The kratom plant material (Green Maeng Da, 4 kg; Figure S8 ) was extracted with 10 L of CHCl3-CH3OH (1:1) and 500 mL of 10% aqueous KOH by maceration over 24 h at room temperature. The mixture was filtered, and the solvent was evaporated under reduced pressure. The dried extract was reconstituted in a solution of 1M HCl and hexanes (1:1), transferred into a separatory funnel, and shaken vigorously. The hexanes phase was drawn off, the pH of the aqueous phase was adjusted to 9.0 with dropwise addition of concentrated NH4OH, and the alkaloids were extracted as the free base with CHCl3; after washing with neutral water, the organic phase was dried to yield 12 g of the alkaloid extract. This material was fractionated by normal phase flash chromatography using a silica column (120 g) and a gradient solvent system of hexanes-CHCl3-CH3OH at a flow rate of 85 mL/min over 67 min to yield 11 pooled fractions.
Fraction 4 (500 mg) was subjected to reverse phase HPLC using a CN column and a gradient system of 40:60 to 100:0 of CH3OH-H2O (10 mM of NH4OAc in both phases) over 20 min with a flow rate of 20 mL/min. This process yielded eight subfractions, and fraction 8 was identified as compound1 (450.5 mg).
Fraction 9 (1.1 g) was fractionated by normal phase flash chromatography using a silica column (12 g) and a gradient system of hexanes-EtOAc-CH3OH using a flow rate of 30 mL/min to generate four subfractions. Subfraction 2 (40 mg) was purified by preparative HPLC over a CN column using a gradient of 70:30 to 100:0 of CH3OH-H2O (10 mM of NH4OAc in both phases) over 20 min with a flow rate of 20 mL/min. This process yielded compounds4 (13.1 mg) and 6 (15.20 mg). Subfraction 4 (800 mg) was subjected to flash chromatography using a silica column (12 g) via a gradient of CHCl3-CH3OH (10 mM of NH4OAc in both phases) over 60 min with a flow rate of 30 mL/min to generate five fractions (F9–4_1 through F9–4_5). Fraction F9–4_3 was subjected to preparative HPLC over a Kinetex column using a gradient of 60:40 to 70:30 of CH3OH-H2O (10 mM of NH4OAc in both phases) over 30 min with a flow rate of 20 mL/min; three fractions were collected and the first fraction was characterized as compound 2 (20.5 mg). Fraction F9–4_4 (300 mg) was subjected to flash chromatography using a silica column (4 g) and a gradient of hexanes-acetone-CH3OH over 20 min at a flow rate of 18 mL/min to generate eight fractions. The second fraction was resolved by preparative HPLC over a CN column using a gradient of 50:50 to 100:0 of CH3OH-H2O (10 mM of NH4OAc in both phases) over 20 min with a flow rate of 20 mL/min; this process yielded 16.5 mg of compound 7 . The seventh fraction (16 mg) from F9–4_4 was subjected to preparative HPLC using a CN column and a gradient of 40:60 to 90:10 of CH3OH-H2O (10 mM of NH4OAc in both phases) over 20 min with a flow rate of 20 mL/min; two fractions were collected and the first fraction was characterized as compound 11 (2.3 mg). Fraction F9–4_5 (40 mg) was subjected to preparative HPLC over a Kinetex column with a gradient of 50:50 to 100:0 of CH3OH-H2O (10 mM of NH4OAc in both phases) over 35 min with a flow rate of 20 mL/min; the third fraction was identified as 8 (10.5 mg).
Fraction 6 (680 mg) was subjected to a second fractionation using flash chromatography over a silica column (12 g) and a gradient of CHCl3-CH3OH (10 mM of NH4OAc in both phases) over 60 min with a flow rate of 30 mL/min to generate five subfractions. Subfraction 1 (47 mg) was subjected to preparative HPLC over a CN column and a gradient system of 55:45 to 100:0 CH3OH-H2O (10 mM of NH4OAc in both phases) over 25 min; the first and second fractions were identified as5 (9.83 mg) and 3 (18.33 mg), respectively.
Fraction 8 (1.3 g) was fractionated by flash chromatography using a silica column (12 g) with a gradient of CHCl3-CH3OH (10 mM of NH4OAc in both phases) over 25 min with a flow rate of 30 mL/min to generate four subfractions. Subfractions 3 (12.8 mg) and 4 (7.0 mg) were purified by preparative HPLC over a CN column and a gradient system of 50:50 to 100:0 CH3OH-H2O (10 mM of NH4OAc in both phases) over 15 min at a flow rate of 20 mL/min to yield compounds9 (5.7 mg) and 10 (4.5 mg), respectively.
Fraction 4 (500 mg) was subjected to reverse phase HPLC using a CN column and a gradient system of 40:60 to 100:0 of CH3OH-H2O (10 mM of NH4OAc in both phases) over 20 min with a flow rate of 20 mL/min. This process yielded eight subfractions, and fraction 8 was identified as compound
Fraction 9 (1.1 g) was fractionated by normal phase flash chromatography using a silica column (12 g) and a gradient system of hexanes-EtOAc-CH3OH using a flow rate of 30 mL/min to generate four subfractions. Subfraction 2 (40 mg) was purified by preparative HPLC over a CN column using a gradient of 70:30 to 100:0 of CH3OH-H2O (10 mM of NH4OAc in both phases) over 20 min with a flow rate of 20 mL/min. This process yielded compounds
Fraction 6 (680 mg) was subjected to a second fractionation using flash chromatography over a silica column (12 g) and a gradient of CHCl3-CH3OH (10 mM of NH4OAc in both phases) over 60 min with a flow rate of 30 mL/min to generate five subfractions. Subfraction 1 (47 mg) was subjected to preparative HPLC over a CN column and a gradient system of 55:45 to 100:0 CH3OH-H2O (10 mM of NH4OAc in both phases) over 25 min; the first and second fractions were identified as
Fraction 8 (1.3 g) was fractionated by flash chromatography using a silica column (12 g) with a gradient of CHCl3-CH3OH (10 mM of NH4OAc in both phases) over 25 min with a flow rate of 30 mL/min to generate four subfractions. Subfractions 3 (12.8 mg) and 4 (7.0 mg) were purified by preparative HPLC over a CN column and a gradient system of 50:50 to 100:0 CH3OH-H2O (10 mM of NH4OAc in both phases) over 15 min at a flow rate of 20 mL/min to yield compounds
