Combination medicaments for treating tumors and their use

Abstract

The present disclosure relates to the combined use of IN10018 and an epidermal growth factor receptor tyrosine kinase inhibitor in the treatment of tumors.

Description

Technical Field

[0001] The present disclosure belongs to the field of medicinal chemistry. Specifically, the present disclosure relates to the use of the focal adhesion kinase (FAK) inhibitor IN10018 in combination with another anti-tumor agent for the treatment of tumors.

Background Art

[0002] EGFR is known as the epidermal growth factor receptor. The EGFR gene is responsible for encoding and producing a receptor protein called the epidermal growth factor receptor. The EGFR receptor protein is a transmembrane protein that is divided into three parts: one end of the protein located outside the cell, one part located within the cell membrane, and the other end located inside the cell. This enables the EGFR receptor to bind to other proteins (called ligands) outside the cell, facilitating the reception of signals by the cell and its response to the stimulation. The binding of the receptor to the ligand is like a key to a lock, and therefore they each have specific binding "partners". When EGFR binds to a ligand, it binds to another nearby EGFR receptor to form a complex (dimer), becoming activated and activating the intracellular signaling pathway. EGFR mutations mainly occur in exons 18 to 21, among which the deletion mutation in exon 19 and the L858R point mutation in exon 21 are the most common types of mutations, accounting for 90% of all mutation types. When a pathogenic mutation occurs in EGFR, the EGFR receptor protein is continuously activated, which results in cells that continuously receive signals for growth and survival, ultimately leading to excessive cell growth and survival (failure of proper apoptosis) and inducing tumor formation.

[0003] Target drugs for currently available EGFR mutations include first-generation icotinib, gefitinib, and erlotinib for exon 19 and exon 21 mutations, second-generation afatinib for exon 8 and exon 20 mutations, and third-generation osimertinib (also known as AZD9291 in this specification), almonertinib, and alflutinib for T790M mutation. Target drugs for ALK mutations include first-generation target drug crizotinib, second-generation target drugs ceritinib, alectinib, and brigatinib, and third-generation target drug lorlatinib. However, resistance to these target drugs often appears about one year after administration. Overcoming resistance to target drugs or delaying the emergence of resistance is a major goal in anti-neoplastic drug development.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0005] In one aspect, the present disclosure provides the use of IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor in the manufacture of a medicament for treating tumors in a subject, wherein IN10018 has

[0006]

Chemical Formula

[0007] the structure of, and provides the use.

[0008] In another aspect, the present disclosure provides a combined pharmaceutical formulation of IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor for use in treating tumors in a subject.

[0009] In another aspect, the present disclosure provides a method for treating a tumor, the method comprising administering to a subject in need thereof a therapeutically effective amount of IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor.

[0010] In another aspect, the present disclosure provides a kit or a pharmaceutically acceptable composition comprising (a) IN10018 or a pharmaceutically acceptable salt thereof, and (b) an epidermal growth factor receptor tyrosine kinase inhibitor.

[0011] In another aspect, the present disclosure provides the use of IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor in the manufacture of a combination drug for the treatment of a tumor.

[0012] In another aspect, the present disclosure provides the use of IN10018 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in combination with an epidermal growth factor receptor tyrosine kinase inhibitor for the treatment of a tumor.

[0013] In another aspect, the present disclosure provides the use of an epidermal growth factor receptor tyrosine kinase inhibitor in the manufacture of a medicament for use in combination with IN10018 or a pharmaceutically acceptable salt thereof for the treatment of a tumor.

[0014] In another aspect, the present disclosure provides a kit comprising IN10018 or a pharmaceutically acceptable salt thereof and an instruction manual stating that IN10018 or a pharmaceutically acceptable salt thereof can be used in combination with an epidermal growth factor receptor tyrosine kinase inhibitor for the treatment of a tumor.

[0015] In another aspect, the present disclosure provides a kit comprising an epidermal growth factor receptor tyrosine kinase inhibitor and an instruction manual stating that the epidermal growth factor receptor tyrosine kinase inhibitor can be used in combination with IN10018 or a pharmaceutically acceptable salt thereof for the treatment of tumors.

[0016] In another aspect, the present disclosure provides a method for treating a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor.

[0017] In another aspect, the present disclosure provides a combined pharmaceutical preparation of IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor for use in the treatment of tumors in a subject in need of treatment of tumors.

[0018] Optionally, the pharmaceutically acceptable salt of IN10018 is the tartrate salt of IN10018.

[0019] Optionally, the epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as flmonertinib), EAI045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163, or a pharmaceutically acceptable salt thereof, preferably osimertinib, almonertinib, alflutinib, or a pharmaceutically acceptable salt thereof.

[0020] The CAS number of gefitinib is 184475-35-2. The CAS number of erlotinib is 183321-74-6. The CAS number of icotinib is 610798-31-7. The CAS number of afatinib is 850140-72-6. The CAS number of crizotinib is 877399-52-5. The CAS number of osimertinib (AZD9291) is 1421373-65-0. The CAS number of almonertinib is 1899921-05-1. The CAS number of alflutinib (also known as fulmonertinib) is 1869057-83-9. The CAS number of EAI045 is 1942114-09-1. The CAS number of JBJ-04-125-02 is 2060610-53-7. The CAS number of BLU945 is 2660250-10-0, and BLU701 is co-developed by Blueprint Medicines Corp. and Zai Lab. The CAS number of TQB3804 is 2267329-76-8. BBT-176 is developed by Bridge Biotherapeutics. ES-072 is developed by Bossan Pharmaceutical Co., Ltd. BPI-361175 is developed by Betta Pharmaceuticals Co., Ltd. CH7233163 is developed by Chugai Pharmaceutical Co., Ltd.

[0021] Optionally, IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor are administered to the subject simultaneously or sequentially.

[0022] Optionally, the tumor is caused by bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, multiple myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, cholangiocarcinoma, leiomyosarcoma, liposarcoma, hypopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland cancer, spindle cell carcinoma, metastases, undifferentiated large cell lymphoma, anaplastic thyroid carcinoma, non-Hodgkin lymphoma, Hodgkin lymphoma, glioma, and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), or alternatively, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer or colorectal cancer, or alternatively, the tumor is lung cancer or colorectal cancer.

[0023] To more clearly show the technical solutions of the examples of the present disclosure, the drawings of the examples are briefly introduced below. Obviously, the drawings in the following description do not limit the present disclosure, but only relate to some examples of the present disclosure.

Brief Description of the Drawings

[0024]

Figure 1

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Figure 12

Figure 13

Best Mode for Carrying Out the Invention

[0025] To make the objectives, technical solutions, and advantages of the examples of the present disclosure clearer, the technical solutions of the examples of the present disclosure will be clearly and completely described below together with the drawings of the examples of the present disclosure. Obviously, the described examples are part of the examples of the present disclosure, but not all of the examples of the present disclosure. All other examples obtained by those skilled in the art without creative efforts based on the described examples of the present disclosure shall be included in the protection scope of the present invention.

