General information
All the reagents were used without further purification unless otherwise specified. Solvents were dried and redistilled prior to use in the usual manner. All starting materials were commercially available. For product purification by flash column chromatography, silica gel (200~300 mesh) was used. The reactions monitoring was accomplished by TLC on silica gel polygram SILG/UV 254 plates. 1H NMR spectra were recorded at 400 MHz or 500 MHz in CDCl3 using TMS as internal standard. 13C NMR spectral measurements were performed at 125 MHz or 101MHz using TMS as an internal standard. All compounds were identified by 1H NMR and are in good agreement with those reported. HRMS(ESI) was determined on a PerkinElmer spectrometer.
- General procedure for the preparation of compounds 2a-2k
To a solution of various secondary amine (1.0 eq.) in 20 mL of DCM was added Et3N (2.0 eq.) and chloroacetyl chloride (1.0 eq.) in an ice bath under N2. The reaction mixture was allowed to warm to room temperature and continued to be stirred for 3-5 h. The reaction was monitored by TLC. After completion, the reaction was quenched by addition of water and extracted with EtOAc (3×20 mL). The organic phase was dried over anhydrous Na2SO4. After condensation under reduced pressure, the obtained compounds 2a-2k were directly used into the next step without further purification.
General procedure for the preparation of compounds 3a-3k
To a solution of curcumol (1.0 eq.) in 25 mL dry THF, NaH (5.0 eq.) was added and the reaction mixture was stirred at room temperature for 2 h. Then, the compounds 2a-2k (1.0 eq.) was added at room temperature for 5-8 h. Reaction was monitored by TLC. The reaction is quenched by water and extracted with EtOAc (3 × 20 ml). The combined organic layer was dried over Na2SO4 and purified through column chromatography to offer the corresponding products.
2-(((3S,3aS,5R,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)-N,N-dimethylacetamide (3a): Yellow oil (395 mg, 58% yield), 1H NMR (500 MHz, CDCl3) δ 4.77 (s, 2H), 4.29 (d, J = 12.5 Hz, 1H), 4.11 (d, J = 12.5 Hz, 1H), 3.01 (s, 3H), 2.85 (s, 3H), 2.50 (d, J = 14.7 Hz, 1H), 2.40 (d, J = 14.7 Hz, 1H), 2.12-2.05 (m, 1H), 2.02-1.94 (m, 1H), 1.90-1.75 (m, 2H), 1.75-1.66 (m, 1H), 1.66-1.50 (m, 3H), 1.31-1.38 (m, 1H), 1.08 (dd, J = 12.5, 6.4 Hz, 1H), 0.87 (t, J = 6.2 Hz, 6H), 0.76 (d, J = 6.4 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 169.24, 144.54, 112.61, 107.26, 87.49, 62.03, 54.36, 49.63, 39.16, 37.10, 36.70, 35.24, 33.97, 30.81, 28.36, 27.97, 22.71, 20.74, 12.04; HRMS m/z (ESI): calcd for C19H32NO3 [M+H]+: 322.2377, found: 322.2379.
N,N-diethyl-2-(((3S,3aS,5R,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)acetamide (3b): Yellow oil ( 385 mg, 52% yield), 1H NMR (500 MHz, CDCl3) δ 4.83 (s, 2H), 4.34 (d, J = 12.1 Hz, 1H), 4.14 (s, 1H), 3.38-3.48 (m, 2H), 3.28-3.37 (m, 2H), 2.55 (d, J = 15.7 Hz, 1H), 2.46 (d, J = 14.8 Hz, 1H), 2.18-2.10 (m, 1H), 2.04 (t, J = 12.3 Hz, 1H), 1.97-1.80 (m, 2H), 1.81-1.72 (m, 1H), 1.71-1.57 (m, 3H), 1.37-1.44 (m, 1H), 1.08-1.17 (m, 7H), 0.94 (d, J = 6.5 Hz, 6H), 0.82 (d, J = 6.4 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 168.64, 144.87, 112.78, 107.52, 87.61, 62.33, 54.62, 49.68, 41.30, 39.90, 39.39, 37.49, 34.19, 31.03, 28.61, 28.22, 22.92, 21.00, 14.27, 12.69, 12.24; HRMS m/z (ESI): calcd for C21H36NO3 [M+H]+: 350.2690, found: 350.2693.
