Chemistry
All reagents were reagent grade quality and obtained from Sigma-Aldrich (Prague, Czech Republic). The reaction process was monitored using thin layer chromatography on the glass-backed silica gel sheets (Silica Gel 60 GF254) and visualized under UV light (254 nm). Column chromatography was performed on silica gel (90–150 mm; Merck Chemical Inc.). 1H and 13C NMR spectra were determined by a Bruker FT-300 MHz spectrometer in DMSO-d6. All the chemical shifts were reported as (δ) values (ppm). Mass spectra were obtained on Agilent 7890A spectrometer at 70 eV. The infrared (IR) spectra were run as KBr disk on Perki-Elmer Spectrum RXI FTIR.
Procedure for the synthesis of methyl 4-hydroxy-3-methoxybenzoate (3)
Methyl 4-hydroxy-3-methoxybenzoate (1, 10 mmol) and hydrazine hydrate (2, 30 mmol) were added to 100 mL EtOH in the presence of catalytic amount of acetic acid. The mixture was refluxed for 24 h. The filtered residue was purified by recrystallization in ethanol. The residues was then washed three times with 5 mL cold ethanol. Finally, the solid was dried in a vacuum at 50 °C to give 3 without further purification. White solid, 93% yield. Melting point: 135.0 °C
General procedure for the synthesis of compounds 4a–k
Methyl 4-hydroxy-3-methoxybenzoate (3, 2 mmol) was then added into 20 mL 2-propanol as a solvent. To the resulting solution different selected aldehyde (2.2 mmol) were added. On completion of reaction (TLC) the precipitate were filtered and recrystallized from ethanol. Subsequently dried under reduced pressure to provide the (4a–i) product.
Synthesis of (E)-Nʹ-(2,4-dihydroxybenzylidene)-4-hydroxy-3-methoxybenzohydrazide (4a)
Compound 4a was prepared by the described procedure to afford pure yellow crystalline solid. Yield: 79%; Melting point: 243.0 °C; FT-IR (KBr): υ = 3524, 3420, 3164, 2875, 1640, 1613, 1259; 1HNMR (300 MHz, DMSO-d6, ppm): δH 11.42 (s, 1H, hydrazine-N–H), 11.37 (s, 1H, 2,4-dihydroxybenzylidene-N=CH), 11.06 (s, 1H, 2,4-dihydroxybenzylidene-OH), 10.18 (s, 1H, 2,4-dihydroxybenzylidene-OH), 9.93 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide-OH), 8.19 (m, 2H, 4-hydroxy-3-methoxybenzohydrazide-C2,6-H), 8.13 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide-C5-H), 7.39 (d, J = 6.0 Hz, 1H, 2,4-dihydroxybenzylidene-C3-H), 7.26 (d, J = 6.0 Hz, 2H, 2,4-dihydroxybenzylidene-C5,6-H), 3.83 (s, 3H, 4-hydroxy-3-methoxy benzohydrazide-OCH3); 13C NMR (DMSO d6, 125 MHz): δc (ppm) = 163.9, 159.7, 150.4, 147.7, 129.2, 125.9, 124.8, 121.6, 121.3, 120.0, 116.2, 115.4, 111.9, 109.7, 56.2; MS (EI) m/z (%): 302 (M+, 5), 194 (40), 151 (30), 135 (100), 123 (18), 60 (35), 43 (30).
Synthesis of (E)-4-hydroxy-Nʹ-(4-hydroxybenzylidene)-3-methoxybenzohydrazide (4b)
Golden powder; Yield: 75%; Melting point: 256 °C; FT-IR (KBr): υ = 3521, 3405, 3024, 2870, 1646, 1605, 1269; 1HNMR (300 MHz, DMSO-d6, ppm): δH 11.45 (s, 1H, hydrazine-N-H), 9.92 (s, 1H, 4-hydroxybenzylidene-N=CH), 9.71 (s, 1H, 4-hydroxybenzylidene-OH), 8.34 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide-OH), 7.48 (m, 2H, 4-hydroxybenzylidene-C2,6-H), 7.44 (m, 2H, 4-hydroxy-3-methoxy-C2,6-H), 6.86 (m, 3H, 4-hydroxy-3-methoxy benzohydrazide-C5-H and 4-hydroxybenzylidene-C3,5-H), 3.85 (s, 3H, 4-hydroxy-3-methoxy benzohydrazide-OCH3); 13C NMR (DMSO d6, 125 MHz): δc (ppm) = 163.9, 159.7, 150.4, 147.7, 129.2, 125.9, 124.8, 121.6, 121.3, 120.0, 116.2, 115.4, 111.9, 109.7, 56.2; MS (EI) m/z (%): 286 (M+, 10), 167 (40), 151 (100), 136 (11), 123 (20), 108 (10), 77 (112).
