Butyric acid-inulin ester, its preparation method, and its application in the preparation of drugs for treating liver cancer.

Abstract

This invention belongs to the field of biomedical technology and discloses a butyric acid-inulin ester, its preparation method, and its application in the preparation of drugs for treating liver cancer. The preparation method of butyric acid-inulin ester includes the following steps: an esterification reaction is carried out by heating butyrylating reagent and inulin in an aprotic solvent under the action of an alkaline catalyst. After the reaction, the butyric acid-inulin ester is obtained through precipitation, purification, and drying. Butyric acid-inulin ester induces phase separation of the Twist1 / YY1 transcriptional complex and forms core-shell-like biomolecule condensates to inhibit the invasion and metastasis of liver cancer cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to butyric acid-inulin ester, its preparation method, and its application in the preparation of drugs for treating liver cancer. Background Technology

[0002] Liver cancer, especially hepatocellular carcinoma (HCC), is one of the most prevalent and deadliest malignant tumors worldwide. Currently, clinical treatments for liver cancer mainly include surgical resection, liver transplantation, local ablation, transarterial chemoembolization (TACE), and molecularly targeted therapy. However, liver cancer has an insidious onset, and most patients are diagnosed at an advanced stage, missing the optimal window for surgery. While systemic chemotherapy and molecularly targeted drugs (such as sorafenib and lenvatinib) can prolong patient survival, they generally suffer from low response rates, significant side effects, and a high risk of drug resistance. Therefore, developing novel, highly effective, and low-toxicity anti-liver cancer drugs remains a hot research topic and an urgent clinical need.

[0003] Short-chain fatty acids (SCFAs), especially butyrate, are important byproducts of dietary fiber metabolism by gut microbiota and have recently been found to have great potential in the field of anti-tumor therapy. Studies have shown that butyrate has histone deacetylase inhibitory (HDACi) activity, which can induce tumor cell cycle arrest, promote apoptosis, and inhibit cell invasion and metastasis. However, as a small molecule, butyrate has significant drawbacks in vivo, such as an extremely short half-life, rapid metabolism, and the requirement of high concentrations to be effective, which severely limits its clinical application value.

[0004] The present invention aims to design and synthesize a novel butyric acid-inulin ester to overcome the inherent defects of butyric acid, and to explore its application and mechanism in the treatment of liver cancer, providing new strategies and candidate compounds for the development of novel liver cancer therapeutic drugs. Summary of the Invention

[0005] The purpose of this invention is to address the technical deficiencies in the prior art by providing a butyric acid-inulin ester and its preparation method.

[0006] Another object of the present invention is to provide the use of the said butyric acid-inulin ester in the preparation of a medicament for treating liver cancer.

[0007] Another object of the present invention is to provide a medicament for treating liver cancer, wherein the active substance comprises butyrate-inulin ester.

[0008] The technical solution adopted to achieve the purpose of this invention is:

[0009] Butyric acid-inulin ester, whose general structural formula is shown in Formula I:

[0010] ;

[0011] In Equation I, R is H or -COCH2CH2CH3, and the average number of -COCH2CH2CH3 in each sugar unit is n, where 0.5≤n≤3 and 40≤m≤60.

[0012] In the above technical solution, the preparation method of butyric acid-inulin ester includes the following steps: esterification reaction of butyrylating reagent and inulin in an aprotic solvent under the action of alkaline catalyst, and after the reaction is completed, the butyric acid-inulin ester is obtained by precipitation, purification and drying steps.

[0013] In the above technical solution, the butyrylating agent is butyryl chloride or butyric anhydride.

[0014] In the above technical solution, the alkaline catalyst is pyridine or 4-dimethylaminopyridine (DMAP).

[0015] In the above technical solution, the aprotic solvent is dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF).

[0016] In the above technical solution, the molar ratio of inulin to butyrylating agent is 1:(2~10).

[0017] In the above technical solution, the heating temperature is 50~80℃.

