阅读说明：本技术 白桦脂酮酸衍生物在制备治疗心血管疾病的药物中的应用 (Application of betulonic acid derivative in preparation of medicine for treating cardiovascular diseases ) 是由 陈广通 吴艳妮 朱心娟 陆游佳 范博义 宋妍 于 2021-09-13 设计创作，主要内容包括：本发明属于医药技术领域,公开了一种白桦脂酮酸衍生物在制备治疗心血管疾病的药物中的应用。本发明利用微生物转化技术,对白桦脂酮酸成功地进行了结构修饰,获得了16个具有母核结构修饰的新型白桦脂酮酸衍生物。通过体外心肌细胞损伤保护试验和心肌细胞缺血再灌注试验证实,这些化合物具有较好的心肌细胞保护活性,可以作为治疗心肌梗死、冠状动脉粥样硬化性心脏病、慢性心力衰竭药物的活性成分,可应用于制备治疗心血管疾病的药物。(The invention belongs to the technical field of medicines, and discloses an application of a betulonic acid derivative in preparation of a medicine for treating cardiovascular diseases. The invention successfully carries out structural modification on the betulonic acid by utilizing a microbial transformation technology to obtain 16 novel betulonic acid derivatives with mother nucleus structural modification. The in vitro myocardial cell injury protection test and the myocardial cell ischemia reperfusion test prove that the compounds have better myocardial cell protection activity, can be used as active ingredients of medicaments for treating myocardial infarction, coronary atherosclerotic heart disease and chronic heart failure, and can be applied to the preparation of medicaments for treating cardiovascular diseases.)

1. An application of betulonic acid derivative or its pharmaceutically acceptable salt with any structural formula shown in the specification in preparing medicine for treating cardiovascular diseases,

2. the use of claim 1, wherein the cardiovascular disease comprises cardiovascular disease caused by apoptosis of cardiac myocytes, 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 of claim 1, wherein the medicament further comprises a pharmaceutically acceptable excipient.

5. The use according to claim 4, wherein the pharmaceutically acceptable excipient is one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier and a lubricant.

Technical Field

The invention relates to the technical field of medicines, in particular to application of a betulonic acid derivative in preparing a medicine for treating cardiovascular diseases.

Background

With the improvement of living standard and the aggravation 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 human mortality. In the occurrence and development processes of various cardiovascular diseases such as myocardial infarction, ischemia-reperfusion injury and the like, myocardial cell injury is often accompanied, and further apoptosis and the like occur to cause the disorder of cardiovascular functions. Myocardial ischemia and oxidative stress are important factors in the development of cardiovascular disease.

Betulonic acid, also known as betulonic acid, is a lupane-type pentacyclic triterpene compound, mainly derived from bark of birch, and also present in plants such as apple, fructus quisqualis, negundo chastetree and buxus. Modern pharmacological research finds that betulonic acid has the biological activities of resisting tumors, viruses and inflammation, and the like, and is known for resisting melanoma. Betulonic acid is an oxidation product of betulinic acid and is an important intermediate for medicinal chemistry research. The research finds that the anti-HIV activity of the product of the betulonic acid after structural modification is enhanced. In the prior art, the sites of chemical structure modification are mainly carbonyl at the 3-position and carboxyl at the 28-position of betulonic acid. Due to the structural particularity of the pentacyclic triterpenoid, the parent nucleus lacks active groups, the number of reaction sites is small, the structure of the parent nucleus is difficult to modify by adopting a conventional chemical reaction method, and the derivative with the modification of hydroxyl, carbonyl and the like on the parent nucleus is obtained, so that the research on the betulonic acid derivative with the modification of the parent nucleus structure is less.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of betulonic acid derivatives or pharmaceutically acceptable salts thereof in the preparation of drugs for treating cardiovascular diseases.

The technical scheme provided by the invention is as follows:

an application of betulonic acid derivative or its pharmaceutically acceptable salt with any structural formula shown in the specification in preparing medicine for treating cardiovascular diseases,

the preparation method of the betulonic acid derivative comprises the following steps:

1) fermenting and culturing microorganism, adding betulonic acid into culture medium with concentration of 2-5000 μ g/mL, performing transformation culture, and removing mycelium to obtain fermentation broth, wherein the microorganism is Rhizopus, Absidia, Mucor or Coptomyces;

2) extracting the fermentation liquor by using an organic solvent, and evaporating the extract to obtain a converted crude extract, wherein the organic solvent is preferably ethyl acetate;

3) the crude extract was subjected to reverse phase silica gel column chromatography with methanol: performing gradient elution by using water as a mobile phase, collecting fractions, analyzing and combining by using HPLC (high performance liquid chromatography) to obtain 5 components, wherein the gradient elution condition preferably adopts methanol: water 20:80-40:60-60:40-80:20-100: 0;

4) purifying the components by reversed phase high performance liquid chromatography to obtain the betulonic acid derivative.

