CN118161618A

CN118161618A - Combination product comprising a Bcl-2 inhibitor or a Bcl-2/Bcl-xL inhibitor and use thereof

Info

Publication number: CN118161618A
Application number: CN202410147423.XA
Authority: CN
Inventors: 翟一帆; 杨大俊; 方东; 邓静; 王光凤
Original assignee: Yasheng Pharmaceutical Group Hong Kong Co ltd; Suzhou Yasheng Pharmaceutical Co ltd
Current assignee: Yasheng Pharmaceutical Group Hong Kong Co ltd; Suzhou Yasheng Pharmaceutical Co ltd
Priority date: 2020-06-18
Filing date: 2021-06-17
Publication date: 2024-06-11
Also published as: CN113813268A

Abstract

The present invention relates to a combination comprising a Bcl-2 inhibitor or a Bcl-2/Bcl-xL inhibitor and an additional agent, which combination provides use in the prevention and/or treatment of a disease (e.g. cancer, autoimmune disease and inflammatory disease).

Description

Combination product comprising a Bcl-2 inhibitor or a Bcl-2/Bcl-xL inhibitor and use thereof

The invention is a divisional application of Chinese patent application 202110670032.2 with application date of 2021, 6-17, and the original application is named as a combined product containing Bcl-2 inhibitor or Bcl-2/Bcl-xL inhibitor and application thereof.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a Bcl-2 inhibitor or a Bcl-2/Bcl-xL inhibitor-containing combination product and application thereof in preventing and/or treating diseases (such as cancers, autoimmune diseases and inflammatory diseases).

Background

Apoptosis (programmed cell death) is a natural pathway of the body to clear abnormal or unwanted cells, which if affected can lead to the development of various diseases such as cancer.

Anti-apoptotic Bcl-2 proteins are associated with a number of diseases. Bcl-2 family proteins are key regulatory factors in the mitochondrial-mediated apoptosis pathway. Escape from apoptosis is one of the features of human cancers and is a common cause of clinical resistance.

Bruton's tyrosine kinase, BTK) belongs to a member of the Tec family. It consists of a unique N-terminal domain, namely PH (pleckstrinhomology) domain, TH (Tec homology) homology region, SH3 (Src homology 3) domain, SH2 (Src homology 2) domain and catalytic domain, also called SH 1/TK (Src homology1/Tyrosine kinase) domain or kinase domain (Akinleye et al:Ibrutinib and novel BTK inhibitors in clinical development,Journal of Hematology&Oncology 2013,6:59)., and the correct expression of different protein regions of BTK gene has critical role in the function of B cell and various transduction pathways during the normal development of B lymphocyte.

BTK functions are downstream of a variety of receptors, including growth factors, B cell antigens, chemokines and innate immune receptors, to initiate a diverse range of cellular processes such as cell proliferation, survival, differentiation, motility, angiogenesis, cytokine production, antigen expression, etc. BTK therefore plays an important role in many hematopoietic cell signaling pathways, as well as in B cell activation, development, survival and signaling (Kurosaki, molecular MECHANISMS IN B CELL ANTIGEN receptor signaling.curr OP Imm,1997,9 (3): 309-18).

Evidence of the role of BTK in autoimmune disease BTK-deficient mice have been provided (Kil LP,et al:Bruton's tyrosine kinase mediated signaling enhances leukemogenesis in a mouse model for chronic lymphocytic leukemia.Am J Blood Res 2013,3(1):71-83.). in a Chronic Lymphocytic Leukemia (CLL) mouse model by BTK-deficient mice and BTK-full mouse model experiments, BTK-deficient mice completely abrogate chronic lymphocytic leukemia, and BTK overexpression accelerates leukemia onset, increasing mortality.

As research advances in molecular biology, molecular targeted therapies have become a hotspot in medical research (particularly in oncology), where the biological behavior of most tumors is not dominated by a single signaling pathway, but rather multiple signaling pathways act together. Thus, there is a need in the art for a combination regimen and product for different target proteins and/or different signal transduction pathways that can reduce the dosage of a single agent, reduce the toxic side effects of a single agent, and/or act in a synergistic manner to achieve the goal of preventing and/or treating a disease.

Disclosure of Invention

In order to meet the needs in the art, the present invention provides a combination product comprising a Bcl-2 inhibitor or a Bcl-2/Bcl-xL inhibitor and its use in the treatment and/or prevention of diseases (e.g. cancer, autoimmune diseases and inflammatory diseases).

In particular, in a first aspect the invention relates to a combination product comprising a Bcl-2 inhibitor or a Bcl-2/Bcl-xL inhibitor and an additional agent.

In some embodiments, the additional agent is selected from one or more of a BTK inhibitor, an HDAC inhibitor, an MDM2 inhibitor, a JAK inhibitor, an antimetabolite, and an enzyme.

In particular, in another aspect the invention relates to a method of treating a disease (e.g., cancer, autoimmune disease, and inflammatory disease) in a subject in need thereof, comprising administering to the subject a combination product comprising a therapeutically effective amount of a Bcl-2 inhibitor or a Bcl-2/Bcl-xL inhibitor, wherein the subject is not responsive or resistant to a BTK inhibitor. In certain embodiments, the subject is unresponsive or resistant to ibrutinib.

In some embodiments, the Bcl-2 inhibitor is selected from the following compounds, or pharmaceutically acceptable salts or solvates thereof:

In some embodiments, the Bcl-2/Bcl-xL inhibitor is selected from the following compounds or pharmaceutically acceptable salts or solvates thereof:

In some embodiments, the BTK inhibitor is selected from the group consisting of: ibrutinib (Ibrutinib), ICP-022, acartinib (Acalabrutinib (ACP-196)), zebutinib (BGB 3111), ONO/GS-4059, spebrutinib (CC-292 or AVL-292), CNX-774, olmutinib (HM 61713, BI 1482694), M7583, HM71224, PCI-32765racemate (PCI-32765 Racemate), GDC-0853, ONO-4059, zalutinib (Zanubrutinib), RN486, PCI-32765, CGI-1746, QL47, LFM-a13, (±) -zalutinib ((±) -Zanubrutinib), SNS-062, BMS-935177, BMS-986195, PCI29732, btk inhibitor 2, evobrutinib, ibrutinin (Ibrutinib-biotin), BMX-IN-1, GDC-0834 and CB1763.

In some embodiments, the HDAC inhibitor is selected from the group consisting of sidamamine (chidamide).

In some embodiments, the MDM2 inhibitor is selected from the group consisting of APG-115.

In some embodiments, the JAK inhibitor is selected from tofacitinib (tofacitinib).

In some embodiments, the antimetabolite is selected from gemcitabine (gemcitabine).

In some embodiments, the enzyme is selected from asparaginase (ASPARAGINASE).

In some embodiments, the combination product is in the form of a pharmaceutical composition.

In some embodiments, the Bcl-2 inhibitor and the additional agent are each in separate formulations, or wherein the Bcl-2/Bcl-xL inhibitor and the additional agent are each in separate formulations.

In some embodiments, the Bcl-2 inhibitor and the additional agent are administered simultaneously or sequentially, or wherein the Bcl-2/Bcl-xL inhibitor and the additional agent are administered simultaneously or sequentially.

In some embodiments, the combination further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

In some embodiments, the combination product is in the form of a tablet, capsule, granule, syrup, powder, lozenge, sachet, cachet, elixir, suspension, emulsion, solution, syrup, aerosol, ointment, cream, or injection.

In a second aspect the invention relates to the use of a combination product comprising a Bcl-2 inhibitor and an additional agent, or a Bcl-2/Bcl-xL inhibitor and an additional agent, for the manufacture of a medicament for the prevention and/or treatment of a disease selected from the group consisting of cancer, autoimmune disease and inflammatory disease. In some embodiments, the additional agent is selected from one or more of a BTK inhibitor, an HDAC inhibitor, an MDM2 inhibitor, a JAK inhibitor, an antimetabolite, and an enzyme.

In a third aspect the present invention relates to a combination product for use in the prevention and/or treatment of a disease, the combination product comprising a Bcl-2 inhibitor, or a Bcl-2/Bcl-xL inhibitor and an additional agent, and the disease is selected from the group consisting of cancer, autoimmune disease and inflammatory disease. In some embodiments, the additional agent is selected from one or more of a BTK inhibitor, an HDAC inhibitor, an MDM2 inhibitor, a JAK inhibitor, an antimetabolite, and an enzyme.

In a fourth aspect the present invention relates to a method of preventing and/or treating a disease comprising administering to a subject in need thereof a combination comprising a prophylactically and/or therapeutically effective amount of a Bcl-2 inhibitor and an additional agent, or a Bcl-2/Bcl-xL inhibitor and an additional agent, wherein the disease is selected from the group consisting of cancer, autoimmune disease and inflammatory disease. In some embodiments, the additional agent is selected from one or more of a BTK inhibitor, an HDAC inhibitor, an MDM2 inhibitor, a JAK inhibitor, an antimetabolite, and an enzyme.

In some embodiments, the cancer is a hematological malignancy.

Preferably, the hematological malignancy is selected from Acute Myelogenous Leukemia (AML), acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular Lymphoma (FL), chronic Lymphoblastic Leukemia (CLL)/Small Lymphoblastic Lymphoma (SLL), marginal Zone Lymphoma (MZL), chronic Myelogenous Leukemia (CML), mantle Cell Lymphoma (MCL), waldenstrom's Macroglobulinemia (WM), multiple Myeloma (MM), T-cell prolymphocytic leukemia (T-PLL), small Cell Lung Cancer (SCLC), and NK/T-cell lymphoma. More preferably, the hematological malignancy is diffuse large B-cell lymphoma (DLBCL), follicular Lymphoma (FL), and NK/T cell lymphoma.

In some embodiments, the method of preventing and/or treating a disease, wherein the Bcl-2 inhibitor, bcl-2/Bcl-xL inhibitor, or pharmaceutically acceptable salts or solvates thereof in the combination is administered in an amount of about 0.0025 to 1500 mg/day.

In some embodiments, the method of preventing and/or treating a disease, wherein the additional agent in the combination product, or a pharmaceutically acceptable salt or solvate thereof, is administered in an amount of about 0.0025 to 1000 mg/day.

Drawings

Figure 1 shows the inhibition of proliferation of ibrutinib, acartinib or zebuttinib single drug and compound 72 in ibrutinib, acartinib or zebuttinib combination, respectively, in a WST experiment in: DOHH-2 (follicular lymphoma (FL)) (FIG. 1A, FIG. 1B), OCI-LY8 (diffuse large B-cell lymphoma (DLBCL)) (FIG. 1C), and Z-138 (mantle cell lymphoma (MCL)) (FIG. 1D, FIG. 1E, and FIG. 1F).

Figure 2 shows that compound 6 in combination with acartinib enhances apoptosis in primary cells derived from CLL patients.

Figure 3 shows that compound 6 in combination with acartinib has a synergistic antiproliferative effect in human ALL cells.

FIG. 4 shows the effect of compound 6 in combination with acartinib on tumor volume in a DLBCL OCI-LY19 mouse xenograft tumor model (FIG. 4A) and the effect of compound 6 in combination with acartinib on body weight in a DLBCL OCI-LY19 mouse xenograft tumor model (FIG. 4B).

Fig. 5 shows the effect of compound 72 in combination with ibrutinib on tumor volume in a human FL DOHH-2 mouse xenograft tumor model (fig. 5A) and the effect of compound 72 in combination with ibrutinib on body weight in a human FL DOHH-2 mouse xenograft tumor model (fig. 5B).

FIG. 6 shows the effect of compound 72 in combination with ibrutinib or acartinib on tumor volume in a human FL DOHH2 mouse xenograft tumor model (FIG. 6A) and the effect of compound 72 in combination with ibrutinib or acartinib on body weight in a human FL DOHH2 mouse xenograft tumor model (FIG. 6B).

FIG. 7 shows the effect of compound 72 in combination with ibrutinib or acartinib on tumor volume in a human MCL Z138 mouse xenograft tumor model (FIG. 7A) and the effect of compound 72 in combination with ibrutinib or acartinib on body weight in a human MCL Z138 mouse xenograft tumor model (FIG. 7B).

FIG. 8 shows that compound 72 and its metabolic (compound 88) compounds induce apoptosis in NK/TCL cells SNK-6.

FIG. 9 shows that compound 72 induces CASPASE-3 and PARP-1 lysis of NK/TCL cells SNK-6 (FIG. 9A); IP detection compound 72 inhibited the binding of BCL-XL to BAK or BAX (FIG. 9B).

FIG. 10 effect of combination of Compound 72 or its metabolite (Compound 88) with Sidamide, APG-115, gemcitabine, asparaginase and tofacitinib, respectively, on NK/TCL cells SNK-6.

FIG. 11 effect of compound 72 and its metabolite (compound 88) or ABT-263 on tumor volume (FIG. 11A) and on body weight (FIG. 11B) in a model of human NK/TCL cell SNK-6 xenograft tumors.

FIG. 12 effect of compound 72 on tumor volume in NK/TCL cell SNK-6 xenograft tumor model in combination with Sidamide or APG-115 (FIG. 12A) and effect on body weight (FIG. 12B) anti-tumor effect of compound 72 in combination with Sidamide or APG-115 in NK/TCL cell SNK-6 xenograft tumor model (FIG. 12C).

