Synthesis of Benzoxazole Derivatives via Zn(OAc)2·2H2O Catalyzed Solvent-Free Reaction and Evaluation of Their Antitumor and Antibacterial Activities

Benzoxazole derivatives are a prominent group of heterocyclic compounds known for their wide-ranging pharmacological activities, including notable anticancer and antimicrobial effects¹. Their significance in biomedical research is largely attributed to the presence of the benzoxazole core in many biologically active compounds and pharmaceutical drugs². The structural flexibility of the benzoxazole skeleton allows for various substitutions, enabling researchers to tailor the physicochemical properties and pharmacological profiles of these compounds, which makes them appealing candidates in medicinal chemistry^3-6. As a result, there has been extensive investigation into developing effective and sustainable synthetic methods for these derivatives. Traditionally, the synthesis of benzoxazoles has relied on the condensation of 2-aminophenols with various carbonyl compounds. However, many of these traditional methods are hampered by harsh reaction conditions, lengthy reaction times, and significant waste generation. Recent advancements have concentrated on environmentally friendly approaches that address these challenges by focusing on atom efficiency, reduced energy requirements, and the avoidance of toxic reagents.^7-10 The crucial role of heterocyclic compounds, especially benzoxazoles, in drug discovery, along with their broad bioactivity spectrum, underscores the need to develop more sustainable synthetic approaches in synthetic organic chemistry. Specifically, benzoxazole derivatives have gained attention as isosteres of natural nucleotides and are frequently incorporated into synthetic analogs with substantial chemotherapeutic potential, showcasing anticancer, antimalarial, antileishmanial, antiviral, and antibacterial activities.^11-13 The  use of the benzoxazole scaffold in drug discovery highlights its importance, promoting ongoing exploration of various synthetic strategies to overcome issues such as low yields, stringent conditions, and costly catalysts. This ongoing research seeks to create novel benzoxazole derivatives with enhanced biological activities through environmentally friendly methods, such as solvent-free catalysis, which adheres to green chemistry principles by minimizing solvent usage and waste. Notably, benzoxazole derivatives exhibit considerable promise as effective antimicrobial agents and potent antitumor compounds, drawing significant attention in the field of medicinal chemistry. Despite their recognized utility, many of the conventional synthetic pathways for these compounds require strong acidic environments, extended heating, and stoichiometric amounts of hazardous reagents and solvents, which emphasizes the urgent need for more eco-friendly and efficient synthetic methodologies.^14-17 The current work outlines an environmentally friendly method for synthesizing 2-substituted benzoxazoles through the condensation of 2-aminophenol with various aldehydes, utilizing Zn(OAc)₂·2H₂O as a catalyst under solvent-free conditions.

2. Methodology

Melting points were determined using an electrothermal melting point apparatus, with the values reported uncorrected to accurately indicate the compounds purity under standardised conditions. The purity and progress of reactions for all compounds were regularly monitored by thin-layer chromatography on silica gel plates using an n-hexane: ethyl acetate (7:3, v/v) solvent system. The visualisation of spots was conducted under UV light at 254 nm and in an iodine-vapour chamber to improve detection. All reagents and solvents used in this study were obtained from Sigma-Aldrich and S.D. Fine-Chem Ltd., and were utilised as received to ensure the high-quality starting materials essential for reproducible synthetic procedures. Infrared spectra were recorded as KBr pellets using a PerkinElmer FT-IR spectrophotometer, spanning 4000-400 cm?¹. Proton NMR spectra were acquired on an Agilent 400 MHz spectrometer in DMSO or CDCl?, with chemical shifts expressed in parts per million (ppm) relative to tetramethylsilane as the internal standard. High-resolution mass spectrometry was performed on a Waters Xevo G2-XS QTOF to confirm molecular identities and isotopic distributions.

2.1 General procedure for the Synthesis of benzoxazole derivatives by Zn(OAc)₂. 2H₂O catalysed solvent-free reaction 10(a-h)

A substituted o-aminophenol (1.0 mmol) and the corresponding substituted aromatic aldehyde (1.0 mmol) were combined in an agate mortar. Zinc acetate dihydrate [Zn(OAc)?·2H?O] (10 mol%) was introduced as a catalyst. The mixture was ground thoroughly at room temperature under solvent-free conditions for 15-25 minutes. The reaction's progress was monitored by thin-layer chromatography (TLC) with an appropriate solvent system. Once the reaction was complete, the solid mixture was washed with cold water to eliminate the catalyst and other soluble impurities. The crude product was then filtered, dried, and recrystallized from ethanol to yield the desired benzoxazole derivatives 10(a–h) in good yields.

