BRD4 inhibitor and preparation method and application thereof

Abstract

The invention provides a BRD4 inhibitor as well as a preparation method and application thereof, and belongs to the field of medicines. The synthesized compound is a BRD4 inhibitor, is novel in structure, excellent in pharmacokinetic property, good in drug effect, good in druggability, good in affinity, high in selectivity and good in blood-brain barrier penetrating capacity, can effectively treat diseases related to BRD4, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to a BRD4 inhibitor and a preparation method and use thereof. Background Art

[0002] Studies have shown that lysine acetylation (Kac) is an important and common epigenetic modification that plays a key regulatory role in gene transcription, metabolic processes and cell signaling, and is crucial in the formation of tumors, neurodegenerative diseases, etc.

[0003] Among them, BET is a class of bromodomain and extra-terminal domain family proteins (BRD), which consists of two highly conserved and tandem bromodomains (BD1 and BD2) at the N-terminus and the terminal extradomain (ET) at the C-terminus, which can recognize acetylated residues on histones H3 and H4. Among the four protein members, BRD2, BRD3, BRD4 and BRDT, BRD4 is known as the "Reader" of acetylated lysine. The BD1 and BD2 domains of BRD4, composed of approximately 110 amino acids, are composed of four antiparallel α-helices (αZ, αA, αB and αC) and two hydrophobic loops (ZA and BC). The crystal structure of BRD4 shows that a hydrophobic cavity is formed between the hydrophobic loops ZA and BC, which can specifically recognize acetylated lysine, which is also a key feature in the development of BRD4 inhibitors.

[0004] As an important cell cycle regulatory protein, BRD4 acts as a key transcriptional regulator that can specifically bind to acetylated histone tails, recruit disease-related target genes, and act on promoter and / or enhancer regions to promote gene expression. Abnormal histone acetylation can affect cell differentiation and apoptosis, thereby causing inflammation, tumors and other diseases. Therefore, diseases can be treated by inhibiting BRD4. Currently, BET inhibitors are used to treat cancer and related diseases, but they still face challenges, such as low specificity leading to side effects, and long-term use may induce drug resistance. In addition, BET inhibitors have been reported to have good therapeutic effects on some central nervous system diseases, but most of the BET inhibitors currently developed cannot pass through the blood-brain barrier and are difficult to achieve therapeutic effects. Therefore, it is necessary to further develop new BET inhibitors with good target affinity, good specificity, and the ability to pass through the blood-brain barrier.

[0005] The literature (DOI: 10.1039 / C2MD20189E) discloses a BRD4 small molecule inhibitor, compound 28a: However, the change in melting temperature (ΔTm / °C) of the compound during the heating process was 3.2 as tested by differential scanning fluorimetry, and the inhibitory activity against BRD4 protein was low (its pIC 50 Its target affinity needs to be further improved. Summary of the invention

[0006] The purpose of the present invention is to provide a BRD4 inhibitor and a preparation method and use thereof.

[0007] The present invention provides a compound represented by formula I, its tautomer, its stereoisomer, its hydrate, its solvate, its pharmaceutically acceptable salt, its prodrug, and its radiolabeled substance:

[0008]

[0009] wherein A1 is selected from CH, N;

[0010] Ring A2 is selected from a 5-6 membered heterocyclic ring which is not substituted or substituted by one or more A3, and A3 is selected from C 1～3 alkyl;

[0011] A4 is selected from hydrogen, carbonyl;

[0012] Ring B is unsubstituted or substituted with one or more B ’ Substituted phenyl or 6-membered nitrogen heteroaryl, B ’ Selected from hydrogen, halogen, nitro, hydroxyl, C 1～3 Alkyl, C 1～3 Alkoxy, 3- to 6-membered heterocycloalkyl;

[0013] Ring C is unsubstituted or substituted with one or more C ’ Substituted 5- to 6-membered heterocyclic ring, C ’ Selected from C 1～3 Alkyl, C 1～3 Alkoxy.

[0014] Furthermore, the structure of the compound is shown in Formula II:

[0015]

[0016] wherein A is selected from NR3, O or CHR3;

[0017] R3 is selected from hydrogen, C 1～3 alkyl;

[0018] R4, R5, R6 are independently selected from hydrogen, halogen, nitro, hydroxyl, C 1～3 Alkyl, C 1～3 Alkoxy, 3- to 6-membered heterocycloalkyl;

[0019] R7 and R8 are independently selected from hydrogen, a 5-6 membered heterocyclic ring which is unsubstituted or substituted by one or more R", and R7 and R8 are not hydrogen at the same time, and R" is selected from C 1～3 Alkyl; the 5-6 membered heterocyclic ring is selected from the following structures:

[0020] Furthermore, the structure of the compound is shown in Formula III:

[0021]

[0022] Among them, R9, R 10 are independently selected from hydrogen, a 5-membered heterocyclic ring which is unsubstituted or substituted with one or more R"', and R9, R 10 are not hydrogen at the same time, R'' is selected from C 1～3 Alkyl; the 5-membered heterocyclic ring is selected from:

[0023]

[0024] X1 is halogen.

[0025] Furthermore, the structure of the compound is shown in Formula IV:

[0026]

[0027] Wherein, B1 and B3 are independently selected from N or CH; B2 is selected from N or CR 13 ;

[0028] R 11 , R 12 , R 13 are independently selected from hydrogen, halogen, nitro, hydroxyl, C 1～3 Alkyl, 3-6 membered heterocycloalkyl;

[0029] R 14 , R 15 are independently selected from hydrogen, a 5-6 membered heterocyclic ring which is unsubstituted or substituted by one or more R"", and R 14 , R 15 are not hydrogen at the same time, R"" is selected from C 1～3 Alkyl; the 5-6 membered heterocyclic ring is selected from the following structures:

[0030] Furthermore, the structure of the compound is shown in Formula V:

[0031]

[0032] Among them, R9, R 10 independently selected from hydrogen, a 5-6 membered heterocyclic ring which is unsubstituted or substituted by one or more R'', wherein R'' is selected from hydrogen, C 1～3 alkyl;

[0033] X1 is halogen.

[0034] Furthermore, the above compound, its tautomer, its stereoisomer, its hydrate, its solvate, its pharmaceutically acceptable salt, its prodrug, and its radiolabeled substance are selected from one of the following compounds:

[0035]

[0036]

[0037]

[0038] The present invention also provides a method for preparing the above compound, the method comprising the following steps:

[0039]

[0040] (1) Compound A reacts with compound B to obtain compound D;

[0041] (2) Compound D reacts with Compound C to obtain the compound; or Compound D reacts with Compound C to obtain an intermediate, and the intermediate reacts with boron tribromide to obtain the compound;

[0042] Among them, X a , X b It is a halogen.

[0043] The present invention also provides the use of the above-mentioned compound, its tautomer, its stereoisomer, its hydrate, its solvate, its pharmaceutically acceptable salt, its prodrug, and its radioactive label in the preparation of BRD4 inhibitors.

[0044] Furthermore, the BRD4 inhibitor is a drug for preventing and / or treating Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, mood disorders, acute myeloid leukemia, non-small cell lung cancer, prostate cancer, myeloma, breast cancer, glioblastoma, renal cell carcinoma or thyroid cancer.

