Flavonoid compound and preparation method and application thereof
阅读说明：本技术 一种黄酮类化合物及其制备方法和用途 (Flavonoid compound and preparation method and application thereof ) 是由 李青山 张朕 韩际宏 段亚君 龚克 于 2021-07-07 设计创作，主要内容包括：本发明公开了一种黄酮类化合物及其制备方法和用途,其中黄酮类化合物包括黄酮类化合物FLM-1-24、黄酮衍生物FLM-25-40和异黄酮衍生物FLM-41-48。本发明黄酮类化合物可用于制备对人肝癌细胞系Huh7细胞的Nogo-B表达具有调节作用的调节剂,具体地说,本发明黄酮类化合物对人肝癌细胞系Huh7细胞的Nogo-B表达具有良好的抑制活性。(The invention discloses a flavonoid compound and a preparation method and application thereof, wherein the flavonoid compound comprises a flavonoid compound FLM _1-24, a flavone derivative FLM _25-40 and an isoflavone derivative FLM _ 41-48. The flavonoid compound can be used for preparing a regulator with a regulating effect on Nogo-B expression of human hepatoma cell line Huh7 cells, and particularly has good inhibitory activity on Nogo-B expression of human hepatoma cell line Huh7 cells.)
1. A flavonoid compound characterized by:
the flavonoid compounds comprise flavone derivatives FLM _25-40 and isoflavone derivatives FLM _ 41-48;
the general structural formula of the flavonoid derivative FLM _25-40 is shown as follows:
wherein R is1Selected from 5-OC2H5、5-OC4H9、5-OC2H4CH＝CH2、5-OCH2CH(CH3)2、5-OCH(CH3)2、5-OCOCH3、5-OCOC2H5、5-OCOC3H7、6-OC2H5、6-OC4H9、6-OC2H4CH＝CH2、6-OCH2CH(CH3)2、6-OCH(CH3)2、6-OCOCH3、6-OCOC2H5Or 6-OCOC3H7；
The structural general formula of the isoflavone derivative FLM _41-48 is shown as follows:
wherein R is2Is selected from C2H5、C4H9、C2H4CH＝CH2、CH2CH(CH3)2、CH(CH3)2、COCH3、COC2H5Or COC3H7。
2. The flavonoid compound according to claim 1, characterized in that said flavonoid derivative FLM _25-40 has the structure:
3. the flavonoid compound according to claim 1, characterized in that said isoflavone derivative FLM _41-48 has the structure:
4. a method for preparing flavonoid compounds according to claim 1, characterized by comprising the steps of:
step 1: adding 2, 6-dihydroxyacetophenone and anhydrous potassium carbonate into a reaction bottle, adding acetone as a solvent, heating to 55 ℃, carrying out reflux reaction, slowly dropwise adding benzoyl chloride under stirring, and carrying out reflux reaction for 12 hours after dropwise adding; cooling, suction filtering, washing the filter cake with a little acetone, collecting the filtrate and vacuum concentrating, adding the concentrated solid into a flask containing 10% acetic acid, and stirring thoroughly, wherein a large amount of CO is in the flask2Gas is discharged, yellow solid precipitates out, and the stirring is continued until the precipitation is complete; and (3) carrying out suction filtration, washing a filter cake with water, drying, and then recrystallizing with acetone to obtain an intermediate A, wherein the structural formula is as follows:
step 2: adding the intermediate A and concentrated sulfuric acid into a reaction bottle, and stirring for reaction for 4 hours under ice-bath cooling; after the reaction is finished, pouring the reaction liquid into ice water, separating out white solid, performing suction filtration, and collecting a filter cake; then adding the filter cake into 5% potassium carbonate solution, heating to 120 ℃ and boiling for 1 hour; cooling, separating out a solid, performing suction filtration, collecting a filter cake, dissolving the filter cake with dichloromethane, performing column chromatography separation, and recrystallizing the obtained crude product with ethanol to obtain an intermediate B, wherein the structural formula is shown as follows:
and step 3: placing the intermediate B in a reaction bottle, taking dichloromethane as a solvent, adding potassium carbonate, adding substituted alkane C1-8, reacting at normal temperature, detecting the reaction progress by TLC, performing silica gel column chromatography separation after the reaction is finished, and recrystallizing the separated crude product by ethanol to obtain a target compound FLM _ 25-32;
the structural formula of the substituted alkane C1-8 is as follows:
I-R or Br-R
Wherein R is selected from OC2H5、OC4H9、OC2H4CH＝CH2、OCH2CH(CH3)2、OCH(CH3)2、OCOCH3、OCOC2H5Or OCOC3H7；
And 4, step 4: placing 6-hydroxyflavone in a reaction bottle, taking dichloromethane as a solvent, adding potassium carbonate, adding substituted alkane C1-8, reacting at normal temperature, detecting the reaction progress by TLC (thin layer chromatography), performing column chromatography separation after the reaction is finished, and recrystallizing the separated crude product by ethanol to obtain a target compound FLM _ 33-40;
the structural formula of the substituted alkane C1-8 is as follows:
I-R or Br-R
Wherein R is selected from OC2H5、OC4H9、OC2H4CH＝CH2、OCH2CH(CH3)2、OCH(CH3)2、OCOCH3、OCOC2H5Or OCOC3H7；
And 5: placing 7-hydroxy-3- (4-methoxyphenyl) -4-hydro-chromen-4-one in a reaction bottle, taking dichloromethane as a solvent, adding potassium carbonate, adding substituted alkane C1-8, reacting at normal temperature, detecting the reaction progress by TLC, performing column chromatography separation after the reaction is finished, and recrystallizing the separated crude product by ethanol to obtain a target compound FLM _ 41-48;
the structural formula of the iodine-substituted or bromine-substituted alkane C1-8 is as follows:
I-R or Br-R
Wherein R is selected from OC2H5、OC4H9、OC2H4CH＝CH2、OCH2CH(CH3)2、OCH(CH3)2、OCOCH3、OCOC2H5Or OCOC3H7。
5. The method of claim 4, wherein:
in the step 1, the mass ratio of the 2, 6-dihydroxyacetophenone to the anhydrous potassium carbonate is 1:3, and the mass ratio of the 2, 6-dihydroxyacetophenone to the benzoyl chloride is 1: 2.
