CN110669034A

CN110669034A - Isoflavone-chalcone dimer and chalcone dimer, preparation method and application

Info

Publication number: CN110669034A
Application number: CN201910940174.9A
Authority: CN
Inventors: 杨学东; 王立娜
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2019-09-30
Filing date: 2019-09-30
Publication date: 2020-01-10
Anticipated expiration: 2039-09-30
Also published as: CN110669034B

Abstract

The invention discloses an isoflavone-chalcone dimer and a chalcone dimer, a preparation method and application thereof, wherein four novel flavone dimer compounds provided by the invention have the activity of inhibiting IL-6, TNF-alpha and NO generation by carrying out extraction and separation on Tibetan medicine caragana and systematic chemical component research and inflammation medium inhibition activity evaluation, and can be used for preparing medicaments for resisting inflammation and treating ischemic cardiovascular and cerebrovascular diseases.

Description

Isoflavone-chalcone dimer and chalcone dimer, preparation method and application

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to isoflavone-chalcone dimers (caraganins A and B) and chalcone dimers (caraganins C and D) in Tibetan medicine caragana, and a preparation method and application thereof.

Background

Caragana tibetana is taken as one of the main Tibetan medicines for blood breaking and stasis removal in Tibetan medicine, and is mainly prepared by using red wood hearts of three plants, namely Caragana phalloidea (Caragana jubata), Caragana changduensis (Caragana gigansis) and Caragana chuanensis (Caragana tibetaca) in Caragana of leguminosae and in Qinghai province of Qinghai-Tibet high-altitude origin. The caragana tibetana has the main effects of breaking blood, dissolving stasis and reducing blood pressure, and is commonly used for treating diseases such as hyperemia, hypertension, irregular menstruation and the like.

Inflammation is a process by which human tissues produce a self-protective response when invading the body by foreign pathogens and harmful stimuli, involving the local vascular system and the immune system, as well as a variety of molecular mediators in the damaged tissue. However, uncontrolled inflammation may lead to a range of diseases. Ischemic cardiovascular and cerebrovascular diseases are one of the most serious diseases in humans, and can cause pathological changes in the body, which are caused by such uncontrolled inflammation. Therefore, controlling the cascade activation of inflammatory mediators such as interleukin-6 (IL-6), tumor necrosis factor (TNF- α) and Nitric Oxide (NO) is a promising approach for the treatment of inflammatory diseases as well as ischemic cardiovascular and cerebrovascular diseases.

Disclosure of Invention

The invention provides an isoflavone-chalcone dimer and a chalcone dimer, a preparation method and application thereof, and solves the problem that few medicines for treating inflammatory diseases can be selected in the prior art.

The technical scheme of the invention is as follows:

a compound represented by the following structure:

the preparation method of the compound comprises the following steps:

(1) pulverizing radix Caraganae Sinicae, extracting with solvent, and recovering extractive solution to obtain total extract;

(2) dispersing the total extract obtained in the step (1) in water, extracting by adopting an organic solvent immiscible with water, and recovering the solvent to obtain an extract;

(3) separating the extract obtained in the step (2) by medium-pressure liquid chromatography, and performing gradient elution by using a methanol/water or acetonitrile/water mixed solvent as a mobile phase;

(4) and (4) separating the fractions obtained in the step (3) by preparative high performance liquid chromatography, and performing gradient elution by using methanol/water or acetonitrile/water as a mobile phase to obtain the compounds 1-4.

The Caragana tibetana is red wood heart of rhizome of Caragana microphylla (Caragaajubata) of Caragana of Leguminosae (Leguminosae), Caragana changduensis (Caragana changduensis) and Caragana chuanensis (Caragana tibetaca).

The extraction method in the step (1) comprises heating reflux extraction, leaching, percolation or ultrasonic extraction, wherein a solvent is at least one of dichloromethane, chloroform, ethyl acetate, methanol, ethanol and methanol-water or ethanol-water with the concentration of more than 60%, and the weight-volume ratio of the medicinal materials to the solvent is 1: 4-1: 15.

In the extraction method in the step (2), the organic solvent is any one of dichloromethane, chloroform, diethyl ether and ethyl acetate, and the volume ratio of the aqueous solution to the organic solvent is 1: 1-1: 2.

In the step (3), gradient elution is carried out by a methanol/water system at a ratio of 25: 75-100: 0, or gradient elution is carried out by an acetonitrile/water system at a ratio of 20: 80-90: 10. Wherein the optimized ratio of the methanol/water system is 40: 60-95: 5, and the optimized ratio of the acetonitrile/water system is 35: 65-80: 20.

