Pharmaceutical composition containing PRMT5 inhibitor and PD-1 / PD-l1 inhibitor

Abstract

A pharmaceutical composition, which is a pharmaceutical composition or a combined pharmaceutical composition containing a protein arginine methyltransferase 5 (PRMT5) inhibitor and a PD-1 / PD-L1 inhibitor. The pharmaceutical composition can be used for treating various cancers, comprising solid tumors.

Description

Pharmaceutical composition comprising a PRMT5 inhibitor and a PD-1 / PD-L1 inhibitor Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to a pharmaceutical composition for protecting PRMT5 inhibitors and PD-1 / PD-L1 inhibitors. Background Art

[0002] Cancer is a leading cause of death worldwide. Popular treatments, such as chemotherapy and immunotherapy, are limited in that their cytotoxic effects are not restricted to cancer cells and can also cause adverse side effects in normal tissues.

[0003] PRMT5 is a type II arginine methyltransferase that regulates important cellular functions, including cell cycle progression, apoptosis, and the DNA damage response, by symmetrically dimethylating proteins involved in transcription and signal transduction. However, data from genome-wide genetic perturbation screens using shRNA revealed a selective requirement for PRMT5 activity in MTAP-deleted cancer cell lines (Kruykov et al., 2016; Marjon et al., 2016; Markarov et al., 2016). The accumulation of MTA caused by MTAP loss in these cell lines partially inhibits PRMT5, making these cells selectively sensitive to additional PRMT5 inhibition.

[0004] Certain PRMT5 inhibitors have been developed, but they do not show selectivity for MTAP-deficient cancer cell lines. This lack of selectivity could be explained by the inhibitors’ mechanism of action, as they are either SAM-uncompetitive or SAM-competitive inhibitors and therefore independent of MTAP (Kruykov et al., 2016; Marjon et al., 2016 and Markarov et al., 2016).

[0005] By using inhibitors that bind PRMT5 non-competitively or cooperatively with MTA, selectivity for MTAP-deficient / MTA-accumulating cells can be improved. PRMT5 inhibitors that bind non-competitively or cooperatively with MTA will exhibit increased binding to PRMT5 in the presence of MTA compared to the binding of the same inhibitor in the absence of MTA. Consequently, such inhibitors will bind with significantly greater potency in the presence of high concentrations of MTA and, therefore, lead to preferential inhibition of PRMT5 in MTA-accumulating cells relative to normal cells.

[0006] Despite significant advances over the years in standard therapies for many different types of cancer, the current standard of care still falls short of the need for improved cancer treatment. Recent clinical use of immuno-oncology drugs targeting programmed cell death receptor 1 (PD-1) and its ligand PD-L1 has resulted in improvements in the treatment of many types of cancer. While these checkpoint inhibitors have improved clinical responses in some cancers, durable clinical responses occur in only approximately 10-45% of patients. Furthermore, many tumors either become resistant to treatment or become difficult to treat. Therefore, new therapies, including combination therapies for cancer, are needed.

[0007] Chromosomal region 9p21, which contains the tumor suppressors CDKN2A / B and methylthioadenosine phosphorylase (MTAP), is one of the most common genetic deletions in cancer. 9p21 deletions are associated with decreased tumor-infiltrating lymphocytes (TILs) and resistance to immune checkpoint inhibitor (ICI) therapy. Previously thought to be caused by CDKN2A / B deletion, Donjeta Gjuka et al. found that MTAP deletion leads to poor ICI efficacy and reduced TIL density. MTAP deletion leads to intracellular and extracellular accumulation of methylthioadenosine (MTA), which severely impairs T cell function by inhibiting protein arginine methyltransferase 5 (PRMT5) and adenosine receptor agonism (Cancer Cell, 2023, DOI: 10.1016 / j.ccell.2023.09.005). Administration of MTA-cooperating PRMT5 inhibitors has the potential to reverse this immunosuppressive effect, increase TILs, and inhibit tumor growth, potentially providing significant benefits for patients with 9p21 / MTAP-null / low expression. Summary of the Invention

[0008] In order to solve the technical problem of the present invention, the present invention provides a pharmaceutical composition comprising a PRMT5 inhibitor having a first active substance and a chemotherapeutic agent having a second active substance, wherein the PRMT5 inhibitor of the first active substance has the following structure:

[0009] Wherein, in formula (I) or formula (II),

[0010] R1 is selected from H, halogen, C1-C6 alkyl, halo(C1-C6 alkyl), CN;

[0011] R2 is selected from H, C1-C6 alkyl, halo(C1-C6 alkyl), deuterated(C1-C6 alkyl), C3-C6 cycloalkyl;

[0012] R3 represents H, halogen, C1-C6 alkyl, halo(C1-C6 alkyl), halo(C1-C6 alkoxy), or SF5;

[0013] In formula II, R4 represents hydrogen or C1-C6 alkyl; X represents CR5 or N;

[0014] Wherein, R5 represents hydrogen, halogen, C1-C6 alkyl, halo(C1-C6 alkyl), hydroxyl, -NH2, or CN.

