CN107141287B

CN107141287B - 2-imine-5-keto-2, 5-dihydro-1-H-dipyridopyrimidine compounds

Info

Publication number: CN107141287B
Application number: CN201610116185.1A
Authority: CN
Inventors: 杨财广; 刘江; 罗成; 郭中强; 巩守哲; 李家飞; 郑桐; 蒋华良
Original assignee: Shanghai Institute of Materia Medica of CAS; Beijing Institute of Genomics of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS; Beijing Institute of Genomics of CAS
Priority date: 2016-03-01
Filing date: 2016-03-01
Publication date: 2021-10-26
Anticipated expiration: 2036-03-01
Also published as: CN107141287A

Abstract

The invention provides a 2-imine-5-keto-2, 5-dihydro-1-H-dipyridyl [3,4-c:1',2' -f]The invention discloses a synthesis method and application of a pyrimidine compound, and particularly discloses a compound shown as a general formula (I), and pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof, wherein the definition of each group is described in the specification. The compound of the invention can be used as an inhibitor targeting SPOP, or can be used for preparing a medicament for treating and/or preventing diseases (such as kidney cancer and the like) targeting SPOP.

Description

2-imine-5-keto-2, 5-dihydro-1-H-dipyridopyrimidine compounds

Technical Field

The invention discloses a 2-imine-5-keto-2, 5-dihydro-1-H-dipyridyl [3,4-c:1',2' -f ] pyrimidine compound shown as the following general formula (I) and pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof, which are used as an inhibitor taking SPOP as a target point and can treat diseases taking SPOP as the target point, such as kidney cancer and the like.

Background

Renal Cell Carcinoma (RCC), Renal cancer for short, is a common malignant tumor of the urinary system, accounting for about 2-3% of adult malignant tumors. According to the pathological type, the kidney cancer is mainly classified into Clear cell carcinoma, papillary carcinoma, chromophobe cell carcinoma, collecting duct carcinoma and the like, wherein Clear cell renal cell carcinoma (ccRCC) is the most common pathological type of the kidney cancer, and accounts for about 80% of the total incidence number of the kidney cancer. Up to 30% of kidney cancers are metastases, while about half of the rest develop into metastases, with over 10 million cases of death worldwide per year.

SPOP (spelle-type POZ protein) belongs to the MATH/BTB protein family, and plays an important role in regulating ubiquitination and degradation of substrate proteins as a linker molecule of E3 ubiquitin ligase CUL-3, such as Daxx, Pdx1, PIPKIIb, SRC-3 and the like. Studies now find that SPOP protein expression is abnormal in a variety of tumor cells, indicating that SPOP plays an important role in maintaining the growth and development of normal cells. Liujiang and the like in 2009 disclose that SPOP is over-expressed in clear cell carcinoma cell strains, and arouse the interest of people in using SPOP as a treatment target and applying the SPOP to targeted treatment of clear cell carcinoma.

Hypoxia inducible factor HIF is a nuclear protein with transcriptional activity, has a quite broad target gene spectrum, and plays an important role in accelerating the growth of various cancer cells. Liujiang et al found that over-activated hypoxia inducible factor HIF could transcriptionally regulate SPOP expression and over-express the nuclear protein SPOP in renal cancer tissue and mis-localize in the cytoplasm. Unlike the pro-apoptotic function of nuclear-localized SPOPs, cytoplasmic SPOPs accelerate cell proliferation. The cytosolic SPOP binds to the tumor suppressor PTEN, ERK phosphatase, Daxx and Gli2 and degrades it through the ubiquitination pathway, resulting in the production of kidney cancer. The SPOP knockout specifically kills renal clear cell carcinoma, but has little effect on normal cells.

The SPOP can be used as a potential molecular probe or a drug target to provide clues and also provide a new theoretical basis for the diagnosis and treatment of the kidney cancer. In view of the foregoing, there remains a need in the art for the development of compounds having SPOP inhibitory activity.

Disclosure of Invention

In order to solve the above technical problems, it is an object of the present invention to provide 2-imine-5-keto-2, 5-dihydro-1-H-bipyridyl [3,4-c:1',2' -f ] pyrimidine compounds and pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof.

Another object of the present invention is to provide the use of the 2-imine-5-keto-2, 5-dihydro-1-H-bipyrido [3,4-c:1',2' -f ] pyrimidines and their pharmaceutically acceptable salts or pharmaceutically acceptable solvates for the preparation of SPOP inhibitors according to the invention.

It is a further object of the present invention to provide the use of the 2-imine-5-keto-2, 5-dihydro-1-H-bipyridino [3,4-c:1',2' -f ] pyrimidines and pharmaceutically acceptable salts or solvates thereof according to the invention for the preparation of a medicament for the treatment of diseases associated with SPOP, in particular for the treatment of renal cancer.

It is still another object of the present invention to provide a pharmaceutical composition comprising as an active ingredient one or more selected from the group consisting of 2-imine-5-keto-2, 5-dihydro-1-H-bipyridino [3,4-c:1',2' -f ] pyrimidines, pharmaceutically acceptable salts or solvates thereof, or mixtures thereof according to the invention in a therapeutically effective amount.

It is still another object of the present invention to provide a method for treating SPOP-related diseases (kidney cancer, etc.) comprising administering to a patient a therapeutically effective amount of one or more selected from the group consisting of 2-imine-5-keto-2, 5-dihydro-1-H-bipyrido [3,4-c:1',2' -f ] pyrimidines, pharmaceutically acceptable salts or solvates thereof, or mixtures thereof according to the present invention as an active ingredient.

