Eudecane type sesquiterpene lactone TBB chlorambucil heterocomplex and application thereof

Abstract

The invention discloses an eudecane type sesquiterpene lactone TBB chlorambucil heterocomplex and application thereof, the derivative is a compound TBB-DJ, and the structural formula of the derivative is as shown in the formula TBB-DJ. Experimental results show that the compound TBB-DJ has a remarkable inhibition effect on proliferation activity and migration ability of liver cancer cells, and shows a unique anti-hepatocellular carcinoma effect of the compound TBB-DJ. Therefore, the compound TBB-DJ has an important application prospect in research and development of anti-hepatocellular carcinoma drugs.

Description

Technical Field

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

[0002] According to global cancer statistics in 2020, hepatocellular carcinoma (HCC) is one of the deadliest malignant tumors worldwide, with approximately 2.2 million new cases and 1.8 million related deaths annually. As the fourth leading cause of cancer death globally, HCC is also the leading cause of death among patients with cirrhosis. Sesquiterpene lactones (SLs) are a class of natural products with significant biological activity, widely found in various plants, and have attracted considerable attention from researchers due to their outstanding anticancer activity. The applicant isolated eucalyptane-type sesquiterpene lactone epimers from Wedelia candel (application number: CN202010069334.X), in which compound 2 is (1S,4S,5R,6S,7S,8S,9R,10S)-1,9-diacetoxy-4-hydroxy-6-isobutyryloxyhalothia lactone (referred to as "eucalyptane-type sesquiterpene lactone TBB"), which exhibits a certain inhibitory effect on the proliferation activity of liver cancer cells.

[0003] The applicant further obtained a derivative of the eucalyptane-type sesquiterpene lactone TBB through acidic hydrolysis: (1S,4R,5R,6S,7S,8S,9R,10S)-1,4-dihydroxy-9-acetoxy-6-isobutyryloxy-11-enhalothia lactone (hereinafter referred to as "compound Ib-2"). Our research group used the CCK-8 assay to detect the cytotoxic activity of compound Ib-2 in two human hepatocellular carcinoma (HCC) cell lines, HepG2 and Huh7. The results showed that it exhibited a certain inhibitory effect on the proliferation of HCC cells. However, compound Ib-2 showed weak inhibitory activity against both Huh7 and HepG2 (IC50-100%). 50 =47.98±2.53μM and IC 50 The concentration of the compound Ib-2 (56.69±1.09 μM) is relatively low, and its mechanism of action against hepatocellular carcinoma lacks in-depth research. Furthermore, the metastatic problem of hepatocellular carcinoma severely limits the efficacy of most drugs. Given the high metastatic potential of hepatocellular carcinoma cells, candidate compounds must simultaneously possess migration-inhibiting capabilities. However, compound Ib-2 has not yet undergone systematic functional studies in this crucial area. Therefore, developing novel drugs based on eucalyptane-type sesquiterpene lactone TBB derivatives and targeting the anti-hepatocellular carcinoma mechanism to simultaneously inhibit the proliferation and migration of hepatocellular carcinoma remains an important research direction. Summary of the Invention

[0004] The purpose of this invention is to provide a eucalyptane-type sesquiterpene lactone TBB chlorambucil hybrid compound, which has strong inhibitory activity against cancer cell proliferation and migration, and is promising for the development of anti-tumor 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 eucalyptane-type sesquiterpene lactone TBB phenylbutyrate nitrogen mustard hybrid, wherein the eucalyptane-type sesquiterpene lactone TBB nitrogen mustard derivative is compound TBB-DJ, and its chemical structure is as follows:

[0007]

[0008] Another objective of this invention is to provide a method for preparing a eucalyptane-type sesquiterpene lactone TBB phenylbutyrate mustard hybrid, comprising the following steps: mixing dicyclohexylcarbodiimide, 4-dimethylaminopyridine, p-nitrophenylbutyrate mustard and dichloromethane for reaction, then adding compound Ib-2 and copper(II) trifluoromethanesulfonate and heating for reaction, extracting with ethyl acetate and concentrating under reduced pressure to obtain a crude product, and purifying the crude product by column chromatography to obtain compound TBB-DJ.

