A proteolysis targeting chimera targeting dopamine d2 receptor, preparation method and application

Abstract

This application discloses a proteolytic targeting chimera targeting the dopamine D2 receptor, its preparation method, and its application, relating to the field of medicinal chemistry. The proteolytic targeting chimera consists of an E3 ubiquitin ligase ligand portion, a dopamine D2 receptor ligand portion, and an alkane chain linker arm containing a quaternary ammonium solubilizing group. In vitro cell experiments confirmed that target compounds 1, 2, and 3 all showed good safety in primary striatal neurons. ELISA and Western blot experiments confirmed that target compounds 1, 2, and 3 could degrade intracellular DRD2 protein in a dose-dependent manner, with PROTAC3 showing the best degradation effect and synergistic effect when used in combination with risperidone. Stereoscopic injection experiments into the striatum of C57 mice further confirmed the in vivo targeted degradation activity of DRD2.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, and in particular to a protein hydrolysis-targeting chimera that targets the dopamine D2 receptor and its preparation method. Background Technology

[0002] The dopamine D2 receptor (DRD2) is an important member of the G protein-coupled receptor superfamily, widely distributed in the striatum, mesolimbic system, and mesocortical system of the central nervous system. DRD2 plays a crucial role in regulating physiological processes such as motor function, emotional cognition, and neuroendocrine function. Its abnormal expression is closely related to various neuropsychiatric disorders, including schizophrenia, Parkinson's disease, bipolar disorder, and drug addiction. Therefore, DRD2 has long been an important target in the development of drugs for neuropsychiatric diseases.

[0003] Current clinical treatments for DRD2 are mainly divided into two categories: one is DRD2 antagonists, represented by first-generation antipsychotics such as haloperidol and chlorpromazine; the other is DRD2 partial agonists, represented by aripiprazole. The mechanism of action of these drugs is based on competitively binding to the orthoautomerism of DRD2. By continuously occupying the receptor binding site, they block dopamine-mediated downstream signal transduction, thereby exerting a therapeutic effect.

[0004] However, the aforementioned target-based inhibition strategies have inherent pharmacological limitations. First, drug molecules must occupy the receptor binding site at a sufficiently high concentration to maintain the therapeutic effect. Once administration is interrupted or the drug concentration decreases, receptor function can recover, which necessitates long-term and frequent drug administration. Second, after the receptor is continuously occupied for a long time, the body may produce compensatory adaptive responses, including receptor upregulation and signaling pathway remodeling, leading to decreased drug efficacy or drug resistance. Third, these site-occupying drugs only act on the functional level of the receptor and cannot fundamentally affect the expression level of the DRD2 protein itself. Therefore, their therapeutic effect on pathological states driven by DRD2 protein overexpression is limited.

[0005] Proteolysis-targeting chimera (PROTAC) technology is a novel strategy for targeted protein degradation that has emerged in the field of medicinal chemistry in recent years. PROTAC molecules typically consist of three parts: a target protein ligand, an E3 ubiquitin ligase ligand, and a linker arm connecting the two. These molecules can simultaneously bind to the target protein and the E3 ubiquitin ligase, inducing polyubiquitination of the target protein, which is then recognized and degraded by the 26S proteasome in the cell. Compared to traditional site-occupying inhibitors, PROTAC technology has event-driven pharmacology; a single PROTAC molecule can be recycled after completing one target protein degradation cycle, theoretically achieving sustained clearance of the target protein with substoichiometric dosages. Currently, PROTAC technology has made significant progress in research on multiple targets, including BRD4, androgen receptor (AR), and estrogen receptor (ER), with several candidate molecules entering clinical trials.

[0006] Patent CN119454988A discloses a proteolytic targeting chimera for the targeted degradation of the YY1 transcription factor. This chimera uses a double-stranded DNA molecule as its structural backbone, with an E3 ubiquitin ligase receptor linked to its 3' end, for use in cancer treatment. Patent CN114891119A discloses a biomacromolecule-targeted proteolytic chimera for the degradation of PD-L1. This chimera degrades PD-L1 via the proteasome pathway, thereby activating the killing power of T cells against tumor cells. Patent CN118987246A discloses a method for repositioning intracellular E3 ubiquitin ligases to achieve membrane protein degradation. This method uses aptamers to reposition the E3 ubiquitin ligase to the inner side of the cell membrane, achieving membrane protein degradation. The aforementioned prior art mainly targets the degradation of tumor-associated target proteins (such as YY1 and PD-L1) or membrane proteins.

