Application of imidazo tetrazine compound

Abstract

The invention relates to novel application of imidazo tetrazine compounds, or solvates, hydrates, polymorphs, prodrugs or pharmaceutically acceptable salts thereof, in particular to application in preparation of drugs for treating and / or preventing diffuse large B-cell lymphoma of subjects. # imgabs0 #

Description

Uses of imidazotetrazine compounds Technical Field

[0001] The present invention belongs to the field of medicine, and more specifically, relates to a novel anti-tumor use of imidazotetrazine compounds and the use of pharmaceutical preparations containing at least the imidazotetrazine compounds as active ingredients in preventing and treating diffuse lymphoma. Background Art

[0002] According to the World Health Organization's Pathological Classification of Lymphatic Tumors, there are currently nearly 70 known pathological types of lymphoma, which can be broadly divided into two categories: Hodgkin's lymphoma and non-Hodgkin's lymphoma. Hodgkin's lymphoma accounts for approximately 10% of all lymphomas, while non-Hodgkin's lymphoma accounts for approximately 90% of all lymphoma cases, with incidence rates increasing annually. Non-Hodgkin's lymphoma is divided into two major types: B-cell lymphoma and T / NK-cell lymphoma. B-cell lymphoma accounts for approximately 70% of cases, while T / NK-cell lymphoma accounts for approximately 30%. With the continuous advancement of basic and clinical research, the classification of lymphomas continues to be further refined and improved.

[0003] Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma (NHL), accounting for approximately 30-40% of all adult NHL cases. It can occur in all age groups, but is more common in the elderly, with a median age of 60 years. Males are slightly more likely to develop DLBCL than females. DLBCL accounts for the majority of clinically "aggressive" or "intermediate-to-high-grade" lymphomas. Approximately 40% of DLBCL patients have a median life expectancy of less than five years after diagnosis due to relapse or refractory disease. Therefore, research into specific treatments for DLBCL is of paramount importance.

[0004] According to relevant research reports, DLBCL may be related to congenital inheritance, dysregulation of cell apoptosis mechanisms, and DNA repair defects, but the specific cause of DLBCL remains unclear. Over the past two decades, the clinical treatment of DLBCL has improved significantly, mainly due to the traditional combination of rituximab and chemotherapy as initial treatment and the refinement of high-dose chemotherapy and autologous stem cell transplantation strategies for the treatment of DLBCL. However, both treatment options are subject to the problem of drug resistance in DLBCL, which has affected the clinical application of related drugs, and new treatment strategies are urgently needed.

[0005] The imidazotetrazine compound 3-methyl-4-oxo-8-imidazo[5,1-d][1,2,3,5]tetrazinecarboxamide (TMZ, see U.S. Patent No. 5,260,291) is a chemotherapeutic agent approved for the treatment of brain tumors (marketed by Schering AG under the trademarks Temodar in the United States and Temodal in Europe). Under biological conditions, TMZ degrades via pH-dependent hydrolysis to its active metabolite MTIC (3-methyl-(triazen-1-yl)imidazole-4-carboxamide). Temodar capsules are currently indicated in the United States for the treatment of adult patients with newly diagnosed glioblastoma multiforme and refractory undifferentiated astrocytomas. Temodal capsules are currently approved in Europe for the treatment of patients with malignant gliomas. In addition, TMZ has been reported to be used in the treatment of various lymphomas, such as the treatment of T / NK cell types (Ma Xuejun, Study on the treatment model, radiotherapy plan and prognostic-related factors of early extranodal NK / T cell lymphoma nasal type, Fudan University doctoral dissertation), the treatment of primary central nervous system lymphoma (O Asif and M Nehal, Temozolomide for Relapsed Primary CNS Lymphoma Journal of the College of Physicians and Surgeons Pakistan 2012, Vol. 22(9): 594-595), and the treatment of leukemia and lymphoma (Wang Chunmei, Synthesis and biological activity of temozolomide derivatives, Guizhou University master's thesis).

[0006] Summary of the Invention

[0007] The present invention aims to provide the use of imidazotetrazine compounds, particularly TMZ, in the preparation of a medicament for preventing or treating diffuse lymphoma. The present invention surprisingly discovered that imidazotetrazine compounds selectively exhibit excellent antagonistic effects against diffuse lymphoma that does not include the primary site or has a metastatic site in the central nervous system.

[0008] In a first aspect, the present invention provides use of an imidazotetrazine compound, or a solvate, hydrate, polymorph, prodrug, or pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating and / or preventing diffuse lymphoma, wherein the imidazotetrazine compound is represented by Formula I:

[0009] In formula I, R1 is hydrogen or C1-C3 alkyl, R2 is amino or amino substituted by C1-C3 alkyl; R3 is hydrogen or C1-C3 alkyl.

[0010] In some embodiments, the DLBCL is selected from diffuse large B-cell lymphoma whose primary site and / or metastatic site is not the central nervous system (DLBCL other than DLBCL whose primary site and / or metastatic site is the central nervous system).

[0011] In some embodiments, the DLBCL is selected from DLBCL not otherwise specified, other large B-cell lymphoma, high-grade B-cell lymphoma, and unclassifiable B-cell lymphoma.

[0012] In some embodiments, the DLBCL is selected from the group consisting of centroblastic variant DLBCL, immunoblastic variant DLBCL, anaplastic variant DLBCL, spindle cell variant DLBCL, signet ring cell variant DLBCL, T cell / histiocyte enriched DLBCL, primary cutaneous DLBCL, EBV-positive DLBCL, large B-cell lymphoma associated with chronic inflammation, lymphomatoid granulomatosis, large B-cell lymphoma with IRF4 rearrangement, large B-cell lymphoma with IRF4 / MUM1 rearrangement, primary mediastinal large B-cell lymphoma, and leukemia. B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-positive DLBCL, primary effusion lymphoma, high-grade B-cell lymphoma with MYC, BCL2, and / or BCL6 rearrangement, high-grade B-cell lymphoma not otherwise specified, unclassifiable B-cell lymphoma intermediate between DLBCL and classical Hodgkin lymphoma, B-cell lymphoma intermediate between primary mediastinal large B-cell lymphoma and nodular sclerosis classical Hodgkin lymphoma, and B-cell lymphoma intermediate between DLBCL and Burkitt lymphoma.

[0013] In some embodiments, the DLBCL is primary DLBCL, or transformed DLBCL, or advanced and / or relapsed, and / or refractory DLBCL, or DLBCL that has received prior systemic therapy, or DLBCL that has failed chemotherapy drugs and / or targeted drugs and / or immunotherapy.

[0014] In some embodiments, the DLBCL cells are selected from one or more of human diffuse tissue lymphoma cells SU-DHL-2, human diffuse lymphoma cell line TMD-8, and human diffuse large cell lymphoma cells SU-DHL-6.

[0015] A second aspect of the present invention provides a use of a pharmaceutical composition for preparing a medicament for treating and / or preventing DLBCL, wherein the pharmaceutical composition comprises an imidazotetrazine compound represented by Formula I, or a solvate, hydrate, polymorph, prodrug, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0016] In formula I, R1 is hydrogen or C1-C3 alkyl, R2 is amino or amino substituted by C1-C3 alkyl; R3 is hydrogen or C1-C3 alkyl.

[0017] In some embodiments, R1 is methyl.

[0018] In some embodiments, R2 is amino.

[0019] In some embodiments, R3 is hydrogen.

[0020] In some embodiments, R1 is methyl, R2 is amino, and R3 is hydrogen.

[0021] In some embodiments, the DLBCL is selected from diffuse large B-cell lymphoma whose primary site and / or metastatic site is not the central nervous system.

[0022] In some embodiments, the DLBCL is selected from DLBCL not otherwise specified, other large B-cell lymphoma, high-grade B-cell lymphoma, and unclassifiable B-cell lymphoma.

[0023] In some embodiments, the DLBCL is selected from the group consisting of centroblastic variant DLBCL, immunoblastic variant DLBCL, anaplastic variant DLBCL, spindle cell variant DLBCL, signet ring cell variant DLBCL, T cell / histiocyte enriched DLBCL, primary cutaneous DLBCL, EBV-positive DLBCL, large B-cell lymphoma associated with chronic inflammation, lymphomatoid granulomatosis, large B-cell lymphoma with IRF4 rearrangement, large B-cell lymphoma with IRF4 / MUM1 rearrangement, primary mediastinal large B-cell lymphoma, and leukemia. B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-positive DLBCL, primary effusion lymphoma, high-grade B-cell lymphoma with MYC, BCL2, and / or BCL6 rearrangement, high-grade B-cell lymphoma not otherwise specified, unclassifiable B-cell lymphoma intermediate between DLBCL and classical Hodgkin lymphoma, B-cell lymphoma intermediate between primary mediastinal large B-cell lymphoma and nodular sclerosis classical Hodgkin lymphoma, and B-cell lymphoma intermediate between DLBCL and Burkitt lymphoma.

[0024] In some embodiments, the DLBCL is primary DLBCL, or transformed DLBCL, or advanced and / or relapsed, and / or refractory DLBCL, or DLBCL that has received prior systemic therapy, or DLBCL that has failed chemotherapy drugs and / or targeted drugs and / or immunotherapy.

