Compound as well as preparation method and application thereof in targeted degradation of GKS-3beta

Abstract

The invention discloses a compound as well as a preparation method and application thereof in targeted degradation of GKS-3beta. The structure of the compound is shown as a formula A, wherein m and n are independently selected from any integer of 3-5. According to the scheme, the environment-sensitive GSK-3beta PTOTAC compound and the pharmaceutically acceptable salt, the crystal form, the solvate, the stereoisomer or the isotope substitution form thereof are provided, and the compound has good biological safety to nerve cells and can precisely degrade GSK-3beta protein in a targeted mode. Meanwhile, the compound also shows neuroprotective activity, and a new convenient tool is provided for clinical treatment of GSK-3beta related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, and in particular to a compound, a preparation method thereof and application of the compound in targeted degradation of GSK-3β. BACKGROUND

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory loss and impairment of cognitive function. The dementia symptoms caused by AD seriously impair the quality of life of patients and bring huge social and economic burden. The pathological mechanism of AD has not been fully elucidated, and the existing therapeutic drugs have limited efficacy.

[0003] Glycogen synthase kinase 3β (GSK-3β) is a key multifunctional serine / threonine kinase involved in the regulation of various intracellular processes. Studies have shown that GSK-3β plays a key role in the core pathological process of AD: it promotes the abnormal hyperphosphorylation of tau protein, increases the production of β-amyloid protein (Aβ), and is involved in the neuroinflammatory response. Therefore, GSK-3β is considered an important therapeutic target for AD and other diseases.

[0004] However, traditional GSK-3β small molecule inhibitors can only target its kinase catalytic activity, and the mechanism of action is relatively simple. In contrast, the new strategy based on the protein degradation targeting chimera (PROTAC) technology provides a breakthrough approach: by specifically recruiting the KEAP1-Cul3 ubiquitin E3 ligase complex, PROTAC molecules can induce ubiquitination of GSK-3β protein and degradation by proteasome, thereby achieving the elimination of target proteins rather than simple inhibition.

[0005] However, current research on fluorescent PROTACs (fluorescent PROTACs) targeting GSK-3β is still insufficient, and the development of such novel probes has important scientific and clinical value. Such degraders not only can real-time visualize monitor the protein expression level and dynamic positioning changes of GSK-3β in AD and other disease models, deepening the understanding of pathological mechanisms, but also can provide a tool with high degradation capacity and convenient visualization monitoring for future precision diagnosis and treatment, exhibiting unique value in basic research and translational application. SUMMARY

[0006] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes a compound with a novel structure, which can target and degrade GSK-3β and has fluorescent properties.

[0007] The present application also proposes a preparation method of the above-mentioned compound.

[0008] The present application also proposes an application of the above-mentioned compound.

[0009] According to one aspect of the invention, a compound and its pharmaceutically acceptable salt, crystal form, solvate, stereoisomer, or isotopically substituted form are provided, the structure of which is shown in Formula A:

[0010]

[0011] In the formula, m and n are independently selected from any integers in 3-5.

[0012] The compounds of the present invention, their pharmaceutically acceptable salts, crystal forms, solvates, stereoisomers, or isotopically substituted forms, have at least the following beneficial effects: The present invention provides an environmentally sensitive GSK-3β POTAC compound, its pharmaceutically acceptable salts, crystal forms, solvates, stereoisomers, or isotopically substituted forms, which exhibit good biocompatibility with nerve cells and can precisely target and degrade GSK-3β proteins. Simultaneously, the above compounds also exhibit neuroprotective activity, providing a new and convenient tool for the clinical treatment of GSK-3β-related diseases.

[0013] According to some embodiments of the present invention, the compound is selected from one of the following structural formulas:

[0014]

[0015] According to another aspect of the present invention, a method for preparing a compound having the structure shown in Formula A is provided. The method comprises the following steps: reacting precursor compounds S3 and S10 to obtain a compound as shown in Formula A.

[0016] The structures of formula A and precursor compounds S3 and S10 are as follows:

[0017]

[0018] In the formula, m and n are independently selected from any integers in 3-5.

[0019] According to some embodiments of the present invention, the reaction of the precursor compounds S3 and S10 is carried out in the presence of a solvent (such as acetonitrile (MeCN) or tetrahydrofuran (THF), a catalyst (such as CuSO4 and / or sodium ascorbate) or a ligand (such as tris(benzyltriazolylmethyl)amine (TBTA)).

[0020] According to some embodiments of the present invention, the reaction of the precursor compounds S3 and S10 is carried out at room temperature (preferably 25±5°C) for a reaction time of 14 to 20 h (preferably 16 h).

[0021] According to some embodiments of the present invention, after the reaction of the precursor compounds S3 and S10 is completed, a post-processing step is further included, specifically including dilution, extraction, washing, drying and column purification.

[0022] According to some embodiments of the present invention, the preparation steps of the precursor compound S3 include:

[0023] Compound S1 and compound S1-1 are reacted as raw materials to obtain compound S2; compound S2 undergoes an azide reaction to obtain the final product; the structural formulas of compounds S1, S1-1, and S2 are as follows:

[0024]

[0025] According to some embodiments of the present invention, the reaction of compound S1 and compound S1-1 is carried out in the presence of a solvent (such as acetonitrile (MeCN) or tetrahydrofuran (THF)) and a base (the base is an organic base or an inorganic base, the organic base including pyridine, the inorganic base including carbonates such as Cs2CO3).

[0026] According to some embodiments of the present invention, the reaction of compound S1 and compound S1-1 is carried out under heating conditions, with a heating temperature of 50-80°C (preferably 60-70°C) and a reaction time of 4-7 h (preferably 5 h).

[0027] According to some embodiments of the present invention, after the reaction of compound S1 and compound S1-1 is completed, a post-processing step is further included, specifically including dilution, extraction, washing, drying and column chromatography purification. Specifically, after the reaction is completed, the reaction mixture is diluted with water, extracted with ethyl acetate, the combined organic layers are washed with brine, dried with anhydrous Na2SO4, and the crude product is purified by column chromatography using PE (petroleum ether) / EA (ethyl acetate) (2 / 1, v / v) as eluent to obtain yellow oily product S2.

[0028] According to some embodiments of the present invention, the azide reaction includes the step of reacting compound S2 with an azide.

[0029] According to some embodiments of the present invention, the azide includes at least one of NaN3 or tetramethylammonium azide (TMAN).

[0030] According to some embodiments of the present invention, the azide reaction is carried out in solution in the presence of a solvent such as dimethyl sulfoxide (DMSO).

[0031] According to some embodiments of the present invention, the azide reaction is carried out at room temperature (preferably 15-40°C) for 1-3 hours (preferably 2 hours).

[0032] According to some embodiments of the present invention, the azide reaction is further complicated by a post-processing step, specifically including dilution, extraction, washing and drying.

[0033] According to some embodiments of the present invention, the preparation steps of the precursor compound S10 include:

[0034] Compound S6 reacts with compound S6-1 to give compound S7; compound S7 undergoes selective deprotection of its Boc protecting group to give compound S8; compound S8 reacts with compound S5 to give compound S9; compound S9 undergoes amidation to give compound S10; wherein the structural formulas of compounds S5, S6, S6-1, S7, S8, and S9 are as follows:

[0035]

[0036] According to some embodiments of the present invention, the reaction between compound S6 and compound S6-1 is carried out at room temperature (preferably 15-40°C) for 14-20 hours (preferably 16-18 hours).

[0037] According to some embodiments of the present invention, after the reaction of compound S6 with compound S6-1 is completed, post-processing is further included, specifically including dilution, extraction, washing, drying and column purification.

[0038] According to some embodiments of the present invention, the selective deprotection reaction of compound S7 is carried out at room temperature (preferably 15-40°C) for 0.5-2 hours (preferably 1 hour).

[0039] According to some embodiments of the present invention, the selective deprotection reaction of compound S7 further includes post-treatment, specifically pH adjustment, filtration, and column chromatography purification. Specifically, the pH is adjusted to 7-8 with sodium hydroxide, a large amount of white solid precipitate is formed, the precipitate is filtered to obtain a crude product, and DCM (dichloromethane) / CH3OH (10 / 1, v / v) is used as the eluent to purify the product into a yellow solid, S8, by column chromatography.

[0040] According to some embodiments of the present invention, the reaction of compound S8 with compound S5 is carried out in the presence of a solvent and a base or a solvent and a catalyst (such as CuSO4 and / or sodium ascorbate).

[0041] According to some embodiments of the present invention, the reaction temperature of compound S8 and compound S5 is room temperature (preferably 25±5℃), and the reaction time is 2 to 8 hours (preferably 4 to 6 hours).

[0042] According to some embodiments of the present invention, after the reaction of compound S8 and compound S5 is completed, a post-processing step is further included, specifically including dilution, extraction, washing, drying and column purification.

