CN113603744A - Betulonic acid derivative and preparation method thereof - Google Patents
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Abstract
The invention belongs to the technical field of medicines, and discloses a betulonic acid derivative and a preparation method thereof. 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, and the compounds can be used as medical intermediates and can also be applied to the preparation of medicaments for treating cardiovascular diseases, thereby having wide application.
Description
Technical Field
The invention relates to the technical field of medicines, and particularly relates to a betulonic acid derivative and a preparation method thereof.
Background
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 derivatives with modifications of hydroxyl, carbonyl and the like on the parent nucleus are obtained, so that the betulonic acid derivatives with the modified parent nucleus structure and the preparation method thereof are less researched.
Disclosure of Invention
In view of the above, the present invention provides a betulonic acid derivative or a pharmaceutically acceptable salt thereof, and a preparation method thereof, wherein the betulonic acid derivative can be used as a pharmaceutical intermediate, and can also be applied to the preparation of a medicament for treating cardiovascular diseases.
The betulonic acid derivative provided by the invention is a compound with a structural formula of formula I-formula XVI:
the compounds with the structural formulas of formula I, formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, formula X, formula XI, formula XII, formula XIII, formula XIV, formula XV and formula XVI are the novel betulonic acid derivatives disclosed for the first time in the invention.
The invention also provides a preparation method of the betulonic acid derivative, which comprises the following steps:
1) fermenting and culturing microorganisms, adding betulonic acid into a culture medium, then performing transformation culture, and removing mycelia to obtain a fermentation broth, wherein the microorganisms are strains of rhizopus, Absidia, Mucor or Coptomyces;
2) extracting the fermentation liquor, and evaporating the extract to obtain a converted crude extract;
3) the crude extract was subjected to reverse phase silica gel column chromatography with methanol: gradient elution is carried out by taking water as a mobile phase, and 5 components are obtained by collecting fractions and then combining the fractions through HPLC analysis.
4) Purifying the components by reversed phase high performance liquid chromatography to obtain the betulonic acid derivative.
Preferably, in the step 1), the concentration of betulonic acid in the medium before the transformation culture is 2 to 5000. mu.g/mL.
Preferably, in step 2), the extraction solvent for extraction is ethyl acetate.
Preferably, in step 3), the gradient elution conditions are preferably methanol: 20:80-40:60-60:40-80:20-100:0 of water.
Compared with the prior art, the invention successfully carries out structural modification on the mother nucleus structure of the betulonic acid by utilizing a microbial transformation technology to obtain a new betulonic acid derivative, and the compounds have better myocardial cell protective activity as proved by an in vitro myocardial cell injury protection test and a myocardial cell ischemia reperfusion test, can be used as active ingredients of medicaments for treating myocardial infarction, coronary atherosclerotic heart disease and chronic heart failure, and have wide application.
Detailed Description
In order to further illustrate the present invention, the betulonic acid derivatives and the preparation method thereof provided by the present invention are described in detail below 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
+24.7 ° (c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrummax：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
-31.5 ° (c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrummax：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
+11.8 ° (c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrummax：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
+17.2 ° (c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrummax：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
-10.1 ° (c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrummax：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
Main absorption peak (KBr) v of infrared spectrummax：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
+20.2 ° (c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrummax：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
+14.9 ° (c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrummax：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
+28.1 ° (c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrummax：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
-16.8 ° (c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrummax：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
+34.4 ° (c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrummax：3455,3046,2936,1724,1706,1388,1215,1024cm-1(ii) a High resolution mass spectrometrym/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
+10.3 ° (c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrummax：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
+26.7 ° (c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrummax：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); the NMR 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
+35.5 ° (c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrummax：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-a)cetoxy-lup-28-oic acid); melting point 311-313 ℃; optical rotation
-13.6 ° (c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrummax：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
+58.3 ° (c ═ 0.1, MeOH); main absorption peak (KBr) v of infrared spectrummax：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, Techniaki, Tianjin) and H9c2 cells (tumor research institute, national academy of medical sciences).
(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. 490nm of enzyme labeling instrumentThe absorbance of each well was measured and the cell viability was calculated: 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 remarkably improve the survival rate of cells, and show that the betulonic acid derivatives I-XVI have remarkable myocardial cell protection effect, show good dose dependence in a certain dose range, and can be used as active ingredients 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 Biotech research institute), DMEM high-sugar medium (Gibcol BRL), fetal bovine serum, dimethyl sulfoxide (DMSO), trypsin (Shanghai bioengineering Co., Ltd.), and H9c2 cell (Chinese Biotechnology engineering Co., Ltd.)Institute of oncology, 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.
Claims (4)
1. The betulonic acid derivative or the pharmaceutically acceptable salt thereof is characterized in that the structural formula of the betulonic acid derivative is selected from any one of the following structural formulas:
2. the method for producing a betulonic acid derivative according to claim 1, comprising the steps of:
1) fermenting and culturing microorganisms in a culture medium, adding betulonic acid for transformation culture, and removing mycelium to obtain a fermentation broth, wherein the microorganisms are strains of rhizopus, Absidia, Mucor or Coptomyces;
2) extracting the fermentation liquor obtained in the step 1) to obtain a converted crude extract;
3) subjecting the transformed crude extract obtained in the step 2) to reversed-phase silica gel ODS-C18 column chromatography, performing gradient elution by adopting a methanol-water two-phase system, and collecting and combining components;
4) further purifying the components obtained in the step 3) by using a reversed phase high performance liquid chromatography to obtain the betulonic acid derivative.
3. The method according to claim 2, wherein the concentration of betulonic acid in the medium before the transformation culture in step 1) is 2 to 5000. mu.g/mL.
4. The method according to claim 2, wherein the extraction solvent used in the step 2) is ethyl acetate.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113712972A (en) * | 2021-09-13 | 2021-11-30 | 南通大学 | Application of betulonic acid derivative in preparation of medicine for treating cardiovascular diseases |
| CN115232187A (en) * | 2022-08-10 | 2022-10-25 | 江西省科学院应用化学研究所 | Method for extracting 7 beta-hydroxy betulinic acid from cinnamomum camphora and application thereof |
-
2021
- 2021-09-13 CN CN202111068956.1A patent/CN113603744A/en not_active Withdrawn
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113712972A (en) * | 2021-09-13 | 2021-11-30 | 南通大学 | Application of betulonic acid derivative in preparation of medicine for treating cardiovascular diseases |
| CN115232187A (en) * | 2022-08-10 | 2022-10-25 | 江西省科学院应用化学研究所 | Method for extracting 7 beta-hydroxy betulinic acid from cinnamomum camphora and application thereof |
| CN115232187B (en) * | 2022-08-10 | 2024-04-12 | 江西省科学院应用化学研究所 | Method for extracting 7 beta-hydroxy betulinic acid from borneol camphor and application thereof |
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Application publication date: 20211105 |