CN104230869A - Substitutive flavonoid compound as well as preparation method and application thereof - Google Patents

Abstract

The present invention relates to the technical field of biomedicine, and firstly provides a class of flavonoid compounds represented by general formula (I) and their use in the preparation of antitumor drugs. In addition, the present invention also provides pharmaceutical compositions containing such compounds. The flavonoids of the present invention can competitively bind to Bcl-2, Bcl-x _L and Mcl-1 proteins, and specifically cause tumor cell apoptosis, and are expected to be developed as safe and efficient anti-inflammatory agents targeting Bcl-2 family proteins. cancer drugs. In addition, the compound of the present invention also has good antifungal activity, and the above data all indicate that the compound of the present invention has a good development prospect.

Description

Substituted flavonoids and their preparation and use

technical field

The present invention relates to the technical field of medicine, more specifically, the present invention relates to a class of flavonoid compounds, and the present invention also relates to the composition, preparation method and preparation of such compounds for the treatment of anti-apoptosis with Bcl-2 protein family Use in medicines, antineoplastics and synergists for diseases or symptoms related to the high expression of dead members. the

the

Background technique

Apoptosis is the programmed death of cells after being stimulated by certain signals, and it is a basic biological phenomenon of cells. The Bcl-2 protein family plays an important regulatory role in the apoptosis pathway. It can be divided into anti-apoptotic members (such as Bcl-2, Bcl-x _L , Mcl-1, etc.) and pro-apoptotic members. Studies have shown that the overexpression of anti-apoptotic members of the Bcl-2 protein family can lead to the blockage of normal apoptosis pathways, which is related to the occurrence of many diseases (such as tumors, autoimmune diseases, etc.), especially the important factors for tumor generation and drug resistance. one of the reasons. ( Nature 2000, 407, 796-801; Nat Rev Cancer 2004, 4, 592-603. )

Studies have shown that anti-apoptotic members of the Bcl-2 protein family are found to be overexpressed in many tumors, and the expression levels are different in different tumors and different tumor subtypes ( Oncogene 2003, 22, 8590-607; Oncogene 2008, 27, 6398 -406 ). By inhibiting the anti-apoptotic effect of overexpressed anti-apoptotic members in tumor cells, it can restore its normal apoptotic pathway and increase its sensitivity to chemotherapy and radiotherapy, which is a new strategy for treating tumors. The anti-cell and anti-apoptotic members of the Bcl-2 protein family interact with the conserved region (BH) 3 of the pro-apoptotic member of the Bcl-2 protein family through the hydrophobic groove on its surface to regulate the normal physiological apoptosis of cells . Small-molecule inhibitors can interfere with the binding of the BH3 region of the pro-apoptotic member to promote cell apoptosis by binding to the hydrophobic groove on the surface of the anti-apoptotic member. ( Nat Rev Cancer 2005, 5, 876-85; Kelly, P. N.; Cell Death Differ 2011, 18, 1414-24. ) In recent years, this type of small molecule inhibitors has aroused widespread interest of researchers, and discovered A series of small molecule inhibitors, four of which (ABT-199, ABT-263, AT-101, GX15-07) have entered clinical research as oral anti-tumor drugs. The research results show that small molecule inhibitors of anti-apoptotic members of the Bcl-2 protein family exhibit good anti-tumor effects and synergistic effects on other anti-tumor drugs or radiotherapy, and have good development prospects. (Nat Rev Drug Discov 2008, 7, 989-100 ; Chinese Journal of New Drugs 2008, 17, 2008-2013.; Pharmaceutical Progress 2004, 28, 97-103; Clin Cancer Res 2012, 18, 1-7; J Thorac Oncol 2011 , 6, 1757-1760; Lung Cancer 2011,74, 481-485 ）

Contents of the invention

The object of the present invention is to provide a class of novel flavonoids or pharmaceutically acceptable salts thereof. The invention also discloses the preparation method, medical application and composition of the compound. the

In the present invention, flavonoids such as luteolin, which have anti-apoptotic member inhibitory activity of the Bcl-2 protein family discovered in the laboratory as lead compounds, are designed and synthesized by computer-aided drug design technology. Anti-apoptosis member small molecule inhibitor of flavonoid Bcl-2 protein family, activity tests on various tumor cells show that this type of compound shows good anti-tumor activity, and is expected to be developed into a new class of anti-tumor drug with a new structure. the

the

In the first aspect of the present invention, there is provided a flavonoid compound represented by the general formula (I) or a pharmaceutically acceptable salt, solvate, prodrug or polymorphic form thereof:

In the formula, R _1a , R _1b , R _1c , R _1d , R _1e , R _1f are independently any of the following groups: a) hydrogen; b) hydroxyl; c) amino; d) substituted C1- 8 linear or branched alkoxy; e) -O(CH ₂ ) _n R ₂ ; f) -(CH ₂ ) _n R ₂ ; g) substituted C1-8 linear or branched alkyl; h) substituted C2-6 straight or branched alkenyl; i) substituted C2-6 straight or branched alkynyl; j) substituted C1-8 straight or branched alkanoyloxy ;

n is 1-8, R is _substituted phenyl or 3-8 membered heterocyclic aryl;

The substitution refers to being substituted by one or more of the following substituents: hydrogen, C1-8 alkyl, C2-6 alkenyl, C2-6 alkynyl, halogenated C1-8 alkyl, C1-8 alkoxy group, halogen, trifluoromethyl group, nitro group, cyano group, sulfonic acid group, carboxyl group, hydroxyl group, amino group.

the

In another preferred example, in the compound, n is 1-3.

the

In another preferred example, in the compound, R _1a and R _1b can be independently hydrogen or -(CH ₂ ) _n R ₂ , but they are not hydrogen or -(CH ₂ ) _n R ₂ at the same time; R _1c , R _1e , R _1f can be independently hydroxyl, amino, C1-8 straight or branched alkoxy, -O(CH ₂ ) _n R ₂ , R _1d can be independently hydroxyl, amino, C1 -8 linear or branched alkoxy;

n is 1-8, R is _substituted phenyl or 3-8 membered heterocyclic aryl;

