Novel CYP1B1 enzymatic degradation agent as well as preparation method and application thereof

Abstract

The invention discloses a novel CYP1B1 enzyme degradation agent as well as a preparation method and application thereof, and particularly discloses a CYP1B1 enzyme targeted hydrophobic tag HyT degradation agent which comprises an affinity ligand, a hydrophobic tag and a connecting chain for connecting the affinity ligand and the hydrophobic tag, each of the affinity ligand and the hydrophobic tag comprises an alpha-naphthylflavone derivative; the connecting chain comprises a plurality of repetitive units, and the repetitive units are composed of ethylene glycol fragments or alkyl groups. The invention also discloses a preparation method of the hydrophobic tag HyT degradation agent and application of the hydrophobic tag HyT degradation agent or a pharmaceutical composition thereof. The novel CYP1B1 enzyme degradation agent provided by the invention has stronger CYP1B1 enzyme targeting capability, and can specifically target tumors, realize sensitization anti-PD-L1 monoclonal antibody immunotherapy and enhance the tumor immunotherapy effect.

Description

Technical Field

[0001] This invention relates to the field of antitumor drug technology, and more specifically, to a novel CYP1B1 enzyme degrading agent, its preparation method, and its uses. Background Technology

[0002] Small molecule inhibitors are a crucial component of cancer chemotherapy drugs. However, traditional small molecule inhibitors primarily employ a site-driven pharmacological mode of action, exerting their therapeutic effects through direct regulation of protein activity. This approach lacks the ability to directly regulate target proteins and suffers from limitations such as high dosage requirements and the development of drug resistance. Therefore, novel therapeutic approaches utilizing intrinsic intracellular protein degradation mechanisms to degrade specific target proteins have become highly promising treatment methods. Protein degradation drugs, due to their unique mechanisms of action, offer numerous advantages and have been widely developed and applied in cancer treatment. For example, in prostate cancer, degradation of the androgen receptor (AR) can effectively block the proliferation of prostate cancer cells, achieving a therapeutic effect. Furthermore, for abnormally activated oncogenic signaling pathways within tumor cells, such as the PI3K-AKT-mTOR signaling pathway, protein degradation agents can directly degrade key proteins in this pathway, interfering with downstream signal transduction and inhibiting tumor cell proliferation and survival, thereby exerting a therapeutic effect. Therefore, selecting appropriate anti-tumor drug targets and designing and developing novel, highly efficient protein degradation drugs are crucial for driving revolutions in cancer treatment.

[0003] The ubiquitin-proteasome pathway is one of the main pathways for protein degradation in eukaryotic cells, primarily clearing short-lived proteins and soluble misfolded proteins. With scientific advancements, target protein degradation strategies based on the ubiquitin-proteasome system have been gradually developed, such as protein hydrolysis-targeting chimeras (PROTACs) and protein degradation technologies induced by hydrophobic tags (HyTs). Hydrophobic tags are bifunctional molecules that induce target protein degradation. Compared to the E3 ubiquitin ligase ligands at the ends of PROTACs, which recruit protein degradation, the hydrophobic tag ends with highly hydrophobic groups that perform the degradation function. After binding to the target protein, the hydrophobic groups are exposed on the protein surface, mimicking the misfolded state of the protein and thus enabling its degradation through protein quality control (PQC). HyT degraders do not rely on the intracellular E3 ubiquitin ligase system and do not require the recruitment of E3 ubiquitinases or the formation of ternary complexes, thus having the advantage of being less prone to inactivation in vivo. Furthermore, compared to commonly used E3 ubiquitin ligands such as VHL in PROTAC drugs, hydrophobic tags exhibit better plasma stability and lower molecular weight, potentially enhancing the cell permeability of degradative drugs. Therefore, developing novel hydrophobic groups suitable for HyT degradative agent design is beneficial for further expanding the design scope of such drugs.

[0004] Cytochrome P450 enzymes (CYPs) are a family of proteins widely distributed in human tissues. Using heme as a cofactor, they participate in the biotransformation of many endogenous and exogenous substances, playing a crucial role in regulating the interaction between the body and its external environment and maintaining homeostasis. CYP1B1, a drug-metabolizing enzyme specifically highly expressed in tumor cells, not only participates in tumorigenesis and development but also mediates resistance to chemotherapeutic drugs and immune checkpoint inhibitors. Therefore, CYP1B1 is an ideal target for anti-tumor drug development. However, current drug designs targeting CYP1B1 are mostly inhibitors, typically only able to bind to the protein's active site or key domains to inhibit its function, while the protein itself remains in the cell, thus limiting its anti-tumor efficacy.

[0005] Immunotherapy has achieved remarkable results in cancer treatment, and the relationship between the CYP1B1 enzyme and immunotherapy is gradually being elucidated. Studies have shown that the CYP1B1 enzyme can mediate increased ferroptosis resistance in tumor cells, inducing resistance to anti-PD-1 immune checkpoint inhibitors. Furthermore, knocking down the CYP1B1 enzyme on mouse colon cancer MC38 cells followed by anti-PD-1 monoclonal antibody therapy achieved better tumor suppression. Based on the negative effects of CYP1B1 enzyme expression on immunotherapy, many immunotherapy strategies targeting the CYP1B1 enzyme have emerged. For example, Luby et al. selected the CYP1B1 enzyme as a tumor-specific antigen and designed the CYP1B1 DNA vaccine ZYC300, which enhances the CYP1B1 enzyme-specific CD8+. + T cells are responsible for killing tumor cells, and their effectiveness in treating tumors has been demonstrated in phase I clinical trials. Therefore, for cancers resistant to immunotherapy, reducing the level of the CYP1B1 enzyme in tumor cells may be key to enhancing the efficacy of immunotherapy. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide a novel CYP1B1 enzyme degrading agent and expand its application in tumor treatment and sensitizing anti-PD-L1 monoclonal antibody immunotherapy resistance. Through research on target protein degradation strategies based on the ubiquitin-proteasome system, the present invention has discovered that the hydrophobic tag β-naphthoflavone used in the HyT degrading agent drug has a very high similarity to the CYP1B1 enzyme targeting ligand α-naphthoflavone. Based on this idea, this invention designs a novel CYP1B1 enzyme degrading agent. Using previously obtained inhibitors with high selectivity for CYP1B1 enzyme as targeting ligands, and linking them with hydrophobic tags via suitable linker chains, this invention utilizes the protein degradation mechanism to design a more efficient "dual-target" CYP1B1 enzyme protein degrading agent with an α-naphthylflavonoid core-targeting ligand-linker chain-α-naphthylflavonoid core-targeting ligand structure. Both sides can serve as ligands binding to the target protein or as hydrophobic tags inducing degradation, thereby obtaining a novel CYP1B1 enzyme degrading agent drug for application in tumor treatment, solving the problems of low ligand affinity and limited application in tumor treatment of existing degrading agents.

[0007] The principle of this invention is as follows: CYP1B1 enzyme, as a drug-metabolizing enzyme specifically highly expressed in tumor cells, not only participates in tumor development and progression but also mediates the development of resistance to chemotherapeutic drugs and immune checkpoint inhibitors in tumor cells, making it an ideal target for anti-tumor drug development. In previous studies, structure-activity relationship studies of α-naphthylflavonoids have yielded derivatives with high inhibitory activity and selectivity against CYP1B1 enzyme. This invention aims to utilize the α-naphthylflavonoids with strong inhibitory activity against CYP1B1 discovered in previous studies. A novel, highly efficient degradative agent targeting the CYP1B1 enzyme was obtained by introducing a linker chain onto the naphthaleneflavonoid nucleus ligand via easily derivatizable hydroxyl functional groups. Two to four ethylene glycol fragments (n=2,3,4,5,6) were selected as the linker chain, and α-naphthaleneflavonoid nucleus ligands with covalent warheads were simultaneously linked to both ends of the linker chain to prepare the "dual-warhead" CYP1B1 protein degradative agent described in this invention. On the one hand, the "dual-warhead" design increases the binding probability, allowing either end to extend into the pocket to bind to the enzyme, while the other end extends out as a hydrophobic tag. On the other hand, the PEG linker chain improves the overall water solubility of the molecule, exploring suitable chain lengths for effective binding and degradation, and developing degradative agents with high affinity and degradation efficiency. This invention further explores and obtains a degradative agent targeting the CYP1B1 enzyme with stronger binding ability, and realizes its application in sensitizing anti-PD-L1 monoclonal antibody therapy for tumors.

