Bicyclic heteroaryl BRAF negative allosteric modulator as well as preparation method and application thereof

Abstract

The invention provides a bicyclic heteroaryl BRAF negative allosteric modulator and a preparation method and application thereof.The structural general formula of the bicyclic heteroaryl BRAF negative allosteric modulator or pharmaceutically acceptable salt of the bicyclic heteroaryl BRAF negative allosteric modulator is shown as I. The bicyclic heteroaryl BRAF negative allosteric modulator targeting BRAF kinase is high in effect and good in druggability and can be used for preparing BRAF kinase. The drug resistance phenomenon caused by BRAF protein dimerization is overcome. A test example verifies that the compound synthesized by the invention shows remarkable cell proliferation inhibition activity on BRAFWT, BRAFV600E and a p61-BRAFV600E tumor drug-resistant cell line constructed by slow virus transient transfer, has remarkable kinase inhibition activity, and has a good application prospect; and # imgabs0 # I is adopted.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a bicyclic heteroaryl BRAF negative allosteric regulator and a preparation method and application thereof. 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 tumors each year, making it a major disease threatening human health. According to research, 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 MAPK pathway signal transduction in malignant tumor cells is excessively upregulated, and the signal is transmitted from the extracellular space to the cell 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, encoding a serine / threonine protein kinase, and belongs to the RAF kinase family. BRAF protein is a key molecule in the mitogen-activated protein kinase (MAPK) signaling pathway. The MAPK signaling pathway regulates a variety of cell response processes such as cell proliferation, differentiation and cycle regulation. After BRAF mutation, the activity of MAPK signaling pathway increases hundreds of times, leading to abnormal cell proliferation and differentiation. BRAF mutation is present in a variety of cancer tissues, including malignant melanoma, thyroid cancer, colorectal cancer, non-small cell lung cancer, etc. Among them, the mutation of T to A at the 1,799th nucleotide of exon 15 of BRAF (causing the 600th valine of its encoded protein to become glutamic acid, i.e. BRAF V600 mutations) are the most common.

[0003] Targeting BRAF V600E The U.S. Food and Drug Administration (FDA) has approved a variety of drugs such as Vemurafenib and Dabrafenib for BRAF mutations. V600E This inhibitor inhibits the monomeric BRAF V600E The protein blocks the abnormal activation of the MAPK signaling pathway, thereby inhibiting the growth and proliferation of tumor cells. Studies have shown that the formation of wild-type and mutant BRAF dimers will over-activate the phosphorylation of downstream signaling pathways, promote the development of cancer and lead to drug resistance. V600E Acquired resistance to inhibitors has become a new research direction.

[0004] Allosteric regulation is a new drug design strategy that regulates protein function by acting on protein allosteric sites. Studies have shown that the FDA-approved multi-target tyrosine kinase inhibitor Ponatinib has good efficacy on drug-resistant strains by acting on BRAF allosteric sites. Through X-ray single crystal diffraction analysis of residues, it was found that Ponatinib N The -methylpiperazine group extends into the BRAF allosteric pocket (allosteric site) to form hydrogen bond interactions with residues H574 and I573 skeletons. However, Ponatinib has side effects in cardiovascular, digestive, blood and bone marrow suppression during clinical use. Therefore, it is urgent to develop new BRAF allosteric modulators targeting the BRAF protein allosteric site to overcome the drug resistance caused by BRAF protein dimerization. Summary of the invention

[0005] In view of this, the present invention proposes a bicyclic heteroaryl BRAF negative allosteric modulator and a preparation method and application thereof.

