CN112494467A - Application of vanillin in preparation of nicotinamide-N-methyltransferase inhibitor - Google Patents
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- CN112494467A CN112494467A CN202011399745.1A CN202011399745A CN112494467A CN 112494467 A CN112494467 A CN 112494467A CN 202011399745 A CN202011399745 A CN 202011399745A CN 112494467 A CN112494467 A CN 112494467A
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- vanillin
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Images
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention discloses an application of vanillin in preparation of a nicotinamide-N-methyltransferase inhibitor. The inhibitor is prepared from a traditional Chinese medicine monomer vanillin and pharmaceutically available auxiliary materials. The invention inhibits the proliferation of various colorectal cancer cell strains by inhibiting NNMT expression and enzyme activity in vivo and in vitro, and plays an anti-tumor role. The research of the invention shows that vanillin can weaken the drug resistance of colorectal cancer cell strains to 5-FU caused by NNMT high expression in vitro, thereby enhancing the chemotherapy sensitivity; the apoptosis of a colorectal cancer cell line with high expression of NNMT is increased by inducing the generation of intracellular ROS and mitochondrial damage; can enhance the inhibition effect and apoptosis rate of 5-fluorouracil on tumor proliferation and enhance the chemotherapy effect. The vanillin can be used as NNMT inhibitor for preparing anti-colorectal cancer drugs or auxiliary chemotherapy drugs, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of medicines, and relates to an application of vanillin in preparation of a nicotinamide-N-methyltransferase inhibitor, in particular to a method for preventing and treating colorectal cancer by taking vanillin serving as a traditional Chinese medicine monomer NNMT inhibitor.
Background
The incidence and mortality rate of colorectal cancer increases year by year in the world, and the incidence and mortality rate of colorectal cancer in China are the top five of all cancers and are still continuously rising. Chemotherapy is one of the important measures for clinically treating malignant tumors, and chemotherapy resistance is the main reason of poor prognosis of colorectal cancer chemotherapy, so that the research and development of new effective anti-colorectal cancer drugs and the search of new treatment schemes are the problems which are urgently needed to be solved in the current colorectal cancer treatment. Chinese medicinal plants have abundant resources, and Chinese medicinal monomers have the advantages of safety, effectiveness and the like, so that the Chinese medicinal plants become important sources of medicaments and lead compounds. Most anticancer drugs used in modern medicine are natural products or derivatives of natural products such as paclitaxel, taxotere, vincristine, vinblastine, camptothecin, irinotecan, and the like. The screening and research on the medicinal molecular mechanism of the active monomer of the traditional Chinese medicine and the search for the molecular target point of the direct action of the active monomer of the traditional Chinese medicine are important research directions for searching novel chemotherapeutic drugs and reducing the drug resistance of colorectal cancer chemotherapy, and have important significance for guiding the formulation of an individualized treatment scheme, improving the effective rate of chemotherapy, prolonging the life cycle of colorectal cancer patients and improving the life quality of the patients.
Nicotinamide-N-methyltransferase (NNMT) is an important metabolic enzyme, and has the main function of catalyzing methylation of Nicotinamide (NA) and pyridine compounds to form pyridine salts by taking S-adenosyl-L-methionine (SAM) as a methyl donor, wherein the main products are 1-Methylnicotinamide (MNA) and S-adenosyl homocysteine (SAH). Nicotinamide is one of the important sources of NAD + through the salvage synthesis pathway, and NAD + is involved in a variety of basic functions of cells such as energy metabolism, resistance to stress and injury, and lifespan of cells. On the other hand, nicotinamide as a metabolite can inhibit NAD + digestive enzymes, which can decompose NAD + into nicotinamide and ADP-ribosyl, which is important for maintaining steady-state equilibrium of NAD + in vivo. Nicotinamide is also an inhibitor of the DNA single-strand repair protein PARP, NA is consumed by NNMT enzymatic reaction, and the reduction of the cellular level of NA may enhance the activity of PARP enzyme, thereby enhancing the repair of DNA damage. NNMT is abnormally expressed in cancer tissues, and is over-expressed in various tumor tissues such as colorectal cancer, breast cancer, pancreatic cancer, lung cancer, gastric cancer and the like. NNMT overexpression can affect various biological functions of tumor cells, is related to the occurrence and development of various tumors, is probably a key protein influencing the curative effect of chemotherapy, and can also be a potential target of tumor molecule targeted therapy.