Preferably, the cardiovascular disease comprises cardiovascular disease caused by myocardial apoptosis, heart failure or myocardial ischemia-reperfusion injury.

Preferably, the cardiovascular disease comprises myocardial infarction, coronary atherosclerotic heart disease or chronic heart failure.

Preferably, the medicine also contains pharmaceutically acceptable auxiliary materials.

Preferably, the pharmaceutically acceptable auxiliary materials are one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers and lubricants.

The betulonic acid derivative obtained by modifying the mother nucleus structure of the betulonic acid by utilizing the microbial transformation technology has better myocardial cell protection activity, can be used as an active ingredient of medicaments for treating myocardial infarction, coronary atherosclerotic heart disease and chronic heart failure, and has wide application in the field of medicines.

Detailed Description

In order to further illustrate the present invention, the following detailed description will be given of the technical solutions provided by the present invention with reference to examples.

Example 1: preparation of compounds of formula I-XVI

The compound is prepared by adopting a microbial conversion method and taking betulonic acid as a raw material through the steps of fermentation, extraction, separation and the like. The strain of Rhizopus can be purchased from China academy of sciences (CGMCC), and is preserved in a solid slant culture medium at 4 deg.C in a refrigerator.

Taking Rhizopus arrhizus CGMCC 3.868 as an example, the process for preparing the compound with the structural formula I-XVI is as follows:

1) fermentation, transformation and extraction

Rhizopus arrhizus CGMCC 3.868 was inoculated into 2 250mL triangular flasks (containing 100mL potato medium) as seed solutions. After shaking culture on a shaking table at 160rpm and 26 ℃ for 1 day, 1mL of seed solution was aspirated by a sterile pipette and added to 20 1000mL shake flasks (containing 400mL potato medium) until the hyphae growth was in vigorous phase. After 1 day of shake culture, 20mg betulonic acid (0.2mL, 100mg/mL DMSO solution) was added to each flask, for a total of 400mg substrate. Continuing to transform for 7 days under the same conditions, filtering the fermentation broth, filtering to remove mycelium, extracting the filtrate with equal volume of ethyl acetate for 3 times, and concentrating the extractive solution under reduced pressure to dryness to obtain about 0.78g of crude extract of the transformed extract.

2) Reversed phase column chromatography

The crude extract was separated by reverse phase silica gel column chromatography ODS-C18(100g, 60X 3cm, 50. mu.M). Methanol: water gradient elution (20:80, 40:60, 60:40, 80:20, 100: 0). Collecting fractions, analyzing by HPLC, and mixing to obtain mixed components A-E.

3) Purification by reversed phase high performance liquid chromatography

The combined fractions A-E were purified by reverse phase high performance liquid chromatography, respectively. The preparation conditions were a column for semi-preparative YMC ODS A-5 μm, 10.0X 250mm, acetonitrile-water (35:65, 42:58, 55:45, 58:42, V/V), flow rate 2.5mL/min, and detection wavelength 203 nm. 16 transformation products of the formulae I to XVI are obtained, the mass spectra and the spectral data of which are shown below.

A compound I: 7 beta-acetoxy betulinic acid (3-oxo-7 beta-acetoxy-lup-20 (29) -en-28-oic acid); melting point 286-288 ℃; optical rotation(c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrum_max：3562,3029,2921,1741,1707,1693,1376,1214,1061cm^-1(ii) a High resolution mass spectrometry M/z 511.3426[ M-H [ ]]^-(calcd.for C₃₂H₄₇O₅511.3423); the NMR data are shown in Table 1.

Compound ii: 11 alpha, 15 alpha-dihydroxy betulinic acid (3-oxo-11 alpha, 15 alpha-dihydroxy-lup-20 (29) -en-28-oic acid); melting point 324-325 ℃; optical rotation(c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrum_max：3457,3035,2943,1745,1712,1382,1241,1076cm^-1(ii) a High resolution mass spectrometry M/z 485.3264[ M-H [ ]]^-(calcd.for C₃₀H₄₅O₅485.3267); the NMR data are shown in Table 1.

