Instruments and Materials
All the organic solvents used for the synthesis were of analytical grade. Unless oth-erwise stated, all chemicals were purchased from Aladdin (Shanghai, China). Melting points were measured on WRX-4 micro melting point instrument (Shanghai YiCe Equipment, China) and were uncorrected. 1H NMR and 13C NMR spectra were recorded on BRUKER DPX-400 and DPX-600 spectrometers (Bruker Company, Germany), using TMS as an in-ternal standard and CDCl3, MeOD-d4, Pyridine-d5 and DMSO-d6 as solvents. Chemical shifts (δ values) and coupling constants (J values) are given in ppm and Hz, respectively. All the solvents and reactants were of analytical grade and were used without further purification unless noted. Column chromatography was accomplished on silica gel (100–200 or 200–300 mesh, Qingdao, China). The purity of compounds was confirmed to be over 95% and was determined by high performance liquid chromatography (HPLC) with a ZORBAX SB-Aq column (250 mm × 4.6 mm, 5 µm, Agilent) using ACN/water (10%, v/v) as the mobile phase (1.0 mL/min).
Synthesis
General Procedure Of Compounds 1a-1d
Esculetin (0.4g, 2.2 mmoL) was dissolved in 10 mL of ACN, and 0.66 mL triethylamine (4.4 mmoL) was added and stirred for 10 minutes. Then 4.4 mmoL different 1, ω-dibromoalkane (1, 3-dibromopropane, 1, 4-dibromobutane, 1, 5-dibromopentane, 1, 6-dibromohexane) were added into the mixture, stirring for 6 h at 65°C. Finally, when the reaction was completed (monitored by TLC analysis), the solvent was removed under reduced pressure to afford the crude product. The residue was applied to ash column chromatography (silica gel) to afford compounds 1a-1d. The physical properties, 1H NMR and 13C NMR data of the compounds are as follows.
General Procedure Of Compounds 2a-2d, 3a-3d And 4a-4d
0.6 mmoL of 1a-1d was dissolved in 10 mL of ACN, and 1.2 mmoL of K2CO3 was added and stirred for 10 mintes. Then 1.2 mmoL of different amine (piperidine, diethylamine and morpholine) were added into the mixture and stirred for 8 hours at room temperature. After the completion of the reaction (monitored by TLC analysis), the reaction was quenched with water. The resulting reaction mixture was extracted with dichloromethane and the dichloromethane was removed under reduced pressure to afford the crude product. The residue was applied to ash column chromatography (silica gel) to afford the compounds 2a-2d, 3a-3d and 4a-4d. The physical properties, 1H NMR and 13C NMR data of the compounds are as follows.
General Procedure Of Compounds 5–6
0.6 mmoL of 1a-1d was dissolved in 10 mL of ACN, 1.2 mmoL of K2CO3 and 0.3 mmoL of KI were added into the reaction mixture, stirred and reacted for 10 minutes, then 1.2 mmoL of different amine (imidazole, 2-methylimidazole) were add into the mixtu;re, stirring at 65°C for 8 hours. After the reaction was completed (monitored by TLC analysis), the reaction was quenched with water. The resulting reaction mixture was extracted with dichloromethane and the dichloromethane was removed under reduced pressure to afford the crude product. The residue was applied to ash column chromatography (silica gel) to afford the compounds 5a-5d and 6a-6d. The physical properties, 1H NMR and 13C NMR data of the compounds are as follows.
General Procedure Of Compounds 7–8
The synthesis of compounds 7a-c and 8a-c (Fig. 2) were carried out according to the method we have published previously [19–20].
Physical properties, HRMS, 1H NMR and 13C NMR data of compounds 1–8.
6-Hydroxyl-7-(3-Bromopropoxy)-2H-benzopyran-2-one (1a). White powder, yield: 23%, MP: 157–160℃. 1H NMR (400 MHz, DMSO) δ 7.93(d, J = 9.5 Hz, 1H, Pyran-H), 7.34 (s, 1H, Benzene-H), 7.01 (s, 1H, Benzene-H), 6.36 (d, J = 9.5 Hz, 1H, Pyran-H), 4.27–4.24 (m, 2H, CH2), 4.18–4.15 (t, J = 5.7 Hz, 2H, CH2), 2.16 (m, 2H, CH2). 13C NMR (101 MHz, DMSO) δ 160.7, 155.0, 150.2, 148.2, 144.2, 120.3, 114.6, 109.1, 71.3, 71.1, 31.4. HRMS(ESI): Calcd. for C12H11BrO4 [M-H]−: 296.9762, found: 296.9765.
