CN114671875A

CN114671875A - Novel dihydropyrimidine compound, isomer or salt, and preparation method and application thereof

Info

Publication number: CN114671875A
Application number: CN202210448077.XA
Authority: CN
Inventors: 曾燕群; 周广林; 朱绪成; 付海霞; 牟霞
Original assignee: Chengdu Shibeikang Biological Medicine Technology Co ltd
Current assignee: Chengdu Shibeikang Biological Medicine Technology Co ltd
Priority date: 2022-04-27
Filing date: 2022-04-27
Publication date: 2022-06-28
Also published as: CN114891003A; CN114891003B

Abstract

The invention discloses a compound shown in formula (I), or a stereoisomer, a geometric isomer, a tautomer, a nitrogen oxide compound, a hydrate, a solvent compound, a metabolite and a medicine of the compound shown in formula (I)An upper acceptable salt. The invention also provides application of the compound, the stereoisomer or the pharmaceutically acceptable salt thereof in preparing medicines for treating and/or preventing diseases related to the TRPA1 receptor, in particular application in preparing medicines for treating and/or preventing cough, asthma, pain and sleep apnea.

Description

Novel dihydropyrimidine compound, isomer or salt, and preparation method and application thereof

Technical Field

The invention relates to the field of pharmaceutical chemistry, in particular to application of dihydropyrimidine compounds or salts and isomers thereof, a preparation method thereof and a pharmaceutical composition thereof in preparation of medicines for treating and/or preventing diseases related to TRPA1 receptors, especially application in treatment and/or prevention of respiratory diseases.

Background

The Transient Receptor Potential (TRP) channel is a non-selective cation channel. TRP ion channels in mammals can be divided into 7 subfamilies based on TRP sequence homology, namely TRPC (7 members), TRPM (8 members), TRPV (6 members), TRPA (ANKTM 1, the only member), TRPML (3 members), TRPP (5 members) and TRPN. The TRP family is involved in a variety of cellular functions, including sensory perception and signal transduction. Among them, the TRPA1 receptor is associated with temperature, pain sensation, hyperalgesia and neurogenic inflammation.

TRPA1 is widely found in trigeminal nerve, dorsal root, nodose, and is expressed on primary sensory neurons of a δ and C fibers. Expression is also found in non-neuronal cells, such as inner ear hair cells, enterochromaffin cells, vascular endothelial cells, dental pulp fibroblasts, keratinocytes, islet cells, and the like. The channel can be activated by nociceptive cold stimulation at a temperature lower than 17 ℃, a series of chemical substance stimulation and inflammatory mediators, generates transmembrane voltage change mainly based on calcium ion influx, participates in cold sensation formation of noxious cold stimulation, and has the functions of regulating inflammatory response, apoptosis and necrosis and mediating pain. Recent studies have shown that receptors for TRPA1 are also "switches" for cough. Thus, activation of TRPA1 receptor has been associated with various diseases, such as pain, neuralgia, asthma, airway inflammation, bronchoconstriction and cough, showing significant therapeutic effects.

Among them, cough is one of the most common clinical symptoms. In 2006, 1087 college students in Guangzhou region of China investigated that the incidence of cough was 10.9%, with a chronic cough incidence of 3.3%, presumably higher for the community population. There is currently no approved drug for the treatment of chronic cough. Common antitussive therapeutic agents include codeine and dextromethorphan, but central antitussives often have side effects such as constipation and somnolence. Pain is one of the most common pains and the most common and unbearable symptoms in clinical. The incidence rate of the world pain is about 35-45%, and the incidence rate of the old people is higher, about 75% -90%. The therapeutic drugs mainly have two types, namely COX inhibitors (weak in analgesic effect and relatively high in safety), opioid receptor agonists (strong in analgesic effect, constipation, addiction and respiratory depression), and have advantages and disadvantages, and clinical requirements cannot be met. In addition, painful diabetic neuropathy occurs in about 16% of diabetic patients. The drugs used to treat painful DPN include tricyclic antidepressants, selective 5-hydroxytryptamine and norepinephrine reuptake inhibitors, opioids, and antiepileptics. And available treatment regimens are not completely effective in all patients, with more than 50% pain relief being achieved in only about one third of patients. Therefore, TRPA1 antagonists are potential therapeutic agents for a variety of diseases and there is a great unmet clinical need in the areas of pain, asthma, cough, etc.

TRPA1 antagonists currently only two varieties are clinically under investigation, clinical stage 2 ISC-17536 (diabetic peripheral neuropathy, pain, respiratory disease) and clinical stage 1 LY-3526318 (pain). The IC50 value of ISC-17536 was about 70 nM when it inhibited calcium current through TRPA 1. In the clinical study of intractable cough carried out in Europe, the terminal point is not reached finally, and the failure is ended, so that the antagonism IC50 of LY-3526318 on TRPA1 is 5-6 uM, and the activity is weak. Therefore, an antagonist with high activity to the TRPA1 is more clinically needed, a higher-activity and safer drug selection opportunity is provided for patients, and the development of the TRPA1 antagonist has great market value and academic value.

Disclosure of Invention

The compound is a novel dihydropyrimidine compound, and most of the compounds in the embodiment show good cough relieving effect and in-vitro affinity of TRPA1 in animals. In a mouse cough-relieving experiment, when 60mg/kg of the compound is orally administered, the compound has a very strong cough-relieving effect, and has statistical significance compared with a model group.

In one aspect, the present invention provides a compound of formula (i), a stereoisomer, or a pharmaceutically acceptable salt thereof:

wherein,

ring A is selected from a substituted or unsubstituted aromatic ring, or a substituted or unsubstituted aromatic heterocycle;

R¹selected from hydrogen, hydroxy, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkylamino, substituted or unsubstituted alkoxy, substituted or unsubstituted alkylthio, substituted or unsubstituted cyclic amino, substituted or unsubstituted aryloxy, or substituted or unsubstituted arylamino;

R²、R³independently hydrogen, deuterium, hydroxy, halogen, cyano, nitro, substituted or unsubstituted amino, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkoxy, C3-C6 cycloalkyl, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, - (CH)₂)fNRR’、—O-(CH₂) fNRR ', -C (= O) fNRR', or carboxyl, wherein:

f is an integer from 1 to 4;

each R is independently selected from hydrogen or lower alkyl;

each R' is independently selected from hydrogen, lower alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted aralkyl.

Further, the ring A is selected from a substituted or unsubstituted benzene ring, or a substituted or unsubstituted 5-to 6-membered aromatic heterocyclic ring.

Preferably, the ring A is selected from a substituted or unsubstituted 5-to 6-membered aromatic heterocyclic ring.

Still further, the above ring a is selected from: substituted or unsubstituted pyrimidine, substituted or substituted thiazole, substituted or unsubstituted isothiazole, substituted or unsubstituted oxazole, substituted or unsubstituted isoxazole, substituted or unsubstituted thiadiazole, substituted or unsubstituted oxadiazole, substituted or unsubstituted imidazole, substituted or unsubstituted pyrazole, substituted or unsubstituted thiophene, substituted or unsubstituted 1,2, 3-triazole, or substituted or unsubstituted 1,2, 4-triazole.

Preferably, the above ring a is selected from: a substituted or unsubstituted pyrimidine, a substituted or substituted thiazole, a substituted or unsubstituted isothiazole, a substituted or unsubstituted oxazole, a substituted or unsubstituted isoxazole, a substituted or unsubstituted thiadiazole, or a substituted or unsubstituted oxadiazole.

Further, the above R¹Selected from the group consisting of hydrogen, hydroxy, halogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkylamino, substituted or unsubstituted lower alkoxy, substituted or unsubstituted lower alkylthio, and substituted or unsubstituted 3-to 10-membered cyclic amino.

Further, the above R¹Selected from hydrogen, halogen, trifluoromethyl, trifluoromethoxy, difluoromethyl, difluoromethoxy, C1-C6 alkyl, or freely selected from the following rings:

wherein:

x is selected from: o, NH or CHR⁷；

Each R⁴、R⁵、R⁶、R⁷Independently selected from the group consisting of: hydrogen, halogen, hydroxy, amino, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted lower alkylamino, substituted or unsubstituted lower alkanoylamino, substituted or unsubstituted ester, C3-C6 cycloalkyl, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, or carboxy;

additionally or alternatively, two R's attached to the same ring carbon⁴Or two R⁵Or two R⁶The substituents may together form an oxo group (i.e.: O) or a C3-C7 spiro ring group; and additionally or alternatively, two R's attached to different ring carbons⁴Or two R⁵Or two R⁶The substituents may together form a ring, two of whichR is⁶When taken together, form a ring having 4 to 7 ring atoms, including 0 to 3 ring heteroatoms;

n is an integer from 0 to 4;

a is selected from an integer of 0 to 3;

b. c is independently an integer selected from 0 to 2;

m and p are independently selected from integers of 1 to 3;

q and r are independently selected from integers of 0 to 3.