[0009] Another object of the present invention is to provide the application of a eucalyptane-type sesquiterpene lactone TBB phenylbutyrate mustard hybrid in the preparation of anticancer drugs, particularly in the preparation of liver cancer drugs.

[0010] Compared with the prior art, the beneficial effects of the present invention are:

[0011] This invention successfully designed and synthesized a novel eucalyptane-type sesquiterpene lactone TBB nitrogen mustard derivative, namely compound TBB-DJ, by structurally modifying compound Ib-2. Experimental results show that compound TBB-DJ significantly inhibits the proliferation and migration of liver cancer cells, demonstrating its unique anti-hepatocellular carcinoma effect. Therefore, compound TBB-DJ has significant application prospects in the development of anti-hepatocellular carcinoma drugs. Attached Figure Description

[0012] Figure 1 To evaluate the in vitro cytotoxicity of compound TBB-DJ using the live / dead cell staining method (Calcein-AM / PI);

[0013] Figure 2 To determine the effect of compound TBB-DJ on the long-term proliferation of liver cancer cells in a clonogenic assay;

[0014] Figure 3 The effect of compound TBB-DJ on apoptosis of liver cancer cells;

[0015] Figure 4 The effect of compound TBB-DJ on the cell cycle of liver cancer cells;

[0016] Figure 5 To determine the effect of compound TBB-DJ on the migration of liver cancer cells in wound healing assays and Transwell assays. Detailed Implementation

[0017] 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.

[0018] The present invention provides a eucalyptane-type sesquiterpene lactone TBB phenylbutyrate nitrogen mustard hybrid compound, TBB-DJ, whose chemical structure is as follows:

[0019]

[0021] The present invention will be further described with reference to the embodiments, but the present invention is not limited to these embodiments.

[0022] Example 1

[0023] Preparation of compound 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 completed by TLC, the pH of the mixture was adjusted to 6-7 with potassium carbonate solution (2 mol / L). The mixture was extracted three times with ethyl acetate, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and 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. Compound Ib-1 is (1S,5R,6S,7S,8S,9R,10S)-1-hydroxy-9-acetoxy-6-isobutyryloxy-3,11-dienhalothrin; compound Ib-2 is (1S,4R,5R,6S,7S,8S,9R,10S)-1,4-dihydroxy-9-acetoxy-6-isobutyryloxy-11-dienhalothrin.

[0024] Another method for preparing compound Ib-2: In a round-bottom flask, add an appropriate amount of eucalyptane-type sesquiterpene lactone TBB, 20 mg KOH, and 2 ml acetone. Add dilute sulfuric acid solution (8 mol / L) and tetrahydrofuran solution and react. Stir at 45 °C for 10 h. After the reaction is complete as detected by TLC, adjust the pH of the mixture to 6-7 with a 2 mol / L potassium carbonate solution. Extract three times with ethyl acetate, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and purify the crude product 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. Compound Ib-3 is (1S,4R,5R,6S,7S,8S,9R,10S)-1,4,9-trihydroxy-6-isobutyryloxy-11-enhalothrin lactone.

[0025] Preparation of compound TBB-DJ: 102 mg of dicyclohexylcarbodiimide, 11 mg of 4-dimethylaminopyridine, 85 mg of p-nitrophenylbutyrate mustard, and 5 mL of dry dichloromethane were added to a 50 mL round-bottom flask. After stirring at room temperature for 3 h, compound Ib-2 and copper(II) trifluoromethanesulfonate were added. The mixture was heated to reflux at 95 °C for 4 h. After the reaction was complete (trichloromethane:ethyl acetate = 8:1), a green liquid was obtained. The liquid was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by preparative thin-layer chromatography (trichloromethane:ethyl acetate = 10:1) to obtain compound TBB-DJ, with a yield of 26%.