[0045] The present invention also provides a pharmaceutical composition, which is a preparation prepared with the above-mentioned compound, its tautomer, its stereoisomer, its hydrate, its solvate, its pharmaceutically acceptable salt, its prodrug, and its radioactive marker as active ingredients, and pharmaceutically acceptable excipients.

[0046] The experimental results show that the compound synthesized in the present invention is a BRD4 inhibitor with novel structure, excellent pharmacokinetic properties, good efficacy, good drugability, good affinity, high selectivity and good blood-brain barrier penetration ability, can effectively treat BRD4-related diseases, and has good application prospects.

[0047] Definitions of terms used in the present invention: Unless otherwise specified, the initial definitions provided for groups or terms herein apply to the groups or terms throughout the specification; for terms that are not specifically defined in the present invention, the meanings that can be given to them by those skilled in the art should be given according to the disclosure and context. Among them:

[0048] A "radiolabel" is a chemical compound that is a radioactive form of an isotope label.

[0049] The minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix C a～b Alkyl refers to any alkyl group containing from "a" to "b" carbon atoms. 1～3 Alkyl refers to straight or branched chain alkyl groups containing 1, 2 or 3 carbon atoms, and so on.

[0050] "Alkyl" refers to a saturated hydrocarbon chain having a specified number of member atoms. The alkyl group may be straight or branched, and the alkyl group may be optionally substituted with one or more substituents as defined herein. Alkyl includes methyl, ethyl, propyl (n-propyl and isopropyl).

[0051] "Alkoxy" represents an alkyl group as described above with a specific number of carbon atoms attached through an oxygen bridge, unless otherwise specified. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy.

[0052] "Heteroaryl" refers to a heteroaromatic group containing one to multiple heteroatoms. The heteroatoms referred to here include, but are not limited to, oxygen, sulfur, and nitrogen. For example, furyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc.

[0053] "Heterocycloalkyl" refers to a saturated cyclic group containing one or more heteroatoms. The heteroatoms referred to here include, but are not limited to, oxygen, sulfur, and nitrogen.

[0054] "Halogen" refers to fluorine, chlorine, bromine or iodine.

[0055] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0056] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1The results of the activity screening of small molecule compounds tested by differential scanning fluorimetry.

[0058] Figure 2 PET / CT imaging results (left) and time-activity curve (TAC, right) of mouse brain. DETAILED DESCRIPTION

[0059] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0060] Example 1: Method for preparing compound No. 1 of the present invention

[0061] Compound No. 1 was prepared according to the following synthetic route:

[0062]

[0063] 1) Step ⅰ:

[0064] Potassium carbonate (2.382 g, 17.25 mmol, 3.00 equiv) and 3-fluorobenzyl bromide (221.32 mg, 6.90 mmol, 1.20 equiv) were added to a solution of 5-bromobenzo[d]oxazol-2(3H)-one (1.0 g, 5.75 mmol, 1.00 equiv) in N,N-dimethylformamide (10 mL) and stirred at 80 °C for 3 hours. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (50 mL*3). The organic layers were combined and dried over sodium sulfate, then filtered and concentrated in vacuo to obtain a residue. The residue was purified by flash chromatography on silica gel (gradient elution, 0% to 35% ethyl acetate-petroleum ether) to obtain the intermediate compound 5-bromo-3-(3-fluorobenzyl)benzo[d]oxazol-2(3H)-one, 1.56 g, 77.56% yield.

[0065] 2) Step ii:

[0066] Potassium carbonate (692.96 mg, 2.13 mmol, 3.00 equiv) and 1,1'-bis(diphenylphosphino)ferrocenepalladium(II) dichloromethane complex (115.79 mg, 0.14 mmol, 0.20 equiv) were added to a solution of 5-bromo-3-(3-fluorobenzyl)benzo[d]oxazol-2(3H)-one (200.0 mg, 0.71 mmol, 1.00 equiv) and 3,5-dimethylisoxazole-4-boropinacol ester (189.77 mg, 0.85 mmol, 1.20 equiv) in 1,4-dioxane (5.0 mL) and water (1.0 mL). The reaction mixture was then stirred at 100°C for 3 hours under nitrogen. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (60 mL*2). The organic layers were combined and dried over sodium sulfate, then filtered and concentrated in vacuo to give a residual ethyl acetate residue. The residue was purified by silica gel flash chromatography (gradient elution, 0% to 75% ethyl acetate petroleum ether) to give Compound No. 1 (5-(3,5-dimethylisoxazol-4-yl)-3-(3-fluorobenzyl)benzo[d]oxazol-2(3H)-one) as a yellow oil, yield: 75.10%, 1 H NMR (400MHz, CDCl3) δ7.38–7.32(m,1H),7.29(d,J=8.0Hz,1H),7.15(d,J=8.0Hz,1H),7.09–7.01( m, 2H), 6.97 (dd, J = 8.0, 2.0Hz, 1H), 6.62 (d, J = 1.6Hz, 1H), 5.03 (s, 2H), 2.29 (s, 3H), 2.14 (s, 3H).

[0067] Example 2: Method for preparing compound No. 2 of the present invention

[0068] Compound No. 2 was prepared according to the following synthetic route:

[0069]

[0070] 1) Step iii:

[0071] Sodium hydride (19.11 mg, 13.26 mmol, 60% purity, 1.5 equivalents) was added in batches to a solution of 6-bromo-3,4-dihydroquinolin-2(1H)-one (200 mg, 0.884 mmol, 1.0 equivalents) in tetrahydrofuran (5 mL) at 0°C. The reaction mixture was stirred at 0°C for 15 minutes, and then 3-fluorobenzyl bromide (250.85 mg, 1.33 mmol, 1.5 equivalents) was added dropwise. The reaction solution was naturally warmed to room temperature, reacted at room temperature for 12 hours, then quenched with water (10 mL) and extracted with ethyl acetate (30 mL*2). The combined organic layer was concentrated to obtain a residue. The residue was purified by silica gel flash chromatography (gradient elution, 0% to 10% ethyl acetate petroleum ether) to give the intermediate compound, 6-bromo-1-(3-fluorobenzyl)-3,4-dihydroquinolin-2(1H)-one (180.0 mg, 61.01% yield) as a yellow oil.

[0072] 2) Step ii:

[0073] Referring to step ii of Example 1, compound No. 2 was finally synthesized, 6-(3,5-dimethylisoxazol-4-yl)-1-(3-fluorobenzyl)-3,4-dihydroquinolin-2(1H)-one, with a yield of 72.23%. 1 H NMR (400MHz, CDCl3) δ7.34–7.27(m,1H),7.08–7.03(m,2H),7.00(dd,J=8.8,2.4Hz,1H),6.98–6.90(m,2 H),6.88(d,J=8.8Hz,1H),5.19(s,2H),3.06–3.01(m,2H),2.87–2.82(m,2H),2.38(s,3H),2.24(s,3H)).