N,N-dibutyl-2-(((3S,3aS,5R,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)acetamide (3c): Yellow oil (515 mg, 60% yield), 1H NMR (500 MHz, CDCl3) δ 4.78 (s, 2H), 4.31 (d, J = 12.1 Hz, 1H), 4.08 (d, J = 12.1 Hz, 1H), 3.36-3.11 (m, 4H), 2.53-2.37 (m, 2H), 2.11 (d, J = 10.3 Hz, 1H), 1.99 (t, J = 12.3 Hz, 1H), 1.91-1.76 (m, 2H), 1.69-1.75 (m, 1H), 1.67-1.55 (m, 3H), 1.42-1.51 (m, 4H), 1.41-1.32 (m, 1H), 1.20-1.29 (m, 5H), 1.10 (dd, J = 12.5, 6.4 Hz, 1H), 0.90 (d, J = 6.6 Hz, 6H), 0.83-0.87 (m, 6H), 0.78 (d, J = 6.3 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 168.76, 144.74, 112.66, 107.38, 87.50, 62.08, 54.54, 49.51, 46.91, 45.27, 39.30, 37.42, 34.12, 30.98, 30.89, 29.42, 28.50, 28.11, 22.82, 20.91, 20.06, 19.89, 13.70, 13.67, 12.1; HRMS m/z (ESI): calcd for C25H44NO3 [M+H]+: 406.3316, found: 406.3315.
2-(((3S,3aS,5R,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)-1-(pyrrolidin-1-yl)ethan-1-one (3d): Yellow oil (391mg, 53% yield), 1H NMR (500 MHz, CDCl3) δ 4.82 (s, 2H), 4.30 (d, J = 12.9 Hz, 1H), 4.13 (d, J = 12.9 Hz, 1H), 3.52 (s, 2H), 3.44 (t, J = 6.7 Hz, 2H), 2.55 (d, J = 14.8 Hz, 1H), 2.46 (d, J = 14.7 Hz, 1H), 2.13 (d, J = 19.9 Hz, 1H), 2.03 (t, J = 13.1 Hz, 1H), 1.96-1.84 (m, 3H), 1.84-1.75 (m, 4H), 1.71-1.56 (m, 3H), 1.45-1.33 (m, 1H), 1.13 (dd, J = 11.4, 5.3 Hz, 1H), 0.93 (d, J = 6.5 Hz, 6H), 0.81 (d, J = 6.3 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 168.03, 144.77, 112.69, 107.37, 87.55, 62.58, 54.50, 49.69, 46.22, 45.86, 39.30, 37.24, 34.12, 30.95, 28.52, 28.13, 26.12, 23.76, 22.85, 20.91, 12.18; HRMS m/z (ESI): calcd for C21H34NO3 [M+H]+: 348.2533, found: 348.2531.
2-(((3S,3aS,5R,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)-1-(piperidin-1-yl)ethan-1-one (3e): Yellow oil (490 mg, 64 yield%), 1H NMR (500 MHz, CDCl3) δ 4.77 (s, 2H), 4.27 (d, J = 12.0 Hz, 1H), 4.09 (d, J = 12.0 Hz, 1H), 3.37-3.52 (m, 4H), 2.48 (d, J = 14.7 Hz, 1H), 2.39 (d, J = 14.7 Hz, 1H), 2.08 (t, J = 10.0 Hz, 1H), 1.98 (t, J = 12.3 Hz, 1H), 1.90-1.75 (m, 2H), 1.66-1.73 (m, 1H), 1.56-1.63 (m, 6H), 1.52-1.42 (m, 5H), 1.34 (m, 1H), 1.08 (dd, J = 12.4, 6.4 Hz, 1H), 0.93-0.83 (m, 6H), 0.76 (d, J = 6.4 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 167.48, 144.48, 112.56, 107.20, 87.38, 62.18, 54.33, 49.44, 46.13, 42.65, 39.13, 37.18, 33.90, 30.79, 28.33, 27.95, 26.09, 25.25, 24.30, 22.66, 20.78, 12.00; HRMS m/z (ESI): calcd for C22H36NO3 [M+H]+: 362.2690, found: 362.2689.