Detailed Description

Definition of the definition

The term "BTK inhibitor" as used herein refers to a substance that inhibits BTK enzyme activity, or a substance that degrades BTK enzyme, or a genetic tool that reduces BTK enzyme levels.

The term "drug resistance" as used herein refers in the present application to resistance or non-response to a therapeutic agent (e.g., a BTK inhibitor). For example, even with treatment by therapeutic agents, the number of tumor cells is still increased.

The term "pharmaceutically acceptable salt" as used herein refers to salts of the free acid or free base, typically prepared by reacting the free base with a suitable organic or inorganic acid or by reacting the acid with a suitable organic or inorganic base. The term may be used for any compound in the present invention. Representative salts include: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camphorsulfonate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, ethanedisulfonate, propionate laurylsulfate (estolate), ethanesulfonate (esylate), fumarate, glucoheptonate, gluconate, glutamate, glycerylarsenate (glycol lylarsanilate), hexylresorcinol (hexylresorcinate), hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandelate, methanesulfonate, methoxybromate, methanesulfonate, meldate (pamoate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, potassium salt, salicylate, sodium salt, stearate, hypohalite, tannic acid, theanine, p-toluenesulfonate, triethyliodide, and trimethamate. When an acidic substituent is present, for example, -COOH, ammonium salts, morpholinium salts, sodium salts, potassium salts, barium salts, calcium salts, and the like may be formed for use in the dosage form. When basic groups are present (e.g. in limonoids or 1, 1-dimethylbiguanides), for example amino groups or basic heteroaryl groups such as pyridyl groups, acidic salts such as hydrochloride, hydrobromide, phosphate, sulfate, trifluoroacetate, trichloroacetate, acetate, oxalate, maleate, pyruvate, malonate, succinate, citrate, tartrate, fumarate, mandelate, benzoate, cinnamate, mesylate, ethanesulfonate, picrate and the like may be formed.

The term "preventing" as used herein refers to a compound or drug that, when used in a disease or disorder (e.g., cancer), reduces the frequency of symptoms of the medical disorder or delays the onset of the medical disorder in a subject as compared to a subject to whom the compound or drug (e.g., a combination product as claimed herein) was not administered.

The term "treating" as used herein refers to alleviating, alleviating or ameliorating a symptom of a disease or disorder, ameliorating a symptom of underlying metabolism, inhibiting a disease or symptom, e.g., preventing a house-hold of a disease or disorder, alleviating a disease or disorder, causing regression of a disease or disorder, alleviating a condition caused by a disease or disorder, or preventing a symptom of a disease or disorder.

The term "cancer" as used herein refers to a neoplasm or tumor caused by abnormal uncontrolled cell growth. Non-limiting examples include those exemplary cancers described in the detailed description of the invention. The term "cancer" includes diseases involving both premalignant and malignant cancer cells.

The term "solvate" as used herein is a combination, physical association, and/or solvate of a compound of the invention with a solvent molecule, e.g., a di-, mono-, hemi-solvate. The compounds of the present invention may be in solvated form with pharmaceutically acceptable solvents such as water, methanol, ethanol, and the like, which do not significantly affect the pharmacological activity or toxicity of the compounds and which as such may function as pharmacological equivalents.

The term "subject" as used herein is meant to include humans (e.g., patients) and animals (e.g., mice, rats, dogs, cats, rabbits, chickens, or monkeys, etc.). When the subject is a human patient (typically weighing 60 kg), the dosages of the present invention can be converted from the conversion factor of the experimental animal (e.g., human dose=mouse dose/12.3) unless otherwise specified (please refer to Kin Tam."Estimating the"First in human"dose-a revisit with particular emphasis on oncology drugs,ADMET&DMPK 1(4)(2013)63-75).. Those skilled in the art can reasonably adjust the dosages according to the general knowledge, the specific weight of the subject, the kind and severity of the disease, and other factors, and the adjusted solutions are within the scope of the claimed solution of the present invention).

The term "effective amount" or "prophylactically and/or therapeutically effective amount" as used herein refers to a sufficient amount (e.g., dose) of a drug or compound administered that will alleviate to some extent one or more symptoms of the disease or disorder being treated. The result may be a reduction and/or alleviation of the cause of a disorder or disease or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic use is an amount of a compound or drug (e.g., a combination product as claimed in the present application) that provides for significant alleviation of the clinical symptoms of a disease or disorder without undue toxic side effects.

The term "dose" as used herein refers to the weight (e.g., milligrams (mg)) of active substance per kilogram (kg) of subject's body weight.

The term "IC ₅₀" as used herein refers to the amount, concentration, or dose of a particular test compound or drug that achieves 50% inhibition of the maximum effect, e.g., inhibition of BCL-2 or BTK, in an assay that measures such effect.

The term "room temperature" as used herein refers to 25 ℃ ± 1 ℃. Meanwhile, unless the experimental temperature is specifically indicated, the experimental temperature is room temperature.

The term "about" as used herein refers to + -10%, more preferably + -5%, and most preferably + -2% of the value modified by the term, so that a person of ordinary skill in the art will be able to determine the scope of the term "about" based on the value modified.

The term "selected from …" as used herein includes one or more of the elements defined in the term. For example, "selected from element a, element B, and element C" may include "selected from one of element a, element B, and element C" and "selected from a plurality of elements a, element B, and element C".

The term "ibrutinib" as used herein is a compound having the structure:

the term "acartinib ((Acalabrutinib (ACP-196)))" as used herein is a compound having the structure:

the term "zebutinib (BGB 3111)" as used herein is a compound having the following structure:

the term "APG-115" as used herein is a compound having the structure:

In a first aspect the invention relates to a combination product comprising or consisting of a Bcl-2 inhibitor and an additional agent. In a first aspect the invention relates to a combination product comprising or consisting of a Bcl-2/Bcl-xL inhibitor and an additional agent. In some embodiments, the additional agent is selected from one or more of a BTK inhibitor, an HDAC inhibitor, an MDM2 inhibitor, a JAK inhibitor, an antimetabolite, and an enzyme.

In another aspect the invention relates to a method of treating a disease (e.g., cancer, autoimmune disease, and inflammatory disease) in a subject in need thereof, comprising administering to the subject a combination comprising a therapeutically effective amount of a Bcl-2 inhibitor or a Bcl-2/Bcl-xL inhibitor, wherein the subject is not responsive or resistant to a BTK inhibitor. IN certain embodiments, the subject does not respond or is resistant to ibrutinib (Ibrutinib), ICP-022, acartinib (Acalabrutinib), zebutinib (BGB 3111), ONO/GS-4059, spebrutinib (CC-292 or AVL-292), CNX-774, olmutinib (HM 61713, BI 1482694), M7583, HM71224, PCI-32765racemate (PCI-32765 Racemate), GDC-0853, ONO-4059, zalutinib (Zanubrutinib), RN486, PCI-32765, CGI-1746, QL47, LFM-a13, (±) -zutinib ((±) -Zanubrutinib), SNS-062, BMS-935177, BMS-986195, PCI 732, btk inhibitor 2, evobrutinib, ibrutinin (Ibrutinib-bin), x-1, GDC-0834, and CB 3. In certain embodiments, the subject is unresponsive or resistant to ibrutinib.

(sometimes simply referred to as "compound 6").

(sometimes simply referred to as "compound 3").

(sometimes simply referred to as "compound 13").

In some embodiments, the Bcl-2/Bcl-xL inhibitor is (R) -2- (1- (3- (4- (N- (4- (4- (3- (2- (4-chlorophenyl) -1-isopropyl-5-methyl-4- (methylsulfonyl) -1H-pyrrol-3-yl) -5-fluorophenyl) piperazin-1-yl) phenyl) sulfamoyl) -2- (trifluoromethylsulfonyl) phenylamino) -4- (phenylthio) butyl) piperidine-4-carbonyloxy) ethyl phosphonic acid (sometimes referred to simply as "compound 72") or a pharmaceutically acceptable salt or solvate thereof, represented by the following structural formula:

Compound 72 binds Bcl-2, bcl-xL, bcl-w protein with high affinity with IC50 of 1.6nM, 4.4nM, 9.3nM, respectively. Compound 72 was able to bind weakly to Mcl-1. Compound 72 effectively reduces the platelet toxicity defect of the first generation BCL-2 inhibitors in the blood circulation by chemical structural modification, but is capable of obtaining specific enzyme activation in the tissues to effectively kill tumor cells. Its platelet toxicity is reduced by 10-30 times, but its activity is about 10 times that of the first-generation BCL-2 inhibitor. Compound 88 is an active metabolite of compound 72. Compound 72 is a second generation new target BCL-2 protein inhibitor.

The Bcl-2/Bcl-xL inhibitor of the invention may also preferably be (R) -1- (3- (4- (N- (4- (4- (3- (2- (4-chlorophenyl) -1-isopropyl-5-methyl-4- (methylsulfonyl) -1H-pyrrol-3-yl) -5-fluorophenyl) piperazin-1-yl) phenyl) -sulfamoyl) -2- (trifluoromethylsulfonyl) phenylamino) -4- (phenylsulfanyl) butyl) piperidine-4-carboxylic acid (sometimes simply referred to as "compound 88") or a pharmaceutically acceptable salt thereof, represented by the following structural formula:

The above-described Bcl-2/Bcl-xL inhibitors in the pharmaceutical compositions of the present invention can be synthesized according to the methods described in WO2014/113413A 1.

In some embodiments, the BTK inhibitor is selected from the group consisting of: ibrutinib (Ibrutinib), ICP-022, acartinib (Acalabrutinib (ACP-196)), zebutinib (BGB 3111), ONO/GS-4059, spebrutinib (CC-292 or AVL-292), CNX-774, olmutinib (HM 61713, BI 1482694), M7583, HM71224, PCI-32765racemate (PCI-32765 Racemate), GDC-0853, ONO-4059, zalutinib (Zanubrutinib), RN486, PCI-32765, CGI-1746, QL47, LFM-a13, (±) -zalutinib ((±) -Zanubrutinib), SNS-062, BMS-935177, BMS-986195, PCI29732, btk inhibitor 2, evobrutinib, ibrutinin (Ibrutinib-biotin), BMX-IN-1, GDC-0834 and CB1763. Further, the BTK inhibitor is selected from: ibrutinib (Ibrutinib), acartinib (Acalabrutinib (ACP-196)) and zebutinib (BGB 3111).

In some embodiments, the HDAC inhibitor is selected from the group consisting of sidamamine.

In some embodiments, the JAK inhibitor is selected from tofacitinib.

In some embodiments, the antimetabolite is selected from gemcitabine.

In some embodiments, the enzyme is selected from asparaginase.

In some embodiments, the Bcl-2 inhibitor and the additional agent are each in separate formulations, or the Bcl-2/Bcl-xL inhibitor and the additional agent are each in separate formulations.

In some embodiments, the time interval for the sequential administration of the Bcl-2 inhibitor or Bcl-2/Bcl-xL inhibitor and the additional agent can be about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 96 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 8 weeks, or about 12 weeks.

In some embodiments, the combination of the invention comprising the Bcl-2 inhibitor or Bcl-2/Bcl-xL inhibitor and the additional agent in the form of a pharmaceutical composition (preferably, each in a separate dosage unit form) may be administered daily as desired, including but not limited to: 1, 2,3, 4, 5 or 6 times.

In some embodiments, the combination of the invention comprising the Bcl-2 inhibitor or Bcl-2/Bcl-xL inhibitor and the additional agent in the form of a pharmaceutical composition (preferably, in dosage unit form) may be administered daily as desired, including but not limited to: 1,2, 3,4, 5 or 6 times.

In some embodiments, the combination product may be applied by: oral, buccal, inhalation spray, sublingual, rectal, transdermal, vaginal mucosal, transmucosal, topical, nasal or enteral administration; injection administration, such as intramuscular, subcutaneous, intramedullary, as well as intrathecal, direct brain administration, in situ, subcutaneous, intraperitoneal, intravenous, intra-articular, intrasternal, intrahepatic, intralesional, intracranial, intraperitoneal, nasal, or intraocular injection or other means of drug delivery.

In some embodiments, the Bcl-2 inhibitor or pharmaceutically acceptable salt or solvate thereof is administered in an amount of about 0.0025-1500 mg/day. Preferably, the daily dosage of the Bcl-2 inhibitor is 1mg、5mg、10mg、20mg、30mg、40mg、50mg、60mg、70mg、80mg、90mg、100mg、150mg、200mg、250mg、300mg、350mg、400mg、450mg、460mg、470mg、480mg、487mg、490mg、500mg、550mg、600mg、650mg、700mg、750mg、800mg、850mg、900mg、950mg、1000mg, and ranges between the amounts, e.g., ,1mg-1000mg、30mg-900mg、30mg-800mg、30mg-900mg、30mg-800mg、30mg-700mg、30mg-600mg、30mg-500mg、30mg-490mg、30mg-487mg, etc., and the additional agent or pharmaceutically acceptable salt or solvate thereof is administered in an amount of about 0.0025-1000 mg/day.