[0006] The technical solution of the present invention is achieved in this way: Bicyclic heteroaryl BRAF negative allosteric modulators, the general structural formula or its pharmaceutically acceptable salt is shown as Ⅰ:

[0007] Ⅰ, Wherein, ring A is a differently substituted aromatic heterocycle, including a benzene ring, a pyridine ring, a pyrrole ring or a quinolinone ring; Ring B is a monocyclic aromatic ring with different substitutions, including a benzene ring, a pyridine ring or a furan ring; Ring C is any one of a differently substituted furan ring, a piperazine ring, a pyridine ring, a piperidine ring, a morpholine ring, a thiomorpholine ring or an amine; X 1 is a nitrogen atom or a carbon atom; Z 1 is an oxygen atom, a sulfur atom or CH; Z 2 , Z 3 are the same or different and are each independently selected from an oxygen atom, a nitrogen atom or CH; Y 1 is a differently substituted alkyl group, -CONH-, -NHCO-, -CH=CH- or COO-; R 1 is a hydrogen atom, a halogen or an alkyl group which may be substituted.

[0008] Furthermore, the general structural formula or its pharmaceutically acceptable salts include: .

[0010] A method for preparing a bicyclic heteroaryl BRAF negative allosteric modulator, comprising the following specific steps: (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 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 mixture to 70-120°C for reaction, collecting and treating the reaction solution to obtain compound iv; or adding a nitrobenzene alkyl 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 solution 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.

[0011] 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; and the molar ratio of the amino-substituted pyridine / benzene / pyrrole compound to ethyl 4-chloroacetoacetate and polyphosphoric acid is 1:1-2:1.

[0012] Furthermore, in step (2), the phenol / thiophenol compound is any one of 3-nitrophenol, 2-methyl-5-nitrophenol, 2-methyl-4-nitrophenol, 2-methyl-5-nitrothiophenol, 2-methyl-5-benzyloxyphenol or 2-methyl-4-benzyloxyphenol; 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.

[0013] Furthermore, 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; and 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, thiomorpholine, methylamine or dimethylamine; 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 - at least one of 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 nitrogen heterocycle is 1-1.2:2-2.5:1.5:1.

[0014] Furthermore, 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. Application of bicyclic heteroaryl BRAF negative allosteric modulators in the preparation of anti-tumor drugs.

[0015] Furthermore, the anti-tumor drug comprises the general structural formula of the bicyclic heteroaryl BRAF negative allosteric modulator or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable excipient.

[0016] Furthermore, the anti-tumor drug is a drug that targets wild-type BRAF. WT BRAF and its mutant V600E Intervention of kinases can treat and / or prevent diseases including thyroid, melanoma, colorectal cancer, and non-small cell lung cancer.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention obtains 19 compound core structures from nearly 1.5 million compounds in the Specs and ChemDiv databases through computer virtual screening, and synthesizes a potent and druggable bicyclic heteroaryl BRAF negative allosteric modulator targeting BRAF kinase, overcoming the drug resistance caused by BRAF protein dimerization. By performing computer molecular matching on Ponatinib and the allosteric modulator designed by the present invention, the allosteric modulator designed by the present invention and Ponatinib have similar regulatory effects on the hotspot residues (I573, H574) of the allosteric site. The experimental examples verify that the compounds synthesized by the present invention have a strong effect on BRAF. WT BRAF V600E and p61-BRAF V600E The tumor cell line exhibits significant activity in inhibiting cell proliferation and has significant kinase inhibitory activity, and has good application prospects. The bicyclic heteroaryl BRAF negative allosteric regulator of the present invention can be used in the prevention and / or treatment of diseases related to thyroid, melanoma, colorectal cancer, and non-small cell lung cancer. DETAILED DESCRIPTION

[0018] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0019] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0020] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0021] The present invention also includes prodrugs of the derivatives according to the present invention. According to the present invention, prodrugs are derivatives of the general formula I which may be active or even inactive themselves, but after administration, are converted into the corresponding biologically active form under physiological conditions (e.g. by metabolism, solvolysis or other pathways).

[0022] The present invention also includes a pharmaceutical composition comprising a bicyclic heterocyclic derivative of general formula I or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient refers to any diluent, adjuvant and / or carrier that can be used in the pharmaceutical field. The derivative of the present invention can be used in combination with other active ingredients as long as other adverse effects, such as allergic reactions, are produced.

[0023] The pharmaceutical composition 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: adding injection water before injection can be used immediately); local preparations (such as ointments, gels or solutions).

