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

Abstract

The invention provides a synthesis of 2-imine-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f]pyrimidine compounds and its use, specifically, the present invention discloses a compound with the following general formula (I), and a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof, wherein the definitions of each group are as in the specification said. The compounds of the present invention can be used as SPOP-targeted inhibitors, or prepared as medicaments for treating and/or preventing SPOP-targeted diseases (such as renal cancer, etc.).

Description

2-Imine-5-keto-2,5-dihydro-1-H-dipyridopyrimidines

technical field

The present invention discloses 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2' having the following general formula (I) -f] Pyrimidine compounds and their pharmaceutically acceptable salts or pharmaceutically acceptable solvates, as inhibitors targeting SPOP, can treat diseases targeting SPOP, such as renal cancer and the like.

Background technique

Renal cell carcinoma (RCC), referred to as renal cancer, is a common malignant tumor of the urinary system, accounting for about 2-3% of adult malignant tumors. According to different pathological types, renal cancer is mainly divided into clear cell carcinoma, papillary carcinoma, chromophobe carcinoma and collecting duct carcinoma, among which clear cell renal cell carcinoma (ccRCC) is the most common pathological type of renal carcinoma. , accounting for about 80% of the total incidence of kidney cancer. Up to 30% of kidney cancers are metastases, while about half of the remaining will gradually develop into metastases. Worldwide, more than 100,000 kidney cancer deaths occur each year.

SPOP (Speckle-type POZ protein) belongs to the MATH/BTB protein family. As the linker molecule of the E3 ubiquitin ligase CUL-3, it plays an important role in regulating the ubiquitination and degradation of substrate proteins, such as Daxx, Pdx1, PIPKIIb , SRC-3 and so on. Current studies have found 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. In 2009, Liu Jiang et al. revealed that SPOP was overexpressed in clear cell cancer cell lines, which aroused people's interest in using SPOP as a therapeutic target for targeted therapy of clear cell carcinoma.

Hypoxia-inducible factor HIF is a transcriptionally active nuclear protein with a wide spectrum of target genes, which plays an important role in accelerating the growth of various cancer cells. Liu Jiang et al. found that the hyperactivated hypoxia-inducible factor HIF can transcriptionally regulate the expression of SPOP, and the nuclear protein SPOP is overexpressed in renal cancer tissue and mislocalized in the cytoplasm. Unlike the pro-apoptotic function of nuclear-localized SPOP, cytoplasmic SPOP accelerates cell proliferation. Cytoplasmic SPOP can bind to tumor suppressors PTEN, ERK phosphatase, Daxx and Gli2, and degrade them through the ubiquitination pathway, leading to the development of renal cancer. Knockout of SPOP can specifically kill renal clear cell carcinoma, but has little effect on normal cells.

SPOP provides clues as potential molecular probes or drug targets, and also provides a new theoretical basis for the diagnosis and treatment of renal cancer. In conclusion, there is still a need in the art to develop compounds with SPOP inhibitory activity.

SUMMARY OF THE INVENTION

In order to solve the above technical problems, one object of the present invention is to provide a 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[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 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f according to the present invention ] Use of pyrimidine compounds and their pharmaceutically acceptable salts or pharmaceutically acceptable solvates in the preparation of SPOP inhibitors.

Yet another object of the present invention is to provide 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f according to the present invention ] The application of pyrimidine compounds and their pharmaceutically acceptable salts or pharmaceutically acceptable solvates in the preparation of medicines for the treatment of diseases related to SPOP, especially in the preparation of medicines for the treatment of renal cancer application.

A further object of the present invention is to provide a therapeutically effective amount of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c: A pharmaceutical composition in which one or more of 1',2'-f]pyrimidine compounds, pharmaceutically acceptable salts or pharmaceutically acceptable solvates or mixtures thereof are used as active ingredients.

Still another object of the present invention is to provide a method of treating diseases associated with SPOP (renal cancer, etc.), the method comprising administering to a patient a therapeutically effective amount selected from 2-imino-5-ones according to the present invention Base-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f]pyrimidines, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof one or more of the compounds or mixtures thereof as the active ingredient.

