A galloyl derivative of resimote, its synthesis method and application

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to a galloyl derivative of resimodil, its synthesis method, and its applications. The derivative is a substance in which resimodil and gallic acid are linked by an ester bond, as shown in Formula 1 below. This invention enhances the affinity of resimodil for antigens by galloyl derivatization, thereby allowing it to be easily mixed with antigens to form nanoparticles. This provides new ideas and opportunities for the development of antigen and adjuvant co-delivery nanoparticles to meet the urgent clinical need for cancer vaccines.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a galloyl derivative of retimote, its synthesis method, and its application. Background Technology

[0002] In today's medical field, cancer poses an extremely serious threat to human health, with its continuously rising incidence and mortality rates becoming a global focus. Tumor vaccines deliver tumor antigens into the patient's body in various forms, exerting their anti-tumor effect by activating an immune response. As a significant breakthrough in cancer immunotherapy, tumor vaccines have demonstrated remarkable potential, with several therapeutic tumor vaccines already on the market abroad, such as Provenge for prostate cancer, Melacine for malignant melanoma, and Oncophage for renal cell carcinoma. Tumor cell lysates (TCLs) provide a wide variety of tumor antigens, making them a comprehensive source capable of inducing a robust and durable anti-tumor immune response. However, traditional TCL-based vaccines have yielded unsatisfactory results in early clinical trials due to low immunogenicity, instability in vivo, and the inability to be co-delivered with adjuvants.

[0003] Toll-like receptor 7 / 8 (TLR7 / 8) agonists can activate antigen-presenting cells and enhance antigen-specific T cell responses by activating the innate immune system. Combining tumor cell lysates with Toll-like receptor 7 / 8 agonists holds significant potential, but simple co-administration methods have limited efficacy. Existing microparticle-based delivery strategies often involve demanding synthetic conditions that may inhibit the bioactivity of conjugates and result in low antigen / adjuvant loading efficiency. Therefore, finding and developing alternative strategies that are simple to design, highly biocompatible, and have high antigen / adjuvant loading efficiency remains a challenge for next-generation drug delivery technologies.

[0004] Polyphenols are widely found in nature and are secondary metabolites of plants, possessing a variety of biological activities, including antioxidant, anti-inflammatory, antibacterial, antiviral, antitumor, and cardiovascular disease prevention. A key characteristic is the presence of multiple galloyl groups, which allows them to form strong interactions (both covalent and non-covalent) with various materials, including biomolecules. This diverse binding capacity makes them valuable in numerous biomedical applications. Furthermore, natural polyphenols, due to their potent anti-inflammatory and antioxidant properties, as well as their ability to regulate key molecular targets and signaling pathways involved in cancer progression, also have potential applications in cancer treatment. However, research on the preparation of antigen-binding nanoparticles through galloylation of retimote has not yet been reported. Summary of the Invention

[0005] In order to overcome the limitations and deficiencies of existing cancer vaccines, the present invention aims to provide a resimot galloyl derivative, its synthesis method and application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A galloyl derivative of resimilarto, wherein resimilarto and gallic acid are linked by an ester bond, and the structure is shown in Formula 1 below.

[0008]

[0009] Gallic acid can be replaced by phenolic acid compounds including caffeic acid, p-coumaric acid, ferulic acid, sinapic acid, rosmarinic acid, 4-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid and 3,4-dihydroxyphenylacetic acid.

[0010] A method for synthesizing the aforementioned resimote galloyl derivative, comprising the following reaction formula:

[0011]

[0012] 3,4,5-tris(benzyloxy)benzoic acid was reacted with retsimot under the catalysis of 4-dimethylaminopyridine (DMAP) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and the intermediate compound was isolated and purified. The intermediate compound was deprotected by palladium on carbon hydrogenation catalysis, and the retsimot galloyl derivative was obtained by isolation and purification.

[0013] A resimodil galloyl derivative antigen nanocomposite, wherein the nanocomposite comprises the resimodil galloyl derivative, antigen, and PEG; the weight ratio of the resimodil galloyl derivative to the antigen is (1:2) to (1:10); the weight ratio of the resimodil galloyl derivative to PEG is (1:1) to (1:8).

