CN112225688A

CN112225688A - 4-hydroxypyridones compounds, and preparation method and application thereof

Info

Publication number: CN112225688A
Application number: CN202011120193.6A
Authority: CN
Inventors: 秦盛莹; 张维阳; 于洋; 陈国栋; 胡丹; 高昊
Original assignee: Jinan University
Current assignee: Jinan University; University of Jinan
Priority date: 2020-10-19
Filing date: 2020-10-19
Publication date: 2021-01-15

Abstract

The invention relates to a novel 4-hydroxypyridones compound, a preparation method and application thereof. The invention discloses a structure and a preparation method thereof, and further discloses an application of the structure and the preparation method in preparation of medicines for treating oxidative stress related diseases.

Description

4-hydroxypyridones compounds, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of natural products, and particularly relates to a 4-hydroxypyridone compound, and a preparation method and application thereof.

Background

Oxidation in an organism refers to the process of losing electrons or gaining oxygen atoms, which raises the oxidation state of biomolecules, atoms or ions in molecules, and forms free radicals in the organism. Imbalance in the human redox process results in the production of excessive free radicals, which in turn trigger a series of antioxidant defense responses in the body, a process known as oxidative stress. Oxidative stress is associated with the development of a number of diseases, such as coronary heart disease, atherosclerosis, parkinson's disease, stroke, and the like. Therefore, antioxidants and radical scavenging agents have been widely used in the medical field.

Natural small molecular compounds from fungi are important sources of medicinal lead compounds. The fungi evolve a unique secondary metabolic pathway in the process of long-term adaptation to the environment or symbiosis and parasitism, so that the secondary metabolites of the fungi have specific chemical structures and various biological activities, and natural products from the fungi have a plurality of precedent cases for preparing medicaments. Therefore, the search for new natural active compounds from fungal secondary metabolites is an important drug discovery approach.

Torticollis (Tolypocladium sp.) is a new genus established by W.gams in 1971, and is characterized in that the base of a phialide is expanded in a spherical or elliptical shape, the neck of the phialide is slender and bent, conidia are single or cohered into a small head shape, and up to now, 43 species of torticollis are recorded in an international fungus directory database. The campylobacter is an important entomogenous fungus, is wide in ecological distribution and various in host types, is found to be an anamorph of cordyceps, is closely related to the cordyceps, and is classified into nematology (Ophiocordipi ceae) by some famous mycologists internationally in 2014 based on a new classification system of molecular biology, and more importantly, the campylobacter can produce various insecticidal and antibacterial active ingredients, such as cyclosporine (cyclosporine), macrocystin (Ophiocordin, a strong protease inhibitor), cyclopeptide (efrapeptin), Ophiostein (tetrapolyaminic acid), various insecticides and the like.

Disclosure of Invention

Object of the Invention

The invention aims to systematically prepare, separate and identify a fermentation extract of a fungus of the genus Tolypocladium sp, and search for a drug or a lead compound having functions of resisting oxidation and scavenging free radicals.

Technical scheme

In a first aspect of the present invention, there is provided a 4-hydroxypyridone compound, or a pharmaceutically acceptable salt, solvate, or metabolite thereof, wherein the 4-hydroxypyridone compound is represented by formula (I):

wherein R is H, optionally substituted C₁-C₄Alkyl, optionally substituted C₁-C₄Alkoxy, -OH, p-hydroxyphenyl (i.e.

) Any one or more of them; preferably, R is H or p-hydroxyphenyl (i.e.

)；

Further, the compound has the following structure shown in formula 1 or formula 2:

further, the pharmaceutically acceptable salt is a salt prepared by reacting the 4-hydroxypyridone compound with a chemically acceptable acid or base; further, the chemically acceptable acid is an inorganic acid or an organic acid; further preferably, the inorganic acid is selected from any one of hydrochloric acid, sulfuric acid, nitric acid and hydrobromic acid; or further preferably, the organic acid is selected from any one of acetic acid, propionic acid, malonic acid, butyric acid, lactic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, maleic acid, benzoic acid, succinic acid, picric acid, tartaric acid, citric acid and fumaric acid;

or further, the pharmaceutically acceptable salt is a salt prepared by reacting the 4-hydroxypyridone compound with a chemically acceptable alkali; further, the chemically acceptable base is an inorganic base or an organic base; further preferably, the inorganic base is any one selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate and potassium bicarbonate; or further preferably, the organic base is selected from any one of trimethylamine, triethylamine and pyridine;

further preferably, the pharmaceutically acceptable salt can be any one or more of potassium salt, sodium salt, ammonium salt, calcium salt, pyridine salt and choline salt;

