Chemistry
General information
All reagents were obtained from commercial sources and utilized without further purification unless otherwise noted. 1H and 13C NMR spectra were recorded on a Varian INOVA spectrometer and referenced to Tetramethylsilane (TMS), using CDCl3 or DMSO-d6 as solvents at 400 MHz and 100 MHz, respectively. Chemical shifts (d) are given in ppm. Splitting patterns are indicated as follow: s, single; d, doublet; dd, double-doublet; t, triplet; m, multi-plet. HPLC analysis was carried out on a Waters e2695 plus system with the use of a Ultimate CQ-C18 column (4.6 mm × 250 mm, 5um) at a flow ratio of 1 mL/min. Mobile phase: A: 0.1%TFA in H2O, B: 0.1% TFA in CH3CN.
General procedure for the preparation of intermediate compounds 3a and 3b. The starting material 2’,4’-dihydroxyacetophenone (3 mmol) with excessive amounts of (R)-(-)-epichlorohydrin or (S)-(+)-epichlorohydrin (3.6 mmol) in the presence of K2CO3 (1.2 mmol) in anhydrous CH3CN (15 mL) under reflux followed by thin layer chromtaography (TLC). Upon completion, the solvent was concentrated by vacuum and K2CO3 was removed by filtration. The crude products were purified by silica gel chromatography using petroleum ether/acetone (50:1) as eluent to obtain the intermediates 3a and 3b.
(R)-1-(2-hydroxy-4-(oxiran-2-ylmethoxy)phenyl)ethan-1-one (3a). White solid, 60.5% yield. 1H NMR (400 MHz, CDCl3) δ 12.73 (s, 1H, OH), 7.65 (d, J = 8.8 Hz, 1H, Ar-H), 6.49 (d, J = 8.8 Hz, 1H, Ar-H), 6.43 (s, 1H, Ar-H), 4.30 (d, J = 9.2 Hz, 1H, 1/2 OCH2), 3.96 (d, J = 6.0 Hz, 1H, 1/2 OCH2), 3.38-3.37 (m, 1H, OCH), 2.94 (t, J = 4.4 Hz, 1H, 1/2 OCH2), 2.78-2.77 (m, 1H, 1/2 OCH2), 2.57 (s, 3H, COCH3).
(S)-1-(2-hydroxy-4-(oxiran-2-ylmethoxy)phenyl)ethan-1-one (3b). White solid, 45.8% yield. 1H NMR (400 MHz, CDCl3) δ 12.73 (s, 1H, OH), 7.65 (d, J = 9.2 Hz, 1H, Ar-H), 6.49 (d, J = 6.4 Hz, 1H, Ar-H), 6.42 (d, J = 2.8Hz, 1H, Ar-H), 4.30 (d, J = 8.4 Hz, 1H, 1/2 OCH2), 3.95 (d, J = 6.0 Hz, 1H, 1/2 OCH2), 3.39-3.36 (m, 1H, OCH), 2.94 (t, J = 4.8 Hz, 1H, 1/2 OCH2), 2.77 (d, J = 2.8 Hz, 1H, 1/2 OCH2), 2.57 (s, 3H, COCH3).
General procedure for the preparation of 2-acetylphenol-O-alkylhydroxyethylamine derivatives 5a-g and 6a-g. The crude intermediate 3a (1 mmol) was dissolved in anhydrous CH3CN (10 mL). K2CO3 and respective secondary amine (4a~4g) (1.5 mmol) was added to the mixture. The reaction mixture was stirred and refluxed. Upon completion, CH3CN was concentrated under reduced pressure. The residue was extracted with water (40 mL) and dichloromethane (20 mL×3). The organic phases were washed with saturated NaCl aqueous, dried over Na2SO4, filtered and evaporated to dryness under reduced pressure. The crude products were purified by silica gel chromatography using petroleum ether/ ethyl acetate (20:1) as eluent to give the target compounds 5a-g. The target derivatives 6a-g were also got under the same experiments condition using intermediate 3b as the starting material.
