Stress granule targeting degraders, methods of making and using the same

Abstract

This invention relates to the field of pharmaceutical technology, and discloses a stress particle targeted degrader, its preparation method, and its applications. The compounds provided by this invention include ternary complexes composed of stress particle protein, a linker group, and an E3 ubiquitin ligand, or ternary complexes composed of stress particle protein, a linker group, and a ribonuclease L ligand. Compounds with different ligands exhibit good targeting and degradation effects on stress particles and can be used as stress particle targeted degraders in the preparation of antitumor drugs and anti-neurodegenerative disease drugs, showing promising application prospects in treating stress particle-induced tumor drug resistance and neurodegenerative diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a stress particle targeted degrader, its preparation method, and its application. Background Technology

[0002] Stress granules (SGs) are dense, non-membrane-bound granular structures in the cytoplasm of eukaryotic cells formed in response to environmental stresses (such as oxidative stress, hypoxic stress, and heat shock). They are primarily composed of untranslated mRNA, translation initiation factors, small ribosomal subunits, and various RNA-binding proteins (such as TIA1 and G3BP). These RNA-binding proteins promote SG formation through self-aggregation and interaction with mRNA. Through their dynamic formation and deaggregation, stress granules regulate overall cellular translation levels and related signaling pathways, rapidly responding to changes in the intracellular environment.

[0003] Under normal physiological conditions, stress granules protect cells from apoptosis by minimizing stress-induced damage through inhibiting overall cellular translation, optimizing resource allocation, and suppressing apoptosis-related signaling pathways. However, pathological mutations in proteins can lead to abnormal formation of stress granules and inhibit their disintegration, resulting in the accumulation of toxic pathological stress granules. Studies have shown that the formation of abnormal toxic pathological stress granules is closely related to chemotherapy drug resistance and the development of neurodegenerative diseases.

[0004] Many antitumor drugs (such as 5-fluorouracil, sorafenib, and bortezomib) can induce the formation of stress granules through multiple mechanisms, including oxidative stress and endoplasmic reticulum stress. In this process, stress granules act as an adaptive protective mechanism of cells, helping to protect tumor cells from apoptosis and thus leading to chemotherapy drug resistance. In the pathological process of neurodegenerative diseases, mutated RNA-binding proteins, such as FUS, hnRNPA1, and TDP-43, are continuously recruited to stress granules, leading to abnormalities in these granules. This further promotes the accumulation of these proteins in the cytoplasm, where they remain and form fibrotic aggregates, causing persistent lesions such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).

[0005] Therefore, stress particles are considered an important potential drug target in disease treatment. Developing drugs targeting stress particles is of great significance and has broad application prospects in the treatment of neurodegenerative diseases and in overcoming tumor drug resistance. Summary of the Invention

[0006] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, one objective of the present invention is to provide a compound; a second objective is to provide a method for preparing such a compound; a third objective is to provide a stress-targeted degrader for particles; and a fourth objective is to provide the application of such a stress-targeted degrader for particles.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A first aspect of the present invention provides a compound having a structural formula as shown in formula (A), formula (B) or formula (C):

[0009]

[0010] Among them, in equations (A) to (C),

[0011] The A - Each of the following is independently selected from methylated anions, iodide ions, bromide ions, p-toluenesulfonate, trifluoroacetate, perchlorate, tetrafluoroborate, methyl sulfate, or trifluoromethanesulfonate;

[0012] R1 is a linker group; R2 is a ligand structural group, and the ligands are independently selected from E3 ubiquitin ligase ligands or ribonuclease L ligands.

[0013] In some embodiments of the present invention, in formulas (A)-(C), the linking group R1 is independently selected from any one of the following groups:

[0014]

[0015] Where n is independently selected from 1, 3 or 5.

[0016] In some embodiments of the present invention, in formulas (A)-(C), the ligand structural group R2 is independently selected from CRBN protein ligand structural groups. VHL protein ligand structural groups or ribonuclease L ligand structural group In some embodiments of the present invention, the compounds are shown in Formulas 1-6:

[0017]

[0018] A second aspect of the present invention provides a method for preparing the compound described in the first aspect of the present invention, wherein when the compound is as shown in formula (A), the method for preparing it includes the following steps:

[0019] The compound of formula (a) is reacted with a ligand to give a compound of formula (A), the structure of which is as follows:

[0020]

[0021] When the compound is as shown in formula (B), its preparation method includes either of the following two methods:

[0022] Method 1: The compound of formula (b) is reacted with a ligand to obtain a compound as shown in formula (B), the structure of which is as follows:

[0023]

[0024] Method 2: The compound of formula (c), the ligand, and 2-(7-azobenzotriazole)-tetramethylurea hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) were reacted to give the compound shown in formula (B), and the structure of the compound of formula (c) is as follows:

[0025]

[0026] When the compound is as shown in formula (C), its preparation method includes the following steps:

[0027] The compound of formula (d) was reacted with a ligand to give the compound of formula (C), the structure of which is as follows:

[0028]

[0029] In some embodiments of the present invention, in the preparation of the compound shown in formula (A), the compound of formula (a) is obtained by a preparation method comprising the following steps:

[0030] 1) The reaction with bromopropyne (1-2 eq) and potassium carbonate (3-4 eq) yields

[0031] 2) Reacting with iodomethane (5-6 eq) upon heating at 65-75 °C yields

[0032] 3) and (1-1.5 eq) were reacted under pyridine catalysis to give the compound of formula (a).

[0033] In some embodiments of the present invention, the molar ratio of the compound of formula (a) to the ligand is 1:(1-2).

[0034] In some embodiments of the present invention, in the preparation of compounds as shown in formula (B), said compound (b) or (c) is obtained by a preparation method comprising the following steps:

[0035] 1) Reaction with bromopropyne or bromoalkyl acid (1.5-2 eq) yields

[0036] 2) and (1-1.2eq) The reaction was carried out under pyridine catalysis to give the compound of formula (b) or (c).

[0037] In some embodiments of the present invention, the molar ratio of the compound of formula (b) to the ligand is 1:(1-2).

