Application of compounds and combination drugs in the preparation of antitumor drugs

Abstract

This application discloses the use of compounds and combination drugs in the preparation of antitumor drugs, wherein the compounds are stereoisomers, tautomers, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs of the compounds shown in formula (I); the drugs are used for antitumor purposes. The compounds and combination drugs of this application can effectively inhibit the replication and transcriptional expression of the KRAS and c-myc genes, and can activate the immune system while inhibiting tumor growth, without significant toxic side effects. They show promising prospects in the preparation of KRAS and c-myc gene inhibitors and in antitumor prevention and treatment.

Description

Technical Field

[0001] This application relates to the pharmaceutical field. Specifically, this application relates to the use of compounds and combinations of drugs in the preparation of antitumor drugs. Background Technology

[0002] Cancer (malignant tumors) has become one of the most serious diseases threatening human health and life. A report from the International Agency for Research on Cancer (IARC) of the United Nations indicates that by 2030, up to 13.2 million people worldwide will die from cancer each year. Drug therapy is a crucial component of cancer treatment, and effective anti-cancer drugs can help patients achieve longer survival times. Targeted anti-cancer drugs are developed to target tumor genes. They can identify characteristic sites in tumor cells determined by genes specific to tumor cells, and by binding to these sites (or similar mechanisms), block the signal transduction pathways controlling cell growth and proliferation within tumor cells, thereby killing tumor cells and preventing their proliferation.

[0003] RAS was the first human oncogene discovered. KRAS belongs to the RAS gene family, which also includes NRAS and HRAS. The full name of the KRAS gene is Kirsten rats arcomaviral oncogene homolog, and it is one of the most common oncogenes in solid tumors. Approximately one in seven cancer patients carry a KRAS mutation, including about 90% of pancreatic cancers, 40% of colorectal cancers, and 30% of non-small cell lung cancers. The KRAS gene acts like a "switch" in the body, playing a crucial regulatory role in signal transduction pathways related to tumor cell growth and angiogenesis. A normal KRAS gene inhibits tumor cell growth, but once mutated, it continuously stimulates cell growth, disrupting the growth pattern and leading to tumor development. Although KRAS was one of the first human proto-oncogenes discovered, it was previously considered an untreatable target.

[0004] The proto-oncogene c-myc is an important member of the myc family, encoding a phosphorylated protein in the cell nucleus. The c-myc gene plays a crucial role in maintaining normal cellular function, participating in cell proliferation, differentiation, and apoptosis. c-myc is closely related to human tumors and is found in various tumor tissues, such as lung cancer, gastric cancer, breast cancer, colon cancer, cervical cancer, certain neuroblastomas, granulocytic leukemia, retinoblastoma, osteosarcoma, chondrosarcoma, chordoma, liposarcoma, rhabdomyosarcoma, Hodgkin's disease, and head tumors. Amplification or overexpression of the c-myc gene can be detected in all these cases, and abnormalities in the proto-oncogene c-myc and its protein expression products can be detected. c-myc exhibits various characteristics that promote tumor development and progression. For example, overexpression of c-myc can accelerate the transition of cells from the G1 phase to the S phase and cause abnormalities in the cell cycle; it can enhance the utilization and absorption of nutrients by tumor cells and reduce apoptosis, thereby increasing cell proliferation. c-myc also participates in various signal transduction pathways, such as Wnt, Hippo, and PI3K / AKT / mTOR, through which it regulates physiological functions such as cell proliferation and differentiation, thereby affecting tumor development and progression.

[0005] Currently, drugs that target the KRAS and c-myc genes to treat tumors still need further investigation. Summary of the Invention

[0006] This application aims to at least partially address the technical problems existing in the prior art. To this end, this application proposes compounds and combination drugs, and their uses, which can effectively inhibit the replication and transcriptional expression of the KRAS and c-myc genes, and can activate the immune system while inhibiting tumor growth, without significant toxic side effects. These compounds and combination drugs show promising prospects in the preparation of KRAS and c-myc gene inhibitors and in anti-tumor prevention and treatment.

[0007] In one aspect of this application, the use of the compound in the preparation of a pharmaceutical remedy is proposed. According to embodiments of this application, the compound is a stereoisomer, tautomer, hydrate, solvate, pharmaceutically acceptable salt, or prodrug of the compound represented by formula (I).

[0008]

[0009] The drug is used for anti-tumor purposes.

[0010] The compounds according to the embodiments of this application can effectively inhibit the replication and transcriptional expression of the KRAS gene and the c-myc gene, and can activate the immune system while inhibiting tumor growth. They have the potential to be used in the clinical treatment of various types of tumors and have good application prospects.

[0011] According to embodiments of this application, the use of the above-mentioned compounds in the preparation of pharmaceuticals may also have the following additional technical features:

[0012] According to embodiments of this application, the tumor is selected from tumors that highly express the KRAS gene and / or the c-myc gene.

[0013] According to embodiments of this application, the drug is used to prevent or treat lung cancer, papillary thyroid carcinoma, lung adenocarcinoma, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, bile duct cancer, leukemia, neuroendocrine carcinoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, uterine cancer, cervical cancer, testicular cancer, nasopharyngeal carcinoma, anal cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma, or gastric cancer.

[0014] According to embodiments of this application, the drug further comprises one or both of nucleic acid-modified iron oxide nanoparticles and doxorubicin.

[0015] According to embodiments of this application, the compound and / or the doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles.

[0016] According to embodiments of this application, the compound is embedded in the nucleic acid-modified iron oxide nanoparticles, and the embedding rate of the compound is 80% to 90%.

[0017] According to an embodiment of this application, the doxorubicin is embedded in the nucleic acid-modified iron oxide nanoparticles, and the embedding rate of the doxorubicin is 85% to 95%.

[0018] According to embodiments of this application, the compound and doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles, with the embedding rate of the compound being 80%–90% and the embedding rate of the doxorubicin being 85%–95%.

[0019] According to embodiments of this application, the nucleic acid has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence having at least 80% homology with it.

[0020] According to embodiments of this application, the medicament further includes pharmaceutically acceptable excipients.

[0021] In another aspect of this application, a combination drug is proposed. According to an embodiment of this application, the combination drug comprises:

[0022] The compound is a stereoisomer, tautomer, hydrate, solvate, pharmaceutically acceptable salt, or prodrug of the compound shown in formula (I).