And (4) carrying out gradient elution on methanol/water or acetonitrile/water in a volume ratio of 30: 70-90: 10, wherein the optimized ratio is 50: 50-80: 10.

A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable excipient.

The compound and the pharmaceutical composition are applied to the preparation of anti-inflammatory and ischemic cardiovascular and cerebrovascular disease drugs.

The anti-inflammatory agent is an agent that inhibits the activity of IL-6, TNF-alpha and NO production.

The invention has the beneficial effects that: through systematic chemical component research and inflammation medium inhibition activity evaluation on Tibetan medicine caragana, the research discovers that the four novel flavone dimer compounds provided by the invention have the activity of inhibiting the generation of IL-6, TNF-alpha and NO, and can be used for preparing medicines for resisting inflammation and treating ischemic cardiovascular and cerebrovascular diseases.

Drawings

FIG. 1 HRESIMS spectra of Compound 1 of the invention;

FIG. 2 Process for preparation of Compound 1 of the present invention¹H-NMR spectrum;

FIG. 3 preparation of Compound 1 of the present invention¹³C-NMR spectrum;

FIG. 4 DEPT-135 spectrum of Compound 1 of the present invention;

FIG. 5 preparation of Compound 1 of the present invention¹H,¹H COSY spectrum;

FIG. 6 HSQC spectra of Compound 1 of the present invention;

FIG. 7 HMBC spectra of Compound 1 of the present invention;

FIG. 8 HRESIMS spectra of Compound 2 of the invention;

FIG. 9 preparation of Compound 2 of the present invention¹H-NMR spectrum;

FIG. 10 preparation of Compound 2 of the present invention¹³C-NMR spectrum;

FIG. 11 DEPT-135 spectrum of Compound 2 of the present invention;

FIG. 12 preparation of Compound 2 of the present invention¹H,¹H COSY spectrum;

FIG. 13 HSQC spectra of Compound 2 of the present invention;

FIG. 14 HMBC spectra of Compound 2 of the present invention;

FIG. 15 HRESIMS spectra of Compound 3 of the invention;

FIG. 16 preparation of Compound 3 of the present invention¹H-NMR spectrum;

FIG. 17 preparation of Compound 3 of the present invention¹³C-NMR spectrum;

FIG. 18 DEPT-135 spectrum of Compound 3 of the present invention;

FIG. 19 preparation of Compound 3 of the present invention¹H,¹H COSY spectrum;

FIG. 20 HSQC spectra of Compound 3 of the present invention;

FIG. 21 HMBC spectrum of compound 3 of the present invention;

FIG. 22 NOESY spectrum of Compound 3 of the present invention;

FIG. 23 HRESIMS spectra of Compound 4 of the invention;

FIG. 24 preparation of Compound 4 of the present invention¹H-NMR spectrum;

FIG. 25 of Compound 4 of the present invention¹³C-NMR spectrum;

FIG. 26 DEPT-135 spectrum of Compound 4 of the present invention;

FIG. 27 of Compound 4 of the present invention¹H,¹H COSY spectrum;

FIG. 28 HSQC spectra of Compound 4 of the present invention;

FIG. 29 HMBC spectrum of compound 4 of the present invention;

FIG. 30 NOESY spectrum of Compound 4 of the present invention;

FIG. 31 HMBC and¹H-¹h COSY correlation scheme;

FIG. 32 is a NOESY-related schematic of compounds 3-4 of the present invention;

FIG. 33 Experimental and calculated ECD spectra of compounds 1-4 of the present invention;

FIG. 34 structural formulas of the compounds 1 to 4 of the present invention.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.

Example 1

(1) 1 kg of Tibetan caragana medicinal material (original plant caragana microphylla), crushing, extracting for 3 times by using 95% ethanol at room temperature, soaking for 24 hours by 5L each time, and recovering the solvent under reduced pressure to obtain a 95% ethanol extract;

(2) dispersing the 95% ethanol extract obtained in the step (1) in water to prepare a suspension, extracting with petroleum ether to remove impurities, then extracting with dichloromethane, and recovering the solvent to obtain a dichloromethane extract;

(3) separating the extract obtained in the step (2) by medium-pressure liquid chromatography (reverse phase silica gel C18), and performing gradient elution (35: 65-100: 0) by using a methanol/water mixed solvent as a mobile phase;

(4) separating the methanol/water (45: 55-65: 35) fractions obtained in the step (3) by preparative high performance liquid chromatography (reverse phase silica gel C18), and performing gradient elution (45: 55-60: 40) using acetonitrile/water as a mobile phase to obtain the novel compounds 1 (yield 0.0012%), 2 (yield 0.001%), 3 (yield 0.0008%) and 4 (yield 0.0005%).