[0014] The antigen is ovalbumin, carcinoembryonic antigen, alpha-fetoprotein, P1AB antigen, gp95 / p97 antigen, breast cancer cell lysate protein, melanoma cell lysate protein, colorectal cancer cell lysate protein, prostate cancer cell lysate protein, liver cancer cell lysate protein, lung cancer cell lysate protein, gastric cancer cell lysate protein, esophageal cancer cell lysate protein, pancreatic cancer cell lysate protein, ovarian cancer cell lysate protein, bladder cancer cell lysate protein, or nasopharyngeal cancer cell lysate protein; the PEG has a molecular weight of 2k-35k.

[0015] A method for preparing the resimod galloyl derivative antigen nanocomposite, comprising dissolving the resimod galloyl derivative in an organic solvent as the organic phase according to the specified ratio, and dissolving the antigen and PEG in deionized water as the aqueous phase; the organic phase and the aqueous phase are rapidly stirred and mixed to spontaneously form a uniform nanocomposite with the resimod galloyl derivative and the antigen; wherein the organic solvent is one or a mixture of several of DMSO, acetone, ethanol, methanol, acetonitrile, or tetrahydrofuran.

[0016] The aforementioned rethimod galloyl derivative antigen nanocomposite was prepared under mild conditions using a stirring and mixing method. This process is simple, easily scaled up for production, and retains the bioactivity of the antigen. It requires no antigen modification and is not limited to specific antigens; it is also suitable for delivering tumor cell lines (TCLs) containing multiple antigens of uncertain properties. Synthesis can be completed within one day, avoiding the time-consuming and expensive antigen screening process typical of neoantigen-based cancer vaccines. Simultaneously, the resulting composite enables the simultaneous release of antigen and adjuvant within dendritic cells (DCs), enhancing cross-presentation of tumor antigens, promoting the production of pro-inflammatory cytokines, activating dendritic cells, and thereby inducing a strong anti-tumor immune response. It exhibits good safety and has significant potential for clinical application.

[0017] The use of one of the derivatives, or the nanocomposite, in the preparation of a drug delivery system.

[0018] The use of one of the derivatives, or the nanocomposite, in the preparation of antitumor drugs.

[0019] The drug can be administered by injection, oral administration, or topical administration.

[0020] The beneficial effects of this invention are:

[0021] This invention enhances the affinity of rethimod for antigens through galloyl derivatization, allowing for simple mixing with antigens to form nanoparticles. No antigen modification is required, making it suitable not only for specific antigens but also for delivering tumor-specific cells (TCLs) containing multiple antigens of uncertain properties. This provides new ideas and opportunities for the development of antigen-adjuvant co-delivery nanoparticles to meet the urgent clinical need for cancer vaccines. The fabricated nanoparticles enable the simultaneous release of antigens and adjuvants within dendritic cells (DCs), enhancing the cross-presentation of tumor antigens, activating dendritic cells, and thereby triggering a robust anti-tumor immune response. Attached Figure Description

[0022] Figure 1 The high-resolution mass spectrum of the resimote galloyl derivative (R848-GA) of phenolic acid moiety 3,4,5-trihydroxybenzoic acid in Example 1 of this invention is shown below. 1 H-NMR spectrum.

[0023] Figure 2 The images show the particle size distribution and morphology of the resimod galloyl derivative ovalbumin nanocomposite, the resimod galloyl derivative colon cancer cell (MC38) lysate protein nanocomposite, the resimod galloyl derivative melanoma cell (B16) lysate protein nanocomposite, and the resimod galloyl derivative breast cancer cell (4T1) lysate protein nanocomposite of Example 2 of the present invention.

[0024] Figure 3 The stability diagrams are for the resimod galloyl derivative colon cancer cell (MC38) lysate protein nanocomplex, the resimod galloyl derivative melanoma cell (B16) lysate protein nanocomplex, and the resimod galloyl derivative breast cancer cell (4T1) lysate protein nanocomplex of Example 3 of the present invention.