The first aspect of the present invention provides a 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c represented by the following general formula (I) :1',2'-f]pyrimidines, or their pharmaceutically acceptable salts or solvates:

in,

X and Y are each independently none, or a substituted or unsubstituted alkylene or alkylene-O- having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms; Wherein, described alkylene is straight-chain or branched alkylene;

环上的一个或多个H或C₁-C₄烷基；R ₁ is located at

One or more H or C ₁ -C ₄ alkyl groups on the ring;

R ₂ , R ₃ are each independently selected from the group consisting of hydrogen, halogen, hydroxy, nitro, amino, cyano, carbonyl, substituted or unsubstituted C ₂ -C ₁₂ alkenyl, substituted or unsubstituted C ₂ -C ₁₂ Alkynyl, carboxyl, substituted or unsubstituted C ₁ -C ₁₂ alkoxy, substituted or unsubstituted C ₁ -C ₁₂ alkylamino, substituted or unsubstituted C ₃ -C ₉ cycloalkyl, substituted or unsubstituted C ₂ - _C9 non-heteroaryl, substituted or unsubstituted 3- to ₁₂ -membered heterocycle, substituted or unsubstituted _C6 -C12 aryl, substituted or unsubstituted _C4 - _C12 heteroaryl;

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

Wherein, the substitution refers to that one or more hydrogen atoms on the group are replaced by a substituent selected from the group consisting of halogen, hydroxyl, nitro, amino, cyano, carbonyl, carboxyl, C ₁ -C ₆ alkane group, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylamino, hydroxy C ₁ -C ₆ alkyl, amino C ₁ -C ₆ alkyl, carbonyl C ₁ -C ₆ alkyl, C ₃ - C ₉ cycloalkyl, C ₆ -C ₁₂ aryl, 3-12-membered heterocycle, C ₄ -C ₁₂ heteroaryl.

In another preferred embodiment, X and Y are each independently none, or an unsubstituted straight-chain alkylene group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms;

R ₂ , R ₃ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, nitro, cyano, carbonyl, carboxyl, amino, or a substituted or unsubstituted group selected from the group consisting of : Methoxy, ethoxy, propoxy, isooxypropyl, n-butoxy, tert-butoxy, N-(N,N-dimethyl) substituent, N-(N,N-di ethyl) 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, tetrahydrofuranyl, tetrahydrofuranyl Hydrothienyl, tetrahydropyrrolyl, tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, phenyl, naphthyl, pyridyl, furanyl, pyrrolyl, thienyl, indolyl, quinoline olinyl, imidazolyl, or benzimidazolyl.

In another preferred embodiment, X and Y are each independently none, or a substituted or unsubstituted alkylene or alkylene-O- having 1, 2, 3 or 4 carbon atoms;

R ₁ is selected from hydrogen, methyl, ethyl, propyl, isopropyl, cyclopropyl, n-butyl or tert-butyl;

R is selected 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) Substituents, N-(N-methyl-N-ethyl) substituents, N-(N-methyl-N-propyl) substituents, N-(N-methyl-N-butyl) substituents , N-(N-ethyl-N-propyl) substituent, N-(N-ethyl-N-butyl) substituent, N-(N-propyl-N-butyl) substituent, ring propyl, 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-tetrahydrofuranyl, 2 or 3-tetrahydrothienyl, N,2 or 3-tetrahydropyrrolyl, 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, the X, Y, R ₁ , R ₂ , R ₃ and M are each independently a group corresponding to the specific compound in the embodiment.

In another preferred embodiment, the R ₁ is selected from hydrogen or methyl.

In another preferred embodiment, the R ₁ is methyl.

环上的一个甲基。In another preferred embodiment, the R ₁ is located in

A methyl group on the ring.

In another preferred embodiment, R ₂ -X- is a group selected from the group consisting of 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 R ₂ is a group selected from the group consisting of N-(N,N-diethyl) substituents, N,2 or 3- Linyl, 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.

The second aspect of the present invention provides the 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 preparing a SPOP target inhibitor, Or for the preparation of treatment and/or prevention of SPOP-targeted diseases.

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

In another preferred embodiment, the inhibition is competitive inhibition.

The third aspect of the present invention provides the use of the compound of formula (I) according to the first aspect of the present invention, or a pharmaceutically acceptable salt or solvate thereof, for inhibiting the proliferation activity of renal cancer cells .

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

The fourth aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the compound described in the first aspect of the present invention, and/or a pharmaceutically acceptable salt or pharmaceutically acceptable salt thereof An acceptable solvate serves as the active ingredient, and a pharmaceutically acceptable carrier.

