Bicyclic heteroaryl class BRAF negative allosteric modulators and their preparation methods and uses

Abstract

The present invention provides bicyclic heteroaryl BRAF negative allosteric modulators, their preparation methods and uses. The general structural formula of the bicyclic heteroaryl BRAF negative allosteric modulators or their pharmaceutically acceptable salts is shown as Ⅰ. The present invention synthesizes bicyclic heteroaryl BRAF negative allosteric modulators that are potent and have good drug-likeness and target the BRAF kinase, overcoming the drug resistance phenomenon caused by BRAF protein dimerization. Verified by test examples, the compounds synthesized by the present invention have significant cell proliferation inhibitory activity against BRAF WT , BRAF V600E and the p61-BRAF V600E tumor drug-resistant cell lines constructed by lentivirus transient transfection, and at the same time have significant kinase inhibitory activity, showing good application prospects; Ⅰ.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly relates to bicyclic heteroaryl BRAF negative allosteric modulators, their preparation methods and applications. Background Art

[0002] Highly invasive, metastatic and recurrent malignant tumors are common and frequently-occurring diseases that seriously threaten human life and health. According to WHO statistics, there are 30 million new cases of malignant tumor patients every year, becoming the main disease threatening human health. According to research, the RAF kinase and its mediated RAS-RAF-MEK-ERK (MAPK) pathway play a significant role in the occurrence, development and metastasis of malignant tumors. The signal transduction of the MAPK pathway in malignant tumor cells is over-upregulated, and the signal is transmitted from outside the cell into the nucleus through the specific cascade phosphorylation of RAS, RAF, MEK and ERK, triggering the malignant proliferation and differentiation of cells. The BRAF gene is located on chromosome 7q34, encodes serine / threonine protein kinase, and belongs to the RAF kinase family. The BRAF protein is a key molecule in the mitogen-activated protein kinase (MAPK) signaling pathway. The MAPK signaling pathway regulates various cellular response processes such as cell proliferation, differentiation and cycle regulation. After BRAF mutation, the activity of the MAPK signaling pathway increases hundreds of times, leading to abnormal cell proliferation and differentiation, and BRAF mutations exist in various cancer tissues such as malignant melanoma, thyroid cancer, colorectal cancer, non-small cell lung cancer, etc. Among them, the most common one is the T to A mutation at the 1,799th nucleotide of exon 15 of BRAF (resulting in the change of the 600th valine of its encoded protein to glutamate, i.e., BRAF V600 mutation).

[0003] Regarding BRAF V600E mutation, the US Food and Drug Administration (FDA) has approved a variety of drugs such as Vemurafenib and Dabrafenib for tumors with BRAF V600E mutation. This inhibitor blocks the abnormal activation of the MAPK signaling pathway by inhibiting the monomeric BRAF V600E protein, thereby inhibiting the growth and proliferation of tumor cells. Research shows that the formation of wild-type and mutant BRAF dimers will over-activate the phosphorylation of downstream signaling pathways, promoting the occurrence and development of cancer and leading to drug resistance. At present, solving the acquired drug resistance of BRAF V600E inhibitors has become a new research direction.

[0004] Allosteric regulation, as a novel drug design strategy, modulates protein function by acting on allosteric sites of proteins. Research indicates that the FDA-approved multi-target tyrosine kinase inhibitor Ponatinib can act on the allosteric site of BRAF and has good efficacy against drug-resistant strains. Through the analysis of residues by X-ray single crystal diffraction, it was found that the N -methylpiperazine group of Ponatinib extends into the BRAF allosteric pocket and forms hydrogen bond interactions with the backbones of residues H574 and I573. However, Ponatinib has side effects in aspects such as cardiovascular, digestive, hematological systems, and bone marrow suppression during clinical application. Therefore, aiming at the allosteric site of the BRAF protein, it is urgent to develop a new BRAF allosteric regulator to overcome the drug resistance phenomenon caused by BRAF protein dimerization. Summary of the Invention

[0005] In view of this, the present invention provides bicyclic heteroaryl-based BRAF negative allosteric regulators, their preparation methods, and applications.

