CN114591282A

CN114591282A - Pyrone compound separated from moldavica dragonhead and preparation method and application thereof

Info

Publication number: CN114591282A
Application number: CN202210181677.4A
Authority: CN
Inventors: 陈明华; 邢建国; 许艳妮; 陈渝川; 雷丽娟; 司书毅; 郑瑞芳; 肖同美; 闫璧滢; 苏冰洁; 李妍; 张晶
Original assignee: XINJIANG INSTITUTE OF MATERIA MEDICA; Institute of Medicinal Biotechnology of CAMS
Current assignee: XINJIANG INSTITUTE OF MATERIA MEDICA; Institute of Medicinal Biotechnology of CAMS
Priority date: 2022-02-25
Filing date: 2022-02-25
Publication date: 2022-06-07
Anticipated expiration: 2042-02-25
Also published as: CN114591282B

Abstract

The invention discloses an alpha-pyrone compound or a medicinal salt thereof, the structure of which is shown as a formula I. The invention also discloses a method for preparing the alpha-pyrone compound from the moldavica dragonhead. The invention adopts a KLF2 screening model and takes KLF2-COS7 cells as experimental objects, and results show that the alpha-pyrone compound has obvious effect on up-regulating the expression of KLF 2. The invention also discloses application of the alpha-pyrone compound or the medicinal salt thereof in preparing a medicament for up-regulating the expression of KLF 2. The invention also discloses the application of the alpha-pyrone compound or the medicinal salt thereof in preparing medicines for treating diseasesCardiovascular diseases or anti-inflammatory drugs.

Description

Pyrone compound separated from moldavica dragonhead and preparation method and application thereof

Technical Field

The invention belongs to the field of natural medicines, and particularly relates to an alpha-pyrone compound or a medicinal salt thereof prepared from plants, particularly labiate plants including moldavica dragonhead, but not limited to moldavica dragonhead, wherein the alpha-pyrone compound or the medicinal salt thereof can remarkably up-regulate the expression of target point KLF2 related to atherosclerosis, anti-inflammation and the like, and can be used for treating cardiovascular diseases including atherosclerosis resistance, thrombosis resistance, coronary heart disease, angina, myocardial infarction, stroke and the like.

Background

In recent years, with the improvement of the living standard of people, the morbidity of cardiovascular and cerebrovascular diseases is on a remarkable rising trend, the number of the cardiovascular and cerebrovascular disease deaths accounts for 1/3 of the number of the deaths of the global population, the cardiovascular diseases become the leading death reasons of urban and rural residents in China, and great economic burden is brought to the society and the residents. Of these, atherosclerotic disease has become the first "killer" in humans. Atherosclerosis (AS) is a chronic inflammatory disease, which is the common pathophysiological basis for ischemic cardiovascular and cerebrovascular diseases (such AS coronary heart disease, angina pectoris, myocardial infarction, stroke, etc.). At present, atherosclerosis in China has a high incidence and a young trend.

Although there are various AS-treating drugs, each drug has certain limitations, which limits the clinical application of the drug to some extent. KLF2(Krueppel-like factor 2) belongs to a member of the zinc finger family of DNA binding transcription factors, and is also called lung Krueppel-like transcription factor 2(LKLF2) because it is highly expressed in lung, and has a highly conserved zinc finger domain at C-terminus for binding to specific DNA sequences and intranuclear localization, and a highly dispersed non-DNA binding domain at N-terminus for transcription activation or transcription repression. KLF2 plays a very important regulatory role as a key "molecular switch" in vascular function and disease. KLF2 has multiple functions to negatively regulate the expression of inflammatory factors so as to play an anti-inflammatory role, and has the functions of maintaining the structure and the function of endothelial cells, preventing thrombosis, resisting oxidation and the like, thereby having close relation with atherosclerosis.

Natural products have been an important source of innovative drugs. Dracocephalum Moldavica L (Dracocephalum Moldavica L.) of Dracocephalum of Labiatae is a medicine commonly used by Uygur nationality in Xinjiang, has effects of benefiting heart and protecting brain, protecting liver and invigorating stomach, and relieving brain occlusion, and can be used for treating heart disease, hypertension and headache, with long history and definite therapeutic effect. In recent years, with the progress of research on moldavica dragonhead, attention has been paid to the effects of moldavica dragonhead on diseases such as coronary heart disease, atherosclerosis, myocardial ischemia, and the like.