[0043] According to some embodiments of the present invention, the amidation reaction is carried out at room temperature (25±5℃) for 3 to 5 hours (preferably 4 hours).

[0044] According to some embodiments of the present invention, the aftermath of the amidation reaction includes post-treatment, specifically pH adjustment and filtration. Specifically, the pH is adjusted to 3-4 with hydrochloric acid, resulting in the precipitation of a large amount of white solid. The precipitate obtained by filtration is the intermediate. Subsequent post-treatment includes dilution, extraction, washing, drying, and column chromatography purification to obtain the yellow solid product S10.

[0045] According to some embodiments of the present invention, the preparation steps of compound S5 include:

[0046] Compound S5 is obtained by reacting compounds S4 and S4-1 as raw materials; wherein the structural formulas of compounds S4 and S4-1 are as follows:

[0047]

[0048] According to some embodiments of the present invention, the reaction temperature between compound S4 and compound S4-1 is 60-90°C (preferably 70-80°C), and the reaction time is 3-5 hours (preferably 4 hours).

[0049] According to some embodiments of the present invention, after the reaction of compound S4 and compound S4-1 is completed, a post-processing step is further included, specifically including dilution, extraction, washing, drying and column purification.

[0050] According to another aspect of the invention, the use of the above-described compounds and their pharmaceutically acceptable salts, crystal forms, solvates, stereoisomers or isotopically substituted forms thereof in the preparation of GSK-3β protein degradation and / or GSK-3β protein imaging products is proposed.

[0051] According to another aspect of the invention, the use of the above-described compounds and their pharmaceutically acceptable salts, crystal forms, solvates, stereoisomers or isotopically substituted forms thereof is proposed in the preparation of reagents for the treatment and / or prevention of diseases associated with GSK-3β protein.

[0052] According to some embodiments of the present invention, the GSK-3β protein-related diseases include neurodegenerative diseases or metabolic diseases.

[0053] According to some embodiments of the present invention, the GSK-3β protein-related diseases include at least one of Alzheimer's disease, Parkinson's disease, bipolar disorder, type 2 diabetes, or diabetic nephropathy.

[0054] According to another aspect of the invention, the use of the above-mentioned compounds and their pharmaceutically acceptable salts, crystal forms, solvates, stereoisomers or isotopic substitutions thereof in the preparation of neuroprotective products is proposed.

[0055] According to some embodiments of the present invention, the neuroprotection includes exhibiting neuroprotective activity in the prevention or treatment of neurodegenerative diseases (such as Alzheimer's disease).

[0056] According to another aspect of the invention, a pharmaceutical composition is provided, the composition comprising one or more of the above-described compound and its pharmaceutically acceptable salts, crystal forms, solvates, stereoisomers, or isotopically substituted forms thereof.

[0057] According to another aspect of the invention, a kit is provided comprising the above-described compound and its pharmaceutically acceptable salts, crystal forms, solvates, stereoisomers, or isotopically substituted forms thereof.

[0058] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0059] Figure 1 The cytotoxicity of compounds A-1, A-2, A-3, A-4 and A-5 in HT-22 cells was evaluated.

[0060] Figure 2 The fluorescence emission spectra of compound A-1 in THF / H2O systems with different ratios are shown.

[0061] Figure 3 The fluorescence emission spectra of compound A-2 in THF / H2O systems with different ratios are shown.

[0062] Figure 4 The fluorescence emission spectra of compound A-3 in THF / H2O systems with different ratios are shown.

[0063] Figure 5 The fluorescence emission spectra of compound A-4 in THF / H2O systems with different ratios are shown.

[0064] Figure 6 The fluorescence emission spectra of compound A-5 in THF / H2O systems with different ratios are shown.

[0065] Figure 7 The figure shows the effect of compounds A-1, A-2, A-3, A-4, and A-5 on the expression level of GSK-3β in HT-22 cells. ***p<0.001 (t-test); n=3.

[0066] Figure 8 The following graphs show the effects of compound A-1 on the expression level of GSK-3β in HT-22 cells: A represents the results of different treatment times; B represents the results of different concentrations. ***p<0.001 (t-test); n=3.

[0067] Figure 9 Figure A shows inverted fluorescence images of compounds A-1, S11, and S12 in HT-22 cells at different time points; Figure B shows the corresponding relative average intensity statistical bar chart. Note that p < 0.001 (t-test); n = 2.

[0068] Figure 10 Figure A shows the neuroprotective activity of compound A-1 (1, 5, and 10 μM), GSK-3β inhibitor S11 (10 μM), KEAP1 inhibitor S12 (10 μM), and their combination therapy (5 μM each) in a glutamate (10 mM)-induced HT-22 cell injury model; Figure B is the corresponding bar chart for Figure A. *p<0.05, ***p<0.001 vs. Model; ### p<0.001 vsCtrl(t-test); n=3. Detailed Implementation

[0069] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available. Unless otherwise specified, the same parameter value is the same in all embodiments. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0070] In some embodiments of the present invention, a GSK-3βPROTAC compound with the following structural formula is provided:

[0071] In the formula, m and n are independently selected from any integers in 3-5.

[0072] Its preparation method includes the following steps:

[0073] Step 1. React compounds S1 and S1-1 as starting materials to obtain compound S2; Step 2. React compound S2 obtained in Step 1 with NaN3 to obtain compound S3; Step 3. React compounds S4 and S4-1 as starting materials to obtain compound S5; Step 4. React compound S6 with compound S6-1 to obtain compound S7; Step 5. Selectively remove the Boc protecting group from compound S7 obtained in Step 4 to obtain compound S8; Step 6. React compound S8 obtained in Step 5 with compound S5 to obtain compound S9; Step 7. Perform an amidation reaction on compound S9 obtained in Step 6 to obtain compound S10; Step 8. React compound S3 obtained in Step 1 with compound S10 obtained in Step 7 as starting materials to obtain the compound shown in Formula A.

[0074]

[0075] Furthermore, the reaction in step 1 is carried out under conditions of solvent and base.

[0076] Furthermore, the reaction with sodium azide in step 2 is carried out under dimethyl sulfoxide (DMSO) conditions.

[0077] Furthermore, in step 3, the reaction operation, solvent, alkali, and feed ratio are all the same as in step 1.

[0078] Furthermore, the reaction in step 4 is carried out under the conditions of tributylphosphine (Bu3P), diethyl azodicarbonate (DEAD), and tetrahydrofuran (THF).

[0079] Furthermore, the selective removal of the Boc protecting group in step 5 is carried out under conditions of dichloromethane and trifluoroacetic acid.

[0080] Furthermore, the reaction in step 6 is carried out under conditions of solvent and base or solvent and catalyst.

[0081] Furthermore, the reaction in step 7 is carried out first under the conditions of solvent and base or solvent and catalyst, and then under the conditions of TBTU, DIPEA and DMF.

[0082] Furthermore, the reaction in step 8 is carried out under the conditions of solvent, catalyst, and ligand.

[0083] Furthermore, the solvent mentioned above is acetonitrile or tetrahydrofuran.

[0084] Furthermore, the aforementioned base can be an organic base or an inorganic base. Organic bases include pyridine, and inorganic bases include carbonates.

[0085] Furthermore, the catalysts mentioned above are CuSO4 and sodium ascorbate.

[0086] Furthermore, the aforementioned ligand is tris(benzyltriazolylmethyl)amine (TBTA).

[0087] Furthermore, the reaction temperature in step 1 is 50–80°C (preferably 60–70°C), and the reaction time is 4–7 h (preferably 5 h).

[0088] Furthermore, after the reaction in step 1 is completed, post-processing is also included. Post-processing includes dilution, extraction, washing, drying, and column chromatography purification. Specifically, after the reaction is completed, the reaction mixture is diluted with water, extracted with ethyl acetate, the combined organic layers are washed with brine, dried with anhydrous Na2SO4, and the crude product is purified by column chromatography using PE (petroleum ether) / EA (ethyl acetate) (2 / 1, v / v) as eluent to obtain a yellow oily product S2.

[0089] Furthermore, the reaction temperature in step 2 is room temperature, and the reaction time is 1 to 3 hours (preferably 2 hours).

[0090] Furthermore, the reaction with sodium azide in step 2 also includes post-treatment, which includes dilution, extraction, washing, and drying.

[0091] Furthermore, the reaction temperature in step 3 is 60–90°C (preferably 70–80°C), and the reaction time is 3–5 h (preferably 4 h).

[0092] Furthermore, after the reaction in step 3 is completed, post-processing is also included, which includes dilution, extraction, washing, drying, and column purification.

[0093] Furthermore, the reaction temperature in step 4 is room temperature, and the reaction time is 14–20 h (preferably 16–18 h).

[0094] Furthermore, after the reaction in step 4 is completed, post-processing is also included, which includes dilution, extraction, washing, drying and column chromatography purification to obtain a yellow solid product S7.

