Application of compound and combined drug in preparation of antitumor drug

Abstract

The invention provides an application of a compound and a combined drug in preparation of antitumor drugs. The compound is a stereoisomer, a tautomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug of the compound as shown in a formula (I), the medicine is used for resisting tumors. The compound and the combined drug can effectively inhibit the replication and transcription expression of the KRAS gene and the c-myc gene, can activate immunity while inhibiting the growth of tumors, have no obvious toxic and side effects, and have good prospects in the application of preparing KRAS gene and c-myc gene inhibitors and anti-tumor prevention and treatment. # imgabs0 #

Description

Technical Field

[0001] The present application relates to the field of medicine, and in particular, to the use of compounds and combined drugs in the preparation of anti-tumor drugs. Background Art

[0002] Cancer (malignant tumor) has become the most serious disease threatening human health and life. According to a report by the United Nations International Cancer Research Center, by 2030, up to 13.2 million people will die of cancer each year worldwide. Drug therapy is a very important part of cancer treatment, and effective anti-cancer drugs can help patients survive longer. Targeted anti-cancer drugs are developed for tumor genes. They can recognize characteristic sites in tumor cells that are determined by genes specific to tumor cells, and by binding to them (or other similar mechanisms), block the signal transduction pathways in tumor cells that control cell growth and proliferation, thereby killing tumor cells and preventing their proliferation.

[0003] RAS is the first human tumor gene discovered. KRAS belongs to the RAS gene family, in addition to NRAS and HRAS. The full name of the KRAS gene is Kirsten rats arcomaviral oncogene homolog, which is translated into Chinese as Kirsten rat sarcoma viral oncogene homolog. It is one of the most common oncogenes in solid tumors. About one in seven cancer patients carry KRAS mutation genes, including about 90% of pancreatic cancer, 40% of colorectal cancer and 30% of non-small cell lung cancer. The KRAS gene is like a "switch" in the body. It plays an important regulatory role in the signal transduction pathway of tumor cell growth and angiogenesis. The normal KRAS gene can inhibit tumor cell growth, but once it mutates, it will continue to stimulate cell growth, disrupt the growth pattern, and thus lead to the occurrence of tumors. Although KRAS is one of the first human proto-oncogenes discovered, it has been considered an undruggable target in the past.

[0004] The proto-oncogene c-myc is an important member of the myc family, which encodes phosphorylated proteins in the cell nucleus. The c-myc gene plays an important role in maintaining normal cell function and is involved in cell proliferation, differentiation and apoptosis. c-myc is closely related to human tumors and is found in various tumors in many 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. There is amplification or overexpression of the c-myc gene, and abnormalities of the proto-oncogene c-myc and its protein expression products can be detected. c-myc has many characteristics that promote the occurrence and development of tumors. For example, overexpression of c-myc can lead to an acceleration of the rate at which cells enter the S phase from the G1 phase and abnormalities in the cell cycle; it can enhance the utilization and absorption of nutrients by tumor cells and reduce the apoptosis process, thereby enhancing the cell's reproductive capacity; c-myc is also involved in a variety of signal transduction pathways, such as Wnt, Hippo and PI3K / AKT / mTOR, etc., through which it regulates physiological functions such as cell proliferation and differentiation, thereby affecting the occurrence and development of tumors.

[0005] Currently, drugs that target KRAS and c-myc genes to treat tumors remain to be studied. Summary of the invention

[0006] The present application aims to solve the technical problems existing in the prior art at least to a certain extent. To this end, the present application proposes compounds and combined drugs and their uses, which can effectively inhibit the replication and transcriptional expression of KRAS gene and c-myc gene, and can activate immunity while inhibiting tumor growth, without obvious toxic side effects, and show good prospects in the preparation of KRAS gene and c-myc gene inhibitors and anti-tumor prevention and treatment applications.

[0007] In one aspect of the present application, the present application proposes the use of a compound in the preparation of a drug. According to an embodiment of the present application, the compound is a stereoisomer, a tautomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug of the compound represented by formula (I),

[0008]

[0009] The medicine is used for anti-tumor.

[0010] The compounds according to the embodiments of the present application can effectively inhibit the replication and transcriptional expression of the KRAS gene and the c-myc gene, and can activate immunity while inhibiting tumor growth. They may be clinically applied to the treatment of multiple types of tumors and have good application prospects.

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

[0012] According to an embodiment of the present application, the tumor is selected from tumors with high expression of KRAS gene and / or high expression of c-myc gene.

[0013] According to an embodiment of the present application, the drug is used to prevent or treat lung cancer, papillary thyroid cancer, 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 cancer, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, uterine cancer, cervical cancer, testicular cancer, nasopharyngeal cancer, anal cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma or gastric cancer.

[0014] According to an embodiment of the present application, the drug further includes one or both of nucleic acid-modified iron oxide nanoparticles and doxorubicin.