Most preferably, R is as defined above¹Selected from hydrogen, halogen, trifluoromethyl, trifluoromethoxy, difluoromethyl, difluoromethoxy, C1-C6 alkyl, or selected from the group consisting of 1 to 2R⁸Substituted of the following rings:

wherein each R is⁸Independently selected from the group consisting of: hydrogen, halogen, hydroxy, amino, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted lower alkylamino, substituted or unsubstituted lower alkanoylamino, substituted or unsubstituted ester, C3-C6 cycloalkyl, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, or carboxy.

Further, the above R⁸Selected from: hydrogen, halogen, hydroxyl, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, substituted or unsubstituted aryl, C3-C6 cycloalkyl, C1-C3 perfluoroalkyl or C1-C3 perfluoroalkoxy.

Further, the above R⁸Selected from: hydrogen, halogen, C1-C4 alkyl, C1-C4 alkoxy, C1-C3 perfluoroalkyl or C1-C3 perfluoroalkoxy.

Further, the above R²、R³Independently selected from: hydrogen, deuterium, hydroxyl, halogen, cyano, nitro, amino, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C1-C3 perfluoroalkyl, or C1-C3 perfluoroalkoxy.

Go further forwardIn one step, the above R²、R³Independently selected from: hydroxyl, halogen, C1-C4 alkyl, C1-C4 alkoxy, C1-C3 perfluoroalkyl or C1-C3 perfluoroalkoxy.

The present invention provides a compound represented by formula (i), a stereoisomer or a pharmaceutically acceptable salt thereof:

wherein,

the ring A is selected from substituted or unsubstituted 5-6 membered aromatic heterocyclic ring;

R¹selected from the group consisting of hydrogen, hydroxy, halogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkylamino, substituted or unsubstituted lower alkoxy, substituted or unsubstituted lower alkylthio, substituted or unsubstituted 3 to 10 membered cyclic amino;

R²、R³independently hydrogen, deuterium, hydroxyl, halogen, cyano, nitro, substituted or unsubstituted amino, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, C3-C6 cyclic alkyl, C1-C3 perfluoroalkyl, or C1-C3 perfluoroalkoxy.

Further, the above compound, its stereoisomers or pharmaceutically acceptable salts thereof, has the structure defined as follows:

ring a is selected from substituted or unsubstituted pyrimidine, substituted or unsubstituted thiazole, substituted or unsubstituted isothiazole, substituted or unsubstituted oxazole, substituted or unsubstituted isoxazole, substituted or unsubstituted thiadiazole, substituted or unsubstituted oxadiazole, substituted or unsubstituted imidazole, substituted or unsubstituted pyrazole, substituted or unsubstituted thiophene, substituted or unsubstituted 1,2, 3-triazole, or substituted or unsubstituted 1,2,4 triazole; preferably, ring a is selected from substituted or unsubstituted pyrimidine, substituted or unsubstituted thiazole, substituted or unsubstituted isothiazole, substituted or unsubstituted oxazole, substituted or unsubstituted isoxazole, substituted or unsubstituted thiadiazole, or substituted or unsubstituted oxadiazole;

R¹selected from hydrogen, halogen, trifluoromethyl, trifluoromethoxy, difluoromethyl, difluoromethoxy, C1-C6 alkyl, or freely selected from the following rings:

wherein:

x is selected from: o, NH or CHR⁷；

Each R⁴、R⁵、R⁶、R⁷Independently selected from: hydrogen, halogen, hydroxy, amino, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted lower alkylamino, substituted or unsubstituted lower alkanoylamino, substituted or unsubstituted ester, C3-C6 cycloalkyl, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, or carboxy;

additionally or alternatively, two R's attached to the same ring carbon⁴Or two R⁵Or two R⁶The substituents may together form an oxo group (i.e.: O) or a C3-C7 spiro group; and additionally or alternatively, two R's attached to different ring carbons⁴Or two R⁵Or two R⁶The substituents may together form a ring, wherein two R' s⁶The rings when taken together have 4 to 7 ring atoms including 0 to 3 ring heteroatoms;

n is an integer from 0 to 4;

a is selected from an integer of 0 to 3;

b. c is independently an integer selected from 0 to 2;

m and p are independently selected from integers of 1 to 3;

q and r are independently selected from integers of 0 to 3;

R²、R³independently selected from: hydrogen, halogen, hydroxyl, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, substituted or unsubstituted aryl, C3-C6 cycloalkyl, C1-C3 perfluoroalkyl or C1-C3 perfluoroalkoxy; preferably, R²、R³Independently selected from: hydroxy, halogen, C1-C4 alkyl, C1-C4 alkoxy, C1EC3 perfluoroalkyl group or C1-C3 perfluoroalkoxy group.

Further, the above R¹Selected from hydrogen, halogen, trifluoromethyl, trifluoromethoxy, difluoromethyl, difluoromethoxy, C1-C6 alkyl, or selected from the group consisting of 1 to 2R⁸Substituted of the following rings:

Further, the above R⁸Selected from the group consisting of: hydrogen, halogen, hydroxyl, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, substituted or unsubstituted aryl, C3-C6 cycloalkyl, C1-C3 perfluoroalkyl or C1-C3 perfluoroalkoxy; preferably, R is as defined above⁸Selected from the group consisting of: hydrogen, halogen, C1-C4 alkyl, C1-C4 alkoxy, C1-C3 perfluoroalkyl or C1-C3 perfluoroalkoxy.

Further, the above substituents of ring a include, but are not limited to: hydrogen, halogen, hydroxy, amino, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted alkylamino, substituted or unsubstituted alkylamido, substituted or unsubstituted ester group, C3-C6 cyclic alkyl, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, or carboxy.

Further, the substituents of the above ring a are selected from: hydrogen, halogen, hydroxyl, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C3-C6 cycloalkyl, C1-C3 perfluoroalkyl or C1-C3 perfluoroalkoxy.

Further, compounds of formula (i) above, stereoisomers or pharmaceutically acceptable salts thereof, exemplary structures are as follows:

。

further, the compounds of the present invention also include geometric isomers, tautomers, nitroxides, hydrates, solvates, metabolites or prodrugs of the above compounds.

Further, the hydrogen in the above compounds, stereoisomers, tautomers, pharmaceutically acceptable salts, hydrates, solvates, nitrogen oxides, metabolites or prodrugs thereof, may be substituted with one or more deuterium.

In another aspect, the present invention provides a process for the preparation of a compound of formula (i), a stereoisomer or a pharmaceutically acceptable salt thereof, comprising, but not limited to the steps of:

wherein, ring A, R¹、R²、R³Is as defined in any one of the preceding paragraphs;

a, step a: adding a protective group into (R) -2-hydroxy methyl propionate to obtain (R) -2- ((trifluoromethyl) sulfonyl) oxypropionic acid methyl ester;

step b: (R) -2- ((trifluoromethyl) sulfonyl) oxypropionic acid methyl ester and theophylline are subjected to substitution reaction to obtain an intermediate

(S) -methyl 2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) propionate;

step c: the intermediate (S) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) methyl propionate is subjected to hydrolysis reaction to obtain a key intermediate b;

step d: and carrying out condensation reaction on the key intermediate b and the key intermediate a to obtain the compound shown in the formula I.

The invention also provides a pharmaceutical composition of the compound, the stereoisomer or the pharmaceutically acceptable salt thereof, and the composition further comprises pharmaceutically acceptable auxiliary materials.

In still another aspect, the present invention also provides a use of the above compound, a stereoisomer or a pharmaceutically acceptable salt thereof for the preparation of a medicament for preventing and/or treating a disease associated with the TRPA1 receptor.

Further, the above-mentioned diseases related to the TRPA1 receptor are respiratory diseases or nervous system diseases; preferably, the above-mentioned TRPA1 receptor-related disease is a respiratory disease.

Further, the above-mentioned diseases related to TRPA1 receptor are cough, asthma, pain or sleep apnea; preferably a cough.

Interpretation of terms:

the above-mentioned "alkyl group" includes straight chain and branched chain alkyl groups.

The above "lower alkyl" is: C1-C16 straight chain or branched chain alkyl.

The "for the alkyl moiety" in the above-mentioned "lower alkoxy", "lower alkylamino", "lower alkylthio", "lower alkanoylamino" is as defined above for the "lower alkyl".

The above-mentioned "C1-C6 alkyl group": refers to a linear or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, isohexyl, as examples.

The alkyl moiety of the "C1-C6 alkoxy group" and the "C1-C6 alkylamino group" is the same as the "C1-C6 alkyl group".

The "perfluoro group" in the above-mentioned "C1-C3 perfluoroalkyl group" means that all the hydrogens on the carbon atoms of the alkyl group are replaced with fluorine. Such as trifluoromethyl, -CF₂CF₃、—CFCF₃CF₃、—CF₂CF₂CF₃。

The "perfluoro" group in the above "C1-C3 perfluoroalkoxy group" is as defined above.

The "substituent" in the above "substituted or unsubstituted" is selected from the group consisting of: hydrogen, halogen, hydroxy, amino, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heterocycle, substituted or unsubstituted alkylamino, substituted or unsubstituted alkylamido, substituted or unsubstituted ester group, C3-C6 cycloalkyl, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, or carboxy; preferably, the "substituents" are selected from: hydrogen, halogen, hydroxyl, amino, substituted or unsubstituted C1-C6 alkyl.

