General Information
All chemical compounds have been reagent grade until in any other case certain. five,five-dithiobis- (2-nitrobenzoic acid) (DTNB), acetylcholinesterase, butylcholinesterase, galantamine hydrobromide, were obtained from Sigma-Aldrich (Steinheim, Germany). Acetylthiocholine iodide reagent was from Applichem (Germany). Butylcholine iodide (Fluka), sodium carbonate, ammonium acetate, sodium hydrogen phosphate and sodium dihydrogen phosphate had been bought from Riedel-de-Haen (Germany). (R)-styrene oxide and (S)-propylene oxide and (S)-glycidyl phenyl ether had been purchased from Sigma-Aldrich. Silica Gel 60 (Merck, 0.040–0.063 mm) and silica gel/TLC-playing cards (F254) had been used for column chromatography and TLC. All reactions were completed under an N2 environment with a dry solvent beneath anhydrous conditions, until in any other case stated. Melting factors have been decided with Gallenkamp model equipment with open capillaries. Optical rotations had been taken on a Perkin Elmer 341 model polarimeter. IR spectra have been recorded on Mattson a thousand ATI Unicam toes-IR spectrophotometer. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on Bruker Avance-400 MHz high overall performance virtual ft-NMR spectrometer, with tetramethylsilane because the inner popular solutions in deuteriochloroform. Elemental analyses have been received with CARLO-ERBA model 1108 device.
Synthesis
Compounds 5, 6, 9 and 2-4, 10 have been synthesized as our previous reports respectively [22, 23].
(R)-1-Phenyl-2-[(R)-α-cyclohexylethyl)amine]ethanol (1): Compounds 1 have been synthesized using a procedure reported [23]. (R)-α-cyclohexylethylamine (10 mmol, 1.21 g (1.2 equiv.)) in 5 mL i-propanol was added to the solution of (R)-styrene oxide (990 mg, 8.33 mmol) in 5 mL of i-propanol at in an ice bath and stirred for 1 h. then refluxed for 12 hours. The reaction monitored by TLC using H:EA:TEA (4:1:0.1). Solvent was evaporated then the crude product was purified by crystallization from n-hexane to give 1.48 g (72 %) of pure product as a white crystal. M.p.: 72-73 oC. 1H NMR(CDCl3): 7.41-7.28(m, 5H), 4.78-4.74(dd, 4Hz, 1H), 2.93-2.89(m, 1H), 2.722.64(m, 1H); 2.51-2.47(p, 1H); 1.79-1.68(m, 5H), 1.34-0.9(m, 10H). 13C NMR (CDCl3): 142.84, 127.55, 127.35, 125.82, 71.55, 57.28, 54.60, 43.22, 29.71, 28.25, 26.75, 17.05. Anal. Calcd. for C16H25NO C: 77.73, H: 10.12, N: 5.67. Found: C: 77.94, H: 9.79, N: 6.03.
(1R,2R)-Dibenzyl-bis[(R)-2-hydroxy(1-phenyl)ethyl]-1,2-diaminocyclohexane (7): This (R)-Styrene oxide (1.63 g, 13.6 mmol) was added to a solution of (R,R)-N,Nꞌ-dibenzyl-1,2-diaminocyclohexane (2 g, 6.8 mmol) in methanol (3 mL) and stirred at 40, 50 and 60oC, 24 h for each temperature. Solvent was evaporated and then unreacted epoxide and amine was removed by Kugelrohr distillation apparatus. Crude product was purified by column chromatograph using petroleum ether (60-80): ethylacetate: triethyl amine (85/10/5) to give 7 as a regioisomer of compound 10 (0.87 g, 24 %) as a colourless solid. M.p.: 80-81 oC; 1H (400 MHz, CDCl3) 7.56-7.25 (20H, m), 4.66-4.63(d, 9.6 Hz, 2H); 4.15(bs, 3H); 3.749-3.715(d, 13.6Hz, 2H); 3.6(bs, 2H); 2.64-2.61(d, 13.6Hz, 3H); 1.63- 1.14(m, 8H). 13C (100 MHz, CDCl3): 142.18, 130.42, 130.03, 129.77, 129.55, 129.02, 128.85, 127.35, 126.01, 71.09, 57.44, 48.00, 42.97, 27.50, 25.29, 25.03. Anal. Cald. for C36H42N2O2: C, 80.89; H, 7.86; N, 5.24, Found: C, 80.25; H, 7.46; N, 5.30.