In a first aspect of the present invention, there is provided a 2-imine-5-keto-2, 5-dihydro-1-H-bipyrido [3,4-c:1',2' -f ] pyrimidine compound represented by the following general formula (I), or a pharmaceutically acceptable salt or solvate thereof:

wherein the content of the first and second substances,

x and Y are each independently absent, or substituted or unsubstituted alkylene or alkylene-O-having 1,2, 3,4, 5, 6, 7, 8, 9, or 10 carbon atoms; wherein the alkylene is a linear or branched alkylene;

R₁to be located at

One or more H or C on the ring₁-C₄An alkyl group;

R₂、R₃each independently selected from the group consisting of: hydrogen, halogen, hydroxy, nitro, amino, cyano, carbonyl, substituted or unsubstituted C₂-C₁₂Alkenyl, substituted or unsubstituted C₂-C₁₂Alkynyl, carboxyl, substituted or unsubstituted C₁-C₁₂Alkoxy, substituted or unsubstituted C₁-C₁₂Alkylamino radical, substituted or unsubstituted C₃-C₉Cycloalkyl, substituted or unsubstituted C₂-C₉Non-heteroaryl, substituted or unsubstituted 3-12 membered heterocycle, substituted or unsubstituted C₆-C₁₂Aryl, substituted or unsubstituted C₄-C₁₂A heteroaryl group;

m is selected from the group consisting of: NH, O or S;

wherein said substitution means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of: halogen, hydroxy, nitro, amino, cyano, carbonyl, carboxyl, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Alkylamino radical, hydroxy radical C₁-C₆Alkyl, amino C₁-C₆Alkyl, carbonyl C₁-C₆Alkyl radical, C₃-C₉Cycloalkyl radical, C₆-C₁₂Aryl, 3-to 12-membered heterocycle, C₄-C₁₂A heteroaryl group.

In another preferred embodiment, X and Y are each independently absent, or unsubstituted, straight chain alkylene having 1,2, 3,4, 5, 6, 7, 8, 9, or 10 carbon atoms;

R₂、R₃each independently selected from the group consisting of: hydrogen, fluoro, chloro, bromo, iodo, hydroxy, nitro, cyano, carbonyl, carboxyl, amino, or a substituted or unsubstituted group selected from the group consisting of: methoxy, ethoxy, propoxy, isopropyl, N-butoxy, tert-butoxy, N- (N, N-dimethyl) substituent, N- (N, N-diethyl) substituent, N- (N, N-dipropyl) substituent, N- (N, N-dibutyl) substituent, N- (N-methyl-N-ethyl) substituent, N- (N-methyl-N-propyl) substituent, N- (N-methyl-N-butyl) substituent, N- (N-ethyl-N-propyl) substituent, N- (N-ethyl-N-butyl) substituent, N- (N-propyl-N-butyl) substituent, cyclopropyl, tert-butoxy, N- (N, N-dimethyl) substituent, N- (N, N-diethyl) substituent, N- (N, N-dipropyl) substituent, N- (N, N-dibutyl) substituent, N- (N-methyl-N-ethyl-N-butyl) substituent, N- (N-propyl-N-butyl) substituent, cyclopropyl-butyl) substituent, N- (N-butyl) substituent, N- (N-propyl-butyl) substituent, N- (N-butyl) substituent, N- (N-butyl) substituent, N- (N-butyl) substituent, N- (N-butyl) substituent, N-butyl, N- (C, N-butyl, C, N-butyl, C, Cyclobutyl, cyclopentyl, cyclohexyl, tetrahydrofuryl, tetrahydrothienyl, tetrahydropyranyl, piperazinoPyridyl, morpholinyl, piperazinyl, phenyl, naphthyl, pyridyl, furanyl, pyrrolyl, thienyl, indolyl, quinolinyl, imidazolyl, or benzimidazolyl.

In another preferred embodiment, X and Y are each independently a substituted or unsubstituted alkylene or alkylene-O-having 1,2, 3 or 4 carbon atoms;

R₁selected from hydrogen, methyl, ethyl, propyl, isopropyl, cyclopropyl, n-butyl or tert-butyl;

R₂selected from the group consisting of: hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, ethynyl, nitro, cyano, carboxyl, methoxy, ethoxy, propoxy, cyclohexyloxy, N- (N, N-dimethyl) substituent, N- (N, N-diethyl) substituent, N- (N, N-dipropyl) substituent, N- (N, N-dibutyl) substituent, N- (N-methyl-N-ethyl) substituent, N- (N-methyl-N-propyl) substituent, N- (N-methyl-N-butyl) substituent, N- (N-ethyl-N-propyl) substituent, N- (N-ethyl-N-butyl) substituent, N- (N-propyl-N-butyl) substituent, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 2,3 or 4-methylcyclohexyl, 2,3 or 4-ethylcyclohexyl, 2,3 or 4-hydroxycyclohexyl, 2,3 or 4-carboxycyclohexyl, 2,3 or 4-cyanocyclohexyl, 2,3 or 4-aminocyclohexyl, 2 or 3-tetrahydrofuryl, 2 or 3-tetrahydrothienyl, N,2 or 3-tetrahydropyranyl, 2,3 or 4-tetrahydropyranyl, N,2,3 or 4-piperidinyl, N,2 or 3-morpholinyl, N or 2-piperazinyl, N,2 or 3- (N-methylpiperazinyl), N,2 or 3- (N-benzylpiperazinyl) phenyl, 2,3 or 4-methylphenyl, 2,3 or 4-methoxyphenyl, 2,3 or 4-aminophenyl, 2,3 or 4-cyanophenyl, 2,3 or 4-carboxyphenyl, 2,3 or 4-nitrophenyl, 2,3 or 4-hydroxyphenyl, naphthyl, 2 or 3-furyl, 2 or 3-thienyl, N,2 or 3-pyrrolyl, 2,3 or 4-pyridyl, N,2 or 3- (N-morpholinyl) phenyl, indolyl.

In another preferred embodiment, said X, Y, R₁、R₂、R₃And M are each independently a group corresponding to the specific compound in the examples.

