Imidazopyridine compounds, processes for their preparation and uses thereof

Abstract

The application discloses an imidazopyridine compound and a preparation method and application thereof, and belongs to the technical field of chemical pharmacy. The imidazopyridine compound is shown as formula I: (I), R is -OH, -H, -OR1 or -NHR2, wherein R1 and R2 are independently selected from hydrogen, C1-C6 alkyl, C1-C8 cycloalkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl. The novel imidazopyridine compound prepared by the application has a simple and easy preparation process. It is found through pharmacological experiments that the compound has strong inhibitory USP5 enzymatic activity and selectivity, can inhibit proliferation of various tumor cells, is extremely sensitive to pancreatic cancer cells, has high efficient pancreatic cancer inhibitory activity in vivo and in vitro, and is suitable for development of anti-pancreatic cancer drugs. The compound has obvious in-vitro and in-vivo anti-cancer activity, and is suitable for development of various anti-tumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of chemical pharmaceutical technology, and specifically relates to the preparation and application of a USP5 inhibitor compound. Background Technology

[0002] The ubiquitin-proteasome system (UPS) is a major pathway for protein degradation in cells, playing a crucial role in maintaining protein homeostasis, regulating the cell cycle, signal transduction, and immune responses. Deubiquitinating enzymes (DUBs) reverse ubiquitination by hydrolyzing ubiquitin chains or precursors, and are important regulatory nodes in the UPS. Among them, USP5 (ubiquitin-specific protease 5), as an important member of the deubiquitinating enzyme family, is mainly responsible for recognizing and cleaving unanchored polyubiquitin chains, maintaining the homeostasis of the free ubiquitin pool, and participating in the regulation of the stability of various substrate proteins.

[0003] Studies have shown that USP5 exhibits abnormally high expression or upregulated activity in various tumor types, promoting tumor cell proliferation, survival, and drug resistance through mechanisms such as stabilizing oncoproactive proteins (e.g., β-catenin, cyclin D1, p53 mutants) or interfering with DNA damage repair. Furthermore, abnormal USP5 activity is closely related to the development of neurodegenerative diseases, inflammation, and autoimmune diseases. For example, patent CN118203576A discloses the application of a combination of a USP5 inhibitor and a PD-1 antibody in the preparation of antitumor drugs. Based on the fact that the YTH protein domain is the main region for ubiquitination modification of the YTHDF1 protein, small molecule inhibitors that can affect YTHDF1 protein homeostasis were screened. It was found that the deubiquitinating enzyme USP5 inhibitor WP1130 can inhibit YTHDF1 protein homeostasis and negatively regulate PD-L1 protein expression. Therefore, USP5 has become a potential drug target, and the development of highly active and selective USP5 small molecule inhibitors has significant clinical translational value.

[0004] Currently, although a small number of USP5 inhibitors have been reported, such as compound EOAI3402143 (also known as "IU1-47"), they still have significant limitations: First, the enzyme activity of existing inhibitors is generally not high, and high concentrations are often required to effectively inhibit USP5 function, which limits their in vivo efficacy; Second, due to the high conservation of the catalytic domain of deubiquitinating enzyme family members, existing compounds generally have insufficient selectivity and are prone to off-target effects on other DUB family members (such as USP4, USP14, etc.), leading to potential off-target toxicity and confusion in mechanism analysis.

[0005] Therefore, developing a class of novel small molecule compounds that can specifically, efficiently, and selectively target USP5 and induce tumor cell death has significant scientific and clinical translational value. Summary of the Invention

[0006] This invention addresses the technical problems of poor selectivity and significant off-target effects of existing USP5 inhibitors by proposing an imidazopyridine compound, its preparation method, and its application.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0008] This invention provides an imidazopyridine compound, the structure of which is shown in general formula (I):

[0009] (I),

[0010] The R is -OH, -H, -OR1 or -NHR2, wherein R1 and R2 are independently selected from hydrogen, C1-C6 alkyl, C1-C8 cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

[0011] Preferably, R is selected from any one of the following 1-18:

[0012]

[0013] This invention provides a method for preparing the aforementioned imidazopyridine compound, the preparation process of which is as follows:

[0014] ;

[0015] Includes the following steps:

[0016] (1) Compound A, compound B, tetra(triphenylphosphine)palladium(0) and cesium carbonate were dissolved in toluene and mixed and heated and stirred to carry out the reaction. After the reaction was complete, the mixture was cooled, filtered to remove insoluble matter, and the filtrate was concentrated and purified to obtain compound C.

[0017] (2) The compound C obtained in step (1) was mixed with dichloromethane and cooled. DMF was added dropwise and oxalyl chloride was introduced. The mixture was stirred to carry out the reaction. After the reaction was complete, the solvent and excess oxalyl chloride were removed by rotary evaporation to obtain compound D.

[0018] (3) The compound D obtained in step (2), ethyl 4-aminocyclohexane carboxylate and dichloromethane were mixed and cooled. Triethylamine was added dropwise and the mixture was heated and stirred to carry out the reaction. After the reaction was completed, the reaction was quenched. After extraction, drying, filtration, concentration and purification, imidazopyridine compound 1 was obtained.

[0019] (4) The imidazopyridine compound 1 obtained in step (3), lithium hydroxide, methanol and water are mixed and stirred to react. After the reaction is complete, methanol is removed by rotary evaporation. The residue is added with water and the pH is adjusted. The obtained solid is filtered, washed and dried to obtain imidazopyridine compound 2.

[0020] (5) The imidazopyridine compound 2 and the amine compound obtained in step (4) are dissolved in DMF, and then HATU and DIPEA are added in sequence. The mixture is stirred to carry out the reaction. After the reaction is complete, the mixture is washed, dried, filtered, concentrated and purified to obtain the imidazopyridine compound.

[0021] In step (1), the molar ratio of compound A, compound B, tetrakis(triphenylphosphine)palladium(0), and cesium carbonate is 1:1.2-1.08:0.02-0.1:1.5-3, and the initial concentration of compound A is 0.05-0.25M. The reaction is carried out under nitrogen protection at a temperature of 80-110℃ for 12-18h. The purification is performed by silica gel column chromatography with a gradient elution of petroleum ether / ethyl acetate. The reaction is monitored by TLC until compound A disappears, with the developing solvent being petroleum ether / ethyl acetate = 1 / 2.