The substitution refers to being substituted by one or more of the following substituents: hydrogen, C1-8 alkyl, halogenated C1-8 alkyl, C1-8 alkoxy, halogen, trifluoromethyl, nitro, Cyano, Sulfonate, Carboxyl.

the

In another preferred example, in the compound, R _1a and R _1b can be independently hydrogen or substituted benzyl, but they are not hydrogen or substituted benzyl at the same time; R _1c , R _1e , R _1f can be Independently be hydroxyl, amino, C1-8 linear or branched alkoxy, substituted benzyloxy, R _1d can be independently hydroxyl, amino, C1-8 linear or branched alkoxy; the Substitution means that one or more of the 2, 3 or 4 positions on the benzene ring is substituted by any of the following groups: hydrogen, C1-8 alkyl, halogenated C1-8 alkyl, C1-8 alkoxy group, halogen, trifluoromethyl group, nitro group, cyano group, sulfonic acid group, carboxyl group.

the

In another preferred embodiment, the compound is any one of the following compounds:

In the second aspect of the present invention, a composition is provided, which contains the compound described in the first aspect of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

In another preferred example, the composition is a pharmaceutical composition. the

the

In the third aspect of the present invention, there is provided the use of the compound described in the first aspect or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating diseases associated with high expression of anti-apoptotic members of the Bcl-2 protein family.

In another preferred example, the disease is a tumor. More specifically, the tumor is leukemia or lung cancer. the

the

In the third aspect, the present invention also provides the use of the compound described in the first aspect or a pharmaceutically acceptable salt thereof in the preparation of an antifungal drug.

the

In the fourth aspect of the present invention, the preparation method of the two types of specific compounds described in the first aspect is provided.

First of all, a preparation method of flavonoids IV and V substituted by benzyl group is provided, which is characterized in that it comprises the following steps:

(1) Flavonoid compound II reacts with benzyl chloride substituted by R on the benzene ring in an inert solvent under alkaline conditions to generate compound III;

(2) Compound III is refluxed in an inert solvent under acidic conditions to generate compound IV;

(3) When one or more of R _1d , R _1e , and R _1f in compound IV is an alkoxy group, add BBr3 in an inert solvent to remove one or more alkoxy groups to generate a hydroxyl group , other groups remain unchanged, compound IV is further converted into compound V;

In compound IV and compound V, the substituent position of the substituted benzyl group is R _1a or R _1b ; R _1d , R _1e , R _1f are as described in claim 1; R _1d , R _1e , R _1f are not alkoxy groups The group remains unchanged after the reaction; the substitution position of R refers to one or more positions in the 2, 3 or 4 positions on the benzene ring, and R is substituted by any of the following groups: hydrogen, C1-8 alkyl, Halogenated C1-8 alkyl, halogen.

In another preferred example, the base used in the synthesis of the compound (III) is an inorganic base. Preferably, the inorganic base is K ₂ CO ₃ .

In another preferred example, the reaction conditions used in the synthesis of the compound (III) are common heating or microwave. the

In another preferred example, the inert solvent used in the synthesis of the compound (III) is DMF, DMSO or a combination thereof. the

In another preferred example, the acid used in the synthesis of the compound (IV) is an organic acid. Preferably, the acid used is methanesulfonic acid or trifluoroacetic acid. the

In another preferred example, the inert solvent used in the synthesis of the compound (IV) is chloroform. the

In another preferred example, the inert solvent used in the synthesis of the compound (V) is dichloromethane. the

the

Secondly, also provide a kind of preparation method of flavonoid compound VIII, IX that substituted benzyl substitutes, it is characterized in that, comprises the following steps:

(1) The flavonoid compound VI reacts with benzyl chloride substituted by R on the benzene ring in an inert solvent under alkaline conditions to generate compound VII;

(2) Compound VII is refluxed in an inert solvent under acidic conditions to generate compound VIII;

(3) When one or more of R _1c , R _1d , and R _1f in compound VIII is an alkoxy group, add BBr3 in an inert solvent to remove one or more alkoxy groups to generate a hydroxyl group , other groups remain unchanged, compound VIII is further converted into compound IX;

R _1c , R _1d , and R _1f are as described in claim 1; groups in R _1c , R _1d , and R _1f that are not alkoxy groups remain unchanged after the reaction; the substitution position of R refers to 2, In one or more of the 3 or 4 positions, R is substituted by any of the following groups: hydrogen, C1-8 alkyl, halogenated C1-8 alkyl, halogen.

In another preferred example, the base used in the synthesis of the compound (VII) is an inorganic base. Preferably, the inorganic base used is K ₂ CO ₃ .

In another preferred example, the reaction conditions used in the synthesis of the compound (VII) are common heating or microwave. the

In another preferred example, the inert solvent used in the synthesis of the compound (VII) is DMF, DMSO or a combination thereof. the

In another preferred example, the acid used in the synthesis of the compound (VIII) is an organic acid. Preferably, the acid used is methanesulfonic acid or trifluoroacetic acid. the

In another preferred example, the inert solvent used in the synthesis of the compound (VIII) is chloroform. the

In another preferred example, the inert solvent used in the synthesis of the compound (IX) is dichloromethane. the

the

The pharmaceutical composition involved in the present invention can be in solid form or liquid form, and the pharmaceutical dosage form can be tablet, capsule, powder, granule, suspension or injection. When the compound of the present invention is used for the above purposes, it can be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, etc., and can be orally administered in the following forms: tablets, pills, Capsules, dispersible powders, granules or suspensions (containing e.g. about 0.05-5% suspending agent), syrups (containing e.g. about 10-50% sugar), and elixirs (containing e.g. about 20-50% ethanol), or topical forms Administration: ointment, gel, medicated adhesive tape, etc., or parenteral administration in the form of a sterile injectable solution or suspension (containing about 0.05-5% suspending agent in an isotonic medium). For example, these pharmaceutical formulations may contain about 0.01-99%, more preferably about 0.1%-90% by weight of the active ingredient in admixture with a carrier.