The above-mentioned "C1 to C16" indicates that the carbon number is 1 to 16. Other similar writing methods are similarly explained.

The "3-to 10-membered cyclic amino group" is: a nitrogen-containing heterocycle having 3 to 10 ring atoms; the heterocyclic ring includes, but is not limited to, a monocyclic ring, a bridged ring, the number of heteroatoms is at least 1, the heteroatoms are all N, or comprise N and S and/or O. As part of the R1 substituent, the moiety is preferably a 5-to 8-membered cyclic amino group; more preferably 5-6 membered cyclic amino; most preferably a 5-membered cyclic amino group.

The above "halogen" is: fluorine, chlorine or bromine.

The above "pharmaceutically acceptable salts" include, but are not limited to, organic acid salts or inorganic acid salts; such acids include, but are not limited to, hydrochloric acid, sulfuric acid, benzenesulfonic acid, p-toluenesulfonic acid, 1, 5-naphthalenedisulfonic acid, trifluoroacetic acid, acetic acid, malic acid, tartaric acid, hydrobromic acid, and the like.

The above "solvate" includes, but is not limited to, organic solvents or inorganic solvents, including, but not limited to, methanol, ethanol, acetone, heptane, and the like.

The "hydrates" mentioned above include, but are not limited to, monohydrate, dihydrate, trihydrate and the like.

The above "nitroxide" includes, but is not limited to, any or at least one nitrogen atom on the parent nucleus that is oxidized to form a N → O bond.

The above "pharmaceutically acceptable excipients" include, but are not limited to, pharmaceutically acceptable additives including, but not limited to, fillers, disintegrants, lubricants, solubilizers, binders, diluents, glidants, and the like.

The above "pharmaceutical composition" includes but is not limited to active ingredients and pharmaceutically acceptable excipients, and is formulated into certain dosage forms by conventional preparation methods in the art, such as tablets, capsules, injections, microparticles, aerosols, ointments, and the like. Routes of administration include, but are not limited to, oral, intravenous, and the like.

Has the advantages that: compared with the prior art, the invention has better cough relieving effect and higher safety.

Detailed Description

The present invention will be described in further detail with reference to examples and experimental examples, which are provided for illustration of the technical solution of the present invention and are not intended to limit the present invention, and any equivalent replacement in the field made in accordance with the disclosure of the present invention is within the scope of the present invention.

The compounds of the present invention, stereoisomers or pharmaceutically acceptable salts thereof can be prepared by selecting the synthetic routes of the examples, and the conventional conditions of the reaction raw materials and the reaction solvent are adjusted according to the requirements of substituents or salt formation, which can be realized by those skilled in the art based on the present disclosure. In addition, the column chromatography of the present invention refers to silica gel column chromatography without specific description, and the elution solvent may be combined with a reaction solvent without specific description to determine a single or mixed elution solvent by common knowledge or common means of those skilled in the art.

The structure of the compound is nuclear magnetic resonance (¹H NMR) or liquid mass spectrometry (LC-MS).

The liquid mass spectrometer (LC-MS) is Agilent G6120B (used with liquid Agilent 1260); nuclear magnetic resonance apparatus (¹H NMR is Bruker AVANCE-400 or Bruker AVANCE-800, nuclear magnetic resonance: (¹H NMR) shifts (δ) Given in parts per million (ppm), the assay solvent is DMSO or CDCl₃Internal standard is Tetramethylsilane (TMS), and chemical shift is 10^-6(ppm) is given as a unit.

The term "room temperature" in the present invention means a temperature of 10 to 25 ℃.

Example 1: preparation of (S) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) -N- (5- (3-fluoro-4- (S) -2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-yl) propionamide:

the method comprises the following steps: preparation of methyl (2S) -2- (1-methyl-2, 6-dioxo-3, 4,5, 6-tetrahydro-1H-purin-7 (2H) -yl) propionate

A25 ml three-necked flask was charged with 1-methyl-3, 4,5, 7-tetrahydro-1H-purine-2, 6-dione (690mg, 4.15mmol) and K₂CO₃(0.573g, 4.15mmol) and DMF (7mL) were stirred and mixed well. Methyl (R) -2- (methylsulfonyloxy) propionate (0.58 g, 3.2 mmoL) was added and the reaction stirred at room temperature overnight, whereupon the reaction was quenched and then saturated NH was added₄Cl (20ml) quench. The resulting mixture was extracted with EA (3X 20 mL). The combined organic phases were washed with water (3X 50mL) and brine. Anhydrous Na for organic phase₂SO₄Dried and concentrated. The residue was purified by column separation (MeOH: DCM = 1: 100) and the product was collected and concentrated to dryness to give the title product as a white solid (500mg, 50%), yield 50%, purity 97.89%.

ESI-MS: m/z = 267.1(M+H)⁺。

Step two: preparation of (2S) -2- (1-methyl-2, 6-dioxo-3, 4,5, 6-tetrahydro-1H-purin-7 (2H) -yl) propionic acid

A25 mL reaction flask was charged with methyl (2S) -2- (1-methyl-2, 6-dioxo-3, 4,5, 6-tetrahydro-1H-purin-7 (2H) -yl) propanoate (0.35g, 1.31mmol), dioxane (4mL), 6N HCl (2 mL). The reaction was refluxed for 3h, cooled to room temperature, concentrated to dryness, added 3ml of water, stirred in an ice bath to precipitate a solid, filtered, and dried to a white solid product of 250mg, yield 75.4%, purity 97.39%.

ESI-MS:m/z=253(M+H)⁺。

Step three: preparation of (S) -3-fluoro-4- (2-methylpyrrolidin-1-yl) benzoic acid methyl ester

A50 ml reaction flask was charged with methyl 3, 4-difluorobenzoate (1 g, 5.81 mmol), (S) -2-methylpyrrolidine (0.54 g, 6.39 mmol), potassium carbonate (0.88 g, 6.39 mmol), and DMSO (10 ml), and heated to 80 ℃ for reaction for 5 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, water (15 ml) was added thereto, and EA (15 ml. times.2) was extracted twice. The organic phases were combined, washed twice (15 ml. times.2) with saturated NaCl solution, separated, and the organic phase was concentrated to dryness to give 1.3g of product in 94.2% yield with 97.50% purity.

ESI-MS:m/z=238.2(M+H)⁺。

Step four: preparation of (S) -3-fluoro-4- (2-methylpyrrolidin-1-yl) benzoic acid

To a 25ml reaction flask were added (S) -3-fluoro-4- (2-methylpyrrolidin-1-yl) benzoic acid methyl ester (1.3 g, 5.48 mmol) and lithium hydroxide monohydrate (0.46 g, 10.96 mmol) dissolved in 5ml water, and the mixture was added dropwise to the reaction flask and reacted at room temperature overnight. After the reaction is finished, adding water, EA and 10% citric acid to adjust the pH value to acidity, layering, drying the organic phase with anhydrous sodium sulfate, filtering, and concentrating to obtain 0.9g of a product, wherein the yield is 95.6%, and the purity is 97.90%.

ESI-MS:m/z=224.1(M+H)⁺。

Step five: preparation of (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine

A25 ml reaction flask was charged with (S) -3-fluoro-4- (2-methylpyrrolidin-1-yl) benzoic acid (0.5 g, 2.24 mmol), thiosemicarbazide (0.25 g, 2.69 mmol), and phosphorus oxychloride (4ml), and heated to 75 ℃ for reaction overnight. After the reaction is finished, concentrating the dry phosphorus oxychloride, and adding DCM and water. Adjusted to pH =8 with sodium hydroxide solution, the layers were separated, the organic phase was concentrated to dryness and purified by silica gel column chromatography (PE: EA =2:1 → 1: 2), the product was collected and concentrated to dryness to give 379mg of product with 60.9% yield and 98.87% purity.

ESI-MS:m/z=279.1(M+H)⁺。

Step six: preparation of (S) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) -N- (5- (3-fluoro-4- (S) -2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-yl) propionamide

A25 ml reaction flask was charged with (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine (155 mg, 0.62 mmol), (2S) -2- (1-methyl-2, 6-dioxo-3, 4,5, 6-tetrahydro-1H-purin-7 (2H) -yl) propionic acid (114 mg, 0.41 mmol), HOAT (112 mg, 0.82 mmol), and 5ml DCM, stirred and cooled to-10 deg.C, N-methylmorpholine (207 mg, 2.05 mmol), EDCI (157 mg, 0.82 mmol) were added, and the reaction was maintained at 0-10 deg.C for 2H. After the reaction, water was added, DCM was added for extraction, the organic phase was dried and concentrated to dryness, purified by silica gel column chromatography (PE: EA = 1: 1 → 1: 5), and the product was collected and concentrated to dryness to yield 170mg of yellow solid product with yield 80.9% and purity 99.10%.

