A protac chimera targeting degradation of alkbh5 and preparation method and application thereof

Abstract

This invention discloses a PROTAC chimera for targeted degradation of ALKBH5, its preparation method, and its applications. The structural formula of the PROTAC chimera is shown in Formula I. The PROTAC chimera provided by this invention achieves specific ubiquitination modification and proteasome-dependent degradation of the ALKBH5 protein, completely eliminating the target protein function from its source. This invention overcomes the inherent limitations of traditional small molecule inhibitors, which can only reversibly block ALKBH5 enzyme activity, cannot eliminate the target protein, easily induce compensatory upregulation of the target protein, and develop drug resistance. It provides a novel and precise targeted therapy strategy for ALKBH5-driven malignant tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a PROTAC chimera that targets and degrades ALKBH5, its preparation method, and its application. Background Technology

[0002] ALKBH5 (AlkB Homolog 5), as a Fe(II) / α-ketoglutarate-dependent RNA demethylase, is an N... 6 -Methyladenine (m 6 A) A key "eraser" in the epigenetic transcriptional regulatory network. It specifically recognizes and catalyzes m in RNA molecules. 6 Demethylation of A-modified RNA regulates the stability, translation efficiency, and nucleocytoplasmic translocation of target RNA, thereby precisely regulating gene expression programs at the posttranscriptional level. Studies have shown that ALKBH5 is abnormally highly expressed in various malignant tumors, including glioblastoma, breast cancer, and acute myeloid leukemia (AML), and enhances... MYC, TACC3, BCL2 The stability and translation efficiency of oncogene mRNAs drive malignant proliferation, stemness maintenance, and chemotherapy resistance in tumor cells. In AML, ALKBH5 is crucial for maintaining the stemness of leukemia stem cells (LSCs) and can directly mediate AML cell resistance to cytarabine, making it a key potential target for AML targeted therapy.

[0003] Currently, small molecule inhibitors targeting ALKBH5 (such as MV1035) reversibly block ALKBH5's enzymatic activity by competitively binding to its catalytically active pocket. However, they cannot eliminate existing ALKBH5 protein within cells and are prone to inducing drug resistance due to compensatory upregulation of the target protein, becoming a significant bottleneck in the clinical translation of this type of inhibitor. In contrast, the Proteolytic Targeted Chimeric (PROTAC) technology, with its unique bifunctional molecular design, can simultaneously bind to the target protein and E3 ubiquitin ligase, catalytically inducing ubiquitination of the target protein and achieving irreversible degradation of the target protein through the proteasome. This completely eliminates the biological function of ALKBH5 at its source, providing a revolutionary targeted intervention strategy to overcome the inherent limitations of traditional enzyme activity inhibitors.

[0004] However, to date, there have been no reports of research on PROTAC molecules targeting ALKBH5 globally, leaving this field largely unexplored. Therefore, developing efficient and highly selective ALKBH5-targeting PROTAC degraders could not only fill this gap... 6 This research fills a technological gap in the development of PROTAC targeting ALKBH5-modified enzymes and is expected to break through the treatment bottleneck of malignant tumors with high ALKBH5 expression (such as AML, glioblastoma, and breast cancer), providing a novel precision targeted degradation therapy with significant scientific research value and clinical translational significance. Summary of the Invention

[0005] This invention aims to provide a novel, highly active, and selective PROTAC chimera that targets and degrades ALKBH5, achieving specific ubiquitination and proteasome-dependent degradation of the ALKBH5 protein. This completely eliminates the target protein's function at its source, overcoming the technical limitations of traditional small-molecule inhibitors that only reversibly block ALKBH5 enzyme activity, cannot clear existing target proteins, and are prone to inducing compensatory upregulation and drug resistance. Furthermore, this invention validates the antitumor activity of this PROTAC molecule in ALKBH5-overexpressing AML leukemia cells (such as KG-1 cells), demonstrating its significant inhibition of tumor cell proliferation, induction of apoptosis, and reversal of chemotherapy resistance phenotype. This invention provides a novel precision treatment strategy and potential drug candidate for ALKBH5-driven acute myeloid leukemia (AML).

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a PROTAC chimera that targets and degrades ALKBH5, the structural formula of which is shown in Formula I:

[0008] In Formula I, n is 1, 2, or 3.

[0009] The PROTAC molecule of this invention adopts a ternary structure design of "two ligands-linking chain", and the functions and selection of each part are as follows: 1. ALKBH5 targeting ligand: The core structure of the known ALKBH5 catalytic inhibitor ALKBH5-IN-5 (compound 18I, CAS No.: 140481-05-6) was optimized. Its core function is to specifically bind to the catalytically active pocket of the ALKBH5 protein, ensuring the precise recognition of the target protein by the PROTAC molecule.