Vanillin (Vanillin), also known as Vanillin, can be extracted directly from plants such as Vanilla, can also be synthesized industrially, and is a widely used edible spice. The existing research shows that vanillin has the activities of resisting cell mutation and tumor, and can inhibit the proliferation and migration of tumor cells, the generation of tumor blood vessels and the like. Vanillin has synergistic effect with other chemotherapeutic drugs or natural drug molecules, and can enhance the curative effects of radiotherapy and chemotherapy. However, the action target and molecular mechanism of vanillin on colorectal cancer and the effect of synergistic colorectal cancer chemotherapy are not researched.
Disclosure of Invention
The invention aims to provide an application of vanillin in preparation of a nicotinamide-N-methyltransferase (NNMT) inhibitor. The inhibitor is prepared from a traditional Chinese medicine monomer vanillin and pharmaceutically available auxiliary materials. The effective concentration range of vanillin in the inhibitor is 0-4 millimoles per liter. The inhibition is to inhibit the proliferation and the cloning formation of various colorectal cancer cell strains and induce the apoptosis of colorectal cancer cells by inhibiting the expression and the activity of NNMT.
The vanillin is white or light yellow needle-shaped or crystalline powder at normal temperature, has poor solubility in polar solution, can be dissolved in organic solvents such as DMSO and ethanol, and is stored in a dark place. The molecular weight of the vanillin is as follows: 152.15, CAS number: 121-33-5, linear formula: 4- (HO) C6H3-3-(OCH3) CHO, having the following structure of formula (I):
the inhibitors of the present invention may be used alone or in combination with other drugs.
The present invention provides a method for inhibiting the expression of NNNT in cells cultured in vitro by adding vanillin to the cell culture medium. The cells are colorectal cancer cell strains SW480 and HT-29, and simultaneously comprise an SW480 cell strain which enables NNMT to be highly expressed through genetic engineering modification and an HT-29 cell strain which enables NNMT to be lowly expressed through a gene knock-down technology.
The invention discloses a new application of a traditional Chinese medicine active monomer small molecule compound vanillin, and in vitro experiments show that vanillin can inhibit the expression and activity of NNMT, further inhibit the proliferation of various colorectal cancer cell strains and induce apoptosis. The mechanism research shows that vanillin can increase the mitochondrial injury and the generation of ROS. In vivo experiments show that the combination of vanillin and 5-fluorouracil can enhance the inhibition of colorectal cancer cell proliferation by utilizing a nude mouse model of human colorectal cancer cell tumor. The invention discusses the anti-tumor activity and the specific action mechanism of vanillin from a plurality of aspects such as in vitro, in vivo and biochemical molecular level. Provides a new lead compound for the NNMT inhibitor and provides theoretical guidance for vanillin serving as the NNMT inhibitor to be used for personalized treatment of colorectal cancer.
Drawings
FIG. 1 shows colorectal cancer cell lines with different expression levels of NNMT.
FIG. 2 shows that vanillin inhibits colorectal cancer cell proliferation and decreases NNMT-associated 5-fluorouracil resistance.
Figure 3 shows that vanillin inhibits NNMT expression and enzyme activity in colorectal cancer cells.
FIG. 4 shows a spatial mimetic of vanillin in combination with NNMT.
FIG. 5 shows that vanillin can induce apoptosis of colorectal cancer cells and reduce the inhibitory effect of NNMT on apoptosis.
FIG. 6 shows that vanillin activates apoptosis-related protein expression.
FIG. 7 shows that vanillin increases 5-fluorouracil-induced apoptosis.