"Safe and effective amount" means: the amount of the compound is sufficient to improve the condition without causing serious side effects. The safe and effective dose is determined according to the age, condition, course of treatment, etc. of the subject to be treated. the

"Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and low enough toxicity. "Compatibility" herein means that the components of the composition can be blended with the compound of the present invention and with each other without significantly reducing the efficacy of the compound. Some examples of pharmaceutically acceptable carriers include sugars (such as glucose, sucrose, lactose, etc.), starches (such as corn starch, potato starch, etc.), cellulose and its derivatives (such as sodium carboxymethylcellulose, ethyl cellulose, etc.) sodium, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as sodium stearate, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tweens), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives agent, pyrogen-free water, etc. the

The flavonoid compound of the present invention shows better binding ability to Bcl-2, Bcl-x _L and Mcl-1 proteins, especially some compounds show better binding ability to Mcl-1 protein. Therefore, these compounds have the potential application of preparing medicines for treating diseases related to the high expression of anti-apoptotic members of Bcl-2 protein family, and preparing synergists to be used in combination with other antitumor drugs or radiotherapy to treat tumors.

The flavonoid compounds of the present invention have better broad-spectrum anti-tumor activity on human solid tumors (lung cancer) and hematological tumors (leukemia), especially some compounds have better cytotoxic activity on human leukemia cell lines. Therefore, the compound of the present invention is expected to have a good development prospect. the

In addition, the flavonoids of the present invention have a certain broad-spectrum antifungal activity in vitro, especially some compounds show better inhibitory activity against Candida glabrata, indicating that the compounds of the present invention have the potential to prepare antifungal drugs. use. the

the

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are usually followed. Percentages and parts are by weight unless otherwise indicated. the

Embodiment 1-6: 2-((3-benzyl-4-hydroxyl-5-methoxy)phenyl)-5,7-dimethoxy- 4H -chromen-4-one ( Compound 1 in Table 1), 2-((2-benzyl-4-hydroxy-5-methoxy)phenyl)-5,7-dimethoxy- 4H -chromen-4-one (Compound 5 in Table 1), 2-((3-benzyl-4,5-dihydroxy)phenyl)-5,7-dihydroxy- 4H -chromen-4-ketone (compound in Table 1 3 ), 2-((3-benzyl-4,5-dihydroxy)phenyl)-5-hydroxy-7-methoxy- 4H -chromen-4-one (compound 4 in Table 1) , 2-((2-benzyl-4,5-dihydroxy)phenyl)-5,7-dihydroxy-4 H -chromen-4-one (compound 7 in Table 1) and 2-(( Preparation of 2-benzyl-4,5-dihydroxy)phenyl)-5-hydroxy-7-methoxy- 4H -chromen-4-one (compound 8 in Table 1):

1, Preparation of 2-hydroxy-4,6-dimethoxy-acetophenone

Add 2,4,6-trihydroxyacetophenone monohydrate (3.72g, 20mmol) into acetone (60ml), add potassium carbonate (6.06g, 40mmol) under stirring, if it is not dissolved, slowly add dimethyl sulfate The ester (4ml, 42mmol) was added dropwise and stirred at room temperature for 4 hours. The degree of reaction was monitored by TLC. After the reaction is finished, filter out the solid insoluble matter, slowly add water (100ml), shake repeatedly, and let it stand for half an hour, a white solid precipitates out. Filtration and drying gave a white solid (3.0 g), yield 75%.

2. Preparation of 4-benzyloxy-3-methoxybenzaldehyde

Dissolve 4-hydroxy-3-methoxybenzaldehyde (4.56g, 30mmol) in DMF (45ml), add potassium carbonate (4.56g, 33mmol) and benzyl chloride (4.18g, 33mmol), and heat to reflux for 6 hours , the extent of the reaction was monitored by TLC. After the reaction was completed, cool to room temperature, filter out solid insoluble matter, slowly add water (150ml) under rapid stirring, and let it stand for a while, a white solid precipitated, filtered and dried to obtain a white solid (5.8g), yield 80%.

3. ( E )-3-(4-(benzyloxy)-3-methoxyphenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-ene-1 - Preparation of ketones

Intermediate 2-hydroxy-4,6-dimethoxy-acetophenone (2 g, 10.2 mmol) and intermediate 4-benzyloxy-3-methoxybenzaldehyde (2.5 g, 10.2 mmol) were dissolved in In methanol (50ml), slowly add 50% potassium hydroxide aqueous solution (6ml) dropwise under stirring, the color of the solution gradually turns yellow, after dropping, stir at room temperature for 36 hours, and the reaction degree is monitored by TLC. After the reaction is finished, add 1mol/L hydrochloric acid solution dropwise under stirring, adjust to PH=5, let stand for a while, filter to obtain a yellow oily solid, wash with methanol (30ml), filter, and dry to obtain a yellow solid (3.0g). The yield is about 70%.

4. Preparation of 2-((4-benzyloxy-3-methoxy)phenyl)-5,7-dimethoxy- 4H -benzopyran-4-one

The intermediate ( E )-3-(4-(benzyloxy)-3-methoxyphenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-ene- 1-Kone (0.42g, 1mmol) was dissolved in pyridine (15ml). After dissolution, iodine (0.3g, 1.2mmol) was added and heated to reflux. The degree of reaction was monitored by TLC. After the reaction was completed, a sufficient amount of saturated sodium thiosulfate solution was added under stirring, a solid precipitated out, filtered, and recrystallized from ethyl acetate to obtain 0.3 g of a white solid, with a yield of 71%.

