CN112552254A - Phenanthrene compound and preparation method and application thereof - Google Patents
Phenanthrene compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of medicines, relates to a phenanthrene compound, a preparation method and medical application thereof, and particularly relates to the phenanthrene compound, the preparation method and the application in preparation of medicines for preventing or treating neurodegenerative diseases, wherein the structure of the compound is as follows: wherein R is1‑R6As described in the claims and specification.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to three phenanthrene compounds in Stephania delavayi Diels, a preparation method and application thereof.
Background
Stephania sinica (Stephania epigaea H.S.Lo) is root tuber of Stephania sinica Diels of Menispermaceae, and has bitter taste and cold nature. Mainly distributed in Guangdong, Guangxi, Yunnan, Sichuan, Hubei, Zhejiang and other places, and has the effects of clearing away heat and toxic materials, invigorating stomach, relieving pain, removing blood stasis and relieving swelling. Folk medicine is often used for treating stomachache, acute gastroenteritis, rheumatic arthritis, malaria, etc.
In recent years, few studies on chemical components and biological activities of Stephania delavayi Diels are reported.
Disclosure of Invention
The invention aims to provide a phenanthrene compound and a pharmaceutically acceptable salt, isomer or solvate thereof:
the phenanthrene compound provided by the invention has a structure shown in a formula 1,2 or 3:
R2r adjacent to it1Or R3Forming an oxazole ring which may be substituted by R7The structure of the compound is as follows:
R7is C1-C4 alkyl, C1-C4 alkoxy, hydroxyl;
R3is C1-C4 alkoxy and hydroxyl;
R4and R5Forming a methylenedioxy group;
R6represents a C1-C4 aldehyde group;
the following 3 specific compounds and pharmaceutically acceptable salts, isomers or solvates thereof are preferred in the present invention:
the invention also provides a preparation method of the phenanthrene compound 1-3, which comprises the following steps:
(1) extracting radix Stephaniae Sinicae (Stephania epigaea) root tuber with ethanol, and recovering extractive solution to obtain ethanol extract;
(2) dissolving the ethanol extract obtained in the step (1) by using methanol, mixing the dissolved ethanol extract with a sample, eluting the sample in macroporous resin by using an ethanol-water-formic acid (20:75: 5-35: 60:5) system and an ethanol-water-triethylamine (65:30: 5-80: 15:5) system in sequence, and recovering to obtain an ethanol-water-triethylamine eluate;
(3) separating the ethanol-water-triethylamine eluate obtained in the step (2) by silica gel column chromatography, and separating by using a mixed solvent of petroleum ether and ethyl acetate, namely 100: 8-1: 1. petroleum ether and acetone mixed solvent 100: 8-1: 1. chloroform-acetone mixed solvent 100: 1-100: 10. dichloromethane and acetone mixed solvent 100: 1-100: 10. chloroform-methanol mixed solvent 100: 1-100: 10. dichloromethane and methanol mixed solvent 100: 1-100: 10 gradient elution;
(4) 100 obtained in the above step (3): 1-100: separating 20 fractions by ODS chromatography, and gradient eluting with mixed solvent of methanol and water, or mixed solvent of acetonitrile and water as mobile phase;
(5) methanol and water obtained in the step (4) 3: 7-9: 1. acetonitrile and water 1: 9-7: the 3 fractions were further separated by preparative HPLC-UV chromatography to give compounds 1-3.
The preparation method of the novel phenanthrene compound 1-3 provided by the invention is characterized in that the extraction method in the step (1) is heating reflux extraction or heating ultrasonic extraction for 1-3 times, and the used solvents are as follows: 50 to 90 percent of ethanol aqueous solution. The medicinal materials are as follows: the weight volume ratio of the solvent is 1: 8-1: 15g/mL, preferably 1: 8-1: 12 g/mL.
The preparation method of the novel phenanthrene compounds 1-3 provided by the invention is characterized in that the macroporous resin adsorption elution method in the step (2) adopts methanol/ethanol to dissolve the extract, and the mass ratio of the macroporous resin to the extract is 3: 1-6: 1 sample is taken and 8-12 column volumes, preferably 10 column volumes, are eluted.
