Galactosyl zinc-porphyrin derivative as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biochemistry, and particularly discloses a galactosyl zinc-porphyrin derivative and a preparation method and application thereof.The galactosyl zinc-porphyrin derivative is formed by coupling a group with double functions of fluorescence and cytotoxic activity and a liver cancer targeting group, can be specifically combined with a liver cancer cell HepG2, emits 608 nm and 659 nm fluorescence signals under the excitation wavelength of 425 nm, and can be used for detecting liver cancer. And accurate tracing can be realized. Meanwhile, IC509.1 mu M + / -2.59 is used for killing liver cancer cells, so that targeted therapy is realized. The chemical disclosed by the invention has the functions of fluorescence tracing and targeted killing, can synchronously realize accurate positioning (fluorescence tracing) and efficient removal (targeted treatment) of liver cancer cells without drug combination, can be used for liver cancer diagnosis, treatment and curative effect evaluation, and has extremely high research and development value.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, and in particular to a galactosyl zinc-porphyrin derivative, its preparation method, and its application. Background Technology

[0002] Liver cancer is one of the most common and serious malignant tumors threatening human health worldwide, and its chemotherapy and treatment drugs have always been a focus of medical research. Currently, commonly used chemotherapy drugs for liver cancer mainly include doxorubicin and fluorouracil. Their main drawbacks are a lack of tumor targeting and high toxicity to normal hepatocytes, resulting in a narrow therapeutic window and strong side effects. Some drugs used to treat liver cancer have certain targeting properties, such as lenvatinib, but they cannot track the distribution of the drug in the body or its binding to the tumor, making it difficult to accurately assess efficacy and adjust dosage. Standalone fluorescent tracers for liver cancer, such as fluorescein and quantum dots, can only achieve tumor imaging and do not have therapeutic functions. They must be used in combination with therapeutic drugs, which presents problems such as complex dosing and poor synergy.

[0003] Therefore, the current lack of an integrated compound that can simultaneously achieve precise fluorescent tracing of liver cancer cells and highly effective targeted killing has, to some extent, hindered the development of precision treatment for liver cancer. Summary of the Invention

[0004] This invention provides a galactosyl zinc-porphyrin derivative, its preparation method, and its application. This derivative has both the functions of specific fluorescent tracing of liver cancer cells and targeted killing activity of liver cancer cells, providing a broader approach for "precision tracing-targeted therapy" of liver cancer.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] The first aspect of this invention provides a galactosyl zinc-porphyrin derivative having the structural formula of formula (I):

[0007] (I).

[0008] Furthermore, the excitation wavelength of the galactosyl zinc-porphyrin derivative is 425 nm, and the emission wavelengths are 608 nm and 659 nm.

[0009] A second aspect of this invention provides a method for preparing the above-described galactosyl zinc-porphyrin derivative, comprising the following steps:

[0010] (1) Azide-based acetylgalactose was added to anhydrous methanol, and sodium methoxide was added to adjust the pH of the system to 9-10. The reaction was carried out under nitrogen or inert gas protection for 8-24 h. After the reaction was completed, ion exchange resin was added to neutralize to neutrality. After stirring, the mixture was filtered to remove the solvent and purified to obtain white solid azide-based galactose. ,spare;

[0011] (2) Methyl p-formylbenzoate and pyrrole were added to propionic acid and refluxed at 120-160°C in the dark for 12-24 hours. After the reaction was completed, the reaction system was allowed to stand at 2-6°C, and the precipitate was collected by centrifugation or filtration, washed, and recrystallized to obtain purple crystal HTPP. The HTPP has the structural formula of formula (II):

[0012] (II);

[0013] (3) HTPP is dissolved in a mixed solvent of tetrahydrofuran and methanol, and then an inorganic base solution is added. The mixture is stirred and refluxed at 50-75°C for 12-24 h. After the reaction is complete, an inorganic acid is added until no more precipitate is formed. The mixture is filtered under reduced pressure, and the precipitate is washed and dried to obtain a purple solid HTPP-1. The HTPP-1 has the structural formula of formula (III):

[0014] (III);

[0015] (4) HTPP-1, N,N-diisopropylethylamine, and 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate were added to anhydrous N,N-dimethylformamide and reacted at 0-4℃ for 0.6-3 h. Then, propargylamine was added, and the reaction was continued at room temperature for 8-24 h. After the reaction was completed, the mixture was extracted and purified to obtain purple solid HTPP-2, which has the structural formula of formula (IV):

