Aza aromatic amine PROTAC compound with targeted NCoR1 degradation activity and preparation method and application thereof
By synthesizing aza-aromatic amine PROTAC compounds, the problem of insufficient targeted degradation activity of NCoR1 in existing technologies has been solved, achieving efficient degradation of NCoR1 protein and exhibiting significant metabolic regulation effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-03-27
AI Technical Summary
The lack of drugs that can effectively target and degrade NCoR1 activity in the current technology leads to significant side effects when treating metabolic syndrome and makes it difficult to effectively regulate metabolic-related symptoms.
A nitrogen-containing aromatic amine-based PROTAC compound was designed and synthesized. An intermediate was prepared by acylation and N-alkylation reactions, and then further reacted with nitrogen-containing aromatic amines to obtain compounds XH-1~XH-20 with targeted degradation activity of NCoR1.
The synthesized compound XH-10 exhibited significant NCoR1 degradation activity at 5 μM, demonstrating advantages such as novel structure and high degradation activity, providing a new option for the development of NCoR1 protein degradation drugs.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of medicinal chemistry, and discloses a kind of nitrogen heterocyclic aromatic amine class PROTAC compound with targeted degradation NCoR1 activity and its preparation method and purpose. BACKGROUND
[0002] Nuclear receptor co-repressor 1 (NCoR1) is a key protein involved in the transcriptional regulation of various nuclear factors, which can bind and inhibit the activity of key nuclear receptors such as androgen receptor (AR), thyroid hormone receptor (TR), and peroxisome proliferator-activated receptors (PPARs) involved in metabolic regulation. It plays an important regulatory role in glucose metabolism, lipid metabolism, and inflammatory response (① Saito T, Kuma A, Sugiura Y, et al. Autophagy regulates lipid metabolism through selective turnover of NCoR1. Nature Communications, 2019, 10(1): 1-16; ② Van Leene, C, De Bosscher, K. NCOR1 / 2 and glucocorticoid receptor orchestrate hepatic function. Nature Metabolism, 2024, 6: 783-784).
[0003] Studies have shown that the deficiency of NCoR1 in intestinal epithelial cells not only increases energy expenditure but also inhibits lipid absorption, thereby comprehensively improving various symptoms of metabolic syndrome. Compared with existing glucagon-like peptide-1 receptor (GLP-1) drugs, treatment targeting NCoR1 mainly affects fat weight and has no effect on non-fat weight, reducing the possible side effects (③ Hou S, Yu H, Liu C, et al. Intestinal epithelial cell NCoR deficiency ameliorates obesity and metabolic syndome. Acta Pharmaceutica Sinica B, 2024,14(12): 5267-5285). Therefore, the development of small molecule drugs targeting the important metabolic factor NCoR1 shows great potential in the treatment of metabolic syndrome.
[0004] Protein degradation-targeting chimeras (PROTACs) are an emerging drug development technology that selectively degrades pathogenic proteins using intracellular protein degradation mechanisms, offering a novel approach to disease treatment. Based on the clinical needs for treating metabolic syndrome, the applicant intends to develop PROTAC compounds that target the degradation of NCoR1 activity. Following extensive literature review, the applicant has conducted design, synthesis, and activity evaluation studies of novel compounds, obtaining valuable research results. Summary of the Invention
[0005] The present invention aims to provide a novel PROTAC compound of the aza-aromatic amine class with good activity and targeted degradation activity of NCoR1; another objective is to provide its preparation method and uses.
[0006] To achieve the objectives of this invention, the aza-aromatic amine compounds of this invention have the structural formulas shown in Formulas I to IV: In equations I-IV, .
[0007] It may also be other pharmaceutically acceptable salts, or solvates, or hydrates, or prodrugs, or metabolites thereof.
[0008] The present invention also provides a method for preparing the aforementioned compound, comprising the following steps: The first step involves using linalidomide and different acyl chlorides as raw materials, and then performing an acylation reaction in an acetic acid / sodium acetate buffer solution to obtain intermediates M-1 to M-4. .
