Compound with HIF-1alpha protein degradation activity as well as synthesis method and application thereof

Abstract

The invention discloses a compound with hypoxia-inducible factor-1 alpha (HIF-1 alpha) protein degradation activity as well as a synthesis method and application thereof. The compound degrades HIF-1alpha protein through a ubiquitin-proteasome pathway, and is suitable for treating diseases such as cancers (such as pancreatic cancer and cervical cancer), encephaledema, atherosclerosis, rheumatoid arthritis, psoriasis, Alzheimer's disease and glaucoma related to overexpression of HIF-1alpha. Experiments show that the compound can significantly degrade HIF-1alpha protein, the synthesis method is simple and convenient, raw materials are easy to obtain, and the compound has wide drug development prospects.

Description

Technical Field

[0001] The present invention belongs to the fields of medicinal chemistry and pharmaceutical technology, and particularly relates to a hypoxia-inducible factor-1α (HIF-1α) protein compound, a synthesis method thereof, and applications thereof. Background Art

[0002] Pancreatic cancer is a highly invasive malignant tumor of the digestive tract system, characterized by a low early diagnosis rate, poor prognosis, high mortality, a single type of clinical drug, and limited efficacy. Cervical cancer shows a trend of becoming younger (Nat. Rev. Clin. Oncol. 2025, 22, 182–199; N. Engl. J. Med. 2025, 392:56-71.). Hypoxia-inducible factor-1 (HIF-1) is a key oxygen homeostasis regulatory protein in cells, composed of an oxygen-sensitive α-subunit (HIF-1α) and a ubiquitously expressed β-subunit (HIF-1β). Under normoxic conditions, the HIF-1α protein is hydroxylated by prolyl hydroxylase (PHDs), and then degraded via the ubiquitin-proteasome pathway mediated by the tumor suppressor protein pVHL. However, in hypoxic pancreatic cancer and cervical cancer tissues, the PHD enzyme is inactivated, resulting in the cytoplasmic accumulation of the HIF-1α protein. After further binding to the HIF-1β protein in the nucleus, a dimer is formed, which binds to the hypoxia response element and activates the overexpression of a variety of downstream HIF-regulated genes and pathway proteins, including genes and proteins related to glycolysis, angiogenesis, erythropoiesis, and invasion and metastasis, and is closely related to tumor proliferation, chemoradiotherapy resistance, connective tissue hyperplasia, and immune escape (Cancer Lett. 2020, 489:50–55; Cancer Cell Int. 2020, 20:273.). However, since HIF-1α belongs to a transcription factor protein and has no ATP binding pocket, it is difficult to find the binding pocket site, and no HIF-1α inhibitor has been approved for marketing clinically. The research and development of HIF-1α inhibitors face many difficulties.

[0003] Targeted protein degradation (TPD) is a rapidly developing biotechnology in recent years. By constructing heterobifunctional molecules, it utilizes the intracellular inherent ubiquitin-proteasome pathway (UPS), endogenous lysosomal pathway, and autophagy-lysosomal pathway to achieve rapid and effective degradation of target proteins (POI) (Sig. Transduct. Target Ther. 2022, 7(1):181; Eur. J. Med. Chem. 2021, 210:112993.). Currently, the TPD technologies that can achieve the degradation of transcription factor proteins mainly include: heterobifunctional proteolysis targeting chimeras (PROTACs), transcription factor-targeted chimeras (TF-PROTACs), heat shock protein-mediated targeting chimeras (HEMTACs) (Prog. Med. Chem. 2021, 60:67–190; J. Am. Chem. Soc. 2021, 143(23):8902–8910; J. Med. Chem. 2023, 66(1):733–751.). PROTACs are the earliest reported and most widely studied TPD technology. By linking POI and E3 ligase ligands such as VHL and CRBN, they mediate the rapid and efficient ubiquitination degradation of POI, showing good therapeutic potential in the anti-tumor field. Degraders represented by ARV-471 and ARV-110 are undergoing clinical studies (Drug Discov. Today. 2023, 28(8):103643; J. Clin. Oncol. 2022, 40, 6suppl; NCT05909397; NCT03888612). PROTACs degrade target proteins in a catalytic dose cycle through a unique mechanism of action, have significant degradation activity against easily mutated proteins and undruggable target proteins, overcome drug resistance, and enhance anti-tumor activity (Cell, 2020, 181(1):102–114; Cell Discovery, 2019, 5:10.). However, there is currently no literature report on HIF-1α degraders. Therefore, promoting the rapid degradation of intracellular overexpressed HIF-1α protein by discovering effective HIF-1α-PROTACs degraders is an important aspect of molecular targeted therapy for malignant tumors such as pancreatic cancer and cervical cancer, and has potential therapeutic effects on hypoxic diseases such as cerebral edema, atherosclerosis, rheumatoid arthritis, psoriasis, Alzheimer's disease, and glaucoma, with a profound market application prospect. Summary of the Invention

[0004] The object of the present invention is to provide a compound having hypoxia-inducible factor-1α (HIF-1α) protein degradation activity, its synthesis method, and its application in the preparation of drugs for treating diseases related to overexpression of HIF-1α, particularly its use in drugs for treating diseases such as cancer, cerebral edema, atherosclerosis, rheumatoid arthritis, psoriasis, Alzheimer's disease, glaucoma, etc.

[0005] In the first aspect of the present invention, there is provided a compound having hypoxia-inducible factor-1α (HIF-1α) protein degradation activity, or a pharmaceutically acceptable salt, stereoisomer, metabolite, solvate, polymorph, isotope-labeled substance or prodrug thereof:

[0006]

[0007] In the formula:

[0008] X is a carbonyl group or a methylene group; preferably a methylene group;

[0009] n is a natural integer between 3 and 10, representing the number of C3-C10 saturated straight-chain alkyl groups; n is preferably 7.

[0010] According to some embodiments of the present invention, the compound represented by the above formula (I) may be the compounds represented by formula II and formula III:

[0011]

[0012] Specifically, the compound represented by formula (I) of the present invention is specifically the following compound:

[0013]

[0014] In the second aspect of the present invention, there is provided a preparation method of the compound having the general formula (Ⅰ) as described in the first aspect of the present invention, and the synthesis route is as follows:

[0015]

[0016] (1) 4-(1-Piperazinyl)phenol (II-1) is subjected to two-step nucleophilic substitution reactions with di-tert-butyl dicarbonate and methyl 2-bromoacetate to obtain intermediate II-2; the solvent for the nucleophilic substitution reaction is selected from dichloromethane, acetonitrile, N,N-dimethylformamide or dimethyl sulfoxide, preferably dichloromethane or acetonitrile;

[0017] (2) The intermediate (II-2) undergoes an ester hydrolysis reaction under strong basic conditions and then an amide condensation reaction with methyl 3-amino-4-hydroxybenzoate to obtain intermediate II-3; the strong base used is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide or potassium tert-butoxide, and lithium hydroxide is most preferred; the coupling reagent for the amide condensation reaction is selected from 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and HATU is preferred;