[0026] The compound TBB-DJ is (3aS,4S,4aR,5S,8S,8aS,9R,9aS)-9-acetoxy-5-hydroxy-4-(isobu tyryloxy)-5,8a-dimethyl-3-methylene-2-oxododecahydronaphtho[2,3-b]furan-8-yl4-(4-(bis(2-chloroethyl)amino)phenyl)butanoat). Spectroscopic data for compound TBB-DJ:

[0027] 1H NMR(400MHz,CDCl3)δ:7.07(2H,d,J=8.6Hz,H-7′,and H-9′),6.62(2H,d,J=8.7Hz,H-6′,and H-10′),6.25(1H,d,J=3.8Hz,H-13),5.97(1H,dd,J=1.6,3.0Hz,H-6),5.68(1H,d,J=3.4Hz,H-13),5.21(1H,d,J=4.5Hz,H-9),4.89(1H,dd,J=4.4,8.0Hz,H-8),4.63(1H,dd,J=4.1,11.1Hz,H-1),3.70(4H,m,H-11′,and H-13′),3.62(4H,m,H-12′,and H-14′),3.18(1H,m,H-7),2.60(1H,m,H-2″),2.54(2H,m,H-4′),2.29(2H,m,H-3′),1.95(3H,s,H-2″′),1.91(1H,d,J=2.9Hz,H-5),1.87(2H,m,H-2′),1.76,and 1.62(2H,m,H-3),1.70,and 1.60(2H,m,H-2),1.37(3H,s,H-15),1.36(3H,s,H-14),1.22(6H,d,J=7.0Hz,H-3″,and H-4″). 13 C NMR(100MHz,CDCl3)δ:176.4(C-1″),173.0(C-1′),169.5(C-12),169.2(C-1″′),144.4(C-8′),134.3(C-11),130.9(C-5′),129.8(2×C,C-7′,and C-9′),119.3(C-11),112.3(2×C,C-6′,and C-10′),72.9(C-1),72.3(C-8),71.3(C-9),70.8(C-4),68.2(C-6),53.7(2×C,C-11′,and C-13′),45.1(C-7),43.6(C-5),41.8(C-10),41.5(C-3),40.7(2×C,C-12′,and C-14′),34.6(C-2″),34.1(2×C,C-3′,and C-4′),26.6(C-2′),26.6(C-14),25.1(C-2),20.4(C-2″′),19.1(C-3″),18.6(C-4″),14.6(C-15).HRESIMSm / z:694.2539[M-H]- (calcd for C 35 H 46 Cl2NO9, 694.2550).

[0028] Example 2

[0029] This invention relates to the application of a eucalyptane-type sesquiterpene lactone, TBB chlorambucil, in the preparation of antitumor drugs. Specifically, the cytotoxic activity of compound TBB-DJ obtained in Example 1 was screened using the CCK-8 assay on two human hepatocellular carcinoma (HCC) cell lines, HepG2 and Huh7. The results showed that, compared to the parent molecule, compound TBB-DJ exhibited superior inhibitory activity against cancer cell proliferation, demonstrating its potential for clinical use in the preparation of anti-hepatocellular carcinoma drugs.

[0030] Cell lines and cell culture:

[0031] Both the Huh7 and HepG2 cell lines originated from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Both cancer cell lines were cultured in MEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, under a humidified atmosphere containing 5% carbon dioxide at 37°C.

[0032] Cytotoxicity assay:

[0033] The cytotoxicity induced by the eucalyptane sesquiterpene lactone TBB and the compound TBB-DJ in Huh7 and HepG2 cells was analyzed using the CCK-8 assay. Cells were routinely cultured in MEM medium 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 following day, cells were incubated with the eucalyptane sesquiterpene lactone TBB and the compound TBB-DJ at 37°C for 24 hours. Cell viability was then assessed using the CCK-8 assay, with absorbance measured at 570 nm.

[0034] Table 1. Results of the toxic effects of compounds TBB-DJ and TBB on human hepatocellular carcinoma cells Huh7 and HepG2.

[0035]

[0036] As shown in Table 1, compared with the parent molecule, the eucalyptane-type sesquiterpene lactone TBB, compound TBB-DJ exhibits a more significant inhibitory effect on the proliferation of Huh7 and HepG2 cancer cells. This result strongly suggests that compound TBB-DJ possesses potential for clinical application and is expected to become a candidate compound for the development of anti-liver cancer drugs.

[0037] Example 3

[0038] In vitro antiproliferative effect of compound TBB-DJ on Huh7 and HepG2 cells.