The macroporous resin is D101, AB-8, HPD-100, DM-301 or DM 130.
According to the preparation method of the novel phenanthrene compounds 1-3 provided by the invention, in the step (3), the volume ratio of the mixed solvent of the eluting solvent petroleum ether and ethyl acetate to the mixed solvent of petroleum ether and acetone is preferably 100: 8-100: 20; the volume ratio of the mixed solvent of dichloromethane and acetone, the mixed solvent of chloroform and acetone, the mixed solvent of dichloromethane and methanol, or the mixed solvent of chloroform and methanol is preferably 100: 1-100: 8.
according to the preparation method of the phenanthrene compound 1-3 provided by the invention, in the step (4), the mixed solvent of methanol and water is 1: 9-9: 1, preferably 3: 7-9: 1; the volume ratio of the acetonitrile-water mixed solvent is 1: 9-9: 1, preferably 1: 9-7: 3.
according to the preparation method of the novel phenanthrene compounds 1-3, the mobile phase of the preparative HPLC-UV chromatogram in the step (5) is a mixture of the following components in a volume ratio of 4: 6-8: 2, a methanol-water mixed solvent; or the volume ratio is 2: 8-6: 4 acetonitrile-water mixed solvent.
The invention evaluates the anti-neuritis activity of the prepared novel phenanthrene compound 1-3 by using an LPS-induced microglia overactivation model. The result shows that when the concentration of the compound 1 is 30 mu M, the survival rate of BV-2 microglia is 91.7 percent, and the inhibition rate of NO release amount is 87.3 percent; at a concentration of 100. mu.M, compounds 2 and 3 did not affect the survival of microglia, and the inhibition rates of NO release amount were 62.2% and 76.1%, respectively. Therefore, the phenanthrene compound prepared by the invention can be applied to the development of medicines for treating neurodegenerative diseases.
The invention provides a method for preparing and identifying 3 novel phenanthrene compounds by taking Stephania delavayi Diels roots as raw materials for the first time, systematically evaluates the activity of the novel phenanthrene compounds in the aspect of neuroprotection and clarifies the application of the novel phenanthrene compounds in the aspect of developing and treating neurodegenerative diseases.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto.
Example 1
(1) Reflux-extracting Stephania delavayi Diels 5kg with 50% ethanol for 1 time, 2 hr each time (dosage: 50L each time), and recovering extractive solution under reduced pressure to obtain ethanol extract;
(2) dissolving the ethanol extract obtained in the step (1) by using ethanol, mixing the sample according to the mass ratio of macroporous resin to the extract of 5:1, drying the mixture at room temperature, sequentially eluting 10 column volumes by using ethanol-water-formic acid (30:65:5) and ethanol-water-triethylamine (70:25:5), and recovering the ethanol-water-triethylamine part eluate under reduced pressure.
(3) Separating the ethanol-water-triethylamine eluate obtained in the step (2) by silica gel column chromatography, and eluting with a mixed solvent of petroleum ether and ethyl acetate at a ratio of 100:5,100:7,100:10,100:15,100:20 and 100: 40;
(4) performing ODS (ODS chromatography) separation on the 100: 10-100: 20 flow obtained in the step (3), and performing gradient elution by using methanol-water 1:9,3:7,5:5,7:3,9:1 as a mobile phase;
(5) separating the fraction of methanol and water (2: 8-5: 5) obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with the flow rate of 4mL/min, and eluting with methanol and water (47:53) as a mobile phase to obtain a compound 1 (t)R29.2min) (yield 0.00003%);
(6) separating the fraction of methanol and water (6: 4-9: 1) obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with the flow rate of 4mL/min, and eluting with methanol and water (66:34) as a mobile phase to obtain a compound 2 (t)R＝40.1min),3(tR24.9min) (yield 0.00004% each);
the structures of compounds 1,2 and 3 were identified based on their physicochemical properties and spectral data.
