Cyclopentane phenylpropyl furan compound Aglatestate E as well as preparation method and application thereof

Abstract

The invention relates to a cyclopentane phenylpropanofuran compound Aglatestate E and a preparation method and application thereof, and belongs to the field of medical chemistry, the cyclopentane phenylpropanofuran compound Aglatestate E is obtained by extracting and separating from Arcusia edulia for the first time, and the structural formula of the cyclopentane phenylpropanofuran compound Aglatestate E is (I). The cyclopentane benzofuran compound Aglatestate E shows excellent anti-dengue virus and anti-tumor activity in BHK-21 cells and tumor cells infected by dengue virus 2, the anti-dengue virus activity is higher than that of a positive control drug ribavirin, and the inhibition activity on human colon cancer cells HCT-116 is higher than that of a positive drug adriamycin; the invention also relates to a pharmaceutical composition, the pharmaceutical composition comprises the Aglatestate E and pharmaceutically acceptable auxiliary materials, and the Aglatestate E has the effects of resisting dengue viruses and tumors.

Description

Technical Field

[0001] The invention belongs to the field of medicinal chemistry, and specifically relates to a cyclopentane benzofuran compound Aglatestate E and a preparation method and application thereof. Background Art

[0002] Dengue virus (DENV) is the pathogen of dengue hemorrhagic fever / dengue shock syndrome (DHF / DSS), which is mainly transmitted by Aedes aegypti and Aedes albopictus. The World Health Organization divides it into two categories: common dengue and severe dengue. Patients may develop symptoms 1-14 days after infection. Common symptoms include fever, severe headache, eye pain, muscle pain and bone and joint pain, fatigue, nausea, vomiting, diarrhea, etc., and patients with severe symptoms may experience severe bleeding and shock, leading to death. It is estimated that 390 million people are infected by DENV every year in more than 100 countries / regions in tropical and subtropical areas. The vast majority of patients infected with DENV will recover on their own, but a small number of them will worsen and may develop dengue hemorrhagic fever / dengue shock syndrome. There are no drugs or vaccines for the treatment of DENV on the market. Therefore, it is imperative to discover new drugs for the treatment of DENV.

[0003] Tumors are common diseases that seriously threaten human health. According to statistics from the World Health Organization, the number of new cancer cases in the world will reach 20 million in 2022, and the number of deaths will reach 9.7 million; about 1 / 5 of the world's people will develop cancer in their lifetime, and about 1 / 9 of men and 1 / 12 of women will die from cancer. There are about 80 common anti-tumor drugs in clinical practice internationally, which can be roughly divided into the following 6 categories: cytotoxic drugs, hormone drugs, biological response modifiers, monoclonal antibody drugs, other drugs, adjuvant drugs, etc. Although there are a large number of tumor treatment drugs on the market, most of them have obvious toxic side effects and are prone to drug resistance. Therefore, it is of great significance to discover new anti-tumor drugs.

[0004] As one of the important sources of innovative drug discovery, natural products have become a hot area for the treatment of DENV and tumors. Therefore, the discovery of new anti-DENV and tumor drugs from natural products has attracted the attention of many medicinal chemists. Cyclopentanebenzofuran ring compounds are a special class of natural products, mainly found in plants of the genus Aglaia in the Meliaceae family. From the biosynthetic pathway, this class of compounds is obtained by the cycloaddition reaction between 3-hydroxyflavone and cinnamic acid amide or ester. So far, more than 100 cyclopentanebenzofuran ring compounds have been isolated and identified from plants of the genus Aglaia, which have significant anti-tumor, anti-neuroinflammatory and antiviral activities. However, this class of ingredients has been less studied in anti-DENV. Therefore, it is of great research value and significance to explore cyclopentanebenzofuran ring compounds with novel structures and anti-DENV and anti-tumor effects from Aglaia edulis of the genus Aglaia. Summary of the invention

[0005] The purpose of the present invention is to discover new cyclopentane benzofuran compounds with anti-DENV and anti-tumor effects from horse kidney fruit, and their application in the preparation of anti-DENV and anti-tumor drugs. A cyclopentane benzofuran compound Aglatestate E and its preparation method and application are provided. To achieve the above purpose, the following technical scheme is adopted:

[0006] A cyclopentane benzofuran compound Aglatestate E, whose structural formula is formula (I):

[0007]

[0008] The preparation method of the cyclopentane benzofuran compound Aglatestate E comprises the following steps:

[0009] Step A: taking 20.5 kg of dried spatholobi branches and leaves, using 70-80 kg of methanol as solvent, extracting 3-4 times at 25° C., each extraction time being 3-4 days, and concentrating under reduced pressure to recover methanol to obtain crude extract A;

[0010] Step B: subjecting the crude extract A obtained in step A to silica gel column chromatography, using a gradient elution with a volume ratio of petroleum ether to acetone of 100:0 to 0:100, detecting different fractions using thin layer chromatography technology, and merging them according to the thin layer chromatography performance to obtain 8 fractions: Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7, and Fr.8;

[0011] Step C: Fraction Fr.6 was separated by using an MCI column, and the eluent was gradient eluted with a methanol to water volume ratio of 60:40 to 100:0. The fractions were combined according to the thin layer chromatography performance to obtain five sub-fractions: Fr.6A, Fr.6B, Fr.6C, Fr.6D, and Fr.6E. According to the thin layer chromatography color development, it was determined that fraction Fr.6C contained a benzofuran compound structure, and then Fr.6C was separated by reverse phase C18, and the methanol to water volume ratio was 60:40 to 100:0. Gradient elution with an eluent ratio of 70-100%, v / v, obtained 9 subfractions: Fr.6C1, Fr.6C2, Fr.6C3, Fr.6C4, Fr.6C5, Fr.6C6, Fr.6C7, Fr.6C8 and Fr.6C9. Finally, Fr.6C5 was eluted by silica gel column chromatography with petroleum ether / acetone (v / v, 5:1) as eluent to obtain cyclopentanebenzofuran compound Aglatestate E.

[0012] The methanol used in the preparation method of the cyclopentane benzofuran compound Aglatestate E is 100% industrial methanol.

[0013] In step A of the preparation method of the cyclopentane benzofuran compound Aglatestate E, the crude extract is extracted by cold soaking extraction.

[0014] In step B of the preparation method of the cyclopentanebenzofuran compound Aglatestate E, the concentrations of the gradient elution of petroleum ether / acetone are volume ratios of 100:0, 80:20, 70:30, 60:40, 50:50, and 0:100, respectively.

[0015] In step C of the preparation method of the cyclopentane benzofuran compound Aglatestate E, fraction Fr.6 is separated using an MCI column, and the concentrations of the methanol / water gradient elution are volume ratios of 60:40, 70:30, 80:20, 90:10, and 100:0, respectively.

[0016] The application of the cyclopentane benzofuran compound Aglatestate E in the preparation of anti-dengue virus and anti-tumor drugs.

[0017] A pharmaceutical composition comprises a cyclopentane benzofuran compound Aglatestate E and a pharmaceutically acceptable excipient.

[0018] The pharmaceutical composition contains 0.1-99% by mass of the cyclopentane benzofuran compound Aglatestate E, and the rest is a pharmaceutical carrier or excipient.

[0019] Experimental studies have shown that the cyclopentane benzofuran compound Aglatestate E exhibits excellent anti-dengue virus and anti-tumor activity in DENV 2-infected BHK-21 cells, and its anti-dengue virus activity is stronger than that of the positive control drug ribavirin, and its inhibitory activity on human colon cancer cells HCT-116 is stronger than that of the positive drug doxorubicin, and can be used to prepare anti-dengue virus and tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the structural formula of cyclopentane benzofuran compound Aglatestate E;

[0021] Figure 2 It is the H NMR spectrum of the cyclopentane benzofuran compound Aglatestate E;

[0022] Figure 3 This is the carbon NMR spectrum of the cyclopentane benzofuran compound Aglatestate E. DETAILED DESCRIPTION

[0023] Example 1

[0024] A cyclopentane benzofuran compound Aglatestate E, whose structural formula is formula (I):

[0025]

[0026] The preparation method of the cyclopentane benzofuran compound Aglatestate E comprises the following steps:

[0027] Step A: taking 20.5 kg of dried spatholobi branches and leaves, using 70-80 kg of methanol as solvent, extracting 3-4 times at 25° C., each extraction time being 3-4 days, and concentrating under reduced pressure to recover methanol to obtain 3.8 kg of crude extract A;

[0028] Step B: subjecting the crude extract A obtained in step A to silica gel column chromatography, using a gradient elution with a volume ratio of petroleum ether to acetone of 100:0 to 0:100, detecting different fractions using thin layer chromatography technology, and merging them according to the thin layer chromatography performance to obtain 8 fractions: Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7, and Fr.8;