a second aspect of the present invention provides a method for preparing any one of the above 4-hydroxypyridones compounds or a pharmaceutically acceptable salt, solvate or metabolite thereof, comprising: the extract is obtained by fermenting fungus of Tolypocladium sp with microorganism, and separating by chromatography;

further, the preparation method of the 4-hydroxypyridone compound or the pharmaceutically acceptable salt, the solvate or the metabolite thereof comprises the following steps:

s1: inoculating a strain of a fungus of the genus Tolypocladium sp in a culture medium for culturing to obtain a fermentation product;

s2: soaking the fermentation product obtained in the step S1 in 70-95% v/v ethanol for 10-15 h, then ultrasonically extracting for 1-3 times, each time for 10-30 min, combining the extracting solutions, concentrating and drying to obtain a crude extract;

s3: separating and identifying the crude extract obtained in the step S2 by silica gel column chromatography and preparative high performance liquid chromatography (namely preparative HPLC);

further, the operation of S1 includes the following steps: activating the strain by a potato dextrose agar slant culture medium (namely PDA slant), culturing in a potato dextrose liquid culture medium (namely PDB culture medium) for 3-7 days at 10-30 ℃, then inoculating into a rice culture medium, and standing and culturing for 24-60 days at 10-30 ℃ to obtain a fermentation product; preferably, the culture temperature in both PDA and PDB is 25 ℃; preferably, the culture is carried out in PDB for 7 days; preferably, standing and culturing in rice culture medium for 60 days;

further, the ethanol concentration in S2 is 95% v/v;

further, the soaking time in the step S2 is 12 hours;

further, in the step S2, the ultrasonic extraction times are 3 times, and the extraction time is 30 min;

further, the operation of separating by silica gel column chromatography in S3 specifically includes: and (4) separating the crude extract obtained in the step S2 through a silica gel column, performing gradient elution by adopting a dichloromethane-methanol solution, and sequentially performing gradient elution by using methanol: the dichloromethane volume ratio of 1:99,2:98,5:95,10:90,15:85,20:80,25:75,30:70,50:50,100:0 are eluted to obtain 12 fractions Fr.1-12, wherein the 9 th sub-fraction is methanol: fraction fr.9 obtained by elution with a dichloromethane volume ratio of 50: 50;

further, the preparative HPLC separation and identification process in S3 specifically includes: the method comprises the following steps of mixing methanol: the volume ratio of water is 75: 25 preparing a solution as a mobile phase fraction Fr.9, performing isocratic elution at a flow rate of 3.0mL/min, detecting with an ultraviolet detector at a wavelength of 210nm or 280nm to obtain compound 1, tolypyridone C, and t of tolypyridone C_RIs 32.0 min; and compound 2 is tolypyridone D, t thereof_RIs 26.9 min;

further, the preparative HPLC column used is Cosmosil 5C18, the specification is phi 10.0X 250mm, and the particle size of the packing is 5 μm);

further, the preparative HPLC employs a column temperature of 30 ℃;

a third aspect of the present invention provides a pharmaceutical composition comprising a 4-hydroxypyridone compound according to any one of the above, or a pharmaceutically acceptable salt, solvate or metabolite thereof;

further, the 4-hydroxypyridone compound or the pharmaceutically acceptable salt, the solvate or the metabolite thereof in the pharmaceutical composition accounts for 0.01-90% of the pharmaceutical composition; preferably, the 4-hydroxypyridone compound or the pharmaceutically acceptable salt, the solvate or the metabolite thereof in the pharmaceutical composition accounts for 0.1-20% of the pharmaceutical composition; further preferably, the content of the 4-hydroxypyridone compound or the pharmaceutically acceptable salt, the solvate or the metabolite thereof in the pharmaceutical composition accounts for 1-10% of the pharmaceutical composition;

further, although the compound of the present invention can be administered directly without any formulation, it is preferably prepared with a pharmaceutically acceptable carrier for use in pharmaceutical preparations; further preferably, the pharmaceutically acceptable carrier comprises diluents, lubricants, binders, disintegrants, stabilizers, solvents; further, the diluents of the present invention include, but are not limited to, starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, glucose, etc.; still further, the lubricants include, but are not limited to, magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamers, and the like; still further, the binder includes, but is not limited to, water, ethanol, starch slurry, syrup, hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose, sodium alginate, polyvinyl pyrrolidone, etc.; further, the disintegrant includes, but is not limited to, starch, sodium bicarbonate and/or citric acid, tartaric acid, low substituted hydroxypropylcellulose, and the like; further, the stabilizing agent includes, but is not limited to, polysaccharides such as acacia gum, agar, alginic acid, cellulose ethers, carboxymethyl chitin ester, and the like; still further, the solvent includes, but is not limited to, water, balanced salt solutions, and the like;

