CN103044392B - A kind of preparation method of efficient DPP-IV inhibitor - Google Patents

Abstract

The invention belongs to medical art, relate to a kind of preparation method of efficient DPP-IV inhibitor, relate to particularly a kind of take uracil as the compound or pharmaceutically acceptable salt thereof of parent nucleus, its preparation method, composition and this compounds to benefit from purposes in the disease that DPP-IV suppresses in prevention or treatment as dipeptidyl peptidase (DPP-IV) inhibitor.The compounds of this invention has extraordinary selective inhibitory to DPP-IV, and cardiac toxic is also very low, is very promising DPP-IV inhibitor class medicine.

Description

A kind of preparation method of efficient DPP-IV inhibitor

technical field

The invention belongs to the technical field of medicine, and in particular relates to a compound with uracil as the core or a pharmaceutically acceptable salt thereof, a preparation method, a composition thereof, and this type of compound as a dipeptidyl peptidase (DPP-IV) inhibitor Use in the prophylaxis or treatment of a disease which would benefit from inhibition of DPP-IV.

Background technique

Diabetes mellitus is caused by absolute or relative lack of insulin, resulting in elevated blood sugar, which can lead to serious complications and eventually lead to disability or death of the patient. Clinically, diabetes is divided into type I and type II. Type I diabetes is caused by the destruction of pancreatic β-cells and lack of insulin secretion, which leads to elevated blood sugar. Such patients can only rely on exogenous insulin; type II diabetes is caused by relatively insufficient insulin secretion or imperfect insulin action. Hyperglycemia, its incidence rate accounts for more than 90% of all diabetic patients. Current drug research is mainly launched for type II diabetes.

There are many kinds of traditional hypoglycemic drugs, mainly divided into insulin sensitizers (such as biguanides, thiazolidinediones, etc.) and insulin secretagogues (such as sulfonylureas and non-sulfonylureas), and so on. But these drugs do not prevent the progression of diabetes, and there are toxic side effects such as weight gain, hypoglycemia, and eventual loss of efficacy. Therefore, it is an urgent task to develop new anti-diabetic drugs, overcome the above-mentioned problems, and prevent or even reverse the deterioration of the disease.

Dipeptidyl peptidase (DPP-IV) is a glycoprotein widely distributed in the human body. Its function is similar to that of serine protease. It inactivates polypeptides by cutting them, so as to regulate physiological functions. The cleavage site of DPP-IV to the substrate is constant, which is the proline or alanine at the penultimate position of its N-terminus. Glucagon-like peptide-1 (GLP-1) is an endogenous hormone that is secreted by L cells in the small intestine as postprandial blood sugar rises, thereby stimulating insulin secretion. Therefore, the secretion of GLP-1 is closely related to the intake of blood sugar. GLP-1-based treatment regimens can effectively control blood sugar without gaining weight, and will not produce adverse reactions such as hypoglycemia. However, as the substrate of DPP-IV, GLP-1 has a very short half-life and will be rapidly cut and inactivated by DPP-IV within 1-2 minutes after secretion. Therefore, two new drug development strategies can be adopted based on the mechanism of action of GLP-1: the development of DPP-IV resistant GLP-1 analogs and the development of DPP-IV inhibitors. Based on this idea, the inventors found that uracil compounds are effective DPP-IV inhibitors that can effectively lower blood sugar without causing risks such as weight gain and hypoglycemia, and based on this, the present invention was completed.

Contents of the invention

One aspect of the present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof

Wherein, R is selected from substituted or unsubstituted alkyl, cycloalkyl, aryl, heteroaryl or heterocycloalkyl _; substituents are selected from alkyl, alkoxy, halogen, cyano, amino, hydroxyl, nitrate radical, carbonyl, sulfonylalkyl, amido, carbonylalkyl, aryl, aryloxy, heterocycloalkyl, heteroaryl, heteroaryloxy, cycloalkyl, cycloalkylalkyl, sulfonyl, or _Sulfinyl ; R preferably substituted or unsubstituted aryl or heteroaryl;

R2 is selected from _:

(1) Hydrogen;

(2) cyano group;

(3) Unsubstituted or substituted alkyl groups;

(4) Phenyl unsubstituted or substituted by 1-5 independent substituents selected from halogen, cyano, OH, alkyl, alkoxy, NHSO ₂ R ₃ , N(alkyl)SO ₂ R ₃ , SO ₂ R ₃ , SO ₂ NR ₄ R ₅ , NR ₄ R ₅ , CONR ₄ R ₅ , COOH, and carboxyalkyl;

(5)OH;

(6) Alkoxy;

(7) NR ₄ R ₅ ;

R2 is preferably _hydrogen , alkyl or alkoxy;

R ₃ is selected from substituted or unsubstituted alkyl groups, and the substituents are selected from 1-5 independent halogen or COOH;

R ₄ and R ₅ are each independently selected from:

(1) hydrogen,

(2) Unsubstituted or substituted phenyl, the substituents are selected from halogen, OH, alkyl or alkoxy,

(3) C _3-6 cycloalkyl groups that are unsubstituted or substituted by independent substituents, the substituents are selected from halogen, OH, alkyl or alkoxy,

(4) Unsubstituted or substituted alkyl, substituents are selected from:

(a) halogen, or

(b) phenyl that is unsubstituted or substituted with 1-5 independent substituents selected from halogen, OH, alkyl or alkoxy.

In the compound of formula _I of the present invention, R is preferably substituted or unsubstituted aryl or heteroaryl, and the substituent is selected from alkyl, alkoxy, halogen, cyano, amino, hydroxyl, nitro or carbonyl;

R ₂ is preferably hydrogen, alkyl or alkoxy, more preferably hydrogen.

For example, in the compound of formula _I provided by the present invention, R is preferably selected from substituted or unsubstituted aryl or heteroaryl, the substituent is selected from alkyl, alkoxy or halogen, and R is selected from _hydrogen .

R ₁ is further preferably substituted or unsubstituted phenyl, quinazolinyl, benzimidazolyl, quinolinyl, pyrimidinyl, indolyl, quinoxalinyl, isoquinoline or azaphenanthrene.

For example R is selected from phenyl, ₄ -methylquinazolin-2-yl, benzimidazol-2-yl, quinolin-2-yl, 6-bromoquinolin-2-yl, 6-chloroquinolin-2 - Base, 6-fluoroquinolin-2-yl, 6-methylquinolin-2-yl, 7-chloroquinolin-2-yl, 7-fluoroquinolin-2-yl, 6-methoxyquinolin -2-yl, 4-chloroquinolin-2-yl, 3-methylquinoxalin-2-yl, quinolin-4-yl, quinolin-3-yl, pyrimidin-2-yl, indole-3 -yl, quinoxalin-2-yl, isoquinolin-1-yl or 1-azepine-2-yl, R ₂ is selected from hydrogen.

The term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms, which is connected to the rest of the molecule by a single bond, which usually has 1 to 6 carbon atoms. Also denoted is a C _1-6 alkyl group, preferably a C _1-4 alkyl group having 1-4 carbon atoms. The alkyl group may be unsubstituted or substituted with one or more substituents selected from alkyl, alkoxy, aryl, halogen, amino, hydroxyl, nitro or carboxyl, and the like. Non-limiting examples of unsubstituted alkyl groups include, but are not limited to, groups such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-methyl Butyl, neopentyl, n-hexyl, or 2-methylhexyl, etc.