Disclosure of Invention

The inventor carries out separation and purification of chemical components on an alcohol/water extract of the moldavica dragonhead based on systematic excavation of active components in the moldavica dragonhead for treating cardiovascular diseases, obtains an alpha-pyrone compound (named as moldapryrone A) from plant moldavica dragonhead for the first time, wherein the moldapyrone A is a novel phenolic acid component, and the compound is different from known alpha-pyrone compounds dumortins A-C in that the compound has one more carboxyl substituent on the carbon skeleton of the dumortins A-C and is a novel skeleton compound. The invention firstly evaluates the cardiovascular activity of the alpha-pyrone compound, and finds that the compound has obvious up-regulation activity on a target KLF2 closely related to atherosclerosis resistance, so the compound has better application prospect in the aspect of treating atherosclerosis related diseases.

The invention aims to provide an alpha-pyrone compound (recorded as moldapyrone A) or a medicinal salt thereof, wherein the structure of the alpha-pyrone compound is shown as a formula I:

the molecular formula of the compound is as follows: c₁₅H₁₂O₇The molecular weight is: 304.

the pharmaceutical salt of the alpha-pyrone compound comprises a salt formed by the alpha-pyrone compound and inorganic acid, organic acid, amino acid or sulfonic acid.

The inorganic acid is hydrochloric acid or sulfuric acid; the organic acid is acetic acid, trifluoroacetic acid, citric acid, maleic acid, oxalic acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, ascorbic acid or malic acid; the amino acid is alanine, aspartic acid and lysine; the sulfonic acid is methanesulfonic acid or p-toluenesulfonic acid.

Another object of the present invention is to provide a method for preparing the α -pyrone compound, comprising: taking dry moldavica dragonhead herb, adding 0-60% ethanol according to the weight-volume ratio of 1:3 of the dry moldavica dragonhead herb to 0-60% ethanol, and performing reflux extraction for 2-3 times to obtain an extracting solution; concentrating the extract until no alcohol smell exists to obtain suspension, and adding distilled water to obtain clear solution; subjecting the clear solution to macroporous adsorbent resin column chromatography, gradient eluting with water, 30% ethanol, 50% ethanol and 95% ethanol to obtain 4 eluting parts: water-eluting part, 30% ethanol-eluting part, 50% ethanol-eluting part, and 95% ethanol-eluting part; extracting the 50% ethanol eluate with ethyl acetate for 3 times to obtain ethyl acetate fraction; purifying the ethyl acetate extraction part by silica gel column chromatography, Sephadex LH-20 column chromatography and semi-preparative HPLC to obtain the compound shown in the formula I.

Preferably, the dried moldavica dragonhead herb is taken, 40% ethanol is added according to the weight-volume ratio of the dried moldavica dragonhead herb to the 40% ethanol of 1:3(kg/L or g/mL), and the reflux extraction is carried out for 3 times.

The volume ratio of the suspension to distilled water is 1: 2.

The macroporous adsorption resin is HP20 macroporous adsorption resin.

The filler of the silica gel column chromatography is 200-300 mesh silica gel, the eluent is dichloromethane and methanol, the ratio of dichloromethane to methanol is 100: 0-0: 100V: V, and 15 fractions are obtained and recorded as: XQL-1 to XQL-15. And (5) carrying out Sephadex LH-20 column chromatography on the fraction XQL-8.

The eluent of Sephadex LH-20 column chromatography is dichloromethane and methanol with the ratio of 1: 1V: V, and 11 sub-streams are obtained and are marked as XQL-8-1 to XQL-8-11. The retentate XQL-8-7 was subjected to semi-preparative HPLC.

The chromatographic column of the semi-preparative HPLC is as follows: CAPCELL PAK PFP (particle size 5 μm, internal diameter 10 mm. times. length 250mm), acetonitrile in 0.1% aqueous trifluoroacetic acid at 36: 64V: V, and flow rate of 1.5 ml/min.

The invention adopts a KLF2 screening model, uses KLF2-COS7 cells as an experimental object, screens the moldavica dragonhead extract, extracts fractions and separated alpha-pyrone compounds shown in formula I to up-regulate the expression activity of KLF 2. Experimental results show that the alpha-pyrone compound has a remarkable effect on up-regulating the expression of KLF 2.

The invention also aims to provide the application of the alpha-pyrone compound or the medicinal salt thereof in preparing medicaments for up-regulating the expression of KLF 2.

The invention also aims to provide the application of the alpha-pyrone compound or the medicinal salt thereof in preparing medicaments for treating cardiovascular diseases or resisting inflammation.

The cardiovascular diseases include atherosclerosis, thrombosis, coronary heart disease, angina pectoris, myocardial infarction and apoplexy.