[0009] In the second step, intermediates M-1 to M-4 undergo N-alkylation reactions with different aza-aromatic amines to obtain the target compounds XH-1 to XH-20.
[0010] .
[0011] The applicant of this invention confirmed the structure of the synthesized target compounds XH-1 to XH-20 and performed content analysis by high performance liquid chromatography. The contents of the target compounds XH-1 to XH-20 were all greater than 99.0%. Based on this, the activity of the obtained compounds was evaluated.
[0012] The present invention has the following beneficial effects: This invention discloses a nitrogen-containing aromatic amine PROTAC compound with targeted NCoR1 degradation activity, its preparation method, and its uses. Liver X Receptor Response Element-Luciferase reporter gene system (LXRE-Luc luciferase reporter gene system) assays showed that this class of compounds exhibited significant liver X Receptor (LXRs) regulatory activity at 10 μM. Western blotting studies demonstrated that these compounds showed significant degradation activity against NCoR1 protein, with the most active compound, XH-10, producing significant NCoR1 degradation at 5 μM. The compounds prepared in this invention, when used for targeted NCoR1 protein degradation, possess novel structures and high degradation activity, providing a new option for developing drugs with NCoR1 protein degradation activity.
[0013] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention. Attached Figure Description
[0014] Figure 1 The image shows the results of LXRE-Luc luciferase reporter gene system assays using the compounds XH-10, XH-12, and XH-17 of this invention. Figure 2 The image shows the results of a Western blot (WB) experiment using the compound XH-10 of this invention. Detailed Implementation
[0015] The following detailed description of specific embodiments, presented in the form of experimental examples, further illustrates the above-described content of the present invention. However, this should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.
[0016] The chemical reagents used in the specific embodiments of the present invention are obtained by purchasing commercially available products.
[0017] The instruments used for detection and analysis in this invention are as follows: Nuclear magnetic resonance imaging (MRI) scanner, specifically the Bruker Advance Digital 400 MRI scanner from Switzerland.
[0018] High-resolution mass spectrometers: Bruker SolanX 70 FT-MS (Switzerland); Agilent 6540 TOF.
[0019] High-performance liquid chromatograph, Agilent Technologies 1260 Infinity II high-performance liquid chromatograph.
[0020] Examples 1-5 are experiments on the synthesis of compounds, and Examples 6-7 are experiments on the activity evaluation of compounds.
[0021] Example 1: Preparation of Intermediate M-1 Under ice-water bath conditions, 2.59 g (0.01 mol) of lenalidomide and 12 mL of distilled water were added to a 100 mL three-necked flask and stirred until homogeneous. Then, 6.4 mL (0.18 mol) of acetic acid, 2.8 g (0.06 mol) of sodium acetate, and 2 mL (0.025 mol) of chloroacetyl chloride were added sequentially, and the mixture was stirred for 6 hours. Next, 20 mL of distilled water was added to the system, and the mixture was stirred for 30 minutes. The mixture was filtered, and the filter cake was washed three times with 90 mL of water and dried to give compound M-1, a white solid, with a yield of 95.3%.
[0022] Compound M-1: 1 H NMR (400 MHz, DMSO- d 6) δ ppm: 11.01 (s, 1H), 10.19 (s,1H), 7.88 - 7.72 (m, 1H), 7.59 - 7.39 (m, 2H), 5.14 (m, 1H), 4.51 - 4.20 (m,3H), 2.91 (m, 1H), 2.60 (m, 1H), 2.34 (m, 1H), 2.12 - 1.95 (m, 2H). 13 C NMR (101 MHz, DMSO-)d 6) δ ppm: 172.90, 171.11, 168.18, 167.79, 133.79, 133.44,132.76, 128.76, 125.28, 119.33, 51.55, 46.49, 40.76, 31.21, 22.66.HRMS (ESI) m / z: [C 15 H 15 ClN3O4] + : 336.0751.