The term "alkoxy" refers to an oxygen-containing moiety having an alkyl substitution, i.e. -O-alkyl group, usually consisting of oxygen and an alkyl group containing _1-6 carbon atoms, i.e. -OC1-6alkyl , preferably -OC _1-4 alkyl, specific examples include but are not limited to methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, n-pentyl oxy, 2-methylbutoxy, neopentyloxy, n-hexyloxy or 2-methylhexyloxy, etc.

The term "heterocycloalkyl" refers to a five-membered or six-membered substituted or unsubstituted monocyclic non-aromatic ring group with one or two heteroatoms such as nitrogen, oxygen, sulfur, etc., specific examples include but not Limited to piperazine, piperidine, tetrahydropyrrole or morpholine, etc., the substituents can be selected from cyano, halogen, hydroxyl, amino or amido, etc.

The term "aryl" refers to an all-carbon monocyclic or bicyclic carbocyclic aromatic ring system containing 6-10 carbon atoms, with a fully conjugated π-electron system, non-limiting examples of aryl are phenyl, biphenyl or naphthyl etc. The aryl group can be substituted or unsubstituted, and the substituents are selected from alkyl, alkoxy, aryl, halogen, amino, hydroxyl, nitro or carboxyl and the like.

The term "heteroaryl" refers to a monocyclic, bicyclic or tricyclic aryl group having 5-14 atoms, which contains at least one heteroatom selected from N, O or S, and the remaining atoms are C. The heteroaryl group may be substituted or unsubstituted, and the substituents are selected from alkyl, alkoxy, aryl, hydroxyl or amino, and the like. Non-limiting examples of unsubstituted heteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole, pyrazole, pyridine, pyrimidine, indole, quinoline, isoquinoline, oxazoline, benzimidazole, quinoxaline phenanthroline, azaphenanthrene or quinazoline, etc.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "amino" refers to a -NH2 group, -NH(alkyl) group or -N(alkyl) ₂ . Specific examples of amino include, but are not limited to, -NH ₂ , -NHCH ₃ , -N(CH ₃ ) ₂ , -NHC ₂ H ₅ or -N(C ₂ H ₅ ) ₂ and the like.

The term "sulfonylalkyl" refers to a straight or branched chain alkyl group containing 1 to 6 carbon atoms substituted by a sulfonyl group, such as sulfonylmethyl, sulfonylethyl or 1-sulfonyl-2-methyl The alkyl group can be substituted or unsubstituted, and the substituent is selected from alkyl, alkoxy, aryl, halogen, amino, hydroxyl, nitro or carboxyl, etc.

The term "cycloalkyl" refers to a saturated cyclic hydrocarbon containing 3-6 carbon atoms, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, cycloalkyl may be substituted or unsubstituted , The substituent is selected from alkyl, alkoxy, aryl, halogen, amino, hydroxyl, carboxyl, amido or cyano and the like.

The term "cycloalkylalkyl" refers to a straight or branched chain alkyl group containing 1 to 6 carbon atoms substituted by a cycloalkyl group, such as cyclopropylmethyl, cyclopentylethyl or 1-cyclohexyl- 3-Ethylbutyl etc.

The term "alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon group containing 2-6 carbon atoms and at least one double bond. The alkenyl group may be unsubstituted or substituted by a substituent selected from the group consisting of: Specific examples of unsubstituted alkenyl groups include, but are not limited to, vinyl, propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1-butenyl, Pentenyl or 1-hexenyl, etc.

The term "alkynyl" refers to a straight-chain or branched unsaturated hydrocarbon group containing 2-6 carbon atoms and at least one triple bond, and the alkynyl group may be unsubstituted or substituted by a substituent selected from the group consisting of: Specific examples of unsubstituted alkynyl include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl or 2-butane Alkynyl etc.

The term "carboxyl" refers to a straight-chain or branched group with 1-7 carbon atoms containing -COO-, including but not limited to HOOC-, CH ₃ OOC- or CH ₃ CH ₂ OOC-, etc.

The specific compounds provided by the present invention are exemplified as follows, but not limited to the following compounds or pharmaceutically acceptable salts thereof:

Another aspect of the present invention provides a preparation method for a compound of formula I, comprising the following steps:

(1) The compound of formula A and the compound of formula B react in an organic solvent to generate the compound of formula C in the presence of a base:

(2) Formula C compound reacts with R-3-aminopiperidine dihydrochloride to generate formula I compound:

Wherein, R ₁ and R ₂ are the same as defined in the compound of formula I above, and Hal is chlorine or bromine.

In step (1), the base is selected from sodium hydride, potassium carbonate or sodium carbonate; preferably sodium hydride; the organic solvent is selected from a mixed solvent of ethylene glycol dimethyl ether and N,N-dimethylformamide.

In a specific embodiment of the present invention, reaction method is exemplified as:

(1) Mix the base with a mixed solvent of ethylene glycol dimethyl ether and N,N-dimethylformamide, stir at 0°C for 10 minutes, add the compound of formula A, stir at 0°C for 20 minutes, add anhydrous lithium bromide , stirred at room temperature for 30 minutes, added the compound of formula B, and stirred overnight at room temperature. After the reaction was completed, crushed ice was added, extracted with dichloromethane, the obtained organic phase was washed with saturated brine, dried, concentrated, and purified by column chromatography to obtain the compound of formula C;

(2) Mix the compound of formula C and R-3-aminopiperidine dihydrochloride, add ethanol, mix well, add sodium bicarbonate, heat and reflux in an oil bath for 6 hours. After the reaction is complete, the reaction solution is concentrated, and the residue is purified by column chromatography to obtain the compound of formula I.

Wherein, the alkali described in step (1) is selected from sodium hydride, potassium carbonate or sodium carbonate; Preferred sodium hydride; Ethylene glycol dimethyl ether and N, N-dimethylformamide are anhydrous solvents, both The volume ratio is 2:1.

Wherein, the compound of formula A can be prepared by methods commonly used in the art. For example, when R is H, _formula A can be prepared from:

The concrete preparation method of formula A compound is exemplified as:

Add N,N-dimethylformamide (DMF) to 6-chlorouracil, add N,N-diisopropylethylamine (DIEA) after dissolving, stir well and add 1-bromo-2-butyne dropwise , and the reaction solution was stirred overnight at 25°C. After the reaction was complete, water was added to precipitate a solid, which was filtered with suction. The filter cake was washed with water and ether, and then dried to obtain the target product.

Wherein, the molar ratio of 6-chlorouracil to 1-bromo-2-butyne is 1:1.05-1:2, preferably 1:1.1; the reaction time is 3-36 hours, preferably 24 hours; the reaction temperature is 10-40°C , preferably 25°C.

Compounds of formula B can be purchased from the market or synthesized by methods commonly used in the art, non-limiting examples are as follows:

The specific reaction method is exemplified as: unsubstituted or substituted methyl compound is mixed with N-bromosuccinimide (NBS), then added azobisisobutyronitrile (AIBN), added carbon tetrachloride, and heated in an oil bath reaction. After the reaction was completed, the insoluble matter was filtered off, and the filtrate was spin-dried and purified by column chromatography to obtain the target compound.

Wherein, the molar ratio of unsubstituted or substituted methyl compound to N-bromosuccinimide is 1:1-1:2, preferably 1:1; the reaction time is 1-12 hours, preferably 6 hours; the reaction The temperature is 50-80°C, preferably 80°C. The molar ratio of unsubstituted or substituted methyl compound to azobisisobutyronitrile is 1:0.01-1:0.1, preferably 1:0.02.