In certain embodiments, the Bcl-2/Bcl-xL inhibitor can be administered in an amount of about 0.005 to about 500 mg/day, preferably about 0.05 to about 365 mg/day, preferably about 0.05 to about 317 mg/day, preferably about 0.05 to about 250 mg/day, more preferably about 0.5 to about 100 mg/day. In certain embodiments, the Bcl-2/Bcl-xL inhibitor is administered in an amount of about 10 mg/week to about 1000 mg/week, about 10 mg/week to about 900 mg/week, about 10 mg/week to about 800 mg/week, about 10 mg/week to about 700 mg/week, about 10 mg/week to about 640 mg/week, about 10 mg/week to about 600 mg/week, about 10 mg/week to about 500 mg/week, about 10 mg/week to about 400 mg/week, about 10 mg/week to about 300 mg/week, about 10 mg/week to about 200 mg/week, or about 20 mg/week to about 100 mg/week, e.g., about 10、15、20、25、30、35、40、50、55、60、65、70、75、80、85、90、95、100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000mg/ weeks, and the additional agent or pharmaceutically acceptable salt or solvate thereof is administered in an amount of about 0.0025-1000 mg/day. In certain embodiments, the Bcl-2/Bcl-xL inhibitor is administered in an amount of about 0.005, 0.05, 0.5, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 milligrams per dose. In certain embodiments, the Bcl-2/Bcl-xL inhibitor is administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, seven times a week.

Preferably, the daily dosage of the additional agent is 10mg、20mg、30mg、40mg、50mg、61mg、70mg、73mg、80mg、90mg、97.6mg、100mg、122mg、150mg、200mg、250mg、300mg、350mg、400mg、450mg、460mg、470mg、480mg、487mg、490mg、500mg,550mg、600mg、650mg、700mg、750mg、800mg、850mg、900mg、950mg、1000mg, and ranges between the amounts, e.g. ,10mg-1000mg、20mg-950mg、30mg-900mg、50mg-650mg、61mg-600mg、70mg-450mg、73mg-400mg、73mg-550mg、73mg-522mg、97.6mg-600mg、97.6mg-600mg、97.6mg-700mg、97.6mg-800mg、97.6mg-950mg、122mg-500mg、122mg-600mg、122mg-700mg、122mg-800mg、97.6mg-900mg、73mg-1000mg, etc.

In a second aspect the invention relates to the use of a combination product comprising a Bcl-2 inhibitor and an additional agent, or a Bcl-2/Bcl-xL inhibitor and an additional agent, for the manufacture of a medicament for the prevention and/or treatment of a disease selected from the group consisting of cancer, autoimmune disease and inflammatory disease. In some embodiments, the disease is not responsive or resistant to BTK inhibitors. IN some embodiments, the disease is unresponsive or resistant to ibrutinib (Ibrutinib), ICP-022, acartinib (Acalabrutinib), zebutinib (BGB 3111), ONO/GS-4059, spebrutinib (CC-292 or AVL-292), CNX-774, olmutinib (HM 61713, BI 1482694), M7583, HM71224, PCI-32765racemate (PCI-32765 Racemate), GDC-0853, ONO-4059, zalutinib (Zanubrutinib), RN486, PCI-32765, CGI-1746, QL47, LFM-a13, (±) -zutinib ((±) -Zanubrutinib), SNS-062, BMS-935177, BMS-986195, PCI 732, btk inhibitor 2, evobrutinib, ibrutinin (Ibrutinib-bin), x-1, GDC-0834, and CB 3. In certain embodiments, the disease is unresponsive or resistant to ibrutinib.

In some embodiments, the Bcl-2 inhibitor is compound 6 or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the Bcl-2/Bcl-xL inhibitor is compound 72 or compound 88 or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the BTK inhibitor is selected from the group consisting of: ibrutinib (Ibrutinib), ICP-022, acartinib (Acalabrutinib), zebutinib (BGB 3111), ONO/GS-4059, spebrutinib (CC-292 or AVL-292), CNX-774, olmutinib (HM 61713, BI 1482694), M7583, HM71224, PCI-32765racemate (PCI-32765 Racemate), GDC-0853, ONO-4059, zalutinib (Zanubrutinib), RN486, PCI-32765, CGI-1746, QL47, LFM-a13, (±) -zalutinib ((-) -Zanubrutinib), SNS-062, BMS-935177, BMS-986195, PCI29732, btk inhibitor 2, evobrutinib, ibrutinib-biotin (Ibrutinib-biotin), BMX-IN-1, GDC-0834 and CB1763. Preferably, the BTK inhibitor is selected from: ibrutinib (Ibrutinib), acartinib (Acalabrutinib) and zebutinib (BGB 3111).

In some embodiments, the JAK inhibitor is selected from tofacitinib.

In some embodiments, the antimetabolite is selected from gemcitabine.

In some embodiments, the enzyme is selected from asparaginase.

In some embodiments, the medicament is in the form of a pharmaceutical composition.

In some embodiments, the Bcl-2 inhibitor and the additional agent are administered simultaneously or sequentially, or the Bcl-2/Bcl-xL inhibitor and the additional agent are administered simultaneously or sequentially.

In some embodiments, the medicament of the invention containing the Bcl-2 inhibitor or Bcl-2/Bcl-xL inhibitor and the additional agent in the form of a pharmaceutical composition (preferably, each in a separate dosage unit form) may be administered daily as desired, including but not limited to: 1, 2, 3,4, 5 or 6 times.

In some embodiments, the medicament of the invention containing the Bcl-2 inhibitor or Bcl-2/Bcl-xL inhibitor and the additional agent in the form of a pharmaceutical composition (preferably, in dosage unit form) may be administered daily as desired, including but not limited to: 1,2, 3, 4, 5 or 6 times.

In some embodiments, the medicament may be administered by: oral, buccal, inhalation spray, sublingual, rectal, transdermal, vaginal mucosal, transmucosal, topical, nasal or enteral administration; injection administration, such as intramuscular, subcutaneous, intramedullary, as well as intrathecal, direct brain administration, in situ, subcutaneous, intraperitoneal, intravenous, intra-articular, intrasternal, intrahepatic, intralesional, intracranial, intraperitoneal, nasal, or intraocular injection or other means of drug delivery.

In some embodiments, the Bcl-2 inhibitor or Bcl-2/Bcl-xL inhibitor or a pharmaceutically acceptable salt or solvate thereof is administered daily for the additional agent or a pharmaceutically acceptable salt or solvate thereof as described in the first aspect of the invention in the above detailed description of the invention.

In some embodiments, the disease is cancer. In some embodiments, the cancer is not responsive or resistant to BTK inhibitors. IN some embodiments, the cancer is unresponsive or resistant to ibrutinib (Ibrutinib), ICP-022, acartinib (Acalabrutinib), zebutinib (BGB 3111), ONO/GS-4059, spebrutinib (CC-292 or AVL-292), CNX-774, olmutinib (HM 61713, BI 1482694), M7583, HM71224, PCI-32765racemate (PCI-32765 Racemate), GDC-0853, ONO-4059, zalutinib (Zanubrutinib), RN486, PCI-32765, CGI-1746, QL47, LFM-a13, (±) -zutinib ((±) -Zanubrutinib), SNS-062, BMS-935177, BMS-986195, PCI 732, btk inhibitor 2, evobrutinib, ibrutinin (Ibrutinib-bin), x-1, GDC-0834, and CB 3. In certain embodiments, the cancer is unresponsive or resistant to ibrutinib.

Further, cancers described in the present invention include, but are not limited to, cancers selected from the group consisting of: adrenal cancer, lymphoepithelial tumors, adenoid cell carcinoma, lymphoma, auditory neuroma, acute lymphoblastic leukemia, acromelanoma, acute myelogenous leukemia, acromioclavine tumor, chronic lymphocytic leukemia, acute eosinophilic leukemia, liver cancer, acute erythrocytic leukemia, small cell lung cancer, acute lymphoblastic leukemia, non-small cell lung cancer, acute megakaryoblastic leukemia, MALT lymphoma, acute monocytic leukemia, malignant fibrous histiocytoma, acute promyelocytic leukemia, malignant peripheral nerve sheath tumor, adenocarcinoma, Malignant hippocampal tumors, adenoid cystic carcinoma, mantle cell lymphoma, adenoma, marginal zone B cell lymphoma, adenomatoid odontogenic tumors, mast cell leukemia, adenosquamous carcinoma, mediastinal germ cell tumors, adipose tissue tumors, breast medullary carcinoma, adrenocortical carcinoma, thyroid medullary carcinoma, adult T cell leukemia/lymphoma, medulloblastoma, invasive NK cell leukemia, melanoma, AIDS-related lymphoma, meningioma, alveolar rhabdomyosarcoma, merck cell carcinoma, alveolar soft tissue sarcoma, mesothelioma, ameloblastic tumor, metastatic urothelial carcinoma, anaplastic large cell lymphoma, mixed Miao's tumor, thyroid undifferentiated carcinoma, Myxoid tumors, angioimmunoblastic T-cell lymphomas, multiple myeloma, angiosmooth muscle lipomas, muscle tissue tumors, angiosarcomas, mycosis fungoides, astrocytomas, myxoid liposarcomas, atypical malformed rhabdomyomas, myxomas, B-cell chronic lymphocytic leukemia, myxosarcoma, B-cell prolymphotic leukemia, nasopharyngeal carcinoma, B-cell lymphomas, schwannomas, basal cell carcinoma, neuroblastomas, biliary tract carcinoma, neurofibromas, bladder carcinoma, neuroma, blastomas, nodular melanoma, bone carcinoma, eye carcinoma, brennan's tumor, oligodendrogliomas, brown tumors, oligodendrogliomas, Burkitt's lymphoma, eosinophil breast cancer, tunica meningioma, brain cancer, optic nerve tumor cancer, carcinoma in situ of oral cavity cancer, osteosarcoma, carcinomatous sarcoma, ovarian cancer, cartilage tumor, suprapulmonary sulcus tumor, cement tumor, papillary thyroid cancer, myeloma, paraganglioma, chondrioma, pineal blastoma, chordoma, pineal tumor, choriocarcinoma, pituitary tumor, choroidal papillary tumor, pituitary adenoma, renal clear cell sarcoma, pituitary tumor, craniopharyngeal tumor, plasmacytoma, cutaneous T cell lymphoma, multiple embryo tumor, cervical cancer, precursor T lymphoblastoma, colorectal cancer, primary central nervous system lymphoma, degoss disease, Primary effusion lymphoma, proliferative microcylinoma, primary peritoneal carcinoma, diffuse large B-cell lymphoma, prostate carcinoma, dysplastic neuroepithelial tumors, pancreatic carcinoma, asexual cell carcinoma, pharyngeal carcinoma, embryonal carcinoma, pseudomyxoma of the peritoneum, endocrine gland tumors, renal cell carcinoma, endoembryo sinus tumor, nephromedullary carcinoma, enteropathy-associated T-cell lymphoma, retinoblastoma, esophageal carcinoma, rhabdomyoma, fetuses, rhabdomyosarcoma, fibroma, richter's syndrome transformation, fibrosarcoma, rectal carcinoma, follicular lymphoma, sarcoma, follicular thyroid carcinoma, schwannoma, gangliocytoma, seminoma, fibromatosis, Gastrointestinal cancer, supporting cell tumor, germ cell tumor, sex cord-gonadal mesothelioma, choriocarcinoma of pregnancy, ring cell carcinoma, giant cell fibroblast tumor, skin carcinoma, bone giant cell tumor, small blue round cell tumor, glioma, small cell carcinoma, glioblastoma multiforme, soft tissue sarcoma, glioma, somatostatin tumor, glioma disease, soot wart, glucagon tumor, spinal tumor, gonadoblastoma, splenic marginal zone lymphoma, granulocytoma, squamous cell carcinoma, estrogenic tumor, synovial sarcoma, gallbladder carcinoma, sezary disease, gastric cancer, small intestine carcinoma, hairy cell leukemia, squamous cell carcinoma, angioblastoma, gastric cancer, Head and neck cancer, T cell lymphoma, vascular epidermoid carcinoma, testicular cancer, hematological malignancy, sarcoma hepatoblastoma, thyroid cancer, hepatosplenic T cell lymphoma, transitional cell carcinoma, hodgkin's lymphoma, laryngeal carcinoma, non-hodgkin's lymphoma, umbilical duct carcinoma, invasive lobular carcinoma, genitourinary system carcinoma, intestinal cancer, urothelial carcinoma, renal carcinoma, uveal melanoma, laryngeal carcinoma, uterine carcinoma, lentigo malignancies, warty carcinoma, lethal midline carcinoma, ocular pathway glioma, leukemia, vulval carcinoma, testicular stromal cell tumor, vaginal carcinoma, liposarcoma, waldenstrom's macroglobulinemia (Waldenstrom macroglobulinemia, WM), lung cancer, adenolymphoma, lymphangioma, nephroblastoma, and lymphangiosarcoma.

Preferably, the cancer is selected from: acute monocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia mixed lineage leukemia, NUT midline cancer, multiple myeloma, small cell lung cancer, neuroblastoma, burkitt lymphoma, cervical cancer, esophageal cancer, ovarian cancer, colorectal cancer, prostate cancer, and breast cancer.

Preferably, the cancer is a hematological malignancy.

More preferably, the hematological malignancy is selected from: acute Myelogenous Leukemia (AML), acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular Lymphoma (FL), chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Lymphoma (SLL), marginal Zone Lymphoma (MZL), chronic Myelogenous Leukemia (CML), mantle Cell Lymphoma (MCL), waldenstrom's Macroglobulinemia (WM), multiple Myeloma (MM), T-prolymphocytic leukemia (T-PLL), small Cell Lung Cancer (SCLC), and NK/T-cell lymphoma. More preferably, the hematological malignancy is selected from diffuse large B-cell lymphoma (DLBCL), follicular Lymphoma (FL), and NK/T cell lymphoma.