[0008] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a hydrophobic tag HyT degrader targeting the CYP1B1 enzyme, comprising an affinity ligand, a hydrophobic tag, and a linking strand for connecting the affinity ligand and the hydrophobic tag; both the affinity ligand and the hydrophobic tag comprise α Naphthaleneflavonoid derivatives; the linking chain comprises multiple repeating units, wherein the repeating units are composed of ethylene glycol fragments or alkyl groups; The structure of the degradation agent is shown in formula (I): (I) Where X is selected from CH2 or O; n is selected from 2, 3, 4, 5 or 6; R is selected from any one of H, halogen, hydroxyl, mercapto, cyano, amino, nitro, carbonyl, vinyl, ethynyl, C1-C6 alkyl, C3-C7 cycloalkyl, and 3-7 membered heterocyclic groups; or, a group formed by chemically connecting or substituting two or more groups selected from the above groups.

[0009] In some specific embodiments of the present invention, X is an O atom, n is 2, and R is an F atom; The structural formula of the degrading agent is: .

[0010] Secondly, the present invention provides a method for preparing the hydrophobic tag HyT degrader as described in any of the preceding claims, using the following synthetic route: .

[0011] As some specific embodiments of the present invention, in step a of the synthetic route, compound S1 and NBS ( N2.5-4.0 equivalents of bromosuccinimide undergoes bromination in acetonitrile; the reaction temperature is 20-30 °C, and the reaction time is 8-12 hours. In step b, compound S2 and CH3ONa (2.5-4.0 equivalents) undergo a substitution reaction in a mixed solvent of DMF and methanol (DMF:MeOH = 2:1 to 1:2) catalyzed by CuI (2.5-4.0 equivalents); the reaction temperature is 75-110 °C and the reaction time is 30-40 hours. In step c, compound S3 undergoes a Vilsmeier formylation reaction with DMF (3.0-5.0 equivalents) and POCl3 (3.0-5.0 equivalents) in dichloromethane; the reaction temperature is 35-55 °C and the time is 8-12 hours. In step d, compound S4 reacts with CH3MgBr (1.5-2.5 equivalents) in tetrahydrofuran via a Grignard reaction; the reaction temperature is 20-30 °C and the reaction time is 2-4 hours. In step e, compound S5 is oxidized by MnO2 in dichloromethane; the reaction temperature is 40-50 °C and the time is 6-8 hours. In step f, compound S6 reacts selectively with AlCl3 (5.0-8.0 equivalents) in acetonitrile. O - Demethylation reaction; reaction temperature 40-60 ℃, time 1.5-3 hours; In step g, compound S8 reacts with MOMCl (chloromethyl methyl ether) and DIPEA ( N , N (-Diisopropylethylamine) undergoes a substitution reaction in dichloromethane; the reaction temperature is 20-30 ℃, and the time is 8-12 hours; In step h, the intermediate obtained in step g is alkalized in methanol with NaOH aqueous solution at a reaction temperature of 60-75 °C for 2-3 hours; then cooled to room temperature and the pH of the reaction solution is adjusted to 5 with dilute hydrochloric acid. In step i, compounds S7 and S9 (1.2-1.5 equivalents) are reacted in DIC (… N , N The esterification reaction of '-diisopropylcarbodiimide (1.3-1.5 equivalents) and DMAP (4-dimethylaminopyridine (0.1-0.2 equivalents) in dichloromethane was carried out at a temperature of 20-30 °C for 8-12 hours. In step j, compound S10 undergoes a Baker-Venkataraman rearrangement reaction with potassium tert-butoxide (tBuOK) in tetrahydrofuran; the reaction temperature is 20-30 °C and the time is 1-3 hours. In step k, the key intermediate obtained in step j undergoes cyclization and deprotection reactions with H2SO4 in acetic acid; the reaction temperature is 60-80 °C and the time is 1-2 hours. In step 1, compound Int and (0.4-0.6 equivalents) and Cs2CO3 (3.0-5.0 equivalents) undergo a substitution reaction in DMF; the reaction temperature is 60-80 °C and the time is 8-12 hours.

[0012] As some specific embodiments of the present invention, the preparation method specifically includes the following steps: a. Compound S1 is subjected to a bromination reaction with N-bromosuccinimide to obtain compound S2; b. Compound S2 is reacted with sodium methoxide under the catalysis of cuprous iodide to undergo a substitution reaction, yielding compound S3; c. Compound S3 was subjected to a Vilsmeier formylation reaction with DMF and POCl3 to obtain compound S4; d. Compound S4 is reacted with methylmagnesium bromide in a Grignard reaction to give compound S5; e. Compound S5 is oxidized by MnO2 to give compound S6; f. Compound S6 exhibits selective reaction with AlCl3. O -Demethylation reaction yields compound S7; g. Compound S8 undergoes a substitution reaction with chloromethyl methyl ether and N,N-diisopropylethylamine; h. The product obtained in step g is treated with alkali to obtain compound S9; i. Compound S7 and compound S9 undergo esterification under the catalysis of N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine to give compound S10; j. Compound S10 undergoes a Baker-Venkataraman rearrangement reaction with potassium tert-butoxide; k. The product obtained in step j undergoes a cyclization and deprotection reaction with H2SO4 to yield the key intermediate Int; l. Key intermediate Int and It undergoes a substitution reaction with Cs2CO3 to obtain the product.

[0013] As some specific embodiments of the present invention, the preparation method includes at least one of the following technical features: (1) In step a, the bromination reaction is carried out in acetonitrile at a temperature of 20-30 °C for 8-12 h. (2) In step b, the substitution reaction is carried out in a mixed solvent of DMF and methanol, wherein the volume ratio of DMF to methanol in the mixed solvent is 2:1-1:2, the reaction temperature is 75-110 °C, and the time is 30-40 h; (3) In step c, the Vilsmeier formylation reaction is carried out in dichloromethane at a temperature of 35-55°C for 8-12 h. (4) In step d, the Grignard reaction is carried out in tetrahydrofuran at a temperature of 20-30 °C for 2-4 h. (5) In step e, compound S5 is oxidized by MnO2 in dichloromethane. The oxidation reaction is carried out at a temperature of 40-50 °C for 6-8 h. (6) In step f, the selective O-demethylation reaction is carried out in acetonitrile at a temperature of 40-60°C for 1.5-3 h. (7) In step g, the substitution reaction is carried out in dichloromethane at a temperature of 20-30 °C for 8-12 h. (8) In step h, the product obtained in step g is alkalized in methanol with NaOH aqueous solution at a reaction temperature of 60-75℃ for 2-3 h; then cooled to room temperature and the pH of the reaction solution is adjusted to 5 with dilute hydrochloric acid to obtain compound S9. (9) In step i, the esterification reaction is carried out in dichloromethane at a temperature of 20-30 °C for 8-12 h. (10) In step j, the Baker-Venkataraman rearrangement reaction is carried out in tetrahydrofuran at a temperature of 20-30 °C for 1-3 h. (11) In step k, the cyclization and deprotection reactions are carried out in acetic acid at a temperature of 60-80 °C for 1-2 h. (12) In step 1, the substitution reaction is carried out in DMF at a temperature of 60-80 °C for 8-12 h.

[0014] As some specific embodiments of the present invention, the preparation method further includes at least one of the following technical features: (1) In step a, the amount of N-bromosuccinimide used is equivalent to 2.5-4.0 molar equivalents of compound S1; (2) In step b, the amounts of sodium methoxide and copper iodide are each equivalent to 2.5-4.0 molar equivalents of compound S2; (3) In step c, the amounts of DMF and POCl3 are each equivalent to 3.0-5.0 molar equivalents of compound S3; (4) In step d, the amount of CH3MgBr used is equivalent to 1.5-2.5 molar equivalents of compound S4; (5) In step f, the amount of AlCl3 used is equivalent to 5.0-8.0 molar equivalents of compound S6; (6) In step i, the amount of compound S9 is equivalent to 1.2-1.5 molar equivalents of compound S7, the amount of N,N'-diisopropylcarbodiimide is equivalent to 1.3-1.5 molar equivalents of compound S7, and the amount of compound 4-dimethylaminopyridine is equivalent to 0.1-0.2 molar equivalents of compound S7. (7) In step 1, the compound The amount used is equivalent to 0.4-0.6 molar equivalents of the key intermediate Int, and the amount used of compound Cs2CO3 is equivalent to 3.0-5.0 molar equivalents of the key intermediate Int.

