CN116082247A - Benzimidazole derivative and application thereof - Google Patents

Abstract

The present invention provides a benzimidazole derivative and its application. The compound is a brand-new compound with targeting MAO-B inhibitory activity, and the diseases with MAO-B overexpression include Alzheimer's disease and Parkinson's disease has potential therapeutic effect. The compounds of the patent WO2017068090 series are predicted by admet.scbdd.com and cannot penetrate the blood-brain barrier; the compounds provided by the present invention have blood-brain barrier permeability and can act on central nervous system diseases including Alzheimer's disease and Parkinson's disease.

Description

A benzimidazole derivative and its application

Technical Field

The present invention provides a benzimidazole derivative and a pharmaceutically acceptable salt thereof and a preparation method thereof, and use thereof as a medicine, in particular as a monoamine oxidase B inhibitor, and more particularly for preparing a medicine for preventing or treating diseases caused by overexpression of monoamine oxidase B, such as Alzheimer's disease and Parkinson's disease.

Background Art

Monoamine oxidases (MAOs) belong to the flavin adenine dinucleotide (FAD)-dependent enzyme family and are located on the outer membrane of mitochondria. MAOs can catalyze the oxidation of various exogenous amines and endogenous neurotransmitters to regulate their levels in the peripheral and central nervous systems. There are two subtypes of MAOs, monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B). They share 70% sequence similarity but differ in substrate specificity, tissue distribution, and inhibitor selectivity. The catalytic mechanism of MAOs is that the deprotonated form of the monoamine substrate binds to the active site of the enzyme, and the amine is oxidized to an imine by reduction of 8α-s-cysteine to a hydroquinone form. The reduced FAD cofactor can then react with _O2 to produce an oxidized form of FAD and hydrogen peroxide, which in turn produces highly toxic hydroxyl radicals, leading to neuronal damage and death.

With age, the expression level of MAO-B in neural tissue, especially in glial cells, increases fourfold, leading to increased levels of dopamine metabolism and an increase in toxic byproducts. Studies have shown that MAO-B overexpression plays an important role in the pathology of neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. MAO-B inhibitors can inhibit the catalytic activity of the enzyme and reduce the metabolism of monoamine neurotransmitters. Therefore, MAO-B inhibitors are considered to be therapeutic drugs for diseases related to MAO-B overexpression. In addition, MAO-B inhibitors can also reduce the production of toxic byproducts such as hydrogen peroxide, toxic aldehydes and hydroxyl radicals by blocking the monoamine oxidation process, thereby preventing damage to neurons. Therefore, MAO-B inhibitors are also considered to be potential neuroprotective agents.

Summary of the invention

In order to solve the above problems, the present invention designs and synthesizes a benzimidazole derivative with potential MAO-B inhibitory activity based on computer-aided drug design, drug-like property prediction, blood-brain barrier permeability experiment and the like, provides a preparation method of benzimidazole derivatives and pharmaceutically acceptable salts thereof, and prevents or treats diseases related to MAO-B overexpression, including Alzheimer's disease and Parkinson's disease, by inhibiting MAO-B activity.

To achieve the above object, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a benzimidazole derivative represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof:

In formula (I):

R ¹ and R ² are each independently H, halogen, C ₁ -C ₅ straight chain or branched chain alkyl or C ₁ -C ₅ straight chain or branched chain alkoxy;

m is 0, 1 or 2; when m is 0, the carbonyl group is directly connected to the C on the imidazole.

In formula (II):

R ³ and R ⁴ are each independently H, halogen, C ₁ -C ₅ haloalkyl, C ₁ -C ₅ straight or branched alkyl, or C ₁ -C ₅ straight or branched alkoxy;

n is 0, 1 or 2. When n is 0, the carbonyl group is directly connected to the C on the imidazole.

Preferably, in formula (I), R ¹ and R ² are each independently H, F, Cl, CH ₃ or OCH ₃ ; and m is 1.

Preferably, in formula (II), R ³ and R ⁴ are each independently H, F, Cl, CF ₃ , CH ₃ or OCH ₃ ; and n is 1.

Specifically, the benzimidazole derivative represented by formula (I) or formula (II) is one of the following compounds:

In a second aspect, the present invention provides the use of the benzimidazole derivatives represented by formula (I) or formula (II) or their pharmaceutically acceptable salts in the preparation of drugs for treating or preventing diseases caused by overexpression of MAO-B.

Furthermore, the disease caused by overexpression of MAO-B is Parkinson's disease or Alzheimer's disease.

Preferably, the benzimidazole derivative represented by formula (I) or formula (II) is a compound represented by formula b2, b3, b5, b6, b8 or b12, and the compound represented by b3 is particularly preferred.

In a third aspect, the present invention also provides a method for preparing the benzimidazole derivatives represented by formula (I) or formula (II) or their pharmaceutically acceptable salts.

1. A method for preparing a benzimidazole derivative represented by formula (I), the method comprising the following steps:

(1) dissolving the compound of Formula 2 in an organic solvent A, adding alkaline substance A and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) in sequence, stirring at room temperature, adding the aniline compound of Formula 9, stirring at room temperature for 3-18 hours (8 hours in one embodiment of the present invention), and subjecting the obtained reaction solution A to post-treatment A to obtain the compound of Formula 3; the molar ratio of the compound of Formula 2, the alkaline substance A, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and the aniline compound of Formula 9 is 1:1-3:1-1.5:0.5-1.5 (preferably 1:2:1.1:1); the alkaline substance A is one or a mixture of two or more of potassium carbonate, sodium carbonate, sodium bicarbonate, potassium hydroxide, sodium hydroxide and triethylamine (preferably triethylamine);

(2) The compound of formula 3 in step (1) is reacted in concentrated hydrochloric acid at 40° C. for 35-50 min, and the resulting reaction solution B is subjected to post-treatment B to obtain a benzimidazole derivative of formula (I);

Furthermore, in step (1), the organic solvent A is one or a mixture of two or more of acetonitrile, N,N-dimethylformamide (DMF), methanol, ethanol, acetonitrile, and dichloromethane (DCM), preferably N,N-dimethylformamide (DMF).

Furthermore, in step (1), the volume of the organic solvent A is 1 to 2 mL/mmol based on the amount of the compound represented by formula 2.

Furthermore, in step (1), the post-treatment A is: adding saturated brine, extracting with dichloromethane (DCM), combining the organic phases, and drying over anhydrous sodium sulfate; removing the solvent by distillation under reduced pressure, performing column chromatography separation using a mixed solution of dichloromethane and methanol in a volume ratio of 30:1 as an eluent, collecting the eluate containing the target intermediate, concentrating, and drying to obtain the compound shown in Formula 3.

Furthermore, in step (2), the volume of the concentrated hydrochloric acid is 2 to 4 mL/mmol based on the amount of the compound represented by formula 3.

Furthermore, in step (2), the post-treatment B is: adjusting the pH of the reaction solution B to neutral with a saturated sodium bicarbonate solution, precipitating a solid, filtering with suction, and separating the filter cake by column chromatography using a mixed solution of dichloromethane and methanol in a volume ratio of 20:1 as an eluent, collecting the eluate containing the target compound, concentrating, and drying to obtain the benzimidazole derivative represented by formula (I).

2. A method for preparing a benzimidazole derivative represented by formula (II), comprising the following steps:

S1: dissolving the compound of formula 5 in solvent D, adding 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and alkaline substance D, stirring at room temperature for 5 minutes, and then adding cyanoacetic acid; stirring at room temperature for 5-10 hours, and subjecting the obtained reaction solution D to post-treatment D to obtain the compound of formula 6; the molar ratio of the compound of formula 5, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), alkaline substance D and cyanoacetic acid is 1:1-2:1.5-2.5:1-1.5 (preferably 1:1.1:2:1.1);

S2: dissolving the compound of formula 6 described in step S1 in a mixed solvent E, adding Zn powder and NH ₄ Cl in sequence, stirring and reacting at 50°C for 10 minutes, and subjecting the obtained reaction solution E to post-treatment E to obtain a compound of formula 7; the molar ratio of the compound of formula 6, Zn powder and NH ₄ Cl is 1:5-10:5-10 (preferably 1:8:8);

S3: dissolving the compound of formula 7 described in step S2 in acetic acid, and refluxing the reaction for 2 hours. The resulting reaction solution G is subjected to post-treatment G to obtain the compound of formula 8;

S4: dissolving the compound of formula 8 described in step S3 in a solvent Q, adding 30% by mass of hydrogen peroxide and alkaline substance Q in sequence, stirring at 25°C for 3-8h, and subjecting the obtained reaction solution Q to post-treatment Q to obtain a benzimidazole derivative of formula (II); the molar ratio of the compound of formula 8, hydrogen peroxide contained in hydrogen peroxide and alkaline substance Q is 1:5-12:0.2-0.5 (preferably 1:8:0.2);

The ranges of R ¹ to R ⁴ in Formulas 1-9 are the same as those described above.

Further, in step S1, the solvent D is one or a mixture of two or more of water, toluene, acetonitrile, dichloromethane (DCM), methanol, ethanol, N,N-dimethylformamide (DMF), preferably dichloromethane (DCM).

Furthermore, in step S1, the volume of the solvent D is 1 to 3 mL/mmol based on the amount of the compound represented by formula 5.

Further, in step S1, the alkaline substance D is one or a mixture of two or more of potassium carbonate, sodium carbonate, sodium bicarbonate, potassium hydroxide, sodium hydroxide, N,N-diisopropylethylamine (DIPEA), and triethylamine, preferably N,N-diisopropylethylamine (DIPEA).

Further, in step S1, the post-treatment D is: distilling the reaction solution D under reduced pressure to remove part of the solvent, filtering with suction, washing the obtained filter cake A with methanol, slurrying the obtained filter cake B with methanol, filtering with suction, and drying the obtained filter cake C to obtain the compound shown in formula 6.

Further, in step S2, the mixed solvent E is a mixed solvent of water and an organic solvent E, and the organic solvent E is a mixture of one or more of toluene, acetonitrile, dichloromethane (DCM), methanol, ethanol, and N,N-dimethylformamide (DMF), preferably a mixed solvent of ethanol, water and acetonitrile in a volume ratio of 2:1:1.

Furthermore, in step S2, the volume of the mixed solvent E is 8 to 12 mL/mmol based on the amount of the compound represented by formula 6.

Further, in step S2, the post-treatment E is: filtering the reaction solution E, distilling the obtained filtrate under reduced pressure, extracting with dichloromethane (DCM) and saturated brine, drying the obtained organic phase with anhydrous sodium sulfate, and distilling under reduced pressure to obtain the compound shown in formula 7.

Furthermore, in step S3, the volume of the acetic acid is 5 to 8 mL/mmol based on the amount of the compound represented by formula 7.

Further, in step S3, the post-treatment G is: extracting the reaction solution G with ethyl acetate (EA) and saturated brine, drying the obtained organic phase with anhydrous sodium sulfate, and distilling under reduced pressure to obtain the compound shown in formula 8.

Further, in step S4, the solvent Q is one or a mixture of two or more of water, methanol, acetonitrile, dichloromethane (DCM), ethanol, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), preferably dimethyl sulfoxide (DMSO).

Furthermore, in step S4, the volume of the solvent Q is 1 to 3 mL/mmol based on the amount of the compound represented by formula 8.

Further, in step S4, the alkaline substance Q is one or a mixture of two or more of potassium carbonate, sodium carbonate, sodium bicarbonate, potassium hydroxide, and sodium hydroxide, preferably potassium carbonate.

Further, in step S4, the post-treatment Q is: adding water to the reaction solution Q to precipitate solids, filtering, and purifying the filter cake by column chromatography using a mixed solution of DCM and methanol in a volume ratio of 20:1 as an eluent, collecting the eluate containing the target compound, concentrating, and drying to obtain the benzimidazole derivative shown in (II).

The present invention also provides a method for preparing the compound represented by formula 2:

The compound represented by Formula 1 is dissolved in an organic solvent C, ethyl cyanoacetate is added, and the mixture is refluxed for 2 hours. The obtained reaction solution C is subjected to post-treatment C to obtain the compound represented by Formula 2; the molar ratio of the compound represented by Formula 1 to ethyl cyanoacetate is 1:1 to 5 (preferably 1:3);

Furthermore, the organic solvent C is one or a mixture of two or more of toluene, acetonitrile, dichloromethane (DCM), methanol, ethanol, N,N-dimethylformamide (DMF), preferably DMF.

Furthermore, the volume of the organic solvent C is 1 to 2 mL/mmol based on the amount of the compound of Formula 1.

Further, the post-treatment C is: adding saturated brine to the reaction solution C, extracting with ethyl acetate (EA), combining the organic phases, drying with anhydrous sodium sulfate, concentrating under reduced pressure, recrystallizing with a mixed solvent of methanol, ethyl acetate (EA) and petroleum ether (PE) in a volume ratio of 1:5:20, filtering with suction, and drying the obtained filter cake to obtain the compound shown in Formula 2.

Further, more specifically, the compound represented by formula 5 of the present invention is prepared according to the following method:

The compound of Formula 4 is dissolved in a mixed solvent H, and alkaline substance I and tetrabutylammonium bromide (TBAB) are added in sequence, and finally the benzyl bromide compound of Formula 10 is added, and the mixture is stirred at room temperature for 2 hours to obtain a reaction solution J, which is then post-treated to obtain the compound of Formula 5; the molar ratio of the compound of Formula 4, benzyl bromide with different substituents, TBAB and alkaline substance is 1:1-2:0.1-0.5:1-3 (preferably 1:1.4:0.1:2);

Further, the mixed solvent H is a mixed solvent of water and an organic solvent H, and the organic solvent H is one or a mixture of two or more of toluene, acetonitrile, DCM, methanol, ethanol, and DMF, preferably a mixed solvent of DCM and water in a volume ratio of 1:1.

Furthermore, the volume of the mixed solvent H is 4 to 8 mL/mmol based on the amount of the compound of formula 4.

Furthermore, the alkaline substance I is one or a mixture of two or more of potassium carbonate, sodium carbonate, sodium bicarbonate, potassium hydroxide, sodium hydroxide or triethylamine, preferably potassium hydroxide.

Furthermore, the post-treatment J is: distilling the reaction solution J under reduced pressure to remove the solvent, adding saturated brine, and extracting with ethyl acetate (EA); combining the organic phases and drying over anhydrous sodium sulfate; concentrating the solvent by distillation under reduced pressure, and performing column chromatography separation using a mixed solvent of PE and DCM in a volume ratio of 1:1 as an eluent, collecting the eluate containing the target compound, concentrating, and drying to obtain the compound shown in Formula 5.

The above letters are used to distinguish different reaction stages or substances in different stages for the convenience of description and have no other special meanings.

