Secondary amine PROTAC compound with NCoR1 targeted degradation activity as well as preparation method and application of secondary amine PROTAC compound

Abstract

The invention belongs to the technical field of medical chemistry, and discloses a secondary amine PROTAC compound with the activity of degrading a nuclear receptor co-inhibitor 1 (NCoR1) in a targeted manner as well as a preparation method and application of the secondary amine PROTAC compound. The secondary amine compound is prepared through acylation and N-alkylation reaction, and the structure of the compound is shown as a formula I to a formula IV. A Liver X Receptor Response Element-Luciferase reporter gene system (LXRE-Luc luciferase reporter gene system) determination experiment shows that when the compound is 10 [mu] M, the compound has an obvious liver receptor (LXR) regulation effect, and the compound has an obvious liver receptor regulation effect when the compound is 10 [mu] M; western blot experiment research shows that the compound has a remarkable degradation effect on NCoR1, and the compound ZD-5 with the highest activity can generate a remarkable NCoR1 degradation effect when the concentration is 5 mu M. The compound is a protein degradation targeting chimera (PROTAC), has the advantages of being high in activity and easy and convenient to prepare, and has great value in the aspect of glycometabolism and ester metabolism regulation medicine development.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry and discloses a secondary amine PROTAC compound with targeted degradation activity of NCoR1, its preparation method and uses. Background Technology

[0002] Nuclear receptor co-repressor 1 (NCoR1) is a key transcriptional regulatory protein that interacts with nuclear receptors and other transcription factors to inhibit the transcriptional expression of target genes and participate in the regulation of various physiological and pathological processes such as metabolic homeostasis, cell differentiation, and inflammatory responses. (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 syndrome. 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-targeted chimeras (PROTACs) are an emerging drug development technology that selectively degrades pathogenic proteins using natural intracellular protein degradation mechanisms, offering a novel approach to disease treatment. The estrogen receptor degrader ARV-471 is the world's first innovative drug designed under the PROTAC concept to enter clinical development, marking a key milestone in the transition of PROTAC technology from the laboratory to clinical application.

[0005] Based on the clinical needs for the treatment of metabolic syndrome, the applicant intends to develop PROTAC compounds with targeted NCoR1 degradation activity. Based on extensive literature review, the applicant has conducted research on the design, synthesis and activity evaluation of novel compounds and obtained beneficial research results. Summary of the Invention

[0006] The present invention aims to provide a novel secondary amine PROTAC compound with good activity and targeted degradation activity of NCoR1; another objective is to provide its preparation method and uses.

[0007] To achieve the objectives of this invention, the secondary amine compounds of this invention have the structural formulas shown in Formulas I to IV: 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: (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 secondary amines to obtain target compounds ZD-1 to ZD-12.

[0009] The applicant of this invention confirmed the structure of the synthesized target compounds ZD-1 to ZD-12 and performed content analysis by high performance liquid chromatography. The contents of the target compounds ZD-1 to ZD-12 were all greater than 99.0%. Based on this, the activity of the obtained compounds was evaluated.

[0010] The present invention has the following beneficial effects: This invention discloses a secondary 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 receptor (LXR) regulatory activity at 10 μM. In Western blotting experiments, these compounds showed significant degradation activity against NCoR1 protein, with the most active compound, ZD-5, 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.

[0011] 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

[0012] Figure 1 The image shows the results of LXRE-Luc luciferase reporter gene system assays using the compounds ZD-2, ZD-5, and ZD-7 of this invention. Figure 2 The image shows the results of a Western blot (WB) experiment using the compound ZD-5 of this invention. Detailed Implementation

[0013] The following detailed embodiments further illustrate the above-described content of the present invention, but should not be construed as limiting the scope of the invention to these examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. The chemical reagents used in the specific embodiments of the present invention are obtained by purchasing commercially available products.

[0014] Examples 1-5 are experiments on the synthesis of compounds, and Examples 6-7 are experiments on the activity evaluation of compounds.

[0015] 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%.

[0016] Compound M-1: 1 H NMR (400 MHz, DMSO-d6) 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-d6) 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.

[0017] Intermediates M-2 to M-4 can be obtained using a preparation method similar to that in Example 1: Example 2 Preparation of compound ZD-1 In a 100 mL three-necked flask, 10 mL of toluene, 3.36 g (0.01 mol) of intermediate M-1, 1.0 mL (0.011 mol) of aniline, 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 ZD-1 was obtained. The crude product was purified by column chromatography with dichloromethane:methanol = 30:1 as the eluent, yielding 2.8 g of product, with a yield of 71.8%.

