Instruments
Unless otherwise noted, all commercial reagents and solvents were obtained from the commercial provider and used without further purification. Melting points (mp) were obtained with CXM-300 melting point apparatus and are uncorrected. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance 400 (400/101 MHz for 1H/13C) spectrometer or Bruker Avance III 500 (500/126 MHz for 1H/13C) spectrometer (Varian, San Francisco, USA) respectively, in DMSO-d
6
with Me4Si as the internal standard. ESI high-resolution mass spectra (HRMS) were recorded on an AutospecUitima-TOF spectrometer (Micromass UK Ltd., Manchester, U.K.). Flash chromatography was performed on Combiflash Rf 200 (Teledyne, Nebraska, USA).
General procedures for methyl 12N-substituted matrinate derivatives 6a–f
Matrine (5.0 g, 20.0 mmol) was added to 5 N NaOH in water (30 mL), and the reaction mixture was refluxed for 9 h, cooled in an ice bath and then acidified with HCl (2 N) to pH 6–7. The solvent was removed in vacuo and the residue was dissolved with 2 N HCl in methanol and then heated at refluxing for 2 h. The solvent methanol was removed under reduced pressure to give crude 5 (5.5 g, yield 77%), which was applied directly in the next step without further purification.
To a stirred solution of 5 (10.0 mmol) and K2CO3 (35.0 mmol) in chloroethane (50 mL), the substituted benzyl halide (10 mmol) was added. The reaction mixture was stirred at room temperature for 5–8 h until TLC analysis showed completion of the reaction. Water (20 mL) was added to the mixture and the organic phase was separated and dried with anhydrous Na2SO4, concentrated, and the gained residue was purified by flash column chromatography on silica gel with CH2Cl2/CH3OH as the eluent to afford the title compounds.
Methyl 12N-(4-methoxybenzyl)matrinate dihydrochloride (6a)
The title compound was prepared from 5 and 4-methoxybenzyl bromide in the same manner as described above followed by an acidification with 2 N hydrochloride/ether (10 mL). Yield: 61%; white solid; mp 208–209 °C; 1H NMR (500 MHz) δ 11.42 (br, 1H), 11.06 (br, 1H), 7.53 (d, J = 8.7 Hz, 2H), 7.01 (d, J = 8.7 Hz, 2H), 4.93–4.89 (m, 1H), 4.22–4.18 (m, 1H), 4.00–3.88 (m, 2H), 3.79 (s, 3H), 3.61 (s, 3H), 3.58 (d, J = 10.4 Hz, 1H), 3.30–3.24 (m, 2H), 2.99–2.87 (m, 2H), 2.68–2.65 (m, 1H), 2.60–2.54 (m, 1H), 2.49–2.46 (m, 3H), 2.05–1.98 (m, 2H), 1.94–1.88 (m, 1H), 1.82–1.58 (m, 8H), 1.47 (d, J = 13.7 Hz, 1H); 13C NMR (126 MHz) δ 173.7, 160.3, 133.4 (2), 122.0, 114.6 (2), 60.7, 60.6, 57.2, 55.7, 54.7, 54.6, 51.8, 48.8, 36.3, 32.9, 30.4, 28.0, 24.5, 23.9, 21.8, 18.3 (2). HRMS: calcd for C24H37O3N2·2HCl [M−2HCl+H]+: 401.2799, found: 401.2790.
Methyl 12N-(4-methylbenzyl)matrinate (6b)
The title compound was prepared from 5 and 4-methylbenzyl bromide in the same manner as described in the general procedures. Yield: 67%; white solid; mp 89–91 °C; 1H NMR (500 MHz) δ 7.17 (d, J = 7.4 Hz, 2H), 7.10 (d, J = 7.4 Hz, 2H), 3.96 (d, J = 13.0 Hz, 1H), 3.55 (s, 3H), 3.01 (d, J = 12.9 Hz, 1H), 2.79 (s, 1H), 2.72 (d, J = 8.6 Hz, 1H), 2.65 (d, J = 9.1 Hz, 1H), 2.55 (d, J = 11.5 Hz, 1H), 2.30 (d, J = 6.3 Hz, 1H), 2.27 (s, 3H), 2.18 (d, J = 8.5 Hz, 1H), 1.96 (s, 1H), 1.85–1.24 (m, 17H); 13C NMR (126 MHz) δ 174.0, 137.6, 135.8, 129.2 (2), 128.9 (2), 64.3, 57.4, 57.1, 56.9, 55.4, 52.0, 51.6 (2), 37.6, 33.8, 28.4, 27.8, 27.4, 21.5, 21.2 (2), 19.0. HRMS: calcd for C24H37O2N2 [M+H]+: 385.2850, found: 385.2844.
Methyl 12N-(4-vinylbenzyl)matrinate (6c)
The title compound was prepared from 5 and 4-vinylbenzyl chloride in the same manner as 6b. Yield: 70%; white solid; mp 75–77 °C. 1H NMR (500 MHz) δ 7.40 (d, J = 7.9 Hz, 2H), 7.27 (d, J = 7.9 Hz, 2H), 6.80–6.68 (m, 1H), 5.79 (d, J = 17.7 Hz, 1H), 5.21 (d, J = 11.1 Hz, 1H), 3.99 (d, J = 13.7 Hz, 1H), 3.54 (s, 3H), 3.10–3.01 (m, 1H), 2.88–2.78 (m, 1H), 2.78–2.67 (m, 1H), 2.70–2.60 (m, 1H), 2.63–2.56 (m, 1H), 2.29 (t, J = 6.7 Hz, 2H), 2.18 (d, J = 8.5 Hz, 1H), 1.96 (s, 1H), 1.85–1.52 (m, 11H), 1.36–1.24 (m, 5H); 13C NMR (126 MHz) δ 174.0, 140.7, 137.0, 135.9, 129.1 (2), 126.4 (2), 114.0, 64.3, 57.4, 57.1, 56.9, 55.4, 52.2, 51.6 (2), 37.6, 33.7, 28.3, 27.9, 27.4, 21.5, 21.2, 19.0. HRMS: calcd for C25H37O2N2 [M+H]+: 397.2850, found: 397.2838.
