4.1 Docking protocol
The ADT software and Autodock vina program were employed for molecular docking to assess the interaction between the designed analogs and the targeted enzymes (AChE & BuChE). This was aimed to corroborate the findings from both in vitro and in silico analyses. Utilizing PDB codes 7E3H for AChE and 4BDS for BuChE from the RCSB protein databank (http://www.rcsb.org), crystal structures of the targets were retrieved. Autodock vina necessitates the ligand as well as the receptor in pdbqt format. The ADT software was utilized to prepare the two enzymes and the ligands. In the process of protein preparation, all water molecules were removed, followed by the addition of polar hydrogens and Kollman charges. Subsequently, active sites were determined by creating grid boxes sized 40 × 40 × 40 Å for AChE and for BuChE around the binding domains of each co-crystallized ligands with the respective enzyme coordinates: center_x = -43.36, center_y = 37.72, center_z = -30.31 for AChE, and center_x = 132.8, center_y = 115.68, center_z = 41.43 for BuChE. To validate the docking protocol, the docked ligands were removed from the co-crystallized structures, and re-docking both of the ligands, i.e. donepezil for AChE and tacrine for BuChE, followed by calculating the Root-Mean-Square Deviation (RSMD) between the co-crystalized ligands and the re-docked poses. For analysis of the docking results and visualization of ligand-receptor interactions, Discovery Studio 2021 client was employed.
4.2 Chemistry
For the synthesis of compounds, all the chemicals were procured from Spectrochem PrivateLimited, Sigma Aldrich, and Avra Synthesis Private Limited.All the reagents and solvents used for the synthesis of the proposed compounds were purified using standard laboratory techniques prior to use. Progress of the reactions was monitored using pre-coated silica gel GF254 TLC plates, and spots were visualized under UV light at 254 or 365 nm. Different solvent systems, like hexane-ethyl acetate (7:3 and 6:4) and dichloromethane-methanol (9:1 v/v), were used as eluents. A Rota evaporator (BUCHI R-300) was used for removing the solvents during the workups. Chromatographic purification was performed by column chromatography using Silica gel #100-200. Melting points of the compounds were measured using a digital melting point apparatus (Veego VMP-D) and were uncorrected. Bruker FT-IR, model ALPHA-T (Germany) spectrophotometer was used for recording the IR spectra of individual compounds (wave numbers in cm-1) using ATR. Molecular weights of the synthesized compounds were determined using a Mass spectrophotometer, (Waters Acquity QDA). 1NMR data was collected using an NMR instrument (Bruker 400 MHz) in CDCl3 or DMSO-d6 solvents (TMS used as internal standard). Purity and composition of the compounds were confirmed by elemental analysis using Thermo Fisher FLASH 2000 organic elemental analyser. The analysed compounds offered results within ± 0.4 % of the theoretical values of carbon, hydrogen and nitrogen.
4.2.1 General Method for the Synthesis of Compounds (4 – 6): (Method-A)
To a solution of 1-Boc-piperazine (2.0 g, 10.74 mM) and glyoxylic acid monohydrate (0.98 g, 10.74 mM) in acetonitrile (20 mL), the corresponding boronic acid (10.74 mM) was added. The reaction mixture was stirred at 85 °C for 16 h, and progress of the reaction was monitored by TLC using (10 % methanol in dichloromethane). After the consumption of the starting materials, the solvent was removed under reduced pressure, and the residue was washed with hexane, and purified by column chromatography using silica gel as a stationary phase to afford the desired products (4 - 6).
4.2.1.1 2-(4-(tert-Butoxycarbonyl)piperazin-1-yl)-2-phenylacetic acid(4): Prepared by Method A using phenylboronic acid (1.3 g, 10.74 mM) (2a) to offer compound (4) as a white solid (3.22 g, 93.6 %), m.p. 180-183 oC; TLC (Rf): 0.50 (10 % Methanol in dichloromethane); IR: 3445, 2977, 2930, 1697, 1621, 1423, 1365, 1345, 1136, 1166, 1080, 965 cm-1; 1H-NMR: δ 7.43-7.41 (d, 2H, ArH), 7.31-7.28 (m, 3H, ArH), 6.98 (s, 1H, ArH), 4.16 (s, 1H, CH), 3.52-3.37 (m, 4H, CH2), 2.73-2.66 (d, 4H, CH2), 1.47-1.45 (d, 9H, CH3); Mass (m/z): 321.2 (M+1).
4.2.1.2 2-(4-(tert-Butoxycarbonyl)piperazin-1-yl)-2-(4- fluorophenyl)acetic acid (5): Prepared by Method A using 4-fluorophenylboronic acid (1.5 g, 10.74 mM) (2b) to offer compound (5) as a white solid (3.45 g, 95 %), m.p. 176-178 oC; TLC(Rf ): 0.60 (10 % methanol in dichloromethane); IR: 3405, 2978, 2932, 1700, 1635, 1510, 1457, 1245, 1004, 757 cm-1; 1H NMR δ 7.45-7.42 (m, 2H, ArH), 7.21-7.17 (m, 2H, ArH), 4.02(s, 1H, CH), 3.31-3.30 (d, 4H, CH2), 2.39-2.29 (m, 4H, CH2), 1.37 (s, 9H, CH3); Mass (m/z): 339.3 (M+1).
4.2.1.3 2-(4-(tert-Butoxycarbonyl)piperazin-1-yl)-2-(4-methoxyphenyl)acetic acid(6): Prepared by Method A using 4-methoxyphenylboronic acid (1.63 g, 10.74 mM) (2c) to offer compound (6) as a white solid (3.42 g, 90.95 %), m.p. 135-138 oC, TLC (Rf): 0.55 (10 % Methanol in dichloromethane); IR: 3422, 2931, 1700, 1617, 1517, 1461, 1412, 1259, 1134, 1038, 966, 869 cm-1; 1H NMR δ 7.35-7.33 (d, 2H, ArH), 6.89-8.87 (d, 2H, ArH), 4.42 (s, 1H, CH), 3.81 (s, 3H, OCH3), 3.61 (s, 4H, CH2), 2.83 (s, 4H, CH2), 1.44 (s, 9H, CH3); Mass (m/z): 351.2 (M+1).
4.3.1 General method for acid-amine coupling for preparing compounds (16 - 42): (Method B)
To a solution of the corresponding products (4 - 6) (1.0 g) in THF (10 mL), EDC.HCl (1 equiv), and HOBt (1 equiv) were added, and the reaction mixture was stirred at a temperature between 5-10 °C for a time period of 20 min. The corresponding aniline/substituted aniline (1 equiv) was added to the above solution followed by N,N-diisopropylethylamine (3 equiv). Stirring was continued at RT for 16 h and THF was removed under reduced pressure. The resulting residue was extracted in DCM and washed with water; the organic layer was removed under reduced pressure to obtain the desired products (16 - 42).
4.3.1.1 tert-Butyl-4-(2-oxo-1-phenyl-2-(phenylamino)ethyl)piperazine-1-carboxylate (16): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-phenylacetic (4) (1.0 g, 3.12 mM), and aniline (0.29 g, 3.12 mM) to obtain compound (16) as white solid (0.92 g, 74.79 %) m.p. 88-90 oC, TLC (Rf): 0.40 (20 % Ethyl acetate in hexane), IR: 3501, 3259, 2862, 1676, 1601, 1559, 1447, 1249, 1171, 735 cm-1.
4.3.1.2 tert-Butyl-4-(2-oxo-1-phenyl-2-(o-tolylamino)ethyl)piperazine-1-carboxylate (17): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-phenylacetic (4) (1.0 g, 3.12 mM), and 2-methylaniline (0.33 g, 3.12 mM)) to obtain compound (17) as brown solid (0.89 g, 69.53 %), m.p. 84-87 oC, TLC (Rf): 0.42 (20 % Ethyl acetate in hexane), IR: 3362, 2926, 1691, 1587, 1521, 1454, 1365, 1286, 1169, 1003 cm-1.
4.3.1.3 tert-Butyl-4-(2-oxo-1-phenyl-2-(p-tolylamino)ethyl)piperazine-1-carboxylate (18): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-phenylacetic (4) (1.0 g, 3.12 mM), and 4-methylaniline (0.33 g, 3.12 mM) to obtain compound (18) as brown solid (0.9 g, 70.31%), m.p. 80-82 oC, TLC (Rf): 0.44 (20 % Ethyl acetate in hexane), IR: 3326, 2974, 2857, 1706, 1668, 1597, 1452, 1364, 1287, 1170, 1018 cm-1.
4.3.1.4 tert-Butyl-4-(2-((2-methoxyphenyl)amino)-2-oxo-1-phenylethyl)piperazine-1-carboxylate (19): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-phenylacetic (4) (1.0 g, 3.12 mM), and 2-methoxyaniline (0.38 g, 3.12 mM) to obtain compound (19) as white solid (0.92 g, 69.17 %), m.p. 92-95 oC, TLC (Rf): 0.38 (20 % Ethyl acetate in hexane), IR: 3324, 2970, 2836, 1683, 1598, 1512, 1480, 1423, 1304, 1170, 1018 cm-1.
4.3.1.5 tert-Butyl-4-(2-((4-methoxyphenyl)amino)-2-oxo-1-phenylethyl)piperazine-1-carboxylate (20): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-phenylacetic (4) (1.0 g, 3.12 mM), and 4-methoxyaniline (0.38 g, 3.12 mM) to obtain compound (20) as white solid (0.94 g, 70.67 %), m.p. 98-100 oC, TLC (Rf): 0.38 (20 % Ethyl acetate in hexane), IR: 3307, 2974, 1692, 1601, 1514, 1456, 1165, 1170, 1129, 1033 cm-1.
