Macrocycles with antioxidant and neuroprotective activities
A technology for compounds, epoxides, used in antioxidant and neuroprotective products to solve problems such as failure
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Embodiment 1
[0213] Embodiment 1: the fermentation of HT-09B-VENT-WQ002
[0214] stock culture :
[0215] Pure cultures of HT-09B-VENT-WQ002 were stored frozen at -70°C in 20% glycerol.
[0216] Inoculum :
[0217] 40 mL of a well-grown agar culture inoculated in a 250 mL shake flask containing 2% oat flour, 2% malt extract, 0.01% KH 2 PO 4 , 0.005% MgSO 4 and tap water and cultured at 24°C on a rotary shaker (200rpm). Flasks were incubated for 48 hours and used as the first stage inoculum.
[0218] fermentation :
[0219] 250 mL of the same medium was inoculated in a 2L Erlenmeyer flask with 10% of the first stage inoculum. Fermentation was carried out on a rotary shaker (200 rpm) for 7 days at 24°C. The production of Compound I can be monitored by HPLC or any other method with sufficient sensitivity.
Embodiment 2
[0220] Example 2: Generation of Compound I and GKK1032
[0221] The fermentation broth (4L) of the fungus HT-09B-VENT-WQ002 was filtered through celite, and the mycelial cake was extracted twice with 2L of EtOAc / MeOH (3:1) mixture. The resulting suspension was filtered and partitioned between EtOAc and water. The organic layer was dried and the crude extract (3.1 g) was fractionated by VFC (vacuum flash chromatography) on silica gel, eluting with a stepwise gradient of hexane / EtOAc / MeOH. Fractions 7-8 (eluted with EtOAc and EtOAc / MeOH 9:1, 0.4 g) were applied to a silica gel column and flash chromatographed by elution with a hexane / EtOAc gradient. Fraction 11 (eluted with hexane / EtOAc 6:4, 32 mg) was finally passed through semi-preparative reverse phase HPLC (Agilent Prep 1100 HPLC, 5-μg C18 column; 30x100 mm; gradient eluted with 40-100 aqueous MeOH 25 Minutes, flow rate is 21.6mL / min, UV detection at 200nm) purification to obtain 19mg of compound I (Rt: 17.64min). Fractio...
Embodiment 3
[0227] Example 3: Generation of Compounds of Formula (I) or (II)
[0228] Using the adsorption resin XAD-1180 as the stationary phase and EtOAc / MeOH 3:1 as the mobile phase, the fermentation broth (8 L) of the fungus HT-09B-VENT-WQ002 was subjected to solid-phase extraction. LCMS allowed the isolation of Compound I (106 mg, 96% purity), GKK1032 (3.5 mg, 70% purity), pyrrocidine A (15.8 mg, 77% purity), pyrrocidine B (5.63 mg, 94% purity) and GKK-1032A2 (7.67 mg, 53% purity).
[0229] Five additional fermentations were performed (total volume 49.5 L) and 3 g of GKK1032 were isolated. Subjecting this compound to various reactions, mainly with low molecular weight organometallics, acids, bases, alkylating agents, and reducing agents, produces 14 compounds that are structurally related to Compound I:
[0230]
[0231]
[0232] Compound CL0652 was prepared as follows:
[0233] Dissolve 13 mg of compound GKK1032 in 10 mL of CH 2 Cl 2 and 1mL CH 3 CN. Then add 2 teaspoon...
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