A natural denudaquinol dimer and its use in the preparation of an anti-hepatoma product

Abstract

This application discloses a natural denudaquinol dimer and its use in the preparation of anti-liver cancer products. The structural formula of the denudaquinol dimer is as follows: This application isolated the denudaquinol dimer from the branches and leaves of *Magnolia longicornis*. Multiple in vitro pharmacological experiments showed that this compound exhibited significant inhibitory effects on three types of liver cancer cells: PLC / PRF5, HepG2, and MHCC-97H. This application is the first to discover that this compound possesses this activity and can be used to prepare inhibitors, drug lead compounds, or active ingredients of anti-liver cancer drugs for PLC / PRF5, HepG2, and MHCC-97H liver cancer cells.

Description

Technical Field

[0001] This invention relates to the field of natural compounds and their pharmaceutical uses, specifically to the use of a natural denudaquinol dimer, alcimaphenol B, in the preparation of inhibitors of human liver cancer cells (PLC / PRF5, HepG2, MHCC-97H) or as their active pharmaceutical ingredient. Background Technology

[0002] Currently, the main treatments for liver cancer include surgical resection, liver transplantation, local ablation, transarterial chemoembolization, and systemic drug therapy. Although recent advances in these treatments have improved patient survival rates, persistent challenges such as limited efficacy, significant side effects, and drug resistance highlight the urgent need for highly effective and low-toxicity novel therapeutic drugs.

[0003] Natural products and their derivatives (such as paclitaxel, vincristine, and camptothecin) have long been the cornerstone of anticancer drug development, with approximately 60% of clinically approved anticancer drugs derived from natural products. Therefore, discovering novel lead compounds with unique mechanisms of action from natural products has become an important direction in the development of anticancer drugs. Among numerous natural sources, the active ingredients contained in Magnoliaceae plants have attracted much attention due to their excellent anticancer biological activity. *Magnolia longistylum* (…) Alcimandra cathcartii As a rare and endangered monotypic genus of Magnoliaceae endemic to China, it is a Class I protected wild plant in China. To date, phytochemical and pharmacological research on this rare species remains very limited. Summary of the Invention

[0004] This application describes the isolation of denudaquinol dimer from the protective harvesting of *Magnolia longicornis* branches and leaves. Multiple in vitro pharmacological experiments have shown that this compound exhibits significant inhibitory effects on three types of liver cancer cells: PLC / PRF5, HepG2, and MHCC-97H. This application is the first to discover this activity in a compound, which can be used to prepare inhibitors, drug lead compounds, or active ingredients for anti-liver cancer drugs targeting PLC / PRF5, HepG2, and MHCC-97H liver cancer cells.

[0005] A natural denudaquinol dimer with the following structural formula:

[0006] .

[0007] This application conducted inhibitory activity experiments on the obtained denudaquinol dimer against PLC / PRF5, HepG2, and MHCC-97H cells, showing that the compound exhibits significant inhibitory activity against PLC / PRF5, HepG2, and MHCC-97H cells, and can be used to prepare PLC / PRF5, HepG2, and MHCC-97H inhibitors or drug lead compounds.

[0008] This application also provides the use of the natural denudaquinol dimer in the preparation of human liver cancer cell inhibitors.

[0009] Optionally, the human liver cancer cells are at least one of PLC / PRF5, HepG2, and MHCC-97H human liver cancer cells. More preferably, the human liver cancer cells are PLC / PRF5.

[0010] This application also provides a human liver cancer cell inhibitor, using the described natural denudaquinol dimer as the active ingredient.

[0011] This application also provides the use of the natural denudaquinol dimer in the preparation of a medicament for treating liver cancer.

[0012] Optionally, the liver cancer is liver cancer associated with PLC / PRF5, HepG2h, or MHCC-97H human liver cancer cells. More preferably, the liver cancer is liver cancer associated with PLC / PRF5. For example, liver cancer associated with PLC / PRF5 includes hepatitis B virus (HBV)-associated liver cancer, liver cancer associated with HepG2h includes ordinary hepatocellular carcinoma (HCC), and liver cancer associated with MHCC-97H includes highly invasive and highly metastatic liver cancer.

