Synthesis, Characterization, and Biological Evaluation of Nano Schiff Base Derivatives and Their Conversion into Oxazepine and Imidazoline Derivatives: A Comprehensive Study

INTRODUCTION 
High electron-donating and strong coordination characteristics have made heterocyclic compounds popular, and their numerous uses continue to draw attention [1]. Many methods have been found to prepare nano Schiff-bases. Schiff-bases have many biological properties and are employed in industry [2]. They are widely used as organic synthesis intermediates, catalysts, dyes, and polymer stabilizers [3]. Green chemistry’s clean energy cycloaddition operations produce oxazepines with less byproducts and shorter reaction times [4]. Oxazepine derivatives are stable and biologically active due to the seven-membered ring’s double bond alternation [5]. This has inspired researchers to develop novel synthesis methods for seven-membered ring compounds. Oxazepine compounds are synthesized by condensing nano Schiff-bases with phthalic anhydride. In recent years, interest in tetrazole chemistry has rapidly increased due to its applications. The first discovery of tetrazole was made by the Swedish chemist Bladin A.J. in 1896, and then, research interest in this heterocyclic system has grown, leading to its chemical and biological development [6]. Tetrazole ring systems have been extensively studied for a variety of applications in organic chemistry, coordination chemistry, photography, agriculture, explosives, and particularly in medicine, for designing numerous bioequivalents of carboxylic acids, such as 5-substituted tetrazoles and 2,5-disubstituted tetrazoles, which serve as bioequivalents of the cis-amide bond in peptides [7]. These bioequivalents play a similar role in various biological activities by enhancing wide metabolic stability, especially for their role in medicine [8]. Beta-lactams have garnered significant attention due to their antibacterial properties, in which those found in penicillin and cephalosporin, making them important classes of antibiotics in both human and veterinary medicine [9]. They represent over 85% of the global antibiotic market [10]. Additionally, they are used in other clinical applications, such as clavulanic acid as beta-lactamase inhibitors and ezetimibe as cholesterol absorption inhibitors. Beta-lactams also demonstrate other biological activities, including anti-cancer, anti-diabetic, anti-tuberculosis, and anti-inflammatory effects [11]. 

MATERIALS AND METHODS
Reagents and instruments 
The preparation of various derivatives involved the use of the following reagents and instruments. Aldehyde and ketone derivatives, amine derivatives, absolute ethanol, glacial acetic acid, dry benzene, succinic anhydride, maleic anhydride, phthalic anhydride, tetrahydrofuran (THF), amino acids (alanine, glycine), 1,4-dioxane, thioglycolic acid, anhydrous zinc chloride, sodium azide, triethylamine, chloroacetyl chloride, and anthracitic acid were used as chemical reagents. The instruments and equipment included round-bottom flasks equipped with magnetic stirrers, conical flasks, reflux setups, thin-layer chromatography (TLC) plates, and appropriate mobile phases (such as benzene in a 4:1 ratio). The reaction mixtures were typically heated, refluxed, and monitored for progress using TLC. The resulting precipitates were filtered, dried, recrystallized from absolute ethanol, and their physical properties were determined.

Synthesis procedure 
General method for preparing derivative (1)
Equimolar amounts (0.005 mol) of aldehyde and ketone derivatives were mixed with 0.005 mol of amine derivatives in a round-bottom flask equipped with a magnetic stirrer containing 30 mL of absolute ethanol. Three drops of glacial acetic acid were added to the mixture, which was then refluxed for 6 h. The reaction mixture was cooled and left to stand for 24 h. The precipitate was filtered, washed, dried, and recrystallized from absolute ethanol. The progress of the reaction was monitored using TLC with an appropriate mobile phase [1]. 

General method for preparing derivatives (2, 3, 4)
Nano Schiff-base derivative (1) (0.00167 mol) was dissolved in 30 mL of dry benzene. Then, 0.00167 mol of succinic anhydride, maleic anhydride, or phthalic anhydride was added. The mixture was heated at 60°C for 39 h. The reaction progress was monitored using TLC with a mobile phase of benzene (4:1). The precipitate was filtered, dried, recrystallized from absolute ethanol, weighed, and its physical properties were determined [12].