[0010] 2. E3 ubiquitin ligase recruitment ligand: Pomalidomide, a CRBN (Cereblon, cerebellar protein) specific ligand, was selected. This ligand can efficiently recruit CRBN-mediated E3 ubiquitin ligase complexes, providing an enzymatic basis for the ubiquitination modification of target proteins.

[0011] 3. Linker: Utilizes flexible polyethylene glycol (PEG) chains. n (n=1, 2, 3) serves as a linker unit, whose core function is to regulate the spatial conformation of the ternary complex formed by the ALKBH5 ligand, the E3 ligase ligand, and the target protein, thereby optimizing intermolecular interactions to improve degradation efficiency.

[0012] Figure 1The chemical structure of the PROTAC compound of this invention is shown, and the binding affinity of the PROTAC molecule to the target protein ALKBH5 was predicted and analyzed using the CSatDTA method. The results showed that the PROTAC with polyethylene glycol (PEG2, n=2) as the linker chain exhibited the strongest binding affinity to ALKBH5, while the analogue with polyethylene glycol (PEG3, n=3) as the linker chain showed relatively low affinity. Based on these predictions, PEG2 (n=2) was selected as the preferred linker chain for the design, synthesis, and functional verification of the PROTAC molecule (DEG-PROTAC). To further evaluate the effect of linker chain length on molecular activity, a corresponding PROTAC with PEG3 (n=3) as the linker chain (TEG-PROTAC) was also synthesized, and the inhibitory effects of DEG-PROTAC and TEG-PROTAC on the proliferation of acute myeloid leukemia cells were systematically compared.

[0013] In a second aspect, the present invention provides a method for preparing the PROTAC chimera that targets and degrades ALKBH5 as described in the first aspect.

[0014] The method for preparing PROTAC chimeras that target the degradation of ALKBH5 provided by this invention has the following synthetic route: Figure 2 As shown, the specific steps include the following: a) Under alkaline conditions, 2-(2-aminoethyl)phenol and di-tert-butyl dicarbonate are reacted to obtain tert-butyl (2-hydroxyphenylethyl) carbamate. b) Under alkaline conditions, tert-butyl (2-hydroxyphenylethyl) carbamate and propynyl bromide are reacted to obtain tert-butyl (2-(prop-2-yn-1-yloxy)phenylethyl) carbamate. c) In the presence of trifluoroacetic acid, deprotection of Boc in tert-butyl (2-(prop-2-yn-1-yloxy)phenethyl)carbamate is carried out to give 2-(2-(prop-2-yn-1-yloxy)phenyl)ethyl-1-amine. d) React 2-(2-(prop-2-yn-1-yloxy)phenyl)ethyl-1-amine with maleic anhydride in glacial acetic acid to give 1-(2-(prop-2-yn-1-yloxy)phenethyl)-1H-pyrrole-2,5-dione; e) 1-(2-(prop-2-yn-1-yloxy)phenyl)-1H-pyrrole-2,5-dione was dissolved in N,N-dimethylformamide (DMF), and copper sulfate and sodium ascorbate were added sequentially at room temperature with stirring to activate the copper catalytic system. Then, pomalidomide-diethylene glycol-azide was added to the reaction system for a click chemical coupling reaction. After the reaction was completed, the PROTAC chimera (DEG-PROTAC) was obtained by purification. Similarly, if pomalidomide-polyethylene glycol-azide is used for the click reaction, a PROTAC with a polyethylene glycol linker (TEG-PROTAC) can be obtained.

[0015] In step a) of the above method, the alkaline condition is provided by sodium bicarbonate (NaHCO3); the reaction is carried out in an organic solvent, the organic solvent being dichloromethane (DCM); the reaction conditions are: stirring overnight at room temperature.

[0016] In step b) of the above method, the alkaline conditions are provided by potassium carbonate (K2CO3); the reaction is carried out in an organic solvent, the organic solvent being N,N-dimethylformamide (DMF); the reaction conditions are: stirring overnight at 60°C.

[0017] In step c) of the above method, the reaction is carried out in an organic solvent, namely dichloromethane (DCM); the reaction conditions are: stirring overnight at room temperature.

[0018] In step d) of the above method, the reaction conditions are: stirring and reacting overnight under reflux conditions.

[0019] In step e) of the above method, the reaction conditions are: stirring and reacting overnight at room temperature.

[0020] Thirdly, the present invention provides the application of the PROTAC chimera that targets and degrades ALKBH5 as described in the first aspect.