Preferably, the cancer is a B cell proliferative disease. More preferably, the B cell proliferative disease is selected from: diffuse large B cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, B cell pre-lymphocytic leukemia, lymphoplasmacytoid lymphoma/Waldenstem macroglobulinemiaMacrolobulinema), splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B cell lymphoma, lymph node marginal zone B cell lymphoma, mantle cell lymphoma, mediastinal (thymus) large B cell lymphoma, intravascular large B cell lymphoma, primary exudative lymphoma, burkitt lymphoma (Burkittlymphoma)/leukemia, and lymphomatoid granulomatosis.

In some embodiments, the disease is an autoimmune disease. In some embodiments, the autoimmune disease is not responsive or resistant to a BTK inhibitor. IN certain embodiments, the subject does not respond or is resistant to ibrutinib (Ibrutinib), ICP-022, acartinib (Acalabrutinib), zebutinib (BGB 3111), ONO/GS-4059, spebrutinib (CC-292 or AVL-292), CNX-774, olmutinib (HM 61713, BI 1482694), M7583, HM71224, PCI-32765racemate (PCI-32765 Racemate), GDC-0853, ONO-4059, zalutinib (Zanubrutinib), RN486, PCI-32765, CGI-1746, QL47, LFM-a13, (±) -zutinib ((±) -Zanubrutinib), SNS-062, BMS-935177, BMS-986195, PCI 732, btk inhibitor 2, evobrutinib, ibrutinin (Ibrutinib-bin), x-1, GDC-0834, and CB 3. In certain embodiments, the autoimmune disease is unresponsive or resistant to ibrutinib.

Further, the autoimmune diseases described in the present invention include, but are not limited to, autoimmune diseases selected from the group consisting of: inflammatory bowel disease, arthritis, lupus, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, still's disease, juvenile arthritis, diabetes, myasthenia gravis, hashimoto' sthyroiditis, ord's thyroiditis, graves' disease, rheumatoid arthritis syndromeSynrome), multiple sclerosis, infectious neuronal inflammation (Guillain-barre syndome), acute disseminated encephalomyelitis, addison's disease, ocular clonus-myoclonus syndrome, ankylosing spondylitis, antiphospholipid antibody syndrome, aplastic anemia, autoimmune hepatitis, celiac disease (coeliac disease), goodpasture's syndome, idiopathic thrombocytopenic purpura, optic neuritis, scleroderma, primary biliary cirrhosis, lyter's syndrome (Reiter's syndome), high-safety arteritis (Takayasu ' S ARTERITIS), temporal arteritis, warm autoimmune hemolytic anemia, wegener ' sgranulomatosis), psoriasis, alopecia, betty's disease, chronic atopic fatigue, familial nerve dysfunction, autonomic nerve dysfunction, bladder dysfunctions, and neuromyelitis, and dystonia.

In some embodiments, the disease is an inflammatory disease. In some embodiments, the inflammatory disease is not responsive or resistant to BTK inhibitors. IN certain embodiments, the subject does not respond or is resistant to ibrutinib (Ibrutinib), ICP-022, acartinib (Acalabrutinib), zebutinib (BGB 3111), ONO/GS-4059, spebrutinib (CC-292 or AVL-292), CNX-774, olmutinib (HM 61713, BI 1482694), M7583, HM71224, PCI-32765racemate (PCI-32765 Racemate), GDC-0853, ONO-4059, zalutinib (Zanubrutinib), RN486, PCI-32765, CGI-1746, QL47, LFM-a13, (±) -zutinib ((±) -Zanubrutinib), SNS-062, BMS-935177, BMS-986195, PCI 732, btk inhibitor 2, evobrutinib, ibrutinin (Ibrutinib-bin), x-1, GDC-0834, and CB 3. In certain embodiments, the inflammatory disease is unresponsive or resistant to ibrutinib.

Further, the inflammatory diseases described in the present invention include, but are not limited to, inflammatory diseases selected from the group consisting of: asthma, appendicitis, blepharitis, bronchiolitis, bronchitis, bursitis, cervicitis, cholangitis, cholecystitis, colitis, conjunctivitis, cystitis, dacryocystitis, dermatitis, dermatomyositis, encephalitis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, hepatitis, suppurative sweat gland, laryngitis, mastitis, meningitis, myelitis myocarditis, myositis, nephritis, oophoritis, orchitis, osteomyelitis, otitis, pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis, pleurisy, phlebitis, localized pneumonia (pneumatinitis), pneumonia (pneumonia), proctitis, prostatitis, pyelonephritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, tendinitis, tonsillitis, uveitis, colpitis, vasculitis or vulvitis.

In a third aspect the present invention relates to a combination product for use in the prevention and/or treatment of a disease selected from the group consisting of cancer, autoimmune diseases and inflammatory diseases, comprising a Bcl-2 inhibitor or a Bcl-2/Bcl-xL inhibitor and said additional agent. Further, the cancers, autoimmune diseases and inflammatory diseases include, but are not limited to, those as described in the second aspect of the invention in the above detailed description of the invention. Further, the disease is not responsive or resistant to BTK inhibitors (e.g., ibrutinib).

In some embodiments, the additional agent is selected from one or more of a BTK inhibitor, an HDAC inhibitor, an MDM2 inhibitor, a JAK inhibitor, an antimetabolite, and an enzyme. In some embodiments, the BTK inhibitor is selected from the group consisting of: ibrutinib (Ibrutinib), ICP-022, acartinib (Acalabrutinib), zebutinib (BGB 3111), ONO/GS-4059, spebrutinib (CC-292 or AVL-292), CNX-774, olmutinib (HM 61713, BI 1482694), M7583, HM71224, PCI-32765racemate (PCI-32765 Racemate), GDC-0853, ONO-4059, zalutinib (Zanubrutinib), RN486, PCI-32765, CGI-1746, QL47, LFM-a13, (±) -zalutinib ((-) -Zanubrutinib), SNS-062, BMS-935177, BMS-986195, PCI29732, btk inhibitor 2, evobrutinib, ibrutinib-biotin (Ibrutinib-biotin), BMX-IN-1, GDC-0834 and CB1763. Preferably, the BTK inhibitor is selected from: ibrutinib (Ibrutinib), acartinib (Acalabrutinib) and zebutinib (BGB 3111).

In some embodiments, the JAK inhibitor is selected from tofacitinib.

In some embodiments, the antimetabolite is selected from gemcitabine.

In some embodiments, the enzyme is selected from asparaginase.

In a fourth aspect the present invention relates to a method of preventing and/or treating a disease selected from the group consisting of cancer, autoimmune diseases and inflammatory diseases, comprising administering to a subject in need thereof a combination comprising a prophylactically and/or therapeutically effective amount of a Bcl-2 inhibitor and said additional agent, or a Bcl-2/Bcl-xL inhibitor and said additional agent. Further, the cancers, autoimmune diseases and inflammatory diseases include, but are not limited to, those as described in the second aspect of the invention in the above detailed description of the invention. Further, the disease is not responsive or resistant to BTK inhibitors (e.g., ibrutinib).

In some embodiments, the JAK inhibitor is selected from tofacitinib.

In some embodiments, the antimetabolite is selected from gemcitabine.

In some embodiments, the enzyme is selected from asparaginase.

In some embodiments, the Bcl-2 inhibitor and the additional agent are in the form of a pharmaceutical composition, or the Bcl-2/Bcl-xL inhibitor and the additional agent are in the form of a pharmaceutical composition.

In some embodiments, the Bcl-2 inhibitor or Bcl-2/Bcl-xL inhibitor and the additional agent in the form of a pharmaceutical composition (preferably, each in separate dosage unit form) may be administered daily as desired, including but not limited to: 1,2, 3, 4, 5 or 6 times.

In some embodiments, the Bcl-2 inhibitor or Bcl-2/Bcl-xL inhibitor in the form of a pharmaceutical composition (preferably, in dosage unit form) and the additional agent may be administered daily as desired, including but not limited to: 1, 2, 3, 4, 5 or 6 times.

In some embodiments, the Bcl-2 inhibitor or Bcl-2/Bcl-xL inhibitor and the additional agent can be administered by: oral, buccal, inhalation spray, sublingual, rectal, transdermal, vaginal mucosal, transmucosal, topical, nasal or enteral administration; injection administration, such as intramuscular, subcutaneous, intramedullary, as well as intrathecal, direct brain administration, in situ, subcutaneous, intraperitoneal, intravenous, intra-articular, intrasternal, intrahepatic, intralesional, intracranial, intraperitoneal, nasal, or intraocular injection or other means of drug delivery.

In some embodiments, the daily dose of the Bcl-2 inhibitor is 0.017mg/kg、0.083mg/kg、0.17mg/kg、0.33mg/kg、0.5mg/kg、0.67mg/kg、0.83mg/kg、1mg/kg、1.16mg/kg、1.33mg/kg、1.5mg/kg、1.67mg/kg、2.5mg/kg、3.33mg/kg、4.17mg/kg、5mg/kg、5.83mg/kg、6.67mg/kg、7.5mg/kg、7.67mg/kg、7.83mg/kg、8mg/kg、8.12mg/kg、8.16mg/kg、8.33mg/kg、9.17mg/kg、10mg/kg、10.83mg/kg、11.66mg/kg、12.5mg/kg、13.33mg/kg、14.17mg/kg、15mg/kg、15.83mg/kg、16.67mg, and ranges between the administered doses, e.g., ,0.017mg-16.67mg/kg、0.083mg-16.67mg/kg、0.17mg-16.67mg/kg、0.33mg-16.67mg/kg、0.5mg-15mg/kg、0.5mg-13.33mg/kg、0.5mg-11.67mg/kg、0.5mg-10mg/kg、0.5mg-8.33mg/kg、0.5mg-8.16mg/kg、0.5mg-8.12mg/kg, etc., and the daily dose of the additional agent is 0.17mg/kg、0.33mg/kg、0.5mg/kg、0.67mg/kg、0.83mg/kg、1mg/kg、1.02mg/kg、1.17mg/kg、1.22mg/kg、1.33mg/kg、1.5mg/kg、1.62mg/kg、1.67mg/kg、2.03mg/kg、2.5mg/kg、3.33mg/kg、4.17mg/kg、5mg/kg、5.83mg/kg、6.67mg/kg、7.5mg/kg、7.67mg/kg、7.83mg/kg、8mg/kg、8.17mg/kg、8.33mg/kg, and ranges between the doses, e.g., ,0.17mg-8.33mg/kg、0.33mg-7.5mg/kg、0.5mg-6.67mg/kg、0.83mg-5.83mg/kg、1mg-5mg/kg、1.02mg-5mg/kg、1.16mg-4.17mg/kg、1.22mg-3.33mg/kg、1.22mg-2.5mg/kg、1.22mg-2.03mg/kg、1.62mg-8.33mg/kg、1.62mg-8mg/kg、1.62mg7.5mg/kg、1.62mg-5mg/kg、1.62mg-2.5mg/kg、1.22mg-1.62mg/kg, etc. In certain embodiments, the Bcl-2/Bcl-xL inhibitor is administered in an amount of about 10 mg/week to about 1000 mg/week, about 10 mg/week to about 900 mg/week, about 10 mg/week to about 800 mg/week, about 10 mg/week to about 700 mg/week, about 10 mg/week to about 640 mg/week, about 10 mg/week to about 600 mg/week, about 10 mg/week to about 500 mg/week, about 10 mg/week to about 400 mg/week, about 10 mg/week to about 300 mg/week, about 10 mg/week to about 200 mg/week, or about 20 mg/week to about 100 mg/week, e.g., about 10、15、20、25、30、35、40、50、55、60、65、70、75、80、85、90、95、100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000mg/ weeks.

Finally, WO 2018/027097 is incorporated herein by reference in its entirety and for all purposes.

Detailed Description

The present invention will be further illustrated by the following specific examples and comparative examples, but it should be understood that these examples and comparative examples are merely for the purpose of more detailed description and should not be construed as limiting the invention in any way.

Example 1 Experimental materials used in the invention and sources thereof

(1) Experimental reagent and general preparation thereof

Ibrutinib was purchased from Selleck (cat.s2680, china) or Aikonchem (south tokyo, cat.2645743, china). For in vivo studies, ibrutinib (Selleck) was formulated in 5% DMSO (Sigma, cat.d 8418) and 95% (20% h- β -CD); for in vitro studies ibrutinib was dissolved in DMSO to a 10mM stock solution and diluted to the indicated concentration in serum-free medium.

Acartinib was purchased from Selleck (China, cat.S8116) or Aikonchem (Nanjing, cat.AK2017-11533-001, china). For in vivo studies, acartinib (Selleck) was formulated in 5% DMSO (Sigma, cat.d 8418) and 95% (20% h- β -CD); for in vitro studies, acartinib was dissolved in DMSO to a 10mM stock solution and diluted to the indicated concentration in serum-free medium.

Zebutinib was purchased from Aikonchem (south tokyo, cat.2645743). For in vitro studies, zebutinib was dissolved in DMSO to a 10mM stock solution and diluted to the indicated concentration in serum-free medium.

Compound 6 (R16 JA450041-A5 s) was synthesized from ASCENTAGE PHARMA (Jiangsu Seisaku). Compound 6 was formulated in 10% ethanol (Sinopharma, shanghai, cat.10009257, china), 30% polyethylene glycol 400 and 60% Phosal 50PG (Lipoid GmbH, germany, cat.368315-31700201006) for in vivo studies. Compound 6 used in vitro was dissolved in DMSO to 10mM stock solution and diluted to the indicated concentration with serum free medium.