5. 2-((3-Benzyl-4-hydroxy-5-methoxy)phenyl)-5,7-dimethoxy- 4H -chromen-4-one (1) and 2 Preparation of -((2-benzyl-4-hydroxy-5-methoxy)phenyl)-5,7-dimethoxy- 4H -chromen-4-one (5)

The intermediate 2-((4-benzyloxy-3-methoxy)phenyl)-5,7-dimethoxy- 4H -chromen-4-one (1.25g, 3mmol) was dissolved Add methanesulfonic acid (4ml) to chloroform (16ml), place in a microwave reactor, and heat to reflux for 13 minutes under the condition of 300W. After the reaction, wash with water (50ml), stand and separate the layers, wash the aqueous phase with a small amount of dichloromethane, combine the organic phases, dry and concentrate, reverse phase (C18) column chromatography (MeOH:H ₂ O), separate and purify Pure compounds 1 and 5 , and 2-(4-hydroxy-3-methoxyphenyl)-5,7-dimethoxy-4 H -benzopyran-4-one were obtained.

6. 2-((3-benzyl-4,5-dihydroxy)phenyl)-5,7-dihydroxy- 4H -chromen-4-one (3) and 2-((3-benzyl Preparation of -4,5-dihydroxy)phenyl)-5-hydroxy-7-methoxy- 4H -chromen-4-one (4)

2-((3-Benzyl-4-hydroxy-5-methoxy)phenyl)-5,7-dimethoxy- 4H -chromen-4-one ( 1 ) (0.5g , 1.19mmol) was dissolved in anhydrous dichloromethane (10ml), and under stirring at -30 ^o C, slowly added 1M boron tribromide dichloromethane solution (10ml) dropwise, the solution slowly turned yellow, and after dropping, naturally The temperature was raised to room temperature, and the reaction progress was monitored by TLC. After the reaction, slowly add water (30ml) to the reaction solution, extract with ethyl acetate, collect the organic phase, concentrate, reverse phase (C18) column chromatography (MeOH:H ₂ O), separation and purification to obtain compound 3 and 4 pure products.

7. 2-((2-benzyl-4,5-dihydroxy)phenyl)-5,7-dihydroxy-4 H -chromen-4-one (7) and 2-((2-benzyl Preparation of 4,5-dihydroxy)phenyl)-5-hydroxy-7-methoxy- 4H -chromen-4-one (8)

The intermediate 2-((2-benzyl-4,5-dihydroxy)phenyl)-5-hydroxy-7-methoxy- 4H -chromen-4-one ( 5 ) (0.5g, 1.19mmol) was dissolved in anhydrous dichloromethane (10ml), under stirring at -30 ^o C, slowly added 1M boron tribromide dichloromethane solution (10ml) dropwise, the solution slowly turned yellow, after dropping, the temperature rose naturally To room temperature, TLC monitors the reaction progress. After the reaction was completed, water (30ml) was slowly added to the reaction liquid, extracted with ethyl acetate, the organic phase was collected, concentrated, purified by reverse phase (C18) column chromatography (MeOH:H ₂ O), and compound 7 was obtained by separation and purification and 8 sterling.

the

Example 7-8: 2-((3-Benzyl-4-hydroxy-5-methoxy)phenyl)-5-hydroxy-7-methoxy- 4H -benzopyran-4-one (Compound 2 in Table 1) and 2-((2-benzyl-4-hydroxy-5-methoxy)phenyl)-5-hydroxy-7-methoxy- 4H -benzopyran-4 - Preparation of ketone (compound 6 in table 1):

1. Preparation of 2-((4-benzyloxy-3-methoxy)phenyl)-5-hydroxy-7-methoxy- 4H -benzopyran-4-one

The intermediate ( E )-3-(4-(benzyloxy)-3-methoxyphenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-ene- 1-Kone (0.42g, 1mmol) was dissolved in pyridine (15ml). After dissolution, iodine (0.5g, 2mmol) was added and heated to reflux. The degree of reaction was monitored by TLC. After the reaction was completed, a sufficient amount of saturated sodium thiosulfate solution was added with stirring, and a solid precipitated out, filtered, and recrystallized from ethyl acetate to obtain a white solid (0.25 g), with a yield of 59%.

2. 2-((3-benzyl-4-hydroxy-5-methoxy)phenyl)-5-hydroxy-7-methoxy- 4H -benzopyran-4-one (2) and Preparation of 2-((2-benzyl-4-hydroxy-5-methoxy)phenyl)-5-hydroxy-7-methoxy- 4H -benzopyran-4-one (6)

The intermediate 2-((4-benzyloxy-3-methoxy)phenyl)-5-hydroxy-7-methoxy- 4H -chromen-4-one (1.25g, 3mmol) Dissolve in chloroform (16ml), add methanesulfonic acid (4ml), place in a microwave reactor, and heat to reflux for 13 minutes under the condition of 300W. After the reaction, wash with water (50ml), stand and separate the layers, wash the aqueous phase with a small amount of dichloromethane, combine the organic phases, dry and concentrate, reverse phase (C18) column chromatography (MeOH:H ₂ O), separate and purify Pure compounds 2 and 6 , and 2-(4-hydroxy-3-methoxyphenyl)-5-hydroxy-7-methoxy- 4H -benzopyran-4-one were obtained.

the

Example 9-12: 2-(3,4-dimethoxyphenyl)-5-benzyloxy-7-methoxy- 4H -benzopyran-4-one (compound 9 in Table 1 ), 2-((2-benzyl-4, 5-dimethoxy)phenyl)-5-hydroxy-7-methoxy-4 H -chromen-4-one (compound 10 in Table 1 ), 2-((2-benzyl-4,5-dihydroxy)phenyl)-5,7-dihydroxy-4 H -chromen-4-one (compound 7 in Table 1) and 2-( Preparation of (2-benzyl-4,5-dihydroxy)phenyl)-5-hydroxy-7-methoxy- 4H -chromen-4-one (compound 8 in Table 1):

