3.1. Chemistry
All chemicals used for the synthesis were purchased from the companies Sigma-Aldrich, AlfaAesar, POCH and used without further purification. Melting points of obtained compounds were determined using Fisher-Johns block and presented without corrections. The
1H and
l3C NMR spectra were recorded on a Bruker Avance 600 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) in DMSO-d6. The chemical shifts are given in δ (ppm) scale using TMS as the standard reference. The attenuated total reflectance ATR-IR spectra were recorded over the range 4000–400 cm
−1 on the Thermo Scientific Nicolet 6700 FTIR spectrophotometer. LC-QTOF HRMS measurements were performed using 1290 HPLC coupled to 6550 ifunnel QTOF LC/MS (Agilent Technologies, Santa Clara, CA, USA). MS data and MS spectrograms are presented in
Supplementary Material (Table S1, Figures S2 and S3). The content of Cu (II) in solid complex was determined by the F-AAS spectrometer with a continuum source of light and using air/acetylene flame (Analityk Jena, contraAA 300, Jena, Germany). Absorbance was measured at analytical spectral line 324.7 nm. Limit of quantification was 0.04 mg/L. Solid sample was decomposed using the Anton Paar Multiwave 3000 closed system instrument. Mineralization was carried out for 45 min at 240 °C under pressure 60 bar. The contents of carbon, hydrogen and nitrogen were determined by a Vario micro company Elementar Analysensysteme GmbH. FTIR spectra were recorded with an IRTracer-100 Schimadzu Spectrometer (4000–600 cm
−1) with accuracy of recording 1 cm
−1 using KBr pellets. The thermolysis of compounds in air atmosphere were studied by TG-DTG techniques in the range of temperature 25–800 °C at a heating rate of 10 °C·min
−1; TG and DTG curves were recorded on Netzsch TG 209 apparatus under air atmosphere v = 20 mL·min
−1 using ceramic crucibles, and as a reference material ceramic crucibles were used.
3.1.1. General Procedure for the Synthesis of Thiosemicarbazone Derivatives (T1–T19)
Thiosemicarbazone derivatives (T1–T19) were synthesized according to the general procedure by condensation of a thiosemicarbazide (0.01 mol) with an appropriate aldehyde or ketone (0.01 mol) in ethanol. Thiosemicarbazide was dissolved in ethanol while warm and the carbonyl compound dissolved in ethanol was added to the warm solution. The resulting mixture was heated to reflux for 1–2 h. Then, the reaction mixture was cooled to room temperature, the resulting precipitate was filtered off and, after drying, it was crystallized from ethanol.
3.1.2. T1. 2-[(3,4-Dimethoxyphenyl)methylidene-N-phenyl- hydrazine-1-carbothioamide
C
16H
17N
3O
2S (315.39 g/mol), yield: 86%, m.p. 161–163°C.
1H NMR (DMSO-d
6) δ: 3.80 (s, 3H, CH
3), 3.83 (s, 3H, CH
3), 6.99–7.58 (m, 8H, CH
aromat), 8.09 (s, 1H, CH), 10.03 (s, 1H, NH), 11.74 (s, 1H, NH).
13C NMR (DMSO-d
6) δ: 56, 56, 109, 111, 122, 125, 126, 127, 128, 139, 143, 149, 151, 176 [
16]. FTIR spectra (KBr, cm
−1): ν(NH) 3334, 3311; ν(CH) 3145–2834; ν(CN) 1598, 1573; δ(NH) 1540, 1507; β(CH) 1266, 1238, 1194, 1160, 1136; ν(NN) 1021; ν(CS) 976–862; γ(CH) 790, 755, 732.
3.1.3. T2. 2-[(4-Bromophenyl)methylidene-N-methyl- hydrazine-1-carbothioamide
C
9H
10N
3SBr (272.16 g/mol), yield: 81%, m.p. 181–183 °C.
1H NMR (DMSO-d
6) δ: 3.02–3.03 (d,
J = 4.8 Hz, 3H, CH
3), 7.59–7.61 (m, 2H, CH
aromat), 7.75–7.77 (m, 2H, CH
aromat), 8.01 (s, 1H, CH), 8.57–8.58 (m, 1H, NH), 11.55 (s, 1H, NH).