[0018] (3) Intermediate II-3 is prepared to obtain the HIF-1α binding ligand compound II-4 through a Boc deprotection reaction under acidic conditions, and the acid used is selected from trifluoroacetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, oxalic acid, and trifluoroacetic acid is most preferred;

[0019] (4) Monomethyl alkanedioates with different alkyl chain lengths (intermediate II-5) and the VHL proteasome inhibitor VH032 undergo an amide condensation reaction to obtain intermediate II-6; the coupling reagent for the amide condensation reaction is selected from HATU, EDCI, O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), and HATU is most preferred;

[0020] (5) Intermediate (II-6) undergoes an ester hydrolysis reaction under strong basic conditions to obtain intermediate II-7, and then an amide condensation reaction with the HIF-1α binding ligand compound II-4 to obtain the compound of formula II, and the strong base used is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, potassium tert-butoxide, cesium carbonate, and lithium hydroxide is most preferred; the coupling reagent for the amide condensation reaction is selected from HATU, EDCI or 1-hydroxybenzotriazole (HOBT), and HATU is most preferred;

[0021] (6) Bromoalkyl acids with different alkyl chain lengths (intermediate III-1) and the VHL proteasome inhibitor VH032 undergo an amide condensation reaction to obtain bromo intermediate III-2, and then a nucleophilic substitution reaction with the HIF-1α binding ligand compound II-4 to obtain the compound of formula III; the solvent for the nucleophilic substitution reaction is selected from N,N-dimethylformamide, acetonitrile, dichloromethane or dimethyl sulfoxide, and N,N-dimethylformamide is most preferred; the base used for the nucleophilic reaction is selected from potassium carbonate, sodium carbonate, sodium hydroxide or cesium carbonate, and potassium carbonate is most preferred.

[0022] The "compounds" described in the present invention include HIF-1α protein degraders and their pharmaceutically acceptable salts, stereoisomers, enantiomers, diastereoisomers, tautomers or mixtures thereof, metabolites, solvates, polymorphs, isotopically labeled compounds and prodrugs.

[0023] The "pharmaceutically acceptable salts" described in the present invention can be inorganic acid salts or organic acid salts of the compounds represented by formula (I). The inorganic acids can include but are not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. The organic acids can include but are not limited to acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, benzenesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

[0024] The "C3-C10 alkyl" described in the present invention refers to a straight-chain or branched-chain alkyl containing 3-10 (3, 4, 5, 6, 7, 8, 9, 10) carbon atoms, such as n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl, isononyl, n-decyl, isodecyl, etc. The "C3-C10 alkyl" described in the present invention refers to the above examples containing 3-10 carbon atoms.

[0025] The HIF-1α protein degradants and pharmaceutically acceptable salts described in the present invention also include solvates or hydrates. Usually, the forms of solvates or hydrates are regarded as equivalent to the non-solvate forms in terms of use and are included in the protection scope of the present invention. In addition, some compounds in the present invention may exist in polymorphic or amorphous crystal forms. Regardless of their physical crystal forms, they are regarded as equivalent and included in the content of the present invention.

[0026] The third aspect of the present invention provides a pharmaceutical composition, comprising the compound represented by formula (I) or its pharmaceutically acceptable salt, stereoisomer, enantiomer, diastereomer, tautomer or a mixture thereof, metabolite, solvate, polymorph, isotope-labeled substance or prodrug, and at least one pharmaceutically acceptable carrier; the pharmaceutical composition may further comprise excipients, odorants, flavorants or diluents, etc.

[0027] The fourth aspect of the present invention provides a pharmaceutical preparation, comprising the compound represented by formula (I) or its pharmaceutically acceptable salt, stereoisomer, enantiomer, diastereomer, tautomer or a mixture thereof, metabolite, solvate, polymorph, isotope-labeled substance or prodrug, and the pharmaceutical composition described in the third aspect. The pharmaceutical preparation is preferably an oral solid preparation, liquid preparation or topical patch.

[0028] The fifth aspect of the present invention provides the use of the compound represented by formula (I), or the pharmaceutical composition, or the pharmaceutical preparation, in the preparation of an HIF-1α protein degradant, especially an HIF-1α-PROTAC degradant.

[0029] In the sixth aspect of the present invention, there is provided the use of a compound of formula (I), or a pharmaceutical composition, or a pharmaceutical preparation, in the preparation of a therapeutic drug for hypoxia-related diseases with overexpression of HIF-1α, said diseases including cancer, cerebral edema, atherosclerosis, rheumatoid arthritis, psoriasis, Alzheimer's disease, glaucoma, etc., and said cancer including cervical cancer, pancreatic cancer, breast cancer, etc.

[0030] Advantages of the present invention over the prior art:

[0031] (1) Targeting undruggable targets: As a transcription factor, HIF-1α lacks traditional drug-binding sites (such as ATP pockets), and it is difficult to directly inhibit in the prior art. The present invention for the first time realizes the ubiquitination and degradation of HIF-1α through PROTAC technology, breaking through the limitation of "undruggable targets".

[0032] (2) Broad-spectrum therapeutic potential: The degrader of the present invention can be applied to a variety of diseases with overexpression of HIF-1α (such as pancreatic cancer, cervical cancer, cerebral edema, atherosclerosis, etc.), covering the fields of tumors and chronic diseases.

[0033] (3) Efficient catalytic degradation mechanism: Traditional inhibitors need to continuously occupy the target, while PROTAC degrader acts in a catalytic dose cycle, with high degradation efficiency and can overcome drug resistance (the degradation rate of compound 12 is significant at 20 μM for 36 hours).

[0034] (2) Structure optimization and druggability: By adjusting the length of the linker chain (such as C7 alkyl chain) to balance the degradation activity and cell permeability, the IC 50 of compound 12 against Hela cells reaches 10.10 μM, which is significantly better than the non-degradable inhibitors in similar studies.

[0035] (4) Simple synthesis process: The synthesis method is simple, the raw materials are cheap and easily available, and the reaction conditions are mild (room temperature condensation, conventional hydrolysis), which is suitable for industrial scale-up production. Description of the drawings

[0036] Figure 1 It is for the study of the in vitro HIF-1α protein degradation activity and degradation mechanism of the target compound 12 against Hela cells. A - B: Degradation activity of compound 12 at different concentrations against HIF-1α protein; C - D: Degradation activity of 20 μM compound 12 against HIF-1α protein at different times; E - F: Mechanism study of the ubiquitination degradation pathway of compound 12 against HIF-1α protein. Detailed implementation manners

[0037] To ensure a clear and complete description of the research plan of the present invention, it is further illustrated by specific examples. It should be particularly noted that the scientific terms involved in the examples are well-known in the field. The preparation methods provided in the examples are only for illustrative purposes and do not constitute a non-exclusive limitation to the present invention. Under the premise of the core concept of the present invention, simple improvements made to the preparation method shall be regarded as within the protection scope of the present invention. The reagents and materials used in the present invention can be obtained through commercial channels.

