BRD4 / NAMPT dual-target inhibitor and its application as an anti-hepatocellular carcinoma drug

Abstract

The present invention discloses a BRD4 / NAMPT dual-target inhibitor and its application as an anti-hepatocellular carcinoma drug, wherein the BRD4 / NAMPT dual-target inhibitor is a compound with a structural general formula as shown in Formula I or its pharmaceutically acceptable salt, isomer, metabolite or prodrug: The dual-target inhibitor described in the present invention can simultaneously inhibit the activities of BRD4 and NAMPT and is used for preparing an anti-hepatocellular carcinoma drug. The dual-target inhibitor provided by the present invention can effectively overcome the problem of resistance to hepatocellular carcinoma of a single NAMPT inhibitor and has good application prospects in the clinical treatment of hepatocellular carcinoma.

Description

Technical Field

[0001] The present invention belongs to the technical field of dual-target compounds and their new pharmaceutical uses, and particularly relates to a BRD4 / NAMPT dual-target inhibitor, a preparation method thereof, and an application as an anti-hepatocellular carcinoma drug. Background Art

[0002] Nicotinamide adenine dinucleotide (NAD + ) is a coenzyme that transfers hydrogen ions and participates in many important physiological activities such as cell metabolism, energy synthesis, and DNA repair. The energy and material requirements of tumor cells are much greater than those of normal cells. Therefore, tumor cells are more dependent on NAD + , and are more sensitive to changes in key enzymes that control the NAD + biosynthetic pathway. Current research shows that tumor cells obtain NAD + through the main salvage synthesis pathway, and nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme of this pathway. NAMPT can directly regulate cell metabolism by controlling the synthesis of NAD + , and can also indirectly regulate cell metabolism by affecting the activity of NAD-dependent enzymes and upregulating the level of NADPH to improve the survival ability of cells. NAMPT is highly expressed in various solid tumors and hematological tumors. Therefore, NAMPT may be a potential target for cancer treatment. In the human body, NAMPT is mainly produced and released from the liver. Compared with normal hepatocytes, the NAMPT activity in hepatocellular carcinoma cells is about 3 times higher. Therefore, targeting NAMPT is an effective means for HCC treatment. From the perspective of the mechanism of action, NAMPT inhibitors can indirectly regulate the activity of NAD-dependent proteins by regulating the intracellular NAD level, and inhibit the proliferation, migration, and invasion of hepatocellular carcinoma cells. Although NAMPT inhibitors are a class of effective anti-HCC drugs, however, hepatocellular carcinoma cells inhibited by NAMPT can acquire drug resistance by activating the NAPRT enzyme in the PH pathway, which greatly reduces the therapeutic effect of NAMPT inhibitors. Developing a combined preparation that dual-inhibits NAMPT and NAPRT is of great significance for the development of new anti-HCC tumor drugs.

[0003] Bromodomain-containing protein 4 (BRD4) can recognize and bind to acetylated lysine residues at the N-terminal tail of histones through two domains (BD1 and BD2), participate in the transmission of epigenetic histone post-translational modification acetylation signals, regulate downstream gene transcription, and play an important role in cell cycle, cell differentiation, and signal transduction processes. BRD4 is highly expressed in most malignant tumors such as lung cancer, breast cancer, acute myeloid lymphoma, and liver cancer. Its downstream genes such as c-Myc, CDK, and BCL2 are closely related to the occurrence and development of tumors. Therefore, it is considered one of the ideal targets for cancer treatment. BRD4 inhibitors can competitively bind to the acetyl-lysine binding site of BRD4, block the binding of BRD4 to histone acetylated lysine, and antagonize the promoting effect of BRD4 on the occurrence and development of tumors, thereby inhibiting tumor proliferation and metastasis. In recent years, many studies have shown that BRD4 inhibitors can inhibit the proliferation, migration, and invasion of HCC cells and induce apoptosis of HCC cells. Targeting BRD4 is a promising HCC treatment strategy. It is noteworthy that while BRD4 inhibitors inhibit the transcription of genes such as c-MYC and YAP1, they can also reduce the expression of NAPRT. When combined with NAMPT inhibitors, they can significantly reduce the NAD + level in HCC cells, showing good synergistic anti-proliferative activity against HCC cells.

[0004] Multi-target drugs can reduce toxicity, increase efficacy, and are expected to solve the problem of drug resistance. In addition, multi-target drugs can avoid the defects of combination drug use. In the present invention, active fragments of BRD4 inhibitors and NAMPT inhibitors are rationally spliced to obtain a BRD4 / NAMPT dual-target inhibitor, which is expected to solve the problem of drug resistance generated by NAMPT inhibitors in the treatment of liver cancer and the problem of relatively high toxicity of NAMPT inhibitors. Summary of the Invention

[0005] The object of the present invention is to provide a highly efficient and low-toxic BRD4 / NAMPT dual-target inhibitor, its preparation method, and its application as an anti-liver cancer drug.

[0006] The present invention adopts the following technical solution to achieve the above object: A BRD4 / NAMPT dual-target inhibitor, characterized in that: the BRD4 / NAMPT dual-target inhibitor is a compound represented by the general structural formula as shown in Formula I or a pharmaceutically acceptable salt thereof:

[0007]

[0008] Where R is selected from

[0009] The L linking group is selected from:

[0010]

[0011] Preferably, the compounds represented by Formula I include:

[0012]

[0013] In a second aspect of the present invention, there is provided a method for preparing the above-mentioned BRD4 / NAMPT dual-target inhibitor. The synthetic route of the dual-target compound in this embodiment is as follows:

[0014] Synthetic Route 1:

[0015]

[0016] Synthetic Route 2:

[0017]

[0018] Synthetic Route 3:

[0019]

[0020] For the specific method of compound synthesis, it will be further described in detail in the following example section.

[0021] In one embodiment, the present invention relates to a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and one or more pharmaceutical carriers. Preferably, the pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, ointment, suppository or patch.

[0022] In one embodiment, the pharmaceutical composition of the present invention further comprises other therapeutic drugs. Preferably, the other therapeutic drugs are one or more of tumor chemotherapy drugs, tumor-targeted drugs, tumor immunotherapy drugs or tumor drug conjugates.

[0023] Another embodiment relates to the use of the compound or pharmaceutical composition represented by Formula I in the BRD4 / NAMPT dual-target inhibitor of the present invention as an anti-hepatocellular carcinoma drug.