[0021] The application includes at least one of the following: (a1) Application in the preparation of ALKBH5 targeted degradative agents; (b1) Application in the preparation of inhibitors for the proliferation of acute myeloid leukemia cells; (c1) Use in the preparation of medicines for the prevention and / or relief and / or treatment of acute myeloid leukemia (AML); According to a specific embodiment of the present invention, the ALKBH5 targeted degrader is an ALKBH5 specific targeted degrader.

[0022] According to a specific embodiment of the present invention, the acute myeloid leukemia cells are KG-1 cells, a human acute myeloid leukemia cell line that highly expresses ALKBH5.

[0023] Fourthly, the present invention provides a product.

[0024] The product provided by this invention comprises the PROTAC chimera that targets and degrades ALKBH5 as described in the first aspect of this invention as its active ingredient; The product has at least one of the following effects: (a2) Targeted degradation of ALKBH5; (b2) Inhibits the proliferation of acute myeloid leukemia cells in vivo or in vitro; (c2) Prevention and / or mitigation and / or treatment of acute myeloid leukemia (AML).

[0025] In this invention, the above-mentioned product may be a drug or a reagent.

[0026] In this invention, the above-mentioned products also include pharmaceutically acceptable excipients.

[0027] In this invention, the pharmaceutically acceptable excipients are selected from one or more of carriers, diluents, binders, lubricants, and wetting agents.

[0028] In this invention, the dosage form of the drug is not limited, as long as the active ingredient can be effectively delivered into the body, including one or more of the following: solution, injection, spray, nasal drops, aerosol, and powder inhaler.

[0029] The PROTAC molecule targeting ALKBH5 developed in this invention has significant advantages over existing small molecule ALKBH5 enzyme activity inhibitors in terms of mechanism of action, target specificity, therapeutic effect, and development prospects. Specific beneficial effects are as follows: 1) Achieve complete clearance of ALKBH5 protein, overcoming the limitation of traditional inhibitors that only inhibit enzyme activity. Existing small-molecule inhibitors of ALKBH5 (such as MV1035) can only reversibly block its catalytic center, failing to reduce the total amount of ALKBH5 protein in cells. Its non-catalytic functions (such as protein-protein interactions and subcellular localization regulation) can still mediate pro-cancer signal transduction, making it difficult to achieve complete intervention on the target molecule. In contrast, the PROTAC molecule of this invention mediates the complete degradation of ALKBH5 protein through the ubiquitin-proteasome system, fundamentally eliminating all its biological functions and significantly improving the depth and duration of targeted intervention on ALKBH5.

[0030] 2) Effectively overcomes compensatory upregulation and drug resistance risks, significantly enhancing treatment durability. Under prolonged drug stress, tumor cells are prone to upregulating ALKBH5 protein expression or activating alternative oncogenic pathways (such as FTO / m). 6 Adaptive resistance, stemming from the A regulatory axis, constitutes a core bottleneck in the clinical translation of traditional enzyme activity inhibitors. After 72 hours of continuous treatment in KG-1 cells, proteomics analysis confirmed that DEG-PROTAC of this invention did not detect a rebound in ALKBH5 protein expression or a compensatory increase in FTO protein expression. This result demonstrates that a degradation-based targeting strategy can effectively overcome the adaptive resistance mechanisms faced by traditional inhibitors.

[0031] 3) It possesses catalytic and substoichiometric effects, reducing drug dosage and potential toxicity. PROTAC molecules function through an "event-driven" mechanism, where a single molecule can act as a catalyst, cyclically mediating the ubiquitination and degradation of multiple target proteins to achieve efficient target protein clearance. In the acute myeloid leukemia (AML) cell line KG-1, this PROTAC can efficiently degrade and clear the target protein ALKBH5 (DC) at micromolar concentrations. 50 =1.42 μM). Compared to traditional small molecule inhibitors 18I (IC50, 1.42 μM), 50 = 1.907 μM), this PROTAC employs a targeted protein degradation mechanism to induce irreversible ubiquitination and proteasome degradation of target proteins, replacing the "occupation-based" inhibitory mechanism that relies on continuous target site occupancy. This mechanism is based on catalytic cycling characteristics, allowing a single PROTAC molecule to participate multiple times in the formation of a ternary complex, mediating the ubiquitination of multiple target proteins and achieving a substoichiometric effect. Therefore, even though its inhibitory ability on cell proliferation is relatively weak (IC50 = 1.907 μM), this PROTAC works by targeting protein degradation to induce irreversible ubiquitination and proteasome degradation of target proteins, replacing the "occupancy-based" inhibitory mechanism that relies on continuous target site occupancy. 50 Even at concentrations of 16.73 μM, functional clearance can still be achieved at levels far below the inhibitory concentration, demonstrating higher functional efficiency and lower drug demand. This "degradation-functional uncoupling" advantage is expected to reduce drug dosage, off-target toxicity, and systemic side effects, thereby significantly improving the persistence and target specificity of drug action, providing a novel, efficient, and long-lasting precise targeted degradation strategy for ALKBH5-overexpressing AML.