[0013] This application also provides a pharmaceutical composition comprising a therapeutically effective amount of an active ingredient and a pharmaceutically acceptable carrier or excipient, wherein the active ingredient is the aforementioned natural denudaquinol dimer.

[0014] Therapeutic effective dose refers to the range of doses in which a drug, in clinical application, can produce a clear therapeutic effect on the target disease while keeping adverse reactions within an acceptable range. This range can vary depending on the severity of the disease and the physical condition, age, weight, and sex of the subject to be treated.

[0015] The compounds described in this application can be used alone or in combination, or combined with pharmaceutically acceptable carriers or excipients, and formulated into oral or non-oral dosage forms using conventional methods.

[0016] Pharmaceutically acceptable carriers or excipients include buffer solutions, emulsifiers, suspending agents, disintegrants, disintegrants, dispersants, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delay agents, liposomes, etc., which can be selected according to different needs. Drug dosage forms include granules, tablets, capsules, powders, oral liquids, drops, microcapsules, or injections.

[0017] The compounds described in this application can be obtained by isolation and purification from natural plants; after isolation, the compounds can also be synthesized by chemical methods well known to those skilled in the art.

[0018] This application also provides a method for extracting the natural denudaquinol dimer, wherein dried Magnolia longistylum branches and leaves are air-dried and pulverized at room temperature, and extracted once or multiple times at room temperature with a methanol-water solution of 70% or higher concentration. The extract is concentrated, suspended in water, and extracted sequentially with petroleum ether and ethyl acetate to obtain a petroleum ether extract and an ethyl acetate extract. The ethyl acetate extract is concentrated under reduced pressure and then sequentially subjected to silica gel column chromatography and MCI column chromatography, and then sequentially separated by ODS reversed-phase column chromatography and reversed-phase semi-preparative high-performance liquid chromatography to obtain the compound shown in Formula 1.

[0019] Specifically, including: (1) Dry the dried branches and leaves of Magnolia longistylum at room temperature, crush them, and extract them once or multiple times with a methanol-water solution of 70% (v / v) or higher at room temperature. Concentrate the extract, suspend it in water, and extract it three times each with equal volumes of petroleum ether, ethyl acetate and n-butanol to obtain four components: petroleum ether, ethyl acetate, n-butanol and water. The ethyl acetate component was subjected to silica gel column chromatography and eluted with a petroleum ether-ethyl acetate gradient to obtain 15 components F1 – F15. (2) The eluent F8 with a petroleum ether-ethyl acetate volume ratio of 3:1 was separated by MCI column chromatography and eluted with a methanol-water gradient to obtain 7 subfractions F8-1–F8-7; the eluent F8-7 with a methanol-water volume ratio of 100:0 was separated by ODS reversed-phase column chromatography and eluted with a methanol-water gradient to obtain 5 subfractions F8-7-1. F8-7-5, and subfraction F8-7-3 of methanol-water volume ratio 90:10, were further separated by semi-preparative high-performance liquid chromatography and collected. t R The fraction obtained at 11.5 min yielded the natural denudaquinol dimer.

[0020] Optionally, in step (1), the methanol-water solution used for methanol extraction is a 90% methanol-water solution, that is, a methanol-water solution with a volume ratio of 90:10 is used for extraction.

[0021] Optionally, in step (1), the methanol-water solution is extracted at room temperature more than 3 times, and can be extracted 5 times, and the extracts are combined. There is no special limit to the extraction time at room temperature, and it can be more than 12 hours per extraction.

[0022] Optionally, in step (1), the elution gradient of the ethyl acetate component during silica gel column chromatography gradient elution is set as follows: the volume ratio of petroleum ether to ethyl acetate is 10:1 → 7:1 → 5:1 → 3:1 → 1:1 → 0:1.

