Biphenyl dimer compounds with cage-like structure, and preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a biphenyl dimer compound with a cage structure and a preparation method and application thereof. Two biphenyl dimer compounds with a cage structure and a preparation method and application thereof are disclosed, and belong to the technical field of medicine. The structural formula of the biphenyl dimer compound with a cage structure is shown as formula I and II: two compounds are new skeleton compounds isolated from Yunnan gamboge branches and leaves, have proliferation inhibition, anti-migration and anti-invasion effects on human lung cancer A549 and Calu-1 cells, and can be applied to preparation of anti-lung cancer drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a biphenyl dimer compound with a cage structure and a preparation method and application thereof. BACKGROUND

[0002] Lung cancer is one of the most common malignant tumors, which poses a great threat to human health, and its morbidity and mortality remain high and become a major cause of death. This phenomenon is closely related to environmental factors and genetic factors, and the 5-year survival rate is extremely low. Traditional treatment methods such as surgery, chemotherapy and radiotherapy also have limitations, so drug research and development has become the key to the treatment of lung cancer.

[0003] Natural products have long played an important role in various drug research and development, and are an important source of antitumor drugs. Biphenyl compounds exhibit structural diversity and complexity due to the presence of multiple substitutable sites and a variety of substituents. Such components not only have novel and diverse structures, but also exhibit a wide range of biological activities, providing multiple options for the research and development of antitumor drugs.

[0004] Garcinia plants are a natural rich source of biphenyl compounds. So far, about 70 structurally different biphenyls have been found in plants of the Garcinia family. Biphenyl compounds have shown certain application potential and important value in the field of anti-lung cancer research. Biphenyl compounds have also been obtained from Garcinia xanthochymus in Yunnan, but the research on biphenyl dimers is very limited, and no biphenyl dimer with a cage structure has been found so far. SUMMARY

[0005] The purpose of the present application is to provide a biphenyl dimer compound with a cage structure and a preparation method and application thereof. Two biphenyl dimer compounds with a cage structure are obtained by systematic separation of the branches and leaves of Garcinia xanthochymus, and it is found that these compounds have anti-proliferative, anti-migratory and anti-invasive effects on lung cancer cells, and can be used as drugs for the preparation of anti-lung cancer drugs.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0007] In a first aspect, the present application provides a biphenyl dimer compound with a cage structure, which has the following structure:

[0008]

[0009] In a second aspect, the present application provides a preparation method of the biphenyl dimer compound with a cage structure according to the first aspect, comprising the following steps:

[0010] (1) The Garcinia is crushed, and an extract is obtained by extraction with ethanol. After being suspended with water, the extract is extracted with petroleum ether and ethyl acetate respectively to obtain a petroleum ether extract and an ethyl acetate extract;

[0011] (2) The petroleum ether extract is subjected to MCI column chromatography, eluted with ethanol / water to obtain eluate 1;

[0012] (3) The eluate 1 is loaded onto a silica gel column, eluted with petroleum ether / ethyl acetate, and each elution fraction is tracked by silica gel thin layer chromatography to obtain eluate 2 and eluate 4;

[0013] (4) The eluate 2 is loaded onto a molecular sieve gel column, eluted with methanol to obtain eluate 3; the eluate 3 is subjected to HPLC analysis to obtain biphenyl dimer cage compound I;

[0014] (5) The eluate 4 is subjected to HPLC analysis to obtain biphenyl dimer cage compound II.

[0015] In some other embodiments, in step (1), the G. elliottii is G. elliottii from Yunnan, and the volume fraction of the ethanol is 85-95%.

[0016] In some other embodiments, in step (1), the G. elliottii is G. elliottii branches and leaves from Yunnan, the amount of the ethanol is 3-8 times the mass of the G. elliottii, and the extraction is performed at least 3 times, each time for 2-3 weeks.

[0017] In some other embodiments, in step (1), the amount of the ethanol is 4 times the mass of the G. elliottii, the extraction is performed 3 times, each time for 2 weeks; and the amount of the water is 3-4 times the mass of the extract.

[0018] In some other embodiments, in step (2), the volume ratio of the ethanol / water is 40:60-100:0; and the eluate 1 is the fraction eluted with ethanol / water at a volume ratio of 50:50-60:40;

[0019] In some other embodiments, in step (3), the volume ratio of the petroleum ether / ethyl acetate is 90:10-0:100; the eluate 2 is the fraction eluted with petroleum ether / ethyl acetate at a volume ratio of 55:45-45:55; and the eluate 4 is the fraction eluted with petroleum ether / ethyl acetate at a volume ratio of 65:35-55:45.

