Arbutiol amino acid derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and discloses an arbutin amino acid derivative as well as a preparation method and application thereof. According to the invention, a microbial conversion technology is utilized, the arbutin is taken as a substrate, pyroglutamic acid is introduced to the 28 site of the arbutin in a regioselective manner, and the arbutin-28beta-pyroglutamate with a novel structure is obtained. An in-vitro anti-tumor cell test proves that the compound has remarkable anti-tumor activity and can be used as an active component of an anti-tumor drug. A mouse in-vivo experiment proves that the compound has a remarkable anti-osteoporosis effect, can be used as an active ingredient of an anti-osteoporosis medicine, and has wide application.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine and relates to a ursolic alcohol amino acid derivative, a preparation method thereof and an application thereof. Background Art

[0002] Ursolic alcohol is a naturally occurring pentacyclic triterpenoid compound, which mainly exists in plants of Saxifragaceae, Ericaceae, etc. in free or glycoside form. Its typical sources include bearberry leaves, gardenia fruits, privet leaves, plantain, pomegranate leaves, etc. Recent studies have shown that ursolic alcohol has rich biological activities, including anti-inflammatory and immunomodulatory, antioxidant, antibacterial and antitumor effects. However, the clinical application of ursolic alcohol is limited due to its low content in plants, poor water solubility and low bioavailability. Therefore, it is necessary to appropriately modify the structure of ursolic alcohol to improve its biological activity and drug-likeness and overcome the above disadvantages. At present, researchers mainly modify the structure of ursolic alcohol through two approaches: chemical synthesis and biotransformation.

[0003] Conventional chemical synthesis methods have two technical bottlenecks in the structural modification of natural products: firstly, the synthesis cost is high and it depends on the chemical active groups of the substrates; secondly, it is difficult to achieve directed functional group modification. In contrast, microbial transformation technology exhibits unique biocatalytic advantages. Through the enzymatic reaction mechanism, it can precisely achieve regio-selective and stereo-selective functionalization modification, with both the characteristics of green chemistry and the advantages of atom economy. This technology has been successfully applied to the skeleton modification of various types of natural products (such as terpenoids, alkaloids, etc.). Its transformation products have dual application values: novel derivatives with enhanced biological activity can be directly obtained, or molecular modification sites can be provided for subsequent chemical modification by introducing active functional groups. Especially for the problem of limited chemical derivatization sites of triterpenoid compounds, this technology can effectively break through the bottleneck of their structural modification.

[0004] However, the microbial transformation system has significant biological characteristics: ① The catalytic reaction shows non-directional characteristics, and it is difficult to achieve the predictability of chemical synthesis for the transformation products; ② The strain-substrate interaction is specific, manifested as the differential catalysis of homologous strains on structural analogs and the diverse transformation of heterologous strains on the same substrate. Therefore, it is crucial to select a suitable strain when using the method of microbial transformation for the structural modification of compounds. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a novel ursolic alcohol amino acid derivative, a preparation method thereof and an application thereof.

[0006] In the first aspect of the present invention, a ursolic alcohol amino acid derivative is provided. The ursolic alcohol amino acid derivative is ursolic alcohol-28β-pyroglutamate ester, and the ursolic alcohol amino acid derivative has the structure shown in Formula I:

[0007]

[0008] The compound with the structural formula of Formula I is a novel arbutinol derivative first disclosed in the present invention.

[0009] In the second aspect of the present invention, a preparation method of the above arbutinol amino acid derivative is further provided. The preparation method is as follows: using Circinella muscae CGMCC 3.2695 as a transformation strain, performing microbial transformation on arbutinol to obtain the arbutinol amino acid derivative.

[0010] In some embodiments of the present invention, the preparation method includes the following steps:

[0011] 1) Fermenting and culturing the microorganism, adding arbutinol to the culture medium, then performing transformation culture, and obtaining the fermentation broth after removing the mycelium. The microorganism is Circinella muscae CGMCC 3.2695;

[0012] 2) Extracting the fermentation broth to obtain the transformant;

[0013] 3) Purifying the transformant by reverse-phase silica gel column chromatography. The reverse-phase silica gel column purification uses a methanol-water two-phase system for gradient elution, and collecting and combining the elution fractions obtained when using a mixed solvent with a volume ratio of methanol to water of 80:20 as the eluent;

[0014] 4) Purifying the elution fraction by reverse-phase high performance liquid chromatography to obtain the arbutinol amino acid derivative.