Methyl 12N-(2,4-difluorobenzyl)matrinate (6d)
The title compound was prepared from 5 and 2,4-difluorobenzyl bromide in the same manner as 6b. Yield: 73%; white solid; mp 68–70 °C; 1H NMR (500 MHz) δ 7.48–7.43 (m, 1H), 7.18–7.14 (m, 1H), 7.08–7.04 (m, 1H), 3.95 (d, J = 13.8 Hz, 1H), 3.55 (s, 3H), 3.15 (d, J = 13.7 Hz, 1H), 2.85–2.83 (m, 1H), 2.72 (d, J = 10.7 Hz, 1H), 2.67–2.61 (m, 2H), 2.31–2.28 (m, 2H), 2.23–2.05 (m, 1H), 1.96 (s, 1H), 1.85–1.73 (m, 4H), 1.67–1.47 (m, 7H), 1.37–1.25 (m, 5H); 13C NMR (126 MHz) δ 173.9, 162.3, 160.3, 132.4, 123.4, 111.7, 103.9, 64.2, 57.3, 57.1 (2), 52.1, 51.6, 47.9, 37.6, 33.7, 33.6, 28.3, 27.9, 27.3, 21.5, 21.2, 19.1. HRMS: calcd for C23H33O2N2 F [M+H]+: 407.2505, found: 407.2488.
Methyl 12N-(4-nitrobenzyl)matrinate dihydrochloride (6e)
The title compound was prepared from 5 and 4-nitrobenzyl bromide in the same manner as 6a. Yield: 75%; white solid; mp 215–217 °C; 1H NMR (500 MHz) δ 11.87 (br, 1H), 11.07 (br, 1H), 8.52–8.52 (m, 1H), 8.32–8.30 (m, 1H), 8.09 (d, J = 7.7 Hz, 1H), 7.77 (t, J = 8.0 Hz, 1H), 5.10 (d, J = 11.7 Hz, 1H), 4.27–4.23 (m, 1H), 4.22–4.16 (m, 1H), 4.00–3.93 (m, 1H), 3.61 (s, 3H), 3.60–3.56 (m, 1H), 3.35–3.10 (m, 2H), 3.00–2.87 (m, 2H), 2.82–2.77 (m, 1H), 2.61–2.57 (m, 1H), 2.53–2.37 (m, 2H), 2.12–1.51 (m, 13H); 13C NMR (126 MHz) δ 173.7, 148.3, 138.7, 132.2, 130.7, 126.9, 124.8, 60.8, 60.6, 56.6, 54.6, 51.8 (2), 49.2, 36.4, 32.9, 30.5, 28.0, 24.3, 23.9, 21.9, 18.3, 18.3. HRMS: calcd for C23H34O4N3·2HCl [M−2HCl+H]+: 416.2544, found: 416.2539.
Methyl 12N-(pyridin-4-ylmethyl)matrinate (6f)
The title compound was prepared from 5 and 4-(chloromethyl)pyridine in the same manner as 6b. Yield: 45%; white solid; mp 84–86 °C; 1H NMR (500 MHz) δ 8.49–8.48 (m, 2H), 7.32 (d, J = 5.9 Hz, 2H), 4.02 (d, J = 14.7 Hz, 1H), 3.53 (s, 3H), 3.15 (d, J = 14.7 Hz, 1H), 2.90–2.87 (m, 1H), 2.74–2.64 (m, 3H), 2.29–2.26 (m, 2H), 2.25–2.08 (m, 1H), 1.98 (s, 1H), 1.85–1.76 (m, 4H), 1.65–1.25 (m, 12H); 13C NMR (126 MHz) δ 173.9, 150.3, 149.9 (2), 123.9 (2), 64.2, 57.3, 57.1, 56.7, 54.3, 52.6, 51.6, 37.6, 33.6, 33.5, 28.2, 27.8, 27.4, 21.5, 21.2, 19.0. HRMS: calcd for C22H34O2N3 [M+H]+: 372.2646, found: 372.2635.
General procedures for 12N-substituted matrinol derivativess 7a–e
A solution of LiAlH4 (12 mmol) in anhydrous THF (20 mL) was added to the solution of compound 6 (10 mmol) in anhydrous THF (3 mL) in an ice bath, the mixture solution was then stirred at room temperature for 30 min before the reaction was quenched with acetone. Saturated ammonium chloride (2 mL) was then added and the mixture was stirred for 30 min, and the precipitation was filtered off. The solvent was evaporated, and the residue was purified by flash column chromatography on silica gel with CH2Cl2/CH3OH as the eluent or followed by an acidification with 2 N hydrochloride/ether (10 mL) to afford target compounds.
12N-(4-Methoxybenzyl)matrinol dihydrochloride (7a)
The title compound was prepared from 6a as described above. Yield: 82%; white solid; mp 241–243 °C; 1H NMR (400 MHz) δ 11.04 (br, 1H), 10.99 (br, 1H), 7.52 (d, J = 8.7 Hz, 2H), 7.02 (d, J = 8.7 Hz, 2H), 4.82 (d, J = 11.2 Hz, 1H), 4.36 (s, 4H), 4.21–4.11 (m, 1H), 4.03–3.87 (m, 2H), 3.79 (s, 3H), 3.55 (d, J = 10.2 Hz, 1H), 3.27 (t, J = 13.0 Hz, 2H), 3.00–2.84 (m, 2H), 2.73–2.63 (m, 1H), 2.41 (d, J = 11.2 Hz, 1H), 1.92 (d, J = 9.3 Hz, 2H), 1.87–1.75 (m, 3H), 1.75–1.64 (m, 3H), 1.65–1.56 (m, 2H), 1.52 (s, 4H); 13C NMR (101 MHz) δ 159.9, 132.9 (2), 121.6 (2), 114.2, 60.4, 60.3, 60.1, 57.0, 55.2, 54.2, 54.2, 48.4, 36.1, 31.8, 30.0, 28.2, 24.1, 23.6, 22.7, 17.9, 17.8. HRMS: calcd for C23H37O2N2 ·2HCl [M−2HCl+H]+: 373.2850, found: 373.2848.