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

[0016] According to an embodiment of the present 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 the present 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 an embodiment of the present application, the compound and doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles, the embedding rate of the compound is 80% to 90%, and the embedding rate of the doxorubicin is 85% to 95%.

[0019] According to an embodiment of the present application, the nucleic acid has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence having at least 80% homology thereto.

[0020] According to an embodiment of the present application, the drug further includes a pharmaceutically acceptable excipient.

[0021] In another aspect of the present application, the present application proposes a combined drug. According to an embodiment of the present application, the combined drug comprises:

[0022] A compound, wherein the compound is a stereoisomer, a tautomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug of the compound represented by 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 the present application can effectively inhibit the replication and transcriptional expression of the KRAS gene and the 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 an embodiment of the present application, the compound and / or the doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles.

[0027] According to an embodiment of the present 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 the present 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 an embodiment of the present application, the compound and doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles, the embedding rate of the compound is 80% to 90%, and the embedding rate of the doxorubicin is 85% to 95%.

[0030] According to an embodiment of the present application, the nucleic acid has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence having at least 80% homology thereto.

[0031] In another aspect of the present application, the present application proposes the use of the above-mentioned combined drug in the preparation of a drug. According to an embodiment of the present application, the drug is used for anti-tumor.

[0032] According to an embodiment of the present application, the tumor is selected from tumors with high expression of KRAS gene and / or high expression of c-myc gene.

[0033] According to an embodiment of the present application, the drug is used to prevent or treat lung cancer, papillary thyroid cancer, 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 cancer, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, uterine cancer, cervical cancer, testicular cancer, nasopharyngeal cancer, anal cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma or gastric cancer.

[0034] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0038] Figure 3 The immunogenic death analysis diagram of compound (I) caused by in vitro cell validation according to Example 3 of the present invention is shown;

[0039] Figure 4 The cGAS-STING signaling pathway validation analysis diagram according to Example 4 of the present invention is shown;

[0040] Figure 5 Analysis diagram of Transwell experiment results according to Example 5 of the present invention;

[0041] Figure 6 Analysis diagram of the in vivo anti-tumor experimental results of Compound I according to Example 6 of the present invention;

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

[0043] Figure 8 The flow cytometry analysis of anti-tumor immune response according to Example 8 of the present invention is shown;

[0044] Fig. 9 The results of the anti-tumor experiment on metastatic tumors according to Example 9 of the present invention are shown;

[0045] Fig.10 The results of the in vivo anti-tumor experiment of the compound I combination drug according to Example 10 of the present invention are shown;

[0046] Fig.11 The graph shows the results of the anti-tumor experiment of the combination drug of Compound I according to Example 11 of the present invention on metastatic tumors. DETAILED DESCRIPTION

[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 understood 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 understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0049] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0050] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0051] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0052] The present application proposes the use of a compound in the preparation of a drug, a combination drug and its use in the preparation of a drug, which will be described in detail below.

[0053] Application of compounds in the preparation of medicines

[0054] In one aspect of the present application, the present application proposes the use of a compound in the preparation of a drug. According to an embodiment of the present application, the compound is a stereoisomer, tautomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug of the compound represented by formula (I),

[0055]

[0056] The medicine is used for anti-tumor.

[0057] The compounds according to the embodiments of the present application (also referred to as "compounds of the present application") can effectively inhibit the replication and transcriptional expression of the KRAS gene and the c-myc gene, and can activate immunity 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" refer to compounds that have the same chemical constitution but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, and the like.

[0059] "Chiral" refers to a molecule that is non-superimposable on its mirror image, whereas "achiral" refers to a molecule that is superimposable on its mirror image.

[0060] "Enantiomers" refer to two non-superimposable isomers of a compound that are mirror images of each other.

[0061] "Diastereoisomers" refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereoisomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivity. Diastereomeric mixtures can be separated by high resolution analytical procedures such as electrophoresis and chromatography, for example HPLC.

[0062] The stereochemical definitions and conventions used in the present invention generally follow SP 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.) of the compounds disclosed herein can exist in a racemic or enantiomerically enriched form, such as in the (R)-, (S)-, or (R,S)-configuration. In certain 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 terms of the (R)- or (S)-configuration.

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

[0065] Various pharmaceutically acceptable salt forms of the compounds of the invention are useful. The term "pharmaceutically acceptable salt" refers to those salt forms that are obvious to the pharmaceutical chemist, 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, are also important for selection, and these are: cost of the raw materials, ease of crystallization, yield, stability, hygroscopicity and flowability of the resulting drug substance. In brief, a pharmaceutical composition can be prepared by combining the active ingredient with a pharmaceutically acceptable carrier.

[0066] The "pharmaceutically acceptable salt" used in the present invention refers to organic salts and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as described in the literature: SM Berge 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, iodine salts or bromide salts.

