Use of compound in preparation of malt1 inhibitor drug

Abstract

Provided in the present invention is the use of a compound of general formula I or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, tautomer or stereoisomer thereof acting as an MALT1 inhibitor. The MALT1 tumor inhibitor can inhibit the expression of an MALT1 gene or inactivate the function of an MALT1 protein. The compound of the general formula achieves the effect of inhibiting the expression of the MALT1 gene or inactivating the function of the MALT1 protein by means of affecting the expression of an MALT1 protein death domain. The screened small molecule compound is capable of inhibiting the RNA expression of a marker in tumor cells and delaying tumor progression in tumor model mice. In addition, the use of the compound in combination with an immune checkpoint inhibitor can produce a synergistic effect and significantly enhance the efficacy.

Description

Application of compounds in the preparation of MALT1 inhibitor drugs Technical Field

[0001] This application relates to the field of pharmaceutical technology, specifically to MALT1 inhibitor compounds, drugs containing them, and their application in the field of oncology treatment. Background Technology

[0002] MALT1 is a cysteine-dependent aspartate-specific protease that shares high homology with members of the Para-caspase family and plays a key role in the activation of the NF-κB signaling pathway.

[0003] The structure of the MALT1 protein is mainly composed of three parts: the Para-caspase domain, the death domain, and the immunoglobulin-like domain.

[0004] MALT1 can cleave multiple substrates, including CYLD, A20, and RelB, all of which are negative regulators of the NF-κB signaling pathway. Therefore, in addition to acting as a backbone in the formation of components within the NF-κB signaling pathway, MALT1 can also function as a proteolytic enzyme, participating in and activating NF-κB. Furthermore, MALT1 can induce lymphocyte growth and stimulate the transcription of the cytokine interleukin-2, participating in immune responses. Therefore, MALT1 inhibitors can effectively suppress intracellular activation of MALT1, which is of great significance for MALT1-mediated immunomodulation and lymphoma treatment.

[0005] Studies have shown that the MALT1 protein is associated with hematologic malignancies, and research on MALT1 includes:

[0006] Cornell University disclosed an aryl triazole compound (2-chloro-N-[4-[5-(3,4-dichlorophenyl)-3-(2-methoxyethoxy)-1H-1,2,4-triazol-1-yl]phenyl]acetamide (hereinafter referred to as MI-2) in patent document WO2014 / 074815, which inhibits MALT1 activity by binding to the Para-caspase fragment. Subsequently, Cornell University disclosed a series of MI-2 derivatives in patent document WO2017 / 040304. These compounds all covalently bind to the Para-caspase fragment of MALT1 and significantly inhibit the development of ABC-DLBCL tumors in in vivo and in vitro experiments.

[0007] Hatcher JM et al. disclosed a series of aryltriazole compounds (WO2014 / 074815). These compounds carry a reactive chloromethyl warhead, which is proposed to covalently bind to the catalytic C464 in the para-aspase domain of MALT1.

[0008] Helmholtz Zentrum München disclosed a series of phenothiazine compounds and their derivatives as MALT1 inhibitors in patent documents WO2013 / 017637, WO2014 / 086478 and WO2014 / 207067, including mepazine, promazine and thioridazine, demonstrating that this class of MALT1 inhibitors has good biochemical and cellular activity.

[0009] Novartis disclosed a series of pyrazolidine derivatives as MALT1 inhibitors in patent documents WO2015 / 181747 and WO2017 / 081641.

[0010] Janssen disclosed a series of trifluoromethylpyrazoles as MALT1 inhibitors in patent documents WO2019 / 243965, US2019 / 0381012 and US2019 / 0381019.

[0011] However, the aforementioned MALT1 inhibitors all target the typical active center region of MALT1, namely the Para-caspase fragment; and there are few research reports on this type of MALT1 inhibitor in solid tumors.

[0012] Furthermore, GE Healthcare disclosed a MALT1 inhibitor targeting the CARMA2,3-BCL10-MALT1 complex in patent document CN111770759A. CARMA, a membrane-associated guanylate kinase protein containing a caspase recruitment domain, belongs to the membrane-associated guanylate kinase family. The CARMA protein family has three members: CARMA1, CARMA2, and CARMA3. CARMA1 is mainly expressed in lymphatic tissues, including the spleen, thymus, and peripheral blood; CARMA2 is mainly expressed in the skin and mucous membranes, with levels in the skin being 5 times higher than in other tissues and organs; CARMA3 is widely expressed and distributed in various organs and tissues, including the heart, kidneys, and liver. Therefore, the CARMA2,3-BCL10-MALT1 complex disclosed in patent document CN111770759A only reveals its function in immune cells.

[0013] The applicant's series of studies (PCT / CN2024 / 097119) found that inhibiting the activity of the BCL10-MALT1 binding region can disrupt the formation of the CARMA3-BCL10-MALT1 complex (hereinafter referred to as the CBM complex). Based on this, the applicant discovered for the first time that the MALT1 protein structure contains an effector region related to tumor therapy (hereinafter referred to as the MALT1 tumor effector region), which plays an important role in the protein-protein interaction between MALT1 and BCL-10. This MALT1 tumor effector region is different from the catalytic active site of MALT1's para-caspase; instead, it is located in the death domain of MALT1.

[0014] The advantages of small molecule compound drugs are generally considered to include:

[0015] Oral availability: It is usually administered to patients orally, which is convenient and has high compliance;

[0016] Drug molecule permeability: Due to their smaller molecular size, drugs can more easily penetrate cell membranes and affect intracellular biological processes.

[0017] Long half-life: It stays in the body for a longer period of time, maintaining a stable therapeutic effect;

[0018] High stability: More stable than biological agents, and easier to store and transport;

[0019] Lower cost: Research and development, production, and costs are relatively low;

[0020] Mature production processes: Production processes are typically more mature and scalable.

[0021] Broad treatment scope: It can usually cover a wider range of diseases;

[0022] Long history of treatment: It has a longer period of application experience in medical history.

[0023] Therefore, more suitable MALT1 inhibitor compounds are needed, especially inhibitor compounds targeting the MALT1 death domain. Summary of the Invention

[0024] This invention addresses the problems existing in related technologies by providing the application of a compound of general formula I as a MALT1 inhibitor. This MALT1 tumor inhibitor can inhibit the expression of the MALT1 gene or inactivate the function of the MALT1 protein. The compound exerts its effect by affecting the expression of the MALT1 protein death domain, specifically by inhibiting the protein-protein interaction between MALT1 and BCL-10. Therefore, the compound of general formula I can effectively treat various solid tumors and can treat tumors that cannot be treated by immune checkpoint inhibitors. Furthermore, when used in combination with immune checkpoint inhibitors, it can have a synergistic effect, significantly enhancing the therapeutic efficacy.

[0025] Firstly,

[0026] This invention provides the use of compounds of general formula I or their pharmaceutically acceptable salts, hydrates, solvates, prodrugs, tautomers, and stereoisomers as MALT1 inhibitors.

[0027] Or the use of compounds of general formula I or their pharmaceutically acceptable salts, hydrates, solvates, prodrugs, tautomers, or stereoisomers in the preparation of MALT1 inhibitor drugs. A1—L—A2

[0028] General Formula I

[0029] Wherein, A1 and A2 are independently selected from aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, fused ring, and groups in which one or more H are substituted. When there are multiple substituents, the multiple substituents can be the same or different; L is the connecting chain.

[0030] As a preferred option for the above applications, the rings selected for A1 and A2 are preferably five-membered or six-membered rings, wherein if the ring contains heteroatoms, the heteroatoms are selected from N, O, and S.

[0031] As a preferred embodiment of the above applications, the compound of formula I or its pharmaceutically acceptable salt, hydrate, solvate, prodrug, tautomer, or stereoisomer can inhibit the expression of the MALT1 gene or inactivate the function of the MALT1 protein.

[0032] As a further preferred option for the above applications, compounds of general formula I or their pharmaceutically acceptable salts, hydrates, solvates, prodrugs, tautomers, and stereoisomers exert their effects by influencing the expression of the MALT1 protein death domain, thereby inhibiting the expression of the MALT1 gene or inactivating the function of the MALT1 protein.

[0033] As a further preferred application, the compound of formula I or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, tautomer, or stereoisomer thereof exerts its effect by inhibiting the protein-protein interaction between MALT1 and BCL-10; furthermore, the compound of formula I or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, tautomer, or stereoisomer thereof reduces or eliminates the ability of MALT1 and BCL-10 to form a complex by inhibiting the death domain of the MALT1 protein.

[0034] As a preferred application of the above, the MALT1 inhibitor has the function of treating cancer, including solid tumors; preferably malignant tumors in the lungs, pancreas, liver, digestive tract, reproductive system, etc.

[0035] Furthermore,

[0036] Lungs: such as lung cancer (including non-small cell lung cancer and small cell lung cancer); Breast: such as breast cancer (including triple-negative breast cancer); Skin: such as melanoma; Digestive tract: such as stomach cancer, colon cancer, rectal cancer; Liver: such as liver cancer, hepatoblastoma; Pancreas: such as pancreatic cancer; Reproductive system: such as ovarian cancer, endometrial cancer, cervical cancer; Head and neck: such as head and neck malignancies; Urinary system: such as urinary system malignancies, nephroblastoma; Bones and soft tissues: such as osteosarcoma, chondrosarcoma, Ewing's sarcoma; Thyroid: such as thyroid cancer; Lymphatic system: such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.

[0037] As a preferred application of the above, the cancer includes hematologic malignancies. As a more preferred application of the above, the cancer includes B-cell lymphoma. As a further preferred application of the above, the cancer includes activated B-cell-like diffuse large B-cell lymphoma.

[0038] For the preferred application described above, the applicable objects are mammals; more specifically, the applicable objects are humans.

[0039] As a preferred application of the above, the MALT1 inhibitor is administered to patients who have already received immune checkpoint inhibitor therapy, or to patients who will receive immune checkpoint inhibitor therapy, or to patients who are simultaneously receiving immune checkpoint inhibitor therapy; furthermore, the immune checkpoint inhibitor includes PD-L1, PD-L2, PD-1, CTLA-4, TIM-3, LAG-3, VISTA, or TIGIT inhibitors, preferably PD-1 inhibitors, PD-L1 inhibitors, or CTLA-4 inhibitors.

[0040] As some preferred embodiments of the present invention, the PD-1 inhibitor is selected from nivolumab, pembrolizumab, avelumab, cimiplimab, BGB-A317, sintilimab, toripalimab, camrelizumab, or tislelizumab.

[0041] As some preferred embodiments of the present invention, the PD-L1 inhibitor is selected from atezolizumab or durvalumab.

[0042] As some preferred embodiments of the present invention, the CTLA-4 inhibitor is selected from ipilimumab or tremelimumab.

[0043] As a preferred embodiment of the above-mentioned compound of general formula I, the aryl group is an aryl group with 6 ring atoms, and the heteroaryl group is a heteroaryl group with 5 or 6 ring atoms.

[0044] As a further preferred option, the heteroatom of the heteroaryl group is selected from N, O, and S.

[0045] As a further preferred embodiment, the linking chain L is selected from M1—L1—M2, wherein M1 and M2 independently include bonds, alkyl groups, alkenyl groups, imino groups, tertiary amino groups, -N=C(H)- groups, and groups in which one or more H groups are substituted; L1 includes -NH-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -NHC(S)NH-, -NHC(NH)NH-, -C(O)O-, alkylene groups, heteroalkylene groups, etc. Alkylene, -C=N-NHC(O)-, -C=NC(O)NH-, -C(O)NH-N=C-, -NHC(O)-N=C-, -NHC(NH)NH-, ring structures containing -NHC(O)NH-, -NHC(NH)NH-, -C(O)NH- or -NHC(S)NH-, and groups after any one or more H atoms of these groups have been substituted; preferably, the ring structure here is a five-membered ring or a six-membered ring. When there are multiple substituents, the multiple substituents can be the same or different.

[0046] As a preferred embodiment of the above-mentioned compound of general formula I, the compound of general formula I is selected from compound of general formula Ia A1-M1-C(O)NH-M2-A2, wherein: A1 includes a five-membered heterocycle, a five-membered heteroaryl or a six-membered aryl; A2 includes a five-membered heterocycle, a five-membered heteroaryl, a six-membered aryl or a fused ring; M1 includes a bond or an alkyl group; M2 includes a bond, an imino group, a tertiary amino group or -N=C(H)-; Optionally, any one or more H groups in A1, A2, M1, and M2 can be independently substituted by substituents, and when there are multiple substituents, the multiple substituents can be the same or different.

[0047] As a preferred embodiment of the above-mentioned compound of general formula Ia, A1 and A2 are independently selected from phenyl, Or, the structure resulting from the substitution of one or more H atoms in these rings by substituents, wherein B1, B2, B3, B4, B5, B6, B7, and B8 are independently selected from C, N, O, and S; M1 includes the structure resulting from the substitution of one or more H atoms in the bond, methyl, ethyl, propyl, or other groups by substituents; M2 includes the structure resulting from the substitution of H atoms in the bond, -N=C(H)-, or -N=C(H)-. When there are multiple substituents, the substituents can be the same or different.

