CN111004220B - 3- (4-phenyl-1H-2-imidazolyl) -1H-pyrazole compound, and preparation method and application thereof - Google Patents

Abstract

The invention relates to a 3- (4-phenyl-1H-2-imidazolyl) -1H-pyrazole compound, a preparation method and application thereof, belonging to the field of pharmaceutical chemistry and pharmacotherapeutics. The invention provides application of a compound shown in a formula I or pharmaceutically acceptable salt thereof in preparing medicaments for treating diseases related to tumors, in particular application in preparing Aurora A kinase specific inhibitors.

Description

3-(4-phenyl-1H-2-imidazolyl)-1H-pyrazole compounds, preparation method and the same application

technical field

The invention belongs to the field of medicinal chemistry and pharmacotherapeutics, in particular to a pyrazoloimidazole compound. The present invention also relates to the preparation method of the compounds and the pharmaceutical combination containing them.

Background technique

With the continuous deepening of molecular biology, tumor pathology, cell biology, tumor genetics and other research, the current therapeutic mechanism based on key molecular pathways of tumors and regulatory factors in cell mitosis has aroused widespread interest.

Mitosis controls the division of a mother cell into two daughter cells with identical chromosomes and cytoplasm throughout the cell cycle. Chromosome segregation is a complex process and involves the formation of a bipolar mitotic spindle as well as chromosome segregation. Given the complexity of mitosis, multiple checkpoint systems have been identified to ensure a proper coordination. The overall progression of mitosis depends on three regulatory mechanisms: protein localization, proteolysis and the regulation of several serine/threonine kinases known as mitotic kinases. Among them, Aurora kinase is a serine/threonine kinase that is important and indispensable in multiple steps of mitotic progression. The Aurora kinase family is artificially divided into three subtypes, Aurora-A, Aurora-B, and Aurora-C. The three members of the Aurora kinase family are highly homogeneous in mammalian cells. As a regulator of mitosis, loss of Aurora kinase results in failure of mitotic spindle assembly and chromosome segregation, followed by markedly increased genetic instability and tumor incidence. Overexpression or amplification of Aurora kinase is routinely detected in many human cancers such as human chronic myelogenous leukemia (K562 cells), colon cancer (HCT116 cells), breast cancer, ovarian cancer, gastric/gastrointestinal cancer and others Tumors are closely related to poor prognosis.

One way to treat cancer is to combine Aurora kinase inhibitors with currently used anticancer therapies, the study suggests. Combination therapy can increase the sensitivity of anti-tumor therapy, so as to better treat cancer. In the process of Aurora kinase inhibitor research, whether to study targeting a single Aurora kinase inhibitor or an inhibitor that can act on three subtypes or even multiple targets at the same time to achieve the best cancer treatment effect, currently There is no clear explanation yet. Based on the important role played by Aurora kinases in mitosis, the research and development prospects of Aurora kinase inhibitors are very optimistic, and they are expected to stand out among many anti-tumor drugs.

Contents of the invention

The object of the present invention is to provide a kind of 3-(4-phenyl-1H-2-imidazolyl)-1H-pyrazole compound on the basis of prior art, and this kind of compound has good Aurora A kinase inhibitory activity , can effectively inhibit the expression of Aurora kinase, and has better inhibitory activity on human chronic myelogenous leukemia cell K562 and human colon cancer cell HCT116, and its inhibition rate is significantly higher than that of the positive control drug.

Another object of the present invention is to provide a preparation method of the above compound.

The third object of the present invention is to provide a use of the above compound in medicine.

Technical scheme of the present invention is as follows:

Compounds, isomers, or pharmaceutically acceptable salts thereof represented by formula I,

in,

R ¹ represents a substituted or unsubstituted heteroalicyclic group whose substituent is selected from C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, phenyl, hydroxyl or halogen;

R ² represents phenyl, benzoyl, phenylacetyl, phenylpropionyl, aromatic heterocycle, substituted phenyl, substituted benzoyl, substituted phenylacetyl, substituted phenylpropionyl or substituted aromatic heterocycle, and the substituted Phenyl, substituted benzoyl, substituted phenylacetyl, substituted phenylpropionyl or substituted aromatic heterocycles may optionally be mono- or polysubstituted with the following substituents: C ₁ -C ₄ alkyl, C ₁ -C ₄ alkane Oxygen, C ₁ -C ₄ haloalkyl, -S(=O) ₂ -R ³ , cyano, hydroxyl, nitro or halogen;

R ³ represents C ₁ -C ₄ alkyl;

X represents -CH ₂ - or O atom;

n represents an integer of 0-6.

In a preferred version, ^R represents a morpholine ring, a piperazine ring, a tetrahydropyrrole ring, a tetrahydrofuran ring, a piperidine ring, a substituted morpholine ring or a substituted piperazine ring, and its substituents are selected from methyl, ethyl , methoxy, ethoxy, hydroxyl, bromine or chlorine.

In a more preferred version, ^R represents a morpholine ring, a piperazine ring, a tetrahydropyrrole ring, a tetrahydrofuran ring or a piperidine ring.

Particularly preferably, R ¹ mentioned in the present invention represents a morpholine ring or a piperidine ring.

In a preferred embodiment, the compound, isomer, or pharmaceutically acceptable salt thereof shown in formula I, wherein R ² represents the following groups:

R ⁴ or R ⁵ independently represent a hydrogen atom, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, C ₁ -C ₄ haloalkyl, -S(=O) ₂ -R ³ , nitro or halogen.

In a more preferred solution, R ⁴ or R ⁵ independently represent a hydrogen atom, methyl, ethyl, methoxy, ethoxy, CF ₃ , -S(=O) ₂ -CH ₃ , -S (=O) _{2 - CH2CH3} , nitro, bromo or chloro.

In a particularly more preferred embodiment, R ⁴ or R ⁵ independently represent a hydrogen atom, methyl, methoxy, CF ₃ , -S(=O) ₂ -CH ₃ , nitro, bromine or chlorine.

In the present invention, m represents an integer of 0-2; preferably, m represents 0 or 1.

In another preferred version, wherein R ² represents pyrimidine, thiazole, thiophene, imidazole, substituted pyrimidine, substituted thiazole or substituted imidazole, and said substituted pyrimidine, substituted thiazole or substituted imidazole can be optionally substituted by the following substituents Or multi-substituted: C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, C ₁ -C ₄ haloalkyl, -S(=O) ₂ -R ³ or halogen.

In another more preferred scheme, wherein R ² represents pyrimidine or substituted pyrimidine, and said substituted pyrimidine can be optionally substituted by the following substituents: methyl, ethyl, CF ₃ , -S(= O) ₂ -CH ₃ , -S(=O) ₂ -CH ₂ CH ₃ , bromine or chlorine.

Particularly preferably, the compound shown in formula I, the isomer, or a pharmaceutically acceptable salt thereof, wherein, ^R represents pyrimidine or substituted pyrimidine, and the substituted pyrimidine can be optionally substituted by the following substituents or Multiple substitutions: methyl, _CF3 , -S(=O) ₂ - _CH3 , bromine or chlorine.

In a preferred embodiment, X represents -CH ₂ - or an O atom. Further preferably, X represents -CH ₂ -.

In a preferred embodiment, n represents an integer of 0-4. Further preferably, n represents 0, 1, 2 or 3.