13C NMR (DMSO-d
6) δ: 31, 123, 129, 132, 134, 140, 178. IR cm
−1: 3152, 2952, 1602 [
17].
3.1.4. T3. 2-[(3,4-Dimethoxyphenyl)methylidene-N-methyl- hydrazine-1-carbothioamide
C
11H
15N
3O
2S (253.32 g/mol), yield: 89%, m.p. 171–173 °C.
1H NMR (DMSO-d
6) δ: 3.04–3.05 (d,
J = 4.8 Hz, 3H, CH
3), 3.79 (s, 3H, CH
3), 3.83 (s, 3H, CH
3), 6.96–6.98 (d,
J = 8.4 Hz, 1H, CH
aromat), 7.19–7.20 (d,
J = 1.8 Hz
, 1H, CH
aromat), 7.46–7.47 (d,
J = 1.8 Hz, 1H, CH
aromat), 7.98 (s, 1H, CH), 8.42–8.43 (q,
J = 4.2 Hz, 1H, NH), 11.39 (s, 1H, NH).
13C NMR (DMSO-d
6) δ: 31, 56, 109, 111, 122, 127, 142, 149, 151, 177. IR cm
−1: 3351, 2990, 1601 [
46]. LC-QTOF HRMS (
m/
z): calculated monoisotopic mass: 253.0885, measured monoisotopic mass: 253.0889.
3.1.5. T4. 2-[1-(4-Aminophenyl)ethylidene]-N-methyl-hydrazine-1-carbothioamide
C10H14N4S (222.31 g/mol), yield: 87%, m.p. 170–172 °C. 1H NMR (DMSO-d6) δ: 2.18 (s, 3H, CH3). 3.00 (s, 3H, CH3), 3.83 (s, 3H, CH3), 5.46 (s, 2H, CH2), 6.54–6.55 (q, J = 8.4 Hz, 2H, CHaromat), 7.63–7.64 (d, J = 8.4 Hz, 2H, CHaromat), 8.26–8.27 (d, J = 4.2 Hz, 1H, NH), 9.96 (s, 1H, NH). LC-QTOF HRMS (m/z): calculated monoisotopic mass: 222.0939, measured monoisotopic mass: 222.0944.
3.1.6. T5. 2-[(3,4-Dichlorophenyl)methylidene]-N-methyl- hydrazine-1-carbothioamide
C
9H
9N
3SCl
2 (262.26 g/mol), yield: 80%, m.p. 192–194 °C.
1H NMR (DMSO-d
6) δ: 3.02 (s, 3H, CH
3), 7.66–7.99 (m, 3H, CH
aromat), 8.21 (s, 1H, CH), 8.69 (q,
J = 4.2 Hz, 1H, NH), 11.64 (s, 1H, NH).
13C NMR (DMSO-d
6) δ: 31, 128, 131, 132, 135, 139, 178. IR cm
−1: 3150, 2930, 1587 [
18]. LC-QTOF HRMS (
m/
z): calculated monoisotopic mass: 260.9894, measured monoisotopic mass: 260.9896.
3.1.7. T6. 2-[(4-Bromophenyl)methylidene]-N-(2-methylphenyl)hydrazine-1-carbothioamide
C15H14N3SBr (348,26 g/mol), yield: 85%, m.p. 178–180 °C. 1H NMR (DMSO-d6) δ: 2.23 (s, 3H, CH3), 7.21–7.28 (m, 4H, CHaromat), 7.61–7.88 (m, 4H, CHaromat), 8.10 (s, 1H, CH), 10.05 (s, 1H, NH), 11.58 (s, 1H, NH). IR cm−1: 3150, 2930, 1587. LC-QTOF HRMS (m/z): calculated monoisotopic mass: 347.0092, measured monoisotopic mass: 347.0096.
