3-deoxidized anthocyanin derivative as well as preparation method and application thereof

Abstract

The invention relates to a fixed-point propynylation modified 3-deoxy anthocyanin derivative, the molecular formula of the derivative is C21H15ClO4, and the chemical name of the derivative is 2-(4-hydroxyphenyl)-5, 7-bis (propyl-2-alkyne-1-yloxy) benzopyran chloride. The invention provides a preparation method of the 3-deoxy anthocyanin derivative, and effectively solves the problems that natural 3-deoxy anthocyanin is low in content and difficult to extract. The invention provides application of the 3-deoxy anthocyanin derivative in preparation of drugs for treating cervical cancer. The invention also provides a pharmaceutical composition for treating cervical cancer. The pharmaceutical composition comprises an effective dose of 3-deoxy anthocyanin derivative and 5-fluorouracil. The 3-deoxy anthocyanin derivative can be used as an effective anti-tumor compound for preparing a medicine for treating cervical cancer or an adjuvant therapy medicine for synergistically enhancing the anti-tumor activity of 5-fluorouracil.

Description

A 3-deoxyanthocyanin derivative, its preparation method and application Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a 3-deoxyanthocyanin derivative, specifically to a site-directed propyne-modified 3-deoxyanthocyanin derivative (named APN-A), as well as the preparation method and application of the 3-deoxyanthocyanin derivative. Background Technology

[0002] Cervical cancer is one of the leading causes of cancer-related deaths among women worldwide, and its pathogenesis is closely related to persistent infection with high-risk human papillomavirus (HPV). Although comprehensive treatments such as surgical resection, radiotherapy, and chemotherapy are widely used in clinical practice, the treatment outcomes for patients with advanced or recurrent / metastatic cervical cancer remain unsatisfactory. Furthermore, the toxic side effects of chemotherapy drugs and tumor drug resistance seriously affect patient adherence to treatment and prognosis.

[0003] 5-Fluorouracil (5-FU) is a first-line chemotherapy drug for cervical cancer, exerting its anti-tumor effect by inhibiting tumor cell DNA synthesis. However, its efficacy is limited when used alone, and it is prone to causing serious side effects such as gastrointestinal reactions and bone marrow suppression, thus limiting its clinical application. Therefore, developing novel and highly effective anti-tumor drugs, or searching for compounds that can produce synergistic effects with 5-fluorouracil to improve therapeutic efficacy and reduce toxic side effects, has become a research hotspot in the field of cervical cancer treatment.

[0004] Apigeninidin (APN) belongs to the 3-deoxyanthocyanin class of compounds and is a unique, naturally occurring red pigment found abundantly in the bran and leaf sheaths of red sorghum, usually in its chloride form. Previous studies have confirmed that APN possesses significant antioxidant activity, antibacterial properties, and antitumor potential. As a proven fungal growth inhibitor, APN has potential value in anti-infection research by interfering with fungal cell membrane synthesis or metabolic processes. Furthermore, the crude extract of the cork layer from sorghum stalks, rich in APN, effectively inhibits the proliferation of colon cancer stem cells and early-stage HCT116 cell lines. APN also inhibits the activity of human hepatocellular carcinoma (HepG2) and promyelocytic leukemia (HL-60) cells, with the inhibitory efficacy increasing in a dose-dependent manner. However, its poor membrane permeability and lipid solubility, limited by its polyhydroxy structure, hinder the bioavailability of APN.

[0005] Modifying the structure of small molecule compounds using chemical synthesis is an effective means of improving drug activity. Alkyne groups, as functional groups with unique electronic effects and spatial structures, can significantly enhance drug bioactivity by adjusting the compound's lipophilicity, targeting ability, and binding affinity to the target site. However, site-directed propyrynylation modification of 3-deoxyanthocyanin (APN) has not been reported. How to obtain activity-enhanced 3-deoxyanthocyanin derivatives (APN-A) through precise structural modification and explore their synergistic effects with chemotherapeutic drugs has become a pressing technical problem in this field. Summary of the Invention

[0006] The first objective of this invention is to provide a 3-deoxyanthocyanin derivative (APN-A).

[0007] The 3-deoxyanthocyanin derivative (APN-A) described in this invention is a site-directed propyne-modified 3-deoxyanthocyanin derivative with the molecular formula C. 21 H 15 ClO4, chemically named 2-(4-hydroxyphenyl)-5,7-bis(prop-2-yn-1-yloxy)benzopyran chloride, has the following structural formula: .