[0074] Example 3: Method for preparing compound No. 3 of the present invention

[0075] Compound No. 3 was prepared according to the following synthetic route:

[0076]

[0077] The preparation method of reference example 2 is different in that 5-bromo-1H-indole is used instead of 6-bromo-3,4-dihydroquinolin-2(1H)-one in step iii: 4-(4,5-dimethyl-1,3,2-dioxaborolan-2-yl)-1-methyl-1H-pyrazole is used instead of 3,5-dimethylisoxazole-4-boronic acid pinacol ester in step ii, and finally compound No. 3 [1-(3-fluorobenzyl)-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)-1H-indole] is obtained, with a yield of 72.37%. 1H NMR (400MHz, CDCl3) δ7.48(dd,J=1.6,0.8Hz,1H),7.34–7.26(m,1H),7.25(s,1H),7.15(d,J=3.2Hz,1H),7.06(dd,J=8.4,1.7Hz,1H),6. 95(dd,J=8.0,2.0Hz,2H),6.83(dd,J=9.6,2.0Hz,1H),6.57(dd,J=3.2,0.8Hz,1H),5.32(s,2H),3.80(s,3H),2.26(s,3H),2.24(s,3H).

[0078] Example 4: Method for preparing compound No. 4 of the present invention

[0079] Compound No. 4 was prepared according to the following synthetic route:

[0080]

[0081] The preparation method of reference example 1 is different in that 5-bromobenzo[d]oxazol-2(3H)-one is used instead of 4-bromopyridin-2(1H)-one in step i; 4-(4,5-dimethyl-1,3,2-dioxaborolan-2-yl)-1-methyl-1H-pyrazole is used instead of 3,5-dimethylisoxazole-4-boronic acid pinacol ester in step ii; finally, compound No. 4 is obtained with a yield of 63.68%. 1 H NMR (400MHz, CDCl3) δ7.63(d,J=0.8Hz,1H),7.52–7.49(m,1H),7.37–7.30(m,1H),7.20(d,J=1. 6Hz, 2H), 7.16–7.12 (m, 1H), 7.09–6.98 (m, 2H), 6.86 (t, J = 1.2Hz, 1H), 5.02 (s, 2H), 3.93 (s, 3H).

[0082] Example 5: Method for preparing compound No. 5 of the present invention

[0083] Compound No. 5 was prepared according to the following synthetic route:

[0084]

[0085] The preparation method of reference example 1 is different in that 1-(bromomethyl)-3,5-difluorobenzene is used instead of 1-(bromomethyl)-3-fluorobenzene in step i, and 6-bromo-1H-imidazo[4,5-b]pyridine is used instead of 4-bromopyridin-2(1H)-one, and other conditions are the same; finally, compound No. 5, 4-(1-(3,5-difluorobenzyl)-1H-imidazo[4,5-b]pyridin-6-yl)-3,5-dimethylisoxazole, is obtained in a yield of 77.46%. 1 H NMR (400MHz, DMSO) δ8.70 (s, 1H), 8.39 (d, J = 2.0Hz, 1H), 8.19 (d, J = 2.0Hz, 1H), 7.20 (tt,J=12.0,2.4Hz,1H),7.14–7.10(m,2H),5.56(s,2H),2.42(s,3H),2.24(s,3H).

[0086] Example 6: Method for preparing compound No. 6 of the present invention

[0087] Compound No. 6 was prepared according to the following synthetic route:

[0088]

[0089] The preparation method of reference example 1 is different in that 1-(bromomethyl)-3,5-difluorobenzene is used instead of 1-(bromomethyl)-3-fluorobenzene in step i, and 6-bromo-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one is used instead of 4-bromopyridin-2(1H)-one, and other conditions are the same; finally, compound No. 6, 4-(1-(3,5-difluorobenzyl)-1H-imidazo[4,5-b]pyridin-6-yl)-3,5-dimethylisoxazole, is obtained in a yield of 77.46%. 1 H NMR (400MHz, DMSO) δ8.70 (s, 1H), 8.39 (d, J = 2.0Hz, 1H), 8.19 (d, J = 2.0Hz, 1H), 7.20 (tt,J=12.0,2.4Hz,1H),7.14–7.10(m,2H),5.56(s,2H),2.42(s,3H),2.24(s,3H).

[0090] Example 7: Method for preparing compound No. 7 of the present invention

[0091] Compound No. 7 was prepared according to the following synthetic route:

[0092]

[0093] The preparation method of reference example 1 is different in that in step i, 1-(bromomethyl)-3,5-difluorobenzene is used instead of 1-(bromomethyl)-3-fluorobenzene, 6-bromo-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one is used instead of 4-bromopyridin-2(1H)-one, and other conditions are the same; in step ii, 5-(4,5-dimethyl-1,3,2-dioxaborolan-2-yl)-1,3-dimethylpyridin-2(1H)-one is used instead of 3,5-dimethylisoxazole-4-boronic acid pinacol ester; finally, compound No. 7 is obtained, [1-(3,5-difluorobenzyl)-5-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-3-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one], with a yield of 81.23%. 1 H NMR(400MHz, CDCl3)δ8.50(s,1H),8.24(s,1H),8.11(s,1H),7.54–7.50(m,1H), 7.42(d,J=2.4Hz,1H),6.86–6.74(m,3H),5.50(s,2H),3.66(s,3H),2.26(s,3H).

[0094] Example 8: Method for preparing compound No. 8 of the present invention

[0095] Compound No. 8 was prepared according to the following synthetic route:

[0096]

[0097] The preparation method of reference example 1 is different in that 6-bromo-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one is used instead of 4-bromopyridin-2(1H)-one in step i, and other conditions are the same; finally, compound No. 8, [5-(3,5-dimethylisoxazol-4-yl)-1-(3-fluorobenzyl)-3-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one], is obtained in a yield of 74.69%. 1 H NMR(400MHz,DMSO)δ7.43–7.36(m,1H),7.23–7.17(m,4H),7.15–7.08(m,1H),7 .00(dd,J=8.4,2.0Hz,1H),5.10(s,2H),3.40(s,3H),2.38(s,3H),2.21(s,3H).

[0098] Example 9: Method for preparing compound No. 9 of the present invention

[0099] Compound No. 9 was prepared according to the following synthetic route:

[0100]

[0101] The preparation method of reference example 1 is different in that 6-bromo-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one is used instead of 4-bromopyridin-2(1H)-one in step i, and other conditions are the same; 5-(4,5-dimethyl-1,3,2-dioxaborolan-2-yl)-1,3-dimethylpyridin-2(1H)-one is used instead of 3,5-dimethylisoxazole-4-boronic acid pinacol ester in step ii; finally, compound No. 9 is obtained, [5-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(3-fluorobenzyl)-3-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one], with a yield of 69.35%. 1 H NMR (400MHz, CDCl3) δ7.48 (s, 1H), 7.34–7.28 (m, 2H), 7.11 (d, J = 2.0Hz, 1H), 7.05–6. 94(m,4H),6.86(d,J=8.4Hz,1H),5.08(s,2H),3.63(s,3H),3.51(s,3H),2.23(s,3H).