[0023]

[0024] One or both of nucleic acid-modified iron oxide nanoparticles and doxorubicin.

[0025] The combined drug according to the embodiments of this application can effectively inhibit the replication and transcriptional expression of KRAS gene and c-myc gene, and can activate immunity while inhibiting tumor growth, which is helpful for clinical application in the treatment of multiple types of tumors and has good application prospects.

[0026] According to embodiments of this application, the compound and / or the doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles.

[0027] According to embodiments of this application, the compound is embedded in the nucleic acid-modified iron oxide nanoparticles, and the embedding rate of the compound is 80% to 90%.

[0028] According to an embodiment of this application, the doxorubicin is embedded in the nucleic acid-modified iron oxide nanoparticles, and the embedding rate of the doxorubicin is 85% to 95%.

[0029] According to embodiments of this application, the compound and doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles, with the embedding rate of the compound being 80%–90% and the embedding rate of the doxorubicin being 85%–95%.

[0030] According to embodiments of this application, the nucleic acid has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence having at least 80% homology with it.

[0031] In another aspect of this application, the use of the aforementioned combined medicaments in the preparation of a medicament is proposed. According to embodiments of this application, the medicament is used for antitumor purposes.

[0032] According to embodiments of this application, the tumor is selected from tumors that highly express the KRAS gene and / or the c-myc gene.

[0033] According to embodiments of this application, the drug is used to prevent or treat lung cancer, papillary thyroid carcinoma, lung adenocarcinoma, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, bile duct cancer, leukemia, neuroendocrine carcinoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, uterine cancer, cervical cancer, testicular cancer, nasopharyngeal carcinoma, anal cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma, or gastric cancer.

[0034] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0035] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0036] Figure 1 The diagram shows the inhibition analysis of the KRAS gene by compound (I) according to Example 1 of the present invention;

[0037] Figure 2 The diagram shows the inhibition analysis of the c-myc gene by compound (I) according to Example 2 of the present invention;

[0038] Figure 3 The diagram shows the immunogenic cell death analysis induced by the in vitro cell-verified compound (I) according to Example 3 of the present invention;

[0039] Figure 4 The diagram showing the verification and analysis of the cGAS-STING signal path according to Embodiment 4 of the present invention is displayed.

[0040] Figure 5 Analysis of Transwell experimental results according to Embodiment 5 of the present invention;

[0041] Figure 6 Graph showing the in vivo antitumor experimental results of compound I according to Example 6 of the present invention;

[0042] Figure 7 In vivo toxicity evaluation chart of the compound according to Example 7 of the present invention;

[0043] Figure 8 The diagram shows a flow cytometry analysis of the antitumor immune response according to Embodiment 8 of the present invention;

[0044] Figure 9 The following graph shows the analysis results of the anti-tumor experiment on metastatic tumors according to Example 9 of the present invention;

[0045] Figure 10 The following graph shows the in vivo antitumor experimental results analysis of the compound I combination drug according to Example 10 of the present invention;

[0046] Figure 11 The diagram shows the experimental results analysis of the combination drug of compound I for metastatic tumors according to Example 11 of the present invention. Detailed Implementation

[0047] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0048] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0049] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0050] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0051] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0052] This application discloses the use of compounds in the preparation of pharmaceuticals, combination drugs, and their use in the preparation of pharmaceuticals, which will be described in detail below.

[0053] Application of compounds in drug preparation

[0054] In one aspect of this application, the application proposes the use of a compound in the preparation of a pharmaceutical product. According to embodiments of this application, the compound is a stereoisomer, tautomer, hydrate, solvate, pharmaceutically acceptable salt, or prodrug of the compound shown in formula (I).

[0055]

[0056] The drug is used for anti-tumor purposes.

[0057] The compounds according to the embodiments of this application (also referred to as "the compounds of this application") can effectively inhibit the replication and transcriptional expression of the KRAS gene and the c-myc gene, and can activate the immune system while inhibiting tumor growth, which is helpful for clinical application in the treatment of various types of tumors and has good application prospects.

[0058] "Stereoisomers" are compounds that have the same chemical structure but whose atoms or groups are arranged differently in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans) isomers, and hindered isomers, etc.

[0059] "Chirality" refers to molecules that have the property that cannot be superimposed on their mirror image; while "chirality" refers to molecules that can be superimposed on their mirror image.

[0060] "Enantiomers" refer to two non-overlapping but mirror-image isomers of a compound.

[0061] A diastereomer is a stereoisomer that has two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. Mixtures of diastereomers can be separated by high-resolution analytical procedures such as electrophoresis and chromatography, for example, HPLC.

[0062] The stereochemical definitions and rules used in this invention generally follow those of S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., “Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., New York, 1994.

[0063] Any asymmetric atom (e.g., carbon, etc.) in the compounds disclosed in this invention can exist in a racemic or enantiomerically enriched form, such as in (R)-, (S)-, or (R,S)- configurations. In some embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in the (R)- or (S)- configuration.

[0064] Any mixture of stereoisomers obtained can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization.

[0065] Various pharmaceutically acceptable salt forms of the compounds of this invention are useful. The term "pharmaceutically acceptable salt" refers to salt forms that are readily apparent to pharmaceutical chemists, i.e., they are substantially non-toxic and provide the desired pharmacokinetic properties, palatability, absorption, distribution, metabolism, or excretion. Other factors, more practical in nature and important for selection, include: cost of the raw materials, ease of crystallization, yield, stability, hygroscopicity, and the resulting flowability of the active pharmaceutical ingredient. Simply put, pharmaceutical compositions can be prepared from the active ingredient with a pharmaceutically acceptable carrier.

[0066] As used in this invention, "pharmaceutically acceptable salts" refers to the organic and inorganic salts of the compounds of this invention. Pharmaceutically acceptable salts are well-known in the field, as described in the literature: SMBerge et al., Describe Pharmaceutically Acceptable Salts in Detail in J. Pharmaceutical Sciences, 66:1-19, 1977. Pharmaceutically acceptable salts include, but are not limited to, iodized or brominated salts.

[0067] In this specification, if there are any differences between chemical names and chemical structures, the structure is preferred.

[0068] According to embodiments of this application, the tumor is selected from tumors that highly express the KRAS gene and / or the c-myc gene. The compounds of this application can effectively inhibit the replication and transcriptional expression of the KRAS gene and the c-myc gene, and are therefore particularly suitable for the prevention and / or treatment of tumors that highly express the KRAS gene and / or the c-myc gene.