Compound iii: 7 beta, 11 beta-dihydroxy betulinic acid (3-oxo-7 beta, 11 beta-dihydroxy-lup-20 (29) -en-28-oic acid); melting point 318-; optical rotation(c＝0.1,MeOH); main absorption peak (KBr) v of infrared spectrum_max：3524,3041,2938,1742,1705,1365,1231,1086cm^-1(ii) a High resolution mass spectrometry M/z 485.3266[ M-H [ ]]^-(calcd.for C₃₀H₄₅O₅485.3267); the NMR data are shown in Table 1.

A compound IV: 7 beta-hydroxy-30-acetoxy betulinic acid (3-oxo-30-acetoxy-7 beta-hydroxy-lup-20 (29) -en-28-oic acid); melting point 304-305 ℃; optical rotation(c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrum_max：3481,3036,2952,1738,1715,1698,1388,1235,1057cm^-1(ii) a High resolution mass spectrometry M/z 527.3372[ M-H [ ]]^-(calcd.for C₃₂H₄₇O₆527.3373); the NMR data are shown in Table 1.

Compound v: 15 alpha-hydroxy-30-acetoxy betulinic acid (3-oxo-30-acetoxy-15 alpha-hydroxy-lup-20 (29) -en-28-oic acid); melting point 297-299 ℃; optical rotation(c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrum_max：3506,3041,2953,1748,1711,1695,1368,1227,1050cm^-1(ii) a High resolution mass spectrometry M/z 527.3370[ M-H [ ]]^-(calcd.for C₃₂H₄₇O₆527.3373); the NMR data are shown in Table 2.

Compound vi: 30-hydroperoxy betulonic acid (3-oxo-30-hydroperoxyl-lup-28-oic acid); melting point 296-; optical rotation(c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrum_max：3577,3039,2963,1735,1701,1371,1243,1075cm^-1(ii) a High resolution mass spectrometry M/z 485.3264[ M-H [ ]]^-(calcd.for C₃₀H₄₅O₅485.3267); the NMR data are shown in Table 2.

And (3) a compound VII: 7 beta, 23-dihydroxy betulinic acid (3-oxo-7 beta, 23-dihydroxy-lup-20(29) -en-28-oic acid); melting point 322-324 ℃; optical rotation(c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrum_max：3497,3044,2962,1753,1712,1384,1207,1055cm^-1(ii) a High resolution Mass Spectrometry M/z 509.3239[ M + Na ]]⁺(calcd.for C₃₀H₄₆O₅Na, 509.3243); the NMR data are shown in Table 2.

Compound viii: 7 beta, 15 alpha, 23-trihydroxy betulonic acid (3-oxo-7 beta, 15 alpha, 23-trihydroxy-lup-20(29) -en-28-oic acid); melting point 343-; optical rotation(c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrum_max：3559,3035,2955,1751,1714,1379,1235,1033cm^-1(ii) a High resolution mass spectrometry M/z 501.3215[ M-H [ ]]^-(calcd.for C₃₀H₄₅O₆501.3216); the NMR data are shown in Table 2.

A compound IX: 7 beta-hydroxy-23-acetoxy betulinic acid (3-oxo-23-acetoxy-7 beta-hydroxy-lup-20 (29) -en-28-oic acid); melting point 311-312 ℃; optical rotation(c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrum_max：3543,3032,2941,1742,1715,1697,1376,1214,1028cm^-1(ii) a High resolution mass spectrometry M/z 527.3373[ M-H [ ]]^-(calcd.for C₃₂H₄₇O₆527.3373); the NMR data are shown in Table 3.

Compound x: 15 alpha-hydroxy-23-acetoxy betulinic acid (3-oxo-23-acetoxy-15 alpha-hydroxy-lup-20 (29) -en-28-oic acid); melting point 305-; optical rotation(c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrum_max：3537,3046,2938,1750,1712,1698,1384,1228,1031cm^-1(ii) a High resolution mass spectrometry M/z 527.3376[ M-H [ ]]^-(calcd.for C₃₂H₄₇O₆527.3373); the NMR data are shown in Table 3.