(R)-1-(4-(3-(4-benzylpiperidin-1-yl)-2-hydroxypropoxy)-2-hydroxyphenyl)ethan-1-one (5a). Light yellow oil, 80.7% yield, 97.8% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 8.6 Hz, 1H, Ar-H), 7.27 (d, J = 6.4 Hz, 2H, 2 ×Ar-H), 7.19 (d, J = 7.1 Hz, 1H, Ar-H), 7.13 (d, J = 6.7 Hz, 2H, 2 ×Ar-H), 6.46 (d, J = 8.8 Hz, 1H, Ar-H), 6.41 (s, 1H, Ar-H), 4.17 (s, 1H, OCH), 3.99 (s, 2H, OCH2), 3.05-3.03 (m, 2H, NCH2), 2.56 (m, 7H, 2 × NCH2, COCH3), 2.33 (m, 1H, 1/2 phCH2), 2.08 (d, J = 11.1 Hz, 1H, 1/2 phCH2), 1.68 (d, J = 12.2 Hz, 2H, CH2), 1.59 (s, 1H, CH), 1.41 (m, 2H, CH2). HR-ESI-MS: Calcd. for C23H29NO4 [M+H]+: 384.2130, found: 384.2175. (R)-1-(4-(3-(4-benzylpiperazin-1-yl)-2-hydroxypropoxy)-2-hydroxyphenyl)ethan-1-one (5b). Light yellow oil, 86.1% yield, 97.6% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.55 (d, J = 8.9 Hz, 1H, Ar-H), 7.26 (d, J = 4.1 Hz, 4H, 4 ×Ar-H), 7.21 (m, 1H, Ar-H), 6.41 (d, J = 8.9 Hz, 1H, Ar-H), 6.37 (s, 1H, Ar-H), 4.05 (d, J = 8.7 Hz, 1H, OCH), 3.94 (t, J = 5.4 Hz, 2H, OCH2), 3.48 (s, 2H, NCH2), 2.66 (s, 2H, NCH2), 2.50 (m, 11H, 4 × NCH2, COCH3). 13C NMR (100 MHz, CDCl3) δ 202.6, 165.1, 165.1, 137.6, 132.3, 129.2 (2C), 128.2 (2C), 127.2, 114.0, 107.8, 101.5, 70.6, 65.3, 62.8, 60.2, 52.9 (4C), 26.2. HR-ESI-MS: Calcd. for C22H28N2O4 [M+H]+: 385.2083, found: 385.2125.
(R)-1-(2-hydroxy-4-(2-hydroxy-3-(4-isopropylpiperazin-1-yl)propoxy)phenyl)ethan-1-one (5c). Light yellow oil, 89.3% yield, 98.1% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.55 (d, J = 8.9 Hz, 1H, Ar-H), 6.40 (d, J = 9.0 Hz, 1H, Ar-H), 6.35 (s, 1H, Ar-H), 4.05 (d, J = 8.7 Hz, 1H, OCH), 3.94 (d J = 19.9 Hz, 2H, OCH2), 2.66 (d, J = 12.7 Hz, 3H, NCH2, NCH), 2.48 (m, 11H, 4 × NCH2, COCH3), 1.02 (d, J = 6.5 Hz, 6H, 2 × CH3). 13C NMR (100 MHz, CDCl3) δ 202.5, 165.1, 165.0, 132.2, 113.9, 107.7, 101.4, 70.6, 65.3, 60.2, 54.5, 53.2, 48.5 (3C), 26.1, 18.4, 18.3. HR-ESI-MS: Calcd. for C18H28N2O4 [M+H]+: 337.2083, found: 337.2124.
