Application of shikonin derivatives in the preparation of anti-pancreatic cancer drugs

Abstract

The present invention relates to the technical field of anticancer drugs and provides the use of shikonin derivatives in the preparation of anti-pancreatic cancer drugs. The present invention has found that shikonin derivatives have a significant inhibitory effect on pancreatic cancer cells and have broad application prospects in the preparation of anti-pancreatic cancer drugs. The results of the embodiment show that shikonin derivatives inhibit the IC of PANC-1 cells. 50 The ion concentration of shikonin derivatives was 1.218 μM, effectively inhibiting the proliferation, invasion, and migration of PANC-1 cells and arresting the cell cycle. Shikonin derivatives significantly promoted apoptosis in PANC-1 cells in a dose-dependent manner. Animal studies demonstrated that shikonin derivatives significantly inhibited the growth of nude mouse xenograft tumors, disrupted their pathological structures, and induced apoptosis in cancer cells, without significant hepatotoxicity or renal toxicity, demonstrating an overall good safety profile. LiP-MS experiments demonstrated that PKM2 is a potential target of shikonin derivatives.

Description

Technical Field

[0001] The present invention relates to the technical field of anticancer drugs, and in particular to the application of shikonin derivatives in the preparation of anti-pancreatic cancer drugs. Background Art

[0002] Pancreatic cancer is one of the common malignant tumors of the digestive system. It originates from transformed cells in pancreatic tissue and is characterized by insidious onset, high malignancy, rapid progression, and high mortality rate.

[0003] For early-stage pancreatic cancer, surgery offers the most promising cure. By removing the tumor tissue, it can directly remove the lesion, alleviate symptoms, and improve patient survival. However, since most patients present to the clinic in the advanced stages of the disease, surgical resection is no longer an option. Therefore, non-surgical treatments play a crucial role in the comprehensive treatment of pancreatic cancer. Chemotherapy is the mainstay of clinical treatment for advanced pancreatic cancer. However, the current range of chemotherapy drugs used to treat pancreatic cancer is limited, and their effectiveness is poor. Therefore, there is an urgent need to research and develop new drugs that can effectively inhibit pancreatic cancer activity. Summary of the Invention

[0004] In view of this, the present invention provides the use of shikonin derivatives in the preparation of anti-pancreatic cancer drugs. The present invention found that shikonin derivatives have high inhibitory activity against pancreatic cancer cells and have broad application prospects in the preparation of drugs for treating pancreatic cancer.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] Use of a shikonin derivative in the preparation of an anti-pancreatic cancer drug, wherein the shikonin derivative is one or more of a compound having a structure represented by Formula I and a pharmaceutically acceptable salt thereof;

[0007] Formula I.

[0008] Preferably, the pancreatic cancer comprises pancreatic ductal adenocarcinoma.

[0009] Preferably, the anti-pancreatic cancer drug comprises an active ingredient and a pharmaceutically acceptable excipient; the active ingredient comprises a shikonin derivative.

[0010] Preferably, the dosage form of the anti-pancreatic cancer drug includes injection, powder, granule, capsule or tablet.

[0011] Preferably, the anti-pancreatic cancer drug is administered by injection or oral administration.

[0012] The present invention provides the use of shikonin derivatives in the preparation of anti-pancreatic cancer drugs. The shikonin derivatives are compounds of the structure represented by Formula I (see above) and one or more pharmaceutically acceptable salts thereof. The present invention has found that shikonin derivatives have a significant inhibitory effect on pancreatic cancer cells and have broad application prospects in the preparation of anti-pancreatic cancer drugs. The results of the examples show that the shikonin derivatives of the structure represented by Formula I inhibit the IC of PANC-1 cells. 50 The shikonin derivatives of Formula I have a 1.218 μM kinase inhibitory activity and are able to effectively inhibit the proliferation, invasion, and migration of PANC-1 cells. Shikonin derivatives with the structure shown in Formula I can arrest the G0 / G1 phase of the PANC-1 cell cycle at low concentrations and the S phase at high concentrations. Shikonin derivatives with the structure shown in Formula I can significantly promote apoptosis in PANC-1 cells in a dose-dependent manner. Animal studies have shown that shikonin derivatives with the structure shown in Formula I can significantly inhibit the growth of nude mouse xenograft tumors, destroy their pathological structures, and induce apoptosis in cancer cells, without significant hepatotoxicity or renal toxicity, demonstrating an overall good safety profile. LiP-MS experiments have shown that PKM2 is a potential target of shikonin derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The IC values of compound 1, shikonin and other shikonin derivatives in Example 1 for inhibiting PANC-1 cells are shown in Table 1. 50 value;