[0006] The technical solution of the present invention is realized as follows:

[0007] Bicyclic heteroaryl-based BRAF negative allosteric regulators, wherein the regulator is a compound represented by the general structural formula I or a pharmaceutically acceptable salt thereof:

[0008]

[0009] Ⅰ,

[0010] The regulator is:

[0011]

[0012] A preparation method of bicyclic heteroaryl-based BRAF negative allosteric regulators, the specific steps include:

[0013] (1) Under an inert atmosphere, an amino-substituted pyridine / benzene / pyrrole compound and ethyl 4-chloroacetoacetate are added to polyphosphoric acid, and heated and stirred at 120 - 130 °C. The reaction solution is collected and processed to obtain compound i;

[0014] The structural formula of the compound i is: ;

[0015] (2) A phenol / benzenethiol compound and a basic substance are added to an organic solvent, stirred at room temperature, compound i is added, and the reaction is continued after heating to 70 - 120 °C. The reaction solution is collected and processed to obtain compound ii;

[0016] The phenol / phenyl mercaptan compound is any one of 3-nitrophenol, 2-methyl-5-nitrophenol, 2-methyl-4-nitrophenol or 2-methyl-5-nitrobenzenethiol;

[0017] The structural formula of the compound ii is ;

[0018] Under an inert atmosphere, the compound ii is added to a mixed solvent, and then a reducing metal is added for reflux reaction. The reaction solution is collected and treated to obtain the compound iii;

[0019] The structural formula of the compound iii is ;

[0020] The nitrobenzene alkyl bromide compound, the azacycle and the basic substance are added to an organic solvent, and the temperature is raised to 70 - 120 °C for reaction. The reaction solution is collected and treated to obtain the compound iv; or the nitrobenzoic acid compound, the condensing agent and 1-hydroxybenzotriazole are added to an organic solvent and stirred at room temperature, and an amine / alcohol compound is added for continuous reaction. The reaction solution is collected and treated to obtain the compound iv;

[0021] The structural formula of the compound iv is ;

[0022] Under an inert atmosphere, Raney nickel, hydrazine hydrate and the compound iv are added to an organic solvent, and the mixture is refluxed at 50 - 150 °C. The reaction solution is collected and treated to obtain the compound v;

[0023] The structural formula of the compound v is ;

[0024] Under an inert atmosphere, the compound v, triphosgene and triethylamine are added to an organic solvent for reaction and then subjected to crude treatment. Under an inert atmosphere, the compound iii and the crude product are dissolved in an organic solvent for reaction. The reaction solution is collected and treated to obtain the target bicyclic heteroaryl BRAF negative allosteric modulator.

[0025] Furthermore, in step (1), the amino-substituted pyridine / benzene / pyrrole compound is any one of 4-methyl-2-aminopyridine, 2-aminopyridine, 5-fluoro-3-aminopyridine, 2-aminopyrrole, aniline or 3-aminopyridine; the molar ratio of the amino-substituted pyridine / benzene / pyrrole compound to ethyl 4-chloroacetoacetate and polyphosphoric acid is 1:1 - 2:1.

[0026] Furthermore, in step (2), the basic substance is any one of sodium hydroxide, potassium carbonate, potassium hydroxide, sodium carbonate or sodium bicarbonate; the organic solvent is acetonitrile, acetone, ethanol, methanol, dichloromethane, ethyl acetate, chloroform, carbon tetrachloride, toluene, dimethyl sulfoxide,N , N -dimethylformamide and N , N -dimethylacetamide; the molar ratio of the phenol / thiophenol compound, the basic substance, and Compound i is 1-2:3:1.

