Synthesis and Evaluation of Benzoxazole Schiff Base Derivatives as Antitumor and Antibacterial Agents

Benzoxazole-containing Schiff bases represent a significant category of heterocyclic compounds that are synthesised through the reaction of benzoxazole-based amines or hydrazides with aldehydes or ketones. Benzoxazole derivatives are an important class of heterocyclic compounds that have attracted significant interest due to their wide-ranging pharmacological effects. These compounds exhibit impressive antitumor and antibacterial properties, which are essential in tackling the challenges posed by increasing drug resistance and the scarcity of effective therapeutic alternatives. ^1,2 Benzoxazole-containing Schiff bases have attracted considerable attention for their wide range of applications in medicinal chemistry, agrochemicals, and materials science. The presence of the azomethine (–C=N–) linkage, together with the benzoxazole heterocycle, contributes to their diverse pharmacological properties and makes them valuable intermediates in the synthesis of more complex bioactive molecules. This wide range of bioactivity results from the unique structural characteristics of the benzoxazole scaffold, which facilitates diverse functionalization and interaction with various biological targets.³ Integrating Schiff base moieties into benzoxazole structures significantly improves their properties. This is mainly due to the introduction of an imine (–C=N–) linkage, which promotes various biological interactions, including hydrogen bonding, π–π stacking, and metal chelation, ultimately enhancing their therapeutic potential.⁴ Investigating new benzoxazole analogues that incorporate Schiff base functionalities is a promising direction for discovering effective anticancer and antimicrobial agents.⁵ Cancer is one of the primary causes of death worldwide, highlighting the need for continuous innovation in new cancer-fighting drugs that offer better selectivity and lower systemic toxicity, especially considering the drawbacks of current chemotherapy treatments.⁶ The growing crisis of antimicrobial resistance highlights the pressing need for new antibacterial compounds capable of overcoming resistance mechanisms and providing effective treatment options against pathogenic microorganisms.⁷ The present work focuses on the design, synthesis, and biological evaluation of novel benzoxazole-Schiff base hybrids, aiming to elucidate the structural determinants underlying their enhanced cytotoxic and antimicrobial activities against a panel of human cancer cell lines and bacterial strains.

2. Methodology

Melting points were assessed using an electrothermal melting point apparatus, with values reported uncorrected to accurately reflect the purity of the compounds under standardised conditions. The purity and progress of reactions for all compounds were consistently evaluated by thin-layer chromatography on silica gel plates using an n-hexane:ethyl acetate (7:3, v/v) solvent system. Visualisation of the spots was performed under UV light at 254 nm and in an iodine-vapour chamber to enhance detection. All reagents and solvents utilised in this research were sourced from Sigma-Aldrich and S.D. Fine-Chem Ltd., and were used as received, ensuring high-quality starting materials necessary for reproducible synthetic processes. Infrared spectra were collected in the form of KBr pellets using a PerkinElmer FT-IR spectrophotometer, covering a spectral range from 4000 to 400 cm?¹. Proton NMR spectra were obtained on an Agilent 400 MHz spectrometer in DMSO or CDCl?, with chemical shifts indicated in parts per million (ppm) relative to tetramethyl silane as the internal standard. High-resolution mass spectrometry was performed using a Waters Xevo G2-XS QTOF instrument to verify molecular identities and isotopic distributions.

2.1 General procedure for the synthesis of 2-[(5-methyl-1,3-benzoxazol-2-yl) sulfanyl]         acetohydrazide derivatives involving Schiff base reaction 2(a-h)

A reaction mixture consisting of 2-((5-methylbenzo[d]oxazol-2-yl)thio)acetohydrazide (1, 1.0 mmol) and a suitable substituted aldehyde (1.0 mmol) was prepared in 20 mL of ethanol within a round-bottom flask. A catalytic amount of p-toluenesulfonic acid (p-TSA, 10 mol%) was introduced to this solution. The mixture was then refluxed for 4 to 6 hours with continuous stirring. The progress of the reaction was assessed by thin-layer chromatography (TLC) on silica gel plates using a mobile phase of ethyl acetate and n-hexane (6:4). Upon completion of the reaction, the mixture was allowed to cool to ambient temperature and then transferred to cold water. The precipitated product was collected through filtration, washed with cold ethanol, and dried under reduced pressure. The crude product was further purified by recrystallisation from ethanol, yielding Schiff base derivatives 2(a–h) in favourable yields.

2.2    2-((5-Methylbenzo[d]oxazol-2-yl)thio)-N'-(thiophen-2-ylmethylene)acetohydrazide

         2a

IR(cm^-1) : 3320 (NH str.), 3050 (Ar–CH); ¹H NMR (400 MH, DMSO, δ ): 2.40 (s, 3H, Ar–CH?), 4.2 (s, 2H, –CH?–CO–), 7.1-7.7 (m, 6H,Ar–H & thiophene–),11.7 s,1H–CONH); ¹³C NMR (DMSO, δ ): 21(Ar–CH?), 35 (–CH?–CO–), 110 –150 (Ar–C and heterocyclic C), 168 (C=O); MS(m/z): 332.02 (M⁺).

