Histone acetyltransferase p300 / CBP degradation compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and relates to a histone acetyltransferase p300 / CBP degradation compound. The invention specifically relates to a histone acetyltransferase p300 / CBP degradation compound with a structure as shown in a formula (P-L-D (I)) or derivatives (including stereoisomers, deuterated compounds, solvates, prodrugs, metabolites, pharmaceutically acceptable salts or co-crystals) of the histone acetyltransferase p300 / CBP degradation compound. The invention also relates to a medicinal composition of the compound or the derivative thereof and application of the compound or the derivative thereof in preparation of medicines for treating p300 / CBP related diseases. The compound comprises a part combined with histone acetyltransferase p300 / CBP and a part combined with E3 ubiquitin ligase, can be combined with and degrade p300 / CBP protein in tumor cells, and has a remarkable inhibition effect on growth of the tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to histone acetyltransferase p300 / CBP degradation compounds, and specifically relates to compounds having a structure shown in formula (I) or their stereoisomers, deuterated substances, solvates, prodrugs, metabolites, pharmaceutically acceptable salts or cocrystals, and their use in diseases related to p300 / CBP. Background Art

[0002] The data document that post-translational modifications of proteins, such as acetylation, phosphorylation, methylation, and ubiquitination, participate in the regulation of protein function. Histone acetyltransferase p300 (EP300) and cAMP response element binding protein binding protein (CBP; CREBBP) are two highly homologous histone acetyltransferases (HATs) that have been widely studied. Their main function is to transfer acetyl groups to the amino groups at the ends of lysine residues on the surface of proteins, thereby affecting protein function. Due to the high homology of their sequences and high similarity of their functions, they are often collectively referred to as p300 / CBP. Histones are the main substrates of p300 / CBP. Acetylation of histones regulates the conformation of chromatin and often leads to gene transcriptional activation. Most transcription factors and transcriptional regulatory proteins recruit p300 and / or CBP to promote gene transcription. Other non-histone proteins are also substrates of p300 / CBP, such as p53, MYC, FOXO1, and NF-κB, all of which have important physiological and pathophysiological functions. Since p300 / CBP interacts with a variety of signaling proteins, they are hub proteins in numerous signal transduction pathways. p300 / CBP is involved in a variety of cellular functions, including cell proliferation, differentiation, development, DNA repair, inflammation, metabolism, and memory.

[0003] Studies have shown that abnormal p300 or CBP function is associated with a variety of human diseases. p300 / CBP participates in the occurrence and development of cancer through a variety of mechanisms. In a subtype of acute myeloid leukemia (AML), t(8;16)(p11;p13) recurrent chromatin translocation generates a fusion of MOZ and CBP genes, and the fusion gene product is an oncogenic MOZ-CBP fusion protein. Compared with p300, CBP often mediates the generation of chemotherapy-resistant mixed lineage leukemia cells. A large number of studies have confirmed that p300 / CBP is a transcriptional coactivator of a variety of oncogenic transcription factors, such as MYC, NF-κB, β-catenin, E2F1, and nuclear receptors. Therefore, knocking out p300 and / or CBP can inhibit the occurrence and development of tumors by blocking the function of these oncogenic transcription factors. In addition, p300 / CBP is involved in the regulation of immune cell function, and p300 and CBP are the main transcriptional coactivators of the STAT and NF-κB transcription factor families. Therefore, inhibition of p300 / CBP function can regulate the activity of the immune system and also regulate the interaction between immune cells and tumor cells. In summary, the development of new therapies targeting p300 and CBP is of great significance for the treatment of cancer, immune diseases and other diseases or disorders.

[0004] Proteolysis targeting chimera (PROTAC) is a class of compounds that can simultaneously bind to target proteins and E3 ubiquitin ligases, induce ubiquitination and proteasome-mediated degradation of target proteins, and effectively reduce the content of target proteins in cells. By introducing different target protein ligands into the molecule, PROTAC technology can be applied to the treatment of various diseases, and this technology has received widespread attention in recent years.

[0005] Based on the foundation and current status of the prior art, the inventors of the present application intend to provide a histone acetyltransferase p300 / CBP degradation compound and a preparation method and application thereof. Summary of the invention

[0006] The purpose of the present invention is to provide a histone acetyltransferase p300 / CBP degradation compound and its preparation method and application based on the foundation and current status of the prior art to overcome the defects of the prior art. Specifically, it relates to a compound having a structure shown in the following formula (I) or its derivative (such as its stereoisomer, deuterated substance, solvate, prodrug, metabolite, pharmaceutically acceptable salt or cocrystal), wherein the compound or its derivative comprises a part that binds to histone acetyltransferase p300 / CBP and a part that binds to E3 ubiquitin ligase. The compound or its derivative involved in the present invention can bind to and degrade p300 / CBP protein in tumor cells, has a significant inhibitory effect on tumor cell growth, and has better activity than the clinical drug CCS1477 under investigation.

[0007] The compounds or derivatives of the present invention can act as proteolysis targeting chimeras and can simultaneously bind to target proteins (e.g., p300 / CBP) and E3 ubiquitin ligases, induce ubiquitination and proteasome-mediated degradation of target proteins, and effectively reduce the intracellular content of target proteins, thereby being used to treat various cancers.

[0008] In the first aspect, the present invention relates to a compound or derivative (such as a stereoisomer, deuterated substance, solvate, prodrug, metabolite, pharmaceutically acceptable salt or cocrystal thereof) having a structure represented by the following formula (I):

[0009] P-L-D(Ⅰ);

[0010] in

[0011] P is selected from P1, P2, P3, P4 or P5;

[0012] P1 is P2 is P3 is P4 is P5 is

[0013] L is selected from L1 or L2;

[0014] L1 is selected from a single bond, -(CH2) 2-10 -NH-, -C(=O)-(CH2) 1-10 -NH-, -C(=O)-(CH2CH2O) 1-5 -CH2CH2-NH-,

[0015] -(CH2) 2-10 -O-, -C(=O)-(CH2) 1-10 -O-, -C(=O)-(CH2CH2O) 1-5 -CH2CH2-O-, -(CH2) 2-10 -NH-C(=O)-(CH2) 1-10 -NH-, -C(=O)-(CH2) 1-10 -NH-C(=O)-(CH2) 1-10 -NH-, -C(=O)-(CH2CH2O) 1-5 -CH2CH2-NH-C(=O)-(CH2) 1-10 -NH-, -(CH2) 2-10 -O-, -C(=O)-(CH2) 1-10 -O- or -C(=O)-(CH2CH2O) 1-5 -CH2CH2-O-;

[0016] L2 is selected from -Ak1-Cy1-, -Ak1-Cy1-Ak2-Cy2- or -Ak1-Cy1-Ak2-Cy2-Ak-3-Cy3-;

[0017] Ak1, Ak2 and Ak3 are each independently selected from CH2, C=O or a single bond;

[0018] Cyl, Cy2 and Cy3 are each independently selected from one of the following substituted or unsubstituted groups:

[0019]

[0020] When substituted, it is optionally substituted by 1 to 4 (e.g., 1, 2, 3 or 4) selected from H, F, Cl, Br, I, OH, NH2, oxo (oxy), CF3, COOH, CN, C 1-4 Alkyl, hydroxy substituted C 1-4 Alkyl, halogen substituted C 1-4 Alkyl or C 1-4 Preferably, the C 1-4 The alkyl group is a straight-chain alkyl group, for example, methyl, ethyl, n-propyl or n-butyl.

[0021] The left side of L is connected to P and the right side of L is connected to D;

[0022] D is selected from X is CH2 or C=O.