Compound 72 (P compound 72-DP-201410B or C13091003-J18001W) was synthesized from ASCENTAGE PHARMA (Jiangsu Seisaku). Compound 72 was placed in a precooled mortar, placed on ice to maintain low temperature, 10% peg-400Sigma, st.louis, mo, cat.91893-1L-F) was added to grind to homogeneity, 5% Cremophor EL (Sigma, cat.c 5135-500G) was added to continue grinding until drug was completely homogeneous in solution, 0.1M NaOH (national pharmaceutical systems chemical company, cat.10019718) was added dropwise to the solution to clarify the solution, pH was adjusted to 6-8 with 0.1M HCl (national pharmaceutical systems chemical company, cat.1011018), volume was set with PBS (genenome, chinese, cat.gnm 14190), transferred to a dispensing bottle after sufficient mixing, placed on ice, filtered with a 0.22 μm filter membrane before use, and used within 1 hour.

Antibodies for use in the present invention were purchased from fomis (BD, china): CD5 stream antibody (PE) (Cat.555353), CD19 stream antibody (PE-Cy 7) (Cat.560728).

APG-115 was synthesized from ASCENTAGE PHARMA (Jiangsu sub-stances) and for in vitro studies APG-115 was dissolved in DMSO (Sigma, cat. D8418) to a 10mM stock solution and diluted to the indicated concentration in serum-free medium.

Sidamine was purchased from Jiangsu Ai Kang Shengwu pharmaceutical development Co., ltd (China, cat.1616493-44-7), and for in vitro studies, sidamine was dissolved in DMSO (Sigma, cat.D8418) to a 10mM stock solution and diluted to the indicated concentration in serum-free medium.

Tofacitinib was purchased from Jiangsu Ai Kang Shengwu pharmaceutical development Co., ltd (China, cat.540737-29-9), and for in vitro studies, tofacitinib was dissolved in DMSO (Sigma, cat.D8418) to a 10mM stock solution and diluted to the indicated concentration in serum-free medium.

Gemcitabine was purchased from calico biotechnology limited (cat.95058-81-4, china) and for in vitro studies, gemcitabine was dissolved in DMSO (Sigma, cat.d 8418) to a 10mM stock solution and diluted to the indicated concentration in serum-free medium.

Asparaginase was purchased from Shanghai Aibixin Biotechnology Co., ltd (China, cat.9015-68-3), was dissolved in PBS (Biotechnology Co., shanghai), cat.SD8117) to a stock solution of 2000U/ml for in vitro studies and diluted to the indicated concentration in serum-free medium.

(2) Cell lines

Human Acute Lymphoblastic Leukemia (ALL) Molt-4 cells were purchased from Nanjac, and the cells were cultured in RPMI 1640 medium (GIBCO, china, cat.C11875500 BT) supplemented with 10% fetal bovine serum (GIBCO, australia, cat.10099-141) and 1% penicillin/streptomycin (GENOME, hangzhou, china, cat.GNM15140).

The human B cell lymphoma cell line DOHH-2 was purchased from Nanjac. The human diffuse large B-cell lymphoma cell line OCI-LY19 was purchased from Nanjac, and OCI-LY8 was obtained from Dajun Yang doctor (university of Zhongshan cancer center). All cell lines were tested and identified by STR (short tandem repeat) analysis. DOHH-2 and OCI-LY8 cells were cultured in RPMI 1640 medium (GIBCO, china, cat.C11875500 BT) supplemented with 10% fetal bovine serum (GIBCO, australia, cat.10099-141) and 1% penicillin/streptomycin (GENOME, hangzhou, china, cat.GNM15140). OCI-LY19 cells were cultured in alpha-MEM (GIBCO, china, cat. C12571500 BT) medium containing 20% fetal bovine serum and 1% penicillin/streptomycin. Z-138 cells were cultured in IMDM (GIBCO, china, cat.12200036) medium containing 20% fetal bovine serum and 1% penicillin/streptomycin. Cells were cultured and maintained at 37℃in a humidified incubator containing 5% CO ₂ and 95% air.

Primary Chronic Lymphocytic Leukemia (CLL) patient cancer cells were from a collaborative project with the university of sulzer medical college, and passed the institutional review board of sulzer medical college review and approval according to the declaration of helsinki. Peripheral blood meeting CLL diagnostic criteria was collected after informed consent was obtained from the patient. The collected blood sample is separated by Ficoll Paque density gradient centrifugation to obtain peripheral blood mononuclear cells, and the peripheral blood mononuclear cells are frozen in fetal bovine serum containing 10% of DMSO and stored in liquid nitrogen.

Molt-4 cells (purchased from southern Beijing Corp.) were cultured in RPMI 1640 medium (GIBCO, china, cat. C11875500 BT) supplemented with 10% fetal bovine serum (GIBCO, australia, cat. 10099-141) and 1% penicillin/streptomycin (GENOME, cat. GNM15140, hangzhou, china).

Z138 was purchased from ATCC and cultured in IMEM medium (GIBCO, china, cat.12200036) supplemented with 10% horse serum (GIBCO, australia, cat.16050130) and 1% penicillin/streptomycin (GENOME, hangzhou, china, cat.GNM15140).

NK/TCL (NK/T cell lymphoma) cell lines SNK-1, SNK-6 and SNT-8 are given by the department of Xinhua Hospital affiliated to Shanghai university medical school.

EXAMPLE 2 general Experimental procedure used in the present invention

(1) In vitro cell antiproliferation assay

(1.1) WST experiment

Antiproliferative effects (purchased from Shanghai Li Ji medicine technologies Co., ltd.) were detected by CCK-8 (Cell Counting Kit-8 ) based on water-soluble tetrazolium salts (WST) (please refer to Ishiyama M,Tominaga H,Shiga M et al.,A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt,neutral red and crystal violet.Biol.Pharm.Bull 19(11)1518-1520(1996),Vol.19,No.11 and Tominaga H,Ishiyama M,Ohseto F et al.,A water-soluble tetrazolium salt useful for colorimetric cell viability assay.Anal.Commun.,1999,36,47-50). cells inoculated in 96-well plates and treated with test substances at different concentrations for 72 hours; by using 9 different concentrations of BTK inhibitor (e.g., ibrutinib, wherein 9 concentrations were selected in a gradient of 3 between 10 ^-2 and 10 ²) and 3 different concentrations of compound 6 (see FIG. 1 in particular) for 72 hours, test compound 6 was combined with the drug; each test dose was 3 multiplexed.

Typically, 9 series of doses of the test substance are selected and 100 μl/well is added to a 96-well plate. For the combined experiments, the final volume of 2 subjects was 100 μl/well. 3 wells were made for each test dose. 100 μl of the dilution was added to 3-6 wells of the same plate as the control group, and 3-6 wells were used as the blank. In addition to the blank wells, 100 μl of cell suspension (containing the appropriate number of cells to ensure that the cells of the cell control group just spread over the bottom of the well when detection is required) was added to the same 96-well plate per well. The plates were incubated at 37℃for 72 hours in a CO2 incubator. At the end of the culture, the old solution in the wells to be tested was removed and 100. Mu.l/well of CCK-8 assay (corresponding medium containing 10% CCK-8,5% FBS) was added to the adherent cells. For suspension cells, 20. Mu.l/well CCK-8 stock was added directly. The plates were incubated at 37℃in a CO2 incubator for 2-4 hours.

The OD was measured with a microplate reader (SpectraMax Plus 384,Molecular Devices,LLC, US) A450 nm. Using the average OD value of 3 wells, the percent cell viability was calculated by the following formula:

(O.D. test well-O.D. control well)/(O.D. cell control well-O.D. control well). Times.100.

IC ₅₀ was calculated using the non-linear regression data analysis method of GRAPHPAD PRISM 6.0.0 software.

For the combination experiments, cell viability was calculated after normalization treatment by means of the average OD value of 3 wells of the single drug control. The synergy of 2 compounds was determined by performing an IC50 comparison of the combination curve with the single drug curve in combination with observing whether the combination drug group curve shifted to the left. Meanwhile, the Combination Index (CI) value was calculated by CalcuSyn software (bio oft, UK). Typically, CI <0.9 represents a synergistic combination. CI <0.1 is marked 5+ indicating a very strong synergistic combination, CI between 0.1 and 0.3 is marked 4+ indicating a strong synergistic combination, CI between 0.3 and 0.7 is marked 3+ indicating a moderate synergistic combination.

(1.2) CTG experiment

CTG by ATP-based quantificationLuminescent Cell Viability Assay) experimental methods to detect antiproliferative effects. In 96-well plates, 9 serial concentrations of the test substance were added and allowed to act for 72 hours after cell seeding. In the combined experiment, 9 series of doses of 2 subjects were used in one-to-one correspondence, and the cells were seeded and allowed to act for 72 hours. 3 wells were made for each test dose. A diluent control and a blank control were placed on the same plate. The plates were incubated at 37℃in a CO2 incubator. At the end of the incubation, each test well was charged with CTG reagent (CellTiter-Lumi ^TM luminescence cell viability assay kit, #C0068L) equilibrated to room temperature and subjected to chemiluminescence assay using a microplate reader (Molecular Devices, spectraMax i 3X). Cell viability% = (o.d. test well-o.d. blank well)/(o.d. cell control well-o.d. blank well) ×100 was calculated. IC50 was calculated using the nonlinear regression data analysis method of GRAPHPAD PRISM 9.1.0 software (GraphPad Software inc., san Diego, CA). The drug combination was calculated using Calcusyn 2.11 software (Biosoft, dr. Tc. Zhou) to calculate CI (combination index) values, CI >1 for antagonism, ci=1 for additive, CI <1 for synergy.

(2) Apoptosis detection

After 24 hours of exposure to the test agent, the cells were collected and washed once with pre-chilled PBS. According to the instructions of Annexin V-Alexa Fluor 647/PI apoptosis detection kit (assist in Saint, #40304ES 20), 100. Mu.l of 1 Xbinding buffer was added to resuspend cells, 5. Mu.l of Annexin V-Alexa Fluor 647 was added, mixed well, 10. Mu.l of Propidium Iodide (PI) was added, mixed well, incubated at room temperature for 10-20 minutes in the absence of light, 400. Mu.l of PBS was added, mixed well, and then placed in an ice bath. Flow cytometry (CytoFLEX, BECKMAN) detects apoptosis.

(3) Immunoblotting (Western blotting) experiment

After the cells were subjected to the test substance, the cells were collected at a set time point and washed once with pre-chilled PBS. Cell pellets were lysed using RIPA lysates (bi-cloudy, # P0013B) containing 1% PMSF (bi-cloudy, # ST 506), 1% phosphatase inhibitor (assist in the holy, #20109-a, # 20109-B) and 1% protease inhibitor (assist in the holy, #20124ES 03). Protein concentration was measured by BCA protein concentration measurement kit (bi yun, #p0011). Tumor lysates (20-50. Mu.g) were separated by 8-12% SDS-PAGE (Acry/Bis 30% Solution (37.5:1), ind. # B546018). The isolated proteins were transferred to PVDF membrane (GE, # 10600023). After blocking the PVDF membrane with 1-4% BSA (GENVIEW, # FA 016) buffer for 30 min to 1h at room temperature, the diluted primary antibodies (PARP, CST, #9532; CASPASE-3, CST, #9665; beta-ACTIN, CST, # 4970) were incubated overnight on a shaker at 4℃with 1 XTBST (TBS, bio-technology, # B548105) containing 1-4% BSA. The membrane was washed 3 times with 1 XTBST. Membranes were incubated with horseradish peroxidase-labeled secondary antibodies (Goat Anti Rabbit, union, # GAR0072; goat Anti Mouse, union, # GAM 0072) for 1 hour at room temperature. The membrane was washed 3 times with 1 XTBST. Signal generation and detection was performed using ECL chemiluminescent hypersensitivity chromogenic kit (YEASEN, #36208ES 76) and chemiluminescent imaging system (c 300, azure).

(4) Co-immunoprecipitation assay (Co-Immunoprecipitation, co-IP)

After the cells were subjected to the test substance, the cells were collected by centrifugation after 24 hours and washed once with pre-chilled PBS. Cell pellets were lysed using IP and western lysates (bi-cloudy, #p0013) containing 1% PMSF, 1% phosphatase inhibitor and 1% protease inhibitor. Protein concentration was detected by BCA protein concentration detection kit. The protein concentration of the cell lysate was uniformly adjusted to 2.5ug/ul, input was removed from the control group, and the remainder was added with antibodies (BCL-XL, CST, # 2764) at a ratio of 1:100, and the mixture was spun overnight at 4 ℃. Washing the Beads with IP and western lysate, adding a mixture of antibody and cell lysate, rotating at 4deg.C for 4 hr, and washing the Beads after rotation. Adding 1xLoading Buffer (Biyun Tian, product number: P0015) into Beads at equal volume, adding 5xLoading Buffer,100 deg.C into Input, decocting for 5min, centrifuging at high speed, and collecting supernatant. Proteins (20-50. Mu.g) were separated by 8-12% SDS-PAGE and transferred to PVDF membrane. The target proteins were detected by conventional Western blotting methods, and the primary antibodies were BAX (CST, # 5023) and BAK (CST, # 12105).