[0018] Compound ZD-1: 1 H NMR (400 MHz, DMSO-d6) δ ppm: 10.99 (s, 1H), 9.81 (s,1H), 7.79 (m, 1H), 7.54 - 7.43 (m, 2H), 7.04 (m, 2H),6.58 - 6.51 (m, 2H),6.48 (t,J= 7.2 Hz, 1H), 5.56 (t,J= 5.7 Hz, 1H), 5.12 (m, 1H), 4.42- 4.25 (m,2H), 2.96 - 2.82 (m, 1H), 2.58 (d,J= 17.2 Hz, 1H), 2.29 (m, 1H), 2.06 - 1.94(m, 1H), 1.91 - 1.78 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ ppm: 172.93, 171.14,171.12, 167.72, 133.84, 132.99, 128.88, 128.85, 125.56, 122.86, 120.52,119.82, 115.83, 114.02,112.56, 108.49,71.57, 46.3, 31.24, 23.44, 22.68. HRMS(ESI) m / z: [C 21 H 21 N4O4] + :393.1554. Compound ZD-2-ZD-3 was obtained using a preparation method similar to that in Example 2: Example 3: Preparation of compound ZD-4 In a 100 mL three-necked flask, 20 mL of toluene, 7.0 g (0.02 mol) of intermediate M-2, 2.0 mL (0.022 mol) of aniline, and 5 mg of KI were added sequentially. The mixture was refluxed for 7 hours and then filtered while hot. The filter cake was added to 40 mL of acetone and refluxed for half an hour. After filtration, crude compound ZD-4 was obtained. The crude product was purified by column chromatography with dichloromethane:methanol = 30:1 as the eluent, yielding 5.6 g of product, with a yield of 69.1%.

[0019] Compound ZD-4: 1 H NMR (400 MHz, DMSO-d6) δ ppm: 11.05 (s, 1H), 9.85 (s,1H), 7.84 (m, 1H), 7.58 – 7.42 (m, 2H), 7.09 (m, 2H), 6.64 – 6.46 (m, 3H), 5.61 (s, 1H), 5.16 (s, 1H), 4.38 (d,J= 7.2 Hz, 2H), 3.09 (m, 2H), 2.94 (m,1H), 2.63 (d,J= 17.2 Hz, 1H), 2.34 (m, 1H), 2.12 - 1.97 (m, 1H), 1.91 (m,2H). 13 C NMR (101 MHz, DMSO-d6) δ ppm: 172.94, 171.14, 168.23, 167.84, 148.65,134.32, 133.84, 133.48, 131.52, 128.84, 128.77, 125.34, 119.35, 117.36,115.66, 113.89,51.59, 51.54, 46.57, 40.80, 31.24, 22.69. HRMS (ESI) m / z:[C 22 H 23 N4O4] + :407.1719. Compounds ZD5~ZD-6 were obtained using a preparation method similar to that in Example 3: Example 4: Preparation of compound ZD-7 In a 100 mL three-necked flask, 30 mL of toluene, 7.3 g (0.02 mol) of intermediate M-3, 2.0 mL (0.022 mol) of aniline, 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 ZD-7 was obtained. The crude product was purified by column chromatography with dichloromethane:methanol = 30:1 as the eluent, yielding 4.6 g of product, with a yield of 54.8%.

[0020] Compound ZD-7: 1 H NMR (400 MHz, DMSO-d6) δ ppm: 11.00 (s, 1H), 9.78 (s,1H), 7.80 (m, 1H), 7.56 - 7.41 (m, 2H), 7.03 (m, 2H),6.56 - 6.51 (m, 2H),6.47 (t, J = 7.2 Hz, 1H), 5.51 (t, J = 5.3 Hz, 1H), 5.11 (m, 1H), 4.34 (d, J= 5.4 Hz, 2H), 3.01 (m = 6.5 Hz, 2H), 2.96 - 2.81 (m, 1H),2.58 (d, J = 17.1Hz, 1H), 2.39 (t, J = 7.3 Hz, 2H), 2.30 (m, 1H), 2.04 - 1.94 (m, 1H), 1.69(m, 2H), 1.59 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ ppm: 172.95, 171.14,170.42, 167.89, 148.64, 133.77, 133.70, 132.73, 128.84, 128.69, 125.32,124.86, 119.13, 117.42, 115.67, 113.90,51.57, 46.51, 45.09, 32.92, 31.24,27.93, 22.67. HRMS (ESI) m / z: [C 23 H 25 N4O4] + :421.1880. Compounds ZD-8 to ZD-9 were obtained using a preparation method similar to that in Example 4: Example 5: Preparation of compound ZD-10 In a 100 mL three-necked flask, 15 mL of toluene, 3.78 g (0.01 mol) of intermediate M-4, 1.0 mL (0.011 mol) of aniline, 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 ZD-10 was obtained. The crude product was purified by column chromatography with dichloromethane:methanol = 30:1 as the eluent, yielding 2.2 g of product, with a yield of 51.2%.