[0024] The carriers used in the pharmaceutical composition of the present invention are common types available in the pharmaceutical field, including binders, fillers, disintegrants, and lubricants in tablets; preservatives, antioxidants, flavoring agents, aromatics, solubilizers, emulsifiers, osmotic pressure regulators, colorants, etc. in liquid preparations.

[0025] The definitions of the terms used in this specification are as follows.

[0026] The term "aromatic ring" refers to a 5-7 membered monocyclic or bicyclic aromatic ring which, in addition to carbon atoms, may contain 1-4 heteroatoms selected from the group consisting of oxygen atoms, sulfur atoms and nitrogen atoms. Specific examples include the above-mentioned aromatic hydrocarbons and heteroaromatic hydrocarbons. More specifically, aromatic hydrocarbons such as benzene, pyridine, piperidine and pyridine may be mentioned.

[0027] The term "alkyl" refers to a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms (C1-C6), and specific examples thereof include methyl, ethyl, propyl, isopropyl, butyl, and various branched isomers thereof.

[0028] The amino-substituted pyridine / benzene / pyrrole compound of the present invention is any one of 4-methyl-2-aminopyridine, 2-aminopyridine, 5-fluoro-3-aminopyridine, 2-aminopyrrole, aniline or 3-aminopyridine.

[0029] The phenol / thiophenol compound of the present invention is any one of 3-nitrophenol, 2-methyl-5-nitrophenol, 2-methyl-4-nitrophenol, 2-methyl-5-nitrothiophenol, 2-methyl-5-benzyloxyphenol or 2-methyl-4-benzyloxyphenol.

[0030] The alkaline substance of the present invention is any one of sodium hydroxide, potassium carbonate, potassium hydroxide, sodium carbonate or sodium bicarbonate.

[0031] The organic solvent of the present invention 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.

[0032] The nitrobenzene alkyl bromide compound of the present invention is 4-nitrobenzene ethyl bromide or 4-nitrobenzene propyl bromide. The nitrogen heterocycle of the present invention is methylpiperazine, N -Boc any one of piperazine, piperidine, morpholine, thiomorpholine, methylamine or dimethylamine.

[0033] Example 1 A method for preparing a bicyclic heteroaryl BRAF negative allosteric modulator, comprising the following specific steps: (1) Under an inert atmosphere, polyphosphoric acid (10 mmol) was preheated at 120°C until it could be stirred, 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 a saturated sodium bicarbonate solution was added dropwise to adjust the pH of the reaction solution to 6.5±0.5. The filter cake was filtered and rinsed, and then dried in vacuo to obtain compound i. The structural formula of compound i is: ; (2) Add phenol / thiophenol compound (5.5 mmol) and alkaline substance (15 mmol) to an organic solvent (25 mL), stir at room temperature for 0.5-1 h, add compound i (5 mmol), heat to 75°C and continue the reaction, and monitor the reaction progress by TLC during the reaction. After the reaction is completed, add 100 mL of water to the cooled reaction solution, filter, wash the filter cake with water, and vacuum dry to obtain compound ii; The structural formula of compound ii is ; (3) Under an inert atmosphere, compound ii (2 mmol) was added to 10 mL of a mixed solvent (ethanol, acetic acid and water in a volume ratio of 2:2:1), followed by the addition of iron powder (12 mmol) for a 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 out, the organic layer was concentrated under reduced pressure, and purified by column chromatography (eluent was dichloromethane, methanol and triethylamine in a volume ratio of 30:1:0.01, gradient elution), the eluate containing the target product was collected, distilled under reduced pressure, and dried in vacuo to obtain compound iii; The structural formula of compound iii is ; (4) Add nitrobenzene alkyl bromide compounds (2 mmol), nitrogen heterocycles (2.4 mmol) and alkaline substances (2 mmol) to 3 mL of organic solvent and heat to 100 °C for reaction; or under an inert atmosphere, dissolve nitrobenzene carboxylic acid compounds (1 mmol), condensing agents (2 mmol) and 1-hydroxybenzotriazole (1.5 mmol) in 25 mL of organic solvent, stir for 2 h and then add amine / alcohol compounds (1 mmol). During the reaction, monitor the reaction progress by TLC. After the reaction is completed, extract with ethyl acetate and water system, separate and purify with silica gel column chromatography (eluent is ethyl acetate: methanol system with a volume ratio of 10:1), collect the eluate containing the target product, distill under reduced pressure, and dry in vacuo to obtain compound iv; The structural formula of compound iv is ; (5) Under an inert atmosphere, add Raney nickel (0.5 mmol), 80% hydrazine hydrate (1 mL) and compound iv (1 mmol) to 5 mL of organic solvent, reflux at 100°C, and monitor the reaction progress by TLC. After the reaction is completed, filter to remove insoluble matter, remove the solvent from the filtrate by vacuum distillation, and purify by silica gel column chromatography (eluent: dichloromethane: methanol: triethylamine in a volume ratio of 15:1:0.01). Collect the eluate containing the target product, vacuum distill it, and vacuum dry it to obtain compound v; The structural formula of compound v is ; (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 the reaction was monitored and the conversion of the raw materials was complete, the solvent was removed by concentrating 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 BRAF negative allosteric modulator.