[0027] Furthermore, in step (3), the mixed solvent is ethanol, acetic acid, and water with a volume ratio of 2:2:1; the reducing metal is iron or zinc; the molar ratio of Compound ii to the reducing metal is 1:5-8;

[0028] In step (4), the nitrobenzenealkyl bromide compound is 4-nitrophenethyl bromide or 4-nitropropyl bromide; the azacyclic ring is methylpiperazine, N -Boc piperazine, hexahydropiperidine, morpholine, or thiomorpholine; the basic substance is at least one of sodium hydroxide, potassium carbonate, potassium hydroxide, sodium carbonate, and sodium bicarbonate; the nitrobenzoic acid compound is any one of p-nitrobenzoic acid, p-nitrophenylacetic acid, 3-nitrophenylacetic acid, 1,4-dioxane-2-carboxylic acid, or p-nitropropionic acid; the condensing agent is any one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, or diisopropylcarbodiimide; the organic solvent is acetonitrile, acetone, ethanol, methanol, dichloromethane, ethyl acetate, chloroform, carbon tetrachloride, toluene, dimethyl sulfoxide, N , N -dimethylformamide and N , N -dimethylacetamide; the molar ratio of the nitrobenzenealkyl bromide compound, the azacyclic ring, and the basic substance is 1:1-2:1; the molar ratio of the nitrobenzoic acid, the condensing agent, 1-hydroxybenzotriazole, and the amine / alcohol compound is 1-1.2:2-2.5:1.5:1.

[0029] Furthermore, in step (5), the organic solvent is methanol, ethanol, dimethyl sulfoxide, N , N -dimethylformamide and N , N -dimethylacetamide; the molar ratio of Raney nickel and Compound iv is 0.5:1;

[0030] In step (6), the organic solvent is acetonitrile, acetone, ethanol, methanol, dichloromethane, ethyl acetate, chloroform, carbon tetrachloride, toluene, dimethyl sulfoxide, N , N -dimethylformamide and N , Nat least one of N, N-dimethylacetamide; the molar ratio of the compound v, triphosgene and triethylamine is 1:1:1.5 - 2; the molar ratio of the compound iii and the crude product is 1:1 - 1.5.

[0031] Use of a bicyclic heteroaryl class BRAF negative allosteric modulator in the preparation of an anti-tumor drug.

[0032] Furthermore, the anti-tumor drug comprises the structural general formula of the bicyclic heteroaryl class BRAF negative allosteric modulator or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable excipient.

[0033] Furthermore, the anti-tumor drug intervenes by targeting wild-type BRAF WT and its mutant BRAF V600E kinase, and can treat and / or prevent diseases related to thyroid tumors, melanoma, colorectal cancer, and non-small cell lung cancer.

[0034] Compared with the prior art, the beneficial effects of the present invention are:

[0035] Through computer virtual screening, the present invention obtained 19 compound parent nuclei from nearly 1.5 million compounds in the Specs and ChemDiv databases, and simultaneously synthesized a bicyclic heteroaryl class BRAF negative allosteric modulator that is potent and has good drug-likeness and targets BRAF kinase, overcoming the drug resistance phenomenon caused by BRAF protein dimerization. By performing computer-aided molecular docking on Ponatinib and the allosteric modulator designed in the present invention respectively: the allosteric modulator designed in the present invention has a similar regulatory effect on the allosteric site hotspot residues (I573, H574) as Ponatinib. Verified by the experimental examples, the compounds synthesized in the present invention have significant cell proliferation inhibitory activity against BRAF WT , BRAF V600E and p61-BRAF V600E tumor cell lines, and at the same time have significant kinase inhibitory activity, with good application prospects. The bicyclic heteroaryl class BRAF negative allosteric modulator of the present invention can be applied in the prevention or / and treatment of diseases related to thyroid, melanoma, colorectal cancer, and non-small cell lung cancer. Detailed implementation manners

[0036] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0037] The experimental methods used in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0038] The materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial channels unless otherwise specified.