[0048] As a preferred embodiment of the above-mentioned compound of general formula I, the compound of general formula I is selected from the compound of general formula Ib: A1-M1-NHC(B9)NH-M2-A2, wherein NHC(B9)NH can be replaced by a ring structure containing NHC(B9)NH;

[0049] B9 is selected from NH, O, and S;

[0050] A1, A2, M1, and M2 are consistent with compound Ia.

[0051] Preferably, the Ib compound can be selected as in:

[0052] B9 is selected from NH, O, and S. Any one or more H in the general formula Ib can be independently replaced by any substituent. When there are multiple substituents, the substituents can be the same or different.

[0053] As a preferred embodiment of the above-mentioned compound of general formula I, the compound of general formula I is selected from compound of general formula IcC A1-M1-NH-M2-A2, wherein A1, A2, M1, and M2 are consistent with those defined in compound Ia, and the H in -NH- can be substituted by any substituent.

[0054] As a preferred embodiment of the above-mentioned compound of general formula I, compound Ic may be... Any one or more H atoms can be independently replaced by any substituent. When there are multiple substituents, the substituents can be the same or different.

[0055] As a further preferred embodiment, the substituents described in any of the above paragraphs include halogens, alkyl groups, strong groups, five-membered cycloyl groups, six-membered cycloyl groups, amino groups, dialkylamino groups, alkoxy groups, alkylthio groups, nitro groups, carbonyl groups, imino groups, tertiary amino groups, ester groups, alkoxy groups, haloalkyl groups, =O, =S, =NH groups, and structures in which one or more of these substituents are replaced by other substituent groups.

[0056] Furthermore, other substituents may include alkyl, alkoxy, aryl, halogen, nitro, amino, imino, and tertiary amino groups.

[0057] The most preferred compounds of the above general formula I are selected from the following compounds:

[0058] As some preferred embodiments of the present invention, the compound of general formula I also includes derivatives of the compound of formula C15 and further substituted or replaced derivatives, which are substituted or replaced on a single benzene ring connected to a pyrimidine ring, having the general formula (C15a). R1 includes at least one of heterocyclic, cycloalkyl, alkyl, and haloalkyl groups, with or without substituents, wherein the substituents include at least one of halogen, methoxy, and alkyl groups; R1 is selected from any of the following:

[0059] Further substitutions or replacements of the derivatives of the C15 compound can also be compounds with the ester bond of the pyrimidine ring replaced, having the general formula (C15b).

[0060] Wherein, R2 is selected from

[0061] Further substituted or replaced derivatives of the C15 compound can also be compounds in which the terminal benzene ring of the non-benzene ring attached to the pyrimidine ring is substituted or replaced, with the general formula (C15c) as follows:

[0062] R3 is selected from

[0063] Further derivatives of the C15 compound, including those with substitutions or replacements, can also be products with substitutions or replacements of the pyrimidine ring. The general formula (C15d) of these derivatives is:

[0064] L2 is selected from

[0065] As some preferred embodiments of the present invention, the compound of general formula I further includes derivative compounds of any of the following C15 compounds:

[0066] In certain preferred embodiments of the invention, the compound of formula I further includes derivatives of formula C17 and further substituted or replaced derivatives of the derivatives, having the general formula (C17a). R4 is selected from

[0067] Or its general formula is (C17b). R5 is selected from

[0068] When R5 in its general formula (C17b) forms a cyclic ring with -NH-, it includes any of the following derivatives:

[0069] A drug comprising a pharmaceutically acceptable salt, hydrate, solvate, prodrug, tautomer, or stereoisomer of general formula I as described in any of the preceding paragraphs.

[0070] As some preferred embodiments of the present invention, the compound of general formula I is any one of C3, C15, and C17.

[0071] As a more preferred embodiment of the present invention, the compound of general formula I is any compound of C15 or C17.

[0072] As a further preferred embodiment of the present invention, the compound of general formula I is C15.

[0073] In the most preferred embodiment of the present invention, the compound of general formula I is a derivative of C15, and the derivative of C15 includes any compound represented by formula I”, formula II”, formula III”, formula IV”, formula V”, formula VI”, formula VII”, formula VIII”, formula IX”, formula X”, formula XI”, formula XII”, formula XIII”, formula XIV”, formula XV”, formula XVI”, formula XVII”, formula XVIII”, formula XIX”, formula XX”, formula XXI”, formula XII”, formula XXIII”, formula XXIV”, formula XXV”, XXVI”, formula XXVII”, formula XXVIII”, formula XXIX”.

[0074] Preferably, the C17 derivative of the general formula I compound includes compounds of general formulas (C17a) and (C17b), as well as compounds C17-I and C17-II.

[0075] A drug combination comprising the compound of general formula I or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, tautomer, stereoisomer, and a second drug, for simultaneous or sequential administration;

[0076] Preferably, the compound of general formula I is C15;

[0077] More preferably, the compound of general formula I is a derivative of C15, and the derivative of C15 includes any compound represented by formula I”, formula II”, formula III”, formula IV”, formula V”, formula VI”, formula VII”, formula VIII”, formula IX”, formula X”, formula XI”, formula XII”, formula XIII”, formula XIV”, formula XV”, formula XVI”, formula XVII”, formula XVIII”, formula XIX”, formula XX”, formula XXI”, formula XXII”, formula XXIII”, formula XXIV”, formula XXV”, XXVI”, formula XXVII”, formula XXVIII”, formula XXIX”.

[0078] Preferably, the second drug comprises an immune checkpoint inhibitor.

[0079] The advantages of this invention are:

[0080] (1) By inhibiting the activity of the BCL10-MALT1 binding region, the formation process of the CARMA3-BCL10-MALT1 complex (hereinafter referred to as the CBM complex) can be disrupted. Based on this, the present invention targets the effector region in the MALT1 protein structure that is related to tumor therapy (hereinafter referred to as the MALT1 tumor effector region), which plays an important role in mediating the protein-protein interaction between MALT1 and BCL-10. This MALT1 tumor effector region is different from the catalytic active site of MALT1's para-caspase, but is located in the death domain of MALT1.

[0081] (2) The location of the MALT1 tumor effector region is far from the catalytic active site of MALT1 Para-caspase. Therefore, the MALT1 tumor inhibitor has the characteristic of non-competitive inhibition with MALT1 Para-caspase enzyme activity inhibitors. Therefore, this MALT1 tumor inhibitor can be used in combination with existing inhibitors targeting the MALT1 Para-caspase active site to achieve better therapeutic effects.

[0082] (3) The small molecule compound of this application can be administered to tumor-bearing mice by intraperitoneal injection at the Cxcl1 and Csf1 RNA levels in Malt1-WT and Malt1-KD cell lines, and has a certain inhibitory effect on the progression of 4T1 mammary tumors in mice.

[0083] (4) After administration of the small molecule compounds of this application, the expression levels of IL-6 and IL-10 in the OCI-Ly3 cell line and / or TMD-8 cell line can be affected. In mouse tumor models, they can effectively delay tumor progression without affecting mouse weight. Attached Figure Description

[0084] Figure 1 illustrates the experimental principle of the compounds of the present invention inhibiting the interaction between MALT1 and BCL10;

[0085] Figure 2 illustrates the determination of the IC50 of some compounds of the present invention. 50 Gradient dilution experimental curves of the values;

[0086] Figure 3 shows the results of small molecule inhibition efficiency detection in vivo and in vitro.

[0087] Figure 4 shows the detection of small molecule functional activity in solid tumors;

[0088] Figure 5 illustrates the role of small molecule inhibitors in the ABC-DLBCL cell line;

[0089] Figure 6 shows the inhibitory efficiency and intracellular activity of the C15 derivative. Detailed Implementation

[0090] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below. However, it should be understood that the description herein is merely illustrative and not intended to limit the scope of the invention.

[0091] Unless otherwise stated, the following definitions are used throughout this specification and the claims. These definitions are identical whether the term is used alone or in combination with other terms. Thus, the definition of "alkyl" can be used for "alkyl" and the "alkyl" portion of "alkoxy," etc. When any variant in any construction appears more than once, its definition in each place is independent of the definitions in other places; that is, the above choices are independent of each other. Moreover, those skilled in the art can combine substituents and / or variants, as long as such combination yields a stable compound.

[0092] "Effective dose" refers to a dose that is effective in treating a disease, such as a dose that provides a clinical response to the disease being treated.

[0093] In this invention, the notation "Cx-Cy", "xy carbon atoms", or "x to y carbon atoms" represents a range of carbon atoms, where x and y are integers. For example, C3-C8 cycloalkyl represents a cycloalkyl group with 3-8 carbon atoms, and C0-C2 alkyl represents an alkyl group with 0-2 carbon atoms, where C0 alkyl refers to a single chemical bond. Therefore, the number of carbon atoms in the groups defined below in this invention can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, depending on the circumstances.

[0094] Similarly, "xy ring atoms" and "x to y ring atoms" represent the range of the number of atoms in the ring system, where x and y are both integers. Therefore, the number of ring atoms in the groups defined below in this invention can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, depending on the circumstances.

[0095] “Bn” represents benzyl.

[0096] The “pharmaceutically acceptable salts” described in this invention are discussed in Berge, et al., “Pharmaceutically Acceptable Salts”, J. Pharm. Sci., 66, 1-19 (1977), and are obvious to medicinal chemists that the salts are substantially nontoxic and provide the desired pharmacokinetic properties, palatability, absorption, distribution, metabolism, or excretion. Typical salts include, but are not limited to, the following: acetates, adipates, alginates, citrates, aspartates, benzoates, benzenesulfonates, hydrogen sulfates, butates, camphorates, camphorsulfonates, digluconate, cyclopentanepropionate, dodecyl sulfates, ethanesulfonates, glucono-p-ethylhexanoates, glycerol phosphates, hemisulfates, heptahydrates, hexanoates, fumarates, hydrochlorides, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactates, maleates, methanesulfonates, nicotinate, 2-naphthalenesulfonate, oxalates, pyruvate, pectinates, persulfates, 3-phenylpropionate, picrates, pentanoates, propionates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, and undecanoates. Pharmaceutically acceptable salts of this invention can be synthesized by conventional chemical methods.

[0097] Generally, salts can be prepared by reacting a free base or acid with an equistoichiometric or excess amount of an acid (inorganic or organic) or base in a suitable solvent or solvent combination.

[0098] "Solvate" refers to the physical association of the compound of the present invention with one or more solvent molecules. This physical association involves varying degrees of bonding, including hydrogen bonding. In some cases, the solvate can be separated. The term "solvate" is intended to cover both solution-phase solvates and separable solvates.

[0099] A "hydrate" is a compound that is bound to water. Typically, it is determined by the ratio of the number of water molecules contained in the hydrate to the number of molecules of the compound in the hydrate. Therefore, a hydrate of a compound can be represented, for example, by the general formula R·xH₂O, 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, e.g., hemihydrates (R·0.5H₂O)), and polyhydrates (x is a number greater than 1, e.g., dihydrates (R·2H₂O) and hexahydrates (R·6H₂O)).

[0100] "Heteroatoms" refers to elements other than carbon, such as nitrogen, oxygen, sulfur, phosphorus, or selenium, preferably nitrogen, oxygen, or sulfur.

[0101] "alkyl" refers to a straight-chain or branched saturated hydrocarbon group having a specified number of carbon atoms. When the number of carbon atoms is not specified, it refers to 1 to 20 carbon atoms. Preferably, the alkyl chain contains 1 to 12 carbon atoms. More preferably, the alkyl chain contains 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

[0102] "Alkenyl" refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond, and can be straight-chain or branched. When the number of carbon atoms is not specified, it indicates 2 to 20 carbon atoms. Preferably, the alkenyl chain has 2 to 12 carbon atoms, and more preferably 2 to 6 carbon atoms. Non-limiting examples of applicable alkenyl groups include vinyl, propenyl, n-butenyl, 3-methylbut-2-enyl, n-pentenyl, octenyl, and decenyl. Alkenylalkyl means an alkenyl group connected to the stated portion via an alkyl group.

[0103] "Alynyl" means an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond and can be straight-chain or branched. When the number of carbon atoms is not specified, it indicates 2 to 15 carbon atoms. Preferably, the alkynyl chain has 2 to 12 carbon atoms, and more preferably 2 to 4 carbon atoms. Non-limiting examples of applicable alkynyl groups include ethynyl, propynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl, and decanynyl. Alynyl group means an alkynyl group connected to the stated portion via an alkyl group.

[0104] "Alkoxy" means alkyl-oxy group, where the alkyl group has the meaning described above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, and n-butoxy.