[0020] In some other embodiments, in step (4), the HPLC analysis employs a C18 column, and the mobile phase is methanol / water at a volume ratio of 50:50.

[0021] In step (5), the HPLC analysis employs a C18 column, and the mobile phase is methanol / water at a volume ratio of 80:20.

[0022] In a third aspect, the present application provides the use of the biphenyl dimer compound with cage structure according to the first aspect in the preparation of a medicament for treating lung cancer.

[0023] In a fourth aspect, the present application provides a pharmaceutical preparation comprising the biphenyl dimer compound with cage structure according to the first aspect 1 or a pharmaceutically acceptable salt thereof.

[0024] Advantages of the present application:

[0025] The two compounds provided by the present application are new skeleton compounds isolated from Yunnan Garcinia branches and leaves, and have anti-proliferation, anti-migration and anti-invasion effects on lung cancer cells, and can be used as a medicament for treating lung cancer. BRIEF DESCRIPTION OF DRAWINGS

[0026] The accompanying drawings, which form a part of the present application, are used to provide further understanding of the present application, and the illustrative embodiments of the present application and their description serve the purpose of explaining the present application, and do not constitute an improper limitation of the present application.

[0027] Figure 1 Effects of compounds I and II on the viability of A549 and Calu-1 cells;

[0028] Figure 2 Effects of compounds I and II on the colony formation of A549 and Calu-1 cells;

[0029] Figure 3 Effects of compounds I and II on the scratch healing of A549 and Calu-1 cells;

[0030] Figure 4 Effects of compounds I and II on the invasion of A549 and Calu-1 cells. DETAILED DESCRIPTION

[0031] Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If specific conditions are not indicated in the examples, conventional conditions or manufacturer's recommended conditions are used. If the manufacturer of the used components is not indicated, it is a conventional product that can be obtained on the market.

[0032] Example 1

[0033] Preparation of biphenyl dimer compounds I and II with cage structure:

[0034] The crushed Yunnan Garcinia branches and leaves 10.0 kg were extracted with 90% ethanol by cold maceration for 3 times, each time with 40.0 kg of ethanol, and each time for 2 weeks. The extract was combined and concentrated to dryness to obtain Yunnan Garcinia extract 1.1 kg. Then the extract was suspended in water 4.0 kg and extracted with petroleum ether, 4.0 L each time, for 9 times. The petroleum ether extract was combined and concentrated to dryness. The remaining water extract was extracted with ethyl acetate, 4.0 L each time, for 7 times. The ethyl acetate extract was combined and concentrated to dryness.

[0035] The petroleum ether extract was further separated by MCI column chromatography, eluted with ethanol / water, and the fractions eluted with ethanol / water at 55:45 (v / v) were combined, concentrated to dryness, and loaded onto a silica gel column, eluted with petroleum ether / ethyl acetate at 90:10-0:100 (v / v), and the fractions eluted with petroleum ether / ethyl acetate at 50:50 (v / v) were combined, concentrated to dryness, and loaded onto a Sephadex LH-20 column, eluted with methanol, and each fraction was followed by silica gel thin layer chromatography. The same fractions were combined and concentrated, and the concentrated solution was further prepared by HPLC with a C 18 column and a mobile phase of methanol / water at 50:50 (v / v) to obtain the biphenyl dimer compound I with a cage structure; and the fractions eluted with petroleum ether / ethyl acetate at 60:40 (v / v) were combined, and the concentrated solution was prepared by HPLC with a C 18 column and a mobile phase of methanol / water at 80:20 (v / v) to obtain the biphenyl dimer compound II with a cage structure.

[0036] The compounds I and II have the following structures:

[0037]

[0038] The 1 H NMR and 13 C NMR data of the compounds I and II are shown in Tables 1 and 2.

[0039] The 1 H NMR (600 MHz) data (δ in ppm, J in Hz) of the compounds I and II are shown in Table 1.

[0040]

[0041]

[0042] a were measured in methanol-d4, b were measured in acetone-d6.

[0043] The13 C NMR (150 MHz) data (δ in ppm)

[0044]

[0045]

[0046] a In methanol-d4, b In acetone-d6.