[0039] The present invention also includes prodrugs of the derivatives of the present invention. According to the present invention, the prodrugs are derivatives of general formula I, which may be active or even inactive themselves, but after administration, they are converted into the corresponding bioactive forms under physiological conditions (e.g., by metabolism, solvolysis or other routes).

[0040] The present invention also includes pharmaceutical compositions which contain the bicyclic heterocyclic derivatives of general formula I or their pharmaceutically acceptable salts as active ingredients, and pharmaceutically acceptable excipients. The said pharmaceutically acceptable excipients refer to any diluents, adjuvants and / or carriers that can be used in the pharmaceutical field. The derivatives of the present invention can be used in combination with other active ingredients as long as no other adverse effects are produced, such as allergic reactions.

[0041] The pharmaceutical compositions of the present invention can be formulated into several dosage forms, which contain some excipients commonly used in the pharmaceutical field: for example, oral preparations (such as tablets, capsules, solutions or suspensions); injectable preparations (such as injectable solutions or suspensions, or injectable dry powders: they can be immediately used after adding water for injection before injection); topical preparations (such as ointments, gels or solutions).

[0042] The carriers used in the pharmaceutical compositions of the present invention are common types available in the pharmaceutical field, including binders, fillers, disintegrants, lubricants in tablets; preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, osmotic pressure regulators, coloring agents, etc. in liquid preparations.

[0043] The definitions of the various terms used in this specification are as described below.

[0044] The term "aromatic ring" means a 5- to 7-membered monocyclic or bicyclic aromatic ring which may contain heteroatoms selected from the group consisting of 1 to 4 oxygen atoms, sulfur atoms and nitrogen atoms in addition to carbon atoms. As specific examples, the above-mentioned aromatic hydrocarbons and heteroaromatic hydrocarbons can be cited. More specifically, benzene, pyridine, piperidine, pyrrole and other aromatic hydrocarbons can be cited.

[0045] The term "alkyl" means a straight-chain or branched-chain saturated hydrocarbon group having 1 to 6 carbon atoms (C1-C6). As specific examples, methyl, ethyl, propyl, isopropyl, butyl, and their various branched isomers can be cited.

[0046] The amino-substituted pyridine / benzene / pyrrole compounds of the present invention are any one of 4-methyl-2-aminopyridine, 2-aminopyridine, 5-fluoro-3-aminopyridine, 2-aminopyrrole, aniline or 3-aminopyridine.

[0047] The phenol / benzenethiol compounds of the present invention are any one of 3-nitrophenol, 2-methyl-5-nitrophenol, 2-methyl-4-nitrophenol or 2-methyl-5-nitrobenzenethiol.

[0048] The basic substance of the present invention is any one of sodium hydroxide, potassium carbonate, potassium hydroxide, sodium carbonate or sodium bicarbonate.

[0049] The organic solvents of the present invention are acetonitrile, acetone, ethanol, methanol, dichloromethane, ethyl acetate, chloroform, carbon tetrachloride, toluene, dimethyl sulfoxide, N , N -dimethylformamide and N , N -dimethylacetamide, at least one of them.

[0050] The nitrobenzylalkyl bromide compounds of the present invention are 4-nitrophenethyl bromide or 4-nitropropyl bromide.

[0051] The azacycle of the present invention is any one of methylpiperazine, N -Boc piperazine, hexahydropiperidine, morpholine or thiomorpholine.