ESI-MS:m/z=513.2(M+H)⁺。

¹H NMR (400 MHz, DMSO-d6) δ:13.13 (s, 1H), 8.33 (s, 1H), 7.60 – 7.56 (dd, 1H), 7.52– 7.49 (dd, 1H), 6.83 – 6.79 (t, 1H), 5.80 – 5.74 (q, 1H), 4.12 – 4.07 (m 1H), 3.59 – 3.53 (m, 1H), 3.46 (s, 3H), 3.30– 3.25 (m, 1H), 3.18(s, 3H), 2.13 – 2.04 (m, 1H), 2.02 – 1.92(m, 1H), 1.91 – 1.80 (m, 4H), 1.65 – 1.58 (m, 1H), 1.10 – 1.08(d, 3H)。

Example 2: preparation of (S) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) -N- (5-fluoro-4- (3-fluoro-4- (S) -2-methylpyrrolidin-1-yl) phenyl) thiazol-2-yl) propionamide

The method comprises the following steps: preparation of (S) -1- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) ethan-1-one

A25 ml reaction flask was charged with (S) -dimethylpyrrolidine (190 mg, 2.23 mmol), 3, 4-difluoroacetophenone (317 mg, 2.03 mmol), and potassium carbonate (309 mg, 2.23 mmol), and heated to 80 ℃ for reaction overnight. After the reaction is finished, the temperature is reduced to room temperature, water is added, EA is used for extraction, layering is carried out, drying and concentration are carried out till dryness, 400mg of yellow oily product is obtained, the yield is 88.9%, and the purity is 98.16%.

ESI-MS:m/z=222.2(M+H)⁺。

Step two: preparation of (S) -2-bromo-1- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) ethan-1-one

A25 ml reaction flask was charged with (S) -1- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) ethan-1-one (400 mg, 1.81 mmol), anhydrous THF (5 ml), acetic acid (1 ml) and stirred to cool to 0 ℃. Pyridinium tribromide (803 mg, 2.51 mmol) was added in portions, and the reaction was allowed to warm to room temperature for 5 h. After the reaction is finished, concentrating the reaction solution, removing THF, adding water, extracting with EA, drying the organic phase, and concentrating to dryness. Silica gel column purification (PE: EA = 15: 1 → 5: 1) and the product was collected and concentrated to dryness to give 370mg of the product as a yellow oil in 68.2% yield.

ESI-MS:m/z=301.2(M+H)⁺。

Step three: preparation of (S) -4- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) thiazol-2-amine

A25 ml reaction flask was charged with (S) -2-bromo-1- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) ethan-1-one (370 mg, 1.23 mmol), thiourea (112.6 mg, 1.48 mmol), ethanol (5 ml) and heated to 80 ℃ for 2 h. After the reaction is finished, concentrating to remove ethanol, adding water, extracting by EA, drying an organic phase, and concentrating to dryness. Silica gel column purification (PE: EA = 10: 1 → 2:1) and the product was collected and concentrated to dryness to give 300mg of the product as a yellow oil in 87.7% yield and 98.59% purity.

ESI-MS:m/z=278.2(M+H)⁺。

Preparation method the same as preparation example 1, substituting (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine in step six with an equimolar amount of (S) -4- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) thiazol-2-amine, gave the title compound as a light yellow solid in yield: 66.3 percent and the purity is 97.20 percent.

ESI-MS: m/z = 512.2(M+H)⁺。

¹HNMR (400 MHz, DMSO-d6) δ: 12.72 (s, 1H), 8.32 (s, 1H), 7.59 – 7.51 (m, 2H), 7.43 (s, 1H), 6.78 (t, 1H), 5.76 (d, 1H), 4.02 (d, 1H), 3.52 (d, 1H), 3.44 (s, 3H), 3.21 (d, 1H), 3.16 (s, 3H), 2.08 (dt, 1H), 1.94 (dt, 1H), 1.85 (d, 4H), 1.58 (dd, 1H), 1.06 (d, 3H)。

Example 3: preparation of (S) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) -N- (4- (3-fluoro-4- (S) -2-methylpyrrolidin-1-yl) phenyl) -5-methylthiazol-2-yl) propionamide

Preparation of intermediate (S) -4- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -5-methylthiazol-2-amine

The preparation method of the intermediate is the same as that of example 2, the 3, 4-difluoroacetophenone in the first step is replaced by the equimolar 3, 4-difluoropropiophenone, the second step is the same as the third step, the intermediate compound is obtained as yellow oil, and the three-step reaction yield is as follows: 43.9% and a purity of 98.11%.

ESI-MS: m/z = 292.2 (M+H)⁺。

The title compound was prepared according to the procedure for example 1 by substituting (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine in step six with an equimolar amount of (S) -4- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -5-methylthiazol-2-amine to give the title compound as a white solid in yield: 66.3 percent and the purity is 97.20 percent.

ESI-MS: m/z = 526.2 (M+H)⁺。

¹HNMR (400 MHz, DMSO-d6) δ:12.54 (s, 1H), 8.31 (s, 1H), 7.35–7.27 (m, 2H), 6.79 (t, 1H), 5.73 (q, 1H), 4.07–3.96 (m, 1H), 3.53 (d, 1H), 3.44 (s, 3H), 3.21 (d, 1H), 3.17 (s, 3H), 2.41 (s, 3H), 2.08 (dq, 1H), 1.95 (d, 1H), 1.83 (d, 4H), 1.59 (dt, 1H), 1.07 (d, 3H)。

Example 4: preparation of (S) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) -N- (2- (3-fluoro-4- (S) -2-methylpyrrolidin-1-yl) phenyl) pyrimidin-4-yl) propionamide

The method comprises the following steps: preparation of (S) -3-fluoro-4- (2-methylpyrrolidin-1-yl) benzimidazole

A25 mL three-necked flask was charged with 3, 4-difluorobenzamide hydrochloride (308 mg, 1.60 mmol), (S) -dimethylpyrrolidine (150 mg)，1.76mmol）、K₂CO₃(664mg, 4.81mmol), DMSO (5 ml), heated to 100 ℃ with stirring and reacted for 5 h. TLC monitors the reaction of the raw materials, the temperature is reduced to room temperature, water is added, EA is used for extraction, and the organic phase is concentrated to dryness. The concentrate was purified by column separation (MeOH: DCM =2: 100) and the product was collected and concentrated to dryness to give the title product 180mg, 51% yield and 98.50% purity.

ESI-MS: m/z = 222.1(M+H)⁺。

Step two: preparation of (S) -2- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) pyrimidin-4-amine

A25 ml three-necked flask was charged with (S) -3-fluoro-4- (2-methylpyrrolidin-1-yl) benzimidazole (130 mg, 0.59 mmol), (E) -3-ethoxyacrylonitrile (286 mg, 2.94 mmol), and DMSO (0.5 ml), heated to 130 ℃ with stirring for 24h, and most of the starting materials were reacted by TLC. The reaction was cooled to room temperature, water was added, DCM was added and the organic phase was concentrated to dryness. The concentrate was purified by column separation (PE: EA =2:1), and the product was collected and concentrated to dryness to give the title product 70mg, yield 44%, purity 96.89%.

ESI-MS: m/z = 273.1(M+H)⁺。

The title compound was prepared according to the procedure for example 1 by substituting (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine in step six with an equimolar amount of (S) -2- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) pyrimidin-4-amine to give the title compound as a white solid in yield: 51.3 percent and the purity is 97.50 percent.

ESI-MS: m/z = 507.2(M+H)⁺。

¹H NMR (400 MHz, DMSO-d6) δ: 12.35 (s, 1H), 8.57 (d, 1H), 8.22 (d, 1H), 7.80 (dd, 1H), 7.72 (dd, 1H), 7.47 (d, 1H), 7.06 (dd, 1H), 5.76 (d, 1H), 3.71–3.60 (m, 2H), 3.46 (s, 4H), 3.38 (s, 3H), 1.96–1.82 (m, 1H), 1.87–1.72 (m, 2H), 1.67 (d, 3H), 1.56–1.44 (m, 1H), 1.22 (d, 3H)。

Example 5: (S) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) -N

Preparation of (5- (3-fluoro-4- (S) -2- (trifluoromethyl) pyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-yl) propionamide

Preparation of intermediate (S) -5- (3-fluoro-4- (2- (trifluoromethyl) pyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine

The preparation method was the same as that of example 1 except that (S) -dimethylpyrrolidine in step three was replaced with equimolar (S) -2- (trifluoromethyl) pyrrolidine, and steps four and five were the same, to give an intermediate compound.

ESI-MS: m/z = 333.1 (M+H)⁺。

The title compound was prepared according to the procedure for example 1 by replacing (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine in step six with an equimolar amount of (S) -5- (3-fluoro-4- (2- (trifluoromethyl) pyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine to give the title compound as a light yellow solid in yield: 45.3 percent and the purity is 96.30 percent.

