A protac compound targeting degradation of pak4 and preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a PAK4-targeted degradation PROTAC compound and a preparation method and application thereof. The application provides a PAK4-targeted degradation PROTAC compound shown in formula I or formula II or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, nitroxide, hydrate, solvate, metabolite or prodrug thereof. In vitro activity test results show that the compound is a PAK4-degrading PROTAC molecule. Compared with existing PAK4 inhibitors, the small-molecule compound involved in the application has the ability to degrade PAK4 protein and then rapidly inhibit tumor migration and invasion, and has the potential to become an effective treatment for malignant tumors.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a PROTAC compound that targets and degrades PAK4, its preparation method, and its application. Background Technology

[0002] p21-activated kinase 4 (PAK4) is a serine / threonine protein kinase that plays a crucial role in cytoskeleton remodeling and cell motility regulation. As an important downstream effector molecule of Cdc42, PAK4 often exhibits amplification, mutation, and upregulated expression levels and activities during the development of various tumors, such as non-small cell lung cancer, breast cancer, and gastric cancer. Under normal physiological conditions, PAK4 precisely regulates cytoskeleton formation and cell cycle progression; its abnormal expression can trigger various pathological processes. PAK4 is a relevant target in the development and functional maintenance of the nervous system, and its inhibition is associated with the pathological progression of neurodegenerative diseases. PAK4 regulation is also related to cardiovascular diseases. In adult tissues, PAK4 is significantly associated with cancer, exhibiting aberrant expression in various tumors, providing a target for cancer treatment. PAK4 regulation of the cytoskeleton is closely related to cancer cell metastasis. It can affect the cytoskeleton and migration through multiple pathways. PI3K / AKT plays a key role in the PAK4 pathway. PAK4 overexpression promotes tumor formation. Therefore, designing PAK4 inhibitors that block its signaling pathway is expected to become a new strategy for cancer treatment.

[0003] PROTAC (Proteolysis-Targeting Chimeras) molecules have significant advantages over traditional drugs. Therefore, designing PROTAC compounds that target the degradation of PAK4 is expected to synthesize highly active and highly selective PAK4 inhibitors.

[0004] Current therapies targeting the regulation of PAK4 protein kinase are still focused on competitive inhibitors of PAK4. Therefore, designing PROTAC compounds that target the degradation of PAK4 is expected to synthesize PAK4 inhibitors with higher activity and selectivity. Summary of the Invention

[0005] The present invention aims to provide a method for preparing and using a PROTAC compound that targets and degrades PAK4, or its pharmaceutically acceptable salts, stereoisomers, geometric isomers, tautomers, nitrides, hydrates, solvates, metabolites, or prodrugs. In vitro activity assays show that the compound of the present invention is a PROTAC molecule capable of degrading PAK4. Compared with existing PAK4 inhibitors, the small molecule compound involved in this invention has the ability to degrade PAK4 protein, thereby rapidly inhibiting tumor migration and invasion, and has the potential to become an effective treatment for malignant tumors.

[0006] The technical solution of the present invention is as follows:

[0007] The primary objective of this invention is to provide a PROTAC compound (PAK4-PROTAC compound) that targets the degradation of PAK4 as shown in Formula I or Formula II, or its pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, nitride, hydrate, solvate, metabolite, or prodrug.

[0008]

[0009] Wherein: E3 ligase ligand refers to the ligand molecule that binds to E3 ligase, and E3 ligase includes CRBN, MDM2, IAP, DCAF or RNF, wherein the E3 ligase ligand includes:

[0010] The linker is a linking group, selected from any one of the following groups:

[0011]

[0012]

[0013] Furthermore, the PAK4-PROTAC compound provided by the present invention is a compound as shown below or its stereoisomers, geometric isomers, tautomers, nitrides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs.

[0014]

[0015]

[0016]

[0017] The pharmacologically or physiologically acceptable salts mentioned in this invention refer to the addition salts formed by the PROTAC compound that targets and degrades PAK4 and a pharmacologically or physiologically acceptable acid or base.

[0018] The present invention also proposes a pharmaceutical composition comprising the PAK4-PROTAC compound or its stereoisomers, geometric isomers, tautomers, nitrides, hydrates, solvates, metabolites, pharmaceutically acceptable salts, or prodrugs;

[0019] The pharmaceutical composition further includes pharmaceutically acceptable carriers, excipients, diluents, excipients, mediators, or combinations thereof;

[0020] The pharmaceutical composition is an injection, an oral preparation, or a mucosal delivery preparation;

[0021] The pharmaceutical composition further includes other drugs with therapeutic or preventative effects against tumors. The PAK4-related disease is lung cancer, specifically non-small cell lung cancer.

[0022] The use of the PROTAC compound that targets and degrades PAK4, or a pharmaceutical composition comprising such a PROTAC compound, in an antitumor drug. The tumor is breast cancer, pancreatic cancer, colon cancer, lung cancer, gastric cancer, or ovarian cancer. Further, the tumor is a tumor with high PAK4 expression, or a PAK4 inhibitor-resistant tumor. Even further, the tumor is a solid lung cancer tumor.

[0023] This invention also proposes a synthetic route for PROTAC compounds targeting the degradation of PAK4, as shown in general formula I or general formula II, specifically including the following steps:

[0024] Based on the designed compounds, pomalidomide, 5-aminopomalidomide, 5-fluoro anhydride phthalide, and N-methylpomalidomide were used as ligands. The compounds were synthesized through nucleophilic substitution, acylation, debonding of the boc group, p-methylbenzenesulfonate formation, azidation, reduction, and click chemistry, according to the structural characteristics of the compounds.

[0025] The benefits of this invention are:

[0026] This invention successfully designed and synthesized a PROTAC compound with significant PAK4 degradation activity. The inventors confirmed the PAK4 degradation effect of the provided PROTAC degrading compound through Western blotting experiments. Further experiments, including in vitro enzyme activity assays, CCK8 cell viability assays, scratch assays, Transwell cell migration and invasion assays, and in vivo pharmacokinetic experiments in mice, demonstrated that this PROTAC degrading compound has the ability to degrade PAK4 protein and thus rapidly inhibit tumor migration and invasion, showing potential as an effective treatment for malignant tumors. Attached Figure Description

[0027] Figure 1These are the results of screening for PAK kinase activity by compounds PS1-PS36 in the embodiments of the present invention. (A) Experimental results of compounds PS1, PS2, PS3, PS4, and PS5 degrading PAK4 protein in A549 cells. (B) Experimental results of compounds PS6, PS7, PS8, PS9, and PS10 degrading PAK4 protein in A549 cells. (C) Experimental results of compounds PS11, PS12, and PS13 degrading PAK4 protein in A549 cells. (D) Experimental results of compounds PS14 and PS15 degrading PAK4 protein in A549 cells. (E) Experimental results of compounds PS23, PS17, PS18, PS19, and PS20 degrading PAK4 protein in A549 cells. (F) Experimental results of compounds PS21, PS22, and PS16 degrading PAK4 protein in A549 cells. (G) Experimental results of compounds PS24, PS25, and PS26 degrading PAK4 protein in A549 cells. (H) Experimental results of compounds PS27 and PS28 degrading PAK4 protein in A549 cells. (I) Experimental results of compounds PS29, PS30, and PS31 degrading PAK4 protein in A549 cells. (J) Experimental results of compounds PS32, PS33, and PS34 degrading PAK4 protein in A549 cells. (K) Experimental results of compounds PS35 and PS36 degrading PAK4 protein in A549 cells.