The invention also aims to provide a pharmaceutical composition which is prepared by taking the alpha-pyrone compound or the pharmaceutical salt thereof as an active ingredient or a main effective ingredient and one or more pharmaceutically acceptable carriers.

The application of the pharmaceutical composition in preparing a medicament for up-regulating the expression of KLF 2.

The application of the medicinal composition in preparing medicaments for treating cardiovascular diseases or resisting inflammation.

The alpha-pyrone compound or the medicinal salt thereof can also form a compound preparation with the known medicine for up-regulating the expression of KLF2 or treating cardiovascular diseases.

In the pharmaceutical composition, the weight percentage of the alpha-pyrone compound or the pharmaceutical salt thereof is 0.1-99.9%, and the weight percentage of the pharmaceutically acceptable carrier is 0.1-99.9%.

The pharmaceutical composition is in a form suitable for pharmaceutical use.

The preparation is tablets, capsules, granules, pills, powder, ointment, suspension, injection, powder injection, suppository, cream, drops or patches. Wherein the tablet is a sugar-coated tablet, a film-coated tablet, an enteric-coated tablet or a sustained-release tablet; the capsule is a hard capsule, a soft capsule or a slow release capsule; the powder injection is freeze-dried powder injection.

The pharmaceutical composition is in a preparation form, and each dose of the pharmaceutical composition contains 0.1-1000 mg of the alpha-pyrone compound or the pharmaceutical salt thereof. Although the amount of active ingredient contained in the dosage unit form may vary, it is generally adjusted within the range of 1 to 800mg, depending on the potency of the active ingredient selected. The preferred dosage for a given situation can be determined by one skilled in the art in a routine manner. Generally, the amount of the active ingredient to be initially treated is lower than the optimum dose of the active ingredient, and then the dose to be administered is gradually increased until the optimum therapeutic effect is achieved. The total daily dose may be administered once or in divided doses for therapeutic purposes.

Each dose means each unit of preparation, such as each tablet of tablet, each capsule, or each dose, such as 100mg per dose.

The pharmaceutical compositions of the present invention may be formulated as solid or semi-solid pharmaceutical preparations in the form of powders, tablets, dispersible powders, capsules, cachets, suppositories, and ointments, using a solid carrier. The solid carrier is selected from one or more of diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, bulking agents, and the like, or may be an encapsulating material. In the powdery preparation, 5 to 70% of micronized active ingredients are contained in a carrier. The solid carrier comprises magnesium carbonate, magnesium stearate, talcum powder, sucrose, lactose, pectin, dextrin, starch, gelatin, methylcellulose, sodium carboxymethylcellulose, low boiling point wax, cacao butter, etc. Because of their ease of administration, tablets, powders, cachets, capsules and the like represent the most advantageous oral solid dosage forms.

Liquid formulations of the present invention include solutions, suspensions and emulsions. For example, parenteral injection preparations may be in the form of water or water-propylene glycol solutions, which are adjusted in isotonicity, pH, etc. to suit the physiological conditions of the living body. The liquid preparation can also be prepared into solution in polyethylene glycol or water solution. Aqueous solutions for oral administration can be prepared by dissolving the active ingredient in water, followed by the addition of suitable amounts of coloring, flavoring, stabilizing and thickening agents. Aqueous suspensions suitable for oral administration can be prepared by dispersing the micronized active ingredient in viscous materials such as natural and synthetic gums, methylcellulose, sodium carboxymethylcellulose, and other known suspending agents.

It is particularly advantageous to formulate the above pharmaceutical preparations in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form of a formulation refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect. Such dosage unit forms may be in the form of a pack, such as a tablet, capsule or powder in a small tube or vial, or an ointment, gel or cream in a tube or bottle.

Detailed Description

Embodiments of the present invention are suitable for the preparation of compounds of formula I from plants, not limited to, moldavica dragonhead. The following examples are intended to assist the skilled person in a better understanding of the technical solutions of the present invention, but are not intended to limit the invention in any way.

Example 1

Extraction:

taking 40kg of dried moldavica dragonhead herb, carrying out reflux extraction for 3 times by 3 times (120L) of 40% ethanol to obtain an extracting solution, concentrating the extracting solution until no alcohol smell exists to obtain 20L of suspension, and adding 40L of distilled water to the suspension to obtain 60L of clear solution.

