Preparation method and application of brucea javanica bitter alcohol derivative

Abstract

The invention provides a preparation method of a brucea javanica picrol derivative, which comprises the following steps: adding anhydrous pyridine into brucea javanica picrol in an ice-water bath, dissolving, stirring at room temperature, dropwise adding propionyl chloride into the system under the protection of nitrogen, stirring at room temperature, completing a thin-layer monitoring reaction, and purifying to obtain the brucea javanica picrol derivative, and also provides application of the brucea javanica picrol derivative. The brucea javanica bitter alcohol derivative is used for preparing drugs for dual targeting inhibition of abnormal lipid metabolism of cells and repairing of mitochondrial functions. The brucea javanica bitter alcohol derivative prepared by the invention can be obtained by performing functional group modification on brucea javanica bitter alcohol, has the activity of dual targeting regulation of cell lipid metabolism and mitochondrial function, and has application potential in preparation of drugs for treating metabolic diseases.

Description

Technical Field

[0001] This invention belongs to the field of natural product modification and pharmaceutical research and development technology, specifically relating to a method for preparing and applying a crotonic acid derivative. Background Technology

[0002] Abnormal cellular lipid metabolism and mitochondrial dysfunction are two core and interconnected pathological links in the development of many major chronic diseases. On the one hand, lipid metabolism disorders, such as excessive fatty acid synthesis, insufficient oxidation, or abnormal lipid accumulation, are fundamental characteristics of metabolic diseases such as non-alcoholic fatty liver disease (NAFLD), obesity, rheumatoid arthritis, atherosclerosis, and type II diabetes. On the other hand, mitochondria, as the cell's "energy factory," are crucial for maintaining cellular homeostasis due to their functional integrity (including ATP synthesis, fatty acid β-oxidation, reactive oxygen species and membrane potential balance, mitochondrial division and fusion). Mitochondrial dysfunction not only leads to energy crises but also exacerbates oxidative stress and lipid metabolism disorders, creating a vicious cycle.

[0003] It is noteworthy that lipid metabolism and mitochondrial function are pathologically intertwined. For example, lipotoxicity (excessive lipids) can directly impair mitochondrial membrane integrity and inhibit electron transport chain complex function, leading to mitochondrial damage. Conversely, impaired mitochondria cannot effectively oxidize fatty acids, resulting in the accumulation of lipid intermediates and further exacerbating metabolic disorders. Therefore, developing single-drug agents that can simultaneously and synergistically inhibit abnormal lipid metabolism and repair damaged mitochondrial function is considered a promising new strategy for treating these diseases. However, current clinical and investigational drugs mostly focus on single targets or pathways (such as FASN inhibitors, ACC inhibitors, or agonists targeting only mitochondrial biosynthesis), lacking single compounds that can integrate and regulate these two key processes, thus making it difficult to completely break the vicious cycle in disease progression.

[0004] Bruceine D, a natural product, and its analogues have attracted considerable attention due to their broad-spectrum antitumor and anti-inflammatory activities. Current research primarily focuses on the direct cytotoxic effects of these compounds, but whether they specifically intervene in the fundamental biological process of cellular lipid energy metabolism remains unresolved. Furthermore, as a natural product, brucelline D often suffers from limitations such as poor solubility, low bioavailability, and significant toxicity, restricting its potential for direct drug development. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing and applying a crotonic acid derivative that addresses the shortcomings of the prior art. This crotonic acid derivative can be obtained by functional group modification of crotonic acid and has dual-targeted regulatory activities on "cellular lipid metabolism" and "mitochondrial function", and has application potential in the preparation of drugs for treating metabolic diseases.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing a crotonic acid derivative, the method being as follows: Anhydrous pyridine was added to crotonic acid (BRU) under ice-water bath conditions. After dissolution, the mixture was stirred at room temperature. Then, propionyl chloride was added dropwise to the system under nitrogen protection, and the mixture was stirred at room temperature. The reaction was monitored for completion by thin-layer chromatography. After purification by high-performance liquid chromatography, crotonic acid derivative (BRUD) was obtained. The structural formula of the crotonic acid derivative is as follows: R is benzoyl group.

