Thiosemicarbazide

In order to determine the cytotoxicity, BJ cells were seeded in flat-bottom 96-well plates in 100 μL of a complete growth medium at a concentration of 1.7 × 104 cells/well and incubated for 24 h at 37 °C in a humidified atmosphere of 5 % CO₂. Immediately before drug treatment, the synthesized compounds (1, 2, 4, 5, 6, 7, 10) were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) and then diluted in cell culture medium supplemented with 2 % FBS. Moreover, cefepime dihydrochloride (Maxipime, Bristol-Myers Squibb Latina), chlorhexidine digluconate ((CLX) Amara Poland) and ethacridine lactate (Rivanolum, PharmaSwiss, Czech Republic) were used as reference antibacterial agents. After incubation, the growth medium was replaced with 100 μL of the appropriate serial dilutions of the investigated compounds. Untreated cells were used as negative controls, and different concentrations of DMSO were used as the solvent control. The cell cultures were incubated at 37 °C for 24 h. The cytotoxicity was estimated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described by Mosmann with some modifications (Mosmann, 1983 (link)). Briefly, the cells were incubated for 3 h with 25 μL of MTT solution (5 μg/mL in PBS buffer) per well. The MTT assay is a rapid colorimetric method based on the conversion of mitochondrial succinate dehydrogenase from yellow, soluble tetrazolium salt to blue formazan crystals to determine the number of viable cells. In order to dissolve formazan crystals in live cells, 100 μL of SDS-HCl solution (10 % SDS in 0.01 N HCl) was added per well. After overnight incubation, absorbance was measured at 570 nm using microplate reader (BioTek ELx50). The MTT assay was repeated in three independent experiments performed in octuplicates. The half-maximal cytotoxic concentration (CC₅₀) was defined as the compound concentration (μg/mL) required to reduce cell viability to 50 %. Therapeutic index (TI) is a widely accepted parameter to represent the specificity of antibacterial agents for human (Begg et al., 1999 (link)). In this study, the in vitro TI values were calculated as the ratio of CC₅₀ (cytotoxic activity) and MIC (antibacterial activity); thus, greater values of in vitro therapeutic index indicate safer specificity for eukaryotic cells.

The initial solutions were prepared at the following concentration: 100 mg/mL. Then, in order to obtain a homogeneous emulsion, sonication was carried out using an ultrasonic homogenizer (Hielscher Ultrasound Technology, Teltow, Germany).
The research involved the use of Rhabditis sp. nematodes, which live in soil in the natural environment. The culturing nematodes and research methodology of a nematocidal properties assessment was developed by the Department and Chair of Biology and Genetics of the Medical University of Lublin, Poland, patent no. 232918, Bogucka-Kocka A, Kołodziej P. The Rhabditis sp. nematodes were cultured in sterile 6-well plates on an agar medium enriched with bovine serum. After 4–5 days of culturing at room temperature, the growth and development of all the nematode development stages were observed.
Rhabditis sp. nematodes were eluted from the agar solid medium using 0.6% NaCl. They were then transferred to new sterile 24-well plates for culturing on a liquid medium. The tested compounds and albendazole were added to the culture prepared in 2 experimental concentrations: 5.56 mg/mL and 11.12 mg/mL. The respective new thiosemicarbazide derivatives were tested in a concentration corresponding to a survival rate of 50% for albendazole (LC₅₀) and half of the former. The culture of nematodes without the addition of the tested compounds constituted the negative control, while the positive control was the culture with the addition of the following reference substance with anthelmintic activity: albendazole (Sigma, Munich, Germany).
After a 24 h exposure, the culture of Rhabditis sp. nematodes was observed in terms of development, deformity, damage, and motility using a stereoscopic and light microscope (Olympus, Tokyo, Japan). To determine the viability of nematodes, they were stained with methylene blue. The live and dead specimens were counted in counting chambers.