[0028] Figure 2 This indicates that the degradation of PAK4 protein by compound PS21 in the embodiments of the present invention is concentration-dependent. (A) The degradation of PAK4 induced by PS21 in a concentration-dependent manner is A549-induced; (B) The degradation of PAK4 induced by PS21 in a concentration-dependent manner is MDA-MB-231-induced.

[0029] Figure 3 This invention demonstrates that compound PS21 induces PAK4 degradation via a cereblon (CRBN)-dependent and proteasome-dependent mechanism. A549 cells were pretreated with 5 μM of a PAK4 inhibitor, pomalidomide, or MG132 for 4 hours, followed by treatment with 2 μM PS21 for 14 hours. PAK4 levels were then detected by Western blotting, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as an internal control.

[0030] Figure 4 These are the scratch test results of compound PS21 in the embodiments of the present invention. (A) Scratch test results of PS21 on A549 cell line; (B) Scratch test results of PS21 on H1299 cell line.

[0031] Figure 5The results of the anti-tumor cell migration and invasion effects of compound PS21 in the embodiments of the present invention are as follows: (A) Transwell experiment results of PS21 on A549, MDA-MB-231, and H1299 cell lines; (B) Statistical graph of the results of the migration experiments of different concentrations of PS21 inhibiting A549, MDA-MB-231, and H1299 cell lines; (C) Statistical graph of the results of the migration experiments of different concentrations of PS21 inhibiting A549, MDA-MB-231, and H1299 cell lines. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0033] The following examples depict methods for preparing some of the compounds described herein. It should be understood that the following methods, as well as other methods known to those skilled in the art, are applicable to the preparation of all compounds described herein. The examples are intended to illustrate, but not limit, the scope of the invention.

[0034] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0035] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0036] Example 1

[0037] Preparation method and structural confirmation of PROTAC for targeted degradation of PAK4.

[0038] Synthesis route:

[0039] Synthesis of compound CPL042

[0040]

[0041] Chemical reagents and reaction conditions: (a) Trifluoroacetic anhydride, N,N-dimethylformamide, reaction at room temperature; (b) 4M sodium hydroxide solution, reflux; (c) Oxalic anhydride, dichloromethane, reaction at room temperature; (d) tert-butanol, potassium tert-butoxide; (e) Cesium carbonate, 2-amino-4-chloropyrimidine, N,N-dimethylformamide, 80℃; (f) Trifluoroacetic acid, dichloromethane, reaction at room temperature; (g) HBTU, piperazine, triethylamine, reaction at room temperature; (h) 1-ethynyl-1-cyclohexanol, tetrakis(triphenylphosphine)palladium, cuprous iodide, triethylamine, N,N-dimethylformamide, 80℃.

[0042] Synthesis of compound CPL042b

[0043] 10.00 g (50.1 mmol) of 6-bromoindole was added to a 100 mL flask, followed by 50 mL of N,N-dimethylformamide. The mixture was sonicated to completely dissolve the 6-bromoindole. The flask was then placed in an ice bath, and trifluoroacetic anhydride (8.60 mL, 61.20 mmol) was added dropwise over half an hour with stirring. After the addition was complete, the reaction mixture was transferred to room temperature and allowed to react for 1.5 h. TLC monitoring was performed until the starting material completely disappeared. The reaction mixture was then poured into 500 mL of water, resulting in the precipitation of a large amount of off-white solid. The mixture was thoroughly stirred and filtered to obtain the off-white solid product. This product was dried by infrared spectroscopy to obtain the target product, with a yield of 92%. ESI-MS (m / z): 292.8 [M+H] + .

[0044] Synthesis of compound CPL042c

[0045] 9.00 g (30.82 mmol) of 1-(6-bromo-1H-indol-3-yl)-2,2,2-trifluoroethyl ketone was placed in a 250 mL flask, and 100 mL of 20% NaOH solution was added. The mixture was sonicated until the solid was completely dispersed. The reaction mixture was then heated under reflux with stirring for 4 h. TLC monitoring was performed until the starting material spot completely disappeared. After the reaction was complete, the reaction mixture was cooled to room temperature in an ice bath. The reaction solution was extracted with 3 × 200 mL of dichloromethane to remove impurities. The organic layer was discarded, and the aqueous layer was retained. The aqueous layer was then adjusted to pH 5-6 with 4 M hydrochloric acid, resulting in the precipitation of a large amount of solid. The mixture was filtered to obtain the solid product. The solid layer was washed with water until the pH was neutral, and the solid product was dried to obtain the final product, with a yield of 84%. ESI-MS (m / z): 237.4 [MH] - .

[0046] Synthesis of compound CPL042e

[0047] 5.00 g (20.92 mmol) of 6-bromoindole-3-carboxylic acid was placed in a 500 mL flask, and 40 mL of dichloromethane was added. The mixture was sonicated until the solid was fully dispersed. After reacting in an ice bath, a drying tube was connected, and 6.64 g (52.3 mmol) of oxalyl chloride solution dissolved in 40 mL of dichloromethane was added dropwise over 15 min with stirring. After the addition was complete, the reaction mixture was transferred to room temperature and reacted for 2 h. TLC was used to monitor the complete conversion of the starting material. The unreacted oxalyl chloride solution in dichloromethane was removed by rotary evaporation until the system was completely dry. This compound is highly susceptible to moisture and deterioration; therefore, the subsequent reaction step can be carried out without purification.

[0048] The product solid from the previous step was transferred to a solution of potassium tert-butoxide (2.58 g, 23.01 mmol) in tert-butanol (40 mL). The reaction was allowed to proceed for 1 h until the system was completely clear. TLC analysis confirmed complete conversion of the starting material. The reaction mixture was then poured into 500 mL of water, resulting in the precipitation of a large amount of white solid. This solid was filtered and washed to obtain the crude product. The crude product was purified by normal-phase silica gel column chromatography (PE:EA = 5:1, v / v) to obtain the purified product. The two-step yield was 85%, and the ESI-MS (m / z) was 296.7 [M+H]. + .

[0049] Synthesis of compound CPL042f

[0050] 6-Bromo-1H-indole-3-carboxylic acid tert-butyl ester (5.00 g, 16.91 mmol) was dissolved in 40 mL of N,N-dimethylformamide and sonicated until the solid starting material was completely dissolved. Cesium carbonate (5.79 g, 17.75 mmol) was added with stirring, and the mixture was stirred at room temperature for half an hour. Then, 2-amino-4-chloropyrimidine (2.41 g, 18.60 mmol) was added. The system was then heated to 80 °C and stirred for 8 hours until the starting material, 1-(2-aminopyrimidin-4-yl)-6-bromo-1H-indole-3-carboxylic acid tert-butyl ester, was completely converted. After the starting material spot disappeared as monitored by TLC, the reaction mixture was poured into 500 mL of water with stirring. A large amount of white solid precipitated out, which was then filtered to obtain the crude product. The crude product was dissolved in dichloromethane:methanol = 20:1 and purified by silica gel column chromatography (dichloromethane:MeOH = 20:1, v / v) to obtain a purified white product in 86% yield. ESI-MS (m / z): 388.8 [M+H] + .

