Disubstituted benzimidazole compound and application thereof

Abstract

The invention provides a disubstituted benzimidazole compound with a structure as shown in a formula I, or pharmaceutically acceptable salt or stereoisomer thereof, and application of the disubstituted benzimidazole compound or the pharmaceutically acceptable salt or stereoisomer thereof. Compared with a 2, 8-disubstituted quinazoline lead compound, the disubstituted benzimidazole compound disclosed by the invention has better PDGFR alpha D842V kinase inhibition activity and anti-tumor effect, also has a better inhibition effect on PDGFR alpha D842V / G680R mutant kinase and PDGFR alpha D842V / T674I mutant kinase, shows very strong anti-proliferative activity in corresponding Ba / F3-PDGFR alpha stable strain cells, and can be used for preparing anti-tumor drugs. The compound can play a good role in inhibiting tumor cells, and can be used for preparing drugs for preventing or treating PDGFRalpha kinase mediated diseases (such as tumors). # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, relates to anti-tumor drug compounds, and specifically relates to disubstituted benzimidazole compounds and their uses. Background Art

[0002] Platelet derived growth factor receptor alpha (PDGFRα) kinase belongs to the type III receptor tyrosine kinase (RTK) family. PDGFR is mainly expressed by mesenchymal-derived cells, such as fibroblasts, pericytes, and vascular smooth muscle cells, and stimulates their proliferation and migration. Early studies found that PDGFR and its ligands may play a role in tumor development, and subsequent receptor gene and genetic variations were found in hematological malignancies, gliomas, and soft tissue tumors. In normal cells, after PDGFRα binds to its ligand platelet-derived growth factor (PDGF), it induces receptor dimerization and transphosphorylation, activates downstream pathways, and regulates cell proliferation and growth. In patients with gastrointestinal stromal tumors (GISTs), after the PDGFRα gene mutates, the receptor phosphorylates itself independently of the ligand, thereby activating the downstream cell signaling pathways (including the SRC / RAC1 / JNK pathway, the PI3K / AKT / mTOR pathway, and the RAS / RAF / MEK / MAPK pathway), ultimately leading to continuous proliferation of malignant tumors.

[0003] Currently, four lines of drugs have been developed clinically for the treatment of gastrointestinal stromal tumors. The first three lines of drugs, imatinib, sunitinib, and regorafenib, belong to multi-target kinase inhibitors and are resistant to GISTs patients carrying PDGFRα D842V mutations. Ripretinib, as a fourth-line drug on the market, solves the clinical drug resistance problem of some GISTs patients. Ayvakit is currently the only first-line drug specifically for the treatment of patients carrying PDGFRα D842V mutations. However, neurocognitive side effects and acquired solvent front and gatekeeper residue resistance mutations in some patients after drug use limit its further clinical use, and the clinical options for patients are still limited. Summary of the Invention

[0004] Based on this, the present invention provides a disubstituted benzimidazole compound, which has strong inhibitory activity against mutant PDGFRα kinases such as PDGFRα D842V mutations, and can be used to prepare drugs for preventing or treating diseases (such as tumors) mediated by PDGFRα kinase.

[0005] The present invention includes the following technical solutions.

[0006] In a first aspect, the present invention provides a disubstituted benzimidazole compound having the structure shown in Formula I, or a pharmaceutically acceptable salt or a stereoisomer thereof,

[0007]

[0008] wherein R is selected from: 5- to 7-membered heterocyclic groups;

[0009] R 1 is selected from: hydrogen, C 1 -C 6 alkyl;

[0010] n is selected from: 1, 2, 3, 4, 5, 6.

[0011] In some embodiments of the present invention, the disubstituted benzimidazole compound has the structure shown in Formula I-1 or Formula I-2:

[0012]

[0013] Further preferably, R is selected from: 5- to 7-membered saturated heterocyclic groups.

[0014] Further preferably, R is selected from: 5- to 7-membered heterocyclic groups with a heteroatom of N.

[0015] Further preferably, R is selected from: 5- to 7-membered heterocyclic groups with 1 heteroatom of N.

[0016] Further preferably, R is selected from: 5-membered heterocyclic groups with 1 heteroatom of N, 6-membered heterocyclic groups with 1 heteroatom of N.

[0017] Further preferably, R is selected from the following groups:

[0018]

[0019] Further preferably, R 1 is selected from: hydrogen, methyl, ethyl, propyl.

[0020] In some embodiments of the present invention, the disubstituted benzimidazole compound is selected from the following compounds:

[0021]

[0022] The present invention also provides the use of the disubstituted benzimidazole compound, or a pharmaceutically acceptable salt or a stereoisomer thereof, including the following:

[0023] The present invention provides the use of the described disubstituted benzimidazole compounds or their pharmaceutically acceptable salts or their stereoisomers in the preparation of PDGFRα kinase inhibitors; wherein, the PDGFRα kinase may be wild-type PDGFRα kinase or mutant PDGFRα kinase, preferably PDGFRα kinase with D842V mutation, G680R mutation and / or T674I mutation.

[0024] The present invention also provides the use of the described disubstituted benzimidazole compounds or their pharmaceutically acceptable salts or their stereoisomers in the preparation of drugs for treating and / or preventing diseases, wherein the diseases are diseases caused by abnormal expression of PDGFRα kinase, preferably PDGFRα D842V mutation, PDGFRα G680R mutation, PDGFRα D842V / G680R mutation, PDGFRα T674I mutation or PDGFRα D842V / T674I mutation-mediated diseases.

[0025] The present invention also provides the use of the described disubstituted benzimidazole compounds or their pharmaceutically acceptable salts or their stereoisomers in the preparation of drugs for treating and / or preventing tumors.

[0026] Further preferably, the tumors are tumors caused by abnormal expression of PDGFRα kinase.

[0027] Further preferably, the tumors are tumors carrying PDGFRα D842V mutation, PDGFRα G680R mutation, PDGFRα D842V / G680R mutation, PDGFRα T674I mutation or PDGFRα D842V / T674I mutation.

[0028] Further preferably, the tumors are gastric cancers, preferably gastric cancers caused by abnormal expression of PDGFRα kinase, more preferably gastric cancers carrying PDGFRα D842V mutation, PDGFRα G680R mutation, PDGFRα D842V / G680R mutation, PDGFRα T674I mutation or PDGFRα D842V / T674I mutation.

[0029] Further preferably, the tumors are gastrointestinal stromal tumors, preferably gastrointestinal stromal tumors caused by abnormal expression of PDGFRα kinase, more preferably gastrointestinal stromal tumors carrying PDGFRα D842V mutation, PDGFRα G680R mutation, PDGFRα D842V / G680R mutation, PDGFRαT674I Mutation or PDGFRα D842V / T674I Mutated gastrointestinal stromal tumor.

[0030] In a third aspect, the present invention provides an anti-tumor pharmaceutical composition prepared from an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises the disubstituted benzimidazole compound described in the present invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

[0031] Based on the 2,8-disubstituted quinazoline lead compound (i.e., compound 8 in the examples) of the present invention, through further optimization and improvement of the parent nucleus and substituents, a new disubstituted benzimidazole compound that can more effectively target PDGFRα D842V protein and downstream signaling pathways, and can overcome PDGFRα D842V mutation resistance has been finally obtained.