[0015] In the preferred technical solution of the present disclosure, wherein, in the structure of formula (I) or formula (II):

[0016] R1 is selected from hydrogen, halogen, C1-C6 alkyl, halo(C1-C6 alkyl);

[0017] R2 is selected from hydrogen, C1-C6 alkyl, halo(C1-C6 alkyl), deuterated(C1-C6 alkyl), C3-C6 cycloalkyl;

[0018] R3 represents hydrogen, halogen, halo(C1-C6 alkyl), halo(C1-C6 alkoxy), or SF5;

[0019] R4 represents hydrogen or methyl;

[0020] In formula II, X represents CH or N.

[0021] In the preferred technical solution of the present disclosure, R1 is selected from hydrogen or fluorine.

[0022] In the preferred technical solution of the present disclosure, R2 is selected from cyclopropyl, methyl or deuterated methyl.

[0023] In the preferred technical solution of the present disclosure, R3 represents CF3.

[0024] In the preferred technical solution of the present disclosure, R4 represents hydrogen or methyl.

[0025] In the preferred technical solution of the present disclosure, X represents N.

[0026] In the preferred technical solution of the present disclosure, the PRMT5 inhibitor of the first active substance is selected from the following compounds or any combination thereof:

[0027] In the preferred technical solution of the present disclosure, the PD-1 / PD-L1 inhibitor of the second active substance can be used to interfere with the interaction between PD-1 and PD-L1 to stimulate an anti-tumor immune response, but is not limited by any binding theory. It is a small molecule compound, nucleic acid, peptide, protein, antibody, peptide antibody, diabody, mini antibody, single-chain variable fragment (ScFv) or its fragment or variant or any combination thereof.

[0028] In the preferred technical solution of the present disclosure, the PD-1 / PD-L1 inhibitor of the second active substance is an antibody selected from anti-PD-1 antibody (or simply "PD-1 antibody") and anti-PD-L1 antibody (or simply "PD-L1 antibody").

[0029] In the preferred technical solution of the present disclosure, the anti-PD-1 / PD-L1 inhibitor is a monoclonal PD-1 antibody selected from nivolumab Pembrolizumab Pidilizumab, tislelizumab Sintilimab Penampril Dostarlimab Cemiplimab Toripalimab Tislelizumab Retifanlimab Spartalizumab (PDR001), Camrelizumab (SHR-1210), MEDI0680, or RMP1-14 (rat IgG), or any combination thereof;

[0030] In the preferred technical solution of the present disclosure, the PD-1 / PD-L1 inhibitor is a monoclonal PD-L1 antibody selected from Atezolizumab Envoylumab ( KN035), Avelumab Durvalumab BMS936559 or any combination thereof.

[0031] In the preferred technical solution of the present disclosure, the anti-PD-1 / PD-L1 inhibitor is a small molecule compound selected from ASC61, BPI-371153, AN4005, INCB086550, MAX-10181 or emdefen (IMMH-010) or any combination thereof.

[0032] In addition, the present disclosure also provides a method for treating cancer or tumor, comprising administering the pharmaceutical composition of the present invention to an individual in need thereof.

[0033] In the preferred technical scheme of the present disclosure, the tumor or cancer is selected from: glioblastoma multiforme, brain cancer, prostate cancer, pancreatic cancer, mantle cell lymphoma, non-Hodgkin's lymphoma and diffuse large B-cell lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, non-small cell lung cancer, small cell lung cancer, breast cancer, triple-negative breast cancer, gastric cancer, colorectal cancer, ovarian cancer, bladder cancer, hepatocellular carcinoma, esophageal cancer, bile duct cancer, mesothelioma, laryngeal cancer, melanoma, malignant peripheral nerve sheath tumor, osteosarcoma, myxochondrosarcoma, soft tissue sarcoma, oropharyngeal squamous cell carcinoma, chronic myeloid leukemia, epidermal squamous cell carcinoma, nasopharyngeal carcinoma, neuroblastoma, endometrial cancer, head and neck cancer and cervical cancer.

[0034] As will be understood by those of ordinary skill in the art, in any embodiment disclosed herein, any feasible combination of a compound of formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof and a compound of formula (II) or a pharmaceutically acceptable salt, solvate or prodrug thereof is included in the present invention, as long as such combination is capable of producing some synergistic effect in the treatment of a subject in need of such treatment.