Figure 8 shows that vanillin causes apoptosis by inducing mitochondrial damage and ROS.
FIG. 9 shows that vanillin can increase apoptosis induced by 5-fluorouracil in tumor tissues of tumor-bearing mice and synergistically inhibit tumor proliferation.
Detailed Description
The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.
Example 1
A technical method
Construction and identification of cell model for NNMT modified expression
In the invention, firstly, a gene engineering method is utilized to establish a colorectal cancer cell strain SW480/NNMT with stable and high expression of NNMT and a control group cell strain SW 480/NC; and establishing an NNMT knock-down expression colorectal cancer cell line HT-29/shNNMT and a control group cell line HT-29/NC. The specific method comprises the following steps:
cloning the full-length cDNA sequence (SEQ ID NO.1-3) of NNMT, connecting with pcDNA3.1+ plasmid vector, and constructing eukaryotic expression vector pcDNA3.1 +/NNMT; transfecting a colorectal cancer cell strain SW480, screening and monoclonalizing cell strains, and identifying the cell strains with stable and high expression of NNMT; meanwhile, SW480 cell line transfected by pcDNA3.1+ empty plasmid was established as a control.
Designing a plurality of pairs of specific NNMT shRNA, constructing NNMT shRNA lentivirus, and infecting a colorectal cancer cell strain HT-29 with high NNMT expression; meanwhile, non-specific shRNA lentivirus transfected HT-29 is established as a negative control.
Detecting the expression of NNMT in the constructed cell model by fluorescent quantitative PCR (SEQ ID NO.4-7) and immunoblotting; NNMT enzyme activity is measured by an HPLC method, and the operation method is briefly described as follows: preparing a reaction buffer system according to substrate-level reaction conditions, adding a certain amount of sample to be detected into the reaction system to start reaction, measuring the variable of the product MNA by using HPLC, and evaluating the change of the enzyme activity according to the change of the amount of the MNA.
2. Cell viability assay
The colorectal cancer cells HT-29 and SW480 used in the invention and the corresponding NNMT modified cell strains are cultured in a constant temperature incubator (humidity 95 percent, CO)2 Concentration 5%). Cell viability was measured using the CCK-8 kit. Cells were cultured at 8X 103The mixture was inoculated into a 96-well plate at a density per well, the monomeric compound was added at different concentrations 24 hours later, and the same amount of DMSO was added to control groups, each group having 3 wells. After further culturing for 48 hours, 10. mu.l of CCK-8 was added thereto and incubated at 37 ℃ for 1 to 2 hours, and the absorbance at 450nm was measured with a microplate reader. The experiment was independently repeated 3 times.
3. Clone formation experiments
Cells were seeded in six well plates at 1000 cells per well, and 24 hours later the medium was changed and complete medium containing the corresponding concentrations of the monomeric compound was added. After one week of culture, the medium was discarded, washed with PBS 3 times, treated with 4% paraformaldehyde solution for 20min to fix the cells, stained with 0.5% crystal violet stain for 5min, then washed away with tap water gently to remove unbound crystal violet stain, and naturally dried at room temperature. The number of cell clones formed was counted by taking pictures under a microscope.
4. Immunoblotting (Western Blotting) experiment
After the cells are treated by drugs with different concentrations for 48 hours, the cells are cracked by a cracking solution, total cell proteins are extracted after low-temperature centrifugation, the denatured proteins are boiled and then are separated by polyacrylamide gel electrophoresis, the proteins are transferred to PVDF from the polyacrylamide gel, the PVDF is incubated overnight by using corresponding primary antibody, and PBST is washed for three times to remove the unbound primary antibody. The secondary antibody was incubated with HPR-labeled antibody at room temperature and then washed three times with PBST to remove unbound secondary antibody. ECL developed and photographed.
5. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) assay
After the cells are treated by drugs with different concentrations for 48 hours, TRIzol is used for separating and extracting RNA, after reverse transcription, corresponding gene specific primers are used for detecting the mRNA level (SEQ ID NO.4-7) of the corresponding gene by a fluorescent quantitative PCR kit.