[0015] Thirdly, the present invention provides a pharmaceutical composition comprising the hydrophobic tag HyT degrader as described in any one of the preceding claims or a pharmacologically or physiologically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, medium or combination thereof.

[0016] Fourthly, the present invention provides the use of the hydrophobic tag HyT degrader or pharmaceutical composition as described in any of the preceding claims in the preparation of a medicament for degrading CYP1B1 enzymes.

[0017] Fifthly, the present invention provides the use of the hydrophobic tag HyT degrader or pharmaceutical composition as described in any of the preceding claims in the preparation of a medicament for sensitizing anti-PD-L1 monoclonal antibody treatment of resistance.

[0018] Compared to degrading agents prepared from other hydrophobic tags, this invention designs a hydrophobic tag (HyT) degrading agent that can bind to the CYP1B1 enzyme, and further applies it to sensitizing anti-PD-L1 monoclonal antibody therapy for resistance.

[0019] In a sixth aspect, the present invention provides the use of the hydrophobic tag HyT degrader or pharmaceutical composition as described in any of the preceding claims in the preparation of an antitumor drug targeting the CYP1B1 enzyme.

[0020] Currently, there are many types of CYP1B1 enzyme inhibitors, but their applications are mainly focused on reversing chemotherapy resistance in tumors, and there are no reports on their synergistic use with immune checkpoint inhibitors in tumor treatment. Furthermore, some small-molecule CYP1B1 enzyme degraders have been disclosed, such as compound P31 described in patent CN115043831B. This compound exhibits a significant "hooking" effect when degrading CYP1B1 enzymes, meaning its degradative activity decreases significantly at higher concentrations (≥1000 nM), resulting in a narrow drug dosage selection window, which limits its application potential. In contrast, compound 2 provided by this invention continues to exhibit highly efficient degradation of CYP1B1 enzymes even at high concentrations, overcoming the problem of activity loss at high concentrations of existing degraders, demonstrating superior stability and application prospects. Moreover, previously reported applications of CYP1B1 enzyme inhibitors or degraders have all demonstrated effects in reversing drug resistance in tumor cells, without achieving success in mouse in vivo experiments. This invention is the first to apply a CYP1B1 enzyme degrader to achieve a sensitizing effect for tumor immunotherapy in a mouse xenograft model.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1) The target CYP1B1 enzyme selected in this invention is an anti-tumor drug target. The designed and synthesized hydrophobic degrading agent has a more novel structure and a stronger ability to target CYP1B1 enzyme, which will effectively promote the CYP1B1 enzyme to play a target role in the occurrence and development of tumors. It will have good application prospects and clinical translational value in tumor treatment.

[0022] 2) Currently, most antitumor compounds designed targeting the CYP1B1 enzyme are inhibitors, which can only block part of the protein's biological function and have limitations. Furthermore, all reported CYP1B1 enzyme degrading agents are PROTAC drugs, which have limitations such as low affinity and degradation efficiency. There are currently no reports of degrading agents based on hydrophobic tags for CYP1B1 enzyme degradation. The degrading agent provided in this invention uses a highly selective and specific CYP1B1 enzyme inhibitor as a ligand. Following the design strategy of previous probes, it introduces protein degradation effector molecules at appropriate sites through a linker strand. The design strategy of the degrading agent is supported by previous experiments and has the potential to enhance tumor therapy through combined use.

[0023] 3) This invention further investigates the application of novel CYP1B1 enzyme-targeting degraders in sensitizing anti-PD-L1 monoclonal antibody therapy. The hydrophobic tagging degraders provided by this invention can specifically target tumors and achieve sensitization of anti-PD-L1 monoclonal antibody immunotherapy, thereby enhancing the efficacy of tumor immunotherapy. Although immune checkpoint inhibitors have achieved significant results in clinical applications, they still have limitations such as treatment resistance in some patients and specific cancers. As a tumor-specific enzyme, CYP1B1 has been shown to participate in the process of tumor treatment resistance to various chemotherapy drugs and is closely related to tumor immune regulation. Therefore, this invention, by directly degrading CYP1B1 enzyme and interrupting its mediated tumor immune escape signaling pathway, can break down the barrier of anti-PD-L1 treatment resistance and enhance the body's anti-tumor immune response. Attached Figure Description

[0024] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This invention relates to a method for synthesizing a HyT degrader with the structure of formula (I) obtained from the key intermediate Int; Figure 2 This study illustrates the degradation effects of degradative agents with different linker chains on CYP1B1 enzyme in the human colorectal cancer HCT-15 cell line. (a) shows the Western blot diagram of CYP1B1 enzyme degradation by compound 1; (b) shows the quantitative results of CYP1B1 enzyme degradation by compound 1 relative to the internal control; (c) shows the Western blot diagrams of CYP1B1 enzyme degradation by compounds 2, 4, and 5; (d) shows the quantitative results of CYP1B1 enzyme degradation by compounds 2, 4, and 5 relative to the internal control; (e) shows the Western blot diagrams of CYP1B1 enzyme degradation by compounds 3 and 6; and (f) shows the quantitative results of CYP1B1 enzyme degradation by compounds 3 and 6 relative to the internal control. Figure 3 The degradation effect of compound 2 on CYP1B1 enzyme in different cell lines is shown. (a) is a Western blot plot of compound 2 degrading CYP1B1 enzyme in the human colon cancer HCT-15 cell line; (b) is a compound concentration-degradation effect curve fitted after quantification of CYP1B1 enzyme expression relative to the internal control in HCT-15 cells, and DC is calculated. 50 =31.34 nM; (c) is a Western Blot diagram of the degradation of CYP1B1 enzyme by compound 2 in mouse colon cancer MC38 cell line; (d) is the quantitative result of the expression level of CYP1B1 enzyme in MC38 cells relative to the internal control after degradation by compound 2; Figure 4This study aims to verify the molecular level and mechanism of compound 2 in degrading CYP1B1 enzyme in HCT-15 cells; (a) shows confocal microscopy images of CYP1B1 levels in cells after co-incubation of different concentrations of compound 2 with HCT-15 for 24 hours; (b) shows the levels of CYP1B1 in cells after co-incubation of different concentrations of compound 2 with HCT-15 for 24 hours. Cyp1b1 (c) Immunoblot imaging results of CYP1B1 protein in cells after co-incubation of 200 nM compound 2 with HCT-15 for different times; (d) Quantification results of CYP1B1 enzyme expression relative to internal control in cells after co-incubation of 200 nM compound 2 with HCT-15 for different times; (e) Immunoblot images of the degradation effect of compound 2 (500 nM) on CYP1B1 enzyme in HCT-15 cells after pretreatment with proteasome inhibitor MG-132 (10 µM) and lysosomal inhibitor Bafilomycin A1 (100 nM); (f) Quantification results of CYP1B1 enzyme expression relative to internal control in HCT-15 cells after pretreatment with proteasome inhibitor MG-132 (10 µM) and lysosomal inhibitor Bafilomycin A1 (100 nM). Figure 5 The concentrations of compound 2 in plasma at different time points after intravenous injection of 5 mg / kg into wild-type C57BL / 6 mice are shown in A, where A is the compound concentration change curve and B is the parameter table. Figure 6 The study aimed to enhance the therapeutic effect of compound 2 on MC38 colon cancer tumors treated with anti-PD-L1 monoclonal antibody. (a) shows the tumor growth curves of mice in different treatment groups after treatment; (b) shows the tumor size of mice in different treatment groups after treatment; (c) shows the Western blot diagram of CYP1B1 enzyme in tumor tissues of mice in different treatment groups after treatment; and (d) shows the quantitative results of CYP1B1 enzyme expression in tumor tissues of mice in different treatment groups compared to the internal control protein after treatment. Detailed Implementation

