Combined application of tricyclic inhibitor and anticancer agent in preparation of antitumor drugs

Abstract

The invention relates to combined application of a tricyclic inhibitor and an anti-cancer agent in preparation of an anti-tumor drug, and particularly provides combined application of the tricyclic inhibitor and the anti-cancer agent in preparation of a drug for preventing or treating tumor diseases.

Description

Combined application of tricyclic inhibitors and anticancer agents in the preparation of antitumor drugs Technical Field

[0001] The present invention belongs to the field of medicine and relates to the use of a mucosa-associated lymphoid tissue lymphoma translocator protein 1 (MALT1) inhibitor in combination with an anticancer agent in the preparation or treatment of tumor disease drugs. Background Art

[0002] Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma, with activated B-cell (ABC) and germinal center B-cell (GCB) subtypes accounting for approximately 85% of all DLBCL cases. The introduction of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) immunochemotherapy has significantly improved overall survival, but the response rate for ABC subtype is significantly lower than that for GCB subtype.

[0003] Mucosa-associated lymphoid tissue protein 1 (MALT1), also known as paracysteine ​​protease 1 (PCASP1), is a human immunoprotease that has attracted considerable attention as an emerging drug target for cancer therapy. MALT1 is not only an oncogenic driver but also performs key functions in the immune system. Recent studies have found that its protease function in Treg cells is crucial for maintaining the immunosuppressive tumor microenvironment (TME) in solid cancers. Several clinical trials are currently exploring the direct targeting of MALT1 in non-Hodgkin lymphoma (NHL) (NCT03900598, NCT04876092, and NCT04657224); and another clinical trial is investigating the role of MALT1 inhibitors in reprogramming tumor-infiltrating Treg cells into proinflammatory effector cells to enhance anti-tumor immune responses in non-hematological cancers (NCT04859777).

[0004] The growth of ABC-DLBCL is strongly dependent on the sustained activation of the NF-κB signaling pathway. MALT1 is downstream of Bruton's tyrosine kinase (BTK) in the NF-κB signaling pathway. MALT1 inhibitors can target ABC-DLBCL patients who do not respond to the BTK inhibitor ibrutinib (primarily those with CARD11 mutations) and those who develop resistance to ibrutinib. Combining MALT1 inhibitors with ibrutinib can provide clinical benefit to patients with ABC-DLBCL.

[0005] B-cell leukemia / lymphoma-2 (BCL-2) is a key anti-apoptotic protein. Approximately 30% of DLBCL patients carry BCL-2 translocations and have enhanced BCL-2 expression, which is associated with a poor prognosis. Venetoclax (ABT-199) is a highly selective BCL-2 inhibitor approved for the treatment of chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL) with potent activity against FL, DLBCL, and MCL cell lines. Therefore, the combination of BCL-2 inhibitors with MALT1 inhibitors could provide clinical benefits for patients with ABC-DLBCL with high BCL-2 expression.

[0006] Mammalian target of rapamycin (mTOR) is a major regulator of growth and survival of normal and tumor cells. mTOR activation is regulated by upstream phosphatidylinositol 3, 4, 5 kinase (PI3K) and Akt signaling, promoting cell growth, survival, and proliferation. Mechanisms of aberrant mTOR activation include PTEN loss in MCL, PIK3CA amplification in DLBCL, and activation of PKCδ or Syk kinases in FL. The first-generation mTOR inhibitor temsirolimus (TEM) has been approved by the FDA for the treatment of metastatic renal cell carcinoma and has shown monotherapy activity in several lymphoma subtypes, including MCL, DLBCL, and FL. Fontan et al. reported that mTOR inhibitors and MALT1 inhibitors can synergistically kill DLBCL cells.

[0007] Therefore, MALT1 inhibitor combination therapy can bring greater efficacy to DLBCL patients. Currently, only JNJ67856633, ABBV-525, SGR-1505, XL114, and MPT-0118 are in Phase I clinical trials worldwide. More combination therapies with novel MALT1 inhibitors are needed to improve the treatment of DLBCL.

[0008] Summary of the Invention

[0009] The present invention provides a use of a MALT1 inhibitor and an anticancer agent in combination in the preparation of a medicament for treating tumor diseases, wherein the MALT1 inhibitor is selected from 1-(2-carbonyl-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-N-(2-(trifluoromethyl)pyridin-4-yl)-1H-pyrazole-4-carboxamide or a pharmaceutically acceptable salt thereof, N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide or a pharmaceutically acceptable salt thereof, 5-cyclopropyl-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-N-(2-(trifluoromethyl)pyridin-4-yl)- 1H-pyrazole-4-carboxamide or a pharmaceutically acceptable salt thereof; preferably N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide or a pharmaceutically acceptable salt thereof, 5-cyclopropyl-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide or a pharmaceutically acceptable salt thereof [cd] indol-6-yl)-N-(2-(trifluoromethyl) pyridin-4-yl)-1H-pyrazole-4-carboxamide or a pharmaceutically acceptable salt thereof; more preferably N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd] indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide;

[0010] Preferably, the pharmaceutically acceptable salt of the compound is selected from sulfate, phosphate, benzenesulfonate, cinnamate, tartrate, ethane-1,2-disulfonate, ethanesulfonate, fumarate, methanesulfonate or p-toluenesulfonate.

[0011] In a preferred embodiment of the present invention, the anticancer agent is selected from one or more of a BTK inhibitor, an XPO1 inhibitor, an m-TOR inhibitor, a PI3K inhibitor, a BCL-2 inhibitor or an Akt inhibitor;

[0012] Preferably, the BTK inhibitor is selected from ibrutinib; the XPO1 inhibitor is selected from selinexor; the m-TOR inhibitor is selected from temsirolimus (CCI-779) or rapamycin; the PI3K inhibitor is selected from dactolisib (BEZ235), copanlisib, alpelisib or idelalisib; the BCL-2 inhibitor is selected from venetoclax or navitoclax (ABT-263); the Akt inhibitor is selected from capasertib (AZD5363);

[0013] Ibrutinib, Temsirolimus (CCI-779), or Venetoclax are preferred.

[0014] In a preferred embodiment of the present invention, the tumor disease is selected from diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma, follicular lymphoma, transformed follicular lymphoma, marginal zone lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue lymphoma, advanced or metastatic / refractory solid tumors or chronic lymphocytic leukemia; preferably diffuse large B-cell lymphoma.

[0015] In a preferred embodiment of the present invention, the single administration dose range of the MALT1 inhibitor is selected from 1-1000 mg; preferably 25-1000 mg; or preferably 25-500 mg; or preferably 25-300 mg; or preferably 25-250 mg; or preferably 25-100 mg; or preferably 50-350 mg; or preferably 50-150 mg; or preferably 75-150 mg; or preferably 100-400 mg; or preferably 150-300 mg; or preferably 150-200 mg; or preferably 200-500 mg; or preferably 250-300 mg; or preferably 300-350 mg; or preferably 350-400 mg. Exemplary dosages are selected from 1 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 120mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 210mg, 220mg, 230mg, 240mg, 250mg, 260mg, 270mg, 280 mg, 290mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1000mg.

[0016] The dosage of the MALT1 inhibitor of the present invention may be in the form of a free base or a salt. However, unless otherwise specified, the dosage is calculated based on the free base form.

[0017] In a preferred embodiment of the present invention, the MALT1 inhibitor is administered once a day, twice a day, three times a day, once a week, twice a week, or three times a week.