[0008] The second objective of this invention is to provide a method for preparing the aforementioned 3-deoxyanthocyanin derivative (APN-A), which effectively solves the problems of low content and difficulty in extraction of natural 3-deoxyanthocyanins.

[0009] The preparation method of the 3-deoxyanthocyanin derivative (APN-A) of the present invention includes the following steps: A. Dissolving 2,4,6-trihydroxybenzaldehyde in sufficient N,N-dimethylformamide, and slowly adding 2 equivalents of propyne bromide to the solution at 15°C to 25°C, using potassium carbonate as a base to react and generate an alkyne-propylated intermediate; B. In 20 mL of ethyl acetate / methanol (volume ratio 3:1) mixed solvent, reacting the alkyne-propylated intermediate obtained in step A with 20 equivalents of trimethylchlorosilane until complete precipitation, to obtain the 3-deoxyanthocyanin derivative.

[0010] A third object of the present invention is to provide the use of the 3-deoxyanthocyanin derivative (APN-A), specifically the use of the APN-A of the present invention in the preparation of a medicament for treating cervical cancer.

[0011] Experiments have shown the synergistic antitumor effect of the combined use of 3-deoxyanthocyanin derivative (APN-A) and 5-fluorouracil.

[0012] A fourth object of the present invention is to provide a pharmaceutical composition for treating cervical cancer.

[0013] The pharmaceutical composition for treating cervical cancer according to the present invention comprises an effective dose of the 3-deoxyanthocyanin derivative (APN-A) and 5-fluorouracil described in the present invention.

[0014] A further feature of the pharmaceutical composition according to the present invention is that the pharmaceutical composition further contains pharmaceutically usable excipients.

[0015] This invention, for the first time, utilizes propyrynolative site-directed modification of 3-deoxyanthocyanin (APN) using chemical methods, resulting in a 3-deoxyanthocyanin derivative named APN-A. Experiments have demonstrated for the first time that the drug prepared from APN-A can significantly induce apoptosis in cervical cancer cells, reduce cervical cancer cell viability, and inhibit cervical cancer cell migration and proliferation. Further experiments have shown that APN-A, used alone or in combination with 5-fluorouracil, can induce apoptosis in HeLa and SiHa cells. Cell viability, migration, and proliferation assays revealed that the 3-deoxyanthocyanin derivative effectively reduces the viability of both types of cervical cancer cells, inhibits cell migration and proliferation, exhibiting a significant dose-dependent effect, while simultaneously enhancing the antitumor activity of 5-fluorouracil. Within a reasonable dosage range, the 3-deoxyanthocyanin derivative (APN-A) can serve as an effective antitumor compound for the preparation of drugs for treating cervical cancer or as an adjuvant therapy to synergistically enhance the antitumor activity of 5-fluorouracil.

[0016] This invention further demonstrates, through chemical proteomics experiments and computer molecular docking experiments, that 3-deoxyanthocyanin derivatives (APN-A) influence the life cycle processes of cervical cancer cells, including protein translation, transport, and mRNA binding, by binding to target proteins such as EIF3J, PARP-1, TCEA1, and QARS1, ultimately inducing apoptosis in cervical cancer cells. Attached Figure Description

[0017] Figure 1 shows the chemical synthesis method of 3-deoxyanthocyanin and its derivatives, as well as the analysis results of liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0018] Figure 2 shows the effects of 3-deoxyanthocyanins and their derivatives on cervical cancer cell viability and apoptosis. The 3-deoxyanthocyanin derivatives also synergistically enhance the antitumor activity of 5-fluorouracil.

[0019] Figure 3 shows the effects of 3-deoxyanthocyanins and their derivatives on the migration and proliferation of cervical cancer cells. The 3-deoxyanthocyanin derivatives synergistically enhance the inhibitory effect of 5-fluorouracil on the migration and proliferation of cervical cancer cells.

[0020] Figure 4 shows the results of chemical proteomics capture of 3-deoxyanthocyanin derivatives binding to target proteins in cervical cancer cells and enrichment analysis.