[0105] "alkyl carbonyloxy" refers to an alkyl-C(O)O group, where the alkyl group has the meaning described above. Preferably, the alkyl carbonyloxy group is a C1-C10 alkyl carbonyloxy group; more preferably, the alkyl carbonyloxy group is a C1-C6 alkyl carbonyloxy group; even more preferably, the alkyl carbonyloxy group is a C1-C4 alkyl carbonyloxy group; non-limiting examples include formyloxy and acetoxy groups.

[0106] "Alkoxycarbonyl" refers to an alkyl-OC(O)- group, where the alkyl group has the meaning described above. Preferably, the alkoxycarbonyl group is a C1-C10 alkoxycarbonyl group; more preferably, the alkoxycarbonyl group is a C1-C6 alkoxycarbonyl group; even more preferably, the alkoxycarbonyl group is a C1-C4 alkoxycarbonyl group; non-limiting examples include methoxycarbonyl and ethoxycarbonyl.

[0107] "alkyl carbonylamino" refers to an alkyl-C(O)NH- group, where the alkyl group has the meaning described above. Preferably, the alkyl carbonylamino group is a C1-C10 alkyl carbonylamino group; more preferably, the alkyl carbonylamino group is a C1-C6 alkyl carbonylamino group; even more preferably, the alkyl carbonylamino group is a C1-C4 alkyl carbonylamino group; non-limiting examples include formamide and acetamido.

[0108] The term "alkylaminocarbonyl" refers to an alkyl-NHC(O)- group, where the alkyl group has the meaning described above. Preferably, the alkylaminocarbonyl group is a C1-C10 alkylaminocarbonyl group; more preferably, the alkylaminocarbonyl group is a C1-C6 alkylaminocarbonyl group; even more preferably, the alkylaminocarbonyl group is a C1-C4 alkylaminocarbonyl group; non-limiting examples include methylaminocarbonyl, ethylaminocarbonyl, butylaminocarbonyl, and tert-butylaminocarbonyl.

[0109] "Aryl" refers to an aromatic monocyclic or polycyclic system comprising 6 to 14 carbon atoms, preferably 6 to 10 carbon atoms. Non-limiting examples of applicable aryl groups include phenyl, naphthyl, indene, tetrahydronaphthyl, indanyl, anthracene, fluorenyl, etc.

[0110] "Arylalkyl" means aryl-alkyl, where both the aryl group and the alkyl group are as defined above. Non-limiting examples of applicable arylalkyl groups include benzyl (i.e., benzyl), phenethyl, and diphenylmethyl. It is formed by the alkyl group being bonded to its paired group.

[0111] "Cycloalkyl" refers to a non-aromatic ring system having one to three rings with 3 to 10 carbon atoms, preferably 5 to 10 carbon atoms. Preferred cycloalkyl rings contain 5 to 7 ring atoms. Non-limiting examples of cycloalkyl include cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, orthobornyl, adamantyl, etc.

[0112] "Cycloalkylalkyl" means cycloalkyl and alkyl as defined above, where the alkyl group is bonded to a paired group via an alkyl group. Non-limiting examples include cyclopropylmethyl, cyclohexylmethyl, etc.

[0113] "Cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic system comprising 3 to 10 carbon atoms, preferably 5 to 10 carbon atoms, and containing at least one carbon-carbon double bond. Preferred cycloalkenyl systems contain 5 to 7 ring atoms. Non-limiting examples of cycloalkenyl systems include cyclopentenyl, cyclohexenyl, cycloheptenyl, and protobornenyl.

[0114] "Cycloalkyl" means that all atoms that make up the ring structure are carbon atoms.

[0115] "Heterocyclic alkyl" means that "cycloalkyl" contains heteroatoms.

[0116] "Cycloalkenyl" means that all atoms that make up the ring structure are carbon atoms, and the ring contains at least one double bond.

[0117] "Heterocyclic alkenyl" means that "cyclic alkenyl" contains heteroatoms.

[0118] "Circular rings" refers to rings formed by two adjacent rings sharing two adjacent atoms, such as naphthalene. wait.

[0119] "Tertiary amino" means that all the H atoms in the amino group are replaced by substituents, which can be of any structure.

[0120] "Halogen" refers to fluorine, chloro, bromine, or iodine groups. Fluorine, chloro, or bromine groups are preferred, and fluorine and chloro groups are more preferred.

[0121] "Halogen" refers to fluorine, chlorine, bromine, or iodine. Fluorine, chlorine, or bromine are preferred, and fluorine and chlorine are more preferred.

[0122] "Haloalkyl" means an alkyl group as defined above, wherein one or more hydrogen atoms on the alkyl group are substituted with a halogen group as defined above. Non-limiting examples include chloromethyl, fluoromethyl, trifluoromethyl, etc.

[0123] "Heterocyclic group" or "heterocyclic system" means a non-aromatic saturated monocyclic or polycyclic system comprising 3 to 10 ring atoms, preferably 5 to 10 ring atoms, wherein one or more atoms in the ring system are heteroatoms. The ring system does not contain adjacent oxygen and / or sulfur atoms. Preferred heterocyclic groups contain 5 to 6 ring atoms. The prefixes of the heterocyclic group name (aza, oxa, or thioza) respectively indicate that it contains at least one nitrogen, oxygen, or sulfur atom as a ring atom. The nitrogen or sulfur atom of the heterocyclic group may be oxidized, as appropriate, to the corresponding N-oxide, S-oxide, or S,S-dioxide. Non-limiting examples of suitable monocyclic heterocyclic groups include piperidinyl, pyrrolyl, piperazine, morpholinyl, thiomorpholinyl, thiazolyl, 1,3-dioxolane, 1,4-dioxane, tetrahydrofuranyl, tetrahydrothiophene, tetrahydrothioranyl, etc.

[0124] The term "heterocyclic acidic functional group" will include groups such as pyrrole, imidazole, triazole, and tetraazole.

[0125] "Heteroaryl" refers to an aromatic monocyclic or polycyclic system comprising 5 to 14 ring atoms, preferably 5 to 10 ring atoms, wherein one or more of the ring atoms are heteroatoms. Preferred heteroaryls contain 5 to 6 ring atoms. The prefixes of the heteroaryl name (aza, oxa, or thia) indicate that it contains at least a nitrogen, oxygen, or sulfur atom as a ring atom, respectively. The nitrogen atom of the heteroaryl group may be oxidized to the corresponding N-oxide, depending on the specific case. Non-limiting examples of applicable heteroaryl groups include furanyl, thiophene, pyrroloyl, thiazolyl, pyrazolyl, oxazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyranyl, pyrazinyl, isoxazolyl, isothiazolyl, triazolyl, 1,2,4-thiadiazolyl, tetrazolyl, quinoxalinyl, 2,3-diazanaphthyl, imidazo[1,2-a]pyridinyl, imidazo[2,1-b]thiazolyl, benzofuranyl, indoleyl, azaindoleyl, benzimidazolyl, benzothiophene, quinolinyl, isoquinolinyl, indoleyl, purine, thiophenepyridinyl, quinazolinyl, thiophenepyrimidinyl, pyrrolopyridinyl, imidazopyridinyl, benzoazaindoleyl, 1,2,4-triazinyl, benzothiazolyl, etc.

[0126] "Heteroarylene alkyl" means that both heteroarylene and alkyl are heteroarylene-alkyl as defined above, bonded to their paired groups via alkyl groups.

[0127] "alkylene" refers to a straight-chain or branched saturated alkylene group with a specified number of carbon atoms, having two terminal monovalent groups, and can be considered as a product of removing two hydrogen atoms from an alkane. When the number of carbon atoms is not specified, it refers to 1 to 20 carbon atoms. Preferably, the alkylene contains 1 to 12 carbon atoms. More preferably, the alkylene contains 1 to 6 carbon atoms. Non-limiting examples include methylene, ethylene, propylene, etc.

[0128] "Alkenyl" refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond in its chain, which can be straight-chain or branched, and has two terminal monovalent groups. It can be considered as a product of removing two hydrogen atoms from an alkene. When the number of carbon atoms is not specified, it refers to 2 to 20 carbon atoms. Preferably, the alkenyl group contains 2 to 12 carbon atoms. More preferably, the alkenyl group contains 2 to 6 carbon atoms. Non-limiting examples include vinylidene, propenylidene, etc.

[0129] "Alynyl group" refers to an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond in its chain, which can be straight-chain or branched, and has two terminal monovalent groups. It can be considered as a product of removing two hydrogen atoms from an alkyne. When the number of carbon atoms is not specified, it refers to 2 to 20 carbon atoms. Preferably, the alynyl group contains 2 to 12 carbon atoms. More preferably, the alynyl group contains 2 to 6 carbon atoms. Non-limiting examples include ethynylene, propynylene, etc.

[0130] "Heteroalkylene" refers to an alkylene group that has heteroatoms in its chain.

[0131] "Substituted" or "substituted" means that one or more hydrogen atoms are replaced by a monovalent or divalent group. The groups described in this article may be substituted or unsubstituted. Suitable substituents include, for example, hydroxyl, nitro, amino, imino, cyano, halogen, thiogroup, sulfonyl, thioamido, amido, imidino, oxo, aminooxime, methoxamido, imidino, guanidino, sulfonylamino, carboxyl, formyl, alkyl, haloalkyl, alkylamino, haloalkylamino, alkoxy, haloalkoxy, alkoxyalkyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroarylalkyl, carbonyl, alkylthio, aminoalkyl, cyanoalkyl, aryl, heteroaryl, etc., provided that the oxo, imidino, or other divalent substituents are not located on the aryl or heteroaryl ring, due to the well-known valence restrictions of such rings. Preferred substituents are halogens, alkyl groups, aryl groups, heteroaryl groups, alkylamino groups, dialkylamino groups, alkoxy groups, alkylthio groups, nitro groups, alkylcarbonyl groups, and haloalkyl groups; more preferably, they are fluorine, chlorine, bromine, methyl, ethyl, phenyl, dimethylamino, methylamino, methoxy, ethoxy, nitro, chloromethyl, fluoromethyl, trifluoromethyl, methylcarbonyl, ethylcarbonyl, furanyl, thiophene, pyrrole, thiazolyl, pyrazolyl, oxazolyl, imidazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyranyl, pyrazinyl, isoxazolyl, isothiazolyl, triazolyl, 1,2,4-thiadiazolyl, and tetrazolyl.

[0132] N-oxides can be formed on the tertiary nitrogen of a compound, or on the =N- of a heteroaryl substituent, and are included in compounds of general formula I.

[0133] As used herein, the term “composition” will include a product comprising a specific amount of a specific ingredient and any product formed directly or indirectly by combining a specific amount of the specific ingredient.

[0134] The “NCBI Gene ID” and “NCBI NP” are provided by the National Center for Biotechnology Information (NCBI), whose website is https: / / www.ncbi.nlm.nih.gov / .

[0135] The MALT1 gene (NCBI Gene ID: 10892) encodes a caspase-like protease that plays a role in BCL10-induced NF-κB activation. This protein is a component of the CARMA3-BCL10-MALT1 (CBM) signaling matrix, which triggers NF-κB signaling and lymphocyte activation upon antigen-receptor stimulation. MALT1 can refer to human MALT1, including its naturally occurring variants, molecules, and alleles. MALT1 can also refer to MALT1 in mammals such as mice, rats, rabbits, dogs, cats, cattle, horses, and pigs.

[0136] The "CARMA3-BCL10-MALT1 signaling complex" refers to a three-molecule protein complex composed of protein 3 (CARMA3) containing CARD and membrane-associated guanylate kinase-like domains, B-cell lymphoma / leukemia factor 10 (BCL10), and mucosa-associated lymphoid tissue lymphoma translocation protein 1, namely CARMA3, BCL10, and MALT1. Following antigen-receptor junction and signal cascade transduction, CARMA3 is released from its self-inhibitory conformation and binds to BCL10 and MALT1 to form the functional CARMA2,3-BCL10-MALT1 complex.

[0137] "Inhibition of the activity of the CARMA2,3-BCL10-MALT1 complex" refers to the formation, function, or expression level of the CARMA2,3-BCL10-MALT1 complex. In some embodiments, the activity is the level of formation of the CARMA2,3-BCL10-MALT1 complex. In some embodiments, to inhibit the activity of the CARMA2,3-BCL10-MALT1 complex, the subject needs to be given a MALT1 tumor inhibitor that inhibits the activity of the CARMA2,3-BCL10-MALT1 complex.

[0138] In some embodiments, the MALT1 tumor inhibitor can inhibit the activity of the CARMA2,3-BCL10-MALT1 complex by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or higher compared to a control level. A control level can be, for example, the activity of the CARMA2,3-BCL10-MALT1 complex before administration of the MALT1 tumor inhibitor, or the activity of the CARMA2,3-BCL10-MALT1 complex in cells that have not been treated (e.g., exposed) with the MALT1 tumor inhibitor.

[0139] In some embodiments, the MALT1 tumor inhibitor inhibits the activity of the CARMA2,3-BCL10-MALT1 complex in T cells or inhibits the activity of the CARMA2,3-BCL10-MALT1 complex in tumor cells. In some embodiments, the tumor cells are tumor-invasive tumor cells. In some embodiments, as measured by the amount of intact complex in cells, the MALT1 tumor inhibitor reduces the amount of intact CARMA2,3-BCL10-MALT1 complex by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or more compared to a control level.