[0035] When any compound is used in the present invention, it includes any pharmaceutically acceptable form thereof, including but not limited to isomers, tautomers, salts, solvates, polymorphs, prodrugs, etc. It should be understood that the term "compound" includes any and all such forms, whether or not explicitly stated, although sometimes only certain terms are explicitly stated, such as "salt" and "prodrug".

[0036] Unless expressly defined otherwise, all terms used herein have the ordinary meaning as would be interpreted or understood by one of ordinary skill in the art.

[0037] The terms "a", "an" or "the" used herein refer to both the singular and the plural forms. Generally, when a singular or plural form of a noun is used, it refers to both the singular and the plural form of the noun.

[0038] When the term "about" is applied to a parameter, it means that the parameter can vary within ±10%, preferably within ±5%, including any number from the lower limit to the upper limit. When the term "about" is applied to a range, it applies to both the lower and upper limits of the range. As will be understood by those skilled in the art, when a parameter is not critical, a number is generally given for illustrative purposes only and is not limiting.

[0039] "Alkoxy" refers to a group -OR, where R is alkyl as defined herein. Representative examples include methoxy, ethoxy, propoxy, isopropoxy, sec-butoxy, tert-butoxy, and the like.

[0040] "Alkyl" refers to a group derived from a straight or branched chain saturated hydrocarbon by removing a hydrogen from one of the saturated carbons. Alkyl groups preferably contain 1 to 8 carbon atoms, sometimes preferably 1 to 6 carbon atoms, and sometimes even more preferably 1 to 4 carbon atoms. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, and the like. "Lower alkyl," "lower alkoxy," or "lower haloalkyl" refers to an alkyl or alkyl moiety having one to four, sometimes preferably one to three or one to two carbon atoms.

[0041] As used herein, the term "cyano" refers to -CN.

[0042] The term "cycloalkyl" as used herein refers to a group derived from a monocyclic saturated carbocyclic ring by removing a hydrogen atom from the saturated carbocyclic ring, preferably having 3 to 8, more preferably 3 to 6 carbon atoms. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl.

[0043] As used herein, the terms "halo" and "halogen" refer to F, Cl, Br, or I.

[0044] The term "haloalkyl" as used herein refers to an alkyl group substituted with at least one halogen atom. A haloalkyl group can be an alkyl group in which all hydrogen atoms are substituted with halogens. Representative examples of haloalkyl groups include, but are not limited to, trifluoromethyl, fluoromethyl, difluoromethyl, bromomethyl, 1-chloroethyl, perchloroethyl, 2-fluoroethyl, etc.

[0045] The term "heterocyclyl" as used herein refers to a 3 to 10-membered monocyclic or bicyclic non-aromatic group containing one or more, preferably 1 to 3, heteroatoms independently selected from nitrogen (N), oxygen and sulfur (S, S(O) or S(O)2) in the non-aromatic ring. The heterocyclyl of the present disclosure can be connected to the parent molecular moiety through a carbon atom or a nitrogen atom in the group. The heterocyclyl group can be saturated or unsaturated, for example, containing one or more double bonds in the ring. Unless otherwise stated, the valence of the group can be located on any atom of any ring within the group where the valence rules permit. Examples may include, but are not limited to, azetidinyl, pyrrolidinyl, 2-oxopyrrolidinyl, 2,5-dihydro-1H-pyrrolyl, piperidinyl, 4-piperidinyl, morpholinyl, piperazinyl, 2-oxopiperazinyl, tetrahydropyranyl, tetrahydrofuranyl, 2-oxopiperidinyl, thiomorpholinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, etc.

[0046] When any group, such as "cycloalkyl" or "heterocyclyl" is referred to as "substituted or unsubstituted" or "optionally substituted", unless otherwise specified, it means that the group is substituted or not substituted by 1 to 5, sometimes preferably 1 to 3 or 1 to 2 substituents independently selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and cyano.

[0047] The term "solvate" as used herein refers to a physical association of a compound of the invention with one or more, preferably one to three, solvent molecules (whether organic or inorganic). This physical association includes hydrogen bonding. In some cases, the solvate is capable of separation, for example when one or more, preferably one to three, solvent molecules are incorporated into the crystal lattice of a crystalline solid. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are generally known in the art.

[0048] "Prodrugs" refer to compounds that can be converted in vivo to produce the active parent compound under physiological conditions, such as by hydrolysis in the blood. Common examples include, but are not limited to, ester and amide forms of compounds having an active form with a carboxylic acid moiety. Amides and esters of the compounds of the present invention can be prepared according to conventional methods. In particular, in the present invention, prodrugs can also be formed by acylation of the amino group or nitrogen atom in the heterocyclyl ring structure, where the acyl group can hydrolyze in vivo. Such acyl groups include, but are not limited to, C1-C6 acyl groups, preferably C1-C4 acyl groups, more preferably C1-C2 (formyl or acetyl) groups, or benzoyl groups.