[0015] The synthesis route of the above preparation method by microbial transformation is shown as follows:

[0016]

[0017] In the third aspect of the present invention, an application of the above arbutinol amino acid derivative as an active ingredient in the following aspects is provided:

[0018] 1) Application in the preparation of anti-tumor drugs;

[0019] 2) Application in the preparation of eukaryotic cancer cell proliferation inhibitors;

[0020] 3) Application in the preparation of drugs for killing leukemia drug-resistant cells;

[0021] 4) Application in the preparation of drugs for treating osteoporosis.

[0022] In some embodiments of the present invention, the tumor is one of cervical cancer, leukemia, neuroblastoma, liver cancer and colon cancer.

[0023] In some embodiments of the present invention, the eukaryote is a mammal.

[0024] In some embodiments of the present invention, the cancer cell is one of cervical cancer cells, leukemia cells, neuroblastoma cells, liver cancer cells, and colon cancer cells.

[0025] In some embodiments of the present invention, the osteoporosis is postmenopausal osteoporosis.

[0026] In some embodiments of the present invention, the drug for treating osteoporosis is a drug for increasing the bone density of postmenopausal osteoporosis mice.

[0027] In the fourth aspect of the present invention, a product is provided. The active ingredient of the product is the above-mentioned arbutinol amino acid derivative, and the product is:

[0028] 1) An anti-tumor drug;

[0029] 2) A eukaryotic cancer cell proliferation inhibitor;

[0030] 3) A drug for killing leukemia drug-resistant cells;

[0031] 4) A drug for treating osteoporosis.

[0032] In some embodiments of the present invention, the drug further contains pharmaceutically acceptable excipients.

[0033] In some embodiments of the present invention, the pharmaceutically acceptable excipients include excipients, diluents, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, etc.

[0034] Compared with the prior art, the present invention utilizes the catalytic action of microbial enzymes to regioselectively introduce pyroglutamic acid produced by the microorganism itself at the 28β position of arbutinol, thereby obtaining the novel-structured arbutinol-28β-pyroglutamate ester type I. It is confirmed by in vitro anti-tumor cell experiments that compound type I has significant anti-tumor activity and can be used as the active ingredient of anti-tumor drugs. It is confirmed by in vivo animal experiments that compound type I has significant anti-osteoporosis activity and can be used as the active ingredient of anti-osteoporosis drugs, and has a wide range of uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 1H-NMR spectrum of compound I prepared in Example 1 of the present invention 1 H-NMR spectrum;

[0036] Figure 2 1H-NMR spectrum of compound I prepared in Example 1 of the present invention 13C-NMR spectrum;

[0037] Figure 3 It is the DEPT-135 spectrum of Compound Ⅰ prepared in Example 1 of the present invention;

[0038] Figure 4 It is the HSQC spectrum of Compound Ⅰ prepared in Example 1 of the present invention;

[0039] Figure 5 It is the HMBC spectrum of Compound Ⅰ prepared in Example 1 of the present invention;

[0040] Figure 6 It is the HR-ESIMS diagram of Compound Ⅰ prepared in Example 1 of the present invention. Detailed implementation mode

[0041] In order to further illustrate the present invention, the arbutinol amino acid derivatives provided by the present invention, their preparation methods and applications will be described in detail below in conjunction with examples.

[0042] Example 1: Preparation of a compound with the structural formula of Formula Ⅰ

[0043] The present invention adopts a microbial transformation method, uses arbutinol as a raw material, and prepares the compound of the present invention through steps such as fermentation, extraction, and separation. Circinella muscae CGMCC 3.2695 was purchased from the China General Microbiological Culture Collection Center (CGMCC), and a potato medium was selected and stored in a 4°C refrigerator on a solid slant medium.

[0044] 1) Fermentation, transformation, and extraction

[0045] Circinella muscae CGMCC 3.2695 was inoculated into a 250 mL conical flask (containing 100 mL of potato liquid medium) as a seed solution. After shaking culture at 160 rpm and 26°C for 12 hours, when the mycelial growth was in the vigorous stage, 1 mL of the seed solution was aspirated with a sterile pipette and added to 10 1000 mL flasks (containing 400 mL of potato liquid medium). After shaking culture for 24 hours, 25 mg of arbutinol (0.25 mL, 100 mg / mL ethanol solution) was added to each flask, and a total of 250 mg of the substrate was used. The transformation was continued for 7 days under the same conditions. The fermentation broth was filtered to remove the mycelium, and the filtrate was extracted 3 times with an equal volume of ethyl acetate. The extract was concentrated under reduced pressure to dryness, and about 0.86 g of the crude extract of the transformed product was obtained.

[0046] 2) ODS-C18 column chromatography separation

[0047] The crude extract was separated by ODS-C18 column chromatography. Methanol: water gradient elution (20:80, 30:70, 60:40, 80:20, 100:0). The fraction of methanol: water 80:20 was collected.