[0140] In some embodiments, the MALT1 tumor inhibitor of this application inhibits the formation of a complex. The MALT1 tumor inhibitor inhibits the formation of the complex by binding to and inhibiting binding sites (such as the MALT1 binding site on BCL10) contained within the complex that are required for higher-order assembly. In some embodiments, the MALT1 tumor inhibitor can inhibit the release of CARMA3 from its self-inhibitory conformation. In some embodiments, the MALT1 tumor inhibitor inhibits upstream factors required for the formation of the CARMA2,3-BCL10-MALT1 complex.

[0141] In some embodiments, the MALT1 tumor inhibitor can reduce the expression level of at least one component of the CARMA2,3-BCL10-MALT1 complex by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or higher compared to a control level. The control level can be, for example, the expression level of at least one component (e.g., CARMA3, BCL10, or MALT1) prior to administration of the MALT1 tumor inhibitor. Those skilled in the art can use, for example, co-immunoprecipitation or sucrose gradient analysis to evaluate the integrity of the complex to determine whether the MALT1 tumor inhibitor effectively inhibits the formation of the CARMA2,3-BCL10-MALT1 complex. For a given component, those skilled in the art can perform PCR-based analysis or Western blotting to evaluate mRNA or protein levels, respectively, to determine whether the level of at least one component in the complex is reduced.

[0142] In some embodiments, the activity is the function of the CARMA2,3-BCL10-MALT1 complex. In some embodiments, the MALT1 tumor inhibitor inhibits the CARMA2,3-BCL10-MALT1 complex from activating its downstream targets (e.g., NF-κB nuclear translocation and activation). In some embodiments, the MALT1 tumor inhibitor reduces the expression level of the CARMA2,3-BCL10-MALT1 complex by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or higher compared to a control level. The control level may be, for example, the level of the CARMA2,3-BCL10-MALT1 complex prior to administration of the MALT1 tumor inhibitor.

[0143] In some embodiments, the MALT1 tumor inhibitor reduces the expression level of at least one gene selected from the CARMA3, BCL10, or MALT1 genes by 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or more compared to a control level. The control level may be, for example, the expression level of the CARMA3, BCL10, or MALT1 genes prior to administration of the MALT1 tumor inhibitor. Methods known in the art can be used to determine whether the MALT1 tumor inhibitor reduces the expression level of genes (e.g., by PCR-based assays) or gene products (e.g., by Western blotting).

[0144] In this article, "MALT1 inhibitor" refers to an active inhibitor targeting the Para-caspase domain. It is known that the compositional activity of the MALT1 protease drives the survival and proliferation of various lymphocytic malignancies. Therefore, MALT1 inhibitors can effectively inhibit tumor survival and growth.

[0145] "Effective amount" refers to the amount of agent required to alleviate at least one symptom of a tumor (e.g., headache). Therefore, the term "therapeutic effective amount" refers to the amount of agent sufficient to provide a specific anticancer effect when administered to a typical subject. The effective amount used in this application will also include the amount of agent sufficient to delay the development of tumor symptoms, alter the course of tumor symptoms (e.g., but not limited to slowing tumor progression), or reverse tumor symptoms. Therefore, specifying a precise "effective amount" is generally impractical. However, for any given situation, an appropriate "effective amount" can be determined by a person skilled in the art using only routine experiments. Effective amounts, toxicity, and therapeutic efficacy can be evaluated using standard pharmaceutical procedures in cell culture or laboratory animals. Dosage can vary depending on the dosage form used and the route of administration employed. The dose ratio between toxic effects and therapeutic effects is the therapeutic index and can be expressed as a proportional LD50. 50 / ED 50 Compositions and methods exhibiting a high therapeutic index are preferred.

[0146] The "therapeutic effective dose" can be initially estimated from cell culture assays. Furthermore, the dose can be formulated in animal models to achieve the IC50 values ​​determined in cell culture or appropriate animal models. 50 The range of circulating plasma concentrations. Plasma levels can be measured, for example, by high-performance liquid chromatography. The effect of any particular dose can be monitored by appropriate bioassays (e.g., non-invasive imaging). The dose can be determined by a physician and adjusted as necessary to suit the observed therapeutic effect.

[0147] "Statistically significant" or "significantly" means statistical significance and typically refers to a difference of 2 standard deviations (2SD) or greater. Unless otherwise indicated in the operational examples or elsewhere, all figures used herein to represent the amount of an ingredient or reaction conditions should be understood to be modified in all cases by the term "about".

[0148] "Effective Treatment / Efficacy": The efficacy of the MALT1 tumor inhibitors described in this application, which inhibit the activity of the CARMA3-BCL10-MALT1 complex, in treating, for example, the conditions described in this application (e.g., solid tumors) or inducing the responses described in this application (e.g., tumor size reduction), can be determined by a skilled clinician. However, if treatment according to the methods described in this application results in a beneficial change in one or more signs or symptoms of the condition described in this application, improvement or even relief of other clinically acceptable symptoms, or induction of at least 10% of the desired response, then the treatment is considered "effective treatment" as used in this application. Efficacy can be evaluated, for example, by measuring biomarkers, indicators, symptoms, and / or the incidence of the condition treated according to the methods described in this application, or any other suitable measurable parameter. Treatment according to the methods described in this application can reduce the levels of symptoms or biomarkers of the condition by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or higher. The efficacy described in this application can also be measured by the failure of individual deterioration or the cessation of medical intervention (i.e., cessation of disease progression) as evaluated by hospitalization. Methods for measuring these indicators are known to those skilled in the art and / or described in this application.

[0149] When used with a percentage, “approximately” can represent ±1%.

[0150] The word "includes / contains / contains" indicates that other elements may exist in addition to the defined element. The use of "includes / contains / contains" indicates inclusion rather than limitation.

[0151] "Composed of" means the compositions, methods and their respective components as described in this application, excluding any elements not listed in the description of this embodiment.

[0152] "Basically composed of..." refers to the elements required for a given implementation. This term allows for the presence of additional elements that do not substantially affect the basic, novel, or functional characteristics of the implementation of the technology.

[0153] "Relief of tumor symptoms" refers to the improvement of any condition or symptom associated with a tumor. Such a reduction, measured using any standard technique known to those skilled in the art, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%, or more, compared to an equivalent untreated control. Various methods for administering the reagents described in this application to subjects are known to those skilled in the art.

[0154] To further understand the present invention, the present invention will be described in detail below with reference to the preferred embodiments.

[0155] Example 1

[0156] Experimental principle:

[0157] Luciferase (Luc) was split into its N-terminus (LucN) and C-terminus (LucC), which were then linked to MALT1 and BCL10, respectively, to construct a luciferase complementary pair (Split-Luc system). In addition, a complete luciferase system (Linked-Luc system) composed of directly linked short peptides was constructed as a control to normalize signal intensity. In the experiments, if a small molecule compound could block the binding of MALT1 and BCL10, the fluorescence signal would be significantly reduced after the addition of the luciferin substrate because the luciferase complementary pair could not recombine to form an active enzyme. Conversely, if a small molecule compound directly inhibited luciferase activity, both the Split-Luc and Linked-Luc system signals would be significantly reduced, and such compounds were excluded. Therefore, only small molecule compounds that specifically inhibit the Split-Luc system without significantly affecting the Linked-Luc system were considered effective inhibitors of the MALT1-BCL10 interaction.

[0158] The specific principle is shown in Figure 1.

[0159] Specific methods:

[0160] 1. Constructing expression carriers

[0161] First, the nanoluciferase (NLuc) was split into its N-terminus (amino acids 1-159) and C-terminus (amino acids 160-170). The N-terminus (amino acids 1-159) of NLuc was linked to the N-terminus (amino acids 1-337) of MALT1 via a linker sequence (Linker amino acid sequence: GSSGGGGSGGGGSGGGGS). Similarly, the C-terminus (amino acids 160-170) of NLuc was linked to the full-length BCL10 via a linker. This resulted in the construction of two recombinant protein expression sequences: NLucN-Linker-MALT1 and NLucC-Linker-BCL10.

[0162] 2. Cloning into the vector

[0163] The nucleic acid sequences encoding NLucN-Linker-MALT1 and NLucC-Linker-BCL10 were cloned into the KpnI and NotI sites of the pCDNA3.1 vector, respectively. Specifically, the pCDNA3.1 vector and the nucleic acid sequences of NLucN-Linker-MALT1 and NLucC-Linker-BCL10 were digested using KpnI and NotI enzyme digestion reactions, and then the two were ligated to construct the expression plasmids pCDNA3.1-NLucN-Linker-MALT1 and pCDNA3.1-NLucC-Linker-BCL10.

[0164] 3. Transfection of 293F cells

[0165] The two constructed plasmids (pCDNA3.1-NLucN-Linker-MALT1 and pCDNA3.1-NLucC-Linker-BCL10) were transfected into 293F cells using PEI transfection reagent. The cells were then cultured in suspension. After transfection, the cells were cultured for another 96 hours to ensure adequate expression of the exogenous gene.

[0166] 4. Cell lysis and preparation of lysis buffer

[0167] Ninety-six hours after transfection, cells were collected and centrifuged. Cell lysis buffer (2 mM EDTA, 20 mM DTT, 10% glycerol, 1% Triton X-100, 25 mM Tris-base, pH 7.8) was added, and cells were lysed at 4°C for 30 minutes. The lysis buffer was designed to maximize protein extraction and maintain its stability. Cell debris was then removed by high-speed centrifugation (12,000 rpm, 10 minutes), and the supernatant was collected.

[0168] 5. Complex formation

[0169] The two lysis buffers (supernatants containing NLucN-Linker-MALT1 and NLucC-Linker-BCL10) were mixed together and reacted at 37°C for 30 minutes. This step promotes the formation of a complex between MALT1 and BCL10, with the linker-linked portion facilitating the interaction between the two.

[0170] 6. Follow-up Analysis

[0171] After the reaction, the formation and binding efficiency of the complex were detected using a luciferase activity assay. The intensity of the fluorescence signal was used to assess the interaction between MALT1 and BCL10.

[0172] 7. Screening of small molecule compounds

[0173] Based on the activity screening platform of the Tsinghua University Pharmaceutical Technology Center, two libraries containing 150,000 small molecule compounds were selected from the drug-like compound library. These compounds underwent screening for physicochemical properties and druggability, including molecular weight, hydrogen bond donors and acceptors, and lipid-water partition coefficient. Each compound was concentrated at 10 mM and dissolved in DMSO. Using an Echo 650 ultrasonic pipette, 50 nL of the compound solution was transferred to each well. Then, using a dispenser, 50 μL of cell lysis buffer containing split-luc or linked-luc protein with luciferin substrate was added to each well. After reacting for 30 minutes, the fluorescence signal was measured using a microplate reader.

[0174] 8. Preliminary screening and identification of active compounds

[0175] Calculate the fluorescence signal change rate (%) for each sample based on the changes in fluorescence signal:

[0176] Fluorescence signal change rate (%) for each sample = (average signal of the whole plate - signal of the well) / average signal of the whole plate × 100%.

[0177] Compound inhibition rate (%) = Inhibition rate of Split-Luc - Inhibition rate of Linked-Luc

[0178] Small molecule compounds with an inhibition rate of not less than 20% were screened out.

[0179] 9. Concentration-dependent inhibition rate assessment

[0180] The concentration-dependent inhibition rates of the initially screened active compounds were evaluated. Different volumes of small molecule compound solutions were transferred to each well using an Echo 650 ultrasonic pipette, ensuring final concentrations of 200, 100, 50, 37.5, 25, 20, 17.5, 12.5, 7.5, 5, and 2.5 μM for each compound. Then, 50 μL of cell lysis buffer containing Split-Luc or Linked-Luc protein and a luciferin substrate was added using a dispenser. After incubation at room temperature for half an hour, fluorescence signals were detected using a microplate reader, and the inhibition rates at different concentrations were calculated.

[0181] 10. Half-maximal inhibitory concentration (IC50) 50 )calculate

[0182] The half-maximal inhibitory concentration (IC50) of each compound was calculated using the "log(inhibitor) vs. response(three parameters)" model in the Nonlinear regression (curve fitting) formula of GraphPad Prism 9. 50 The values ​​are shown in Table 1 below.

[0183] Table 1 Experimental Results:

[0184] A gradient dilution experiment was performed on the compound, and its IC50 value was determined by plotting the dilution curve. 50 Values. The curves for some compounds are shown in Figure 2.

[0185] It is evident that the compounds in this application can target the MALT1-BCL10 interaction and act as small molecule inhibitors.

[0186] Example 2 Synthesis of compound C15

[0187] When R1 in compound 1 is phenyl, the general formula (C15a) after the reaction is compound C15, and the structural formula is...