[0025] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0026] Example 1 This embodiment synthesizes the key intermediate Int via the following route:

[0027] The reaction conditions for each step in the above synthetic route are as follows: (a) NBS (3.0 equivalent), CH3CN, stirred overnight at room temperature; (b) A mixture of CH3ONa (3.0 equivalents), CuI (3.0 equivalents), DMF and CH3OH (volume ratio 1:1), 90°C, 36 hours; (c) POCl3 (5.0 equivalent), DMF (5.0 equivalent), DCM, 45 °C, overnight; (d) CH3MgBr (2.0 equivalents), THF, stirred at room temperature for 2 hours; (e) MnO2 (12.0 equivalent), DCM, 42 °C, 6 hours; (f) AlCl3 (6.0 equivalent), CH3CN, 60 °C, 2 hours; (g) MOMCl (5.0 equivalents), DIPEA (5.0 equivalents), DCM, stirred overnight at room temperature; (h) NaOH (10.0 equivalent), CH3OH:H2O=1:1, 70 °C, 3 hours, pH adjusted to 5; (i) DIC (1.3 equivalents), DMAP (0.1 equivalents), DCM, stirred overnight at room temperature; (j) tBuOK (1.5 equivalent), THF, room temperature, 1.5 hours; (k) H2SO4-CH3COOH (10%) V / V ), 75 °C, 1 hour.

[0028] In this embodiment, the R group is a fluorine atom.

[0029] The key intermediate synthesized in this embodiment is 2-(5-fluoro-3-hydroxyphenyl)-6,7,10-trimethoxy-4H-benzo[h]chromene-4-one (compound 1), a yellow solid.

[0030] The proton NMR and mass spectrometry data of compound 1 are shown below: 1 H NMR (400 MHz, DMSO- d 6) δ 10.38 (s, 1H), 7.53 (dd, J = 9.8, 2.3 Hz, 1H), 7.50 (t, J= 1.9 Hz, 1H), 7.29 (s, 1H), 7.29 (s, 2H), 7.16 (s, 1H), 6.81(dt, J = 10.5, 2.4 Hz, 1H), 4.07 (s, 3H), 3.94 (s, 3H), 3.84 (s, 3H). HRMS(ESI-TOF) calculated m / z for [M+H] + C 22 H 18 FO6 + 397.1082, found 397.1091. The structural formula of compound 1 is shown below: .

[0031] Based on the key intermediate synthesized in Example 1, Examples 2-6 are referenced. Figure 1 Different HyT degrading agents were further synthesized along the indicated pathway.

[0032] Example 2 At room temperature, the key intermediate compound 1 (9.9 mg, 0.025 mmol), cesium carbonate (33 mg, 0.1 mmol), and 1,8-dibromo-3,6-dioxane (3.5 mg, 0.013 mmol) synthesized in Example 1 were sequentially dissolved in N,N-dimethylformamide (1.5 mL). After purging the container with argon, the mixture was stirred overnight at 65 °C. After the reaction was complete, the reaction solution was diluted with water and extracted with ethyl acetate (3 × 10 mL). The resulting organic phase was washed sequentially with water and saturated brine, dried over sodium sulfate, filtered, and concentrated by rotary evaporation. The concentrate was purified by silica gel column chromatography (2.5% to 5% methanol / dichloromethane) to give 2-{3-fluoro-5-[(8-{[3-fluoro-5-(6,7,10-trimethoxy-4-oxonylbenzo[ h [Creno-2-yl)phenyl]oxy}-3,6-dioxaoct-1-yl)oxy]phenyl]6,7,10-trimethoxy-4 H -benzo[ h Chromene-4-one (compound 2, 4.7 mg, 0.0052 mmol), yellow solid, yield 42%.

[0033] The proton NMR, carbon NMR, and mass spectrometry data of compound 2 are shown below: 1 H NMR (400 MHz, CDCl3) δ 7.37 (d, J= 9.5 Hz, 1H), 7.35 (s, 1H), 7.32(s, 1H), 7.00 (d, J = 8.9 Hz, 1H), 6.89 (d, J = 9.0 Hz, 1H), 6.81 (s, 1H), 6.74(dt, J = 10.1, 2.4 Hz, 1H), 4.23 – 4.15 (m, 2H), 4.01 (s, 3H), 3.99 (s, 3H), 3.95 – 3.91 (m, 2H), 3.90 (s, 3H), 3.81 (s, 2H). 13 C NMR (101 MHz, CDCl3) δ 177.5, 163.8 (d, J = 245.2 Hz), 161.0 (d, J = 3.1 Hz), 160.5 (d, J = 10.9 Hz),154.5, 151.5, 151.1, 148.9, 134.5 (d, J = 10.2 Hz), 122.2, 121.5, 118.0,112.9, 109.1 (d, J = 2.5 Hz), 108.6, 107.2, 105.9 (d, J = 24.3 Hz), 104.5 (d, J = 25.1 Hz), 98.7, 71.3, 69.9, 68.3, 58.0, 56.6, 56.3. HRMS (ESI-TOF)calculated m / z for [M+H] + C 50 H 45 F2O 14 + 907.2772, found 907.2784. The structural formula of compound 2 is shown below: .

[0034] Example 3 At room temperature, intermediate 1 (9.9 mg, 0.025 mmol), cesium carbonate (33 mg, 0.1 mmol), and 1,11-dibromo-3,6,9-trioxaundecanane (4.0 mg, 0.0125 mmol) were dissolved sequentially in...N , N In 1.5 mL of dimethylformamide, after purging the container with argon, the mixture was stirred overnight at 65 °C. After the reaction was complete, the reaction solution was diluted with water and extracted with ethyl acetate (3 × 10 mL). The resulting organic phase was washed successively with water and saturated brine, dried over sodium sulfate, filtered, and concentrated by rotary evaporation. The concentrate was purified by silica gel column chromatography (2.5% to 5% methanol / dichloromethane) to give 2-{5-fluoro-3-[(11-{[5-fluoro-3-(6,7,10-trimethoxy-4-oxomethylenebenzo[ h [Creno-2-yl)phenyl]oxy}-3,6,9-trioxaundecan-1-yl)oxy]phenyl}-6,7,10-trimethoxy-4 H -benzo[ h Chromene-4-one (compound 3, 5.8 mg, 0.0061 mmol), yellow solid, 49% yield.

[0035] The proton NMR, carbon NMR, and mass spectrometry data of compound 3 are shown below: 1 H NMR (400 MHz, CDCl3) δ 7.37 (dt, J = 9.7, 1.9 Hz, 1H), 7.35 (s, 1H), 7.27 (s, 1H), 7.02 (d, J = 8.8 Hz, 1H), 6.92 (d, J = 9.0 Hz, 1H), 6.81 (s, 1H), 6.72 (dt, J = 10.3, 2.4 Hz, 1H), 4.15 (t, J = 4.7 Hz, 2H), 4.00 (s, 3H), 3.99(s, 3H), 3.92 (d, J = 4.8 Hz, 2H), 3.90 (s, 3H), 3.78 (td, J = 4.4, 2.2 Hz, 2H), 3.74 (td, J = 4.4, 2.2 Hz, 2H). 13 C NMR (101 MHz, CDCl3) δ 177.5, 163.8 (d, J = 244.9 Hz), 161.0 (d, J = 3.3 Hz), 160.4 (d, J= 10.9 Hz), 154.5, 151.5,151.1, 148.9, 134.5 (d, J = 10.5 Hz), 122.2, 121.4, 118.0, 113.0, 108.9 (d, J =2.9 Hz), 108.7, 107.2, 105.9 (d, J = 25.1 Hz), 104.5 (d, J = 25.1 Hz), 98.6,71.1, 70.9, 69.7, 68.3, 58.1, 56.6, 56.3. HRMS (ESI-TOF) calculated m / z for [M+H] + C 52 H 49 F2O 15 + 951.3034, found 951.3041. The structural formula of compound 3 is shown below: .