[0069] According to embodiments of this application, the drug is used to prevent or treat lung cancer, papillary thyroid carcinoma, lung adenocarcinoma, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, bile duct cancer, leukemia, neuroendocrine carcinoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, uterine cancer, cervical cancer, testicular cancer, nasopharyngeal carcinoma, anal cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma, or gastric cancer.

[0070] In this document, the term "treatment" means the use of a drug to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" covers diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in individuals susceptible to disease but not yet diagnosed with the disease; (b) suppression of disease, such as inhibiting disease progression; or (c) alleviating disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any use of a drug to treat, cure, alleviate, improve, reduce, or suppress a disease in an individual, including but not limited to administering a drug containing the drugs described herein to an individual in need.

[0071] As used herein, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The compounds of the present invention can be administered via any common route, as long as it reaches the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, dermal, oral, local, nasal, pulmonary, and rectal administration, but the invention is not limited to these exemplified routes of administration.

[0072] The drug can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient in a single dose is generally the amount of the compound that produces the therapeutic effect. Generally, in units of one percent, this amount is from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0073] According to embodiments of this application, the drug further comprises one or both of nucleic acid-modified iron oxide nanoparticles and doxorubicin.

[0074] Doxorubicin, abbreviated as DOX, is an antibiotic with the chemical formula C64-320. 27 H 29 NO 11 Doxorubicin has a broad antitumor spectrum and is suitable for acute leukemia (lymphocytic and granulocytic), malignant lymphoma, breast cancer, bronchogenic carcinoma (undifferentiated small cell and non-small cell), ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, gastric cancer, liver cancer, etc. The inventors have discovered that the compound of this application, when administered in combination with doxorubicin, can achieve a better therapeutic effect.

[0075] Iron oxide nanoparticles (IONPs) can serve as drug carriers for targeted drug delivery, enhancing efficacy and reducing side effects. The nucleic acids modified on IONPs can bind to drug molecules, thereby loading the drug, and the nucleic acid sequences are rich in CpG, possessing immune-activating functions.

[0076] According to embodiments of this application, the compound and / or doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles. This allows for better delivery of the compound and / or doxorubicin into cells, improving bioavailability and enhancing therapeutic efficacy. Furthermore, embedding the compound and doxorubicin in the same or different nucleic acid-modified iron oxide nanoparticles yields even better results.

[0077] According to embodiments of this application, the compound is embedded in the nucleic acid-modified iron oxide nanoparticles, with an embedding rate of 80%–90%. This helps to better exert the therapeutic effect.

[0078] According to embodiments of this application, the doxorubicin is embedded in the nucleic acid-modified iron oxide nanoparticles, with an embedding rate of 85%–95%. This helps to better exert the therapeutic effect.

[0079] According to embodiments of this application, the compound and doxorubicin are encapsulated in the nucleic acid-modified iron oxide nanoparticles, with an encapsulation rate of 80%–90% for the compound and 85%–95% for the doxorubicin. This contributes to better therapeutic efficacy.

[0080] According to embodiments of this application, the nucleic acid has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence having at least 80% homology with it. This nucleic acid can bind to drug molecules, thereby loading drugs, and the nucleic acid sequence is rich in CpG, thus having an immune-activating function.

[0081] 5'-GGGTGTCGTTTGGGTGTCGTTTGGGTGTCGTTTGGGTT-3' (SEQ ID NO: 1)

[0082] According to embodiments of this application, the medicament further includes pharmaceutically acceptable excipients.

[0083] In this document, the term "pharmaceuticalally acceptable excipient" refers to a pharmaceutically acceptable excipient, carrier, excipient, solvent, or combination thereof used to deliver the active component of a medicament of the present invention to an animal or human. Exemplary excipients may be liquids or solids, including but not limited to: pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, flow aids, sweeteners, dyes / coloring agents, flavor enhancers, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, emulsifiers, sprays, compressed air or other suitable gases, or other suitable inactive ingredients used in conjunction with the pharmacodynamic compound. Examples of excipients include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, rubber arabic, alginate, gel, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0084] combination drugs

[0085] In another aspect of this application, a combination drug is proposed. According to an embodiment of this application, the combination drug comprises:

[0086] The compound is a stereoisomer, tautomer, hydrate, solvate, pharmaceutically acceptable salt, or prodrug of the compound shown in formula (I).

[0087]

[0088] One or both of nucleic acid-modified iron oxide nanoparticles and doxorubicin.

[0089] The combined drug according to the embodiments of this application can effectively inhibit the replication and transcriptional expression of KRAS gene and c-myc gene, and can activate immunity while inhibiting tumor growth, which is helpful for clinical application in the treatment of multiple types of tumors and has good application prospects.

[0090] According to embodiments of this application, the compound and / or doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles. This allows for better delivery of the compound and / or doxorubicin into cells, improving bioavailability and enhancing therapeutic efficacy.

[0091] According to embodiments of this application, the compound is embedded in the nucleic acid-modified iron oxide nanoparticles, with an embedding rate of 80%–90%. This helps to better exert the therapeutic effect.

[0092] According to embodiments of this application, the doxorubicin is embedded in the nucleic acid-modified iron oxide nanoparticles, with an embedding rate of 85%–95%. This helps to better exert the therapeutic effect.

[0093] According to embodiments of this application, the compound and doxorubicin are encapsulated in the nucleic acid-modified iron oxide nanoparticles, with an encapsulation rate of 80%–90% for the compound and 85%–95% for the doxorubicin. This contributes to better therapeutic efficacy.

[0094] According to embodiments of this application, the nucleic acid has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence having at least 80% homology with it. This nucleic acid can bind to drug molecules, thereby loading drugs, and the nucleic acid sequence is rich in CpG, thus having an immune-activating function.

[0095] It should be noted that the characteristics and advantages described above regarding the application of the compound in drug preparation also apply to this combination drug, and will not be repeated here.

[0096] Application of combination drugs in drug preparation

[0097] In another aspect of this application, the use of the aforementioned combined medicaments in the preparation of a medicament is proposed. According to embodiments of this application, the medicament is used for antitumor purposes.

[0098] According to embodiments of this application, the tumor is selected from tumors that highly express the KRAS gene and / or the c-myc gene.