[0038] In some embodiments of the present invention, the molar ratio of the compound of formula (c), the ligand, 2-(7-azobenzotriazole)-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine is 1:(1-2):(1-2):(3-4).

[0039] In some embodiments of the present invention, in the preparation of the compound shown in formula (C), the compound of formula (d) is obtained by a preparation method comprising the following steps:

[0040] 1) N-Boc piperazine (1.5-2 eq), cesium carbonate (3-4 eq), tris(dibenzylacetone)dipalladium (0.01-0.1 eq) and 2-dicyclohexylphosphino-2'-(N,N-dimethylamine)-biphenyl (0.01-0.1 eq) were reacted by heating at 90-100 °C to obtain

[0041] 2) It reacts with iodomethane (5-6 eq) to give

[0042] 3) and (1-1.5 eq) The reaction was carried out under pyridine catalysis to give the compound of formula (d).

[0043] In some embodiments of the present invention, the molar ratio of the compound of formula (d) to the ligand is 1:(1-2).

[0044] In some embodiments of the present invention, in the preparation of compounds as shown in formulas (A)-(C), the ligands are each independently selected from any one of the following:

[0045] (n = 1, 3 or 5) (n = 1 or 2)

[0046] (n = 1, 3 or 5)

[0047] (n = 1, 3 or 5) (n = 1, 3 or 5) (n = 1 or 2) (n = 1 or 2)

[0048] (n = 1 or 2)

[0049] (n = 1 or 3)

[0050] (n = 1 or 2).

[0051] In some embodiments of the present invention, the ligand (n = 1, 3, or 5) or (n = 1 or 2) is prepared by a method including the following steps:

[0052] 1) Reacting with dibromoalkane (1-1.5 eq) or dibromo-substituted polyethylene glycol chains (1-1.2 eq) and potassium carbonate (3-4 eq) yields

[0053] 2) The ligand was reacted with sodium azide (3-5 eq) to give the ligand. (n = 1, 3, or 5) or (n = 1 or 2).

[0054] In some embodiments of the present invention, the ligand (n = 1 or 3) or ligands (n = 1, 2, or 3) are prepared by a method including the following steps:

[0055] 1) and The reaction of (1-1.5 eq), palladium acetate (0.01-0.1 eq), and potassium acetate (3-4 eq) yields... Remove Boc;

[0056] 2) Combine the product obtained after removing Boc in 1) with... The mixture was reacted with (1-1.5 eq), 2-(7-azobenzotriazole)-tetramethylurea hexafluorophosphate (1-1.5 eq), and N,N-diisopropylethylamine (3-4 eq) to give Remove Boc;

[0057] 3) Combine the product obtained after removing Boc in step 2) with... The reaction of (1-2 eq), 2-(7-azobenzotriazole)-tetramethylurea hexafluorophosphate (1-1.5 eq), and N,N-diisopropylethylamine (3-4 eq) yields Remove Boc;

[0058] 4) Combine the product obtained after removing Boc in step 3) with... (1-2eq) or (n = 1, 2 or 3) (1-2 eq) are mixed to obtain the ligand. (n = 1 or 3) or the ligands (n = 1 or 2).

[0059] In some embodiments of the present invention, the ligand (n = 1, 3 or 5) (n = 1 or 2) is prepared by a method including the following steps:

[0060] 1) Reacting with dibromoalkane (1-1.2 eq) or dibromo-substituted polyethylene glycol chains (1-1.2 eq) and potassium carbonate (3-4 eq) yields

[0061] 2) Reacting with sodium azide (3-5 eq) yields

[0062] 3) and The ligand was obtained by a (1-1.2 eq) reaction. (n = 1, 3, or 5) or (n = 1 or 2).

[0063] In some embodiments of the present invention, the ligand (n = 1, 3 or 5) (n = 1 or 2) is prepared by a method including the following steps:

[0064] Will The mixture was reacted with tert-butyl bromide (1-1.2 eq) or tert-butyl bromide (1-1.2 eq) and potassium carbonate (3-4 eq) to prepare the following product: After de-Boc, the ligand is obtained. (n = 1, 3, or 5) or (n = 1 or 2).

[0065] In some embodiments of the present invention, in the preparation of the compound as shown in formula (A), the ligand is selected from any one of the following:

[0066] (n = 1, 3 or 5) (n = 1 or 2) (n = 1, 3 or 5)

[0067] (n = 1 or 2)

[0068] (n = 1 or 3)

[0069] (n = 1 or 2).

[0070] In some embodiments of the present invention, in the preparation of the compound as shown in formula (B), the ligand is selected from any of the following:

[0071] (n = 1, 3 or 5) (n = 1, 3 or 5) (n = 1 or 2) (n = 1 or 2).

[0072] In some embodiments of the present invention, in the preparation of the compound as shown in formula (C), the ligand is selected from any one of the following:

[0073] (n = 1, 3 or 5) (n = 1, 3 or 5)

[0074] (n = 1 or 2)

[0075] (n = 1 or 2).

[0076] A third aspect of the invention provides a stress particle targeted degrader, comprising the compound described in the first aspect of the invention or a pharmaceutically acceptable salt thereof.

[0077] The fourth aspect of the present invention provides the use of the stress particle targeting degrader described in the third aspect of the present invention in the preparation of antitumor drugs and / or drugs for treating neurodegenerative diseases.

[0078] In some embodiments of the present invention, the tumor includes pancreatic cancer, colorectal cancer, osteosarcoma, glioma, cervical cancer, breast cancer, prostate cancer, liver cancer, non-small cell lung cancer, or melanoma.

[0079] In some embodiments of the present invention, the neurodegenerative disease includes Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington's disease (HD), or Parkinson's disease (PD).