Compound xi: 2-carbonyl-3 beta, 7 beta-dihydroxy betulinic acid (2-oxo-3 beta, 7 beta-dihydroxy-lup-20 (29) -en-28-oic acid); melting point 332 and 334 ℃; optical rotation(c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrum_max：3455,3046,2936,1724,1706,1388,1215,1024cm^-1(ii) a High resolution mass spectrometry M/z 485.3263[ M-H [ ]]^-(calcd.for C₃₀H₄₅O₅485.3267); the NMR data are shown in Table 3.

Compound XII: 2 alpha, 7 beta-dihydroxy betulinic acid (3-oxo-2 alpha, 7 beta-dihydroxy-lup-20 (29) -en-28-oic acid); melting point 315-; optical rotation(c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrum_max：3527,3046,2981,1746,1701,1382,1237,1022cm^-1(ii) a High resolution mass spectrometry M/z 485.3262[ M-H [ ]]^-(calcd.for C₃₀H₄₅O₅485.3267); the NMR data are shown in Table 3.

Compound XIII: 7 beta, 22 beta-dihydroxy betulinic acid (3-oxo-7 beta, 22 beta-dihydroxy-lup-20 (29) -en-28-oic acid); melting point 324-325 ℃; optical rotation(c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrum_max：3471,3053,2973,1757,1711,1379,1213,1027cm^-1(ii) a High resolution mass spectrometry M/z 485.3261[ M-H [ ]]^-(calcd.for C₃₀H₄₅O₅485.3267); nuclear magnetic resonanceThe hydrogen and carbon spectra data are shown in table 4.

Compound XIV: 20(S) -7 beta-hydroxy-29-acetoxy betulinic acid (20(S) -3-oxo-7 beta-hydroxy-29-acetoxy-lup-28-oic acid); melting point 302-; optical rotation(c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrum_max：3538,2963,1745,1713,1702,1369,1223,1033cm^-1(ii) a High resolution mass spectrometry M/z 529.3531[ M-H [ ]]^-(calcd.for C₃₂H₄₉O₆529.3529); the NMR hydrogen and carbon spectra data are shown in Table 4.

Compound XV: 20(S) -7 beta-hydroxy-29-acetoxy betulinic acid (20(R) -3-oxo-7 beta-hydroxy-29-acetoxy-lup-28-oic acid); melting point 311-313 ℃; optical rotation(c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrum_max：3533,2977,1744,1716,1701,1361,1227,1039cm^-1(ii) a High resolution mass spectrometry M/z 529.3533[ M-H [ ]]^-(calcd.for C₃₂H₄₉O₆529.3529); the NMR hydrogen and carbon spectra data are shown in Table 4.

Compound XVI: 7 beta-hydroxybetulinic acid-28-O-beta-D-glucopyranoside (3-oxo-7 beta-hydroxy-lup-20 (29) -en-28-oic acid-beta-D-glucopyranosyl ester); melting point 386-388 ℃; optical rotation(c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrum_max：3575,3047,2967,1741,1709,1355,1237,1035cm^-1(ii) a High resolution mass spectrometry M/z 631.3847[ M-H [ ]]^-(calcd.for C₃₆H₅₅O₉631.3846); the NMR hydrogen and carbon spectra data are shown in Table 4.

TABLE 1 nuclear magnetic hydrogen and carbon spectra data (deuterated chloroform) of Compound I, Compound II, Compound III, and Compound IV

TABLE 2 nuclear magnetic hydrogen and carbon spectra data (deuterated chloroform) of Compound V, Compound VI, Compound VII, and Compound VIII

TABLE 3 nuclear magnetic hydrogen and carbon spectra data (deuterated chloroform) of Compound IX, Compound X, Compound XI, and Compound XII

TABLE 4 nuclear magnetic hydrogen and carbon spectra data (deuterated chloroform) of Compound XIII, Compound XIV, Compound XV, and Compound XVI

The above results indicate that the obtained compound has the correct structure.

Example 2: protective Activity of Compounds I-XVI against myocardial cell injury by Hydrogen peroxide

(1) Experimental Material

CO₂Incubator (Joean IGO 150); microplate reader (Bio-TEK ELx 800); fluorescence inverted microscope (Olympus IX 51); MTT cell proliferation and cytotoxicity detection kit (Biyuntian Biotech research institute), DMEM high-sugar medium (Gibcol BRL), fetal bovine serum, dimethyl sulfoxide (DMSO), trypsin (Shanghai bioengineering Co., Ltd.), and 30% hydrogen peroxide (H)₂O₂) (Renjite Chemicals Co., Tianjin) H9c2 cell (swollen college of Chinese medical science)Institute for tumor).