[0102] Example 10: Method for preparing compound No. 10 of the present invention

[0103] Compound No. 10 was prepared according to the following synthetic route:

[0104]

[0105] The preparation method of reference example 1 is different in that 6-bromo-1H-imidazo[4,5-b]pyridine is used instead of 4-bromopyridin-2(1H)-one in step i, and other conditions are the same; finally, compound No. 10, [4-(1-(3-fluorobenzyl)-1H-imidazo[4,5-b]pyridin-6-yl)-3,5-dimethylisoxazole], is obtained, with a yield of 82.44%. 1 H NMR(400MHz, CDCl3)δ8.48(d,J=2.0Hz,1H),8.27(s,1H),7.43–7.32(m,2H),7.10–7.03(m, 1H),7.02–6.98(m,1H),6.91(dt,J=8.0,2.0Hz,1H),5.42(s,2H),2.35(s,3H),2.18(s,3H).

[0106] Example 11: Method for preparing compound No. 11 of the present invention

[0107] Compound No. 11 was prepared according to the following synthetic route:

[0108]

[0109] The preparation method of reference example 1 is different in that in step i, 6-bromo-1H-imidazo[4,5-b]pyridine is used instead of 4-bromopyridin-2(1H)-one, 1-(bromomethyl)-3,5-difluorobenzene is used instead of 1-(bromomethyl)-3-fluorobenzene, and other conditions are the same; in step ii, 4-(4,5-dimethyl-1,3,2-dioxaborolan-2-yl)-1-methyl-1H-pyrazole is used instead of 3,5-dimethylisoxazole-4-boronic acid pinacol ester; finally, compound No. 11, 1-(3,5-difluorobenzyl)-6-(1-methyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-b]pyridine, is obtained in a yield of 67.48%. 1 H NMR (400MHz, CDCl3) δ8.52(s,1H),8.08(s,2H),7.75(s,1H),7.62(s,1H),6.80–6.67(m,3H),5.44(s,2H),3.94(s,3H).

[0110] Example 12: Method for preparing compound No. 12 of the present invention

[0111] Compound No. 12 was prepared according to the following synthetic route:

[0112]

[0113] The preparation method of reference example 1 is different in that 6-bromo-1H-imidazo[4,5-b]pyridine is used instead of 4-bromopyridin-2(1H)-one in step i, and other conditions are the same; 4-(4,5-dimethyl-1,3,2-dioxaborolan-2-yl)-1-methyl-1H-pyrazole is used instead of 3,5-dimethylisoxazole-4-boronic acid pinacol ester in step ii; finally, compound No. 12, 1-(3-fluorobenzyl)-6-(1,3,5-trimethyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-b]pyridine, is obtained in a yield of 70.18%. 1 HNMR(400MHz, CDCl3)δ8.33(s,1H),8.10(s,1H),7.96(s,1H),7.38–7.30(m,1H), 7.13(d,J=7.6Hz,1H),7.07–6.99(m,2H),5.51(s,2H),3.82(s,3H),2.26(s,6H).

[0114] Example 13: Method for preparing compound No. 13 of the present invention

[0115] Compound No. 13 was prepared according to the following synthetic route:

[0116]

[0117] The preparation method of reference example 1 is different in that 6-bromo-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one is used instead of 4-bromopyridin-2(1H)-one in step i, and other conditions are the same; 6-bromo-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one is used instead of 3,5-dimethylisoxazole-4-boronic acid pinacol ester in step ii; finally, compound No. 13, 1-(3-fluorobenzyl)-3-methyl-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one, is obtained in a yield of 64.67%. 1 H NMR(400MHz, CDCl3)δ7.33–7.26(m,1H),7.15(d,J=8.0Hz,1H),7.07–7.01(m,1H),7.00–6 .93(m,1H),6.88(s,2H),6.85(s,1H),5.08(s,2H),3.78(s,3H),3.48(s,3H),2.22(s,6H).

[0118] Example 14: Method for preparing compound No. 14 of the present invention

[0119] Compound No. 14 was prepared according to the following synthetic route:

[0120]

[0121] The preparation method of reference example 1 is different in that in step i, 6-bromo-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one is used instead of 4-bromopyridin-2(1H)-one, and 4-(bromomethyl)-2-fluoro-1-methoxybenzene is used instead of 1-(bromomethyl)-3-fluorobenzene, and other conditions are the same; finally, compound No. 14, 5-(3,5-dimethylisoxazol-4-yl)-1-(3-fluoro-4-methoxybenzyl)-3-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one, is obtained, with a yield of 63.79%. 1 H NMR (400MHz, CDCl3) δ7.12–7.06(m,2H),6.95–6.86(m,3H),6.85–6.82(m,1H),5.02(s,2H),3.86(s,3H),3.48(s,3H),2.38(s,3H),2.25(s,3H).

[0122] Example 15: Method for preparing compound No. 15 of the present invention

[0123] Compound No. 15 was prepared according to the following synthetic route:

[0124]

[0125] The preparation method of reference example 1 is different in that in step i, 6-bromo-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one is used instead of 4-bromopyridine-2(1H)-one, and 4-(bromomethyl)-2-fluoro-1-methoxybenzene is used instead of 1-(bromomethyl)-3-fluorobenzene; in step iv, boron tribromide (985.23 mg, 3.93 mmol, 15 equiv) is added dropwise to a solution of 5-(3,5-dimethylisoxazol-4-yl)-1-(3-fluoro-4-methoxybenzyl)-3-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (100 mg, 0.262 mmol, 1.0 equiv) in dichloromethane at -78°C under nitrogen protection. The reaction solution was reacted at -78°C for 30 minutes, then naturally warmed to room temperature, reacted at room temperature for 1 hour, then quenched with methanol (4 mL), and concentrated to obtain a residue. The residue was purified by reverse phase chromatography (gradient elution, 0% to 90% methanol-water), with other conditions being the same; finally, compound No. 15 was obtained as a white solid, [5-(3,5-dimethylisoxazol-4-yl)-1-(3-fluoro-4-hydroxybenzoyl)-3-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one, 57 mg, 57.79% yield], 1 H NMR (400MHz, DMSO) δ9.86 (s, 1H), 7.24–7.16 (m, 3H), 7.05–6.98 (m, 2H), 6.91 (t, J = 8.0Hz, 1H), 4.96 (s, 2H), 3.40 (s, 3H), 2.38 (s, 3H), 2.21 (s, 3H).

[0126] Example 16: Method for preparing compound No. 16 of the present invention

[0127] Compound No. 16 was prepared according to the following synthetic route:

[0128]

[0129] The preparation method of reference example 1 is different in that in step i, 6-bromo-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one is used instead of 4-bromopyridin-2(1H)-one, and 4-(bromomethyl)-2-fluoro-1-nitrobenzene is used instead of 1-(bromomethyl)-3-fluorobenzene; finally, compound No. 16, 5-(3,5-dimethylisoxazol-4-yl)-1-(3-fluoro-4-nitrobenzyl)-3-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one, is obtained in a yield of 62.46%. 1H NMR(400MHz, CDCl3)δ8.06(d,J=8.0Hz,1H),7.30–7.27(m,1H),7.26–7.22(m,1H),6.92(dd ,J=8.0,1.6Hz,1H),6.90–6.85(m,2H),5.16(s,2H),3.51(s,3H),2.39(s,3H),2.25(s,3H).

