CN116077508A - Application of oleanane-type triterpene derivative in preparation of cardiovascular disease drugs - Google Patents

Application of oleanane-type triterpene derivative in preparation of cardiovascular disease drugs Download PDF

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CN116077508A
CN116077508A CN202211106110.7A CN202211106110A CN116077508A CN 116077508 A CN116077508 A CN 116077508A CN 202211106110 A CN202211106110 A CN 202211106110A CN 116077508 A CN116077508 A CN 116077508A
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oleanane
cardiovascular disease
type triterpene
compound
preparation
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陈广通
吴艳妮
易颖
陆游佳
范博义
宋妍
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Nantong University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

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Abstract

The invention belongs to the technical field of medicines, and discloses application of oleanane-type triterpene derivatives in preparation of cardiovascular disease medicines. The invention converts betulinic acid into a novel oleanane type triterpene derivative with 3-carbonyl, 7 beta, 15 alpha, 29-hydroxyl, 19 beta and 28 beta lactone substituted structures by utilizing a microbial conversion technology. The compound has better myocardial cell protection activity, can be used as an active ingredient of a medicament for treating myocardial infarction, coronary atherosclerotic heart disease and chronic heart failure, and can be applied to preparing medicaments for treating cardiovascular diseases.

Description

Application of oleanane-type triterpene derivative in preparation of cardiovascular disease drugs
Technical Field
The invention belongs to the field of medicines, and in particular relates to an application of oleanane-type triterpene derivative in preparing a cardiovascular disease medicine.
Background
With the improvement of living standard and the increase of population aging, the incidence of cardiovascular diseases is increased year by year under the influence of social and environmental factors, and the cardiovascular diseases become one of the diseases with the highest mortality rate of human beings. Myocardial cell injury is often accompanied in the occurrence and development processes of various cardiovascular diseases such as myocardial infarction, ischemia reperfusion injury and the like, and further apoptosis and the like are generated, so that cardiovascular dysfunction is caused. Myocardial ischemia and oxidative stress are important factors in the development of cardiovascular disease.
Betulonic acid is also called betulonic acid, and is a lupeane pentacyclic triterpene compound mainly derived from bark of birch, and also exists in apple, fructus quisqualis, negundo chastetree, and radix Buxae. Modern pharmacological studies have found that betulonic acid has anti-tumor, antiviral and anti-inflammatory activities, among which anti-melanoma is known. Betulonic acid is an important intermediate in pharmaceutical chemistry research as an oxidation product of betulinic acid. It has been found that betulonic acid has enhanced anti-HIV activity after structural modification. In the aspect of chemical structure modification, the research on the anti-tumor aspect of the chemical synthesis derivative of the betulonic acid is rich, and the sites of chemical structure modification mainly comprise the carbonyl group at the 3 position and the carboxyl group at the 28 position of the betulonic acid. However, due to the structural specificity of pentacyclic triterpene compounds, the parent nucleus lacks active groups, reaction sites are few, and derivatives with modification such as hydroxyl, carbonyl and the like on the parent nucleus are difficult to obtain by adopting a conventional chemical reaction method. Thus, the structural diversity of the reaction sites and derivatives of modification of the chemical structure of betulonic acid is severely limited. Microbial transformation is a process in which enzymes in microbial cells are used to structurally modify a substrate to convert it into other compounds. The special enzymes in cells or outside cells generated in the growth process of the microorganisms such as fermentation fungi, actinomycetes and the like can catalyze self substances and specific chemical reactions such as hydrolysis, oxidation, reduction, cracking, framework rearrangement and the like on external compounds, so that the method becomes a powerful tool for modifying and