Application of dihydroquinazolinone compound with BET / AKT dual-channel activity in preparation of anti-prostatic cancer drugs

Abstract

The invention discloses an application of a dihydroquinazolinone compound with BET / AKT dual-channel activity in preparation of an anti-prostatic cancer drug. The dihydroquinazolinone compound provided by the invention is applied to preparation of an inhibitor of a BET and / or AKT mediated pathway. The novel dihydroquinazolinone compound and the pharmaceutical composition thereof are provided for the first time, and have a remarkable inhibition effect on cancers. Meanwhile, a pharmacological test shows that the dihydroquinazolinone compound disclosed by the invention simultaneously has the inhibitory activity of BET and AKT dual pathways, and obviously inhibits the functional activity of BET and AKT proteins, while the contrast drug Capivasertib only has the inhibitory activity on the AKT protein. The compound provided by the invention can significantly inhibit the growth of prostate cells 22RV1 and VCaP, the inhibitory activity is significantly superior to that of a control drug Capivasertib, and the compound provided by the invention has a significant treatment effect on prostate cancer, and is significantly superior to that of an existing control drug.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of a dihydroquinazolinone compound with BET / AKT dual-pathway activity in the preparation of an anti-prostate cancer drug. Background Art

[0002] Since the discovery of tumors, they have always been the main factor endangering human health. From the discovery of the first anti-tumor drug, nitrogen mustard, in the 1940s of the last century to the discovery of the first tumor-targeted drug in the 1990s of the last century, the survival rates of most types of tumor patients have been continuously increasing. The full name of BET is bromodomain and extraterminal domain. There are 4 members in the BET family of proteins, namely BRD2, BRD3, BRD4, and BRDT, and BRD4 has three different forms of splice variants. The RBET family of proteins has two BRD / BDs and also contains an ET domain. The proteins of the BET family have powerful functions and play an indispensable role in regulating gene expression. Due to the high affinity of the BRD domain for acetylated histones, BET proteins are mainly distributed near histone acetylation sites on chromatin. BRD4 is a protein with relatively well-understood functions at present. In addition to being able to bind acetylated modified histones through the BRD domain, BRD4 can also form protein complexes with other proteins such as P-TEFb and Mediator. The P-TEFb protein can interact with CDK9 and increase the transcriptional activity of RNA polymerase II (Pol II) through phosphorylation to activate the expression of downstream genes. The dysregulation of BRD4 expression level or function is related to the occurrence of tumors such as acute myeloid leukemia, melanoma, colon cancer, and breast cancer. Akt is a serine / threonine-specific protein kinase and plays an important role in cell survival and apoptosis. AKT is at the core node of the PI3K / AKT / mTOR signaling pathway (abbreviated as the PAM pathway). This signaling pathway is activated by various types of cell stimuli or toxic injuries and regulates basic cell functions. Growth factors bind to their receptor tyrosine kinases (RTKs), stimulating PI3K to catalyze the production of PIP3. PIP3, as a second messenger, helps activate AKT. AKT regulates key cellular processes by phosphorylating various kinases and transcription factors. PI3K / AKT / mTOR is the most frequently altered pathway in human cancers, and abnormalities exist in 38% of cancer patients. The abnormal activation of this pathway can cause a series of diseases such as cancer, neuropathy, autoimmune diseases, and hematolymphatic system diseases. AKT is overactivated in more than 50% of tumors, including breast cancer, lung cancer, head and neck tumors, endometrial cancer, prostate cancer, colorectal cancer, etc.

[0003] Although current tumor treatment drugs and strategies are diverse, there are still some problems in the field of tumor treatment that need to be solved urgently. For example, the efficacy of existing tumor drugs is poor, the drug toxicity and side effects are serious, and drug resistance is prone to occur. How to improve the treatment effect of anti-tumor drugs and develop new anti-tumor drugs with novel mechanisms has become an urgent problem in the clinical treatment of tumors. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides the use of dihydroquinazolinone compounds with BET / AKT dual pathway activity in the preparation of anti-prostate cancer drugs. The present invention provides dual inhibitors of BET and AKT with high activity and high specificity, which are used for the treatment of various tumors and have a huge market demand.

[0005] The present invention provides the use of dihydroquinazolinone compounds, pharmaceutically acceptable salts, stereoisomers, deuterated compounds thereof, or the above drug compositions in the preparation of drugs for preventing or treating abnormal cell growth in a subject. Further preferably, the cancer is prostate cancer.

[0006] The present invention provides the use of dihydroquinazolinone compounds in the preparation of anti-prostate cancer drugs, and the dihydroquinazolinone compounds are selected from:

[0007]

[0008]

[0009] The present invention provides the use of dihydroquinazolinone compounds in the preparation of inhibitors of BET and / or AKT-mediated pathways.

[0010] Advantages: The novel dihydroquinazolinone compounds and their drug compositions of the present invention are proposed for the first time and can significantly inhibit cancer. At the same time, pharmacological tests show that the dihydroquinazolinone compounds of the present invention have inhibitory activities on both BET and AKT pathways, significantly inhibiting the functional activities of BET and AKT proteins, while the control drug Capivasertib only has inhibitory activity on AKT protein.

[0011] Due to the active characteristics of the compounds of the present invention that can simultaneously inhibit both BET and AKT signaling pathways, the present invention can prepare dual inhibitors of BET / AKT. The present invention has better tumor treatment effects compared with single-target drugs. In particular, pharmacological tests show that the compounds of the present invention can significantly inhibit the growth of prostate cells 22RV1 and VCaP, and the inhibitory activity is significantly better than that of the control drug Capivasertib. The present invention has a significant therapeutic effect on prostate cancer and is significantly better than existing control drugs. Description of the Drawings

[0012] Figure 1The compound inhibits the growth of prostate cancer 22RV1 and VCaP cells. Detailed implementation mode

[0013] The present invention will be further described below in conjunction with specific examples and test examples, but the scope of the present invention is not limited in any form.