[0032] The disubstituted benzimidazole compound of the present invention has better PDGFRα D842V kinase inhibitory activity and anti-tumor effect compared with the 2,8-disubstituted quinazoline lead compound, and some compounds also have good inhibitory effects on PDGFRα D842V / G680R mutant kinase and PDGFRα D842V / T674I mutant kinase, show strong anti-proliferative activity in the corresponding Ba / F3-PDGFRα stable cell line, can play a good inhibitory role on tumor cells, and can be used to prepare drugs for preventing or treating diseases mediated by PDGFRα kinase (such as tumors), and for treating tumors (especially gastric cancer) in humans and other mammals. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Results of the inhibitory activity of compound 1 on the cell proliferation of gastric cancer cells SNU-16 and KATO III. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0035] The experimental methods without specific conditions noted in the following examples are generally carried out under conventional conditions or according to the conditions recommended by the manufacturers. All kinds of commonly used chemical reagents used in the examples are commercially available products.

[0036] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present invention pertains. The terms used in the description of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0037] In addition, as used in the present invention, the term "or" is the inclusive "or" symbol and is equivalent to the term "and / or", unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for other factors not described, unless the context clearly dictates otherwise. Further, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in" includes "in" and "on".

[0038] The term "alkyl" as used in the present invention refers to saturated aliphatic hydrocarbon groups, including branched and straight-chain ones, having a specific number of carbon atoms. For example, in " 1 -C 6 alkyl", the definition of " 1 -C 6 " includes groups having 1, 2, 3, 4, 5, or 6 carbon atoms arranged in a straight-chain or branched-chain manner. For example, " 1 -C 6 alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0039] The term "heterocyclic group" as used in the present invention is a saturated or partially unsaturated monocyclic or polycyclic ring substituent, in which one or more ring atoms are heteroatoms selected from N, O, or S(O)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. For example: morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, tetrahydrofuryl, tetrahydrothienyl, etc., and their N-oxides. The connection of the heterocyclic substituent can be achieved through a carbon atom or through a heteroatom.

[0040] The present invention includes the free form of the compound of formula I, as well as its pharmaceutically acceptable salts and stereoisomers. The term "free form" refers to an amine compound in non-salt form. The pharmaceutically acceptable salts included not only the exemplary salts of the specific compounds described herein, but also the typical pharmaceutically acceptable salts of all free forms of the compounds of formula I. The free form of the specific salts of the compounds can be isolated using techniques known in the art. For example, the free form can be regenerated by treating the salt with a dilute aqueous solution of a suitable base such as dilute aqueous NaOH, dilute aqueous potassium carbonate, dilute aqueous ammonia, and dilute aqueous sodium bicarbonate. The free form is somewhat different from its respective salt form in certain physical properties such as solubility in polar solvents, but for the purposes of the invention, such acid salts and base salts are equivalent to their respective free forms in other pharmaceutical aspects.

[0041] The pharmaceutically acceptable salts of the present invention can be synthesized from the compounds of the present invention containing a basic moiety by conventional chemical methods. Generally, salts of basic compounds are prepared by ion exchange chromatography or by reacting the free base with a stoichiometric or excess amount of the desired salt form of an inorganic or organic acid in a suitable solvent or a combination of solvents.

[0042] Accordingly, the pharmaceutically acceptable salts of the compounds of the present invention include the conventional non-toxic salts of the compounds of the present invention formed by reacting the basic compounds of the present invention with inorganic or organic acids. For example, the conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc., and also salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxy-benzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, 2-hydroxyethanesulfonic acid, trifluoroacetic acid, etc.

[0043] The present invention also provides a pharmaceutical composition which comprises an active ingredient within a safe and effective amount range, as well as pharmaceutically acceptable excipients.

[0044] The "active ingredient" as described in the present invention refers to the compound of formula I as described in the present invention, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule.

[0045] The "active ingredient" and the pharmaceutical composition as described in the present invention can be used as inhibitors of PDGFRα (especially PDGFRα D842V ), and can be used for preparing drugs for preventing and / or treating tumors and the like.

[0046] "Safe and effective amount" means that the amount of the active ingredient is sufficient to significantly improve the condition without causing serious side effects.

[0047] "Pharmaceutically acceptable excipients" refer to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity.

[0048] "Compatibility" herein refers to the ability of the components in the composition to blend with the active ingredient of the present invention and with each other without significantly reducing the efficacy of the active ingredient.

[0049] Some examples of pharmaceutically acceptable carriers or excipients include cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium dodecyl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0050] In another preferred embodiment, the compound of formula I of the present invention can form a complex with a macromolecular compound or polymer through non-bonding interactions. In another preferred embodiment, the compound of formula I of the present invention, as a small molecule, can also be linked to a macromolecular compound or polymer through chemical bonds. The macromolecular compound can be a biological macromolecule such as polysaccharide, protein, nucleic acid, polypeptide, etc.

[0051] There is no particular limitation on the mode of administration of the active ingredient or pharmaceutical composition of the present invention. Representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

[0052] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.

[0053] In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dibasic calcium phosphate, or is mixed with the following components:

[0054] (a) Fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol and silicic acid;

[0055] (b) Binders, such as hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic;

[0056] (c) Humectants, such as glycerol;

[0057] (d) Disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;

[0058] (e) Retardants, such as paraffin;

[0059] (f) Absorption accelerators, e.g., quaternary amine compounds;

[0060] (g) Wetting agents, e.g., cetyl alcohol and glycerol monostearate;

[0061] (h) Adsorbents, e.g., kaolin; and

[0062] (i) Lubricants, e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0063] The solid dosage forms described above can also be prepared with coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the release of the active ingredient in such compositions can be delayed and released in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric materials and wax materials.

[0064] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizing agents, and emulsifying agents, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3 - butanediol, dimethylformamide, and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances. In addition to these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, and fragrances.

[0065] In addition to the active ingredient, suspensions may contain suspending agents, e.g., ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, and agar, or mixtures of these substances.

[0066] Compositions for parenteral injection may contain physiologically acceptable sterile aqueous or non - aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for re - dissolving into sterile injectable solutions or dispersions. Suitable aqueous and non - aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0067] The compounds of the present invention can be administered alone or in combination with other therapeutic agents.

[0068] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal in need of treatment (such as a human), wherein the dosage during administration is an effective dosage considered pharmaceutically. For a person weighing 60 kg, the daily dosage is usually 1 - 2000 mg, preferably 20 - 500 mg. Of course, the specific dosage should also consider factors such as the administration route and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0069] The following are specific examples.