12N-(4-Methylbenzyl)matrinol dihydrochloride (7b)
The title compound was prepared from 6b as described above. Yield: 85%; white solid; mp 111–113 °C; 1H NMR (400 MHz) δ 11.20 (s, 1H), 11.06 (s, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 4.85–4.80 (m 1H), 4.21–4.14 (m, 1H), 3.99–3.89 (m, 2H), 3.55 (d, J = 10.0 Hz, 1H), 3.26 (t, J = 13.6 Hz, 2H), 3.16 (s, 1H), 2.95 (m, 2H), 2.67–2.62 (m, 1H), 2.55 (m, 1H), 2.47–2.42 (m, 1H), 2.33 (s, 3H), 1.92–1.41 (m, 16H); 13C NMR (101 MHz) δ 138.8, 131.4 (2), 129.4 (2), 126.8, 60.6, 60.2, 60.1, 57.2, 54.2, 54.2, 48.6, 36.1, 31.8, 30.0, 28.2, 24.1, 23.6, 22.7, 20.8, 17.9, 17.8. HRMS: calcd for C23H37ON2·2HCl [M−2HCl+H]+: 357.2900, found: 357.2898.
12N-(4-Vinylbenzyl)matrinol dihydrochloride (7c)
The title compound was prepared from 6c as described above. Yield: 75%; white solid; mp 121–123 °C; 1H NMR (400 MHz) δ 11.02 (br, 2H), 7.92–7.50 (m, 4H), 6.78 (dd, J = 17.6, 10.8 Hz, 1H), 5.93 (d, J = 17.6 Hz, 1H), 5.34 (d, J = 11.2 Hz, 1H), 4.87 (d, J = 11.6 Hz, 1H), 3.54 (d, J = 10.0 Hz, 1H), 3.45 (m, 2H), 3.33–3.20 (m, 2H), 3.16 (s, 1H), 3.08–2.81 (m, 3H), 2.78–2.57 (m, 2H), 2.45–2.40 (m, 1H), 2.11–1.33 (m, 16H); 13C NMR (101 MHz) δ 138.0, 136.0, 131.7 (2), 129.3, 126.4 (2), 115.6, 60.6, 60.2, 60.1, 57.2, 54.2, 48.7, 36.1, 31.8, 30.0, 28.2, 24.0, 23.6, 22.7, 18.6, 17.9, 17.8. HRMS: calcd for C24H37ON2·2HCl [M−2HCl+H]+: 369.2900, found: 369.2900.
12N-(2,4-Difluorobenzyl)matrinol dihydrochloride (7d)
The title compound was prepared from 6d as described above. Yield: 80%; white solid, mp 124–126 °C; 1H NMR (400 MHz) δ 11.13 (br, 1H), 10.87 (br, 1H), 7.92–7.80 (m, 1H),7.45–7.38 (m, 1H), 7.28–7.18 (m, 1H), 4.77 (d, J = 13.0 Hz, 1H), 4.28–4.08 (m, 2H), 4.08–3.92 (m, 2H), 3.54 (d, J = 10.2 Hz, 2H), 3.28 (t, J = 12.0 Hz, 2H), 2.99–2.87 (m, 4H), 2.42–2.38 (m, 1H), 2.02–1.87 (m, 3H), 1.87–1.83 (m, 2H), 1.79–1.68 (m, 3H), 1.68–1.58 (m, 3H), 1.52 (s, 4H); 13C NMR (101 MHz) δ 135.5, 132.6, 113.4, 112.1, 111.9, 104.4, 60.4, 60.1, 60.1, 54.2, 54.2, 49.8, 48.7, 36.0, 31.8, 30.0, 28.1, 23.9, 23.7, 22.4, 17.9, 17.8. HRMS: calcd for C22H33ON2F2·2HCl [M−2HCl+H]+: 379.2556, found: 379.2551.
12N-(Pyridin-4-ylmethyl)matrinol dihydrochloride (7e)
The title compound was prepared from 6f as described above. Yield: 77%; white solid; mp 205–206 °C; 1H NMR (400 MHz) δ 12.40 (br, 1H), 11.08 (br, 1H), 9.00 (d, J = 5.5 Hz, 2H), 8.34 (d, J = 5.5 Hz, 2H), 5.17 (s, 1H), 4.42–4.16 (m, 2H), 3.99–3.95 (m, 1H), 3.62 (d, J = 9.7 Hz, 1H), 3.43 (t, J = 5.6 Hz, 2H), 3.30–3.17 (m, 3H), 2.95–2.89 (m, 3H), 2.71 (d, J = 9.8 Hz, 1H), 2.07 (s, 1H), 1.95–1.39 (m, 14H); 13C NMR (101 MHz) δ 148.3 (2), 143.5, 129.4 (2), 61.4, 60.6, 56.1, 54.6, 49.9,49.0, 39.6 (2), 36.5, 32.3, 30.5, 28.8, 24.2, 24.1, 23.3, 18.3. HRMS: calcd for C21H34ON3·2HCl [M−2HCl+H]+: 344.2696, found: 344.2694.
General procedures for 12N-substituted matrinol derivatives 7f–i
To a stirred solution of 5 (5.0 mmol) and K2CO3 (17.0 mmol) in dichloroethane (50 mL), substituted pyridylmethyl halide or phenylcarbamic chloride (5 mmol) was added. The reaction mixture was stirred at room temperature for 8 h until TLC analysis showed completion of the reaction. Water (20 mL) was added to the mixture and the organic phase was separated and dried with anhydrous Na2SO4, concentrated. To a solution of the gained residue in anhydrous THF (3 mL) in an ice bath, a solution of LiAlH4 (6 mmol) in anhydrous THF (10 mL) was added, the mixture solution was stirred at room temperature for 30 min before the reaction was quenched with acetone. The saturated ammonium chloride (2 mL) was then added and the mixture was stirred for 30 min, and the precipitation was filtered off. Then the solvent was evaporated, and the residue was purified by flash column chromatography on silica gel with CH2Cl2/CH3OH as the eluent to afford the target compounds.