[0025] Figure 4 This is an in vivo efficacy experiment of the rethimod galloyl derivative colon cancer cell (MC38) lysate protein nanocomposite of Example 4 of the present invention, showing the tumor volume-time growth curve of MC38 tumor-bearing mice.

[0026] Figure 5 This is a graph showing the in vivo efficacy experiment of the rethimod galloyl derivative colon cancer cell (MC38) lysate protein nanocomposite of Example 4 of the present invention, and the change in body weight over time of MC38 tumor-bearing mice.

[0027] Figure 6 This is a graph showing the changes in blood parameters of MC38 tumor-bearing mice in the in vivo efficacy experiment of the rethimod galloyl derivative colon cancer cell (MC38) lysate protein nanocomposite of Example 4 of the present invention.

[0028] Figure 7 This is an in vivo efficacy experiment of the rethimod galloyl derivative colon cancer cell (MC38) lysate protein nanocomposite of Example 5 of the present invention. The expression of the protein in immune cells of MC38 tumor-bearing mice is shown.

[0029] Figure 8 This is a graph showing the changes in serum cytokines in MC38 tumor-bearing mice during the in vivo efficacy experiment of the rethimod galloyl derivative colon cancer cell (MC38) lysate protein nanocomplex of Example 5 of the present invention. Detailed Implementation

[0030] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. These embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the content described herein are within the scope of protection of the present invention.

[0031] This invention synthesizes a galloyl derivative of resimod with high protein affinity, and then prepares an antigen-resimod galloyl derivative nanocomposite with simple design and good stability by simply mixing the galloyl derivative with the antigen, thereby achieving co-delivery of antigen and adjuvant and inducing a strong anti-tumor immune response.

[0032] Example 1

[0033] Synthesis of Resimotol Galloyl Derivative (R848-GA)

[0034] Accurately weigh 3,4,5-tris(benzyloxy)benzoic acid (210.1 mg, 0.477 mmol), EDC (74.0 mg, 0.477 mmol), and DMAP (58.3 mg, 0.477 mmol) into a 100 mL round-bottom flask, dissolve in 5 mL of dichloromethane, and stir in an ice bath for 2 hours. Accurately weigh resimolec (100 mg, 0.318 mmol), dissolve in 5 mL of dichloromethane, and slowly add dropwise to the above reaction solution with stirring in an ice bath. Transfer to room temperature and continue the reaction for 24 hours. Monitor the reaction progress by thin-layer chromatography. After the reaction is complete, evaporate the reaction solution to dryness, dissolve the sample in acetonitrile, and separate and purify by preparative liquid chromatography (using a YMC-Pack ODS-A column, acetonitrile:water = 85:15 mobile phase, flow rate 5 mL / min, collecting the fraction at 20 minutes) to obtain the intermediate product (white powder, yield 70%). The intermediate product (100 mg) was added to a 100 mL round-bottom flask and dissolved in 15 mL of anhydrous ethanol. Then, 10% Pd / C (10 mg, 10% w / w) was added, and the mixture was reacted at room temperature for 8 h in the presence of H2. The reaction was monitored by thin-layer chromatography. After the reaction was complete, Pd / C was removed by microfiltration. The filtrate was purified by preparative liquid chromatography (using a YMC-Pack ODS-A column, acetonitrile:water = 80:20 mobile phase, flow rate 5 mL / min, collecting the fraction at the 10th minute) to obtain the target product R848-GA (yellow solid, 90% yield). High-resolution mass spectrometry and... 1 The structure of the target compound was confirmed by H-NMR spectroscopy, and the results are as follows: Figure 1As shown, the NMR spectral analysis results are as follows: δ 10.20 (s, 1H, Ar-OH), 9.19 (s, 2H, Ar-OH), 8.57 (d, J = 8.3 Hz, 1H, H-4), 8.05-7.91 (d, J = 8.3 Hz, 1H, H-1), 7.65 (t, J = 7.6 Hz, 1H, H-3), 7.58 (t, J = 7.7 Hz, 1H, H-2), 7.06 (s, 2H, H-5 and 6), 4.95 (s, 2H, 8-CH2), 4.77 (s, 2H, 7-CH2), 3.52 (q, J = 6.9 Hz, 2H, -O CH2 CH3),1.22(s,6H,-C(CH3)2),1.13(t,J=7.0Hz,3H,-OCH2 CH3 ).