[0026] The present disclosure may be embodied in other specific forms without departing from the essential attributes of the present disclosure. It should be understood that any embodiment of the present disclosure can be combined with another embodiment or technical features in other embodiments to obtain additional embodiments on the premise of no contradiction. The additional embodiments resulting from such combinations are also included in the present disclosure.

[0027] All publications and patents mentioned in the present disclosure are hereby incorporated by reference in their entirety into the present disclosure. If there is a contradiction between the usage or terms used in any incorporated publication or patent by reference and the usage or terms used in the present disclosure, the usage and terms in the present disclosure shall prevail.

[0028] The section headings used in this specification are only for the purpose of organizing items and should not be construed as limitations or interpretations of the subject matter described.

[0029] Unless otherwise defined, all technical and scientific terms used in this specification have their ordinary meanings in the technical field to which the claimed subject matter belongs. If there are two or more definitions for a term, the definition in this specification shall prevail.

[0030] Unless otherwise indicated in the Examples or elsewhere, all numbers expressing quantitative properties such as dosages recited in this specification and the claims are to be understood as being modified in all instances by the term "about". Also, any numerical range recited in this specification is intended to include all sub-ranges within that range and any combination of the range or sub-ranges with their respective endpoints.

[0031] As used in this disclosure, the words "comprising", "including", or "containing", and similar words, mean that the elements appearing before these words include the elements recited after these words and their equivalents, and do not exclude any elements not recited. The terms "comprising" or "including" (or "containing") as used herein can be non-limiting, semi-limiting, and limiting. In other words, these terms also include "consisting essentially of" or "consisting of".

[0032] Definitions The following terms and symbols used in this application have the meanings set forth below unless otherwise indicated in the context.

[0033] As used herein, the term "epidermal growth factor receptor tyrosine kinase inhibitor" refers to an agent that selectively and effectively inhibits epidermal growth factor receptor tyrosine kinase. Examples of epidermal growth factor receptor tyrosine kinase inhibitors include, but are not limited to, gefitinib, erlotinib, icotinib, afatinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as fulmonertinib), EAI045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163, or a pharmaceutically acceptable salt thereof. In some embodiments, the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib, almonertinib, alflutinib, or a pharmaceutically acceptable salt thereof.

[0034] The CAS number of gefitinib is 184475-35-2. The CAS number of erlotinib is 183321-74-6. The CAS number of icotinib is 610798-31-7. The CAS number of afatinib is 850140-72-6. The CAS number of crizotinib is 877399-52-5. The CAS number of osimertinib (AZD9291) is 1421373-65-0. The CAS number of almonertinib is 1899921-05-1. The CAS number of alflutinib (also known as fulmonertinib) is 1869057-83-9. The CAS number of EAI045 is 1942114-09-1. The CAS number of JBJ-04-125-02 is 2060610-53-7. The CAS number of BLU945 is 2660250-10-0, and BLU701 is co-developed by Blueprint Medicines Corp. and Zai Lab. The CAS number of TQB3804 is 2267329-76-8. BBT-176 is developed by Bridge Biotherapeutics. ES-072 is developed by Bossan Pharmaceutical Co., Ltd. BPI-361175 is developed by Betta Pharmaceuticals Co., Ltd. CH7233163 is developed by Chugai Pharmaceutical Co., Ltd.

[0035] As used herein, "combination drug" or "combination pharmaceutical" can refer to any of a fixed combination in the form of a single dosage unit (e.g., all active ingredients present in one dosage form), or a kit for administering the combined pharmaceuticals, or a combination of a drug and an instruction indicating that the drug can be used in combination with one or more other drugs.

[0036] As used herein, the terms "combination treatment" or "combination drug" refer to the use of one or more drugs in combination with one or more other drugs to treat a disease, including both the combination of one drug with one or more other drugs and the combination of one drug with a label indicating that the drug can be used in combination with one or more other drugs.

[0037] As used in this application, "simultaneous or sequential administration" means that two or more drugs are administered simultaneously or sequentially at regular intervals within an administration cycle (e.g., within 4 weeks, 3 weeks, 2 weeks, 1 week, or 24 hours), and their administration modes (e.g., oral, intravenous, intramuscular, or subcutaneous) may be the same or different, and the frequencies / cycles of administration of two or more drugs may be the same or different. When the treatment methods, formulations, or uses of the present disclosure involve two drugs, the two drugs may be administered simultaneously or separately at regular time intervals.

[0038] As used herein, the terms "treat", "treating", or "treatment" refer to the administration of one or more drugs to an individual having a disease or a symptom of a disease for the purpose of curing, alleviating, reducing, modifying, treating, remitting, or improving the disease or the symptom of the disease, or for the purpose of affecting the disease or the symptom of the disease. In some embodiments, the disease is a tumor or cancer.

[0039] As used herein, the term "tumor" refers to an abnormal lesion formed by clonal proliferation of cells of a local tissue that has lost normal genetic-level control over growth under the action of various tumorigenic factors. Examples include, but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, cholangiocarcinoma, leiomyosarcoma, liposarcoma, hypopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland cancer, metastasis caused by spindle cell carcinoma, anaplastic large cell lymphoma, anaplastic thyroid carcinoma, non-Hodgkin lymphoma, Hodgkin lymphoma, glioma, and hematological malignancies, for example, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML). Alternatively, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer or colon cancer, or alternatively, the tumor is lung cancer or colon cancer.

[0040] As used herein, the terms "individual" or "subject" refer to mammals and non-mammals. Mammals include, but are not limited to, humans; non-human primates such as chimpanzees and other apes and monkey species; domestic animals such as cows, horses, sheep, goats, and pigs; companion animals such as rabbits, dogs, and cats; experimental animals such as rodents including rats, mice, and guinea pigs, and mean any member of the class Mammalia. Examples of non-mammals include, but are not limited to, birds. The term "individual" does not denote a particular age or sex. In some embodiments, the individual is a human.

[0041] As used herein, the term "pharmaceutically acceptable" means non-toxic, biologically tolerated, and suitable for administration to an individual.

[0042] As used herein, the term "pharmaceutically acceptable salt" refers to acid addition salts that are non-toxic, biologically tolerable, and suitable for administration to an individual, and includes, but is not limited to, acid addition salts of inorganic acids such as hydrochloride, hydrobromide, carbonate, bicarbonate, phosphate, sulfate, sulfite, nitrate, etc.; and acid addition salts of organic acids such as formate, acetate, malate, maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate, benzoate, salicylate, stearate, and salts of alkane-dicarboxylic acids of the formula HOOC-(CH2) n -COOH (wherein n is from 0 to 4), etc. are included.

[0043] In addition, pharmaceutically acceptable acid addition salts may be formed by dissolving the free base in a suitable solvent and treating the solution with an acid according to conventional procedures for preparing acid addition salts from basic compounds. One of ordinary skill in the art can determine the various synthetic methods used to prepare non-toxic pharmaceutically acceptable acid addition salts without undue experimentation. In some embodiments, the pharmaceutically acceptable salt of IN10018 is the tartrate salt.