[0032] PROTAC molecules function through an "event-driven" mechanism, where a single molecule can act as a catalyst, cyclically mediating the ubiquitination and degradation of multiple target proteins to achieve efficient target protein clearance. In the acute myeloid leukemia (AML) cell line KG-1, this PROTAC can achieve highly efficient degradation (DC) of the target protein ALKBH5 at micromolar concentrations. 50 =1.42 μM). Compared to traditional small molecule inhibitors 18I (IC50, 1.42 μM), 50= 1.907 μM), the PROTAC of this invention employs a targeted protein degradation mechanism to induce irreversible ubiquitination and proteasome degradation of target proteins, replacing the "occupation-type" inhibitory mechanism that relies on continuous target site occupation. This mechanism is based on catalytic cycling characteristics, allowing a single PROTAC molecule to participate multiple times in the formation of a ternary complex, mediating the ubiquitination of multiple target proteins and achieving a substoichiometric effect. Therefore, even though its inhibitory ability on cell proliferation is relatively weak (IC50 = 1.907 μM), it still achieves a significant inhibitory effect. 50 Even at concentrations of 16.73 μM, functional clearance can still be achieved at levels far below the inhibitory concentration, demonstrating higher functional efficiency and lower drug requirements. This characteristic significantly improves the persistence and target specificity of the drug's action, providing technical advantages such as reduced drug dosage, reduced off-target effects, and systemic toxicity, offering a novel, efficient, and sustainable strategy for targeting protein degradation in the treatment of acute myeloid leukemia (AML).

[0033] 4) It exhibits high selectivity for ALKBH5, with manageable off-target risk. After treatment of the acute myeloid leukemia cell line KG-1 with DEG-PROTAC of the present invention, except for the target protein ALKBH5, its homologous m 6 The protein expression level of FTO, a member of the A demethylase family, did not change significantly, demonstrating that the PROTAC molecule has high targeting specificity for ALKBH5, which is significantly better than some existing multi-target small molecule inhibitors, and the off-target risk for clinical application is controllable.

[0034] 5) It can significantly inhibit the malignant phenotype of leukemia cells and has clear clinical therapeutic potential. In an ALKBH5-overexpressing KG-1 cell model of acute myeloid leukemia, the DEG-PROTAC of this invention efficiently degrades the ALKBH5 target protein, thereby significantly inhibiting the malignant proliferation of leukemia cells. This achievement provides a highly promising drug candidate for the treatment of ALKBH5-driven acute myeloid leukemia and has clear clinical translational value.

[0035] 6) Modular structure design and strong platform versatility, with good prospects for industrialization development. The PROTAC molecule of this invention adopts a modular design of ALKBH5 ligand – Linker – E3 ligand. The molecular structure can be iterated by optimizing the length and type of the linker or by changing the E3 ubiquitin ligase ligand. This study has verified that the combination of PEG2 linker and CRBN ligand (pomalidomide) is the optimal matching scheme, which lays a solid technical foundation for the subsequent structural optimization of PROTAC molecules, development of new dosage forms (such as VHL ligand version) and patent layout, and has good prospects for industrialization development. Attached Figure Description

[0036] Figure 1 The molecular structure of PROTAC; Figure 2 The synthetic route for PROTAC molecules; Figure 3 The NMR spectrum of tert-butyl (2-hydroxyphenylethyl) carbamate; Figure 4 The NMR spectrum of tert-butyl (2-(prop-2-yn-1-yloxy)phenethyl)carbamate; Figure 5 The NMR spectrum of 1-(2-(prop-2-yn-1-yloxy)phenethyl)-1H-pyrrole-2,5-dione; Figure 6 Liquid phase triple quadrupole mass spectrum of the target DEG-PROTAC; Figure 7 The spectrum was detected by a high-resolution liquid chromatography-mass spectrometry (Orbitrap Exploris 120) instrument targeting DEG-PROTAC. Figure 8 The degradation effect of DEG-PROTAC molecules; Figure 9 To detect the inhibitory effects of DEG-PROTAC and TEG-PROTAC on the proliferation of KG-1 cells using the CCK-8 assay; Figure 10 Liquid phase triple quadrupole mass spectrum of the target TEG-PROTAC; Figure 11 The chromatogram of the target TEG-PROTAC was detected by a high-resolution liquid chromatography-mass spectrometry system (Orbitrap Exploris 120). Detailed Implementation

[0037] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0039] Example 1: Synthesis of PROTAC Molecular The synthetic route of PROTAC molecules is as follows: Figure 2As shown in the figure. The reagents, solvents, reaction temperatures, and times used in this route are all conventional organic synthesis conditions, demonstrating good reproducibility and industrial feasibility.

