Polyhydroxysubstituted arbutin derivatives and their preparation methods and applications through microbial transformation

Abstract

This invention belongs to the field of pharmaceutical technology and discloses a class of polyhydroxy-substituted arbutin derivatives, their microbial transformation preparation method, and their applications. This invention utilizes microbial transformation technology, using arbutin as a substrate, and employing the microorganism *Helicobacter pylori* for catalytic transformation. Hydroxyl groups are introduced at multiple positions in the arbutin core with regioselectivity and stereoselectivity, thereby obtaining novel polyhydroxy-substituted arbutin derivatives. In vitro antitumor cell experiments have confirmed that the polyhydroxy-substituted arbutin derivatives obtained by this invention possess significant antitumor activity and can be used as active ingredients in antitumor drugs, exhibiting broad applications.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to a class of polyhydroxy substituted arbutin derivatives and their preparation methods and applications through microbial transformation. Background Technology

[0002] Arbutin, also known as ursolicol, is a natural ursane-type pentacyclic triterpenoid compound widely distributed in olives, including olive leaves, bark, fruit, and extra virgin olive oil. The arbutin content is one of the important indicators for evaluating the quality of olive oil. It can also be isolated from different parts of plants such as bayberry, camellia, oleander, lavender, and apple. Preliminary studies have shown that arbutin possesses various pharmacological activities, including anti-inflammatory, cardiotonic, antiarrhythmic, and antitumor properties, as well as the ability to promote skin wound healing.

[0003] However, arbutin's poor solubility, low bioavailability, and limited number of hydrogen bond donors in its parent nucleus and rotatable bonds in its rigid framework have restricted further chemical and biological research. To improve these properties, researchers primarily use chemical synthesis and biotransformation to modify its structure. Conventional chemical synthesis methods are costly, and introducing functional groups onto saturated CH bonds is particularly difficult. Microbial transformation, on the other hand, is a simple and effective technique for modifying the structure of natural compounds, allowing for regioselectivity and stereoselectivity in introducing functional groups. It offers advantages such as high efficiency, economy, and environmental friendliness, which are difficult to achieve with conventional chemical modifications. As a highly efficient modification tool, microbial transformation technology can modify the structures of naturally active compounds with different skeletons and has been widely applied to the structural modification of various compounds. The transformation products obtained through microbial transformation can, on the one hand, yield derivatives with stronger biological activity that can be directly used in drug development. On the other hand, the newly introduced chemically active groups on the parent nucleus after microbial transformation increase the sites for chemical modification and alteration, thus solving the problem of limited reaction sites in the organic chemical preparation of triterpenoid derivatives. However, compared to chemical synthesis, microbial transformation is highly stochastic; the results are not as predictable or designed as in chemical synthesis. Furthermore, even similar substrates transformed by the same or different strains can yield significantly different products. Conversely, the same substrate transformed by different strains may produce significantly different transformation products. Therefore, selecting appropriate strains is crucial when using microbial transformation for compound structural modification. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a series of polyhydroxy-substituted arbutin derivatives and their preparation methods by microbial transformation, which can be used to prepare antitumor drugs.

[0005] This invention provides a series of polyhydroxy substituted arbutin derivatives, namely 7β,15α,19α,21α-tetrahydroxyarbutin, 7β,15α,20β,21β-tetrahydroxyarbutin, and 7β,15α,20β,23-tetrahydroxyarbutin, with structures shown in Formulas I-III, respectively:

[0006]

[0007] This invention also provides a method for preparing the above-mentioned polyhydroxy-substituted arbutin derivative by microbial transformation, comprising the following steps:

[0008] 1) Fermentation culture of microorganisms, adding arbutin, followed by transformation culture, and after removing the mycelium, the fermentation broth was obtained. The microorganism was Circinellamuscae CGMCC3.2695.

[0009] 2) The fermentation broth is extracted to obtain the transformed product;

[0010] 3) The transformed product was purified by reversed-phase silica column chromatography using a methanol-water two-phase system with gradient elution, and the components were collected and combined.

[0011] 4) The components were purified by reversed-phase high-performance liquid chromatography to obtain 7β,15α,19α,21α-tetrahydroxyarbutin, 7β,15α,20β,21β-tetrahydroxyarbutin and 7β,15α,20β,23-tetrahydroxyarbutin.