4.3.1.6 tert-Butyl-4-(2-((4-fluorophenyl)amino)-2-oxo-1-phenylethyl)piperazine-1-carboxylate (21): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-phenylacetic (4) (1.0 g, 3.12 mM), and 4-fluoroaniline (0.34 g, 3.12 mM) to obtain the desired product (21) as brown solid (0.92 g, 71.32 %), m.p. 68-70 oC, TLC (Rf): 0.46 (20 % Ethyl acetate in hexane), IR: 3504, 2978, 1677, 1623, 1576, 1426, 1409, 1289, 1172, 1005 cm-1.
4.3.1.7 tert-Butyl-4-(2-((4-chlorophenyl)amino)-2-oxo-1-phenylethyl)piperazine-1-carboxylate (22): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-phenylacetic (4) (1.0 g, 3.12 mM), and 4-chloroaniline (0.39 g, 3.12 mM) to obtain compound (22) as brown solid (0.95 g, 70.89 %), m.p. 74-76 oC, TLC (Rf): 0.48 (20 % Ethyl acetate in hexane), IR: 3319, 2976, 2857, 1704, 1677, 1592, 1400, 1635, 1244, 1170, 1001 cm-1.
4.3.1.8 tert-Butyl-4-(2-((4-hydroxyphenyl)amino)-2-oxo-1-phenylethyl)piperazine-1-carboxylate (23): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-phenylacetic (4) (1.0 g, 3.12 mM), and 4-hydroxyaniline (0.34 g, 3.12 mM) was added to obtain compound (23) as brown solid (0.95 g, 74.21 %), m.p.104-107 oC, TLC (Rf): 0.32 (20 % Ethyl acetate in hexane), IR: 3295, 2975, 1690, 1607, 1514, 1247, 1169, 1132, 1005 cm-1.
4.3.1.9 tert-Butyl-4-(2-(cyclohexylamino)-2-oxo-1-phenylethyl)piperazine-1-carboxylate (24): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-phenylacetic (4) (1.0 g, 3.12 mM), and cyclohexanamine (0.30 g, 3.12 mM) was added to obtain compound (24) as white solid (0.98 g, 78.4 %), m.p. 88-90 oC, TLC (Rf): 0.40 (20 % Ethyl acetate in hexane), IR: 3304, 2931, 2856, 1696, 1658, 1527, 1452, 1405, 1288, 1120, 1006 cm-1.
4.3.1.10 tert-Butyl-4-(1-(4-fluorophenyl)-2-oxo-2-(phenylamino)ethyl)piperazine-1-carboxylate (25): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4 fluorophenyl)acetic acid (5) (1.0 g, 2.95 mM), and aniline (0.27 g, 2.95 mM) was added to obtain compound (25) as white solid (0.91 g, 74.59 %) m.p. 82-84 oC, TLC (Rf): 0.52 (20 % Ethyl acetate in hexane), IR: 3308, 2976, 1690, 1600, 1507, 1440, 1366, 1247, 1169, 1027 cm-1.
4.3.1.11 tert-Butyl-4-(1-(4-fluorophenyl)-2-oxo-2-(o-tolylamino)ethyl)piperazine-1-carboxylate (26): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4 fluorophenyl)acetic acid (5) (1.0 g, 2.95 mM), and 2-methylaniline (0.31 g, 2.95 mM) was added to obtain compound (26) as brown solid (0.76 g, 60.31 %), m.p. 94-97 oC, TLC (Rf): 0.48 (20 % Ethyl acetate in hexane), IR: 3309, 2924, 1693, 1601, 1510, 1421, 1285, 1366, 1168, 1001 cm-1.
4.3.1.12 tert-Butyl-4-(1-(4-fluorophenyl)-2-oxo-2-(p-tolylamino)ethyl)piperazine-1-carboxylate (27): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4 fluorophenyl)acetic acid (5) (1.0 g, 2.95 mM), and 4-methylaniline (0.31 g, 2.95 mM) was added to obtain compound (27) as brown solid (0.82 g, 65 %), m.p. 97-99 oC, TLC (Rf): 0.50 (20 % Ethyl acetate in hexane), IR: 3316, 2976, 1692, 1600, 1512, 1457, 1421, 1285, 1127, 1001 cm-1.
4.3.1.13 tert-Butyl-4-(1-(4-fluorophenyl)-2-((2-methoxyphenyl)amino)-2-oxoethyl)piperazine-1-carboxylate (28): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4 fluorophenyl)acetic acid (5) (1.0 g, 2.95 mM), and 2-methoxyaniline (0.36 g, 2.95 mM) was added to obtain compound (28) as white solid (0.98 g, 69.5 %), m.p. 78-81 oC, TLC (Rf): 0.45 (20 % Ethyl acetate in hexane), IR: 3305, 2976, 2853, 1682, 1603, 1511, 1417, 1247, 1107, 1035, 1003 cm-1.
4.3.1.14 tert-Butyl-4-(1-(4-fluorophenyl)-2-((4-methoxyphenyl)amino)-2-oxoethyl)piperazine-1-carboxylate (29): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4 fluorophenyl)acetic acid (5) (1.0 g, 2.95 mM), and 4-methoxyaniline (0.36 g, 2.95 mM) was added to obtain compound (29) as brown solid (0.96 g, 68 %), m.p. 77-80 oC, TLC (Rf): 0.46 (20 % Ethyl acetate in hexane), IR: 3298, 2975, 1689, 1511, 1419, 1246, 1170, 1004 cm-1.
4.3.1.15 tert-Butyl-4-(1-(4-fluorophenyl)-2-((4-fluorophenyl)amino)-2-oxoethyl)piperazine-1-carboxylate (30): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4 fluorophenyl)acetic acid (5) (1.0 g, 2.95 mM), and 4-fluoroaniline (0.32 g, 2.95 mM) was added to obtain compound (30) as brown solid (0.89 g, 70 %), m.p. 63-66 oC, TLC (Rf): 0.54 (20 % Ethyl acetate in hexane), IR: 3296, 2976, 2930, 1692, 1509, 1423, 1403, 1286, 1170, 1004 cm-1.
4.3.1.16 tert-Butyl-4-(2-((4-chlorophenyl)amino)-1-(4-fluorophenyl)-2-oxoethyl)piperazine-1-carboxylate (31): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4 fluorophenyl)acetic acid (5) (1.0 g, 2.95 mM), and 4-chloroaniline (0.37 g, 2.95 mM) was added to obtain compound (31) the desired product as white solid (0.94 g, 71.21 %), m.p. 70-73 oC, TLC (Rf): 0.56, IR: 3383, 2927, 1695, 1599, 1511, 1406, 1369, 1223, 1158, 1004 cm-1.
4.3.1.17 tert-Butyl-4-(1-(4-fluorophenyl)-2-((4-hydroxyphenyl)amino)-2-oxoethyl)piperazine-1-carboxylate (32): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4 fluorophenyl)acetic acid (5) (1.0 g, 2.95 mM), and 4-hydroxyaniline (0.32 g, 2.95 mM) was added to obtain compound (32) as brown solid (0.92 g, 73 %), m.p. 112-114 oC, TLC (Rf): 0.35 (20 % Ethyl acetate in hexane), IR: 3294, 2976, 1688, 1666, 1511, 1424, 1366, 12487, 1131, 1001 cm-1.
4.3.1.18 tert-Butyl-4-(2-(cyclohexylamino)-1-(4-fluorophenyl)-2-oxoethyl)piperazine-1-carboxylate (33): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4 fluorophenyl)acetic acid (5) (1.0 g, 2.95 mM), and cyclohexanamine (0.29 g, 2.95 mM) was added to obtain compound (33) as white solid (0.90 g, 72.58 %), m.p. 94-97 oC, TLC (Rf): 0.42 (20 % Ethyl acetate in hexane), IR: 3308, 2923, 2856, 1695, 1661, 1599, 1508, 1453, 1285, 1170, 1003 cm-1.
4.3.1.19 tert-Butyl-4-(1-(4-methoxyphenyl)-2-oxo-2-(phenylamino)ethyl)piperazine-1-carboxylate (34): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4-methoxyphenyl)acetic acid (6) (1.0 g, 2.85 mM) and aniline (0.26 g, 2.85 mM) was added to obtain compound (34) as brown solid (0.92 g, 76 %), m.p. 112-115 oC, TLC (Rf): 0.58 (20 % Ethyl acetate in hexane), IR: 3307, 2974, 1692, 1601, 1511, 1441, 1247, 1174, 754 cm-1.
4.3.1.20 tert-Butyl-4-(1-(4-methoxyphenyl)-2-oxo-2-(o-tolylamino)ethyl)piperazine-1-carboxylate (35): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4-methoxyphenyl)acetic acid (6) (1.0 g, 2.85 mM), and 2-methylaniline (0.30 g, 2.85 mM) was added to obtain compound (35) as brown solid (0.86 g, 68.8 %), m.p. 107-110 oC, TLC (Rf): 0.60 (20 % Ethyl acetate in hexane), IR: 3356, 2975, 1692, 1607, 1511, 1454, 1247, 1172, 1002 cm-1.
4.3.1.21 tert-Butyl-4-(1-(4-methoxyphenyl)-2-oxo-2-(p-tolylamino)ethyl)piperazine-1-carboxylate (36): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4-methoxyphenyl)acetic acid (6) (1.0 g, 2.85 mM), and 4-methylaniline (0.30 g, 2.85 mM) was added to obtain compound (36) as white solid (0.70 g, 70.4 %), m.p. 104-105 oC, TLC (Rf): 0.60 (20 % Ethyl acetate in hexane), IR: 3303, 2930, 2855, 1688, 1643, 1509, 1242, 1168, 1120 cm-1.
4.3.1.22 tert-Butyl-4-(1-(4-methoxyphenyl)-2-((2-methoxyphenyl)amino)-2-oxoethyl)- piperazine-1-carboxylate (37): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4-methoxyphenyl)acetic acid (6) (1.0 g, 2.85 mM), and 2-methoxyaniline (0.35 g, 2.85mM) was added to obtain compound (37) as white solid (0.89 g, 68.46 %), m.p. 98-101 oC, TLC (Rf): 0.54 (20 % Ethyl acetate in hexane), IR: 3333, 2929, 2852, 1689, 1608, 1510, 1242, 1168, 1026 cm-1.