[0044] As used herein, the term "pharmaceutically acceptable composition" means that it must be chemically and / or toxicologically compatible with other ingredients contained in the formulation and / or the subject being treated. The term "therapeutically effective amount" as used herein refers to an amount that is generally sufficient to produce a beneficial therapeutic effect in a subject. The therapeutically effective amount of the present disclosure can be determined by conventional methods (e.g., modeling, dose escalation studies, or clinical trials) in combination with conventional influencing factors (e.g., mode of administration, pharmacokinetics of the compound, severity and duration of the disease, medical history of the subject, health status of the subject, responsiveness of the subject to the drug, etc.).

[0045] As used herein, the terms "inhibit", "inhibiting", or "inhibition" refer to a decrease in the baseline activity of a biological function or biological process.

[0046] As used herein, the term "kit" refers to a box for containing chemical reagents for detecting chemical components, residual drugs, virus species, etc. The kits described herein can include (i) one or two of IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor, and (ii) an instruction manual stating that IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor can be used to treat tumors in a subject. In one embodiment, the kit includes (i) IN10018 or a pharmaceutically acceptable salt thereof, and (ii) an instruction manual stating that IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor can be used to treat tumors in a subject. In one embodiment, the kit includes (i) an epidermal growth factor receptor tyrosine kinase inhibitor, and (ii) an instruction manual stating that IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor can be used to treat tumors in a subject. In one embodiment, the kit includes (i) IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor, and (ii) an instruction manual stating that IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor can be used to treat tumors in a subject.

[0047] The compounds of the kit may be contained in separate containers. Alternatively, two or more compounds may be contained in the same container. For example, the kit can include a first container, a second container, and an accompanying document, where the first container contains a medicament comprising at least one dose of IN10018 or a pharmaceutically acceptable salt thereof, the second container contains at least one dose of an epidermal growth factor receptor tyrosine kinase inhibitor, and the accompanying document includes instructions for using the medicament to treat tumors in an individual. The first container and the second container may have the same or different shapes (e.g., vials, syringes, and bottles) and / or materials (e.g., plastic or glass). The kit may further include other materials useful for administering the medicament, such as diluents, filters, IV bags and tubes, needles, and syringes.

[0048] The exact amounts of IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor to be administered to a subject can depend on various factors, such as a given drug or compound, pharmaceutical preparation, route of administration, type of disease, condition, attributes of the subject or host being treated, etc., but nonetheless can be routinely determined by those skilled in the art. For example, the determination of an effective amount can also depend on the degree, severity, and type of cell growth. Those skilled in the art will be able to determine an appropriate dosage based on these and other factors.

[0049] IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor can be administered by suitable means such as oral, intravenous, intramuscular, or subcutaneous administration.

[0050] For example, in the case of oral administration, the drug may be orally administered together with a pharmaceutically acceptable carrier such as an inert diluent or an absorbable edible carrier. These can be enclosed in gelatin capsules with hard or soft shells, compressed into tablets, or directly mixed with the patient's diet. For example, the drug can be used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, or cachets in combination with one or more excipients. Tablets, lozenges, pills, capsules, etc. may further contain a binder such as tragacanth, gum arabic, corn starch or gelatin; an excipient such as dicalcium phosphate; a disintegrant such as corn starch, potato starch, alginic acid, etc.; a lubricant such as magnesium stearate; or a sweetening agent such as sucrose, fructose, lactose or aspartame; or a flavoring agent.

[0051] For example, in the case of infusion or injection by intravenous or intraperitoneal administration, a solution of the drug is prepared in water and may optionally be mixed with a non-toxic surfactant.

[0052] Exemplary pharmaceutical dosage forms for injection or infusion include sterile aqueous solutions, dispersions, or sterile powders containing an active ingredient suitable for the extemporaneous preparation of a sterile injection or infusion solution or dispersion. In any case, the final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage.

[0053] A sterile injection solution can be prepared by placing the required amount of the drug and the other desired ingredients listed above in a suitable solvent and then filtering and sterilizing. In the case of sterile powders for preparing sterile injection solutions, a preferred preparation method may be vacuum drying and lyophilization techniques that yield a powder of the active ingredient and any other desired ingredients after prior sterile filtration.

[0054] The amounts of IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor may vary depending not only on the particular agent selected, but also on the route of administration, the nature of the disease being treated, and the age and condition of the patient, and are ultimately left to the discretion of the attending physician or clinician. However, the dosage can generally range from about 0.1 to about 50 mg / kg body weight per day.

[0055] In some embodiments, IN10018 or a pharmaceutically acceptable salt thereof is administered to adults at a dosage of 5 mg / day to 100 mg / day, such as 20 mg / day, calculated on the basis of the free base.

[0056] The epidermal growth factor receptor tyrosine kinase inhibitor is administered to adults in a dosage range of 2 to 500 mg per day. In a specific embodiment, osimertinib or a pharmaceutically acceptable salt thereof is administered to adults in a dosage range of 2 to 500 mg per day, such as 80 mg per day, calculated on the basis of osimertinib; amonertinib or a pharmaceutically acceptable salt thereof is administered to adults in a dosage range of 2 to 250 mg per day, such as 110 mg per day, calculated on the basis of amonertinib; and alflutinib or a pharmaceutically acceptable salt thereof is administered to adults in a dosage range of 2 to 250 mg per day, such as 80 mg per day, calculated on the basis of alflutinib.

[0057] Technical and scientific terms used herein that are not specifically defined have the meanings commonly understood by those of ordinary skill in the art to which this invention belongs.

[0058] In some embodiments, the present disclosure also discloses the following:

[0059] 1. In the manufacture of a medicament for the treatment of tumors in a subject,

[0060]

Chemical formula

[0061] Use of IN10018 having the structure of or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor.

[0062] 2. For use in the treatment of tumors in a subject,

[0063]

Chemical formula

[0064] Combined pharmaceutical preparation of IN10018 having the structure of or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor.

[0065] 3. A method of treating a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of

[0066]

Chemical formula

[0067] IN10018 having the structure of or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor.

[0068] 4. The use, combined pharmaceutical preparation, or method according to any one of embodiments 1 to 3, wherein the pharmaceutically acceptable salt of IN10018 is a tartrate.

[0069] 5. The use, combined pharmaceutical preparation, or method according to any one of embodiments 1 to 4, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as fulmonertinib), EAI045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163, or a pharmaceutically acceptable salt thereof; alternatively, the use, combined pharmaceutical preparation, or method according to any one of embodiments 1 to 4, wherein the inhibitor is osimertinib, almonertinib, alflutinib, or a pharmaceutically acceptable salt thereof.

[0070] 6. The use, combined pharmaceutical preparation, or method according to any one of embodiments 1 to 5, wherein IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor are administered to a subject simultaneously or sequentially.