[0025] In some embodiments, the DLBCL cells are selected from one or more of human diffuse tissue lymphoma cells SU-DHL-2, human diffuse lymphoma cell line TMD-8, and human diffuse large cell lymphoma cells SU-DHL-6.

[0026] The third aspect of the present invention further provides a pharmaceutical combination comprising an imidazotetrazine compound and a second therapeutic agent that can be used to treat DLBCL, wherein the imidazotetrazine compound is represented by Formula I:

[0027] Wherein, R1 is hydrogen or C1-C3 chain alkyl, R2 is amino or C1-C3 chain alkyl substituted amino; R3 is hydrogen or C1-C3 chain alkyl.

[0028] In some embodiments, R1 is methyl.

[0029] In some embodiments, R2 is amino.

[0030] In some embodiments, R3 is hydrogen.

[0031] In some embodiments, R1 is methyl, R2 is amino, and R3 is hydrogen, that is, the imidazotetrazine compound is temozolomide (3-methyl-4-oxo-8-imidazo[5,1-d][1,2,3,5]tetrazinecarboxamide).

[0032] In some embodiments, the second therapeutic agent is one or more of a chemotherapy drug, a small molecule targeted anti-tumor drug, an immunotherapy drug, and an antibody drug. In some preferred embodiments, the second therapeutic agent is cyclophosphamide.

[0033] The fourth aspect of the present invention relates to use of the drug combination described in the third aspect in the preparation of a medicament for treating and / or preventing DLBCL.

[0034] In some embodiments, the DLBCL is selected from DLBCL other than DLBCL whose primary site and / or metastatic site is the central nervous system.

[0035] In some embodiments, the DLBCL is selected from DLBCL not otherwise specified, other large B-cell lymphoma, high-grade B-cell lymphoma, and unclassifiable B-cell lymphoma.

[0036] In some embodiments, the DLBCL is selected from the group consisting of centroblastic variant DLBCL, immunoblastic variant DLBCL, anaplastic variant DLBCL, spindle cell variant DLBCL, signet ring cell variant DLBCL, T cell / histiocyte enriched DLBCL, primary cutaneous DLBCL, EBV-positive DLBCL, large B-cell lymphoma associated with chronic inflammation, lymphomatoid granulomatosis, large B-cell lymphoma with IRF4 rearrangement, large B-cell lymphoma with IRF4 / MUM1 rearrangement, primary mediastinal large B-cell lymphoma, and leukemia. B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-positive DLBCL, primary effusion lymphoma, high-grade B-cell lymphoma with MYC, BCL2, and / or BCL6 rearrangement, high-grade B-cell lymphoma not otherwise specified, unclassifiable B-cell lymphoma intermediate between DLBCL and classical Hodgkin lymphoma, B-cell lymphoma intermediate between primary mediastinal large B-cell lymphoma and nodular sclerosis classical Hodgkin lymphoma, and B-cell lymphoma intermediate between DLBCL and Burkitt lymphoma.

[0037] In some embodiments, the DLBCL is primary DLBCL, or transformed DLBCL, or advanced and / or relapsed, and / or refractory DLBCL, or DLBCL that has received prior systemic therapy, or DLBCL that has failed chemotherapy drugs and / or targeted drugs and / or immunotherapy.

[0038] In some embodiments, the DLBCL cells are selected from one or more of human diffuse tissue lymphoma cells SU-DHL-2, human diffuse lymphoma cell line TMD-8, and human diffuse large cell lymphoma cells SU-DHL-6.

[0039] Another aspect of the present invention relates to a method for treating and / or preventing DLBCL in a subject, comprising administering to the subject an effective amount of an imidazotetrazine compound or a solvate, hydrate, polymorph, prodrug or pharmaceutically acceptable salt thereof, or a pharmaceutical combination comprising the same, wherein the imidazotetrazine compound is represented by Formula I:

[0040] Wherein, R1 is hydrogen or C1-C3 chain alkyl, R2 is amino or amino substituted by C1-C3 chain alkyl; R3 is hydrogen or C1-C3 chain alkyl.

[0041] In some embodiments, R1 is methyl.

[0042] In some embodiments, R2 is amino.

[0043] In some embodiments, R3 is hydrogen.

[0044] In some embodiments, R1 is methyl, R2 is amino; and R3 is hydrogen.

[0045] In some embodiments, the imidazotetrazine compound is 3-methyl-4-oxo-8-imidazo[5,1-d][1,2,3,5]tetrazinecarboxamide.

[0046] In some embodiments, the drug combination further comprises a second therapeutic agent that can be used to treat DLBCL.

[0047] In some embodiments, the second therapeutic agent is one or more of a chemotherapy drug, a small molecule targeted anti-tumor drug, an immunotherapy drug, and an antibody drug. Preferably, the second therapeutic agent is cyclophosphamide.

[0048] In some embodiments, the subject is a mammal, preferably a human.

[0049] In some embodiments, the DLBCL is selected from DLBCL other than DLBCL whose primary site and / or metastatic site is the central nervous system.

[0050] In some embodiments, the DLBCL is selected from DLBCL not otherwise specified, other large B-cell lymphoma, high-grade B-cell lymphoma, and unclassifiable B-cell lymphoma.

[0051] In some embodiments, the DLBCL can be selected from the group consisting of centroblastic variant DLBCL, immunoblastic variant DLBCL, anaplastic variant DLBCL, spindle cell variant DLBCL, signet ring cell variant DLBCL, T cell / histiocyte-enriched DLBCL, primary cutaneous DLBCL, EBV-positive DLBCL, large B-cell lymphoma associated with chronic inflammation, lymphomatoid granuloma, large B-cell lymphoma with IRF4 rearrangement, large B-cell lymphoma with IRF4 / MUM1 rearrangement, primary mediastinal large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-positive DLBCL, primary effusion lymphoma, High-grade B-cell lymphoma with MYC, BCL2, and / or BCL6 rearrangement, unspecified high-grade B-cell lymphoma, unclassifiable B-cell lymphoma intermediate between DLBCL and classical Hodgkin lymphoma, B-cell lymphoma intermediate between primary mediastinal large B-cell lymphoma and nodular sclerosis classical Hodgkin lymphoma, and B-cell lymphoma intermediate between DLBCL and Burkitt lymphoma.

[0052] In some embodiments, the DLBCL can be primary DLBCL, transformed DLBCL, advanced and / or recurrent, and / or refractory DLBCL, DLBCL that has previously received systemic therapy, or DLBCL that has failed chemotherapy and / or targeted therapy and / or immunotherapy. In some embodiments, the DLBCL cells are selected from one or more of human diffuse histiocytic lymphoma cells SU-DHL-2, human diffuse lymphoma cell line TMD-8, and human diffuse large cell lymphoma cells SU-DHL-6. DETAILED DESCRIPTION

[0053] definition

[0054] "Subjects" to be administered include, but are not limited to, humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults)) and / or non-human animals, e.g., mammals, such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cows, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human," "patient," and "subject" are used interchangeably herein.

[0055] "Disease," "disorder," and "condition" are used interchangeably herein.

[0056] As used herein, and unless otherwise indicated, the term "treating" includes actions that occur while a subject has a particular disease, disorder, or condition that reduces the severity of, or delays or slows the development of, the disease, disorder, or condition ("therapeutic treatment"), as well as actions that occur before a subject develops a particular disease, disorder, or condition ("prophylactic treatment").

[0057] Generally, an "effective amount" of a compound is an amount sufficient to elicit the desired biological response. As will be appreciated by those skilled in the art, the effective amount of a compound of the invention can vary depending on factors such as the biological target, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, health, and symptoms of the subject. An effective amount includes both a therapeutically effective amount and a prophylactically effective amount.

[0058] As used herein, and unless otherwise specified, a "therapeutically effective amount" of a compound is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder, or condition, or to delay or minimize one or more symptoms associated with the disease, disorder, or condition. A therapeutically effective amount of a compound refers to an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a disease, disorder, or condition. The term "therapeutically effective amount" can include an amount that improves overall treatment, reduces or avoids symptoms or causes of a disease or condition, or enhances the therapeutic effects of other therapeutic agents.

[0059] As used herein, unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease, disorder, or condition, or an amount sufficient to prevent one or more symptoms associated with a disease, disorder, or condition, or an amount to prevent the recurrence of a disease, disorder, or condition. A prophylactically effective amount of a compound refers to an amount of a therapeutic agent, alone or in combination with other agents, that provides a prophylactic benefit in preventing a disease, disorder, or condition. The term "prophylactically effective amount" can include an amount that improves overall prevention, or an amount that enhances the prophylactic effect of other prophylactic agents.

[0060] Those skilled in the art will appreciate that organic compounds can form complexes with solvents in which they react or from which they precipitate or crystallize. These complexes are referred to as "solvates." When the solvent is water, the complex is referred to as a "hydrate." The present invention encompasses all solvates of the compounds of the present invention.

[0061] The term "solvate" refers to a form of a compound or its salt that is associated with a solvent, typically formed by a solvolysis reaction. Physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein can be prepared, for example, in crystalline form and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. In some cases, the solvate will be capable of separation, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. "Solvate" includes solvates in solution and separable solvates. Representative solvates include hydrates, ethanolates, and methanolates.

