A cyclopentanephenylpropionate furan compound, Aglatestate E, its preparation method and application

Abstract

This invention relates to a cyclopentanephenylpropionate furan compound, Aglatestate E, its preparation method, and its applications, belonging to the field of pharmaceutical chemistry. Aglatestate E was first extracted and isolated from *Aglaia edulis*, and its structural formula is (I). The cyclopentanephenylpropionate furan compound Aglatestate E of this invention exhibits excellent anti-dengue virus and anti-tumor activity against dengue virus 2-infected BHK-21 cells and tumor cells. Its anti-dengue virus activity is stronger than that of the positive control drug ribavirin, and its inhibitory activity against human colon cancer cells HCT-116 is stronger than that of the positive control drug doxorubicin. It can be used to prepare anti-dengue virus and anti-tumor drugs. This invention also relates to a pharmaceutical composition comprising the above-mentioned Aglatestate E and pharmaceutically acceptable excipients, which has anti-dengue virus and anti-tumor effects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry, specifically relating to a cyclopentanephenylpropionate furan compound AglatestateE, its preparation method, and its applications. Background Technology

[0002] Dengue virus (DENV) is the pathogen of dengue hemorrhagic fever / dengue shock syndrome (DHF / DSS), primarily transmitted by Aedes aegypti and Aedes albopictus mosquitoes. The World Health Organization classifies it into common dengue and severe dengue. Symptoms typically appear 1-14 days after infection, including fever, severe headache, eye pain, muscle and joint pain, fatigue, nausea, vomiting, and diarrhea. Severe cases may involve severe bleeding and shock, leading to death. It is estimated that 390 million people are infected with DENV annually in over 100 countries in tropical and subtropical regions. The vast majority of DENV infections resolve spontaneously; however, a small percentage develop severe illness, potentially leading to dengue hemorrhagic fever / dengue shock syndrome. Currently, there are no marketed drugs or vaccines for treating DENV. Therefore, the development of new treatments for DENV is urgently needed.

[0003] Cancer is a common disease that seriously threatens human health. According to statistics from the World Health Organization, in 2022, there were 20 million new cancer cases and 9.7 million cancer deaths worldwide; approximately one in five people globally will develop cancer in their lifetime, and about one in nine men and one in twelve women will die from it. There are approximately 80 commonly used anti-cancer drugs internationally, which can be broadly classified into six categories: cytotoxic drugs, hormonal drugs, biological response modifiers, monoclonal antibody drugs, other drugs, and adjuvant drugs. Although many cancer treatment drugs are currently on the market, most have significant toxic side effects and are prone to drug resistance. Therefore, the discovery of new anti-cancer drugs is of great importance.

[0004] Natural products, as a crucial source of innovative drug discovery, have become a hot area in the treatment of DENV and tumors. Therefore, the discovery of novel anti-DENV and anti-tumor drugs from natural products has attracted the attention of numerous medicinal chemists. Cyclopentanebenzofuran compounds are a special class of natural products, mainly found in plants of the genus *Aglaia* in the family Meliaceae. Biosynthetically, these compounds are obtained through a cycloaddition reaction between 3-hydroxyflavonoids and cinnamic acid amides or esters. To date, more than 100 cyclopentanebenzofuran compounds have been isolated and identified from *Aglaia* plants, exhibiting significant antitumor, anti-neuroinflammatory, and antiviral activities. However, research on their anti-DENV effects is limited. Therefore, the discovery of novel cyclopentanebenzofuran compounds with anti-DENV and antitumor activities from *Aglaia edulis* is of significant research value and importance. Summary of the Invention

[0005] The purpose of this invention is to discover novel cyclopentanebenzofuran compounds with anti-DENV and antitumor effects from *Pterocarya stenoptera*, and their application in the preparation of anti-DENV and antitumor drugs. This invention provides a cyclopentanebenzofuran compound, Aglatestate E, its preparation method, and its application. The following technical solution is adopted to achieve the above objective:

[0006] A cyclopentanephenylpropionate furan compound, Aglatestate E, has the structural formula (I):

[0007]

[0008] The method for preparing the cyclopentanephenylpropionate furan compound Aglatestate E includes the following steps:

[0009] Step A: Take 20.5 kg of dried purslane branches and leaves, use 70-80 kg of methanol as solvent, and extract 3-4 times at 25℃, with each extraction lasting 3-4 days. Concentrate under reduced pressure to recover methanol and obtain crude extract A.