further, the pharmaceutical composition is an oral preparation or an injection; further preferably, the oral preparation is selected from any one of common tablets, dispersible tablets, enteric-coated tablets, granules, capsules, dripping pills, powder, oral liquid and emulsion; or further preferably, the injection is small water injection, infusion solution or freeze-dried powder injection;

the fourth aspect of the present invention provides a use of any one of the above 4-hydroxypyridones compounds, or pharmaceutically acceptable salts, solvates, or metabolites thereof, or pharmaceutical compositions thereof, in the preparation of a medicament for treating a disease associated with oxidative stress;

further, the oxidative stress related diseases include, but are not limited to, coronary heart disease, atherosclerosis, parkinson's disease, or stroke.

Drawings

FIG. 1 oxygen radical absorbance Capacity plots for Compounds 1 and 2

Advantageous effects

According to the invention, two new compounds are obtained by separating and structurally identifying the extract of the fungus of the torticola (Tolypocladium sp.), and pharmacological activity tests are carried out on the two new compounds, so that the two new compounds have good free radical scavenging activity and antioxidant effect, can be used for preparing medicines for treating oxidative stress related diseases, and have good pharmaceutical application prospects.

Detailed Description

The present invention will be illustrated in detail below. It is noted that the examples illustrate some methods of preparation or use, however, it is to be understood that these examples do not limit the invention. The scope of the present invention is defined by the appended claims.

1. The source of the strain

Is derived from a fungus of the genus Tolypocladium sp.

2. Laboratory apparatus

EYELA rotary evaporator N-1001 (Rikakikai, Tokyo); dionex analytical and preparative HPLC (Thermo Fisher Scientific, USA); cosmosil 5C18 analytical chromatography columns (10 × 250mm,5 μm) and preparative chromatography columns (20 × 250mm,5 μm) (Nacalai Tesque, japan); JASCO V-550 UV tester available from Jasco International, Japan; JASCO FT/IR-480plus Infrared tester available from Jasco International, Japan; JASCO P-1020 optical rotation measuring instrument is available from Jasco International, Japan; bruker AV-600MHz NMR spectrometer from Bruker BioSpin, Switzerland; waters Synapt G2 TOF high resolution mass spectrometer was purchased from Waters corporation, USA.

3. Experimental Material

Analytically pure cyclohexane, ethyl acetate and ethanol are purchased from Fuyu fine chemical Co., Ltd, Tianjin; deuterated dimethyl sulfoxide was purchased from Sigma-Aldrich, Germany; chromatographic grade methanol was purchased from Shandong Yuwang industries, Inc.; ODS (50 μm) was purchased from YMC, Japan; silica gel GF254 and column chromatography silica gel (200-300 mesh) for thin layer chromatography are products of Qingdao ocean chemical plant;

the following abbreviations are used throughout the specification:

t_R: retention time

DMSO-d₆: deuterated dimethyl sulfoxide

δ_H: chemical shift of hydrogen

δ_C: compound shift of carbon

Example 1: fermentation and extraction of a fungus of the genus Tolypocladium sp

(1) Activating Tolypocladium sp strain with PDA slant, inoculating into PDB culture medium, and culturing at 25 deg.C for 200r min^-1Performing shake culture for 3d to prepare seed solution, inoculating the seed solution into 20 bottles of rice culture medium (the rice culture medium comprises 70 g/bottle of rice and 110 mL/bottle of purified water) according to the inoculation amount of 5 mL/bottle, and standing and culturing at 25 deg.C for 60 days to obtain fermented product;

(2) soaking the fermented product in 95% ethanol for 12h, ultrasonic extracting for 3 times (30 min each time), and concentrating the extractive solution under reduced pressure to dry to obtain crude extract (20.1g)

Example 2: preparation of Compounds 1(tolypyridone C) and 2(tolypyridone D)

(1) The crude extract was isolated by silica gel column chromatography using a dichloromethane-methanol gradient eluting with methanol: the dichloromethane volume ratio is 1:99,2:98,5:95,10:90,15:85,20:80,25:75,30:70,50:50 and 100:0, and 12 fractions Fr.1-12 are obtained.

(2) Preparing sub-fraction Fr.9 by HPLC, and collecting the fraction with Cosmosil 5C18 and specification of phi 10.0250mm, the grain diameter of the filler is 5 mu m), and the column temperature is 30 ℃; and (3) eluting by using a methanol-water solution as a mobile phase, wherein the ratio of methanol: the volume ratio of water is 75: 25 at a flow rate of 3mL/min, and detecting with an ultraviolet detector at a wavelength of 210nm or 280nm to obtain compound 1 as lyopyridone C (t)_R32.0min, about 30 mg), compound 2 is tolypyridone D (t)_R26.9 minutes, about 27 mg).