1. Preparation of ( E )-3-(3,4-dimethoxyphenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one

Dissolve 2-hydroxy-4,6-dimethoxy-acetophenone (0.5 g, 2.5mmol) in 9ml methanol, add 3,4-dimethoxybenzaldehyde (0.42g, 2.5mmol), 3 After 4-dimethoxybenzaldehyde was dissolved, 50% potassium hydroxide aqueous solution (4.5ml) was added and stirred at room temperature for 36 hours. After the reaction is finished, adjust the pH to <7 with 1mol/L hydrochloric acid solution. The reaction solution was filtered with a Buchner funnel to collect a yellow solid. The filtrate was extracted with ethyl acetate (30ml×2). The combined organic phases were dried over anhydrous MgSO4 and distilled under reduced pressure. The yellow solids were combined, methanol (50 ml) was added thereto, stirred for 5 minutes, filtered, and the filter cake was collected to obtain 0.64 g of yellow solids, with a yield of 72.7%.

the

2. Preparation of 2-(3,4-dimethoxy)phenyl)-5-hydroxy-7-methoxy- 4H -benzopyran-4-one

The intermediate ( E )-3-(3,4-dimethoxyphenyl)-1-(2-hydroxyl-4,6-dimethoxyphenyl)prop-2-en-1-one (0.6 g, 1.7mmol) was dissolved in 8ml of pyridine, then iodine (0.86g, 3.4mmol) was added, heated to reflux and stirred for 13 hours. After the reaction was completed, 30ml of sodium thiosulfate solution was added, filtered, and the crude product was recrystallized with ethyl acetate (20ml) to obtain 0.41g of a yellow solid, with a yield of 72%.

the

3. Preparation of 2-(3,4-dimethoxyphenyl)-5-benzyloxy-7-methoxy- 4H -benzopyran-4-one (9)

Dissolve 2-(3,4-dimethoxy)phenyl)-5-hydroxy-7-methoxy- 4H -chromen-4-one (1.72 g, 5.2 mmol) in 30ml DMF, Then potassium carbonate (0.8 g, 5.72 mmol) was added, and benzyl chloride (1.33 g, 10.4 mmol) was added, heated under reflux and stirred for 6 hours. After the reaction, 100ml of water was added to the reaction solution, filtered, and the filtrate was extracted with ethyl acetate (20ml×3). The combined organic phases were dried over anhydrous MgSO4 and distilled under reduced pressure. The combined crude products were purified by silica gel column chromatography (ethyl acetate/n-hexane=1:4) to obtain 9 pure products.

 

4. Preparation of 2-((2-benzyl-4, 5-dimethoxy)phenyl)-5-hydroxy-7-methoxy- 4H -chromen-4-one (10)

The intermediate 2-(3,4-dimethoxyphenyl)-5-benzyloxy-7-methoxy- 4H -chromen-4-one (1 g, 2.4 mmol) was dissolved in 8 ml In chloroform, pour it into a 30ml microwave reaction bottle, add 2ml of methanesulfonic acid, and react at 65°C for 1 hour. Add 20ml of dichloromethane to the reaction bottle, wash with 20ml of water, dry the organic phase with anhydrous MgSO4, and distill under reduced pressure to obtain the crude product, which is purified by reverse phase (C18) column chromatography (MeOH:H ₂ O), and the compound is obtained by separation and purification 10 pure product and 2-(3,4-dimethoxy)phenyl)-5-hydroxy-7-methoxy- 4H -chromen-4-one pure product.

the

5. 2-((2-benzyl-4,5-dihydroxy)phenyl)-5,7-dihydroxy-4 H -chromen-4-one (7) and 2-((2-benzyl Preparation of 4,5-dihydroxy)phenyl)-5-hydroxy-7-methoxy- 4H -chromen-4-one (8)

The intermediate 2-((2-benzyl-4,5-dimethoxy)phenyl)-5-hydroxy-7-methoxy- 4H -chromen-4-one (0.4g, 0.96 mmol) was dissolved in 10ml DCM, the solution was cooled to -30°C, 1ml BBr3 was dissolved in 9ml DCM, the prepared BBr3 solution was added dropwise to the above solution, and the reaction solution was stirred at room temperature for 3 hours. Then 30ml of water was added, 20ml of ethyl acetate was added, the organic phases were combined, dried over anhydrous MgSO4, and evaporated under reduced pressure. The crude product was purified by reverse phase (C18) column chromatography (MeOH:H ₂ O), separated and purified to obtain pure compounds 7 and 8 . Taste.

the

Examples 13-19: 2-((3-Methoxy-4-(2-chlorobenzyl))phenyl)-5,7-dimethoxy- 4H -benzopyran-4-one (Compound 11 in Table 1), 2-((3-(2-chlorobenzyl)-4-hydroxy-5-methoxy)phenyl)-5,7-dimethoxy- 4H -benzo Pyran-4-one (compound 20 in Table 1), 2-((2-(2-chlorobenzyl)-4-hydroxy-5-methoxy)phenyl)-5,7-dimethoxy -4 H -benzopyran-4-one (compound 47 in Table 1), 2-((3-(2-chlorobenzyl)-4,5-dihydroxy)phenyl)-5,7-di Hydroxy-4 H -benzopyran-4-one (compound 29 in Table 1), 2-((3-(2-chlorobenzyl)-4,5-dihydroxy)phenyl)-5-hydroxy- 7-Methoxy-4 H -benzopyran-4-one (compound 38 in Table 1), 2–((2–(2-chlorobenzyl)-4,5-dihydroxy)phenyl)- 5,7-dihydroxy- 4H -chromen-4-one (compound 56 in Table 1) and 2-((2-(2-chlorobenzyl)-4,5-dihydroxy)phenyl) - Preparation of 5-hydroxy-7-methoxy- 4H -benzopyran-4-one (compound 65 in Table 1):

1, 2-((3-methoxy-4-(2-chlorobenzyl))phenyl)-5,7-dimethoxy- 4H -chromen-4-one (11) preparation