[0022] In step (2), based on the molar amount of compound C, 10-15 mL of dichloromethane is added for every 0.7 mmol of compound C, and the cooling temperature is 0-5℃; the molar ratio of compound C to oxaloyl chloride is 1:3-10; the molar ratio of compound C to DMF is 1:0.1-2; the stirring time is 0.5-3 h; the reaction is monitored by TLC until compound C disappears, and the developing solvent is petroleum ether / ethyl acetate = 3 / 1.

[0023] In step (3), the molar ratio of compound D, ethyl 4-aminocyclohexanecarboxylate, and triethylamine is 1:1-1.8:2-3. Based on the molar amount of compound D, 5-15 mL of dichloromethane is added for every 0.7 mmol of compound D. The temperature for heating and stirring is 25-40℃, and the time is 6-12 h. The purification is carried out by column chromatography, with petroleum ether / ethyl acetate gradient elution as the eluent. The reaction is monitored by TLC until compound D disappears, and the developing solvent is petroleum ether / ethyl acetate = 2 / 1.

[0024] In step (4), the molar ratio of imidazopyridine compound 1 to lithium hydroxide is 1:2-10. Based on the molar amount of imidazopyridine compound 1, 3-8 mL of methanol and 2-6 mL of water are added per mmol of imidazopyridine compound 1. The stirring temperature is 25-40℃ and the stirring time is 0-5h. The pH is adjusted to 3-5 using 0.5-2M hydrochloric acid solution. The reaction is monitored by TLC until imidazopyridine compound 1 disappears. The developing solvent is DCM / MeOH=6 / 1.

[0025] In step (5), the molar ratio of imidazopyridine compound 2, amine compound, HATU, and DIPEA is 1:1-2:1-3:2-4. The initial concentration of imidazopyridine compound 2 is 0.05-0.2M. The amine compound is a substituted or unsubstituted amine, and the substituted or unsubstituted amine is selected from: substituted or unsubstituted C3-C8 cycloalkylamines, substituted or unsubstituted aromatic amines, and substituted or unsubstituted heteroaromatic amines. The stirring time is 8-24h. The washing is performed using saturated sodium bicarbonate solution and water. The drying is performed using anhydrous sodium sulfate. The purification is performed using column chromatography with CH2Cl2 / MeOH = 20:1 as the eluent.

[0026] The amine compound is selected from any one of cyclopropylamine, cyclobutylamine, cyclopentylamine, cyclohexylamine, aniline, 4-aminopyridine, 4-cyanoaniline, 4-nitroaniline, 4-methylaniline, 4-fluoroaniline, 4-chloroaniline, 4-bromoaniline, 1-naphthylamine, 4-fluoro-1-naphthylamine, 8-fluoro-1-naphthylamine, and 5-fluoro-1-naphthylamine.

[0027] This invention provides the use of the aforementioned imidazopyridine compound or a pharmaceutically acceptable salt thereof in the preparation of a drug that inhibits deubiquitinase USP5.

[0028] This invention provides the use of the imidazopyridine compound or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention or treatment of cancers associated with the deubiquitinase USP5.

[0029] The cancers are preferably leukemia, lymphoma, lung cancer, colorectal cancer, liver cancer, stomach cancer, pancreatic cancer, cervical cancer, and neuroblastoma.

[0030] The present invention provides a pharmaceutical composition comprising the imidazopyridine compound or a pharmaceutically acceptable salt thereof, and further comprising a pharmaceutically acceptable excipient; said excipient being selected from at least one of gum arabic, syrup, lanolin and starch.

[0031] The excipient is stable, has no incompatibility with the active ingredient, does not produce side effects, does not affect efficacy, is not easily deformed, cracked, moldy, or infested by insects at room temperature, is harmless to the human body, has no physiological effects, does not produce chemical or physical reactions with the active ingredient, and does not affect the content determination of the active ingredient.

[0032] The beneficial effects of this invention are:

[0033] The preparation method provided by this invention is simple and easy to implement, and the prepared imidazopyridine compound or its pharmaceutically acceptable salt has a highly efficient and selective inhibitory effect on deubiquitinase USP5. This provides new perspectives and ideas for broadening our understanding of the USP family, studying the mechanism of deubiquitinase USP5 in the process of protein deubiquitination, and developing novel small molecule drugs for pancreatic cancer. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 Tumor growth curves in vivo for pancreatic cancer in subcutaneous xenograft models of imidazopyridine compounds 12 and 18. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1

[0038] A method for preparing imidazopyridine compound 1, the preparation flow chart is as follows:

[0039] ;

[0040] The specific steps are as follows:

[0041] (1) Compound A (1 mmol), compound B (1.2 mmol), tetrakis(triphenylphosphine)palladium(0) (0.05 mmol), cesium carbonate (2 mmol), and toluene (10 mL) were added to a 250 mL three-necked flask. Under nitrogen protection, the reaction system was heated to reflux temperature of 110 °C and stirred for 12 h. The reaction was monitored by TLC (eluent: petroleum ether / ethyl acetate = 1 / 2) until compound A disappeared. After the reaction was complete, the mixture was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting agent: petroleum ether / ethyl acetate gradient elution) to obtain compound C as a white solid with a yield of 72%. Compound C was characterized, and the results are as follows: HRMS (ESI): m / z calcd for C 14 H 12 N3O2 + [M+H] + : 254.0924; found 254.0922.

[0042] (2) Add compound C (0.7 mmol) and dichloromethane (10 mL) to a 100 mL three-necked flask, cool to 0 °C with stirring, add N,N-dimethylformamide (DMF, 50 μL) dropwise, and then oxalyl chloride (COCl2, 3.5 mmol) is introduced. The reaction system is stirred at room temperature for 0.5 h, and the reaction is monitored by TLC (electrolyte: petroleum ether / ethyl acetate = 3 / 1) until the starting material C disappears. After the reaction is complete, remove the solvent and excess oxalyl chloride under reduced pressure to obtain compound D as a colorless oil (which can be used directly in the next step).

