Pharmaceutical composition comprising PRMT5 inhibitor and EGFR inhibitor

Abstract

Disclosed is a pharmaceutical composition, comprising a protein arginine methyltransferase 5 (PRMT5) inhibitor and an epidermal growth factor receptor tyrosine kinase (EGFR) inhibitor. The pharmaceutical composition can be used to treat various cancers, including solid tumors. The combination product can be used to treat any number of diseases associated with PRMT5 and / or EGFR.

Description

Pharmaceutical composition comprising a PRMT5 inhibitor and an EGFR inhibitor Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a pharmaceutical composition for protecting a PRMT5 inhibitor and an EGFR inhibitor. 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] Recently, Thierry et al. reported that first-generation PRMT5 inhibitors and EGFR inhibitors exhibited significant synergistic effects on the anti-proliferative activity of triple-negative breast cancer cells, providing a new and promising clinical treatment strategy for triple-negative breast cancer (Dovepress, 2023, https: / / doi.org / 10.2147 / BCTT.S430513). Second-generation PRMT5 inhibitors are known to have better selectivity and safety than first-generation PRMT5 inhibitors, so the study of their combination therapy and indications is of great value. Summary of the Invention

[0007] 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 an EGFR inhibitor compound having a second active substance, wherein the PRMT5 inhibitor of the first active substance has the following structure:

[0008] Wherein, in formula (I) or formula (II),

[0009] R1 is selected from H, halogen, C1-C6 alkyl, halo(C1-C6 alkyl), CN;

[0010] R2 is selected from H, C1-C6 alkyl, halo(C1-C6 alkyl), C3-C6 cycloalkyl;

[0011] R3 represents H, halogen, C1-C6 alkyl, halo(C1-C6 alkyl), halo(C1-C6 alkoxy), or SF5;

[0012] In formula II, R4 represents hydrogen or C1-C6 alkyl; X represents CR5 or N;

[0013] Wherein, R5 represents hydrogen, halogen, C1-C6 alkyl, halo(C1-C6 alkyl), hydroxyl, -NH2, or CN.

[0014] In the preferred technical solution of the present disclosure, wherein, in the structure of formula (I) or formula (II):

[0015] R1 is selected from hydrogen, halogen, C1-C6 alkyl, halo(C1-C6 alkyl);

[0016] R2 is selected from hydrogen, C1-C6 alkyl, halo(C1-C6 alkyl), C3-C6 cycloalkyl;

[0017] R3 represents hydrogen, halogen, halo(C1-C6 alkyl), halo(C1-C6 alkoxy), or SF5;

[0018] R4 represents hydrogen or methyl;

[0019] In formula II, X represents CH or N.

[0020] In the preferred technical solution of the present disclosure, R1 is selected from hydrogen or fluorine.

[0021] In the preferred technical solution of the present disclosure, R2 is selected from cyclopropyl or methyl.

[0022] In the preferred technical solution of the present disclosure, R3 represents CF3.

[0023] In the preferred technical solution of the present disclosure, R4 represents hydrogen or methyl.

[0024] In the preferred technical solution of the present disclosure, X represents N.

[0025] 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:

[0026] In the preferred technical solution of the present disclosure, the EGFR inhibitor of the second active substance is selected from any one of the following compounds or their pharmaceutical salts: Osimertinib Almonertinib Dacomitinib Afatinib Icotinib Erlotinib Gefitinib Zorifertinib (AZD3759), Lazertinib, Nazartinib (EGF816), Rocieletinib (CO1686), HM61713, Naquotinib (ASP8273), Mavelertinib (PF-06747775), Abivertinib (avitinib), Alflutinib (AST2 818), Olafertinib (CX-101; RX-518), Almonertinib (aumolertinib; HS-10296), Rezivertinib (BPI-7711), Mobocertinib (TAK-788), BLU-945 (Bluprint), BLU-701 (Bluprint), BBT-176 (Bridge) Biotherapeutics), TQB3804 (Zhengda Tianqing), BPI-361175 (Beida), QLH11811 (Qilu), HS10375 (Hausen), H002 (Hongyun Bio), DAJH-1050766 (Chengdu Diao Jiuhong), BI-4020 (Boehringer Ingelheim), CH7233163 (Zhongwai Pharmaceutical), JBI-09-063 or any combination thereof.