[0036] Example 4 At room temperature, intermediate 1 (9.9 mg, 0.025 mmol), cesium carbonate (33 mg, 0.1 mmol), and 1,14-dibromo-3,6,9,12-tetraoxatetradecane (4.7 mg, 0.013 mmol) were dissolved sequentially in... N , N In 1.5 mL of dimethylformamide, after purging the container with argon, the mixture was stirred overnight at 65 °C. After the reaction was complete, the reaction solution was diluted with water and extracted with ethyl acetate (3 × 10 mL). The resulting organic phase was washed successively with water and saturated brine, dried over sodium sulfate, filtered, and concentrated by rotary evaporation. The concentrate was purified by silica gel column chromatography (2% to 6% methanol / dichloromethane) to give 2-{5-fluoro-3-[(14-{[5-fluoro-3-(6,7,10-trimethoxy-4-oxomethylenebenzo[ h [Creno-2-yl)phenyl]oxy}-3,6,9,12-tetraoxatetradecane-1-yl)oxy]phenyl}-6,7,10-trimethoxy-4 H -benzo[ h Chromene-4-one (compound 4, 5.0 mg, 0.0050 mmol), yellow solid, 40% yield.

[0037] The proton NMR, carbon NMR, and mass spectrometry data of compound 4 are shown below: 1 H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 9.8 Hz, 1H), 7.39 (s, 1H), 7.31(s, 1H), 7.06 (d, J = 8.8 Hz, 1H), 6.97 (d, J = 8.9 Hz, 1H), 6.86 (s, 1H), 6.73(dt, J = 10.3, 2.6 Hz, 1H), 4.16 (t, J = 4.8 Hz, 2H), 4.02 (s, 6H), 3.92 (s,3H), 3.89 (t, J = 4.3 Hz, 2H), 3.76 (dd, J = 5.8, 3.1 Hz, 2H), 3.72 (dd, J =5.4, 2.6 Hz, 2H), 3.70 (s, 2H). 13 C NMR (101 MHz, CDCl3) δ 177.6, 163.8 (d, J =245.2 Hz), 161.2 (d, J = 3.1 Hz), 160.5 (d, J = 11.3 Hz), 154.6, 151.6, 151.2,149.0, 134.6 (d, J = 10.5 Hz), 122.3, 121.5, 118.1, 113.1, 108.9 (d, J = 2.7Hz), 108.8, 107.3, 106.0 (d, J = 24.7 Hz), 104.6 (d, J = 25.4 Hz), 98.7, 71.1,70.9, 70.8, 69.7, 68.3, 58.1, 56.6, 56.4. HRMS (ESI-TOF) calculated m / z for [M+H] + C 54 H 53 F2O 16 + 995.3296, found 995.3319. The structural formula of compound 4 is shown below: .

[0038] Example 5 At room temperature, intermediate 1 (9.9 mg, 0.025 mmol), cesium carbonate (33 mg, 0.1 mmol), and 1,17-dibromo-3,6,9,12,15-pentaheptadecane (4.9 mg, 0.012 mmol) were dissolved sequentially in... N , N In 1.5 mL of dimethylformamide, after purging the container with argon, the mixture was stirred overnight at 65 °C. After the reaction was complete, the reaction solution was diluted with water and extracted with ethyl acetate (3 × 10 mL). The resulting organic phase was washed successively with water and saturated brine, dried over sodium sulfate, filtered, and concentrated by rotary evaporation. The concentrate was purified by silica gel column chromatography (2.5% to 5% methanol / dichloromethane) to give 2-{5-fluoro-3-[(17-{[5-fluoro-3-(6,7,10-trimethoxy-4-oxomethylenebenzo[ h [Creno-2-yl)phenyl]oxy}-3,6,9,12,15-pentaheptadecane-1-yl)oxy]phenyl}-6,7,10-trimethoxy-4 H -benzo[ h Chromene-4-one (compound 5, 6.2 mg, 0.0060 mmol), yellow solid, 48% yield.

[0039] The proton NMR, carbon NMR, and mass spectrometry data of compound 5 are shown below: 1 H NMR (400 MHz, CDCl3) δ 7.45 (d, J = 9.5 Hz, 1H), 7.41 (s, 1H), 7.33(s, 1H), 7.07 (d, J = 8.9 Hz, 1H), 6.99 (d, J = 8.9 Hz, 1H), 6.88 (s, 1H), 6.75(dt, J = 10.1, 2.3 Hz, 1H), 4.21 – 4.13 (m, 2H), 4.04 (s, 3H), 4.03 (s, 3H), 3.92 (s, 3H), 3.90 (t, J = 4.7 Hz, 2H), 3.75 (dd, J= 6.0, 3.0 Hz, 2H), 3.73 –3.69 (m, 2H), 3.68 (s, 4H). 13 C NMR (101 MHz, CDCl3) δ 177.6, 163.8 (d, J = 245.2Hz), 161.2 (d, J = 3.0 Hz), 160.5 (d, J = 10.9 Hz), 154.6, 151.6, 151.2, 149.0,134.6 (d, J = 10.5 Hz), 122.3, 121.5, 118.2, 113.2, 108.9 (d, J = 2.5 Hz),108.9, 107.4, 106.0 (d, J = 25.1 Hz), 104.6 (d, J = 25.1 Hz), 98.7, 71.1, 70.8,70.8, 69.7, 68.3, 58.2, 56.6, 56.4. HRMS (ESI-TOF) calculated m / z for [M+H] + C 56 H 57 F2O 17 + 1039.3558, found 1039.3582. The structural formula of compound 5 is shown below: .

[0040] Example 6 At room temperature, intermediate 1 (9.9 mg, 0.025 mmol), cesium carbonate (33 mg, 0.1 mmol), and 1,20-dibromo-3,6,9,12,15,18-hexaoxaecoane (5.8 mg, 0.0128 mmol) were dissolved sequentially in... N , NIn 1.5 mL of dimethylformamide, after purging the container with argon, the mixture was stirred overnight at 65 °C. After the reaction was complete, the reaction solution was diluted with water and extracted with ethyl acetate (3 × 10 mL). The resulting organic phase was washed successively with water and saturated brine, dried over sodium sulfate, filtered, and concentrated by rotary evaporation. The concentrate was purified by silica gel column chromatography (2.5% to 5% methanol / dichloromethane) to give 2-{5-fluoro-3-[(20-{[5-fluoro-3-(6,7,10-trimethoxy-4-oxomethylenebenzo[ h [Creno-2-yl)phenyl]oxy}-3,6,9,12,15,18-hexaoxaeco-1-yl)oxy]phenyl}-6,7,10-trimethoxy-4 H -benzo[ h Chromene-4-one (compound 6, 7.3 mg, 0.0067 mmol), yellow solid, yield 54%.

[0041] The proton NMR, carbon NMR, and mass spectrometry data of compound 6 are shown below: 1 H NMR (400 MHz, CDCl3) δ 7.48 (d, J = 9.6 Hz, 1H), 7.43 (s, 1H), 7.35(d, J = 1.9 Hz, 1H), 7.09 (d, J = 8.9 Hz, 1H), 7.01 (d, J = 8.9 Hz, 1H), 6.90(s, 1H), 6.77 (dt, J = 10.1, 2.3 Hz, 1H), 4.18 (t, J = 4.8 Hz, 2H), 4.06 (s,3H), 4.04 (s, 3H), 3.93 (s, 3H), 3.90 (t, J = 4.7 Hz, 2H), 3.75 (dd, J = 6.4,3.5 Hz, 2H), 3.73 – 3.69 (m, 2H), 3.67 (s, 4H), 3.65 (s, 2H). 13 C NMR (101 MHz, CDCl3) δ 177.6, 163.9 (d, J = 245.2 Hz), 161.3 (d, J = 3.3 Hz), 160.5 (d, J=11.3 Hz), 154.6, 151.7, 151.3, 149.0, 134.7 (d, J = 10.5 Hz), 122.4, 121.6,118.2, 113.2, 109.0, 108.9, 107.4, 106.0 (d, J = 24.7 Hz), 104.7 (d, J = 25.1Hz), 98.8, 71.0, 70.8, 70.7, 69.7, 68.3, 58.2, 56.7, 56.4. HRMS (ESI-TOF)calculated m / z for [M+H] + C 58 H 61 F2O 18 + 1083.3820, found 1083.3812. The structural formula of compound 6 is shown below: .

