PROTAC compound for targeted degradation of PAK4 as well as preparation method and application of PROTAC compound

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a PROTAC compound for targeted degradation of PAK4 as well as a preparation method and application of the PROTAC compound. The invention provides a PROTAC compound for targeted degradation of PAK4 as shown in a formula I or a formula II or pharmaceutically acceptable salts, stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites or prodrugs of the PROTAC compound. An in-vitro activity test result shows that the compound is a PROTAC molecule capable of degrading PAK4, and compared with an existing PAK4 inhibitor, the small molecule compound has the capacity of degrading PAK4 protein and further rapidly inhibiting tumor migration and invasion and has the potential to become an effective treatment mode for treating malignant tumors. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to a PROTAC compound for targeted degradation of PAK4, and a preparation method and application thereof. Background Art

[0002] P21-activated kinase 4 (PAK4) is a serine / threonine protein kinase that plays a key role in cytoskeleton reorganization and regulation of cell motility. As an important downstream effector molecule of Cdc42, PAK4 often shows abnormal states such as amplification, mutation, and up-regulation of its expression level and activity 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 generation, cell cycle progression, etc., and abnormal expression of it will trigger various pathological processes. In the development and function maintenance of the nervous system, PAK4 is a related target, and inhibition of its protein kinase activity is related to the pathological process of neurodegenerative diseases. The regulation of PAK4 is also related to cardiovascular diseases. In adult tissues, PAK4 is significantly associated with cancer, and there are phenomena such as abnormal expression in various tumors, providing a target for cancer treatment. The regulation of the cytoskeleton by PAK4 is closely related to cancer cell metastasis, and it can affect the cytoskeleton and migration through various pathways. PI3K / AKT plays a key role in the PAK4 pathway, and overexpression of PAK4 promotes tumor formation. Therefore, designing PAK4 inhibitors that block its signaling pathway is expected to become a new strategy for tumor treatment.

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

[0004] Currently, the therapies related to the regulation of PAK4 protein kinase still focus on PAK4 competitive inhibitors. Therefore, designing PROTAC compounds for targeted degradation of PAK4 is expected to synthesize PAK4 inhibitors with higher activity and higher selectivity. Summary of the Invention

[0005] The object of the present invention is to provide a method for preparing a PROTAC compound targeting the degradation of PAK4 or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, N-oxide, hydrate, solvate, metabolite or prodrug thereof, and its application. The results of in vitro activity tests show that the compounds of the present invention are PROTAC molecules capable of degrading PAK4. Compared with existing PAK4 inhibitors, the small molecule compounds involved in the present invention have the ability to degrade PAK4 protein and thus rapidly inhibit tumor migration and invasion, and have the potential to become an effective treatment for malignant tumors.

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

[0007] The primary object of the present invention is to provide a PROTAC compound targeting the degradation of PAK4 (PAK4-PROTAC compound) represented by the following formula I or formula II, or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, N-oxide, hydrate, solvate, metabolite or prodrug thereof.

[0008]

[0009] Wherein: The E3 ligase ligand refers to a ligand molecule that binds to an E3 ligase. The E3 ligase includes CRBN, MDM2, IAP, DCAF or RNF, and the E3 ligase ligands include:

[0010] 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 stereoisomer, geometric isomer, tautomer, N-oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug;

[0014]

[0015]

[0016]

[0017] The pharmaceutically or physiologically acceptable salt referred to in the present invention means an addition salt formed by the PROTAC compound targeting the degradation of PAK4 described in the present invention and a pharmaceutically or physiologically acceptable acid or base;

[0018] The present invention also provides a pharmaceutical composition, which comprises the PAK4-PROTAC compound or its stereoisomers, geometric isomers, tautomers, N-oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs;

[0019] The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle or a combination thereof;

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

[0021] The pharmaceutical composition further comprises other drugs having a therapeutic or prophylactic effect on tumors. The PAK4-related disease is lung cancer, and further, it is non-small cell lung cancer;

[0022] Use of the PROTAC compound for targeted degradation of PAK4 or a pharmaceutical composition comprising the PROTAC compound for targeted degradation of PAK4 in anti-tumor drugs. The tumors are breast cancer, pancreatic cancer, colon cancer, lung cancer, gastric cancer, ovarian cancer. Further, the tumors are tumors with high expression of PAK4, or tumors resistant to PAK4 inhibitors. Still further, the tumors are lung cancer solid tumors;

[0023] The present invention also provides a synthetic route of the PROTAC compound for targeted degradation of PAK4 represented by General Formula I or General Formula II, which specifically comprises the following steps:

[0024] According to the designed compound, pomalidomide, 5-aminopomalidomide, 5-fluoroanhydride phthalide and N-methylpomalidomide are used as ligand raw materials, and are synthesized by methods such as nucleophilic substitution, acylation reaction, deprotection of boc group, formation of p-toluenesulfonate, azidation, reduction, and click chemical reaction, in accordance with the structural characteristics of the compound.

[0025] Effective benefits of the present invention:

[0026] The present invention has successfully designed and synthesized a PROTAC compound with significant PAK4 degradation activity. The inventors have confirmed the degradation effect of the PROTAC degrading agent compound provided by the present invention on PAK4 through Western Blot experiments, and further proved through in vitro enzyme activity tests, CCK8 cell viability tests, scratch tests, Transwell cell migration and invasion tests, and mouse in vivo pharmacokinetic experiments that the PROTAC degrading agent compound has the ability to degrade PAK4 protein, and thus rapidly inhibit tumor migration and invasion, and has the potential to become an effective treatment method for malignant tumors. Description of the Drawings

[0027] Figure 1It is the activity screening result of the compounds PS1 - PS36 of the embodiments of the present invention against PAK kinase. (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 It shows that the compound PS21 of the embodiments of the present invention degrades PAK4 protein in a concentration - dependent manner. (A) PS21 induces concentration - dependent degradation of PAK4 in A549; (B) PS21 induces concentration - dependent degradation of PAK4 in MDA - MB - 231.