[0016] (Ⅳ);

[0017] (5) HTPP-2 is dissolved in anhydrous chloroform to obtain solution A, and zinc acetate is dissolved in anhydrous methanol to obtain solution B. Solutions A and B are mixed and refluxed at 50-70°C for 3-10 h with stirring. After the reaction is completed, the extraction solvent is extracted and recovered to obtain a purple-red solid ZnTPP. The ZnTPP has the structural formula of formula (V):

[0018] (V);

[0019] (6) ZnTPP is dissolved in ethanol to obtain solution C, and azidogalactose is dissolved in methanol to obtain solution D. Solution C and solution D are mixed to obtain a reaction solution. Copper sulfate pentahydrate and sodium ascorbate are dissolved in water to obtain solution E. Solution E is added to the reaction solution, and the reaction is carried out under nitrogen or inert gas protection and stirred for 8-24 h. After the reaction is completed, the crude product is concentrated under vacuum. The crude product is further purified to obtain the galactosyl zinc-porphyrin derivative.

[0020] Furthermore, in step (1), the purification specifically includes: the solid obtained after removing the solvent is purified by silica gel column chromatography, and the eluent is dichloromethane and methanol in a volume ratio of 10:1-20:1.

[0021] Furthermore, in step (2), washing and recrystallization specifically include: washing with ethanol at 2-10°C, and then recrystallizing with a mixed solvent of dichloromethane and ethanol in a volume ratio of 1-3:1.

[0022] Furthermore, in step (4), the extraction and purification specifically include: extraction with chloroform, followed by separation and purification with silica gel column, using petroleum ether and ethyl acetate in a volume ratio of 1:1 to 1:10 as the eluent.

[0023] Furthermore, in step (6), the purification specifically includes: separation and purification using a silica gel column, with dichloromethane and methanol used as the eluent in a volume ratio of 3:1 to 20:1.

[0024] The third aspect of this invention provides the application of the above-described galactosyl zinc-porphyrin derivative in the preparation of drugs that possess both fluorescence tracing and targeted anti-hepatocellular carcinoma cell functions.

[0025] The fourth aspect of this invention provides the application of the above-described galactosyl zinc-porphyrin derivative in the preparation of antitumor drugs.

[0026] The galactosyl zinc-porphyrin derivatives described above have the following advantages:

[0027] (1) Dual-function integration: The "fluorescence tracing" and "targeted killing" functions are integrated into a single compound, which can simultaneously achieve precise localization (fluorescence tracing) and efficient clearance (targeted therapy) of liver cancer cells without the need for combination therapy. It can be used in the whole process of liver cancer diagnosis, treatment and efficacy evaluation, and simplifies the drug administration process and improves the convenience of treatment.

[0028] (2) High targeting and low toxicity: Through the four-pronged galactosyl specific targeting group, selective binding to liver cancer cells is achieved, with low toxicity to other cells, significantly reducing the side effects of traditional chemotherapy.

[0029] (3) The mechanism is clear and highly optimizable: The functional protein and regulatory pathway were identified by mass spectrometry. The regulatory pathway and core functional protein include EGFR, Rho, JNK, ASK1, Cat D, RagB and Rheb. It is clear that they inhibit cell proliferation, growth and survival, mainly by activating apoptosis through the endoplasmic reticulum stress pathway and mitochondrial pathway, but do not activate autophagy. This provides a development idea for the development of targeted killing drugs and also provides clear guidance for subsequent compound structure modification, such as enhancing the binding affinity with functional proteins, and combination therapy, such as combining with other pathway inhibitors.

[0030] (4) Wide range of applications: It can be used for targeted therapy of liver cancer, and can also monitor the accumulation of drugs in tumors through fluorescence tracing and fluorescence intensity, so as to realize the early diagnosis of liver cancer and the evaluation of drug efficacy, which has extremely high value. Attached Figure Description

[0031] Figure 1 This is the mass spectrum of a galactosyl zinc-porphyrin derivative.

[0032] Figure 2 It is a galactosyl zinc-porphyrin derivative. 1 H NMR spectrum.

[0033] Figure 3 It is a galactosyl zinc-porphyrin derivative. 13 C10 NMR spectrum.

[0034] Figure 4 A is the absorption spectrum of HTTP and ZnTPPGal; Figure 4 B is the absorption spectrum of HTTP, ZnTPP, and ZnTPPGal; Figure 4 C is a comparison of fluorescence imaging of ZnTPPGal and ZnTPP in HepG2 and A549 cells (scale bar 20 μm).