[0023] Using a preparation method similar to that of Experimental Example 1, intermediates M-2 to M-4 can be obtained: Example 2 Preparation of compound XH-1 In a 100 mL three-necked flask, 10 mL of toluene, 3.35 g (0.01 mol) of intermediate M-1, 1.0 g (0.011 mol) of 2-aminopyridine, and 5 mg of KI were added sequentially. The mixture was refluxed for 6 hours and filtered while hot. The filter cake was added to 20 mL of acetone and refluxed for half an hour. After filtration, crude compound XH-1 was obtained. The crude product was purified by column chromatography with dichloromethane:methanol = 10:1 as the eluent, yielding 2.9 g of product, with a yield of 73.7%.
[0024] Compound XH-1: 1 H NMR (400 MHz, DMSO- d 6) δ ppm: 10.95 (s, 1H), 7.75 (d, J =7.3 Hz, 1H), 7.60 - 7.32 (m, 3H), 7.14 (m, 1H), 6.23 (s, 1H), 5.08 (d, J = 13.2Hz, 1H), 4.30 (d, J = 21.1 Hz, 4H), 2.84 (m, 1H), 2.53 (d, J = 19.5 Hz, 1H), 2.33- 2.22 (m, 1H), 2.01 (s, 3H). 13 C NMR (101 MHz, DMSO- d6) δ ppm: 172.97, 171.15,168.85, 167.91, 150.79, 142.80, 133.87, 132.86, 131.35, 128.90, 127.57,125.58, 123.45, 119.8, 113.54, 51.61,46.44, 31.25, 23.45, 22.69. HRMS (ESI)m / z: [C 20 H 20 N5O4] + :394.1518.
[0025] Compounds XH-2 to XH-5 were obtained using a preparation method similar to that in Example 2:
[0026] Example 3: Preparation of compound XH-6 In a 100 mL three-necked flask, 20 mL of toluene, 7.0 g (0.02 mol) of intermediate M-2, 2.1 g (0.022 mol) of 2-aminopyridine, and 5 mg of KI were added sequentially. The mixture was refluxed for 7 hours and filtered while hot. The filter cake was added to 40 mL of acetone and refluxed for half an hour. After filtration, crude compound XH-6 was obtained. The crude product was purified by column chromatography using dichloromethane:methanol = 10:1 as the eluent, yielding 5.4 g of product, with a yield of 66.2%.
[0027] Compound XH-6: 1 H NMR (400 MHz, DMSO- d 6) δ ppm: 10.92 (s, 1H), 7.71 (m,1H), 7.40 (m, 2H), 7.31 – 7.07 (m, 1H), 6.52 – 6.20 (m, 1H), 5.71 (s, 1H),5.16 – 4.93 (m, 1H),4.22 (d, J = 9.4 Hz, 4H), 2.88 – 2.68 (m, 1H), 2.48 (d, J =17.6 Hz, 1H), 2.30 – 2.14 (m, 1H), 2.05 – 1.84 (m, 3H). 13 C NMR (101 MHz, DMSO- d6) δ ppm: 172.98, 171.17, 168.53, 167.7, 159.78, 147.69, 137.03, 133.87,132.86, 128.92,125.60, 123.56, 119.85, 111.85, 108.03, 51.61, 46.52, 43.16,31.27, 23.46, 22.70. HRMS (ESI) m / z: [C 21 H 22 N5O4] + :408.1673.
[0028] Compounds XH-7 to XH-10 were obtained using a preparation method similar to that in Example 3:
[0029] Example 4: Preparation of compound XH-11 In a 100 mL three-necked flask, 30 mL of toluene, 7.3 g (0.02 mol) of intermediate M-3, 2.1 g (0.022 mol) of 2-aminopyridine, and 5 mg of KI were added sequentially. The mixture was refluxed for 7 hours and filtered while hot. The filter cake was added to 40 mL of acetone and refluxed for half an hour. After filtration, crude compound XH-11 was obtained. The crude product was purified by column chromatography with dichloromethane:methanol = 10:1 as the eluent, yielding 4.4 g of product, with a yield of 54.8%.