Preparation of oxazepine derivative (2)
This derivative was prepared using the general method with 0.179 g of derivative (1) and 0.24 g of phthalic anhydride under reaction time of 39 h. A yellow precipitate was obtained [13]. 

Preparation of oxazepine derivative (3)
This derivative was prepared using the general method with 0.5 g of derivative (1) and 0.163 g of maleic anhydride under reaction time of 39 h. An orange precipitate was obtained.

Preparation of oxazepine derivative (4)
This derivative was prepared using the general method with 0.5 g of derivative (1) and 0.190 g of succinic anhydride under reaction time of 39 h. A yellow precipitate was obtained.

Preparation of imidazoline derivatives (5)
Derivative (1) (0.926 g, 0.0031 mol) was dissolved in 30 mL of THF, along with amino acids (alanine, glycine) in a conical flask equipped with a magnetic stirrer. The mixture was refluxed for 42-48 h, and the reaction progress was monitored using TLC with a mobile phase of benzene (4:1). The precipitate was allowed to stand for a day, recrystallized from absolute ethanol, dried, and weighed (Fig. 1) [13]. 

RESULTS AND DISCUSSION
FT-IR, 1H-NMR, and 13C-NMR results
The nano Schiff-base derivative (1) was characterized by FT-IR spectroscopy, as illustrated in Fig. 2a. The NH2 stretching band disappeared, and a new band at 1641 cm-1, corresponding to the azomethine group (C=N) stretching vibration, was observed. Additionally, a signal at 3369 cm-1 attributed to the phenolic (O-H) stretch and a signal at 1679 cm-1 corresponding to the C=O stretch were detected. The characterization of compound (1) was further confirmed using 1H-NMR spectroscopy with (DMSO-d6) as the solvent. The spectrum, depicted in Fig. 2b, showed a singlet at 9.8 ppm corresponding to the hydroxyl group proton, a triplet at 1.9 ppm for the six protons of the CH3 group, a singlet at 3.8 ppm for the three protons of the OCH3 group, and multiple signals in the range of 6.6-7.2 ppm corresponding to the aromatic ring protons. A signal at 2.5 ppm was attributed to the solvent, (DMSO-d6). Additionally, compound (1) was characterized using 13C-NMR spectroscopy, as illustrated in Fig. 2c, which revealed a distinct signal. A signal at 180 ppm was assigned to C1, while a signal at 164 ppm corresponded to C11. The signal at 55.98 ppm was attributed to C15, and the signal at 14.70 ppm was assigned to C17. Additionally, a signal at 56.12 ppm was attributed to C8. Signals in the range of 111-166 ppm were observed for the aromatic rings [14, 15]. 
FT-IR spectrum of derivative (2) is depicted in Fig. 3a. The spectrum revealed a band at 3369 cm⁻1 corresponding to the phenolic O-H stretch. The disappearance of the C=N stretch band for the azomethine group and the appearance of a band at 1679 cm⁻1 for the C=O stretch of the oxazepine ring were observed. Additionally, a band at 1642 cm⁻1 was assigned to the C=O stretch. Compound (2) was further characterized by 1H-NMR spectroscopy using DMSO-d₆ as the solvent. The spectrum in Fig. 3b displayed a singlet at 9.7 ppm attributed to the hydroxyl group proton, a triplet at 1.3 ppm corresponding to the six protons of the CH3 group, and a singlet at 3.87 ppm for the three protons of the OCH3 group. Multiplets in the range of 7.3-6.5 ppm were assigned to the aromatic ring protons. An additional signal at 2.5 ppm was due to the solvent (DMSO-d₆). 13C-NMR spectroscopy was also employed to characterize compound (2). The spectrum in Fig. 3c exhibited a signal at 195 ppm corresponding to C1, signals in the range of 174-191 ppm for C9 and C12, and a signal at 166 ppm for C19. Signals at 14.56 and 14.28 ppm were assigned to C23 and C25, respectively. A signal at 55 ppm corresponded to C24, while signals in the range of 112-165 ppm were attributed to the aromatic rings [16]. 