[0070] Synthesis of Compound 1 in Example 1

[0071] Compound 1 was synthesized using the following reaction formula:

[0072]

[0073] 1. Synthesis methods of Intermediate 1 - Intermediate 5

[0074] Dissolve 5 - methoxy - 2 - nitroaniline (550 mg, 3.17 mmol) and 2,8 - dichloroquinoline (520 mg, 2.64 mmol) in a two - necked flask containing 7.5 mL of ultra - dry tert - butanol (t - BuOH) solvent. Then add palladium acetate (35.6 mg, 0.16 mmol), 4,5 - bis(diphenylphosphino) - 9,9 - dimethyloxanthrene (Xantphos) (91.7 mg, 0.16 mmol) and cesium carbonate (2.07 g, 6.34 mmol). Replace the reaction system with argon three times repeatedly and seal the reaction system. Stir at 80 °C for 4 hours. Monitor by TLC. After the reaction is complete, filter the reaction solution through diatomaceous earth and wash the filter cake repeatedly with ethyl acetate. Then add 7.5 mL of water for extraction three times, collect the organic phase, wash with saturated brine, and then dry with anhydrous Na 2 SO 4 dry, filter, concentrate under reduced pressure and purify by silica gel column chromatography. The mobile phase is petroleum ether:ethyl acetate = 5:1 to obtain Intermediate 1 (887 mg, yield: 85%).

[0075] Dissolve Intermediate 1 (550 mg, 1.74 mmol) in 7.5 mL of acetic acid, add trimethyl orthoformate (1.14 mL, 10.5 mmol) and iron powder (0.39 g, 6.9 mmol). Stir the reaction solution at 70 °C for 3 hours. Monitor by TLC. After the reaction is complete, filter the reaction solution through diatomaceous earth, concentrate the reaction solution and purify by silica gel column chromatography. The mobile phase is dichloromethane:methanol = 100:1 to obtain Intermediate 2 (340 mg, yield: 63.2%).

[0076] Intermediate 2 (464 mg, 1.5 mmol) and 1,4-dioxo-8-azaspiro[4.5]decane (0.23 mL, 1.8 mmol) were dissolved in a two-necked flask containing 5 mL of ultradry 1,4-dioxane solvent, and then Pd 2 (dba) 3 (0.14 g, 0.15 mmol), 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)-biphenyl (60 mg, 0.15 mmol) and sodium tert-butoxide (215 mg, 2.25 mmol) were added. The reaction system was repeatedly purged with argon three times and the reaction system was sealed. The reaction was carried out at 100 °C overnight. Monitored by TLC, after the reaction was complete, the reaction solution was filtered through diatomaceous earth and the filter cake was washed repeatedly with dichloromethane. Then 7.5 mL of water was added for extraction three times, the organic phase was collected, washed with saturated brine, and then dried with anhydrous Na 2 SO 4 , filtered, concentrated under reduced pressure and purified by silica gel column chromatography. The mobile phase was dichloromethane:methanol = 100:1 to obtain Intermediate 3 (476 mg, yield 76.3%).

[0077] Intermediate 3 (1.1 g, 2.68 mmol) was dissolved in 15 mL of anhydrous dichloromethane, and after the system was replaced with argon, it was cooled to -78 °C. Boron tribromide (1.55 mL, 16.1 mmol) was slowly added to this solution with stirring. After the addition was complete, the mixture was stirred at room temperature for 5 hours. Monitored by TLC, after the reaction was complete, the system was cooled to 0 °C, and saturated aqueous sodium bicarbonate solution was slowly added dropwise until the pH was 7. The solid was filtered and washed with methanol, and the filter cake was dried under vacuum to obtain a white solid. The white solid was then dissolved in 7.5 mL of tetrahydrofuran solvent, 5 mL of 2N HCl solution was slowly added dropwise, and the reaction solution was reacted at 50 °C for 24 hours. Monitored by TLC, after the reaction was complete, the reaction solution was concentrated, saturated sodium bicarbonate solution was added to adjust the pH to 7, then dichloromethane was added for extraction, the organic phase was collected, and dried with anhydrous Na 2 SO 4 , filtered, and concentrated under reduced pressure to obtain Intermediate 4 (595 mg, total two-step yield: 62%).

[0078] Intermediate 4 (50 mg, 0.14 mmol) was dissolved in 5 mL of N,N-dimethylformamide (DMF) solvent, tert-butyl 2-bromoethylcarbamate (46.8 mg, 0.21 mmol) and cesium carbonate (90.9 mg, 0.28 mmol) were added, and the mixture was stirred at 80 °C overnight. Monitored by TLC, after the reaction was complete, it was cooled to room temperature, 5 mL of water was added to dilute the reaction solution, then 5 mL of ethyl acetate was added for extraction, the organic phase was washed twice with saturated brine, and then dried with anhydrous Na 2 SO 4After drying and concentration, it was purified by silica gel column chromatography with the mobile phase of dichloromethane:methanol = 100:1 to obtain Intermediate 5 (58.4 mg, yield: 81%).

[0079] 2. Synthesis method of Compound 1

[0080] Intermediate 5 (58.4 mg, 0.11 mmol) was dissolved in 2.5 mL of dichloromethane solvent, 0.25 mL of trifluoroacetic acid was slowly added dropwise, and the mixture was stirred at room temperature for 3 hours. After monitoring by TLC plate, after the reaction was complete, the reaction solution was diluted with dichloromethane, and saturated sodium bicarbonate solution was added to adjust the pH to 9. The organic phase was washed twice with saturated brine, dried over anhydrous Na2SO4, and the solvent was evaporated to obtain a light yellow oily liquid. The obtained crude product was dissolved in 40 mL of anhydrous dichloromethane solvent, a small amount of 4A molecular sieve, 2 drops of glacial acetic acid were added, and the mixture was stirred at room temperature for 20 minutes, then sodium triacetoxyborohydride (95.8 mg, 0.46 mmol) was added, and the stirring was continued at room temperature for 6 hours. After monitoring by TLC, after the reaction was complete, saturated sodium bicarbonate solution was added, the organic phase was separated, the organic phase was washed twice with saturated brine, and then with anhydrous Na 2 SO 4 After drying and concentration, it was purified by silica gel column chromatography with the mobile phase of dichloromethane:methanol = 20:1 to obtain light green solid Compound 1 (6.1 mg, total two-step yield 14.3%).

[0081] Compound 1, light green solid (yield 14.3%). 1 HNMR (400 MHz, DMSO-d 6 ) δ9.43(s,1H),9.15(s,1H),8.58(d,J=8.9Hz,1H),8.14(d,J=8.9Hz,1H),7.73(d,J=8.7Hz,1H),7.67(d,J=8.1Hz,1H),7.51(t,J=7.8Hz,1H),7.32(d,J=7.1Hz,1H),7.15(dd,J=8.6,2.5Hz,1H),4.51(t,J=4.4Hz,2H),3.81-3.54(m,4H),3.50(t,J=4.3Hz,2H),3.31(s,1H),3.16(d,J=7.8Hz,1H),2.86(t,J=11.2Hz,2H),1.98-1.88(m,2H).

[0082] Example 2 Synthesis of Compound 2

[0083] Replace tert-butyl-2-bromoethylcarbamate in Example 1 with tert-butyl-4-bromobutylcarbamate, and synthesize the following Compound 2 by the same method as in Example 1.

