Hydrophobic tag compound for targeted degradation of Tau protein as well as preparation and application of hydrophobic tag compound

Abstract

The invention discloses a hydrophobic tag compound for targeted degradation of Tau protein as well as preparation and application of the hydrophobic tag compound, the compound is a small-molecule degradation agent with low molecular weight, good degradation effect and good blood brain barrier permeability, and the preparation method of the compound is characterized in that a hydrophobic tag technology is adopted to prepare the hydrophobic tag compound for targeted degradation of Tau protein. A small molecule Tau protein ligand is connected with a hydrophobic tag structure fragment with hydrophobicity through a proper linking chain (Linker), so that the blood-brain barrier permeability of the compound is improved on the basis of generating Tau protein degradation activity, and the compound has important application potential in treatment of neurodegenerative diseases.

Description

Technical Field

[0001] The present invention relates to a hydrophobic tag compound, in particular to a hydrophobic tag compound for targeted degradation of Tau protein, and also to a preparation method and application of the above compound. Background Art

[0002] Microtubule-associated protein Tau (MAPT) plays a role in stabilizing tubulin in the brain and helps regulate the transport of intracellular substances. In some neurodegenerative diseases such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), dementia with Lewy bodies (DLB), corticobasal degeneration (CBD), Pick's disease (PiD), and frontotemporal dementia (FTD), the normal function of Tau protein is destroyed, leading to its aggregation and pathology. Therefore, how to reduce or even eliminate Tau pathology has become the direction of treating these neurodegenerative diseases (NDD). The main challenges in the development of NDD drugs currently come from the pathogenicity of aggregated proteins and the blood-brain barrier permeability of drugs.

[0003] Targeted protein degradation (TPD) technology is a disease treatment strategy that has developed rapidly since the 21st century. This technology can overcome the shortcomings of small molecule drugs that are prone to drug resistance, and has unparalleled catalytic activity of small molecule drugs, and also opens up new treatment strategies for some "undruggable" targets. Using TPD technology to reduce or even eliminate Tau pathology is a strategy for treating neurodegenerative diseases that has emerged in recent years. So far, protein degradation targeted chimeric PROTACs capable of degrading Tau protein have been reported in the literature, such as TH006 (Cell Chem. Biol. 2016; 23(4): 453-461), QC-01-175 (eLife. 2019; 8: e45457), C004019 (Theranostics. 2021; 11(11): 5279-5295) and T3 (J. Med. Chem. 2024; 67(5): 3448-3466); as well as a peptide hydrophobic tag HyT-Tau-CPP (RSC Adv. 2017; 7: 40362-40366) that has the function of degrading Tau protein. At present, the literature reports that Tau protein degraders, whether PROTACs or peptide hydrophobic tag molecules, have the disadvantage of large molecular weight. Since drugs for the treatment of neurodegenerative diseases need to have good blood-brain barrier permeability, large molecular weight compounds have poor blood-brain barrier permeability and it is difficult to achieve effective therapeutic effects. At present, the only Tau protein degraders that have been discovered are PROTAC and peptide hydrophobic tags. The principle of PROTAC is to connect the target protein ligand and E3 ubiquitin enzyme ligands such as CRBN or Vhl through a linker. E3 ubiquitin enzyme ligands such as CRBN and Vhl often have the disadvantages of large molecular weight and large topological polar surface area. After being connected with the target protein ligand, the molecular weight and topological polar surface area will become larger, and they cannot effectively penetrate the blood-brain barrier; the target protein ligand of the peptide hydrophobic tag is a short peptide sequence, which also has similar defects as PROTAC. The small molecule hydrophobic tag has a small molecular weight and a small topological polar surface area, which makes it easier to penetrate the blood-brain barrier and has greater drugability potential.

[0004] Among the two major markers of AD, Tau protein aggregates are more strongly correlated with AD cognitive impairment than Aβ, and targeted therapy for Tau protein is expected to produce better clinical effects. Therefore, exploring new methods and strategies to improve the blood-brain barrier permeability of Tau protein degraders while retaining their excellent degradation effect on Tau protein is expected to achieve better therapeutic effects for AD-related diseases. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a hydrophobic tag compound for targeted degradation of Tau protein, and also to provide a preparation method and application of the above compound.

[0006] Technical solution: The hydrophobic tag compound for targeted degradation of Tau protein of the present invention is composed of a Tau protein binding ligand and a hydrophobic tag group connected by a linker, as shown in Formula I, and also includes stereoisomers, tautomers, deuterated compounds, pharmaceutically acceptable salts or mixtures thereof:

[0007]

[0008] Wherein: X is CH or N;

[0009] Y is a substituted or unsubstituted piperidine ring, a substituted or unsubstituted piperazine ring, a substituted or unsubstituted bridged ring, a substituted or unsubstituted spiro ring, a substituted or unsubstituted 6- to 10-membered aromatic ring, a substituted or unsubstituted 5- to 10-membered aromatic heterocyclic ring, -CH 2 -, -NH-, -O- or -S-;

[0010] Linker is a connecting chain, which is a straight or branched alkylene chain with a total length of 1 to 15 atoms, or no group, wherein the straight or branched alkylene chain is composed of -CH 2 -, -O-, -CONH-, -NHCO-, -NHCONH-, -NH-, -S-, one or more of sulfinyl, sulfonyl, aminosulfonylamino, alkynylene, alkenylene, cycloalkylene, arylene, heterocyclylene or heteroarylene, wherein the linear or branched alkylene chain is optionally substituted with one or more substituents;

[0011] HyT is selected from:

[0012]

[0013] Preferably, Y is the following group:

[0014]

[0015] Preferably, the Linker is the following group:

[0016]

[0017] Preferably, the compound is selected from any of the following compounds:

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025] Preferably, the pharmaceutically acceptable salt is a salt of the compound formed with any one of the following acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, phosphorous acid, carbonic acid, methanesulfonic acid, benzoic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, propionic acid, malonic acid, butyric acid, isobutyric acid, fumaric acid, suberic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, mandelic acid, amino acids, glucuronic acid and ferulic acid.

[0026] The method for preparing the hydrophobic tag compound comprises the following steps:

[0027] (1) Synthesis of Tau-targeting ligands

[0028]

[0029] The synthesis steps are as follows: Take Ⅰ-1 and Ⅰ-2 as raw materials, in anhydrous copper acetate and Cs 2 CO 3 In the presence of Et, anhydrous DMF was used as solvent and cyclization was carried out under argon protection to obtain ligand Ⅰ-3. The synthesis method of ligand Ⅰ-8 is to use Ⅰ-4 and (E)-1,1,1-trichloro-4-ethoxybut-3-ene-2-one Ⅰ-5 as raw materials, 3 In the presence of N, toluene is used as a solvent for cyclization to obtain intermediate Ⅰ-6, and then compound Ⅰ-6 is reacted with a mixed solution of 1N NaOH solution and acetonitrile as a solvent to obtain intermediate Ⅰ-7. Finally, compound Ⅰ-7 is brominated in the presence of phosphorus oxybromide with DCE and DMF as a mixed solvent to obtain ligand Ⅰ-8.

[0030] (2) Synthesis route 1 of hydrophobic tag compound for targeted degradation of Tau protein:

[0031]

[0032] The synthesis steps are as follows: Use Ⅰ-9 and Ⅰ-10 as raw materials, in K 2 CO 3In the presence of DMA as solvent, intermediate Ⅰ-11 is reacted, and then compound Ⅰ-11 is reacted with a mixed solution of 2.5N NaOH solution and THF as solvent to obtain intermediate Ⅰ-12. Then compound Ⅰ-12 and compound Ⅰ-13 are reacted in the presence of DIPEA and HATU with DMF as solvent to obtain hydrophobic tag compound Ⅰ-14 targeted to degrade Tau protein.

[0033] (3) Synthesis route 2 of hydrophobic tag compounds for targeted degradation of Tau protein:

[0034]

[0035] The synthesis steps are as follows: Use Ⅰ-9 and Ⅰ-15 as raw materials, in K 2 CO 3 In the presence of , DMA is used as a solvent to react to obtain a Boc-protected intermediate Ⅰ-16, and then the Boc protecting group is removed in the presence of TFA and DCM to obtain intermediate Ⅰ-16, and then compounds Ⅰ-16 and Ⅰ-17 are reacted in the presence of DIPEA and DMSO as a solvent to obtain intermediate Ⅰ-18 with a protecting group, and then the protecting group is removed in the presence of TFA and DCM to obtain intermediate Ⅰ-18, and compounds Ⅰ-18 and Ⅰ-19 are reacted in the presence of DIPEA and HATU with DMF as a solvent to obtain a hydrophobic tag compound Ⅰ-20 for targeted degradation of Tau protein. The present invention also discloses a pharmaceutical composition, comprising the above-mentioned hydrophobic tag compound, stereoisomer, tautomer, deuterated compound, pharmaceutically acceptable salt or mixture for targeted degradation of Tau protein, and pharmaceutically acceptable excipients or carriers.

[0036] Wherein, the excipient is a material used in pharmaceutical preparations to enhance drug stability, improve drug performance or achieve specific preparation properties. The pharmaceutical excipient may include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0037] Wherein, the carrier is a material or system that has been verified and meets the safety, effectiveness and quality standards of the drug, and is used to improve the delivery effect of the drug. Wherein the carrier includes liposomes, nanoparticles, solid lipid nanoparticles, polymer microspheres, hydrogels, nanocapsules, and microemulsions. "Pharmaceutically acceptable carriers" can also be excipients or diluents that do not cause obvious irritation to the organism and do not interfere with the biological activity and properties of the administered compound. The excipients include flavoring agents, aromatics, cosolvents, emulsifiers, solubilizers, adhesives, preservatives, antioxidants, osmotic pressure regulators, colorants, fillers, disintegrants, lubricants, etc., and the diluents include starch, saline, sucrose, lactose, dextrin, etc.

[0038] The hydrophobic tag compound or pharmaceutical composition for targeted degradation of Tau protein can also be used in the preparation of drugs for treating neurodegenerative diseases.

[0039] Among them, the neurodegenerative diseases include Alzheimer's disease (AD), progressive supranuclear palsy (PSP), dementia with Lewy bodies (DLB), corticobasal degeneration (CBD), Pick's disease (PiD), frontotemporal dementia (FTD), etc.

[0040] Among them, the hydrophobic tag compound for targeted degradation of Tau protein connects the small molecule Tau protein ligand with the hydrophobic tag structure fragment with hydrophobicity through a connecting chain. On the basis of producing Tau protein degradation activity, it is used in drugs for degenerative diseases to improve the blood-brain barrier permeability of the drug.

[0041] Principle of the invention: The hydrophobic tag compound of the present invention that targets the degradation of Tau protein connects the small molecule Tau protein ligand with the hydrophobic tag group through a Linker to synthesize a class of small molecule degraders with low molecular weight, good degradation effect, and good blood-brain barrier permeability. By binding to the Tau protein, the compound of the present invention simulates misfolded proteins, so that the target protein is recognized by the ubiquitin proteasome system or the lysosome system, and thus degraded. By degrading the Tau protein, the content of pathological phosphorylated Tau protein can be effectively reduced. The compound of the present invention can target the degradation of Tau protein and alleviate the pathological state of Tau. In addition, the compound of the present invention is expected to overcome the defect of low blood-brain barrier permeability of the existing Tau protein PROTAC molecules.

[0042] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the compound of the present invention has the ability to degrade Tau protein, such as degrading more than 50% of Tau protein at a dosage concentration of 10 μM, and has high blood-brain barrier permeability, and can better penetrate the blood-brain barrier into the brain to exert a degradation effect; when used in therapeutic drugs for neurodegenerative diseases, it can simultaneously achieve the effect of degrading Tau protein and improving blood-brain barrier permeability. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The Western Blot results of the hydrophobic tag compound 31 for targeted degradation of Tau protein of the present invention on the concentration-dependent degradation of Tau protein;

[0044] Figure 2 The quantitative analysis of the Western Blot results of the hydrophobic tag compound 31 for targeted degradation of Tau protein of the present invention on the concentration-dependent degradation of Tau protein;

[0045] Figure 3 The Western Blot results of the hydrophobic tag compound 43 for targeted degradation of Tau protein of the present invention on Tau protein concentration-dependent degradation;

[0046] Figure 4 This is a quantitative analysis of the Western Blot results of the hydrophobic tag compound 43 for targeted degradation of Tau protein in the present invention, which is in a concentration-dependent manner. DETAILED DESCRIPTION

[0047] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0048] Example 1: Synthesis of 2-(1-(benzo[4,5]imidazo[1,2-a]pyridin-3-yl)piperidin-4-yl)-N-(1,2,3,5,6,7-tetrahydro-s-indacen-4-yl)acetamide (H1)

[0049]

[0050] Step a: Preparation of 3-bromobenzo[4,5]imidazole[1,2-a]pyridine (Z-1-1)

[0051] In a 500 mL eggplant-shaped bottle, 4-bromopyridin-2-amine (2.79 g, 16.14 mmol) and (2-iodophenyl)boronic acid (4 g, 16.14 mmol), anhydrous copper acetate (879 mg, 4.84 mmol), Cs 2 CO 3 (15.78 g, 48.42 mmol) were added to anhydrous DMF (150 mL) in sequence and stirred at 120 °C for 20 hours under argon protection. After the reaction was completed, water was added to quench the reaction, EA was extracted (400 mL × 3), the organic layers were combined, washed with saturated brine, and anhydrous Na 2 SO 4After drying and concentration, separation by silica gel column chromatography gave Z1-1 (light yellow solid, 2.54 g, yield 63.7%). ESI-MS m / z: 247.10 [M+H] + .