[0018] In a preferred embodiment of the present invention, the MALT1 inhibitor is administered once a day, with a single dose of 100-1000 mg; preferably 100-400 mg; or preferably 150-300 mg; or preferably 150-200 mg; or preferably 200-500 mg; or preferably 250-300 mg; or preferably 300-350 mg; or preferably 350-400 mg.

[0019] In a preferred embodiment of the present invention, the MALT1 inhibitor is administered twice a day, with a single dose of 25-1000 mg; preferably 25-100 mg; or preferably 50-150 mg; or preferably 75-150 mg.

[0020] In a preferred embodiment of the present invention, the single administration dose range of the anticancer agent is selected from 1-1000 mg; preferably 50-1000 mg; or preferably 50-500 mg; or preferably 50-350 mg; or preferably 50-300 mg; or preferably 50-200 mg; or preferably 75-300 mg; or preferably 100-200 mg. Exemplary dosages are selected from 1 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 120mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 210mg, 220mg, 230mg, 240mg, 250mg, 260mg, 270mg, 280 mg, 290mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1000mg.

[0021] In a preferred embodiment of the present invention, the anticancer agent is administered once a day, twice a day, three times a day, once a week, twice a week, or three times a week.

[0022] In a preferred embodiment of the present invention, the anticancer agent is administered once a day, and the single dose is 50-1000 mg; preferably 50-500 mg; or preferably 50-350 mg; or preferably 50-300 mg; or preferably 75-300 mg;

[0023] In a preferred embodiment of the present invention, the anticancer agent is administered twice a day, and the single dose is 50-1000 mg; preferably 50-300 mg; preferably 50-200 mg; or preferably 100-200 mg.

[0024] In a preferred embodiment of the present invention, the anticancer agent is selected from ibrutinib, and the single administration dose ranges from 50 to 1000 mg; preferably 50 to 500 mg. Exemplary dosages are selected from 50 mg, 52.5 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg.

[0025] In a preferred embodiment of the present invention, the administration frequency of ibrutinib is once a day, twice a day, three times a day, once a week, twice a week, or three times a week; preferably once a day or twice a day.

[0026] In a preferred embodiment of the present invention, ibrutinib is administered once a day with a single dose of 50-500 mg; or twice a day with a single dose of 50-500 mg.

[0027] In a preferred embodiment of the present invention, the anticancer agent is selected from venetoclax, and the single dose range is 20-1000 mg; preferably 20-500 mg. Exemplary doses are selected from 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, g, 160mg, 170mg, 180mg, 190mg, 200mg, 210mg, 220mg, 230mg, 240mg, 250mg, 260mg, 270mg, 280mg, 290mg, 30 0mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1000mg.

[0028] In a preferred embodiment of the present invention, the administration frequency of venetoclax is once a day, twice a day, three times a day, once a week, twice a week or three times a week; preferably once a day or twice a day;

[0029] In a preferred embodiment of the present invention, venetoclax is administered once a day with a single dose of 20-500 mg; or administered twice a day with a single dose of 20-500 mg.

[0030] In a preferred embodiment of the present invention, the MALT1 inhibitor and the anticancer agent are administered simultaneously, concurrently, independently or sequentially.

[0031] The combined administration route described in the present application is selected from oral administration, parenteral administration, and transdermal administration. The parenteral administration includes but is not limited to intravenous injection, subcutaneous injection or intramuscular injection; the administration route is preferably oral administration.

[0032] In the scheme described in the present invention, the combination optionally further comprises other components, and the other components include but are not limited to the use in other drugs for treating tumor diseases.

[0033] The present invention also provides a pharmaceutical composition comprising:

[0034] (a) one or more MALT1 inhibitors;

[0035] (b) one or more anticancer agents;

[0036] The MALT1 inhibitor is selected from N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide or a pharmaceutically acceptable salt thereof, 5-cyclopropyl-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-N-(2-(trifluoromethyl)pyridin-4-yl)-1H-pyrazole-4-carboxamide or a pharmaceutically acceptable salt thereof; preferably N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide;

[0037] Preferably, the pharmaceutically acceptable salt of the compound is selected from sulfate, phosphate, benzenesulfonate, cinnamate, tartrate, ethane-1,2-disulfonate, ethanesulfonate, fumarate, methanesulfonate or p-toluenesulfonate;

[0038] The anticancer agent is selected from BTK inhibitors, XPO1 inhibitors, m-TOR inhibitors, PI3K inhibitors, BCL-2 inhibitors or Akt inhibitors;

[0039] The BTK inhibitor is selected from ibrutinib; the XPO1 inhibitor is selected from selinexor; the m-TOR inhibitor is selected from temsirolimus (CCI-779) or rapamycin; the PI3K inhibitor is selected from dactolisib (BEZ235), copanlisib, alpelisib or idelalisib; the BCL-2 inhibitor is selected from venetoclax or navitoclax (ABT-263); the Akt inhibitor is selected from capivasertib (AZD5363);

[0040] Ibrutinib, Temsirolimus (CCI-779), or Venetoclax are preferred.

[0041] When the combination is in the form of a pharmaceutical composition, it also contains one or more pharmaceutically acceptable carriers, excipients or diluents.

[0042] In a preferred embodiment of the present invention, the pharmaceutical composition can be prepared in any pharmaceutically acceptable dosage form, for example, tablets, capsules, pills, granules, solutions, suspensions, syrups, injections (including injections, sterile powders for injection, and concentrated solutions for injection), suppositories, inhalants, or sprays.

[0043] The present invention also provides a combined preparation comprising:

[0044] (a) N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide or a pharmaceutically acceptable salt thereof, 5-cyclopropyl-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-N-(2-(trifluoromethyl)pyridin-4-yl)-1H-pyrazole-4-carboxamide or a pharmaceutically acceptable salt thereof; preferably N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide;

[0045] Preferably, the pharmaceutically acceptable salt of the compound is selected from sulfate, phosphate, benzenesulfonate, cinnamate, tartrate, ethane-1,2-disulfonate, ethanesulfonate, fumarate, methanesulfonate or p-toluenesulfonate;

[0046] (b) one or more of BTK inhibitors, XPO1 inhibitors, m-TOR inhibitors, PI3K inhibitors, BCL-2 inhibitors, or Akt inhibitors;

[0047] The BTK inhibitor is selected from ibrutinib; the XPO1 inhibitor is selected from selinexor; the m-TOR inhibitor is selected from temsirolimus (CCI-779) or rapamycin; the PI3K inhibitor is selected from dactolisib (BEZ235), copanlisib, alpelisib or idelalisib; the BCL-2 inhibitor is selected from venetoclax or navitoclax (ABT-263); the Akt inhibitor is selected from capivasertib (AZD5363);

[0048] Ibrutinib, Temsirolimus (CCI-779), or Venetoclax are preferred.

[0049] In a preferred embodiment of the present invention, the combined preparation or pharmaceutical composition can be used to prevent or treat tumor diseases.

[0050] In a preferred embodiment of the present invention, the combined preparation or pharmaceutical composition is used to prevent or treat diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma, follicular lymphoma, transformed follicular lymphoma, marginal zone lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue lymphoma, advanced or metastatic / refractory solid tumors or chronic lymphocytic leukemia; preferably diffuse large B-cell lymphoma.