3.1.8. T7. 2-[(3,4-Dimethoxyphenyl)methylidene]-N-(2-methylphenyl)hydrazine-1-carbothioamide
C17H19N3O2S (329.41 g/mol), yield: 85%, m.p. 205–206°C. 1H NMR (DMSO-d6) δ: 2.25 (s, 3H, CH3), 3.80 (s, 3H, CH3), 3.82 (s, 3H, CH3), 6.98–6.99 (m, 1H, CHaromat), 7.21–7.24 (m, 4H, CHaromat), 7.28–7.29 (m, 1H, CHaromat), 7.35–7.37 (m, 1H, CHaromat), 7.59–7.60 (m, 1H, CHaromat), 8.08 (s, 1H, CH), 9.87 (s, 1H, NH), 11.70 (s, 1H, NH). LC-QTOF HRMS (m/z): calculated monoisotopic mass: 329.1198, measured monoisotopic mass: 329.1202.
3.1.9. T8. 2-[(3,4-Dichlorophenyl)methylidene]-N-(2-methylphenyl)hydrazine-1-carbothioamide
C15H13N3SCl2 (338,25 g/mol), yield: 84%, m.p. 200–202 °C. 1H NMR (DMSO-d6) δ: 2.24 (s, 3H, CH3), 7.22–7.30 (m, 4H, CHaromat), 7.65–7.67 (m, 1H, CHaromat), 7.77–7.78 (m, 1H, CHaromat), 7.79–8.10 (m, 1H, CHaromat), 8.37 (s, 1H, CH), 10.15 (s, 1H, NH), 11.94 (s, 1H, NH). LC-QTOF HRMS (m/z): calculated monoisotopic mass: 337.0207, measured monoisotopic mass: 337.0211.
3.1.10. T9. 2-[(4-Bromophenyl)methylidene]-N-(2-chlorophenyl)hydrazine-1-carbothioamide
C14H11N3SBrCl (368,68 g/mol), yield: 86%, m.p. 209–211 °C. 1H NMR (DMSO-d6) δ: 7.31–7.86 (m, 8H, CHaromat), 8.13 (s, 1H, CH), 10.15 (s, 1H, NH), 12.04 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 123, 127, 128, 129, 130, 131, 132, 133, 137, 142, 177. IR cm−1: 3137, 2970, 1738. LC-QTOF HRMS (m/z): calculated monoisotopic mass: 366.9546, measured monoisotopic mass: 366.9545.
3.1.11. T10. 2-[(3,4-Dimethoxyphenyl)methylidene-N-(2-chlorophenyl)hydrazine-1-carbothioamide
C16H16N3O2SCl (349.84 g/mol), yield: 78%, m.p. 179–180°C. 1H NMR (DMSO-d6) δ: 3.80 (s, 3H, CH3), 3.82 (s, 3H, CH3), 7.00–7.86 (m, 7H, CHaromat), 8.10 (s, 1H, CH), 10.03 (s, 1H, NH), 11.94 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 56, 109, 111, 123, 126, 127, 128, 129, 130, 136, 143, 149, 151, 176. FTIR spectra (KBr, cm−1): ν(NH) 3246; ν(CH) 3144–2827; ν(CN) 1595, 1579; δ(NH) 1552, 1507; β(CH) 1267, 1238, 1194, 1162, 1134; ν(NN) 1023; ν(CS) 978–858; γ(CH) 799, 753, 723. LC-QTOF HRMS (m/z): calculated monoisotopic mass: 349.0652, measured monoisotopic mass: 349.0653.
3.1.12. T11. N-(2-Chlorophenyl)-2-(1-phenylethylidene)hydrazine-1-carbothioamide
C15H14N3SCl (303.80 g/mol), yield: 78%, m.p. 115–117°C. 1H NMR (DMSO-d6) δ: 2.41 (s, 3H, CH3), 7.28–8.01 (m, 8H, CHaromat), 10.12 (s, 1H, NH), 10.89 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 125, 126, 127, 129, 136, 179. IR cm−1: 3599, 2970, 1594. LC-QTOF HRMS (m/z): calculated monoisotopic mass: 303.0597, measured monoisotopic mass: 303.0601.