[0040] 1. General Experimental Methods All commercial chemicals were used directly in the experiments without further purification. Rapid column chromatography was performed using 200–300 mesh silica gel as the stationary phase, with eluents optimized based on compound polarity. The target final product was purified by high-performance liquid chromatography (HPLC) using a mobile phase of chromatographically pure acetonitrile (mobile phase A) and ultrapure water (mobile phase B) with a linear gradient elution program: the proportion of mobile phase A increased linearly from 5% to 100% within 20 minutes. The eluted fraction was collected and detected using a 254 nm UV detector.

[0041] Nuclear magnetic resonance (NMR) spectra were measured on a Bruker 400 MHz NMR spectrometer at a default experimental temperature of 298 K (unless otherwise specified). The chemical shift of the residual peak in the solvent used was used as an internal standard. Chemical shifts (δ) are expressed in parts per million (ppm), coupling constants (J) are expressed in Hertz (Hz), and peak types are labeled as follows: (s) singlet, (d) doublet, (t) triplet, (q) quartet, (m) multiplet, (br) broad peak. High-resolution mass spectrometry (HRMS) data were acquired using a Thermo Scientific Orbitrap Exploris 120 HPLC-MS system equipped with a Z-type spray ionization source, with positive ion electrospray ionization (ESI+) detection mode and a mass scan range of 100–1200 Da.

[0042] 2. Reaction conditions (corresponding to) Figure 2 (Synthetic route) a. The reaction system consisted of di-tert-butyl dicarbonate (Boc2O), sodium bicarbonate (NaHCO3), and dichloromethane (DCM), and the mixture was stirred overnight at room temperature. b. The reaction system consisted of propyne bromide, potassium carbonate (K2CO3), and N,N-dimethylformamide (DMF), and the mixture was stirred overnight at 60°C. c. Trifluoroacetic acid (TFA) and dichloromethane (DCM) were used as the reaction system, and the mixture was stirred overnight at room temperature. d. Maleic anhydride and acetic acid are used as the reaction system, and the mixture is stirred and reacted overnight under reflux conditions; e. Copper sulfate, sodium ascorbate, pomalidomide-diethylene glycol-azide derivative (or pomalidomide-triethylene glycol-azide derivative), stirred overnight at room temperature.

[0043] 3. Synthesis and characterization of a series of target products 3.1 Synthesis and Characterization of Tert-Butyl (2-Hydroxyphenylethyl) Carbamate tert-butyl (2-hydroxyphenylethyl) carbamate Add 2-(2-aminoethyl)phenol (1.37 g, 10 mmol, 1.0 eq), sodium bicarbonate (NaHCO3, 1.68 g, 20 mmol, 2.0 eq), and di-tert-butyl dicarbonate (Boc2O, 1.68 g, 20 mmol, 2.0 eq) to a dry reaction vessel. Add an appropriate amount of dichloromethane (DCM) to fully dissolve the mixture. Stir the mixture magnetically overnight at room temperature (TLC monitoring the reaction endpoint). After the reaction is complete, add DCM to dilute the mixture. Transfer the mixture to a separatory funnel, wash the organic phase three times with saturated brine, allowing it to separate into layers after each wash, and collect the organic phase. The combined organic phases were dried over anhydrous sodium sulfate (and allowed to stand for 1–2 h), filtered to remove the desiccant, concentrated by rotary evaporation to remove the solvent, and the crude product was purified by silica gel column chromatography (eluting gradient optimized, such as petroleum ether / ethyl acetate system) to obtain the target product as a white solid (2.2 g, yield 92.8%). Its structural characterization data are as follows: ¹H NMR (400 MHz, CD3OD) δ 7.09–6.97 (m, 2H), 6.76 (t, J = 7.4 Hz, 2H), 3.27 (t, J = 7.3 Hz, 2H), 2.77 (t, J = 7.3 Hz, 2H), 1.43 (s, 9H).

[0044] ¹³C NMR (100 MHz, CD3OD) δ 130.29, 127.10, 125.47, 119.15, 114.53, 40.36, 30.29, 27.39.