The characterization of derivative (3) was carried out using FT-IR spectroscopy, as illustrated in Fig. 4a. The spectrum indicated the disappearance of the C=N stretch band of the azomethine group and the appearance of a band at 3307 cm⁻1 corresponding to the phenolic O-H stretch. A band at 1708.8 cm⁻1 was assigned to the C=O stretch, and another band at 1673 cm⁻1 was attributed to the C=O stretch vibration. Further analysis was conducted using 1H-NMR spectroscopy with DMSO-d₆ as the solvent. The ¹H-NMR spectrum in Fig. 4b exhibited a singlet at 10 ppm attributed to the hydroxyl group proton, a triplet at 1.3 ppm corresponding to the six protons of the CH3 group, and a singlet at 3.87 ppm for the three protons of the OCH3 group. Multiplets in the range of 6.2-7.08 ppm were assigned to the aromatic ring protons. An additional signal at 2.5 ppm was due to the solvent (DMSO-d₆). 13C-NMR spectroscopy was also utilized to characterize compound (3). The 13C-NMR spectrum in Fig. 4c showed a signal at 191.9 ppm corresponding to C1, signals at 188 ppm and 182 ppm for C9 and C12, respectively, and a signal at 170 ppm for C14. A signal at 14.82 ppm was assigned to C23, while another at 14.66 ppm was attributed to C21. A signal at 56 ppm corresponded to C20, with additional signals in the range of 112-165 ppm are attributed to the aromatic rings [17, 18]. 
FT-IR spectrum of derivative (4) is depicted in Fig. 5a. The spectrum indicated the disappearance of the C=N stretch band of the azomethine group and the appearance of a band at 3368 cm⁻1 corresponding to the phenolic O-H stretch. A band at 1684 cm⁻1 is attributed to the C=O stretch. Compound (4) was further characterized by 1H-NMR spectroscopy using DMSO-d₆ as solvent. The spectrum in Fig. 5b exhibited a singlet at 10.33 ppm attributed to the hydroxyl group proton, a triplet at 2.6 ppm corresponding to the protons of the CH3 group, and a singlet at 3.87 ppm for the three protons of the OCH3 group. Multiplets in the range of 6.5-7.28 ppm were assigned to the aromatic ring protons. An additional signal at 2.5 ppm was due to the solvent (DMSO-d₆). 13C-NMR spectroscopy was also utilized to characterize compound 4. The spectrum in Fig. 5c showed a signal at 191.9 ppm corresponding to C1, signals at 174.1 ppm and 174.2 ppm for C9 and C12, respectively, and a signal at 165 ppm for C8. A signal at 14.82 ppm was assigned to C21, while another at 14.66 ppm was also attributed to C21. A signal at 56 ppm corresponded to C20, with additional signals in the range of 112-171 ppm attributed to the aromatic rings [19]. 
FT-IR spectrum of derivative (5) is depicted in Fig. 6a. The spectrum indicated the disappearance of the C=N stretch band of the azomethine group and the appearance of a band at 3369 cm⁻¹ corresponding to the phenolic O-H stretch [20-22]. Additionally, a band at 3083 cm⁻1 was observed for the aromatic C-H stretch, a band at 2935 cm⁻1 for the aliphatic C-H stretch, and a band at 1679 cm⁻1 for the C=O stretch. Compound (5) was further characterized by 1H-NMR spectroscopy using DMSO-d₆ as the solvent. The spectrum in Fig. 6b exhibited a singlet at 9.79 ppm attributed to the hydroxyl group proton, a triplet at 1.26 ppm corresponding to the six protons of the CH3 group, and a singlet at 3.87 ppm for the three protons of the OCH3 group. Multiples in the range of 6.6-7.37 ppm were assigned to the aromatic ring protons. An additional signal at 2.5 ppm was due to the solvent (DMSO-d₆). 13C-NMR spectroscopy was also utilized to characterize compound (5). The spectrum in Fig. 6c showed a signal at 191 ppm for C1, a signal at 185 ppm for C10, and a signal at 174 ppm for C14. Additional signals in the range of 111-166 ppm were attributed to the aromatic rings [23]. 