[0042] Example 7 In this embodiment, the degradation effect of the candidate CYP1B1 hydrophobic degrader on CYP1B1 protein in human colon cancer HCT-15 cell line and mouse colon cancer MC38 cell line was determined by Western blot (WB).

[0043] 1. Validation of the degradation effect of degradative agents with different linked chains on CYP1B1 enzyme in human colorectal cancer HCT-15 cell line. HCT-15 cells, which highly express CYP1B1 enzyme, were used at a rate of 5 × 10⁻⁶. 5 Cells were seeded at a density of 1000 mcg in 6-well plates and incubated at 37°C for 24 hours. After incubation, the culture medium was discarded, and the cells were washed three times with PBS. Then, the candidate degradation agents prepared in Examples 1-6 (concentrations of 0.01 nM, 1 nM, 100 nM, and 10 µM) were added. After incubation for another 24 hours, the cell culture supernatant was removed, and the cells were washed thoroughly with PBS. Then, 200 µL of cell lysis buffer (SDS cell lysis buffer containing 1% PMSF protease inhibitor) was added to extract proteins from the cells.

[0044] After determining the protein concentration of each sample using the Bicinchoninic Acid Assay (BCA), Loading Buffer was added and the sample was heated in a 90°C metal bath for 10 minutes to fully reduce the protein, thus obtaining protein samples treated with the degradation agent for Western blotting (WB) assay. Subsequently, using a 4-12% gel electrophoresis pre-prepared gel, 50 μg of total protein was loaded into each well and electrophoresed at 130V for 50 minutes. After electrophoresis, the protein was transferred to a PVDF membrane using the wet transfer method and kept at 100V for 70 minutes. After that, the PVDF membrane was blocked at room temperature for 1 hour using TPST in 5% skim milk. After that, it was incubated overnight at 4°C with rabbit anti-mouse / human GAPDH (1:1000) and rabbit anti-human / mouse CYP1B1 (1:1000). After overnight incubation, the bands were washed 3 times with TBST. Then, HRP-labeled anti-rabbit IgG (H+L) (1:5000) prepared with 5% skim milk and TBST was added and incubated at room temperature for 1 hour. After washing the bands 3 times with TBST, the bands were developed. Subsequently, ImageJ software was used to compare the gray values ​​of the CYP1B1 enzyme band and the total protein GAPDH band between different candidate degrading agents and the negative control group, in order to preliminarily screen and evaluate candidate degrading agents with strong degradation effects on CYP1B1 enzyme.

[0045] The results are as follows Figure 2 The figures show the degradation effects of the key intermediate compound 1 obtained in Example 1 and the degrading agent compounds 2-6 with different linker chains obtained in Examples 2-6 on the CYP1B1 enzyme in the human colorectal cancer HCT-15 cell line. (a) is a Western Blot diagram of the degradation of CYP1B1 enzyme by compound 1; (b) is the quantitative result of the expression level of CYP1B1 enzyme after degradation by compound 1 relative to the internal control; (c) is a Western Blot diagram of the degradation of CYP1B1 enzyme by compounds 2, 4, and 5; (d) is the quantitative result of the expression level of CYP1B1 enzyme after degradation by compounds 2, 4, and 5 relative to the internal control; (e) is a Western Blot diagram of the degradation of CYP1B1 enzyme by compounds 3 and 6; and (f) is the quantitative result of the expression level of CYP1B1 enzyme after degradation by compounds 3 and 6 relative to the internal control.

[0046] Depend on Figure 2 The results showed that incubating HCT-15 cells with the single-ligand compound 1 did not significantly change the CYP1B1 protein level, indicating that this symmetrical bivalent structure is essential for degradation activity. Subsequently, other compounds were tested, with compound 2 exhibiting the most significant inhibitory effect, reducing CYP1B1 protein levels in a concentration-dependent manner. Other compounds, at higher concentrations, actually increased CYP1B1 expression, demonstrating a clear "hooking" effect.

[0047] 2. The degradation effect of compound 2 on CYP1B1 enzyme in different cell lines HCT-15 cells and mouse MC38 cells were co-incubated with gradient concentrations of candidate degradative compound 2 for 24 hours. Proteins were extracted and subjected to Western blotting (WB). The ratio of CYP1B1 enzyme to GAPDH grayscale values ​​was plotted on the ordinate, and the degradative concentration on the x-axis. GraphPad Prism5 was used to process and plot the graphs, and the DC values ​​of the degradative agent were obtained. 50 value.

[0048] The results are as follows Figure 3 As shown, (a) is a Western blot diagram of compound 2 degrading CYP1B1 enzyme in human colon cancer HCT-15 cell line, and (b) is a compound concentration-degradation effect curve fitted relative to the internal control expression level after compound 2 degrades CYP1B1 enzyme in HCT-15 cells, and the DC value was calculated. 50 =31.34 nM; (c) is a Western Blot diagram of the degradation of CYP1B1 enzyme by compound 2 in mouse colon cancer MC38 cell line, and (d) is the quantitative result of the expression level of CYP1B1 enzyme in MC38 cells relative to the internal control after compound 2 degrades CYP1B1 enzyme.

[0049] The results above indicate that, consistent with its activity in human colon cancer cells, compound 2 effectively degrades CYP1B1 in MC38 cells in a concentration-dependent manner, confirming its degradation efficacy against the CYP1B1 enzyme in different cell lines and supporting its suitability for further in vivo evaluation in the MC38 subcutaneous tumor mouse model.

[0050] Example 8 This embodiment evaluated the specific degradation of CYP1B1 enzyme by degrading agent compound 2 at the cellular level.

[0051] Human colon cancer HCT-15 cell line, which highly expresses CYP1B1 enzyme, was used at a rate of 2×10⁻⁶. 4Cells were seeded at a density in confocal dishes and cultured overnight to allow them to adhere. Three treatment groups were set up: a solvent control group, a 20 nM compound 2 treatment group, and a 200 nM compound 2 treatment group. All cells were incubated at 37°C for 24 hours. After washing three times with PBS buffer, cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, followed by permeabilization with PBS containing 5% Triton for 20 minutes. Subsequently, cells were blocked with 5% BSA in PBS at room temperature for 1 hour. After blocking, cells were co-incubated overnight at 4°C with primary antibody (rabbit anti-mouse / human CYP1B1, 1:500). After three washes with PBS, cells were incubated at room temperature for 1 hour with secondary antibody (Alexa Fluor® 488-labeled goat anti-rabbit IgG, 1:1000) in the dark. After three washes with PBS, cells were stained with DAPI (1:1000) for 20 minutes in the dark. After thorough rinsing, the confocal dishes were observed under a confocal fluorescence microscope, and fluorescence images were acquired.

[0052] The results are as follows Figure 4 As shown, (a) is a confocal microscopy image of the CYP1B1 level in cells after co-incubating different concentrations of compound 2 with HCT-15 for 24 hours.

[0053] The results above show that compound 2 can significantly reduce the level of CYP1B1 protein in HCT-15 cells in a dose-dependent manner: effective degradation can be achieved with treatment of 20 nM, and CYP1B1 signaling is almost completely eliminated at 200 nM, providing intuitive evidence of its effective degradation activity at the cellular level.

[0054] Example 9 This embodiment verifies that compound 2 degrades CYP1B1 protein without affecting the Cyp1b1 gene level.

[0055] HCT-15 cells, which highly express CYP1B1 enzyme, were used at a rate of 5 × 10⁻⁶. 5 The cells were seeded at a density of 250 mg / L in 6-well plates and incubated at 37°C for 24 hours to allow adhesion. Then, compound 2 at varying concentrations was added and co-cultured for another 24 hours. After incubation, the culture medium was discarded, and the cells were thoroughly washed with PBS buffer. Total RNA was extracted from the cells using 1 mL of TRIzol reagent. Subsequently, complementary DNA was synthesized using a reverse transcription kit. Quantitative RT-PCR was performed using the SYBR Green Premix Pro Taq HS qPCR kit according to standard operating procedures. The results were obtained by transfection via 2000 μL of PCR. -△△CT Method analysis of each group Cyp1b1 Genes relative to internal reference Gapdh Gene expression levels.

