Eudesmane-type sesquiterpene lactone tbb hydrolysis derivative and application thereof

Abstract

The application discloses eudesmane type sesquiterpene lactone TBB hydrolysis derivatives, a preparation method and application thereof. The hydrolysis derivatives are compounds Ib-1 to Ib-3, and structural formulas of the compounds Ib-1 to Ib-3 are shown in formulas Ib-1 to Ib-3. Anti-human hepatocellular carcinoma (HCC) activity evaluation shows that the compounds Ib-1 to Ib-3 all have good cancer cell proliferation inhibition activity, and can be used for preparing anti-tumor drugs in clinic. In addition, the compound Ib-1 has time and dose dependent inhibitory effect on growth and colony formation of HCC cells in vitro, and inhibits migration of HCC cells, and is a promising lead compound for treating HCC.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a eucalyptane-type sesquiterpene lactone TBB hydrolysis derivative and its applications. Background Technology

[0002] According to a 2020 report by the World Health Organization, liver cancer accounts for 4.7% of the incidence rate and 8.3% of the mortality rate among 36 types of cancer. Hepatocellular carcinoma (HCC) is the most common primary liver cancer and one of the most common and deadliest tumors. Therefore, conducting relevant research is a long and arduous task.

[0003] Eucalyptane-type sesquiterpene lactones (TBBs) are compounds isolated from *Wedelia candel*, possessing an α-methylene-γ-lactone group. Studies have shown that eucalyptane-type sesquiterpene lactones (TBBs) can inhibit the growth of hepatocellular carcinoma (HCC) by suppressing the IL-6 / STAT3 signaling pathway. The inhibitory activity of eucalyptane-type sesquiterpene lactones (TBBs) and their derivatives on tumor cell proliferation varies. This invention, through further structural modification of eucalyptane-type sesquiterpene lactones (TBBs), obtains a series of novel analogs, and studies the antitumor cell activity of these analogs, laying a foundation for the next step in the preparation of antitumor drugs. Summary of the Invention

[0004] The purpose of this invention is to provide a eucalyptane-type sesquiterpene lactone TBB hydrolysate derivative, which has strong inhibitory activity against cancer cell proliferation and is promising for the development of antitumor drugs, especially for use in anti-hepatocellular carcinoma drugs.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution:

[0006] A hydrolyzed derivative of eucalyptane-type sesquiterpene lactone TBB, wherein the hydrolyzed derivative of eucalyptane-type sesquiterpene lactone TBB is compound Ib-1, compound Ib-2 and / or compound Ib-3, and their chemical structures are as follows:

[0007]

[0008] Another object of the present invention is to provide a method for preparing hydrolyzed derivatives of eucalyptane-type sesquiterpene lactones TBB, comprising the following steps: adding eucalyptane-type sesquiterpene lactones TBB into an acidic tetrahydrofuran solution for reaction, adjusting the pH with an alkaline solution, extracting with ethyl acetate solution, and then purifying by column chromatography to obtain compounds Ib-1, Ib-2 and / or Ib-3.

[0009] Preferredly, the specific steps are as follows: Eucalyptane-type sesquiterpene lactones TBB are added to hydrochloric acid solution and tetrahydrofuran solution for reaction. After adjusting the pH with potassium carbonate solution, the mixture is extracted with ethyl acetate solution and then purified by column chromatography to obtain compounds Ib-1 and Ib-2.

[0010] Preferredly, the specific steps are as follows: Eucalyptane-type sesquiterpene lactones TBB are added to sulfuric acid solution and tetrahydrofuran solution for reaction. After adjusting the pH with potassium carbonate solution, the mixture is extracted with ethyl acetate solution and then purified by column chromatography to obtain compounds Ib-1, Ib-2 and Ib-3.

[0011] Another object of the present invention is to provide the application of eucalyptane-type sesquiterpene lactone TBB hydrolysis derivatives in the preparation of anticancer drugs, particularly in the preparation of drugs for treating hepatocellular carcinoma.