[0095] Furthermore, the reaction temperature in step 5 is room temperature, and the reaction time is 0.5–2 hours (preferably 1 hour).

[0096] Furthermore, after selectively removing the Boc protecting group in step 5, post-processing is also included. The post-processing includes pH adjustment, filtration, and column chromatography purification. Specifically, the pH is adjusted to 7-8 with sodium hydroxide, and a large amount of white solid precipitates out. The precipitate is filtered to obtain the crude product. DCM (dichloromethane) / CH3OH (10 / 1, v / v) is used as the eluent, and the product is purified by column chromatography to obtain the yellow solid product S8.

[0097] Furthermore, the reaction temperature in step 6 is room temperature, and the reaction time is 2 to 8 hours (preferably 4 to 6 hours).

[0098] Furthermore, after the reaction in step 6 is completed, post-processing is also included, which includes dilution, extraction, washing, drying, and column purification.

[0099] Furthermore, the reaction temperature in step 7 is room temperature, and the reaction time is 3–5 hours (preferably 4 hours).

[0100] Furthermore, step 7 includes post-processing after the reaction is complete. This post-processing includes pH adjustment and filtration. Specifically, the pH is adjusted to 3-4 with hydrochloric acid, resulting in the precipitation of a large amount of white solid. The precipitate obtained by filtration is the intermediate. Subsequent post-processing involves dilution, extraction, washing, drying, and column chromatography purification to obtain the yellow solid product S10.

[0101] Furthermore, the reaction temperature in step 8 is room temperature, and the reaction time is 14–20 h (preferably 16 h).

[0102] Furthermore, after the reaction in step 8 is completed, post-processing is also included, which includes dilution, extraction, washing, drying, and column purification.

[0103] THF stands for tetrahydrofuran; Py stands for pyridine; TBTU stands for O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid; DIPEA stands for N,N-diisopropylethylenediamine; DMF stands for N,N-dimethylformamide; DCM stands for dichloromethane; DMSO stands for dimethyl sulfoxide; Bu3P stands for tributylphosphine.

[0104] The CAS number for compound S1 is 261914-52-7; the CAS number for compound S4 is 142246-48-8; the CAS number for compound S6 is 1702428-88-3; the CAS number for compound S1-1a is 109-64-8; the CAS number for compound S1-1b is 110-52-1; the CAS number for compound S1-1c is 111-24-0; the CAS number for compound S6-1a is 58885-58-8; the CAS number for compound S6-1b is 75178-87-9; and the CAS number for compound S6-1c is 75178-90-4.

[0105] In the following examples, the room temperature is specifically 25°C, and the ambient temperature is 30°C.

[0106] Preparation of intermediates:

[0107] The structural formula of the prepared intermediate compound is as follows:

[0108]

[0109] (1) Preparation of intermediates S3a-S3c

[0110] The synthetic pathways for the aforementioned intermediates S3a-S3c are shown below:

[0111]

[0112] Specifically, the above intermediates S3a-S3c can be prepared by the following method:

[0113] Step 1-1: Preparation of (E)-1-(3-(4-ethoxy-3,5-dimethoxyphenyl)acryloyl)-5,6-dihydropyridine-2(1H)-one-bromomethane S2a:

[0114] Compound S1 (0.10 g, 0.34 mmol) was weighed into a reaction flask and dissolved in 2.0 mL of dry MeCN. Cs₂CO₃ (0.14 g, 0.44 mmol) was then added to the reaction mixture and stirred at room temperature. After 15 min, 1,3-dibromopropane (50 μL, 0.51 mmol) was added to the reaction mixture, and the system was heated to 60 °C and stirred for 3 h. The aqueous layer was extracted twice with ethyl acetate, and the combined organic phases were washed successively with brine and dehydrated with sodium sulfate, followed by rotary evaporation for concentration. A pale yellow oil was obtained using petroleum ether / ethyl acetate (2 / 1, v / v) as the developing solvent. The yield was 59%.

[0115] The obtained intermediate compound S2 was characterized by NMR, and the characterization data are as follows:

[0116] 1 H NMR (500MHz, CDCl3) δ7.69(d,J=15.5Hz,1H),7.44(d,J=15.5Hz,1H),6.97(dt,J=9.8,4.2Hz,1H),6.81(s,2H),6.06(dt,J=9.8,1 .8Hz,1H),4.14(t,J=5.7Hz,2H),4.06(t,J=6.6Hz,2H),3.89(s,6H),3.73(t,J=6.6Hz,2H),2.52–2.48(m,2H),2.29–2.24(m,2H).

[0117] Steps 1-2: Preparation of (E)-1-(3-(3-(3,5-dimethoxy-4-propoxyphenyl)acryloyl)-5,6-dihydropyridine-2(1H)-one-bromomethane S2b:

[0118] The specific preparation method is the same as that for S2a, except that 1,3-dibromopropane is replaced with an equimolar amount of 1,3-dibromobutane. The characterization data for the obtained intermediate S2b are as follows:

[0119] 1 H NMR (500MHz, CDCl3) δ7.68(d,J=15.5Hz,1H),7.43(d,J=15.5Hz,1H),6.96(dt,J=9.7,4.1Hz,1H),6.06(dt,J=9.7,1.8H z,1H),4.06–4.02(m,4H),3.88(s,6H),3.54(t,J=6.8Hz,2H),2.51–2.47(m,2H),2.17–2.10(m,2H),1.93–1.86(m,2H).

[0120] Steps 1-3: Preparation of (E)-1-(3-(4-butoxy-3,5-dimethoxyphenyl)acryloyl)-5,6-dihydropyridine-2(1H)-one-bromomethane S2c:

[0121] The specific preparation method is the same as that for S2a, except that 1,3-dibromopropane is replaced with an equimolar amount of 1,3-dibromopentane. The characterization data for the obtained intermediate S2c are as follows:

[0122] 1 H NMR (400MHz, CDCl3) δ7.69(d,J=15.5Hz,1H),7.43(d,J=15.5Hz,1H),6.96(dt,J=9.7,4.2Hz,1H),6.06(dt,J=9.7,1.8Hz,1H),4. 07–4.00(m,4H),3.88(s,6H),3.45(t,J=6.9Hz,2H),2.52–2.47(m,2H),1.99–1.91(m,2H),1.82–1.76(m,2H),1.68–1.62(m,2H).

[0123] Step 2-1: Preparation of (E)-1-(3-(4-ethoxy-3,5-dimethoxyphenyl)acryloyl)-5,6-dihydropyridine-2(1H)-one-azidomethane S3a:

[0124] At room temperature, NaN3 (3.6 mg, 0.060 mmol) was added to a DMSO (1.0 mL) solution of compound S2a (19 mg, 0.050 mmol). The reaction was stirred for 1.5 h, then diluted with water and extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was used in subsequent reactions without purification.

[0125] Step 2-2: Preparation of (E)-1-(3-(3-(3,5-dimethoxy-4-propoxyphenyl)acryloyl)-5,6-dihydropyridine-2(1H)-one-azidomethane S3b:

[0126] The specific preparation method is the same as that for S3a. The difference is that compound S2a is replaced with an equimolar amount of S2b.

[0127] Steps 2-3: Preparation of (E)-1-(3-(4-butoxy-3,5-dimethoxyphenyl)acryloyl)-5,6-dihydropyridine-2(1H)-one-azidomethane S3c:

[0128] The specific preparation method is the same as that for S3a. The difference is that compound S2a is replaced with an equimolar amount of S2c.

[0129] (2) Preparation of intermediates S10a-S10c

[0130] The synthetic pathways for the aforementioned intermediates S10a-S10c are shown below:

[0131]

[0132] Step 1: Preparation of 7-chloro-N-methyl-N-(prop-2-yn-1-yl)benzo[c][1,2,5]oxadiazole-4-sulfonamide S5

[0133] The synthetic route for 7-chloro-N-methyl-N-(prop-2-yn-1-yl)benzo[c][1,2,5]oxadiazole-4-sulfonamide S5 is shown below:

[0134]

[0135] Specifically, 7-chloro-N-methyl-N-(prop-2-yn-1-yl)benzo[c][1,2,5]oxadiazole-4-sulfonamide S5 can be prepared by the following method:

[0136] N-methylpropargyl-2-yn-1-amine S4 (0.14 mL, 1.7 mmol) was dissolved in acetonitrile (5.0 mL), and anhydrous pyridine (0.33 mL, 3.1 mmol) was added to the system under ice bath conditions. After 30 min, N-methylpropargylamine (0.36 g, 1.4 mmol) was added dropwise to the system, and the mixture was stirred at room temperature for 4 h. After the reaction was complete, the mixture was diluted with water and extracted with ethyl acetate. The organic layer was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation. The solution was purified by rapid column chromatography (petroleum ether / ethyl acetate = 10 / 1, v / v) to give a yellow solid, compound S5, in 89% yield.

