Five-membered dihydroheterocyclic ketone derivatives and applications as Dhodh inhibitors
A technology of heterocyclic group, dihydroorotic acid, applied in the direction of anti-inflammatory agent, drug combination, non-central analgesic agent, etc., can solve the problems of mucositis side effect, narrow therapeutic window, limited wide application and the like
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Embodiment 1
[0269] The in vitro inhibitory effect of compound provided by the invention on dihydroorotate dehydrogenase (DHODH) activity:
[0270] The catalytic region 159-565 of PfDHODH (Plasmodium falciparum dihydroorotatedehydrogenase) was cloned into the vector pET101 / D (The Journal of Biological Chemistry, 2008, 283, 35078-35085), and transferred into the expression strain E.coliBL21, and 1.5ml of pET101 / D-PfDHODH containing liquid was inoculated for 5 In 2×YT medium with 250 μM ampicillin, culture on a shaker at 37° C. at 230 rpm for about 4 hours. When the OD value of the bacteria reached 0.8-1, IPTG was added to the culture medium so that the final concentration of IPTG was 0.5 mM, and induced at 25°C for 4-6 hours. Centrifuge at 4000rpm for 20min at 4°C to collect the induced cells, resuspend the cells in 15ml of deionized water, and centrifuge at 10,000rpm for 30min to collect the cell pellet. 40mMimidazole, 0.1% TritonX-100 resuspended, sonicated to lyse the bacteria. After e...
Embodiment 2
[0274] The inhibitory effect of the compound of the present invention on the in vitro activity of two kinds of Plasmodium falciparum cell lines:
[0275]Chloroquine is purchased from Sigma Company, and SYBRGreen is purchased from Invitrogen Company, and the Plasmodium falciparum Dd2 cell line of chloroquine-sensitive Plasmodium falciparum Dd2 cell line is used for testing antimalarial activity in vitro, replaces human with 0.5%AlbumaxII (Invitrogen Company) Serum, others were cultured according to the culture method of Trager and Jensen. Both Plasmodium falciparum strains were from MR4 (Malaria Research and Reference Reagent Resource Center) in the United States.
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