In another preferred embodiment, R is₁Selected from hydrogen or methyl.

In addition toIn a preferred embodiment, R is₁Is methyl.

In another preferred embodiment, R is₁To be located at

One methyl group on the ring.

In another preferred embodiment, R₂-X-is a group selected from: methyl, ethyl, propyl, cyclohexyl, or N,2 or 3- (N-morpholinyl) phenyl;

or X is an alkylene group having 1,2, 3 carbon atoms, and R₂Is a group selected from: an N- (N, N-diethyl) substituent, an N,2 or 3-morpholinyl group, a phenyl group, a 2,3 or 4-methylphenyl group, a 2,3 or 4-fluorophenyl group, a 2,3 or 4-methoxyphenyl group, a 2,3 or 4-hydroxyphenyl group, a 2 or 3-furyl group, a 2 or 3-thienyl group, an indolyl group.

In a second aspect of the present invention, there is provided a use of a compound of formula (I) as described in the first aspect of the present invention, or a pharmaceutically acceptable salt or solvate thereof, for the preparation of an inhibitor of SPOP targets, or for the preparation of a medicament for the treatment and/or prevention of diseases targeted to SPOP.

In another preferred embodiment, said compound of formula (I) is used to inhibit SPOP activity.

In another preferred embodiment, the inhibition is competitive inhibition.

In a third aspect of the invention, there is provided the use of a compound of formula (I) as described in the first aspect of the invention, or a pharmaceutically acceptable salt or solvate thereof, for inhibiting the proliferative activity of renal cancer cells.

In another preferred embodiment, the disease is selected from the group consisting of: kidney cancer, endometrial cancer, germ cell tumor.

In a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of a compound according to the first aspect of the present invention, and/or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, and a pharmaceutically acceptable carrier.

In a fifth 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, said process comprising the steps of:

reacting a compound of formula Ia with a compound of formula Ib in an inert solvent to obtain a compound of formula I.

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.

Drawings

FIG. 1 shows that cell proliferation experiments verify the inhibitory activity of 2-imine-5-keto-2, 5-dihydro-1-H-dipyridyl [3,4-c:1',2' -f ] pyrimidine compounds on renal cancer cells A498;

FIG. 2 shows that cell proliferation experiments verify the inhibitory activity of 2-imine-5-keto-2, 5-dihydro-1-H-bipyridyl [3,4-c:1',2' -f ] pyrimidines on 6 renal cancer cell lines and 1 normal tubular epithelial cell line;

FIG. 3 shows that Western blot experiment (Western blot) verifies the inhibitory activity of 2-imine-5-keto-2, 5-dihydro-1-H-dipyridyl [3,4-c:1',2' -f ] pyrimidine compounds on SPOP downstream signal channels;

FIG. 4 shows that Co-immunoprecipitation assay (Co-IP) verifies that 2-imine-5-keto-2, 5-dihydro-1-H-bipyridino [3,4-c:1',2' -f ] pyrimidines competitively inhibit the interaction of SPOP with substrates in cells;

FIG. 5 shows In Vivo Ubiquitination experiments (In Vivo Ubiquitination Assay) to verify that 2-imine-5-keto-2, 5-dihydro-1-H-bipyridino [3,4-c:1',2' -f ] pyrimidines inhibit Ubiquitination of SPOP on substrates In cells;

FIG. 6 shows that Surface Plasmon Resonance (SPR) experiments verify the interaction of 2-imine-5-keto-2, 5-dihydro-1-H-bipyrido [3,4-c:1',2' -f ] pyrimidines with SPOP proteins;

FIG. 7 shows fluorescence polarization assay (FP) to verify the interaction of 2-imine-5-keto-2, 5-dihydro-1-H-bipyrido [3,4-c:1',2' -f ] pyrimidines with SPOP proteins;

FIG. 8 shows that the body weight and organ weight of normal nude mice and organ section staining experiments verify that 2-imine-5-keto-2, 5-dihydro-1-H-dipyridyl [3,4-c:1',2' -f ] pyrimidine compounds have small influence on the body weight and organ of normal nude mice.

FIG. 9 shows that the compound in-plasma metabolism experiment verifies that the 2-imine-5-keto-2, 5-dihydro-1-H-dipyridyl [3,4-c:1',2' -f ] pyrimidine compound has good metabolic stability.

FIG. 10 shows that the in vivo nude mouse tumorigenesis experiment verifies the inhibition effect of 2-imine-5-keto-2, 5-dihydro-1-H-dipyridyl [3,4-c:1',2' -f ] pyrimidine compounds on the growth of subcutaneous tumors of nude mice.

Detailed Description

The inventors of the present invention have conducted long-term and intensive studies to prepare a compound having a structure represented by formula I and found that it has SPOP inhibitory activity. And said compounds are at very low concentrations (IC)₅₀A value as low as 50nmol/L), namely, an inhibitory effect on the SPOP activity, which is quite excellent, and thus, it can be used for the treatment of diseases associated with the SPOP activity or expression amount, such as kidney cancer and the like. Based on the above findings, the inventors have completed the present invention.

Term(s) for

As used herein, the term "C_1-6The "alkyl group" means a straight or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like. Expressions like "C1-C6" are intended to include the corresponding group having 1,2, 3,4, 5, or 6 carbon atoms, e.g., "C_1-6Alkyl "refers to an alkyl group having 1,2, 3,4, 5, or 6 carbon atoms," C_2-9Non-heteroaryl "refers to non-heteroaryl having 2,3, 4, 5, 6, 7, 8, 9 carbon atoms.

As used herein, the term "C₁-C₆The alkoxy group means a substituent of a structure such as "straight-chain or branched alkyl-oxy group having 1 to 6 carbon atoms", such as ethoxy, propoxy, butoxy, or the like.