The fifth aspect of the present invention provides a method for preparing a compound of formula (I) as described in the first aspect of the present invention, the method comprising the steps of:

Compounds of formula I are obtained by reacting a compound of formula Ia with a compound of formula Ib in an inert solvent.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

Figure 1 shows the verification of cell proliferation experiments with 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f]pyrimidine compounds Inhibitory activity on renal cancer cell A498;

Figure 2 shows the verification of cell proliferation experiments for 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f]pyrimidine compounds Inhibitory activity against 6 renal cancer cell lines and 1 normal renal tubular epithelial cell line;

Figure 3 shows the verification of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-dipyrido[3,4-c:1',2'- f] The inhibitory activity of pyrimidine compounds on the downstream signaling pathway of SPOP;

Figure 4 shows co-immunoprecipitation experiments (Co-IP) validation of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2' -f] Pyrimidine compounds competitively inhibit the interaction between SPOP and substrate;

Figure 5 shows the verification of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1', 2'-f]pyrimidines inhibit the ubiquitination of substrates by SPOP;

Figure 6 shows surface plasmon resonance (SPR) verification of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f ] Pyrimidines interact with SPOP proteins;

Figure 7 shows fluorescence polarization experiments (FP) verification of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f] Pyrimidines interact with SPOP proteins;

Figure 8 shows the body weight and organ weight of normal nude mice and the verification of organ section staining experiments for 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1' ,2'-f]pyrimidine compounds had little effect on the body weight and organs of normal nude mice.

Figure 9 shows the compound plasma metabolism experiment to verify 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f]pyrimidine The compounds have good metabolic stability.

Figure 10 shows the validation of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f] in vivo tumorigenic experiments in nude mice Inhibitory effect of pyrimidine compounds on subcutaneous tumor growth in nude mice.

Detailed ways

After long-term and in-depth research, the inventors prepared a class of compounds with the structure shown in formula I, and found that they have SPOP inhibitory activity. And the compound has an inhibitory effect on SPOP activity at a very low concentration (IC ₅₀ value can be as low as ≤50nmol/L), and the inhibitory activity is quite excellent, so it can be used to treat diseases related to SPOP activity or expression. , such as kidney cancer. Based on the above findings, the inventors have completed the present invention.

the term

As used herein, the term "C _1-6 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 similar groups. Expressions of the form "C1-C6" are intended to include corresponding groups having 1, 2, 3, 4, 5, or 6 carbon atoms, eg, " _C1-6 alkyl" refers to groups having Alkyl of 1, 2, 3, 4, 5 or 6 carbon atoms, "C _2-9 non-heteroaryl" means having 2, 3, 4, 5, 6, Non-heteroaryl groups of 7, 8, and 9 carbon atoms.

As used herein, the term "C ₁ -C ₆ alkoxy" refers to a substituent in the form of a "straight-chain or branched-chain alkyl-oxy group having 1 to 6 carbon atoms", such as ethoxy, propoxy group, butoxy group, or similar group.

As used herein, the term "C ₁ -C ₆ alkylamino" refers to a substituent in the form of a "straight-chain or branched-chain alkyl-amino group having 1 to 6 carbon atoms", such as ethylamino, propylamino , dimethylamino, or similar groups.

The term "alkylene" refers to a group formed by the loss of a hydrogen atom from an alkyl group as described above, eg, _-CH2- , _{-CH2 -} CH2-, or the like.

The term "C ₂ -C ₁₂ alkenyl" refers to a group formed by the loss of one hydrogen atom from an alkene having 2 to 12 carbon atoms, including vinyl, propenyl, n-butenyl, or the like.

The term "C3 _{- C9} cycloalkyl" refers to a cycloalkyl group having 3-9 carbon atoms, such as cyclopropane, cyclohexane, and the like.

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

The term " _C4 -C12heteroaryl" refers to unsaturated ring system substituents having _4-12 carbon atoms and one or more heteroatoms selected from O, S, N or P, such as pyridyl, thienyl, or similar groups.

The term "3-12-membered heterocycle" refers to a saturated ring system substituent with one or more heteroatoms selected from O, S, N or P on the ring system with 3-12 members, such as piperidinyl, pyrrolyl , or similar groups.

The term "halogen" refers to F, Cl, Br and I.

In the present invention, the terms "comprising", "comprising" or "including" mean that the various ingredients can be used together in the mixture or composition of the present invention. Thus, the terms "consisting essentially of" and "consisting of" are encompassed by the term "comprising".

In the present invention, the term "pharmaceutically acceptable" ingredients refers to substances that are suitable for use in humans and/or animals without excessive adverse side effects (such as toxicity, irritation and allergy), ie, have 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 depends on 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 useless to prespecify the exact effective amount. However, for a given situation, routine experimentation can be used to determine the effective amount, as is the judgment of the clinician.