[0023] Preferably, the eluent fraction (F8) with a petroleum ether:ethyl acetate ratio of 3:1 is subjected to MCI column chromatography with a methanol:water gradient (MeOH / H2O, 50:50→70:30→90:10→100:0, v / v), and the eluents are collected separately. Then, the eluent fraction F8-7 with a methanol:water volume ratio of 100:0 is subjected to ODS reversed-phase column chromatography with a methanol-water gradient (70:30→80:20→90:10→100:0, v / v), and the eluents are collected separately to obtain 5 subfractions F8-7-1. F8-7-5 was used to separate the subfraction F8-7-3, which had a methanol-water volume ratio of 90:10, by reversed-phase semi-preparative high-performance liquid chromatography (RP-HPLC) (mobile phase: MeOH-H2O, 80:20, volume ratio; flow rate: 3 mL / min). t R After purification (11.5 min), the compound shown in Formula 1 was obtained. Compared with the prior art, the present invention has the following advantages: The denudaquinol dimer compounds isolated in this application exhibit significant inhibitory activity against PLC / PRF5, HepG2, and MHCC-97H cells, and show promising application prospects. Attached Figure Description

[0024] Figure 1 The 1H NMR spectrum of compound 1 (denudaquinol dimer) (400 MHz, in CD3OD).

[0025] Figure 2 The carbon spectrum of compound 1 (denudaquinol dimer) (100 MHz, in CD3OD).

[0026] Figure 3 This is the high-resolution mass spectrum of compound 1 (denudaquinol dimer).

[0027] Figure 4 The graph shows the inhibition of PLC / PRF5 cell proliferation by compound 1 (denudaquinol dimer).

[0028] Figure 5 The graph shows the inhibition of PLC / PRF5 cell migration by compound 1 (denudaquinol dimer). Detailed Implementation

[0029] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0031] Mass spectrometry (ESI-MS) was performed using a Thermo Fisher Q-Exactive mass spectrometer, employing an ESI ion source to simultaneously acquire positive and negative ion modes; NMR data were obtained from a Bruker Avance II 400 nuclear magnetic resonance spectrometer, with chemical shifts referenced to the residual solvent peaks of the undeuterated system. δ (ppm) is used for analysis; the thin-layer chromatography (TLC) plates used were purchased from Yantai Jiangyou Silica Gel Development Co., Ltd., and UV chromatography was employed. λ Colorimetric analysis was performed using sulfuric acid-vanillin solution (254 nm, 365 nm); column chromatography primarily used MCI microporous resin CHP20P (Japan, Mitsubishi Chemical Industries, 75-150). μ m), silica gel (200-300 mesh, Ji-Yi-Da silysia chemical Ltd, Qingdao PR China), ODS reversed-phase column (China, Beijing Yinglaike Technology Development Co. LTD, 50–75 μ m) and Sephadex LH-20 (Sweeden, GE Healthcare BioSciences AB); the analytical grade solvents used in the experiment, such as petroleum ether, acetone, ethyl acetate, methanol, and acetonitrile, were purchased uniformly by Taizhou University, and Magnolia longistylum was collected from Yunnan Province.

[0032] Example 1: Preparation of denudaquinol dimer 45.2 kg of dried Magnolia longistylum branches and leaves were pulverized and extracted five times (24 hours each time) with 90% methanol (60 L) at room temperature. The extract was concentrated under reduced pressure to obtain a dark brown semi-dry extract (9.1 kg). This extract was suspended in water (10 L) and successively extracted with petroleum ether (PE, 3 × 10 L), ethyl acetate (EtOAc, 3 × 10 L), and n-butanol (…). n Liquid-liquid partition extraction was performed using BuOH (3×10 L).

[0033] The obtained ethyl acetate fraction extract (1.5 kg, semi-dry) was separated by silica gel column chromatography, eluted with a petroleum ether-ethyl acetate gradient (10:1→7:1→5:1→3:1→1:1→pure ethyl acetate, v / v), yielding fifteen fractions (F1). F15).