[0188] Procedure: In a dry round-bottom flask, add starting compound 1 (1.0 equiv), compound 2 (1.0 equiv), compound 3 (thiourea) (3.0 equiv), and catalyst ZrOCl2·8H2O (0.2 equiv) sequentially. Under solvent-free conditions, heat the mixture to 80-100 °C and stir magnetically for 4-6 hours. The reaction progress can be monitored by thin-layer chromatography (TLC) (e.g., using petroleum ether / ethyl acetate = 3:1 as the developing solvent). After the reaction is complete, cool the mixture to room temperature to obtain a solid or viscous substance. Add ice water (approximately 20 mL) to the reaction mixture and stir for 30 minutes to precipitate the product. Filter to collect the solid, wash with water (3 × 10 mL) to remove residual catalyst and byproducts. Recrystallize the crude product from ethanol (e.g., dissolve in hot ethanol and cool to crystallize) to obtain a pure C15 compound as a white or pale yellow solid. Yield: approximately 70-80%, melting point: approximately 150-152 °C. 1H NMR(400MHz,DMSO-d6)δ10.43(d,J=1.8Hz,1H),9.70(dd,J=3.9,1.8Hz,1H),7.49–7.26(m,12H),7 .08–7.00(m,2H),5.22(d,J=3.8Hz,1H),5.11(s,2H),3.74(q,J=7.1Hz,2H),0.74(t,J=7.1Hz,3H).

[0189] Example 3: Synthesis of Derivative II of Compound C15

[0190] The preparation method of "Compound II" is the same as in Example 2, except that R1 in Compound 1 of the reactants is different, wherein when R1 is At the same time, with all other reaction conditions being the same, the derivative II of compound C15 was prepared as follows: Sample name B-82-b(C15-4), molecular weight 480.53; 1H NMR (400MHz, DMSO-d6) δ 10.60 (d, J=1.8Hz, 1H), 9.73 (dd, J=3.7, 1.9Hz, 1H), 7.49–7.37 (m, 5H), 7.36–7.30 (m, 2H), 7.30–7.26 (m, 2H), 7.13 (td, J=8.5, 2.5Hz, 1H), 7.08–7.01 (m, 2H), 5.24 (d, J=3.7Hz, 1H), 5.11 (s, 2H), 3.79 (q, J=7.1Hz, 2H), 0.81 (t, J=7.1Hz, 3H).

[0191] Example 4: Synthesis of Derivative III of Compound C15

[0192] The preparation method of "Compound III" is the same as in Example 2, except that R1 in Compound 1 of the reactants is different, wherein when R1 is At the same time, with all other reaction conditions being the same, the derivative III of compound C15 prepared was Sample name B-83-a(C15-5), molecular weight 478.99; 1H NMR (400MHz, DMSO-d6) δ 10.58 (d, J = 4.5Hz, 1H), 9.68 (dt, J = 23.0, 2.7Hz, 1H), 7.55–7.28 (m, 11H), 7.04 (dd, J = 8.5, 5.9Hz, 2H), 5.27 (d, J = 3.6Hz, 1H), 5.12 (d, J = 3.7Hz, 2H), 3.83–3.63 (m, 2H), 0.71 (td, J = 7.1, 3.2Hz, 3H).

[0193] Example 5: Synthesis of Derivative IV of Compound C15

[0194] The preparation method of "Compound IV" is the same as in Example 2, except that R1 in Compound 1 of the reaction raw materials is different, wherein when R1 is When all other reaction conditions are the same, the derivative IV of compound C15 prepared is Sample name B-83-a(C15-6), molecular weight 462.54; ¹H NMR (400MHz, DMSO-d6) δ 10.47 (d, J=1.8Hz, 1H), 9.71 (dd, J=4.0, 1.9Hz, 1H), 7.49–7.29 (m, 7H), 7.33–7.24 (m, 2H), 7.27–7.18 (m, 2H), 7.08–7.00 (m, 2H), 5.22 (d, J=3.8Hz, 1H), 5.11 (s, 2H), 3.77 (q, J=7.1Hz, 2H), 0.78 (t, J=7.1Hz, 3H).

[0195] Example 6: Synthesis of Derivative VII of Compound C15

[0196] The preparation method of compound VII” is the same as in Example 2, except that R1 in compound 1 of the reactants is different, wherein when R1 is When all other reaction conditions are the same, the derivative VII” of compound C15 is prepared as follows: Sample name 85-b(△C15-9), molecular weight 408.52; 1 H NMR(400MHz,DMSO-d6)δ9.69(d,J=4.1Hz,1H),9.14(s,1H),7.45–7.31(m,5H) ,7.12(d,J=8.3Hz,2H),6.99(d,J=8.3Hz,2H),5.13(d,J=4.0Hz,1H),5.08(s, 2H),4.05(q,J=7.1Hz,2H),2.85(td,J=8.9,4.5Hz,1H),1.21(dq,J=10.4,5.0 Hz,1H),1.13(t,J=7.1Hz,3H),0.96–0.83(m,2H),0.79(tt,J=6.7,2.6Hz,1H).

[0197] Example 7 Synthesis of Derivative VIII of Compound C15

[0198] The preparation method of compound VIII is the same as in Example 2, except that R1 in compound 1 of the reactants is different, wherein when R1 is At the same time, with all other reaction conditions being the same, the derivative VIII of compound C15 was prepared as follows: Sample name 86-a(-CF3, C15-10), molecular weight 454.46. 1H NMR (400MHz, DMSO-d6) δ9.04(s,1H),8.92(s,1H),7.85(s,1H),7.39(ddd,J=25.3,17.6,7.3Hz,6H),7.24(d,J=8.2Hz,2H),6. 99(d,J=8.2Hz,2H),5.12(s,2H),4.73(d,J=11.8Hz,1H),3.81(q,J=7.1Hz,2H),3.01(d,J=11.9Hz,1H),0.83(t,J=7.1Hz,3H).

[0199] Example 8: Synthesis of Derivative IX of Compound C15

[0200] The preparation method of "Compound IX" is the same as in Example 2, except that R1 in Compound 1 of the reactants is different, wherein when R1 is When all other reaction conditions are the same, the derivative IX of compound C15 prepared is Sample name: 86-b(-CH3, C15-11), molecular weight: 382.48; 1 H NMR (400MHz, DMSO-d6) δ7.64–7.28(m,7H),7.07(d,J=8.3Hz,2H),6.90(d,J=8.3Hz,2H ),5.24(s,1H),5.05(s,2H),4.03(p,J=6.8Hz,2H),2.37(s,3H),1.14(t,J=7.1Hz,3H).

[0201] Example 9: Synthesis of Derivative X of Compound C15

[0202] The preparation method of compound X is the same as in Example 2, except that R1 in compound 1 of the reaction raw materials is different, wherein when R1 is When all other reaction conditions are the same, the derivative X” of compound C15 is prepared as follows: Sample name: 86-c(-CH3, C15-12), molecular weight: 445.54; 1H NMR (400MHz, DMSO-d6) δ10.61(s,1H),9.76(t,J=2.4Hz,1H),8.65–8.59(m,2H),7.46(d,J=7.5Hz,2H),7.40(t,J=7.4Hz,2H),7.35(q,J=4.3 ,3.0Hz,3H),7.29(d,J=8.2Hz,2H),7.04(d,J=8.2Hz,2H),5.23(d,J=3.6Hz,1H),5.12(s,2H),3.76(q,J=7.0Hz,2H),0.74(t,J=7.1Hz,3H).

[0203] Example 10 Synthesis of Derivative XI of Compound C15

[0204] The preparation method of compound XI is the same as in Example 2, except that R1 in compound 1 of the reaction raw materials is different, wherein when R1 is When all other reaction conditions are the same, the derivative XI of compound C15 prepared is Sample name: 86-d(-Ph-O-, C15-13), molecular weight: 474.57; 1 H NMR (400MHz, DMSO-d6) δ10.36(s,1H),9.74–9.69(m,1H),7.45(d,J=7.5Hz,2H),7.40(t,J=7.4Hz,2H),7.34(d,J=7.1Hz,1H),7.26(dd,J=11.4,8. 5Hz,4H),7.03(d,J=8.2Hz,2H),6.95(d,J=8.3Hz,2H),5.19(d,J=3.9Hz, 1H), 5.11 (s, 2H), 3.80 (s, 4H), 3.84–3.75 (m, 1H), 0.82 (t, J = 7.1Hz, 3H).

[0205] Example 11 Synthesis of Derivative XII of Compound C15

[0206] The preparation method of compound XII is the same as in Example 2, except that R1 in compound 1 of the reactants is different, wherein when R1 is When all other reaction conditions are the same, the derivative XII of compound C15 prepared is Sample name: 87-a(-thiophene, C15-14), molecular weight: 450.57; 1H NMR (400MHz, DMSO-d6) δ10.51(s,1H),9.76(t,J=2.6Hz,1H),7.72(d,J=5.0Hz,1H),7.45(d,J=7.5Hz,2H),7.40(t,J=7.3Hz,2H),7.34(d,J=7.1Hz,1 H),7.29–7.22(m,3H),7.10(t,J=4.4Hz,1H),7.04(d,J=8.3Hz,2H),5.18( d,J=3.9Hz,1H),5.11(s,2H),3.86(q,J=7.1Hz,2H),0.89(t,J=7.1Hz,3H).

[0207] Example 12 Synthesis of Derivative XIV of Compound C15

[0208] The preparation method of compound XIV is the same as in Example 2, except that R1 in compound 1 of the reaction raw materials is different, wherein when R1 is When all other reaction conditions are the same, the derivative XIV of compound C15 prepared is Sample name 90-b(C15-16), molecular weight 452.57; 1 H NMR(400MHz, DMSO-d6)δ9.79(s,1H),9.63(d,J=3.9Hz,1H),7.46–7.28(m,5H),7.12(d,J=8.2Hz,2H),6.98(d,J=8.2Hz,2H),5.12(d,J=3.9Hz,1H), 5.08(s,2H),4.03(q,J=6.8Hz,3H),3.93(d,J=11.2Hz,2H),3.35(s,1H),3 .29(s,0H),2.22–1.97(m,2H),1.40(d,J=12.2Hz,2H),1.16–1.08(m,3H).

[0209] Example 13 Synthesis of Derivative XV of Compound C15

[0210] The preparation method of compound XV is the same as in Example 2, except that R1 in compound 1 of the reaction raw materials is different, wherein when R1 is When all other reaction conditions are the same, the derivative XV” of compound C15 is prepared as follows: Sample name 91-c(C15-17), molecular weight 450.60; 1H NMR(400MHz,DMSO-d6)δ9.68(d,J=1.8Hz,1H),9.58(dd,J=4.0,1.7Hz,1H),7.47–7.36( m,4H),7.36–7.28(m,1H),7.16–7.08(m,2H),7.02–6.94(m,2H),5.11(d,J=3.8Hz,1H),5 .08(s,2H),4.01(q,J=7.1Hz,2H),3.83–3.72(m,1H),1.89(q,J=13.1,12.2Hz,1H),1.7 8(s,3H),1.60(s,1H),1.49(d,J=11.7Hz,2H),1.33–1.20(m,3H),1.11(t,J=7.1Hz,3H).

[0211] Example 14 Synthesis of Derivative XVI of Compound C15

[0212] Preparation steps: Weigh 0.445 g (1.0 mmol) of compound C15 into a dry 50 mL round-bottom flask. Measure a mixture of methanol and water (volume ratio 3:1, total volume 20 mL, e.g., 15 mL methanol and 5 mL water) and add it to the round-bottom flask. Stir until C15 is completely dissolved or suspended. Weigh 0.067 g (1.2 mmol) of potassium hydroxide and carefully add it to the reaction mixture, stirring until completely dissolved. Connect the round-bottom flask to a condenser and place it on a heated magnetic stirrer. Heat the reaction mixture to 80 °C (temperature controlled by an oil bath or heating mantle) and maintain gentle reflux (solvent boiling) for 12 hours. Stir continuously during this period. Every 2-3 hours, take a small amount of the reaction mixture (approximately 0.1 mL) and analyze it by TLC. Dichloromethane / methanol (10:1, v / v) is recommended as the developing solvent. The Rf value of C15 (ester) is relatively high (approximately 0.6-0.7), while the Rf value of compound XVI (carboxylic acid) is relatively low (approximately 0.1-0.2, possibly remaining at the origin). The reaction is complete when the C15 spot has essentially disappeared. After the reaction is complete, the reaction mixture is cooled to room temperature. Most of the methanol is removed by rotary evaporation under reduced pressure (concentrating to approximately 1 / 3 of the original volume), yielding a solution. The concentrated solution is poured into a 50 mL beaker, and 1M methanol is slowly added dropwise while cooling in an ice bath. Add HCl solution dropwise while stirring until the pH reaches 3-4 (test with pH paper). At this point, the carboxylic acid will precipitate. Filter the precipitate through a Buchner funnel and wash with ice water (3 × 10 mL) to remove excess salt and impurities. Recrystallize the crude product from hot ethanol: Dissolve the solid in a minimum volume of hot ethanol (approximately 10-15 mL), heat until completely dissolved, then slowly cool to room temperature to precipitate crystals. Collect the crystals by filtration, wash with cold ethanol, and dry to obtain product XVI”, yield: 0.35-0.40 g (yield 80-90%). 1H NMR (400MHz, DMSO-d6) δ11.87 (s, 1H), 10.30 (s, 1H), 9.66 (d, J = 3.4Hz, 1H), 7 .49–7.26(m,12H),7.04(d,J=8.3Hz,2H),5.22(d,J=3.9Hz,1H),5.11(s,2H).