As used herein, unless otherwise specified, the term "substituted" refers to the replacement of one or more hydrogen atoms on a group with a substituent selected from the group consisting of halogen, unsubstituted or halogenated C1-C6 alkyl, unsubstituted Substituted or halogenated C2-C6 acyl, unsubstituted or halogenated C1-C6 alkyl-hydroxy.

Unless otherwise specified, in the present invention, all occurrences of compounds are intended to include all possible optical isomers, such as single chiral compounds, or mixtures of various chiral compounds (ie, racemates). In all compounds of the present invention, each chiral carbon atom can optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.

As used herein, the term "compounds of the present invention" refers to compounds of formula I. The term also includes the 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 suitable for use as a medicament. Pharmaceutically acceptable salts include inorganic and organic salts. A preferred class of salts are the salts of the compounds of the present invention with acids. Acids suitable for forming salts include, but are not limited to, inorganic acids such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, nitric, phosphoric, formic, acetic, propionic, oxalic, malonic, succinic, fumaric, Maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenemethanesulfonic acid, benzenesulfonic acid and other organic acids; and acidic amino acids such as aspartic acid and glutamic acid.

Compounds of formula I and their preparation

The present invention provides a 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2 represented by the following general formula (I) '-f]pyrimidine compounds, or pharmaceutically acceptable salts or solvates thereof:

Wherein, the definition of each group is as described above.

Preferably said compound is a compound selected from the following compounds:

Pharmaceutical compositions and methods of administration

Since the compound of the present invention has excellent inhibitory activity against SPOP, the compound of the present invention and its various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and containing the compound of the present invention as the main active ingredient The pharmaceutical composition can be used for the treatment, prevention and alleviation of diseases related to SPOP activity or expression level. According to the prior art, the compounds of the present invention are useful in 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 and a pharmacologically acceptable excipient or carrier within a safe and effective amount. The "safe and effective amount" refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 5-200 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。"Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid filler or gelling substances which are suitable for human use and which must be of sufficient purity and sufficiently low toxicity. "Compatibility" as used herein means that the components of the composition can be admixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid) , magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (

), wetting agents (such as sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

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 (a) fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators such as quaternary amine compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostearate; (h) adsorbents such as kaolin; and (i) lubricants such as talc, hard Calcium fatty acid, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared using coatings and shell materials, 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 in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric substances and waxes. 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 compound, liquid dosage forms may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, and the like.

Besides these inert diluents, the compositions can 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 such as 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 non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration of the compounds of this 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 that may be required if necessary.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) in need of treatment, and the dose is the effective dose considered pharmaceutically, for a 60kg body weight, the daily dose is The administration dose is usually 1 to 2000 mg, preferably 5 to 500 mg. Of course, the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.

The main advantages of the present invention include:

1. A compound of formula I is provided.

2. A novel SPOP inhibitor with novel structure and preparation and application thereof are provided. The inhibitor can inhibit the activity of various SPOPs at a very low concentration.

3. A pharmaceutical composition for treating diseases associated with SPOP activity is provided.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

The present invention will be further described below in conjunction with specific embodiments. These examples are for illustrative purposes only and are not intended to limit the scope and spirit of the invention.

The sources of the medicines in the following examples: DMEM/HG medium was purchased from gibco, 10% fetal bovine serum was purchased from lifetechnology, penicillin, streptomycin and thiazole blue were purchased from Shanghai Sangon Bioengineering Co., Ltd., and the reagents in the preparation examples were purchased from Sinopharm Chemical Reagent Co., Ltd. and Bailingwei Technology Co., Ltd. SPF-grade BALD/c null female nude mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd. and kept in the SPF-grade animal room of Shanghai Institute of Materia Medica. Animal management and use followed the IACUC policies and guidelines of Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

Preparation Examples

The 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f]pyrimidine compounds of the present invention and their pharmacy The above acceptable salts or pharmaceutically acceptable solvates can be prepared by the following routes:

The compound N-(3-(4-morpholinyl)propyl)-1-(2-phenylethyl)-2-imino-10-methyl-5-ketobipyridine[4,3 -b:1',2'-f]lopyrimidine-3-carboxamide as an example brief description of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4 Synthesis of -c:1',2'-f]pyrimidines.

