CN108570039A - One kind, which has, inhibits the active compound of anti-apoptotic proteins and its preparation and application - Google Patents

One kind, which has, inhibits the active compound of anti-apoptotic proteins and its preparation and application Download PDF

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CN108570039A
CN108570039A CN201810379582.7A CN201810379582A CN108570039A CN 108570039 A CN108570039 A CN 108570039A CN 201810379582 A CN201810379582 A CN 201810379582A CN 108570039 A CN108570039 A CN 108570039A
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bcl
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CN108570039B (en
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刘海
陈春麟
周南梅
张秀芳
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Shanghai Medicilon Inc
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

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Abstract

Have the present invention provides one kind and inhibits the active compound of anti-apoptotic proteins and its preparation and application.Specifically, the present invention provides a kind of such as following formula I compound represented；Wherein, the definition of each group is as noted in the discussion.The compound of the present invention has 2 family protein inhibitory activity of good Bcl, can be used for preparing a series of drug of the relevant disease for the treatment of Bcl 2 family protein activity.

Description

Compound with anti-apoptosis protein activity inhibition function and preparation and application thereof
Technical Field
The invention belongs to the field of medicines, and particularly relates to a quinazoline compound serving as a Bcl-2 family protein inhibitor, and preparation and application thereof.
Background
Apoptosis is a highly regulated process that directs cells to self-degrade to eliminate dysfunctional cells. During the progression of tumors to a dangerous state, it must suppress the effects of apoptosis. Specifically activating apoptosis in tumor cells provides a novel approach to tumor-targeted therapy. The biological target of the product developed in the project is the main control point in the process of cell apoptosis.
Apoptosis is divided into intrinsic and extrinsic pathways. The intrinsic pathway is regulated by the interaction between two classes of Bcl-2 proteins that control both pro-apoptosis and anti-apoptosis of the mitochondrial outer membrane integrity. Pro-apoptotic members (Bax, Bak, Bad, Bik, Bid, Bim, Hrk, Bmf, Noxa and Puma) induce an increase in mitochondrial membrane permeability, release cytochrome c, activate caspases and thus initiate the apoptotic process. Anti-apoptotic members (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and Bcl2-A1) prevent the occurrence of apoptosis by binding to and sequestering pro-apoptotic members. Cancer cells often overexpress anti-apoptotic Bcl-2 family members, inhibit apoptosis, increase cell survival, and develop resistance during chemotherapy. The inhibition of the effects of anti-apoptotic Bcl-2 family members has been investigated as an effective means of re-inducing apoptosis in cancer cells and has been identified as an important biological target for clinical therapeutic intervention. Cancers that overexpress the anti-apoptotic Bcl-2 protein in the cell include Acute Lymphocytic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), multiple myeloma, human follicular lymphoma, Small Cell Lung Cancer (SCLC), uterine cancer, ovarian cancer, bladder cancer, prostate cancer, pancreatic cancer, gastric cancer, colorectal cancer, and breast cancer, among others.
Therefore, protein-protein interaction (PPI) inhibitors that develop tumor cell apoptosis pathways have become a leading hotspot for the research of antitumor drugs.
Disclosure of Invention
The invention aims to provide a novel targeted Bcl-2 family protein inhibitor.
In a first aspect of the present invention, there is provided a compound of formula I, or a pharmaceutically acceptable salt thereof:
wherein,
ring a is a group selected from:
R1and R2Each independently selected from: H. halogen, NO2、CN、CF3SO2Or CF3；
R3And R4Each independently selected from: H. substituted OR unsubstituted C1-C8 alkyl, C (O) OR5、CONR6R7、C(O)R8Substituted or unsubstituted 5-8 membered aryl, substituted or unsubstituted 5-8 membered heteroaryl, substituted or unsubstituted 3-12 membered saturated or unsaturated heterocycle or carbocycle; wherein said heteroaryl group contains 1 to 3 heteroatoms selected from the group consisting of: n, O or S; said heterocycle comprising 1 to 3 heteroatoms selected from the group consisting of: n, O or S;
R5、R6、R7and R8Each independently selected from: H. substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C1-C6 alkylene-amino, substituted or unsubstituted C1-C6 alkylene-hydroxy, substituted or unsubstituted 5-to 8-membered aryl, or substituted or unsubstituted 5-to 8-membered heteroaryl; a substituted or unsubstituted 3-to 12-membered saturated heterocyclic or carbocyclic ring; wherein said heteroaryl group comprises at least one heteroatom selected from the group consisting of: n, O or S, the heterocyclic ring comprising at least one heteroatom selected from the group consisting of: n, O or S;
any "substitution" described above means that one or more hydrogen atoms on the group is substituted with a substituent selected from the group consisting of: halogen, OH, NH2CN, unsubstituted or halogenated C1-C8 alkanesA C1-C8 alkoxy group, an unsubstituted or halogenated C2-C6 alkenyl group, an unsubstituted or halogenated C2-C6 alkynyl group, an unsubstituted or halogenated C2-C6 acyl group, an unsubstituted or halogenated 5-to 8-membered aryl group, an unsubstituted or halogenated 5-to 8-membered heteroaryl group, an unsubstituted or halogenated 3-to 12-membered saturated heterocyclic or carbocyclic ring; wherein said heteroaryl group contains 1 to 3 heteroatoms selected from the group consisting of: n, O or S, the heterocycle comprising 1 to 3 heteroatoms selected from: n, O or S.
In another preferred embodiment, ring A is
In another preferred embodiment, when R is1Selected from: halogen, NO2、CN、CF3SO2Or CF3When R is2Is H.
In another preferred embodiment, when R is1When is H, R2Selected from: halogen, NO2、CN、CF3SO2Or CF3。
In another preferred embodiment, when R is1Is NO2Or CF3SO2When R is2Is H.
In another preferred embodiment, when R is2Is NO2Or CF3SO2When R is1Is H.
In another preferred embodiment, R3And R4Each independently selected from: H. substituted or unsubstituted C1-C8 alkyl.
In another preferred embodiment, R3Or R4Wherein one is H and the other is substituted C1-C8 alkyl.