Synthesis of (E)-4-hydroxy-3-methoxy-Nʹ-(4-methoxybenzylidene)benzohydrazide (4c)
White powder; Yield: 76%; Melting point: 110.0 °C; FT-IR (KBr): υ = 3523, 3393, 3016, 2841, 1509, 1364, 1209. 1HNMR (300 MHz, DMSO-d6, ppm): δH 11.55 (s, 1H, hydrazine-N–H), 9.76 (s, 1H, 4-methoxybenzylidene-N=CH), 8.40 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide-OH), 7.67 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide-C2-H), 7.50 (brs, 1H, 4-hydroxy-3-methoxy benzohydrazide-C6-H), 7.46 (d, J = 6.0 Hz, 2H, 4-methoxybenzylidene-C2,6-H), 7.01 (brs, 1H, 4-hydroxy-3-methoxy benzohydrazide-C5-H), 6.89 (d, J = 6.0 Hz, 2H, 4-methoxybenzylidene-C3,5-H), 3.85 (s, 3H, 4-hydroxy-3-methoxybenzohydrazide-OCH3), 3.80 (s, 3H, 4-methoxybenzylidene-OCH3); 13CNMR (DMSO-d6, 125 MHz): δc (ppm) = 163.1, 161.1, 150.5, 149.7, 148.4, 138.3, 129.0, 127.5, 124.7, 121.7, 118.4, 115.4, 114.8, 112.0, 56.1, 55.7; MS (EI) m/z (%): 300 (M+, 50), 268 (10), 168 (85), 151 (100), 123 (15), 105 (12), 77 (10).
Synthesis of (E)-Nʹ-(3,4-dimethoxybenzylidene)-4-hydroxy-3-methoxy benzohydrazide (4d)
White powder; Yield: 75%; Melting point: 231.0 °C; FT-IR (KBr): υ = 3313, 3009, 2964, 2838, 1639, 1597, 1364; 1HNMR (300 MHz, DMSO-d6, ppm): δH 11.58 (s, 1H, hydrazine-N–H), 9.74 (s, 1H, dimethoxybenzylidene-N=CH), 8.39 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide-OH), 7.50 (s, 1H, 3,4-dimethoxybenzylidene-C2-H), 7.39 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide-C2-H), 7.18 (brs, 1H, 4-hydroxy-3-methoxy benzohydrazide-C6-H), 7.02 (m, 2H, 3,4-dimethoxybenzylidene-C6-H and 4-hydroxy-3-methoxy benzohydrazide-C5-H), 6.89 (d, J = 6.0 Hz, 1H, 3,4-dimethoxybenzylidene-C5-H), 3.85 (s, 3H, 4-hydroxy-3-methoxy benzohydrazide-OCH3), 3.81 (s, 6H, 3,4-dimethoxybenzylidene-2(OCH3)); 13C NMR (DMSO-d6, 125 MHz): δc (ppm) = 163.1, 151.7, 150.5, 149.5, 147.7, 127.6, 124.7, 122.2, 122.0, 121.7, 115.4, 112.0, 111.9, 108.58, 56.2, 56.0, 55.9; MS (EI) m/z (%): 330 (M+, 18), 207 (5), 180 (20), 167 (40), 151 (100), 123 (20), 77 (10), 65 (12).