[0029] Figure 3 It shows that the compound PS21 of the embodiments of the present invention induces PAK4 degradation through a mechanism dependent on cereblon (CRBN) and the proteasome. A549 cells were pretreated with 5 μM of PAK4 inhibitor, pomalidomide, or MG132 for 4 hours, then treated with 2 μM of PS21 for 14 hours, and finally the level of PAK4 was detected by Western blotting, with glyceraldehyde - 3 - phosphate dehydrogenase (GAPDH) as the internal reference control.

[0030] Figure 4 It is the wound - healing assay result of the compound PS21 of the embodiments of the present invention. (A) Wound - healing assay result of PS21 on A549 cell line; (B) Wound - healing assay result of PS21 on H1299 cell line.

[0031] Figure 5It is the result of the anti-tumor cell migration and invasion of the compound PS21 in the embodiments of the present invention. (A) Transwell assay results of PS21 on A549, MDA-MB-231, and H1299 cell lines; (B) Statistical chart of the migration assay results of different concentrations of PS21 inhibiting A549, MDA-MB-231, and H1299 cell lines; (C) Statistical chart of the migration assay results of different concentrations of PS21 inhibiting A549, MDA-MB-231, and H1299 cell lines. Detailed implementation manners

[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 only used to explain the present invention and are not used to limit the scope of the present invention.

[0033] In the following embodiments, methods for preparing some of the compounds described above are depicted. It should be understood that the following methods and other methods known to those of ordinary skill in the art can be applied to the preparation of all the compounds described in the present invention. The embodiments are intended to illustrate rather than limit the scope of the present invention.

[0034] For those without specific technical or conditions noted in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through regular channels.

[0035] In the following embodiments, the experimental methods, unless otherwise specified, are all conventional methods. The test materials used in the following embodiments, unless otherwise specified, are all commercially available products.

[0036] Example 1

[0037] Preparation method and structure confirmation of PROTAC targeting the degradation of PAK4.

[0038] Synthetic route:

[0039] Synthesis of compound CPL042

[0040]

[0041] Chemical reaction 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 °C; (f) Trifluoroacetic acid, dichloromethane, reaction at room temperature (g) HBTU, piperazine, triethylamine, reaction at room temperature (h) 1-Ethynyl-1-cyclohexanol, tetrakis(triphenylphosphine)palladium, copper(I) iodide, triethylamine, N,N-dimethylformamide, 80 °C.

[0042] Synthesis of Compound CPL042b

[0043] Add 6-bromoindole (10.00 g, 50.1 mmol) into a 100 mL eggplant-shaped flask, then add 50 mL of N,N-dimethylformamide, and ultrasonically dissolve 6-bromoindole completely. Place the eggplant-shaped flask in an ice bath, and dropwise add trifluoroacetic anhydride (8.60 mL, 61.20 mmol) within half an hour under stirring. After the addition is completed, transfer the reaction system to room temperature and react for 1.5 h. Monitor the reaction system by TLC until the raw material spot completely disappears. Then pour the reaction solution into 500 mL of water, a large amount of off-white solid precipitates. Stir the mixture thoroughly and filter it by suction to obtain the off-white solid product. The target product is obtained by infrared drying, with a yield of 92%. ESI-MS (m / z): 292.8 [M+H] + 。

[0044] Synthesis of Compound CPL042c

[0045] Place 1-(6-bromo-1H-indol-3-yl)-2,2,2-trifluoroethanone (9.00 g, 30.82 mmol) into a 250 mL eggplant-shaped flask, then add 100 mL of 20% NaOH solution, and ultrasonically disperse the solid completely. Heat the reaction system under reflux for 4 h under stirring, and monitor it by TLC until the raw material spot completely disappears. After the reaction is completed, cool the reaction system to room temperature in an ice bath, and extract and remove impurities from the reaction solution with 3×200 mL of dichloromethane. Discard the organic layer and retain the aqueous layer. Then adjust the pH of the aqueous layer to 5-6 with 4M hydrochloric acid, a large amount of solid precipitates. Filter the mixture by suction to obtain the solid product. Wash the solid layer with water until the pH is neutral, and dry the solid product to obtain the product, with a yield of 84%. ESI-MS (m / z): 237.4 [M-H] - 。

[0046] Synthesis of Compound CPL042e

[0047] Place 6-bromoindole-3-carboxylic acid (5.00 g, 20.92 mmol) into a 500 mL eggplant-shaped flask, add 40 mL of dichloromethane, and ultrasonically disperse the solid sufficiently. After placing the reaction system in an ice bath, connect a drying tube, and dropwise add oxalyl chloride solution (6.64 g, 52.3 mmol) dissolved in 40 mL of dichloromethane within 15 min under stirring. After the addition is completed, transfer the reaction system to room temperature and react for 2 h. Detect by TLC until the raw materials are completely converted. Rotate and evaporate the reaction system to remove the unreacted oxalyl chloride dichloromethane solution until the system is completely dry. This compound is extremely prone to moisture and deterioration and can be used in the next step without purification.

