A compound and uses thereof

Abstract

This invention belongs to the field of pharmaceutical chemistry technology, and discloses a compound and its uses. Specifically, it relates to a CRBN small molecule ligand compound based on a pyridazine-glutarimide core skeleton (PDG), its composition, and its application. It also relates to a protein degrading agent based on a CRBN small molecule ligand compound based on a pyridazine-glutarimide core skeleton (PDG) and its application. Specifically, the CRBN small molecule ligand compound is shown in (Ⅰ): wherein R... 1 and R 2 Selected from one or more of the following: fused rings attached to a pyridazine core, H, alkyl, OR, F, CN, CF3, NR2, Ph, 4-Py; Y selected from one or more of CH, CD, CF, and N; R 3 Selected from one or more of OR, NR2, CHR2, and C≡CR; R 4 Selected from H or alkyl.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology, specifically relating to a CRBN small molecule ligand compound based on a pyridazine-glutarimide core skeleton (PDG), its composition and its application, and also to a protein degrading agent based on a CRBN small molecule ligand compound based on a pyridazine-glutarimide core skeleton (PDG) and its application. Background Technology

[0002] Cereblon (CRBN) is the substrate receptor protein of the CRL4CRBN isoform of the E3 ubiquitin ligase complex. It, along with DDB1 (Damage-specific DNA Binding protein 1), zinc finger domain protein RBX1, and Cullin4 (Cul4) scaffold protein, forms the DDB1–Cul4–Rbx1–CRBN E3 ubiquitin ligase complex, which determines the substrate specificity of the CRL4 E3 ubiquitin ligase. It is widely expressed in the cytoplasm, nucleus, and peripheral membranes of the prostate, liver, pancreas, placenta, kidney, lung, peripheral blood leukocytes, and brain. E3 ubiquitin ligases specifically recognize substrate proteins and induce polyubiquitination of these proteins. This leads to the degradation of ubiquitinated substrate proteins via the ubiquitin-proteasome pathway, thereby affecting various physiological activities such as cellular energy metabolism, membrane potential regulation, and transcription factor degradation.

[0003] In 2010, Ito et al. first confirmed that the direct target of thalidomide is CRBN, a substrate receptor protein of an E3 ubiquitin ligase. Following increasing evidence indicating that immunomodulators exert their antitumor effects by targeting CRBN, three articles in the same issue of *Science* in 2014 [34-36] elucidated at the molecular level that immunomodulators exert their anti-multiple myeloma effects through a protein ubiquitination degradation mechanism. The CRL4CRBN (CUL4-Roc1-DDB1-CRBN) E3 ubiquitin ligase is composed of Cullin-4A (Cul4A) protein, regulator of cullins 1 (Roc1) protein, damaged DNA binding protein 1 (DDB1) protein, and CRBN (cereblon) protein. Cul4A functions as a scaffold, while Roc1 contains a ring finger domain associated with E2 ubiquitin-binding enzymes. CRBN acts as a substrate receptor within this domain, directly binding to specific substrates and mediating their ubiquitination. This ubiquitin ligase labels specific proteins via ubiquitin (Ub). Ubiquitin-labeled proteins are recognized as damaged or defective by the proteasome, leading to their hydrolysis. The interaction of immunomodulators with this E3 ubiquitin ligase activates its activity, which is fundamental to the cytotoxic and immunomodulatory effects of immunomodulators.

[0004] The ubiquitination-proteasome degradation pathway is a common and important endogenous protein degradation process that requires several consecutive steps: 1. ATP provides energy to covalently bind the C-terminus of ubiquitin to the cysteine ​​residue of E1 ubiquitin activator; 2. E1 ubiquitin activator transfers ubiquitin to E2 ubiquitin conjugate, which then transfers ubiquitin to the -NH2 residue of the target protein's lysine residue; 3. E3 ubiquitin ligase recruits the substrate protein and catalyzes the binding of ubiquitin on E2 ubiquitin conjugate to the substrate protein; 4. The 26S proteasome specifically recognizes the ubiquitin-tagged substrate protein and hydrolyzes it.

[0005] When immunomodulators such as thalidomide bind to CRBN, they activate the activity of E3 ubiquitin ligase, which in turn recruits the substrate proteins IKZF1 / IKZF3 (Ikaros / Aiolos are zinc finger transcription factors that play an important role in blood cell differentiation). Subsequently, the E3 ubiquitin ligase, the substrate proteins IKZF1 / IKZF3, and the immunomodulator form a stable ternary complex, thereby transferring ubiquitin to the substrate proteins IKZF1 / IKZF3, which then leads to the degradation of IKZF1 / IKZF3 by the 26S proteasome. IKZF1 and IKZF3 are essential transcriptional regulators for the proliferation and development of B cells and T cells [John LB, Ward AC. The ikaros gene family: transcriptional regulators of hematopoiesis and immunity[J]. Molecular immunology, 2011, 48(9):1272-1278. and Dijon M, Bardin F, Murati A, et al. The role of ikaros in human erythroid differentiation[J]. Blood, 2008, 111(3):1138-1146]. Under normal circumstances, IKZF3 inhibits the gene encoding IL-2 in T cells and stimulates the expression of IRF4 (a transcription factor that has a stress response to infection). Therefore, the degradation of IKZF1 and IKZF3 directly reduces the expression of transcription factors such as IRF4 and Myc, thereby exerting cytotoxic effects on myeloma cells. On the other hand, the degradation of IKZF1 and IKZF3 increases the expression of IL-2 in T cells, thereby activating the immune response of T cells and inhibiting the function of B cells, achieving tumor killing and tumor proliferation inhibition. At the same time, it reduces the expression of tumor necrosis factor TNF and promotes the secretion of the anti-inflammatory factor IL-10 by human peripheral blood mononuclear cells. This imbalance leads to apoptosis of multiple myeloma cells. Based on this, in recent years, protein degradation-targeting chimeras (PROTACs) designed using the chemical structures of amine immunomodulators such as thalidomide, lenalidomide, and pomalidomide have emerged continuously.

[0006] PROTAC, or Proteolysis Targeting Chimera, is a bifunctional molecule consisting of three parts: an E3 ligand unit that binds to a specific E3 ubiquitin ligase, a target protein ligand unit that binds to the target protein, and a linker connecting the two ligands. PROTAC recruits a specific E3 ubiquitin ligase to form a [E3 ubiquitin ligase-PROTAC-target protein] ternary complex, inducing polyubiquitination of the target protein by the recruited E3 ubiquitin ligase, thereby causing degradation of the target protein via the ubiquitin-proteasome pathway.

[0007] Currently, over 100 proteins have been successfully degraded by PROTAC. These targets include (1) kinases, such as RIPK2, BCR-ABL, EGFR, HER2, c-Met, TBK1, CDK2 / 4 / 6 / 9, ALK, Akt, CK2, ERK1 / 2, FLT3, PI3K, BTK, Fak, etc.; (2) BET proteins, such as BRD2 / 4 / 6 / 9; (3) nuclear receptors, such as AR, ER, etc.; (4) other proteins, such as MetAp-2, Bcl-xL, Sirt2, HDAC6, Pirin, SMAD3, ARNT, PCAF / GCN5, Tau, FRS2, etc. It even includes transcription factor regulatory proteins such as Pirin, epigenetic-related proteins such as PCAF / GCN5, and KRAS-G12C, which are considered "undruggable targets".

[0008] In the development of PROTACs, CRBN ligands are the most widely used E3 ubiquitin ligands. By selecting different target protein ligands and CRBN ligands, the designed PROTAC molecules can achieve different effects. However, in reality, there are too few CRBN ligands available to meet the needs of PROTAC development. In view of the problems of immunomodulatory drug resistance, the limited number of CRBN ligands and lack of structural diversity, and the poor chemical stability, easy epimerization in vivo, large number of hydrogen bond donors and acceptors, poor solubility, and poor binding activity of widely used amine CRBN ligands, there is an urgent need to develop more CRBN ligands to solve the above problems. Summary of the Invention

[0009] In a first aspect, the present invention provides a compound capable of binding to the CRBN protein, said compound as shown in (I):

[0010]

[0011] Among them, R 1 and R 2It is selected from one or more of the following: pyridazine core-connected cyclic rings, H, alkyl, OR, F, CN, CF3, NR2, Ph, and 4-Py.

[0012] Furthermore, the fused ring may be selected from fused ring, cyclopentane, cyclohexane, benzene, and / or pyrazine.

[0013] Furthermore, R in the compound 1 and R 2 It can be selected from alkyl groups.

[0014] Furthermore, R in the compound 1 and R 2 It cannot be selected simultaneously from H or one or more of alkyl, OR, F, CN, CF3, NR2, Ph, and 4-Py.

[0015] Furthermore, when R 1 When it is H, R 2 It is selected from one or more of alkyl, OR, F, CN, CF3, NR2, Ph, and 4-Py.

[0016] Furthermore, when R 2 When it is H, R 1 It is selected from one or more of alkyl, OR, F, CN, CF3, NR2, Ph, and 4-Py.

[0017] Y is selected from one or more of CH, CD, CF, CCH3, and N;

[0018] R 3 Selected from one or more of OR, NR2, CHR2, and C≡CR;

[0019] R 4 Selected from H or alkyl.

[0020] Secondly, the present invention also provides a pharmaceutically acceptable salt of the compound (I) described in the first aspect.

[0021] Furthermore, the pharmaceutically acceptable salt refers to a salt formed by the addition of a non-toxic acid or its base to the parent compound.

[0022] Thirdly, the present invention provides a protein degrading agent, wherein the protein degrading agent is: a target protein ligand (POI ligand) + a linker + a CRBN protein ligand; wherein the CRBN protein ligand is the compound described in the first aspect of the present invention, and the structural formula of the protein degrading agent is (II):

[0023]

[0024] L of the Linker substructure in Formula II 1 Can be connected to R 2 The connected C, at this time R 2 That is, L 1 ;R 3 Selected from one or more of OR, NR2, CHR2, and C≡CR;

[0025] L of the Linker substructure in Formula II 1 Can be connected to R 3 The connected C, at this time R 3 That is, L 1 R 2 It is selected from one or more of the following: pyridazine core-connected cyclic rings, H, alkyl, OR, F, CN, CF3, NR2, Ph, and 4-Py.

[0026] In formula II, R 1 It is selected from one or more of the following: pyridazine core-connected cyclic rings, H, alkyl, OR, F, CN, CF3, NR2, Ph, and 4-Py.

[0027] R 4 Selected from H or alkyl.

[0028] Y is selected from one or more of CH, CD, CF, CCH3, and N.

[0029] The substructure L of Linker in Equation II 1 L 3 and L 5 One or more can be selected freely; as shown below:

[0030]

[0031] Among them, R a It is selected from one or more of H, alkyl, SO2 (alyl) or SO2 (aryl).

[0032] The substructure L of Linker in Equation II 2 L 4 You may choose from one of the following:

[0033]

[0034] Furthermore, the target protein ligand is a ligand of a target protein that causes the related disease.

[0035] Furthermore, the diseases mentioned include, but are not limited to, prostate cancer, breast cancer, non-small cell lung cancer, chronic myeloid leukemia, acute myeloid leukemia, T-cell acute lymphoblastic leukemia, Alzheimer's disease, gout, autoimmune diseases, inflammatory bowel disease, B-cell lymphoma, androgenetic alopecia, acne, synovial sarcoma, solid tumors, lung cancer, multiple myeloma, lymphoma, tumor metastasis, cervical cancer, neuroblastoma, hepatocellular carcinoma, colorectal cancer, pancreatic cancer, malignant rhabdomyosarcoma, oral squamous cell carcinoma, and other cancers or diseases.

[0036] Furthermore, the target protein ligand, i.e., the POI ligand in Formula II, can be selected from:

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045] One or more of them.

[0046] Fourthly, the present invention provides a pharmaceutical composition comprising a combination of a compound of formula (I) of the first aspect or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable diluent or carrier.

[0047] Furthermore, the pharmaceutical composition may also comprise an enantiomer, diastereomer, stereoisomer, or pharmaceutically acceptable salt of a compound of formula (I) combined with a pharmaceutically acceptable diluent or carrier.

[0048] Fifthly, the present invention provides a pharmaceutical composition for targeting protein degradation, the pharmaceutical composition comprising a combination of the protein degrading agent (II) described in the second aspect of the present invention or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable diluent or carrier.

[0049] Furthermore, the pharmaceutical composition targeting the degradation protein may also comprise a combination of its enantiomer, diastereomer, stereoisomer, or pharmaceutically acceptable salt with a pharmaceutically acceptable diluent or carrier.

[0050] In a sixth aspect, the present invention provides a regulator for regulating transcriptional regulatory factors, said regulator containing a compound of formula (I) or a pharmaceutically acceptable salt or protein degrader (II) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer.

[0051] Furthermore, the transcriptional regulatory factor is an essential regulatory factor for the proliferation and development of B cells and T cells.

[0052] Furthermore, the adjustment can be positive adjustment and / or negative adjustment.