[0051] The present invention also provides a method for preventing or treating tumor diseases, comprising administering to a patient a combination of the following compounds at an effective dose:

[0052] (a) one or more MALT1 inhibitors;

[0053] (b) one or more anticancer agents;

[0054] The MALT1 inhibitor is selected from N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide or a pharmaceutically acceptable salt thereof, 5-cyclopropyl-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-N-(2-(trifluoromethyl)pyridin-4-yl)-1H-pyrazole-4-carboxamide or a pharmaceutically acceptable salt thereof; preferably N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide;

[0055] Preferably, the pharmaceutically acceptable salt of the compound is selected from sulfate, phosphate, benzenesulfonate, cinnamate, tartrate, ethane-1,2-disulfonate, ethanesulfonate, fumarate, methanesulfonate or p-toluenesulfonate;

[0056] The anticancer agent is selected from BTK inhibitors, XPO1 inhibitors, m-TOR inhibitors, PI3K inhibitors, BCL-2 inhibitors or Akt inhibitors;

[0057] The BTK inhibitor is selected from ibrutinib; the XPO1 inhibitor is selected from selinexor; the m-TOR inhibitor is selected from temsirolimus (CCI-779) or rapamycin; the PI3K inhibitor is selected from dactolisib (BEZ235), copanlisib, alpelisib or idelalisib; the BCL-2 inhibitor is selected from venetoclax or navitoclax (ABT-263); the Akt inhibitor is selected from capivasertib (AZD5363);

[0058] Ibrutinib, Temsirolimus (CCI-779), or Venetoclax are preferred.

[0059] The MALT1 inhibitor and the anticancer agent are used simultaneously, concurrently, independently or sequentially.

[0060] The MALT1 inhibitor and anticancer agent described in the present application can be administered alone or in combination with one or more therapeutic agents.

[0061] The present application also provides a pharmaceutical kit for use in treating tumor diseases, which contains the pharmaceutical composition of the MALT1 inhibitor and anticancer agent described in the present application.

[0062] The present application administers a MALT1 inhibitor in combination with an anticancer agent, thereby enhancing the use of the drug in treating tumor diseases and improving the therapeutic effect.

[0063] As used herein, "combination" refers to a regimen that includes administering at least one dose of a MALT1 inhibitor and at least one dose of an anticancer agent within a timeframe, wherein both agents exhibit a pharmacological effect. The timeframe can be within a single dosing cycle, preferably within 4 weeks, 3 weeks, 2 weeks, 1 week, or 24 hours. The MALT1 inhibitor and anticancer agent can be administered simultaneously or sequentially. This timeframe includes treatments in which the MALT1 inhibitor and anticancer agent are administered via the same or different routes of administration.

[0064] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention that are safe and effective when used in mammals and have the desired biological activity.

[0065] The term "effective amount" refers to an amount of a drug that is effective in treating a disease or condition in a mammal. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., slow down to a certain extent and preferably prevent) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down to a certain extent and preferably prevent) tumor metastasis; inhibit tumor growth to a certain extent; and / or alleviate one or more symptoms associated with the condition to a certain extent. Depending on the extent to which the drug can prevent the growth of existing cancer cells and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer treatment, in vivo efficacy can be measured by assessing duration of survival, progression-free survival (PFS) duration, response rate (RR), duration of response and / or quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 shows the tumor volume growth in the OCI-LY3 mouse model after treatment with Compound A alone, Venetoclax alone, and combination treatment with Compound A and Venetoclax.

[0067] Figure 2 shows the changes in mouse body weight in the OCI-LY3 mouse model after treatment with Compound A alone, Venetoclax alone, and combined treatment with Compound A and Venetoclax.

[0068] FIG3 shows the tumor volume growth in the TMD-8 mouse model after treatment with Compound A alone, Venetoclax alone, and a combination of Compound A and Venetoclax.

[0069] Figure 4 shows the changes in mouse body weight in the TMD-8 mouse model after treatment with Compound A alone, Venetoclax alone, and combined treatment with Compound A and Venetoclax.

[0070] FIG5 shows the tumor volume growth in the TMD-8 mouse model after treatment with Compound A alone, Ibrutinib alone, and combination treatment with Compound A and Ibrutinib.

[0071] FIG6 shows the changes in mouse body weight in the TMD-8 mouse model after treatment with Compound A alone, Ibrutinib alone, and combined treatment with Compound A and Ibrutinib.

[0072] Figure 7 shows the results of human induced leukemia after treatment with compound A alone, ibrutinib alone, and combination of compound A and ibrutinib. Tumor volume growth in the lymphoma LY2298 mouse model.

[0073] Figure 8 shows the results of human induced leukemia after treatment with compound A alone, ibrutinib alone, and combination of compound A and ibrutinib. Body weight changes in mice in the LY2298 mouse model of lymphoma.

[0074] Figure 9 shows the tumor volume growth in the patient-derived lymphoma LY-24-0179 mouse model after Compound A alone, Ibrutinib alone, Venetoclax alone, Compound A and Ibrutinib combination therapy, and Compound A and Venetoclax combination therapy.

[0075] Figure 10 shows the tumor volume growth in the patient-derived lymphoma LY-24-0179 mouse model after discontinuation of Compound A alone, Ibrutinib alone, Venetoclax alone, Compound A and Ibrutinib combination therapy, and Compound A and Venetoclax combination therapy.

[0076] Figure 11 shows the changes in mouse body weight in the patient-derived lymphoma LY-24-0179 mouse model after treatment with Compound A alone, Ibrutinib alone, Venetoclax alone, combination treatment with Compound A and Ibrutinib, and combination treatment with Compound A and Venetoclax. DETAILED DESCRIPTION

[0077] The present application will be explained in more detail below with reference to the embodiments. The embodiments of the present application are only used to illustrate the technical solutions of the present application and are not intended to limit the essence and scope of the present application.

[0078] Biological evaluation methods

[0079] Example 1. Exploration of the synergistic effect of compound A of the present invention and other drugs in cell lines

[0080] 1. Purpose of the experiment

[0081] The cell proliferation inhibition experiment was conducted to explore the proliferation inhibition effect of compound A in combination with other drugs in DLBCL cell lines.

[0082] 2. Experimental instruments and reagents:

[0083] 2.1 Instruments:

[0084] 2.2 Experimental reagents:

[0085] Compound A: N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide, prepared by the method of PCT / CN2022 / 143665.

[0086] Reagents:

[0087] 3. Experimental Methods

[0088] The CellTiter-Glo method was used to detect the inhibitory effects of compound A and other drugs on the proliferation of HBL1, TMD8, OCI-Ly3, and OCI-LY10 cells. Cells were plated at an appropriate density and incubated for 3 hours. Compound A and other drugs were then added to the cell culture plates in an orthogonal manner. Eight concentration gradients were set for the compound and each drug. The starting dilution concentration and dilution factor are shown in the table below. After drug addition, the cells were incubated for another 6 days. The cells were then detected using the CellTiter-Glo Luminescebt Assay Kit and the values ​​were read using an EnVision plate reader.

[0089] 4. Experimental data processing method:

[0090] 1. Survival rate (%):

[0091] Survival rate % = [(sample well value - negative control well average) / (positive control well average - negative control well average)] × 100, where the positive control wells are compound-free wells and the negative control wells are PBS wells.

[0092] 2. Synergistic effect evaluation: The survival rate was input into the Combenifit software template and analyzed using the HSA, BLISS and LOEWE models respectively.