Example 2

(1) 2 kg of Tibetan caragana (original plant of Changdu caragana), crushing, soaking in 85% ethanol at room temperature for 8 hours, then refluxing and extracting for 3 times, refluxing for 8L multiplied by 1 hour each time, and recovering the solvent under reduced pressure to obtain 85% ethanol extract;

(2) dispersing the 85% ethanol extract obtained in the step (1) in water to prepare a suspension, extracting with petroleum ether to remove impurities, extracting with ethyl acetate, and recovering the solvent to obtain an ethyl acetate extract;

(3) separating the extract obtained in the step (2) by medium-pressure liquid chromatography (reverse phase silica gel C18), and performing gradient elution (25: 75-80: 20) by using an acetonitrile/water mixed solvent as a mobile phase;

(4) separating the methanol/water (40: 60-60: 40) fractions obtained in the step (3) by preparative high performance liquid chromatography (reverse phase silica gel C18), and performing gradient elution (50: 50-90: 10) with acetonitrile/water as a mobile phase to obtain the new compound 1 (yield 0.0015%), 2 (yield 0.0011%), 3 (yield 0.0009%) and 4 (yield 0.0008%).

Example 3

(1) 1 kg of Tibetan caragana (original plant caragana microphylla), crushing, soaking in 75% ethanol for 4 hours at room temperature, extracting for 3 times by ultrasonic oscillation (ultrasonic frequency: 40KHz, ultrasonic power: 500W) for 4.5L multiplied by 1 hour each time, and recovering the solvent under reduced pressure to obtain 75% ethanol extract;

(2) dispersing the 75% ethanol extract obtained in the step (1) in water to prepare a suspension, extracting with petroleum ether to remove impurities, extracting with chloroform, and recovering the solvent to obtain a chloroform extract;

(3) separating the extract obtained in the step (2) by medium-pressure liquid chromatography (reverse phase silica gel C18), and performing gradient elution (40: 60-95: 5) by using a methanol/water mixed solvent as a mobile phase;

(4) separating the methanol/water (47: 53-68: 32) fractions obtained in the step (3) by preparative high performance liquid chromatography (reverse phase silica gel C18), and performing gradient elution (30: 70-65: 35) with acetonitrile/water as a mobile phase to obtain the new compounds 1 (yield 0.0010%), 2 (yield 0.0009%), 3 (yield 0.0010%) and 4 (yield 0.0003%).

Experimental example 1

The structure of the compound 1-4 is identified according to the physicochemical property and the spectrum data of the compound 1-4 (the spectrum analysis map of the compound 1-4 is shown in the figure 1-34). The structural identification data for compound 1 is as follows:

yellow powder (MeOH); [ alpha ] to]²² _D-1.2(c 0.09,MeOH)；UV(MeOH)λ_max(logε)261(4.38)nm；ECD(MeOH)λ_max(Δε)206(+10.49),220(+5.06),240(-7.00)nm；IR(KBr)ν_max3439,2921,1653,1610,1567,1549,1513,1464,1450,1438,1309,1265,1208,1112,1030,667,660cm^-1；¹H NMR(DMSO-d₆600MHz) and¹³C NMR(DMSO-d₆150MHz) data are shown in Table 1; HRESIMS M/z 591.1614[ M + Na ]]⁺,calcd for C₃₃H₂₈NaO₉,591.1626. The HMBC associated signals of the compounds are shown in FIG. 31 (1). The absolute configuration of the compound is determined by an ECD map determined by a comparative experiment and an ECD map of an enantiomer obtained by calculation by adopting a TDDFT (time density functional theory) method. The absolute configuration of this compound was determined as 9' R and the ECD pattern is shown in FIG. 33 (A).