[0062] The term "hydrate" refers to a compound that is combined with water. Generally, the ratio of the number of water molecules contained in the hydrate of a compound to the number of molecules of the compound in the hydrate is determined. Therefore, the hydrate of a compound can be represented by the general formula R·x H2O, for example, where R is the compound and x is a number greater than 0. A given compound can form more than one type of hydrate, including, for example, monohydrates (x is 1), lower hydrates (x is a number greater than 0 and less than 1, for example, hemihydrate (R·0.5H2O)) and polyhydrates (x is a number greater than 1, for example, dihydrates (R·2H2O) and hexahydrates (R·6H2O)).

[0063] The compounds of the present invention can be in amorphous or crystalline form (polymorph). In addition, the compounds of the present invention can exist in one or more crystalline forms. Therefore, the present invention includes all amorphous or crystalline forms of the compounds of the present invention within its scope. The term "polymorph" refers to the crystalline form (or its salt, hydrate or solvate) of a compound with a specific crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms typically have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, photoelectric properties, stability and solubility. Recrystallization solvent, crystallization rate, storage temperature and other factors can cause one crystalline form to dominate. The various polymorphs of a compound can be prepared by crystallization under different conditions.

[0064] In addition, prodrugs are also included in the context of the present invention. The term "prodrug" as used herein refers to a compound that is converted in vivo, for example by hydrolysis in the blood, into its active form that has a medical effect. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the ACS Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, and D. Fleisher, S. Ramon and H. Barbra, "Improved oral drug delivery: solubility limitations overcome by the use of prodrugs", Advanced Drug Delivery Reviews (1996) 19 (2) 115-130, which are incorporated herein by reference.

[0065] A prodrug is any covalently bonded compound of the present invention that releases the parent compound in vivo when such a prodrug is administered to a patient. Prodrugs are typically prepared by modifying functional groups in such a way that the modification can be cleaved to produce the parent compound by conventional manipulation or in vivo. Prodrugs include, for example, compounds of the present invention in which a hydroxyl, amino, or sulfhydryl group is bonded to any group that, when administered to a patient, can be cleaved to form a hydroxyl, amino, or sulfhydryl group. Thus, representative examples of prodrugs include, but are not limited to, amide derivatives of amino functional groups that may be present in the compound. Additionally, in the case of carboxylic acids (-COOH), esters such as methyl esters, ethyl esters, etc. may be used. Esters themselves may be active and / or hydrolyzable under human in vivo conditions. Suitable pharmaceutically acceptable in vivo hydrolyzable ester groups include those that readily decompose in the human body to release the parent acid or a salt thereof.

[0066] "Pharmaceutically acceptable" means that it is used to prepare a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes that it is acceptable for human pharmaceutical use. "Pharmaceutically acceptable salts" include, but are not limited to, acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or acid addition salts formed with organic acids such as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, p-toluenesulfonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, dodecylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and the like.

[0067] Imidazolotetrazine compounds

[0068] The imidazotetrazine compounds described in the present invention are shown in Formula I:

[0069] Wherein, R1 is hydrogen or C1-C3 chain alkyl, R2 is amino or amino substituted by C1-C3 chain alkyl; R3 is hydrogen or C1-C3 chain alkyl.

[0070] In some embodiments, R1 is methyl.

[0071] In some embodiments, R2 is amino.

[0072] In some embodiments, R3 is hydrogen.

[0073] In some embodiments, R1 is methyl, R2 is amino, and R3 is hydrogen, that is, the imidazotetrazine compound is temozolomide.

[0074] Pharmaceutical compositions and kits

[0075] On the other hand, the present invention provides a pharmaceutical composition comprising an imidazo tetrazine compound of Formula I of the present invention (also referred to as an "active ingredient") and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of an imidazo tetrazine compound of Formula I of the present invention. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an imidazo tetrazine compound of Formula I of the present invention. In some embodiments, the pharmaceutical composition comprises a prophylactically effective amount of an imidazo tetrazine compound of Formula I of the present invention.

[0076] Pharmaceutically acceptable excipients used in the present invention refer to non-toxic carriers, adjuvants or vehicles that do not destroy the pharmacological activity of the compound formulated together. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions of the present invention include (but are not limited to) ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin.

[0077] The present invention also includes kits (e.g., pharmaceutical packaging). The kits provided may include a compound of the invention, other therapeutic agents, and first and second containers (e.g., vials, ampoules, bottles, syringes, and / or dispersible packaging or other suitable containers) containing the compound of the invention and other therapeutic agents. In some embodiments, the kit provided may also optionally include a third container containing a pharmaceutical excipient for diluting or suspending the compound of the invention and / or other therapeutic agents. In some embodiments, the compound of the invention and other therapeutic agents provided in the first and second containers are combined to form a unit dosage form.

[0078] The present invention also provides a pharmaceutical combination comprising the above-mentioned imidazotetrazine compound and a second therapeutic agent that can be used to treat DLBCL. In the combination, the imidazotetrazine compound and the second therapeutic agent can be administered to a subject separately or sequentially.

[0079] The second therapeutic agent can be one or more of chemotherapy drugs, small molecule targeted anti-tumor drugs, immunotherapy drugs, and antibody drugs.

[0080] Drug administration

[0081] Compound provided by the invention or pharmaceutical composition can be administered by many approaches, including but not limited to: oral administration, parenteral administration, inhalation administration, topical administration, rectal administration, nasal administration, oral administration, vaginal administration, administration by implant or other modes of administration. For example, parenteral administration used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intraarticular administration, intraarterial administration, intrasynovial administration, intrasternal administration, intrathecal administration, intralesional administration and intracranial injection or infusion technology.

[0082] Typically, an effective amount of the compounds provided herein is administered. The amount of compound actually administered can be determined by a physician based on the relevant circumstances, including the condition being treated, the route of administration selected, the compound actually administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

[0083] When used to prevent the conditions described herein, the compounds provided herein are administered to a subject at risk of developing the condition, typically based on the advice and under the supervision of a physician, at dosage levels as described above. Subjects at risk of developing a particular condition typically include those with a family history of the condition, or those identified by genetic testing or screening as being particularly susceptible to developing the condition.

[0084] The pharmaceutical compositions provided herein can also be administered long-term ("chronic administration"). Long-term administration refers to administration of a compound or pharmaceutical composition thereof over an extended period of time, e.g., 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or administration can continue indefinitely, e.g., for the remainder of the subject's life. In some embodiments, long-term administration is intended to provide a constant level of the compound in the blood over an extended period of time, e.g., within the therapeutic window.

[0085] Various methods of administration can be used to further deliver the pharmaceutical composition of the present invention. For example, in some embodiments, the pharmaceutical composition can be administered by injection, for example, in order to increase the concentration of the compound in the blood to an effective level. The bolus dose depends on the target systemic level of the active ingredient by the body, for example, an intramuscular or subcutaneous bolus dose slowly releases the active ingredient, and the bolus (for example, by IV intravenous drip) delivered directly to the vein can be delivered more quickly so that the concentration of the active ingredient in the blood is rapidly increased to an effective level. In other embodiments, the pharmaceutical composition can be given in a continuous infusion form, for example, by IV intravenous drip, so as to provide a steady-state concentration of the active ingredient in the subject's body. In addition, in other embodiments, the pharmaceutical composition of the bolus dose can be first given, and then continuous infusion.

[0086] Oral compositions can be in the form of bulk liquid solutions or suspensions or bulk powders. However, more generally, in order to facilitate accurate dosing, the compositions are provided in unit dosage form. The term "unit dosage form" refers to a physically discrete unit suitable as a unit dose for human patients and other mammals, each unit containing a predetermined amount of active substance suitable for producing the desired therapeutic effect and a suitable pharmaceutical excipient. Typical unit dosage forms include pre-filled, pre-measured ampoules or syringes of liquid compositions, or pills, tablets, capsules, etc. in the case of solid compositions. In such compositions, the compound is typically a minor component (about 0.1 to about 50% by weight, or preferably about 1 to about 40% by weight), with the remainder being various carriers or excipients and processing aids useful for forming the desired dosage form.

[0087] Liquid forms suitable for oral administration may include a suitable aqueous or non-aqueous carrier and buffers, suspending and dispersing agents, colorants, flavorings, etc. Solid forms may include, for example, any of the following components, or compounds of similar properties: binders, such as microcrystalline cellulose, tragacanth, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, sodium starch glycolate, or corn starch; lubricants, such as magnesium stearate; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as mint, methyl salicylate, or orange flavor; stabilizers, such as tartaric acid, aspartic acid, glutamic acid, etc.

[0088] Injectable compositions are typically based on sterile saline or phosphate buffered saline for injection, or other injectable excipients known in the art. As previously mentioned, in such compositions, the active compound is typically a minor component, often about 0.05 to 10% by weight, with the remainder being injectable excipients and the like.