[0067] In this specification, if there is any discrepancy between a chemical name and a chemical structure, the structure prevails.

[0068] According to the embodiments of the present application, the tumor is selected from tumors with high KRAS gene expression and / or high c-myc gene expression. The compounds of the present application can effectively inhibit the replication and transcriptional expression of KRAS gene and c-myc gene, and are therefore particularly suitable for preventing and / or treating tumors with high KRAS gene expression and / or high c-myc gene expression.

[0069] According to an embodiment of the present application, the drug is used to prevent or treat lung cancer, papillary thyroid cancer, 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 cancer, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, uterine cancer, cervical cancer, testicular cancer, nasopharyngeal cancer, anal cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma or gastric cancer.

[0070] As used herein, the term "treatment" refers to the use of drugs to obtain the desired pharmacological and / or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug to an individual to treat, cure, alleviate, improve, mitigate or inhibit an individual's disease, including but not limited to administering a drug described herein to an individual in need.

[0071] The term "administration" as used herein refers to introducing a predetermined amount of material into a patient by a suitable manner. The compound of the present invention can be administered by any common route as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, cortical, oral, topical, nasal, pulmonary and rectal administration, but the present invention is not limited to these exemplified modes of administration.

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

[0073] According to an embodiment of the present application, the drug further includes one or both of nucleic acid-modified iron oxide nanoparticles and doxorubicin.

[0074] Doxorubicin, referred to as DOX, is an antibiotic drug with a chemical formula of C 27 H 29 NO 11 Adriamycin has a wide anti-tumor spectrum and is suitable for acute leukemia (lymphocytic and granulocytic), malignant lymphoma, breast cancer, bronchial lung cancer (undifferentiated small cell and non-small cell), ovarian cancer, soft tissue sarcoma, osteogenic sarcoma, 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 found that the compound of the present application can better exert the therapeutic effect when it is administered in combination with adriamycin.

[0075] Iron oxide nanoparticles (IONPs) can be used as drug carriers for targeted drug delivery, improving drug efficacy and reducing side effects. The modified nucleic acids on IONPs can bind to drug molecules to load drugs, and the nucleic acid sequences are rich in CpG and have the function of activating immunity.

[0076] According to an embodiment of the present application, the compound and / or the doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles. Thus, the compound and / or doxorubicin can be better delivered into cells, the bioavailability can be improved, and the therapeutic effect can be better exerted. Among them, the compound and doxorubicin are respectively embedded in the same or different nucleic acid-modified iron oxide nanoparticles for better effect.

[0077] According to an embodiment of the present application, the compound is embedded in the nucleic acid-modified iron oxide nanoparticles, and the embedding rate of the compound is 80% to 90%, thereby helping to better exert the therapeutic effect.

[0078] According to an embodiment of the present application, the doxorubicin is embedded in the nucleic acid-modified iron oxide nanoparticles, and the embedding rate of the doxorubicin is 85% to 95%, thereby helping to better exert the therapeutic effect.

[0079] According to the embodiment of the present application, the compound and doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles, and the embedding rate of the compound is 80% to 90%, and the embedding rate of doxorubicin is 85% to 95%, thereby helping to better exert the therapeutic effect.

[0080] According to an embodiment of the present application, the nucleic acid has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence having at least 80% homology thereto. The nucleic acid can bind to drug molecules to load the drug, and the nucleic acid sequence is rich in CpG and has the function of activating immunity.

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

[0082] According to an embodiment of the present application, the drug further includes a pharmaceutically acceptable excipient.

[0083] In this article, the term "pharmaceutically acceptable adjuvant" is a pharmaceutically acceptable adjuvant, carrier, excipient, solvent or combination thereof for transmitting the active ingredient in the medicine of the present invention to an animal or human. Exemplary adjuvants can be liquid or solid, including but not limited to: pH regulators, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes / colorants, 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 combination with pharmacodynamic compounds. Examples of adjuvants include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gel, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0084] Combination drugs

[0085] In another aspect of the present application, the present application proposes a combined drug. According to an embodiment of the present application, the combined drug comprises:

[0086] A compound, wherein the compound is a stereoisomer, a tautomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug of the compound represented by 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 the present application can effectively inhibit the replication and transcriptional expression of the KRAS gene and the 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 the embodiments of the present application, the compound and / or the doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles, thereby better delivering the compound and / or doxorubicin into cells, improving bioavailability and better exerting therapeutic effects.

[0091] According to an embodiment of the present application, the compound is embedded in the nucleic acid-modified iron oxide nanoparticles, and the embedding rate of the compound is 80% to 90%, thereby helping to better exert the therapeutic effect.

[0092] According to an embodiment of the present application, the doxorubicin is embedded in the nucleic acid-modified iron oxide nanoparticles, and the embedding rate of the doxorubicin is 85% to 95%, thereby helping to better exert the therapeutic effect.