(2) Experimental methods

Determination of H for each test Compound pair Using the MTT method₂O₂Effect of damaged H9c2 cell activity: after digestion with pancreatin, cell counts were performed, and the cell density of the cell suspension was adjusted to 5X 10⁴one/mL, adding 200. mu.L/well of 96-well culture plate, and placing in 5% CO₂Constant temperature of 37 ℃ CO₂Culturing in an incubator for 12 h. Grouping treatment after cell adherence: control group, model group (H)₂O₂600 μmol/L lesion 6h), model + test compound (10, 20, 40 μ M) groups. The final volume of each well was 200. mu.L, 3 replicates for each concentration. After 24h drug treatment, 10. mu.L of MTT solution (5mg/mL, i.e., 0.5% MTT) was added to each well and incubation continued for 4 h. Measuring the light absorption value of each hole at 490nm of the microplate reader, and calculating the cell survival rate: the cell survival rate is the OD value of the drug-added group/the OD value of the control group.

(3) Results of the experiment

According to the MTT method test result, the betulonic acid derivatives I-XVI to H are calculated₂O₂The results of the viability of the damaged H9c2 cells are shown in table 5.

Table 5 test compounds vs H₂O₂Effect of injured H9c2 cell survival

(in comparison to the control group,^#P<0.05; in comparison with the set of models,^*P<0.05，^**P<0.01)

comparison with the control group, H₂O₂The cell survival rate of the treated group was significantly reduced, indicating successful cell modeling. And H₂O₂Compared with treatment groups, the betulonic acid derivatives I-XVI can obviously improve the survival rate of cells, and the betulonic acid derivatives I-XVI have obvious effectHas good myocardial cell protection effect and shows good dose dependence in a certain dose range, and can be used as an active ingredient of medicaments for treating myocardial infarction, coronary atherosclerotic heart disease and chronic heart failure.

EXAMPLE 3 protective Effect of Compounds I-XVI of the present invention on myocardial cell ischemia-reperfusion injury

1) Experimental Material

CO₂Incubator (Joean IGO 150); microplate reader (Bio-TEK ELx 800); fluorescence inverted microscope (Olympus IX 51); MTT cell proliferation and cytotoxicity detection kit (Biyuntian Biotechnology research institute), DMEM high-sugar medium (Gibcol BRL), fetal bovine serum, dimethyl sulfoxide (DMSO), trypsin (Shanghai bioengineering Co., Ltd.), and H9c2 cells (tumor research institute of Chinese medical academy of sciences).

Test samples: betulonic acid and the compounds I to XVI synthesized in example 1 were found to have a purity of 95% or more, and each compound was dissolved in DMSO and then diluted.

2) Experimental methods

Collecting H9c2 cells in logarithmic growth phase, and adjusting cell concentration to 5 × 10 with DMEM culture solution containing 10% calf serum and 1% penicillin-streptomycin double antibody⁴And (2) inoculating the cells/mL, inoculating the cells/mL into a 96-well culture plate, replacing a new sugar-free and serum-free fresh culture solution after about 24 hours, adding a compound, placing the cells in an anaerobic workstation for hypoxia injury for 1 hour, taking out the cells, adding sugar and serum for re-culture for 24 hours, and detecting the cell survival rate of each well by using an MTT staining method after 24 hours.

3) Results of the experiment

The results of calculating the effect of betulonic acid derivatives i to XVI on the survival rate of H9c2 cells of ischemia-reperfusion injury according to the MTT method test results are shown in table 6.

TABLE 6 Effect of test samples on H9c2 cell viability in ischemia reperfusion injury

(in comparison to the control group,^#P<0.05; in comparison with the set of models,^*P<0.05，^**P<0.01)

the results show that the survival rate of the myocardial cells can be obviously improved after the treatment of the compounds I-XVI with different concentrations compared with a model group, and the compounds I-XVI can effectively protect the injury of the H9c2 myocardial cells caused by hypoxia/reoxygenation, have certain dose dependence and can be used as active ingredients of medicaments for treating myocardial infarction, coronary atherosclerotic heart disease and chronic heart failure.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.