[0053] Furthermore, the transcription factors include, but are not limited to, IRF4 and Myc.

[0054] In a seventh aspect, the present invention provides a treatment method for treating or preventing pathological conditions or symptoms of diseases such as those caused by limiting or inhibiting the expression of IRF4 and Myc, comprising administering to a patient in need of treatment an effective amount of at least one compound of formula I or formula II or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof.

[0055] Furthermore, the pathological conditions or symptoms include, but are not limited to, bone marrow failure, anemia, immunoparesis and infection, fractures and bone pain, high calcium levels and renal failure. Detailed Implementation

[0056] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0057] The present invention will be described below through specific embodiments, but the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0059] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0060] The compound of the CRBN protein described in this invention is as shown in (Ⅰ):

[0061]

[0062] If it contains aliphatic amines, the structure represented includes, but is not limited to, its free amine form, hydrochloride form, and trifluoroacetate form;

[0063] In some implementations, R 1 It is one of the following structures:

[0064]

[0065] R 2 When it is one of the following structures:

[0066]

[0067] Then R 3 Choose any one of the following structures:

[0068]

[0069] R 2 When it is one of the following structures:

[0070]

[0071] R 3 It can be any of the following structures:

[0072]

[0073] Y can be one of the following structures: CCH3, CD, CF, N;

[0074] R 4 It can be one of the following structures: H, CH3, Boc, Cbz, Ph.

[0075]

[0076] If the above structure contains aliphatic amines, the structure represented includes, but is not limited to, its free amine form, hydrochloride form, and trifluoroacetate form;

[0077] AB parallel ring can be one of the following structures:

[0078]

[0079] R 1 It is one of the following structures:

[0080]

[0081] Y can be one of the following structures: CCH3, CD, CF, N;

[0082] R 4It can be one of the following structures: H, CH3, Boc, Cbz, Ph.

[0083] Table 1. Exemplary compound structures combining CRBN small molecules.

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092] Table 2. Exemplary compound structures of PROTAC protein degraders

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] The term "alkyl" as used herein refers to both branched and straight-chain saturated aliphatic hydrocarbon groups, and has a specified number of carbon atoms, generally from 1 to 12 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, 3-methylbutyl, tert-butyl, n-pentyl, and sec-pentyl.

[0103] The term “stereoisomer” as used in this article refers to compounds that have the same chemical composition but different spatial arrangements of atoms or groups, including “diastereomers” and “enantiomers”.

[0104] The term "diastereomer" as used herein refers to a stereoisomer having two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers possess different physical properties, such as melting point, boiling point, spectral characteristics, and reactivity. In the presence of resolving agents or chromatography, mixtures of diastereomers can be separated using high-resolution analytical steps such as electrophoresis and crystallization, in the presence of chiral HPLC columns.

[0105] The term "enantiomer" as used herein refers to two stereoisomers of a compound that do not overlap as mirror images of each other. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur during chemical reactions or processes where stereoselectivity or stereoorientation has ceased.

[0106] The terms “pharmaceutically acceptable salt” and “salt of a compound” used herein are interchangeable and both refer to derivatives of the disclosed compound, wherein the parent compound is prepared by the addition of a non-toxic acid or its base to form a salt. Examples of pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts: salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid. Other pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentylpropionate, diglucuronate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucohepanoate, glyceryl phosphate, glucuronate, hemisulfate, heptaate, hexanoate, hydroiodate, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl groups having 1 to 6 carbon atoms, sulfonate, and arylsulfonate.

[0107] Example

[0108] Example 1: Synthesis of Compound 1

[0109] [Step A] In a round-bottom flask, add the following raw materials in sequence: 1-(6-chloropyridazin-3-yl)ethan-1-one (1.50 g, 9.58 mmol, 1.0 equiv.), 2,6-bis(benzyloxy)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (4.80 g, 11.50 mmol, 1.2 equiv.), K3PO4 (6.22 g, 28.74 mmol, 3.0 equiv.), DavePhos (1.131 g, 2.87 mmol, 30 mol%), and Pd(OAc)2 (215 mg, 0.96 mmol, 10 mol%). Then add 50 mL of isopropanol-water (2:1, v / v) mixed solution. Stir the mixture and stir it at 80 °C (oil bath) under an argon atmosphere for 9 h. TLC analysis showed no residual starting material. After the reaction system cooled to room temperature, it was diluted with 40 mL of water and extracted with EtOAc. The combined organic phases were washed with saturated brine and dried. After filtration and solvent removal under reduced pressure, 2.68 g of compound 44 was obtained by chromatographic purification, yielding 68% (MS (ESI) m / z 412.2).

[0110] [Step B] In a round-bottom flask, add compound 44 (1.00 g, 2.43 mmol), 20 mL of anhydrous ethanol, and Pd / C (200 mg, 20 wt%, 10% on carbon). Stir the mixture and allow it to react for 8 hours under hydrogen balloon pressure. TLC showed no remaining starting material, and LC-MS showed no unreacted intermediates. Filter the reaction mixture and evaporate to dryness under reduced pressure to obtain 612 mg of crude product compound 45, which was used directly in the next step without purification. MS (ESI) m / z 234.1.

[0111] [Step C] To a 15 mL solution of crude product 45 (612 mg, ~2.62 mmol, ~1.0 equiv.) in acetone, PMBCl (360 μL, 2.62 mmol, 1.0 equiv.), K₂CO₃ (724 mg, 5.24 mmol, 2.0 equiv.), and TBAI (193 mg, 0.52 mmol, 0.2 equiv.) were added, respectively. The reaction mixture was stirred for 12 h. TLC showed no residual starting material. The mixture was diluted with 20 mL of water, extracted with EtOAc, and the combined organic phases were washed with saturated brine and dried. After filtration and solvent removal under reduced pressure, the product was purified by chromatographic chromatography to obtain 515 mg of compound 46, with a two-step yield of 60% and MS (ESI) m / z 354.1.

[0112] [Step D] At -78°C under an argon atmosphere, TMSCHN2 (trimethylsilyl)diazomethane (360 μL, 1.90 min hexanes, 0.68 mmol, 1.2 equiv.) was added dropwise to an anhydrous THF (tetrahydrofuran) solution of LDA (diisopropylaminolithium) (~0.68 mmol, 7.0 mL, ~1.2 equiv.). The reaction mixture was stirred at the same temperature for 30 min. Then, an anhydrous THF (tetrahydrofuran) solution of compound 46 (200 mg, 0.57 mmol, 1.0 equiv.) (5 mL) was added dropwise. The reaction mixture was stirred at the same temperature for 1 h, then heated to room temperature and refluxed for 3 h. TLC showed no reactants remaining. After the reaction system cooled to room temperature, it was diluted with 10 mL of ice water, extracted with EtOAc, and the combined organic phases were washed with saturated brine and dried. After filtration and solvent removal under reduced pressure, 103 mg of compound 47 was obtained by chromatographic purification, yielding 52% and MS (ESI) m / z 350.1.

[0113] [Step E] Under an argon atmosphere, CAN (cerium ammonium nitrate) (941 mg, 1.72 mmol, 10.0 equiv.) was added in portions to a MeCN-H2O (20 mL, 10:1, v / v) solution of compound 47 (60 mg, 0.17 mmol, 1.0 equiv.). The reaction mixture was stirred at 0 °C for 15 min, followed by stirring at room temperature for 2 h. TLC analysis showed no starting material remaining. A saturated sodium bicarbonate aqueous solution (10 mL) was added, and the mixture was extracted with EtOAc. The combined organic phases were washed with saturated brine and dried. After filtration, solvent removal under reduced pressure, and purification by chromatographic chromatography, 34 mg of compound 1 was obtained, with a yield of 85% (see synthetic route 1).

[0114] UPLC-MS(ESI) m / z 230.1,t R 4.516 min. 1 H NMR (400MHz, Chloroform-d) δ7.69 (dd, J=7.8, 0.6Hz, 1H), 7.64 (d, J=7.8Hz, 1H), 4.05–3.91 (m, 1H), 2.68–2.45 (m, 2H), 2.26–2.01 (m, 5H)ppm.

[0115]

[0116] Synthetic route 1: Synthetic route of compound 1

[0117] Example 2: Preparation of compounds 2-43

[0118] [Step A]:

[0119] Negishi reaction (Rt.2):

[0120] Diatomaceous earth (10 wt% of Zn) was added to a round-bottom flask and heated under high vacuum with a hot air gun (400 °C) for 15 min. After cooling to room temperature, zinc powder (200 mesh, 2.0 equiv.) was added. Dry DMA (N,N-dimethylacetamide) (1 M) and 1,2-dibromoethane (0.12 equiv.) were added sequentially. The reaction system was heated at 70 °C for 15 min. After cooling to room temperature, TMSCl (trimethylchlorosilane) (0.12 equiv.) was added dropwise. The mixture was stirred at room temperature for 1 h. Subsequently, a dry DMA (0.4 M) solution of tert-butyl3-iodoazetidine-1-carboxylate (1-Boc-3-iodoazacyclobutane) (1.5 equiv.) was added dropwise while maintaining the reaction system temperature below 40 °C. After the addition was complete, the reaction system was stirred at 40 °C for 2 h. After cooling to room temperature and standing for 30 min, the entire supernatant was transferred to a round-bottom flask. Pd₂(dba)₃ (2 mol%) and P(o-furyl)₃ (4 mol%) were added sequentially. After purging with argon gas, a dry DMA (0.4 M) solution of 1.0 equiv. of 3,6-dichloropyridazine was injected. The reaction mixture was stirred at 70 °C for 2 h. TLC analysis showed no residual reactants. After cooling to room temperature, the reaction was quenched with ammonium chloride solution and extracted with EtOAc. The combined organic phases were washed with brine and dried. The product was purified by silica gel column chromatography after filtration, solvent removal under reduced pressure, and final purification.

[0121] Suzuki reaction (Rt.3):

[0122] In a round-bottom flask, 3,6-dichloropyridazine (1.0 equiv.), tert-butyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate (1.1 equiv.), and Pd(PPh3)4 (5 mol%) were added. The flask was then purged with argon gas, and dried DME (ethylene glycol dimethyl ether) (0.2 M) and pre-deoxygenated sodium bicarbonate aqueous solution (50 v% of DME) were injected. The reaction mixture was stirred at 80 °C for 8 h. TLC analysis showed no residual feedstock. After cooling to room temperature, the mixture was diluted with water and extracted with EtOAc (3×). The combined organic phases were washed with saturated brine and dried. After filtration, solvent removal under reduced pressure, and purification by silica gel column chromatography, the Suzuki coupling product was obtained.

[0123] [Step B](Rt.2~6)

[0124] In a round-bottom flask, 6-chloropyridazine derivative (1.0 equiv.), 2,6-bis(benzyloxy)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (1.2 equiv.), K3PO4 (3.0 equiv.), DavePhos (30 mol%), and Pd(OAc)2 (10 mol%) were added. The argon atmosphere was replaced, and the flask was sealed with a rubber stopper. A deoxygenated isopropanol-water mixture (0.2 M, 2:1, v / v) was then injected. The argon atmosphere was carefully replaced with stirring, and the reaction mixture was stirred at 80 °C under an argon atmosphere for 9 h. TLC analysis showed no residual starting material. After the reaction mixture cooled to room temperature, it was diluted with water, extracted with EtOAc, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The mixture was filtered, the solvent was removed under reduced pressure, and the product was purified by silica gel column chromatography to obtain the Suzuki coupling product.

[0125] [Step C](Rt.2~6)

[0126] A dibenzyl-protected pyridinide derivative was dissolved in MeOH-THF (0.1 M, 1:1, v / v), and 10% Pd / C (20 wt%) was added. After purging with argon, the mixture was purged with hydrogen under stirring. The reaction system was vigorously stirred at room temperature for 8–16 h. TLC monitoring showed no remaining starting material, and LC-MS monitoring showed no unreacted intermediates. The reaction solution was purified by silica gel cake filtration, solvent removal under reduced pressure, and rapid silica gel column chromatography to obtain the glutarimide derivative.

[0127] [Step D](Rt.2, 3, 5)

[0128] The glutarimide derivative obtained in the previous step was dissolved in DCM (1,2-dichloroethane) (0.2 M), and HCl dioxane solution (4.0 M, ~6 equiv.) was slowly added dropwise at 0 °C. The reaction system was stirred at room temperature for 2 h. TLC monitoring showed no residual raw material. An equal volume of diethyl ether was added to the reaction solution, and the mixture was stirred for 0.5 h. The solid obtained was filtered, washed with PE, and then removed from the filter under vacuum to remove residual solvent, yielding a white solid secondary amine hydrochloride final product.

[0129]

[0130] Synthetic route 2: Synthetic routes of compounds 2 to 8

[0131] Following the synthetic route Rt.2, the Step A-Negishi reaction used 3,6-dichloropyridazine (200 mg, 1.34 mmol) as the equivalence base to yield 268 mg of the Negishi conjugate 48, with a yield of 74%. MS (ESI) m / z 214.0 ([M+H–t-Bu]). + ).