[0014] Figure 2 The results of the CCK-8 assay of compound 1 in Example 1 on PANC-1 cells are shown below;

[0015] Figure 3 The figure shows the microscopic observation results of the clone formation experiment in Example 2;

[0016] Figure 4 The microscopic observation results of the cell invasion experiment in Example 3;

[0017] Figure 5 The statistical results of the number of invading cells in Example 3;

[0018] Figure 6 The results of microscopic observation of the cell migration experiment in Example 4 are shown;

[0019] Figure 7 The statistical results of cell migration rate in Example 4;

[0020] Figure 8 This is a flow cytometric analysis of the effects of different concentrations of compound 1 on the PANC-1 cell cycle in Example 5;

[0021] Figure 9is the relative ratio of the cell cycle at different concentrations of compound 1 in Example 5;

[0022] Figure 10 The Ki67 cell immunofluorescence staining results in Example 6;

[0023] Figure 11 is the Ki67 positive rate of the cells in Example 6;

[0024] Figure 12 This is the EdU staining result in Example 7;

[0025] Figure 13 is the EdU positive rate of the cells in Example 7;

[0026] Figure 14 This is the live / dead cell staining result in Example 8;

[0027] Figure 15 is the PI-positive cell ratio in Example 8;

[0028] Figure 16 This is a flow cytometric analysis of the effects of different concentrations of compound 1 on PANC-1 cell apoptosis in Example 9;

[0029] Figure 17 is the apoptosis rate of PANC-1 cells after being treated with different concentrations of Compound 1 in Example 9;

[0030] Figure 18 This is a photo of the tumor appearance in the animal experiment of Example 10;

[0031] Figure 19 The statistical results of the weight of mice in the animal experiment of Example 10;

[0032] Figure 20 The statistical results of tumor volume in the animal experiment of Example 10;

[0033] Figure 21 The H&E staining results of the tumor in the animal experiment of Example 10;

[0034] Figure 22 The Ki67 immunohistochemical staining results of the tumor in the animal experiment of Example 10;

[0035] Figure 23 The p53 immunohistochemical staining results of the tumor in the animal experiment of Example 10;

[0036] Figure 24 The results of TUNEL tissue immunofluorescence staining of tumors in the animal experiment of Example 10 are shown in FIG.

[0037] Figure 25The biochemical index test results of serum in the animal experiment of Example 10;

[0038] Figure 26 These are the H&E staining results of the liver and kidneys in the animal experiment of Example 10. DETAILED DESCRIPTION

[0039] The present invention provides an application of a shikonin derivative in the preparation of an anti-pancreatic cancer drug, wherein the shikonin derivative is one or more of a compound (Arnebidin) having a structure shown in Formula I and a pharmaceutically acceptable salt thereof;

[0040] Formula I.

[0041] The present invention has no special requirements on the source of the compound of the structure shown in Formula I, and can be prepared by using commercially available shikonin derivatives or by methods well known to those skilled in the art; in a specific embodiment of the present invention, the shikonin derivative of the structure shown in Formula I is extracted from Lithospermum erythrorhizon.

[0042] The present invention has no special requirements on the type of pharmaceutically acceptable salt of the compound represented by the structure of Formula I, and any salt familiar to those skilled in the art can be used.

[0043] In the present invention, the pancreatic cancer preferably includes pancreatic ductal adenocarcinoma.

[0044] In the present invention, the anti-pancreatic cancer drug preferably includes an active ingredient and a pharmaceutically acceptable excipient; the active ingredient includes a shikonin derivative; the present invention has no special requirements for the pharmaceutically acceptable excipient, and those familiar to those skilled in the art can be used, specifically pharmaceutically acceptable carriers, excipients, etc.

[0045] In the present invention, the dosage form of the anti-pancreatic cancer drug preferably includes injection, powder, granule, capsule or tablet.

[0046] In the present invention, the anti-pancreatic cancer drug is administered by injection or oral administration.

[0047] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0048] In the following examples, the shikonin derivative (Arnebidin) with the structure shown in Formula I is referred to as Compound 1.