Synthesis of (E)-Nʹ-(4-bromobenzylidene)-4-hydroxy-3-methoxybenzohydrazide (4e)
Yellow powder; Yield: 91%; Melting point: 115.0 °C; FT-IR (KBr): υ = 3331, 3046, 2929, 2839, 1654, 1593, 1438; 1HNMR (300 MHz, DMSO-d6,ppm): δH 12.46 (s, 1H, hydrazine-N–H), 11.85 (s, 1H, 4-bromobenzylidene-N=CH), 8.43 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide-OH), 7.90 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide-C2-H), 7.68 (m, 4H, 4-bromobenzylidene C2,3,5,6-H), 6.53 (brs, 2H, 4-hydroxy-3-methoxy benzohydrazide-C5,6-H), 3.80 (s, 3H, 4-hydroxy-3-methoxy benzohydrazide-OCH3); 13C NMR (DMSO-d6, 125 MHz): δc (ppm) = 164.8, 163.4, 161.7, 150.0, 149.9, 146.5, 132.9, 131.3, 129.0, 128.5, 122.9, 106.9, 105.9, 100.8, 54.9; MS (EI) m/z (%): 348 (M+2, 6), 348 (M, 6), 167 (40), 151 (100), 123 (20), 89 (12).
Synthesis of (E)-4-hydroxy-3-methoxy-Nʹ-(4-nitrobenzylidene)benzohydrazide (4f)
Yellow powder; Yield: 81%; Melting point: 195.0 °C; FT-IR (KBr): υ = 3093, 2970, 2927, 2840, 1643, 1621, 1523, 1477; 1HNMR (300 MHz, DMSO-d6, ppm): δH 12.22 (s, 1H, hydrazine-N-H), 11.98 (s, 1H, 4-nitrobenzylidene-N=CH), 8.55 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide-OH), 8.30 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide-C2-H), 7.99 (m, 2H, 4-nitrobenzylidene-C3,5-H), 7.91 (m, 2H, 4-nitrobenzylidene-C2,6-H), 6.57 (m, 2H, 4-hydroxy-3-methoxy-benzohydrazide-C5,6-H), 3.80 (s, 3H, 4-hydroxy-3-methoxy benzohydrazide-OCH3); 13C NMR (DMSO-d6, 125 MHz): δc (ppm) = 163.5, 163.5, 162.5, 161.5, 147.4, 145.1, 144.9, 140.0, 129.3, 127.5, 123.5, 106.9, 106.0, 101.0, 54.9; MS (EI) m/z (%): 315 (M+, 10), 167 (20), 151 (100), 135 (10), 123 (18), 108 (18), 59 (24).
Synthesis of (E)-4-hydroxy-Nʹ-(4-hydroxy-3,5-dimethoxybenzylidene)-3-methoxy benzohydrazide (4g)
White powder; Yield: 85%, Melting point:151 °C; FT-IR (KBr): υ = 3521, 3434, 3227, 2965, 1643, 1558, 1426; 1HNMR (300 MHz, DMSO-d6, ppm): δH 11.52 (s, 1H, hydrazine-N–H), 9.73 (s, 1H, 4-hydroxy-3,5-dimethoxybenzylidene-N=CH), 8.91 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide-OH), 8.34 (s, 1H, 4-hydroxy-3,5-dimethoxybenzylidene-OH), 7.46 (s, 1H, 4-hydroxy-3-methoxybenzohydrazide–C2–H), 7.44 (s, 1H, 4-hydroxy-3-methoxybenzohydrazide–C6–H), 6.89 (brs, 2H, 4-hydroxy-3,5-dimethoxybenzylidene –C2,6–H), 6.87 (brs, 1H, 4-hydroxy-3-methoxybenzohydrazide–C5–H), 3.85 (s, 3H, 4-hydroxy-3-methoxy benzohydrazide-OCH3), 3.82 (s, 6H, 4-hydroxy-3,5-dimethoxybenzylidene-2(OCH3)); 13C NMR (DMSO-d6, 125 MHz): δc (ppm) = 163.0, 157.7, 150.5, 148.6, 148.2, 147.7, 138.3, 125.2, 124.8, 121.7, 115.4, 112.1, 112.0, 104.9, 56.5, 56.4, 56.2; MS (EI) m/z (%): 346 (M+, 15), 196 (10), 179 (15), 167 (30), 151 (100), 135 (10), 123 (20), 108 (10).