12N-(Pyridin-3-ylmethyl)matrinol (7f)
The title compound was prepared from 5 and 3-chloromethylpyridine as described above. Yield: 43%; yellow oil; 1H NMR δ (500 MHz) 9.13 (s, 1H), 8.96 (d, J = 5.5 Hz, 1H), 8.78 (d, J = 5.5 Hz, 1H), 8.07 (t, J = 5.5 Hz, 1H), 5.04–4.97(m, 1H), 4.37–4.17 (m, 2H), 3.95–3.92 (m, 1H), 3.64–3.62 (m, 1H), 3.45 (t, J = 5.9 Hz, 2H), 3.29–3.18 (m, 3H), 3.07–2.86 (m, 4H), 2.68–2.66 (m, 1H), 2.09–2.08 (m, 1H), 1.93–1.91 (m, 2H), 1.86–1.47 (m, 11H); 13C NMR (126 MHz) δ 148.1, 145.8, 143.8, 129.5, 127.1, 61.2, 60.8, 60.6, 54.6, 53.8, 49.8, 49.6, 36.6, 32.3 (2), 30.6, 28.5, 24.3, 24.1, 22.9, 18.3. HRMS: calcd for C21H34ON3 [M+H]+: 344.2696, found: 344.2694.
12N-(5-Chloropyridin-2-ylmethyl)matrinol dihydrochloride (7g)
The title compound was prepared from 5 and 5-chloro-2-(chloromethyl)pyridine in the same manner as 7f followed by an acidification with 2 N hydrochloride/ether (3 mL). Yield: 48%; light yellow solid; mp: 91–92 °C; 1H NMR (500 MHz) δ 11.98 (br, 1H), 11.06 (br, 1H), 8.61 (d, J = 2.4 Hz, 1H), 8.19 (dd, J = 8.2, 2.4 Hz, 1H), 7.64 (d, J = 8.2 Hz, 1H), 5.01–4.95 (m, 1H), 4.33–4.22 (m, 2H), 3.99–3.95 (m, 1H), 3.64–3.62 (m, 1H), 3.43–3.41 (m, 2H), 3.30–3.17 (m, 3H), 2.95–2.89 (m, 3H), 2.71 (d, J = 9.8 Hz, 1H), 2.07 (s, 1H), 1.97–1.35 (m, 14H); 13C NMR (126 MHz) δ 152.9, 151.7, 143.3, 125.8, 124.8, 60.7, 60.3, 54.7, 53.9, 51.8, 49.2, 39.5, 36.5, 33.2, 32.9, 30.5, 28.0, 24.3, 23.9, 21.5, 18.4. HRMS: calcd for C21H33ON3Cl·2HCl [M−2HCl+H]+: 378.2307, found: 378.2304.
12N-(2-Oxo-2-(phenylamino)ethyl)matrinol (7h)
The title compound was prepared from 5 and phenylcarbamic chloride in the same manner as 7f. Yield: 46%; white solid; mp: 136–137 °C; 1H NMR (400 MHz) δ 9.64 (br, 1H), 7.64–7.57 (m, 2H), 7.31 (t, J = 7.9 Hz, 2H), 7.06 (t, J = 7.4 Hz, 1H), 4.41–4.25 (m, 1H), 3.41–3.37 (m, 2H), 3.03 (s, 2H), 2.75–2.72 (m, 2H), 2.44–2.31 (m, 1H), 2.00–1.93 (m, 2H), 1.85–1.76 (m, 3H), 1.70–1.48 (m, 4H), 1.47–1.20 (m, 12H); 13C NMR (101 MHz) δ 170.1, 138.9, 131.1 (2), 123.9, 119.8 (2), 64.2, 61.2, 61.1, 57.3, 56.4, 55.7, 53.9, 37.9, 33.2, 29.7 (2), 28.7, 28.1, 27.4, 21.5, 20.8. HRMS: calcd for C23H36O2N3 [M+H]+: 386.2802, found: 386.2800.
12N-(2-Oxo-2-((4-(trifluoromethyl)phenyl)amino)ethyl)matrinol dihydrochloride (7i)
The title compound was prepared from 5 and 4-(trifluoromethyl)phenylcarbamic chloride in the same manner as 7 g. Yield: 62%; white solid; mp: 185–187 °C; 1H NMR (400 MHz) δ 11.20 (br, 1H), 10.48 (br, 1H), 10.08 (br, 1H), 8.44 (d, J = 1.6 Hz, 1H), 7.56–7.46 (m, 1H), 7.30 (d, J = 8.6 Hz, 1H), 4.65–4.55 (m, 1H), 4.32–4.16 (m, 2H), 4.09 (d, J = 9.4 Hz, 1H), 3.46–3.30 (m, 2H), 3.26 (t, J = 9.5 Hz, 2H), 3.03–2.87 (m, 2H), 2.60–2.56 (m, 1H), 2.46–2.42 (m, 1H), 1.95–1.60 (m, 12H), 1.50–1.30 (m, 6H); 13C NMR (101 MHz) δ 164.5, 152.8, 127.2 (2), 126.2, 118.9 (2), 61.2, 60.7, 60.4, 56.8, 54.7, 54.6, 52.3, 36.6, 32.3, 30.7, 29.7, 29.2, 24.3, 24.1, 23.7, 18.4, 18.3. HRMS: calcd for C24H35O2N3F3·2HCl [M−2HCl+H]+: 454.2676, found: 454.2679.