6. High Performance Liquid Chromatography-mass spectrometry (High Performance Liquid Chromatography-MS/MS, HPLC-MS/MS) method for detecting 1-MNA level
Collecting 100ul biological sample or standard, adding 200ul 1% zinc sulfate solution to precipitate protein, shaking for 15min, centrifuging at 14000rpm for 15min, and analyzing the supernatant with AB-SCIEX-TRIPLE-QUADTM4500MD liquid chromatography tandem mass spectrometry system. mu.L of the above supernatant was injected into a Jasper-connected Eclipse XDB-C18(4.6X150 mm, 5 μm) column. Chromatographic conditions are as follows: mobile phase A: 0.1% formic acid water by volume percentage, mobile phase B: 0.1% by volume of formic acid methanol; gradient elution is carried out at the flow rate of 0.6 mL/min; the column temperature is 35 ℃; and taking the marked concentration of the standard as a horizontal coordinate, taking the ratio of the actual detection peak area to the respective internal standard peak area as a vertical coordinate, drawing a standard curve and calculating a curve regression equation, and substituting the ratio of the actual detection peak area to the internal standard peak area in the sample into the standard curve regression equation to calculate the concentration of the 1-MNA in the sample.
7. Apoptosis detection
After the cells are treated by drugs with different concentrations for 48 hours, cell culture medium and the cells are collected, the culture medium is washed away by PBS after centrifugation, and then the number of apoptotic cells is counted by a flow cytometer after staining by an Annexin V-PE/7-AAD apoptosis detection kit and an Annexin V-FITC/PI apoptosis detection kit of BD company according to instructions. Tumor tissue apoptosis was detected by TUNEL in situ apoptosis detection kit from Roche (Roche) according to the instructions, followed by staining and photographing with a fluorescence microscope to count the number of apoptotic cells.
8. Reactive Oxygen Species (ROS) detection
After the cells are treated by the drugs with different concentrations for 48 hours, the cells are collected, centrifuged, washed by PBS and washed off the culture medium, stained by an active oxygen detection kit of Shanghai Biyuntian biotechnology limited company according to the instruction, detected by a flow cytometer, and the fluorescence intensity of ROS is counted.
9. Mitochondrial membrane potential detection
After the cells are treated by the drugs with different concentrations for 48 hours, the cells are collected, centrifuged, washed by PBS and washed off the culture medium, stained by a mitochondrial membrane potential detection kit of Shanghai Bintian biotechnology limited company according to the instruction, detected by a flow cytometer, and counted in proportion of red fluorescence and green fluorescence.
10. Animal tumor model experiment
The procedure of the animal experiment was in accordance with the care and use of NIH test animals and animal theory. Male BALB/c mice of about four weeks of age were purchased from Shanghai Spiker laboratory animals, and were housed in the SPF-class animal research laboratory center. Mice were inoculated subcutaneously with SW480/NNMT and SW480/NC cells, and after about 10 days when the solid tumors were about 5mm in diameter, they were randomly divided into two groups, i.e., 5 fluorouracil (30mg/kg) was intraperitoneally injected every three days, followed by increasing the treatment with vanillin in combination with vanillin, and vanillin (100mg/kg) was intraperitoneally injected every other day. Treatment was continued for three weeks. After the animal pharmacology experiment is finished, the mouse is sacrificed and stripped, the tumor body size is weighed and counted, the slicing experiment operation is carried out in a pathology room, the tumor block is fixed, dehydrated and embedded in the paraffin block, and the slicing, dyeing and observation are carried out.