[0024] The compounds of the present invention were tested for NAMPT and BRD4 inhibitory activities, and it was demonstrated that most compounds showed high inhibitory activities against both targets. Among them, the preferred compounds showed high and balanced inhibitory activities against both targets. Further, through in vitro anti-proliferative activity tests, the preferred compound A2 showed good inhibitory activity against HCC cells and high selectivity for normal hepatocytes (LX-2). Compared with the prior art, the present invention has the following remarkable advantages: The dual-target compounds described in the present invention can effectively overcome the problem of resistance to hepatocellular carcinoma of single NAMPT inhibitors and have good application prospects in clinical hepatocellular carcinoma treatment. Detailed Embodiments

[0025] The present invention will be further described below in conjunction with embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0026] Synthesis Route 1:

[0027]

[0028] Synthesis Route 2:

[0029]

[0030] Synthesis Route 3:

[0031]

[0032] Example 1

[0033] Preparation of (6S)-4-(4-chlorophenyl)-N-[2-[[(2E)-3-(3-pyridinyl)-1-oxo-2-propen-1-yl]amino]ethyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-acetamide (A1):

[0034] Using Route 1, add trans-3-(3-pyridinyl)acrylic acid (596 mg, 4 mmol), mono-Boc-ethylenediamine (640 mg, 4 mmol), EDCI (1.15 g, 6 mmol), HOBt (810 mg, 6 mmol), anhydrous DIPEA (12 mmol) and DMF (10 mL) to a 100 mL round-bottom flask, and react at room temperature for 24 hours. After the reaction is completed, add 20 mL of water, extract three times with 60 mL of CH2Cl2, and the organic layer is washed successively with saturated NaHCO3 and brine, dried over anhydrous Na2SO4, filtered, concentrated and purified by column chromatography to obtain tert-butyl 2-((E)-3-(pyridin-3-yl)acrylamido)ethylcarbamate (565 mg, yield 51%).

[0035] Add tert-butyl 2-((E)-3-(pyridin-3-yl)acrylamido)ethylcarbamate (554 mg, 2 mmol) and ethyl acetate solution of 4M HCl (10 mL) to a 100 mL round-bottom flask, and react at room temperature for 8 hours. After the reaction is completed, filter and dry to obtain the compound (E)-N-(2-aminoethyl)-3-(pyridin-3-yl)acrylamide (227 mg, yield 50%), and the product is directly used in the next reaction without purification.

[0036] In a 100 mL round-bottom flask, (E)-N-(2-aminoethyl)-3-(pyridin-3-yl)acrylamide (48 mg, 0.25 mmol), JQ1-COOH (100 mg, 0.25 mmol), HATU (143 mg, 0.375 mmol), anhydrous DIPEA (0.75 mmol) and DMF (5 mL) were added, and the reaction was carried out at room temperature for 24 hours. After the reaction was completed, 5 mL of water was added, and the mixture was extracted three times with 15 mL of CH2Cl2. The organic layer was washed successively with saturated NaHCO3 and brine, dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography to obtain (6S)-4-(4-chlorophenyl)-N-[2-[[(2E)-3-(3-pyridinyl)-1-oxo-2-propen-1-yl]amino]ethyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A1, 125 mg, yield 87%).

[0037] 1 H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.77 (d, 1H, J = 2.4 Hz), 8.55 (dd, 1H, J = 4.8, 1.6 Hz), 8.36 (t, 1H, J = 5.6 Hz), 8.21 (t, 1H, J = 4.8 Hz), 8.01–7.99 (m, 1H), 7.50–7.41 (m, 6H), 6.81 (d, 1H, J = 16.0 Hz), 4.52 (t, 1H, J = 7.6 Hz), 3.32–3.25 (m, 6H), 2.60 (s, 3H), 2.41 (s, 3H), 1.61 (s, 3H); 13 C NMR (DMSO-d6, 100 MHz) δ (ppm): 170.37, 165.22, 163.58, 155.66, 150.60, 150.38, 149.60, 137.22, 135.92, 135.70, 134.46, 132.73, 131.19, 131.16, 130.65, 130.33, 130.07, 128.95, 124.65, 124.45, 54.27, 39.20, 38.78, 38.18, 14.52, 13.15, 11.79; HRMS-ESI (m / z): calcd for C 29 H 29 ClN7O2S [M + H] + 574.1786, found: 574.1779.

[0038] Example 2

[0039] (6S)-4-(4-Chlorophenyl)-N-[3-[[(2E)-3-(3-pyridinyl)-1-oxo-2-propen-1-yl]amino]propyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A2) Preparation:

[0040] Using the synthesis method of Example 1 and replacing the corresponding raw materials, the target compound can be synthesized.

[0041] 1 H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.76 (d, 1H, J = 2.2 Hz), 8.56–8.55 (m, 1H), 8.30–8.21 (m, 2H), 8.00–7.98 (m, 1H), 7.49–7.41 (m, 6H), 6.77 (d, 1H, J = 16.0 Hz), 4.52 (dd, 1H, J = 8.4, 6.0 Hz), 3.31–3.11 (m, 6H), 2.60 (s, 3H), 2.41 (s, 3H), 1.68–1.62 (m, 5H); 13 C NMR (DMSO-d6, 100 MHz) δ (ppm): 170.08, 165.02, 163.58, 155.59, 150.46, 150.36, 149.46, 137.39, 137.22, 135.69, 134.56, 132.73, 131.24, 131.20, 130.63, 130.32, 130.03, 128.96, 124.72, 124.52, 54.33, 38.11, 37.21, 36.94, 29.75, 14.53, 13.15, 11.77; HRMS-ESI (m / z): calcd for C 30 H 31 ClN7O2S [M+H] + 588.1943, found: 588.1975.

[0042] Example 3

[0043] (6S)-4-(4-Chlorophenyl)-N-[4-[[(2E)-3-(3-pyridinyl)-1-oxo-2-propen-1-yl]amino]butyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A3) Preparation:

[0044] Using the synthesis method of Example 1 and replacing the corresponding raw materials, the target compound can be synthesized.

[0045] 1 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.74 (d, 1H, J = 2.0 Hz), 8.55 (dd, 1H, J = 4.8, 1.6 Hz), 8.26 - 8.20 (m, 2H), 7.97 (dt, 1H, J = 8.0, 2.0 Hz), 7.50–7.40 (m, 6H), 6.73 (d, 1H, J = 15.6 Hz), 4.50 (dd, 1H, J = 8.4, 6.0 Hz), 3.26–3.10 (m, 6H), 2.59 (s, 3H), 2.40 (s, 3H), 1.60 (s, 3H), 1.50 (d, 4H, J = 5.2 Hz); 13 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 169.95, 164.94, 163.53, 155.60, 150.53, 150.33, 149.52, 137.22, 135.70, 135.66, 134.38, 132.72, 131.19, 130.57, 130.30, 130.03, 128.95, 126.42, 124.70, 124.44, 54.35, 38.96, 38.71, 38.12, 27.24, 27.04, 14.50, 13.13, 11.76; HRMS-ESI (m / z): calcd for C 31 H 32 ClN7NaO2S [M+Na] + 624.1919, found: 624.1932.

[0046] Example 4

[0047] (6S)-4-(4-Chlorophenyl)-N-[5-[[(2E)-3-(3-Pyridinyl)-1-oxo-2-propen-1-yl]amino]pentyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A4) Preparation:

[0048] Using the synthesis method of Example 1 and replacing the corresponding raw materials can synthesize the target compound.