ESI-MS: m/z = 567.2(M+H)⁺。

¹H NMR (400 MHz, DMSO-d6)δ:12.07 (s, 1H), 8.22 (d, 1H), 7.66 (dd, 1H), 7.49 (dd, 1H), 7.04 (dd, 1H), 5.73 (q, 1H), 4.33 (m, 1H), 3.79–3.71 (m, 1H), 3.71–3.62 (m, 1H), 3.46 (s, 3H), 3.38 (s, 3H), 2.10–1.82 (m, 4H), 1.67 (d, 3H)。

Example 6: preparation of (S) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) -N- (4- (3-fluoro-4- (S) -2- (trifluoromethyl) pyrrolidin-1-yl) phenyl) -5-methylthiazol-2-yl) propionamide

Preparation of intermediate (S) -4- (3-fluoro-4- (2- (trifluoromethyl) pyrrolidin-1-yl) phenyl) -5-methylthiazol-2-amine

The preparation method is the same as that of example 2, in the first step, (S) -dimethylpyrrolidine is replaced by equimolar (S) -2- (trifluoromethyl) pyrrolidine, 3, 4-difluoroacetophenone is replaced by equimolar 3, 4-difluoropropiophenone, the second step is the same as the third step, an intermediate compound is obtained, and the reaction yield in the third step is as follows: 33.5 percent.

ESI-MS: m/z = 346.1 (M+H)⁺。

The title compound was prepared according to the procedure for example 1 by replacing in step six (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine with an equimolar amount of (S) -4- (3-fluoro-4- (2- (trifluoromethyl) pyrrolidin-1-yl) phenyl) -5-methylthiazol-2-amine to give the title compound as a light yellow solid in yield: 52.3 percent and the purity is 97.80 percent.

ESI-MS: m/z = 580.2(M+H)⁺。

¹H NMR(400 MHz, DMSO-d6)δ: 12.30 (s, 1H), 8.32 (s, 1H), 7.50 (dd, 1H), 7.38 (dd, 1H), 7.00 (dd, 1H), 5.73 (q, 1H), 4.33 (m, 1H), 3.79–3.71 (m, 1H), 3.71–3.62 (m, 1H), 3.46 (s, 3H), 3.38 (s, 3H), 2.48 (s, 3H), 2.10–1.82 (m, 4H), 1.67 (d, 3H)。

Example 7: preparation of (S) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) -N- (5- (3-fluoro-4- (S) -2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-oxadiazol-2-yl) propionamide

Preparation of intermediate (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-oxadiazole-2-amine

The preparation method was the same as that of example 1, and steps three and four were the same, and thiosemicarbazide was replaced with an equimolar amount of thiosemicarbazide in step five to obtain an intermediate compound, and the yield was obtained in three steps: 17.9 percent.

ESI-MS: m/z = 263.1 (M+H)⁺。

The title compound was prepared according to the procedure for example 1 by replacing in step six (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine with an equimolar amount of (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-oxadiazol-2-amine to give the title compound as a white solid in yield: 60.5 percent and the purity of 98.30 percent.

ESI-MS: m/z = 497.2(M+H)⁺。

¹H NMR (400 MHz, DMSO-d6) δ: 12.42 (s, 1H), 8.32 (s, 1H), 7.59 – 7.51 (m, 2H), 6.78 (t, 1H), 5.76 (d, 1H), 4.02 (d, 1H), 3.52 (d, 1H), 3.44 (s, 3H), 3.21 (d, 1H), 3.16 (s, 3H), 2.08 (m, 1H), 1.94 (dt, 1H), 1.85 (d, 4H), 1.58 (dd, 1H), 1.06 (d, 3H)。

Example 8: preparation of (S) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) -N- (4- (3-fluoro-4- (S) -2-methylpyrrolidin-1-yl) phenyl) -5-methyloxazol-2-yl) propionamide

Preparation of intermediate (S) -4- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -5-methyloxazol-2-amine

The preparation method is the same as that of example 2,3, 4-difluoroacetophenone in the first step is replaced by equimolar 3, 4-difluoropropiophenone, the second step is the same, thiourea in the third step is replaced by equimolar urea, an intermediate compound is obtained, and the reaction yield in the three steps is as follows: 56.9 percent.

ESI-MS: m/z = 276.1 (M+H)⁺。

The title compound was prepared by the same method as example 1 except substituting (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine in step six with an equimolar amount of (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-oxadiazol-2-amine to give the title compound as a white solid in yield: 76.3 percent and the purity of 98.50 percent.

ESI-MS: m/z = 510.2(M+H)⁺。

¹H NMR (400 MHz, DMSO-d6) δ:12.64 (s, 1H), 8.31 (s, 1H), 7.35–7.27 (m, 2H), 6.79 (t, 1H), 5.73 (q, 1H), 4.07–3.96 (m, 1H), 3.53 (d, 1H), 3.44 (s, 3H), 3.21 (d, 1H), 3.17 (s, 3H), 2.51 (s, 3H), 2.08 (dq, 1H), 1.95 (d, 1H), 1.83 (d, 4H), 1.59 (dt, 1H), 1.07 (d, 3H)。

Example 9: preparation of (S) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) -N- (4- (3-fluoro-4- (S) -2-methylpyrrolidin-1-yl) phenyl) thiazol-2-yl) propionamide

Preparation of intermediate (S) -5-fluoro-4- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) thiazol-2-amine

A10 ml reaction flask is added with (S) -4- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) thiazol-2-amine (250 mg, 0.89 mmol) and acetonitrile (2.5 ml), cooled to-25 ℃, and added with Selectfluor (349 mg, 0.98 mmol), TLC monitors until the raw materials are reacted completely, water is added, DCM is added for extraction, layers are separated, and the organic phase is concentrated to dryness. Silica gel column purification (PE: EA = 8: 1 → 1: 1) and product was collected and concentrated to dryness to give 100mg of product as a yellow oil in 37.6% yield.

ESI-MS:m/z=296.1(M+H)⁺。

The title compound was prepared according to the procedure for example 1 by substituting (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine in step six with an equimolar amount of (S) -5-fluoro-4- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) thiazol-2-amine to give the title compound as a yellow solid in yield: 57.6 percent and the purity of 98.80 percent.

ESI-MS: m/z = 530.2 (M+H)⁺。

¹H NMR (400 MHz, DMSO-d6) δ: 12.86 (s, 1H), 8.32 (s, 1H), 7.59–7.51 (m, 2H), 6.78 (t, 1H), 5.76 (d, 1H), 4.02 (d, 1H), 3.52 (d, 1H), 3.44 (s, 3H), 3.21 (d, 1H), 3.16 (s, 3H), 2.08 (dt, 1H), 1.94 (dt, 1H), 1.85 (d, 4H), 1.58 (dd, 1H), 1.06 (d, 3H)。

Example 10: preparation of (S) -N- (4- (2, 4-difluoro-3- (trifluoromethyl) phenyl) thiazol-2-yl) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) propionamide

Preparation of intermediate 4- (2, 4-difluoro-3- (trifluoromethyl) phenyl) thiazol-2-amine

The preparation method was the same as that of example 2, except that (S) -1- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) ethan-1-one in the second step was replaced with equimolar 1- (2, 4-difluoro-3- (trifluoromethyl) phenyl) ethan-1-one, and the procedure was the same as in the third step, to obtain an intermediate compound as a reddish brown solid, with two-step reaction yield: 65.9 percent.

ESI-MS: m/z = 281.0 (M+H)⁺。

The title compound was prepared according to the procedure for example 1 by substituting (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine in step six with an equimolar amount of 4- (2, 4-difluoro-3- (trifluoromethyl) phenyl) thiazol-2-amine to give the title compound as a white solid in yield: 60.3 percent and the purity is 96.90 percent.

ESI-MS:m/z=515.1(M+H)⁺。

¹H NMR (400 MHz, DMSO-d6) δ: 12.87 (s, 1H), 8.32 (d, 2H), 7.67 (d, 1H), 7.50 (t, 1H), 5.78 (q, 1H), 3.44 (s, 3H), 3.16 (s, 3H), 1.86 (d, 3H)。

Example 11: preparation of (S) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) -N- (4- (3-fluoro-4- (piperidin-1-yl) phenyl) thiazol-2-yl) propionamide

Preparation of intermediate 4- (3-fluoro-4- (piperidin-1-yl) phenyl) thiazol-2-amine

The preparation method was the same as that of example 2, except that (S) -dimethylpyrrolidine was replaced with an equimolar piperidine hydrochloride in the first step, and that in the second step and the third step, a yellow oily intermediate compound was obtained, in three-step reaction yield: 52.4 percent and the purity of 98.90 percent.

ESI-MS: m/z =278.1 (M+H)⁺。

The title compound was prepared according to the procedure for example 1 by substituting (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine in step six with an equimolar amount of 4- (3-fluoro-4- (piperidin-1-yl) phenyl) thiazol-2-amine to give the title compound as a white solid in yield: 66.3 percent and the purity is 97.20 percent.