[0084]

[0085] Compound 2, a light green solid (yield 15.3%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.16 (s, 1H), 8.70 (d, J = 2.4 Hz, 1H), 8.54 (d, J = 8.9 Hz, 1H), 8.19 (d, J = 8.9 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.50 (t, J = 7.8 Hz, 1H), 7.30 (d, J = 7.5 Hz, 1H), 7.11 (dd, J = 8.8, 2.5 Hz, 1H), 4.14 (t, J = 5.2 Hz, 2H), 3.78 (d, J = 11.2 Hz, 2H), 2.96 - 2.91 (m, 1H), 2.87 (t, J = 7.7 Hz, 2H), 2.73 - 2.67 (m, 2H), 2.25 (dd, J = 12.0, 4.1 Hz, 2H), 1.82 (d, J = 6.7 Hz, 2H), 1.79 - 1.65 (m, 4H).

[0086] Synthesis of Compound 3 in Example 3

[0087] Replace tert-butyl-2-bromoethylcarbamate in Example 1 with tert-butyl-5-bromopentylcarbamate, and synthesize the following Compound 3 in the same method as in Example 1.

[0088]

[0089] Compound 3, a light green solid (yield 17.2%). 1 H NMR (400 MHz, DMSO-d 6)δ9.11(s,1H),8.50(d,J=9.0Hz,1H),8.42(d,J=2.4Hz,1H),8.15(d,J=8.9Hz,1H),7.69(d,J=8.8Hz,1H),7.60(d,J=7.9Hz,1H),7.48(t,J=7.8Hz,1H),7.31-7.25(m,1H),7.03(dd,J=8.8,2.4Hz,1H),4.00(t,J=4.5Hz,2H),3.83(d,J=11.4Hz,2H),2.76-2.69(m,2H),2.68(d,J=5.1Hz,2H),2.63(dd,J=10.7,4.3Hz,1H),2.06-1.97(m,2H),1.94-1.82(m,2H),1.78-1.63(m,4H),1.40(q,J=5.4Hz,2H).

[0090] Synthesis of Compound 4 in Example 4

[0091] Replace 5-methoxy-2-nitroaniline in Example 1 with 4-methoxy-2-nitroaniline, replace tert-butyl 2-bromoethylcarbamate in Example 1 with tert-butyl 5-bromopentylcarbamate, and synthesize the following Compound 4 in the same method as in Example 1.

[0092]

[0093] Compound 4, light green solid (yield 21%). 1 H NMR(400MHz,DMSO-d 6 )δ9.04(s,1H),9.00(d,J=8.9Hz,1H),8.54(d,J=8.8Hz,1H),8.08(d,J=8.8Hz,1H),7.58(d,J=8.1Hz,1H),7.47(t,J=7.8Hz,1H),7.19(q,J=2.7Hz,2H),7.15(dd,J=9.1,2.5Hz,1H),4.22(t,J=6.5Hz,2H),3.59(d,J=11.3Hz,2H),2.65(dd,J=11.1,4.1Hz,2H),2.61(d,J=5.2Hz,1H),2.53(d,J=5.3Hz,2H),1.88-1.81(m,4H),1.59-1.49(m,6H).

[0094] Synthesis of Compound 5 in Example 5

[0095]

[0096] Compound 1 (30 mg, 0.08 mmol) was dissolved in 30 mL of anhydrous dichloromethane, and then paraformaldehyde (7.0 mg, 0.23 mmol) and sodium triacetoxyborohydride (65.9 mg, 0.31 mmol) were added to this solution. The resulting mixed solution was stirred at room temperature for 6 hours. Saturated NaHCO 3 solution was used to quench the reaction, and then it was extracted with dichloromethane. The organic layer was separated and dried with anhydrous Na 2 SO 4 , filtered and concentrated. It was purified by silica gel column chromatography with the mobile phase of dichloromethane:methanol = 20:1 to obtain a light green solid (10 mg, yield: 32.4%).

[0097] Compound 5, light green solid (yield 32.4%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.33 (s, 1H), 9.13 (s, 1H), 8.56 (d, J = 8.8 Hz, 1H), 8.13 (d, J = 8.8 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.50 (t, J = 7.8 Hz, 1H), 7.30 (d, J = 7.3 Hz, 1H), 7.09 (dd, J = 8.7, 2.5 Hz, 1H), 4.48 (d, J = 5.1 Hz, 2H), 3.64 (d, J = 11.5 Hz, 2H), 3.30 - 3.23 (m, 3H), 2.83 - 2.74 (m, 2H), 2.72 (s, 3H), 2.44 - 2.34 (m, 2H), 2.00 - 1.92 (m, 2H).

[0098] Synthesis of Compound 6 in Example 6

[0099] Compound 6 was synthesized according to the following reaction formula:

[0100]

[0101] 1. Synthesis method of intermediates 6-1 to 6-6

[0102] 3-Amino-4-nitrophenol (986 mg, 6.4 mmol) was dissolved in 20 mL of N,N-dimethylformamide solvent, potassium carbonate (1.77 g, 12.8 mmol) and benzyl bromide (0.79 mL, 19.44 mmol) were added, and it was stirred at 100 °C for 2 hours. Monitored by TLC, after the reaction was complete, it was cooled to room temperature, 20 mL of water was added to dilute the reaction solution, and then 20 mL of ethyl acetate was added for extraction. The organic phase was washed twice with saturated brine, and then with anhydrous Na 2 SO 4After drying and concentration, it was purified by silica gel column chromatography with the mobile phase of petroleum ether: ethyl acetate = 8:1 to obtain intermediate 6-1 (625 mg, yield: 40%).

[0103] Intermediate 6-1 (773 mg, 3.17 mmol) and 2,8-dichloroquinoline (0.52 g, 2.64 mmol) were dissolved in a two-necked flask containing 15 mL of ultradry tert-butanol (t-BuOH) solvent. Then palladium acetate (35.9 mg, 0.16 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos) (91.7 mg, 0.16 mmol) and cesium carbonate (2.07 g, 6.34 mmol) were added. The reaction system was repeatedly purged with argon three times and the reaction system was sealed. It was stirred at 80 °C for 4 hours. Monitored by TLC, after the reaction was complete, the reaction solution was filtered through diatomaceous earth and the filter cake was washed repeatedly with ethyl acetate. Then 15 mL of water was added for extraction three times, the organic phase was collected, washed with saturated brine, and then with anhydrous Na 2 SO 4 It was dried, filtered, concentrated under reduced pressure and purified by silica gel column chromatography with the mobile phase of petroleum ether: ethyl acetate = 5:1 to obtain intermediate 6-2 (321 mg, yield: 25%).

[0104] Intermediate 6-2 (352 mg, 0.87 mmol) was dissolved in 15 mL of acetic acid, and trimethyl orthoformate (0.57 mL, 5.2 mmol) and iron powder (0.20 g, 3.5 mmol) were added. The reaction solution was stirred at 70 °C for 3 hours. Monitored by TLC, after the reaction was complete, the reaction solution was filtered through diatomaceous earth, the reaction solution was concentrated and purified by silica gel column chromatography with the mobile phase of dichloromethane: methanol = 100:1 to obtain intermediate 6-3 (214 mg, yield: 64%).