3.1.13. T12. 2-[(3,4-Dichlorophenyl)methylidene]-N-(2-chlorophenyl)hydrazine-1-carbothioamide
C14H10N3SCl3 (358.67 g/mol), yield: 81%, m.p. 210–211 °C. 1H NMR (DMSO-d6) δ: 7.33–8.11 (m, 7H, CHaromat), 8.34 (s, 1H, CH), 10.24 (s, 1H, NH), 12.11 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 127, 128, 129, 131, 132, 135, 137, 140, 177. FTIR spectra (KBr, cm−1): ν(NH) 3315, 3258; ν(CH) 3140–2983; ν(CN) 1591; δ(NH) 1545, 1512; β(CH) 1265, 1204, 1197, 1114; ν(NN) 1031; ν(CS) 960–825; γ(CH) 787, 736, 723. LC-QTOF HRMS (m/z): calculated monoisotopic mass: 356.9661, measured monoisotopic mass: 356.9654.
3.1.14. T13. 2-[(4-Bromophenyl)methylidene]-N-(3-chlorophenyl)hydrazine-1-carbothioamide
C14H11N3SBrCl (368.68 g/mol), yield: 86%, m.p. 162–164 °C. 1H NMR (DMSO-d6) δ: 7.26–7.89 (m, 8H, CHaromat), 8.14 (s, 1H, CH), 10.23 (s, 1H, NH), 12.02 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 123, 124, 125, 130, 132, 132, 133, 141, 142, 176. FTIR spectra (KBr, cm−1): ν(NH) 3330; ν(CH) 3128–2972; ν(CN) 1578; δ(NH) 1536, 1499; β(CH) 1277, 1253, 1204, 1129; ν(NN) 1009; ν(CS) 964–867; γ(CH) 780, 746, 703. LC-QTOF HRMS (m/z): calculated monoisotopic mass: 366.9546, measured monoisotopic mass: 366.9551.
3.1.15. T14. 2-[(4,5-Dimethoxyphenyl)methylidene-N-(3-chlorophenyl)hydrazine-1-carbothioamide
C
16H
16N
3O
2SCl (349.84 g/mol), yield: 80%, m.p. 180–182 °C.
1H NMR (DMSO-d
6) δ: 3.81 (s, 3H, CH
3), 3.84 (s, 3H, CH
3), 6.99–7.79 (m, 7H, CH
aromat), 8.11 (s, 1H, CH), 10.10 (s, 1H, NH), 11.88 (s, 1H, NH).
13C NMR (DMSO-d
6) δ:. 56, 109, 111, 122, 124, 125, 126, 130, 132, 141, 144, 149, 151, 175. IR cm
−1: 3140, 2834, 1597 [
18].
3.1.16. T15. N-(3-Chlorophenyl)-2-[(3,4-dichlorophenyl)methylidene]hydrazine-1-carbothioamide
C
14H
10N
3SCl
2 (358.67 g/mol), yield: 77%, m.p. 210–212 °C.
1H NMR (DMSO-d
6) δ: 7.28–8.12 (m, 7H, CH
aromat), 8.33 (s, 1H, CH), 10.30 (s, 1H, NH), 12.09 (s, 1H, NH) [
20].
3.1.17. T16. 2-[(4,5-Dimethoxyphenyl)methylidene-N-(4-chlorophenyl)hydrazine-1-carbothioamide
C
16H
16N
3O
2SCl (349.84 g/mol), yield: 76%, m.p. 200–202 °C.
1H NMR (DMSO-d
6) δ: 3.80 (s, 3H, CH
3), 3.83 (s, 3H, CH
3), 6.99–7.64 (m, 7H, CH
aromat), 8.10 (s, 1H, CH), 10.07 (s, 1H, NH), 11.83 (s, 1H, NH).
13C NMR (DMSO-d
6) δ: 56, 109, 111, 122, 126, 128, 129, 138, 144, 149, 151, 176 [
19]. FTIR spectra (KBr, cm
−1): ν(NH) 3331, 3326; ν(CH) 3142–2828; ν(CN) 1599, 1574; δ(NH) 1545, 1505; β(CH) 1265, 1235, 1210, 1140; ν(NN) 1019; ν(CS) 976–832; γ(CH) 795, 768, 746, 716.