[0049] 11H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.74 (d, 1H, J = 2.4 Hz), 8.54 (dd, 1H, J = 4.8, 1.6 Hz), 8.21 (dt, 2H, J = 15.6, 5.6 Hz), 7.96 (d, 1H, J = 8.0 Hz), 7.51–7.40 (m, 6H), 6.73 (d, 1H, J = 16.0 Hz), 4.51 (dd, 1H, J = 8.0, 6.0 Hz), 3.29–3.07 (m, 6H), 2.59 (s, 3H), 2.40 (s, 3H), 1.61 (s, 3H), 1.51–1.44 (m, 4H), 1.38–1.32 (m, 2H); 13 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 169.93, 164.90, 163.53, 155.59, 150.50, 150.33, 149.51, 137.22, 135.72, 135.57, 134.38, 132.72, 131.21, 131.19, 130.58, 130.30, 130.03, 128.95, 124.74, 124.45, 54.37, 39.14, 38.83, 38.12, 29.37, 29.18, 24.23, 14.54, 13.14, 11.77; HRMS-ESI (m / z): calcd for 32 C 35 H + ClN7O2S [M + H]

[0050] Example 5

[0051] (6S)-4-(4-chlorophenyl)-N-[6-[[(2E)-3-(3-pyridinyl)-1-oxo-2-propen-1-yl]amino]hexyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A5) Preparation:

[0052] Using the synthesis method of Example 1 and replacing the corresponding raw materials can synthesize the target compound.

[0053] 11H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.74 (d, 1H, J = 2.4 Hz), 8.54 (dd, 1H, J = 4.8, 1.6 Hz), 8.19 (dt, 2H, J = 16.4, 5.6 Hz), 7.96 (dt, 1H, J = 8.0, 2.0 Hz), 7.50–7.40 (m, 6H), 6.72 (d, 1H, J = 16.0 Hz), 4.50 (dd, 1H, J = 8.0, 4.0 Hz), 3.35–3.07 (m, 6H), 2.59 (s, 3H), 2.40 (s, 3H), 1.61 (s, 3H), 1.45 (s, 4H), 1.33–1.32 (m, 4H); 13 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 169.93, 164.88, 163.53, 155.59, 150.51, 150.32, 149.51, 137.21, 135.73, 135.59, 134.38, 132.70, 131.20, 130.57, 130.30, 130.03, 128.93, 124.72, 124.44, 54.37, 39.07, 38.84, 38.12, 29.65, 29.52, 26.58, 26.46, 14.50, 13.12, 11.74; HRMS-ESI (m / z): calcd for 33 C 36 H + ClN7NaO2S [M+Na]

[0054] Example 6

[0055] (6S)-4-(4-Chlorophenyl)-N-[5-[[(2E)-3-(3-Pyridinyl)-1-oxo-2-propen-1-yl]amino]ethoxyethyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A6) Preparation:

[0056] Using the synthesis method of Example 1, replacing the corresponding raw materials can synthesize the target compound.

[0057] 11H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.70 (d, 1H, J = 24 Hz), 8.53 (dd, 1H, J = 4.8, 1.6 Hz), 8.36 (t, 1H, J = 5.6 Hz), 8.25 (t, 1H, J = 5.6 Hz), 7.92 (dt, 1H, J = 8.0, 2.0 Hz), 7.49–7.38 (m, 6H), 6.78 (d, 1H, J = 15.6 Hz), 4.53 (dd, 1H, J = 8.4, 5.6 Hz), 3.55–3.3.48 (m, 4H), 3.43 - 3.38 (m, 2H), 3.37–3.31 (m, 2H), 3.26–3.20 (m, 2H), 2.59 (s, 3H), 2.35 (s, 3H), 1.58 (d, 3H, J = 0.8 Hz); 13 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 170.42, 165.12, 163.56, 155.56, 150.52, 150.33, 149.55, 137.25, 135.85, 135.70, 134.27, 132.73, 131.13, 130.64, 130.32, 129.98, 128.91, 124.46, 124.41, 69.53, 69.27, 54.32, 39.28, 38.99, 38.08, 14.57, 13.10, 11.78; HRMS-ESI (m / z): calcd for C 31 H 33 ClN7NaO3S [M+Na] + 640.1868 found: 640.1873.

[0058] Example 7

[0059] (6S)-4-(4-Chlorophenyl)-N-[8-[[(2E)-3-(3-Pyridyl)-1-oxo-2-propen-1-yl]amino]ethoxyethoxyethyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-acetamide (A7) Preparation:

[0060] Using the synthesis method of Example 1, replacing the corresponding raw materials can synthesize the target compound.

[0061] 11H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.74 (d, 1H, J = 4.0 Hz), 8.54 (dd, 1H, J = 4.8, 2.0 Hz), 8.29 (dt, 2H, J = 24.8, 5.6 Hz), 7.97 (dt, 1H, J = 8.0, 2.0 Hz), 7.50–7.41 (m, 6H), 6.79 (d, 1H, J = 16 Hz), 4.51 (dd, 1H, J = 8.0, 6.4 Hz), 3.56–3.46 (m, 9H), 3.32–3.21 (m, 5H), 2.59 (s, 3H), 2.40 (s, 3H), 1.61 (s, 3H); 13 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 170.23, 165.10, 163.53, 155.58, 150.49, 150.33, 149.52, 137.22, 135.82, 135.71, 134.45, 132.73, 131.19, 130.63, 130.30, 128.93, 124.59, 124.46, 70.07, 69.68, 69.58, 54.29, 40.34, 39.30, 39.11, 37.97, 14.52, 13.14, 11.76; HRMS-ESI (m / z): calcd for C 33 H 37 ClN7O4S [M+H] + 662.2311 found: 662.2319.

[0062] Example 8

[0063] (6S)-4-(4-Chlorophenyl)-N-[1-[[(2E)-3-(3-Pyridinyl)-1-oxo-2-propen-1-yl]amino]piperidin-4-yl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A8) Preparation:

[0064] Using the synthesis method of Example 1, the target compound can be synthesized by replacing the corresponding raw materials. 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.78–8.76 (m, 1H), 8.56 (d, 1H, J = 4.8 Hz), 8.28 (dd, 1H, J = 10.4, 7.6 Hz), 8.01–7.97 (m, 1H), 7.52–7.44 (m, 6H), 6.75 (dd, 1H, J = 16.0, 2.4 Hz), 4.59 (t, 1H, J = 6.8 Hz), 4.25 (d, 1H, J = 12.0 Hz), 4.05 (d, 2H, J = 48.8 Hz), 3.70–3.62 (m, 1H), 3.46–3.41 (m, 1H), 3.37–3.29 (m, 1H), 2.91–2.83 (m, 1H), 2.61 (s, 3H), 2.42 (s, 3H), 1.89 (d, 2H, J = 48.0 Hz), 1.64 (s, 3H), 1.53–1.47 (m, 1H), 1.31–1.23 (m, 1H); 13 C NMR (DMSO-d6, 100 MHz) δ (ppm): 168.41, 164.29, 163.35, 155.81, 150.58, 150.24, 149.55, 137.29, 137.24, 135.93, 135.87, 135.68, 134.42, 132.67, 131.22, 131.17, 130.64, 130.38, 130.14, 128.97, 124.74, 124.48, 54.75, 46.53, 4,4.29, 44.17, 35.22, 32.44, 31.74, 14.50, 13.17, 11.75, HRMS-ESI (m / z): calcd for C 32 H 33 ClN7O4S [M + H] + 614.2099.