[0132] In Step B, compound 48 (80 mg, 0.30 mmol) was used as an equivalent starting material to yield 103 mg of Suzuki conjugate 49, with a yield of 66% and MS (ESI) m / z 469.2 ([M+H–t-Bu]). + ).

[0133] In Step C, compound 49 (100 mg, 0.19 mmol) was used as an equivalent starting material to yield 31 mg of product 50, with a yield of 47%. MS (ESI) m / z 291.1 ([M+H–t-Bu]). + ).

[0134] In Step D, compound 50 (31 mg, 0.09 mmol) was used as an equivalent starting material to obtain 23 mg of target product 2, with a yield of 92%.

[0135] UPLC-MS(ESI) m / z 247.1,t R 0.972 min. 1 H NMR(400MHz,D2O)δ7.67(dd,J=8.3,0.6

[0136] Hz,1H),7.57(dd,J=8.2,0.5Hz,1H),4.03–3.92(m,1H),3.55–3.38(m,5H),2.67–2.52(m,2H),2.25–2.03(m,2H)ppm.

[0137]

[0138] Following the procedures described above, and according to the synthetic route in Rt.3, the Step A-Suzuki reaction used 3,6-dichloropyridazine (200 mg, 1.34 mmol) as the equivalent starting material to yield 301 mg of the Suzuki conjugate 51, with a yield of 76%. MS (ESI) m / z 240.1 ([M+H–t-Bu]). + ).

[0139] In Step B, compound 51 (250 mg, 0.85 mmol) was used as an equivalent starting material to yield 224 mg of Suzuki conjugate 52, with a yield of 48% and an MS (ESI) m / z of 495.2 ([M+H–t-Bu]). + ).

[0140] In Step C, compound 52 (200 mg, 0.36 mmol) was used as an equivalent starting material to yield 55 mg of product 53, with a yield of 40%. MS (ESI) m / z 319.1 ([M+H–t-Bu]). + ).

[0141] In Step D, compound 53 (55 mg, 0.15 mmol) was used as an equivalent reference starting material to yield 43 mg of the target product 3, with a yield of 95%. UPLC-MS (ESI) m / z 275.2, t R 0.941 min. 1 H NMR (400MHz, D2O) δ7.92–7.82(m,2H),4.04–3.90(m,4H),3.41(dd,J=7.0,3.4Hz,5H),2.78–2.70(m,2H),2.41–2.22(m,2H)ppm.

[0142]

[0143] (ii) Aromatic nucleophilic

[0144] Aromatic nucleophilic substitution reactions (Rt. 4, 5):

[0145] To a dry NMP (N-methylpyrrolidone) (0.2 M) solution of 1.0 equiv. of 3,6-dichloropyridazine, a secondary amine (1.0 equiv.) and K₂CO₃ (3.0 equiv.) were added, and the reaction mixture was purged with argon. The mixture was stirred at 110 °C for 8 h. TLC analysis showed no residual reactants. After cooling to room temperature, the mixture was diluted with water, extracted with EtOAc (3×), and the combined organic phases were washed with saturated brine and dried. The product was purified by chromatographic chromatography after filtration, solvent removal under reduced pressure, and finally the aromatic nucleophilic substitution product.

[0146] Following the above procedure, according to the synthetic route in Rt.4, Step A-aromatic nucleophilic substitution reaction used 3,6-dichloropyridazine (200 mg, 1.34 mmol) as the equivalence base and 3-(benzyloxy)azetidine hydrochloride (268 mg, 1.47 mmol, 1.1 equiv.) as the reactant to yield 303 mg of 3-(3-(benzyloxy)azetidin-1-yl)-6-chloropyridazine (54a), yield 82%, MS (ESI) m / z 276.1.

[0147] In Step B, compound 54a (250 mg, 0.91 mmol) was used as an equivalent reference to obtain 289 mg of product 55a, with a yield of 60% and MS (ESI) m / z 531.6.

[0148]

[0149] Step C used compound 55a (200 mg, 0.36 mmol) as an equivalent reference to obtain 43 mg of the target product 4, with a yield of 44%. UPLC-MS (ESI) m / z 263.1,t R 2.689 min. 1 H NMR (400MHz, Methanol-d4) δ7.51–7.36(m,1H),6.95(d,J=8.6Hz,1H),4.20(q,J=4.3Hz,1H),4.07–4.01(m ,1H),3.93(dd,J=12.3,4.4Hz,2H),3.83(dd,J=12.4,4.4Hz,2H),2.65–2.51(m,2H),2.20–2.05(m,2H)ppm.

[0150]

[0151] Following the above procedure, according to the synthetic route in Rt.4, in Step A-aromatic nucleophilic substitution reaction, 3,6-dichloropyridazine (200 mg, 1.34 mmol) was used as the equivalence base starting material, and (R)-2-((benzyloxy)methyl)pyrrolidine ((R)-2-(benzyloxy)methyl)pyrrolidine (256 mg, 1.34 mmol, 1.0 equiv.) was used as the reactant to give 346 mg of product 54b, with a yield of 85% and MS (ESI) m / z 304.1; using (S)-2-((benzyloxy)methyl)pyrrolidine (256 mg, 1.34 mmol, 1.0 equiv.) as the reactant, 334 mg of product 54c was given, with a yield of 82% and MS (ESI) m / z 304.1.

[0152] In Step B, compound 54b (250 mg, 0.82 mmol) was used as an equivalent starting material to yield 267 mg of product 55b, with a yield of 58% and an MS (ESI) m / z of 559.3. Compound 54c (250 mg, 0.82 mmol) was used as an equivalent starting material to yield 276 mg of product 55c, with a yield of 60% and an MS (ESI) m / z of 559.3.

[0153]

[0154] In Step C, compound 55b (200 mg, 0.36 mmol) was used as an equivalent reference to obtain 54 mg of the target product 5, with a yield of 52%. UPLC-MS (ESI) m / z 291.2, t R 2.323 min. 1 H NMR (400MHz, Methanol-d4) δ7.66–7.46(m,1H),6.92(d,J=8.1Hz,1H),4.07–3.67(m,6H),2.69–2.49(m,2H),2.22–1.71(m,6H)ppm.

[0155]

[0156] Using compound 55c (200 mg, 0.36 mmol) as the equivalent standard starting material, 51 mg of the target product 6 was obtained, with a yield of 49%. UPLC-MS (ESI) m / z 291.2, t R 2.323 min. 1H NMR (400MHz, Methanol-d4) δ7.66–7.36(m,1H),6.92(d,J=8.1Hz,1H),4.12–3.55(m,6H),2.69–2.40(m,2H),2.25–1.75(m,6H)ppm.

[0157]

[0158] Following the procedures outlined above, and according to the synthetic route in Rt. 5, Step A – the aromatic nucleophilic substitution reaction – used 3,6-dichloropyridazine (200 mg, 1.34 mmol) as the equivalence starting material and tert-butyl piperazine-1-carboxylate (250 mg, 1.34 mmol, 1.0 equiv.) as the reactant, yielding 344 mg of product over 54 days, with a yield of 86%. MS (ESI) m / z 199.1 ([M+H–Boc]). + ).

[0159]

[0160] In Step B, compound 54d (250 mg, 0.84 mmol) was used as an equivalent starting material to yield 255 mg of product 55d, with a yield of 55%. MS (ESI) m / z 454.2 ([M+H–Boc]). + ).

[0161] In Step C, compound 55d (200 mg, 0.36 mmol) was used as an equivalent starting material to yield 70 mg of product 56, with a yield of 52%. MS (ESI) m / z 276.4 ([M+H–Boc]). + ).

[0162] In Step D, compound 56 (70 mg, 0.13 mmol) was used as an equivalent starting material to obtain 34 mg of the target product 7, with a yield of 88%.

[0163] UPLC-MS(ESI) m / z 276.1,t R 0.982 min. 1 H NMR(400MHz,D2O)δ7.97(d,J=1.3Hz,

[0164] 2H),3.52(d,J=12.9Hz,2H),3.41–3.30(m,1H),3.21–3.09(m,2H),2.81-2.60(m,2H),2.52–2.15(m,4H),2.09–1.93(m,2H)ppm.

[0165]

[0166] Following the above procedure, according to the synthetic route of Rt.6, in Step B, 133 mg of Suzuki coupling product 57 was obtained with 3-chloro-6-methoxypyridazine (100 mg, 0.69 mmol) as the equivalent standard starting material, yielding 48% and MS (ESI) m / z 400.2.

[0167] In Step C, compound 57 (100 mg, 0.25 mmol) was used as an equivalent starting material to yield 18 mg of the target product 8, with a yield of 32%.

[0168] UPLC-MS(ESI) m / z 222.1,t R 3.567 min. 1 HNMR (400MHz, Methanol-d4) δ7.58–7.52(m,1H),7.03(d,J=8.1Hz,1H),4.07–3.99(m,1H),3.90(s,3H),2.65–2.50(m,2H),2.23–2.05(m,2H)ppm.

[0169]

[0170] Following the synthetic route 3-Eq.1, compounds CMP-F, CMP-CN, and CMP-CF3 were synthesized using starting materials (1.0 equiv.) and MeONa (1.5 equiv.).

[0171]

[0172] Synthetic route 3: Synthetic reaction formula for 4-substituted pyridazine derivatives

[0173]

[0174] Following synthetic route 3-Eq.2, compounds CMP-Et, CMP-IP, CMP-Cp, CCP-Cp, and CMP-NC were synthesized. The starting material (1.0 equiv.) was added to ultrapure water, followed by the addition of carboxylic acid (1.2 equiv.), TFA (trifluoroacetic acid) (1.0 equiv.), AgNO3 (0.2 equiv.), and Na2S2O8 (1.2 equiv.). After purging with argon, the reaction was carried out at 60°C for 6–12 h under an argon atmosphere. TLC showed no remaining starting material. After cooling to room temperature, Na2CO3 was added to neutralize the reaction, and saturated sodium thiosulfate solution was added to quench the reaction. The mixture was extracted with EtOAc, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The resulting compounds were purified by silica gel column chromatography after filtration, solvent removal under reduced pressure, and extraction with saturated silica gel column chromatography.

[0175]

[0176] Following synthetic route 3-Eq.3, compounds CMP-Ph, CMP-Py, CCP-Ph, and CCP-Py were synthesized. Starting materials (6-chloro-4-iodo-3-methoxypyridazine or 3,6-dichloro-4-iodopyridazine) (1.0 equiv.), arylboronic acid (1.05 equiv.), KF (2.5 equiv.), Pd(OAc)₂ (5 mol%), and QPhos (5 mol%) were added to a dry, argon-filled side-port reaction tube, respectively. After purging with argon three times, deoxygenated ultrapure water (4:1, v / v) was injected. The reaction was then carried out under argon atmosphere at 70°C for 10–18 h. TLC analysis showed no residual reactants. After cooling to room temperature, EtOAc was added to dilute the reaction solution. The mixture was filtered, and the filtrate was diluted with water and extracted with EtOAc. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, solvent removal under reduced pressure, and purification by silica gel column chromatography, the desired compound was obtained.

[0177]

[0178] [Rt.7]

[0179] Following synthetic route 4-Rt.7, in the first step, 6-chloropyridazine derivative (1.0 equiv.), benzyl piperazine-1-carboxylate (1.2 equiv.), Cs₂CO₃ (1.0 equiv.), BINAP (10 mol%), and Pd(OAc)₂ (10 mol%) were added sequentially to a side-port reaction tube that was dried in an oven and filled with argon. The mixture was evacuated by an oil pump for 10 min and then purged with argon. Dry toluene (0.1 M) was injected, and the resulting mixture was stirred at room temperature for 10 min, followed by heating at 100 °C under an argon atmosphere for 8–15 h. TLC analysis showed no remaining starting material. After the reaction system cooled to room temperature, the solvent was removed under reduced pressure. The mixture was dissolved in DCM (1,2-dichloroethane), filtered, and the solvent was removed again under reduced pressure. The resulting product was purified by silica gel column chromatography to obtain a series of intermediates, CbzNN-PM-R.