In one scheme, the compound, isomer, or pharmaceutically acceptable salt thereof represented by formula I, wherein,

^R represents a morpholine ring, a piperazine ring, a tetrahydropyrrole ring, a tetrahydrofuran ring or a piperidine ring; preferably, ^R represents a morpholine ring or a piperidine ring;

R ² represents phenyl, benzoyl, pyrimidine, substituted benzoyl or substituted pyrimidine, and the substituted benzoyl or substituted pyrimidine can be optionally mono-substituted or multi-substituted by the following substituents: methyl, ethyl , methoxy, ethoxy, CF ₃ , -S(=O) ₂ -CH ₃ , -S(=O) ₂ -CH ₂ CH ₃ , nitro, bromine or chlorine; preferably, the substituted Benzoyl or substituted pyrimidine, optionally monosubstituted or polysubstituted by the following substituents: methyl, methoxy, CF ₃ , -S(=O) ₂ -CH ₃ , nitro or chlorine;

X represents -CH ₂ - or O atom; preferably, X represents -CH ₂ -;

n represents an integer of 0-4, preferably, n represents 0, 1, 2 or 3.

Further, in the compound described in general formula I or a pharmaceutically acceptable salt thereof, the compound is selected from the following compounds:

Further, the compound described in general formula I is further selected from the following compounds:

The invention discloses a preparation method of a compound represented by general formula I, which uses 4-nitropyrazole-3-carboxylic acid, p-methylacetophenone and p-hydroxyacetophenone as raw materials, undergoes N-alkylation, free Base reaction, α-halogenation of ketone, esterification, closing imidazole ring, reduction, amidation and other reaction steps can finally obtain pyrazoloimidazole compounds. The specific synthetic route is as follows:

When X represents -CH ₂ -, the preparation method of the compound shown in general formula I comprises the following steps:

Preferably, when X represents -CH ₂ - and n represents 0, the preparation method of the compound represented by the general formula I comprises the following steps:

When X represents O atom, the preparation method of compound shown in general formula I comprises the following steps:

The intermediates or target compounds mentioned in the present invention can be purified according to conventional separation techniques, and converted into addition salts with pharmaceutically acceptable acids as required.

Unless otherwise stated, the following terms used in the specification and claims have the meanings discussed below:

"Pharmaceutically acceptable salts" means those salts that retain the biological effectiveness and properties of the parent compound. Such salts include:

(1) Salt formation with acid, obtained by reacting the free base of the parent compound with inorganic or organic acids. Inorganic acids include hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, metaphosphoric acid, sulfuric acid, sulfurous acid and perchloric acid, etc. , organic acids include acetic acid, trifluoroacetic acid, propionic acid, acrylic acid, caproic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, oxalic acid, (D) or (L) malic acid, fumaric acid, maleic acid, Ascorbic acid, camphoric acid, benzoic acid, hydroxybenzoic acid, gamma-hydroxybutyric acid, methoxybenzoic acid, phthalic acid, methanesulfonic acid, ethanesulfonic acid, naphthalene-1-sulfonic acid, naphthalene-2-sulfonic acid , p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, lactic acid, cinnamic acid, lauryl sulfate, gluconic acid, glutamic acid, aspartic acid, stearic acid, mandelic acid, succinic acid, pentanoic acid Diacid or malonic acid, etc.

(2) The acidic proton present in the parent compound is replaced by a metal ion or a salt formed by coordination with an organic base, such as an alkali metal ion, an alkaline earth metal ion or an aluminum ion, and an organic base such as ethanolamine, diethanolamine, three Ethanolamine, tromethamine, N-methylglucamine, quinine, etc.

"Pharmaceutical composition" refers to mixing one or more of the compounds of the present invention or their pharmaceutically acceptable salts, solvates, hydrates or prodrugs with other chemical ingredients, such as pharmaceutically acceptable carriers. . The purpose of the pharmaceutical composition is to facilitate the process of administration to animals.

"Pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" refers to an inactive ingredient in a pharmaceutical composition that does not cause significant irritation to the organism and does not interfere with the biological activity and properties of the administered compound, such as but not limited to : Calcium carbonate, calcium phosphate, various sugars (such as lactose, mannitol, etc.), starch, cyclodextrin, magnesium stearate, cellulose, magnesium carbonate, acrylic acid polymer or methacrylic acid polymer, gel, water , polyethylene glycol, propylene glycol, ethylene glycol, castor oil or hydrogenated castor oil or polyethoxylated hydrogenated castor oil, sesame oil, corn oil, peanut oil, etc.

"Alkyl" means a saturated aliphatic group of 1-20 carbon atoms, including straight-chain and branched-chain groups (the numerical range mentioned in this application, such as "1-20", refers to the group, here is an alkyl group, which may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms). More preferably, the alkyl group is a medium-sized alkyl group having 1-10 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl Base etc. Preferably, the alkyl group is a lower alkyl group having 1-8 or 1-6 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl or tert-butyl, etc. . Alkyl groups can be substituted or unsubstituted. When substituted alkyl, the substituent is preferably one or more, more preferably 1-3, most preferably 1 or 2 substituents.

"Heteroaromatic ring" or "heteroaryl" means a monocyclic or fused ring group of 5 to 12 ring atoms containing one, two, three or four ring heteroatoms selected from N, O or S , the remaining ring atoms are C, and additionally have a fully conjugated π-electron system. Non-limiting examples of unsubstituted heteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine, tetrazole, triazine and carbazole. Heteroaryl groups can be substituted or unsubstituted. When substituted, the substituents are preferably one or more, more preferably one, two or three, even more preferably one or two, independently selected from the following groups, including: lower alkyl, tri Haloalkyl, halogen, hydroxy, lower alkoxy, mercapto, (lower alkyl)thio, cyano, acyl, thioacyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl Acyl, N-thiocarbamoyl, C-amido, N-amido, nitro, N-sulfonylamino, S-sulfonylamino. Preferred heteroaryl groups are optionally substituted with one or two substituents independently selected from halogen, lower alkyl, trihaloalkyl, hydroxy, mercapto, cyano, N-acylamino, mono- or dialkyl Amino, carboxyl or N-sulfonylamino.

"Heteroalicyclic" means a monocyclic or fused ring group having 5 to 9 ring atoms in the ring, one or two of which are selected from N, O or S(O) _m (where m is integer of 0 to 2), and the remaining ring atoms are C. These rings may have one or more double bonds, but these rings do not have a fully conjugated pi-electron system. Non-limiting examples of unsubstituted heteroalicyclic groups are pyrrolidinyl, piperidino, piperazino, morpholino, and the like.

"Hydroxy" means a -OH group.

"-S(=O) ₂ -R" represents a sulfonyl group.

"Nitro" means a _-NO2 group.

"Cyano"means a -CN group.

"Alkoxy" means -O- (unsubstituted alkyl) and -O- (unsubstituted cycloalkyl). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, and the like.

"Halogen" means fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.

"Haloalkyl" means a halogen substituted alkyl, preferably a halogen substituted lower alkyl as defined above, which is substituted by one or more of the same or different halogen atoms, for example _-CH2Cl , _-CF3 , _-CH2 CF ₃ , -CH ₂ CCl ₃ , etc.

The invention provides a pharmaceutical composition, which uses the compound of the invention or a pharmaceutically acceptable salt thereof as an active component or main active component, supplemented with a pharmaceutically acceptable carrier.

The compound of the present invention or a pharmaceutically acceptable salt thereof can be applied to the preparation of medicines for tumor-related diseases, for example, medicines for specific inhibitors of Aurora A kinase.

Adopt technical scheme of the present invention, advantage is as follows:

The pyrazoloimidazole compounds provided by the present invention have good Aurora A kinase inhibitory activity, can effectively inhibit the expression of Aurora kinase, and have good inhibitory activity on human chronic myelogenous leukemia cell K562 and human colon cancer cell HCT116, and the inhibition rate Significantly higher than the positive control drug, it can be applied to the preparation of drugs related to tumors.