[0065] Example 9

[0066] (6S)-4-(4-Chlorophenyl)-N-[1-[[(2E)-3-(3-Pyridyl)-1-oxo-2-propen-1-yl]amino]methyl]piperidin-4-yl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-acetamide (A9) Preparation:

[0067] Using the synthesis method of Example 1, the target compound can be synthesized by replacing the corresponding raw materials.

[0068] 1 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.77 (d, 1H, J = 2.4 Hz), 8.55 (dd, 1H, J = 4.8, 1.6 Hz), 8.28 (t, 1H, J = 5.6 Hz), 8.00–7.97 (m, 1H), 7.52–7.42 (m, 6H), 6.78 (d, 1H, J = 15.6 Hz), 4.58 (t, 1H, J = 6.4 Hz), 4.38 (d, 1H, J = 12.8 Hz), 4.17 (d, 1H, J = 13.6 Hz), 3.64–3.57 (m, 1H), 3.44–3.39 (m, 1H), 3.16–3.11 (m, 3H), 2.63–2.55 (m, 4H), 2.42 (s, 3H), 1.81–1.63 (m, 3H), 1.63 (s, 3H), 1.27–1.20 (m, 1H), 1.04–0.93 (m, 1H); 13 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 168.34, 165.13, 165.10, 155.83, 150.55, 150.23, 149.55, 137.30, 137.25, 135.77, 135.74, 135.67, 134.42, 132.68, 131.24, 131.16, 130.63, 130.37, 130.13, 128.97, 124.76, 124.71, 124.46, 54.46, 45.42, 44.55, 41.61, 36.48, 35.27, 30.68, 29.96, 14.50, 13.16, 11.75; HRMS-ESI (m / z): calcd for C 33 H 35 ClN7O2S [M + H] + 628.2256 found: 628.2239.

[0069] Example 10

[0070] (6S)-4-(4-Chlorophenyl)-N-[1-[[[(2E)-3-(3-Pyridyl)-1-oxo-2-propen-1-yl]amino]ethyl]piperidin-4-yl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A10) Preparation:

[0071] Using the synthesis method of Example 1, replacing the corresponding raw materials can synthesize the target compound.

[0072] 11H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.77 (d, 1H, J = 2.0 Hz), 8.56 (dd, 1H, J = 4.8, 1.6 Hz), 8.22 (d, 1H, J = 4.8 Hz), 7.99 (dt, 1H, J = 8.0, 2.0 Hz), 7.51–7.42 (m, 6H), 6.74 (dd, 1H, J = 16.0, 1.6 Hz), 4.58 (t, 1H, J = 6.4 Hz), 4.37 (d, 1H, J = 13.6 Hz), 4.15 (d, 1H, J = 13.6 Hz), 3.65–3.58 (m, 1H), 3.42–3.39 (m, 1H), 3.29–3.23 (m, 2H), 3.13–3.07 (m, 1H), 2.63–2.55 (m, 4H), 2.42 (s, 3H), 1.82 (d, 1H, J = 12.8 Hz), 1.71 (s, 1H), 1.62 (d, 4H, J = 11.2 Hz), 1.44 (t, 2H, J = 6.0 Hz), 1.26–1.17 (m, 1H), 1.00–0.93 (m, 1H); 13 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 168.24, 164.89, 163.36, 163.28, 155.83, 150.52, 150.22, 149.54, 137.30, 137.25, 135.68, 135.64, 134.43, 132.67, 131.24, 131.15, 130.62, 130.38, 130.35, 130.13, 128.96, 124.78, 124.46, 54.76, 45.76, 41.94, 36.65, 36.23, 35.25, 33.47, 32.55, 32.05, 14.49, 13.16, 11.75; HRMS-ESI (m / z): calcd for C 34 H 37 ClN7O2S [M + H] + 642.2412 found: 642.2410.

[0073] Example 11

[0074] (6S)-4-(4-Chlorophenyl)-N-[1-[[[(2E)-3-(3-Pyridinyl)-1-oxo-2-propen-1-yl]amino]propyl]piperidin-4-yl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A11) Preparation:

[0075] Using the synthesis method of Example 1, the target compound can be synthesized by replacing the corresponding raw materials.

[0076] 1 H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.76 (d, 1H, J = 2.4 Hz), 8.55 (dd, 1H, J = 4.8, 1.6 Hz), 8.21 (d, 1H, J = 4.4 Hz), 7.98 (d, 1H, J = 8.0 Hz), 7.50–7.42 (m, 6H), 6.73 (d, 1H, J = 16.0 Hz), 4.57 (t, 1H, J = 6.8 Hz), 4.37 (d, 1H, J = 12.8 Hz), 4.13 (d, 1H, J = 13.6 Hz), 3.64–3.56 (m, 1H), 3.39 (d, 1H, J = 6.8 Hz), 3.20–3.16 (m, 2H), 3.10 (d, 1H, J = 4.0 Hz), 2.62–2.55 (m, 4H), 2.41 (s, 3H), 1.77 (d, 1H, J = 12.4 Hz), 1.65 (d, 4H, J = 20.0 Hz), 1.53–1.48 (m, 3H), 1.26 (s, 2H), 1.21–1.15 (m, 1H), 0.96–0.85 (m, 1H); 13 C NMR (DMSO-d6, 100 MHz) δ (ppm): 168.19, 164.87, 163.35, 155.81, 150.54, 150.23, 149.56, 135.66, 135.61, 134.40, 132.67, 131.23, 131.15, 130.64, 130.37, 130.11, 128.96, 126.44, 124.78, 124.47, 54.73, 45.77, 41.97, 40.58, 35.61, 35.24, 33.75, 32.93, 32.19, 26.74, 14.51, 13.17, 11.76; HRMS-ESI (m / z): calcd for C 35 H 39 ClN7O2S [M + H] + 656.2569 found: 656.2572.

[0077] Example 12

[0078] (6S)-4-(4-Chlorophenyl)-N-[1-[[[(2E)-3-(3-Pyridinyl)-1-oxo-2-propen-1-yl]amino]butyl]piperidin-4-yl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A12) Preparation:

[0079] Using the synthesis method of Example 1 and replacing the corresponding raw materials can synthesize the target compound.