[0099] According to embodiments of this application, the drug is used to prevent or treat lung cancer, papillary thyroid carcinoma, lung adenocarcinoma, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, bile duct cancer, leukemia, neuroendocrine carcinoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, uterine cancer, cervical cancer, testicular cancer, nasopharyngeal carcinoma, anal cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma, or gastric cancer.

[0100] It should be noted that the features and advantages described above for the application of combination drugs and compounds in drug preparation also apply to the application of this combination drug in drug preparation, and will not be repeated here.

[0101] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0102] All oligonucleotide sequences used in the embodiments of this invention were synthesized and modified by Shanghai Sangon Biotech Co., Ltd., and purified using high-performance liquid chromatography. Cell lines HeLa, MCF-7, and A549 were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0103] Compound I in the examples is

[0104] Example 1 verifies the inhibitory effect of compound I on the KRAS gene.

[0105] (1) Experimental procedure:

[0106] Control group (blank group) cells (culture medium without compound) and experimental group cells (HeLa, A549, MCF-7) were cultured in culture medium containing compound (I) at a final concentration of 4 μM for 48 hours, and then the cells (approximately 5 × 10⁶ cells) were collected. 7 Culture medium composition: RPMI 1640 medium (HyClone) containing 10% (v / v) fetal bovine serum (Gibco), 0.1 mg / mL streptomycin (Sigma-Aldrich), and 100 U / mL penicillin (Sigma-Aldrich).

[0107] Total RNA was extracted from cells using the Trizol method, and after concentration calibration, it was reverse transcribed into cDNA. The mRNA transcription level of the KRAS gene was further determined using quantitative real-time PCR. The loading amount and the concentration of each reagent in the system were calculated according to experimental requirements. The PCR program was set as follows: 37℃, 25–60 min, 98℃, 5 min, 4℃ hold. The program was run; after the program was completed, the cDNA was stored at -20℃.

[0108] Primers for real-time PCR:

[0109] KRAS-For: 5'-ACACAAACAGGCTCAGGACT-3', SEQ ID NO: 2;

[0110] KRAS-Rev: 5'-TGTCGGATCTCCCTCACCAA-3', SEQ ID NO: 3;

[0111] β-actin-For: 5'-AGCACTGTGTTGGCGTACAG-3', SEQ ID NO: 4;

[0112] β-actin-Rev: 5'-TCCCTGGAGAAGAGCTACGA-3', SEQ ID NO: 5.

[0113] (2) Experimental results:

[0114] Real-time PCR, such as Figure 1 As shown, after compound I was incubated with three types of cancer cells for 48 hours, the mRNA transcription level of the KRAS gene was significantly downregulated, showing a significant difference compared to the control group. This indicates that compound I can effectively inhibit KRAS gene expression.

[0115] Example 2 verifies the inhibitory effect of compound I on the c-myc gene.

[0116] (1) Experimental procedure:

[0117] Control group (blank group) cells (cells without compound + culture medium) and experimental group cells (HeLa, A549, MCF-7) were cultured in culture medium containing compound I at a final concentration of 4 μM for 48 hours, and then the cells (approximately 5 × 10⁻⁶) were collected. 7 Culture medium composition: RPMI 1640 medium (HyClone) containing 10% (v / v) fetal bovine serum (Gibco), 0.1 mg / mL streptomycin (Sigma-Aldrich), and 100 U / mL penicillin (Sigma-Aldrich).

[0118] Total RNA was extracted from cells using the Trizol method, and after concentration calibration, it was reverse transcribed into cDNA. The mRNA transcription level of the c-myc gene was further determined using quantitative real-time PCR. The loading amount and the concentration of each reagent in the system were calculated according to experimental requirements. The PCR program was set as follows: 37℃, 25–60 min, 98℃, 5 min, 4℃ hold. The program was run; after the program was completed, the cDNA was stored at -20℃.

[0119] Primers for real-time PCR:

[0120] c-myc-For: 5'-GAGAGGCAGAGGGAGCGAGCGGGC-3'SEQ ID NO: 6;

[0121] c-myc-Rev: 5'-TGTCGTTGAGAGGGTAGGGGAAGA-3'SEQ ID NO:7;

[0122] β-actin-For: 5'-AGCACTGTGTTGGCGTACAG-3'SEQ ID NO: 4;

[0123] β-actin-Rev: 5'-TCCCTGGAGAAGAGCTACGA-3' SEQ ID NO: 5.

[0124] (2) Experimental results:

[0125] Real-time PCR, such as Figure 2 As shown, after compound I was incubated with three types of cancer cells for 48 hours, the mRNA transcription level of the c-myc gene was significantly downregulated, showing a significant difference compared to the control group. This indicates that compound I can effectively inhibit c-myc gene expression.

[0126] Example 3: In vitro cell validation of immunogenic cell death induced by compound I

[0127] The immunogenic death induced by compound I in 4T1 tumor cells was verified by detecting the surface expression of calreticulin (CRT) and the release of high-migration protein 1 (HMGB1).

[0128] 1. Calretin

[0129] 4T1 cells were fed at a dose of 1×10 6Cells were seeded at a density of cells / well in confocal dishes and incubated overnight in a cell culture incubator. The original culture medium was then discarded, and the cells were divided into two groups: one group received culture medium containing PBS, and the other group received culture medium containing compound I (0.2 μg / mL). Cells were incubated for another 24 hours. After incubation, the culture media containing different materials were discarded, and the cells were washed three times with PBS. Cells were then fixed with freshly prepared cell fixation solution at room temperature for 7 min, followed by permeabilization with PBS solution containing 0.2% Triton X-100 for 5 min. After washing the cells three times with PBS, they were incubated with freshly prepared immunofluorescence blocking solution at room temperature for 30 min to reduce non-specific antibody binding. Simultaneously with blocking, the rabbit monoclonal antibody against calreticulin was diluted 1:1000 (v / v) and the Alexa Fluor 647-labeled goat anti-rabbit secondary antibody was diluted 1:500 (v / v) using immunofluorescence antibody dilution buffer. After blocking, the prepared primary antibody solution was added to the confocal dishes and incubated overnight at 4°C. The following day, the primary antibody solution was aspirated from the confocal dish, and the cells were washed three times with PBS. Then, diluted secondary antibody solution was added, and the cells were incubated at room temperature in the dark for 1 hour. Finally, Hoechst 33342 was added, and the cells were incubated at room temperature for 5 minutes. The cells were then observed under a laser confocal microscope. The excitation wavelength selected for the Hoechst 33342 channel was 405 nm, and the excitation wavelength selected for the calreticulin channel was 635 nm.