[0038] Example 1 Synthesis of 6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexanoic acid (Synthesis of Compound 1)

[0039] 1. Synthesis of methyl 4-hydroxy-3-(2-(4-(piperazin-1-yl)phenoxy)acetamido)benzoate trifluoroacetate (Compound LW6-L), and its synthetic route is as follows:

[0040]

[0041] (1) Synthesis of tert-butyl 4-(4-(2-methoxy-2-oxoethoxy)phenyl)piperazin-1-ylcarbamate (Intermediate L-2)

[0042] Weigh 4-(1-piperazinyl)phenol (L-1) (500 mg, 2.8 mmol) and 4-dimethylaminopyridine (103 mg, 0.8 mmol) into a 50 mL reaction flask, add 10 mL of tetrahydrofuran solution, and slowly dropwise add a tetrahydrofuran solution of di-tert-butyl dicarbonate (643 mg, 3.0 mmol). React at 0 °C for 4 h. After the reaction is completed, concentrate the solvent, add 20 mL of purified water, extract twice with ethyl acetate, combine the organic phases, dry over anhydrous Na2SO4, evaporate the solvent, add 15 mL of acetonitrile to dissolve, then add methyl bromoacetate (430 mg, 2.8 mmol) and potassium carbonate (775 mg, 5.6 mmol), and reflux at 90 °C for 4 h. After the reaction is completed, concentrate the solvent, extract with ethyl acetate-water, and purify by column chromatography (DCM:MeOH = 120:1 - 80:1) to obtain 600 mg of a white solid, with an overall yield of 73% in two steps. 1 H NMR (400 MHz, DMSO-d6) δ 6.98 - 6.73 (m, 4H), 4.70 (s, 2H), 3.68 (s, 3H), 3.44 (s, 4H), 3.03 - 2.87 (m, 4H), 1.41 (s, 9H).

[0043] (2) Synthesis of tert-butyl 4-(4-(2-((2-hydroxy-5-(methoxycarbonyl)phenyl)amino)-2-oxoethoxy)phenyl)piperazin-1-ylcarbamate (Intermediate L-3)

[0044] Weigh intermediate L-2 (600 mg, 1.7 mmol) into a 50 mL reaction flask, add 16 mL of a tetrahydrofuran-distilled water solution (3:1), add lithium hydroxide monohydrate (359 mg, 8.5 mmol), and stir the reaction at room temperature for 10 h. After the reaction is completed, remove some of the solvent by evaporation, adjust the pH to 5 with 1 M HCl solution, extract with ethyl acetate, and concentrate. Dissolve in 10 mL of N,N-dimethylformamide solution, and successively add 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATU (780 mg, 2.1 mmol) and triethylamine (259 mg, 2.5 mmol), and stir the reaction at room temperature for 5 h. After the reaction is completed, add 50 mL of purified water, extract with ethyl acetate, combine the organic phases, dry over anhydrous Na2SO4, and purify by column chromatography (DCM:MeOH = 120:1 - 80:1) to obtain 480 mg of a white solid, with an overall yield of 58% for the two steps. 1 H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 9.20 (s, 1H), 8.70 (s, 1H), 7.62 (dd, J = 8.4, 2.1 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.94 (s, 4H), 4.70 (s, 2H), 3.80 (s, 3H), 3.57 - 3.36 (m, 4H), 3.05 - 2.84 (m, 4H), 1.42 (s, 9H).

[0045] (3) Synthesis of methyl 4-hydroxy-3-(2-(4-(piperazin-1-yl)phenoxy)acetamido)benzoate trifluoroacetate (Intermediate LW6-L)

[0046] Weigh intermediate L-3 (480 mg, 1.0 mmol) into a 50 mL reaction flask, dissolve it in 12 mL of dichloromethane solution, and slowly add 4.5 mL of trifluoroacetic acid solution, then stir the reaction at room temperature for 3 h. After the reaction is completed, remove the solvent by evaporation to obtain 470 mg of a light red solid, with a yield of 95%. 1 H NMR (400 MHz, DMSO-d6) δ 11.20 (s, 1H), 9.23 (s, 1H), 8.88 (s, 2H), 8.69 (s, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 6.97 (s, 4H), 4.72 (s, 2H), 3.80 (s, 3H), 3.23 (s, 8H).

[0047] 2. Synthesis of Compound 1, and its synthetic route is as follows:

[0048]

[0049] (1) Synthesis of methyl 6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexanoate (Intermediate 1-2)

[0050] Weigh methyl adipate (Intermediate 1-1) (64 mg, 0.4 mmol) and the VHL ligand inhibitor (2S,4R)-1-((S)-2-amino-3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (150 mg, 0.3 mmol) into a 50 mL reaction flask, add 10 mL of N,N-dimethylformamide solution to dissolve, and successively add HATU (158 mg, 0.4 mmol) and triethylamine (98 mg, 1.0 mmol). Stir the reaction at room temperature for 5 h. After the reaction is completed, add 50 mL of purified water, extract with ethyl acetate, combine the organic phases, dry over anhydrous Na2SO4, and purify by column chromatography (DCM:MeOH = 120:1 - 80:1) to obtain 114 mg of a white solid, with a yield of 65%. 1 1H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.57 (t, J = 6.1 Hz, 1H), 7.88 (d, J = 9.3 Hz, 1H), 7.40 (q, J = 8.2 Hz, 4H), 5.14 (d, J = 3.5 Hz, 1H), 4.53 (d, J = 9.3 Hz, 1H), 4.47 - 4.39 (m, 2H), 4.35 (s, 1H), 4.23 - 4.16 (m, 1H), 3.66 (q, J = 5.1, 3.3 Hz, 2H), 3.57 (s, 3H), 2.44 (s, 3H), 2.31 (d, J = 6.8 Hz, 2H), 2.24 (d, J = 7.1 Hz, 2H), 2.07 - 1.98 (m, 1H), 1.90 (ddd, J = 13.0, 8.6, 4.7 Hz, 1H), 1.53 - 1.46 (m, 4H), 0.93 (s, 9H).

[0051] (2) Synthesis of 6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexanoic acid (Intermediate 1-3)

[0052] Weigh intermediate 1-2 (114 mg, 0.2 mmol) into a 25 mL reaction flask, add 12 mL of a tetrahydrofuran-distilled water solution (3:1), add lithium hydroxide monohydrate (46 mg, 1.1 mmol), and stir the reaction at room temperature for 6 h. After the reaction is completed, evaporate part of the solvent, adjust the pH to 5 with 1 M HCl solution, filter with suction after the solid precipitates, wash with water, and dry to obtain 95 mg of a pale yellow solid with a yield of 85%. 1H NMR (400 MHz, DMSO-d6) δ 11.94 (s, 1H), 8.96 (s, 1H), 8.57 (t, J = 6.1 Hz, 1H), 7.84 (d, J = 9.3 Hz, 1H), 7.39 (q, J = 8.3 Hz, 4H), 5.19 (d, J = 3.4 Hz, 1H), 4.52 (d, J = 9.3 Hz, 1H), 4.41 (t, J = 8.0 Hz, 2H), 4.34 (s, 1H), 4.21 (dd, J = 15.9, 5.5 Hz, 1H), 3.65 (d, J = 4.0 Hz, 2H), 2.43 (s, 3H), 2.27 - 2.04 (m, 4H), 2.07 - 1.98 (m, 1H), 1.89 (ddd, J = 13.0, 8.7, 4.6 Hz, 1H), 1.46 (p, J = 7.6, 7.1 Hz, 4H), 0.92 (s, 9H).