[0052] Example 1

[0053] Preparation method of bicyclic heteroaryl BRAF negative allosteric modulator, the specific steps include:

[0054] (1) Under an inert atmosphere, polyphosphoric acid (10 mmol) was preheated to a stirrable state at 120 °C, and an amino-substituted pyridine / benzene / pyrrole compound (10 mmol) was added and stirred until completely dissolved. Ethyl 4-chloroacetoacetate (14 mmol) was added dropwise to the system, and the reaction progress was monitored by TLC. After the reaction was completed, ice water was added to the system, and saturated sodium bicarbonate solution was added dropwise to adjust the pH of the reaction solution to 6.5 ± 0.5. The mixture was filtered and the filter cake was washed, and then dried in vacuo to obtain compound i;

[0055] The structural formula of compound i is: ;

[0056] (2) A phenol / benzenethiol compound (5.5 mmol) and a basic substance (15 mmol) were added to an organic solvent (25 mL), and the mixture was stirred at room temperature for 0.5 - 1 h. Then compound i (5 mmol) was added, and the temperature was raised to 75 °C and the reaction was continued. The reaction progress was monitored by TLC during the reaction. After the reaction was completed, 100 mL of water was added to the cooled reaction solution, and the mixture was filtered, and the filter cake was washed with water and dried in vacuo to obtain compound ii;

[0057] The structural formula of compound ii is ;

[0058] Under an inert atmosphere, compound ii (2 mmol) was added to 10 mL of a mixed solvent (ethanol, acetic acid, and water with a volume ratio of 2:2:1), and then iron powder (12 mmol) was added for reflux reaction at 80 °C. The reaction progress was monitored by TLC during the reaction. After the reaction was completed, the iron powder was filtered off, the organic layer was concentrated under reduced pressure, purified by column chromatography (the eluent was dichloromethane, methanol, and triethylamine with a volume ratio of 30:1:0.01, gradient elution), the eluate containing the target product was collected, and after distillation under reduced pressure and vacuum drying, compound iii was obtained;

[0059] The structural formula of compound iii is ;

[0060] (4) A nitrobenzene alkyl bromide compound (2 mmol), an azacycle (2.4 mmol), and a basic substance (2 mmol) were added to 3 mL of an organic solvent, and the temperature was raised to 100 °C for reaction; or under an inert atmosphere, a nitrobenzoic acid compound (1 mmol), a condensing agent (2 mmol), and 1-hydroxybenzotriazole (1.5 mmol) were dissolved in 25 mL of an organic solvent, and after stirring for 2 h, an amine / alcohol compound (1 mmol) was added. The reaction progress was monitored by TLC during the reaction. After the reaction was completed, extraction was carried out with an ethyl acetate and water system, and separation and purification were carried out by silica gel column chromatography (the eluent was an ethyl acetate:methanol system with a volume ratio of 10:1). The eluate containing the target product was collected, and after distillation under reduced pressure and vacuum drying, compound iv was obtained;

[0061] The structural formula of compound iv is ;

[0062] Under an inert atmosphere, Raney nickel (0.5 mmol), 80% hydrazine hydrate (1 mL), and compound iv (1 mmol) were added to 5 mL of an organic solvent, and reflux reaction was carried out at 100 °C. The reaction progress was monitored by TLC during the reaction. After the reaction was completed, the insoluble matter was filtered off, the solvent was removed from the filtrate by distillation under reduced pressure, purified by silica gel column chromatography (the eluent was dichloromethane:methanol:triethylamine with a volume ratio of 15:1:0.01), the eluate containing the target product was collected, and after distillation under reduced pressure and vacuum drying, compound v was obtained;

[0063] The structural formula of compound v is ;

[0064] (6) Under an inert atmosphere, a solution of compound v (1 mmol) in dichloromethane (5 mL) was slowly added dropwise to a solution of triphosgene (1 mmol) and triethylamine (2 mmol) in dichloromethane (5 mL) at 0 °C. After the addition was complete, the mixture was transferred to room temperature and stirred continuously. After monitoring that the raw materials were completely converted, the solvent was removed by concentration under reduced pressure. Under an inert atmosphere, compound iii (1 mmol) and the crude product (1 mmol) were dissolved in an organic solvent for reaction to obtain a bicyclic heteroaryl class BRAF negative allosteric modulator.

[0065] Example 2

[0066] Based on the preparation method of the bicyclic heteroaryl class BRAF negative allosteric modulator in Example 1, compounds I-1 to I-17 were prepared, as specifically shown in Table 1-2.