[0089] Typically, transdermal compositions are formulated as topical ointments or creams containing the active ingredient. When formulated as an ointment, the active ingredient is typically combined with a paraffin or water-miscible ointment base. Alternatively, the active ingredient can be formulated into a cream together with, for example, an oil-in-water cream base. Such transdermal formulations are well known in the art and typically include other components that enhance the stable skin penetration of the active ingredient or formulation. All such known transdermal formulations and components are included within the scope provided by the present invention.

[0090] The compounds of the present invention may also be administered by transdermal devices.Thus, transdermal administration may be achieved using patches of the reservoir or porous membrane type, or various solid matrices.

[0091] The above components for oral administration, injection or topical administration are representative only. Other materials and processing techniques are described in Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which is incorporated herein by reference.

[0092] The compounds of the invention can also be administered in sustained release form or from a sustained release delivery system. Descriptions of representative sustained release materials can be found in Remington's Pharmaceutical Sciences.

[0093] treat

[0094] The imidazotetrazine compounds of Formula I have value as anti-tumor agents. In particular, the compounds of the present invention have value as anti-proliferative, apoptotic, and / or anti-invasive agents in the containment and / or treatment of solid and / or liquid tumor diseases. In particular, the compounds of the present invention are expected to be useful in the prevention or treatment of diffuse lymphomas, etc. Anti-cancer effects useful in treating cancer in patients include, but are not limited to, anti-tumor effects, response rates, time to disease progression, and survival rates. Anti-tumor effects of the treatment methods of the present invention include, but are not limited to, inhibition of tumor growth, delay of tumor growth, tumor regression, tumor shrinkage, prolonged tumor regrowth after cessation of treatment, and slowed disease progression. Anti-cancer effects include preventive treatment as well as treatment of existing diseases.

[0095] The effective amount of the compounds of this invention is usually 0.01mg to 100mg compound / kg patient weight at an average daily dose, preferably 0.1mg to 30mg compound / kg patient weight, with single or multiple administration. Usually, the compounds of this invention can be administered to the patient with this treatment need with a daily dose range of about 1mg to about 3500mg per patient, preferably 10mg to 1000mg. For example, the daily dose of each patient can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 160, 180, 200, 240, 250, 300, 350, 360, 400, 500, 600, 700, 800, 900 or 1000mg. Can be administered once or repeatedly every day, every week (or at intervals of several days) or with an intermittent schedule. For example, the compound may be administered once or more daily on a weekly basis (e.g., every Monday) for an indefinite period or for several weeks, e.g., 4-10 weeks. Alternatively, the compound may be administered daily for a few days (e.g., 2-10 days), followed by a few days (e.g., 1-30 days) without administration of the compound, with the cycle repeated indefinitely or for a given number of times, e.g., 4-10 cycles. For example, the compound of the invention may be administered daily for 5 days, followed by a 9-day break, then administered daily again for 5 days, followed by a 9-day break, and so on, with the cycle repeated indefinitely or for a total of 4-10 times.

[0096] The compounds of the present invention can be prepared in any dosage form and administered to cancer patients via an appropriate route of administration. Those skilled in the art can appropriately select the dosage of the compounds of the present invention based on the patient's symptoms and physical condition. The daily dose can be administered all at once or divided into multiple doses. The dosage varies depending on the symptoms, age, and other factors.

[0097] The preferred dosage is, in the case of oral administration to adults, with a dose of 0.1 mg / kg (preferably 0.1 mg / kg) as the lower limit and a dose of 20 mg / kg (preferably 10 mg / kg) as the upper limit, and in the case of parenteral administration, with a dose of 0.02 mg / kg (preferably 0.01 mg / kg) per time as the lower limit and a dose of 10 mg / kg (preferably 2 mg / kg) as the upper limit, and the dosage is administered 1 to 6 times a day depending on the symptoms.

[0098] The following examples specifically describe the biological function tests and experimental tumor treatment of the compounds of the present invention. However, it should be understood that the scope of protection of the present invention is not limited to these examples.

[0099] Example

[0100] The materials and reagents used herein are either commercially available or prepared by synthetic methods generally known in the art.

[0101] Example 1: Cell test

[0102] The compound of the present invention (TMZ) was tested for its inhibitory effect on the proliferation of human tumor cells cultured in vitro, including human glioma U87 and U251, human lymphocytoma Mino, human Burkitt's tumor Raji, human B lymphocytoma Ramos, human multiple myeloma peripheral blood B lymphocytoma RPMI8226, human diffuse tissue lymphoma SU-DHL-2, (ABC type) diffuse lymphoma TMD-8, and human diffuse large cell lymphoma cells SU-DHL-6.

[0103] Tumor cell lines

[0104] A total of 9 tumor cell lines were used in this experiment, as shown in Table 1:

[0105] Table 1. List of cell lines

[0106] Cells were routinely cultured according to the method in Table 2.

[0107] Table 2. Cell culture conditions

[0108] On day 0 (D0) of the plating experiment, cells in the logarithmic growth phase were harvested, a single-cell suspension was prepared, and the cells were counted. The cell concentration was adjusted to the desired concentration, and 100 μL was added to each well of a 96-well cell culture plate. See Table 3 for cell plating number and cell concentration.

[0109] Table 3. Cell plating information

[0110] Drug preparation:

[0111] U87, U251: Dissolve the TMZ raw material (hereinafter referred to as TMZ) in DMSO and dilute to 364.5 μM, 121.5 μM, 40.5 μM, 13.5 μM, 4.5 μM, 1.5 μM, 0.5 μM, 0.167 μM, and 0.056 μM as drug stock solutions. Refrigerate at 4°C until use.

[0112] SU-DHL-2, Mino, Raji, Ramos, RPMI 8226, TMD-8, SU-DHL-6: Add appropriate volume of DMSO and vortex to completely dissolve TMZ to make a 243 mM stock solution. Dilute to 486 μM, 162 μM, 54.0 μM, 18.0 μM, 6.00 μM, 2.00 μM, 0.667 μM, 0.222 μM, and 0 μM, and store in a -90°C to -60°C refrigerator.

[0113] Detection and data analysis

[0114] IC50 assay

[0115] On D7, the fluorescence intensity of each well was detected using CCK8 solution for U87 and U251, and the other 7 strains were detected using Alamarblue method, and the IC50 was calculated.

[0116] Data Analysis

[0117] IC50 is calculated by the following formula

[0118] Y=Min+(Max-Min) / [1+〖10〗^((LogIC_50-X)*Slope)]

[0119] Min, Max, and Slope represent the minimum value, maximum value, and slope, respectively.

[0120] Quality Control

[0121] Any experimental phenomena, problems, and data generated during the experiment should be recorded in detail. The entire process of the experiment, all original data, records, and receipts generated should be recorded.

[0122] The test results are shown in Table 4.

[0123] Table 4. Inhibitory effects of the compounds of the present invention on the proliferation of 9 human tumor cell lines

[0124] The data in Table 4 show that the compound of the present invention has a good anti-tumor effect only on brain glioma and diffuse lymphocytic tumor, and has poor activity on other lymphoma or leukemia cells.

[0125] Example 2: Inhibitory effect of TMZ on tumor growth in a human diffuse histiocytic lymphoma SU-DHL-2 transplanted tumor model

[0126] Objective: To test the inhibitory effect of TMZ compared with doxorubicin on the growth of human diffuse histiocytic lymphoma SU-DHL-2 xenografts in nude mice.

[0127] Reagents: RPMI-1640 powder (Gibco; Catalog No.: 31800-022), fetal bovine serum (FBS, Gibco; Catalog No.: 10099-141C), blue-chain bispecific antibody (HyClone; Catalog No.: SV30010; Lot No.: J220018), PBS (0.01 M, pH 7.4, Sangon Biotech; Catalog No.: B040100-0005), NN-dimethylacetamide (aladdin; Catalog No.: D108095), sterile water for injection (Zhejiang Dubang Pharmaceutical).

[0128] experimental animals

[0129] Female NOD / SCID mice (62 mice, 6-7 weeks old) were purchased from Weitong Lihua and housed in the SPF animal facility at Suzhou Shengsu New Drug Development Co., Ltd. at a temperature of 20-25°C, a relative humidity of 40%-70%, and a 12-hour light and dark cycle. The animals had free access to water and food. After approximately 5 days of normal feeding, mice that were found to be in good physical condition after veterinary examination were enrolled in this experiment. Before grouping, the animals were identified using a marker at the base of their tails. After grouping, each animal was identified by ear notching.

[0130] Transplantable tumor lines

[0131] Human diffuse histiocytic lymphoma SU-DHL-2 cells were obtained from Kebai Biotechnology (frozen in liquid nitrogen in our laboratory).

[0132] Cell culture

[0133] SU-DHL-2 cells were routinely cultured in RPMI-1640 medium containing 10% fetal bovine serum under 5% CO2 and 37°C conditions; the cells were passaged according to their growth conditions at a passage ratio of 1:2 to 1:5.

