A kind of anti-hbv oxygen-containing heterocyclic compound
A compound and composition technology, applied in the field of oxygen-containing heterocyclic compounds, to achieve the effects of overcoming toxic side effects, well inhibiting HBV activity, and low physiological toxicity
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Embodiment 1
[0029] The preparation of embodiment 1 formula I compound
[0030]
[0031] Step 1): In a three-necked flask, add o-trifluoromethylbenzaldehyde (8.7g, 50mmol), thiazol-2-ylformamidine hydrochloride (8.2g, 50mmol), ethyl acetoacetate (7.8g, 60mmol) ), sodium acetate (5.3g, 65mmol) and absolute ethanol (100mL), heated to reflux reaction under nitrogen protection, TLC detection reaction process, after the reaction was complete (about 11-13h), the reaction solution was vacuum filtered, and the filtrate The solvent was removed under reduced pressure to obtain a yellow oil, which was purified by column chromatography (gradient elution with petroleum ether and ethyl acetate as eluents) to obtain a yellow solid (i.e. Intermediate 1) (13.35 g, 67.6 %).
[0032] m / z:396.1(M+1);
[0033] 1 H NMR (400MHz, CDCl 3 )δ7.78(d,1H),7.73(d,1H),7.64-7.28(m,4H),6.18(s,1H),5.13(bts,1H),3.97(q,2H),2.59(s ,3H), 1.02(t,3H).
[0034] Step 2): Add Intermediate 1 (9.9g, 25mmol) to 100ml methanol ...
Embodiment 2
[0041] Embodiment 2: In vitro anti-HBV pharmacodynamic activity assay experiment
[0042] The cell line is HBV HepG2.2.15 cell line
[0043] qPCR detects the viral DNA content of the cell culture fluid and calculates the inhibitory percentage (%, Inh) of the compound to the virus. The specific experimental method is as follows:
[0044] Inoculate HepG2.2.15 cells into a 96-well microplate (40,000 cells / well). The next day, add cell culture medium containing the compound to be tested to treat the cells. The compound to measure the inhibition percentage is used as a pair of secondary wells. The final concentration of the compound is 500nmol. On the fifth day, the drug-containing culture medium was replaced, and on the eighth day, the culture supernatant was collected and the DNA in the supernatant was extracted.
[0045] Viral DNA extraction: refer to QIAamp96DNABlood Kit (QIAGEN51161).
[0046] Quantitative PCR: configure the reaction mixture according to the PCR system; add ...
Embodiment 3
[0051] Example 3 In Vitro Test for Toxic and Side Effects of Human Normal Liver Cell Line L02
[0052] 1) L02 cells were cultured in 1640 medium containing 1% double antibody and 10% FBS in 5% CO 2 , In a saturated humidity incubator at 37°C, subculture once every 2 days.
[0053] Select the L02 cells in the logarithmic growth phase, and use the medium containing 10% FBS to prepare a concentration of 5 × 10 4 / mL of cell suspension, inoculate into 96-well culture plate, add 100 μL of cell suspension to each well. at 5% CO 2 , in a saturated humidity incubator at 37°C for 24 hours, discard the culture medium, and then add 200 μL of medium with a compound concentration of 8, 16, 32, 64, and 128 uM (set 3 auxiliary wells), and continue to cultivate for 48 hours. , add 20 μL MTT, put it in the incubator for 4 hours, discard the medium, add 150uL DMAO to each well, shake on a shaker at low speed and avoid light for 10 minutes to completely dissolve the crystals, and measure the ...
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