Chemistry
Copper-nickel oxide nanoparticles were analyzed using a UV-visible spectrophotometer (UV 1800 Shimadzu), a Fourier transform infrared spectrophotometer (FTIR Affinity 1 Shimadzu), a thermogravimetric analyzer (TGA 50 Thermoanalyzer Shimadzu), a high-end X-ray diffractometer, scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS), and transmission electron microscopy elected area electron diffraction (TEM-SAED) techniques. Flash chromatography was used to purify the titled compounds. Melting points (m.p.) were obtained in uncorrected open capillary tubes using a Gallenkamp electric melting point device. Thin-layer chromatography (TLC, Silica gel 60 F254, Merck) was used to monitor the reaction's course and determine the purity of the chemicals identified via UV light and iodine vapor absorption. IR, 1H NMR, and 13C NMR spectroscopy were used to characterize all compounds, where as LC-HRMS/MS (small molecules) spectroscopy was used to characterize two compounds. On a Jasco IR Affinity-1 spectrophotometer, IR spectra (KBr discs) were recorded. Bruker 500 MHz 1H NMR spectra were acquired in chloroform (CDCl3) and dimethyl sulfoxide (DMSO-d6) solvents, with tetramethylsilane (TMS) serving as an internal standard. Chemical shifts are expressed in terms of values (ppm). Signals are denoted by the letters s (singlet), d (doublet), t (triplet), q (quintet), and m (multiplet) (multiplet). Bruker 500 MHz 13C NMR spectra were acquired in chloroform (CDCl3) and dimethyl sulfoxide (DMSO-d6) solvents, with tetramethylsilane (TMS) used as an internal standard. Chemical shifts are expressed in terms of values (ppm). The LC-HRMS/MS data were acquired using an Impact II UHR-TOF Mass Spectrometer System and a Dionex UHPLC Ultimate 3000 System spectrometer. The molecular ion for the majority of the substances was identified as (M+1) +. Commercially available compounds were obtained from Sigma Aldrich, S. D. Fine Chemicals, Loba, and Spectrochem.
Electrochemical synthesis:
To prepare copper-nickel oxide nanoparticles, cetyltrimethyl ammonium bromide (CTAB) was dissolved in water to obtain 0.01 N solutions. A 50 mL electrolysis cell vessel was filled with the solution mentioned above as one of the electrodes, a 1 cm x 1 cm sheet of copper and nickel was used. Another 1 cm x 1 cm platinum sheet serves as an inert electrode. Two electrode sheets were immersed in a 0.01 N solution of CTAB. CTAB works as a supporting electrolyte and stabilizing agent during this synthesis process, preventing the generated nanoparticles from growing further. The copper and nickel technique metals are oxidized and transformed to copper and nickel ions during this electrochemical process to create nanoparticles. Copper ions generated in this way migrate towards the cathode. They produce nanoscale oxides of copper-nickel in the electrolyte solution and at the contact between the cathode's surface and the electrolyte solution. Electrolysis was carried out at various current densities. The applied current densities in mA/cm2 were 5, 10, 15, and 20, and the electrolysis was carried out for 2 hours. The color of the solution varies during this electrolysis process. The colorless solution first changed to a bright blue hue, a dirty green hue, and eventually a dark brown residue. After a two-hour interval, the electrolysis process was ended. The brown precipitate-containing solution was then collected in a bottle. Permitted the solid particles to settle and decantation was used to separate the solid obtained in this manner. Three to four times with water washed the separated solid to achieve complete elimination of excess capping agent.
General procedure for the synthesis of 2-(2-substituted-4-oxo-thiazolidin-3-yl)-1, 9-dihydro-purin-6-ones (GB-1 to GB-12)
In a round bottom flask, guanine (0.01 mol), substituted aldehydes (0.01 mol), thioglycolic acid (0.01 mol), and sodium hydroxide (0.01 mol) were combined in ethanol (10 ml). The mixture was vigorously agitated and then refluxed for 30 minutes at 40W using a microwave synthesizer system (CEM, USA). Once the reaction mixture was reduced to ambient temperature, it was placed in crushed ice and stirred for 5 minutes. The resulting compounds were filtered. Purification of the crude product was accomplished using flash chromatography. The completeness of the reaction was monitored using thin-layer chromatography.