[0134] Animal model preparation

[0135] SU-DHL-2 cells in the logarithmic growth phase were collected, counted, and resuspended in serum-free RPMI-1640 culture medium to adjust the cell concentration to 1.5×10 8 cells / mL; pipette the cells to disperse them evenly and then put them into a 50-mL centrifuge tube, which was placed in an ice box; use a 1-mL syringe to draw up the cell suspension and inject it into the subcutaneous part of the right axilla of the mouse. Each animal was inoculated with 100 μL (1.5×10 7 SU-DHL-2 mouse transplant tumor model was established. The animal status and tumor growth were regularly observed after inoculation. The tumor diameter was measured using an electronic vernier caliper and the data was entered into an Excel spreadsheet to calculate the tumor volume. When the tumor volume reached 100-300 mm 3 Forty-two animals in good health and with similar tumor volumes were selected and randomly divided into six groups (n=7). The day of grouping was designated the first day of the experiment (D1). After the start of the experiment, tumor diameters were measured twice a week, and tumor volumes were calculated. Animal body weights were also recorded.

[0136] The formula for calculating tumor volume (TV) is as follows: TV (mm 3 )=l×w 2 / 2

[0137] Wherein, l represents the long diameter of the tumor (mm); w represents the short diameter of the tumor (mm).

[0138] Preparation of drug delivery preparations

[0139] Blank solvent preparation

[0140] Pipette appropriate volumes of DMA and sterile water for injection respectively, mix well, and use as blank solvent. Store at room temperature for future use. The ratio of DMA to sterile water for injection in the mixed solvent is 10% and 90%.

[0141] Preparation of TMZ dosing preparation

[0142] Before each administration, an appropriate amount of TMZ was weighed and an appropriate volume of DMA was added to it. After vortexing to completely dissolve the compound, an appropriate volume of sterile water for injection was added and vortexed to mix the liquid evenly. The ratio of DMA to sterile water for injection in the dosing preparation was 10% and 90%, respectively, to obtain final concentrations of 1.0, 2.0, and 3.0 mg mL, respectively. -1 The dosage form is prepared and used immediately.

[0143] Animal grouping and drug administration

[0144] The animal groups and dosing schedule are shown in Table 5. The test samples were administered starting on the day of grouping (D1) for a total of 3 weeks (the specific dosing schedule can be adjusted based on the progress of the experiment). The dosage, route of administration, and time of administration of the above-mentioned compounds can be adjusted as appropriate based on the progress of the experiment.

[0145] Table 5. Dosage regimen for efficacy experiments in mouse transplant tumor models Note: “ig” means oral administration, “QD” means once a day, and “QW” means once a week.

[0146] End of experiment

[0147] On the last day of the experiment, the animals were euthanized by CO2 inhalation after being weighed and the tumor diameter was measured. The tumors were removed, weighed, and photographed.

[0148] Data recording, calculation formula

[0149] The formula for calculating relative tumor volume (RTV) is: RTV = 100 × TV t / TV initial

[0150] Among them, TV initial TV is the tumor volume measured at the time of group administration; t The tumor volume was measured at each time during the drug administration period.

[0151] The calculation formula of relative tumor proliferation rate (%T / C) is: %T / C = 100% × (RTV T / RTV C )

[0152] Among them, RTVT Indicates the treatment group RTV; RTV C represents the solvent control group RTV.

[0153] The calculation formula of tumor growth inhibition rate TGI (%) is: TGI (%) = 100% × [1–(TV t(T) –TV initial(T) ) / (TV t(C) –TV initial(C) )]

[0154] Among them, TV t(T) TV represents the tumor volume of each treatment group; initial(T) TV represents the tumor volume of the treatment group at the time of drug administration; t(C) TV represents the tumor volume of the solvent control group at each measurement; initial(C) It represents the tumor volume of the solvent control group at the time of group administration.

[0155] The formula for calculating the relative body weight (RBW) of an animal is: RBW = 100 × BW t / BW initial

[0156] Among them, BW initial BW is the body weight of animals when they were grouped and dosed; t The animals were weighed at each time during the dosing period.

[0157] The formula for calculating the animal weight loss rate is:

[0158] Animal weight loss rate = 100% × (BW initial -BW t ) / BW initial

[0159] Among them, BW t Indicates the body weight of the animals measured at each time during the dosing period; BW initial Indicates the body weight of animals at the time of grouping and dosing.

[0160] The calculation formula of tumor weight inhibition rate IR (%) is: IR (%) = 100% × (W C -W T ) / W C

[0161] Among them, W C W represents the tumor weight of the solvent control group; T Indicates tumor weight in treatment group.

[0162] Statistical analysis methods

[0163] The experimental data were calculated and statistically processed using Microsoft Office Excel 2007. Unless otherwise specified, the data were expressed as mean ± standard error (mean ± SE), and the t-test was used for comparison between the two groups.

[0164] Experimental observation

[0165] During the experiment, the experimenter and veterinarian must continuously monitor the physical signs and health of the experimental animals. Any abnormal behavior of the animals, such as pain, depression, or decreased activity, must be recorded in the original experimental records. If the abnormal behavior of the experimental animals exceeds the requirements of the relevant IACUC animal welfare documents, the veterinarian may determine whether to terminate the experiment.

[0166] Experimental results

[0167] The effects on the growth of SU-DHL-2 nude mouse transplanted tumors are shown in Table 6.

[0168] Table 6 Average tumor volume ± SE (mm) of each group of animals during the administration period 3 ) and tumor growth inhibition rate (TGI) Note: "*" indicates that the tumor volume is significantly different from that of the solvent control group (P<0.05); "**" indicates that the tumor volume is extremely significantly different from that of the solvent control group (P<0.01).

[0169] Group A was the solvent control group, which was given blank solvent by gavage. The tumors of the animals grew well and no spontaneous regression was found. By D22, the average tumor volume reached 1163±133mm 3 .

[0170] Group B was the positive control drug doxorubicin group, with a dose of 7.5 mg·kg -1 Severe toxicity was produced during QW treatment. The animals initially experienced a severe weight loss and all died later. The tumor growth inhibition rate was 109% on D11 (P<0.01), indicating a significant inhibitory effect on tumor growth.

[0171] Group C was the low-dose TMZ group, with a dose of 10 mg·kg -1 QD, the tumor growth inhibition rate was 114% on D22 (P<0.05), showing an extremely excellent anti-tumor effect. By D11, the anti-tumor effect was better than the positive control drug, and the safety was significantly better than the positive control drug group.

[0172] Group D was the medium-dose TMZ group, with a dose of 20 mg·kg -1QD, although the tumor growth inhibition rate at D22 was the same as that of the low-dose group, both 114% (P<0.05), it took effect faster than the low-dose group, showing an extremely excellent anti-tumor effect. By D8, the anti-tumor effect had reached that of the positive control drug, and by D11, the anti-tumor effect was better than the positive control drug, and the safety was significantly better than the positive control drug group.

[0173] Group E was the high-dose TMZ group, with a dose of 30 mg·kg -1 QD, although the tumor growth inhibition rate was 116% (P<0.05) on D22, and the tumor completely disappeared, and it was more effective than the low- and medium-dose groups, showing an extremely excellent anti-tumor effect. In particular, by D4, the anti-tumor effect was better than that of the positive control drug, and the safety was significantly better than that of the positive control drug group.

[0174] The effects on the body weight of SU-DHL-2 tumor-bearing mice are shown in Table 7.

[0175] Table 7 Body weight and weight loss rate of animals in each group during administration Note: “-” indicates that the animal has gained weight.

[0176] During the dosing period, animals in the solvent control group (Group A) and the TMZ low, medium, and high groups (Groups C, D, and E) were in good condition, with body weight increases of 7.5%, 2.1%, -4.4%, and -11%, respectively, by Day 22. The positive control, the doxorubicin group (Group B), experienced significant weight loss early in the treatment period and all animals died by Day 15 due to excessive drug dosage.

[0177] As can be seen from Tables 6-7 above, the compounds of the present invention not only have good effects but also have better safety even when compared with the commonly used anti-lymphoma drug doxorubicin at extremely high doses.

[0178] Example 3: Inhibitory effect of TMZ on tumor growth in a human diffuse histiocytic lymphoma TMD-8 transplanted tumor model

[0179] Objective: This study evaluated the antitumor effect of TMZ using a human diffuse large B-cell lymphoma TMD-8 mouse xenograft model, with cyclophosphamide as a control.

[0180] Reagents: RPMI-1640 powder (Gibco; Catalog No.: 31800-022); fetal bovine serum (FBS) (Gibco; Catalog No.: 10099-141C); cyan-chain bispecific antibody (HyClone; Catalog No.: SV30010); PBS (Beyotime; Catalog No.: ST447); sterile water for injection (Zhejiang Dubang Pharmaceutical); normal saline (Guizhou Tiandi Pharmaceutical); NN-dimethylacetamide (aladdin; Catalog No.: D108095).

[0181] experimental animals

[0182] Female CB17 / SCID mice (75 mice, 6-8 weeks old) were purchased from Weitong Lihua and housed in the SPF animal facility at Suzhou Shengsu New Drug Development Co., Ltd. at a temperature of 20-25°C, a relative humidity of 40%-70%, and a 12-hour light and dark cycle. The animals had free access to water and food. After 7 days of normal feeding, mice that were found to be in good physical condition after veterinary examination were enrolled in this experiment. Before grouping, the animals were identified at the base of their tails with a marker pen. After grouping, each animal was identified by ear notching.