3.1.18. T17. N-(4-Chlorophenyl)-2-[(3,4-dichlorophenyl)methylidene]hydrazine-1-carbothioamide
C14H10N3SCl3 (358.67 g/mol), yield: 83%, m.p. 202–204 °C, 1H NMR (DMSO-d6) δ: 7.43–8.12 (m, 7H, CHaromat), 8.34 (s, 1H, CH), 10.29 (s, 1H, NH), 12.05 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 128, 129, 130, 131, 132, 135, 138, 141, 177. IR cm−1: 3122, 2970, 1589. LC-QTOF HRMS (m/z): calculated monoisotopic mass: 356.9661, measured monoisotopic mass: 356.9664.
3.1.19. T18. 2-[(4-Bromophenyl)methylidene]-N-(4-chlorophenyl)hydrazine-1-carbothioamide
C
14H
11N
3SBrCl (368.68 g/mol), yield: 89%, m.p. 182–184 °C.
1H NMR (DMSO-d
6) δ: 7.42–7.89 (m, 8H, CH
aromat), 8.13 (s, 1H, CH), 10.21 (s, 1H, NH), 11.98 (s, 1H, NH).
13C NMR (DMSO-d
6) δ: 123, 128, 129, 130, 132, 133, 138, 142, 176, IR cm
−1: 3128, 2971, 1589 [
20].
3.1.20. T19. N-(3-Chlorophenyl)-2-(1-phenylethylidene) hydrazine-1-carbothioamide
C
15H
14N
3SCl (303,81 g/mol), yield: 80%, m.p. 162–165 °C.
1H NMR (DMSO-d
6) δ: 2.39 (s, 3H, CH
3), 7.26–7.69, 8,02 (m, 9H, CH
aromat), 10.11 (s, 1H, NH), 10.77 (s, 1H, NH).
13C NMR (DMSO-d
6) δ: 122, 123, 124, 130, 132, 141, 176. IR cm
−1: 3138, 2970, 1586 [
21].
3.2. X-ray Structure Determination
X-ray data of T2, T3 and T5 were collected on the KUMA Diffraction KM-4 CCD diffractometer; MoKα (λ = 0.71073 Å) radiation, ω scans, T = 296(2) K; crystal sizes 0.50 × 0.50 × 0.10 mm for T2, 0.50 × 0.50 × 0.30 mm for T3 and 0.50 × 0.40 × 0.30 mm for T5, absorption correction: multi-scan CrysAlisPro [
47], Tmin/Tmax of 0.0054/1.0000, 0.7525/1.0000 and 0.2426/1.0000 for T2, T3, and T5, respectively. The structures were solved by direct methods using SHELXS97 [
48] and refined by full-matrix least-squares with SHELXL-2014/7 [
48]. The N-bound H atoms were located by difference Fourier synthesis and refined freely. The remaining H atoms were positioned geometrically and treated as riding on their parent C atoms with C-H distances of 0.93 Å (aromatic) and 0.96 Å (CH
3). All H atoms were refined with isotropic displacement parameters taken as 1.5 times those of the respective parent atoms. All calculations were performed using WINGX version 1.64.05 package [
49]. CCDC-2058475 for T2, 2058476 for T3, and 2058477 for T5 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge at
www.ccdc.cam.ac.uk/conts/retrieving.html (accessed on 15 Mar 2021) [or from the Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0)-1223-336-033; email:
[email protected]].
Crystal data of T2: C9H10N3SBr, M = 272.17, orthorhombic, space group Pbca, a = 13.580(15), b = 8.5494(8), c = 19.0926(18) Å, V = 2216.7(4) Å3, Z = 8, dcalc = 1.631 Mg m−3, F(000) = 1088, μ(MoKα) = 3.862 mm−1, T = 296K, 12,588 measured reflections (θ range 2.13–28.10°), 2431 unique reflections, final R = 0.049, wR = 0.129, S = 1.009 for 1713 reflections with I > 2σ(I).
Crystal data of T3: C11H15N3O2S, M = 253.32, monoclinic, space group P21/c, a = 8.5409(7), b = 8.1329(5), c = 18.8922(12) Å, β = 102.099(7)°, V = 1283.15(16) Å3, Z = 4, dcalc = 1.311Mg m−3, F(000) = 536, μ(Mo Kα) = 0.247 mm−1, T = 296K, 8182 measured reflections (θ range 2.20–27.78°), 2666 unique reflections, final R = 0.031, wR= 0.085, S = 1.045 for 2366 reflections with I > 2σ(I).