(5) In vivo pharmacodynamic experiment evaluation method

Establishing a subcutaneous xenograft tumor model of a human tumor immunodeficiency mouse by a cell inoculation method: tumor cells in logarithmic growth phase were collected, resuspended in1 XPBS after counting, and the cell suspension concentration was adjusted to 2.5-5X 10 ⁷/mL. Tumor cells were inoculated subcutaneously into the right dorsal aspect of immunodeficient mice with a 1mL syringe (No. 4 needle), 5-10X 106/0.2 mL/mouse. All animal experimental procedures strictly follow Ji Ma gene stock company and the laboratory animal use and management specifications of the su state sub-flourishing pharmaceutical industry. The calculation of the related parameters refers to the technical guidelines of non-clinical research of cytotoxic anti-tumor drugs of CFDA in China. The experimental animal sources are shown in the following table:

Table 1 animal sources for experiments

Animal body weight and tumor size were measured twice a week during the experiment. Tumor growth was observed periodically and when tumors grew to an average volume of 100-200mm ³, they were administered randomly in groups according to tumor size and mouse weight. The animal is observed every day for death. Conventional monitoring includes the effect of tumor growth and administration on normal animal behavior, including activity, feeding and drinking conditions, weight gain or loss, eyes, hair and other abnormalities in experimental animals. Both the death and clinical symptoms observed during the experiment are recorded in the raw data. The whole administration, the measurement of the body weight of the mice and the tumor volume were performed in an ultra clean bench. Plasma and tumor tissue were collected after the end of the last dose, weighed and photographed for recording, as required by the protocol. The plasma and tumor samples were stored frozen at-80℃until use.

The calculation formula of Tumor Volume (TV) is: tv=a×b ²/2. Wherein a and b represent tumor measurement length and width, respectively. The relative tumor volume (relative tumor volume, RTV) was calculated as: rtv=v _t/V₁. Where V ₁ is the tumor volume at the time of group administration and V _t is the tumor volume at the time of day of administration. The evaluation index of the anti-tumor activity is relative tumor proliferation rate T/C (%), and the calculation formulas are respectively as follows: relative tumor proliferation rate T/C (%) = (T _RTV/C_RTV)×100％,T_RTV is RTV in treatment group, C _RTV is RTV in vehicle control group; tumor remission rate (%) is the number of SD (disease stability), PR (partial regression of tumor) and CR (complete regression of tumor) occurring in tumor bearing mice after treatment divided by the total number of mice in this group x 100%.

Animal weight change (Change of body weight,%) = (measured weight-weight at group)/weight at group x 100%.

Efficacy evaluation criteria: according to the guidelines of non-clinical research technology of cytotoxic antitumor drugs (11 months in 2006) of CFDA of China, the T/C (%) value is less than or equal to 40%, and p is effective by statistical analysis. A drug dose is considered severely toxic if the mice lose more than 20% weight or the drug-related mortality exceeds 20%.

The synergy analysis uses the following formula: cofactor= ((a/C) × (B/C))/(AB/C); RTV values for a = a drug single group; RTV values for B = B drug single group; RTV values for c=vehicle control, RTV values (Clarke R.Issues in experimental design and endpoint analysis in the study of experimental cytotoxic agents in vivo in breast cancer and other models[J].Breast Cancer Research&Treatment,1997,46(2-3):255-278). for ab=ab combination, if the cofactor >1, have a synergistic effect; if the cofactor=1, then there is an additive effect; if the cofactor is <1, then antagonism is provided.

(6) Tumor xenograft model experiments

NCG mice, 4-6 week old, female, weighing 16-18+ -20% g, were offered by Jiangsu Jiuyaokang biotechnology Co., ltd (production license number: SCXK (Su) 2018-0008, animal eligibility number: 202105885, 202107138). The right back of NCG immunodeficient mice was subcutaneously injected with SNK-6 tumor cells 3X 10 ⁶/mouse (30% Matrigel; corning, # 354234) to model xenograft tumors. When the tumors reached the appropriate size (100-200 mm ³), animals were randomized according to the tumor volume of the animals, 5 animals per group, and dosing was started on the day of the grouping. Animal body weight and tumor size were measured twice a week during the experiment. Daily observations record clinical symptoms. Laboratory animals were kept in SPF-grade laboratories, inc. of Shanghai Bikeside biotechnology, inc. (use license number: SYXK (Shanghai) 2018-0026). The protocols involved in animal management and Use were all approved and approved by the Institutional ANIMAL CARE AND Use Committee (IACUC) of Shanghai Bikeside biosciences, inc. laboratory animal management and Use Committee. Tumor volume, animal weight change, efficacy evaluation criteria, and synergy analysis refer to example item (5).

EXAMPLE 3 preparation of Bcl-2 inhibitors (Compounds 3, 6 and 13)

(1) Synthesis of 2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((6- (4-chlorophenyl) spiro [3.5] non-6-en-7-yl) methyl) piperazin-1-yl) -N- ((3-nitro-4- (((tetrahydro-2H-pyran-4-yl) methyl) amino) phenyl) sulfonyl) benzamide (Compound 3)

2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((6- (4-chlorophenyl) spiro [3.5] non-6-en-yl) mixture 7-yl) methyl) piperazin-1-yl) benzoic acid (1.75 g,3 mmol), 3-nitro-4- (((tetrahydro-2H-pyran-4-yl) methyl) amino) benzenesulfonamide (1.43 g, 4.5) EDCI (1.15 g,6 mmol) and 4- (N, N-dimethylamino) pyridine (550 mg,4.5 mmol) were reacted with dichloromethane (40 ml) overnight at room temperature, then water was added. The aqueous layer was extracted with dichloromethane. The combined organic layers were washed with brine, concentrated and purified by silica gel column to give 2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((6- (4-chlorophenyl) spiro [3.5] non-6-en-7-yl) methyl) piperazin-1-yl) -N- ((3-nitro-4- (((tetrahydro-2H-pyran-4-yl) methyl) amino) phenyl) sulfonyl) benzamide (1.7 g, 64.4%) as a yellow solid.

¹ H NMR (400 MHz, methanol -d4)δ8.70(d,J＝2.3Hz,1H),8.01(d,J＝2.7Hz,1H),7.87(d,J＝9.2,2.3Hz,1H),7.66(d,J＝8.9Hz,1H),7.55(d,J＝2.7Hz,1H),7.47(d,J＝3.4Hz,1H),7.38(d,J＝8.4Hz,2H),7.10(d,J＝8.4Hz,2H),6.97(d,J＝9.2Hz,1H),6.77(dd,J＝8.9,2.4Hz,1H),6.44(d,J＝3.4Hz,1H),6.34(d,J＝2.4Hz,1H),4.02-3.94(m,3H),3.66(s,3H),3.49-3.38(m,2H),3.41-3.25(m,7H),2.42(s,3H),2.26(s,3H),2.00-1.67(m,4H),1.45-1.38(m,2H).)

(2) (R) -N- ((4- (((1, 4-bis) s)Synthesis of Alkan-2-yl-methyl) amino) -3-nitrophenyl-sulfonyl) -2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy) -4- (4- ((6- (4-chlorophenyl) spiro [3.5] non-6-en-7-yl) methyl) piperazin-1-yl) benzamide (Compound 13)

The title compound was prepared using a procedure analogous to that described for the synthesis of compound 3.

¹ H NMR (400 MHz, methanol -d4)δ8.66(d,J＝2.4Hz,1H),7.99(d,J＝2.4Hz,1H),7.84(dd,J＝9.2,2.4Hz,1H),7.64(d,J＝8.9Hz,1H),7.51(d,J＝2.4Hz,2H),7.45(d,J＝3.3Hz,1H),7.37(d,J＝8.4Hz,2H),7.10(d,J＝8.4Hz,2H),6.94(d,J＝9.2Hz,1H),6.76(dd,J＝8.9,2.3Hz,1H),6.40(d,J＝3.3Hz,1H),6.36(d,J＝2.3Hz,1H),3.87(dd,J＝11.8,4.2Hz,3H),3.83-3.70(m,3H),3.67(s,2H),3.62(dd,J＝11.7,2.9Hz,1H),3.51-3.41(m,2H),3.40-3.35(m,1H),3.29(dq,J＝3.2,1.6Hz,1H),2.41(s,2H),2.26(s,2H),2.00-1.77(m,6H).)

Likewise, preparation is effected analogously to the process described for the synthesis of compound 13(Compound 6), see in particular WO2018/027097.

EXAMPLE 4 preparation of Bcl-2/Bcl-xL inhibitors (Compounds 72 and 88)

Compound 72: (R) -2- (1- (3- (4- (N- (4- (4- (3- (2- (4-chlorophenyl) -1-isopropyl-5-methyl-4- (methylsulfonyl) -1H-pyrrol-3-yl) -5-fluorophenyl) piperazin-1-yl) phenyl) sulfamoyl) -2- (trifluoromethylsulfonyl) phenylamino) -4- (phenylthio) butyl) piperidine-4-carbonyloxy) ethylphosphonic acid. The method for synthesizing the compound 72 can be prepared by the following way according to the description of WO2014/113413A 1:

¹H NMR(300MHz,CD3OD):δ7.93(d,J＝1.9Hz,1H),7.72(dd,J＝9.2,1.8Hz,1H),7.30-7.12(m,12H),6.83-6.42(m,5H),4.46-4.33(m,3H),3.96(s,1H),3.54-2.93(m,16H),2.82(s,3H),2.72(s,3H),2.71-2.55(m,1H),2.24-1.65(m,8H),1.41(d,J＝7.1Hz,6H).MS(ESI):m/z1268.58(M+H)⁺.

Compound 88: (R) -1- (3- (4- (N- (4- (4- (4- (3- (2- (4-chlorophenyl) -1-isopropyl-5-methyl-4- (methylsulfonyl) -1H-pyrrol-3-yl) -5-fluorophenyl) piperazin-1-yl) phenyl) -sulfamoyl) -2- (trifluoromethylsulfonyl) phenylamino) -4- (phenylthio) butyl) piperidine-4-carboxylic acid. The method for synthesizing the compound 88 can be prepared by the following way with reference to the description of WO2014/113413A 1:

¹H NMR(400MHz,DMSO-d6)δ9.84(s,1H),7.82(s,1H),7.65(d,J＝8.7Hz,1H),7.38(d,J＝8.5Hz,2H),7.32-7.14(m,7H),7.11-6.81(m,6H),6.63-6.47(m,2H),6.43-6.30(m,1H),4.33(p,J＝7.1Hz,1H),4.07(s,1H),3.32-3.22(m,4H),3.19-3.03(m,9H),2.89(s,4H),2.67(s,4H),2.31-1.55(m,8H),1.35(d,J＝7.0Hz,6H).

Example 5 Effect of Compound 72 on different malignant tumor cells by combination administration of ibrutinib, acartinib or zebutinib, respectively

(1) Purpose of experiment

Compound 72 is a novel, intravenous BCL-2/BCL-xL dual-target inhibitor. In this experiment, it was evaluated whether combination therapy of compound 72 with ibrutinib, acartinib or zebutinib could further enhance the antiproliferative activity of single agents in these hematological malignancy cell lines.

(2) Experimental materials and sources thereof

As described in example 1.

(3) Experimental method

As described in example 2, part (1). In a WST experiment, the cell viability (%) of ibrutinib, acartinib or zebutitinib single drug and compound 72, respectively, in combination with ibrutinib, acartinib or zebutitinib in the following malignant tumor cells was determined: OCI-LY8 (diffuse large B-cell lymphoma (DLBCL)), DOHH-2 (follicular lymphoma (FL)) and Z-138 (mantle cell lymphoma (MCL)).

(4) Experimental results

As shown in fig. 1, the combination of compound 72 with the BTK inhibitor ibrutinib, acartinib or zebutinib in a variety of hematologic malignant cells has an enhancing effect on the antiproliferative activity in hematologic malignant cell lines.

In the experiments, proliferation of FL (fig. 1a,1 b), DLBCL (fig. 1C) and MCL (fig. 1D, fig. 1E and fig. 1F) cells was assessed by WST assay 72 hours after treatment with the indicated concentrations of compound 72, ibrutinib, acartinib, zebutinib or a combination of both drugs. Dose-response curves for cell viability are shown. CI values for combination therapy with compound 72 and ibrutinib, acartinib or zebutinib at the indicated concentrations were calculated by CalcuSyn (BIOSOFT). CI <0.9 represents the synergistic effect of the two single agents. The drug dose matrix represents the percent growth inhibition of the treated cells relative to the vehicle control group.

Specifically, cell viability assays from human FL DOHH-2 (fig. 7a,7 b) and DLBCL OCI-LY8 (fig. 1C) and MCL (fig. 1D, fig. 1E and fig. 1F) cells showed that the dose-response curve of compound 72 in combination with the BTK inhibitors ibrutinib, acartinib and zebutinib shifted to the left compared to single drug, indicating enhanced anti-tumor proliferation effects.

As shown, the Combination Index (CI) of compound 72 and BTK inhibitor was calculated for the combination of different doses, CI <0.9 considered synergistic (table below). Compound 72 (0.6 uM) and ibrutinib (0.037 uM) began to have a synergistic effect (CI < 0.9) in the DOHH-2 cell line; compound 72 (0.2 uM) and zebutinib (0.037 uM) started to have a synergistic effect (CI < 0.9). In the OCI-LY8 cell line, the dose response curve for compound 72 in combination with acartinib shifted to the left, indicating an enhanced anti-tumor proliferation effect. In the Z138 cell line, compound 72 (0.03 uM) and ibrutinib (0.411 uM) began to have a synergistic effect (CI < 0.9); compound 72 (0.03 uM) and acartinib (3.70 uM) started to have a synergistic effect (CI < 0.9); compound 72 (0.15 uM) and zebutinib (3.70 uM) started to have a synergistic effect (CI < 0.9).