[0007] Preferably, the ratio of crotonic acid, anhydrous pyridine, and propionyl chloride is 0.02 mmol: 1 mL: 60 μL.

[0008] This invention also provides the application of the croton picrol derivative prepared by the above preparation method, wherein the croton picrol derivative is used to prepare a drug that dual-targets the inhibition of abnormal lipid metabolism in cells and the repair of mitochondrial function.

[0009] Preferably, the croton picrol derivative is used to inhibit the expression of the lipid transport molecule CD36 and the expression of carnitine palmitoyltransferase 1A; The croton picrol derivative is used to inhibit the production of reactive oxygen species in mitochondria, repair mitochondrial membrane potential, inhibit the expression of mitochondrial fission molecules, and / or target and bind to the key mitochondrial fission protein Drp1.

[0010] Preferably, the mitochondrial fission molecules include mitochondrial fission-related MiD49, MiD51, Drp1, Fis1, and / or Mff.

[0011] Compared with the prior art, the present invention has the following advantages: 1. The croton icteryl derivative prepared by this invention not only has significantly better activity than the croton icteryl parent nucleus, but its unique feature is that it can exert a therapeutic effect by inhibiting cellular lipid metabolism and repairing mitochondrial function, providing a brand-new candidate compound to overcome the limitations of existing clinical drugs.

[0012] 2. The croton icteryl derivative prepared by this invention can be obtained by functional group modification of croton icteryl and has dual-targeted regulation of "cellular lipid metabolism" and "mitochondrial function", which has application potential in the preparation of drugs for treating metabolic diseases.

[0013] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0014] Figure 1 This is a visualized three-dimensional molecular structure of the croton picrol derivative prepared in Example 1 of this invention.

[0015] Figure 2 This is the hydrogen spectrum of the croton picrol derivative prepared in Example 1 of this invention.

[0016] Figure 3 This is the carbon spectrum of the croton picrol derivative prepared in Example 1 of this invention.

[0017] Figure 4 This describes the screening of the activity concentration of the crotonol derivative in Example 2 of this invention. A represents the drug efficacy assay, specifically the cell viability assay of MH7A cells treated with 50 μM of the drug; B represents the effective drug concentration analysis, specifically the cell viability assay of MH7A cells treated with different drugs; and C represents the half-maximal inhibitory concentration (WMC) analysis of the drug. In the figure, ns indicates no statistically significant difference, * indicates P < 0.05, and *** indicates P < 0.001.

[0018] Figure 5 This describes the inhibitory effect of the croton picrol derivative on cellular lipid metabolism in Example 2 of this invention. In the figure, A represents the inhibitory effect of the drug on CD36 (human leukocyte differentiation antigen 36, which mainly recognizes and transports oxidized low-density lipoprotein and long-chain fatty acids); B represents the inhibitory effect of the drug on CPT1A (carnitine palmitoyltransferase 1A, which mainly catalyzes the process of long-chain fatty acids entering mitochondria); C represents the inhibitory effect of the drug on ACADVL (very long chain acyl-CoA dehydrogenase, which mainly catalyzes the first step of the mitochondrial fatty acid β-oxidation pathway); and D represents the inhibitory effect of the drug on CRAT (carnitine acetyltransferase, which mainly binds acetyl-CoA to carnitine to form acetylcarnitine, thereby helping cells transport fatty acids to mitochondria for oxidative decomposition). In the figure, ns indicates no statistically significant difference, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0019] Figure 6This describes the inhibitory effect of the croton picrol derivative on mitochondrial reactive oxygen species in Example 2 of this invention. (a) is a representative chromatogram of the flow cytometry samples, and (b) is a statistical graph. In the graph, ns indicates no statistical difference, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0020] Figure 7 This describes the repair effect of the croton picrol derivative on mitochondrial membrane potential in Example 2 of this invention. (a) is a representative chromatogram of the flow cytometry samples, and (b) is a statistical graph. In the graph, ns indicates no statistical difference, ** indicates P < 0.01, and *** indicates P < 0.001.

