Benzoazepine and pomalidomide combined compound and preparation method and application thereof

Abstract

The invention relates to a compound of benzoazepine combined with pomalidomide, and a preparation method, a pharmaceutical composition and application thereof. Specifically disclosed is a compound represented by formula I or a pharmaceutically acceptable salt thereof. The compound provided by the invention has good tumor inhibition activity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for preparing a benzozazepine-bound pomalidomide compound and its application. Background Technology

[0002] SKF-83566, chemically named 8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzozazepine-7-ol hydrobromide, belongs to the benzozazepine compound family. SKF-83566 is a D1-like dopamine receptor antagonist with blood-brain penetration and oral activity, and a weaker competitive 5-HT2 receptor antagonist. It is a competitive dopamine transporter inhibitor and may be used in studies related to Parkinson's disease and nicotine craving relief; its therapeutic effects on cancer are rarely reported.

[0003] Summary of the Invention

[0004] Objectives of the invention: The first objective of this invention is to provide a compound of benzozazepine bound to pomalidomide; the second objective of this invention is to provide a method for preparing the benzozazepine bound to pomalidomide compound; and the third objective of this invention is to provide applications of the benzozazepine bound to pomalidomide compound.

[0005] Technical solution: The benzozazepine-bound pomalidomide compound of the present invention has the following structural formula:

[0006]

[0007] Among them, Linker is Where n is 2 to 6.

[0008] Preferably, the benzo[a]azepine-bound pomalidomide compound has the following structural formula:

[0009]

[0010] The preparation method of benzozazepine-bound pomalidomide according to the present invention includes the following steps:

[0011] (1) Compound 1 and Compound 2 undergo a nucleophilic substitution reaction to give Compound 3;

[0012] (2) Compound 3 and compound 4 undergo a nucleophilic substitution reaction to obtain target compound I.

[0013] The synthesis route is as follows:

[0014]

[0015] Where n is 2 to 6.

[0016] The solvent used in step (1) is an organic solvent, such as tetrahydrofuran, N,N-dimethylformamide, dichloromethane, or N-methylpyrrolidone, preferably N,N-dimethylformamide. The alkaline conditions used in step (1) are potassium carbonate, cesium carbonate, or triethylamine, preferably cesium carbonate, with potassium iodide as the catalyst. The reaction temperature in step (1) is 10℃ to 30℃, preferably 15℃ to 25℃.

[0017] The solvent used in step (2) is an organic solvent, such as tetrahydrofuran, N,N-dimethylformamide, dichloromethane, or triethylamine, preferably N,N-dimethylformamide. The alkaline conditions used in step (2) are potassium carbonate, cesium carbonate, or triethylamine, preferably potassium carbonate, with potassium iodide as the catalyst. The reaction temperature in step (2) is 30℃~60℃, preferably 45℃~55℃.

[0018] The application of the benzozazepine-pomalidomide compound described in this invention in the treatment of tumors.

[0019] The tumors mentioned are cervical cancer, breast cancer, and kidney cancer.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The compound of benzozazepine combined with pomalidomide of the present invention has good antitumor activity, and exhibits better antitumor activity than benzozazepine and pomalidomide. Attached Figure Description

[0021] Figure 1 This is a graph showing the inhibitory effect of Example 3 on tumor angiogenesis in a chicken embryo model; where A is a schematic diagram of the effect of Example 3 on tumor angiogenesis; B is a quantitative graph showing the tumor angiogenesis rate after treatment of chicken embryos with Example 3 (2-8 μM) and 5-Fu (8 μM);

[0022] Figure 2 A is a graph showing the inhibitory effect of Example 3 on tumor growth in a chicken embryo model; B is a schematic diagram showing the effect of Example 3 on tumor growth; C is a quantitative graph showing the tumor weight (mg) of chicken embryos after treatment with Example 3 (2-8 μM) and 5-Fu (8 μM). Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the embodiments.

[0024] Example 1

[0025] 4-((2-((8-bromo-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1H-benzo[d]azapyro-7-yl)oxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione, with the following structural formula:

[0026]

[0027] Its preparation method includes the following steps:

[0028] (1) Synthesis of 4-((2-bromoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindololin-1,3-dione

[0029]

[0030] 500 mg (1.83 mmol) of pomalidomide, 1193 mg (3.66 mmol) of Cs₂CO₃, and 30 mg (0.18 mmol) of KI were added sequentially to the reaction flask. 5 mL of DMF was used as the solvent, followed by 157 μL (1.83 mmol) of 1,2-dibromoethane. The mixture was reacted at 15 °C–25 °C for 3 h. 10 mL of water was added, and the mixture was extracted with ethyl acetate. The extract was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane:methanol = 50:1) to obtain 180 mg of the target product, with a yield of 25.9%.

