Preparation of a coumarin derivative metabolite and its application in the prevention and treatment of cerebral ischemia and Alzheimer's disease
A technology of coumarin derivatives and metabolites, applied in the field of medicine, can solve the problems of IMM-H004-7-O-β-glucuronic acid and other problems that have not been seen before, and achieves improvement of cerebral infarction area and behavioral damage, good health Pharmacokinetic advantages, structurally novel effects
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Embodiment 1
[0048] Glycosyltransferase UGT88D4 catalyzes glucuronidation of IMM-H004
[0049] The reaction system is IMM-H004 304mg (1.0mM), glycosyl donor UDP-glucuronic acid 930mg (1.5mM), glycosyltransferase (UGT88D4) 80mg, pH 7.4 Tris-HCl buffer solution to 80ml. The reaction conditions were 30°C water bath, shaking (60rpm) for 24h. Through HPLC-UV detection, the conversion rate of glycosylation reaction can reach more than 85%.
Embodiment 2
[0051] Isolation, purification and identification of IMM-H004 glucuronide
[0052] The enzymatic reaction solution was repeatedly extracted 5 times with 160ml of ethyl acetate to remove the unreacted substrate IMM-H004. Subsequently, the raffinate was evaporated to dryness under reduced pressure and resuspended with 2 ml of methanol. Centrifuge at 15,000 g for 30 min, take the supernatant, and use reverse semi-preparative HPLC to separate and prepare glycosylated products. The initial HPLC mobile phase (methanol / water) ratio is 15 / 85, and the mobile phase ratio gradient increases to 100 / 0 within 30min; the flow rate is 3ml / min; the semi-preparative column is Shiseido capcell pak C18 column (250mm×10mm I.D., Shiseido Co., Ltd., Tokyo, Japan); glycosylation product t R It is 7.1min. After UV, MS, 1 HNMR, 13 It was identified as IMM-H004-7-O-β-glucuronic acid by means of spectroscopy such as C NMR, HSQC and HMBC, and its structure was as in formula I. The compound has a nov...
experiment example 1
[0058] Metabolism of IMM-H004 citrate in rat and human liver microsomes
[0059] IMM-H004 citrate (100μM) and rat and human liver microsomes (1mg / mL protein) were incubated for 30min after adding UDPGA (3mM) and alamethicin (50μg / mL) and all decreased by 99.8%, and could A glucuronide-conjugated product was detected. The glucuronic acid conjugation product was collected for NMR analysis, and the product was IMM-H004-7-O-β-glucuronic acid (IMM-H004G).
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