1-(azepan-1-yl)-2-(((3S,3aS,5R,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)ethan-1-one (3f): Yellow oil (548 mg, 69% yield), 1H NMR (500 MHz, CDCl3) δ 4.76 (s, 2H), 4.29 (d, J = 12.0 Hz, 1H), 4.07 (d, J = 12.0 Hz, 1H), 3.51-3.36 (m, 4H), 2.47 (d, J = 14.2 Hz, 1H), 2.39 (d, J = 14.7 Hz, 1H), 2.12-2.03 (m, 1H), 1.97 (t, J = 12.3 Hz, 1H), 1.74-1.80 (m, 2H), 1.61-1.72 (m, 9H), 1.47 (s, 5H), 1.38-1.27 (m, 1H), 1.07 (dd, J = 12.5, 6.4 Hz, 1H), 0.87 (dd, J = 6.5, 2.4 Hz, 6H), 0.75 (d, J = 6.4 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 168.66, 144.50, 112.53, 107.20, 87.30, 62.04, 54.32, 49.38, 46.93, 46.10, 39.10, 37.17, 33.89, 30.77, 29.31, 28.32, 27.92, 27.22, 26.70, 26.29, 22.66, 20.74, 11.99; HRMS m/z (ESI): calcd for C23H38NO3 [M+H]+: 376.2846, found: 376.2847.
2-(((3S,3aS,5R,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)-1-morpholinoethan-1-one (3g): Yellow oil ( 385 mg, 50% yield), 1H NMR (500 MHz, CDCl3) δ 4.78 (s, 2H), 4.27 (d, J = 12.2 Hz, 1H), 4.11 (d, J = 12.2 Hz, 1H), 3.66-3.55 (m, 7H), 3.51 (d, J = 4.4 Hz, 2H), 2.33-2.52 (m, 2H), 2.09 (t, J = 10.1 Hz, 1H), 1.99 (t, J = 12.3 Hz, 1H), 1.92-1.74 (m, 2H), 1.66-1.72 (m, 1H), 1.65-1.53 (m, 3H), 1.29-1.36 (m, 1H), 1.09 (dd, J = 12.5, 6.4 Hz, 1H), 0.86 (dd, J = 6.4, 3.6 Hz, 6H), 0.77 (d, J = 6.4 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 167.87, 144.17, 112.83, 107.32, 87.61, 66.53, 66.45, 62.07, 54.26, 49.42, 45.76, 42.02, 39.09, 37.15, 33.83, 30.80, 28.30, 27.92, 22.65, 20.82, 12.07; HRMS m/z (ESI): calcd for C21H34NO4 [M+H]+: 364.2482, found: 364.2481.
1-(3,4-dihydroisoquinolin-2(1H)-yl)-2-(((3S,3aS,5R,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)ethan-1-one (3h): Brown oil ( 347 mg, 40% yield), 1H NMR (500 MHz, CDCl3) δ 6.95-7.15 (m, 4H), 4.82 (s, 2H), 4.76 (s, 1H), 4.65 (s, 1H), 4.47-4.38 (m, 1H), 4.20 (dd, J = 18.2, 12.2 Hz, 1H), 3.62-3.90 (m, 2H), 2.85 (t, J = 5.3 Hz, 1H), 2.79 (s, 1H), 2.63-2.35 (m, 2H), 2.13 (t, J = 9.9 Hz, 1H), 2.09-1.99 (m, 1H), 1.97-1.73 (m, 4H), 1.55-1.71 (m, 4H), 1.13 (dd, J = 12.4, 6.3 Hz, 1H), 0.89-0.92 (m, 6H), 0.81 (t, J = 5.7 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 168.22, 144.23, 133.87, 132.68, 128.09, 126.16, 112.73, 107.28, 87.45, 62.36, 54.23, 49.17, 46.82, 44.19, 42.60, 39.92, 39.03, 37.13, 33.81, 30.81, 29.12, 28.27, 27.90, 22.64, 20.69, 12.04 ppm; HRMS m/z (ESI): calcd for C26H36NO3 [M+H]+: 410.2690, found: 410.2692.