[0130] Example 17: Method for preparing compound No. 17 of the present invention

[0131] Compound No. 17 was prepared according to the following synthetic route:

[0132]

[0133] The preparation method of reference example 1 is different in that 6-bromo-1H-imidazo[4,5-b]pyridine is used instead of 4-bromopyridin-2(1H)-one in step i, and 4-(bromomethyl)-2-fluoro-1-methoxybenzene is used instead of 1-(bromomethyl)-3-fluorobenzene; other conditions are the same; finally, compound No. 17 is obtained as a white solid, 5-(3,5-dimethylisoxazol-4-yl)-1-(3-fluoro-4-hydroxybenzoyl)-3-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one, with a yield of 61.55%. 1 H NMR (400MHz, CDCl3) δ8.33(d,J=2.0Hz,1H),8.13(s,1H),7.98(d,J=2.0Hz,1H),7.14–7 .08(m,2H),6.95(d,J=8.0Hz,1H),5.44(s,2H),3.88(s,3H),2.44(s,3H),2.30(s,3H).

[0134] Example 18: Method for preparing compound No. 18 of the present invention

[0135] Compound No. 18 was prepared according to the following synthetic route:

[0136]

[0137] The preparation method of reference example 15 is different in that 6-bromo-1H-imidazo[4,5-b]pyridine is used instead of 6-bromo-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one in step i; finally, compound No. 18 is obtained with a yield of 59.68%. 1HNMR (400MHz, DMSO) δ10.01(s,1H),8.66(s,1H),8.40(d,J=1.6Hz,1H),8.16(d,J=2.0Hz,1H),7.26(dd, J=12.0,2.4Hz,1H),7.06(d,J=8.0Hz,1H),6.91(d,J=8.0Hz,1H),5.41(s,2H),2.42(s,3H),2.24(s,3H).

[0138] Example 19: Method for preparing compound No. 19 of the present invention

[0139] Compound No. 19 was prepared according to the following synthetic route:

[0140]

[0141] The preparation method of reference example 16 is different in that 6-bromo-1H-imidazo[4,5-b]pyridine is used instead of 6-bromo-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one in step i; finally, compound No. 19, 4-(1-(3-fluoro-4-nitrobenzyl)-1H-imidazo[4,5-b]pyridin-6-yl)-3,5-dimethylisoxazole, is obtained in a yield of 58.95%. 1 H NMR(400MHz, CDCl3)δ8.33(d,J=4.0Hz,1H),8.24(s,1H),8.12–8.03(m,1H),8. 02(d,J=2.0Hz,1H),7.30–7.22(m,3H),5.62(s,2H),2.45(s,3H),2.30(s,3H).

[0142] Example 20: Method for preparing compound No. 20 of the present invention

[0143] Compound No. 20 was prepared according to the following synthetic route:

[0144]

[0145] The preparation method of reference example 1 is different in that 1-(bromomethyl)-4-fluorobenzene is used instead of 1-(bromomethyl)-3-fluorobenzene in step i, and other conditions are the same; finally, compound No. 20, 5-(3,5-dimethylisoxazol-4-yl)-1-(4-fluorobenzyl)-3-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one, is obtained in a yield of 70.04%. 1H NMR (400MHz, CDCl3) δ7.38–7.32(m,2H),7.05–6.99(m,2H),6.94–6.83(m,3H),5.07(s,2H),3.48(s,3H),2.38(s,3H),2.24(s,3H).

[0146] Example 21. Method for preparing compound No. 21 of the present invention

[0147] Compound No. 21 was prepared according to the following synthetic route:

[0148]

[0149] The preparation method of reference example 10 is different in that 1-(bromomethyl)-4-fluorobenzene is used instead of 1-(bromomethyl)-3-fluorobenzene in step i, and other conditions are the same; finally, compound No. 21, 4-(1-(4-fluorobenzyl)-1H-imidazo[4,5-b]pyridin-6-yl)-3,5-dimethylisoxazole, is obtained in a yield of 76.22%. 1 H NMR (400MHz, CDCl3) δ8.34(d,J=2.0Hz,1H),8.15(s,1H),7.98(d,J=2.0Hz,1H), 7.39–7.34(m,2H),7.06(t,J=8.0Hz,2H),5.49(s,2H),2.44(s,3H),2.30(s,3H).

[0150] Example 22: Method for preparing compound No. 22 of the present invention

[0151] Compound No. 22 was prepared according to the following synthetic route:

[0152]

[0153] The preparation method of reference example 10 is different in that 5-(4,5-dimethyl-1,3,2-dioxaborolan-2-yl)-1,3-dimethylpyridin-2(1H)-one is used instead of 3,5-dimethylisoxazole-4-boronic acid pinacol ester in step ii; finally, compound No. 22, 5-(1-(3-fluoro-4-methoxybenzyl)-1H-imidazo[4,5-b]pyridin-6-yl)-1,3-dimethylpyridin-2(1H)-one is obtained, with a yield of 57.85%. 1H NMR (400MHz, CDCl3) δ8.49(d,J=2.0Hz,1H),8.15(s,1H),8.09(d,J=2.0Hz,1H),7.53–7.50(m,1H),7.41(d ,J=2.8Hz,1H),7.12–7.06(m,2H),6.96–6.91(m,1H),5.43(s,2H),3.87(s,3H),3.66(s,3H),2.26(s,3H).

[0154] Example 23. Method for preparing compound No. 23 of the present invention

[0155] Compound No. 23 was prepared according to the following synthetic route:

[0156]

[0157] The preparation method of reference example 12 is different in that 5-(4,5-dimethyl-1,3,2-dioxaborolan-2-yl)-1,3-dimethylpyridin-2(1H)-one is used instead of 4-(4,5-dimethyl-1,3,2-dioxaborolan-2-yl)-1-methyl-1H-pyrazole in step ii; finally, compound No. 23, 5-(1-(3-fluoro-4-methoxybenzyl)-1H-imidazo[4,5-b]pyridin-6-yl)-1,3-dimethylpyridin-2(1H)-one, is obtained in a yield of 61.35%. 1 H NMR(400MHz, CDCl3)δ8.50(s,1H),8.24(s,1H),8.11(s,1H),7.54–7.50(m,1H), 7.42(d,J=2.4Hz,1H),6.86–6.74(m,3H),5.50(s,2H),3.66(s,3H),2.26(s,3H).

[0158] Example 24: Method for preparing compound No. 24 of the present invention

[0159] Compound No. 24 was prepared according to the following synthetic route:

[0160]

[0161] The preparation method of reference example 22 is different in that 6-bromo-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one is used instead of 6-bromo-1H-imidazo[4,5-b]pyridine in step i; finally, compound No. 24, 5-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(3-fluoro-4-methoxybenzyl)-3-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one, is obtained in a yield of 63.12%.1 H NMR (400MHz, CDCl3) δ7.49–7.46(m,1H),7.32(d,J=3.2Hz,1H),7.09–7.02(m,3H),6.98(d,J=1 .6Hz,1H),6.93–6.86(m,2H),5.01(s,2H),3.86(s,3H),3.63(s,3H),3.50(s,3H),2.23(s,3H).