[0093] According to the embodiment of the present application, the compound and doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles, and the embedding rate of the compound is 80% to 90%, and the embedding rate of doxorubicin is 85% to 95%, thereby helping to better exert the therapeutic effect.

[0094] According to an embodiment of the present application, the nucleic acid has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence having at least 80% homology thereto. The nucleic acid can bind to drug molecules to load the drug, and the nucleic acid sequence is rich in CpG and has the function of activating immunity.

[0095] It should be noted that the characteristics and advantages described above for the use of the compound in the preparation of drugs are also applicable to the combination drug and will not be repeated here.

[0096] Application of combined drugs in the preparation of drugs

[0097] In another aspect of the present application, the present application proposes the use of the above-mentioned combined drug in the preparation of a drug. According to an embodiment of the present application, the drug is used for anti-tumor.

[0098] According to an embodiment of the present application, the tumor is selected from tumors with high expression of KRAS gene and / or high expression of c-myc gene.

[0099] According to an embodiment of the present application, the drug is used to prevent or treat lung cancer, papillary thyroid cancer, 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 cancer, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, uterine cancer, cervical cancer, testicular cancer, nasopharyngeal cancer, 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 use of the combination drug and the compound in the preparation of the drug are also applicable to the use of the combination drug in the preparation of the drug and will not be repeated here.

[0101] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

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

[0103] The compound I in the embodiment is

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

[0105] (1) Experimental process:

[0106] The control group (blank group) cells (medium without compound) and the experimental group cells (HeLa, A549, MCF-7) were cultured in medium containing compound (I) at a final concentration of 4 μM for 48 hours and then the cells (approximately 5×10 7 The culture medium composition was 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 in cells was extracted using the Trizol method, and then reverse transcribed into cDNA after calibration. The mRNA transcription level of the KRAS gene was further determined using the fluorescence quantitative PCR method. The loading amount and the content of each reagent in the system were calculated according to the experimental requirements. The program was set in the PCR instrument: 37°C, 25-60min, 98°C, 5min, 4°C hold. The program was run; after the program ended, the cDNA was stored at -20°C.

[0108] Fluorescence quantitative PCR primers:

[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] Fluorescence quantitative PCR Figure 1 As shown, after compound I was incubated with three cancer cells for 48 hours, the mRNA transcription level of the KRAS gene was significantly downregulated, and there was a significant difference with the blank group. This shows that compound I can effectively inhibit the expression of the KRAS gene.

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

[0116] (1) Experimental process:

[0117] The control group (blank group) cells (cells without compound + medium) and experimental group cells (HeLa, A549, MCF-7) were cultured in medium containing compound I at a final concentration of 4 μM for 48 hours and then the cells (approximately 5×10 7 The culture medium composition was 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 in cells was extracted using the Trizol method, and then reverse transcribed into cDNA after calibration. The mRNA transcription level of the c-myc gene was further determined using the fluorescence quantitative PCR method. The loading amount and the content of each reagent in the system were calculated according to the experimental requirements. The program was set in the PCR instrument: 37°C, 25-60min, 98°C, 5min, 4°C hold. The program was run; after the program ended, the cDNA was stored at -20°C.

[0119] Fluorescence quantitative PCR primers:

[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] Fluorescence quantitative PCR Figure 2 As shown, after compound I was incubated with three cancer cells for 48 hours, the mRNA transcription level of c-myc gene was significantly downregulated, and there was a significant difference with the blank group. Therefore, it was shown that compound I can effectively inhibit the expression of c-myc gene.

[0126] Example 3 In vitro cell verification of immunogenic death caused by compound I

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

[0128] 1. Calreticulin

[0129] 4T1 cells were cultured at 1×10 6The cells were inoculated into the confocal dish at a density of 100 / well and placed in a cell culture incubator for overnight culture. The original culture medium was then poured out and divided into two groups of experiments, with culture medium containing PBS and culture medium containing compound I (0.2 μg / mL) added respectively and continued to be cultured for 24 hours. After the incubation, the culture medium containing different materials was discarded and the cells were washed 3 times with PBS. Subsequently, the cells were fixed at room temperature for 7 minutes with the prepared cell fixative and incubated with a PBS solution containing 0.2% Triton X-100 for 5 minutes to permeabilize the cells. After washing the cells 3 times with PBS, the cells were incubated at room temperature for 30 minutes with the prepared cell immunofluorescence blocking solution to reduce the nonspecific binding of the antibody. While blocking, the calreticulin rabbit monoclonal antibody was diluted with a cell immunofluorescence antibody diluent at a volume ratio of 1:1000, and the Alexa Fluor 647-labeled goat anti-rabbit secondary antibody was diluted at a volume ratio of 1:500. After the blocking was completed, the prepared primary antibody solution was added to the confocal dish and incubated overnight at 4°C. The next day, the primary antibody solution in the confocal dish was aspirated, the cells were washed 3 times with PBS, and the diluted secondary antibody solution was added, and incubated at room temperature in the dark for 1 hour. Finally, Hoechst33342 was added and incubated at room temperature for 5 minutes, and then observed under a laser confocal microscope. The excitation wavelength selected for the Hoechst 33342 channel was 405nm, and the excitation wavelength selected for the calreticulin channel was 635nm.