[0080] 1 H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.76 (s, 1H), 8.55 (d, 1H, J = 4.8 Hz), 8.19 (t, 1H, J = 6.0 Hz), 7.98 (dt, 1H, J = 8.0, 2.0 Hz), 7.50–7.42 (m, 6H), 6.73 (d, 1H, J = 16.0 Hz), 4.57 (t, 1H, J = 6.8 Hz), 4.36 (d, 1H, J = 12.8 Hz), 4.13 (d, 1H, J = 13.2 Hz), 3.64–3.56 (m, 1H), 3.38 (d, 1H, J = 8.8 Hz), 3.19 (dd, 2H, J = 13.6, 6.8 Hz), 3.11 (d, 1H, J = 14.4 Hz), 2.62–2.54 (m, 4H), 2.42 (s, 3H), 1.77 (d, 1H, J = 12.8 Hz), 1.63 (s, 4H), 1.46 (d, 3H, J = 7.2 Hz), 1.35–1.33 (m, 2H), 1.27–1.14 (m, 3H), 0.96–0.85 (m, 1H); 13 C NMR (DMSO-d6, 100 MHz) δ (ppm): 168.18, 164.85, 163.34, 155.82, 150.54, 150.23, 149.56, 137.23, 135.66, 135.57, 134.39, 132.67, 131.24, 131.15, 130.64, 130.38, 130.34, 130.11, 128.96, 124.80, 124.47, 54.69, 45.80, 41.98, 36.10, 35.81, 35.25, 32.93, 32.77, 32.24, 29.78, 24.03, 14.51, 13.17, 11.76; HRMS-ESI (m / z): calcd for C 36 H 41 ClN7O2S [M + H] + 670.2725 found: 670.2738.

[0081] Example 13

[0082] (6S)-4-(4-Chlorophenyl)-N-[4-[[[(2E)-3-(3-Pyridyl)-1-oxo-2-propen-1-yl]amino]methyl]benzyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A16) Preparation:

[0083] Using the synthesis method of Example 1 and replacing the corresponding raw materials can synthesize the target compound.

[0084] 1 H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.78–8.70 (m, 3H), 8.56 (dd, 1H, J = 4.8, 1.6 Hz), 7.99 (dt, 1H, J = 8.0, 2.0 Hz), 7.54–7.38 (m, 6H), 7.30–7.25 (m, 4H), 6.80 (d, 1H, J = 16.0 Hz), 4.55 (dd, 1H, J = 8.4, 5.2 Hz), 4.44–4.38 (m, 3H), 4.26 (dd, 1H, J = 15.2, 5.6 Hz), 3.33–3.21 (m, 2H), 2.60 (s, 3H), 2.39 (s, 3H), 1.60 (s, 3H); 13 C NMR (DMSO-d6, 100 MHz) δ (ppm): 170.12, 165.00, 163.66, 155.56, 150.60, 150.35, 149.59, 138.76, 138.28, 137.24, 136.16, 135.76, 134.48, 132.76, 131.17, 130.57, 130.30, 130.06, 128.94, 127.82, 127.59, 124.47, 54.43, 42.59, 142.21, 38.14, 14.54, 13.14, 11.79; HRMS-ESI (m / z): calcd for C 35 H 33 ClN7O2S [M + H] + 650.2099 found: 650.2102.

[0085] Example 14

[0086] (6S)-4-(4-Chlorophenyl)-N-[3-(1H-pyrrolo[3,2-c]pyridine-2-carboxamido)propyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A17) Preparation:

[0087] Using the synthesis method of Example 1, replacing the corresponding raw materials can synthesize the target compound.

[0088] 1 H NMR (DMSO-d6, 400 MHz) δ (ppm): 12.04 (s, 1H), 8.92 (s, 1H), 8.66 (t, 1H, J = 5.6 Hz), 8.33 (t, 1H, J = 5.6 Hz), 8.22 (d, 1H, J = 6.0 Hz), 7.47–7.36 (m, 5H), 7.26 (s, 1H), 4.53 (dd, 1H, J = 8.4, 6.0 Hz), 3.35–3.16 (m, 6H), 2.59 (s, 3H), 2.40 (s, 3H), 1.75 (t, 2H, J = 7.2 Hz), 1.59 (s, 3H); 13 C NMR (DMSO-d6, 100 MHz) δ (ppm): 169.08, 162.50, 159.84, 154.50, 149.26, 143.85, 140.25, 138.96, 136.14, 134.59, 132.76, 131.67, 130.11, 129.53, 129.23, 128.95, 127.86, 123.76, 106.91, 101.04, 53.26, 37.04, 36.17, 35.85, 28.74, 13.42, 12.06, 10.69; HRMS-ESI (m / z): calcd for C 30 H 30 ClN8O2S [M+H] + 601.1895 found: 601.1898.

[0089] Example 15

[0090] (6S)-4-(4-Chlorophenyl)-N-[4-(1H-pyrrolo[3,2-c]pyridine-2-carboxamido)butyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A18) Preparation:

[0091] Using the synthesis method of Example 1, replacing the corresponding raw materials can synthesize the target compound.

[0092] 1 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 12.01 (s, 1H), 8.91 (s, 1H), 8.67 (t, 1H, J = 5.6 Hz), 8.27–8.20 (m, 2H), 7.47–7.36 (m, 5H), 7.27 (s, 1H), 4.52 (dd, 1H, J = 8.4, 6.0 Hz), 3.37–3.14 (m, 6H), 2.59 (s, 3H), 2.39 (s, 3H), 1.61–1.50 (m, 7H); 13 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 169.93, 163.51, 160.94, 155.60, 150.33, 145.16, 141.69, 139.90, 137.21, 135.69, 133.70, 132.74, 131.17, 130.57, 130.29, 130.03, 128.93, 124.88, 107.87, 101.98, 54.35, 39.06, 38.69, 38.11, 27.23, 27.05, 14.50, 13.13, 11.77; HRMS-ESI (m / z): calcd for C 31 H 32 ClN8O2S [M+H] + 615.2052 found: 615.2062.

[0093] Example 16

[0094] (6S)-4-(4-Chlorophenyl)-N-[5-(1H-pyrrolo[3,2-c]pyridine-2-carboxamido)pentyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A19) Preparation:

[0095] Using the synthesis method of Example 1, replacing the corresponding raw materials can synthesize the target compound.

[0096] 11H NMR (DMSO-d6, 400 MHz) δ (ppm): 12.02 (s, 1H), 8.91 (d, 1H, J = 1.6 Hz), 8.63 (t, 1H, J = 5.6 Hz), 8.26–8.20 (m, 2H), 7.50–7.36 (m, 5H), 7.28 (s, 1H), 4.52 (dd, 1H, J = 8.0, 6.0 Hz), 3.34–3.09 (m, 6H), 2.59 (s, 3H), 2.39 (s, 3H), 1.60–1.36 (m, 9H); 13 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 169.97, 163.52, 160.87, 155.59, 150.31, 145.02, 141.49, 139.93, 137.22, 135.72, 133.80, 132.72, 131.16, 130.59, 130.27, 130.00, 128.95, 124.86, 107.89, 102.04, 54.38, 40.58, 38.82, 38.13, 29.45, 29.22, 24.27, 14.50, 13.13, 11.77; HRMS-ESI (m / z): calcd for C 32 H 34 ClN8O2S [M + H] + 629.2208 found: 629.2205.

[0097] Example 17

[0098] (6S)-4-(4-Chlorophenyl)-N-[6-(1H-pyrrolo[3,2-c]pyridine-2-carboxamido)hexyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A20) Preparation:

[0099] Using the synthesis method of Example 1, replacing the corresponding raw materials can synthesize the target compound.