[0007] Currently, there are no reports in publicly available literature or patents regarding PROTAC compounds specifically targeting DRD2. How to apply PROTAC technology to the DRD2 target, design and synthesize bifunctional chimeric molecules that can effectively induce DRD2 protein degradation, and establish reliable preparation methods are urgent technical problems to be solved in this field. Summary of the Invention

[0008] The primary objective of this invention is to provide a proteolytic targeting chimera that targets the dopamine D2 receptor, thereby addressing the problems of existing therapeutic drugs targeting the dopamine D2 receptor (DRD2) that can only block signal transduction by continuously occupying the receptor's orthotopic site, cannot reduce DRD2 expression at the protein level, require long-term and frequent administration, and are prone to drug resistance, as well as the current lack of a proteolytic targeting chimera (PROTAC) compound specifically targeting DRD2.

[0009] To achieve the aforementioned first objective, the present invention adopts the following technical solution: A proteolytic targeting chimera comprises an E3 ubiquitin ligase ligand portion, a target protein ligand portion, and a linker arm for connecting the two portions. The target protein ligand portion is a dopamine D2 receptor ligand portion for specifically binding to the dopamine D2 receptor. The linker arm is composed of an alkane chain with a carbon chain length of 5-12 carbon atoms, and includes a quaternary ammonium salt solubilizing group and a terminal reactive group. The terminal reactive group is used to covalently link the E3 ubiquitin ligase ligand portion and the dopamine D2 receptor ligand portion.

[0010] Preferably, the E3 ubiquitin ligase ligand portion is a lenalidomide derivative.

[0011] Preferably, the dopamine D2 receptor ligand portion is bromocriptine or a derivative thereof.

[0012] Preferably, the quaternary ammonium salt solubilizing group is a quaternary ammonium salt group.

[0013] The second objective of this invention is to provide a method for preparing the above-mentioned protein hydrolysis-targeted chimera, so as to solve the problem of the lack of reliable modular synthetic routes for DRD2-targeted PROTAC compounds in the prior art.

[0014] To achieve the second objective mentioned above, the present invention adopts the following technical solution: A method for preparing a protein hydrolysis-targeted chimera includes the following steps: Step 1: A hydroxyl compound containing a dopamine D2 receptor ligand backbone is subjected to an O-alkylation reaction with a dihaloalkane with a carbon chain length of 5-12 carbon atoms under alkaline conditions to obtain intermediate A; wherein the reaction temperature of Step 1 is 40°C. Step 2: Remove the terminal protecting group from intermediate A under deprotection conditions to expose the terminal carboxyl group, thereby obtaining intermediate B; wherein the reaction temperature in step 2 is room temperature; Step 3: In the presence of an amide coupling reagent and an organic base, intermediate B is subjected to an amidation coupling reaction with a lenalidomide derivative to obtain the protein hydrolysis targeting chimera.

[0015] Preferably, in step 1, the alkaline conditions are provided by potassium hydroxide, and 18-crown ether-6 is used as a phase transfer catalyst, and the reaction solvent is a mixture of tetrahydrofuran and water.

[0016] Preferably, in step 3, the amide coupling reagent for the amidation coupling reaction is HATU, the organic base is N,N-diisopropylethylamine (DIEA), the solvent is N,N-dimethylformamide, and the reaction temperature is 25°C.

[0017] Preferably, the coupling product of step 3 is purified by preparative high performance liquid chromatography, and the eluent is an acetonitrile / water solution containing 0.1% ammonium bicarbonate, with an acetonitrile volume fraction gradient of 0-60%.

[0018] A third objective of this invention is to provide applications of the aforementioned protein hydrolysis-targeting chimera to expand the application scope of PROTAC technology in the field of dopamine D2 receptor-related disease research.

[0019] To achieve the third objective mentioned above, the present invention adopts the following technical solution: The use of the protein hydrolysis targeting chimera according to any one of claims 1-5 in the preparation of a degradation reagent for dopamine D2 receptor.

[0020] Preferably, the reagent is used to study the in vitro mechanism of dopamine hypersensitivity symptoms, wherein the dopamine hypersensitivity symptoms are selected from antipsychotic drug resistance, rebound syndrome, or tardive dyskinesia.

[0021] Preferably, the protein hydrolysis-targeting chimera is used in combination with traditional antipsychotic drugs to enhance the degradation effect of dopamine D2 receptors.