[0071] 7. The use, combined pharmaceutical preparation, or method according to any one of embodiments 1 to 6, wherein the tumor is caused by bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, cholangiocarcinoma, leiomyosarcoma, liposarcoma, hypopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland cancer, spindle cell carcinoma metastasis, undifferentiated large cell lymphoma, anaplastic thyroid carcinoma, non-Hodgkin lymphoma, Hodgkin lymphoma, glioma, and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), or chronic myeloid leukemia (CML); alternatively, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer, or colon cancer; alternatively, the tumor is lung cancer or colon cancer.

[0072] 8. (a)

[0073] [Chemical]

[0074] IN10018 having the structure or a pharmaceutically acceptable salt thereof, and (b) an epidermal growth factor receptor tyrosine kinase inhibitor A kit or pharmaceutically acceptable composition comprising the same.

[0075] 9. The kit or composition according to embodiment 8, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as fulmonertinib), EAI045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163, or a pharmaceutically acceptable salt thereof; alternatively, osimertinib, almonertinib, alflutinib, or a pharmaceutically acceptable salt thereof.

[0076] 10. The kit or composition according to embodiment 8 or 9, for use as a medicament.

[0077] 11. The kit or composition according to any one of embodiments 8 to 10, wherein the pharmaceutically acceptable salt of IN10018 is a tartrate.

[0078] 12. A method for treating a tumor in a subject, the method comprising administering the compounds in the kit or composition according to any one of embodiments 8 to 11 to the subject simultaneously or sequentially.

[0079] 13. The method according to embodiment 12, wherein the tumor is caused by metastasis from bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, multiple myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, cholangiocarcinoma, leiomyosarcoma, liposarcoma, hypopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland cancer, spindle cell carcinoma, undifferentiated large cell lymphoma, anaplastic thyroid carcinoma, non-Hodgkin lymphoma, Hodgkin lymphoma, glioma, and hematological malignancies such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), or alternatively, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer or colon cancer, or alternatively, the tumor is lung cancer or colon cancer.

[0080] 14. A method of treating a tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of

[0081]

Chemical formula

[0082] IN10018 having the structure of or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor.

[0083] 15. The method according to embodiment 14, wherein the pharmaceutically acceptable salt of IN10018 is IN10018 tartrate.

[0084] 16. The method according to embodiment 14 or 15, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as fulmonertinib), EAI045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163, or a pharmaceutically acceptable salt thereof; alternatively, the method is osimertinib, almonertinib, alflutinib, or a pharmaceutically acceptable salt thereof.

[0085] 17. The method according to any one of embodiments 14 to 16, wherein IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor are administered to the subject simultaneously or sequentially.

[0086] 18. The tumor is caused by bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, cholangiocarcinoma, leiomyosarcoma, liposarcoma, hypopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland cancer, spindle cell carcinoma metastasis, undifferentiated large cell lymphoma, anaplastic thyroid carcinoma, non-Hodgkin lymphoma, Hodgkin lymphoma, glioma, or hematological malignancy, for example, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), or chronic myeloid leukemia (CML); alternatively, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer, or colon cancer; alternatively, the tumor is lung cancer or colon cancer. The method according to any one of embodiments 14 to 17.

[0087] 19.

[0088] [Chem.]

[0089] In the manufacture of a medicament for the treatment of tumors in a subject, wherein IN10018 having the structure or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor are administered to the subject, Use of IN10018 or a pharmaceutically acceptable salt thereof.

[0090] 20. The use according to embodiment 19, wherein the pharmaceutically acceptable salt of IN10018 is IN10018 tartrate.

[0091] 21. The use according to embodiment 19 or 20, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as flumonertinib), EAI045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163, or a pharmaceutically acceptable salt thereof; alternatively, osimertinib, almonertinib, alflutinib, or a pharmaceutically acceptable salt thereof.

[0092] 22. The use according to any one of embodiments 19 to 21, wherein IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor are administered to the subject simultaneously or sequentially.

[0093] 23. Use according to any one of embodiments 19 to 22, wherein the tumor is a metastasis caused by bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, cholangiocarcinoma, leiomyosarcoma, liposarcoma, hypopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland cancer, spindle cell carcinoma, undifferentiated large cell lymphoma, anaplastic thyroid carcinoma, non-Hodgkin lymphoma, Hodgkin lymphoma, glioma, or a hematological malignancy, for example, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), or chronic myeloid leukemia (CML); alternatively, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer or colorectal cancer; alternatively, the tumor is lung cancer or colorectal cancer.

[0094] 24. Use of an epidermal growth factor receptor tyrosine kinase inhibitor in the manufacture of a medicament for the treatment of a tumor in a subject, wherein the subject is administered IN10018 having the structure of

[0095]

Chemical formula

[0096] or a pharmaceutically acceptable salt thereof.

[0097] 25. The use according to embodiment 24, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as flumonertinib), EAI045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163, or a pharmaceutically acceptable salt thereof; alternatively, the use according to embodiment 24, wherein the inhibitor is osimertinib, almonertinib, alflutinib, or a pharmaceutically acceptable salt thereof.

[0098] 26. The use according to embodiment 24 or 25, wherein the pharmaceutically acceptable salt of IN10018 is IN10018 tartrate.

[0099] 27. The use according to any one of embodiments 24 to 26, wherein IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor are administered to a subject simultaneously or sequentially.

[0100] 28. Use according to any one of embodiments 24 to 27, wherein the tumor is a metastasis caused by bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, multiple myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, cholangiocarcinoma, leiomyosarcoma, liposarcoma, hypopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland cancer, spindle cell carcinoma, undifferentiated large cell lymphoma, anaplastic thyroid carcinoma, non-Hodgkin lymphoma, Hodgkin lymphoma, glioma, or hematological malignancy, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), or chronic myelogenous leukemia (CML); alternatively, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer or colon cancer; alternatively, the tumor is lung cancer or colon cancer.

[0101] 29. In the manufacture of a medicament for the combined treatment of tumors,

[0102]

Chemical formula

[0103] Use of IN10018 having the structure of or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor.

[0104] 30. In the manufacture of a medicament for use in combination with an epidermal growth factor receptor tyrosine kinase inhibitor for the treatment of tumors,

[0105]

Chemical formula

[0106] Use of IN10018 having the structure of or a pharmaceutically acceptable salt thereof.