[0180]

[0181] Synthetic route 4: Synthetic routes for compounds 9–15, 19, and 20

[0182] Table 3. A series of compounds for intermediate CbzNN-PM-R

[0183]

[0184]

[0185] In the second step, NaI (3.0 equiv.) was added to the dried MeCN of CbzNN-PM-R (1.0 equiv.) under stirring. The suspension was placed in an ice-water bath at 0°C, and TMSCl (trimethylchlorosilane) (3.0 equiv.) was slowly added dropwise to the reaction system. After the addition was complete, the reaction system was moved to room temperature and stirred for 6–14 h. TLC showed no remaining raw material. The reaction was quenched by adding sodium bicarbonate solution and sodium thiosulfate solution, and stirring was continued for 30 min. The mixture was extracted with DCM-MeOH (5–10:1, v / v), and the organic phases were combined and dried. The crude 3(2H)-pyridazinone derivative was obtained by filtration and solvent removal under reduced pressure. Anhydrous toluene (0.2 M) was added to a round-bottom flask containing the crude 3(2H)-pyridazinone derivative at 0 °C in an ice-water bath. POCl3 (1.5 equiv.) was then slowly added, followed by dropwise addition of DIPEA (diisopropylethylamine) (3.0 equiv.). The reaction mixture was stirred at room temperature for 15 min, then at 100 °C for 4–8 h. TLC analysis showed no residual reactants. After cooling to room temperature, all volatile components were removed under reduced pressure. The residue was dissolved in DCM (1,2-dichloroethane), and the DCM phase was rapidly washed with ice water (<1 min). The resulting organic phase was washed with ice-cold saturated brine and dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, the 3-chloropyridazin derivative was purified by rapid silica gel column chromatography.

[0186] Table 4 3-Chloropyridazine Derivatives

[0187]

[0188]

[0189] The third step involves following Step B of synthetic route 2, using the 3-chloropyridazine derivative obtained in the second step as an equivalent reference raw material to synthesize a series of intermediate CbzNN-P(Py)-R compounds.

[0190] Table 5. A series of compounds for the intermediate CbzNN-P(Py)-R

[0191]

[0192] In the fourth step, following Steps C and D in synthetic route 2, the CbzNN-P(Py)-R intermediate obtained in the third step was used as the equivalent reference raw material to complete the synthesis of target products 9-15, 19, and 20.

[0193] Table 6 Synthesis of products 9–15, 19, and 20

[0194]

[0195]

[0196]

[0197]

[0198] Synthetic routes for compounds 21–24, 31, and 32 (Synthetic route 5)

[0199] [Rt.8]

[0200] In the synthetic route 5-Rt.8, the first step is to follow Step B in synthetic route 2, using a 3,6-dichloropyridazine derivative as the equivalent base material to synthesize a series of intermediates C-(R)P-Py.

[0201] Table 7. A series of compounds of intermediate C-(R)P-Py

[0202]

[0203] In the second step, benzyl piperazine-1-carboxylate (1.5 equiv.) and Cs₂CO₃ (3.0 equiv.) were added to a dry NMP (N-methylpyrrolidone) solution of C-(R)P-Py (1.0 equiv.) under stirring. The suspension was then stirred at 110 °C under an argon atmosphere for 8–15 h. TLC analysis showed no remaining reactants. After the reaction system cooled to room temperature, it was diluted with water, and the reaction was quenched with saturated sodium bicarbonate and saturated sodium thiosulfate solutions. Stirring continued for 30 min. Extraction was performed with DCM-MeOH (5–10:1, v / v) (4×). The organic phases were combined and dried. After filtration and solvent removal under reduced pressure, the CbzNN-(R)P-Py series compounds were purified by silica gel rapid column chromatography.

[0204] Table 8. CbzNN-(R)P-Py series compounds

[0205]

[0206]

[0207] In the third step, following the Step C and D operations in synthetic route 2, the CbzNN-(R)P-Py series compounds obtained in the second step were used as equivalent reference raw materials to complete the synthesis of target products 21-24, 31, and 32.

[0208] Table 9 Synthesis of target products 21–24, 31, and 32

[0209]

[0210]

[0211]

[0212]

[0213] Synthetic routes 6: Synthetic routes for compounds 16 and 28

[0214]

[0215] Add KH (2.24 g, 30 wt%, 16.76 mmol, 2.0 equiv.) to a round-bottom flask, add anhydrous n-hexane (5 mL), stir, let stand, discard the supernatant, repeat twice, remove residual solvent with an oil pump and replace with argon gas, inject dry THF (tetrahydrofuran) (20 mL), place in an ice-water bath at 0 °C, add a THF (tetrahydrofuran) (5 mL) solution of PMBOH (p-methoxybenzyl alcohol) (1.50 g, 10.89 mmol, 1.3 equiv.) dropwise while stirring, then add 18-C-6 (111 mg, 0.42 mmol, 0.05 equiv.), and continue stirring the reaction system at room temperature for 2 h. A dry THF solution (20 mL) of 3,6-dichloro-4-methoxypyridazine (1.50 g, 8.38 mmol, 1.0 equiv.) was added dropwise to the above reaction solution at 0 °C. The reaction mixture was stirred at room temperature for 6 h after the addition was complete. TLC analysis showed no remaining starting material. The reaction mixture was poured onto crushed ice, and a small amount of water (20 mL) was added. The aqueous phase was extracted with DCM (1,2-dichloroethane) (3 × 80 mL). The combined organic phases were washed with sodium chloride solution and dried. After filtration, solvent removal under reduced pressure, and purification by chromatographic chromatography, 1.08 g of compound 58 was obtained, with a yield of 46%.

[0216] MS(ESI)m / z 281.0. 1 H NMR (400MHz, Chloroform-d) δ7.37 (d, J = 8.2Hz, 2H), 7.17–6.77 (m, 3H), 5.32 (s, 2H), 3.98 (s, 3H), 3.78 (s, 3H) ppm.

[0217] [Synthesis Route 6Rt.9]:

[0218] In the first step, following the method of the first step of synthetic route 4-Rt.7, 1.07 g of compound 59 was synthesized using compound 58 (1.00 g, 3.56 mmol) as the equivalent starting material, with a yield of 65% and MS (ESI) m / z 465.2.

[0219] In the second step, compound 59 (1.00 g, 2.15 mmol, 1.0 equiv.) was dissolved in MeCN (20 mL), and H2O (5 mL) was added. While stirring, 1.0 equiv. CAN (cerium ammonium nitrate) (total 9.13 g, 17.22 mmol, 8.0 equiv.) was added to the reaction solution every 1 hour. TLC analysis showed no remaining starting material. The reaction was quenched with saturated sodium thiosulfate solution. The aqueous phase was extracted with DCM-MeOH (10:1, v / v) (3 × 80 mL). The combined organic phases were washed with saturated sodium chloride solution (1 × 80 mL) and dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, 511 mg of crude compound 60 was obtained, which could be used directly in the next reaction without further purification. MS (ESI) m / z 345.1.

[0220] In the third step, following the method of the third step of synthetic route 4-Rt.7, 258 mg of compound 61 was synthesized using crude product 60 (511 mg, ~1.48 mmol) as the equivalent starting material. The two-step yield was 33%, and the MS (ESI) m / z was 363.1.

[0221] In the fourth step, following the method of the fourth step of the synthetic route 4-Rt.7, 312 mg of compound 62 was synthesized using compound 61 (300 mg, 0.83 mmol) as the equivalent starting material, with a yield of 61% and MS (ESI) m / z 618.3.

[0222] In steps five and six, following the methods of steps five and six of synthetic route 4-Rt.7, 52 mg of target compound 16 was synthesized using compound 62 (300 mg, 0.83 mmol) as the equivalent starting material, with a yield of 38%.

[0223] UPLC-MS(ESI) m / z 306.2,t R 0.911 min. 1 H NMR (400MHz, Methanol-d4) δ6.54 (s, 1H), 4.14 (t, J = 6.4Hz, 1H), 3.86 (s, 3H), 3 .65–3.60(m,4H),2.97–2.89(m,4H),2.62–2.54(m,2H),2.27–2.08(m,2H)ppm.

[0224]

[0225] [Synthesis Route 6Rt.10]

[0226] In the first step, following the method of the fourth step of the synthetic route 4-Rt.7, 1.03 g of compound 64 was synthesized using compound 58 (1.00 g, 3.56 mmol) as the equivalent starting material, with a yield of 54% and MS (ESI) m / z 536.2.

[0227] In the second step, compound 64 (1.00 g, 1.87 mmol, 1.0 equiv.) was dissolved in anhydrous DCM (1,2-dichloroethane) (10 mL). TFA (trifluoroacetic acid) (10 mL) was slowly added to the solution at 0 °C with stirring. The reaction system was continued to be stirred at room temperature for 2–3 h. TLC analysis showed no remaining starting material. All volatile components were removed under reduced pressure, and the resulting 780 mg residue was crude product 65, which was directly used in the next reaction. MS (ESI) m / z 416.2.

[0228] In the third step, following the method of the third step of synthetic route 4-Rt.7, using crude product 65 (780 mg, ~1.87 mmol) as the equivalent starting material, 381 mg of compound 66 was synthesized, with a two-step yield of 47% and MS (ESI) m / z 434.1.

[0229] In the fourth step, following the method of the first step of synthetic route 4-Rt.7, 378 mg of compound 67 was synthesized using compound 66 (300 mg, 0.69 mmol) as the equivalent starting material, with a yield of 74% and MS (ESI) m / z 618.3.

[0230] Steps 5 and 6 were performed following steps 5 and 6 of synthetic route 4-Rt.7, using compound 67 (300 mg, 0.83 mmol) as the equivalence base, to synthesize 62 mg of the target compound 16, with a yield of 45%. UPLC-MS (ESI) m / z 306.2, t R 0.921 min. 1 H NMR (400MHz, Methanol-d4) δ6.99(s,1H),4.04–3.86(m,8H),3.29–3.20(m,4H),2.65–2.49(m,2H),2.22–2.11(m,2H)ppm.

[0231] Boc₂O (5.32 g, 24.40 mmol, 4.0 equiv.) and DMAP (4-dimethylaminopyridine) (74 mg, 0.61 mmol, 0.1 equiv.) were added to an anhydrous THF (tetrahydrofuran) solution (30 mL). The reaction mixture was stirred under reflux for 18 h. TLC showed no reactants remaining. After cooling to room temperature, the solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography to obtain 1.91 g of compound CCP-NDB, with a yield of 86% and MS (ESI) m / z 364.2.

[0232]

[0233] Following the first step of Rt.11 in synthetic route 7, and referring to the first step of synthetic routes 4-Rt.7, 1.40 g of compound CbzNN-CP-NDB (1.50 g, 4.12 mmol) was synthesized using compound CCP-NDB as the equivalent starting material, with a yield of 62% and MS (ESI) m / z 548.2.

[0234] In the second step, compound CbzNN-CP-NDB (1.2 g, 2.19 mmol, 1.0 equiv.) was dissolved in dry DCM (1,2-dichloroethane) (10 mL). HCl dioxane solution (5 mL) was added to the solution at 0 °C with stirring. The reaction system was stirred at room temperature for 2–4 h. TLC analysis showed no residual starting material. All volatile components were removed under reduced pressure. The resulting residue was dispersed in water-EtOAc, and the pH was adjusted to 8 with sodium bicarbonate. The aqueous phase was extracted with EtOAc (3 × 40 mL). The combined organic phases were washed with saturated sodium chloride solution and dried. After filtration and solvent removal under reduced pressure, 678 mg of compound CbzNN-CP-N was purified by chromatographic chromatography, yielding 89% (MS (ESI) m / z 348.1).

[0235] In the third step, following the method of the fourth step of the synthetic route 4-Rt.7, 582 mg of compound CbzNN-P(Py)-N was synthesized using compound CbzNN-CP-N (600 mg, 1.72 mmol) as the equivalent starting material, with a yield of 56% and MS (ESI) m / z 536.2.

[0236] In the fourth step, following Steps C and D in synthetic route 2, using the compound CbzNN-P(Py)-N (500 mg, 0.83 mmol) obtained in the third step as the equivalent reference starting material, 81 mg of the target product 17 was obtained, with a yield of 30%.

[0237]

[0238] Synthetic routes 7: Synthetic routes for compounds 17 and 29

[0239] 17: UPLC-MS(ESI) m / z 291.2,t R 0.758 min. 1 H NMR (400MHz, Methanol-d4) δ6.44(s,1H),4.13(t,J=6.6Hz,1H),3.65–3.57(m,4H),2.92(m,4H),2.62–2.53(m,2H),2.22–2.07(m,2H)ppm.

[0240]

[0241] Following the first step of Rt.12 in synthetic route 7, and referring to the fourth step of synthetic route 4-Rt.7, using compound CCP-NDB (1.5 g, 4.12 mmol) as the equivalent starting material, 1.86 g of compound NDB-CP-Py was synthesized with a yield of 73% and MS (ESI) m / z 619.2.

[0242] In the second step, following the second step of Rt.11 in synthetic route 7, using compound NDB-CP-Py (1.5 g, 2.42 mmol) as the equivalent starting material, 913 mg of compound N-CP-Py was synthesized, with a yield of 90% and MS (ESI) m / z 419.1.

[0243] In the third step, following the method of the first step of synthetic route 4-Rt.7, using compound N-CP-Py (800 mg, 1.91 mmol) as the equivalent starting material, 610 mg of compound N-(CbzNN)P-Py was synthesized with a yield of 53% and MS (ESI) m / z 603.2.