[0022] The beneficial effects of this invention are: (1) The protein hydrolysis targeting chimera provided by the present invention specifically recognizes and binds to DRD2 via the dopamine D2 receptor ligand, while recruiting E3 ubiquitin ligase to induce DRD2 to undergo polyubiquitination modification and degradation via the intracellular 26S proteasome pathway, thereby eliminating the pathogenic target at the protein level. This overcomes the inherent defect of traditional DRD2 antagonists that can only block receptor function and cannot reduce the expression level of DRD2 protein. The ubiquitination analysis experiment (Western Blot) confirmed that target compound 3 (PROTAC3) can induce DRD2 ubiquitination degradation in a dose-dependent manner. There was a significant difference in the gray value of the DRD2 protein band between the control group (Con) and the treatment group (low P3+, medium P3+, high P3+). (2) The protein hydrolysis targeting chimera provided by the present invention demonstrated the activity of targeting DRD2 degradation in vivo in the bilateral striatal stereotactic injection experiment of C57 mice. 24 hours after injecting PROTAC3 (0.6 μg / side or 1.2 μg / side, concentration 3.4 mg / mL, injection volume 150 μL / side) into the bilateral striatal regions of mice, Western Blot analysis of brain tissue showed a decrease in striatal DRD2 protein levels, proving that the compound has the ability to target DRD2 degradation in vivo. (3) The preparation method provided by the present invention adopts a modular three-step synthetic route. By introducing a linker arm through O-alkylation, exposing the terminal carboxyl group through deprotection, and coupling the E3 ligand with HATU / DIEA amide, target compound 1 (LCMS m / z = 1011.4 (M+H)⁺, LC purity 100%, yield 14%), target compound 2 (LCMS m / z = 1067.5 (M+H)⁺, LC purity 98.43%, yield 18%) and target compound 3 (LCMS m / z = 1109.5 (M+H)⁺, LC purity 95.29%, yield 14%) were prepared. The LC purity of each target compound reached more than 95%, which provided a material basis for subsequent structure-activity relationship studies and lead compound optimization. (4) The protein hydrolysis targeting chimera provided by the present invention can be used to prepare a degradation reagent for dopamine D2 receptor, which can be applied to the in vitro mechanism study of dopamine hypersensitivity symptoms (including antipsychotic drug resistance, rebound and tardive dyskinesia), and can be used in combination with traditional antipsychotic drugs to enhance the degradation effect of DRD2, providing an important tool molecule for basic research on DRD2-related neuropsychiatric diseases and the development of new treatment strategies; (5) The protein hydrolysis targeting chimera provided by the present invention showed in the safety evaluation of primary rat striatal neurons that, within a 24-hour dosing window, the target compound 3 at a concentration of 1 μM inhibited the primary neurons by less than 10%, indicating good cell safety, and provided a reasonable safe concentration reference for its further in vivo and in vitro functional verification experiments. (6) When the protein hydrolysis-targeting chimera provided by the present invention is used in combination with traditional antipsychotic drugs (risperidone), it can produce a synergistic effect: ELISA results showed that the DRD2 content in cells of the group using risperidone in combination with compound 3 (risperidone + high P3) decreased to about 80 pg / mg protein. The protein hydrolysis-targeting chimera provided by the present invention can enhance the degradation effect of DRD2 when used in combination with traditional antipsychotic drugs, providing experimental evidence for the combined treatment strategy of dopamine hypersensitivity symptoms of schizophrenia. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the synthetic route for target compound 1.

[0024] Figure 2 This is a schematic diagram of the synthetic route for target compound 2.

[0025] Figure 3 This is a schematic diagram of the synthetic route for target compound 3.

[0026] Figure 4 This is an optical microscope image of the morphology of primary rat striatal neurons after isolation.

[0027] Figure 5A The effect of target compound 1 (PROTAC1) on the activity of primary striatal neurons. Figure 5B The effect of target compound 2 (PROTAC2) on the activity of primary striatal neurons.

[0028] Figure 5C The effect of target compound 3 (PROTAC3) on the activity of primary striatal neurons.

[0029] Figure 6A The bar chart shows the effect of target compound 1 (PROTAC1) on the content of cellular dopamine D2 receptors under different dosing regimens using ELISA.

[0030] Figure 6B The bar chart shows the effect of target compound 2 (PROTAC2) on the content of cellular dopamine D2 receptors under different dosing regimens using ELISA.

[0031] Figure 6C The bar chart shows the effect of target compound 3 (PROTAC3) on the content of cellular dopamine D2 receptors under different dosing regimens using ELISA.

[0032] Figure 7 This is a schematic diagram showing the effect of Western blotting on the expression level of cellular dopamine D2 receptor protein.

[0033] Figure 8 This is a schematic diagram of the Western blot results for the dopamine D2 receptor ubiquitination degradation induced by target compound 3 (PROTAC3).

[0034] Figure 9 This is a schematic diagram of the Western blot results for detecting the level of dopamine D2 receptor protein in brain tissue after bilateral striatal injection of PROTAC3 in C57 mice. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to specific embodiments. All reagents used in the following embodiments are commercially available analytical grade or chemically pure reagents, and all instruments used have been calibrated.

[0036] Example 1 Synthesis of target compound 1 This embodiment follows Figure 1 The synthetic route shown is used to prepare target compound 1, and the specific steps are as follows.

[0037] Step 1: Synthesis of intermediate C Compound A (685.0 mg, 1 mmol, 1.0 eq.) was dissolved in a mixture of THF and water. Then, compound B (325.0 mg, 1.3 mmol, 1.3 eq.), 18-crown ether-6 (26.0 mg, 0.1 mmol, 0.1 eq.), and potassium hydroxide (101 mg, 1.2 mmol, 1.2 eq.) were added sequentially to the reaction mixture. The reaction mixture was stirred overnight at 40 °C. After the reaction was complete, the mixture was filtered, the solvent was evaporated under reduced pressure, and then purified by silica gel column chromatography (PE / EA = 0–30%) to give a white solid compound C (456 mg, yield 55.5%).