[0130] For flow cytometric analysis of cell surface CRT, 4T1 cells were plated at 1 × 10 6 The cells were inoculated into 6-well plates at a density of 100 / well and placed in a cell culture incubator for overnight culture. The next day, a culture medium containing PBS and compound I (0.2 μg / mL) was added to the 6-well plate and cultured for 24 hours. Subsequently, the culture medium containing the material was discarded, the cells were washed with PBS, and 200 μL of trypsin was added to digest the cells, transferred to a 1.5 mL centrifuge tube, and the supernatant was removed by centrifugation to obtain a cell pellet. While centrifuging, the CRT rabbit monoclonal antibody was diluted at a ratio of 1:100 with the prepared flow cytometry antibody diluent, and the Alexa Fluor 647-labeled goat anti-rabbit secondary antibody was diluted at a ratio of 1:200. After centrifugation, the diluted CRT antibody was added and incubated at 4°C for 30 minutes. After washing with PBS, the diluted secondary antibody was added and incubated at 4°C for 30 minutes. Finally, the cells were washed with PBS and placed in an Agilent NovoCyte flow cytometer for analysis.

[0131] 2. High mobility group protein 1

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

[0133] The specific operations are as follows:

[0134] 4T1 cells were cultured at 1×10 6 The cells were inoculated into 6-well plates at a density of 100 cells / well and placed in a cell culture incubator for overnight culture. The next day, the experiment was divided into two groups. The culture medium containing PBS and the culture medium containing compound I (0.2 μg / mL) were added to the 6-well plates and cultured for 24 hours. Subsequently, the cell supernatant was collected and the released HMGB1 was quantitatively analyzed according to the operating instructions of the ELISA kit.

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

[0136] Example 4 Validation of cGAS-STING Signaling Pathway

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

[0138] 4T1 cells were cultured at 1×10 6The cells were inoculated into the confocal dish at a density of 100 / well and placed in a cell culture incubator for overnight culture. The original culture medium was then poured out and divided into two groups of experiments, with culture medium containing PBS and culture medium containing compound I (0.2 μg / mL) added respectively and continued to be cultured for 24 hours. After the incubation, the culture medium containing different materials was discarded and the cells were washed 3 times with PBS. Subsequently, the cells were fixed at room temperature for 7 minutes with the prepared cell fixative and incubated with a PBS solution containing 0.2% Triton X-100 for 5 minutes to permeabilize the cells. After washing the cells 3 times with PBS, the cells were incubated at room temperature for 30 minutes with the prepared cell immunofluorescence blocking solution to reduce the nonspecific binding of the antibody. While blocking, the p-STING rabbit monoclonal antibody was diluted with a cell immunofluorescence antibody diluent at a volume ratio of 1:1000, and the Alexa Fluor 647-labeled goat anti-rabbit secondary antibody was diluted at a volume ratio of 1:500. After the blocking was completed, the prepared primary antibody solution was added to the confocal dish and incubated overnight at 4°C. The next day, the primary antibody solution in the confocal dish was aspirated, the cells were washed 3 times with PBS, and the diluted secondary antibody solution was added, and incubated at room temperature in the dark for 1 hour. Finally, Hoechst 33342 was added and incubated at room temperature for 5 minutes, and then observed under a laser confocal microscope. The excitation wavelength selected for the Hoechst 33342 channel was 405nm, and the excitation wavelength selected for the p-STING channel was 635nm.

[0139] 2. Western Blot (WB)