6-Hydroxyl-7-(4-Bromobutoxy)-2H-benzopyran-2-one (1b). White powder, yield: 44%, MP: 117–121℃. 1H NMR (400 MHz, CDCl3) δ 7.53 (d, J = 9.5 Hz, 1H, Pyran-H), 6.91 (s, 1H, Benzene-H), 6.74 (s, 1H, Benzene-H), 6.22 (d, J = 9.5 Hz, 1H, Pyran-H), 4.09 (t, J = 5.7 Hz, 2H, CH2), 3.44 (t, J = 6.1 Hz, 2H, CH2), 2.01 (m, 4H, 2CH2). 13C NMR (101 MHz, CDCl3) δ 165.5, 159.7, 155.0, 153.0, 148.9, 125.1, 119.3, 113.9, 76.0, 75.9, 36.2. HRMS(ESI): Calcd. for C13H13BrO4 [M-H]−: 310.9919, found: 310.9924.
6-Hydroxyl-7-(5-Bromopentyloxy)-2H-benzopyran-2-one (1c). White powder, yield: 55%, MP: 114–119℃. 1H NMR (400 MHz, CDCl3) δ 7.53 (d, J = 9.5 Hz, 1H, Pyran-H), 6.90 (s, 1H, Benzene-H), 6.74 (s, 1H, Benzene-H), 6.22 (d, J = 9.5 Hz, 1H, Pyran-H), 4.06 (t, J = 6.4 Hz, 2H, CH2), 3.39 (t, J = 6.6 Hz, 2H, CH2), 1.92–1.81 (m, 4H, 2CH2), 1.63–1.58 (m, 2H, CH2). 13C NMR (101 MHz, CDCl3) δ 161.5, 149.3, 149.2, 143.4, 142.7, 113.9, 112.2, 111.1, 99.9, 69.1, 33.4, 32.2, 28.0, 24.6. HRMS(ESI): Calcd. for C14H15BrO4 [M-H]−: 325.0075, found: 325.0076.
6-Hydroxyl-7-(6-Bromohexyloxy)-2H-benzopyran-2-one (1d). White powder, yield: 59%, MP: 107–114℃. 1H NMR (400 MHz, CDCl3) δ 7.53 (d, J = 9.5 Hz, 1H, Pyran-H), 6.90 (s, 1H, ArH), 6.74 (s, 1H, ArH), 6.22(d, J = 9.5 Hz, 1H, Pyran-H), 4.05 (t, J = 6.5 Hz, 2H, CH2), 3.37 (t, J = 6.7 Hz, 2H, CH2), 1.81–1.86 (m, 4H, 2CH2), 1.48–1.46 (m, 4H, 2CH2). 13C NMR (101 MHz, CDCl3) δ 160.4, 148.3, 148.2, 142.4, 141.7, 112.8, 111.1, 110.0, 98.9, 68.3, 32.6, 31.5, 27.7, 26.8, 24.2. HRMS(ESI): Calcd. for C15H17BrO4 [M-H]−: 339.0232, found: 339.0235.
6-Hydroxyl-7-(3-(Piperidine-1-yl)propoxy)-2H-benzopyran-2-one (2a). Brown powder, yield: 18%, MP: 130–132℃. 1H NMR (400 MHz, MeOD) δ 7.80 (d, J = 9.5 Hz, 1H, Pyran-H), 6.96 (d, J = 4.3 Hz, 2H, ArH), 6.24 (d, J = 9.5 Hz, 1H, Pyran-H), 4.15 (t, J = 6.0 Hz, 2H, CH2), 3.31 (dt, J = 3.3, 1.6 Hz, 2H, CH2), 2.73–2.69 (m, 2H, CH2), 2.12–2.02 (m, 2H, CH2), 1.54 (dt, J = 11.2, 5.7 Hz, 4H, 2CH2), 1.34 (m, 2H, CH2), 1.29 (m, 2H, CH2). 13C NMR (101 MHz, MeOD) δ 162.6, 151.4, 148.4, 145.2, 144.5, 112.6, 112.3, 111.9, 101.1, 68.0, 55.6, 53.9, 25.2, 24.6, 23.3. HRMS(ESI): Calcd. for C17H21NO4 [M-H]−: 302.1392, found: 302.1401.
6-Hydroxyl-7-(4-(Piperidine-1-yl)butoxy)-2H-benzopyran-2-one (2b). Brown powder, yield: 19%, MP: 85–88℃. 1H NMR (400 MHz, MeOD) δ 7.71 (d, J = 9.4 Hz, 1H, Pyran-H), 6.86 (d, J = 14.4 Hz, 2H, ArH), 6.14 (d, J = 9.4 Hz, 1H, Pyran-H), 4.06 (d, J = 5.8 Hz, 2H, CH2), 2.61 (dd, J = 16.5, 8.0 Hz, 6H, 3CH2), 1.80–1.73 (m, 4H, 2CH2), 1.61 (d, J = 5.0 Hz, 4H, 2CH2), 1.45 (s, 2H, CH2). 13C NMR (101 MHz, MeOD) δ 162.7, 151.4, 148.7, 144.5, 144.2, 112.0, 111.6, 100.1, 100.0, 68.6, 57.9, 53.6, 26.3, 24.3, 23.0, 21.9. HRMS(ESI): Calcd. for C18H23NO4 [M-H]−: 316.1549, found: 316.1555.