Compared with the prior art, the beneficial effects of the present invention are as follows: the compounds provided by the present invention are a new class of compounds, which have targeted MAO-B inhibitory activity and have potential therapeutic effects on diseases with MAO-B overexpression, including Alzheimer's disease and Parkinson's disease. The compounds of patent WO2017068090 are predicted by admet.scbdd.com to be unable to pass through the blood-brain barrier; the compounds provided by the present invention have blood-brain barrier permeability and can act on central nervous system diseases including Alzheimer's disease and Parkinson's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of the pharmacokinetic test.

Specific implementation methods

The present invention is further described below with reference to specific examples, but the present invention is not limited to the following examples.

Example 1 Preparation method of a1

Add 3,4-diaminobenzoic acid (1.522g, 10mmol), DMF (12mL), ethyl cyanoacetate (3.394g, 30mmol) to a single-mouth bottle, heat to 160℃ and react for 2h; after TLC monitoring, the reaction was stopped when the raw materials reacted completely; add saturated brine (200mL), extract with EA (100mL); combine the organic phases, dry over anhydrous sodium sulfate, concentrate under reduced pressure, recrystallize with a mixed solvent of methanol, EA, and PE (v/v/v＝1:5:20), filter, collect the filter cake, and dry. The brown solid is 1.368g of crude 2-cyanomethyl-5-carboxyl-1H-benzimidazole. The yield is 68%.

2-Cyanomethyl-5-carboxyl-1H-benzimidazole (0.805 g, 4 mmol), DMF (5 mL), triethylamine (0.810 g, 8 mmol), HATU (1.673 g, 4.4 mmol) obtained in the above reaction were added to a round-bottom flask, stirred evenly at room temperature, and then o-fluoroaniline (0.444 g, 4 mmol) was added, and stirring was continued for 8 h; after the reaction was completed, saturated brine (200 mL) was added, extracted with DCM (120 mL), the organic phases were combined, and dried over anhydrous sodium sulfate; the solvent was removed by distillation under reduced pressure, and column chromatography was performed for separation (DCM: methanol v/v = 30:1), and the eluent containing the target intermediate was collected, concentrated, and dried to obtain 0.259 g of a reddish brown intermediate. The yield was 22%.

The reddish brown intermediate (0.259 g, 0.88 mmol) obtained in the above reaction was added to a round-bottom flask, and 3 mL of concentrated hydrochloric acid (w/v = 36%) was added, and the reaction was carried out at 40°C for 35 min. After the reaction was completed, the pH was adjusted to neutral with a saturated sodium bicarbonate solution, and the solid was precipitated. The filter cake was collected by suction and separated by column chromatography (DCM: methanol v/v = 20:1). The eluate containing the target compound was collected, concentrated, and dried to obtain a white solid a1 (0.107 g). The yield was 39%. mp＝259.1-260.8℃; ¹ H NMR (600MHz, DMSO-d ₆ ) δ12.62(s,1H),10.05(s,1H),8.20(s,1H),7.83(dd,J＝8.4,1.8Hz,1H),7.75(s,1H),7.65–7.57(m,2H),7.32 –7.18 (m, 4H), 3.79 (s, 2H). ¹³ C NMR (100MHz, DMSO-d ₆ , TFA) δ166.82, 164.88, 158.72 (q, ² J _CF = 37.8Hz, TFA), 158.08 (d, ¹ J _CF ＝245.5Hz),151.00,133.20,131.66,130.87,127.41,127.40,127.30(d, ³ J _CF ＝7.6Hz),125.82(d, ² J _CF ＝12.2Hz),125.57,124.50(d, ⁴ J _CF ＝3.5 Hz), 116.03 (d, ² J _CF = 19.7Hz), 114.41 (q, ¹ J _CF = 287.1Hz, TFA), 114.17 (d, ³ J _CF = 11.0Hz), 33.60. HRMS (ESI): m/z calcd for C ₁₆ H ₁₄ FN ₄ O ₂ [M+H] ⁺ 313.10 95,found 313.1097; HPLC purity:100%.

Example 2 Preparation method of a2

2-Cyanomethyl-5-carboxyl-1H-benzimidazole (0.805 g, 4 mmol), DMF (5 mL), triethylamine (0.810 g, 8 mmol), and HATU (1.673 g, 4.4 mmol) prepared according to Example 1 were added to a round-bottom flask. After stirring at room temperature, m-fluoroaniline (0.444 g, 4 mmol) was added and stirring was continued for 8 h. After the reaction was completed, saturated brine (200 mL) was added, and DCM was used for extraction (120 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and column chromatography was performed for separation (DCM: methanol v/v = 30:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 0.283 g of a reddish brown intermediate. The yield was 24%.

The reddish brown intermediate (0.283 g, 0.96 mmol) obtained in the above reaction was added to a round-bottom flask, and 3 mL of concentrated hydrochloric acid (w/v = 36%) was added, and the reaction was carried out at 40°C for 35 min. After the reaction was completed, the pH was adjusted to neutral with a saturated sodium bicarbonate solution, and the solid was precipitated. The filter cake was collected by suction and separated by column chromatography (DCM: methanol v/v = 20:1). The intermediate containing the target compound was collected, concentrated, and dried to obtain a khaki solid a2 (0.120 g). The yield was 40%. mp＝141.8-156.1℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.57(d,J＝28.8Hz,1H),10.36(s,1H),8.18(d,J＝64.8Hz,1H),7.81(m,2H),7.72(s,1H),7.68–7.51(m,2 H), 7.38 (q, J＝7.9Hz, 1H), 7.19 (s, 1H), 6.91 (td, J＝8.4, 2.8Hz, 1H), 3.79 (s, 2H). ¹³ C NMR (100MHz, DMSO-d ₆ , TFA) δ 166.90, 165.22, 162.37 (d, ¹ J _CF ＝239.7Hz),158.80(q, ² J _CF ＝37.8Hz, TFA),151.09,141.04(d, ³ J _CF ＝11.0Hz),133.20,132.37,130.88,130.47(d, ³ J _CF ＝9.3Hz),125.57,116.40( d, ⁴ J _CF = 2.5Hz), 115.46 (q, ¹ J _CF = 287.0Hz, TFA), 114.23, 114.15, 110.57 (d, ² J _CF = 21.0Hz), 107.40 (d, ² J _CF = 26.1Hz), 33.64. HRMS (ESI): m/z calcd for C ₁₆ H ₁₄ FN ₄ O ₂ [M+H] ⁺ 313.1095, found 313.1088; HPLC purity: 100%.

Example 3 Preparation method of a3

2-Cyanomethyl-5-carboxyl-1H-benzimidazole (0.805 g, 4 mmol), DMF (5 mL), triethylamine (0.810 g, 8 mmol), and HATU (1.673 g, 4.4 mmol) prepared according to Example 1 were added to a round-bottom flask. After stirring at room temperature, p-fluoroaniline (0.444 g, 4 mmol) was added and stirring was continued for 9 h. After the reaction was completed, saturated brine (200 mL) was added, and DCM was used for extraction (120 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and column chromatography was performed for separation (DCM: methanol v/v = 30:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 0.353 g of a reddish brown intermediate. The yield was 30%.

The reddish brown intermediate (0.353 g, 1.2 mmol) obtained in the above reaction was added to a round-bottom flask, and 4 mL of concentrated hydrochloric acid (w/v = 36%) was added, and the reaction was carried out at 40°C for 40 min. After the reaction was completed, the pH was adjusted to neutral with a saturated sodium bicarbonate solution, and the solid was precipitated. The filter cake was collected by suction and separated by column chromatography (DCM: methanol v/v = 20:1). The eluate containing the target compound was collected, concentrated, and dried to obtain an off-white solid a3 (0.146 g). The yield was 39%. mp＝132.0-139.5℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.52(s,1H),10.24(s,1H),8.17(d,J＝59.6Hz,1H),7.82(m,3H),7.71(s,1H),7.59(d,J＝20.8Hz,1H),7.23 –7.13 (m, 3H), 3.78 (s, 2H). ¹³ C NMR (100MHz, DMSO-d ₆ , TFA) δ166.92, 164.88, 158.82 (q, ² J _CF = 38.0Hz, TFA), 158.80 (d, ¹ J _CF = 239.2Hz), 151.01, 135.62 (d, ⁴ J _CF ＝2.6Hz),133.11,132.63,130.91,125.56,122.78(d, ³ _JCF ＝7.8Hz), _{115.47(q, 1JCF＝286.9Hz,TFA),115.43(d, 2JCF} ^＝ _22.1Hz ⁾ ,114.13,114.10,3 3.66.HRMS(ESI): m/zcalcd for C ₁₆ H ₁₄ FN ₄ O ₂ [M+H] ⁺ 313.1095, found 313.1090; HPLC purity: 99.84%.

Preparation method of embodiment 4a4

2-Cyanomethyl-5-carboxyl-1H-benzimidazole (0.805 g, 4 mmol), DMF (5 mL), triethylamine (0.810 g, 8 mmol), and HATU (1.673 g, 4.4 mmol) prepared according to Example 1 were added to a round-bottom flask. After stirring at room temperature, aniline (0.373 g, 4 mmol) was added and stirring was continued for 5 h. After the reaction was completed, saturated brine (200 mL) was added, and DCM was used for extraction (120 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and column chromatography was performed for separation (DCM: methanol v/v = 30:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 0.287 g of a reddish brown intermediate. The yield was 26%.

The reddish brown intermediate (0.287 g, 1.04 mmol) obtained in the above reaction was added to a round-bottom flask, and 4 mL of concentrated hydrochloric acid (w/v = 36%) was added, and the reaction was carried out at 40°C for 40 min. After the reaction was completed, the pH was adjusted to neutral with a saturated sodium bicarbonate solution, and the solid was precipitated. The filter cake was collected by suction and separated by column chromatography (DCM: methanol v/v = 20:1). The eluate containing the target compound was collected, concentrated, and dried to obtain an off-white solid a4 (0.125 g). The yield was 41%. mp＝150.5-153.2℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.55(s,1H),10.21(s,1H),8.22(s,1H),7.88–7.69(m,4H),7.59(s,1H),7.35(t,J＝7.6Hz,2H),7.22(s,1 H), 7.09 (t, J = 7.2Hz, 1H), 3.78 (s, 2H). ¹³ C NMR (100MHz, DMSO-d ₆ , TFA) δ 167.02, 165.05, 158.93 (q, ² J _CF =38.0Hz,TFA),151.05,139.37,133.14,132.89,130.96,128.95,125.69,124.25,120.99,115.53(q, ¹ J _CF =286.8Hz,TFA),114.23,114.15,33.72.H RMS (ESI): m/z calcd for C ₁₆ H ₁₅ N ₄ O ₂ [M+H] ⁺ 295.1190, found 295.1195; HPLC purity: 100%.

Preparation method of embodiment 5a5

2-Cyanomethyl-5-carboxyl-1H-benzimidazole (0.805 g, 4 mmol), DMF (5 mL), triethylamine (0.810 g, 8 mmol), and HATU (1.673 g, 4.4 mmol) prepared according to Example 1 were added to a round-bottom flask. After stirring at room temperature, m-chloroaniline (0.510 g, 4 mmol) was added and stirring was continued for 10 h. After the reaction was completed, saturated brine (200 mL) was added, and DCM was used for extraction (120 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and column chromatography was performed for separation (DCM: methanol v/v = 30:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 0.286 g of a reddish brown intermediate. The yield was 23%.

The reddish brown intermediate (0.286 g, 0.92 mmol) obtained in the above reaction was added to a round-bottom flask, and 3.5 mL of concentrated hydrochloric acid (w/v = 36%) was added, and the reaction was carried out at 40°C for 45 min. After the reaction was completed, the pH was adjusted to neutral with a saturated sodium bicarbonate solution, and the solid was precipitated. The filter cake was collected by suction and separated by column chromatography (DCM: methanol v/v = 20:1). The intermediate containing the target compound was collected, concentrated, and dried to obtain an off-white solid a5 (0.118 g). The yield was 39%. mp＝227.8-231.1℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.56(m,1H),10.34(s,1H),8.18(d,J＝52.4Hz,1H),8.01(m,1H),7.87–7.67(m,3H),7.60(s,1H),7.38( t,J＝8.0Hz,1H),7.25–7.01(m,2H),3.78(s,2H). ¹³ CNMR(150MHz,DMSO-d ₆ ,TFA)δ166.94,165.23,158.87(q, ² J _CF ＝37.5Hz,TFA),151.10,140.77,133.33,133.27,132.33,130.95,130.54,125.59,123.85,120.27,119.11,115.55(q, ¹ J _CF ＝287.1Hz,TFA),114.25, 114.18,33.67.HRMS (ESI): m/z calcd for C ₁₆ H ₁₄ ClN ₄ O ₂ [M+H] ⁺ 329.0800, found 329.0806; HPLC purity: 100%.

Preparation method of embodiment 6a6

2-Cyanomethyl-5-carboxyl-1H-benzimidazole (0.805 g, 4 mmol), DMF (5 mL), triethylamine (0.810 g, 8 mmol), and HATU (1.673 g, 4.4 mmol) prepared according to Example 1 were added to a round-bottom flask. After stirring at room temperature, p-chloroaniline (0.510 g, 4 mmol) was added and stirring was continued for 8 h. After the reaction was completed, saturated brine (200 mL) was added, and DCM was used for extraction (120 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and column chromatography was performed for separation (DCM: methanol v/v = 30:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 0.298 g of a reddish brown intermediate. The yield was 24%.

The reddish brown intermediate (0.298 g, 0.96 mmol) obtained in the above reaction was added to a round-bottom flask, and 3.5 mL of concentrated hydrochloric acid (w/v = 36%) was added, and the reaction was carried out at 40°C for 50 min. After the reaction, the pH was adjusted to neutral with saturated sodium bicarbonate solution, and the solid was precipitated. The filter cake was collected by suction and separated by column chromatography (DCM: methanol v/v = 20:1). The eluate containing the target compound was collected, concentrated, and dried to obtain a white solid a6 (0.126 g). The yield was 40%. mp＝251.3-254.7℃; ¹ H NMR (600MHz, DMSO-d ₆ ) δ12.59 (s, 1H), 10.33 (s, 1H), 8.18 (s, 1H), 7.89–7.83 (m, 2H), 7.80 (dd, J＝8.4, 1.8Hz, 1H), 7.74 (s, 1H), 7. 60(d,J＝8.4Hz,1H),7.43–7.38(m,2H),7.20(s,1H),3.78(s,2H). ¹³ CNMR(100MHz,DMSO-d ₆ ,TFA)δ166.88,165.02,158.79(q, ² J _CF ＝38.0Hz,TFA),151.04,138.25,133.15,132.48,130.89,128.77,127.88,125.57,122.33,115.45(q, ¹ J _CF ＝287.0Hz,TFA),114.19,114.10,33.64.H RMS (ESI): m/z calcd for C ₁₆ H ₁₄ ClN ₄ O ₂ [M+H] ⁺ 329.0800, found 329.0796; HPLC purity: 100%.