[0010] Step B: The crude extract A obtained in Step A was subjected to silica gel column chromatography, using a gradient elution of petroleum ether and acetone in a volume ratio of 100:0 to 0:100. Thin-layer chromatography was used to detect different fractions, and the fractions were combined according to their thin-layer chromatographic properties 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 using an MCI column with a methanol-to-water gradient elution ratio of 60:40 to 100:0 (v / v). The fractions were combined based on thin-layer chromatography (TLC) results, yielding five subfractions: Fr.6A, Fr.6B, Fr.6C, Fr.6D, and Fr.6E. TLC colorimetric analysis confirmed that fraction Fr.6C contained a phenylpropanofuranoid compound structure. Fr.6C was then separated using a reversed-phase C18 column with a methanol-to-water volume ratio of... Gradient elution with 70-100% v / v eluent yielded nine 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 obtain the cyclopentanephenylpropanefuran compound Aglatestate E.

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

[0013] In step A of the preparation method of the cyclopentanephenylpropionate furan compound Aglatestate E, the crude extract is extracted by cold maceration.

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

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

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

[0017] A pharmaceutical composition comprising a cyclopentanephenylpropionate furan compound, Aglatestate E, and pharmaceutically acceptable excipients.

[0018] The pharmaceutical composition contains 0.1-99% by mass of a cyclopentanephenylpropionate furan compound Aglatestate E, with the remainder being a pharmaceutical carrier or excipient.

[0019] Experimental studies have shown that the cyclopentanephenylpropionate furan compound Aglatestate E exhibits excellent anti-dengue virus and anti-tumor activity against DENV 2-infected BHK-21 cells. Its anti-dengue virus activity is stronger than that of the positive control drug ribavirin, and its inhibitory activity against human colon cancer cells HCT-116 is stronger than that of the positive drug doxorubicin. It can be used to prepare anti-dengue virus and anti-tumor drugs. Attached Figure Description

[0020] Figure 1 It is the structural formula of Aglatestate E, a cyclopentanephenylpropanefuran compound;

[0021] Figure 2 This is the 1H NMR spectrum of Aglatestate E, a cyclopentanephenylpropionuran compound.

[0022] Figure 3 This is the carbon NMR spectrum of Aglatestate E, a cyclopentanephenylpropionate furan compound. Detailed Implementation

[0023] Example 1

[0024] A cyclopentanephenylpropionate furan compound, Aglatestate E, has the structural formula (I):

[0025]

[0026] The method for preparing the cyclopentanephenylpropionate furan compound Aglatestate E includes the following steps:

[0027] Step A: Take 20.5 kg of dried purslane branches and leaves, use 70-80 kg of methanol as solvent, and extract 3-4 times at 25℃, with each extraction lasting 3-4 days. Concentrate under reduced pressure to recover methanol and obtain 3.8 kg of crude extract A.

[0028] Step B: The crude extract A obtained in Step A was subjected to silica gel column chromatography, using a gradient elution of petroleum ether and acetone in a volume ratio of 100:0 to 0:100. Thin-layer chromatography was used to detect different fractions, and the fractions were combined according to their thin-layer chromatographic properties 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 using an MCI column with a gradient elution of methanol to water at a volume ratio of 60:40 to 100:0. The fractions were then combined according to thin-layer chromatography (TLC) results, yielding five sub-fractions: Fr.6A, Fr.6B, Fr.6C, Fr.6D, and Fr.6E. Based on TLC colorimetric analysis, fraction Fr.6C was determined to contain a phenylpropanoid furan analogue structure. Fr.6C was then separated using reversed-phase C18 column chromatography, separating methanol and water. Gradient elution with eluents of 70%-100% v / v yielded nine 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 obtain the cyclopentanephenylpropionate furan compound Aglatestate E (15 mg).

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

[0031] In step A of the preparation method of the cyclopentanephenylpropionate furan compound Aglatestate E, the crude extract is extracted by cold maceration.

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

[0033] In step C of the preparation method of the cyclopentanephenylpropanefuran compound Aglatestate E, the distillate Fr.6 is separated using a reverse-phase C18 column, and the concentrations of the methanol / water gradient elution are successively 60:40, 70:30, 80:20, 90:10, and 100:0 (volume ratios).

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

[0035] A pharmaceutical composition comprising a cyclopentanephenylpropionate furan compound, Aglatestate E, and pharmaceutically acceptable excipients.

[0036] The pharmaceutical composition contains 0.1-99% by mass of a cyclopentanephenylpropionate furan compound Aglatestate E, with the remainder being a pharmaceutical carrier or excipient.