2.2    2-(4-Chlorophenyl)-5-methylbenzo[d]oxazole  10a

IR(cm^-1) :3056 (Ar–CH), 2924 (aliphatic C–H), 1618 (C=N), 1501 ; ¹H NMR (400 MH, DMSO, δ ):2.20 (s, 3H, Ar–CH?),7.18–7.86 (m, 8H, Ar–H). ; ¹³C NMR (DMSO, δ ):20 (Ar–CH?),114–155 (Ar–C and heterocyclic C),  ; MS(m/z): 246.04(M⁺), 248.04

(M+2)

2.3    4-(5-Methylbenzo[d]oxazol-2-yl)phenol  10b

IR(cm^-1) :3352 (O–H str., phenolic), 3050 (Ar–CH), ; ¹H NMR (400 MH, DMSO, δ ):2.21 (s, 3H, Ar–CH?), 6.5–7.60 (m,7H, Ar–H), 8.7 (br, 1H, Ar–OH). ; ¹³C NMR (DMSO, δ ): 21(Ar–CH?), 115–150 (Ar–C & heterocyclic C),158.8(CO,phenolic),

162.3(C=N).  ; MS(m/z): 228.08 (M⁺).

2.4    5-Methyl-2-(3-nitrophenyl)benzo[d]oxazole  10c

IR(cm^-1) :3364 (Ar–CH), 1599 (C=N), 1518 & 1334 (NO? asym. & sym. str.) ; ¹H NMR (400 MH, DMSO, δ ):2.24 (s, 3H, Ar–CH?),6.8–7.90 (m, 7H, Ar–H), ; ¹³C NMR (DMSO, δ ): 21 (Ar–CH?),114–155 (Ar–C and heteroaromatic carbons),148.3–150.6 (C–NO?). ; MS(m/z): 257.06 (M⁺).

2.5    5-Methyl-2-(2-nitrophenyl)benzo[d]oxazole  10d

IR(cm^-1) :3056 (Ar–CH), 2924 (aliphatic C–H),1608 (C=N),  ; ¹H NMR (400 MH, DMSO, δ ):2.45 (s, 3H, Ar–CH?),7.16–7.69 (m, 7H, Ar–H) ; ¹³C NMR (DMSO, δ ): 21.3 (Ar–CH?), 109.3–133.2 (Ar–C, benzoxazole ring),124.4–135.3 (Ar–C, nitrophenyl ring),141.4 (C=N), 146.9 (C–NO?), 162.7 (C–O, oxazole carbon).; MS(m/z): 257.04 (M⁺).

2.6    2-(2-Chlorophenyl)-5-methylbenzo[d]oxazole  10e

IR(cm^-1) :3060 (Ar–CH), 2920 (aliphaticC–H), 1618 (C=N), ; ¹H NMR (400 MH, DMSO, δ ):2.45 (s, 3H, Ar–CH?),7.38–7.69 (m, 7H, benzoxazole Ar–H), ; ¹³C NMR (DMSO, δ ): 21.3(Ar–CH?), 109.3-147.0 (benzoxazole ring C),127.3-136.9 (chlorophenyl ring C),162.7(C=N), 147.0(C–O, benzoxazole). ;MS(m/z): 246.01(M⁺), 248.01 (M+2)

2.7    2-(3-Bromophenyl)-5-methylbenzo[d]oxazole  10f

IR(cm^-1) :3060 (Ar–CH), 2920 (aliphatic C–H), 1625 (C=N), ; ¹H NMR (400 MH, DMSO, δ ):2.45 (s, 3H, Ar–CH?),7.16 -7.99 (m. 7H, Ar–H,) ; ¹³C NMR (DMSO, δ ):21.3 (Ar–CH?),109.3-133.2 (benzoxazole Ar-C),141.4 (C=N),147.0 (C–O),126.5-133.1 (Br-substituted phenyl carbons), 122.2 (C–Br).  ; MS(m/z): 290.01(M⁺),292.01

(M+2)

2.8    5-Methyl-2-phenylbenzo[d]oxazole  10g

IR(cm^-1) :3052 (Ar–CH), 2924 (aliphatic C–H), 1618 (C=N), ; ¹H NMR (400 MH, DMSO, δ ):2.45 (s, 3H, Ar–CH?), 7.17–7.65 (m, 8H,Ar–H) ; ¹³C NMR (DMSO, δ ): 21.3(Ar–CH?),109.3(C–O),119.1, 125.7, 127.5, 128.7, 129.2 (Ar–CH),130.6 (C=N),

133.2, 141.4, 147.0 (Ar–C). ; MS(m/z): 210.03 (M⁺).