[0043] (3) Compound D (0.7 mmol), compound E (ethyl 4-aminocyclohexanecarboxylate, 0.9 mmol), and dichloromethane (8 mL) were added to a 50 mL round-bottom flask. The flask was cooled in an ice bath, and triethylamine (1.5 mmol) was added dropwise. The mixture was then heated to room temperature and stirred for 12 h. The reaction was monitored by TLC (eluent: petroleum ether / ethyl acetate = 2 / 1) until the starting material D disappeared. After the reaction was complete, water (10 mL) was added to quench the reaction, and the mixture was extracted with DCM (10 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: petroleum ether / ethyl acetate gradient) to obtain imidazopyridine compound 1 as a white solid with a yield of 81%. The results of the detection of imidazopyridine compound 1 are as follows:

[0044] 1H NMR (500 MHz, Chloroform-d) δ 8.46 (dd, J = 3.8, 1.8 Hz, 1H), 7.93 (dd, J = 6.6, 1.8 Hz, 1H), 7.89 (t, J = 1.9 Hz, 1H), 7.64 (dd, J = 7.8, 6.5Hz, 1H), 7.55 (ddd, J = 7.7, 1.9, 1.2 Hz, 1H), 7.45 – 7.30 (m, 2H), 4.13 (qd,J = 6.3, 0.7 Hz, 2H), 3.78 (dd, J = 12.4, 5.4 Hz, 1H), 3.63 (dd, J = 12.4,5.4 Hz, 1H), 3.58 – 3.26 (m, 2H), 2.78 – 2.60 (m, 1H), 2.47 (s, 3H), 2.14 –1.67 (m, 4H), 1.24 (t, J = 6.4 Hz, 3H).

[0045] HRMS (ESI): m / z calcd for C 22 H 25 N4O3 + [M+H] + : 393.1921; found 393.1924.

[0046] Example 2

[0047] A method for preparing imidazopyridine compound 2, the preparation process is as follows:

[0048] ;

[0049] The specific steps are as follows:

[0050] (1) Compound A (1 mmol), compound B (1.2 mmol), tetrakis(triphenylphosphine)palladium(0) (0.05 mmol), cesium carbonate (2 mmol) and toluene (10 mL) were added to a 250 mL three-necked flask. Under nitrogen protection, the reaction system was heated to reflux temperature of 110 °C and stirred for 12 h. The reaction was monitored by TLC (eluent: petroleum ether / ethyl acetate = 1 / 2) until compound A disappeared. After the reaction was complete, the mixture was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting agent: petroleum ether / ethyl acetate gradient elution) to give compound C as a white solid with a yield of 72%.

[0051] (2) Add compound C (0.7 mmol) and dichloromethane (10 mL) to a 100 mL three-necked flask, cool to 0 °C with stirring, add N,N-dimethylformamide (DMF, 50 μL) dropwise, and then oxalyl chloride (COCl2, 3.5 mmol) is introduced. The reaction system is stirred at room temperature for 0.5 h, and the reaction is monitored by TLC (electrolyte: petroleum ether / ethyl acetate = 3 / 1) until the starting material C disappears. After the reaction is complete, remove the solvent and excess oxalyl chloride under reduced pressure to obtain compound D as a colorless oil (which can be used directly in the next step).

[0052] (3) Compound D (0.7 mmol), compound E (ethyl 4-aminocyclohexane carboxylate, 0.9 mmol) and dichloromethane (8 mL) were added to a 50 mL round-bottom flask. The flask was cooled in an ice bath, and triethylamine (1.5 mmol) was added dropwise. The mixture was heated to room temperature and stirred for 12 h. The reaction was monitored by TLC (eluent: petroleum ether / ethyl acetate = 2 / 1) until the starting material D disappeared. After the reaction was complete, water (10 mL) was added to quench the reaction. The mixture was extracted with DCM (10 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: petroleum ether / ethyl acetate gradient) to obtain imidazopyridine compound 1 as a white solid with a yield of 81%.

[0053] (4) Add imidazopyridine compound 1 (0.5 mmol), lithium hydroxide (LiOH, 2.5 mmol), methanol (MeOH, 3 mL), and water (H2O, 2 mL) to a 50 mL round-bottom flask. Stir at room temperature for 2 hours, and monitor the reaction by TLC (DCM / MeOH = 6 / 1) until the reactant 1 disappears. After the reaction is complete, remove the methanol by rotary evaporation, add water (5 mL) to the residue, and adjust the pH to 3 with 1 M hydrochloric acid (a solid precipitates). Filter, wash the solid with cold water, and dry to obtain imidazopyridine compound 2 as a white solid with a yield of 95%. The results of the detection of imidazopyridine compound 2 are as follows:

[0054] 1H NMR (500 MHz, Chloroform-d) δ 8.46 (dd, J = 3.8, 1.8 Hz, 1H), 7.93 (dd, J = 6.6, 1.8 Hz, 1H), 7.89 (t, J = 1.9 Hz, 1H), 7.64 (dd, J = 7.8, 6.5Hz, 1H), 7.55 (ddd, J = 7.7, 1.9, 1.2 Hz, 1H), 7.45 – 7.32 (m, 2H), 3.80 (dd,J = 12.5, 5.1 Hz, 1H), 3.66 – 3.50 (m, 2H), 3.43 – 3.32 (m, 1H), 2.66 (tt, J= 6.6, 5.0 Hz, 1H), 2.47 (s, 3H), 2.03 – 1.68 (m, 4H).

[0055] HRMS (ESI): m / z calcd for C 20 H 21 N4O3 + [M+H] + : 365.1608; found 365.1603.

[0056] Example 3

[0057] A method for preparing an imidazopyridine compound 3-18, the preparation process is as follows:

[0058] ;

[0059] The specific steps are as follows:

[0060] (1) Compound A (1 mmol), compound B (1.2 mmol), tetrakis(triphenylphosphine)palladium(0) (0.05 mmol), cesium carbonate (2 mmol) and toluene (10 mL) were added to a 250 mL three-necked flask. Under nitrogen protection, the reaction system was heated to reflux temperature of 110 °C and stirred for 12 h. The reaction was monitored by TLC (eluent: petroleum ether / ethyl acetate = 1 / 2) until compound A disappeared. After the reaction was complete, the mixture was cooled to room temperature, filtered to remove insoluble matter, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting agent: petroleum ether / ethyl acetate gradient elution) to give compound C as a white solid with a yield of 72%.

[0061] (2) Add compound C (0.7 mmol) and dichloromethane (10 mL) to a 100 mL three-necked flask, cool to 0 °C with stirring, add N,N-dimethylformamide (DMF, 50 μL) dropwise, and then oxalyl chloride (COCl2, 3.5 mmol) is introduced. The reaction system is stirred at room temperature for 0.5 h, and the reaction is monitored by TLC (electrolyte: petroleum ether / ethyl acetate = 3 / 1) until the starting material C disappears. After the reaction is complete, remove the solvent and excess oxalyl chloride under reduced pressure to obtain compound D as a colorless oil (which can be used directly in the next step).