[0023] Some embodiments of the present invention relate to a compound having a structure as shown in formula (I) or a derivative thereof (such as a stereoisomer, deuterated substance, solvate, prodrug, metabolite, pharmaceutically acceptable salt or cocrystal thereof), wherein

[0024] P is selected from P1, P2, P3, P4 or P5;

[0025] L is selected from -(CH2) 2-10 -、-(CH2) 2-10 -C(=O)-, -C(=O)-(CH2) 2-10 -、-C(=O)-(CH2) 1-10 -C(=O)-,

[0026] -(CH2CH2O) 1-5 -CH2CH2-, -C(=O)-(CH2CH2O) 1-5 -CH2CH2-C(=O)-, -(CH2CH2O) 1-5 -CH2CH2-C(=O)-, -C(=O)-(CH2CH2O)1-5 -CH2CH2-、-(CH2) 2-10 -NH-C(=O)-(CH2) 1-10 -、-(CH2) 2-10 -NH-C(=O)-(CH2) 1-10 -C(=O)-, -C(=O)-(CH2) 1-10 -NH-C(=O)-(CH2) 1-10 -NH- or -C(=O)-(CH2CH2O) 1-5 -CH2CH2-NH-C(=O)-(CH2) 1-10 -C(=O)-;

[0027] The left side of L is connected to P and the right side of L is connected to D.

[0028] In some preferred embodiments, D is Wherein Z is CH3 or H.

[0029] In the present invention, "-(CH2) 2-10 -" means that the number of "CH2" units can be any case from 2 to 10, for example, the number of "CH2" units can be 2, 3, 4, 5, 6, 7, 8, 9 or 10. Similarly, "-(CH2) 1-10 -" means that the number of "CH2" units can be any case from 1 to 10, for example, the number of "CH2" units can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Similarly, "-(CH2CH2O) 1-5 -" indicates that the number of "CH2CH2O" units therein can be 1, 2, 3, 4 or 5.

[0030] Some preferred embodiments of the present invention disclose a compound having a structure represented by formula (I) or a derivative thereof (e.g., a stereoisomer, deuterated substance, solvate, prodrug, metabolite, pharmaceutically acceptable salt or cocrystal thereof), wherein the compound is selected from one of the following structures:

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040] In a second aspect, the present invention provides a pharmaceutical composition comprising the compound described in the first aspect of the present invention or its derivative (such as its stereoisomer, deuterated substance, solvate, prodrug, metabolite, pharmaceutically acceptable salt or cocrystal), and a pharmaceutically acceptable carrier.

[0041] In a third aspect, the present invention provides the use of the compound or its derivative (such as its stereoisomer, deuterated product, solvate, prodrug, metabolite, pharmaceutically acceptable salt or cocrystal) or the pharmaceutical composition described in the second aspect of the present invention in the preparation of a drug for treating diseases related to p300 / CBP.

[0042] In some preferred embodiments, the disease associated with p300 / CBP is a disease associated with the activity or expression of p300 / CBP. In other preferred embodiments, the disease associated with p300 / CBP is a disease associated with the inhibition or degradation of p300 / CBP.

[0043] In some more preferred embodiments, the disease associated with p300 / CBP, such as a disease associated with the activity or expression of p300 / CBP, or a disease associated with the inhibition or degradation of p300 / CBP, is selected from hematological malignancies, leukemia, multiple myeloma, diffuse large B-cell lymphoma, liver cancer, prostate cancer, melanoma, rhabdomyosarcoma, colon cancer, rectal cancer, gastric cancer, cervical cancer, ovarian cancer, breast cancer or pancreatic cancer.

[0044] In a fourth aspect, the present invention provides a method for treating the disease using the composition of the present invention or its derivatives.

[0045] In the present invention, p300 / CBP protein means "p300 protein and / or CBP protein", which includes the following three situations: (1) p300 protein; (2) CBP protein; (3) p300 protein and CBP protein.

[0046] The compound or its derivative (such as its stereoisomer, deuterated product, solvate, prodrug, metabolite, pharmaceutically acceptable salt or cocrystal) described in the present invention comprises a portion that binds to histone acetyltransferase p300 / CBP and a portion that binds to E3 ubiquitin ligase, can bind to and degrade p300 / CBP protein in tumor cells, has a significant inhibitory effect on tumor cell growth, and has better activity than the clinical drug CCS1477 under investigation. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is a schematic diagram of the structure shown in the formula P-L-D (Ⅰ) of the present invention.

[0048] Figure 2 Images showing Western blot analysis of p300 protein in SK-HEP-1 tumor bearing mice treated with 50 mg / kg compound ip at the indicated time points (24 hours post treatment). DETAILED DESCRIPTION

[0049] The following examples further describe the present invention. It should be understood that the above invention content and the following specific embodiments are only exemplary and illustrative, and are not intended to limit the invention to be protected. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

[0050] Example 1: 2-(2,6-dioxypiperidin-3-yl)-4-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)isoindole-1,3-dione (Compound 1)

[0051] Step 1: 4-(4-Fluoro-3-nitrophenyl)-3,5-dimethylisoxazole (Intermediate 1-1)

[0052]

[0053] Pd(dppf)Cl2 (1.81 g, 2.47 mmol), 4-bromo-1-fluoro-2-nitrobenzene (10.87 g, 49.4 mmol) and 3,5-dimethylisoxazole-4-boronic acid pinacol ester (12.68 g, 56.8 mmol) were dissolved in 100 mL of DME. The reaction system was evacuated and filled with nitrogen for degassing 3 times, and then heated at 80 ° C for 10 hours. After the reaction was completed, the reactants were cooled to room temperature, diluted with water (100 mL), and then extracted with EtOAc (100 mL × 3 times). The organic phases were combined, then dried over anhydrous sodium sulfate, the solids were filtered, and the filtrate was evaporated. The residue obtained by filtration was purified by column chromatography (EtOAc: PE = 1: 5) to give a light yellow solid product (8.57 g, yield 73%). 1 HNMR (400MHz, CDCl3) δ7.96(dd,J=7.0,2.5Hz,1H),7.54(ddd,J=8.5,4.0,2.5Hz,1H),7.41(dd,J=10.5,8.5Hz,1H),2.44(s,3H),2.29(s,3H).LC-MS m / z=237.1[M+H] + .

[0054] Step 2: 4-{[4-(3,5-dimethylisoxazol-4-yl)-2-nitrophenyl]amino}piperidine-1-carboxylic acid benzyl ester (Intermediate 1-2)

[0055]

[0056] 4-(4-fluoro-3-nitrophenyl)-3,5-dimethylisoxazole (23.4 g, 99.1 mmol), 4-aminopiperidine-1-carboxylic acid benzyl ester (27.9 g, 118.9 mmol) and potassium carbonate (27.4 g, 198.2 mmol) were dissolved in 500 mL of acetonitrile and heated under reflux for 3 hours. After the reaction was completed, the reactants were cooled, and the reaction system was diluted dropwise with water (100 mL) under vigorous stirring. After the dilution was completed, it was allowed to stand, filtered, and the precipitate was collected to obtain a bright orange solid (45.2 g, yield 79%). 1H NMR (400MHz, CDCl3) δ7.43–7.30(m,4H),7.29–7.23(m,2H),6.73–6.64(m,2H),6.62(d,J=1.9Hz,1H),5.15(s,2H),4.14 (brs,2H),3.58–3.19(m,4H),3.16–2.96(m,1H),2.38(s,3H),2.26(s,3H),2.17–1.99(m,2H),1.54–1.36(m,2H).LC-MS m / z=451.2[M+H] + .