[0018] Another aspect of the present invention includes the use of the butyric acid-inulin ester in the preparation of a drug for treating liver cancer.

[0019] Another aspect of the invention includes a medicament for treating liver cancer, comprising the butyrate-inulin ester and pharmaceutically acceptable excipients.

[0020] In the above technical solution, the pharmaceutically acceptable excipients include one or more of the following: fillers, diluents, absorbents, wetting agents, binders, disintegrants, lubricants, flavoring agents, or transdermal absorption enhancers.

[0021] In the above technical solution, the dosage form of the drug is drops, oral liquid, tablets, capsules, granules, powders, films, gels, powders, emulsions, pills, suppositories, aerosols, sprays, powder mists, patches, plasters, solutions, ointments, or creams.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] 1. Butyric acid-inulin ester releases butyric acid in vivo to exert an anti-liver cancer effect. Compared with butyric acid, the butyric acid-inulin ester has a longer in vivo half-life and / or lower systemic toxicity.

[0024] 2. Butyrate-inulin exerts its effects through one or more mechanisms, including inhibiting the proliferation, invasion, and metastasis of hepatocellular carcinoma cells. Specifically, it inhibits the invasion and metastasis of hepatocellular carcinoma cells by suppressing the function of the Twist1-YY1 transcriptional complex. In particular, butyrate-inulin induces phase separation of the Twist1 / YY1 transcriptional complex, leading to the formation of core-shell-like biomolecular condensates. Attached Figure Description

[0025] Figure 1 The image shows the infrared spectrum of butyric acid-inulin ester.

[0026] Figure 2 This is the NMR spectrum of butyric acid-inulin ester.

[0027] Figure 3 This is a mass spectrum of butyrylation modification in hepatocellular carcinoma cells MHCC-97H.

[0028] Figure 4 The number of Twist1-YY1 "core-shell" phase-separated droplets in liver cancer cells MHCC-97H.

[0029] Figure 5 This study was conducted to investigate the clonogenic formation of liver cancer cells MHCC-97H.

[0030] Figure 6 Gelatin footprinting assay for liver cancer cells MHCC-97H.

[0031] Figure 7 The curves showing the changes in tumor fluorescence intensity in mice with orthotopic liver xenografts are shown.

[0032] Figure 8 Survival curves for mice with orthotopic liver xenografts.

[0033] Figure 9 Immunohistochemical staining and pathological scoring of liver tissue from mice with orthotopic liver xenografts. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0035] Example 1

[0036] Artificially synthesized butyrate-inulin ester:

[0037] Reaction principle: Under alkaline conditions, butyryl chloride acts as the acylation reagent, reacting with the hydroxyl groups on inulin molecules to undergo esterification, producing butyric acid-inulin ester and releasing hydrogen chloride. Pyridine is used as a solvent and also as a base to neutralize the HCl produced in the reaction, promoting the forward reaction.

[0038] The preparation process of butyric acid-inulin ester includes the following steps:

[0039] Step 1, Setup of the reaction apparatus and preparation of materials:

[0040] Take a dry round-bottom flask and install a reflux condenser, a constant-pressure dropping funnel, and a thermometer. Connect a drying tube containing anhydrous calcium chloride to the upper end of the condenser to prevent moisture from the air from entering.

[0041] Weigh 2.0g of inulin into a round-bottom flask, add 20mL of anhydrous pyridine to the flask, and add 5mL of DMSO at the same time to help the inulin dissolve better;

[0042] Turn on stirring and heating, and stir at 65°C until the inulin is completely dispersed and forms a uniform suspension. Then cool to room temperature and then cool the system to 0-5°C with an ice bath.

[0043] Step 2, Dropping reaction and reflux:

[0044] Measure 30 mL of butyryl chloride, add butyryl chloride and pyridine to a dropping funnel and mix. Under ice-water bath cooling and vigorous stirring, slowly add the butyryl chloride / pyridine mixture dropwise to the inulin solution. After the addition is complete, remove the ice-water bath and allow the reaction mixture to slowly return to room temperature. Then heat to 60°C and reflux for 24 hours.

