Eucalyptane type sesquiterpene lactone TBA dimethylamine adduct and application thereof

Abstract

The invention discloses an eudecane type sesquiterpene lactone TBA dimethylamine adduct, namely a compound TBA-DMA, and the structural formula of the compound TBA-DMA is as shown in the formula TBA-DMA. Experimental results show that the compound TBA-DMA has a remarkable inhibiting effect on proliferation activity of liver cancer cells, the targeting effect on the liver cancer cells is more selective than that on normal cells, the specific anti-tumor effect of the compound TBA-DMA is shown, and damage of anti-cancer drugs to the normal liver cells can be reduced. Therefore, the compound can be used as a candidate molecule for the development of anti-cancer drugs, and particularly has an important application prospect in the research and development of anti-hepatocellular carcinoma (HCC) drugs.

Description

Technical Field

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

[0002] The global incidence of malignant tumors is on the rise, seriously threatening human health. Liver cancer is the sixth most common cancer worldwide and the third leading cause of cancer-related deaths, with hepatocellular carcinoma (HCC) accounting for approximately 90% of primary liver cancers. 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 tricuspidata* (application number: CN202010069334.X), in which compound 1 is (1S,4S,5S,6S,7S,8S,9R,10S)-1,9-diacetoxy-4-hydroxy-6-isobutyryloxyhalothia lactone (referred to as "eucalyptane-type sesquiterpene lactone TBA"), which showed certain inhibitory effects on the proliferation activity of breast cancer and liver cancer cells.

[0003] Because hepatocellular carcinoma (HCC) has an insidious onset and rapid progression, most patients are diagnosed at an intermediate or advanced stage, and systemic drug therapy is the primary treatment. However, existing anti-hepatocellular carcinoma drugs (such as regorafenib, sorafenib, and donafenib) all have certain hepatotoxicity, which may cause serious side effects such as liver and kidney dysfunction and proteinuria. Therefore, developing anti-HCC drugs with fewer side effects and higher specificity is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a eucalyptane-type sesquiterpene lactone TBA dimethylamine adduct, which has strong inhibitory activity against the proliferation of liver cancer cells and is more selective in targeting liver cancer cells than normal cells, showing promise for the development of specific anti-tumor drugs, especially for application 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 TBA dimethylamine adduct, wherein the eucalyptane-type sesquiterpene lactone TBA dimethylamine adduct is compound TBA-DMA, 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 TBA dimethylamine adduct, comprising the following steps: mixing eucalyptane-type sesquiterpene lactone TBA with a methanol solution of dimethylamine and reacting them; after the reaction is complete, transferring the mixture to a suitable round-bottom flask and evaporating it under reduced pressure using a rotary evaporator; and separating and purifying the crude product by column chromatography to obtain the compound TBA-DMA.

[0009] Another object of the present invention is to provide an application of eucalyptane-type sesquiterpene lactone TBA dimethylamine adduct 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 dimethylamine adduct of eucalyptane-type sesquiterpene lactone TBA (compound TBA-DMA) by modifying the structure of TBA. Experimental results show that compound TBA-DMA has a significant inhibitory effect on the proliferation activity of liver cancer cells and its targeting effect on liver cancer cells is more selective than that on normal cells. It demonstrates the specific anti-tumor effect of compound TBA-DMA and can reduce the damage of anticancer drugs to normal liver cells. Attached Figure Description

[0012] Figure 1 To determine the effect of compound TBA-DMA on the proliferation of liver cancer cells using the CCK-8 assay;

[0013] Figure 2 To evaluate the in vitro cytotoxicity of compound TBA-DMA using the live / dead cell staining method (Calcein-AM / PI);

[0014] Figure 3 To determine the effect of compound TBA-DMA on the long-term proliferation of liver cancer cells in a clonogenic assay;

[0015] Figure 4 The effect of compound TBA-DMA on apoptosis of liver cancer cells;

[0016] Figure 5 The effect of compound TBA-DMA on the cell cycle of liver cancer cells;

[0017] Figure 6 To determine the effect of TBA-DMA on the migration of liver cancer cells in a wound healing experiment;

[0018] Figure 7 Transcriptome sequencing was used to analyze the apoptosis mechanism of TBA-DMA against liver cancer cells. 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 TBA dimethylamine adduct, namely compound TBA-DMA, and its synthetic route is as follows:

[0021] Detailed Implementation

[0022] The present invention will be further described with reference to the embodiments, but the present invention is not limited to these embodiments.