6-Hydroxyl-7-(5-(Piperidine-1-yl)pentyloxy)-2H-benzopyran-2-one (2c). Brown powder, yield: 15%, MP: 115–121℃. 1H NMR (400 MHz, MeOD) δ 7.70 (d, J = 9.5 Hz, 1H, Pyran-H), 6.87 (s, 1H, ArH), 6.82 (s, 1H, ArH), 6.13 (d, J = 9.5 Hz, 1H, Pyran-H), 4.03 (t, J = 6.3 Hz, 2H, CH2), 2.84 (s, 4H, 2CH2), 2.75–2.71 (m, 2H, CH2), 1.83– 1.80 (m, 2H, CH2), 1.67–1.66 (m, 6H, 3CH2), 1.51–1.47 (m, 4H, 2CH2). 13C NMR (101 MHz, MeOD) δ 163.9, 152.8, 150.1, 145.8, 145.4, 113.4,113.3, 112.9, 101.3, 70.0, 58.9, 54.6, 29.4, 25.5, 25.0, 24.6, 23.6. HRMS(ESI): Calcd. for C19H25NO4 [M-H]−: 330.1705, found: 330.1713.
6-Hydroxyl-7-(6-(Piperidine-1-yl)hexyloxy)-2H-benzopyran-2-one (2d). Brown powder, yield: 16%, MP: 51–54℃. 1H NMR(400 MHz, MeOD) δ 7.71 (d, J = 9.5 Hz, 1H, Pyran-H), 6.88 (s, 1H, ArH), 6.84 (s, 1H, ArH), 6.14 (d, J = 9.5 Hz, 1H, Pyran-H), 4.03 (t, J = 6.4 Hz, 2H, CH2), 2.92 (s, 4H, 2CH2), 2.79 (dd, J = 9.8, 6.8 Hz, 2H, CH2), 1.70 (m, 2H, CH2), 1.62–1.61 (m, 4H, 2CH2), 1.53–1.52 (m, 2H, CH2), 1.51–1.49 (m, 4H, 2CH2), 1.47–1.34 (d, J = 8.2 Hz, 2H, CH2). 13C NMR (101 MHz, MeOD) δ 162.6, 151.5, 148.9, 144.5, 144.0, 112.1, 112.0, 111.5, 100.0, 68.8, 57.4, 53.2, 28.2, 26.2, 25.2, 24.1, 23.4, 22.0. HRMS(ESI): Calcd. for C20H27NO4 [M-H]−: 344.1862, found: 344.1874.
6-Hydroxyl-7-(3-(Diethylamino-1-yl)propoxy)-2H-benzopyran-2-one (3a). Yellow powder, yield: 19%, MP: 76–79℃. 1H NMR (400 MHz, MeOD) δ 7.71 (d, J = 9.5 Hz, 1H, Pyran-H), 6.86 (s, 2H, ArH), 6.14 (d, J = 9.5 Hz, 1H, Pyran-H), 4.08 (t, J = 5.9 Hz, 2H, CH2), 2.85–2.82 (m, 2H, CH2), 2.77–2.71 (q, J = 7.2 Hz, 4H, 2CH2), 2.02–1.99 (m, 2H, CH2), 1.07 (t, J = 7.2 Hz, 6H, 2CH3). 13C NMR (101 MHz, MeOD) δ 162.6, 151.5, 148.3, 145.55, 144.6, 112.7, 112.3, 112.0, 101.2, 67.8, 49.4, 46.5, 29.4 24.8, 9.0. HRMS(ESI): Calcd. for C16H21NO4 [M-H]−: 290.1392, found: 290.1400.
6-Hydroxyl-7-(4-(Diethylamino-1-yl)butoxy)-2H-benzopyran-2-one (3b). Yellow powder, yield: 20%, MP: 84–87℃. 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 9.5 Hz, 1H, Pyran-H), 6.96 (s, 1H, ArH), 6.80 (s, 1H, ArH), 6.26 (d, J = 9.5 Hz, 1H, Pyran-H), 4.13 (t, J = 5.9 Hz, 2H, CH2), 2.70 (dt, J = 13.1, 7.1 Hz, 6H, 3CH2), 1.91 (dt, J = 12.5, 6.2 Hz, 2H, CH2), 1.80 (dd, J = 13.9, 7.0 Hz, 2H, CH2), 1.12 (t, J = 7.2 Hz, 6H, 2CH3). 13C NMR (101 MHz, CDCl3) δ 161.6, 150.2, 148.9, 143.8, 143.5, 113.6, 112.4, 111.9, 100.7, 69.5, 52.1, 46.6, 26.8, 23.0, 10.3. HRMS(ESI): Calcd. for C17H23NO4 [M-H]−: 304.1549, found: 304.1564.
