectively. Moreover, the hydroxyl proton possesses a high positive charge of 0.4900. The MEP map revealed the high electron density related to the oxygen atoms and the high positive charge density at the OH proton. The calculated dipole moment is 2.1554 Debye, indicating less polarity compared to 3. In addition, the HOMO and LUMO energies are calculated to be −5.8442 and −1.5252 eV, respectively. The energy separation between the HOMO and LUMO levels is 4.3190 eV. Moreover, the ionization potential, electron affinity, chemical potential, hardness, and electrophilicity index are calculated to be 5.8442, 1.5252, −3.6847, 4.3190, and 1.5718 eV, respectively.

Calculated NMR Spectra
The calculated 1 H and 13

General Methods
Reagents and solvents were obtained from commercial sources and used as received. 1 H and 13 C{ 1 H} NMR spectra were recorded on a Bruker Avance III 400 (9.4 T, 400.13 MHz for 1 H, 100.62 MHz for 13 C{ 1 H}) spectrometer with a 5-mm BBFO probe at 298 K. Chemical shifts (δ in ppm) are given relative to internal solvent CDCl 3 7.25 for 1 H and 77.7 for 13 C{ 1 H}. Fourier transform infrared spectroscopy (FT-IR) spectra were recorded on an Alpha Bruker FT-IR spectrophotometer, where samples were prepared with KBr pellets, and the wavenumbers are in cm −1 .

Synthesis of p-Dimethylaminobenzaldoxime 1 and m-Nitrobenzaldoxime 2
To a solution of sodium carbonate (63.38 mg, 0.598 mmol) in MeOH (10 mL), hydroxylamine hydrochloride (83.18 mg, 1.196 mmol) was added. The mixture was stirred at room temperature for 5 min. p-dimethylaminobenzaldehyde (162.3 mg, 1.088 mmol) or m-nitrobenzaldehyde (164.4 mg, 1.088 mmol) was then added, and the reaction mixture was stirred at room temperature for 12 h. The precipitate formed was then filtered off, and the filtrate was evaporated in vacuo to produce the aryl aldoximes 1 and 2, respectively.  13

X-ray Structure Determinations
The X-ray diffraction data were collected on a Bruker D8 QUEST diffractometer using MoKα radiation ( Table 3). The Apex3 [46] program package was used for cell refinements and data reductions. Multi-scan absorption correction (SADABS) [47] was applied to the intensities before the structure solution. The structures of compounds 3 and 4 were solved by the intrinsic phasing method using the SHELXT [48] software. Structural refinement was carried out using SHELXL-2017 [48].  [52,53]. Natural charges were calculated using the NBO 3.1 program as implemented in the Gaussian 09W package [54]. Structures were optimized in solutions of the compounds in CHCl 3 using the self-consistent reaction field (SCRF) method [55,56]. Then, the NMR spectra of the studied compounds were computed using the GIAO method [57].

Biological Experiments
Assessment of cytotoxicity of compounds 1-4 against the HepG2 and MCF-7 cell lines: The Tissue Culture Unit, Department of Biochemistry, Faculty of Science, King Abdulaziz University, provided HepG2 and MCF-7 human cell lines. In complete media, Dulbecco's Modified Eagle's Medium (DMEM), which contains 10% fetal bovine serum and 1% antibiotic, was used to cultivate the human cell lines at 37 • C and 95% humidity in a 5% CO 2 incubator for 24-48 h. After 70-90% of the confluent cells were completed, the cells were gathered, then 4 mL of 0.25% trypsin with EDTA were incubated in a CO 2 incubator for 5 min. The addition of 5 mL of the complete medium was made to stop the activity of the trypsin process. Unattached cell-containing medium was centrifuged; the pellets were then twice washed in sterile phosphate buffer saline (PBS). After 20 µL of this cell-containing media were stained with 20 µL of 0.4% trypan blue, the numbers of cells were counted using a hemocytometer in the four major squares. The following equation was used to compute the number of cells per milliliter : A 96-well microplate was filled with 0.1 mL of 5000 cells suspended in complete media, and the plate was then cultured in the incubator for 24 h. Anticancer effects of compounds 1-4 were assessed. The cisplatin was used as a positive control against the two human cell lines. Each compound was added to the media at different concentrations, ranging from 6.25 to 100 µg/mL and 3.125 to 50 µg/mL for cisplatin, once 70% of the cells in each well had reached confluence. The 96-well plates were placed in the incubator for 48 h before the media in each well was changed to 100 µL of free media with 0.5 mg of MTT/mL for 4 h. Each well received 100 µL of dimethylsulfoxide (DMSO), which was added and kept at room temperature for 15 min before being detected by a microplate reader at 595 nm (Bio-RAD microplate reader, Japan). The 50% inhibitory concentrations (ICs 50 ) of 1, 2, 3, and 4 treated with two attached cell lines were computed using the GraphPad Prism program version 9, utilizing the curve of cell viability vs. various concentrations [58,59].