<p>3,6-Disubstituted 1,2,4-Triazolo[3,4-<em>b</em>]Thiadiazoles with Anticancer Activity Targeting Topoisomerase II Alpha</p>

Abstract

Background: Topoisomerase IIα (topIIα) maintains the topology of DNA in order to ensure the proper functioning of numerous DNA processes. Inhibition of topIIα leads to the killing of cancer cells thus constituting such inhibitors as useful tools in cancer therapeutics. Triazolo[3,4-b]thiadiazole derivatives are known for their wide range of pharmacological activities while previous studies have documented their in vitro anticancer activity. The purpose of the current study was to investigate if these chemical compounds can act as topIIα inhibitors in cell-free and cell-based systems. Materials and Methods: The MTT assay was performed in DLD-1, HT-29, and LoVo cancer cells so as to evaluate the antiproliferative activity of KA25, KA26, and KA39 triazolo[3,4-b]thiadiazole derivatives. The KA39 compound was tested as a potential topIIα inhibitor using the plasmid-based topoisomerase II drug screening kit. The inhibitory effect of the three derivatives on topIIα phosphorylation was studied in HT-29 and LoVo cancer cells according to Human Phospho-TOP2A/Topoisomerase II Alpha Cell-Based Phosphorylation ELISA Kit. Moreover, flow cytometry was utilized in order to explore apoptotic induction and cell cycle growth arrest, upon treatment with KA39, in DLD-1 and HT-29 cells, respectively. In silico studies were also carried out for further investigation. Results: All three triazolo[3,4-b]thiadiazole derivatives showed an in vitro antiproliferative effect with the KA39 compound being the most potent one. Our results indicated that KA39 induced both early and late apoptosis as well as cell cycle growth arrest in S phase. In addition, the compound blocked the relaxation of supercoiled DNA while it also inhibited topIIα phosphorylation (upon treatment; P<0.001). Conclusion: Among the three triazolo[3,4-b]thiadiazole derivatives, KA39 was shown to be the most potent anticancer agent and catalytic inhibitor of topIIα phosphorylation as well.

Anticancer Activity of Triazolo-Thiadiazole Derivatives and Inhibition of AKT1 and AKT2 Activation

“…The residue was purified by silica gel column chromatography (eluent; hexane/ethyl acetate = 2/1) to yield compound 2 as a white solid (88%). The spectral data were in accordance with those reported in the literature [ 23 ]. 2: 1 H NMR (500 MHz, CDCl 3 ) δ 7.52 (s, 1H), 6.88 (s, 1H), 4.11 (s, 2H), 3.97 (s, 3H), 3.95 (s, 3H), 3.73 (s, 3H); 13 C NMR (126 MHz, CDCl 3 ) δ 170.5, 154.1, 148.0, 134.6, 127.8, 115.3, 111.3, 56.4, 56.4, 52.4, 38.0.…”

“…Synthesis of the 1,2,4-triazolo-[3,4- b ]-[1,3,4]-thiadiazoles was accomplished according to the synthetic methodology developed by our group ( Scheme 1 and Scheme 2 ) [ 23 ]. After a reaction with oxalyl chloride, 2-(3,4-dimethoxyphenyl)acetic acid 1 was esterified and the corresponding methyl ester reacted with chlorosulfonic acid in CHCl 3 to give methyl 2-(2-(chlorosulfonyl)-4,5-dimethoxyphenyl)acetate 2 in 88% yield.…”

“…The LC-MS spectra were recorded on a LC20AD Shimadzu connected to Shimadzu LCMS-2010EV equipped with C18 analytical column (SUPELCO Discovery (C18, 25 cm × 4.6 mm, 5 μm)). Compound 2, KA25, KA26, and KA39 were synthesized according to a previously reported procedure [ 23 ].…”

“…After 24 h of cell growth, the culture medium was discarded and replaced with 2 mL of fresh medium. Then, the cells were treated with KA25 and KA39 derivatives at GI50 and TGI concentrations (μΜ) for 3 and 24 h. The required GI50 and TGI concentrations (μΜ) of the KA25 and KA39 derivatives in HT-29 and LoVo cells have been determined in previous studies [ 23 ]. To estimate the TGI concentration (μΜ) of the KA25 compound in SW403 cells, further MTT assays were carried out as previously described.…”

“…Furthermore, 1,2,4-triazolo[3,4- b ][1,3,4]thiadiazole derivatives containing 3-methyl or benzyl moiety showed moderate anti-HIV-1 activity at subcytotoxic concentrations [ 22 ]. In previous work, we have shown that triazolo[3,4- b ]thiadiazole derivatives show potent in vitro antiproliferative activities [ 23 ]. Our studies resulted in the identification of three bioactive compounds (KA39, KA25, and KA26) against three human colorectal cancer cell lines ( Figure 1 ).…”