[0038] Step 2: Synthesis of intermediate D Compound C (456.0 mg, 0.55 mmol, 1.0 eq.) was dissolved in 10 mL of dioxane hydrochloride solution and stirred at room temperature for 2 hours. After the reaction was complete, the mixture was filtered, and the solvent was evaporated under reduced pressure to obtain crude compound D, which was used directly in the next reaction without purification.

[0039] Step 3: Synthesis of target compound 1 Compound D (424.0 mg, 0.55 mmol, 1.0 eq.) was dissolved in N,N-dimethylformamide (DMF) solution. Then, compounds E (142.0 mg, 0.55 mmol, 1.0 eq.), HATU (313.0 mg, 0.825 mmol, 1.5 eq.), and N,N-diisopropylethylamine (DIEA, 212 mg, 1.65 mmol, 3.0 eq.) were added sequentially to the reaction mixture. The reaction mixture was stirred overnight at 25 °C. After the reaction was complete, the mixture was concentrated under reduced pressure and purified by preparative high-performance liquid chromatography (HPLC) by elution with an acetonitrile / water solution containing 0.1% NH4HCO3 (acetonitrile volume fraction gradient 0–60%) to give a white solid, target compound 1 (80.0 mg, 0.08 mmol, yield 14%). LC purity: 100% (UV wavelength 254 nm); LCMS m / z = 1011.4 (M+H)⁺.

[0040] Example 2 Synthesis of target compound 2 This embodiment follows Figure 2 The synthetic route shown is used to prepare target compound 2, and the specific steps are as follows.

[0041] Step 1: Synthesis of intermediate C Compound A (653.0 mg, 1.0 mmol, 1.0 eq.) was dissolved in a mixture of THF and water. Then, compound B (325.0 mg, 1.3 mmol, 1.3 eq.), 18-crown ether-6 (26.0 mg, 0.1 mmol, 0.1 eq.), and potassium hydroxide (101 mg, 1.2 mmol, 1.2 eq.) were added sequentially to the reaction mixture. The reaction mixture was stirred overnight at 40 °C. After the reaction was complete, the mixture was filtered, the solvent was evaporated under reduced pressure, and then purified by silica gel column chromatography (PE / EA = 0–30%) to give a white solid compound C (163.0 mg, yield 15.2%); LCMS m / z = 1067.5 (M+H)⁺. The obtained compound C was used directly in the next coupling reaction.

[0042] Step 2: Synthesis of target compound 2 Compound C (163.0 mg, 0.15 mmol, 1.0 eq.) was dissolved in N,N-dimethylformamide (DMF) solution. Then, compound E (38.0 mg, 0.15 mmol, 1.0 eq.), HATU (85.0 mg, 0.225 mmol, 1.5 eq.), and N,N-diisopropylethylamine (DIEA, 58 mg, 0.45 mmol, 3.0 eq.) were added sequentially to the reaction mixture. The reaction mixture was stirred overnight at 25 °C. After the reaction was complete, the mixture was concentrated under reduced pressure and purified by preparative high-performance liquid chromatography (HPLC) by elution with an acetonitrile / water solution containing 0.1% NH4HCO3 (acetonitrile volume fraction gradient 0–55%) to give a white solid, target compound 2 (30.0 mg, 0.03 mmol, yield 18%). LC purity: 98.43% (UV wavelength 254 nm); LCMS m / z = 1067.5 (M+H)⁺.

[0043] Step 1: Synthesis of intermediate C Example 3 Synthesis of target compound 3 This embodiment follows Figure 3 The synthetic route shown is used to prepare target compound 3, and the specific steps are as follows.

[0044] Compound A (685.0 mg, 1 mmol, 1.0 eq.) was dissolved in a mixture of THF and water. Then, compound B (397.0 mg, 1.3 mmol, 1.3 eq.), 18-crown ether-6 (26.0 mg, 0.1 mmol, 0.1 eq.), and potassium hydroxide (101 mg, 1.2 mmol, 1.2 eq.) were added sequentially to the reaction mixture. The reaction mixture was stirred overnight at 40 °C. After the reaction was complete, the mixture was filtered, the solvent was evaporated under reduced pressure, and then purified by silica gel column chromatography (PE / EA = 0–30%) to give a white solid compound C (480 mg, yield 54.5%).

[0045] Step 2: Synthesis of intermediate D Compound C (480.0 mg, 0.54 mmol, 1.0 eq.) was dissolved in a mixture of THF and water and stirred at room temperature for 2 hours. After the reaction was complete, the mixture was filtered, the THF was evaporated under reduced pressure, and dilute hydrochloric acid was added dropwise to the remaining reaction solution to precipitate the product. The solid was collected by filtration to obtain crude compound D, which was directly used in the next reaction step.