In another preferred embodiment, A, R1、R2、R3Or R4Are the corresponding groups in the specific compounds described in the examples.
In another preferred embodiment, the compound of formula I is a compound as shown below:
in a second aspect of the invention, there is provided a process for the preparation of a compound of formula I as described in the first aspect of the invention, comprising the steps of:
(a) reacting a compound of formula I-13 with a compound of formula I-14 in an inert solvent to obtain a compound of formula I;
in the above formulae, each group is as defined above.
In another preferred embodiment, in the step (a), the inert solvent is selected from the group consisting of: dichloromethane, methanol, ethanol, isopropanol, n-butanol, t-butanol, isobutanol, dioxane, THF, DMF, DMSO, NMP, or combinations thereof.
In another preferred embodiment, in the step (a), the reaction is carried out in the presence of a base.
In another preferred embodiment, in the step (a), the alkali is selected from the group consisting of: triethylamine, diisopropylamine, diisopropylethylamine, Na2CO3、K2CO3、Cs2CO3LiHMDS, NaHMDS, KHMDS, sodium tert-butoxide, potassium tert-butoxide, or a combination thereof.
In another preferred embodiment, the preparation method further comprises the following steps:
(b) reacting a compound of formula I-12 with a compound of formula I-5 in an inert solvent to obtain a compound of formula I-13;
in the above formulae, each group is as defined above.
In another preferred embodiment, in the step (b), the inert solvent is selected from the group consisting of: toluene, dioxane, THF, DMF, or a combination thereof.
In another preferred embodiment, in the step (b), the reaction is carried out in the presence of a palladium catalyst.
In another preferred embodiment, in the step (b), the palladium catalyst is selected from the group consisting of: pd (PPh)3)4、Pd2(dba)3、Pd(dba)2、Pd(OAc)2、Pd(PPh3)2Cl2、Pd(dppe)Cl2、Pd(dppf)Cl2、Pd(dppf)Cl2·CH2Cl2Or a combination thereof.
In another preferred embodiment, in the step (b), the reaction is performed in the presence of a copper salt.
In another preferred embodiment, in the step (b), the copper salt is selected from the group consisting of: CuI, Cu, CuCl, Cu2O、CuO、Cu(OAc)2、CuSO4·5H2O、Cu(acac)2、CuCl2CuSCN, or a combination thereof.
In another preferred embodiment, in the step (b), the reaction is performed in the presence of a base.
In another preferred embodiment, in the step (b), the alkali is selected from the group consisting of: k2CO3、K3PO4、Cs2CO3Or a combination thereof.
In a third aspect of the invention, there is provided the use of a compound according to the first aspect of the invention for:
(a) preparing a medicament for treating diseases related to the activity or expression quantity of Bcl-2 family protein;
(b) preparing a Bcl-2 family protein targeted inhibitor;
(c) non-therapeutically inhibiting the activity of a Bcl-2 family protein in vitro;
(d) non-therapeutically inhibiting tumor cell proliferation in vitro; and/or
(e) Treating diseases related to the activity or expression level of Bcl-2 family protein.
In another preferred embodiment, the Bcl-2 family protein is selected from the group consisting of: bcl-2, Bcl-xL, Bcl-w, Mcl-1, Bcl2-A1, or a combination thereof.
In another preferred embodiment, the tumor cell is a leukemia cell strain; preferably a lymphocyte leukemia cell line; more preferably acute lymphoblastic leukemia cell strain MV-4-11 cells.
In a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising: (i) an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.
In another preferred embodiment, the effective amount refers to a therapeutically effective amount or an inhibitory effective amount, preferably 0.01 to 99.99%.
In another preferred embodiment, the pharmaceutical composition is used for inhibiting the activity of a Bcl-2 family protein.
In another preferred embodiment, the pharmaceutical composition is used for treating diseases related to the activity or expression amount of Bcl-2 family proteins.
In a fifth aspect of the present invention, there is provided a method of inhibiting the activity of a Bcl-2 family protein, comprising the steps of: administering to the subject an inhibitory effective amount of a compound of formula I as described in the first aspect of the invention or a pharmaceutically acceptable salt thereof, or administering to the subject an inhibitory effective amount of a pharmaceutical composition as described in the fourth aspect of the invention.
In another preferred embodiment, the inhibition is an in vitro non-therapeutic inhibition.
In another preferred embodiment, when an inhibitory effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, as described above, is administered to a subject, said inhibitory effective amount is 0.001-500nmol/L, preferably 0.01-200 nmol/L.
In a sixth aspect of the invention, there is provided a method of inhibiting tumor cells in vitro, the method comprising: administering to the subject an inhibitory effective amount of a compound of formula I as described in the first aspect of the invention, or a pharmaceutical composition as described in the fourth aspect of the invention.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Detailed Description
The inventor prepares a compound with a structure shown in a formula I through long-term and intensive research, and finds that the compound has Bcl-2 family protein inhibitory activity. The compound has an inhibiting effect on a series of Bcl-2 family proteins at an extremely low concentration (which can be as low as less than or equal to 100nmol/L), and the inhibiting activity is quite excellent, so that the compound can be used for treating diseases related to the activity or expression of the Bcl-2 family proteins, such as tumors. Based on the above findings, the inventors have completed the present invention.
Term(s) for
As used herein, the term "C1-C6 alkyl" refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like. The term "C1-C8 alkyl" refers to a straight or branched chain alkyl group having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like. The term "C1-C8 alkoxy" refers to C1-C8 straight or branched chain alkoxy groups and refers to alkoxy groups containing 1-8 carbon atoms, including but not limited to methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy, heptyloxy or octyloxy.
As used herein, the term "C2-C6 acyl" refers to a substituent such as a "straight or branched alkyl-carbonyl group having 1 to 6 carbon atoms" structure, such as acetyl, propionyl, butyryl, or the like.