[0057] Step 3: Benzyl 4-{[2-amino-4-(3,5-dimethylisoxazol-4-yl)phenyl]amino}piperidine-1-carboxylate (Intermediate 1-3)

[0058]

[0059] Dissolve 4-{[4-(3,5-dimethylisoxazol-4-yl)-2-nitrophenyl]amino}piperidine-1-carboxylic acid benzyl ester (35.3g, 78.4mmol) in a mixed solvent of 1000mL water and 1000mL tetrahydrofuran, add 29mL concentrated ammonia and sodium dithionite (81.8g, 469.8mmol) to the reaction system in turn, and stir at room temperature for 2 hours. After the reaction is completed, add 500mL ethyl acetate for extraction, and transfer the diluted mixture to a separatory funnel, and wash with 1M NaOH (150mL) and saturated brine (100mL) in turn. Then add anhydrous sodium sulfate to dry, filter the solid, and then evaporate the filtrate to obtain a pink solid, which is directly used in the next step. 1 HNMR (400MHz, CDCl3) δ7.43–7.30(m,4H),7.29–7.23(m,2H),6.73–6.64(m,2H),6.62(d,J=1.9Hz,1H),5.15(s,2H),4.1 4(brs,2H),3.58–3.19(m,4H),3.16–2.96(m,1H),2.38(s,3H),2.26(s,3H),2.17–1.99(m,2H),1.54–1.36(m,2H).LC-MS m / z=421.3[M+H] + .

[0060] Step 4: Benzyl 4-{[4-(3,5-dimethylisoxazol-4-yl)-2-({[(2S)-6-oxyylidenepiperidin-2-yl]carbonyl}amino)phenyl]amino}piperidine-1-carboxylate (Intermediate 1-4)

[0061]

[0062] Benzyl 4-{[2-amino-4-(3,5-dimethylisoxazol-4-yl)phenyl]amino}piperidine-1-carboxylate (32.1 g, 76.4 mmol), (S)-6-oxapiperidine-2-carboxylic acid (10.7 g, 74.8 mmol) and DIPEA (37 mL, 213.9 mmol) were dissolved in 300 mL DMF, and then HATU (29.8 g, 78.4 mmol) was added and stirred at room temperature overnight. After the reaction was completed, 500 mL of water was added, and then extracted with ethyl acetate (500 mL×3). The combined organic phase was washed with 1M HCl, saturated sodium bicarbonate solution and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography (DCM:MeOH=50:1) to obtain a white solid product (38.4 g, yield 92%). 1 H NMR(400MHz, CDCl3)δ8.27(s,1H),7.43–7.30(m,4H),7.29–7.23(m,1H),7.22(s,1H),7.03(dd,J=8 .4,2.0Hz,1H),6.86(d,J=8.4Hz,1H),6.62(d,J=1.9Hz,1H),5.13(s,2H),4.25–4.17(m,1H),4.17–4 .02(brs,2H),4.00–3.87(m,1H),3.52–3.33(m,1H),3.17–2.90(m,2H),2.44–2.38(m,2H),2.38(s, 3H),2.25(s,3H),2.18–2.10(m,2H),2.07–1.97(m,2H),1.92–1.76(m,2H),1.51–1.35(m,2H).LC-MS m / z=546.3[M+H] + .

[0063] Step 5: Benzyl 4-[5-(3,5-dimethylisoxazol-4-yl)-2-[(2S)-6-oxypiperidin-2-yl]benzo[d]imidazol-1-yl]piperidine-1-carboxylate (Intermediate 1-5)

[0064]

[0065] Benzyl 4-{[4-(3,5-dimethylisoxazol-4-yl)-2-({[(2S)-6-oxyylidenepiperidin-2-yl]carbonyl}amino)phenyl]amino}piperidine-1-carboxylate (35.8 g, 65.6 mmol) was dissolved in 328 mL of glacial acetic acid and stirred at 80°C for 16 hours. After the reaction was completed, the reactant was cooled, diluted with 200 mL of water, and then extracted with ethyl acetate (200 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography (DCM:MeOH=50:1) to give a white solid product (27.7 g, yield 80%). 1 H NMR (400MHz, CDCl3) δ7.62(d,J=1.6Hz,1H),7.48(d,J=8.5Hz,1H),7.29–7.23(m,1H),7.22(s,1H) ,7.03(dd,J=8.4,2.0Hz,1H),6.86(d,J=8.4Hz,1H),6.62(d,J=1.9Hz,1H),5.10(s,2H),4.20–4.1 3(m,1H),4.13–3.96(brs,2H),3.52–3.39(m,1H),3.13–2.94(m,2H),2.32(s,3H),2.32–2.24(m,4 H),2.19(s,3H),2.18–2.10(m,2H),2.07–1.97(m,2H),1.92–1.76(m,2H),1.51–1.35(m,2H).LC-MS m / z=528.3[M+H] + .

[0066] Step 6: Benzyl 4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidine-1-carboxylate (Intermediate 1-6)

[0067]

[0068] Benzyl 4-[5-(3,5-dimethylisoxazol-4-yl)-2-[(2S)-6-oxypiperidin-2-yl]benzo[d]imidazol-1-yl]piperidine-1-carboxylate (27.7 g, 52.5 mmol), 3,4-difluorophenylboronic acid (24.9 g, 157.5 mmol), copper acetate monohydrate (12.6 g, 63.0 mmol) and pyridine (21 mL, 262.5 mmol) were dissolved in 2000 mL DCM and stirred at room temperature overnight. After the reaction was completed, the solvent was dried, 200 mL 1M HCl was added, and DCM (200 mL×3) was extracted. The organic phases were combined, washed with saturated brine, then dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography (DCM: MeOH = 50: 1) to obtain a white solid product (30.9 g, yield 92%). 1 HNMR(400MHz, CDCl3)δ7.69(d,J=1.6Hz,1H),7.43–7.34(m,6H),7.16–6.99(m,3H),6 .98–6.92(m,1H),5.24(t,J=5.0Hz,1H),5.20(s,2H),4.42(brs,1H),4.24–4.07(m,1 H),3.01–2.74(m,3H),2.73–2.56(m,1H),2.45(s,3H),2.32–2.24(m,2H),2.32(s,3H ),2.29–2.12(m,3H),1.97–1.85(m,1H),1.85–1.72(m,1H),1.71–1.55(m,2H).LC-MS m / z=640.3[M+H] + .

[0069] Step 7: (6S)-1-(3,4-difluorophenyl)-6-[5-(3,5-dimethylisoxazol-4-yl)-1-(piperidin-4-yl)benzo[d]imidazol-2-yl]piperidin-2-one (Intermediate 1-7)

[0070]

[0071] Benzyl 4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidine-1-carboxylate (24.4 g, 38.1 mmol) and 10% palladium on carbon (2.4 g) were dissolved in 724 mL MeOH and stirred at room temperature for 3 hours under hydrogen. After the reaction was completed, the mixture was filtered through diatomaceous earth and dried to obtain a light gray solid product (19.1 g, yield 99%). 1HNMR(400MHz, CDCl3)δ7.68(d,J=2.1Hz,1H),7.64(d,J=8.1Hz,1H),7.17–7.08(m,2 H),7.07–6.99(m,1H),6.99–6.93(m,1H),5.29(dd,J=6.9,4.2Hz,1H),4.10(p,J=4. 3Hz,1H),3.35–3.18(m,2H),2.93–2.58(m,2H),2.50–2.26(m,5H),2.42–2.32(m,5H ),2.31–2.15(m,4H),1.95–1.82(m,1H),1.80–1.67(m,1H),1.41–1.27(m,1H).LC-MS m / z=506.3[M+H] + .