reforming the structure of complex natural products. The subject group has long been under study for microbial transformation of natural active ingredients, especially for microbial transformation of natural active triterpenes. The conversion studies of common triterpenes of natural origin, including dammarane type, cycloartane type, ursane type, oleanane type triterpenes, lupeane type, etc., were carried out using a variety of microorganisms, and it was found that microbial enzyme systems were able to selectively catalyze a plurality of non-chemically reactive sites on the triterpene parent nucleus, thereby obtaining derivatives having chemically reactive groups such as hydroxyl groups, carbonyl groups, etc., on the parent nucleus (Journal of natural products 2021,84:2664-2674;Phytochemistry 2021,182:112608;Natural Product Research 2021,35 (16): 2685-2690;Phytochemistry 2019, 166:112076;Planta Medica 2019,85 (1): 56-61). On the one hand, the microbial transformation product can obtain a derivative with stronger biological activity to be directly used for drug research and development, and on the other hand, the newly introduced chemical active group on the mother nucleus after microbial transformation increases the sites for chemical modification and transformation, thereby solving the problem of few reaction sites for preparing the derivative by organic chemistry of the triterpene compound.
Disclosure of Invention
The invention aims to provide an application of oleanane-type triterpene derivative or pharmaceutically acceptable salt thereof in preparing a medicine for treating cardiovascular diseases, which can prevent or treat the cardiovascular diseases.
The invention provides an application of oleanane-type triterpene derivative with a structure shown in formula I or pharmaceutically acceptable salt thereof in preparing a cardiovascular disease medicament,
Figure BDA0003840572620000011
the compound with the structural formula of formula I is the first disclosed oleanane type triterpene derivative.
The invention also provides a preparation method of the oleanane-type triterpene derivative, which comprises the following steps:
1) Fermenting and culturing microorganisms, namely adding betulinic acid into a culture medium, then performing transformation culture, and removing mycelium to obtain fermentation liquor, wherein the microorganisms are strain of the genus Mucor;
2) Extracting the fermentation liquor, and evaporating the extract liquor to obtain a converted crude extract;
3) The crude conversion extract was chromatographed on a silica gel column with methylene chloride: methanol was used as mobile phase for gradient elution, and the fractions were collected and combined by TLC analysis to give 5 fractions.
4) Purifying the components by reverse phase high performance liquid chromatography to obtain the oleanane-type triterpene derivative.
Preferably, in step 1), the transformation culture temperature is 30 ℃, the rotation speed of the shaking table is 130rpm, and the culture time is 120 hours.
Preferably, in step 2), the extraction solvent of the extraction is dichloromethane.
Preferably, in step 3), the gradient elution conditions are dichloromethane: methanol 100:0-50:1-30:1-20:1-10:1.
Further, the cardiovascular disease includes cardiovascular disease caused by myocardial apoptosis, heart failure or myocardial ischemia reperfusion injury.
Further, the cardiovascular disease includes myocardial infarction, coronary atherosclerotic heart disease or chronic heart failure.
Furthermore, the medicine also contains pharmaceutically acceptable auxiliary materials.
Further, the pharmaceutically acceptable auxiliary materials are one or more of diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers and lubricants.
Compared with the prior art, the invention utilizes the microbial transformation technology to transform lupin alkane type triterpenoid betulinic acid into oleanane type triterpenoid, simultaneously successfully carries out structural modification on the 7 th site, the 15 th site and the 29 th site of the non-chemical reaction active site on the mother nucleus, introduces the hydroxyl of the active reaction active group, and obtains the oleanane type triterpenoid derivative with obvious protection activity on myocardial cell injury, which can be used as an active ingredient of a medicament for treating cardiovascular diseases and has wide application.
Figure BDA0003840572620000021
Detailed Description