N,N'-Bis-[(L)-N-Boc-proline]-(1R,2R)-1,2-cyclohexanediamide (11) : To an ice cold (S)-N-(tert-butyloxycarbonyl)proline (3.77g, 17.54 mmol), (1R,2R)-1,2-diaminocyclohexane (1g, 8.77 mole) in dicholorometane (20 mL) a solution of dicyclohexylcarbodiimide (3.7g, 18 mmole) in 10 mL DCM was added dropwise. The resulting mixture was stirred and the temperature of the reaction mixture was allowed to raise the room temperature. The progress of the reaction was monitored by TLC n-hexane:ethyl acetate (Rf 0.25). After completition of reaction dicyclohexylurea and solvent were separated by filtration and evaporation respectively. Remain mixture was separated by column chromatograph by using n-hexane:ethyl acetate (3:1) with silica gel packed column to give 2.8 g (62.85 %) of compound 11 (Rf=0.25) as a white solid. M.p.: 87-88 oC. [α]D24= -55.5 (c=1, MeOH). 1H NMR (CDCl3): 6.67(m, 1H, -C=ONH-), 4.136 (bs, 1H), 3.56(bs, 1H); 3.42(m, 2H, -CH2-); 1.99(m, 3H, -CH2-); 1.76(m, 3H, 1.41(s, 9H, (CH3)3C-) ; 1.27-1.17(m, 2H). 13C NMR(CDCl3): 172.98, 154.63, 80.36, 61.54, 53.22, 47.07, 32.45, 31.14, 28.35, 24.32, 23.83. Anal. Calcd for C26H44N4O6 C: 61.42, H: 8.66, N: 11.02. Found: C: 60.94, H: 8.39, N: 11.18.
Cyclohexyltetrahydro-1H-Pyrolo-[1,2-c]imidazole-1,3-(2H)-dione, (12): Compound 12 is a by product of compound 13. Thus compound 13 (1.12g, 2.56 mmol) was deprotected by using trifloroacetic acid (2.55 mL) in DCM (3 mL) according to the literature [29]. Solvent evaporated and remain mixture was separated by column chromatograph by using n-hexane: ethyl acetate (4:1) with silica gel packed column to give 250 mg of compound 12 (Rf = 0.2) as a white solid. M.p.: 102-104 oC. [α]D22= -62. 0 (c=1, MeOH) for compound 12. 1H NMR(CDCl3): 6.67(m, 1H, -C=ONH-), 4.136 (bs, 1H), 3.56(bs, 1H); 3.42(m, 2H, -CH2-); 1.99(bm, 3H, -CH2-); 1.76(bm, 3H, 1.41(s, 9H, (CH3)3C-) ; 1.27-1.17(m, 2H). 13C NMR(CDCl3): 172.98, 154.63, 80.36, 61.54, 53.22, 47.07, 32.45, 31.14, 28.35, 24.32, 23.83 Anal. Calcd for C12H18N2O2 C: 64.86, H: 8.11, N: 12.61. Found: C: 65.12, H: 8.03, N: 12.87.
(S)-1-Pyrrolidinecarboxylicacid,2-[[cyclohexyl[(cyclohexylamino)-carbonyl]-amino]carbonyl]-1,1-dimethylethyl ester (13): 1.2 g (% 32.78) of compound 13 (Rf = 0.7)
was obtained as a by product of compound 11 as above description. M.p.: 73-74 oC. [α]D24= +11.9 (c=1, MeOH) for compound 13. 1H NMR (CDCl3): 8.08-8.06(d, 8Hz, 1H); 4.49-4.47(m, 1H); 4.46-4.45(m, 1H); 3.57-3.55(m, 1H); 3.54-3.52(m, 1H); 3.41-3.40(m, 1H); 1.97-1.60(m, 18H); 1.45(s, 9H); 1.37-1.11(m, 6H). 13C NMR (CDCl3): 171.6, 154.9, 154.2, 58.43, 54.44, 49.99, 47.45, 32.67, 31.79, 31.62, 31.28, 29.12, 28.49, 26.12, 25.95, 25.57, 25.40, 24.57. Anal. Calcd. for C23H39N3O4: C: 65.55, H: 9.26, N: 9.97. Found: C: 64.96, H: 9.32, N: 9.83.