Crystal data of T5:C9H9N3SCl2, M = 262.15, monoclinic, space group C2/c, a = 12.8992(10), b = 10.0640(8), c = 18.4826(13) Å, β = 96.754(7), V = 2382.7(3) Å3, Z = 8, dcalc = 1.462 Mg m−3, F(000) = 1072, μ(MoKα) = 0.690 mm−1, T = 296K, 6897 measured reflections (θ range 2.22–28.06°), 2493 unique reflections, final R = 0.044, wR= 0.127, S = 1.066 for 1981 reflections with I > 2σ(I).
3.4. General Procedure for the Synthesis of Cu (II) Complex of Thiosemicarbazone Derivatives
Cu(T1)Cl2, Cu(T10)2Cl2, Cu(T12)2Cl2, Cu(T13)Cl2, Cu(T16)Cl2) Synthesis of complexes was carried out in MeOH/EtOH (v/v = 1/1) mixture. In all cases molar ratio of organic ligand and copper(II) chloride dihydrate was 1:1. Total volume of reaction mixtures did not exceed 60 mL. After 6 h, green precipitates of Cu(T1)Cl2, Cu(T10)2Cl2, Cu(T13)Cl2, Cu(T16)Cl2, and yellow precipitate of Cu(T12)2Cl2 were filtered and washed three times with small amounts of MeOH/EtOH. Solid compounds were then dried in open air for several days and weighted.
Cu(T1)Cl2 (C16H17N3SO2CuCl2) (449.87 g/mol), yield (47%), anal. calculated (%): Cu, 14.12; C, 42.72; H, 3.81; N, 9.34. Found (%): Cu, 14.39; C, 42.50; H, 3.79; N, 8.85. FTIR spectra (KBr, cm−1): ν(NH) 3442, 3343; ν(CH) 3056–2833; ν(CN) 1590; δ(NH) 1500; β(CH) 1266, 1168, 1139; ν(NN) 1021; ν(CS) 853, 808; γ(CH) 764, 728.
Cu(T10)2Cl2 (C32H32N6S2O4CuCl4) (834.18 g/mol), yield (75%), anal. calculated (%): Cu, 7.62; C, 46.07; H, 3.87; N, 10.08. Found (%): Cu, 8.08; C, 46.25; H, 3.83; N, 9.66. FTIR spectra (KBr, cm−1): ν(NH) 3251; ν(CH) 3116–2828; ν(CN) 1597, 1576; δ(NH) 1555, 1508; β(CH) 1267, 1240; ν(NN) 1021; ν(CS) 939, 851; γ(CH) 738, 722.
Cu(T12)2Cl2 (C28H20N6S2CuCl8) (851.82 g/mol), yield (69%), anal. calculated (%): Cu, 7.46; C, 39.48; H, 2.37; N, 9.87. Found (%): Cu, 6.86; C, 39.69; H, 2.40; N, 9.71. FTIR spectra (KBr, cm−1): ν(NH) 3305, 3272; ν(CH) 3092, 2923; ν(CN) 1592; δ(NH) 1549, 1520; β(CH) 1278, 1205, 1197, 1128; ν(NN) 1031; ν(CS) 941, 900, 877; γ(CH) 745, 731.
Cu(T13)Cl2 (C14H11N3SCuCl3Br) (503.14 g/mol), yield (62%), anal. calculated (%): Cu, 12.63; C, 33.42; H, 2.20; N, 8.35. Found (%): Cu, 12.35; C, 33.26; H, 2.16; N, 8.23. FTIR spectra (KBr, cm−1): ν(NH) 3382, 3279; ν(CH) 3167–3067; ν(CN) 1589; δ(NH) 1537, 1505; β(CH) 1278, 1253, 1205, 1128; ν(NN) 1010; ν(CS) 959, 912, 871; γ(CH) 774, 745.