[0051] Synthesis of compound CPL042g

[0052] Intermediate 1-(2-aminopyrimidin-4-yl)-6-bromo-1H-indole-3-carboxylic acid tert-butyl ester (3.00 g, 7.71 mmol) was added to a 250 mL flask, followed by 50 mL of dichloromethane until completely dissolved. Over 15 min, 10 mL of trifluoroacetic acid dissolved in 50 mL of dichloromethane was added with stirring, and the reaction was allowed to proceed at room temperature. The reaction was monitored by TLC until the starting material disappeared. The reactants were then poured into a 20% sodium hydroxide solution and extracted with 3 × 200 mL of ethyl acetate, retaining the aqueous layer. The pH of the aqueous layer was adjusted to 5-6, precipitating a large amount of white solid. The solid was filtered, and the filter cake was washed with water to pH 7.0 to obtain the target product in 80% yield. ESI-MS (m / z): 330.6 [MH] - .

[0053] Synthesis of compound CPL042h

[0054] Intermediate 1-(2-aminopyrimidin-4-yl)-6-bromo-1H-indole-3-carboxylic acid (2.00 g, 6.04 mmol) was added to 20 mL of N,N-dimethylformamide. The mixture was sonicated until completely dissolved. Then, N-boc piperazine (1.15 g, 6.16 mmol), HBTU (2.34 g, 6.16 mmol), and 1 mL of triethylamine were added with stirring. The reaction mixture was stirred at room temperature for 3 h, and TLC was used to monitor the reaction until the starting material spot completely disappeared. After the reaction was complete, the mixture was poured into 250 mL of water, and a white solid precipitated. After filtration, the boc-protected intermediate was obtained. This intermediate was filtered, washed, dried, and then removed. 25 mL of hydrogen chloride-ethyl acetate solution was added to remove the boc protecting group, yielding the hydrochloride salt of the target product. The intermediate was added to 1M NaOH and stirred. Extraction was performed with 3 × 200 mL of ethyl acetate, followed by rotary evaporation and vacuum drying to obtain the target product (1-(2-aminopyrimidin-4-yl)-6-bromo-1H-indol-3-yl)(piperazin-1-yl) methyl ketone, yield 90%, ESI-MS (m / z): 401.2 [M+H]. + .

[0055] Synthesis of compound CPL042

[0056] The intermediate 1-(2-aminopyrimidin-4-yl)-6-bromo-1H-indole-3-carboxylic acid (2.00 g, 4.97 mmol) was dissolved in 20 mL of ultradry N,N-dimethylformamide and 1 mL of ultradry triethylamine, followed by the addition of etynylcyclohexanol (1.23 g, 9.94 mmol). The reaction mixture was sonicated until completely dissolved. The air in the system was replaced with a nitrogen balloon. Under nitrogen protection, tetrakis(triphenylphosphine)palladium (287.16 mg, 0.2485 mmol) and cuprous iodide (189.30 mg, 0.994 mmol) were added. The reaction mixture was stirred at 80 °C for 12 hours, and TLC monitoring was performed until the starting material disappeared. After the reaction was complete, the reaction mixture was poured into 200 mL of water, and 200 mL of ethyl acetate was added and stirred thoroughly. The mixture was then filtered through diatomaceous earth, and the organic layer was separated using a separatory funnel. The organic layer was then separated by silica gel column chromatography with a mobile phase of dichloromethane:MeOH = 8:1 (v / v). The yield was 60%, and the ESI-MS (m / z) result was 445.5 [M+H]. + .

[0057] Synthetic routes for compounds PS1, PS2, PS3, PS4, PS5, PS6, and PS15

[0058]

[0059] Chemical reagents and reaction conditions: (a) tetrahydrofuran, bromosubstituted acyl chloride, reflux; (b) potassium iodide, CPL042, triethylamine, N,N-dimethylformamide, 80℃.

[0060] Synthesis of compound 2a

[0061] The starting material, 4-amino-2-(2,6-dioxadiazine-3-yl)isoindoline-1,3-dione (2.00 g, 7.32 mmol), was added to 20 mL of tetrahydrofuran and stirred until completely dissolved. Then, 5-bromopentanoyl chloride (7.30 g, 36.6 mmol) was added, and the mixture was heated under reflux until the starting material spot disappeared. After the reaction was complete, the reaction solution was evaporated to dryness, dissolved in ethyl acetate, and then added to silica gel for column chromatography. The product was separated by column chromatography to obtain a white product in 72% yield. ESI-MS (m / z): 424.5 [M+H] + Compounds 2b-2g were synthesized using the same method as 2a, with yields of 48-75%.

[0062] Synthesis of compound PS1

[0063] The starting material 5-bromo-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl]pentanamide (200 mg, 0.46 mmol) and CPL042 (204.5 mg, 0.46 mmol) were dissolved in 10 mL of N,N-dimethylformamide. Potassium iodide (76.36 mg, 0.46 mmol) and 0.5 mL of triethylamine were added, and the mixture was stirred at 80 °C for 5 h. The reaction was monitored by TLC until one of the starting material spots completely disappeared. After the reaction was completed, the reaction mixture was poured into 100 mL of water, and extracted with 3 × 50 mL of ethyl acetate. The organic layers were combined, concentrated, and then the target product was separated by thin-layer chromatography using dichloromethane:MeOH = 5:1 (v / v). The purified product was a white solid with a yield of 12%.

[0064] Compounds PS2, PS3, PS4, PS5, PS6, and PS15 were synthesized using the same method as PS1.

[0065] Synthetic routes for compounds PS7, PS9, PS10, PS11, PS12, PS13, and PS16.

[0066]

[0067] Chemical reagents and reaction conditions: (a) 3-amino-2,6-piperidinidone hydrochloride, sodium acetate, acetic acid, reflux; (b) N-boc-1,6-hexanediamine, potassium iodide, N,N-dimethylformamide, triethylamine, 80℃; (c) 1M hydrogen chloride-ethyl acetate solution; (d) bromosubstituted fatty acid, HBTU, triethylamine, N,N-dimethylformamide, reaction at room temperature; (e) potassium iodide, CPL042, N,N-dimethylformamide, triethylamine, 80℃.

[0068] Synthesis of Compound 4

[0069] In a 100 mL flask, 4-fluoroisoindoline-1,3-dione (3.00 g, 18.07 mmol), 3-aminopiperidine-2,6-dione hydrochloride (3.27 g, 19.88 mmol), and sodium acetate (1.92 g, 23.47 mmol) were added sequentially, followed by 30 mL of acetic acid. The mixture was stirred thoroughly until combined, and then heated to 110 °C. The reaction was carried out for 8 h, monitored by TLC until the starting material was completely converted. After the reaction was complete, the mixture was evaporated to dryness to remove the acetic acid, and then purified by column chromatography to obtain a white solid product, compound 4, in 59% yield. 1 H NMR (400MHz, DMSO-d6) δ11.12(s,1H),7.96–7.91(m,1H),7.78(d,J=7.3Hz,1H),7.72(t,J=8.9Hz,1H),5.15(dd,J=12. 8,5.4Hz,1H),2.88(ddd,J=17.1,13.9,5.5Hz,1H),2.66–2.50(m,2H),2.11–2.01(m,1H).MS(ESI)m / z(%):299.8[M+Na] + .

[0070] Synthesis of Compound 5

[0071] Intermediate 2-(2,6-dioxadiazine-3-yl)-4-fluoroisoindoline-1,3-dione (1 equivalent v) was placed in ultradry DMSO, and Boc-N-hexanediamine (1.2 equivalent v) and DIPEA (2 equivalent v) were added. The reaction system was heated to 90 °C and reacted for 8 h. The reaction system was monitored by TLC until the starting material was completely converted. After the reaction was completed, the reaction solution was poured into water and extracted three times with ethyl acetate. The organic layers were then combined and evaporated to dryness with silica gel for column chromatography. Intermediate 5 was obtained by column chromatography. MS (ESI) m / z (%): 473.8 [M+H] + .