[0049] Example 1 CCK-8 assay

[0050] PANC-1 cells were plated at 8,000 cells / well in a 96-well plate. After the cells recovered to normal, they were treated with compound 1 (DMSO) at different concentrations (0, 0.5, 1, 1.5, 2, 2.5, 5, 10, 20, and 40 μM). After 48 hours, the cells were incubated with CCK-8 solution at 37°C for 1 hour. The absorbance was measured at 450 nm, and statistical analysis was performed using Graphad Prism. The IC value of compound 1 for inhibiting PANC-1 cell proliferation was determined. 50 value.

[0051] At the same time, the same experiment was performed using shikonin and other shikonin derivatives to test the IC of shikonin and its derivatives in inhibiting PANC-1 cells. 50 The other shikonin derivatives used included deoxyshikonin, β,β-dimethylacryloylshikonin, 1,4-dihydroxy-5-methylanthracene-9,10-dione, shikonin furan A, shikonin furan E, β-acetoxyisovaleryl akanin, (+)-(R)-de-O-methyllasiodine, acetylshikonin, compound 2, and rod lactone A.

[0052] Structural formulas and ICs of compound 1, shikonin, and other shikonin derivatives 50 The test results are as follows Figure 1 As shown, the IC of compound 1 on PANC-1 cells 50 The value is 1.218 μM. The specific test results of compound 1 are as follows Figure 2 In addition, according to Figure 1 It can be seen that the IC of shikonin and other shikonin derivatives on PANC-1 cells 50 The values were significantly higher than those of compound 1.

[0053] Example 2 Clone formation experiment

[0054] PANC-1 cells were plated at 500 cells / well. The medium was changed every three days and the cell status was observed. After the number of cell clones exceeded 5, different concentrations of compound 1 (0, 0.5, 1, and 1.5 μM, solvent: DMSO) were added for treatment. When the cell clones were visible to the naked eye, the culture was terminated. The cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes and then stained with crystal violet solution for 10 minutes. After staining, the colony formation was recorded under an inverted microscope. The results are shown in Figure 2. Figure 3 As shown. Figure 3 The results show that compound 1 at different concentrations has a significant inhibitory effect on PANC-1 cells, and no clonal colonies can be formed at 1.5 μM.

[0055] Example 3 Cell invasion assay

[0056] After diluting Matrigel with pre-chilled serum-free medium at a volume ratio of 1:8 on ice, 60 μL of the dilution was evenly spread onto the upper chamber surface of the bottom membrane of the Transwell chamber. The wells were incubated in a 37°C incubator for 3 h to allow the Matrigel to polymerize into a thin film. The remaining liquid in the upper chamber was aspirated, and 100 μL of serum-free medium was added to each well. The wells were incubated in a 37°C incubator for 30 min to hydrate the basement membrane. The liquid in the upper chamber was aspirated, and PANC-1 cells in the logarithmic growth phase were digested, resuspended, and adjusted to a concentration of 5×10 4 / mL, 100 μL of PANC-1 cell suspension was inoculated into each well of the Transwell chamber and compound 1 was added to make the final concentrations of 0 μM, 1 μM, 2.5 μM, and 5 μM. 500 μL of culture medium containing 10% FBS was added to each well of the lower chamber. It was placed in a 37°C incubator and cultured for 48 h. The culture medium in the upper chamber was discarded, and the Matrigel gel and cells in the upper chamber were gently wiped with a cotton swab, and fixed with 4% paraformaldehyde for 20 min. Stained with 0.1% crystal violet stain for 3 min. The cells and excess stain in the chamber were wiped with a cotton swab, and the cell invasion was recorded under a microscope. The results are shown as follows. Figure 4 The number of invading cells was counted, and the results were as shown in Figure 5 As shown. Figure 4~Figure 5 As can be seen from the results, different concentrations of compound 1 can inhibit the invasion ability of PANC-1 cells.

[0057] Example 4 Cell migration experiment

[0058] The cell scratch insert was placed in a 12-well plate and fixed, and then PANC-1 cells were seeded in the wells. When the cell density reached 90%, the insert was removed with tweezers to generate a 500 μm wide scratch. The T0 time was recorded using an inverted microscope. Then, 0 μM, 1 μM, 2.5 μM, and 5 μM of compound 1 (solvent: DMSO) were added for 48 hours, and cell migration was recorded using an inverted microscope. The results are shown in Figure 2. Figure 6 As shown; the cell migration rate was statistically analyzed, and the results were as follows Figure 7 shown. Figure 6 and Figure 7 The results showed that compound 1 at different concentrations could inhibit the migration ability of PANC-1 cells with significant differences.