[0021] Figure 8 In Example 2 of this invention, the croton picrol derivative inhibits the expression of mitochondrial fission molecules (MiD49, MiD51, Drp1, Fis1, Mff).

[0022] Figure 9 This is an example of the crotonol derivative of the present invention, which can target and bind to the mitochondrial splitting molecule Drp1. In this example, A shows a schematic diagram of the binding of crotonol (BRU) and its derivative (BRUD) to Drp1; B shows the binding energy analysis of crotonol (BRU) and its derivative (BRUD) to Drp1; and C shows a schematic diagram of the dynamic and equilibrium analysis of the binding of crotonol (BRU) and its derivative (BRUD) to Drp1. Detailed Implementation

[0023] Example 1 The method for preparing the croton picrol derivative of this embodiment is as follows: Under ice-water bath conditions, 1 mL of anhydrous pyridine was added to crotonic acid (BRU) (11.1 mg, 0.02 mmol) and dissolved. The mixture was stirred at room temperature for 5 min, and then 60 μL of propionyl chloride was added dropwise under nitrogen protection. The mixture was stirred at room temperature for 4 h, and the reaction was monitored for completion by thin-layer chromatography. After purification by high-performance liquid chromatography (YMC-Pack ODS-A column, 50% MeCN, 3 mL / min), the crotonic acid derivative (BRUD) was obtained (yield 30.0%, 3.0 mg, tR = 13 min). The structural formula of the crotonic acid derivative is as follows: R is benzoyl group; The structural formula of the crotonic acid is as follows: .

[0024] like Figure 1 The image shows the visualized three-dimensional molecular structure of the croton picrol derivative prepared in this embodiment.

[0025] The identification data is as follows: Amorphous gelatinous substance of broodstock bitter alcohol derivative (BRUD). For example... Figure 2 As shown, the ¹H NMR (600 MHz, MeOD) values ​​are δ 8.12 (d, J = 7.5 Hz, 2H), 7.60 (t, J = 7.5 Hz, 1H), 7.47 (t, J = 7.5 Hz, 2H), 5.63 (s, 1H), 4.82 (s, 1H), 4.74 (d, J = 7.6 Hz, 1H), 4.26 (brs, 1H), 4.20 (s, 1H), 3.78 (d, J = 7.8 Hz, 1H), 2.17 (s, 3H), 1.91 (s, 3H), and 1.84 (s, 3H). Figure 3 As shown, the 13C NMR (151MHz, MeOD) values ​​are δ189.37, 171.65, 167.18, 164.53, 160.85, 146.04, 142.27, 133.65, 130.22, 128.62, 128.52, 114.05, 82.31, 81.23, 75.64, 73.72, 70.88, 65.88, 52.94, 51.12, 50.03, 45.26, 42.91, 41.51, 40.73, 28.81, 27.60, 20.55, 15.51, and 14.55.

[0026] Example 2 This embodiment illustrates the application of the croton sorbitol derivative (BRUD) prepared in Example 1, which is used to prepare a drug that dual-targets the inhibition of abnormal lipid metabolism in cells and the repair of mitochondrial function.

[0027] BRUD inhibits cellular lipid metabolism Drug activity screening MH7A cells were fed at 5 × 10 3 Cells were seeded per well into 96-well culture plates. After cell attachment, the cells were pretreated with serum-free DMEM medium for 12 hours to promote homogeneous cell proliferation and division. Figure 4 As shown in (A), two groups were set up: the Ctrl group (untreated group) and the BRU group (…). Brucea javanica treated with arugula and BRUD group ( (Treatment with Brucea javanica bitter alcohol derivatives), cell viability was assessed using CCK-8 assays after 48 hours, revealing that both BRU and BRUD effectively inhibited cell proliferation; Figure 4 As shown in (B), different drug concentrations were used ( After homogenizing cells were treated with 100 µL of 10% FBS DMEM, cell viability was assessed using a CCK-8 assay after 48 hours. The results showed that even at reduced concentrations, BRUD still inhibited cell proliferation, and the effect was significantly higher than that of its crotonic acid nucleus, demonstrating statistically significant inhibitory effects even at 10 nM. Figure 4 As shown in (C), analysis of the half-inhibitory concentration of the drug revealed that the inhibitory effect of BRUD (242.8 nM) was significantly greater than that of BRU (575.8 nM).