[0027] In the preferred technical solution disclosed herein, the EGFR inhibitor of the second active substance is selected from Trastuzumab Pertuzumab Cetuximab Amivantamab (JNJ-6372).

[0028] In the preferred technical solution disclosed herein, the EGFR inhibitor of the second active substance is selected from Osimertinib Almonertinib Dacomitinib Afatinib Icotinib Erlotinib Gefitinib or its pharmaceutically acceptable salt and Trastuzumab Pertuzumab Cetuximab Amivantamab (JNJ-6372).

[0029] 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.

[0030] 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.

[0031] In the preferred technical solution disclosed herein, the tumor is non-small cell lung cancer.

[0032] In the preferred technical solution disclosed herein, the non-small cell lung cancer is osimertinib-resistant non-small cell lung cancer.

[0033] 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.

[0034] 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".

[0035] 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.

[0036] 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 forms of the noun.

[0037] 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.

[0038] "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.

[0039] "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.

[0040] As used herein, the term "cyano" refers to -CN.

[0041] 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.

[0042] As used herein, the terms "halo" and "halogen" refer to F, Cl, Br, or I.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] "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.

[0048] 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."

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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 an EGFR inhibitor, or lower doses of both therapeutic agents than would normally be required when either agent is used alone. The present invention includes any and all such "synergistic" effects.

[0058] The pharmaceutical composition may comprise a PRMT5 inhibitor and an EGFR 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 EGFR 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.

[0059] 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.

[0060] 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.

[0061] In a preferred technical solution of the present disclosure, the tumor is non-small cell lung cancer.

[0062] In the preferred technical solution of the present disclosure, the non-small cell lung cancer is osimertinib-resistant non-small cell lung cancer.

[0063] In a preferred technical solution of the present disclosure, the cancer is metastatic cancer.

[0064] In a preferred technical solution of the present disclosure, the metastatic cancer is brain metastatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1A-1B: Anti-tumor inhibition curves and inhibition rates of compound A, osimertinib, and their combination in the NCI-H292 MTAP(- / -) deficient cell model

[0066] Figure 2A-2B: Anti-tumor inhibition curves and inhibition rates of compound C, osimertinib, and their combination in the NCI-H292 MTAP(- / -) deficient cell model

[0067] Figure 3: In vivo antitumor efficacy of compound A, osimertinib, and their combination in the osimertinib-resistant non-small cell lung cancer PDXNU / NU mouse model Specific embodiments

[0068] 1. Experimental compounds

[0069] The compound used in this study, osimertinib, is a drug developed by AstraZeneca, with the trade name Tagrisso, and has the following chemical structure:

[0070] 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:

[0071] 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:

[0072] 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:

[0073] 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:

[0074] 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:

[0075] 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:

[0076] 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:

[0077] 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:

[0078] 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:

[0079] 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:

[0080] 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:

[0081] 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:

[0082] 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:

[0083] 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:

[0084] 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:

[0085] 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;

[0086] The preparation of compound B, compound C, compound N and compound O is described in international patent application PCT / CN2023 / 111604.

[0087] are incorporated herein by reference in their entirety, as if fully incorporated herein.

[0088] 2. Combination of compounds in the NCI-H292 MTAP(- / -) deficient cell model

[0089] Cell lines and cell culture:

[0090] NCI-H292 MTAP(- / -) cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. The cells were cultured in a cell culture medium containing 90% RPMI1640 and 10% fetal bovine serum at 37°C and 5% CO2. Experiments were performed only when the cells were in the logarithmic growth phase.