[0062] (3) Compound D (0.7 mmol), compound E (ethyl 4-aminocyclohexane carboxylate, 0.9 mmol) and dichloromethane (8 mL) were added to a 50 mL round-bottom flask. The flask was cooled in an ice bath, and triethylamine (1.5 mmol) was added dropwise. The mixture was heated to room temperature and stirred for 12 h. The reaction was monitored by TLC (eluent: petroleum ether / ethyl acetate = 2 / 1) until the starting material D disappeared. After the reaction was complete, water (10 mL) was added to quench the reaction. The mixture was extracted with DCM (10 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: petroleum ether / ethyl acetate gradient) to obtain imidazopyridine compound 1 as a white solid with a yield of 81%.

[0063] (4) Add imidazopyridine compound 1 (0.5 mmol), lithium hydroxide (LiOH, 2.5 mmol), methanol (MeOH, 3 mL), and water (H2O, 2 mL) to a 50 mL round-bottom flask. Stir at room temperature for 2 h, and monitor the reaction by TLC (DCM / MeOH = 6 / 1) until starting material 1 disappears. After the reaction is complete, remove methanol by rotary evaporation, add water (5 mL) to the residue, and adjust the pH to 3 with 1 M hydrochloric acid (a solid precipitates). Filter, wash the solid with cold water, and dry to obtain imidazopyridine compound 2 as a white solid with a yield of 95%.

[0064] (5) Under nitrogen protection, imidazopyridine compound 2 (1 mmol) and cyclopropylamine (1.5 mmol) were added to a reaction flask containing DMF (10 mL). HATU (1.5 mmol) and DIPEA (3 mmol) were added sequentially, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, post-processing was performed. The reaction mixture was washed with saturated sodium bicarbonate solution and water, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: CH2Cl2 / MeOH = 20:1) to give imidazopyridine compound 3. The chemical structure is as follows:

[0065] ;

[0066] The results of the detection of imidazopyridine compound 3 are as follows:

[0067] 1 H NMR (500 MHz, Chloroform-d) δ 8.46 (dd, J = 3.8, 1.8 Hz, 1H), 7.99– 7.83 (m, 2H), 7.64 (dd, J = 7.8, 6.5 Hz, 1H), 7.55 (ddd, J = 7.7, 1.9, 1.2Hz, 1H), 7.45 – 7.32 (m, 2H), 6.79 (d, J = 7.5 Hz, 1H), 3.76 (dd, J = 12.3,4.4 Hz, 1H), 3.64 – 3.47 (m, 2H), 3.35 (ddd, J = 12.4, 6.0, 3.7 Hz, 1H), 2.80– 2.59 (m, 2H), 2.47 (s, 3H), 1.95 – 1.58 (m, 4H), 0.72 – 0.51 (m, 4H).

[0068] HRMS (ESI): m / z calcd for C 23 H 26 N5O2 + [M+H] + : 404.2081; found 404.2083.

[0069] Replacing cyclopropylamine with cyclobutylamine in step (5) yields imidazopyridine compound 4. Its chemical structural formula is:

[0070] ;

[0071] The results of the detection of imidazopyridine compound 4 are as follows:

[0072] 1H NMR (500 MHz, Chloroform-d) δ 8.46 (dd, J = 3.8, 1.8 Hz, 1H), 7.99– 7.84 (m, 2H), 7.64 (dd, J = 7.8, 6.5 Hz, 1H), 7.55 (ddd, J = 7.7, 1.9, 1.2Hz, 1H), 7.45 – 7.29 (m, 2H), 6.39 (d, J = 7.3 Hz, 1H), 3.82 – 3.71 (m, 2H), 3.64 – 3.47 (m, 2H), 3.35 (ddd, J = 12.4, 6.0, 3.7 Hz, 1H), 2.66 (ddd, J =6.0, 4.4, 1.5 Hz, 1H), 2.47 (s, 3H), 1.97 – 1.54 (m, 10H).

[0073] HRMS (ESI): m / z calcd for C 24 H 28 N5O2 + [M+H] + : 418.2238; found 418.2235.

[0074] Replacing cyclopropylamine with cyclopentylamine in step (5) yields imidazopyridine compound 5. Its chemical structural formula is:

[0075] ;

[0076] The results of the detection of imidazopyridine compound 5 are as follows:

[0077] 1H NMR (500 MHz, Chloroform-d) δ 8.46 (dd, J = 3.8, 1.8 Hz, 1H), 8.06– 7.84 (m, 2H), 7.72 – 7.48 (m, 2H), 7.47 – 7.26 (m, 2H), 6.55 (d, J = 7.3Hz, 1H), 3.87 (dt, J = 7.2, 3.6 Hz, 1H), 3.76 (dd, J = 12.3, 4.4 Hz, 1H), 3.67 – 3.46 (m, 2H), 3.35 (ddd, J = 12.4, 6.0, 3.7 Hz, 1H), 2.66 (tt, J =6.0, 4.4 Hz, 1H), 2.47 (s, 3H), 1.99 – 1.45 (m, 12H).

[0078] HRMS (ESI): m / z calcd for C 25 H 30 N5O2 + [M+H] + : 432.2394; found 432.2396.

[0079] Replacing cyclopropylamine with cyclohexylamine in step (5) yields imidazopyridine compound 6. Its chemical structural formula is:

[0080] ;

[0081] The results of the detection of imidazopyridine compound 6 are as follows:

[0082] 1 H NMR (500 MHz, Chloroform-d) δ 8.46 (dd, J = 3.8, 1.8 Hz, 1H), 8.02– 7.78 (m, 2H), 7.73 – 7.48 (m, 2H), 7.46 – 7.24 (m, 2H), 6.33 (d, J = 8.1Hz, 1H), 4.11 – 3.48 (m, 4H), 3.35 (ddd, J = 12.4, 6.0, 3.7 Hz, 1H), 2.66(tt, J = 5.9, 4.4 Hz, 1H), 2.47 (s, 3H), 1.97 – 1.19 (m, 14H).

[0083] HRMS (ESI): m / z calcd for C 26 H 32 N5O2 + [M+H] + : 446.2551; found 446.2554.