[0072] Synthesis of Compound 6

[0073] The intermediate tert-butyl(6-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)hexyl)carbamate was placed in 20 ml of ethyl hydrochloride and stirred. The reaction was monitored by TLC until the starting material was completely converted. After 3 h, the mixture was filtered and dried under vacuum to obtain the target product. MS (ESI) m / z (%): 372.3 [M+H]+.

[0074] Synthesis of compound 7a

[0075] The reaction conditions were the same as CPL0 42h, but the starting materials were changed to compound 6 and 5-bromopentanoic acid. The MS (ESI) m / z (%) of the product was 521.1 [M+H]+.

[0076] Synthesis of compound PS7

[0077] The reaction conditions were the same as for PS1, but the reactants were replaced with 7a and CPL042. The product was a bright yellow solid with a yield of 32%.

[0078] Compounds PS9, PS10, PS11, PS12, PS13, and PS16 were synthesized using the same method as PS7.

[0079] Synthetic route of compound PS23.

[0080]

[0081] Chemical reagents and reaction conditions: (a) N,N-dimethylformamide, N-Boc glycine, HBTU, triethylamine, 80℃; (b) 1M hydrogen chloride-ethyl acetate solution, reaction at room temperature; (c) bromosubstituted fatty acids, HBTU, triethylamine, N,N-dimethylformamide, reaction at room temperature; (d) potassium iodide, CPL042, triethylamine, N,N-dimethylformamide, 80℃.

[0082] Synthesis of Compound 9

[0083] In a 50 mL flask, 5-amino-2-(2,6-dioxadiazine-3-yl)isoindoline-1,3-dione (1 equivalent), HBTU (1.05 equivalent), and Boc glycine (1.01 equivalent) were added sequentially. Then, 20 mL of N,N-dimethylformamide was added until the solid was completely dissolved. 1 mL of triethylamine was added, and the mixture was stirred at room temperature for 5 min. The temperature was then increased to 50 °C and stirred again. TLC monitoring showed that the starting material had disappeared. The reaction mixture was then poured into 200 mL of water, and the aqueous phase was extracted with ethyl acetate at 50 × 3. The combined organic phases were evaporated to dryness to obtain the target product, with a yield of 72%. ESI-MS (m / z): 431.1 [M+H] + .

[0084] Synthesis of Compound 10

[0085] The intermediate tert-butyl{2-{2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl]amino}-2-oxoethyl}carbamate was placed in 20 mL of ethyl hydrochloride and stirred at room temperature for 3 h. After the starting material spot completely disappeared, the reaction mixture was filtered to obtain the solid, which is compound 10. This compound can be used for the next reaction without purification. ESI-MS (m / z): 330.3 [M+H] + .

[0086] Synthesis of Compound 11

[0087] The reaction conditions were the same as CPL0 42h, but the starting materials were replaced with 2-amino-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl]acetamide and 10-bromodecanoic acid, with a yield of 95%. ESI-MS (m / z): 562.4 [M+H] + .

[0088] Synthesis of compound PS23

[0089] The reaction conditions were the same as PS1, but the starting materials were replaced with CPL042 and compound 11. The product was a bright yellow solid with a yield of 24%.

[0090] Synthetic route of compound PS8

[0091]

[0092] Chemical reagents and reaction conditions: (a) Chloroacetyl chloride, N,N-dimethylformamide, triethylamine, 50℃; (b) Sodium azide, triethylamine, tetrahydrofuran, room temperature reaction; (c) Hydrogen, Pd / C, tetrahydrofuran; (d) Brominated fatty acids, HBTU, triethylamine, N,N-dimethylformamide, room temperature reaction; (e) Potassium iodide, CPL042, triethylamine, N,N-dimethylformamide, 80℃.

[0093] Synthesis of Compound 12

[0094] Pomalidomide (1 equiv) was dissolved in dry DMSO, followed by the sequential addition of triethylamine (2 equiv) and chloroacetyl chloride (5 equiv). The mixture was stirred until completely dissolved, then heated to 80 °C and reacted for 5 h. TLC was used to monitor the reaction until the starting material was completely converted. After the reaction was complete, the reaction solution was poured into water and stirred thoroughly. The mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic layers yielded the product, with a yield of 62%. ESI-MS (m / z): 349.3 [M+H] + .

[0095] Synthesis of Compound 13

[0096] The intermediate 2-chloro-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindololin-4-yl]acetamide (800 mg, 2.28 mmol) was placed in 10 mL of N,N-dimethylformamide, and sodium azide (300 mg, 2.30 mmol) was added. The reaction mixture was heated to 80 °C and stirred for 5 h. The reaction mixture was then poured into water, extracted with ethyl acetate (3 × 50 mL), and the organic layers were combined, dried over a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate to obtain the final product. ESI-MS (m / z): 379.4 [MH] - .

[0097] Synthesis of Compound 14

[0098] Intermediate 2-azido-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl]acetamide (0.74 g, 2.10 mmol) and triphenylphosphine (1.66 g, 6.30 mmol) were added to 20 mL of tetrahydrofuran:water = 7:3, and the mixture was heated to 80 °C with stirring for 8 h. TLC monitoring was maintained until complete conversion of the starting material. After the reaction, the non-aqueous solvent was removed by rotary evaporation, and the mixture was extracted with 3 × 50 mL of ethyl acetate. The organic phases were combined, dried over saturated sodium chloride solution and anhydrous sodium sulfate, and then concentrated by filtration to obtain intermediate 14. ESI-MS (m / z): 331.9 [M+H] + .

[0099] Synthesis of Compound 15

[0100] The reaction conditions were the same as CPL0 42h, but the starting materials were changed to 2-amino-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl]acetamide and 10-bromodecanoic acid, with a yield of 90%. ESI-MS (m / z): 562.7 [M+H] + .

[0101] Synthesis of compound PS8

[0102] The reaction conditions were the same as PS1, but the starting materials were changed to 10-bromo-N-{2-{2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl]amino}-2-oxoethyl}decanoamide and CPL042, with a yield of 25%.

[0103] Synthetic routes for compounds PS14 and PS17.

[0104]

[0105] Chemical reagents and reaction conditions: (a) ethylene glycol chain with 2-3 repeating units corresponding to amino substitution, triethylamine, N,N-dimethylformamide, 80℃; (b) p-methylbenzenesulfonyl chloride, triethylamine, dichloromethane, reaction at room temperature; (c) potassium iodide, CPL042, triethylamine, N,N-dimethylformamide, 80℃.

[0106] Synthesis of compound 16a

[0107] The reaction conditions were the same as those for the synthesis of compound 5, but the starting material was replaced with 2-[2-(2-aminoethoxy)ethoxy]ethylene-1-ol. ESI-MS (m / z): 427.9 [M+Na] + .

[0108] Synthesis of compound 17a

[0109] The intermediate 2-(2,6-dioxadiazine-3-yl)-4-{2-[2-(2-hydroxyethoxy)ethoxy]amino}isoindoline-1,3-dione (1 equiv) was dissolved in dichloromethane. Then, p-toluenesulfonyl chloride (1.5 equiv) and triethylamine (3 equiv) were added sequentially, and the reaction was carried out at room temperature for 5 h. TLC monitoring continued until the starting material was completely converted. After the reaction was complete, the reaction mixture was poured into water, extracted three times with ethyl acetate, and the organic layers were combined. After drying in saturated sodium chloride solution, the mixture was stirred into silica gel for column chromatography. The target product was separated by column chromatography in 45% yield. ESI-MS (m / z): 582.6 [M+Na] + .

