Synthesis of thia-Michael-Type Adducts between Naphthoquinones and N-Acetyl- L-Cysteine and Their Biological Activity

Abstract

A series of naphthoquinones, namely, 1,4-naphthoquinone, menadione, plumbagin, juglone, naphthazarin, and lawsone, were reacted with N-acetyl-L-cysteine, and except for lawsone, which did not react, the related adducts were obtained. After the tuning of the solvent and reaction conditions, the reaction products were isolated as almost pure from the complex reaction mixture via simple filtration and were fully characterized. Therefore, the aim of this work was to evaluate whether the antitumor activity of new compounds of 1,4-naphthoquinone derivatives leads to an increase in ROS in tumor cell lines of cervical carcinoma (HeLa), neuroblastoma (SH-SY5Y), and osteosarcoma (SaOS2, U2OS) and in normal dermal fibroblast (HDFa). The MTT assay was used to assay cell viability, the DCF-DA fluorescent probe to evaluate ROS induction, and cell-cycle analysis to measure the antiproliferative effect. Compounds 8, 9, and 12 showed a certain degree of cytotoxicity towards all the malignant cell lines tested, while compound 11 showed biological activity at higher IC₅₀ values. Compounds 8 and 11 induced increases in ROS generation after 1 h of exposure, while after 48 h of treatment, only 8 induced an increase in ROS formation in HeLa cells. Cell-cycle analysis showed that compound 8 caused an increase in the number of G0/G1-phase cells in the HeLa experiment, while for the U2OS and SH-SY5Y cell lines, it led to an accumulation of S-phase cells. Therefore, these novel 1,4-naphthoquinone derivatives may be useful as antitumoral agents in the treatment of different cancers.

Keywords: N-acetyl-L-cysteine; cancer; juglone; menadione; naphthazarin; naphthoquinone; plumbagin; thia-Michael.

Figure 1

Quinone classification and related reduced forms.

Figure 2

Naphthoquinones 1–6 reacted with N-acetyl-L-cysteine (7) in the current study.

Scheme 1

Reaction pathway for thia-Michael type addition of N-acetyl-L-cysteine to quinones.

Scheme 2

Possible redox pathways that might occur during thia-Michael reaction between N-acetyl-L-cysteine and quinones.

Scheme 3

Thia-Michael addition of N-acetyl-L-cysteine (7) to naphthoquinone derivatives 1–6 and relative products.

Scheme 4

Regioisomers obtained via reaction between 4 and 7 in methanol.

Figure 3

Keto–enol tautomerism in lawsone.

Figure 4

Effects of compounds on the viability of transformed and non-transformed human cells. HeLa, SH-SY5Y, SaOS2, U2OS, and HDFa were incubated for 48 h with increasing concentrations of compounds 8, 9, 11, and 12 (from 0.01 to 200 μM). Viability was evaluated using MTT tests as reported in Materials and Methods. (A) Compound 8. (B) Compound 9. (C) Compound 11. (D) Compound 12. The white bars correspond to the control. Results were expressed as means ± SDs of three independent experiments.

Figure 5

Induction of intracellular ROS in HeLa, U2OS, and SH-SY5Y cells treated with 8 and 11 under physiological conditions at 37 °C for 1 h (A) and 48 h (B). Data represent the means (SDs) of three determinations. *** indicates statistically significant differences at p < 0.0001, ** at p < 0.001 respectively, compared with control (0.1% DMSO). ## p < 0.001 relative to HeLa cells treated with 8; ††† p < 0.0001 and † p < 0.05 relative to HeLa cells treated with 11.

Figure 6

Cell cycle analysis. (A). Representative data showed cell cycle distribution with PI staining followed by flow cytometry analysis of HeLa, U2OS and SH-SY5Y cells treated whit 8 (5 μM) for 24 (sx) or 48 h (dx). (B). Histograms show the G1, S and G2/M phase distribution of HeLa, U2OS and SH-SY5Y cells from three separate experimental analyzes, after 24 h (above) and 48 h (below) of treatment. Error bars based on the mean ± SD of three independent experiments.