[0093] 5. Experimental Results

[0094] Table 1-1 In vitro results of the combined use of compound A and anticancer agents in TMD8 cells

[0095] Table 1-2 In vitro results of the combined use of compound A and anticancer agents in HBL1 cells

[0096] Table 1-3 In vitro combination results of compound A and anticancer agents in OCI-LY3 cells

[0097] Table 1-4 In vitro combination results of compound A and anticancer agents in OCI-LY10 cells

[0098] ++: All three analysis models showed synergy and there were regions with scores >40

[0099] +: Synergistic effects were observed in all three analysis models

[0100] Additive: synergistic in one or two models and additive in the remaining models

[0101] 6. Experimental Conclusion

[0102] The combination of compound A and other drugs can synergistically inhibit the proliferation of HBL1, TMD8, OCI-Ly3 and OCI-Ly10 cells.

[0103] Example 2. Evaluation of the in vivo inhibitory effect of compound A combined with the Bcl-2 inhibitor Venetoclax on OCI-LY3 human diffuse large B-cell lymphoma cell transplanted tumors in mice

[0104] 1. Experimental Materials

[0105] Compound A: N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide, prepared using the method described in PCT / CN2022 / 143665, using 0.5% HPMC for drug formulation;

[0106] Venetoclax: The drug is formulated with 0.5% HPMC;

[0107] Human diffuse large B-cell lymphoma (OCI-LY3) cells were cultured in suspension in IMDM medium supplemented with 15% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. Cells were routinely digested and passaged twice weekly. When cell saturation reached 80%-90% and the desired number of cells was reached, cells were harvested, counted, and plated.

[0108] NCG mice, female, weighing 17–20 g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0109] 2. Experimental Methods

[0110] OCI-LY3 (density: 10×10 7 / mL) cell suspension was inoculated subcutaneously on the back of the right forelimb of each mouse, and the tumor was grown to an average volume of 100-200mm. 3 The mice were divided into groups (D0) and dosed (D1). The mice were gavage-administered (po) once daily (QD) at a volume of 10 mL / kg. The solvent group received the same volume of solvent (0.5% HPMC). The specific dosage and dosing schedule are shown in Table 2-1. Tumor volume was measured, mice were weighed, and the data were recorded.

[0111] The experimental indicators are to examine the effect of drugs on tumor growth, and the specific indicators are T / C% or tumor inhibition rate TGI (%).

[0112] The tumor diameter was measured with a vernier caliper, and the tumor volume (V) was calculated as follows:

[0113] V=1 / 2×a×b 2 , where a and b represent length and width respectively.

[0114] T / C (%) = (T-T0) / (C-C0) × 100, where T and C are the average tumor volumes of the drug-treated group and the solvent control group at the end of the experiment, and T0 and C0 are the average tumor volumes of the drug-treated group and the solvent control group at the beginning of the experiment.

[0115] Tumor inhibition rate (TGI) (%) = 100-T / C ​​(%).

[0116] When the tumor regresses, the tumor inhibition rate (TGI) (%) = 100-(T-T0) / T0×100

[0117] The experiment ends, the experimental endpoint is reached, or the tumor volume reaches 2000mm 3 The animals were killed by CO2 anesthesia, and then the tumors were removed by dissection and photographed.

[0118] Experimental data were analyzed and graphed using GraphPad Prism 9. Tumor volume data for each group at each time point are presented as the mean and standard deviation (SEM). Differences between groups were analyzed using Dunnett's multiple comparisons test in a two-way ANOVA. A p < 0.05 was considered statistically significant.

[0119] Table 2-1. Dosing regimen of compound A combined with venetoclax in the OCI-LY3 model Note: Compound A and Venetoclax were administered simultaneously.

[0120] 3. Experimental Results

[0121] The pharmacodynamic effects of compound A combined with venetoclax in the CARD11 mutant OCI-LY3 xenograft tumor model are shown in Table 2-2 and Figure 1:

[0122] Table 2-2. Evaluation of the antitumor efficacy of compound A combined with venetoclax in the OCI-LY3 xenograft tumor model Note: Data in the table are from Day 21; a. Mean ± SD; b. Compared with the vehicle control group, statistical analysis was performed using the two-way Dunnett's multiple comparisons test.

[0123] The experimental results showed that the average tumor volumes of the Compound A monotherapy group (20 mg / kg), the Venetoclax monotherapy group (40 mg / kg), and the Compound A & Venetoclax combination therapy group were 528 mm and 133 mm, respectively, on the 21st day after administration. 3 , 963mm 3 and 333mm 3The tumor inhibition rates (TGI) of the two groups were 73.12%, 28.81% and 92.94%, respectively. Compared with the vehicle group, the combined effect of compound A and venetoclax was superior to that of single drug, with statistical significance (p<0.01).

[0124] The body weight and weight changes of the animals in each group in this experiment are shown in Figure 2. The results showed that the tumor-bearing mice tolerated the drug dose well throughout the experiment, and no significant weight loss symptoms occurred.

[0125] 3. Experimental Conclusion

[0126] Compared with either treatment alone, compound A combined with venetoclax showed stronger anti-tumor effects and no obvious toxic side effects.

[0127] Example 3. Evaluation of the in vivo inhibitory effect of Compound A combined with the mTOR inhibitor Temsirolimus (CCI-779) on OCI-LY3 human diffuse large B-cell lymphoma cell transplanted tumors in mice

[0128] 1. Experimental Materials

[0129] Compound A: N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide, prepared using the method described in PCT / CN2022 / 143665, using 0.5% HPMC for drug formulation;

[0130] Temsirolimus (CCI-779): 0.5% HPMC is used for drug preparation;

[0131] Human diffuse large B-cell lymphoma (OCI-LY3) cells were cultured in suspension in IMDM medium supplemented with 15% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. Cells were routinely digested and passaged twice weekly. When cell saturation reached 80%-90% and the desired number of cells was reached, cells were harvested, counted, and plated.

[0132] NSG mice, female, weighing 17-23 g, were purchased from the Experimental Animal Management Department of Shanghai Institute of Family Planning Science.

[0133] 2. Experimental Methods

[0134] OCI-LY3 (density: 10×10 7 / mL) cell suspension was inoculated subcutaneously on the back of the right forelimb of each mouse, and the tumor was grown to an average volume of 100-200mm. 3 Grouping was performed at 4 pm (D0) and dosing was performed at 4 pm (D1). Mice were administered orally (po) twice daily (BID) at a volume of 10 mL / kg. The solvent group received the same volume of solvent (0.5% HPMC). Specific dosing schedules are shown in Table 2-1. Tumor volume was measured, mice were weighed, and data were recorded.

[0135] The experimental indicators are to examine the effect of drugs on tumor growth, and the specific indicators are T / C% or tumor inhibition rate TGI (%).

[0136] The tumor diameter was measured with a vernier caliper, and the tumor volume (V) was calculated as follows:

[0137] V=1 / 2×a×b 2 , where a and b represent length and width respectively.

[0138] T / C (%) = (T-T0) / (C-C0) × 100, where T and C are the average tumor volumes of the drug-treated group and the solvent control group at the end of the experiment, and T0 and C0 are the average tumor volumes of the drug-treated group and the solvent control group at the beginning of the experiment.

[0139] Tumor inhibition rate (TGI) (%) = 100-T / C ​​(%).

[0140] When the tumor regresses, the tumor inhibition rate (TGI) (%) = 100-(T-T0) / T0×100

[0141] The experiment ends, the experimental endpoint is reached, or the tumor volume reaches 2000mm 3 The animals were killed by CO2 anesthesia, and then the tumors were removed by dissection and photographed.