[0014] Example 1: Synthesis of Compound 1

[0015]

[0016] Synthesis route:

[0017]

[0018] Step 1: Synthesis of Compound 1-2

[0019] Compound 1-1 (0.5 g, 2.051 mmol, 1 eq), 4-(Boc-amino)piperidine-4-carboxylic acid (0.314 g, 2.05 mmol, 1 eq) and sodium bicarbonate (0.808 g, 9.62 mmol, 4.7 eq) were added to a mixed solvent of 2 ml of acetonitrile and 6 ml of water. The reaction was carried out at 80 °C for 24 h under nitrogen protection. After monitoring the completion of the reaction by thin layer chromatography, ethyl acetate was added to the reaction solution for extraction, and the organic phase was discarded. A certain amount of methanol was added to the aqueous phase, and the aqueous phase was concentrated by rotary evaporation. The pH was adjusted to 4.5 with 3 mol / L hydrochloric acid aqueous solution, and the mixture was stirred overnight at room temperature. A large amount of solid precipitated out. The white solid compound 1-2 (0.366 g, 1.01 mmol, yield 49.48%) was obtained by filtration. (ESI) m / z = 262.1 [M - Boc] + . 1 H NMR (300 MHz, DMSO-d 6 ) δ 12.44 (s, 1H), 11.71 (s, 1H), 8.13 (s, 1H), 7.35 (s, 1H), 7.17 (t, J = 2.9 Hz, 1H), 6.65–6.55 (m, 1H), 4.30 (d, J = 13.4 Hz, 2H), 3.57–3.24 (m, 4H), 2.10–1.77 (m, 4H), 1.38 (s, 9H).

[0020] Step 2: Synthesis of Compound 1-4

[0021] Compound 1-3 (0.5 g, 2.69 mmol, 1 eq) was added to 5 ml of dichloromethane and dissolved completely. Then di-tert-butyl dicarbonate (0.705 g, 3.23 mmol, 1.2 eq) was added and the reaction was carried out at room temperature for 2 h. After monitoring the completion of the reaction by thin-layer chromatography, the reaction was stopped. The reaction solution was concentrated in vacuo, then 100 mL of water was added to the reaction solution, and it was extracted with ethyl acetate (80 mL×2), washed with saturated brine (70 mL×2), dried over anhydrous sodium sulfate, filtered, concentrated in vacuo and purified by silica gel column chromatography to obtain white solid compound 1-4 (0.765 g, 2.68 mmol, yield 99.4%). (ESI) m / z = 186.1 [M - Boc] + . 1 H NMR (300 MHz, DMSO-d 6 ) δ 7.43 (s, 1H), 7.40–7.34 (m, 2H), 7.28 (d, J = 8.5 Hz, 2H), 4.66–4.47 (m, 2H), 3.28 (tt, J = 10.9, 6.0 Hz, 2H), 1.35 (s, 9H).

[0022] Step 3: Synthesis of compound 1-5

[0023] Triphenylphosphine (0.367 g, 2 mmol, 1 eq) and iodine (0.355 g, 1.4 mmol, 2 eq) were dissolved in 3 ml of dichloromethane. After stirring at room temperature for 10 minutes, imidazole (0.19 g, 2.8 mmol, 4 eq) was added to the above reaction solution. After stirring at room temperature for 30 min, the intermediate 1-4 (0.2 g, 0.7 mmol, 1 eq) was added and stirred at room temperature for 2 h. After monitoring the completion of the reaction, 10% sodium sulfite and saturated sodium bicarbonate aqueous solution were added to the reaction solution. The mixture was extracted with dichloromethane (10 ml×2), washed with saturated brine (10 mL×2), concentrated in vacuo and purified by silica gel column chromatography to obtain white solid compound 1-5 (0.268 g, 0.68 mmol, yield 97.14%). (ESI) m / z = 296.1 [M - Boc] + .

[0024] Step 4: Synthesis of compound 1-6

[0025] Compound 1-5 (0.39 g, 0.98 mmol, 1 eq) was dissolved in acetonitrile (5 mL), stirred at room temperature, and then potassium carbonate (0.54 g, 3.91 mmol, 4 eq) and benzyl-1-piperazine carbonate (0.43 g, 1.95 mmol, 2 eq) were added to the above reaction solution. After stirring at room temperature for 10 minutes, it was transferred to an oil bath and heated to 80 °C for overnight reaction. After monitoring the completion of the reaction, the reaction solution was cooled to room temperature, the reaction solution was rotary evaporated to dryness, and then purified by silica gel column chromatography to obtain compound 1-6 as a yellow oil (0.44 g, 0.90 mmol, yield 91.4%). (ESI) m / z = 388.2 [M-Boc] + . 1 H NMR (300 MHz, DMSO-d 6 ) δ 7.48 (d, J = 8.5 Hz, 1H), 7.36 (s, 6H), 7.30 (d, J = 8.6 Hz, 3H), 5.07 (s, 2H), 4.52 (q, J = 7.8 Hz, 1H), 2.25 (dt, J = 24.1, 6.1 Hz, 7H), 1.76 (dh, J = 26.9, 7.0 Hz, 3H), 1.35 (s, 9H), 1.25 (d, J = 9.8 Hz, 2H).

[0026] Step 5: Synthesis of compound 1-7

[0027] Compound 1-6 (0.055 g, 0.113 mmol, 1 eq), palladium chloride (0.002 g, 0.011 mmol, 0.1 eq), and triethylamine (0.034 g, 0.338 mmol, 3 eq) were added to a two-necked flask and stirred at room temperature for ten minutes. Subsequently, under nitrogen protection, 1.5 mL of dichloromethane was slowly added to the above reaction flask, and triethylsilane (0.039 g, 0.338 mmol, 3 eq) dissolved in 0.5 mL of dichloromethane was slowly added to the two-necked flask. After reacting at room temperature for 40 minutes, the reaction was completed, and the reaction solution was rotary evaporated to dryness and directly used in the next step. (ESI) m / z = 354.2 [M-Boc] + .