The structural identification data for compound 1 is as follows:
yellow amorphous powder (CH)3OH), reacts positively with the improved bismuth potassium iodide; HR-ESI-MS gives the peak of the excimer ion [ M + H ]]+m/z:324.1230(calcd.324.1236for C19H18NO4) In combination with it1H-NMR and13C-NMR data (see tables 1 and 2) suggest that the molecular formula is C19H17NO4The unsaturation degree was 12.1H-NMR(600MHz,DMSO-d6) Giving out the characteristic proton signal delta of the phenanthrene nucleusH7.78(1H, d, J ═ 8.8Hz, H-9), 8.08(1H, d, J ═ 8.8Hz, H-10); three isolated aromatic proton signals deltaH8.12(1H, s, H-5), 8.07(1H, s, H-4), 7.48(1H, s, H-8); three methoxy signals deltaH 4.21(3H,s,3-OCH 3)，4.06(3H,s,6-OCH 3)，3.93(3H,s,7-OCH 3) (ii) a A carbon-bonded A to one olefinBase signal deltaH 2.72(s,3H,12-CH 3)。13C-NMR(150MHz,DMSO-d6) Gives 19 carbon signals including 14 phenanthrene nucleus skeleton carbon signals deltaC149.4(C-6), 149.0(C-7), 144.0(C-3), 138.9(C-2), 138.5(C-1), 127.3(C-4a), 126.9(C-8a), 124.8(C-9), 124.4(C-4b), 118.1(C-10), 117.9(C-1a), 108.8(C-8), 104.3(C-5), 100.4 (C-4); three methoxy carbon signals deltaC 56.5(6-OCH3)，56.0(3-OCH3)，55.5(7-OCH3) (ii) a A signal of unsaturated carbon δC163.5 (C-11); one methyl carbon signal δC14.2 (C-12). Delta observed in HMBCH7.78(H-9) is remotely related to 108.8(C-8), 124.4(C-4 b); deltaH7.48(H-8) is remotely related to 124.8(C-9), 149.4 (C-6); deltaH8.12(H-5) is remotely related to 127.3(C-4a), 126.9(C-8a), 149.0 (C-7); deltaH 4.06(6-OCH 3)，3.93(7-OCH 3) Respectively is equal to deltaC149.4(C-6), 149.0 (C-7); the connection mode of the B and C rings is determined. In HMBC spectra, deltaH8.08(H-10), 8.07(H-4) and deltaC138.5(C-1) remote correlation, δH7.78(H-9) and δC117.9(C-1a) remote correlation confirmed the attachment of the A, B rings of the compounds. In combination with the degree of unsaturation and the molecular weight of compound 1, it is presumed that one oxazole ring is present in compound 1. But due to the compound 11The H-NMR signals have an overlap of 8.08 and 8.07, and the signals related to the H-NMR signals are difficult to accurately distinguish in two-dimensional nuclear magnetism. Therefore, the substitution positions of the oxazole ring and the methoxyl group in the compound 1 are determined by ACD-Lab's software prediction and carbon spectrum chemical shift calculation. We use density function B3LYP, select 6-311G as the basis set to perform four prediction structures given by ACD-Lab' s13C-NMR data were calculated using DMSO as the solvent model, and the results were averaged using the Boltzman average method. Measured by experiment13The C-NMR data were compared with the calculated carbon spectrum data and the planar structure of Compound 1, designated epigaea-A, was determined by DP4+ and linear curve analysis.