[0047] Spectral data of compounds I and II are as follows:

[0048] Compound I: light yellow crystal. (c 0.05, MeOH); UV (MeOH) λmax 220, 330 nm; 1 H and 13 C NMR data are shown in Tables 1 and 2; HRESIMS m / z 559.2096 [M+Na] + (calcd for C 34 H 32 O6Na, 559.2097).

[0049] Compound II: yellow amorphous powder. (c 0.05, MeOH); UV (MeOH) λmax 330 nm; 1 H and 13 C NMR data are shown in Tables 1 and 2; HRESIMS m / z 549.2282 [M-H] - (calcd for C 35 H 33 O6, 549.2277).

[0050] Example 2

[0051] Inhibition of lung cancer cell proliferation experiment of compounds I and II prepared in Example 1

[0052] Experimental materials: Human lung cancer A549 and Calu-1 cell lines were purchased from ATCC, USA; A549 cells were cultured with Dulbecco's Modified Eagle Medium (DMEM) medium (Servicebio Company), and Calu-1 cells were cultured with McCoy's 5A medium (Servicebio Company). Both media were supplemented with 10% (v / v) fetal bovine serum (FBS, Servicebio Company), penicillin and streptomycin (100 U / ml). All cells were cultured in an incubator at 37°C, 5% CO2. MTT was purchased from Solarbio Company.

[0053] Experimental method: A549 and Calu-1 cells in logarithmic growth phase were inoculated in 96-well plates at a density of 8000 cells per well, and cultured overnight to adhere to the cells. Compound I and II DMSO solution was added to treat the cells, and the final concentration was as shown in Figure 1 After 48 hours of culture in the drug-containing medium, 10 μL of 5.0 mg / mL MTT solution was added to each well, and the culture was continued for 4 hours. Then the medium was removed, 100 μL of DMSO was added to each well, and the crystals were dissolved by shaking. The absorbance was read at 570 nm wavelength using an enzyme-labeled instrument, and the cell viability was calculated.

[0054] Experimental results: As shown in Figure 1 , compounds I and II showed dose-dependent inhibition of A549 cell viability, with IC 50 values of 5.37 μM for compound I and 26.84 μM for compound II; compounds I and II showed dose-dependent inhibition of Calu-1 cell viability, with IC 50 values of 6.09 μM for compound I and 4.97 μM for compound II.

[0055] Example 3

[0056] Example 1 prepared compounds I and II inhibit lung cancer cell proliferation experiment

[0057] Experimental materials: Human lung cancer A549 and Calu-1 cell lines were purchased from ATCC, USA; A549 cells were cultured with DMEM medium (Servicebio Company), and Calu-1 cells were cultured with McCoy's 5A medium (Servicebio Company). Both media were supplemented with 10% (v / v) fetal bovine serum (FBS, Servicebio Company), penicillin and streptomycin (100 U / ml). All cells were cultured in an incubator at 37°C, 5% CO2.

[0058] Experimental method: Cells were seeded in 6-well plates at a density of 500 cells per well and incubated at 37°C overnight. Then DMSO or compounds I and II were added and incubated for 10 days. Cells were washed with PBS and fixed with ice-cold methanol, and then stained with 0.1% crystal violet solution.

[0059] Experimental results: As shown in Table 1, compared with the blank control group, the cell colony formation rate showed a dose-dependent decrease after treatment with compounds I and II, respectively. This indicates that long-term (10 days) use of compounds I and II can reduce the colony formation ability of A549 and Calu-1 cells. Figure 2

[0060] Example 4

[0061] Experiment of anti-tumor migration effect of compounds I and II prepared in Example 1

[0062] Experimental materials: Human lung cancer A549 and Calu-1 cell lines were purchased from ATCC, USA. A549 cells were cultured in DMEM medium (Servicebio Company), and Calu-1 cells were cultured in McCoy's 5A medium (Servicebio Company). Both media were supplemented with 10% (v / v) fetal bovine serum (FBS, Servicebio Company), penicillin and streptomycin (100 U / ml). All cells were cultured in an incubator at 37°C, 5% CO2.

[0063] Experimental method: Cells were seeded in 6-well plates at a density of 5x10 5 cells per well and incubated at 37°C overnight. The cells were gently scratched with a 10 μl tip, then washed with PBS, and then cultured in medium containing DMSO or compounds I and II. After 72 h, the cells were photographed.