6-Hydroxyl-7-(5-(Diethylamino-1-yl)pentyloxy)-2H-benzopyran-2-one (3c). Yellow powder, yield: 16%, MP: 56–58℃. 1H NMR(400 MHz, CDCl3) δ 7.51 (d, J = 9.5 Hz, 1H, Pyran-H), 6.89 (s, 1H, ArH), 6.70 (s, 1H, ArH), 6.17 (d, J = 9.5 Hz, 1H, Pyran-H), 4.03 (t, J = 5.9 Hz, 2H, CH2), 2.72 (q, J = 7.2 Hz, 4H, 2CH2), 2.65–2.58 (m, 2H, CH2), 1.84 (m, 2H, CH2), 1.64 (dt, J = 14.9, 7.3 Hz, 2H, CH2), 1.53–1.49 (m, 2H, CH2), 1.09 (t, J = 7.2 Hz, 6H, 2CH3). 13C NMR (101 MHz, CDCl3) δ 160.7, 149.4, 148.0, 142.61, 142.5, 112.4, 111.2, 111.1, 99.0, 68.2, 50.3, 45.2, 26.7, 24.0, 23.0, 8.6. HRMS(ESI): Calcd. for C18H25NO4 [M-H]−: 318.1705, found: 318.1712.
6-Hydroxyl-7-(6-(Diethylamino-1-yl)hexyloxy)-2H-benzopyran-2-one (3d). Yellow powder, yield: 35%, MP: 72–75℃. 1H NMR(400 MHz, CDCl3) δ 7.57 (d, J = 9.5 Hz, 1H, Pyran-H), 6.95 (s, 1H, ArH), 6.77 (s, 1H, ArH), 6.24 (d, J = 9.5 Hz, 1H, Pyran-H), 4.07 (t, J = 6.1 Hz, 2H, CH2), 2.77 (q, J = 7.1 Hz, 4H, 2CH2), 2.67–2.63 (m, 2H, CH2), 1.90–1.80 (m, 2H, CH2), 1.63 (dt, J = 15.0, 7.4 Hz, 2H, CH2), 1.56–1.50 (m, 2H, CH2), 1.40 (td, J = 14.1, 6.4 Hz, 2H, CH2), 1.14 (t, J = 7.2 Hz, 6H, 2CH3). 13C NMR (101 MHz, CDCl3) δ 160.6, 149.2, 148.1, 142.5, 142.3, 112.5, 111.0, 110.6, 99.0, 68.3, 50.7, 45.1, 28.7, 27.4, 25.6, 24.5, 8.9. HRMS(ESI): Calcd. for C19H27NO4 [M-H]−: 332.1862, found: 332.1874.
6-Hydroxyl-7-(3-(Morpholine-1-yl)propoxy)-2H-benzopyran-2-one (4a). Brown powder, yield: 46%, MP: 170–171℃. 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 9.5 Hz, 1H, Pyran-H), 6.94 (d, J = 7.6 Hz, 2H, ArH), 6.30 (d, J = 9.5 Hz, 1H, Pyran-H), 4.09 (t, J = 5.3 Hz, 2H, CH2), 3.79 (t, J = 4.6 Hz, 4H, 2CH2), 2.64 (t, J = 6.0 Hz, 2H, CH2), 2.58 (s, 4H, 2CH2), 2.05–2.00 (m, 2H, CH2). 13C NMR (101 MHz, CDCl3) δ 161.5, 150.2, 148.0, 146.6, 143.4, 114.8, 114.7, 112.8, 106.9, 72.4, 66.0, 57.1, 53.7, 25.1. HRMS(ESI): Calcd. for C16H19NO5 [M-H]−: 304.1185, found: 304.1194.
6-Hydroxyl-7-(4-(Morpholine-1-yl)butoxy)-2H-benzopyran-2-one (4b). Brown powder, yield: 53%, MP: 92–95℃.1H NMR (400 MHz, MeOD) δ 7.79 (d, J = 9.5 Hz, 1H, Pyran-H), 6.94 (d, J = 14.3 Hz, 2H, ArH), 6.22 (d, J = 9.5 Hz, 1H, Pyran-H), 4.14 (t, J = 6.3 Hz, 2H, CH2), 3.71 (m, 4H, 2CH2), 2.54 (s, 4H, 2CH2), 2.50 (d, J = 7.7 Hz, 2H, CH2), 1.90–1.85 (m, 2H, CH2), 1.79–1.71 (m, 2H, CH2). 13C NMR (101 MHz, MeOD) δ 162.6, 151.3, 148.9, 144.4, 144.0, 112.1, 112.0, 111.6, 100.0, 68.7, 66.0, 58.1, 53.2, 29.4, 22.1. HRMS(ESI): Calcd. for C17H21NO5 [M-H]−: 318.1341, found: 318.1352.