[0048] Transfer the solid product of the previous step to a solution of potassium tert-butoxide (2.58 g, 23.01 mmol) in tert-butanol (40 mL). React for 1 h until the system becomes completely clear, and monitor by TLC until the raw materials are completely converted. Pour the reaction system into 500 mL of water, and a large amount of white solid will precipitate. Filter by suction and wash to obtain the crude product. The crude product is purified by normal-phase silica gel column chromatography (PE:EA = 5:1, v / v) to obtain the purified product. The two-step yield is 85%, and ESI-MS (m / z): 296.7 [M+H] + 。

[0049] Synthesis of compound CPL042f

[0050] Dissolve tert-butyl 6-bromo-1H-indole-3-carboxylate (5.00 g, 16.91 mmol) in 40 mL of N,N-dimethylformamide. Sonicate until the solid raw material is completely dissolved. Add cesium carbonate (5.79 g, 17.75 mmol) with stirring at room temperature and stir for half an hour. Then add 2-amino-4-chloropyrimidine (2.41 g, 18.60 mmol). After that, heat the system to 80 °C and stir for 8 h until the raw material tert-butyl 1-(2-aminopyrimidin-4-yl)-6-bromo-1H-indole-3-carboxylate is completely converted. After monitoring by TLC until the raw material spot disappears, pour the reaction system into 500 mL of water with stirring, and a large amount of white solid will precipitate. Filter by suction to obtain the crude product. Dissolve the crude product in dichloromethane:methanol = 20:1 and purify it by silica gel column chromatography (dichloromethane:MeOH = 20:1, v / v) to obtain the purified white product. The yield is 86%, and ESI-MS (m / z): 388.8 [M+H] + 。

[0051] Synthesis of compound CPL042g

[0052] Add the intermediate tert-butyl 1-(2-aminopyrimidin-4-yl)-6-bromo-1H-indole-3-carboxylate (3.00 g, 7.71 mmol) to a 250 mL eggplant-shaped flask. Add 50 mL of dichloromethane until it is completely dissolved. Add 10 mL of trifluoroacetic acid dissolved in 50 mL of dichloromethane with stirring within 15 min and react at room temperature. After monitoring the reaction progress by TLC until the raw material spot disappears, pour the reactant into 20% sodium hydroxide solution and extract with 3×200 mL of ethyl acetate. Retain the aqueous layer, then adjust the pH value of the aqueous layer to 5-6, and a large amount of white solid will precipitate. Filter by suction and wash the filter cake with water until pH = 7.0 to obtain the target product. The yield is 80%, and ESI-MS (m / z): 330.6 [M-H] - 。

[0053] Synthesis of compound CPL042h

[0054] The 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 reaction system was sonicated until completely dissolved, and N-boc piperazine (1.15 g, 6.16 mmol), HBTU (2.34 g, 6.16 mmol), and 1 mL of triethylamine were added under stirring. The reaction system was stirred at room temperature for 3 h, and TLC was used to monitor the reaction system until the raw material spots completely disappeared. After the reaction was completed, the reaction system was poured into 250 mL of water, and a white solid precipitated. The boc-protected intermediate was obtained by suction filtration. After suction filtration, washing, and drying, the intermediate was taken out, and the boc protecting group was removed by adding 25 mL of hydrogen chloride-ethyl acetate solution to obtain the hydrochloride salt of the target product. The intermediate was added to 1 M NaOH and stirred, then extracted with 3 × 200 mL of ethyl acetate, concentrated by rotation, and dried under vacuum to obtain the target product (1-(2-aminopyrimidin-4-yl)-6-bromo-1H-indol-3-yl)(piperazin-1-yl)methanone, with a yield of 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 ultra-dry N,N-dimethylformamide and 1 mL of ultra-dry triethylamine, and then ethynylcyclohexanol (1.23 g, 9.94 mmol) was added. The reaction system was sonicated until completely dissolved, and the air in the system was replaced with a nitrogen balloon. Under nitrogen protection, tetrakis(triphenylphosphine)palladium (287.16 mg, 0.2485 mmol) and copper(I) iodide (189.30 mg, 0.994 mmol) were added. The reaction system was stirred at 80 °C for 12 h, and TLC was used to monitor until the raw material spots disappeared. After the reaction was completed, the reaction system was poured into 200 mL of water, 200 mL of ethyl acetate was added and stirred thoroughly, then filtered through diatomaceous earth, and the organic layer was separated using a separatory funnel. After mixing with silica gel for column chromatography, silica gel column chromatography separation was carried out with the mobile phase dichloromethane:MeOH = 8:1 (v / v), with a yield of 60%, ESI-MS (m / z): 445.5 [M+H] + 。

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

[0058]

[0059] Chemical reaction reagents and reaction conditions: (a) Tetrahydrofuran, bromo-substituted acyl chloride, reflux; (b) Potassium iodide, CPL042, triethylamine, N,N-dimethylformamide, 80 °C.

[0060] Synthesis of Compound 2a

[0061] The raw material 4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (2.00 g, 7.32 mmol) was added to 20 mL of tetrahydrofuran, stirred until completely dissolved, and 5-bromovaleryl chloride (7.30 g, 36.6 mmol) was added. The reaction was heated to reflux until the raw material spot disappeared. After the reaction was completed, the reaction solution was evaporated to dryness, dissolved in ethyl acetate, and then column chromatography silica gel was added. The off-white product was obtained by column chromatography separation, with a yield of 72%, ESI-MS (m / z): 424.5 [M+H] + . The synthesis methods of Compounds 2b - 2g are the same as that of 2a, with yields of 48 - 75%.