[0049] As used herein, the term "subject" refers to a human or other mammal, such as a monkey, dog, cat, horse, etc. The term is intended to encompass and is sometimes interchangeable with "patient."

[0050] As used herein, the terms "administering" or "administering" refer to providing a compound or pharmaceutical composition to a subject having or at risk for a disease or condition to be treated or prevented.

[0051] Any route of administration is suitable for the present invention. In one embodiment, the compounds of the present invention can be administered to a subject in a solid dosage form such as a tablet, capsule, or the like. In one embodiment, the compounds of the present invention can be administered to a subject by intravenous injection. In another embodiment, the compounds of the present invention can be administered to a subject by any other suitable systemic delivery method, such as oral, parenteral, intranasal, sublingual, rectal, or transdermal administration.

[0052] As used herein, the term "therapeutically effective amount" refers to that amount of a compound or composition that will elicit the desired or intended biological or medical response in a subject that is being sought by a physician, veterinarian, or researcher. The therapeutically effective amount of the compound and the specific pharmaceutically acceptable carrier will vary depending on, for example, the age, weight, sex of the subject, the mode of administration, and the disease or condition being treated.

[0053] The term "pharmaceutically acceptable" when used before a compound, salt, prodrug, composition, or carrier means that such compound, salt, prodrug, composition, or carrier is suitable for administration to a subject for treatment without causing intolerable side effects to the subject considering the desired treatment.

[0054] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is compatible with the compounds used in the present invention and can be used to administer the compounds in the methods of the present invention, and is preferably non-toxic, or inert and pharmaceutically acceptable. Pharmaceutically acceptable carriers can be solid, liquid or gaseous substances, including any and all dry powders, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like. Examples of such carriers include oils such as corn oil, buffers such as phosphate buffered saline (PBS), saline, polyethylene glycol, glycerol, polypropylene glycol, dimethyl sulfoxide, amides such as dimethylacetamide, proteins such as albumin, detergents such as Tween 80, monosaccharides and oligosaccharides such as glucose, lactose, cyclodextrins, starch, and the like.

[0055] As described herein, some embodiments of the compounds of the present invention can contain basic functional groups, such as amino or alkylamino, and therefore can form pharmaceutically acceptable salts with pharmaceutically acceptable acids. In this respect, the term "pharmaceutically acceptable salts" refers to relatively nontoxic inorganic and organic acid addition salts of the compounds of the present invention. These salts can be prepared on site during the administration of carriers or dosage form production processes, or by reacting the purified compounds of the present invention in free alkali form with suitable organic or inorganic acids alone, and separating the salts so formed in subsequent purification processes to prepare. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate.

[0056] The formulations used in the present invention may also contain stabilizers, preservatives, buffers, antioxidants or other additives known to those skilled in the art. The use of such media and agents for pharmaceutically active substances is well known in the art.

[0057] The terms "synergistic" and the like as used herein refer to an effect caused by a combination of two or more agents that is greater than the cumulative effect of the two or more agents used alone. This synergistic effect of combination therapy includes higher efficacy, lower side effects, or both. In some embodiments, the synergistic effect includes a significant reduction in the side effects of the two therapeutic inhibitors due to a reduction in the dosage of the two therapeutic inhibitors, while the overall therapeutic efficacy remains at approximately the same or improved levels. In some embodiments, the synergistic effect includes a significant improvement in the efficacy of inhibiting cancer cell proliferation, while the side effects caused by the two drugs remain at approximately the same or lower levels. The synergistic effect allows the use of a lower dose of a single drug to effectively treat the disease. In general, the synergistic combination of two or more drugs can lead to improvements in disease treatment compared to monotherapy.

[0058] Combination therapy can allow the use of a lower dose of a first therapeutic agent, such as a PRMT5 inhibitor, or a second therapeutic agent, such as a PD-1 / PD-L1 inhibitor, or lower doses of both therapeutic agents than would normally be required when either agent is used alone. The present invention encompasses any and all such "synergistic" effects.

[0059] The pharmaceutical composition may comprise a PRMT5 inhibitor and a PD-1 / PD-L1 inhibitor for use in the methods of the present invention in an amount ranging from 0.01% to 99% by weight of the total composition, preferably from 0.1% to 80% by weight of the total composition, and more preferably from 0.1% to 50% by weight of the total composition. The weight ratio between the PRMT5 inhibitor and the PD-1 / PD-L1 inhibitor may be in the range of 1:20 to 20:1, sometimes preferably from 1:15 to 15:1, and sometimes more preferably from 1:10 to 10:1.