[0048] 3) Purification by reverse-phase high performance liquid chromatography

[0049] The fraction of methanol: water 80:20 was purified by reverse-phase high performance liquid chromatography to obtain Compound Ⅰ. The preparation conditions were a semi-preparative chromatographic column YMC ODSA-5μm, 12.0×250mm, acetonitrile-water (40:60, V / V), flow rate 3.0 mL / min, detection wavelength 203 nm.

[0050] The mass spectrometry and spectroscopy data of Compound Ⅰ are shown below.

[0051] Compound Ⅰ: Arbutol-28β-pyroglutamate; specific rotation +28.2° (c = 0.22, MeOH); high resolution mass spectrometry m / z 576.4048 [M+Na] + (calcd. C 35 H 55 NO4Na, 576.4028); the carbon nuclear magnetic resonance spectrum data are shown in Table 1.

[0052] Table 1 Signal assignment of 13 C-NMR (150 MHz, CDCl3) of Compound Ⅰ

[0053] Number <![CDATA[δ C > Number <![CDATA[δ C > 1 38.8 19 39.2 2 27.3 20 39.4 3 79.0 21 32.8 4 36.9 22 35.8 5 54.3 23 28.1 6 18.3 24 15.7 7 30.4 25 15.6 8 40.0 26 16.7 9 47.6 27 23.4 10 38.8 28 72.4 11 23.4 29 17.3 12 125.9 30 21.2 13 137.9 1' 171.9 14 42.0 2' 55.4 15 26.0 3' 29.2 16 23.5 4' 26.0 17 37.1 5' 177.5 18 55.2

[0054] For Compound Ⅰ 1 the H-NMR spectrum, 13 C-NMR spectrum, DEPT-135 spectrum, HSQC spectrum, HMBC spectrum and HR-ESIMS diagram are as Figures 1 - 6 shown.

[0055] The above results indicate that Compound Ⅰ prepared in this example has the structure of Formula Ⅰ.

[0056] Example 2: Antitumor activity of Compound of Formula Ⅰ

[0057] 1) Experimental materials

[0058] Instruments and reagents: CO2 incubator (Jouan IGO150); fluorescence inverted microscope (Olympus IX51); MTT cell proliferation and cytotoxicity detection kit (Beyotime Biotechnology Research Institute), RPMI 1640 medium (GibcolBRL), RnaseA, fetal bovine serum, dimethyl sulfoxide (DMSO), trypsin (Shanghai Bioengineering Co., Ltd.), arbutinol (Shanghai Macklin Biochemical Co., Ltd.), L-pyroglutamic acid (Shanghai Macklin Biochemical Co., Ltd.).

[0059] Tumor cell lines for testing: 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), CT26 cells (colon cancer cells), purchased from the Cancer Institute of the Chinese Academy of Medical Sciences.

[0060] Test samples: Arbutinol, L-pyroglutamic acid, and the compound of formula I synthesized in Example 1, with a purity of over 95%; at the same time, cisplatin was selected as the positive control drug, and each compound was dissolved in DMSO and diluted.

[0061] 2) Experimental method

[0062] The half inhibitory concentration IC 50 value of each test compound on tumor cell lines was determined by the MTT method: Tumor cells in the logarithmic growth phase were adjusted to a cell concentration of 5×10 5 / mL with RPMI 1640 culture medium containing 10% calf serum, inoculated into 96-well culture plates, 100 μL of cell suspension was added to each well in the drug treatment group and the cell control group, with 3 replicates in each group. The blank control group only added 100 μL of RPMI 1640 complete medium, with 3 replicates in each well. After culturing the 96-well culture plates in a 37°C, 5% CO2 incubator for 24 h, different concentrations of test samples were added to make the final concentration 0.1 - 100 μM, and the culture was continued for 72 h. According to the MTT method, the absorbance (A) value at 570 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the inhibition rate was calculated [inhibition rate = (1 - A value of the experimental group / A value of the control group) × 100%]. The experiment was repeated 3 times. The SPSS11.5 software was used to establish a regression equation to calculate the half inhibitory concentration (IC 50 ) of each test sample on tumor cells after 72 h of action.

[0063] 3) Experimental results

[0064] According to the test results of the MTT method, the IC 50The values are shown in Table 2 as follows.

[0065] Table 2. Screening results of in vitro cytotoxic activity of test samples ( n = 3)

[0066]

[0067] The results show that the compound shown in Formula I of the present invention has stronger killing activity against cervical cancer, leukemia, neuroblastoma, liver cancer and colon cancer compared with arbutinol, and can be used as an active ingredient of anti-tumor drugs. However, it does not show killing activity against prostate cancer and breast cancer, showing a certain selectivity. At the same time, the compound shown in Formula I of the present invention can effectively kill human leukemia drug-resistant cells.