[0029] Step C: Fraction Fr.6 was separated by using an MCI column, and the eluent was gradient eluted with a volume ratio of methanol to water of 60:40 to 100:0. The fractions were combined according to the thin layer chromatography performance to obtain five sub-fractions: Fr.6A, Fr.6B, Fr.6C, Fr.6D, and Fr.6E. According to the thin layer chromatography color development, it was determined that fraction Fr.6C contained a benzofuran analog structure, and then Fr.6C was separated by reverse phase C18 column chromatography. The methanol and water were separated. Gradient elution with an eluent volume ratio of 70%-100% and a v / v ratio gave 9 subfractions: Fr.6C1, Fr.6C2, Fr.6C3, Fr.6C4, Fr.6C5, Fr.6C6, Fr.6C7, Fr.6C8 and Fr.6C9. Finally, Fr.6C5 was eluted by silica gel column chromatography with petroleum ether / acetone (v / v, 5:1) as the eluent to give cyclopentanebenzofuran compound Aglatestate E (15 mg).

[0030] The methanol used in the preparation method of the cyclopentane benzofuran compound Aglatestate E is 100% industrial methanol.

[0031] In step A of the preparation method of the cyclopentane benzofuran compound Aglatestate E, the crude extract is extracted by cold soaking extraction.

[0032] In step B of the preparation method of the cyclopentanebenzofuran compound Aglatestate E, the concentrations of the gradient elution of petroleum ether / acetone are volume ratios of 100:0, 80:20, 70:30, 60:40, 50:50, and 0:100, respectively.

[0033] In step C of the preparation method of the cyclopentane benzofuran compound Aglatestate E, a reverse phase C18 column is used to separate the fraction Fr.6, and the concentrations of the methanol / water gradient elution are volume ratios of 60:40, 70:30, 80:20, 90:10, and 100:0, respectively.

[0034] The application of the cyclopentane benzofuran compound Aglatestate E in the preparation of anti-dengue virus and anti-tumor drugs.

[0035] A pharmaceutical composition comprises a cyclopentane benzofuran compound Aglatestate E and a pharmaceutically acceptable excipient.

[0036] The pharmaceutical composition contains 0.1-99% by mass of the cyclopentane benzofuran compound Aglatestate E, and the rest is a pharmaceutical carrier or excipient.

[0037] Example 2

[0038] The structure of cyclopentane benzofuran compound Aglatestate E was identified by high resolution mass spectrometry (HRESIMS), nuclear magnetic resonance spectroscopy ( 1 H NMR, 13 C NMR, 2D-NMR), infrared spectrum (IR), optical rotation and other data were comprehensively analyzed, and its physical and chemical properties are as follows:

[0039] Aglatestate E: yellow oil, [α] 24 D +67.2 (c 0.36, MeOH); molecular formula is C 36 H 40 O8 N2Na;IR(KBr)v max 3334, 2927, 1748, 1616, 1517, 1418, 1369, 1357, 1251, 1200, 1149, 1116, and 1078 cm -1 ;(+)-HRESIMS:m / z 651.2688[M+Na] + (The calculated value is C 36 H 40 O8N2Na, 651.2677). NMR spectrum ( 1 HNMR and 13 C NMR), the test solvent is deuterated chloroform, see Table 1 below:

[0040] Table 1 Cyclopentane benzofuran compounds Aglatestate E 1 HNMR and 13 C NMR data

[0041]

[0042]

[0043] Example 3

[0044] In order to further verify the beneficial effects of the compounds of the present invention, the inhibitory activity of the cyclopentane benzofuran compound Aglatestate E on dengue virus was studied in Syrian hamster kidney cells (BHK-21). The specific experiment is as follows:

[0045] (1) MTT assay for drug cytotoxicity: Syrian hamster kidney cells (BHK-21) were inoculated into 96-well microplates (5 × 10 3cells), cultured at 37°C, 5% CO2 for 24 hours. After the cells grow into a monolayer, the culture supernatant is discarded, and DMEM medium containing gradient dilutions of the test compound is added. Three replicate wells are set for each concentration, and normal cell control, solvent control, and blank control are set. After culturing for 2 days, 10 μL of 5 mg / mL MTT is added to each well, and the cells are incubated in a 37°C constant temperature incubator for 4 hours. 100 μL of supernatant is discarded, and 160 μL of DMSO solution is added. The cells are shaken at 37°C for 15 minutes. After the crystals are completely dissolved, the OD value is detected by Bio-TEK microplate reader (detection wavelength 490 nm); a dose-response curve is drawn based on the experimental results, and the 50% inhibition of cell growth concentration (CC 50 )value.