[0012] The present invention also provides the application of the above-mentioned polyhydroxy substituted arbutin derivatives or the polyhydroxy substituted arbutin derivatives prepared by the above-mentioned microbial transformation preparation method in the preparation of antitumor drugs, wherein the tumor includes one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer and colon cancer.

[0013] The present invention also provides the application of the above-mentioned polyhydroxy substituted arbutin derivatives or the polyhydroxy substituted arbutin derivatives prepared by the above-mentioned microbial transformation preparation method in the preparation of drugs for killing drug-resistant leukemia cells.

[0014] The present invention also provides an antitumor drug, wherein the active ingredient of the antitumor drug is the above-mentioned polyhydroxy substituted arbutin derivative or a pharmaceutically acceptable salt thereof; the polyhydroxy substituted arbutin derivative is one or more of 7β,15α,19α,21α-tetrahydroxyarbutin, 7β,15α,20β,21β-tetrahydroxyarbutin and 7β,15α,20β,23-tetrahydroxyarbutin; the tumor includes one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer and colon cancer.

[0015] Compared with the prior art, the present invention utilizes the catalytic action of microbial enzymes to introduce hydroxyl or carbonyl groups at the 7β, 15α, 19α, 20β, 21β(α) or 23 positions of the arbutin core in a regioselective and stereoselective manner, thereby obtaining novel polyhydroxy substituted arbutin derivatives of formulas I to III. In vitro antitumor cell experiments have confirmed that compounds of formulas I to III have significant antitumor activity and can be used as active ingredients in antitumor drugs, with a wide range of applications. Detailed Implementation

[0016] To further illustrate the present invention, the preparation method of the polyhydroxy arbutin derivative provided by the present invention and its application in the preparation of antitumor drugs are described in detail below with reference to embodiments.

[0017] Example 1: Preparation of compounds with structural formulas I-III

[0018] Using a microbial transformation method, arbutin was converted from the substrate *Circinella muscae* CGMCC 3.2695 to prepare compounds with structural formulas I-III. The preparation process of the compounds is as follows:

[0019] After 7 days of transformation with *Pheretima aspergillum*, 500 mg of arbutin was extracted with an equal volume of ethyl acetate to obtain approximately 0.92 g of crude extract. The crude extract was separated by ODS-C18 column chromatography with a methanol:water gradient elution (20:80, 30:70, 60:40, 80:20, 100:0). The fractions were then combined after HPLC analysis to obtain combined fractions Fr.1-Fr.3. Fraction Fr.2 was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) with acetonitrile-water (55:45, V / V) at a flow rate of 3.0 mL / min and a detection wavelength of 203 nm. The transformation products with structural formulas I-III were obtained. The mass spectrometry and wavelength spectrometry data of compounds I-III are shown below.

[0020] Compound I: 7β,15α,19α,21α-tetrahydroxyarbutin; optical rotation High-resolution mass spectrometry m / z 529.3506 [M+Na] +(calcd.C 30 H 50 O6Na, 529.3505); the proton and carbon NMR spectra are shown in Table 2.

[0021] Compound II: 7β,15α,20β,21β-tetrahydroxyarbutin; optical rotation +11.3° (c=0.1, MeOH); High-resolution mass spectrometry m / z 529.3507 [M+Na] + (calcd.forC 30 H 50 O6Na, 529.3505); the proton and carbon NMR spectra are shown in Table 2.

[0022] Compound III: 7β,15α,20β,23-Tetrahydroxyarbutin; Optical rotation High-resolution mass spectrometry m / z 529.3506 [M+Na] + (calcd.for C 30 H 50 O6Na, 529.3505); the proton and carbon NMR spectra are shown in Table 1.

[0023] Table 1. 1H and 1C NMR spectra of compounds I through III

[0024]

[0025]

[0026] The above results indicate that the structures of the obtained compounds, formulas I-III, are correct.

[0027] Example 2: Antitumor activity of compounds of formulas I-III

[0028] 1) Experimental materials

[0029] Instruments and reagents: CO2 incubator (Jouan IGO150); fluorescence inverted microscope (Olympus IX51); MTT cell proliferation and cytotoxicity assay kit (Beyotime Biotechnology Research Institute), RPM I 1640 medium (Gibcol BRL), RNase A, fetal bovine serum, dimethyl sulfoxide (DMSO), and trypsin (Shanghai Bioengineering Co., Ltd.).