The term "C1-C6 alkylene" refers to a group formed after a C1-C6 alkyl group has lost one hydrogen atom, as described above, for example-CH2-、-CH2-CH2-, or the like. The term "C2-C6 alkenyl" refers to a straight or branched chain hydrocarbon group having 2 to 6 carbon atoms and containing at least one double bond. The term "C2-C6 alkynyl" refers to a straight or branched chain hydrocarbon group having 2 to 6 carbon atoms and containing at least one triple bond.
The term "C6-C10 arylene" refers to an aryl group having 6 to 10 carbon atoms with one hydrogen atom removed to form a group, and includes monocyclic or bicyclic arylene groups such as phenylene, naphthylene, or the like.
The term "6-membered aryl" refers to phenyl.
The term "5-to 8-membered aryl" refers to a substituent of a carbon unsaturated series of a 5-to 8-membered ring, such as phenyl, or the like.
The term "5-to 8-membered heteroaryl" refers to a non-saturated ring system substituent having one or more heteroatoms selected from O, S, N or P in a ring system having 5-to 8-membered rings, such as pyridyl, thienyl, or the like.
The term "3 to 12 membered saturated carbocyclic ring" refers to a saturated carbocyclic ring having 3 to 12 carbon atoms, such as cyclohexyl, or the like. The term "3-12 membered unsaturated carbocyclic ring" refers to an unsaturated carbocyclic ring having 3-12 carbon atoms, such as cyclohexenyl, or the like.
The term "3-12 membered saturated heterocyclic ring" refers to a saturated ring system substituent having 3-12 membered ring system with one or more heteroatoms selected from O, S, N or P, such as piperidinyl, tetrahydropyranyl, morpholino, or the like. The term "3-12 membered unsaturated heterocyclic ring" refers to an unsaturated ring system substituent having one or more heteroatoms selected from O, S, N or P on a ring system having 3-12 members, such as pyrrolyl, or the like.
The term "halogen" refers to F, Cl, Br and I.
As used herein, the terms "comprising," "including," or "including" mean that the various ingredients may be used together in a mixture or composition of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "comprising.
In the present invention, the term "pharmaceutically acceptable" ingredient refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio.
In the present invention, the term "effective amount" refers to an amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect. The precise effective amount for a subject will depend upon the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. Therefore, it is not useful to specify an exact effective amount in advance. However, for a given condition, the effective amount can be determined by routine experimentation and can be determined by a clinician.
Herein, unless otherwise specified, the term "substituted" means that one or more hydrogen atoms on a group are replaced with a substituent selected from the group consisting of: halogen, unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C2-C6 acyl, unsubstituted or halogenated C1-C6 alkyl-hydroxy, OH, NH2CN, unsubstituted or halogenated C1-C8 alkyl, C1-C8 alkoxy, unsubstituted or halogenated C2-C6 alkenyl, unsubstituted or halogenated C2-C6 alkynyl, unsubstituted or halogenated 5-8 membered aryl, unsubstituted or halogenated 5-8 membered heteroaryl, unsubstituted or halogenated 3-12 membered saturated heterocycle or carbocycle; wherein said heteroaryl group contains 1 to 3 heteroatoms selected from the group consisting of: n, O or S, the heterocycle comprising 1 to 3 heteroatoms selected from: n, O or S.
Unless otherwise specified, all occurrences of a compound in the present invention are intended to include all possible optical isomers, such as a single chiral compound, or a mixture of various chiral compounds (i.e., a racemate). In all compounds of the present invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.
As used herein, the term "compounds of the invention" refers to compounds of formula I. The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of formula I.
As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention with an acid or base that is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with an acid. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, phenylmethanesulfonic acid, benzenesulfonic acid, etc.; and acidic amino acids such as aspartic acid and glutamic acid.
A compound of formula I
Wherein,
ring a is a group selected from:
R1and R2Each independently selected from: H. halogen, NO2、CN、CF3SO2Or CF3；
R3And R4Each independently selected from: H. substituted OR unsubstituted C1-C8 alkyl, C (O) OR5、CONR6R7、C(O)R8Substituted or unsubstituted 5-8 membered aryl, substituted or unsubstituted 5-8 membered heteroaryl, substituted or unsubstituted 3-12 membered saturated or unsaturated heterocycle or carbocycle; wherein said heteroaryl group contains 1 to 3 heteroatoms selected from the group consisting of: n, O or S; said heterocycle comprising 1 to 3 heteroatoms selected from the group consisting of: n, O or S;
R5、R6、R7and R8Each independently selected from: H. substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C1-C6 alkylene-amino, substituted or unsubstituted C1-C6 alkylene-hydroxy, substituted or unsubstituted 5-to 8-membered aryl, or substituted or unsubstituted 5-to 8-membered heteroaryl; a substituted or unsubstituted 3-to 12-membered saturated heterocyclic or carbocyclic ring; wherein said heteroaryl group comprises at least one heteroatom selected from the group consisting of: n, O or S, the heterocyclic ring comprising at least one heteroatom selected from the group consisting of: n, O or S;
any "substitution" described above means that one or more hydrogen atoms on the group is substituted with a substituent selected from the group consisting of: halogen, OH, NH2CN, unsubstituted or halogenated C1-C8 alkyl, C1-C8 alkoxy, unsubstituted or halogenated C2-C6 alkenyl, unsubstituted or halogenated C2-C6 alkynyl, unsubstituted or halogenated C2-C6 acyl, unsubstituted or halogenated 5-8 membered aryl, unsubstituted or halogenated 5-8 membered heteroaryl, unsubstituted or halogenated 3-12 membered saturated heterocycle or carbocycle; wherein said heteroaryl group contains 1 to 3 groups selected fromHeteroatom: n, O or S, the heterocycle comprising 1 to 3 heteroatoms selected from: n, O or S.
In another preferred embodiment, ring A is
In another preferred embodiment, when R is1Selected from: halogen, NO2、CN、CF3SO2Or CF3When R is2Is H.
In another preferred embodiment, when R is1When is H, R2Selected from: halogen, NO2、CN、CF3SO2Or CF3。
In another preferred embodiment, when R is1Is NO2Or CF3SO2When R is2Is H.
In another preferred embodiment, when R is2Is NO2Or CF3SO2When R is1Is H.