[0028] Step 6: Synthesis of compound 1-9

[0029] 1 - 8 (8 g, 40.56 mmol) was dissolved in 80 mL of dichloromethane, stirred for 15 min under an ice bath condition, and N - bromosuccinimide (7.58 g, 42.59 mmol) was slowly added in portions to the reaction flask, and the reaction was continued in the ice bath for 2 hours. 40 mL of water was added to the reaction solution, and it was extracted with dichloromethane. The organic layer was washed twice with saturated brine, the organic layer was collected and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and purified by silica gel column chromatography to obtain the yellow solid compound 1 - 9 (9.83 g, yield 88%). (ESI) m / z = 276.1 [M + H] + . 1 H NMR (300 MHz, DMSO - d 6 ) δ 7.63–7.43 (m, 5H), 7.40 (dd, J = 8.7, 2.4 Hz, 1H), 7.31 (d, J = 2.4 Hz, 1H), 7.24 (s, 2H), 6.86 (d, J = 8.9 Hz, 1H).

[0030] Step 7: Synthesis of compound 1 - 10

[0031] Compound 1 - 9 (9.83 g, 35.60 mmol) and tert - butylsulfinamide (12.94 g, 106.80 mmol) were dissolved in 60 mL of tetrahydrofuran. After dissolution, tetraethyl titanate (24.36 g, 106.80 mmol) was added. After addition, the reaction was carried out at 70 °C for 48 hours. The reaction solution was cooled to room temperature, 35 mL of water and 70 mL of ethyl acetate were added dropwise, and a large amount of solid precipitated. The reaction solution was filtered through diatomaceous earth to remove the solid. The filtrate was extracted with ethyl acetate, the organic layer was collected and washed with saturated brine, the organic layer was collected and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and purified by silica gel column chromatography to obtain the yellow solid compound 1 - 10 (12.33 g, yield 91%). (ESI) m / z = 379.2 [M + H] + .

[0032] Step 8: Synthesis of compound 1 - 11

[0033] Compound 1 - 10 (12.33 g, 32.51 mmol) was dissolved in a mixed solution of 49 mL of tetrahydrofuran and 1 mL of water, stirred at room temperature, and sodium borohydride (4.92 g, 130.02 mmol) was slowly added in portions. The reaction was carried out at room temperature for 3 hours. 25 mL of water was added to the reaction solution, and it was extracted with ethyl acetate. The organic layer was collected and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure to obtain the white solid compound 1 - 11 (12.14 g, yield 98%). (ESI) m / z = 381.1 [M + H] + . 1 H NMR (300 MHz, DMSO - d 6)δ 7.42–7.20 (m, 5H), 7.12–7.00 (m, 2H), 6.61 (d, J = 8.6 Hz, 1H), 5.96 (d, J = 5.9 Hz, 1H), 5.48 (d, J = 6.1 Hz, 1H), 5.24 (s, 2H), 1.14 (s, 9H).

[0034] Step 9: Synthesis of Compound 1-12

[0035] Dissolve intermediate 1-11 (12.14 g, 31.84 mmol) in 50 mL of tetrahydrofuran, stir at room temperature, slowly add trichloromethyl carbonate (14.17 g, 47.75 mmol), and react at room temperature for 3 hours. Rotate the reaction mixture to dryness under reduced pressure. Add 20 mL of water dropwise to the reaction flask, and add saturated sodium carbonate solution dropwise to adjust the pH of the reaction mixture to 7. Extract the reaction mixture with ethyl acetate, extract the aqueous layer with ethyl acetate twice, combine the ethyl acetate layers, wash with saturated brine, collect the organic layer, dry it over anhydrous sodium sulfate, filter, rotate the filtrate to dryness under reduced pressure, and purify it by silica gel column chromatography to obtain yellow solid compound 1-12 (9.28 g, yield 96%). (ESI) m / z = 303.2 [M+H] + .

[0036] Step 10: Synthesis of Compound 1-13

[0037] Dissolve compound 1-12 (0.56 g, 1.85 mmol) and cesium carbonate (1.81 g, 5.54 mmol) in acetonitrile solution, stir under ice bath conditions, dissolve dimethyl sulfate (0.28 g, 2.22 mmol) in acetonitrile and slowly add it dropwise to the reaction flask. After the addition is complete, remove the ice bath and stir at room temperature for 3 hours. Rotate the reaction mixture to dryness under reduced pressure. Add 10 mL of water dropwise to the reaction flask, extract with ethyl acetate, collect the organic layer, dry it over anhydrous sodium sulfate, filter, rotate the filtrate to dryness under reduced pressure, and purify it by silica gel column chromatography to obtain white solid 1-13 (0.41 g, yield 70%). (ESI) m / z = 317.2 [M+H] + . 1 H NMR (300 MHz, DMSO-d 6 ) δ 7.47–7.43 (m, 4H), 7.29–7.25 (m, 2H), 7.15 (dd, J = 11.6, 3.4 Hz, 2H), 6.95 (d, J = 8.1 Hz, 1H), 5.77 (s, 1H), 2.97 (s, 3H).

[0038] Step 11: Synthesis of Compound 1-14

[0039] Compound 1-13 (0.50 g, 1.58 mmol), 3,5-dimethylisoxazole-4-boronic acid pinacol ester (0.51 g, 1.73 mmol), sodium carbonate (0.50 g, 4.73 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.18 g, 0.16 mmol) were dissolved in a mixed solution of 3 mL of toluene, 1 mL of ethanol and 3 mL of water, and stirred at 80 °C for 18 hours under nitrogen protection. The reaction solution was filtered through diatomaceous earth, the filtrate was extracted with ethyl acetate, the organic layer was collected and washed once with saturated sodium bicarbonate and saturated brine respectively, the organic layer was collected and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to obtain white solid compound 1-14 (0.36 g, yield 70%). (ESI) m / z = 334.2 [M+H] + . 1 H NMR (300 MHz, DMSO-d 6 ) δ 7.53–7.48 (m, 4H), 7.31–7.26 (m, 2H), 7.17 (dd, J = 11.6, 3.4 Hz, 2H), 6.92 (d, J = 8.1 Hz, 1H), 5.73 (s, 1H), 2.92 (s, 3H), 2.47 (s, 3H), 2.27 (s, 3H).