[0053] (3) Synthesis of 6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexanoic acid (Compound 1)

[0054] Weigh intermediate 1-2 (80 mg, 0.14 mmol) and intermediate LW6-L (78 mg, 0.16 mmol) into a 25 mL reaction flask, add 8 mL of N,N-dimethylformamide solution to dissolve, successively add HATU (71 mg, 0.19 mmol) and triethylamine (36 mg, 0.36 mmol), and stir the reaction at room temperature for 7 h. After the reaction is completed, add 15 mL of purified water, extract with ethyl acetate, combine the organic phases, dry over anhydrous Na2SO4, and purify by column chromatography (DCM:MeOH = 80:1 - 15:1) to obtain 47 mg of a white solid with a yield of 36%. 11H NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H), 9.25 (s, 1H), 8.98 (s, 1H), 8.69 (d, J = 2.1 Hz, 1H), 8.63 (t, J = 6.2 Hz, 1H), 7.88 (d, J = 9.3 Hz, 1H), 7.59 (dd, J = 8.4, 2.2 Hz, 1H), 7.39 (q, J = 8.3 Hz, 4H), 7.13 (d, J = 8.5 Hz, 1H), 6.93 (s, 4H), 5.22 (d, J = 3.5 Hz, 1H), 4.69 (s, 2H), 4.53 (d, J = 9.4 Hz, 1H), 4.47 - 4.38 (m, 2H), 4.34 (s, 1H), 4.25 - 4.20 (m, 1H), 3.78 (s, 3H), 3.65 (dd, J = 10.9, 7.0 Hz, 2H), 3.56 (t, J = 5.2 Hz, 4H), 2.98 (d, J = 23.2 Hz, 4H), 2.43 (s, 3H), 2.33 (s, 2H), 2.29 - 2.13 (m, 2H), 2.04 (t, J = 10.3 Hz, 1H), 1.92 - 1.86 (m, 1H), 1.54 - 1.44 (m, 4H), 0.91 (d, J = 8.2 Hz, 9H). HRMS (ESI) m / z calculated for C 48 H 59 N7NaO 10 S [M+Na] + 948.3936, found 948.3924.

[0055] Example 2: Preparation of methyl 4-hydroxy-3-(2-(4-(4-(7-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-7-oxoheptanoyl)piperazin-1-yl)phenoxy)acetamido)benzoate (Compound 2) in the present invention

[0056]

[0057] Adapt to change the raw material compound (replace the monomethyl adipate raw material in step 2 of Example 1 with the monomethyl pimelate raw material), and the remaining steps are the same as in Example 1 to obtain a white solid product with a yield of 42%. 11H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 9.21 (d, J = 1.8 Hz, 1H), 8.98 (s, 1H), 8.70 (d, J = 2.1 Hz, 1H), 8.57 (t, J = 6.1 Hz, 1H), 7.87 (d, J = 9.3 Hz, 1H), 7.62 (dd, J = 8.5, 2.2 Hz, 1H), 7.40 (q, J = 8.3 Hz, 4H), 6.98 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 3.4 Hz, 4H), 5.14 (d, J = 3.4 Hz, 1H), 4.70 (d, J = 2.3 Hz, 2H), 4.55 (d, J = 9.3 Hz, 1H), 4.43 (t, J = 8.0 Hz, 2H), 4.35 (s, 1H), 4.22 (dd, J = 15.9, 5.5 Hz, 1H), 3.66 (d, J = 4.3 Hz, 2H), 3.59 - 3.55 (m, 4H), 3.02 - 2.94 (m, 4H), 2.45 (s, 3H), 2.32 (t, J = 7.6 Hz, 2H), 2.29 - 2.11 (m, 2H), 2.04 (ddd, J = 13.0, 7.7, 2.3 Hz, 1H), 1.90 (ddd, J = 12.9, 8.6, 4.6 Hz, 1H), 1.50 (q, J = 6.7, 5.3 Hz, 4H), 1.25 (dd, J = 13.7, 6.2 Hz, 2H), 0.94 (s, 9H). HRMS (ESI) m / z calculated for C 49 H 61 N7NaO 10 S [M+Na] + 962.4093, found 962.4079.

[0058] Example 3: Preparation of methyl 4-hydroxy-3-(2-(4-(4-(8-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-8-oxooctanoyl)piperazin-1-yl)phenoxy)acetamido)benzoate (Compound 3) in the present invention

[0059]

[0060] Adapt to change the starting compound (replace the monomethyl adipate starting material in Step 2 of Example 1 with the monomethyl suberate starting material), and the remaining steps are the same as in Example 1 to obtain a white solid product with a yield of 52%. 11H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1H), 9.19 (s, 1H), 8.97 (s, 1H), 8.69 (d, J = 2.1 Hz, 1H), 8.57 (t, J = 6.1 Hz, 1H), 7.85 (d, J = 9.3 Hz, 1H), 7.61 (dd, J = 8.5, 2.2 Hz, 1H), 7.39 (q, J = 8.2 Hz, 4H), 6.97 (d, J = 8.5 Hz, 1H), 6.93 (d, J = 3.4 Hz, 4H), 5.13 (d, J = 3.4 Hz, 1H), 4.68 (s, 2H), 4.54 (d, J = 9.3 Hz, 1H), 4.47 - 4.39 (m, 2H), 4.34 (d, J = 5.2 Hz, 1H), 4.21 (dd, J = 15.9, 5.5 Hz, 1H), 3.79 (s, 3H), 3.65 (d, J = 4.0 Hz, 2H), 3.56 (q, J = 5.0 Hz, 4H), 2.98 (dt, J = 21.4, 5.2 Hz, 4H), 2.44 (s, 3H), 2.31 (t, J = 7.6 Hz, 2H), 2.28 - 2.09 (m, 2H), 2.07 - 2.00 (m, 1H), 1.89 (ddd, J = 12.9, 8.6, 4.6 Hz, 1H), 1.47 (p, J = 7.0, 5.9 Hz, 4H), 1.30 - 1.23 (m, 4H), 0.93 (s, 9H). HRMS (ESI) m / z calculated for C 50 H 63 N7NaO 10 S [M+Na] + 976.4249, found 976.4254.