Cu(T16)Cl2 (C16H16N3SO2CuCl3) (484.31 g/mol), yield (35%), anal. calculated (%): Cu, 13.12; C, 39.68; H, 3.34; N, 8.68. Found (%): Cu, 13.15; C, 39.93; H, 3.30; N, 8.21. FTIR spectra (KBr, cm−1): ν(NH) 3424, 3265; ν(CH) 3096–2834; ν(CN) 1590; δ(NH) 1528, 1492; β(CH) 1268, 1177; ν(NN) 1020; ν(CS) 939, 858, 828; γ(CH) 768, 746, 718.
3.5. Biological Assays
3.5.1. Cell Culturing
The evaluation of cytotoxicity of tested compounds was carried out on the G361, A375, and SK-MEL-28 human melanoma cells and BJ human normal fibroblast cells. The cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured at 37 °C in the presence of 5% CO2 in atmosphere. The culture medium (Dulbecco’s Modified Eagle’s Medium for G361 and A375 and Eagle’s Minimum Essential Medium for SK-MEL-28 and BJ) was supplemented with 10% foetal bovine serum. The cells morphology was examined under inverted phase contrast microscope Nikon Eclipse Ti (Nikon, Tokyo, Japan). The authenticity of tested cell lines was verified by short tandem repeat (STR) genotyping in the Department of Forensic Medicine (Medical University of Lublin, Lublin, Poland).
3.5.2. Cell Viability Assay
The cells were seeded into 96-well plates (in concentration: BJ, A375, and SK-MEL-28—1 × 10
5 cells/mL, G361—1.5 × 10
5 cells/mL) and cultured until they reach 70–80% confluency. Then the cells were incubated with tested compounds and dacarbazine in concentrations ranging from 10 to 500 µM or DMSO as vehicle in control cultures for next 24 h (max. DMSO concentration <0.5%). The compounds cytotoxicity was evaluated with the MTT colorimetric method based on the ability of viable cells to the transformation of tetrazolium salts (3-[4,5-dimethylthiazol-2-yl]-2,5-) diphenyltetrazolium bromide, MTT) to purple formazan, by cellular dehydrogenases. After 24-h-incubation cell cultures were supplemented with 20 µL of 5 mg/mL MTT (Thermofisher, Waltham, MA, USA) stock in PBS (Corning, NY, USA), and the incubation was continued for 4 h at 37 °C. Next, the medium with MTT was removed, and the formed crystals were dissolved in 200 µL of DMSO (dimethyl sulfoxide, Avantor, Gliwice, Poland). The solution absorbency was measured at 570 nm, using a PowerWave™ xs microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA). The experiment was performed three times with three replicates for each concentration of tested compounds. IC
50 values were determined using the AAT Bioquest IC
50 calculator [
51]. On the basis of obtained IC
50 values for normal and malignant cells, A375 cell line and three tested compounds (CuT1, Cu10, CuT16) were chosen for further investigation.
3.5.3. Cell Cycle Assay
Cell-cycle assay was performed using NC 3000 system (ChemoMetec, Lillerød, Denmark) according to manufacturer’s protocol for the two-step cell cycle analysis. The A375 cells were seeded in 12-well plates at density of 1 × 105 cells/mL and after 70–80% confluency achievement the cells were treated with CuT1, Cu10, CuT16 in concentrations corresponding to IC50 values or DMSO as vehicle in control cultures and analysed after 24h incubation. The cells were washed with PBS (Corning, New York, NY, USA), incubated with lysis buffer (Solution 10) supplemented with 10 µg/mL of DAPI for 5 min at 37 °C. After addition of stabilization buffer (Solution 11) the samples were analysed. Fluorescence signal was quantified using a NucleoCounter® NC-3000™ image cytometer (ChemoMetec USA Inc., Lillerød, Denmark). Data were derived from three independent experiments with three replicates for each concentration of tested compound.