Preparation method of embodiment 7a7

2-Cyanomethyl-5-carboxyl-1H-benzimidazole (0.805 g, 4 mmol), DMF (5 mL), triethylamine (0.810 g, 8 mmol), and HATU (1.673 g, 4.4 mmol) prepared according to Example 1 were added to a round-bottom flask. After stirring at room temperature, o-methylaniline (0.429 g, 4 mmol) was added and stirring was continued for 4 h. After the reaction was completed, saturated brine (200 mL) was added, and DCM was used for extraction (120 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and column chromatography was performed for separation (DCM: methanol v/v = 30:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 0.372 g of a reddish brown intermediate. The yield was 32%.

The reddish brown intermediate (0.372 g, 1.28 mmol) obtained in the above reaction was added to a round-bottom flask, and 4 mL of concentrated hydrochloric acid (w/v = 36%) was added, and the reaction was carried out at 40°C for 40 min. After the reaction was completed, the pH was adjusted to neutral with a saturated sodium bicarbonate solution, and the solid was precipitated. The filter cake was collected by suction and separated by column chromatography (DCM: methanol v/v = 20:1). The eluate containing the target compound was collected, concentrated, and dried to obtain an off-white solid a7 (0.162 g). The yield was 41%. mp＝232.9-236.4℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.53(s,1H),9.30(s,1H),8.15(s,1H),7.87(dd,J＝7.6,1.6Hz,1H),7.79(dd, _J ＝8.4,1.6Hz,1H),7.71(s,1H ¹³ C NMR ,TFA)δ167.01,164.66,158.95(q, ² J _CF =37.3Hz,TFA),152.24,150.99,133.15,132.27,130.99,126.80,126.50,125.41,120.48,115.66(q, ¹ J _CF =286.1Hz, TFA), 114.23, 114.20, 114.08, 111.68, 55.85, 33.64. HRMS (ESI): m/z calcd for C ₁₇ H ₁₇ N ₄ O ₂ [M+H] ⁺ 309.1346, found309.1359; HPLC purity: 100%.

Preparation method of embodiment 8a8

In a round-bottom flask, 2-cyanomethyl-5-carboxyl-1H-benzimidazole (0.805 g, 4 mmol), DMF (5 mL), triethylamine (0.810 g, 8 mmol), and HATU (1.673 g, 4.4 mmol) prepared in Example 1 were added, stirred evenly at room temperature, and then m-methylaniline (0.429 g, 4 mmol) was added, and stirring was continued for 4.5 h. After the reaction was completed, saturated brine (200 mL) was added, and DCM was used for extraction (120 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and the mixture was separated by column chromatography (DCM: methanol v/v = 30:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 0.383 g of a reddish brown intermediate. The yield was 33%.

The reddish brown intermediate (0.383 g, 1.32 mmol) obtained in the above reaction was added to a round-bottom flask, and 4 mL of concentrated hydrochloric acid (w/v = 36%) was added, and the reaction was carried out at 40°C for 40 min. After the reaction was completed, the pH was adjusted to neutral with a saturated sodium bicarbonate solution, and the solid was precipitated. The filter cake was collected and separated by column chromatography (DCM: methanol v/v = 20:1). The eluate containing the target intermediate was collected, concentrated, and dried to obtain a white solid a8 (0.155 g). The yield was 38%. mp＝187.2-195.3℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.57(s,1H),10.13(s,1H),8.18(s,1H),7.80(dd,J＝8.8,2.0Hz,1H),7.74(s,1H),7.65(m,1H),7.59(m, 2H),7.22(m,2H),6.91(d,J＝7.2Hz,1H),3.78(s,2H),2.31(s,3H). ¹³ CNMR(100MHz,DMSO-d ₆ ,TFA)δ166.97,164.90,158.88(q, ² J _CF ＝37.9Hz,TFA),150.99,139.22,138.16,133.08,132.84,130.87,128.75,125.61,124.90,121.47,118.11,115.52(q, ¹ J _CF ＝287.0Hz,TFA),114.13, 114.09,33.68,21.35.HRMS(ESI):m/z calcd forC ₁₇ H ₁₇ N ₄ O ₂ [M+H] ⁺ 309.1346, found 309.1344; HPLC purity: 100%.

Preparation method of embodiment 9a9

In a round-bottom flask, 2-cyanomethyl-5-carboxyl-1H-benzimidazole (0.805 g, 4 mmol), DMF (5 mL), triethylamine (0.810 g, 8 mmol), and HATU (1.673 g, 4.4 mmol) prepared in Example 1 were added, stirred evenly at room temperature, and p-methylaniline (0.429 g, 4 mmol) was added, and stirring was continued for 4 h. After the reaction was completed, saturated brine (200 mL) was added, and DCM was used for extraction (120 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and column chromatography was performed for separation (DCM: methanol v/v = 30:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 0.372 g of a reddish brown intermediate. The yield was 32%.

The reddish brown intermediate (0.372 g, 1.28 mmol) obtained in the above reaction was added to a round-bottom flask, and 4 mL of concentrated hydrochloric acid (w/v = 36%) was added, and the reaction was carried out at 40°C for 40 min. After the reaction was completed, the pH was adjusted to neutral with a saturated sodium bicarbonate solution, and the solid was precipitated. The filter cake was collected by suction and separated by column chromatography (DCM: methanol v/v = 20:1). The eluate containing the target compound was collected, concentrated, and dried to obtain an off-white solid a9 (0.162 g). The yield was 41%.

mp＝235.7-238.9℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.50 (s, 1H), 10.10 (s, 1H), 8.22 (s, 1H), 7.80 (d, J＝8.4Hz, 1H),7.74–7.63(m,3H),7.58(s,1H),7.16(m,3H),3.78(s,2H),2.28(s,3H). ¹³ C NMR(100MHz,DMSO-d ₆ ,TFA)δ167.06,164.85,159.00(q, ² J _CF =38.0Hz,TFA),150.97,136.81,133.39,133.11,132.97,130.99,129.34,125.66,121.08,115.58(q, ¹ J _CF =286.6Hz,TFA),114.15,114.10,33.72,2 0.64.HRMS(ESI ): m/z calcd forC ₁₇ H ₁₇ N ₄ O ₂ [M+H] ⁺ 309.1346, found 309.1360; HPLC purity: 100%.

Preparation method of embodiment 10a10

2-Cyanomethyl-5-carboxyl-1H-benzimidazole (0.805 g, 4 mmol), DMF (5 mL), triethylamine (0.810 g, 8 mmol), and HATU (1.673 g, 4.4 mmol) prepared according to Example 1 were added to a round-bottom flask. After stirring at room temperature, o-anisidine (0.493 g, 4 mmol) was added and stirring was continued for 6 h. After the reaction was completed, saturated brine (200 mL) was added, and the mixture was extracted with dichloromethane (120 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and the mixture was separated by column chromatography (DCM: methanol v/v = 30:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 0.343 g of a reddish brown intermediate. The yield was 28%.

The reddish brown intermediate (0.343 g, 1.12 mmol) obtained in the above reaction was added to a round-bottom flask, and 4 mL of concentrated hydrochloric acid (w/v = 36%) was added, and the reaction was carried out at 40°C for 40 min. After the reaction was completed, the pH was adjusted to neutral with a saturated sodium bicarbonate solution, and the solid was precipitated. The filter cake was collected by suction and separated by column chromatography (DCM: methanol v/v = 20:1). The intermediate containing the target compound was collected, concentrated, and dried to obtain an off-white solid a10 (0.127 g). The yield was 35%. mp＝226.2-229.9℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.51 (s, 1H), 9.79 (s, 1H), 8.19 (s, 1H), 7.82 (dd, J＝8.4 ^, 2.0Hz, 1H), 7.70 ( _s , 1H), 7.58 (d, J＝6.4Hz, 1H), ⁷ ... _2JCF ＝37.8Hz,TFA),150.97,136.57,134.25,133.13,132.38,131.00,130.67,127.07,126.54,126.36,125.57,115.55(q, ¹ J _CF ＝287.1Hz,TFA),114.11, 33.68,18.12.HRMS(ESI):m/z calcd for C ₁₇ H ₁₇ N ₄ O ₃ [M+H] ⁺ 325.1295, found325.1300; HPLCpurity:100%.

Preparation method of embodiment 11a11

2-Cyanomethyl-5-carboxyl-1H-benzimidazole (0.805 g, 4 mmol), DMF (5 mL), triethylamine (0.810 g, 8 mmol), and HATU (1.673 g, 4.4 mmol) prepared according to Example 1 were added to a round-bottom flask. After stirring at room temperature, m-methoxyaniline (0.493 g, 4 mmol) was added and stirring was continued for 6 h. After the reaction was completed, saturated brine (200 mL) was added, and DCM was used for extraction (120 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and column chromatography was performed for separation (DCM: methanol v/v = 30:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 0.343 g of a reddish brown intermediate. The yield was 28%.

The reddish brown intermediate (0.343 g, 1.12 mmol) obtained in the above reaction was added to a round-bottom flask, and 4 mL of concentrated hydrochloric acid (w/v = 36%) was added, and the reaction was carried out at 40°C for 40 min. After the reaction was completed, the pH was adjusted to neutral with a saturated sodium bicarbonate solution, and the solid was precipitated. The filter cake was collected by suction and separated by column chromatography (DCM: methanol v/v = 20:1). The eluate containing the target compound was collected, concentrated, and dried to obtain an off-white solid a11 (0.141 g). The yield was 39%. mp＝140.0-141.4℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.57(s,1H),10.18(s,1H),8.17(s,1H),7.84–7.77(m,1H),7.74(s,1H),7.59(d,J＝8.4Hz,1H),7.52(m ,1H),7.44–7.37(m,1H),7.28–7.17(m,2H),6.67(dd,J＝8.0,2.4Hz,1H),3.78(s,2H),3.76(s,3H). ¹³ C NMR(100MHz,DMSO-d ₆ ,TFA)δ166.95,165.00,159.7 2,158.86(q, ² J _CF = 37.4Hz, TFA), 151.00, 140.45, 133.10, 132.68, 130.89, 129.69, 125.53, 115.58 (q, ¹ J _CF = 287.6 Hz, TFA) 114.08, 113.00, 109.61, 106.49, 55. 21,33.64.HRMS(ESI):m/z calcd for C ₁₇ H ₁₇ N ₄ O ₃ [M+H] ⁺ 325.1295, found325.1294; HPLCpurity:100%.

Preparation method of embodiment 12a12

2-Cyanomethyl-5-carboxyl-1H-benzimidazole (0.805 g, 4 mmol), DMF (5 mL), triethylamine (0.810 g, 8 mmol), and HATU (1.673 g, 4.4 mmol) prepared according to Example 1 were added to a round-bottom flask. After stirring at room temperature, p-methoxyaniline (0.493 g, 4 mmol) was added and stirring was continued for 6 h. After the reaction was completed, saturated brine (200 mL) was added, and DCM was used for extraction (120 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and the mixture was separated by column chromatography (DCM: methanol v/v = 30:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 0.306 g of a reddish brown intermediate. The yield was 25%.

The reddish brown intermediate (0.306 g, 1 mmol) obtained in the above reaction was added to a round-bottom flask, and 4 mL of concentrated hydrochloric acid (w/v = 36%) was added, and the reaction was carried out at 40°C for 40 min. After the reaction, the pH was adjusted to neutral with a saturated sodium bicarbonate solution, and the solid was precipitated. The filter cake was collected by suction and separated by column chromatography (DCM: methanol v/v = 20:1). The eluate containing the target compound was collected, concentrated, and dried to obtain a light yellow solid a12 (0.156 g). The yield was 48%. mp＝230.3-244.1℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.54(s,1H),10.08(s,1H),8.16(s,1H),7.80(dd,J＝8.4,1.6Hz,1H),7.71(m,3H),7.58(d,J＝8.4Hz,1H),7 .21(s,1H),6.97–6.88(m,2H),3.77(s,2H),3.75(s,3H). ¹³ CNMR(100MHz,DMSO-d ₆ ,TFA)δ167.12,164.68,159.00(q, ² J _CF =37.2Hz,TFA),156.19,151.12,133.22,132.96,132.47,131.15,125.61,122.66,115.83(q, ¹ J _CF =287.9Hz,TFA),114.24,114.21,114.16,55.58,3 3.85.HRMS (ESI): m/z calcd for C ₁₇ H ₁₇ N ₄ O ₃ [M+H] ⁺ 325.1295, found 325.1294; HPLC purity: 100%.

Preparation method of embodiment 13a13

2-Cyanomethyl-5-carboxyl-1H-benzimidazole (0.805 g, 4 mmol), DMF (5 mL), triethylamine (0.810 g, 8 mmol), and HATU (1.673 g, 4.4 mmol) prepared according to Example 1 were added to a round-bottom flask. After stirring at room temperature, 2,3-dimethylaniline (0.485 g, 4 mmol) was added and stirring was continued for 6 h. After the reaction was completed, saturated brine (200 mL) was added, and DCM was used for extraction (120 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and column chromatography was performed for separation (DCM: methanol v/v = 30:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 0.341 g of a reddish brown intermediate. The yield was 28%.

The reddish brown intermediate (0.341 g, 1.12 mmol) obtained in the above reaction was added to a round-bottom flask, and 4 mL of concentrated hydrochloric acid (w/v = 36%) was added, and the reaction was carried out at 40°C for 40 min. After the reaction was completed, the pH was adjusted to neutral with a saturated sodium bicarbonate solution, and the solid was precipitated. The filter cake was collected by suction and separated by column chromatography (DCM: methanol v/v = 20:1). The eluate containing the target compound was collected, concentrated, and dried to obtain an off-white solid a13 (0.141 g). The yield was 39%. mp＝218.5-223.8℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.57 (s, 1H), 9.90 (s, 1H), 8.19 (s, 1H), 7.82 (dd, J＝8.4, 1.6Hz, 1H), 7.76 (s, 1H), 7.58 (d, J＝8.4Hz, 1H), 7. 24(s,1H),7.20–7.00(m,3H),3.78(s,2H),2.28(s,3H),2.11(s,3H). ¹³ CNMR(100MHz,DMSO-d ₆ ,TFA)δ167.13,165.10,159,04(q, ² J _CF =38.0Hz,TFA),151.06,137.60,136.55,133.35,133.21,132.54,131.11,128.20,125.74,125.71,125.25,115.61(q, ¹ J _CF =286.7Hz,TFA),114.26, 114.20,33.78,20.37,14.56.HRMS(ESI):m/zcalcd for C ₁₈ H ₁₉ N ₄ O ₂ [M+H] ⁺ 323.1503, found 323.1509; HPLC purity: 100%.