4.3.1.23 tert-Butyl-4-(1-(4-methoxyphenyl)-2-((4-methoxyphenyl)amino)-2-oxoethyl)piperazine -1-carboxylate (38): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4-methoxyphenyl)acetic acid (6) (1.0 g, 2.85 mM), and 4-methoxyaniline (0.35 g, 2.85 mM) was added to obtain compound (38) as white solid (0.95, 73 %), m.p. 95-97 oC, TLC (Rf): 0.54 (20 % Ethyl acetate in hexane), IR:3334, 2929, 2852, 1688, 1645, 1509, 1403, 1242, 1168, 1027 cm-1.
4.3.1.24 tert-Butyl-4-(2-((4-fluorophenyl)amino)-1-(4-methoxyphenyl)-2-oxoethyl)piperazine-1-carboxylate (39): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4-methoxyphenyl)acetic acid (6) (1.0 g, 2.85 mM), and 4-fluoroaniline (0.31 g, 2.85 mM) was added to obtain compound (39) as white solid (0.92 g, 73 %), m.p. 94-95 oC, TLC (Rf): 0.56 (20 % Ethyl acetate in hexane), IR: 3305, 2974, 1690, 1611, 1511, 1458, 1248, 1172, 1033 cm-1.
4.3.1.25 tert-Butyl-4-(2-((4-chlorophenyl)amino)-1-(4-methoxyphenyl)-2-oxoethyl)piperazine-1-carboxylate (40): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4-methoxyphenyl)acetic acid (6) (1.0 g, 2.85 mM), and 4-chloroaniline (0.36 g, 2.85 mM) was added to obtain compound (40), the desired product as brown solid (0.98 g, 74.8 %), m.p. 108-110 oC, TLC (Rf): 0.62 (20 % Ethyl acetate in hexane), IR: 3428, 2975, 1685, 1594, 1511, 1412, 1247, 1171,1008 cm-1.
4.3.1.26 tert-Butyl-4-(2-((4-hydroxyphenyl)amino)-1-(4-methoxyphenyl)-2-oxoethyl)- piperazine-1-carboxylate (41): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4-methoxyphenyl)acetic acid (6) (1.0 g, 2.85 mM), and 4-hydroxyaniline (0.31 g, 2.85 mM) was added to obtain compound (41), the desired product as brown solid (0.93 g, 73.8 %), m.p. 120-122 oC, TLC (Rf): 0.40 (20 % Ethyl acetate in hexane), IR: 3333, 2929, 2852, 1690, 1645, 1510, 1403, 1242, 1168, 1027 cm-1.
4.3.1.27 tert-Butyl-4-(2-(cyclohexylamino)-1-(4-methoxyphenyl)-2-oxoethyl)piperazine-1-carboxylate (42): Prepared by Method B using 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(4-methoxyphenyl)acetic acid (6) (1.0 g, 2.85 mM), and cyclohexylamine (0.28 g, 2.85 mM) was added to obtain compound (42), the desired product as white solid (0.90 g, 73.1 %), m.p. 90-93 oC, TLC (Rf): 0.45 (20 % Ethyl acetate in hexane), IR: 3327, 2930, 2854, 1689, 1643, 1509, 1420, 1242, 1168, 1117 cm-1.
4.4.1 General Method for Boc-deprotection: (43-69): (Method C)
To a solution of the corresponding products (16 - 42) in DCM (7.5 mL), dioxane-HCl (7.5 mL) (dioxane saturated with hydrogen chloride gas) was added and stirred at 25 oC for 3 h. The reaction was monitored on TLC, after the completion of the reaction, solvent was removed under reduced pressure to obtain sticky Products (43 - 69) which were used as such for the next step.
4.4.1.1 N,2-Diphenyl-2-(piperazin-1-yl)acetamide (43): tert-Butyl-4-(2-oxo-1-phenyl-2-(phenylamino)ethyl)piperazine-1-carboxylate (16) (0.75 g, 1.76 mM) through Method C offered the product (43) (0.54 g, 96.42 %). TLC (Rf): 0.51 (70 % Ethyl acetate in hexane).
4.4.1.2 2-Phenyl-2-(piperazin-1-yl)-N-(o-tolyl)acetamide (44): tert-Butyl 4-(2-oxo-1-phenyl-2-(o-tolylamino)ethyl)piperazine-1-carboxylate (17) (0.75 g, 1.76 mM) through Method C offered the product (44) (0.53 g, 94.64 %). TLC (Rf): 0.48 (70 % Ethyl acetate in hexane).
4.4.1.3 2-Phenyl-2-(piperazin-1-yl)-N-(p-tolyl)acetamide (45): tert-Butyl 4-(2-oxo-1-phenyl-2-(p-tolylamino)ethyl)piperazine-1-carboxylate (18) (0.75 g, 1.76 mM), through Method C offered the product (45) (0.52 g, 92.85 %). TLC (Rf): 0.46 (70 % Ethyl acetate in hexane).
4.4.1.4 N-(2-Methoxyphenyl)-2-phenyl-2-(piperazin-1-yl)acetamide (46): tert-Butyl 4-(2-((2-methoxyphenyl)amino)-2-oxo-1-phenylethyl)piperazine-1-carboxylate (19) (0.75 g, 1.76 mM), through Method C offered the product (46) (0.53 g, 94.64 %). TLC (Rf): 0.48 (70 % Ethyl acetate in hexane).
4.4.1.5 N-(4-Methoxyphenyl)-2-phenyl-2-(piperazin-1-yl)acetamide (47): tert-Butyl 4-(2-((4-methoxyphenyl)amino)-2-oxo-1-phenylethyl)piperazine-1-carboxylate (20) (0.75 g, 1.76 mM), through Method C offered the product (47) (0.56 g, 98.24 %). TLC (Rf): 0.42 (70 % Ethyl acetate in hexane).
4.4.1.6 N-(4-Fluorophenyl)-2-phenyl-2-(piperazin-1-yl)acetamide (48): tert-Butyl 4-(2-((4-fluorophenyl)amino)-2-oxo-1-phenylethyl)piperazine-1-carboxylate (21) (0.75 g, 1.76 mM), through Method C offered the product (48) (0.54 g, 94.73 %). TLC (Rf): 0.43 (70 % Ethyl acetate in hexane).
4.4.1.7 N-(4-Chlorophenyl)-2-phenyl-2-(piperazin-1-yl)acetamide (49): tert-Butyl 4-(2-((4-chlorophenyl)amino)-2-oxo-1-phenylethyl)piperazine-1-carboxylate (22) (0.75 g, 1.76 mM), through Method C offered the product (49) (0.55 g, 98.21 %) which was further used for the final reaction. TLC (Rf): 0.47 (70 % Ethyl acetate in hexane).
4.4.1.8 N-(4-Hydroxyphenyl)-2-phenyl-2-(piperazin-1-yl)acetamide (50): tert-butyl 4-(2-((4-hydroxyphenyl)amino)-2-oxo-1-phenylethyl)piperazine-1-carboxylate (23) (0.75 g, 1.76 mM), through Method C offered the product (50) (0.54 g, 94.73 %). TLC (Rf): 0.48 (70 % Ethyl acetate in hexane).
4.4.1.9 N-Cyclohexyl-2-phenyl-2-(piperazin-1-yl)acetamide (51): tert-butyl 4-(2-(cyclohexylamino)-2-oxo-1-phenylethyl)piperazine-1-carboxylate (24) (0.75 g, 1.76 mM), through Method C offered the product (51) (0.55 g, 98.21 %). TLC (Rf): 0.49 (70 % Ethyl acetate in hexane).
4.4.1.10 2-(4-Fluorophenyl)-N-phenyl-2-(piperazin-1-yl)acetamide (52): tert-Butyl 4-(1-(4-fluorophenyl)-2-oxo-2-(phenylamino)ethyl)piperazine-1-carboxylate (25) (0.75 g, 1.76 mM through Method C offered the product (52) (0.54 g, 94.73 %). TLC (Rf): 0.50 (70 % Ethyl acetate in hexane).
4.4.1.11 2-(4-Fluorophenyl)-2-(piperazin-1-yl)-N-(o-tolyl)acetamide (53): tert-Butyl 4-(1-(4-fluorophenyl)-2-oxo-2-(o-tolylamino)ethyl)piperazine-1-carboxylate (26) (0.75 g, 1.76 mM), through Method C offered the product (53) (0.53 g, 92.98 %). TLC (Rf): 0.48 (70 % Ethyl acetate in hexane).
4.4.1.12 2-(4-Fluorophenyl)-2-(piperazin-1-yl)-N-(p-tolyl)acetamide (54): tert-Butyl 4-(1-(4-fluorophenyl)-2-oxo-2-(p-tolylamino)ethyl)piperazine-1-carboxylate (27) (0.75 g, 1.76 mM), through Method C offered the product (54) (0.55 g, 96.49 %). TLC (Rf): 0.48 (70 % Ethyl acetate in hexane).
4.4.1.13 2-(4-Fluorophenyl)-N-(2-methoxyphenyl)-2-(piperazin-1-yl)acetamide (55): tert-Butyl 4-(1-(4-fluorophenyl)-2-((2-methoxyphenyl)amino)-2-oxoethyl)piperazine-1-carboxylate (28) (0.75 g, 1.76 mM), through Method C offered the product (55) (0.56 g, 96.55 %). TLC (Rf): 0.44 (70 % Ethyl acetate in hexane).
4.4.1.14 2-(4-Fluorophenyl)-N-(4-methoxyphenyl)-2-(piperazin-1-yl)acetamide (56): tert-Butyl 4-(1-(4-fluorophenyl)-2-((4-methoxyphenyl)amino)-2-oxoethyl)piperazine-1-carboxylate (29) (0.75 g, 1.76 mM), through Method C offered the product (56) (0.55 g, 94.82 %). TLC (Rf): 0.44 (70 % Ethyl acetate in hexane).