Example 3:

the structural identification of compounds 1 and 2 in this example is as follows:

compound 1

Light yellow needle crystal with melting range 168-171 deg.c;

+7.5(c 2.00MeOH)；UV(MeOH)λ_max(logε)246nm(1.31),344nm(0.77)；IR(KBr)v_max 2962,2923,1647,1610,1547,1509,1455,1437,1261,1219,832cm^-1；HR-ESI-MS m/z[M+H]⁺356.1870(calcd for C₂₁H₂₆NO₄,356.1862).¹h and¹³c NMR data are shown in Table 1;

compound 2

A light yellow oil;

-36.3(c 3.00MeOH)；UV(MeOH)λ_max(logε)229nm(1.33),269nm(0.86),325nm(1.26)；IR(KBr)v_max 2959,2929,1647,1636,1553,1467,1375,1348,1231,973cm^-1；HR-ESI-MS m/z 264.1595[M+H]⁺(calculated for C₁₅H₂₂NO₃,264.1600).¹h and¹³c NMR data are shown in Table 1;

TABLE 1 NMR spectra of Compounds 1 and 2 (DMSO-d)₆,600MHz for ¹H and 150MHz for ¹³C,J in Hz)

Example 4: oxygen radical absorbance Capacity test of Compounds 1 and 2

(1) Principle of experiment

Oxygen radical absorbance test total antioxidant capacity of a material was determined using Trolox (6-hydroxy-2, 5,7, 8-tetramethylchroman-2-carboxylic acid), a water-soluble analog of alpha-tocopherol, as a standard control. The fluorescent substance, namely, the phosphor, can emit 527nm fluorescence under the excitation of 485nm light. AAPH [2,2' -azo (diamidinopropane) dihydrochloride ] releases peroxy radicals (ROO.) and oxidizes disodium Fluoroscein (FL), thereby eliminating the fluorescent property. Antioxidants compete with discodium fluoroscein for oxidizing agents, slowing the rate of fluorescence decay. The oxygen radical absorbing ability of the compound was determined based on this characteristic.

75mM pH 7.4 Phosphate Buffer (PBS): weighing 5.103g KH respectively₂PO₄，8.56g K₂HPO₄·3H₂O, mixed, deionized water to 500mL and adjusted to pH 7.4 with 1mol/L HCl.

Solution of iodine Fluorochein (FL): 0.237mg of FL was mixed with 1mL of the above potassium phosphate buffer solution to prepare a 0.63mM stock solution, which was stored at 4 ℃ in the dark. When the solution is used, the solution is diluted 1000 times by the potassium phosphate buffer solution to prepare 630nM working solution.

Trolox solution: 1mg of Trolox was added to 199.8. mu.L of the above potassium phosphate buffer to obtain 0.02M of a mother liquor. mu.L of the mother solution was taken, and 799. mu.L of the buffer solution was added to dilute the solution to 25. mu.M, and 50. mu.L of the 25. mu.M solution was further added to 50. mu.L of the buffer solution to obtain 100. mu.L of a 12.5. mu.M working solution.

AAPH solution: 0.248g of AAPH was dissolved in 50mL of the above potassium phosphate buffer to obtain 18.3mM working solution.

Test monomer compounds 1 and 2:taking appropriate amount of monomer compound, respectively preparing into 2 × 10^-2And taking a dimethyl sulfoxide solution in mol/L as a mother solution. mu.L of the mother solution was added to 799.5. mu.L of buffer solution to dilute the solution to 12.5. mu.M.

(2) Experimental methods

The experiments were divided into 5 groups: the blank control group, the negative control group, the standard control group, the positive drug group and the test drug group, all tests were 4 replicates, and the detailed experimental design is shown in table 2 below.

Table 2 detailed experimental design

(3) Results of the experiment

Calculation and comparison of Experimental results reference is made to the method published by Amorti et al^[1]. As shown in fig. 1, when an oxygen radical absorbance plot is plotted with a fluorescence value at 527nm as an abscissa and a time as an ordinate, the oxygen radical absorbance capacity (3.14) of compound 1 was comparable to that of epigallocatechin gallate (EGCG 3.96), and compound 2(0.95) exhibited the corresponding activity, but to a lesser extent than compound 1.

In the description herein, references to the description of the term "example," "some embodiments," or "examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the example or example is included in at least one example or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction. The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the concept of the present invention, and these improvements and modifications should also be considered as within the protection scope of the present invention.