[0105] Intermediate 6-3 (193 mg, 0.5 mmol) and tert-butyl pyrrolidin-3-ylcarbamate (0.08 mL, 0.6 mmol) were dissolved in a two-necked flask containing 10 mL of ultradry 1,4-dioxane solvent. Then Pd 2 (dba) 3 palladium (45 mg, 0.05 mmol), 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)-biphenyl (20 mg, 0.05 mmol) and sodium tert-butoxide (72 mg, 0.75 mmol) were added. The reaction system was repeatedly purged with argon three times and the reaction system was sealed. It was reacted at 100 °C overnight. Monitored by TLC, after the reaction was complete, the reaction solution was filtered through diatomaceous earth and the filter cake was washed repeatedly with dichloromethane. Then 15 mL of water was added for extraction three times, the organic phase was collected, washed with saturated brine, and then with anhydrous Na 2 SO 4It was dried, filtered, concentrated under reduced pressure and purified by silica gel column chromatography with the mobile phase of dichloromethane:methanol = 100:1 to obtain intermediate 6-4 (212 mg, yield 79%).

[0106] Intermediate 6-4 (150 mg, 0.28 mmol) was dissolved in 10 mL of methanol solvent, and 10% palladium on carbon (containing 55% water) (29.8 mg, 0.028 mmol) was added. The mixture was evacuated and the system was replaced with hydrogen, and it was stirred at room temperature overnight under a hydrogen atmosphere. Monitored by TLC, after the reaction was complete, the reaction solution was filtered through diatomaceous earth and washed with methanol, and the filtrate was dried with anhydrous Na 2 SO 4 After drying and concentration, it was purified by silica gel column chromatography with the mobile phase of dichloromethane:methanol = 50:1 to obtain intermediate 6-5 (91.1 mg, yield: 73%).

[0107] Intermediate 6-5 (77.3 mg, 0.14 mmol) was dissolved in 10 mL of N,N-dimethylformamide (DMF) solvent, 1,2-dibromoethane (39.5 mg, 0.21 mmol) and cesium carbonate (90.9 mg, 0.28 mmol) were added, and it was stirred at 80 °C overnight. Monitored by TLC, after the reaction was complete, it was cooled to room temperature, 10 mL of water was added to dilute the reaction solution, then 10 mL of ethyl acetate was added for extraction, the organic phase was washed twice with saturated brine, and then dried with anhydrous Na 2 SO 4 After drying and concentration, it was purified by silica gel column chromatography with the mobile phase of dichloromethane:methanol = 100:1 to obtain intermediate 6-6 (55.6 mg, yield: 72%).

[0108] 2. Synthesis method of compound 6

[0109] Intermediate 6-6 (55.2 mg, 0.10 mmol) was dissolved in 5 mL of dichloromethane solvent, 0.5 mL of trifluoroacetic acid was slowly added dropwise, and it was stirred at room temperature for 3 hours. Monitored by TLC, after the reaction was complete, dichloromethane was added to dilute the reaction solution, and saturated sodium bicarbonate solution was added to adjust the pH to 9. The organic phase was washed twice with saturated brine, and then dried with anhydrous Na 2 SO 4 It was dried, and the solvent was rotary evaporated to obtain a light green oily liquid. The obtained crude product was dissolved in 60 mL of ultra-dry N,N-dimethylformamide solvent, potassium carbonate (41.4 mg, 0.3 mmol) was added, and it was stirred at 80 °C for 6 hours. Monitored by TLC, after the reaction was complete, it was cooled to room temperature, 15 mL of water was added to dilute the reaction solution, then 15 mL of dichloromethane was added for extraction, the organic phase was washed twice with saturated brine, and then dried with anhydrous Na 2 SO 4After drying and concentration, it was purified by silica gel column chromatography with a mobile phase of dichloromethane:methanol = 25:1 to obtain Compound 6 (21.5 mg, total yield in two steps: 58%).

[0110] Compound 6, light green solid (yield: 58%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.61 (s, 1H), 8.53 (d, J = 2.3 Hz, 1H), 8.30 (dd, J = 8.8, 1.3 Hz, 1H), 7.85 (d, J = 8.7 Hz, 1H), 7.67 (dd, J = 8.8, 1.9 Hz, 1H), 7.47 (t, J = 7.8 Hz, 1H), 7.44 - 7.39 (m, 1H), 7.19 - 7.11 (m, 2H), 5.35 (d, J = 11.0 Hz, 1H), 4.52 (t, J = 11.6 Hz, 1H), 4.09 (d, J = 11.8 Hz, 1H), 3.59 - 3.50 (m, 2H), 3.18 - 3.04 (m, 2H), 2.75 (dd, J = 11.0, 6.3 Hz, 1H), 2.67 - 2.58 (m, 2H), 1.97 - 1.88 (m, 1H).

[0111] Synthesis of Compound 7 in Example 7

[0112] Compound 7 was synthesized according to the following reaction formula:

[0113]

[0114] 1. Synthesis method of Intermediate 7-1 to Intermediate 7-7

[0115] Take 2,4,5-trichloropyrimidine (2 g, 10.9 mmol) and dissolve it in 30 mL of isopropanol solvent, add 1,4-dioxane-8-azaspiro[4.5]decane (1.68 mL, 13.0 mmol) and N,N-diisopropylethylamine (2.16 mL, 13.0 mmol), and stir the reaction solution at 80 °C for 6 hours. Monitored by TLC, after the reaction was complete, it was cooled to room temperature, 20 mL of water was added to dilute the reaction solution, and then 20 mL of ethyl acetate was added for extraction. The organic phase was washed twice with saturated brine, and then with anhydrous Na 2 SO 4 After drying and concentration, it was purified by silica gel column chromatography with a mobile phase of petroleum ether:ethyl acetate = 5:1 to obtain Intermediate 7-1 (2.34 g, yield: 74%).

[0116] Dissolve intermediate 7-1 (2.34 g, 8.06 mmol) and 5-methoxy-2-nitroaniline (1.62 g, 9.67 mmol) in a two-necked flask containing 30 mL of anhydrous dioxane solvent, and then add Pd 2 (dba) 3 (738.5 mg, 0.8 mmol), 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)biphenyl (Davephos) (466.6 mg, 0.8 mmol) and cesium carbonate (5.25 g, 16.1 mmol). Replace the reaction system with argon three times repeatedly and seal the reaction system. Stir overnight at 100 °C. Monitor by TLC. After the reaction is complete, filter the reaction solution through diatomaceous earth and wash the filter cake repeatedly with dichloromethane. Then add 15 mL of water for extraction three times, collect the organic phase, wash with saturated brine, and then dry with anhydrous Na 2 SO 4 , filter, concentrate under reduced pressure and purify by silica gel column chromatography. The mobile phase is dichloromethane:methanol = 200:1 to obtain intermediate 7-2 (1.12 g, yield: 33%).

[0117] Dissolve intermediate 7-2 (1.47 g, 3.48 mmol) in 15 mL of acetic acid, add trimethyl orthoformate (2.28 mL, 20.9 mmol) and iron powder (0.78 g, 13.9 mmol). Stir the reaction solution at 70 °C for 3 hours. Monitor by TLC. After the reaction is complete, filter the reaction solution through diatomaceous earth, concentrate the reaction solution and purify by silica gel column chromatography. The mobile phase is dichloromethane:methanol = 100:1 to obtain intermediate 7-3 (852 mg, yield: 61%).