[0060] For systemic administration, the daily dosage range for adult human treatment of a PRMT5 inhibitor is about 0.01 to about 150 mg / kg, preferably about 0.05 to about 100 mg / kg, and sometimes more preferably about 0.1 to about 50 mg / kg.

[0061] The present disclosure provides pharmaceutical compositions that can be used to treat and / or prevent various cancers that may include or exclude the following cancers: glioblastoma multiforme, brain cancer, prostate cancer, pancreatic cancer, mantle cell lymphoma, non-Hodgkin lymphoma and diffuse large B-cell lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, non-small cell lung cancer, small cell lung cancer, breast cancer, triple-negative breast cancer, gastric cancer, colorectal cancer, ovarian cancer, bladder cancer, hepatocellular carcinoma, esophageal cancer, bile duct cancer, mesothelioma, laryngeal cancer, melanoma, malignant peripheral nerve sheath tumor, osteosarcoma, myxochondrosarcoma, soft tissue sarcoma, oropharyngeal squamous cell carcinoma, chronic myeloid leukemia, epidermal squamous cell carcinoma, nasopharyngeal carcinoma, neuroblastoma, endometrial cancer, head and neck cancer, and cervical cancer.

[0062] In a preferred technical solution of the present disclosure, the cancer is metastatic cancer.

[0063] In a preferred technical solution of the present disclosure, the metastatic cancer is brain metastatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 In vivo pharmacodynamic study of compound D in the C3H mouse model with orthotopic tibial transplantation of DuNN MTAP KO cells Specific embodiments

[0065] The PD-1 antibody information used in this experiment is: 29F.1A12 TM Monoclonal antibody (inVivoMab anti-mouse PD-1) was purchased from BioXCell, catalog number: #BE0273.

[0066] The chemical name of Compound A used in this test is (S)-4-amino-N-methyl-N-(6-(trifluoromethyl)-2,3-dihydrobenzofuran-3-yl)imidazo[1,5-a]quinoxaline-8-carboxamide, which has the following chemical structure:

[0067] The chemical name of compound B used in this test is: 4-amino-N-cyclopropyl-7-fluoro-N-(5-(trifluoromethyl)pyridin-2-ylmethyl)imidazo[1,5-a]quinoxaline-8-carboxamide, which has the following chemical structure:

[0068] The chemical name of compound C used in this test is: (R)-4-amino-N-cyclopropyl-7-fluoro-N-(1-(5-trifluoromethyl)pyridin-2-yl)ethyl)imidazo[1,5-a]quinoxaline-8-carboxamide, which has the following chemical structure:

[0069] The chemical name of compound D used in this test is: (S)-4-amino-7-fluoro-N-methyl-N-(6-trifluoromethyl)-2,3-dihydrobenzofuran-3-yl)imidazo[1,5-a]quinoxaline-8-carboxamide, which has the following chemical structure:

[0070] The chemical name of compound E used in this test is: (S)-4-amino-N-methyl-N-(6-(trifluoromethyl)-2,3-dihydrobenzofuran-3-yl)imidazo[1,5-a]pyrido[3,4-e]pyrazine-8-carboxamide, which has the following chemical structure:

[0071] The chemical name of compound F used in this test is: (S)-4-amino-N-(methyl-d3)-N-(6-(trifluoromethyl)-2,3-dihydrobenzofuran-3-yl)imidazo[1,5-a]quinoxaline-8-carboxamide, which has the following chemical structure:

[0072] The chemical name of compound G used in this test is: (S)-4-amino-N-methyl-N-(6-(pentafluoro-λ6-sulfane)-2,3-dihydrobenzofuran-3-yl)imidazo[1,5-a]quinoxaline 8-carboxamide, which has the following chemical structure:

[0073] The chemical name of compound H used in this test is: (S)-4-amino-N-methyl-N-(6-(perfluoroethane)-2,3-dihydrobenzofuran-3-yl)imidazo[1,5-a]quinoxaline-8-carboxamide, which has the following chemical structure:

[0074] The chemical name of Compound I used in this test is: (S)-4-amino-7-fluoro-N-methyl-N-(6-trifluoromethyl)-2,3-dihydrobenzofuran-3-yl)imidazo[1,5-a]quinoxaline-8-carboxamide, which has the following chemical structure:

[0075] The chemical name of compound J used in this test is: (S)-4-amino-N-methyl-N-(6-(trifluoromethyl)-2,3-dihydrobenzofuran-3-yl)imidazo[1,5-a]quinoxaline-8-carboxamide-1-d, which has the following chemical structure:

[0076] The chemical name of compound K used in this test is: (S)-4-amino-N-methyl-N-(6-(trifluoromethyl)-2,3-dihydrobenzofuran-3-yl)imidazo[1,5-a]quinoxaline-8-carboxamide-3-d, which has the following chemical structure:

[0077] The chemical name of compound L used in this test is: (S)-4-amino-N-(methyl-d3)-N-(6-(trifluoromethyl)-2,3-dihydrobenzofuran-3-yl)imidazo[1,5-a]pyrido[3,4-e]pyrazine-8-carboxamide, which has the following chemical structure:

[0078] The chemical name of the compound M used in this test is: (S)-4-amino-7-cyano-N-methyl-N-(6-(trifluoromethyl)-2,3-dihydrobenzofuran-3-yl)imidazo[1,5-a]quinoxaline-8-carboxamide, which has the following chemical structure:

[0079] The chemical name of the compound N used in this test is: (R)-4-amino-N-methyl-d3-N-(1-(5-trifluoromethyl)pyridin-2-yl)ethyl)imidazo[1,5-a]quinoxaline-8-carboxamide, which has the following chemical structure:

[0080] The chemical name of Compound O used in this test is (R)-4-amino-N-methyl-N-(1-(5-(trifluoromethyl)pyridin-2-yl)ethyl)imidazo[1,5-a]quinoxaline-8-carboxamide, which has the following chemical structure:

[0081] Among them, the preparation of compound A, compound D, compound E, compound F, compound G, compound H, compound I, compound J, compound K, compound L and compound M refers to international patent application PCT / CN2023 / 120923;

[0082] Among them, the preparation of compound B, compound C, compound N and compound O refers to international patent application PCT / CN2023 / 111604, which is incorporated herein by reference in its entirety, as if they were fully cited herein.

[0083] Example 1: In vivo pharmacodynamic study of compound D in a C3H mouse model with orthotopic tibial transplantation of DuNN MTAPKO cells

[0084] Cell Culture: Human osteosarcoma DuNN MTAP KO cells (provided by the Experimental Animal Center of Shanghai First People's Hospital) were cultured as monolayers in DMEM supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic in a 37°C, 5% CO2 incubator. Twice weekly, cells were routinely digested and passaged using trypsin-EDTA. When cell saturation reached 80%-90% and the desired cell number was reached, cells were harvested, counted, and plated.

[0085] Animals: C3H normal female mice, 4-6 weeks old, weighing 18-22 grams. A total of 30 mice are required (24 mice are included in each group, with a surplus).

[0086] Tumor inoculation: Dunn cells were digested with 0.25% trypsin-EDTA solution to make a single-cell suspension, washed with PBS buffer and adjusted to a cell density of 1 × 107 / ml, for future use. On the day of the experiment, mice were anesthetized by intraperitoneal injection of 0.5% sodium pentobarbital solution at a dose of 40 mg / kg, and a bone hole with a diameter of about 1 mm was drilled at the upper end of the tibia of the right hind limb with a 1 ml syringe to reach the bone marrow cavity. Be careful not to penetrate the bone cortex of the opposite side to cause fractures or bleeding. Use a 1 ml syringe and a 25G needle to draw up the DUNN cell suspension, and slowly inject it into the tibial bone marrow cavity of the experimental group mice at a dose of 30 μl. Be careful not to cause reflux or leakage of cells. After injection, disinfect the surgical field with 75% alcohol, return the mouse to the cage, cover it with sterile dressing, maintain its body temperature until it wakes up, and then continue to feed and observe. When the average tumor volume reaches about 150 mm 3 Randomization and dosing began at 1:00 PM. At the end of the experiment, mice were euthanized with an intraperitoneal injection of 120 mg / kg of 0.5% sodium pentobarbital solution. Tibiae and tumor tissues were removed and fixed with 10% formaldehyde. Paraffin sections were prepared and stained with hematoxylin and eosin. Tumor morphology and invasion were observed microscopically, and the expression of proliferation markers such as Ki-67 and PCNA in tumor cells was assessed by immunohistochemistry. The experimental groups and dosing schedule are shown in the table below.

[0087] Animal experimental groups and dosing regimen:

[0088] Table 1: Drug efficacy experiments Note: 1. N: Number of mice per group; 2. Dosing volume: 10 μL / g based on mouse body weight. If body weight loss exceeds 15%, discontinue dosing immediately and resume dosing when body weight loss returns to within 10%. 3. Experimental duration may be adjusted based on tumor volume.