[0035] Figure 5 A is a schematic diagram illustrating the cytotoxicity of HTTP, ZnTPP, and ZnTPPGal on HepG2 cells (horizontal axis: derivative concentration, vertical axis: cell viability). Figure 5 B is the absorption spectrum of the final product in the MTT assay for the detection of ZnTPP and ZnTPPGal.

[0036] Figure 6 A is a volcano plot of differential protein expression (DEPs) based on mass spectrometry analysis (horizontal axis is log2 (fold change), vertical axis is -log2). 10 (P-value, red, blue, and gray dots represent upregulation, downregulation, and no significant change in protein expression, respectively). Figure 6B is a bubble chart of the KEGG pathway in DEPs (the horizontal axis represents -log). 10 (p-value), the vertical axis represents the enrichment pathway, the size of each bubble represents the average number of proteins enriched in a specific pathway, and the color of the bubble represents log2 (fold enrichment)). Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer and more concise, the invention is described using the following specific embodiments, but the invention is by no means limited to these examples. The following descriptions are merely preferred embodiments of the invention and are used only to describe the invention; they should not be construed as limiting the scope of the invention. It should be noted that any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

[0038] Example 1: Preparation and identification of the target compound

[0039] A method for preparing galactosyl zinc-porphyrin derivatives includes the following steps:

[0040] (1) Azide-based acetylgalactose was added to anhydrous methanol, and sodium methoxide was added to adjust the pH of the system to 9-10. The reaction was carried out under nitrogen or inert gas protection for 16 h. After the reaction was completed, ion exchange resin was added to neutralize to neutrality. After stirring for 30 min, the mixture was filtered to remove the solvent and purified to obtain white solid azide-based galactose. (C6H) 11 N3O5), for later use.

[0041] In this embodiment, the purification specifically includes: the solid obtained after removing the solvent is purified by silica gel column chromatography, and the eluent is dichloromethane and methanol in a volume ratio of 20:1-10:1.

[0042] (2) Methyl p-formylbenzoate and pyrrole were added to propionic acid at a molar ratio of methyl p-formylbenzoate to pyrrole of 1:1.1. The mixture was refluxed at 140°C in the dark for 16 h. After the reaction was completed, the reaction system was allowed to stand at 4°C. The precipitate was collected by filtration, washed, and recrystallized to obtain purple crystals of HTPP (C 52 H 38 N4O8), the HTPP has the structural formula (II):

[0043] (Ⅱ).

[0044] In this embodiment, washing and recrystallization specifically include: washing with ethanol at 4°C, and then recrystallizing with a mixed solvent of dichloromethane and ethanol in a volume ratio of 2:1.

[0045] (3) HTPP was dissolved in a mixed solvent of tetrahydrofuran and methanol, and then 2 mol / L potassium hydroxide aqueous solution was added. The mixture was stirred and refluxed at 66°C for 16 h. After the reaction was completed, 1 mol / L HCl was added until no more precipitate was formed. The precipitate was washed with ice water, collected, and dried under vacuum to obtain purple solid HTPP-1 (C 48 H 30 N4O8), the HTPP-1 has the structural formula of formula (Ⅲ):

[0046] (III).

[0047] In this embodiment, the volume ratio of tetrahydrofuran to methanol is 1:1.

[0048] (4) HTPP-1, N,N-diisopropylethylamine (DIPEA) and 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) were added to anhydrous N,N-dimethylformamide (DMF) and reacted at 0 °C for 1 h. Then, propargylamine was added. The molar ratio of HTPP-1, DIPEA, HATU and propargylamine was 1:3:1.2:2. The reaction was continued at room temperature for 16 h. After the reaction was completed, the mixture was extracted and purified to obtain purple solid HTPP-2 (C 60 H 42 N8O4), the HTPP-2 has the structural formula of formula (Ⅳ):

[0049] (Ⅳ).

[0050] In this embodiment, the extraction and purification specifically include: extraction with chloroform, followed by separation and purification using a silica gel column, with petroleum ether and ethyl acetate used as the eluent in a volume ratio of 1:1.