[0130] For flow cytometry analysis of cell surface CRT, 4T1 cells were analyzed at a concentration of 1×10⁻⁶. 6 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured overnight in a cell culture incubator. The next day, culture medium containing PBS and Compound I (0.2 μg / mL) was added to the 6-well plates and cultured for another 24 hours. Subsequently, the culture medium containing the material was discarded, cells were washed with PBS, and 200 μL of trypsin was added to digest the cells. The cells were transferred to 1.5 mL centrifuge tubes, and the supernatant was removed by centrifugation to obtain a cell pellet. Simultaneously, the CRT rabbit monoclonal antibody was diluted 1:100 with freshly prepared flow cytometry antibody dilution buffer, and the Alexa Fluor 647-labeled goat anti-rabbit secondary antibody was diluted 1:200. After centrifugation, the diluted CRT antibody was added and incubated at 4°C for 30 min. After washing with PBS, the diluted secondary antibody was added again and incubated at 4°C for 30 min. Finally, the cells were washed with PBS and analyzed using an Agilent NovoCyte flow cytometer.

[0131] 2. High-mobility protein 1

[0132] The release of high-mobility protein 1 (HMGB1) was measured using an ELISA kit from Wuhan Elite Biotech Co., Ltd. In addition to the control and experimental groups, a standard curve was also determined during the test.

[0133] The specific steps are as follows:

[0134] 4T1 cells were fed at a dose of 1×10 6 Cells were seeded at a density of 1 cell / well into 6-well plates and incubated overnight. The following day, two groups were divided into two experiments: one group was incubated with PBS, and the other group was incubated with compound I (0.2 μg / mL) in 6-well plates for 24 hours. Subsequently, the cell supernatant was collected, and the released HMGB1 was quantitatively analyzed according to the ELISA kit instructions.

[0135] The experimental results of Example 3 are as follows: Figure 3 As shown, Figure 3 Figure A shows the laser confocal imaging of calreticulin (CRT), where the expression level of calreticulin was significantly enhanced after the addition of compound I compared to the PBS group. Figure B shows the flow cytometry results of calreticulin, where the expression level of calreticulin was significantly enhanced in the group with compound I compared to the PBS group. Figure C shows the detection results of high-migration protein 1 (HMGB1), where the expression level was significantly enhanced in the group with compound I compared to the PBS group. This indicates that compound I can upregulate the expression of calreticulin and high-migration protein 1 in solid tumors, activate immunogenic cell death (ICD), and improve the anti-tumor effect.

[0136] Example 4: cGAS-STING Signal Path Verification

[0137] 1. Immunofluorescence staining of phosphorylated STING (p-STING) protein

[0138] 4T1 cells were fed at a dose of 1×10 6Cells were seeded at a density of cells / well in confocal dishes and incubated overnight in a cell culture incubator. The original culture medium was then discarded, and the cells were divided into two groups: one group received culture medium containing PBS, and the other group received culture medium containing compound I (0.2 μg / mL). Cells were incubated for another 24 hours. After incubation, the culture media containing different materials were discarded, and the cells were washed three times with PBS. Cells were then fixed with freshly prepared cell fixation solution at room temperature for 7 min, followed by permeabilization with PBS solution containing 0.2% Triton X-100 for 5 min. After washing the cells three times with PBS, they were incubated with freshly prepared immunofluorescence blocking solution at room temperature for 30 min to reduce non-specific antibody binding. Simultaneously with blocking, p-STING rabbit monoclonal antibody was diluted 1:1000 (v / v) and Alexa Fluor 647-labeled goat anti-rabbit secondary antibody was diluted 1:500 (v / v) using immunofluorescence antibody dilution buffer. After blocking, the prepared primary antibody solution was added to the confocal dishes and incubated overnight at 4°C. The following day, the primary antibody solution was aspirated from the confocal dish, and the cells were washed three times with PBS. Then, diluted secondary antibody solution was added, and the cells were incubated at room temperature in the dark for 1 hour. Finally, Hoechst 33342 was added, and the cells were incubated at room temperature for 5 minutes. The cells were then observed under a laser confocal microscope. The excitation wavelength selected for the Hoechst 33342 channel was 405 nm, and the excitation wavelength selected for the p-STING channel was 635 nm.

[0139] 2. Western blot (WB)

[0140] 4T1 cells were fed at a dose of 1×10 6Cells were seeded at a density of [number] cells / well in 6-well plates and incubated overnight in a cell culture incubator. The original culture medium was then discarded, and the cells were divided into two groups: one group received culture medium containing PBS, and the other group received culture medium containing compound I (0.2 μg / mL). Cells were incubated for another 24 h. After incubation, the culture media containing different materials were discarded, and the cells were washed once with PBS. Next, 200 μL of high-efficiency RIPA lysis buffer containing protease / phosphatase inhibitors was added to each well of the 6-well plate, and the plates were incubated on ice for 30 min. The lysed samples were centrifuged at 12000g for 5 min, and the supernatant was collected. The extracted proteins were quantified using a BCA protein assay kit. 10% and 12% SDS-PAGE gels were prepared according to the size of the target proteins, and the proteins were separated using a vertical electrophoresis tank, with Rainbow 180 broad-spectrum protein markers used as band indicators. After electrophoresis, the proteins from the SDS-PAGE gels were transferred to a PVDF membrane via a transfer process. After transfer, the PVDF membrane was removed, placed in an antibody incubation chamber, and freshly prepared Western blotting (WB) blocking buffer was added. The membrane was incubated overnight at 4°C on a shaker. The next day, the membrane was washed at least three times with pre-prepared TBST solution. The interferon regulatory factor 3 (p-IRF3) monoclonal antibody and p-STING monoclonal antibody were diluted 1:1000 with freshly prepared WB antibody dilution buffer, and the internal control protein GAPDH antibody was diluted 1:5000. Subsequently, the prepared primary antibody solution was added to the antibody incubation chamber, and the membrane was incubated overnight at 4°C on a shaker. The following day, the membrane was washed at least three times with pre-prepared TBST solution, and horseradish peroxidase (HRP)-labeled secondary antibody was diluted 1:1000 with freshly prepared WB antibody dilution buffer. The prepared secondary antibody solution was added to the antibody incubation chamber, incubated for 1 hour at room temperature on a shaker, and excess secondary antibody solution was removed with TBST solution. Finally, the PVDF membrane was placed in an ECL chemiluminescence reagent and imaged using a fluorescence imaging analyzer.