Detailed ways

The pyrazoloimidazole compounds of the present invention are further illustrated by the following examples, but these examples do not constitute any limitation to the present invention.

The synthesis of embodiment 1 compound 1

Add 4-nitropyrazole-3-carboxylic acid (5.0g, 31.8mmol) and 40ml tetrahydrofuran (THF) into a 100ml three-necked flask, stir at room temperature, then add 3,4-dihydro-2H-pyran (DHP) (8.04 g, 95.5 mmol) and p-toluenesulfonic acid monohydrate (PTSA) (0.30 g, 0.15 mmol). After stirring at room temperature for 16 hours, the reaction solution was concentrated under reduced pressure, then water (30ml) and ethyl acetate (40ml) were added, mixed evenly and transferred to a 100ml separatory funnel, the organic layer was collected, and the aqueous layer was extracted with ethyl acetate (40ml ×3), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was concentrated under reduced pressure to obtain a brown oil. Purification by column chromatography (CH ₂ Cl ₂ :CH ₃ OH, 50:1 v/v) yielded 7.29 g of the target compound with a yield of 95%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 15:1 v/v): Rf=0.4.

The synthesis of embodiment 2 compound 2

Under nitrogen protection, p-methylacetophenone (10ml, 74.5mmol) was dissolved in 80ml of acetonitrile. NBS (14.6 g, 82.0 mmol) and AIBN (1.23 g, 7.49 mmol) were added, and the reaction mixture was stirred at 90° C. for 4 h, and the reaction was completed to obtain a pale yellow solution. Concentrate under reduced pressure, dissolve the residue in 80ml of dichloromethane, add water, mix evenly, separate the layers, collect the organic layer, extract the aqueous layer with dichloromethane (50ml×3), combine the organic layers, and then wash the organic layer with 1M hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated brine, washed three times, dried over anhydrous sodium sulfate, and then suction-filtered, and the filtrate was concentrated under reduced pressure to obtain a brown oil. Recrystallization: Add ethyl acetate dropwise (according to 1g:1.5ml) under heating, then add petroleum ether with 10 times the amount of ethyl acetate, mix well, put it into the freezer to cool and crystallize. 15.1 g of white solid was obtained with a yield of 95%. TLC (silica, petroleum ether: ethyl acetate, 10:1 v/v): Rf = 0.6.m.p.32-33°C.

The synthesis of embodiment 3 compound 3a

Compound 2 (5.0g, 23.5mmol) was dissolved in 30ml of acetonitrile, potassium carbonate (3.24g, 23.5mmol) and a catalytic amount of potassium iodide were added under stirring, then morpholine (2.04g, 23.4mmol) was added, and the reaction solution was The temperature was raised to 90°C and the reaction was refluxed for 4h. After the reaction was completed, it was lowered to room temperature, filtered with suction, and the filtrate was concentrated under reduced pressure to obtain a light yellow oil. Separation and purification by column chromatography (petroleum ether: ethyl acetate, 40:1 v/v) yielded 4.65 g of the target compound with a yield of 90%. TLC (silica, petroleum ether: ethyl acetate, 4:1v/v): Rf=0.3.

The synthesis of embodiment 4 compound 3b

Compound 2 (5.0g, 23.5mmol) was dissolved in 30ml of acetonitrile, potassium carbonate (3.24g, 23.5mmol) and a catalytic amount of potassium iodide were added under stirring, then piperidine (2.0g, 23.5mmol) was added, and the reaction solution was The temperature was raised to 90°C and the reaction was refluxed for 4h. After the reaction was completed, it was lowered to room temperature, filtered with suction, and the filtrate was concentrated under reduced pressure to obtain a reddish-brown oil. Separation and purification by column chromatography (petroleum ether: ethyl acetate, 40:1 v/v) yielded 4.67 g of the target compound with a yield of 91.3%. TLC (silica, petroleum ether: ethyl acetate, 4:1v/v): Rf = 0.3.

The synthesis of embodiment 5 compound 3c

Compound 2 (5.0g, 23.5mmol) was dissolved in 30ml of acetonitrile, potassium carbonate (3.24g, 23.5mmol) and a catalytic amount of potassium iodide were added under stirring, tetrahydropyrrole (1.67g, 23.5mmol) was then added, and the reaction The solution was heated to 90°C and refluxed for 4h. After the reaction was completed, it was lowered to room temperature, filtered with suction, and the filtrate was concentrated under reduced pressure to obtain a brown oil. After separation and purification by column chromatography (petroleum ether: ethyl acetate, 40:1 v/v), 4.02 g of the target compound was obtained with a yield of 84%. TLC (silica, petroleum ether: ethyl acetate, 4:1v/v): Rf = 0.3.

The synthesis of embodiment 6 compound 4a

Compound 3a (0.11 g, 0.48 mmol) was dissolved in 2 ml of 48% HBr, and the temperature was raised to 60°C. Then bromine (0.96ml, 0.5M acetic acid solution) was slowly added dropwise to the above reaction solution, and kept overnight for reaction. The reaction solution was cooled to room temperature, concentrated under reduced pressure, added toluene to continue to concentrate under reduced pressure, and then dried in a vacuum oven at 100°C to obtain 0.14 g of the target compound with a yield of 75%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 200:1 v/v): Rf=0.3.

The synthesis of embodiment 7 compound 4b

Compound 3b (0.104 g, 0.48 mmol) was dissolved in 2 ml of 48% HBr, and the temperature was raised to 60°C. Then bromine (0.96ml, 0.5M acetic acid solution) was slowly added dropwise to the above reaction solution, and kept overnight for reaction. The reaction liquid was cooled to room temperature, concentrated under reduced pressure, added toluene to continue to concentrate under reduced pressure, and then dried in a vacuum oven at 100°C to obtain 0.125 g of the target product with a yield of 70%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 200:1 v/v): Rf=0.3.

The synthesis of embodiment 8 compound 4c

Compound 3c (0.09 g, 0.48 mmol) was dissolved in 2 ml of 48% HBr, and the temperature was raised to 60°C. Then bromine (0.96ml, 0.5M acetic acid solution) was slowly added dropwise to the above reaction solution, and kept overnight for reaction. The reaction solution was cooled to room temperature, concentrated under reduced pressure, added toluene to continue concentrated under reduced pressure, and then dried in a vacuum oven at 100°C to obtain 0.11 g of the target compound with a yield of 74%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 200:1 v/v): Rf=0.3.

The synthesis of embodiment 9 compound 5a

At room temperature, compound 1 (2.41g, 10mmol) was dissolved in 10ml of acetonitrile, potassium carbonate (1.38g, 10mmol) and a catalytic amount of potassium iodide were added under stirring; compound 4a (3.78g, 10mmol) was dissolved in 15ml of acetonitrile , slowly added to the above reaction solution, and reacted overnight at room temperature. After the reaction, suction filtered, the filter cake was washed with acetonitrile, and the filtrate was concentrated under reduced pressure to obtain an orange-yellow oil, which was subjected to column chromatography (petroleum ether: ethyl acetate, 5:1 v/v) separation and purification to obtain 3.11 g of the target compound with a yield of 68%. TLC (silica, petroleum ether: ethyl acetate, 1:1v/v): Rf＝0.3.

The synthesis of embodiment 10 compound 5b

At room temperature, compound 1 (2.41g, 10mmol) was dissolved in 10ml of acetonitrile, potassium carbonate (1.38g, 10mmol) and a catalytic amount of potassium iodide were added under stirring; compound 4b (3.74g, 10mmol) was dissolved in 15ml of acetonitrile , slowly added to the above reaction solution, and reacted overnight at room temperature. After the reaction, suction filtered, the filter cake was washed with acetonitrile, and the filtrate was concentrated under reduced pressure to obtain an orange-yellow oil, which was analyzed by column chromatography (CH ₂ Cl ₂ :CH ₃ OH, 100:1, v/v) separation and purification to obtain 3.32 g of the target compound, with a yield of 73%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 30:1 v/v): Rf=0.2.