[0051] (5) HTPP-2 was dissolved in anhydrous chloroform to obtain solution A, and zinc acetate was dissolved in anhydrous methanol to obtain solution B. Solutions A and B were mixed, with a molar ratio of HTPP-2 to zinc acetate of 1:10. The mixture was stirred and refluxed at 64°C for 4 h. After the reaction was completed, the solvent was extracted and recovered to obtain a purple-red solid ZnTPP (C 60 H 40 N8O4Zn), the ZnTPP has the structural formula (V):

[0052] (V).

[0053] In this embodiment, the extraction solvent used is chloroform.

[0054] (6) ZnTPP is dissolved in ethanol to obtain solution C, and galactosyl azidophosphate is dissolved in methanol to obtain solution D. Solution C and solution D are mixed to obtain a reaction solution. Copper sulfate pentahydrate and sodium ascorbate are dissolved in water to obtain solution E. Solution E is added to the reaction solution. The molar ratio of ZnTPP, galactosyl azidophosphate and copper sulfate pentahydrate is 1:2:0.3. The reaction is carried out under nitrogen or inert gas protection and stirred at room temperature for 16 h. After the reaction is completed, the solution is purified to obtain the target compound.

[0055] In this embodiment, the purification specifically includes: separation and purification using a silica gel column, with dichloromethane and methanol used as the eluent in a volume ratio of 3:1 to 20:1.

[0056] The target compound ZnTPPGal, bound Figure 1-3 As shown, HPLC-MS (ESI+): [M-2H] 2- The theoretical value is C 84 H 82 N 20 O 24 Zn, m / z 909.25457, detected value m / z 909.25562; 1 H NMR (500 MHz, DMSO) δ9.30 (d, J = 5.9 Hz, 4H), 8.71 (d, J = 5.0 Hz, 8H), 6.23 (s, 2H), 5.81 (s,4H), 4.58 (d, J = 9.5 Hz, 8H), 4.33 – 4.25 (m, 4H), 4.13 (s, 4H), 4.00 (s, 4H), 3.75 (s, 8H), 3.67 (s, 8H), 3.49 (s, 4H); 13 C NMR (126 MHz, DMSO) δ168.36, 152.35, 148.32, 138.57, 136.78, 135.68, 129.31, 123.38, 104.05,84.33, 77.43, 61.87, 43.55.

[0057] Example 2: Fluorescence Tracing and Targeted Killing Activity Experiments of the Compound

[0058] 2.1 Materials

[0059] HepG2 and A549 cells, cell culture medium (DMEM), fetal bovine serum (FBS), trypsin-EDTA digestion solution, penicillin-streptomycin mixture, DAPI, fixative, phosphate buffered saline (PBS), MTT, dimethyl sulfoxide (DMSO).

[0060] 2.2 Experimental Methods

[0061] 2.2.1 Fluorescence Tracing Experimental Method

[0062] HepG2 and A549 cells were seeded into confocal culture dishes, respectively. ZnTPP and ZnTPPGal were added to a final concentration of 10 μM, respectively. After incubation for 20 min, the cells were washed three times with PBS, fixed with fixative at room temperature for 15 min, washed three times with PBS, and the cell nuclei were counterstained with 5 µg / m LDAPI at room temperature for 5 min. Cell fluorescence imaging was detected using laser confocal microscopy (λEx=405 nm, λEm=440-470 nm for DAPI imaging; λEx=552 nm, λEm=605-670 nm for compound imaging) to achieve qualitative identification of liver cancer cells.

[0063] 2.2.2 Experimental Methods for Targeted Killing Activity

[0064] Cell viability was assessed and the killing effect was evaluated using the 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

[0065] HepG2 cells were seeded in 96-well plates (1×10⁶ cells per well). 4 Cells / well were prepared by adding different concentrations (5, 10, 20, 40, 60 μM) of HTPP, ZnTPP, and ZnTPPGal, with each concentration configured in triplicate. After incubation for 24 h, 5 mg / mL LMT reagent was added, and incubation continued for another 4 h to produce a purple formazan solid. The absorbance at 490 nm was measured within 10 min using a microplate reader, and the IC50 was calculated. 50 .

[0066] 2.3 Results

[0067] 2.3.1 Results of Fluorescence Tracing Experiment

[0068] like Figure 4 As shown in Figure A, the excitation wavelength of the target compound ZnTPPGal is 425 nm. Figure 4 As shown in B, the emission wavelengths are 608 nm and 659 nm.