[0059] Example 5 Cell cycle experiment

[0060] PANC-1 cells were cultured at 2×10 5The cells were plated in 6-well plates. After the cells returned to normal, they were treated with 0 μM, 1 μM, 2.5 μM, and 5 μM of compound 1 (solvent was DMSO). After 48 h, PANC-1 cells treated with different concentrations of compound 1 were collected with 0.25% trypsin, and 1 mL of pre-cooled (0°C) 70% ethanol was added to each sample to resuspend the cells. The cell samples were fixed in an ice bath for 30 minutes and allowed to stand at 4°C overnight. The samples were centrifuged at 2500 rpm for 10 minutes, the cold ethanol was discarded, and the samples were washed twice with PBS. A mixed stain containing 5 μg / mL PI, 0.2% Triton X-100, and 100 μg / mL RNase A was added to each sample, and the cell samples were incubated at room temperature in the dark. The results were analyzed by flow cytometry. Figures 8 and 9 As shown, Figure 8 Flow cytometric analysis of the effects of different concentrations of compound 1 on the PANC-1 cell cycle; Figure 9 The relative ratios of cell cycles at different concentrations of compound 1. Figures 8 and 9 The results in the results showed that compound 1 could arrest the G0 / G1 phase of the cell cycle at low concentrations and the S phase of the cell cycle at high concentrations.

[0061] Example 6 Ki67 cell immunofluorescence assay

[0062] PANC-1 cells treated with compound 1 at different concentrations (0 μM, 1 μM, 2.5 μM and 5 μM, solvent was DMSO) were fixed with 4% paraformaldehyde at room temperature for 20 min, then permeabilized with 0.3% Triton X-100 for 15 min and blocked with 3% BSA for 30 min. After discarding the blocking solution, the samples were incubated with Ki67 overnight and then incubated with CoraLite 488-labeled goat anti-rabbit IgG (H + L) for 1.5 h. The nucleus was stained with DAPI and the cytoskeleton was stained with Actin-Tracker Red 594 (F-Actin). The number of Ki67 cell proliferation markers was observed and recorded using a fluorescence inverted microscope at a wavelength of 550 nm. Ki67 cell immunofluorescence staining is shown in Figure 2. Figure 10 As shown; the Ki67 positive cell rate was statistically analyzed, and the results were as follows Figure 11 As shown, according to Figure 10 and Figure 11 It can be seen that compound 1 significantly reduced the Ki67 positive ratio, indicating that compound 1 can inhibit the proliferation of PANC-1 cells.

[0063] Example 7 EdU experiment

[0064] In order to study the effect of compound 1 on DNA proliferation in PANC-1 cells, the EdU (5-ethynyl-2'-deoxyuridine) method was used to detect DNA replication. PANC-1 cell samples treated with different concentrations (0 μM, 1 μM, 2.5 μM and 5 μM, solvent is DMSO) of compound 1 were incubated with EdU at a final concentration of 10 μM for 30 min, washed once with PBS, and fixed with 1 mL of 4% paraformaldehyde solution per sample, and incubated at room temperature for 15 min; the paraformaldehyde was discarded and washed once with PBS, and permeabilized with 1 mL of 0.3% Triton-X-100 solution per sample, and incubated at room temperature for 15 min; the Triton-X-100 was discarded and washed twice with 1 mL of 3% BSA solution per sample; click reaction solution was prepared according to the instructions of the EdU cell proliferation detection kit, and the cell samples were protected from light for click reaction; washed twice with 3% BSA, and Hoechst 33342 staining solution was added to each sample, and incubated at room temperature in the dark to stain the cell nucleus; washed twice with PBS, and observed and photographed under an inverted fluorescence microscope under PBS environment. The results are shown in Figure 2. Figure 12 Quantification was performed using ImageJ software, and the quantitative statistical results are shown in Figure 13 As shown, according to Figure 12 and Figure 13 It can be seen that the DNA proliferation rate (EdU / H33342) of PANC-1 cells was significantly reduced after treatment with compound 1, and the compound 1 treatment group showed a significant difference compared with the control group.

[0065] Example 8 Live / dead cell staining experiment

[0066] PANC-1 cells were plated at 1×10 5 Cells were seeded in 12-well plates at a density of 100 μM. When the cell density reached approximately 50%, different concentrations of compound 1 (0 μM, 1 μM, 2.5 μM, and 5 μM) were added for treatment. Cell viability was detected using the Calcein AM cell viability assay kit, and the stained cells were recorded under a microscope. The results are shown in Figure 2. Figure 14 Quantitative statistics were performed using ImageJ software, as shown in Figure 15 As shown, according to Figure 14 and Figure 15 It can be seen that the number of PI-positive cells gradually increased, indicating that compound 1 can induce PANC-1 cell death.