The synthesis of embodiment 11 compound 5c

At room temperature, compound 1 (2.41g, 10mmol) was dissolved in 10ml of acetonitrile, potassium carbonate (1.38g, 10mmol) and a catalytic amount of potassium iodide were added under stirring; compound 4c (3.60g, 10mmol) was dissolved in 15ml of acetonitrile , slowly added to the above reaction solution, and reacted overnight at room temperature. After the reaction, suction filtered, the filter cake was washed with acetonitrile, and the filtrate was concentrated under reduced pressure to obtain an orange-yellow oil, which was analyzed by column chromatography (CH ₂ Cl ₂ :CH ₃ OH, 100:1, v/v) separation and purification to obtain 2.78g of the target compound. Yield 63%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 25:1 v/v): Rf=0.3.

The synthesis of embodiment 12 compound 6a

Compound 5a (4.58g, 10mmol) was dissolved in 30ml of acetic acid, ammonium acetate (9.24g, 120mmol) was added with stirring, and the temperature of the reaction solution was raised to 130°C for 4h. After the reaction was completed, it was concentrated under reduced pressure and the residue was poured into into a saturated sodium bicarbonate solution, adjust the pH to neutral, and precipitate a large amount of yellow solid, stir for 1 h, filter with suction, place the filter cake in a vacuum oven to dry, and obtain a yellow solid product, which is separated by column chromatography (petroleum ether: Ethyl acetate, 5:1 v/v) was purified to obtain 1.63 g of the target compound with a yield of 46.2%. TLC (silica, petroleum ether: ethyl acetate, 1:2v/v): Rf＝0.3.

The synthesis of embodiment 13 compound 6b

Compound 5b (4.56g, 10mmol) was dissolved in 30ml of acetic acid, ammonium acetate (9.24g, 120mmol) was added under stirring, and the temperature of the reaction solution was raised to 130°C for 4h. After the reaction was completed, it was concentrated under reduced pressure, and the residue was poured into into a saturated sodium bicarbonate solution, adjust the pH to neutral, and precipitate a large amount of yellow solid, stir for 1 h, filter with suction, and place the filter cake in a vacuum oven to dry to obtain a yellow solid product, which is purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH, 100:1, v/v) separation and purification to obtain 1.80 g of the target compound with a yield of 51.3%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 25:1 v/v): Rf=0.2.

The synthesis of embodiment 14 compound 6c

Compound 5c (4.42g, 10mmol) was dissolved in 30ml of acetic acid, ammonium acetate (9.24g, 120mmol) was added under stirring, and the temperature of the reaction solution was raised to 130°C for 4h. After the reaction was completed, it was concentrated under reduced pressure and the residue was poured into into a saturated sodium bicarbonate solution, adjust the pH to neutral, and precipitate a large amount of yellow solid, stir for 1 h, filter with suction, and place the filter cake in a vacuum oven to dry to obtain a yellow solid product, which is purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH, 100:1, v/v) separation and purification to obtain 1.59 g of the target compound with a yield of 47.3%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 20:1 v/v): Rf=0.3.

The synthesis of embodiment 15 compound 7a

Compound 6a (1.42 g, 4 mmol) was dissolved in 40 ml of methanol, and palladium carbon (10% on Carbon (wetted with ca. 55% Water)) (0.14 g, 1.3 mmol) was added. Filled with hydrogen, heated to 40°C and reacted overnight. After the reaction, the reaction liquid was cooled to room temperature, suction filtered, and the filtrate was concentrated under reduced pressure to obtain the target product 7a (1.1 g) with a yield of 85.2%, which was directly carried out to the next reaction. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 30:1 v/v): Rf=0.3.

The synthesis of embodiment 16 compound 7b

Compound 6b (1.4 g, 4 mmol) was dissolved in 40 ml of methanol, and palladium carbon (10% on Carbon (wetted with ca. 55% Water)) (0.14 g, 1.3 mmol) was added. Filled with hydrogen, heated to 40°C and reacted overnight. After the reaction, the reaction liquid was cooled to room temperature, suction filtered, and the filtrate was concentrated under reduced pressure to obtain the target product 7b (0.91 g) with a yield of 71.1%, which was directly carried out to the next reaction. TLC (silica, _CH2Cl2 :CH3OH, 25:1v/v): _Rf =0.3.

The synthesis of embodiment 17 compound 7c

Compound 6c (1.35 g, 4 mmol) was dissolved in 40 ml of methanol, and palladium carbon (10% on Carbon (wetted with ca. 55% Water)) (0.14 g, 1.3 mmol) was added. Filled with hydrogen, heated to 40°C and reacted overnight. After the reaction, the reaction solution was cooled to room temperature, filtered with suction, and the filtrate was concentrated under reduced pressure to obtain the target product 7c (0.85 g) with a yield of 69.1%, which was directly carried out to the next reaction. TLC (silica, _CH2Cl2 :CH3OH, 30:1v/v): _Rf =0.3.

The synthesis of embodiment 18 compound MZ-1A

Dissolve benzoic acid (0.24g, 2mmol) in SOCl ₂ (3ml), heat up to 85°C for reflux reaction for 3h, after the reaction is complete, concentrate under reduced pressure to remove excess SOCl ₂ to obtain the product acid chloride for later use; Compound 7a (0.54g, 1.67 mmol) was dissolved in 5ml of dry dichloromethane, added triethylamine (0.28ml, 2mmol) and stirred in an ice bath, then slowly added the prepared acid chloride dropwise to the above reaction solution, and the temperature was controlled at 0-5 ℃. After the dropwise addition was completed, the reaction was incubated for 30 minutes, then raised to room temperature for reaction, and the reaction was monitored by TLC. After completion of the reaction, the reaction solution was concentrated under reduced pressure, 10ml of water and 20ml of ethyl acetate were added to the residue, and after mixing evenly, the liquid was separated in a separatory funnel, the aqueous layer was extracted with ethyl acetate several times, the organic layers were combined, and then Wash with saturated brine, dry over anhydrous sodium sulfate for 1 h, filter with suction, and concentrate the filtrate under reduced pressure to obtain a black oil, which is separated and purified by column chromatography ( _CH2Cl2 : _CH3OH , 100:1v/v) to obtain 0.13g of the target compound , yield 31.0%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 25:1 v/v): Rf = 0.2.mp 268.3-270.7°C. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 13.02(s, 1H), 12.92(s, 1H), 11.55(s, 1H), 8.29(s, 1H), 8.04(d, J= 7.2Hz, 2H), 7.82(d, J=8.4Hz, 2H), 7.66(d, J=7.6Hz, 2H), 7.61(t, J=7.2Hz, 2H), 7.34(d, J=9.2Hz ,2H),3.56(s,4H),3.46(s,2H),2.34(s,4H). ¹³ C NMR(100MHz,DMSO-d6)δ(ppm): 163.233,142.610,140.344,134.552,134.418, 132.561,131.954,129.889,129.509,127.427,127.300,124.947,124.723,120.159,120.030,66.492,62.616,53.444.HR-MS(calculated for C ₂₄ H ₂₄ N ₆ O ₂ (M+H) ⁺ 429.2039；found 429.2028 ).