3.5.4. Cell Apoptosis Assay
Cell apoptosis assay was performed using NC 3000 system (ChemoMetec USA Inc., Lillerød, Denmark) according to the manufacturer’s protocol for Annexin V Assay. The A375 cells were seeded in 6-well culture plates at a density 1 × 105 cells/mL and after 70–80% confluency achievement the cells were treated with CuT1, Cu10, CuT16 in concentrations corresponding to IC50 values or DMSO as vehicle in control cultures and analysed after 24h incubation. The cells were dissociated into single-cell suspensions in PBS (Corning, New York, NY, USA) and app. 4 × 105 cells were resuspended in 100µl of Annexin V binding buffer and incubated with Annexin V—CF488A conjugate and Hoechst 33,342 for 15 min at 37 °C. Next, the cells were washed with Annexin V binding buffer and the cell pellets were resuspended in 100 mL of Annexin V binding buffer supplemented with 10 µg/mL of propidium iodide (PI) and Annexin V Assay was performed immediately. Cellular fluorescence was quantified using a NucleoCounter® NC-3000™ image cytometer (ChemoMetec USA Inc., Lillerød, Denmark). Data were derived from three independent experiments with three replicates for each concentration of tested compounds.
3.5.5. DNA Damage Evaluation
DNA damage was evaluated on the basis of a basic (AP) sites presence, that arose in response to oxidative damage or as an intermediate in base excision repair pathway. The A375 cells were seeded in 6-well culture plates at a density 1 × 105 cells/mL and after 70–80% confluency achievement the cells were treated with CuT1, Cu10, CuT16 in concentrations corresponding to IC50 values or DMSO as vehicle in control cultures. After 24 h of incubation genomic DNA was isolated using Syngen DNA Mini Kit (Syngen Biotech, Wroclaw, Poland) according to the manufacturer’s instruction. The concentrations of isolated DNA for all samples were equalized to 100 µg/mL. The measurements of the number of AP sites were evaluated with DNA Damage Quantification Kit (Dojindo, Japan) according to the manufacturer’s instruction. The DNA was treated with Aldehyde Reactive Probe (ARP) to tag all of the AP sites with a biotin residue. Then all of the AP sites were quantified using avidin—biotin assay. The coloured product of horseradish peroxidase conjugated to the avidin was detected at 650 nm using a PowerWave™ xs microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA).
3.5.6. Gene Expression Evaluation
The expression level of genes connected with DNA repair and oxidative stress response was evaluated with quantitative real-time PCR (qRT-PCR) method. The A375 cells were seeded in 6-well culture plates at a density 1 × 10
5 cells/mL and after 70–80% confluency achievement the cells were treated with CuT1, Cu10, CuT16 in concentrations corresponding to IC
50 values or DMSO as vehicle in control cultures. After 24h of incubation total RNA was isolated using the Syngen Blood/Cell RNA Mini Kit (Syngen Biotech, Wroclaw, Poland). All samples were transcribed using NG dART RT-PCR reagents (EURx, Gdansk, Poland) according to the manufacturer’s instructions. The relative mRNA expression level was determined by relative quantification method (ΔΔCt) using 18S ribosomal N5 (RNA18SN5) and beta-actin (ACTB) as endogenous controls. The reference genes were selected on the basis of our preliminary studies, where
RNA18SN5 and
ACTB remained unaffected by the experimental conditions. The PCR was conducted in triplicate using Applied Biosystems
® 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) and SG qPCR Master Mix (2×) (EURx, Gdansk, Poland) in accordance with the manufacturer’s protocol. Data was presented and analysed as RQ values (RQ = 2
−ΔΔCt). The genes and sequences of used primers were listed in
Table 5.
3.5.7. X-ray Radiation
G361, A375 and SK-MEL-28 were seeded in 6-well plates at following density: A375 and SK-MEL-28—1 × 105 cells/mL, G361—1.5 × 105 cells/mL and were irradiated using the RS-2000 Biological Research Irradiator (Rad Source Technologies, Buford, GE, USA). In the studies radiation at the dose of 10 Gy was used. X-ray treated cells were incubated for further 48 h under standard culturing conditions (characterised in Cells culturing section). Control cells that had not been exposed to X-rays were also cultured for 48 h. To compare all three cell lines responses, cell cycle analysis was conducted using NC 3000 system as described above.
3.5.8. Statistical Analysis
Statistical analysis was performer using STATISTICA 13 software (StatSoft, Krakow, Poland). Comparison of values was performed by one-way analysis of variance (ANOVA) and post hoc multiple comparisons with Tukey’s honest significant difference test (Tukey’s HSD test). The data were calculated as mean ± SD. If the p-value was under 0.05, the results were considered statistically significant.