Preparation method of embodiment 14a14

2-Cyanomethyl-5-carboxyl-1H-benzimidazole (0.805 g, 4 mmol), DMF (5 mL), triethylamine (0.810 g, 8 mmol), and HATU (1.673 g, 4.4 mmol) prepared according to Example 1 were added to a round-bottom flask. After stirring at room temperature, 3,4-dimethylaniline (0.485 g, 4 mmol) was added and stirring was continued for 6 h. After the reaction was completed, saturated brine (200 mL) was added, and DCM was used for extraction (120 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and column chromatography was performed for separation (DCM: methanol v/v = 30:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 0.317 g of a reddish brown intermediate. The yield was 26%.

The reddish brown intermediate (0.317 g, 1.04 mmol) obtained in the above reaction was added to a round-bottom flask, and 4 mL of concentrated hydrochloric acid (w/v = 36%) was added, and the reaction was carried out at 40°C for 40 min. After the reaction was completed, the pH was adjusted to neutral with a saturated sodium bicarbonate solution, and the solid was precipitated. The filter cake was collected by suction and separated by column chromatography (DCM: methanol v/v = 20:1). The eluate containing the target compound was collected, concentrated, and dried to obtain an off-white solid a14 (0.127 g). The yield was 38%. mp＝252.1-254.8℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.59 (s, 1H), 10.07 (s, 1H), 8.17 (s, 1H), 7.83–7.74 (m, 2H), 7.58 (m, 2H), 7.52 (dd, J＝8.0, 2.0Hz, 1H), 7. 25(s,1H),7.09(d,J=8.4Hz,1H),3.78(s,2H),2.22(s,3H),2.19(s,3H). ¹³ C NMR(100MHz,DMSO-d ₆ ,TFA)δ167.13,164.80,159.05(q, ² J _CF ＝37.9Hz,TFA),151.05,137.11,136.69,133.13,133.03,132.21,131.02,129.89,125.72,122.32,118.64,115.62(q, ¹ J _CF ＝286.7Hz,TFA),114.16, 33.78,19.86,19.07.HRMS(ESI):m/zcalcd for C ₁₈ H ₁₉ N ₄ O ₂ [M+H] ⁺ 323.1503, found 323.1507; HPLC purity: 100%.

Preparation method of Example 15b1

3-amino-4-nitrophenol (1.233 g, 8 mmol) of Formula 4, 40 mL of mixed solvent (DCM:H ₂ O v/v＝1:1), potassium hydroxide (0.898 g, 16 mmol), TBAB (0.258 g, 0.8 mmol) and 2-fluorobenzyl bromide (2.117 g, 11.2 mmol) were added to a round-bottom flask in sequence and stirred at room temperature for 2 h. After the reaction, DCM was removed by vacuum distillation, saturated brine (200 mL) was added, and EA (80 mL) was used for extraction. The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was concentrated by vacuum distillation, and then separated by column chromatography (PE:DCM v/v＝1:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 1.930 g of an orange solid intermediate of Formula 5. The yield was 92%.

The above-obtained orange solid intermediate (1.834 g, 7 mmol) was added to DCM (15 mL) in a round-bottom flask, and then DIPEA (1.809 g, 14 mmol) and HATU (2.927 g, 7.7 mmol) were added. After stirring at room temperature for 5 min, cyanoacetic acid (0.595 g, 7 mmol) was added and continued to stir for 8 h. After the reaction was completed, an orange solid was precipitated. Part of the solvent was removed by vacuum distillation, and the filter cake was collected by suction filtration. The filter cake was washed with methanol, pulped, suction filtered, and dried to obtain 1.175 g of the yellow solid intermediate shown in Formula 6. The yield was 51%.

The yellow solid intermediate (0.988 g, 3 mmol) was dissolved in 32 mL of a mixed solvent (ethanol: acetonitrile: water v/v/v = 2:1:1), and ammonium chloride (1.284 g, 24 mmol) and zinc powder (1.569 g, 24 mmol) were added, and the mixture was vigorously stirred at 50°C for 10 min; after the reaction was completed, the mixture was filtered and the filtrate was collected; the filtrate was distilled under reduced pressure, and then extracted with DCM (300 mL) and saturated brine (400 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain 0.790 g of an off-white solid intermediate shown in Formula 7. The yield was 88%.

The above white solid intermediate (0.748 g, 2.5 mmol) was dissolved in glacial acetic acid (20 mL) and heated under reflux for 2 h. After the reaction was completed, it was extracted with EA (30 mL) and saturated brine (80 mL). The organic phase was collected, dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain 0.640 g of a reddish brown solid intermediate shown in formula 8. The yield was 91%.

The above reddish brown solid intermediate (0.563 g, 2 mmol) was dissolved in DMSO (6 mL), and 30% hydrogen peroxide (1.812 g containing 16 mmol H ₂ O ₂ ) and potassium carbonate (0.056 g, 0.4 mmol) were added in sequence, and the mixture was reacted at 25°C for 5 h. After the reaction, water (20 mL) was added to precipitate a pink solid, which was filtered and the filter cake was collected. The filter cake was purified by column chromatography (DCM: methanol v/v = 20:1), and the eluent containing the target compound was collected, concentrated, and dried to obtain a white solid b1 (0.293 g). The yield was 49%. mp＝194.7-199.3℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.12(s,1H),7.71(s,1H),7.57(td,J＝7.6,1.6Hz,1H),7.46–7.33(m,2H),7.29–7.15(m,3H),7.11(s,1 H), 6.87–6.79 (m, 1H), 5.14 (s, 2H), 3.67 (s, 2H). ¹³ C NMR (100MHz, DMSO-d ₆ , TFA) δ 167.22, 160.82 (d, ¹ J _CF = 244.7Hz), 158.93 (q, ² J _CF ＝37.9Hz, TFA),156.99,148.41,132.03,131.06(d, ³ J _CF ＝3.9Hz),130.84(d, ³ J _CF ＝8.2Hz),125.63,124.85(d, ⁴ J _CF ＝3.4Hz),123.71(d, ² J _CF ＝1 4.4Hz), 116.21, 115.73 (d, ² J _CF = 20.9Hz), 115.53 (q, ¹ J _CF = 286.8Hz, TFA), 115.15, 97.97, 64.69 (d, ³ J _CF = 3.5Hz), 33.44. HRMS (ESI): m/z calcd for C ₁₆ H _{1 5FN3O 2} [M+H] ⁺ 300.1143,found 300.1141; HPLCpurity:100%.

Preparation method of Example 16b2

3-Amino-4-nitrophenol (1.233 g, 8 mmol) of Formula 4, 40 mL of mixed solvent (DCM:H ₂ O v/v＝1:1), potassium hydroxide (0.898 g, 16 mmol), TBAB (0.258 g, 0.8 mmol) and 3-fluorobenzyl bromide (2.117 g, 11.2 mmol) were added to a round-bottom flask in sequence and stirred at room temperature for 2 h. After the reaction, DCM was removed by vacuum distillation, saturated brine (200 mL) was added, and EA (80 mL) was used for extraction. The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was concentrated by vacuum distillation, and then separated by column chromatography (PE:DCM v/v＝1:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 1.888 g of an orange solid intermediate of Formula 5. The yield was 90%.

The above-obtained orange solid intermediate (1.834 g, 7 mmol) was added to DCM (15 mL) in a round-bottom flask, and then DIPEA (1.809 g, 14 mmol) and HATU (2.927 g, 7.7 mmol) were added. After stirring at room temperature for 5 min, cyanoacetic acid (0.654 g, 7.7 mmol) was added and stirring was continued for 8 h. After the reaction was completed, an orange solid was precipitated. Part of the solvent was removed by vacuum distillation, and the filter cake was collected by suction filtration. The filter cake was washed with methanol, pulped, suction filtered, and dried to obtain 1.314 g of an orange-yellow solid intermediate shown in Formula 6. The yield was 57%.

The above orange-yellow solid intermediate (0.988 g, 3 mmol) was dissolved in 32 mL of a mixed solvent (ethanol: acetonitrile: water v/v/v = 2:1:1), and ammonium chloride (1.284 g, 24 mmol) and zinc powder (1.569 g, 24 mmol) were added, and the mixture was vigorously stirred at 50°C for 10 min; after the reaction was completed, the mixture was filtered and the filtrate was collected; the filtrate was distilled under reduced pressure, and then extracted with DCM (300 mL) and saturated brine (400 mL), the organic phase was collected, dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain 0.763 g of an off-white solid intermediate shown in Formula 7. The yield was 85%.

The above white solid intermediate (0.748 g, 2.5 mmol) was dissolved in glacial acetic acid (20 mL) and heated under reflux for 2 h. After the reaction was completed, it was extracted with EA (30 mL) and saturated brine (80 mL). The organic phase was collected, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to obtain 0.675 g of a reddish brown solid intermediate shown in formula 8. The yield was 96%.

The above reddish brown solid intermediate (0.563 g, 2 mmol) was dissolved in DMSO (6 mL), and 30% hydrogen peroxide (1.812 g containing 16 mmol H ₂ O ₂ ) and potassium carbonate (0.056 g, 0.4 mmol) were added in sequence, and the mixture was reacted at 25°C for 5 h. After the reaction, water (20 mL) was added to precipitate a pink solid, which was filtered and the filter cake was collected. The filter cake was purified by column chromatography (DCM: methanol v/v = 20:1), and the eluent containing the target compound was collected, concentrated, and dried to obtain a slightly yellow solid b2 (0.323 g). The yield was 54%. mp＝181.8-182.6℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.07 (s, 1H), 7.68 (s, 1H), 7.47–7.35 (m, 2H), 7.35–7.25 (m, 2H), 7.20–7.10 (m, 2H), 7.08 (d, J＝2.4Hz, 1 H), 6.85 (dd, J = 8.8, 2.4Hz, 1H), 5.14 (s, 2H), 3.67 (s, 2H). ¹³ C NMR (100MHz, DMSO-d ₆ , TFA) δ 167.27, 162.69 (d, ¹ J _CF = 242.4Hz), 158.97 (q, ² J _CF =38.0Hz, TFA),156.98,148.41,139.93(d, ³ J _CF =7.4Hz),132.06,130.84(d, ³ J _CF =8.3Hz),125.63,123.85(d, ⁴ J _CF =2.7Hz),116.34,115.54(q, ¹ J _CF = 286.6Hz, TFA), 115.18, 115.04 (d, ² J _CF = 20.9Hz), 114.64 (d, ² J _CF = 21.8Hz), 98.09, 69.50 (d, ⁴ J _CF = 1.2Hz), 33.48. HRMS (ESI): m/z calcd for C ₁₆ H _{1 5FN3O 2} [M+H] ⁺ 300.1143, found 300.1145; HPLC purity: 100%.

Preparation method of Example 17b3

3-amino-4-nitrophenol (1.233 g, 8 mmol) of Formula 4, 40 mL of mixed solvent (DCM:H ₂ O v/v＝1:1), potassium hydroxide (0.898 g, 16 mmol), TBAB (0.258 g, 0.8 mmol) and 4-fluorobenzyl bromide (2.117 g, 11.2 mmol) were added to a round-bottom flask in sequence, and stirred at room temperature for 2 h; after the reaction, DCM was removed by vacuum distillation, saturated brine (200 mL) was added, and EA (80 mL) was used for extraction; the organic phases were combined and dried over anhydrous sodium sulfate; the solvent was concentrated by vacuum distillation, and then separated by column chromatography (PE:DCM v/v＝1:1), and the eluent containing the target intermediate was collected, concentrated, and dried to obtain 2.014 g of an orange solid intermediate of Formula 5. The yield was 96%.

The above-obtained orange solid intermediate (1.834 g, 7 mmol) was added to a round-bottom flask in a DCM (15 mL) solvent, and then DIPEA (1.809 g, 14 mmol) and HATU (2.927 g, 7.7 mmol) were added. After stirring at room temperature for 5 min, cyanoacetic acid (0.654 g, 7.7 mmol) was added and continued to stir for 8 h. After the reaction was completed, an orange solid was precipitated. Part of the solvent was removed by vacuum distillation, and the filter cake was collected. The filter cake was washed with methanol, pulped, filtered, and dried to obtain 1.267 g of an orange-yellow solid intermediate shown in Formula 6. The yield was 55%.

The above orange-yellow solid intermediate (0.988 g, 3 mmol) was dissolved in 32 mL of a mixed solvent (ethanol: acetonitrile: water v/v/v = 2:1:1), and ammonium chloride (1.284 g, 24 mmol) and zinc powder (1.569 g, 24 mmol) were added, and the mixture was vigorously stirred at 50°C for 10 min; after the reaction was completed, the mixture was filtered and the filtrate was collected; the filtrate was evaporated under reduced pressure, and then extracted with DCM (300 mL) and saturated brine (400 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain 0.826 g of an off-white solid intermediate shown in Formula 7. The yield was 92%.

The above white solid intermediate (0.748 g, 2.5 mmol) was dissolved in glacial acetic acid (20 mL) and heated under reflux for 2 h. After the reaction was completed, it was extracted with EA (30 mL) and saturated brine (80 mL). The organic phase was collected, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to obtain 0.633 g of a reddish brown solid intermediate shown in formula 8. The yield was 90%.

The above reddish brown solid intermediate (0.563 g, 2 mmol) was dissolved in DMSO (6 mL), and 30% hydrogen peroxide (1.812 g containing 16 mmol H ₂ O ₂ ) and potassium carbonate (0.056 g, 0.4 mmol) were added in sequence, and the mixture was reacted at 25° C. for 5 h. After the reaction, water (20 mL) was added to precipitate a pink solid, which was filtered and the filter cake was collected. The filter cake was purified by column chromatography (DCM: methanol v/v = 20:1), and the eluent containing the target compound was collected, concentrated, and dried to obtain an off-white solid b3 (0.377 g). The yield was 63%. mp＝213.9-217.0℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.09(s,1H),7.68(s,1H),7.55–7.46(m,2H),7.37(d,J＝8.4Hz,1H),7.24–7.18(m,2H),7.16(s,1H),7. 08 (d, J＝2.4Hz, 1H), 6.83 (dd, J＝8.8, 2.4Hz, 1H), 5.09 (s, 2H), 3.66 (s, 2H). ¹³ C NMR (100MHz, DMSO-d ₆ , TFA) δ 167.14, 162.17 ( _d , ¹ J _CF ＝ 242.5Hz), 158.85 (q, ^2JCF ＝37.6Hz, TFA),156.98,148.26,133.04(d, ⁴ J _CF ＝3.0Hz),131.96,130.28(d, ³ J _CF ＝8.3Hz),125.45,116.21,115.54(d, ² J _CF ＝21.2Hz),115.52(q, ¹ J _CF = 287.2Hz, TFA), 115.03, 97.94, 69.57, 33.37. HRMS (ESI): m/z calcd for C ₁₆ H ₁₅ FN ₃ O ₂ [M+H] ⁺ 300.1143, found 300.1145; HPLC purity: 100%.