[0023] Example 1

[0024] Preparation of compound TBA-DMA: Eucalyptane-type sesquiterpene lactone TBA was added to a 25 mL round-bottom flask, followed by 2 mL of dimethylamine methanol solution. After complete dissolution, a magnetic stir bar was added, and the flask was fixed on a temperature-controlled magnetic stirrer. The mixture was stirred at room temperature. The reaction was monitored by silica gel thin-layer chromatography (chloroform:acetone = 1:1). After stirring for 10 min until complete, the mixture was transferred to a suitable round-bottom flask and evaporated to dryness under reduced pressure using a rotary evaporator. The crude product was purified by column chromatography (chloroform:ethyl acetate = 1:1) to obtain compound TBA-DMA in 96% yield.

[0025] The compound TBA-DMA is (3S,3aS,4S,4aS,5S,8S,8aS,9R,9aS)-3-((dimethylamino)methyl)-5-hydroxy-4-(isobutyryloxy)-5,8a-dimethyl-2-oxododecah-ydronaphtho[2,3-b]furan-8,9-diyl diacetate. Spectroscopic data for compound TBA-DMA:

[0026] 11H NMR (400 MHz, CDCl3) δ: 5.80 (1H, dd, J = 8.1, 10.0 Hz, H-6), 5.41 (1H, t, J = 7.8 Hz, H-1), 5.29 (1H, d, J = 3.2 Hz, H-9), 4.87 (1H, dd, J = 3.2, 10.1 Hz, H-8), 3.02 (1H, m, H-7), 2.83 (1H, m, H-11), 2.66, and 2.49 (2H, m, H-13), 2.51 (1H, m, H-2″), 2.21 (6H, s, -NCH3CH3), 2.16, and 1.46 (2H, m, H-2), 2.02 (3H, s, H-2′), 1.97 (3H, s, H-2″′), 1.87, and 1.67 (2H, m, H-3), 1.71 (1H, d, J = 10.0 Hz, H-5), 1.35 (3H, s, H-14), 1.27 (3H, s, H-15), 1.21 (6H, dd, J = 7.0, 12.7 Hz, H-3″, 4″). 13 13C NMR (100 MHz, CDCl3) δ: 177.8 (C-12), 177.0 (C-1″), 170.5 (C-1′), 169.1 (C-1″′), 74.0 (C-8), 72.8 (C-6), 72.7 (C-9), 70.5 (C-4), 69.1 (C-1), 59.3 (C-13), 53.4 (C-5), 46.6 (2×C, -NCH3CH3), 44.3 (C-11), 41.9 (C-7), 40.5 (C-10), 35.2 (C-3), 34.6 (C-2″), 32.3 (C-14), 22.6 (C-15), 22.4 (C-2), 21.4 (C-2″′), 21.0 (C-2′), 19.3 (C-3″), 18.7 (C-4″).

[0027] Single crystal data of compound TBA-DMA:

[0028] C 25 H 39 NO9,f w = 497.57, 293(2) K, α = 90°, β = 106.418(3)°, γ = 90°, space group P21, Z = 2, μ(MoK α ) = 0.762 mm -1 , F(000) = 536.0, ρ calc = 1.216 g / cm 3;15758reflectionsmeasured,4800were unique(R int =0.0452),2Θrange for data collection:7.98to133.76°.Flack 0.1(2).The final R1=0.0489,wR2=0.1306(I>=2σ(I)).