[0213] Example 15 Synthesis of “XXIV”, a derivative of compound C15

[0214] When R in compound 4 is a methylene group, R2 is... At that time, the C15 derivative of the compound with the general formula (C15b) after the reaction has the following structural formula:

[0215] Preparation steps: In a 25 mL round-bottom flask, dissolve compound XXIV (0.416 g, 1.0 mmol) in 10 mL of dry DMF and stir until completely dissolved. Add HATU (0.456 g, 1.2 mmol) and DIPEA (0.258 g, 2.0 mmol) and stir at room temperature to activate the carboxylic acid for 10–15 minutes. The mixture may become slightly turbid or remain clear. Add compound 4, amine R-NH2 (1.2 mmol), and continue stirring at room temperature for 2 hours. Take a small amount of the reaction solution (about 0.1 mL) and analyze by TLC. The developing solvent is recommended to be dichloromethane / methanol (10:1, v / v). Compound 4 has a low Rf value (about 0.1–0.2), while the amide product C15b / C15b-1 has a high Rf value. The value is relatively high (approximately 0.5-0.7, depending on the R group). The reaction is complete when spot 4 of compound 4 has essentially disappeared. Pour the reaction mixture into ice water (50 mL) and stir for 10 minutes to precipitate the amide. If the product does not precipitate, it can be extracted with ethyl acetate (3 × 20 mL). If a precipitate forms, filter through a Buchner funnel, wash with water (3 × 10 mL), and dry. If extraction is used, combine the organic phases, wash with saturated brine (20 mL), dry to anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to obtain the crude product. The crude product can be purified by column chromatography (silica gel, eluent dichloromethane / methanol gradient, from 100:1 to 10:1, v / v) or by recrystallization (e.g., using ethanol or ethyl acetate / n-hexane) to obtain the pure compound XXIV” in 70-90% yield. 1H NMR (400MHz, DMSO-d6) δ9.93 (s, 1H), 9.36 (d, J = 2.7Hz, 1H), 7.48–7.26 (m, 12H), 6.99 (dd, J = 1 4.0,7.2Hz,3H),5.14(d,J=3.4Hz,1H),5.11(s,2H),2.87–2.67(m,2H),0.59(t,J=7.2Hz,3H).

[0216] Example 16 Synthesis of “XXV”, a derivative of compound C15

[0217] The preparation method of compound XXV is the same as in Example 15, except that R in reactant compound 4 is different, wherein when R is When all other reaction conditions are the same, R2 is The prepared compound C15 derivative "XXV" is 1H NMR (400MHz, DMSO-d6) δ9.98 (s, 1H), 9.42 (s, 1H), 7.48–7.26 (m, 13H), 7.01 (d, J = 8.4Hz, 2H), 6.67 (t, J = 5.5Hz, 1H), 5.16 (d, J = 3.5Hz, 1H), 5.10 (s, 2H), 2.90 (q, J = 6.7Hz, 2H), 2.17 (d, J = 27.2Hz, 10H), 1.93 (t, J = 6.6Hz, 2H).

[0218] Example 17 Synthesis of “XXVI”, a derivative of compound C15

[0219] The preparation method of compound XXVI is the same as in Example 15, except that R in reactant compound 4 is different, wherein when R is When all other reaction conditions are the same, R2 is The prepared compound C15 derivative "XXVI" is 1 H NMR (400MHz, DMSO-d6) δ10.02(d,J=1.8Hz,1H),9.48(dd,J=3.5,1.7Hz,1H),7.52(t,J=5.8Hz,1H),7.47–7.28(m,12H),7. 05–6.98(m,2H),5.19(d,J=3.4Hz,1H),5.11(s,2H),3.21–3.04(m,J=6.8,6.1Hz,2H),2.74(t,J=6.5Hz,2H),2.55(s,6H).

[0220] Example 18 Synthesis of derivative "XXVII" of compound C15

[0221] The preparation method of compound XXVII” is the same as that in Example 17, except that the reactant is compound XIV” prepared in Example 12. All other reaction conditions are the same. The resulting derivative of compound C15, XXVII”, is… 1H NMR (400MHz, DMSO-d6) δ9.56 (s, 1H), 9.44 (s, 1H), 7.98 (t, J = 5.7Hz, 1H), 7.47–7.36 (m, 4H),7.34(d,J=7.0Hz,1H),7.15(d,J=8.2Hz,2H),6.99(d,J=8.3Hz,2H),5.13(d,J=3.4H z,1H),5.09(s,2H),3.90(d,J=10.1Hz,2H),3.28(q,J=10.4,8.2Hz,6H),2.93(t,J=6.8 Hz,2H),2.64(s,5H),2.15–1.94(m,2H),1.50(d,J=12.3Hz,1H),1.33(d,J=12.3Hz,1H).

[0222] Example 19 Synthesis of “XXVIII”, a derivative of compound C15

[0223] The preparation method of compound XXVIII is the same as that in Example 15, except that the reactant is compound XIV prepared in Example 12. All other reaction conditions are the same. The resulting derivative of compound C15, XXVIII, is... 1 H NMR(400MHz, DMSO-d6)δ9.39(s,1H),9.25(d,J=3.2Hz,1H),7.83(t,J=5.7Hz,1H),7.47–7.28(m,5 H),7.13(d,J=8.2Hz,2H),6.98(d,J=8.2Hz,2H),5.11(d,J=3.1Hz,1H),5.09(s,2H),3.93–3.85(m, 2H),3.25(t,J=11.5Hz,2H),3.18–3.03(m,2H),3.00(dq,J=13.4,6.7Hz,1H),2.70(s,1H),2.00(dd d,J=41.1,12.2,4.0Hz,1H),1.52(d,J=12.3Hz,1H),1.31(d,J=12.6Hz,1H),0.93(t,J=7.2Hz,3H).

[0224] Example 20 Synthesis of “XXIX”, a derivative of compound C15

[0225] The preparation method of "compound XXX" is the same as that in Example 16, except that the reactant is the same as that used in Example 12 to prepare compound XIV". All other reaction conditions are the same. The resulting derivative of compound C15, "XXX", is... 1H NMR (400MHz, DMSO-d6) δ9.45 (s, 1H), 9.29 (t, J = 2.3Hz, 1H), 7.66 (t, J = 5.5Hz, 1H), 7.5 0–7.29(m,5H),7.15(d,J=8.4Hz,2H),6.98(d,J=8.4Hz,2H),5.09(d,J=3.9Hz,3H),3.8 9(dd,J=10.9,4.1Hz,2H),3.30–3.16(m,4H),3.07(dt,J=13.6,6.3Hz,1H),2.32(d,J=3 3.5Hz,8H),2.21(s,3H),2.14–1.91(m,3H),1.50(d,J=12.3Hz,1H),1.37–1.22(m,2H).

[0226] Example 21 Synthesis of Derivative VI of Compound C15

[0227] The preparation method of compound VI” is the same as in Example 2, except that the selection of compound 3 in the reaction raw materials is different. All other reaction conditions are the same. The derivative VI” of compound C15 prepared is Sample name: B-84-b(C15-8), molecular weight: 428.49; 1 H NMR(400MHz, DMSO-d6)δ9.26(s,1H),7.79(d,J=3.1Hz,1H),7.49–7.27(m,12H),7.02(d,J=8 .2Hz,2H),5.18(d,J=3.4Hz,1H),5.11(s,2H),3.71(q,J=7.1Hz,2H),0.72(t,J=7.1Hz,3H).

[0228] Example 22 Synthesis of Derivative XVII of Compound C15

[0229] The preparation method of compound XVII' is the same as in Example 2, except that the selection of compound 3 in the reaction raw materials is different; all other reaction conditions are the same. Compound 3 is... When R6 is -CN, the derivative XVII” of compound C15 prepared is

[0230] Example 23 Synthesis of Derivative XVIII of Compound C15

[0231] The preparation method of compound XVIII is the same as in Example 22, except that the choice of compound 3 in the reaction raw materials is different; all other reaction conditions are the same. Compound 3 is... When R6 is -CH3, the derivative XVIII of compound C15 prepared is... 1H NMR (400MHz, DMSO-d6) δ9.21 (s, 1H), 7.48–7.29 (m, 10H), 7.26 (d, J = 8.2Hz, 2H), 7.00 (d, J = 8. 3Hz,2H),5.36(s,1H),5.09(s,2H),3.76(q,J=7.1Hz,2H),2.36(s,3H),0.77(t,J=7.1Hz,3H).

[0232] Example 24 Synthesis of Derivative XIX of Compound C15

[0233] The preparation method of compound XIX is the same as in Example 22, except that the selection of compound 3 in the reaction raw materials is different. All other reaction conditions are the same. Compound 3 is... R6 is At that time, the derivative of compound C15, "XIX", was prepared as...

[0234] Example 25 Synthesis of Derivative V” of Compound C15

[0235] When R3 in compound 6 is The general formula (C15c) after the reaction is compound V”, and the structural formula is... Sample name B-84-a(C15-7), molecular weight 498.52.

[0236] Preparation steps: In a dry 50 mL round-bottom flask, add the following ingredients sequentially: thiourea (3.0 mmol, 0.228 g), compound 5 (1.1 mmol), compound 6 (1.0 mmol), and zirconium oxychloride octahydrate (ZrOCl2·8H2O, 0.2 mmol, 0.064 g), along with 10 mL of anhydrous ethanol as solvent. Connect the round-bottom flask to a reflux condenser. Place the reaction apparatus in a heating mantle, turn on magnetic stirring, and heat to reflux (ethanol boiling point approximately 78 °C). Maintain vigorous reflux for 24 to 72 hours. Reaction monitoring: Monitor the reaction progress using TLC. Take samples every 12 hours, using petroleum ether / ethyl acetate (e.g., 3:1, v / v) as the developing solvent. The reaction is considered complete when the spot of the starting aldehyde (compound 6) essentially disappears. After the reaction is complete, cool the reaction mixture to room temperature. A solid product usually precipitates at this point. Further cool the mixture in an ice-water bath for 1 hour to allow complete precipitation of the product. The solid product was collected by suction filtration using a Buchner funnel. The solid was washed with cold anhydrous ethanol (approximately 2 × 5 mL) to remove residual catalyst and soluble impurities. The resulting crude product usually had high purity. The crude product was dissolved in hot ethanol (or ethyl acetate), filtered while hot, and then slowly cooled to room temperature or an ice bath to precipitate crystals again, which were then collected by filtration. The purified solid was placed in a vacuum drying oven and dried at 40-50 °C for 6-8 hours to obtain the final product V". Yield: 60%-85%. 1H NMR (400MHz, DMSO-d6) δ10.63–10.58(m,1H),9.74(s,1H),7.50(dd,J=8.4,5.4Hz,2H),7.42(q,J=8.0Hz,1H),7.30(d,J=8.2Hz,3H),7.22( t,J=8.8Hz,2H),7.18–7.09(m,1H),7.04(d,J=8.3Hz,2H),5.26(d,J=3.7Hz,1H),5.10(s,2H),3.79(q,J=7.1Hz,2H),0.81(t,J=7.1Hz,3H).

[0237] Example 26 Synthesis of Derivative XIII of Compound C15

[0238] The preparation method of "compound XIII" is the same as that in Example 25, except that R3 in compound 6 of the reactants is different, wherein when R3 is When all other reaction conditions are the same, the derivative XIII of compound C15 prepared is Sample name B-82-b(C15-4), molecular weight 480.53; 1H NMR (400MHz, DMSO-d6) δ10.40–10.35(m,1H),9.65(dd,J=4.0,1.9Hz,1H),9.46(s,1H),7.48–7.35(m,3H),7.31(dq,J=6. 4,2.2Hz,2H),7.21–7.13(m,2H),6.81–6.73(m,2H),5.16(d,J=3.8Hz,1H),3.74(q,J=7.1Hz,2H),0.74(t,J=7.1Hz,3H).

[0239] Example 27 Synthesis of Derivative XX of Compound C15

[0240] When R7 in compound 10 is -CH3, the general formula (C15d) after the reaction is "compound XX", and its structural formula is:

[0241] Preparation steps: Step 1: In a dry round-bottom flask, dissolve compound 5 (1.0 mmol) and compound 2 (1.0 mmol) in anhydrous dichloromethane (DCM) or N,N-dimethylformamide (DMF). Add a coupling agent such as HATU (1.2 mmol) and a base such as DIPEA (2.0 mmol). Stir the reaction at room temperature for 2–12 hours, monitoring the reaction progress by TLC. After the reaction is complete, quench with water, extract with DCM, wash the organic phase with brine, dry (Na2SO4), and concentrate. Purification: Compound 7 is obtained by column chromatography.