[0067] Example 9 Cell apoptosis experiment

[0068] In order to explore whether compound 1 can induce apoptosis of PANC-1 cells, a cell apoptosis experiment was performed: PANC-1 cells treated with compound 1 were digested with 0.25% Trypsin without EDTA and centrifuged at 1000 rpm for 4 min. The cells were collected and washed once with PBS. The staining solution was prepared using the Annexin V-FITC cell apoptosis detection kit according to the instructions. 500 μL of staining solution was added to each sample and incubated at room temperature in the dark for 20 min. The cell samples were detected by flow cytometry. The results are shown in Figure 2. Figure 16-17 As shown, Figure 16-17 The results showed that PANC-1 cells showed apoptosis after treatment with compound 1, and the cell apoptosis rate gradually increased with the increase of treatment concentration.

[0069] Example 10 Animal Experiment

[0070] Eight 4-week-old female Balb / c nude mice were acclimated for one week and each mouse was subcutaneously injected with 400 μL (1×10 7 / mL) of PANC-1 cells. When the nude mouse transplanted tumor grows to 50 mm 3 Afterwards, 8 nude mice were randomly divided into 2 groups, 4 in each group. The drug-treated group received a 0.75 mg / kg solution of compound 1 (the solvent was a PBS solution containing 5% DMSO) and was injected once every 2 days, 100 μL / mouse each time. The control group received an injection of the same volume of solvent. The administration method was intraperitoneal injection, and the tumor size was measured before each injection. After 8 doses of administration, there was a significant difference in the volume of the transplanted tumors in the two groups. The mice were weighed, and then dissected to observe the tumor size and calculate the tumor volume. The results are shown in Figure 2. Figures 18 to 20 As shown, Figure 18 This is a photo of the tumor's appearance. Figure 19 The statistical results of mouse weight are shown in Figure 2. Figure 20 is the statistical result of tumor volume. Figures 18 to 20 The results in Figure 3 show that, compared with the control group, treatment with compound 1 can inhibit the growth of nude mouse transplanted tumors without obvious side effects.

[0071] The nude mouse transplanted tumor was fixed with 10% neutral formalin, dehydrated and embedded into paraffin blocks. The paraffin blocks were cut into 4 μm thick wax slices using a microtome. After the slices were dewaxed and hydrated, they were stained with hematoxylin and eosin (H&E). The changes in the pathological sections were then observed under a microscope. The results are as follows: Figure 21 shown. Figure 21 The results in the present study showed that treatment with compound 1 could not only inhibit the proliferation of transplanted tumors, but also destroy their pathological structures.

[0072] Immunohistochemistry was used to detect biomarkers in nude mouse xenograft tumors. Paraffin blocks were cut into 4 μm sections and oven-baked at 60°C for 1 h. Sections were removed, deparaffinized, hydrated, and then antigen retrieval was performed using a modified sodium citrate antigen retrieval solution. Endogenous peroxidase was inactivated by incubation in 3% H₂O₂ for 30 min in the dark. The sections were then blocked with ready-to-use normal goat serum for 30 min. After discarding the blocking solution, primary antibodies were added, including Ki67 (Rabbit, 1:800, ab15580, abcam, UK) and p53 (mouse, 1:2000, 60283-2-Ig, proteintech, China), respectively, at 4°C overnight. After washing with PBS, poly(HRP)-conjugated anti-rabbit / mouse IgG was added and incubated at room temperature in the dark for 30 min. Development was performed using DAB colorimetric solution. After development, the sections were counterstained with hematoxylin. The sections were then bluing in alkaline PBS. Finally, the sections were dehydrated and mounted. Scanning and observation were performed using a tissue slice scanner. The results were as follows: Figure 22-23 As shown, Figure 22 The results of Ki67 immunohistochemical staining are shown in Figure 2. Figure 23 The results of p53 immunohistochemical staining are shown. Figure 22-23 The results showed that compared with the control group, the level of proliferation marker Ki67 in the compound 1-treated group was significantly decreased, and the level of tumor suppressor p53 was significantly increased, indicating that compound 1 can inhibit the proliferation of transplanted tumors.