2-(4-Oxo-2-phenyl-thiazolidin-3-yl)-1, 9-dihydro-purin-6-one (GB-1)
White solid; Yield: 64.07%; mp: 160–1620C; FT-IR (KBr) Vmax: 3323, 2927, 2561, 1714, 1659, 1356 cm-1; 1H NMR (DMSO-d6, 500 MHz): δ 4.28 (s, 2H), 7.06–7.14 (m, 5H), 7.97(s, 1H), 5.92(s, 1H), 3.33(s, 2H); 13C NMR (DMSO-d6, 500 MHz) δ 171.216, 167.290, 159.544, 139.544, 129.135, 128.677, 127.943, 120.172, 114.629, 53.173, 40.263, 39.929, 39.763, 34.466; LC-HRMS/MS (ESI): m/z, calcd (M +) 314.06, found 314.0071.
2-[2-(4-Chloro-phenyl)-4-oxo-thiazolidin-3-yl]-1, 9-dihydro-purin-6-one (GB-2)
Yellow solid; Yield: 59.22%; mp: 144–1460C; FT-IR (KBr) Vmax: 3323, 2927, 2615, 1714, 1651, 1348, 757 cm-1; 1H NMR (CDCl3, 500 MHz): δ 4.28 (s, 2H, NH), 7.00–7.15 (m, 4H), 7.97(s, 1H), 5.92(s, 1H, CH), 3.33(s, 2H); 13C NMR (CDCl3 500 MHz) δ 130.035, 129.156, 128.081, 127.834, 127.262, 127.118, 126.505, 77.269, 77.016, 76.742, 43.599, 39.788, 33.179, 24.345; LC-HRMS/MS (ESI): m/z, calcd (M +) 348.02, found 348.019.
2-[2-(3-Methoxy-phenyl)-4-oxo-thiazolidin-3-yl]-1, 9-dihydro-purin-6-one (GB-3)
Black solid; Yield: 47.36%; mp: 139–1410C; FT-IR (KBr) Vmax: 3329, 2920, 2619, 1718, 1656, 1340, 1240 cm-1; 1H NMR (CDCl3, 500 MHz): δ 4.28 (s, 2H), 6.50–7.03 (m, 4H), 7.97(s, 1H), 5.92(s, 1H), 3.33(s, 2H), 3.73(s, 3H); 13C NMR (CDCl3, 500 MHz) δ 191.199, 154.532, 149.557, 148.954, 133.724, 130.757, 129.846, 126.964, 126.447, 119.311, 111.850, 110.199, 77.483, 66.464, 64.883; LC-HRMS/MS (ESI): m/z, calcd (M +) 344.07, found 344.01.
2-[2-(4-Nitro-phenyl)-4-oxo-thiazolidin-3-yl]-1, 9-dihydro-purin-6-one (GB-4)
Black solid; Yield: 68.03%; mp: 167–1690C; FT-IR (KBr) Vmax: 3306, 2928, 2735, 1725, 1656, 1533, 1340 cm-1; 1H NMR (CDCl3, 500 MHz): δ 4.28 (s, 2H), 7.32–8.07 (m, 4H), 7.97(s, 1H), 5.92(s, 1H), 3.33(s, 2H); 13C NMR (CDCl3, 500 MHz) δ 153.894, 131.695, 131.322, 131.082, 130.683, 130.497, 129.892, 124.357, 124.252, 116.492, 113.092, 112.600, 40.124, 39.791; LC-HRMS/MS (ESI): m/z, calcd (M +) 359.05, found 359.024.
2-[2-(4-Dimethylamino-phenyl)-4-oxo-thiazolidin-3-yl]-1, 9-dihydro-purin-6-one (GB-5)
Yellow solid; Yield: 52.29%; mp: 128–1300C; FT-IR (KBr) Vmax: 3329, 2928, 2619, 1725, 1656, 1340 cm-1; 1H NMR (CDCl3, 500 MHz): δ 4.28 (s, 2H), 6.47–6.88 (m, 4H), 7.97(s, 1H), 5.92(s, 1H), 3.33(s, 2H), 2.85(m, 6H); 13C NMR (CDCl3, 500 MHz) δ 190.290, 176.992, 154.336, 131.960, 131.960, 125.123, 124.870, 111.198, 110.976, 110.715, 77.327, 77.073, 76.819, 45.299, 40.052, 34.582; LC-HRMS/MS (ESI): m/z, calcd (M +) 357.11, found 357.27.