[0141] Figure 4 A shows the immunofluorescence staining of p-STING protein observed under a laser confocal microscope. The fluorescence of the group with added compound I was significantly stronger than that of the control group. Figure 4 B shows Western blotting (WB) fluorescence imaging, where the fluorescence of both p-IRF3 antibody and p-STING antibody in the compound I group was stronger than that in the PBS control group. This indicates that compound I can activate the STING pathway and promote T cell activation and recruitment.

[0142] Example 5: Transwell Experiment

[0143] 4T1 cells were fed at a dose of 1×10 6Cells were seeded at a density of 1 cell / well into the upper chamber of a 0.4 μm transwell and cultured overnight in a cell culture incubator. The next day, two groups were divided into two experiments: one group was incubated with medium containing PBS, and the other group was incubated with medium containing compound I (0.2 μg / mL) for 12 h. Subsequently, the original medium was discarded, fresh medium was added, and the 4T1 cells in the upper chamber and the immature mouse myeloid dendritic cells (BMDCs) in the lower chamber were co-incubated and cultured for another 24 h in a cell culture incubator. Next, the cell culture medium was collected, and the expression levels of interleukin-6 (IL-6) and α-tumor necrosis factor (TNF-α) were measured using an ELISA kit. In addition, the BMDCs in the lower chamber of the transwell were collected, and a flow cytometry antibody dilution containing PE-CD11c, FITC-CD80, and APC-CD86 antibodies was added. The cells were incubated at 4 °C on a shaker for 30 min, washed with PBS, and analyzed using an Agilent NovoCyte flow cytometer.

[0144] Figure 5 A shows the flow cytometry results of CD86 and CD80 in the Transwell assay. Figure 5 B shows the percentage of mature DC. Figure 5 C and 5D show the expression levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), respectively. These data support the immune-enhancing effect of compound I.

[0145] Example 6

[0146] 1. Preparation of IONP-G2

[0147] 700 mg of ferric acetylacetone was accurately weighed and added to a three-necked flask, followed by 3 mL of oleic acid, 2 mL of oleylamine, and 10 mL of 1-octadecene. The flask was placed in a temperature-controlled heating device, magnetically stirred, evacuated, and maintained at 90 °C for 15 minutes. After evacuation, the nitrogen gas was turned on to bring the entire reaction system under nitrogen protection, and the mixture was heated to 200 °C and refluxed for half an hour. The mixture was then gradually cooled to room temperature to obtain IONP. The synthesized IONP was washed by centrifugation with cyclohexane and anhydrous ethanol, and the washed nanoparticles were dispersed in cyclohexane. The IONP was then surface-modified with 50 mg of bisphosphonate-PEG-maleimide (DP-PEG-MAL) to ensure uniform dispersion in the aqueous phase. Using the Michael addition reaction between maleimide and thiol groups, thiol-modified nucleic acids were coupled to the IONP surface to prepare IONP-G2. After ultrafiltration with ultrapure water, each IONP contained approximately 300 nucleic acid molecules.

[0148] 2. Preparation of nanoparticles

[0149] DOX and Compound I were added separately or together to the above IONP-G2 solution. The mixture was shaken overnight at room temperature. The sample was then transferred to an ultrafiltration tube and ultrafiltered at 6000 rpm for 10 minutes. The sample was washed three times with a physiological buffer solution containing potassium ions. The liquid in the upper layer of the ultrafiltration tube was collected to obtain IONP-G2-DOX (DOX encapsulation rate 92%, liquid contains 3 mg / kg Fe and 1.2 mg / kg DOX), IONP-G2-Compound I (Compound I encapsulation rate 87%, liquid contains 3 mg / kg Fe, 1.2 mg / kg DOX and 0.25 mg / kg Compound I), and IONP-G2-DOX / Compound I (DOX and Compound I encapsulation rates 89% and 85%, respectively, liquid contains 3 mg / kg Fe, 1.2 mg / kg DOX and 0.25 mg / kg Compound I). The samples were stored at 4°C for later use.

[0150] Example 7: In vivo antitumor experiment

[0151] To evaluate the in vivo tumor-suppressing effect, a subcutaneous mouse breast cancer tumor model was constructed. The specific procedures were as follows: Well-grown 4T1 cells were digested with 1 mL of trypsin and incubated at 37°C for 3 min. Immediately after incubation, 5 mL of culture medium was added to terminate the digestion. The cells were gently pipetted from the bottom of the culture dish, centrifuged at 3000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in PBS. 100 μL of the cell suspension (approximately 5 × 10⁶ cells / mL) was then collected. 5 (Number of cells) were injected into the upper right hip area of ​​BALB / c mice to construct a subcutaneous tumor model. One week later, the tumor had grown to approximately 100 mm. 3 The size of the tumor was then determined. Subsequently, 15 tumor-bearing mice were randomly divided into three groups of five: PBS, IONP-G2, DOX (1.2 mg / kg), and compound I (0.25 mg / kg). On days 1, 3, and 5 of treatment, 100 μL of the corresponding material was injected intravenously into the tumor-bearing mice. The mouse weight and tumor length and width were recorded every other day, according to the formula V(mm). 3 = length × width 2 The tumor volume was calculated by multiplying the result by 0.5. After the experiment, mice were sacrificed, tumor tissue was removed, photographed, and weighed. The isolated tumor tissue was then subjected to hematoxylin-eosin (H&E) staining, terminal deoxynucleotidyl transferase-mediated dUTP in situ end-of-cut labeling (TUNEL) fluorescent staining, immunofluorescence staining, and immunohistochemical staining to further evaluate the in vivo antitumor effect.

[0152] Figure 6 A shows the body weight of the experimental mice. Figure 6 B represents the change in the size of the tumor. Figure 6C represents the difference in tumor weight. Figure 6 D represents the staining of a section of tumor tissue. (Through...) Figure 6 The data from each group show that compound I has a significant inhibitory effect on tumors. When used at doses lower than DOX, its inhibitory effect is comparable to that of DOX, while compound I has no obvious toxic side effects. Figure 6 As can be seen from D, compound I not only kills tumors but also promotes the activation of the immune system.