[0140] 4T1 cells were cultured at 1×10 6The cells were inoculated into 6-well plates at a density of 100 cells / well and placed in a cell culture incubator for overnight culture. The original culture medium was then poured out and divided into two groups of experiments, with culture medium containing PBS and culture medium containing compound I (0.2 μg / mL) added to continue culturing for 24 hours. After the incubation, the culture medium containing different materials was 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 placed on ice for 30 minutes. The lysed sample was centrifuged at 12000g for 5 minutes, the supernatant was taken, and the extracted protein was quantified using the BCA protein concentration determination kit. 10% and 12% SDS-PAGE gels were prepared according to the size of the target protein, the proteins were separated by a vertical electrophoresis tank, and the Rainbow 180 broad-spectrum protein marker was used as a band indicator. After the electrophoresis, the proteins in the SDS-PAGE were transferred to the PVDF membrane through the membrane transfer process. After the transfer, the PVDF membrane was removed and placed in an antibody incubation box, and the freshly prepared WB blocking solution was poured in, and incubated on a shaker at 4°C overnight. The next day, the membrane was washed at least 3 times with the prepared TBST solution, and the interferon regulatory factor 3 (p-IRF3) monoclonal antibody and p-STING monoclonal antibody were diluted at a ratio of 1:1000 with the freshly prepared WB antibody diluent, and the internal reference protein GAPDH antibody was diluted at a ratio of 1:5000. Subsequently, the prepared primary antibody solution was added to the antibody incubation box and incubated on a shaker at 4°C overnight. The next day, the membrane was washed at least 3 times with the prepared TBST solution, and the horseradish peroxidase (HRP)-labeled secondary antibody was diluted at a ratio of 1:1000 with the freshly prepared WB antibody diluent. The prepared secondary antibody solution was added to the antibody incubation box, incubated on a shaker at room temperature for 1h, and the excess secondary antibody solution was removed with TBST solution. Finally, the PVDF membrane was placed in the ECL chemiluminescent reagent and photographed with a fluorescent 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 compound I was significantly stronger than that of the control group. Figure 4 B shows WB fluorescence imaging, and the fluorescence of p-IRF3 antibody and p-STING antibody in the group with compound I 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 cultured at 1×10 6The cells were inoculated into the upper chamber of a 0.4 μm transwell at a density of 100 cells / well and placed in a cell culture incubator for overnight culture. The next day, the experiment was divided into two groups, and the cells were incubated for 12 hours with a culture medium containing PBS and a culture medium containing compound I (0.2 μg / mL). Subsequently, the original culture medium was discarded, fresh culture 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 placed in a cell culture incubator for further culture for 24 hours. Next, the cell culture medium was collected and the expression levels of interleukin-6 (IL-6) and α tumor necrosis factor (TNF-α) were determined using an ELISA kit. In addition, the BMDCs cells in the lower chamber of the transwell were collected, and flow antibody diluent containing PE-CD11c, FITC-CD80 and APC-CD86 flow antibodies were added, incubated on a shaker at 4°C for 30 minutes, and analyzed using an Agilent NovoCyte flow cytometer after washing with PBS.

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

[0145] Example 6

[0146] 1. Preparation of IONP-G2

[0147] Accurately weigh 700 mg of ferric acetylacetonate and add it to a three-necked flask, then add 3 mL of oleic acid, 2 mL of oleylamine and 10 mL of 1-octadecene. Place the flask in a temperature-controlled heating device, stir magnetically, evacuate, and keep it at 90 ° C for 15 minutes. After the evacuation, turn on the nitrogen switch to put the entire reaction system in a nitrogen-protected state, heat to 200 ° C and continue to reflux for half an hour, and gradually cool to room temperature to obtain IONP. The synthesized IONP was centrifuged and washed with cyclohexane and anhydrous ethanol respectively, and finally the washed nanoparticles were dispersed with cyclohexane, and 50 mg of bisphosphoric acid-PEG-maleimide (DP-PEG-MAL) was used to modify the surface of IONP so that IONP can be evenly dispersed in the aqueous phase. Using the Michael addition reaction between the maleimide group and the thiol group, the thiol-modified nucleic acid was coupled to the surface of IONP to prepare IONP-G2. After ultrafiltration with ultrapure water, there were about 300 nucleic acid molecules on each IONP.

[0148] 2. Preparation of Nanoparticles

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

[0150] Example 7 In vivo anti-tumor experiment

[0151] In order to evaluate the tumor inhibition effect in vivo, a mouse breast cancer subcutaneous tumor model was constructed. The specific operation is as follows: 1 mL of trypsin was added to the well-growing 4T1 cells for digestion, incubated at 37°C for 3 minutes, and 5 mL of culture medium was immediately added to terminate the digestion. The cells were gently blown down from the bottom of the culture dish, centrifuged at 3000 rpm for 5 minutes, the supernatant was discarded, and PBS was added for resuspending. 100 μL of the cell suspension (about 5×10 5 The subcutaneous tumor model was constructed by injecting 100 cells into the upper right hip of BALB / c mice. One week later, the tumor grew to about 100 mm. 3 Subsequently, 15 tumor-bearing mice were randomly divided into 3 groups, namely PBS, IONP-G2, DOX (1.2 mg / kg), and compound I (0.25 mg / kg), with 5 mice in each group. On the 1st, 3rd, and 5th days of treatment, 100 μL of the corresponding material was injected into the tumor-bearing mice by intravenous injection, and the weight of the mice and the length and width of the tumor were recorded every other day. According to the formula V (mm 3 ) = length × width 2 ×0.5 to calculate the tumor volume. After the experiment, the mice were sacrificed, the tumor tissues were removed, photographed and recorded, and the weight of the tumor tissues was weighed. The ex vivo tumor tissues were subjected to hematoxylin-eosin (H&E) staining, terminal deoxynucleotidyl transferase-mediated dUTP in situ nick end labeling (TUNEL) fluorescence staining, immunofluorescence staining and immunohistochemistry staining to further evaluate the in vivo antitumor effect.

