1. Introduction
Benzothiazole derivatives have attracted extensive attention in medicinal chemistry due to their diverse pharmacological activities, including antimicrobial, anti-inflammatory, and anticancer effects. The benzothiazole nucleus is frequently employed as a pharmacophore in drug discovery. Modifications at the 2- and 6-positions of benzothiazole have been shown to significantly influence biological activity. While 2-substituted benzothiazoles are well explored, relatively few studies have addressed the combined effects of 2,6-disubstitution on anticancer properties.
In this study, we synthesized a series of 2,6-disubstituted benzothiazole derivatives and evaluated their anticancer activities against three different human cancer cell lines. The structure–activity relationship (SAR) was investigated based on variations of substituents at the 2- and 6-positions.
2. Materials and Methods
2.1. Chemicals and reagents
All chemicals and solvents were of analytical grade and used without further purification. 2-Aminothiophenol, substituted benzaldehydes, iodine, potassium carbonate, and other reagents were purchased from commercial suppliers.
2.2. General synthetic procedure
Equimolar quantities of 2-aminothiophenol and substituted benzaldehyde were mixed in ethanol and stirred at room temperature for 30 min. Iodine and potassium carbonate were added, and the reaction mixture was refluxed at 60°C for 3 h. The crude product was extracted, dried, and purified by column chromatography. Substitution at the 6-position was introduced using electrophilic substitution reactions (nitration, halogenation, cyanation) under standard conditions.
Scheme 1. Synthetic route to 2,6-disubstituted benzothiazole derivatives.
2.3. Characterization
NMR spectra were recorded on a 400 MHz spectrometer using CDCl₃ or DMSO-d₆ as solvent. Chemical shifts are reported in ppm. IR spectra were recorded using a FTIR spectrophotometer. HRMS were obtained using electrospray ionization (ESI) techniques.
2.4. Anticancer activity assay
MCF-7, HeLa, and A549 cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% CO₂. Cytotoxicity was assessed by the MTT assay. Cells were seeded in 96-well plates and treated with various concentrations (0.1–100 µM) of test compounds for 72 h. The IC₅₀ values were calculated from dose–response curves.
3. Results and Discussion
3.1. Synthesis
All target compounds (1a–1h) were obtained in good yields (45–82%). Table 1 shows the structures, substituents, and physical properties.
Table 1. Yields and physical data of synthesized compounds
| Compound | R (C-2) | R’ (C-6) | Yield (%) | MP (°C) |
| 1a | Phenyl | H | 78 | 142–144 |
| 1b | 4-CH₃-phenyl | H | 72 | 136–138 |
| 1c | 4-F-phenyl | H | 65 | 150–152 |
| 1d | 4-CF₃-phenyl | 6-NO₂ | 58 | 168–170 |
| 1e | 4-OCH₃-phenyl | 6-Cl | 82 | 154–156 |
| 1f | n-Butyl | 6-CF₃ | 47 | oil |
| 1g | 2-Thienyl | 6-NO₂ | 50 | 126–128 |
| 1h | 4-Cl-phenyl | 6-CN | 60 | 162–164 |
3.2. Anticancer screening
The IC₅₀ values obtained from MTT assays are summarized in Table 2.
Table 2. IC₅₀ values (µM) of synthesized compounds
| Compound | MCF-7 | HeLa | A549 |
| 1a | 24.6 ± 2.1 | 29.4 ± 2.8 | 38.2 ± 3.5 |
| 1b | 18.4 ± 1.7 | 21.0 ± 1.9 | 26.8 ± 2.4 |
| 1c | 12.2 ± 1.1 | 13.8 ± 1.2 | 15.6 ± 1.3 |
| 1d | 2.8 ± 0.3 | 3.2 ± 0.4 | 4.6 ± 0.5 |
| 1e | 9.6 ± 0.9 | 10.2 ± 0.9 | 12.4 ± 1.1 |
| 1f | 48.0 ± 4.6 | 52.3 ± 4.9 | 60.1 ± 5.8 |
| 1g | 7.5 ± 0.7 | 8.3 ± 0.7 | 9.1 ± 0.8 |
| 1h | 11.0 ± 1.0 | 12.6 ± 1.2 | 14.0 ± 1.3 |
| Doxorubicin | 0.67 ± 0.05 | 0.41 ± 0.03 | 0.83 ± 0.06 |
Compound 1d exhibited the highest cytotoxic activity across all cell lines, showing IC₅₀ values close to low micromolar range.
3.3. Structure–activity relationships (SAR)
- Aryl groups at the 2-position enhance anticancer activity more than alkyl groups (compare 1a–1e vs 1f).
- Electron-withdrawing groups (CF₃, NO₂, CN) at 2- or 6-position improve potency.
- Electron-donating groups reduce activity (1e vs 1c).
3.4. Proposed mechanism
Flow cytometry of MCF-7 cells treated with 1d showed G2/M phase arrest and increased Annexin V positive cells, suggesting apoptosis induction. Caspase-3 assays revealed a 3.5-fold increase in activity, confirming apoptotic pathways.
4. Conclusion
A series of novel 2,6-disubstituted benzothiazole derivatives were successfully synthesized and evaluated for anticancer activity. Compound 1d emerged as the most promising lead, showing potent cytotoxic activity across multiple cancer cell lines. These findings support further optimization and mechanistic studies of this scaffold as a potential anticancer therapeutic.
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