[0021] Figure 5 shows the computer-aided molecular docking results of 3-deoxyanthocyanin derivatives with target proteins EIF3J, PARP-1, TCEA1, and QARS1. Detailed Implementation

[0022] Commercially available human cervical cancer cells (HeLa), human cervical squamous cell carcinoma cells (SiHa), and human renal epithelial cells (HK-2) were cryopreserved by the applicant's laboratory. 3-Deoxyanthocyanin was chemically synthesized by the applicant's laboratory, with a purity >97%. 5-Fluorouracil was purchased from Hengrui Medicine Co., Ltd., with a purity ≥99%.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0024] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0025] Example 1: Chemical synthesis of 3-deoxyanthocyanin (APN) and 3-deoxyanthocyanin propyne derivative (APN-A) (1) APN synthesis (Figure A in Figure 1): 2,4,6-trihydroxybenzaldehyde and 4-hydroxyacetophenone were reacted in a methanol / ethyl acetate (volume ratio 1:3) mixed solvent, and then trimethylchlorosilane (TMSCl) was slowly added until a red powder product was precipitated, and APN was finally obtained with a yield of 86%.

[0026] (2) Synthesis of APN-A (Figure B in Figure 1): First, 2,4,6-trihydroxybenzaldehyde (1.52 g, 10 mmol) was dissolved in N,N-dimethylformamide (DMF). Propylene bromide (20 mmol, 2 equivalents) was slowly added to the solution at room temperature (15 °C to 25 °C) to generate a propargylated intermediate with potassium carbonate (K2CO3) as the base, in a yield of 58.6%. Subsequently, the propargylated intermediate was reacted with trimethylchlorosilane (20 equivalents) in an ethyl acetate / methanol (volume ratio 2:1) mixed solvent until complete precipitation, to obtain a 3-deoxyanthocyanin derivative (named APN-A).

[0027] APN-A was purified by medium-pressure C18 reversed-phase chromatography, and its purity was analyzed by high-performance liquid chromatography (HPLC). The final yield was 55%, and the purity reached 98%. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis in positive ion mode (Figure 1, C): APN showed a molecular ion peak at a mass-to-charge ratio (m / z) of 255.06, while APN-A showed a characteristic peak at m / z 331.95. Hydrogen nuclear magnetic resonance (¹H NMR) spectroscopy confirmed the presence of the propargyl group structure, specifically as evidenced by the characteristic chemical shifts of the propargyl protons and carbon atoms. The NMR spectra of APN and APN-A are as follows: 3-Deoxyanthocyanin (APN): 1H NMR (400MHz, Methanol-d6) δ 9.04 (d, J=8.6Hz, 1H), 8.27 (d, J=8.9Hz, 2H), 7.99 (d, J=8.7Hz, 1H), 7.05 (d, J=8.9Hz, 2H), 6.94–6.87 (m, 1H), 6.63 (d, J=2.0Hz, 1H), 3.43–3.14 (m, 2H).

[0028] 3-Deoxyanthocyanin derivative (APN-A): 1H NMR (400MHz, Methanol-d6) δ 9.23–9.16 (m, 1H), 8.50–8.39 (m, 2H), 8.30 (d, J=9.0Hz, 1H), 7.49 (dd, J=2.1, 0.8Hz, 1H), 7.19–7.02 (m, 3H), 5.13 (dd, J=2.5, 1.1Hz, 4H), 3.22 (t, J=2.4Hz, 2H).

[0029] The prepared 3-deoxyanthocyanin derivative (APN-A) is a site-directed propyne-modified 3-deoxyanthocyanin derivative with the molecular formula C3. 21 H 15 ClO4, chemically named 2-(4-hydroxyphenyl)-5,7-bis(prop-2-yn-1-yloxy)benzopyran chloride, has the structural formula shown below: .

[0030] The 3-deoxyanthocyanin (APN) and 3-deoxyanthocyanin derivative (APN-A) prepared in this embodiment were used in the following experiments.

[0031] Example 2: Inhibitory effects of 3-deoxyanthocyanin (APN) and 3-deoxyanthocyanin derivative (APN-A) on the viability, migration and proliferation of cervical cancer cells (1) Cell viability: HeLa, SiHa and HK-2 cells were divided into 1×10 4Cells were seeded per well in a 96-well plate. After complete cell adhesion, APN-A was administered at the specified concentration. 24 hours after administration, 10 μL of CCK-8 solution was added to each well. The plate was incubated in an incubator for 1 hour, and the absorbance at 450 nm was measured using a microplate reader.