[0028] Inhibit lipid metabolism in MH7A cells MH7A cells were fed at a rate of 1×10 5 Cells were seeded per well in 6-well plates, with three groups: Ctrl (untreated), BRU (treated with 100 nM crotonol for 48 h), and BRUD (treated with 100 nM crotonol derivative for 48 h). After treatment, cells were collected by trypsin digestion, and total RNA was extracted. cDNA was then obtained by reverse transcription using a reverse transcription kit. The mRNA expression of key lipid metabolism molecules was detected by quantitative PCR. Figure 5 As shown in (A), when detecting the expression of CD36 (human leukocyte differentiation antigen 36, which mainly recognizes and transports oxidized low-density lipoprotein and long-chain fatty acids), a key molecule in lipid transport, it was found that both BRUD and BRU could significantly inhibit the expression of CD36 in cells, and BRUD had a better inhibitory effect than BRU; Figure 5 As shown in (B), when detecting CPT1A (carnitine palmitoyltransferase 1A, mainly responsible for catalyzing the entry of long-chain fatty acids into mitochondria) expression, it was found that both BRUD and BRU could significantly inhibit CPT1A expression in cells, and BRUD had a better inhibitory effect than BRU; Figure 5 As shown in (C), when detecting ACADVL (extremely long chain acyl-CoA dehydrogenase, mainly responsible for catalyzing the first step of the mitochondrial fatty acid β-oxidation pathway) expression, it was found that both BRUD and BRU could significantly inhibit CPT1A expression in cells, and BRUD had a better inhibitory effect than BRU; Figure 5 As shown in (D), when detecting CRAT (carnitine acetyltransferase, which is mainly responsible for binding acetyl-CoA to carnitine to form acetylcarnitine, thereby helping cells transport fatty acids to mitochondria for oxidative decomposition) expression, it was found that BRUD and BRU had no significant effect on CRAT expression in cells. These results indicate that BRUD can inhibit cellular lipid metabolism to a certain extent, and its effect is due to the presence of crotonic acid in the nucleus.

[0029] BRUD repairs mitochondrial function in cells BRUD inhibits the production of reactive oxygen species in MH7A mitochondria. MH7A cells were fed at a rate of 1×105 Cells were seeded per well into 6-well culture plates, with three groups: Ctrl (untreated), BRU (treated with 100 nM crotonol for 48 h), and BRUD (treated with 100 nM crotonol derivative for 48 h). After 48 h of treatment, cells were collected by trypsin digestion and stained with diluted MitoSOX fluorescent probe. Mitochondrial reactive oxygen species (MtROS) levels were detected by flow cytometry 30 min later. Figure 6 As shown, analysis of the mean fluorescence intensity (MFI) of cells (Figure a is a representative graph of the flow cytometry samples, and Figure b is its statistical graph) revealed that BRUD and BRU can significantly inhibit the production of reactive oxygen species (ROS) in intracellular mitochondria, as evidenced by a nearly 40% reduction in mean fluorescence intensity. Increased mitochondrial ROS impairs mitochondrial function and affects cellular energy supply; drugs can inhibit mitochondrial ROS, thereby repairing mitochondrial function.