2-[2-(3, 4-Dimethoxy-phenyl)-4-oxo-thiazolidin-3-yl]-1, 9-dihydro-purin-6-one (GB-6)
Black solid; Yield: 42.02%; mp: 155–1570C; FT-IR (KBr) Vmax: 3346, 2935, 2615, 1729, 1656, 1348, 1240 cm-1. 1H NMR (500 MHz, CDCl3): δ 4.28 (s, 2H), 6.46–6.54 (m, 3H), 7.97(s, 1H), 5.92(s, 1H), 3.33(s, 2H), 3.73(s, 6H); 13C NMR (CDCl3, 500 MHz) δ 190.355, 177.324, 169.748, 154.357, 144.642, 141.120, 131.999, 125.172, 110.998, 77.283, 77.029, 76.776, 45.712, 43.243, 40.087, 8.560; LC-HRMS/MS (ESI): m/z, calcd (M +) 374.08, found 374.019.
2-(2-Methyl-4-oxo-thiazolidin-3-yl)-1, 9-dihydro-purin-6-one (GB-7)
Brown solid; Yield: 58.44%; mp: 121–1230C; FT-IR (KBr) Vmax: 3346, 2927, 2615, 1714, 1651, 1348 cm-1; 1H NMR (CDCl3, 500 MHz): δ 4.28 (s, 2H), 7.97(s, 1H), 4.81(s, 1H), 3.33(s, 2H), 1.54(s, 3H); 13C NMR (CDCl3, 500 MHz) δ 144.854, 144.015, 129.835, 123.618, 114.477, 112.788, 112.560, 63.676, 40.292; LC-HRMS/MS (ESI): m/z, calcd (M +) 252.05, found 252.045.
2-[2-(2-Nitro-phenyl)-4-oxo-thiazolidin-3-yl]-1, 9-dihydro-purin-6-one (GB-8)
Yellow solid; Yield: 38.51%; mp: 173–1750C; FT-IR (KBr) Vmax: 3346, 2927, 2615, 1714, 1651, 1533, 1341 cm-1. 1H NMR (CDCl3, 500 MHz): δ 4.28 (s, 2H), 7.32–8.07 (m, 4H), 7.97(s, 1H), 5.92(s, 1H), 3.33(s, 2H); 13C NMR (CDCl3, 500 MHz) δ 163.244, 155.310, 149.314, 139.148, 121.698, 121.181, 111.198, 110.976, 110.715, 77.282, 77.028, 76.774, 28.317; LC-HRMS/MS (ESI): m/z, calcd (M +) 359.05, found 359.014.
2-[2-(2-Chloro-phenyl)-4-oxo-thiazolidin-3-yl]-1, 9-dihydro-purin-6-one (GB-9)
Yellow solid; Yield: 56.11%; mp: 166–1680C; FT-IR (KBr) Vmax: 3351, 3095, 2735, 1718, 1625, 1348, 824 cm-1. 1H NMR (CDCl3, 500 MHz): δ 4.28 (s, 2H), 7.00–7.15 (m, 4H), 7.97(s, 1H), 5.92(s, 1H), 3.33(s, 2H); 13C NMR (CDCl3, 500 MHz) δ 198.834, 161.638, 141.351, 140.202, 133.832, 131.060, 129.785, 128.098, 127.275, 126.617, 125.512, 77.296, 45.303, 37.250; LC-HRMS/MS (ESI): m/z, calcd (M +) 348.02, found 348.28.
2-[2-(3-Chloro-phenyl)-4-oxo-thiazolidin-3-yl]-1, 9-dihydro-purin-6-one (GB-10)
Yellow solid; Yield: 61.35%; mp: 141–1430C; FT-IR (KBr) Vmax: 3346, 2919, 2615, 1722, 1628, 1341, 718 cm-1; 1H NMR (CDCl3, 500 MHz): δ 4.28 (s, 2H), 6.94–7.08 (m, 4H), 7.97(s, 1H), 5.92(s, 1H), 3.33(s, 2H); 13C NMR (CDCl3, 500 MHz) δ 198.545, 161.367, 145.244, 134.582, 130.179, 129.602, 128.099, 127.397, 126.994, 125.412, 77.296, 77.043, 76.789, 40.434; LC-HRMS/MS (ESI): m/z, calcd (M +) 348.02, found 348.087.