[0084] Replacing cyclopropylamine with aniline in step (5) yields imidazopyridine compound 7. Its chemical structural formula is:

[0085] ;

[0086] The results of the detection of imidazopyridine compound 7 are as follows:

[0087] 1 H NMR (500 MHz, Chloroform-d) δ 9.22 (s, 1H), 8.46 (dd, J = 3.8, 1.8Hz, 1H), 8.01 – 7.83 (m, 2H), 7.78 – 7.50 (m, 4H), 7.47 – 7.26 (m, 4H), 7.08(tt, J = 7.0, 1.2 Hz, 1H), 3.84 (dd, J = 12.4, 4.5 Hz, 1H), 3.65 (dd, J =12.5, 4.4 Hz, 1H), 3.58 – 3.23 (m, 2H), 2.90 (tt, J = 6.0, 4.5 Hz, 1H), 2.47(s, 3H), 1.96 – 1.72 (m, 4H).

[0088] HRMS (ESI): m / z calcd for C 26 H 26 N5O2 + [M+H] + : 440.2081; found 440.2085.

[0089] Replacing cyclopropylamine with 4-aminopyridine in step (5) yields imidazopyridine compound 8. Its chemical structural formula is:

[0090] ;

[0091] The results of the detection of imidazopyridine compound 8 are as follows:

[0092] 1H NMR (500 MHz, Chloroform-d) δ 9.34 (s, 1H), 8.46 (dd, J = 3.8, 1.8Hz, 1H), 8.36 – 8.31 (m, 2H), 8.05 – 7.78 (m, 2H), 7.71 – 7.48 (m, 4H), 7.45– 7.14 (m, 2H), 3.83 (dd, J = 12.5, 4.4 Hz, 1H), 3.63 (dd, J = 12.4, 4.5 Hz, 1H), 3.57 – 3.49 (m, 1H), 3.39 – 3.31 (m, 1H), 2.91 – 2.83 (m, 1H), 2.47 (s, 3H), 2.15 – 1.68 (m, 4H).

[0093] HRMS (ESI): m / z calcd for C 25 H 25 N6O2 + [M+H] + : 441.2034; found 441.2036.

[0094] Replacing cyclopropylamine with 4-cyanoaniline in step (5) yields imidazopyridine compound 9. Its chemical structural formula is:

[0095] ;

[0096] The results of the detection of imidazopyridine compound 9 are as follows:

[0097] 1 H NMR (500 MHz, Chloroform-d) δ 9.19 (s, 1H), 8.46 (dd, J = 3.8, 1.8Hz, 1H), 8.07 – 7.83 (m, 2H), 7.80 – 7.50 (m, 6H), 7.44 – 7.17 (m, 2H), 3.84(dd, J = 12.4, 4.5 Hz, 1H), 3.65 (dd, J = 12.5, 4.4 Hz, 1H), 3.57 – 3.49 (m,1H), 3.40 – 3.29 (m, 1H), 2.90 (tt, J = 5.9, 4.5 Hz, 1H), 2.47 (s, 3H), 2.01– 1.68 (m, 4H).

[0098] HRMS (ESI): m / z calcd for C 27 H 25 N6O2 + [M+H] + : 465.2034; found 465.2038.

[0099] Replacing cyclopropylamine with 4-nitroaniline in step (5) yields imidazopyridine compound 10. Its chemical structural formula is:

[0100] ;

[0101] The results of the detection of imidazopyridine compound 10 are as follows:

[0102] 1 H NMR (500 MHz, Chloroform-d) δ 9.17 (s, 1H), 8.46 (dd, J = 3.8, 1.8Hz, 1H), 8.35 – 8.07 (m, 2H), 8.04 – 7.78 (m, 4H), 7.77 – 7.49 (m, 2H), 7.45– 7.17 (m, 2H), 3.84 (dd, J = 12.4, 4.5 Hz, 1H), 3.65 (dd, J = 12.5, 4.4 Hz, 1H), 3.57 – 3.49 (m, 1H), 3.40 – 3.31 (m, 1H), 2.90 (tt, J = 6.0, 4.5 Hz, 1H), 2.47 (s, 3H), 2.04 – 1.68 (m, 4H).

[0103] HRMS (ESI): m / z calcd for C 26 H 25 N6O4 + [M+H] + : 485.1932; found 485.1936.

[0104] Replacing cyclopropylamine with 4-methylaniline in step (5) yields imidazopyridine compound 11. Its chemical structural formula is:

[0105] ;

[0106] The results of the detection of imidazopyridine compound 11 are as follows:

[0107] 1H NMR (500 MHz, Chloroform-d) δ 9.15 (s, 1H), 8.46 (dd, J = 3.8, 1.8Hz, 1H), 8.05 – 7.81 (m, 2H), 7.78 – 7.51 (m, 2H), 7.49 – 7.27 (m, 4H), 7.27– 7.05 (m, 2H), 3.84 (dd, J = 12.4, 4.5 Hz, 1H), 3.65 (dd, J = 12.5, 4.4 Hz, 1H), 3.57 – 3.49 (m, 1H), 3.40 – 3.31 (m, 1H), 2.90 (tt, J = 5.8, 4.4 Hz, 1H), 2.47 (s, 3H), 2.35 (d, J = 0.9 Hz, 3H), 1.95 – 1.72 (m, 4H).

[0108] HRMS (ESI): m / z calcd for C 27 H 28 N5O2 + [M+H] + : 454.2238; found 454.2239.

[0109] Replacing cyclopropylamine with 4-fluoroaniline in step (5) yields imidazopyridine compound 12. Its chemical structural formula is:

[0110] ;

[0111] The results of the detection of imidazopyridine compound 12 are as follows:

[0112] 1H NMR (500 MHz, Chloroform-d) δ 9.04 (s, 1H), 8.46 (dd, J = 3.8, 1.8Hz, 1H), 8.03 – 7.80 (m, 2H), 7.74 – 7.53 (m, 2H), 7.50 – 7.26 (m, 4H), 7.23– 6.91 (m, 2H), 3.84 (dd, J = 12.4, 4.5 Hz, 1H), 3.65 (dd, J = 12.5, 4.4 Hz, 1H), 3.57 – 3.49 (m, 1H), 3.39 – 3.31 (m, 1H), 2.90 (tt, J = 5.9, 4.5 Hz, 1H), 2.47 (s, 3H), 2.18 – 1.62 (m, 4H).

[0113] HRMS (ESI): m / z calcd for C 26 H 25 FN5O2 + [M+H] + : 458.1987; found 458.1988.