The intermediate 2-(4-hydroxy-3-methoxyphenyl)-5,7-dimethoxy- 4H -chromen-4-one (0.98g, 3mmol) was dissolved in DMF (15ml) In , under stirring, potassium carbonate (0.46g, 3.3mmol) was added, and the solid was not dissolved. Add 2-chlorobenzyl chloride (0.53 g, 3.3 mmol) and heat to reflux for 2 hours, and monitor the progress of the reaction by spotting a TLC plate. After the reaction is over, filter out the solid insoluble matter, slowly add 75ml of water under rapid stirring, let it stand for a while, a white solid precipitates, filter and dry to obtain a white solid 2-(4-(2-chlorobenzyloxy)-3-methoxy Phenyl)-5,7-dimethoxy- 4H -chromen-4-one (1.09g), yield 80%. ¹ H NMR (600 MHz, DMSO) δ 7.64 – 7.59 (m, 2H, -Ar-H), 7.56 (d, J = 2.2 Hz, 1H), 7.53 – 7.51 (m, 1H, -Ar-H), 7.41 – 7.39 (m, 2H, -Ar-H), 7.20 (d, J = 8.6 Hz, 1H), 6.85 (d, J = 2.3 Hz, 1H, H-8), 6.77 (s, 1H, H- 3), 6.49 (d, J = 2.3 Hz, 1H, H-6), 5.23 (s, 2H, -CH ₂ -), 3.89 (s, 3H, -OCH ₃ ), 3.89 (s, 3H, -OCH ₃ ), 3.82 (s, 3H, -OCH ₃ ). MS (ESI): m/z 453.43 (M +H) ⁺ .

2, 2-((3-(2-chlorobenzyl)-4-hydroxy-5-methoxy)phenyl)-5,7-dimethoxy- 4H -chromen-4-one (9) and 2-((2-(2-chlorobenzyl)-4-hydroxy-5-methoxy)phenyl)-5,7-dimethoxy- 4H -benzopyran-4 - Preparation of ketone (12)

The intermediate 2-(4-(2-chlorobenzyloxy)-3-methoxyphenyl)-5,7-dimethoxy- 4H -chromen-4-one (1.36g, 3mmol ) was dissolved in 16ml of anhydrous chloroform, 4ml of methanesulfonic acid was added, placed in a microwave reactor, and heated to reflux for 13 minutes under the condition of 300W. After the reaction, wash with water (50ml), stand and separate the layers, wash the aqueous phase with a small amount of dichloromethane, combine the organic phases, dry and concentrate, reverse phase (C18) column chromatography (MeOH:H ₂ O), separate and purify Pure compounds 9 and 12 , and 2-(4-hydroxy-3-methoxyphenyl)-5,7-dimethoxy-4 H -benzopyran-4-one were obtained.

3. 2-((3-(2-chlorobenzyl)-4,5-dihydroxy)phenyl)-5,7-dihydroxy- 4H -chromen-4-one (10) and 2 – Preparation of ((3-(2-chlorobenzyl)-4,5-dihydroxy)phenyl)-5-hydroxy-7-methoxy- 4H -benzopyran-4-one (11)

The intermediate 2-(3-(2-chlorobenzyl)-4-hydroxyl-5-methoxyphenyl)-5,7-dimethoxy- 4H -chromen-4-one ( 0.6g, 1.32mmol) was dissolved in 10ml of anhydrous dichloromethane, under stirring at -30 ^o C, slowly added 1M boron tribromide dichloromethane solution (10ml) dropwise, the solution slowly turned yellow, and after dropping, naturally The temperature was raised to room temperature, and the reaction progress was monitored by TLC. After the reaction was completed, water (30ml) was slowly added to the reaction solution at -30 ^o C, and after dropping, it was extracted with ethyl acetate, the organic phase was collected, concentrated, and purified by reverse phase (C18) column chromatography (MeOH:H ₂ O), separated and purified to obtain pure compounds 10 and 11 .

4. 2–((2–(2-chlorobenzyl)-4,5-dihydroxy)phenyl)-5,7-dihydroxy- 4H -chromen-4-one (13) and 2 – Preparation of ((2-(2-chlorobenzyl)-4,5-dihydroxy)phenyl)-5-hydroxy-7-methoxy- 4H -benzopyran-4-one (14)

The intermediate 2-(2-(2-chlorobenzyl)-4-hydroxyl-5-methoxyphenyl)-5,7-dimethoxy- 4H -chromen-4-one ( 0.6g, 1.32mmol) was dissolved in 10ml of anhydrous dichloromethane, under stirring at -30 ^o C, slowly added 1M boron tribromide dichloromethane solution (10ml) dropwise, the solution slowly turned yellow, and after dropping, naturally The temperature was raised to room temperature, and the reaction progress was monitored by TLC. After the reaction was completed, water (30ml) was slowly added to the reaction solution at -30 ^o C, and after dropping, it was extracted with ethyl acetate, the organic phase was collected, concentrated, and purified by reverse phase (C18) column chromatography (MeOH:H ₂ O), separated and purified to obtain pure compounds 13 and 14 .

the

Examples 20-73

Repeat Example 13-19, the difference is: using different raw materials, thereby preparing compounds 12-19, 21-28 , 48-55, 30-37, 39-46, 57-64, 66-73 in Table 1 . details as follows:

(1) The intermediate 2-(4-hydroxy-3-methoxyphenyl)-5,7-dimethoxy-4 H -benzopyran-4-one and 3-chlorobenzyl chloride, 4- Chlorobenzyl chloride, 2-methylbenzyl chloride, 3-methylbenzyl chloride, 4-methylbenzyl chloride, 2-trifluoromethylbenzyl chloride, 3-trifluoromethylbenzyl chloride, 4-trifluoromethylbenzyl chloride Chlorine reaction can synthesize compounds 12-19 in Table 1 respectively.

(2) Compounds 12-19 in Table 1 were dissolved in chloroform, added methanesulfonic acid, and reacted in a microwave reactor to synthesize three products, specifically compounds 21-28 in Table 1 and compounds 48-55 in Table 1, and 2-(4-hydroxy-3-methoxyphenyl)-5,7-dimethoxy-4 H -chromen-4-one.