In a specific embodiment of the present invention, the preparation method is exemplified as follows:

(1) The compound of formula A' and the compound of formula B react in the mixed solvent of ethylene glycol dimethyl ether and N,N-dimethylformamide in the presence of sodium hydride to generate the compound of formula C':

(2) Formula C' compound reacts with R-3-aminopiperidine dihydrochloride to generate formula I' compound:

Wherein, R ₁ is the same as defined in the compound of formula I above, and Hal is chlorine or bromine.

In a specific embodiment of the present invention, reaction method is exemplified as:

(1) Mix sodium hydride with a mixed solvent of ethylene glycol dimethyl ether/N,N-dimethylformamide, stir at 0°C for 10 minutes, add the compound of formula A', stir at 0°C for 20 minutes, add Lithium bromide in water, stirred at room temperature for 30 minutes, added the compound of formula B, stirred overnight at room temperature. After the reaction was completed, crushed ice was added, extracted with dichloromethane, the obtained organic phase was washed with saturated brine, dried, concentrated, and purified by column chromatography to obtain the compound of formula C'.

(2) Add ethanol after mixing the compound of formula C' and R-3-aminopiperidine dihydrochloride, add sodium bicarbonate after mixing evenly, and heat to reflux in an oil bath for 6 hours. After completion of the reaction, the reaction solution was concentrated, and the residue was purified by column chromatography to obtain the compound of formula I'.

Wherein, both ethylene glycol dimethyl ether and N,N-dimethylformamide are anhydrous solvents, and the volume ratio of the two is 2:1.

The uracil compound provided by the present invention can exist in its free or salt form. When the compound of the present invention has a free base form, the free base form of the compound is reacted with a pharmaceutically acceptable inorganic or organic acid to prepare the present invention. Acid addition salts of compounds of the invention including, but not limited to: hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, tosylate and benzenesulfonate salts, acetates, maleates, tartrates, succinates, citrates, benzoates, ascorbates and salicylates, malonates, adipates, caproates, Arginate, Fumarate, Niacinate, Phthalate, Oxalate, etc.

Yet another aspect of the present invention provides the use of the compound of formula I in the manufacture of a medicament for the treatment or prevention of diseases benefited from DPP-IV inhibition. The disease benefiting from DPP-IV inhibition is selected from the group consisting of type II diabetes, diabetic dyslipidemia, impaired glucose tolerance (IGT), decreased fasting plasma glucose (IFG), metabolic acidosis, ketosis, appetite Regulation, obesity, various cancers, neurological disorders, immune system disorders, etc., preferably include type II diabetes and obesity, more preferably type II diabetes.

Another aspect of the present invention provides a pharmaceutical composition, comprising the compound of formula I of the present invention or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients. The compositions of the present invention may be in liquid, semi-liquid or solid form, formulated in a manner suitable for the intended route of administration. The composition of the present invention can be administered according to the following administration methods: oral, parenteral, intraperitoneal, intravenous, transdermal, sublingual, intramuscular, rectal, buccal, intranasal, liposome and other methods.

Oral compositions may be solid, gel or liquid. Examples of solid formulations include, but are not limited to, tablets, capsules, granules, and bulk powders. These preparations may optionally contain binders, diluents, disintegrants, lubricants, glidants, sweeteners, flavoring agents and the like. Examples of binders include, but are not limited to, microcrystalline cellulose, dextrose solution, acacia mucilage, gelatin solution, sucrose, and starch paste; examples of lubricants include, but are not limited to, talc, starch, magnesium stearate, calcium stearate , stearic acid; examples of diluents include but not limited to lactose, sucrose, starch, mannitol, dicalcium phosphate; examples of glidants include but not limited to silicon dioxide; Sodium carboxymethylcellulose, sodium starch glycolate, alginic acid, corn starch, potato starch, methylcellulose, agar, and carboxymethylcellulose.

The composition of the present invention is administered parenterally, generally by injection, including subcutaneous, intramuscular or intravenous injection. Injectables can be prepared in any conventional form, such as liquid solutions or suspensions, solid forms suitable for solution in or suspension in liquid prior to injection, or emulsions. Examples of pharmaceutically acceptable carriers that can be used in the injection of the present invention include, but are not limited to, aqueous carriers, non-aqueous carriers, antimicrobial agents, isotonic agents, buffers, antioxidants, suspending and dispersing agents, emulsifying agents, and chelating agents and other pharmaceutically acceptable substances. Examples of aqueous vehicles include Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringer's Injection; examples of non-aqueous vehicles include fixed oils of vegetable origin, Cottonseed oil, corn oil, sesame oil, and peanut oil; examples of antimicrobial agents include m-cresol, benzyl alcohol, chlorobutanol, benzalkonium chloride, etc.; examples of isotonic agents include sodium chloride and dextrose; buffering agents include phosphate and citrate.

The composition of the present invention can also be prepared as sterile freeze-dried powder injection, the compound is dissolved in sodium phosphate buffer solution, which contains glucose or other suitable excipients, and then the solution is dissolved under standard conditions known to those skilled in the art. Sterile filtration followed by lyophilization yields the desired formulation.

The above-mentioned heterocyclic pyrimidinone compounds provided by the present invention have a simple preparation process, readily available raw materials, and are suitable for large-scale industrial production, and it has been verified by in vitro and in vivo experiments that the compound of the present invention has a very good selective inhibitory effect on DPP-IV. While effectively inhibiting the activity of DPP-IV, it has almost no effect on the activities of DPP-VIII and DPP-IX. It can be predicted that the compound of the present invention will have relatively low toxicity after being developed into a drug, and has a very good application prospect.

detailed description

The compounds provided by the present invention can be synthesized by various preparation methods, and the examples only provide representative methods for synthesizing these compounds. It should be noted here that no matter how the compounds of the present invention are developed, the free acid and/or base form, or the salt form, all belong to the scope of the present invention. The purpose of the specific examples is to further illustrate the content of the present invention but not to limit the present invention.

The initial raw materials and reaction reagents used in the specific examples of the present invention are all commercially available products.

The reagent abbreviations used in the present invention are expressions commonly used in the art, and the meanings are as follows:

DME: Ethylene glycol dimethyl ether

DMF: N,N-Dimethylformamide

DIEA: N,N-Diisopropylethylamine

Embodiment 1. Synthesis of Compound 4

Synthesis of Compound 4-0:

Add 6-chlorouracil (15g, 102.4mmol) and 250mL DMF into a 500mL eggplant-shaped bottle, add 15mL DIEA after dissolving, and dropwise add 1-bromo-2-butyne (9.85mL, 112.64mmol) after stirring, react The solution was stirred overnight at 25°C. After the reaction is complete, add ice water, filter with suction, wash the filter cake with water and ether, and then dry it to obtain the target product.

1H NMR (400MHz, CDCl ₃ ) δ8.85(s, 1H), 5.91(s, 1H), 4.75(d, J=2.0Hz, 2H), 1.82(t, J=2.4Hz, 3H).MS 199.82 [M+H]+.