6-Hydroxyl-7-(5-(Morpholine-1-yl)pentyloxy)-2H-benzopyran-2-one (4c). Brown powder, yield: 48%, MP: 131–133℃.1H NMR(400 MHz, CDCl3) δ 7.57 (d, J = 9.5 Hz, 1H, Pyran-H), 6.93 (s, 1H, ArH), 6.76 (s, 1H, ArH), 6.24 (d, J = 9.5 Hz, 1H, Pyran-H), 4.09 (t, J = 6.2 Hz, 2H, CH2), 3.74–3.71 (m, 4H, 2CH2), 2.45 (s, 4H, 2CH2), 2.41–2.37 (m, 2H, CH2), 1.89 (m, 2H, CH2), 1.85 (m, 2H, CH2), 1.64–1.48 (dt, J = 13.8, 6.8 Hz, 2H, CH2). 13C NMR (101 MHz, CDCl3) δ 160.6, 148.9, 148.1, 142.5, 142.0, 112.6, 111.1, 110.7, 98.9, 68.4, 65.6, 57.1, 52.6, 26.9, 24.8, 22.9. HRMS(ESI): Calcd. for C18H23NO5 [M-H]−: 332.1498, found: 332.1508.
6-Hydroxyl-7-(6-(Morpholine-1-yl)hexyloxy)-2H-benzopyran-2-one (4d). Brown powder, yield: 51%, MP: 41–43℃.1H NMR(400 MHz, CDCl3) δ 7.57 (d, J = 9.5 Hz, 1H, Pyran-H), 6.93 (s, 1H, ArH), 6.77 (s, 1H, ArH), 6.25 (d, J = 9.5 Hz, 1H, Pyran-H), 4.07 (t, J = 6.5 Hz, 2H, CH2), 3.72–3.69 (m, 4H, 2CH2), 2.44 (s, 4H, 2CH2), 2.36–2.32 (m, 2H, CH2), 1.85–1.81 (m, 2H, CH2), 1.56–1.50 (dt, J = 16.5, 8.2 Hz, 4H, 2CH2), 1.48–1.36 (m, 2H, CH2). 13C NMR (101 MHz, CDCl3) δ 161.6, 149.8, 149.2, 143.5, 142.9, 113.6, 112.1, 111.3, 100.0, 69.4, 66.8, 58.9, 53.7, 28.7, 27.1, 26.2, 25.8. HRMS(ESI): Calcd. for C19H25NO5 [M-H]−: 346.1654, found: 346.1663.
6-Hydroxyl-7-(3-(Imidazole-1-yl)propoxy)-2H-benzopyran-2-one (5a). Brown powder, yield: 32%, MP: 100–102℃. 1H NMR (400 MHz, MeOD) δ 7.83 (s, 1H, CH), 7.82 (d, J = 9.5 Hz, 1H, Pyran-H), 7.25 (s, 1H, CH), 7.06 (s, 1H, CH), 7.01 (s, 1H, ArH), 6.93 (s, 1H, ArH), 6.25 (d, J = 9.5 Hz, 1H, Pyran-H), 4.35 (t, J = 7.1 Hz, 2H, CH2), 4.08 (t, J = 5.8 Hz, 2H, CH2), 2.35 (dt, J = 12.8, 6.3 Hz, 2H, CH2). 13C NMR (101 MHz, MeOD) δ 162.5, 150.9, 148.8, 144.3, 143.9, 126.6, 119.7, 112.4, 112.35, 111.8, 100.3, 65.5, 43.6, 29.9. HRMS(ESI): Calcd. for C15H14N2O4 [M-H]−: 285.0875, found: 285.0883.
6-Hydroxyl-7-(4-(Imidazole-1-yl)butoxy)-2H-benzopyran-2-one (5b). Brown powder, yield: 47%, MP: 142–144℃. 1H NMR(400 MHz, MeOD) δ 7.61 (d, J = 9.5 Hz, 1H, Pyran-H), 7.58 (s, 1H, CH), 7.56 (d, J = 1.2 Hz, 1H, CH), 6.97 (d, J = 1.2 Hz, 1H, CH), 6.94 (s, 1H, ArH), 6.78 (s, 1H, ArH), 6.26 (d, J = 9.5 Hz, 1H, Pyran-H), 4.08 (t, J = 4.9 Hz, 2H, CH2), 4.01 (t, J = 3.9 Hz, 2H, CH2), 1.89 (dt, J = 19.8, 7.2 Hz, 2H, CH2), 1.52 − 1.48 (m, 2H, CH2). 13C NMR (101 MHz, MeOD) δ 161.6, 150.0, 149.0, 143.3, 137.1, 128.9, 118.9, 113.6, 111.5, 100.1, 68.8, 46.9, 30.6, 28.2, 23.1. HRMS(ESI): Calcd. for C16H16N2O4 [M-H]−: 299.1032, found: 299.1038.