[0100] 1 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 12.05 (s, 1H), 8.93 (d, 1H, J = 1.2 Hz), 8.63 (s, 1H), 8.23–8.20 (m, 2H), 7.49–7.38 (m, 5H), 7.27 (s, 1H), 4.52 (dd, 1H, J = 7.6, 6.4 Hz), 3.31–3.09 (m, 6H), 2.59 (s, 3H), 2.37 (s,13 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 169.93, 163.51, 160.81, 155.60, 150.30, 144.92, 141.33, 139.97, 137.20, 135.73, 133.91, 132.72, 131.15, 130.58, 130.26, 130.01, 128.93, 124.87, 107.93, 102.03, 54.39, 39.14, 38.80, 38.13, 29.70, 29.63, 26.56, 26.44, 14.47, 13.10, 11.75; HRMS-ESI (m / z): calcd for C 33 H 36 ClN8O2S [M+H] + 643.2365 found: 643.2362。

[0101] Example 18

[0102] (6S)-4-(4-Chlorophenyl)-N-[5-(1H-pyrrolo[3,2-c]pyridine-2-carboxamido)ethoxyethyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A21) Preparation:

[0103] Using the synthesis method of Example 1 and replacing the corresponding raw materials can synthesize the target compound.

[0104] 1 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 12.01 (s, 1H), 8.85 (d, 1H, J = 1.2 Hz), 8.66 (t, 1H, J = 5.2 Hz), 8.38 (t, 1H, J = 5.6 Hz), 8.20 (d, 1H, J = 6.0 Hz), 7.48–7.33 (m, 6H), 4.54 (dd, 1H, J = 8.0, 6.0 Hz), 3.63–3.36 (m, 7H), 3.34–3.22 (m, 3H), 2.59 (s, 3H), 2.34 (s, 3H), 1.54 (s, 3H); 1313C NMR (DMSO-d6, 100 MHz) δ (ppm): 170.49, 163.56, 161.11, 155.56, 150.33, 145.16, 141.69, 139.90, 137.25, 135.68, 133.43, 132.68, 131.09, 130.63, 130.24, 129.92, 128.89, 124.83, 107.83, 102.38, 69.68, 69.13, 54.29, 40.36, 38.98, 38.07, 14.43, 13.08, 11.77; HRMS-ESI (m / z): calcd for C 31 H 32 ClN8O3S [M+H] + 631.2001 found: 631.1996。

[0105] Example 19

[0106] (6S)-4-(4-Chlorophenyl)-N-[8-(1H-pyrrolo[3,2-c]pyridine-2-carboxamido)ethoxyethoxyethyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A22) Preparation:

[0107] Using the synthesis method of Example 1, replacing the corresponding raw materials can synthesize the target compound.

[0108] 1 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 12.02 (s, 1H), 8.92 (s, 1H), 8.73 (t, 1H, J = 5.2 Hz), 8.25 (dd, 2H, J = 8.0, 5.6 Hz), 7.49 - 7.36 (m, 5H), 7.28 (s, 1H), 4.51 (dd, 1H, J = 6.4 Hz), 3.57 (d, 6H, J = 5.2 Hz), 3.47 (dd, 4H, J = 9.6, 4.8 Hz), 3.29–3.19 (m, 4H), 2.59 (s, 3H), 2.40 (s, 3H), 1.61 (s, 3H); 1313C NMR (DMSO-d6, 100 MHz) δ (ppm): 170.22, 163.52, 161.12, 155.58, 150.33, 145.24, 141.74, 139.91, 137.22, 135.71, 133.50, 132.74, 131.17, 130.63, 130.29, 130.01, 128.94, 124.86, 107.86, 102.24, 70.11, 70.08, 69.69, 69.47, 54.30, 40.36, 39.39, 39.09, 37.97, 14.51, 13.13, 11.77; HRMS-ESI (m / z): calcd for C 33 H 36 ClN8O4S [M+H] + 675.2263 found: 675.2262。

[0109] Example 20

[0110] (6S)-4-(4-Chlorophenyl)-N-[1-[(1H-Pyrrolo[3,2-c]pyridine-2-carboxamido)ethyl]piperidin-4-yl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-acetamide (A23) Preparation:

[0111] Using the synthesis method of Example 1 and replacing the corresponding raw materials can synthesize the target compound.

[0112] 1 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 12.02 (s, 1H), 8.94 (s, 1H), 8.65 (t, 1H, J = 6.0 Hz), 8.22 (d, 1H, J = 6.0 Hz), 7.50–7.37 (m, 5H), 7.27 (s, 1H), 4.59 (t, 1H, J = 6.8 Hz), 4.38 (d, 1H, J = 12.8 Hz), 4.15 (d, 1H, J = 13.6 Hz), 3.64–3.58 (m, 1H), 3.40 (s, 3H), 3.11 (d, 1H, J = 4.8 Hz), 2.58 (d, 4H, J = 15.2 Hz), 2.42 (s, 3H), 1.86 (d, 1H, J = 12.8 Hz), 1.74 (s, 1H), 1.65 (s, 4H), 1.55–1.52 (m, 2H), 1.28–1.15 (m, 1H), 1.03–0.96 (m, 1H); 1313C NMR (DMSO-d6, 100 MHz) δ (ppm): 168.23, 163.36, 163.28, 160.91, 155.82, 150.24, 145.20, 141.72, 139.89, 137.28, 137.23, 135.67, 133.71, 132.66, 131.16, 130.63, 130.37, 130.34, 130.11, 128.96, 124.89, 107.85, 101.99, 54.68, 45.75, 41.93, 36.79, 36.33, 35.24, 33.50, 32.75, 32.03, 14.50, 13.16, 11.75; HRMS-ESI (m / z): calcd for C 34 H 36 ClN8O2S [M+H] + 655.2365 found: 655.2355。

[0113] Example 21

[0114] (6S)-4-(4-Chlorophenyl)-N-[4-[(1H-pyrrolo[3,2-c]pyridine-2-carboxamido)methyl]benzyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A24) Preparation:

[0115] Using the synthesis method of Example 1 and replacing the corresponding raw materials, the target compound can be synthesized.

[0116] 1 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 12.05 (s, 1H), 9.23 (t, 1H, J = 6.0 Hz), 8.93 (s, 1H), 8.75 (t, 1H, J = 12.0 Hz), 8.22 (d, 1H, J = 5.6 Hz), 7.48–7.27 (m, 10H), 4.56–4.50 (m, 3H), 4.41–4.26 (m, 2H), 3.32–3.21 (m, 2H), 2.59 (s, 3H), 2.37 (s, 3H), 1.58 (s, 3H); 1313C NMR (DMSO-d6, 100 MHz) δ (ppm): 170.12, 163.65, 160.98, 155.54, 150.35, 145.09, 141.53, 140.07, 138.71, 138.33, 137.20, 135.75, 133.57, 131.18, 130.56, 130.28, 130.04, 128.92, 127.66, 127.58, 124.86, 107.95, 102.41, 54.43, 42.49, 42.22, 38.14, 14.49, 13.10, 11.76; HRMS-ESI (m / z): calcd for C 35 H 32 ClN8O2S [M+H] + 663.2052 found 663.2543。

[0117] Example 22

[0118] (6S)-4-(4-Chlorophenyl)-N-[4-[[(2E)-3-(3-pyridinyl)-1-oxo-2-propen-1-yl]amino]phenyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A13) Preparation:

[0119] Using Route 2, add trans-3-(3-pyridinyl)acrylic acid (596 mg, 4 mmol), tert-butyl (4-aminophenyl)carbamate (832 mg, 4 mmol), HATU (2.28 g, 6 mmol), anhydrous DIPEA (12 mmol) and DMF (10 mL) to a 100 mL round-bottom flask and react at room temperature for 24 hours. After the reaction is completed, add 20 mL of water, extract three times with 60 mL of CH2Cl2, wash the organic layer successively with saturated NaHCO3 and brine, dry over anhydrous Na2SO4, filter, concentrate and perform column chromatography to obtain tert-butyl 4-((E)-3-(pyridin-3-yl)acrylamido)phenylcarbamate (488 mg, yield 36%).