[0052] Step b: Preparation of ethyl 2-(1-(benzo[4,5]imidazo[1,2-a]pyridin-3-yl)piperidin-4-yl)acetate (Z-1-2)

[0053] In a 250 mL eggplant-shaped bottle, 3-bromobenzo[4,5]imidazole[1,2-a]pyridine (2.54 g, 10.28 mmol), ethyl 2-(piperidin-4-yl)acetate (3.52 g, 20.56 mmol) and K 2 CO 3 (2.84 g, 20.56 mmol) was added to DMA (100 mL). Stirred at 130 °C for 6 hours. After the reaction was completed, it was cooled to room temperature, water was added to quench the reaction, and EA was extracted (200 mL × 3). The organic layers were combined, washed with saturated brine, and anhydrous Na 2 SO 4 After drying and concentration, separation by silica gel column chromatography gave Z-1-2 (yellow solid, 2.97 g, yield 85.4%). ESI-MS m / z: 338.35 [M+H] + .

[0054] Step c: Preparation of 2-(1-(benzo[4,5]imidazo[1,2-a]pyridin-3-yl)piperidin-4-yl)acetic acid (Z-1-3)

[0055] In a 500 mL eggplant-shaped bottle, ethyl 2-(1-(benzo[4,5]imidazo[1,2-a]pyridin-3-yl)piperidin-4-yl)acetate (2.97 g, 8.8 mmol) was added to a mixed solution of 2.5 N NaOH solution (100 mL) and THF (100 mL), and the mixture was refluxed for 1 h. After the reaction, the reaction solution was concentrated, water was added to quench the reaction, and EA was extracted (200 mL × 3). The aqueous phase was adjusted to pH 1 with 2 N HCl solution, extracted with DCM (200 mL × 3), and the organic layers were combined and anhydrous Na 2 SO 4 Drying and concentration gave Z-1-3 (light yellow oily liquid, 2.72 g, yield 74.6%). ESI-MS m / z: 308.28 [MH] - .

[0056] Step d: Preparation of 2-(1-(benzo[4,5]imidazo[1,2-a]pyridin-3-yl)piperidin-4-yl)-N-(1,2,3,5,6,7-tetrahydro-s-indacen-4-yl)acetamide (H1)

[0057] In a 50 mL eggplant-shaped bottle, 2-(1-(benzo[4,5]imidazo[1,2-a]pyridin-3-yl)piperidin-4-yl)acetic acid (100 mg, 323 μmol), HATU (160 mg, 420 μmol), and DIPEA (169 μL, 970 μmol) were added to DMF (5 mL) in sequence. After stirring at room temperature for 15 minutes, 1,2,3,5,6,7-hexahydro-s-indacene-4-amine (84 mg, 485 μmol) was added and reacted at room temperature overnight. After the reaction was completed, water was added to quench the reaction and EA was extracted (20 mL×3). The organic layers were combined, washed with saturated brine, and anhydrous Na 2 SO 4 After drying and concentration, H1 (white solid, 85 mg, yield 56.6%) was isolated by silica gel column chromatography. 1 H NMR (400 MHz, DMSO-d 6 )δ9.23(s,1H),8.20(d,J=6.8Hz,1H),7.95-7.91(m,1H),7.90-7.85(m,1H),7.41(td,J=7.2 ,1.6Hz,1H),7.38(dd,J=4.9,1.0Hz,1H),6.87(s,1H),6.77(dd,J=6.9,1.3Hz,1H),6.69(d,J =1.4Hz,1H),3.28-3.21(m,4H),2.83(t,J=6.4Hz,4H),2.75(t,J=6.4Hz,4H),2.38(d,J=7.3 Hz,2H),2.16-2.09(m,4H),2.05-1.97(m,1H),1.83-1.71(m,4H).HR-MS(ESI)m / z:calcd.for C 30 H 32 N 4 O[M+Na] + 487.2468, found:487.2472.

[0058] Example 2: Preparation of 2-(1-(benzo[4,5]imidazole[1,2-a]pyridin-3-yl)piperidin-4-yl)-N-(9H-fluorene-9-yl)acetamide (H2)

[0059] The preparation method refers to the operation of Example 1, using 9H-fluorene-9-amine instead of 1,2,3,5,6,7-hexahydro-s-indacene-4-amine as the reactant to prepare H2 (light yellow solid, 77 mg, yield 50.4%). 1 H NMR (400 MHz, DMSO-d 6)δ8.20(d,J=6.8Hz,1H),7.93(d,J=6.9Hz,1H),7.88(d,J=6.2Hz,1H),7.74(dd,J=7.8, 1.4Hz,2H),7.65(d,J=7.6Hz,1H),7.52(dd,J=7.6,1.7Hz,2H),7.44-7.31(m,6H),6.77 (dd,J=6.9,1.3Hz,1H),6.69(s,1H),5.99(dd,J=7.6,1.0Hz,1H),3.30-3.20(m,4H),2. 36(d,J=7.7Hz,2H),2.08-1.96(m,1H),1.84-1.70(m,4H).HR-MS(ESI)m / z:calcd.forC 31 H 28 N 4 O[M+Na] + 495.2155, found:495.2158.

[0060] Example 3: Preparation of 2-(1-(benzo[4,5]imidazo[1,2-a]pyridin-3-yl)piperidin-4-yl)-N-(bicyclo[2.2.1]hept-5-en-2-ylmethyl)acetamide (H3)

[0061] The preparation method was prepared by referring to the procedure of Example 1, except that bicyclo[2.2.1]hept-5-en-2-ylmethylamine was used instead of 1,2,3,5,6,7-hexahydro-s-indacene-4-amine as the reactant to prepare H3 (beige solid, 96 mg, yield 71.6%). 1 H NMR (400 MHz, DMSO-d 6)δ8.20(d,J=6.8Hz,1H),7.95-7.91(m,1H),7.89-7.86(m,1H),7.44-7.40(m,1H),7.40-7.35(m,1H),7.29( t,J=6.2Hz,1H),6.77(dd,J=6.9,1.3Hz,1H),6.69(d,J=1.4Hz,1H),6.03-5.97(m,1H),5.92-5.86(m,1H),3. 25(m,4H),3.08-3.02(m,2H),2.81-2.70(m,2H),2.21(d,J=7.5Hz,2H),2.17-2.10(m,1H),2.02-1.92(m,1H) ,1.87-1.69(m,4H),1.56(t,J=4.9Hz,2H),1.36-1.28(m,1H),1.26-1.19(m,1H).HR-MS(ESI)m / z:calcd.for C 26 H 30 N 4 O[M+Na] + 437.2312, found:437.2307.

[0062] Example 4: Preparation of N-(((3R,5R,7R)-adamantan-1-yl)methyl)-2-(1-(benzo[4,5]imidazo[1,2-a]pyridin-3-yl)piperidin-4-yl)acetamide (H4)

[0063] The preparation method was prepared by referring to the procedure of Example 1, except that ((3R,5R,7R)-adamantan-1-yl)methylamine was used instead of 1,2,3,5,6,7-hexahydro-s-indacene-4-amine as the reactant to prepare H4 (white solid, 121 mg, yield 82.0%). 1 H NMR (400 MHz, DMSO-d 6)δ8.20(d,J=6.8Hz,1H),7.95-7.91(m,1H),7.90-7.86(m,1H),7.44-7.40(m,1H),7.4 0-7.36(m,1H),7.25(t,J=5.8Hz,1H),6.77(dd,J=6.9,1.3Hz,1H),6.69(d,J=1.3Hz,1 H),3.25(m,4H),3.00(d,J=5.7Hz,2H),2.20(d,J=7.5Hz,2H),2.05-1.90(m,4H),1.84 -1.69(m,4H),1.64(t,J=5.7Hz,6H),1.49(d,J=5.1Hz,6H).HR-MS(ESI)m / z:calcd.for C 29 H 36 N 4 O[M+Na] + 479.2781,found:479.2786

[0064] Example 5: Preparation of N-diphenylmethyl-2-(1-(benzo[4,5]imidazo[1,2-a]pyridin-3-yl)piperidin-4-yl)acetamide (H5)

[0065] The preparation method was similar to that of Example 1, except that diphenylmethane was used instead of 1,2,3,5,6,7-hexahydro-s-indacene-4-amine as the reactant to prepare H5 (light yellow solid, 67 mg, yield 43.8%). 1 H NMR (400 MHz, DMSO-d 6 )δ8.25(d,J=8.5Hz,1H),8.20(d,J=6.9Hz,1H),7.96-7.90(m,1H),7.90-7.86(m,1H) ,7.44-7.40(m,1H),7.40-7.36(m,1H),7.32(d,J=4.7Hz,8H),7.29-7.23(m,2H),6.77 (dd,J=6.9,1.3Hz,1H),6.69(d,J=1.5Hz,1H),6.03(d,J=8.5Hz,1H),3.20(m,4H),2. 28(d,J=7.8Hz,2H),2.03-1.93(m,1H),1.83-1.67(m,4H).HR-MS(ESI)m / z:calcd.for C 31 H 30 N 4 O[M+Na] + 497.2312, found:497.2314.

[0066] Example 6: Preparation of 2-(1-(benzo[4,5]imidazo[1,2-a]pyridin-3-yl)piperidin-4-yl)-N-(dicyclohexylmethyl)acetamide (H6)

[0067] The preparation method refers to the operation of Example 1, using dicyclohexylmethylamine instead of 1,2,3,5,6,7-hexahydro-s-indacene-4-amine as the reactant to prepare H6 (light yellow solid, 101 mg, yield 64.2%). 1 H NMR (400 MHz, DMSO-d 6 )δ8.20(d,J=6.8Hz,1H),7.95-7.91(m,1H),7.90-7.85(m,1H),7.44-7.40(m,1 H),7.40-7.36(m,1H),6.77(dd,J=6.9,1.3Hz,1H),6.71(d,J=8.7Hz,1H),6.69( d,J=1.4Hz,1H),3.46-3.43(m,1H),3.25-3.14(m,4H),2.25-2.15(m,2H),2.02- 1.93(m,1H),1.83-1.71(m,6H),1.51-1.25(m,20H).HR-MS(ESI)m / z:calcd.for C 31 H 42 N 4 O[M+Na] + 509.3251, found:509.3246.

[0068] Example 7: Preparation of (1R,2S,5R)-2-isopropyl-5-methylcyclohexyl 2-(1-(benzo[4,5]imidazo[1,2-a]pyridin-3-yl)piperidin-4-yl)acetate (H7)

[0069] The preparation method refers to the operation of Example 1, using (1R, 2S, 5R)-2-isopropyl-5-methylcyclohexyl-1-ol instead of 1,2,3,5,6,7-hexahydro-s-indacene-4-amine as the reactant, and H7 (white solid, 63 mg, yield 43.5%) was prepared under appropriate conditions understood in the art. 1 H NMR (400 MHz, DMSO-d 6)δ8.20(d,J=6.8Hz,1H),7.95-7.91(m,1H),7.90-7.87(m,1H),7.44-7.40(m,1H),7.40-7.36(m, 1H),6.77(dd,J=6.9,1.3Hz,1H),6.69(d,J=1.3Hz,1H),4.71-4.59(m,1H),3.46-3.00(m,4H),2.4 1-2.20(m,2H),2.10-1.95(m,1H),1.89-1.80(m,4H),1.80-1.75(m,1H),1.74-1.69(m,1H),1.69- 1.64(m,2H),1.58-1.48(m,1H),1.46-1.25(m,4H),0.94-0.69(m,9H).HR-MS(ESI)m / z:calcd.for C 28 H 37 N 3 O 2 [M+Na] + 470.2778,found:470.2785.