[0045] 3.2 Synthesis and Characterization of Tert-Butyl(2-(Prop-2-yn-1-yloxy)phenethyl)carbamate tert-butyl (2-(prop-2-yn-1-yloxy)phenethyl)carbamate tert-butyl (2-hydroxyphenylethyl) carbamate (2.37 g, 10 mmol, 1.0 eq) was dissolved in an appropriate amount of N,N-dimethylformamide (DMF), and potassium carbonate (2.76 g, 20 mmol, 2.0 eq) was added. The mixture was activated by stirring at room temperature for 30 min. Then, propargyl bromide (1.19 g, 10 mmol, 1.0 eq) was added, and the mixture was heated to 60 °C and magnetically stirred overnight in an oil bath (TLC monitoring the reaction endpoint). After the reaction was complete, the mixture was cooled to room temperature, diluted with ethyl acetate, transferred to a separatory funnel, and the organic phase was washed three times with saturated brine. After standing and separating the layers, the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated to remove the solvent, and the crude product was purified by silica gel column chromatography (elutant gradient optimized) to obtain a pale yellow oily target product (2.5 g, yield 90.9%). Its characterization data are as follows: ¹H NMR (400 MHz, CD3OD) δ 7.26–7.11 (m, 2H), 7.08–7.01 (m, 1H), 6.92 (td, J = 7.4, 1.1 Hz, 1H), 4.78 (d, J = 2.4 Hz, 2H), 3.26 (dd, J = 7.9, 6.6 Hz, 2H), 2.94 (t, J =2.4 Hz, 1H), 2.80 (dd, J = 7.9, 6.6 Hz, 2H), 1.43 (s, 9H).

[0046] ¹³C NMR (101 MHz, CD3OD) δ 130.38, 127.20, 120.96, 111.88, 78.64, 75.25, 55.39, 40.22, 27.38.

[0047] 3.3 Synthesis of 2-(2-(prop-2-yn-1-yloxy)phenyl)ethyl-1-amine (intermediate) 2-(2-(prop-2-yn-1-yloxy)phenyl)ethyl-1-amine 2.75 g (10 mmol, 1.0 eq) of tert-butyl(2-(prop-2-yn-1-yloxy)phenethyl)carbamate was dissolved in 20 mL of dichloromethane (DCM), and 10 mL of trifluoroacetic acid (TFA) was slowly added. The mixture was stirred overnight at room temperature. After the reaction was complete, all solvent (including excess TFA) in the system was completely removed by rotary evaporation, yielding the crude product 2-(2-(prop-2-yn-1-yloxy)phenyl)ethyl-1-amine. Since this intermediate is a key precursor for the next reaction and the subsequent cyclization reaction has low purity requirements, the crude product did not require further purification and characterization and was directly added to the subsequent reaction at the starting molar amount (10 mmol).

[0048] 3.4 Synthesis and Characterization of 1-(2-(prop-2-yn-1-yloxy)phenethyl)-1H-pyrrole-2,5-dione 1-(2-(prop-2-yn-1-yloxy)phenethyl)-1H-pyrrole-2,5-dione 2-(2-(prop-2-yn-1-yloxy)phenyl)ethyl-1-amine (10 mmol, 1.0 eq) was dissolved in glacial acetic acid and reacted with maleic anhydride (1.0 g, 10 mmol, 1.0 eq) under reflux overnight. After the reaction was complete, all solvents were removed by rotary evaporation, and the crude product was purified by silica gel column chromatography to give the target product (2.5 g, yield 90.9%). Its characterization data are as follows: ¹H NMR (400 MHz, CDCl3) δ 7.11 (ddd, J = 8.2, 7.4, 1.8 Hz, 1H), 6.98 (dd, J = 7.4, 1.7 Hz, 1H), 6.86 (dd, J = 8.3, 1.0 Hz, 1H), 6.79 (dd, J = 7.4, 1.1 Hz, 1H), 6.52 (s, 2H), 4.63 (d, J = 2.4 Hz, 2H), 3.70 (t, J = 7.0 Hz, 2H), 2.84 (t, J = 7.0 Hz, 2H), 2.43 (t, J = 2.4 Hz, 1H).

[0049] ¹³C NMR (101 MHz, CDCl3) δ 170.65, 155.87, 133.93, 130.79, 127.99, 127.05, 121.43, 111.89, 78.81, 75.51, 56.00, 37.67, 29.51.

[0050] 3.5 Structure of the target PROTAC molecule DEG-PROTAC: Synthesis and characterization of 4-((2-(2-(2-(4-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0051] 4-((2-(2-(4-((2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 1-(2-(prop-2-yn-1-yloxy)phenyl)-1H-pyrrole-2,5-dione (25.5 mg, 0.1 mmol, 1.0 eq) was dissolved in an appropriate amount of DMF, and copper sulfate (80 mg, 0.5 mmol, 5.0 eq) and sodium ascorbate (118 mg, 0.6 mmol, 6.0 eq) were added sequentially. The catalytic system was activated by stirring at room temperature for 15 min. Then, pomalidomide-diethylene glycol-azide derivative (43 mg, 0.1 mmol, 1.0 eq) was added, and the reaction was carried out overnight with magnetic stirring at room temperature (TLC monitoring showed complete 1,3-dipolar cycloaddition). After the reaction, the reaction solution was purified by high-performance liquid chromatography (HPLC) (chromatographic grade acetonitrile-ultrapure water gradient elution) to obtain a pale yellow solid target PROTAC molecule (8 mg, yield 11.7%). Its structure was verified by mass spectrometry, and the characterization data are shown below. Figure 6 and Figure 7 .