2-(((3S,3aS,5R,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)-1-(4-phenylpiperazin-1-yl)ethan-1-one (3i): Yellow oil (724 mg, 78% yield), 1H NMR (500 MHz, CDCl3) δ 7.34-7.21 (m, 2H), 7.02-6.80 (m, 3H), 4.89 (s, 2H), 4.43 (d, J = 12.2 Hz, 1H), 4.25 (d, J = 12.2 Hz, 1H), 3.73-3.90 (m, 4H), 3.29-3.05 (m, 4H), 2.58 (d, J = 14.8 Hz, 1H), 2.51 (d, J = 14.7 Hz, 1H), 2.23-2.16 (m, 1H), 2.10 (t, J = 12.3 Hz, 1H), 2.02-1.86 (m, 3H), 1.79-1.84 (m, 1H), 1.75-1.64 (m, 3H), 1.50-1.40 (m, 1H), 1.19 (dd, J = 12.5, 6.5 Hz, 1H), 0.98 (dd, J = 11.8, 6.5 Hz, 6H), 0.87 (d, J = 6.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 167.82, 150.83, 144.29, 128.89 (Ar-overlap), 120.06, 116.33(Ar-overlap), 112.88, 107.38, 87.63, 62.24, 54.31, 49.61, 49.41, 48.93, 45.06, 41.58, 39.15, 37.29, 33.90, 30.89, 28.38, 28.02, 22.75, 20.94, 12.20 ppm. HRMS m/z (ESI): calcd for C27H39N2O3 [M+H]+: 439.2955, found: 439.2957.
tert-butyl(2-(2-(((3S,3aS,5R,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)-N-methylacetamido)ethyl)(methyl)carbamate (3j): Yellow oil ( 580 mg, 59% yield), 1H NMR (500 MHz, CDCl3) δ 4.78 (s, 2H), 4.34-4.21 (m, 1H), 4.21-4.01 (m, 1H), 3.60-3.20 (m, 4H), 3.03 (s, 2H), 2.87 (s, 1H), 2.81 (s, 3H), 2.55-2.33 (m, 2H), 2.10 (t, J = 9.9 Hz, 1H), 1.92-2.05 (m, 1H), 1.77-1.86(m, 2H), 1.57-1.63 (m, 4H), 1.38 (s, 9H), 1.09 (dd, J = 12.6, 6.2 Hz, 1H), 0.88 (d, J = 6.3 Hz, 6H), 0.77 (d, J = 5.7 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 169.33, 155.67, 144.56, 112.78, 107.32, 87.59, 61.78, 54.45, 49.69, 45.36, 39.23 (overlap), 37.40, 37.24, 34.70, 34.05, 30.86 (overlap), 28.44, 28.20 (overlap), 28.06, 22.81, 20.92, 12.13; HRMS m/z (ESI): calcd for C26H43N2O5 [M+H]+: 465.3323, found: 465.3324.
