Thiadiazoles are an important group of five-membered heterocycles demonstrating extraordinary physiochemical properties including dual fluorescence emission [1
], crystal solvatomorphism [4
], and keto-enol-like tautomerism [5
]. Numerous reports have highlighted the antimicrobial, anticancer, antioxidative, or anticonvulsant activities as characteristic of 1,3,4-thiadiazoles [6
]. Due to these interesting features the thiadiazole-derived compounds were extensively studied in our group [9
], with several 1,3,4-thiadiazole derivatives reported to possess significant acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibition activities rendering them as potential anti-neurodegenerative agents [14
Inhibition of AChE and BuChE enzymes is one of the existing approaches taken in the design of novel anti-neurodegenerative agents. Given that neurodegenerative disorders may result from a perturbed homeostasis of essential metals such as Cu(II) and Zn(II) [15
], engineering of novel metal chelators possessing AChE and BuChE inhibitory ability has been proposed as a new approach to the treatment of neurodegenerative disorders such as Alzheimer’s or Parkinson’s diseases [17
Our previous studies on thiadiazole derivatives have focused on examination of the metal-binding ability of 1,3,4-thiadiazoles bearing the o
-hydroxyphenyl moiety at C5 carbon, which were used for the isolation of a series of Zn(II), Cu(II), and Pd(II) complexes [21
]. The spectroscopic characterization of these complexes revealed significant structural differences in their metal–ligand ratios depending on the central metal type; however, in all those complexes the o
-hydroxyphenyl moiety together with the neighboring thiadiazole nitrogen were identified as the metal binding sites. These findings were consistent with widely reported fact that thiadiazole-derived ligands may demonstrate versatile coordination modes which strongly depend on the presence of additional substituents [8
]. This diversity is particularly high in case of the coordination to Cu(II) and Zn(II) ions, which are well-known for their ability to adopt a wide variety of coordination modes [25
Our current studies focus on the isolation of 1,3,4-thiadiazole ligands 1
, which would keep their metal-chelating ability, while offering the possibility for additional structural modifications (Figure 1
). Therefore in this work, the o
-hydroxyphenyl attached to the C5 carbon of the thiadiazole ring remained a main structural motif, while the C2 position was substituted by the simple -NH2
group, as the family of 2-amino-1,3,4-thiadiazoles belong to the most extensively studied thiadiazole derivatives. Secondly, regardless of the fact that the most biologically active 2-amino-1,3,4-thiadiazoles are usually substituted with an aromatic ring at their C5 carbon, such tandems with polyphenolic moieties are limited. This relative scarcity prompted us to revisit the classical synthetic route in the 1,3,4-thiadiazloes synthesis aiming at obtaining 2-amino-2-(2,4-dihydroxy)-1,3,4-thiadiazole 1
as a model ligand for subsequent reaction with Zn(II) and Cu(II) salts. Introduction of additional reactive substituents, and especially the lone electron pair donors such as -OH and -NH2
, is associated with an increase in the number of potential metal-binding sites. In order to assess the possibility for alternative coordination modes, compound 1
was modified by acetylating its -NH2
and both its -OH groups. The metal coordination ability of the resulting mono-acetylated and tri-acetylated derivatives (2
, respectively) was compared to that of 1
). The structures of all compounds were elucidated using spectroscopic methods. Moreover, antioxidant activity testing and antimicrobial screening against a limited number of bacterial strains was performed on the thiadiazole free ligands and their Zn(II) complexes. Therefore, the main aim of our current work was an isolation and structural elucidation of newly synthesized thiadiazole derivatives and spectroscopic examination of their metal-binding ability. Secondly, given the fact that the thiadiazole derivatives and especially the metal complexes obtained are novel, their antibacterial and antioxidant activity was assessed for a first time. Thirdly, based on the synergistic antifungal effects that are characteristic of the structurally similar thiadiazoles [12
], a possibility for the synergism with commercial antibacterial agent, kanamycin, was examined. Our studies were driven by hypotheses that determination of the metal-binding ability of thiadiazole ligands obtained may be a new approach to treatment of neurodegenerative disorders, while the assessment of their synergistic interactions with known antibiotics may shed new light on the formulation of more effective antibacterial medicines.