(R)-1-(2-hydroxy-4-(2-hydroxy-3-(4-(pyrimidin-2-yl)piperazin-1-yl)propoxy)phenyl)ethan-1-one (5d). Light yellow oil, 78.5% yield, 97.5% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 12.63 (s, 1H, ph-OH), 8.23 (d, J = 4.7 Hz, 2H, 2 ×Ar-H), 7.55 (d, J = 8.9 Hz, 1H, Ar-H), 6.40 (m, 3H, 3 ×Ar-H), 4.12 (m, 1H, OCH), 3.97 (m, 2H, OCH2), 3.79 (s, 4H, 2 ×NCH2), 3.03 (q, J = 7.3 Hz, 2H, NCH2), 2.66 (m, 2H, NCH2), 2.50 (m, 2H, NCH2), 2.46 (s, 3H2, COCH3). HR-ESI-MS: Calcd. for C19H24N4O4 [M+H]+: 373.1831, found: 373.1871.
(R)-1-(2-hydroxy-4-(2-hydroxy-3-(piperidin-1-yl)propoxy)phenyl)ethan-1-one (5e). Light yellow oil, 83.7% yield, 98.8% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 8.9 Hz, 1H, Ar-H), 6.41 (d, J = 8.9 Hz, 1H, Ar-H), 6.36 (s, 1H, Ar-H), 4.07 (dd, J = 8.9, 4.6 Hz, 1H, OCH), 3.94 (d, J = 4.6 Hz, 2H, OCH2), 2.59 (d, J = 4.3 Hz, 2H, NCH2), 2.47 (m, 5H, NCH2, COCH3), 2.39 (s, 2H, NCH2), 1.57 (d, J = 10.4 Hz, 4H, 2 × CH2), 1.41 (m, 2H, CH2). 13C NMR (100 MHz, CDCl3) δ 202.6, 165.2, 165.0, 132.3, 114.0, 107.8, 101.5, 70.7, 65.1, 61.0 (2C), 54.7, 26.1, 25.8 (2C), 24.0. HR-ESI-MS: Calcd. for C19H24N4O4 [M+H]+: 294.1661, found: 294.1700.
(R)-1-(4-(3-(benzyl(ethyl)amino)-2-hydroxypropoxy)-2-hydroxyphenyl)ethan-1-one (5f). Light yellow oil, 88.6% yield, 98.3% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 12.73 (s, 1H, ph-OH), 7.59 (d, J = 8.9 Hz, 1H, Ar-H), 7.31 (d, J = 3.3 Hz, 4H, 4 ×Ar-H), 7.27 (m, 1H, Ar-H), 6.43 (d, J = 8.9 Hz, 1H, Ar-H), 6.38 (s, 1H, Ar-H), 4.04 (d, J = 7.6 Hz, 1H, OCH), 3.96 (d, J = 9.7 Hz, 2H, OCH2), 3.78 (d, J = 13.5 Hz, 1H, 1/2NCH2), 3.57 (d, J = 13.5 Hz, 1H, 1/2NCH2), 2.63 (m, 4H, 2 × NCH2), 2.51 (s, 3H, COCH3), 1.09 (t, J = 7.0 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 202.6, 165.3, 165.1, 138.3, 132.4, 129.1 (2C), 128.5 (2C), 127.4, 114.0, 107.8, 101.6, 70.6, 66.0, 58.3, 55.7, 47.9, 26.2, 11.7. HR-ESI-MS: Calcd. for C20H25N4O4 [M+H]+: 344.1817, found: 344.1856.
(R)-1-(4-(3-(diethylamino)-2-hydroxypropoxy)-2-hydroxyphenyl)ethan-1-one (5g). Light yellow oil, 83.7% yield, 97.9% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 8.9 Hz, 1H, Ar-H), 6.40 (t, J = 10.1 Hz, 1H, Ar-H), 6.33 (d, J = 15.0 Hz, 1H, Ar-H), 4.04 (d, J = 22.7 Hz, 1H, OCH), 3.95 (d, J = 4.7 Hz, 2H, OCH2), 2.67 (m, 6H, 3 × NCH2), 2.46 (d, J = 17.1 Hz, COCH3), 1.05 (t, J = 7.1 Hz, 6H, 2 × CH3). 13C NMR (100 MHz, CDCl3) δ 202.6, 165.1, 164.9, 132.3, 114.0, 107.7, 101.5, 70.5, 65.6, 55.6, 47.3 (2C), 26.1, 11.3 (2C). HR-ESI-MS: Calcd. for C15H23NO4 [M+H]+: 282.1661, found: 282.1698.