Synthesis of 12N-4-methoxybenzyl matrinic butane 9
To a solution of 7a (5 mmol) in anhydrous CH2Cl2 (20 mL), TsCl (5 mmol), TEA (10 mmol) and dimethylamino pyridine (0.5 mmol) were added and stirred at room temperature until the TLC showed completion of the reaction. The solution was washed successively by water (10 mL), saturated ammonium chloride solution (10 mL) and brine (10 mL), dried over anhydrous sodium sulfate, and concentrated to obtain crude 8. To a solution of the crude 8 in anhydrous THF, a solution of LiAlH4 (6 mmol) in anhydrous THF was added in an ice bath, then the mixture was stirred at room temperature for 30 min, the reaction was then quenched with acetone, 2 ml saturated ammonium chloride was added and stirred for 30 min, and the precipitation was filtrated. The gained residue was purified by flash column chromatography on silica gel with CH2Cl2/CH3OH as the eluent to afford the title compound 9 as a yellow solid. Yield: 56%; mp: 73–74 °C; 1H NMR (400 MHz) δ 7.22 (d, J = 8.8 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 3.93–3.88 (m, 1H), 3.74 (s, 3H), 3.43–3.21 (m, 1H), 3.00 (d, J = 10.3 Hz, 1H), 2.88–2.61 (m, 3H), 2.53 (d, J = 11.7 Hz, 1H), 2.22-2.15 (m, 1H), 1.96 (s, 1H), 1.90–1.73 (m, 3H), 1.64 (s, 2H), 1.50-1.37 (m, 12H), 0.87 (s, 3H); 13C NMR (101 MHz) δ 158.4, 129.9, 128.4, 114.1 (2), 113.9, 64.3, 63.0, 57.1, 55.5, 55.4, 55.1, 52.0, 37.8, 37.5, 33.7, 28.4, 27.5, 25.9, 23.0, 21.6, 21.2, 14.5. HRMS: calcd for C23H37ON2 [M+H]+: 357.2900, found: 357.2899.
General procedures for 1′,1′-dialkyl-12N-substituted matrinol derivatives 10a–e
To a solution of compound 6 (5 mmol) in anhydrous THF (10 mL), a solution of 2 N alkylmagnesium chloride in THF (25 mmol) was added in an ice bath, and the mixture solution was heated at refluxing for 2 h. After reaction completed, the reaction was quenched with a solution of saturated aqueous ammonium chloride (2 mL). The residue was purified by flash column chromatography on silica gel with CH2Cl2/CH3OH as the eluent followed by the acidification with 2 N hydrochloride/ether (3 mL) to afford the title compounds.
1′,1′-Dimethyl-12N-(4-methoxybenzyl)matrinol dihydrochloride (10a)
The title compound was prepared from 6a and methylmagnesium chloride using the same method as described above. Yield: 67%; white solid; mp: 125–127 °C; 1H NMR (400 MHz) δ 11.35 (br, 1H), 11.05 (br, 1H), 7.53 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H), 4.78 (d, J = 11.2 Hz, 1H), 4.22–4.12 (m, 1H), 3.94–3.89 (m, 2H), 3.77 (s, 3H), 3.59 (d, J = 10.0 Hz, 1H), 3.25 (t, J = 13.6 Hz, 2H), 3.00–2.87 (m, 2H), 2.68–2.57 (m, 2H), 2.46 (d, J = 12.4 Hz, 1H), 2.02–1.51 (m, 12H), 1.46–1.39 (m, 4H), 1.11-1.07 (m, 5H); 13C NMR (126 MHz) δ 159.8, 132.9 (2), 121.6, 114.1 (2), 72.4, 68.7, 57.0, 55.2, 54.2, 54.1, 44.8, 42.9, 35.7, 32.3, 32.0, 29.9, 29.6, 29.2, 27.6, 25.5, 24.1, 23.6, 21.3. HRMS: calcd for C25H41O2N2·2HCl [M−2HCl+H]+: 401.3163, found: 401.3163.
1′,1′-Dimethyl-12N-(4-methylbenzyl)matrinol dihydrochloride (10b)
The title compound was prepared from 6b and methylmagnesium chloride using the same method as described above. Yield 63%; white solid; mp: 120–122 °C; 1H NMR (400 MHz) δ 11.12 (br, 1H), 10.98 (br, 1H), 7.48 (dd, J = 8.0, 5.6 Hz, 2H), 7.27 (d, J = 7.6 Hz, 2H), 4.91–4.74 (m, 1H), 4.33–4.14 (m, 2H), 4.04 (s, 5H), 3.62–3.54 (m, 1H), 3.29–3.24 (m, 2H), 2.99–2.88 (m, 2H), 2.73–2.68 (m, 1H), 2.34 (s, 3H), 2.04–1.56 (m, 15H), 1.49–1.42 (m, 2H), 1.10 (d, J = 3.2 Hz, 2H); 13C NMR (126 MHz) δ 132.1, 123.1 (2), 121.5 (2), 117.9, 62.3, 61.7, 53.3, 53.0, 50.1, 46.9, 41.5, 36.8, 34.4, 28.4, 23.4, 23.3, 22.9 (2), 19.9 (2), 15.9, 15.6, 11.8. HRMS: calcd for C25H41ON2·2HCl [M−2HCl+H]+: 385.3213, found: 385.3214.
1′,1′-Diethyl-12N-(4-methylbenzyl)matrinol dihydrochloride (10c)
The title compound was prepared from 6b and ethylmagnesium chloride using the same method as described above. Yield 72%; yellow oil; 1H NMR (400 MHz) δ 10.91 (br, 1H), 10.54 (br, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 7.6 Hz, 2H), 4.85–4.80 (m, 1H), 4.56–4.14 (m, 3H), 4.06–3.82 (m, 3H), 3.61–3.53 (m, 3H), 3.35–3.24 (m, 2H), 3.12–2.85 (m, 3H), 2.78 (d, J = 6.8 Hz, 1H), 2.34 (s, 3H), 2.09–1.99 (m, 3H), 1.95–1.69 (m, 9H), 1.69–1.53 (m, 3H), 1.53–1.33 (m, 2H), 0.96 –0.92 (m, 4H); 13C NMR (126 MHz) δ 138.9, 131.3 (2), 129.3 (2), 126.8, 80.6, 60.3, 60.2, 57.3, 54.2 (2), 48.9, 35.6, 32.9, 32.8, 30.0, 24.0, 23.6, 20.8 (2), 19.9, 17.8, 13.2, 12.6, 8.6 (2). HRMS: calcd for C27H45ON2·2HCl [M−2HCl+H]+: 413.3526, found: 413.3524.