3. Materials and Methods
All chemicals used for the syntheses were of reagent grade or higher. 2,4-dihydroxybenzaldehyde, thiosemicarbazide, acetic anhydride, phosphorous oxychloride, Trolox (6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl), DMSO-d6, and MS grade methanol were purchased from Aldrich (Darmstadt, Germany). Concentrated HCl and solid NaOH were purchased from ChemPur (Piekary Śląskie, Poland). Ethanol, methanol, acetonitrile, formic acid, Zn(CH3COOH)2, and Cu(CH3COOH)2 were purchased from Avantor (Gliwice, Poland). All solvents were of 99% purity or higher (HPLC grade).
The NMR spectra, were acquired on a Bruker Avance III spectrometer (500 MHz) (Bruker, Coventry, UK), using d6
-DMSO as solvent. Signal assignments were made using standard techniques including HSQC and HMBC experiments. The infrared spectra were recorded in the region of 4000 cm−1
to 450 cm−1
on a Thermo Scientific Nicolet iS5 Fourier-transform infrared spectrophotometer equipped with the iD7 ATR adapter (Shimadzu, Kyoto, Japan). The electronic absorption and steady-state fluorescence measurements, antioxidant, and antibacterial assays were performed in 96-well plates on a Tecan Infinite 200 microplate reader (Tecan Austria GmbH, Grödig/Salzburg, Austria). HPLC-ESI-MS analyses were performed on a Shimadzu 8030 ESI-Triple Quad mass spectrometer (Shimadzu, Kyoto, Japan). All HPLC-MS analyses were performed in positive ion mode. The HPLC solvents gradient was 40% B in A at 0 min to 90% B in A at 15 min (A: 2% v/v
formic acid in water; B: methanol). Helium (He) was used as a collision gas during collision-induced (CID) MS/MS experiments and collision energy (CE) was set at −35 V. The X-ray data collection for single crystal (T = 120K) was carried out on a SuperNova diffractometer (Oxford Diffraction, Oxford, UK), with micro-focusing source of CuKα radiation. Indexing, integration, and scaling was done using CrysAlis RED software [44
]. The structure was solved with direct methods and then successive least-square refinements were carried out, based on the full-matrix least-squares on F2
using the SHELX program package [45
]. All heavy atoms were refined anisotropically. Hydrogen atoms were fitted isotropically with geometry idealized positions except those forming intermolecular H-bonds. Table S1
includes experimental details for a measured single crystal. Presented structure has been deposited in the CCDC with no. 1845297. Melting point values were recorded on a Stuart SMP20 apparatus within the range of 25–300 °C, and were uncorrected.
|Synthesis of 2-amino-5-(2,4-dihydroksyphenyl)-1,3,4-thiadiazole||(1)|
2,4-dihydroxybenzoic acid (5.00 g, 32.00 mmol) was suspended in POCl3
(15 mL) and stirred at room temperature for 20 min. Thiosemicarbazide (2.95 g, 32.00 mmol) was then added and the reaction mixture was refluxed at 75 °C and stirred for 12 hours. The thick, yellow slurry that formed was cooled down to 30 °C followed by quenching the excess POCl3
by slow addition of small aliquots of water. The mixture was then refluxed at 105 °C for 5 h and then it was cooled down to ambient temperature and the pH was then brought to 8.5 with saturated NaOH. The precipitate formed was filtered off, washed with water, and allowed to dry in air. The dry solid was washed thoroughly with methanol and the solution was evaporated to dry under reduced pressure yielding compound 1
. Single crystals suitable for X-ray diffraction were grown in ethanol. Yield: 5.32 g (79%); C8
S (209.22 g/mol); calc: C 45.93, H 3.37, N 20.08%, found: C 42.96, H 3.30, N 17.84%; M.P.: 252–255 °C; 1
H-NMR (DMSO): δ = 10.91 ppm (s, 1H, H9, (-OH)), 9.84 (s, 1H, H7, (-OH)), 7.53 (d, 1H, H11, J
= 8.51 Hz), 7.15 (s, 2H, H12 (-NH2
)), 6.38 (d, 1H, H8, J
= 2.28 Hz), 6.36 (dd, 1H, H10, J1
= 8.51, J2
= 2.28 Hz); 13
C-NMR (DMSO): 167.84 ppm (C5), 160.22 (C9), 156.43 (C7), 156.03 (C2), 129.20 (C11), 108.99 (C6), 108.35 (C10), 102.99 (C8); IR (ATR): 3385, 3320, 3206, 2656, 2584, 1628, 1604, 1530, 1514, 1472, 1317, 1268, 1174, 1125,1057, 983, 967, 831, 761, 655, 459 cm−1
; UV-Vis (MeOH): λ1
= 294, λ2
= 324 nm; Fluorescence (MeOH): λEm(Ex290)
= 380 nm.