(S)-1-(4-(3-(4-benzylpiperidin-1-yl)-2-hydroxypropoxy)-2-hydroxyphenyl)ethan-1-one (6a). Light yellow oil, 84.7% yield, 98.2% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 12.74 (s, 1H, ph-OH), 7.64 (d, J = 8.9 Hz, 1H, Ar-H), 7.30 (t, J = 7.4 Hz, 2H, 2 ×Ar-H), 7.22 (d, J = 7.1 Hz, 1H, Ar-H), 7.16 (d, J = 7.4 Hz, 2H, 2 ×Ar-H), 6.50 (d, J = 8.9 Hz, 1H, Ar-H), 6.44 (s, 1H, Ar-H), 4.08 (m, 1H, OCH), 4.01 (d, J = 5.8 Hz, 2H, OCH2), 3.00 (d, J = 11.2 Hz, 1H, 1/2NCH2), 2.83 (d, J = 11.2 Hz, 1H, 1/2NCH2), 2.56 (d, J = 5.2 Hz, 5H, NCH2, COCH3), 2.49 (d, J = 13.4, 6.8 Hz, 2H, NCH2,), 2.29 (d, J = 11.5 Hz, 1H, 1/2 phCH2), 1.98 (d, J = 11.4 Hz, 1H, 1/2 phCH2), 1.68 (d, J = 13.0 Hz, 2H, CH2), 1.32 (d, J = 10.1 Hz, 3H, CH, CH2). 13C NMR (100 MHz, CDCl3) δ 202.7, 165.3, 165.2, 140.5, 132.4, 129.2 (2C), 128.3 (2C), 126.0, 114.1, 108.0, 101.6, 70.7, 65.2, 60.5, 55.6, 52.7, 43.1, 37.8, 32.3, 32.0, 26.3. HR-ESI-MS: Calcd. for C23H29NO4 [M+H]+: 384.2130, found: 384.2172.
(S)-1-(4-(3-(4-benzylpiperazin-1-yl)-2-hydroxypropoxy)-2-hydroxyphenyl)ethan-1-one (6b). Light yellow oil, 78.4% yield, 98.0% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 12.73 (s, 1H, ph-OH), 7.64 (d, J = 8.8 Hz, 1H, Ar-H), 7.32 (t, J = 12.1 Hz, 5H, 5 ×Ar-H), 6.49 (d, J = 8.9 Hz, 1H, Ar-H), 6.44 (s, 1H, Ar-H), 4.12 (m, 1H, OCH), 4.01 (d, J = 5.1 Hz, 2H, OCH2), 3.55 (s, 2H, NCH2), 2.75 (s, 2H, NCH2), 2.58 (m, 11H, 4 × NCH2, COCH3). HR-ESI-MS: Calcd. for C22H28N2O4 [M+H]+: 385.2083, found: 385.2126.
(S)-1-(2-hydroxy-4-(2-hydroxy-3-(4-isopropylpiperazin-1-yl)propoxy)phenyl)ethan-1-one (6c). Light yellow oil, 86.2% yield, 97.6% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 8.9 Hz, 1H, Ar-H), 6.44 (d, J = 8.9 Hz, 1H, Ar-H), 6.39 (s, 1H, Ar-H), 4.07 (d, J = 4.4 Hz, 1H, OCH), 3.97 (d, J = 8.6 Hz, 2H, OCH2), 2.67(m, 3H, NCH2, NCH), 2.49 (m, 11H, 4 × NCH2, COCH3), 1.03 (d, J = 6.5 Hz, 6H, 2 × CH3). HR-ESI-MS: Calcd. for C18H28N2O4 [M+H]+: 337.2083, found: 337.2117.