[0061] Example 4: Preparation of methyl 4-hydroxy-3-(2-(4-(4-(9-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-9-oxononanoyl)piperazin-1-yl)phenoxy)acetamido)benzoate (Compound 4) in the present invention

[0062]

[0063] Adapt to change the starting compound (replace the monomethyl adipate starting material in Step 2 of Example 1 with monomethyl azelate starting material), and the remaining steps are the same as in Example 1 to obtain a white solid product with a yield of 42%. 11H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 9.20 (d, J = 1.8 Hz, 1H), 8.98 (s, 1H), 8.70 (d, J = 2.1 Hz, 1H), 8.58 (t, J = 6.1 Hz, 1H), 7.86 (d, J = 9.3 Hz, 1H), 7.40 (q, J = 8.3 Hz, 4H), 6.98 (d, J = 8.5 Hz, 1H), 6.94 (d, J = 4.0 Hz, 4H), 5.13 (d, J = 3.4 Hz, 1H), 4.69 (d, J = 2.4 Hz, 2H), 4.54 (d, J = 9.4 Hz, 1H), 4.44 (q, J = 7.3 Hz, 2H), 4.34 (d, J = 5.7 Hz, 1H), 4.21 (dd, J = 15.9, 5.5 Hz, 1H), 3.79 (s, 3H), 3.66 (d, J = 3.9 Hz, 2H), 3.57 (d, J = 10.0 Hz, 4H), 2.99 (s, 4H), 2.44 (s, 3H), 2.32 (t, J = 7.6 Hz, 2H), 2.27 - 2.09 (m, 2H), 2.08 - 1.97 (m, 1H), 1.90 (ddd, J = 12.9, 8.8, 4.6 Hz, 1H), 1.48 (h, J = 7.4 Hz, 4H), 1.24 (d, J = 11.0 Hz, 6H), 0.93 (s, 9H). HRMS (ESI) m / z calculated for C 51 H 65 N7NaO 10 S [M+Na] + 990.4406, found 990.4425.

[0064] Example 5: Preparation of methyl 4-hydroxy-3-(2-(4-(4-(10-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-10-oxodecanoyl)piperazin-1-yl)phenoxy)acetamido)benzoate (Compound 5) in the present invention

[0065]

[0066] Adapt to change the starting compound (replace the monomethyl adipate starting material in Step 2 of Example 1 with monomethyl sebacate starting material), and the remaining steps are the same as in Example 1 to obtain a white solid product with a yield of 47%. 11H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.21 (s, 1H), 8.99 (s, 1H), 8.71 (d, J = 2.2 Hz, 1H), 8.59 (t, J = 6.1 Hz, 1H), 7.86 (d, J = 9.3 Hz, 1H), 7.63 (dd, J = 8.5, 2.2 Hz, 1H), 7.41 (q, J = 8.4 Hz, 4H), 6.99 (d, J = 8.5 Hz, 1H), 6.94 (s, 4H), 5.15 (s, 1H), 4.70 (s, 2H), 4.56 (d, J = 9.4 Hz, 1H), 4.45 (q, J = 7.3 Hz, 2H), 4.36 (s, 1H), 4.22 (dd, J = 15.9, 5.5 Hz, 1H), 3.80 (s, 3H), 3.67 (d, J = 4.0 Hz, 2H), 3.57 (q, J = 5.1 Hz, 4H), 2.99 (dt, J = 22.2, 5.2 Hz, 4H), 2.45 (s, 3H), 2.32 (t, J = 7.5 Hz, 2H), 2.28 - 2.10 (m, 2H), 2.08 - 1.98 (m, 1H), 1.91 (d, J = 2.4 Hz, 1H), 1.49 (t, J = 8.1 Hz, 4H), 1.24 (d, J = 5.0 Hz, 8H), 0.94 (s, 9H). HRMS (ESI) m / z calculated for C 52 H 67 N7NaO 10 S [M+Na] + 1004.4562, found 1004.4543.

[0067] Example 6: Preparation of methyl 4-hydroxy-3-(2-(4-(4-(11-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-11-oxoundecanoyl)piperazin-1-yl)phenoxy)acetamido)benzoate (Compound 6) in the present invention

[0068]

[0069] Adapt to change the starting compound (replace the monomethyl adipate starting material in Step 2 of Example 1 with monomethyl undecanedioate starting material), and the remaining steps are the same as in Example 1 to obtain a white solid product with a yield of 44%. 11H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 9.21 (d, J = 1.7 Hz, 1H), 8.99 (s, 1H), 8.70 (d, J = 2.2 Hz, 1H), 8.58 (t, J = 6.1 Hz, 1H), 7.86 (d, J = 9.4 Hz, 1H), 7.62 (dd, J = 8.5, 2.2 Hz, 1H), 7.40 (q, J = 8.3 Hz, 5H), 6.98 (d, J = 8.5 Hz, 1H), 6.94 (s, 4H), 5.14 (d, J = 3.5 Hz, 1H), 4.69 (s, 2H), 4.55 (d, J = 9.4 Hz, 1H), 4.47 - 4.40 (m, 2H), 4.36 (d, J = 5.1 Hz, 1H), 4.26 - 4.18 (m, 1H), 3.80 (s, 3H), 3.70 - 3.63 (m, 2H), 3.58 - 3.54 (m, 4H), 2.98 (dt, J = 15.8, 5.5 Hz, 4H), 2.45 (s, 3H), 2.32 (t, J = 7.5 Hz, 2H), 2.28 - 2.09 (m, 2H), 2.07 - 2.01 (m, 1H), 1.92 (dd, J = 8.7, 4.4 Hz, 1H), 1.50 (dd, J = 14.5, 7.3 Hz, 4H), 1.24 (d, J = 5.0 Hz, 10H), 0.94 (s, 9H). HRMS (ESI) m / z calculated for C 53 H 69 N7NaO 10 S [M+Na] + 1018.4719, found 1018.4724.

[0070] Example 7: Preparation of methyl 4-hydroxy-3-(2-(4-(4-(12-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-12-oxododecanoyl)piperazin-1-yl)phenoxy)acetamido)benzoate (Compound 7) in the present invention

[0071]

[0072] Adapt to change the starting compound (replace the monomethyl adipate starting material in Step 2 of Example 1 with monomethyl dodecanedioate), and the remaining steps are the same as in Example 1 to obtain a white solid product with a yield of 42%. 11H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 9.20 (s, 1H), 8.98 (s, 1H), 8.69 (d, J = 2.2 Hz, 1H), 8.57 (t, J = 6.1 Hz, 1H), 7.85 (d, J = 9.3 Hz, 1H), 7.61 (dd, J = 8.4, 2.2 Hz, 1H), 7.39 (q, J = 8.2 Hz, 4H), 6.98 (d, J = 8.5 Hz, 1H), 6.93 (s, 4H), 5.12 (d, J = 3.5 Hz, 1H), 4.68 (s, 2H), 4.54 (d, J = 9.4 Hz, 1H), 4.42 (td, J = 8.8, 4.7 Hz, 2H), 4.34 (s, 1H), 4.21 (dd, J = 15.9, 5.5 Hz, 1H), 3.79 (s, 3H), 3.65 (d, J = 5.5 Hz, 2H), 3.56 (dt, J = 7.6, 3.6 Hz, 4H), 2.98 (dq, J = 15.1, 5.0 Hz, 4H), 2.44 (s, 3H), 2.31 (t, J = 7.5 Hz, 2H), 2.17 (dd, J = 55.5, 7.1 Hz, 2H), 2.00 (d, J = 14.9 Hz, 1H), 1.94 - 1.85 (m, 1H), 1.52 - 1.41 (m, 4H), 1.27 - 1.20 (m, 12H), 0.93 (s, 9H). HRMS (ESI) m / z calculated for C 54 H 71 N7NaO 10 S [M+Na] + 1032.4875, found 1032.4848.