6. The method of claim 4, wherein:
in the step 3, the mass ratio of the intermediate B to the substituted alkane C1-8 is 1: 1.5.
7. The method of claim 4, wherein:
in the step 4, the mass ratio of the 6-hydroxyflavone to the substituted alkane C1-8 is 1: 1.5.
8. The method of claim 4, wherein:
in step 5, the mass ratio of the 7-hydroxy-3- (4-methoxyphenyl) -4-hydro-chromen-4-one to the substituted alkane C1-8 is 1: 1.5.
9. The application of the flavonoid compound is characterized in that:
the flavonoid compound is used for preparing a regulator having a regulating effect on Nogo-B expression of human hepatoma cell line Huh7 cells;
the flavonoid compounds comprise 5-hydroxyflavone FLM _1, 6-hydroxyflavone FLM _2, 7-hydroxyflavone FLM _3, 3-methoxyflavone FLM _4, 5-methoxyflavone FLM _5, 6-methoxyflavone FLM _6, 7-methoxyflavone FLM _7, 3-methyl-8-carboxyflavone FLM _8, 6-methylflavone FLM _9, 6-aminoflavone FLM _10, 7-aminoflavone FLM _11, 3 ', 5, 7-trihydroxy-4-methoxyflavone FLM _12, resveratrol FLM _13, flavonol FLM _14, 7-hydroxyisoflavone FLM _15, 7-isopropoxyisoflavone FLM _16, glycinin FLM _17, 3 ', 7-dimethoxyisoflavone FLM _18, 3 ', 6, 7-trimethoxyisoflavone FLM _19, and beta-hydroxyisoflavone FLM _15, 3 ', 7-dihydroxyisoflavone FLM _20, 3 ' -methoxy-7-hydroxyisoflavone FLM _21, 3 ' -nitro-7-hydroxyisoflavone FLM _22, 3 ' -methoxy-5, 7-dihydroxyisoflavone FLM _23, 3 ', 5, 7-trihydroxyisoflavone FLM _24, flavone derivatives FLM _25-40 and isoflavone derivatives FLM _ 41-48.
10. Use according to claim 9, characterized in that:
the flavonoid compound has a remarkable inhibition effect on the expression of Nogo-B of Huh7 cells.
Technical Field
The invention relates to a flavonoid compound and a preparation method and application thereof. The flavonoid compound disclosed by the invention has good inhibitory activity on Nogo-B expression of human liver cancer cell line Huh7 cells.
Background
Flavonoid (flavanon)oids) refers to the generic name given to a series of compounds in which two benzene rings are connected to one another via three carbon atoms, i.e. having C6-C3-C6Generic term for a class of compounds of structure. It is widely existed in plants in nature, belongs to plant secondary metabolites, and exists in the form of glycoside or carbon sugar combined with sugar in most parts in plant body, or exists in free form. The structural types can be divided into: flavones, isoflavones, flavonols, flavanones, biflavones, and the like.
Researches show that the flavonoid compounds, derivatives and analogues thereof have multiple biological functions. The flavonoids compounds have the effects of improving the oxidation resistance and free radical scavenging capability of animal organisms, and have good effects on preventing and treating cardiovascular diseases, such as preventing arteriosclerosis, reducing blood fat and cholesterol, reducing blood sugar, relaxing blood vessels, improving vascular permeability, reducing the incidence rate of coronary heart disease and the like. At the same time, almost all flavonoids have varying degrees of bacteriostatic activity against many microorganisms. The flavonoids compounds can also induce apoptosis of cancer cells and tumor cells, play a role in resisting cancers and tumors and play a role in delaying apoptosis of normal tissue cells. The flavonoids can improve immunity and promote health.
Nogo-B is one of the members of the Nogo superfamily of reticulins. The Nogo family is, in addition to Nogo-B, a member of Nogo-A, Nogo-C and the like. There is a clear specificity in the distribution of these members in tissues in vivo: Nogo-A is highly expressed in the central nervous system, Nogo-C is highly expressed in skeletal muscle, and Nogo-B is expressed in most tissues, such as liver, blood vessels, heart, kidney, nervous system, etc. Nogo-B is functionally found as an Endoplasmic Reticulum (ER) resident protein that regulates the structure and function of the ER. Nogo-B is involved in various pathological processes in the liver, and is involved in hepatocyte proliferation and liver regeneration, alcoholic liver disease, hepatic fibrosis and end-stage cirrhosis thereof, and the like. In 2020, research finds that the deficiency of Nogo-B expression can obviously relieve the symptoms of cholestatic liver diseases; Nogo-B participates in the process of hepatic glycolipid metabolism by regulating carbohydrate acting element binding protein (ChREBP) and insulin pathway, reduces Nogo-B expression, can reduce inflammation, apoptosis and endoplasmic reticulum stress phenomena induced by high glucose or high fructose food, and activates energy metabolism process, thereby effectively antagonizing metabolic disorder induced by high glucose or fructose food, so Nogo-B can be an important target for potential treatment of glycolipid metabolic disorder and can be used as a target gene for drug design and screening.
