A kind of pyrroloquinolinone alkaloid and its preparation and application as ido1 inhibitor
A kind of technology of pyrroloquinolinone and IDO1, applied in the field of medicine
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Embodiment 1
[0037] The separation and purification of embodiment 1 pyrroloquinolinone alkaloid (compound 1)
[0038] Streptomyces albogriseolus MGR072 bacterial strain was carried out oat medium liquid fermentation culture (10 liters), 30 ℃ cultivated for 7 days, the fermented liquid was combined and soaked in the same volume of ethyl acetate solvent for two days, and after shaking and extracting twice with a separatory funnel, The organic phase solvents were combined, concentrated and dried with a rotary evaporator to obtain about 4 grams of the total extract of the strain. Get the total extract (2 grams), dissolve it with an appropriate amount of chloroform-methanol mixed solvent, add 4 grams of 200-300 mesh reversed-phase silica gel (product of Qingdao Ocean Chemical Group Co., Ltd.) On the column, gradient elution with methanol-water [30:70, 40:60, 45:55, 50:50, 60:40, (volume ratio) to 100% methanol], the polarity of the elution solvent depends on Increase the amount of methanol in ...
Embodiment 2
[0044] Example 2 Binding activity experiment of pyrroloquinolinone alkaloids and IDO1 protein
[0045] The materials are as follows:
[0046] Protein: IDO1 protein derived from mice (references for the preparation method of the IDO1 protein "Guo Zhan, Li Juanjuan, Yang Qing, the establishment of a detection method for mouse indoleamine 2, 3-dioxygenase activity, Fudan Journal (Medical Edition) ), 2016, 43(3), 350-356)"); drug: compound 1 obtained from the above examples, control: epacadostat.
[0047] Methods: SPR (Surface Plasmon Resonance, Surface Plasmon Resonance) was used to measure the dissociation and binding constants of pyrroloquinolinone substrate and IDO1 protein, and finally the affinity between the substrate and protein was obtained. The Biacore T200 instrument and CM5 sensor chip (GE Healthcare) from School of Pharmacy, Fudan University were used, and the experimental temperature was room temperature. Activate the chip: use 0.2M N-ethyl-N'-(3-dimethylaminopropy...
Embodiment 3
[0049] Example 3 Experiment of pyrroloquinolinone alkaloids inhibiting activity of IDO1 protein in vitro
[0050] The materials are as follows: protein: IDO1 protein; drug: compounds 1 and 1' obtained from the above examples, control: 1-methyltryptophan (1-MT), epacadostat.
[0051] Methods: In order to further study the inhibitory effect of compound 1 and 1' on IDO1 protein, the enzyme reaction catalyzed by IDO1 protein was carried out in vitro, and the inhibitory activity of compound 1 and 1' was detected. Through in vitro reaction verification, compounds 1 and 1' can inhibit the enzymatic activity of IDO in vitro reaction. The reaction system was set as follows: 20nM IDO1 protein, 2mM D-tryptophan, 20mM ascorbic acid (need to be neutralized to neutral with NaOH during preparation), 3.5μM methylene blue, 200μg / mL catalase, 50mM phosphate buffer (pH 6.5) A total of 90 μL of the system. At room temperature, the reaction was started when the IDO1 protein was finally added, an...
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