(R,R)-Methoxy-{N,N',N''-1,2',3-[(1-phenylethanolato)-(2-phenylethanolato)propyl]amino}silane (14): 3-Aminopropyltrimetoxysilan (0.755g, 5 mmol) and (R)-phenyloxyrane (1.3g, 10.5 mmol) refluxed in methanol for 24 h. Crude product was purified by column chromatograph using H:EA (6:1) after evaporation of solvent. M.p.: 178-149 oC. IR (cm-1): 3033, 2914, 2874, 2821, 1490, 1452, 1268, 1205, 1124, 1077, 937, 811, 756, 639. 1H NMR (CDCl3): 7.47-7.28(m, 10H); 4.99-4.91(m, 2H); 3.60(s, 3H); 3.58-3.12(m,1H); 3.0-2.9(m, 2H); 2.67-2.49(m, 3H); 1.84-1.67(m, 2H); 1.06-1.02(m,1H); 0.76-0.71(m, 1H). 13C NMR (CDCl3): 141.66, 141.57, 128.44, 128.39, 127.55, 125.42, 125.30, 70.76, 70.16, 60.41, 60.15, 52.87, 50.17, 20.69, 8.14. Anal. Calculated for C20H37NO3Si: C: 65.50, H: 10.08, N: 3.82. Found: C: 64.96, H: 9.78, N: 3.69.
Anticholinesterase inhibition activity
All samples were dissolved in ethanol to prepare their stock solution at 100 and 1000 g/mL concentration. Aliquots of 140, 150, 155, 158 µL, of 100 mM sodium phosphate buffer (pH 8.0), 20, 10, 5, 2 μL of sample solution (10, 100 and 1000 μg/mL) and 20 μL BuChE (or AChE) solution were mixed and incubated for 15 min at 25 ºC, and DTNB (10 μL) is added. The reaction was then initiated by the addition of butyrylthiocholine iodide (or acetylthiocholine iodide) (10 μL). Final concentration of the tested solutions was 100, 50, 25, 10, 5, 2.5, 1, 0.5, 0.25 and 0.1 μg/mL [24]. The hydrolysis of these substrates were monitored using a BioTek Power Wave XS at 412 nm.
Computational methodology
Resumption of goal proteins from PDB the 3D structure of AcHE and BuChE with PDB IDs 2X8B and 4BBZ were accessed from Protein information bank (PDB) (www.rcsb.org). Crystallographic water molecules were eliminated from all of the systems and the lacking coordinates of the atoms were modelled the usage of xLeAP and an ff99SB force field. Atoms on proteins were assigned the PARM99 expenses, and all ionizable residues were set at their default protonation states at impartial pH. All structures were in addition processed with the aid of the xLeAP module of AMBER. The molecular structures were neutralized through the addition of counterions. the chosen goal proteins were minimized with Amber force field [32-35] by employing conjugate gradient algorithm in UCSF Chimera 1.10.1 [36]. The discovery Studio 4.0 a visualizing tool became used to generate the hydrophobicity graphs and graphical depiction of target proteins. The protein Ramachandran graph become accessed thru PDB. the discovery Studio 2.1 customer [37] become used to view 3D conformation of goal proteins.
Docking Studies
Dock 6.5 [38] module permits all tiers of a docking procedure to be done with the technology of ligand conformations, ligand docking, and the scoring of the binding modes. As in this situation, in which a inflexible receptor approximation changed into used, it is predicted that one hundred thirty the extraordinary receptors taken into consideration will result in one-of-a-kind ligand binding modes relying at the preliminary size of the enzyme-binding hollow space. for that reason, 14 designed molecules were docked onto available receptors following a multistep technique. To be able to describe receptor-binding residences, a grid of potential strength became calculated for atoms taking part within the binding pocket. these atoms had been acquired from the evaluation of each protein-ligand complicated. on this step, default parameters have been used. The ligand changed into then docked using the calculated grid to vicinity it into the binding site and score the proposed binding mode.