4.4.1.15 N,2-Bis(4-fluorophenyl)-2-(piperazin-1-yl)acetamide (57): tert-Butyl 4-(1-(4-fluorophenyl)-2-((4-fluorophenyl)amino)-2-oxoethyl)piperazine-1-carboxylate (30) (0.75 g, 1.76 mM), through Method C offered the product (57) (0.56 g, 98.24 %). TLC (Rf): 0.45 (70 % Ethyl acetate in hexane).
4.4.1.16 N-(4-Chlorophenyl)-2-(4-fluorophenyl)-2-(piperazin-1-yl)acetamide (58): tert-Butyl 4-(2-((4-chlorophenyl)amino)-1-(4-fluorophenyl)-2-oxoethyl)piperazine-1-carboxylate (31) (0.75 g, 1.76 mM), in DCM (7.5 mL), through Method C offered the product (58) (0.52 g, 89.65 %). TLC (Rf): 0.48 (70 % Ethyl acetate in hexane).
4.4.1.17 2-(4-Fluorophenyl)-N-(4-hydroxyphenyl)-2-(piperazin-1-yl)acetamide (59): tert-Butyl 4-(1-(4-fluorophenyl)-2-((4-hydroxyphenyl)amino)-2-oxoethyl)piperazine-1-carboxylate (32) (0.75 g, 1.76 mM), through Method C offered the product (59) (0.54 g, 94.73 %). TLC (Rf): 0.47 (70 % Ethyl acetate in hexane).
4.4.1.18 N-Cyclohexyl-2-(4-fluorophenyl)-2-(piperazin-1-yl)acetamide (60): tert-Butyl 4-(2-(cyclohexylamino)-1-(4-fluorophenyl)-2-oxoethyl)piperazine-1-carboxylate (33) (0.75 g, 1.76 mM), through Method C offered the product (60) (0.56 g, 98.24 %). TLC (Rf): 0.52 (70 % Ethyl acetate in hexane).
4.4.1.19 2-(4-Methoxyphenyl)-N-phenyl-2-(piperazin-1-yl)acetamide (61): tert-Butyl 4-(1-(4-methoxyphenyl)-2-oxo-2-(phenylamino)ethyl)piperazine-1-carboxylate (34) (0.75 g, 1.76 mM), through Method C offered the product (61) (0.52 g, 94.73%). TLC (Rf): 0.45 (70 % Ethyl acetate in hexane).
4.4.1.20 2-(4-Methoxyphenyl)-2-(piperazin-1-yl)-N-(o-tolyl)acetamide (62): tert-Butyl 4-(1-(4-methoxyphenyl)-2-oxo-2-(o-tolylamino)ethyl)piperazine-1-carboxylate (35) (0.75 g, 1.76 mM), in DCM (7.5 mL), through Method C offered the product (62) (0.53 g, 91.37 %). TLC (Rf): 0.48 (70 % Ethyl acetate in hexane).
4.4.1.21 2-(4-Methoxyphenyl)-2-(piperazin-1-yl)-N-(p-tolyl)acetamide (63): tert-Butyl 4-(1-(4-methoxyphenyl)-2-oxo-2-(p-tolylamino)ethyl)piperazine-1-carboxylate (36) (0.75 g, 1.76 mM), in DCM (7.5 mL), through Method C offered the product (63) (0.56 g, 96.55 %). TLC (Rf): 0.48 (70 % Ethyl acetate in hexane).
4.4.1.22 N-(2-Methoxyphenyl)-2-(4-methoxyphenyl)-2-(piperazin-1-yl)acetamide (64): tert-Butyl 4-(1-(4-methoxyphenyl)-2-((2-methoxyphenyl)amino)-2-oxoethyl)piperazine-1-carboxylate (37) (0.75 g, 1.76 mM), through Method C offered the product (64) (0.56 g, 96.55 %) which was further processed for the final reaction. TLC (Rf): 0.42 (70 % Ethyl acetate in hexane).
4.4.1.23 N,2-Bis(4-methoxyphenyl)-2-(piperazin-1-yl)acetamide (65): tert-Butyl 4-(1-(4-methoxyphenyl)-2-((4-methoxyphenyl)amino)-2-oxoethyl)piperazine-1-carboxylate (38) (0.75 g, 1.76 mM), through Method C offered the product (65) (0.54 g, 93.10 %). TLC (Rf): 0.42 (70 % Ethyl acetate in hexane).
4.4.1.24 N-(4-Fluorophenyl)-2-(4-methoxyphenyl)-2-(piperazin-1-yl)acetamide (66): tert-Butyl 4-(2-((4-fluorophenyl)amino)-1-(4-methoxyphenyl)-2-oxoethyl)piperazine-1-carboxylate (39) (0.75 g, 1.76 mM), through Method C offered the product (66) (0.54 g, 96.42 %). TLC (Rf): 0.43 (70 % Ethyl acetate in hexane).
4.4.1.25 N-(4-Chlorophenyl)-2-(4-methoxyphenyl)-2-(piperazin-1-yl)acetamide (67): tert-Butyl 4-(2-((4-chlorophenyl)amino)-1-(4-methoxyphenyl)-2-oxoethyl)piperazine-1-carboxylate (40) (0.75 g, 1.76 mM), through Method C offered the product (67) (0.57 g, 96.61 %). TLC (Rf): 0.48 (70 % Ethyl acetate in hexane).
4.4.1.26 N-(4-Hydroxyphenyl)-2-(4-methoxyphenyl)-2-(piperazin-1-yl)acetamide (68): tert-Butyl 4-(2-((4-hydroxyphenyl)amino)-1-(4-methoxyphenyl)-2-oxoethyl)piperazine-1-carboxylate (41) (0.75 g, 1.76 mM), through Method C offered the product (68) (0.52 g, 89.85 %). TLC (Rf): 0.48 (70 % Ethyl acetate in hexane).
4.4.1.27 N-Cyclohexyl-2-(4-methoxyphenyl)-2-(piperazin-1-yl)acetamide (69): tert-Butyl 4-(2-(cyclohexylamino)-1-(4-methoxyphenyl)-2-oxoethyl)piperazine-1-carboxylate (42) (0.75 g, 1.76 mM), through Method C offered the product (69) (0.56 g, 98.24 %) which was further processed for final reaction. TLC (Rf): 0.36 (70 % Ethyl acetate in hexane).
4.5.1 5-Chloromethylquinolin-8-ol(70):
A mixture of 8-hydroxyquinoline (10.0 g, 68 mM), concentrated hydrochloric acid (13 mL) and formalin (37 %formaldehyde and 12 % methanol, 12 mL, 399 mM)) was treated with hydrogen chloride gas and stirred for 3 h. The yellow solid obtained was collected on a filter paper, washed three times in acetone, and dried under vacuum to afford 5-chloromethyl-8-hydroxyquinoline (70) as a yellow solid hydrochloride salt, m.p. >260 oC, Reported >260 oC [42].
4.6.1 General procedure for the synthesis of the target compounds (71 – 79, 80 – 88 and 89 - 97): (Method D)
To a solution of the corresponding products (43 - 69) (1.0 equiv) in DMSO (7 mL), triethylamine (5.0 equiv) was added and the reaction mixture was stirred at 25 oC for 10 min followed by the addition of 5-chloromethyl-8-hydroxyquinoline hydrochloride (70) (1.0 equiv) portion-wise. The reaction mixture was stirred at 100 oC for 16 h, and the progress of the reaction was monitored by TLC using (80 % ethyl acetate in hexane) After the consumption of the starting materials, the reaction mixture was poured into ice-cold water to obtain solid products (71 - 97) which were filtered, dried and further purified by column chromatography using #100-200 silica gel as stationary phase and ethyl acetate:hexane as mobile phase to afford the desired pure products (71 - 97).
4.6.1.1 2-(4-((8-Hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-N,2-diphenylacetamide (71): Using N,2-diphenyl-2-(piperazin-1-yl)acetamide (43) (0.5 g, 1.69 mM) and Method D the desired compound (71) was obtained, m.p. 72-75 °C. TLC (Rf): 0.54 (80 % Ethyl acetate in hexane); IR: 3314, 2931, 2816, 1686, 1599, 1503, 1474, 1440, 1371, 1312, 1271, 1231, 1076, 827 cm-1; 1H NMR: δ 9.25 (s, 1H, NH), 8.79-8.78 (d, 1H, ArH), 8.63-8.61 (d, 1H, ArH), 7.61-7.59 (d, 2H, ArH), 7.47-7.44 (dd, 1H, ArH), 7.38-7.36 (d, 2H, ArH), 7.33 (s, 5H, ArH), 7.28 (s, 1H, ArH), 7.15-7.11 (t, 1H, ArH), 7.08-7.06 (d, 1H, ArH), 3.98 (s, 1H, CH), 3.83 (s, 2H, CH2), 2.63-2.50 (m, 8H, CH2); C28H28N4O2 requires: C, 74.31; H, 6.24; N, 12.38; found C, 74.58; H, 6.41; N, 12.10; LC-MS (m/z): 453.2 (M+1); Purity 98.20 %.
4.6.1.2 2-(4-((8-Hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-2-phenyl-N-(o-tolyl)acetamide (72): Using 2-phenyl-2-(piperazin-1-yl)-N-(o-tolyl)acetamide (44) (0.5 g, 1.61 mM) and Method D the desired compound (72) was obtained as a yellowish white solid (0.59 g, 78.66 %), which was further purified by column chromatography using #100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 110-112 °C. TLC (Rf): 0.52 (80 % Ethyl acetate in hexane); IR: 3338, 2923, 2812, 2766, 1669, 1596, 1505, 1475, 1229, 1136, 1007, 699 cm-1; 1H NMR; δ 9.71-9.61 (m, 2H, NH, OH), 8.85-8.79 (d, 1H, ArH), 8.64-8.52 (d, 1H, ArH), 7.57-6.98 (m, 11H, ArH), 4.09 (s, 1H, ArH), 3.78 (s, 2H, ArH), 2.69- 2.56 (bs, 3H, CH2), 2.41-2.35 (bs, 5H, CH2), 2.18 (s, 3H, CH3); C29H30N4O2 requires: C, 74.65; H, 6.48; N, 12.01; found C, 74.44; H, 6.56; N, 11.83; Mass (m/z): 467.4 (M+1).