[0183] Transplantable tumor lines

[0184] Human diffuse large B-cell lymphoma TMD-8 cells were purchased from Shanghai Huzhen Industrial Co., Ltd. and stored in liquid nitrogen in our laboratory.

[0185] Cell culture

[0186] TMD-8 cells were routinely cultured in RPMI-1640 medium containing 10% fetal bovine serum under 5% CO2, 37°C, and saturated humidity. Cells were digested and passaged with 0.25% trypsin, 1 to 2 times per week, at a passage ratio of 1:3 to 1:4, depending on cell growth.

[0187] Animal model preparation

[0188] TMD-8 cells in the logarithmic growth phase were collected, counted, and resuspended in 30% serum-free RPMI-1640 culture medium and 70% Matrigel to adjust the cell concentration to 5.0×10 7 cells / mL; the cells were placed in an ice box, and the cell suspension was drawn up with a 1-mL syringe and injected into the subcutaneous tissue of the right axilla of the mouse. Each animal was inoculated with 200 μL (1.0×10 7 Cells / mouse) were used to establish a TMD-8 transplant tumor model. The animal status was regularly observed, and the tumor diameter was measured using an electronic vernier caliper. The data were entered into an Excel spreadsheet to calculate the tumor volume and monitor tumor growth. 3 Forty-eight tumor-bearing mice in good health with similar tumor volumes were selected and randomly divided into six groups (n=8) based on tumor volume, while ensuring that the average body weight within each group remained consistent. The day of grouping was designated the first day of the experiment (D1). Tumor diameters were measured and tumor volumes calculated twice weekly thereafter, and animal weights were also recorded.

[0189] The formula for calculating tumor volume (TV) is as follows: TV (mm 3 )=l×w 2 / 2

[0190] Wherein, l represents the long diameter of the tumor (mm); w represents the short diameter of the tumor (mm).

[0191] Preparation of drug delivery preparations

[0192] Blank solvent preparation

[0193] Take appropriate volumes of DMA (NN-dimethylacetamide) and sterile water for injection, mix them evenly, and use them as blank solvents. Store at room temperature for future use. The ratios of DMA and sterile water for injection in the mixed solvent are 10% and 90% respectively.

[0194] Preparation of TMZ dosing preparation

[0195] Before each administration, weigh an appropriate amount of TMZ, add an appropriate volume of DMA, vortex, and sonicate for 10-20 minutes to evenly disperse the compound. Add an appropriate volume of sterile water for injection and vortex to completely dissolve the drug. The ratio of DMA to sterile water for injection in the dosing preparation is 10% and 90%, respectively, to obtain dosing preparations with final concentrations of 0.125, 0.25, and 0.5 mg / mL, respectively. Prepare and use immediately.

[0196] CTX drug delivery preparation

[0197] Weigh an appropriate amount of CTX and place it in a glass bottle; add an appropriate volume of normal saline to the bottle, vortex and sonicate for 15 minutes to completely dissolve the drug; prepare a dosage preparation with a concentration of 2.5 mg / mL, prepare it before each administration, and use it immediately.

[0198] Animal grouping and drug administration

[0199] The animal groups and dosing schedule are shown in Table 8. The first dosing began on the day of grouping (D1), and the dosing volume was 10 mL / kg.

[0200] Table 8. Dosage regimen for efficacy experiments in mouse transplant tumor models Note: “iv” means intravenous injection, “ig” means oral administration, “QD” means once a day, and “QW” means once a week.

[0201] The experimental end processing, data recording, calculation formula, statistical analysis method, and experimental observation are the same as those in Example 2.

[0202] Experimental results

[0203] The effects on the growth of TMD-8 nude mouse transplanted tumors are shown in Table 9.

[0204] Table 9 Average tumor volume ± SE (mm) of each group of animals during the administration period 3 ) and tumor growth inhibition rate (TGI) Note: "*" indicates that the tumor volume is significantly different from that of the solvent control group (P<0.05); "**" indicates that the tumor volume is extremely significantly different from that of the solvent control group (P<0.01).

[0205] The effects on the body weight of TMD-8 tumor-bearing mice are shown in Table 10.

[0206] Table 10 Body weight and weight loss rate of animals in each group during administration Note: “-” indicates that the animal has gained weight.

[0207] The experimental results in Table 9 show that at doses of 1.25, 2.5 and 5.0 mg / kg (ig, QD), the tumor volume inhibition rate TGI was greater than 60%, and p < 0.01, showing a significant inhibitory effect on the growth of transplanted tumors. The effective dose was lower than 1.25 mg / kg (ig, QD); TMZ at 1.25 mg·kg -1 The combined use of TMZ (1.25 mg / kg, ig, QD) and CTX (25 mg / kg, iv, QW) significantly improved the antitumor efficacy of CTX (25 mg / kg, iv, QW) alone against TMD-8 xenografts.

[0208] The experimental results in Table 10 show that TMZ, at the dose set in this experiment, had no significant effect on the body weight of mice bearing human diffuse large B-cell lymphoma TMD-8, and no obvious drug-related adverse reactions were observed during administration. The body weight loss rate of animals treated with TMZ (1.25 mg / kg, ig, QD) in combination with CTX (25 mg / kg, iv, QW) was less than 5%, also demonstrating good safety.

[0209] As can be seen from Tables 9-10 above, compared with the positive control drug CTX, the compound of the present invention not only has a good effect, but also has better safety.

[0210] Example 4: Inhibitory Effects of TMZ and Its Preparations on Tumor Growth in Human Diffuse Histiocytic Lymphoma SU-DHL-6 Transplanted Tumor Model

[0211] Objective: This study evaluated the antitumor effects of TMZ and its preparations alone and in combination with cyclophosphamide using a human diffuse large B-cell lymphoma SU-DHL-6 mouse xenograft tumor model, with cyclophosphamide as a control.

[0212] Reagents: RPMI-1640 powder (Gibco; catalog number: 31800-022); fetal bovine serum (FBS) (Gibco; catalog number: 10099-141C); cyan-chain bispecific antibody (HyClone; catalog number: SV30010; batch number: J220018); PBS (Sangon Biotech; catalog number: B040100-0005); sterile water for injection (Zhejiang Dubang Pharmaceutical); normal saline (Guizhou Tiandi Pharmaceutical); NN-dimethylacetamide (aladdin).

[0213] experimental animals

[0214] Female NOD / SCID mice (85 mice, 6-8 weeks old) were purchased from Weitong Lihua and housed in the SPF animal facility at Suzhou Shengsu New Drug Development Co., Ltd. at a temperature of 20-25°C, a relative humidity of 40%-70%, and a 12-hour light and dark cycle. The animals had free access to water and food. After 7 days of normal feeding, mice that were found to be in good physical condition after veterinary examination were enrolled in this experiment. Before grouping, the animals were identified using a marker at the base of their tails. After grouping, each animal was identified by ear notching.

[0215] Transplantable tumor lines

[0216] Human large cell lymphoma SU-DHL-6 cells were obtained from the Kunming Cell Bank of the Chinese Academy of Sciences (CAS(KM) and stored in liquid nitrogen in our laboratory).

[0217] Cell culture

[0218] SU-DHL-6 cells were routinely cultured in RPMI-1640 medium containing 10% fetal bovine serum under 5% CO2, 37°C, and saturated humidity. Cells were digested and passaged with 0.25% trypsin, 1 to 2 times per week, at a passage ratio of 1:3 to 1:4, depending on cell growth.

[0219] SU-DHL-6 cells were routinely cultured in RPMI-1640 medium containing 10% fetal bovine serum under 5% CO2, 37°C, and saturated humidity. Cells were digested and passaged with 0.25% trypsin, 1 to 2 times per week, at a passage ratio of 1:3 to 1:4, depending on cell growth.

[0220] Animal model preparation

[0221] SU-DHL-6 cells in the logarithmic growth phase were collected, counted, and resuspended in 50% serum-free RPMI-1640 culture medium and 50% Matrigel to adjust the cell concentration to 3.0×10 7cells / mL; the cells were placed in an ice box, and the cell suspension was drawn up with a 1-mL syringe and injected into the subcutaneous tissue of the right axilla of the mouse. Each animal was inoculated with 200 μL (6.0×10 6 SU-DHL-6 transplant tumor model was established. The animal status was regularly observed, and the tumor diameter was measured using an electronic vernier caliper. The data were entered into an Excel spreadsheet to calculate the tumor volume and monitor tumor growth. 3 Forty-eight tumor-bearing mice in good health with similar tumor volumes were selected and randomly divided into seven groups (n=8) based on tumor volume, while ensuring that the average body weight within each group remained consistent. The day of grouping was designated Day 1 (D1). Tumor diameters were measured and tumor volumes calculated twice weekly thereafter, and animal weights were recorded.

[0222] The formula for calculating tumor volume (TV) is as follows: TV (mm 3 )=l×w 2 / 2

[0223] Wherein, l represents the long diameter of the tumor (mm); w represents the short diameter of the tumor (mm).