[0046] Step 3: Synthesis of target compound 3 Compound D (465.0 mg, 0.54 mmol, 1.0 eq.) was dissolved in N,N-dimethylformamide (DMF) solution. Then, compounds E (142.0 mg, 0.55 mmol, 1.0 eq.), HATU (313.0 mg, 0.825 mmol, 1.5 eq.), and N,N-diisopropylethylamine (DIEA, 212 mg, 1.65 mmol, 3.0 eq.) were added sequentially to the reaction mixture. The reaction mixture was stirred overnight at 25 °C. After the reaction was complete, the mixture was concentrated under reduced pressure and purified by preparative high-performance liquid chromatography (HPLC) by elution with an acetonitrile / water solution containing 0.1% NH4HCO3 (acetonitrile volume fraction gradient 0–55%) to give a white solid, target compound 3 (40.0 mg, 0.04 mmol, yield 14%). LC purity: 95.29% (UV wavelength 254 nm); LCMS m / z = 1109.5 (M+H)⁺.

[0047] The preparation data of target compounds 1, 2, and 3 are summarized in the table below.

[0048] Example 4: Isolation and Culture of Primary Rat Striatal Neurons This embodiment establishes a primary rat striatal neuron cell model for subsequent functional verification experiments. Experimental results are detailed below. Figure 4 .

[0049] Brain tissue was extracted from newborn rats (1-3 days after birth) under aseptic conditions, and the bilateral striatum was dissected and isolated. The striatal tissue was placed in pre-chilled D-Hank's buffer, minced, and digested with 0.25% trypsin at 37°C for 15 minutes. Digestion was terminated by adding serum-containing culture medium. The cells were gently pipetted to prepare a single-cell suspension, filtered through a 200-mesh sieve, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in complete neuronal culture medium (Neurobasal medium supplemented with 2% B27, 0.5 mM glutamine, and penicillin / streptomycin) and seeded into culture plates pre-coated with poly-L-lysine (PLL). The cells were incubated at 37°C in a 5% CO2 incubator. Half of the medium was replaced on day 3, and then the entire medium was replaced every 2 days thereafter. Cell morphology was observed under an optical microscope on day 7.

[0050] like Figure 4 As shown, under an optical microscope, the cells are plump and have clear outlines. Multiple slender neural processes extend from the cell bodies and intertwine to form a network, exhibiting a typical neuronal morphology. This indicates that the primary striatal neurons were successfully isolated and cultured, and the cells are in good condition and can be used for subsequent experiments.

[0051] Example 5 In this embodiment, the cell safety of target compounds 1, 2, and 3 was evaluated using a cell activity assay. The experimental results are shown in Figure 5.

[0052] Primary rat striatal neurons cultured for 7 days were harvested, and after adjusting the cell density, they were seeded into 96-well plates at 100 μL per well and cultured at 37°C in a 5% CO2 incubator for 24 hours until the cells adhered to the plate.

[0053] A series of working solutions with concentration gradients (0, 0.1, 0.5, 1, 2, 4, 8, 16, 32 μM) for target compounds 1, 2, and 3 were prepared and added to the corresponding wells of each group, with 3 replicates for each concentration.

[0054] At 6, 24, 48 and 72 hours after drug administration, the absorbance of each well was measured according to the instructions of the cell viability assay kit, and the cell inhibition rate was calculated.

[0055] Figure 5A To investigate the effect of target compound 1 (PROTAC1) on the activity of primary striatal neurons, Figure 5B To investigate the effect of target compound 2 (PROTAC2) on the activity of primary striatal neurons, Figure 5C The effect of target compound 3 (PROTAC3) on the activity of primary striatal neurons.

[0056] The inhibitory effects of the three compounds on primary striatal neurons were both dose- and time-dependent.

[0057] At the 6-hour time point, the cell inhibition rate of each concentration group was close to 0, indicating that the cytotoxicity was extremely low in a short period of time.

[0058] At the 24-hour time point, the cell inhibition rate of all three compounds was less than 10% at a concentration of 1 μM, indicating good cell safety within this concentration range.

[0059] With extended time to 48 hours and 72 hours, the inhibition rate of the high concentration group (≥16 μM) increased significantly.

[0060] The above results indicate that, within a 24-hour dosing window, target compounds 1, 2, and 3 exhibit acceptable safety for primary neurons at low to moderate concentrations, providing a reasonable reference for dosing concentrations for subsequent functional experiments.

[0061] Example 6 This embodiment uses enzyme-linked immunosorbent assay (ELISA) to quantitatively evaluate the effects of target compounds 1, 2, and 3, used alone or in combination with risperidone, on the intracellular dopamine D2 receptor protein content. Experimental results are available in [reference needed]. Figure 6A , 6B 6C.