[0068] Example 3: Effect of Compound Formula I on BMD of OVX Mice

[0069] A postmenopausal osteoporosis model of mice was prepared by bilateral gonad (ovary) removal method (ovariectomized, OVX). Female C57BL / 6J mice at 12 weeks of age were randomly divided into sham operation group, model group, positive control group, arbutinol treatment group (2 mg / kg), L-pyroglutamic acid group (2 mg / kg) and compound formula I treatment group (2 mg / kg), with 8 mice in each group. A postmenopausal osteoporosis model was established by bilateral ovariectomy. After the operation, the sham operation group and the model group were given intragastric administration of 0.9% sodium chloride solution, the positive control group was given intragastric administration of 0.2 mg / kg estradiol valerate solution, and the drug treatment groups were given corresponding drugs by intragastric administration once a day for 8 consecutive weeks. After the animals were anesthetized, the bone mineral density (BMD) values of the fourth lumbar vertebra and the right femur of the rats were measured by a bone densitometer. SPSS 22.0 software was used for variance analysis and LSD-t test, and the results are shown in Table 2.

[0070] Table 2. Effect of Compound Formula I on Bone Mineral Density (BMD) of OVX Mice ( n = 3)

[0071]

[0072]

[0073] Compared with the blank group * P < 0.05; compared with the model group # P < 0.05

[0074] Compared with the blank control group, the bone mineral density values of the fourth lumbar vertebra and the right femur in the model group were significantly decreased (P < 0.05). Compared with the model control group, the bone mineral density values of the fourth lumbar vertebra and the right femur in the positive control group and the compound formula I treatment group were significantly increased (P < 0.05), and the therapeutic effect of compound formula I was better than that of the positive control group. However, no obvious therapeutic effects were observed for both arbutinol and L-pyroglutamic acid. The experimental results indicate that compound formula I can significantly increase the bone mineral density of postmenopausal osteoporosis mice and can be used as an active ingredient of anti-osteoporosis drugs.

[0075] Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A bearberry alcohol amino acid derivative, characterized in that, The arbutinol amino acid derivative is arbutinol-28β-pyroglutamate ester, and the arbutinol amino acid derivative has the structure shown in Formula I:

2. A method for preparing the arbutin amino acid derivative according to claim 1, characterized in that, The preparation method is as follows: Using Circinella muscae CGMCC 3.2695 as the transformation strain, arbutinol is subjected to microbial transformation to obtain the arbutinol amino acid derivative.

3. The preparation method according to claim 2, characterized in that, The preparation method includes the following steps: 1) Ferment and culture the microorganism, add arbutinol to the culture medium, then carry out transformation culture, and obtain the fermentation broth after removing the mycelium. The microorganism is Circinella muscae CGMCC 3.2695; 2) Extract the fermentation broth to obtain the transformant; 3) Purify the transformant by reverse-phase silica gel column chromatography. The reverse-phase silica gel column purification uses a gradient elution of a methanol-water two-phase system, and collect and combine the elution fractions obtained when using a mixed solvent with a volume ratio of methanol to water of 80:20 as the eluent; 4) Purify the elution fraction by reverse-phase high performance liquid chromatography to obtain the arbutinol amino acid derivative.

4. Application of the arbutinol amino acid derivative according to claim 1 as an active ingredient in the following aspects: 1) Application in the preparation of anti-tumor drugs; 2) Application in the preparation of eukaryotic cancer cell proliferation inhibitors; 3) Application in the preparation of drugs for killing leukemia drug-resistant cells; 4) Application in the preparation of drugs for treating osteoporosis.

5. The application according to claim 4, wherein, The tumor is one of cervical cancer, leukemia, neuroblastoma, liver cancer and colon cancer.

6. The application according to claim 4, wherein The eukaryote is a mammal.

7. The application according to claim 4, wherein The cancer cell is one of cervical cancer cells, leukemia cells, neuroblastoma cells, liver cancer cells and colon cancer cells.

8. The application according to claim 4, characterized in that, The osteoporosis is postmenopausal osteoporosis.

9. A product, characterized in that, The active ingredient of the product is the arbutinol amino acid derivative according to claim 1, and the product is: 1) Anti-tumor drugs; 2) Eukaryotic cancer cell proliferation inhibitors; 3) Drugs for killing leukemia drug-resistant cells; 4) Drugs for treating osteoporosis.

10. The medicament according to claim 9, wherein, The drug also contains pharmaceutically acceptable excipients.

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