[0091] Combination drug testing:

[0092] Adjust the cell density using complete medium and then seed the cells in a 96-well cell culture plate. Culture the cells in the 96-well plate at 37°C and 5% CO2. Add the two test compounds to the cell plate using gradient dilutions. Continue culturing the cells in the 96-well plate at 37°C and 5% CO2 for 144 hours. Thaw the CTG reagent and equilibrate the cell plate to room temperature for 30 minutes. Add an equal volume of CTG solution to each well. Shake on an orbital shaker for 5 minutes to lyse the cells. Place the cell plate at room temperature for 20 minutes to stabilize the luminescence signal. Read the luminescence value and collect the data.

[0093] Data analysis: Cell inhibition rate (%) = 100-(Lum test drug-Lum culture medium control) / (Lum solvent control-Lum culture medium control) × 100%

[0094] Calcusyn was used to analyze the combination index (CI) values.

[0095] Example 1: Testing of single-drug compounds in the NCI-H292 MTAP(- / -) deficient cell model

[0096] Table 1

[0097] Table 1 shows the IC values ​​of each compound in NCI-H292 MTAP(- / -) deficient cells. 20 and IC 50 , and the maximum inhibition rate is given.

[0098] Example 2: Compound A combined with Osimertinib in the NCI-H292 MTAP(- / -) deficient cell model

[0099] The experimental results of the combination of Compound A and Osimertinib on NCI-H292 MTAP(- / -) cells are shown in Table 2 for inhibition analysis, Figure 1A for inhibition curves, and Figure 1B for inhibition rates:

[0100] Table 2

[0101] FIG1B shows the inhibition rates of compound A (16.0 nM) and osimertinib (12.3 nM) alone and in combination, indicating that the two compounds have a synergistic effect under these conditions.

[0102] The analysis of the CI combined index is shown in Table 3:

[0103] Table 3

[0104] Table 3 shows the combined effect index CI values ​​of compound A and osimertinib.

[0105] Example 3 Compound C and Osimertinib were combined and tested in the NCI-H292 MTAP(- / -) deficient cell model

[0106] The experimental results of the combination of Compound C and Osimertinib in the NCI-H292 MTAP(- / -) deficient cell model are shown in Table 4 for inhibition analysis, Figure 2A for inhibition curves, and Figure 2B for inhibition rates.

[0107] Table 4

[0108] FIG2B shows the inhibition rates of compound C (16.0 nM) and osimertinib (12.3 nM) alone and in combination, indicating that the two compounds have an additive effect under this condition.

[0109] Analysis of CI combined index

[0110] Table 5

[0111] Table 5 shows the combined effect index CI value of compound C and osimertinib.

[0112] Conclusion: The combination index of compound A and compound C with the EGFR inhibitor osimertinib ranged from 0.32 to 2.23, showing an additive effect.

[0113] 3. Combination therapy of compounds in osimertinib-resistant non-small cell lung cancer PDX NU / NU mouse model

[0114] Example 1: In vivo pharmacodynamic study of the compound in an osimertinib-resistant non-small cell lung cancer PDXNU / NU mouse model Animal modeling and grouping:

[0115] NU / NU mice, female, 6-8 weeks old, weighing approximately 18-22 g, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. All experimental mice were housed in Xi'an Lidi SPF animal room and acclimatized to the environment for at least 3 days in advance. Lung cancer tumor-bearing mice were selected and, when the tumor grew to 800-1000 mm, the mice were randomly divided into two groups. 3 When the average tumor volume reached approximately 150 mm 3 (100-200mm 3 ), 18 tumor-bearing mice were selected and randomly divided into 6 groups according to tumor volume. The day of grouping was designated as day 0. The drugs were administered according to the following schedule:

[0116] Table 6. Dosage and grouping N: number of animals; dosing volume: 10 μL / g; PO: oral administration. Osimertinib or osimertinib vehicle was administered first, followed by Compound A or Compound A vehicle 1 hour later.

[0117] Table 7. PDX model information

[0118] Animal husbandry:

[0119] All mice were housed in an SPF-grade animal room with an IVC constant temperature and pressure system, maintaining a temperature of 20-26°C, a humidity of 40-70%, and a 12-hour light-dark cycle. No more than five mice were housed in each cage, and the cages were littered with autoclaved corn cobs, which were changed once or twice weekly. Throughout the experiment, all mice had free access to food. Their feed was sterilized by Co60 irradiation, and their drinking water was autoclaved. Both feed and drinking water were kept plentiful. All personnel entering and exiting the animal room or performing experimental procedures were required to wear sterile lab coats, disposable medical masks, and rubber gloves. Each cage was clearly labeled with the number of animals, sex, strain, date of receipt, project number, group, current experimental phase, and the person in charge. Animals were numbered using a mouse ear punch. All animals were acclimated for three days before use.