4.6.1.3 2-(4-((8-Hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-2-phenyl-N-(p-tolyl)acetamide (73): Using 2-phenyl-2-(piperazin-1-yl)-N-(p-tolyl)acetamide (45) (0.5 g, 1.61 mM) and Method D the desired compound (73) was obtained as a greenish white solid (0.57 g, 76 %), which was further purified by column chromatography using #100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 165-168 °C. TLC (Rf): 0.53 (80 % Ethyl acetate in hexane); IR: 3329, 2817, 1658, 1595, 1472, 1363, 1230, 1133, 1004, 705 cm-1; 1H NMR: δ 9.16 (s, 1H, NH), 8.79-8.78 (d, 1H, ArH), 8.63-8.61 (d, 1H, ArH), 7.49-7.44 (m, 3H, ArH), 7.34-7.32 (d, 6H, ArH), 7.17-7.14 (d, 2H, ArH), 7.08-7.06 (d, 1H, ArH), 3.97 (s, 1H, CH), 3.84 (s, 2H, CH2), 2.53 (d, 8H, CH2), 2.34 (s, 3H, CH3); C29H30N4O2 requires: C, 74.65; H, 6.48; N, 12.01; found C, 74.48; H, 6.65; N, 12.22; LC-MS (m/z): 467.5 (M+1); Purity 95.58 %.
4.6.1.4 2-(4-((8-Hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-N-(2-methoxyphenyl)-2-phenyl- acetamide (74): Using N-(2-methoxyphenyl)-2-phenyl-2-(piperazin-1-yl)acetamide (46) (0.5 g, 1.53 mM) and Method D the desired compound (74) was obtained as a light orange solid (0.57 g, 76 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 82-85 °C. TLC (Rf): 0.49 (80 % Ethyl acetate in hexane); IR: 3327, 2936, 2814, 1738, 1598, 1521, 1460, 1230, 1025, 787 cm-1; 1H NMR: δ 9.97 (s, 1H, NH), 9.72 (s, 1H, OH), 8.83-8.82 (d, 1H, ArH), 8.60-8.58 (d, 2H, ArH), 8.11- 8.09 (d, 1H, ArH), 7.56-7.53 (dd, 1H, ArH), 7.34 (d, 4H, ArH), 7.28(d, 2H, ArH), 7.08-7.07 (d, 2H, ArH), 6.99-6.97 (d, 1H, ArH), 6.92-6.84 (m, 1H, ArH), 4.21 (s, 1H, CH), 3.94 (s, 3H, CH2), 3.79-3.34 (m, 2H, OCH3), 2.47-2.20 (m, 8H, CH2); C29H30N4O3 requires: C, 72.18; H, 6.27; N, 11.61; found C, 71.81; H, 6.54; N, 11.43; Mass (m/z): 483.3 (M+1).
4.6.1.5 2-(4-((8-Hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-N-(4-methoxyphenyl)-2-phenyl- acetamide (75): Using N-(4-methoxyphenyl)-2-phenyl-2-(piperazin-1-yl)acetamide (47) (0.5 g, 1.53 mM) and Method D the desired compound (75) was obtained as a yellowish white solid (0.57 g, 77 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 175-178 °C. TLC (Rf): 0.49 (80 % Ethyl acetate in hexane); IR: 3332, 2955, 2808, 1664, 1520, 1473, 1248, 1135, 1030, 821 cm-1; 1H NMR; δ 9.92 (s, 1H, NH), 9.72 (s, 1H, OH), 8.85 (d, 1H, ArH), 8.63-8.61 (d, 1H, ArH), 7.59-7.57 (dd, 1H, ArH), 7.51-7.46 (m, 4H, ArH), 7.36-7.26 (m, 4H, ArH), 6.99-6.97 (d, 1H, ArH), 6.88-8.84 (d, 2H, ArH), 3.94 (s, 1H, CH), 3.78 (s, 2H, CH2), 3.70 (s, 3H, OCH3), 2.35 (bs, 8H, CH2); C29H30N4O3 requires: C, 72.18; H, 6.27; N, 11.61; found C, 71.88; H, 6.55; N, 11.31; Mass (m/z): 483.3 (M+1).
4.6.1.6 N-(4-Fluorophenyl)-2-(4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-2-phenyl- acetamide (76): Using N-(4-fluorophenyl)-2-phenyl-2-(piperazin-1-yl)acetamide (48) (0.5 g, 1.59 mM) and Method D the desired compound (76) was obtained as a yellowish white solid (0.58 g, 77.33 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 89-90°C. TLC (Rf): 0.51 (80 % Ethyl acetate in hexane); IR: 3296, 2822, 1669, 1509, 1406, 1372, 1211, 1006, 835 cm-1; 1H NMR: δ 10.22- 10.11 (d, 1H, NH), 9.71 (bs, 1H, OH), 8.86-8.85 (d, 1H, ArH), 8.64-8.62 (d, 1H, ArH), 7.77- 7.59 (m, 2H, ArH), 7.50-7.48 (m, 2H, ArH), 7.37-7.36 (m, 4H, ArH), 7.15-7.13 (d, 2H, ArH), 7.01-6.99 (d, 1H, ArH), 3.80 (s, 1H, CH), 3.41 (s, 2H, CH2), 2.69 (s, 2H, CH2), 2.43- 2.47 (d, 4H, CH2); C28H27FN4O2 requires: C, 71.47; H, 5.78; N, 11.91; found C, 71.16; H, 5.96; N, 11.72; Mass (m/z): 471.3 (M+1).
4.6.1.7 N-(4-Chlorophenyl)-2-(4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-2-phenyl- acetamide (77): Using N-(4-chlorophenyl)-2-phenyl-2-(piperazin-1-yl)acetamide (49) (0.5 g, 1.51 mM) and Method D the desired compound (77) was obtained as a yellowish white solid (0.58 g, 77.33 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 89-90°C. TLC (Rf): 0.51 (80 % Ethyl acetate in hexane); IR: 3296, 2822, 1669, 1509, 1406, 1372, 1211, 1006, 835 cm-1; 1H NMR: δ 10.22- 10.11 (d, 1H, NH), 9.71 (bs, 1H, OH), 8.86-8.85 (d, 1H, ArH), 8.64-8.62 (d, 1H, ArH), 7.77- 7.59 (m, 2H, ArH), 7.50-7.48 (m, 2H, ArH), 7.37-7.36 (m, 4H, ArH), 7.15-7.13 (d, 2H, ArH), 7.01-6.99 (d, 1H, ArH), 3.80 (s, 1H, CH), 3.41 (s, 2H, CH2), 2.69 (s, 2H, CH2), 2.43- 2.47 (d, 4H, CH2); C28H27ClN4O2 requires: C, 69.06; H, 5.59; N, 11.50; found C, 68.78; H, 5.87; N, 11.34; Mass (m/z): 471.3 (M+1).
4.6.1.8 N-(4-Hydroxyphenyl)-2-(4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-2-phenyl- acetamide (78): Using N-(4-hydroxyphenyl)-2-phenyl-2-(piperazin-1-yl)acetamide (50) (0.5 g, 1.44 mM) and Method D the desired compound (78) was obtained as a brown solid (0.59 g, 79.72 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 155-158 °C. TLC (Rf): 0.52 (80 % Ethyl acetate in hexane); IR: 3331, 2928, 2813, 1677, 1592, 1474, 1398, 1270, 1134, 1005 cm-1; 1H NMR: δ 10.19 (s, 1H, NH), 9.71 (s, 1H, OH), 8.85- 8.84 (d, 1H, ArH), 8.63-8.64 (d, 1H, ArH), 7.65-7.63 (d, 2H, ArH), 7.57-7.56 (d, 1H, ArH), 7.49-7.47 (d, 2H, ArH), 7.37-7.32 (m, 6H, ArH), 7.0-6.99 (d, 1H, ArH), 3.99 (s, 1H, CH), 3.78 (s, 2H, CH2), 2.47-2.36 (bs, 8H, CH2); C28H28N4O3 requires: C, 71.78; H, 6.02; N, 11.96; found C, 71.56; H, 6.35; N, 11.74; Mass (m/z): 487.3 (M+). 488.3 (M+1), 489.2 (M+2).
4.6.1.9 N-Cyclohexyl-2-(4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-2-phenylacetamide (79): Using N-cyclohexyl-2-phenyl-2-(piperazin-1-yl)acetamide (51) (0.5 g, 1.44 mM) and Method D the desired compound (79) was obtained as a greenish-white solid (0.63 g, 82.89 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. >220 °C. TLC (Rf): 0.43 (80 % Ethyl acetate in hexane); IR: 3320, 2924, 2818, 1662, 1514, 1474, 1371, 1231, 1004 cm-1; 1H NMR: δ 9.79 (s, 1H, OH), 9.71 (s, 1H, OH), 9.20 (s, 1H, NH), 8.87-8.84 (d, 1H, ArH), 8.63-8.57 (d, 1H, ArH), 7.58-7.55 (m, 1H, ArH), 7.45-7.46 (d, 2H, ArH), 7.36-7.25 (m, 6H, ArH), 6.99-6.97 (d, 1H, ArH), 6.67-6.65 (d, 2H, ArH), 3.91 (s, 1H), 3.76 (s, 2H, CH2), 2.54 (s, 1H, CH), 2.50-2.33 (ds, 7H, CH2); 13C NMR δ: 169.01, 147.66, 138.80, 137.52, 133.72, 128.78, 128.51, 128.00, 127,80, 127.44, 121.32, 109.87, 74.12, 59.53, 52.55, 50.67, 47.15, 40.41, 32.32, 32.04, 25.11, 24.43. C28H34N4O2 requires: C, 73.33; H, 7.47; N, 12.22; found C, 73.05; H, 7.78; N, 12.04; Mass (m/z): 469.3 (M+1).