The isothiocyanates, 2,2-dimethyl-3-(isothiocyanomethyl) norbornane isothiocyanate (ISO L) and R-1-methyl-4-(1-isothiocyano-4-isopropyl)-cyclohexene isothiocyanate (ISO C) were prepared according to the method of Silva [33 (link)], as shown in Scheme 1. A solution of hydrazine (5.15 g, 0.0491 mol) was dissolved in ethanol (25 mL), and then the isothiocyanomonoterpene (9.57 g, 0.0491 mol) was added. The mixture was stirred for 5 h at 90 °C, extracted with CHCl₃, and washed with hexane.
N(4)-[R-(1-Methyl-4-isopropylcyclohexene]-thiosemicarbazide (TIO L): White crystals; yield 85%; mp. 125 -127 °C; [α]_D +24; IR (KBr): (NH) 3370, (C=S) 1504; EI-MS m/z 227 (M^+•); ¹H-NMR: δ 5.36 (1H, brs, H-2), 1.83-1.98 (2H, m, H-3), 2.56 (1H, m, H-4), 1.75-1.79 (2H, m, H-5), 1.92-2.05 (2H, m, H-6), 1.50 (3H, s, H-8), 1.48 (3H, s, H-9), 1.64 (3H, s, H-10); ¹³C-NMR: δ 134.3 (C-1), 120.7 (C-2), 26.7 (C-3), 41.4 (C-4), 24.3 (C-5), 30.8 (C-6), 58.2 (C-7), 24.2 (C-8), 24.6 (C-9), 23.5 (C-10) and 180.7 (C-3’) (C=S).
N(4)-[2,2-Dimethyl-3-methylnorbornane]- thiosemicarbazide (TIO C): White crystals; yield 85%; mp. 106 -109 °C; [α]_D -105; IR (KBr): (NH) 3261, (C=S) 1532; EI-MS m/z 227 (M^+•); ¹H-NMR: δ 5.36 (1H, brs, H-2 ), 1.83-1.98 (2H, m, H-3), 2.56 (1H, m, H-4), 1.75-1.79 (2H, m, H-5), 1.92-2.05 (2H, m, H-6), 1.50 (3H, s, H-8), 1.48 (3H, s, H-9), 1.64 (3H, s, H-10); ¹³C-NMR: δ 134.3 (C-1), 120.7 (C-2), 26.7 (C-3), 41.4 (C-4), 24.3 (C-5), 30.8 (C-6), 58.2 (C-7), 24.2 (C-8), 24.6 (C-9), 23.5 (C-10) and 180.7 (C-3’) (C=S).

A total of 0.001 mol of 3-trifluoromethylbenzoic acid hydrazide (2) was dissolved hot in 5 mL of anhydrous ethanol. Then, 0.001 mol of isothiocyanate was added and the mixture was refluxed for 1/2 h. After this time, the contents of the flasks were cooled to room temperature, the precipitates were filtered, washed with water and diethyl ether, and allowed to air dry. The resulting thiosemicarbazide derivatives were purified by crystallization from 96% ethanol.

Yield 67%, m.p. 161.6 °C, ¹H NMR (DMSO-d₆) δ (ppm): 0.87 (t, 3H, CH₃, J = 7.3 Hz), 1.27–1.32 (m, 2H, CH₂), 1.43–1.52 (m, 2H, CH₂), 3.43 (d, 2H, CH₂, J = 6.7 Hz), 7.73–7.78 (m, 1H, ArH), 7.94–7.97 (m, 1H, ArH), 9.15–8.20 (m, 2H, ArH), 8.25 (s, 1H, NH), 9.30 (s, 1H, NH), 10.57 (s, 1H, NH). ¹³C NMR (DMSO-d₆) δ (ppm): 14.27, 19.92, 31.39, 48.89, 123.35, 124.94, 128.79, 129.48, 129.52, 130.10, 131.46, 132.36, 165.07, 177.78. Elemental analysis for C₁₃H₁₆F₃N₃OS. Calculated: C 48.89; H 5.05; N 13.16. Found: C 48.98; H 5.30; N 13.45.

Yield 69%, m.p. 176.4 °C, ¹H NMR (DMSO-d₆) δ (ppm): 7.17 (t, 1H, ArH, J = 7.2 Hz), 7.33 (t, 2H, ArH, J = 7.8 Hz), 7.41 (bs, 2H, ArH), 7.77 (t, 1H, ArH, J = 7.8 Hz), 7.97 (d, 1H, ArH, J = 7.8 Hz), 8.23 (d, 1H, ArH, J = 7.9 Hz), 9.78 (s, 1H, NH), 9.87 (s, 1H, NH), 10.83 (s, 1H, NH). ¹³C NMR (DMSO-d₆) δ (ppm): 123.35, 125.04, 125.35, 126.67, 128.53, 128.86, 129.32, 130.12, 132.44, 134.01, 139.64, 165.17, 181.60. Elemental analysis for C₁₅H₁₂F₃N₃OS. Calculated: C 53.09; H 3.56; N 12.38. Found: C 53.30; H 3.90; N 12.45.