[0080] Compared with the prior art, the beneficial effects of the present invention are:

[0081] The compounds provided by this invention consist of three parts: a stress granule protein, a linker group, and an E3 ubiquitin ligand or a ribonuclease L ligand. In the ternary complex composed of the stress granule protein, the linker group tagged the stress granule protein with ubiquitination via the linker group, which then recognized and degraded the ubiquitinated stress granule protein via the proteasome 26S. In the ternary complex composed of the stress granule protein, the linker group, and the ribonuclease L ligand, the linker group recruited the ribonuclease L ligand to the stress granule, activating the ribonuclease L ligand and inducing selective cleavage of the stress granule RNA, thereby exerting a degradation effect on the stress granule. Compounds with different ligands exhibit good targeting and degradation effects on stress granules, and can be used as stress granule-targeting degradative agents in the preparation of anti-tumor drugs and anti-neurodegenerative disease drugs, showing promising application prospects in the treatment of stress granule-induced tumor drug resistance and neurodegenerative diseases. Attached Figure Description

[0082] Figure 1 This is an experimental diagram showing the targeting behavior of the compound of formula (II) prepared in Example 2 to the stress particles;

[0083] Figure 2 The figure shows the experimental results of the targeting of the stress particles to the compound of formula (II) prepared in Example 2;

[0084] Figure 3 This is an experimental diagram showing the targeting behavior of the compound of formula (Ⅲ) prepared in Example 3 to the stress particles;

[0085] Figure 4 The figure shows the experimental results of the targeting of the stress particles to the compound of formula (Ⅲ) prepared in Example 3;

[0086] Figure 5 The following are experimental diagrams showing the degradation effects of compounds of formula (II) and formula (III) prepared in Examples 2 and 3 on the stress particles.

[0087] Figure 6 The results of degradation experiments on stress particles by compounds of formula (II) and formula (III) prepared in Examples 2 and 3 are shown.

[0088] Figure 7 The diagram shows the experimental mechanism of the degradation of stress particles by the compound of formula (II) prepared in Example 2.

[0089] Figure 8 The diagram shows the experimental mechanism of the degradation of stress particles by the compound of formula (Ⅲ) prepared in Example 3. Detailed Implementation

[0090] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments can be obtained from conventional commercial sources or by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0091] Example 1

[0092] In this embodiment, a compound is prepared with the structural formula shown in formula (Ⅰ):

[0093] Where A is Br.

[0094] The preparation method is as follows:

[0095] (1) Synthesis of CRBN protein ligands:

[0096] 2-(2,6-dioxadiazine-3-yl)-4-hydroxyisoindoline-1,3-dione (300 mg, 1.09 mmol) and 1,3-dibromopropane (231 mg, 1.15 mmol) were dissolved in 3.0 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (453 mg, 3.28 mmol) was added under stirring. The reaction was carried out at room temperature for 6 h, quenched with water, extracted three times with ethyl acetate, washed three times with saturated brine, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, mixed with silica gel, and eluted with petroleum ether / ethyl acetate (5:1, v / v). The purified solid was obtained by silica gel column chromatography with a yield of 57%.

[0097] 200 mg (506 mmol) of 4-(3-bromopropoxy)-2-(2,6-dipyridinone-3-yl)isoindoline-1,3-dione was added to sodium azide (97 mg, 1.50 mmol), and the mixture was stirred at room temperature for 24 h to give a pale yellow clear solution. The solution was quenched with water, extracted with ethyl acetate, washed three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to give a white solid, designated SL3-N3, with a yield of 91%.

[0098] (2) Synthesis of compound (b):

[0099] 2-Methylbenzothiazole (3.90 g, 26.1 mmol) and bromopropyne (6.30 g, 52.9 mmol) were dissolved in acetonitrile and stirred under reflux at 80 °C for 24 h. After cooling to room temperature, a large amount of pale yellow precipitate was precipitated. The precipitate was filtered under reduced pressure, washed with cold anhydrous ethanol, and dried in a vacuum drying oven to give a pale yellow solid with a yield of 43%.

[0100] 2-Methyl-3-(prop-2-ynyl-1-yl)benzothiazole iodide (300 mg, 1.1 mmol) and julonidinaldehyde (270 mg, 1.3 mmol) were dissolved in 3.0 mL of ethanol, refluxed and stirred at 75 °C for 6 h, cooled to room temperature, and a large amount of purple solid precipitated. The solid was filtered under reduced pressure, washed with cold anhydrous ethanol, and dried under vacuum to obtain the purple solid of compound (b), denoted as TASG-8-C, with a yield of 71%.

[0101] (3) Synthesis of compound (Ⅰ):

[0102] TASG-8-C (25 mg, 67.29 μmol) and SL3-N3 (29 mg, 81 μmol) were dissolved in 4.0 mL of dichloromethane / methanol (1:10, v / v). Then, copper sulfate (10 mg, 62.66 μmol) and sodium ascorbate (22.20 mg, 112.07 μmol) were dissolved in 0.3 mL of water, respectively, and added to the above reaction solution under stirring. The mixture was stirred at room temperature for 24 h, the solvent was removed under reduced pressure, and the mixture was reconstituted in dichloromethane / methanol (10:1, v / v). The mixture was purified by preparative chromatography with dichloromethane / methanol (50:1, v / v) as the developing solvent to obtain a purple solid of compound (I) in a yield of 36%.

[0103] The compound of formula (Ⅰ) in Example 1 was analyzed using nuclear magnetic resonance. 1 HNMR and 13 The results of the C NMR analysis are as follows:

[0104] 1H NMR(500MHz,DMSO-d6)δ11.12(s,1H),8.47(s,1H),8.21(d,J=8.0H z,1H),8.10(d,J=8.5Hz,1H),7.89(d,J=14.8Hz,1H),7.76(t,J=7.9Hz,1H),7.70–7.64(m,2H),7.5 7(t,J=7.6Hz,1H),7.48(s,2H),7.44(d,J=7.2Hz,1H),7.41(d,J=8.6Hz,1H),6.09(s,2H),5.08(dd ,J=12.8,5.5Hz,1H),4.58(t,J=7.0Hz,2H),4.15(t,J=6.1Hz,2H),3.35(s,4H),2.91–2.84(m,1H), 2.69(t,J=6.2Hz,4H),2.61–2.53(m,2H),2.32–2.26(m,2H),2.04–1.99(m,1H),1.90–1.84(m,4H).;

[0105] 13 C NMR(126MHz,DMSO-d6)δ172.82,170.48,169.97,166.80,165.39,155.48, 150.84,148.15,140.98,140.14,137.12,133.16,130.83,128.65,126.92 ,126.36,124.60,123.78,121.29,120.73,119.80,116.42,115.59,103.9 3,65.67,49.74,48.78,46.47,42.50,30.96,29.19,26.92,22.05,20.54..