[0224] Preparation of drug delivery preparations

[0225] Preparation of TMZ preparations for administration

[0226] Before each administration, take out one TMZ capsule (i.e., the aforementioned TMZ preparation, the same below), remove the contents into a glass bottle, add 75 ml of sterile water for injection, vortex, and sonicate for 20 min to 30 min to prepare a dosing preparation containing 0.5 mg / ml (API); then add an appropriate volume of sterile water for injection and dilute to obtain dosing preparations with final concentrations of 0.125, 0.25, and 0.5 mg / mL, respectively, which are prepared and used immediately.

[0227] The TMZ drug substance dosing formulation, cyclophosphamide (CTX) dosing formulation and blank solvent were prepared in the same manner as in Example 3.

[0228] Animal grouping and drug administration

[0229] The animal groups and dosing schedule are shown in Table 11. The first dosing began on the day of grouping (D1), and the dosing volume was 10 mL / kg.

[0230] Table 11. Dosage regimen for efficacy experiments in mouse transplant tumor models

[0231] Note: “iv” means intravenous injection, “ig” means oral administration, “QD” means once a day, and “QW” means once a week.

[0232] The experimental end processing, data recording, calculation formula, statistical analysis method, and experimental observation are the same as those in Example 2.

[0233] Experimental results

[0234] The effects on the growth of SU-DHL-6 nude mouse transplanted tumors are shown in Table 12.

[0235] Table 12 Average tumor volume ± SE (mm) of each group of animals during the administration period 3 ) and tumor growth inhibition rate (TGI) Note: "*" indicates that the tumor volume is significantly different from that of the solvent control group (P<0.05); "**" indicates that the tumor volume is extremely significantly different from that of the solvent control group (P<0.01).

[0236] The effects on the body weight of SU-DHL-6 tumor-bearing mice are shown in Table 13.

[0237] Table 13 Body weight and weight loss rate of animals in each group during administration Note: “-” indicates that the animal has gained weight.

[0238] The experimental results in Tables 12 and 13 show that the use of TMZ API and TMZ preparation alone, as well as the combination of TMZ preparation and cyclophosphamide, at the doses set in this experiment, had no significant effect on the body weight of mice bearing human large cell lymphoma SU-DHL-6 tumors, and no obvious drug-related adverse reactions were observed during the administration period. Cyclophosphamide (CTX) at a dose of 25 mg / kg (iv, QW) had no significant inhibitory effect on xenograft tumor growth, with a tumor volume inhibition rate (TGI) of less than 60%. However, TMZ at doses of 1.25, 2.5, and 5.0 mg / kg (ig, QD) significantly inhibited xenograft tumor growth, with TGIs exceeding 60%, with p < 0.01. The combination of TMZ (1.25 mg / kg, ig, QD) and CTX (25 mg / kg, iv, QW) significantly inhibited xenograft tumor growth, and the tumor inhibition rate in the combination group was significantly higher than that of TMZ (1.25 mg / kg, ig, QD) or CTX (25 mg / kg, iv, QW) alone, demonstrating a clear synergistic and / or additive effect. TMZ at a dose of 5 mg / kg (ig, QD) significantly inhibited xenograft tumor growth. While the ultimate tumor suppression effect was comparable to that of TMZ (2.5 mg / kg, ig, QD), the onset of action was more rapid. TMZ API at a dose of 2.5 mg / kg (ig, QD) significantly inhibited xenograft tumor growth, comparable to that of the TMZ formulation (2.5 mg / kg, ig, QD) alone.

[0239] Example 5: Inhibitory effect of TMZ on tumor growth in a human brain glioma cell U87 transplanted tumor model

[0240] Objective: To test the inhibitory effect of TMZ on the growth of human glioma cell U87 xenograft tumor in nude mice.

[0241] Experimental instruments: centrifuge, Thermo Corporation, USA, model ThermoSorvall ST 40; Hitachi 7600 fully automatic biochemical analyzer equipped with corresponding reagents; electronic balance, Sartorius, model: BSA3202S-CW; electronic balance, METTLER-TOLEDO, model: LE204; small animal weight balance, Shanghai Yueping Scientific Instrument Co., Ltd., model: YP1002; pipette, Eppendorf; IVC mouse cage, Suzhou Teaching Cage Factory products.

[0242] Experimental site and U87 cells: Beijing Yongxin Kangtai Technology Development Co., Ltd.

[0243] Preparation of tumor-bearing mouse model:

[0244] U87 cells are human malignant glioma cells with an epithelial-like morphology and adherent growth. The culture medium consists of 90% DMEM and 10% Fetal Bovine Serum; culture conditions are 37°C and 5% CO2; digestion conditions are 0.25% (w / v) Trypsin-0.53mM EDTA solution for 5-10 minutes; the subculture ratio is 1:2 to 1:4; and the medium is changed every 2-3 days until the inoculation number is reached. Tumors are then inoculated into nude mice for passage. After two passages, the tumors are removed for formal experiments. Reagents include RPMI-1640 culture medium, fetal bovine serum (FBS), cyan-chain anti-antibody, PBS (0.01M, pH 7.4), Matrigel, and sterile water for injection.

[0245] Nude mice weighing 16±2g were taken and adapted to be raised in the experimental animal center for one week. Tumor-bearing mice with vigorous tumor growth and no ulceration were selected and killed by cervical dislocation. Under sterile conditions in a clean bench, the animal skin was disinfected with alcohol, the skin was cut open, and the tumor was removed. The tumor tissue was cut into 1.5mm 3 The vaccine was inoculated subcutaneously in the right axilla of the animal using a trocar.

[0246] After the tumor was inoculated into nude mice, the diameter of the transplanted tumor (long diameter a, short diameter b) was measured with a vernier caliper and the tumor volume was calculated. The calculation formula of tumor volume (TV) is: V = 1 / 2 × a × b 2When the tumor volume grew to about 100 mm3, the animals were randomly divided into two groups, 8 in each group, namely the control group and the TMZ treatment group. After the start of medication, the animals in each group were weighed twice a week and the tumor volume was measured twice a week.

[0247] Experimental drug: It is a white powder provided by Beijing Zimeng Pharmaceutical Company and the dosage is calculated. The dosage is 40 mg / kg, which is equivalent to the dosage for clinical patients. It is stored in a dry place away from light.

[0248] Dosage: Oral administration, once a day; Dosage cycle: 19 days; Solvent: Animal drinking water

[0249] Calculation and statistical methods: After the experiment, tumor tissue was collected and weighed, and the in vitro tumor weight was measured. The average body weight, average tumor volume, and tumor inhibition rate of each group were calculated.

[0250] The relative tumor volume (RTV) was calculated based on the measurement results using the formula: RTV = Vt / V0, where V0 is the tumor volume measured at the time of cage administration (ie, d0), and Vt is the tumor volume at each measurement.

[0251] Tumor growth inhibition rate (%), calculated as follows:

[0252] Animal grouping and drug administration

[0253] The animal groups and dosing schedule are shown in Table 14. The first dosing began on the day of grouping (D1).

[0254] Table 14. Dosage regimen for efficacy experiments in mouse transplant tumor models

[0255] Experimental results

[0256] The effects on the growth of U87 nude mouse transplanted tumors are shown in Table 15.

[0257] Table 15 Average tumor volume ± SE (mm) of each group of animals during the administration period 3 ) and tumor growth inhibition rate (TGI) Note: "*" indicates that the tumor volume is significantly different from that of the solvent control group (P<0.05); "**" indicates that the tumor volume is extremely significantly different from that of the solvent control group (P<0.01).

[0258] The effects on the body weight of U87 tumor-bearing mice are shown in Table 16.

[0259] Table 16 Body weight and weight loss rate of animals in each group during administration Note: “-” indicates that the animal has gained weight.

[0260] As shown in Tables 15-16 above, the compounds of the present invention have a good anti-proliferative effect on glioma. During the administration period, the body weight of the animals in the TMZ group continued to decrease, at one point decreasing by 7.9%. Compared with the blank control group, the decrease was at one point about 15%.

[0261] Based on Examples 1 to 5, although in vitro experiments showed that TMZ had comparable and good inhibitory effects on glioma cells and large B lymphoma cells, the in vivo effectiveness of TMZ on the two cells was very different: the dose was as low as 2.5 mg·kg -1 It has produced significant anti-tumor activity against various types of DLBCL. At the end of the trial, the tumor volume was smaller than the initial volume, and even the tumor volume was close to disappearing. When the dose reached 30 mg kg -1 When the TMZ dose reached 40 mg·kg, all tumors disappeared, and all animals grew normally with no significant changes in body weight. -1 Even when administered intracerebrally, glioma tumor volume continued to grow slowly, and some animals died, with significant weight loss. Considering that glioma animal models are not intracranial, while TMZ has a significant advantage in its ability to penetrate the blood-brain barrier among anti-tumor drugs, its exposure in brain tissue is less than half that in the circulatory system. Therefore, it is reasonable to infer that TMZ is less effective against intracranial gliomas. Although glioma is the only globally approved indication for TMZ, its efficacy and safety profile far exceed those of gliomas in DLBCL. For example, primary central nervous system lymphoma, also known as microglioma cells, has not shown significant efficacy with TMZ, solely leveraging its superior blood-brain barrier penetration (M Kazuhiko, N Ryo et al. Randomized phase III study of high-dose methotrexate and whole-brain radiotherapy with / without temozolomide for newly diagnosed primary CNS lymphoma: JCOG 1114C, Neuro-Oncology, 1–12, 2022). Therefore, the present invention mainly provides the use of TMZ in treating and / or preventing diffuse large B-cell lymphoma (DLBCL) other than DLBCL whose primary site or metastatic site is the central nervous system. Industrial Applicability

[0262] The imidazotetrazine compounds proposed by the present invention have excellent antagonistic effects and excellent safety on diffuse lymphomas whose primary sites or metastatic sites are not the central nervous system.