[0040] Step 11: Synthesis of compound 1-15

[0041] Compound 1-14 (0.15 g, 0.45 mmol) was dissolved in 2 mL of DMF, stirred in an ice bath, and sodium hydride (0.016 g, 0.67 mmol) was slowly added portionwise. After 10 minutes, ethyl 3-bromopropionate I-f13 (0.16 g, 0.90 mmol) was added dropwise to the reaction solution. After the addition was complete, the ice bath was removed and the mixture was stirred at room temperature for 2 hours. 10 mL of saturated ammonium chloride solution was added, and the mixture was extracted with ethyl acetate. The organic layer was collected and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to obtain pale yellow solid compound 1-15 (0.17 g, yield 89%). (ESI) m / z = 420.3 [M+H] + .

[0042] Step 12: Synthesis of compound 1-16

[0043] Compound 1-15 (0.17 g, 0.39 mmol) and lithium hydroxide monohydrate (0.02 g, 0.78 mmol) were dissolved in a mixed solution of 2 mL of methanol and 1 mL of water and reacted at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, the pH was adjusted to 3 with 1 mol / L hydrochloric acid solution, and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was rotary evaporated to dryness under reduced pressure to obtain a pale yellow viscous liquid compound 1-16 (0.14 g, yield 88%). (ESI) m / z = 406.2 [M+H] + . 1 H NMR (300 MHz, CDCl 3 ) δ 11.92 (s, 1H), 7.37–7.27 (m, 4H), 7.11 (dd, J = 8.4, 1.8 Hz, 1H), 6.98–6.90 (m, 2H), 5.61 (s, 1H), 3.97–3.85 (m, 1H), 3.41 (s, 3H), 2.77 (dd, J = 15.3, 8.2 Hz, 1H), 2.55 (dd, J = 11.3, 6.0 Hz, 1H), 2.31 (s, 3H), 2.17 (s, 3H), 1.36–1.18 (m, 2H).

[0044] Step 13: Synthesis of Compound 1-17

[0045] Compound 5-9 (0.11 g, 0.271 mmol, 1 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.078 g, 0.407 mmol, 1.5 eq), 1-hydroxy-7-azabenzotriazole (0.055 g, 0.407 mmol, 1.5 eq) and N-methylmorpholine (0.082 g, 0.814 mmol, 3 eq) were added to 2 ml of DMSO and stirred at room temperature for 20 minutes. Then, compound 1-7 (0.101 g, 0.285 mmol, 1.1 eq) was added to the above reaction solution and reacted at room temperature for 3 h. After monitoring the completion of the reaction, the reaction was stopped, and the reaction solution was quenched with water (10 mL), extracted with ethyl acetate (10 mL × 2), washed with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and rotary evaporated to dryness. The residue was purified by silica gel column chromatography to obtain a pale yellow oily compound 1-17 (0.115 g, 0.16 mmol, yield 57.2%). (ESI) m / z = 641.2 [M - Boc] + . 1 H NMR (300 MHz, CDCl 3)δ 7.39–7.29 (m, 6H), 7.28–7.23 (m, 3H), 7.11 (dd, J = 8.3, 2.0 Hz, 1H), 7.00 (d, J = 1.9 Hz, 1H), 6.93 (dd, J = 8.4, 4.3 Hz, 1H), 6.02 (s, 1H), 5.76 (s, 1H), 5.32 (s, 1H), 4.77 (s, 1H), 3.88 (ddd, J = 12.7, 7.0, 4.6 Hz, 1H), 3.42 (d, J = 1.9 Hz, 3H), 2.86 (dt, J = 15.2, 7.5 Hz, 1H), 2.58–2.44 (m, 1H), 2.34 (d, J = 1.5 Hz, 8H), 2.20 (d, J = 1.4 Hz, 3H), 1.97 (s, 1H), 1.53–1.14 (m, 13H).

[0046] Step 14: Synthesis of Compound 1-18

[0047] Trifluoroacetic acid (2 mL) was added dropwise to a solution of Compound 1-17 (100 mg, 0.14 mmol, 1 eq) in dichloromethane (10 mL) at 0 °C. The reaction mixture was stirred at 20 °C for 1 h. The reaction was monitored by LCMS until the starting material completely disappeared and the main peak corresponded to the target product. The reaction mixture was directly concentrated under reduced pressure to obtain Compound 1-18 (0.1 g, crude product), which was directly used in the next step. (ESI) m / z = 641.1 [M+H] + .

[0048] Step 15: Synthesis of Compound 1-19

[0049] Compound 1-2 (0.068 g, 0.188 mmol, 1 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.052 g, 0.269 mmol, 1.4 eq), 1-hydroxy-7-azabenzotriazole (0.037 g, 0.269 mmol), and N-methylmorpholine (0.064 g, 0.628 mmol, 3.3 eq) were added to a round-bottom flask. Then, 2 mL of DMSO was added to the above mixture to dissolve it. After stirring at room temperature for 15 min, Compound 1-18 was added, and the reaction was carried out at room temperature for 6 h. After monitoring the completion of the reaction, the reaction was stopped. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL × 2), washed with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain a pale yellow solid, Compound 1-19 (0.117 g, yield 66.3%). (ESI) m / z = 884.2 [M-Boc] + .