[0137] The above intermediate S5 was subjected to NMR characterization, and the characterization data are as follows:

[0138] 1 H NMR (500MHz, CDCl3) δ8.01(d,J=7.4Hz,1H),7.58(d,J=7.4Hz,1H),4.24(d,J=2.5Hz,2H),3.09(s,3H),1.97(s,1H).

[0139] Step 2-1: 4-(3-((tert-butoxycarbonyl)amino)propoxy)-2-(2-(cyclopropanecarboxamide)pyridin-4-yl)thiazol-5-carboxylic acid ester S7a can be prepared by the following method:

[0140] Under nitrogen protection and an ice bath, DEAD (0.32 mL, 2.0 mmol) was added dropwise to a mixture of anhydrous tetrahydrofuran (10 mL) containing compound S6 (0.33 g, 1.0 mmol), tert-butyl(3-hydroxypropyl)carbamate (0.34 mL, 2.0 mmol), and tributylphosphine (0.47 mL, 2.0 mmol). The reaction was terminated by stirring at room temperature for 18 h. The reaction mixture was diluted with water, extracted with ethyl acetate, and the combined organic phases were washed with brine, dried over anhydrous Na2SO4, and concentrated by rotary evaporation to obtain the crude product. Purification was performed by silica gel column chromatography (petroleum ether / ethyl acetate = 1:1) to obtain a pale yellow solid compound S7a in 75% yield.

[0141] The above intermediate S7a was subjected to NMR characterization, and the characterization data are as follows:

[0142] 1H NMR (500MHz, CDCl3) δ8.97(s,1H),8.76(s,1H),8.36(d,J=5.3Hz,1H),7.56(d,J=5.3Hz,1H),5.67(t,J=5.9Hz,1H),4.65(t,J=5.9Hz,2H),4.36– 4.32(m,2H),3.42–3.39(m,2H),2.07–2.02(m,2H),1.68–1.63(m,1H),1 .46(s,9H),1.37(t,J=7.1Hz,3H),1.18–1.15(m,2H),0.96–0.92(m,2H).

[0143] Step 2-2: Preparation of ethyl 4-(4-(tert-butoxycarbonyl)amino)butoxy)-2-(2-(cyclopropanecarboxyl)pyridin-4-yl)thiazol-5-carboxylic acid S7b:

[0144] The specific preparation method is the same as that for S7a, except that tert-butyl (3-hydroxypropyl) carbamate is replaced with an equimolar amount of tert-butyl (4-hydroxybutyl) carbamate. The characterization data for the resulting intermediate S7b are as follows:

[0145] 1 H NMR (400MHz, CDCl3) δ8.76(s,1H),8.38(s,1H),8.37(s,1H),7.57(d,J=5.2Hz,1H),4.93(s,1H),4.61(t,J=6.2Hz,2H),4.36–4.32(m,2H),3.27– 3.21(m,2H),1.97–1.87(m,2H),1.78–1.71(m,2H),1.62–1.58(m,1H),1 .45(s,9H),1.37(t,J=7.1Hz,3H),1.21–1.15(m,2H),0.98–0.94(m,2H).

[0146] Steps 2-3: Preparation of 4-((5-((tert-butoxycarbonyl)amino)pentyl)oxy)-2-(2-(cyclopropanecarboxamide)pyridin-4-yl)thiazol-5-carboxylic acid ester S7c:

[0147] The specific preparation method is the same as that for S7a, except that tert-butyl (3-hydroxypropyl) carbamate is replaced with an equimolar amount of tert-butyl (5-hydroxypentyl) carbamate. The characterization data for the resulting intermediate S7c are as follows:

[0148] 1H NMR (500MHz, CDCl3) δ8.76(s,1H),8.65(s,1H),8.37(d,J=5.2Hz,1H),7.56(d,J= 5.2Hz,1H),4.76(s,1H),4.59(t,J=6.5Hz,2H),4.35–4.31(m,2H),3.16(t,J=6.5 Hz,2H),1.82–1.86(m,2H),1.66–1.62(m,1H),1.62–1.57(m,2H),1.55(t,J=6.0H z,2H),1.45(s,9H),1.37(t,J=7.1Hz,3H),1.20–1.15(m,2H),0.97–0.93(m,2H).

[0149] Step 3-1: Preparation of ethyl 4-(3-aminopropoxy)-2-(2-(cyclopropanecarboxamide)pyridin-4-yl)thiazole-5-carboxylic acid S8a:

[0150] Compound S7a (0.37 g, 0.75 mmol) was dissolved in anhydrous dichloromethane (5.0 mL), and trifluoroacetic acid (2.5 mL) was slowly added dropwise under ice bath conditions. After stirring the reaction system at room temperature for 1 h, the pH of the reaction solution was adjusted to neutral (pH = 7) with sodium bicarbonate aqueous solution, and the product was obtained by filtration as a yellow solid, S8a, in 44% yield.

[0151] The above intermediate S8a was subjected to NMR characterization, and the characterization data are as follows:

[0152] 1 H NMR(500MHz,DMSO-d6)δ11.10(s,1H),8.72–8.64(m,1H),8.48(d,J=5.6Hz,1H),7.63–7.59(m,1H),4.59(t,J=5.9Hz,2H), 4.28–4.23(m,2H),3.02(t,J=7.0Hz,2H),2.09–2.05(m,2H),2.05–2.02(m,1H),1.29(t,J=7.0Hz,3H),0.88–0.83(m,4H).

[0153] Step 3-2: Preparation of ethyl 4-(4-aminobutoxy)-2-(2-(cyclopropanecarboxamide)pyridin-4-yl)thiazole-5-carboxylic acid S8b:

[0154] The specific preparation method is the same as that for S8a, except that compound S7a is replaced with an equimolar amount of S7b. The characterization data for the resulting intermediate S8b are as follows:

[0155] 1H NMR (500MHz, DMSO-d6) δ8.66(d,J=1.7Hz,1H),8.46(d,J=5.1Hz,1H),7.59(dd,J=5.1,1.7Hz,1H),4.52(t,J=6.2Hz,2H),4.26–4.2 2(m,2H),2.86(t,J=7.5Hz,2H),2.07–2.01(m,1H),1.90–1.76(m,2H),1.77–1.68(m,2H),1.28(t,J=7.1Hz,3H),0.87–0.84(m,4H).

[0156] Step 3-3: Preparation of ethyl 4-((5-aminopentyl)oxy)-2-(2-(cyclopropanecarboxamide)pyridin-4-yl)thiazolyl-5-carboxylic acid S8c:

[0157] The specific preparation method is the same as that for S8a, except that compound S7a is replaced with an equimolar amount of S7c. The characterization data for the resulting intermediate S8c are as follows:

[0158] 1 H NMR (400MHz, DMSO-d6) δ11.09(s,1H),8.68(s,1H),8.48(d,J=5.1Hz,1H),7.61(d,J=5.1Hz,1H),4.52(t,J=6.3Hz,2H),4.28–4.23(m,2H),2 .82(t,J=7.5Hz,2H),2.10–2.02(m,1H),1.84–1.77(m,2H),1.66–1.59(m,2H),1.54–1.48(m,2H),1.29(t,J=7.1Hz,3H),0.89–0.83(m,4H).

[0159] Step 4-1: Preparation of ethyl 2-(2-(cyclopropanecarbamoyl)pyridin-4-yl)-4-(3-((7-(N-methyl-N-(prop-2-yn-1-yl)sulfonamido)benzo[c][1,2,5]oxadiazol-4-yl)amino)propoxy)thiazole-5-carboxylic acid S9a:

[0160] Compound S8a (0.12 g, 0.31 mmol) was dissolved in anhydrous acetonitrile (3.0 mL), followed by the sequential addition of potassium carbonate (0.17 g, 1.2 mmol) and compound S5 (89 mg, 0.31 mmol). The reaction mixture was then heated to 78 °C and maintained for 3 h. After the reaction was complete, the mixture was quenched with water and extracted three times with ethyl acetate. The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by column chromatography (PE / EA = 2:1) to give a yellow oil, S9a, in 91% yield.

[0161] The above intermediate S9a was subjected to NMR characterization, and the characterization data are as follows:

[0162] 1 H NMR (500MHz, DMSO-d6) δ11.08(s,1H),8.66(s,1H),8.52(t,J=5.7Hz,1H),8.45(d,J=5.2H z,1H),7.81(d,J=8.2Hz,1H),7.57–7.55(m,1H),6.38(d,J=8.2Hz,1H),4.64(t,J=6.0Hz,2 H),4.27–4.23(m,2H),4.03(d,J=2.5Hz,2H),3.66–3.57(m,2H),3.01(t,J=2.5Hz,1H),2. 79(s,3H),2.21–2.16(m,2H),2.07–2.02(m,1H),1.29(t,J=7.1Hz,3H),0.87–0.83(m,4H).