ESI-MS: m/z = 512.6 (M+H)⁺。

¹H NMR (400 MHz, DMSO-d6)δ: 12.72 (s, 1H), 8.32 (s, 1H), 7.59–7.51 (m, 2H), 7.43 (s, 1H), 6.78 (t, 1H), 5.76 (d, 1H), 3.46 (s, 3H), 3.42–3.36 (m, 6H), 1.77 – 1.61 (m, 8H), 1.66–1.56 (m, 2H)。

Example 12: preparation of (S) -N- (4- (3, 5-difluoro-4- (S) -2-methylpyrrolidin-1-yl) -5-methylthiazol-2-yl) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) -propionamide

Preparation of intermediate (S) -4- (3, 5-difluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -5-methylthiazol-2-amine

The preparation method is the same as that of example 2, the 3, 4-difluoroacetophenone in the first step is replaced by equimolar 3 ', 4 ', 5 ' -trifluoropropiophenone, the second step is the same as the third step, a yellow oily intermediate compound is obtained, and the three-step reaction yield is as follows: 16.3% and the purity is 96.90%.

ESI-MS: m/z = 310.1 (M+H)⁺。

The title compound was prepared according to the procedure for example 1 by substituting (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine in step six with an equimolar amount of (S) -4- (3, 5-difluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -5-methylthiazol-2-amine to give the title compound as a white solid in yield: 49.3 percent and the purity is 97.60 percent.

ESI-MS: m/z = 544.2 (M+H)⁺。

¹H NMR(400 MHz, DMSO-d6)δ:12.84 (s, 1H), 8.31 (s, 1H), 7.35–7.27 (m, 2H), 5.73 (q, 1H), 4.07–3.96 (m, 1H), 3.53 (d, 1H), 3.44 (s, 3H), 3.21 (d, 1H), 3.17 (s, 3H), 2.41 (s, 3H), 2.08 (dq, 1H), 1.95 (d, 1H), 1.83 (d, 4H), 1.59 (dt, 1H), 1.07 (d, 3H)。

Example 13: preparation of (2S) -N- (4- (4- (2, 5-diazabicyclo [2.2.1] hept-2-yl) -3-fluorophenyl) thiazol-2-yl) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) -propionamide

Preparation of intermediate tert-butyl 5- (4- (2-aminothiazol-4-yl) -2-fluorophenyl) -2, 5-diazacyclo [2.2.1] heptane-2-carboxylate

The intermediate was prepared in the same manner as in example 2 by replacing (S) -dimethylpyrrolidine with equimolar tert-butyl 2, 5-diazacyclo [2.2.1] heptane-2-carboxylate in step one, and in step two in the same manner as in step three, to give a yellow oily intermediate compound, with a total yield of three steps: 46.1% and the purity is 99.20%.

ESI-MS: m/z = 391.2(M+H)⁺。

The title compound was prepared by the same method as in example 1 except that in step six, (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazole-2-amine was replaced with equimolar amounts of tert-butyl 5- (4- (2-aminothiazol-4-yl) -2-fluorophenyl) -2, 5-diazacyclo [2.2.1] heptane-2-carboxylate to give tert-butyl 5- (4- (2- ((S) -2- (1, 3-dimethyl-2, 6-dioxy-1, 2,3, 6-tetrahydro-7H-purin-7-yl) propionamide) thiazol-4-yl) -2-fluorophenyl) -2, 5-diazacyclo [2.2.1] heptane-2-carboxylate, Boc protection removal with trifluoroacetic acid gave the title compound in two-step yield: 32.9% and the purity is 97.60%.

ESI-MS: m/z = 525.2 (M+H)⁺。

¹H NMR(400 MHz, DMSO-d6) δ:12.72 (s, 1H), 8.32 (s, 1H), 7.59–7.51 (m, 2H), 7.43 (s, 1H), 6.78 (t, 1H), 5.76 (d, 1H), 4.04 (dd, 1H), 3.99 (tt, 1H), 3.68 (dd, 1H), 3.51 (m, 1H), 3.46 (s, 3H), 3.38 (s, 3H), 3.16–3.08 (m, 1H), 3.06–2.97 (m, 1H), 2.15 (dt, 1H), 2.06–1.97 (m, 1H), 1.84–1.75 (m, 1H), 1.67 (d, 3H)。

Example 14: preparation of (2S) -N- (4- (4- (3, 8-diazabicyclo [3.2.1] octan-8-yl) -3-fluorophenyl) thiazol-2-yl) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) propionamide

Preparation of intermediate tert-butyl 8- (4- (2-aminothiazol-4-yl) -2-fluorophenyl) -3, 8-diazacyclo [3.2.1] octane-3-carboxylate

The intermediate preparation method was the same as that of example 2, except that (S) -dimethylpyrrolidine was replaced with equimolar 3, 8-diazacyclo [3.2.1] octane-3-carboxylic acid tert-butyl ester in the first step, and that in the second step and the third step, a yellow oily intermediate compound was obtained, with three-step reaction yield: 39.7% and the purity is 98.64%.

ESI-MS: m/z = 405.2(M+H)⁺。

The title compound was prepared by the same method as in example 1 except that in step six, (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazole-2-amine was replaced with equimolar tert-butyl 8- (4- (2-aminothiazol-4-yl) -2-fluorophenyl) -3, 8-diazacyclo [3.2.1] octane-3-carboxylate to give tert-butyl 8- (4- (2- ((S) -2- (1, 3-dimethyl-2, 6-dioxy-1, 2,3, 6-tetrahydro-7H-purin-7-yl) propionamide) thiazol-4-yl) -2-fluorophenyl) -3, 8-diazacyclo [3.2.1] octane-3-carboxylate, Boc protecting group was removed with trifluoroacetic acid to give the title compound in two-step yield: 29.6 percent and the purity of 98.80 percent.

ESI-MS: m/z = 539.2 (M+H)⁺。

¹H NMR(400 MHz, DMSO-d6) δ:12.65 (s, 1H), 8.32 (s, 1H), 7.59–7.51 (m, 2H), 7.43 (s, 1H), 6.78 (t, 1H), 5.76 (d, 1H), 3.89 (m, 2H), 3.46 (s, 3H), 3.38 (s, 3H), 3.01 (m, 2H), 2.90 (m, 2H), 2.78 (q, 1H), 2.02–1.87 (m, 2H), 1.86–1.71 (m, 2H), 1.67 (d, 3H)。

Example 15: preparation of (S) -N- (4- (4- (4-aza-1-yl) -3-fluorophenyl) thiazol-2-yl) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) propionamide

Preparation of intermediate 4- (4- (aza ring-1-yl) -3-fluorophenyl) thiazole-2-amine

The preparation method is the same as that of example 2, the (S) -dimethylpyrrolidine in the first step is replaced by heptamethine with the same mole, the second step is the same as the third step, and the intermediate compound is obtained as yellow oil, and the yield is obtained in three steps: 57.5 percent.

ESI-MS: m/z =292.1(M+H)⁺。

The title compound was prepared according to the procedure for example 1 by substituting (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine in step six with an equimolar amount of 4- (4- (azepin-1-yl) -3-fluorophenyl) thiazol-2-amine to give the title compound as a white solid in yield: 66.3 percent and the purity is 97.20 percent.

ESI-MS: m/z = 526.2 (M+H)⁺。

¹H NMR (400 MHz, DMSO-d6)δ: 12.78 (s, 1H), 8.32 (s, 1H), 7.59–7.51 (m, 2H), 7.43 (s, 1H), 6.78 (t, 1H), 5.70 (d, 1H), 3.46 (s, 3H), 3.38 (s, 3H), 3.33 (m, 4H), 1.83–1.72 (m, 4H), 1.67 (d, 3H), 1.63–1.50 (m, 4H)。

Example 16: preparation of (2S) -N- (4- (4- (3, 6-diazabicyclo [3.1.1] hept-6-yl) -3-fluorophenyl) thiazol-2-yl) -2- (1, 3-dimethyl-2, 6-dioxo-1, 2,3, 6-tetrahydro-7H-purin-7-yl) -propionamide

Preparation of intermediate tert-butyl 6- (4- (2-aminothiazol-4-yl) -2-fluorophenyl) -3, 6-diazacyclo [3.1.1] heptane-3-carboxylate

The intermediate was prepared in the same manner as in example 2 except that (S) -dimethylpyrrolidine in the first step was replaced with equimolar 6- (tert-butoxycarbonyl) -3, 6-diazabicyclo [3.1.1] heptane and the second step was performed in the same manner as in the third step to give a yellow oily intermediate compound, in a three-step total yield: 45.1 percent and the purity is 97.76 percent.

ESI-MS: m/z = 391.2(M+H)⁺。

The title compound was prepared by the same method as in example 1 except that in step six, (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazole-2-amine was replaced with equimolar of tert-butyl 6- (4- (2-aminothiazol-4-yl) -2-fluorophenyl) -3, 6-diazacyclo [3.1.1] heptane-3-carboxylate to give tert-butyl 6- (4- (2- ((S) -2- (1, 3-dimethyl-2, 6-dioxy-1, 2,3, 6-tetrahydro-7H-purin-7-yl) propionamide) thiazol-4-yl) -2-fluorophenyl) -3, 6-diazacyclo [3.1.1] heptane-3-carboxylate, Boc protecting group removal with trifluoroacetic acid gave the title compound in two-step reaction yield: 36.9 percent and the purity of 98.83 percent.