[0106] Example 2

[0107] In this embodiment, a compound is prepared with the structural formula shown in formula (II):

[0108] Where A is CF3COO.

[0109] The preparation method is as follows:

[0110] (1) Synthesis of CRBN protein ligands:

[0111] 2-(2,6-dioxadiazine-3-yl)-4-hydroxyisoindoline-1,3-dione (300 mg, 1.09 mmol) and tert-butyl 6-bromohexanoate (224 mg, 1.15 mmol) were dissolved in 3.0 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (453 mg, 3.28 mmol) was added under stirring. The reaction was carried out at room temperature for 6 h, quenched with water, extracted three times with ethyl acetate, washed three times with saturated brine, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, mixed with silica gel, and eluted with petroleum ether / ethyl acetate (5:1, v / v). The purified solid was obtained by silica gel column chromatography with a yield of 33%.

[0112] 100 mg (257 mmol) of tert-butyl 2-((2-(2,6-dipyridinone-3-yl)-1,3-dioxindololin-4-yl)oxy)acetate was dissolved in dichloromethane / trifluoroacetic acid (2:1, v / v), stirred at room temperature for 2 h, and concentrated by rotary evaporation after TLC to obtain a white solid, denoted as SL2-COOH.

[0113] (2) Synthesis of compound (d):

[0114] 6-Bromo-2-methylbenzothiazole (2 g, 8.77 mmol), N-methylpiperazine (2.45 g, 13.15 mmol), cesium carbonate (8.57 g, 26.30 mmol), tris(dibenzylacetone)palladium (402 mg, 0.44 mmol), and 2-dicyclohexylphosphino-2'-(N,N-dimethylamine)-biphenyl (173 mg, 0.44 mmol) were dissolved in 20 mL of anhydrous 1,4-dioxane. The mixture was purged three times with nitrogen and heated at 95 °C for 24 h. The reaction solution was filtered through diatomaceous earth, extracted with dichloromethane and water, and the organic phase was washed with saturated brine, concentrated by rotary evaporation, mixed with silica gel, eluted with dichloromethane / methanol (200:1, v / v), and purified by silica gel column chromatography to give a pale yellow solid in 65% yield.

[0115] 6-(4-tert-Butoxycarbonylpiperazin-1-yl)-2-methylbenzothiazole (1 g, 3 mmol) was added to 12 mL of acetonitrile, and iodomethane (1.28 g, 9 mmol) was added dropwise. The mixture was stirred and refluxed in a pressure-resistant tube at 70 °C for 12 h. After cooling to room temperature, a large amount of white solid precipitated. The solid was filtered under reduced pressure, washed with isopropanol, and dried in a vacuum drying oven to obtain a white solid with a yield of 68%.

[0116] 6-(4-tert-Butoxycarbonylpiperazin-1-yl)-2,3-dimethylbenzothiazole iodide (800 mg, 1.68 mmol) and juulonidinaldehyde (321 mg, 1.60 mmol) were dissolved in 5.0 mL of anhydrous ethanol and stirred under reflux at 80 °C for 6 h in a pressure-resistant tube. The solution turned purple. After cooling to room temperature, a large amount of purple solid precipitated. The solid was filtered under reduced pressure, washed with cold anhydrous ethanol, and dried in a vacuum drying oven to obtain the purple solid with a yield of 48%.

[0117] (E)-6-(4-tert-butoxycarbonylpiperazin-1-yl)-3-methyl-2-(2-(2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-yl)vinyl)benzothiazole iodide (300 mg, 564 μmol) was dissolved in dichloromethane / trifluoroacetic acid (2:1, v / v), stirred at room temperature for 2 h, and concentrated by rotary evaporation after TLC to obtain the purple solid of compound (d), designated TASG-8-D.

[0118] (3) Synthesis of compound (II):

[0119] TASG-8-D (25 mg, 57.92 μmol), SL2-COOH (27 mg, 81.26 μmol), 2-(7-azobenzotriazole)-tetramethylurea hexafluorophosphate (33 mg, 86.88 μmol), and N,N-diisopropylethylamine (22.5 mg, 173.76 μmol) were dissolved in 4.0 mL of N,N-dimethylformamide and stirred at room temperature for 12 h. The mixture was extracted with dichloromethane and water, and the organic phase was washed three times with saturated brine. The mixture was concentrated by rotary evaporation and purified by preparative chromatography using dichloromethane / methanol (30:1, v / v) containing 0.1% triethylamine as the developing solvent. The resulting compound (II) was a purple solid with a yield of 26%.

[0120] The compound of formula (II) in Example 2 was analyzed using nuclear magnetic resonance. 1 H NMR and 13 The results of the C NMR analysis are as follows:

[0121] 1H NMR(500MHz,DMSO-d6)δ11.12(s,1H),7.88(d,J=9.3Hz,1H),7.77–7.72(m,2H),7.65–7.6 0(m,1H),7.44(s,2H),7.40(d,J=15.2Hz,1H),7.14(d,J=8.6Hz,1H),7.09(d,J=7.3Hz,2H ),5.11–5.05(m,1H),4.28(d,J=4.1Hz,2H),4.12(s,3H),3.70(s,4H),3.35(d,J=4.9Hz,6 H),2.72(t,J=5.9Hz,4H),2.64–2.52(m,4H),1.92–1.86(m,4H),1.21(d,J=13.9Hz,2H).;

[0122] 13 C NMR (126MHz, DMSO-d6) δ174.86,173.33,170.57,169.30,167.82,167.48,167.13,150.37,147.56,145.94,136.69,135.09,132 .48,130.28,121.46,117.93,116.54,111.38,110.05,108.07,50.00,49.06,46.05,35.67,31.46,27.45,22.62,21.14,9.01.