[0110] Synthesis of compounds PS14 and PS17

[0111] The reaction conditions were the same as for PS1, but the starting material was changed to ethyl 2-{2-{2-{{2-[2-(2-6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl]amino}ethoxy}ethoxy}-4-methylbenzenesulfonate, a bright yellow solid with a yield of 18%. The synthesis of compound PS17 was performed under the same conditions as PS14, but the starting material was replaced with ethyl 2-{2-{2-{{2-{{2-[2-(2-6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl]amino}ethoxy}ethoxy}ethoxy}-4-methylbenzenesulfonate, a bright yellow solid with a yield of 23%.

[0112] Synthetic route of compound PS18.

[0113]

[0114] Chemical reagents and reaction conditions: (a) N-Boc glycine, N,N-dimethylformamide, triethylamine, 80℃; (b) trifluoroacetic acid, dichloromethane, room temperature reaction; (c) ethylene glycol chain with corresponding amino-substituted 2-3 repeating units, HBTU, triethylamine, N,N-dimethylformamide, room temperature reaction; (d) p-Toluenesulfonyl chloride, triethylamine, dichloromethane, room temperature reaction; (e) CPL042, triethylamine, potassium iodide, N,N-dimethylformamide, 80℃.

[0115] Synthesis of Compound 18

[0116] The reaction conditions were the same as for compound 2, but the starting material was replaced with alanine tert-butyl ester (1.05 equiv). The product was a white solid in 65% yield. ESI-MS (m / z): 389.1 [M+H] + .

[0117] Synthesis of Compound 19

[0118] The intermediate tert-butyl[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl]glycine ester was placed in dichloromethane, and trifluoroacetic acid dissolved in dichloromethane was added. The mixture was stirred at room temperature for 4 h. After the starting material was completely converted, the reaction solution was poured into saturated sodium bicarbonate and extracted with ethyl acetate. The organic layer was discarded, and the aqueous layer was retained. The pH of the aqueous layer was then adjusted to 4-5, and the mixture was extracted three more times with ethyl acetate. The organic layers were combined, evaporated to dryness, and then dried under vacuum to obtain the product with a yield of 92%. ESI-MS (m / z): 329.2 [M+H] + .

[0119] Synthesis of Compound 20

[0120] The reaction conditions were the same as for compound CPL042h, but the starting material was replaced with 2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy}ethane-1-ol (1.01 equiv), with a yield of 82%. ESI-MS (m / z): 507.8 [M+H] + .

[0121] Synthesis of Compound 21

[0122] The reaction conditions were the same as for compound 17a, but the starting material was replaced with 2-{{2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl]amino)-N-{2-{2-[2-(2-)hydroxyethoxy]ethoxy}ethyl}acetamide}acetamide (1.00 equiv), yield 73%. ESI-MS (m / z): 662.1 [M+H] + .

[0123] Synthesis of compound PS18

[0124] The reaction conditions were the same as for compound PS14, but the starting material was replaced with 1-{{2-{2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl]amino}-2-oxo-6,9,12-trioxa-3-azatetradecane-14-yl 4-methylbenzenesulfonate (1.00 equiv), a bright yellow solid, in 12% yield.

[0125] Synthetic route of compound PS19

[0126]

[0127] Chemical reagents and reaction conditions: (a) i) tert-butyl 4-aminobutyrate, N,N-dimethylformamide, triethylamine, 80℃; ii) trifluoroacetic acid, dichloromethane; (b) ethylene glycol chain with corresponding amino-substituted 2-3 repeating units, HBTU, triethylamine, N,N-dimethylformamide, room temperature reaction; (c) p-Toluenesulfonyl chloride, triethylamine, dichloromethane, room temperature reaction; (d) CPL042, triethylamine, potassium iodide, N,N-dimethylformamide, room temperature reaction.

[0128] Synthesis of Compound 22

[0129] The reaction conditions were the same as those for compounds 18 and 19, but the starting material was changed to tert-butyl γ-aminobutyrate (1.5 equiv). The yield was 75%, and the ESI-MS (m / z) was 360.6 [M+H]. + .

[0130] Synthesis of Compound 23

[0131] The reaction conditions were the same as for compound 20, but the starting material was changed to 4-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl]amino}butyric acid (1 equiv), yield 83%, ESI-MS (m / z): 491.3 [M+H] + .

[0132] Synthesis of Compound 24

[0133] The reaction conditions were the same as for compound 23, but the reactant was changed to 4-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl]amino}-N-{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}butyramide, yield 42%, ESI-MS (m / z): 638.9 [M+Na] + .

[0134] Synthesis of compound PS19

[0135] The reaction conditions were the same as those for compound PS18, but the starting material was changed to 2-{2-{2-{4-{[2-(2-6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl]amino}butamido}ethoxy}ethoxy}4-methylbenzenesulfonate, a bright yellow solid, with a yield of 16%.

[0136] Synthetic routes for compounds PS20 and PS21.

[0137]

[0138] Chemical reagents and reaction conditions: (a)i) tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate, N,N-dimethylformamide, triethylamine, 80℃; (b) 1M hydrogen chloride-ethyl acetate solution; (c) bromine-substituted fatty acid, HBTU, triethylamine, N,N-dimethylformamide, reaction at room temperature; (d) CPL042, triethylamine, potassium iodide, N,N-dimethylformamide, reaction at room temperature.

[0139] Synthesis of Compound 25

[0140] The reaction conditions were the same as for compound 18, but the starting material was replaced with tert-butyl-4-(2-aminoethyl)piperazine-1-carboxylic acid ester (1.2 equiv). The yield was 76%, and the ESI-MS (m / z) result was 486.4 [M+H]. + .

[0141] Synthesis of Compound 26

[0142] The reaction conditions were the same as for compound 6, but the starting material was replaced with tert-butyl 4-{2-{{2-[2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindoline-4-yl]amino}ethyl}piperazine-1-carboxylate (1.1 equiv), yield 76%, ESI-MS (m / z): 386.2 [M+H] + .

[0143] Synthesis of compound 27a

[0144] The reaction conditions were the same as for compound 7a, but the starting material was replaced with 2-(2,6-dioxadiazin-3-yl)-4-{[2-(piperazin-1-yl)ethyl]amino}isoindoline-1,3-dione (1.1 equiv), yield 76%, ESI-MS (m / z): 576.3 [M+H] + .

[0145] Synthesis of compounds PS20 and PS21

[0146] The synthesis of compound PS20 was performed under the same conditions as compound PS7, but the starting material was replaced with 4-{{2-[4-(7-bromoheptanoyl)piperazin-1-yl]ethyl}amino}-2-(2,6-dioxadiazin-3-yl)isoindoline-1,3-dione (1.1 equiv), a bright yellow solid, in 36% yield. The synthesis of compound PS21 was performed under the same conditions as compound PS7, but the starting material was replaced with 4-{{2-[4-(8-bromooctanoyl)piperazin-1-yl]ethyl}amino}-2-(2,6-dioxadiazin-3-yl)isoindoline-1,3-dione (1.1 equiv), a bright yellow solid, in 45% yield. The synthetic route for PS21M was the same as for PS21.

[0147] Synthetic route of compound PS22.

