Design, Synthesis, and Biological Evaluation of Novel Dihydropyridine and Pyridine Analogs as Potent Human Tissue Nonspecific Alkaline Phosphatase Inhibitors with Anticancer Activity: ROS and DNA Damage-Induced Apoptosis

Abstract

Small molecules with nitrogen-containing scaffolds have gained much attention due to their biological importance in the development of new anticancer agents. The present paper reports the synthesis of a library of new dihydropyridine and pyridine analogs with diverse pharmacophores. All compounds were tested against the human tissue nonspecific alkaline phosphatase (h-TNAP) enzyme. Most of the compounds showed excellent enzyme inhibition against h-TNAP, having IC50 values ranging from 0.49 ± 0.025 to 8.8 ± 0.53 µM, which is multi-fold higher than that of the standard inhibitor (levamisole = 22.65 ± 1.60 µM) of the h-TNAP enzyme. Furthermore, an MTT assay was carried out to evaluate cytotoxicity against the HeLa and MCF-7 cancer cell lines. Among the analogs, the most potent dihydropyridine-based compound 4d was selected to investigate pro-apoptotic behavior. The further analysis demonstrated that compound 4d played a significant role in inducing apoptosis through multiple mechanisms, including overproduction of reactive oxygen species, mitochondrial dysfunction, DNA damaging, and arrest of the cell cycle at the G1 phase by inhibiting CDK4/6. The apoptosis-inducing effect of compound 4d was studied through staining agents, microscopic, and flow cytometry techniques. Detailed structure–activity relationship (SAR) and molecular docking studies were carried out to identify the core structural features responsible for inhibiting the enzymatic activity of the h-TNAP enzyme. Moreover, fluorescence emission studies corroborated the binding interaction of compound 4d with DNA through a fluorescence titration experiment.

Levamisole suppresses activation and proliferation of human T cells by the induction of a p53-dependent DNA damage response

“…In our study, LMS showed relatively low intercalation abilities, and only in high dosages (Supplemental Fig 3F ), making a direct intercalation mechanism in our experiments unlikely. Similarily to our data, the compound 4d, a synthetic manufactured metabolite of LMS, has been shown to induce cell-cycle arrest in HeLa cells and was found to induce DNA damage, although the direct mechanism remains unknown (66).…”

Levamisole suppresses activation and proliferation of human T cells by the induction of a p53‐dependent DNA damage response

“…S4F), making a direct intercalation mechanism in our experiments unlikely. Similar to our data, the compound 4d, a synthetic manufactured metabolite of LMS, has been shown to induce cell-cycle arrest in HeLa cells and was found to induce DNA damage, although the direct mechanism remains unknown [63].…”

Design, Synthesis and Bioactivity Evaluation of Novel 2-(pyrazol-4-yl)-1,3,4-oxadiazoles Containing an Imidazole Fragment as Antibacterial Agents

“…Permeability of the cell membrane has important physiological functions for the movement of water inside and outside the cell, the exchange of various substances, and the maintenance of pH and osmotic pressure [ 51 , 52 ]. To study the membrane permeability of the tested phytopathogenic bacteria affected by our designed compounds, the Xoo cells were detected by using a typical PI staining assay [ 53 ], in which the nonfluorescent dye PI can bind to DNA and RNA in cells with a damaged cell membrane and produce strong red fluorescence, but it cannot pass through cells with intact cell membranes, so the intensity and distribution of red fluorescence indirectly reflects the membrane permeability [ 54 ]. Clearly, the progressively elevated red fluorescence intensity and increased number of fluorescent cells ( Figure 7 b–f) showed that enhanced membrane permeability of Xoo cells occurred after incubation with compound 7c for 15 h when compared with the cells in the negative control ( Figure 7 a).…”