[0107] 31. For the treatment of tumors

[0108] [Chemical formula]

[0109] Use of an epidermal growth factor receptor tyrosine kinase inhibitor in the manufacture of a medicament for use in combination with IN10018 having the structure or a pharmaceutically acceptable salt thereof

[0110] 32. Use according to any one of embodiments 29 to 31, wherein IN10018 or a pharmaceutically acceptable salt thereof is IN10018 tartrate

[0111] 33. Use according to any one of embodiments 29 to 32, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as flumonertinib), EAI045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163, or a pharmaceutically acceptable salt thereof; alternatively, osimertinib, almonertinib, alflutinib, or a pharmaceutically acceptable salt thereof

[0112] 34. Use according to any one of embodiments 29 to 33, wherein IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor are administered to the subject simultaneously or sequentially

[0113] 35. Use according to any one of embodiments 29 to 34, wherein the tumor is a metastasis caused by bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, cholangiocarcinoma, leiomyosarcoma, liposarcoma, hypopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland cancer, spindle cell carcinoma, undifferentiated large cell lymphoma, anaplastic thyroid carcinoma, non-Hodgkin lymphoma, Hodgkin lymphoma, glioma, or hematological malignancy, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), or chronic myeloid leukemia (CML); alternatively, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer, or colon cancer; alternatively, the tumor is lung cancer or colon cancer.

Examples

[0114] The following examples are provided to further illustrate the present disclosure. It should be understood that these examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure.

[0115] The experimental methods without specific conditions in the following examples can be carried out according to the conventional conditions of this type of reaction or the conditions suggested by the manufacturer.

[0116] The experimental materials and reagents used in the following examples can be obtained from commercial suppliers unless otherwise specified.

[0117] The meanings of the abbreviations used in the examples are as follows:

[0118]

Table 1

[0119] (Example 1) Study of IN10018 and AZD9291 in Lung Cancer KPL Cells KPL cells (Institute of Cellular Sciences, CAS) were cultured using RPMI1640 (Shanghai BasalMedia Technology Co., Ltd., Catalog Number: L210KJ, Lot Number: F210916) + 10% FBS (Gibco, Catalog Number: 10099 - 141c, Lot Number: 2158737cp) and passaged twice. When the cell state was good, the culture was seeded into 24 - well plates. Four groups were set up 24 hours after cell seeding. The first group was the negative control group with culture medium, the second group was 10 μM of IN10018, the third group was 10 μM of AZD9291, and the fourth group was the combination group of IN10018 (10 μM) and AZD9291 (10 μM). The drugs were mixed with the cells and incubated at 37 °C in a 5% CO2 incubator for 48 hours.

[0120] 48 hours after treatment with the drugs, the cells were observed under a microscope, photographed, and the photos were saved. Then, the cells were collected for flow analysis and washed twice with flow buffer (PBS + 2% FBS). 0.5 μl of CoraLite® 488 conjugated GRP94 polyclonal antibody (Proteintech, Catalog Number: CL488 - 14700, Lot Number: 21006579) was added to each well, mixed, and incubated in the dark at 4 °C for 20 minutes. After 20 minutes, the cells were washed twice with flow buffer (PBS + 2% FBS). Then, the cells were analyzed on a flow cytometer.

[0121] The cells were observed under a microscope. The cell conditions in the AZD9291 monotherapy group and the combination group of the two drugs were poor, and the combination group of the two drugs was the worst with more cell death, while the cell conditions in the negative control group and the IN10018 group were better. For details, please refer to Figure 1 and Figure 2.

[0122] (Example 2) Study of IN10018 and Almonertinib in Lung Cancer KPL Cells KPL cells (Institute of Cellular Sciences, CAS) were cultured using RPMI1640 (Shanghai BasalMedia Technology Co., Ltd., classification number: L210KJ, lot number: F210916) + 10% FBS (Gibco, classification number: 10099 - 141c, lot number: 2158737cp) and passaged twice. When the cell state was good, the culture was seeded into 24 - well plates. Four groups were set up 24 hours after cell seeding. The first group was the negative control group with culture medium, the second group was 10 μM of IN10018, the third group was 4.7 μM of almonertinib, and the fourth group was the combination group of IN10018 (10 μM) and almonertinib (4.7 μM). The drugs were mixed with the cells and incubated at 37 °C for 48 hours in a 5% CO2 incubator.

[0123] 48 hours after treatment with the drugs, the cells were observed under a microscope, photographed, and the photos were saved. Then, the cells were collected for flow analysis and washed twice with flow buffer (PBS + 2% FBS). 0.5 μl of CoraLite® 488 conjugated GRP94 polyclonal antibody (Proteintech, classification number: CL488 - 14700, lot number: 21006579) was added to each well, mixed, and incubated in the dark at 4 °C for 20 minutes. After 20 minutes, the cells were washed twice with flow buffer (PBS + 2% FBS). Then, the cells were analyzed on a flow cytometer.

[0124] The cells were observed under a microscope. The cell conditions in the almonertinib monotherapy group and the combination group of the two drugs were poor, and the combination group of the two drugs was the worst with more cell death, while the cell conditions in the negative control group and the IN10018 group were better. For details, please refer to Figures 3 and 4.

[0125] (Example 3) Study of IN10018 and afatinib in lung cancer KPL cells KPL cells (Institute of Cellular Sciences, CAS) were cultured using RPMI1640 (Shanghai BasalMedia Technology Co., Ltd., classification number: L210KJ, lot number: F210916) + 10% FBS (Gibco, classification number: 10099 - 141c, lot number: 2158737cp) and subcultured twice. When the cell state was good, the culture was seeded into 24-well plates. Four groups were set up 24 hours after cell seeding. The first group was the negative control group with culture medium, the second group was 10 μM of IN10018, the third group was 4.7 μM of afatinib, and the fourth group was the combination group of IN10018 (10 μM) and afatinib (4.7 μM). The drugs were mixed with the cells and incubated at 37 °C in a 5% CO2 incubator for 48 hours.

[0126] Forty-eight hours after treatment with the drugs, the cells were observed under a microscope, photographed, and the photos were saved. Then, the cells were collected for flow analysis and washed twice with flow buffer (PBS + 2% FBS). 0.5 μl of CoraLite® 488 conjugated GRP94 polyclonal antibody (Proteintech, classification number: CL488 - 14700, lot number: 21006579) was added to each well, mixed, and incubated at 4 °C in the dark for 20 minutes. After 20 minutes, the cells were washed twice with flow buffer (PBS + 2% FBS). Then, the cells were analyzed on a flow cytometer.

[0127] The cells were observed under a microscope. The cell conditions in the afatinib single-drug group and the combination group of the two drugs were poor, and the combination group of the two drugs was the worst with more cell death, while the cell conditions in the negative control group and the IN10018 group were better. For details, please refer to Figure 5 and Figure 6.