[0062] Primary rat striatal neurons were seeded into 24-well plates and cultured for 7 days. Then, the following groups were treated with the following drugs: control group (no drug), bromocriptine group (0.1 μM), risperidone group (20 μM), low concentration group of target compound 1 (P1 low, 0.25 μM), medium concentration group of target compound 1 (P1 medium, 0.5 μM), high concentration group of target compound 1 (P1 high, 1 μM), low concentration group of target compound 2 (P2 low, 0.25 μM), medium concentration group of target compound 2 (P2 medium, 0.5 μM), high concentration group of target compound 2 (P2 high, 1 μM), low concentration group of target compound 3 (P3 low, 0.25 μM), medium concentration group of target compound 3 (P3 medium, 0.5 μM), and high concentration group of target compound 3 (P3 high, 1 μM). The groups included: μM, low concentration of risperidone + target compound 3 (risperidone + P3 low), medium concentration of risperidone + target compound 3 (risperidone + P3 medium), and high concentration of risperidone + target compound 3 (risperidone + P3 high), with 3 replicates per group.

[0063] Twenty-four hours after drug administration, cell lysates were collected, and the DRD2 protein content (pg / mg protein) in the cell lysates of each group was detected according to the instructions of the DRD2 ELISA kit, and statistical analysis was performed.

[0064] like Figure 6A , 6B As shown in Figures 6C, compared with the control group (approximately 840 pg / mg protein), the DRD2 content in the bromocriptine group and the risperidone group showed no significant change (approximately 830–850 pg / mg protein), indicating that traditional space-occupying drugs do not affect the total amount of DRD2 protein.

[0065] The DRD2 content in cells decreased significantly in a dose-dependent manner in the groups treated alone with target compounds 1, 2, and 3.

[0066] Among them, the DRD2 content in the high concentration group of target compound 3 (P3 high) decreased to about 230 pg / mg protein (p<0.01), and the degradation effect was better than that of target compound 1 and target compound 2.

[0067] The degradation effect of risperidone combined with target compound 3 was further enhanced. The DRD2 content in the high concentration group of risperidone + P3 decreased to about 80 pg / mg protein (p<0.01), which was significantly better than the target compound 3 alone group.

[0068] The above results demonstrate that the protein hydrolysis-targeting chimera provided by this invention can effectively degrade DRD2 protein at the cellular level, and can produce a synergistic effect when used in combination with traditional antipsychotic drugs.

[0069] Based on the comparison of experimental data, target compound 3 showed the best degradation effect, therefore target compound 3 was selected as the first choice for subsequent in vivo experiments.

[0070] Example 7 This embodiment uses Western blotting to further verify the effect of target compound 3 on the expression level of dopamine D2 receptor protein in cells. The experimental results are shown in [link to experimental results]. Figure 7 .

[0071] Primary rat striatal neurons were seeded into 6-well plates and cultured for 7 days. Then, they were treated according to the following groups: control group, bromocriptine group (0.1 μM), risperidone group (20 μM), low concentration of target compound 3 group (low P3, 0.25 μM), medium concentration of target compound 3 group (medium P3, 0.5 μM), high concentration of target compound 3 group (high P3, 1 μM), risperidone + low concentration of target compound 3 combined group (risperidone + low P3), risperidone + medium concentration of target compound 3 combined group (risperidone + medium P3), and risperidone + high concentration of target compound 3 combined group (risperidone + high P3).

[0072] 24 hours after drug administration, cells were collected, total protein was extracted, and protein concentration was determined using the BCA protein quantification kit.

[0073] Equal amounts of protein samples were separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane.

[0074] Block the membrane with 5% skim milk powder blocking buffer at room temperature for 2 hours, add DRD2 primary antibody (1:1000 dilution) and incubate overnight at 4°C. After washing the membrane with TBST, add HRP-labeled secondary antibody (1:5000 dilution) and incubate at room temperature for 2 hours. After washing the membrane with TBST, develop the color with ECL chemiluminescence reagent, using GAPDH as the internal control protein.

[0075] like Figure 7 As shown, the internal reference GAPDH bands exhibited consistent brightness across all lanes, indicating that the sample loading amounts were equal in each group.

[0076] Compared with the control group, bromocriptine group and risperidone group, the DRD2 protein bands in each treatment group of target compound 3 decreased sequentially with increasing drug concentration, and the bands almost disappeared in the high concentration group of P3, which proves that target compound 3 can significantly reduce the intracellular DRD2 protein level in a dose-dependent manner.

[0077] The combination of risperidone and target compound 3 also showed a trend of decreasing DRD2 band with increasing concentration of target compound 3.

[0078] Figure 7 The grayscale analysis data showed that the PROTAC / GAPDH ratio was 3.574 in the control group, 3.778 in the bromocriptine group, 3.266 in the risperidone group, 1.872 in the low concentration P1 group, 1.118 in the medium concentration P3 group, 0.375 in the high concentration P3 group, 1.414 in the risperidone + low concentration P3 group, 0.548 in the risperidone + medium concentration P3 group, and 0.062 in the risperidone + high concentration P3 group.