[0030] Compound XH-11: 1 H NMR (400 MHz, DMSO- d 6) δ ppm: 10.84 (s, 1H), 9.82 (s,1H), 7.64 (m, 2H), 7.42 – 7.23 (m, 2H), 7.22 – 7.07 (m, 2H), 6.26 – 6.19 (m,1H), 5.63 (s, 1H), 4.93 (m,1H), 4.39 – 4.03 (m, 2H), 3.68 (t, J = 6.2 Hz, 2H), 2.69 (m, 3H), 2.53 – 2.31 (m, 2H), 2.19 – 2.05 (m, 1H), 1.91 – 1.76 (m, 2H). 13 CNMR (101 MHz, DMSO-d 6) δ ppm: 172.98, 171.17, 168.26, 167.88,159.77, 147.69,137.02, 133.85, 133.48, 132.79, 128.81, 125.37, 119.39, 111.85, 108.03,51.61, 46.57, 40.81, 33.45, 31.26, 22.71,21.36. HRMS (ESI) m / z: [C 22 H 24 N5O4] + :422.1826.
[0031] Compounds XH-12 to XH-15 were obtained using a preparation method similar to that in Example 4:
[0032] Example 5: Preparation of compound XH-16 In a 100 mL three-necked flask, 15 mL of toluene, 3.8 g (0.01 mol) of intermediate M-4, 1.0 g (0.011 mol) of 2-aminopyridine, and 5 mg of KI were added sequentially. The mixture was refluxed for 6 hours and filtered while hot. The filter cake was added to 20 mL of acetone and refluxed for half an hour. After filtration, crude compound XH-16 was obtained. The crude product was purified by column chromatography using dichloromethane:methanol = 10:1 as the eluent, yielding 2.5 g of product, with a yield of 59.3%.
[0033] Compound XH-16: 1 H NMR (400 MHz, DMSO- d 6) δ ppm: 11.00 (s, 1H), 10.14(s, 1H), 8.30 (s, 2H), 8.03 (s, 1H), 7.81 (s, 1H), 7.49(s, 2H), 6.86 (s, 1H), 4.27 (d, J = 69.3 Hz, 3H), 2.92 (s, 2H), 2.63 (s, 2H), 2.32 (s, 2H) 2.18 (d, J =7.7 Hz, 2H), 1.91 (s, 2H), 1.82 (s, 2H). 13 C NMR (101 MHz, DMSO- d6) δ ppm:173.54, 172.13, 171.51, 162.3, 146.58, 137.10, 136.32, 135.21, 128.68,127.52, 125.32, 124.36, 118.32, 114.36, 113.47, 51.56,46.51, 45.08, 33.61,32.15, 28.32, 23.53, 22.28. HRMS (ESI) m / z: [C 23 H 26 N5O4] + :436.1987.
[0034] Compounds XH-17 to XH-20 were obtained using a preparation method similar to that in Example 5:
[0035] Example 6: Systematic Evaluation of LXRE-Luc Luciferase Reporter Genes of Compounds XH-1 to XH-20 The LXR regulatory effects of compounds XH-1 to XH-20 synthesized in this invention were evaluated using the LXRE-Luc luciferase reporter gene system. The experimental procedures are as follows: (1) Cell preparation: Commonly used tool cells, human embryonic kidney cells HEK293T, were selected and cultured in DMEM + 10% fetal bovine serum FBS + 1% penicillin-streptomycin double antibiotic solution PS in a constant temperature incubator at 37℃ and 5% CO2. (2) Preparation of experimental drugs: Dissolve compounds XH-1 to XH-20 (purity greater than 99%) in DMSO to prepare a 10 mM stock solution and store at -20℃. When using, dilute compounds XH-1 to XH-20 to a final concentration of 10 μM in PS medium containing DMEM + 10% fetal bovine serum FBS + 1% penicillin-streptomycin double antibiotic solution.