[0050] Step 16: Synthesis of Compound 1

[0051] To a solution of Compound 1-19 (100 mg, 0.10 mmol, 1 eq) in dichloromethane (10 mL) was added dropwise trifluoroacetic acid (2 mL) at 0 °C. The reaction mixture was stirred at 20 °C for 1 hour. The reaction was monitored by LCMS until the starting material was completely consumed and the main peak corresponded to the target product. The reaction mixture was concentrated under reduced pressure to afford Compound 1. (ESI) m / z = 884.7 [M+H] + 。 1 H NMR (300 MHz, Chloroform-d) δ 10.20 (s, 1H), 8.64 (dd, J = 17.9, 6.9 Hz, 1H), 8.25 (d, J = 2.4 Hz, 1H), 7.31 (d, J = 6.5 Hz, 7H), 7.24 (t, J = 6.5 Hz, 1H), 7.17 (d, J = 8.3 Hz, 1H), 7.12 (dt, J = 8.3, 1.8 Hz, 1H), 7.07 (d, J = 3.7 Hz, 1H), 7.00 (t, J = 2.1 Hz, 1H), 6.97–6.89 (m, 1H), 6.50 (d, J = 3.6 Hz, 1H), 5.77 (d, J = 2.3 Hz, 1H), 5.02 (d, J = 8.0 Hz, 1H), 4.57 (t, J = 13.6 Hz, 2H), 3.98–3.82 (m, 1H), 3.72–3.56 (m, 3H), 3.55–3.38 (m, 6H), 3.25 (s, 2H), 2.74 (d, J = 7.8 Hz, 1H), 2.49–1.95 (m, 18H), 1.59 (d, J = 15.2 Hz, 2H).

[0052] Referring to the synthetic method of Compound 1, Compounds 3, 11-13, 17-20 were synthesized.

[0053]

[0054]

[0055]

[0056] Example 2: Synthesis of Compound 2

[0057]

[0058] Synthetic route:

[0059]

[0060] Step 1: Synthesis of Compound 2-1

[0061] Dissolve compound 1-4 (0.6 g, 1.8 mmol, 1 eq) in 2 mL of DMF, stir in an ice bath, and slowly add sodium hydride (0.64 g, 2.7 mmol, 1.5 eq) portionwise. After stirring for 10 minutes, add ethyl bromoacetate (0.60 g, 3.6 mmol, 2 eq) dropwise. Stir at room temperature for 2 hours, add 10 mL of saturated ammonium chloride solution, extract with ethyl acetate, collect the organic layer, dry it over anhydrous sodium sulfate, filter, rotary evaporate the filtrate under reduced pressure, and purify by silica gel column chromatography to obtain a pale yellow solid 2-1 (0.71 g, 1.69 mmol, yield 94%). (ESI) m / z = 420.3 [M+H] + 。

[0062] Step 2: Synthesis of compound 2-2

[0063] Dissolve compound 2-1 (0.71 g, 1.69 mmol, 1 eq) and lithium hydroxide monohydrate (0.08 g, 3.39 mmol, 2 eq) in a mixed solution of 2 mL of methanol and 1 mL of water, and react at room temperature for 2 hours. Concentrate the reaction solution under reduced pressure, adjust the pH to 3 with 1 mol / L hydrochloric acid solution, let it stand overnight, and filter to obtain a white solid compound 2-2 (0.63 g, 1.61 mmol, yield 96%). (ESI) m / z = 392.2 [M+H] + 。 1 H NMR (300 MHz, DMSO-d 6 ) δ 12.59 (s, 1H), 7.47–7.14 (m, 7H), 7.09 (d, J = 8.5 Hz, 1H), 5.74 (s, 1H), 3.48 (d, J = 17.4 Hz, 5H), 2.32 (d, J = 3.4 Hz, 3H), 2.15 (d, J = 3.4 Hz, 3H).

[0064] Step 3: Synthesis of compound 2

[0065] 1-Hydroxy-7-azabenzotriazole (0.052 g, 0.383 mmol, 1.5 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.074 g, 0.383 mmol, 1.5 eq), N-methylmorpholine (0.09 g, 0.894 mmol, 3.5 eq) and compound 2-2 (0.1 g, 0.256 mmol, 1 eq) were added to a eggplant-shaped flask, dissolved in 3 mL of DMSO. After stirring at room temperature for 30 min, 4-(1-piperazinyl)-1H-pyrrolo[2,3-d]pyrimidine (0.052 g, 0.256 mmol, 1 eq) was added and the reaction was carried out at room temperature for 5 h. After monitoring the completion of the reaction, the reaction was stopped. The reaction solution was quenched with water (10 mL), and a white solid precipitated. After suction filtration and drying, compound 2 (0.112 g, 0.19 mmol, yield 76.2%) was obtained. (ESI) m / z = 577.3 [M+H] + 。 1 H NMR (300 MHz, DMSO-d 6 ) δ 11.75 (s, 1H), 8.16 (s, 1H), 7.42–7.32 (m, 4H), 7.29 (d, J = 1.8 Hz, 2H), 7.27 (q, J = 1.8 Hz, 1H), 7.21 (dd, J = 3.6, 2.4 Hz, 1H), 7.11–7.06 (m, 1H), 6.61 (dd, J = 3.7, 1.9 Hz, 1H), 5.69 (s, 1H), 4.87 (d, J = 16.6 Hz, 1H), 3.88 (d, J = 5.7 Hz, 4H), 3.68–3.51 (m, 5H), 2.33 (s, 3H), 2.17 (s, 3H).

[0066] Referring to the synthesis method of compound 2, compounds 10, 16, and 21 were synthesized.

[0067]

[0068] Example 3: Synthesis of compound 4

[0069]

[0070] Synthesis route:

[0071]

[0072] Step 1: Synthesis of compound 4-2

[0073] 4-1 (0.4 g, 1.57 mmol, 1 eq), 1-methyl-1H-pyrazole-5-boronic acid (0.391 g, 1.88 mmol, 1.2 eq) and potassium carbonate (0.649 g, 4.7 mmol, 3 eq) were added to a three-necked flask. Subsequently, 10 mL of dioxane and 1 mL of water were added as a mixed solvent to dissolve them, and the mixture was stirred at room temperature for 5 minutes under nitrogen protection. Bis(tri-tert-butylphosphine)palladium (0.04 g, 0.078 mmol, 0.5 eq) was added to the above reaction solution. The above reaction solution was heated to 80 °C under nitrogen protection and reacted for 4 hours. After monitoring the completion of the reaction by thin-layer chromatography, the oil bath was removed, the reaction solution was cooled to room temperature, the insoluble substances were removed by suction filtration, the reaction solution was rotary evaporated, and then purified by silica gel column chromatography to obtain white solid compound 4-2 (0.24 g, 0.94 mmol, yield 59.7%). (ESI) m / z = 257.1 [M + H] + 。 1 H NMR (400 MHz, Chloroform-d) δ 7.65 (s, 1H), 7.55 (d, J = 2.0 Hz, 1H), 6.39 (d, J = 2.0 Hz, 1H), 3.91 (s, 3H), 3.83 (s, 3H).