[0114] Replacing cyclopropylamine with 4-chloroaniline in step (5) yields imidazopyridine compound 13. Its chemical structural formula is:

[0115] ;

[0116] The results of the detection of imidazopyridine compound 13 are as follows:

[0117] 1 H NMR (500 MHz, Chloroform-d) δ 9.10 (s, 1H), 8.46 (dd, J = 3.8, 1.8Hz, 1H), 8.11 – 7.85 (m, 2H), 7.75 – 7.52 (m, 4H), 7.49 – 7.24 (m, 4H), 3.84(dd, J = 12.4, 4.5 Hz, 1H), 3.65 (dd, J = 12.5, 4.4 Hz, 1H), 3.57 – 3.49 (m,1H), 3.39 – 3.31 (m, 1H), 2.90 (tt, J = 5.9, 4.5 Hz, 1H), 2.47 (s, 3H), 1.96– 1.72 (m, 4H).

[0118] HRMS (ESI): m / z calcd for C 26 H 25 ClN5O2 + [M+H] + : 474.1691; found474.1694.

[0119] Replacing cyclopropylamine with 4-bromoaniline in step (5) yields imidazopyridine compound 14. Its chemical structural formula is:

[0120] ;

[0121] The results of the detection of imidazopyridine compound 14 are as follows:

[0122] 1 H NMR (500 MHz, Chloroform-d) δ 9.17 (s, 1H), 8.46 (dd, J = 3.8, 1.8Hz, 1H), 8.08 – 7.80 (m, 2H), 7.77 – 7.07 (m, 8H), 4.22 – 3.18 (m, 4H), 2.90(tt, J = 6.0, 4.5 Hz, 1H), 2.47 (s, 3H), 1.95 – 1.72 (m, 4H).

[0123] HRMS (ESI): m / z calcd for C 26 H 25 BrN5O2 + [M+H] + : 518.1186; found 518.1189.

[0124] Replacing cyclopropylamine with 1-naphthylamine in step (5) yields imidazopyridine compound 15. Its chemical structural formula is:

[0125] ;

[0126] The results of the detection of imidazopyridine compound 15 are as follows:

[0127] 1H NMR (500 MHz, Chloroform-d) δ 9.03 (s, 1H), 8.46 (dd, J = 3.8, 1.8Hz, 1H), 8.10 – 8.05 (m, 1H), 8.02 – 7.73 (m, 4H), 7.72 – 7.25 (m, 8H), 3.84(dd, J = 12.4, 4.5 Hz, 1H), 3.65 (dd, J = 12.5, 4.4 Hz, 1H), 3.57 – 3.49 (m,1H), 3.39 – 3.31 (m, 1H), 2.93 (tt, J = 6.0, 4.5 Hz, 1H), 2.47 (s, 3H), 1.95– 1.72 (m, 4H).

[0128] HRMS (ESI): m / z calcd for C 30 H 28 N5O2 + [M+H] + : 490.2238; found 490.2242.

[0129] Replacing cyclopropylamine with 4-fluoro-1-naphthylamine in step (5) yields imidazopyridine compound 16. Its chemical structural formula is:

[0130] ;

[0131] The results of the detection of imidazopyridine compound 16 are as follows:

[0132] 1H NMR (500 MHz, Chloroform-d) δ 9.14 (s, 1H), 8.46 (dd, J = 3.8, 1.8Hz, 1H), 8.18 (dd, J = 7.8, 1.4 Hz, 1H), 8.07 – 8.00 (m, 1H), 7.93 (dd, J =6.6, 1.8 Hz, 1H), 7.89 (t, J = 1.9 Hz, 1H), 7.64 (dd, J = 7.8, 6.5 Hz, 1H), 7.61 – 7.31 (m, 8H), 7.23 (t, J = 8.0 Hz, 1H), 3.84 (dd, J = 12.4, 4.5 Hz,1H), 3.65 (dd, J = 12.5, 4.4 Hz, 1H), 3.57 – 3.49 (m, 1H), 3.39 – 3.31 (m,1H), 2.93 (tt, J = 5.9, 4.4 Hz, 1H), 2.47 (s, 3H), 1.96 – 1.72 (m, 4H).

[0133] HRMS (ESI): m / z calcd for C 30 H 27 FN5O2 + [M+H] + : 508.2143; found 508.2147.

[0134] Replacing cyclopropylamine with 8-fluoro-1-naphthylamine in step (5) yields imidazopyridine compound 17. Its chemical structural formula is:

[0135] ;

[0136] The results of the detection of imidazopyridine compound 17 are as follows:

[0137] 1H NMR (500 MHz, Chloroform-d) δ 9.16 (s, 1H), 8.46 (dd, J = 3.8, 1.8Hz, 1H), 8.00 – 7.83 (m, 3H), 7.80 – 7.59 (m, 3H), 7.59 – 7.45 (m, 2H), 7.44– 7.29 (m, 3H), 7.19 (td, J = 7.9, 1.0 Hz, 1H), 3.84 (dd, J = 12.4, 4.5 Hz, 1H), 3.65 (dd, J = 12.5, 4.4 Hz, 1H), 3.57 – 3.49 (m, 1H), 3.40 – 3.31 (m,1H), 2.47 (s, 3H), 2.01 – 1.68 (m, 4H).

[0138] HRMS (ESI): m / z calcd for C 30 H 27 FN5O2 + [M+H] + : 508.2143; found 508.2143.

[0139] Replacing cyclopropylamine with 5-fluoro-1-naphthylamine in step (5) yields imidazopyridine compound 18. Its chemical structural formula is:

[0140] ;

[0141] The results of the detection of imidazopyridine compound 18 are as follows:

[0142] 1H NMR (500 MHz, Chloroform-d) δ 9.17 (s, 1H), 8.46 (dd, J = 3.8, 1.8Hz, 1H), 8.07 (dd, J = 7.6, 1.1 Hz, 1H), 8.00 – 7.84 (m, 3H), 7.73 – 7.68 (m,1H), 7.64 (dd, J = 7.8, 6.5 Hz, 1H), 7.55 (ddd, J = 7.7, 1.9, 1.2 Hz, 1H),7.48 – 7.32 (m, 4H), 7.23 (td, J = 7.8, 1.2 Hz, 1H), 3.84 (dd, J = 12.4, 4.5Hz, 1H), 3.65 (dd, J = 12.5, 4.4 Hz, 1H), 3.57 – 3.49 (m, 1H), 3.40 – 3.29(m, 1H), 2.93 (tt, J = 5.9, 4.5 Hz, 1H), 2.47 (s, 3H), 2.04 – 1.68 (m, 4H).

[0143] HRMS (ESI): m / z calcd for C 30 H 27 FN5O2 + [M+H] + : 508.2143; found 508.2144.