[0079] Table 2. Example 7 Western Blot Gray-Scale Analysis Data The Western Blot results above corroborate the ELISA detection results in Example 6, jointly demonstrating that the protein hydrolysis targeting chimera provided by the present invention can effectively induce intracellular DRD2 protein degradation.

[0080] Example 8 This embodiment uses Western blotting to verify the ubiquitination and degradation activity of target compound 3 on dopamine D2 receptor. The experimental results are shown in [link to experimental results]. Figure 8 .

[0081] The specific experimental steps are as follows.

[0082] Just before the electrophoresis was finished, the PVDF membrane was soaked in methanol for 15 seconds, then rinsed with ultrapure water for 2 minutes, and then soaked in transfer buffer for 5 minutes for later use.

[0083] After removing the adhesive from the water and trimming the film, immerse it in transfer buffer for 15 minutes to equilibrate.

[0084] Prepare the transfer membrane "sandwich" in the following order: black side (negative electrode) - sponge - filter paper - gel - PVDF membrane - filter paper - sponge - red side (positive electrode). After each layer is laid, remove the air bubbles before laying the next layer. Prepare the sandwich in the transfer buffer to avoid the generation of air bubbles.

[0085] Connect the positive and negative electrodes, place the transfer box into the electroporator with the membrane facing the positive electrode, and add the transfer buffer.

[0086] Place the electrotransfer instrument in an ice-water bath and transfer the membrane at a constant current of 200 mA for 70 minutes.

[0087] After the transfer was completed, the PVDF membrane was quickly removed and placed in a 5% BSA solution for 2 hours at room temperature.

[0088] Remove the membrane and wash it with TBST for 5 minutes x 3 times on a shaker.

[0089] Add the DRD2 polyclonal antibody primary antibody diluted with blocking buffer into the incubation bag and incubate overnight at 4°C.

[0090] Wash the membrane with TBST for 5 minutes × 3 times, then add horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:5000 dilution) and / or HRP-labeled goat anti-mouse secondary antibody (1:5000 dilution), and incubate at room temperature for 2 hours.

[0091] TBST wash film for 15 minutes x 5 times.

[0092] The membrane was placed in a chemiluminescent detection reagent (reagent A: reagent B = 1:1) and reacted for 2 minutes. The membrane was then removed, excess liquid was shaken off, and the membrane was placed flat in the BLT imaging instrument. The door was closed, and the instrument was photographed using its software. The gray values ​​of the DRD2 protein bands in each lane were calculated using image analysis software.

[0093] The experiment included a control group (without target compound 3), a low concentration of target compound 3 administration group (P3+ low), a medium concentration of target compound 3 administration group (P3+ medium), and a high concentration of target compound 3 administration group (P3+ high).

[0094] like Figure 8 As shown, compared with the control group, the gray value of the DRD2 protein band in each treatment group of target compound 3 decreased significantly with the increase of the dosage, proving that target compound 3 can induce DRD2 ubiquitination degradation in a dose-dependent manner.

[0095] Example 9 This embodiment uses a bilateral striatal stereotactic injection experiment in C57 mice to verify the in vivo targeted degradation activity of target compound 3 against DRD2. The experimental results are shown below. Figure 9 .

[0096] The experimental design is as follows.

[0097] Normal wild-type C57 mice were used, and target compound 3 was injected into the bilateral striatums using stereotactic injection (injection dose of 0.6 μg / side or 1.2 μg / side, target compound 3 concentration of 3.4 mg / mL, injection volume of 150 μL / side). A saline control group was set up at the same time.

[0098] Animals were sacrificed 24 hours after drug administration, and brain tissue was collected. The level of DRD2 protein in the striatum was detected by Western blotting to evaluate the in vivo degradation effect of target compound 3 on DRD2 in the striatum of normal wild-type mice.

[0099] In addition, to construct an animal model of DRD2 overexpression, C57 mice were divided into a saline group and a drug administration group (n=3 in each group). The mice were orally administered haloperidol (1 mg / kg, equivalent to 0.025 mg per 25 g mouse; drug preparation method: 6.65 μL of stock solution was diluted with 244.7 μL of 0.1 M glacial acetic acid to 0.25 mL) for 14 consecutive days. After the drug administration, the expression level of DRD2 receptor was detected by Western Blot to confirm the successful establishment of the DRD2 overexpression model, providing a suitable animal model for subsequent verification of PROTAC functional molecules.

[0100] like Figure 9 As shown, compared with the saline control group, the DRD2 protein level in the striatum of mice treated with target compound 3 was reduced, demonstrating that target compound 3 has the ability to target and degrade DRD2 in vivo.