[0242] Step 2: Dissolve compounds 7 (1.0 mmol) and 8 (1.0 mmol) in ethanol or acetonitrile. Add a Lewis acid such as ZnCl2 or Br2 as a catalyst. Heat under reflux for 2–6 hours, monitored by TLC. After cooling, concentrate the reaction solution, add water to precipitate the solid, filter, and dry. Purify by recrystallization or column chromatography to obtain compound 9.

[0243] Step 3: Dissolve compound 9 (1.0 mmol) in DCM, add compound 10 (5 mL), and stir at 0°C to room temperature for 1–4 hours. Neutralize with NaHCO3, extract, dry, and concentrate to obtain the thiol intermediate. Dissolve the thiol intermediate in methanol, expose to air or add iodine (catalytic amount), and stir overnight. After concentration and purification, obtain C15d.

[0244] Example 28 Synthesis of “XXI”, a derivative of compound C15

[0245] The preparation method of compound XXI is the same as in Example 27, except that the choice of compound 10 in the reaction raw materials is different. All other reaction conditions are the same. In compound 10, R7 is... At that time, the derivative of compound C15, "XXI", was prepared as

[0246] Example 29 Synthesis of “XXII”, a derivative of compound C15

[0247] Preparation steps: In a dry 25 mL round-bottom flask, add compound CL5 (1.0 mmol) and anhydrous potassium carbonate (2.0 mmol). Evacuate the flask and purge with an inert gas (such as nitrogen or argon), repeating this process 2-3 times to remove air and moisture. Under an inert gas atmosphere, add anhydrous DMF (10 mL) via syringe. While stirring, slowly add 1,2-dibromoethane (5.0 mmol, ~0.40 mL) via syringe. Seal the reaction apparatus and place it in a preheated oil or metal bath, heating to 60-80 °C. At this temperature, stir vigorously for 12-24 hours. Reaction monitoring: Monitor the reaction progress using TLC. Take samples every few hours, using petroleum ether / ethyl acetate (e.g., 2:1 or 1:1, v / v) as the developing solvent. Under UV light, the reaction is complete when the spots of the CL5 starting material significantly weaken or disappear, and a new spot with slightly lower polarity (XXII” product) appears. After the reaction is complete, cool the mixture to room temperature. Slowly pour the reaction solution into an ice-water mixture (about 50 mL). Note: This process may be exothermic and must be carried out in an ice bath. At this time, solids or oily substances may precipitate.

[0248] The aqueous phase was repeatedly extracted with ethyl acetate (3 × 30 mL). All organic phases were combined. The organic phases were washed successively with cold water (2 × 20 mL) and saturated brine (20 mL) to remove DMF and residual salts. The organic phases were dried over anhydrous sodium sulfate for 15–20 minutes. The mixture was filtered, and the residue was washed with a small amount of ethyl acetate. The filtrate was concentrated under reduced pressure using a rotary evaporator to obtain the crude product. The crude product may be a yellow or brown oil or solid. Since product XXII” contains bromine atoms and may contain trace amounts of dibromoethane and dialkylation byproducts, it must be purified by column chromatography. A silica gel column was used, with a gradient elution of petroleum ether and ethyl acetate (e.g., gradually increasing from 100% petroleum ether to 20%–30% ethyl acetate). The fraction rich in the target product (XXII”) was tracked and collected by TLC. All fractions containing the target product were combined and concentrated using a rotary evaporator to obtain a purified white to off-white solid or a pale yellow oil XXII”. Yield: 50%–75%.

[0249] Example 30 Synthesis of “XXIII”, a derivative of compound C15

[0250] When compounds R8 in At that time, the derivative of compound C15, "XXIII", was prepared as... The preparation steps are as follows: In a dry 50 mL round-bottom flask, add compound C15 (1.0 mmol), bromide R8-Br (1.2 mmol), and anhydrous potassium carbonate (2.0 mmol). Connect the flask to a reflux condenser. Purge the system with an inert gas (such as nitrogen or argon) through the top of the condenser for a short time to purge air. (This step is optional, but it prevents oxidation of some sensitive compounds and promotes the reaction). Under an inert gas atmosphere, add anhydrous acetonitrile (10-15 mL) using a syringe or directly, ensuring the solid is fully submerged and dispersed. Turn on the stirrer, place the reaction apparatus in a heating mantle, and heat to reflux (acetonitrile's boiling point is approximately 82°C). Maintain a gentle reflux condition for 6-12 hours. Reaction monitoring: Monitor the reaction progress using TLC. Take samples every 2-3 hours, using petroleum ether / ethyl acetate (e.g., 2:1, v / v) as the developing solvent. Under UV light, the reaction is complete when the spot of the C15 starting material (usually with a low Rf value) significantly diminishes or disappears, and a new spot with lower polarity (XXIII” product) appears. After the reaction, the mixture is cooled to room temperature. Insoluble inorganic salts (such as potassium carbonate and the generated potassium bromide) are removed by filtration or centrifugation. The filter residue or precipitate is washed with a small amount of acetonitrile, and the filtrate and washings are combined. The combined organic phases are concentrated under reduced pressure using a rotary evaporator to remove most of the acetonitrile, yielding the crude product. The crude product usually needs to be purified by column chromatography to separate unreacted C15, excess R8-Br, and possible polyalkylation byproducts. A silica gel column is used with a gradient elution of petroleum ether and ethyl acetate (e.g., from 100% petroleum ether to 30%-50% ethyl acetate). The fraction rich in the target product (XXIII”) is tracked and collected by TLC. All fractions containing the target product are combined and concentrated using a rotary evaporator to give XXIII”, a purified white or off-white solid, in yields of 60%-85%.

[0251] Example 31 Synthesis of Derivative I of Compound C15

[0252] The preparation method of compound I is the same as that of Example 25, except that R3 in compound 6 of the reactants is different, wherein when R3 is When all other reaction conditions are the same, the derivative XIII of compound C15 prepared is Sample name B-82-a(C15-3), molecular weight 352.45;

[0253] Example 32 Synthesis of compound C17

[0254] When R in the compound is At that time, through C17(a), we can identify compound C17, with the following structural formula:

[0255] Preparation process: Step 1: Compound 11 (bromoaromatic heterocycle) and Compound 12 (diamino compound) are reacted in ethanol under reflux with sodium bicarbonate as a base. One amino group (possibly an aliphatic amine) of Compound 12 replaces the bromine to generate Compound 13.

[0256] Step 2: React compounds 13 and 14 under suitable conditions (may not require additional conditions, or may require base catalysis). The other amino group in compound 13 (possibly an aromatic amine or another amino group) attacks the acyl chloride or halocarbonyl group in compound 14, forming an amide bond or an amine bond, to generate compound C17a or compound C17. 1 H NMR(400MHz,DMSO-d6)δ12.54(s,1H),7.65–7.58(m,2H),7.51(d,J=5.9Hz,3H),3.25 (d,J=7.6Hz,4H),2.61(s,3H),2.12(dt,J=13.8,6.9Hz,2H),0.88(d,J=6.6Hz,12H).

[0257] Example 33

[0258] During the initial screening process, the applicant completed the screening of drug-like compound libraries provided by the active screening platform of the Center for Pharmaceutical Technology at Tsinghua University. This involved screening 150,000 small molecule compounds that had already undergone drug-likeness screening in terms of physicochemical properties and structures, such as molecular weight, hydrogen bond donors and acceptors, and lipid-water partition coefficient.

[0259] Ultimately, the applicant obtained 17 small molecule compounds with inhibition rates of not less than 20%, including 12 compounds listed in Table 1. For these compounds, the applicant conducted IC50 assays at the extracellular protein lysate level (Figure 3A) and assays of the intracellular small molecule inhibition efficiency of MDA-MB231 and MDA-MB468 stably expressed Split-Luc systems (Figure 3B). The results showed that C3, C15, and C17 exhibited good performance.

[0260] Furthermore, based on previous findings that MALT1 inhibits T cell function by upregulating M2 macrophage polarization through CXCL1, CSF1, and PTGES, the applicant treated 4T1 and E0771 mouse breast cancer tumor cell lines with 50 μM of the compounds, respectively, and compared the Cxcl1 and Csf1 RNA levels in the Malt1-WT and Malt1-KD cell lines treated with the small molecules (Figures 4A-C). In both cell analyses, compounds C15 and C17 showed strong intracellular inhibitory effects. In addition, CCK-8 cell viability assays showed that neither C15 nor C17 exhibited detectable cytotoxicity at concentrations up to 80 μM (Figures 4D-E). Intraperitoneal injection in tumor-bearing mice also revealed that C15 and C17 had a certain inhibitory effect on the progression of 4T1 breast tumors in mice (Figure 4F). These findings indicate that C15 and C17 are potential small molecule inhibitor lead compounds, and further optimization of their metabolic stability and water solubility will be conducted based on their structures.

[0261] Furthermore, considering the important role of the CARD11-BCL10-MALT1 (CBM) complex in B-cell lymphomas, particularly activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL), it is important to understand that in ABC-DLBCL, the CBM complex and its downstream NF-κB pathway are continuously activated, inhibiting apoptosis and enhancing the tumor cells' resistance to chemotherapy. 1 Although the rituximab-based first-line immunochemotherapy regimen R-CHOP can achieve long-term survival for some patients, the prognosis of the ABC-DLBCL subtype remains worse than that of the germinal center B-cell-like (GCB) subtype. 1,2 Currently available targeted therapies, such as BTK inhibitors (e.g., ibrutinib), are effective for MCD, but some patients still experience rapid relapse due to resistance mutations (e.g., CARD11 mutations). 1 Therefore, the applicant also wants to explore the potential efficacy of small molecule inhibitors targeting the BCL10 and MALT1 interaction sites in ABC-DLBCL.

[0262] The OCI-Ly3 cell line contains a CARD11 mutation, and the TMD-8 cell line contains a CD79B mutation in the BCR-binding linker. In these two ABC-DLBCL cell lines, the BCR-CBM signaling axis is continuously activated. 3 IL-6 and IL-10 are NF-κB-induced cytokines that promote the proliferation and survival of malignant B cells through autocrine and paracrine mechanisms, and are one of the factors contributing to the poor prognosis of ABC-DLBCL. 4The applicant found that the small molecule inhibitor C15 reduced IL-6 and IL-10 expression levels in OCI-Ly3 and TMD-8 in a dose-dependent manner, while C17 only showed good performance in OCI-Ly3 (Figures 5A-D). In NOD / ShiLtJGpt-Prkdc em26Cd52 Il2rg em26Cd22 In Gpt(NCG) mice, 2.5 × 10⁶ Gpt (NCG) mice were subcutaneously injected. 6 A tumor model was established using individual cells, and daily intraperitoneal administration of a C15 small molecule inhibitor at a dose of 50 mg / kg effectively delayed tumor progression without affecting the body weight of the mice (Figure 5E-G).

[0263] Considering the poor metabolic stability and water solubility of small molecule compounds C15 and C17, the applicant is currently modifying the structure of these small molecule compounds (see Table 2 for details). The structural formulas of these C15 derivatives are shown in Table 2.

[0264] The IC50 values ​​of the C15 compound derivatives in the Split-Luc screening system are shown in Figures 6A-C. Their IL-6 downregulation ability in the ABC-DLBCL cell line is shown in Figures 6D-E, and their effect on cell viability is shown in Figure 6F.

[0265] Example 34: RNA expression levels after treatment of cancer cell lines with various small molecule inhibitors of the present invention

[0266] 1. Experimental materials and reagents

[0267] Cell lines: mouse breast cancer cell line 4T1, melanoma cell line (E0771), and B-cell lymphoma cell line (OCI-Ly3).

[0268] Compounds: C15, C15 derivatives, C17, and C17 derivatives; C15 derivatives include compounds II”, V”, X”, XII”, XVI”, XVII”, XIX”, XX”, XXIII”; C17 derivatives include C17-I and C17-II. All compound stock solutions were prepared with DMSO.

[0269] Control group: The same amount of DMSO as the treatment group.

[0270] Culture medium: RPMI-1640 or DMEM complete medium (containing 10% fetal bovine serum FBS and 1% penicillin-streptomycin).

[0271] Main reagents: RNA extraction kit (such as TRIzol reagent).

[0272] Nuclease-free water.

[0273] Reverse transcription kit (containing reverse transcriptase, random primers, dNTPs, etc.).

[0274] qPCR premix (such as SYBR Green qPCR Master Mix).

[0275] Specific primers for the target gene (Cxcl1, Csf1) and the internal reference gene (Gapdh or Actb).