Synthesis of (E)-4-hydroxy-Nʹ-(3-hydroxy-4-methoxybenzylidene)-3-methoxybenzohydrazide (4h)
White powder; Yield: 90%; Melting point: 98.0 °C; FT-IR (KBr): υ = 3246, 3087, 3227, 2838, 1598, 1356, 1212; 1HNMR (300 MHz, DMSO-d6, ppm): δH 11.49 (s, 1H, hydrazine-N–H), 9.74 (s, 1H, 3-hydroxy-4-methoxybenzylidene-N=CH), 9.35 (s, 1H, OH), 8.30 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide-OH), 7.49 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide-C2–H), 7.45 (d, J = 6.0 Hz, 1H, 4-hydroxy-3-methoxy benzohydrazide-C6–H), 7.28 (s, 1H, 3-hydroxy-4-methoxybenzylidene-C2–H), 7.05 (d, J = 6.0 Hz, 1H, 4-hydroxy-3-methoxy benzohydrazide-C5–H), 6.97 (d, J = 6.0 Hz, 1H, 3-hydroxy-4-methoxybenzylidene-C6–H), 6.88 (d, J = 6.0 Hz, 1H, 3-hydroxy-4-methoxybenzylidene-C5–H), 3.85 (brs, 3H, 3-hydroxy-4-methoxybenzylidene-OCH3), 3.81 (brs, 3H, 4-hydroxy-3-methoxy benzohydrazide-OCH3); 13C NMR (DMSO-d6, 125 MHz): δc (ppm) = 163.0, 150.5, 150.1, 147.7, 147.6, 147.3, 127.8, 124.8, 121.7, 120.6, 115.4, 112.7, 112.3, 112.0, 56.2, 56.0; MS (EI) m/z (%): 316 (M+, 12), 180 (10), 167 (40), 151 (100), 134 (15), 123 (20), 106 (10).
Synthesis of (E)-Nʹ-(3-ethoxy-4-hydroxybenzylidene)-4-hydroxy-3-methoxybenzohydrazide (4i)
White powder; Yield: 76%; Melting point: 114.0 °C; FT-IR (KBr): υ = 3424, 3301, 3080, 2975, 1504, 1371, 1271; 1HNMR (300 MHz DMSO-d6, ppm): δH 12.63 (s, 1H, hydrazine-N–H), 11.65 (s, 1H, 3-ethoxy-4-hydroxybenzylidene-N=CH), 9.55 (s, 1H, 3-ethoxy-4-hydroxybenzylidene-OH), 8.34 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide-OH), 7.91 (m, 2H, 4-hydroxy-3-methoxy benzohydrazide–C2–H and 3-ethoxy-4-hydroxybenzylidene-C2–H), 7.31 (brs, 1H, 4-hydroxy-3-methoxy benzohydrazide–C6–H), 7.11 (brs, 1H, 3-ethoxy-4-hydroxybenzylidene-C6–H), 6.87 (s, 1H, 3-ethoxy-4-hydroxybenzylidene-C5–H), 6.51 (brs, 1H, 3-ethoxy-4-hydroxybenzylidene-C5–H), 4.07 (q, J = 6 Hz, 2H, 3-ethoxy-4-hydroxybenzylidene-OCH2CH3), 3.80 (s, 3H, 4-hydroxy-3-methoxy benzohydrazide-OCH3), 1.37 (t, J = 6.0 Hz, 3H, 3-ethoxy-4-hydroxybenzylidene-OCH2CH3); 13C NMR (DMSO-d6, 125 MHz): δc (ppm) = 164.6, 163.3, 161.8, 148.9, 146.6, 128.7, 124.9, 121.7, 115.0, 109.7, 106.8, 105.8, 100.7, 100.4, 63.3, 55.5, 54.9; MS (EI) m/z (%): 328 (M+, 15), 222 (10), 207 (5), 163 (35), 151 (25), 135 (100), 123 (10), 105 (15).
Synthesis of (E)-4-hydroxy-3-methoxy-Nʹ-(2-nitrobenzylidene)benzohydrazide (4j)
White powder; Yield: 78%, Melting point: 151 °C; FT-IR (KBr): υ = 3093, 2970, 2970, 2840, 1643, 1630, 1523, 1477; 1HNMR (300 MHz DMSO-d6, ppm): δH 12.22 (s, 1H, hydrazine-N–H), 11.98 (s, 1H, 2-nitrobenzylidene-N=CH), 8.55 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide-OH), 8.31 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide–C2–H), 7.91 (m, 4H, 2-nitrobenzylidene–C3,4,5,6–H), 6.51 (brs, 2H, 4-hydroxy-3-methoxy benzohydrazide–C5,6–H), 3.81 (s, 3H, 4-hydroxy-3-methoxy benzohydrazide-OCH3); 13C NMR (DMSO-d6, 125 MHz): δc (ppm) = 164.8, 163.5, 162.5, 161.5, 147.4, 145.1, 144.9, 139.9, 129.3, 127.5, 123.5, 106.9, 105.9, 100.8, 54.9; MS (EI) m/z (%): 315 (M+, 15), 167 (10), 151 (100), 135 (10), 107 (10), 95 (8).