2.9    5-Methyl-2-(4-nitrophenyl)benzo[d]oxazole  10h

IR(cm^-1) :3045 (Ar–CH), 2892 (aliphatic C–H),1610 (C=N), 1533, 1347 (NO? asym. & sym. str.) ; ¹H NMR (400 MH, DMSO, δ ):2.35 (s, 3H, Ar–CH?),7.18–7.70 (m, 7H, Ar–H) ; ¹³C NMR (DMSO, δ ): 21.9 (Ar–CH?),110.3–133.2 (Ar–C,126–136.3 (Ar–C, nitrophenylring),142.4(C=N),147.9C–NO?),163.7(C–O, oxazole carbon). ; MS(m/z): 257.03(M⁺).

3. Biological Activity

3.1 Antitumor Activity

The assessment of the antitumor effects of the synthesized compounds was conducted using the HepG2 human liver cancer cell line, utilizing the MTT assay (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) for quantitative analysis. HepG2 cells were initially seeded into a sterile 96-well microplate at a density of 5 × 10? cells per well and incubated at 37 °C to facilitate cell attachment and proliferation. After ensuring proper adherence of the cells, they were treated with different concentrations of the synthesized compounds, which were dissolved in dimethyl sulfoxide (DMSO). The cells then underwent a 48-hour incubation in serum-free medium to isolate the compound effects from factors present in serum. Following incubation, the culture medium was carefully aspirated to minimize disturbance of the cell layer, and 40 µL of freshly prepared MTT solution (2.5 mg/mL) was added to each well. The mixture was incubated with the cells for an additional 4 hours to allow viable cells to convert the yellow MTT into insoluble purple formazan crystals. To quantify cell viability, the formazan crystals were dissolved by adding 200 µL of DMSO to each well, resulting in a measurable purple solution. The absorbance of this solution was recorded at 570 nm using a multimode microplate reader, serving as an indicator of cell viability. The relative cell viability was determined by comparing the absorbance values from the treated cells to those of the untreated control cells. All experiments were performed in triplicate for reliability, with multiple replicates carried out over three separate days to ensure consistency. Results were reported as mean values along with their corresponding standard deviations. The half-maximal inhibitory concentration (IC??) for the compounds was calculated by analyzing the dose-response curves generated from the data.^18-21

3.2 Antibacterial Activity

The antibacterial efficacy of the synthesized compounds was extensively evaluated against a range of bacterial strains, including Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa. The agar well diffusion method, a widely used microbiological approach to assess antimicrobial activity, was employed for this testing.Initially, fresh bacterial cultures were prepared in nutrient broth and incubated at 37 °C for 18 to 24 hours to promote growth. Once adequately developed, these cultures were adjusted to match the turbidity of the 0.5 McFarland standard, ensuring a consistent bacterial concentration from the start. Subsequently, sterile nutrient agar plates were inoculated with the bacterial suspension to achieve even distribution across the surface.Using a sterile cork borer, 6-mm wells were created in the agar to hold the test compounds, which were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mg/mL. Tetracycline was used as a positive control to confirm the presence of antibacterial activity, while DMSO acted as a negative control to evaluate any growth-inhibiting effects from the solvent alone. After the compounds were introduced into the wells, the plates were incubated at 37 °C for an additional 24 hours.

After incubation, the zones of inhibition surrounding each well were measured in millimeters to determine the antibacterial potency of the synthesized compounds. All experiments were conducted in triplicate to ensure accurate results, and the mean values along with standard deviations were calculated and presented to underscore the reliability and significance of the findings.^22-25

Table 1 Antitumor Evaluation of Synthesised Compounds 10(a–h)

Fig. 1 Graphical representation of % inhibition of benzoxazole derivatives 10(a–h).

Table 2 Antibacterial Activity of Synthesised Compounds 10(a–h)

Fig. 2 Graphical representation of Antibacterial Activity of Synthesized Compounds 10(a–h)

Table 3: Physical data of compounds 10(a-h)

Scheme 1

Plausible Mechanism

Scheme 2