[0029] Example 2

[0030] This invention relates to the application of a eucalyptane-type sesquiterpene lactone TBA dimethylamine adduct in the preparation of antitumor drugs. Specifically, the cytotoxic activity of the compound TBA-DMA obtained in Example 1 was screened using the CCK-8 assay on two human hepatocellular carcinoma (HCC) cell lines, HepG2 and Huh7, as well as normal hepatocytes HL-7701. The results showed that compound TBA-DMA exhibited superior inhibitory activity against cancer cell proliferation and showed some selectivity for normal cells, demonstrating its potential for clinical use in the preparation of anti-hepatocellular carcinoma drugs.

[0031] Cell lines and cell culture:

[0032] The Huh7, HepG2, and HL-7701 cell lines were all derived from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. All three cell lines were cultured in MEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, under a humidified atmosphere containing 5% CO2 at a temperature of 37°C.

[0033] The cytotoxicity induced by the compounds on Huh7, HepG2, and HL-7702 cells was evaluated using a CCK-8 assay. The procedure was as follows: 100 μL of the prepared cell suspension was added to each well of a 96-well plate. The plate was then placed in a 37°C incubator with 5% CO2 to allow cell adhesion for 24 h. The culture medium was changed the following day. Subsequently, the cell culture was co-incubated with all test compounds, TBA-DMA, at 37°C for 24 h, 48 h, and 72 h, respectively. After incubation, the supernatant was carefully removed from each well, and 100 μL of fresh culture medium containing 10% CCK-8 solution was added to each well. The cells were then incubated for approximately 2–4 h, and absorbance was measured at 570 nm.

[0034] The results showed that the compound TBA-DMA could also inhibit the viability of Huh7, HepG2, and HL-7702 cells in a time- and dose-dependent manner (e.g., Figure 1As shown in Table 1, different concentrations of the compound TBA-DMA (10, 20, 40, 80, 160 μM) were applied to Huh7, HepG2, and HL-7702 cells, and their survival rates were measured at 24, 48, and 72 h. For the normal cell line HL-7702, after 24 h of treatment, the selectivity of compound TBA-DMA for Huh7 cells was 3-fold, and the selectivity for HepG2 cells was 2-fold. The results indicate that with increasing concentrations of compound TBA-DMA, the survival rates of Huh7, HepG2, and HL-7702 cells decreased significantly, with a relatively smaller impact on HL-7702 cells. With prolonged treatment time, the survival rates of Huh7 and HepG2 cells decreased more significantly compared to HL-7702 cells, further demonstrating that compound TBA-DMA has a more selective targeting effect on liver cancer cells than on normal cells.

[0035] Table 1. Cell growth inhibitory activity of compound TBA-DMA.

[0036]

[0037] The in vitro cytotoxicity of compound TBA-DMA was evaluated using a live / dead cell staining assay performed with a Calcein-AM / PI double staining kit.

[0038] Huh7 and HepG2 cells were co-incubated with the compound TBA-DMA. Then, 100 μL of 1× detection buffer was added to each well to eliminate residual esterase activity from dead cells. Next, Calcein-AM / PI staining solution was added to each well, and the samples were incubated at 37°C for 30 min. The distribution of live and dead cells was observed under a fluorescence microscope using excitation light at a wavelength of 490 nm, and images were captured for recording.

[0039] like Figure 2 As shown, compared with the control group, after treatment with compound TBA-DMA, obvious bright green fluorescence (generated by the live cell probe calcein-AM) and red fluorescent spots (generated by the dead cell probe propidine iodide) were observed in Huh7 and HepG2 cells.

[0040] The effect of compound TBA-DMA on the proliferation of Huh7 and HepG2 cells was examined using a colony formation assay.

[0041] Huh7 and HepG2 cells in good logarithmic growth phase were cultured at 1×10⁻⁶ cells per well. 3Cells were seeded at a density of [number] cells per well in 6-well plates. The cells were then incubated at 37°C with 5% CO2 for 24 h. Afterward, the cells were treated with the compound TBA-DMA. After 14 days, the cells were washed, fixed with 4% paraformaldehyde for 30 min, and then stained with 0.1% crystal violet solution for 15 min. The crystal violet staining solution was thoroughly rinsed off for further observation, and cell images were taken.