|Synthesis of 2-acetamido-5-(2,4-dihydroksyphenyl)-1,3,4-thiadiazole||(2)|
(1.00 g, 4.78 mmol) was refluxed in the mixture of acetic anhydride (10 mL) and water (4 mL) for 6 hours. The reaction mixture was then cooled to ambient temperature and the solid was filtered off, washed with water, and dried. The product was recrystallized from ethanol yielding 0.86 g (72%) of 2. Yield: 0.91 g (72%); C10
S (251.26 g/mol); calc: C 47.80, H 3.61, N 16.72%, found: C 46.34, H 3.41, N 16.21%, M.P.: >300 °C; 1
H-NMR (DMSO): δ = 12,32 (s, 1H, H12 (-NH-)), 10.90 (s, 1H, H7(-OH)), 9.92 (s, 1H, H9 (-OH)), 7.91 (d, 1H, H11, J
= 8.74 Hz), 6.45 (d, 1H, H8, J
= 2.30 Hz), 6.40 (dd, 1H, H10, J1
= 8.74, J2
= 2.30 Hz), 2.18 (s, 3H, H14); 13
C-NMR (DMSO): 168.79 ppm (C2), 160.94 (C9), 158. 89 (C13), 158. 87 (C5), 156.35 (C7), 129.03 (C11), 109.15 (C6), 108.62 (C10), 102.88 (C8), 22.88 (C14); IR (ATR): 3309, 3158, 2885, 2791,1680,1626, 1597,1557, 1527, 1483, 1415, 1310, 1217, 1180, 1129, 974, 841, 804, 709, 681, 659, 623, 518, 467 cm−1
; UV-Vis (MeOH): λ1
= 292, λ2
= 324 nm; Fluorescence (MeOH): λEm(Ex290)
= 377 nm.
|Synthesis of 2-acetamido-5-((phenyl-2,4-diacetate)-yl)-1,3,4-thiadiazole||(3)|
(0.36 g, 1.70 mmol) was suspended in acetic anhydride (10 mL) and three drops of concentrated H2
was added. The mixture was refluxed for 6 h and then cooled to ambient temperature and the solid was filtered off, washed with water, and allowed to dry in air. The crude product was recrystallized from ethanol yielding 0.48 g (58%) of 3
. Yield: 0.48 g (58%); C14
S (335.33 g/mol); calc: C 50.15, H 3.91, N 12.53%, found: C 49.02, H 3.66, N 12.50%; M.P.: 269–271 °C; 1
H-NMR (DMSO): δ = 12,68 (s, 1H, H12 (-NH-)), 8.23 (d, 1H, H11, J
= 8.56 Hz), 7.29 (d, 1H, H8, J
= 2.27 Hz), 7.26 (dd, 1H, H10, J1
= 8.56, J2
= 2.27 Hz), 2.38 (s, 3H, H18), 2.31 (s, 3H, H16), 2.22 (s, 3H, H14); 13
C-NMR (DMSO): 169.33 ppm (C17), 169.24 (C2), 168. 97 (C15), 160.03 (C13), 155.92 (C5), 152.53 (C9), 147.89 (C7), 129.72 (C11), 121.36 (C6), 121.02 (C10), 118.21 (C8), 22.86 (C14), 21.68 (C18), 21.31 (C16); IR (ATR): 3154, 2899, 2782, 1771, 1695, 1612, 1588, 1563, 1504, 1440, 1336, 1321, 1213, 1185, 1150, 1117, 1105, 1014, 991, 900, 882, 820, 686, 672, 609, 551, 473 cm−1
; UV-Vis (MeOH): λ1
= 293 nm; Fluorescence (MeOH): λEm(Ex290)
= 361 nm.