2.3    N'-(2-Hydroxybenzylidene)-2-((5-methylbenzo[d]oxazol-2-yl)thio)acetohydrazide

        2b

IR(cm^-1) : 3050 (Ar–CH), 2920 (aliphatic C–H), 1672 (C=O amide); ¹H NMR (400 MH, DMSO, δ ):2.50 (s, 3H, Ar–CH?), 4.40 (s, 2H, –CH?–CO–7.2-7.7 (m, 7H, Ar–H), 12.07 (s, 1H, –CONH) ; ¹³C NMR (DMSO, δ ):21 (Ar–CH?), 35 (–CH?–CO–),48.5 (–NH–CH?–),110–157 (Ar–C& hetero carbons),168 (C=O). ; MS(m/z): 342.06 (M⁺).

2.4    2-((5-Methylbenzo[d]oxazol-2-yl)thio)-N'-(2-nitrobenzylidene)acetohydrazide 2c

IR(cm^-1) : 3320 (NH str.), 3050 (Ar–CH), 2925 (–CH), 1670 (C=O amide), 1615 (C=N),; ¹H NMR (400 MH, DMSO, δ ): 2.39 (s, 3H, Ar–CH?), 4.3 (s, 2H, –CH?–CO),7.1-7.9 (m, 7H, Ar–H), 8.1 (s, 1H, –CONH), 12.03 (s, 1H, –NH–).; ¹³C NMR (DMSO, δ ): 21 (Ar–CH?), 35 (–CH?–CO–), 110–150 (Ar–C & heteroaromatic C),

168 (C=O).; MS(m/z): 371.04 (M⁺).

2.5    N'-Benzylidene-2-((5-methylbenzo[d]oxazol-2-yl)thio)acetohydrazide 2d

IR(cm^-1) : 3320 (NH str.), 3050 (Ar–CH), 2925 (–CH), 1680 (C=O amide) ; ¹H NMR (400 MH, DMSO, δ ):2.45 (s, 3H, Ar–CH?),4.00 (s, 2H, –CH?–CO–),7.16–7.93 (m, 8H, Ar–H), 8.47 (s, 1H, –H=N–),11.07 (br s, 1H, –NH–),11.75 (br s, 1H, –CONH–) ; ¹³C NMR (DMSO, δ ): 21.3 (Ar–CH?), 40.9 (–CH?–CO–), 65.0 (C–S),109.3–148.9 (Ar–C and heteroaromatic C),171.0 (C=O) ; MS(m/z): 325.06 (M⁺).

2.6    N'-(4-Chlorobenzylidene)-2-((5-methylbenzo[d]oxazol-2-yl)thio)acetohydrazide

         2e

IR(cm^-1) :3320 (NH str.), 3050 (Ar–CH), 2930 (aliphatic C–H), 1670 (C=O, amide), 1615 (C=N), 1500, 1475  ; ¹H NMR (400 MH, DMSO, δ ): 2.45 (s, 3H, Ar–CH?),4.00 (s, 2H, –S–CH?–CO–),7.47–7.82 (m, 7H, Ar–H), 8.41 (s, 1H, –NH–N=),11.07 (s, 1H, –CONH).; ¹³C NMR (DMSO, δ):21.3 (Ar–CH?),40.9 (–S–CH?–),109.3–148.9 (Ar–C and heterocyclic), 30.6–136.6 (Ar–C, p-chlorophenyl), 144.1 (C=N), 171.0 (C=O); MS(m/z): 359.05 (M⁺).361.05 (M+2)

2.7    N'-(2-Chlorobenzylidene)-2-((5-methylbenzo[d]oxazol-2-yl)thio)acetohydrazide

         2f

IR(cm^-1) : 3320 (NH str.), 3050 (Ar–CH), 2930 (aliphatic C–H), 1670 (C=O, amide), 1615 (C=N), 1520–1480 (Ar C=C) ; ¹H NMR (400 MH, DMSO, δ ):2.45 (s, 3H, Ar–CH?), 4.00 (s, 2H, –CH?–CO–),7.15–7.75 (m, 7H, Ar–H),8.40–9.00 (s, 1H, –NH–N=),11.05 (br s, 1H, –CONH) ; ¹³C NMR (DMSO, δ ):21.3 (Ar–CH?), 40.9 (–CH?–CO–), 109–149 (Ar–C & heterocyclic C),171.0 (C=O) ; MS(m/z): 359.08 (M⁺), 361.08 (M+2).