[0128] (Example 4) In Vivo Antitumor Efficacy Study of AZD9291 in a Subcutaneous Allograft Tumor Model of Colon Cancer CT-26 Cells in BALB / c Mice Assay Materials: Mice: Female BALB / c mice, 6 - 8 weeks old, were purchased from Shanghai Slack Laboratory Animal Co., Ltd. The assay was started after the animals arrived and were acclimated to the assay environment. The animals were housed in IVC (Individually Ventilated Cages) in a breeding room at the SPF level (5 animals per cage). All cages, bedding, and drinking water were sterilized before use. All laboratory personnel should wear protective clothing and latex gloves during operations in the breeding room. Cages, food, and drinking water were changed twice a week. The breeding environment and lighting conditions were as follows:

[0129] Temperature: 20 - 26°C Humidity: 40 - 70% Lighting hours: 12 hours of light and 12 hours of darkness Cages: Made of polycarbonate, with a volume of 300 mm × 180 mm × 150 mm. The bedding is corn cob, which is changed twice a week. Feed: The assay animals were allowed to freely consume irradiated and sterilized dry pellet feed throughout the experimental stage. Drinking water: The assay animals were allowed to freely drink sterilized water. Cage identification: The animal information card for each cage recorded the number of animals, gender, strain, receipt date, administration schedule, experimental number, group, and start date of the assay in that cage. Animal identification: The assay animals were identified by ear tags.

[0130] The information of the compound is shown in Table 1 (Table 2).

[0131]

Table 2

[0132] Colorectal cancer cell line CT-26 (Supplier: Nanjing Cobioer Biosciences Co., Ltd., Stock number: CBP60043) was maintained by Inxmed (Nanjing) Co., Ltd. The cells were cultured in monolayer in vitro in RPMI-1640 medium containing 10% fetal bovine serum in a 5% CO2 incubator at 37°C. The cells were passaged by normal digestion with trypsin-EDTA 2-3 times a week. When the cells entered the exponential growth phase and reached a saturation of 80%-90%, the cells were harvested, counted, and seeded.

[0133] Cell seeding and grouping 3×10 5 Individual cells in 0.1 mL of cell suspension were subcutaneously inoculated into the right dorsal side of each mouse. When the tumor volume reached approximately 59 mm 3 (on the 12th day after cell inoculation), the mice were randomized into multiple treatment groups according to the tumor volume, and the grouping information is shown in Table 2 (Table 3).

[0134]

Table 3

[0135] Note: 1. Number of mice per group; 2. Dosage: 10 mL / kg based on the body weight of the mouse. When the body weight decreased by more than 15%, the administration to the animal was stopped and resumed until the body weight recovered to 10% or less.

[0136] Preparation of test substance For details, please refer to Table 3 (Table 4).

[0137]

Table 4

[0138] Daily observation of assay animals Animals were monitored daily for health status and mortality. Regular examinations included observation of tumor growth and the effects of drug treatment on the daily behavior of the animals, such as locomotor activity, food and water intake (visual inspection only), body weight changes, and observation of appearance signs or other abnormalities. The number of animal deaths and side effects in each group were recorded based on the number of animals in each group.

[0139] End of assay If the health status of the animal continued to deteriorate, or if the tumor volume exceeded 3,000 mm 3 or if the animal had severe illness or pain, the animal had to be euthanized. In the following situations: significantly emaciated, weight loss exceeding 20%, unable to eat and drink freely, the average tumor volume in the control group reached 2,000 mm 3 the veterinarian was notified, the animal was euthanized, and the experiment was terminated. The animals presented the following clinical findings and continued to deteriorate: piloerection, arched back, pallor of ears, nose, eyes or feet, dyspnea, convulsions, continuous diarrhea, dehydration, slow movement, or sound.

[0140] Tumor measurement and assay indicators The tumor diameter was measured three times a week using calipers. The tumor volume was calculated by the formula: V = 0.5 × a × b 2 (where a and b represent the major and minor diameters of the tumor, respectively).

[0141] The tumor growth inhibition rate TGI (%) was calculated with reference to the tumor volume on the first day after grouping according to the following formula: TGI (%) = [1 - (average tumor volume of the treatment group - average tumor volume at the start of treatment of that treatment group) / (average tumor volume of the vehicle control group - average tumor volume at the start of treatment of that vehicle control group)] × 100%.

[0142] Statistical analysis Statistical analysis was performed using Prism Graphpad software based on tumor volume and tumor weight at the end of the experiment. The comparison results of multiple groups were analyzed using two-way analysis of variance and Fisher's LSD test. A P < 0.05 was considered statistically significant.

[0143] Results of the assay In vivo efficacy of the test substance AZD9291 in combination with IN10018 in a BALB / c mouse subcutaneous syngeneic tumor model of CT-26 mouse colon cancer cells. Tumor growth was observed daily after cell inoculation. Grouping was based on tumor volume on day 11 after inoculation, and the average tumor volume at the time of group registration was approximately 38 mm 3 Since the mice had tumors, the control group was euthanized on day 26 after inoculation, i.e., 15 days after administration for each group, and the entire assay was terminated.

[0144] On day 15 after administration for each group, the tumor volume in the control group was 1546.6 ± 1038.8 mm 3 The tumor volumes in the individual treatment groups were 1766.9 ± 732.5 mm, 1046.3 ± 407.8 mm, and 540.4 ± 218.0 mm for IN10018 (25 mg / kg), AZD9291 (20 mg / kg), and AZD9291 + IN10018 (20 + 25 mg / kg), respectively, as detailed in Table 4 (Table 5). 3 1046.3 ± 407.8 mm 3 and 540.4 ± 218.0 mm 3It was as follows. When the tumor volume was compared with that of the control group, the tumor growth inhibition rate (TGI) was -14.5% (p = 0.3079), 33.2% (p = 0.0216), and 66.6% (p < 0.0001) for the IN10018 (25 mg / kg) group, the AZD9291 (20 mg / kg) group, and the AZD9291 + IN10018 (20 + 25 mg / kg) group, respectively. Details are shown in Table 4 (Table 5). The tumor volume was compared with that of the AZD9291 + IN10018 (20 + 25 mg / kg) combination group and statistically analyzed, showing p-values of p < 0.0001, p < 0.0001, and p = 0.0202 for the control group, the IN10018 (25 mpk) monotherapy group, and the AZD9291 (20 mpk) monotherapy group, respectively. The tumor volumes at different time points for each dose group are shown in Figure 7.

[0145]

Table 5

[0146] Note: 1. Calculated according to the number of days after administration for each group, and the data were mean ± standard error. 2. *: p < 0.05, ****: p < 0.0001, compared with the control group, two-way ANOVA. 3. *: p < 0.05, ****: p < 0.0001, compared with the AZD9291 + IN10018 (20 + 25 mg / kg) group, two-way ANOVA.

[0147] The assay was performed according to the dosing regimen, during which the animals were observed daily for diet and water intake, and the body weights of the animals were recorded three times a week. The body weight curves of the animals are shown in Figure 8. Throughout the dosing cycle, the animals in all groups did not show significant weight loss and were in good condition.

[0148] Conclusion Compared with the blank control group, both the AZD9291 (20 mg / kg) group and the AZD9291 + IN10018 (20 + 25 mg / kg) group showed obvious tumor growth inhibition with a statistical difference from the control group. Generally, throughout the administration cycle, the AZD9291 + IN10018 (20 + 25 mg / kg) group consistently had a statistically significant smaller tumor volume than both the IN10018 (25 mg / kg) group and the AZD9291 (20 mg / kg) group, and had better tumor growth inhibition compared to the IN10018 (25 mg / kg) group and the AZD9291 (20 mg / kg) group. At the same time, the animals showed good body weight changes throughout the administration cycle, showed no abnormalities in activity, water intake, and mental state, indicating that the animals tolerated the combination of AZD9291 + IN10018 (20 + 25 mg / kg).