[0062] Synthesis of Compound PS1

[0063] The raw material 5-bromo-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-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 reaction was stirred at 80 °C for 5 h. The reaction was monitored by TLC until one of the raw material spots completely disappeared. After the reaction was completed, the reaction solution system was poured into 100 mL of water, and extracted with 3 × 50 mL of ethyl acetate. The organic layers were combined, concentrated, and the target product was separated by preparative thin-layer chromatography. The mobile phase was dichloromethane:MeOH = 5:1 (v / v), and the refined product was obtained as a white solid, with a yield of 12%.

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

[0065] Synthetic routes of Compounds PS7, PS9, PS10, PS11, PS12, PS13, PS16.

[0066]

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

[0068] Synthesis of Compound 4

[0069] In a 100 mL eggplant-shaped 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 successively added. Finally, 30 mL of acetic acid was added. After stirring well until mixed, the temperature of the system was raised to 110 °C and the reaction was carried out for 8 h. The reaction system was monitored by TLC until the raw materials were completely converted. After the reaction was completed, the reaction system was evaporated to dryness to remove acetic acid, and then purified by column chromatography to obtain the white solid product Compound 4 with a yield of 59%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.12 (s, 1H), 7.96–7.91 (m, 1H), 7.78 (d, J = 7.3 Hz, 1H), 7.72 (t, J = 8.9 Hz, 1H), 5.15 (dd, J = 12.8, 5.4 Hz, 1H), 2.88 (ddd, J = 17.1, 13.9, 5.5 Hz, 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] The intermediate 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (1 equiv) was placed in ultra-dry DMSO, Boc-N-hexanediamine (1.2 equiv) and DIPEA (2 equiv) were added, and the temperature of the reaction system was raised to 90 °C and the reaction was carried out for 8 h. The reaction system was monitored by TLC until the raw materials were completely converted. After the reaction was completed, the reaction solution was poured into water, extracted three times with ethyl acetate, and then the organic layers were combined and added with column chromatography silica gel and evaporated to dryness. The intermediate 5 was separated 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-dioxoisoindolin-4-yl)amino)hexyl)carbamate was placed in 20 ml of ethyl acetate hydrochloride and stirred. The reaction was monitored by TLC until the raw materials were completely converted. After 3 h, filtration was carried out by suction, and vacuum drying gave the target product MS(ESI) m / z(%): 372.3 [M+H]+.

[0074] Synthesis of compound 7a

[0075] The reaction conditions were the same as those of CPL042h, but the raw materials were changed to compound 6 and 5-bromopentanoic acid. The product MS(ESI) m / z(%): 521.1 [M+H]+.

[0076] Synthesis of compound PS7

[0077] The reaction conditions were the same as those of PS1, but the raw materials 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 in the same manner as PS7.

[0079] Synthetic route of compound PS23.

[0080]

[0081] Chemical reaction reagents and reaction conditions: (a) N,N-dimethylformamide, N-Boc glycine, HBTU, triethylamine, 80 °C; (b) 1 M hydrochloric acid-ethyl acetate solution, reaction at room temperature; (c) bromo-substituted fatty acid, HBTU, triethylamine, N,N-dimethylformamide, reaction at room temperature; (d) potassium iodide, CPL042, triethylamine, N,N-dimethylformamide, 80 °C.

[0082] Synthesis of compound 9

[0083] In a 50 ml eggplant-shaped flask, 5-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (1 equiv), HBTU (1.05 equiv), and Boc glycine (1.01 equiv) were sequentially added. 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 and then heated to 50 °C and stirred for reaction. After monitoring by TLC until the raw material spots disappeared, the reaction solution was poured into 200 ml of water, and the aqueous phase was extracted 50×3 with ethyl acetate. The organic phases were combined and 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-dioxoisoindolin-5-yl]amino}-2-oxoethyl}carbamate was placed in 20 mL of ethyl acetate hydrochloride and stirred at room temperature for 3 h. After monitoring by TLC until the raw material spot completely disappeared, the reaction system was filtered by suction to obtain a solid, which was 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 those of CPL042h, but the raw materials were replaced with 2-amino-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl]acetamide and 10-bromodecanoic acid, and the yield was 95%. ESI-MS (m / z): 562.4 [M+H] + 。

[0088] Synthesis of compound PS23

[0089] The reaction conditions were the same as those of PS1, but the raw 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 reaction reagents and reaction conditions: (a) chloroacetyl chloride, N,N-dimethylformamide, triethylamine, 50 °C.; (b) sodium azide, triethylamine, tetrahydrofuran, reaction at room temperature; (c) hydrogen, Pd / C, tetrahydrofuran; (d) bromo-substituted fatty acid, HBTU, triethylamine, N,N-dimethylformamide, reaction at room temperature; (e) potassium iodide, CPL042, triethylamine, N,N-dimethylformamide, 80 °C.

[0093] Synthesis of compound 12

[0094] The raw material pomalidomide (1 equiv) was dissolved in dry DMSO, and triethylamine (2 equiv) and chloroacetyl chloride (5 equiv) were sequentially added. After the reactants were completely dissolved under stirring, the temperature was raised to 80 °C and reacted for 5 h. The reaction system was monitored by TLC until the raw material spot was completely converted. After the reaction was completed, the reaction solution was poured into water, stirred well, and extracted with ethyl acetate (3×50 mL). The organic layers were combined to obtain 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-dioxoisoindolin-4-yl]acetamide (800 mg, 2.28 mmol) was placed in 10 mL of N,N-dimethylformamide, sodium azide (300 mg, 2.30 mmol) was added, and the reaction system was heated to 80 °C and stirred for 5 h. The reaction system was poured into water, extracted with ethyl acetate 3×50 mL, the combined organic layers were dried over saturated sodium chloride solution, and then dried over anhydrous sodium sulfate to obtain the product. ESI-MS (m / z): 379.4 [M-H] - 。