[0042] like Figure 3 As shown, after 14 days of culture, the number of clone clusters formed by Huh7 and HepG2 cells treated with the compound TBA-DMA was significantly reduced, indicating that TBA-DMA has a significant inhibitory effect on the clonogenic ability of liver cancer cells.

[0043] Example 4

[0044] Effects of compound TBA-DMA on promoting apoptosis in Huh7 and HepG2 cells.

[0045] Cells were spaced at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in six-well plates and incubated for 24 h. Subsequently, cells were treated with TBA-DMA and incubated for another 24 h. After treatment, cells were collected and washed twice with phosphate-buffered saline (PBS). Cells were then resuspended in 1× binding buffer, and Annexin V-FITC and propidium iodide (PI) staining solution were added. Staining was performed for 15 min at room temperature in the dark. The stained cell sample was diluted with 400 μL of 1× binding buffer and then analyzed using a flow cytometer (Sysmex-Partec CyFlow). TM The data was analyzed using Cube 6. The acquired data was processed and analyzed using FlowJoV1 software.

[0046] like Figure 4 As shown, compared with the blank control group, the TBA-DMA treatment group significantly induced apoptosis in Huh7 and HepG2 cells. Flow cytometry results showed that in HepG2 cells, the proportion of apoptotic cells significantly increased from 0.58% in the control group to 38.57% in the TBA-DMA treatment group; while in Huh7 cells, the proportion of apoptotic cells significantly increased from 2.94% in the control group to 54.4% in the TBA-DMA treatment group. These data indicate that TBA-DMA can significantly induce apoptosis in liver cancer cells, and its pro-apoptotic effect is more pronounced in Huh7 cells.

[0047] Example 5

[0048] Effects of compound TBA-DMA on the cell cycle of Huh7 and HepG2 cells.

[0049] Huh7 and HepG2 cells in logarithmic growth phase were digested with trypsin and then divided into 2 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates. After overnight incubation in a constant temperature incubator, cells were treated with the compound TBA-DMA (dissolved in MEM medium containing 10% fetal bovine serum). After 24 h of exposure, cells were collected and resuspended in 300 μL phosphate-buffered saline (PBS), followed by the addition of 700 μL anhydrous ethanol, and fixed at 4 °C for 12 h. After 12 h, cells were washed twice with PBS, and cell cycle analysis was performed using propidium iodide (PI) staining. Fluorescence signals were acquired by flow cytometry. Data analysis was performed using FlowJo V1 software.

[0050] The results showed that, compared with untreated control cells (the proportions of HepG2 and Huh7 cells in S phase were 43.57% and 36.74%, respectively), the compound TBA-DMA significantly increased the proportions of HepG2 and Huh7 cells in S phase, reaching 50.49% and 49.69%, respectively (e.g., ...). Figure 5 (As shown). This result confirms that the compound TBA-DMA arrests the cell cycle progression of HepG2 and Huh7 cells in the G2 / M and S phases.

[0051] Example 6

[0052] Effects of compound TBA-DMA on cell migration in Huh7 and HepG2 cells.

[0053] Huh7 cells were distributed at a rate of 1 × 10⁶ cells per well. 6 Cells were seeded at a density of [number] cells / mL and cultured at 37°C in a 5% CO2 incubator until 90% confluence. A linear scratch was created on the cell monolayer using a sterile 200 μL pipette tip, followed by washing with PBS to remove detached cells. Cells were then treated with the test compound TBA-DMA in a medium containing 5% fetal bovine serum (FBS) and incubated for 48 h. Microscopic images were taken at 0 h and 48 h post-scraping to monitor wound healing. Wound area was quantified using ImageJ software, and the data were the average of three independent experiments.

[0054] like Figure 6 As shown, the compound TBA-DMA significantly inhibited the wound healing rate of Huh7 cells after treatment at a specified concentration for 48 hours.

[0055] Example 7

[0056] The apoptosis mechanism of compound TBA-DMA was investigated using RNA sequencing analysis.