[0142] Experimental data were analyzed and graphed using GraphPad Prism 9. Tumor volume data for each group at each time point are presented as the mean and standard deviation (SEM). Differences between groups were analyzed using Dunnett's multiple comparisons test in a two-way ANOVA. A p < 0.05 was considered statistically significant.

[0143] Table 2-1. Dosing regimen of compound A combined with CCI-779 in the OCI-LY3 model Note: Compound A and CCI-779 were administered simultaneously.

[0144] 3. Experimental results and conclusions

[0145] Compared with either treatment alone, compound A combined with temsirolimus showed a stronger anti-tumor effect without exhibiting obvious toxic side effects.

[0146] Example 4. Evaluation of the in vivo inhibitory effect of compound A combined with the Bcl2 inhibitor Venetoclax on TMD-8 human diffuse large B-cell lymphoma cell transplanted tumors in mice

[0147] 1. Experimental Materials

[0148] Compound A: N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide, prepared using the method described in PCT / CN2022 / 143665, using 0.5% HPMC for drug formulation;

[0149] Venetoclax: The drug is formulated with 0.5% HPMC;

[0150] Human diffuse large B-cell lymphoma TMD-8 cells were cultured in suspension in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. Cells were passaged twice weekly. When cell saturation reached 80%-90% and the desired number of cells was reached, cells were harvested, counted, and plated.

[0151] CB17 SCID mice, female, weighing 16-19 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0152] 2. Experimental Methods

[0153] TMD-8 (density: 5.0×10 7 / mL) cell suspension was inoculated subcutaneously on the back of the right forelimb of each mouse, and the tumor was grown to an average volume of 100mm 3 The mice were divided into groups (D0) and dosed (D1). The mice were gavage-administered (po) once daily (QD) at a volume of 10 mL / kg. The solvent group received the same volume of solvent (0.5% HPMC). The specific dosage and dosing schedule are shown in Table 3-1. Tumor volume was measured, mice were weighed, and the data were recorded.

[0154] The experimental indicators are to examine the effect of drugs on tumor growth, and the specific indicators are T / C% or tumor inhibition rate TGI (%).

[0155] The tumor diameter was measured with a vernier caliper, and the tumor volume (V) was calculated as follows:

[0156] V=1 / 2×a×b 2 , where a and b represent length and width respectively.

[0157] T / C (%) = (T-T0) / (C-C0) × 100, where T and C are the average tumor volumes of the drug-treated group and the solvent control group at the end of the experiment, and T0 and C0 are the average tumor volumes of the drug-treated group and the solvent control group at the beginning of the experiment.

[0158] Tumor inhibition rate (TGI) (%) = 100-T / C ​​(%).

[0159] When the tumor regresses, the tumor inhibition rate (TGI) (%) = 100-(T-T0) / T0×100

[0160] The experiment ends, the experimental endpoint is reached, or the tumor volume reaches 2000mm 3 The animals were killed by CO2 anesthesia, and then the tumors were removed by dissection and photographed.

[0161] Tumor volume data for each group at each time point are presented as the mean and standard error (SEM). GraphPad Prism 9 was used to analyze and plot the experimental data. Tumor volume data for each group at each time point are presented as the mean and standard error (SEM). Two-way ANOVA was used to analyze intergroup differences in tumor volume data at different time points using Dunnett's multiple comparisons test. A p < 0.05 was considered statistically significant.

[0162] Table 4-1. Dosing regimen of compound A combined with venetoclax in the TMD-8 model Note: Compound A and Venetoclax were administered simultaneously.

[0163] 3. Experimental Results

[0164] The pharmacodynamic effects of compound A combined with venetoclax in the MYD88 mutant TMD-8 xenograft tumor model are shown in Table 4-2 and Figure 3:

[0165] Table 4-2. Evaluation of the antitumor efficacy of compound A combined with venetoclax in TMD-8 xenograft tumor model

[0166] Note: Data in the table are from Day 21; c. Mean ± SD; d. Compared with the vehicle control group, statistical analysis was performed using the two-way Dunnett's multiple comparisons test.

[0167] The experimental results showed that on the 21st day of the experiment, the average tumor volume of the tumor-bearing mice in the control group reached 1,234 mm 3 The tumor surface was intact and without ulceration. After oral administration of compound A at a dose of 30 mg / kg once a day for 3 consecutive weeks, the average tumor volume was 593 mm 3 The tumor inhibition rate (TGI) was 57.36%. The BCL2 small molecule inhibitor Venetoclax was administered orally once a day at a dose of 40 mg / kg for 3 consecutive weeks, and the average tumor volume was 1,126 mm 3 The average tumor volume of the Compound A & Venetoclax combination treatment group on day 21 after administration was 470 mm 3 The TGI was 68.33%. Compared with the Vehicle group, the combination of Compound A and Venetoclax had a better tumor inhibition effect than either drug alone, with statistical significance (p<0.01).

[0168] In this experiment, the body weight and weight changes of animals in each group are shown in Figure 4. The results showed that the tumor-bearing mice tolerated the drug dose well throughout the experiment.

[0169] 4. Experimental Conclusion

[0170] Compared with either treatment alone, compound A combined with venetoclax showed stronger anti-tumor effects and no obvious toxic side effects.

[0171] Example 5. Evaluation of the in vivo inhibitory effect of compound A combined with the BTK inhibitor ibrutinib on TMD-8 human diffuse large B-cell lymphoma cell transplanted tumors in mice

[0172] 1. Experimental Materials

[0173] Compound A: N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide, prepared using the method described in PCT / CN2022 / 143665, using 0.5% HPMC for drug formulation;

[0174] Ibrutinib: 0.5% HPMC is used for drug preparation;

[0175] Human diffuse large B-cell lymphoma TMD-8 cells were cultured in suspension in RPMI 1640 medium supplemented with 15% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. Cells were routinely digested and passaged twice weekly. When cell saturation reached 80%-90% and the desired number of cells was reached, cells were harvested, counted, and plated.

[0176] CB17 SCID mice, female, weighing 16-19 g, were purchased from Zhejiang Weitonglihua Laboratory Animal Technology Co., Ltd.

[0177] 2. Experimental Methods

[0178] TMD-8 (density: 3.5×10 7 / mL) cell suspension was inoculated subcutaneously on the back of the right forelimb of each mouse, and the tumor was grown to an average volume of 200mm 3 The mice were divided into groups (D0) and dosed (D1). The mice were gavage-administered (po) once daily (QD) at a volume of 10 mL / kg. The solvent group received the same volume of solvent (0.5% HPMC). The specific dosage and dosing schedule are shown in Table 5-1. Tumor volume was measured, mice were weighed, and the data were recorded.

[0179] The experimental indicators are to examine the effect of drugs on tumor growth, and the specific indicators are T / C% or tumor inhibition rate TGI (%).

[0180] The tumor diameter was measured with a vernier caliper, and the tumor volume (V) was calculated as follows:

[0181] V=1 / 2×a×b 2 , where a and b represent length and width respectively.

[0182] T / C (%) = (T - T0) / (C - C0) × 100, where T and C are the average tumor volumes of the drug-treated and vehicle-controlled groups at the end of the experiment, and T0 and C0 are the average tumor volumes of the drug-treated and vehicle-controlled groups at the beginning of the experiment. Tumor inhibition rate (TGI) (%) = 100 - T / C (%).

