阅读说明：本技术 白桦脂酮酸衍生物在制备抗肿瘤药物中的应用 (Application of betulonic acid derivative in preparation of antitumor drugs ) 是由 陈广通 吴艳妮 李哲 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,公开了一类白桦脂酮酸衍生物或其药学上可成的盐及其制备抗肿瘤药物中的应用。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的白桦脂酮酸衍生物,通过体外抗肿瘤细胞试验证实,这些化合物具有较好的抗肿瘤活性,可以作为抗肿瘤药物的活性成分,具有广泛的用途。(The invention belongs to the technical field of medicines, and discloses betulonic acid derivatives or pharmaceutically acceptable salts thereof and application thereof in preparing antitumor medicines. 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 in-vitro anti-tumor cell tests prove that the compounds have better anti-tumor activity, can be used as active ingredients of anti-tumor drugs and have wide application.)

1. The application of betulonic acid derivatives with any structural formula or pharmaceutically acceptable salts thereof in preparing antitumor drugs,

2. use according to claim 1 wherein the antineoplastic medicament comprises an active ingredient selected from one or more betulonic acid derivatives 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 or a pharmaceutically acceptable salt thereof,

3. the use of claim 1 or 2, wherein the tumor is cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer or colon cancer.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an application of betulonic acid derivatives in preparation of antitumor drugs.

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 terms of chemical structure modification, the chemical synthesis derivatives of betulonic acid are rich in research on anti-tumor aspects, but the sites for chemical structure modification are few, and the carbonyl group at the 3-position and the carboxyl group at the 28-position of betulonic acid are mainly subjected to derivatization. However, due to the structural particularity of the pentacyclic triterpenoid, the parent nucleus lacks active groups, and the derivative with hydroxyl, carbonyl and other modifications on the parent nucleus is difficult to obtain by adopting a conventional chemical reaction method. Therefore, the chemical and pharmacological studies of betulonic acid derivatives with mother nucleus structure modification are not comprehensive.

Microbial transformation is an enzyme-catalyzed reaction carried out by utilizing an enzyme system with the self-specificity of an organism, has multiple reaction types and high stereoselectivity and regioselectivity, and becomes an important tool in organic synthesis. When the derivative is used for preparing the betulonic acid derivative, active reaction groups such as hydroxyl, carbonyl, acetyl and the like can be introduced into a triterpene mother nucleus, so that the biological activity and the bioavailability of the compound are improved. Meanwhile, the active group introduced on the parent nucleus can provide a new active site for chemical reaction, thereby providing a large amount of samples for the subsequent research of the compounds.

Disclosure of Invention

In view of the above, the present invention provides an application of betulonic acid derivatives in the preparation of antitumor drugs, and particularly provides an application of betulonic acid derivatives or pharmaceutically acceptable salts thereof in the preparation of antitumor drugs, and these betulonic acid derivatives have good antitumor activity. In the invention, the betulonic acid derivative is a compound with a structural formula of formula I-formula XVI:

the invention also provides a preparation method of the betulonic acid derivative, which 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.

Experiments prove that the betulonic acid derivative has good anti-tumor activity and can be used as an active ingredient of an anti-tumor medicament. The active ingredient of these antitumor drugs may be one or more compounds selected from the group consisting of compounds of the formulae I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV and XVI.

In the medicine taking the compound as the active ingredient, one or more pharmaceutically acceptable carriers can be added when necessary. The carrier comprises a diluent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant and the like which are conventional in the pharmaceutical field, and can be prepared according to a conventional method in the pharmaceutical field.

Further, the tumor is cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer or colon cancer.

The invention successfully carries out structural modification on the betulonic acid by utilizing a microbial transformation technology to obtain a new betulonic acid derivative, and the compounds have better antitumor activity and can be used as active ingredients of antitumor drugs and have wide application as proved by in vivo animal experiments and in vitro antitumor cell experiments.

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 rotationMain 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 rotationMain 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 rotationMain 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 betulonic acid (3-oxo-30-acetoxy-7 beta-hydroxy-lup-20 (29) -en-28-oic acid); melting point 304-305 ℃; optical rotationMain 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 rotationMain 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 rotationMain 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 rotationMain absorption peak (KBr) v of infrared spectrum_max：3497,3044,2962,1753,1712,1384,1207,1055cm^-1(ii) a High resolution mass spectrometrym/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 rotationMain 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 rotationMain 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 rotationMain 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 rotationMain 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 rotationMain 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 rotationMain 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); 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 rotationMain 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 rotationMain 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 rotationMain 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 antitumor Activity of Compound I of the present invention-Compound XVI

1) Experimental Material

Instruments and reagents: CO 2₂Incubator (Joean IGO 150); microplate reader (Bio-TEK ELx 800); fluorescence inverted microscope (Olympus IX 51); MTT cell proliferation and cytotoxicity detection kit (Biyuntian biotechnological research institute), RPM I1640 culture medium (Gibcol BRL), RNase A, fetal bovine serum, dimethyl sulfoxide (DMSO), and trypsin (Shanghai bioengineering Co., Ltd.).

Tumor cell lines were tested: hela cells (human cervical cancer cells), K562 cells (human leukemia cells), K562/ADR cells (human leukemia drug-resistant cells), SH-SY5Y cells (human neuroblastoma cells), Du-145 (human prostate cancer cells), HePG2 cells (human liver cancer cells), MCF-7 cells (human breast cancer cells), and CT26 cells (colon cancer cells), which were purchased from the institute of tumor research of the Chinese academy of medicine and sciences.

Test samples: the purity of the ursolic acid and the compounds I-IV synthesized in the embodiment 1 is more than 95 percent; meanwhile, cisplatin is selected as a positive control drug, and each compound is dissolved in DMSO and then diluted.

2) Experimental methods

Determination of half inhibition rate IC of each tested compound on tumor cell strain by MTT method₅₀The value: taking tumor cells in logarithmic growth phase, adjusting cell concentration to 5 × 10 with RPM I1640 culture solution containing 10% calf serum⁵and/mL, inoculating the cells in a 96-well culture plate, adding 100 mu L of cell suspension into each well of a drug treatment group and a cell control group, setting 3 multiple wells in each group, adding only RPM I1640 full culture medium into a blank control group, setting 100 mu L of cell suspension into each well, and setting 3 multiple wells. Placing 96-well culture plate at 37 deg.C and 5% CO₂After 24h of incubation in an incubator, test samples of different concentrations were added to a final concentration of 0.1-100. mu.M, and incubation was continued for 72 h. Measuring absorbance (A) value at 570nm by MTT method in microplate reader, and calculating inhibition rate (1-experiment group A value/control group A value) × 100%]. The experiment was repeated 3 times. Using SPSS 11.5 software as regression equation, calculate half maximal Inhibitory Concentration (IC) of each test sample for 72h on tumor cells₅₀)。

3) Results of the experiment

Calculating IC of betulonic acid and compounds I-XVI of the present invention on the above cells based on MTT assay results₅₀The results are shown in Table 5.

TABLE 5 screening results for in vitro cytotoxic Activity of test samples

The results show that the compounds I-XVI have good antitumor activity and can be used as the active ingredients of antitumor drugs.

It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.