[0163] Step 4-2: Preparation of ethyl 2-(2-(cyclopropanecarbamoyl)pyridin-4-yl)-4-(4-((7-(N-methyl-N-(prop-2-yn-1-yl)sulfonamido)benzo[c][1,2,5]oxadiazol-4-yl)amino)butoxy)thiazole-5-carboxylic acid S9b:

[0164] The specific preparation method is the same as that for S9a, except that compound S8a is replaced with an equimolar amount of S8b. The characterization data for the resulting intermediate S9b are as follows:

[0165] 1 H NMR (400MHz, CDCl3) δ9.75 (s, 1H), 8.85 (s, 1H), 8.29 (d, J = 5.3Hz, 1H), 7.90 (d, J = 8.0Hz, 1H), 7.54(d,J=5.3Hz,1H),6.53(t,J=5.6Hz,1H),6.18(d,J=8.0Hz,1H),4.71–4.63(m,2H),4.37–4 .31(m,2H),4.15(d,J=2.5Hz,2H),3.64–3.60(m,2H),2.97(s,3H),2.06–2.03(m,4H),2.01(t, J=2.5Hz,1H),1.76–1.72(m,1H),1.37(t,J=7.1Hz,3H),1.16–1.12(m,2H),0.97–0.92(m,2H).

[0166] Step 4-3: Preparation of ethyl 2-(2-(cyclopropanecarbamoyl)pyridin-4-yl)-4-((5-((7-(N-methyl-N-(prop-2-en-1-yl)sulfonamido)benzo[c][1,2,5]oxadiazol-4-yl)amino)pentyl)oxy)thiazole-5-carboxylic acid S9c:

[0167] The specific preparation method is the same as that for S9a, except that compound S8a is replaced with an equimolar amount of S8c. The characterization data for the resulting intermediate S9c are as follows:

[0168] 1 H NMR (400MHz, CDCl3) δ8.77(d,J=1.5Hz,1H),8.46(s,1H),8.36(d,J=5.2Hz,1H),7.90(d,J=8.0Hz,1H), 7.53(dd,J=5.2,1.5Hz,1H),6.13(d,J=8.0Hz,1H),6.08(t,J=5.6Hz,1H),4.64(t,J=6.1Hz,2H),4.36– 4.32(m,2H),4.16(d,J=2.4Hz,2H),3.50–3.45(m,2H),2.98(s,3H),2.01–1.99(m,1H),1.99–1.91(m,4 H),1.78–1.74(m,2H),1.65–1.60(m,1H),1.37(t,J=7.1Hz,3H),1.18–1.14(m,2H),0.98–0.93(m,2H).

[0169] Step 5-1: Preparation of 2-(2-(cyclopropanecarboxamide)pyridin-4-yl)-N-methyl-4-(3-((7-(N-methyl-N-(prop-2-yn-1-yl)sulfonyl)benzo[c][1,2,5]oxadiazol-4-yl)amino)propoxy)thiazole-5-carboxamide S10a:

[0170] Compound S9a (0.18 g, 0.28 mmol) was dissolved in 4.0 mL of a tetrahydrofuran / methanol (3 / 1, v / v) mixture. A 1.5 mol / L lithium hydroxide aqueous solution (2.0 mL) was slowly added dropwise under ice bath conditions. After the addition was complete, the reaction was continued in the ice bath for 5 min, then the reaction system was slowly raised to room temperature and stirred continuously for 6 h. After the reaction was complete, the organic solvent was removed by vacuum distillation, and the residue was diluted with an appropriate amount of ice water. The pH of the mixture was adjusted to 2-3 using 1.0 mol / L hydrochloric acid, and a large amount of solid was observed to precipitate. The solid was collected by filtration and used directly in subsequent reactions. The obtained solid was dissolved in DMF (2.0 mL), and DIPEA (90 μL, 0.560 mmol), TBTU (0.11 g, 0.34 mmol), and methylamine hydrochloride (23 mg, 0.34 mmol) were added sequentially. The reaction solution was stirred at room temperature for 24 h, diluted with water, and extracted with EA. The organic layer was dried with anhydrous sodium sulfate and then concentrated by rotary evaporation. The crude product was separated by rapid chromatography (PE / EA, 5 / 1, v / v) to obtain a light yellow solid S10a, with a yield of 46%.

[0171] The above intermediate S10a was subjected to NMR characterization, and the characterization data are as follows:

[0172] 1 H NMR (400MHz, DMSO-d6) δ11.03(s,1H),8.63–8.61(m,1H),8.51(s,1H),8.42(d,J=6.0Hz,1H ),7.81(d,J=8.2Hz,1H),7.55(d,J=4.8Hz,1H),7.51(d,J=5.2Hz,1H),6.34(d,J=8.2Hz,1H ),4.67(t,J=6.1Hz,2H),4.03–4.01(m,2H),3.59(t,J=2.4Hz,2H),3.01(t,J=2.4Hz,1H),2 .82(d,J=4.7Hz,3H),2.78(s,3H),2.28–2.21(m,2H),2.05–2.02(m,1H),0.86–0.83(m,4H).

[0173] Step 5-2: Preparation of 2-(2-(cyclopropanecarbamoyl)pyridin-4-yl)-N-methyl-4-(4-((7-(N-methyl-N-(prop-2-yn-1-yl)sulfanyl)benzo[c][1,2,5]oxadiazol-4-yl)amino)butoxy)thiazole-5-carboxamide S10b:

[0174] The specific preparation method is the same as that for S10a, except that S9a is replaced with an equimolar amount of S9b. The characterization data for the obtained intermediate S10b are as follows:

[0175] 1 H NMR (500MHz, CDCl3) δ8.82(s,1H),8.33(d,J=5.4Hz,1H),7.91(d,J=7.9Hz,1H),7.46(d,J=5.4 Hz,1H),6.98(d,J=4.9Hz,1H),6.16(d,J=8.0Hz,1H),6.02(s,1H),4.72(t,J=6.5Hz,2H),4.16( d,J=2.4Hz,2H),3.58–3.54(m,2H),3.01(d,J=4.9Hz,3H),2.99(s,3H),2.12–2.07(m,2H),2.0 4–2.00(m,2H),1.99(t,J=2.4Hz,1H),1.70–1.66(m,1H),1.15–1.12(m,2H),0.98–0.94(m,2H).

[0176] Step 5-3: Preparation of 2-(2-(cyclopropanecarboxamide)pyridin-4-yl)-N-methyl-4-((5-((7-(N-methyl-N-(prop-2-yn-1-yl)sulfonyl)benzo[c][1,2,5]oxadiazol-4-yl)amino)pentyl)oxy)thiazole-5-carboxamide S10c:

[0177] The specific preparation method is the same as that for S10a, except that S9a is replaced with an equimolar amount of S9c. The characterization data for the obtained intermediate S10c are as follows:

[0178] 1 H NMR (500MHz, CDCl3) δ8.83(s,1H),8.74(d,J=1.6Hz,1H),8.32(d,J=5.3Hz,1H),7.88(d,J=8.0Hz,1H),7.43 (dd,J=5.3,1.6Hz,1H),7.04–7.01(m,1H),6.11(d,J=8.0Hz,1H),6.07(t,J=5.5Hz,1H),4.65(t,J=6.7Hz,2H ),4.14(d,J=2.4Hz,2H),3.46–3.42(m,2H),2.99(d,J=4.9Hz,3H),2.96(s,3H),2.00(t,J=4.9Hz,1H),1.99– 1.94(m,2H),1.94–1.88(m,2H),1.69–1.66(m,1H),1.66–1.62(m,2H),1.14–1.10(m,2H),0.94–0.90(m,2H).

[0179] Example 1

[0180] This example provides a GSK-3βPTOTAC compound (compound A-1) and its preparation method. The compound is (E)-2-(2-(cyclopropanecarbamoyl)pyridin-4-yl)-4-(3-((7-(N-((1-((2,6-dimethoxy-4-(3-oxo-3-(6-oxo-3-)prop-1-en-1-yl)phenoxy)methyl)-1,1-dimethyl-1H-1λ 5 The compound, 2,3-triazol-4-yl)methyl)-N-methylsulfonyl)benzo[c][1,2,5]oxazol-4-yl)amino)propoxy)-N-methylthiazol-5-carboxamide)-N-methylthiazol-5-carboxamide, has the following structural formula:

[0181]

[0182] The specific preparation process is as follows:

[0183] Compound S10a (23 mg, 0.050 mmol), compound S3a (23 mg, 0.050 mmol), and tris(benzyltriazolylmethyl)amine (TBTA, 2.6 mg, 0.0050 mmol) were dissolved in tetrahydrofuran (1.0 mL). Then, aqueous solutions of copper sulfate (4.0 mg, 0.025 mmol) and sodium ascorbate (ASC, 12 mg, 0.060 mmol) (0.50 mL) were added sequentially. After stirring at room temperature for 6 h, the mixture was quenched with water, extracted with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent. The residue was purified by rapid column chromatography using dichloromethane / methanol (15 / 1, v / v) as eluent to give a yellow oily product A-1 in 22% yield.