[0045] Step 3, Product precipitation and crude separation:

[0046] After the reaction is complete, cool the reaction solution to room temperature. Slowly pour the reaction solution into 200 mL of ice-cold ethanol / acetone mixture while stirring vigorously. Butyrate-inulin ester will precipitate as a white or pale yellow fibrous or flocculent precipitate. Let stand for a moment to allow complete precipitation. Then filter and collect the solid product.

[0047] Step 4, Product purification:

[0048] Dissolve the crude product obtained in the previous step in 2 mL of water. Transfer the solution into a pretreated 1000 Da dialysis bag. Place the dialysis bag in a large volume of deionized water for dialysis, changing the water every 8 hours, for at least 72 hours, until the dialysate shows no chloride ions (Cl) when tested with silver nitrate solution. - Continue until the final solution in the dialysis bag is freeze-dried to obtain a white, fluffy solid of butyrate-inulin ester.

[0049] like Figure 1 The infrared spectrum shows that the phenolic hydroxyl groups of the polysaccharide are at 3328 cm⁻¹. -1 There is a characteristic peak at 2851 cm⁻¹, with the butyryl group at that point. -1There is a characteristic peak at 1735 cm⁻¹, with the carbonyl group at this point. -1 There is a characteristic peak at 1244 cm⁻¹, indicating that the ether bond is at this point. -1 and 1271cm -1 There are two characteristic peaks, with the methyl group at 1384 cm⁻¹. -1 There is a characteristic peak at this point. The appearance of a strong carbonyl peak confirms the completion of the esterification reaction.

[0050] like Figure 2 The 1H NMR spectrum showed that the triplet at chemical shift δ0.9 (t) represents the three protons in the methyl group at the butyryl end; the multiplet at chemical shift δ1.6 (m) represents the protons in the methylene group far from the acyl group in the -CH2-CH2-C=O of the butyryl group; the triplet at chemical shift δ2.3 (t) represents the protons in the methylene group of the -CH2-C=O of the butyryl group; and the complex multiplet between chemical shifts δ4 and δ5 represents the hydrogen on the inulin backbone and the protons of the esterified methylene group. The presence of novel aliphatic chain signals at δ0.9 (t), 1.6 (m), and 2.3 (t) in the 1H NMR spectrum confirms the presence of the butyryl group; the continued presence of proton signals on the inulin backbone indicates the completion of the esterification reaction. These results demonstrate the successful synthesis of butyric acid-inulin ester.

[0051] Example 2

[0052] In this embodiment, butyrate-inulin ester was used to treat liver cancer cells MHCC-97H to identify butyrylation sites of transcription factor Twist1.

[0053] MHCC-97H hepatocellular carcinoma cells were treated with 5 mM butyrate-inulin for 48 hours to induce butyrylation modification of proteins. Total protein was then extracted, and Twist1 protein and its modified forms were enriched from the whole protein lysate using immunoprecipitation with a Twist1 antibody. The enriched Twist1 protein was denatured, reduced, alkylated, and digested with trypsin in solution to generate a peptide mixture. The peptides were separated by liquid chromatography and sequenced by high-resolution mass spectrometry (MS / MS). Butyrylation modification (+85.05276 Da) caused a peptide mass shift; the modification site was precisely identified as K73 by analyzing the mass fragmentation pattern (b / y ion series). Figure 3 The mass spectrum showed a triple-charged precursor ion [M+3H]. 3+ -H,[M+3H] 3+ [M+3H] 3+ The m / z of +H is 903.55. The molecular weight of the parent ion = 3 × 903.55 − 3 × 1.0078 ≈ 2707.63 Da. (Note: The MH is not explicitly stated.) +The value was 2708.30326 Da, which is in high agreement with the calculated value (within a matching tolerance of 0.5 Da). The amino acid sequence fragment (positions 50-73) of the Twist1 protein is shown in SEQ ID NO: 1, specifically GGGAGPGGAAGGGVGGGDEPGSPAQGKRGK, and its unmodified molecular weight was calculated to be approximately 2419.13 Da. There is a difference of approximately 288.5 Da between the actual measured value (2707.63 Da) and the unmodified molecular weight, while butyrylation (Bu) modification increases the molecular weight by 70.04 Da. The multiples of 288.5 and 70.04 are similar, and the overall mass increase is consistent with butyrylation.