[0073] To detect apoptosis in tumor tissue, paraffin was cut into 4 μm slices and oven-baked at 60°C for 1 hour. After dewaxing and hydration, the slices were incubated with proteinase K working solution at 37°C for 20 minutes. They were then incubated with TUNEL reaction mixture at 37°C in the dark for 60 minutes. The staining solution was removed, and the cells were washed three times with PBS. Hoechst 33342 stain was added to stain the cell nuclei for 10 minutes. After washing three times with PBS, TUNEL-positive cells were observed under a fluorescence microscope. The results are shown in Figure 2. Figure 24 As shown. Figure 24 As can be seen from the results, the level of apoptosis marker TUNEL in the compound 1-treated group was significantly increased, indicating that compound 1 can induce apoptosis.

[0074] In order to detect the hepatotoxicity of compound 1, serum biochemical analysis and hematoxylin and eosin pathological staining were used for analysis. Blood was collected by eyeball sampling, and after the blood was completely coagulated, it was centrifuged (3000 rpm, centrifugation for 10 min) to separate the serum. The biochemical indicators related to liver and kidney function (AST, UREA, CREA-K) were then detected using an automatic biochemical analyzer. The results are shown as follows: Figure 25The liver and kidney pathological sections were stained with hematoxylin and eosin, and then the changes in the pathological sections were observed under a microscope. The results are shown in the figure below. Figure 26 shown. Figure 25 and 26 The results showed that compound 1 did not exhibit significant hepatotoxicity and renal toxicity in the tumor model and had good overall safety.

[0075] Example 11 Lip-MS experiment

[0076] PANC-1 cells were treated with different concentrations of compound 1 (0 μM and 2.5 μM). After 24 hours, the medium was discarded, the cells were washed with PBS, and then harvested. The cells were centrifuged at 1000 g for 10 minutes and washed three times with PBS. The supernatant was discarded, leaving a volume of approximately 50 μL per tube. Pre-chilled PBS (containing cocktail) was added to the cell pellet, followed by cryo-grinding and centrifugation at 12,000 g for 5 minutes at 4°C. The supernatant was collected. Protein quantification was performed by BCA. For the compound 1-treated group, 50 μg of protein was added with 100 μM of the small molecule drug and incubated at 25°C for 15 minutes. For the control group, 50 μg of protein was added with the same volume of solvent as the small molecule drug and incubated at 25°C for 15 minutes. Proteinase K (1 μg / μL, enzyme: sample ratio, 1:100) was added to the protein solutions of the compound 1-treated and control groups, and the digestion reaction was performed at 25°C for 3 minutes. The digestion reaction was immediately terminated by heating at 95°C for 5 minutes. After the previous treatment, the sample was removed and cooled to room temperature. An equal volume of 2% SDC (20 mM, in Tris-HCl) was then added, heated at 98°C for 5 minutes, and cooled to room temperature. Then, 5 μL of 0.1 M TCEP and 5 μL of 0.4 M CAM were added and incubated at 45°C at 1500 rpm in the dark for 5 minutes. The sample was removed and cooled to room temperature. Then, trypsin was added at a 1:50 enzyme-to-sample ratio and digested overnight at 37°C. After desalting using a C18 desalting column, the final peptide concentration was determined using a peptide quantification kit and lyophilized. The desalted, lyophilized peptides were reconstituted in phase A (0.1% formic acid in water) and analyzed by LC-MS / MS. DIA data were analyzed using Spectronaut19 default parameters (BGS Factory Settings (default)). The results are shown in Table 1, which indicate that PKM2 is a potential target of compound 1.

[0077] Table 1 Test results

[0078]

[0079] The results of the above examples show that the shikonin derivatives of the present invention have significant anti-tumor effects on pancreatic cancer PANC-1 cells. The present invention uses them in the preparation of anti-pancreatic cancer drugs and has broad application prospects.

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of shikonin derivatives in the preparation of anti-pancreatic cancer drugs, characterized in that: The shikonin derivative is one or more compounds of the structure shown in Formula I and pharmaceutically acceptable salts thereof; Formula I.

2. The use according to claim 1, characterized in that The pancreatic cancer includes pancreatic ductal adenocarcinoma.

3. The use according to claim 1, characterized in that The anti-pancreatic cancer drug comprises an active ingredient and pharmaceutically acceptable excipients; the active ingredient comprises a shikonin derivative having a structure shown in Formula I.

4. The use according to claim 1, characterized in that The dosage form of the anti-pancreatic cancer drug includes injection, powder, granule, capsule or tablet.

5. The use according to claim 1, characterized in that The anti-pancreatic cancer drug is administered by injection or oral administration.

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