(3) Compounds 21-28 in Table 1 are dissolved in dichloromethane, and two products can be synthesized under the action of boron tribromide, specifically compounds 30-37 in Table 1 and compounds 39-46 in Table 1.

(4) Compounds 48-55 in Table 1 were dissolved in dichloromethane, and two products could be synthesized under the action of boron tribromide, specifically compounds 57-64 in Table 1 and compounds 66-73 in Table 1.

the

The chemical structure of the target product in the general formula ( I ) synthesized by the present invention is shown in Table 1. The chemical structure of the target product was characterized by H NMR and mass spectrometry systems, and the specific data are shown in Table 2.

the

Table 1 Structure of target compounds in general formula ( I )

Table 2 Proton spectrum and mass spectrum data of the target compound in general formula ( Ⅰ )

Example 74: Bcl-2, Bcl-x _L and Mcl-1 protein affinity test

Experimental method: referring to previous work and related literature ( Bioorg Med Chem Lett 2012, 22, 39-44; ChemMedChem 2011, 6, 904-2 1), gossypol acetate (AT-101) and luteolin (control 1) As a control drug, the fluorescence polarization (FP) method was used to investigate the ability of the target compound to competitively inhibit the binding of Bcl-2, Bcl-x _L and Mcl-1 proteins to the BH3 peptide (fluorescein-labeled) of the pro-apoptotic protein Bim or Bid. Evaluate its affinity to the target protein. The fluorescence polarization signal was detected by a fluorescence spectrophotometer under the conditions of excitation light wavelength 485nm and emission light wavelength 535nm. After incubation at room temperature for 20 minutes with serial concentrations of the target compound, fluorescein-labeled Bim or Bid BH3 peptide, and Bcl-2, Bcl-x _L or Mcl-1 protein, detect the fluorescence polarization signal, and calculate the IC50 values _. And according to the total protein concentration used in the measurement, the total concentration of the fluorescent polypeptide, the dissociation constant of the protein-polypeptide complex, and the IC ₅₀ value of the test compound, the competitive inhibition constant K _i of the test compound is calculated. (See Table 3 for test results)

Table 3 Affinity of some samples in general formula ( I ) to Bcl-2, Bcl-x _L and Mcl-1 proteins

IR: Inhibition Rate

The results show that the flavonoid compound of the present invention has better binding ability to Bcl-2, Bcl-x _L and Mcl-1 proteins, especially some compounds have better binding ability to Mcl-1 protein. Therefore, these compounds have the potential application of preparing medicines for treating diseases related to the high expression of anti-apoptotic members of Bcl-2 protein family, and preparing synergists to be used in combination with other antitumor drugs or radiotherapy to treat tumors.

the

Example 75: In vitro anti-tumor activity test

1. Experimental strains:

The tumor cell lines used in this experiment are: A549 (human lung cancer cells), HL-60 (human leukemia cells) (purchased from Shanghai Institute of Pharmaceutical Industry).

2. Sample preparation:

After dissolving with DMSO (Merck), add PBS (-) to make a 1000 mg/mL solution or a uniform suspension, and then dilute with DMSO-containing PBS (-). Positive control drugs were ABT-737 developed by Abbott, luteolin (control 1) and 2-(3-methoxy-4-benzyloxy)phenyl)-5,7-dimethoxy- 4H - benzopyran-4-one (control 2).

3. Test method

According to the experimental detection and related literature ( Bioorg Med Chem Lett 2012, 22, 39-44; Nature. 2005, 437, 677-681 ), the MTT method was used. Add 100 mL of cell suspension with a concentration of 4-5× ¹⁰⁴ cells/mL to each well of a 96-well plate, and place in a 37°C, 5% _CO2 incubator. After 24 h, add the sample solution, 10 mL/well, set up duplicate wells, and act at 37 °C, 5% CO ₂ for 72 h. Add 20 mL of 5 mg/mL MTT solution to each well, add the dissolving solution after 4 hours of action, 100 mL/well, put it in an incubator, measure the OD value at 570 nm with an MK-2 automatic microplate reader after dissolution. (See Table 4 for antitumor activity in vitro)

Table 4 Inhibitory effect of some samples in general formula ( I ) on human tumor cell proliferation in vitro

IR: Inhibition Rate

It can be seen from Table 4 that the flavonoids of the present invention have shown better broad-spectrum anti-tumor activity to human solid tumors (lung cancer) and hematological tumors (leukemia), especially some compounds have shown better cell Toxic activity indicates that the compound of the present invention is expected to have a good development prospect.

the

Example 76: In vitro antifungal activity test

1. Experimental strains

In this experiment, the following two common human pathogenic standard fungal strains were selected as screening objects, and the fungal strains were provided by the fungal laboratory of Changhai Hospital. These include Candida glabrata and Cryptococcus neoformans.

2. Test method

Use conventional in vitro antibacterial test methods (see: Antimicrob Agents Chemother 1995,39(5): 1169).

Bacterial solution preparation: cocci were cultured in YEPD liquid medium at 35°C for 16 hours, activated twice, counted with a hemocytometer, and adjusted to 1×103-5×103/ml with RPMI 1640 culture solution; filamentous bacteria Inoculate on the SDA slope, culture at 35°C for one week, activate twice, make the strain cover the SDA slope, add an appropriate amount of RPMI1640 culture solution, blow the colony with a straw, make the fungal spores free in the RPMI1640 culture solution, and then filter through four layers of sterile gauze . After the culture solution was counted by a hemocytometer, add RPMI1640 culture solution to adjust the spore concentration to 1×103-5×103/ml. the

Preparation of liquid medicine: the tested medicines were made into 6.4 g·L ^-1 with DMSO

Inoculation: Add 100 μl of RPMI1640 to well 1 of a 96-well plate as a blank control, add 100 μl of bacterial suspension to each well of No. Stepwise dilution, the drug concentrations in each well were 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 μg/mL. Well No. 12 was not added with the drug solution, as a positive control, and the drug controls were fluconazole and luteolin (control 1).