[0118] Dissolve intermediate 7-3 (852 mg, 2.12 mmol) in 10 mL of tetrahydrofuran solvent, slowly add 10 mL of 2N HCl solution dropwise, and react the reaction solution at 50 °C for 24 hours. Monitor by TLC. After the reaction is complete, concentrate the reaction solution, adjust the pH to 7 with saturated sodium bicarbonate solution, then add dichloromethane for extraction, collect the organic phase, and dry with anhydrous Na 2 SO 4 , filter, concentrate under reduced pressure to obtain intermediate 7-4 (660 mg, yield: 87%).

[0119] Dissolve intermediate 7-4 (300 mg, 0.83 mmol) in 15 mL of anhydrous dichloromethane solvent. Add a small amount of 4A molecular sieve and 2 drops of glacial acetic acid. After stirring at room temperature for 20 minutes, add 3-aminopropanol (75.7 mg, 0.99 mmol) and sodium cyanoborohydride (210 mg, 3.32 mmol), and continue stirring at room temperature for 6 hours. Monitor by TLC. After the reaction is complete, adjust the pH to 7 with saturated sodium bicarbonate solution, separate the organic phase, wash the organic phase twice with saturated brine, and then use anhydrous Na 2 SO 4 Dry and concentrate, and purify by silica gel column chromatography. The mobile phase is dichloromethane:methanol = 20:1 to obtain intermediate 7-5 (111 mg, yield: 32%).

[0120] Dissolve intermediate 7-5 (111 mg, 0.26 mmol) in 10 mL of anhydrous dichloromethane solvent, replace the system with argon, and cool to -78 °C. Slowly add boron tribromide (0.15 mL, 1.59 mmol) to this solution with stirring. After the addition is complete, continue stirring at room temperature for 5 hours. Monitor by TLC. After the reaction is complete, cool the system to 0 °C and slowly add saturated aqueous sodium bicarbonate solution until the pH reaches 7. Filter the solid and wash with methanol, and dry the filter cake under vacuum to obtain intermediate 7-6 (38.6 mg, yield: 36%).

[0121] Dissolve intermediate 7-6 (100 mg, 0.25 mmol) in 10 mL of ultra-dry methanol solvent. Slowly add triethylamine (50 mg, 0.5 mmol) and di-tert-butyl dicarbonate (59.5 mg, 0.27 mmol) dropwise in an ice bath. After the addition is complete, place the system at room temperature and continue stirring for 6 hours. Monitor by TLC. After the reaction is complete, add dichloromethane to the system, and then wash with water three times repeatedly. Separate the organic phase, and use anhydrous Na 2 SO 4 Dry and concentrate, and purify by silica gel column chromatography. The mobile phase is dichloromethane:methanol = 30:1 to obtain intermediate 7-7 (62.4 mg, yield: 51%).

[0122] 2. Synthesis method of compound 7

[0123] Intermediate 7-7 (60 mg, 0.12 mmol) and triphenylphosphine (312 mg, 1.2 mmol) were dissolved in 60 mL of dry dichloromethane solvent, and the gas in the reaction system was replaced with argon. The reaction system was placed in an environment of 0 °C, and diisopropyl azodicarboxylate (0.2 mL, 1.2 mmol) was slowly added thereto. The reaction system was allowed to react at room temperature for 3 hours. Monitored by TLC, after the reaction was complete, the reaction system was concentrated under reduced pressure to obtain a yellow oily liquid. The resulting crude product was continuously dissolved in 10 mL of dichloromethane solvent, 0.5 mL of trifluoroacetic acid was added, and the mixture was continuously stirred at room temperature for 3 hours. Monitored by TLC, after the reaction was complete, the reaction solution was diluted with dichloromethane, and saturated sodium bicarbonate solution was added to adjust the pH to 9. The organic phase was washed twice with saturated brine, and then with anhydrous Na 2 SO 4 dried, purified by silica gel column chromatography, and the mobile phase was dichloromethane:methanol = 30:1 to obtain white solid compound 7 (33 mg, total yield in two steps: 71.7%).

[0124] Compound 7, white solid (yield: 71.7%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.58 (s, 1H), 8.56 (s, 1H), 7.82 (s, 1H), 7.60 (d, J = 8.7 Hz, 1H), 6.86 (d, J = 8.8 Hz, 1H), 4.28 (t, J = 8.0 Hz, 2H), 4.16 (d, J = 13.4 Hz, 2H), 3.20 (d, J = 10.7 Hz, 2H), 3.04 (t, J = 6.8 Hz, 1H), 2.69 (t, J = 5.9 Hz, 2H), 1.94 - 1.75 (m, 6H).

[0125] Synthesis of Compound 8 in Example 8

[0126] Compound 8 was synthesized according to the following reaction formula:

[0127]

[0128] 1. Synthesis methods of intermediates 8-1 to 8-4

[0129] Dissolve 3-nitrophenol (100 mg, 0.28 mmol) in 10 mL of N,N-dimethylformamide (DMF) solvent, add tert-butyl 3-bromopropylcarbamate (99.6 mg, 0.42 mmol) and cesium carbonate (181.8 mg, 0.55 mmol), stir overnight at 80 °C, monitor by TLC. After the reaction is complete, cool to room temperature, add 10 mL of water to dilute the reaction solution, then add 10 mL of ethyl acetate for extraction. The organic phase is washed twice with saturated brine, and then with anhydrous Na 2 SO 4 After drying and concentration, purify by silica gel column chromatography with the mobile phase of dichloromethane:methanol = 100:1 to obtain intermediate 8-1 (75.2 mg, yield: 90.7%).

[0130] Dissolve intermediate 8-1 (1.5 g, 5.06 mmol) in 20 mL of methanol solvent, add 10% palladium on carbon (containing 55% water) (269 mg, 0.25 mmol). The mixture is evacuated and the system is replaced with hydrogen, and stirred at room temperature for 2 hours under a hydrogen atmosphere. Monitor by TLC. After the reaction is complete, filter the reaction solution through diatomaceous earth and wash with methanol. The filtrate is dried with anhydrous Na 2 SO 4 After drying and concentration, purify by silica gel column chromatography with the mobile phase of dichloromethane:methanol = 100:1 to obtain intermediate 8-2 (1.21 g, yield: 89.9%).

[0131] Dissolve intermediate 8-2 (1.21 g, 4.54 mmol) in 10 mL of sec-butanol solvent, add 8-bromo-2-chloroquinazoline (0.92 g, 3.78 mmol) and trifluoroacetic acid (144 μL, 1.88 mmol). The mixture is heated to 110 °C under an argon atmosphere and stirred at this temperature for 12 hours. Monitor by TLC. After the reaction is complete, cool the system to room temperature, add 10 mL of saturated aqueous sodium bicarbonate solution. The mixture is extracted with 15 mL of dichloromethane solvent, and the organic phase is dried with anhydrous Na 2 SO 4 After drying and concentration, purify by silica gel column chromatography with the mobile phase of dichloromethane:methanol = 100:1 to obtain intermediate 8-3 (471.5 mg, yield: 22%).