Preparation method of Example 18b4

3-amino-4-nitrophenol (1.233 g, 8 mmol) of Formula 4, 40 mL of mixed solvent (DCM:H ₂ O v/v＝1:1), potassium hydroxide (0.898 g, 16 mmol), TBAB (0.258 g, 0.8 mmol) and 2-chlorobenzyl bromide (2.301 g, 11.2 mmol) were added to a round-bottom flask in sequence, and stirred at room temperature for 2 h; after the reaction, DCM was removed by vacuum distillation, saturated brine (200 mL) was added, and extracted with EA (80 mL); the organic phases were combined and dried over anhydrous sodium sulfate; the solvent was concentrated by vacuum distillation, and then separated by column chromatography (PE:DCM v/v＝1:1), and the eluent containing the target intermediate was collected, concentrated, and dried to obtain 1.984 g of an orange solid intermediate of Formula 5. The yield was 89%.

The above-obtained orange solid intermediate (1.834 g, 7 mmol) was added to a round-bottom flask in a DCM (15 mL) solvent, and then DIPEA (1.809 g, 14 mmol) and HATU (2.927 g, 7.7 mmol) were added. After stirring at room temperature for 5 min, cyanoacetic acid (0.654 g, 7.7 mmol) was added and continued to stir for 8 h. After the reaction was completed, an orange solid precipitated, and part of the solvent was removed by vacuum distillation. The filter cake was collected by suction filtration, washed with methanol, slurried, suction filtered, and dried to obtain 1.355 g of an orange-yellow solid intermediate shown in Formula 6. The yield was 56%.

The above orange-yellow solid intermediate (0.988 g, 3 mmol) was dissolved in 32 mL of a mixed solvent (ethanol: acetonitrile: water v/v/v = 2:1:1), and ammonium chloride (1.284 g, 24 mmol) and zinc powder (1.569 g, 24 mmol) were added, and the mixture was vigorously stirred at 50°C for 10 min; after the reaction was completed, the mixture was filtered and the filtrate was collected; the filtrate was evaporated under reduced pressure, and then extracted with DCM (300 mL) and saturated brine (400 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain 0.890 g of an off-white solid intermediate shown in Formula 7. The yield was 95%.

The above white solid intermediate (0.748 g, 2.5 mmol) was dissolved in glacial acetic acid (20 mL) and heated under reflux for 2 h. After the reaction was completed, it was extracted with EA (50 mL) and saturated brine (80 mL). The organic phase was collected, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to obtain 0.670 g of a reddish brown solid intermediate shown in formula 8. The yield was 90%.

The above reddish brown solid intermediate (0.563 g, 2 mmol) was dissolved in DMSO (6 mL), and 30% hydrogen peroxide (1.812 g containing 16 mmol H ₂ O ₂ ) and potassium carbonate (0.056 g, 0.4 mmol) were added in sequence, and the mixture was reacted at 25°C for 5 h. After the reaction, water (20 mL) was added to precipitate a pink solid, which was filtered and the filter cake was collected. The filter cake was purified by column chromatography (DCM: methanol v/v = 20:1), and the eluent containing the target compound was collected, concentrated, and dried to obtain a white solid b4 (0.360 g). The yield was 57%. mp＝204.9-220.2℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.11(s,1H),7.69(s,1H),7.64–7.58(m,1H),7.55–7.48(m,1H),7.43–7.33(m,3H),7.17(s,1H),7.09 (d, J＝2.4Hz, 1H), 6.86 (dd, J＝8.4, 2.4Hz, 1H), 5.17 (s, 2H), 3.67 (s, 2H). ¹³ C NMR (100MHz, DMSO-d ₆ , TFA) δ 167.29, 159.00 (q, ² J _CF ＝38.1Hz,TFA),157.10,148.49,134.31,133.17,132.13,130.51,130.32,129.83,127.73,125.77,116.27,115.56(q, ¹ J _CF ＝286.5Hz,TFA),115.24, 98.15,68.12,33.51.HRMS(ESI):m/z calcd for C ₁₆ H ₁₅ ClN ₃ O ₂ [M+H] ⁺ 316.0847, found 316.0841; HPLCpurity: 100%.

Preparation method of Example 19b5

3-Amino-4-nitrophenol (1.233 g, 8 mmol) of Formula 4, 40 mL of mixed solvent (DCM:H ₂ O v/v＝1:1), potassium hydroxide (0.898 g, 16 mmol), TBAB (0.258 g, 0.8 mmol) and 3-chlorobenzyl bromide (2.301 g, 11.2 mmol) were added to a round-bottom flask in sequence and stirred at room temperature for 2 h. After the reaction, DCM was removed by vacuum distillation, saturated brine (200 mL) was added, and EA (80 mL) was used for extraction. The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was concentrated by vacuum distillation, and then separated by column chromatography (PE:DCM v/v＝1:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 1.984 g of an orange solid intermediate of Formula 5. The yield was 89%.

The above-obtained orange solid intermediate (1.834 g, 7 mmol) was added to a round-bottom flask in a DCM (15 mL) solvent, and then DIPEA (1.809 g, 14 mmol) and HATU (2.927 g, 7.7 mmol) were added. After stirring at room temperature for 5 min, cyanoacetic acid (0.654 g, 7.7 mmol) was added and continued to stir for 7 h. After the reaction was completed, an orange solid precipitated, and part of the solvent was removed by vacuum distillation. The filter cake was collected by suction filtration, washed with methanol, slurried, suction filtered, and dried to obtain 1.306 g of an orange-yellow solid intermediate shown in Formula 6. The yield was 54%.

The above orange-yellow solid intermediate (0.988 g, 3 mmol) was dissolved in 32 mL of a mixed solvent (ethanol: acetonitrile: water v/v/v = 2:1:1), and ammonium chloride (1.284 g, 24 mmol) and zinc powder (1.569 g, 24 mmol) were added, and the mixture was vigorously stirred at 50°C for 10 min; after the reaction was completed, the mixture was filtered and the filtrate was collected; the filtrate was evaporated under reduced pressure, and then extracted with DCM (300 mL) and saturated brine (400 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain 0.853 g of an off-white solid intermediate shown in Formula 7. The yield was 90%.

The above white solid intermediate (0.748 g, 2.5 mmol) was dissolved in glacial acetic acid (20 mL) and heated under reflux for 2 h. After the reaction was completed, it was extracted with EA (50 mL) and saturated brine (90 mL). The organic phase was collected, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to obtain 0.670 g of a reddish brown solid intermediate shown in formula 8. The yield was 90%.

The above reddish brown solid intermediate (0.563 g, 2 mmol) was dissolved in DMSO (6 mL), and 30% hydrogen peroxide (1.812 g containing 16 mmol H ₂ O ₂ ) and potassium carbonate (0.056 g, 0.4 mmol) were added in sequence, and the mixture was reacted at 25°C for 5 h. After the reaction, water (20 mL) was added to precipitate a pink solid, which was filtered and the filter cake was collected. The filter cake was purified by column chromatography (DCM: methanol v/v = 20:1), and the eluent containing the target compound was collected, concentrated, and dried to obtain a white solid b5 (0.398 g). The yield was 63%. mp＝215.7-217.5℃; ¹ H NMR(400MHz, DMSO-d ₆ )δ ¹ H NMR(400MHz, DMSO-d ₆ )δ12.12(s,1H),7.69(s,1H),7.55–7.52(m,1H),7.44–7.36(m,4H),7.17(s,1H),7. 08 (d, J＝2.4Hz, 1H), 6.85 (dd, J＝8.4, 2.4Hz, 1H), 5.14 (s, 2H), 3.67 (s, 2H). ¹³ C NMR (100MHz, DMSO-d ₆ , TFA) δ 167.23, 158, 93 (q, ² J _CF ＝38.2Hz,TFA),156.94,148.41,139.56,133.66,132.04,130.68,128.22,127.72,126.48,125.63,116.30,115.52(q, ¹ J _CF ＝286.6Hz,TFA),115.18, 98.10,69.43,33.47.HRMS(ESI):m/z calcd for C ₁₆ H ₁₅ ClN ₃ O ₂ [M+H] ⁺ 316.0847, found 316.0849; HPLCpurity: 100%.

Preparation method of Example 20b6

3-amino-4-nitrophenol (1.233 g, 8 mmol) of Formula 4, 40 mL of mixed solvent (DCM:H ₂ O v/v＝1:1), potassium hydroxide (0.898 g, 16 mmol), TBAB (0.258 g, 0.8 mmol) and 4-chlorobenzyl bromide (2.301 g, 11.2 mmol) were added to a round-bottom flask in sequence, and stirred at room temperature for 2 h; after the reaction, DCM was removed by vacuum distillation, saturated brine (200 mL) was added, and EA (70 mL) was used for extraction; the organic phases were combined and dried over anhydrous sodium sulfate; the solvent was concentrated by vacuum distillation, and then separated by column chromatography (PE:DCM v/v＝1:1), and the eluent containing the target intermediate was collected, concentrated, and dried to obtain 2.118 g of an orange solid intermediate of Formula 5. The yield was 95%.

The above-obtained orange solid intermediate (1.834 g, 7 mmol) was added to a round-bottom flask in a DCM (15 mL) solvent, and then DIPEA (1.809 g, 14 mmol) and HATU (2.927 g, 7.7 mmol) were added. After stirring at room temperature for 5 min, cyanoacetic acid (0.654 g, 7.7 mmol) was added and continued to stir for 8 h. After the reaction was completed, an orange solid was precipitated. Part of the solvent was removed by vacuum distillation, and the filter cake was collected by suction filtration. The filter cake was washed with methanol, pulped, suction filtered, and dried to obtain 1.331 g of an orange-yellow solid intermediate shown in Formula 6. The yield was 55%.

The above orange-yellow solid intermediate (0.988 g, 3 mmol) was dissolved in 32 mL of a mixed solvent (ethanol: acetonitrile: water v/v/v = 2:1:1), and ammonium chloride (1.284 g, 24 mmol) and zinc powder (1.569 g, 24 mmol) were added, and the mixture was vigorously stirred at 50°C for 10 min; after the reaction was completed, the mixture was filtered and the filtrate was collected; the filtrate was evaporated under reduced pressure, and then extracted with DCM (300 mL) and saturated brine (400 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain 0.843 g of an off-white solid intermediate shown in Formula 7. The yield was 89%.

The above white solid intermediate (0.748 g, 2.5 mmol) was dissolved in glacial acetic acid (20 mL) and heated under reflux for 2 h. After the reaction was completed, it was extracted with EA (50 mL) and saturated brine (90 mL). The organic phase was collected, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to obtain 0.670 g of a reddish brown solid intermediate shown in formula 8. The yield was 90%.

The above reddish brown solid intermediate (0.563 g, 2 mmol) was dissolved in DMSO (6 mL), and 30% hydrogen peroxide (1.812 g containing 16 mmol H ₂ O ₂ ) and potassium carbonate (0.056 g, 0.4 mmol) were added in sequence, and the mixture was reacted at 25°C for 5 h. After the reaction, water (20 mL) was added to precipitate a pink solid, which was filtered and the filter cake was collected. The filter cake was purified by column chromatography (DCM: methanol v/v = 20:1), and the eluent containing the target compound was collected, concentrated, and dried to obtain a white solid b6 (0.278 g). The yield was 44%. mp＝230.2-238.4℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.11(s,1H),7.70(s,1H),7.52–7.42(m,4H),7.38(d,J＝8.4Hz,1H),7.18(s,1H),7.07(d,J＝2.4Hz,1H),6 .83 (dd, J＝8.8, 2.4Hz, 1H), 5.11 (s, 2H), 3.66 (s, 2H). ¹³ C NMR (100MHz, DMSO-d ₆ , TFA) δ 167.13, 158.85 (q, ² J _CF =37.9 Hz, TFA), 156.93, 148.29, 135.91, 132.94, 131.98, 129.78, 128.76, 125.53, 116.25, 115.50 (q, ¹ J _CF =286.9 Hz, TFA), 115.07, 98.07, 69.50, 33.39. HRMS (ESI): m/z calcd for C ₁₆ H ₁₅ ClN ₃ O ₂ [M+H] ⁺ 316.0847, found 316.0846; HPLC purity: 100%. Preparation method of Example 21b7

3-Amino-4-nitrophenol (1.233 g, 8 mmol) of Formula 4, 40 mL of mixed solvent (DCM:H ₂ O v/v＝1:1), potassium hydroxide (0.898 g, 16 mmol), TBAB (0.258 g, 0.8 mmol) and 2-methylbenzyl bromide (2.072 g, 11.2 mmol) were added to a round-bottom flask in sequence and stirred at room temperature for 2 h. After the reaction, DCM was removed by vacuum distillation, saturated brine (200 mL) was added, and EA (50 mL) was used for extraction. The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was concentrated by vacuum distillation, and then separated by column chromatography (PE:DCM v/v＝1:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 1.900 g of an orange solid intermediate of Formula 5. The yield was 92%.

The above-obtained orange solid intermediate (1.834 g, 7 mmol) was added to a round-bottom flask in a DCM (15 mL) solvent, and then DIPEA (1.809 g, 14 mmol) and HATU (2.927 g, 7.7 mmol) were added. After stirring at room temperature for 5 min, cyanoacetic acid (0.654 g, 7.7 mmol) was added and continued to stir for 9 h. After the reaction was completed, an orange solid precipitated, and part of the solvent was removed by vacuum distillation. The filter cake was collected by suction filtration, washed with methanol, slurried, suction filtered, and dried to obtain 1.161 g of an orange-yellow solid intermediate shown in Formula 6. The yield was 51%.

The above orange-yellow solid intermediate (0.988 g, 3 mmol) was dissolved in 32 mL of a mixed solvent (ethanol: acetonitrile: water v/v/v = 2:1:1), and ammonium chloride (1.284 g, 24 mmol) and zinc powder (1.569 g, 24 mmol) were added, and the mixture was vigorously stirred at 50°C for 10 min; after the reaction was completed, the mixture was filtered and the filtrate was collected; the filtrate was evaporated under reduced pressure, and then extracted with DCM (350 mL) and saturated brine (400 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain 0.780 g of an off-white solid intermediate shown in Formula 7. The yield was 88%.