Second, results and analysis
1. Traditional Chinese medicine monomer vanillin remarkably inhibits colorectal cancer cell proliferation and reduces NNMT-related chemotherapy drug resistance
The immunoblotting experiment detects the expression level of NNMT of various colorectal cancer cell lines, and cell lines SW480 with negative expression of NNMT, cell lines HT-29 with positive expression of NNMT and HCE8693 are screened (figure 1A). The eukaryotic expression vector pcDNA3.1+/NNMT (SEQ ID NO.1-3) is constructed, the SW480 cell NNMT high-expression clone strain (figure 1B) is successfully constructed, shRNA lentivirus is used for interfering the expression of the HT-29 cell strain NNMT, and the cell strain which stably interferes the expression of the NNMT is successfully established (figure 1C).
The SW480 and HT-29 cell lines constructed above, CCK-8 and clonogenic experiments were treated with different concentrations of vanillin, and the results of the clone formation experiments showed that vanillin had significantly different IC50 in cell lines with different expression levels of NNMT. Vanillin has a more significant effect of inhibiting cell proliferation in cell lines with high NNMT expression (FIGS. 2A-D).
Cells were treated with a combination of vanillin and 5-fluorouracil at low toxic concentrations, and CCK-8 examined cell viability and calculated the IC50 for 5-fluorouracil. The result shows that the high expression of NNMT can obviously increase the IC50 of colorectal cancer cell line to 5-fluorouracil, namely increase the drug resistance to 5-fluorouracil. After the combination of vanillin and 5-fluorouracil, the IC50 of colorectal cancer cell lines to 5-fluorouracil can be obviously reduced, namely the sensitivity to 5-fluorouracil is increased (fig. 2E and F).
2. Vanillin can remarkably inhibit expression and activity of NNMT in colorectal cancer cells HT-29
HT-29 cells were treated with two vanillin concentrations (2.5mM and 3.5mM) respectively, and NNMT gene expression (SEQ ID NO.4-7) was detected by a fluorescence quantitative method using GAPDH as an internal reference, and NNMT protein expression was detected by an immunoblot assay using β -Actin as an internal reference. The results show that vanillin can inhibit the expression of NNMT at both the transcriptional and protein levels (fig. 3A-C). We assessed changes in NNMT enzyme activity using high phase liquid chromatography-mass spectrometry techniques to detect the levels of the metabolite 1-MNA of NNMT. The results show that vanillin can inhibit NNMT enzyme activity (fig. 3D). We simulated the three-dimensional structure of the NNMT enzyme after binding to the substrate in the active state (fig. 4A), and the three-dimensional structure of vanillin with NNMT in the active state, with the small molecule indicated by the arrow representing vanillin (fig. 4B). Vanillin is presumed to inhibit NNMT enzyme activity sterically.
3. Vanillin can induce apoptosis of colorectal cancer cells and increase apoptosis induced by 5-fluorouracil
Treating colorectal cancer cell strains with different NNMT expression levels by using vanillin with different concentrations, and detecting apoptosis by using flow cytometry. The results show that high expression of NNMT can inhibit apoptosis, vanillin can increase apoptosis, and the inhibition effect of NNMT on apoptosis is weakened (figure 5). The apoptosis-related protein was detected by immunoblotting, and the result showed that vanillin could activate the expression of apoptosis-related protein (FIG. 6). The result of treating colorectal cancer cells by combining vanillin and 5-fluorouracil shows that NNMT high expression can reduce apoptosis induced by 5-fluorouracil, and chemotherapy resistance is caused. Vanillin can increase apoptosis induced by 5-fluorouracil and increase drug sensitivity (FIG. 7).
4. Vanillin can induce apoptosis by inducing mitochondrial damage and ROS
Vanillin is used for treating the colorectal cancer SW480 and a SW480/NNMT cell strain with high expression of NNMT, and the intracellular ROS level and the mitochondrial damage level are detected by flow cytometry. The vanillin-induced mitochondrial damage and ROS levels can be attenuated with the ROS scavenger NAC. Also, vanillin-induced apoptosis could be attenuated after scavenging ROS with NAC (fig. 8). The results are combined to conjecture that vanillin can induce the ROS level in the cells to be increased by inducing the mitochondrial injury, and then induce the apoptosis.