[0046] (2) Virus plaque assay to detect the effect of compounds on virus plaques: BHK-21 cells were cultured at 2×10 5 100 μL / well were plated in a 12-well plate and cultured overnight in a 37°C, 5% CO2 incubator; DMEM medium was added to the virus-free control group, and DENV-2 (MOI = 0.01) was added to the drug group and the virus group. After adsorption at 37°C for 2 hours, the supernatant was removed and washed twice with PBS. 1 mL of maintenance medium containing aglaiastatin (0.25, 0.5, 1, 2, 4 μM) was added to the drug group, and 1 mL of maintenance medium containing DMSO was added to the virus group. After 48 hours of culture, the supernatant was frozen at -80°C. BHK-21 cells were cultured at 2×10 5 100 μL / well of the plate was plated in a 12-well plate and cultured in a 37°C, 5% CO2 incubator overnight; the supernatant was discarded, washed with PBS, and 300 μL of the supernatant sample was added to each well. Placed in an incubator at 37°C for adsorption for 2 hours; the supernatant was discarded and washed with PBS, and 1 mL of a mixed culture medium of 4% FBSDMEM culture medium and 2% low-melting-point agarose was added to each well. After solidification, it was placed in a 37°C incubator and cultured for 6 to 7 days; 4% paraformaldehyde was used for fixation for 30 minutes, and the agarose covering was discarded upside down; 1% crystal violet staining solution was used for staining for 20 minutes, and the plate was washed with running water; the plate was dried at room temperature. The number of plaques was counted by enzyme-linked fluorescent spot analyzer (CTL), and a dose-response curve was drawn according to the experimental results to calculate the EC of the sample to be tested. 50 .

[0047] (3) Evaluation of anti-DENV efficacy: The therapeutic index (TI) is the half inhibitory concentration CC of the drug on cells. 50 and the half effective concentration (EC) of the virus 50 The ratio represents the safety of the drug. The larger the value, the safer it is.

[0048] (4) The compounds separated by the above method were subjected to drug cytotoxicity and anti-DENV virus activity experiments using the clinical therapeutic drug Ribavirin as a positive control. The therapeutic index of the compound Aglatestate E in Table 2 was higher than that of the positive control Ribavirin. The therapeutic index of the compound was 10 times higher than that of Ribavirin, and it can be used as a lead compound for anti-DENV.

[0049] The cytotoxicity and anti-dengue virus activity of the compounds (Table 2) are shown below:

[0050] Table 2: Cytotoxicity and anti-dengue virus activity of compounds

[0051]

[0052] Note: Ribavirin positive control ribavirin

[0053] (5) Experimental conclusion: The cyclopentane benzofuran compound Aglatestate E has significant inhibitory activity against DENV virus, and its activity is stronger than that of the positive control drug Ribavirin. Therefore, the cyclopentane benzofuran compound Aglatestate E described in the present invention can be used to prepare anti-dengue virus drugs.

[0054] Example 4

[0055] In order to further verify the anti-tumor effect of the compound of the present invention, the anti-tumor activity of the cyclopentane benzofuran compound Aglatestate E was determined by MTT method. The specific method is as follows:

[0056] (1) Cell culture: The cells were cultured in DMEM medium (containing 5% fetal bovine serum) in a CO2 incubator at 37°C, 5% CO2 and 95% humidity. The cells used in the experiment were revived and passaged at least 3 times. Human erythroleukemia cells HEL, human colon cancer cells HCT-116, and human colorectal adenocarcinoma cells COLO 320DM in the logarithmic growth phase were cultured at 5×10 3 Up to 6×10 3 Each well was inoculated in a 96-well plate. Then 0.0625, 0.125, 0.25, 0.5, 1, 5, 10 and 20 μM of the test compound were added, and a positive control group and a blank control group were set up. After 72 hours of culture, 10 μL of MTT was added to each well, and the culture was continued at 37°C for 4 hours. 160 μL of DMSO was added to each well, and the micro-oscillator was oscillated for 10 minutes. The OD value of each well was measured at a wavelength of 490 nm using an enzyme marker. The inhibition rate of the compound on different tumor cells was calculated according to the following formula:

[0057] Inhibition rate = (1-OD value of drug group / OD value of cell control group) × 100%

[0058] At the same time, calculate its IC 50 value.