[0184] The characterization data of the obtained compound A-1 are as follows:

[0185] 11H NMR (400 MHz, CDCl3) δ 8.73 (s, 1H), 8.59 (s, 1H), 8.32 (d, J = 5.2 Hz, 1H), 7.90 (d, J = 7.9 Hz, 1H), 7.86 (s, 1H), 7.69 (d, J = 15.5 Hz, 1H), 7.47–7.44 (m, 1H), 7.43 (d, J = 15.5 Hz, 1H), 7.01–6.96 (m, 1H), 6.96–6.91 (m, 1H), 6.83 (s, 2H), 6.24 (t, J = 5.8 Hz, 1H), 6.17 (d, J = 8.0 Hz, 1H), 6.07 (dt, J = 9.7, 1.8 Hz, 1H), 4.83 (t, J = 6.1 Hz, 2H), 4.70 (t, J = 6.7 Hz, 2H), 4.57 (s, 2H), 4.06 (t, J = 6.7 Hz, 2H), 4.01–3.98 (m, 2H), 3.92 (s, 6H), 3.67–3.63 (m, 2H), 3.01 (d, J = 4.9 Hz, 3H), 2.83 (s, 3H), 2.53–2.48 (m, 2H), 2.40–2.35 (m, 2H), 2.34–2.28 (m, 2H), 1.65–1.62 (m, 1H), 1.14–1.10 (m, 2H), 0.96–0.92 (m, 2H); 13 13C NMR (125 MHz, CDCl3) δ 172.8, 168.9, 165.9, 163.3, 161.1, 160.0, 153.5, 152.5, 148.8, 146.0, 145.7, 144.4, 143.7, 143.6, 141.6, 140.7, 139.2, 138.3, 131.1, 125.8, 123.9, 121.3, 115.7, 111.2, 110.9, 109.8, 105.4, 99.0, 69.0, 68.7, 56.2, 47.1, 45.7, 41.7, 40.1, 34.7, 30.8, 29.7, 26.5, 24.8, 15.9, 8.7; HR-ESI-MS for C 46 H 51 N 12 O 11 S2 ([M + H] + ) calcd: 1011.3236; found: 1011.3232.

[0186] Example 2

[0187] This example provides a GSK-3βPTOTAC compound (compound A-2) and its preparation method. The compound is (E)-2-(2-(cyclopropanecarboxamide)pyridin-4-yl)-4-((7-(N-((1-((2,6-dimethoxy-4-(3-oxo-3-(6-oxo-3,6-dihydropyridin-1(2H)-yl)prop-1-en-1-yl)phenoxy)methyl)-1,1,1-trimethyl-1H-1λ 6 The compound, 2,3-triazol-4-yl)methyl)-N-methylsulfonyl)benzo[c][1,2,5]oxadiazol-4-yl)amino)propoxy)-N-methylthiazol-5-carboxamide, has the following structural formula:

[0188]

[0189] The specific preparation process is as follows:

[0190] The specific preparation method is the same as that for A-1, except that compound 3a is replaced with an equimolar amount of 3b. The characterization data for the resulting compound A-2 are as follows:

[0191] 1 H NMR (400MHz, CDCl3) δ8.81(s,1H),8.33(d,J=5.4Hz,1H),7.93(d,J=8.0Hz,1H),7.86(s,1H),7.70(d,J=15.6Hz,1H),7.46–7.44(m ,1H),7.44(d,J=15.6Hz,1H),6.99–6.95(m,2H),6.83(s,2H),6.17(d,J=8.0Hz,1H),6.07(dt,J=9.7,1.8Hz,1H),5.97(s,1H),4.74 –4.69(m,4H),4.58(s,2H),4.07(t,J=6.5Hz,2H),4.01(t,J=5.5Hz,2H),3.93(s,6H),3.59–3.54(m,2H),3.02(d,J=4.9Hz,3H),2.8 6(s,3H),2.55–2.46(m,2H),2.35–2.29(m,2H),2.10(t,J=7.2Hz,2H),1.99(t,J=7.2Hz,2H),1.15–1.11(m,2H),0.97–0.94(m,2H); 13C NMR (100MHz, CDCl3) δ172.9,168.9,165.9,163.0,161.2,160.3,153.5,152.5,1 48.7,146.0,145.7,144.4,143.7,143.6,141.8,140.9,139.3,138.3,131.1,12 5.8,123.9,121.3,115.7,110.9,110.4,109.7,105.4,98.9,70.7,69.0,56.2,4 7.1,45.7,43.1,41.7,34.8,30.8,27.0,26.4,24.9,24.8,15.9,8.7; HR-ESI-MS forC 47 H 53 N 12 O 11 S2([M+H)) + )calcd:1025.3393; found:1025.3387.

[0192] Example 3

[0193] This example provides a GSK-3βPTOTAC compound (compound A-3) and its preparation method. The compound is (E)-2-(2-(cyclopropanecarbamoyl)pyridin-4-yl)-4-(3-((7-(N-((1-((2,6-dimethoxy-4-(3-oxo-3-(6-oxo-3-(6-oxo-3,6-dihydropyridin-1(2H)-yl)prop-1-en-1-yl)phenoxy)methyl)-1,1,1,1-tetramethyl-1H-1λ 7 The compound, 2,3-triazol-4-yl)methyl)-N-methylsulfanyl)benzo[c][1,2,5]oxazol-4-yl)amino)propoxy)-N-methylthiazol-5-carboxamide, has the following structural formula:

[0194]

[0195] The specific preparation process is as follows:

[0196] The specific preparation method is the same as that for A-1, except that compound 3a is replaced with an equimolar amount of 3c. The characterization data for the obtained compound A-3 are as follows:

[0197] 11H NMR (400 MHz, CDCl3) δ 8.80 (s, 1H), 8.28 (d, J = 5.4 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.86 (s, 1H), 7.70 (d, J = 15.5 Hz, 1H), 7.46 (s, 1H), 7.45 (d, J = 15.5 Hz, 1H), 7.02–7.01 (m, 1H), 7.00–6.95 (m, 1H), 6.84 (s, 2H), 6.12 (d, J = 8.0 Hz, 1H), 6.07 (dt, J = 9.7, 1.8 Hz, 1H), 5.91 (s, 1H), 4.73–4.71 (m, 4H), 4.59 (s, 2H), 4.07 (t, J = 6.5 Hz, 2H), 4.01 (t, J = 5.6 Hz, 2H), 3.93 (s, 6H), 3.48–3.43 (m, 2H), 3.02 (d, J = 4.9 Hz, 3H), 2.85 (s, 3H), 2.54–2.47 (m, 2H), 2.35–2.29 (m, 2H), 2.05–1.89 (m, 6H), 1.65–1.62 (m, 1H), 1.16–1.13 (m, 2H), 0.97–0.94 (m, 2H); 13 13C NMR (100 MHz, CDCl3) δ 172.9, <168.9>, <165.9>, <163.0>, <161.3>, <160.5>, <153.5>, <152.5>, <148.7>, <146.0>, <145.6>, <144.4>, <143.7>, <143.6>, <141.9>, <140.8>, <139.4>, <138.3>, <131.1>, <125.8>, <123.9>, <121.3>, <115.7>, <110.8>, <110.4>, <109.7>, <105.4>, <98.7>, <70.9>, <69.0>, <56.2>, <47.2>, <45.7>, <43.4>, <41.7>, <34.7>, <30.8>, <29.1>, <28.1>, <26.4>, <24.8>, <2 <23.2>>, <16.0>, <8.6>; HR-ESI-MS for C 48 H 55 N 12 O 11 S2 ([M + H] + ) calcd: 1039.3539; found: 1039.3546.