The dual-luciferase reporter gene reporter system has the advantages of high sensitivity, wide dynamic range, flexible application and the like, and is widely applied to the research fields of gene regulation, non-coding RNA targeted interaction and the like. Usually, the transcription regulation and control element of the target gene is constructed into an expression vector with luciferase (Fireforciferase) to construct a reporter gene plasmid, so that the sequence regulates and controls the transcription expression of the Fireforciferase. Then the reporter gene plasmid is transfected into the cells, the cells are lysed after different treatments, and a substrate fluorescein (luciferin) is added, so that the Firefly luciferase can catalyze the luciferin to emit fluorescence (the strongest wavelength is about 560 nm). The influence of different processing groups on the transcription regulation and control original can be judged according to the detected fluorescence value. In order to avoid errors caused by the difference in efficiency when the plasmid transfects cells, the reporter plasmid of Renilla luciferase is usually transferred as an internal reference (the strongest wavelength is around 465 nm), and by this method, the experimental accuracy weakened by the inherent variation factors can be reduced. For example, differences in the number and viability of cultured cells, efficiency of cell transfection and lysis. Usually, an experimental report gene is coupled to a regulated promoter, and the structure and physiological basis of the regulated gene are researched, so that the transcriptional activation effect of a drug on a related gene can be judged from the level of the promoter by comparing the fluorescence value of the reporter gene coupled with the promoter after different drug treatments, and the drug for promoting or inhibiting the expression of a target gene is screened.
Disclosure of Invention
The invention aims to provide a flavonoid compound and a preparation method and application thereof. The flavonoid compound disclosed by the invention has good inhibitory activity on Nogo-B expression of human liver cancer cell line Huh7 cells.
The Huh7 cell is a well-differentiated hepatocyte-derived cell cancer cell line and is often used for in vitro research of hepatocellular carcinoma pathogenesis, liver metabolic diseases, drug metabolism, in vitro hepatotoxicity experiments and the like. Meanwhile, Huh7 cells were able to highly express Nogo-B, which is not expressed in normal parenchymal hepatocytes, in order to confirm the inhibitory activity of flavonoids against the expression of Nogo-B in hepatocytes.
The flavonoid compounds comprise 5-hydroxyflavone (FLM _1), 6-hydroxyflavone (FLM _2), 7-hydroxyflavone (FLM _3), 3-methoxyflavone (FLM _4), 5-methoxyflavone (FLM _5), 6-methoxyflavone (FLM _6), 7-methoxyflavone (FLM _7), 3-methyl-8-carboxyflavone (FLM _8), 6-methylflavone (FLM _9), 6-aminoflavone (FLM _10), 7-aminoflavone (FLM _11), 3', 5, 7-trihydroxy-4-methoxyflavone (FLM _12), resveratrol (FLM _13), flavonol (FLM _14), 7-hydroxyisoflavone (FLM _15), 7-isopropoxyisoflavone (FLM _16), glycitin (FLM _17), 3 ', 7-dimethoxyisoflavone (FLM _18), 3 ', 6, 7-trimethoxyisoflavone (FLM _19), 3 ', 7-dihydroxyisoflavone (FLM _20), 3 ' -methoxy-7-hydroxyisoflavone (FLM _21), 3 ' -nitro-7-hydroxyisoflavone (FLM _22), 3 ' -methoxy-5, 7-dihydroxyisoflavone (FLM _23), 3 ', 5, 7-trihydroxyisoflavone (FLM _24), flavone derivatives (FLM _25-40), isoflavone derivatives (FLM _ 41-48). The compounds FLM _1-24 are available from MCE (MedChemExpress) official or research reagents, and their structures are correct.
The flavone derivative (FLM _25-40) in the invention has the following general formula:
wherein R is1Selected from 5-OC2H5、5-OC4H9、5-OC2H4CH＝CH2、5-OCH2CH(CH3)2、5-OCH(CH3)2、5-OCOCH3、5-OCOC2H5、5-OCOC3H7、6-OC2H5、6-OC4H9、6-OC2H4CH＝CH2、6-OCH2CH(CH3)2、6-OCH(CH3)2、6-OCOCH3、6-OCOC2H5Or 6-OCOC3H7。
Specifically, the structural formula of the flavone derivative (FLM _25-40) is preferably as follows:
isoflavone derivatives (FLM _41-48) of the present invention have the following general formula:
wherein R is2Is selected from C2H5、C4H9、C2H4CH＝CH2、CH2CH(CH3)2、CH(CH3)2、COCH3、COC2H5Or COC3H7。
Specifically, the structural formula of the isoflavone derivative (FLM _41-48) is preferably as follows:
the preparation method of the flavonoid derivative comprises the following steps:
step 1: adding 2, 6-dihydroxy acetophenone (20mmol) and anhydrous potassium carbonate (140mmol) into a reaction bottle, adding acetone (120mL) as a solvent, heating to 55 ℃, carrying out reflux reaction, slowly dropwise adding benzoyl chloride (40mmol) under stirring, and carrying out reflux reaction for 12 hours after dropwise adding; cooling, suction filtering, washing filter cake with small amount of acetone, collecting filtrate and filteringIt was concentrated in vacuo, and the concentrated solid was added to a flask containing 10% acetic acid (300mL) and stirred well, in which case a large amount of CO was present2Gas is discharged, yellow solid precipitates out, and stirring is continued for 2 hours until the precipitates are complete; suction filtering, washing the filter cake with water, drying and recrystallizing with acetone to obtain intermediate A, namely 3-hydroxy-2- (3-oxo-3-phenylpropionyl) benzoic acid phenyl ester which is yellow solid. In the step 1, the dripping speed of benzoyl chloride cannot be too high, and the benzoyl chloride is slowly dripped, wherein the dripping speed is controlled to be 2 mL/min.
In step 1, the percentage of acetic acid is not too high, and 10% is the best.
Step 2: adding the intermediate A obtained in the step 1 and concentrated sulfuric acid (30mL) into a reaction bottle, and stirring for reacting for 4 hours under the cooling of an ice bath; after the reaction is finished, pouring the reaction solution into a large amount of ice water, placing the ice water in a low-temperature environment, separating out white solid, performing suction filtration, and collecting a filter cake; the filter cake was then added to a 5% potassium carbonate solution (130mL) and boiled at 120 ℃ for 1 hour; cooling, precipitating solid, suction filtering, collecting filter cake, dissolving the filter cake with dichloromethane, then performing column chromatography separation with a proper eluent (ethyl acetate: petroleum ether ═ 1: 15, v/v), and recrystallizing the separated crude product with ethanol to obtain intermediate B-5-hydroxyflavone as yellow solid.