(5) Knot (S)

As can be seen, in vitro experiments, when compound 72 was used in combination with a targeted therapeutic (i.e., BTK inhibitor), the in vitro antiproliferative activity of compound 72 was further enhanced in hematological malignancies, and by comparing the IC50 of the combination versus that of the single drug profile, a shift to the left of the combination profile was observed, with the IC50 values of the combination being smaller than those of the respective single drug, and CI <0.9 in most of the combination. Thus, compound 72 has a synergistic effect in combination with ibrutinib, acartinib or zebutinib.

Example 6 Effect of Albuminib in combination with Compound 6 on Chronic Lymphocytic Leukemia (CLL) or Acute Lymphoblastic Leukemia (ALL)

1. Effect of acartinib or compound 6 alone or in combination with compound 6 on CLL

(1) Experimental materials and sources thereof

As described in example 1.

(2) Experimental method

Cell viability assay prior to the assay, patient peripheral blood cancer cell surface markers were assayed to confirm they were B-CLL cells. A portion of the cancer cells (0.5x106 cells/assay) were centrifuged and resuspended in staining buffer without antibody (negative control), 1.25 μl of anti-CD 19 and/or anti-CD 5 antibodies, respectively. After incubation for 30min on ice in the dark, centrifugation and re-suspension in 400 μl of PBS. Cd19+cd5+ (or cd19+) cells in cancer cells account for about 90% and more of the total cell count for subsequent experiments as measured on Attune NxT flow cytometer (Thermo FISHER SCIENTIFIC).

Apoptosis was detected using an Annexin V-PI (propidium iodide) staining kit. Briefly, cells were collected 24 or 48 hours after treatment, stained with surface markers (CD 5 and CD19 for CLL), annexin-V and PI for 30 minutes at room temperature, and analyzed using Attune NxT flow cytometry according to the manufacturer's instructions. Apoptosis data of 20,000 cells under each experimental condition were analyzed.

(3) Experimental results

As shown in fig. 2, 3.3nM compound 6 single-drug treatment of CLL primary cells, 10 μm acartinib single-drug treatment or two-drug combination treatment of CLL primary cells after 24 hours resulted in less than 10% apoptosis even in the 10 μm acartinib single-drug treatment group; 3.3nM Compound 6 alone can result in about 26% of apoptosis. When 3.3nM compound 6 was combined with acartinib, the percentage of apoptosis was about 40%.

(4) Knot (S)

Treatment of CLL primary cells with acartinib in combination with compound 6 had an enhanced apoptotic effect compared to each single treatment. This suggests that the administration of acartinib with compound 6 may benefit clinically Chronic Lymphocytic Leukemia (CLL) patients.

2. Effect of acartinib or Compound 6 alone or in combination with Compound 6 on ALL

(1) Experimental materials and sources thereof

As described in example 1.

(2) Experimental method

As described in example 2, part (1).

(3) Experimental results

As shown in fig. 3, the cell viability assay from human ALL MOLT-4 (cells showed that the dose response curve for compound 6 in combination with acartinib shifted to the left compared to single drug and started to show synergy at higher concentrations of compound 6 (3 μm) and acartinib (11.111 μm) (CI < 0.9).

(4) Knot (S)

The combination of acartinib with compound 6 may benefit patients with clinical Acute Lymphoblastic Leukemia (ALL).

Example 7 Effect of acartinib in combination with Compound 6 on model of human OCI-LY19 cell line DLBCL mouse xenograft tumors

(1) Experimental method

The experimental procedure is as described in example 2, section (5). In brief, the effect of combination therapy of compound 6 and acartinib was evaluated in DLBCL xenograft models derived from human OCI-LY19 cell lines (see Donnou S,Galand C,Touitou V et al.,Murine Models of B-Cell Lymphomas:Promising Tools for Designing Cancer Therapies.Advances in Hematology,Volume 2012,Article ID 701704,13pages and Benet Pera,Tiffany Tang,Rossella Marullo et al.,Combinatorial epigenetic therapy in diffuse large B cell lymphoma pre-clinical models and patients.Clin Epigenetics.2016;8:79)). Briefly, the dose of OCI-LY19 tumor cells was randomized according to mouse body weight the following day, divided into vehicle control group, compound 6 single group, acartinib single group, and acartinib-and compound 6 combination group starting on day of randomized group (defined as day 1.) on day 14, when the average tumor volume of tumor-bearing mice of vehicle control group reached 142mm ³ time, starting all groups at a dose of 12.5mg/kg, p.o., twice daily starting at day of group administration, 64 times total dose of compound 6 starting at day of 100mg/kg, p.o., 1 time daily starting at day of group 14, 19 times total, additionally, setting the combination of acartinib and compound 6 (12.5 mg, q.5 mg, q.1) +d.1.day).

(2) Experimental results

As shown in fig. 4A and 4B and table 2, compound 6 alone showed weak antitumor effect, T/C (%) value was 61.4, acartinib alone showed no antitumor effect, the combination group had significant antitumor effect compared to both vehicle control group and acartinib alone group, P values were <0.05, and enhanced antitumor effect compared to compound 6, and the cofactor was 2.27. No significant weight loss was seen for each dosing group (fig. 4B).

TABLE 2 anti-tumor effect of Compound 6 alone or in combination with acartinib in human OCI-LY1 (DLBCL) mouse xenograft tumor models

(3) Knot (S)

The combined administration of the compound 6 and the acartinib has no obvious side effect (figure 4B), can increase the anti-tumor effect of the single drug in a DLBCL model of OCI-LY19 cells, and has obvious synergistic effect (the synergistic factor is 2.27> 1). Thus, compound 6 in combination with acartinib may benefit clinically Diffuse Large B Cell Lymphoma (DLBCL) patients.

Example 8 Effect of ibrutinib and Compound 72 combination on model of human DOHH-2 cell line FL mouse xenograft tumors

(1) Experimental method

The experimental procedure is as described in example 2, section (5). This experiment evaluated the antitumor effect of compound 72 in combination with the BTK inhibitor ibrutinib in a FL mouse xenograft tumor model derived from human DOHH-2 cells (please refer to Donnou S,Galand C,Touitou V et al.,Murine Models of B-Cell Lymphomas:Promising Tools for Designing Cancer Therapies.Advances in Hematology,Volume 2012,Article ID 701704,13pages and Ackler S,Mitten MJ,Chen J et al.,Navitoclax(ABT-263)and bendamustine±rituximab induce enhanced killing of non-Hodgkin's lymphoma tumours in vivo.British Journal of Pharmacology(2012)167 881-891.)).

(2) Experimental results

As shown in fig. 5A and table 3, ibrutinib (25 mg/kg) alone group showed weak antitumor effect at day 32, with T/C value (%) of 89.8%; compound 72 (75 mg/kg) alone showed no antitumor effect, with a T/C (%) value of 137.9%; the ibrutinib and compound 72 combined drug showed remarkable antitumor effect, the T/C (%) value was 39.2% (P <0.05 compared with vehicle control group, P <0.001 compared with compound 72 single drug group, P <0.05 compared with ibrutinib single drug group), and the synergistic factor was 3.16, and the combined drug group still showed synergistic antitumor effect at 46 days after 12 days of drug withdrawal, the T/C (%) value was 36.2%, and the synergistic factor was 1.44. No significant weight loss was seen for each dosing group (fig. 5B).

TABLE 3 anti-tumor effects of Compound 72 alone or in combination with ibrutinib in human DOHH-2 (FL) mice xenograft tumor models

(3) Knot (S)

The compound 72 and ibrutinib combined administration have no obvious side effect (figure 5B), remarkably increase the anti-tumor effect of the single drug in a human DOHH-2 (FL) mouse xenograft tumor model, and have remarkable synergistic effect (the synergistic factors of 32 days, 39 days and 46 days are respectively 3.16, 2.44 and 1.44 and are all more than 1). Thus, the combination of compound 72 with ibrutinib may benefit patients with clinical Follicular Lymphoma (FL).

Example 9 effects of ibrutinib or acartinib in combination with Compound 72 on the model of human DOHH-2 cell line FL mouse xenograft tumors

(1) Experimental method

The experimental procedure is as described in example 2, section (5). This experiment evaluated the antitumor effect of compound 72 in combination with the BTK inhibitor ibrutinib or acartinib in a FL mouse xenograft tumor model derived from human DOHH2 cells (please refer to Donnou S,Galand C,Touitou V et al.,Murine Models of B-Cell Lymphomas:Promising Tools for Designing Cancer Therapies.Advances in Hematology,Volume 2012,Article ID 701704,13pages and Ackler S,Mitten MJ,Chen J et al.,Navitoclax(ABT-263)and bendamustine±rituximab induce enhanced killing of non-Hodgkin's lymphoma tumours in vivo.British Journal of Pharmacology(2012)167 881-891.)).

(2) Experimental results

As shown in fig. 6A and table 4, ibrutinib (25 mg/kg) alone group showed weak antitumor effect at 46 days of treatment, with T/C (%) value of 89.6%. Compound 72 (75 mg/kg) alone showed no antitumor effect, with a T/C (%) value of 118.9%, compound 72 and ibrutinib in combination had an enhanced antitumor effect relative to the alone, and a T/C value (%) of 74.3% (P <0.01 compared to vehicle control). No significant weight loss was seen for each dosing group (fig. 6B).

TABLE 4 anti-tumor effect of Compound 72 alone or in combination with ibrutinib or acartinib in human DOHH2 (FL) mouse xenograft tumor models

(3) Knot (S)

Compound 72 had no significant side effects in combination with ibrutinib or acartinib (fig. 6B). The combination of the compound 72 and ibrutinib can increase the anti-tumor effect of two single drugs in a human DOHH2 (FL) mouse xenograft tumor model, and has a synergistic effect (the synergistic factor 1.43 is larger than 1). Thus, the combination of compound 72 with ibrutinib may benefit patients with clinical Follicular Lymphoma (FL).

Example 10 effects of ibrutinib or acartinib in combination with Compound 72 on a model of human Z138 cell-derived MCL mouse xenograft tumors

(1) Experimental method

The experimental procedure is as described in example 2, section (5). This experiment evaluated the antitumor effect of compound 72 in combination with the BTK inhibitors ibrutinib and acartinib in a MCL mouse xenograft tumor model derived from human Z138 cells (please refer to Donnou S,Galand C,Touitou V et al.,Murine Models of B-Cell Lymphomas:Promising Tools for Designing Cancer Therapies.Advances in Hematology,Volume 2012,Article ID 701704,13pages and Ackler S,Mitten MJ,Chen J et al.,Navitoclax(ABT-263)and bendamustine±rituximab induce enhanced killing of non-Hodgkin's lymphoma tumours in vivo.British Journal of Pharmacology(2012)167 881-891.)).

(2) Experimental results

As shown in fig. 7A and table 5, ibrutinib (25 mg/kg) alone group showed weak antitumor effect at 29 days of treatment, with T/C (%) value of 65.59%. Compound 72 (65 mg/kg) showed no significant antitumor effect in the single drug group, a T/C (%) value of 40.11% (P <0.001 compared to vehicle control group), compound 72 had significant antitumor effect in the ibrutinib-combined drug group, and a T/C value (%) of 41.04% (P <0.001 compared to vehicle control group). The acartinib (12.5 mg/kg) single drug group showed no antitumor effect, the T/C (%) value was 101.51%, the compound 72 and acartinib combined drug group showed significant antitumor effect, and the T/C value (%) was 29.59% (P <0.001 compared with the vehicle control group; P <0.001 compared with the acartinib single drug group). No significant weight loss was seen for each dosing group (fig. 7B).

TABLE 5 anti-tumor effects of Compound 72 alone or in combination with ibrutinib or acartinib in human Z138 (MCL) mouse xenograft tumor models

(3) Knot (S)

Compound 72 had no significant side effects in combination with ibrutinib or acartinib (fig. 7B). Compound 72 showed a significant synergistic anti-tumor effect in combination with acartinib (cofactor 1.38 is greater than 1). Thus, the combination of compound 72 with acartinib may benefit clinically in patients with Mantle Cell Lymphoma (MCL).

EXAMPLE 11 antiproliferative effect of Compound 72 and its metabolite (Compound 88) and ABT-263 in NK/TCL cell lines

(1) Purpose of experiment

Compound 72 is a novel, intravenous BCL-2/BCL-xL dual-target inhibitor, while ABT-263 is an inhibitor of BCL-2/BCL-xL/BCL-w. In this experiment, the antiproliferative activity of compound 72 and its metabolite (compound 88) as well as ABT-263 in NK/TCL cell lines was evaluated.

(2) Experimental materials and sources thereof

As described in example 1.

(3) Experimental method

As described in example 2, part (1). In the CTG experiment, IC50 values of the epcompound 72 and its metabolite and ABT-263 in the following malignant cells, respectively, were determined: SNK-1, SNK-6 and SNK-8.

As described in example 2, part (2). In apoptosis assays, apoptosis of NK/TCL cells SNK-6 is induced by Ecompound 72 and its metabolites and ABT-263 are assayed.