The synthesis of embodiment 19 compound MZ-1B

Dissolve p-methoxybenzoic acid (0.30 g, 2 mmol) in SOCl ₂ (3 ml), heat up to 85° C. for reflux reaction for 3 h, after the reaction is complete, concentrate under reduced pressure to remove excess SOCl ₂ to obtain the product acid chloride for later use; compound 7a ( 0.54g, 1.67mmol) was dissolved in 5ml of dry dichloromethane, triethylamine (0.28ml, 2mmol) was added and stirred in an ice bath, then the prepared acid chloride was slowly added dropwise to the above reaction solution, and the temperature was controlled throughout the process at 0-5°C. After the dropwise addition was completed, the reaction was incubated for 30 minutes, then raised to room temperature for reaction, and the reaction was monitored by TLC. After completion of the reaction, the reaction solution was concentrated under reduced pressure, 10ml of water and 20ml of ethyl acetate were added to the residue, and after mixing evenly, the liquid was separated in a separatory funnel, the aqueous layer was extracted with ethyl acetate several times, the organic layers were combined, and then Wash with saturated brine, dry over anhydrous sodium sulfate for 1 h, filter with suction, and concentrate the filtrate under reduced pressure to obtain a black oil, which is separated and purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH, 100:1 v/v) to obtain the target compound 0.105 g, 14% yield. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 40:1 v/v): Rf = 0.3.mp 265.3-267.8°C. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 12.98(s, 1H), 12.97(s, 1H), 11.42(s, 1H), 8.26(s, 1H), 7.99(d, J= 8.8Hz, 2H), 7.81(d, J=8.4Hz, 2H), 7.67(s, 1H), 7.35(d, J=8.0Hz, 2H), 7.12(d, J=8.8Hz, 2H), 3.87 (s,3H),3.55(t,J＝4.0 4H),3.47(s,2H),2.35(s,4H).HR-MS(calculated for C ₂₅ H ₂₆ N ₆ O ₃ (M+H) ⁺ 459.2144; found 459.2137).

The synthesis of embodiment 20 compound MZ-1C

Dissolve p-nitrobenzoic acid (0.33g, 2mmol) in SOCl2 (3ml), heat up to 85°C for reflux reaction for 3h, after the reaction is complete, concentrate under reduced pressure to remove excess _SOCl2 to obtain the product acid chloride for later use; Compound 7a (0.54 g, 1.67mmol) was dissolved in 5ml of dry dichloromethane, added triethylamine (0.28ml, 2mmol) and stirred in an ice bath, then slowly added dropwise to the prepared acid chloride in the above reaction solution, and the temperature was controlled during the whole process. 0-5°C. After the dropwise addition was completed, the reaction was incubated for 30 minutes, then raised to room temperature for reaction, and the reaction was monitored by TLC. After completion of the reaction, the reaction solution was concentrated under reduced pressure, 10ml of water and 20ml of ethyl acetate were added to the residue, and after mixing evenly, the liquid was separated in a separatory funnel, the aqueous layer was extracted with ethyl acetate several times, the organic layers were combined, and then Wash with saturated brine, dry over anhydrous sodium sulfate for 1 h, filter with suction, and concentrate the filtrate under reduced pressure to obtain a yellow oil, which is separated and purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH, 100:1 v/v) to obtain the yellow target Compound 0.21g, yield 26.6%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 40:1 v/v): Rf = 0.25.mp 258.4-261.7°C. ¹ H NMR (400MHz,DMSO-d ₆ )δ(ppm): 13.09(s,1H),12.96(s,1H),11.71(s,1H),8.39(d,J=8.4Hz,1H),8.32 (s,1H),8.24(d,J=8.4Hz,1H),7.78(d,J=7.6Hz,2H),7.67(s,1H),7.32(d,J=7.6Hz,2H),3.56 (s,4H),3.46(s,2H),2.35(s,4H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ(ppm):161.634,149.991,142.412,140.465,140.091,136.640,133.318, 132.171, 129.767, 128.976, 124.862, 124.611, 120.472, 119.786, 113.710, 66.511, 62.897, 53.718. HR-MS (calculated for C ₂₄ H ₂₃ N ₇ O ₄ (M+H) ⁺ 474.18174.found);

The synthesis of embodiment 21 compound MZ-1D

Dissolve p-chlorobenzoic acid (0.31g, 2mmol) in SOCl ₂ (3ml), heat up to 85°C for reflux reaction for 3h, after the reaction is complete, concentrate under reduced pressure to remove excess SOCl ₂ to obtain the product acid chloride for later use; Compound 7a (0.54g , 1.67mmol) was dissolved in 5ml of dry dichloromethane, added triethylamine (0.28ml, 2mmol) and stirred in an ice bath, then the acid chloride prepared was slowly added dropwise to the above reaction solution, and the temperature was controlled during the whole process at 0 -5°C. After the dropwise addition was completed, the reaction was incubated for 30 minutes, then raised to room temperature for reaction, and the reaction was monitored by TLC. After completion of the reaction, the reaction solution was concentrated under reduced pressure, 10ml of water and 20ml of ethyl acetate were added to the residue, and after mixing evenly, the liquid was separated in a separatory funnel, the aqueous layer was extracted with ethyl acetate several times, the organic layers were combined, and then Wash with saturated brine, dry over anhydrous sodium sulfate for 1 h, filter with suction, and concentrate the filtrate under reduced pressure to obtain a black oil, which is separated and purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH, 100:1 v/v) to obtain the target compound 0.18 g, yield 23.4%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 40:1 v/v): Rf = 0.3.mp 265.3-268.7°C. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 11.52(s, 1H), 8.28(s, 1H), 8.03(d, J=8.4Hz, 2H), 7.78(d, J=8.0Hz ¹³ C NMR(100MHz,DMSO-d6)δ(ppm)：162.281,142.653,140.452,137.353,133.436,133.282,131.982,129.906,129.547.129.315,129.208,124.789,120.244,119.999,113.619,66.629,62.795,53.670. HR-MS (calculated for C ₂₄ H ₂₃ ClN ₆ O ₂ (M+H) ⁺ 463.1649; found 463.1639).

The synthesis of embodiment 22 compound MZ-1E

Dissolve m-chlorobenzoic acid (0.31g, 2mmol) in SOCl ₂ (3ml), heat up to 85°C for reflux reaction for 3h, after the reaction is complete, concentrate under reduced pressure to remove excess SOCl ₂ to obtain the product acid chloride for later use; Compound 7a (0.54g , 1.67mmol) was dissolved in 5ml of dry dichloromethane, added triethylamine (0.28ml, 2mmol) and stirred in an ice bath, then the acid chloride prepared was slowly added dropwise to the above reaction solution, and the temperature was controlled during the whole process at 0 -5°C. After the dropwise addition was completed, the reaction was incubated for 30 minutes, then raised to room temperature for reaction, and the reaction was monitored by TLC. After completion of the reaction, the reaction solution was concentrated under reduced pressure, 10ml of water and 20ml of ethyl acetate were added to the residue, and after mixing evenly, the liquid was separated in a separatory funnel, the aqueous layer was extracted with ethyl acetate several times, the organic layers were combined, and then Wash with saturated brine, dry over anhydrous sodium sulfate for 1 h, filter with suction, and concentrate the filtrate under reduced pressure to obtain a black oil, which is separated and purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH, 100:1 v/v) to obtain the target compound 0.21 g, yield 27.3%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 30:1 v/v): Rf = 0.3.mp 266.4-269.2°C. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 13.07(s, 1H), 12.94(s, 1H), 11.47(s, 1H), 8.31(s, 1H), 7.99(d, J= 9.2Hz, 2H), 7.81(d, J=6.4Hz, 2H), 7.74(d, J=8.8Hz, 1H), 7.66(dd, J=7.6, 5.4Hz, 2H), 7.33–7.31(m, 2H), 3.55(s, 4H), 3.45(s, 2H), 2.34(s, 4H). ¹³ C NMR (100MHz, DMSO-d ₆ ) δ (ppm): 161.839, 142.631, 140.498, 136.441, 134.326, 133.403,132.435,132.039,131.540,129.891,126.917,126.458,125.019,124.711,120.341,119.857,113.741,66.492,62.714,53.653.HR-MS(calculated for C ₂₄ H ₂₃ ClN ₆ O ₂ (M+H) ⁺ 463.1649; found 463.1630).