[0162] Example 25: Method for preparing compound No. 25 of the present invention

[0163] Compound No. 25 was prepared according to the following synthetic route:

[0164]

[0165] Based on Example 24, the difference is that the reference step iv of Example 18 is used; finally, compound No. 25, 5-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(3-fluoro-4-hydroxybenzoyl)-3-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one, is obtained, with a yield of 66.21%. 1 H NMR (400MHz, DMSO) δ9.83(s,1H),7.93(d,J=3.2Hz,1H),7.76–7.73(m,1H),7.38(d,J=2.0Hz,1H),7.21(dd,J=8.0,2.4Hz,1H) ,7.17–7.12(m,2H),6.97(dd,J=8.8,2.0Hz,1H),6.89(d,J=12.0Hz,1H),4.95(s,2H),3.51(s,3H),3.40(s,3H),2.08(s,3H).

[0166] Example 26: Method for preparing compound No. 26 of the present invention

[0167] Compound No. 26 was prepared according to the following synthetic route:

[0168]

[0169] The preparation method of reference example 23 is different in that 5-(bromomethyl)-2-fluoropyridine is used instead of 1-(bromomethyl)-3,5-difluorobenzene in step i; finally, compound No. 26 is obtained with a yield of 59.67%. 1H NMR (400MHz, CDCl3) δ8.49(d,J=2.0Hz,1H),8.36–8.30(m,1H),8.22(s,1H),8.09(d,J=1.6Hz,1H),7.84(td,J=8.0,2. 4Hz,1H),7.53–7.49(m,1H),7.41(d,J=2.8Hz,1H),6.93(dd,J=8.0,2.0Hz,1H),5.52(s,2H),3.66(s,3H),2.26(s,3H).

[0170] Example 27. Method for preparing compound No. 27 of the present invention

[0171] Compound No. 27 was prepared according to the following synthetic route:

[0172]

[0173] The preparation method of reference example 24 is different in that 1-(bromomethyl)-3-fluoro-5-methoxybenzene is used instead of 6-bromo-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one in step i; finally, compound No. 27, 5-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(3-fluoro-5-methoxybenzyl)-3-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one, is obtained in a yield of 65.20%. 1 H NMR (400MHz, DMSO) δ9.93(s,1H),7.94(d,J=2.8Hz,1H),7.78–7.73(m,1H),7.41(d,J=1.6Hz,1H),7.22(dd,J=8.0,1.6Hz,1H),7.13(d,J=8. 0Hz,1H),6.59(dt,J=9.2,1.6Hz,1H),6.54–6.50(m,1H),6.45(dt,J=10.8,2.4Hz,1H),4.99(s,2H),3.52(s,3H),3.42(s,3H),2.09(s,3H).

[0174] Example 28: Method for preparing compound No. 28 of the present invention

[0175] Compound No. 28 was prepared according to the following synthetic route:

[0176]

[0177] The preparation method of reference example 3 is different in that 1,3-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-pyridin-2(1H)-one is used in step ii to finally obtain compound No. 28 [1-(3-fluorobenzyl)-5-(1,3-dimethyl-pyridin-2(1H)-one)-1H-indole] with a yield of 75.53%. 1 H NMR(400MHz,Chloroform-d)δ7.67(d,J=1.6Hz,1H),7.59–7.56(m,1H),7.38(d,J=2.5Hz,1H),7.31–7.24(m,2H),7.22(d,J=1.6Hz,1H),7.17(d, J=3.2Hz,1H),6.96(d,J=2.6Hz,1H),6.90(d,J=7.8Hz,1H),6.77(d,J=9.6Hz,1H),6.59(d,J=3.2Hz,1H),5.34(s,2H),3.64(s,3H),2.25(s,3H).

[0178] Example 29: Method for preparing compound No. 29 of the present invention

[0179] Compound No. 29 was prepared according to the following synthetic route:

[0180]

[0181] The preparation method of reference example 28 is different in that 3,5-difluorobenzyl bromide is used instead of 3-fluorobenzyl bromide in step iii, and finally compound No. 29 [1-(3,5-difluorobenzyl)-5-(1,3-dimethyl-pyridin-2(1H)-one)-1H-indole] is obtained, and the yield is 69.66%. 1 H NMR(400MHz,Chloroform-d)δ7.68(s,1H),7.60–7.56(m,1H),7.38(s,1H)7.26(s,2H),7 .23(s,1H),6.71-6.73(m,1H),6.66-6.50(m,3H),5.32(s,2H),3.64(s,3H),2.25(s,3H).

[0182] Example 30: Method for preparing compound No. 30 of the present invention

[0183] Compound No. 30 was prepared according to the following synthetic route:

[0184]

[0185] The preparation method of reference example 24 is different in that 3-fluoro-4-methoxybenzyl bromide is used instead of 3-fluorobenzyl bromide in step iii, and finally compound No. 30 [1-(3-fluoro-4-methoxybenzyl)-5-(1,3-dimethyl-pyridin-2(1H)-one)-1H-indole] is obtained, and the yield is 67.87%. 1 H NMR(400MHz,Chloroform-d)δ8.12(s,1H),7.54(s,1H),7.45(s,1H),7.40(d,J=8.4Hz,1H),7.34(s,1H),7.23( d,J=8.4Hz,1H),7.01–6.95(m,3H),6.88(t,J=8.4Hz,1H),4.07(s,2H),3.86(s,3H),3.64(s,3H),2.25(s,3H).

[0186] Example 31: Method for preparing compound No. 31 of the present invention

[0187] Compound No. 31 was prepared according to the following synthetic route:

[0188]

[0189] The preparation method of reference example 24 is different in that 2-(4-(bromomethyl)-2-fluorophenyl)oxirane is used instead of 3-fluoro-4-methoxybenzyl bromide in step i; finally, compound No. 31, 5-(1,5-dimethyl-6-oxo-1,6-dihydropyridin-3-yl)-1-(3-fluoro-4-(oxirane-2-yl)benzyl)-3-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one, is obtained in a yield of 61.52%. 1 H NMR(400MHz, CDCl3-d)δ8.01-8.00(m,1H),7.72-7.70(m,1H),7.62(d,J=2.0Hz,1H),7.51(d,J=8.0Hz,1H),7.38–7.29(m,2H),7.16–7.05(m,2H),5 .23(t,J=1.2Hz,2H),4.29–4.24(m,1H),3.53(s,3H),3.53(dd,J=7.7,2. 4Hz, 1H), 3.39 (d, J = 1.2Hz, 3H), 3.28 (dd, J = 7.6, 2.2Hz, 1H), 2.10 (s, 2H).