As used herein, the term "C₁-C₆Alkylamino refers to a substituent having a structure of a straight-chain or branched alkyl-amino group having 1 to 6 carbon atoms, such as ethylamino, propylamino, dimethylamino, or the like.

The term "alkylene" refers to a group formed after an alkyl group as described above has lost one hydrogen atom, e.g., -CH₂-、-CH₂-CH₂-, or the like.

The term "C₂-C₁₂The "alkenyl group" means a group formed by losing one hydrogen atom of an olefin having 2 to 12 carbon atoms, and includes an ethenyl group, a propenyl group, a n-butenyl group, or the like.

The term "C₃-C₉Cycloalkyl "refers to cycloalkyl groups having 3 to 9 carbon atoms, such as cyclopropane, cyclohexane, and the like.

The term "C₆-C₁₂Aryl "refers to an aromatic substituent having 6 to 12 carbon atoms and no heteroatoms in the backbone, such as phenyl, or the like.

The term "C₄-C₁₂Heteroaryl "refers to a non-saturated ring system substituent having 4 to 12 carbon atoms and having one or more heteroatoms selected from O, S, N or P, such as pyridyl, thienyl, or the like.

The term "3-12 membered heterocyclic ring" refers to a saturated ring system substituent having one or more heteroatoms selected from O, S, N or P in a 3-12 membered ring system, such as piperidinyl, 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.

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.

Compounds of formula I and their preparation

The invention provides a 2-imine-5-keto-2, 5-dihydro-1-H-dipyridyl [3,4-c:1',2' -f ] pyrimidine compound shown as the following general formula (I), or a pharmaceutically acceptable salt or solvate thereof:

wherein each group is as defined above.

Preferably, the compound is one selected from the following compounds:

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 of the present invention as a main active ingredient are useful for the treatment, prevention and alleviation of diseases associated with SPOP activity or expression level, since the compound of the present invention has excellent inhibitory activity against SPOP. According to the prior art, the compounds of the invention are useful for the treatment of the following diseases: renal cancer, clear cell carcinoma, 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, glycerol, mannitol, sorbitol, etc.), emulsifiers (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.)

) Wetting agent (such as sodium lauryl sulfate), colorant, flavoring agent, stabilizer, antioxidant, antiseptic, and pyrogen-free agentWater, 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, especially 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 an SPOP inhibitor with a novel structure, and preparation and application thereof, wherein the inhibitor can inhibit the activity of various SPOPs at an extremely low concentration.

3. Pharmaceutical compositions for treating diseases associated with SPOP activity are provided.

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.

The invention will be further illustrated with reference to specific examples. These examples are for illustrative purposes only and do not limit the scope and spirit of the present invention.

The following examples are the sources of drugs, DMEM/HG medium from gibco, 10% fetal bovine serum from life technology, penicillin, streptomycin and thiazole blue from Shanghai Biotechnology, Inc., and the reagents from pharmaceutical group chemical reagents, Inc. and Bailingwei science and technology, Inc. in the preparation examples. SPF grade BALD/c null female nude mice were purchased from Shanghai Slek laboratory animals Co., Ltd and were housed in SPF grade animal houses at Shanghai Hospital, and animal management and use followed the IACUC policy and guidelines of Shanghai Hospital, Japan.

Preparation examples

The 2-imine-5-keto-2, 5-dihydro-1-H-dipyridyl [3,4-c:1',2' -f ] pyrimidine compound and the pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof can be prepared by the following routes:

the synthesis of 2-imine-5-keto-2, 5-dihydro-1-H-bipyridino [3,4-c:1',2' -f ] pyrimidines is briefly described below by way of example with the compound N- (3- (4-morpholinyl) propyl) -1- (2-phenylethyl) -2-imine-10-methyl-5-ketobipyridine [4,3-b: 1',2' -f ] pyrimidine-3-carboxamide.

Step 1, adding 10.81g (100mmol) of 2-amino-3-methylpyridine and 24.03g (150mmol) of diethyl malonate into a 250mL eggplant-shaped bottle, and reacting at 200 ℃ for 2h, wherein a large amount of solid products are generated in the reaction process. After the reaction was complete, it was cooled to room temperature, added 200ml ethanol and refluxed overnight, cooled to room temperature and filtered to give 13g of product in 76% yield.

¹H NMR(400MHz,DMSO)δ8.79(dd,J＝7.2,1.6Hz,1H),7.83(d,J＝6.9Hz,1H),7.17(t,J＝7.0Hz,1H),5.40(s,1H),2.42(s,3H).

Step 2, 200mL (calcium hydride dehydration) of N, N-Dimethylformamide (DMF) was added to a 500mL eggplant-shaped bottle, cooled in an ice bath, and then 13mL (140mmol) of phosphorus oxychloride (P (O) Cl₃) The reaction was continued for 1h under ice-bath and 7.05g (40mmol) of 2-hydroxy-4-hydroxy-9-methylpyridine [1, 2-. alpha.were added in portions]And (3) pyridine amine, after the addition is finished, the mixture is subjected to ice bath reaction for 1h, room temperature reaction for 2h, then the mixture is subjected to reaction at 95 ℃ for 2h, after the reaction is finished, the mixture is added into 200mL of ice water, the reaction is performed for 1h, the filtration is performed, the solid is pulped with ethanol, the filtration and the drying are performed, and 6.2g of a product is obtained, wherein the yield is 70%.

¹H NMR(400MHz,CDCl₃)δ10.43(s,1H),9.12(ddd,J＝7.0,1.6,0.8Hz,1H),7.94(ddd,J＝7.1,1.7,0.9Hz,1H),7.35(t,J＝7.0Hz,1H),2.66(s,3H).