[0148]

[0149] Chemical reagents and reaction conditions: (a) 5-Aryntohexanoic acid, CPL042, triethylamine, HBTU, N,N-dimethylformamide, reaction at room temperature; (b) sodium azide, triethylamine, tetrahydrofuran, reaction at room temperature; (c) copper sulfate, L-ascorbic acid, ethanol:water = 1:1, v / v, reaction at room temperature.

[0150] Synthesis of Compound 28

[0151] The reaction conditions were the same as for compound 7a, but the reactants were replaced with CPL042 (1.00 equiv) and 5-pentyneic acid, with a yield of 89% and ESI-MS (m / z): 538.6 [M+H]. + .

[0152] Synthesis of Compound 30

[0153] The reaction conditions were the same as for compound 13, but the reactant was replaced with 7-bromo-N-[2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindoline-4-yl]heptanamide (1.00 equiv), ESI-MS (m / z): 449.4 [M+Na] + .

[0154] Synthesis of compound PS22

[0155] In a 25 ml flask, 1-{4-{1-(2-aminopyrimidin-4-yl)-6-[(1-hydroxycyclohexyl)ethynyl]-1H-indol-3-carbonyl}piperazin-1-yl}hex-5-yn-1-one (75 mg, 0.14 mmol), 7-azido-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindololin-4-yl)heptamide (60 mg, 0.14 mmol), copper sulfate (35 mg, 0.14 mmol), and sodium isoascorbate (83 mg, 0.42 mmol) were added sequentially. Then, a mixed solution of tert-butanol and water (10 mL + 1 mL) was added. After sonicating until fully mixed, the reaction system was placed at 60 °C and stirred for 12 h. After the reaction was completed, the reaction system was dried by rotary evaporation, dissolved completely in ethyl acetate, filtered to remove solid impurities, and then mixed with silica gel for column chromatography to separate and purify the target product. The product was a white solid with a yield of 56%.

[0156] Synthetic routes for compounds PS24, PS25, PS26, PS27, and PS28.

[0157]

[0158] Chemical reagents and reaction conditions: (a) tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate, N,N-dimethylformamide, triethylamine, 80℃; (b) 1M hydrogen chloride-ethyl acetate solution, reaction at room temperature; (c) bromosubstituted fatty acids, HBTU, triethylamine, N,N-dimethylformamide, reaction at room temperature; (d) potassium iodide, CPL042, triethylamine, N,N-dimethylformamide, 80℃.

[0159] Synthesis of Compound 32

[0160] The reaction conditions were the same as 25a, but the starting material was replaced with 2-(2,6-dioxadiazine-3-yl)-5-fluoroisoindoline-1,3-dione, with a yield of 53% and MS (ESI) m / z (%) of 486.4 [M+H]+.

[0161] Synthesis of Compound 33

[0162] The reaction conditions were the same as 26a, but the starting material was replaced with tert-butyl 4-{2-{[2-(2-6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl]amino}ethyl}piperazine-1-carboxylic acid ester. The product could be used for subsequent reactions without purification. ESI-MS (m / z): 385.8 [M+H]+.

[0163] Synthesis of compound 34a

[0164] The reaction conditions were the same as 27a, but the starting material was replaced with 2-(2,6-dioxadiazin-3-yl)-5-((2-(piperazin-1-yl)ethyl)amino)isoindoline-1,3-dione, with a yield of 83% and ESI-MS (m / z): 561.7 [M+H]+.

[0165] Synthesis of compound PS24

[0166] The reaction conditions were the same as for P20, but the starting material was replaced with 5-{{2-[4-(6-bromohexanoyl)piperazin-1-yl]ethyl}amino}-2-(2,6-dioxadiazin-3-yl)isoindoline-1,3-dione, with a yield of 72%.

[0167] Synthesis of compounds PS25-PS28

[0168] The synthesis reaction conditions for compounds PS25-PS28 are the same as those for P20, but the starting materials are replaced with 5-{{2-[4-(7-bromoheptanoyl)piperazin-1-yl]ethyl}amino}-2-(2,6-dioxadiazin-3-yl)isoindoline-1,3-dione and 5-{{2-[4-(8-bromooctanoyl)piperazin-1-yl]ethyl}amino}-2-(2,6-dioxadiazin-3-yl)isoindoline-1,3-dione, respectively. Indoline-1,3-dione, 5-{{2-[4-(9-bromononanoyl)piperazin-1-yl]ethyl}amino}-2-(2,6-dioxopiridine-3-yl)isoindoline-1,3-dione, 5-{{2-[4-(10-bromodecanoyl)piperazin-1-yl]ethyl}amino}-2-(2,6-dioxopiridine-3-yl)isoindoline-1,3-dione, yield 34-65%.

[0169] Synthetic routes of compounds PS29, PS30, and PS31

[0170]

[0171] Chemical reagents and reaction conditions: (a) tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate, N,N-dimethylformamide, triethylamine, 80℃; (b) 1M hydrogen chloride-ethyl acetate solution, reaction at room temperature; (c) bromosubstituted fatty acid, HBTU, triethylamine, N,N-dimethylformamide, reaction at room temperature; (d) potassium iodide, CPL042, triethylamine, N,N-dimethylformamide, 80℃.

[0172] Synthesis of Compound 35

[0173] The reaction conditions were the same as those for the synthesis of compounds 9 and 10, but the starting materials were replaced with N-boc piperidinecarboxylic acid (1.02 equiv) and compound 26a (1.00 equiv). The yield was 85%, and the ESI-MS (m / z) was 496.5 [M+H].+ .

[0174] Synthesis of compound 36a

[0175] The reaction conditions were the same as those for the synthesis of compounds 9 and 10, but the starting materials were replaced with 2-(2,6-dioxadiazin-3-yl)-4-{4-{4-[4-(piperidin-4-carbonyl)piperazin-1-yl]methyl}amino}isoindoline-1,3-dione (1.00 equiv) and N-Boc aminovaleric acid (1.01 equiv). The yield was 89%, and the MS (ESI) m / z (%) was 596.3 [M+H]. + .

[0176] Synthesis of compound PS29

[0177] The synthesis of compound PS29 was performed under the same conditions as that of compound 9, but the reactants were replaced with 4-{{{4-[1-(5-aminopentanoyl)piperidin-4-carbonyl]piperazin-1-yl}methyl}amino}-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (1.00 equiv) and 1-(2-aminopyrimidin-4-yl)-6-[(1-hydroxycyclohexyl)ethynyl]-1H-indole-3-carboxylic acid (1.00 equiv), a white solid, in 15% yield. The synthesis of compound PS30 was performed under the same conditions as that of compound 9, but the starting materials were replaced with 4-{{{4-[1-(6-aminohexanoyl)piperidin-4-carbonyl]piperazin-1-yl}methyl}amino}-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (1.00 equiv) and 1-(2-aminopyrimidin-4-yl)-6-[(1-hydroxycyclohexyl)ethynyl]-1H-indole-3-carboxylic acid (1.00 equiv), a white solid, in 20% yield. The synthesis of compound PS31 was carried out under the same reaction conditions as that of compound 9, but the starting materials were replaced with 4-{{{4-[1-(7-aminoheptanoyl)piperidin-4-carbonyl]piperazin-1-yl}methyl}amino}-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (1.00 equiv) and 1-(2-aminopyrimidin-4-yl)-6-[(1-hydroxycyclohexyl)ethynyl]-1H-indole-3-carboxylic acid (1.00 equiv), a white solid, in 17% yield.