[0031] (2) Synthesis of 4-((2-((8-bromo-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1H-benzo[d]azapyro-7-yl)oxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0032] 73 mg (0.22 mmol) of 8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzozazepine-7-ol hydrobromide, 376 mg (0.55 mmol) of potassium carbonate, 4 mg (0.02 mmol) of KI, and 5 mL of DMF were added sequentially to the reaction flask. Then, 100 mg (0.26 mmol) of 4-((2-bromoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione was added. The reaction was carried out at 45℃~55℃ for 8 h. After adding 10 mL of water, the mixture was extracted with ethyl acetate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 30:1) to obtain 74 mg of the target product, with a yield of 53.4%.

[0033] The target compound is a yellow solid; 1 H NMR(400MHz, DMSO-d6)δ 8.03 (t, J=5.6Hz, 1H), 7.45 (dt, J=13.4, 7.7Hz, 2H), 7.05-6.93 (m, 3H), 6.48 (d, J= 31.5Hz, 3H), 4.91-4.33(m, 3H), 3.47(s, 7H), 3.24-2.64(m, 4H), 2.50-1.97(m, 7H). 13C NMR(101MHz, DMSO-d6)δ 173.03, 172.12, 170.11, 169.58, 169.03, 168.60, 168.31, 167.83, 147.23, 146.95, 135.98, 135.55, 132.91, 132.45, 122.23, 1 21.72, 111.50, 111.18, 109.77, 108.96, 60.02, 58.01, 52.12, 51.71, 49.59, 42.32, 42.08, 40.41, 31.72, 30.92, 24.18, 21.88.

[0034] Example 2

[0035] 4-((3-((8-bromo-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1H-benzo[d]azapyro-7-yl)oxy)propyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione, with the following structural formula:

[0036]

[0037] Its preparation method is as follows:

[0038] 70 mg (0.21 mmol) of 8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzozazepine-7-ol hydrobromide, 373 mg (0.53 mmol) of potassium carbonate, 3 mg (0.02 mmol) of KI, and 5 mL of DMF were added sequentially to the reaction flask. Then, 100 mg (0.25 mmol) of 4-((3-bromopropyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (prepared using step 1 of Example 1, except that 1,2-dibromoethane was replaced with 1,3-dibromopropane) was added. The reaction was carried out at 45 °C–55 °C for 9 h. After adding 10 mL of water, the mixture was extracted with ethyl acetate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 30:1) to obtain 47 mg of the target product, with a yield of 36.4%.

[0039] The target compound is a yellow solid; 1 H NMR (400MHz, DMSO-d6) δ7.57-6.88 (m, 11H), 6.54 (d, J = 10.6Hz, 1H), 6.31 (s, 1H), 4.24 (dd, J = 28.9, 7. 6Hz, 2H), 3.81 (q, J=5.5Hz, 2H), 3.20-2.56 (m, 9H), 2.25 (s, 3H), 2.08-1.79 (m, 2H), 1.35-1.16 (m, 2H). 13C NMR (101MHz, DMSO-d6) δ172.04, 153.21, 152.40, 145.40, 143.28, 135.96, 135.85, 135.82, 134.01, 133.95, 133.52, 132.45, 128.92, 128.7 0, 128.64, 128.42, 126.63, 126.45, 122.23, 117.10, 106.40, 62.60, 5 7.48, 57.39, 49.79, 49.54, 49.26, 47.87, 47.67, 34.50, 31.68, 21.92.

[0040] Example 3

[0041] 4-((4-((8-bromo-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1H-benzo[d]azapyro-7-yl)oxy)butyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione, with the following structural formula, is prepared by the following steps:

[0042]

[0043] Its preparation method is as follows:

[0044] To the reaction flask, 68 mg (0.20 mmol) of 8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzozazepine-7-ol hydrobromide, 369 mg (0.52 mmol) of potassium carbonate, 3 mg (0.02 mmol) of KI, and 5 mL of DMF were added sequentially. Then, 100 mg (0.24 mmol) of 4-((4-bromobutyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (prepared using step 1 of Example 1, except that 1,2-dibromoethane was replaced with 1,4-dibromobutane) was added. The reaction was carried out at 45°C–55°C for 7 h. After that, 10 mL of water was added, and the mixture was extracted with ethyl acetate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 30:1) to obtain 56 mg of the target product, with a yield of 42.5%.