The structural identification data for compound 2 is as follows:

yellow powder (MeOH); [ alpha ] to]²² _D-1.0(c 0.09,MeOH)；UV(MeOH)λ_max(logε)261(4.26)nm；ECD(MeOH)λ_max(Δε)227(-26.82),239(-14.46),254(+6.41),284(+10.21)nm；IR(KBr)ν_max3433,2922,1612,1508,1459,1263,1207,1158,1039,559cm^-1；¹H NMR(DMSO-d₆400MHz) and¹³C NMR(DMSO-d₆100MHz) data are shown in Table 1; HRESIMS M/z 567.1666[ M-H ]]^-,calcd for C₃₃H₂₇O₉,567.1661. The HMBC associated signals of the compounds are shown in FIG. 31 (2). The absolute configuration of the compound is determined by an ECD (equivalent-density-distribution) map determined by a contrast experiment and an enantiomer obtained by calculating by adopting a TDDFT (time density functional theory) methodECD mapping of (a). The absolute configuration of this compound was determined as 9' R and the ECD pattern is shown in FIG. 33 (B).

The structural identification data for compound 3 is as follows:

light yellow oil (MeOH); [ alpha ] to]²² _D-1.0(c 0.08,MeOH)；UV(MeOH)λ_max(logε)274(3.99)nm；ECD(MeOH)λ_max(Δε)214(+3.59),221(+10.00),239(-12.70)nm；IR(KBr)ν_max3432,2928,2378,1617,1508,1453,1378,1265,1208,1157,1119,1037,929,832cm^-1；¹H NMR(DMSO-d₆600MHz) and¹³C NMR(DMSO-d₆150MHz) data are shown in Table 2; HRESIMS M/z 547.2072[ M + Na ]]⁺,calcd for C₃₃H₃₂NaO₆,547.2091. The HMBC related signals of the compounds are shown in FIG. 31(3), and the NOESY related signals are shown in FIG. 32 (3). The absolute configuration of the compound is determined by an ECD map determined by a comparative experiment and an ECD map of an enantiomer obtained by calculation by adopting a TDDFT (time density functional theory) method. The absolute configuration of the compound was determined as 2R, 3R, 4R, and the ECD pattern is shown in FIG. 33 (C).

The structural identification data for compound 4 is as follows:

light yellow powder (MeOH); [ alpha ] to]²² _D+1.2(c 0.10,MeOH)；UV(MeOH)λ_max(logε)267(3.79)nm；ECD(MeOH)λ_max(Δε)219(-4.50),231(+24.10),243(-20.00),267(+16.65),290(+10.85)nm；IR(KBr)ν_max3438,2923,2853,1616,1513,1443,1261,1159,1024,804,578cm^-1；¹H NMR(DMSO-d₆600MHz) and¹³C NMR(DMSO-d₆150MHz) data are shown in Table 2; HRESIMS M/z 507.1811[ M-H ]]^-,calcd for C₃₂H₂₇O₆,507.1813. The HMBC related signals of the compounds are shown in FIG. 31(4), and the NOESY related signals are shown in FIG. 32 (4). The absolute configuration of the compound is determined by an ECD map determined by a comparative experiment and an ECD map of an enantiomer obtained by calculation by adopting a TDDFT (time density functional theory) method. The absolute configuration of the compound was determined as 2S, 3R, 4R, and the ECD pattern is shown in FIG. 33 (D).

TABLE 1 of Compounds 1 and 2¹H and¹³c NMR data(DMSO-d₆)

TABLE 2 of Compounds 3 and 4¹H and¹³c NMR data (DMSO-d)₆)

Experimental example 2

Anti-inflammatory Activity assay of novel Compounds 1-4

Lipopolysaccharide (LPS) is adopted to induce macrophage of a RAW264.7 mouse to generate a model of NO, IL-6 and TNF-alpha, and the anti-inflammatory activity of the model is evaluated by detecting the expression levels of three inflammatory mediators in RAW264.7 cells after a test compound is added, so that potential drugs for resisting inflammation and treating ischemic cardiovascular and cerebrovascular diseases are discovered.

1. Method for preparing test compound solution

Test compounds were dissolved in DMSO to prepare a stock solution at a concentration of 50mM and stored at-20 ℃. The test was carried out by diluting the medium with DMEM in the order of 10mM, 5mM, 3mM, 1mM, 0.1mM, and 0.01 mM.

2. Culture of mouse RAW264.7 macrophage

DMEM medium is used as the base to prepare cell culture medium containing 10% fetal calf serum and 1% double antibody (penicillin: streptomycin 1: 1), and 5% CO is added at 37 deg.C₂And (5) culturing in an incubator, and changing the culture solution once in 2-3 days until the cells are fully paved at the bottom of the culture bottle for testing.