The synthesis of embodiment 23 compound MZ-3B

Dissolve p-methoxybenzoic acid (0.30 g, 2 mmol) in SOCl ₂ (3 ml), heat up to 85° C. for reflux reaction for 3 h, after the reaction is complete, concentrate under reduced pressure to remove excess SOCl ₂ to obtain the product acid chloride for later use; Compound 7b ( 0.54g, 1.67mmol) was dissolved in 5ml of dry dichloromethane, triethylamine (0.28ml, 2mmol) was added and stirred in an ice bath, then the prepared acid chloride was slowly added dropwise to the above reaction solution, and the temperature was controlled throughout the process at 0-5°C. After the dropwise addition was completed, the reaction was incubated for 30 minutes, then raised to room temperature for reaction, and the reaction was monitored by TLC. After completion of the reaction, the reaction solution was concentrated under reduced pressure, 10ml of water and 20ml of ethyl acetate were added to the residue, and after mixing evenly, the liquid was separated in a separatory funnel, the aqueous layer was extracted with ethyl acetate several times, the organic layers were combined, and then Wash with saturated brine, dry over anhydrous sodium sulfate for 1 h, filter with suction, and concentrate the filtrate under reduced pressure to obtain a black oil, which is separated and purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH, 100:1 v/v) to obtain the target compound 0.14 g, yield 18.3%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 30:1 v/v): Rf=0.3.mp 240.3-243.1°C. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 11.42(s, 1H), 8.26(s, 1H), 8.00(d, J=8.4Hz, 2H), 7.81(d, J=8.0Hz ,2H),7.66(s,1H),7.34(d,J=7.6Hz,2H),7.11(d,J=8.4Hz,2H),3.86(s,3H),3.46(s,2H),2.34 (s,4H),1.48(s,4H),1.36(s,2H) ^.13 CNMR(100MHz,DMSO-d ₆ )δ(ppm):162.901,162.610,142.747,140.420,133.382,131.788,129.812,129.299 , ^{129.177,126.726,124.704,120.377,119.821,114.652,113.575,62.625,56.042,54.298,25.991,24.451.HR} -MS(calculated for C ₂₆ H ₂₈ N ₆ O ₂ (M+H) 2345 found.2); .

The synthesis of embodiment 24 compound MZ-3D

Dissolve p-chlorobenzoic acid (0.31g, 2mmol) in SOCl ₂ (3ml), heat up to 85°C for reflux reaction for 3h, after the reaction is complete, concentrate under reduced pressure to remove excess SOCl ₂ to obtain the product acid chloride for later use; Compound 7b (0.54g , 1.67mmol) was dissolved in 5ml of dry dichloromethane, added triethylamine (0.28ml, 2mmol) and stirred in an ice bath, then the acid chloride prepared was slowly added dropwise to the above reaction solution, and the temperature was controlled during the whole process at 0 -5°C. After the dropwise addition was completed, the reaction was incubated for 30 minutes, then raised to room temperature for reaction, and the reaction was monitored by TLC. After completion of the reaction, the reaction solution was concentrated under reduced pressure, 10ml of water and 20ml of ethyl acetate were added to the residue, and after mixing evenly, the liquid was separated in a separatory funnel, the aqueous layer was extracted with ethyl acetate several times, the organic layers were combined, and then Wash with saturated brine, dry over anhydrous sodium sulfate for 1 h, filter with suction, and concentrate the filtrate under reduced pressure to obtain a black oil, which is separated and purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH, 100:1 v/v) to obtain the target compound 0.11 g, yield 14.2%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 30:1 v/v): Rf=0.3.mp 253.3-256.0°C. ¹ H NMR (400MHz, DMSO-d ₆ )δ(ppm): 11.43(s,1H), 10.68(s,1H), 8.34(s,1H), 8.05–8.01(m,2H), 7.96(d, J＝8.0Hz, 2H), 7.84(s, 1H), 7.76(d, J＝8.0Hz, 1H), 7.71(d, J＝8.0Hz, 1H), 7.66(d, J＝7.6Hz, 2H) ,4.25(s,2H),3.29(s,2H),2.84(s,2H),1.78(s,4H),1.67(s,1H),1.36(s,1H). ¹³ C NMR(100MHz,DMSO -d6)δ(ppm)：161.145,142.202,139.217,135.668,134.789,133.698,131.668,131.241,130.861,126.197,125.756,124.204,119.687,119.157,114.040,58.602,51.347,22.047,21.351.HR-MS( calculated for C ₂₅ H ₂₅ ClN ₆ O(M+H) ⁺ 461.1856; found461.1850).

The synthesis of embodiment 25 compound MZ-3E

Dissolve m-chlorobenzoic acid (0.31g, 2mmol) in SOCl ₂ (3ml), heat up to 85°C for reflux reaction for 3h, after the reaction is complete, concentrate under reduced pressure to remove excess SOCl ₂ to obtain the product acid chloride for later use; Compound 7b (0.54g , 1.67mmol) was dissolved in 5ml of dry dichloromethane, added triethylamine (0.28ml, 2mmol) and stirred in an ice bath, then the acid chloride prepared was slowly added dropwise to the above reaction solution, and the temperature was controlled during the whole process at 0 -5°C. After the dropwise addition was completed, the reaction was incubated for 30 minutes, then raised to room temperature for reaction, and the reaction was monitored by TLC. After completion of the reaction, the reaction solution was concentrated under reduced pressure, 10ml of water and 20ml of ethyl acetate were added to the residue, and after mixing evenly, the liquid was separated in a separatory funnel, the aqueous layer was extracted with ethyl acetate several times, the organic layers were combined, and then Wash with saturated brine, dry over anhydrous sodium sulfate for 1 h, filter with suction, and concentrate the filtrate under reduced pressure to obtain a black oil, which is separated and purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH, 150:1 v/v) to obtain the target compound 0.15 g, yield 19.4%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 30:1 v/v): Rf = 0.3.mp 255.2-258.4°C. ¹ H NMR (400MHz, DMSO-d ₆ )δ(ppm): 11.43(s,1H), 10.68(s,1H), 8.34(s,1H), 8.05–8.01(m,2H), 7.96(d, J＝8.0Hz, 2H), 7.84(s, 1H), 7.76(d, J＝8.0Hz, 1H), 7.71(d, J＝8.0Hz, 1H), 7.66(d, J＝7.6Hz, 2H) ,4.25(s,2H),3.29(s,2H),2.84(s,2H),1.78(s,4H),1.67(s,1H),1.36(s,1H). ¹³ C NMR(100MHz,DMSO -d6)δ(ppm)：161.145,142.202,139.217,135.668,134.789,133.698,131.668,131.241,130.861,126.197,125.756,124.408,124.204,119.687,119.477,119.259,114.040,58.602,51.347,22.047,21.351. HR-MS (calculated for C ₂₅ H ₂₅ ClN ₆ O (M+H) ⁺ 461.1856; found 461.1836).