[0097] Synthesis of Compound 14

[0098] The intermediate 2-azido-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-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 temperature was raised to 80 °C with stirring and reacted for 8 h. The reaction was monitored by TLC until the raw materials were completely converted. After the reaction was completed, the non-aqueous solvent was removed by rotary evaporation under reduced pressure, extracted with 3×50 mL of ethyl acetate, the combined organic phases were dried over saturated sodium chloride solution and anhydrous sodium sulfate, and then filtered and concentrated to obtain the intermediate 14. ESI-MS (m / z): 331.9 [M+H] + 。

[0099] Synthesis of Compound 15

[0100] The reaction conditions were the same as those of CPL042h, but the raw materials were changed to 2-amino-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]acetamide and 10-bromodecanoic acid, and the yield was 90%. ESI-MS (m / z): 562.7 [M+H] + 。

[0101] Synthesis of Compound PS8

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

[0103] Synthetic routes of Compounds PS14 and PS17

[0104]

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

[0106] Synthesis of Compound 16a

[0107] The reaction conditions are the same as those for the synthesis of Compound 5, but the raw material is replaced with 2-[2-(2-aminoethoxy)ethoxy]ethan-1-ol, ESI-MS (m / z): 427.9 [M+Na] + 。

[0108] Synthesis of Compound 17a

[0109] Dissolve the intermediate 2-(2,6-dioxopiperidin-3-yl)-4-{2-[2-(2-hydroxyethoxy)ethoxy]amino}isoindoline-1,3-dione (1 equiv) in dichloromethane, sequentially add p-toluenesulfonyl chloride (1.5 equiv) and triethylamine (3 equiv), and then react at room temperature for 5 h. Monitor the reaction by TLC until the raw materials are completely converted. After the reaction is complete, pour the reaction system into water, extract with ethyl acetate three times, combine the organic layers, dry with saturated sodium chloride solution, and then mix with column chromatography silica gel. Separate the target product by column chromatography, with a yield of 45%. ESI-MS (m / z): 582.6 [M+Na] + 。

[0110] Synthesis of Compounds PS14 and PS17

[0111] The reaction conditions are the same as those for PS1, but the raw material is changed to ethyl 2-{2-{2-{{2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]amino}ethoxy}ethoxy}-4-methylbenzenesulfonate, a bright yellow solid, with a yield of 18%. The reaction conditions for the synthesis of Compound PS17 are the same as those for PS14, but the raw material is replaced with 2-{2-{2-{2-{{2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-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 reaction reagents and reaction conditions: (a) N-Boc glycine, N,N-dimethylformamide, triethylamine, 80 °C; (b) trifluoroacetic acid, dichloromethane, reaction at room temperature; (c) corresponding amino-substituted ethylene glycol chain with 2-3 repeating units, HBTU, triethylamine, N,N-dimethylformamide, reaction at room temperature; (d) p-toluenesulfonyl chloride, triethylamine, dichloromethane, reaction at room temperature; (e) CPL042, triethylamine, potassium iodide, N,N-dimethylformamide, 80 °C.

[0115] Synthesis of Compound 18

[0116] The reaction conditions were the same as those for Compound 2, but the raw material was replaced with tert-butyl alaninate (1.05 equiv), off-white solid, yield 65%. 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] glycinate was placed in dichloromethane, and trifluoroacetic acid dissolved in dichloromethane was added. The mixture was stirred at room temperature for 4 h. After monitoring until the raw materials were completely converted, the reaction solution was poured into saturated sodium bicarbonate, extracted with ethyl acetate, the organic layer was discarded, and the aqueous layer was retained. Then, the pH of the aqueous layer was adjusted to 4-5, and it was extracted with ethyl acetate three times again. The organic layers were combined, dried by rotary evaporation, and then dried under vacuum to obtain the product, yield 92%. ESI-MS (m / z): 329.2 [M+H] + 。

[0119] Synthesis of Compound 20

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

[0121] Synthesis of Compound 21

[0122] The reaction conditions were the same as those for Compound 17a, but the raw 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 those for compound PS14, but the raw material was replaced with 4-methylbenzenesulfonate of 1-{{2-{2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]amino}-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl (1.00 equiv), a bright yellow solid, with a yield of 12%.

[0125] Synthetic route of compound PS19

[0126]

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

[0128] Synthesis of compound 22

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

[0130] Synthesis of compound 23

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

[0132] Synthesis of compound 24

[0133] The reaction conditions were the same as those for compound 23, but the raw material was changed to 4-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]amino}-N-{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}butyramide, with a yield of 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 raw material was changed to 2-{2-{2-{4-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]amino}butanamido}ethoxy}ethoxy}-4-methylbenzenesulfonate, a bright yellow solid, with a yield of 16%.

[0136] Synthetic routes of compounds PS20 and PS21.

[0137]

[0138] Chemical reaction reagents and reaction conditions: (a) i) tert-Butyl 4-(2-aminoethyl)piperazine-1-carboxylate, N,N-dimethylformamide, triethylamine, 80 °C; (b) 1 M hydrogen chloride-ethyl acetate solution; (c) bromo-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 those for compound 18, but the raw material was replaced with tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate (1.2 equiv), with a yield of 76%, ESI-MS (m / z): 486.4 [M + H] + 。

[0141] Synthesis of compound 26

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

[0143] Synthesis of compound 27a

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

[0145] Synthesis of compounds PS20 and PS21

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

[0147] Synthetic route of compound PS22.