Synthesis of (E)-4-hydroxy-3-methoxy-Nʹ-(3-nitrobenzylidene)benzohydrazide (4k)
White powder; Yield: 88%, Melting point:151 °C; FT-IR (KBr): υ = 3091, 2970, 2970, 2840, 1643, 1630, 1523, 1477; 1HNMR (300 MHz DMSO-d6, ppm): δH 11.62 (s, 1H, hydrazine-N–H), 11.47 (s, 1H, 3-nitro benzylidene-N=CH), 8.48 (s, 1H, 4-hydroxy-3-methoxy benzohydrazide-OH), 8.42 (s, 1H, 3-nitrobenzylidene–C2–H), 8.22 (m, 2H, 4-hydroxy-3-methoxy benzohydrazide–C2–H and 3-nitrobenzylidene–C4–H), 8.08 (m, 2H, 3-nitrobenzylidene–C5,6–H), 7.71 (m, 2H, 4-hydroxy-3-methoxy benzohydrazide-C5,6-H), 3.37 (s, 3H, 4-hydroxy-3-methoxy benzohydrazide-OCH3); 13C NMR (DMSO-d6, 125 MHz): δc (ppm) = 171.6, 165.4, 147.6, 142.6, 139.6, 135.6, 132.6, 132.1, 129.8, 123.5, 123.3, 120.3, 120.1, 21.1, 19.7; MS (EI) m/z (%):315 (M+, 15), 167 (10), 151 (100), 135 (10), 107 (10), 95 (8).
Mushroom tyrosinase inhibition assay
All test samples were first dissolved in DMSO at 50 mM and diluted to the required concentrations. First, 10 ml of tyrosinase (0.5 mg ml) was mixed with 160 μl of 50 mM phosphate buffer (pH = 6.8) in 96-well microplates and then 10 μl of different concentration of the test sample was added. After 20 min incubation at 28 °C, 20 ml of L-Dopa solution (0.5 mM) was added to the phosphate buffer and the enzymatic activity was monitored by observing dopa quinone formation at 475 nm. DMSO without test compounds and kojic acid were used as the control and positive control respectively. The tyrosinase activity without inhibitor was defined as 100%. Each concentration was analyzed in three independent experiments run in triplicate. The inhibitory activity of the tested compounds was expressed as the concentration that inhibited 50% of the enzyme activity (IC50).
Determining the inhibition type
To determine the inhibition kinetics of vaniline-benzylidenehydrazine a series of experiments were performed. Different concentrations of the 4i (0, 10 and 25 µM) was chosen to get a series of straight lines. Pre-incubation and measurement time were the same as discussed in mushroom tyrosinase inhibition assay protocol. The maximal velocity (Vmax) and the Michaelis constant (Km) of the tyrosinase activity were determined by the Line weaver Burk plot at various concentrations of L-DOPA (0.25, 0.5, 0.75 and 1 mM) as a substrate. The inhibition type of the enzyme was assayed by Line weaver Burk plots of inverse of velocities (1/V) versus inverse of substrate concentrations 1/[S] mM.
Molecular docking study
The X-ray crystal structure of tyrosinase (PDB code: 2Y9X) containing tropolone as the innate ligand in the binding site were obtained from protein data bank (http://www.rcsb.org). Water molecules and cognate ligand were excluded from 2Y9X, hydrogens were added, nonpolar hydrogens were merged and Gasteiger charges were calculated for protein. 3D structures of ligands were sketched and optimized (by molecular mechanics, MM+ and AM1, methods) using Hyperchem software. The PDBQT files were created by adding charges and defining the degree of torsions. The three-dimensional grids 60 * 60 * 60 (x, y, z) were created with a grid spacing of 0.375 Å and the cubic grids were centered on the binding site of native ligand comprise copper metal ions [23]. Lamarckian genetic algorithm (LGA) was applied to model the interaction/binding between then ligand and the tyrosinase active site. For Lamarckian GA, 27,000. The other parameters were left at program default values. The final binding mode described in the manuscript was selected taking into account the best-ranked scoring functions.