[0070] Example 8: Preparation of 2-(1-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperidin-4-yl)-N-(1,2,3,5,6,7-tetrahydro-s-indacen-4-yl)acetamide (H8)

[0071]

[0072] Step a: Preparation of 2-(trichloromethyl)benzo[4,5]imidazo[1,2-a]pyrimidine (Z-2-2)

[0073] In a 500 mL eggplant-shaped bottle, 1H-benzo[d]imidazol-2-amine (6.43 g, 45.99 mmol), Et 3 N (6.39 mL, 45.99 mmol) and (E)-1,1,1-trichloro-4-ethoxy-3-en-2-one (10 g, 45.99 mmol) were added to toluene (200 mL). The reaction was refluxed for 2 h. After the reaction was completed, the solvent was evaporated under reduced pressure. After washing with water, the filter was filtered and the residue was dried to obtain Z-2-2 (yellow solid, 12.85 g, yield 97.5%). ESI-MS m / z: 286.11 [M+H] + .

[0074] Step b: Preparation of benzo[4,5]imidazo[1,2-a]pyrimidin-2-ol (Z-2-3)

[0075] In a 500mL eggplant-shaped bottle, add 2-(trichloromethyl)benzo[4,5]imidazo[1,2-a]pyrimidine (12.85g, 44.85mmol) to a mixed solution of 1N NaOH (60ml) solution and acetonitrile (200mL), heat and reflux for 30 minutes, and cool and concentrate the reaction after the reaction is completed. Add water, adjust pH to 8 with 1N HCl, let stand, filter, and dry the residue to obtain Z-2-3 (yellow solid, 5.86g, yield 70.6%). ESI-MS m / z: 186.17[M+H] + .

[0076] Step c: Preparation of 2-bromobenzo[4,5]imidazo[1,2-a]pyrimidine (Z-2-4)

[0077] In a 250mL eggplant-shaped bottle, benzo[4,5]imidazo[1,2-a]pyrimidin-2-ol (5.86g, 31.66mmol) and phosphorus oxybromide (18.15g, 63.32mmol) were added to a mixed solution of DCE (100mL) and DMF (1mL) in sequence and heated under reflux for 3 hours. After the reaction was complete, it was cooled to room temperature, concentrated, added with water, adjusted to pH=8 with 1N NaOH solution, filtered, and the residue was washed with ether and dried to obtain Z-2-4 (yellow solid, 5.05g, yield 64.4%). ESI-MS m / z: 248.09[M+H] + .

[0078] Step d: Preparation of ethyl 2-(1-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperidin-4-yl)acetate (Z-2-5)

[0079] In a 100 mL eggplant-shaped bottle, 2-bromobenzo[4,5]imidazo[1,2-a]pyrimidine (2 g, 8.06 mmol), ethyl 2-(piperidin-4-yl)acetate (2.76 g, 16.12 mmol) and K 2 CO 3 (2.23 g, 16.12 mmol) were added to DMA (30 mL) in sequence and reacted at 130 °C for 8 hours. After the reaction was completed, the mixture was cooled to room temperature, water was added to quench the reaction, and EA was extracted (60 mL × 3). The organic layers were combined, washed with saturated brine, and anhydrous Na 2 SO 4 After drying and concentration, separation by silica gel column chromatography gave Z-2-5 (yellow solid, 2.49 g, yield 91.3%). ESI-MS m / z: 339.18 [M+H] + .

[0080] Step e: Preparation of 2-(1-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperidin-4-yl)acetic acid (Z-2-6)

[0081] In a 250 mL eggplant-shaped bottle, ethyl 2-(1-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperidin-4-yl)acetate (2.49 g, 7.36 mmol) was added to a mixed solution of 2.5 N NaOH solution (50 mL) and THF (50 mL), and refluxed for 2 h. After the reaction, water was added for dilution, and EA was extracted (100 mL × 3). The aqueous phase was adjusted to pH = 1 with 2 N HCl solution, and extracted with DCM (100 mL × 3). The organic layers were combined and anhydrous Na 2 SO 4 Drying and concentration gave Z-2-6 (light yellow oily liquid, 1.96 g, yield 85.8%). ESI-MS m / z: 309.31 [MH] - .

[0082] Step f: Preparation of 2-(1-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperidin-4-yl)-N-(1,2,3,5,6,7-tetrahydro-s-indacen-4-yl)acetamide (H8)

[0083] In a 50 mL eggplant-shaped bottle, 2-(1-(benzo[4,5]imidazole[1,2-a]pyrimidin-2-yl)piperidin-4-yl)acetic acid (100 mg, 322 μmol), HATU (159 mg, 419 μmol), and DIPEA (225 μL, 1.29 mmol) were added to DMF (5 mL) in sequence. After stirring at room temperature for 15 minutes, 1,2,3,5,6,7-hexahydro-s-indoxane-4-amine (84 mg, 484 μmol) was added and reacted at room temperature overnight. After the reaction was completed, water was added to quench the reaction and EA was extracted (20 mL×3). The organic layers were combined, washed with saturated brine, and anhydrous Na 2 SO 4 After drying and concentration, H8 (light yellow solid, 75 mg, yield 50.0%) was isolated by silica gel column chromatography. 1 H NMR (400 MHz, DMSO-d 6)δ9.23(s,1H),8.14(d,J=7.2Hz,1H),7.86(td,J=6.9,1.4Hz,2H),7.40(td,J=7.2 ,1.5Hz,1H),7.35(td,J=6.9,1.6Hz,1H),6.87(s,1H),6.59(d,J=7.0Hz,1H),3.67- 3.45(m,4H),2.82(t,J=6.2Hz,4H),2.75(t,J=6.4Hz,4H),2.38(d,J=7.3Hz,2H),2 .19-2.07(m,4H),2.05-1.96(m,1H),1.91-1.76(m,4H).HR-MS(ESI)m / z:calcd.for C 29 H 31 N 5 O[M+Na] + 488.2421, found:488.2424.

[0084] Example 9: Preparation of 2-(1-(benzo[4,5]imidazole[1,2-a]pyrimidin-2-yl)piperidin-4-yl)-N-(9H-fluorene-9-yl)acetamide (H9)

[0085] The preparation method was prepared by referring to the procedure of Example 8, using 9H-fluorene-9-amine instead of 1,2,3,5,6,7-hexahydro-s-indacene-4-amine as the reactant to prepare H9 (beige solid, 103 mg, yield 67.5%). 1 H NMR (400 MHz, DMSO-d 6 )δ8.14(d,J=7.2Hz,1H),7.86(td,J=6.9,1.4Hz,2H),7.74(dd,J=7.8,1.4 Hz,2H),7.65(d,J=7.6Hz,1H),7.52(dd,J=7.6,1.7Hz,2H),7.43-7.31(m,6 H),6.59(d,J=7.0Hz,1H),5.99(d,J=7.6Hz,1H),3.69-3.43(m,4H),2.36(d,J=7.7Hz,2H),2.03(m,1H),1.92-1.76(m,4H).HR-MS(ESI)m / z:calcd.for C 30 H 27 N 5 O[M+Na] + 496.2108, found:496.2111.

[0086] Example 10: Preparation of 2-(1-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperidin-4-yl)-N-(bicyclo[2.2.1]hept-5-en-2-ylmethyl)acetamide (H10)

[0087] The preparation method was prepared by referring to the procedure of Example 8, except that bicyclo[2.2.1]hept-5-en-2-ylmethylamine was used instead of 1,2,3,5,6,7-hexahydro-s-indacen-4-amine as the reactant to obtain H10 (white solid, 96 mg, yield 71.7%). 1 H NMR (400 MHz, DMSO-d 6 )δ8.14(d,J=7.2Hz,1H),7.86(td,J=6.9,1.4Hz,2H),7.40(td,J=7.2,1.5Hz,1H),7.35(td,J=6.9, 1.6Hz,1H),7.29(t,J=6.2Hz,1H),6.59(d,J=7.0Hz,1H),6.03-5.97(m,1H),5.92-5.87(m,1H),3.6 6-3.45(m,4H),3.14-3.00(m,2H),2.79-2.70(m,2H),2.21(d,J=7.4Hz,2H),2.17-2.10(m,1H),2.0 3-1.92(m,1H),1.90-1.77(m,4H),1.59-1.53(m,2H),1.36-1.19(m,2H).HR-MS(ESI)m / z:calcd.for C 25 H 29 N 5 O[M+Na] + 438.2264,found:438.2268.

[0088] Example 11: Preparation of N-(((3R,5R,7R)-adamantan-1-yl)methyl)-2-(1-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperidin-4-yl)acetamide (H11)

[0089] The preparation method was similar to that of Example 8, except that ((3R,5R,7R)-adamantan-1-yl)methylamine was used instead of 1,2,3,5,6,7-hexahydro-s-indacene-4-amine as the reactant to prepare H11 (white solid, 121 mg, yield 82.1%). 1 H NMR (400 MHz, DMSO-d 6)δ8.14(d,J=7.2Hz,1H),7.86(td,J=6.9,1.3Hz,2H),7.40(td,J=7.2,1.5Hz,1H), 7.35(td,J=6.8,1.5Hz,1H),7.25(t,J=5.8Hz,1H),6.59(d,J=7.0Hz,1H),3.68-3.4 2(m,4H),3.00(d,J=5.7Hz,2H),2.20(d,J=7.3Hz,2H),2.05-1.92(m,4H),1.91-1.7 9(m,4H),1.64(t,J=5.7Hz,6H),1.49(d,J=5.0Hz,6H).HR-MS(ESI)m / z:calcd.forC 28 H 35 N 5 O[M+Na] + 480.2734,found:480.2731.

[0090] Example 12: Preparation of N-diphenylmethyl-2-(1-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperidin-4-yl)acetamide (H12)

[0091] The preparation method was similar to that of Example 8, except that diphenylamine was used instead of 1,2,3,5,6,7-hexahydro-s-indacene-4-amine as the reactant to prepare H12 (beige solid, 66 mg, yield 43.1%). 1 H NMR (400 MHz, DMSO-d 6 )δ8.25(d,J=8.5Hz,1H),8.14(d,J=7.2Hz,1H),7.86(td,J=6.9,1.4Hz,2H),7.40 (td,J=7.2,1.5Hz,1H),7.35(td,J=6.8,1.6Hz,1H),7.32(d,J=4.7Hz,8H),7.29-7 .22(m,2H),6.59(d,J=7.0Hz,1H),6.03(d,J=8.5Hz,1H),3.66-3.45(m,4H),2.28( d,J=7.7Hz,2H),2.06-1.96(m,1H),1.91-1.77(m,4H).HR-MS(ESI)m / z:calcd.for C 30 H 29 N 5 O[M+Na] + 498.5852, found:498.5855.

[0092] Example 13: Preparation of 2-(1-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperidin-4-yl)-N-(dicyclohexylmethyl)acetamide (H13)

[0093] The preparation method was prepared by referring to the procedure of Example 8, using dicyclohexylmethylamine instead of 1,2,3,5,6,7-hexahydro-s-indacene-4-amine as the reactant to prepare H13 (white solid, 88 mg, yield 56.0%). 1 H NMR (400 MHz, DMSO-d 6 )δ8.14(d,J=7.2Hz,1H),7.86(td,J=6.9,1.4Hz,2H),7.40(td,J=7.2,1.5Hz,1H) ,7.35(td,J=6.9,1.6Hz,1H),6.71(d,J=8.6Hz,1H),6.59(d,J=7.0Hz,1H),3.70-3 .45(m,4H),3.42(t,J=8.5,7.8Hz,1H),2.28-2.10(m,2H),2.06-1.94(m,1H),1.9 1-1.81(m,4H),1.80-1.74(m,2H),1.54-1.25(m,20H).HR-MS(ESI)m / z:calcd.for C 30 H 41 N 5 O[M+Na] + 510.3203, found:510.3199.

[0094] Example 14: Preparation of (1R,2S,5R)-2-isopropyl-5-methylcyclohexyl 2-(1-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperidin-4-yl)acetate (H14)

[0095] The preparation method refers to the operation of Example 8, using (1R, 2S, 5R)-2-isopropyl-5-methylcyclohexyl-1-ol instead of 1,2,3,5,6,7-hexahydro-s-indacene-4-amine as the reactant, and H14 (beige solid, 57 mg, yield 39.4%) was prepared under appropriate conditions understood in the art. 1 H NMR (400 MHz, DMSO-d 6)δ8.14(d,J=7.2Hz,1H),7.86(td,J=6.9,1.4Hz,2H),7.40(td,J=7.2,1.5Hz,1H),7.35(td, J=6.9,1.6Hz,1H),6.59(d,J=7.0Hz,1H),4.68-4.60(m,1H),3.67-3.39(m,4H),2.43-2.18(m ,2H),2.11-1.97(m,1H),1.94-1.83(m,4H),1.82-1.75(m,1H),1.74-1.69(m,1H),1.69-1.63 (m,2H),1.59-1.48(m,1H),1.46-1.23(m,4H),0.92-0.82(m,9H).HR-MS(ESI)m / z:calcd.for C 27 H 36 N 4 O 2 [M+Na] + 471.2730,found:471.2733.