[0183] When the tumor regresses, the tumor inhibition rate (TGI) (%) = 100-(T-T0) / T0×100

[0184] The experiment ends, the experimental endpoint is reached, or the tumor volume reaches 2000mm 3 The animals were killed by CO2 anesthesia, and then the tumors were removed by dissection and photographed.

[0185] Experimental data were analyzed and graphed using GraphPad Prism 9. Tumor volume data for each group at each time point are presented as the mean and standard deviation (SEM). Differences between groups were analyzed using Dunnett's multiple comparisons test in a two-way ANOVA. A p < 0.05 was considered statistically significant.

[0186] Table 5-1. Dosing regimen of compound A combined with Ibrutinib in the TMD-8 model Note: Compound A and Ibrutinib were administered simultaneously.

[0187] 3. Experimental Results

[0188] The pharmacodynamic effects of compound A combined with Ibrutinib in the MYD88 mutant TMD-8 xenograft tumor model are shown in Table 5-2 and Figure 5:

[0189] Table 5-2. Evaluation of the antitumor efficacy of compound A combined with Ibrutinib in TMD-8 xenograft tumor model Note: Data in the table are from Day 22; e. Mean ± SD; f. Compared with the vehicle control group, statistical analysis was performed using the two-way Dunnett's multiple comparisons test.

[0190] The experimental results showed that the average tumor volumes of the Compound A monotherapy group (30 mg / kg), the Ibrutinib monotherapy group (3 mg / kg), and the Compound A & Ibrutinib combination therapy group were 501 mm and 100 mm, respectively, on the 22nd day after administration. 3 , 762mm 3 and 11mm 3 , with tumor inhibition rates (TGI) of 69.42%, 43.62%, and 194.49%, respectively. At the efficacy endpoint, two mice in the Compound A & ibrutinib combination treatment group experienced complete tumor regression. Compared to the vehicle group, the combination of Compound A and ibrutinib demonstrated statistically significant superior tumor inhibition compared to either agent alone (p<0.00001).

[0191] The body weights and weight changes of the animals in each group in this experiment are shown in Figure 6. The results showed that tumor-bearing mice tolerated the drug doses well throughout the experiment. The average body weight of mice in the Compound A & Ibrutinib combination treatment group decreased by 5% during the dosing period, but this weight was restored after nutritional supplementation.

[0192] 4. Experimental Conclusion

[0193] Compared with either treatment alone, compound A combined with Ibrutinib showed stronger anti-tumor effects and no obvious toxic side effects.

[0194] Example 6 Evaluation of Compound A combined with BTK inhibitor Ibrutinib in human Antitumor Effects of NPG in a Female Lymphoma LY2298 Subcutaneous Xenograft Mouse Model 1. Experimental Materials

[0195] Compound A: N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide, prepared using the method described in PCT / CN2022 / 143665, using 0.5% HPMC for drug formulation;

[0196] Ibrutinib: The drug was formulated with 0.5% HPMC;

[0197] Model Information:

[0198] NPG mice, female, 6-7 weeks old, were purchased from Beijing Weitongda Biotechnology Co., Ltd.

[0199] 2. Experimental Methods

[0200] from Tumor tissue was harvested from LY2298 lymphoma xenograft model mice, cut into 2-3 mm diameter tumor masses, and inoculated subcutaneously at the right anterior scapula of NPG mice. Before dosing, all animals were weighed and tumor volumes were measured with a vernier caliper. Tumors were grown to an average volume of 181.55 mm. 3 Dosing was performed by randomization based on tumor size. Given that tumor volume can affect treatment effectiveness, mice were randomly assigned to groups based on tumor volume to ensure similar tumor volumes across groups. Grouping was performed using StudyDirector™ (version 3.1.399.19, supplier: Studylog System, Inc., San Francisco, CA, USA) using the matched distribution method. Dosing began on the day of grouping (Day 0) according to the experimental design. Detailed dosing methods, dosages, and routes of administration are shown in Table 6-1.

[0201] Table 6-1. Dosing regimen of compound A combined with Ibrutinib in the LY2298 model

[0202] Note: Compound A and Ibrutinib were administered simultaneously.

[0203] The test drugs were prepared twice a week. Each test drug solution was thoroughly mixed before each administration, and then administered by gavage according to body weight in a clean bench, once a day.

[0204] The experimental indicator is to examine whether tumor growth is inhibited, delayed, or cured. Tumor diameter is measured twice a week with a vernier caliper. Tumor volume is calculated using the formula: V = 0.5 × D × d², where V represents tumor volume and D and d represent tumor length and width, respectively.

[0205] The tumor inhibition efficacy of the compounds was evaluated by TGI (%) and T / C (%).

[0206] Tumor growth inhibition, TGI

[0207] Formula: TGI% = {[1-(average tumor volume of a treatment group at the end of drug administration - average tumor volume of the treatment group at the beginning of drug administration)] / (average tumor volume of the solvent control group at the end of treatment - average tumor volume of the solvent control group at the beginning of treatment)} × 100%

[0208] Tumor proliferation rate: The T / C value (%),

[0209] Formula: T / C%=TmTV / CmTV×100%;

[0210] TmTV: mean tumor volume of the treatment group; CmTV: mean tumor volume of the control group;

[0211] Or T / C% = TmTW / CmTW × 100%

[0212] TmTW: mean tumor weight at the end of the experiment in the treatment group; CmTW: mean tumor weight at the end of the experiment in the control group

[0213] To compare tumor volumes on a given day between treatment groups, the assumption of homogeneity of variance among all groups was first tested using the Bartlett test. When the p-value from the Bartlett test was ≥0.05, one-way ANOVA was used to test the equality of the means across all groups. If the p-value from the one-way ANOVA was less than 0.05, the Tukey HSD test was used for pairwise comparisons between all groups, or the Dunnett's t-test was used for pairwise comparisons between each treatment group and the control group. If the p-value from the Bartlett test was less than 0.05, the Kruskal-Wallis test was used to test the equality of the medians across all groups. If the p-value from the Kruskal-Wallis test was less than 0.05, the Conover test was used for pairwise comparisons between all groups or between each treatment group and the control group, with the p-value adjusted for multiple testing.

[0214] All statistical analyses and graphics were performed in the R language environment (version 3.3.1). Unless otherwise specified, all tests were two-tailed, and p values ​​less than 0.05 were considered statistically significant.

[0215] 3. Experimental Results

[0216] Compound A and Ibrutinib combined in human The pharmacodynamic effects in the lymphoma LY2298 xenograft tumor model are shown in Table 6-2 and Figure 7:

[0217] Table 6-2. Effects of Compound A and Ibrutinib on human Evaluation of anti-tumor efficacy in the lymphoma LY2298 xenograft tumor model Note: All data in the table are from Day 14; g. Mean ± SD; h. Analysis method: Conover test was used for pairwise comparisons among all groups, and the p-value was adjusted accordingly based on the number of groups for multiple testing.

[0218] The experimental results showed that the average tumor volumes of the Compound A monotherapy group (20 mg / kg), the Ibrutinib monotherapy group (25 mg / kg), and the Compound A & Ibrutinib combination therapy group were 810.85 mm and 810.85 mm, respectively, on the 14th day after administration. 3 、885.83mm 3 and 0mm 3 The tumor inhibition rates (TGI) were 64.23%, 59.95%, and 110.33%, respectively. Compared with Compound A alone, the combined effect of Compound A and Ibrutinib was statistically significant (p<0.001). Tumor regression was observed in all animals (7 / 7) after 14 days of dosing.