2.8    N'-(2-Bromobenzylidene)-2-((5-methylbenzo[d]oxazol-2-yl)thio)acetohydrazide 

         2g

IR(cm^-1) : 3320–3270 (NH str.), 3050 (Ar–CH), 2920 (C–H),1670 (C=O, amide); ¹H NMR (400 MH, DMSO, δ ):2.45 (s, 3H, Ar–CH?), 4.00 (s, 2H, –CH?–CO–),7.15–7.70 (m, 7H,Ar–H), 8.39 (s, 1H,–CH=N–),9.02 (br s, 1H, –NH–),11.07 (s, 1H, –CONH) ; ¹³C NMR (DMSO, δ ):21.3 (Ar–CH?), 40.9 (–CH?–CO–), 109–148 (Ar–C& heteroaromatic C), 143–145(C=N), 171.0(C=O, amide) ; MS(m/z): 403 (M⁺), 405

(M+2).

2.9    2-((5-Methylbenzo[d]oxazol-2-yl)thio)-N'-(4-nitrobenzylidene)acetohydrazide 2h

IR(cm^-1) : 3320 (NH str.), 3050 (Ar–CH), 2930 (–CH), 1670 (C=O, amide),1615 (C=N); ¹H NMR (400 MH, DMSO, δ ):2.45 (s, 3H, Ar–CH?) 4.00 (s, 2H, –S–CH?–CO–),8.47 (br s, 1H, –NH–),11.07 (s, 1H, –CONH),7.16–7.69 (m, 7H, Ar–H). ; ¹³C NMR (DMSO, δ ):21.3 (Ar–CH?),40.9 (–CH?–CO),171.0 (C=O),109.3–148.9 (benzoxazole,Ar–C & C=N) 124–144 (NO?–phenyl,Ar–C) 150.2 (C–NO?) ; MS(m/z): 369.69(M⁺).

3. Biological Activity

3.1 Antitumor Activity

The evaluation of the antitumor activity of the synthesised compounds was performed using the HepG2 human liver cancer cell line, employing the MTT assay (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) as a quantitative measure. Initially, HepG2 cells were carefully seeded into a sterile 96-well microplate at a consistent density of 5 × 10? cells per well and incubated at 37 °C to promote cellular attachment and growth.^9-12 Once the cells were properly attached, they were treated with varying concentrations of the synthesised test compounds, dissolved in dimethyl sulfoxide (DMSO). The cells were then incubated for 48 hours in a serum-free medium to isolate the effects of the compounds from serum-derived factors. After incubation, the culture medium was gently aspirated to avoid disturbing the cell layer, and 40 µL of freshly prepared MTT solution (2.5 mg/mL) was added to each well. This mixture was incubated with the cells for an additional 4 hours, allowing viable cells to reduce the yellow MTT to insoluble formazan purple crystals. To quantify cell viability, formazan crystals were dissolved by adding 200 µL of DMSO to each well, resulting in a clearly measurable purple solution. The absorbance of this solution was then recorded at 570 nm using a multimode microplate reader, providing a metric for cell viability. The relative cell viability was calculated by comparing the absorbance readings of treated cells to those of the untreated control cells. All experiments were performed in triplicate to ensure reliability, with multiple replicates conducted over three different days to establish consistency. The outcomes were reported as mean values with their corresponding standard deviations. The half-maximal inhibitory concentration (IC??) values for the compounds were determined by analysing the dose-response curves generated from the data.^13-15

3.2 Antibacterial Activity

The antibacterial properties of the synthesised compounds were thoroughly tested against various bacterial strains, including Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa. This testing was performed using the agar well diffusion method, a standard microbiological technique for measuring antimicrobial activity.^16-18 To start the experiment, fresh bacterial cultures were carefully prepared in nutrient broth and incubated at 37 °C for 18 to 24 hours. After adequate growth, the cultures were adjusted to a turbidity equivalent to the 0.5 McFarland standard, ensuring a uniform initial bacterial concentration. Next, sterile nutrient agar plates were uniformly inoculated with the bacterial suspension to ensure even distribution across the surface. Using a sterile cork borer, 6-mm-diameter wells were carefully created in the agar to hold the test compounds. The compounds, dissolved in dimethyl sulfoxide (DMSO) at 1 mg/mL, were then added to the wells. Tetracycline was used as a positive control to demonstrate antibacterial activity, while DMSO served as the negative control to assess any growth-inhibitory effects attributable to the solvent alone. After adding the compounds, the plates were incubated again at 37 °C for 24 hours. Following incubation, the zones of inhibition around each well were measured in millimetres to evaluate the antibacterial effectiveness of the synthesised compounds. All tests were performed in triplicate to ensure reliable results, and the mean ± standard deviation was calculated and presented to highlight the consistency and significance of the findings.^19-20

Table 1 Antitumor Evaluation of Synthesised Compounds 2(a–h)

Fig. 1 Graphical representation of % inhibition of benzoxazole derivatives 2(a–h)

Table 2 Antibacterial Evaluation of Synthesised Compounds 2(a–h)

Fig. 2 Graphical representation of Antibacterial Activity of benzoxazole derivatives 2(a–h)

Table-3: Physical data of compounds 2(a-h)

Scheme 1

Scheme 2