[0096] Example 15: Preparation of 2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)-N-(1,2,3,5,6,7-tetrahydro-s-indacen-4-yl)acetamide (H15)

[0097]

[0098] Step a: Preparation of tert-butyl 4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazine-1-carboxylate (Z-3-1)

[0099] In a 250 mL eggplant-shaped bottle, 2-bromobenzo[4,5]imidazo[1,2-a]pyrimidine (2.9 g, 11.69 mmol), tert-butyl piperazine-1-carboxylate (4.35 g, 23.38 mmol) and K 2 CO 3 (3.23 g, 23.38 mmol) were added to DMA (50 mL) in sequence, and reacted at 130 °C for 3 hours. After the reaction was completed, it was cooled to room temperature, water was added to quench the reaction, and EA was extracted (100 mL × 3). The organic layers were combined, washed with saturated brine, and anhydrous Na 2 SO 4 After drying and concentration, Z-3-1 (yellow solid, 3.51 g, yield 85.0%) was obtained by silica gel column chromatography. ESI-MS m / z: 354.19 [M+H] + .

[0100] Step b: Preparation of 2-(piperazin-1-yl)benzo[4,5]imidazo[1,2-a]pyrimidine (Z-3-2)

[0101] In a 500 mL eggplant-shaped bottle, tert-butyl 4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazine-1-carboxylate (3.51 g, 9.93 mmol) and dioxane hydrochloride solution (100 mL) were added respectively and reacted at room temperature for 4 hours. After the reaction was complete, the solvent was evaporated under reduced pressure, water was added, and it was extracted with DCM (40 mL × 3). Then the aqueous layer was adjusted to alkaline with 1N NaOH solution and extracted with DCM (40 mL × 3). The organic layers were combined and anhydrous Na 2 SO 4 After drying, DCM was evaporated under reduced pressure to obtain Z-3-2 (yellow solid, 2.38 g, yield 94.6%). ESI-MS m / z: 254.14 [M+H] + .

[0102] Step c: Preparation of tert-butyl 2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)acetate (Z-3-3)

[0103] In a 100 mL eggplant-shaped bottle, 2-(piperazine-1-yl)benzo[4,5]imidazo[1,2-a]pyrimidine (300 mg, 1.18 mmol), tert-butyl 2-bromoacetate (181 μL, 1.24 mmol) and DIPEA (619 μL, 3.55 mmol) were added to DMSO (10 mL) in sequence, and reacted at room temperature overnight. After the reaction was complete, water was added to quench the reaction, and EA was extracted (30 mL × 3). The organic layers were combined, washed with saturated brine, and anhydrous Na 2 SO 4 After drying and concentration, Z-3-3 (light yellow solid, 351 mg, yield 80.7%) was obtained by silica gel column chromatography. ESI-MS m / z: 368.21 [M+H] + .

[0104] Step d: Preparation of 2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)acetic acid (Z-3-4)

[0105] In a 50 mL eggplant-shaped bottle, tert-butyl 2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)acetate (120 mg, 327 μmol) was dissolved in DCM (4 mL), TFA (3 mL) was added, and the reaction was carried out at room temperature for 4 hours. After the reaction was complete, the solvent was evaporated under reduced pressure to obtain Z-3-4 (light yellow oily liquid, 102 mg, yield 100.0%). ESI-MS m / z: 310.13 [MH] -.

[0106] Step e: Preparation of 2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)-N-(1,2,3,5,6,7-tetrahydro-s-indacen-4-yl)acetamide (H15)

[0107] In a 50 mL eggplant-shaped bottle, 2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazine-1-yl)acetic acid (102 mg, 328 μmol), HATU (162 mg, 426 μmol) and DIPEA (228 μL, 1.31 mmol) were added to DMF (6 mL) in sequence. After stirring at room temperature for 15 minutes, 1,2,3,5,6,7-hexahydro-s-indacene-4-amine (85 mg, 491 mmol) was added. The reaction was allowed to proceed overnight at room temperature. After the reaction was completed, water was added to quench the reaction and EA was extracted (20 mL×3). The organic layers were combined, washed with saturated brine, and anhydrous Na 2 SO 4 After drying and concentration, H15 (white solid, 89 mg, yield 58.2%) was isolated by silica gel column chromatography. 1 H NMR (400 MHz, DMSO-d 6 )δ9.35(s,1H),8.14(d,J=7.2Hz,1H),7.86(td,J=6.9,1.4Hz,2H),7.40(td,J=7.3,1.5Hz,1H),7.35(td,J=7.9,1.6Hz,1H),6.87(s,1H),6. 58(d,J=7.0Hz,1H),3.58(t,J=4.3Hz,4H),3.24(s,2H),2.86-2.79(m,4H),2.78-2.70(m,8H),2.19-2.07(m,4H).HR-MS(ESI)m / z:calcd.for C 28 H 30 N 6 O[M+Na] + 489.2373, found:489.2368.

[0108] Example 16: Preparation of 2-(4-(benzo[4,5]imidazole[1,2-a]pyrimidin-2-yl)piperazine-1-yl)-N-(9H-fluorene-9-yl)acetamide (H16)

[0109] The preparation method was prepared by referring to the procedure of Example 15, using 9H-fluorene-9-amine instead of 1,2,3,5,6,7-hexahydro-s-indacene-4-amine as the reactant to prepare H16 (white solid, 100 mg, yield 64.3%). 1H NMR (400 MHz, DMSO-d 6 )δ9.54(d,J=7.8Hz,1H),9.30(d,J=7.7Hz,1H),8.33(d,J=8.1Hz,1H),7.87(d ,J=7.5Hz,2H),7.69(d,J=8.0Hz,1H),7.57(t,J=7.9Hz,3H),7.50(ddd,J=7.5, 5.7,1.9Hz,2H),7.44(t,J=7.5Hz,2H),7.34(td,J=7.5,1.1Hz,2H),6.05(d,J= 8.1Hz,1H),4.10(s,4H),3.57(s,4H),3.15(s,2H).HR-MS(ESI)m / z:calcd.for C 29 H 26 N 6 O[M+Na] + 497.2060,found:497.2064.

[0110] Example 17: Preparation of 2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)-N-(bicyclo[2.2.1]hept-5-en-2-ylmethyl)acetamide (H17)

[0111] The preparation method refers to the operation of Example 15, using bicyclo[2.2.1]hept-5-en-2-ylmethylamine instead of 1,2,3,5,6,7-hexahydro-s-indacen-4-amine as the reactant to prepare H17 (light yellow solid, 77 mg, yield 56.4%). 1 HNMR (300 MHz, DMSO-d 6)δ9.10(d,J=7.8Hz,1H),8.05(d,J=8.0Hz,1H),7.86(t,J=5.9Hz,1H),7.56(d,J=8.0Hz,1H),7.40(td,J=7. 5,0.9Hz,1H),7.28(td,J=7.9,1.0Hz,1H),7.06(d,J=7.8Hz,1H),6.21-6.09(m,1H),6.09-5.97(m,1H),3.89 (s,4H),3.04(s,2H),2.93-2.80(m,1H),2.79-2.74(m,2H),2.61(t,J=4.9Hz,4H),2.26(qt,J=8.6,3.6Hz,1 H),1.78(ddd,J=12.4,9.1,3.8Hz,1H),1.38-1.28(m,2H),1.24(d,J=5.7Hz,2H).HR-MS(ESI)m / z:calcd.for C 24 H 28 N 6 O[M+Na] + 439.2217,found:439.2223.

[0112] Example 18: Preparation of N-(((3R,5R,7R)-adamantan-1-yl)methyl)-2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)acetamide (H18)

[0113] The preparation method was prepared by referring to the procedure of Example 15, except that ((3R,5R,7R)-adamantan-1-yl)methylamine was used instead of 1,2,3,5,6,7-hexahydro-s-indacene-4-amine as the reactant to prepare H18 (white solid, 91 mg, yield 60.6%). 1 H NMR (300 MHz, DMSO-d 6 )δ9.07(d,J=7.6Hz,1H),8.01(d,J=8.0Hz,1H),7.63(t,J=4.6Hz,2H),7.3 3(t,J=7.7Hz,1H),7.22(t,J=7.8Hz,1H),6.98(d,J=7.7Hz,1H),3.75(s,4 H),3.37(s,4H),2.99(s,2H),2.82(d,J=6.4Hz,2H),1.92(d,J=5.3Hz,3H),1.62(q,J=12.2Hz,6H),1.42(d,J=2.8Hz,6H).HR-MS(ESI)m / z:calcd.for C 27 H 34N 6 O[M+Na] + 481.2686, found:481.2689.

[0114] Example 19: Preparation of N-diphenylmethyl-2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)acetamide (H19)

[0115] The preparation method was prepared by referring to the procedure of Example 15, except that diphenylamine was used instead of 1,2,3,5,6,7-hexahydro-s-indacene-4-amine as the reactant to obtain H19 (light yellow solid, 71 mg, yield 45.5%). 1 H NMR (400 MHz, DMSO-d 6 )δ9.23(d,J=7.7Hz,1H),8.93(d,J=8.8Hz,1H),8.14(d,J=8.1Hz,1H),7.58(d,J=8.0Hz,1H),7.43(t,J=7.7Hz,1H),7.33(d,J=4.3Hz,10H) ,7.26-7.22(m,1H),7.17(d,J=7.9Hz,1H),6.16(d,J=8.7Hz,1H),3.89(s,4H),3.26(s,2H),2.73-2.61(m,4H).HR-MS(ESI)m / z:calcd.for C 29 H 28 N 6 O[M+Na] + 499.2217,found:499.2213.

[0116] Example 20: Preparation of 2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)-N-(dicyclohexylmethyl)acetamide (H20)

[0117] Preparation method: Referring to the operation of Example 15, dicyclohexylmethylamine was used instead of 1,2,3,5,6,7-hexahydro-s-indacene-4-amine as the reactant to prepare H20 (white solid, 97 mg, yield 60.6%). 1 H NMR (400 MHz, DMSO-d 6)δ8.14(d,J=7.1Hz,1H),7.86(ddd,J=8.0,7.0,1.3Hz,2H),7.52-7.27(m ,2H),6.69(d,J=8.6Hz,1H),6.58(d,J=7.0Hz,1H),3.60-3.57(m,4H),3.4 2(dt,J=8.6,7.8Hz,1H),3.13(d,J=13.9Hz,1H),3.06(d,J=13.9Hz,1H),2.71(m,4H),1.79(m,2H),1.51-1.25(m,20H).HR-MS(ESI)m / z:calcd.for C 29 H 40 N 6 O[M+Na] + 511.3156, found:511.3148.

[0118] Example 21: Preparation of (1R,2S,5R)-2-isopropyl-5-methylcyclohexyl 2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)acetate (H21)

[0119] The preparation method refers to the operation of Example 15, using (1R, 2S, 5R)-2-isopropyl-5-methylcyclohexyl-1-ol instead of 1,2,3,5,6,7-hexahydro-s-indacene-4-amine as the reactant to prepare H21 (white solid, 77 mg, yield 52.3%). 1 H NMR (400 MHz, DMSO-d 6 )δ8.14(d,J=7.2Hz,1H),7.86(td,J=6.9,1.4Hz,2H),7.40(td,J=7.2,1.5Hz,1H),7.35 (td,J=7.2,6.6,1.6Hz,1H),6.58(d,J=7.0Hz,1H),4.69-4.60(m,1H),3.64-3.52(m,4H) ,3.36(s,2H),2.74-2.67(m,4H),1.82-1.74(m,1H),1.74-1.59(m,3H),1.58-1.50(m,1 H),1.46-1.39(m,1H),1.36-1.25(m,3H),0.93-0.76(m,9H).HR-MS(ESI)m / z:calcd.for C 26 H 35 N 5 O 2 [M+Na] +472.2683,found:472.2689.

