Microwave-Assisted Synthesis of New N1,N4-Substituted Thiosemicarbazones

We present an efficient procedure for the synthesis of thirty-six N1,N4-substituted thiosemicarbazones, including twenty-five ones that are reported for the first time, using a microwave-assisted methodology for the reaction of thiosemicarbazide intermediates with aldehydes in the presence of glacial acetic acid in ethanol and under solvent free conditions. Overall reaction times (20–40 min when ethanol as solvent, and 3 min under solvent free conditions) were much shorter than with the traditional procedure (480 min); satisfactory yields and high-purity compounds were obtained. The thiosemicarbazide intermediates were obtained from alkyl or aryl isothiocyanates and hydrazine hydrate or phenyl hydrazine by stirring at room temperature for 60 min or by microwave irradiation for 30 min, with lower yields for the latter. The preliminary in vitro antifungal activity of thiosemicarbazones was evaluated against Aspergillus parasiticus and Candida albicans

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2).

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO2, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma

Effects of 1,3,4-thiadiazolium derivatives on hepatocytes morphology (the experimental conditions are described in the Materials and Methods section 2.6).

<p>Hepatocytes were incubated with the derivatives at 25 μM for 24 h. The images were obtained using inverted microscope. A: control (untreated cells); B-E: treatments by MI-D, MI-J, MI-4F and MI-2,4diF, respectively. The scale is indicated by black bars representing 50 μm. The photographs represent three different experiments in triplicate.</p

Annexin V-FITC and propidium iodide staining of HepG2 treated with 1,3,4-thiadiazolium derivatives (the experimental conditions are described in the Materials and Methods section 2.5.3).

<p>The cells were seeded with or without 1,3,4-thiadiazolium derivatives at 25 μM for 18–24 h. Then, the cells were collected with trypsin and 10.000 events were analyzed by flow cytometry by FL2 and FL1 filters. (A) control, (B) MI-D, (C) MI-J, (D) MI-4F and (E) MI-2,4diF. The figures show representative dot-plot with the different cell populations: left-bottom = labeled cells; left-top = PI labeled; right-top = doubly labeled; right-bottom = annexin V labeled. The results were expressed as mean ± SD of three independents experiments.</p

Effects of 1,3,4-thiadiazolium derivatives on HepG2 cell morphology (the experimental conditions are described in the Materials and Methods section 2.6).

<p>The cells were seeded with or without 1,3,4-thiadiazolium derivatives at 5 μM for 3 h. The images were captured with a 100X magnification; they correspond to control (A), MI-D (B), MI-J (C), MI-4F (D) and MI-2,4diF (E). The scale is indicated by black bars representing 0.02 mm. The arrows show morphological modifications as blebs, increased volume and vacuolization. The photographs represent three different experiments in triplicate.</p

Annexin V-FITC and propidium iodide staining of hepatocytes treated by 1,3,4-thiadizolium derivatives (the experimental conditions are described in the Materials and Methods section 2.5.3).

<p>Hepatocytes were incubated with derivatives at 25 μM for 20 h. The images (10X magnification) were captured with an AXIOVERT 40CSFL fluorescence microscope. The scale is indicated by white bars representing 100 μm. The annexin V-FITC-positive cells are stained in green, and the PI-positive cells in red. The images represent (A) control (untreated cells), (B) ASA positive control at 20 mM, (C) MI-D, (D) MI-J, (E) MI-4F and (F) MI-2,4diF. The figures represent three different experiments in triplicate.</p

Cytotoxic effects of 1,3,4-thiadiazolium derivatives on HepG2 cells.

<p>A. MTT assay (the experimental conditions are described in the Materials and Methods section 2.5.1). The cells were seeded with or without 1,3,4-thiadiazolium derivatives at 5, 25 or 50 μM for 24 h. The results were expressed as % of viability in comparison to control. B. LDH release assay (the experimental conditions are described in the Materials and Methods section 2.5.2). Under the same treatment conditions, as described above, LDH activity was measured in supernatants. Data represent means of four different experiments in quadruplicate The results were expressed as % of viability in comparison to control. * and *** denote values significantly different from the control or between the different treatments at <i>P</i>< 0.05 and <i>P</i>< 0.0001, respectively.</p