[0073] Example 8: Preparation of methyl 4-hydroxy-3-(2-(4-(4-(6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexyl)piperazin-1-yl)phenoxy)acetamido)benzoate (Compound 8) in the present invention, and its synthetic route is as follows:

[0074]

[0075] (1) Synthesis of (2S,4R)-1-((S)-2-(4-bromobutanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (Compound 8-2)

[0076] Weigh 4-bromobutyric acid (intermediate 8-1) (88 mg, 0.5 mmol) and the VHL ligand inhibitor (2S,4R)-1-((S)-2-amino-3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (172 mg, 0.4 mmol) into a 25 mL reaction flask, add 10 mL of N,N-dimethylformamide solution to dissolve, and successively add HATU (190 mg, 0.5 mmol) and triethylamine (131 mg, 1.3 mmol), then stir the reaction at room temperature for 5 h. After completion, add 20 mL of purified water, extract with ethyl acetate, combine the organic phases, dry over anhydrous Na2SO4, and purify by column chromatography (DCM:MeOH = 120:1 - 60:1) to obtain 98 mg of a white solid with a yield of 42%. 1 H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.58 (t, J = 6.1 Hz, 1H), 8.01 (d, J = 9.3 Hz, lH), 7.40 (q, J = 8.3 Hz, 4H), 5.14 (s, 1H), 4.54 (d, J = 9.4 Hz, 1H), 4.47 - 4.39 (m, 2H), 4.35 (s, 1H), 4.24 - 4.18 (m, 1H), 3.65 (t, J = 5.8 Hz, 2H), 3.61 (dt, J = 6.7, 3.4 Hz, 2H), 2.44 (s, 3H), 2.41 - 2.27 (m, 2H), 2.03 (dd, J = 13.5, 7.9 Hz, 1H), 1.98 - 1.84 (m, 3H), 0.94 (s, 9H).

[0077] (2) Synthesis of methyl 4-hydroxy-3-(2-(4-(4-(6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexyl)piperazin-1-yl)phenoxy)acetamido)benzoate (Compound 8)

[0078] Weigh intermediate 8-2 (98 mg, 0.17 mmol), intermediate LW6-L (100 mg, 0.2 mmol), and potassium carbonate (69 mg, 0.5 mmol) into a 25 mL reaction flask, add 6 mL of N,N-dimethylformamide solution to dissolve, and reflux the reaction at 80 °C for 4 h. After the reaction is completed, add 15 mL of purified water, extract with ethyl acetate, combine the organic phases, dry over anhydrous Na2SO4, and purify by column chromatography (DCM:MeOH = 60:1 - 15:1) to obtain 27 mg of a white solid with a yield of 18%. 11H NMR (400 MHz, DMSO-d6) δ 9.16 (s, 1H), 8.99 (d, J = 1.7 Hz, 1H), 8.80 (d, J = 2.3 Hz, 1H), 8.60 (t, J = 6.1 Hz, 1H), 7.97 (d, J = 9.4 Hz, 1H), 7.74 (dd, J = 8.6, 2.2 Hz, 1H), 7.40 (q, J = 8.2 Hz, 4H), 7.19 (d, J = 8.7 Hz, 1H), 6.97 (dd, J = 7.9, 5.1 Hz, 4H), 5.17 (d, J = 3.9 Hz, 1H), 4.71 (d, J = 6.5 Hz, 2H), 4.57 (d, J = 9.3 Hz, 1H), 4.48 - 4.39 (m, 2H), 4.35 (s, 1H), 4.22 (dd, J = 15.8, 5.4 Hz, 1H), 4.14 (d, J = 6.4 Hz, 2H), 3.82 (s, 3H), 3.66 (d, J = 4.8 Hz, 2H), 3.23 (s, 4H), 3.17 (s, 2H), 3.09 (d, J = 7.3 Hz, 2H), 2.44 (s, 3H), 2.41 - 2.33 (m, 1H), 2.27 - 2.20 (m, 1H), 2.04 (t, J = 10.3 Hz, 1H), 1.90 (ddd, J = 12.9, 8.4, 4.4 Hz, 1H), 1.76 - 1.69 (m, 2H), 0.93 (s, 9H). HRMS (ESI) m / z calculated for C 46 H 58 N7O9S [M + H] + 884.4011, found 884.4002.

[0079] Example 9: Preparation of methyl 4-hydroxy-3-(2-(4-(4-(5-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)piperazin-1-yl)phenoxy)acetamido)benzoate (Compound 9) in the present invention

[0080]

[0081] Adapt to change the starting compound (replace the 4-bromobutyric acid starting material in Step 1 of Example 8 with a 5-bromovaleric acid starting material), and the remaining steps are the same as in Example 8 to obtain a white solid product with a yield of 31%. 11H NMR (400 MHz, DMSO-d6) δ 9.16 (s, 1H), 8.99 (d, J = 1.7 Hz, 1H), 8.80 (d, J = 2.3 Hz, 1H), 8.60 (t, J = 6.1 Hz, 1H), 7.97 (d, J = 9.4 Hz, 1H), 7.74 (dd, J = 8.6, 2.2 Hz, 1H), 7.40 (q, J = 8.2 Hz, 4H), 7.19 (d, J = 8.7 Hz, 1H), 6.97 (dd, J = 7.9, 5.1 Hz, 4H), 5.17 (d, J = 3.9 Hz, 1H), 4.71 (d, J = 6.5 Hz, 2H), 4.57 (d, J = 9.3 Hz, 1H), 4.48 - 4.39 (m, 2H), 4.35 (s, 1H), 4.22 (dd, J = 15.8, 5.4 Hz, 1H), 4.14 (d, J = 6.4 Hz, 2H), 3.82 (s, 3H), 3.66 (d, J = 4.8 Hz, 2H), 3.23 (s, 4H), 3.17 (s, 2H), 3.09 (d, J = 7.3 Hz, 2H), 2.44 (s, 3H), 2.41 - 2.33 (m, 1H), 2.27 - 2.20 (m, 1H), 2.04 (t, J = 10.3 Hz, 1H), 1.90 (ddd, J = 12.9, 8.4, 4.4 Hz, 1H), 1.76 - 1.69 (m, 2H), 1.26 (ddd, J = 8.4, 6.8, 3.1 Hz, 2H), 0.93 (s, 9H). HRMS (ESI) m / z calculated for C 47 H 60 N7O9S [M + H] + 898.4168, found 898.4159.