tert-butyl4-(2-(((3S,3aS,5R,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)acetyl)piperazine-1-carboxylate (3k): Yellow solid (617mg, 63% yield), m.p. 118-120 °C; 1H NMR (500 MHz, CDCl3) δ 4.83 (s, 2H), 4.33 (d, J = 12.0 Hz, 1H), 4.16 (d, J = 12.0 Hz, 1H), 3.76-3.18 (m, 8H), 2.55-2.33 (m, 2H), 2.08-2.17 (m, 1H), 2.08-1.98 (m, 1H), 1.95-1.78 (m, 2H), 1.69-1.75 (m, 1H), 1.61-1.67 (m, 3H), 1.43 (s, 9H), 1.13 (dd, J = 12.5, 6.4 Hz, 1H), 0.91 (t, J = 6.5 Hz, 6H), 0.81 (d, J = 6.3 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 168.31, 154.62, 144.48, 113.14, 107.64, 87.94, 80.14, 62.52, 54.56 (overlap), 45.32, 41.82, 39.38 (overlap), 37.47, 34.13, 31.08, 28.60, 28.35 (overlap), 28.23, 22.93, 21.11 (overlap), 12.38; HRMS m/z (ESI): calcd for C26H43N2O5 [M+H]+: 463.3166, found: 463.3168.
General procedure for the preparation of compounds 3l-3m
To a solution of compound 3j (1.0 eq.) in DCM (10 mL), TFA (1 mL) as added at 0°C and the mixture was stirred for 1 h at 0°C. Reaction was monitored by TLC. Saturated aqueous Na2CO3 was added and the mixture was extracted with DCM. The combined organic layer was dried over Na2SO4 and purified by silica gel column chromatography to provide the corresponding products.
2-(((3S,3aS,5R,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)-N-methyl-N-(2-(methylamino)ethyl)acetamide (3l) : Yellow oil (168 mg, 71% yield), 1H NMR (500 MHz, CDCl3) δ4.80 (s, 2H), 4.36 (dd, J = 26.5, 12.3 Hz, 1H), 4.10-4.17 (m, 1H), 3.56-3.37 (m, 2H), 3.04 (d, J = 10.3 Hz, 2H), 2.88 (d, J = 3.6 Hz, 2H), 2.74 (t, J = 6.6 Hz, 2H), 2.40 (s, 3H), 2.17-2.07 (m, 1H), 2.01 (t, J = 12.3 Hz, 1H), 1.92-1.68 (m, 4H), 1.59-1.66 (m, 3H), 1.35-1.41 (m, 1H), 1.09-1.14 (m, 1H), 0.93-0.87 (m, 6H), 0.79 (d, J = 6.3 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 169.81, 144.55, 112.74, 107.34, 87.60, 54.40, 49.67, 49.16, 48.52, 47.16, 39.19, 37.23, 35.64, 35.37, 34.01, 30.83, 28.41, 28.02, 22.77, 20.89, 12.11; HRMS m/z (ESI): calcd for C21H37N2O3 [M+H]+: 365.2799, found: 363.2797.
2-(((3S,3aS,5R,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)-1-(piperazin-1-yl)ethan-1-one (3m): Yellow oil (179 mg, 76% yield), 1H NMR (400 MHz, CDCl3) δ 4.87 (d, J = 2.4 Hz, 2H), 4.37 (d, J = 12.1 Hz, 1H), 4.20 (d, J = 12.1 Hz, 1H), 3.79-3.54 (m, 4H), 3.09 (s, 2H), 2.80-2.95 (m, 4H), 2.59-2.41 (m, 2H), 2.18 (t, J = 10.0 Hz, 1H), 2.08 (t, J = 12.2 Hz, 1H), 2.01-1.83 (m, 2H), 1.83-1.73 (m, 1H), 1.73-1.62 (m, 3H), 1.47-1.37 (m, 1H), 1.17 (dd, J = 12.5, 6.3 Hz, 1H), 0.95 (dd, J = 6.4, 3.4 Hz, 6H), 0.86 (d, J = 6.3 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 168.18, 144.63, 113.12, 107.63, 87.90, 62.42, 54.58, 49.66, 46.36, 46.05, 45.56, 42.63, 39.41, 37.52, 34.16, 31.10, 28.65, 28.26, 22.99, 21.16, 12.41; HRMS m/z (ESI): calcd for C21H35N2O3 [M+H]+: 363.2642, found: 363.2640.