1′,1′-Dimethyl-12N-(2,4-difluorobenzyl)matrinol dihydrochloride (10d)
The title compound was prepared from 6d and methylmagnesium chloride using the same method as described above. Yield: 75%; white solid; mp: 237–238 °C; 1H NMR (400 MHz) δ 11.02 (s, 1H), 10.40 (s, 1H), 7.93–7.81 (m, 1H),7.47–7.40 (m, 1H), 7.29–7.19 (m, 1H), 4.78 (d, J = 13.2 Hz, 1H), 4.30–4.20 (m, 1H), 4.20–4.05 (m, 1H), 4.10–3.85 (m, 1H), 3.53 (d, J = 8.4 Hz, 3H), 3.29 (t, J = 12.0 Hz, 2H), 3.06–2.82 (m, 4H), 2.00–1.51 (m, 11H), 1.47–1.44 (m, 3H), 1.11 (d, J = 3.6 Hz, 6H); 13C NMR (126 MHz) δ 164.2, 162.2, 135.6, 113.5, 112.0, 104.3, 68.6, 60.5, 60.1, 54.2 (2), 49.8, 48.8, 42.9, 35.9, 29.9, 29.5, 29.2, 28.7, 23.9, 23.7, 20.4, 17.8 (2). HRMS: calcd for C24H37ON2F2·2HCl [M−2HCl+H]+: 407.2868, found: 407.2863.
1′,1′-Dimethyl-12N-(4-pyridylmethyl)matrinol (10e)
The title compound was prepared from 6f and methylmagnesium chloride using the same method as described above without acidification. Yield: 68%; yellow solid; mp: 243–245 °C; 1H NMR (400 MHz) δ 8.47 (d, J = 5.6 Hz, 2H), 7.33 (d, J = 5.6 Hz, 2H), 4.02 (s, 1H), 3.94 (d, J = 15.2 Hz, 1H), 3.32 (s, 1H), 3.19 (d, J = 14.4 Hz, 1H), 2.89 (d, J = 8.4 Hz, 1H), 2.72 (t, J = 11.2 Hz, 3H), 2.17 (d, J = 8.8 Hz, 1H), 2.00 (s, 1H), 1.81 (d, J = 12.0 Hz, 3H), 1.64–1.54 (m, 4H), 1.42–1.24 (m, 10H), 1.01 (d, J = 2.8 Hz, 6H); 13C NMR (126 MHz) δ 149.7, 149.4 (2), 123.3 (2), 68.7, 63.8, 56.5, 56.4, 53.7, 51.9, 43.9, 37.0, 34.7, 32.7, 29.4, 29.1, 29.0, 27.4, 26.7, 20.8, 20.5, 18.1. HRMS: calcd for C23H38ON3 [M+H]+: 372.3009, found: 372.3008.
General procedures for methyl (Z)-12N-substituted Δβγ-matrinic crotonate derivatives 13a–c
Lehmannine (3.0 g, 12.2 mmol) was added to a solution of 5 N HCl (30 mL). The reaction mixture was heated at reflux for 9 h. The solvent was then removed in vacuo, and the residue was recrystallized by methanol and ethyl acetate to afford the intermediate 11 (2.5 g, 60%) as white solid. mp: 191–193 °C. 1H NMR (400 MHz) δ 12.39 (s, 1H), 11.21 (d, J = 8.0 Hz, 1H), 10.27 (d, J = 9.3 Hz, 1H), 9.30 (d, J = 9.0 Hz, 1H), 6.01 (dt, J = 10.8, 7.3 Hz, 1H), 5.49 (t, J = 10.4 Hz, 1H), 5.04–4.92 (m, 1H), 3.99–3.764 (m, 1H), 3.65 (d, J = 10.1 Hz, 1H), 3.44–3.33 (m, 2H), 3.25–3.20 (m, 2H), 3.20–3.02 (m, 1H), 2.97–2.89 (m, 2H), 2.55–2.51 (m, 1H), 2.40–2.23 (m, 1H), 1.89–1.56 (m, 8H); 13C NMR (101 MHz) δ 172.4, 132.4, 125.7, 60.4, 54.8, 54.7, 49.8, 41.5, 35.5, 33.8, 30.8, 24.6, 23.6, 18.5(2); HRMS: calcd for C15H25N2O2·2HCl [M−2HCl+H]+: 265.1911, found: 265.1909.
Compound 11 (1.0 g, 3.0 mmol) was dissolved in 2 N MeOH/HCl (30 mL), and the reaction mixture was refluxed for 2 h. Compound 12 was obtained by evaporation and used in the next reaction without further purification. Anhydrous K2CO3 (3.5 equiv) and substituted benzyl bromide (1.5 equiv) were added to a solution of compound 12 in acetonitrile (30 mL), and the reaction solution was then stirred at room temperature until TLC analysis showed completion of the reaction. The reaction mixture was filtered, and the filtrate was washed by water and brine, dried with anhydrous Na2SO4, filtrated, and concentrated to afford crude compound 13. The title compounds were obtained by purifying with flash column chromatography on silica gel with dichloromethane and methanol as the eluent.
(Z)-Methyl 12N-(4-methoxybenzyl)-Δβγ-matrinic crotonate (13a)
The title compound was prepared from 12 and 4-methoxybenzyl bromide using the same method as described above. Yield: 62%; white solid; mp: 98–100 °C. 1H NMR (400 MHz) δ 7.14 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H), 5.81–5.75 (m, 1H), 5.33 (t, J = 10.4 Hz, 1H), 3.92 (d, J = 13.2 Hz, 1H), 3.73 (s, 3H), 3.61 (s, 3H), 3.34–3.15 (m, 3H), 2.89–2.86 (m, 1H), 2.72–2.75(m, 2H), 2.51–2.44 (m, 1H), 2.21–2.18 (m, 1H), 1.98 (s, 1H),1.84–1.75 (m, 2H), 1.65–1.24 (m, 10H); 13C NMR (126 MHz) δ 171.3, 158.0, 136.0, 131.5, 129.7 (2), 124.8, 113.4 (2), 62.5, 58.2, 56.8, 56.7, 54.9 (2), 51.6 (2), 50.8, 34.7, 33.3, 28.1, 26.8, 21.4, 21.2. HRMS: calcd for C24H35N2O3 [M+H]+: 399.2642, found: 399.2642.