[0012] Compared with the prior art, the beneficial effects of the present invention are:

[0013] This invention involves structural modification of eucalyptane-type sesquiterpene lactones (TBB) to obtain novel compounds, namely compounds Ib-1, Ib-2, and Ib-3. Evaluation of their activity against human hepatocellular carcinoma (HCC) showed that compounds Ib-1, Ib-2, and Ib-3 all exhibit inhibitory activity against cancer cell proliferation, and can be further developed for the preparation of anticancer drugs, particularly for use in the preparation of anti-HCC drugs. Attached Figure Description

[0014] Figure 1 To determine the effect of compound Ib-1 on the proliferation of liver cancer cells using the CCK-8 assay;

[0015] Figure 2 To determine the effect of Ib-1 on the long-term proliferation of liver cancer cells in a clonogenic assay;

[0016] Figure 3 The effect of compound Ib-1 on apoptosis of liver cancer cells;

[0017] Figure 4 The effect of compound Ib-1 on the cell cycle of liver cancer cells;

[0018] Figure 5 To determine the effect of Ib-1 on the migration of liver cancer cells in wound healing assays and Transwell assays. Detailed Implementation

[0019] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional experimental conditions.

[0020] The present invention provides a eucalyptane-type sesquiterpene lactone TBB hydrolysis derivative, compounds Ib-1 to Ib-3, whose chemical structures are as follows:

[0021]

[0022] The present invention will be further described below with reference to specific analysis.

[0023] Example 1

[0024] Preparation of compounds Ib-1 and Ib-2: An appropriate amount of eucalyptane-type sesquiterpene lactone TBB was added to a round-bottom flask, followed by the addition of dilute hydrochloric acid solution (2 mol / L) and tetrahydrofuran solution. The mixture was stirred at 45 °C for 9 h. After the reaction was complete as detected by TLC, the pH of the mixture was adjusted to 6-7 with a 2 mol / L potassium carbonate solution. The mixture was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (chloroform:ethyl acetate = 6:1) to obtain compounds Ib-1 and Ib-2, with yields of 7% and 48%, respectively.

[0025] Compound Ib-1 is (1S,5R,6S,7S,8S,9R,10S)-1-hydroxy-9-acetoxy-6-isobutyryloxy-3,11-dienhalothrin. Spectral data for compound Ib-1 are as follows:

[0026] 1 H NMR (400MHz, CDCl3) δ: 6.32 (1H, d, J = 4Hz, H-13), 5.77 (1H, d, J = 3.2Hz, H-13), 5.53 (1H, dd, J = 1.6, 4.0Hz, H-6), 5.41 (1H, br s,H-3),5.31(1H,d,J=4.4Hz,H-9),5.03(1H,dd,J=4.4,8.0Hz,H-8),3.43(1H,dd,J=6.0,10.4Hz,H-1),3.31(1H,m,H-7),2.56(1H,m,H-2′) ,2.48(1H,H-5),2.24,2.07(2H,m,H-2),2.03(3H,s,H-2″),1.60(3H,d,J=1.2Hz,H-14),1.17(6H,d,J=7.2Hz,H-3′,4′),1.11(3H,s,H-15); 13C NMR (100MHz, CDCl3) δ: 176.6 (C-1′), 172.3 (C-1″), 169.6 (C-12), 134.9 (C- 11),130.2(C-4),122.4(C-3),119.8(C-13),72.8(C-8),72.2(C-9),69.7(C -6),68.4(C-1),43.5(C-7),41.4(C-10),37.9(C-5),34.4(C-2′),29.7(C-2 ),20.7(C-2″),20.4(C-14),19.0(C-3′),18.8(C-4′),11.0(C-15).HRESIMS m / z415.1725[M+Na] + (calcd for C 21 H 28 O7+Na, 415.1727).

[0027] Compound Ib-2 is (1S,4R,5R,6S,7S,8S,9R,10S)-1,4-dihydroxy-9-acetoxy-6-isobutyryloxy-11-enhalothrin lactone. Spectral data for compound Ib-2 are as follows:

[0028] 1 H NMR(400MHz,Methanol-d4)δ:6.18(1H,d,J=4.0Hz,H-13),5.90(1H,dd,J=1.6,3.2Hz,H-6),5.72(1H,d ,J=4.0Hz,H-13),5.34(1H,d,J=4.4Hz,H-9),5.08(1H,dd,J=4.4,8.4Hz,H-8),3.39(1H,dd,J=6.4,8.4 Hz,H-1),3.31(1H,m,H-7),2.61(1H,m,H-2′),1.94(3H,s,H-2″),1.86(1H,d,J=3.2Hz,H-5),1.68,1.5 0(2H,m,H-3),1.64(2H,m,H-2),1.34(3H,s,H-14),1.30(3H,s,H-15),1.21(6H,d,J=7.0Hz,H-3′,4′); 13C NMR(100MHz,Methanol-d4)δ:177.5(C-1′),171.8(C-12),170.9(C-1″),136 .8(C-11),119.0(C-13),74.5(C-8),73.6(C-9),72.2(C-4),72.1(C-1),70.0 (C-6),45.8(C-7),44.5(C-5),43.7(C-3),43.1(C-10),35.7(C-2′),28.6(C- 14),26.3(C-2),20.5(C-2″),19.3(C-3′),18.8(C-4′),13.8(C-15).HRESIMS m / z 428.2272[M+NH4] + (calcd for C 21 H 30 O8+NH4,428.2278).

[0029] Example 2

[0030] Preparation of compound Ib-3: An appropriate amount of eucalyptane-type sesquiterpene lactone TBB, 20 mg KOH, and 2 ml acetone were added to a round-bottom flask. A dilute sulfuric acid solution (8 mol / L) and tetrahydrofuran solution were then added, and the mixture was stirred at 45 °C for 10 h. After the reaction was complete as detected by TLC, the pH of the mixture was adjusted to 6-7 with a 2 mol / L potassium carbonate solution. The mixture was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (chloroform:acetone = 8:1) to obtain compounds Ib-1, Ib-2, and Ib-3, with yields of 6%, 48%, and 43%, respectively.

[0031] Compound Ib-3 is (1S,4R,5R,6S,7S,8S,9R,10S)-1,4,9-trihydroxy-6-isobutyryloxy-11-enhalothrin lactone. Spectral data for compound Ib-3 are as follows:

[0032] 1H NMR(400MHz,Methanol-d4)δ:6.56(1H,d,J=1.2Hz,H-13),5.82(1H,d,J=1.2Hz,H-13),5.41(1H,d d,J=2.4,3.6Hz,H-6),4.51(1H,d,J=2.4Hz,H-9),4.25(1H,dd,J=2.4,4Hz,H-8),3.79(1H,dd,J=6. 4,9.6Hz,H-1),3.16(1H,m,H-7),2.62(1H,m,H-2′),1.72,1.60(2H,m,H-2),1.67,1.44(2H,m,H-2) ,1.35(1H,d,J=2.8Hz,H-5),1.29(3H,s,H-14),1.26(3H,s,H-15),1.22(6H,d,J=7.2Hz,H-3′,4′); 13 C NMR (100MHz, Methanol-d4) δ: 177.3 (C-1′), 166.5 (C-12), 135.2 (C-11), 132.8 (C-13), 86.1 (C-9), 76.1 (C-6), 71.8 (C-1), 71.6 (C-4), 62. 2(C-8),47.2(C-7),45.3(C-5),44.6(C-10),42.8(C-3),35.7(C-2′),28.8(C-2),25(C-14),19.5(C-3′),19(C-4′),15.2(C-15).HRESIMS m / z 386.2166[M+NH4] + (calcd for C 19 H 28 O7+NH4,386.2173).

[0033] Example 3

[0034] Toxicity experiments of TBB hydrolyzed derivatives of eucalyptane-type sesquiterpene lactones on two HCC cell lines, Huh7 and HepG2.

[0035] Cell lines and cell culture: Huh7 and HepG2 cells were obtained from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Both Huh7 and HepG2 cancer cells were grown in MEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and cultured at 37°C in a humid atmosphere containing 5% carbon dioxide.

[0036] Cytotoxicity assay: Compound-induced cytotoxicity in HepG2 and Huh7 cells was analyzed using the CCK-8 assay. HepG2 and Huh7 cells were cultured in MEM supplemented with 10% fetal bovine serum at 37°C. Cells were seeded at a density of 8000 cells per well in 96-well plates. The next day, cells were incubated with compounds Ib-1–Ib-3 at 37°C for 24 h. Cell viability was assessed using the CCK-8 assay, and absorbance was measured at 570 nm.

[0037] Table 1. Results of the antitumor activity of compounds Ib-1 to Ib-3 against HepG2 and Huh7 cells.