2-[2-(3-Bromo-phenyl)-4-oxo-thiazolidin-3-yl]-1, 9-dihydro-purin-6-one (GB-11)
Yellow solid; Yield: 71.09%; mp: 183–1850C; FT-IR (KBr) Vmax: 3385, 3082, 2732, 1722, 1620, 1333, 672 cm-1; 1H NMR (CDCl3, 500 MHz): δ 4.28 (s, 2H), 7.00–7.24 (m, 4H), 7.97(s, 1H), 5.92(s, 1H), 3.33(s, 2H); 13C NMR (CDCl3, 500 MHz) δ 198.471, 161.293, 145.541, 131.197, 130.446, 130.135, 129.935, 126.614, 125.880, 123.021, 122.841, 77.297, 77.043, 40.320; LC-HRMS/MS (ESI): m/z, calcd (M +) 391.45, found 391.97.
2-[2-(3-Nitro-phenyl)-4-oxo-thiazolidin-3-yl]-1, 9-dihydro-purin-6-one (GB-12)
Black solid; Yield: 67.19%; mp: 153–1550C; FT-IR (KBr) Vmax: 3354, 2926, 2618, 1725, 1623, 1536, 1339; 1H NMR (CDCl3, 500 MHz): δ 4.28 (s, 2H), 7.40–8.00 (m, 4H), 7.97(s, 1H), 5.92(s, 1H), 3.33(s, 2H); 13C NMR (CDCl3, 500 MHz) δ 190.297, 151.153, 140.060, 130.901, 130.499, 129.495, 124.328, 124.009, 121.861, 77.289, 77.035, 76.781, 45.736, 8.547; LC-HRMS/MS (ESI): m/z, calcd (M +) 359.26, found 359.48.
Anticancer activity (Sulforhodamine B assay)
RPMI 1640 medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine was used to culture the cells. For the current screening experiment, cells were injected into 96 well microtiter plates in 90 L at a density of 5000 cells per well. Before adding experimental drugs, the microtiter plates were incubated for 24 hours at 37°C, 5% CO2, 95% air, and 100% relative humidity. The experimental drugs were dissolved in a suitable solvent to form a stock solution with a concentration of 10-2. Four 10-fold serial dilutions of full media were conducted during the experiment. Aliquots of 10 µl of each of these various drug dilutions were added to the appropriate microtiter wells that had been previously filled with 90 µl of a medium, yielding in the required final drug concentrations. Plates were incubated under usual conditions for 48 hours after drug addition, and the test was stopped by adding cold TCA. Cells were gently fixed in situ for 60 minutes at 4°C using 50 µl of cool 30% (w/v) TCA (final concentration, 10% TCA). After discarding the supernatant, the plates were rinsed and dried five times with tap water. Sulforhodamine B (SRB) solution (50 µl) was added to each well at a concentration of 0.4 percent (w/v) in 1% acetic acid, and plates were incubated at room temperature for 20 minutes. Following staining, the unbound dye was recovered, and the remaining dye was removed using a five-step wash with 1% acetic acid. The plates were dried in the air. After eluting the bound dye with 10 mM trizma base, the absorbance was measured using an Elisa plate reader at a wavelength of 540 nm with a reference wavelength of 690 nm.
On a plate-by-plate basis, percent rise was assessed for test wells relative to control wells. Percent Growth was computed as the average absorbance of the test well to the average absorbance of the control well multiplied by 100. The percentage growth rate at each of the four drug concentration levels was calculated using the six absorbance measurements [time zero (Tz), control growth (C), and test growth in the presence of the four-drug concentration levels (Ti)]. For each test article, dosage response parameters were computed. The 50% growth inhibition (GI50) was calculated as [(Ti-Tz)/(C-Tz)] x 100 = 50, which is the drug concentration that results in a 50% reduction in the net protein increase (as shown by SRB staining) in control cells during drug incubation. The medication concentration required to suppress total growth (TGI) was determined using Ti = Tz. The LC50 value (drug concentration that results in a 50% drop in the measured protein at the end of treatment compared to the beginning) indicates a net loss of cells following treatment when [(Ti-Tz)/Tz] x 100 = -50. If the desired level of activity was not achieved or was surpassed, values for each of these three parameters were stated as higher or less than the maximum or minimum concentration tested [19,20].