[0032] Cell viability (%) = [A(drug-treated) - A(blank)] / [A(0-drug-treated) - A(blank)] × 100% After co-incubating HeLa, SiHa, and HK-2 cells with different concentrations of APN-A for 24 hours, compared with the control group (0 μM), APN-A significantly reduced the cell viability (IC50) of HeLa cells. 50 =10.44μM) and SiHa cells (IC 50 =2.94μM) activity. Furthermore, APN-A showed activity against the non-tumor cell line HK-2 (CC) 50 =43.42 μM) showed reduced cytotoxicity, indicating that APN-A can significantly reduce the viability of cervical cancer cells, but has low cytotoxicity to normal cells (AC plot in Figure 2).

[0033] (2) Apoptosis: Apoptosis was detected using the FITC Annexin V apoptosis detection kit from Beijing Solarbio Biotechnology Co., Ltd., following the manufacturer's instructions. HeLa cells treated with the specified concentrations of APN and APN-A for 24 hours were collected, washed twice with sulfate-buffered saline, and resuspended in binding buffer. FITC-labeled Annexin V and propidium iodide (PI) were added, and the cells were incubated at room temperature in the dark for 15 minutes. Finally, 1×10⁶ cells were collected. 4 Cells were analyzed for apoptosis rate using a flow cytometer from BDBiosciences (FG plot in Figure 2).

[0034] (3) Cell migration: The migration ability of cervical cancer cells was analyzed using a wound healing assay. HeLa and SiHa cells were used at a concentration of 5 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of cells / well in 6-well plates. After complete confluence, a 200 μL sterile pipette tip was used to vertically scratch the cell monolayer in a cross pattern. The cells were gently washed three times with sulfate-buffered saline to remove detached cells. Serum-free medium containing APN and APN-A was then added, and the cells were cultured for another 24 hours. Images of the same field of view were taken at 0 hours (W0) and 24 hours (W24) post-scraping (AD images in Figure 3). The scratch width was measured using ImageJ software, and cell migration was calculated using the following formula: Migration (%) = [(W0 - W24)] t(4) Cell proliferation: HeLa and SiHa cells were seeded in 6-well plates at a density of 500 cells / well and cultured for 7-14 days. Half of the intermediate fluid containing different drugs was replaced every 3 days to maintain a stable growth environment. When cell colonies were visible, the plates were fixed with fixative for 5 minutes and stained with 0.5% crystal violet solution for 20 minutes at room temperature. The culture plates were gently rinsed with distilled water 3-5 times until the background was clear. After the culture plates were air-dried upside down, the colony morphology was observed under a microscope and photographed. The number of colonies with a diameter ≥0.5 mm was counted using ImageJ software, and the colony formation rate was calculated (EG diagram in Figure 3).

[0035] Example 3: Synergistic Enhancement of the Antitumor Effect of 5-Fluorouracil by 3-Deoxyanthocyanin Derivative (APN-A) The pretreatment and detection steps for HeLa and SiHa cells treated with 5-fluorouracil and APN-A alone or in combination were consistent with those described in Example 2. The results showed that APN-A (5 μM) significantly enhanced the ability of 5-fluorouracil (10 μM) to inhibit the viability of HeLa and SiHa cells, with a stronger inhibition rate than that of 5-fluorouracil (20 μM) alone. Furthermore, APN-A (5 μM) increased the apoptosis rate of HeLa cells induced by 5-fluorouracil (10 μM) to 71.6%, which was higher than the 45.9% induced by 5-fluorouracil (20 μM) alone.

[0036] In experiments inhibiting cervical cancer cell migration, treatment with APN-A (5 μM) combined with 5-fluorouracil (10 μM) significantly enhanced the migration rate of HeLa and SiHa cells (AD plot in Figure 3), with a stronger inhibition rate than treatment with 5-fluorouracil alone (20 μM). Similarly, treatment with APN-A (5 μM) combined with 5-fluorouracil (10 μM) significantly enhanced the inhibitory effect of 5-fluorouracil on the proliferation of HeLa and SiHa cells, with an inhibition rate higher than treatment with 5-fluorouracil alone (20 μM) (EG plot in Figure 3). These experimental results collectively indicate that APN-A has a synergistic enhancing effect on the antitumor efficacy of 5-fluorouracil.