6-Hydroxyl-7-(5-(Imidazole − 1-yl)pentyloxy)-2H-benzopyran-2-one (5c). Brown powder, yield: 56%, MP: 145–148℃. 1H NMR (400 MHz, CDCl3) δ 7.61 (s, 1H, CH), 7.58 (d, J = 9.5 Hz, 1H, Pyran-H), 7.56 (s, 1H, CH), 6.97 (s, 1H, ArH), 6.94 (s, 1H, CH), 6.78 (s, 1H, ArH), 6.26 (d, J = 9.5 Hz, 1H, Pyran-H), 4.08 (t, J = 6.2 Hz, 2H, CH2), 4.01 (t, J = 7.0 Hz, 2H, CH2), 1.90–1.86 (m, 4H, 2CH2), 1.52–1.48 (m, 2H, CH2). 13C NMR (101 MHz, CDCl3) δ 161.6, 150.0, 149.0, 143.3, 137.1, 128.9, 118.9, 113.6, 111.5, 100.1, 68.8, 46.9, 30.6, 28.2, 23.1. HRMS(ESI): Calcd. for C17H18N2O4 [M-H]−: 313.1188, found: 313.1195.
6-Hydroxyl-7-(6-(Imidazole-1-yl)hexyloxy)-2H-benzopyran-2-one (5d). Brown powder, yield: 49%, MP: 133–135℃. 1H NMR(400 MHz, CDCl3) δ 7.61 (d, J = 9.4 Hz, 1H, Pyran-H), 7.11 (s, 1H, CH), 7.11 (s, 1H, CH), 7.01 (s, 1H, ArH), 6.97 (s, 1H, CH), 6.81 (s, 1H, ArH), 6.28 (d, J = 9.4 Hz, 1H, Pyran-H), 4.09 (t, J = 6.3 Hz, 2H, CH2), 4.01 (t, J = 6.9 Hz, 2H, CH2), 1.85 (dd, J = 12.5, 6.5 Hz, 4H, CH2, CH2), 1.53–1.42 (m, 2H, CH2), 1.39 (dd, J = 17.7, 10.9 Hz, 2H, CH2). 13C NMR (101 MHz, CDCl3) δ 160.7, 149.3, 148.0, 142.5, 142.5, 135.9, 127.7, 118.0, 112.4, 110.6, 99.1, 68.0, 46.2, 29.7, 27.6, 25.3, 24.5. HRMS(ESI): Calcd. for C18H20N2O4 [M-H]−: 327.1345, found: 327.1352.
6-Hydroxyl-7-(3-(2-methylimidazole-1-yl)propoxy)-2H-benzopyran-2-one (6a). Brown powder, yield: 37%, MP: 110–112℃. 1H NMR (400 MHz, MeOD) δ 7.82 (d, J = 9.5 Hz, 1H, Pyran-H), 7.06 (s, 1H, CH), 7.02 (s, 1H, ArH), 6.93 (s, 1H, ArH), 6.85 (s, 1H, CH), 6.25 (d, J = 9.5 Hz, 1H, Pyran-H), 4.23 (t, J = 6.9 Hz, 2H, CH2), 4.05 (t, J = 5.7 Hz, 2H, CH2), 2.35 (s, 3H, CH3), 2.30 (dt, J = 12.7, 6.5 Hz, 2H, CH2). 13C NMR (101 MHz, MeOD) δ 162.5, 150.9, 148.8, 144.4, 144.0, 125.4, 119.4, 112.4, 111.8, 100.2, 65.2, 42.1, 29.4, 11.8. HRMS(ESI): Calcd. for C16H16N2O4 [M-H]−: 299.1032, found: 299.1038.
6-Hydroxyl-7-(4-(2-methylimidazole-1-yl)butoxy)-2H-benzopyran-2-one (6b). Brown powder, yield: 41%, MP: 70–74℃.1H NMR(400 MHz, MeOD) δ 7.81 (d, J = 9.4 Hz, 1H, Pyran-H), 7.13 (d, J = 1.4 Hz, 1H, CH), 6.98 (d, J = 2.7 Hz, 2H, ArH), 6.90 (d, J = 1.3 Hz, 1H, CH), 6.24 (d, J = 9.4 Hz, 1H, Pyran-H), 4.15 (t, J = 6.0 Hz, 2H, CH2), 4.06 (t, J = 7.2 Hz, 2H, CH2), 1.37 (s, 3H, CH3), 0.89 (t, J = 6.8 Hz, 4H, 2CH2). 13C NMR (101 MHz, MeOD) δ 162.6, 151.3, 148.9, 144.4, 144.0, 124.2, 122.9, 119.8, 112.2, 111.6, 100.2, 78.1, 68.5, 29.4, 13.1. HRMS(ESI): Calcd. for C17H18N2O4 [M-H]−: 313.1188, found: 313.1196.