[0072] Step 8: 2-(2,6-dioxypiperidin-3-yl)-4-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)isoindole-1,3-dione (Compound 1)

[0073]

[0074] (6S)-1-(3,4-difluorophenyl)-6-[5-(3,5-dimethylisoxazol-4-yl)-1-(piperidin-4-yl)benzo[d]imidazol-2-yl]piperidin-2-one (50 mg, 0.1 mmol) and 2-(2,6-dioxo-3-piperidinyl)-4-fluoroisoindole-1,3-dione (30 mg, 0.11 mmol) were dissolved in 2 mL of DMF, and DIPEA (0.87 mL, 0.5 mmol) was added. The reaction mixture was stirred at 90°C for 12 hours. After the reaction was completed, the reaction solution was cooled to room temperature, 50 mL of water was added, and then extracted with ethyl acetate (100 mL×3). The organic phase was washed with 100 mL of water, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (DCM:MeOH=50:1-20:1) to give a yellow solid product (54 mg, yield 71%). 1H NMR (600MHz, DMSO-d6) δ11.09(s,1H),7.79–7.67(m,2H),7.61(d,J=8.5Hz,1H),7.45(d,J=8.4Hz,1H),7.42–7.29(m, 3H),7.22(dd,J=8.4,1.7Hz,1H),7.10–7.03(m,1H),5.81(t,J=4.5Hz,1H),5.13(dd,J=12.9,5.5Hz,1H),4.66(td,J=1 2.3,5.9Hz,1H),3.89(d,J=11.2Hz,1H),3.80(d,J=12.1Hz,1H),3.14(q,J=12.8Hz,2H),2.92–2.80(m,1H),2.59(d,J= 4.5Hz,3H),2.57–2.51(m,1H),2.44–2.36(m,5H),2.27–2.14(m,4H),2.15–1.90(m,5H),1.81(d,J=10.1Hz,1H).LC-MS m / z=762.3[M+H] + .

[0075] Example 2: 2-(2,6-dioxypiperidin-3-yl)-5-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)isoindole-1,3-dione (Compound 2)

[0076]

[0077] The synthesis method is similar to that of Example 1, except that the raw material 2-(2,6-dioxo-3-piperidinyl)-4-fluoroisoindole-1,3-dione is replaced by 2-(2,6-dioxo-3-piperidinyl)-5-fluoroisoindole-1,3-dione. 1H NMR(600MHz,DMSO-d6)δ11.09(s,1H),7.75–7.68(m,2H),7.46–7.40(m,2H),7.40–7.29(m,3H),7.12(dd ,J=8.4,1.7Hz,1H),7.08–7.04(m,1H),5.80(t,J=4.5Hz,1H),5.08(dd,J=12.9,5.5Hz,1H),4.75(td,J=1 2.3,5.9Hz,1H),4.26(d,J=11.2Hz,1H),3.83(d,J=12.1Hz,1H),3.13(q,J=12.8Hz,2H),2.92–2.80(m,1 H),2.63–2.54(m,4H),2.47–2.32(m,5H),2.22–2.16(m,4H),2.14–2.06(m,5H),1.87–1.74(m,1H).LC-MS m / z=762.3[M+H] + .

[0078] Example 3: 2-(2,6-dioxypiperidin-3-yl)-4-[(2-{[3-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)-3-oxyylidenepropyl]oxy}ethyl)amino]isoindole-1,3-dione (Compound 3)

[0079]

[0080] Dissolve tert-butyl 3-[(2-{[2-(2,6-dioxypiperidin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-4-yl]amino}ethyl)oxy]propanoate (45 mg, 0.1 mmol) in 1 mL DCM, add 1 mL TFA, and stir at room temperature for 1 hour. After the reaction is completed, spin dry directly and use it directly in the next step. LC-MS m / z=388.3[M–H] +The above crude product (39 mg, 0.1 mmol) and (6S)-1-(3,4-difluorophenyl)-6-[5-(3,5-dimethylisoxazol-4-yl)-1-(piperidin-4-yl)benzo[d]imidazol-2-yl]piperidin-2-one (55 mg, 0.11 mmol) were dissolved in 2 mL DMF, and DIPEA (0.87 mL, 0.5 mmol) and HTAU (46 mg, 0.12 mmol) were added and stirred at room temperature for 2 hours. After the reaction was completed, 50 mL of water was added, and then extracted with ethyl acetate (100 mL×3). The organic phase was washed with 100 mL of water, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (DCM:MeOH=50:1–20:1) to obtain a yellow solid product (53 mg, two-step yield 61%). 1 H NMR (600MHz, DMSO-d6) δ11.08(s,1H),7.70(d,J=2.1Hz,1H),7.62(d,J=8.5Hz,1H),7.54(q,J=7.9Hz,1H),7.38–7.30(m ,2H),7.18–7.03(m,3H),7.03–6.95(m,1H),6.60(t,J=5.6Hz,1H),5.77(p,J=3.8Hz,1H),5.02(dd,J=12.9,5.5Hz,1H), 4.63(d,J=10.4Hz,2H),4.13–3.91(m,2H),3.79–3.68(m,2H),3.68–3.56(m,3H),3.50–3.44(m,2H),2.86–2.51(m,5H), 2.41–2.28(m,5H),2.28–2.18(m,5H),2.18–2.10(m,1H),2.10–1.99(m,2H),1.99–1.87(m,2H),1.87–1.71(m,2H).LC-MS m / z=877.3[M+H] + .

[0081] Example 4: 2-(2,6-dioxypiperidin-3-yl)-5-[(2-{[3-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)-3-oxyylidenepropyl]oxy}ethyl)amino]isoindole-1,3-dione (Compound 4)

[0082]

[0083] The synthesis method is similar to that of Example 3, except that the raw material tert-butyl 3-[(2-{[2-(2,6-dioxypiperidin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-4-yl]amino}ethyl)oxy]propanoate is replaced by tert-butyl 3-[(2-{[2-(2,6-dioxypiperidin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-5-yl]amino}ethyl)oxy]propanoate. 1 H NMR(600MHz, DMSO-d6)δ11.05(s,1H),7.70(dd,J=5.9,1.7Hz,1H),7.66–7.61(m,1H),7.57–7.50(m,1H),7.38–7.29(m, 2H),7.16–7.08(m,2H),7.08–6.96(m,2H),6.92–6.82(m,1H),5.79–5.75(m,1H),5.79–5.75(m,1H),5.01(dd,J=12.8,5 .5Hz,1H),4.70–4.45(m,3H),4.05(dd,J=36.1,13.7Hz,1H),3.77–3.69(m,2H),3.66–3.58(m,4H),2.86–2.51(m,5H),2 .41–2.29(m,5H),2.28–2.18(m,5H),2.18–2.10(m,1H),2.10–1.99(m,2H),1.99–1.87(m,2H),1.87–1.71(m,2H).LC-MS m / z=877.3[M+H] + .