Step 3, adding 222.6mg (1mmol) of 2-chloro-3-aldehyde-4-carbonyl-4H-9-methylpyridine [1, 2-alpha ] pyrimidine into 5mL of absolute ethyl alcohol, dropwise adding 484.7mg (4mmol) of phenethylamine, reacting at room temperature for 12H, filtering after the reaction is finished, and fully washing the solid with ethyl alcohol to obtain 320.2mg of light yellow solid with the yield of 78%.

¹H NMR(400MHz,CDCl₃)δ10.69(s,1H),8.86(s,1H),8.79(d,J＝7.0Hz,1H),7.47(d,J＝6.7Hz,1H),7.43–7.05(m,10H),6.77(t,J＝7.0Hz,1H),3.90–3.71(m,4H),2.91(t,J＝7.4Hz,2H),2.86(t,J＝7.3Hz,2H),2.47(s,3H).

And 4, step 4: 1.44g (10mmol) of N- (2-aminopropyl) morpholine and 1.36g (1.2mmol) of ethyl cyanoacetate were put into a 25mL eggplant-shaped bottle and reacted for 48 hours, and after the reaction was completed, the bottle was washed three times with anhydrous diethyl ether, dried and put into a-20 ℃ refrigerator to obtain 1.7g of a pale yellow solid with a yield of 80%.

¹H NMR(400MHz,CDCl₃)δ7.90(s,1H),3.78–3.75(m,4H),3.40(dd,J＝11.5,5.8Hz,2H),3.35(s,2H),2.52–2.48(m,6H),1.72(ddd,J＝12.2,7.6,3.7Hz,2H).

Step 5, 108mg (0.5mmol) of 9-methyl-2- (2-phenylethylamino) -3- (2-phenylethyliminomethyl) pyridin [1,2-a ] opyrimidin-4-one and 103.5mg (0.55mmol) of 2-cyano-N- (3- (4-morpholinyl) propyl) acetamide were added to 2mL of chloroform, the reaction was refluxed for 8 hours, ether was added after the reaction was completed, the reaction was filtered, and the solid was recrystallized from absolute ethanol to give about 60mg of the product in 32% yield.

¹H NMR(400MHz,CDCl₃)δ8.88(d,J＝6.8Hz,1H),7.65(dt,J＝6.9,1.4Hz,1H),7.35–7.27(m,4H),7.24(d,J＝4.8Hz,1H),7.01(t,J＝7.0Hz,1H),4.62(s,2H),3.72(t,J＝4.7Hz,4H),3.52(q,J＝6.2Hz,2H),3.05(t,J＝8.1Hz,2H),2.59(s,3H),2.49(d,J＝6.6Hz,5H),1.82(q,J＝6.7Hz,3H).

Other compounds of formula I may be prepared by analogous methods using the corresponding starting materials.

The nuclear magnetic data of the 2-imine-5-keto-2, 5-dihydro-1-H-dipyridyl [3,4-c:1',2' -f ] pyrimidine compounds are shown in the following table.

The following is an SPOP activity inhibition experiment of 2-imine-5-keto-2, 5-dihydro-1-H-dipyridyl [3,4-c:1',2' -f ] pyrimidine compounds shown in a general formula (I).

First experimental example:cell proliferation assay (MTT) on 2-Imin-5-keto-2, 5-dihydro-1-H-bipyrido [3,4-c:1',2' -f]Evaluation of SPOP inhibition Activity of pyrimidine Compounds

The renal cancer cell line A498 was prepared from culture medium containing 10% fetal bovine serum (DMEM/HG medium, 100U penicillin, 100U streptomycin, 10% fetal bovine serum) at a concentration of 5X 10⁴-1×10⁵Cell suspension/mL. Each well of a 96-well cell culture plate was inoculated with 100. mu.L of cell suspension at 37 ℃ and 5% CO₂After culturing overnight under the condition until adherent growth, the compound in the concentration gradient table 1 is added, and culturing is continued for 68h, 10 μ L of thiazole blue solution (5mg/mL) is added into each hole, and culturing is continued for 4 h. After the culture is finished, completely absorbing and removing the culture medium in the wells, adding 150 mu L of dimethyl sulfoxide (DMSO) into each well, and shaking for 10min to fully melt the crystals. And (3) measuring the light absorption value at 490nm by using an enzyme-labeling instrument, recording the result and calculating the cell survival rate. Plotting the concentration of the compound as abscissa and the cell survival rate as ordinate, and obtaining IC by nonlinear regression fitting curve of GraphPad Prism 5.0 software₅₀The value is obtained. 2-imino-5-keto-2, 5-dihydro-1-H-bipyridino [3,4-c:1',2' -f]Pyrimidines IC₅₀The values are given in Table 1. As can be seen from Table 1, 2-imine-5-keto-2, 5-dihydro-1-H-bipyridino [3,4-c:1',2' -f]The pyrimidine compounds can inhibit SPOP activity.

TABLE 12-Imin-5-oxo-2, 5-dihydro-1-H-bipyrido [3,4-c:1',2' -f]Pyrimidines IC₅₀Value of

Experimental example two:cell proliferation experiment proves that 2-imine-5-keto-2, 5-dihydro-1-H-dipyridyl [3,4-c:1',2' -f]Inhibitory activity of pyrimidine compounds on 6 renal cancer cell lines and 1 normal renal tubular epithelial cell line

Experiments are carried out with SPOP-B-30 and SPOP-B-88 in 2-imine-5-keto-2, 5-dihydro-1-H-bipyridino [3,4-c:1',2' -f ] pyrimidines.

The renal cancer cell lines selected in the experiment comprise A498, Caki-2, Ketr-3, 769-P, OS-RC-2 and 786-O, and the normal renal tubular epithelial cells are HK-2 cells. Cells were cultured in DMEM/HG medium (A498, Ketr-3) or McCoy's 5a medium (Caki-2) or RPMI1640 medium (786-O, 769-P, OS-RC-2) or DMEM/F121:1 medium (HK-2) containing 10% (vol/vol) fetal bovine serum, 100U/ml penicillin and 100ng/ml streptomycin. The cell proliferation assay was performed as in experimental example one. As can be seen from FIG. 2, SPOP-B-30 and SPOP-B-8 can significantly inhibit the proliferative activity of renal cancer cells, but have no significant inhibitory activity against HK-2 (non-tumor cells) of normal renal tubular epithelial cells.