[0123] Example 3

[0124] In this embodiment, a compound is prepared with the structural formula shown in formula (Ⅲ):

[0125] Where A is CF3COO.

[0126] The preparation method is as follows:

[0127] (1) Synthesis of L-ribonuclease ligand:

[0128] 3,4-Dihydroxybenzaldehyde (300 mg, 2.17 mmol), tert-butyl bromoacetate (444 mg, 2.28 mmol), and potassium carbonate (900 mg, 6.52 mmol) were dissolved in 3.0 mL of N,N-dimethylformamide and stirred at room temperature for 12 h. The mixture was then extracted with ethyl acetate and water. The organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, mixed with silica gel, and eluted with petroleum ether / ethyl acetate (10:1, v / v). The purified solid was obtained by silica gel column chromatography with a yield of 52%.

[0129] 200 mg (634.50 μmol) of tert-butyl-2-(4-formaldehyde-2-hydroxyphenoxy)acetate and 171 mg (650.86 μmol) of 4-oxo-2-(phenylamino)-4,5-dihydrothiophene-3-carboxylic acid ethyl ester were dissolved in 4.0 mL of ethanol. One drop of piperidine was added as a catalyst. The mixture was stirred and refluxed at 80 °C for 12 h in a pressure-resistant tube. The solution was then cooled to room temperature, and a large amount of pale yellow solid precipitated. The solid was filtered under reduced pressure, washed with cold anhydrous ethanol, and dried in a vacuum drying oven to obtain a pale yellow solid with a yield of 63%.

[0130] Ethyl(Z)-5-(4-(2-(tert-butoxy)-2-oxoethoxy)-3-hydroxybenzylmethyl)-4-oxo-2-(phenylamino)-4,5-dihydrothiophene-3-carboxylate (100 mg, 200 μmol) was dissolved in dichloromethane / trifluoroacetic acid (2:1, v / v), stirred at room temperature for 2 h, and the reaction was confirmed to be complete by TLC. The solution was then concentrated by rotary evaporation to give a white solid, which was designated as R2-COOH.

[0131] (2) Synthesis of compound (III):

[0132] TASG-8-D (25 mg, 57.92 μmol, compound (d) prepared in Example 2), R2-COOH (36 mg, 81.26 μmol), 2-(7-azobenzotriazole)-tetramethylurea hexafluorophosphate (33 mg, 86.88 μmol), and N,N-diisopropylethylamine (22.5 mg, 173.76 μmol) were dissolved in 4.0 mL of N,N-dimethylformamide and stirred at room temperature for 12 h. The mixture was extracted with dichloromethane and water, and the organic phase was washed three times with saturated brine. The mixture was concentrated by rotary evaporation and purified by preparative chromatography using dichloromethane / methanol (30:1, v / v) containing 0.1% triethylamine as the developing solvent. The resulting compound (III) was a purple solid with a yield of 16%.

[0133] The compound of formula (III) in Example 3 was analyzed using nuclear magnetic resonance. 1 H NMR and 13 The results of the C NMR analysis are as follows:

[0134] 1H NMR (500MHz, DMSO-d6) δ11.25(s,1H),9.67(d,J=25.5Hz,1H),7.86(d,J=9.3Hz,1H),7.72(d,J=14. 3Hz,1H),7.69(s,1H),7.53(dd,J=13.4,6.0Hz,4H),7.48(s,1H),7.46–7.41(m,4H),7.37(d,J=15. 3Hz,1H),6.97(d,J=6.9Hz,3H),4.94(s,2H),4.27(q,J=7.0Hz,2H),4.11(s,3H),3.63(s,4H),3.33 (d,J=5.7Hz,6H),2.71(t,J=5.9Hz,4H),1.89(d,J=4.9Hz,4H),1.29(t,J=7.1Hz,3H),1.22(s,2H).

[0135] 13 C NMR (126MHz, DMSO) δ167.42,166.66,165.30,150.33,148.67,147.75,147.55,135.04,130.19,128.75,127.57,126.05,125.5 8,121.45,121.07,117.84,116.57(d,J=16.3Hz),115.34,107.98,105.25,97.34,59.99,49.99,35.64,27.45,21.13,14.88.

[0136] Example 4

[0137] In this embodiment, a compound is prepared with the structural formula shown in formula (Ⅳ):

[0138] Where A is I.

[0139] The preparation method is as follows:

[0140] (1) Synthesis of L-ribonuclease ligand:

[0141] 3,4-Dihydroxybenzaldehyde (300 mg, 2.17 mmol), 1,7-dibromoheptane (588 mg, 2.28 mmol), and potassium carbonate (900 mg, 6.52 mmol) were dissolved in 3.0 mL of N,N-dimethylformamide and stirred at room temperature for 12 h. The mixture was then extracted with ethyl acetate and water. The organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, mixed with silica gel, and eluted with petroleum ether / ethyl acetate (10:1, v / v). The purified solid was obtained by silica gel column chromatography with a yield of 37%.

[0142] 200 mg (634.50 μmol) of 4-(7-bromoheptyloxy)-3-hydroxybenzaldehyde was dissolved in 5.0 mL of N,N-dimethylformamide, and 123 mg (1.90 mmol) of sodium azide was added. The mixture was stirred at room temperature and reacted for 24 h to obtain a pale yellow clear solution. The solution was quenched with water, extracted with ethyl acetate, washed three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to give a white solid with a yield of 83%.

[0143] 4-(7-azidoheptyloxy)-3-hydroxybenzaldehyde (190 mg, 685.12 μmol) and ethyl 4-oxo-2-(phenylamino)-4,5-dihydrothiophene-3-carboxylate (171 mg, 650.86 μmol) were dissolved in 4.0 mL of ethanol. One drop of piperidine was added as a catalyst. The mixture was stirred and refluxed at 80 °C for 12 h in a pressure-resistant tube. The solution was then cooled to room temperature, and a large amount of pale yellow solid precipitated. The solid was filtered under reduced pressure, washed with cold anhydrous ethanol, and dried in a vacuum drying oven to obtain a pale yellow solid, designated as RIBO7-N3, with a yield of 78%.