Step 1: 10.81 g (100 mmol) of 2-amino-3-methylpyridine and 24.03 g (150 mmol) of diethyl malonate were added to a 250 mL eggplant-shaped flask, and the reaction was carried out at 200° C. for 2 h, and a large amount of solid products were generated during the reaction. After the reaction was completed, it was cooled to room temperature, 200 ml of ethanol was added to reflux overnight, cooled to room temperature, and filtered to obtain 13 g of product with a yield of 76%.

¹ H NMR (400MHz, DMSO) δ 8.79 (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: Add 200mL (calcium hydride to remove water) N,N-dimethylformamide (DMF) into a 500mL eggplant-shaped bottle, cool in an ice bath, then slowly dropwise add 13mL (140mmol) phosphorus oxychloride (P(O) Cl ₃ ), continue to react for 1 h under ice bath, add 7.05 g (40 mmol) of 2-hydroxy-4-hydroxy-9-methylpyridine[1,2-α]-pyridinamine in batches, and react in ice bath for 1 h after the addition , reacted at room temperature for 2 hours, then reacted at 95°C for 2 hours, added 200 mL of ice water after the reaction, reacted for 1 hour, filtered, the solid was slurried with ethanol, filtered and dried to obtain 6.2 g of product with a yield of 70%.

¹ H NMR (400MHz, CDCl ₃ ) δ 10.43 (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: 222.6 mg (1 mmol) of 2-chloro-3-aldol-4-carbonyl-4H-9-methylpyridine[1,2-α]pyrimidine was added to 5 mL of absolute ethanol, and 484.7 mg (4 mmol) were added dropwise. ) phenethylamine, reacted at room temperature for 12 h, filtered after the reaction, and washed the solid with ethanol to obtain 320.2 mg of a pale yellow solid with a yield of 78%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.69 (s, 1H), 8.86 (s, 1H), 8.79 (d, J=7.0 Hz, 1H), 7.47 (d, J=6.7 Hz, 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).

Step 4: Add 1.44g (10mmol) N-(2-aminopropyl)morpholine and 1.36g (1.2mmol) ethyl cyanoacetate into a 25mL eggplant-shaped flask, react for 48h, and wash with anhydrous ether after the reaction Three times, dried, and placed in a -20°C refrigerator to obtain 1.7 g of a pale yellow solid with a yield of 80%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.90 (s, 1H), 3.78–3.75 (m, 4H), 3.40 (dd, J=11.5, 5.8 Hz, 2H), 3.35 (s, 2H), 2.52– 2.48(m,6H),1.72(ddd,J=12.2,7.6,3.7Hz,2H).

Step 5: 108 mg (0.5 mmol) of 9-methyl-2-(2-phenylethylamino)-3-(2-phenethyliminomethyl)pyridine[1,2-a]pyrimidine- 4-ketone and 103.5 mg (0.55 mmol) 2-cyano-N-(3-(4-morpholinyl) propyl) acetamide were added to 2 mL of chloroform, refluxed for 8 h, ether was added after the reaction, filtered, solid Recrystallization from absolute ethanol gave about 60 mg of product in 32% yield.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.88 (d, J=6.8 Hz, 1H), 7.65 (dt, J=6.9, 1.4 Hz, 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 can be prepared by analogous methods using corresponding starting materials.

The NMR data of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f]pyrimidines are listed in the table below.

The following is 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f]pyrimidine represented by general formula (I) Experiments on the inhibition of SPOP activity by compounds.

Experimental Example 1: Cell proliferation test (MTT) for 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f ] Evaluation of pyrimidine compounds inhibiting SPOP activity

Renal cancer cell line A498 was prepared with a culture medium containing 10% fetal bovine serum (DMEM/HG medium, 100U penicillin, 100U streptomycin, 10% fetal bovine serum) at a concentration of 5×10 ⁴ -1×10 ⁵ cells /mL cell suspension. Inoculate 100 μL of cell suspension in each well of a 96-well cell culture plate, incubate overnight at 37°C and 5% CO ₂ until adherent growth, then add the compounds listed in the concentration gradient Table 1, and continue to culture for 68 h, add 10 μL of thiazole to each well blue solution (5mg/mL), and continue to cultivate for 4h. After the incubation, the medium in the wells was completely aspirated, 150 μL of dimethyl sulfoxide (DMSO) was added to each well, and the crystals were fully dissolved by shaking for 10 min. The absorbance at 490 nm was measured by a microplate reader, the results were recorded and the cell viability was calculated. Taking the compound concentration as the abscissa and the cell viability as the ordinate, the _IC50 value was obtained by nonlinear regression fitting curve of GraphPad Prism 5.0 software. The _IC50 values of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f]pyrimidines are listed in Table 1. As can be seen from Table 1, 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f]pyrimidines Can inhibit SPOP activity.