[0054] Example 3

[0055] Treatment of MHCC-97H and Hep 3B cells with butyrate-inulin esters revealed phase separation of the Twist1-YY1 transcription complex, transforming it into nucleus-shell-like phase-separated droplets.

[0056] MHCC-97H and Hep 3B cells with stable growth and Twist1-YY1 fluorescent plasmid were cultured at 5 × 10⁻⁶ cells / year. 4 Cells / dish were seeded at a density of 35 mm confocal culture dishes.

[0057] Experimental group: The culture medium was replaced with fresh medium containing 1 mM and 5 mM butyrate-inulin ester.

[0058] Control group: The culture medium was replaced with a fresh medium containing an equal volume of DMSO solvent.

[0059] After culturing the cells for another 48 hours, images were acquired using a confocal microscope and subsequently analyzed using ImageJ software. The percentage of cells in each group that exhibited nucleus-shell-like phase separation droplets was counted out of the total transfected cells.

[0060] Data are expressed as mean ± standard deviation. Statistical differences between the experimental and control groups were analyzed using a t-test. Results showed that, compared to the control group, the experimental group with added butyrate-inulin ester produced "core-shell" phase-separated droplets within the cells. Figure 4 This shows that butyrate-inulin can inhibit the Twist1-YY1 transcription complex, thereby inhibiting the invasion and metastasis of liver cancer cells.

[0061] Example 4

[0062] Clonalization experiments were conducted on hepatocellular carcinoma cells MHCC-97H and Hep 3B treated with butyrate-inulin ester.

[0063] Weigh 10 mg of butyrate-inulin ester powder, dissolve it in DMSO to prepare a 50 mg / mL stock solution, and filter the stock solution through a 0.22 μm sterile filter membrane for sterilization. Take MHCC-97H and Hep 3B cells in good growth condition, digest them with trypsin and resuspend them into a single-cell suspension. After cell counting, dilute to the appropriate density with complete culture medium.

[0064] Gently seed the cell suspension into 6cm cell culture dishes, typically 800 cells per well, and gently agitate to ensure even distribution. Incubate at 37°C with 5% CO2. After 24 hours of culture and cell attachment, aspirate the old culture medium.

[0065] Experimental group: Fresh complete culture medium containing different concentrations (1 mM and 5 mM) of butyric acid-inulin ester was added.

[0066] Control group: Added complete culture medium containing an equal volume of DMSO.

[0067] Each concentration group was set up in triplicate, and the cells were returned to the incubator for further culture. The culture medium containing the corresponding drug was replaced every 3 days to maintain drug concentration and nutrient supply. Culture was continued for approximately 10 days until clearly visible cell clones (usually >50 clones) appeared in the control group culture dishes. The culture medium was carefully aspirated, and the cells were gently washed twice with pre-cooled PBS. The cells were fixed with 4% paraformaldehyde or methanol for 20 minutes. The fixative was aspirated, and the cells were washed once with PBS. The cells were stained with 0.1% crystal violet for 30 minutes. The staining solution was carefully aspirated, and excess stain was washed away very slowly with running water and allowed to air dry. The culture dishes were photographed. Cell counts were then performed using ImageJ analysis software. The inhibitory effect of the drug on cell proliferation was evaluated. Data are expressed as mean ± standard deviation and statistically analyzed using t-tests. Results are as follows: Figure 5 As shown, butyrate-inulin significantly inhibited the proliferation of MHCC-97H and Hep 3B cells.