[0038]

[0039] As shown in Table 1, compounds Ib-1 to Ib-3 all exhibited varying degrees of inhibitory effects on the proliferation activity of Huh7 and HepG2 cancer cells. They all demonstrated inhibitory activity on Huh7 and HepG2 cancer cells that was comparable to or better than that of the parent molecule (eucalyptane-type sesquiterpene lactone TBB), and could be used in the clinical preparation of drugs for hepatocellular carcinoma.

[0040] Example 4

[0041] Experiment on the antiproliferative effect of compound Ib-1 on Huh7 and HepG2 cells.

[0042] The effects of different concentrations of compound Ib-1 on two human HCC cell lines (Huh7 and HepG2) were detected using the CCK-8 assay. Huh7 and HepG2 cells were cultured in MEM supplemented with 10% fetal bovine serum at 37°C. Cells were seeded at a density of 8000 cells per well in 96-well plates. The next day, Huh7 cells were incubated with different concentrations (3.125, 6.25, 12.5, 25, 50 μM) of compound Ib-1 at 37°C for 24 h, and with compound Ib-1 for 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h at 37°C. Cell viability was assessed using the CCK-8 assay, and absorbance was measured at 570 nm. The results showed that compound Ib-1 dose-dependently reduced the viability of both HCC cell lines. Ib-1 also inhibited the viability of Huh7 cells in a time- and dose-dependent manner (e.g., ...). Figure 1 (As shown).

[0043] The in vitro cytotoxicity of compound Ib-1 was evaluated using a live / dead cell staining assay (Calcein-AM / PI double staining kit): Huh7 and HepG2 cells were seeded in 6-well cell culture plates and incubated with compound Ib-1 at 37°C for 24 h the next day. Cells were washed three times with PBS, trypsinized, centrifuged, and resuspended in 100 μL of 1×Assay Buffer. Live and dead cells were then separated by a mixture of calcein (2 mM) and PI (1.5 mM) solution for 20 min. The cells were then imaged using an inverted fluorescence microscope. Compared to the control group, the number of live Huh7 and HepG2 cells in the live cells treated with compound Ib-1 was significantly reduced, while the number of live dead cells was significantly increased. Therefore, the fluorescence imaging results were consistent with the cytotoxicity assay results in Example 3.

[0044] The effect of compound Ib-1 on the proliferation of Huh7 and HepG2 cells was investigated using a colony formation assay: Huh7 and HepG2 cells were seeded at 1000 cells per well in 6-well plates and cultured for 24 h. Different concentrations of compound Ib-1 were then added to the cells. Cells were cultured in an incubator for 14 days, with the culture medium changed every 3 days. Cells were then fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 30 min. After washing, the plates were photographed and counted using ImageJ software. The colony formation rate relative to the solvent control group was calculated. Visible cell clusters were observed after 14 days. The results showed that Huh7 and HepG2 cells treated with compound Ib-1 had significantly fewer colony clusters (e.g., ...). Figure 2 As shown in the figure, Ib-1 significantly inhibited clonogenic ability.

[0045] Example 5

[0046] Effect of compound Ib-1 on promoting apoptosis in Huh7 and HepG2 cells.

[0047] Huh7 and HepG2 cells were seeded in 6-well plates. Cells were then cultured with different concentrations of Ib-1 for 24 h, and subsequent treatment was performed according to the instructions of the Annexin V-FITC / PI apoptosis detection kit. Cells were first digested with trypsin, centrifuged at 1500 rpm for 5 min at 4 °C, soaked twice in pre-chilled PBS, resuspended in 100 μL of 1× Binding Buffer, and then incubated in the dark for 10–15 min with 5 μL Annexin-V-FITC and 10 μL of LPI staining solution. After dilution with 400 μL of 1× Binding Buffer, samples were analyzed by flow cytometry. Data were analyzed using FlowJo V1 software.

[0048] Compound Ib-1 significantly induced apoptosis in both Huh7 and HepG2 cells in the treatment groups. Figure 3 As shown, the total apoptotic cell proportions of 6.3% (control, Huh7 cells) and 6.51% (control, HepG2 cells) significantly increased to 67.8% (40 μM, Huh7 cells) and 24% (10 μM, HepG2 cells), respectively. Compound Ib-1 induced approximately 30% late apoptosis in Huh7 cells, with the late apoptosis being more pronounced. These results indicate that compound Ib-1 can significantly induce apoptosis in both Huh7 and HepG2 cells.