The above white solid intermediate (0.748 g, 2.5 mmol) was dissolved in glacial acetic acid (20 mL) and heated under reflux for 2 h. After the reaction was completed, it was extracted with EA (50 mL) and saturated brine (80 mL). The organic phase was collected, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to obtain 0.631 g of a reddish brown solid intermediate shown in formula 8. The yield was 91%.

The above reddish brown solid intermediate (0.563 g, 2 mmol) was dissolved in DMSO (6 mL), and 30% hydrogen peroxide (1.812 g containing 16 mmol H ₂ O ₂ ) and potassium carbonate (0.056 g, 0.4 mmol) were added in sequence, and the mixture was reacted at 25°C for 4 h. After the reaction, water (20 mL) was added to precipitate a pink solid, which was filtered and the filter cake was collected. The filter cake was purified by column chromatography (DCM: methanol v/v/v = 20:1), and the eluent containing the target compound was collected, concentrated, and dried to obtain a white solid b7 (0.289 g). The yield was 49%. mp＝225.7-233.7℃; ¹ H NMR (600MHz, DMSO-d ₆ ) δ12.12(s,1H),7.68(s,1H),7.42(d,J＝7.2Hz,1H),7.38(d,J＝8.4Hz,1H),7.26–7.17(m,3H),7.15(s,1H),7 .11(d,J＝2.4Hz,1H),6.85(dd,J＝9.0,2.4Hz,1H),5.08(s,2H),3.68(s,2H),2.34(s,3H). ¹³ C NMR (100MHz, DMSO-d ₆ ,TFA) δ167.12,158.85(q, ² J _CF ＝37.7Hz,TFA),157.24,148.20,137.01,134.72,132.01,130.47,128.94,128.49,126.06,125.46,116.17,115.54(q, ¹ J _CF ＝287.0Hz,TFA),115.02, 97.94,69.12,33.38,18.65.HRMS(ESI):m/z calcd for C ₁₇ H ₁₈ N ₃ O ₂ [M+H] ⁺ 296.1394, found296.1395; HPLC purity: 100%.

Preparation method of Example 22b8

3-Amino-4-nitrophenol (1.233 g, 8 mmol) of Formula 4, 40 mL of mixed solvent (DCM:H ₂ O v/v＝1:1), potassium hydroxide (0.898 g, 16 mmol), TBAB (0.258 g, 0.8 mmol) and 3-methylbenzyl bromide (2.072 g, 11.2 mmol) were added to a round-bottom flask in sequence, and stirred at room temperature for 2 h; after the reaction, DCM was removed by vacuum distillation, saturated brine (200 mL) was added, and extracted with EA (70 mL); the organic phases were combined and dried over anhydrous sodium sulfate; the solvent was concentrated by vacuum distillation, and then separated by column chromatography (PE:DCM v/v＝1:1), and the eluent containing the target intermediate was collected, concentrated, and dried to obtain 1.942 g of an orange solid intermediate of Formula 5. The yield was 94%.

The above-obtained orange solid intermediate (1.808 g, 7 mmol) was added to DCM (15 mL) in a round-bottom flask, and then DIPEA (1.809 g, 14 mmol) and HATU (2.927 g, 7.7 mmol) were added. After stirring at room temperature for 5 min, cyanoacetic acid (0.654 g, 7.7 mmol) was added and stirring was continued for 8 h. After the reaction was completed, an orange solid was precipitated. Part of the solvent was removed by vacuum distillation, and the filter cake was collected. The filter cake was washed with methanol, pulped, filtered, and dried to obtain 1.344 g of an orange-yellow solid intermediate shown in Formula 6. The yield was 59%.

The above orange-yellow solid intermediate (0.976 g, 3 mmol) was dissolved in 32 mL of a mixed solvent (ethanol: acetonitrile: water v/v/v = 2:1:1), and ammonium chloride (1.284 g, 24 mmol) and zinc powder (1.569 g, 24 mmol) were added, and the mixture was vigorously stirred at 50°C for 10 min; after the reaction was completed, the mixture was filtered and the filtrate was collected; the filtrate was distilled under reduced pressure, and then extracted with DCM (300 mL) and saturated brine (400 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain 0.824 g of an off-white solid intermediate shown in Formula 7. The yield was 93%.

The above white solid intermediate (0.738 g, 2.5 mmol) was dissolved in glacial acetic acid (20 mL) and heated under reflux for 2 h. After the reaction was completed, it was extracted with EA (50 mL) and saturated brine (100 mL). The organic phase was collected, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to obtain 0.631 g of a reddish brown solid intermediate shown in formula 8. The yield was 91%.

The above reddish brown solid intermediate (0.555 g, 2 mmol) was dissolved in DMSO (6 mL), and 30% hydrogen peroxide (1.812 g containing 16 mmol H ₂ O ₂ ) and potassium carbonate (0.056 g, 0.4 mmol) were added in sequence, and the mixture was reacted at 25°C for 4 h. After the reaction, water (20 mL) was added to precipitate a pink solid, which was filtered and the filter cake was collected. The filter cake was purified by column chromatography (DCM: methanol v/v = 20:1), and the eluent containing the target compound was collected, concentrated, and dried to obtain an off-white solid b8 (0.295 g). The yield was 50%. mp＝223.1-227.5℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.10(s,1H),7.70(s,1H),7.37(d,J＝8.8Hz,1H),7.30–7.23(m,3H),7.18(s,1H),7.12(d,J＝6.4Hz,1H), 7.07 (d, J＝2.4Hz, 1H), 6.83 (dd, J＝8.8, 2.4Hz, 1H), 5.06 (s, 2H), 3.66 (s, 2H), 2.31 (s, 3H). ¹³ C NMR (100MHz, DMSO-d ₆ , TFA) δ 167.19, 158.91 (q, ² J _CF ＝38.0Hz,TFA),157.16,148.23,138.04,136.81,132.01,128.90,128.68,128.61,125.42,125.13,116.27,115.54(q, ¹ J _CF ＝287.0Hz,TFA),115.05, 97.87,70.33,33.40,21.15.HRMS(ESI):m/z calcd for C ₁₇ H ₁₈ N ₃ O ₂ [M+H] ⁺ 296.1394, found296.1399; HPLC purity: 99.32%.

Preparation method of Example 23b9

3-Amino-4-nitrophenol (1.233 g, 8 mmol) of Formula 4, 40 mL of mixed solvent (DCM:H ₂ O v/v＝1:1), potassium hydroxide (0.898 g, 16 mmol), TBAB (0.258 g, 0.8 mmol) and 4-methylbenzyl bromide (2.072 g, 11.2 mmol) were added to a round-bottom flask in sequence and stirred at room temperature for 2 h. After the reaction, DCM was removed by vacuum distillation, saturated brine (200 mL) was added, and EA (40 mL) was used for extraction. The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was concentrated by vacuum distillation, and then separated by column chromatography (PE:DCM v/v＝1:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 1.922 g of an orange solid intermediate of Formula 5. The yield was 93%.

The above-obtained orange solid intermediate (1.808 g, 7 mmol) was added to a round-bottom flask in a DCM (15 mL) solvent, and then DIPEA (1.809 g, 14 mmol) and HATU (2.927 g, 7.7 mmol) were added. After stirring at room temperature for 5 min, cyanoacetic acid (0.654 g, 7.7 mmol) was added and stirred at room temperature for 8 h. After the reaction, an orange solid was precipitated. Part of the solvent was removed by vacuum distillation, and the filter cake was collected by suction filtration. The filter cake was washed with methanol, pulped, suction filtered, and dried to obtain 1.230 g of an orange-yellow solid intermediate shown in Formula 6. The yield was 54%.

The above orange-yellow solid intermediate (0.976 g, 3 mmol) was dissolved in 32 mL of a mixed solvent (ethanol: acetonitrile: water v/v/v = 2:1:1), and ammonium chloride (1.284 g, 24 mmol) and zinc powder (1.569 g, 24 mmol) were added, and the mixture was vigorously stirred at 50°C for 10 min; after the reaction was completed, the mixture was filtered and the filtrate was collected; the filtrate was distilled under reduced pressure, and then extracted with DCM (300 mL) and saturated brine (450 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain 0.842 g of an off-white solid intermediate shown in Formula 7. The yield was 95%.

The above white solid intermediate (0.738 g, 2.5 mmol) was dissolved in glacial acetic acid (20 mL) and heated under reflux for 2 h. After the reaction was completed, it was extracted with EA (30 mL) and saturated brine (80 mL). The organic phase was collected, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to obtain 0.645 g of a reddish brown solid intermediate shown in formula 8. The yield was 93%.

The above reddish brown solid intermediate (0.555 g, 2 mmol) was dissolved in DMSO (6 mL), and 30% hydrogen peroxide (1.812 g containing 16 mmol H ₂ O ₂ ) and potassium carbonate (0.056 g, 0.4 mmol) were added in sequence, and the mixture was reacted at 25°C for 5 h. After the reaction, water (20 mL) was added to precipitate a pink solid, which was filtered and the filter cake was collected. The filter cake was purified by column chromatography (DCM: methanol v/v = 20:1), and the eluent containing the target compound was collected, concentrated, and dried to obtain an off-white solid b9 (0.354 g). The yield was 60%. mp=208.4-213.9℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.08 (s, 1H), 7.69 (s, 1H), 7.45–7.28 (m, 3H), 7.18 (d, J = 8.0Hz, 3H), 7.06 (d, J = 2.0Hz, 1H), 6.81 (dd, J = 8. 8, 2.4Hz, 1H), 5.05 (s, 2H), 3.66 (s, 2H), 2.30 (s, 3H). ¹³ C NMR (100MHz, DMSO-d ₆ , TFA) δ 167.10, 158.81 (q, ² J _CF =37.7Hz,TFA),157.06,148.14,137.54,133.76,131.93,129.26,128.09,125.33,116.25,115.52(q, ¹ J _CF =287.3Hz,TFA),114.96,97.88,70.16,33 .33,20.88.HRMS(ESI):m/z calcd forC ₁₇ H ₁₈ N ₃ O ₂ [M+H] ⁺ 296.1394, found 296.1391; HPLC purity: 100%.

Preparation method of Example 24b10

3-Amino-4-nitrophenol (1.233 g, 8 mmol) of Formula 4, 40 mL of mixed solvent (DCM:H ₂ O v/v＝1:1), potassium hydroxide (0.898 g, 16 mmol), TBAB (0.258 g, 0.8 mmol) and 3-methoxybenzyl bromide (2.251 g, 11.2 mmol) were added to a round-bottom flask in sequence and stirred at room temperature for 2 h. After the reaction, DCM was removed by vacuum distillation, saturated brine (200 mL) was added, and EA (80 mL) was used for extraction. The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was concentrated by vacuum distillation, and then separated by column chromatography (PE:DCM v/v＝1:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 2.018 g of an orange solid intermediate of Formula 5. The yield was 92%.

The above-obtained orange solid intermediate (1.808 g, 7 mmol) was added to a round-bottom flask in a DCM (15 mL) solvent, and then DIPEA (1.920 g, 14 mmol) and HATU (2.927 g, 7.7 mmol) were added. After stirring at room temperature for 5 min, cyanoacetic acid (0.654 g, 7.7 mmol) was added and continued to stir for 8 h. After the reaction was completed, an orange solid was precipitated. Part of the solvent was removed by vacuum distillation, and the filter cake was collected by suction filtration. The filter cake was washed with methanol, pulped, suction filtered, and dried to obtain 1.219 g of an orange-yellow solid intermediate shown in Formula 6. The yield was 51%.

The above orange-yellow solid intermediate (1.024 g, 3 mmol) was dissolved in 32 mL of a mixed solvent (ethanol: acetonitrile: water v/v/v = 2:1:1), and ammonium chloride (1.284 g, 24 mmol) and zinc powder (1.569 g, 24 mmol) were added, and the mixture was vigorously stirred at 50°C for 10 min; after the reaction was completed, the mixture was filtered and the filtrate was collected; the filtrate was evaporated under reduced pressure, and then extracted with DCM (300 mL) and saturated brine (400 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain 0.778 g of an off-white solid intermediate shown in Formula 7. The yield was 88%.

The above white solid intermediate (0.778 g, 2.5 mmol) was dissolved in glacial acetic acid (20 mL) and heated under reflux for 2 h. After the reaction was completed, it was extracted with EA (60 mL) and saturated brine (90 mL). The organic phase was collected, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to obtain 0.660 g of a reddish brown solid intermediate shown in formula 8. The yield was 90%.

The above reddish brown solid intermediate (0.587 g, 2 mmol) was dissolved in DMSO (6 mL), and 30% hydrogen peroxide (1.812 g containing 16 mmol H ₂ O ₂ ) and potassium carbonate (0.056 g, 0.4 mmol) were added in sequence, and the mixture was reacted at 25°C for 4 h. After the reaction, water (20 mL) was added to precipitate a pink solid, which was filtered and the filter cake was collected. The filter cake was purified by column chromatography (DCM: methanol v/v = 20:1), and the eluent containing the target compound was collected, concentrated, and dried to obtain an off-white solid b10 (0.324 g). The yield was 52%. mp＝197.7-198.9℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.09(s,1H),7.69(s,1H),7.37(d,J＝8.8Hz,1H),7.29(t,J＝8.0Hz,1H),7.16(s,1H),7.06(d,J＝2.4Hz,1H), 7.04–7.00(m,2H),6.88(ddd,J＝8.0,2.4,1.2Hz,1H),6.84(dd,J＝8.4,2.4Hz,1H),5.08(s,2H),3.75(s,3H),3.66(s,2H). ¹³ C NMR(100MHz,DMSO-d ₆ ,TFA)δ167.29,159.93,159.03(q, ² J _CF =38.1Hz,TFA),157.30,148.28,138.62,132.17,130.03,125.63,120.12,116.51,115.63(q, ¹ J _CF =286.7 Hz, TFA), 115.14, 113.72, 113.63, 98.18, 70.37, 55.32, 33.53. HRMS (ESI): m/z calcd for C ₁₇ H ₁₈ N ₃ O ₃ [M+H] ⁺ 312.1343, found 312.1345; HPLC purity: 100%.

Preparation method of Example 25b11

3-Amino-4-nitrophenol (1.233 g, 8 mmol) of Formula 4, 40 mL of mixed solvent (DCM:H ₂ O v/v＝1:1), potassium hydroxide (0.898 g, 16 mmol), TBAB (0.258 g, 0.8 mmol) and benzyl bromide (1.915 g, 11.2 mmol) were added to a round-bottom flask in sequence and stirred at room temperature for 2 h. After the reaction, DCM was removed by vacuum distillation, saturated brine (200 mL) was added, and the mixture was extracted with ethyl acetate (60 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was concentrated by vacuum distillation, and then separated by column chromatography (petroleum ether:DCM v/v＝1:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 1.798 g of an orange solid intermediate of Formula 5. The yield was 92%.