[0244] In the fourth step, following Steps C and D of synthetic route 2, using the compound N-(CbzNN)P-Py (600 mg, 1.00 mmol) obtained in the third step as the equivalent reference starting material, 75 mg of the target product 29 was obtained, with a yield of 23%. UPLC-MS (ESI) m / z 29 1.2, t R 0.788 min. 1 H NMR (400MHz, Methanol-d4) δ6.84(s,1H),4.03–3.87(m,5H),3.30–3.15(m,4H),2.70–2.46(m,2H),2.24–2.04(m,2H)ppm.

[0245]

[0246] Following the synthetic route Rt.13 in Synthetic Route 8, and referring to steps one, two, three, and four of synthetic routes 4-Rt.7 and Step C of synthetic route 2, starting with compound CMP-NC (2.00 g, 6.22 mmol), 138 mg of compound BocNN-GP-NH was obtained through five steps, with an MS (ESI) m / z of 419.2.

[0247] In step six, compound BocNN-GP-NH (100 mg, 0.24 mmol, 1.0 equiv.) was dissolved in DCE (5 mL), and 37 wt% HCHO aqueous solution (28 μL, 0.36 mmol, 1.5 equiv.) was added. The mixture was stirred at room temperature for 30 min, then cooled to 0 °C. NaBH(OAc)3 (253 mg, 1.20 mmol, 5.0 equiv.) was added to the reaction system, and the reaction mixture was heated to room temperature and stirred for 12 h. TLC showed no residual starting material. The reaction was quenched with saturated sodium bicarbonate solution, and the aqueous phase was extracted with DCM-MeOH (5:1, v / v) (3 × 5 mL). The organic phases were combined and dried. After filtration and solvent removal under reduced pressure, crude BocNN-GP-NM product was obtained, which could be used directly in the next reaction without further purification. MS (ESI) m / z 433.2.

[0248] Step 7: Referring to Step D in synthetic route 2, using the crude product obtained in step 6 as the equivalent raw material, 37 mg of target product 18 was obtained, with a yield of 38%.

[0249]

[0250] Synthetic route 8: Synthetic route of compound 18

[0251] 18: UPLC-MS(ESI) m / z [M+2H] 2+ 167.1,t R 0.781 min. 1 H NMR(400MHz, Methanol-d4)δ6.74(s,1H),4.12(t,J=6.4Hz,1H),3.84(s,2H),3.64–3 .58(m,4H),2.96–2.88(m,4H),2.62–2.57(m,2H),2.41(s,6H),2.26–2.09(m,2H)ppm.

[0252]

[0253] Following the first step of Rt.14 in synthetic route 9, referencing the fourth step of synthetic routes 4-Rt.7; the second and third steps of Rt.14 in synthetic route 9, referencing the second and third steps of synthetic routes 4-Rt.7; the fourth step of Rt.14 in synthetic route 9, referencing the first step of synthetic routes 4-Rt.7; and the fifth to seventh steps of Rt.14 in synthetic route 9, referencing the fifth to seventh steps of synthetic route 8, Rt.13; using compound CMP-NC (2.00 g, 6.22 mmol) as the starting material, 22 mg of compound 30 was finally synthesized.

[0254] 30: UPLC-MS(ESI) m / z [M+2H] 2+ 167.1,t R 0.741 min. 1 H NMR(400MHz, Methanol-d4)δ7.68–7.59(m,3H),7.51–7.45(m,2H),7.43–7.37(m,1H),4.09(t, J=6.2Hz,1H),4.03–3.89(m,4H),3.30–3.19(m,4H),2.65–2.48(m,2H),2.24–2.08(m,2H)ppm.

[0255]

[0256] Synthetic route 9: Synthetic route of compound 30

[0257]

[0258] Following the synthetic route 2-Rt.5, compounds 25, 26, and 27 were synthesized using 3,6-dichloro-4-fluoropyridazine, 3,6-dichloropyridazine-4-carbonitrile, and 3,6-dichloro-4-(trifluoromethyl)pyridazine as stoichiometric starting materials, respectively.

[0259] 25: UPLC-MS(ESI) m / z 294.1,t R 0.915 min. 1H NMR (400MHz, Methanol-d4) δ7.11(d,J=7.9Hz,1H),4.07(t,J=6.1Hz,1H),3.98–3.88(m,4H),3.29–3.18(m,4H),2.63–2.49(m,2H),2.23–2.09(m,2H)ppm.

[0260] 26: UPLC-MS(ESI) m / z 301.1,t R 0.932 min. 1 H NMR (400MHz, Methanol-d4) δ7.67(s,1H),4.06(t,J=6.2Hz,0H),4.01–3.90(m,4H),3.28–3.19(m,4H),2.65–2.48(m,2H),2.24–2.07(m,2H)ppm.

[0261] 27: UPLC-MS(ESI) m / z 344.1,t R 0.942 min. 1 H NMR (400MHz, Methanol-d4) δ7.62(s,1H),4.05(t,J=6.2Hz,1H),4.00–3.89(m,4H),3.32–3.20(m,4H),2.67–2.48(m,2H),2.24–2.04(m,2H)ppm.

[0262]

[0263] Following the synthetic route 2-Rt.5, compounds 33, 34, 35, 36, and 37 were synthesized using 3,6-dichloro-4,5-dimethylpyridazine, 1,4-dichloro-6,7-dihydro-5H-cyclopenta[d]pyridazine, 1,4-dichloro-5,6,7,8-tetrahydrophthalazine, 1,4-dichlorophthalazine, and 5,8-dichloropyrazino[2,3-d]pyridazine as stoichiometric starting materials.

[0264] 33: UPLC-MS(ESI) m / z 304.2,t R 1.103 min. 1H NMR(400MHz,Methanol-d4)δ4.05(t,J=6.0Hz,1H),3.93–3.79(m,4H),3.32–3.18(m,4H),2.69–2.52(m,2H),2.43(s,3H),2.24–2.10(m,5H)ppm.

[0265] 34:UPLC-MS(ESI)m / z 316.2,t R 1.116min. 1 H NMR(400MHz,Methanol-d4)δ4.06(t,J=6.4Hz,1H),3.95–3.84(m,4H),3.33–3.22(m,4H),2.99–2.82(m,4H),2.67–2.52(m,2H),2.38–2.10(m,4H)ppm.

[0266] 35:UPLC-MS(ESI)m / z 330.2,t R 1.120min. 1 H NMR(400MHz,Methanol-d4)δ4.06(t,J=6.4Hz,1H),3.96–3.84(m,4H),3.34–3.21(m,4H),3.14–2.87(m,4H),2.67–2.51(m,2H),2.26–2.09(m,2H),1.93–1.78(m,4H)ppm.

[0267] 36:UPLC-MS(ESI)m / z 326.1,t R 1.019min. 1 H NMR(400MHz,Methanol-d4)δ8.31(dd,J=8.8,1.3Hz,1H),8.10(dd,J=9.1,1.4Hz,1H),7.86(ddd,J=9.1,7.8,1.3Hz,1H),7.72(ddd,J=9.0,7.9,1.3Hz,1H),4.05–3.95(m,4H),3.79(t,J=6.2Hz,1H),3.28–3.21(m,4H),2.70–2.49(m,2H),2.26–2.11(m,2H)ppm.

[0268] 37:UPLC-MS(ESI)m / z 328.1,t R 1.120min. 1H NMR(400MHz, Methanol-d4)δ8.73(d,J=3.5Hz,1H),8.72(d,J=3.5Hz,1H),4.03–3.91(m, 4H),3.84(t,J=6.6Hz,1H),3.29–3.17(m,4H),2.69–2.50(m,2H),2.38–2.17(m,2H)ppm.

[0269]

[0270] According to the synthetic route Rt.15 in synthetic route 10

[0271] In the first step, compound 56 (200 mg, 0.53 mmol, 1.0 equiv.) was dissolved in 10 mL of dry DCM (1,2-dichloroethane). Et3N (0.22 mL, 1.59 mmol, 3.0 equiv.), Boc2O (349 mg, 1.59 mmol, 3.0 equiv.), and DMAP (4-dimethylaminopyridine) (6 mg, 0.05 mmol, 0.1 equiv.) were added sequentially, and the reaction was stirred at room temperature for 5 h. TLC showed no remaining starting material. The reaction was quenched with saturated sodium bicarbonate solution. The aqueous phase was extracted with DCM-MeOH (10:1, v / v) (3 × 10 mL). The organic phases were combined and dried. After filtration and solvent removal under reduced pressure, compound 69 was purified by silica gel column chromatography to obtain 192 mg of compound 69, with a yield of 76% and MS (ESI) m / z 476.2.

[0272] In the second step, compound 69 (85 mg, 0.18 mmol, 1.0 equiv.) was dissolved in dry THF (tetrahydrofuran) (4 mL). The solution was cooled to -78 °C, and LiHMDS (0.27 mL, 1.0 M in THF, 0.27 mmol, 1.5 equiv.) was added dropwise to the solution. After the addition was complete, the reaction system was stirred at the same temperature for 1 h. (A) At the same temperature, NFSI (91 mg, 0.29 mmol, 1.6 equiv.) in dry THF (2.0 mL) was added dropwise to the reaction solution, and the reaction was stirred at the same temperature for 2–5 h. LC-MS showed no remaining starting material. The reaction was quenched by adding saturated ammonium chloride solution. After the reaction was heated to room temperature, the aqueous phase was extracted with DCM-MeOH (10:1, v / v) (3 × 10 mL), and the combined organic phases were dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, 61 mg of compound 70 was obtained by chromatographic purification, yielding 69% (MS (ESI) m / z 494.2). (B) Alternatively, at the same temperature, AcOD (17 μL, 0.29 mmol, 1.6 equiv.) was added dropwise to the reaction solution, and the reaction was continued with stirring at the same temperature for 2–5 h. LC-MS showed no residual starting material. After the reaction was heated to room temperature, water was added for dilution, and the aqueous phase was extracted with DCM-MeOH (10:1, v / v) (3 × 10 mL). The combined organic phases were dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, 75 mg of compound 71 was obtained by chromatographic purification, yielding 88% (MS (ESI) m / z 477.2).

[0273] In the third step, referring to Step D in synthetic route 2, using the compounds 70 (50 mg, 0.10 mmol) or 71 (60 mg, 0.13 mmol) obtained in the second step as equivalent starting materials, 24 mg and 23 mg of target products 38 and 39 were obtained, respectively, with yields of 72% and 59%.

[0274] 38: UPLC-MS(ESI) m / z 294.1,t R 0.965 min. 1 H NMR (400MHz, Methanol-d4) δ7.45(d,J=7.2Hz,1H),6.99(d,J=7.3Hz,1H),4.01–3.82(m,5H),3.34–3.17(m,4H),2.74–2.49(m,2H),2.44–2.08(m,2H)ppm.

[0275] 39: UPLC-MS(ESI) m / z 277.2,t R 0.977 min. 1H NMR (400MHz, Methanol-d4) δ7.28(d,J=8.6Hz,1H),6.93(d,J=8.6Hz,1H),3.96–3.83(m,4H),3.41–3.15(m,4H),2.78–2.47(m,2H),2.28–2.06(m,2H)ppm.

[0276]

[0277] Synthetic routes 10: Synthetic routes of compounds 38 and 39

[0278] Following the synthetic route Rt.16 in synthetic route 11, in the first step, Boc2O (2.92 g, 13.38 mmol, 3.0 equiv.) and DMAP (4-dimethylaminopyridine) (163 mg, 1.34 mmol, 0.3 equiv.) were added sequentially to a stirred DME (20 mL) solution of uracil (500 mg, 4.46 mmol, 1.0 equiv.). The reaction system was heated under reflux overnight. TLC showed no remaining starting material. After cooling the reaction system to room temperature, all volatile components were removed by vacuum rotation to obtain the crude product N1Boc-N3Boc-PM. No further purification was required, and it was directly used in the next step. MS (ESI) m / z 257.1 ([M+Ht-Bu) + ).

[0279] In the second step, the crude product obtained in the previous step was dissolved in DCM-MeOH (9:1, v / v, 10 mL), and silica gel powder (300 mg, 300–400 mesh, 60 wt%) was added. The reaction mixture was stirred at 60 °C, and the reaction progress was monitored by TLC. After filtration and solvent removal under reduced pressure, 786 mg of compound N1H-N3Boc-PM was obtained by chromatographic purification, with a yield of 83% and MS (ESI) m / z 213.1.

[0280]

[0281] Synthetic route 11: Synthetic route of compound N1H-N3Boc-PM

[0282] Following the synthetic route in 12, the first step involved adding N1H-N3Boc-PM (100 mg, 0.47 mmol, 1.0 equiv.), chiral proline amide cat-1 (31 mg, 0.09 mmol, 0.3 equiv.), and 3-chloro-6-iodopyridazine (170 mg, 0.71 mmol, 1.5 equiv.) to an argon-filled reaction tube. Then, 2.5 mL of deoxygenated ultrapure water was added, followed by CuI (9 mg, 0.05 mmol, 0.1 equiv.) and K2CO3 (130 mg, 0.94 mmol, 2.0 equiv.). The gas was then purged with argon. The reaction system was stirred at 100 °C for 24 h under an argon atmosphere. TLC analysis showed no remaining starting material. After cooling to room temperature, the aqueous phase was extracted with EtOAc (3 × 5 mL), and the combined organic phases were dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, compound 72 was purified by chromatographic chromatography to obtain 89 mg of compound 72, with a yield of 58% and MS (ESI) m / z 325.1.