[0057] Huh7 cells were pretreated with 40 μM TBA-DMA for 24 h. Cells were then collected, total RNA was extracted using TRIzol reagent, and cDNA libraries were sequenced on the Illumina sequencing platform. Four independent RNA samples were prepared for each experimental group for RNA sequencing (RNA-Seq) analysis. The obtained sequencing data were filtered using FastP software. High-quality reads were mapped to a reference genome using the HISAT alignment tool. Heatmaps were generated using the pheatmap R package to visualize gene expression patterns among different samples. Differential expression analysis was performed using the DESeq2 R package, with a significance criterion of Q ≤ 0.05. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were performed using the clusterProfiler R package and the iDEP integrated network platform.

[0058] To investigate the potential biological activity of compound TBA-DMA, RNA sequencing was used to detect the transcriptome levels of Huh7 cells treated with TBA-DMA. Total RNA was extracted after treatment for RNA sequencing analysis. Figure 7 As shown in Figures 7A and 7B, a total of 7496 single genes showed differential expression, of which 4973 genes were upregulated and 2523 genes were downregulated.

[0059] Figure 7 The 7C sequence presents the gene ontology (GO) enrichment analysis results for compound TBA-DMA. Differentially expressed genes were primarily enriched in three distinct categories: biological processes (BP), cellular components (CC), and molecular functions (MF). These categories encompass cellular processes, biological regulation, regulation of biological processes, cellular anatomy, and binding activity.

[0060] At the same time, such as Figure 7 As shown in Figure 7D, Kyoto Encyclopedia of Genetics and Genomes (KEGG) enrichment analysis revealed significant enrichment of multiple pathways associated with antitumor activity. These pathways include the mitogen-activated protein kinase (MAPK) signaling pathway, the arachidonic acid metabolism pathway, the tumor necrosis factor (TNF) signaling pathway, the cytokine-cytokine receptor interaction pathway, and the adipokines signaling pathway. Figure 7 As shown in E, the study selected the nine KEGG pathways with the lowest q values ​​and constructed enrichment analysis and chord diagrams. The results showed significant gene differences, such as those observed in genes ALOXE3, HSPA6, LTA, MSTN, IL-31, CCL24, and IL5RA.

[0061] Furthermore, gene set enrichment analysis (GSEA) showed that, compared with the control group, the cytokine-cytokine receptor interaction pathway (CCR) was significantly enhanced in the treatment group. Figure 7 The F) and MAPK signaling pathways, as well as the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, were significantly upregulated. The occurrence and development of hepatocellular carcinoma (HCC) involves the abnormal regulation of multiple signaling pathways, among which the MAPK, TNF, and PI3K-Akt pathways form a core regulatory network. They promote HCC progression by influencing phenotypes such as cell proliferation, apoptosis, and cell cycle. Specifically, the MAPK signaling pathway accelerates the G1 / S phase transition by upregulating cyclin D1, leading to cell cycle dysregulation; the TNF signaling pathway induces overexpression of B-cell lymphoma-2 (Bcl-2), blocking the mitochondrial apoptosis pathway; in the PI3K-Akt pathway, Akt can relieve the inhibition of cyclin-dependent kinase (Cyclin-CDK) by inhibiting p21 / p27, thereby promoting the G1 / S phase progression. The changes observed in these pathways, combined with cell phenotype experiments, suggest that the compound TBA-DMA may have potential anti-HCC activity.

[0062] 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 TBA dimethylamine adduct, characterized in that, The eucalyptane-type sesquiterpene lactone TBA dimethylamine adduct is compound TBA-DMA, and its chemical structure is as follows:

2. The preparation method of the eucalyptane-type sesquiterpene lactone TBA dimethylamine adduct as described in claim 1, characterized in that, Includes the following steps: The compound TBA-DMA was obtained by reacting a eucalyptane-type sesquiterpene lactone (TBA) with a methanol solution of dimethylamine and then separating and purifying the mixture.

3. The use of the eucalyptane-type sesquiterpene lactone TBA dimethylamine adduct as described in claim 1 in the preparation of anticancer drugs.

4. The use of the eucalyptane-type sesquiterpene lactone TBA dimethylamine adduct as described in claim 3 in the preparation of anti-hepatocellular carcinoma drugs.

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