Table 1 IC ₅₀ values of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f]pyrimidine compounds

Experimental Example 2: Cell Proliferation Experiment Verification of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f]pyrimidines Inhibitory activity of compounds on 6 renal cancer cell lines and 1 normal renal tubular epithelial cell line

Experiments based on SPOP-B- 30 and SPOP-B-88 for example.

The renal cancer cell lines selected in this experiment include 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's5a medium (Caki-2) containing 10% (vol/vol) fetal bovine serum, 100U/ml penicillin and 100ng/ml streptomycin or Cultured in RPMI1640 medium (786-O, 769-P, OS-RC-2) or DMEM/F121:1 medium (HK-2). The implementation method of the cell proliferation experiment is as in Experimental Example 1. As can be seen from Figure 2, SPOP-B-30 and SPOP-B-8 can significantly inhibit the proliferation activity of renal cancer cells, but the inhibition activity of normal renal tubular epithelial cells HK-2 (non-tumor cells) is not obvious.

Experimental Example 3: Validation of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2 Inhibitory activity of '-f]pyrimidines on downstream signaling pathways of SPOP

Experiments based on SPOP-B- 30 and SPOP-B-88 for example.

2 mL of A498 cell line resuspension (5×10 ⁴ -1×10 ⁵ cells/mL) was added to a 6-well cell culture plate, and cultured overnight until the cells adhered. Concentration gradient SPOP-B-30 and SPOP-B-88 were added, and the culture was continued for 10 h. After the incubation, the medium was aspirated, washed three times with phosphate buffer, 150 μL of RIPA cell lysate and 1 mM phenylmethylsulfonyl fluoride (PMSF) were added, and the cells were lysed on ice for 30 min. The lysate was scraped into a 1.5 mL centrifuge tube with a cell scraper, centrifuged at 15,000 g for 20 min at 4°C, and the supernatant was transferred to a 1.5 mL centrifuge tube, and the protein concentration of the supernatant was determined by BCA method. 100 μL of supernatant was added to 25 μL of 5× loading buffer, heated at 100 °C for 10 min, and centrifuged at 12000 g for 1 min. The loading amount was adjusted according to the measured concentration, and polyacrylamide denaturing electrophoresis (SDS-PAGE) was performed. The separated protein bands were transferred to nitrocellulose membrane (NC membrane) by transfer electrophoresis, then blocked by 5% skim milk, incubated with primary antibody (rabbit-derived PTEN antibody), washed with TBST buffer, and incubated with secondary antibody (horseradish peroxide). Enzyme HRP-labeled goat anti-rabbit antibody), washed with TBST buffer, and finally detected the protein amount by enhanced chemiluminescence (ECL) luminescence development step. It can be clearly seen from Figure 3 that with the increase of SPOP-B-30 and SPOP-B-8 concentrations, the content of SPOP substrates PTEN and DUSP7 increases, and the phosphorylation of downstream signaling pathway active molecules AKT and ERK1/2 weakens , indicating that SPOP-B-30 and SPOP-B-8 can inhibit the activity of SPOP, thereby increasing the content of tumor suppressor substrate proteins PTEN and DUSP7.

Experimental Example 4: Co-immunoprecipitation experiment (Co-IP) verification of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2 '-f]pyrimidines competitively inhibit the interaction between SPOP and substrates

Experiments based on SPOP-B- 30 and SPOP-B-88 for example.