[0178] Synthetic routes for compounds PS32, PS33, and PS34.

[0179]

[0180] Chemical reagents and reaction conditions: (a) tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate, N,N-dimethylformamide, triethylamine, 80℃; (b) 1M hydrogen chloride-ethyl acetate solution, reaction at room temperature; (c) bromosubstituted fatty acid, HBTU, triethylamine, N,N-dimethylformamide, reaction at room temperature; (d) potassium iodide, CPL042, triethylamine, N,N-dimethylformamide, 80℃.

[0181] Synthesis of Compound 37

[0182] In a 25 mL flask, compound 12 (500 mg, 1.43 mmol), N-boc piperazine (293.0 mg, 1.57 mmol), and 1 mL triethylamine were added sequentially, followed by 10 mL N,N-dimethylformamide. The mixture was stirred until the solid mixture was completely dissolved. The reaction system was stirred at 80 °C, and TLC was used to monitor the reaction until the starting material was completely converted. After the reaction was complete, the reaction system was poured into 100 mL of water, and 3 × 50 mL of ethyl acetate was added for extraction. The organic layers were combined and dried to obtain the boc-protected N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)-2-(piperazin-1-yl)acetamide intermediate. The intermediate was redissolved in 10 mL of ethyl acetate, and then the boc protecting group was removed by adding 10 mL of 1 mol / L ethyl hydrochloride solution to give the final product N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)-2-(piperazin-1-yl)acetamide. The product was a white solid with a yield of 72% and ESI-MS (m / z): 400.9 [M+H]+.

[0183] Synthesis of compound 38a

[0184] The reaction conditions were the same as for compound 9, but the reactants were replaced with N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl]-2-(piperazin-1-yl)acetamide (1.00 equiv) and 7-bromoheptanoic acid (1.00 equiv). The product was a yellow oily liquid with a yield of 84% and ESI-MS (m / z): 591.3 [M+H]+.

[0185] Synthesis of compounds PS32, PS33, and PS34

[0186] The synthesis of compound PS32 was performed under the same conditions as PS24, but the starting materials were replaced with CPL042 (1.00 equiv) and 2-[4-(7-bromoheptanoyl)piperazin-1-yl]-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl]acetamide (1.00 equiv). The product was a white solid in 25% yield. The synthesis of compound PS33 was performed under the same conditions as PS24, but the starting materials were replaced with CPL042 (1.00 equiv) and 2-[4-(8-bromooctanoyl)piperazin-1-yl]-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl]acetamide (1.00 equiv). The product was a white solid in 32% yield. The synthesis of compound PS34 was carried out under the same reaction conditions as PS24, but the starting materials were replaced with CPL042 (1.00 equiv) and 2-[4-(9-bromononanoyl)piperazin-1-yl]-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl]acetamide (1.00 equiv). The product was a white solid in 30% yield.

[0187] Synthetic routes of compounds PS35 and PS36

[0188]

[0189] Chemical reagents and reaction conditions: (a) tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate, N,N-dimethylformamide, triethylamine, 80℃; (b) 1M hydrogen chloride-ethyl acetate solution, reaction at room temperature; (c) bromosubstituted fatty acid, HBTU, triethylamine, N,N-dimethylformamide, reaction at room temperature; (d) potassium iodide, CPL042, triethylamine, N,N-dimethylformamide, 80℃.

[0190] Synthesis of compound 39a

[0191] The reaction conditions were the same as 28a, but the starting materials were replaced with CPL042 (1 equiv) and 8-bromooctanoic acid (1 equiv). The product was a white solid in 82% yield. ESI-MS (m / z): 648.9 [M+H] + .

[0192] Synthesis of compounds PS35 and PS36

[0193] The reaction conditions were the same as for PS32, but the reactants were replaced with 1-{4-{1-(2-aminopyrimidin-4-yl)-6-[(1-hydroxycyclohexyl)ethynyl]-1H-indole-3-carbonyl}piperazin-1-yl}-8-bromopyrimidin-1-one (1 equiv) and compound 26a. The product was an off-white solid in 32% yield. The synthesis conditions for compound PS36 were the same as for PS32, but the reactants were replaced with 1-{4-{1-(2-aminopyrimidin-4-yl)-6-[(1-hydroxycyclohexyl)ethynyl]-1H-indole-3-carbonyl}piperazin-1-yl}-9-bromopyrimidin-1-one (1 equiv) and compound 26a. The product was an off-white solid in 30% yield. Spectral data of the compounds:

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201] Example 2

[0202] Activity screening of the PROTAC compound prepared in Example 1 above at the level of Western blotting assay.

[0203] Experimental methods:

[0204] Experimental Procedure: A549 and H1299 cells in logarithmic growth phase were cultured at 37°C in a 5% CO2 incubator. A 10mM stock solution of the drug was prepared using DMSO, and then diluted to a specific concentration using complete culture medium. After the cells filled the culture dish, the cell culture medium was replaced with drug-containing medium for incubation for the specified time. After incubation, the drug-containing medium was aspirated, and the cells were washed twice with pre-chilled PBS, removing the PBS solution. A cell lysis buffer was prepared by diluting Western blotting (WB) lysis buffer with 100mM PMSF at a ratio of 100:1, or by preparing a phosphorylated protein lysis buffer using WB lysis buffer and a mixed protease inhibitor solution. The cell lysis buffer was added to the cell culture dish, and cells were collected using a scraper and transferred to a 1.5mL epoxide tube. The cells were incubated on ice for 30 min, followed by centrifugation at 12000rpm for 10 min at 4°C. The supernatant was transferred to a new centrifuge tube to obtain the total tumor cell protein solution. The whole protein solution was quantified using the BCA method. All protein solutions were diluted to the same concentration according to the test results. The protein solutions were then mixed with 5X loading buffer and boiled in boiling water for 10 minutes to completely denature the proteins. After cooling, SDS-PAGE electrophoresis was performed.

[0205] Based on the protein molecular weight, prepare a vertical separation gel consisting of a 10% separating gel and a 5% stacking gel. Calculate the solution volume of 10-50 μg of protein as the loading volume, ensuring consistent volume across all wells. Then proceed with the electrophoresis process. During electrophoresis, the stacking gel is set to 80V for 30 min; the separating gel is set to 120-150V for 1 h. Electrophoresis is stopped when the bromophenol blue layer reaches the bottom of the gel, and the transfer process begins. After cutting the PVDF membrane, activate it by soaking it in pure methanol. Place the sponge, filter paper, gel segment, and PVDF membrane sequentially in the transfer holder, set up the transfer sandwich, and immerse it in transfer buffer. Transfer at a constant current of 200 mA for 2 h. After immersion, remove the PVDF membrane and proceed with the blocking process. Block using 5% BSA or skim milk, soaking the PVDF membrane for 1 h. After blocking, cut the PVDF membrane according to the marker instructions, rinse three times in TBST for 5 min each time, and finally incubate overnight in the corresponding primary antibody solution. After overnight incubation, the PVDF membrane segment was removed, rinsed three times in TBST, and then incubated in secondary antibody dilution solution at room temperature for 1 hour. After removal, it was rinsed three times in TBST, developed with ECL developer, photographed using Tanon gel development system, and semi-quantitatively analyzed for luminescent bands using ImageJ to obtain experimental results.