The structural identification data for compound 2 is as follows:
white amorphous powder (CH)3OH), 10% sulfuric acid-ethanol is pale yellow. HR-ESI-MS gives the peak of the excimer ion [ M-H ]]-at m/z 537.1555(calcd 537.1549for C32H25O8). Combined with it1H-NMR and13C-NMR data (see tables 1 and 2) suggest that the molecular formula is C32H26O8The unsaturation degree was 20. Process for preparation of Compound 21H-NMR(400MHz,DMSO-d6) A group of phenanthrene nucleus characteristic hydrogen signals delta is givenH7.18(1H, d, J ═ 9.2Hz, H-9), 6.84(1H, d, J ═ 9.2Hz, H-10); three isolated aromatic proton signals deltaH9.46(1H, s, H-5), 7.29(1H, s, H-2), 7.12(1H, s, H-8); two methoxy signals deltaH3.96(3H,s,6-OCH 3)，3.95(3H,s,3-OCH 3)。13C-NMR(100MHz,DMSO-d6) Gives 16 carbon signals, including 14 phenanthrene mother nucleus carbon signals: deltaC147.3(C-6), 146.2(C-7), 143.9(C-3), 142.7(C-4), 130.0(C-1a), 127.7(C-4a), 126.2(C-1), 124.1(C-9), 123.7(C-4b), 123.7(C-10), 119.1(C-8a), 114.0(C-2), 111.7(C-8), 110.7 (C-5); two sets of methoxy carbon signals deltaC 56.9(3-OCH3)，55.5(6-OCH3). And (3) conjecturing the structure of two molecules of phenanthrene nucleus by combining HR-ESI-MS information. In NOESY spectrum, H-2 'is related to H-10, H10' is related to 3-OCH3In relation, the structure of Compound 2 was identified and designated epigaea-B.
The structural identification data for compound 3 is as follows:
white amorphous powder (CH)3OH). The 10% sulfuric acid-ethanol reaction is pale yellow. HR-ESI-MS gives the peak of the excimer ion [ M + Na ]]+m/z:303.0639(calcd.303.0633for C17H12O4Na) in combination with it1H-NMR and13C-NMR data (see tables 1 and 2) suggest that the molecular formula is C17H12NO4The unsaturation degree was 14. Process for preparation of Compound 31H-NMR(600MHz,DMSO-d6) Giving an active hydrogen signal deltaH10.47(1H, s, H-11); a set of ortho coupled hydrogen signals deltaH9.02(1H, d, J ═ 9.7Hz, H-10), 8.20(1H, d, J ═ 9.7Hz, H-9); aGroup 1,2, 3-trisubstituted benzene Ring Hydrogen Signal δH8.66(1H, d, J ═ 8.1Hz, H-5), 7.68(1H, t, J ═ 8.1Hz, H-6), 7.28(1H, d, J ═ 8.1Hz, H-7); an aromatic proton signal deltaH7.98(1H, s, H-2); methylenedioxy signal deltaH6.50(2H, s, H-12); one methoxy hydrogen signal deltaH 4.02(3H,s,8-OCH 3)。13C-NMR(150MHz,DMSO-d6) Giving 17 carbon signals, including a ketocarbonyl signal deltaC191.6(C-11), 14 phenanthrene nucleus skeleton carbon signals deltaC154.8(C-8), 148.7(C-4), 145.3(C-3), 128.8(C-1a), 128.2(C-6), 127.9(C-4b), 125.5(C-1),121.9(C-4a), 121.7(C-9), 120.9(C-10), 118.6(C-5), 115.3(C-8a), 114.7(C-2), 107.7 (C-7); one methylene dioxy carbon signal δC102.8 (C-12); one methoxy carbon signal deltaC 55.8(8-OCH3). The planar structure of compound 3 was further determined by HMBC spectroscopy. δ in HMBCH10.47(s,1H, C-11) and δC125.5(C-1),114.7(C-2) and 128.8(C-1a) are remotely related, suggesting that the formaldehyde group is substituted at the C-1 position of the compound. Delta observable in HMBC spectraH9.02(H-10) and δC125.5(C-1),121.9(C-4a) remote association, suggesting the attachment of the A, B ring of the compound. DeltaH8.20(H-9) and δC154.8(C-8) remote correlation, determination of methoxy attached at C-8, and determination of the structure of Compound 3, designated epigaea-C.