[0132] Dissolve intermediate 8-3 (1.23 g, 3.0 mmol) and 1,4-dioxa-8-azaspiro[4.5]decane (0.46 mL, 3.6 mmol) in a two-necked flask equipped with 10 mL of ultra-dry 1,4-dioxane solvent, then add Pd 2 (dba) 3Palladium (0.27 g, 0.3 mmol), 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)-biphenyl (0.12 g, 0.3 mmol), and sodium tert-butoxide (0.43 g, 4.5 mmol). The reaction system was purged with argon three times repeatedly and the reaction system was sealed. The reaction was carried out overnight at 100 °C. Monitored by TLC, after the reaction was complete, the reaction solution was filtered through diatomaceous earth, and the filter cake was washed repeatedly with dichloromethane. Then 15 mL of water was added for extraction three times, the organic phase was collected, washed with saturated brine, and then with anhydrous Na 2 SO 4 dried, filtered, concentrated under reduced pressure and purified by silica gel column chromatography. The mobile phase was dichloromethane:methanol = 100:1 to obtain intermediate 8-4 (698.5 mg, yield 43.5%).

[0133] 2. Synthetic method of compound 8

[0134] Intermediate 8-4 (112 mg, 0.2 mmol) was dissolved in 5 mL of tetrahydrofuran solvent, 5 mL of 2N HCl solution was slowly added dropwise, and the reaction solution was reacted at 50 °C for 24 hours. Monitored by TLC, after the reaction was complete, the reaction solution was concentrated, saturated sodium bicarbonate solution was added to adjust the pH to 7, then dichloromethane was added for extraction, the organic phase was collected, and anhydrous Na 2 SO 4 dried, filtered, concentrated under reduced pressure to obtain a light green oily liquid. The crude product was continued to be dissolved in 80 mL of anhydrous dichloromethane solvent, a small amount of 4A molecular sieve was added, 2 drops of glacial acetic acid were added and stirred at room temperature for 20 minutes, then sodium triacetoxyborohydride (177.2 mg, 0.83 mmol) was added, and stirring was continued at room temperature for 6 hours. Monitored by TLC, after the reaction was complete, saturated sodium bicarbonate solution was added, the organic phase was separated, the organic phase was washed twice with saturated brine, and then with anhydrous Na 2 SO 4 dried and concentrated, purified by silica gel column chromatography. The mobile phase was dichloromethane:methanol = 20:1 to obtain light green solid compound 8 (15.7 mg, total two-step yield: 20%).

[0135] Compound 8, light green solid, yield 20%; 11H NMR (400 MHz, DMSO-d6) δ 9.88 (s, 1H), 9.25 (d, J = 4.3 Hz, 1H), 8.46 (s, 1H), 7.55–7.46 (m, 1H), 7.31–7.19 (m, 3H), 6.88 (t, J = 6.1 Hz, 1H), 6.77 (t, J = 6.3 Hz, 1H), 4.22–4.11 (m, 2H), 3.75 (s, 2H), 3.57–3.49 (m, 2H), 3.18 (s, 1H), 2.70 (s, 2H), 2.31 - 2.16 (m, 2H), 2.07 (s, 2H), 1.90 (d, J = 11.5 Hz, 2H).

[0136] Example 9 Compound against PDGFRα D842V Kinase IC 50 Test

[0137] Using a FRET (Fluorescence Resonance Energy Transfer)-based Z’-Lyte assay system (detected by fluorescence, enzyme-coupled form, based on the difference in sensitivity of proteolytic cleavage between phosphorylated and non-phosphorylated polypeptides), according to the manufacturer's instructions (Life Technologies, Carlsbad, California, USA), the inhibitory activity of the compound against PDGFRα D842V kinase was tested.

[0138] Enzymatic reaction: Add 5 μL of enzyme-substrate system to a 384-well plate: 30 nM kinase, 5 mM magnesium chloride (MgCl 2 ), 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5, 2 mM manganese chloride (MnCl 2 ), 1 mM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 0.01% Brij-35, 2 μM Tyr 04 peptide substrate. Then transfer 5 μM ATP and different concentration gradients of the test compound using an echo520 ultra-micro liquid pipetting system, shake and mix well, and centrifuge. Incubate at room temperature for 1 hour.

[0139] Detection reaction: Add 2.5 μL of developer to each well, incubate at room temperature for 1 hour, and then add 2.5 μL of termination solution.

[0140] Plate reading and calculation: A multi-label microplate reader (Perkin Elmer EnVision Multimode Plate Reader) was used to detect the fluorescence signal (the fluorescence signal ratio of 445 nm (coumarin) / 520 nm (fluorescein)), and the phosphorylation ratio was calculated. Then, the inhibition rate of each well was calculated through the fully active wells and the control signal wells. Inhibition rate = 100×(1 - phosphorylation ratio of the compound / phosphorylation ratio of the negative control). Graphpad Prism 5.0 (Graphpad Software, Inc) was used to analyze the experimental data and calculate the IC50 value.

[0141] The test results of kinase activity are shown in Table 1.

[0142] Table 1 Inhibitory activity test results of compounds against PDGFRα D842V kinase (IC 50 : nM)

[0143] Compound Number <![CDATA[PDGFRα D842V > Compound 1 23.8 Compound 2 33.1 Compound 3 770.0 Compound 4 605.4 Compound 5 59.4 Compound 6 ＞1000 Compound 7 ＞10000 Compound 8 1497

[0144] As can be seen from the data in Table 1, the disubstituted benzimidazole compounds of the present invention have strong inhibitory activity against PDGFRα D842V kinase, and compound 1 exhibits the optimal activity.

[0145] Example 10 Test for cell proliferation inhibitory activity

[0146] The Ba / F3-PDGFRα D842V cell line used in this experiment, the Ba / F3-PDGFRα D842V / G680R cell line, the Ba / F3-PDGFRα D842V / T674I cell line can be obtained commercially or constructed by the following method. Among them, the mouse Ba / F3 cell line was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ).

[0147] Establish a Ba / F3 cell line stably expressing PDGFRα D842V 、Ba / F3-PDGFRα D842V / G680R 、or Ba / F3-PDGFRα D842V / T674I : Synthesize the wild-type PDGFRα fusion gene FIP1L1-PDGFRα and clone it into the pCDNA3.1(+) vector (Invitrogen, Carlsbad, CA). Use the QuickChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) to introduce point mutations into the wild-type construct. Use Cell Line Kit V Kit (VCA-1003) was used to electrotransfer the pcDNA3.1 plasmid containing the PDGFRα fusion gene into Ba / F3 cells; two weeks after transfection, geneticin (G418) (Merck, Whitehouse Station, NJ) with a final concentration of 800 mg / mL was added for cell screening, and interleukin-3 (IL-3) was withdrawn within the next two weeks to establish a stable polyclonal cell line, and then a stable monoclonal cell line was obtained by limiting dilution. All Ba / F3 stable cell lines were verified by DNA sequencing, protein expression, and anti-proliferation effects on positive drugs. Parental Ba / F3 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 10 ng / mL IL-3, while the PDGFRα fusion-transformed Ba / F3 stable cell line was cultured in the same medium without IL-3. Monoclonal cells with completely correct identification were used for the cell proliferation inhibition activity test of the inhibitor.