[0084] Example 5: 2-(2,6-dioxypiperidin-3-yl)-4-{[2-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)-2-oxyylideneethyl]amino}isoindole-1,3-dione (Compound 5)

[0085]

[0086] The synthesis method is similar to that of Example 3, except that the raw material tert-butyl 3-[(2-{[2-(2,6-dioxypiperidin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-4-yl]amino}ethyl)oxy]propanoate is replaced by tert-butyl {[2-(2,6-dioxypiperidin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-4-yl]amino}acetate. 1HNMR(600MHz,DMSO-d6)δ11.11(s,1H),7.77–7.72(m,1H),7.70–7.65(m,1H),7.65–7.59(m,1H), 7.39–7.31(m,4H),7.19–7.12(m,1H),7.12–7.02(m,2H),5.79(p,J=3.8Hz,1H),5.08(dd,J=12.9 ,5.5Hz,1H),4.66(d,J=5.3Hz,2H),4.44–3.89(m,4H),2.94–2.81(m,1H),2.71–2.51(m,4H),2.4 5–2.33(m,5H),2.27–2.17(m,4H),2.15–1.94(m,4H),1.94–1.83(m,1H),1.82–1.72(m,1H).LC-MS m / z=819.3[M+H] + .

[0087] Example 6: 2-(2,6-dioxypiperidin-3-yl)-5-{[2-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)-2-oxyylideneethyl]amino}isoindole-1,3-dione (Compound 6)

[0088]

[0089] The synthesis method is similar to that of Example 3, except that the raw material tert-butyl 3-[(2-{[2-(2,6-dioxypiperidin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-4-yl]amino}ethyl)oxy]propanoate is replaced by tert-butyl {[2-(2,6-dioxypiperidin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-5-yl]amino}acetate. 1HNMR(600MHz,DMSO-d6)δ11.06(s,1H),7.75–7.65(m,2H),7.65–7.57(m,1H),7.38–7.30(m, 2H),7.20–7.12(m,2H),7.12–7.02(m,2H),5.79(p,J=3.8Hz,1H),5.08(dd,J=12.8,5.5Hz,1 H),4.63(d,J=5.3Hz,2H),4.37–3.91(m,4H),2.96–2.85(m,1H),2.70–2.52(m,4H),2.44–2. 36(m,5H),2.26–2.18(m,4H),2.12–1.93(m,4H),1.92–1.83(m,1H),1.82–1.73(m,1H).LC-MS m / z=819.3[M+H] + .

[0090] Example 7: 2-(2,6-dioxypiperidin-3-yl)-4-{[12-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)-12-oxy-3,6,9-trioxadodecane-1-yl]amino}isoindole-1,3-dione (Compound 7)

[0091]

[0092] The synthesis method is similar to that of Example 3, except that the raw material tert-butyl 3-[(2-{[2-(2,6-dioxypiperidin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-4-yl]amino}ethyl)oxy]propanoate is replaced by tert-butyl 3-[(8-{[2-(2,6-dioxypiperidin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-4-yl]amino}-3,6-dioxaoct-1-yl)oxy]propanoate. 1H NMR (600MHz, DMSO-d6) δ11.08(s,1H),7.70(d,J=1.6Hz,1H),7.65(t,J=8.8Hz,1H),7.56(d,J=8.3Hz,1H),7.37–7.28(m,2H),7.17–7.08( m,2H),7.07–7.04(m,2H),7.02(d,J=2.1Hz,1H),6.59(dd,J=8.5,2.1Hz,1H),5.77(q,J=5.4Hz,1H),5.05(dd,J=12.8,5.4Hz,1H),4.68–4. 56(m,2H),4.01(dd,J=31.3,13.6Hz,1H),3.70–3.62(m,2H),3.62–3.55(m,3H),3.55–3.47(m,9H),3.47–3.39(m,4H),2.92–2.81(m,1H),2 .76–2.63(m,2H),2.63–2.51(m,4H),2.43–2.29(m,5H),2.28–2.19(m,5H),2.09–1.90(m,3H),1.89–1.80(m,1H),1.80–1.70(m,1H).LC-MS m / z=965.4[M+H] + .

[0093] Example 8: 2-(2,6-dioxypiperidin-3-yl)-5-{[12-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)-12-oxy-3,6,9-trioxadodecane-1-yl]amino}isoindole-1,3-dione (Compound 8)

[0094]

[0095] The synthesis method is similar to that of Example 3, except that the raw material tert-butyl 3-[(2-{[2-(2,6-dioxypiperidin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-4-yl]amino}ethyl)oxy]propanoate is replaced by tert-butyl 3-[(8-{[2-(2,6-dioxypiperidin-3-yl)-1,3-dioxy-2,3-dihydro-1H-isoindol-5-yl]amino}-3,6-dioxaoct-1-yl)oxy]propanoate. 1H NMR (600MHz, DMSO-d6) δ11.05(s,1H),7.70(d,J=1.6Hz,1H),7.64(dd,J=10.9,8.5Hz,1H),7.54(d,J=8.4Hz,1H),7.42–7.29(m,2H),7.19–7.11(m,2H ),7.08–7.04(m,1H),7.00(d,J=2.0Hz,1H),6.88(dd,J=8.5,2.1Hz,1H),5. 77(q,J=5.4Hz,1H),5.02(dd,J=12.8,5.5Hz,1H),4.62(d,J=21.3Hz,2H),4 .04(dd,J=34.1,13.9Hz,1H),3.70–3.63(m,2H),3.63–3.54(m,3H),3.54–3 .43(m,9H),3.43–3.39(m,4H),2.90–2.81(m,1H),2.76–2.63(m,2H),2.63– 2.51(m,4H),2.43–2.30(m,5H),2.30–2.17(m,5H),2.09–2.01(m,1H),2.00 –1.90(m,1H),1.88–1.81(m,1H),1.81–1.70(m,1H).LC-MSm / z=965.4[M+H] + .

[0096] Example 9: 3-(4-{[2-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxyylidenepiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)-2-oxyylideneethyl]oxy}phenyl)piperidine-2,6-dione (Compound 9)

[0097]

[0098] {[4-(2,6-dioxypiperidin-3-yl)phenyl]oxy}acetic acid (26 mg, 0.1 mmol) and (6S)-1-(3,4-difluorophenyl)-6-[5-(3,5-dimethylisoxazol-4-yl)-1-(piperidin-4-yl)benzo[d]imidazol-2-yl]piperidin-2-one (55 mg, 0.11 mmol) were dissolved in 2 mL DMF, and DIPEA (0.87 mL, 0.5 mmol) and HTAU (46 mg, 0.12 mmol) were added and stirred at room temperature for 2 hours. After the reaction was completed, 50 mL of water was added, and then extracted with ethyl acetate (100 mL×3). The organic phase was washed with 100 mL of water, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (DCM:MeOH=50:1–20:1) to give a white solid product (53 mg, yield 87%). 1 HNMR (600MHz, DMSO-d6) δ11.08(s,1H),7.65(d,J=2.3Hz,1H),7.58(d,J=8.0Hz,1H),7.49(ddd,J=8.0,5.0,2.0 Hz,1H),7.34–7.27(m,2H),7.26–7.20(m,2H),7.07(ddd,J=9.3,5.0,1.9Hz,1H),6.89–6.83(m,2H),5.63(dd,J =6.8,4.2Hz,1H),4.78(s,2H),4.44(p,J=3.8Hz,1H),3.94(ddd,J=12.1,7.3,4.7Hz,2H),3.84–3.78(m,1H),3. 39–3.31(m,2H),2.47–2.40(m,6H),2.30(s,3H),2.20(dtd,J=12.3,7.1,4.2Hz,1H),2.06–1.82(m,10H).LC-MS m / z=751.3[M+H] + .