Claims

1. Use of an imidazotetrazine compound or a solvate, hydrate, polymorph, prodrug, or pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing diffuse large B-cell lymphoma (DLBCL), wherein the imidazotetrazine compound is represented by Formula I: in, R1 is hydrogen or C1-C3 alkyl, R2 is amino or amino substituted by C1-C3 alkyl; R3 is hydrogen or C1-C3 alkyl.

2. Use of a pharmaceutical composition in the preparation of a medicament for treating and / or preventing diffuse large B-cell lymphoma (DLBCL), the pharmaceutical composition comprising an imidazotetrazine compound of Formula I or a solvate, hydrate, polymorph, prodrug, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. in, R1 is hydrogen or C1-C3 alkyl, R2 is amino or amino substituted by C1-C3 alkyl; R3 is hydrogen or C1-C3 alkyl.

3. The use according to claim 1 or 2, wherein R1 is methyl, R2 is amino, and R3 is hydrogen.

4. The use according to any one of claims 1 to 3, wherein The DLBCL is selected from diffuse large B-cell lymphoma whose primary site and / or metastatic site is not the central nervous system, Preferably, the diffuse large B-cell lymphoma is selected from unspecified DLBCL, other large B-cell lymphoma, high-grade B-cell lymphoma, and unclassifiable B-cell lymphoma.

5. The use according to any one of claims 1 to 4, wherein The DLBCL is selected from the group consisting of centroblastic variant DLBCL, immunoblastic variant DLBCL, anaplastic variant DLBCL, spindle cell variant DLBCL, signet ring cell variant DLBCL, T cell / histiocyte enriched DLBCL, primary cutaneous DLBCL, EBV-positive DLBCL, large B cell lymphoma associated with chronic inflammation, lymphomatoid granuloma, large B cell lymphoma with IRF4 rearrangement, large B cell lymphoma with IRF4 / MUM1 rearrangement, primary mediastinal large B cell lymphoma, vascular Internal large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-positive DLBCL, primary effusion lymphoma, high-grade B-cell lymphoma with MYC, BCL2, and / or BCL6 rearrangement, unspecified high-grade B-cell lymphoma, unclassifiable B-cell lymphoma between DLBCL and classical Hodgkin lymphoma, B-cell lymphoma between primary mediastinal large B-cell lymphoma and nodular sclerosis classical Hodgkin lymphoma, and B-cell lymphoma between DLBCL and Burkitt lymphoma; Optionally, the DLBCL cells are selected from human diffuse histiocytic lymphoma cells SU-DHL-2, human diffuse lymphoma cell line TMD-8 and one or more of human diffuse large cell lymphoma cells SU-DHL-6.

6. The use according to any one of claims 1 to 5, wherein The DLBCL is primary DLBCL, or transformed DLBCL, or advanced and / or recurrent, and / or refractory DLBCL, or DLBCL that has previously received systemic treatment, or DLBCL that has failed chemotherapy and / or targeted drugs and / or immunotherapy.

7. A pharmaceutical combination comprising an imidazotetrazine compound and a second therapeutic agent that can be used to treat DLBCL, wherein the imidazotetrazine compound is represented by Formula I: in, R1 is hydrogen or C1-C3 alkyl, R2 is amino or C1-C3 alkyl substituted amino; R3 is hydrogen or C1-C3 alkyl.

8. The pharmaceutical combination according to claim 7, wherein R1 is methyl, R2 is amino; and R3 is hydrogen.

9. The pharmaceutical combination according to claim 7 or 8, wherein The second therapeutic agent is one or more of chemotherapy drugs, small molecule targeted anti-tumor drugs, immunotherapy drugs, and antibody drugs; preferably, the second therapeutic agent is cyclophosphamide.

10. Use of the pharmaceutical combination according to any one of claims 7 to 9 in the preparation of a medicament for treating and / or preventing DLBCL.

11. The use according to claim 10, wherein The DLBCL is selected from DLBCL whose primary site and / or metastatic site is not central nervous system DLBCL, Preferably, the DLBCL is selected from unspecified DLBCL, other large B-cell lymphomas, high-grade B-cell lymphomas, and unclassifiable B-cell lymphomas.

12. The use according to claim 10, wherein The DLBCL is selected from the group consisting of centroblastic variant DLBCL, immunoblastic variant DLBCL, anaplastic variant DLBCL, spindle cell variant DLBCL, signet ring cell variant DLBCL, T cell / histiocyte enriched DLBCL, primary cutaneous DLBCL, EBV-positive DLBCL, large B cell lymphoma associated with chronic inflammation, lymphomatoid granuloma, large B cell lymphoma with IRF4 rearrangement, large B cell lymphoma with IRF4 / MUM1 rearrangement, primary mediastinal large B cell lymphoma, vascular Internal large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-positive DLBCL, primary effusion lymphoma, high-grade B-cell lymphoma with MYC, BCL2, and / or BCL6 rearrangement, unspecified high-grade B-cell lymphoma, unclassifiable B-cell lymphoma between DLBCL and classical Hodgkin lymphoma, B-cell lymphoma between primary mediastinal large B-cell lymphoma and nodular sclerosis classical Hodgkin lymphoma, and B-cell lymphoma between DLBCL and Burkitt lymphoma; Optionally, the DLBCL cells are selected from one or more of human diffuse histiocytic lymphoma cells SU-DHL-2, human diffuse lymphoma cell line TMD-8, and human diffuse large cell lymphoma cells SU-DHL-6.

13. The use according to any one of claims 10 to 12, wherein The DLBCL is primary DLBCL, or transformed DLBCL, or advanced and / or recurrent, and / or refractory DLBCL, or DLBCL that has previously received systemic treatment, or DLBCL that has failed chemotherapy and / or targeted drugs and / or immunotherapy.

14. A method for treating and / or preventing DLBCL in a subject, comprising administering to the subject an effective amount of an imidazotetrazine compound or a solvate, hydrate, polymorph, prodrug or pharmaceutically acceptable salt thereof, or a pharmaceutical combination comprising the same, wherein the imidazotetrazine compound is represented by Formula I: in, R1 is hydrogen or C1-C3 alkyl, R2 is amino or C1-C3 alkyl substituted amino; R3 is hydrogen or C1-C3 alkyl.

15. The method according to claim 14, wherein R1 is methyl, R2 is amino; and R3 is hydrogen.

16. The method according to claim 14, wherein The pharmaceutical combination also includes a second therapeutic agent that can be used to treat DLBCL.

17. The method according to claim 16, wherein The second therapeutic agent is one or more of chemotherapy drugs, small molecule targeted anti-tumor drugs, immunotherapy drugs, and antibody drugs. Preferably, the second therapeutic agent is cyclophosphamide.

18. The method according to any one of claims 14 to 17, wherein: The DLBCL is selected from DLBCL whose primary site and / or metastatic site is not central nervous system DLBCL, Preferably, the DLBCL is selected from unspecified DLBCL, other large B-cell lymphomas, high-grade B-cell lymphomas, and unclassifiable B-cell lymphomas.

19. The method according to any one of claims 14 to 17, wherein: The DLBCL is selected from the group consisting of centroblastic variant DLBCL, immunoblastic variant DLBCL, anaplastic variant DLBCL, spindle cell variant DLBCL, signet ring cell variant DLBCL, T cell / histiocyte enriched DLBCL, primary cutaneous DLBCL, EBV-positive DLBCL, large B cell lymphoma associated with chronic inflammation, lymphomatoid granuloma, large B cell lymphoma with IRF4 rearrangement, large B cell lymphoma with IRF4 / MUM1 rearrangement, primary mediastinal large B cell lymphoma, vascular Internal large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-positive DLBCL, primary effusion lymphoma, high-grade B-cell lymphoma with MYC, BCL2, and / or BCL6 rearrangement, unspecified high-grade B-cell lymphoma, unclassifiable B-cell lymphoma between DLBCL and classical Hodgkin lymphoma, B-cell lymphoma between primary mediastinal large B-cell lymphoma and nodular sclerosis classical Hodgkin lymphoma, and B-cell lymphoma between DLBCL and Burkitt lymphoma; Optionally, the DLBCL cells are selected from one or more of human diffuse histiocytic lymphoma cells SU-DHL-2, human diffuse lymphoma cell line TMD-8, and human diffuse large cell lymphoma cells SU-DHL-6.

20. The method according to any one of claims 14 to 17, wherein: The DLBCL is primary DLBCL, or transformed DLBCL, or advanced and / or recurrent, and / or refractory DLBCL, or DLBCL that has previously received systemic treatment, or DLBCL that has failed chemotherapy and / or targeted drugs and / or immunotherapy.

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