[0120] Example 22: Preparation of 1-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)-2-(tert-butyloxycarbonylamino)-5-(N-tert-butyloxycarbonylamino-N'-tert-butyloxycarbonyliminoguanidino)pentan-1-one (H22)

[0121] Preparation method: Referring to the operation of Example 15, H22 (white solid, 61 mg, yield 21.3%) was prepared under appropriate conditions understood in the art. 1 H NMR (400 MHz, DMSO-d 6 )δ11.50(s,1H),9.04(d,J=7.7Hz,1H),8.30(t,J=5.7Hz,1H),7.97(d,J=7.9Hz,1H),7.53(d, J=8.0Hz,1H),7.31(t,J=7.7Hz,1H),7.17(t,J=7.6Hz,1H),7.08(d,J=8.2Hz,1H),6.89(d,J=7 .8Hz,1H),4.46(td,J=8.1,3.2Hz,1H),3.79(s,4H),3.64(ddd,J=26.7,19.5,11.5Hz,2H),3. 30(s,4H),1.64-1.50(m,4H),1.45(s,9H),1.38(d,J=2.2Hz,18H).HR-MS(ESI)m / z:calcd.for C 35 H 51 N 9 O 7 [M+Na] + 732.3804,found:732.3796.

[0122] Example 23: Preparation of N-(2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)ethyl)-9H-fluorene-9-carboxamide (H23)

[0123] Preparation method: Referring to the operation of Example 16, using (2-bromoethyl)carbamic acid tert-butyl ester instead of 2-bromoacetate as the reactant, H23 (white solid, 60 mg, yield 45.5%) was prepared under appropriate conditions understood in the art. 1 H NMR (300 MHz, DMSO-d 6)δ9.01(d,J=7.7Hz,1H),8.32(t,J=5.6Hz,1H),7.98(d,J=8.0Hz,1H),7.88(d ,J=7.4Hz,2H),7.55(dd,J=13.8,7.7Hz,3H),7.41(t,J=7.4Hz,2H),7.36-7.2 7(m,3H),7.18(t,J=7.6Hz,1H),6.93(d,J=7.8Hz,1H),4.84(s,1H),3.79(s,4 H),3.31(d,J=6.1Hz,4H),2.53(d,J=5.7Hz,4H).HR-MS(ESI)m / z:calcd.forC 30 H 28 N 6 O[M+Na] + 511.2217, found:511.2213.

[0124] Example 24: Preparation of 4-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)-N-(9H-fluoren-9-yl)butanamide (H24)

[0125] The preparation method was prepared by referring to the procedure of Example 16, using tert-butyl 4-bromobutyrate instead of tert-butyl 2-bromoacetate as the reactant to obtain H24 (white solid, 59 mg, yield 46.4%). 1 H NMR (400 MHz, DMSO-d 6 )δ9.13(d,J=7.8Hz,1H),8.48(d,J=8.4Hz,1H),8.07(d,J=8.0Hz,1H),7.86(d,J=7.5Hz,2H) ,7.57(d,J=8.0Hz,1H),7.49(d,J=7.4Hz,2H),7.42(t,J=7.7Hz,3H),7.33(qd,J=8.4,7.9,1. 1Hz,3H),7.11(d,J=7.8Hz,1H),6.05(d,J=8.3Hz,1H),3.89(s,4H),2.70(t,J=4.4Hz,4H),2. 55(t,J=7.5Hz,2H),2.29(t,J=7.3Hz,2H),1.87(p,J=7.4Hz,2H).HR-MS(ESI)m / z:calcd.for C 31 H 30 N 6 O[M+Na] + 525.2373, found:525.2374.

[0126] Example 25: Preparation of N-(4-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)butyl)-9H-fluorene-9-carboxamide (H25)

[0127] The preparation method refers to the operation of Example 16, using (4-bromobutyl)carbamic acid tert-butyl ester instead of 2-bromoacetate as the reactant, and H25 (light yellow solid, 48 mg, yield 39.4%) was prepared under appropriate conditions understood in the art. 1 H NMR (400 MHz, DMSO-d 6 )δ8.14(d,J=7.2Hz,1H),7.98(t,J=4.8Hz,1H),7.86(td,J=6.9,1.4Hz,2H),7.79(d d,J=7.9,1.2Hz,2H),7.60(dd,J=7.4,1.7Hz,2H),7.46-7.31(m,6H),6.58(d,J=7.0 Hz,1H),4.76(d,J=0.7Hz,1H),3.62-3.50(m,4H),3.22(td,J=5.7,4.7Hz,2H),2.74 -2.64(m,4H),2.46(t,J=5.7Hz,2H),1.65-1.49(m,4H).HR-MS(ESI)m / z:calcd.for C 32 H 32 N 6 O[M+Na] + 539.2530,found:517.2525.

[0128] Example 26: Preparation of 6-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)-N-(9H-fluorene-9-yl)hexanamide (H26)

[0129] The preparation method was similar to that of Example 16, except that tert-butyl 6-bromohexanoate was used instead of tert-butyl 2-bromoacetate as the reactant to obtain H26 (white solid, 61 mg, yield 48.7%). 1 H NMR (400 MHz, DMSO-d 6)δ9.01(d,J=7.8Hz,1H),8.42(d,J=8.4Hz,1H),7.97(d,J=8.0Hz,1H),7.85(d,J=7.5Hz,2H),7.52(d,J=8.1Hz ,1H),7.48(d,J=7.4Hz,2H),7.42(t,J=7.4Hz,2H),7.32(q,J=7.1Hz,3H),7.18(t,J=7.6Hz,1H),6.91(d,J=7. 8Hz,1H),6.05(d,J=8.3Hz,1H),3.85-3.74(m,4H),2.52(d,J=5.8Hz,4H),2.37(t,J=7.3Hz,2H),2.23(t,J=7. 2Hz,2H),1.65(p,J=7.4Hz,2H),1.51(p,J=7.5Hz,2H),1.36(td,J=8.3,4.0Hz,2H).HR-MS(ESI)m / z:calcd.for C 33 H 34 N 6 O[M+Na] + 553.2686,found:553.2682.

[0130] Example 27: Preparation of N-(6-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)hexyl)-9H-fluorene-9-carboxamide (H27)

[0131] The preparation method was similar to that of Example 16, except that tert-butyl (6-bromohexyl)carbamate was used instead of tert-butyl 2-bromoacetate as the reactant to obtain H27 (light yellow solid, 32 mg, yield 26.6%). 1 H NMR (400 MHz, DMSO-d 6 )δ8.14(d,J=7.2Hz,1H),7.98(t,J=4.8Hz,1H),7.86(td,J=6.9,1.4Hz,2H),7.82-7.7 7(m,2H),7.60(dd,J=7.5,1.7Hz,2H),7.47-7.32(m,6H),6.58(d,J=7.0Hz,1H),4.76( d,J=0.8Hz,1H),3.63-3.50(m,4H),3.21(td,J=5.8,4.8Hz,2H),2.77-2.63(m,4H),2. 42(t,J=6.1Hz,2H),1.60-1.44(m,4H),1.40-1.25(m,4H).HR-MS(ESI)m / z:calcd.for C 34 H36 N 6 O[M+Na] + 567.2843,found:567.2841.

[0132] Example 28: Preparation of 8-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)-N-(9H-fluorene-9-yl)octanamide (H28)

[0133] The preparation method was prepared by referring to the procedure of Example 16, using 8-bromooctanoic acid tert-butyl ester instead of 2-bromoacetate tert-butyl ester as the reactant to prepare H28 (light yellow solid, 49 mg, yield 39.6%). 1 H NMR (400 MHz, DMSO-d 6 )δ8.99(d,J=7.6Hz,1H),8.42(d,J=8.5Hz,1H),7.90(dd,J=43.4,7.7Hz,3H),7.51(d,J=7.7Hz, 1H),7.47(d,J=7.3Hz,2H),7.44-7.38(m,2H),7.34(d,J=7.5Hz,2H),7.31(s,1H),7.16(t,J=7.7 Hz,1H),6.88(d,J=7.8Hz,1H),6.04(d,J=8.4Hz,1H),3.78(s,4H),2.47(s,4H),2.31(d,J=7.4H z,2H),2.21(t,J=7.3Hz,2H),1.61(s,2H),1.47(s,2H),1.32(s,6H).HR-MS(ESI)m / z:calcd.for C 35 H 38 N 6 O[M+Na] + 581.2999,found:581.2991.

[0134] Example 29: Preparation of N-(2-(2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)ethoxy)ethyl)-9H-fluorene-9-carboxamide (H29)

[0135] The preparation method refers to the operation of Example 16, using tert-butyl (2-(2-bromoethoxy)ethyl)carbamate instead of tert-butyl 2-bromoacetate as the reactant, and H29 (colorless oily liquid, 43 mg, yield 35.6%) is prepared under appropriate conditions understood in the art. 1 H NMR (300 MHz, DMSO-d 6)δ9.01(d,J=7.7Hz,1H),8.53-8.46(m,1H),7.98(d,J=8.0Hz,1H),7.87(d,J=7.4Hz,2H ),7.52(d,J=7.4Hz,3H),7.40(t,J=7.3Hz,2H),7.32(t,J=7.4Hz,3H),7.19(t,J=7.6Hz, 1H),6.90(d,J=7.8Hz,1H),4.86(s,1H),3.77(s,4H),3.58(t,J=5.6Hz,2H),3.48(t,J= 5.8Hz,2H),3.32(q,J=5.7Hz,2H),3.17(s,2H),2.57(s,4H).HR-MS(ESI)m / z:calcd.for C 32 H 32 N 6 O 2 [M+Na] + 555.2479, found:555.2485.

[0136] Example 30: Preparation of N-(2-(2-(2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)ethoxy)ethoxy)ethyl)-9H-fluorene-9-carboxamide (H30)

[0137] The preparation method refers to the operation of Example 16, using tert-butyl (2-(2-(2-bromoethoxy)ethoxy)ethyl)carbamate instead of tert-butyl 2-bromoacetate as the reactant, and H30 (light yellow oily liquid, 67 mg, yield 56.3%) is prepared under appropriate conditions understood in the art. 1 H NMR (300 MHz, DMSO-d 6)δ8.96(d,J=7.8Hz,1H),8.48(t,J=5.7Hz,1H),7.96(d,J=7.9Hz,1H),7.86(d,J=7.3Hz,2H),7.5 2(d,J=7.5Hz,3H),7.40(td,J=7.5,1.3Hz,2H),7.33(dd,J=7.4,1.3Hz,2H),7.31-7.25(m,1H),7 .17(d,J=7.4Hz,1H),6.86(d,J=7.8Hz,1H),4.84(s,1H),3.75(t,J=5.0Hz,4H),3.58(t,J=5.8Hz ,6H),3.48(t,J=5.8Hz,2H),3.31(q,J=6.0Hz,2H),2.61-2.51(m,6H).HR-MS(ESI)m / z:calcd.for C 34 H 36 N 6 O 3 [M+Na] + 599.2741,found:599.2745.

[0138] Example 31: Preparation of N-(2-(2-(2-(2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-9H-fluorene-9-carboxamide (H31)

[0139] The preparation method refers to the operation of Example 16, using tert-butyl (2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate instead of tert-butyl 2-bromoacetate as the reactant, and H31 (light yellow oily liquid, 24 mg, yield 18.4%) is prepared under appropriate conditions understood in the art. 1 H NMR (400 MHz, DMSO-d 6)δ8.99(d,J=7.7Hz,1H),8.55(t,J=5.7Hz,1H),7.98(d,J=8.0Hz,1H),7.86(d,J=7.4H z,2H),7.51(d,J=7.4Hz,2H),7.39(t,J=7.4Hz,3H),7.31(dt,J=8.6,4.2Hz,3H),7.20 (t,J=7.7Hz,1H),6.91(d,J=7.8Hz,1H),4.84(s,1H),3.76(s,4H),3.62-3.48(m,12H) ,3.48(t,J=5.8Hz,2H),3.31(q,J=5.8Hz,2H),2.55(s,4H).HR-MS(ESI)m / z:calcd.for C 36 H 40 N 6 O 4 [M+Na] + 643.3003,found:643.3006.

[0140] Example 32: Preparation of N-(2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)ethyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide (H32)

[0141] The preparation method refers to the operation of Example 17, using (2-bromoethyl)carbamic acid tert-butyl ester instead of 2-bromoacetate as the reactant, and H32 (light yellow solid, 36 mg, yield 26.2%) was prepared under appropriate conditions understood in the art. 1 H NMR (400 MHz, DMSO-d 6 )δ9.10(d,J=7.8Hz,1H),8.06(d,J=8.0Hz,1H),7.55(t,J=6.0Hz,2H),7.39(t,J=7.8Hz,1H) ,7.29(t,J=7.7Hz,1H),7.07(d,J=7.8Hz,1H),6.10(dd,J=5.7,3.0Hz,1H),5.83(dd,J=5.7, 2.8Hz,1H),3.81(s,4H),3.25-3.16(m,1H),3.13(s,2H),2.85-2.75(m,2H),2.54(s,4H),2. 40(s,1H),1.73(ddd,J=11.1,9.2,3.7Hz,1H),1.42-1.19(m,4H).HR-MS(ESI)m / z:calcd.for C 24 H 28 N 6O[M+Na] + 439.2217,found:439.2222.