[0099] Example 10: 2-(2,6-dioxypiperidin-3-yl)-4-[4-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)piperidin-1-yl]isoindole-1,3-dione (Compound 10)

[0100]

[0101] Step 1: 2-methylpropan-2-yl 4-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)piperidine-1-carboxylate (Intermediate 10-1)

[0102]

[0103] (6S)-1-(3,4-difluorophenyl)-6-[5-(3,5-dimethylisoxazol-4-yl)-1-(piperidin-4-yl)benzo[d]imidazol-2-yl]piperidin-2-one (50 mg, 0.1 mmol) and tert-butyl 4-oxopiperidine-1-carboxylate (22 mg, 0.11 mmol) were dissolved in 2 mL DMF, acetic acid (6 μL, 0.1 mmol) was added, and after stirring at room temperature for 10 minutes, NaBH(OAc)3 (64 mg, 0.3 mmol) was added, and stirring at room temperature was continued for 12 hours. After the reaction was completed, 50 mL of saturated sodium bicarbonate solution was added, and then extracted with ethyl acetate (100 mL×3). The organic phase was washed with 100 mL of water, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (DCM:MeOH=50:1) to obtain a white solid product (56 mg, yield 81%). 1 H NMR(400MHz,Chloroform-d)δ7.68(d,J=1.6Hz,1H),7.64(d,J=8.4Hz,1H),7.11(d,J=8.6H z,2H),7.08–6.99(m,1H),6.98–6.92(m,1H),5.25(dd,J=7.0,4.2Hz,1H),4.17–4.06(m,4H ),3.13–2.95(m,2H),2.90–2.79(m,2H),2.68–2.58(m,2H),2.57–2.45(m,2H),2.44–2.33( m,7H),2.32–2.12(m,7H),1.97–1.83(m,2H),1.82–1.55(m,8H),1.54–1.35(m,11H).LC-MS m / z=689.4[M+H] + .

[0104] Step 2: 2-(2,6-dioxypiperidin-3-yl)-4-[4-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)piperidin-1-yl]isoindole-1,3-dione (Compound 10)

[0105]

[0106] Dissolve 4-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)piperidine-1-carboxylic acid-2-methylpropan-2-yl ester in 1 mL DCM, add 1 mL TFA, and stir at room temperature for 1 hour. After the reaction is completed, spin dry directly and use it directly in the next step. LC-MS m / z=589.3[M+H] + .The above crude product (47 mg, 0.08 mmol) and 2-(2,6-dioxo-3-piperidinyl)-4-fluoroisoindole-1,3-dione (30 mg, 0.11 mmol) were dissolved in 2 mL DMF, DIPEA (0.87 mL, 0.5 mmol) was added and dissolved in 2 mL DMF, DIPEA (0.87 mL, 0.5 mmol) was added, and the reaction was stirred at 90 ° C for 12 hours. After the reaction was completed, the reaction solution was cooled to room temperature, 50 mL of water was added, and then extracted with ethyl acetate (100 mL×3). The organic phase was washed with 100 mL of water, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography (DCM: MeOH = 50: 1–20: 1) to obtain a yellow solid product (46 mg, two-step yield 68%). 1 HNMR(600MHz,DMSO-d6)δ11.09(s,1H),7.73–7.64(m,1H),7.40–7.30(m,4H),7.19(dd,J=8.4,1.7Hz, 1H),7.06(dd,J=7.0,2.0Hz,1H),5.77(t,J=4.7Hz,1H),5.09(dd,J=12.8,5.5Hz,1H),4.35(s,1H),4. 03(q,J=7.1Hz,2H),3.78(d,J=11.5Hz,2H),3.04–2.83(m,4H),2.63–2.51(m,4H),2.47–2.29(m,7H), 2.29–2.16(m,4H),2.07–1.95(m,4H),1.93–1.85(m,2H),1.85–1.75(m,2H),1.75–1.66(m,2H).LC-MS m / z=845.4[M+H] + .

[0107] Example 11: 2-(2,6-dioxypiperidin-3-yl)-5-[4-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)piperidin-1-yl]isoindole-1,3-dione (Compound 11)

[0108]

[0109] The synthesis method is similar to that of Example 10, except that the raw material 2-(2,6-dioxo-3-piperidinyl)-4-fluorodihydroisoindole-1,3-dione is replaced by 2-(2,6-dioxo-3-piperidinyl)-5-fluorodihydroisoindole-1,3-dione. 1 HNMR(600MHz,DMSO-d6)δ11.08(s,1H),7.70(d,J=1.7Hz,1H),7.66(dd,J=11.9,8.5Hz,2H),7.40–7.29(m,4H),7.28 –7.23(m,1H),7.18(dd,J=8.4,1.7Hz,1H),7.07–7.03(m,1H),5.75(t,J=4.6Hz,1H),5.07(dd,J=12.8,5.5Hz,1H),4 .35–4.32(m,1H),4.12(d,J=12.8Hz,2H),3.03–2.94(m,4H),2.88(ddd,J=16.9,13.8,5.5Hz,1H),2.69(s,1H),2.63 LC-MS m / z=845.4[M+H] + .

[0110] Example 12: 2-(2,6-dioxypiperidin-3-yl)-4-[3-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)azetidin-1-yl]isoindole-1,3-dione (Compound 12)

[0111]

[0112] The synthesis method is similar to that of Example 10, except that the raw material 4-oxopiperidine-1-carboxylic acid tert-butyl ester is replaced by 3-oxoazetidine-1-carboxylic acid tert-butyl ester.1 H NMR(600MHz,DMSO-d6)δ11.06(s,1H),7.70(d,J=1.7Hz,1H),7.67(d,J=8.5Hz,1H),7.61–7.5 7(m,1H),7.42–7.29(m,2H),7.20–7.12(m,2H),7.05(d,J=9.3Hz,1H),6.82(d,J=8.5Hz,1H), 5.76(t,J=4.6Hz,1H),5.05(dd,J=12.8,5.6Hz,1H),4.02(s,2H),3.03–2.81(m,4H),2.63–2. 51(m,4H),2.44–2.33(m,6H),2.28–2.17(m,5H),2.16–1.91(m,6H),1.86–1.72(m,2H).LC-MS m / z=817.3[M+H] + .

[0113] Example 13: 2-(2,6-dioxypiperidin-3-yl)-5-[3-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)azetidin-1-yl]isoindole-1,3-dione (Compound 13)

[0114]

[0115] The synthesis method is similar to that of Example 12, except that the raw material 2-(2,6-dioxo-3-piperidinyl)-4-fluorodihydroisoindole-1,3-dione is replaced by 2-(2,6-dioxo-3-piperidinyl)-5-fluorodihydroisoindole-1,3-dione. 1HNMR(600MHz,DMSO-d6)δ11.06(s,1H),7.70(d,J=1.7Hz,1H),7.65(dd,J=21.0,7.9Hz,1H),7.41–7.29(m,2H),7 .16(dd,J=8.5,1.7Hz,1H),7.05(ddd,J=9.4,4.1,2.1Hz,1H),6.80(dd,J=17.7,2.1Hz,1H),6.66(ddd,J=18.9,8 .3,2.1Hz,2H),5.80(d,J=6.4Hz,1H),5.09–5.02(m,1H),4.28–4.23(m,1H),3.75(dd,J=9.1,4.4Hz,1H),3.04–2 .81(m,4H),2.62–2.51(m,4H),2.43–2.34(m,6H),2.34–2.18(m,5H),2.17–1.92(m,6H),1.92–1.71(m,2H).LC-MS m / z=817.3[M+H] + .