3. Cytotoxicity of test Compounds

Adjusting the cell density of the cells in logarithmic growth phase to 1 × 10⁵one/mL, inoculated in a 96-well plate, placed at 37 ℃ in 5% CO₂After 24 hours of culture in an incubator, test compounds at different concentrations were added, and after 20 hours, the survival of cells was observed, and the toxicity of the compounds to the cells was quantitatively determined by the MTT method to determine the test concentration of the test compound.

Detection of NO inhibitory Activity

RAW264.7 cells in logarithmic growth phase were seeded in 96-well plates (1X 10) at an adjusted cell density⁴One/well), culturing for 24 hours, adding the compounds to be treated with different concentrations after the cells are completely attached to the wall, pretreating for 30min, adding LPS until the final concentration is 1.0 mu g/mL, and continuously culturing for 24 hours. And (3) taking 160 mu L of cell culture supernatant, adding 80 mu L of Griess reagent, measuring absorbance at the wavelength of 550nm according to a Griess method, and calculating the inhibition rate of each compound to NO according to the absorbance value and a standard curve.

Detection of IL-6 and TNF-alpha inhibitory Activity

RAW264.7 cells in logarithmic growth phase were seeded in 96-well plates (1X 10)⁵One/hole), culturing for 24 hours, adding the compounds to be treated with different concentrations after the cells are completely attached to the wall, pretreating for 30min, adding LPS until the final concentration is 10ng/mL, and continuously culturing for 24 hours. The extracted RNA was reverse transcribed into cDNA using ELISA kits for IL-6 and TNF- α determination, and mRNA expression level detection of IL-6 and TNF- α was performed using a real-time quantitative PCR method to determine the inhibitory effect of each compound on the inflammatory response induced by LPS.

6. Statistical method

All data were processed and analyzed using the SPSS (V17.0) statistical software package. Results are expressed as standard error of the mean. Calculating IC of each compound by nonlinear regression fitting of parameters such as each dose and inhibition rate₅₀The value is obtained.

TABLE 3 IC for inhibiting NO production and IL-6, TNF-alpha secretion by novel compounds 1-4₅₀Value (μ M)

^aDexamethasone (dexamethasone) was used as a positive control drug.

The data in Table 3 show that the new compounds 1-4 can obviously inhibit the generation of NO and/or the expression level of IL-6, TNF-alpha mRNA in mouse macrophage RAW264.7 induced by Lipopolysaccharide (LSP) and show dose dependence, and the compounds have obvious anti-inflammatory activity and can be used for preparing new anti-inflammatory activity medicaments.

The invention is not limited to the embodiments described above, many variations in detail are possible without departing from the scope and spirit of the invention.

Claims

1. A compound represented by the following structure:

2. a process for the preparation of a compound according to claim 1, comprising the steps of:

3. The method for producing a compound according to claim 2, wherein the Caragana tibetana is a red wood heart of a rhizome of Caragana microphylla (Caragana jubata) of Caragana of Leguminosae (Leguminosae), Caragana changii (Caragana changduensis) and Caragana chuanensis (Caragana tiebetica).

4. The preparation method of the compound according to claim 2, wherein the extraction method in the step (1) is heating reflux extraction, leaching, percolation or ultrasonic extraction, the used solvent is at least one of dichloromethane, chloroform, ethyl acetate, methanol, ethanol, methanol-water with the concentration of more than 60% or ethanol-water, and the weight-volume ratio of the medicinal materials to the solvent is 1: 4-1: 15.

5. The method for preparing the compound according to claim 2, wherein the organic solvent used in the extraction method of step (2) is any one of dichloromethane, chloroform, diethyl ether and ethyl acetate, and the volume ratio of the aqueous solution to the organic solvent is 1: 1-1: 2.

6. The method for preparing the compound according to claim 2, wherein the elution is carried out in a gradient of 25: 75 to 100:0 in a methanol/water system or in a gradient of 20: 80 to 90: 10 in an acetonitrile/water system in the step (3).

7. The method for preparing the compound according to claim 2, wherein the methanol/water or acetonitrile/water in the step (4) is eluted at a gradient of 30: 70 to 90: 10 by volume ratio.

8. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable excipient.

9. The use of the compound of claim 1 and the pharmaceutical composition of claim 8 in the preparation of anti-inflammatory and ischemic cardiovascular and cerebrovascular diseases drugs.

10. The use according to claim 9, wherein the anti-inflammatory agent is an agent that inhibits the activity of IL-6, TNF-a and NO production.