Reference to the literature

[1]Amorati,R.；Valgimigli,L.,Advantages and limitations of common testing methods for antioxidants.Free Radical Research 2015,49,(5),633-649.

Claims

1. A4-hydroxypyridone compound or a pharmaceutically acceptable salt, solvate or metabolite thereof, wherein the hydroxypyridone compound is represented by formula (I):

) Any one or more of them.

2. The 4-hydroxypyridone compound according to claim 1, wherein the 4-hydroxypyridone compound is represented by formula 1 or formula 2:

3. the 4-hydroxypyridone compound according to claim 1 or 2, wherein the pharmaceutically acceptable salt is a salt obtained by reacting the 4-hydroxypyridone compound with a chemically acceptable acid or base; further, the chemically acceptable acid is an inorganic acid or an organic acid; further preferably, the inorganic acid is selected from any one of hydrochloric acid, sulfuric acid, nitric acid and hydrobromic acid; or more preferably, the organic acid is selected from any one of acetic acid, propionic acid, malonic acid, butyric acid, lactic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, maleic acid, benzoic acid, succinic acid, picric acid, tartaric acid, citric acid and fumaric acid.

4. The 4-hydroxypyridone compound and the pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the pharmaceutically acceptable salt is a salt obtained by reacting the 4-hydroxypyridone compound with a chemically acceptable base; further, the chemically acceptable base is an inorganic base or an organic base; further preferably, the inorganic base is any one selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate and potassium bicarbonate; or more preferably, the organic base is selected from any one of trimethylamine, triethylamine and pyridine.

5. The 4-hydroxypyridone compound according to claim 1 or 2, wherein the pharmaceutically acceptable salt is any one or more of potassium salt, sodium salt, ammonium salt, calcium salt, pyridinium salt, and choline salt.

6. A method for preparing a 4-hydroxypyridone compound or a pharmaceutically acceptable salt, solvate or metabolite thereof according to any one of claims 1 to 5, comprising: the extract is obtained by fermenting a fungus of the genus Tolypocladium sp with a microorganism, and separating the extract by chromatography.

7. The method of claim 6, wherein the method comprises the steps of:

s1: inoculating a strain of a fungus of the genus Tolypocladium sp in a culture medium for culturing to obtain a fermentation product; further, the operation of S1 includes the following steps: activating the strain by a potato dextrose agar slant culture medium (namely PDA slant), culturing in a potato dextrose liquid culture medium (namely PDB culture medium) for 3-7 days at 10-30 ℃, then inoculating into a rice culture medium, and standing and culturing for 24-60 days at 10-30 ℃ to obtain a fermentation product;

s3: separating and identifying the crude extract obtained in the step S2 by silica gel column chromatography and preparative high performance liquid chromatography (namely preparative HPLC); further, the operation of separating by silica gel column chromatography in S3 specifically includes: and (4) separating the crude extract obtained in the step S2 through a silica gel column, performing gradient elution by adopting a dichloromethane-methanol solution, and sequentially performing gradient elution by using methanol: the dichloromethane volume ratio of 1:99,2:98,5:95,10:90,15:85,20:80,25:75,30:70,50:50,100:0 are eluted to obtain 12 fractions Fr.1-12, wherein the 9 th sub-fraction is methanol: fraction Fr.9 obtained by elution with dichloromethane in a volume ratio of 50: 50; (ii) a Further, the preparative HPLC separation and identification process in S3 specifically includes: the method comprises the following steps of mixing methanol: the volume ratio of water is 75: 25 preparing a solution as a mobile phase fraction Fr.9, performing isocratic elution at a flow rate of 3.0mL/min, detecting with an ultraviolet detector at a wavelength of 210nm or 280nm to obtain compound 1, tolypyridone C, and t of tolypyridone C_RIs 32.0 min; and compound 2 is tolypyridone D, t thereof_RIt is 26.9 min.

8. A pharmaceutical composition comprising the 4-hydroxypyridone compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, solvate or metabolite thereof;

further, the 4-hydroxypyridone compound or the pharmaceutically acceptable salt thereof in the pharmaceutical composition accounts for 0.01-90% of the pharmaceutical composition;

further, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier;

further, the pharmaceutical composition is an oral preparation or an injection.

9. Use of the 4-hydroxypyridones compound or the pharmaceutically acceptable salt, solvate or metabolite thereof according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating a disease associated with oxidative stress.

10. The use according to claim 9, wherein the oxidative stress related diseases include, but are not limited to, coronary heart disease, atherosclerosis, parkinson's disease or stroke.