In order to further illustrate the present invention, the following describes in detail the preparation process of the oleanane-type triterpene derivative and the application thereof in the cardiovascular disease drug provided by the present invention in combination with examples.
Example 1: preparation of Compounds of formula I
The invention adopts a microbial transformation method, takes betulinic acid as a raw material, and prepares the compound through steps of fermentation, extraction, separation and the like. The strain of genus Rhizopus (Circiella) can be purchased from China center for culture collection (CGMCC), potato culture medium is selected, and stored in a refrigerator at 4deg.C on solid slant culture medium.
Taking Mucor Circinella muscae CGMCC 3.2695 as an example, the process for preparing the compound of formula I is as follows:
1) Fermentation, conversion and extraction
Mucor miehei Circinella muscae CGMCC 3.2695 was inoculated into 2 250mL Erlenmeyer flasks (containing 100mL potato culture medium) as seed solutions. After shaking culture for 1 day at 30℃at 130rpm on a shaker, 1mL of the seed solution was pipetted with a sterile pipette and added to 20 1000mL shake flasks (containing 400mL potato culture medium) until hyphae grew to be vigorous. After 1 day of shaking culture, 20mg of betulonic acid (0.2 mL of 100mg/mL DMSO solution) was added to each flask, together with 400mg of substrate. The conversion was continued for 5 days under the same conditions, the fermentation broth was filtered to remove the mycelium, the filtrate was extracted 3 times with an equal volume of dichloromethane, and the extract was concentrated to dryness under reduced pressure to give about 0.52g of crude extract of the conversion.
2) Silica gel column chromatography
The crude conversion extract was separated by silica gel column chromatography. Dichloromethane: methanol gradient elution (100:0, 50:1, 30:1, 20:1, 10:1). Fractions were collected and pooled after TLC analysis to give pooled fractions A-E.
3) Reversed phase high performance liquid chromatography purification
The combined fractions C were purified by reverse phase high performance liquid chromatography. The preparation conditions were a semi-preparative chromatographic column YMC ODSA-5 μm, 10.0X1250 mm, acetonitrile-water (55:45, V/V), flow rate 2.5mL/min, detection wavelength 203nm. The conversion product of formula I is obtained, and mass spectrum and spectrum data are shown below.
Compound i: 3-carbonyl-7β,15α,29-trihydroxy oleanane-28β,19β -lactone (3-oxo-7β,15α, 29-trihydroxyolenan-28,19 β -lactone); melting point 304-306 ℃;
Figure BDA0003840572620000032
+37.5 ° (c=0.1, meoh); the main absorption peak (KBr) v of infrared spectrum max ：3551,3032,2968,1734,1695,1381,1226,1048cm -1 The method comprises the steps of carrying out a first treatment on the surface of the High resolution mass spectrum m/z 525.3192[ M+Na ]] + (calcd.525.3187 for C 30 H 46 O 6 Na); the nmr hydrogen spectrum and carbon spectrum data are shown in table 1.
TABLE 1 Nuclear magnetic Hydrogen Spectrometry and carbon Spectrometry data for Compound I (deuterated chloroform)
Figure BDA0003840572620000031
Figure BDA0003840572620000041
The above results indicate that the resulting compounds are structurally correct.
EXAMPLE 2 protective Activity of Compound I against myocardial cells injured by Hydrogen peroxide
(1) Experimental materials
CO 2 Incubator (Jouan IGO 150); microplate reader (Bio-TEK ELx 800); fluorescence inverted microscope (Olympus IX 51); MTT cell proliferation and cytotoxicity detection kit (Biyun Biotechnology institute), DMEM high sugar culture medium (Gibcol BRL), fetal bovine serum, dimethyl sulfoxide (DMSO), trypsin (Shanghai Biotechnology Co., ltd.), 30% hydrogen peroxide (H) 2 O 2 ) (Tianjin Ruijin Chemicals Co., ltd.) H9c2 cells (tumor institute of China medical science center).
(2) Experimental method
Determination of the pairs of H of each test Compound by MTT method 2 O 2 Damaged H9Effects of c2 cell activity: cell count was performed after digestion with pancreatin to adjust the cell density of the cell suspension to 5×10 4 mu.L of each well was added to a 96-well plate at a concentration of 5% CO 2 Constant temperature CO at 37 DEG C 2 Culturing in an incubator for 12h. Grouping treatment after cell adhesion: control group, model group (H) 2 O 2 600. Mu. Mol/L injury 6 h), model+test compound (10, 20, 40. Mu.M) group, positive control group, pinacol. The final volume per well was 200 μl, 3 replicates per concentration. After 24h of drug treatment, 10. Mu.L of MTT solution (5 mg/mL, i.e., 0.5% MTT) was added to each well and incubation was continued for 4h. The absorbance of each well was measured at 490nm with a microplate reader and cell viability was calculated: cell viability = dosing/control OD.