[0037] Example 2

[0038] Structure identification of Aglatestate E, a cyclopentanephenylpropionate furan compound: High-resolution mass spectrometry (HRESIMS) and nuclear magnetic resonance spectroscopy were used. 1 H NMR, 13 Based on a comprehensive analysis of data including C NMR, 2D-NMR, infrared spectroscopy (IR), and optical rotation, its physicochemical properties are as follows:

[0039] Aglatestate E: Yellow oily substance, [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 1078cm -1 ;(+)-HRESIMS:m / z 651.2688[M+Na] + (The calculated value is C) 36 H 40 O8N2Na, 651.2677). Nuclear magnetic resonance spectrum ( 1 HNMR and 13 (C NMR), the test solvent was deuterated chloroform, as shown in Table 1 below:

[0040] Table 1. Aglatestate E of cyclopentanephenylpropionate furans 1 HNMR and 13 C NMR data

[0041]

[0042]

[0043] Example 3

[0044] To further verify the beneficial effects of the compounds described in this invention, the inhibitory activity of the cyclopentanephenylpropionate furan compound Aglatestate E on dengue virus was studied using Syrian hamster kidney cells (BHK-21). The specific experiments are as follows:

[0045] (1) MTT assay for drug cytotoxicity: Syrian hamster kidney cells (BHK-21) were seeded into 96-well microplates (5 × 10⁶ cells per well). 3Cells were cultured at 37°C and 5% CO2 for 24 hours. After the cells grew into a monolayer, the culture supernatant was discarded, and DMEM medium containing serially diluted test compounds was added. Each concentration was used in triplicate, with normal cell control, solvent control, and blank control included. After 2 days of culture, 10 μL of 5 mg / mL MTT was added to each well, and the cells were incubated at 37°C for another 4 hours. 100 μL of supernatant was discarded, and 160 μL of DMSO solution was added. The cells were then shaken at 37°C for 15 minutes until the crystals were completely dissolved. The OD value was measured using a Bio-TEK microplate reader (detection wavelength 490 nm). A dose-response curve was plotted based on the experimental results, and the 50% inhibitory concentration (Cd) was calculated using the Reed & Muench method. 50 )value.

[0046] (2) Viral plaque assay to detect the effect of compounds on viral plaques: BHK-21 cells were prepared at 2×10⁶ 5 BHK-21 cells were seeded per well in 12-well plates and incubated overnight at 37°C with 5% CO2. The virus-free control group was treated with DMEM medium, while the drug and virus groups were treated with DENV-2 (MOI = 0.01). After adsorption at 37°C for 2 hours, the supernatant was removed and the cells were washed twice with PBS. The drug groups were treated with 1 mL of maintenance medium containing aglaiastatin (0.25, 0.5, 1, 2, 4 μM), and the virus groups were treated with 1 mL of maintenance medium containing DMSO. After 48 hours of incubation, the supernatant was collected and stored at -80°C. BHK-21 cells were then cultured at a rate of 2 × 10⁶ cells / well. 5 Plasma was seeded per well in 12-well plates and incubated overnight at 37°C with 5% CO2. The supernatant was discarded, and the plates were washed with PBS. 300 μL of the supernatant was added to each well. The plates were then incubated at 37°C for 2 hours for adsorption. The supernatant was discarded, and the plates were washed with PBS. 1 mL of a mixture of 4% FBSDMEM and 2% low-melting-point agarose was added to each well. After solidification, the plates were incubated at 37°C for 6–7 days. Fixation was performed with 4% paraformaldehyde for 30 minutes, and the agarose covering was removed by inverting the plate. Staining was performed with 1% crystal violet for 20 minutes, followed by washing with running water. The plates were then air-dried at room temperature. Plaque counts were performed using a cytokine speckle analyzer (CTL). A dose-response curve was plotted based on the experimental results, and the EC50 of the sample was calculated. 50 .

[0047] (3) Evaluation of anti-DENV efficacy: The therapeutic index (TI) is the half-maximal inhibitory concentration (CMC) of the drug on cells. 50 and the half-maximal effective concentration (EC50) against the virus 50 The ratio of the drug's safety to its safety index represents the safety of the drug; the higher the value, the safer the drug.

[0048] (4) Using the clinical treatment drug ribavirin as a positive control, the compounds isolated by the above method were subjected to drug cytotoxicity and anti-DENV virus activity experiments. In Table 2, the therapeutic index of compound Aglatestate E was higher than that of the positive control Ribavirin. Among them, the therapeutic index of compound Aglatestate E 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 activities of the compounds (Table 2) are shown below:

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

[0051]

[0052] Note: Ribavirin is a positive control for ribavirin.

[0053] (5) Experimental conclusion: The cyclopentane phenylpropionate furan 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 phenylpropionate furan compound Aglatestate E described in this invention can be used to prepare anti-dengue virus drugs.