Of compounds 1 to 3 of Table 11H-NMR data (DMSO-d)6)
aThe detection frequency of 600MHz is adopted,b400MHz detection
TABLE 2 preparation of compounds 1 to 313C NMR data (DMSO-d)6)
Example 2
(1) Reflux-extracting Stephania delavayi Diels 5kg with 80% ethanol for 3 times, each time for 2 hr (dosage: 60L), and recovering extractive solution under reduced pressure to obtain ethanol extract;
(2) dissolving the ethanol extract obtained in the step (1) by using ethanol, mixing the sample according to the mass ratio of macroporous resin to the extract of 3:1, drying the mixture at room temperature, sequentially eluting 12 column volumes by using ethanol-water-formic acid (20:75:5) and ethanol-water-triethylamine (80:15:5), and recovering the ethanol-water-triethylamine part eluate under reduced pressure.
(3) Separating the ethanol-water-triethylamine eluate obtained in the step (2) by silica gel column chromatography, and eluting with a mixed solvent of petroleum ether and acetone 100:5,100:7,100:10,100:15,100:20 and 100: 40;
(4) performing ODS (ODS chromatography) separation on the 100: 10-100: 20 flow obtained in the step (3), and performing gradient elution by using methanol-water 1:9,3:7,5:5,7:3,9:1 as a mobile phase;
(5) separating the fraction of methanol and water (2: 8-5: 5) obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with the flow rate of 4mL/min, and eluting with methanol and water (47:53) as a mobile phase to obtain a compound 1 (t)R29.2min) (yield 0.00003%);
(6) separating the fraction of methanol and water (6: 4-9: 1) obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with the flow rate of 4mL/min, and eluting with methanol and water (66:34) as a mobile phase to obtain a compound 2 (t)R＝40.1min),3(tR24.9min) (yield 0.00004% each);
structural data for compounds 1,2,3 were identified in example 1.
Example 3
(1) Reflux-extracting Stephania delavayi Diels 5kg with 90% ethanol for 2 times, each time for 2 hr (40L per time), and recovering extractive solution under reduced pressure to obtain ethanol extract;
(2) dissolving the ethanol extract obtained in the step (1) by using ethanol, mixing the sample according to the mass ratio of macroporous resin to the extract of 4:1, drying the mixture at room temperature, sequentially eluting 8 column volumes by using ethanol-water-formic acid (35:60:5) and ethanol-water-triethylamine (65:30:5), and recovering the ethanol-water-triethylamine part eluate under reduced pressure.
(3) Separating the ethanol-water-triethylamine eluate obtained in the step (2) by silica gel column chromatography, and eluting with a mixed solvent of dichloromethane and methanol at a ratio of 100:1,100:3,100:5,100:7 and 100: 10;
(4) performing ODS (ODS chromatography) separation on the 100: 5-100: 10 flow obtained in the step (3), and performing gradient elution by using acetonitrile-water 1:9,3:7,5:5,7:3,9:1 as a mobile phase;
(5) separating the acetonitrile-water (3: 7-7: 3) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm, wherein the flow rate is 4mL/min, and mixing acetonitrile: eluting with water (34: 66) as mobile phase to obtain compound 1 (t)R21.6min) (yield 0.00003%);
(6) separating the acetonitrile-water (6: 4-9: 1) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with the flow rate of 4mL/min, and eluting with acetonitrile-water (54:46) as a mobile phase to obtain a compound 2 (t)R＝35.5min),3(tR19.9min) (yield 0.00004% each);
structural data for compounds 1,2,3 were identified in example 1.
Example 4
(1) Reflux-extracting Stephania delavayi Diels 5kg with 60% ethanol for 3 times, each time for 2 hr (each dosage: 75L), and recovering extractive solution under reduced pressure to obtain ethanol extract;
(2) dissolving the ethanol extract obtained in the step (1) by using ethanol, mixing the sample according to the mass ratio of macroporous resin to extract being 6:1, drying the mixture at room temperature, sequentially eluting the mixture for 10 column volumes by using ethanol-water-formic acid (25:70:5) and ethanol-water-triethylamine (75:20:5), and recovering the ethanol-water-triethylamine part eluate under reduced pressure.