[0052] 3.6 Structure of the target PROTAC molecule TEG-PROTAC: Synthesis and characterization of 5-((2-(2-(2-(2-(4-((2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0053] 5-((2-(2-(2-(4-((2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 1-(2-(prop-2-yn-1-yloxy)phenyl)-1H-pyrrole-2,5-dione (25.5 mg, 0.1 mmol, 1.0 eq) was dissolved in an appropriate amount of DMF, and copper sulfate (80 mg, 0.5 mmol, 5.0 eq) and sodium ascorbate (118 mg, 0.6 mmol, 6.0 eq) were added sequentially. The catalytic system was activated by stirring at room temperature for 15 min. Then, pomalidomide-polyethylene glycol-azide derivative (47.4 mg, 0.1 mmol, 1.0 eq) was added, and the reaction was carried out overnight with magnetic stirring at room temperature (TLC monitoring showed complete 1,3-dipolar cycloaddition). After the reaction, the reaction solution was purified by high-performance liquid chromatography (HPLC) (chromatographic grade acetonitrile-ultrapure water gradient elution) to obtain a pale yellow solid target PROTAC molecule (13 mg, yield 17.8%). Its structure was verified by mass spectrometry, and the characterization data are shown below. Figure 10 and Figure 11 .

[0054] Example 2: Functional Verification and Cell Biology Applications of PROTAC Molecules (Biochemical and Cell Biology Experiments) This study used the human acute myeloid leukemia (ALKBH5) high-expressing cell line KG-1 (ATCC, Cat# CCL-246) as a model, and systematically verified the target protein degradation activity, targeting specificity, and anti-leukemia biological function of the PROTAC molecule designed in this invention through a series of biochemical and cell biology experiments. The specific experimental contents are as follows: 1. Detection of the degradation activity and specificity of PROTAC molecules against ALKBH5 protein: KG-1 cells in logarithmic growth phase were seeded into 12-well cell culture plates. After cell attachment, PROTAC molecules (DEG-PROTAC) were added at final concentrations of 0, 0.1, 0.5, 1, 2, 5, 10, and 15 μM, respectively, with three replicates per group, and the treatment lasted for 48 h. After treatment, the culture medium was discarded, and the cells were washed twice with pre-chilled PBS. Then, RIPA cell lysis buffer containing protease inhibitors was added, and the cells were lysed on ice for 30 min. The cell lysates were collected, and the protein concentration was determined. The expression levels of ALKBH5 protein (primary antibody: Abcam, Cat# ab195377) and FTO protein (primary antibody: Abcam, Cat# ab126605) in the cell lysates were detected by Western blot, with GAPDH (Proteintech, Cat# 60004-1) used as an internal control protein. The relative protein expression levels were calculated by grayscale analysis. Figure 8 ).

[0055] Experimental results show that the DEG-PROTAC molecule of the present invention has a significant degradation effect on ALKBH5 protein, and the degradation effect shows a clear dose-dependent effect, with a half-maximal concentration (DC) of 100%. 50 The concentration was 1.42 μM. Meanwhile, this PROTAC molecule showed almost no degradation activity against FTO proteins of the same family, confirming its good targeted degradation specificity against ALKBH5 protein. Figure 8 ).

[0056] 2. PROTAC molecules inhibit the proliferation of leukemia cells. The inhibitory effects of DEG-PROTAC and TEG-PROTAC on the proliferation of human acute myeloid leukemia KG-1 cells were detected using the CCK-8 assay. The experimental procedure was as follows: KG-1 cells in logarithmic growth phase were collected and the cell density was adjusted to 5 × 10⁻⁶ cells / year. 4 Cells / mL were seeded at 100 μL per well in 96-well cell culture plates, with blank control, negative control, and drug treatment groups, each group having 3 replicates. After cell attachment, serially diluted DEG-PROTAC or TEG-PROTAC was added to the drug treatment groups to final concentrations of 0, 2.5, 10, 20, 30, 40, 60, and 80 μM, respectively. The culture plates were incubated at 37 ℃ and 5% CO2 for 64 hours. After incubation, 10 μL of CCK-8 solution was added to each well, and the plates were cultured for another 2–4 hours. The absorbance (OD) of each well was measured at 450 nm using a multi-mode microplate reader. 450 () Figure 9 ).