[0069] like Figure 4As shown in Figure C, after HepG2 cells were treated with porphyrin derivatives, the fluorescence response of ZnTPPGal was significantly stronger than that of ZnTPP. This is attributed to the galactosyl group in the ZnTPPGal molecular structure, which can be specifically recognized by ASGPR, promoting the rapid uptake of the compound and its subsequent diffusion throughout the cell, exhibiting excellent fluorescence tracking capabilities. However, when the porphyrin derivatives were co-incubated with A549 cells, the fluorescence signal was extremely weak and showed no significant difference. Compared with ZnTPP, the fluorescence intensity of ZnTPPGal in HepG2 cells was much higher than that in A549 cells, indicating that ZnTPPGal is more easily internalized by HepG2 cells. ASGPR is highly expressed on the surface of HepG2 cells but not on the surface of A549 cells; this difference allows HepG2 cells to better recognize the tetra-terminal galactosyl group in ZnTPPGal, thus exhibiting excellent targeting ability.

[0070] 2.3.2 Targeted Kill Experiment Results

[0071] Combination Figure 5 As shown, for HepG2 cells, the IC50 of HTPP was 41.3 µM ± 3.78, the IC50 of ZnTPP was 19.2 µM ± 2.80, and the IC50 of ZnTPPGal was 9.1 µM ± 2.59. This confirms that ZnTPPGal with a tetra-terminal galactosyl group can exhibit strong specific recognition ability for HepG2 cells through ligand-receptor interaction, thereby selectively accumulating in the cells and having the strongest ability to induce cell death.

[0072] Example 3: Analysis of the Mechanism of Action Based on Mass Spectrometry

[0073] 3.1 Materials

[0074] HepG2 cells, urea and RIPA lysis buffer, reagents for SDS-PAGE analysis, trypsin, chromatographic formic acid, ultrafiltration tubes, protein quantification kit, iTRAQ labeling kit (4 plex); the iTRAQ kit includes tris(2-carboxyethyl)phosphine solution (TCEP), iodoacetamide (IAM), and TEAB.

[0075] 3.2 Methods

[0076] 3.2.1 Grouping

[0077] HepG2 cells were divided into "ZnTPPGal sample group" (hereinafter referred to as "sample group") and "solvent control group" (hereinafter referred to as "control group"), with 3 replicates in each group.

[0078] 3.2.2 Protein extraction and quantification:

[0079] (1) HepG2 cells were incubated with 20 µM ZnTPPGal for 12 h, and trypsin-EDTA digestion solution was added until the cells detached from the cell wall. The resulting cell suspension was centrifuged at 800 rpm for 5 min at 4°C, the supernatant was discarded, and the precipitate was added to urea lysis buffer. The cells were intermittently shaken for 5 min to disperse the cell clusters. The cells were then intermittently sonicated at a frequency of 1 s for 1 min under ice bath conditions. The cells were then incubated at 4°C for 2 h for lysis. The cells were intermittently shaken for 1 min every 30 min to promote complete lysis. The cells were then centrifuged at 12000 rpm for 30 min at 0°C, and the supernatant was collected. The incompletely lysed precipitate was added to RIPA lysis buffer and incubated at 4°C for 30 min for further lysis. The cells were then centrifuged at 0°C for 15 min, and the supernatants were combined to obtain the total cell protein solution. Five volumes of pre-chilled acetone were added to this solution and the solution was incubated at -20°C overnight. Centrifuge at 8000×g for 20 min at 0°C, discard the supernatant, air-dry the protein precipitate at room temperature, and dissolve it in urea lysis buffer.

[0080] (2) The Bradford method was used for quantification, and a series of bovine serum albumin (BSA) standard solutions with concentrations of 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, and 0.50 µg / µL were prepared. G250 dye was added to the BSA solution, and the mixture was thoroughly mixed and allowed to stand at room temperature for 10 min. The absorbance was immediately measured at a wavelength of 595 nm using an ELISA reader. A standard curve was constructed based on the linear relationship between BSA concentration and absorbance. The absorbance of the protein sample solution was measured using the same method, and the concentration of the sample solution was calculated based on the standard curve.

[0081] 3.2.3 Proteolysis and iTRAQ labeling

[0082] Take an appropriate volume of solution based on the protein sample concentration to ensure that the protein content of each sample is 100 µg, and add urea lysis buffer to make the final volume 100 µL.