[0036] (3) Preparation of control drugs: negative control (DMSO, final concentration = 0.1%), positive control (LXR agonist GW3965, working concentration 5 μM).
[0037] (4) Plasmid preparation: LXRE-Luc reporter gene plasmid and pCMV5-hLXR overexpression plasmid.
[0038] (5) Experimental procedure: ① Add 2 mL of logarithmic-phase human embryonic kidney cells HEK293T (approximately 8 × 10⁶ cells per well) to each well. 5① Seeds of 1000 cells were seeded into 6-well plates and incubated in DMEM medium containing 10% fetal bovine serum for 24 hours at 37°C and 5% CO2 until cell confluence reached 60%–70%. ② Transfection: 2 μg / well of LXRE-Luc reporter gene plasmid and pCMV5-hLXR overexpression plasmid were co-transfected into the cells. After transfection, the cells were cultured for 6–8 hours and then replaced with fresh complete medium. ③ After digestion, the transfected human embryonic kidney cells HEK293T were seeded into 96-well all-white plates (approximately 5 × 10⁶ cells per well). 4 (1) For each cell, compounds XH-1~XH-20 at a concentration of 10 μM were added to the corresponding wells. A DMSO solvent control group (final concentration ≤0.1%) and a positive control group (GW3965, 5 μmol / L) were set up, with 4 replicates per group, and cultured for 24 h. ④ Fluorescence detection: Firefly luciferase substrate was added, and firefly luciferase activity (Luc) was measured sequentially. ⑤ Data processing: The relative fluorescence activity percentage of the experimental groups was calculated using the formula = (average fluorescence activity of experimental groups / average fluorescence activity of control groups) × 100%. The relative activity ratio of the negative control group (DMSO) was 100%, and the relative activity ratio of the positive control group (GW3965 group) was 146.8%.
[0039] The compounds XH-1 to XH-20 synthesized in this invention were measured using the LXRE-Luc luciferase reporter gene system method. The relative activity percentages obtained are shown in Table 1. The evaluation results of compounds XH-10, XH-12, and XH-17 synthesized in this invention on the LXRE-Luc luciferase reporter gene system are shown in [link to table]. Figure 1 .
[0040] .
[0041] As shown in Table 1, compounds XH-1 to XH-20 of this invention all exhibit a certain degree of LXR regulation, which can be summarized as follows: (1) All synthesized compounds XH-1 to XH-20 have a clear regulatory effect on LXR. At 10 μM, the relative fluorescence activity percentage of the synthesized compounds XH-1 to XH-20 is 113.1-119.3% (the relative fluorescence activity percentage of the positive control drug GW3965 at 5 μM is 146.8%). (2) Among the synthesized compounds XH-1 to XH-20, compound XH-10 has the most significant regulatory effect on LXR.
[0042] Evaluation of Western Blot (WB) Results of Experiment Example 7: Compounds XH-1 to XH-20 The degradation activity of the compounds XH-1 to XH-20 synthesized in this invention on NCoR1 protein was evaluated using Western blotting (WB). The experimental procedures are as follows: (1) Cell preparation: Human colon cancer cells HCT116 were selected and cultured in a 37℃, 5% CO2 constant temperature incubator with DMEM + 10% fetal bovine serum FBS + 1% penicillin-streptomycin double antibiotic solution PS.