[0148]

[0149] Chemical reaction reagents and reaction conditions: (a) 5-ynoic 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 those of compound 7a, but the raw materials were replaced with CPL042 (1.00 equiv) and 5-pentynoic acid, with a yield of 89%, ESI-MS (m / z): 538.6 [M+H] + 。

[0152] Synthesis of compound 30

[0153] The reaction conditions were the same as those of compound 13, but the raw material was replaced with 7-bromo-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]heptanamide (1.00 equiv), ESI-MS (m / z): 449.4 [M+Na] + 。

[0154] Synthesis of compound PS22

[0155] In a 25 mL eggplant-shaped flask, sequentially add 1-{4-{1-(2-aminopyrimidin-4-yl)-6-[(1-hydroxycyclohexyl)ethynyl]-1H-indole-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-dioxoisoindolin-4-yl)heptanamide (60 mg, 0.14 mmol), copper sulfate (35 mg, 0.14 mmol) and sodium erythorbate (83 mg, 0.42 mmol). Then add a mixed solution of tert-butanol and water (10 mL + 1 mL). After ultrasonic mixing until fully homogeneous, place the reaction system in a 60 °C water bath and stir for 12 h. After the reaction is completed, rotary evaporate the reaction system to dryness, add ethyl acetate to fully dissolve it, filter off the solid impurities by suction, and mix with silica gel for column chromatography to separate and purify the target product. The product is a white solid with a yield of 56%.

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

[0157]

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

[0159] Synthesis of compound 32

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

[0161] Synthesis of compound 33

[0162] The reaction conditions are the same as 26a, but the raw material is replaced with tert-butyl 4-{2-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl]amino}ethyl}piperazine-1-carboxylate. The product can be used in the subsequent reaction without purification. ESI-MS(m / z): 385.8[M + H]+.

[0163] Synthesis of compound 34a

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

[0165] Synthesis of Compound PS24

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

[0167] Synthesis of Compounds PS25 - PS28

[0168] The reaction conditions for the synthesis of Compounds PS25 - PS28 were the same as those of P20, but the raw materials were replaced with 5-{{2-[4-(7-bromoheptanoyl)piperazin-1-yl]ethyl}amino}-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione, 5-{{2-[4-(8-bromooctanoyl)piperazin-1-yl]ethyl}amino}-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione, 5-{{2-[4-(9-bromononanoyl)piperazin-1-yl]ethyl}amino}-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione, and 5-{{2-[4-(10-bromodecanoyl)piperazin-1-yl]ethyl}amino}-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione, respectively, and the yields were 34 - 65%.

[0169] Synthetic Routes of Compounds PS29, PS30, and PS31

[0170]

[0171] Chemical Reaction Reagents and Reaction Conditions: (a) tert-Butyl 4-(2-aminoethyl)piperazine-1-carboxylate, N,N-dimethylformamide, triethylamine, 80 °C; (b) 1 M hydrogen chloride - ethyl acetate solution, reaction at room temperature; (c) bromo-substituted fatty acid, HBTU, triethylamine, N,N-dimethylformamide, reaction at room temperature; (d) potassium iodide, CPL042, triethylamine, N,N-dimethylformamide, 80 °C.

[0172] Synthesis of Compound 35

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

[0174] Synthesis of Compound 36a

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

[0176] Synthesis of Compound PS29

[0177] The reaction conditions for the synthesis of Compound PS29 were the same as those for the synthesis of Compound 9, but the starting materials were replaced with 4-{{{4-[1-(5-aminopentanoyl)piperidine-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, with a yield of 15%. The reaction conditions for the synthesis of Compound PS30 were the same as those for the synthesis of Compound 9, but the starting materials were replaced with 4-{{{4-[1-(6-aminohexanoyl)piperidine-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, with a yield of 20%. The reaction conditions for the synthesis of Compound PS31 were the same as those for the synthesis of Compound 9, but the starting materials were replaced with 4-{{{4-[1-(7-aminoheptanoyl)piperidine-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, with a yield of 17%.

[0178] Synthetic routes of Compounds PS32, PS33, and PS34.

[0179]

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

[0181] Synthesis of Compound 37

[0182] In a 25 mL eggplant-shaped flask, successively add Compound 12 (500 mg, 1.43 mmol), N-boc piperazine (293.0 mg, 1.57 mmol) and 1 mL of triethylamine, then add 10 mL of N,N-dimethylformamide, and stir until the solid mixture is completely dissolved. Place the reaction system in a water bath at 80 °C and stir for reaction. Monitor the reaction system by TLC until the raw material spots are completely converted. After the reaction is completed, pour the reaction system into 100 mL of water, add 3 × 50 mL of ethyl acetate for extraction, combine the organic layers, and dry to obtain the intermediate of boc-protected N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-2-(piperazin-1-yl)acetamide. After redissolving this intermediate with 10 mL of ethyl acetate, add 10 mL of 1 mol / L hydrogen chloride-ethyl acetate solution to remove the boc protecting group, obtaining the final product N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-2-(piperazin-1-yl)acetamide. The product is a white solid with a yield of 72%, ESI-MS (m / z): 400.9 [M+H]+.

[0183] Synthesis of Compound 38a

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

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

[0186] The synthesis reaction conditions of compound PS32 were the same as those of PS24, but the raw 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-dioxoisoindolin-4-yl]acetamide (1.00 equiv). The product was a white solid with a yield of 25%. The synthesis reaction conditions of compound PS33 were the same as those of PS24, but the raw 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-dioxoisoindolin-4-yl]acetamide (1.00 equiv). The product was a white solid with a yield of 32%. The synthesis reaction conditions of compound PS34 were the same as those of PS24, but the raw 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-dioxoisoindolin-4-yl]acetamide (1.00 equiv). The product was a white solid with a yield of 30%.