[0144] Example of implementation effect 1

[0145] The USP5 enzyme inhibitory activity of the imidazopyridine compounds prepared in Examples 1-3 was studied, as follows:

[0146] The in vitro enzyme activity of USP was assessed using the Ub-AMC (C-terminal-7-acylamino-labeled ubiquitin protein-4-methylcoumarin-labeled) method. The direct interaction between the small molecule and deubiquitinating enzymes was evaluated to assess the activity of the small molecule, with commercially available USP5 inhibitors WP1130 and USP5-IN-1 used as positive controls. The fluorescent substrate AMC does not emit fluorescence when bound to the ubiquitin chain. When USP cleaves the fluorescent substrate AMC from the ubiquitin chain, AMC emits fluorescence. The degree of inhibition by USP was analyzed by detecting the fluorescence intensity. Experiments were performed in 96-well black polypropylene microplates (TECAN) at a volume of 200 μL. Fluorescence measurements were performed using a multi-functional microplate detector (TECAN) with an excitation wavelength of 345 nm and an emission wavelength of 445 nm. 2 nM of USP5 was added to each well. 1−858Different concentrations of the compound were incubated at room temperature for 1 hour. Finally, 250 nM Ub-AMC was added, and fluorescence readings were immediately taken. The data were analyzed using GraphPad Prism 8.2.0 software to calculate the IC50. 50 Values. Specific test results are shown in Table 1; IC 50 Less than 1 μM, marked as ++++, IC 50 At 1 μM-5 μM, marked as +++, active IC 50 In the range of 5 μM to 20 μM, marked as ++, IC 50 A value greater than 20 μM was marked as +; the results are shown in Table 1:

[0147] Table 1 USP5 enzyme activity IC 50

[0148]

[0149] As shown in Table 1, compounds 1-18 all exhibited good USP5 enzyme inhibitory activity. Among them, compounds 1 / 2 / 4 / 6 / 7 / 9 / 11 / 14 / 16 showed USP5 enzyme inhibitory activity comparable to the positive control, while compounds 3 / 5 / 8 / 10 / 12 / 13 / 15 / 17 / 18 showed superior USP5 inhibitory activity compared to the positive control. These compounds hold promise for development as potential small molecule inhibitors of USP5.

[0150] Example 2 of implementation results

[0151] The USP5 enzymatic selectivity of imidazopyridine compound 12 and imidazopyridine compound 18 prepared in Example 3 was studied, as follows:

[0152] The in vitro enzyme activity of deubiquitinating enzymes was assessed using the Ub-AMC (C-terminal-7-acylamino-labeled ubiquitin protein-4-methylcoumarin-labeled) method. The selectivity of the small molecules was evaluated by detecting their direct interaction with the deubiquitinating enzymes, with commercially available USP5 inhibitors WP1130 and USP5-IN-1 used as positive controls. The fluorescent substrate AMC does not emit fluorescence when bound to the ubiquitin chain. AMC emits fluorescence after USP cleaves it from the ubiquitin chain. The degree of inhibition by USP was analyzed by detecting the fluorescence intensity. Experiments were performed in 200 μL volumes in 96-well black polypropylene microplates (TECAN). Fluorescence measurements were performed using a multi-functional microplate detector (TECAN) with an excitation wavelength of 345 nm and an emission wavelength of 445 nm. Add 2 nM of different DUB proteins and different concentrations of compounds to each well and incubate at room temperature for 1 h. Finally, add 250 nM of Ub-AMC, and immediately take fluorescence readings. Analyze the data using GraphPad Prism 8.2.0 software to calculate IC50.50 Values. Specific test results are shown in Table 1; IC 50 Less than 1 μM, marked as ++++, IC 50 At 1 μM-5 μM, marked as +++, active IC 50 In the range of 5 μM to 20 μM, marked as ++, IC 50 A value greater than 20 μM was marked as +; the results are shown in Table 2:

[0153] Table 2 Enzymatic Selectivity

[0154]

[0155] As shown in Table 2, compounds 12 and 18 both exhibited good selectivity for inhibiting USP5 enzyme activity, showing excellent enzymatic inhibitory activity only against USP5, and no significant inhibitory activity against the other deubiquitination enzymes. In contrast, the control compounds WP1130 and USP5-IN-1 showed significantly poor selectivity.

[0156] Example of implementation effect 3

[0157] The in vitro inhibitory effects of the imidazopyridine compounds prepared in Examples 1-3 on cancer cells were studied, as follows:

[0158] The inhibitory effect of imidazopyridine compounds on cancer cells was detected by the MTT assay.

[0159] MTT colorimetric assay procedure: Inoculate cancer cells in the logarithmic growth phase at a concentration of 5 × 10⁻⁶ cells per milliliter. 4 Cells were seeded at the specified density into 96-well cell culture plates, with the zero well containing cell-free normal medium. After 12 hours, the medium was replaced with different concentration gradients of imidazopyridine compounds (or positive controls WP1130 and USP5-IN-1), and the zero wells were replaced with normal medium. Five replicates were set up for each concentration gradient, and the plates were incubated at 37°C in a 5% CO2 incubator. Cell status and growth changes were observed under a microscope after 24 hours. After 48 hours, tetramethylazazole blue (Kaiji Biotechnology, 5 mg / mL) solution was added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator. After 4 hours, the medium was aspirated, and 100 μL of DMSO was added to each well. The absorbance was measured at 570 nm using a microplate reader, and the data were statistically analyzed using Graphpad software to calculate the half-maximal effective concentration (IC50). 50 (Unit: μM). The inhibition effect is shown in Table 3 below. IC50 50 Less than 1 μM, marked as ++++, IC 50 At 1 μM-5 μM, marked as +++, active IC 50 In the range of 5 μM to 20 μM, marked as ++, IC50 A value greater than 20 μM is marked as +;

[0160] Table 3. Effects of imidazopyridine compounds on the proliferation of different cancer cell lines.

[0161]

[0162] In summary, compared with the positive control compounds WP1130 and USP5-IN-1, these imidazopyridine compounds generally exhibited enhanced antiproliferative activity. Furthermore, they were more sensitive to pancreatic cancer cells, demonstrating extremely low IC50 values. 50 It has value for further in-depth research and possesses broad-spectrum anti-tumor activity.