[0030] The tumor cell lines used for testing were: HeLa cells (human cervical cancer cells), K562 cells (human leukemia cells), K562 / ADR cells (human drug-resistant leukemia cells), SH-SY5Y cells (human neuroblastoma cells), Du-145 cells (human prostate cancer cells), HePG2 cells (human liver cancer cells), MCF-7 cells (human breast cancer cells), and CT26 cells (colon cancer cells), purchased from the Cancer Institute of the Chinese Academy of Medical Sciences.

[0031] Test samples: arbutin and compounds I-III synthesized in Example 1, with a purity of over 95%; cisplatin was selected as a positive control drug, and all compounds were dissolved in DMSO and then diluted.

[0032] 2) Experimental methods

[0033] The half-maximal inhibitory rate (IC50) of each tested compound against tumor cell lines was determined using the MTT assay. 50 Value: Tumor cells in the logarithmic growth phase were collected and their concentration adjusted to 5 × 10⁻⁶ cells using RPM I 1640 culture medium containing 10% fetal bovine serum. 5 Cells were seeded in 96-well plates at a concentration of 100 μL / well. The drug treatment group and cell control group received 100 μL of cell suspension per well, with three replicates per group. The blank control group received only 100 μL of RPM I 1640 complete culture medium per well, also with three replicates. After incubating the 96-well plates at 37°C and 5% CO2 for 24 h, different concentrations of the test sample were added to achieve a final concentration of 0.1-100 μM, and incubation continued for 72 h. The absorbance (A) at 570 nm was measured using the MTT assay, and the inhibition rate was calculated [Inhibition rate = (1 - A value of experimental group / A value of control group) × 100%]. The experiment was repeated three times. SPSS 11.5 software was used to perform regression equations to calculate the half-maximal inhibitory concentration (IC50) of each test sample against tumor cells after 72 h. 50 ).

[0034] 3) Experimental Results

[0035] Based on the MTT assay results, the IC50 of the test sample for the above-mentioned cells was calculated. 50 The values ​​are shown in Table 2.

[0036] Table 2. Screening results of in vitro cytotoxic activity of test samples

[0037]

[0038]

[0039] The results show that the compounds represented by Formulas I-III of this invention possess good antitumor activity and can be used as active ingredients in antitumor drugs. Furthermore, the compounds represented by Formulas I-III of this invention can effectively kill drug-resistant human leukemia cells.

[0040] Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A multi-hydroxyl substituted ursolic acid derivative, characterized in that, the ursolic acid derivative is one of 7β, 15α, 19α, 21α-tetrahydroxy ursolic acid, 7β, 15α, 20β, 21β-tetrahydroxy ursolic acid and 7β, 15α, 20β, 23-tetrahydroxy ursolic acid; the 7β, 15α, 19α, 21α-tetrahydroxy ursolic acid has a structure of Formula I: ； the 7β, 15α, 20β, 21β-tetrahydroxy ursolic acid has a structure of Formula II: ； the 7β, 15α, 20β, 23-tetrahydroxy ursolic acid has a structure of Formula III:

2. A process for the microbial conversion of a polyhydroxy-substituted urocanic acid derivative according to claim 1, characterized in that comprising the following steps: 1) fermenting a microorganism, adding ursolic acid, then performing a transformation culture, removing mycelium to obtain a fermentation liquor, wherein the microorganism is Circinella muscae CGMCC 3.2695; 2) extracting the fermentation liquor to obtain a transformation product; 3) purifying the transformation product through a reversed-phase silica gel column chromatography, wherein the reversed-phase silica gel column chromatography adopts a methanol-water two-phase system gradient elution, and after collecting and combining components, a reversed-phase high performance liquid chromatography is used for purification to obtain the multi-hydroxyl substituted ursolic acid derivative. 3.Use of the multi-hydroxyl substituted ursolic acid derivative of claim 1 in the preparation of an anti-tumor drug, wherein the tumor is one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer and colon cancer.

4. Use according to claim 3, characterized in that, The anti-tumor drug is a drug for killing leukemia drug-resistant cells.

5. A pharmaceutical composition for use against tumors, characterized in that, The active ingredient of the pharmaceutical composition is the multi-hydroxyl substituted ursolic acid derivative of claim 1 or a pharmaceutically acceptable salt thereof, and the tumor is one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer and colon cancer.

6. The pharmaceutical composition of claim 5, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant, which is one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier and a lubricant.

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