[0187] Synthetic routes of compounds PS35 and PS36

[0188]

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

[0190] Synthesis of compound 39a

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

[0192] Synthesis of compounds PS35 and PS36

[0193] The reaction conditions were the same as those in PS32, but the raw materials 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 with a yield of 32%. The synthesis reaction conditions of compound PS36 were the same as those in PS32, but the raw materials 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 with a yield of 30%. Compound spectral data:

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201] Example 2

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

[0203] Experimental method:

[0204] Experimental procedures: A549 and H1299 cells in the logarithmic growth phase were cultured in an incubator at 37°C with 5% carbon dioxide. The drug was prepared as a 10 mM stock solution using DMSO and then diluted to a specific concentration with complete medium. After the cells covered the culture dish, the cell medium was replaced with the drug-containing medium for incubation, and the cells were incubated for a specific time. After the incubation was completed, the drug-containing medium was aspirated, and the cells were washed twice with pre-cooled PBS and the PBS solution was removed. The cell lysate was obtained by diluting WB lysis buffer and 100 mM PMSF solution at a ratio of 100:1, or the cell phosphorylated protein lysate was prepared using WB lysis buffer and a mixed protease inhibitor solution for standby. The cell lysate was added to the cell culture dish, the cells were collected with a scraper and added to a 1.5 mL ep tube, lysed on ice for 30 min, then centrifuged at 12,000 rpm at 4°C for 10 min, and the supernatant was taken out and transferred to a new centrifuge tube to obtain the whole protein solution of tumor cells. The whole protein solution was quantified by the BCA method, and all the protein solutions were diluted to the same concentration according to the test results. Then the protein solution was mixed with 5X loading buffer and boiled in boiling water for 10 min to completely denature the protein. After cooling, SDS-PAGE electrophoresis could be carried out.

[0205] According to the protein molecular weight, a protein vertical separation gel composed of 10% separating gel and 5% stacking gel was prepared. The volume of the protein solution of 10 - 50 μg was calculated as the loading volume to keep the volume of each loading well consistent, and then the electrophoresis process could be carried out. During electrophoresis, the electrophoresis conditions for the stacking gel layer were 80 V for 30 min; the electrophoresis conditions for the separating gel layer were 120 - 150 V for 1 h. Electrophoresis was stopped when the bromophenol blue layer reached the bottom of the gel, and the transfer membrane process was carried out. After the PVDF membrane was cut and soaked in pure methanol for activation, the sponge, filter paper, gel segment, and PVDF membrane were sequentially placed in the transfer clip. After setting up the transfer sandwich, it was soaked in the transfer buffer. After transferring for 2 h at a constant current of 200 mA, the PVDF membrane was taken out for the blocking process. Blocking was carried out using 5% BSA or skim milk, and the PVDF membrane was soaked for 1 h. After the blocking was completed, the PVDF was cut according to the marker, rinsed 3 times in TBST for 5 min each time, and finally placed in the corresponding primary antibody solution for overnight incubation. After the overnight incubation was completed, the PVDF membrane segment was taken out, rinsed 3 times in TBST, then the PVDF membrane was placed in the secondary antibody dilution solution and incubated at room temperature for 1 h. After taking it out, it was rinsed 3 times in TBST, developed with ECL developer, photographed with a Tanon gel imaging system, and semi-quantified for the luminescent bands with ImageJ to obtain the experimental results.

[0206] Experimental results:

[0207] The inventors evaluated the ability of some PROTAC compound molecules to degrade PAK4 using immunoblotting and performed immunoblot analysis in the non-small cell lung cancer cell line A549. The results showed that the PAK4 ligand CPL055 did not have the activity to degrade PAK4, while the compound PS21 provided by the present invention exhibited the best degradation activity. However, the compound PS21 could degrade 42% of the PAK4 protein at 200 nM (see Figure 1 , Table 1).

[0208] The present invention detected the degradation of PAK4 protein by compound PS21 after adding different concentrations of compound PS21 into A549 cells. As Figure 2 shown: As shown by the Western Blot degradation results, in A549 cells, the content of the PAK4 protein kinase decreased significantly with the increase of the administration concentration, and 72% of the PAK4 protein kinase could be degraded at 5 μM. The concentration point for degrading 50% of PAK4 fell within the range of 0.5 - 1.5 μM, and the DC 50 value was approximately 1 μM. After 14 hours of treatment, in MDA-MB-231 cells treated with 5 μM of PS21, the content of the PAK4 protein decreased to 12%, and even when co-incubated at the lowest concentration of 50 nM, the content of the PAK4 protein in MDA-MB-231 cells did not reach 50%, indicating that PS21 could effectively degrade PAK4 in MDA-MB-231 and the protein content could not be restored to the state before administration.

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

[0210]

[0211]

[0212] Based on the above results, the inventors further investigated whether compound PS21 degraded PAK4 through the PAK4-PROTAC-E3 ligase ternary complex and the ubiquitin-proteasome-dependent degradation pathway. The present invention used the PAK4 inhibitor CPL055, the E3 ligase inhibitor pomalidomide, the proteasome inhibitor MG132, and the negative analysis compound PS21M that did not have affinity for the CRBN-E3 ligase to pretreat the cells for 2 hours, then added compound PS21 for treatment, and finally detected the change results of the PAK4 protein band by WB. As Figure 3 shown: The above treatments could all reverse the degradation effect of PS21 on PAK4, and the negative ligand PS21M also had no degradation effect, indicating that the degrader PS21 indeed degraded PAK4 through the PAK4-PROTAC-CRBN ternary complex and the ubiquitin-proteasome-dependent degradation pathway.