[0163] Example of implementation effect 4

[0164] The imidazopyridine compounds 12 and 18 prepared in Example 3 were evaluated in vivo in animal experiments as follows:

[0165] The effects of the selected imidazopyridine compounds 12 and 18 on pancreatic cancer in vivo were evaluated using a subcutaneous xenograft model. Nu / Nu mice (24 mice) were used and purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The specific method was as follows: Pan02 cells were injected into Nu / Nu immunodeficient mice. When the average tumor diameter reached 3 mm, the mice were randomly divided into a control group (6 mice), a low-dose intraperitoneal injection group of compound 12 (10 mpk, 6 mice), a high-dose intraperitoneal injection group of compound 12 (25 mpk, 6 mice), a low-dose intraperitoneal injection group of compound 18 (10 mpk, 6 mice), a high-dose intraperitoneal injection group of compound 18 (25 mpk, 6 mice), and a positive control group of WP1130 intraperitoneal administration (25 mpk, 6 mice). After 12 days, there were no significant fluctuations in the body weight of the mice in the treatment groups and the control group, and the mice showed no significant abnormalities, indicating that compounds 12 and 18 had good safety. The tumor weight and tumor volume of the mice in the treatment groups were significantly lower than those in the control group. The tumor inhibition rate ((1 - tumor weight in the treatment group / tumor weight in the control group) * 100%) is shown in Table 4.

[0166] Table 4 Pan02 cell xenograft model TGI

[0167]

[0168] The results showed that, compared with the positive control WP1130, both high- and low-dose groups of compounds 12 and 18 exhibited strong activity.

[0169] Tumor growth curve diagram (see) Figure 1As can be seen, the high-dose group of compound 12 showed the best performance, with significant inhibition of tumor growth. The high-dose group of compound 18 also showed a strong tumor inhibition rate. In addition, the low-dose groups of compounds 12 and 18 also showed stronger antitumor activity than the positive control high-dose group. The overall results indicate that compounds 12 and 18 exhibit strong in vivo antitumor activity.

[0170] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An imidazopyridine compound, characterized in that: The structure of the compound is shown in general formula I: （Ⅰ）， The R is -OH, -H, -OR1 or -NHR2, wherein R1 and R2 are independently selected from hydrogen, C1-C6 alkyl, C1-C8 cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

2. The imidazopyridine compound according to claim 1, characterized in that: The R is selected from any of the following structures: -OEt,-OH, , , , , , , , , , , , , , , , 。 3. The method for preparing the imidazopyridine compound according to claim 1 or 2, characterized in that, Includes the following steps: (1) Compound A, compound B, tetrakis(triphenylphosphine)palladium(O) and cesium carbonate were dissolved in toluene, mixed, heated and stirred to carry out the reaction. After the reaction was complete, the mixture was cooled, filtered, concentrated, and purified to obtain compound C. The structural formula of compound A is [insert structural formula here]. The compound B is ; (2) The compound C obtained in step (1) was mixed with dichloromethane and cooled. DMF was added dropwise, followed by the introduction of oxaloyl chloride. The mixture was stirred to carry out the reaction. After the reaction was complete, compound D was obtained by rotary evaporation. The structural formula of compound C is as follows: ; (3) The compound D obtained in step (2), ethyl 4-aminocyclohexanecarboxylate, and dichloromethane were mixed and cooled. Triethylamine was added dropwise, and the mixture was heated and stirred to carry out the reaction. After the reaction was completed, the reaction was quenched. After extraction, drying, filtration, concentration, and purification, imidazopyridine compound 1 was obtained. The structural formula of compound D is as follows: ; (4) The imidazopyridine compound 1 obtained in step (3), lithium hydroxide, methanol and water are mixed and stirred to react. After complete reaction, the mixture is rotary evaporated, water is added to adjust the pH, and the solid obtained by filtration is washed and dried to obtain imidazopyridine compound 2. (5) The imidazopyridine compound 2 and the amine compound obtained in step (4) are dissolved in DMF, and then HATU and DIPEA are added in sequence. The mixture is stirred to carry out the reaction. After the reaction is complete, the mixture is washed, dried, filtered, concentrated and purified to obtain the imidazopyridine compound.

4. The preparation method according to claim 3, characterized in that: In step (1), the molar ratio of compound A, compound B, tetrakis(triphenylphosphine)palladium(0) and cesium carbonate is 1:1.2-1.8:0.02-0.1:1.5-3, and the initial concentration of compound A is 0.05-0.25M. The reaction is carried out under nitrogen protection at a temperature of 80-110℃ for 12-18h.

5. The preparation method according to claim 4, characterized in that: In step (2), based on the molar amount of compound C, 10-15 mL of dichloromethane is added for every 0.7 mmol of compound C, and the cooling temperature is 0-5℃; the molar ratio of compound C to oxalyl chloride is 1:3-10; and the stirring time is 0.5-3 h.

6. The preparation method according to claim 5, characterized in that: In step (3), the molar ratio of compound D, ethyl 4-aminocyclohexanecarboxylate, and triethylamine is 1:1-1.8:2-3. Based on the molar amount of compound D, 5-15 mL of dichloromethane is added for every 0.7 mmol of compound D. The temperature for heating and stirring is 25-40℃, and the time is 6-12 h.

7. The preparation method according to claim 6, characterized in that: In step (4), the molar ratio of imidazopyridine compound 1 to lithium hydroxide is 1:2-10. Based on the molar amount of imidazopyridine compound 1, 3-8 mL of methanol and 2-6 mL of water are added per mmol of imidazopyridine compound 1. The stirring temperature is 25-40℃ and the stirring time is 0.5-5h. The pH is adjusted to 3-5 using 0.5-2M hydrochloric acid solution.

8. The preparation method according to claim 7, characterized in that: In step (5), the molar ratio of imidazopyridine compound 2, amine compound, HATU and DIPEA is 1:1-2:1-3:2-4. The initial concentration of imidazopyridine compound 2 is 0.05-0.2M. The amine compound is a substituted or unsubstituted amine, and the substituted or unsubstituted amine is selected from: substituted or unsubstituted C3-C8 cycloalkylamines, substituted or unsubstituted aromatic amines, and substituted or unsubstituted heteroaromatic amines. The stirring time is 8-24h.

9. The preparation method according to claim 8, characterized in that: The amine compound in step (5) is selected from any one of cyclopropylamine, cyclobutylamine, cyclopentylamine, cyclohexylamine, aniline, 4-aminopyridine, 4-cyanoaniline, 4-nitroaniline, 4-methylaniline, 4-fluoroaniline, 4-chloroaniline, 4-bromoaniline, 1-naphthylamine, 4-fluoro-1-naphthylamine, 8-fluoro-1-naphthylamine, and 5-fluoro-1-naphthylamine.

10. The use of the imidazopyridine compound of claims 1-2 or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting deubiquitinase USP5.

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