(3) Separating the ethanol-water-triethylamine eluate obtained in the step (2) by silica gel column chromatography, and eluting with a mixed solvent of chloroform and methanol at a ratio of 100:1,100:3,100:5,100:7 and 100: 10;
(4) performing ODS (ODS chromatography) separation on the 100: 5-100: 10 flow obtained in the step (3), and performing gradient elution by using acetonitrile-water 1:9,3:7,5:5,7:3,9:1 as a mobile phase;
(5) separating the acetonitrile-water (3: 7-7: 3) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm, wherein the flow rate is 4mL/min, and mixing acetonitrile: eluting with water (34: 66) as mobile phase to obtain compound 1 (t)R21.6min) (yield 0.00003%);
(6) obtained in the above step (4)Separating acetonitrile-water (6: 4-9: 1) fractions by HPLC-UV chromatography, detecting at 210nm with the flow rate of 4mL/min, and eluting with acetonitrile-water (54:46) as a mobile phase to obtain a compound 2 (t)R＝35.5min),3(tR19.9min) (yield 0.00004% each);
structural data for compounds 1,2,3 were identified in example 1.
Example 5
(1) Reflux-extracting Stephania delavayi Diels 5kg with 70% ethanol for 3 times, each time for 2 hr (dosage: 50L), and recovering extractive solution under reduced pressure to obtain ethanol extract;
(2) dissolving the ethanol extract obtained in the step (1) by using ethanol, mixing the sample according to the mass ratio of macroporous resin to the extract of 3:1, drying the mixture at room temperature, sequentially eluting 12 column volumes by using ethanol-water-formic acid (30:65:5) and ethanol-water-triethylamine (70:25:5), and recovering the ethanol-water-triethylamine part eluate under reduced pressure.
(3) Separating the ethanol-water-triethylamine eluate obtained in the step (2) by silica gel column chromatography, and eluting with a mixed solvent of dichloromethane and methanol at a ratio of 100:1,100:3,100:5,100:7 and 100: 10;
(4) performing ODS (ODS chromatography) separation on the 100: 10-100: 20 flow obtained in the step (3), and performing gradient elution by using methanol-water 1:9,3:7,5:5,7:3,9:1 as a mobile phase;
(5) separating the fraction of methanol and water (2: 8-5: 5) obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with the flow rate of 4mL/min, and eluting with methanol and water (47:53) as a mobile phase to obtain a compound 1 (t)R29.2min) (yield 0.00003%);
(6) separating the fraction of methanol and water (6: 4-9: 1) obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with the flow rate of 4mL/min, and eluting with methanol and water (66:34) as a mobile phase to obtain a compound 2 (t)R＝40.1min),3(tR24.9min) (yield 0.00004% each);
structural data for compounds 1,2,3 were identified in example 1.
EXAMPLE 6 test of inhibitory Activity of Compounds 1,2,3 prepared in examples 1-5 on excessive activation of microglia
(1) The experimental principle is as follows: the chronic inflammatory reaction mediated by the microglia activation is an important link in the generation and development process of neurodegenerative diseases, and the inhibition of the microglia activation can become a new target point for drug discovery. LPS activates microglia to release NO, proinflammatory cytokines, active oxygen and the like. In the experiment, the anti-inflammatory activity of the novel phenanthrene compounds 1,2 and 3 obtained from Stephania sinica Diels is evaluated by establishing a screening model for abnormal activation of BV2 microglia activated by in vitro LPS and taking NO released by activated microglia as an index.
(2) The experimental method comprises the following steps:
culture of mouse microglia line BV2
All glassware and metal instruments (culture bottles, pipettes, solution bottles, etc.) used in cell culture and model building were autoclaved at 121 ℃ for 30min to completely remove the contaminated LPS. A cell culture solution containing 10% fetal calf serum was prepared on the basis of DMDM medium. Microglia at about 2 × 105cells/ml at 5% CO2And subculturing in a culture bottle at 37 ℃, wherein the adherent cells account for about 70-80% of the bottom area of the culture bottle by the third day, digesting the adherent cells by pancreatin, and subculturing to another culture bottle. BV-2 after frozen and thawed in an ultra-low temperature refrigerator at minus 80 ℃ is taken as the first generation, and BV-2 cells of 3 rd to 8 th generations are selected for experiments.