Isolation, preparation of Compounds of formula I:

passing the clear solution through HP-20 macroporous adsorbent resin column, discarding unadsorbed solution, gradient eluting with water, 30% ethanol, 50% ethanol and 95% ethanol to obtain 4 eluting parts: water-eluting part, 30% ethanol-eluting part, 50% ethanol-eluting part, and 95% ethanol-eluting part; extracting the 50% ethanol eluate with ethyl acetate for 3 times to obtain ethyl acetate eluate (41.1 g); separating the ethyl acetate extraction part by silica gel (200-300 meshes) column chromatography, and performing gradient elution by using dichloromethane/methanol (100: 0-0: 100 (V: V)) as an eluent to obtain 15 fractions (marked as XQL-1-XQL-15); subjecting fraction XQL-8 to Sephadex LH-20 column chromatography, eluting with dichloromethane/methanol at ratio of 1: 1 (V: V) to obtain 11 sub-fractions (marked as XQL-8-1-XQL-8-11); subfraction XQL-8-7 was subjected to semi-preparative HPLC, column: CAPCELL PAK PFP (5 μm,10 mm. times.250 mm), mobile phase: acetonitrile/0.1% trifluoroacetic acid water 36: 64 (V: V), flow rate: 1.5ml/min to give Compound 1.

Structural characterization of compound 1:

compound 1 is a white amorphous powder, ESI-MS gives the peak of the excimer ion M/z 305.0[ M + H ]]⁺HRESIMS m/z 305.0663, suggesting that its molecular composition is C₁₅H₁₂O₇(calcd for C₁₅H₁₃O₇305.0656) having an unsaturation of 10. IR Spectrum suggests that Compound 1 contains a hydroxyl group(3177cm^-1) Carboxylic acid (3000 + 2500 cm)^-1Strong and broad absorption peak), carbonyl (1720 cm)^-1) And benzene rings (1600,1451 cm)^-1) Characteristic absorption peak of (2). Process for preparation of Compound 1¹H NMR spectrum of the compound shows an ortho-trisubstituted benzene ring [ delta ]_H6.74(d, J-7.8 Hz, H-4'), 6.71(d, J-7.8 Hz, H-6'), and 7.18(dd, J-7.8 Hz, H-5')]Two isolated aromatic hydrogens [ delta ]_H7.06(s, H-3) and 6.30(s, H-5)]And two methylene groups [ delta ] linked_H2.89(brt, J-9.0 Hz, H-7') and 2.73(brt, J-9.0 Hz, H-8')]The proton resonance signal of (1). According to¹³The C NMR spectrum and HSQC spectrum confirmed that in addition to the carbons corresponding to the above hydrogen protons in Compound 1, 8 quaternary carbon signals were observed (see Table 1). The HMBC spectrum of compound 1 shows heteronuclear remote correlation signals of H-4' with C-3' and C-6', H-5' with C-1' and C-3', H-6' with C-1', C-2', C-4' and C-7', H-7' with C-1', C-2' and C-3', and H-8' with C-1', and the chemical shifts of these carbons and protons are very similar to the salicylic acid analogues reported in the reference, indicating that the compound contains a 2' -carboxy-3' -hydroxyphenoxynetyl unit. Meanwhile, heteronuclear remote correlation signals of H-3 and C-2 and C-5, and H-5 and C-3, C-6 and C-7 can be observed in an HMBC spectrum, and the compound contains a 3-substituted 6-carboxy-alpha-pyrone unit by combining the chemical shifts of the protons and the carbon and the molecular composition. Furthermore, the signals associated with H-7 'and C-4, H-8' and C-3, C-4 and C-5, and H-3 and H-5 and C-8 'in the HMBC spectra show that the two units are linked by a C-4-C-8' linkage. Therefore, the compound is determined to be 4- (2 '-methoxy-3' -hydroxypropyl) -2-oxo-2H-pyran-6-carboxylic acid and is named moldapyrone A.

The structural formula of the compound 1 is shown as the formula I:

TABLE 1 preparation of Compound 1¹H NMR and¹³c NMR data (DMSO-d)₆,600MHz)

EXAMPLE 3 test of the ability of Compound 1 to upregulate the expression of KLF2

This experiment was carried out using KLF2 screening model, COS7 cells (KLF2-COS7) transfected with KLF2 plasmid as the subject, Luciferase (Bright-Lite Luciferase Assay System, Nanjing Nodezam Biotech Co., Ltd.; batch No. 7E512L1) as the reporter gene, Luminescence intensity of the reaction between Luciferase and the substrate measured using an EnVision multifunctional microplate reader, and the Luminescence intensity was expressed as the value of Relative Luminescence Unit (RLU). Blank control wells (no test sample added) were set during the experiment, and the ratio of fluorescence intensity of the drug added wells to that of the blank control wells was the fold value of the drug upregulation KLF 2.