[0034] Example 2 Based on the preparation method of the bicyclic heteroaryl BRAF negative allosteric modulator in Example 1, compounds I-1 to I-30 were prepared, as shown in Table 1-2.

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045] Test example 1. Cell Counting Kit-8 (CCK-8) assay (1) Experimental principle: CCK-8 reagent contains water-soluble tetrazolium salt WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazolyl monosodium salt), which can be reduced by dehydrogenase in cell mitochondria to generate highly water-soluble orange-yellow formazan dye, which is soluble in culture medium. The depth of 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 reader. Therefore, this characteristic can be used to directly analyze cell proliferation and toxicity.

[0046] (2) Experimental method: BRAF WT BRAF V600E and p61-BRAF V600E Tumor cell lines were used to evaluate the in vitro proliferation inhibition ability of the compounds. Among them, human malignant melanoma A375 cells belong to BRAF WT type cells; human thyroid cancer cell 8505C cells belong to BRAF V600E Mutant cells; Lentivirus-transfected cells were constructed to overexpress spliceosome p61-BRAF V600E System is used to construct a Vemurafenib-resistant cell model.

[0047] 100 μL of cell suspension (3.0×10 4 / mL) were distributed in a 96-well plate, and the plate was placed in a 5% CO2, 37°C incubator for pre-incubation. After 24 hours, 200 μL of culture medium containing gradient drug concentrations (0-30 μM) was added to the designated wells, where the positive drug was Vemurafenib, and then placed in a 5% CO2, 37°C incubator for 24 hours. The cytotoxicity was determined by the CCK-8 method. The CCK-8 detection method was as follows: remove the medium containing the drug, add 100 μL of serum-free medium containing 10% CCK-8 to each well, place in a 5% CO2, 37°C incubator for 2 hours, and measure the absorbance at 450 nm with an enzyme reader.

[0048] (3) Data processing: Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100% Note: As is the drug-administered group (culture medium containing cells, CCK-8, and the drug to be tested); Ac is the control group (culture medium containing cells, CCK-8, and no drug to be tested); Ab was the blank group (culture medium without cells and the drug to be tested, CCK-8).

[0049] Cell IC 50 The results are shown in Table 3.

[0050] Table 3

[0051] As can be seen from Table 3, the compounds of the present invention showed significant activity in inhibiting cell proliferation in all three cell lines. In 8505C and A375 cell lines, the inhibitory activity of compounds I-10, I-14, I-16, I-20, and I-24 was better than that of the positive drug vemurafenib. In the drug-resistant cell line p61-8505C, the inhibitory activity of the positive drug vemurafenib decreased significantly, while the inhibitory activity of the tested compounds decreased slightly, but compounds I-10, I-14, I-16, and I-24 still showed the best inhibitory activity.