[0190] Example 32: Method for preparing compound No. 32 of the present invention

[0191] Compound No. 32 was prepared according to the following synthetic route:

[0192]

[0193] The preparation method of reference example 30 is different in that 2-(4-(bromomethyl)-2-fluorophenyl)oxirane is used instead of 3-fluorobenzyl bromide in step i, and other conditions are the same, and finally compound No. 32 5-(1-(3-fluoro-4-(oxirane-2-yl)benzyl)-1H-indol-5-yl)-1,3-dimethylpyridin-2(1H)-one is obtained, with a yield of 66.28%. 1 H NMR (400MHz, CDCl3-d) δ8.19 (dd, J=2.4, 1.2Hz, 1H), 7.98 (t, J=1.2Hz, 1H), 7.7 2–7.68(m,1H),7.65(d,J=8.4Hz,1H),7.37–7.24(m,3H),7.15–7.10(m,1H),7. 01(ddt,J=12.0,1.6,0.8Hz,1H),6.53(dd,J=4.8,2.0Hz,1H),5.44(s,2H),4.2 9–4.24(m,1H),3.56–3.50(m,1H),3.39(s,3H),3.31–3.25(m,1H),2.10(s,3H).

[0194] Example 33: Method for preparing compound No. 33 of the present invention

[0195] Compound No. 33 was prepared according to the following synthetic route:

[0196]

[0197] The preparation method of reference example 32 is different in that 6-bromo-1H-imidazo[4,5-b]pyridine is used instead of 5-bromo-1H-indole in step i, and other conditions are the same, and finally compound No. 33 5-(1-(3-fluoro-4-(oxiran-2-yl)benzyl)-1H-imidazo[4,5-b]pyridin-6-yl)-1,3-dimethylpyridin-2(1H)-one is obtained, with a yield of 61.21%. 1 H NMR(400MHz, CDCl3-d)δ8.47(d,J=1.6Hz,1H),8.23–8.19(m,1H),8.07–8.03(m,2H),7.79–7.74(m,1H),7.33–7.27(m,1H),7.15–7.09(m,1 H),7.04–6.97(m,1H),5.61(s,2H),4.29–4.24(m,1H),3.53(dd,J=7.7,2.2Hz,1H),3.39(s,3H),3.28(dd,J=7.6,2.4Hz,1H),2.10(s,3H).

[0198] Example 34: Method for preparing compound No. 34 of the present invention

[0199] Compound No. 34 was prepared according to the following synthetic route:

[0200]

[0201] The preparation method of reference example 17 is different in that 2-(4-(bromomethyl)-2-fluorophenyl)oxirane is used instead of 3-fluoro-4-methoxybenzyl bromide in step i, and other conditions are the same; finally, compound No. 34, 4-(1-(3-fluoro-4-(oxirane-2-yl)benzyl)-1H-imidazo[4,5-b]pyridin-6-yl)-3,5-dimethylisoxazole, is obtained in a yield of 61.55%. 1 H NMR (400MHz, CDCl3) δ8.54(d,J=1.6Hz,1H),8.12(d,J=1.6Hz,1H),8.05(d,J=0.8Hz,1H),7.33–7.27(m,1H),7.15–7.09(m,1H),7.0 4–6.97(m,1H),5.61(s,2H),4.29–4.24(m,1H),3.53(dd,J=7.6,2.4Hz,1H),3.28(dd,J=7.6,2.4Hz,1H),2.55(s,3H),2.42(s,3H).

[0202] Example 35. Method for preparing compound No. 35 of the present invention

[0203] Compound No. 35 was prepared according to the following synthetic route:

[0204]

[0205] The preparation method of reference example 34 is different in that 5-bromobenzo[d]oxazol-2(3H)-one is used instead of 6-bromo-1H-imidazo[4,5-b]pyridine in step i, and other conditions are the same; finally, compound No. 35, 5-(3,5-dimethylisoxazol-4-yl)-3-(3-fluoro-4-(oxiran-2-yl)benzyl)benzo[d]oxazol-2(3H)-one, is obtained in a yield of 59.95%. 1HNMR (400MHz, CDCl3) δ7.51(d,J=2.0Hz,1H),7.45(d,J=8.8Hz,1H),7.32(ddd,J=8.4,4.8,0.8Hz,1H),7.17–7.06(m,3 H),5.40(s,2H),4.29–4.24(m,1H),3.53(dd,J=7.6,2.4Hz,1H),3.28(dd,J=7.7,2.2Hz,1H),2.55(s,3H),2.42(s,3H).

[0206] The compounds shown in Table 1 were prepared by the above Examples 1 to 35.

[0207] Table 1. Compound structures and characterization

[0208]

[0209]

[0210]

[0211]

[0212]

[0213] Note: CLogP in the table is calculated by ChemDaw2022.0.0.

[0214] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0215] Experimental Example 1: Determination of small molecule activity by differential scanning fluorimetry

[0216] 1. Experimental methods

[0217] Differential scanning fluorescence (DSF) is an efficient screening method that uses a traditional real-time quantitative PCR instrument to measure the change in the melting temperature (ΔTm / °C) of the protein during the heating process in the presence and absence of the ligand to determine the small molecule ligand that can bind and stabilize the protein.

[0218] The specific steps are as follows:

[0219] 1) BRD4 protein was added to a buffer solution containing 20 mM HEPES, 150 nM NaCl, pH 7.5, and finally prepared into a 2 μM / 40 μL solution in a 96-well plate, and containing (5X) SYPRO fluorescent dye.

[0220] 2) Fill the reference wells and control wells of a 96-well storage plate with 100% (v / v) DMSO and distilled water, respectively.

[0221] 3) Use a pipette to transfer a sufficient volume of each compound stock solution (DMSO solvent) into the 96-well tubes so that the final concentration of protein is 1:10 to the final concentration of each compound, vortex to ensure complete dissolution, and centrifuge to allow the liquid to fall to the bottom of the tube.

[0222] 4) Real-time quantitative polymerase chain reaction (PCR) instrument program: 25°C (1.6°C / s, 2:00 min); 95°C (0.05°C / s, 2:00 min), 25°C (2.5°C / s, 2:00 min).

[0223] 2. Experimental results

[0224] The results show that the compounds of the present invention can effectively bind to BRD4-BD1 and BD2, among which the document (DOI: 10.1039 / C2MD20189E) discloses compound 28a, The compound was tested by differential scanning fluorescence method, and the change in melting temperature (ΔTm / °C) of the small molecule compound during the heating process was 3.2, while the ΔTm / °C of the compounds 7, 9, 15, 17, 22-35 of the present invention can reach above 3.2, and the stability of binding to the BRD4 protein is significantly improved. At the same time, the affinity (Kd) determination results of the compounds and proteins in Table 2-3 show that the Kd values ​​of multiple compounds with BRD4-BD1 / BD2 are less than 100nM, and the affinity is good. The above results show that the compounds of the present invention can effectively bind to BRD4-BD1 and BD2, and can be used to prepare BRD4 inhibitors.

[0225] Table 2. Affinity determination of compounds for BRD4-BD1 (Kd)

[0226]

[0227] Table 3. Affinity determination of compounds for BRD4-BD2 (Kd)

[0228]

[0229]

[0230] Experimental Example 2: Pharmacodynamic Evaluation Experiment

[0231] 1. Experimental methods

[0232] Collect tumor cells in the logarithmic growth phase, inoculate them into 96-well plates at 3500-4500 cells / 100 μL per well, and culture them in an incubator for 24 hours. Observe the cells under an inverted microscope. If the cells are in good condition, add 100 μL of the required compound at different concentrations to each well. Add only 100 μL of complete culture medium to the control wells and continue to culture them in the incubator. After incubation for 72 hours, add 20 μL of CCK-8 to each well at a ratio of 1:100, incubate for 2-4 hours until the color turns orange, and detect the absorbance value at a wavelength of 450 nm.