Synthesis of Compound 4-1:

Under nitrogen protection, 60% NaH (54mg, 3.78mmol) was added to a 100mL eggplant-shaped flask, anhydrous DME/DMF (2:1) solution was added at 0°C, and after stirring at 0°C for 10 minutes, 1-(2- Butyne)-6-chlorouracil (500mg, 2.52mmol) in 10ml of anhydrous DME/DMF (2:1) solution. After the addition was completed, anhydrous lithium bromide (263mg, 3.02mmol) was added after stirring at 0°C for 20 minutes, and after stirring at room temperature for 30 minutes, 2-chloromethyl-4-methylquinazoline (534mg, 2.77mmol) was added, and the reaction solution was stirred at room temperature overnight. After the reaction was complete, crushed ice was added, extracted with dichloromethane, the combined organic phases were washed with saturated brine, the washed organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography (petroleum Ether/ethyl acetate = 1/1 elution, UV color development) purification to obtain compound 4-1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.03(d, J=8.0Hz, 1H), 7.89(d, J=8.0Hz, 1H), 7.80(t, J=7.2Hz, 1H), 7.55(t , J=8.0Hz, 1H), 6.06(s, 1H), 5.47(s, 2H), 4.83(d, J=2.0Hz, 2H), 2.90(s, 3H), 1.83(t, J=2.4Hz , 3H).MS 355.80[M+H] ⁺ .

Synthesis of compound 4:

Add compound 4-1 (306mg, 0.86mmol), sodium bicarbonate (361mg, 4.30mmol), R-3-aminopiperidine dihydrochloride (224mg, 1.29mmol) and 25mL ethanol, oil The bath was heated to reflux with stirring for 6 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol=15/1+1% ammonia water elution, UV color development) to obtain compound 4.

¹ H NMR (400MHz, CDCl ₃ ) δ7.94(d, J=8.0Hz, 1H), 7.81(d, J=8.4Hz, 1H), 7.70(t, J=7.2Hz, 1H), 7.45(t , J=7.2Hz, 1H), 5.41(s, 2H), 5.26(s, 1H), 4.52(d, J=1.6Hz, 2H), 3.33(m, 1H), 3.22(m, 1H), 3.00 (m, 1H), 2.81(s, 3H), 2.72(m, 1H), 2.50(m, 1H), 1.92(m, 1H), 1.82(m, 1H), 1.74(s, 3H), 1.64( m, 1H), 1.26(m, 1H). MS 419.50[M+H] ⁺ .

Embodiment 2. Synthesis of compound 3

Synthesis of compound 3-1:

Using benzyl bromide as a raw material, refer to the synthetic method of compound 4-1 in Example 1 to prepare compound 3-1.

¹ H NMR (400MHz, CDCl ₃ ) δ7.43-7.19(m, 5H), 5.97(s, 1H), 5.42(s, 2H), 4.60(s, 2H), 1.77(s, 3H).MS 289.07 [M+H] ⁺ .

Synthesis of compound 3:

Compound 3-1 obtained in the above step was used as a raw material, and with reference to the synthetic method of compound 4 in Example 1, compound 3.

¹ H NMR (400 MHz, CDCl ₃ ) δ7.46-7.22 (m, 5H), 5.33 (s, 2H), 5.20 (s, 1H), 4.40 (s, 2H), 3.22 (m, 3H), 2.96 ( m, 1H), 2.62(m, 1H), 2.42(m, 1H), 1.90(m, 1H), 1.77(s, 3H), 1.60(m, 1H), 1.22(m, 1H).MS 353.2[ M+H] ⁺ .

Embodiment 3. Synthesis of compound 5

Synthesis of Compound 5-1:

Using 2-chloromethylbenzimidazole as raw material, refer to the synthetic method of compound 4-1 in Example 1 to prepare compound 5-1.

¹ H NMR (400MHz, CDCl ₃ ) δ7.58(m, 2H), 7.23(m, 2H), 5.99(s, 1H), 5.40(s, 2H), 4.75(d, J=2.4Hz, 2H) , 1.79(t, J=2.4Hz, 3H). MS 329.10[M+H] ⁺ .

Synthesis of compound 5:

Compound 5-1 obtained in the above step was used as a raw material, and with reference to the synthetic method of compound 4 in Example 1, compound 5.

¹ H NMR (400MHz, CDCl ₃ ) δ7.47(m, 2H), 7.14(m, 2H), 5.31(s, 2H), 5.18(s, 1H), 4.40(s, 2H), 3.21(m, 3H), 2.95(m, 1H), 2.61(m, 1H), 2.43(m, 1H), 1.92(m, 1H), 1.75(s, 3H), 1.61(m, 1H), 1.21(m, 1H ).MS 393.45[M+H] ⁺ .

Embodiment 4. Synthesis of compound 6

Synthesis of Compound 6-1:

Taking 2-chloromethylquinoline as raw material, the synthetic method of compound 4-1 in the reference example 1 was prepared to obtain compound 6-1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.10(d, J=8.4Hz, 1H), 8.00(d, J=8.4Hz, 1H), 7.77(d, J=8.4Hz, 1H), 7.65(m , 1H), 7.48(m, 1H), 7.32(d, J=8.4Hz, 1H), 6.05(s, 1H), 5.43(s, 2H), 4.81(m, 2H), 1.82(t, J= 2.4Hz，3H).MS 340.90[M+H] ⁺ .

Synthesis of Compound 6:

Compound 6-1 obtained in the above step was used as a raw material, and with reference to the synthetic method of compound 4 in Example 1, compound 6.

¹ H NMR (400MHz, CDCl ₃ ) δ8.05(d, J=8.8Hz, 1H), 8.00(d, J=8.8Hz, 1H), 7.73(d, J=8.0Hz, 1H), 7.62(t , J=7.2Hz, 1H), 7.44(t, J=7.2Hz, 1H), 7.29(t, J=8.4Hz, 1H), 5.40(s, 2H), 5.28(s, 1H), 4.53(d , J=2.4Hz, 2H), 3.34(m, 1H), 3.23(m, 1H), 3.00(m, 1H), 2.72(m, 1H), 2.50(m, 1H), 1.95(m, 1H) , 1.77(m, 1H), 1.72(s, 3H), 1.66(m, 1H), 1.27(m, 1H). MS 404.49[M+H] ⁺ .

Embodiment 5. Synthesis of compound 7

Synthesis of compound 7-1:

Using 2-chloromethylpyrimidine as a raw material, refer to the synthetic method of compound 4-1 in Example 1 to prepare compound 7-1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.56(d, J=4.8Hz, 2H), 7.09(t, J=4.8Hz, 1H), 5.94(s, 1H), 5.28(s, 2H), 4.72 (m, 2H), 1.73 (t, J=2.4Hz, 3H). MS 291.78[M+H] ⁺ .

Synthesis of compound 7:

Compound 7-1 obtained in the above step was used as a raw material, and with reference to the synthetic method of compound 4 in Example 1, compound 7.

¹ H NMR (400MHz, CDCl ₃ ) δ8.59(d, J=4.8Hz, 2H), 7.09(t, J=4.8Hz, 1H), 5.32(s, 2H), 5.25(s, 1H), 4.51 (d, J=2.0Hz, 2H), 3.35(m, 1H), 3.24(m, 1H), 2.99(m, 1H), 2.72(m, 1H), 2.50(m, 1H), 1.95(m, 1H), 1.84(m, 1H), 1.76(s, 3H), 1.64(m, 1H), 1.28(m, 1H). MS 355.45[M+H] ⁺ .

Embodiment 6. Synthesis of compound 8

Synthesis of Compound 8-1:

^1H NMR (400MHz, CDCl ₃ ) 7.55 (m, 2H), 7.28 (m , 2H), 7.22(s, 1H), 5.97(s, 1H), 5.41(s, 2H), 4.74(d, J=2.4Hz, 2H), 1.80(t, J=2.4Hz, 3H).MS 328.08[M+H] ⁺ .