Experimental example three:western blotting experiments (Western blot) verify that 2-imine-5-keto-2, 5-dihydro-1-H-dipyridyl [3,4-c:1',2' -f)]Inhibitory Activity of pyrimidines on SPOP downstream Signaling pathways

2mL of A498 cell line resuspension (5X 10)⁴-1×10⁵one/mL) was added to a 6-well cell culture plate and cultured overnight to a cell-adherent state. Adding concentration gradient SPOP-B-30 and SPOP-B-88, and continuing to culture for 10 h. After the incubation was completed, the medium was aspirated, washed 3 times with phosphate buffer, and 150. mu.L of RIPA cell lysate and 1mM phenylmethylsulfonyl fluoride (PMSF) were added thereto and lysed on ice for 30 min. The lysate was scraped off with a cell scraper into a 1.5mL centrifuge tube, centrifuged at 15000g at 4 ℃ for 20min, the supernatant was transferred to a 1.5mL centrifuge tube, and the protein concentration of the supernatant was determined by the BCA method. mu.L of the supernatant was added to 25. mu.L of 5 Xloading buffer, heated at 100 ℃ for 10min, and centrifuged at 12000g for 1 min. The amount of the sample was adjusted according to the measured concentration, and polyacrylamide gel electrophoresis (SDS-PAGE) was performed. The separated protein bands were transferred to nitrocellulose membrane (NC membrane) by transfer electrophoresis, then blocked with 5% skim milk, incubated with primary antibody (rabbit-derived PTEN antibody), washed with TBST buffer, and incubated with secondary antibodyAnti (horseradish peroxidase HRP labeled goat anti-rabbit antibody), TBST buffer wash, and finally protein amount was detected by Enhanced Chemiluminescence (ECL) luminescence development step. As is evident from FIG. 3, as the concentrations of SPOP-B-30 and SPOP-B-8 increase, the contents of SPOP substrates PTEN and DUSP7 increase, and the phosphorylation of downstream signaling pathway active molecules AKT and ERK1/2 decreases, indicating that SPOP-B-30 and SPOP-B-8 can inhibit SPOP activity, thereby increasing the contents of anticancer substrate proteins PTEN and DUSP 7.

Experimental example four:co-immunoprecipitation assay (Co-IP) verification of 2-imine-5-keto-2, 5-dihydro-1-H-bipyridino [3,4-c:1',2' -f]Intracellular competitive inhibition of SPOP (Spinositide-phosphate-binding protein) and substrate interaction of pyrimidine compounds

HEK293 cells were grown in 60mm dishes at a density of about 30% -50%. The next day the corresponding plasmid myc-PTEN, Flag-SPOP was transfected. Transfection was carried out for 40h, corresponding amounts of small molecule compounds were added to each group, and after 8h the medium was aspirated off and washed twice with pre-cooled PBS. After the residual PBS was blotted, 200. mu.l of the RIPA cell lysate was added to each dish of cells, the cells were scraped from the dish using a precooled cell scraper, the suspension was pipetted into an EP tube, and placed on ice for 10-30 min. The supernatant was transferred to a new EP tube by centrifugation at 12000rpm for 10min at 4 ℃ and the protein concentration was quantified by BCA method. And (3) reserving 30 mu l of total protein after the protein concentration is leveled, adding 5 Xloading buffer solution, and boiling for 5 minutes in boiling water for denaturation. 20 μ l of antibody-conjugated agarose beads suspension was taken for each sample, washed three times with PBST, and finally resuspended at 1:1 by volume. Mu.l of beads resuspension was added to each sample, followed by 300. mu.l of RIPA lysate and rotary incubation at 4 ℃ for 4h or overnight. Centrifugation was carried out at 5000g for 1 minute at 4 ℃ to remove the supernatant. Add 500. mu.l PBST buffer, spin wash at 4 ℃ for 10min, centrifuge at 4 ℃ for 1min at 5000g, remove supernatant. The washing was repeated three times. After the final centrifugation, the supernatants were aspirated to dryness, and then 30. mu.l of 2 XP buffer was added and denatured in boiling water for 5 minutes. The denatured sample was centrifuged at 8000g at room temperature, and the supernatant was aspirated for SDS-PAGE, taking care not to aspirate the beads. As is evident from FIG. 4, SPOP-B-30 and SPOP-B-88 inhibit the binding of SPOP to the substrate PTEN in cells.

Experimental example five:in Vivo Ubiquitination experiments (In Vivo Ubiquitination Assay) verified 2-imine-5-keto-2, 5-dihydro-1-H-bipyridino [3,4-c:1',2' -f]Intracellular inhibition of ubiquitination of substrates by SPOPs using pyrimidines

HEK293 cells were grown in 60mm dishes at a density of about 30% -50%. The next day, myc-PTEN, Flag-SPOP and HA-Ub were co-transfected into HEK cells, respectively. 40h after transfection, corresponding amount of small molecule compound was added to each group, and 4h later, 10. mu.M MG132 medium was added for further culture for 4 h. After the incubation was complete, the medium was removed, the dish was placed on an ice-box, washed once with pre-chilled PBS and 100. mu.l of pre-chilled denaturing lysis buffer (1% SDS; 0.5mM EDTA; 1mM DTT; 50mM Tris, pH 7.5) was added. Cells were scraped from the culture dish into 1.5ml EP tubes quickly with cell scraping and subsequently denatured at 100 ℃ for 5 min. After denaturation, the lysate was cooled on ice and finally diluted with 900. mu.l of RIPA lysate. After dilution, carrying out ultrasonic treatment under the ultrasonic conditions: amplitude 10%, switch intermittently for 10s, total sonication time 1 min. The supernatant was transferred to a new EP tube by centrifugation at 12000g for 5min at 4 ℃. And leveling the protein concentration among the samples according to the protein quantification result. 100 μ l of the buffer solution was reserved as input, and the protein loading buffer was added for 5min at 100 ℃. The remaining lysates were immunoprecipitated with myc antibody-conjugated agarose beads and the final eluate was immunoblotted with HA antibody. As is evident from the results in FIG. 5, SPOP-B-30 and SPOP-B-88 can inhibit ubiquitination of the substrate PTEN by SPOP in cells.