[0233] Inhibition rate = (1-OD value of experimental well / OD value of control well) × 100%

[0234] After calculating the inhibition rate of cell proliferation at each concentration of the compound, Graphpad prism 7.0 software was used for statistical analysis to fit the IC 50 value.

[0235] 2. Experimental results

[0236] Table 4. Antiproliferative activity (IC) of representative compounds against various tumor cell lines 50 )

[0237]

[0238] The above results (Table 4) show that the compounds of the present invention can effectively inhibit the proliferation of various tumor cells, and the effect is even better than that of the positive control small molecule JQ1, indicating that the compounds of the present invention can be used to prepare anti-tumor drugs.

[0239] Experimental Example 3: Evaluation of blood-brain barrier permeability

[0240] 1. Experimental methods

[0241] Compound 7 was labeled with radioactive fluorine-18 and injected into male C57 / BL mice via the tail vein. Dynamic positron emission tomography (PET) scanning was performed for 60 minutes and CT scanning was performed for 3 minutes. The dynamic PET data were reconstructed and analyzed. 18 F] Uptake and distribution of compound No. 7 in the mouse brain.

[0242] 2. Experimental results

[0243] As shown in the figure, [ 18 F] Compound 7 rapidly crosses the blood-brain barrier and enters the brain after intravenous injection. The time-activity curve (TAC) shows that [ 18 F] Compound 7 rapidly reached maximum uptake (<1 min after injection) with a standardized uptake value (SUV) of 1.5, followed by rapid washout from the brain (SUV 0.3 at 60 min), demonstrating [ 18F] Rapid elution kinetics of compound 7 in mouse brain.

[0244] In summary, the present invention provides a BRD4 inhibitor and its preparation method and use. The compound synthesized by the present invention is a BRD4 inhibitor with novel structure, excellent pharmacokinetic properties, good efficacy, good drugability, good affinity, high selectivity and good blood-brain barrier penetration ability, which can effectively treat BRD4-related diseases and has good application prospects.

Claims

1. The compound represented by formula I, its tautomer, its stereoisomer, its hydrate, its solvate, its pharmaceutically acceptable salt, its prodrug, and its radiolabeled substance: in, A1 is selected from CH, N; Ring A2 is selected from a 5-6 membered heterocyclic ring which is not substituted or substituted by one or more A3, and A3 is selected from C 1～3 alkyl; A4 is selected from hydrogen, carbonyl; Ring B is unsubstituted or substituted with one or more B ’ Substituted phenyl or 6-membered nitrogen heteroaryl, B ’ Selected from hydrogen, halogen, nitro, hydroxyl, C 1～3 Alkyl, C 1～3 Alkoxy, 3- to 6-membered heterocycloalkyl; Ring C is unsubstituted or substituted with one or more C ’ Substituted 5- to 6-membered heterocyclic ring, C ’ Selected from C 1～3 Alkyl, C 1～3 Alkoxy.

2. The compound according to claim 1, its tautomer, its stereoisomer, its hydrate, its solvate, its pharmaceutically acceptable salt, its prodrug, its radiolabeled substance, characterized in that: The structure of the compound is shown in Formula II: wherein A is selected from NR3, O or CHR3; R3 is selected from hydrogen, C 1～3 alkyl; R4, R5, R6 are independently selected from hydrogen, halogen, nitro, hydroxyl, C 1～3 Alkyl, C 1～3 Alkoxy, 3- to 6-membered heterocycloalkyl; R7 and R8 are independently selected from hydrogen, a 5-6 membered heterocyclic ring which is unsubstituted or substituted by one or more R", and R7 and R8 are not hydrogen at the same time, and R" is selected from C 1～3 Alkyl; the 5-6 membered heterocyclic ring is selected from the following structures:

3. The compound according to claim 1, its tautomer, its stereoisomer, its hydrate, its solvate, its pharmaceutically acceptable salt, its prodrug, its radiolabeled substance, characterized in that: The structure of the compound is shown in Formula III: Among them, R9, R 10 are independently selected from hydrogen, a 5-membered heterocyclic ring which is unsubstituted or substituted with one or more R"', and R9, R 10 are not hydrogen at the same time, R'' is selected from C 1～3 Alkyl; the 5-membered heterocyclic ring is selected from: X1 is halogen.

4. The compound according to claim 1, its tautomer, its stereoisomer, its hydrate, its solvate, its pharmaceutically acceptable salt, its prodrug, and its radiolabeled substance, characterized in that: The structure of the compound is shown in Formula IV: Wherein, B1 and B3 are independently selected from N or CH; B2 is selected from N or CR 13 ; R 11 , R 12 , R 13 are independently selected from hydrogen, halogen, nitro, hydroxyl, C 1～3 Alkyl, 3-6 membered heterocycloalkyl; R 14 , R 15 are independently selected from hydrogen, a 5-6 membered heterocyclic ring which is unsubstituted or substituted by one or more R"", and R 14 , R 15 are not hydrogen at the same time, R"" is selected from C 1～3 Alkyl; the 5-6 membered heterocyclic ring is selected from the following structures:

5. The compound according to claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt, prodrug, radiolabeled substance, characterized in that: The structure of the compound is shown in Formula V: Among them, R9, R 10 independently selected from hydrogen, a 5-6 membered heterocyclic ring which is unsubstituted or substituted by one or more R'', wherein R'' is selected from hydrogen, C 1～3 alkyl; X1 is halogen.

6. The compound according to any one of claims 1 to 5, its tautomer, its stereoisomer, its hydrate, its solvate, its pharmaceutically acceptable salt, its prodrug, or its radiolabeled substance, characterized in that: The compound, its tautomer, its stereoisomer, its hydrate, its solvate, its pharmaceutically acceptable salt, its prodrug, and its radiolabeled substance are selected from one of the following compounds:

7. A method for preparing the compound according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: (1) Compound A reacts with compound B to obtain compound D; (2) Compound D reacts with Compound C to obtain the compound; or Compound D reacts with Compound C to obtain an intermediate, and the intermediate reacts with boron tribromide to obtain the compound; Among them, X a , X b It is a halogen.

8. Use of the compound according to any one of claims 1 to 6, its tautomer, its stereoisomer, its hydrate, its solvate, its pharmaceutically acceptable salt, its prodrug, or its radiolabeled substance in the preparation of a BRD4 inhibitor.

9. The use according to claim 8, characterized in that The BRD4 inhibitor is a drug for preventing and / or treating Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, mood disorders, acute myeloid leukemia, non-small cell lung cancer, prostate cancer, myeloma, breast cancer, glioblastoma, renal cell carcinoma or thyroid cancer.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition is a preparation prepared with the compound according to any one of claims 1 to 6, its tautomer, its stereoisomer, its hydrate, its solvate, its pharmaceutically acceptable salt, its prodrug, or its radioactive marker as the active ingredient, and pharmaceutically acceptable excipients.

Images