[0120] Aseptic operation:

[0121] The test compounds will be prepared and used in a biosafety cabinet. All experimental operations, including drug administration, tumor volume and body weight measurement, will be completed in the biosafety cabinet in the animal room.

[0122] Group design:

[0123] All tumor-bearing mice had their tumor volumes and weights measured before grouping. They were then randomly divided into groups based on the measured tumor volumes. Using a randomized block design, mice were first divided into blocks based on tumor volume, and then randomly assigned within each block to treatment groups. This approach was used to reduce systematic error.

[0124] Experimental observation:

[0125] The experimental protocol and the use of experimental animals were reviewed, discussed, revised, and approved by the LIDI Biotech (IACUC) Committee. Throughout the experimental process, the use and observation of experimental animals were carried out in accordance with AAALAC guidelines. After tumor tissue inoculation, the animals were observed daily, and morbidity and mortality were recorded. During routine experimental procedures, all animals were monitored for behavior, food intake, water intake, weight changes, and other abnormalities.

[0126] Evaluation Metrics:

[0127] The main purpose is to detect the growth inhibitory effect or complete cure ability of the test drug compound A as a single drug or in combination with osimertinib on the human lung cancer PDX model LD1-0025-200729.

[0128] Tumor volume and weight of tumor-bearing mice were measured using a vernier caliper twice a week. The tumor volume was calculated using the formula V = 0.5a × b 2 , a, b represent the long diameter and short diameter of the tumor, respectively;

[0129] Tumor growth inhibition rate TGI (%) = [1-(T i -T0) / (V i -V0)]×100

[0130] T i is the average tumor volume of the compound group after the start of administration, T0 is the average tumor volume of the compound group at the first administration, V0 is the average tumor volume of the vehicle control group at the first administration, and V i The mean tumor volume of the vehicle control group after the start of drug administration.

[0131] ●Relative tumor growth rate T / C (%): The calculation formula is as follows: T / C% = T RTV / C RTV ×100%(T RTV :RTV in treatment group; C RTV : RTV of negative control group). Relative tumor volume (RTV) was calculated based on the results of tumor measurement. The calculation formula is RTV=V t / V0, where V0 is the average tumor volume measured at the time of group administration (i.e., d0), V t is the average tumor volume at a certain measurement, T RTV with C RTV Get data for the same day.

[0132] ● The weight of all tumor-bearing mice was measured once a day at the same time within a 2-hour window. The weight gain ratio of mice after drug administration was calculated: RCBW (%) = (BW i –BW0) / BW0×100, BW i BW0 is the body weight after the start of drug administration, and BW1 is the body weight at the first drug administration.

[0133] ●After the experiment, weigh the tumor mass and take photos.

[0134] Experiment termination:

[0135] When some tumor-bearing mice became extremely emaciated and on the verge of death or their tumor volume reached 2500 mm 3 Tumor-bearing mice will be euthanized in advance.

[0136] Safety evaluation of the test drug:

[0137] During the experiment, if the treated mice continue to lose weight and their RCBW reaches 15% or above, the dosage will be adjusted. The specific adjustment will depend on the condition of the tumor-bearing mice and will be determined after consultation. Simultaneously, the behavior, fur, and other abnormalities of the tumor-bearing mice during treatment will be observed and recorded, and the project leader will be promptly informed. If the RCBW reaches 20% or above, the drug treatment will be discontinued for observation until the mice recover and the RCBW is <10%, at which point dosing will be resumed.

[0138] Experimental data analysis:

[0139] All data are expressed as mean ± SEM and analyzed using one-way ANOVA using Graphpad10. *P < 0.05 was considered statistically significant. The experimental results are shown in Table 8.