[0283] In the second step, following the first step of synthetic route 4-Rt.7, using compound 72 (70 mg, 0.22 mmol) as the equivalence base, 64 mg of compound 73 was synthesized, with a yield of 63% and MS (ESI) m / z 419.2 ([M+Ht-Bu]). + ).

[0284] In steps three and four, following Steps C and D of synthetic route 2, compound 73 (50 mg, 1.00 mmol) obtained in step two was used as the equivalent starting material to yield 24 mg of the target product 40, with a yield of 72%. UPLC-MS (ESI) m / z 277.1, t R 0.768 min. 1 H NMR (400MHz, Methanol-d4) δ7.52(d,J=7.9Hz,1H),7.09(d,J=7.9Hz,1H),4.12–3.82(m,6H),3.47–3.14(m,4H),2.74(dd,J=7.0,4.2Hz,2H)ppm.

[0285]

[0286] Synthetic route 12 Synthetic route of compound 40

[0287] Following the synthetic route 13, in the first step, 3,4,6-trichloropyridazine (1.50 g, 8.18 mmol, 1.0 equiv.) was dissolved in dry DMF (40 mL). K2CO3 (2.26 g, 16.36 mmol, 2.0 equiv.) was added to this solution at 0 °C. Subsequently, at the same temperature, a solution of tert-butylpiperazine-1-carboxylate (1.98 g, 10.63 mmol, 1.3 equiv.) in dry DMF (20 mL) was slowly added dropwise to the above reaction mixture. The reaction mixture was automatically heated to room temperature and stirred for 3 h. TLC analysis showed no remaining starting material. The reaction system was diluted with water (100 mL), and the aqueous phase was extracted with EtOAc (4 × 100 mL). The combined organic phases were dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, 2.45 g of compound 74 was obtained by chromatographic purification, yielding 90% and MS (ESI) m / z 333.1.

[0288] In the second step, following Step B of synthetic route 2, 1.08 g of compound 75 was synthesized using compound 74 (1.00 g, 3.00 mmol) as the equivalent starting material, with a yield of 61% and MS (ESI) m / z 588.2.

[0289] Thirdly, compound 75 (450 mg, 0.76 mmol, 1.0 equiv.) was dissolved in anhydrous MeOH (8 mL). (A) MeONa (62 mg, 1.15 mmol, 1.5 equiv.) was added to this solution at 0 °C. The reaction system was stirred at room temperature for 10 min, then refluxed and stirred overnight. TLC showed no residual starting material. The reaction system was poured into ice water, and the aqueous phase was extracted with EtOAc (4 × 20 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The solution was then purified by silica gel column chromatography to obtain 344 mg of compound 76, yield 77%, MS (ESI) m / z 584.3; (B) Alternatively, using a sealed reaction tube, dimethylamine methanol solution (0.58 mL, 2.0 M in) was added to this solution at 0 °C. MeOH (1.15 mmol, 1.5 equiv.) was added, and the reaction system was stirred at room temperature for 10 min. The tube was then sealed and heated at 100 °C for 4 h with stirring. The reaction system was cooled to room temperature, depressurized, and TLC analysis showed no residual starting material. The reaction solution was diluted with water, and the aqueous phase was extracted with EtOAc (4 × 20 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The solution was then purified by chromatographic chromatography to obtain 269 mg of compound 77, with a yield of 59% and MS (ESI) m / z 597.3.

[0290] In steps four and five, following Steps C and D of synthetic route 2, compound 76 (250 mg, 0.43 mmol) obtained in step two was used as an equivalent starting material to yield 51 mg of target product 41, with a yield of 35%; or 77 (200 mg, 0.34 mmol) was used as an equivalent starting material to yield 31 mg of target product 42, with a yield of 26%.

[0291] 41: UPLC-MS(ESI) m / z 306.2,t R 0.682 min. 1 H NMR (400MHz, Methanol-d4) δ7.02(d,J=0.6Hz,1H),4.02–3.94(m,4H),3.49–3.38(m,9H),2.62–2.51(m,2H),2.16(dtd,J=9.7,6.2,2.1Hz,2H)ppm.

[0292] 42: UPLC-MS(ESI) m / z 319.2,t R 0.595 min. 1 H NMR(400MHz, Methanol-d4)δ6.79(d,J=0.6Hz,1H),3.98(td,J=6.2,0.7Hz,1H),3.80–3.71(m,2H),3.54 –3.48(m,2H),3.46–3.36(m,4H),3.15(s,6H),2.63–2.52(m,2H),2.16(dtd,J=9.7,6.2,2.1Hz,2H)ppm.

[0293]

[0294] Synthetic routes 13: Synthetic routes of compounds 41 and 42

[0295] TMSCHN2 (trimethylsilyl)diazomethane (0.35 mL, 2.0 M in Hexanes, 0.71 mmol, 3.0 equiv.) was slowly added dropwise to a dry THF (tetrahydrofuran) (3 mL) solution of compound 56 (100 mg, 0.27 mmol, 1.0 equiv.). The reaction system was stirred at room temperature for 12 h. TLC showed no reactants remaining. The reaction was quenched with water, and the aqueous phase was extracted with DCM-MeOH (10:1, v / v) (3 × 5 mL). The combined organic phases were dried over anhydrous sodium sulfate. After filtration, solvent removal under reduced pressure, and purification by silica gel column chromatography, 78 mg of compound 82 was obtained, yield 75%, MS (ESI) m / z 334.1 ([M+Ht-Bu)). + ).

[0296] Following Step D of synthetic route 2, using compound 82 (50 mg, 0.13 mmol) obtained in the previous step as the equivalent starting material, 33 mg of the target product 43 was obtained, with a yield of 80%.

[0297] UPLC-MS(ESI) m / z 290.2,t R 1.021 min. 1 H NMR (400MHz, Methanol-d4) δ7.47 (dd, J=8.6, 0.6Hz, 1H), 6.95 (d, J=8.3Hz, 1H), 4.03 (td, J=6.5, 0.7 Hz,1H),3.94–3.81(m,4H),3.31–3.18(m,4H),3.12(s,3H),2.68–2.51(m,2H),2.26–2.09(m,2H)ppm.

[0298]

[0299] Synthetic route 14 Synthetic route of compound 43

[0300] Example 3: Preparation of compounds PRTB-01 to PRTB-16

[0301]

[0302] Synthetic route 15: Synthesis of compound JQ1 acid

[0303] TFA (trifluoroacetic acid) (8 mL) was slowly added dropwise to a 20 mL solution of dried DCM (1,2-dichloroethane) containing JQ1 (1.00 g, 2.19 mmol, 1.0 equiv.) at 0 °C. The reaction system was stirred at room temperature for 6 h. TLC analysis showed no residual starting material. The reaction solution was concentrated under reduced pressure to obtain the residue, which was dissolved in EtOAc, washed with saturated brine, and dried over anhydrous sodium sulfate. After filtration, solvent removal under reduced pressure, and purification by silica gel column chromatography, 805 mg of compound JQ1 acid was obtained, with a yield of 92%. MS (ESI) m / z 399.1 ([M+H)). + ).

[0304]

[0305] Synthetic route 16: Synthesis of compound JQ1-LB1

[0306] Compounds JQ1 acid (60 mg, 0.15 mmol, 1.0 equiv.), tert-butyl (4-aminobutyl)carbamate (30 mg, 0.16 mmol, 1.05 equiv.), HATU (85 mg, 0.22 mmol, 1.5 equiv.), and DIPEA (diisopropylethylamine) (78 μL, 0.45 mmol, 3.0 equiv.) were dissolved in anhydrous DMF (2 mL). The reaction mixture was stirred overnight at room temperature under argon protection. TLC analysis showed no residual starting material. The reaction was quenched with water, and the aqueous phase was extracted with EtOAc. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, solvent removal under reduced pressure, and purification by silica gel column chromatography, 72 mg of compound JQ1-LB1 was obtained, with a yield of 84%. MS (ESI) m / z 472.0 ([M+H-Boc]). + ).

[0307]

[0308] Following the synthetic method for compound JQ1-LB1, tert-butyl(4-aminobutyl)carbamate was substituted for tert-butyl(5-aminopentyl)carbamate to yield 68 mg of compound JQ1-LB2LB13, with a yield of 78%. MS (ESI) m / z 486.0 ([M+H-Boc)). + ).

[0309]

[0310] Following the synthetic method for compound JQ1-LB1, tert-butyl(4-aminobutyl)carbamate was substituted for tert-butyl(6-aminohexyl)carbamate to yield 65 mg of compound JQ1-LB3, with a yield of 73%. MS (ESI) m / z 500.1 ([M+H-Boc) + ).

[0311]

[0312] Synthetic route 17 Compound 7-LB4

[0313] 4-((tert-butoxycarbonyl)amino)butanoic acid (91 mg, 0.45 mmol, 2.0 equiv.), EDCI (129 mg, 0.67 mmol, 3.0 equiv.), and HOBt (61 mg, 0.45 mmol, 2.0 equiv.) were dissolved in dry DCM (1,2-dichloroethane) (3 mL), and the resulting solution was stirred at room temperature for 1 h. An anhydrous DCM (1,2-dichloroethane) solution (2 mL) of compound 7 (70 mg, 0.22 mmol, 1.0 equiv.) and TEA (187 μL, 1.35 mmol, 6.0 equiv.) were added to the reaction mixture, and the reaction system was stirred at room temperature for 3 h. TLC showed no remaining starting material. The reaction was quenched at 0 °C with saturated sodium bicarbonate solution. The resulting aqueous phase was extracted with a DCM-MeOH (5:1) mixed solvent, and the combined organic phases were dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, the compound 7-LB4 was purified by rapid silica gel column chromatography to obtain 68 mg of compound 7-LB4, with a yield of 66%. MS (ESI) m / z 361.4 ([M+H-Boc)). + ).

[0314]

[0315] Following the synthetic method for compound 7-LB4, 4-((tert-butoxycarbonyl)amino)butanoic acid was replaced with 5-(tert-butoxycarbonyl)amino)butanoic acid to yield 77 mg of compound 7-LB5, with a yield of 72% and MS (ESI) m / z 375.4 ([M+H-Boc)). + ).

[0316]

[0317] Following the synthetic method for compound 7-LB4, 4-((tert-butoxycarbonyl)amino)ethoxy)acetic acid was substituted for 4-(tert-butoxycarbonyl)amino)butanoic acid to yield 70 mg of compound 7-LB6, with a yield of 65% and MS (ESI) m / z 377.4 ([M+H-Boc]). + ).

[0318]

[0319] Following the synthetic method for compound 7-LB4, 4-((tert-butoxycarbonyl)amino)ethoxy)acetic acid was substituted for 4-(tert-butoxycarbonyl)amino)butanoic acid to yield 67 mg of compound 7-LB7, with a yield of 61%. MS (ESI) m / z 391.4 ([M+H-Boc]). + ).

[0320]

[0321] Following the synthetic method for compound 7-LB6, compound 7 was replaced by compound 40 to yield 75 mg of compound 40-LB8, with a yield of 70%. MS (ESI) m / z 377.4 ([M+H-Boc]). + ).

[0322]

[0323] Following the synthetic method for compound 7-LB6, compound 7 was replaced by compound 9 to obtain 76 mg of compound 9-LB9, with a yield of 69%. MS (ESI) m / z 391.4 ([M+H-Boc]). + ).

[0324]

[0325] Following the synthetic method for compound 7-LB6, compound 7 was replaced by compound 35 to obtain 74 mg of compound 35-LB10, with a yield of 62% and MS (ESI) m / z 431.5 ([M+H-Boc]). + ).

[0326]

[0327] Following the synthetic method for compound 7-LB6, compound 7 was replaced by compound 36 to yield 84 mg of compound 36-LB11, with a yield of 71%. MS (ESI) m / z 427.5 ([M+H-Boc]). + ).

[0328]

[0329] Following the synthetic method for compound 7-LB6, compound 7 was replaced by compound 41 to obtain 74 mg of compound 41-LB16, with a yield of 65%. MS (ESI) m / z 407.4 ([M+H-Boc]). + ).

[0330]

[0331] Synthetic route 18: Synthesis of compounds 55a-OH, 55a-LB12, and 4-LB12

[0332] [Step A] Following the synthetic method for compound 55a, 3-(benzyloxy)azetidine was substituted for 3-(tert-butyldimethylsilyl)oxy)azetidine to yield 1.21 g of compound 55a-TBS. Compound 55a-TBS (800 mg, 1.44 mmol, 1.0 equiv.) was dissolved in anhydrous THF (10 mL). A THF (tetrahydrofuran) solution of TBAF (tetrabutylamine fluoride) (2.16 mL, 2.16 mmol, 1.0 M in THF, 1.5 equiv.) was added dropwise to this solution. The reaction mixture was stirred at room temperature for 4 h. TLC showed no remaining starting material. The mixture was quenched with saturated sodium bicarbonate solution, and the resulting aqueous phase was extracted with EtOAc. The combined organic phases were dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, 594 mg of compound 55a-OH was obtained by chromatographic purification, with a yield of 93% and MS (ESI) m / z 441.5 ([M+H)). + ).