Synthesis of Compound 8:

Compound 8-1 obtained in the above step was used as a raw material, and compound 8.1 H NMR (400MHz, CDCl ₃ ) 7.46 (m, 2H), 7.22 (m, 2H) was prepared with reference to the synthesis method of compound 4 in Example ¹ . ), 7.20(s, 1H), 5.34(s, 2H), 5.19(s, 1H), 4.41(s, 2H), 3.25(m, 3H), 2.97(m, 1H), 2.61(m, 1H) , 2.42(m, 1H), 1.93(m, 1H), 1.73(s, 3H), 1.62(m, 1H), 1.25(m, 1H). MS 392.2[M+H] ⁺ .

Embodiment 7. Synthesis of compound 9

Synthesis of compound 9-1:

Using 2-bromomethylquinoxaline as raw material, the compound 9-1 was prepared with reference to the synthetic method of compound 4-1 in Example 1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.34(s, 1H), 8.07(m, 1H), 8.00(m, 1H), 7.71(m, 2H), 6.04(s, 1H), 5.47(s, 2H), 4.79(m, 2H), 1.81(t, J=2.4Hz, 3H). MS 341.78[M+H] ⁺ .

Synthesis of compound 9:

Compound 9-1 obtained in the above step was used as a raw material, and with reference to the synthetic method of compound 4 in Example 1, compound 9.

¹ H NMR (400MHz, CDCl ₃ ) δ8.77(s, 1H), 7.98(m, 2H), 7.64(m, 2H), 5.41(s, 2H), 5.25(s, 1H), 4.49(d, J=1.2Hz, 2H), 3.31(m, 1H), 3.20(m, 1H), 2.96(m, 1H), 2.68(m, 1H), 2.47(m, 1H), 1.91(m, 1H), 1.80(m, 1H), 1.74(s, 3H), 1.63(m, 1H), 1.23(m, 1H).MS 405.50[M+H] ⁺ .

Example 8. Synthesis of Compound 10

Synthesis of compound 10-1:

Using 4-bromomethylquinoline as raw material, with reference to the synthetic method of compound 4-1 in Example 1, compound 10-1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.82(d, J=4.8Hz, 1H), 8.18(d, J=8.4Hz, 1H), 8.13(d, J=8.4Hz, 1H), 7.73(m , 1H), 7.61(m, 1H), 7.16(d, J=4.4Hz, 1H), 6.06(s, 1H), 5.61(s, 2H), 4.79(m, 2H), 1.82(t, J= 2.4Hz，3H).MS 340.90[M+H] ⁺ .

Synthesis of Compound 10:

Compound 10-1 obtained in the above step was used as a raw material, and with reference to the synthetic method of compound 4 in Example 1, compound 10.

¹ H NMR (400MHz, CDCl ₃ ) δ8.78(d, J=4.4Hz, 1H), 8.19(d, J=8.4Hz, 1H), 8.09(d, J=8.0Hz, 1H), 7.69(t , J=7.2Hz, 1H), 7.57(t, J=7.2Hz, 1H), 7.16(d, J=4.4Hz, 1H), 5.58(s, 2H), 5.28(s, 1H), 4.52(d , J=2.0Hz, 2H), 3.33(m, 1H), 3.23(m, 1H), 2.99(m, 1H), 2.71(m, 1H), 2.51(m, 1H), 1.95(m, 1H) , 1.86(m, 1H), 1.78(s, 3H), 1.67(m, 1H), 1.88(m, 1H). MS 404.50[M+H] ⁺ .

Example 9. Synthesis of Compound 11

Synthesis of compound 11-1:

Using 3-bromomethylquinoline as raw material, with reference to the synthetic method of compound 4-1 in Example 1, compound 11-1 was prepared.

¹ H NMR (400MHz, CDCl ₃ ) δ9.04(d, J=2.0Hz, 1H), 8.25(d, J=1.2Hz, 1H), 8.06(d, J=8.4Hz, 1H), 7.76(t , J=8.0Hz, 1H), 7.66(m, 1H), 7.49(m, 1H), 6.00(s, 1H), 5.24(s, 2H), 4.72(d, J=2.4Hz, 2H), 1.77 (t, J=2.4Hz, 3H).MS 340.78[M+H] ⁺ .

Synthesis of Compound 11:

Compound 11-1 obtained in the above step was used as a raw material, and compound 11.1 was prepared with reference to the synthetic method of compound 4 in Example 1.

¹ H NMR (400MHz, CDCl ₃ ) δ9.05(d, J=2.0Hz, 1H), 8.28(d, J=1.6Hz, 1H), 8.03(d, J=8.4Hz, 1H), 7.76(t , J=8.0Hz, 1H), 7.64(m, 1H), 7.47(m, 1H), 5.24(s, 2H), 5.22(s, 1H), 4.48(d, J=1.6Hz, 2H), 3.29 (m, 1H), 3.18(m, 1H), 2.97(m, 1H), 2.66(m, 1H), 2.47(m, 1H), 1.94(m, 1H), 1.84(m, 1H), 1.77( s, 3H), 1.64(m, 1H), 1.27(m, 1H). MS 404.51[M+H] ⁺ .

Example 10. Synthesis of Compound 12

Synthesis of compound 12-1:

Using 1-bromomethylisoquinoline as raw material, with reference to the synthetic method of compound 4-1 in Example 1, compound 12-1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.34(d, J=5.6Hz, 1H), 8.15(d, J=8.4Hz, 1H), 7.83(d, J=8.0Hz, 1H), 7.69(m , 1H), 7.63(m, 1H), 7.52(d, J=6.0Hz, 1H), 6.06(s, 1H), 5.78(s, 2H), 4.82(m, 2H), 1.84(t, J= 2.4Hz，3H).MS 340.90[M+H] ⁺ .

Synthesis of Compound 12:

Compound 12-1 obtained in the above step was used as a raw material, and compound 12.1 was prepared with reference to the synthetic method of compound 4 in Example 1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.32(d, J=5.6Hz, 1H), 8.14(d, J=8.4Hz, 1H), 7.77(d, J=7.6Hz, 1H), 7.63(m , 1H), 7.58(m, 1H), 7.47(d, J=5.6Hz, 1H), 5.75(s, 2H), 5.30(s, 1H), 4.54(d, J=2.4Hz, 2H), 3.35 (m, 1H), 3.25(m, 1H), 2.98(m, 1H), 2.72(m, 1H), 2.49(m, 1H), 1.93(m, 1H), 1.83(m, 1H), 1.77( t, J=2.0Hz, 3H), 1.65(m, 1H), 1.26(m, 1H). MS 404.49[M+H] ⁺ .

Example 11. Synthesis of Compound 13

Synthesis of compound 13-1:

Using 2-bromomethyl-6-bromoquinoline as raw material, refer to the synthetic method of compound 4-1 in Example 1 to prepare compound 13-1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.01(d, J=8.8Hz, 1H), 7.93(d, J=2.0Hz, 1H), 7.86(d, J=8.8Hz, 1H), 7.71(m , 1H), 7.34(d, J=8.4Hz, 1H), 6.05(s, 1H), 5.40(s, 2H), 4.81(m, 2H), 1.82(t, J=2.4Hz, 3H).MS 419.77[M+H] ⁺ .

Synthesis of compound 13:

Compound 13-1 obtained in the above step was used as a raw material, and compound 13.1 was prepared with reference to the synthetic method of compound 4 in Example 1.