In another preferred embodiment, R3And R4Each independently selected from: H. substituted or unsubstituted C1-C8 alkyl.
In another preferred embodiment, R3Or R4Wherein one is H and the other is substituted C1-C8 alkyl.
In another preferred embodiment, A, R1、R2、R3Or R4Are the corresponding groups in the specific compounds described in the examples.
In another preferred embodiment, the compound of formula I is a compound as shown below:
process for the preparation of compounds of formula I
The present invention provides a process for the preparation of a compound of formula I, said process comprising the steps of:
(a) reacting a compound of formula I-13 with a compound of formula I-14 in an inert solvent to obtain a compound of formula I;
in the above formulae, each group is as defined above.
In another preferred embodiment, in the step (a), the inert solvent is selected from the group consisting of: dichloromethane, methanol, ethanol, isopropanol, n-butanol, t-butanol, isobutanol, dioxane, THF, DMF, DMSO, NMP, or combinations thereof.
In another preferred embodiment, said step is carried out in the presence of a base.
In another preferred embodiment, the base is selected from the group consisting of: triethylamine, diisopropylamine, diisopropylethylamine, Na2CO3、K2CO3、Cs2CO3LiHMDS, NaHMDS, KHMDS, sodium tert-butoxide, potassium tert-butoxide, or a combination thereof.
In another preferred embodiment, the preparation method further comprises the following steps:
(b) reacting a compound of formula I-12 with a compound of formula I-5 in an inert solvent to obtain a compound of formula I-13;
in the above formulae, each group is as defined above.
In another preferred embodiment, in the step (b), the inert solvent is selected from the group consisting of: toluene, dioxane, THF, DMF, or a combination thereof.
In another preferred embodiment, said step is carried out in the presence of a palladium catalyst.
In another preferred embodiment, the palladium catalyst is selected from the group consisting of: pd (PPh)3)4、Pd2(dba)3、Pd(dba)2、Pd(OAc)2、Pd(PPh3)2Cl2、Pd(dppe)Cl2、Pd(dppf)Cl2、Pd(dppf)Cl2·CH2Cl2Or a combination thereof.
In another preferred embodiment, said step is carried out in the presence of a copper salt.
In another preferred embodiment, the copper salt is selected from the group consisting of: CuI, Cu, CuCl, Cu2O、CuO、Cu(OAc)2、CuSO4·5H2O、Cu(acac)2、CuCl2CuSCN, or a combination thereof.
In another preferred embodiment, said step is carried out in the presence of a base.
In another preferred embodiment, the base is selected from the group consisting of: k2CO3、K3PO4、Cs2CO3Or a combination thereof.
In another preferred embodiment, the preparation method further comprises the following steps:
(c) reacting a compound of formula I-10 with a compound of formula I-11 in an inert solvent to obtain a compound of formula I-12;
in the above formulae, each group is as defined above.
In another preferred embodiment, in the step (c), the inert solvent is selected from the group consisting of: toluene, dioxane, THF, DMF, H2O, or a combination thereof.
In another preferred embodiment, said step is carried out in the presence of a palladium catalyst.
In another preferred embodiment, the palladium catalyst is selected from the group consisting of: pd (PPh)3)4、Pd2(dba)3、Pd(dba)2、Pd(OAc)2、Pd(PPh3)2Cl2、Pd(dppe)Cl2、Pd(dppf)Cl2、Pd(dppf)Cl2·CH2Cl2Or a combination thereof.
In another preferred embodiment, said step is carried out in the presence of a ligand.
In another preferred embodiment, the ligand is a monodentate phosphine ligand or a bidentate phosphine ligand; more preferably, the ligand is selected from the group consisting of: triphenylphosphine, tritolylphosphine, tricyclohexylphosphine, tri-tert-butylphosphine, X-Phos, S-Phos, binaphthyldiphenylphosphine, 1' -bis (diphenylphosphino) ferrocene, 1, 2-bis (diphenylphosphino) ethane, Xant-Phos, or a combination thereof.
In another preferred embodiment, said step is carried out in the presence of a base.
In another preferred embodiment, the base is selected from the group consisting of: na (Na)2CO3、K2CO3、Cs2CO3、K3PO4Or a combination thereof.
In another preferred embodiment, the preparation method further comprises the following steps:
(d) reacting a compound of formula I-8 with a compound of formula I-9 in an inert solvent to obtain a compound of formula I-10;
in the above formulae, each group is as defined above.
In another preferred embodiment, in the step (d), the inert solvent is selected from the group consisting of: dichloromethane, methanol, ethanol, isopropanol, n-butanol, t-butanol, isobutanol, dioxane, THF, DMF, DMSO, NMP, or combinations thereof.
In another preferred embodiment, said step is carried out in the presence of a base.
In another preferred embodiment, the base is selected from the group consisting of: triethylamine, diisopropylamine, diisopropylethylamine, Na2CO3、K2CO3、Cs2CO3LiHMDS, NaHMDS, KHMDS, sodium tert-butoxide, potassium tert-butoxide, or a combination thereof.
Use of compounds of formula I
The compounds of formula I may be used for one or more of the following uses:
(a) preparing a medicament for treating diseases related to the activity or expression quantity of Bcl-2 family protein;
(b) preparing a Bcl-2 family protein targeted inhibitor;
(c) non-therapeutically inhibiting the activity of a Bcl-2 family protein in vitro;
(d) non-therapeutically inhibiting tumor cell proliferation in vitro; and/or
(e) Treating diseases related to the activity or expression level of Bcl-2 family protein.
In another preferred embodiment, the Bcl-2 family protein is selected from the group consisting of: bcl-2, Bcl-xL, Bcl-w, Mcl-1, Bcl2-A1, or a combination thereof.
In another preferred embodiment, the tumor cell is a leukemia cell strain; preferably a lymphocyte leukemia cell line; more preferably acute lymphoblastic leukemia cell strain MV-4-11 cells.
The compounds of formula I of the present invention are useful for preparing a pharmaceutical composition comprising: (i) an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.
In another preferred embodiment, the effective amount refers to a therapeutically effective amount or an inhibitory effective amount.