[0142] Example 33: Preparation of 4-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)-N-(bicyclo[2.2.1]hept-5-en-2-ylmethyl)butanamide (H33)

[0143] The preparation method was prepared by referring to the procedure of Example 17, using tert-butyl 4-bromobutyrate instead of tert-butyl 2-bromoacetate as the reactant to obtain H33 (white solid, 72 mg, yield 64.1%). 1 H NMR (400 MHz, DMSO-d 6 )δ9.06(d,J=7.7Hz,1H),8.01(d,J=8.0Hz,1H),7.85(t,J=5.7Hz,1H),7.54(d,J=8.0Hz,1H),7.34(t,J=7. 6Hz,1H),7.21(t,J=7.6Hz,1H),6.97(d,J=7.8Hz,1H),6.19-6.06(m,1H),6.06-5.93(m,1H),3.84(s,4H),3 .45(s,2H),2.86-2.78(m,1H),2.76(d,J=14.5Hz,2H),2.70-2.62(m,1H),2.60(d,J=6.4Hz,4H),2.17(dt,J =10.4,4.1Hz,1H),2.12(t,J=7.2Hz,2H),1.77-1.70(m,2H),1.41-1.07(m,4H).HR-MS(ESI)m / z:calcd.for C 26 H 32 N 6 O[M+Na] + 467.2530,found:467.2535.

[0144] Example 34: Preparation of 4-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)-N-(bicyclo[2.2.1]hept-5-en-2-ylmethyl)butanamide (H34)

[0145] The preparation method refers to the operation of Example 17, using (4-bromobutyl)carbamic acid tert-butyl ester instead of tert-butyl 2-bromoacetate as the reactant, and H34 (light yellow solid, 56 mg, yield 53.5%) was prepared under appropriate conditions understood in the art. 1 H NMR (300 MHz, DMSO-d 6)δ9.09(d,J=7.8Hz,1H),8.04(d,J=8.0Hz,1H),7.62(t,J=5.6Hz,1H),7.56(d,J=8.0Hz,1H),7.42 -7.34(m,1H),7.26(t,J=7.7Hz,1H),7.04(d,J=7.8Hz,1H),6.11(dd,J=5.7,3.0Hz,1H),5.81(dd,J =5.6,2.8Hz,1H),3.86(s,4H),3.15(s,1H),3.03(dp,J=19.1,6.4Hz,2H),2.86-2.74(m,2H),2.62( s,4H),2.46(s,1H),1.80-1.62(m,1H),1.45(s,4H),1.44-1.13(m,4H).HR-MS(ESI)m / z:calcd.for C 26 H 32 N 6 O[M+Na] + 467.2530,found:467.2533.

[0146] Example 35: Preparation of 6-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)-piperazin-1-yl)-N-(bicyclo[2.2.1]hept-5-en-2-ylmethyl)hexanamide (H35)

[0147] The preparation method was prepared by referring to the procedure of Example 17, using tert-butyl 6-bromohexanoate instead of tert-butyl 2-bromoacetate as the reactant to obtain H35 (white solid, 49 mg, yield 43.9%). 1 H NMR (300 MHz, DMSO-d 6)δ8.96(d,J=7.6Hz,1H),7.91(d,J=8.0Hz,1H),7.67(t,J=5.7Hz,1H),7.47(s,1H),7.25(t,J=7.6Hz,1H) ,7.13(t,J=7.7Hz,1H),6.88(d,J=7.8Hz,1H),6.04(dd,J=5.7,3.0Hz,1H),5.85(dd,J=5.7,2.9Hz,1H),3. 70(s,4H),2.72(dd,J=13.3,6.4Hz,1H),2.65(d,J=8.7Hz,2H),2.61-2.49(m,1H),2.46(d,J=15.3Hz,4H), 2.30(s,2H),2.06(d,J=9.2Hz,1H),1.96(t,J=7.2Hz,2H),1.72-0.98(m,10H).HR-MS(ESI)m / z:calcd.for C 28 H 36 N 6 O[M+Na] + 495.2843,found:495.2842.

[0148] Example 36: Preparation of N-(6-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)hexyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide (H36)

[0149] The preparation method was similar to that of Example 17, except that tert-butyl (6-bromohexyl)carbamate was used instead of tert-butyl 2-bromoacetate as the reactant to obtain H36 (white solid, 26 mg, yield 24.9%). 1 H NMR (300 MHz, DMSO-d 6 )δ8.99(d,J=7.7Hz,1H),7.96(d,J=7.9Hz,1H),7.52(d,J=8.4Hz,2H),7.30(t,J=7.7Hz,1 H),7.16(t,J=7.6Hz,1H),6.89(d,J=7.8Hz,1H),6.16-6.06(m,1H),5.84-5.73(m,1H),3.7 9(s,4H),3.13(s,1H),3.09-2.88(m,1H),2.84-2.73(m,2H),2.47(s,4H),2.33(t,J=7.3H z,2H),1.71(td,J=10.4,9.7,3.6Hz,1H),1.52-1.21(m,12H).HR-MS(ESI)m / z:calcd.forC 28H 36 N 6 O[M+Na] + 495.2843,found:495.2839.

[0150] Example 37: Preparation of N-(6-(2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)acetamide)hexyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide (H37)

[0151] Preparation method: Referring to the operation of Example 17, H37 (light yellow solid, 100 mg, yield 49.0%) was prepared under appropriate conditions understood in the art. 1 H NMR (400 MHz, DMSO-d 6 )δ8.14(d,J=7.2Hz,1H),7.86(td,J=6.9,1.4Hz,2H),7.50(t,J=4.9Hz,1H),7.40(td,J=7.2,1.5Hz,1H),7.37-7.33(m,1H), 7.33-7.31(m,1H),6.58(d,J=7.0Hz,1H),6.12-6.07(m,1H),5.92(dd,J=7.6,4.6Hz,1H),3.58(t,J=4.3Hz,4H),3.26-3.19( m,1H),3.16(td,J=5.3,1.3Hz,2H),3.13-3.11(m,2H),3.11-3.05(m,2H),2.87(m,J=4.7,1.8,1.0Hz,1H),2.74-2.69(m,4H) ,2.63-2.58(m,1H),1.98-1.91(m,1H),1.83-1.76(m,1H),1.54-1.46(m,4H),1.40-1.30(m,6H).HR-MS(ESI)m / z:calcd.for C 30 H 39 N 7 O 2 [M+Na] + 552.3057,found:552.3057.

[0152] Example 38: Preparation of N-(8-(2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)acetamide)octyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide (H38)

[0153] Preparation method: Referring to the operation of Example 17, H38 (light yellow solid, 108 mg, yield 37.7%) was prepared under appropriate conditions understood in the art. 1 H NMR (300 MHz, DMSO-d 6 )δ8.99(d,J=7.7Hz,1H),7.96(d,J=8.0Hz,1H),7.82(t,J=6.0Hz,1H),7.59-7.47(m,2H),7.30(t,J=7.6H z,1H),7.16(t,J=7.5Hz,1H),6.89(d,J=7.8Hz,1H),6.13-6.05(m,1H),5.84-5.73(m,1H),3.83(t,J=4.8, 4.3Hz,4H),3.14-3.05(m,3H),3.02-2.94(m,3H),2.82-2.71(m,2H),2.55(t,J=5.0Hz,4H),1.70(td,J=10 .3,9.3,3.7Hz,1H),1.48-1.38(m,2H),1.38-1.30(m,2H),1.30-1.12(m,12H).HR-MS(ESI)m / z:calcd.for C 32 H 43 N 7 O 2 [M+Na] + 580.3370,found:580.3368.

[0154] Example 39: Preparation of N-(10-(2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)acetamide)decyl)bicyclo[2.2.1]hept-5-ene-2-carboxamide (H39)

[0155] Preparation method: Referring to the operation of Example 17, H39 (light yellow solid, 50 mg, yield 22.2%) was prepared under appropriate conditions understood in the art. 1 H NMR (400 MHz, DMSO-d 6)δ9.00(d,J=7.7Hz,1H),7.96(d,J=7.9Hz,1H),7.82(t,J=5.9Hz,1H),7.67(dt,J=86.9,5.7Hz,1H),7.52(d,J=8 .0Hz,1H),7.33-7.26(m,1H),7.16(t,J=7.6Hz,1H),6.89(d,J=7.8Hz,1H),6.09(td,J=6.6,5.7,3.2Hz,1H),5.7 7(dd,J=5.7,2.8Hz,1H),3.82(s,4H),3.09(q,J=6.6Hz,2H),3.06-2.88(m,2H),2.98(s,2H),2.83-2.71(m,1H), 2.55(t,J=5.0Hz,4H),2.47(d,J=4.5Hz,1H),1.74-1.65(m,1H),1.54-1.18(m,20H).HR-MS(ESI)m / z:calcd.for C 34 H 47 N 7 O 2 [M+Na] + 608.3683, found: 608.3678.

[0156] Example 40: Preparation of 2-(3-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)3,8-diazabicyclo[3.2.1]octan-8-yl)-N-(bicyclo[2.2.1]hept-5-en-2-ylmethyl)acetamide (H40)

[0157] Preparation method: Referring to the procedure of Example 17, tert-butyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate was used instead of tert-butyl piperazine-1-carboxylate as the reactant to prepare H40 (white solid, 45 mg, yield 40.0%). 1 H NMR (400 MHz, DMSO-d 6)δ8.14(d,J=7.2Hz,1H),7.86(td,J=6.9,1.4Hz,2H),7.44(t,J=6.4Hz,1H),7.40(td,J=7.2,1.5Hz,1H),7.35(td ,J=7.2,6.6,1.6Hz,1H),6.74(d,J=7.2Hz,1H),6.03-5.97(m,1H),5.90(dd,J=7.5,4.5Hz,1H),3.90(dd,J=12.3,3 .1Hz,2H),3.59(dd,J=12.4,3.0Hz,2H),3.28-3.20(m,2H),3.16(d,J=8.6Hz,2H),3.14-3.05(m,2H),2.79-2.71( m,2H),2.17-2.10(m,1H),1.94-1.74(m,4H),1.56(t,J=5.1Hz,2H),1.36-1.19(m,2H).HR-MS(ESI)m / z:calcd.for C 26 H 30 N 6 O[M+Na] + 465.2373, found:465.2376.

[0158] Example 41: Preparation of 2-(6-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)2,6-diazaspiro[3.3]heptane-2-yl)-N-(bicyclo[2.2.1]hept-5-en-2-ylmethyl)acetamide (H41)

[0159] Preparation method: Referring to the procedure of Example 17, 2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester was used as the reactant instead of piperazine-1-carboxylic acid tert-butyl ester to prepare H41 (white solid, 66 mg, yield 58.4%). 1 H NMR (300 MHz, DMSO-d 6)δ8.14(d,J=7.2Hz,1H),7.86(td,J=7.3,6.6,1.3Hz,2H),7.46(t,J=6.4Hz,1H),7.40(td,J=7. 2,1.5Hz,1H),7.37-7.32(m,1H),7.03(d,J=7.2Hz,1H),6.02-5.97(m,1H),5.92-5.87(m,1H),3. 85(s,2H),3.74(s,2H),3.17-3.06(m,4H),2.92(s,2H),2.84(s,2H),2.78-2.71(m,2H),2.17-2 .10(m,1H),1.58-1.54(m,2H),1.36-1.29(m,1H),1.26-1.19(m,1H).HR-MS(ESI)m / z:calcd.for C 25 H 28 N 6 O[M+Na] + 451.2217, found:451.2214.

[0160] Example 42: Preparation of 2-(7-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)2,7-diazaspiro[4.4]nonan-2-yl)-N-(bicyclo[2.2.1]hept-5-en-2-ylmethyl)acetamide (H42)

[0161] Preparation method: Referring to the procedure of Example 17, 2,7-diazaspiro[4.4]nonane-2-carboxylic acid tert-butyl ester was used as the reactant instead of piperazine-1-carboxylic acid tert-butyl ester to prepare H42 (white solid, 76 mg, yield 67.8%). 1 H NMR (400 MHz, DMSO-d 6)δ8.14(d,J=7.2Hz,1H),7.86(td,J=6.9,1.4Hz,2H),7.46(t,J=6.4Hz,1H),7.40(td,J=7.3,1.5H z,1H),7.37-7.32(m,1H),6.71(d,J=7.0Hz,1H),6.02-5.97(m,1H),5.93-5.88(m,1H),3.65-3.59( m,2H),3.55-3.49(m,2H),3.17-3.05(m,4H),2.79-2.72(m,2H),2.71-2.57(m,4H),2.17-2.10(m, 1H),1.83-1.70(m,4H),1.56(dd,J=5.3,4.5Hz,2H),1.36-1.19(m,2H).HR-MS(ESI)m / z:calcd.for C 27 H 32 N 6 O[M+Na] + 479.2530,found:479.2533.