[0052] 2. Kinase activity detection experiment (ADP-Glo TM Kinase Assay (1) Experimental principle: ADP-Glo TM 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 the kinase reaction to terminate the kinase reaction and consume the remaining ATP, then a kinase detection reagent is added to convert ADP into ATP, and the newly synthesized ATP is detected by the luciferase / luciferin reaction. The light signal generated is detected using a luminescence detector, and the relationship between the luminescence signal and the ADP concentration is determined by the ATP-ADP conversion curve. The luminescence signal generated is proportional to the ADP concentration generated and is correlated with the enzyme activity.

[0053] (2) Experimental method: Based on a 384-well plate, kinase: ADP-GloTM Regent: KinaseDetection Regent = 10:10:20 is particularly preferred. Use 1× kinase reaction buffer to gradient dilute the drug, take 1 μL of the gradient concentration of the drug and add it to the designated well. Select Vemurafenib as the positive drug, take 2.5× the optimal concentration of enzyme and add it to all wells, incubate at 37°C for 15 min, add 2× the optimal concentration of ATP and 2× the optimal concentration of substrate and incubate for 60 min. After the kinase or ATP is fully reacted, add ADP-GloTM Regent and incubate for 60 min to terminate the kinase reaction and consume the remaining ATP. Add 20 μL Kinase Detection Regent and incubate for 60 min to convert ADP into ATP. Detect the RU luminescence value using an enzyme reader.

[0054] (3) Data processing: Kinase inhibition rate = [1-(As-Ab) / (Ac-Ab)] × 100% Note: As is the drug administration 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, but not the drug to be tested); Ab was the blank group (containing only ATP, substrate, and kinase reaction buffer).

[0055] Kinase IC 50 The results are shown in Table 4.

[0056] Table 4

[0057] It can be seen from Table 4 that the activities of the compounds I-10, I-14, I-16 and I-24 synthesized in the present invention are significantly better than those of the positive drugs.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A bicyclic heteroaryl BRAF negative allosteric modulator, characterized in that: The general structural formula or its pharmaceutically acceptable salt is shown as Ⅰ: Ⅰ， Wherein, ring A is a differently substituted aromatic heterocycle, including a benzene ring, a pyridine ring, a pyrrole ring or a quinolinone ring; Ring B is a monocyclic aromatic ring with different substitutions, including a benzene ring, a pyridine ring or a furan ring; Ring C is any one of a differently substituted furan ring, a piperazine ring, a pyridine ring, a piperidine ring, a morpholine ring, a thiomorpholine ring or an amine; X 1 is a nitrogen atom or a carbon atom; Z 1 is an oxygen atom, a sulfur atom or CH; Z 2 , Z 3 are the same or different and are each independently selected from an oxygen atom, a nitrogen atom or CH; Y 1 is a differently substituted alkyl group, -CONH-, -NHCO-, -CH=CH- or COO-; R 1 is a hydrogen atom, a halogen or an alkyl group which may be substituted.

2. The bicyclic heteroaryl BRAF negative allosteric modulator according to claim 1, characterized in that: The general structural formula or its pharmaceutically acceptable salts include: 。 3. 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 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 mixture to 70-120°C for reaction, collecting and treating the reaction solution to obtain compound iv; or adding a nitrobenzene alkyl 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 solution 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.

4. The method for preparing the bicyclic heteroaryl BRAF negative allosteric modulator according to claim 3, 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.

5. The method for preparing the bicyclic heteroaryl BRAF negative allosteric modulator according to claim 3, characterized in that: In step (2), the phenol / thiophenol compound is any one of 3-nitrophenol, 2-methyl-5-nitrophenol, 2-methyl-4-nitrophenol, 2-methyl-5-nitrothiophenol, 2-methyl-5-benzyloxyphenol or 2-methyl-4-benzyloxyphenol; 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.

6. The method for preparing the bicyclic heteroaryl BRAF negative allosteric modulator according to claim 3, 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, thiomorpholine, methylamine or dimethylamine; 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 - at least one of 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 nitrogen-containing compound is 1-1.2:2-2.5:1.5:

1.

7. The method for preparing the bicyclic heteroaryl BRAF negative allosteric modulator according to claim 3, 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.

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

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

10. The use according to claim 8, 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, melanoma, colorectal cancer, and non-small cell lung cancer.

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