ESI-MS: m/z = 525.2 (M+H)⁺。

¹H NMR(400 MHz, DMSO-d6) δ:12.98 (s, 1H), 8.36 (s, 1H), 7.59–7.51 (m, 2H), 7.43 (s, 1H), 6.78 (t, 1H), 5.79 (d, 1H), 4.01 (m, 2H), 3.46 (s, 3H), 3.38 (s, 3H), 3.01 –2.86 (m, 4H), 2.51–2.43 (m, 1H), 2.13 (dt, 1H), 1.99 (dt, 1H), 1.67 (d, 3H)。

Example 17: preparation of (S) -2- (1, 3-dimethyl-2, 6-dioxy-1, 2,3, 6-tetrahydro-7H-purin-7-yl) -N- (4- (3-fluoro-4- ((S) -3-methoxypyrrolidin-1-yl) phenyl) -5-methylthiazol-2-yl) propionamide

Preparation of intermediate (S) -4- (3-fluoro-4- (3-methoxypyrrolidin-1-yl) phenyl) -5-methylthiazol-2-amine

The preparation method is the same as that of example 2, in the first step, (S) -dimethylpyrrolidine is replaced by equimolar (S) -3-methoxypyrrolidine, 3, 4-difluoroacetophenone is replaced by equimolar 3, 4-difluoropropiophenone, the second step is the same as the third step, an intermediate compound is obtained, and the reaction yield in the third step is as follows: 33.5 percent and the purity is 98.43 percent.

ESI-MS: m/z = 308.1 (M+H)⁺。

The title compound was prepared according to the procedure for example 1 by substituting (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine in step six with an equimolar amount of (S) -4- (3-fluoro-4- (3-methoxypyrrolidin-1-yl) phenyl) -5-methylthiazol-2-amine to give the title compound as a light yellow solid in yield: 67.9% and 98.40% purity.

ESI-MS: m/z = 542.2(M+H)⁺。

¹H NMR(400 MHz, DMSO-d6)δ: 12.20 (s, 1H), 8.38 (s, 1H), 7.50 (dd, 1H), 7.38 (dd, 1H), 7.00 (dd, 1H), 5.74 (q, 1H), 4.18 (m, 1H), 3.73 (m, 1H), 3.55–3.43 (m, 2H), 3.46 (s, 3H), 3.42–3.39 (m, 1H), 3.38 (s, 3H), 3.20 (d, 3H), 2.48 (s, 3H), 2.01–1.90 (m, 1H), 1.83 (m, 1H), 1.67 (d, 3H)。

Example 18: preparation of (S) -2- (1, 3-dimethyl-2, 6-dioxy-1, 2,3, 6-tetrahydro-7H-purin-7-yl) -N- (4- (3-fluoro-4- ((S) -3- (trifluoromethoxy) pyrrolidin-1-yl) phenyl) -5-methylthiazol-2-yl) propionamide

Preparation of intermediate (S) -4- (3-fluoro-4- (3- (trifluoromethoxy) pyrrolidin-1-yl) phenyl) -5-methylthiazol-2-amine

The preparation method is the same as that of example 2, in the first step, (S) -dimethylpyrrolidine is replaced by equimolar (S) -3-trifluoromethoxy pyrrolidine, 3, 4-difluoroacetophenone is replaced by equimolar 3, 4-difluoropropiophenone, the second step is the same as the third step, an intermediate compound is obtained, and the reaction yield in the third step is as follows: 25.5% and the purity is 98.09%.

ESI-MS: m/z = 362.1 (M+H)⁺。

The title compound was prepared according to the procedure for example 1 by replacing in step six (S) -5- (3-fluoro-4- (2-methylpyrrolidin-1-yl) phenyl) -1,3, 4-thiadiazol-2-amine with an equimolar amount of (S) -4- (3-fluoro-4- (3- (trifluoromethoxy) pyrrolidin-1-yl) phenyl) -5-methylthiazol-2-amine to give the title compound as a light yellow solid in yield: 62.2 percent and the purity is 98.70 percent.

ESI-MS: m/z = 596.2(M+H)⁺。

¹H NMR(400 MHz, DMSO-d6)δ: 12.27 (s, 1H), 8.38 (s, 1H), 7.52 (dd, 1H), 7.38 (dd, 1H), 7.00 (dd, 1H), 5.78 (q, 1H), 4.63 (m, 1H), 3.74 (m, 1H), 3.60–3.43 (m, 2H), 3.46 (s, 3H), 3.38 (s, 3H), 3.31–3.23 (m, 1H), 2.48 (s, 3H), 2.07–1.89 (m, 2H), 1.67 (d, 3H)。

Comparative example 1: preparation of N- (4- (2, 4-difluoro-3- (trifluoromethyl) phenyl) thiazol-2-yl) -2- (1, 3-dimethyl-2, 4-dioxo-1, 2,3, 4-tetrahydrothiopheno [2,3-d ] imidin-5-yl) acetamide

Synthesized according to the method described in patent WO2013183035a2, purity: 98.9 percent.

ESI-MS: m/z = 517.1(M+H)⁺。

¹H NMR (300 MHz, DMSO-d6)δ:3.19 (s, 3H), 3.46 (s, 3H), 4.07 (s, 2H), 7.07 (s, 1H), 7.48-7.54 (t, 1H), 7.61 (s, 1H), 8.30-8.37 (q, 1H), 12.48 (br s, 1H)。

Comparative example 2: preparation of (S) -3- (3- (4-chlorobenzyl) -4- (4- (3-fluoropyridin-2-yloxy) phenyl) amino) -2, 6-dioxa-3, 6-dihydropyrimidin-1 (2H) -yl) -2-methylpropionic acid

Synthesized according to the method described in patent WO2010075353a1, purity: 98.5 percent.

ESI-MS: m/z = 498.2(M+H)⁺。

¹H NMR (400 MHz, DMSO-d6) δ: 12.87 (s, 1H), 8.03 (s, 1H), 7.68 (dd, 1H), 7.45 (dd, 1H), 7.42 (s, 1H), 7.06 (dd, 1H), 5.32 (d, 2H), 3.71–3.60 (m, 2H), 3.46 (m, 4H), 3.38 (s, 3H), 1.96–1.82 (m, 1H), 1.87–1.72 (m, 2H), 1.56–1.44 (m, 1H), 1.22 (d, 3H)。

Test example 1: cough test in mice

Working test material

Test article basic information

Examples 1-18 (synthesized by the inventors ' laboratory), comparative example 1 (CRC 17536, positive control, synthesized by the inventors ' laboratory), and comparative example 2 (synthesized by the inventors ' laboratory).

A reagent for experiments

Normal saline and ammonia water.

Perfect animal experiment

Healthy adult KM mice are half male and female, 6 mice in each group and have the weight of about 28-30 g.

⒊ test method

First dosage design and test article use amount

The animal cough model reported in the literature at present mostly adopts methods such as mechanical, chemical and electrical stimulation to stimulate nerves and receptors of animals to cause cough. According to the characteristics of the candidate compound and the existing similar target compounds as references, a mouse cough modeling test is established by primarily selecting a strong ammonia water induction method.

Preparation method of test articles

The preparation method of the 50% ammonia water solution comprises the following steps: 2.5ml of ammonia water is measured and dissolved in 5ml of 0.9 percent sodium chloride injection, and the mixture is fully and evenly mixed.

Comparative example 1 solution preparation method: 18mg of comparative example 1 was dissolved in 3ml of 0.5% CMC-Na solution and mixed well to prepare a solution of 6 mg/ml.

Comparative example 2 solution preparation method: 18mg of comparative example 2 was dissolved in 3ml of 0.5% CMC-Na solution and mixed well to prepare a solution of 6 mg/ml.

Examples solution formulation methods: 18mg of the example was dissolved in 3ml of 0.5% CMC-Na solution and mixed well to prepare a solution of 6 mg/ml.

Experimental operation method for obtaining three

6 KM mice were taken per group: comparative example 1 group, comparative example 2 group, example group, vehicle group. Mice in the comparative example 1 group, comparative example 2 group and example group were intragastrically administered with the compound of comparative example 1 (60 mg/kg), the compound of comparative example 2 (60 mg/kg) and the compound of example (60 mg/kg), respectively, and the vehicle group was administered with an equal volume of 0.5% CMC-Na solution. After administration for 30min, the mice were placed in 500ml beakers, into which 1 cotton ball (100. + -.5 mg in weight) containing 0.3ml of 50% ammonia water was placed. The number of typical coughs that occurred within 3min was observed in the mice (typical coughing action: contraction of abdominal muscles or chest contraction while mouth enlargement with coughing sound).

⒋ results and discussion

First result judgment standard

Judging a cough standard:

the manifestations of cough are: the abdominal muscles contract or contract the chest while the mouth is enlarged, with a cough.

Secondly, a stopwatch is used for timing, the number of coughing of the mice within 3min is recorded, statistical analysis is carried out by software, all groups of data are statistically described by means of mean values plus or minus standard deviations, single-factor variance analysis is carried out among multiple groups, and P <0.05 is a difference which has statistical significance.