[0116] Example 14: (2S, 4R)-4-hydroxy-1-[(2S)-2-{[4-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)-1,4-dioxybutyl]amino}-3,3-dimethyl-1-oxybutyl]-N-{[4-(4-methyl-1,3-thiazolyl-5-yl)phenyl]methyl}tetrahydropyrrole-2-carboxamide (Compound 14)

[0117]

[0118] Step 1: tert-Butyl 4-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)-4-oxobutanoate (Intermediate 14-1)

[0119]

[0120] 4-(tert-Butoxy)-4-oxobutanoic acid (18 mg, 0.1 mmol) and (6S)-1-(3,4-difluorophenyl)-6-[5-(3,5-dimethylisoxazol-4-yl)-1-(piperidin-4-yl)benzo[d]imidazol-2-yl]piperidin-2-one (55 mg, 0.11 mmol) were dissolved in 2 mL of DMF, and DIPEA (0.87 mL, 0.5 mmol) and HTAU (46 mg, 0.12 mmol) were added and stirred at room temperature for 2 hours. After the reaction was completed, 50 mL of water was added, and then extracted with ethyl acetate (100 mL×3). The organic phase was washed with 100 mL of water, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (DCM:MeOH=50:1–20:1) to give a white solid product (61 mg, yield 92%). LC-MS m / z=662.3[M+H] + .

[0121] Step 2: (2S,4R)-4-hydroxy-1-[(2S)-2-{[4-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxydimidenepiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)-1,4-dioxydimidenebutyl]amino}-3,3-dimethyl-1-oxydimidenebutyl]-N-{[4-(4-methyl-1,3-thiazolyl-5-yl)phenyl]methyl}tetrahydropyrrole-2-carboxamide (Compound 14)

[0122]

[0123] Dissolve tert-butyl 4-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)-4-oxobutanoate (61 mg, 0.092 mmol) in 1 mL DCM, add 1 mL TFA, and stir at room temperature for 1 hour. After the reaction is completed, spin dry directly and use it directly in the next step. LC-MS m / z=604.2[M–H] +.The above crude product (56 mg, 0.092 mmol) and (2S, 4R)-1-[(2S)-2-amino-3,3-dimethyl-1-oxobutyl]-4-hydroxy-N-{[4-(4-methyl-1,3-thiazolyl-5-yl)phenyl]methyl}tetrahydropyrrole-2-carboxamide (43 mg, 0.1 mmol) were dissolved in 2 mL of DMF, and DIPEA (0.87 mL, 0.5 mmol) and HTAU (46 mg, 0.12 mmol) were added, and stirred at room temperature for 2 hours. After the reaction was completed, 50 mL of water was added, and then extracted with ethyl acetate (100 mL×3). The organic phase was washed with 100 mL of water, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (DCM: MeOH = 50: 1–20: 1) to obtain a white solid product (69 mg, yield 74%). 1 HNMR (600MHz, DMSO-d6) δ8.71(s,1H),8.21(t,J=5.8Hz,1H),7.92(d,J=9.9Hz,1H),7.65(d,J=2.3Hz,1H),7.62–7.55(m,3H),7.49(dd d,J=8.0,5.0,1.9Hz,1H),7.35–7.26(m,4H),7.07(ddd,J=9.2,5.0,1.9Hz,1H),5.45(dd,J=6.9,4.2Hz,1H),4.51(d,J=9.9Hz,1H),4. 44(p,J=3.8Hz,1H),4.40(dd,J=2.2,0.9Hz,4H),4.02(d,J=5.5Hz,1H),3.62(ddd,J=12.1,7.3,4.6Hz,2H),3.47(s,0H),3.40–3.34(m ,1H),3.33–3.25(m,2H),2.60–2.40(m,12H),2.30(s,2H),2.26–2.09(m,3H),2.06–1.94(m,3H),1.94–1.82(m,4H),0.95(s,9H).LC-MS m / z=1018.4[M+H] + .

[0124] Example 15: (2S, 4R)-4-hydroxy-1-[(2S)-2-{[4-(4-{2-[(2S)-1-(3,4-difluorophenyl)-6-oxypiperidin-2-yl]-5-(3,5-dimethylisoxazol-4-yl)benzo[d]imidazol-1-yl}piperidin-1-yl)-1,4-dioxybutyl]amino}-3,3-dimethyl-1-oxybutyl]-N-[(1S)-1-[4-(4-methyl-1,3-thiazolyl-5-yl)phenyl]ethyl]tetrahydropyrrole-2-carboxamide (Compound 15)

[0125]

[0126] The synthesis method is similar to that of Example 14, except that the raw material (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-1-oxybutylene]-4-hydroxy-N-{[4-(4-methyl-1,3-thiazolyl-5-yl)phenyl]methyl}tetrahydropyrrole-2-carboxamide is replaced by (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-1-oxybutylene]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazolyl-5-yl)phenyl]ethyl]tetrahydropyrrole-2-carboxamide. 1 HNMR (600MHz, DMSO-d6) δ8.71(s,1H),8.02(d,J=8.4Hz,1H),7.92(d,J=9.9Hz,1H),7.65(d,J=2.3Hz,1H),7.62–7.55(m,3H),7.49(ddd,J=7. 9,5.0,1.9Hz,1H),7.35–7.22(m,4H),7.07(ddd,J=9.2,5.0,1.9Hz,1H),5.45(dd,J=6.9,4.2Hz,1H),5.04(dq,J=8.7,6.0Hz,1H),4.51(d,J=9 .9Hz,1H),4.44(p,J=3.8Hz,1H),4.40–4.30(m,2H),4.02(d,J=5.5Hz,1H),3.62(ddd,J=12.1,7.3,4.6Hz,2H),3.45(dd,J=12.5,4.6Hz,1H), 3.40–3.33(m,1H),3.33–3.25(m,2H),2.57–2.39(m,12H),2.30(s,3H) ,2.25–2.08(m,3H),2.06–1.83(m,7H),1.48(s,3H),0.95(s,9H).LC-MS m / z=1032.5[M+H] + .

[0127] Example 16: Test of the inhibitory activity of compounds on histone acetyltransferase p300 bromodomain

[0128] TR-FRET technology is used to detect the inhibitory activity of the compounds of the present invention on the bromodomain of p300 / CBP protein. FRET technology utilizes the energy transfer of the fluorescent groups of the energy donor Donor and the energy acceptor Acceptor, and the emission spectrum of the former overlaps with the excitation spectrum of the latter. When the Donor is excited by an external energy source and it is within a sufficiently close distance (1-10nm) with the Acceptor, the energy resonance can be transferred to the Acceptor. When the Acceptor is excited, it will emit emission light of a specific wavelength. The detection is performed using a PerkinElmer kit. In the detection system, 5μL 1X TR-FRETAssay Buffer is used to dilute the compound or negative control (using an equimolar amount of DMSO (dimethyl sulfoxide) to replace the compound as a negative control) to obtain a 3-fold diluted series of compound concentration solutions (3μM, 1μM, 333.3nM, 111.1nM, 37.0nM, 12.3nM, 4.1nM, 1.4nM, 0.4nM). Add 10 μL CBP bromodomain Europium Chelate and incubate in dark for 15 minutes. Add 5 μL CBP bromodomain Ligand / APC Acceptor mixture and incubate in dark for 1 hour. Use a microplate reader to read the fluorescence signal at 340 nm excitation light / 620 nm / 670 nm emission light to calculate IC 50 CCS1477 (CAS: 2222941-37-7) is a positive compound. The experimental results are shown in Table 1.

[0129] Table 1: Inhibitory activity of compounds against the bromodomain of p300 / CBP protein

[0130] Compound <![CDATA[IC 50 ]]> Compound 1 +++ Compound 2 +++ Compound 3 +++ Compound 4 +++ Compound 5 +++ Compound 6 +++ Compound 7 +++ Compound 8 +++ Compound 9 +++ Compound 10 +++ Compound 11 +++ Compound 12 +++ Compound 13 +++ Compound 14 +++ Compound 15 +++ CCS1477 +++

[0131] +++ represents IC 50 <30nM, ++ represents 30nM≤IC 50 <300nM, + represents 300nM≤IC 50 <3000nM.