[0153] Example 8 In vivo toxicity evaluation

[0154] To evaluate the potential toxicity of compound I, tumor tissue and major organs such as the heart, liver, spleen, lungs, and kidneys were collected from mice sacrificed in Example 6 and preserved in 4% tissue fixative for hematoxylin-eosin (H&E) staining. Serum biological factors were simultaneously measured to evaluate the effects of the material on renal and hepatic function, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE).

[0155] Figure 7 A shows stained sections of various mouse organs. Figure 7 B shows the differences in the levels of ALT, AST, BUN, and CRE under different drug treatments. Figure 7 The data from each group show that the compound has no significant toxic side effects.

[0156] Example 9: Flow Cytometry Analysis of Antitumor Immune Response

[0157] To evaluate the immune-activating effect of the material on the host, BALB / c tumor-bearing mice were injected with 100 μL of PBS, 1.2 mg / kg DOX, and 0.25 mg / kg compound I on days 1 and 3, respectively. On day 7, the mice were sacrificed, and tumor tissue, tumor draining lymph nodes (TDLN), and spleen were harvested. The collected organs were homogenized using freshly prepared tissue homogenate to obtain a cell suspension. Subsequently, the cells were filtered through a 70 μm cell filter to obtain a single-cell suspension. Splenic cells were treated with erythrocyte lysis buffer, and lysis was terminated with homogenate after 5 min. Cells were collected by centrifugation at 3000 rpm / min for 5 min, and fresh homogenate was added to obtain a single-cell suspension free of erythrocytes. The obtained single-cell suspension was stained with flow cytometry antibody for qualitative analysis of mature dendritic cells (CD45) in tumor tissue and TDLN. + CD11c + CD80 + CD86 + CD8 in tumor tissue + T cells (CD3) + CD8 +) and regulatory T cells (Tregs, CD45) + CD3 + CD4 + FOXP3 + ), Central memory CD8 in spleen tissue + T cells (CD3) + CD8 + CD44 + CD62L + The flow cytometry antibodies used were: ER780 Anti-Mouse CD45, PerCP / Cy5.5 Anti-Mouse CD3, FITC Anti-Mouse CD4, PE Anti-Mouse CD8a, PE / Cy7 Anti-Mouse CD44, PE Anti-Mouse CD11c, FITC Anti-Mouse CD80, APC Anti-Mouse CD86, APC Anti-Mouse CD62L, and PE Anti-Mouse FOXP3.

[0158] Figure 8 Demonstrates activation of the immune response in vivo, including representative flow cytometry plots of TDLNs and mature dendritic cells in tumor tissue; CD8+ in tumor tissue. + Representative flow cytometry plots of T cells; representative flow cytometry plots of Treg cells in tumor tissue; (E) representative flow cytometry plots of central memory CD8+ T cells in the spleen. The data in the figures demonstrate the activation of the immune response by compound I in a mouse tumor model. This indicates that the compound has an immune-activating effect in vivo.

[0159] Example 10: Anti-tumor experiment on metastatic tumors

[0160] To evaluate the inhibitory effect of the material on metastatic or recurrent tumors, a metastatic tumor model was constructed. 100 μL of a suspension of 4T1 cells (approximately 5 × 10⁻⁶ cells) was collected. 5 (Number of cells) were injected into the upper right hip area of ​​BALB / c mice to construct a subcutaneous tumor model. One week later, the tumor had grown to approximately 100 mm. 3 The size of the tumor was determined. Subsequently, 15 tumor-bearing mice were randomly divided into three groups: PBS, 1.2 mg / kg DOX, and 0.25 mg / kg compound I, with 5 mice in each group. On days 1, 3, and 5 of treatment, 100 μL of the corresponding material was injected intravenously into the tumor-bearing mice. On day 7, another 100 μL of 4T1 cell suspension (approximately 5 × 10⁻⁶ cells) was collected. 5 (Number of cells) were injected into the upper left hip area of ​​BALB / c mice to artificially construct a metastatic tumor model. The mouse's body weight and the length and width of the metastatic tumor were recorded every other day, according to the formula V(mm).3 = length × width 2 The volume of the metastatic tumor was calculated by multiplying the result by 0.5. After the experiment, the mice were sacrificed, and the metastatic tumor tissue was extracted for flow cytometry analysis and immunohistochemical staining.

[0161] Figure 9 This study demonstrates the antitumor effect of compound I on a metastatic 4T1 tumor model, including (A) tumor growth curves of metastatic tumors (n=5); and (B) CD8+ in metastatic tumor tissue. + (C) Immunohistochemical staining of CD8 and granzyme B in metastatic tumor tissue. Granzyme B can activate apoptosis by activating Caspase-3, and can also reflect the state of immune activation in the body. Figure 9 It can be seen that compound I inhibited metastatic tumors after activating immunity.

[0162] Example 11: In vivo antitumor experiment of compound I combination drug

[0163] Compound I was combined with DOX and nucleic acid-modified iron oxide (IONP) nanoparticles to prepare a combination drug. The tumor-inhibiting effects of different materials were evaluated using a mouse subcutaneous breast cancer tumor model. The specific procedures were as follows: Well-grown 4T1 cells were digested with 1 mL of trypsin and incubated at 37°C for 3 min. Immediately after incubation, 5 mL of culture medium was added to terminate the digestion. The cells were gently pipetted from the bottom of the culture dish, centrifuged at 3000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in PBS. 100 μL of the cell suspension (approximately 5 × 10⁻⁶ cells / mL) was collected. 5 (Number of cells) were injected into the upper right hip area of ​​BALB / c mice to construct a subcutaneous tumor model. One week later, the tumor had grown to approximately 100 mm. 3 The size of the tumor was determined. Subsequently, 35 tumor-bearing mice were randomly divided into 7 groups: PBS, IONP-G2, DOX, Compound I, IONP-G2-DOX, IONP-G2-Compound I, and IONP-G2-DOX / Compound I, with 5 mice in each group. On days 1, 3, and 5 of treatment, 100 μL of the corresponding material was injected intravenously into the tumor-bearing mice. The mouse weight and tumor length and width were recorded every other day, according to the formula V(mm). 3 = length × width 2 The tumor volume was calculated by multiplying the result by 0.5. After the experiment, the mice were sacrificed, the tumor tissue was removed, photographed, and weighed.