[0149] (Example 5) In vivo antitumor efficacy study of AZD9291 and IN10018 in a subcutaneous xenograft tumor model of human non-small cell lung cancer HCC827 cells in BALB / c nude mice Assay materials

[0150] [Table 6]

[0151] [Table 7]

[0152] [Table 8]

[0153] [Table 9]

[0154] [Table 10]

[0155] Assay method and procedure Human non-small cell lung cancer cell HCC827 (Supplier: Shanghai Cell Bank, Stock number: TCHu153) was maintained in passage by Nangjing ClinBridge Biotech Co., Ltd. The cells were cultured in monolayer in vitro in RPMI-1640 medium + 10% FBS in a 5% CO2 incubator at 37°C. The cells were passaged by normal digestion using trypsin-EDTA 2-3 times a week. When the cells entered the exponential growth phase and reached a saturation of 80% - 90%, the cells were harvested, counted, and inoculated. 0.1 mL of cell suspension containing 5×10 6 cells was subcutaneously inoculated into the right dorsum of each mouse. When the tumor volume reached approximately 147 mm 3 (on the 16th day after cell inoculation), the mice were randomized into multiple dosing groups according to the tumor volume, and the grouping information is shown in Table 5 (Table 11).

[0156] [Table 11]

[0157] Note: 1. N indicates the number of mice per group; 2. The dosing volume was 10 mL / kg based on the body weight of the mice. When the body weight decreased by more than 15%, the dosing to the animals was stopped and restarted until the body weight recovered to 10% or less.

[0158] Daily observation of assay animals The use and welfare of experimental animals complied with the regulations of AAALAC. The animals were monitored daily for health status and death. Periodic examinations included observation of tumor growth and the effects of drug treatment on the daily behavior of the animals, such as locomotor activity, food and water intake (by visual inspection only), body weight changes, and observation of external signs or other abnormalities. The number of animal deaths and side effects in each group were recorded based on the number of animals in each group.

[0159] End of assay If the animal's health continued to deteriorate, or if the tumor volume exceeded 3,000 mm 3 or if the animal had a serious illness or pain, it was necessary to euthanize the animal. In the following situations: severely emaciated, with weight loss exceeding 20%, unable to eat and drink freely, when the average tumor volume in the control group reached 2,000 mm 3 the veterinarian was notified, the animal was euthanized, and the experiment was terminated. The animals presented the following clinical findings and continued to deteriorate: piloerection, arched back, pallor of the ears, nose, eyes or feet, dyspnea, convulsions, continuous diarrhea, dehydration, slow movement, or vocalization.

[0160] Tumor measurement and assay indicators The experimental criterion was to examine whether tumor growth was inhibited, delayed or cured. The tumor diameter was measured three times a week using calipers. The tumor volume was calculated according to the following formula: V = 0.5 × a × b 2 (where a and b represent the long diameter and short diameter of the tumor, respectively).

[0161] The tumor suppression efficacy of the compound was evaluated by TGI (%) reflecting the tumor growth inhibition rate. The tumor growth inhibition rate TGI (%) was calculated according to the following formula with reference to the tumor volume on the first day after grouping: TGI (%) = [1 - (average tumor volume of the treatment group - average tumor volume at the start of treatment of that treatment group) / (average tumor volume of the vehicle control group - average tumor volume at the start of treatment of that vehicle control group)] × 100%.

[0162] Statistical analysis Statistical analysis was performed using Prism Graphpad software based on the tumor volume at the end of the test. The comparison results of multiple groups were analyzed using two-way analysis of variance and Fisher's LSD test method, and P < 0.05 was considered a significant difference. The comparison results of two groups were analyzed using t-test and Mann-Whitney test method, and P < 0.05 was considered a significant difference.

[0163] Results of the assay After cell inoculation, tumor growth was observed daily. Grouping was based on the tumor volume on day 16 after inoculation, and the average tumor volume when registered in the group was approximately 147 mm 3 . Due to carrying tumors, the control group was euthanized on day 48 after inoculation, that is, on day 32 after administration for each group. The AZD9291 high-dose (3 mg / kg) group and the combination group related to IN10018 25 mg / kg were euthanized on day 53 after inoculation, that is, on day 37 after administration for each group, and the entire assay was terminated.

[0164] On day 32 after administration for each group, the tumor volume in the control group was 1805.6 ± 722.8 mm 3 . The tumor volumes in each treatment group were 20.3 ± 4.7 mm, 79.2 ± 51.8 mm, and 9.0 ± 7.0 mm for AZD9291 (3 mg / kg), IN10018 (25 mg / kg), and AZD9291 + IN10018 (3 + 25 mg / kg), respectively. 3 3 3 . When the tumor volumes were compared with those of the control group, the tumor growth inhibition rate TGI was 107.7% (p < 0.0001), 104.1% (p < 0.0001), and 108.4% (p < 0.0001) for the AZD9291 (3 mg / kg) group, IN10018 (25 mg / kg) group, and AZD9291 + IN10018 (3 + 25 mg / kg) group, respectively. Details are shown in Table 5-1 (Table 12). The tumor volumes at different time points for each dose group are shown in Figure 9.

[0165] On day 32 after administration for each group, the control group was euthanized due to excessive tumor volume, and the AZD9291 (3 mg / kg) group, IN10018 (25 mg / kg) group, and AZD9291 + IN10018 (3 + 25 mg / kg) group continued administration and observation until day 37. On day 37 after administration for each group, the tumor volumes in the AZD9291 (3 mg / kg) group and IN10018 (25 mg / kg) group were 17.0 ± 6.7 mm 3 and 69.6 ± 46.6 mm 3 ​​It was 5.8±6.5 mm in the combination group of AZD9291+IN10018 (3+25 mg / kg). 3 When the tumor volume was compared with that of the combination group of AZD9291+IN10018 (3+25 mg / kg), the p-values were p = 0.0317 and p = 0.0079 for the AZD9291 (3 mg / kg) group and the IN10018 (25 mg / kg) group, respectively, and both were statistically significant. Details are shown in Table 5-2 (Table 13). The tumor volumes at different time points in each dose group are shown in Figure 9. The tumor volumes in each dose group at the end of the assay are shown in Figure 10.

[0166]

Table 12

[0167] Note: 1. Calculated according to the number of days after administration for each group, and the data are mean ± standard deviation (mean ± SD); 2. ****: p<0.0001, compared with the control group, two-way ANOVA.

[0168]

Table 13

[0169] Note: 1. Calculated according to the number of days after administration for each group, and the data are mean ± standard deviation (mean ± SD); 2. *: p<0.05, **: p<0.01, compared with AZD9291+IN10018 (3+25 mg / kg), t-test, Mann-Whitney test.