[0089] Animal husbandry: The experiment can only begin after the animals have been kept in the experimental environment for 3-7 days after arrival. The animals are kept in IVC (independent ventilation system) cages (3 per cage) in an SPF animal room. All cages, bedding and drinking water must be sterilized before use. All experimental personnel should wear protective clothing and latex gloves when operating in the animal room. The animal information card for each cage should indicate the number of animals in the cage, gender, strain, receipt date, dosing regimen, experimental number, group and start date of the experiment. Cages, feed and drinking water are changed twice a week. The breeding environment and lighting conditions are as follows:

[0090] Temperature: 20-26°C

[0091] Humidity: 40-70%

[0092] Photoperiod: 12 hours of light, 12 hours of no light

[0093] Feed ingredients: Feed meets the standards for laboratory animal food. Maximum contaminant levels are within controllable limits and are routinely inspected by the manufacturer. High-pressure sterilized drinking water is used.

[0094] Animal grouping: Before administration, animals were weighed and tumor volumes were measured, and the animals were randomly grouped according to tumor volume (randomized block design).

[0095] Observation: The design and any modifications to this experimental protocol will be evaluated and approved by the Shanghai WuXi AppTec Institutional Animal Care Committee (IACUC) before implementation. The use and welfare of experimental animals will comply with the rules of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health and mortality of the animals will be monitored daily. Routine examinations will include observing the effects of tumor growth and drug treatment on the animals' daily behavior, such as behavioral activity, food and water intake, weight changes (measured twice a week), physical signs, or other abnormalities. The number of deaths and side effects of animals within each group will be recorded based on the number of animals in each group.

[0096] Experimental indicators: The experimental indicators are used to examine whether tumor growth is inhibited, delayed, or cured. Tumor diameter is measured twice a week with a vernier caliper. The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively.

[0097] The tumor inhibition efficacy of a compound was evaluated using TGI (%) or tumor growth rate (T / C) (%). TGI (%) reflects the rate of tumor growth inhibition. TGI (%) is calculated as follows: TGI (%) = [1 - (average tumor volume at the end of dosing in a given treatment group - average tumor volume at the start of dosing in that treatment group) / (average tumor volume at the end of treatment in the solvent control group - average tumor volume at the start of treatment in the solvent control group)] × 100%.

[0098] Tumor proliferation rate T / C (%): Calculated by the following formula: T / C (%) = Ti / Vi × 100%, where Vi is the average tumor volume of the solvent control group at a certain measurement, and Ti is the average tumor volume of the drug-treated group at the same measurement.

[0099] Termination of the experiment: If the animal's health condition continues to deteriorate or the tumor volume exceeds 3,000 mm 3 If the animal is seriously ill or in pain, it must be euthanized. If the animal has any of the following conditions, notify the veterinarian and euthanize it:

[0100] Obvious emaciation, with weight loss greater than 20%;

[0101] No free access to food and water;

[0102] The animal developed the following clinical manifestations and continued to deteriorate:

[0103] Piloerection

[0104] Arched back

[0105] White ears, nose, eyes, or feet

[0106] shortness of breath

[0107] twitch

[0108] Continuous diarrhea

[0109] dehydration

[0110] Slow movement

[0111] Voice

[0112] Data Analysis: All data are expressed as mean ± SEM and analyzed using one-way ANOVA using Graphpad10. *P < 0.05 was considered statistically significant. The results are shown in Table 2.

[0113] Table 2 Tumor volume statistics

[0114] In a 25-day in vivo efficacy study in a C3H mouse DUNN cell tibial orthotopic homograft tumor model, the changes in tumor volume in each group during the dosing period are detailed in Table 2 and Figure 1. The tumor growth inhibition rate (TGI) was 30.9% in the Compound D monotherapy group (1 mg / kg, oral, once daily); 38.6% in the PD-1 antibody monotherapy group (5 mg / kg, intraperitoneal injection, once daily) (*P < 0.05); and 80.4% in the Compound D (1 mg / kg, oral, once daily) combined with the PD-1 antibody (5 mg / kg, intraperitoneal injection, once daily) group (***P < 0.001).

[0115] The results showed that there was no statistically significant difference in the Compound D monotherapy group compared to the control group; there was a statistically significant difference in the PD-1 antibody monotherapy group compared to the control group; and there was a statistically significant difference in the combination of Compound D and PD-1 antibody compared to the control group. Compared to the monotherapy group, the combination of Compound D and PD-1 antibody demonstrated stronger anti-tumor activity.

[0116] Although the preferred embodiment has been described above, it will be apparent to those skilled in the art that modifications may be made without departing from the invention. Such modifications are considered to be possible variations within the scope of the invention.