[0034] The eluent F8 (163 g) with a petroleum ether:ethyl acetate volume ratio of 3:1 was further separated by MCI gel column chromatography with a methanol-water gradient elution (50:50→70:30→90:10→100:0, v / v) to give seven sub-fractions (F8-1). F8-7).

[0035] Subfraction F8-7 (23.7 g) with a methanol:water volume ratio of 100:0 was separated by ODS reversed-phase column chromatography with methanol-water gradient elution (70:30→80:20→90:10→100:0, v / v) to obtain five secondary fractions (F8-7-1). F8-7-5).

[0036] The methanol-water ratio (90:10) fraction F8-7-3 (83 mg) was purified by semi-preparative high-performance liquid chromatography (Cosmosil πNAP C18 column, 5...). μ m, 250 × 10 mm; mobile phase: methanol-water, 80:20, v / v), yielded denudaquinol dimer, compound 1 (13.2 mg, retention time). t R = 11.5 min).

[0037] .

[0038] The physicochemical data of compound 1 are as follows: Alcimaphenol B (1): colorless oil. UV (MeOH) λmax (log ε) 204 (1.26),301 (0.16) nm; IR (KBr) ν max : 3432, 2975, 2922, 2848, 1802, 1734, 1661, 1618,1453, 1316, 1265, 1179, 1077, 995, 926, 859 cm -1 ; 1 H NMR (in CD3OD, 400 MHz): δ H 6.77 (1H, d, J = 2.5 Hz, H-4), 6.73 (1H, d, J = 2.5 Hz, H-6), 3.34 (2H, d, J = 7.3Hz, H-7), 5.32 (1H, t, J = 7.3 Hz, H-8), 2.06 (2H, m, H-10), 2.12 (2H, m, H-11), 5.10 (1H, m, H-12), 1.64 (3H, s, H-14), 1.57 (3H, s, H-15), 1.75 (3H, s,H-16),6.85 (1H, d, J = 2.4 Hz, H-4 ), 6.53 (1H, d, J = 2.4 Hz, H-6 ), 3.17 (2H, d, J = 7.3 Hz, H-7 ), 5.24 (1H, t, J = 7.3 Hz, H-8 ), 1.99 (2H, m, H-10 ), 2.07 (2H,m, H-11 ), 5.08 (1H, m, H-12 ), 1.64 (3H, s, H-14 ), 1.58 (3H, s, H-15 ), 1.63(3H, s, H-16 ), 5.02 (1H, s, H-17 ), 3.71 (3H, s, H-OCH3). 13 C NMR (in CD3OD,100 MHz): δ C 127.4 (C-1), 146.5 (C-2), 127.4 (C-3), 109.2 (C-4), 156.1 (C-5),119.6 (C-6), 28.7 (C-7), 122.2 (C-8), 138.4 (C-9), 40.8 (C-10), 27.7 (C-11),125.3 (C-12), 132.3 (C-13), 25.9 (C-14), 17.8 (C-15), 16.3 (C-16), 87.3 (C-17), 175.2 (C-18), 125.7 (C-1 ), 150.7 (C-2 ), 124.1 (C-3 ), 111.3 (C-4 ),153.6 (C-5 ), 116.9 (C-6 ), 28.7 (C-7 ), 122.7 (C-8 ), 137.6 (C-9 ), 40.8 (C-10 ), 27.7 (C-11 ), 125.2 (C-12 ), 132.4 (C-13 ), 25.9 (C-14 ), 17.8 (C-15 ), 16.3(C-16 ), 57.7 (C-17 ), 170.4 (C-18 ), 53.1 (OCH3). HRESIMS m / z 641.3093 [M + Na] + (calcd. for C 37 H 46 O8Na, 641.3090, Δ = 0.5 ppm)。

[0039] The proton NMR spectrum (400 MHz, in CD3OD) of compound 1 (denudaquinol dimer) is as follows: Figure 1 As shown; the carbon spectrum (100 MHz, in CD3OD) of compound 1 (denudaquinol dimer) is as follows. Figure 2 As shown; the high-resolution mass spectrum of compound 1 (denudaquinol dimer) is as follows. Figure 3 As shown.