[0198] Example 4

[0199] This example provides a GSK-3βPTOTAC compound (compound A-4) and its preparation method. The compound is (E)-2-(2-(cyclopropanecarboxamide)pyridin-4-yl)-4-((7-(N-((1-((2,6-dimethoxy-4-(3-oxo-3-(6-oxo-3,6-dihydropyridin-1(2H)-yl)prop-1-en-1-yl)phenoxy)methyl)-1,1-dimethyl-1H-1λ 5 The compound, 2,3-triazol-4-yl)methyl)-N-methylsulfonyl)benzo[c][1,2,5]oxadiazol-4-yl)amino)butoxy)-N-methylthiazol-5-carboxamide, has the following structural formula:

[0200]

[0201] The specific preparation process is as follows:

[0202] The specific preparation method is the same as that for A-1, except that compound 10a is replaced with an equimolar amount of 10b. The characterization data for the resulting compound A-4 are as follows:

[0203] 1 H NMR (500MHz, CDCl3) δ8.73(s,1H),8.59(s,1H),8.32(d,J=5.2Hz,1H),7.91(d,J=8.0Hz,1H),7.72(s,1H),7.69(d,J=15.5Hz,1H),7.45–7.44(m ,1H),7.43(d,J=15.5Hz,1H),6.99–6.97(m,1H),6.96–6.93(m,1H),6.8 2(s,2H),6.24(t,J=5.7Hz,1H),6.17(d,J=8.0Hz,1H),6.07(d,J=9.7,1. 9Hz,1H),4.83(t,J=6.1Hz,2H),4.57(s,2H),4.50(t,J=7.3Hz,2H),4.0 7–4.04(m,4H),3.89(s,6H),3.67–3.63(m,2H),3.02(d,J=4.9Hz,3H),2. 85(s,3H),2.52–2.49(m,2H),2.39–2.34(m,2H),2.21–2.17(m,2H),1.8 1–1.77(m,2H),1.67–1.63(m,1H),1.13–1.10(m,2H),0.96–0.92(m,2H); 13C NMR (125MHz, CDCl3) δ172.9,169.0,165.9,163.3,161.1,160.0,153.5,152.5,1 48.8,145.9,145.7,144.5,143.8,143.4,141.6,140.7,139.2,138.8,130.8,12 5.8,123.0,121.2,115.7,111.2,110.9,109.8,105.4,99.0,72.3,68.7,56.2,5 0.2,45.6,41.7,40.1,34.8,28.6,27.2,26.9,26.5,24.8,15.9,8.7; HR-ESI-MS for C 47 H 53 N 12 O 11 S2([M+H)) + )calcd:1025.3393; found:1025.3387.

[0204] Example 5

[0205] This example provides a GSK-3βPTOTAC compound (compound A-5) and its preparation method. The compound is (E)-2-(2-(cyclopropanecarboxamide)pyridin-4-yl)-4-((5-((7-(N-((1-((2,6-dimethoxy-4-(3-oxo-3-(6-oxo-3,6-dihydropyridin-1(2H)-yl)prop-1-en-1-yl)phenoxy)methyl)-1,1-dimethyl-1H-1λ 5 The compound, 2,3-triazol-4-yl)methyl)-N-methylsulfonyl)benzo[c][1,2,5]oxadiazol-4-ylamino)pentyl)oxo)-N-methylthiazol-5-carboxamide, has the following structural formula:

[0206]

[0207] The specific preparation process is as follows:

[0208] The specific preparation method is the same as that for A-1, except that compound 10a is replaced with an equimolar amount of 10c. The characterization data for the resulting compound A-5 are as follows:

[0209] 11H NMR (400 MHz, CDCl3) δ 8.74 (s, 1H), 8.42 (s, 1H), 8.34 (d, J = 5.2 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.71 (s, 1H), 7.68 (d, J = 15.5 Hz, 1H), 7.46–7.44 (m, 1H), 7.43 (d, J = 15.5 Hz, 1H), 6.99–6.97 (m, 1H), 6.96–6.93 (m, 1H), 6.81 (s, 2H), 6.17 (d, J = 8.0 Hz, 1H), 6.13 (t, J = 5.6 Hz, 1H), 6.07 (d, J = 9.8 Hz, 1H), 4.85–4.82 (m, 2H), 4.57 (s, 2H), 4.38 (t, J = 7.3 Hz, 2H), 4.05 (t, J = 6.4 Hz, 2H), 4.01 (d, J = 6.4 Hz, 2H), 3.89 (s, 6H), 3.68–3.64 (m, 2H), 3.02 (d, J = 4.8 Hz, 3H), 2.85 (s, 3H), 2.52–2.48 (m, 2H), 2.39–2.34 (m, 2H), 2.02–1.97 (m, 2H), 1.86–1.79 (m, 2H), 1.56–1.53 (m, 2H), 1.14–1.10 (m, 2H), 0.95–0.93 (m, 2H); 13 13C NMR (150 MHz, CDCl3) δ 172.8, 169.0, 165.9, 163.3, 161.0, 160.0, 153.6, 152.5, 148.8, 145.9, 145.6, 144.4, 143.8(9), 143.8(7), 141.6, 140.7, 139.2, 139.1, 130.6, 125.8, 122.9, 121.0, 115.7, 111.2, 111.0, 109.8, 105.5, 99.0, 72.8, 68.7, 56.2, 50.5, 45.6, 41.7, 40.2, 34.7, 30.0, 29.4, 28.6, 26.5, 24.8, 23.0, 15.9, 8.7; HR-ESI-MS for C 48 H 54 N 12 O 11 S2Na ([M+Na] + ) calcd: 1061.3369; found: 1061.3367.

[0210] Comparative Example 1

[0211] The structural formula of the commercially available GSK-3β inhibitor S11 (CAS: 1702428-31-6) is as follows:

[0212] Comparative Example 2

[0213] The structure of the commercially available KEAP1 inhibitor S12 (CAS: 20069-09-4) is as follows:

[0214] Application Example 1: Cytotoxicity Study of Compound

[0215] 1. Experimental Methods

[0216] The cytotoxicity of the compounds in Examples 1-5 above was tested using the MTT assay.

[0217] The reagents used were dimethyl sulfoxide (Beyotime), tetramethyl azozazolium blue (Beyotime), DMEM medium (Gibco), and fetal bovine serum (Gibco).

[0218] Operation steps: (1) Take mouse hippocampal neurons (HT-22) in the logarithmic growth phase, digest them, and adjust the cell number concentration to 8×10⁻⁶. 4 / mL, seeded into 96-well plates at 100μL / well. Incubate overnight at 37℃ in a 5% CO2 cell culture incubator until cells adhere. (2) Aspirate the original culture medium and add different concentrations of compounds A-1, A-2, A-3, A-4 and A-5 provided by this invention to each group, with compound concentrations of 10μM. Use 0.05% DMSO as the control group and continue culturing in the cell culture incubator for 24h. (3) Add 10μL MTT solution to each well and incubate in the incubator for 4h. (4) Discard the culture medium and add 150μL LDMSO to each well, shake for 10min to fully dissolve formazan crystals. (5) Measure the absorbance value at 570nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0219] 2. Experimental Results

[0220] The results are as follows Figure 1 As shown, the cell viability of this series of compounds at a concentration of 10 μM is over 80%, with no obvious cytotoxicity, indicating that they have good biosafety.

[0221] Application Example 2: Environmental Sensitivity Assessment of Compounds

[0222] 1. Experimental Methods

[0223] The environmental sensitivity of the compounds in Examples 1 to 5 above was tested using a Horiba FluoroMax+ fluorescence spectrometer.

[0224] The reagents used were tetrahydrofuran (Anaiji), ultrapure water, and dimethyl sulfoxide (Anaiji).

[0225] Compounds A-1, A-2, A-3, A-4, and A-5 were dissolved in DMSO to obtain 1 mM stock solutions. The stock solutions of each compound were diluted to a concentration of 10 μM using different proportions of THF and H₂O. The fluorescence properties of the compounds were measured using a HoribaFluoroMax+ fluorescence spectrometer.

[0226] 2. Experimental Results

[0227] The results are as follows Figures 2-6 As shown, this series of compounds all exhibit excellent environmental sensitivity; the fluorescence gradually weakens as the H2O content in the system increases.

[0228] Application Example 3: Degradation Activity Test of Compound on GSK-3β

[0229] 1. Experimental Methods

[0230] The degradation ability of the compounds prepared in the above examples on GSK-3β was tested by Western blot experiments.

[0231] The reagents used were protein lysis buffer (White Shark), GSK-3β antibody (Beyotime), GAPDH antibody (Beyotime), HRP-labeled secondary antibody (Beyotime), and TBST solution (White Shark).

[0232] Operation steps: (1) Preparation of protein samples: (1-1) HT22 cells were seeded into 12-well plates and incubated overnight for about 24 hours. Then, all compounds prepared in the above examples (A-1 to A-5, 10 μM each) and A-1 (working concentrations of 1, 2.5, 5, 7.5 and 10 μM) were added to different wells. After the compounds were incubated for 18 hours, an appropriate amount of protein lysis buffer was added and the samples were collected. The samples were denatured at 100°C for 15 minutes to obtain protein sample 1; (1-2) HT22 cells were seeded into 12-well plates and incubated overnight for about 24 hours. Then, compound (A-1, 10 μM) was added to different wells. After the compounds were incubated for 12, 18, 24 and 36 hours, an appropriate amount of protein lysis buffer was added and the samples were collected. The samples were denatured at 100°C for 10 minutes to obtain protein sample 2. (2) Preparation of polyacrylamide separation gel: After standing, the prepared polyacrylamide stacking gel was added. Add protein samples extracted from HT22 cells (containing GSK-3β and internal reference protein GAPDH). (3) Perform electrophoresis at 70V for about 30 min in TGS buffer, then at 110V for about 60 min for protein separation. (4) After electrophoresis, transfer the protein to transfer buffer in the order of filter paper-gel-NC membrane-filter paper, and transfer at 230mA for about 90 min. After transfer, block the protein bands with 5% skim milk for about 1 h. After blocking, incubate the protein bands with a specific primary antibody overnight at 4°C. After incubation, recover the primary antibody and wash three times with TBST for 10 min each time. After washing, incubate the protein bands with the corresponding secondary antibody for about 2 h. Wash three times with TBST for 10 min each time. Finally, perform ECL luminescence and develop the color in a dark room, and visualize using a bioanalytical imaging system.