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] Test Example

[0074] 1. Cell Counting Kit-8 (CCK-8) detection experiment

[0075] (1) Experimental principle: The CCK-8 reagent contains a water-soluble tetrazolium salt WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt), which can be reduced by dehydrogenases in the mitochondria of cells to form a highly water-soluble orange-yellow formazan dye product that is soluble in the culture medium. The intensity of the color is proportional to cell proliferation and inversely proportional to cell toxicity. The OD (absorbance) value is measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Therefore, this property can be used to directly analyze cell proliferation and toxicity.

[0076] (2) Experimental method: Tumor cell lines of BRAF WT and BRAF V600E and p61-BRAF V600E were selected to evaluate the in vitro proliferation inhibitory ability of the compounds. Among them, the human malignant melanoma cell line A375 cells belong to BRAF WTType cells; Human thyroid cancer cell line 8505C cells belong to BRAF V600E mutant cells; A lentivirus transfection system was used to construct an overexpression splicing variant p61 - BRAF V600E system for constructing a Vemurafenib - resistant cell model.

[0077] Dispense 100 μL of cell suspension (3.0×10 4 cells / mL) into a 96 - well plate. Place the plate in a 5% CO2, 37°C incubator for pre - culture. After 24 h, add 200 μL of medium containing gradient drug concentrations (0 - 30 μM) to the designated wells, where the positive drug is Vemurafenib, and then place it in a 5% CO2, 37°C incubator for 24 h. The CCK - 8 method was used to measure cell cytotoxicity. The CCK - 8 detection method is as follows: Remove the medium containing the drug, add 100 μL of serum - free medium containing 10% CCK - 8 to each well, place it in a 5% CO2, 37°C incubator for 2 h, and measure the absorbance at 450 nm using a microplate reader.

[0078] (3) Data processing: Cell survival rate = [(As - Ab) / (Ac - Ab)]×100%

[0079] Note: As is the drug - treated group (medium containing cells, CCK - 8, and the test drug);

[0080] Ac is the control group (medium containing cells, CCK - 8, and no test drug);

[0081] Ab is the blank group (medium without cells and test drug, CCK - 8).

[0082] Cell IC 50 The results are shown in Table 3.

[0083] Table 3

[0084]

[0085] As can be seen from Table 3, the compounds of the present invention showed significant activities in inhibiting cell proliferation in all three cell lines. In the 8505C and A375 cell lines, the inhibitory activities of Compounds I - 9 and I - 11 were better than those of the positive drug Vemurafenib. In the resistant cell line p61 - 8505C, the inhibitory activity of the positive drug Vemurafenib decreased significantly, while the inhibitory activities of the tested compounds decreased slightly, but Compound I - 9 still showed the best inhibitory activity.

[0086] 2. Kinase activity detection experiment (ADP - Glo TM Kinase Assay)

[0087] (1) Experimental principle: ADP-Glo TM The Kinase Assay measures the activity of ADP-generating enzymes by quantifying the ADP produced during the kinase reaction. The detection process is carried out in two steps: First, after the kinase reaction, an equal volume of ADP-GloTM reagent is added to terminate the kinase reaction and consume the remaining ATP. Then, the kinase detection reagent is added to convert ADP to ATP, and the newly synthesized ATP is detected through the luciferase / luciferin reaction. A luminometer is used to detect the generated light signal, and through the ATP-ADP conversion curve, the relationship between the luminescence signal and the ADP concentration is determined. The generated luminescence signal is proportional to the generated ADP concentration and is correlated with the enzyme activity.