In another preferred embodiment, the pharmaceutical composition is used for inhibiting the activity of a Bcl-2 family protein.
In another preferred embodiment, the pharmaceutical composition is used for treating diseases related to the activity or expression amount of Bcl-2 family proteins.
The compounds of formula I of the present invention may also be used in a method of inhibiting the activity of a Bcl-2 family protein comprising the steps of: administering to the subject an inhibitory effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, as described above, or administering to the subject an inhibitory effective amount of a pharmaceutical composition, as described above.
In another preferred embodiment, the inhibition is an in vitro non-therapeutic inhibition.
In another preferred embodiment, when an inhibitory effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, as described above, is administered to a subject, said inhibitory effective amount is 0.001-500nmol/L, preferably 0.01-200 nmol/L.
The compounds of formula I of the present invention may also be used in a method for inhibiting tumor cells in vitro, comprising: administering to the subject an inhibitory effective amount of a compound of formula I, as described above, or a pharmaceutical composition, as described above.
Pharmaceutical compositions and methods of administration
The compound of the present invention and various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compound as a main active ingredient can be used for treating, preventing and relieving diseases related to the activity or expression amount of Bcl-2 family proteins because the compound has excellent inhibitory activity against Bcl-2 family proteins such as Bcl-2 and Bcl-xL. According to the prior art, the compounds of the invention are useful for the treatment of the following diseases: endometrial cancer, breast cancer, gastric cancer, bladder cancer, myeloma, liver cancer, and the like.
The pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmacologically acceptable salt thereof in a safe and effective amount range and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000mg of a compound of the invention per dose, more preferably, 5-200mg of a compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.
"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and with the compounds of the present invention without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), and the like) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.
The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.
In addition to these inert diluents, the compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.
Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.
The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, and for a human body with a weight of 60kg, the daily administration dose is usually 1 to 2000mg, preferably 5 to 500 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The main advantages of the invention include:
1. provides a compound shown as a formula I.
2. Provides a Bcl-2 family protein inhibitor with novel structure, preparation and application thereof, wherein the inhibitor can inhibit the activity of various Bcl-2 family proteins at extremely low concentration.
3. Provides a kind of medicine composition for treating diseases related to the activity of Bcl-2 family protein.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
Example 1
Compound 1(6.3g, 41.0mmol) and compound 2(8.5g, 82.0mmol) were added to ethylene glycol monomethyl ether (40mL) and stirred at 140 ℃ under reflux for 18 hours. After the reaction, the reaction mixture was concentrated to dryness, diluted with 0.01 mol of aqueous ammonia for neutralization, and a solid precipitated, filtered, washed with water, and dried to obtain Compound 3(5.6g, 83%). LC MS 165(M + H)+)，RT＝1.10min。
Compound 3(5.6g, 34.1mmol) was added to fuming nitric acid (12mL) and concentrated sulfuric acid (12mL), stirred at 100 ℃ for 1 hour, and then cooled to room temperature. The reaction solution was poured into ice water, and the crude product was filtered and recrystallized from acetic acid. Compound 4(3.48g, 49%) was obtained. LC MS 210(M + H)+)，RT＝1.26min。
Compound 4(200mg, 1mmol) was added to thionyl chloride (4mL) followed by 1 drop of DMF. Stirring was carried out at 85 ℃ under reflux for 3 hours. The reaction was complete by LCMS, concentrated and the residual thionyl chloride was taken up in toluene to give compound 5(200mg, crude for next step). LC MS 228(M + H)+)，RT＝1.58min。
Compound 6(1.40g, 10.0mmol) and compound 7(4.6g, 53.1mmol) were added to water (10mL) and stirred at 100 ℃ overnight. After the reaction was completed, the reaction mixture was cooled to room temperature, and a solid precipitated, which was then filtered, washed with water, washed with toluene, and dried to obtain Compound 8(1.68g, 82%). LC MS 206(M + H)+)，RT＝0.30min。