[0213] Example 3

[0214] For the compound PS21 in Example 3, a cell scratch migration experiment was carried out.

[0215] Experimental procedure:

[0216] Take A549, H1299 and MDA-MB-231 cells in the logarithmic growth phase, digest the cells with 0.25 mM trypsin solution, count them, and adjust the cell concentration to 5.0×10 5 cells / mL using complete medium. Add the cell solution with adjusted cell density to a 6-well plate, with a volume of 2 mL per well. Then place the 6-well plate in an incubator at 37°C and 5% CO 2 2 for 24 h until the cells adhere and cover the bottom of the wells. Take out the 6-well plate, use a 10 μL cell scraper to draw a straight line on the cell layer, then wash away the scraped cell debris with PBS solution, take a photo record at a magnification of 20X, and record it as the 0 h control. Then aspirate the PBS, add basic medium with different drug concentrations for drug incubation, and take photo records of the well plate at 24 h and 48 h. After the experiment, measure the length of the pictures at each time point with ImageJ, calculate the scratch healing ratio, and analyze it using a bar chart.

[0217] Experimental results:

[0218] The experimental results are as Figure 4 shown. The results of the scratch experiment show that at concentrations of 10 μM, 2.5 μM, and 0.64 μM, the compound PS21 can effectively inhibit the migration ability of A549, H1299, and MDA-MB-231 tumor cells, and PS21 shows similar inhibitory abilities against the three cell lines.

[0219] Example 4

[0220] For the compound PS21 in Example 4, a cell Transwell migration and invasion experiment was carried out.

[0221] Experimental procedure:

[0222] Take A549, H1299 and MDA-MB-231 cells in the logarithmic growth phase, digest the cells with 0.25 mM trypsin solution, count them, and adjust the cell concentration to 5.0×10 5 cells / mL using complete medium. Add the cell solution with adjusted cell density to a 6-well plate, with a volume of 2 mL per well. Then place the 6-well plate in an incubator at 37°C and 5% CO 2 2 for 24 h. Then discard the culture medium and replace it with complete medium containing the specified drug concentration for another 24 h of incubation. After the incubation, digest the cells in each test well with trypsin and centrifuge them. After removing the medium, adjust the cell density to 1.5×105 A cell suspension of cells / mL. Then, add the cell suspension to a Transwell chamber coated with Matrigel to detect the invasion ability of the cells, or add it to a Transwell chamber without Matrigel to detect the migration ability of the cells. Add 600 μL of complete medium with 20% FBS to the 24-well plate in the lower chamber. After incubating for 16 h, terminate the experiment. Take out the Transwell chamber, rinse it with PBS solution, soak the chamber in 4% paraformaldehyde for 20 min for fixation, rinse the Transwell chamber again with PBS solution to wash away the paraformaldehyde, soak the chamber in 0.1% methyl violet solution for 20 min for staining, take out the chamber, wash it with PBS, wipe off the cells on the inner side of the chamber with a cotton swab, and then take a photo to count the number of cells.

[0223] Experimental results:

[0224] The Transwell experiment further verified the inhibitory ability of compound PS21 on the migration and invasion activities of A549, MDA-MB-231 cells and H1299. The experimental results are as Figure 5 shown. It can be seen that compound PS21 can effectively inhibit the migration and invasion abilities of the three cell lines at concentrations of 10 μM, 5 μM, and 1 μM. At the same time, the migration and invasion abilities of the three cell lines showed the same concentration-dependent decrease after treatment with PS21, indicating that PS21 can effectively inhibit the migration and invasion activities of various tumor cells.

[0225] In summary, the inventors confirmed the degradation effect of this type of degrader on PAK4 through Western Blot experiments. Further, through scratch experiments and Transwell experiments, it was confirmed that this type of PAK4 degrader can effectively inhibit the migration and invasion of malignant tumor cells.

[0226] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A PROTAC compound targeting degradation of PAK4 as shown in Formula I or Formula II, or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite or prodrug thereof, characterized in that: The chemical structure of Formula I or Formula II is shown below: Wherein: E3 ligase ligand refers to a ligand molecule that binds to E3 ligase, E3 ligase includes CRBN, MDM2, IAP, DCAF or RNF, and the E3 ligase ligand includes:

2. The PROTAC compound targeting degradation of PAK4 according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite or prodrug thereof, characterized in that: The Linker is a connecting group selected from any one of the following groups:

3. The PROTAC compound targeting degradation of PAK4 according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite or prodrug thereof, characterized in that: The PROTAC compound targeting degradation of PAK4 is any one of the following compounds PS1-PS36 and PS21M:

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a PROTAC compound targeting degradation of PAK4 according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite or prodrug thereof, and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle or a combination thereof.

5. Use of the PROTAC compound targeting PAK4 degradation according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite or prodrug thereof, or the pharmaceutical composition according to claim 4 in the preparation of a drug for degrading PAK4 or inhibiting PAK4.

6. Use of a PROTAC compound targeting degradation of PAK4 according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite or prodrug thereof, or a pharmaceutical composition according to claim 4 in the preparation of a drug for treating or preventing PAK4-related diseases.

7. The use according to claim 6, characterized in that: The PAK4-related disease is a solid tumor.

8. Use of the PROTAC compound targeting degradation of PAK4 according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite or prodrug thereof, or the pharmaceutical composition according to claim 4 in the preparation of an anti-tumor drug.

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

10. The use according to claim 9, characterized in that: The tumor is a tumor with high expression of PAK4, or a tumor resistant to PAK4 inhibitor.

Images