[0120] Add tert-butyl 4-((E)-3-(pyridin-3-yl)acrylamido)phenylcarbamate (678 mg, 2 mmol) and ethyl acetate solution of 4M HCl (10 mL) to a 100 mL round-bottom flask and react at room temperature for 8 hours. After the reaction is completed, filter and dry to obtain the compound (E)-N-(4-aminophenyl)-3-(pyridin-3-yl)acrylamide hydrochloride (308 mg, yield 56%), and the product is directly used in the next reaction without purification.

[0121] (E)-N-(4-Aminophenyl)-3-(pyridin-3-yl)acrylamide (60 mg, 0.25 mmol), JQ1-COOH (100 mg, 0.25 mmol), HATU (143 mg, 0.375 mmol), anhydrous DIPEA (0.75 mmol) and DMF (5 mL) were added to a 100 mL round-bottom flask and reacted at room temperature for 24 h. After the reaction, 5 mL of water was added, and the mixture was extracted three times with 15 mL of CH2Cl2. The organic layer was washed successively with saturated NaHCO3 and brine, dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography to obtain (6S)-4-(4-chlorophenyl)-N-[4-[[(2E)-3-(3-pyridinyl)-1-oxo-2-propen-1-yl]amino]phenyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A13, 109 mg, yield 70%).

[0122] 1 H NMR (DMSO-d6, 400 MHz) δ (ppm): 10.30 (d, 2H, J = 26.0 Hz), 8.82 (d, 1H, J = 2.4 Hz), 8.59 (dd, 1H, J = 4.8, 1.6 Hz), 8.05–8.02 (m, 1H), 7.67–7.59 (m, 5H), 7.50–7.41 (m, 5H), 6.93 (d, 1H, J = 16.0 Hz), 4.61 (t, 1H, J = 7.2 Hz), 3.50 (d, 2H, J = 7.2 Hz), 2.61 (s, 3H), 2.43 (s, 3H), 1.64 (s, 3H); 13 C NMR (DMSO-d6, 100 MHz) δ (ppm): 168.85, 163.72, 163.27, 155.54, 150.84, 150.42, 149.75, 137.20, 137.01, 135.74, 135.47, 134.99, 134.56, 132.79, 131.25, 131.07, 130.63, 130.35, 130.04, 129.01, 124.77, 124.55, 120.20, 120.01, 54.25, 39.02, 14.56, 13.18, 11.80; HRMS-ESI (m / z): calcd for C 33 H 29 ClN7O2S [M + H] + 622.1786 found: 622.1779.

[0123] Example 23

[0124] Preparation of (6S)-4-(4-chlorophenyl)-N-[4-[[(2E)-3-(3-pyridyl)-1-oxo-2-propen-1-yl]amino]benzyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A14):

[0125] The target compound can be synthesized by adopting the synthesis method of Example 22 and replacing the corresponding raw materials.

[0126] 1 H NMR (DMSO-d6, 400MHz) δ (ppm): 10.32 (s, 1H), 8.83 (d, 1H, J = 2.4Hz), 8.77 (t, 1H, J = 6.0Hz), 8.59 (dd, 1 H,J=4.8,1.6Hz),8.05(dt,1H,J=8.0,2.0Hz),7.70–7.62(m,3H),7.51–7.46(m,3H),7.38–7.29(m,4H) ,6.95(d,1H,J=15.6Hz), 4.56(dd,1H,J=9.2,5.6Hz), 4.41(dd,1H,J=15.2,6.4Hz), 4.24(dd,1H,J=15. 2,5.6Hz),3.39(d,1H,J=8.8Hz),3.23(dd,1H,J=14.8,5.6Hz),2.61(s,3H),2.41(s,3H),1.62(s,3H); 13 C NMR(DMSO-d6,100MHz)δ(ppm):170.10,163.64,163.52,155.57,150.87,150.35,149.75,138.36,137.24,135.74,135.25,134.61,132.78,131. 19,131.04,130.60,130.30,130.05,128.92,128.21,124.74,124.56,119.56,54.49,42.12,38.17,14.56,13.16,11.80; HRMS-ESI(m / z):calcd for C 34 H 30 ClN7NaO2S[M+Na] + 658.1762found:658.1759.

[0127] Example 24

[0128] (6S)-4-(4-Chlorophenyl)-N-[4-[[[(2E)-3-(3-pyridinyl)-1-oxo-2-propen-1-yl]amino]ethyl]phenyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A15) Preparation:

[0129] Using Route 3, add trans-3-(3-pyridinyl)acrylic acid (596 mg, 4 mmol), 4-aminophenethylamine (548 mg, 4 mmol), EDCI (1.15 g, 6 mmol), HOBt (810 mg, 6 mmol), anhydrous DIPEA (12 mmol) and DMF (10 mL) into a 100 mL round-bottom flask and react at room temperature for 24 hours. After the reaction, add 20 mL of water, extract three times with 60 mL of CH2Cl2. The organic layer is washed successively with saturated NaHCO3 and brine, dried over anhydrous Na2SO4, filtered, concentrated and purified by column chromatography to obtain (E)-N-(4-aminophenethyl)-3-(pyridin-3-yl)acrylamide (391 mg, yield 37%).

[0130] Add (E)-N-(4-aminophenethyl)-3-(pyridin-3-yl)acrylamide (67 mg, 0.25 mmol), JQ1-COOH (100 mg, 0.25 mmol), HATU (143 mg, 0.375 mmol), anhydrous DIPEA (0.75 mmol) and DMF (5 mL) into a 100 mL round-bottom flask and react at room temperature for 24 hours. After the reaction, add 5 mL of water, extract three times with 15 mL of CH2Cl2. The organic layer is washed successively with saturated NaHCO3 and brine, dried over anhydrous Na2SO4, filtered, concentrated and purified by column chromatography to obtain (6S)-4-(4-chlorophenyl)-N-[4-[[[(2E)-3-(3-pyridinyl)-1-oxo-2-propen-1-yl]amino]ethyl]phenyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide (A15, 124 mg, yield 76%).