And step 3: putting the intermediate B (2mmol) into a reaction bottle, taking dichloromethane (50mL) as a solvent, adding potassium carbonate (6mmol), adding substituted alkane C1-8(3mmol), reacting at normal temperature, detecting the reaction progress by TLC, completing the reaction after 4-6 hours of reaction, performing column chromatography separation by using an eluent (the eluent is determined according to the polarity of different substituents, ethyl acetate: petroleum ether ═ 1:8-1:4, v/v), and recrystallizing the separated crude product by using ethanol to obtain the target compound FLM _ 25-32.
The structural formula of the substituted alkane C1-8 is as follows:
I-R or Br-R,
wherein R is selected from OC2H5、OC4H9、OC2H4CH＝CH2、OCH2CH(CH3)2、OCH(CH3)2、OCOCH3、OCOC2H5Or OCOC3H7。
And 4, step 4: placing 6-hydroxyflavone (D) (2mmol) in a reaction bottle, using dichloromethane (50mL) as a solvent, adding potassium carbonate (6mmol), adding substituted alkane C1-8(3mmol), reacting at normal temperature, detecting the reaction progress by TLC, completing the reaction after 4-6 hours of reaction, performing column chromatography separation by using an eluent (the eluent is determined according to the polarity of different substituents, ethyl acetate: petroleum ether ═ 1:8-1:4, v/v), and recrystallizing the separated crude product by using ethanol to obtain the target compound FLM _ 33-40.
The structural formula of the substituted alkane C1-8 is as follows:
I-R or Br-R
Wherein R is selected from OC2H5、OC4H9、OC2H4CH＝CH2、OCH2CH(CH3)2、OCH(CH3)2、OCOCH3、OCOC2H5Or OCOC3H7。
The structural formula of the 6-hydroxyflavone (D) is as follows:
and 5: placing 7-hydroxy-3- (4-methoxyphenyl) -4-hydro-chromen-4-one (E) (2mmol) in a reaction bottle, taking dichloromethane (50mL) as a solvent, adding potassium carbonate (6mmol), adding substituted alkane C1-8(3mmol), reacting at normal temperature, detecting the reaction progress by TLC, after reacting for 4-6 hours, completing the reaction, performing column chromatography separation by using an eluent (the eluent is determined according to the polarity of different substituents, ethyl acetate: petroleum ether ═ 1:8-1:4, v/v), and recrystallizing the separated crude product by using ethanol to obtain the target compound FLM _ 41-48.
The structural formula of the substituted alkane C1-8 is as follows:
I-R or Br-R
Wherein R is selected from OC2H5、OC4H9、OC2H4CH＝CH2、OCH2CH(CH3)2、OCH(CH3)2、OCOCH3、OCOC2H5Or OCOC3H7。
The structural formula of the 7-hydroxy-3- (4-methoxyphenyl) -4-hydro-chromen-4-one (E) is as follows:
in the step 1, the mass ratio of the 2, 6-dihydroxyacetophenone to the anhydrous potassium carbonate is 1: 3.
In the step 1, the mass ratio of the 2, 6-dihydroxyacetophenone to the benzoyl chloride is 1: 2.
In the step 3, the mass ratio of the intermediate B to the substituted alkane C1-8 is 1: 1.5.
In the step 4, the mass ratio of the 6-hydroxyflavone (D) to the substituted alkane C1-8 is 1: 1.5.
In step 5, the mass ratio of the 7-hydroxy-3- (4-methoxyphenyl) -4-hydro-chromen-4-one (E) to the substituted alkane C1-8 is 1: 1.5.
In steps 3, 4 and 5, ethanol solvent is selected for recrystallization, and a small amount of 1-2mL ethyl acetate can be added for dissolution assistance.
The flavonoid compound disclosed by the invention is used for preparing a regulation inhibitor with inhibitory activity on Nogo-B expression of human hepatoma cell line Huh7 cells.
The flavonoid compound has a regulating effect on the expression of Nogo-B of Huh7 cells. Specifically, the flavonoid compound has a remarkable inhibition effect on the expression of Nogo-B of Huh7 cells.
Drawings
FIG. 1 is a graph of the effect of flavonoid FLM _1-24 on Nogo-B transcriptional activity of Huh7 cells.
FIG. 2 is a graph of the effect of flavonoids FLM _25-48 on Nogo-B transcriptional activity of Huh7 cells.
Detailed Description
The present invention is further illustrated in detail by the following examples, but it should be noted that the scope of the present invention is not limited by these examples at all.
Example 1: preparation of 5-ethoxy-2-phenyl-4H-chromen-4-one (compound FLM. RTM. 25)
1. A250 mL round bottom flask was taken, 2, 6-dihydroxy acetophenone (3.04g, 20mmol) and anhydrous potassium carbonate (19.34g, 140mmol) were dissolved in 120mL acetone solvent, heated to 55 deg.C under reflux and benzoyl chloride (5.62g, 40mmol) was slowly added dropwise with stirring and allowed to reflux for 12 h. Then cooled, filtered with suction, the filter cake washed with a little acetone, the filtrate obtained is collected and concentrated in vacuo. The concentrated solid was then added to a flask containing 10% acetic acid (300mL) and stirred well at ambient temperature, in which case a large amount of CO was present2Gas is released, yellow solid is precipitated, and stirring is continued for 2 hours until the precipitation is complete. Suction filtration, washing of the filter cake with water, drying and recrystallization with acetone gave a yellow solid intermediate, phenyl 3-hydroxy-2- (3-oxo-3-phenylpropionyl) benzoate (A).
2. A150 mL round-bottomed flask was taken, and the intermediate 3-hydroxy-2- (3-oxo-3-phenylpropionyl) benzoic acid phenyl ester (A) was dissolved in concentrated sulfuric acid (30mL) and allowed to react for 4 hours with stirring under cooling in an ice bath. After the reaction is finished, pouring the reaction solution into a large amount of ice water, placing the ice water in a low-temperature environment, standing the ice water, separating out white solid, performing suction filtration, and collecting a filter cake. The filter cake was then added to a 5% potassium carbonate solution (130mL) and boiled for 1 hour with heating to 120 ℃. Cooling, precipitating solid, suction filtering, collecting filter cake, dissolving the filter cake with dichloromethane, then performing column chromatography separation with a proper eluent (ethyl acetate: petroleum ether ═ 1: 15, v/v), and recrystallizing the separated crude product with ethanol to obtain a yellow solid intermediate 5-hydroxyflavone (B).