[0144] (2) Synthesis of compound (a):

[0145] 2-Methyl-6-hydroxybenzothiazole (1 g, 6.05 mmol), 3-bromopropyne (792 mg, 6.66 mmol), and potassium carbonate (1.25 g, 9.08 mmol) were added to 8 mL of N,N-dimethylformamide. The mixture was stirred at room temperature for 8 h, quenched with water, extracted with dichloromethane, and the organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, mixed with silica gel, and eluted with petroleum ether / ethyl acetate (5:1, v / v). The purified solid was obtained by silica gel column chromatography with a yield of 72%.

[0146] 2-Methyl-6-(2-propynoxy)benzothiazole (700 mg, 3.44 mmol) was added to 5 mL of acetonitrile, and iodomethane (977 mg, 6.89 mmol) was added dropwise. The mixture was stirred and refluxed in a pressure-resistant tube at 70 °C for 12 h. After cooling to room temperature, a large amount of white solid precipitated. The solid was filtered under reduced pressure, washed with isopropanol, and dried in a vacuum drying oven to obtain a white solid with a yield of 93%.

[0147] 2,3-Dimethyl-6-(2-propynoxy)benzothiazole iodide (350 mg, 1.60 mmol) and julonidinaldehyde (306 mg, 1.52 mmol) were dissolved in 3.0 mL of anhydrous ethanol and stirred under reflux at 80 °C for 6 h in a pressure-resistant tube. The solution turned purple. After cooling to room temperature, a large amount of purple solid precipitated. The solid was filtered under reduced pressure, washed with cold anhydrous ethanol, and dried under vacuum to obtain the purple solid of compound (a), denoted as TASG-8-A, with a yield of 59%.

[0148] (3) Synthesis of compound (Ⅳ):

[0149] TASG-8-A (30 mg, 74.71 μmol) and RIBO7-N3 (42.79 mg, 82.18 μmol) were dissolved in 4.0 mL of dichloromethane / methanol (1:10, v / v). Then, copper sulfate (10 mg, 62.66 μmol) and sodium ascorbate (22.20 mg, 112.07 μmol) were dissolved in 0.3 mL of water, respectively, and added to the above reaction solution under stirring. The mixture was stirred at room temperature for 24 h, the solvent was removed under reduced pressure, and the mixture was reconstituted in dichloromethane / methanol (10:1, v / v). The mixture was purified by preparative chromatography using dichloromethane / methanol (30:1, v / v) as the developing solvent to obtain a purple solid of compound (IV) in a yield of 17%.

[0150] The compound of formula (Ⅳ) in Example 4 was analyzed using nuclear magnetic resonance. 1 HNMR and 13 The results of the C NMR analysis are as follows:

[0151] 1 H NMR (400MHz, DMSO-d6) δ11.29–11.17(m,1H),9.38–9.30(m,1H),8.30(s,1H),7.94(d,J=2.4Hz,1H),7.91(s, 1H),7.73(s,1H),7.53(s,1H),7.52(s,1H),7.50(s,2H),7.48(s,1H),7.43(s,4H),7.39(s,1H),7.37–7.34(m ,1H),6.95(d,J=8.0Hz,2H),5.25(s,2H),4.37(s,2H),4.26(d,J=7.1Hz,2H),4.12(s,3H),3.92(s,2H),2.70( t,J=5.9Hz,4H),1.92–1.86(m,4H),1.73(d,J=72.4Hz,6H),1.33(s,2H),1.29(t,J=7.1Hz,6H),1.23(s,3H).;

[0152] 13C NMR(126MHz,DMSO-d6)δ181.37,169.05,165.34,157.59,149.42,147.83,147.41,14 2.32,136.68,130.46,130.10,128.52,126.46,125.92,125.25,124.90,123.58,121. 47,121.02,117.93,116.92,116.27,113.78,108.40,104.97,97.30,68.55,62.38,59.97,50.03,49.86,35.75,29.97,28.84,28.42,27.42,26.12,25.64,21.08,14.87.

[0153] Performance testing

[0154] 1. To investigate the targeting effects of compounds of formula (II) and formula (III) prepared in Examples 2 and 3 on stress particles:

[0155] HeLa cells in logarithmic growth phase were seeded at 3500 cells per well in 96-well MatTek laser confocal microscopy plates and cultured at 37°C for 48 h in a 5% CO2 incubator. Sodium arsenite was prepared to a working concentration of 500 μmol / L using medium containing 10% fetal bovine serum. The medium in the 96-well plates was discarded, and 100 μL of medium containing 500 μmol / L sodium arsenite was added to each well for 1 h of stress. The medium was discarded, and 50 μL of 4% paraformaldehyde was added to each well for 20 min at room temperature to fix the cells. The paraformaldehyde was discarded, and the cells were washed three times with 1×PBS buffer. 50 μL of 0.5% PBST was added to each well, and the cells were incubated at 37°C for 30 min for permeabilization. The PBST was discarded, and the cells were washed three times with 1×PBS buffer. 50 μL of 5% BSA / PBS was added to each well, and the cells were incubated at 37°C for 30 min for blocking. Discard the BSA and add 25 μL of a solution containing G3BP antibody (prepared with 1% BSA / PBS, diluted 1:1000) to each well. Incubate overnight at 4°C. Discard the primary antibody, wash three times with 1×PBS, and add 30 μL of a solution containing fluorescent secondary antibody (prepared with 1% BSA / PBS) to each well. Incubate at 37°C for 1 h. Take the compounds of formula (II) and formula (III) prepared in Examples 2 and 3, prepare them to a working concentration of 1 μmol / L with 1×PBS, discard the secondary antibody, add 100 μL of the above compounds to each well, and incubate at room temperature in the dark for 2 h. Then, image the cells using an FV3000 laser confocal microscope (Olympus).