[0049] Example 6

[0050] Effects of compound Ib-1 on the cell cycle of Huh7 and HepG2 cells.

[0051] Huh7 and HepG2 cells were seeded in 6-well plates. Cells were then incubated for 24 h in different concentrations of Ib-1. Cells were collected, washed three times with PBS, and then fixed in 70% ethanol at 4°C for at least 12 h. Afterward, all cells were removed and washed three times with PBS to remove any remaining ethanol. Cells were then resuspended in propidium iodide staining solution and incubated in the dark at room temperature for 30 min. Samples were analyzed using a NovoCyte flow cytometer. Figure 4 As shown, compared with the untreated control group, the proportion of cells arrested in S phase increased from 39.76% and 33.22% to 46.27% and 36.91%, respectively. This result indicates that Ib-1 inhibits the cell cycle progression of Huh7 and HepG2 cells in S phase.

[0052] Wound healing test: Cells (1×10⁻⁶) 6 Cells were seeded in 6-well plates and cultured until each well was covered. A monolayer of cells was vertically scraped using the tip of a 200 μL sterile pipette and washed three times with sterile PBS. Then, 5% fetal bovine serum medium was added, followed by the test compound, and incubation was performed for 48 h. Images were taken under a microscope at 0 and 48 h. Edge-by-edge measurements at 0 h and 48 h were compared using ImageJ software.

[0053] Transwell assay: The Transwell assay was performed in 24-well Transwell chambers. In the Transwell assay, starved cells were suspended in serum-free culture medium at a density of 2 × 10⁶ cells / well. 4 / ml. 200 μL of the prepared cell suspension and different concentrations of compound Ib-1 were added to the upper chamber of the culture dish, and 600 μL of culture medium (20% fetal bovine serum) was added to the lower chamber. The culture dishes were incubated at 37°C for 48 h. The cells in the upper chamber were then wiped clean, and the remaining cells were fixed with 4% paraformaldehyde for 30 min, stained with 0.1% crystal violet, and photographed for observation. (See attached image.) Figure 5 As shown, compound Ib-1 significantly inhibited the wound healing rate of Huh7 cells after treatment at a specified concentration for 48 h. In Transwell assays, the number of cells passing through the chambers also decreased in a dose-dependent manner. These results indicate that compound Ib-1 effectively inhibits the migration of Huh7 cells in a dose-dependent manner.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A eucalyptane-type sesquiterpene lactone TBB hydrolysis derivative, characterized in that, The eucalyptane-type sesquiterpene lactone TBB hydrolysis derivatives are compounds Ib-1, Ib-2, and / or Ib-3, whose chemical structures are as follows:

2. The method for preparing the eucalyptane-type sesquiterpene lactone TBB hydrolysis derivative as described in claim 1, characterized in that, Includes the following steps: Eucalyptane-type sesquiterpene lactones TBB were reacted in an acidic tetrahydrofuran solution. After adjusting the pH with an alkaline solution, the mixture was extracted with ethyl acetate and purified by column chromatography to obtain compounds Ib-1, Ib-2, and / or Ib-3.

3. The method for preparing the eucalyptane-type sesquiterpene lactone TBB hydrolysis derivative as described in claim 2, characterized in that, Includes the following steps: The eucalyptane-type sesquiterpene lactone TBB, hydrochloric acid, and tetrahydrofuran solution were mixed and reacted. After adjusting the pH with potassium carbonate solution, the mixture was extracted with ethyl acetate solution and then purified by column chromatography to obtain compounds Ib-1 and Ib-2.

4. The method for preparing the eucalyptane-type sesquiterpene lactone TBB hydrolysis derivative as described in claim 2, characterized in that, Includes the following steps: Eucalyptane-type sesquiterpene lactones TBB were reacted with sulfuric acid and tetrahydrofuran solution, and after pH adjustment with potassium carbonate solution, they were extracted with ethyl acetate solution and purified by column chromatography to obtain compounds Ib-1, Ib-2 and Ib-3.

5. The application of the eucalyptane-type sesquiterpene lactone TBB hydrolysis derivative as described in claim 1 in the preparation of anticancer drugs.

6. The use of the sesquiterpene lactone TBB hydrolysis derivative as described in claim 5 in the preparation of an anti-hepatocellular carcinoma drug.

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