XRD study
The XRD patterns provided the structural analysis of five synthesized organic compounds, labeled as derivatives 1-5 (Fig. 7). These patterns displayed the intensity of diffracted X-rays as a function of the diffraction angle (2 Theta), revealing crucial information about the crystalline structure and phase purity of the compounds [24-28]. Derivatives (1) and (2) exhibit sharp and intense peaks, indicating high crystallinity and phase purity. Derivative (3) shows sharp peaks with slightly lower intensity, suggesting minor differences in crystal quality. Derivative (4) has multiple peaks with reduced intensity, indicating a semi-crystalline structure. Derivative (5) features numerous low-intensity peaks, signifying reduced crystallinity and increased amorphous content. Recent research extensively uses XRD analysis to characterize synthesized organic compounds, confirming successful synthesis and purity by comparing experimental patterns with standard references. Sharp peaks correlate with high crystallinity, associated with better stability and performance. Each derivative is uniquely identified by its structural formula. This synthesis and structural characterization align with global research practices, where XRD is fundamental in elucidating the structural properties of newly synthesized compounds [29-31]. 

FESEM analysis
The FESEM images in Fig. 8 provide detailed insights into the surface morphology and particle size distribution of the five synthesized nano organic compounds. Derivative (1) shows a relatively uniform surface with well-defined particles ranging from approximately 50 to 321 nm, suggesting a controlled synthesis process with some variation in particle size. Derivative (2) has a slightly rougher surface with irregularly shaped particles ranging from about 44 nm to 173 nm, indicating slight variations in synthesis parameters or chemical composition. Derivative (3) displays a broad size distribution of particles from 54 nm to 170 nm, suggesting a less controlled synthesis environment and different crystalline phases. Derivative (4) exhibits a more aggregated structure with interconnected particles forming a porous network, with sizes ranging from approximately 53 nm to 70 nm, indicating a rough and textured surface potentially beneficial for catalysis or adsorption. Derivative (5) has a highly irregular and rough surface morphology with particle sizes ranging from 49 nm to 97 nm, indicating the least uniformity among the samples and potential challenges in the synthesis process. Overall, the FESEM analysis provides critical information about the surface characteristics and particle size of the synthesized compounds, essential for understanding their potential applications and performance in various fields. The observed variations in particle size and morphology reflect the different synthesis conditions and potential applications of each derivative [32-36]. 

Anticancer activity
The effects of derivative (5) on the growth of the MCF-7 cell line and the WRL-68 cells were studied, showing a similar trend with the lowest growth inhibition at 10 µg/mL and the highest at 500 µg/mL for both cell lines [37, 38]. The WRL-68 cells were used for comparison and potential medical application. The percentage of live cells remaining after treatment with derivative (5) ranged from 7.14285% to 90.53287% for the MCF-7 cell line, and from 67.02271% to 98.20537% for the WRL-68 cell line. The highest inhibition rate for the MCF-7 cell line was 92.85714% at 500 µg/mL, while for the WRL-68 cell line, it was 32.97728% at the same concentration. The IC50 for derivative (5) with the MCF-7 cell line was 56 µg/mL, and for the WRL-68 cells, it was 116.5 µg/mL, as summarized in Table 1 and Fig. 9 [39-41]. 

CONCLUSION 
This study used FT-IR, 1H-NMR, and 13C-NMR to characterize chemical derivatives. These compounds have unique chemical and physical properties, making them promising for industrial and medical uses. These derivatives’ surface morphology and particle size distribution were examined using FESEM. Shapes and sizes varied due to synthesis circumstances. This information is essential for understanding these compounds’ catalytic and adsorption applications. Compound (5) was tested for anticancer activities in MCF-7 breast cancer and WRL-68 normal liver cells. Results demonstrated substantial anticancer action with reduced influence on normal cells, suggesting therapeutic potential. The purity and crystallinity of these derivatives were examined using XRD patterns. This clarifies the link between crystalline structure and compound physical and chemical properties. This study shows that these compounds have great industrial and medical potential. It emphasizes the need for ongoing R&D to improve characteristics and find new uses.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.