The synthesis of embodiment 26 compound MZ-2C

Dissolve p-nitrobenzoic acid (0.33g, 2mmol) in SOCl ₂ (3ml), heat up to 85°C for reflux reaction for 3h, after the reaction is complete, concentrate under reduced pressure to remove excess SOCl ₂ to obtain the product acid chloride for later use; Compound 7c (0.51 g, 1.67mmol) was dissolved in 5ml of dry dichloromethane, added triethylamine (0.28ml, 2mmol) and stirred in an ice bath, then slowly added dropwise to the prepared acid chloride in the above reaction solution, and the temperature was controlled during the whole process. 0-5°C. After the dropwise addition was completed, the reaction was incubated for 30 minutes, then raised to room temperature for reaction, and the reaction was monitored by TLC. After completion of the reaction, the reaction solution was concentrated under reduced pressure, 10ml of water and 20ml of ethyl acetate were added to the residue, and after mixing evenly, the liquid was separated in a separatory funnel, the aqueous layer was extracted with ethyl acetate several times, the organic layers were combined, and then Wash with saturated brine, dry over anhydrous sodium sulfate for 1 h, filter with suction, and concentrate the filtrate under reduced pressure to obtain a yellow oil, which is separated and purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH, 150:1v/v) to obtain the yellow target Compound 0.18g, yield 23.6%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 30:1 v/v): Rf=0.3.mp 261.3-264.0°C. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 13.10(s, 1H), 11.60(s, 1H), 10.78(s, 1H), 8.43(d, J=8.4Hz, 2H), 8.37 (s,1H),8.29(d,J=8.0Hz,2H),7.93(d,J=8.0Hz,2H),7.84(s,1H),7.64(d,J=8.0Hz,2H),4.37 (d,J=4.4Hz,2H),3.08(s,2H),2.50(s,2H),2.03(s,1H),1.89(s,1H). ¹³ C NMR(100MHz,DMSO-d6)δ (ppm): 161.645, 149.978, 142.616, 140.138, 140.074, 134.527, 132.130, 130.729, 129.003, 124.997, 124.789, 124.419, 120.720, 119.890, 114.3368, 51

The synthesis of embodiment 27 compound MD-1

Compound 7a (0.32g, 1.0mmol), 4,6-dichloro-2-methylpyrimidine (0.13g, 0.83mmol), sodium iodide (0.15g, 1.0mmol) and N,N-diisopropyl Ethylamine (0.13g, 1.0mmol) was dissolved in 3ml of DMF, protected by N ₂ , heated to 90°C, and reacted for 4h. After the reaction, the reaction solution was cooled, poured into 30 ml of ice water, stirred, and suction filtered. After separation and purification by column chromatography (CH ₂ Cl ₂ :CH ₃ OH, 100:1 v/v), 0.131 g of the target compound was obtained, with a yield of 29.5%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 30:1 v/v): Rf=0.3.mp 268.3-271.1°C. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 10.02(s, 1H), 8.37(s, 1H), 7.84(d, J=7.2Hz, 1H), 7.78(d, J=8.0Hz ,1H),7.63(s,1H),7.30(d,J＝7.6Hz,2H),6.90(s,1H),3.55(s,4H),3.44(s,2H),2.47–2.46(m, 3H), 2.34(s,4H). HR-MS (calculated for C ₂₂ H ₂₃ ClN ₈ O(M+H) ⁺ 451.1761; found 451.1753).

Synthesis of Example 28 Compound 8

Dissolve p-hydroxyacetophenone (1.36g, 10mmol), 1-bromo-3-chloropropane (1.56g, 10mmol) in 10ml of acetonitrile, add potassium carbonate (1.38g, 10mmol) and catalytic amount of potassium iodide under stirring, 85 Reaction at ℃ for 4h, after the reaction, cooled to room temperature, suction filtration, and the filtrate was concentrated under reduced pressure to obtain a brown oil, which was separated and purified by column chromatography (petroleum ether: ethyl acetate, 40:1v/v) to obtain the target compound 2.02 g, yield 95.2%. TLC (silica, petroleum ether: ethyl acetate, 10:1, v/v): Rf＝0.6.

Synthesis of Example 29 Compound 9

Dissolve compound 8 (2.12g, 10mmol), morpholine (0.87g, 10mmol) in 10ml of acetonitrile, add potassium carbonate (1.38g, 10mmol) and catalytic amount of potassium iodide under stirring, react at 85°C for 4h, after the reaction, Cool to room temperature, filter with suction, and concentrate the filtrate under reduced pressure to obtain a brown oil, which is separated and purified by column chromatography (petroleum ether: ethyl acetate, 10:1 v/v) to obtain 2.2 g of the target compound with a yield of 83.6%. TLC (silica, petroleum ether: ethyl acetate, 1:1, v/v): Rf＝0.3.

Synthesis of Example 30 Compound 10

Compound 9 (2.63g, 10mmol) was dissolved in 20ml of 48% HBr, and the temperature was raised to 60°C, then bromine (20ml, 0.5M acetic acid solution) was added dropwise, and the reaction was incubated overnight after addition. After the reaction, the reaction solution was concentrated under reduced pressure, and the residue was dried in a vacuum oven to obtain 3.06 g of the target compound with a yield of 73%. TLC (CH ₂ Cl ₂ :CH ₃ OH, 150:1 v/v): Rf=0.3.

Synthesis of Example 31 Compound 11

Compound 1 (2.41g, 10mmol) was dissolved in 10ml of acetonitrile, potassium carbonate (1.38g, 10mmol) and a catalytic amount of potassium iodide were added under stirring; compound 10 (4.21g, 10mmol) was dissolved in 10ml of acetonitrile, and added dropwise Add to the above reaction solution, add and react overnight at room temperature. After the reaction, suction filtration was performed, and the filtrate was concentrated under reduced pressure to obtain a yellow oil, which was separated and purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH, 100:1 v/v) to obtain 3.26 g of the target compound, with a yield of 65.0%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 30:1 v/v): Rf=0.3.

Synthesis of Example 32 Compound 12

Compound 11 (5.02g, 10mmol) was dissolved in 25ml of acetic acid, ammonium acetate (9.24g, 120mmol) was added, and reacted at 125°C for 4h. After the reaction, the reaction solution was concentrated under reduced pressure, and the residue was poured into saturated bicarbonate In the sodium solution, a yellow solid was precipitated, filtered with suction, dried the filter cake, separated and purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH, 100:1 v/v) to obtain 2.0 g of the target compound with a yield of 50.3%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 30:1 v/v): Rf=0.3.

Synthesis of Example 33 Compound 13

Compound 12 (1.59 g, 4 mmol) was dissolved in 40 ml of methanol, and palladium carbon (10% on Carbon (wetted with ca. 55% Water)) (0.14 g, 1.3 mmol) was added. Filled with hydrogen, heated to 40°C and reacted overnight. After the reaction, the reaction liquid was cooled to room temperature, suction filtered, and the filtrate was concentrated under reduced pressure to obtain 1.0 g of the target compound with a yield of 68.0%, which was directly carried out to the next reaction. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 30:1 v/v): Rf=0.25.