The above-obtained orange solid intermediate (1.709 g, 7 mmol) was added to a round-bottom flask in a DCM (15 mL) solvent, and then DIPEA (1.920 g, 14 mmol) and HATU (2.927 g, 7.7 mmol) were added. After stirring at room temperature for 5 min, cyanoacetic acid (0.654 g, 7.7 mmol) was added and stirred at room temperature for 7 h. After the reaction, an orange solid was precipitated. Part of the solvent was removed by vacuum distillation, and the filter cake was collected by suction filtration. The filter cake was slurried with methanol, suction filtered, and dried to obtain 1.264 g of an orange-yellow solid intermediate shown in Formula 6. The yield was 58%.

The above orange-yellow solid intermediate (0.934 g, 3 mmol) was dissolved in 32 mL of a mixed solvent (ethanol: acetonitrile: water v/v/v = 2:1:1), and ammonium chloride (1.284 g, 24 mmol) and zinc powder (1.569 g, 24 mmol) were added, and the mixture was vigorously stirred at 50°C for 10 min; after the reaction was completed, the mixture was filtered and the filtrate was collected; the filtrate was distilled under reduced pressure, and then extracted with DCM (400 mL) and saturated brine (400 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain 0.768 g of an off-white solid intermediate shown in Formula 7. The yield was 91%.

The above white solid intermediate (0.703 g, 2.5 mmol) was dissolved in glacial acetic acid (20 mL) and heated under reflux for 2 h. After the reaction was completed, it was extracted with EA (40 mL) and saturated brine (90 mL). The organic phase was collected, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to obtain 0.592 g of a reddish brown solid intermediate shown in formula 8. The yield was 90%.

The above reddish brown solid intermediate (0.526 g, 2 mmol) was dissolved in DMSO (6 mL), and 30% hydrogen peroxide (1.812 g containing 16 mmol H ₂ O ₂ ) and potassium carbonate (0.056 g, 0.4 mmol) were added in sequence, and the mixture was reacted at 25°C for 4 h. After the reaction, water (20 mL) was added to precipitate a pink solid, which was filtered and the filter cake was collected. The filter cake was purified by column chromatography (DCM: methanol v/v = 20:1), and the eluent containing the target compound was collected, concentrated, and dried to obtain a white solid b11 (0.366 g). The yield was 65%. mp＝202.2-204.8℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.11(s,1H),7.70(s,1H),7.47(d,J＝7.6Hz,2H),7.39(t,J＝8.0Hz,3H),7.32(m,1H),7.18(s,1H),7.08( d, J＝2.4Hz, 1H), 6.84 (dd, J＝8.8, 2.4Hz, 1H), 5.11 (s, 2H), 3.66 (s, 2H). ¹³ C NMR (100MHz, DMSO-d ₆ , TFA) δ 167.12, 158.92 (q, ² J _CF ＝36.8Hz,TFA),156.99,148.18,136.80,131.96,128.70,128.19,127.99,125.40,116.16,115.73(q, ¹ J _CF ＝288.2Hz,TFA),114.99,97.84,70.20,33 .32.HRMS(ESI):m/zcalcd for C ₁₆ H ₁₆ N ₃ O ₂ [M+H] ⁺ 282.1237, found 282.1235; HPLC purity: 100%.

Preparation method of Example 26b12

3-Amino-4-nitrophenol (1.233 g, 8 mmol) of Formula 4, 40 mL of mixed solvent (DCM:H ₂ O v/v＝1:1), potassium hydroxide (0.898 g, 16 mmol), TBAB (0.258 g, 0.8 mmol) and 4-trifluoromethylbenzyl bromide (2.677 g, 11.2 mmol) were added to a round-bottom flask in sequence and stirred at room temperature for 2 h. After the reaction, DCM was removed by vacuum distillation, saturated brine (200 mL) was added, and EA (80 mL) was used for extraction. The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was concentrated by vacuum distillation, and then separated by column chromatography (PE:DCM v/v＝1:1). The eluent containing the target intermediate was collected, concentrated, and dried to obtain 2.198 g of an orange solid intermediate of Formula 5. The yield was 88%.

The above-obtained orange solid intermediate (2.185 g, 7 mmol) was added to a round-bottom flask in a DCM (15 mL) solvent, and then DIPEA (1.920 g, 14 mmol) and HATU (2.927 g, 7.7 mmol) were added. After stirring at room temperature for 5 min, cyanoacetic acid (0.654 g, 7.7 mmol) was added and continued to stir for 8 h. After the reaction was completed, an orange solid precipitated, and part of the solvent was removed by vacuum distillation. The filter cake was collected by suction filtration, washed with methanol, slurried, suction filtered, and dried to obtain 1.513 g of an orange-yellow solid intermediate shown in Formula 6. The yield was 57%.

The above orange-yellow solid intermediate (1.138 g, 3 mmol) was dissolved in 32 mL of a mixed solvent (ethanol: acetonitrile: water v/v/v = 2:1:1), and ammonium chloride (1.284 g, 24 mmol) and zinc powder (1.569 g, 24 mmol) were added, and the mixture was vigorously stirred at 50°C for 10 min; after the reaction was completed, the mixture was filtered and the filtrate was collected; the filtrate was evaporated under reduced pressure, and then extracted with DCM (300 mL) and saturated brine (400 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain 0.891 g of an off-white solid intermediate shown in Formula 7. The yield was 85%.

The above white solid intermediate (0.873 g, 2.5 mmol) was dissolved in glacial acetic acid (20 mL) and heated under reflux for 2 h. After the reaction was completed, it was extracted with EA (30 mL) and saturated brine (80 mL). The organic phase was collected, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to obtain 0.779 g of a reddish brown solid intermediate shown in formula 8. The yield was 94%.

The above reddish brown solid intermediate (0.663 g, 2 mmol) was dissolved in DMSO (6 mL), and 30% hydrogen peroxide (1.812 g containing 16 mmol H ₂ O ₂ ) and potassium carbonate (0.056 g, 0.4 mmol) were added in sequence, and the mixture was reacted at 25°C for 6 h. After the reaction, water (20 mL) was added to precipitate a pink solid, which was filtered and the filter cake was collected. The filter cake was purified by column chromatography (DCM: methanol v/v = 20:1), and the eluent containing the target compound was collected, concentrated, and dried to obtain a light yellow solid b12 (0.426 g). The yield was 61%. mp＝210.7-211.8℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.12 (s, 1H), 7.75 (d, J＝8.4Hz, 2H), 7.69 (d, J＝8.0Hz, 3H), 7.39 (d, J＝8.8Hz, 1H), 7.17 (s, 1H), 7.08 (d, J＝ 2.4Hz, 1H), 6.86 (dd, J＝8.8, 2.8Hz, 1H), 5.24 (s, 2H), 3.67 (s, 2H). ¹³ C NMR (100MHz, DMSO-d ₆ , TFA) δ 167.30, 159.03 (q, ² J _CF =38.2Hz, TFA),157.05,148.48,141.85,132.17,129.18(q, ² J _CF =31.9Hz),128.37,125.80,125.70(q, ³ J _CF =3.9Hz),124.59(q, ¹ J _CF =270.1Hz), 116.38,115.60(q, ¹ J _CF =286.5Hz,TFA),115.25,98.29,69.62,33.54.HRMS(ESI):m/z calcd for C ₁₇ H ₁₅ F ₃ N ₃ O ₂ [M+H] ⁺ 350.1111, found 350.1110; HPLC purity: 100 %.

Preparation method of Example 27b13

4-Methoxy-o-phenylenediamine (0.691 g, 5 mmol) and ethyl cyanoacetate (1.696 g, 15 mmol) were added to the sealed tube in sequence and stirred at 210°C for 1.5 h. After the reaction, the reaction solution was purified by column chromatography (DCM: methanol v/v = 50:1), and the eluate containing the target compound was collected, concentrated, and dried to obtain 0.271 g of a reddish brown solid intermediate of 2-(5-methoxy-1H-benzo[d]imidazol-2-yl)acetonitrile. The yield was 29%.

The above reddish brown solid intermediate (0.234 g, 1.25 mmol) was dissolved in DMSO (3 mL), and 30% hydrogen peroxide (1.133 g containing 10 mmol H ₂ O ₂ ) and potassium carbonate (0.034 g, 0.25 mmol) were added in sequence, and the mixture was reacted at 25°C for 3 h. After the reaction, water (15 mL) was added to precipitate a pink solid, which was filtered and the filter cake was collected. The filter cake was purified by column chromatography (DCM: methanol v/v = 20:1), and the eluent containing the target compound was collected, concentrated, and dried to obtain a white solid b13 (0.154 g). The yield was 60%. mp＝191.7-194.5℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.06(s,1H),7.69(s,1H),7.40–7.31(m,1H),7.17(s,1H),6.99(d,J＝2.4Hz,1H),6.75(dd,J＝8.8,2.4Hz,1H ),3.76(s,3H),3.67(s,2H). ¹³ CNMR(100MHz, _DMSO -d ₆ ,TFA)δ167.05,158.80(q, ² J _CF =37.5Hz,TFA),158.15,148.02,132.56,125.24,115.58,115.56(q , ^1JCF =287.6Hz, TFA), 114.86, 96.55, 55.98, 33.31. HRMS (ESI): m/z calcd for C ₁₀ H ₁₂ N ₃ O ₂ [M+H] ⁺ 206.0924, found206.0920; HPLCpurity: 100%.

Preparation method of Example 28b14

4-Fluoro-o-phenylenediamine (0.630 g, 5 mmol) and ethyl cyanoacetate (1.696 g, 15 mmol) were added to the sealed tube in sequence and stirred at 210°C for 1.5 h. After the reaction, the reaction solution was purified by column chromatography (DCM: methanol v/v = 50:1), and the eluate containing the target intermediate was collected, concentrated, and dried to obtain 0.280 g of a reddish brown solid intermediate of 2-(5-fluoro-1H-benzo[d]imidazol-2-yl)acetonitrile. The yield was 32%.

The above reddish brown solid intermediate (0.219 g, 1.25 mmol) was dissolved in DMSO (3 mL), and 30% hydrogen peroxide (0.850 g containing 7.5 mmol H ₂ O ₂ ) and potassium carbonate (0.034 g, 0.25 mmol) were added in sequence, and the mixture was reacted at 25°C for 3 h. After the reaction, water (15 mL) was added to precipitate a pink solid, which was filtered and the filter cake was collected. The filter cake was purified by column chromatography (DCM: methanol v/v = 20:1), and the eluent containing the target compound was collected, concentrated, and dried to obtain an off-white solid b14 (0.133 g). The yield was 55%. mp＝203.3-206.1℃; ESI-HRMS m/z calc for C ₉ H ₉ FN ₃ O[M+H] ⁺ 194.0724, found 194.0721; ¹ HNMR (400MHz, DMSO-d ₆ ) δ12.18 (s, 1H), 7.55 (s, 1H), 7.41 (dd, J＝8.7, 2.0 Hz,1H),6.97(s,1H),6.99(d,J＝2.4Hz,1H),6.75(dd,J＝8.7,2.4Hz,1H),3.44(d,J＝2.9Hz,2H).

Preparation method of Example 29b15

In a round-bottom flask, the orange solid intermediate of formula 5 prepared according to Example 17 (1.311 g, 5 mmol) was added to DCM (15 mL) solvent, and then acetyl chloride (0.589 g, 7.5 mmol) was slowly added dropwise; after the addition was completed, the reaction was allowed to proceed overnight at room temperature; after the reaction was completed, the filtrate was filtered and collected; the filtrate was concentrated under reduced pressure, slurried with methanol, and dried to obtain 1.111 g of an orange-yellow solid intermediate of N-(4-((3-fluorobenzyl)oxy)-2-nitrophenyl)acetamide. The yield was 73%.

The above orange-yellow solid intermediate (0.913 g, 3 mmol) was dissolved in 32 mL of a mixed solvent (ethanol: acetonitrile: water v/v/v = 2:1:1), and ammonium chloride (1.284 g, 24 mmol) and zinc powder (1.569 g, 24 mmol) were added, and the mixture was vigorously stirred at 50°C for 10 min; after the reaction was completed, the mixture was filtered and the filtrate was collected; the filtrate was distilled under reduced pressure, and then extracted with DCM (100 mL) and saturated brine (200 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain 0.667 g of an off-white solid intermediate shown in Formula 7. The yield was 81%.

The above white solid intermediate (0.549 g, 2 mmol) was dissolved in glacial acetic acid (10 mL) and heated under reflux for 2 h. After the reaction was completed, it was extracted with EA (30 mL) and saturated brine (80 mL). The organic phase was collected, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to obtain a light pink solid b15 (0.492 g). The yield was 96%. mp＝145.8-149.6℃; ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.61 (d, J＝8.8Hz, 1H), 7.48–7.40 (m, 1H), 7.36–7.28 (m, 2H), 7.27 (d, J＝2.4Hz, 1H), 7.20–7.11 (m, 2H), 5.22 (s, 2H), 2.73 (s, 3H). ¹³ C NMR (100MHz, DMSO-d ₆ , TFA) δ162.39 (d, ¹ J _CF = 242.3Hz), 158.56 (q, ² J _CF = 37.9Hz, TFA), 156.49, 150.73, 139.76 (d, ³ J _CF ＝7.3Hz),131.76,130.63(d, ³ _JCF ＝8.4Hz),125.33,123.65(d, 4JCF＝2.7Hz),115.59,115.30(q, ^{1JCF ＝286.8Hz,TFA),114.80(d, 2JCF＝20.8Hz), 114.54, 114.40 (d, 2} _{J CF =} ^{21.8Hz )} _, 97.71, 69.25 ₍ d, ⁴ J _CF = 2.0Hz), 12.26. HRMS (ESI): m/z calcd for C ₁₅ H ₁₄ FN ₂ O [M+H] ⁺ 257.1085, found 257.1083; HPLC purity: 100 %.