[0043] (2) Preparation and addition of experimental drugs: Compounds XH-1 to XH-20 (purity greater than 99%) were dissolved in DMSO to prepare a 10 mM stock solution and stored at -20℃. When using, compounds XH-1 to XH-20 were diluted to the final concentrations (1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 1 μM, 5 μM, 10 μM) in DMEM + 10% fetal bovine serum FBS + 1% penicillin-streptomycin double antibiotic solution PS medium. The solutions of compounds XH-1 to XH-20 at different concentrations were added to the human colon cancer cell HCT116 culture medium prepared above and incubated at room temperature for 24 h.
[0044] (3) Preparation of control drugs: negative control (DMSO, final concentration ≤0.1%), there is no commercially available NCoR1 protein PROTAC.
[0045] (4) Preparation of cell lysis samples: After washing with PBS, add ice-cold RIPA (strong) lysis buffer (containing protease inhibitor and phosphatase inhibitor) to the cells prepared above, lyse on ice for 10 min, remove cell debris by centrifugation at 12000r for 10 min, collect protein supernatant, add 5× loading buffer, and denature at 98℃ for 10 min.
[0046] (5) Western blot (WB) detection procedure: Electrophoresis was performed using a 6% separating gel-SDS-PAGE (80V→120V). The PVDF membrane was activated with methanol, transferred at 400mA for 1.5h, and blocked with 5% skim milk for 2h. 1:500 diluted NCoR1 polyclonal antibody (Rabbit) was added, with HSP90 selected as the internal control protein. The membrane was incubated overnight at 4℃ and washed 3 times with TBST. 1:2000 diluted HRP-conjugated Goat Anti-Rabbit IgG (H+L) secondary antibody was added, and the membrane was incubated at room temperature for 1.5h and washed 3 times with TBST. The membrane was developed and exposed using ECL chemiluminescence buffer.
[0047] Western blotting (WB) was used to determine the properties of compounds XH-1 to XH-20 synthesized in this invention. The experimental results are shown in Table 2. A demonstration of the WB evaluation results for compound XH-10 synthesized in this invention is shown below. Figure 2 .
[0048] The results of Western blot analysis on the compounds XH-1 to XH-20 synthesized in this invention show that: (1) The compounds XH-1 to XH-20 of the present invention all exhibit a certain degree of NCoR1 degradation. Under the experimental conditions, the compounds XH-1 to XH-20 of the present invention do not degrade HSP90 protein, and show targeting of NCoR1 protein.
[0049] (2) Among the synthesized compounds XH-1 to XH-20, compound XH-10 showed the most significant degradation effect on NCoR1. Compound XH-10 exhibited significant NCoR1 degradation at a concentration of 5 μM, and the degree of NCoR1 degradation was significantly positively correlated with the concentration of the compound. .
[0050] It can be seen that this type of compound has a significant regulatory effect on liver receptor (LXR) and has a significant degradation effect on NCoR1, which is of great value in the development of drugs for regulating glucose and ester metabolism.
Claims
1. Aza-aromatic amine compounds, characterized in that, Compounds having the formulas shown in Formulas I to IV: 。 2. The aza-aromatic amine compound according to claim 1, characterized in that, The compound is one of the following compounds: 。 3. The method for preparing the aza-aromatic amine compound according to claim 1, characterized in that, This can be achieved through the following steps: (1) Using linalidomide and different acyl chlorides as raw materials, intermediates M-1 to M-4 were obtained by acylation reaction in acetic acid / sodium acetate buffer solution; (2) Intermediates M-1 to M-4 undergo N-alkylation reactions with different azaaromatic amines to obtain target compounds XH-1 to XH-20. 。 4. The use of the azaaromatic amine compound according to claim 1 or 2 in the preparation of a pharmaceutical, characterized in that, As an active ingredient, it is prepared into a drug for treating metabolic syndrome.
5. The use of the azaaromatic amine compound according to claim 4 in the preparation of a drug, characterized in that, It was prepared into a lipid-regulating drug.
6. The use of the azaaromatic amine compound according to claim 4 in the preparation of a pharmaceutical, characterized in that, It was prepared into a blood sugar regulating drug.