[0037] Example 4: Capture and enrichment analysis of target proteins in cervical cancer cells by 3-deoxyanthocyanin derivative (APN-A) (1) Magnetic bead enrichment: HeLa cell lysis buffer (2 mg / mL) was aliquoted into centrifuge tubes, 1 mL per tube, and divided into experimental, control, and competitive groups. The experimental group was treated with 100 μM APN-A, the control group was treated with dimethyl sulfoxide, and the competitive group was treated with 100 μM APN-A and 3-deoxyanthocyanin (APN). All groups were incubated at 37°C for 2 hours. After incubation, 9 mL of pre-cooled methanol was added to each tube, and the protein was precipitated at -20°C for 30 minutes. After high-speed centrifugation, the supernatant was discarded, and the precipitate was redissolved with 1 mL of 1.0% SDS solution. 100 μL of click reaction solution was added, and the reaction was carried out at room temperature for 2 hours. After centrifugation again, the precipitate was redissolved with 1.0% SDS. The sample was gently mixed with 4 mL of phosphate-buffered saline and 200 μL of streptavidin magnetic beads and incubated by rotation at room temperature for 3 hours. The magnetic beads were then washed three times with phosphate-buffered saline containing 0.1% SDS and then three times with Milli-Q water.

[0038] (2) Protein digestion and mass spectrometry analysis: Magnetic beads used for protein enrichment were successively reduced with dithiothreitol and alkylated with iodoacetamide, followed by trypsin digestion overnight at 37°C. The enzymatically hydrolyzed peptides were desalted using a C18 column, freeze-dried, and dissolved in HPLC-grade 0.1% formic acid aqueous solution. Analysis was then performed using a QExactivePlus mass spectrometer coupled with an EASYnLC1200 HPLC system. Raw mass spectrometry data were analyzed using MaxQuant software for database retrieval. The human proteome database used was UniProt (uniprot-proteome2018). Differential protein screening and statistical analysis (FC value, p-value) were performed using Perseus 2.0.3.0 software. Volcano plots, Venn diagrams, and bubble diagrams were generated using the online tool sangerbox.com (Figure 4). Functional annotation and information retrieval of target proteins were performed based on the UniProt database.

[0039] Example 5: Simulated Molecular Docking of 3-Deoxyanthocyanin Derivative (APN-A) with Target Proteins EIF3J, PARP-1, TCEA1, and QARS1 To evaluate the binding characteristics of APN-A with the core target proteins, molecular docking simulations were performed using AutoDock software (Figure 5). The 3D crystal structures of the target proteins (including EIF3J, TCEA1, QARS1, and PARP-1) were obtained from the PDB database (https: / / www.rcsb.org / ). Simultaneously, the PDB file for APN-A was constructed and optimized using ChemDraw and Chem3D software. The intermolecular interactions between APN-A and each target protein were visualized and analyzed using PyMOL software.

[0040] Conclusion: 3-Deoxyanthocyanin derivative (APN-A) can induce apoptosis in cervical cancer cells and can be used as a chemotherapy drug to treat cervical cancer in women.

[0041] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. It should be noted that for those skilled in the art and any person skilled in the art, any equivalent substitutions or changes made to the technical solution and inventive concept of the present invention without departing from the overall concept of the present invention, as well as any changes and improvements made, should also be considered within the scope of protection of the present invention.

Claims

1. A 3-deoxyanthocyanin derivative (APN-A), characterized in that, The 3-deoxyanthocyanin derivative is a site-directed propyne-modified 3-deoxyanthocyanin derivative with the molecular formula C. 21 H 15 ClO4, chemically named 2-(4-hydroxyphenyl)-5,7-bis(prop-2-yn-1-yloxy)benzopyran chloride, has the structural formula shown below: 。 2. The method for preparing the 3-deoxyanthocyanin derivative (APN-A) as described in claim 1, characterized in that, Includes the following steps: A. Dissolve 2,4,6-trihydroxybenzaldehyde in sufficient N,N-dimethylformamide, and slowly add 2 equivalents of propyne bromide to the solution at 15°C to 25°C, using potassium carbonate as a base to react and generate the propylated intermediate; B. In 20 mL of ethyl acetate / methanol (volume ratio 3:1) mixed solvent, react the propylated intermediate obtained in step A with 20 equivalents of trimethylchlorosilane until complete precipitation, to obtain the APN-A.

3. The use of the 3-deoxyanthocyanin derivative (APN-A) as described in claim 1 in the preparation of a medicament for treating cervical cancer.

4. A pharmaceutical composition for treating cervical cancer, characterized in that, Includes an effective dose of the 3-deoxyanthocyanin derivative (APN-A) and 5-fluorouracil as described in claim 1.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition also contains pharmaceutically usable excipients.

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