[0074] Step 2: Synthesis of compound 4-3

[0075] Compound 4-2 (0.24 g, 0.935 mmol, 1 eq) and N-chlorosuccinimide (0.15 g, 1.12 mmol, 1.2 eq) were added to a sealed tube and heated to 70 °C in an oil bath for 3 h. The oil bath was removed, the reaction solution was cooled to room temperature, the reaction solution was rotary evaporated, and insoluble substances were removed by stirring with methyl tert-butyl ether. The filtrate was rotary evaporated, 2 mL of methanol, 2 mL of tetrahydrofuran and 1 mL of 6N aqueous sodium hydroxide solution were added to the residue, and the mixture was stirred at room temperature overnight. After monitoring the completion of the reaction by thin-layer chromatography, the reaction was stopped, the reaction solution was rotary evaporated, 3N HCl was added to the residue to adjust the pH to 3, and the mixture was extracted with dichloromethane (10 mL × 2), washed with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, and rotary evaporated to obtain white solid product compound 4-3 (0.196 g, 0.71 mmol, yield 72%). (ESI) m / z = 277.1 [M + H] + 。 1 H NMR (300 MHz, DMSO-d 6 ) δ 7.84 (s, 1H), 7.71 (s, 1H), 3.73 (s, 3H).

[0076] Step 3: Synthesis of compound 4-4

[0077] Compound 1-14 (0.05 g, 0.15 mmol, 1 eq) was dissolved in 2 ml of N,N-dimethylformamide. The above reaction solution was transferred to an ice bath, and 60% sodium hydride (0.009 g, 0.225 mmol, 1.5 eq) was slowly added. After stirring for 5 minutes, N-Boc-3-aminopropyl bromide (0.071 g, 0.3 mmol, 2 eq) was added to the above reaction solution. After the addition was completed, the ice bath was removed, and the mixture was stirred at room temperature for 1.5 h. After monitoring the reaction by thin-layer chromatography until completion, saturated ammonium chloride solution (10 mL) was added, and the mixture was extracted with ethyl acetate (10 mL×2), washed with saturated brine (10 mL×2), dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel column chromatography to obtain a transparent oily product 4-4 (0.064 g, 0.13 mmol, yield 86.6%). (ESI) m / z = 491.3 [M+H] + . 1 H NMR (300 MHz, Chloroform-d) δ 7.38–7.24 (m, 6H), 7.14 (dd, J = 8.4, 2.0 Hz, 1H), 7.00–6.93 (m, 2H), 5.49 (s, 1H), 3.95 (dt, J = 14.2, 7.0 Hz, 1H), 3.43 (s, 3H), 3.27 (dd, J = 13.6, 7.0 Hz, 1H), 3.00 (dt, J = 15.0, 5.8 Hz, 2H), 2.35 (s, 3H), 2.21 (s, 3H), 1.70 (s, 2H), 1.44 (s, 9H).

[0078] Step 4: Synthesis of Compound 4-5

[0079] Compound 4-4 (0.25 g, 0.52 mmol) was dissolved in 2 ml of 4N HCl / EA solution, and the mixture was stirred at room temperature for 1 h. After monitoring the reaction to completion, the reaction was stopped, and the reaction solution was concentrated in vacuo. Saturated sodium bicarbonate solution (10 mL) was added to the residue, and the mixture was extracted with ethyl acetate (10 mL×2), washed with saturated brine (10 mL×2), dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain 0.22 g of a crude product, which was used directly in the subsequent reaction without further purification. (ESI) m / z = 391.2 [M+H] + .

[0080] Step 5: Synthesis of Compound 4

[0081] Compound 4-3 (0.064 g, 0.23 mmol, 1 eq), 1-hydroxy-7-azabenzotriazole (0.047 g, 0.346 mmol, 1.5 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.066 g, 0.346 mmol, 1.5 eq), and N-methylmorpholine (0.07 g, 0.69 mmol, 3 eq) were added to a eggplant-shaped flask and dissolved in 3 mL of DMSO. After stirring the above reaction solution at room temperature for 20 min, compound 4-5 (0.09 g, 0.23 mmol, 1 eq) was added, and the reaction was carried out at room temperature for 4 h. After monitoring the completion of the reaction, water (10 mL) was added to the reactants, and a white solid precipitated. The solid was filtered by suction to obtain white solid compound 4 (0.075 g, 0.11 mmol, yield 50.1%). (ESI) m / z = 649.2 [M+H] + 。 1 H NMR (300 MHz, DMSO-d6) δ 8.69 (t, J = 5.7 Hz, 1H), 7.73 (s, 2H), 7.40–7.20 (m, 7H), 7.03 (d, J = 8.5 Hz, 1H), 5.74 (d, J = 5.6 Hz, 1H), 3.92–3.81 (m, 1H), 3.73 (s, 2H), 3.22 (dd, J = 13.1, 6.5 Hz, 1H), 2.85 (ddd, J = 25.2, 16.2, 7.3 Hz, 1H), 2.36 (s, 3H), 2.19 (s, 3H), 1.78 (dp, J = 21.0, 6.8 Hz, 2H).

[0082] With reference to the synthesis method of compound 4, compounds 7, 8, and 14 were synthesized.