General procedure for the preparation of compound 4a-4g
To a solution of compound 3 (1.0 eq.) in 30 mL DCM and placed in an ice bath, and acyl chloride (1.0 eq.) was introduced slowly. The reaction was allowed to come to RT and stirred 2-5 h. Reaction was monitored by TLC. DCM was removed via rotary evaporation. The remaining oil was dissolved in EtOAc and washed with distilled water and the crude mixture was extracted with EtOAc (3×20 mL). The organic phase was dried over anhydrous Na2SO4. After filtration, EtOAc was removed by rotary evaporation and purified through column chromatography to offer the corresponding products.
1-(4-acetylpiperazin-1-yl)-2-(((3S,3aS,5S,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)ethan-1-one (4a): Yellow oil (295 mg, 83% yield), 1H NMR (500 MHz, CDCl3) δ 4.76 (s, 2H), 4.28 (d, J = 12.4 Hz, 1H), 4.10 (dd, J = 12.4, 5.3 Hz, 1H), 3.47-3.61 (m, 6H), 3.42-3.32 (m, 2H), 2.41 (t, J = 15.4 Hz, 2H), 2.07 (t, J = 10.3 Hz, 1H), 2.02 (s, 3H), 1.98-1.89 (m, 1H), 1.89-1.71 (m, 2H), 1.71-1.63 (m, 1H), 1.57 (dd, J = 12.5, 5.4 Hz, 3H), 1.38-1.25 (m, 1H), 1.07 (dd, J = 12.6, 6.2 Hz, 1H), 0.83 (t, J = 7.5 Hz, 6H), 0.74 (d, J = 6.2 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 168.98, 168.09, 143.95, 112.90, 107.27, 87.64, 62.00, 54.15, 49.33, 45.59, 44.83, 41.65, 41.34, 38.99, 37.08, 33.73, 30.75, 28.21, 27.87, 22.59, 20.94, 20.76, 12.07; HRMS m/z (ESI): calcd for C23H37N2O4 [M+H]+: 405.2748, found: 405.2748.
3-chloro-1-(4-(2-(((3S,3aS,5S,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxy azulen-6-yl)oxy)acetyl)piperazin-1-yl)propan-1-one (4b): Yellow oil (284 mg, 68% yield), 1H NMR (500 MHz, CDCl3) δ 4.85 (s, 2H), 4.43-4.33 (m, 1H), 4.19 (d, J = 12.0 Hz, 1H), 3.82 (t, J = 6.9 Hz, 2H), 3.59-3.73 (m, 6H), 3.40-3.53 (m, 2H), 2.82 (t, J = 6.9 Hz, 2H), 2.47 (dd, J = 17.3, 12.0 Hz, 2H), 2.16 (dd, J = 12.7, 7.5 Hz, 1H), 2.11-2.00 (m, 1H), 1.83-1.96 (m, 2H), 1.82-1.58 (m, 4H), 1.48-1.32 (m, 1H), 1.16 (dd, J = 12.4, 6.1 Hz, 1H), 0.92 (t, J = 6.6 Hz, 6H), 0.83 (d, J = 6.2 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 168.39, 168.20, 144.23, 113.18, 107.59, 87.96, 62.34, 54.44, 53.37, 49.65, 45.10, 41.90, 39.72, 39.27, 37.34, 35.85, 34.01, 31.03, 28.50, 28.16, 22.85, 21.03 (overlap), 12.35; HRMS m/z (ESI): calcd for C24H37ClN2O4Na [M+Na]+: 475.2334, found: 475.2334.
1-(4-(2-(((3S,3aS,5S,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)acetyl)piperazin-1-yl)prop-2-en-1-one (4c): Yellow oil (316 mg, 82% yield), 1H NMR (500 MHz, CDCl3) δ 6.48 (dd, J = 16.8, 10.6 Hz, 1H), 6.36-6.04 (m, 1H), 5.61-5.64 (m, 1H), 4.76 (s, 2H), 4.28 (d, J = 12.3 Hz, 1H), 4.13-4.06 (m, 1H), 3.75-3.36 (m, 8H), 2.49-2.30 (m, 2H), 2.12-2.04 (m, 1H), 2.01-1.92 (m, 1H), 1.75-1.85 (m, 2H), 1.63-1.68 (m, 1H), 1.62-1.51 (m, 3H), 1.40-1.24 (m, 1H), 1.07 (dd, J = 12.4, 6.2 Hz, 1H), 0.84 (t, J = 6.7 Hz, 6H), 0.75 (d, J = 6.3 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 167.99, 165.16, 143.92, 128.00, 126.84, 112.89, 107.28, 87.63, 62.05, 54.13, 53.06, 49.31, 45.09, 41.78, 38.97, 37.09, 33.72, 30.74, 28.19, 27.85, 22.57, 20.75 (overlap), 12.05; HRMS m/z (ESI): calcd for C24H36N2O4Na [M+Na]+: 439.2567, found: 439.2568.