[0059] (2) Anti-tumor efficacy evaluation: IC 50 It is the half inhibitory concentration of the drug on cells. The smaller this value is, the better the anti-tumor effect is.

[0060] (3) The compounds separated by the above method were subjected to drug anti-tumor experiments using the clinical drug doxorubicin as a positive control. The compound Aglatestate E in Table 3 showed good anti-tumor effects on HEL, HCT-116 and COLO 320DM tumor cells, especially on HCT-116 cells. Its inhibitory activity was stronger than that of the positive drug doxorubicin, and it can be used as a lead anti-tumor compound.

[0061] The antitumor activities of the compounds (Table 3) are shown below:

[0062] Table 3: Antitumor activity of compounds

[0063]

[0064] Note: Doxorubicin positive control

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for researchers in this technical field, any other improvements and modifications made without departing from the method and content of the present invention should also be regarded as within the scope of protection of the present invention.

Claims

1. A cyclopentane benzofuran compound Aglatestate E, having the structural formula (I):

2. A method for preparing the cyclopentane benzofuran compound Aglatestate E as claimed in claim 1, characterized in that: The following steps are involved: Step A: taking 20.5 kg of dried spatholobi branches and leaves, using 70-80 kg of methanol as solvent, extracting 3-4 times at 25° C., each extraction time being 3-4 days, and concentrating under reduced pressure to recover methanol to obtain crude extract A; Step B: subjecting the crude extract A obtained in step A to silica gel column chromatography, using a gradient elution with a volume ratio of petroleum ether to acetone of 100:0 to 0:100, detecting different fractions using thin layer chromatography technology, and merging them according to the thin layer chromatography performance to obtain 8 fractions: Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7, and Fr.8; Step C: Fr.6 (162.4 g) was separated into five subcomponents (Fr.6A-Fr.6E) using an MCI column and gradient eluted with a MeOH / H2O (60:40-0:100, v / v) solvent system. Step D: Fr.6C (5.4 g) was further purified using a reverse phase C18 column with a gradient of MeOH / H2O (70-100%, v / v) to obtain 9 subfractions (Fr.6C1-Fr.6C9). Step E: The subcomponent Fr.6C5 (314.2 mg) was purified by silica gel column chromatography with petroleum ether / acetone (v / v, 5:1) as the eluent to obtain cyclopentanebenzofuran compound Aglatestate E.

3. The preparation method of cyclopentane benzofuran compound Aglatestate E according to claim 2, characterized in that, The methanol is 100% industrial methanol.

4. The preparation method of cyclopentane benzofuran compound Aglatestate E according to claim 2, characterized in that: In step A, the crude extract is extracted by cold soaking extraction.

5. The method for preparing cyclopentane benzofuran compound Aglatestate E according to claim 2, characterized in that: In step B, the concentrations of the gradient elution of the elution solvent system petroleum ether / acetone in the silica gel column chromatography are volume ratios of 100:0, 80:20, 70:30, 60:40, 50:50, and 0:100, respectively.

6. The method for preparing cyclopentane benzofuran compound Aglatestate E according to claim 2, characterized in that: In step C, Fr.6 was separated using an MCI column, and the concentrations of the methanol / water gradient elution were volume ratios of 60:40, 70:30, 80:20, 90:10, and 100:0, respectively.

7. The method for preparing cyclopentane benzofuran compound Aglatestate E according to claim 2, characterized in that: In step D, Fr.6C was separated by a reverse phase C18 column, and the concentrations of the gradient elution of methanol / water were 70:30, 75:25, 80:20, 90:10, and 100:0 by volume ratio, respectively; the subcomponent Fr.6C5 (314.2 mg) was separated by silica gel column chromatography with petroleum ether / acetone (v / v, 5:1) as the eluent to obtain the cyclopentane benzofuran compound Aglatestate E.

8. Use of the cyclopentane benzofuran compound Aglatestate E as claimed in claim 1 in the preparation of anti-dengue virus and anti-tumor drugs.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the cyclopentane benzofuran compound Aglatestate E according to claim 1 and pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition contains 0.1-99% by mass of the cyclopentanebenzofuran compound Aglatestate E according to claim 1, and the rest is a pharmaceutical carrier or excipient.

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