[0045] The target compound is a yellow solid; 1H NMR(400MHz, DMSO-d6)δ 7.56-6.92 (m, 11H), 6.53 (d, J=6.8Hz, 1H), 6.31 (s, 1H), 4.25 (dd, J=34.1, 7.4Hz, 1H), 3.91-3.57 (m, 3H), 3.1 1-2.58 (m, 9H), 2.25 (s, 3H), 2.13-1.93 (m, 1H), 1.57 (dq, J=21.3, 7.7Hz, 3H), 1.39 (s, 1H), 0.88-0.79 (m, 1H). 13 C NMR (101MHz, DMSO-d6) δ172.10, 170.11, 153.24, 152.38, 147.23, 145.40 ,143.28,142.76,135.75,134.02,133.52,128.91,128.72,128.64,128.4 4, 126.63, 126.48, 122.21, 117.09, 111.49, 108.54, 106.39, 68.70, 62.6 0, 57.48, 49.25, 47.84, 47.67, 45.02, 34.49, 31.65, 26.81, 26.41, 21.95.

[0046] Example 4

[0047] 4-((5-((8-bromo-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1H-benzo[d]azapyro-7-yl)oxy)pentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione, with the following structural formula, is prepared by the following steps:

[0048]

[0049] Its preparation method is as follows:

[0050] To the reaction flask, 66 mg (0.20 mmol) of 8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzozazepine-7-ol hydrobromide, 369 mg (0.52 mmol) of potassium carbonate, 3 mg (0.02 mmol) of KI, and 5 mL of DMF were added sequentially. Then, 100 mg (0.24 mmol) of 4-((5-bromopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (prepared using step 1 of Example 1, except that 1,2-dibromoethane was replaced with 1,5-dibromopentane) was added. The reaction was carried out at 45-55°C for 8 h. After adding 10 mL of water, the mixture was extracted with ethyl acetate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 30:1) to obtain 85 mg of the target product, with a yield of 63.1%.

[0051] The target compound is a yellow solid; 1 H NMR(400MHz,DMSO-d6)δ 9.91 (s, 1H), 7.59-6.92 (m, 11H), 6.53 (s, 1H), 6.30 (d, J=41.4Hz, 1H), 3.91-3.60 (m, 4H), 3.16-2.66 (m, 9H), 2.20-0.80 (m, 10H). 13 C NMR (101MHz, DMSO-d6) δ172.05, 170.04, 167.82, 153.42, 152.80, 147.25 , 135.95, 134.02, 133.69, 132.44, 129.20, 129.02, 128.79, 128.58, 127. 22, 127.05, 122.25, 116.84, 111.48, 108.85, 106.84, 68.69, 61.05, 60.7 6, 56.43, 49.58, 36.26, 31.67, 31.26, 29.47, 28.40, 27.39, 22.99, 21.95.

[0052] Example 5

[0053] 3-(4-((6-((8-bromo-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1H-benzo[d]azapyro-7-yl)oxy)hexyl)amino)-1,3-dioxoisoindoline-2-yl)-2,6-dioxopiperidine-4-yl, with the following structural formula, is prepared by the following steps:

[0054]

[0055] Its preparation method is as follows:

[0056] To the reaction flask, 66 mg (0.20 mmol) of 8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzozazepine-7-ol hydrobromide, 369 mg (0.52 mmol) of potassium carbonate, 3 mg (0.02 mmol) of KI, and 5 mL of DMF were added sequentially. Then, 100 mg (0.23 mmol) of 4-((6-bromohexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (prepared using step 1 of Example 1, except that 1,2-dibromoethane was replaced with 1,6-dibromohexane) was added. The reaction was carried out at 45-55°C for 7 h. After that, 10 mL of water was added, the mixture was extracted with ethyl acetate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 30:1) to obtain 42 mg of the target product, with a yield of 30.5%.

[0057] The target compound is a yellow solid; 1 H NMR (400MHz, DMSO-d6) δ7.55-6.94 (m, 11H), 6.52 (d, J=8.5Hz, 1H), 6.35 (s, 1H), 4.25 (dd, J=34.0, 7.5Hz, 1H ), 3.83-3.60(m, 3H), 3.10-2.59(m, 9H), 2.28-2.24(m, 3H), 2.00(dt,J=12.8, 6.7Hz, 1H), 1.42-1.20(m, 8H). 13 C NMR (101MHz, DMSO-d6) δ172.05, 170.03, 167.82, 153.29, 152.43, 147.24, 14 3.24, 135.67, 133.89, 133.52, 132.45, 128.89, 128.73, 128.62, 128.46, 126. 61, 122.23, 117.19, 115.27, 111.48, 109.00, 106.42, 68.87, 62.55, 57.49, 49.57, 49.29, 47.81, 34.49, 31.66, 28.76, 27.74, 26.81, 26.33, 25.48, 21.95.