5. Vanillin can inhibit proliferation of colorectal cancer of nude mice bearing tumor, and increase apoptosis of tumor cells
A tumor-bearing nude mouse model is constructed by using colorectal cancer SW480 and a SW480/NNMT cell strain with corresponding high expression of NNMT, 5-fluorouracil is used for single or combined with vanillin for intraperitoneal injection for three weeks, and the result shows that the high expression of NNMT can enable tumor tissues to resist the 5-fluorouracil. The 5-fluorouracil combined vanillin can enhance the inhibition effect of 5-fluorouracil on tumors and increase the chemotherapy sensitivity. The TUNEL method is used for detecting the apoptosis condition in tumor tissues, and the result shows that NNMT can reduce the apoptosis induced by 5-fluorouracil, and vanillin can increase the apoptosis after combining with 5-fluorouracil (figure 9).
Sequence listing
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Application of vanillin in preparation of nicotinamide-N-methyltransferase inhibitor
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Claims (5)
1. Use of vanillin in the preparation of a nicotinamide-N-methyltransferase inhibitor, wherein the effective concentration of vanillin in the inhibitor is in the range of 0 to 4 mmoles/l.
2. The use of claim 1, wherein the inhibitor is prepared from a traditional Chinese medicine monomer vanillin and pharmaceutically acceptable excipients.
3. The use of claim 1, wherein the inhibition is inhibition of the proliferation, clonality and induction of apoptosis of a plurality of colorectal cancer cell lines by inhibiting the expression and activity of NNMT.
4. The use of claim 1, wherein the inhibitor is administered alone or in combination with other drugs.
5. The use of claim 3, wherein the cells are colorectal cancer cell lines SW480 and HT-29, and the cell lines SW480 and HT-29 are genetically modified to express NNMT in high levels and genetically modified to express NNMT in low levels.
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| CN112710767A (en) * | 2020-12-07 | 2021-04-27 | 浙江大学 | Tandem mass spectrometry kit for measuring NNMT catalytic substrate and NNMT catalytic product |
| CN113413386A (en) * | 2021-07-07 | 2021-09-21 | 兰州大学 | Application of vanillin derivative in preparation of medicine for treating colorectal cancer combined with fusobacterium nucleatum infection |
| WO2023020561A1 (en) * | 2021-08-20 | 2023-02-23 | 南京施江医药科技有限公司 | Application of benzisoquinoline diketone compound in treatment of tumor |
| WO2023020539A1 (en) * | 2021-08-19 | 2023-02-23 | 南京施江医药科技有限公司 | Application of benzanilide compound in tumor treatment |
| WO2023025011A1 (en) * | 2021-08-23 | 2023-03-02 | 南京施江医药科技有限公司 | Application of hydrazide compound in tumor treatment |
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| CA2709441A1 (en) * | 1998-05-19 | 1999-11-25 | Research Development Foundation | Triterpene compositions and methods for use thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112710767A (en) * | 2020-12-07 | 2021-04-27 | 浙江大学 | Tandem mass spectrometry kit for measuring NNMT catalytic substrate and NNMT catalytic product |
| CN113413386A (en) * | 2021-07-07 | 2021-09-21 | 兰州大学 | Application of vanillin derivative in preparation of medicine for treating colorectal cancer combined with fusobacterium nucleatum infection |
| CN113413386B (en) * | 2021-07-07 | 2022-04-08 | 兰州大学 | Application of vanillin derivative in preparation of medicine for treating colorectal cancer combined with fusobacterium nucleatum infection |
| WO2023020539A1 (en) * | 2021-08-19 | 2023-02-23 | 南京施江医药科技有限公司 | Application of benzanilide compound in tumor treatment |
| WO2023020561A1 (en) * | 2021-08-20 | 2023-02-23 | 南京施江医药科技有限公司 | Application of benzisoquinoline diketone compound in treatment of tumor |
| WO2023025011A1 (en) * | 2021-08-23 | 2023-03-02 | 南京施江医药科技有限公司 | Application of hydrazide compound in tumor treatment |
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