[0333] [Step B] NaH (54 mg, 1.36 mmol, 60 wt%, 1.5 equiv.) was added to an anhydrous DMF (5 mL) solution of compound 55a-OH (400 mg, 0.91 mmol, 1.0 equiv.) at 0 °C. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was placed in an ice-water mixing bath, and an anhydrous DMF (2 mL) solution of tert-butyl(5-bromopentyl)carbamate (266 mg, 1.00 mmol, 1.1 equiv.) was added dropwise. The reaction system was stirred at room temperature for another 5 h. TLC showed no remaining starting material. The reaction was quenched with saturated ammonium chloride solution. The resulting aqueous phase was extracted with EtOAc, and the combined organic phases were dried over anhydrous sodium sulfate. After filtration, solvent removal under reduced pressure, and purification by silica gel column chromatography, 330 mg of compound 55a-LB12 was obtained, with a yield of 58%. MS (ESI) m / z 526.7 ([M+H-Boc)). + ).

[0334] [Step C] Compound 55a-LB12 (300 mg, 0.48 mmol, 1.0 equiv.) was dissolved in a MeOH-THF (4:1, 5 mL) mixture. Pd / C (60 mg, 10 wt% on Carbon) was added, and the reaction mixture was stirred overnight at room temperature under a hydrogen atmosphere (balloon). TLC analysis showed no residual starting material. After filtration, solvent removal under reduced pressure, and purification by chromatographic chromatography, 173 mg of compound 4-LB12 was obtained, with a yield of 81%. MS (ESI) m / z 348.4 ([M+H-Boc)). + ).

[0335]

[0336] Synthetic route 19: Synthesis of compounds 83, 84, 85, 86, and 87

[0337] [Step A] Following the synthetic method for compound 44, 1-(6-chloropyridazin-3-yl)ethan-1-one was replaced with 6-chloropyridazine-3-carbaldehyde to obtain 1.42 g of compound 44-Aldehyde. Compound 44-Aldehyde (1.00 g, 2.52 mmol, 1.0 equiv.) was dissolved in a MeOH-THF (5:2, 14 mL) mixed solvent. After adding Pd / C (100 mg, 10 wt% on Carbon), the reaction system was stirred overnight at room temperature under a hydrogen atmosphere (balloon). TLC showed no residual starting material. After filtration, solvent removal under reduced pressure, and purification by chromatographic chromatography, 427 mg of compound 83 was obtained, with a yield of 77%. MS (ESI) m / z 220.2 ([M+H)). + ).

[0338] [Step B] Following the synthetic method for compound 46, using compound 83 as the equivalent starting material (400 mg, 1.82 mmol, 1.0 equiv.), 530 mg of compound 84 was obtained, with a yield of 86%. MS (ESI) m / z 340.4 ([M+H]). + ).

[0339] [Step C] At 0°C, a solution of tert-butyl 2-(diethoxyphosphoryl)acetate (82 mg, 0.32 mmol, 1.1 equiv.) in anhydrous THF (tetrahydrofuran) (1 mL) was added dropwise to a suspension of NaH (14 mg, 0.35 mmol, 60 wt%, 1.2 equiv.) in anhydrous THF (1 mL). The reaction mixture was stirred at the same temperature for 30 min. I2 (89 mg, 0.35 mmol, 1.2 equiv.) was added to the above mixture at 0°C. The reaction mixture was stirred at the same temperature for 3 h. Compound 84 (100 mg, 0.29 mmol, 1.0 equiv.) in anhydrous THF (1 mL) was added dropwise at the same temperature. After the addition was complete, the reaction system was stirred at the same temperature for 15 min. Then, NaH (14 mg, 0.35 mmol, 60 wt%, 1.2 equiv.) was added, and the mixture was automatically heated to room temperature and stirred for 1 h. TLC analysis showed no remaining raw material. The reaction was quenched with water, and the resulting aqueous phase was extracted with a DCM-MeOH (5:1) mixed solvent. The combined organic phases were dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, the solution was purified by silica gel column chromatography to give 97 mg of compound 85, yield 76%, MS (ESI) m / z 380.4 ([M+Ht-Bu)). + ).

[0340] [Step D] At 0°C, 2 mL of TFA (trifluoroacetic acid) was added dropwise to an anhydrous DCM (1,2-dichloroethane) (3 mL) solution of compound 85 (90 mg, 0.21 mmol, 1.0 equiv.). The reaction system was automatically heated to room temperature and stirred for 4 h. TLC showed no residual starting material. All volatile components were removed under reduced pressure to obtain 46 mg of crude product 86, which could be used directly in the next step without purification.

[0341] [Step E] At -78°C, TMSCHN2 (trimethylsilyl)diazomethane (1.90 M in hexanes, 0.18 mL, 0.35 mmol, 1.2 equiv.) was added dropwise to a 1 mL solution of LDA (diisopropylaminolithium) in THF (tetrahydrofuran) (~1.20 mmol, ~1.20 equiv.), and the reaction mixture was stirred at the same temperature for 30 min. At -78°C, an anhydrous THF (1 mL) solution of compound 84 (100 mg, 0.29 mmol, 1.0 equiv.) was added dropwise to the solution, and the reaction mixture was stirred at -78°C for 1 h, followed by reflux for 2 h. TLC showed no reactants remaining. After cooling to room temperature, the reaction was quenched with water. The resulting aqueous phase was extracted with a DCM (1,2-dichloroethane)-MeOH (5:1) mixed solvent, and the combined organic phases were dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, the compound was purified by silica gel rapid column chromatography to obtain 61 mg of compound 87, with a yield of 63% and MS (ESI) m / z 336.4 ([M+H)). + ).

[0342]

[0343] Synthesis of compound 88 via synthetic route 20

[0344] Compounds 3-bromo-1H-pyrazole (100 mg, 0.68 mmol, 1.0 equiv.), tert-butyl(3-bromopropyl)carbamate (243 mg, 1.02 mmol, 1.5 equiv.), and Cs₂CO₃ (443 mg, 1.36 mmol, 2.0 equiv.) were suspended in 10 mL of dry MeCN. The reaction mixture was stirred overnight under reflux. TLC analysis showed no residue. After dilution with MTBE (methyl tert-butyl ether), the mixture was filtered, and the solvent was removed under reduced pressure. Purification was achieved by rapid silica gel column chromatography to give 104 mg of compound 88, a yield of 50%. MS (ESI) m / z 205.1 ([M+H-Boc]). + ).

[0345]

[0346] Following the synthetic method for compound 88, 3-bromo-1H-pyrazole was substituted with 4-bromo-1H-pyrazole to yield 187 mg of compound 89, with a yield of 90%. MS (ESI) m / z 205.1 ([M+H-Boc]). + ).

[0347]

[0348] Synthetic route 20: Synthesis of compounds 90-LB14-PMB and 90-LB14

[0349] [Step A] Compound 87 (80 mg, 0.24 mmol, 1.0 equiv.), compound 88 (87 mg, 0.29 mmol, 1.2 equiv.), CuI (9 mg, 0.05 mmol, 0.2 equiv.), Et3N (66 μL, 0.48 mmol, 2.0 equiv.), and Pd(PPh3)2Cl2 (17 mg, 0.02 mmol, 0.1 equiv.) were added sequentially to 2 mL of dry THF (tetrahydrofuran). The reaction mixture was purged with argon three times and stirred at 50 °C under an argon atmosphere for 3 h. TLC analysis showed no remaining starting material. After cooling to room temperature, the reaction mixture was diluted with MTBE (methyl tert-butyl ether), filtered, and the solvent was removed under reduced pressure. The mixture was then purified by chromatographic chromatography to obtain 86 mg of compound 90-LB14-PMB, with a yield of 64%. MS (ESI) m / z 459.5 ([M+H-Boc]). + ).

[0350] [Step B] Under an argon atmosphere, CAN (cerium ammonium nitrate) (766 mg, 1.40 mmol, 10.0 equiv.) was added in portions to a MeCN-H2O (10 mL, 10:1, v / v) solution of compound 90-LB14-PMB (80 mg, 0.14 mmol, 1.0 equiv.). The reaction mixture was stirred at 0 °C for 15 min, followed by stirring at room temperature for 2 h. TLC analysis showed no remaining starting material. Saturated sodium bicarbonate solution was added, and the aqueous phase was extracted with a DCM (1,2-dichloroethane)-MeOH (5:1, v / v) mixed solvent. The combined organic phases were dried over anhydrous sodium sulfate. After filtration, solvent removal under reduced pressure, and purification by TLC, 45 mg of compound 90-LB14 was obtained, with a yield of 72% and MS (ESI) m / z 339.2 ([M+H-Boc)). + ).

[0351]

[0352] Following the synthetic method for compound 90-LB14, 42 mg of compound 91-LB15 was obtained, with a yield of 68% and an MS (ESI) m / z of 339.2 ([M+H-Boc]). + ).

[0353]

[0354] Synthesis of compound 92 via synthetic route 21

[0355] NaH (644 mg, 16.11 mmol, 60 wt%, 1.2 equiv.) was added to 2 mL of dried DMF at 0 °C. Then, tert-butyl 2-hydroxyacetate (1.77 g, 13.43 mmol, 1.0 equiv.) was added to the suspension. After stirring at the same temperature for 30 min, 3,6-dichloropyridazine (2.00 g, 13.43 mmol, 1.0 equiv.) was added in a single batch. The resulting mixture was automatically heated to room temperature under an argon atmosphere and stirred overnight. TLC analysis showed no residual starting material. The reaction was carefully quenched with ice water. The resulting aqueous phase was extracted with EtOAc. The combined organic phases were washed with saturated brine, filtered, and the solvent was removed under reduced pressure. The mixture was then purified by chromatographic chromatography to obtain 2.06 g of compound 92, with a yield of 63%. MS (ESI) m / z 189.6 ([M+Ht-Bu)). + ).

[0356]

[0357] Synthesis of compounds 93 and 94 in synthetic route 22

[0358] Following the synthetic method for compound 8, compound 93 was synthesized from compound 92. Compound 93 (1.00 g, 3.11 mmol, 1.0 equiv.) was dissolved in dry DCM (1,2-dichloroethane) (20 mL), and then TFA (trifluoroacetic acid) (5 mL) was slowly added upwards at 0 °C. The reaction system was automatically heated to room temperature under an argon atmosphere and stirred for 4 h. TLC showed no residual starting material. After solvent removal under reduced pressure, 842 mg of crude product compound 94 was obtained, which was directly used in the next step without further purification. MS (ESI) m / z 266.2 ([M+H)). + ).

[0359]

[0360] Synthesis of protein degrading agents PRTB-01, PRTB-02, PRTB-03, and PRTB-13 via route 23

[0361] The protein degrading agents PRTB-01, PRTB-02, PRTB-03, and PRTB-13 were synthesized according to the synthesis strategy in synthesis route 23.

[0362] [Step A] Dissolve JQ1-LB1, JQ1-LB2LB13, or JQ1-LB3 in dry DCM (1,2-dichloroethane) (0.2 M). Slowly add TFA (trifluoroacetic acid) (1 / 3 volume of DCM) to the solution at 0°C. The reaction system is automatically heated to room temperature under an argon atmosphere and stirred for 4–6 hours. LC-MS analysis shows no remaining raw material. After solvent removal under reduced pressure, dissolve in methanol, adjust pH to 8 with saturated sodium bicarbonate solution, and then extract with a DCM-MeOH (5:1, v / v) mixed solvent. Combine the organic phases and dry with anhydrous sodium sulfate. Filter, remove solvent under reduced pressure, and continue to evaporate under reduced pressure at 45°C and 0.6 mbar for 30 min to obtain the crude product JQ1-LBx-NH2, which is directly used in the next step.

[0363] [Step B] Dissolve JQ1-LB1 (1.0 equiv.) and 94 (1.0 equiv.), or JQ1-LB2LB13 (1.0 equiv.) and 94 (1.0 equiv.), or JQ1-LB3 (1.0 equiv.) and 94 (1.0 equiv.), or JQ1-LB2LB13 (1.0 equiv.) and 86 (1.0 equiv.) in dry DMF (0.2 M), then add HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) (1.5 equiv.) and DIPEA (diisopropylethylamine) (3.0 equiv.) in sequence. Stir the reaction system overnight at room temperature under argon protection. When one of the raw materials was consumed by LC-MS, the reaction was quenched with water. The aqueous phase was extracted with a DCM-MeOH (5:1, v / v) mixed solvent, and the combined organic phases were dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, the products were purified by preparative HPLC to obtain PRTB-01, PRTB-02, PRTB-03, and PRTB-13, respectively.