[0164] like Figure 10As shown, compared with the control group PBS, tumor growth in the IONP-G2-DOX group, the IONP-G2-compound I group, and the IONP-G2-DOX / compound I group was inhibited to varying degrees. Among them, the IONP-G2-DOX / compound I group showed the best anti-tumor effect, with a significantly lower average tumor mass (0.39±0.09g) than that in the PBS group (1.87±0.20g). This indicates that the combination of compound I and other therapeutic adjuvants has a synergistic tumor therapeutic effect. Furthermore, there was no significant change in mouse body weight recorded during treatment, indicating that the treatment regimen had no obvious toxic side effects.

[0165] Example 12: Antitumor Experiment of Combination Drugs in Metastatic Tumors

[0166] Take 100 μL of 4T1 cell suspension (approximately 5 × 10⁻⁶ cells). 5 (Number of cells) were injected into the upper right hip area of ​​BALB / c mice to construct a subcutaneous tumor model. One week later, the tumor had grown to approximately 100 mm. 3 The size of the tumor. Subsequently, 35 tumor-bearing mice were randomly divided into 7 groups: PBS, IONP-G2, DOX, compound I, IONP-G2-DOX, IONP-G2-compound I, and IONP-G2-DOX / compound I, with 5 mice in each group. On days 1, 3, and 5 of treatment ( Figure 11 A) 100 μL of the corresponding material was injected intravenously into tumor-bearing mice. On day 7, another 100 μL of 4T1 cell suspension (approximately 5 × 10⁻⁶ cells) was collected. 5 (Number of cells) were injected into the upper left hip area of ​​BALB / c mice to artificially construct a metastatic tumor model. The mouse's body weight and the length and width of the metastatic tumor were recorded every other day, according to the formula V(mm). 3 = length × width 2 The volume of the metastatic tumor was calculated by multiplying the result by 0.5. After the experiment, the mice were sacrificed, the metastatic tumor tissue was removed, the tumor was weighed, and flow cytometry analysis was performed.

[0167] Studies have found that the IONP-G2-DOX / compound I combination drug exhibits significant tumor-suppressive effects compared to other treatment groups. Figure 11 B). After treatment, the mice were sacrificed, and the metastatic tumor tissue was extracted and analyzed by flow cytometry for CD8+. + The proportion of T cells. For example... Figure 11As shown in Figure C, the expression level of CD8+ T cells in metastatic tumor tissues treated with IONP-G2-DOX / compound I was significantly increased, being 2.45, 1.52, and 1.82 times higher than that in the IONP-G2, DOX, and compound I groups, respectively. These results indicate an effective immunotherapy effect in metastatic tumors. In summary, these results suggest that IONP-G2-DOX / compound I treatment can elicit a strong immune response and has an inhibitory effect on metastatic or recurrent tumors.

[0168] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The application of the compound in the preparation of a drug, characterized in that, The compound is the compound shown in formula (I) or a pharmaceutically acceptable salt. Equation (I); The drug is used to prevent or treat tumors, wherein the tumor is selected from lung adenocarcinoma.

2. The application according to claim 1, characterized in that, The tumor was selected from KRAS Gene overexpression and / or c-myc Tumors with high gene expression.

3. The application according to claim 1, characterized in that, The drug is used to prevent or treat metastatic or recurrent cancer.

4. The application according to claim 1, characterized in that, The drug further comprises one or both of nucleic acid-modified iron oxide nanoparticles and doxorubicin.

5. The application according to claim 4, characterized in that, The compound and / or the doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles; Optionally, the compound is embedded in the nucleic acid-modified iron oxide nanoparticles, and the embedding rate of the compound is 80% to 90%. Optionally, the doxorubicin is embedded in the nucleic acid-modified iron oxide nanoparticles, and the embedding rate of the doxorubicin is 85%~95%. Optionally, the compound and doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles, wherein the embedding rate of the compound is 80%~90% and the embedding rate of the doxorubicin is 85%~95%. Optionally, the nucleic acid has a nucleotide sequence as shown in SEQ ID NO:

1.

6. The application according to claim 1, characterized in that, The drug further includes pharmaceutically acceptable excipients.

7. The application of the compound in the preparation of a drug, characterized in that, The compound is the compound shown in formula (I) or a pharmaceutically acceptable salt. Equation (I); The drug is used to prevent or treat tumors, wherein the tumor is selected from breast cancer. The drug comprises nucleic acid-modified iron oxide nanoparticles and doxorubicin; The nucleic acid has a nucleotide sequence as shown in SEQ ID NO:

1.

8. The application according to claim 7, characterized in that, The compound and / or the doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles; Optionally, the compound is embedded in the nucleic acid-modified iron oxide nanoparticles, and the embedding rate of the compound is 80% to 90%. Optionally, the doxorubicin is embedded in the nucleic acid-modified iron oxide nanoparticles, and the embedding rate of the doxorubicin is 85%~95%. Optionally, the compound and doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles, with the encapsulation rate of the compound being 80% to 90% and the encapsulation rate of the doxorubicin being 85% to 95%.

9. The application according to claim 7, characterized in that, The tumor was selected from KRAS Gene overexpression and / or c-myc Tumors with high gene expression.

10. A combination therapy for treating tumors, wherein the tumor is selected from breast cancer, characterized in that, include: The compound is a compound of formula (I) or a pharmaceutically acceptable salt. Equation (I); Nucleic acid-modified iron oxide nanoparticles and doxorubicin; The nucleic acid has a nucleotide sequence as shown in SEQ ID NO:

1.

11. The combined drug according to claim 10, characterized in that, The compound and / or the doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles; Optionally, the compound is embedded in the nucleic acid-modified iron oxide nanoparticles, and the embedding rate of the compound is 80% to 90%. Optionally, the doxorubicin is embedded in the nucleic acid-modified iron oxide nanoparticles, and the embedding rate of the doxorubicin is 85%~95%. Optionally, the compound and doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles, with the encapsulation rate of the compound being 80% to 90% and the encapsulation rate of the doxorubicin being 85% to 95%.

12. The combined drug according to claim 10, characterized in that, The tumor was selected from KRAS Gene overexpression and / or c-myc Tumors with high gene expression.

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