The synthesis of embodiment 34 compound MD-4

Compound 4-chloro-2-(methylsulfonyl)-6-(trifluoromethyl)pyrimidine (0.22g, 0.83mmol), compound 13 (0.37g, 1.0mmol), sodium iodide (0.15g, 1.0mmol ) and DIPEA (0.13g, 1.0mmol) were dissolved in 5ml of DMF, protected by nitrogen, and reacted at 90°C for 4h. After the reaction, the reaction solution was cooled to room temperature, then poured into ice water, stirred, and no obvious solid appeared , extracted with ethyl acetate (40ml×3), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was concentrated under reduced pressure to obtain an orange-yellow oil, which was subjected to column chromatography (CH ₂ Cl ₂ :CH ₃ OH, 100:1 v/v) separation and purification to obtain 0.11 g of the target compound with a yield of 18.5%. TLC (silica, CH ₂ Cl ₂ :CH ₃ OH, 30:1 v/v): Rf=0.3.mp 258.3-260.1°C. ¹ H NMR (400MHz,DMSO-d ₆ )δ(ppm): 10.79(s,1H),8.43(s,1H),7.82(s,2H),7.72(s,1H),7.55(s,1H) ,6.92(d,J=8.0Hz,2H),4.00(t,J=6.2Hz,2H),3.55(t,J=4.2Hz,4H),3.29(s,2H),2.42(t,J= 7.2Hz,2H),2.36(s,4H),1.87(s,3H).HR-MS(calculated for C ₂₅ H ₂₇ F ₃ N ₈ O ₄ S(M+H) ⁺ 593.1906; found

593.1900).

Embodiment 35 The following are some pharmacological tests and results of representative compounds of the present invention:

1. Detection of compound's inhibitory activity against Aurora A kinase

1.1 Determination method

In this experiment, the Kinase-Glo Plus Luminescent Kinase Assay Kit was used. This assay method measures the kinase activity by quantifying the remaining ATP content in the solution after the kinase reaction. The luminescence signal in the assay is related to the amount of ATP present in solution and is inversely related to the activity of the kinase. The specific experimental conditions are as follows: the reaction system of all enzymes contains the following contents: 40mM Tris, pH 7.4, 10mM MgCl ₂ , 0.1mg/mL BSA, 1mM DTT, 10μM ATP, kinase and kinase substrate, and at the same time add Compounds to be screened at different concentrations constituted a 50 μl reaction mixture. Compounds were diluted in 10% DMSO and 5 μl of the dilution was added to 50 μl of the reaction solution to ensure a final concentration of 1% DMSO in all reactions. The reactant mixture was reacted at 30° C. for 40 min. Then the content of ATP in the reaction mixture was detected by luciferase method, and then the chemiluminescence signal was detected on the MD-SpectraMax M5 multifunctional microplate reader, and the chemiluminescence signal intensity value was inversely proportional to the inhibitory activity of the enzyme. The detected chemiluminescent signal value is substituted into the following formula to calculate the inhibition rate of the compound against Aurora A kinase.

1.2 Results and discussion

This experiment mainly detects the inhibitory rate of the compound on Aurora A kinase when the concentration of the compound is 0.5 μM and 1 μM respectively, and the results are shown in Table 1.

The inhibitory rate of the compound of table 1 to Aurora A kinase

a. MZ-1Q is the positive control drug AT9283

It can be seen from Table 1 that most of the tested compounds have good inhibitory activity against Aurora A kinase, and the inhibitory rate of compound MD-1 to Aurora A kinase at the two concentrations of 0.5 μM and 1 μM is comparable to that of the positive control When the compound concentration is 0.5 μM, there are 4 compounds with an inhibition rate exceeding 50%, and when the compound concentration is 1 μM, there are 8 compounds with an inhibition rate exceeding 50% (including 50%), indicating that with the compound concentration The inhibitory activity of the compound on Aurora A kinase increases accordingly.

When the compound concentration is 1 μM, the IC50 value of the compound whose inhibition rate exceeds 50% is determined again. The test results are shown in Table 2.

The _IC50 value of the compound of table 2 to Aurora A kinase

a. MZ-1Q is the positive control drug AT9283

As can be seen from Table 2, the compounds of the present invention have better inhibitory activity to Aurora A kinase, wherein the IC50 values of compounds MD-1, MZ-1B, MZ-1C, and MZ-1E are in the range of 0.1-1.0 μM , and the IC50 values of the rest of the compounds against Aurora A kinase were not more than 3.0 μM. Among them, compound MD-1 has the best inhibitory activity, IC50=0.145±0.016 μM.

2. To detect the effect of compounds on the proliferation of human tumor cells in vitro

2.1 Experimental method

experimental method:

1) Digest and count the cells, prepare a cell suspension of 3.5×104 cells/ml, and add 100 μl of the cell suspension to each well of a 96-well cell culture plate;

2) The 96-well cell culture plate was placed in a 37°C, 5%, _CO2 incubator for 24 hours;

3) Dilute the drug with the medium to the required concentration of the working solution, add 100 μl of the corresponding drug-containing medium to each well, and set up a negative control group at the same time;

4) The 96-well cell culture plate was placed in a 37°C, 5%, _CO2 incubator for 72 hours;

5) Stain the 96-well plate with CCK-8, λ=450nm, and measure the OD value;

a) Add 10 μl CCK-8 to each well, and continue culturing in the incubator for 2-3 hours;

b) Shake gently for 10 minutes to remove air bubbles in the 96-well plate;

c) λ=450nm, read the OD value of each well with a microplate reader, and calculate the inhibition rate.

6) Calculate the inhibition rate of each group.

Sample concentration: 10 μM. According to the inhibition rate, the LOGIT method was used to calculate the half inhibitory concentration IC50. The experiment was repeated more than 3 times, and the data were expressed as Mean ± SD.

2.2 Data processing

Data were expressed as mean ± standard deviation (Mean ± SD), and statistical analysis was performed using SPSS software.

2.3 Results and discussion

In the experiment of detecting the effect of compounds on the proliferation of human tumor cells in vitro, human chronic myelogenous leukemia cells K562 and human colon cancer cells HCT116 were selected for activity detection. Add 100 μl of cell suspension to each well of the culture plate, incubate for 24 hours, and administer the drug. After 72 hours of drug action, perform CCK-8 staining, measure the OD value, and calculate the inhibition rate. The results are shown in Table 3

The compound of table 3 is to the proliferation of K562 and HCT116 cells

a. MZ-1Q is the positive control drug AT9283

As can be seen from Table 3: when the compound concentration is 10 μM, generally speaking, the inhibitory activity of the compound on K562 cells is better than that on HCT116 cells, indicating that the compound has a better selectivity to different cell lines. In the K562 cell line, only compound MZ-3B has poor inhibitory activity, and the inhibitory rates of other compounds are all over 50%, and the inhibitory rates of compounds MZ-1D, MZ-1E, and MD-4 are all over 50%. In addition to the positive control drug, the compound MZ-3E was very close to the inhibitory activity of the positive control drug; in the HCT116 cell line, most of the compounds had poor inhibitory activity on the cell, but the inhibitory activity of the compound MZ-3D and MZ-1C on the cell They all exceeded the positive control drug, and the inhibition rates were as high as 99.66% and 97.62%, respectively, and the inhibition rate of compound MZ-1E against HCT116 cells was close to 50%, and it also had certain inhibitory activity.

The compounds with an inhibition rate of over 50% in K562 cells were selected, and the compounds MZ-3D, MZ-1E, and MZ-1C in HCT116 cells were selected for the determination of the IC50 values of the two cells. Three replicate wells were set for each drug concentration. The test results are shown in the table 4.

The _IC50 value of table 4 compound to K562 and HCT116 cells

a. MZ-1Q is the positive control drug AT9283

Although, the present invention has been described in detail with general description, specific implementation and test above, but on the basis of the present invention, some modifications or improvements can be made to it, which will be obvious to those skilled in the art . Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (4)

1. A compound, or a pharmaceutically acceptable salt thereof, selected from:

2. a pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient or as a major active ingredient, together with a pharmaceutically acceptable carrier.

3. The use of a compound of claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a disease associated with a tumor.

4. Use according to claim 3, wherein the compound or a pharmaceutically acceptable salt thereof is for the preparation of an inhibitor specific for Aurora A kinase.