[0219] The body weight and weight changes of the animals in each group in this experiment are shown in Figure 8. The results showed that the tumor-bearing mice tolerated the drug dose well throughout the experiment, and no significant weight loss symptoms occurred.

[0220] 4. Experimental Conclusion

[0221] Compound A at 20 mg / kg and Ibrutinib at 25 mg / kg had significant anti-tumor effects in the human lymphoma LY2298 xenograft tumor model. Compared with the monotherapy group, the combination treatment group had a significantly improved tumor inhibition effect and showed no obvious toxic side effects.

[0222] Example 7. In vivo pharmacodynamics of Compound A alone and in combination with Ibrutinib and Venetoclax against patient-derived lymphoma LY-24-0179 in a NOD SCID mouse subcutaneous xenograft tumor PDX model

[0223] 1. Experimental Materials

[0224] Compound A: N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide, prepared using the method described in PCT / CN2022 / 143665, using 0.5% HPMC for drug formulation;

[0225] Ibrutinib: 0.5% HPMC is used for drug preparation;

[0226] Venetoclax: The drug is formulated with 0.5% HPMC;

[0227] The human lymphoma LY-24-0179 model (containing the MYD88 mutation) was originally derived from a tumor sample removed during clinical surgery, and its pathological identification was diffuse large B-cell lymphoma (DLBCL) of the activated B cell type. The collection and use of specimens strictly abide by the ethical laws and regulations of the country, hospital, and company. The passage naming rule is that the tumor sample is inoculated into nude mice as the P0 generation, and further passaged as the P1 generation, and so on. The revived specimen is named FP. The FP3 generation tumor tissue was inoculated subcutaneously into mice, that is, the FP4 generation, which was used for this efficacy test. The patient from whom the sample was derived was a 62-year-old female, and her pathological diagnosis was diffuse large B-cell lymphoma.

[0228] NPG mice, female, 6-7 weeks old, were purchased from Beijing Weitongda Biotechnology Co., Ltd.

[0229] 2. Experimental Methods

[0230] The volume is about 20-30mm 3 The FP3 generation human lymphoma LY-24-0179 tumor tissue was subcutaneously inoculated into the right forelimb axilla of each mouse. On the 30th day after inoculation, the average tumor volume reached 197 mm 3 The group medication was started at 9:00 pm. The specific experimental plan is shown in Table 7-1.

[0231] Table 7-1. Dosing regimen of compound A combined with Ibrutinib or Venetoclax in the LY-24-0179 model Note: Compound A and Ibrutinib or Venetoclax were administered simultaneously.

[0232] The experimental indicator is to determine whether tumor growth is inhibited, delayed, or cured. Tumor diameter is measured twice weekly with a vernier caliper. Tumor volume is calculated using the formula: V = 0.5a × b², where a and b represent the major and minor diameters of the tumor, respectively.

[0233] The tumor inhibition efficacy of a compound was evaluated using TGI (%) or relative tumor growth rate (T / C) (%). TGI (%) reflects the rate of tumor growth inhibition. TGI (%) is calculated as follows: TGI (%) = {[1 - (mean tumor volume at the end of dosing in a given treatment group - mean tumor volume at the start of dosing in that treatment group)] / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)} × 100%.

[0234] Relative tumor growth rate (T / C) (%): Calculated as follows: T / C% = TRTV / CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group). Relative tumor volume (RTV) was calculated based on tumor measurement results using the formula: RTV = Vt / V0, where V0 is the average tumor volume measured at the time of group dosing (i.e., PG-D0), and Vt is the average tumor volume at a single measurement. TRTV and CRTV data were collected on the same day.

[0235] Statistical analysis included the mean and standard error (SEM) of tumor volume at each time point for each group. On day 21 after administration, mice in the treatment group had tumors exceeding 3000 mm 3Euthanasia was performed on PG-D21, and statistical analysis was performed based on this data to assess intergroup differences. The experiment was terminated 2 weeks after drug withdrawal (PG-D42), and tumor recurrence analysis and comparison were performed based on this data. Intergroup differences in tumor volume were analyzed using Dunnett's multiple comparisons test in a one-way ANOVA. Differences in tumor volume between the two groups were analyzed using the t-test. All data were analyzed using GraphPad Prism 9, and p < 0.05 was considered significant.

[0236] 3. Experimental Results

[0237] The pharmacodynamic effects of Compound A alone and in combination with ibrutinib and venetoclax on the patient-derived lymphoma LY-24-0179 xenograft tumor model are shown in Table 7-2 and Figure 9 ; the tumor volume analysis after discontinuation of Compound A alone and in combination with ibrutinib and venetoclax is shown in Table 7-3 and Figure 10 :

[0238] Table 7-2. Evaluation of the anti-tumor efficacy of compound A in combination with ibrutinib or venetoclax in the patient-derived lymphoma LY-24-0179 xenograft tumor model Note: Data in the table are from Day 21; i. Mean ± SD; jp values ​​were calculated based on tumor volume in each animal in the different groups, with the vehicle group as the control. Dunnett's multiple comparisons test was used in a two-way ANOVA. In addition, the comparison between the Compound A + ibrutinib 30 + 25 mg / kg group and the Compound A 30 mg / kg group was statistically analyzed using a t-test, with p=0.07. The comparison between the Compound A + venetoclax 30 + 40 mg / kg group and the Compound A 30 mg / kg group was statistically analyzed using a t-test, with p values ​​less than 0.05. The comparison between the Compound A + ibrutinib 30 + 25 mg / kg group and the ibrutinib 25 mg / kg group was statistically analyzed using a t-test, with p values ​​less than 0.05. The comparison between the Compound A + Venetoclax 30 + 40 mg / kg group and the Venetoclax 40 mg / kg group was performed using t-test for statistical analysis, and the p value was less than 0.05.

[0239] Table 7-3. Analysis of tumor volume two weeks after discontinuation of treatment with Compound A alone and in combination with Ibrutinib and Venetoclax

[0240] k. Mean ± standard deviation of the mean; lp values ​​were statistically analyzed using t-test based on the tumor volume of each animal in different groups, with the compound A 30 mg / kg group as the control.

[0241] The results showed that the average tumor volumes of the Compound A 30 mg / kg, Compound A & Ibrutinib combination treatment group (30 mg / kg + 25 mg / kg) and Compound A & Venetoclax combination treatment group (30 mg / kg + 40 mg / kg) on ​​day 21 after administration were 124 mm 3 , 53mm 3 and 17mm 3 The tumor inhibition rates (TGI) were 102.73%, 105.02%, and 106.51%, respectively. Compared with the vehicle group, the mean tumor volume in each group decreased to varying degrees, with statistically significant differences (p < 0.001). The combination of Compound A and ibrutinib and the combination of Compound A and venetoclax significantly outperformed the ibrutinib and venetoclax alone groups in tumor inhibition (p < 0.05, t-test).