[0056] The primers used in this study are as follows: CYP1B1 forward primer: 5'-ACACCTGTCTTGGGCTAC-3' (SEQ ID NO.1); Reverse primer: 5'-ATCACTCTGCTGGTCAGGTC-3' (SEQ ID NO.2); GAPDH forward primer: 5'-ACTCTTCCACCTTCGATGCC-3' (SEQ ID NO.3); Reverse primer: 5'-TGGGATAGGGCCTCTTGC-3' (SEQ ID NO.4).

[0057] The results are as follows Figure 4 As shown in (b), the cells after co-incubating HCT-15 cells with different concentrations of compound 2 for 24 hours... Cyp1b1 The relative change in mRNA levels.

[0058] The results above show that compound 2 has the following effect: Cyp1b1 The impact on gene expression was minimal, effectively ruling out the role of transcriptional regulation as a promoting factor and strongly supporting the mechanism of direct protein degradation.

[0059] Example 10 This embodiment verifies that compound 2 can efficiently and rapidly degrade CYP1B1 protein in cells.

[0060] HCT-15 cells, which highly express CYP1B1 enzyme, were used at a rate of 5 × 10⁻⁶. 5 The cells were seeded at a density of 1000 nM into 6-well plates and incubated at 37°C for 24 hours to allow them to adhere to the plate. Then, 200 nM of compound 2 was added and co-cultured for 2, 4, 6, 10, 24, and 48 hours. After the culture was completed, total cellular protein was extracted according to the method described in Example 7 and the degradation effect of compound 2 on CYP1B1 protein was evaluated by Western blotting.

[0061] The results are as follows Figure 4 As shown in (c) and (d), Figure 4 (c) shows the immunoblotting imaging results of CYP1B1 protein in cells after co-incubation of 200 nM compound 2 with HCT-15 for different times; Figure 4 The middle (d) shows the quantitative results of the expression level of CYP1B1 enzyme relative to the internal control in cells after co-incubation of 200 nM compound 2 with HCT-15 for different times.

[0062] The results above show that compound 2 induces rapid consumption of CYP1B1, with most of the protein degrading within 2 hours. Notably, this degradation effect persists for up to 48 hours after compound removal, demonstrating not only rapid onset of action but also prolonged target inhibition.

[0063] Example 11 This embodiment verifies the mechanism by which compound 2 degrades CYP1B1 protein in HCT-15 cells.

[0064] HCT-15 cells, which highly express CYP1B1 enzyme, were used at a rate of 5 × 10⁻⁶. 5 Cells were seeded at a density of 100 μL in 6-well plates and incubated at 37°C for 24 hours to allow them to adhere to the plates. Then, they were pretreated for 1 hour with DMSO, proteasome inhibitor MG-132 (10 µM), and lysosome inhibitor Bafilomycin A1 (100 nM), respectively. After removal of these pretreatments, the cells were thoroughly washed. Then, 500 nM of compound 2 was added to each well and the cells were co-cultured for 24 hours. After the culture was completed, total cellular protein was extracted according to the method described in Example 7 and the degradation effect of compound 2 on CYP1B1 protein was evaluated by Western blotting.

[0065] The results are as follows Figure 4 As shown in (e) and (f), Figure 4 In the middle (e), the immunoblot image shows the degradation effect of compound 2 (500 nM) on CYP1B1 enzyme in HCT-15 cells after pretreatment with protease inhibitor MG-132 (10 µM) and lysosomal inhibitor Bafilomycin A1 (100 nM). Figure 4 In the middle (f), the expression level of compound 2 (500 nM) relative to the internal control of CYP1B1 enzyme was quantified after HCT-15 cells were pretreated with the proteasome inhibitor MG-132 (10 µM) and the lysosome inhibitor Bafilomycin A1 (100 nM).

[0066] The results above show that the proteasome inhibitor MG-132 completely blocked the degradation of CYP1B1, while the effect of the lysosomal inhibitor Bafilomycin A1 on the CYP1B1 enzyme was negligible. This result clearly indicates that compound 2 mainly mediates the degradation of CYP1B1 through the proteasome pathway.

[0067] Example 12 This example evaluated the pharmacokinetic parameters of compound 2 in C57BL / 6 mice.

[0068] Compound 2 was dissolved in a solvent consisting of 10% DMSO, 10% ethanol-polyoxyethylene castor oil mixture, and 80% physiological saline. Subsequently, 6-8 week old C57BL6 / J mice were administered the drug via tail vein injection at a dose of 5 mg / kg. Blood samples were collected at 0.25, 0.5, 1, 2, 4, and 6 hours post-injection, and plasma was separated. After standardized pretreatment, plasma drug concentrations were determined using ultra-high performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry (VION). Concentration-time curves were plotted, and pharmacokinetic parameters were calculated using Win Nolin software.

[0069] The results are as follows Figure 5 The figure shows the plasma concentration of compound 2 at different time points after intravenous injection of 5 mg / kg into wild-type C57BL / 6 mice. A represents the compound concentration change curve, and B is the parameter table.

[0070] The results above show that compound 2 rapidly reaches its peak concentration in plasma, then gradually decreases, with a half-life (t0). 1 / 2 The time to survival was 3.44 hours. These pharmacokinetic characteristics support the good in vivo stability of compound 2.

[0071] Example 13 This embodiment evaluates the application of compound 2 in enhancing the treatment of MC38 tumors with anti-PD-L1.

[0072] 1×10 6 MC38 mouse colon cancer cells were subcutaneously inoculated into the backs of 6-8 week old C57BL / 6 mice. Seven days after inoculation, mice were randomly divided into four groups (n=5 per group) according to tumor size: a solvent control group, a compound 2 monotherapy group, an anti-PD-L1 monoclonal antibody monotherapy group, and a compound 2 combined with anti-PD-L1 monoclonal antibody group. Compound 2 was dissolved in a solvent composed of 10% DMSO, 10% ethanol-polyoxyethylene castor oil mixture, and 80% physiological saline. Starting from day 8 post-inoculation, compound 2 (5 mg / kg, intravenous injection) or an equivalent volume of solvent was administered every other day for a total of 6 doses. Simultaneously, anti-PD-L1 monoclonal antibody (2.5 mg / kg, intraperitoneal injection) was administered every other day for a total of 3 doses. Tumor volume and mouse body weight were measured every two days, and tumor growth curves and body weight change curves were plotted. Tumor volume (mm) 3 The calculation method is (length × width × height) / 2. Eighteen days after tumor cell inoculation, mice were euthanized, and tumor tissue was collected and ground. Afterward, protein lysis buffer was added for thorough lysis, and total cellular protein was extracted according to the method described in Example 7. The expression level of CYP1B1 protein in the tumor tissue of each group of mice was evaluated by Western blotting.

[0073] The results are as follows Figure 6 The image shows the therapeutic effect of compound 2-sensitized MC38 colon cancer tumors treated with anti-PD-L1 monoclonal antibody. Figure 6 (a) shows the tumor growth curves of mice in different treatment groups after treatment (Vehicle: injected with only the same dose of solvent as compound 2; 2: injected via tail vein at 5 mg / kg of compound 2; Anti-PD-L1+Vehicle: injected intraperitoneally with 2.5 mg / kg of anti-PD-L1 monoclonal antibody and injected via tail vein at the same dose of solvent as compound 2; Anti-PD-L1+2: injected intraperitoneally with 2.5 mg / kg of anti-PD-L1 monoclonal antibody and injected via tail vein at 5 mg / kg of compound 2). Figure 6 (b) shows the tumor size of mice in different treatment groups after treatment; Figure 6 (c) shows the Western blot diagram of CYP1B1 enzyme in tumor tissues of mice in different treatment groups after treatment; Figure 6 (d) shows the quantitative results of CYP1B1 enzyme expression in tumor tissues of mice in different treatment groups after treatment, compared with the internal reference protein.

[0074] The results above indicate that in the MC38 syngeneic mouse model, neither compound 2 nor subtherapeutic doses of anti-PD-L1 antibody monotherapy showed significant antitumor activity; however, the combination of the two significantly inhibited tumor growth, with some mice even achieving complete remission. Western blot analysis showed that compound 2 significantly reduced CYP1B1 protein levels in tumor tissues, and its degradation effect was further enhanced and nearly eliminated when combined with anti-PD-L1 antibody. Notably, anti-PD-L1 antibody monotherapy also significantly downregulated CYP1B1 expression, suggesting that it may regulate this protein through an independent pathway.