[0152] Figure 6 A shows the weight of experimental mice. Figure 6 B is the change in the volume of the tumor. Figure 6C is the difference in tumor weight, Figure 6 D is a stained section of tumor tissue. Figure 6 The data from each group show that compound I has a significant inhibitory effect on tumors. When used at a dose lower than that of DOX, its inhibitory effect is equivalent to that of DOX. At the same time, compound I has no obvious toxic side effects. Figure 6 D shows that compound I not only kills tumors, but also promotes the activation of immune response.

[0153] Example 8 In vivo toxicity evaluation

[0154] In order to evaluate the potential toxicity of Compound I, the tumor tissues and major organs such as heart, liver, spleen, lung, and kidney were collected after sacrificing the mice in Example 6, and stored in 4% tissue fixative for hematoxylin-eosin (H&E) staining. At the same time, serum biological factors were measured to evaluate the effects of the material on renal and liver functions, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN), and creatinine (CRE).

[0155] Figure 7 A shows the staining of sections of various organs of mice. Figure 7 B shows the differences in the levels of ALT, AST, BUN and CRE in different drug treatments. Figure 7 It can be seen from each group of data that the compound has no significant toxic side effects.

[0156] Example 9 Analysis of anti-tumor immune response by flow cytometry

[0157] In order to evaluate the immune activation effect of the material on the body, 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 the 1st and 3rd days, respectively. On the 7th day, the mice were sacrificed and the tumor tissues, tumor-draining lymph nodes (TDLN) and spleens of the mice were removed. The collected organs were ground with freshly prepared tissue grinding fluid to obtain a cell suspension. Subsequently, the cells were filtered with a 70 μm cell strainer to obtain a single cell suspension. The spleen cells were treated with red blood cell lysis solution, and the lysis was terminated with grinding solution after 5 minutes. The cells were collected by centrifugation at 3000 rpm / min for 5 minutes, and new grinding solution was added to obtain a single cell suspension without red blood cells. The obtained single cell suspension was stained with flow cytometry antibodies for qualitative analysis of mature DC cells (CD45 + 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 are: ER780 Anti-Mouse CD45, PerCP / Cy5.5 Anti-Mouse CD3, FITC Anti-Mouse CD4, PE Anti-Mouse CD8a, PE / Cy7 Anti-Mouse CD44, PEAnti-Mouse CD11c, FITC Anti-Mouse CD80, APCAnti-Mouse CD86, APC Anti-MouseCD62L, PE Anti-Mouse FOXP3.

[0158] Figure 8 Representative flow cytometry plots showing activation of in vivo immune responses, including mature DCs in TDLN and tumor tissues; CD8 + Representative flow cytometry of T cells; Representative flow cytometry of Tregs cells in tumor tissue; (E) Representative flow cytometry of spleen central memory CD8+T cells. The data in the figure show the activation of the immune response of compound I in the mouse tumor model. Thus, it is shown that the compound has an immune activation effect in vivo.

[0159] Example 10 Metastatic Tumor Anti-tumor Experiment

[0160] In order to evaluate the inhibitory effect of the material on metastatic or recurrent tumors, a metastatic tumor model was constructed. 100 μL of 4T1 cell suspension (about 5×10 5 The subcutaneous tumor model was constructed by injecting 100 cells into the upper right hip of BALB / c mice. One week later, the tumor grew to about 100 mm. 3 Subsequently, 15 tumor-bearing mice were randomly divided into 3 groups, PBS, 1.2 mg / kg DOX, and 0.25 mg / kg compound I, with 5 mice in each group. On the 1st, 3rd, and 5th days of treatment, 100 μL of the corresponding material was injected into the tumor-bearing mice by intravenous injection. On the 7th day, another 100 μL of 4T1 cell suspension (about 5×10 5 The metastatic tumor model was artificially constructed by injecting 100 cells into the upper left hip of BALB / c mice. The weight of the mice and the length and width of the metastatic tumor were recorded every other day. The formula V (mm3 ) = length × width 2 × 0.5 to calculate the volume of metastatic tumors. After the experiment, the mice were sacrificed, and the metastatic tumor tissues were removed for flow cytometric analysis and immunohistochemical staining.