[0206] Experimental results:

[0207] The inventors evaluated the ability of some PROTAC compounds to degrade PAK4 using Western blotting. Immunoblotting analysis in the non-small cell lung cancer cell line A549 showed that the PAK4 ligand CPL055 did not exhibit PAK4 degradation activity, while the compound PS21 provided by this invention showed the best degradation activity. However, compound PS21 could degrade 42% of the PAK4 protein at 200 nM (see...). Figure 1 (Table 1)

[0208] This invention investigated the degradation of PAK4 protein by compound PS21 after its addition to A549 cells at different concentrations. Figure 2 As shown in the Western blot degradation results, the PAK4 protein kinase content in A549 cells decreased significantly with increasing drug concentration. At 5 μM, 72% of PAK4 protein kinase was degraded, and the concentration at which 50% PAK4 was degraded fell within the 0.5-1.5 μM range. 50 The value was approximately 1 μM. After 14 hours of treatment, the PAK4 protein content in MDA-MB-231 cells treated with 5 μM PS21 decreased to 12%, and even under the lowest concentration of 50 nM co-incubation, the PAK4 protein content in MDA-MB-231 cells did not reach 50%, proving that PS21 can effectively degrade PAK4 in MDA-MB-231 cells and the protein content cannot be restored to the pre-drug state.

[0209] Table 1: Western Blot results of different compounds

[0210]

[0211]

[0212] Based on the above results, the inventors further investigated whether compound PS21 is degraded via a PAK4-PROTAC-E3 ligase ternary complex and ubiquitin-proteasome-dependent degradation pathway. This invention used the PAK4 inhibitor CPL055, the E3 ligase inhibitor pomalidomide, the proteasome inhibitor MG132, and the negative assay compound PS21M, which lacks CRBN-E3 ligase affinity. Cells were pretreated for 2 hours, then treated with compound PS21, and finally, changes in the PAK4 protein band were detected by Western blotting. Figure 3 As shown, the above treatments can all reverse the degradation effect of PS21 on PAK4, and the negative ligand PS21M also has no degradation effect, indicating that the degrading agent PS21 does indeed degrade PAK4 through the PAK4-PROTAC-CRBN ternary complex and ubiquitin-proteasome-dependent degradation mechanism.

[0213] Example 3

[0214] The compound PS21 was used in a cell scratch migration assay.

[0215] Experimental steps:

[0216] A549, H1299, and MDA-MB-231 cells in logarithmic growth phase were collected, digested with 0.25 mM trypsin solution, counted, and the cell concentration was adjusted to 5.0 × 10⁻⁶ cells using complete culture medium. 5 Cell density adjusted cell solution was added to 6-well plates, 2 mL per well. The plates were then incubated at 37°C, 5% CO2 for 24 h until cells adhered and covered the bottom of the wells. The plates were then removed, and a 10 μL stencil was used to draw a straight line on the cell layer. Cell debris was washed away with PBS, and the image was photographed at 20x magnification as the 0 h control. The PBS was then removed, and basal culture medium with different drug concentrations was added for incubation. Images were taken at 24 h and 48 h. After the experiment, the length of the images at each time point was measured using ImageJ, the wound healing rate was calculated, and a bar chart was used for analysis.

[0217] Experimental results:

[0218] Experimental results are as follows Figure 4 As shown, the scratch assay results indicate that at concentrations of 10 μM, 2.5 μM, and 0.64 μM, compound PS21 can effectively inhibit the migration ability of A549, H1299, and MDA-MB-231 tumor cells, and PS21 exhibits similar inhibitory activity against the three cell lines.

[0219] Example 4

[0220] Example: Compound PS21 was used in a Transwell migration and invasion assay for cells.

[0221] Experimental steps:

[0222] A549, H1299, and MDA-MB-231 cells in logarithmic growth phase were collected, digested with 0.25 mM trypsin solution, counted, and the cell concentration was adjusted to 5.0 × 10⁻⁶ cells using complete culture medium. 5 Cells / mL. Cell solution adjusted to the specified cell density was added to 6-well plates, 2 mL per well, and then the plates were incubated at 37°C in a 5% CO2 incubator for 24 h. The culture medium was then discarded and replaced with complete drug-containing medium at the specified drug concentration, and incubated again for 24 h. After incubation, cells in each well were digested with trypsin and centrifuged. The culture medium was removed, and the cell density was adjusted to 1.5 × 10⁻⁶ cells / mL using basal medium. 5Cell suspension was prepared at a density of 1 cell / mL. This cell suspension was then added to a Transwell chamber lined with Matrigel to assess cell invasion, or to a Transwell chamber without Matrigel to assess cell migration. 600 μL of 20% FBS complete culture medium was added to the lower chamber of a 24-well plate, and the experiment was terminated after 16 h of incubation. The PBS chambers were removed, and the Transwell chambers were rinsed with PBS solution. The chambers were then fixed in 4% paraformaldehyde for 20 min, rinsed again with PBS solution to remove the paraformaldehyde, and then stained with 0.1% methyl violet solution for 20 min. After removing the chambers, they were washed with PBS, and the cells inside the chambers were wiped off with a cotton swab before photographing and counting the cells.

[0223] Experimental results:

[0224] Transwell assays further validated the inhibitory effect of compound PS21 on the migration and invasion activities of A549, MDA-MB-231, and H1299 cells. Experimental results are as follows: Figure 5 As shown, compound PS21 can effectively inhibit the migration and invasion of three cell lines at concentrations of 10 μM, 5 μM, and 1 μM. At the same time, the migration and invasion of the three cell lines showed the same concentration-dependent decrease after PS21 treatment, indicating that PS21 can effectively inhibit the migration and invasion activities of various tumor cells.

[0225] In summary, the inventors demonstrated the degradation effect of this type of degrading agent on PAK4 using Western blotting experiments. Further investigation using scratch and transwell assays confirmed that this type of PAK4 degrading agent can effectively inhibit the migration and invasion of malignant tumor cells.

[0226] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A PROTAC compound of Formula I or Formula II that targets and degrades PAK4, or a pharmaceutically acceptable salt thereof, characterized in that, The chemical structures of Formula I or Formula II are shown below: Formula I Formula II Wherein: the E3 ligase ligand is: or ; In Formula I, the Linker is a linking group, which is selected from any one of the following groups: , , , , , , , , , , , , , , , , , , , , , , , , , or ; In Formula II, the Linker is a linking group selected from any one of the following groups: , , , , , , , or .

2. The PROTAC compound or its pharmaceutically acceptable salt that targets and degrades PAK4 according to claim 1, characterized in that, The PROTAC compound that targets and degrades PAK4 is any one of the following compounds: PS1-PS36: 。 3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the PROTAC compound for targeting and degrading PAK4 as described in claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

4. The use of the PROTAC compound of claim 1 or claim 2 that targets and degrades PAK4, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 3, in the preparation of a PAK4-degrading or PAK4-inhibiting drug.

5. The use of the PROTAC compound of claim 1 or claim 2 that targets and degrades PAK4, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 3, in the preparation of a medicament for the treatment or prevention of PAK4-related diseases.

6. The application according to claim 5, characterized in that, The PAK4-related diseases mentioned are solid tumors.

7. The use of the PROTAC compound of claim 1 or claim 2 that targets and degrades PAK4, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 3, in the preparation of an antitumor drug.

8. The application according to claim 7, characterized in that, The tumor is selected from non-small cell lung cancer, breast cancer, pancreatic cancer, colon cancer, ovarian cancer, or gastric cancer.

9. The application according to claim 7, characterized in that, The tumor is a PAK4-overexpressing tumor or a PAK4 inhibitor-resistant tumor.

Images