[0088] (2) Experimental method: Based on a 384-well plate, the ratio of kinase: ADP-GloTM Regent: Kinase Detection Regent is particularly preferably 10:10:20. The drug is serially diluted with 1× kinase reaction buffer, and 1 μL of the serially diluted drug is added to the designated wells. Vemurafenib is used as the positive drug, and 2.5× the optimal concentration of the enzyme is added to all wells. The mixture is incubated at 37 °C for 15 min, then 2× the optimal concentration of ATP and 2× the optimal concentration of the substrate are added and incubated statically for 60 min. After the kinase or ATP has fully reacted, ADP-GloTM Regent is added and incubated statically for 60 min to terminate the kinase reaction and consume the remaining ATP. Then, 20 μL of Kinase Detection Regent is added and incubated statically for 60 min to convert ADP to ATP, and the RU luminescence value is detected using a microplate reader.

[0089] (3) Data processing: Kinase inhibition rate = [1 - (As - Ab) / (Ac - Ab)] × 100%

[0090] Note: As is the drug-administered group (containing ATP, substrate, kinase, kinase reaction buffer, and the drug to be tested); Ac is the control group (containing ATP, substrate, kinase, kinase reaction buffer, without the drug to be tested);

[0091] Ab is the blank group (only containing ATP, substrate, kinase reaction buffer).

[0092] Kinase IC 50 The results are shown in Table 4.

[0093] Table 4

[0094]

[0095] As can be seen from Table 4, the activity of compound I-9 synthesized in the present invention is significantly better than that of the positive drug.

[0096] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A bicyclic heteroaryl BRAF negative allosteric modulator, characterized in that: The regulator is a compound represented by the general structural formula I or a pharmaceutically acceptable salt thereof: Ⅰ， The regulator is:

2. The method for preparing the bicyclic heteroaryl BRAF negative allosteric modulator according to claim 1, characterized in that: The specific steps include: (1) Under an inert atmosphere, an amino-substituted pyridine / benzene / pyrrole compound and ethyl 4-chloroacetoacetate are added to polyphosphoric acid, heated at 120-130° C. with stirring, and the reaction solution is collected and treated to obtain compound i; The structural formula of the compound i is: ; (2) adding a phenol / thiophenol compound and an alkaline substance to an organic solvent, stirring at room temperature, adding compound i, heating to 70-120° C. and continuing the reaction, collecting and treating the reaction solution to obtain compound ii; The phenol / thiophenol compound is any one of 3-nitrophenol, 2-methyl-5-nitrophenol, 2-methyl-4-nitrophenol or 2-methyl-5-nitrothiophenol; The structural formula of the compound ii is ; (3) Under an inert atmosphere, compound ii is added to a mixed solvent, and then a reducing metal is added to carry out a reflux reaction, and the reaction solution is collected and treated to obtain compound iii; The structural formula of the compound iii is ; (4) adding a nitrobenzene alkyl bromide compound, a nitrogen heterocycle and an alkaline substance to an organic solvent, heating the temperature to 70-120°C for reaction, collecting and treating the reaction liquid to obtain compound iv; or adding a nitrobenzene carboxylic acid compound, a condensing agent and 1-hydroxybenzotriazole to an organic solvent, stirring at room temperature, adding an amine / alcohol compound to continue the reaction, collecting and treating the reaction liquid to obtain compound iv; The structural formula of the compound iv is ; (5) Under an inert atmosphere, adding Raney nickel, hydrazine hydrate and compound iv into an organic solvent, reflux at 50-150° C., collecting and treating the reaction solution to obtain compound v; The structural formula of the compound v is ; (6) Under an inert atmosphere, compound v, triphosgene and triethylamine are added to an organic solvent for reaction and then crudely treated. Under an inert atmosphere, compound iii and the crude product are dissolved in an organic solvent for reaction, and the reaction liquid is collected and treated to obtain the target bicyclic heteroaryl BRAF negative allosteric modulator.