(S)-1-(2-hydroxy-4-(2-hydroxy-3-(4-(pyrimidin-2-yl)piperazin-1-yl)propoxy)phenyl)ethan-1-one (6d). Light yellow oil, 72.7% yield, 97.4% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 12.59 (s, 1H, ph-OH), 8.18 (d, J = 4.6 Hz, 2H, 2 ×Ar-H), 7.51 (d, J = 8.9 Hz, 1H, Ar-H), 6.36 (m, 3H, 3 ×Ar-H), 4.08 (m, 1H, OCH), 3.94 (m, 2H, OCH2), 3.74 (s, 4H, 2 ×NCH2), 2.99 (q, J = 7.2 Hz, 2H, NCH2), 2.61 (m, 2H, NCH2), 2.46 (m, 2H, NCH2), 2.42 (s, 3H2, COCH3). HR-ESI-MS: Calcd. for C19H24N4O4 [M+H]+: 373.1831, found: 373.1866.
(S)-1-(2-hydroxy-4-(2-hydroxy-3-(piperidin-1-yl)propoxy)phenyl)ethan-1-one (6e). Light yellow oil, 85.8% yield, 98.8% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 12.68 (s, 1H, ph-OH), 7.60 (d, J = 8.9 Hz, 1H, Ar-H), 6.45 (d, J = 8.9 Hz, 1H, Ar-H), 6.39 (s, 1H, Ar-H), 4.15 (d, J = 8.7 Hz, 1H, OCH), 3.98 (d, J = 4.7 Hz, 2H, OCH2), 2.69 (d, J = 3.6 Hz, 2H, NCH2), 2.55 (m, 7H, 2 ×NCH2, COCH3), 1.64 (d, J = 5.0 Hz, 4H, 2 × CH2), 1.48 (d, J = 5.3 Hz, 2H, CH2). HR-ESI-MS: Calcd. for C16H23NO4 [M+H]+: 294.1661, found: 294.1705.
(S)-1-(4-(3-(benzyl(ethyl)amino)-2-hydroxypropoxy)-2-hydroxyphenyl)ethan-1-one (6f). Light yellow oil, 88.1% yield, 97.3% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 12.72 (s, 1H, ph-OH), 7.63 (d, J = 8.9 Hz, 1H, Ar-H), 7.30 (d, J = 3.3 Hz, 4H, 4 ×Ar-H), 7.23 (m, 1H, Ar-H), 6.49 (d, J = 8.9 Hz, 1H, Ar-H), 6.44 (s, 1H, Ar-H), 4.22 (d, J = 7.6 Hz, 1H, OCH), 4.03 (d, J = 9.7 Hz, 2H, OCH2), 3.78 (d, J = 13.5 Hz, 1H, 1/2NCH2), 3.68 (d, J = 13.5 Hz, 1H, 1/2NCH2), 2.79 (m, 4H, 2 × NCH2), 2.55 (s, 3H, COCH3), 1.12 (t, J = 7.0 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ 202.7, 165.1, 132.5, 132.3, 129.0, 128.5, 127.4, 107.9, 101.5, 70.6, 65.8, 58.2, 55.6, 47.8, 26.3, 11.7. HR-ESI-MS: Calcd. for C20H25NO4 [M+H]+: 344.1817, found: 344.1862.
(S)-1-(4-(3-(diethylamino)-2-hydroxypropoxy)-2-hydroxyphenyl)ethan-1-one (6g). Light yellow oil, 88.9% yield, 98.5% HPLC purity. 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 8.9 Hz, 1H, Ar-H), 6.44 (d, J = 8.9 Hz, 1H, Ar-H), 6.38 (m, 1H, Ar-H), 4.01 (m, 1H, OCH), 3.96 (d, J = 5.1 Hz, 2H, OCH2), 2.61 (d, J = 12.8 Hz, 6H, 3 × NCH2), 2.51 (s, 3H, COCH3), 1.04 (t, J = 7.1 Hz, 6H, 2 × CH3). HR-ESI-MS: Calcd. for C15H23NO4 [M+H]+: 282.1661, found: 282.1687.