[0131] 11H NMR (DMSO-d6, 400 MHz) δ (ppm): 10.27 (s, 1H), 8.75 (d, 1H, J = 2.4 Hz), 8.55 (dd, 1H, J = 4.8, 1.6 Hz), 8.24 (t, 1H, J = 5.6 Hz), 7.97 (dt, 1H, J = 8.0, 2.0 Hz), 7.57–7.54 (m, 2H), 7.49–7.41 (m, 6H), 7.18 (d, 2H, J = 8.4 Hz), 6.72 (d, 1H, J = 15.6 Hz), 4.59 (t, 1H, J = 7.2 Hz), 3.49 (d, 2H, J = 7.2 Hz), 3.43–3.39 (m, 2H), 2.74 (t, 2H, J = 7.2 Hz), 2.60 (s, 3H), 2.42 (s, 3H), 1.63 (s, 3H); 13 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 168.95, 164.94,163.71, 155.53, 150.59, 150.41, 149.60, 137.87, 137.20, 135.74, 134.59, 134.40, 132.78, 131.24, 131.18, 130.62, 130.35, 130.03, 129.38, 129.00, 124.66, 124.46, 119.63, 54.24, 40.89, 39.05, 35.01, 14.56, 13.17, 11.79; HRMS-ESI (m / z): calcd for C 35 H 33 ClN7O2S [M + H] + 650.2105 found: 650.2115.

[0132] Example 25

[0133] Enzyme inhibitory activity of the target compound:

[0134] In vitro BRD4 enzyme inhibitory activity assay: This assay was performed by TR-FRET technology using recombinant BRD4 (BD1 + BD2) protein and its corresponding ligand (BET). The measured TR-FRET signal is related to the binding amount of the ligand and the bromodomain. In all reactions, the final concentration of DMSO in the reaction system was 1 wt%. All binding reactions were carried out at room temperature. The 20 μL reaction mixture in the assay buffer contained the bromodomain (18 ng), BET ligand (8 nM) and the screening drug at the specified concentration. For the negative control, 5 μL of the assay buffer was added instead of the BET ligand. The reaction mixture was incubated at room temperature for 120 minutes. After incubation with the ligand, the TR-FRET signal was measured using a microplate reader.

[0135] In vitro experiment for inhibiting NAMPT enzyme activity: In all reactions, the drug was serially diluted with 10 wt% aqueous DMSO solution, and 5 μL of the drug dilution was added to 50 μL of the reaction system, so that the final concentration of DMSO in all reaction systems was 1 wt%. All enzymatic reactions were carried out in a 30 °C water bath for 90 minutes. The 50 μL reaction mixture contained 50 mM Tris-HCl, pH 8.0, 12.5 mM MgCl2, 20 μM Nicotinamide, 0.4 mM pyrophosphate phospholipid, 2 mM ATP, 30 μg / mL alcohol dehydrogenase, 10 μg / mL NMNAT, 1.5 Vol% alcohol, 1 mM DTT, 0.02 wt% BSA, 0.01 wt% Tween 20, NAMPT enzyme and the screened drug. The fluorescence intensity was measured using a microplate reader under the conditions of excitation at 360 nm and emission at 460 nm. The NAMPT activity assay was performed in duplicate for each concentration. The fluorescence intensity data was analyzed using the computer software Graphpad Prism. In the absence of the compound, the fluorescence intensity (Ft) in each group of data was defined as 100% activity. In the absence of NAMPT, the fluorescence intensity (Fb) in each group of data was defined as 0% activity. The percentage of activity in the presence of each compound was calculated according to the following formula: % activity = (F - Fb) / (Ft - Fb), where F = the fluorescence intensity in the presence of the screened drug.

[0136] Table 1 Inhibitory IC 50 value of compounds on BRD4 and NAMPT

[0137]

[0138] Example 26

[0139] Antitumor activity of preferred compounds in vitro:

[0140] The human tumor cell lines Hep3B, HuH7 and HCCLM3 were selected, and the normal liver cell line LX-2 was used for culture. FK866 and JQ1 were used as positive control drugs, and the inhibitory effect of each compound at different concentrations on cell proliferation was determined by the MTT method. After routine trypsin digestion of the tumor cells in the logarithmic growth phase, they were evenly inoculated into a 96-well culture plate, 2×10 5Cells / well were placed in a 37 °C, 5% CO2 cell culture incubator and cultured overnight. A negative control group, a positive control group and an experimental drug administration group were set up. Different concentrations of the test compound or positive control drug diluted in a serial dilution were added to the drug administration group, with 3-5 parallel wells for each concentration. The control wells were added with a culture medium containing an equal amount of DMSO and placed in a 37 °C CO2 incubator. After 120 hours of culture, the supernatant was discarded, 100 μL of freshly prepared MTT (0.5 mg / mL) solution was added, and the culture was continued at 37 °C for 4 hours. Then the culture medium was discarded, 200 μL of DMSO was added to each well, and the micro oscillator was used to shake until the crystals dissolved. The absorbance (OD) value of each well at a wavelength of 570 nm was measured on an enzyme-labeled instrument. The cell proliferation inhibition rate (%) = (1 - OD value of the experimental group / OD value of the control group) × 100%. Plotting the growth inhibition rate of tumor cells against different concentrations of the test compound can obtain a dose-response curve, and thus calculate the half-lethal concentration IC 50 .

[0141] Table 2 Anti-HCC activities of some compounds

[0142]

[0143] The above has shown and described the basic principles, main features and advantages of the present invention. Without departing from the spirit and scope of the present invention, there are various changes and improvements to the present invention, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. A dual-targeted inhibitor of BRD4 / NAMPT, characterized by: The BRD4 / NAMPT dual-targeting inhibitor is a compound having the general structural formula shown in Formula I or a pharmaceutically acceptable salt thereof: Where R is selected from The L linking group is selected from:

2. The BRD4 / NAMPT dual-targeting inhibitor according to claim 1, characterized in that: The compounds shown in formula I include:

3. A method for preparing the BRD4 / NAMPT dual-targeting inhibitor according to claim 2, characterized in that The synthetic route of the compound shown in Formula I is as follows: Synthesis route 1 corresponding to compounds A1-A12, A16, A17-A24: Synthesis route 2 corresponding to compounds A13-A14: Synthesis route 3 corresponding to compound A15:

4. A pharmaceutical composition, characterized in that Comprising the compound represented by formula I in claim 1 or 2 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers.

5. The pharmaceutical composition according to claim 4, characterized in that: The dosage form of the pharmaceutical composition is capsule, powder, tablet, granule, pill, injection, syrup, oral solution, inhalant, ointment, suppository or patch.

6. The pharmaceutical composition according to claim 4, characterized in that: The pharmaceutical composition further comprises other therapeutic drugs, which are one or more of tumor chemotherapy drugs, tumor targeting drugs, tumor immunotherapy drugs or tumor drug conjugates.

7. Use of the compound represented by formula I in the BRD4 / NAMPT dual-targeting inhibitor according to claim 1 or 2 or the pharmaceutical composition according to any one of claims 4 to 6 in the preparation of an anti-liver cancer drug.

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