[0083] The protein samples were digested using the FASP (filter-aided sample preparation) method, with the following steps: TCEP solution (5 µL, 200 mM) was added to the protein solution and reacted at 55°C for 1 h to reduce disulfide bonds. Then, IAM solution (5 µL, 400 mM) was added and reacted at room temperature in the dark for 30 min to alkylate thiol groups. After the reaction, the solution was transferred to a 30 kDa ultrafiltration tube and centrifuged at 14000 × g for 10 min at 4°C. The liquid was discarded. Urea lysis buffer was added and centrifuged at 14000 × g for 10 min at 4°C. The liquid was discarded. TEAB solution (100 mM) was added to the ultrafiltration tube and centrifuged at 14000 × g for 15 min at 4°C. The liquid was discarded. Then, TEAB solution (100 mM) and 4 µL of trypsin solution (1 µg / µL) were added, vortexed for 1 min to mix, and incubated at 37°C for 16 h. After enzymatic hydrolysis, centrifuge at 14000×g for 10 min at 4°C, and then replace with a clean collection tube to recover the liquid. Add TEAB solution (10 mM) to the ultrafiltration tube, centrifuge at 14000×g for 15 min at 4°C, collect and combine the results to obtain the peptide solution, and concentrate under vacuum to remove the liquid. Mix the iTRAQ solution with the peptide solution and react at room temperature for 1 h. When the pH reaches approximately 8.0, add 0.1% formic acid solution to terminate the labeling reaction, ensuring that the organic phase accounts for ≥ 60% of the total solvent.

[0084] The control group was labeled with iTRAQ-114, and the sample group was labeled with iTRAQ-116.

[0085] 3.2.4 Mass spectrometry detection

[0086] (1) Pre-separation

[0087] High-performance liquid chromatography (HPLC) was used to pre-separate the labeled peptide samples. Mobile phase A for HPLC pre-separation of the labeled peptide samples consisted of 2% acetonitrile, 98% water, pH 10; mobile phase B consisted of 98% acetonitrile, 2% water, pH 10. The labeled peptide samples were dissolved in mobile phase A, with the following mobile phase gradients: 0–10 min, 2% B; 10–11 min, 2–5% B; 11–46 min, 5–20% B; 46–56 min, 20–32% B; 56–57 min, 32–98% B; 57–67 min, 98% B. The flow rate was 0.5 mL / min, and the detection wavelength was 214 nm.

[0088] During the 10-57 min time period, one tube of effluent was collected every 3 min, and finally cross-mixed into 12 fractions. Each fraction was then concentrated and dried under vacuum.

[0089] (2) Each fraction was separated and detected by nano-liquid chromatography-mass spectrometry (NanoLC-MS).

[0090] The mobile phases used for separation and detection by NanoLC-MS were: Phase A, consisting of 2% acetonitrile, 0.1% formic acid, and 97.9% water; and Phase B, consisting of 98% acetonitrile, 0.1% formic acid, and 1.9% water. Each fraction was dissolved in mobile phase A, with the following mobile phase gradients: 0–5 min, 2–12% B; 5–30 min, 12–20% B; 30–43 min, 20–32% B; 43–48 min, 32–98% B; 48–58 minutes, 98% B. The flow rate was 300 nL / min.

[0091] Mass spectrometry was performed in positive ion scanning mode. The primary MS scan range was 300-1800 m / z with a resolution of 70,000. Secondary MS / MS analysis was conducted using data-dependent acquisition (DDA). The precursor ions in the MS were sorted from strongest to weakest according to their response signals. The top 20 precursor ions were selected for high-energy collisional dissociation (HCD) fragmentation with a normalized collision energy (NCE) of 30, an MS / MS resolution of 17,500, and a dynamic exclusion time of 20 s. The quantitative information of the iTRAQ reagent reporter ions was obtained from the mass spectra acquired by MS / MS, and the protein expression differences were then calculated.

[0092] 3.2.5 Data Analysis

[0093] Mass spectrometry data were analyzed using ProteomeDiscoverer 2.1 (PD2.1) software. The theoretical protein database used was the human protein database downloaded from uniprot (http: / / www.uniprot.org / proteomes / UP000005640). Differentially expressed proteins (DEPs, |log2FC|≥0.585, P<0.05) were screened. GO enrichment analysis was performed using DAVID online analysis software (http: / / david.ncifcrf.gov, version 6.8). Regulatory pathways were analyzed using the KEGG pathway database (http: / / www.kegg.jp / ). Targeted parallel response monitoring (PRM) was used to verify the differential expression of functional proteins. Data acquired by PRM were processed using Skyline software (version 24.1).