Experimental example six:surface Plasmon Resonance (SPR) experiments to verify that 2-imine-5-keto-2, 5-dihydro-1-H-bipyridyl [3,4-c:1',2' -f]Interaction of pyrimidines with SPOP proteins

Freshly purified SPOP^MATHThe protein (purity > 95%, concentration 5mg/mL) was diluted to 0.1mg/mL with 10mM sodium acetate solution (pH 4.5). SPOP was coupled by BIAcore T200 (GE) using standard amino coupling procedures^MATHThe proteins were coupled to CM5 chips. The CM5 chip was equilibrated with HBS-EP buffer for 2h before the experiment. SPOP-B-30 and SPOP-B-88 were diluted with HBS-EP buffer gradient, equilibrated at 30. mu.L/min for 60s, combined for 120s, and washed for 120 s. The corresponding curves obtained were fitted by BIA data analysis software (GE Corp.) to obtain the corresponding K_DThe value is obtained. As is apparent from FIG. 6, SPOP-B-30 and SPOP-B-88 are shown with SPOP^MATHThe protein has certain binding effect.

Experimental example seven:fluorescence polarization experiment (FP) verification of 2-imine-5-keto-2, 5-dihydro-1-H-dipyrido [3,4-c:1',2' -f]Interaction of pyrimidines with SPOP proteins

Fluorescence Polarization (FP) experiments were performed on a multi-functional microplate reader (PerkinElmer). The instrument model is an EnVision Multilabel Plate Reader. The experiment uses a Fluorescein Isothiocyanate (FITC) -labeled polypeptide as a tracer substrate, the sequence of which is FITC-LACDEVTSTTSSSTA (produced by Gill Biochemical Co., Ltd.). mu.M SPOP was added to 100. mu.L of hydroxyethylpiperazine ethanethiosulfonic acid buffer (50mM, pH 7.5)^MATH20nM polypeptide substrate, SPOP-B-30 and SPOP-B-88, incubating at 4 deg.C for 1h, and detecting its fluorescence polarization value. The excitation and emission light used were 480nm and 535nm, respectively. The experiment was repeated three times and the experimental data used was analyzed using GraphPad Prism 5.0 software. As can be seen in FIG. 7, SPOP-B-30 and SPOP-B-88 interact with SPOP proteins.

Experimental example eight:mouse animal experiment proves that 2-imine-5-keto-2, 5-dihydro-1-H-dipyridyl [3,4-c:1',2' -f]Toxicity of pyrimidine compounds

Compound in vivo toxicity experiments 6 to 8 week old BALB/c and ICR mice were purchased from Shanghai Spiker laboratory animals Co., Ltd (SLAC) and Shanghai Spikekia laboratory animals Co., Ltd, respectively. Randomly grouped, 3 per group. The solvent, 80mg/kg, 120mg/kg compound were intraperitoneally injected once a day, and the body weight was weighed and checked for activity. Blood is collected in the orbit after 6 to 7 days, and hematology analysis and plasma biochemical detection are carried out. After anesthesia sudden death of mice, viscera (heart, liver, spleen, lung and kidney) were taken for HE staining and pathological section detection. As can be seen in FIG. 8, the compound injection concentration of 80mg/kg is a safe concentration.

Experimental example nine:mouse animal experiment detection of 2-imine-5-keto-2, 5-dihydro-1-H-dipyridyl [3,4-c:1',2' -f]In vivo metabolism of pyrimidines

The experiment takes SPOP-B-30 in 2-imine-5-keto-2, 5-dihydro-1-H-dipyridyl [3,4-c:1',2' -f ] pyrimidine compounds as an example.

BALB/c mice at 4-6 weeks of age were randomly grouped into 3 mice each. The compound was administered by intraperitoneal injection using a suspension prepared from normal saline, 0.5% 2-phenylethyl alcohol and 1.5% tween-80 as a solvent to give a compound concentration of 10 mg/kg. Tail vein bleeds were performed at 0.25, 0.5, 1,2, 4, 6, 10 and 24 hour time points post compound administration, 0.15mL each. Blood samples are anticoagulated by EDTA, and supernatant is centrifuged to detect the content of SPOP-B-30 compounds in serum by an LC-MS/MS method. From FIG. 9, it can be calculated that the blood drug concentration occurs at the highest 0.5 hour post-administration, 1070ng/ml, with a half-life of 6.25 hours.