[0030] BRUD repairs MH7A mitochondrial membrane potential MH7A cells were fed at a rate of 1×10 5 Cells were seeded per well in 6-well plates, with three groups: Ctrl (untreated), BRU (treated with 100 nM crotonol for 48 h), and BRUD (treated with 100 nM crotonol derivative for 48 h). After 48 h of treatment, cells were collected after trypsin digestion and stained with JC-10 staining working solution. Mitochondrial membrane potential (ΔΨm) levels were measured by flow cytometry. Under normal conditions, JC-10 selectively aggregates in the mitochondrial matrix to form a reversible red fluorescent polymer; in unhealthy mitochondria, due to a decrease or loss of membrane potential, JC-10 changes from a polymer to a monomeric form in the cytoplasm, producing green fluorescence. Figure 7 As shown, the analysis of mitochondrial membrane potential in drug-treated cells (Figure a is a representative flow cytometry sample, Figure b is its statistical graph) shows that BRUD significantly restores mitochondrial membrane potential, manifested by a decrease in green fluorescence and an increase in red fluorescence in the detected cells. Decreased mitochondrial membrane potential is associated with autophagy, apoptosis, or necrosis. Dysfunction of mitochondrial membrane potential, even subtle abnormalities, can significantly affect intracellular biological activity and cause various diseases. Drugs can restore mitochondrial function by regulating mitochondrial membrane potential.

[0031] BRUD inhibits the expression of mitochondrial fission molecules Mitochondrial dysfunction is also accompanied by changes in mitochondrial division. To study the effects of invented drugs on mitochondrial division molecules, we first established a CIA model. The specific procedure was as follows: Immuno-grade bovine type II collagen (Chondrex, USA) was emulsified with an equal volume of complete Freund's adjuvant. On day 0, 0.2 mL of the emulsion was subcutaneously injected into the tail of the rats in the treatment group. Then, on day 14, a booster injection of 0.1 mL of the emulsion of immuno-grade bovine type II collagen emulsified with complete Freund's adjuvant was administered. After the onset of arthritis, the rats were divided into the following 4 groups (n=5 / 6): healthy control group (saline group), CIA model group, BRU (CIA 0.5 mg / kg) treatment group, and BRUD (CIA 0.5 mg / kg) treatment group. Treatment was administered every 3 days. The rats were sacrificed on day 38, and their joints were collected for analysis. Immunohistochemistry was used to detect the synovial mitochondrial division in the CIA rats. Figure 8 As shown, after CIA induction, the levels of MiD49 (49 kDa mitochondrial kinetic protein), MiD51 (51 kDa mitochondrial kinetic protein), Drp1 (kinetic protein-associated protein 1), Fis1 (mitochondrial outer membrane protein 1, the receptor for Drp1), and Mff (integrated membrane protein of the mitochondrial outer membrane, responsible for regulating mitochondrial division) in the synovial tissue were significantly increased, indicating that mitochondrial division was increased at this time. However, after drug treatment, compared with the CIA group, BRUD treatment significantly reduced the levels of MiD49, MiD51, Drp1, Fis1, and Mff molecules related to mitochondrial division in the synovium, suggesting that BRUD can repair excessive mitochondrial division in cells and inhibit disease progression.

[0032] BRUD can target and bind to the mitochondrial fission molecule Drp1. To further investigate the regulatory effects of compounds on mitochondrial function-related molecules, we performed a 100 ns molecular dynamics simulation using GROMACS 2025 software. The specific analysis process is as follows: (1) Simulation parameter settings: Force field: The protein uses AMBER99SB-ILDN force field parameters, and the ligand topology is constructed based on GAFF2 force field parameters. The topology files of the protein and ligand are merged, and the atom types are ensured to be conflict-free; Periodic boundary conditions: A cubic box is used, and the minimum distance between the complex surface and the box boundary is set to 1.2 nm; Solvent and ions: Solubilization is performed using the TIP3P water model, and ions are added. Counterions were used to neutralize the net charge of the system while maintaining an ion concentration of 0.15 M to simulate a physiological environment. The simulation ensemble and duration were performed under the NPT ensemble (constant particle number, pressure, and temperature), with the temperature and pressure set at 310 K and 1 bar, respectively. The total simulation duration was 100 ns, with an integration step size of 2 fs, and a total of 50,000,000 steps were executed.

[0033] (2) System equilibrium process: First, energy minimization is performed to eliminate unreasonable atomic contact; then, NVT (isothermal and isochoric) and NPT (isothermal and isobaric) ensemble equilibrium simulations are performed in sequence, with the coupling constant set to 0.1 ps. Each simulation is performed for 1,000,000 steps (total 2 ns) to bring the system to the set temperature, density and pressure equilibrium.