3. A150 mL round bottom flask was taken, intermediate 5-hydroxyflavone (B) (0.48g, 2mmol) was dissolved in dichloromethane (50mL) solvent and potassium carbonate (0.83g, 6mmol) was added followed by iodoethane (0.47g, 3mmol) and the reaction was carried out at ambient temperature and checked by TLC for progress, after about 4-6 hours, the reaction was complete. Column chromatography was performed with an appropriate eluent (ethyl acetate: petroleum ether ═ 1: 5, v/v), and the separated crude product was recrystallized from ethanol to give the objective compound FLM _ 25. Product FLM 25 was a white solid with a yield of 32.6% and a melting point of 99-102 ℃.1H NMR(400MHz,CDCl3)δ(ppm)7.92–7.89(m,2H),7.58–7.49(m,4H),7.13(dd,J＝8.4,1.0Hz,1H),6.83(dd,J＝8.3,0.9Hz,1H),6.72(s,1H),4.22(q,J＝7.0Hz,2H),1.59(t,J＝7.0Hz,3H).HR-MS(ESI):calcd for C17H14O3,[M+H]+,267.1021；found 267.1010.
Example 2: preparation of 5-butoxy-2-phenyl-4H-chromen-4-one (compound FLM. RTM. 26)
The preparation method is the same as example 1. Iodobutane was used instead of iodoethane to give a pale yellow solid in 29.7% yield, m.p. 92-95 ℃.1H NMR(400MHz,CDCl3)δ(ppm)7.96–7.86(m,2H),7.64–7.47(m,4H),7.12(dd,J＝8.4,0.9Hz,1H),6.83(dd,J＝8.3,1.0Hz,1H),6.71(s,1H),4.14(t,J＝6.6Hz,2H),2.01–1.89(m,2H),1.63(m,J＝14.8,7.4Hz,2H),1.03(t,J＝7.4Hz,3H).HR-MS(ESI):calcd for C19H18O3,[M+H]+,295.1334；found 295.1313.
Example 3: preparation of 5- (but-3-en-1-yloxy) -2-phenyl-4H-chromen-4-one (Compound FLM-27)
The preparation method is the same as example 1. Iodobutene was used instead of iodoethane to give a pale yellow solid in 34.2% yield, mp 94-97 ℃.1H NMR(400MHz,CDCl3)δ(ppm)7.92(dd,J＝6.8,2.9Hz,2H),7.64–7.44(m,4H),7.15(d,J＝8.4Hz,1H),6.83(d,J＝8.2Hz,1H),6.72(s,1H),6.06(m,J＝17.1,10.3,6.8Hz,1H),5.26(dd,J＝17.1,1.9Hz,1H),5.17(dd,J＝10.4,1.8Hz,1H),4.18(t,J＝6.9Hz,2H),2.74(q,J＝6.9Hz,2H).HR-MS(ESI):calcd for C19H16O3,[M+H]+,293.1178；found 293.1154.
Example 4: preparation of 5-isobutoxy-2-phenyl-4H-chromen-4-one (Compound FLM-28)
The preparation method is the same as example 1. 1-iodo-2-methylpropane was used instead of iodoethane to give a pale yellow solid with a yield of 31.6% and a melting point of 76-79 ℃.1H NMR(400MHz,CDCl3)δ(ppm)7.98–7.86(m,2H),7.60–7.47(m,4H),7.12(dd,J＝8.4,1.0Hz,1H),6.81(dd,J＝8.3,1.0Hz,1H),6.70(s,1H),3.89(d,J＝6.7Hz,2H),2.31(m,J＝13.4,6.7Hz,1H),1.15(d,J＝6.8Hz,6H).HR-MS(ESI):calcd for C19H18O3,[M+H]+,295.1334；found 295.1330.
Example 5: preparation of 5-isopropoxy-2-phenyl-4H-chromen-4-one (compound FLM-29)
The preparation method is the same as example 1. Isopropane iodide was used instead of ethyl iodide to give a pale yellow solid with a yield of 33.6% and a melting point of 85-87 ℃.1H NMR(400MHz,CDCl3)δ(ppm)8.00–7.81(m,2H),7.53(m,J＝7.2,4.9,2.2Hz,4H),7.13(d,J＝8.1Hz,1H),6.86(d,J＝8.2Hz,1H),6.69(d,J＝2.1Hz,1H),4.66(m,J＝6.0,2.1Hz,1H),1.48(dd,J＝6.0,2.1Hz,6H).HR-MS(ESI):calcd for C18H16O3,[M+H]+,281.1178；found 281.1118.
Example 6: preparation of 4-oxo-2-phenyl-4H-chromen-5-yl acetate (Compound FLM-30)
The preparation method is the same as example 1. Acetyl chloride was used instead of iodoethane to give a white solid with a yield of 31.3% and a melting point of 130-.1H NMR(400MHz,CDCl3)δ(ppm)7.96–7.86(m,2H),7.69(t,J＝8.2Hz,1H),7.64–7.47(m,4H),7.06(dd,J＝7.9,1.1Hz,1H),6.71(s,1H),2.48(s,3H).HR-MS(ESI):calcd for C17H12O4,[M+H]+,281.0814；found 281.0791.
Example 7: preparation of 4-oxo-2-phenyl-4H-chromen-5-yl propionate (Compound FLM-31)
The preparation method is the same as example 1. Propionyl chloride was used instead of iodoethane to give a white solid in 29.3% yield, mp 124-.1H NMR(400MHz,CDCl3)δ(ppm)7.97–7.85(m,2H),7.68(t,J＝8.2Hz,1H),7.61–7.45(m,4H),7.05(dd,J＝7.8,1.1Hz,1H),6.70(s,1H),2.82(q,J＝7.5Hz,2H),1.36(t,J＝7.5Hz,3H).HR-MS(ESI):calcd for C18H14O4,[M+H]+,295.0970；found 295.0963.