[0058] Bioactivity evaluation

[0059] 1. MTT assay for antitumor activity

[0060] Cell culture: Human cancer cell lines MDA-MB-231 and HeLa cells were purchased from the National Biomedical Laboratory in Beijing and cultured in DMEM (KGM12800-500) or MEM (KGM41500-500) medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, in a Thermo Fisher Scientific (BB150) incubator at 37°C with 5% CO2. When the cell confluence reached 70%–80%, 0.25% trypsin was added for digestion, resuspending, and culturing. Cells in the logarithmic growth phase and in good growth condition were selected for further study.

[0061] Methyl thiazolyl tetrazolium (MTT) was used to determine cell viability. Hemocytometer counting was used for cell counting, and cell viability was greater than 95% in all experiments. MDA-MB231 and HeLa cells were counted at 1 × 10⁻⁶ cells / mL. 4 Cells were seeded in 96-well plates. 100 μL of medium (containing 1% FBS) was added to each well to dissolve different concentrations of the drug (0–50 μM), and the cells were incubated for 24 h. After centrifugation (5 min, 2000 rpm), the supernatant was discarded, and 10 μL of MTT (5 mg / mL) solution was added to each well. The cells were incubated at 37°C for 4 h, centrifuged again, and the supernatant was discarded. 100 μL of DMSO was added to each well, and the cells were shaken for 10 min to fully dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader (BioTek, USA). Cytotoxicity was assessed compared to the control group (DMSO). The concentration at which the drug induced 50% cell growth inhibition (IC50) was determined using a curve fitting algorithm in GraphPad Prism 9 (GraphPad software, La Jolla, CA, USA) via nonlinear regression. 50 ).

[0062] To investigate the effects of the compounds on tumor cell activity, cytotoxicity was detected using the MTT assay. Table 1 shows the effects of the compounds on the activity of different tumor cell lines, and the half-maximal inhibitory concentration (IC50) of the drugs was calculated for each cell line. s0 (48h).

[0063] Table 1. Inhibitory activity of compounds against different tumor cells (IC50) 50 )

[0064]

[0065] " / " indicates that it was not measured.

[0066] The results in Table 1 show that the compound has a significant inhibitory effect on HeLa and MDA-MB-231 tumor cells, and has great research value.

[0067] 2. Example 3: Inhibitory effect on tumor growth in the MDA-MB-231 cell xenograft model

[0068] This invention evaluates the inhibitory effect of Example 3 on in vivo tumor growth using an MDA-MB-231 cell xenograft model. This model utilizes the chicken embryo chorioallantoic membrane assay (CAM). After 8 days of chicken embryo incubation, MDA-MB-231 cells were inoculated. On day 11, the chicken embryos were randomly divided into 5 groups: 3 groups received 2 μM, 4 μM, and 8 μM concentrations, 1 group received 0.6 μM 5-FU, and the remaining group served as a blank control group. On day 14, the tumor was removed, photographed, and weighed. Figure 1 The results showed that Example 5 had a strong inhibitory effect on angiogenesis (2μM: 74.03±4.11%; 4μM: 64.62±6.88%; 8μM: 50.28.55±6.52%), which was better than 5-FU (8μM: 66.55±1.52%) at the same concentration.

[0069] Meanwhile, the embodiments were able to significantly inhibit tumor growth in a dose-dependent manner. Figure 2 The results showed that the volume and weight of chicken embryo tumors decreased significantly after administration. The weights of chicken embryo tumors treated with low, medium, and high concentrations of Example 3 were 32.83±2.14 mg, 14.17±1.47 mg, and 8.17±1.47 mg, respectively. This data was superior to the treatment group with the same concentration (8 μM) of 5-Fu (16.17±1.47 mg).

Claims

1. A compound of formula (I), or a salt thereof, or a hydrate thereof: in, Linker is Where n is 2 to 6.

2. The compound of benzo[a]zo[a]zo[b]cyclo[a]zo[b][pomalidomide] according to claim 1, characterized in that, It is a compound or a pharmaceutically acceptable salt thereof, as shown below:

3. A method for preparing the benzo[a]zo ... The following steps are included in which compounds 3 and 4 undergo a nucleophilic substitution reaction to yield compound I: Where: n is 2 to 6.

4. A pharmaceutical composition, characterized in that, It includes the compounds as described in claims 1 to 2 and at least one pharmaceutically acceptable excipient.

5. The use of a substance in the preparation of a medicament for treating diseases, characterized in that, The substance is a compound as described in claims 1-2, and the disease is cancer; preferably cervical cancer, breast cancer, and kidney cancer.

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