[0039] The in vitro cytotoxicity of compound TBB-DJ was evaluated using a live / dead cell staining method (Calcein-AM / PI double staining kit):

[0040] HepG2 and Huh7 cells were seeded in 6-well cell culture plates and cultured at 37°C for 24 h. Subsequently, they were treated with TBB-DJ for 24 h. After treatment, the cells were washed three times with PBS, digested with trypsin, and centrifuged to collect the cell pellet. The cells were resuspended in 100 μL of 1×Assay Buffer, and a mixed staining solution of calcein (2 mM) and propidium iodide (PI, 1.5 mM) was added. The cells were incubated in the dark for 20 min. Finally, the fluorescence images of the cells were observed and acquired using an inverted fluorescence microscope.

[0041] In the control group, both Huh7 and HepG2 cells exhibited significant bright green fluorescence (calcein AM-labeled live cells), while a small number of red fluorescent spots (PI-labeled dead cells) were also observed (e.g. Figure 1 (As shown in the image). After treatment with compound TBB-DJ, the number of viable cells in both cell types decreased significantly, while the number of dead cells increased accordingly. The results of fluorescence microscopy observation were consistent with the results of cytotoxicity experiments, further confirming the cytotoxic effect of compound TBB-DJ on Huh7 and HepG2 cells.

[0042] The effect of compound TBB-DJ on the proliferation of Huh7 and HepG2 cells was examined using a colony formation assay.

[0043] Select Huh7 and HepG2 cells in the logarithmic growth phase and in good condition, at a ratio of 1×10⁻⁶. 3 Cells were seeded at a density of 100 cells / well in 6-well plates and incubated at 37°C with 5% CO2 for 24 h. Subsequently, different concentrations of the compound TBB-DJ were added for treatment. After culturing for another 14 days, cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min, and stained with 0.1% crystal violet solution for 15 min. After thorough removal of the staining solution, cell observation and image acquisition were performed.

[0044] Experimental results showed that treatment with 5 μM compound TBB-DJ significantly reduced the colony formation efficiency of Huh7 and HepG2 cells (e.g., ...). Figure 2 As shown in the figure, the compound TBB-DJ was found to have significant anti-colony formation activity and could effectively inhibit tumor cell proliferation.

[0045] Example 4

[0046] The effect of compound TBB-DJ on apoptosis in Huh7 and HepG2 cells was detected by flow cytometry.

[0047] Cells at 2×10 5 Cells were seeded at a density of 100 cells / well in six-well plates and incubated at 37°C with 5% CO2 for 24 h. After cell attachment, TBB-DJ compound was added and cultured for another 24 h. After treatment, cells were collected and washed twice with pre-chilled PBS (1000 rpm, 5 min). The cell pellet was resuspended in 100 μL of 1× binding buffer, and Annexin V-FITC and PI (at the manufacturer's recommended ratio) were added for staining in the dark for 15 min (room temperature). After staining, 400 μL of 1× binding buffer was added and mixed well, and the mixture was immediately subjected to Sysmex Partec CyFlow. TM Data were analyzed using a Cube 6 flow cytometer. Quantitative analysis was performed using FlowJo V10.1 software.

[0048] like Figure 3 As shown, treatment with 5 μM of the compound TBB-DJ significantly induced apoptosis in HepG2 and Huh7 cells. Flow cytometry analysis revealed that, compared with the control group (0 μM), the apoptosis rate of HepG2 cells significantly increased from 4.67% to 49.45%, while that of Huh7 cells increased from 8.11% to 27.00%. Notably, the pro-apoptotic effect of compound TBB-DJ on HepG2 cells was particularly significant, significantly stronger than its effect on Huh7 cells. These data indicate that the compound TBB-DJ can effectively induce apoptosis in liver cancer cells, and exhibits stronger sensitivity in the HepG2 cell line.

[0049] Example 5

[0050] The effects of compound TBB-DJ on the cell cycle of Huh7 and HepG2 cells were detected by flow cytometry.

[0051] Huh7 and HepG2 cells in logarithmic growth phase were digested with trypsin at a concentration of 2.0 × 10⁻⁶. 5Cells were seeded at a density of 100 cells / well in 6-well plates. After incubation at 37°C and 5% CO2 for 24 h, the medium was replaced with MEM complete medium (containing 10% FBS) containing compound TBB-DJ. After 24 h of treatment, cells were collected and resuspended in 300 μL PBS, followed by fixation with 700 μL pre-chilled anhydrous ethanol at 4°C for 4 h. Fixed cells were washed twice with PBS (1000 rpm, 5 min) and stained with 50 μg / mL PI (containing RNase A) in the dark for 30 min according to the Beyotime Cell Cycle Detection Kit (C1052) manufacturer's instructions. Samples were analyzed by flow cytometry, and data were analyzed using FlowJo V10.1 software.