¹ H NMR (400MHz, CDCl ₃ ) δ7.95(d, J=8.8Hz, 1H), 7.87(m, 2H), 7.67(m, 1H), 7.31(d, J=8.4Hz, 1H), 5.37 (s, 2H), 5.28(s, 1H), 4.54(d, J=2.4Hz, 2H), 3.35(m, 1H), 3.24(m, 1H), 3.01(m, 1H), 2.72(m, 1H), 2.53(m, 1H), 1.96(m, 1H), 1.86(m, 1H), 1.77(t, J=2.4Hz, 3H), 1.69(m, 1H), 1.30(m, 1H). MS 483.48[M+H] ⁺ .

Example 12. Synthesis of Compound 14

Synthesis of compound 14-1:

Using 2-bromomethyl-6-chloroquinoline as raw material, refer to the synthetic method of compound 4-1 in Example 1 to prepare compound 14-1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.00(d, J=8.4Hz, 1H), 7.92(d, J=8.8Hz, 1H), 7.74(d, J=2.4Hz, 1H), 7.57(m , 1H), 7.34(d, J=8.4Hz, 1H), 6.04(s, 1H), 5.40(s, 2H), 4.80(m, 2H), 1.82(t, J=2.4Hz, 3H).MS 357.21[M+H] ⁺ .

Synthesis of Compound 14:

Compound 14-1 obtained in the above step was used as a raw material, and compound 14.1 was prepared with reference to the synthetic method of compound 4 in Example 1.

¹ H NMR (400MHz, CDCl ₃ ) δ7.95(d, J=8.8Hz, 1H), 7.91(d, J=8.8Hz, 1H), 7.70(d, J=2.4Hz, 1H), 7.53(m , 1H), 7.30(d, J=8.4Hz, 1H), 5.37(s, 2H), 5.28(s, 1H), 4.53(d, J=2.4Hz, 2H), 3.34(m, 1H), 3.23 (m, 1H), 3.00(m, 1H), 2.71(m, 1H), 2.52(m, 1H), 1.95(m, 1H), 1.85(m, 1H), 1.76(t, J=2.4Hz, 3H), 1.67(m, 1H), 1.29(m, 1H). MS438.80[M+H] ⁺ .

Example 13. Synthesis of Compound 15

Synthesis of compound 15-1:

Using 2-bromomethyl-6-fluoroquinoline as raw material, refer to the synthetic method of compound 4-1 in Example 1 to prepare compound 15-1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.04(d, J=8.4Hz, 1H), 7.99(dd, J=5.2Hz, 1H), 7.39(m, 3H), 6.05(s, 1H), 5.41 (s, 2H), 4.81 (m, 2H), 1.82 (t, J=2.0Hz, 3H). MS 358.78[M+H] ⁺ .

Synthesis of Compound 15:

Compound 15-1 obtained in the above step was used as a raw material, and compound 15.1 was prepared with reference to the synthetic method of compound 4 in Example 1.

¹ H NMR (400MHz, CDCl ₃ ) δ7.98(m, 2H), 7.35(m, 3H), 5.37(s, 2H), 5.28(s, 1H), 4.53(d, J=2.4Hz, 2H) , 3.34(m, 1H), 3.23(m, 1H), 3.01(m, 1H), 2.71(m, 1H), 2.51(m, 1H), 1.95(m, 1H), 1.85(m, 1H), 1.77(t, J=2.0Hz, 3H), 1.66(m, 1H), 1.28(m, 1H). MS 422.50[M+H] ⁺ .

Example 14. Synthesis of Compound 16

Synthesis of compound 16-1:

Using 2-bromomethyl-6-methylquinoline as raw material, refer to the synthetic method of compound 4-1 in Example 1 to prepare compound 16-1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.01(d, J=8.4Hz, 1H), 7.95(d, J=8.8Hz, 1H), 7.87(d, J=1.6Hz, 1H), 7.79(m , 1H), 7.26(d, J=8.4Hz, 1H), 5.99(s, 1H), 5.25(s, 2H), 4.72(m, 2H), 2.72(s, 3H), 1.80(t, J= 2.4Hz, 3H).MS 354.89[M+H] ⁺ .

Synthesis of compound 16:

Compound 16-1 obtained in the above step was used as a raw material, and compound 16.1 was prepared with reference to the synthetic method of compound 4 in Example 1.

¹ H NMR (400MHz, CDCl ₃ ) δ7.98(d, J=8.4Hz, 1H), 7.89(m, 2H), 7.81(m, 1H), 7.21(d, J=8.4Hz, 1H), 5.23 (m, 3H), 4.49(d, J=1.2Hz, 2H), 3.30(m, 1H), 3.19(m, 1H), 2.98(m, 1H), 2.68(s, 3H), 2.47(m, 1H), 1.94(m, 1H), 1.83(m, 1H), 1.78(t, J=2.4Hz, 3H), 1.64(m, 2H), 1.28(m, 1H). MS 418.66[M+H] ⁺ .

Example 15. Synthesis of Compound 17

Synthesis of compound 17-1:

Using 2-bromomethyl-6-methoxyquinoline as raw material, refer to the synthetic method of compound 4-1 in Example 1 to prepare compound 17-1.

¹ H NMR (400MHz, CDCl ₃ ) δ7.98(d, J=8.4Hz, 1H), 7.90(d, J=9.2Hz, 1H), 7.29(m, 2H), 7.03(d, J=2.8Hz , 1H), 6.04(s, 1H), 5.39(s, 2H), 4.80(m, 2H), 3.90(s, 3H), 1.82(t, J=2.4Hz, 3H).MS 370.89[M+H ] ⁺ .

Synthesis of compound 17:

Compound 17-1 obtained in the above step was used as a raw material, and compound 17.1 was prepared with reference to the synthetic method of compound 4 in Example 1.

¹ H NMR (400MHz, CDCl ₃ ) δ7.94(d, J=8.4Hz, 1H), 7.89(d, J=9.2Hz, 1H), 7.26(m, 2H), 7.00(d, J=2.4Hz , 1H), 5.37(s, 2H), 5.28(s, 1H), 4.53(d, J=1.6Hz, 2H), 3.86(s, 3H), 3.33(m, 1H), 3.23(m, 1H) , 3.01(m, 1H), 2.71(m, 1H), 2.52(m, 1H), 1.96(m, 1H), 1.85(m, 1H), 1.78(s, 3H), 1.67(m, 1H), 1.28(m,1H).MS 434.49[M+H] ⁺ .

Example 16. Synthesis of Compound 18

Synthesis of compound 18-1:

Using 2-bromomethyl-7-fluoroquinoline as raw material, refer to the synthetic method of compound 4-1 in Example 1 to prepare compound 18-1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.08(d, J=8.8Hz, 1H), 7.75(m, 1H), 7.61(m, 1H), 7.27(m, 2H), 6.05(s, 1H) , 5.41(s, 2H), 4.82(m, 2H), 1.83(t, J=2.4Hz, 3H). MS 358.78[M+H] ⁺ .

Synthesis of Compound 18:

Compound 18-1 obtained in the above step was used as a raw material, and compound 18.1 was prepared with reference to the synthetic method of compound 4 in Example 1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.05(d, J=8.8Hz, 1H), 7.73(m, 1H), 7.63(m, 1H), 7.25(m, 2H), 5.40(s, 2H) , 5.31(s, 1H), 4.56(d, J=1.6Hz, 2H), 3.37(m, 1H), 3.26(m, 1H), 3.04(m, 1H), 2.75(m, 1H), 2.55( m, 1H), 1.99(m, 1H), 1.88(m, 1H), 1.80(s, 3H), 1.74(m, 1H), 1.32(m, 1H). MS 422.50[M+H] ⁺ .