[0156] 2. To investigate the degradation effect of compounds of formula (II) and formula (III) prepared in Examples 2 and 3 on stress particles:

[0157] Logarithmic growth phase HeLa cells were seeded at 3500 cells per well in a 96-well MatTek laser confocal microscopy plate and cultured at 37°C for 48 h in a 5% CO2 incubator. Sodium arsenite was prepared to a working concentration of 500 μmol / L using medium containing 10% fetal bovine serum. The medium in the 96-well plate was discarded, and 100 μL of medium containing 500 μmol / L sodium arsenite was added to each well for 1 h of stress. Sodium arsenite was prepared to a working concentration of 50 μmol / L using medium containing 10% fetal bovine serum. Compounds of formula (II) and formula (III) prepared in Examples 2 and 3, respectively, were prepared to working concentrations of 5 μmol / L and 20 μmol / L using the above 50 μmol / L sodium arsenite medium. Discard the culture medium. Add 100 μL of the above-mentioned culture medium containing sodium arsenite and the compound to each well. The control group is 100 μL of 50 μmol / L sodium arsenite culture medium without the compound. After incubation for 6 h, discard the culture medium. Add 50 μL of 4% paraformaldehyde to each well and incubate at room temperature for 20 min to fix the cells. Discard the paraformaldehyde, wash three times with 1×PBS buffer, add 50 μL of 0.5% PBST to each well, and incubate at 37℃ for 30 min to permeabilize the cells. Discard the PBST, wash three times with 1×PBS buffer, add 50 μL of 5% BSA to each well, and incubate at 37℃ for 30 min to block the cells. Discard the BSA, add 25 μL of a solution containing G3BP antibody (prepared with 1% BSA / PBS, diluted 1:1000) to each well, and incubate overnight at 4℃. Discard the primary antibody, wash three times with 1×PBS, add 30 μL of a solution containing fluorescent secondary antibody (prepared with 1% BSA / PBS) to each well, and incubate at 37°C for 1 h. Discard the secondary antibody, add 0.5 mg / L 4',6-diamidinyl-2-phenylindole solution to each well, and incubate at room temperature for 30 min. Then, image the cells using an FV3000 laser confocal microscope (Olympus) and analyze the results using Fiji / ImageJ (statistical analysis of the ratio of stress granule area to nuclear area).

[0158] 3. To investigate the degradation mechanism of the compounds of formula (II) and formula (III) prepared in Examples 2 and 3 on the stress particles:

[0159] HeLa cells in logarithmic growth phase were seeded at 200,000 per well in 6-well plates and cultured for 48 hours in a cell culture incubator at 37°C with 5% CO2. Sodium arsenite was prepared to a working concentration of 500 μmol / L using medium containing 10% fetal bovine serum. The medium in the 6-well plates was discarded, and 2 mL of medium containing 500 μmol / L sodium arsenite was added to each well for 1 hour of stress. Sodium arsenite was prepared to a working concentration of 50 μmol / L using medium containing 10% fetal bovine serum. Compound of formula (II) prepared in Example 2 was prepared to a concentration of 20 μmol / L using the above 50 μmol / L sodium arsenite medium. The medium was discarded, and 2 mL of the above medium containing sodium arsenite and the compound was added to each well. The control group consisted of 2 mL of 50 μmol / L sodium arsenite medium without the compound. After discarding the medium, the cells were washed three times with 1×PBS, and then scraped off on ice and collected in EP tubes. After centrifugation at 3000 rpm for 5 min, discard 1×PBS and collect the cell pellet. Add an appropriate amount of RIPA lysis buffer and lyse on ice for 3 min. Centrifuge at 12000 rpm for 15 min at 4℃, and collect the supernatant as the total protein sample. After determining the protein concentration using the BCA method, add protein loading buffer to the protein sample, heat at 95℃ for 10 min to denature, cool to room temperature, and store at -20℃. Electrophoretic separation: Load 10-20 μg of protein sample onto an SDS-PAGE gel for electrophoresis at 80V for 30 min, then at 120V for 1 h. Transfer: Activate the polyvinylidene fluoride membrane with anhydrous methanol and soak it in transfer buffer. Then, under ice bath conditions, use a Bio-Red wet transfer apparatus to transfer the protein from the SDS-PAGE onto the polyvinylidene fluoride membrane at 400 mA for 42 min. Blocking: Place the polyvinylidene fluoride (PVDF) membrane in 5% BSA (prepared with TBST) blocking solution and block at room temperature for 1.5 h. Primary antibody incubation: Dilute G3BP and Actin primary antibodies 1:1000 with antibody diluent and incubate the PVDF membrane overnight at 4°C with shake. Washing: Wash the PVDF membrane three times with 1×TBST, 5 min each time. Secondary antibody incubation: Dilute the secondary antibody 1:5000 with antibody diluent and incubate with the PVDF membrane at room temperature with slow shaking for 1 h. Washing: Wash the PVDF membrane three times with 1×TBST, 5 min each time. Development: Place the PVDF membrane in a dark room, add chemiluminescence solution to the membrane, and photograph using a Tanon-4200SF chemiluminescence analyzer. Analyze the experimental results using ImageJ software.

[0160] HeLa cells in logarithmic growth phase were seeded at 200,000 cells / well in 6-well plates and cultured for 48 hours in a cell culture incubator containing 5% CO2 at 37°C. Sodium arsenite was prepared to a working concentration of 500 μmol / L using medium containing 10% fetal bovine serum. The medium in the 6-well plates was discarded, and 2 mL of medium containing 500 μmol / L sodium arsenite was added to each well for 1 hour of stress. Sodium arsenite was prepared to a working concentration of 50 μmol / L using medium containing 10% fetal bovine serum. The compound of formula (III) prepared in Example 3 was prepared to a concentration of 5 μmol / L using the above 50 μmol / L sodium arsenite medium. The medium was discarded, and 2 mL of the above medium containing sodium arsenite and the compound was added to each well. The control group consisted of 2 mL of 50 μmol / L sodium arsenite medium without the compound. After washing the cells several times with 1×PBS, total RNA was extracted using a rapid total RNA extraction kit. RNA concentration was determined using an ultra-micro UV spectrophotometer, and samples were stored at -80°C. 1000 ng of total RNA extract was placed in an EP tube, followed by 1 μL of gDNAClean Reagent, 2 μL of 5×gDNA Clean Buffer, and finally, RNase-free water was added to bring the volume to 10 μL. The mixture was incubated at 42°C for 2 min, then cooled to 4°C. To the 10 μL reaction solution obtained in the previous step, 1 μL of EvoMMLVRTase Enzyme Mix, 1 μL of RT Primer Mix, and 4 μL of 5×RTase Reaction Buffer Mix I were added, and finally, 4 μL of RNase-free water was added to bring the total volume to 20 μL. The mixture was incubated at 37°C for 15 min, followed by 85°C for 5 s, and finally cooled to 4°C. cDNA samples were stored at 4°C. 1 μL of cDNA, 0.4 μL of 10 μmol / L forward primer, 0.4 μL of 10 μmol / L reverse primer, 8.2 μL of RNase-free water, and 10 μL of 2×RealStar Green Fast Mixture were added to a PCR plate. The plate was then sealed and centrifuged at 2000 rpm for 1-2 min. Finally, the PCR plate was placed in a real-time quantitative PCR instrument and amplified forty times at 95℃×30s, 95℃×5s, 62℃×1 min, and 40℃×30s. After amplification, the plate was incubated at 4℃, and the relative change in RNA levels was calculated using Ct values.