[0035] Example 2

[0036] Preparation of resimilar galloyl derivative antigen nanocomposites

[0037] 1 mL of a solution of different antigens (ovalbumin (OVA), colon cancer cell (MC38) lysate protein, melanoma cell (B16) lysate protein, and breast cancer cell (4T1) lysate protein) (concentration 2 mg / mL, solvent: water) and 80 μL of PEG20000 solution (concentration 20 mg / mL, solvent: water) were accurately measured and mixed in a 2 mL vial and stirred. Then, 60 μL of R848-GA (concentration 10 mg / mL, solvent: DMSO) prepared in the above example was added dropwise to the vial for assembly, and the mixture was stirred at room temperature for 20 minutes to obtain stable nanoparticles (R848-GA@OVA, R848-GA@MC38-L, R848-GA@B16-L, R848-GA@4T1-L) (see [link to documentation]). Figure 2 The particle sizes and particle size distributions obtained after measurement are shown in Table 1.

[0038] Table 1. Particle size and PDI of different resimilar antigen nanocomposites

[0039]

[0040] Depend on Figure 2 It is evident that resimot galloyl derivatives can effectively bind to antigens to form nanocomposites with uniform particle size.

[0041] Example 3

[0042] Stability test of resimot galloyl derivative antigen nanocomposite

[0043] 1 mL of the different R848-GA@MC38-L, R848-GA@B16-L, and R848-GA@4T1-L samples prepared in the above examples were added to 9 mL of phosphate-buffered saline (PBS, pH 7.4) containing 10% fetal bovine serum. The samples were placed in a water bath at 37°C, and samples were collected at specified times and analyzed using a Malvern particle size analyzer to evaluate stability.

[0044] The results are as follows Figure 3 As shown, R848-GA@MC38-L, R848-GA@B16-L, and R848-GA@4T1-L exhibited excellent physical stability when incubated in phosphate-buffered saline (PBS, 10% fetal bovine serum, pH 7.4) at 37°C. This indicates that R848-GA@TCLs can overcome antigen instability and prevent its degradation during delivery.

[0045] Example 4

[0046] In vivo pharmacodynamic studies of rethimod galloyl derivative antigen nanocomplex

[0047] Take 100 μL of MC38 cells (2 × 10⁻⁶) 6 Injected subcutaneously above the right buttock of mice to establish an MC38 mouse ectopic tumor animal model. When the tumor volume of the mouse reaches 100 mm, the tumor is allowed to grow. 3 Mice were then randomly divided into 6 groups of 5 animals each: PBS group, R848 group, R848+MC38-L group, R848-GA@OVA group, R848-GA+MC38-L group, and R848-GA@MC38-L group. The equivalent dose of rasimort was 2 mg / kg for all groups (except the PBS group). Administration was subcutaneous injection, administered every three days for a total of three doses. Body weight and tumor size were measured and recorded every two days after administration. Serum was collected after treatment to detect the levels of white blood cells, lymphocytes, monocytes, red blood cells, and platelets.

[0048] The results are as follows Figure 4 As shown, the R848-GA@MC38-L group exhibited a significantly enhanced tumor suppression effect, with an average tumor size only 7% of that in the PBS group. Furthermore, the effects of free R848 and the R848-GA+MC38-L combination were significantly less than those of R848-GA@MC38-L, highlighting the advantage of co-delivering antigen and adjuvant for maximizing anti-tumor efficacy. Throughout the study, the body weight of mice in all groups remained essentially constant. Figure 5 This indicates that these treatments have good in vivo safety. No changes were detected in peripheral blood cell subsets (including leukocytes, lymphocytes, monocytes, erythrocytes, and platelets), further confirming the safety of the nanovaccine. Figure 6 ).