Example 8: preparation of 4-oxo-2-phenyl-4H-chromen-5-ylbutanoate (Compound FLM-32)
The preparation method is the same as example 1. Butyryl chloride was used instead of iodoethane to give a white solid in 33.6% yieldPoint 117-.1HNMR(400MHz,CDCl3)δ(ppm)7.91–7.87(m,2H),7.67(dd,J＝8.5,7.9Hz,1H),7.60–7.45(m,4H),7.04(dd,J＝7.9,1.1Hz,1H),6.69(s,1H),2.77(t,J＝7.5Hz,2H),1.89(m,J＝7.4Hz,2H),1.11(t,J＝7.4Hz,3H).HR-MS(ESI):calcd for C19H16O4,[M+H]+,309.1127；found 309.1107.
Example 9: preparation of 6-ethoxy-2-phenyl-4H-chromen-4-one (compound FLM-33)
1. A150 mL round bottom flask was taken, 6-hydroxyflavone (D) (0.48g, 2mmol) was dissolved in dichloromethane (50mL) solvent and potassium carbonate (0.83g, 6mmol) was added followed by iodoethane (0.47g, 3mmol) and the reaction was carried out at ambient temperature and checked by TLC for progress, after about 4-6 hours, the reaction was complete. Column chromatography was performed with an appropriate eluent (ethyl acetate: petroleum ether ═ 1: 6, v/v), and the separated crude product was recrystallized from ethanol to give the objective compound FLM _ 33. The product FLM-33 was a white solid with a yield of 30.5%, m.p. 126-129 ℃.1H NMR(600MHz,CDCl3)δ(ppm)7.95–7.89(m,2H),7.57(d,J＝3.1Hz,1H),7.55–7.47(m,4H),7.31–7.24(m,2H),6.81(s,1H),4.14(q,J＝7.0Hz,2H),1.45(t,J＝7.0Hz,3H).HR-MS(ESI):calcd for C17H14O3,[M+H]+,267.1021；found 267.1013.
Example 10: preparation of 6-butoxy-2-phenyl-4H-chromen-4-one (Compound FLM-34)
The preparation method is the same as example 9. Iodobutane was used instead of iodoethane to give a white solid in 29.6% yield, m.p. 84-87 ℃.1HNMR(600MHz,CDCl3)δ(ppm)7.93–7.88(m,2H),7.57(d,J＝3.1Hz,1H),7.55–7.46(m,4H),7.30–7.24(m,1H),6.80(s,1H),4.06(t,J＝6.5Hz,2H),1.84–1.75(m,2H),1.51(m,J＝7.4Hz,2H),0.98(t,J＝7.4Hz,3H).HR-MS(ESI):calcd for C19H18O3,[M+H]+,295.1334；found 295.1321.
Example 11: preparation of 6- (but-3-en-1-yloxy) -2-phenyl-4H-chromen-4-one (Compound FLM-35)
The preparation method is the same as example 9. Iodobutene was used instead of iodoethane to give a pale yellow solid with a yield of 32.6% and a melting point of 98-101 ℃.1HNMR(400MHz,CDCl3)δ(ppm)7.94(m,J＝6.7,2.8,2.2Hz,2H),7.61(d,J＝3.1Hz,1H),7.57–7.51(m,4H),7.32(dd,J＝9.1,3.1Hz,1H),6.84(s,1H),5.94(m,J＝17.0,10.2,6.7Hz,1H),5.27–5.11(m,2H),4.15(t,J＝6.7Hz,2H),2.61(m,J＝6.7,1.4Hz,2H).HR-MS(ESI):calcd for C19H16O3,[M+H]+,293.1178；found 293.1187.
Example 12: preparation of 6-isobutoxy-2-phenyl-4H-chromen-4-one (Compound FLM-36)
The preparation method is the same as example 9. 1-iodo-2-methylpropane was used instead of iodoethane to give a pale yellow solid with a yield of 35.2% and a melting point of 92-96 ℃.1H NMR(400MHz,CDCl3)δ(ppm)7.96–7.92(m,2H),7.59(d,J＝3.1Hz,1H),7.56–7.51(m,4H),7.32(dd,J＝9.1,3.1Hz,1H),6.84(s,1H),3.85(d,J＝6.6Hz,2H),2.14(m,J＝13.3,6.7Hz,1H),1.07(d,J＝6.7Hz,6H).HR-MS(ESI):calcd for C19H18O3,[M+H]+,295.1334；found 295.1318.
Example 13: preparation of 6-isopropoxy-2-phenyl-4H-chromen-4-one (compound FLM. RTM. 37)
The preparation method is the same as example 9. Isopropane iodide was used instead of ethyl iodide to give a pale yellow solid with a yield of 33.7% and a melting point of 91-94 ℃.1HNMR(400MHz,CDCl3)δ(ppm)7.96–7.92(m,2H),7.62(d,J＝3.2Hz,1H),7.58–7.50(m,4H),7.30–7.25(m,1H),6.83(s,1H),4.72(m,J＝6.1Hz,1H),1.40(d,J＝6.0Hz,6H).HR-MS(ESI):calcd for C18H16O3,[M+H]+,281.1178；found 281.1132.
Example 14: preparation of 4-oxo-2-phenyl-4H-chromen-6-yl acetate (Compound FLM-38)
The preparation method is the same as example 9. Acetyl chloride was used instead of iodoethane to give a pale yellow solid with a yield of 33.7% and a melting point of 91-94 ℃.1H NMR(600MHz,CDCl3)δ(ppm)7.94–7.91(m,3H),7.60(d,J＝9.0Hz,1H),7.58–7.50(m,3H),7.45(dd,J＝9.0,2.8Hz,1H),6.82(s,1H),2.35(s,3H).HR-MS(ESI):calcd for C17H12O4,[M+H]+,281.0814；found 281.0800.