[0040] Example 2: Assay for antitumor activity 1. Assay of inhibitory activity of PLC / PRF5, MHCC97H and HepG2 cells The viability of PLC / PRF5, MHCC97H and HepG2 cells was assessed using the WST-8 assay (using the Cell Counting Kit-8 kit from Dalian Meilun Biotechnology Co., Ltd.).

[0041] Approximately 3 × 10⁸ cells were used for each cell line. 3 Cells were seeded at a concentration of 100 μ In 96-well plates of 10% FBS DMEM medium, compound treatments were performed (concentration gradients from 0.01 to 100 μL). μ In the cell viability assay, DMSO was used as a negative control and sorafenib as a positive control. Cells were incubated in a 37°C humidified incubator for 24 hours, and then 10 μL of the solution was added to each well. μ L CCK-8 solution was incubated at 37°C for 2 hours. The optical density was measured at 450 nm using a 96-well plate reader (BMGLABTECH), and the IC50 value was obtained by fitting with GraphpadPrism 10.1.2 software. The results are shown in Table 1.

[0042] The three-day cell proliferation assay was similar to the viability assay: PLC / PRF5 cells were treated with 15 µM compound 1 (IC50 concentration) on day 1 (24 hours), and the treatment was repeated on day 2 (48 hours) and day 3 (72 hours). Cell viability curves were plotted and data analyzed using GraphPad™ Prism 8 software. The results are as follows: Figure 1 As shown.

[0043] Table 1. Inhibitory activity of compound 1 against PLC / PRF5, MHCC97H and HepG2 a

[0044] annotation: a The result is the average of three independent experiments. b Sorafenib was used as a positive control.

[0045] Table 1 shows that compound 1 significantly inhibited the proliferation of all three types of liver cancer cells, although slightly weaker than the positive control sorafenib, but still within the same order of magnitude. Among the three types of liver cancer cells, compound 1 showed the best inhibitory effect on PLC / PRF5 cells (IC50). 50 =10.9 μM), with relatively lower inhibitory activity against MHCC97H and HepG2.

[0046] Figure 4 The results showed that the absorbance value of compound 1 was consistently lower than that of the negative control DMSO throughout the 3-day observation period, indicating that compound 1 could significantly inhibit cell proliferation, and the inhibitory effect on proliferation on day 3 was statistically significant.

[0047] 2. PLC / PRF5 cell scratch assay Cell migration ability was assessed using a scratch assay. 5 × 10⁶ cells were used. 5 PLC / PRF5 cells were seeded in 6-well plates. After the cells formed a confluent monolayer, 200 μL of water was used to treat the cells. μ L-type pipette tips were used to create scratches on a monolayer of cells. After washing the cells three times with PBS, compound 1 (10 g / L) was administered. μ M) and DMSO (control) were used for processing. A time-lapse microscopy system (Nikon) was used for continuous monitoring for 48 hours, with images acquired at 0, 6, 12, 24, and 48 hours. The scratch closure percentage was calculated by measuring the scratch area at each time point (6, 12, 24, and 48 hours) and comparing it with the scratch area at the initial time (0 hours).

[0048] The results of the inhibitory effect of compound 1 on the migration of PLC / PRF5 cells are as follows: Figure 5 As shown in the figure, (A) is a representative image comparison of the migration of PLC / PRF5 cells after scratching. Cells were seeded in 6-well plates, and scratches were created after they had fully congregated. Subsequently, they were treated with compound 1 (10... μ Cell migration was monitored and recorded at 0, 6, 12, 24, and 48 hours after treatment with either M or DMSO for 48 hours using time-lapse microscopy. The bar chart in Figure (B) compares the relative coverage area of ​​the scratched region of PLC / PRF5 cells after treatment with DMSO and compound 1 (n = 3).