[0057] Cell viability was calculated based on the measured OD values, and a dose-response curve was plotted with the final drug concentration on the x-axis and cell viability on the y-axis. The curve was fitted using GraphPad Prism software, and the half-maximal inhibitory concentrations (IC50) of the two PROTAC molecules (DEG-PROTAC and TEG-PROTAC) on KG-1 cell proliferation were calculated. 50 () Figure 9 ).

[0058] In summary, this invention successfully developed a PROTAC degrader targeting ALKBH5. This strategy not only overcomes the limitations of traditional ALKBH5 enzyme activity inhibitors by specifically degrading the ALKBH5 protein, but also demonstrated significant anti-leukemic activity at the cellular level. DEG-PROTAC exhibited excellent tumor-suppressive effects, highlighting its enormous clinical translational potential. This research combines outstanding novelty, inventiveness, and practicality, providing a new technological paradigm for the development of innovative drugs targeting RNA epigenetic regulatory pathways.

[0059] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A PROTAC chimera that targets and degrades ALKBH5, the structural formula of which is shown in Formula I: In formula I, n is 1, 2, or 3.

2. The method for preparing the PROTAC chimera targeting ALKBH5 degradation according to claim 1, comprising the following steps: a) Under alkaline conditions, 2-(2-aminoethyl)phenol and di-tert-butyl dicarbonate are reacted to obtain tert-butyl (2-hydroxyphenylethyl) carbamate. b) Under alkaline conditions, tert-butyl (2-hydroxyphenylethyl) carbamate and propynyl bromide are reacted to obtain tert-butyl (2-(prop-2-yn-1-yloxy)phenylethyl) carbamate. c) In the presence of trifluoroacetic acid, deprotection of Boc in tert-butyl (2-(prop-2-yn-1-yloxy)phenethyl)carbamate is carried out to give 2-(2-(prop-2-yn-1-yloxy)phenyl)ethyl-1-amine. d) React 2-(2-(prop-2-yn-1-yloxy)phenyl)ethyl-1-amine with maleic anhydride in glacial acetic acid to give 1-(2-(prop-2-yn-1-yloxy)phenethyl)-1H-pyrrole-2,5-dione; e) 1-(2-(prop-2-yn-1-yloxy)phenyl)-1H-pyrrole-2,5-dione was dissolved in N,N-dimethylformamide (DMF), and copper sulfate and sodium ascorbate were added sequentially at room temperature with stirring to activate the copper catalytic system; subsequently, pomalidomide-diethylene glycol-azide was added to the reaction system for a click chemical coupling reaction, and after the reaction was completed, the PROTAC chimeric compound (DEG-PROTAC) was obtained by purification; wherein... When pomalidomide-diethylene glycol-azide is replaced with pomalidomide-polyethylene glycol-azide, TEG-PROTAC can be obtained; 。 3. The preparation method according to claim 2, characterized in that: In step a), the alkaline conditions are provided by sodium bicarbonate; the reaction is carried out in an organic solvent, namely dichloromethane; the reaction conditions are: stirring overnight at room temperature. And / or, in step b), the alkaline conditions are provided by potassium carbonate; the reaction is carried out in an organic solvent, the organic solvent being N,N-dimethylformamide; the reaction conditions are: stirring overnight at 60°C.

4. The preparation method according to claim 2 or 3, characterized in that: In step c), the reaction is carried out in an organic solvent, namely dichloromethane; the reaction conditions are: stirring overnight at room temperature. And / or, in step d), the reaction conditions are: stirring and reacting overnight under reflux conditions; And / or, in step e), the reaction conditions are: stirring overnight at room temperature.

5. The application of the PROTAC chimera for targeted degradation of ALKBH5 as described in claim 1 in the preparation of ALKBH5 targeted degrading agents.

6. The use of the PROTAC chimera that targets and degrades ALKBH5 as described in claim 1 in the preparation of an inhibitor of acute myeloid leukemia cell proliferation.

7. The application according to claim 6, characterized in that: The acute myeloid leukemia cells mentioned are KG-1 cells, a human acute myeloid leukemia cell line that highly expresses ALKBH5.

8. The use of the PROTAC chimera that targets and degrades ALKBH5 as described in claim 1 in the preparation of a medicament for the prevention and / or relief and / or treatment of acute myeloid leukemia.

9. A product, wherein the active ingredient comprises the PROTAC chimera for targeted degradation of ALKBH5 as described in claim 1; The product has at least one of the following effects: (a2) Targeted degradation of ALKBH5; (b2) Inhibits the proliferation of acute myeloid leukemia cells in vivo or in vitro; (c2) Prevention and / or relief and / or treatment of acute myeloid leukemia.

10. The product according to claim 9, characterized in that: The product is a drug or reagent; And / or, the product may also include pharmaceutically acceptable excipients.

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