Example 15: preparation of 4-oxo-2-phenyl-4H-chromen-6-yl propionate (Compound FLM. sup. 39)
The preparation method is the same as example 9. Propionyl chloride was used instead of iodoethane to give a white solid with a yield of 32.6% and a melting point of 109-.1HNMR(600MHz,DMSO-d6)δ(ppm)8.30(s,1H),7.94(d,J＝8.7Hz,1H),7.54–7.44(m,2H),7.00–6.95(m,2H),6.91(dd,J＝8.7,2.2Hz,1H),6.82(d,J＝2.2Hz,1H),3.77(s,3H),2.48(m,J＝1.9Hz,2H).HR-MS(ESI):calcd for C18H14O4,[M+H]+,295.0970；found295.0959.
Example 16: preparation of 4-oxo-2-phenyl-4H-chromen-6-ylbutyric ester (Compound FLM-40)
The preparation method is the same as example 9. Butyryl chloride was used instead of iodoethane to give a white solid in 35.3% yield, mp 124-127 ℃.1H NMR(400MHz,CDCl3)δ(ppm)7.96–7.92(m,3H),7.62(d,J＝9.0Hz,1H),7.60–7.52(m,3H),7.46(dd,J＝9.0,2.8Hz,1H),6.84(s,1H),2.60(t,J＝7.4Hz,2H),1.83(m,J＝7.4Hz,2H),1.09(t,J＝7.4Hz,3H).HR-MS(ESI):calcd for C19H16O4,[M+H]+,309.1127；found 309.1097.
Example 17: preparation of 7-ethoxy-3- (4-methoxyphenyl) -4H-chromen-4-one (compound FLM _41)
1. A150 mL round bottom flask was taken, 7-hydroxy-3- (4-methoxyphenyl) -4 h-chromen-4-one (E) (2mmol) (0.54g, 2mmol) was dissolved in dichloromethane (50mL) solvent, and potassium carbonate (0.83g, 6mmol) was added, iodoethane (0.47g, 3mmol) was further added, the reaction was carried out at room temperature, and the progress of the reaction was checked by TLC, and after about 4-6 hours, the reaction was completed. Column chromatography was performed with an appropriate eluent (ethyl acetate: petroleum ether ═ 1: 5, v/v), and the separated crude product was recrystallized from ethanol to give the objective compound FLM _ 41. The product FLM-41 was a pale yellow solid with a yield of 29.5%, m.p. 122-125 ℃.1H NMR(600MHz,Chloroform-d)δ8.19(d,J＝8.9Hz,1H),7.90(s,1H),7.52–7.47(m,2H),6.98–6.94(m,3H),6.82(d,J＝2.4Hz,1H),4.12(q,J＝7.0Hz,2H),3.83(s,3H),1.47(t,J＝7.0Hz,3H).HR-MS(ESI):calcd for C18H16O4,[M+H]+,297.1127；found 297.1123.
Example 18: preparation of 7-butoxy-3- (4-methoxyphenyl) -4H-chromen-4-one (compound FLM. RTM. 42)
The preparation method is the same as example 17. Iodobutane was used instead of iodoethane to give a white solid in 37.5% yield, mp 124-127 ℃.1H NMR(600MHz,CDCl3)δ(ppm)8.19(d,J＝8.9Hz,1H),7.91(s,1H),7.53–7.46(m,2H),7.00–6.94(m,3H),6.83(d,J＝2.4Hz,1H),4.06(t,J＝6.5Hz,2H),3.84(s,3H),1.82(m,J＝7.9,6.5Hz,2H),1.56–1.50(m,2H),1.00(t,J＝7.4Hz,3H).HR-MS(ESI):calcd for C20H20O4,[M+H]+,325.1440；found 325.1395.
Example 19: preparation of 7- (but-3-en-1-yloxy) -3- (4-methoxyphenyl) -4H-chromen-4-one (compound FLM-43)
The preparation method is the same as example 17. Iodobutene was used instead of iodoethane to give a white solid in 32.9% yield, melting point 128-.1H NMR(400MHz,CDCl3)δ(ppm)8.23(d,J＝8.9Hz,1H),7.94(s,1H),7.60–7.45(m,2H),7.05–6.95(m,3H),6.87(d,J＝2.4Hz,1H),5.94(m,J＝17.0,10.2,6.7Hz,1H),5.26–5.15(m,2H),4.14(t,J＝6.7Hz,2H),3.87(s,3H),2.63(m,J＝6.7,1.4Hz,2H).HR-MS(ESI):calcd for C20H18O4,[M+H]+,323.1283；found 323.1274.
Example 20: preparation of 7-isobutoxy-3- (4-methoxyphenyl) -4H-chromen-4-one (compound FLM. RTM. 44)
The preparation method is the same as example 17. 1-iodo-2-methylpropane was used instead of iodoethane to give a pale yellow solid with a yield of 32.9%, melting point 129-.1H NMR(400MHz,CDCl3)δ(ppm)8.22(d,J＝8.9Hz,1H),7.93(s,1H),7.60–7.46(m,2H),7.09–6.92(m,3H),6.85(d,J＝2.4Hz,1H),3.86(s,3H),3.84(d,J＝6.5Hz,2H),2.16(m,J＝13.2,6.6Hz,1H),1.09(d,J＝6.7Hz,6H).HR-MS(ESI):calcd for C20H20O4,[M+H]+,325.1440；found 325.1413.
Example 21: preparation of 7-isobutoxy-3- (4-methoxyphenyl) -4H-chromen-4-one (compound FLM-45)
The preparation method is the same as example 17. 1-iodo-2-methylpropane was used instead of iodoethane to give a pale yellow solid with a yield of 32.9%, melting point 129-.1H NMR(600MHz,CDCl3)δ(ppm)8.20(d,J＝8.9Hz,1H),7.92(s,1H),7.51(d,J＝8.4Hz,2H),7.02–6.93(m,3H),6.83(s,1H),4.68(m,J＝5.9Hz,1H),3.85(s,3H),1.41(d,J＝6.0Hz,6H).HR-MS(ESI):calcd for C19H18O4,[M+H]+,311.1283；found 311.1281.