(Z)-Methyl 12N-(4-fluorobenzyl)-Δβγ-matrinic crotonate dihydrochloride (13b)
The title compound was prepared from 12 and 4-fluorobenzyl bromide using the same method as described above. Yield: 68%; white solid; mp: 151–153 °C. MS–ESI m/s: 387; 1H NMR (400 MHz) δ 11.93 (d, J = 8.0 Hz, 1H), 11.08 (d, J = 7.6 Hz, 1H), 7.64–7.61 (m, 2H), 7.32–7.27 (m, 2H), 6.27–6.20 (m, 1H), 5.88–5.78 (m, 1H), 5.28 (t, J = 11.2 Hz, 1H), 4.63 (d, J = 12.0 Hz, 1H), 4.00–3.85 (m, 2H), 3.68–3.17 (m, 9H), 3.00–2.79 (m, 3H), 2.63 (s, 1H), 1.83–1.56 (m, 8H); 13C NMR (126 MHz) δ 170.7, 161.7, 133.7, 133.6, 133.2, 126.0, 124.9, 115.9, 115.8, 59.6, 58.6, 56.7, 54.2, 54.1, 51.9, 47.3, 35.1, 33.3, 30.2, 24.0, 23.9, 18.0, 17.9. HRMS: calcd for C23H32FN2O2·2HCl [M−2HCl+H]+: 387.2442, found: 387.2446.
(Z)-Methyl 12N-(3-nitrobenzyl)-Δβγ-matrinic crotonate dihydrochloride (13c)
The title compound was prepared from 12 and 3-nitrobenzyl bromide using the same method as described above. Yield: 70%; white solid; mp: 185–187 °C; 1H NMR (400 MHz) δ 12.33 (s, 1H), 11.07 (s, 1H), 8.41 (s, 1H), 8.26 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 7.5 Hz, 1H), 7.71 (t, J = 8.0 Hz, 1H), 6.22 (dt, J = 15.1, 7.5 Hz, 1H), 5.84 (t, J = 10.6 Hz, 1H), 5.39–5.21 (m, 1H), 4.70 (d, J = 12.9 Hz, 1H), 4.15–3.78 (m, 3H), 3.64 (s, 3H), 3.47–3.41 (m, 1H), 3.26 (d, J = 11.7 Hz, 2H), 2.99–2.83 (m, 3H), 2.61 (s, 1H), 2.56–2.48 (m, 1H), 1.80–1.76 (m, 2H), 1.70–1.49 (m, 7H); 13C NMR (126 MHz) δ 170.7, 147.8, 138.1, 133.4, 131.6, 130.4, 126.3, 124.9, 124.4, 59.6, 58.7, 56.5, 54.3, 54.1, 51.9, 47.7, 35.2, 33.3, 30.3, 23.9, 23.9, 18.0, 17.9. HRMS: calcd for C23H32N3O4·2HCl [M−2HCl+H]+: 414.2387, found: 414.2391.
General procedures for (Z)-12N-substituted Δβγ-matrinic crotonol derivatives 14a–b
A solution of the LiAlH4 in THF (2.4 N, 1.2 equiv) was added to the solution of compound 13 in anhydrous THF in ice bath, then the mixture solution was stirred at room temperature for 30 min, the reaction was then quenched with acetone, 2 mL saturated ammonium chloride solution was added and stirred for 30 min, and the precipitation was filtrated off. The filtrate was concentrated, and the residue was purified by flash column chromatography on silica gel with dichloromethane and methanol as the eluent followed by the acidification by 2 N hydrochloride/ether to afford compounds.
(Z)-12N-(4-Methoxybenzyl)-Δβγ-matrinic crotonol dihydrochloride (14a)
The title compound was prepared from 13a using the same method as described above. Yield: 86%; white solid; mp: 175–177 °C; 1H NMR (400 MHz) δ 11.46 (br, 1H), 11.15 (br, 1H), 7.48–7.45 (d, J = 8.8 Hz, 2H), 7.01–6.99 (d, J = 8.8 Hz, 2H), 6.17–6.11 (m, 1H), 5.68 (t, J = 10.8 Hz, 1H), 5.16 (dd, J = 10.4 Hz, 1H), 4.66 (d, J = 11.6 Hz, 1H), 4.02–3.93 (m, 1H), 3.89–3.78 (m, 1H), 3.74 (s, 3H), 3.69–3.43 (m, 5H), 3.39–3.28 (m, 3H), 2.99–2.88 (m, 2H), 2.78–2.75 (m, 1H), 2.55–2.49 (m, 1H), 2.37–2.31 (m, 1H), 1.89–1.53 (m, 8H); 13C NMR (126 MHz) δ 159.9, 139.2, 132.8 (2), 123.5, 121.6, 114.2 (2), 59.8, 59.7, 58.6, 57.1, 55.2 (2), 54.2, 47.1, 35.3, 31.8, 30.2, 24.2, 24.0, 18.0, 17.9. HRMS: calcd for C23H35N2O2·2HCl [M−2HCl+H]+: 371.2693, found: 371.2698.
(Z)-12N-4-(Fluorobenzyl)-Δβγ-matrinic crotonol dihydrochloride (14b)
The title compound was prepared from 13b using the same method as described above. Yield: 87%; white solid; mp: 194–196 °C; 1H NMR (400 MHz) δ 11.77 (br, 1H), 11.15 (br, 1H), 7.64 (dd, J = 5.6 Hz, 2H), 7.28 (t, J = 8.8 Hz, 2H), 6.18–6.11 (m, 1H), 5.71 (t, J = 10.8 Hz, 1H), 5.18 (dd, J = 10.8 Hz, 1H), 4.70 (d, J = 12.4 Hz, 1H), 4.04–3.87 (m, 2H), 3.77–3.41 (m, 5H), 3.41–3.28 (m, 2H), 3.00–2.76 (m, 3H), 2.58–2.51 (m, 2H), 2.37–2.29 (m, 1H), 1.90–1.56 (m, 8H); 13C NMR (126 MHz) δ 163.6, 139.4, 133.7, 133.6, 126.2, 123.4, 115.9, 115.7, 59.8, 59.6, 58.8, 56.7, 54.2 (2), 47.3, 35.3, 31.8, 30.2, 24.1, 24.0, 18.0, 17.9. HRMS: calcd for C22H32FN2O·2HCl [M−2HCl+H]+: 359.2493, found: 359.2492.