Biological activity
Inhibition experiments of AChE and BuChE. AChE and BuChE inhibitory potency of the target compounds were tested by slightly modified Ellman assay [25]. Briefly, for AChE inhibition assays, a reaction mixture (100 µL) included substrate ATC (1 mmol/L, 30 µL), phosphate-buffered solution (0.1 M KH2PO4/K2HPO4, PH = 8.0, 40 µL), diverse concentrations (DMSO < 1%) of test compounds (20 µL) and 10 µL enzyme (eeAChE 0.45 U/mL) was incubated at 37°C for 15 min. After that 5,5’dithiobis-2-nitrobenzoic acid (DTNB, 0.2%, 30 µL) was added to the mixture. Using a Varioskan Flash Multimode Reader at 412 nm to determine the activities. The inhibition percent was calculated by the following expression: (1−Ai/Ac) × 100, in which Ai and Ac are the absorbance obtained for AChE in the presence and absence of inhibitors after subtracting the background. All samples were performed in triplicate. The procedure of BuChE assay was similar as described above using BTC and BuChE instead of ATC and AChE.
Antioxidant activity assay. The antioxidant activity was assessed by the oxygen radical absorbance capacity fluorescein (ORAC-FL) assay. The detailed procedure referenced our previous work [27, 28].
Recombinant human MAO-A and MAO-B inhibition studies [31]. Recombinant human MAO-A and MAO-B, purchased from Sigma-Aldrich, were prealiquoted and stored at -80 °C. The test compounds were dissolved in DMSO (2.5 mM) and diluted with potassium phosphate buffer (100 mM, pH 7.40, containing KCl 20.2 mM) to a final volume of 500 μL composed of different concentrations of test compounds (0-100 μM) and kynuramine (45 μM for MAO-A and 30 μM for MAO-B). The reactions were triggered via the addition of the enzyme (7.5 μg/mL) and then incubated for 30 min at 37°C. After that 400 μL NaOH (2N) and 1000 μL water were added to terminate the reactions and finally the mistures were centrifuged at 16000g for 10 min. The reactions were quantified on a Varioskan Flash Multimode Reader (PerkinElmer) based on the fluorescence of the supernatant with excitation and emission wavelengths at 310 nm and 400 nm, respectively. IC50 values were calculated using GraphRad Prism 5. IC50 values were determined in triplicate and expressed as mean ± SD.
Metal chelation studies. The UV absorption of the target compound, alone or in the presence of CuCl2, ZnCl2, FeSO4, and AlCl3, was recorded with Shimadzu UV-2450 spectrophotometer with wavelength ranging from 200 to 600 nm after incubating for 30 min at room temperature [27]. The final concentrations of tested compound and metals were 37.5 μM, and the final volume of reaction mixture was 1 mL. The spectra of the metal alone and the compound alone numerically subtracted from the spectra of the mixture to give the difference UV-vis spectra due to complex formation. The procedure referenced our previous work.
BACE-1 inhibition studies. BACE-1 in vitro inhibition assay was tested as described in the assay procedure by utilizing the β-secretase Inhibitor Screening Assay Kit [32]. Reaction mixture comprised of test compound (2.5 µL) or Donepezil (control), BACE-1 enzyme (15 µL) and the substrate was incubated for 1 hour at 25 °C. The fluorescence signal was recorded at an excitation wavelength of 340 nm and an emission wavelength 490 nm by microplate reader (EnSpire Multimode; PerkinElmer). The inhibition percentage was calculated by the equation: Percent inhibition (%) = 100 − [AFi/AF0] × 100%, where AFi and AF0 mean the fluorescence intensities obtained for BACE-1 in the presence and absence of an inhibitor, respectively.
Neuroprotective effect. The neuroprotective effects were evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. The detailed procedure referenced our previous work [27, 28].
In vitro blood-brain barrier permeation assay. The parallel artificial membrane permeation assay (PAMPA) was used to evaluate the blood-brain barrier penetration of compounds [33, 34]. The donor microplate (PVDF membrane, pore size 0.45 mm) and acceptor microplate were both from Millipore. The 96-well UV plate (COSTAR) was from Corning Incorporated. According to the detailed procedure in our previous work, we concluded that compounds with Pe values above 3.44 ×10−6 cm/s could cross the blood- brain barrier.