[0094] Specific operation: PD2.1 software search parameters were set as follows: maximum missed cleavage site 2, MS precursor ion mass deviation range ±12 ppm, MS / MS fragment ion mass deviation range ±0.02 Da. Each peptide was allowed a maximum of 4 modification types, including 3 dynamic modifications: methionine (M) oxidation (+15.995 Da), iTRAQ4plex modification of lysine (K) and tyrosine (Y) (+144.102 Da), and iTRAQ4plex modification at the N-terminus of the peptide (+144.102 Da); and 1 fixed modification, namely cysteine ​​(C) acetylation (+57.021 Da). A high confidence level of ≥99% was set for the peptide, with a false discovery rate (FDR) <0.01.

[0095] The DAVID analysis software used Bonferroni correction to determine the p-values ​​for significant enrichment, setting a threshold of p < 0.05 for GO entries considered to represent the primary biological function of the gene product. The Uniprot accession numbers of proteins were compared with the KEGG pathway database, and the p-values ​​were calculated using a hypergeometric distribution test. Enriched pathways with a significance level of p < 0.05 and a differential protein count ≥ 2 were extracted.

[0096] PRM technology was used for quantitative validation of DEPs based on iTRAQ results. NanoLC-MS was used to detect unlabeled peptides without HPLC pre-separation, employing a 135-minute HPLC elution gradient and PRM mass spectrometry acquisition mode. For PRM relative quantification, the peptides used for quantification must meet specific criteria: 1. They must be specific peptides containing 8-15 amino acids, unmodified, without unexploded fragments, and possess high mass spectrometric response. 2. They must not interfere with other co-eluted peptides and must exhibit consistent quantitative fragment ion MS / MS patterns in all detections.

[0097] 3.3 Results

[0098] Table 1. Relative quantitative results of functional DEPs analyzed using iTRAQ and PRM techniques

[0099]

[0100] Combination Figure 6 As shown in Table 1, differentially expressed proteins: a total of 475 DEPs were identified, of which 174 proteins were upregulated and 301 proteins were downregulated (|log2FC| ≥ 0.585); the core differentially expressed protein included: EGFR (log2FC = -0.62, p = 3.09 × 10⁻⁶). -4 ), Rho (log2FC=-0.79, p=1.57×10 -3), JNK (log2FC=-1.18, p=6.33×10 -3 ), Cat D (log2FC=0.63, p=1.43×10 -5 ), ASK1 (log2FC=1.07, p=1.62×10 -2 ), RagB (log2FC= 0.64, p=1.99×10 -2 ) and Rheb (log2FC=0.63, p=5.14×10) -5 ).

[0101] Regulatory Pathways: Enrichment analysis using KEGG and GO databases revealed that ZnTPPGal induces downregulation of EGFR expression, inhibits the Ras / Raf / MEK / EPK signaling pathway, thereby suppressing cell proliferation; it also induces downregulation of EGFR expression, blocks the PI3K / Akt / mTOR signaling pathway, and promotes apoptosis; it induces endoplasmic reticulum stress, induces upregulation of ASK1 expression, and activates the ASK1 / MKK7 / JNK apoptosis signaling pathway; it induces upregulation of CatD expression, cleavage and activates Bid, which in turn activates Bax and Bak, leading to mitochondrial membrane permeability and inducing apoptosis; and it induces downregulation of Rho and downstream JNK expression, blocking stress transduction and thus inhibiting the Rho / JNK signaling pathway, promoting apoptosis. Furthermore, ZnTPPGal induces upregulation of RagB and Rheb expression, positively regulating and activating the mTOR signaling pathway and inhibiting autophagy. It was confirmed that ZnTPPGal inhibits cell proliferation, growth and survival, mainly by activating apoptosis through the endoplasmic reticulum stress pathway and mitochondrial pathway, but does not activate autophagy, thus achieving targeted killing.

[0102] Validation: The PRM method detected a total of 9 peptides, which correspond to 7 DEPs. The results showed that the expression levels of EGFR, Rho, and JNK proteins in the sample group were downregulated compared with the control group, while the expression levels of ASK1, CatD, RagB, and Rheb proteins were upregulated. The quantitative ratios of differentially expressed proteins in the PRM validation showed good correlation with the iTRAQ analysis results, indicating that the PRM detection results were consistent with the iTRAQ results.