[0161] Fig. 9 The anti-tumor effect of compound I on the metastatic 4T1 tumor model is shown, where (A) the tumor growth curve of metastatic tumor (n=5); (B) the CD8 + The proportion of T cells; (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. Fig. 9 It can be seen that compound I has an inhibitory effect on 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, and the tumor inhibition effect of different materials was evaluated using a mouse breast cancer subcutaneous tumor model. The specific operation is as follows: 1 mL of trypsin was added to the well-growing 4T1 cells for digestion, incubated at 37°C for 3 minutes, and 5 mL of culture medium was immediately added to terminate the digestion. The cells were gently blown down from the bottom of the culture dish, centrifuged at 3000 rpm for 5 minutes, the supernatant was discarded, and PBS was added for resuspending. Take 100 μL of the cell suspension (about 5×10 5 The subcutaneous tumor model was constructed by injecting 100 cells into the upper right hip of BALB / c mice. One week later, the tumor grew to about 100 mm. 3 Subsequently, 35 tumor-bearing mice were randomly divided into 7 groups, namely PBS, IONP-G2, DOX, compound I, IONP-G2-DOX, IONP-G2-compound I, IONP-G2-DOX / compound I, with 5 mice in each group. On the 1st, 3rd, and 5th days of treatment, 100 μL of the corresponding material was injected into the tumor-bearing mice by intravenous injection, and the weight of the mice and the length and width of the tumor were recorded every other day. According to the formula V (mm 3 ) = length × width 2 × 0.5 to calculate the tumor volume. After the experiment, the mice were sacrificed, the tumor tissues were removed, photographed, and weighed.

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

[0165] Example 12 Anti-tumor experiment of combined drugs on metastatic tumors

[0166] Take 100 μL of 4T1 cell suspension (about 5×10 5 The subcutaneous tumor model was constructed by injecting 100 cells into the upper right hip of BALB / c mice. One week later, the tumor grew to about 100 mm. 3 Subsequently, 35 tumor-bearing mice were randomly divided into 7 groups, namely PBS, IONP-G2, DOX, compound I, IONP-G2-DOX, IONP-G2-compound I, IONP-G2-DOX / compound I, with 5 mice in each group. Fig.11 A), 100 μL of the corresponding material was injected into the tumor-bearing mice by intravenous injection. On the 7th day, another 100 μL of 4T1 cell suspension (about 5×10 5 The metastatic tumor model was artificially constructed by injecting 100 cells into the upper left hip of BALB / c mice. The weight of the mice and the length and width of the metastatic tumor were recorded every other day. The formula V (mm 3 ) = length × width 2 × 0.5 to calculate the volume of metastatic tumors. After the experiment, the mice were sacrificed, the metastatic tumor tissues were removed, the tumors were weighed and analyzed by flow cytometry.

[0167] The study found that the IONP-G2-DOX / Compound I combination drug showed significant tumor inhibition compared with other treatment groups ( Fig.11 B) After the treatment, the mice were sacrificed and the metastatic tumor tissues were removed. The CD8 + The proportion of T cells. Fig.11As shown in C, the expression level of CD8+T cells in metastatic tumor tissues treated with IONP-G2-DOX / Compound I was significantly increased, which was 2.45, 1.52 and 1.82 times that of the IONP-G2, DOX and Compound I groups, respectively. These results all indicate that there is an effective immunotherapy effect in metastatic tumors. In general, these results show that IONP-G2-DOX / Compound I treatment can induce a strong immune response and has an inhibitory effect on metastatic or recurrent tumors.

[0168] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Use of a compound in the preparation of a drug, characterized in that: The compound is a stereoisomer, tautomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug of the compound represented by formula (I), The medicine is used for anti-tumor.

2. The use according to claim 1, characterized in that: The tumor is selected from tumors with high KRAS gene expression and / or high c-myc gene expression.

3. The use according to claim 1, characterized in that: The drug is used to prevent or treat lung cancer, papillary thyroid cancer, 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 cancer, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, uterine cancer, cervical cancer, testicular cancer, nasopharyngeal cancer, anal cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma or gastric cancer.

4. The use 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 use 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% to 95%; Optionally, the compound and doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles, the embedding rate of the compound is 80% to 90%, and the embedding rate of the doxorubicin is 85% to 95%; Optionally, the nucleic acid has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence having at least 80% homology thereto.

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

7. A combined drug, characterized in that: include: A compound, wherein the compound is a stereoisomer, a tautomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug of the compound represented by formula (I), One or both of nucleic acid-modified iron oxide nanoparticles and doxorubicin.

8. The combined drug 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% to 95%; Optionally, the compound and doxorubicin are embedded in the nucleic acid-modified iron oxide nanoparticles, the embedding rate of the compound is 80% to 90%, and the embedding rate of the doxorubicin is 85% to 95%; Optionally, the nucleic acid has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence having at least 80% homology thereto.

9. Use of the combined drug according to claim 7 or 8 in preparing a drug, characterized in that: The medicine is used for anti-tumor.

10. The use according to claim 9, characterized in that: The tumor is selected from tumors with high KRAS gene expression and / or high c-myc gene expression; Optionally, the medicament is used to prevent or treat lung cancer, papillary thyroid cancer, 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 cancer, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, uterine cancer, cervical cancer, testicular cancer, nasopharyngeal cancer, anal cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma or gastric cancer.

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