[0049] Example 5

[0050] Investigation on the induction of antitumor immune response by resimot galloyl derivative antigen nanocomplex

[0051] Take 100 μL of MC38 cells (2 × 10⁻⁶) 6 Injected subcutaneously above the right buttock of mice to establish an MC38 mouse ectopic tumor animal model. When the tumor volume of the mouse reaches 100 mm, the tumor is allowed to grow. 3 Mice were then randomly divided into three groups: the PBS group, the R848-GA+MC38-L group, and the R848-GA@MC38-L group. The equivalent dose of rasimod was 2 mg / kg in all groups (except the PBS group). Administration was subcutaneous injection, administered every three days for a total of three doses. After the experiment, lymph nodes and tumor tissues were collected from each group to detect immune cell infiltration. Serum was also collected from mice, and the expression levels of TNF-α, IFN-γ, and IL-12 were detected using an ELISA kit.

[0052] The results are as follows Figure 7 As shown, in lymph nodes and tumors, the percentage of CD80+CD86+ dendritic cells in the R848-GA@MC38-L treatment group was significantly higher than that in the R848-GA@MC38-L group, indicating that R848-GA@TCLs induced stronger dendritic cell maturation. Furthermore, R848-GA@MC38-L treatment significantly increased the percentage of CD8+ T cells and anti-tumor M1-like macrophages (F4 / 80+CD11c+) in lymph nodes, and the percentage of CD25+CD8+ T cells and NK cells in tumor tissue. Serum cytokine TNF-α, IFN-γ, and IL-12 levels further indicated that the R848-GA@MC38-L group had the strongest ability to induce an immune response. Figure 8 ).

Claims

1. A resimodil galloyl derivative, characterized in that, The derivative is a substance in which resimote and gallic acid are linked by an ester bond, and the structure is shown in Formula 1 below; Formula 1.

2. A method for synthesizing the resimilar galloyl derivative of claim 1, characterized in that, The reaction formula is as follows: ； 3,4,5-tris(benzyloxy)benzoic acid was reacted with retsimot under the catalysis of 4-dimethylaminopyridine (DMAP) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and the intermediate compound was isolated and purified. The intermediate compound was deprotected by palladium on carbon hydrogenation catalysis, and the retsimot galloyl derivative was obtained by isolation and purification.

3. A resimilar galloyl derivative antigen nanocomposite, characterized in that, The nanocomposite is the resimot galloyl derivative, antigen, and PEG as described in claim 1; the weight ratio of the resimot galloyl derivative to the antigen is (1:2) to (1:10); the weight ratio of the resimot galloyl derivative to PEG is (1:1) to (1:8). The antigens are ovalbumin, carcinoembryonic antigen, alpha-fetoprotein, gp95 / p97 antigen, breast cancer cell lysate protein, melanoma cell lysate protein, colorectal cancer cell lysate protein, prostate cancer cell lysate protein, liver cancer cell lysate protein, lung cancer cell lysate protein, gastric cancer cell lysate protein, esophageal cancer cell lysate protein, pancreatic cancer cell lysate protein, ovarian cancer cell lysate protein, bladder cancer cell lysate protein, or nasopharyngeal cancer cell lysate protein.

4. The resimilar galloyl derivative antigen nanocomposite according to claim 3, characterized in that, The molecular weight of the PEG is 2k-35k.

5. A method for preparing the resimilar galloyl derivative antigen nanocomposite according to claim 1, characterized in that, According to the proportions described in claim 3, the resimot galloyl derivative of claim 1 is dissolved in an organic solvent as the organic phase, and the antigen and PEG are dissolved in deionized water as the aqueous phase. The organic phase and the aqueous phase are rapidly stirred and mixed to spontaneously form a uniform nanocomposite with the resimot galloyl derivative and the antigen. The organic solvent is one or a mixture of several of DMSO, acetone, ethanol, methanol, acetonitrile, or tetrahydrofuran.

6. The use of the derivative of claim 1 or the nanocomposite of claim 3 in the preparation of a drug delivery system.

7. The use of the derivative of claim 1 or the nanocomposite of claim 3 in the preparation of an antitumor drug.

8. The application according to claim 6, characterized in that: The drug can be administered by injection, oral administration, or topical administration.

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