[0161] Figure 1 This is an experimental diagram showing the targeting behavior of the compound of formula (II) prepared in Example 2 to the stress particles. Figure 1In the middle, from left to right, are the G3BP stress particle signal (G3BP), the compound signal of formula (II) (compound II), the stress particle signal, and the compound signal of formula (II) (Merge). Figure 2 The figure shows the experimental results of the targeting of the stress particles to the compound of formula (II) prepared in Example 2; Figure 3 This is an experimental diagram showing the targeting behavior of the compound of formula (Ⅲ) prepared in Example 3 to the stress particles. Figure 3 In the middle, from left to right, are the G3BP stress particle signal (G3BP), the compound signal of formula (III) (compound III), the stress particle signal, and the compound signal of formula (III) (Merge). Figure 4 This is a graph showing the experimental results of the targeting of the stress particles to the compound of formula (Ⅲ) prepared in Example 3. Figures 1-4 It can be seen that both compounds of formula (II) and formula (III) can effectively recognize stress particles and have good targeting properties for stress particles.

[0162] Figure 5 The figures show the experimental effects of compounds of formula (II) and formula (III) prepared in Examples 2 and 3 on the degradation of stress particles. Figure 6 The graph shows the degradation results of the stress particles by the compounds of formula (II) and formula (III) prepared in Examples 2 and 3. Figure 5 and Figure 6 It can be seen that, compared with the control group, when the concentration of compound (II) or compound (III) is 5 μmol / L, the fluorescent bright spots are reduced. As the concentration of compounds (II) and (III) increases to 20 μmol / L, the number of stress particles gradually decreases, indicating that compounds (II) and (III) prepared in Examples 2 and 3 have a good degradation effect on stress particles.

[0163] Figure 7 The diagram shows the experimental mechanism of the degradation of stress particles by the compound of formula (II) prepared in Example 2. Figure 7 It can be seen that, compared with the control group, when the concentration of compound (II) is 5 μmol / L, it can reduce the content of GCBP in cells. As the concentration increases, the reduction effect on G3BP is stronger, indicating that compound (II) can degrade stress particles by degrading the stress particle cytoskeleton protein G3BP.

[0164] Figure 8 The diagram shows the experimental mechanism of the degradation of stress particles by the compound of formula (Ⅲ) prepared in Example 3. Figure 8 It can be seen that the compound of formula (III) can degrade the stress particles by degrading the RNA that makes up the stress particles.

Claims

1. A stress-targeted particle degrading agent, characterized in that, This includes compounds of formula (A), formula (B) or formula (C) or their pharmaceutically acceptable salts: Formula (A) Formula (B) Formula (C); Among them, in equations (A) to (C), The A - Each is independently selected from iodide ions, bromide ions, or trifluoroacetate ions; R1 is a linking group, which is independently selected from... or n is independently selected from 1, 3, or 5; R2 is a ligand structural group, which is independently selected from CRBN protein ligand structural groups. or ribonuclease L ligand structural group .

2. The stress particle targeted degradation agent according to claim 1, characterized in that, The compounds are shown as those of Formula 1, Formula 2, Formula 5 or Formula 6: Formula 1 Formula 2 Formula 5 Formula 6.

3. The stress particle targeted degradation agent according to claim 1 or 2, characterized in that, When the compound is as shown in formula (A), its preparation method includes the following steps: The compound of formula (a) is reacted with a ligand to give a compound of formula (A), the structure of which is as follows: Equation (a); When the compound is as shown in formula (B), its preparation method includes the following steps: The compound of formula (b) was reacted with a ligand to give a compound of formula (B), the structure of which is as follows: Equation (b); When the compound is as shown in formula (C), its preparation method includes the following steps: The compound of formula (d) was reacted with a ligand to give the compound of formula (C), the structure of which is as follows: Equation (d); The ligands are each independently selected from... (n=1, 3, or 5) (n=1, 3 or 5) (n=1, 3 or 5) or (n=1, 3 or 5).

4. The stress particle targeted degradation agent according to claim 3, characterized in that, The molar ratio of the compound of formula (a) to the ligand is 1:(1-2).

5. The stress particle targeted degradation agent according to claim 3, characterized in that, The molar ratio of the compound of formula (b) to the ligand is 1:(1-2).

6. The stress particle targeted degradation agent according to claim 3, characterized in that, The molar ratio of the compound of formula (d) to the ligand is 1:(1-2).

7. The use of the stress particle targeting degrader according to any one of claims 1-6 in the preparation of antitumor drugs and / or drugs for treating neurodegenerative diseases.

8. The application according to claim 7, characterized in that, The tumors include pancreatic cancer, colorectal cancer, osteosarcoma, glioma, cervical cancer, breast cancer, prostate cancer, liver cancer, non-small cell lung cancer, or melanoma.

9. The application according to claim 7, characterized in that, The neurodegenerative diseases mentioned include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's disease, or Parkinson's disease.

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