2-(((3S,3aS,5S,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)-1-(4-(pyrimidin-2-yl)piperazin-1-yl)ethan-1-one (4d): Yellow oil (333 mg, 86% yield), 1H NMR (500 MHz, CDCl3) δ 8.29 (dd, J = 4.7, 1.1 Hz, 2H), 6.49 (t, J = 4.6 Hz, 1H), 4.83 (s, 2H), 4.39 (d, J = 12.0 Hz, 1H), 4.20 (d, J = 12.0 Hz, 1H), 3.95-3.49 (m, 9H), 2.57-2.40 (m, 2H), 2.19-2.11 (m, 1H), 2.05 (t, J = 12.3 Hz, 1H), 1.96-1.72 (m, 3H), 1.72-1.57 (m, 3H), 1.46-1.36 (m, 1H), 1.14 (dd, J = 12.5, 6.5 Hz, 1H), 0.93 (dd, J = 11.1, 6.6 Hz, 6H), 0.82 (d, J = 6.3 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 168.24, 161.41, 157.60 (Ar-overlap), 144.43, 113.00, 110.16, 107.51, 87.80, 62.40, 54.46, 49.47, 45.18, 43.85, 43.27, 41.77, 39.30, 37.45, 34.04, 30.99, 28.51, 28.16, 22.85, 21.03, 12.31; HRMS m/z (ESI): calcd for C25H36N4O3SNa [M+Na]+: 463.2680, found: 463.2681.
1-(4-(2-hydroxyethyl)piperazin-1-yl)-2-(((3S,3aS,5S,6R,8aS)-5-isopropyl-3-methyl-8-methyleneocta hydro-6H-3a,6-epoxyazulen-6-yl)oxy)ethan-1-one (4e): Yellow oil (243mg, 68% yield), 1H NMR (500 MHz, CDCl3) δ 4.79 (s, 2H), 4.29 (dd, J = 12.0, 2.9 Hz, 1H), 4.13-4.04 (m, 1H), 3.71-3.47 (m, 8H), 3.18 (s, 2H), 2.43 (d, J = 13.1 Hz, 5H), 2.10 (t, J = 10.0 Hz, 1H), 2.00 (t, J = 12.3 Hz, 1H), 1.74-1.92 (m, 2H), 1.74-1.54 (m, 4H), 1.41-1.29 (m, 2H), 1.10 (dd, J = 12.5, 6.3 Hz, 1H), 0.87 (t, J = 5.6 Hz, 6H), 0.78 (d, J = 6.4 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 167.92, 144.34, 112.90, 107.37, 87.65, 62.06, 59.36, 57.73, 54.33, 52.97, 52.43, 49.32, 45.10, 41.62, 39.16, 37.33, 33.90, 30.86, 28.38, 28.02, 22.74, 20.91, 12.17; HRMS m/z (ESI): calcd for C23H39N2O4 [M+H]+: 407.2904, found: 407.2906.