Example 30 Pharmacological Experimental Data of Some Compounds

1. Determination of the inhibitory activity of compounds on MAO-B

Experimental methods:

The MAO-B inhibitor screening kit (MAK296) used in this experiment was purchased from Sigma and stored at -20°C for future use. Before the experiment, the enzyme, buffer, substrate, color developer, etc. in the kit were moved to room temperature for thawing, and the enzyme and substrate solutions were prepared according to the instructions. The sample was dissolved in DMSO solution to prepare a mother solution, and then gradually diluted with buffer solution to the required concentration (50μM, 25μM, 3.125μM, 0.781μM, 0.390μM, 0.195μM, 0.098μM, 0.049μM, 0.012μM). Take 10 μL of the test compound or positive drugs Pargyline and Safinamide and 50 μL of enzyme solution, mix them and add them to a 96-well black flat-bottom plate, shake and incubate at 37°C for 10 minutes, then add 40 μL of substrate solution (containing 1 μL substrate tyrosine, 1 μL color developer, 1 μL horseradish peroxidase and 37 μL buffer), and continuously detect the fluorescence value within 40 minutes (excitation: 535 nm; emission: 587 nm).

The results show that the compounds prepared in Examples 1 to 29 of the present invention have a good inhibitory effect on MAO-B. When the drug concentration is 1 μM, the inhibitory activity can reach up to 78.74%, which is significantly higher than the positive drug Pargyline; the IC ₅₀ value of the best compound b3 is 67.3 nM, which is significantly better than the positive control drug Pargyline and is comparable to Safinamide, the only reversible MAO-B inhibitor currently in clinical use.

Table 1. Inhibition rate of MAO-B in Examples 1 to 29 at 1 μM

Table 2. _IC50 values of inhibitory activity of some compounds

The determination of IC ₅₀ values revealed that Example b2 (IC ₅₀ =125.5±7.1 nM) and Example b3 (IC ₅₀ =67.3±1.5 nM) performed best among all the compounds.

2. Parallel artificial membrane permeability test

The test compounds b2 and b3 were dissolved in DMSO at a concentration of 5 mg/mL, and then gradually diluted to 25 μg/mL with phosphate buffered saline (PBS) buffer solution (pH = 7.4) for testing. An appropriate amount of porcine polar brain lipids (PBL, purchased from Avanti Polar Lipids, Inc) was directly dissolved in dodecane at a concentration of 20 mg/mL. After the entire filter membrane (area = 0.28 cm ² ) was pre-treated, the prepared PBL solution was added to the filter membrane surface of the donor plate (4 μL per well), and then the test compound solution (V _d ) was added to the donor plate (150 μL), and 300 μL PBS buffer (V _a ) was added to the receiving well. After the solution was added, the donor plate was carefully placed on the receiving plate so that the membrane and the upper and lower liquid surfaces were in contact with each other. Cover the lid and let it stand in the dark at 25°C for 6 hours, and repeat eight times. The absorbance was measured using an enzyme reader. According to the Pe equation, the concentration and permeability of the compound in the well were calculated. A is the filter area, t is the permeation time, drug _acceptor is the absorbance obtained in the receptor well, and drug _equilibrium is the theoretical equilibrium absorbance.

Pe＝-ln(1–drug _acceptor /drug _equilibrium )/[At(V _d +V _a )/V _d V _a ]

Table 3. _IC50 values of inhibitory activity of some examples

Pe≥4, BBB high permeability; Pe≤2, BBB low permeability; 2<Pe<4, BBB permeability is uncertain.

Parallel artificial membrane permeability experiments show that both Examples b2 and b3 can penetrate the blood-brain barrier.

3. Pharmacokinetic studies

The pharmacokinetic study of compound b3 was conducted. All rats (190±10g) were divided into three groups (blank group, b3 gavage group, b3 intravenous injection group), and were raised in strict accordance with the requirements of the experimental animal operation procedures, with a temperature (22±2°C) and a day and night cycle every 12h in a fixed environment. Rats were fasted 12h before the experiment, but had free access to water. Retroorbital blood samples (0.3mL) were taken from each rat at 0.083, 0.25, 0.50, 0.70, 1.0, 4.0, 6.0, 6.0, 1.0, 1.0, 10, and 24h after administration and placed in EDTA-soaked polyethylene tubes. The collected blood samples were centrifuged at 4000rpm for 10min at 4°C, 100μL of supernatant was taken, and stored in a -80°C refrigerator before use. Chromatographic separation experiments of biological samples were performed on a 1260HPLC system (Agilent, California, USA), which was equipped with a Triple Quad 4500 system (SCIEX, Bremen, Germany) using composite electrospray ionization (ESI) in positive ion mode. Separation was performed on a BEH C18 column (50×2.1 mm, 1.8 μm, Waters, Milford, MA, USA), and the column temperature was maintained at 35°C. Samples were eluted using an isocratic elution at a flow rate of 0.2 mL/min using 30% A (0.2% formic acid in water) and 70% B (0.3% formic acid in methanol). The total run time for each sample was 5 minutes. The volume of the injection solution was 5 μL. The mass spectrometer was operated in positive ion mode, and multiple reaction monitoring (MRM) mode was selected for all analytes. The precursor-product ion pair for Y-3 was m/z 300.1→191.0 (DP 114.6 eV, CE 35.5 eV). The optimized MS parameters were set as follows: 9 psi collision gas (CAD), 30 psi curtain gas (CUR), 45 psi nebulizer gas (GS1), 40 psi _N2 gas (GS2) heating, 5500 V ion spray voltage, and 500° C. The results are shown in FIG1 and Table 4.

Table 4. Pharmacokinetic parameters in rats (n=4, mean ± SD)

Analysis of pharmacokinetic data showed that compound B3 exhibited good pharmacokinetic properties, wide absorption distribution, not easy to accumulate in the body, and high bioavailability.

4. In vivo blood-brain barrier permeability test

B3 was prepared into a PBS clear solution containing 0.2% DMSO and 10% cyclodextrin, and the blood-brain barrier permeability of compound b3 in SD rats was studied. The rats were administered the compound by oral gavage (17.5 mg/kg) and tail vein injection (4 mg/kg), and the brain tissue was removed at 0.75 h (i.v. group) and 4 h (p.o. group) according to the pharmacokinetic data, washed with saline until there was no blood stain, and the surface moisture was absorbed with filter paper; and the weight of the brain tissue was weighed. After the brain tissue was homogenized, 0.5 mL of the homogenate was added to 1.5 mL of acetonitrile for protein precipitation, followed by centrifugation twice at 4°C and 12,000 rpm for 10 min each time, and the supernatant was taken for sampling. The content of b3 in brain tissue was detected by the method of 3 in Example 29. The results are shown in Table 5.

Table 5. Blood-brain barrier permeability of compound b3 in rats (mean ± SD)

The content was analyzed and it was found that compound b3 had good blood-brain barrier permeability in the rat brain.

Claims (10)

1. A benzimidazole derivative represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof:

In formula (I): R ¹ and R ² are each independently H, halogen, C ₁ -C ₅ straight chain or branched chain alkyl or C ₁ -C ₅ straight chain or branched chain alkoxy; m is 0, 1 or 2; In formula (II): R ³ and R ⁴ are each independently H, halogen, C ₁ -C ₅ haloalkyl, C ₁ -C ₅ straight or branched alkyl, or C ₁ -C ₅ straight or branched alkoxy; n is 0, 1 or 2. 2. The benzimidazole derivative represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: in formula (I), R ¹ and R ² are each independently H, F, Cl, CH ₃ or OCH ₃ ; m is 1; in formula (II), R ³ and R ⁴ are each independently H, F, Cl, CF ₃ , CH ₃ or OCH ₃ ; n is 1. 3. The benzimidazole derivative represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that the benzimidazole derivative represented by formula (I) or formula (II) is one of the following compounds:

4. Use of a benzimidazole derivative represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 3 in the preparation of a medicament for treating or preventing a disease caused by overexpression of MAO-B. 5. The use according to claim 5, characterized in that the disease caused by the overexpression of MAO-B is Parkinson's disease or Alzheimer's disease. 6. The use according to claim 4, characterized in that the benzimidazole derivative represented by formula (I) or formula (II) is a compound represented by formula b2, b3, b5, b6, b8 or b12. 7. A method for preparing a benzimidazole derivative represented by formula (I), characterized in that the method comprises the following steps: (1) dissolving 2-cyanomethyl-5-carboxyl-1H-benzimidazole as shown in Formula 2 in an organic solvent A, adding alkaline substance A and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate in sequence, stirring at room temperature, adding an aniline compound as shown in Formula 9, stirring at room temperature for 3-18 hours, and subjecting the obtained reaction solution A to post-treatment A to obtain a compound as shown in Formula 3; the molar ratio of 2-cyanomethyl-5-carboxyl-1H-benzimidazole as shown in Formula 2, alkaline substance A, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and aniline compound as shown in Formula 9 is 1:1-3:1-1.5:0.5-1.5; the alkaline substance A is one or a mixture of two or more of potassium carbonate, sodium carbonate, sodium bicarbonate, potassium hydroxide, sodium hydroxide and triethylamine;

(2) The compound of formula 3 in step (1) is reacted in concentrated hydrochloric acid at 40° C. for 35-50 min, and the resulting reaction solution B is subjected to post-treatment B to obtain a benzimidazole derivative of formula (I);

In formula 9, 3, (I), R ¹ and R ² are each independently H, halogen, C ₁ -C ₅ straight chain or branched alkyl or C ₁ -C ₅ straight chain or branched alkoxy; m is 0, 1 or 2. 8. The method for preparing the benzimidazole derivative of formula (I) as claimed in claim 7, characterized in that: in step (1), the organic solvent A is one or a mixture of two or more selected from acetonitrile, N,N-dimethylformamide, methanol, ethanol, acetonitrile and dichloromethane; the volume of the organic solvent A is 1-2 mL/mmol based on the amount of the compound of formula 2; the post-treatment A comprises: adding saturated brine, extracting with dichloromethane, combining the organic phases, and drying over anhydrous sodium sulfate; removing the solvent by reduced pressure distillation, performing column chromatography separation using a mixed solution of dichloromethane and methanol in a volume ratio of 30:1 as an eluent, collecting the eluate containing the target intermediate, concentrating, and drying to obtain the compound of formula 3; In step (2), the volume of the concentrated hydrochloric acid is 2 to 4 mL/mmol based on the amount of the compound of formula 3; the post-treatment B is: adjusting the pH of the reaction solution B to neutral with a saturated sodium bicarbonate solution, precipitating a solid, filtering with suction, and separating the filter cake by column chromatography using a mixed solution of dichloromethane and methanol in a volume ratio of 20:1 as an eluent, collecting the eluate containing the target compound, concentrating, and drying to obtain a benzimidazole derivative represented by formula (I). 9. A method for preparing a benzimidazole derivative represented by formula (II), characterized in that the method comprises the following steps: S1: dissolving the compound of Formula 5 in solvent D, adding 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and alkaline substance D, stirring at room temperature for 5 minutes, and then adding cyanoacetic acid; stirring at room temperature for 5-10 hours, and subjecting the obtained reaction solution D to post-treatment D to obtain the compound of Formula 6; the molar ratio of the compound of Formula 5, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, alkaline substance D and cyanoacetic acid is 1:1-2:1.5-2.5:1-1.5;

S2: dissolving the compound of formula 6 described in step S1 in a mixed solvent E, adding Zn powder and NH ₄ Cl in sequence, stirring and reacting at 50° C. for 10 min, and subjecting the obtained reaction solution E to post-treatment E to obtain a compound of formula 7; the molar ratio of the compound of formula 6, Zn powder and NH ₄ Cl is 1:5-10:5-10;

S3: dissolving the compound of formula 7 described in step S2 in acetic acid, and refluxing the reaction for 2 hours. The obtained reaction solution G is subjected to post-treatment G to obtain the compound of formula 8;

S4: dissolving the compound of formula 8 described in step S3 in a solvent Q, adding 30% by mass hydrogen peroxide and alkaline substance Q in sequence, stirring at 25°C for 3-8h, and subjecting the obtained reaction solution Q to post-treatment Q to obtain a benzimidazole derivative represented by formula (II); the molar ratio of the compound of formula 8, hydrogen peroxide contained in hydrogen peroxide and alkaline substance Q is 1:5-12:0.2-0.5;

In formula 5, 6, 7, 8, (II), R ³ and R ⁴ are each independently H, halogen, C ₁ -C ₅ haloalkyl, C ₁ -C ₅ straight or branched alkyl, or C ₁ -C ₅ straight or branched alkoxy; n is 0, 1 or 2. 10. The method for preparing the benzimidazole derivatives of formula (II) as claimed in claim 9, characterized in that: in step S1, the solvent D is one or a mixture of two or more selected from water, toluene, acetonitrile, dichloromethane, methanol, ethanol, and N,N-dimethylformamide; the volume of the solvent D is 1 to 3 mL/mmol based on the amount of the compound of formula 5; the alkaline substance D is one or a mixture of two or more selected from potassium carbonate, sodium carbonate, sodium bicarbonate, potassium hydroxide, sodium hydroxide, N,N-diisopropylethylamine, and triethylamine; the post-treatment D is: distilling the reaction solution D under reduced pressure to remove part of the solvent, filtering with suction, washing the obtained filter cake A with methanol, beating the obtained filter cake B with methanol, filtering with suction, and drying the obtained filter cake C to obtain the compound of formula 6; In step S2, the mixed solvent E is a mixed solvent of water and an organic solvent E, and the organic solvent E is one or a mixture of two or more of toluene, acetonitrile, dichloromethane, methanol, ethanol, and N,N-dimethylformamide; the volume of the mixed solvent E is 8 to 12 mL/mmol based on the amount of the compound represented by formula 6; the post-treatment E is: filtering the reaction solution E, distilling the obtained filtrate under reduced pressure, extracting with dichloromethane and saturated brine, and drying the obtained organic phase with anhydrous sodium sulfate, and distilling under reduced pressure to obtain the compound represented by formula 7; In step S3, the volume of the acetic acid is 5 to 8 mL/mmol based on the amount of the compound of formula 7; the post-treatment G is: extracting the reaction solution G with ethyl acetate and saturated brine, drying the obtained organic phase with anhydrous sodium sulfate, and distilling under reduced pressure to obtain the compound of formula 8; In step S4, the solvent Q is one or a mixture of two or more selected from water, methanol, acetonitrile, dichloromethane, ethanol, dimethyl sulfoxide, and N,N-dimethylformamide; the volume of the solvent Q is 1 to 3 mL/mmol based on the amount of the compound represented by formula 8; the alkaline substance Q is one or a mixture of two or more selected from potassium carbonate, sodium carbonate, sodium bicarbonate, potassium hydroxide, and sodium hydroxide; the post-treatment Q is: adding water to the reaction solution Q to precipitate solids, filtering, and purifying the obtained filter cake by column chromatography using a mixed solution of dichloromethane and methanol in a volume ratio of 20:1 as an eluent, collecting the eluate containing the target compound, concentrating, and drying to obtain the benzimidazole derivative represented by (II).

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