[0075] In summary, this invention develops a highly efficient hydrophobic degrader of CYP1B1 protein based on hydrophobic interactions. Compound 2 with the best degradation effect was screened using Western blotting experiments, and its degradation was successfully demonstrated in the human colon cancer HCT-15 cell line (DC). 50Compound 2 was used to verify its efficient degradation effect on CYP1B1 protein in mice (31.34 nM) and the MC38 colon cancer cell line. Confocal microscopy was used to directly verify the degradation effect of compound 2 on CYP1B1 protein at the cellular level. qPCR confirmed that compound 2 exerts its degradation effect by directly targeting CYP1B1 protein without affecting its mRNA level. Western blotting confirmed that compound 2 can exert a highly efficient degradation effect on CYP1B1 protein within a short time (2 h), and the degradation process depends on the cellular proteasome degradation pathway. Subsequently, C57BL / 6 mice were injected with compound 2 via the tail vein, and blood samples were collected at different time points to separate plasma and characterize its pharmacokinetic parameters after intravenous administration. Finally, a mouse M38 subcutaneous tumor model was constructed to evaluate the efficacy of compound 2 in enhancing anti-PD-L1 monoclonal antibody immunotherapy, and Western blotting was used to evaluate the CYP1B1 protein level in tumor tissues of mice in different administration groups. The results show that the hydrophobic degrader of CYP1B1 protein designed in this invention has the ability to efficiently degrade CYP1B1 protein, and can also enhance the immunotherapy effect of anti-PD-L1 monoclonal antibody in MC38 tumors.

[0076] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A hydrophobic tag HyT degrading agent targeting CYP1B1 enzyme, characterized in that, It includes an affinity ligand, a hydrophobic tag, and a connecting chain for connecting the affinity ligand and the hydrophobic tag; both the affinity ligand and the hydrophobic tag include α Naphthaleneflavonoid derivatives; the linking chain comprises multiple repeating units, wherein the repeating units are composed of ethylene glycol fragments or alkyl groups; The structure of the degradation agent is shown in formula (I): ， Where X is selected from CH2 or O; n is selected from 2, 3, 4, 5 or 6; R is selected from any one of H, halogen, hydroxyl, mercapto, cyano, amino, nitro, carbonyl, vinyl, ethynyl, C1-C6 alkyl, C3-C7 cycloalkyl, and 3-7 membered heterocyclic groups; or, a group formed by chemically connecting or substituting two or more groups selected from the above groups.

2. The hydrophobic label HyT degrading agent according to claim 1, characterized in that, X is an O atom, n is 2, and R is a fluorine atom; The structural formula of the degrading agent is: 。 3. A method for preparing a hydrophobic label HyT degrading agent as described in claim 1 or 2, characterized in that, The following synthetic route was used for preparation: 。 4. The preparation method according to claim 3, characterized in that, The preparation method specifically includes the following steps: a. Compound S1 is subjected to a bromination reaction with N-bromosuccinimide to obtain compound S2; b. Compound S2 is reacted with sodium methoxide under the catalysis of cuprous iodide to undergo a substitution reaction, yielding compound S3; c. Compound S3 was subjected to a Vilsmeier formylation reaction with DMF and POCl3 to obtain compound S4; d. Compound S4 is reacted with methylmagnesium bromide in a Grignard reaction to give compound S5; e. Compound S5 is oxidized by MnO2 to give compound S6; f. Compound S6 undergoes a selective O-demethylation reaction with AlCl3 to give compound S7; g. Compound S8 undergoes a substitution reaction with chloromethyl methyl ether and N,N-diisopropylethylamine; h. The product obtained in step g is treated with alkali to obtain compound S9; i. Compound S7 and compound S9 undergo esterification under the catalysis of N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine to give compound S10; j. Compound S10 undergoes a Baker-Venkataraman rearrangement reaction with potassium tert-butoxide; k. The product obtained in step j undergoes a cyclization and deprotection reaction with H2SO4 to yield the key intermediate Int; l. Key intermediate Int and It undergoes a substitution reaction with Cs2CO3 to obtain the product.

5. The preparation method according to claim 4, characterized in that, Includes at least one of the following technical features: (1) In step a, the bromination reaction is carried out in acetonitrile at a temperature of 20-30 °C for 8-12 h. (2) In step b, the substitution reaction is carried out in a mixed solvent of DMF and methanol, wherein the volume ratio of DMF to methanol in the mixed solvent is 2:1-1:2, the reaction temperature is 75-110 °C, and the time is 30-40 h; (3) In step c, the Vilsmeier formylation reaction is carried out in dichloromethane at a temperature of 35-55 °C for 8-12 h. (4) In step d, the Grignard reaction is carried out in tetrahydrofuran at a temperature of 20-30 °C for 2-4 h. (5) In step e, compound S5 is oxidized by MnO2 in dichloromethane. The oxidation reaction is carried out at a temperature of 40-50 °C for 6-8 h. (6) In step f, the selectivity O - The demethylation reaction is carried out in acetonitrile at a temperature of 40-60 °C for 1.5-3 h. (7) In step g, the substitution reaction is carried out in dichloromethane at a temperature of 20-30 °C for 8-12 h. (8) In step h, the product obtained in step g is alkalized in methanol with NaOH aqueous solution at a reaction temperature of 60-75 °C for 2-3 h; then cooled to room temperature and the pH of the reaction solution is adjusted to 5 with dilute hydrochloric acid to obtain compound S9. (9) In step i, the esterification reaction is carried out in dichloromethane at a temperature of 20-30 °C for 8-12 h. (10) In step j, the Baker-Venkataraman rearrangement reaction is carried out in tetrahydrofuran at a temperature of 20-30 °C for 1-3 h. (11) In step k, the cyclization and deprotection reactions are carried out in acetic acid at a temperature of 60-80 °C for 1-2 h. (12) In step 1, the substitution reaction is carried out in DMF at a temperature of 60-80 °C for 8-12 h.

6. The preparation method according to claim 4, characterized in that, It also includes at least one of the following technical features: (1) In step a, the amount of N-bromosuccinimide used is equivalent to 2.5-4.0 molar equivalents of compound S1; (2) In step b, the amounts of sodium methoxide and copper iodide are each equivalent to 2.5-4.0 molar equivalents of compound S2; (3) In step c, the amounts of DMF and POCl3 are each equivalent to 3.0-5.0 molar equivalents of compound S3; (4) In step d, the amount of CH3MgBr used is equivalent to 1.5-2.5 molar equivalents of compound S4; (5) In step f, the amount of AlCl3 used is equivalent to 5.0-8.0 molar equivalents of compound S6; (6) In step i, the amount of compound S9 is equivalent to 1.2-1.5 molar equivalents of compound S7, the amount of N,N'-diisopropylcarbodiimide is equivalent to 1.3-1.5 molar equivalents of compound S7, and the amount of compound 4-dimethylaminopyridine is equivalent to 0.1-0.2 molar equivalents of compound S7. (7) In step 1, the compound The amount used is equivalent to 0.4-0.6 molar equivalents of the key intermediate Int, and the amount used of compound Cs2CO3 is equivalent to 3.0-5.0 molar equivalents of the key intermediate Int.

7. A pharmaceutical composition, characterized in that, Includes the hydrophobic label HyT degrader as described in claim 1 or 2, or a pharmacologically or physiologically acceptable salt thereof, as well as a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, medium, or combination thereof.

8. Use of a hydrophobic tag HyT degrader as described in claim 1 or 2, or a pharmaceutical composition as described in claim 7, in the preparation of a medicament for degrading CYP1B1 enzyme.

9. Use of a hydrophobic tag HyT degrader as described in claim 1 or 2, or a pharmaceutical composition as described in claim 7, in the preparation of a medicament for sensitizing resistance to PD-L1 monoclonal antibody therapy.

10. Use of a hydrophobic tag HyT degrader as described in claim 1 or 2, or the pharmaceutical composition as described in claim 7, in the preparation of an antitumor drug targeting the CYP1B1 enzyme.

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