The specific experimental method is as follows: COS7 cells in logarithmic growth phase were digested, diluted in 10% fetal bovine serum DMEM medium and the cells counted at 5X 10⁵And (2) simultaneously transiently transfecting a KLF2 plasmid, inoculating a 96-well plate at a rate of 150 mu L/well, replacing the culture medium with a DMEM culture medium containing 5% fetal bovine serum at a rate of 200 mu L/well after cells adhere to the wall 4-6h, respectively adding 1 mu L of compound 1 dissolved in DMSO, wherein the concentration of the compound 1 in the adding well is 100 mu M, and detecting after 18-24 h. Compounds with a fold-up greater than 1.5 were identified as having activity of up-regulating KLF2, with a fold-up greater than 2 for Compound 1, and were further tested for EC₅₀: diluting with 5% fetal bovine serum DMEM medium to concentration gradients of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 and 0.078 μ M at 200 μ L/well, detecting, and preparing EC with GraphPad Prism 8 software₅₀Curve line.

The experimental results are as follows: on a KLF2 screening model, the compound 1 has a remarkable up-regulation effect on KLF2, and can up-regulate the expression of KLF2 by 5.1 times at the concentration of 100 mu M, and the EC of the compound is₅₀It was 59.5. mu.M.

Claims

1. An alpha-pyrone compound or a medicinal salt thereof with a structure shown as formula I:

2. the α -pyrone compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein: the medicinal salt of the alpha-pyrone compound comprises a salt formed by the alpha-pyrone compound and inorganic acid, organic acid, amino acid or sulfonic acid.

3. The α -pyrone compound or a pharmaceutically acceptable salt thereof according to claim 2, wherein: the inorganic acid is hydrochloric acid or sulfuric acid; the organic acid is acetic acid, trifluoroacetic acid, citric acid, maleic acid, oxalic acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, ascorbic acid or malic acid; the amino acid is alanine, aspartic acid and lysine; the sulfonic acid is methanesulfonic acid or p-toluenesulfonic acid.

4. A method for producing an α -pyrone compound according to claim 1, characterized in that: the method comprises the following steps: taking dry moldavica dragonhead herb, adding 0-60% ethanol according to the weight-volume ratio of 1:3 of the dry moldavica dragonhead herb to 0-60% ethanol, and performing reflux extraction for 2-3 times to obtain an extracting solution; concentrating the extract until no alcohol smell exists to obtain suspension, and adding distilled water to obtain clear solution; subjecting the clear solution to macroporous adsorbent resin column chromatography, and gradient eluting with water, 30% ethanol, 50% ethanol and 95% ethanol to obtain 4 eluting parts: water-eluting part, 30% ethanol-eluting part, 50% ethanol-eluting part, and 95% ethanol-eluting part; extracting the 50% ethanol eluate with ethyl acetate for 3 times to obtain ethyl acetate fraction; purifying the ethyl acetate extraction part by silica gel column chromatography, Sephadex LH-20 column chromatography and semi-preparative HPLC to obtain the compound shown in the formula I.

5. The method for producing an α -pyrone compound according to claim 4, characterized in that: the macroporous adsorption resin is HP20 macroporous adsorption resin.

6. The method for producing an α -pyrone compound according to claim 4, characterized in that: the eluent of the silica gel column chromatography is dichloromethane and methanol, wherein the ratio of dichloromethane to methanol is 100: 0-0: 100V: V;

the eluent of Sephadex LH-20 column chromatography is dichloromethane and methanol with the ratio of 1: 1V: V;

the chromatographic column of the semi-preparative HPLC is as follows: CAPCELL PAK PFP (5 μm,10 mm. times.250 mm) and a mobile phase of acetonitrile in 0.1% aqueous trifluoroacetic acid at 36: 64V: V and a flow rate of 1.5 ml/min.

7. The use of the α -pyrone compound according to claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a medicament that upregulates KLF2 expression.

8. Use of the α -pyrone compound according to claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating cardiovascular diseases or for anti-inflammatory treatment.

9. Use according to claim 8, characterized in that: the cardiovascular diseases include atherosclerosis, thrombosis, coronary heart disease, angina pectoris, myocardial infarction and apoplexy.

10. A pharmaceutical composition comprising the α -pyrone compound according to claim 1 or a pharmaceutically acceptable salt thereof.