4-(2-(((3S,3aS,5S,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)acetyl)-1,1-dimethylpiperazin-1-ium (4f): Yellow oil (279 mg, 72% yield), 1H NMR (500 MHz, CDCl3) δ 4.87 (s, 2H), 4.39 (d, J = 12.2 Hz, 1H), 4.18 (d, J = 12.2 Hz, 1H), 3.92-3.64 (m, 4H), 3.33-3.12 (m, 4H), 2.78 (s, 3H), 2.47 (s, 2H), 2.20-2.14 (m, 1H), 2.06 (t, J = 12.0 Hz, 1H), 1.98-1.83 (m, 2H), 1.78-1.61 (m, 4H), 1.19-1.13 (m, 1H), 0.92 (t, J = 6.6 Hz, 6H), 0.84 (d, J = 6.3 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 168.62, 144.05, 113.35, 107.67, 88.01, 62.14, 54.41, 49.26, 45.90, 45.42, 45.12, 41.56, 39.25, 37.54, 34.65, 33.95, 31.03, 28.48, 28.15, 22.82, 20.98, 12.35; HRMS m/z (ESI): calcd for C22H37N2O5S [M+H]+: 441.2418, found: 441.2417.
2-(((3S,3aS,5S,6R,8aS)-5-isopropyl-3-methyl-8-methyleneoctahydro-6H-3a,6-epoxyazulen-6-yl)oxy)-1-(4-tosylpiperazin-1-yl)ethan-1-one (4g): Yellow oil (383 mg, 81% yield), 1H NMR (500 MHz, CDCl3) δ 7.62 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.1 Hz, 2H), 4.86 (dd, J = 7.2, 1.9 Hz, 2H), 4.29 (d, J = 12.2 Hz, 1H), 4.10 (d, J = 12.2 Hz, 1H), 3.84-3.57 (m, 4H), 3.11-2.86 (m, 4H), 2.43 (s, 3H), 2.39 (s, 1H), 2.15 (dd, J = 11.5, 8.7 Hz, 1H), 2.03 (t, J = 12.2 Hz, 1H), 1.94-1.78 (m, 2H), 1.73-1.49 (m, 4H), 1.27 (d, J = 14.5 Hz, 3H), 1.14 (dd, J = 12.6, 6.1 Hz, 1H), 0.89 (d, J = 6.5 Hz, 3H), 0.82 (d, J = 6.0 Hz, 6H); 13C NMR (126 MHz, CDCl3) δ 168.16, 144.29, 143.96, 132.22, 129.77 (Ar-overlap), 127.68 (Ar-overlap), 113.21, 107.61, 87.97, 62.40, 54.44, 49.41, 46.25, 45.78, 44.82, 41.24, 39.29, 37.43, 34.00, 31.04, 28.51, 28.20, 22.89, 21.48, 20.98, 12.35; HRMS m/z (ESI): calcd for C28H40N2O5SNa [M+Na]+: 539.2550, found: 539.2549.
Cell viability assay
Cell viability was evaluated using an MTT assay. SW620 cell were seeded into 96-well plates at a density of 1.5 × 104 cells per well and stabilized at 37 °C for 24 h without CO2. HCT116 cells and CaCo2 cells were seeded into 96-well plates at a density of 5 × 103 cells per well and stabilized at 37 °C for 24 h. Compounds were added to each well at various concentrations, and then the cells were incubated for 72 h. The MTT solution (10 μL 5 mg mL-1) was added to each well, and the cells were incubated for another 4 h. Formazan crystals were dissolved in 150 μL of DMSO. Cell viability was assessed by measuring the absorbance at 490 nm wavelength using a microplate reader (BioTek ELx800 USA).
Flow cytometric analysis of cell cycle arrest and apoptosis.
SW620 cells were plated in 6-well plates (8x105 cells/well) and treated with different dose (0, 1, 10 and 20 μM) of 3l for 24 h. For cell cycle analysis, cells were washed with ice-cold PBS and fixed in 70% ethanol overnight at 4˚C, and then stained with PI/RNase for 15 min at room temperature before analysis. To analyze the apoptotic rate, the treated cells were washed with ice-cold PBS twice and stained with 5 μl Annexin-V-FITC for 5 minutes and 5 μl 7AAD for another 10 minutes. After that, apoptosis was determined by flow cytometer (Guava Technologies; Merck KGaA, Darmstadt, Germany) and cell cycle distributions were analyzed by using ModFit software.