As described in example 2, part (3). In immunoblotting experiments, CASPASE-3 and PARP-1 lysis of NK/TCL cells SNK-6 induced by Compound 72 was determined.

As described in example 2, part (4). In co-immunoprecipitation assays, the binding of BCL-XL to BAK or BAX is assayed by compound 72.

(4) Experimental results

The in vitro anti-proliferation assay results show that the BCL-2/BCL-XL inhibitor compound 72 and the active metabolite compound 88 thereof show good cell proliferation inhibition in NK/TCL cell lines. The IC ₅₀ values of compound 72 for SNK-1, SNK-6 and SNT-8 cell lines were 2.652 + -2.606, 1.568+ -1.109, 0.557 + -0.383 μM, respectively (Table 6).

Table 6: anti-proliferative effects of BCL-2/BCL-XL inhibitor compound 72 and compound 88 in NK/TCL cell lines

The results of flow cytometry on apoptotic cells showed that compound 72 and compound 88 induced apoptosis of SNK-6 cells (FIG. 8). Co-IP assays showed that compound 72 promoted the onset of apoptosis by competitively binding to the BCL-XL/BAX and BCL-XL/BAK complexes to release BAX and BAK, thereby inducing dose-dependent lytic activation of the apoptosis-related markers CASPASE-3 and PARP-1 (FIG. 9).

(5) Knot (S)

It can be seen that compound 72 has an antiproliferative effect in NK/TCL cell lines in vitro experiments.

EXAMPLE 12 Effect of Compound 72 or its metabolite (Compound 88) on NK/TCL cells SNK-6 by combination administration with Simultaneous, APG-115, gemcitabine, asparaginase and Tofacitinib, respectively

(1) Purpose of experiment

In this experiment, it was evaluated whether the combination therapy of compound 72 or a metabolite thereof with cetosteanamine, APG-115, gemcitabine, asparaginase and tofacitinib could further enhance the antiproliferative activity of a single agent in these cell lines.

(2) Experimental materials and sources thereof

As described in example 1.

(3) Experimental method

As described in example 2, part (1). In CTG experiments, compound 72 or its metabolite was administered in combination with cetabamine, APG-115, gemcitabine, asparaginase and tofacitinib, respectively, for cell viability (%) in NK/TCL cells SNK-6.

(4) Experimental results

As shown in FIG. 10, compound 72 or its metabolite, in combination with cetabamine, APG-115, gemcitabine, asparaginase and tofacitinib, respectively, has an enhanced single drug inhibition of antiproliferative activity in cell lines in NK/TCL cells SNK-6 (CTG assay CI < 1).

In the experiment, proliferation of NK/TCL cells SNK-6 was evaluated by CTG assay 72 hours after treatment with the indicated concentrations of compound 72 or its metabolite, cetosteanamide, APG-115, gemcitabine, asparaginase and tofacitinib or a combination of both. Dose-response curves showing cell viability, CI values were calculated for combination therapy with compound 72 or its metabolite at the indicated concentrations with cetadalimide, APG-115, gemcitabine, asparaginase and tofacitinib. CI <1 represents the synergistic effect of two single agents. The drug dose matrix represents the percent growth inhibition of the treated cells relative to the vehicle control group.

Specifically, cell viability assays from NK/TCL cells SNK-6 showed that the dose response curve of compound 72 or its metabolite (compound 88) in combination with the administration of cibutamine, APG-115, gemcitabine, asparaginase and tofacitinib shifted to the left compared to single drug, indicating an enhanced anti-tumor proliferation effect.

As shown, the Combination Index (CI) of the combination of compound 72 or its metabolite (compound 88) with cidamine, APG-115, gemcitabine, asparaginase and tofacitinib was calculated for the combination of different doses, CI <1 being considered synergistic (data below).

(5) Knot (S)

It follows that in vitro antiproliferative activity of compound 72 or its metabolite (compound 88) is further enhanced when compound 72 or its metabolite (compound 88) is used in combination with additional agents (i.e., cibutamine, APG-115, gemcitabine, asparaginase, and tofacitinib), and CI <1 in most combination groups. Thus, compound 72 or a metabolite thereof has a synergistic effect with cetadamine, APG-115, gemcitabine, asparaginase and tofacitinib in NK/TCL cell lines. Thus, compound 72 or a metabolite thereof, with cidamine, APG-115, gemcitabine, asparaginase and tofacitinib would likely benefit clinically in NK/T cell lymphoma patients.

Example 13 Effect of Compound 72 or metabolite thereof (Compound 88) or ABT-263 on the model of human NK/TCL cells SNK-6 mouse xenograft tumor

(1) Experimental method

The experimental procedure is as described in example 2, section (6). The effect of compound 72 or its metabolite (compound 88) or ABT-263 on the human NK/TCL cell SNK-6 mouse xenograft tumor model was evaluated in this experiment. Briefly, SNK-6 tumor cells were randomly grouped according to mouse body weight the next day after inoculation, and were divided into vehicle control, compound 72 different dosing regimen (65 mg/kg, IV, BIW×2W;100mg/kg, IV, BIW×2W;65mg/kg, IV, QW×2W;100mg/kg, IV, QW×2W), compound 72M1 (40 mg/kg, IV, BIW×2W) and ABT-263 (50 mg/kg, PO, QD×14D).

(2) Experimental results

As shown in fig. 11A and 11B and table 7, each of the dosing regimen group of compound 72, the compound 88, and ABT-263 group showed antitumor effect with respect to the vehicle control group, and P values were <0.01. Meanwhile, compound 72 (100 mg/kg, IV, BIW. Times.2W) had better antitumor effect than the ABT-263 group.

Specifically, the antitumor effect of compound 72 and its active metabolite compound 88 was examined in an NK/TCL cell SNK-6 xenograft tumor model. Compound 72 was administered intravenously, twice weekly (BIW) or once weekly (QW) at a dose of 65 or 100mg/kg for 2 weeks. Compound 88 was administered intravenously at a dose of 40mg/kg, twice weekly (BIW) for 2 weeks. ABT-263 was administered at a dose of 50mg/kg by gavage, once daily (QD) for 2 weeks as a control. At the end of the dosing, 65 and 100mg/kg dose groups of compound 72 exhibited dose and dosing regimen (BIW, QW) dependent antitumor effects with T/C values (%) of 23.6% (p < 0.05), 13.7% (p < 0.01), 30.7% (p < 0.05) and 27.6% (p < 0.05), respectively (fig. 11A). The T/C value (%) of 88 40mg/kg BIW of compound was 27.3% (p < 0.05). Of these, the tumor growth inhibition effect of the compound 72 100mg/kg BIW group was superior to that of the ABT-263 50mg/kg QD group (T/C% was 30.1%, p <0.05vs vehicle control), and there was a significant difference between the groups (p < 0.05) (FIG. 11A). No significant weight loss was seen for each dosing group (fig. 11B).

TABLE 7 influence of Compound 72 or its metabolite (Compound 88) or ABT-263 on the model of human NK/TCL cells SNK-6 mouse xenograft tumors

^*:p<0.05,^** P <0.01, vs. vehicle control

⁺:p<0.05,vs.ABT-263 50mg/kg

(3) Knot (S)

Compound 72 each dosing regimen, compound 88, and ABT-263 showed anti-tumor effects (e.g., NK/T cell lymphoma) relative to the vehicle control.

Example 14 Effect of Compound 72 combination on human NK/TCL cell SNK-6 mouse xenograft tumor model

(1) Experimental method

The experimental procedure is as described in example 2, section (6). The combined therapeutic effect of compound 72 and cetostelamine or APG-115 was evaluated in this experiment. Briefly, human NK/TCL cells SNK-6 were randomized according to the body weight of mice the following day of inoculation, and divided into vehicle control group (IV, BIW.times.2W), compound 72 single drug group (65 mg/kg, IV, BIW.times.2W), sidamide single drug group (5 mg/kg (D1, 5), 15mg/kg (D6-14), PO, QD), APG-115 single drug group (50 mg/kg, QD.times.2W), compound 72 (65 mg/kg, IV, BIW.times.2W) and Sidamide combination group (5 mg/kg (D1, 5), 15mg/kg (D6-14), PO, QD) or compound 72 (65 mg/kg, IV, BIW.times.2W) and APG-115 combination group (50 mg/kg, QD.times.2W).

(2) Experimental results

As shown in FIGS. 12A-C and Table 8, compound 72 alone showed antitumor effect with a T/C (%) value of 24.7. The combined drug group has remarkable anti-tumor effect relative to the vehicle control group and each single drug group, the P value is less than 0.05, and the combined drug group has synergistic effect relative to the single drug group. No significant weight loss was seen for each dosing group (fig. 12B).

TABLE 8 influence of Compound 72 combination on human NK/TCL cell SNK-6 mouse xenograft tumor model

(3) Knot (S)

The compound 72 can increase the inhibition effect of single medicine when combined with the HDAC inhibitor of the Sidamide or the MDM2 inhibitor of the APG-115, and shows synergistic anti-tumor effect. Thus, the administration of compound 72 in combination with ciladalimine or with APG-115 may benefit clinically from NK/T cell lymphoma patients.

Claims

1. A combination comprising a Bcl-2 inhibitor and an additional agent, or a Bcl-2/Bcl-xL inhibitor and an additional agent,

Wherein the Bcl-2 inhibitor is the following compound or a pharmaceutically acceptable salt or solvate thereof:

The Bcl-2/Bcl-xL inhibitor is a compound or a pharmaceutically acceptable salt or solvate thereof:

And is also provided with

The additional agent is selected from one or more of a BTK inhibitor, an HDAC inhibitor, an MDM2 inhibitor, a JAK inhibitor, an antimetabolite, and an enzyme, wherein the BTK inhibitor is selected from the group consisting of: ibrutinib (Ibrutinib), ICP-022, acartinib (Acalabrutinib), zebutinib (BGB 3111), ONO/GS-4059, spebrutinib (CC-292 or AVL-292), CNX-774, olmutinib (HM 61713,

BI 1482694), M7583, HM71224, PCI-32765racemate (PCI-32765 Racemate), GDC-0853, ONO-4059, RN486, CGI-1746, QL47, LFM-A13, (+ -) -Zlutidine ((+ -) -Zanubrutinib), SNS-062, BMS-935177, BMS-986195, PCI29732, btk inhibitor 2, evobrutinib, ibrutinib-biotin (Ibrutinib-biotin), BMX-IN-1, GDC-0834, and CB1763.

2. The combination product according to claim 1, wherein the Bcl-2/Bcl-xL inhibitor is the following compound or a pharmaceutically acceptable salt or solvate thereof:

3. The combination product according to claim 1, wherein the BTK inhibitor is selected from the group consisting of: ibrutinib (Ibrutinib), acartinib (Acalabrutinib (ACP-196)) and zebutinib (BGB 3111).

4. A combination product according to claim 1, wherein the HDAC inhibitor is cidamine; and/or the MDM2 inhibitor is APG-115; and/or the JAK inhibitor is tofacitinib; and/or the antimetabolite is gemcitabine; and/or the enzyme is an asparaginase.

5. The combination according to any one of claims 1-4, wherein the combination is in the form of a pharmaceutical composition.

6. The combination of any one of claims 1-4, wherein the Bcl-2 inhibitor and the additional agent are each in separate formulations, or wherein the Bcl-2/Bcl-xL inhibitor and the additional agent are each in separate formulations.

7. The combination of any one of claims 1-4, wherein the Bcl-2 inhibitor and the additional agent are administered simultaneously or sequentially, or wherein the Bcl-2/Bcl-xL inhibitor and the additional agent are administered simultaneously or sequentially.

8. The combination product according to any one of claims 1-4, further comprising a pharmaceutically acceptable carrier, diluent or excipient.

9. The combination according to claim 9, wherein the combination is in the form of a tablet, capsule, granule, syrup, powder, lozenge, sachet, cachet, elixir, suspension, emulsion, solution, syrup, aerosol, ointment, cream or injection.

10. Use of a combination product according to any one of claims 1-9 for the manufacture of a medicament for the prevention and/or treatment of a disease selected from the group consisting of cancer, autoimmune diseases and inflammatory diseases.

11. A combination product as defined in any one of claims 1 to 9 for use in the prevention and/or treatment of a disease selected from cancer, autoimmune diseases and inflammatory diseases.

12. A method of preventing and/or treating a disease selected from cancer, autoimmune diseases and inflammatory diseases, comprising administering a combination product according to any one of claims 1-9 to a subject in need thereof.

13. The method of claim 12, wherein the cancer is selected from Acute Myelogenous Leukemia (AML), acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular Lymphoma (FL), chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Lymphoma (SLL), marginal Zone Lymphoma (MZL), chronic Myelogenous Leukemia (CML), mantle Cell Lymphoma (MCL), waldenstrom's Macroglobulinemia (WM), multiple Myeloma (MM), T-prolymphocytic leukemia (T-PLL), small Cell Lung Cancer (SCLC), and NK/T cell lymphoma.

14. The method of claim 12 or 13, wherein the Bcl-2 inhibitor, bcl-2/Bcl-xL inhibitor, or pharmaceutically acceptable salts or solvates thereof in the combination is administered in an amount of about 0.0025-1500 mg/day.

15. The method of claim 12 or 13, wherein the additional agent in the combination product, or a pharmaceutically acceptable salt or solvate thereof, is administered in an amount of about 0.0025-1000 mg/day.

16. The method of claim 12 or 13, wherein the subject is unresponsive or resistant to a BTK inhibitor.