[0132] Conclusion: The compounds synthesized using the technology of the present invention have significant inhibitory activity on the bromodomain of p300 / CBP protein.

[0133] Example 17: Intracellular p300 / CBP protein degradation experiment

[0134] The degradation activity of the compounds of the present invention on cell p300 protein was detected. SK-HEP-1 cells (human hepatocellular carcinoma cell line, purchased from ATCC) in the logarithmic growth phase were seeded into 96-well plates at an appropriate density. 1 μM of the test compound was added after 24 hours. After 24 hours, the cells were lysed and protein immunoblot analysis was performed. The degradation rate was calculated based on the results. The experimental results are shown in the "p300 degradation rate D%" column of Table 2.

[0135] Conclusion: The compounds synthesized by the technology of the present invention have significant degradation activity on p300 / CBP protein.

[0136] Example 18: CCK8 method to detect tumor cell proliferation inhibition experiment

[0137] The inhibitory effect of the compounds of the present invention on tumor cell proliferation was detected. SK-HEP-1 cells (human hepatocellular carcinoma cell line) in the logarithmic growth phase were seeded into 96-well plates at an appropriate density. After 24 hours, gradient dilutions of the compounds were added. After 6 days of action, 10 μL of cell counting kit-8 (CCK-8) was added to each well. After incubation at 37°C for 2 hours, the OD value at a wavelength of 450 nm was measured with an ELISA reader to calculate the IC 50 The experimental results are shown in Table 2. "SK-HEP-1 cells, IC 50 "List.

[0138] Table 2: Inhibitory activity of compounds on tumor cell proliferation and p300 protein degradation activity

[0139]

[0140]

[0141] +++ represents IC 50 <1μM, ++ represents 1μM≤IC 50 <10μM, + represents 10μM≤IC 50 <100μM;

[0142] A represents D%>60%, B represents 30%≤D%<60%, C represents D%<30%. ND represents not detected.

[0143] Conclusion: The compound synthesized by the technology of the present invention has significant inhibitory activity on the proliferation of tumor cells, and its inhibitory activity is better than that of the clinical drug CCS1477.

[0144] Example 19: Kinetic evaluation of drug effects on tumor tissue protein degradation in a mouse SK-HEP-1 transplanted tumor model

[0145] The pharmacodynamic properties of the compounds of the present invention on the degradation of tumor tissue protein in the mouse SK-HEP-1 cell (human hepatocellular carcinoma cell line) transplanted tumor model were tested. 5×10 6 SK-HEP-1 cells were injected subcutaneously into female nude mice and the tumors were grown to 100 mm 3 After that, the compound was intraperitoneally injected at a dose of 50 mg / kg. After 24 hours of treatment, the tumor tissue was taken for p300 protein immunoblotting analysis, and the degradation rate was calculated based on the obtained results. It was calculated that the degradation rate of the compound of the present invention on the p300 / CBP protein in the tumor tissue of the mouse SK-HEP-1 transplanted tumor model was 33%. The experimental results are shown in Figure 1 .

[0146] Conclusion: The compound synthesized by the technology of the present invention has significant degradation activity on p300 / CBP protein in tumor tissue of mouse SK-HEP-1 transplanted tumor model.

[0147] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details of the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A compound having the structure represented by formula P-L-D (I) or a derivative thereof: ； in P is P1, P2, P3, P4 or P5; P1 is P2 is P3 is P4 is P5 is L is L1 or L2; where, L1 is selected from a single bond, -(CH2) 2-10 -NH-, -C(=O)-(CH2) 1-10 -NH-, -C(=O)-(CH2CH2O) 1-5 -CH2CH2-NH-, -(CH2) 2-10 -O-, -C(=O)-(CH2) 1-10 -O-, -C(=O)-(CH2CH2O) 1-5 -CH2CH2-O-, -(CH2) 2-10 -NH-C(=O)-(CH2) 1-10 -NH-, -C(=O)-(CH2) 1-10 -NH-C(=O)-(CH2) 1-10 -NH-, -C(=O)-(CH2CH2O) 1-5 -CH2CH2-NH-C(=O)-(CH2) 1-10 -NH-, -(CH2) 2-10 -O-, -C(=O)-(CH2) 1-10 -O- or -C(=O)-(CH2CH2O) 1-5 -CH2CH2-O-; L2 is -Ak1-Cy1-, -Ak1-Cy1-Ak2-Cy2- or -Ak1-Cy1-Ak2-Cy2-Ak-3-Cy3-; wherein, Ak1, Ak2 and Ak3 are each independently CH2, C=O or a single bond; Cyl, Cy2 and Cy3 are each independently substituted or unsubstituted one of the following groups: In the case of substitution, the substituents are 1 to 4 selected from H, F, Cl, Br, I, OH, NH2, oxo, CF3, COOH, CN, C 1-4 Alkyl, hydroxy substituted C 1-4 Alkyl, halogen substituted C 1-4 Alkyl or C 1-4 Substituents in an alkoxy group; The left side of L is connected to P and the right side of L is connected to D; D is selected from X is CH2 or C=O.

2. The compound or derivative thereof according to claim 1, wherein: L is selected from -(CH2) 2-10 -、-(CH2) 2-10 -C(=O)-, -C(=O)-(CH2) 2-10 -、-C(=O)-(CH2) 1-10 -C(=O)-, -(CH2CH2O) 1-5 -CH2CH2-, -C(=O)-(CH2CH2O) 1-5 -CH2CH2-C(=O)-, -(CH2CH2O) 1-5 -CH2CH2-C(=O)-, -C(=O)-(CH2CH2O) 1-5 -CH2CH2-, -(CH2) 2-10 -NH-C(=O)-(CH2) 1-10 -, -(CH2) 2-10 -NH-C(=O)-(CH2) 1-10 -C(=O)-, -C(=O)-(CH2) 1-10 -NH-C(=O)-(CH2) 1-10 -NH- or -C(= O)-(CH2CH2O) 1-5 -CH2CH2-NH-C(=O)-(CH2) 1-10 -C(=O)-。 3. The compound or derivative thereof according to claim 2, wherein: D is Z is CH3 or H.

4. The compound or derivative thereof according to any one of claims 1 to 3, wherein The compound is a compound selected from the following compounds:

5. The compound or derivative thereof according to any one of claims 1 to 4, characterized in that The derivative is a stereoisomer, a deuterated substance, a solvate, a prodrug, a metabolite, a pharmaceutically acceptable salt or a co-crystal.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the compound or derivative thereof according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

7. Use of the compound or derivative according to any one of claims 1 to 5 in the preparation of a medicament for treating a disease associated with p300 / CBP.

8. The use according to claim 7, characterized in that: The p300 / CBP-related disease is a disease related to the activity or expression level of p300 / CBP.

9. The use according to claim 7, characterized in that: The p300 / CBP-related disease is a disease related to the inhibition or degradation of p300 / CBP.

10. The use according to any one of claims 7 to 9, characterized in that The p300 / CBP-related disease is selected from hematological malignancies, leukemia, multiple myeloma, diffuse large B-cell lymphoma, liver cancer, prostate cancer, melanoma, rhabdomyosarcoma, colon cancer, rectal cancer, gastric cancer, cervical cancer, ovarian cancer, breast cancer or pancreatic cancer.

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