Example 22: preparation of 3- (4-methoxyphenyl) -4-oxo-4H-chromen-7-yl acetate (compound FLM-46)
The preparation method is the same as example 17. Acetyl chloride was used instead of iodoethane to give a white solid with a yield of 36.5% and a melting point of 155-.1H NMR(600MHz,CDCl3)δ(ppm)8.33(dd,J＝8.7,1.0Hz,1H),7.99(d,J＝1.1Hz,1H),7.54–7.48(m,2H),7.31(dd,J＝2.3,1.0Hz,1H),7.17(m,J＝8.8,2.2,1.1Hz,1H),7.01–6.96(m,2H),3.85(d,J＝1.1Hz,3H),2.37(d,J＝1.1Hz,3H).HR-MS(ESI):calcd for C18H15O5,[M+H]+,311.0919；found 311.0902.
Example 23: preparation of 3- (4-methoxyphenyl) -4-oxo-4H-chromen-7-yl propionate (compound FLM-47)
The preparation method is the same as example 17. Propionyl chloride was used instead of iodoethane to give a white solid with a yield of 33.5%, melting point 111-.1H NMR(600MHz,CDCl3)δ(ppm)8.33(d,J＝8.7Hz,1H),7.99(s,1H),7.51(d,J＝8.6Hz,2H),7.31(d,J＝2.1Hz,1H),7.17(dd,J＝8.7,2.1Hz,1H),7.01–6.96(m,2H),3.85(s,3H),2.66(m,J＝7.5Hz,2H),1.30(t,J＝7.5Hz,3H).HR-MS(ESI):calcd for C19H16O5,[M+H]+,325.1076；found 325.1034.
Example 24: preparation of 3- (4-methoxyphenyl) -4-oxo-4H-chromen-7-yl butyrate (compound FLM-48)
The preparation method is the same as example 17. Butyryl chloride was used instead of iodoethane to give a white solid in 29.3% yield, mp 112-.1H NMR(600MHz,CDCl3)δ(ppm)8.33(d,J＝8.7Hz,1H),7.98(s,1H),7.52–7.49(m,2H),7.30(d,J＝1.9Hz,1H),7.19–7.13(m,1H),7.02–6.96(m,2H),3.85(s,3H),2.61(t,J＝7.4Hz,2H),1.82(m,J＝7.3Hz,2H),1.08(t,J＝7.4Hz,3H).HR-MS(ESI):calcd for C20H18O5,[M+H]+,339.1232；found 339.1209.
Example 22: culture of Huh7 cells
We selected human hepatoma cell line Huh7 cells for culture. Huh7 cells were cultured in DMEM high-sugar medium containing 10% newborn calf serum, 100U/mL penicillin and streptomycin under 5% CO2At 37 ℃, the liquid is changed every other day, and the growth condition of the cells is observed every day. When the Huh7 cells grow to 70-80% fusion degree, discarding old cell culture solution, washing the cells for 2 times by PBS, adding 0.25% trypsin, observing cell morphology change under an inverted microscope, discarding digestive juice when the cells have cytoplasm retraction, cell body rounding and cell gap enlargement, and immediately discarding the digestive juiceAdding cell culture fluid containing 10% serum to stop digestion, sucking the culture fluid with a pipette, repeatedly and gently blowing adherent cells to make them fall off and suspend, adjusting the cells to proper density, inoculating into a new culture dish, placing in 5% CO2And culturing in an incubator at 37 ℃.
Example 23: detection of transcriptional activity of flavonoid compound on Nogo-B of Huh7 cell
We used the dual luciferase reporter system to determine the effect of flavonoids and isoflavonoids on Nogo-B transcriptional activity of Huh7 cells. Huh7 cells were seeded in 48-well plates and cultured for about 10-24h until the cells in the plates reached 80% confluence. According to the instruction, a certain amount of reporter gene blank plasmid or reporter gene plasmid containing Nogo-B promoter is mixed with internal reference plasmid (pGL4.70) and transfection reagent, and is kept stand for 30min to form transfection complex, and the transfection complex is uniformly dripped on the corresponding cell sap surface on the orifice plate. Cells were cultured in a 37 ℃ incubator. After about 6-8h of transfection, the medium containing the transfection complex was discarded and replaced with fresh serum-free medium, and the cells were treated with 10. mu.M flavonoids and isoflavonoids for about 24 h. Cell lysis solution provided by the luciferase reporter gene detection kit is used for cell lysis, and lysate supernatant is used for luciferase activity detection. Diluting the cell protein extract by a certain amount, adding the diluted cell protein extract into a 96-well plate, then placing the 96-well plate into a chemiluminescence apparatus, setting a program, and starting to measure the activity of luciferase. Relative fluorescence intensities were calculated and compared to empty controls to calculate the effect of each drug on Nogo-B transcriptional activity of Huh7 cells.
A total of 24 flavonoids from FLM _1-24 were screened first, and based on the results of the dual-luciferase reporter system (FIG. 1), the effects of FLM _25-48 on Nogo-B transcriptional activity of Huh7 cells were next determined again by the dual-luciferase reporter system. As shown in FIG. 2, compounds FLM _33 and FLM _34 also have significant inhibitory effects on Nogo-B transcriptional activity of Huh7 cells. As can be seen from FIG. 1, flavonoids FLM _5, FLM _6, FLM _9 and FLM _18 all have different degrees of inhibitory effects on Nogo-B transcriptional activity of Huh7 cells. As can also be seen from FIG. 2, at the action concentration of 10. mu.M, the compounds FLM _33 and FLM _34 also have good inhibitory effect on Nogo-B transcriptional activity, so that the compounds can be used as Nogo-B inhibitors to develop drugs for treating related liver diseases.
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