New N-Adducts of Thiadiazole and Thiazoline with Levoglucosenone and Evaluation of Their Significant Cytotoxic (Anti-Cancer) Activity

Abstract

The conjugate N-adducts of thio-1,3,4-diazole and 2-thiazoline with levoglucosenone were synthesized via a stereoselective, base-catalyzed conjugate N-Michael addition to levoglucosenone at C-4. Structural assignments were established using 1H and 13C NMR analysis, and X-ray single-crystal analysis for one of the compounds. The biological properties of the novel compounds were tested on a cell model. Cytotoxicity was analyzed via colorimetric assay. Two distinct types of cell death, apoptosis and necrosis, were analyzed by determining the phosphatidylserine levels from the outer leaflet of the plasma membrane, caspase activation, and lactate dehydrogenase release. We also evaluated DNA damage using an alkaline comet assay. The level of oxidative stress was measured with a modified comet assay and an H2DCFDA probe. The thio-1,3,4-diazole adduct (FCP23) and the 2-thiazoline adduct (FCP26) exhibit similar cytotoxicity values for cancer cells (ovarian (A2780), breast (MCF-7), cervix (HeLa), colon (LoVo), and brain (MO59J and MO59K)), but their mechanism of action is drastically different. While FCP23 induces oxidative stress, DNA damage, and necrosis, FCP26 induces apoptosis through caspase activation.

Keywords: DNA damage; apoptosis; levoglucosenone; thiadiazole; thiazoline.

Figure 1

Chemical structure of (1–4)-S-thiodisaccharide.

Scheme 1

New N-thiadiazole (FCP23) and N-thiazoline (FCP26) scaffolds and crucial features of their molecular structures.

Scheme 2

Tautomerism of 1,3,4-thiadiazole (2) and 2-thiazoline (3).

Scheme 3

Conjugate aza-N-Michael addition of 1,3,4-thiadiazole 2 and 2-thiazoline 3 to levoglucosenone 1.

Figure 2

ORTEP diagram of FCP26.

Figure 3

Molecular models for FCP23 (a) and FCP26 (b) with stereochemistry and atomic distances.

Scheme 4

FCP23 and FCP26 represent classic geminal diheteroatomic motifs marked in red.

Figure 4

Apoptosis and necrosis induced by FCP23 and FCP26 in ovarian cancer cell lines (A2780). Cell lines were incubated with compounds for 72 h at 37 °C, 5% CO₂, and then the translocation of phosphatidylserine from the inner leaflet to the outer leaflet of the plasma membrane (A), activation of executioner caspases 3/7 (B), and release of lactate dehydrogenase (C) were measured. Figures shown as mean derived from three independent measurements ± SD. *** p < 0.001.

Figure 5

Analysis of the level of DNA damage induced by FCP23 and FCP26 in the A2780 cell line. (A) Cells were incubated with the compound for a period of 0 min to 120 min at 37 °C. Measurements were made using the alkaline version of the comet test. (B) The effect of hOGG1 on FCP-induced DNA damage. DNA damage resulted from the alkaline comet assay in A2780 cell lines after 1 h incubation with FCP23 or FCP26 at 37 °C in the presence of the hOGG1 enzyme (red) at 2 μM compared with negative controls and cells incubated with FCPs only (blue). The positive control included cells incubated with H₂O₂ at 10 μM. (C) The effect of PBN and tocopherol on FCP-induced DNA damage. The DNA damage was measured using the alkaline comet assay in A2780 cell lines after 1 h incubation with FCP23 or FCP26 at 37 °C in the presence of PBN at 100 μM (blue), and tocopherol at 25 μM (dark blue) compared with cells incubated with FCPs only (light blue). Graphs present the mean value for 100 cells analyzed per sample. All experiments were replicated three times. The error bars represent SEM, *** p < 0.001.

Figure 6

ROS detection using fluorescent probe 2’,7’-dichlorodihydrofluorescin diacetate (H2DCF-DA) in A2780 cells treated with FCP23 (light green) and FCP26 (dark green) in their concentrations at IC₂₀. Cells were exposed to compounds from 15 min to 102 min at 37 °C before pre-incubation with the H2DCF-DA probe for 30 min at 37 °C. The value of negative control fluorescence was brought to 100. Other values were established relative to this control. Each bar represents the mean value of three independent experiments ± SEM.