[0233] 2. Experimental Results

[0234] The results are as follows Figure 7 and 8 As shown. From Figure 7 As can be seen, compounds A-1, A-2, A-3, A-4, and A-5 all significantly reduced the expression level of GKS-3β at an addition amount of 10 μM. Compared with other compounds, A-1 exhibited better degradation activity. Figure 8 As can be seen, compound A-1 exhibits concentration-dependent GSK-3β degradation activity. Furthermore, the GSK-3β protein level after treatment with 10 μM compound A-1 showed a continuous decreasing trend from 12 to 18 h, reaching its lowest value at 18 h (a decrease of approximately 81.4%), and subsequently rebounded from 18 to 36 h. These results indicate that the degradation effect of compound A-1 on GSK-3β is most significant at 18 h.

[0235] Application Example 4: Imaging Activity Study of Compound

[0236] 1. Experimental Methods

[0237] The imaging activity of compound A-1 in Example 1 was tested using inverted fluorescence imaging.

[0238] The reagents used were Hoechst 33342 (Beyotime), PBS buffer (White Shark), DMEM medium (Gibco), and fetal bovine serum (Gibco).

[0239] Operation steps: (1) Take mouse hippocampal neurons (HT-22) in the logarithmic growth phase, digest them, and adjust the cell number concentration to 5×10⁻⁶. 4 / mL, seeded into 12-well plates at 1.5mL / well. Incubate overnight at 37°C in a 5% CO2 cell culture incubator until cells adhere. (2) Aspirate the original culture medium, add compound A-1 provided by this invention to one group to a final concentration of 10μM. Continue culturing in the cell culture incubator for 3, 9 and 18 h. (3) Incubate another group with GSK-3β inhibitor S11 (10μM) and S12 for 9 h. (4) Discard the culture medium, wash the cells carefully 2-3 times with PBS buffer and add 400μL PBS. (5) Observe the cells using a fluorescence microscope (OLYMPUS, IX73P2F).

[0240] 2. Experimental Results

[0241] The results are as follows Figure 9 As shown, when cells were treated with 10 μM compound A-1, the fluorescence intensity gradually decreased with prolonged incubation time, while the fluorescence of the pretreated group with inhibitors S11 / S12 showed no significant change. The decrease in fluorescence intensity in the compound A-1 treatment group is consistent with the GSK-3β degradation results shown by Western blotting, and this consistency further indicates that the fluorescence change is correlated with the degradation process of GSK-3β over time.

[0242] Study on the neuroprotective activity of compound in application example 5

[0243] 1. Experimental Methods

[0244] The survival rate of HT-22 cells after administration of different concentrations of compound A-1 under glutamate stimulation was determined by the MTT assay, thereby evaluating the neuroprotective effect of compound A-1 on HT22 cells.

[0245] The reagents used were dimethyl sulfoxide (Beyotime), tetramethyl azozazolium blue (Beyotime), DMEM medium (Gibco), glutamate (Glu), and fetal bovine serum (Gibco).

[0246] Operation steps: (1) Take mouse hippocampal neurons (HT-22) in the logarithmic growth phase, digest them, and adjust the cell number concentration to 8×10⁻⁶.4 / mL, seeded into 96-well plates at 100μL / well. Incubate overnight at 37℃ in a 5% CO2 cell culture incubator until cells adhere. (2) Aspirate the original culture medium and add culture medium containing 10mMGlu, incubate for 24h. (3) Add culture medium containing compound A-1 (1, 5 and 10μM), control compound S11 (10μM), S12 (10μM) and their combination drug group (5μM each of S11 and S12). Add culture medium without A-1 (containing 0.5% DMSO) to the blank control group. (4) Add 10μL MTT solution to each well and incubate for 4h in an incubator. (5) Discard the culture medium, add 150μL LDMSO to each well, shake for 10min to fully dissolve formazan crystals. (6) Measure the absorbance value at 570nm using a multi-functional microplate reader.

[0247] 2. Experimental Results

[0248] The results are as follows Figure 10 As shown, under Glu stimulation, the cell viability in the model group decreased to approximately 40%, while the cell viability in the comparative groups of compounds S11, S12, and Comb was all below 60%. In contrast, the A-1 administration group significantly alleviated Glu-induced neurotoxicity in a dose-dependent manner between 1 and 10 μM. At 10 μM, compound A-1 could achieve cell viability of over 85%, and its neuroprotective efficacy was significantly higher than that of compound S11. These results indicate that compound A-1 can reverse Glu-induced cellular neurotoxicity and increase cell viability.

[0249] In summary, this invention designed and synthesized a series of PROTAC degrading agents targeting GSK-3β protein and using KEAP1 as the E3 ligase, with a fluorescent group introduced onto the linker strand. This PROTAC molecule not only effectively degrades GSK-3β protein, but also allows for real-time monitoring of GSK-3β protein levels in living cells via fluorescent labeling, enabling visualization of the degradation process and its application in GSK-3β protein imaging products. In an Alzheimer's disease (AD) model, this PROTAC molecule exhibited significant neuroprotective activity, enhancing its therapeutic potential. In conclusion, the fluorescently labeled PROTAC degrading agent developed in this invention expands the range of therapeutic tools available for studying the role of GSK-3β in the pathological mechanisms of AD.

[0250] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A compound and its pharmaceutically acceptable salt, its crystal form, its solvate, its stereoisomer, or its isotopically substituted form, characterized in that: The structure of the compound is shown in Formula A: In the formula, m and n are independently selected from any integers in 3-5.

2. The compound according to claim 1 and its pharmaceutically acceptable salt, crystal form, solvate, stereoisomer, or isotopically substituted form thereof, characterized in that: The compound is selected from one of the following structural formulas:

3. A method for preparing a compound, characterized in that: The structure of the compound is shown in Formula A. The preparation method includes the following steps: reacting precursor compounds S3 and S10 to obtain the compound shown in Formula A. The structures of formula A and precursor compounds S3 and S10 are as follows: In the formula, m and n are independently selected from any integers in 3-5.

4. The preparation method according to claim 3, characterized in that: The preparation steps of the precursor compound S3 include: Compound S1 and compound S1-1 are reacted as raw materials to obtain compound S2; compound S2 undergoes an azide reaction to obtain the final product; the structural formulas of compounds S1, S1-1, and S2 are as follows: And / or, The preparation steps of the precursor compound S10 include: Compound S6 reacts with compound S6-1 to give compound S7; compound S7 undergoes selective deprotection of its Boc protecting group to give compound S8; compound S8 reacts with compound S5 to give compound S9; compound S9 undergoes amidation to give compound S10; wherein the structural formulas of compounds S5, S6, S6-1, S7, S8, and S9 are as follows:

5. The use of the compound of claim 1 or 2, its pharmaceutically acceptable salt, its crystal form, its solvate, its stereoisomer, or its isotopically substituted form in the preparation of GSK-3β protein degradation and / or GSK-3β protein imaging products.

6. The use of the compound of claim 1 or 2, its pharmaceutically acceptable salt, its crystal form, its solvate, its stereoisomer, or its isotopically substituted form, in the preparation of reagents for the treatment and / or prevention of diseases associated with GSK-3β protein.

7. The application according to claim 6, characterized in that: The GSK-3β protein-related diseases include at least one of Alzheimer's disease, Parkinson's disease, bipolar disorder, type 2 diabetes, or diabetic nephropathy.

8. The use of the compound of claim 1 or 2, its pharmaceutically acceptable salt, its crystal form, its solvate, its stereoisomer, or its isotopic substituted form in the preparation of neuroprotective products.

9. A pharmaceutical composition, characterized in that: The composition comprises one or more of the compounds as described in claim 1 or 2, their pharmaceutically acceptable salts, crystal forms, solvates, stereoisomers, or isotopically substituted forms thereof.

10. A reagent kit, characterized in that: The kit comprises the compound as described in claim 1 or 2, its pharmaceutically acceptable salt, its crystal form, its solvate, its stereoisomer, or its isotopic substituted form.

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