Example 17. Synthesis of Compound 19

Synthesis of compound 19-1:

Using 2-bromomethyl-7-chloroquinoline as raw material, refer to the synthetic method of compound 4-1 in Example 1 to prepare compound 19-1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.06(d, J=8.4Hz, 1H), 8.00(s, 1H), 7.69(d, J=8.8Hz, 1H), 7.42(m, 1H), 7.32 (d, J=8.4Hz, 1H), 6.04(s, 1H), 5.40(s, 2H), 4.81(m, 2H), 1.83(t, J=2.4Hz, 3H).MS 375.21[M+H ] ⁺ .

Synthesis of Compound 19:

Compound 19-1 obtained in the above step was used as a raw material, and compound 19.1 was prepared with reference to the synthesis method of compound 4 in Example 1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.03(d, J=8.4Hz, 1H), 7.99(s, 1H), 7.67(d, J=8.4Hz, 1H), 7.40(m, 1H), 7.30 (d, J=8.4Hz, 1H), 5.39(s, 2H), 5.30(s, 1H), 4.56(d, J=2.0Hz, 2H), 3.37(m, 1H), 3.26(m, 1H) , 3.04(m, 1H), 2.75(s, 3H), 2.55(m, 1H), 1.98(m, 1H), 1.88(m, 1H), 1.79(s, 3H), 1.73(m, 1H), 1.32(m,1H).MS 438.98[M+H] ⁺ .

Example 18. Synthesis of Compound 20

Synthesis of compound 20-1:

Using 2-chloromethyl-4-chloroquinoline as raw material, refer to the synthetic method of compound 4-1 in Example 1 to prepare compound 20-1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.17(d, J=8.0Hz, 1H), 8.02(d, J=8.4Hz, 1H), 7.70(m, 1H), 7.58(m, 1H), 7.41 (s, 1H), 6.05(s, 1H), 5.39(s, 2H), 4.81(m, 2H), 1.82(t, J=2.4Hz, 3H). MS 375.21[M+H] ⁺ .

Synthesis of compound 20:

Compound 20-1 obtained in the above step was used as a raw material, and compound 20.1 was prepared with reference to the synthetic method of compound 4 in Example 1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.13(d, J=8.4Hz, 1H), 8.02(d, J=8.4Hz, 1H), 7.68(t, J=7.6Hz, 1H), 7.55(t , J=7.6Hz, 1H), 7.38(s, 1H), 5.37(s, 2H), 5.30(s, 1H), 4.55(s, 2H), 3.37(m, 1H), 3.25(m, 1H) , 3.03(m, 1H), 2.74(m, 1H), 2.53(m, 1H), 1.97(m, 1H), 1.88(m, 1H), 1.79(s, 3H), 1.70(m, 1H), 1.30(m,1H).MS 438.90[M+H] ⁺ .

Example 19. Synthesis of Compound 21

Synthesis of Compound 21-1:

Using 2-chloromethyl-3-methylquinoxaline as raw material, refer to the synthetic method of compound 4-1 in Example 1 to prepare compound 21-1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.18(d, J=8.0Hz, 1H), 8.06(d, J=8.4Hz, 1H), 7.69(m, 1H), 7.57(m, 1H), 6.00 (s, 1H), 5.41(s, 2H), 4.82(m, 2H), 1.80(t, J=2.4Hz, 3H). MS 354.09[M+H] ⁺ .

Synthesis of Compound 21:

Compound 21-1 obtained in the above step was used as a raw material, and compound 21.1 was prepared with reference to the synthetic method of compound 4 in Example 1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.16(d, J=8.4Hz, 1H), 8.07(d, J=8.4Hz, 1H), 7.66(m, 1H), 7.55(m, 1H), 5.37 (s, 2H), 5.32(s, 1H), 4.58(s, 2H), 3.37(m, 1H), 3.25(m, 1H), 3.03(m, 1H), 2.74(m, 1H), 2.53( m, 1H), 2.43(s, 3H), 1.95(m, 1H), 1.86(m, 1H), 1.78(s, 3H), 1.73(m, 1H), 1.32(m, 1H).MS 419.2[ M+H] ⁺ .

Example 20. Synthesis of Compound 22

Synthesis of compound 22-1:

Using 1-aza-2-chloromethylphenanthrene as raw material, refer to the synthetic method of compound 4-1 in Example 1 to prepare compound 22-1.

¹ H NMR (400MHz, CDCl ₃ ) δ8.34(m, 1H), 8.21(m, 1H), 8.06(m, 1H), 7.88(m, 1H), 7.69(m, 2H), 7.57(m, 2H), 5.99(s, 1H), 5.38(s, 2H), 4.83(m, 2H), 1.79(t, J=2.4Hz, 3H). MS 390.10[M+H] ⁺ .

Synthesis of Compound 22:

Compound 22-1 obtained in the above step was used as a raw material, and compound 22.1 was prepared with reference to the synthesis method of compound 4 in Example 1.

¹ H NMR (400MHz, CDCl ₃ ) 8.35(m, 1H), 8.23(m, 1H), 8.08(m, 1H), 7.89(m, 1H), 7.70(m, 2H), 7.58(m, 2H) , 6, 00(s, 1H), 5.36(s, 2H), 4.81(m, 2H), 3.37(m, 1H), 3.25(m, 1H), 3.01(m, 1H), 2.76(m, 1H ), 2.53(m, 1H), 2.41(s, 3H), 1.94(m, 1H), 1.86(m, 1H), 1.79(s, 3H), 1.72(m, 1H), 1.31(m, 1H) .MS 454.2[M+H] ⁺ .

Example 21. In vitro activity test

The inhibitory rate of the compound provided by the present invention to DPP-IV can be determined by the homogeneous luminescence detection system of DPP-IV-Glo protease (DPP-IV-Glo Protease Assay, Promega cat#G8350). The system contains the DPP-IV substrate Gly-Pro-aminoluciferin and a buffer system for the detection of luciferase activity. After DPPIV-Glo is cut by DPP-IV, the luciferase reaction will be activated to produce "glow-type "Type luminescence signal, and then use Turner Veritas microplate luminescence photometer to detect the luminescence signal to characterize the activity of DPP-IV.

1. Purpose of the experiment

The inhibitory activity and selective inhibitory effect of the compounds of the present invention on DPP-IV enzyme were determined.

2. Experimental materials

Embodiment compound of the present invention;

DPP-IV enzyme, DPP-VIII enzyme, DPP-IX enzyme, GP-AMC (BioMol), black 96-well plate, super microplate reader;

Analysis buffer for DPP-IV and DPP-VIII: 100mmol/l Tris/HCl buffer, pH 8.0, 0.1mg/ml BSA;

DPP-IX analysis buffer: 100mmol/l Tris/HCl buffer, pH 7.4, 0.1mg/mlBSA.

3. Experimental method

a. Determination of enzyme activity:

Dilute GP-AMC in their respective buffers, the concentration is 100umol/L, 25ul per well; the enzyme is serially diluted, and the initial concentration is DPP-VIII, DPP-IX: 0.01ug/ul, DPP-IV: 0.01mU /ul, diluted by 5 times, 25ul per well, mixed well; 37°C, 360/460nm, measure the dynamic change of fluorescence value, measure for 30 minutes; use the concentration of enzyme whose absorbance rises linearly and S/B≥5.

b. Determination of inhibitor activity:

All enzymes, inhibitors, and GP-AMC were prepared with assay buffer, and no compound control and no enzyme solution control were set.