(3) Experimental results
Calculating the ratio of compound I to H according to the test result of MTT method 2 O 2 The results of the survival of damaged H9c2 cells are shown in table 2.
Table 2 test compounds vs H 2 O 2 Effect of damaged H9c2 cell viability
Figure BDA0003840572620000042
(in comparison with the control group, # p is less than 0.05; in comparison with the set of models, * P＜0.05， ** P＜0.01)
compared with the control group, H 2 O 2 The cell viability of the treated group was significantly reduced, indicating successful cell modeling. And H is 2 O 2 Compared with the treatment group, the compound I can obviously improve the survival rate of cells, shows that the compound I has obvious myocardial cell protection effect, shows good dose dependency in a certain dose range, and can be used as an active ingredient of a medicament for treating myocardial infarction, coronary atherosclerotic heart disease and chronic heart failure.
EXAMPLE 3 protective Effect of Compound I of the invention on myocardial ischemia reperfusion injury
1) Experimental materials
CO 2 Incubator (Jouan IGO 150); microplate reader (Bio-TEK ELx 800); fluorescence inverted microscope (Olympus IX 51); MTT cell proliferation and cytotoxicity detection kit (Biyun Biotechnology institute), DMEM high sugar medium (Gibcol BRL), fetal bovine serum, dimethyl sulfoxide (DMSO), trypsin (Shanghai Biotechnology Co., ltd.) and H9c2 cells (China medical science sciences tumor institute).
Test sample: betulonic acid and compound I synthesized in example 1 have a purity of 95% or more, and each compound is dissolved in DMSO and diluted.
2) Experimental method
Taking H9c2 cells in logarithmic growth phase, adjusting cell concentration to 5×10 with DMEM culture solution containing 10% calf serum and 1% penicillin-streptomycin double antibody solution 4 Inoculating the culture medium to a 96-well culture plate at a volume of one liter per mL, replacing fresh sugar-free and serum-free culture medium after about 24 hours, adding a compound, putting the culture medium into an anaerobic working station for hypoxia injury for 1 hour, taking out, adding sugar and serum for further culture for 24 hours, and detecting the survival rate of cells in each well by using an MTT staining method.
3) Experimental results
The results of calculating the effect of compound i on H9c2 cell survival of ischemia reperfusion injury based on MTT assay result are shown in table 3.
TABLE 3 influence of test samples on H9c2 cell survival from ischemia reperfusion injury
Figure BDA0003840572620000051
(in comparison with the control group, # p is less than 0.05; in comparison with the set of models, * P＜0.05， ** P＜0.01)
the result shows that compared with a model group, the compound I with different concentrations can obviously raise the survival rate of myocardial cells, and the compound I provided by the invention can effectively protect the damage of hypoxia/reoxygenation to H9c2 myocardial cells, has certain dose dependency, and can be used as an active ingredient of a medicament for treating myocardial infarction, coronary atherosclerotic heart disease and chronic heart failure.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (5)

1. An application of oleanane-type triterpene derivative with a structure shown in formula I or pharmaceutically acceptable salt thereof in preparing medicaments for treating cardiovascular diseases,
Figure FDA0003840572610000011
2. the use according to claim 1, wherein the cardiovascular disease comprises cardiovascular disease caused by myocardial apoptosis, heart failure or myocardial ischemia reperfusion injury.
3. The use according to claim 1, wherein the cardiovascular disease comprises myocardial infarction, coronary atherosclerotic heart disease or chronic heart failure.
4. The use according to claim 1, wherein the medicament further comprises pharmaceutically acceptable excipients.
5. The use according to claim 4, wherein the pharmaceutically acceptable auxiliary material is one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers and lubricants.
CN202211106110.7A 2022-09-09 2022-09-09 Application of oleanane-type triterpene derivative in preparation of cardiovascular disease drugs Pending CN116077508A (en)

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