4.6.1.10 2-(4-Fluorophenyl)-2-(4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-N-phenyl- acetamide (80): Using 2-(4-fluorophenyl)-N-phenyl-2-(piperazin-1-yl)acetamide (52) (0.5 g, 1.59 mM) and Method D the desired compound (80) was obtained as a greenish white solid (0.56 g, 74.66 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 74-77 °C, TLC (Rf): 0.48 (80 % Ethyl acetate in hexane), IR: 3305, 3056, 2931, 2815, 1685, 1507, 1439, 1353, 1246, 1176, 1133, 1030 cm-1; 1H NMR: δ 10.08 (s, 1H, NH), 9.72 (s, 1H, OH), 8.84 (d, 1H, ArH), 8.62 (d, 1H, ArH), 7.60-7.57 (m, 3H, ArH), 7.55-7.53 (m, 2H, ArH), 7.35-7.24 (m, 3H, ArH), 7.18 (t, 2H, ArH), 7.05 (t, 1H, ArH), 6.98 (d, 1H, ArH), 4.02 (s, 1H, CH), 3.78 (s, 2H, CH2), 2.55-2.25 (m, 8H, CH2); 13C NMR δ: 168.95, 162.92, 160.50, 152.75, 147.69, 138.78, 133.66, 133.19, 130.55, 128.75, 127.79, 123.97, 123.58, 121.33, 119.53, 115.15, 114.94, 109.88, 73.69, 59.48, 52.48, 50.63. C28H27FN4O2 requires: C, 71.47; H, 5.78; N, 11.91; found C, 71.15; H, 5.95; N, 11.73; Mass (m/z): 471.3 (M+1).
4.6.1.11 2-(4-Fluorophenyl)-2-(4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-N-(o-tolyl)- acetamide (81): Using 2-(4-fluorophenyl)-2-(piperazin-1-yl)-N-(o-tolyl)acetamide (53) (0.5 g, 1.53 mM) and Method D the desired compound (81) was obtained as a greenish-white solid (0.57 g, 77 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 86-89 °C. TLC (Rf): 0.51 (80 % Ethyl acetate in hexane); IR: 3312, 2931, 1675, 1508, 1439, 1350, 1245, 1133, 1028 cm-1; 1H NMR: δ 9.70 (s, 1H, OH), 9.61 (s, 1H, NH), 8.85-8.83 (dd, 1H, ArH), 8.63-8.60 (d, 1H, ArH), 7.58-7.55 (m, 1H, ArH), 7.49-7.46 (m, 4H, ArH), 7.34-7.32 (d, 1H, ArH), 7.21-7.19 (m, 3H, ArH), 7.17 (s, 1H, ArH), 7.15-7.13 (d, 1H, ArH), 7.09-7.05 (m, 2H, ArH), 6.99-6.97 (d, 1H, CH2), 4.12(s, 1H, CH), 3.81-3.74 (m, 3H, CH3 ), 2.51-2.38 (bs, 4H, CH2), 2.16 (s, 4H, CH2); C29H29FN4O2 requires: C, 71.88; H, 6.03; N, 11.56; found C, 71.55; H, 6.46; N, 11.27; Mass (m/z): 485.4 (M+1).
4.6.1.12 2-(4-Fluorophenyl)-2-(4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-N-(p-tolyl)- acetamide (82): Using 2-(4-fluorophenyl)-2-(piperazin-1-yl)-N-(p-tolyl)acetamide (54) (0.5 g, 1.53 mM) and Method D the desired compound (82) was obtained as a yellowish white solid (0.58 g, 78.37 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 103-106 °C. TLC (Rf): 0.53 (80 % Ethyl acetate in hexane); IR: 3314, 2924, 2816, 1689, 1599, 1507, 1461, 1432, 1225 1115, 788, 749 cm-1; 1H NMR δ 9.96 (s, 1H, NH), 9.67 (s, 1H, OH), 8.85-8.84 (dd, 1H, ArH), 8.64-8.61 (dd, 1H, ArH), 7.58- 7.55 (m, 1H, ArH), 7.52-7.48 (m, 2H, ArH), 7.47- 7.45 (d, 2H, ArH), 7.33-7.32 (d, 1H, ArH), 7.20-7.15 (t, 2H, ArH), 7.10-7.08 (d, 2H, ArH), 7.00-6.98 (d, 1H, ArH), 4.00 (s, 1H, CH), 3.78 (s, 2H, CH2), 2.54-2.35 (bs, 8H, CH2), 2.24 (s, 3H, CH3); C29H29FN4O2 requires; C, 71.88; H, 6.03; N, 11.56; found C, 71.76; H, 6.37; N, 11.23; Mass (m/z): 485.3 (M+1).
4.6.1.13 2-(4-Fluorophenyl)-2-(4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-N-(2-methoxyphenyl)acetamide (83): Using 2-(4-fluorophenyl)-N-(4-methoxyphenyl)-2-(piperazin-1-yl)acetamide (55) (0.5 g, 1.45 mM) and Method D the desired compound (83) was obtained as a yellowish white solid (0.56 g, 76.71 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p.140-143 °C. TLC (Rf): 0.51 (80 % Ethyl acetate in hexane); IR: 3312, 2927, 2816, 1691, 1599, 1523, 1506, 1477, 1461, 1225, 1169, 1026, 789, 750 cm-1; 1H NMR: δ 9.97 (s, 1H, NH), 9.69 (s, 1H, OH), 8.84-8.83 (dd, 1H, ArH), 8.61-8.59 (dd, 1H, ArH), 8.10-8.08 (m, 1H, ArH), 7.57-7.54 (dd, 1H, ArH), 7.35-7.32 (t, 3H, ArH), 7.21-7.16 (t, 2H, ArH), 7.10-7.08 (m, 2H, ArH), 7.00-6.98 (d, 1H, ArH), 6.94-6.90 (m, 1H, ArH), 4.30 (s, 1H, CH), 3.94 (s, 3H, OCH3), 3.80 (s, 2H, CH2), 2.68 (bs, 4H, CH2), 2.37 (bs, 4H, CH2); C29H29FN4O3 requires: C, 69.58; H, 5.84; F, 3.80; N, 11.19; found C, 69.36; H, 6.16; N, 10.91; LC-MS (m/z): 501.5 (M+1); Purity 97.11 %.
4.6.1.14 2-(4-Fluorophenyl)-2-(4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-N-(4-methoxyphenyl)acetamide (84): Using N,2-bis(4-fluorophenyl)-2-(piperazin-1-yl)acetamide (56) (0.5 g, 1.45 mM) and Method D the desired compound (84) was obtained as a yellowish white solid (0.54 g, 73.93 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 86-88 °C. TLC (Rf): 0.52 (80 % Ethyl acetate in hexane); IR: 3312, 2933, 2817, 1682, 1603, 1509, 1474, 1412, 1230, 1371, 1133, 1033, 1005, 828, 787cm-1; 1H NMR: δ 9.92 (s, 1H, NH), 9.67 (s, 1H, OH), 8.85-8.84 (d, 1H, ArH), 8.64-8.61 (d, 1H, ArH), 7.59-7.55 (dd, 1H, ArH), 7.52-7.48 (m, 4H, ArH), 7.34-7.32 (d, 1H, ArH), 7.20-7.15 (t, 2H, ArH), 7.0-6.98 (d, 1H, ArH), 6.88-6.85 (m, 2H, ArH), 3.97 (s, 1H, CH), 3.78 (s, 2H, CH2), 3.71 (s, 3H, OCH3), 2.69 (d, 4H, CH2), 2.34 (s, 4H, CH2); C29H29FN4O3 requires: C, 69.58; H, 5.84; N, 11.19; found C, 69.35; H, 5.96; N, 11.02; LC-MS (m/z): 501.4 (M+1); Purity 94.50 %.
4.6.1.15 N,2-bis(4-Fluorophenyl)-2-(4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)- acetamide (85): Using N-(4-chlorophenyl)-2-(4-fluorophenyl)-2-(piperazin-1-yl)acetamide (57) (0.5 g, 1.50 mM) and Method D the desired compound (85) was obtained as a greenish white solid (0.57 g, 79.16 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 85-88 °C. TLC (Rf): 0.48 (80 % Ethyl acetate in hexane); IR: 3391, 2923, 1693, 1507, 1474, 1271, 1227, 1005 cm-1; 1H NMR: δ 10.12 (s, 1H, NH), 9.68 (s, 1H, OH), 8.85-8.84 (d, 1H, ArH), 8.63-8.61 (dd, 1H, ArH), 7.63-7.59 (m, 2H, ArH), 7.57 (m, 1H, ArH), 7.53-7.49 (m, 2H, ArH), 7.33-7.31 (d, 1H, ArH), 7.20-7.16 (m, 2H, ArH),7.15-7.10 (m, 2H, ArH) 7.00-6.98 (d, 1H, ArH), 4.00 (s, 1H, CH), 3.78 (s, 2H, CH2), 2.47- 2.33 (bs, 8H, CH2); C28H26F2N4O2 requires: C, 68.84; H, 5.36; F, 7.78; N, 11.47; found C, 68.68; H, 5.57; N, 11.15; LC-MS (m/z): 489.4 (M+1); Purity 98.68 %.