[0162] Example 43: Preparation of 2-(9-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)3,9-diazaspiro[5.5]undec-3-yl)-N-(bicyclo[2.2.1]hept-5-en-2-ylmethyl)acetamide (H43)

[0163] Preparation method: Referring to the procedure of Example 17, tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate was used instead of tert-butyl piperazine-1-carboxylate as the reactant to prepare H43 (white solid, 47 mg, yield 42.3%). 1 H NMR (300 MHz, DMSO-d 6)δ8.14(d,J=7.2Hz,1H),7.93-7.80(m,2H),7.46(t,J=6.4Hz,1H),7.40(td,J=7.2,1.5Hz,1H),7.37-7.3 2(m,1H),6.58(d,J=7.2Hz,1H),6.02-5.97(m,1H),5.92-5.87(m,1H),3.58-3.44(m,4H),3.17-3.05(m,4H ),2.79-2.71(m,2H),2.66-2.59(m,2H),2.57-2.49(m,2H),2.18-2.10(m,1H),1.74(dd,J=5.7,3.2Hz,2H) ,1.66-1.61(m,4H),1.61-1.54(m,4H),1.36-1.29(m,1H),1.26-1.19(m,1H).HR-MS(ESI)m / z:calcd.forC 29 H 36 N 6 O[M+Na] + 507.2843,found:507.2845.

[0164] Example 44: Preparation of 2-(2-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)2,7-diazaspiro[3.5]nonan-7-yl)-N-(bicyclo[2.2.1]hept-5-en-2-ylmethyl)acetamide (H44)

[0165] Preparation method: Referring to the procedure of Example 17, 2,7-diazaspiro[3.5]nonane-7-carboxylic acid tert-butyl ester was used as the reactant instead of piperazine-1-carboxylic acid tert-butyl ester to prepare H44 (white solid, 29 mg, yield 25.9%). 1 H NMR (300 MHz, DMSO-d 6)δ8.14(d,J=7.2Hz,1H),7.90-7.82(m,2H),7.46(t,J=6.4Hz,1H),7.40(td,J=7.3,1.5Hz,1H), 7.35(ddd,J=7.2,6.4,1.4Hz,1H),7.03(d,J=7.2Hz,1H),6.03-5.97(m,1H),5.93-5.87(m,1H),3 .74(s,4H),3.20-3.01(m,4H),2.78-2.72(m,2H),2.72-2.66(m,2H),2.62-2.54(m,2H),2.18-2 .10(m,1H),1.82-1.72(m,4H),1.58-1.53(m,2H),1.36-1.19(m,2H).HR-MS(ESI)m / z:calcd.for C 27 H 32 N 6 O[M+Na] + 479.2530,found:479.2534.

[0166] Example 45: Preparation of (3R,5R,7R)-N-(2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)ethyl)adamantane-1-carboxamide (H45)

[0167] Preparation method: Referring to the operation of Example 18, using (2-bromoethyl)carbamic acid tert-butyl ester instead of tert-butyl 2-bromoacetate as the reactant, H45 (light green solid, 69 mg, yield 55.7%) was prepared under appropriate conditions understood in the art. 1 H NMR (300 MHz, DMSO-d 6 )δ9.17(d,J=7.7Hz,1H),8.11(d,J=8.0Hz,1H),7.58(d,J=7.9Hz,1H),7.47(t,J=7.6Hz,1H),7.41-7.31(m,2H),7.20(d,J=8.0H z,1H),3.88(s,4H),3.23(d,J=6.5Hz,4H),2.64(s,4H),1.96(s,3H),1.79-1.73(m,6H),1.65(s,6H).HR-MS(ESI)m / z:calcd.for C 27 H 34 N 6 O[M+Na] + 481.2686,found:481.2686.

[0168] Example 46: Preparation of N-(((3R,5R,7R)-adamantan-1-yl)methyl)-4-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)butanamide (H46)

[0169] Preparation method: Referring to the procedure of Example 18, tert-butyl 4-bromobutyrate was used instead of tert-butyl 2-bromoacetate as the reactant to prepare H46 (white solid, 35 mg, yield 30.5%). 1 H NMR (300 MHz, DMSO-d 6 )δ9.29(d,J=7.7Hz,1H),8.17(d,J=8.0Hz,1H),7.74-7.57(m,2H),7.48(t, J=7.7Hz,1H),7.39(t,J=7.6Hz,1H),7.25(d,J=7.8Hz,1H),4.14(s,4H),3. 20(s,4H),2.91(s,2H),2.75(d,J=6.0Hz,2H),2.13(t,J=7.3Hz,2H),1.91( s,3H),1.73-1.51(m,8H),1.41(d,J=3.0Hz,6H).HR-MS(ESI)m / z:calcd.for C 29 H 38 N 6 O[M+Na] + 509.2999,found:509.2995.

[0170] Example 47: Preparation of (3R,5R,7R)-N-(4-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)butyl)adamantane-1-carboxamide (H47)

[0171] The preparation method refers to the operation of Example 18, using (4-bromobutyl)carbamic acid tert-butyl ester instead of tert-butyl 2-bromoacetate as the reactant, and H47 (light green solid, 107 mg, yield 71.5%) was prepared under appropriate conditions understood in the art. 1 H NMR (300 MHz, DMSO-d 6)δ9.06(d,J=7.7Hz,1H),8.00(d,J=8.0Hz,1H),7.54(d,J=8.0Hz,1H),7.42-7.35(m, 1H),7.32(d,J=7.8Hz,1H),7.20(t,J=7.7Hz,1H),6.96(d,J=7.8Hz,1H),3.87(s,4H) ,3.11-2.99(m,2H),2.64(s,4H),2.47(d,J=7.5Hz,2H),1.95(t,J=2.9Hz,3H),1.75( d,J=3.0Hz,6H),1.65(d,J=3.5Hz,6H),1.55-1.43(m,4H).HR-MS(ESI)m / z:calcd.for C 29 H 38 N 6 O[M+Na] + 509.2999,found:509.2993.

[0172] Example 48: Preparation of N-(((3R,5R,7R)-adamantan-1-yl)methyl)-6-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)hexanamide (H48)

[0173] The preparation method was prepared by referring to the procedure of Example 18, using tert-butyl 6-bromohexanoate instead of tert-butyl 2-bromoacetate as the reactant to obtain H48 (light yellow solid, 39 mg, yield 30.9%). 1 H NMR (400 MHz, DMSO-d 6 )δ8.98(d,J=7.8Hz,1H),7.95(d,J=8.0Hz,1H),7.62(t,J=6.3Hz,1H),7.51(d,J=8.0Hz,1H),7.35-7 .24(m,1H),7.18-7.11(m,1H),6.88(d,J=7.8Hz,1H),3.76(d,J=5.6Hz,4H),2.75(d,J=6.2Hz,2H),2 .45(t,J=5.0Hz,4H),2.30(t,J=7.3Hz,2H),2.11(t,J=7.3Hz,2H),1.90(t,J=3.0Hz,3H),1.69-1.54 (m,6H),1.54-1.42(m,4H),1.43-1.38(m,6H),1.28(p,J=7.5,7.0Hz,2H).HR-MS(ESI)m / z:calcd.for C 31 H 42 N 6O[M+Na] + 537.3312, found:537.3316.

[0174] Example 49: Preparation of (3R,5R,7R)-N-(6-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)hexyl)adamantane-1-carboxamide (H49)

[0175] The preparation method refers to the operation of Example 18, using (6-bromohexyl)carbamic acid tert-butyl ester instead of tert-butyl 2-bromoacetate as the reactant, and H49 (light yellow solid, 110 mg, yield 75.3%) was prepared under appropriate conditions understood in the art. 1 H NMR (300 MHz, DMSO-d 6 )δ8.98(d,J=7.6Hz,1H),7.95(d,J=8.0Hz,1H),7.51(d,J=8.0Hz,1H),7.29( t,J=7.5,6.2Hz,2H),7.15(t,J=7.5Hz,1H),6.87(d,J=7.7Hz,1H),3.77(s,4H ),3.03(dt,J=13.0,6.5Hz,2H),2.45(s,4H),2.30(t,J=6.8Hz,2H),1.94(s, 3H),1.74(s,6H),1.64(s,6H),1.42-1.09(m,8H).HR-MS(ESI)m / z:calcd.for C 31 H 42 N 6 O[M+Na] + 537.3312, found:537.3309.

[0176] Example 50: Preparation of (3R,5R,7R)-N-(2-(2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)ethoxy)ethyl)adamantane-1-carboxamide (H50)

[0177] The preparation method refers to the operation of Example 18, using tert-butyl (2-(2-bromoethoxy)ethyl)carbamate instead of tert-butyl 2-bromoacetate as the reactant, and H50 (light green solid, 55 mg, yield 37.3%) is prepared under appropriate conditions understood in the art. 1 H NMR (300 MHz, DMSO-d 6)δ9.07(d,J=7.7Hz,1H),8.03(d,J=8.0Hz,1H),7.55(d,J=8.0Hz,1H),7.37(q,J=6. 6,5.5Hz,2H),7.26(t,J=7.6Hz,1H),7.02(d,J=7.8Hz,1H),3.84(t,J=4.9Hz,4H),3. 56(t,J=5.5Hz,4H),3.40(t,J=6.1Hz,2H),3.20(q,J=6.0Hz,2H),2.63(t,J=6.1,5.0 Hz,4H),1.95(s,3H),1.75(d,J=3.0Hz,6H),1.64(s,6H).HR-MS(ESI)m / z:calcd.for C 29 H 38 N 6 O 2 [M+Na] + 525.2948,found:525.2942.

[0178] Example 51: Preparation of (3R,5R,7R)-N-(2-(2-(2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)ethoxy)ethoxy)ethyl)adamantane-1-carboxamide (H51)

[0179] The preparation method refers to the operation of Example 18, using tert-butyl (2-(2-(2-bromoethoxy)ethoxy)ethyl)carbamate instead of tert-butyl 2-bromoacetate as the reactant, and H51 (light green solid, 65 mg, yield 45.7%) is prepared under appropriate conditions understood in the art. 1 H NMR (300 MHz, DMSO-d 6 )δ8.98(d,J=7.7Hz,1H),7.95(d,J=8.0Hz,1H),7.51(d,J=8.0Hz,1H),7.37-7.31(m,1H),7. 28(d,J=7.8Hz,1H),7.15(t,J=7.6Hz,1H),6.88(d,J=7.7Hz,1H),3.77(t,J=5.0Hz,4H),3.56 (t,J=5.8Hz,2H),3.51(s,4H),3.39(t,J=6.2Hz,4H),3.18(q,J=6.1Hz,2H),2.55(d,J=5.0H z,4H),1.93(s,3H),1.74(d,J=3.0Hz,6H),1.63(d,J=3.5Hz,6H).HR-MS(ESI)m / z:calcd.for C 31 H42 N 6 O 3 [M+Na] + 569.3211, found:569.3204.

[0180] Example 52: Preparation of (3R,5R,7R)-N-(2-(2-(2-(2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)ethyl)adamantane-1-carboxamide (H52)

[0181] The preparation method refers to the operation of Example 18, using tert-butyl (2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate instead of tert-butyl 2-bromoacetate as the reactant, and H52 (green solid, 65 mg, yield 52.4%) is prepared under appropriate conditions understood in the art. 1 H NMR (300 MHz, DMSO-d 6 )δ9.17(d,J=7.8Hz,1H),8.11(d,J=8.0Hz,1H),7.58(d,J=7.9Hz,1H),7.46(t,J=7.6Hz, 1H),7.40-7.31(m,2H),7.18(d,J=7.8Hz,1H),3.92(s,4H),3.62(t,J=5.5Hz,2H),3.54(s ,4H),3.51(s,4H),3.39(t,J=6.4Hz,2H),3.19(q,J=6.1Hz,2H),2.79-2.72(m,6H),1.94 (s,3H),1.73(d,J=3.0Hz,6H),1.63(dd,J=694.1,5.9Hz,6H).HR-MS(ESI)m / z:calcd.for C 33 H 46 N 6 O 4 [M+Na] + 613.3473,found:613.3475.