[0101] The above in vitro and in vivo experimental data collectively demonstrate that the protein hydrolysis targeting chimera provided by this invention can achieve catalytic targeted degradation of dopamine D2 receptors by hijacking the intracellular ubiquitin-proteasome system, overcoming the inherent defect of traditional DRD2 antagonists that can only block receptor signal transduction and cannot eliminate the target at the protein level.

[0102] It is understood that the E3 ubiquitin ligase ligand moiety is not limited to lenalidomide derivatives, but may also be other ligands that recruit CRBN-E3 ligases, such as pomalidomide derivatives.

[0103] Obviously, the dopamine D2 receptor ligand moiety is not limited to bromocriptine, but can also be other DRD2 high-affinity ligands, such as derivatives of risperidone or aripiprazole.

[0104] It is understood that the carbon chain length of the connecting arm is not limited to n=5, n=9, or n=12, but can be other carbon chain lengths in the range of 5-12.

[0105] Obviously, the solubilizing group of the quaternary ammonium salt is not limited to a specific quaternary ammonium salt structure, but can also be other positively charged hydrophilic groups, such as quaternary ammonium phosphate salts or quaternary ammonium sulfonic acid salts.

[0106] It is understood that the base used in the O-alkylation reaction in the preparation method is not limited to potassium hydroxide, but can also be other strong bases such as potassium carbonate and potassium tert-butoxide.

[0107] Obviously, the phase transfer catalyst is not limited to 18-crown ether-6, but can also be other crown ethers or phase transfer catalysts.

[0108] The above description only illustrates the preferred embodiments of the present invention and should not be construed as limiting the scope of the claims. The present invention is not limited to the above embodiments, and variations in its specific structure are permitted. All modifications made within the scope of the independent claims of this invention are also within the scope of protection of this invention.

Claims

1. A proteolysis targeting chimera consisting of an E3 ubiquitin ligase ligand moiety, a target protein ligand moiety, and a linker arm for linking the first two moieties, characterized in that, The target protein ligand portion is a dopamine D2 receptor ligand portion, used for specific binding to the dopamine D2 receptor; the linker arm is composed of an alkane chain with a carbon chain length of 5-12 carbon atoms, and the linker arm contains a quaternary ammonium salt solubilizing group and a terminal reactive group, the terminal reactive group being used to covalently link the E3 ubiquitin ligase ligand portion and the dopamine D2 receptor ligand portion.

2. The proteolysis targeting chimera of claim 1, wherein, The ligand portion of the E3 ubiquitin ligase is a lenalidomide derivative.

3. The protein hydrolysis-targeting chimera according to claim 1, characterized in that, The dopamine D2 receptor ligand portion is bromocriptine or a derivative thereof.

4. The protein hydrolysis-targeting chimera according to claim 1, characterized in that, The quaternary ammonium salt solubilizing group is a quaternary ammonium salt group.

5. A method for preparing the protein hydrolysis targeting chimera of claim 1, characterized in that, The method includes the following steps: Step 1: A hydroxyl compound containing a dopamine D2 receptor ligand backbone is subjected to an O-alkylation reaction with a dihaloalkane with a carbon chain length of 5-12 carbon atoms under basic conditions to obtain intermediate A; wherein the basic conditions in Step 1 are provided by potassium hydroxide, and 18-crown ether-6 is used as a phase transfer catalyst, the reaction solvent is a mixed solvent of tetrahydrofuran and water, and the reaction temperature is 40°C; Step 2: Remove the terminal protecting group from intermediate A under deprotection conditions to expose the terminal carboxyl group, thereby obtaining intermediate B; Step 3: In the presence of an amide coupling reagent and an organic base, intermediate B is subjected to an amidation coupling reaction with a lenalidomide derivative to obtain the protein hydrolysis targeting chimera.

6. The method according to claim 5, characterized in that, In step 1, the alkaline conditions are provided by potassium hydroxide, and 18-crown ether-6 is used as a phase transfer catalyst. The reaction solvent is a mixture of tetrahydrofuran and water, and the reaction temperature is 40°C.

7. The method according to claim 5, characterized in that, In step 3, the amide coupling reagent is HATU, the organic base is N,N-diisopropylethylamine, the reaction solvent is N,N-dimethylformamide, and the reaction temperature is 25°C. The coupling product of step 3 is purified by preparative high performance liquid chromatography, and the eluent is an acetonitrile / water solution containing 0.1% ammonium bicarbonate, with an acetonitrile volume fraction gradient of 0-60%.

8. The use of the protein hydrolysis targeting chimera according to any one of claims 1-5 in the preparation of a reagent for the dopamine D2 receptor.

9. The application according to claim 8, characterized in that, The reagent is used to study the in vitro mechanism of dopamine hypersensitivity symptoms, which are selected from antipsychotic drug resistance, rebound symptoms, or tardive dyskinesia.

10. The application according to claim 8, characterized in that, The proteolytic targeting chimera is used in combination with traditional antipsychotic drugs to enhance the degradation effect of dopamine D2 receptors.

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