Compound 8(205mg, 1.0mmol), compound 9(251mg, 1.0mmol) and DIEA (193mg, 1.5mmol) were added to DMF (5mL) and the reaction was stirred at room temperature overnight. After the reaction was completed, concentration was performed, and the crude product was purified by column chromatography to give compound 10(255mg, 68%). LC MS:374(M + H)+)，RT＝1.28min。
Compound 10(155mg, 0.41mmol), compound 11(80mg, 0.5mmol), palladium acetate (10mg, 0.041mmol) and sodium carbonate (65mg, 0.61mmol) were added to DMF (2mL) and water (1mL), and the reaction was stirred at room temperature for 20 hours. The reaction was determined to be complete by LCMS, extracted with ethyl acetate and saturated ammonium chloride, and the organic phase was washed with saturated brine, dried, filtered and concentrated. Purifying the crude product by column chromatography to obtain compound 12(138mg, 83%))。LC MS:406(M+H+)，RT＝1.19min。
Compound 12(183mg, 0.45mmol), compound 5(103mg, 0.45mmol), tetratriphenylphosphine palladium (104mg, 0.09mmol), cuprous iodide (34mg, 0.18mmol), and cesium carbonate (220mg, 0.68mmol) were added to dried dioxane (10 mL). Microwave reaction (160 ℃,1 hour), LCMS check reaction complete. Filtration, washing with a mixture of DCM: MeOH (10:1), and purification of the crude product by column chromatography gave compound 13(58mg, 22%). LCMS 597(M + H)+)，RT＝1.36min。
Compound 13(30mg, 0.05mmol), compound 14(8mg, 0.07mmol) and triethylamine (12mg, 0.12mmol) were added to ethanol (8 mL). The reaction was stirred at 100 ℃ for 5 hours and LCMS checked for completion. Concentration and purification of the crude product by preparative TLC yielded compound 15(12mg, 35%). LC MS 692(M + H)+)，RT＝1.53min。
Compound 15: 4- (4- ((4' -chloro-biphenyl-2-yl) methyl) piperazin-1-yl) -N- (6-nitro-7- (((tetrahydro-2H-pyran-4-yl) methyl) amino) quinazolin-4-yl) benzamide
1H NMR(CDCl3,400MHz)δ(ppm)14.66(s,1H),9.54(s,1H),8.30(d,J＝3.6Hz,2H),8.26(s,1H),7.52(d,J＝4.4Hz,1H),7.38(s,6H),6.99(s,1H),6.91(d,J＝7.2Hz,2H),5.35(s,1H),4.01(s,2H),3.43(s,4H),3.33(s,6H),2.52(s,4H),1.80～1.20(m,5H)。
Using a similar procedure the following compounds were obtained:
4- (4- ((4' -chloro-biphenyl-2-yl) methyl) piperazin-1-yl) -N- (7- (((tetrahydro-2H-pyran-4-yl) methyl) amino) -6- (trifluoromethanesulfonyl) quinazolin-4-yl) benzamide
1H NMR(CDCl3,400MHz)δ(ppm)14.64(s,1H),8.54(s,1H),8.31(d,J＝3.6Hz,2H),8.26(s,1H),7.52(d,J＝4.4Hz,1H),7.38(s,6H),7.18(s,1H),6.91(d,J＝7.2Hz,2H),5.36(s,1H),4.01(s,2H),3.42(s,4H),3.35(s,6H),2.53(s,4H),1.80～1.20(m,5H)
16。
4- (4- ((4' -chloro-biphenyl-2-yl) methyl) piperazin-1-yl) -N- (7- ((3-morphinylpropyl) amino) -6- (trifluoromethanesulfonyl) quinazolin-4-yl) benzamide
1H NMR(CDCl3,400MHz)δ(ppm)14.63(s,1H),8.55(s,1H),8.31(d,J＝4.0Hz,2H),8.26(s,1H),7.51(d,J＝4.4Hz,1H),7.36(s,6H),7.18(s,1H),6.92(d,J＝7.2Hz,2H),5.36(s,1H),4.02(s,2H),3.42(s,4H),3.35(s,6H),2.71(s,4H),2.57(m,2H),2.45(s,4H),1.67(m,2H).
17。
4- (4- ((4' -chloro-biphenyl-2-yl) methyl) piperazin-1-yl) -N- (8-nitro-7- (((tetrahydro-2H-pyran-4-yl) methyl) amino) quinazolin-4-yl) benzamide
1H NMR(CDCl3,400MHz)δ(ppm)14.66(s,1H),8.37(d,J＝3.6Hz,2H),8.28(s,1H),8.05(d,J＝7.2Hz,1H),7.52(d,J＝4.4Hz,1H),7.37(s,6H),7.05(d,J＝7.6Hz,1H),6.90(d,J＝7.2Hz,2H),5.35(s,1H),4.01(s,2H),3.41(s,4H),3.32(s,6H),2.53(s,4H),1.80～1.20(m,5H)
18。
4- (4- ((4' -chloro-biphenyl-2-yl) methyl) piperazin-1-yl) -N- (7- (((tetrahydro-2H-pyran-4-yl) methyl) amino) -8- (trifluoromethanesulfonyl) quinazolin-4-yl) benzamide
1H NMR(CDCl3,400MHz)δ(ppm)14.64(s,1H),8.37(d,J＝3.6Hz,2H),8.28(s,1H),7.88(d,J＝7.2Hz,1H),7.53(d,J＝4.4Hz,1H),7.36(s,6H),7.09(d,J＝7.6Hz,1H),6.90(d,J＝7.2Hz,2H),5.35(s,1H),4.02(s,2H),3.41(s,4H),3.34(s,6H),2.52(s,4H),1.80～1.20(m,5H)
19。
4- (4- ((2- (4-chlorophenyl) -5, 5-dimethyl-cyclohex-1-enyl) methyl) piperazin-1-yl) -N- (7- (((tetrahydro-2H-pyran-4-yl) methyl) amino) -8- (trifluoromethanesulfonyl) quinazolin-4-yl) benzamide
1H NMR(CDCl3,400MHz)δ(ppm)14.65(s,1H),8.35(d,J＝4.0Hz,2H),8.28(s,1H),7.89(d,J＝7.2Hz,1H),7.53(d,J＝4.4Hz,1H),7.37(s,2H),7.08(d,J＝7.6Hz,1H),6.91(d,J＝7.2Hz,2H),5.35(s,1H),3.51(s,2H),3.32(s,4H),3.21(s,6H),2.46(s,4H),2.09(s,2H),2.03(s,2H),1.97(s,2H),1.68(m,3H),1.35(s,2H),1.04(s,6H).
20。
4- (4- ((2- (4-chlorophenyl) -5, 5-dimethyl-cyclohex-1-enyl) methyl) piperazin-1-yl) -N- (8-nitro-7- (((tetrahydro-2H-pyran-4-yl) methyl) amino) quinazolin-4-yl) benzamide
1H NMR(CDCl3,400MHz)δ(ppm)14.65(s,1H),8.36(d,J＝4.0Hz,2H),8.28(s,1H),8.05(d,J＝7.2Hz,1H),7.53(d,J＝4.4Hz,1H),7.37(s,2H),7.04(d,J＝7.2Hz,1H),6.89(d,J＝7.6Hz,2H),5.34(s,1H),3.51(s,2H),3.32(s,4H),3.23(s,6H),2.44(s,4H),2.08(s,2H),2.01(s,2H),1.96(s,2H),1.68(m,3H),1.35(s,2H),1.05(s,6H).
21。
4- (4- ((2- (4-chlorophenyl) -5, 5-dimethyl-cyclohex-1-enyl) methyl) piperazin-1-yl) -N- (7- ((3-morphinylpropyl) amino) -8-nitroquinazolin-4-yl) benzamide
1H NMR(CDCl3,400MHz)δ(ppm)14.66(s,1H),8.35(d,J＝4.0Hz,2H),8.28(s,1H),8.03(d,J＝6.8Hz,1H),7.52(d,J＝4.4Hz,1H),7.37(s,2H),7.05(d,J＝7.2Hz,1H),6.86(d,J＝7.2Hz,2H),5.34(s,1H),3.52(s,2H),3.34(s,4H),3.23(s,6H),2.68(s,4H),2.59(m,2H),2.46(s,4H),2.05(s,2H),2.00(s,2H),1.69(m,4H),1.03(s,6H).