6-Hydroxyl-7-(5-(2-methylimidazole-1-yl)pentyloxy)-2H-benzopyran-2-one (6c). Brown powder, yield: 46%, MP: 62–65℃.1H NMR(400 MHz, MeOD) δ 7.80 (d, J = 9.4 Hz, 1H, Pyran-H), 7.05 (d, J = 1.5 Hz, 1H, CH), 6.97 (s, 1H, ArH), 6.93 (s, 1H, ArH), 6.85 (d, J = 1.4 Hz, 1H, CH), 6.23 (d, J = 9.4 Hz, 1H, Pyran-H), 4.11 (t, J = 6.3 Hz, 2H, CH2), 3.97 (t, J = 7.2 Hz, 2H, CH2), 1.40 (s, 2H, CH2), 1.26 (s, 3H, CH3), 0.89 (t, J = 6.8 Hz, 4H, 2CH2). 13C NMR (101 MHz, MeOD) δ 164.0, 152.7, 150.2, 145.8, 145.3, 125.9, 124.2, 120.9, 114.6, 113.4, 112.8, 101.4, 70.1, 47.0, 30.7, 24.1, 23.7, 14.4. HRMS(ESI): Calcd. for C18H20N2O4 [M-H]−: 327.1345, found: 327.1352.
6-Hydroxyl-7-(6-(2-methylimidazole-1-yl)hexyloxy)-2H-benzopyran-2-one (6d). Yellow powder, yield: 57%, MP: 62–65℃.1H NMR(600 MHz, Pyr) δ 7.72 (d, J = 9.4 Hz, 1H, Pyran-H), 7.32 (s, 1H, ArH), 7.26 (d, J = 1.0 Hz, 1H, CH), 7.09 (d, J = 1.0 Hz, 1H, CH), 7.05 (s, 1H, ArH), 6.38 (d, J = 9.4 Hz, 1H, Pyran-H), 4.02 (t, J = 6.4 Hz, 2H, CH2), 3.89 (q, J = 7.0 Hz, 2H, CH2), 3.69 (t, J = 7.2 Hz, 2H, CH2), 2.42 (s, 3H, CH3), 1.60–1.45 (m, 2H, CH2), 1.31 (dd, J = 15.1, 7.5 Hz, 2H, CH2), 1.10 (dt, J = 15.4, 7.9 Hz, 2H, CH2). 13C NMR (151 MHz, Pyr) δ 162.5, 153.0, 146.2, 145.3, 144.9, 127.5, 125.0, 120.7, 114.3, 114.0, 113.4, 101.9, 70.2, 58.3, 50.6, 46.9, 27.3, 20.1, 13.7. HRMS(ESI): Calcd. for C19H22N2O4 [M-H]−: 341.1501, found: 341.1507.
6,7-dihydroxy-4-(hydroxymethyl)-2H-benzopyran-2-one (7a). Brown solid, yield: 83%, MP: >300℃. 1H NMR (400 MHz, MeOD) δ 6.96 (s, 1H, ArH), 6.76 (s, 1H, ArH), 6.36 (t, J = 1.4 Hz, 1H, Pyran-H), 4.76 (d, J = 1.4 Hz, 2H, CH2). 13C NMR (101 MHz, MeOD) δ 163.3, 156.9, 150.3, 148.4, 143.1, 109.5, 107.4, 106.5, 102.4, 59.5. HRMS(ESI): Calcd. for C10H8O5 [M-H]−: 207.0293, found: 207.0298.
6,7-dihydroxy-4-phenyl-2H-benzopyran-2-one (7b). White solid, yield: 91%, MP: 54–56℃. 1H NMR (400 MHz, MeOD) δ 7.55–7.47 (m, 5H, ArH), 6.85 (s, 1H, ArH), 6.83 (s, 1H, ArH), 6.13 (s, 1H, Pyran-H). 13C NMR (101 MHz, MeOD) δ 162.7, 157.0, 150.7, 149.2, 143.0, 135.9, 129.3, 128.6, 128.1, 110.9, 110.6, 109.9, 102.7. HRMS(ESI): Calcd. for C15H10O4 [M-H]−: 253.0501, found:253.0508.
6,7-dihydroxy-4-carboxymethyl-2H-benzopyran-2-one (7c). White solid, yield: 70%, MP: 210–212℃. 1H NMR (400 MHz, MeOD) δ 7.04 (s, 1H, ArH), 6.76 (s, 1H, ArH), 6.21 (s, 1H, Pyran-H), 3.78 (d, J = 7.2 Hz, 2H, CH2). 13C NMR (101 MHz, MeOD) δ 171.3, 162.7, 150.5, 148.6, 143.1, 111.9, 111.4, 108.7, 102.5, 37.4. HRMS(ESI): Calcd. for C11H8O6 [M-H]−: 236.0243, found: 236.0300.