Synthesis of (Z)-12N-(3-nitrobenzyl)-Δβγ-matrinic crotonol dihydrochloride 20
The compound 12 (1.0 g, 4.0 mmol) was dissolved in 2 N HCl/MeOH (30 mL). The reaction mixture was refluxed for 2 h, then anhydrous K2CO3 (3.5 equiv) and Boc2O (1.5 equiv) were added to the reaction solution, and the mixture solution was stirred at room temperature until TLC analysis showed completion of the reaction. The reaction mixture was filtered, and the filtrate was washed by water and brine, dried with anhydrous Na2SO4, filtrated and concentrated to afford the crude 15.
A solution of the LiAlH4 in THF (2.4 N, 1.2 equiv) was added to the solution of compound 15 in anhydrous THF in ice-bath, then the mixture solution was stirred at room temperature for 30 min, the reaction was then quenched with acetone, 2 mL saturated ammonium chloride solution was added and stirred for 30 min, and the precipitation was filtrated off. The filtrate was concentrated, and the residue of compound 16 was dissolved in ethyl acetate, and washed with water and brine, dried with anhydrous Na2SO4, filtrated and concentrated. The residue was stirred in 2 N HCl/Et2O (20 mL) to remove the Boc protection group, then the mixute was filtrated to give the crude 17.
The crude 17 (1.0 equiv), TBSCl (1.2 equiv) and imidazole (1.5 equiv) were used to synthesize compound 18 in CH2Cl2, after reaction was complete, 3-nitrobenzyl bromide (3.0 equiv) and TEA (3.0 equiv) were added to the reaction solution, which was stirred at room temperature until TLC analysis showed completion of the reaction. The reaction solution was washed by water and brine, dried over anhydrous Na2SO4, filtrated and concentrated to afford the crude compound 19.
The crude 19 was dissolved in 2 N HCl (15 mL), and the mixture was stirred until TLC analysis showed completion of the reaction. The pH of the reaction solution was then adjusted to 7–8 by addition of ammonium hydroxide. The solvent was removed under reduced pressure, and the residue was dissolved in MeOH and filtered to remove the organic salts. The solution was concentrated, and the residue was purified by flash column chromatography on silica gel with dichloromethane and methanol as the eluent to afford compound 20 as white solid. Yield: 30%; mp: 143–145 °C; 1H NMR (400 MHz) δ 12.8 (br, 1H), 11.17 (br, 1H), 8.44 (s, 1H), 8.30 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 6.20–6.14 (m, 1H), 5.73 (t, J = 10.4 Hz, 1H), 5.27–5.20 (m,1H), 4.85(d, J = 12.8 Hz, 1H), 4.10–4.02 (m, 3H), 3.65 (d, J = 10.4 Hz, 1H), 3.57–3.49 (m, 2H), 3.29 (d, J = 12.0 Hz, 2H), 3.00–2.88 (m, 3H), 2.60–2.53 (m, 3H), 2.37–2.32 (m, 1H), 1.91–1.56 (m, 8H); 13C NMR (126 MHz) δ 147.8, 139.7, 138.0, 131.9, 130.4, 126.3, 124.3, 123.3, 59.8, 59.6, 58.9, 56.5, 54.2, 47.6, 35.3, 31.8, 30.3, 29.2, 24.1, 23.9, 18.0, 17.9. HRMS: calcd for C22H32N3O3·2HCl [M−2HCl+H]+: 386.2438, found: 386.2440.
Biology assay
Cell culture
Human liver cell line Huh7.5 cells (kindly provided by Vertex Pharmaceuticals, Inc., Boston, MA) were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% inactivated fetal bovine serum and 1% penicillin–streptomycin (invitrogen). Cells were digested with 0.05% trypsin-ethylene diamine tetraacetic acid (EDTA) and split twice a week.
Anti-HCV effect in vitro
Huh7.5 cells were seeded into 96-well or 6-well plates (Costar) at a density of 3 × 104 cells cm−2. After 24 h incubation, the cells were infected with HCV viral stock (45 IU cell−1) and simultaneously treated with the test compounds at various concentrations or solvent as control. The culture medium was removed after 72 h inoculation, the intracellular total RNA (in 96-well plates) was extracted with RNeasy Mini Kit (Qiagen), and total intracellular proteins (in 6-well plates) were extracted with Cyto-Buster Protein Extraction Reagent added with 1 mM protease inhibitor cocktail. The intracellular HCV RNA was quantified with a real time one-step reverse-transcription polymerase chain reaction (RT-PCR).
Cytotoxicity assay
Huh7.5 cells were seeded into 96-well plates (Costar) at a density of 3 × 104 cells cm−2. After 24 h incubation, fresh culture medium containing test compounds at various concentrations were added. 72 h later, cytotoxicity was evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).
PK studies
Three male SD mice were used in each study. Each of them was dosed with a tested compound at 25 mg kg−1 via oral administration. Eight blood samples were respectively collected at 0, 0.25, 0.5, 1.0, 2.0, 4.0, and 6.0 h and were immediately centrifuged to separate the plasma fractions. The separated plasma samples were stored at −20 °C for analysis. Concentration-versus-time profiles were obtained for each analyte, and standard non-compartmental analysis was performed on the data using WinNonlin software, version 5.3, to recover the AUC and other non-compartmental parameters.
Acute toxicity
Female Kunming mice with weight of 20.0 ± 1.0 g were fed with regular rodent chow and housed in an air conditioned room. The mice were randomly divided into different groups with six mice each. Each compound was given orally in a single-dosing experiment at 0, 250, 500, 750 or 1000 mg kg−1 (ddH2O as control), respectively. The mice were closely monitored for 7 days. Body weight as well as survival was monitored.