Claims

1. A galactosyl zinc-porphyrin derivative, characterized in that, The structural formula with formula (Ⅰ) is: (Ⅰ)。 2. The galactosyl zinc-porphyrin derivative according to claim 1, characterized in that, The excitation wavelength of the galactosyl zinc-porphyrin derivative is 425 nm, and the emission wavelengths are 608 nm and 659 nm.

3. The method for preparing the galactosyl zinc-porphyrin derivative as described in claim 1, characterized in that, Includes the following steps: (1) Azide-based acetylgalactose was added to anhydrous methanol, and sodium methoxide was added to adjust the pH of the system to 9-10. The reaction was carried out under nitrogen or inert gas protection for 8-24 h. After the reaction was completed, ion exchange resin was added to neutralize to neutrality. After stirring, the mixture was filtered to remove the solvent and purified to obtain white solid azide-based galactose. ,spare; (2) Methyl p-formylbenzoate and pyrrole were added to propionic acid and refluxed at 120-160°C in the dark for 12-24 h. After the reaction was completed, the reaction system was allowed to stand at 2-6°C, and the precipitate was collected by centrifugation or filtration, washed, and recrystallized to obtain purple crystal HTPP. The HTPP has the structural formula of formula (II): (Ⅱ)； (3) HTPP is dissolved in a mixed solvent of tetrahydrofuran and methanol, and then an inorganic base solution is added. The mixture is stirred and refluxed at 50-75°C for 12-24 h. After the reaction is complete, an inorganic acid is added until no more precipitate is formed. The precipitate is washed and dried to obtain a purple solid HTPP-1. The HTPP-1 has the structural formula of formula (III): (Ⅲ)； (4) HTPP-1, N,N-diisopropylethylamine, and 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate were added to anhydrous N,N-dimethylformamide and reacted at 0-4℃ for 0.6-3 h. Then, propargylamine was added, and the reaction was continued at room temperature for 8-24 h. After the reaction was completed, the mixture was extracted and purified to obtain purple solid HTPP-2, which has the structural formula of formula (IV): (Ⅳ)； (5) HTPP-2 is dissolved in anhydrous chloroform to obtain solution A, and zinc acetate is dissolved in anhydrous methanol to obtain solution B. Solutions A and B are mixed and refluxed at 50-70°C for 3-10 h with stirring. After the reaction is completed, the extraction solvent is extracted and recovered to obtain a purple-red solid ZnTPP. The ZnTPP has the structural formula of formula (V): (Ⅴ)； (6) ZnTPP is dissolved in ethanol to obtain solution C, and azidogalactose is dissolved in methanol to obtain solution D. Solution C and solution D are mixed to obtain a reaction solution. Copper sulfate pentahydrate and sodium ascorbate are dissolved in water to obtain solution E. Solution E is added to the reaction solution, and the reaction is carried out under nitrogen or inert gas protection and stirred for 8-24 h. After the reaction is completed, the crude product is concentrated under vacuum. The crude product is further purified to obtain the galactosyl zinc-porphyrin derivative.

4. The method for preparing the galactosyl zinc-porphyrin derivative according to claim 3, characterized in that, In step (1), the purification specifically includes: the solid obtained after removing the solvent is purified by silica gel column chromatography, and the eluent is dichloromethane and methanol with a volume ratio of 10:1-20:

1.

5. The method for preparing the galactosyl zinc-porphyrin derivative according to claim 3, characterized in that, In step (2), washing and recrystallization specifically include: washing with ethanol at 2-10℃, and then recrystallizing with a mixed solvent of dichloromethane and ethanol in a volume ratio of 1-3:

1.

6. The method for preparing the galactosyl zinc-porphyrin derivative according to claim 3, characterized in that, In step (4), the extraction and purification specifically include: extraction with chloroform, followed by separation and purification with silica gel column, using petroleum ether and ethyl acetate in a volume ratio of 1:1 to 1:10 as the eluent.

7. The method for preparing the galactosyl zinc-porphyrin derivative according to claim 3, characterized in that, In step (6), purification specifically includes: separation and purification using a silica gel column, with dichloromethane and methanol used as eluents in a volume ratio of 3:1 to 20:

1.

8. The use of the galactosyl zinc-porphyrin derivative according to claim 1 or 2 in the preparation of a drug that has both fluorescent tracing function for liver cancer tumor cells and anti-liver cancer tumor function.

9. The use of the galactosyl zinc-porphyrin derivative according to claim 1 or 2 in the preparation of antitumor drugs.

Images