Seed HEK293 cells in 60mm dishes at a density of about 30%-50%. The following day, the corresponding plasmids myc-PTEN, Flag-SPOP were transfected. After 40 hours of transfection, the corresponding amount of small molecule compound was added to each group. After 8 hours, the medium was aspirated and washed twice with pre-cooled PBS. After blotting the residual PBS, add pre-cooled 200 μl RIPA cell lysate to the cells in each dish, scrape the cells from the culture dish with a pre-cooled cell scraper, pipette the suspension into an EP tube, and place on ice for 10-30 min. Centrifuge at 12,000 rpm for 10 min at 4°C, transfer the supernatant to a new EP tube, and quantify the protein concentration by BCA method. After leveling the protein concentration, reserve 30 μl of total protein, add 5× loading buffer, boil in boiling water for 5 minutes to denature. A 20 μl suspension of antibody-conjugated agarose beads was taken per sample, washed three times with PBST, and finally resuspended 1:1 by volume. Add 30 μl of beads resuspension and 300 μl of RIPA lysis buffer to each sample, and incubate at 4°C for 4 h or overnight with rotation. Centrifuge at 5000g for 1 minute at 4°C and remove the supernatant. Add 500 μl of PBST buffer, spin and wash at 4°C for 10 minutes, centrifuge at 5000 g for 1 minute at 4°C, and remove the supernatant. Repeat the wash three times. After the last centrifugation to remove the supernatant, try to aspirate the participating supernatant as much as possible, then add 30 μl of 2× protein loading buffer, and denature in boiling water for 5 minutes. The denatured samples were centrifuged at 8000g at room temperature, and the supernatant was aspirated for SDS-PAGE electrophoresis, taking care not to absorb the beads. It is evident from Fig. 4 that SPOP-B-30 and SPOP-B-88 can inhibit the mutual binding of SPOP to the substrate PTEN in cells.

Experimental Example 5: In Vivo Ubiquitination Assay Verification of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f]pyrimidines inhibit intracellular ubiquitination of substrates by SPOP

Experiments based on SPOP-B- 30 and SPOP-B-88 for example.

Seed HEK293 cells 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. 40h after transfection, the corresponding amount of small molecule compound was added to each group, and after 4h, 10 μ MMG132 medium was added to continue culturing for 4h. After the incubation, the medium was removed, the culture dish was placed on an ice box, washed once with pre-cooled PBS, and then 100 μl of pre-cooled denaturing lysate (1% SDS; 0.5 mM EDTA; 1 mM DTT; 50 mM Tris, pH 7. 5). Cells were quickly scraped from the dish into 1.5 ml EP tubes with a cell scraper, followed by denaturation at 100°C for 5 min. After denaturation, the lysate was placed on ice to cool, and 900 μl of RIPA lysate was added for dilution. After the dilution, ultrasonic treatment was performed, and the ultrasonic conditions were as follows: the amplitude was 10%, the intermittent 10s switch, and the total ultrasonic time was 1 minute. Centrifuge at 12000g for 5 min at 4°C and transfer the supernatant to a new EP tube. Adjust the protein concentration between samples according to the protein quantification results. Reserve 100 μl as input, add protein loading buffer and denature at 100°C for 5 min. The remaining lysates were immunoprecipitated with myc antibody-conjugated agarose beads, and the final eluate was immunoblotted with HA antibody. It can be clearly seen from the results in Figure 5 that SPOP-B-30 and SPOP-B-88 can inhibit the ubiquitination of SPOP on the substrate PTEN in cells.

Experimental Example VI: Surface Plasmon Resonance (SPR) Validation of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'- f] Pyrimidines interact with SPOP proteins

Experiments based on SPOP-B- 30 and SPOP-B-88 for example.

Freshly purified SPOP ^MATH protein (>95% purity, concentration 5 mg/mL) was diluted to 0.1 mg/mL with 10 mM sodium acetate solution (pH 4.5). The SPOP ^MATH protein was coupled to the CM5 chip by BIAcore T200 (GE) using standard amino coupling procedures. Before the experiment, the CM5 chip was equilibrated with HBS-EP buffer for 2h. SPOP-B-30 and SPOP-B-88 were serially diluted with HBS-EP buffer, equilibrated at a flow rate of 30 μL/min for 60 s, bound for 120 s, and washed for 120 s. The corresponding curve obtained was fitted by BIA data analysis software (GE Company) to obtain the corresponding K _D value. It can be clearly seen from Fig. 6 that SPOP-B-30 and SPOP-B-88 have a certain binding effect with SPOP ^MATH protein.

Experimental Example 7: Fluorescence polarization experiment (FP) verification of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f ] Pyrimidines interact with SPOP proteins

Experiments based on SPOP-B- 30 and SPOP-B-88 for example.