[0082] Example 10: Preparation of methyl 4-hydroxy-3-(2-(4-(4-(6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexyl)piperazin-1-yl)phenoxy)acetamido)benzoate (Compound 10) in the present invention

[0083]

[0084] Adapt to change the starting compound (replace the 4-bromobutyric acid raw material in step 1 of Example 8 with 6-bromohexanoic acid raw material), and the remaining steps are the same as in Example 8, to obtain a white solid product with a yield of 22%. 11H NMR (400 MHz, DMSO-d6) δ 9.26 - 9.10 (m, 1H), 8.99 (s, 1H), 8.75 (d, J = 41.0 Hz, 1H), 8.58 (s, 1H), 7.90 (t, J = 12.2 Hz, 1H), 7.67 (dd, J = 48.5, 8.6 Hz, 1H), 7.40 (q, J = 8.1 Hz, 4H), 7.19 (d, J = 8.6 Hz, 1H), 6.97 - 6.86 (m, 4H), 5.14 (s, 1H), 4.69 (d, J = 10.2 Hz, 2H), 4.55 (d, J = 9.2 Hz, 1H), 4.43 (dd, J = 16.3, 6.9 Hz, 2H), 4.35 (s, 1H), 4.26 - 4.17 (m, 1H), 3.81 (d, J = 12.1 Hz, 3H), 3.65 (s, 2H), 3.19 - 2.93 (m, 8H), 2.44 (s, 3H), 2.34 - 2.23 (m, 2H), 2.20 - 2.10 (m, 1H), 2.07 - 1.99 (m, 1H), 1.93 - 1.85 (m, 1H), 1.80 - 1.71 (m, 1H), 1.50 (dt, J = 27.8, 14.2 Hz, 4H), 1.31 - 1.17 (m, 2H), 0.93 (s, 9H). HRMS (ESI) m / z calculated for C 48 H 62 N7O9S [M + H] + 912.4324, found 912.4309.

[0085] Example 11: Preparation of methyl 4-hydroxy-3-(2-(4-(4-(7-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-7-oxoheptyl)piperazin-1-yl)phenoxy)acetamido)benzoate (Compound 11) in the present invention

[0086]

[0087] Adapt to change the starting compound (replace the 4-bromobutyric acid starting material in Step 1 of Example 8 with 7-bromoheptanoic acid starting material), and the remaining steps are the same as in Example 8 to obtain a white solid product with a yield of 25%. 11H NMR (400 MHz, DMSO-d6) δ 9.14 (d, J = 3.6 Hz, 0.5H), 8.99 (d, J = 1.7 Hz, 1H), 8.81 (t, J = 2.1 Hz, 0.5H), 8.62 (s, 1H), 8.07 (s, 1H), 7.92 - 7.84 (m, 1H), 7.73 (d, J = 8.5 Hz, 1H), 7.40 (q, J = 8.4 Hz, 4H), 7.19 (d, J = 8.7 Hz, 1H), 7.01 - 6.78 (m, 4H), 5.18 (s, 1H), 4.74 - 4.63 (m, 3H), 4.58 - 4.38 (m, 5H), 4.38 - 4.33 (m, 1H), 4.22 (dd, J = 8.1, 5.8 Hz, 1H), 4.12 (t, J = 6.2 Hz, 1H), 3.82 (s, 2H), 3.68 - 3.61 (m, 2H), 3.57 (s, 2H), 3.27 (d, J = 5.0 Hz, 1H), 3.18 (dd, J = 8.9, 4.6 Hz, 1H), 2.44 (s, 2H), 2.28 (dd, J = 14.4, 7.6 Hz, 1H), 2.16 - 2.10 (m, 1H), 2.04 (t, J = 10.6 Hz, 1H), 1.89 (d, J = 4.4 Hz, 1H), 1.74 (d, J = 7.7 Hz, 2H), 1.55 - 1.43 (m, 4H), 1.36 - 1.23 (m, 6H), 0.93 (d, J = 5.5 Hz, 9H). HRMS (ESI) m / z calculated for C 49 H 64 N7O9S [M + H] + 926.4481, found 926.4471.

[0088] Example 12: Preparation of methyl 4-hydroxy-3-(2-(4-(4-(8-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-8-oxooctyl)piperazin-1-yl)phenoxy)acetamido)benzoate (Compound 12) in the present invention

[0089]

[0090] Adapt to change the starting compound (replace the 4-bromobutyric acid starting material in Step 1 of Example 8 with 8-bromooctanoic acid starting material), and the remaining steps are the same as in Example 8, to obtain a white solid product with a yield of 21%. 11H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 0.5H), 8.98 (s, 1H), 8.86 - 8.75 (m, 0.5H), 8.58 (d, J = 6.1 Hz, 1H), 7.87 (d, J = 9.3 Hz, 1H), 7.79 - 7.64 (m, 1H), 7.40 (q, J = 8.0 Hz, 4H), 7.18 (d, J = 8.7 Hz, 1H), 7.01 - 6.71 (m, 4H), 5.15 (s, 1H), 4.68 (s, 2H), 4.55 (d, J = 9.3 Hz, 1H), 4.43 (q, J = 7.7, 7.2 Hz, 2H), 4.35 (s, 1H), 4.21 (dd, J = 15.9, 5.4 Hz, 1H), 4.12 (t, J = 6.3 Hz, 1H), 3.82 (s, 2H), 3.72 - 3.56 (m, 3H), 3.05 (t, J = 6.9 Hz, 4H), 2.81 (d, J = 11.5 Hz, 2H), 2.60 (s, 2H), 2.44 (s, 3H), 2.25 (q, J = 8.1, 7.4 Hz, 1H), 2.07 (ddd, J = 31.6, 13.3, 7.5 Hz, 3H), 1.90 (td, J = 8.7, 4.3 Hz, 1H), 1.75 (h, J = 6.8, 6.4 Hz, 1H), 1.47 (dt, J = 20.3, 9.7 Hz, 4H), 1.25 (d, J = 7.3 Hz, 6H), 0.92 (d, J = 6.2 Hz, 9H). HRMS (ESI) m / z calculated for C 50 H 66 N7O 9S [M + H] + 940.4637, found 940.4604.

[0091] Example 13: Evaluation of the in vitro cytotoxic activity of the compounds synthesized in the present invention against cancer cells

[0092] The anti - proliferative activities of the compounds of the present invention against four pancreatic cancer cells (PANC - 1, SW1990, CFPAC - 1, MIAPaCa - 2) and cervical cancer Hela cells were detected by the CCK - 8 method.

[0093] Experimental procedure: PANC - 1, SW1990, CFPAC - 1, MIA PaCa - 2, and Hela cells were cultured to the logarithmic phase, and at 7×10 per well 3Cells were seeded in a 96-well plate and incubated in a 37 °C cell culture incubator with 5% CO2 for 24 h until the cells adhered. Then, the cells were co-cultured with media containing 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM of the target compound for 72 h, with three parallel replicates. After the culture was completed, 10 μL of CCK-8 solution was added and incubated for another 3 h. The suspension was discarded, and the absorbance of the cell lysate at a wavelength of 450 nm was measured using a multifunctional microplate reader. The IC 50 value was calculated using GraphPad Prism software.