Experimental example ten:mouse animal experiment proves that 2-imine-5-keto-2, 5-dihydro-1-H-dipyridyl [3,4-c:1',2' -f]Activity of pyrimidine compound

A498 cells were cultured in DMEM/HG + 10% fetal bovine serum at 5% CO₂And cultured at 37 ℃. Collecting pancreatically digested A498 cells, preparing cell suspension with fetal calf serum-free culture medium, and counting to 10⁸Cell/ml concentration. Storing at-20 deg.CThe matrigel (Martrigel, BD Co.) was spontaneously thawed at 4 ℃ and the cell suspension: the matrix gel Martrigel 3:1(v/v) was mixed and injected immediately with 100. mu.L (10)⁷Individual cells) to 6-week-old nude mice were subcutaneously placed in the right forelimb axilla. Tumor growth was observed and tumor length (a mm) and width (b mm) were measured with a vernier caliper according to v ═ a × b ²2 calculating the tumor volume to be about 125mm³The groups were divided, and 40 experimental nude mice were divided into 4 groups of 10 mice each. The compound SPOP-B-30 was dissolved in a solvent (physiological saline, 0.5% 2-phenylethyl alcohol, 1.5% Tween-80) to 5mg/mL, and SPOP-B-88 was dissolved in physiological saline to 5 mg/mL. Intraperitoneal injections were performed in amounts of 0, 40, 60, 80mg/kg daily, and body weight weighing and tumor measurements were performed every 3 days, with corresponding care and documentation. And observing the growth conditions of the tumors of the control group and the experimental group, stopping administration for about 25 days, carrying out anesthesia sudden death on the nude mice and carrying out dissection, and respectively taking the heart, the liver, the spleen, the lung and the kidney of 2 nude mice in each group for tissue HE staining observation. Meanwhile, separating out the tumor, weighing, photographing and freezing by liquid nitrogen, wherein the preserved tumor tissue is used for detecting the PTEN amount of the tissue by the Western blot.

As can be seen in FIG. 10, the tumor volumes of mice administered with SPOP-B-30 and SPOP-B-88 were significantly smaller than those of mice injected with solvent alone, and a significant reduction in SPOP content in the tissues was also observed from Western blot analysis of tumor tissues. Animal experiments have shown that SPOP-B-30 and SPOP-B-88 can slow tumor growth by inhibiting SPOP.

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

1. 2-imine-5-keto-2, 5-dihydro-1-one represented by the following general formula (I)H-bipyridino [3,4-c:1',2' -f]A pyrimidine compound, or a pharmaceutically acceptable salt thereof:

general formula (I)

Wherein the content of the first and second substances,

x is-CH₂-CH₂-

Y is-CH₂-CH₂-or- (CH)₂)₃-；

R₁To be located at

One or more C's on the ring₁-C₄An alkyl group;

R₂selected from the group consisting of: substituted or unsubstituted C₃-C₉Cycloalkyl, substituted or unsubstituted C₆-C₁₂Aryl, substituted or unsubstituted C₄-C₁₂A heteroaryl group;

R₃selected from the group consisting of: morpholinyl, piperazinyl;

m is selected from the group consisting of: NH, O or S;

wherein said substitution means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of: halogen, hydroxy, nitro, amino, cyano, carbonyl, carboxyl, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Alkylamino radical, hydroxy radical C₁-C₆Alkyl, amino C₁-C₆Alkyl, carbonyl C₁-C₆Alkyl radical, C₃-C₉Cycloalkyl radical, C₆-C₁₂Aryl, 3-to 12-membered heterocycle, C₄-C₁₂A heteroaryl group;

and said compound is not a structure selected from the group consisting of:

；

wherein, C₄-C₁₂Heteroaryl refers to an unsaturated ring system substituent having one or more heteroatoms selected from O, S, N or P; the term 3-12 membered heterocyclic ring refers to a saturated ring system substituent having one or more heteroatoms selected from O, S, N or P on the ring system.

2. A compound of formula (I), or a pharmaceutically acceptable salt thereof, as claimed in claim 1,

R₂selected from the group consisting of: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, pyridyl, furyl, pyrrolyl, thienyl, indolyl, quinolinyl, imidazolyl, or benzimidazolyl.

3. A compound of formula (I), or a pharmaceutically acceptable salt thereof, as claimed in claim 1 wherein R is₁Is methyl R₂Selected from the group consisting of: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 2,3 or 4-methylcyclohexyl, 2,3 or 4-ethylcyclohexyl, 2,3 or 4-hydroxycyclohexyl, 2,3 or 4-carboxycyclohexyl, 2,3 or 4-cyanocyclohexyl, 2,3 or 4-aminocyclohexyl, N,2 or 3- (N-benzylpiperazinyl) phenyl, 2,3 or 4-methylphenyl, 2,3 or 4-methoxyphenyl, 2,3 or 4-aminophenyl, 2,3 or 4-cyanophenyl, 2,3 or 4-carboxyphenyl, 2,3 or 4-nitrophenyl, 2,3 or 4-hydroxyphenyl, naphthyl, 2 or 3-furyl, 2 or 3-thienyl, N,2 or 3-pyrrolyl, 2,3 or 4-pyridyl, N,2 or 3- (N-morpholinyl) phenyl, indolyl.

4. A compound of formula (I), or a pharmaceutically acceptable salt thereof, as claimed in claim 1 wherein R is₂Is a group selected from: phenyl, 2,3 or 4-methylphenyl, 2,3 or 4-fluorophenyl, 2,3 or 4-methoxyphenyl, 2,3 or 4-hydroxyphenyl, 2 or 3-furyl, 2 or 3-thienyl, indolyl.

5. A compound selected from the following compounds, or a pharmaceutically acceptable salt thereof:

。

6. a compound selected from the group consisting of:

。

7. use of a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, for the preparation of an inhibitor of the SPOP target, or for the preparation of a pharmaceutical composition for the treatment and/or prevention of a disease targeted to SPOP.

8. The use according to claim 7, wherein the pharmaceutical composition is for inhibiting the proliferative activity of renal cancer cells.

9. The use according to claim 7, wherein the disease is selected from the group consisting of: kidney cancer, endometrial cancer, germ cell tumor.

10. A pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of a compound according to any one of claims 1 to 6, and/or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

11. A process for the preparation of a compound according to claim 1, comprising the steps of:

reacting a compound shown in formula Ia with a compound shown in formula Ib in an inert solvent to obtain a compound shown in formula I;

wherein the groups are as defined in claim 1.