[0242] Each group was observed for 2 weeks after drug administration after 4 weeks of discontinuation. Based on the tumor volume on day 42 after grouping, the tumor recurrence of the Compound A group, the Compound A & Ibrutinib combination group, and the Compound A & Venetoclax combination group 2 weeks after drug withdrawal was compared. The changes in tumor volume of each mouse in the group at different time points are shown in Figure 6-3. At a dose of 30 mg / kg, after 4 weeks of administration, the average tumor volume was 40 mm 3 , no mouse in the whole group had complete tumor regression (0 / 6). After drug withdrawal, the tumors in the compound A group continued to grow. The average tumor volume at the end of the efficacy reached 324mm 3 For the combination of Compound A and Ibrutinib, the average tumor volume was 4 mm after 4 weeks of administration. 3 , 3 mice in the whole group had complete tumor regression (3 / 6). After drug withdrawal, the tumors of 2 mice relapsed and the average tumor volume at the end point of drug efficacy was 58mm 3 For the combination of Compound A and Venetoclax, after 4 weeks of administration, the tumors of all mice in the group completely regressed (6 / 6). After drug withdrawal, the tumors of 4 mice recurred and grew, and the average tumor volume at the end point of the efficacy was 93mm. 3Based on the tumor volume on day 42 after grouping as shown in Table 6-3, the tumor volume after discontinuation and recurrence growth in the Compound A and Ibrutinib combination group and the Compound A and Venetoclax combination group was smaller than that in the Compound A alone group, and there was a statistically significant difference (p values ​​were less than 0.05, t-test).

[0243] Figure 11 shows the body weight and weight changes of each group during this experiment. During the experiment, one mouse in the Compound A + Ibrutinib group experienced a weight loss of over 10%, which subsequently recovered. All other mice showed no significant weight loss. These results demonstrate that tumor-bearing mice tolerated the drug doses well throughout the experiment.

[0244] 4. Experimental Conclusion

[0245] Compound A combined with ibrutinib and Compound A combined with venetoclax showed stronger anti-tumor effects than either treatment alone, without exhibiting significant toxic side effects. Furthermore, Compound A combined with ibrutinib and Compound A combined with venetoclax showed stronger effects in preventing tumor recurrence and growth compared to Compound A alone.

Claims

1. A use of a MALT1 inhibitor and an anticancer agent in combination in the preparation or treatment of a medicament for a tumor disease, wherein: The MALT1 inhibitor is N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide or a pharmaceutically acceptable salt thereof.

2. The use according to claim 1, characterized in that The pharmaceutically acceptable salt is selected from sulfate, phosphate, benzenesulfonate, cinnamate, tartrate, ethane-1,2-disulfonate, ethanesulfonate, fumarate, methanesulfonate or p-toluenesulfonate.

3. The use according to claim 1 or 2, characterized in that The anticancer agent is selected from one or more of BTK inhibitors, XPO1 inhibitors, m-TOR inhibitors, PI3K inhibitors, BCL-2 inhibitors or Akt inhibitors.

4. The use according to any one of claims 1 to 3, characterized in that: The anticancer agent is selected from ibrutinib, selinexor, temsirolimus, rapamycin, datolicoxib, venetoclax, navitoclax, cupanisib, apellisib, idelalib or capasitinib; preferably ibrutinib, temsirolimus or venetoclax; Preferably, the BTK inhibitor is selected from ibrutinib; the XPO1 inhibitor is selected from selinexor; the m-TOR inhibitor is selected from temsirolimus or rapamycin; the PI3K inhibitor is selected from datolicoxib, cupanisib, apellisib or adranib; the BCL-2 inhibitor is selected from venetoclax or navitoclax; and the Akt inhibitor is selected from capasitinib.

5. The use according to any one of claims 1 to 4, characterized in that: The tumor disease is selected from diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma, follicular lymphoma, transformed follicular lymphoma, marginal zone lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue lymphoma, advanced or metastatic / refractory solid tumors or chronic lymphocytic leukemia; preferably diffuse large B-cell lymphoma.

6. The use according to any one of claims 1 to 5, characterized in that: The single dose of MALT1 inhibitor ranges from 1 to 1000 mg; preferably 25 to 1000 mg; Alternatively, the MALT1 inhibitor is administered once a day, twice a day, three times a day, once a week, twice a week, or three times a week; Preferably, the MALT1 inhibitor is administered once a day, with a single dose of 25-1000 mg; or administered twice a day, with a single dose of 25-1000 mg.

7. The use according to any one of claims 1 to 6, characterized in that: The single dose of the anticancer agent ranges from 1 to 1000 mg; preferably 50 to 1000 mg; Alternatively, the anticancer agent is administered once a day, twice a day, three times a day, once a week, twice a week, or three times a week; Preferably, the anticancer agent is administered once a day, with a single dose of 50-1000 mg; or twice a day, with a single dose of 50-1000 mg; More preferably, the anticancer agent is selected from ibrutinib, and the single administration dose ranges from 50 to 1000 mg; preferably 50 to 500 mg; Alternatively, the administration frequency of ibrutinib is once a day, twice a day, three times a day, once a week, twice a week, or three times a week; preferably once a day or twice a day; More preferably, ibrutinib is administered once a day, with a single dose of 50-500 mg; or twice a day, with a single dose of 50-500 mg; Or, more preferably, the anticancer agent is selected from venetoclax, and the single administration dose ranges from 20 to 1000 mg; preferably 20 to 500 mg; Alternatively, the administration frequency of venetoclax is once a day, twice a day, three times a day, once a week, twice a week, or three times a week; preferably once a day or twice a day; More preferably, venetoclax is administered once a day, with a single dose of 20-500 mg; or twice a day, with a single dose of 20-500 mg.

8. The use according to any one of claims 1 to 7, characterized in that The MALT1 inhibitor and the anticancer agent are used simultaneously, concurrently, independently or sequentially.

9. A combined preparation or pharmaceutical composition comprising: (a) one or more MALT1 inhibitors; (b) one or more anticancer agents; The MALT1 inhibitor is N-(3-chloro-4-(2H-1,2,3-triazol-2-yl)phenyl)-1-(2-oxo-1,2-dihydrobenzo[cd]indol-6-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide or a pharmaceutically acceptable salt thereof; Preferably, the pharmaceutically acceptable salt is selected from sulfate, phosphate, benzenesulfonate, cinnamate, tartrate, ethane-1,2-disulfonate, ethanesulfonate, fumarate, methanesulfonate or p-toluenesulfonate; The anticancer agent is selected from BTK inhibitors, XPO1 inhibitors, m-TOR inhibitors, PI3K inhibitors, BCL-2 inhibitors or Akt inhibitors; Ibrutinib, selinexor, temsirolimus, rapamycin, datolicoxib, venetoclax, navitoclax, cupanisib, apellisib, idelalib or capasitinib are preferred; ibrutinib, temsirolimus or venetoclax are preferred; More preferably, the BTK inhibitor is selected from ibrutinib; the XPO1 inhibitor is selected from selinexor; the m-TOR inhibitor is selected from temsirolimus or rapamycin; the PI3K inhibitor is selected from datolicoxib, cupanisib, apellisib or adranib; the BCL-2 inhibitor is selected from venetoclax or navitoclax; and the Akt inhibitor is selected from capasitinib. When the combination is in the form of a pharmaceutical composition, it also contains one or more pharmaceutically acceptable carriers, excipients or diluents.

10. Use of the combined preparation or pharmaceutical composition according to claim 9 in the preparation of a drug for preventing or treating a tumor disease, wherein: The tumor disease is selected from diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma, follicular lymphoma, transformed follicular lymphoma, marginal zone lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue lymphoma, advanced or metastatic / refractory solid tumors or chronic lymphocytic leukemia; preferably diffuse large B-cell lymphoma.

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