[0054] Example 4

[0055] To further verify the antitumor effect of the compounds described in this invention, the antitumor activity of the cyclopentanephenylpropionate furan compound Aglatestate E was determined using the MTT assay. The specific method is as follows:

[0056] (1) Cell Culture: Cells were cultured in DMEM medium (containing 5% fetal bovine serum) and placed in a CO2 incubator at 37°C, 5% CO2, and 95% humidity. Cells used in the experiment were passaged at least three times after revival. Human erythroleukemia cells (HEL), human colon cancer cells (HCT-116), and human colorectal adenocarcinoma cells (COLO 320DM) in logarithmic growth phase were cultured at 5 × 10⁻⁶ cells per cell line. 3 Up to 6×10 3 Each well was seeded with 1 micron / sample. Then, 0.0625, 0.125, 0.25, 0.5, 1, 5, 10, and 20 μM of the test compound were added, with positive and blank control groups included. After 72 h of incubation, 10 μL of MTT was added to each well, and the cells were incubated at 37°C for another 4 h. Then, 160 μL of DMSO was added to each well, and the cells were shaken for 10 min on a microplate reader. The OD values ​​of each well were measured at 490 nm using a microplate reader. The inhibitory rates of the compounds against different tumor cells were calculated using the following formula:

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

[0058] Simultaneously calculate its IC 50 value.

[0059] (2) Evaluation of antitumor drug efficacy: IC 50 This is the half-maximal inhibitory concentration (MCC) of the drug on cells; the lower this value, the better the anti-tumor effect.

[0060] (3) Using the clinical treatment drug doxorubicin as a positive control, the compounds isolated by the above method were subjected to antitumor drug experiments. The compound Aglatestate E in Table 3 showed good antitumor effects against HEL, HCT-116 and COLO 320DM tumor cells. In particular, its inhibitory activity against HCT-116 cells was stronger than that of the positive control drug doxorubicin, and it can be used as a lead compound for antitumor treatment.

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

[0062] Table 3: Antitumor activity of the compounds

[0063]

[0064] Note: Doxorubicin positive control

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that any other improvements and modifications made by researchers in this field without departing from the method and content of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a cyclopentanephenylpropionate furan compound, Aglatestate E, characterized in that, Includes the following steps: Step A: Take 20.5 kg of dried purslane branches and leaves, use 70-80 kg of methanol as solvent, and extract 3-4 times at 25℃, with each extraction lasting 3-4 days. Concentrate under reduced pressure to recover methanol and obtain crude extract A. Step B: The crude extract A obtained in Step A was subjected to silica gel column chromatography, using a gradient elution of petroleum ether and acetone in a volume ratio of 100:0 to 0:

100. Thin-layer chromatography was used to detect different fractions, and the fractions were combined according to their thin-layer chromatographic properties 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 was separated using an MCI column and eluted with a gradient of methanol / water solvent system with a volume ratio of 60:40 to 100:0 to obtain five sub-fractions Fr.6A-Fr.6E; Step D: Fr.6C was further purified using a reverse-phase C18 column and eluted with a methanol / water gradient of 70%–100% by volume to obtain nine subfractions Fr.6C1–Fr.6C9. Step E: Subfraction Fr. 6C5 was purified by silica gel column chromatography with petroleum ether / acetone in a volume ratio of 5:1 to give the cyclopentanephenylpropionate furan compound Aglatestate E. Among them, the cyclopentanephenylpropionuran compound Aglatestate E has the structural formula (I): 。 2. The method for preparing the cyclopentanephenylpropionate furan compound Aglatestate E according to claim 1, characterized in that, The methanol in question is 100% industrial methanol.

3. The method for preparing the cyclopentanephenylpropionate furan compound Aglatestate E according to claim 1, characterized in that, In step A, the crude extract is extracted using cold maceration.

4. The method for preparing the cyclopentanephenylpropionate furan compound Aglatestate E according to claim 1, characterized in that, In step B, the volume ratios of petroleum ether to acetone in silica gel column chromatography are 100:0, 80:20, 70:30, 60:40, 50:50, and 0:100, respectively.

5. The method for preparing the cyclopentanephenylpropionate furan compound Aglatestate E according to claim 1, characterized in that, In step C, Fr.6 was separated using an MCI column with methanol / water volume ratios of 60:40, 70:30, 80:20, 90:10, and 100:0, respectively.

6. The method for preparing the cyclopentanephenylpropanefuran compound Aglatestate E according to claim 1, characterized in that, In step D, Fr.6C is separated using a reverse-phase C18 column with methanol / water volume ratios of 70:30, 75:25, 80:20, 90:10, and 100:0, respectively.

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