Triple-negative breast cancer is a highly aggressive subtype characterized by the absence of three receptors—estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER2)—which eliminates the possibility of conventional targeted therapies. In this context, Photodynamic Therapy (PDT) emerges as a promising alternative, provided that efficient photosensitizers (PS) are employed. This study evaluated the photophysical properties and biological activity of two novel methylene blue (MB) derivatives, AM-NMP, derivative in which one dimethylamino substituent is replaced by a naphthyl-methyl-piperazine group and AM-I (3-(dimethylamino)phenothiazin-5-ium chloride), against the triple-negative breast cancer cell line MDA-MB-231. Spectroscopic assays revealed that AM-NMP displayed a high molar absorptivity coefficient (ε ≈ 5.1 × 10⁴ L·mol⁻¹·cm⁻¹), whereas AM-I showed a significantly lower value (ε ≈ 1.0 × 10⁴ L·mol⁻¹·cm⁻¹), corresponding to nearly five-fold lower photon absorption efficiency. Both derivatives exhibited reduced fluorescence quantum yields (Φf) compared to MB (Φf = 0.04), with AM-NMP being about ten times lower (Φf = 0.0041) and AM-I about two times lower (Φf = 0.020). The singlet oxygen quantum yield (ΦΔ) was also low for both derivatives (≈ 0.021), in contrast with MB (0.52). Despite these limitations, both compounds showed good photostability up to 22 minutes of irradiation, ensuring adequate performance under the biological experimental conditions, which required only 12 minutes of light exposure. Cell viability assays demonstrated no dark cytotoxicity for any compound at the tested concentrations (0.1–10 µM) in MDA-MB-231 cells. Under red LED irradiation (λ = 633 nm, dose ≈ 5 J·cm⁻²), MB exhibited IC₅₀ ≈ 3.1 µM, AM-NMP ≈ 6.7 µM, while AM-I showed no phototoxicity (IC₅₀ > 10 µM). These results indicate that AM-NMP retains promising photoactivity (IC₅₀ 10 µM). Esses resultados indicam que o AM-NMP mantém fotoatividade promissora (IC₅₀ < 10 µM), ainda que inferior ao AM, enquanto o AM-I se mostrou ineficiente para a aplicação proposta. De forma geral, as modificações estruturais não resultaram em maior eficácia fotodinâmica em relação ao azul de metileno, mas os dados obtidos fornecem subsídios importantes para a compreensão das relações estrutura-atividade e apontam caminhos para otimizações futuras