[0034] (3) Production simulation and trajectory saving: After balancing, a production simulation lasting 100 ns is performed. The trajectory file is saved every 1000 steps (i.e. every 2 ps) for subsequent analysis.

[0035] (4) Trajectory analysis indicators: The saved trajectories were calculated and analyzed using the GROMACS tool. The main evaluation indicators included: root mean square deviation, which assesses the overall stability of the protein or complex structure; root mean square fluctuation, which analyzes the flexibility of each amino acid residue in the protein; radius of gyration, which characterizes the compactness of the protein structure; solvent-accessible surface area, which reflects the degree of solvent exposure of the protein or ligand; and the number of hydrogen bonds between the protein and ligand, which assesses the stability of the key interactions between them.

[0036] like Figure 9 As shown in (A), both derivatives BRUD and BRU can target and bind to Drp1, a key protein in mitochondrial division, with BRUD exhibiting stronger binding affinity and more stable action. Specifically: Figure 9 As shown in (B), molecular docking indicates that the binding energy between BRUD and Drp1 is -7.5 kcal / mol, lower than that of BRU (-6.7 kcal / mol), suggesting a stronger theoretical affinity. Molecular dynamics simulations further confirm the stability of the binding between both and Drp1. Figure 9 As shown in (C), in the 100 ns simulation, the BRU system reached dynamic equilibrium after approximately 10 ns, experienced a slight fluctuation after 70 ns, and then re-equilibrated. The BRUD system reached equilibrium after approximately 20 ns and performed better throughout the simulation, maintaining high stability in key indicators such as structural fluctuation (RMSF), molecular compactness (Rg), solvent surface area (SASA), and number of hydrogen bonds (HB). Theoretical simulation results indicate that BRUD can potentially regulate the function of Drp1 by stably binding to it, thereby intervening in the mitochondrial division process. This provides an important molecular mechanism basis for its role in repairing mitochondrial function.

[0037] The croton icteryl derivative prepared by this invention not only has significantly better activity than the croton icteryl parent nucleus, but its unique feature is that it can exert a therapeutic effect by inhibiting cellular lipid metabolism and repairing mitochondrial function, providing a novel candidate compound to overcome the limitations of existing clinical drugs.

[0038] The croton picrol derivative prepared by this invention can be obtained by functional group modification of croton picrol, and has dual-targeted regulatory activities on "cellular lipid metabolism" and "mitochondrial function", which has application potential in the preparation of drugs for treating metabolic diseases.

[0039] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a crotonic acid derivative, characterized in that, The method is as follows: Anhydrous pyridine was added to crotonic acid under ice-water bath conditions. After dissolution, the mixture was stirred at room temperature. Then, propionyl chloride was added dropwise to the system under nitrogen protection, and the mixture was stirred at room temperature. The reaction was monitored by thin-layer chromatography until completion. After purification, a crotonic acid derivative was obtained. The structural formula of the crotonic acid derivative is as follows: R stands for benzoyl group.

2. The method for preparing a crotonic acid derivative according to claim 1, characterized in that, The ratio of the amount of crotonic acid, anhydrous pyridine and propionyl chloride used is 0.02 mmol: 1 mL: 60 μL.

3. The application of a crotonic acid derivative prepared by the method described in claim 1 or 2, characterized in that, The croton picrol derivative is used to prepare a drug that dual-targets the inhibition of abnormal lipid metabolism in cells and the repair of mitochondrial function.

4. The application according to claim 3, characterized in that, The croton picrol derivative is used to inhibit the expression of the lipid transport molecule CD36 and the expression of carnitine palmitoyltransferase 1A. The croton picrol derivative is used to inhibit the production of reactive oxygen species in mitochondria, repair mitochondrial membrane potential, inhibit the expression of mitochondrial fission molecules, and / or target and bind to the key mitochondrial fission protein Drp1.

5. The application according to claim 4, characterized in that, The mitochondrial fission molecules include mitochondrial fission-related MiD49, MiD51, Drp1, Fis1, and / or Mff.

Images