② process for preparing medicine
All three compounds were in powder form and dissolved in DMSO. The stock solution was prepared at a concentration of 100mM and stored at-20 ℃. It was diluted with DMEM medium at the time of use to 100. mu.M, 30. mu.M, 10. mu.M and 1. mu.M in this order. The final concentration of DMSO is less than 1 ‰.
③ Griess method for detecting inhibition of compound to LPS activated microglia
Taking BV2 microglia in logarithmic growth phase, and adjusting the cell density to 0.2X 10 by using fresh DMEM medium containing 10% fetal calf serum5cells/ml, seeded in 96-well plates, 100. mu.l/well, 5% CO at 37 ℃2Culturing in the incubator. And replacing the cells with serum-free fresh culture solution after 24 hours of adherent culture, and simultaneously adding drugs. The 3 compounds were co-administered with LPS at 1,10, 30, 100. mu.M. Blank control was also set. LPS termination in each administration groupThe concentration was 100 ng/ml. Continuously culturing for 24h after adding medicine into cells, collecting supernatant, and detecting NO in the supernatant by Griess colorimetric method2-And (4) content.
MTT method for detecting influence of compound on survival rate of microglia cell
Taking BV2 microglia cultured in logarithmic growth phase, and adjusting the cell density to 2 x 10 by using fresh DMEM medium containing 10% fetal calf serum5cells/ml, seeded in 96-well plates, 100. mu.l/well, 5% CO at 37 ℃2Culturing in the incubator. After the cells are cultured for 24 hours adherent, the cells are changed into fresh culture solution, and meanwhile, the cells are treated by adding medicine. The 3 compounds were co-administered with LPS at 1,10, 30, 100. mu.M. Blank control was also set. The final concentration of LPS in each administration group was 100 ng/ml. After adding the drug, the cells were cultured for 24 hours, MTT solution (10. mu.l/well) was added to the cell fluid, the cells were incubated with 0.25mg/ml MTT at 37 ℃ for 3 hours, the culture fluid was aspirated, and 150. mu.l of DMSO solution was added to determine the OD value of the optical density. And (3) processing data, namely processing the data by using corresponding software of a microplate reader, calculating an average value of OD values of 3 holes of each sample, and calculating the cell survival rate (CV%) by using the average value according to the following formula.
Percent cell survival%
Fifthly, statistical method
All data were examined using the SPSS (27.0) statistical software package. Results are expressed as mean ± standard error, and the global differences were evaluated, and the means between groups was analyzed by One-Way ANOVA analysis for homogeneity of variance and by Dunnett's test analysis for comparison between groups. The multiple sample homogeneity of variance test was conducted using a Leven test, where the variances were uniform when p >0.05, the differences in mean among the groups were tested using Dunnett's two-sided T, and the differences in mean among the groups were tested using Dunnett T3 when p <0.05 and the variances were not uniform.
⑥IC50Is calculated by
Calculating IC by nonlinear regression fitting of parameters such as each dosage and inhibition rate50. The experimental results are as follows: see tables 3-1, 3-2.
TABLE 3-1 Effect of New phenanthrene Compounds 1,2,3 on LPS-activated BV-2 microglia survival (%) (Mean. + -. SE)
TABLE 3-2 Effect of New phenanthrenes 1,2,3 on nitric oxide (%) Release from LPS-activated BV-2 microglia (Mean. + -. SE)
Significance is shown in<0.05,**P<0.01,***P<0.001 compared to LPS-induced group;###P<0.001 compared to the control group.
According to experimental results, the novel phenanthrene compounds 1,2 and 3 prepared from the Stephania delavayi Diels root can obviously inhibit NO release of excessive activated BV2 cells induced by LPS under the concentration of not influencing the survival rate of the microglia BV2, thereby playing roles in preventing and treating neuroinflammation.
Claims (10)
1. The phenanthrene compound and pharmaceutically acceptable salt, isomer or solvate thereof are characterized by having the following structural general formula:
R2r adjacent to it1Or R3Forming an oxazole ring which may be substituted by R7The structure of the compound is as follows:
R7is C1-C4 alkyl, C1-C4 alkoxy, hydroxyl;
R3is C1-C4 alkoxy and hydroxyl;
R4and R5Forming a methylenedioxy group;
R6represents a C1-C4 aldehyde group.