Prepare the enzyme solution according to the concentration of the enzyme, 25ul per well; serially dilute the inhibitor (10-fold or 5-fold dilution), 25ul per well, mix well; add 50ul of the diluted GP-AMC solution, mix well; react at 37°C for 20 Minutes, measure the fluorescence value at 360/460nm.

c. Data analysis: analyzed by GraphPad-Prism software.

4. Experimental results

The inhibitory activity data of compound 3-22 of the present invention on three enzymes are shown in Table 1 below.

Table 1 In vitro activity and selectivity data

While common DPP-IV inhibitors inhibit the activity of DPP-IV, they also inhibit the activity of some DPP-IV related enzymes, such as DPP-II, DPP-VIII, and DPP-IX, wherein DPP-VIII and DPP-IX are inhibited by Considered to be cytosolic enzymes, their inhibition may cause the toxic effects of some DPP-IV inhibitors, such as baldness, thrombocytopenia (syndrome), anemia, splenomegaly, and multi-tissue lesions. A more ideal state is to develop a compound that has a highly selective inhibitory effect on DPP-IV and has no effect on the activities of DPP-VIII and DPP-IX.

The experimental results show that the compounds of the examples of the present invention have a very good selective inhibitory effect on DPP-IV. While effectively inhibiting the activity of DPP-IV, they have almost no effect on the activities of DPP-VIII and DPP-IX. It is foreseeable that the compounds of the present invention After the compound is developed into a drug, the side effect is relatively low, and has very good application value.

Example 22. Inhibition of hERG in vitro

Noncardiac drugs may prolong the duration of myocardial action potentials by inhibiting hERG (IKr) channels, increasing the possibility of life-threatening torsades de pointes (TdP) ventricular arrhythmia. In this experiment, the HEK293 cell line without endogenous IKr current was used as the host cell, which is widely used in the detection of hERG.

The hERG cell line was routinely cultured and subcultured in DMEM medium containing 10% fetal bovine serum and 250g/ml G418. One culture dish was taken out for each experiment, washed twice with extracellular fluid, and placed on the stage of an inverted microscope. Whole-cell patch-clamp experiments were performed at room temperature, and the tip resistance of borosilicate glass microelectrodes used was 3-5 MΩ.

After forming the whole-cell recording mode, the membrane potential was clamped at -80mV, and the cells were stimulated with +50mV depolarization voltage every 30s, and then repolarized to -50mV for 2s, and then the hERG tail current was elicited. Before the depolarization voltage stimulation, the cells were given a repolarization voltage of -50mV for 50ms, and the current recorded at this voltage was used as the baseline for calculating the hERG tail current. hERG tail currents were recorded stably in extracellular fluid for at least 3 min prior to compound addition. The drug effect was considered to reach a steady state when the hERG tail current amplitude changed less than 5% after perfusion administration. If the current did not reach a steady state within 6 minutes, the detection of the compound at that concentration was also terminated.

The inhibitory rate of the test compound on the hERG current is calculated according to the following equation: % inhibitory rate={1-(current residual amplitude)/(control current amplitude)}*100

According to the above calculation method, the inhibitory rate of hERG current at multiple concentrations of the compound of the embodiment of the present invention was obtained, and the data were fitted using the logistic equation to obtain the IC ₅₀ value. The inhibition rate and IC ₅₀ data of the compounds of the examples of the present invention on hERG are shown in Table II.

Table 2. Inhibition of hERG in vitro

Compound name The highest detection concentration Highest Concentration Inhibition Rate _IC50 value standard deviation Compound 4 300μM 79.0±1.2% 114.2μM 7.3μM Compound 6 300μM 91.7±1.6% 47.9μM 4.5μM Compound 9 300μM 84.9±8.3% 62.7μM 9.2μM

In this study, the effect of compounds on hERG current was observed in HEK293 cells exogenously expressing hERG potassium channel. The experimental results showed that compound 4, compound 6 and compound 9 inhibited hERG weakly, suggesting that each compound could prolong the duration of myocardial action potential and increase life-threatening torsades de pointes (TdP) by inhibiting the hERG (IKr) channel. The possibility of arrhythmia is very low, that is, after the present invention is developed into a drug for treating diabetes, the cardiotoxicity will be low.

Claims (7)

1. A preparation method for a compound of formula I, comprising the steps of: (1) The compound of formula A and the compound of formula B react in an organic solvent to generate the compound of formula C in the presence of a base: (2) Formula C compound reacts with R-3-aminopiperidine dihydrochloride to generate formula I compound: Wherein, Hal is chlorine or bromine, R is selected from substituted or unsubstituted quinazolinyl, benzimidazolyl, quinolinyl, pyrimidinyl, indolyl, quinoxalinyl, isoquinoline or _{azaphenanthrene} ; substituents are selected from alkyl, Alkoxy, halogen; R2 is selected from _hydrogen . 2. the preparation method of formula I compound described in claim ₁ , wherein, R is selected from 4-methylquinazolin-2-yl, benzimidazol-2-yl, quinoline-2-yl, 6- Bromoquinolin-2-yl, 6-chloroquinolin-2-yl, 6-fluoroquinolin-2-yl, 6-methylquinolin-2-yl, 7-chloroquinolin-2-yl, 7-fluoro Quinolin-2-yl, 6-methoxyquinolin-2-yl, 4-chloroquinolin-2-yl, 3-methylquinoxalin-2-yl, quinolin-4-yl, quinoline- 3-yl, pyrimidin-2-yl, indol-3-yl, quinoxalin-2-yl, isoquinolin-1-yl or 1-azaphenanthrene-2-yl. 3. The preparation method of the compound of formula I described in any one of claims 1-2, wherein, in step (1), the base is selected from sodium hydride, potassium carbonate, sodium carbonate. 4. The preparation method of the compound of formula I described in any one of claims 1-2, wherein, in step (1), organic solvent is selected from the group consisting of ethylene glycol dimethyl ether and N,N-dimethylformamide Mixed solvents. 5. The preparation method of the compound of formula I described in any one of claims 1-2, comprising the steps of: (1) Mix the base with the mixed solvent of ethylene glycol dimethyl ether/N,N-dimethylformamide, stir at 0°C for 10 minutes, add the compound of formula A, stir at 0°C for 20 minutes, add anhydrous lithium bromide , stirred at room temperature for 30 minutes, added the compound of formula B, stirred overnight at room temperature; after the reaction was completed, added crushed ice, extracted with dichloromethane, the obtained organic phase was washed with saturated brine, dried, concentrated, and purified by column chromatography to obtain the compound of formula C ; (2) After mixing the compound of formula C and R-3-aminopiperidine dihydrochloride, add ethanol, add sodium bicarbonate after mixing evenly, and heat and reflux in an oil bath for 6 hours; after the reaction is completed, the reaction solution is concentrated, and the residue is washed Purify by column chromatography to obtain the compound of formula I. 6. The preparation method of the formula I compound described in claim 5, wherein, in step (1), alkali is selected from sodium hydride, potassium carbonate, sodium carbonate; Ethylene glycol dimethyl ether and N,N-dimethylformamide Both are anhydrous solvents, and the volume ratio of the two is 2:1. 7. A preparation method of a formula I' compound, comprising the steps of: (1) The compound of formula A' and the compound of formula B are reacted in the mixed solvent of ethylene glycol dimethyl ether and N,N-dimethylformamide in the presence of sodium hydride to generate the compound of formula C': (2) Formula C' compound reacts with R-3-aminopiperidine dihydrochloride to generate formula I' compound: Wherein, R ₁ is the same as defined in claim 1, and Hal is chlorine or bromine.