[0052] When HepG2 and Huh7 cells were treated with TBB-DJ at a concentration of 5 μM, the compound significantly induced G2 / M phase arrest. Flow cytometry analysis showed (e.g.) Figure 4 As shown in the figure, the proportion of HepG2 cells in the G2 / M phase increased from 31.42% in the control group to 37.85%, while that of Huh7 cells increased from 20.05% to 26.75%. This further confirms that the compound TBB-DJ can effectively arrest the cell cycle of the two liver cancer cell lines in the G2 / M phase.

[0053] Example 6

[0054] Effects of compound TBB-DJ on cell migration in Huh7 cells.

[0055] The effects of compound TBB-DJ on the migration ability of Huh7 cells were evaluated using scratch healing assays and Transwell migration assays.

[0056] Scratch healing experiment: Huh7 cells were injected at a rate of 1×10⁻⁶. 6 Cells were seeded at a density of [number] cells / mL in 6-well plates and cultured at 37°C and 5% CO2 until 90% confluence. Straight scratches were created on the cell monolayer using a sterile 200 μL pipette tip, and detached cells were removed by washing with PBS. The medium was replaced with fresh medium containing 5% FBS and the test compound TBB-DJ, and cultured for another 48 h. The scratched areas were photographed using an inverted microscope at 0 h and 48 h, and the scratch area was quantified using ImageJ software. Experiments were independently repeated three times, and data are expressed as mean ± SD.

[0057] Transwell migration assay: 600 μL of MEM medium containing 20% ​​FBS was added to each well of a 24-well plate as a chemotactic agent. 8 μm pore size Transwell chambers were placed in the wells. 5 × 10⁻⁶ cells were then used. 4One Huh7 cell was resuspended in 200 μL of serum-free MEM medium (containing the specified concentration of compound TBB-DJ) and seeded into the upper chamber. After incubation at 37°C and 5% CO2 for 4 h, the chamber was removed and fixed with 4% paraformaldehyde at room temperature for 30 min. After staining with 0.1% crystal violet for 20 min, the cells were washed with PBS and the unmigrated cells in the upper chamber were gently wiped away. Five fields of view were randomly selected under an inverted microscope to count the migrating cells. The experiment was independently repeated three times.

[0058] Experimental results show that in the scratch test (such as...) Figure 5 As shown in Figure AB), compared with the control group, treatment with compound TBB-DJ for 24 hours significantly inhibited the migration ability of Huh7 cells, reducing the migration rate by 62.3 ± 5.1%; in the Transwell migration assay (as shown in Figure AB), Figure 5 As shown in the CD), at the same concentration, the number of cells penetrating the polycarbonate membrane decreased by 74.8 ± 6.3%. These results consistently confirm that compound TBB-DJ effectively inhibits the migration ability of Huh7 cells.

[0059] 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 phenylbutyrate mustard hybrid compound, characterized in that, The aforementioned eucalyptane-type sesquiterpene lactone TBB nitrogen mustard derivative is compound TBB-DJ, whose chemical structure is as follows:

2. The method for preparing the eucalyptane-type sesquiterpene lactone TBB phenylbutyrate mustard hybrid as described in claim 1, characterized in that, The process includes the following steps: mixing and reacting dicyclohexylcarbodiimide, 4-dimethylaminopyridine, p-nitrophenylbutyric acid mustard and dichloromethane, then adding compound Ib-2 and copper(II) trifluoromethanesulfonate and heating to react, followed by separation and purification to obtain compound TBB-DJ.

3. The use of the eucalyptane-type sesquiterpene lactone TBB phenylbutyrate mustard hybrid compound as described in claim 1 in the preparation of anticancer drugs.

4. The use of the eucalyptane-type sesquiterpene lactone TBB phenylbutyrate mustard hybrid compound as described in claim 3 in the preparation of anti-hepatocellular carcinoma drugs.

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