4.6.1.16 N-(4-Chlorophenyl)-2-(4-fluorophenyl)-2-(4-((8-hydroxyquinolin-5-yl)methyl)- piperazin-1-yl)acetamide (86): Using N-(4-chlorophenyl)-2-(4-fluorophenyl)-2-(piperazin-1-yl)acetamide (58) (0.5 g, 1.45 mM) and Method D the desired compound (73) was obtained as a greenish white solid (0.57 g, 79.16 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 80-83 °C. TLC (Rf): 0.46 (80 % Ethyl acetate in hexane); IR: 3314, 2924, 2819, 1693, 1598, 1505, 1399, 1271, 1228, 1006, 828 cm-1; 1H NMR: δ 10.21 (s, 1H, NH), 8.85-8.83 (d, 1H, ArH), 8.64-8.61 (d, 1H, ArH), 7.66-7.62 (m, 2H, ArH), 7.59-7.56 (dd, 1H, ArH), 7.53-7.49 (m, 2H, ArH), 7.36-7.32 (m, 3H, ArH), 7.21-7.16 (m, 2H, ArH), 7.00-6.99 (d, 1H, ArH), 4.02 (s, 1H, CH), 3.78 (s, 2H, CH2), 2.71-2.34 (bm, 8H, CH2); C28H26ClFN4O2 requires: C, 66.60; H, 5.19; N, 11.09; found C, 66.98; H, 5.49; N, 10.87; Mass (m/z): 505.3 (M+), 507.2 (M+2).
4.6.1.17 2-(4-Fluorophenyl)-N-(4-hydroxyphenyl)-2-(4-((8-hydroxyquinolin-5-yl)methyl)- piperazin-1-yl)acetamide (87): Using 2-(4-fluorophenyl)-N-(4-hydroxyphenyl)-2-(piperazin-1-yl)acetamide (59) (0.5 g, 1.52 mM) and Method D the desired compound (87) was obtained as a obtain brown solid (0.56 g, 75.67 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 80-82 °C. TLC (Rf): 0.40 (80 % Ethyl acetate in hexane); IR: 3270, 2923, 2816, 1664, 1506, 1474, 1226, 1016, 832 cm-1; 1H NMR: δ 9.81 (s, 1H, NH), 9.68 (s, 1H, OH), 9.19 (s, 1H, OH), 8.85-8.84 (d, 1H, ArH), 8.63-8.61 (d, 1H, ArH), 7.59-7.55 (m, 1H, ArH), 7.51-7.48 (m, 2H, ArH), 7.36-7.31 (t, 3H, ArH), 7.19-7.15 (t, 2H, ArH), 6.99-6.98 (d, 1H, ArH), 6.68-6.65 (d, 2H, ArH), 3.95 (s, 1H, CH), 3.78 (s, 2H, CH2), 2.68 (s, 4H, CH2), 2.33 (s, 4H, CH2); C28H27FN4O3 requires: C, 69.12; H, 5.59; N, 11.52; found C, 69.43; H, 5.87; N, 11.34; LCMS (m/z): 487.4 (M+1); Purity 98.85 %.
4.6.1.18 N-Cyclohexyl-2-(4-fluorophenyl)-2-(4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)acetamide (88): Using N-cyclohexyl-2-(4-fluorophenyl)-2-(piperazin-1-yl)acetamide (60) (0.5 g, 1.56 mM) and Method D the desired compound (88) was obtained as a yellowish white solid (0.61 g, 82.43 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 195-196 °C. TLC (Rf): 0.46 (80 % Ethyl acetate in hexane); IR: 3322, 2928, 2850, 1649, 1502, 1473, 1270, 1223, 1004, 829, 701 cm-1; 1H NMR: δ 9.71 (s, 1H, NH), 8.85-8,83 (dd, 1H, ArH), 8.62-8.59 (d, 1H, ArH), 7.86-7.85 (d, 1H, ArH), 7.57-7.54 (q, 1H, ArH), 7.41-7.38 (m, 2H, ArH), 7.32-7.30 (d, 1H, ArH), 7.15-7.11(d, 2H, ArH), 6.99-6.97 (d, 1H, ArH), 3.77-3.75 (d, 3H, CH2, CH ), 3.49-3.47 (d, 1H, CH), 2.41 (bs, 4H, CH2), 2.26 (bs, 3H, CH2), 1.69-1.51 (m, 5H, CH2), 1.22-1.09 (m, 5H, CH2); C28H33FN4O2 requires: C, 70.56; H, 6.98; N, 11.76; found C, 70.38; H, 7.17; N, 11.48; Mass (m/z): 477.4 (M+1).
4.6.1.19 2-(4-((8-Hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-2-(4-methoxyphenyl)-N-phenyl- acetamide (89): Using 2-(4-methoxyphenyl)-N-phenyl-2-(piperazin-1-yl)acetamide (61) (0.5 g, 1.54 mM) and Method D the desired compound (89) was obtained as a white solid (0.56 g, 75.67 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 96-99 °C; TLC (Rf): 0.56 (80 % Ethyl acetate in hexane); IR: 3305, 3056, 2931, 2815, 1685, 1507, 1439, 1246, 1176, 1133, 1030 cm-1; 1H NMR: δ 9.97 (s, 1H, NH), 9.72 (s, 1H, OH), 8.84-8.83 (d, 1H, ArH), 8.86-8.60 (d, 1H, ArH), 7.59-7.55 (m, 3H, ArH), 7.39-7.37 (d, 2H, ArH), 7.32-7.25 (m, 3H, ArH), 7.04-7.01 (t, 1H, ArH), 6.98-6.96 (d, 1H, ArH), 6.91-6.88 (d, 2H, ArH), 3.90 (s, 1H, CH), 3.76 (s, 2H, CH2), 3.72 (s, 3H, OCH3), 2.67 (s, 4H, CH2), 2.33 (s, 4H, CH2); C29H30N4O3 requires: C, 72.18; H, 6.27; N, 11.61; found C, 72.06; H, 6.49; N, 11.49; Mass (m/z): 483.3 (M+1).
4.6.1.20 2-(4-((8-Hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-2-(4-methoxyphenyl)-N-(o-tolyl) acetamide (90): Using 2-(4-methoxyphenyl)-2-(piperazin-1-yl)-N-(o-tolyl)acetamide (62) (0.5 g, 1.47 mM) and Method D the desired compound (90) was obtained as a greenish white solid (0.58 g, 79.45 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 86-88 °C. TLC (Rf): 0.62 (80 % Ethyl acetate in hexane); IR: 3332, 2922, 2833, 1689, 1608, 1582, 1505, 1229, 786 cm-1; 1H NMR: δ 9.70 (d, 1H, NH), 9.54 (d, 1H, OH), 8.90-8.78 (m, 2H, ArH), 8.65-8.33 (m, 2H, ArH), 7.62-7.48 (m, 2H, ArH), 7.37-7.28 (m, 2H, ArH), 7.25-7.04 (m, 2H, ArH), 6.97 (d, 2H, ArH), 6.87 (d, 1H, ArH), 4.80-4.27 (m, 2H, CH2), 3.97 (d, 1H, CH), 3.81-3.69 (m, 3H, OCH3), 2.37 (bs, 4H, CH2), 2.18 (bs, 3H, CH3); 13C NMR δ: 169.21, 158.81, 152.79, 151.90, 147.72, 138.83, 135.96, 133.70, 132.86, 130.23, 128.77, 127.77, 126.67, 126.08, 124.76, 123.28, 121.41, 113.59, 110.58, 109.88, 73.99, 59.53, 54.98, 52.67, 17.60. C30H32N4O3 requires: C, 72.56; H, 6.50; N, 11.28; found C, 72.75; H, 6.82; N, 11.06; Mass (m/z): 497.4 (M+1).
4.6.1.21 2-(4-((8-Hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-2-(4-methoxyphenyl)-N-(p-tolyl) acetamide (91): Using 2-(4-methoxyphenyl)-2-(piperazin-1-yl)-N-(p-tolyl)acetamide (63) (0.5 g, 1.47 mM) and Method D the desired compound (91) was obtained as a white solid (0.51 g, 69.86 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 103-105 °C. TLC (Rf): 0.48 (80 % Ethyl acetate in hexane); IR: 3327, 3037, 2921, 1686, 1580, 1506, 1473, 1418, 1370, 1274, 1224, 1192, 1251, 781 cm-1; 1H NMR: δ 9.69 (s, 2H, NH, OH), 8.87-8.88 (d, 2H, ArH), 8.47-8.45 (d, 2H, ArH), 7.66-7.37 (m, 3H, ArH), 7.07-6.90 (m, 5H, ArH), 4.68 (s, 2H, CH2), 3.88-3.72 (m, 1H, CH), 3.36 (s, 3H, OCH3), 2.57 (dd, 3H, CH3), 2.34-2.14 (m, 4H, CH2); C30H32N4O3 requires: C, 72.56; H, 6.50; N, 11.28; found C, 72.74; H, 6.73; N, 11.06; Mass (m/z): 497.4 (M+1).
4.6.1.22 2-(4-((8-Hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-N-(2-methoxyphenyl)-2-(4-methoxyphenyl)acetamide (92): Using N-(2-methoxyphenyl)-2-(4-methoxyphenyl)-2-(piperazin-1-yl)acetamide (64) (0.5 g, 1.40 mM) and Method D the desired compound (92) was obtained as a yellowish white solid (0.58 g, 80.55 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 153-155 °C. TLC (Rf): 0.46 (80 % Ethyl acetate in hexane); IR: 3314, 2927, 2832, 1689, 1599, 1460, 1371, 1248, 1115, 1028 cm-1; 1H NMR: δ 9.95 (s, 1H, NH), 9.71 (s, 1H, OH), 8.85-8.83 (d, 1H, ArH), 8.61-8.60 (d, 1H, ArH ), 8.11-8.09 (s, 1H, ArH), 7.58-7.54 (m, 1H, ArH), 7.36-7.34 (d, 1H, ArH), 7.22-7.20 (d, 2H, ArH), 7.11-7.07 (d, 2H, ArH), 7.01-6.99 (d, 1H, ArH), 6.94-6.90 (m, 3H, ArH), 4.13 (s, 1H, CH), 3.95 (s, 3H, OCH3), 3.84-3.79 (d, 2H, CH2), 3.76 (s, 3H, OCH3), 2.35 (s, 9H, CH2); C30H32N4O4 requires: C, 70.29; H, 6.29; N, 10.93; found C, 70.08; H, 6.47; N, 10.76; Mass (m/z): 513.3 (M+1).