[0148] Cell proliferation inhibition activity test: Cells in the logarithmic growth phase were seeded into 96-well plates at 5000 - 8000 cells per well. After 24 hours of seeding, the cells were treated with medium containing the test compound and incubated for an additional 72 hours. After the treatment, 10 μL of Cell Counting Kit-8 cell counting reagent (CCK-8, CK04, Dojindo Laboratories, Kumamoto, Japan) was added to each well and incubated with the cells for 3 hours. Then, the absorbance values at 450 nm and 650 nm were measured using a microplate reader. The absorbance value (A) of each well was calculated as OD450 - OD650, and the cell viability of each well was calculated as V% = (As - Ac) / (Ab - Ac) × 100%. The data are the average of at least three independent experiments. The half-maximal inhibitory concentration (IC 50 ) was calculated using the log(inhibitor) vs. response - variable slope (four parameters) with the medical drawing software (GraphPad Prism 8.0). As is the absorbance value of the well with the test compound, Ac is the absorbance value of the well without cells and the test compound, and Ab is the absorbance value of the well with cells and the vehicle control. The test results are shown in Table 2.

[0149] Table 2 Inhibitory activity test results of compounds on cell proliferation (IC 50 : nM)

[0150] Compound Number <![CDATA[PDGFRα D842V > <![CDATA[PDGFRα D842V / G680R > <![CDATA[PDGFRα D842V / T674I > Compound 1 2.1±1.2 64.1±20.0 27.6±1.9 Compound 2 22.8±4.3 295.9±75.7 Not Tested Compound 3 889.1±158.6 ＞1000 Not Tested Compound 4 374.0±61.2 ＞1000 Not Tested Compound 5 7.2±1.8 121.6±1.1 Not Tested Compound 6 165.5±12.7 ＞1000 Not Tested Compound 7 >10000 Not Tested Not Tested Compound 8 201.3 ＞1000 Not Tested

[0151] As can be seen from the data in Table 2, the disubstituted benzimidazole compounds of the present invention have an inhibitory effect on Ba / F3-PDGFRα D842VThe cell proliferation of the stable strain has strong inhibitory activity, and some compounds have inhibitory activity against PDGFRα D842V / G680R and PDGFRα D842V / T674I The cell proliferation of the stable strain also has strong inhibitory activity, and compound 1 shows the optimal activity among them.

[0152] Example 11 Test the inhibitory activity of compounds on the cell proliferation of gastric cancer cells SNU-16 and KATO III

[0153] Prepare cell suspensions of gastric cancer cells (SNU-16, KATO III) with a density of 80% and count them. Inoculate 100 μl of cell suspension in a 96-well plate and plate at a density of 5000 cells per well. Administer drugs according to the set drug concentration gradient and incubate for 72 h. After the reaction time ends, add the culture medium containing 10% CCK-8. Place the culture plate in the incubator and incubate for 3 h for color development. Finally, measure the absorbance value (OD450nm) at 450 nm with an enzyme-labeled instrument, and process the data according to the formula: cell inhibition rate % = [(control well - experimental well) / (control well - blank well)] × 100%, where the control well is the well containing cells, culture medium, CCK-8 but no compound to be tested, the experimental well is the well containing cells, culture medium, CCK-8, and the compound to be tested, and the blank well is the well containing no cells and the compound to be tested, only culture medium and CCK-8.

[0154] The test results of compound 1 are as Figure 1 shown: This compound has strong anti-proliferative activity against gastric cancer cells, and the half-maximal inhibitory concentration (IC50) values for SNU-16 and KATO III cells are 1.46 ± 0.22 μM and 1.72 ± 0.11 μM, respectively.

[0155] Based on the above experiments, the disubstituted benzimidazole compounds provided by the present invention can efficiently inhibit the activity of PDGFRα D842V mutant kinase, and can also significantly inhibit the growth of Ba / F3-PDGFRα D842V , PDGFRα D842V / G680R and PDGFRα D842V / T674I stable strain cells, thereby effectively inhibiting the proliferation of tumor cells and showing good inhibitory effects on tumors (especially gastric cancer) in humans and other mammals.

[0156] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the following embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0157] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention shall be subject to the appended claims.

Claims

1. A disubstituted benzimidazole compound having a structure as shown in formula I or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, in, R is selected from: 5-7 membered heterocyclic group; R1 is selected from: hydrogen, C1-C6 alkyl; n is selected from: 1, 2, 3, 4, 5, 6.

2. The disubstituted benzimidazole compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1, characterized in that: The disubstituted benzimidazole compound has a structure shown in Formula I-1 or Formula I-2:

3. The disubstituted benzimidazole compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1 or 2, characterized in that: R is selected from: 5-7 membered saturated heterocyclic group.

4. The disubstituted benzimidazole compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1 or 2, characterized in that: R is selected from: a 5-7 membered heterocyclic group in which the heteroatom is N, preferably a 5-7 membered heterocyclic group in which the heteroatom is 1 N, more preferably a 5-membered heterocyclic group in which the heteroatom is 1 N, or a 6-membered heterocyclic group in which the heteroatom is 1 N; Preferably, R is selected from the following groups:

5. The disubstituted benzimidazole compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1 or 2, characterized in that: R1 is selected from the group consisting of: hydrogen, methyl, ethyl, propyl.

6. The disubstituted benzimidazole compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1, characterized in that: The disubstituted benzimidazole compound is selected from the following compounds:

7. Use of the disubstituted benzimidazole compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof in the preparation of a PDGFRα kinase inhibitor; the PDGFRα kinase is preferably a PDGFRα kinase with a D842V mutation, a G680R mutation and / or a T674I mutation.

8. Use of the disubstituted benzimidazole compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof in the preparation of a drug for treating and / or preventing a disease, wherein the disease is caused by abnormal expression of PDGFRα kinase, preferably PDGFRα D842V Mutation, PDGFRα G680R Mutation, PDGFRα D842V / G680R Mutation, PDGFRα T674I Mutation or PDGFRα D842V / T674I Mutation-mediated diseases.

9. Use of the disubstituted benzimidazole compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof in the preparation of a drug for treating and / or preventing tumors; Preferably, the tumor is a tumor caused by abnormal expression of PDGFRα kinase; Preferably, the tumor carries PDGFRα D842V Mutation, PDGFRα G680R Mutation, PDGFRα D842V / G680R Mutation, PDGFRα T674I Mutation or PDGFRα D842V / T674I Mutated tumors; Preferably, the tumor is gastric cancer; Preferably, the tumor is a gastrointestinal stromal tumor, preferably a gastrointestinal stromal tumor caused by abnormal expression of PDGFRα kinase, more preferably a gastrointestinal stromal tumor carrying PDGFRα D842V Mutation, PDGFRα G680R Mutation, PDGFRα D842V / G680R Mutation, PDGFRα T674I Mutation or PDGFRα D842V / T674I Mutation in gastrointestinal stromal tumors.

10. An anti-tumor pharmaceutical composition, characterized in that: The invention is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredients include the disubstituted benzimidazole compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

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