3. The method for preparing the bicyclic heteroaryl BRAF negative allosteric modulator according to claim 2, characterized in that: In step (1), the amino-substituted pyridine / benzene / pyrrole compound is any one of 4-methyl-2-aminopyridine, 2-aminopyridine, 5-fluoro-3-aminopyridine, 2-aminopyrrole, aniline or 3-aminopyridine; and the molar ratio of the amino-substituted pyridine / benzene / pyrrole compound to ethyl 4-chloroacetoacetate and polyphosphoric acid is 1:1-2:

1.

4. The method for preparing the bicyclic heteroaryl BRAF negative allosteric modulator according to claim 2, characterized in that: In step (2), the alkaline substance is any one of sodium hydroxide, potassium carbonate, potassium hydroxide, sodium carbonate or sodium bicarbonate; the organic solvent is acetonitrile, acetone, ethanol, methanol, dichloromethane, ethyl acetate, chloroform, carbon tetrachloride, toluene, dimethyl sulfoxide, N , N -dimethylformamide and N , N - at least one of dimethylacetamide; the molar ratio of the phenol / thiophenol compound to the alkaline substance, compound i is 1-2:3:

1.

5. The method for preparing the bicyclic heteroaryl BRAF negative allosteric modulator according to claim 2, characterized in that: In step (3), the mixed solvent is ethanol, acetic acid and water in a volume ratio of 2:2:1; the reducing metal is iron or zinc; the molar ratio of the compound ii to the reducing metal is 1:5-8; In step (4), the nitrobenzene alkyl bromide compound is 4-nitrobenzene ethyl bromide or 4-nitrobenzene propyl bromide; the nitrogen heterocycle is methyl piperazine, N -Boc piperazine, hexahydropiperidine, morpholine or thiomorpholine; the alkaline substance is at least one of sodium hydroxide, potassium carbonate, potassium hydroxide, sodium carbonate and sodium bicarbonate; the nitrobenzene carboxylic acid compound is any one of p-nitrobenzoic acid, p-nitrophenylacetic acid, 3-nitrophenylacetic acid, 1,4-dioxane-2-carboxylic acid or p-nitrophenylpropionic acid; the condensing agent is any one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide or diisopropylcarbodiimide; the organic solvent is acetonitrile, acetone, ethanol, methanol, dichloromethane, ethyl acetate, chloroform, carbon tetrachloride, toluene, dimethyl sulfoxide, N , N -Dimethylformamide and N , N -dimethylacetamide; the molar ratio of the nitrobenzene alkyl bromide compound to the nitrogen heterocycle and the alkaline substance is 1:1-2:1; the molar ratio of the nitrobenzene carboxylic acid to the condensation agent, 1-hydroxybenzotriazole, and the amine / alcohol compound is 1-1.2:2-2.5:1.5:

1.

6. The method for preparing the bicyclic heteroaryl BRAF negative allosteric modulator according to claim 2, characterized in that: In step (5), the organic solvent is methanol, ethanol, dimethyl sulfoxide, N , N -Dimethylformamide and N , N - at least one of dimethylacetamide; the molar ratio of Raney nickel to compound iv is 0.5:1; In step (6), the organic solvent is acetonitrile, acetone, ethanol, methanol, dichloromethane, ethyl acetate, chloroform, carbon tetrachloride, toluene, dimethyl sulfoxide, N , N -dimethylformamide and N , N -dimethylacetamide; the molar ratio of the compound v, triphosgene and triethylamine is 1:1:1.5-2; the molar ratio of the compound iii and the crude product is 1:1-1.

5.

7. Use of the bicyclic heteroaryl BRAF negative allosteric modulator according to claim 1 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that The anti-tumor drug comprises the structural general formula of the bicyclic heteroaryl BRAF negative allosteric regulator or a pharmaceutically acceptable salt thereof as an active component and a pharmaceutically acceptable excipient.

9. The use according to claim 7, characterized in that The anti-tumor drug is by targeting wild-type BRAF WT BRAF and its mutant V600E Intervention of kinases can treat and / or prevent diseases including thyroid tumors, melanoma, colorectal cancer, and non-small cell lung cancer.

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