Claims

1. A pharmaceutical composition comprising a PRMT5 inhibitor having a first active substance and a PD-1 / PD-L1 inhibitor having a second active substance, wherein: The PRMT5 inhibitor of the first active substance has the following structure: Wherein, in formula (I) or formula (II), R1 is selected from H, halogen, C1-C6 alkyl, halo(C1-C6 alkyl), CN; R2 is selected from H, C1-C6 alkyl, halo(C1-C6 alkyl), deuterated(C1-C6 alkyl), C3-C6 cycloalkyl; R3 represents H, halogen, C1-C6 alkyl, halo(C1-C6 alkyl), halo(C1-C6 alkoxy), or SF5; In formula II, R4 represents hydrogen or C1-C6 alkyl; X represents CR5 or N; Wherein, R5 represents hydrogen, halogen, C1-C6 alkyl, halo(C1-C6 alkyl), hydroxyl, -NH2, or CN.

2. The pharmaceutical composition according to claim 1, wherein In the structure of formula (I) or formula (II): R1 is selected from hydrogen, halogen, C1-C6 alkyl, halo(C1-C6 alkyl); R2 is selected from hydrogen, C1-C6 alkyl, halo(C1-C6 alkyl), deuterated(C1-C6 alkyl), C3-C6 cycloalkyl; R3 represents hydrogen, halogen, halo(C1-C6 alkyl), halo(C1-C6 alkoxy), or SF5; R4 represents hydrogen or methyl; In formula II, X represents CH or N.

3. The pharmaceutical composition according to claim 1, wherein R1 is selected from hydrogen or fluorine.

4. The pharmaceutical composition according to claim 1, wherein R2 is selected from cyclopropyl, methyl or deuterated methyl.

5. The pharmaceutical composition according to claim 1, wherein R3 represents CF3.

6. The pharmaceutical composition according to claim 1, wherein R4 represents hydrogen or methyl.

7. The pharmaceutical composition according to claim 1, wherein X represents N.

8. The pharmaceutical composition according to claim 1, wherein The PRMT5 inhibitor of the first active substance is selected from the following compounds or any combination thereof:

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the PD-1 / PD-L1 inhibitor of the second active substance is a small molecule compound, nucleic acid, peptide, protein, antibody, peptide antibody, diabody, minibody, single-chain variable fragment (ScFv) or fragment or variant thereof or any combination thereof that can be used to interfere with the interaction between PD-1 and PD-L1 to stimulate an anti-tumor immune response, but is not limited by any binding theory.

10. The pharmaceutical composition according to claim 9, wherein the PD-1 / PD-L1 inhibitor of the second active substance is an antibody selected from anti-PD-1 antibody (or simply "PD-1 antibody") and anti-PD-L1 antibody (or simply "PD-L1 antibody").

11. The pharmaceutical composition according to any one of claims 9-10, wherein the anti-PD-1 / PD-L1 inhibitor is a monoclonal PD-1 antibody selected from Pembrolizumab Pidilizumab, tislelizumab Sintilimab Penampril Spartalizumab (PDR001), Camrelizumab (SHR-1210), MEDI0680 or RMP1-14 (rat IgG2A) or any combination thereof.

12. The pharmaceutical composition according to claim 9, wherein the PD-1 / PD-L1 inhibitor is a monoclonal PD-L1 antibody selected from Envoylumab ( KN035), BMS936559 or any combination thereof.

13. The pharmaceutical composition according to claim 9, wherein the anti-PD-1 / PD-L1 inhibitor is a small molecule compound selected from ASC61, BPI-371153, AN4005, INCB086550, MAX-10181 or emdefene (IMMH-010) or any combination thereof.

14. A method for treating cancer or tumors, comprising administering the pharmaceutical composition of any one of claims 1 to 13 to a subject in need thereof.

15. The method according to claim 14, wherein The tumor or cancer is selected from the group consisting of glioblastoma multiforme, brain cancer, prostate cancer, pancreatic cancer, mantle cell lymphoma, non-Hodgkin lymphoma and diffuse large B-cell lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, non-small cell lung cancer, small cell lung cancer, breast cancer, triple-negative breast cancer, gastric cancer, colorectal cancer, ovarian cancer, bladder cancer, hepatocellular carcinoma, esophageal cancer, bile duct cancer, mesothelioma, laryngeal cancer, melanoma, malignant peripheral nerve sheath tumor, osteosarcoma, myxochondrosarcoma, soft tissue sarcoma, oropharyngeal squamous cell carcinoma, chronic myeloid leukemia, epidermal squamous cell carcinoma, nasopharyngeal carcinoma, neuroblastoma, endometrial cancer, head and neck cancer and cervical cancer.

16. The method according to claim 14, wherein The cancer is a metastatic cancer.

17. The method according to claim 15, wherein: The metastatic cancer is brain metastatic cancer.

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