22。
4- (4- ((2- (4-chlorophenyl) -4, 4-dimethyl-cyclohex-1-enyl) methyl) piperazin-1-yl) -N- (6-nitro-7- (((tetrahydro-2H-pyran-4-yl) methyl) amino) quinazolin-4-yl) benzamide
1H NMR(CDCl3,400MHz)δ(ppm)14.64(s,1H),9.53(s,1H),8.32(d,J＝4.0Hz,2H),8.25(s,1H),7.53(d,J＝4.4Hz,1H),7.37(s,2H),6.98(s,1H),6.87(d,J＝7.2Hz,2H),5.34(s,1H),3.51(s,2H),3.32(s,4H),3.23(s,6H),2.44(s,4H),2.09(s,2H),2.02(s,2H),1.96(s,2H),1.68(m,3H),1.35(s,2H),1.02(s,6H).
23。
4- (4- ((2- (4-chlorophenyl) -4, 4-dimethyl-cyclohex-1-enyl) methyl) piperazin-1-yl) -N- (8-nitro-7- (((tetrahydro-2H-pyran-4-yl) methyl) amino) quinazolin-4-yl) benzamide
1H NMR(CDCl3,400MHz)δ(ppm)14.66(s,1H),8.34(d,J＝3.6Hz,2H),8.28(s,1H),8.04(d,J＝6.8Hz,1H),7.52(d,J＝4.0Hz,1H),7.37(s,2H),7.06(d,J＝7.2Hz,1H),6.88(d,J＝7.6Hz,2H),5.32(s,1H),3.51(s,2H),3.32(s,4H),3.23(s,6H),2.45(s,4H),2.08(s,2H),2.00(s,2H),1.96(s,2H),1.67(m,3H),1.36(s,2H),1.03(s,6H).
24。
4- (4- ((2- (4-chlorophenyl) -4, 4-dimethyl-cyclohex-1-enyl) methyl) piperazin-1-yl) -N- (7- ((3-morphinylpropyl) amino) -8-nitroquinazolin-4-yl) benzamide
1H NMR(CDCl3,400MHz)δ(ppm)14.64(s,1H),8.34(d,J＝4.0Hz,2H),8.28(s,1H),8.03(d,J＝6.8Hz,1H),7.54(d,J＝4.0Hz,1H),7.37(s,2H),7.07(d,J＝7.6Hz,1H),6.86(d,J＝7.2Hz,2H),5.34(s,1H),3.52(s,2H),3.33(s,4H),3.23(s,6H),2.69(s,4H),2.58(m,2H),2.44(s,4H),2.05(s,2H),1.98(s,2H),1.70(m,4H),1.03(s,6H).
25。
4- (4- ((2- (4-chlorophenyl) -4, 4-dimethyl-cyclohex-1-enyl) methyl) piperazin-1-yl) -N- (7- (((tetrahydro-2H-pyran-4-yl) methyl) amino) -8- (trifluoromethanesulfonyl) quinazolin-4-yl) benzamide
1H NMR(CDCl3,400MHz)δ(ppm)14.65(s,1H),8.36(d,J＝3.6Hz,2H),8.28(s,1H),7.88(d,J＝7.2Hz,1H),7.53(d,J＝4.4Hz,1H),7.35(s,2H),7.08(d,J＝7.2Hz,1H),6.93(d,J＝7.6Hz,2H),5.35(s,1H),3.52(s,2H),3.32(s,4H),3.21(s,6H),2.46(s,4H),2.10(s,2H),2.03(s,2H),1.98(s,2H),1.67(m,3H),1.36(s,2H),1.02(s,6H).
26。
EXAMPLE 2 binding affinity assay of Compounds to Bcl-2 and Bcl-xL at the molecular level
1. Test methods (fluorescence polarization assay)
In vitro Bcl-2 binding assay
A21-residue Bid BH3 peptide (QEDIIRNIARHLAQVGDSMDR) (SEQ ID NO:1) labeled at the N-terminus with 6-6-carboxyfluorescein succinimidyl ester (FAM) was used as a fluorescent label (F1 u-Bid-21). The fluorescent peptide has been demonstrated to have KdHigh binding affinity of 15.74 nM. Bcl-2 used in this assay is a recombinant His-fusion soluble protein.
Test compounds dissolved in DMSO and 5. mu.l samples of Bcl-2 protein (0.120. mu.M) pre-incubated with Flu-Bid-21 peptide (0.010. mu.M) in assay buffer (100mM phosphoric acid clock, pH 7.5; 100. mu.g/ml bovine serum gamma globulin, 0.02% sodium azide, available from Invitrogen Corporation, Life Technologies) were added to Dynex 96-well black round bottom plates (Fisher Scientific) to produce a final volume of 125. mu.1. For each assay, a binding peptide containing Bcl-2 and Flu-Bid-21 peptides (corresponding to 0% inhibition) and a free peptide control containing Flu-Bid-21 alone (corresponding to 100% inhibition) were included on each assay plate. After 4 hours incubation when binding reached equilibrium, the polarization value (mP) in millipolarization (mi1ipo1 ionization) units was determined using an Ultra plate reader (Tecan U.S. Inc., Research Triangle Park, NC) at an excitation wavelength of 485nm and an emission wavelength of 530 nm. Analysis using nonlinear least squaresAnd IC was determined from the plot using a curve fitted with GraphPad Prism @ software50I.e. the concentration of inhibitor at which 50% of the bound peptide is replaced.
In vitro BcI-xL binding assays
To determine binding affinity to Bcl-xL proteins, a human Bcl-xL recombinant His-tagged protein without a C-terminal hydrophobic tail and a Bak-16mer BH3 peptide labeled with 6-carboxyfluorescein succinimidyl ester (FAM) were used. This peptide has been shown to KdBinding affinity of 9.79 nM. In the same manner as described for the Bcl-2 protein, complexes preincubated with 60nMBcl-xL and 5nM Flu-Bak peptide were used in the presence of 50mM Tris-Bis, pH 7.4; competitive binding assays were performed in assay buffer with 0.01% bovine serum gamma globulin.