[0068] Example 5

[0069] Gelatin degradation experiments were conducted on hepatocellular carcinoma cells MHCC-97H and Hep 3B treated with butyrate-inulin ester.

[0070] Preparation of gelatin-coated culture plates: Add 100 μL of diluted FITC-gelatin solution (2 mg / mL) to a 24-well plate containing cell spreaders, ensuring complete coverage of the bottom. Incubate the plate at 37°C for 1 hour to allow the gelatin to fully solidify. Discard excess gelatin solution and wash the plate three times with PBS.

[0071] Cell seeding and drug treatment: MHCC-97H and Hep 3B cells were digested and resuspended, and the cell density was adjusted to 6 × 10⁶ cells / year using serum-free medium. 5cells / mL. Gently seed 500 μL of cell suspension into coated 24-well plates and incubate the plates at 37°C with 5% CO2 for 6 hours to allow the cells to adhere fully. Carefully aspirate the supernatant and add complete culture medium containing different concentrations of butyrate-inulin esters.

[0072] Experimental group: 1 mM and 5 mM butyrate-inulin esters were added respectively;

[0073] Control group: Complete culture medium containing only an equal volume of DMSO;

[0074] The cells were then returned to the incubator and cultured for another 24 hours.

[0075] Fixation, staining, and mounting: Carefully aspirate the culture medium, add 4% paraformaldehyde, and fix for 20 minutes at room temperature in the dark. Gently wash three times with PBS, 5 minutes each time. Add TRITC-phalloidin and incubate at room temperature in the dark for 20 minutes. Wash three times with PBS in the dark, 5 minutes each time. Add 5 μL of DAPI (1 μg / mL) to a glass slide, invert the cell slide onto the slide, and mount. Image using a confocal microscope. Analyze using ImageJ software.

[0076] Convert the FITC images to 8-bit grayscale images and adjust the threshold to make the degraded black areas clearly visible. Use the "Analyze Particles" function in the software to calculate the total area of ​​the black degraded area in each field of view. Each group was repeated in triplicate. Data are expressed as mean ± standard deviation. Results are shown below. Figure 6 As shown, gelatin degradation in the experimental group was significantly inhibited, indicating that butyrate-inulin ester inhibited the migration and invasion abilities of MHCC-97H and Hep 3B cells.

[0077] Example 6

[0078] Evaluation of the therapeutic efficacy of butyrate-inulin ester treatment in BAlb / c mice with orthotopic liver tumors.

[0079] Establishing a mouse model of orthotopic liver xenograft:

[0080] Harvest well-grown H22-luc cells and resuspend them in physiological saline. Adjust the cell density to 1×10⁻⁶. 7 Cells / mL, kept on ice for later use. Mice were anesthetized with isoflurane inhalation and fixed on a surgical board. Abdominal hair was removed with depilatory cream, and the skin was disinfected (iodine + 75% alcohol). A longitudinal incision of about 1 cm was made below the left costal margin, and the muscles and peritoneum were dissected layer by layer to expose the liver. 50 μL of H22 cell suspension (containing 5 × 10⁶ cells / mL) was aspirated from the ice using an insulin syringe or microsyringe. 5(Cells). Gently grasp the left lobe of the liver and insert the needle obliquely into the liver capsule about 2-3 mm, slowly injecting cells. After removing the needle, gently press the puncture site with a sterile cotton swab for about 30 seconds to stop bleeding. Return the liver to the abdominal cavity, and suture the peritoneum and muscle layer continuously with absorbable sutures. Suture the skin with surgical clips or non-absorbable sutures. Place the mouse on a 37°C heating pad to awaken it, and return it to its cage after it is fully awake. Add antibiotics to the drinking water for 3 consecutive days after surgery to prevent infection. After the mice have recovered from the surgery, they are randomly divided into the following 3 groups (n=6 / group):

[0081] Control group: Inoculated with tumor cells and given an equal volume of solvent (such as PBS);

[0082] Low-dose treatment group of butyrate-inulin ester (100 mg / kg / day);

[0083] High-dose butyrate-inulin treatment group (200 mg / kg / day).