In vitro Anti-inflammatory activity
The anti-inflammatory efficacy of the synthesized compounds was determined employing the inhibition of the albumin denaturation procedure. Aceclofenac was used as the standard drug. The standard and test substances were dissolved in a small amount of dimethylformamide (DMF) and diluted with phosphate buffer (0.2 M, pH 7.4). In all solutions, the final concentration of DMF was less than 2.5 percent. A test solution (1 ml) containing various drug doses was mixed with 1 ml of a 1% mM albumin solution in phosphate buffer and incubated for 15 minutes at 27 ± 10 0C in a BOD incubator. Denaturation was induced by maintaining the reaction mixture on a water bath at 60 ± 10 0C for 10 minutes. After cooling, the turbidity was determined using a double beam UV-visible spectrophotometer at 660 nm. The percentage inhibition of denaturation was determined by comparing it to a control condition in which no medication was added. Each experiment was repeated twice, and an average was calculated. The percent inhibition can be computed as follows: percent inhibition = 100 ((Vc / Vt) - 1), where Vt represents the absorbance value of the test group and Vc represents the absorbance value of the control group. The 50% Inhibitory Concentration (IC50) was derived using the percentage inhibition [21].
Angiogenesis activity by chorioallantoic membrane (CAM) of chick embryos assay
The chicken chorioallantoic membrane (CAM) experiment was performed using fertilized chicken eggs that were eight days old. Each egg's shell was drilled with a 1-cm diameter hole, and the dermic sheet's surface was removed to expose the CAM. A filter paper with a diameter of 0.5 cm was placed on top of the CAM, and a volume of 1 nM, 10 nM, or 100 nM drug (control, Thalidomide) was deposited in the center. The shell's windows were then sealed with sterile bandages. For 48 hours, the eggs were incubated at 370C and 90% relative humidity. Following 15 minutes of fixation with stationary solution (a 1:1 combination of methanol and acetone), the CAM was removed and photographed using a digital camera. The morphology of chicken blood arteries was determined following various treatments [22].
Antioxidant activity
DPPH radical scavenging assay
The standard solution was made by dissolving 100 mg ascorbic acid in methanol to get 10, 20, 30, 40, and 50 μg/mL concentrations. The tests solutions were prepared by dissolving 10 mg of compounds (GB-1 to GB-12) in 10 mL of methanol to give 100 μg/mL of stock solution of each compound. The 10, 20, 30, 40 and 50 μg/mL concentrations were prepared using this stock solution. To each dilution, 150 μL of DPPH was added and kept in the dark for 30 min. The 150 μL DPPH solution was added to 10 mL methanol, and the absorbance at 517 nm was measured immediately as a control. 10 mL of different test sample concentrations (10, 20, 30, 40, 50 μL) prepared with methanol were taken, and 150 μL DPPH solution was added to each test tube. After 15 minutes, absorbance at 517 nm was determined using methanol as a blank in a UV visible spectrophotometer (Jasco, UV-630). The following equation was used to compute the free radical scavenging activity (FRSA) (% inhibition). The % inhibition for different log concentrations was plotted to obtain a concentration vs. % inhibition graph from which IC50 value was calculated [23].
KMnO4 radical scavenging assay
The experiment was conducted using a Jasco 630 UV-Spectrophotometer. A fresh stock solution of potassium permanganate (0.05N) was produced and stored in a dark place until use. The sulphuric acid stock solution (2N) was prepared for Mn2+ ion separation. L-ascorbic acid (70 mg/100 ml) was produced in distilled water as a positive control, and successive dilutions [10, 20, 30, 40, 50 μg/ml] were made. The compounds were dissolved in methanol (10 mg/10 ml) and serially diluted. At room temperature and in a dark environment, test solutions were allowed to react with KMnO4 solution, and absorbance values at 528 nm were determined compared to a blank. The radical scavenging activity (percent inhibition) was computed and expressed as a percentage of KMnO4 radical elimination [24].
Molecular docking studies
The docking approach is used to determine the proper binding poses and interactions inside the protein's binding site. The docking of molecules is carried out using the VLife MDS 4.4 program. Polo-like kinase 1 crystal structure (PDB code 1q4k) was obtained from the protein data bank database (http://www.rcsb.org/pdb). The initial crystal structure contained the bound ligand; this was removed, and missing loops were inserted using the same software's homology modeling package. The two-dimensional structures were drawn and translated to three-dimensional structures. The three-dimensional structure was optimized using MMFF up to an rms gradient of 0.01. The structure of the protein was optimized. Cavity number one is chosen for the docking procedure. All atoms within a 5A0 radius were defined as the active site of a protein. Batch docking was used to determine the docking score and interactions between the ligand and target protein.