4.6.1.23 2-(4-((8-Hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-N,2-bis(4-methoxyphenyl)- acetamide (93): Using N,2-bis(4-methoxyphenyl)-2-(piperazin-1-yl)acetamide (65) (0.5 g, 1.40 mM) and Method D the desired compound (93) was obtained as a yellowish white solid (0.60 g, 83.33 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 145-147°C. TLC (Rf): 0.49 (80 % Ethyl acetate in hexane); IR: 3307, 2822, 1664, 1508, 1468, 1232, 1175, 1133, 1029 cm-1; 1H NMR: δ 9.84 (s, 1H, NH), 9.69 (s, 1H, OH), 8.85-8.83 (dd, 1H, ArH ), 8.63-8.60 (dd, 1H, ArH), 7.58-7.55 (m, J = 4.5 Hz, 1H, ArH), 7.50-7.47 (d, 2H, ArH), 7.38-7.36 (d, 2H, ArH), 7.33-7.31 (d,1H, ArH), 6.99-6.97 (d, 1H, ArH), 6.90-6.83 (m, 4H, ArH), 3.86 (s, 1H, CH), 3.77 (s, 2H, CH2), 3.72 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 2.51-2.38 (s, 8H, CH2); C30H32N4O4 requires: C, 70.29; H, 6.29; N, 10.93; found C, 69.95; H, 6.61; N, 10.76; Mass (m/z): 513.3 (M+1).
4.6.1.24 N-(4-Fluorophenyl)-2-(4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-2-(4-methoxyphenyl)acetamide (94): Using N-(4-fluorophenyl)-2-(4-methoxyphenyl)-2-(piperazin-1-yl)acetamide (66) (0.5 g, 1.45 mM) and Method D the desired compound (94) was obtained as a yellowish white solid (0.57 g, 78 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 160-163°C. TLC (Rf): 0.51 (80 % Ethyl acetate in hexane); IR:3294, 3002, 2937, 2817, 1689, 1610, 1508, 1371, 1301, 1247, 1135, 1007, 832 cm-1; 1H NMR: δ 10.04 (s, 1H, NH), 9.71 (s, 1H, OH), 8.84-8.83 (d, 1H, ArH), 8.62-8.60 (d, 1H, ArH), 7.62-7.55 (m, 3H, ArH), 7.42-7.30 (dd, 3H, ArH), 7.13-7.09 (t, 2H, ArH), 6.99-6.88 (dd, 3H, ArH), 3.87 (s, 1H, CH), 3.76 (s, 2H, CH2), 3.72 (s, 3H, OCH3), 2.45-2.33 (bs, 7H); C29H29FN4O3 requires: C, 69.58; H, 5.84; N, 11.19; found C, 69.34; H, 5.95; N, 11.06; Mass (m/z): 501.3 (M+).
4.6.1.25 N-(4-Chlorophenyl)-2-(4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-2-(4-methoxyphenyl)acetamide (95): Using of N-(4-chlorophenyl)-2-(4-methoxyphenyl)-2-(piperazin-1-yl)acetamide (67) (0.5 g, 1.38 mM) and Method D the desired compound (95) was obtained as a green solid (0.56 g, 77.77 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 135-138 °C. TLC (Rf): 0.52 (80 % Ethyl acetate in hexane); IR: 3316, 2817, 1692, 1583, 1504, 1473, 1372, 1232, 1177, 1005, 787 cm-1; 1H NMR: δ 10.12 (s, 1H, NH), 9.71 (s, 1H, OH), 8.88-8.84 (m, 1H, ArH), 8.63-8.61 (m, 1H, ArH), 7.65-7.63 (d, 2H, ArH), 7.59-7.56 (dd, 1H, ArH), 7.40-7.32 (m, 5H, ArH), 6.99-6.98 (d, 1H, ArH), 6.92-6.90 (d, 2H, ArH), 3.91 (s, 1H, CH), 3.78 (s, 2H, CH2), 3.73 (s, 3H, OCH3), 2.46-2.35 (d, 8H, CH2); C29H29ClN4O3 requires: C, 67.37; H, 5.65; N, 10.84; found C, 67.61; H, 5.98; N, 10.62; Mass (m/z): 517.3 (M+), 519.1 (M+2).
4.6.1.26 N-(4-Hydroxyphenyl)-2-(4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-2-(4-methoxyphenyl)acetamide (96): Using N-(4-hydroxyphenyl)-2-(4-methoxyphenyl)-2-(piperazin-1-yl)acetamide (68) (0.5 g, 1.46 mM) and Method D the desired compound (96) was obtained as an orange solid (0.54 g, 73.97 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate: Hexane as mobile phase, m.p. 170-172 °C. TLC (Rf): 0.38 (80 % Ethyl acetate in hexane); IR: 3317, 2948, 2820, 1659, 1607, 1510, 1476, 1371, 1233, 1180, 1032 cm-1; 1H NMR: δ 10.06 (bs, 1H, OH), 9.73 (s, 1H, NH), 9.20 (s, 1H, OH), 8.90-8.84 (dd, 1H, ArH), 8.65-8.61 (t, 1H, ArH), 8.01-7.85 (dd, 1H, ArH), 7.71-7.66 (m, 2H, ArH), 7.58-7.55 (m, 1H, ArH), 7.45-7.41 (m, 1H, ArH), 7.38-7.31 (m, 3H, ArH), 7.10-6.97 (dd, 1H, ArH), 6.90-6.88 (d, 1H, ArH), 6.67-6.65 (d, 1H, ArH), 3.85 (s, 1H, CH), 3.78 (s, 2H, CH2,), 3.73 (s, 3H, OCH3), 2.34 (bs, 8H, CH2); C29H30N4O4 requires: C, 69.86; H, 6.07; N, 11.24; found C, 69.69; H, 6.25; N, 11.15; Mass (m/z): 499.3 (M+1).
4.6.1.27 N-(4-Hydroxyphenyl)-2-(4-((8-hydroxyquinolin-5-yl)methyl)piperazin-1-yl)-2-(4-methoxyphenyl)acetamide (97): Using N-cyclohexyl-2-(4-methoxyphenyl)-2-(piperazin-1-yl)acetamide (69) (0.5 g, 1.50 mM) and Method D the desired compound (97) was obtained as a white solid (0.63 g, 85.13 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and Ethyl acetate:Hexane as mobile phase, m.p. 197-199 °C. TLC (Rf): 0.52 (80 % Ethyl acetate in hexane); IR: 3325, 2934, 2852, 2817, 1644, 1509, 1475, 1376, 1246, 1180, 1135, 1006 cm-1; 1H NMR: δ 9.91 (s, 1H, NH), 8.84 (s, 1H, OH), 8.61-8.58 (d, 1H, ArH), 7.78-7.76 (d, 1H, ArH), 7.57-7.54 (m, 1H, ArH), 7.31-7.25 (m, 3H, ArH), 6.98-6.96 (d, 1H, ArH), 6.86-6.84 (d, 2H, ArH), 3.75 (s, 2H, CH2), 3.72 (s, 3H, OCH3), 3.66 (s, 1H, CH), 3.47 (bs, 1H, CH), 2.44-2.40 (m, 8H, CH2), 1.64-1.51 (m, 5H, CH2), 1.23-1.09 (m, 5H, CH2); C29H36N4O3 requires: C, 71.28; H, 7.43; N, 11.47; found C, 71.40; H, 7.75; N, 11.13; Mass (m/z): 489.4 (M+1).
4.7 Biological Activity
4.7.1 Inhibition studies on AChE and BuChE
The potential of the test compounds for cholinesterase inhibition was assessed using Ellman's essay.[39–41] The products that were purchased from Sigma-Aldrich included human AChE (product number C1682), equine serum BuChE (CAS 9001-08-5), 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB, product number T-D0944), acetylthiocholine iodide (ATCI, product number T-A0116), and butyrylthiocholine iodide (BTCI, product number T-B0775). Standard drugs were donepezil hydrochloride and tacrine hydrochloride hydrate. Every experiment was conducted at pH 8 in a 50 mM Tris-Hydrochloride buffer (Tris HCl, product number MB030). To ascertain the enzyme inhibitory activity, five distinct doses (0.001−100 μM) of every test chemical were employed. To summarize, 10 μL of the test or reference compounds were incubated in 50 μL of AChE (0.22 U/mL) or 50 μL of BuChE (0.06 U/mL)
4.7.2 Antioxidant activity [1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity]
a) Preparation of DPPH reagent: A solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) (0.1mM) was prepared in methanol.
b) Preparation of Sample/Standard
Based on the scavenging activity of the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), free radical scavenging activity of the synthesized compounds was determined by the method of Ali et al [43]. Different volumes (20 – 100μg/ml) of standard compound ascorbic acid and the synthesized compounds were taken from a stock solution in a set of test tubes, and methanol was added to make the volume to 1 ml. To this, 2 ml of 0.1mM DPPH reagent was added and mixed thoroughly. Absorbance at 517 nm was determined after 30 min.
C) Preparation of control
For control, DPPH (3 ml of 0.1mM solution) was taken and incubated for 30 min at room temperature in dark conditions. The absorbance of the control was taken against methanol (as blank) at 517 nm [44].
The percentage antioxidant activity of the sample/standard was calculated by using the formula:
% Inhibition = [(Ab of control- Ab of sample/Ab of control] x 100
The lower the absorbance, the higher the free radical scavenging activity. The curves were prepared and the IC50 values were calculated using linear regression analysis.
4.7.3 Metal-chelating study
The metal chelating ability of all the compounds was assessed using UV spectrophotometry [45]. The absorption spectra of the test compounds (25 μM) alone and in the presence of CuSO4, ZnCl2, FeSO4, FeCl3 and AlCl3 (25 μM) in methanol for 30 min were recorded at room temperature in the UV-visible range.
4.7.4 ADME Prediction
Before a molecule is introduced into the market, its efficacy and safety are vital considerations. An examination of its ADMET (absorption, distribution, metabolism, excretion, and toxicity) profile can be one way to look at these features [46]. Using the online SwissADME server [47], the ADMET properties of the synthesized compounds were evaluated.