[0083]

[0084]

[0085] Example 4: Synthesis of compound 5

[0086]

[0087] Synthesis route:

[0088]

[0089] Step 1: Synthesis of compound 4

[0090] Compound 1-2 (0.093 g, 0.256 mmol, 1 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.074 g, 0.384 mmol, 1.5 eq), 1-hydroxy-7-azabenzotriazole (0.052 g, 0.384 mmol, 0.2 eq) and N-methylmorpholine (0.078 g, 0.768 mmol, 3 eq) were added to 3 ml of DMSO. After stirring at room temperature for 15 minutes, compound 4-5 (0.1 g, 0.256 mmol, 1 eq) was added to the above reaction solution. Subsequently, the reaction was continued at room temperature for 4 h. After monitoring the completion of the reaction, water (10 mL) was added to the reactants, and a white solid precipitated. Filtration by suction gave white solid compound 5 (0.145 g, 0.23 mmol, yield 91%). (ESI) m / z = 634.2 [M+H] + 。 1 H NMR (300 MHz, Chloroform-d) δ 10.43 (s, 1H), 8.34 (s, 1H), 8.11 (t, J = 6.2 Hz, 1H), 7.41–7.24 (m, 7H), 7.19–7.05 (m, 2H), 7.01–6.90 (m, 2H), 6.52 (d, J = 3.7 Hz, 1H), 5.50 (s, 1H), 4.53 (d, J = 13.5 Hz, 2H), 3.94 (dt, J = 14.2, 7.1 Hz, 1H), 3.61 (td, J = 11.1, 3.4 Hz, 2H), 3.44 (s, 4H), 3.07 (ddd, J = 25.6, 13.8, 6.2 Hz, 2H), 2.64 (s, 1H), 2.34 (s, 4H), 2.29 (d, J = 4.3 Hz, 1H), 2.20 (s, 3H), 1.54 (t, J = 13.4 Hz, 2H), 1.29–1.25 (m, 1H), 0.96–0.85 (m, 1H).

[0091] Referring to the synthesis method of compound 5, compounds 9, 15, and 22 were synthesized.

[0092]

[0093] Example 6: Synthesis of compound 6

[0094]

[0095] Synthesis route:

[0096]

[0097] Step 1: Synthesis of compound 6

[0098] Compound 2-2 (0.060 g, 0.15 mmol, 1 eq), N-((S)-1-amino-3-(3-fluorophenyl)propan-2-yl)-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamide (0.063 g, 0.15 mmol, 1 eq) and N-methylimidazole (0.038 g, 0.46 mmol, 3 eq) were added to acetonitrile (10 mL). Then N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate (0.065 g, 0.23 mmol, 1.5 eq) was added to the above reaction solution. After reacting at room temperature for 4 hours, the reaction was monitored by thin layer chromatography to be complete. The above reaction solution was concentrated to dryness, saturated ammonium chloride solution (10 mL) was added to the residue, and extraction was carried out with ethyl acetate (10 mL × 2), washed with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, and concentrated to dryness. Purification by silica gel column chromatography gave the white solid product 6 (0.076 g, 0.095 mmol, yield 63.3%). (ESI) m / z = 800.3 [M + H] + . 1 H NMR (300 MHz, Chloroform-d) δ 7.50 (d, J = 3.6 Hz, 1H), 7.32 (d, J = 4.6 Hz, 1H), 7.30–7.26 (m, 2H), 7.23 (dd, J = 6.7, 2.6 Hz, 3H), 7.18 (dd, J = 3.4, 2.2 Hz, 1H), 7.17–7.13 (m, 1H), 7.11–6.84 (m, 5H), 6.80–6.69 (m, 1H), 5.48 (d, J = 23.9 Hz, 1H), 4.18 (dd, J = 42.3, 16.2 Hz, 2H), 3.98–3.79 (m, 1H), 3.73 (d, J = 8.9 Hz, 3H), 3.43 (d, J = 4.2 Hz, 3H), 3.32–3.04 (m, 3H), 2.69–2.41 (m, 1H), 2.29 (d, J = 17.3 Hz, 3H), 2.14 (d, J = 18.1 Hz, 3H).

[0099] Compounds 23-25 were synthesized by referring to the synthetic method of Compound 6.

[0100]

[0101]

[0102] The pharmaceutical composition involved in the present invention has undergone the following strict and systematic pharmacological tests to comprehensively evaluate its activity and therapeutic effect. These test data provide a strong scientific basis for the further research and development and clinical application of the compounds described in the present invention. Test Example 1. Test for cell proliferation antagonistic effect

[0103] Quickly transfer the cryopreserved 22RV1 or Vcap cells taken out from liquid nitrogen to a water bath at 37 °C and shake rapidly to thaw. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend the cell pellet with RPMI-1640 complete medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin) and transfer it to a cell culture dish containing 10 mL of medium, and culture it in an incubator at 37 °C, 5% CO 2 After the cells adhere to the wall, passage is carried out.

[0104] Take cells in the logarithmic growth phase with good condition, digest them with trypsin cell digestive solution, resuspend them with a certain amount of RPMI-1640 complete medium into a single-cell suspension, count them using a cell counting plate, and inoculate them into a 96-well white plate at a density of 800 cells / well. After standing for 5 min, place it in an incubator at 37 °C, 5% CO 2 Cultivate overnight. The next day, add compounds at different concentrations to act on the cells, and the control group is treated with DMSO at the corresponding concentration. Dilute the stock solution with RPMI-1640 complete medium to a final concentration of 30.00 μM, 10.00 μM, 3.33 μM, 1.11 μM, 0.37 μM, 0.12 μM, 0.04 μM, 0.01 μM. Set 3 parallel wells in each group, add 100 μL of the dosing solution to each well, and place it in an incubator at 37 °C, 5% CO 2 Cultivate for 72 h.

[0105] After the drug acts for 72 h, perform CellTitle-Glo detection. Add 100 μL of CellTitle-Glo solution to each well in the control group and the dosing group, mix on an oscillator for 2 minutes to induce cell lysis. Incubate the 96-well plate at room temperature for 10 minutes to stabilize the fluorescence signal value. Use a microplate reader to detect the luminescence signal. Take the cell group treated with DMSO as the control group, and calculate the half-maximal inhibitory concentration (50% Inhibitory Concentration, IC 50 ) according to the median-effect equation: Inhibition rate (%) = (L value of the control group - L value of the drug-treated group) / A value of the control group × 100%. Use the GraphPad non-linear fitting formula to calculate the IC 50 of the compound. The results are shown in Table 1.