[0084] Administration route: Intraperitoneal injection, starting from day 3 post-surgery, once daily at a fixed time for 4 consecutive weeks. Subsequently, tumor growth in mice was recorded using small animal imaging. D-fluorescein potassium solution was administered intraperitoneally at a dose of 150 mg / kg, with imaging performed every 5 days. (See attached image.) Figure 7 The time of death of mice in each group was recorded, survival curves were plotted, and the differences between groups were compared using the Log-rank test. (See attached table.) Figure 8 On day 60 of tumor bearing, the patient was euthanized via cervical dislocation. The liver was dissected, fixed in formalin, and used for paraffin sectioning. Immunohistochemical staining was performed to detect the expression of Ki-67, E-cadherin, and vimentin. (See attached image) Figure 9 .

[0085] Depend on Figures 7-9 As can be seen, compared with the control group, the tumor volume of mice treated with low-dose butyrate-inulin and the tumor growth rate of mice treated with high-dose butyrate-inulin were significantly reduced, and the survival time of mice was significantly prolonged. Meanwhile, histopathological analysis showed that the expression levels of protein markers associated with malignant tumor progression, including Ki67 and Vimentin, were significantly decreased, while the expression of the epithelial marker E-cadherin was significantly increased. Additionally, the butyrylation modification level at the K73 site of Twist1 was also significantly increased.

[0086] 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 principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of butyric acid-inulin ester in the preparation of a medicament for treating liver cancer, characterized in that, The structural general formula of butyric acid-inulin ester is shown as formula I: I In formula I, R is H or -COCH2CH2CH3, and the number of -COCH2CH2CH3 in each sugar unit is n, 0.5<=n<=3, and 40<=m<=60.

2. Use according to claim 1, wherein The preparation method of butyric acid-inulin ester comprises the following steps: heating butyrylating agent and inulin in aprotic solvent under the action of an alkaline catalyst to perform esterification reaction, and after the reaction is completed, the butyric acid-inulin ester is obtained through precipitation, purification and drying steps.

3. Use according to claim 2, wherein the compound is ###0002### The butyrylating agent is butyryl chloride or butyric anhydride.

4. The use according to claim 2, wherein the compound is ###0002### The alkaline catalyst is pyridine or 4-dimethylaminopyridine.

5. The use according to claim 2, wherein the compound is ###0002### The aprotic solvent is dimethyl sulfoxide or N,N-dimethylformamide.

6. The use according to claim 2, wherein The molar ratio of inulin to butyrylating agent is 1:(2~10).

7. The use according to claim 2, wherein the compound is ###0002### The heating temperature is 50~80℃.

8. A medicament for treating liver cancer, characterized by comprising the compound according to claim 1. The butyric acid-inulin ester and pharmaceutically acceptable adjuvants; The structural general formula of butyric acid-inulin ester is shown as formula I: I In formula I, R is H or -COCH2CH2CH3, and the number of -COCH2CH2CH3 in each sugar unit is n, 0.5<=n<=3, and 40<=m<=60.

9. The medicament for treating liver cancer according to Claim 8, wherein The pharmaceutically acceptable adjuvants include one or more of fillers, diluents, absorbents, wetting agents, binders, disintegrants, lubricants, flavoring agents or transdermal absorption promoters; The dosage form of the drug is drops, oral liquid, tablets, capsules, granules, powder, film, gel, powder, emulsion, drop pills, suppositories, aerosol, spray, powder spray, patches, pastes, solutions, ointments or creams.

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