2. The phenanthrene compounds, and pharmaceutically acceptable salts, isomers, or solvates thereof, of claim 1, characterized in that it is one or more of the following structures,
3. the process for the preparation of the phenanthrene compounds and pharmaceutically acceptable salts thereof according to claim 2, comprising the steps of:
(1) extracting Stephania sinica (Stephania sinia H.S.Lo) tuberous root with ethanol, and recovering the extractive solution to obtain crude extract;
(2) dissolving the ethanol crude extract obtained in the step (1) by using methanol, mixing the dissolved ethanol crude extract with macroporous resin, sequentially eluting by using an ethanol-water-formic acid system with a ratio of 20:75: 5-35: 60:5 and an ethanol-water-triethylamine system with a ratio of 65:30: 5-80: 15:5, and recovering to obtain an ethanol-water-triethylamine eluate;
(3) separating the ethanol-water-triethylamine eluate obtained in the step (2) by silica gel column chromatography, and performing gradient elution by using a mixed solvent of petroleum ether and ethyl acetate, a mixed solvent of petroleum ether and acetone, a mixed solvent of chloroform and acetone, a mixed solvent of dichloromethane and acetone, a mixed solvent of chloroform and methanol, and a mixed solvent of dichloromethane and methanol;
(4) separating the flow obtained in the step (3) by ODS chromatography, and carrying out gradient elution by using a mixed solvent of methanol and water or a mixed solvent of acetonitrile and water as a mobile phase;
(5) methanol and water obtained in the step (4) 3: 7-9: 1. acetonitrile and water 1: 9-7: fractions 3 were further separated by preparative HPLC-UV chromatography to give compounds 1,2 and 3.
4. The method of claim 3, wherein: the extraction method in the step (1) is heating reflux extraction or heating ultrasonic extraction for 1-3 times, the volume concentration of ethanol is 50-90%, and the extraction method is as follows: the weight-volume ratio of the ethanol is 1: 8-1: 15 g/mL.
5. The method of claim 3, wherein: and (3) dissolving the crude extract by using ethanol in the macroporous resin adsorption elution method in the step (2), mixing the crude extract according to the mass ratio of the macroporous resin to the mass ratio of 3: 1-6: 1, drying at room temperature, and then sequentially eluting 8-12 column volumes by using ethanol-water-formic acid and ethanol-water-triethylamine.
6. The method of claim 3, wherein: in the step (3), the volume ratio of the mixed solvent of the eluting solvent petroleum ether and ethyl acetate to the mixed solvent of the petroleum ether and acetone is 100: 8-1: 1, preferably 100: 8-100: 20; the volume ratio of the mixed solvent of dichloromethane and acetone, the mixed solvent of chloroform and acetone, the mixed solvent of dichloromethane and methanol, or the mixed solvent of chloroform and methanol is 100: 1-100: 10, preferably 100:1 to 100: 8.
7. The method of claim 3, wherein: the volume ratio of the methanol and water mixed solvent in the step (4) is 1: 9-9: 1, preferably 3: 7-9: 1; the volume ratio of the acetonitrile-water mixed solvent is 1: 9-9: 1, preferably 1: 9-7: 3.
8. the method of claim 3, wherein: the mobile phase of the preparative HPLC-UV chromatogram in the step (5) is prepared from the following components in a volume ratio of 4: 6-8: 2, a methanol-water mixed solvent; or the volume ratio is 2: 8-6: 4 acetonitrile-water mixed solvent.
9. A pharmaceutical composition comprising the phenanthrene compound of claim 1 or 2, and a pharmaceutically acceptable salt, isomer, or solvate thereof, and a pharmaceutically acceptable carrier.
10. Use of the phenanthrene compound of claim 1 or 2 and a pharmaceutically acceptable salt, isomer or solvate thereof or the pharmaceutical composition of claim 9 in the preparation of a medicament for preventing or treating neurodegenerative diseases.
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