[0106] Table 1 Results of the inhibitory effect of the compounds of the present invention on cell proliferation

[0107] Compound Number <![CDATA[22RV1IC 50 (μM)]]> <![CDATA[VcapIC 50 (μM)]]> Capivasertib 12.42 >50 Compound 1 3.91 3.96 Compound 2 1.12 0.72 Compound 3 11.91 4.37 Compound 9 1.18 0.99 Compound 11 0.89 0.33 Compound 12 4.78 0.86 Compound 13 5.34 2.02 Compound 16 1.07 1.96 Compound 17 0.49 0.49 Compound 18 0.58 0.88 Compound 19 1.99 2.99 Compound 22 13.3 4.21

[0108] Results: The compounds of the present invention show good anti-proliferative activity in 22RV1 and Vcap cells, and the activities are all superior to the existing control drug Capivasertib. Among them, the activity results of compounds 2, 9, and 16 are shown in the appendixFigure 1 In particular, the anti-tumor activities of Compounds 1, 2, 9, 11, 12, 13, 16, 17, 18, and 19 are significantly superior to that of the prior art control drug Capivasertib, and they have significant prospects for drug application. Therefore, the present invention provides that the above-mentioned compounds, their pharmaceutically acceptable salts, stereoisomers, deuterated compounds, or the above-mentioned pharmaceutical compositions can be used to prepare anti-prostate cancer drugs.

[0109] Test Example 2. BRD4 Antagonistic Activity Test

[0110] Prepare 1X buffer (modified HEPES buffer) for BRD4 AlphaScreen protein activity test. Transfer the small molecule compounds to be tested for activity to a 384-well assay plate at a single concentration or gradient concentration for testing the single concentration inhibition rate or IC 50 value. The final concentration of DMSO is 0.1%. Then prepare the BRD4 protein solution with 1X buffer (5 nM). Then prepare the histone (H4) into a substrate solution with 1X buffer. Transfer 5 μL of the protein solution to the 384-well plate as the experimental group or transfer 5 μL of 1X assay buffer to the assay plate as the control group. Then, incubate these assay plates at room temperature for 15 minutes. Add 5 μL of the substrate solution to each well to start the reaction. Next, incubate these 384-well assay plates at room temperature for one hour. In a light-proof environment, add 15 μL of the receptor and donor solutions to each well and incubate at room temperature for another 1 hour. Read the endpoint fluorescence value through the Alpha mode in the full-wavelength microplate reader EnSpire. Finally, import the data into Excel and use Equation 1 to obtain the inhibition value. Equation 1: Inh% = (maximum signal value - compound signal value) / (maximum signal value - minimum signal value) × 100. At the same time, the maximum signal is generated by the action of the enzyme and the substrate, and the minimum signal is obtained from the matrix.

[0111] Test Example 3. AKT1 Antagonistic Activity Test

[0112] Compounds were prepared with DMSO and serially diluted 11-fold at 3-fold intervals. 100 nL of the compound was added to a 384-well plate, and 5 μL of 2× AKT1 enzyme mixture was added. In the complete inhibition control wells, 5 μL of buffer was added instead. After centrifugation at 1000 rpm for 30 s, incubation was carried out at 23 °C for 15 min. Then, 5 μL of 2× ΜLight-MBP peptide (containing 10 μM ATP) was added to all wells of each assay plate, followed by centrifugation at 1000 rpm for 30 s and incubation at 23 °C for 90 min. After incubation, the reaction was terminated with a detection buffer containing 15 mM EDTA and 2 nM Eu-anti-P-MBP antibody, followed by centrifugation at 1000 rpm for approximately 1 min and incubation at 23 °C for 60 min. The plate was read on a Perkin Elmer Envision. The TR-FRET ratio (665 nm value / 615 nm value) was automatically calculated by the Envision; the normalized TR-FRET ratio was calculated, and the % inhibition rate was calculated as follows: Inhibition rate = (100% inhibition control - sample data) / (100% inhibition control - 0% inhibition control) * 100, where the 0% inhibition control is a mixture of enzyme and peptide with ATP without the compound; the 100% inhibition control is a mixture of assay buffer and peptide with ATP without the enzyme solution. The test results of the BRD4 and AKT1 antagonistic activities are shown in Table 2.

[0113] Table 2 Kinase Activity Assay

[0114] Compound BRD4 inh% (3 μM) AKT1 inh% (3 μM) Capivasertib 0 99.2 Compound 1 99.4 98.3 Compound 2 99.8 21.4 Compound 3 99.3 99.2 Compound 11 100 98.7 Compound 12 99.6 100 Compound 13 100 99.3 Compound 17 99.5 98.7

[0115] Results: The compounds showed good dual inhibitory activities against BRD4 and AKT1.

[0116] The above experimental data indicate that the compounds of the present invention have dual inhibitory activities against BRD4 and AKT1. In particular, compounds 1, 3, 11, 12, 13, and 17 showed significant inhibitory activities against the dual-mediated pathways of BRD4 and AKT1, and the inhibitory activities of some compounds against the pathways were significantly better than or equivalent to those of the existing control drugs. However, the advantage of the compounds of the present invention lies in their dual-mediated pathway inhibitory activities. Compared with single-target drugs, the present invention can be effective against different mediated pathways.

[0117] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various equivalent transformations can be made to the technical solutions of the present invention, and these equivalent transformations all fall within the protection scope of the present invention. Additionally, it should be noted that in the above specific embodiments, the various specific technical features described can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods. Furthermore, any combination can be made between various different embodiments of the present invention as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. The use of dihydroquinazolinone compounds in the preparation of anti-prostate cancer drugs, characterized in that The dihydroquinazolinone compound is selected from:

2. Use of the dihydroquinazolinone compound according to claim 1 in the preparation of inhibitors of BET and / or AKT mediated pathways.

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