Fluorescence polarization (FP) experiments were performed on a multi-function microplate reader (PerkinElmer). The instrument model is EnVision Multilabel Plate Reader. Fluorescein isothiocyanate (FITC)-labeled polypeptide was used as the tracer substrate in the experiment, and the sequence was FITC-LACDEVTSTTSSSTA (synthesized by Gill Biochemical Company). Add 2 μM SPOP ^MATH , 20 nM polypeptide substrate, concentration gradient SPOP-B-30 and SPOP-B-88 to 100 μL hydroxyethylpiperazine ethanethiosulfonic acid buffer (50 mM, pH 7.5) buffer, incubate at 4°C for 1 h, Detect its fluorescence polarization value. The excitation and emission light used were 480 nm and 535 nm, respectively. The experiment was repeated three times, and the experimental data were analyzed using GraphPad Prism 5.0 software. As can be seen in Figure 7, SPOP-B-30 and SPOP-B-88 interact with SPOP proteins.

Experimental Example 8: Validation of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f]pyrimidine in mouse animal experiments Compound toxicity

Experiments based on SPOP-B- 30 and SPOP-B-88 for example.

The 6- to 8-week-old BALB/c and ICR mice used in the in vivo toxicity experiments were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Randomly grouped with 3 animals in each group. Solvent, 80 mg/kg, 120 mg/kg compound were injected intraperitoneally once a day, and body weight and activity status were checked. Orbital blood was collected 6-7 days later for hematological analysis and plasma biochemical testing. The internal organs (heart, liver, spleen, lung and kidney) of the mice were taken for HE staining and pathological section detection after sudden death from anesthesia. It can be seen from Fig. 8 that the injection concentration of the compound is 80 mg/kg, which is a safe concentration.

Experimental Example 9: Detection of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f]pyrimidine in mouse animal experiments metabolized in vivo

Experiments based on SPOP-B- 30 for example.

BALB/c mice aged 4-6 weeks were randomly divided into three groups. Compounds were prepared by intraperitoneal injection using physiological saline, 0.5% 2-phenylethanol, and 1.5% Tween-80 as a solvent, and the compound concentration was 10 mg/kg. Tail vein blood was collected at time points 0.25, 0.5, 1, 2, 4, 6, 10 and 24 hours after compound administration, and 0.15 mL of blood was collected from each animal. Blood samples were anticoagulated with EDTA, and the supernatant was collected by centrifugation to detect the content of SPOP-B-30 compound in serum by LC-MS/MS method. It can be calculated from Fig. 9 that the blood drug concentration is the highest at 0.5 hours after administration, which is 1070 ng/ml, and the half-life is 6.25 hours.

Experimental Example 10: Validation of 2-imino-5-keto-2,5-dihydro-1-H-dipyrido[3,4-c:1',2'-f]pyrimidine in mouse animal experiments compound activity

A498 cells were cultured in DMEM/HG+10% fetal bovine serum medium in 5% CO ₂ at 37°C. The trypsinized A498 cells were collected, and a cell suspension was prepared in a medium without fetal bovine serum, and the number of cells reached a concentration of 10 ⁸ cells/ml. Take the Matrigel (Martrigel, BD company) stored at -20 °C to dissolve naturally at 4 °C, and inject 100 μL (10 ⁷ cells) to 6 weeks of age immediately after mixing the cell suspension: Matrigel Martrigel 3:1 (v/v). The right forelimb axilla of nude mice was subcutaneously. Observe the tumor growth and measure the tumor length (a mm) and width (b mm) with vernier calipers, calculate the tumor volume according to v=a*b ² /2, and divide into groups when the tumor volume is about 125 mm ³ , 40 experimental nude mice were divided into 4 groups, 10 animals in each group. Compound SPOP-B-30 was dissolved in a solvent (physiological saline, 0.5% 2-phenylethanol, 1.5% Tween-80) to 5 mg/mL, and SPOP-B-88 was dissolved in physiological saline to 5 mg/mL. Daily intraperitoneal injection was performed at 0, 40, 60, and 80 mg/kg, and body weight and tumor measurements were performed every 3 days, and corresponding care and records were performed. The tumor growth in the control group and the experimental group was observed. The administration was stopped about 25 days later. The nude mice were anesthetized and died suddenly and dissected. The heart, liver, spleen, lung and kidney of 2 nude mice in each group were collected for tissue HE staining observation. . At the same time, the tumor was isolated for weighing, photographing and cryopreservation in liquid nitrogen, and the preserved tumor tissue was used to detect the amount of PTEN in the tissue by western blot.

As can be seen from Figure 10, the tumor volume of mice administered with SPOP-B-30 and SPOP-B-88 was significantly smaller than that of mice injected with only solvent. Significant reduction of SPOP content in tissues was observed. Animal experiments show that SPOP-B-30 and SPOP-B-88 can slow tumor growth by inhibiting SPOP.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (11)

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,

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.

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