Cultivation and detection: Candida fungi were cultured for 24 hours to determine the results. The OD value of each well was measured with a microplate analyzer at 630 nm. Compared with the positive control well, the drug concentration in the lowest concentration well whose OD value drops by more than 80% is MIC ₈₀ . (The results of the in vitro antibacterial test are shown in Table 5)

Table 5 In vitro antifungal minimum inhibitory concentration (MIC, mg·L ^-1 ) of some samples in general formula ( Ⅰ )

As can be seen from Table 5, the flavonoids of the present invention have a certain broad-spectrum antifungal activity in vitro, especially some compounds have shown better inhibitory activity to Candida glabrata, indicating that the compounds of the present invention have the ability to prepare antifungal infections. current use of the drug.

In summary, the present invention provides a class of substituted flavonoids, which have good antitumor activity and antifungal activity, and have good development prospects. the

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the method of the present invention, some improvements and supplements can also be made, and these improvements and supplements should also be considered Be the protection scope of the present invention. the

Claims (12)

1. A class of flavonoids represented by general formula ( I ) or pharmaceutically acceptable salts, solvates, prodrugs or polymorphs thereof:                                                   ( I ) In the formula, R _1a , R _1b , R _1c , R _1d , R _1e , R _1f are independently any of the following groups: a) hydrogen; b) hydroxyl; c) amino; d) substituted C1- 8 linear or branched alkoxy; e) -O(CH ₂ ) _n R ₂ ; f) -(CH ₂ ) _n R ₂ ; g) substituted C1-8 linear or branched alkyl; h) substituted C2-6 straight or branched alkenyl; i) substituted C2-6 straight or branched alkynyl; j) substituted C1-8 straight or branched alkanoyloxy ; n is 1-8, R is _substituted phenyl or 3-8 membered heterocyclic aryl; The substitution refers to being substituted by one or more of the following substituents: hydrogen, C1-8 alkyl, C2-6 alkenyl, C2-6 alkynyl, halogenated C1-8 alkyl, C1-8 alkoxy group, halogen, trifluoromethyl group, nitro group, cyano group, sulfonic acid group, carboxyl group, hydroxyl group, amino group. 2. The compound of claim 1, wherein n is 1-3. 3. The compound according to claim 1, wherein R _1a and R _1b can independently be hydrogen or -(CH ₂ ) _n R ₂ , but both are not hydrogen or -(CH ₂ ) _n R at the same time ₂ ; R _1c , R _1e , R _1f can be independently hydroxyl, amino, C1-8 straight or branched alkoxy, -O(CH ₂ ) _n R ₂ , R _1d can be independently hydroxyl, amino , C1-8 linear or branched alkoxy; n is 1-8, R is _substituted phenyl or 3-8 membered heterocyclic aryl; The substitution refers to being substituted by one or more of the following substituents: hydrogen, C1-8 alkyl, halogenated C1-8 alkyl, C1-8 alkoxy, halogen, trifluoromethyl, nitro, Cyano, Sulfonate, Carboxyl. 4. The compound according to claim 1, wherein R _1a and R _1b can be independently hydrogen or substituted benzyl, but the two are not hydrogen or substituted benzyl at the same time; R _1c , R _1e , R _1f Can be independently hydroxyl, amino, C1-8 linear or branched alkoxy, substituted benzyloxy, R _1d can be independently hydroxyl, amino, C1-8 linear or branched alkoxy; The above substitution means that one or more of the 2, 3 or 4 positions on the benzene ring are substituted by any of the following groups: hydrogen, C1-8 alkyl, halogenated C1-8 alkyl, C1-8 alkane Oxy, halogen, trifluoromethyl, nitro, cyano, sulfonate, carboxy. 5. The compound of claim 1, wherein the compound is any one of the following compounds: . 6. A composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. 7. The use of a compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating diseases related to the high expression of anti-apoptotic members of the Bcl-2 protein family. 8. The use according to claim 7, wherein the disease is a tumor. 9. The use according to claim 8, characterized in that the tumor is leukemia or lung cancer. 10. A compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof is used in the preparation of an antifungal drug. 11. A preparation method for substituted benzyl-substituted flavonoids IV and V, characterized in that it comprises the following steps: (1) Flavonoid compound II reacts with benzyl chloride substituted by R on the benzene ring in an inert solvent under alkaline conditions to generate compound III; (2) Compound III is refluxed in an inert solvent under acidic conditions to generate compound IV; (3) When one or more of R _1d , R _1e , and R _1f in compound IV is an alkoxy group, add BBr3 in an inert solvent to remove one or more alkoxy groups to generate a hydroxyl group , other groups remain unchanged, compound IV is further converted into compound V; In compound IV and compound V, the substituent position of the substituted benzyl group is R _1a or R _1b ; R _1d , R _1e , R _1f are as described in claim 1; R _1d , R _1e , R _1f are not alkoxy groups The group remains unchanged after the reaction; the substitution position of R refers to one or more positions in the 2, 3 or 4 positions on the benzene ring, and R is substituted by any of the following groups: hydrogen, C1-8 alkyl, Halogenated C1-8 alkyl, halogen. 12. A preparation method for benzyl substituted flavonoids VIII, IX, characterized in that it comprises the following steps: (1) The flavonoid compound VI reacts with benzyl chloride substituted by R on the benzene ring in an inert solvent under alkaline conditions to generate compound VII; (2) Compound VII is refluxed in an inert solvent under acidic conditions to generate compound VIII; (3) When one or more of R _1c , R _1d , and R _1f in compound VIII is an alkoxy group, add BBr3 in an inert solvent to remove one or more alkoxy groups to generate a hydroxyl group , other groups remain unchanged, compound VIII is further converted into compound IX; R _1c , R _1d , and R _1f are as described in claim 1; groups in R _1c , R _1d , and R _1f that are not alkoxy groups remain unchanged after the reaction; the substitution position of R refers to 2, In one or more of the 3 or 4 positions, R is substituted by any of the following groups: hydrogen, C1-8 alkyl, halogenated C1-8 alkyl, halogen.