2. Results
Part of the IC is provided in the table below50Data is obtained. Symbol + represents IC50Less than or equal to 1uM, the symbol ++ representing IC50Greater than 1uM, N/A representing temporary data absence
EXAMPLE 3 Effect of Compounds on proliferation potency of acute lymphoblastic leukemia cell line MV-4-11 test 1 and test method
Growth inhibition assay CCK-8 cell counting kit (Dojindo) detection of compounds on acute lymphoblastic leukemia cell line MV-4-11 cells (purchased from ATCC cell bank). The method comprises the following specific steps: MV-4-11 cells in the logarithmic growth phase were seeded into 96-well culture plates at an appropriate density at 90. mu.L per well, cultured overnight, and then a compound at various concentrations was added for 72hr, and a solvent control group (negative control) was set. After the compound acts on the cells for 72h, the influence of the compound on the cell proliferation is detected by using a CCK-8 cell counting kit (Dojindo), 10 mu L of CCK-8 reagent is added into each hole, and the mixture is cultured at 37 DEG CAfter being placed in the box for 2-4 hours, the reading is carried out by a SpectraMax 190 reader of a full-wavelength type micro-pore plate microplate reader, and the measuring wavelength is 450 nm. The inhibition (%) of the tumor cell growth by the compound was calculated using the following formula: the inhibition ratio (%) (OD negative control well-OD administration well)/OD negative control well × 100%. IC (integrated circuit)50The values were determined by regression with a four parameter method using a microplate reader random plus software.
2. Results
The IC of some of the compounds is provided in the table below50The value is obtained. Symbol + represents IC50Less than or equal to 1uM and the symbol ++ represents IC50Is 1uM to 10uM, and the symbol +++ represents IC50Greater than 10uM
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (9)

1. A compound of formula I, or a pharmaceutically acceptable salt thereof:
wherein,
ring a is a group selected from:
R1and R2Each independently selected from: H. halogen, NO2、CN、CF3SO2Or CF3；
R3And R4Each independently selected from: H. substituted OR unsubstituted C1-C8 alkyl, C (O) OR5、CONR6R7、C(O)R8Substituted or unsubstituted 5-8 membered aryl, substituted or unsubstituted 5-8 membered heteroaryl, substituted or unsubstituted 3-12 membered saturated or unsaturated heterocycle or carbocycle; wherein said heteroaryl group contains 1 to 3 heteroatoms selected from the group consisting of: n, O or S; said heterocycle comprising 1 to 3 heteroatoms selected from the group consisting of: n, O or S;
R5、R6、R7and R8Each independently selected from: H. substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C1-C6 alkylene-amino, substituted or unsubstituted C1-C6 alkylene-hydroxy, substituted or unsubstituted 5-to 8-membered aryl, or substituted or unsubstituted 5-to 8-membered heteroaryl; a substituted or unsubstituted 3-to 12-membered saturated heterocyclic or carbocyclic ring; wherein said heteroaryl group comprises at least one heteroatom selected from the group consisting of: n, O or S, the heterocyclic ring comprising at least one heteroatom selected from the group consisting of: n, O or S;
any "substitution" described above means that one or more hydrogen atoms on the group is substituted with a substituent selected from the group consisting of: halogen, OH, NH2CN, unsubstituted or halogenated C1-C8 alkyl, C1-C8 alkoxy, unsubstituted or halogenated C2-C6 alkenyl, unsubstituted or halogenated C2-C6 alkynyl, unsubstituted or halogenated C2-C6 acyl, unsubstituted or halogenated 5-8 membered aryl, unsubstituted or halogenated 5-8 membered heteroaryl, unsubstituted or halogenated 3-12 membered saturated heterocycle or carbocycle; wherein said heteroaryl group contains 1 to 3 heteroatoms selected from the group consisting of: n, O or S, the heterocycle comprising 1 to 3 heteroatoms selected from: n, O or S.
2. The compound of formula I according to claim 1, wherein the compound of formula I is a compound represented by:
3. a process for the preparation of a compound of formula I according to claim 1, comprising the steps of:
(a) reacting a compound of formula I-13 with a compound of formula I-14 in an inert solvent to obtain a compound of formula I;
in the above formulae, the groups are as defined in claim 1.
4. The method of claim 3, wherein the method further comprises the steps of:
(b) reacting a compound of formula I-12 with a compound of formula I-5 in an inert solvent to obtain a compound of formula I-13;
in the above formulae, the groups are as defined in claim 1.
5. Use of a compound according to claim 1 for:
(a) preparing a medicament for treating diseases related to the activity or expression quantity of Bcl-2 family protein;
(b) preparing a Bcl-2 family protein targeted inhibitor;
(c) non-therapeutically inhibiting the activity of a Bcl-2 family protein in vitro;
(d) non-therapeutically inhibiting tumor cell proliferation in vitro; and/or
(e) Treating diseases related to the activity or expression level of Bcl-2 family protein.
6. The use of claim 5, wherein the Bcl-2 family protein is selected from the group consisting of: bcl-2, Bcl-xL, Bcl-w, Mcl-1, Bcl2-A1, or a combination thereof; and/or
The tumor cell is a leukemia cell strain; preferably a lymphocyte leukemia cell line; more preferably acute lymphoblastic leukemia cell strain MV-4-11 cells.
7. A pharmaceutical composition, comprising: (i) an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.
8. A method of inhibiting Bcl-2 protein activity comprising the steps of: administering to a subject an inhibitory effective amount of a compound of formula I as described in claim 1 or a pharmaceutically acceptable salt thereof, or administering to a subject an inhibitory effective amount of a pharmaceutical composition as described in claim 7.
9. A method of inhibiting tumor cells in vitro, comprising: administering to a subject in need thereof an inhibitory effective amount of a compound of formula I as defined in claim 1, or a pharmaceutical composition as defined in claim 7.
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