[0182] Example 53: Preparation of 2-((3R,5R,7R)-adamantan-1-yl)-N-(2-(2-((6-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)hexyl)oxy)ethoxy)ethyl)acetamide (H53)

[0183]

[0184] Step a: Preparation of 2-(2-((6-chlorohexyl)oxy)ethoxy)eth-1-amine (Z-4-2)

[0185] In a 50 mL eggplant-shaped bottle, tert-butyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate (300 mg, 926 μmol) was dissolved in DCM (3 mL), and then TFA (2 mL) was added. The reaction was carried out at room temperature for 1 h, and the solvent was evaporated under reduced pressure. Water was added and extracted with DCM (20 mL × 3). The aqueous phase was adjusted to alkaline with saturated sodium bicarbonate solution, and then extracted with DCM (20 mL × 3). The organic layers were combined and anhydrous Na 2 SO 4 After drying, DCM was evaporated under reduced pressure to obtain Z-4-2 (light yellow oily liquid, 177 mg, yield 85.4%). ESI-MS m / z: 224.28 [M+H] + .

[0186] Step b: Preparation of 2-((3R,5R,7R)-adamantan-1-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)acetamide (Z-4-3)

[0187] In a 50 mL eggplant-shaped bottle, 2-((3R, 5R, 7R)-adamantan-1-yl)acetic acid (169 mg, 870 μmol), HATU (421 mg, 1.11 mmol), and DIPEA (413 μL, 2.37 mmol) were added to DMF (5 mL) in sequence. After stirring at room temperature for 15 minutes, 2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl-1-amine (177 mg, 791 μmol) was added and reacted at room temperature overnight. After the reaction was completed, water was added to quench the reaction and EA was extracted (20 mL×3). The organic layers were combined, washed with saturated brine, and anhydrous Na 2 SO 4 After drying and concentration, separation by silica gel column chromatography gave Z-4-3 (colorless oily liquid, 312 mg, yield 98.6%). ESI-MS m / z: 400.45 [M+H] + .

[0188] Step c: Preparation of 2-((3R,5R,7R)-adamantan-1-yl)-N-(2-(2-((6-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)hexyl)oxy)ethoxy)ethyl)acetamide (H53)

[0189] In a 50 mL eggplant-shaped bottle, 2-(piperazine-1-yl)benzo[4,5]imidazo[1,2-a]pyrimidine (197 mg, 780 μmol), 2-((3R,5R,7R)-adamantan-1-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)acetamide (312 mg, 780 μmol), DIPEA (606 μL, 3.9 mmol) and KI (194 mg, 1.17 mmol) were added to a DMF (9 mL) solution and reacted at 80 °C overnight under argon protection. After the reaction was complete, the mixture was cooled to room temperature, water was added to quench the reaction, extracted with EA (30 mL × 3), washed with saturated brine, and anhydrous Na 2 SO 4 After drying and concentration, separation by silica gel column chromatography afforded H53 (yellow solid, 221 mg, yield 45.9%). 1 H NMR (300 MHz, DMSO-d 6 )δ8.14(d,J=7.2Hz,1H),7.91-7.83(m,2H),7.40(td,J=7.2,1.5Hz,1H),7.37-7.32(m,2H), 6.58(d,J=7.0Hz,1H),3.62-3.58(m,2H),3.58-3.52(m,8H),3.47(t,J=6.1Hz,2H),3.30(q,J =4.4Hz,2H),2.74-2.65(m,4H),2.41(t,J=6.1Hz,2H),2.04-1.94(m,5H),1.65(t,J=5.7Hz, 6H),1.61(d,J=5.1Hz,6H),1.57-1.45(m,4H),1.42-1.29(m,4H).HR-MS(ESI)m / z:calcd.for C 36 H 52 N 6 O 3 [M+Na] + 639.3993,found:639.3984.

[0190] Example 54: Preparation of 2-(4-(benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)piperazin-1-yl)-N-(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)acetamide (H54)

[0191] The preparation method refers to the operation of Example 15, using 1,7,7-trimethylbicyclo[2.2.1]hept-2-amine instead of 1,2,3,5,6,7-hexahydro-s-indacene-4-amine as the reactant to prepare H54 (white solid, 64 mg, yield 52.7%). 1HNMR (400 MHz, DMSO-d 6 )δ8.14(d,J=7.2Hz,1H),7.86(td,J=6.9,1.4Hz,2H),7.40(td,J=7.2,1.5Hz,1H),7.36( d,J=7.0Hz,1H),7.35(td,J=6.5,1.4Hz,1H),6.58(d,J=7.0Hz,1H),3.58(t,J=4.3Hz,4H ),3.57-3.51(m,1H),3.12(s,2H),2.74-2.69(m,4H),1.82-1.76(m,1H),1.76-1.69(m,1 H),1.55-1.44(m,3H),1.42-1.34(m,2H),0.93-0.75(m,9H).HR-MS(ESI)m / z:calcd.for C 26 H 34 N 6 O[M+Na] + 469.2686, found:469.2690.

[0192] Example 55: Experimental test on the ability of hydrophobic tag compounds to degrade Tau protein in SH-SY5Y cell line

[0193] 1. Experimental methods

[0194] Western blot experiments were used to screen the ability of compounds to induce Tau protein degradation. Cells in the logarithmic growth phase were digested and inoculated. After the cells adhered to the wall, different concentrations of hydrophobic label compounds were added, and a negative control was set up at the same time. After 24 hours of action, the culture medium was aspirated, washed with pre-cooled PBS, and RIPA:PMSF=100:1 pre-cooled lysis buffer was added. The cells were lysed for 30 minutes and then transferred to a centrifuge tube and centrifuged at 13000r / min for 30 minutes at 4°C. After the centrifugation, an appropriate amount of protein supernatant was aspirated, and an appropriate amount of protein sample was added to 5×Loading Buffer and mixed evenly. The sample was placed in 100°C boiling water for denaturation for 15 minutes. After the sample returned to room temperature, it was loaded or placed in a -80°C refrigerator. Another appropriate amount of protein sample was taken for BCA quantification. The protein sample was electrophoresed at 200V for 45 minutes, and then transferred to a PVDF membrane at 300mA for 70 minutes. The membrane was then blocked in 5% skim milk TBST buffer at room temperature for 2 hours, and then the membrane was incubated with the primary antibody at 4°C overnight. The membrane was washed with TBST buffer at room temperature for 5 min × 5 times on a shaker, and then the membrane was incubated with the secondary antibody at 37°C for 1 hour, and then washed with TBST buffer at room temperature for 5 min × 5 times on a shaker. The PVDF membrane was exposed using chemiluminescent solution, and quantitative analysis was performed using Image J software to calculate D max value.

[0195] 2. Experimental results

[0196] Table 1. Degradation activity of compounds in various examples

[0197]

[0198]

[0199] D max :40%<A<100%, 20%<B≤40%, 0%≤C≤20%;

[0200] Note: The highest dosing concentration is 20 μM.

[0201] As shown in Table 1, the hydrophobic tag compounds of the present invention have a strong ability to degrade Tau protein in SH-SY5Y cell line. Nearly 1 / 3 of the compounds have a D max (Maximum degradation rate) is greater than 40%.

[0202] Example 56: Parallel Artificial Membrane Permeation (PAMPA) Assay to Test Blood-Brain Barrier Permeability of Hydrophobic Tag Compounds

[0203] 1. Experimental methods

[0204] Prepare a 10mM test compound stock solution in DMSO and dilute 1000-fold to 10μM with PBS; sonicate a 20mg / ml porcine lecithin dodecane solution. Add 10μM test compound solution to the donor chamber (bottom) in triplicate, 300μL per well. Add the porcine lecithin dodecane solution to the receptor chamber (top), 5μL per well, and then add PBS (pH=7.4) to the receptor chamber within 10 minutes, 300μL per well. Transfer 50μL of the 10μM test compound solution to a sample plate containing 200μL of cold methanol (containing 50nM alprazolam, 50nM labetalol, and 100nM ketoprofen). Insert the receptor chamber into the donor chamber and incubate at 25°C for 16 hours. After 16 h of incubation, transfer 50 μL from the acceptor and donor chambers to a sample plate containing 200 μL of cold methanol (containing 50 nM alprazolam, 50 nM labetalol, and 100 nM ketoprofen). Centrifuge the sample plate at 3220 g for 40 min at 4 °C. Transfer 100 μL of the supernatant to a 4% flask containing an appropriate volume of HO. 2 O analysis plate for LC-MS / MS analysis. PAMPA effective permeability Pe (cm·s-1): Pe = -ln[1-C A (t) / C equilibrium ] / [A×(1 / V D +1 / V A )×t]. Wherein, t represents the incubation time; C A (t) represents the concentration of the test compound in the receptor compartment at time t; C equilibrium =[C D (t)×V D +C A (t)×V A ] / (V D +V A ), f is the apparent porosity of the filter, which is 0.76; A is the effective filtration area, which is f×0.3cm 2 ; V D V represents the donor pore volume, which is 0.3 mL; A represents the volume of the receptor pore, which is 0.3 mL; C D (t) represents the concentration of the test compound in the donor chamber at time t.

[0205] 2. Experimental results

[0206] Table 2. Blood-brain barrier permeability of representative compounds

[0207]

[0208]

[0209] High permeability: 4×10-6 <Pe, medium permeability: 2×10 -6 <Pe<4×10 -6 , low permeability: Pe<2×10 -6 .

[0210] As shown in Table 2, the hydrophobic tag compound of the present invention has a high blood-brain barrier permeability, which is comparable to the positive control testosterone, and can better penetrate the blood-brain barrier into the brain to exert a degradation effect.

[0211] Therefore, the hydrophobic tag compound for targeted degradation of Tau protein of the present invention has the ability to degrade Tau protein and penetrate the blood-brain barrier, and has the potential to be developed for the treatment of neurodegenerative diseases.

Claims

1. A hydrophobic tag compound for targeted degradation of Tau protein, characterized in that: The compound is shown in Formula I: Wherein: X is CH or N; Y is composed of one or more of a substituted or unsubstituted piperidine ring, a substituted or unsubstituted piperazine ring, a substituted or unsubstituted bridged ring, a substituted or unsubstituted spiro ring, a substituted or unsubstituted 6- to 10-membered aromatic ring, a substituted or unsubstituted 5- to 10-membered aromatic heterocycle, -CH2-, -NH-, -O- or -S-; Linker is a linker chain, which is a straight or branched alkylene chain with a total length of 1 to 15 atoms, or no group, wherein the straight or branched alkylene chain is composed of one or more of -CH2-, -O-, -CONH-, -NHCO-, -NHCONH-, -NH-, -S-, sulfinyl, sulfonyl, aminosulfonylamino, alkynylene, alkenylene, cycloalkylene, arylene, heterocyclylene or heteroarylene, wherein the straight or branched alkylene chain is optionally substituted by one or more substituents; HyT is selected from:

2. The hydrophobic tag compound for targeting degradation of Tau protein according to claim 1, characterized in that: Y is the following group:

3. The hydrophobic tag compound for targeting degradation of Tau protein according to claim 1, characterized in that: The Linker is the following group:

4. The hydrophobic tag compound for targeted degradation of Tau protein according to claim 1, characterized in that: The compound is selected from any of the following compounds:

5. The hydrophobic tag compound for targeted degradation of Tau protein according to claim 1, characterized in that: It also includes stereoisomers, tautomers, deuterated compounds, pharmaceutically acceptable salts or mixtures thereof.

6. The hydrophobic tag compound for targeted degradation of Tau protein according to claim 5, characterized in that: The pharmaceutically acceptable salt is a salt formed by the compound and any one of the following acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, phosphorous acid, carbonic acid, methanesulfonic acid, benzoic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, propionic acid, malonic acid, butyric acid, isobutyric acid, fumaric acid, suberic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, mandelic acid, amino acids, glucuronic acid or ferulic acid.

7. A pharmaceutical composition, characterized in that Comprising the hydrophobic tag compound, stereoisomer, tautomer, deuterated compound, pharmaceutically acceptable salt or mixture thereof and pharmaceutically acceptable excipient or carrier for targeting degradation of Tau protein as claimed in claim 1.

8. Use of the hydrophobic tag compound for targeting degradation of Tau protein according to claim 1 or the pharmaceutical composition according to claim 7 in the preparation of a drug for treating neurodegenerative diseases.

9. The application according to claim 8, characterized in that: The neurodegenerative disease is Alzheimer's disease, progressive supranuclear palsy, dementia with Lewy bodies, corticobasal degeneration, Pick's disease or frontotemporal dementia.

10. The application according to claim 8, characterized in that: The hydrophobic tag compound for targeted degradation of Tau protein connects a small molecule Tau protein ligand with a hydrophobic tag structural fragment having hydrophobicity through a connecting chain, thereby increasing the blood-brain barrier permeability of the drug on the basis of producing Tau protein degradation activity, and is applied in drugs for degenerative diseases.

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