[0021] Compound ZD-10: 1 H NMR (400 MHz, DMSO-d6) δ ppm: 11.03 (s, 1H), 9.81 (s,1H), 7.83 (m, 1H), 7.60 – 7.46(m, 2H), 7.06 (m, 2H), 6.68 – 6.46 (m, 3H),5.55 (t,J= 5.5 Hz, 1H), 5.15 (m, 1H), 4.38 (d,J= 5.4 Hz, 2H), 3.04 (m, 2H),2.99 – 2.87 (m, 1H),2.62 (d,J= 17.1 Hz, 1H), 2.43 (t,J= 7.3 Hz, 2H), 2.34 (m, 1H), 2.08 – 1.98 (m, 1H), 1.72 (m, 2H), 1.62 (m,2H). 13 C NMR (101 MHz, DMSO-d6) δ ppm: 172.93, 171.13, 171.05, 167.88, 148.64, 133.76, 133.35, 132.72,128.83, 128.68, 125.31, 119.09, 118.34, 115.65, 115.46, 113.88,51.55, 46.49,45.14, 34.88, 31.57, 22.68, 22.46, 21.11. HRMS (ESI) m / z: [C 24 H 27 N4O4] + :435.2037. Compounds ZD-11 to ZD-12 were obtained using a preparation method similar to that in Example 5: Example 6 Systematic evaluation of LXRE-Luc luciferase reporter gene of compound ZD-1-ZD-12 The LXR regulatory effects of compounds ZD-1 to ZD-12 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 ZD-1 to ZD-12 (purity greater than 99%) in DMSO to prepare a 10 mM stock solution and store at -20℃. When using, dilute compounds ZD-1 to ZD-12 to a final concentration of 10 μM in PS medium containing DMEM + 10% fetal bovine serum FBS + 1% penicillin-streptomycin double antibiotic solution.

[0022] (3) Preparation of control drugs: negative control (DMSO, final concentration = 0.1%), positive control (LXR agonist GW3965, working concentration 5 μM).

[0023] (4) Plasmid preparation: LXRE-Luc reporter gene plasmid and pCMV5-hLXR overexpression plasmid.

[0024] (5) Experimental procedure: ① Add 2 mL of logarithmic-phase human embryonic kidney cells HEK293T (approximately 8 × 10⁶ cells per well) to each well. 5 ① 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 ZD-1 to ZD-12 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%.

[0025] The compounds ZD-1 to ZD-12 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 ZD-1, ZD-5, and ZD-7 synthesized in this invention against the LXRE-Luc luciferase reporter gene system are shown in [link to table]. Figure 1 .

[0026] As shown in Table 1, compounds ZD-1 to ZD-12 of this invention all exhibit LXR regulation effects, which can be summarized as follows: (1) All synthesized compounds ZD-1 to ZD-12 have a clear regulatory effect on LXR. At 10 μM, the relative fluorescence activity percentage of the synthesized compounds ZD-1 to ZD-12 is 113.8-119.2% (the relative fluorescence activity percentage of the positive control drug GW3965 at 5 μM is 146.8%). (2) Among the synthesized compounds ZD-1 to ZD-12, compound ZD-5 has the most significant regulatory effect on LXR.

[0027] Example 7: Western Blot (WB) Evaluation of Compounds ZD-1 to ZD-12 The degradation activity of the synthesized compounds ZD-1 to ZD-12 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.

[0028] (2) Preparation and addition of experimental drugs: Compounds ZD-1 to ZD-12 (purity greater than 99%) were dissolved in DMSO to prepare a 10 mM stock solution and stored at -20℃. When using, compounds ZD-1 to ZD-12 were diluted to the final concentrations (1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 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 ZD-1 to ZD-12 at different concentrations were added to the human colon cancer cell HCT116 culture medium prepared above and incubated at room temperature for 24 h.

[0029] (3) Preparation of control drugs: negative control (DMSO, final concentration ≤0.1%), there is no commercially available NCoR1 protein PROTAC.

[0030] (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, 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.

[0031] (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.

[0032] Western blotting (WB) was used to determine the WB results of compounds ZD-1 to ZD-12 synthesized in this invention. The results are shown in Table 2. The WB evaluation results of compound ZD-10 synthesized in this invention are also shown in [the table]. Figure 2 .

[0033] The results of Western blot analysis on the compounds ZD-1 to ZD-12 synthesized in this invention show that: (1) The compounds ZD-1 to ZD-12 of the present invention all exhibit NCoR1 degradation activity, and under the experimental conditions, the compounds ZD-1 to ZD-12 of the present invention do not degrade HSP90 protein, showing targeting of NCoR1 protein.

[0034] (2) Among the synthesized compounds ZD-1 to ZD-12, compound ZD-5 showed the most significant degradation effect on NCoR1. Compound ZD-5 exhibited significant NCoR1 degradation at a concentration of 5 μM, and the degree of NCoR1 degradation was significantly positively correlated with the compound concentration.

[0035] It can be seen that this type of compound has a significant regulatory effect on liver receptor (LXR) and a significant degradation effect on NCoR1, which is of great value in the development of drugs for regulating glucose and ester metabolism.

Claims

1. A secondary amine compound, characterized in that, Compounds having the formulas shown in Formulas I to IV: In equations I-IV, .

2. The secondary amine compound according to claim 1, characterized in that, The compound is one of the following compounds: 。 3. The method for preparing the secondary 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 secondary amines to obtain target compounds ZD-1 to ZD-12; 。 4. The use of the secondary amine compound according to claim 1 or 2 in the preparation of a drug, characterized in that, As an active ingredient, it is prepared into a drug for treating metabolic syndrome.

5. The use of the secondary 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 secondary amine compound according to claim 4 in the preparation of a drug, characterized in that, It was prepared into a blood sugar regulating drug.

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