[0094] The results of the in vitro cytotoxic activity experiments of the target compound against four pancreatic cancer cells (PANC-1, SW1990, CFPAC-1, MIA PaCa-2) and cervical cancer Hela cells are shown in Table 1.

[0095] Table 1 Evaluation of the in vitro anti-tumor cell proliferation activity of the target compound

[0096]

[0097] a The absorbance of the cells treated with the target compound for 72 h was detected by the CCK-8 method. After three independent parallel experiments, the IC 50 was calculated.

[0098] As can be seen from Table 1, the compounds described in the present invention have weak anti-proliferative activities against the four pancreatic cancer cells. However, compound 8 and compound 12 have strong cytotoxic activities against Hela cells.

[0099] Example 14: Study on the in vitro HIF-1α protein degradation activity and degradation mechanism of compound 12 described in the present invention against cervical cancer Hela cells

[0100] The protein immunoblotting method (Western blot) was used to evaluate the in vitro HIF-1α protein degradation activity of compound 12 against cervical cancer Hela cells, and its degradation mechanism was elucidated by studying the HIF-1α protein degradation pathway.

[0101] Experimental procedures of Western blot: Cultivate cervical cancer Hela cells until the logarithmic phase, routinely digest them with trypsin, count about 50,000 cells, inoculate them in a 6-well plate for incubation, and add a medium containing 5 μM, 10 μM, and 20 μM of the target compound for co-culture for 24 h. After collecting the cells, wash them with cold PBS, and then homogenize the cells in RIPA lysis buffer. Scrape the cells and incubate them on ice for 30 min, collect the lysate, pre-cool the centrifuge to 4 °C, centrifuge at 12,000 rmp for 15 min, aspirate the supernatant, measure the protein concentration through a BCA protein assay kit, and reconstruct the protein extract by adding the supernatant and protein loading buffer (containing Tris-HCl, SDS, glycerol, and β-mercaptoethanol) in a ratio of 3:1. Boil the mixture at 100 °C for 15 min, separate equal amounts of proteins by 8-12% SDS-polyacrylamide gel electrophoresis, transfer them to a PVDF membrane, then block the membrane with 5% non-fat milk containing 1% Tween-20 at room temperature for 120 min, incubate with a specific primary antibody overnight at 4 °C, wash the membrane three times in TBST the next day, and then co-incubate the membrane with an HRP-conjugated secondary antibody at room temperature for 2 h. Finally, develop the blot with enhanced chemiluminescence, detect the results with a Tanon 5200 imager, and statistically analyze the bands of three independent experiments using Image J software.

[0102] Study on the in vitro HIF-1α protein degradation activity and degradation mechanism of target compound 12 on Hela cells is as Figure 1 shown. The experimental results show that compound 12 can induce the degradation of HIF-1α protein in Hela cells in a concentration- and time-dependent manner, and has the maximum degradation efficiency after acting for 36 h at a concentration of 20 μM. When the proteasome inhibitor MG-132 or the NAE inhibitor MLN4924 is added, the degradation process of HIF-1α protein will be significantly inhibited. Only adding MG-132 will induce the degradation of HIF-1α protein, and MLN4924, whether administered alone or in combination with compound 12, will not cause the degradation of HIF-1α protein, indicating that compound 12 realizes the degradation of HIF-1α protein through the ubiquitin-proteasome pathway (UPS).

[0103] The synthesis method of this type of compound is simple, the raw materials are cheap and easily available, and the HIF-1α protein degradation activity is significant. It is expected to become a new type of drug for anti-tumor, anti-cerebral edema, atherosclerosis, rheumatoid arthritis, psoriasis, Alzheimer's disease, glaucoma, etc.

[0104] The above embodiments are only exemplary embodiments of the present invention, and professionals in the technical field can make improvements or adjustments according to needs. It should be particularly noted that without departing from the core idea or basic principle of the present invention, these improvements or adjustments should be regarded as falling within the protection scope of the present invention.

Claims

1. A compound having hypoxia-inducible factor-1α (HIF-1α) protein degradation activity, or a pharmaceutically acceptable salt, stereoisomer, metabolite, solvate, polymorph, isotope-labeled compound or prodrug thereof, said compound having the structure shown in Formula I: Wherein: X is a carbonyl group or a methylene group; n is a natural integer between 3 and 10, representing the number of saturated straight-chain alkyl groups of C3-C10.

2. The compound according to claim 1, wherein The compound is a structure shown in Formula II or Formula III:

3. The compound according to claim 1 or 2, wherein, Specifically selected from one of the following compounds:

4. The synthesis method of the compound according to any one of claims 1-3, comprising the following steps: (1) 4-(1-Piperazinyl)phenol (II-1) reacts with di-tert-butyl dicarbonate and methyl 2-bromoacetate through two-step nucleophilic substitution reactions to obtain intermediate II-2; (2) Intermediate II-2 undergoes an ester hydrolysis reaction under strong basic conditions and then undergoes an amide condensation reaction with methyl 3-amino-4-hydroxybenzoate to obtain intermediate II-3; (3) Intermediate II-3 undergoes a Boc deprotection reaction under acidic conditions to prepare the HIF-1α binding ligand compound II-4; (4) Monomethyl alkane-1,ω-dioate with different alkyl chain lengths (intermediate II-5) reacts with the VHL proteasome inhibitor VH032 through an amide condensation reaction to obtain intermediate II-6; (5) Intermediate II-6 undergoes an ester hydrolysis reaction under strong basic conditions to obtain intermediate II-7, and then undergoes an amide condensation reaction with the HIF-1α binding ligand compound II-4 to obtain the compound of Formula II; (6) Bromoalkyl acids with different alkyl chain lengths (intermediate III-1) react with the VHL proteasome inhibitor VH032 through an amide condensation reaction to obtain bromo intermediate III-2, and then undergo a nucleophilic substitution reaction with the HIF-1α binding ligand compound II-4 to obtain the compound of Formula III; 5. A pharmaceutical composition comprising the compound according to any one of claims 1-3 or a pharmaceutically acceptable salt, stereoisomer, metabolite, solvate, polymorph, isotope-labeled compound or prodrug thereof, and at least one pharmaceutically acceptable carrier.

6. A pharmaceutical preparation comprising the compound according to any one of claims 1-3 or the pharmaceutical composition according to claim 5.

7. Use of the compound according to any one of claims 1-3, the pharmaceutical composition according to claim 5 or the pharmaceutical preparation according to claim 6 in the preparation of an HIF-1α protein degrader.

8. The application according to claim 7, wherein The degrader is an HIF-1α-PROTAC degrader.

9. Use of the compound according to any one of claims 1-3, the pharmaceutical composition according to claim 5 or the pharmaceutical preparation according to claim 6 in the preparation of a drug for treating diseases related to HIF-1α overexpression.

10. The application according to claim 9, characterized in that, The diseases include cancer, cerebral edema, atherosclerosis, rheumatoid arthritis, psoriasis, Alzheimer's disease, glaucoma; cancers include cervical cancer, pancreatic cancer, breast cancer; The compound degrades HIF-1α protein through the ubiquitin-proteasome pathway.

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