[0049] Figure 5 The results in (A) show that the yellow area represents cells that migrated into the scratches. As the culture time was extended from 0 h to 48 h, the cells in the DMSO control group were able to migrate rapidly and gradually cover the scratches; while the cells in the compound 1 treatment group migrated much slower and did not completely cover the scratches at 48 h. Figure 5 The quantitative statistical results in (B) further confirmed this phenomenon: at 48 h, the scratch coverage area in the DMSO group reached approximately 75%, while that in the compound 1 group was only approximately 50%, and there was a significant statistical difference between the two groups (p<0.05). This indicates that compound 1 can significantly inhibit cell migration.

[0050] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A natural denudaquinol dimer, characterized in that, The structural formula is: 。 2. Use of the natural denudaquinol dimer as described in claim 1 in the preparation of human liver cancer cell inhibitors.

3. The use according to claim 1, characterized in that, The human liver cancer cells are at least one of PLC / PRF5, HepG2, and MHCC-97H human liver cancer cells.

4. A human liver cancer cell inhibitor, characterized in that, The active ingredient is the natural denudaquinol dimer as described in claim 1.

5. Use of the natural denudaquinol dimer as described in claim 1 in the preparation of a medicament for treating liver cancer.

6. The use according to claim 5, characterized in that, The liver cancer mentioned refers to liver cancer associated with PLC / PRF5, HepG2h, or MHCC-97H human liver cancer cells.

7. A pharmaceutical composition, characterized in that, It includes a therapeutically effective amount of an active ingredient and a pharmaceutically acceptable carrier or excipient, wherein the active ingredient is the natural denudaquinol dimer as described in claim 1.

8. The method for extracting natural denudaquinol dimer as described in claim 1, characterized in that, include: (1) Dry the dried branches and leaves of Magnolia longistylum at room temperature, crush them, and extract them once or multiple times with a methanol-water solution of 70% or higher at room temperature. Concentrate the extract, suspend it in water, and extract it three times each with equal volumes of petroleum ether, ethyl acetate and n-butanol to obtain four components: petroleum ether, ethyl acetate, n-butanol and water. The ethyl acetate component was subjected to silica gel column chromatography and eluted with a petroleum ether-ethyl acetate gradient to obtain 15 components F1 – F15. (2) The eluent F8 with a petroleum ether-ethyl acetate volume ratio of 3:1 was separated by MCI column chromatography and eluted with a methanol-water gradient to obtain 7 subfractions F8-1–F8-7; the eluent F8-7 with a methanol-water volume ratio of 100:0 was separated by ODS reversed-phase column chromatography and eluted with a methanol-water gradient to obtain 5 subfractions F8-7-1. F8-7-5, and subfraction F8-7-3 of methanol-water volume ratio 90:10, were further separated by semi-preparative high-performance liquid chromatography and collected. t R The fraction obtained at 11.5 min yielded the natural denudaquinol dimer.

9. The extraction method according to claim 8, characterized in that, In step (1), extraction was performed using a methanol-water solution with a volume ratio of 90:10; the elution gradient for the ethyl acetate fraction was set as follows: the volume ratio of petroleum ether to ethyl acetate was 10:1 → 7:1 → 5:1 → 3:1 → 1:1 → 0:

1.

10. The extraction method according to claim 8, characterized in that, In step (2), the elution gradient for MCI column chromatography is set as follows: methanol-water volume ratio 50:50 → 70:30 → 90:10 → 100:0; for ODS reversed-phase column chromatography, the elution gradient is set as follows: methanol-water volume ratio 70:30 → 80:20 → 90:10 → 100:0; and for semi-preparative high-performance liquid chromatography, the eluent is methanol-water with a volume ratio of 80:20.

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