Molecular mechanism of action of new 1,4-naphthoquinones tethered to 1,2, 3-1H-triazoles with cytotoxic and selective effect against oral squamous cell carcinoma

The oral squamous cell carcinoma (OSCC) stands out as a public health problem due to its high incidence and low survival rate, despite advances in diagnosis and treatment. Moreover, the most commonly chemotherapeutic agents for OSCC, such as carboplatin and cisplatin, generate important side effects, evidencing the urgency in developing new drugs. Naphthoquinones are an important class of natural products or synthetic compounds with cytotoxic effect demonstrated on different cancer types. In the present study, thirty-five 1,4-naphthoquinones tethered to 1,2,3-1H-triazoles were synthesized and the antitumor activity and molecular mechanisms were evaluated in several assays including in vitro and in vivo models of OSCC and normal oral human cells. Compounds 16a, 16b and 16 g were able to induce cytotoxicity in three different tumor cell lines of human OSCC (SCC4, SCC9 and SCC25) and were more toxic and selective to tumor cells (Selective Index, SI 2) than classical and chemically similar controls (Carboplatin and Lapachol). Compound 16 g showed the higher SI value. Besides, compounds 16a, 16b and 16 g significantly reduced colony formation of SCC9 cells in the tested concentrations. Hemolytic assay using compounds 16a, 16b and 16 g at high concentrations showed no com-pound exhibited hemolysis higher than 5%, similar to controls. In vivo acute toxicity study showed that 16 g was the only one, among the three compounds, with no apparent limiting toxic effects on mice in the tested con-centrations. Thus, the investigation of cell death mechanisms was conducted with this compound. 16 g does not trigger ROS production nor binds to DNA. On the other hand, compound 16 g induced microtubule dis-organization, and molecular modeling studies suggests a potential mechanism of action related to inhibition of topoisomerases and/or hPKM2 activities. Cell morphology, pyknotic nuclei presence, cleaved caspase-3 staining and viability assays using caspase-3 inhibitors demonstrate compound 16 g induced cell death through apop-tosis. Among the 35 synthesized triazole naphthoquinones, compound 16 g was the most effective compound against OSCC cells, presenting high cytotoxicity (similar to 35 mu M), selectivity (SI similar to 6) and low acute toxicity on animals, and therefore might be considered for future cancer therapy.