|Synthesis of Zn(II) and Cu(II) complexes||(4–7)|
The Zn(II) complexes were synthesized according to the previously reported procedure [21
]: Typically, the free ligand (1.70 mmol) was dissolved in a hot mixture of 30 mL MeOH and H2
O (1:1 v/v) and equimolar amount of Zn(II) acetate monohydrate was added. The mixture was heated under reflux for 6 h and cooled down to the ambient temperature. A fine solid formed was then collected with the centrifuge, rinsed with water, and dried. The crude product was recrystallized from methanol. The syntheses of Cu(II) complexes were carried out in a similar manner, except that Cu(II) acetate monohydrate (0.85 mmol) was used. The compound 1
was used as a substrate in the synthesis of complexes 4
, while the compound 2
was applied for the synthesis of 6
(4) Yield: 38%; C16H16CuN6O6S2 (516.01 g/mol); calc: C 37.24, H 3.13, N 16.29, Cu 12.31%, found: C 26.85, H 2.34, N 10.69, Cu 24.67%; M.P.: >300 °C; IR (ATR): 3447, 3311, 1607, 1553, 1475,1428,1239,1187, 1172, 1128, 1080, 994, 979, 825, 728, 684, 619, 456 cm−1; UV-Vis (MeOH): λ1 = 322 nm; Fluorescence (MeOH): λEm(Ex290) = 380 nm.
(5) Yield: 46%; C10H13N3O6SZn (368.67 g/mol); calc: C 32.58, H 3.55, N 11.40, Zn 17.73%, found: C 30.98, H 2.74, N 12.02, Zn 16.93%; M.P.: >300 °C; IR (ATR): 3413, 3233, 1610, 1558, 1477, 1221, 1178, 1128, 1085, 992, 977, 886, 835, 675, 604, 451cm−1; UV-Vis (MeOH): λ1 = 294, λ2 = 342 nm; Fluorescence (MeOH): λEm(Ex290) = 413 nm.
(6) Yield: 41%; C20H20CuN6O8S2 (600.08 g/mol); calc: C 40.03, H 3.36, N 14.01, Cu 10.59%, found: C 37.67, H 2.95, N 12.90, Cu 12.04%; M.P.: >300 °C; IR (ATR): 3159, 2910, 2770, 1681, 1598, 1540, 1476, 1413, 1368, 1311, 1219, 1179, 1142, 1130, 976, 833, 798, 760, 706, 682, 625, 603, 551, 517 cm−1; UV-Vis (MeOH): λ1 = 292, λ2 = 322 nm; Fluorescence (MeOH): λEm(Ex290) = 374 nm.
(7) Yield: 42%; C12H15N3O7SZn (410.71 g/mol); calc: C 35.09, H 3.68, N 10.23, Zn 15.92%, found: C 37.01, H 3.02, N 11.55, Zn 13.21%; M.P.: >300 °C; IR (ATR): 3033, 2893, 2770, 1681, 1625, 1598, 1559, 1480, 1418, 1371, 1329, 1312, 1220, 1180, 1130, 1043, 991, 975, 842, 804, 758, 681, 631, 519, 468 cm−1; UV-Vis (MeOH): λ1 = 293, λ2 = 328 nm; Fluorescence (MeOH): λEm(Ex290) = 372 nm.
3.1. Antioxidant Assay
To a transparent, 96-well plate an increasing concentrations of individual ligands solutions were added. Next, 40 μL of 1.0 mM DPPH· radicals methanolic solution was applied. The concentration of each ligand (1–3) was set so that the decrease of absorption intensity of DPPH˙ radicals solution at λmax 519 nm after 30 minutes of the reaction kept in the dark was in the range of 10–90% of its initial value. The total volume of all samples was 200 μL. The plate was shaken for 10 s on the reader shaker to obtain homogeneous solutions and then the absorbance measurement at λmax 519 nm started. All data were collected for 30 min at 25 °C. The final values were the average of five exposures of the sample to a beam of light. Each sample was repeated three times in independent experiments.
3.2. Antibacterial Activity Assay
The bacterial strains were incubated in Mueller–Hinton Broth medium at 37 °C over 24 h in aerobic conditions. The number of cells in the suspension was adjusted to that of 0.5 McFarland standard, which was an equivalent of 108 colony-forming units (CFU). The antibacterial activities of thiadiazole ligands and their Zn(II) complexes were determined as minimal inhibitory concentration (MIC) using broth dilution method. All experiments were performed based on the standard protocol [39
]. The experiments were carried out in 96-well plate and the thiadiazole derivatives were tested within the concentration range of 7.9 µg/mL to 1 mg/mL. All experiments were run in triplicate.
Additionally, the possibility of synergistic antibacterial action was assessed with use of the checkerboard method, in which the compounds 1–3, 5, and 7 were combined with kanamycin and the mixtures were examined for their activity against S. aureus.