Discussion of the results

The frequency of coughing in mice 30min after administration of 60mg/kg of the compound of example is shown in table 1 below:

note: (1)^*representing P compared to the model set<0.05；^**Representing P compared to the model set<0.01；^#Represents P in comparison with the group of comparative example 1<0.05; (2) group of comparative example 1, group of example 1 the compounds used for representing this group are the compound of comparative example 1, the compound of example 1, respectively, and the others are explained similarly.

As shown in the above table, the compounds of the examples according to the present invention all had a significantly reduced number of coughs compared to the model group, and the compounds of the examples 2,3,6, 9, 10 and 12 had a significantly reduced number of coughs compared to the compounds of the comparative example 1, and also had a statistically significant reduction in the number of coughs compared to the compounds of the comparative example 2.

Test example 2: rat hepatotoxicity serum biomarker study

Working test material

And (3) testing the sample: examples 2,3,6, 9, 12 groups of compounds, comparative example 2 compound;

test reagents: 0.5% CMC-Na solution (batch No. G1226001).

Perfect animal experiment

Healthy adult SD rats weighing 180-200 g, 6-9 weeks old per week, all females, and 6 rats per group.

⒊ test method

Preparation method of test products

Example compounds and comparative example compounds formulation methods: accurately weighing appropriate amount of the medicine, adding 0.5% CMC-Na, ultrasonic treating, and mixing; the drug concentration of 12.5mg/ml is prepared.

Method for experimental operation

Healthy adult SD rats, 6 rats in each group, after fasting overnight (free drinking water), collecting blank serum of 200 microliters respectively for blood supply biochemical detection from jugular veins, respectively injecting and administering tail veins after blood collection, administering 50mg/kg once, observing the toxic reaction condition and death condition of each rat after administration, recording, collecting blood from jugular veins again 24h after administration to detect blood biochemical indexes (AST and ALT), and after blood collection, placing the rats back to the rearing cage to continuously observe the condition after administration.

⒋ results and discussion

The biochemical blood indicators of the rats in each group before and after administration are counted as follows:

the table shows that there is no significant change in blood biochemical markers (AST, ALT) before and after administration in the mice of the examples 2,3,6, 9, and 12, while the blood biochemical markers (AST, ALT) 24h after administration in the comparative example 2 are increased by 4.22 times and 9.52 times respectively compared with those before administration. It is shown that the compounds of examples 2,3,6, 9, 12 of the present invention do not cause hepatotoxicity and are significantly safer than the compound of comparative example 2.

Test example 3: safety study of 14-day repeat dosing in mice

Working test material

Example 3 group of compounds.

Components of capsule wall

Healthy KM mice weigh 18-20 g, are 6-9 weeks old, and are 40 in total.

⒊ test method

First dosage design and test article use amount

Preparation method of test articles

Example 3 group formulation method: accurately weighing appropriate amount of the medicine, adding appropriate amount of 0.5% CMC-Na, mixing with ultrasound and vortex, and respectively preparing into medicine concentrations of 120mg/ml, 60mg/ml or 30 mg/ml.

Experimental operation method for obtaining three

40 healthy KM mice are divided into 8 groups by random, namely a blank female group, a blank male group, an example 3 group low dose female (300 mg/kg) group, an example 3 group low dose male (300 mg/kg) group, an example 3 group medium dose female (600 mg/kg) group, an example 3 group medium dose male (600 mg/kg) group, an example 3 group high dose female (1200 mg/kg) group and an example 3 group high dose male (1200 mg/kg) group, 10 mice in each test group and the blank group are respectively administrated by gastric lavage every day for 14 days, the blank group is administrated with the same volume of solvent, the toxicity reaction condition and death condition of the mice in each group are observed and recorded every day after administration, and the blood serum and the serum of the rest of the mice in each group are taken out, separated and the blood biochemical indexes (AST, ALT) of the blood are detected.

⒋ results and discussion

Mice in each group were counted for mortality and observed for clinical symptoms after administration as shown in the following table:

the table shows that the compound of example 3 did not die in both the low and medium dose groups, whereas 40% of the males and none of the females died in the high dose group. Meanwhile, the swelling condition of the genitals of a part of male mice is found in the medium-dose group and the high-dose group; after the experiment was completed, all surviving mice were dissected and observed without significant change in the remaining mice.

On the last day of the experiment, the eye sockets of the rest mice in each group were bled, serum was separated, and the results of detecting blood biochemical indicators (AST, ALT) are shown in the following table, wherein no obvious change of the blood biochemical indicators (AST, ALT) of the mice is caused by the compound in the group 3 of the example in low dose, medium dose and high dose compared with the blank group after the compound is continuously administered for 14 days, which indicates that the compound in the group 3 of the example does not cause liver injury of the mice.

The above-mentioned embodiment is only one of the preferred embodiments of the present invention, and should not be used to limit the scope of the present invention, but all the insubstantial modifications or changes made within the spirit and scope of the main design of the present invention, which still solve the technical problems consistent with the present invention, should be included in the scope of the present invention.

Claims

1. A compound of formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof:

wherein,

R²、R³independently selected from hydrogen, deuterium, hydroxy, halogen, cyano, nitro, substituted or unsubstituted amino, substituted or unsubstituted lower alkyl, substituted or unsubstitutedSubstituted lower alkoxy, C3-C6 cycloalkyl, C1-C3 perfluoroalkyl, C1-C3 perfluoroalkoxy, - (CH)₂)fNRR’、—O-(CH₂) fNRR ', -C (= O) fNRR', or carboxyl, wherein:

f is an integer from 1 to 4;

each R is independently selected from hydrogen or lower alkyl;

2. The compound, stereoisomer or pharmaceutically acceptable salt thereof according to claim 1, wherein in formula (1):

R¹selected from the group consisting of hydrogen, hydroxy, halogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkylamino, substituted or unsubstituted lower alkoxy, substituted or unsubstituted lower alkylthio, substituted or unsubstituted 3-to 10-membered cyclic amino;

3. A compound, a stereoisomer or pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, wherein in formula (1):

wherein:

x is selected from: o, NH or CHR⁷；

additionally or alternatively, two R's attached to the same ring carbon⁴Or two R⁵Or two R⁶The substituents may together form an oxo group (i.e.: O) or a C3-C7 spiro group; and additionally or alternatively, two R's attached to different ring carbons⁴Or two R⁵Or two R⁶The substituents may together form a ring, wherein two R' s⁶When taken together, form a ring having 4 to 7 ring atoms, including 0 to 3 ring heteroatoms;

n is an integer from 0 to 4;

a is selected from an integer of 0 to 3;

b. c is independently an integer selected from 0 to 2;

m and p are independently selected from integers of 1 to 3;

q and r are independently selected from integers of 0 to 3;

R²、R³independently selected from: hydrogen, halogen, hydroxyGroup, amino group, C1-C6 alkyl group, C1-C6 alkoxy group, C1-C6 alkylamino group, substituted or unsubstituted aryl group, C3-C6 cyclic alkyl group, C1-C3 perfluoroalkyl group or C1-C3 perfluoroalkoxy group; preferably, R²、R³Independently selected from: hydroxyl, halogen, C1-C4 alkyl, C1-C4 alkoxy, C1-C3 perfluoroalkyl or C1-C3 perfluoroalkoxy.

4. A compound, stereoisomer or pharmaceutically acceptable salt thereof according to claim 3, wherein R is¹Selected from hydrogen, halogen, trifluoromethyl, trifluoromethoxy, difluoromethyl, difluoromethoxy, C1-C6 alkyl, or selected from the group consisting of 1 to 2R⁸Substituted of the following rings:

5. The compound, stereoisomer or pharmaceutically acceptable salt thereof according to claim 4, wherein R is⁸Independently selected from the group consisting of: hydrogen, halogen, hydroxyl, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, substituted or unsubstituted aryl, C3-C6 cycloalkyl, C1-C3 perfluoroalkyl or C1-C3 perfluoroalkoxy; preferably, said R is⁸Independently selected from the group consisting of: hydrogen, halogen, C1-C4 alkyl, C1-C4 alkoxy, C1-C3 perfluoroalkyl or C1-C3 perfluoroalkoxy.

6. The compound, stereoisomer or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, which is:

。

7. a compound, stereoisomer or pharmaceutically acceptable salt thereof, according to any one of claims 1 to 6, wherein hydrogen in the compound is substituted by one or more deuterium.

8. A process for the preparation of a compound according to any one of claims 1 to 7, a stereoisomer or a pharmaceutically acceptable salt thereof, comprising the steps of:

wherein, ring A, R¹、R²、R³Is as defined in any one of claims 1 to 7;

step a: adding a protective group into (R) -2-hydroxy methyl propionate to obtain (R) -2- ((trifluoromethyl) sulfonyl) oxypropionic acid methyl ester;

9. The pharmaceutical composition of any one of the compounds of claims 1-8, stereoisomers or pharmaceutically acceptable salts thereof, wherein the composition further comprises a pharmaceutically acceptable excipient.

10. Use of a compound, a stereoisomer or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9 for the preparation of a medicament for the prevention and/or treatment of a disease associated with the TRPA1 receptor, wherein the disease associated with the TRPA1 receptor is a respiratory disease or a neurological disease; preferably, the disease associated with the TRPA1 receptor is cough, asthma, pain or sleep apnea.