[0170] The assay was performed according to the dosing regimen, during which the animals were observed daily for diet and water intake, and the body weights of the animals were recorded three times a week. Twenty-eight days after dosing for each group, the average body weight of the control group changed from 20.0 g on the day of dosing for each group (day 0) to 19.4 g, and the weight gain rate was -3.3%. The average body weight of the IN10018 (25 mg / kg) treatment group changed from 21.3 g on day 0 to 20.7 g on day 28, and the weight change rate was -2.6%.

[0171] Thirty-seven days after dosing for each group, the average body weights in the AZD9291 (3 mg / kg) group and the IN10018 (25 mg / kg) group changed from 19.9 g and 21.3 g on day 0 to 19.4 g and 20.3 g on day 37, respectively, and the weight change rates were -2.2% and -4.5%. The average body weight in the AZD9291 + IN10018 (3 + 25 mg / kg) group changed from 20.0 g on day 0 to 19.5 g on day 37, and the weight change rate was -2.3%.

[0172] Animals in all groups did not show significant weight loss throughout the dosing cycle and were in good condition. Details are shown in Table 5-3 (Table 14). The changes in body weight at different time points in the relevant groups of AZD9291 (3 mg / kg) and IN10018 are shown in Figure 11.

[0173]

Table 14

[0174] Note: 1. Number of animals surviving on day 0 / Number of animals surviving on day 15 after dosing for each group; 2. Data are mean ± standard deviation (mean ± SD); 3. Weight change rate = [1 - (W t - W0) / W0] × 100%.

[0175] Conclusion Compared with the blank control group, each of the AZD9291 (3 mg / kg) group, IN10018 (25 mg / kg) group, and AZD9291 + IN10018 (3 + 25 mg / kg) group showed significant tumor growth inhibition with a statistical difference from the control group. Throughout the dosing cycle, the AZD9291 + IN10018 (3 + 25 mg / kg) group consistently had a smaller tumor volume than both monotherapy groups with a statistical difference from the AZD9291 (3 mg / kg) group and IN10018 (25 mg / kg) group, indicating that the combination of AZD9291 + IN10018 (3 + 25 mg / kg) produced better inhibition of tumor growth.

[0176] (Example 6) Synergistic tumor killing effect caused by the combination of IN10018 and AZD-9291 In an in vitro assay, the killing curve of AZD9291 in lung cancer cell HCC827 was detected and the IC50 value was determined. The cytotoxic effects of various doses of AZD-9291 combined with 3 μM and 5 μM of IN10018 respectively were explored. The cellular apoptosis responses to the monotherapy and combination drugs were also detected using flow cytometry together with annexin V staining.

[0177] Assay antibodies: Recombinant Alexa Fluor® 647 fluorescent anti-calreticulin antibody (Abcam, ab196159), FAK antibody (CST, 3285S), phospho-eIF2α (Ser51) (CST, 3398), eif2α (CST, 5324), DDIT3 (HUABIO, ET1703-05), HRP-tagged alpha-tubulin (α-microtubulin) antibody (Proteintech, HRP-66031).

[0178] 1. Synergistic tumor killing effect caused by the combination of IN10018 and AZD-9291 5000 HCC827 cells / well were seeded in a 96-well plate. After 24 hours, fixed concentrations of IN10018 were added in combination with various concentrations of AZD9291. The concentrations of IN10018 were 3 μM and 5 μM, and a total of 9 concentrations of AZD9291 were prepared by three-fold serial dilution with a maximum concentration of 1 μM. After 72 hours of co-incubation of the drugs and cells, 10 μL of CCK8 solution was added to each well, and the mixture was incubated at 37 °C for 2 hours in a 5% CO2 incubator, and a microplate reader was used at an absorption wavelength of 450 nm. The reading results were compared with the DMSO control group and plotted using Graphpad 8.0. For details, please refer to Figure 12.

[0179] 2. Apoptosis promoted by the combination of IN10018 and AZD-9291 HCC827 cells were treated with 0.3 nM AZD9291 combined with 3 μM IN10018. After 48 hours, the cells were harvested for cell staining using an annexin V kit and detected using flow cytometry. Early and late apoptotic cell values were counted for comparison with the DMSO control group and plotted using Graphpad 8.0. For details, please refer to Figure 13. The results showed that early and late apoptosis were significantly enhanced in the combination group compared with the single-drug group.

[0180] All references mentioned in this specification are incorporated by reference in their entirety as if each were individually recited. After reading the disclosure of the present invention, those skilled in the art should understand that various changes or modifications can be made to the present invention, and these equivalent forms are also included within the scope defined by the appended claims of this application.

[0181] This application claims the priority of Chinese Patent Application No. 202210730136.2, filed on June 24, 2022, which is incorporated herein by reference in its entirety as part of the disclosure of this application.

Claims

1. Use of IN10018 having the structure of 【Chemical 1】 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor in the manufacture of a medicament for the treatment of tumors in a subject.

2. A combined pharmaceutical preparation of IN10018 having the structure of [Chemical 2] or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor for use in the treatment of tumors in a subject.

3. A method of treating a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of 【Chemical Formula 3】 IN10018 having the structure of or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor simultaneously or sequentially.

4. The use, combined pharmaceutical preparation, or method according to any one of claims 1 to 3, wherein the pharmaceutically acceptable salt of IN10018 is a tartrate.

5. The epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as flmonertinib), EAI045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163, or a pharmaceutically acceptable salt thereof; alternatively, osimertinib, almonertinib, alflutinib, or a pharmaceutically acceptable salt thereof. The use, combined pharmaceutical preparation, or method according to any one of claims 1 to 4.

6. The use, combined pharmaceutical preparation, or method according to any one of claims 1 to 5, wherein IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor are administered to the subject simultaneously or sequentially.

7. The metastasis caused by a tumor selected from bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, cholangiocarcinoma, leiomyosarcoma, liposarcoma, hypopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland cancer, spindle cell carcinoma, undifferentiated large cell lymphoma, anaplastic thyroid carcinoma, non-Hodgkin lymphoma, Hodgkin lymphoma, glioma, and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML); alternatively, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer or colon cancer; alternatively, the tumor is lung cancer or colon cancer, the use, combination pharmaceutical preparation, or method according to any one of claims 1 to 6.

8. (a) 【Chemical 4】 IN10018 having the structure of or its pharmaceutically acceptable salt, and (b) an epidermal growth factor receptor tyrosine kinase inhibitor

9. The epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as flmonertinib), EAI045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163, or their pharmaceutically acceptable salts; alternatively, the kit or composition according to claim 8, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib, almonertinib, alflutinib, or their pharmaceutically acceptable salts.

10. The kit or composition according to claim 8 or 9, which is used as a drug.

11. The kit or composition according to any one of claims 8 to 10, wherein the pharmaceutically acceptable salt of IN10018 is a tartrate.

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