[0276] 2. Experimental Procedure

[0277] Cell lines in the logarithmic growth phase were digested with trypsin and resuspended in complete culture medium. Cell counting was performed, and the cell density was adjusted to an appropriate concentration (e.g., 1 × 10⁻⁶). 5 (cells / mL). Seed the cell suspension into 24-well or 6-well plates, adding a certain volume to each well to ensure a uniform cell count (e.g., 5 × 10⁴ cells per well in a 24-well plate). Incubate the cell culture plates at 37°C and 5% CO₂ for approximately 24 hours to allow the cells to adhere fully and enter the logarithmic growth phase (cell density reaching approximately 70%-80%).

[0278] Compound treatment:

[0279] Prepare working solutions of the compounds: Dilute the stock solutions of each compound (and DMSO) with complete culture medium to prepare a series of working solutions of different concentrations. Ensure that the final concentration of DMSO in all working solutions is consistent (e.g., ≤0.1%) and below the toxicity threshold. The final working concentration used in Table 2-4 is 20 μM.

[0280] According to the experimental groups, fresh complete culture medium containing different compounds was added to the corresponding cell wells.

[0281] Control group: Add complete culture medium containing an equal amount of DMSO (the same amount of DMSO as the highest concentration compound group).

[0282] Treatment group: Each group was given a complete culture medium containing 20 μM of different small molecule inhibitors.

[0283] The cell culture plates were returned to the incubator and cultured for another 24 hours. RNA was detected using qRT-PCR, and the results are shown in Tables 2, 3, and 4.

[0284] Table 2. RNA expression levels of various small molecule inhibitors after treatment of the breast cancer cell line (4T1)

[0285] Table 3. RNA expression levels of various small molecule inhibitors after treatment of melanoma cell line (E0771)

[0286] Table 4. RNA expression levels of various small molecule inhibitors after treatment of the B-cell lymphoma cell line (OCI-Ly3)

[0287] Example 35: Apoptosis detection after treatment of B-cell lymphoma cell line (OCI-Ly3) with multiple small molecule inhibitors.

[0288] The control and experimental groups of the B-cell lymphoma cell line (OCI-Ly3) cultured in the above experiment were used to detect cell apoptosis using Annexin V-FITC / PI flow cytometry. The results are shown in Table 5.

[0289] Table 5. Apoptosis detection results of B-cell lymphoma cell line (OCI-Ly3) after treatment with various small molecule inhibitors.

[0290] Example 36

[0291] This invention describes the effect of various small molecule inhibitors on tumor volume in a 4T1 mouse mammary tumor model (BALB / c female mice, 6-8 weeks old). 4T1 cells (1×10⁵) were injected bilaterally into the mammary fat pads of mice. Injections began one week after inoculation, and tumor size was assessed on days 0, 3, 7, 10, 14, 17, and 21 following the start of treatment.

[0292] The small molecule inhibitor was administered via intraperitoneal injection at a dose of 50 mg / kg once daily.

[0293] The control group was treated with 10% DMSO + 30% PEG300 + 5% Tween 80 + 45% Saline, administered intraperitoneally once daily.

[0294] α-PD1: 3 mg / kg / dose

[0295] The treatment of combining small molecule inhibitors with α-PD1 was as follows: simultaneous administration in the same manner as the single-agent administration described above. Specific results are shown in Figure 6.

[0296] Table 6. Tumor volume of various small molecule inhibitors of the present invention after treatment in a 4T1 mouse mammary tumor model.

[0297] Example 37: Tumor volume of mouse B-cell lymphoma after treatment with various small molecule inhibitors of the present invention.

[0298] Five-week-old female NCG mice (NOD / ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22 / Gpt) were injected with OCI-Ly3 cells (2.5×106). Treatment began one week after inoculation, and tumor size was measured on days 0, 3, 7, 10, 14, 17, and 21 after the start of treatment.

[0299] The small molecule inhibitor was administered via intraperitoneal injection at a dose of 50 mg / kg once daily.

[0300] The control group was treated with 10% DMSO + 30% PEG300 + 5% Tween 80 + 45% Saline, administered intraperitoneally once daily. The results are shown in Table 7.

[0301] Table 7. Tumor volume of mouse B-cell lymphoma after treatment with various small molecule inhibitors.

[0302] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Compounds of general formula I A1—L—A2 General Formula I Or its pharmaceutically acceptable salts, hydrates, solvates, prodrugs, tautomers, or stereoisomers as MALT1 inhibitors; in, A1 and A2 are independently selected from aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, fused ring, and groups in which any one or more H are substituted, and L is the linking chain; When there are multiple substituents, the substituents can be the same or different.

2. The application according to claim 1, characterized in that, The rings selected from A1 and A2 are preferably five-membered or six-membered rings. If the ring contains heteroatoms, the heteroatoms are selected from N, O, and S. The linking chain L is selected from M1-L1-M2, wherein M1 and M2 independently include bonds, alkyl, alkenyl, imino, tertiary amino, -N=C(H)- and groups in which any one or more H are substituted; L1 includes -NH-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -NHC(S)NH-, -NHC(NH)NH-, -C(O)O-, alkylene, heteroalkylene, -C=N-NHC(O)-, -C=NC(O)NH-, -C(O)NH-N=C-, -NHC(O)-N=C-, -NHC(NH)NH-, ring structures containing -NHC(O)NH-, -NHC(S)NH-, or -NHC(NH)NH-, -C(O)NH- and groups in which any one or more H are substituted; preferably, the ring structure here is a five-membered ring or a six-membered ring; When there are multiple substituents, the substituents can be the same or different.

3. The application according to claim 1, which exerts the effect of inhibiting the expression of the MALT1 gene or inactivating the function of the MALT1 protein by affecting the expression of the MALT1 protein death domain.

4. The application according to claim 3, which reduces or eliminates the ability of MALT1 to form a complex with BCL-10 by inhibiting the death domain of the MALT1 protein.

5. The application according to any one of claims 1-4, wherein the MALT1 inhibitor has the function of treating cancer; Preferably, the cancer includes solid tumors, and more preferably malignant tumors in the lungs, pancreas, liver, digestive tract, reproductive system, etc. The solid tumors include Hodgkin lymphoma, non-Hodgkin lymphoma, lung cancer, breast cancer (including triple-negative breast cancer), melanoma, ovarian cancer, gastric cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, head and neck malignant tumors, urinary system malignant tumors, endometrial cancer, cervical cancer, osteosarcoma, chondrosarcoma, Ewing's sarcoma, thyroid cancer, hepatoblastoma, and nephroblastoma. Preferably, the cancer includes blood cancer, more preferably B-cell lymphoma; more preferably activated B-cell-like diffuse large B-cell lymphoma.

6. The application according to claim 5, characterized in that, The MALT1 inhibitor is administered to patients who have already received immune checkpoint inhibitor therapy, or to patients who will receive immune checkpoint inhibitor therapy, or to patients who are receiving immune checkpoint inhibitor therapy concurrently. The immune checkpoint inhibitors include PD-L1, PD-L2, PD-1, CTLA-4, TIM-3, LAG-3, VISTA, or TIGIT inhibitors.

7. The application according to any one of claims 1-6, wherein the compound of general formula I is selected from the compound of general formula Ia Al-M1-C(O)NH-M2-A2, wherein: A1 includes five-membered heterocyclic rings, five-membered heteroaryl groups, or six-membered aryl groups; A2 includes five-membered heterocyclic rings, five-membered heteroaryl rings, six-membered aryl rings, or fused rings; M1 includes bonds and alkyl groups; M2 includes bonds, imino groups, tertiary amino groups, or -N=C(H)-; Optionally, any one or more H in A1, A2, M1, and M2 can be independently substituted by substituents. When there are multiple substituents, the multiple substituents can be the same or different.

8. The application according to claim 7, characterized in that: A1 and A2 are independently selected from phenyl, Or the structure after any one or more H in these rings are replaced by substituents, wherein B1, B2, B3, B4, B5, B6, B7, and B8 are independently selected from C, N, O, and S; M1 includes structures consisting of a bond, methyl, ethyl, propyl, or any one or more H atoms of these groups that have been substituted with substituents; M2 includes groups in which the H in -N=C(H)- or -N=C(H)- is substituted; When there are multiple substituents, the substituents can be the same or different.

9. The application according to any one of claims 1-6, wherein the compound of general formula I is selected from the compound of general formula Ib: Al-M1-NHC(B9)NH-M2-A2, wherein, NHC(B9)NH can be replaced by a ring structure containing NHC(B9)NH; B9 is selected from NH, O, and S; A1, A2, M1, and M2 are consistent with those defined in claim 8.

10. In the application according to claim 9, the Ib compound may be selected as... in: B9 is selected from NH, O, and S. Any one or more H in the general formula Ib can be independently replaced by any substituent. When there are multiple substituents, the substituents can be the same or different.

11. The application according to any one of claims 1-6, wherein the compound of general formula I is selected from the compound of general formula Ic: Al-M1-NH-M2-A2, wherein, A1, A2, M1, and M2 are consistent with those defined in claim 8, wherein the H in -NH- can be substituted by any substituent; Furthermore, this Ic compound can be Any one or more H atoms can be independently replaced by any substituent. When there are multiple substituents, the substituents can be the same or different.

12. The application according to any one of claims 1-11, wherein the substituent comprises halogen, alkyl, hydroxyl, five-membered cycloyl group, six-membered cycloyl group, amino, dialkylamino, alkoxy, alkylthio, nitro, carbonyl, imino, tertiary amino, ester group, alkoxy, haloalkyl, =O, =S, =NH and any one or more of these substituents being substituted by other substituents; Furthermore, other substituents may include alkyl, alkoxy, aryl, halogen, nitro, amino, imino, and tertiary amino groups.

13. The application according to any one of claims 1-12, wherein the compound of general formula I comprises the following compounds:

14. The application according to claim 13, wherein the compound of general formula I further comprises derivatives of the compound of formula C15 and further substituted or replaced derivatives, wherein the single benzene ring attached to the pyrimidine ring is substituted or replaced, having the general formula (C15a). R1 includes at least one of heterocyclic, cycloalkyl, alkyl, and haloalkyl groups, with or without substituents, wherein the substituents include at least one of halogen, methoxy, and alkyl groups; R1 is selected from any of the following: Further substitutions or replacements of the derivatives of the C15 compound can also be compounds with the ester bond of the pyrimidine ring replaced, having the general formula (C15b). in, R2 is selected from Further substituted or replaced derivatives of the C15 compound can also be compounds in which the terminal benzene ring of the non-benzene ring attached to the pyrimidine ring is substituted or replaced, with the general formula (C15c) as follows: R3 is selected from Further derivatives of the C15 compound, including those with substitutions or replacements, can also be products with substitutions or replacements of the pyrimidine ring. The general formula (C15d) of these derivatives is: L2 is selected from 15. The application according to claim 13, characterized in that, Compounds of general formula I also include derivatives of any of the following C15 compounds:

16. The application according to claim 13, characterized in that, Compounds of general formula I also include derivatives of compounds of formula C17, as well as derivatives of the derivatives with further substitutions or replacements, having the general formula (C17a). R4 is selected from Or its general formula is (C17b). R5 is selected from When R5 in its general formula (C17b) forms a cyclic ring with -NH-, it includes any of the following derivatives:

17. A drug, characterized in that, The drug comprises a compound of general formula I as described in any one of claims 1-16, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, tautomer, or stereoisomer thereof; Preferably, the compound of general formula I is any one of C3, C15, and C17; More preferably, the compound of general formula I is any compound of C15 or C17; More preferably, the compound of general formula I is C15; Most preferably, the compound of general formula I is a derivative of C15, and the derivative of C15 includes any compound represented by formula I”, formula II”, formula III”, formula IV”, formula V”, formula VI”, formula VII”, formula VIII”, formula IX”, formula X”, formula XI”, formula XII”, formula XIII”, formula XIV”, formula XV”, formula XVI”, formula XVII”, formula XVIII”, formula XIX”, formula XX”, formula XXI”, formula XXII”, formula XXIII”, formula XXIV”, formula XXV”, XXVI”, formula XXVII”, formula XXVIII”, formula XXIX”. Preferably, the C17 derivative of the general formula I compound includes compounds of general formulas (C17a) and (C17b), as well as compounds C17-I and C17-II.

18. A drug combination, characterized in that, The drug combination comprises a compound of general formula I as described in any one of claims 1-16 or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, tautomer, stereoisomer, and a second drug, for simultaneous or sequential administration. Preferably, the compound of general formula I is C15; More preferably, the compound of general formula I is a derivative of C15, and the derivative of C15 includes any compound represented by formula I”, formula II”, formula III”, formula IV”, formula V”, formula VI”, formula VII”, formula VIII”, formula IX”, formula X”, formula XI”, formula XII”, formula XIII”, formula XIV”, formula XV”, formula XVI”, formula XVII”, formula XVIII”, formula XIX”, formula XX”, formula XXI”, formula XXII”, formula XXIII”, formula XXIV”, formula XXV”, XXVI”, formula XXVII”, formula XXVIII”, formula XXIX”. Preferably, the second drug comprises an immune checkpoint inhibitor.

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