[0064] Experimental results: As shown in Table 2, compared with the blank control group, the cell scratch healing rate showed a dose-dependent decrease after treatment with compounds I and II, respectively. This indicates that compounds I and II can effectively inhibit the migration ability of A549 and Calu-1 cells. Figure 3

[0065] Example 5

[0066] Experiment of anti-tumor invasion effect of compounds I and II prepared in Example 1

[0067] ​​Experimental materials: Human lung cancer A549 and Calu-1 cell lines were purchased from ATCC, USA; A549 cells were cultured in DMEM medium (Servicebio Company), and Calu-1 cells were cultured in McCoy's 5A medium (Servicebio Company). Both of the two culture mediums were supplemented with 10% (v / v) fetal bovine serum (FBS, Servicebio Company), penicillin and streptomycin (100 U / ml). All cells were cultured in an incubator at 37℃, 5% CO2.

[0068] Experimental method: Cells were seeded in transwell chambers coated with Matrigel at a density of 20000 cells per well in a 24-well plate containing complete culture medium, and after 24h, the medium was replaced with serum-free medium containing DMSO or compounds I and II, and the cells were cultured for another 24h. The cells in the lower chamber were washed with PBS, fixed with paraformaldehyde, and stained with 0.1% crystal violet solution.

[0069] Experimental results: As shown in Table 1, compared with the blank control group, the invasion rate of cells decreased in a dose-dependent manner after treatment with compounds I and II, respectively. This indicates that compounds I and II can effectively inhibit the invasion ability of A549 and Calu-1 cells. Figure 4

[0070] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.​

Claims

1. A biphenyl dimer cage compound having a structure as shown in the following formula: 。 2. A method for producing the compound of the biphenyl dimer having a cage structure according to claim 1, characterized by, comprising the following steps: (1) crushing G. elliottia, extracting with ethanol to obtain an extract, suspending in water, and then extracting with petroleum ether and ethyl acetate to obtain a petroleum ether extract and an ethyl acetate extract; (2) subjecting the petroleum ether extract to MCI column chromatography, eluting with ethanol / water to obtain eluate 1; (3) subjecting eluate 1 to a silica gel column, eluting with petroleum ether / ethyl acetate, and tracking each elution fraction with silica gel thin layer chromatography to obtain eluate 2 and eluate 4; (4) subjecting eluate 2 to a molecular sieve gel column, eluting with methanol to obtain eluate 3; and subjecting eluate 3 to HPLC analysis to obtain biphenyl dimer cage compound I; (5) subjecting eluate 4 to HPLC analysis to obtain biphenyl dimer cage compound II.

3. The method for preparing the compound of the biphenyl dimer with a cage structure according to claim 2, characterized in that, In step (1), the G. elliottia is G. elliottia from Yunnan, and the volume fraction of the ethanol is 85-95%.

4. The method of claim 2, wherein the method is characterized by, In step (1), the G. elliottia is G. elliottia branches and leaves from Yunnan, the amount of the ethanol is 3-8 times the mass of the G. elliottia, and the extraction is performed at least 3 times, each time for 2-3 weeks.

5. The method for preparing biphenyl dimer compounds with a cage-like structure according to claim 2, characterized in that, In step (1), the amount of the ethanol is 4 times the mass of the G. elliottia, the extraction is performed 3 times, each time for 2 weeks, and the amount of the water is 3-4 times the mass of the extract.

6. The method of claim 2, wherein the method is characterized by, In step (2), the volume ratio of the ethanol / water is 40:60-100:0; and eluate 1 is the fraction eluted with ethanol / water at a volume ratio of 50:50-60:

40.

7. The method for preparing biphenyl dimer compounds with a cage-like structure according to claim 2, characterized in that, In step (3), the volume ratio of the petroleum ether / ethyl acetate is 90:10-0:100; eluate 2 is the fraction eluted with petroleum ether / ethyl acetate at a volume ratio of 55:45-45:55; and eluate 4 is the fraction eluted with petroleum ether / ethyl acetate at a volume ratio of 65:35-55:

45.

8. The method of claim 2, wherein the method is characterized by, In step (4), the HPLC analysis employs a C18 column, and the mobile phase is methanol / water at a volume ratio of 50:

50. In step (5), the HPLC analysis employs a C18 column, and the mobile phase is methanol / water at a volume ratio of 80:

20.

9. Use of the biphenyl dimer cage compound having a structure as shown in the formula of claim 1 in the preparation of a drug for treating lung cancer.

10. A pharmaceutical preparation, characterized in that, The drug comprises the biphenyl dimer cage compound having a structure as shown in the formula of claim 1 or a pharmaceutically acceptable salt thereof.

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