[0140] Table 8 Tumor volume data statistics

[0141] In a 56-day in vivo efficacy study in the osimertinib-resistant PDXNU / NU mouse model of non-small cell lung cancer, detailed changes in tumor volume during dosing in each group are shown in Table 8 and Figure 3. The tumor growth inhibition rate (TGI) was 43.1% in the osimertinib monotherapy group (10 mg / kg, orally, once daily); 85.8% in the Compound A monotherapy group (10 mg / kg, orally, once daily) (**P<0.01); and 104.9% in the Compound A (10 mg / kg, orally, once daily) combined with osimertinib (10 mg / kg, orally, once daily) group (***P<0.001).

[0142] The results showed that compared with the control group, the osimertinib monotherapy group had no statistically significant difference; compared with the control group, the compound A monotherapy group had a statistically significant difference; and compared with the control group, the combination of compound A and osimertinib had a statistically significant difference. Compared with the monotherapy group, the combination of compound A and osimertinib demonstrated stronger anti-tumor activity.

[0143] 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 an EGFR inhibitor compound having a second active substance, wherein, The PRMT5 inhibitor of the first active substance has the following structural formula (I) or formula (II): Among them, 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), C3-C6 cycloalkyl; R3 represents H, halogen, C1-C6 alkyl, halo(C1-C6 alkyl), halo(C1-C6 alkoxy), SF5; In formula II, R4 represents hydrogen or C1-C6 alkyl; X represents CR5 or N; Among them, R5 represents hydrogen, halogen, C1-C6 alkyl, halo(C1-C6 alkyl), hydroxy, -NH2, 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), C3-C6 cycloalkyl; R3 represents hydrogen, halogen, halo(C1-C6 alkyl), halo(C1-C6 alkoxy), SF5; R4 represents hydrogen, 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 or 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 inhibitors of the first active substance are selected from the following compounds or any combination thereof:

9. The pharmaceutical composition according to any one of claims 1-8, wherein the EGFR inhibitor of the second active substance is selected from any one of the following compounds or its pharmaceutically acceptable salts: Zorifertinib (AZD3759), Lazertinib, Nazartinib (EGF816), Rociletinib (CO1686), HM61713, Naquotinib (ASP8273), Mavelertinib (PF-06747775), Abivertinib (avitinib), Alflutinib (AST2818), Olafertinib (CX-101; RX-518), Almonertinib (aumolertinib; HS-10296), Rezivertinib (BPI-7711), Mobocertinib (TAK-788), BLU-945 (Bluprint), BLU-701 (Bluprint), BBT-176 (Bridge Biotherapeutics), TQB3804 (Chia Tai Tianqing), BPI-361175 (BeiGene), QLH11811 (Qilu), HS10375 (Hansoh), H002 (Hongyun Biotech), DAJH-1050766 (Chengdu Diao Nine Hong), BI-4020 (Boehringer Ingelheim), CH7233163 (Chugai Pharmaceutical), JBI-09-063, or any combination thereof.

10. The pharmaceutical composition according to claim 9, wherein the EGFR inhibitor of the second active substance is selected from Amivantamab (JNJ-6372) or a pharmaceutically acceptable salt thereof.

11. The pharmaceutical composition according to claim 9, wherein the EGFR inhibitor of the second active substance is selected from or a pharmaceutically acceptable salt thereof, and Amivantamab (JNJ-6372).

12. A method for treating cancer or tumor, comprising administering the pharmaceutical composition according to any one of claims 1-11 to an individual in need.

13. The method according to claim 12, wherein, The tumor or cancer is selected from: 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, cholangiocarcinoma, 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 cancer, neuroblastoma, endometrial cancer, head and neck cancer, and cervical cancer.

14. The method according to claim 13, wherein the tumor is non-small cell lung cancer.

15. The method according to claim 14, wherein the non-small cell lung cancer is osimertinib-resistant non-small cell lung cancer.

16. The method according to claim 12, wherein, The cancer is metastatic cancer.

17. The method according to claim 16, wherein, The metastatic cancer is brain metastatic cancer.

Images