6,7-dihydroxy-8-((4-hydroxypiperidine-1-yl) methyl)-2H-benzopyran-2-one (8a). Brown powder, yield: 12%, MP: 129–131℃. 1H NMR (600 MHz, MeOD) δ 7.76 (d, J = 9.3 Hz, 1H, Pyran-H), 6.86 (s, 1H, ArH), 6.04 (d, J = 9.3 Hz, 1H, Pyran-H), 4.23 (s, 2H, CH2), 3.34 (d, J = 8.3 Hz, 1H, CHOH)), 3.23–3.21 (m, 2H, CH2), 2.86 (s, 2H, CH2), 1.99 (s, 2H, CH2), 1.73 (m, 2H, CH2). 13C NMR (151 MHz, MeOD) δ 163.2, 148.6, 145.6, 144.8, 109.3, 108.2, 107.6, 104.9, 51.8, 48.5, 48.2, 32.0. HRMS(ESI): Calcd. for C15H17NO5 [M-H]−: 290.1028, found: 290.1033.
6,7-dihydroxy-8-(pyrrolidin-1-ylmethyl)-2H-benzopyran-2-one (8b). Light brown solid, yield: 18%, MP: 125–127℃. 1H NMR (600 MHz, MeOD) δ 7.73 (d, J = 9.2 Hz, 1H, Pyran-H), 6.81 (s, 1H, ArH), 5.94 (d, J = 9.2 Hz, 1H, Pyran-H), 4.44 (d, J = 11.6 Hz, 2H, CH2), 3.31– 3.30 (m, 4H, 2CH2), 2.08–2.06 (m, 4H, 2CH2). 13C NMR (151 MHz, MeOD) δ 163.8, 162.2, 149.7, 146.2, 145.9, 108.1, 106.2, 105.2, 103.7, 53.3, 49.0, 22.7. HRMS(ESI): Calcd. for C14H15NO4 [M-H]−: 260.0923, found: 260.0927.
6,7-dihydroxy-8-((dimethylamino)methyl)-2H-benzopyran-2-one(8c). Brown powder, yield: 17%, MP: 131–133℃. 1H NMR (600 MHz, MeOD) δ 8.40 (s, 1H, ArOH), 7.81 (d, J = 9.4 Hz, 1H, Pyran-H), 7.01 (s, 1H, ArH), 6.19 (d, J = 9.4 Hz, 1H, Pyran-H), 4.46 (s, 2H, CH2), 3.31 (s, 6H, 2CH3). 13C NMR (151 MHz, MeOD) δ 167.3, 162.2, 148.2, 145.0, 143.7, 111.6, 110.0, 109.8, 104.0, 50.6, 42.2. HRMS(ESI): Calcd. for C12H13NO4 [M-H]−: 234.0766, found: 234.0772.
Cell cytotoxicity test
Cytotoxicity of esculetin derivatives were measured by MTT assay [11, 21–22]. Take the cells in logarithmic growth phase to prepare a cell suspension with a density of 2 × 104 cells/mL, inoculate 200µL per well in a 96-well plate, and culture in 37°C. Lamivudine was used as the positive control and the concentration gradient was 20, 40, 80, 160, 320 µM. After 24 h, 200 µl of drug-containing medium was added to each well, and the culture was continued for 9 days. Then the absorbance value (OD) was measured using a microplate reader at a wavelength of 450 nm. Reed-Muench method was used to calculate half the cytotoxicity level (CC50).
Evaluation of HBsAg and HBeAg expression
Cell supernatant was collected on day 6 and day 9. The levels of HBsAg and HBeAg were simultaneously detected using ELISA kits according to the manufacturer’s instructions, and the IC50 and selected index (SI) of each compound were calculated, respectively [23].
Metabolic stability assays of compounds in both phase I and phase II metabolic systems
For phase I metabolism, the incubation mixture (0.6 mL) was consisted of human liver microsomes (0.1 mg/mL), MgCl2 (5 mM), glucose-6-phosohate (1 mM), glucose-6-phosohate dehydrogenase (1 U/mL), target compounds, testosterone (10 mM) and 0.1 M potassium phosphate buffer (pH = 7.4). The mixture was pre-incubated at 37°C for 3 min and then added with NADP+ (1 mM) to initiate the oxidative reaction. At the different time points (0-, 15-, 30-, 60-, 90-min), 100 mL of the aliquots were transferred to a tube containing 100 mL ice-cold ACN to stop the reaction. The mixtures were then vortexed for 30 s at room temperature and centrifuged at 20,000 g at 4°C for 20 min. The supernatants were analyzed by liquid chromatography coupled with an UV detector.
For phase II metabolism, the incubation mixture (0.6 mL) was consisted of human liver microsomes (0.1 mg/mL), MgCl2 (5 mM), target compounds, reference drug (10 mM), and 50mMTris-HCl buffer (pH = 7.4). The reaction mixture was incubated at 37°C for 3 min and then added with UDPGA (2 mM) to initiate the reaction. At the different time points (0-, 15-, 30-, 60-, 90-min), 100 mL of the aliquots were transferred to a tube containing 100 mL ice-cold ACN to stop the reaction. The mixtures were then vortexed for 30 s at room temperature and centrifuged at 20,000 g at 4°C for 20 min. The supernatants were analyzed by liquid chromatography coupled with an UV detector.