[0364]

[0365] The protein degrader PRTB-01 was synthesized and purified using the general synthesis strategy described above to obtain 12 mg of the target product. 1HNMR(400MHz,DMSO-d6)δ11.07(s,1H),7.74–7.62(m,4H),7.62–7.55(m,1 H),7.55–7.48(m,2H),7.09(d,J=8.1Hz,1H),5.78(t,J=9.3Hz,1H),4.69(d ,J=0.6Hz,2H),3.73–3.65(m,1H),3.24–2.98(m,6H),2.63(s,3H),2.59–2 .48(m,2H),2.21–2.04(m,2H),1.45(p,J=2.8Hz,4H)ppm.UPLC-MS(ESI)m / z 718.2,t R 0.842 min, 96% purity.

[0366]

[0367] The protein degrader PRTB-02 was synthesized and purified using the general synthesis strategy described above to obtain 7 mg of the target product. 1 H NMR(400MHz,DMSO-d6)δ11.07(s,1H),7.71(t,J=4.8Hz,1H),7.71–7.62(m,3H),7.6 2–7.55(m,1H),7.55–7.48(m,2H),7.09(d,J=8.1Hz,1H),5.78(t,J=9.3Hz,1H),4.6 9(d,J=0.6Hz,2H),3.73–3.65(m,1H),3.22–2.98(m,7H),2.63(s,3H),2.59–2.48(m ,2H),2.23–2.04(m,2H),1.53–1.41(m,4H),1.39–1.27(m,2H)ppm.UPLC-MS(ESI)m / z 732.2,t R 0.855 min, 98% purity.

[0368]

[0369] The protein degrader PRTB-03 was synthesized and purified using the general synthesis strategy described above to obtain 12 mg of the target product. 1HNMR(400MHz, DMSO-d6)δ11.07(s,1H),7.70(t,J=4.7Hz,1H),7.71–7.62(m,3H),7.62–7.55( m,1H),7.55–7.48(m,2H),7.09(d,J=8.1Hz,1H),5.78(t,J=9.3Hz,1H),4.69(d,J=0.7Hz,2H), 3.73–3.65(m,1H),3.23–3.10(m,4H),3.13–3.04(m,1H),3.08–2.98(m,1H),2.63(s,3H),2.5 9–2.48(m,2H),2.23–2.04(m,2H),1.58–1.45(m,4H),1.40–1.27(m,4H)ppm.UPLC-MS(ESI)m / z 746.3,t R 0.851 min, 96% purity.

[0370]

[0371] The protein degrader PRTB-13 was synthesized and purified according to the general synthesis strategy described above to obtain 15 mg of the target product. 1 HNMR(400MHz, DMSO-d6)δ11.07(s,1H),7.97(t,J=4.6Hz,1H),7.74–7.62(m,5H),7.55–7.48(m,2H),5.78(t,J=9.3Hz,1H),3.69(t,J=6.2Hz,1 H),3.25–2.98(m,6H),2.63(s,3H),2.59–2.48(m,2H),2.22–2.04(m,2H),1.60–1.41(m,4H),1.38–1.26(m,2H)ppm.UPLC-MS(ESI)m / z726.2,t R 0.780 min, 98% purity.

[0372]

[0373] Synthesis route 24: Synthesis of protein degraders PRTB-04~PRTB-12, PRTB-14~PRTB-16

[0374] The protein degrading agents PRTB-04~PRTB-12, PRTB-14~PRTB-16 were synthesized according to the synthesis strategy in synthetic route 24.

[0375] [Step A] Dissolve 7-LB4 or 7-LB5 or 7-LB6 or 7-LB7 or 40-LB8 or 9-LB9 or 35-LB10 or 36-LB11 or 4-LB12 or 90-LB14 or 91-LB15 or 41-LB16 in dry DCM (1,2-dichloroethane) (0.2M). Slowly add TFA (trifluoroacetic acid) (1 / 2 volume of DCM) to the solution at 0°C. The reaction system is automatically heated to room temperature under an argon atmosphere and stirred for 4 hours. LC-MS showed no residual raw material. After removing the solvent under reduced pressure, dissolve in methanol, adjust the pH to 8 with saturated sodium bicarbonate solution, and then extract with a DCM-MeOH (5:1, v / v) mixed solvent. Combine the organic phases and dry with anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, the product was further evaporated under reduced pressure at 45°C and 0.6 mbar for 30 min to obtain the crude product u-LBz-NH2, which could be used directly in the next step without further purification.

[0376] [Step B] JQ1-Acid (1.0 equiv.) and one of the crude products u-LBz-NH2 obtained in the previous step (1.0 equiv.) were dissolved in dry DMF (0.2 M). Then, HATU (1.5 equiv.) and DIPEA (diisopropylethylamine) (3.0 equiv.) were added sequentially. The reaction system was stirred overnight at room temperature under argon protection. When one of the starting materials was consumed by LC-MS, the reaction was quenched with water. The aqueous phase was extracted with a DCM-MeOH (5:1, v / v) mixed solvent, and the combined organic phases were dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, the products were purified by preparative HPLC to obtain PRTB-04, PRTB-05, PRTB-06, PRTB-07, PRTB-08, PRTB-09, PRTB-10, PRTB-11, PRTB-12, PRTB-14, PRTB-15, and PRTB-16, respectively.

[0377]

[0378] The protein degrader PRTB-04 was synthesized and purified using the general synthesis strategy described above to obtain 18 mg of the target product. 1HNMR(400MHz,DMSO-d6)δ11.07(s,1H),7.78(d,J=9.5Hz,1H),7.78(s,1H),7.69–7.62(m,2H),7.55 –7.46(m,2H),7.46(d,J=0.7Hz,1H),6.92(d,J=8.3Hz,1H),5.78(t,J=9.3Hz,1H),3.72–3.52(m,11 H),3.13(td,J=6.0,4.7Hz,2H),3.07(d,J=5.9Hz,1H),3.08–2.98(m,1H),2.63(s,3H),2.59–2.48( m,2H),2.29(d,J=0.9Hz,1H),2.21–2.04(m,2H),1.76(tt,J=7.7,5.9Hz,2H)ppm.UPLC-MS(ESI)m / z 743.3,t R 0.671 min, 98% purity.

[0379]

[0380] The protein degrader PRTB-05 was synthesized and purified using the general synthesis strategy described above to obtain 10 mg of the target product. 1 HNMR(400MHz,DMSO-d6)δ11.07(s,1H),7.71–7.62(m,2H),7.55–7.44(m,2H), 6.92(d,J=8.3Hz,1H),5.78(t,J=9.3Hz,1H),3.72–3.52(m,7H),3.20–3.12(m ,1H),3.16–3.06(m,1H),3.09–2.98(m,1H),2.63(s,2H),2.58–2.49(m,1H),2 .28–2.21(m,1H),2.25–2.04(m,2H),1.60–1.46(m,3H)ppm.UPLC-MS(ESI)m / z 757.3,t R 0.685 min, 98% purity.

[0381]

[0382] The protein degrader PRTB-06 was synthesized and purified using the general synthesis strategy described above to obtain 7 mg of the target product. 1H NMR(400MHz,DMSO-d6)δ11.07(s,1H),7.76(t,J=4.8Hz,1H),7.69–7.62(m,2H),7 .55–7.48(m,2H),7.51–7.44(m,1H),6.92(d,J=8.3Hz,1H),5.78(t,J=9.3Hz,1H) ,4.13(s,2H),3.72–3.58(m,9H),3.62–3.55(m,2H),3.40–3.24(m,2H),3.14–2.9 8(m,2H),2.63(s,3H),2.59–2.48(m,2H),2.21–2.04(m,2H)ppm.UPLC-MS(ESI)m / z 759.3,t R 0.601 min, 98% purity.

[0383]

[0384] The protein degrader PRTB-07 was synthesized and purified using the general synthesis strategy described above to obtain 12 mg of the target product. 1 HNMR(400MHz,DMSO-d6)δ11.07(s,1H),7.69(t,J=4.6Hz,1H),7.69–7.62(m,2H ),7.55–7.44(m,3H),6.92(d,J=8.3Hz,1H),5.78(t,J=9.3Hz,1H),4.14(s,2H) ,3.72–3.51(m,11H),3.19(td,J=6.1,4.6Hz,2H),3.14–2.98(m,2H),2.59–2.4 8(m,2H),2.22–2.04(m,2H),1.80(pd,J=6.0,2.5Hz,2H)ppm.UPLC-MS(ESI)m / z 773.3,t R 0.618 min, 98% purity.

[0385]

[0386] The protein degrader PRTB-08 was synthesized and purified using the general synthesis strategy described above to obtain 12 mg of the target product. 1HNMR(400MHz,DMSO-d6)δ10.64(s,1H),7.76(t,J=4.8Hz,1H),7.69–7.62(m,2H),7.55–7.4 8(m,2H),7.06(d,J=7.9Hz,1H),7.01(d,J=7.9Hz,1H),5.78(t,J=9.3Hz,1H),4.13(s,2H), 3.95(ddd,J=16.5,7.0,4.2Hz,2H),3.71–3.55(m,11H),3.39–3.25(m,2H),3.14–2.98(m,2 H),2.71(ddd,J=7.3,4.2,3.3Hz,2H),2.31(d,J=1.5Hz,6H)ppm.UPLC-MS(ESI)m / z760.2,t R 0.511 min, 99% purity.

[0387]

[0388] The protein degrader PRTB-09 was synthesized and purified using the general synthesis strategy described above to obtain 15 mg of the target product. 1 HNMR(400MHz,DMSO-d6)δ11.13(s,1H),7.76(t,J=4.8Hz,1H),7.69–7.62(m,2H),7.55–7.48(m,2H ),6.67(d,J=0.9Hz,1H),5.78(t,J=9.3Hz,1H),4.15–4.07(m,3H),3.72(dd,J=6.7,3.8Hz,2H),3. 69–3.63(m,4H),3.60(ddd,J=11.9,6.7,3.8Hz,4H),3.39–3.25(m,2H),3.14–2.98(m,2H),2.64–2 .48(m,2H),2.40(d,J=0.7Hz,3H),2.31(d,J=1.5Hz,6H),2.22–2.05(m,2H)ppm.UPLC-MS(ESI)m / z 773.3,t R 0.624 min, 98% purity.

[0389]

[0390] The protein degrader PRTB-10 was synthesized and purified using the general synthesis strategy described above to obtain 8 mg of the target product. 1H NMR(400MHz,DMSO-d6)δ11.12(s,1H),7.76(t,J=4.8Hz,1H),7.69–7.62(m,2H),7.5 5–7.48(m,2H),5.78(t,J=9.3Hz,1H),4.13(s,2H),4.06(t,J=6.4Hz,1H),3.75–3.54 (m,11H),3.39–3.25(m,2H),3.14–2.96(m,4H),2.95–2.85(m,2H),2.64–2.48(m,2H ),2.31(d,J=1.5Hz,6H),2.25–2.07(m,2H),1.90–1.78(m,4H)ppm.UPLC-MS(ESI)m / z 813.3,t R 0.701 min, 96% purity.

[0391]

[0392] The protein degrader PRTB-11 was synthesized and purified using the general synthesis strategy described above to obtain 5 mg of the target product. 1 H NMR (400MHz, DMSO-d6) δ11.17(s,1H),8.33(dd,J=8.8,1.3Hz,1H),8.13(dd,J=8.6,1.3Hz,1H),7.83(ddd,J=9.1, 7.8,1.3Hz,1H),7.76(t,J=4.8Hz,1H),7.71(ddd,J=9.0,7.7,1.3Hz,1H),7.69–7.62(m,2H),7.55–7.48(m,2H),5 .78(t,J=9.3Hz,1H),4.13(s,2H),3.83–3.70(m,5H),3.67(t,J=4.3Hz,2H),3.63–3.54(m,4H),3.40–3.24(m,2H) ,3.14–2.98(m,2H),2.63(s,3H),2.62–2.48(m,2H),2.31(s,3H),2.25–2.08(m,2H)ppm.UPLC-MS(ESI)m / z809.3,t R 0.695 min, 96% purity.

[0393]

[0394] The protein degrader PRTB-12 was synthesized and purified using the general synthetic strategy described above to obtain 11 mg of the target product. 1H NMR (400MHz, DMSO-d6) δ11.07(s,1H),7.71–7.62(m,3H),7.55–7.48(m,2H),7.51–7.44(m,1H),6.94(d,J=8.6Hz,1 H),5.78(t,J=9.3Hz,1H),4.21(p,J=3.7Hz,1H),3.93(dd,J=12.3,3.7Hz,2H),3.79(dd,J=12.5,3.8Hz,2H),3.68(t d,J=6.2,0.7Hz,1H),3.48(t,J=6.2Hz,2H),3.13(qd,J=5.4,1.6Hz,2H),3.12–3.04(m,1H),3.08–2.98(m,1H),2.6 3(s,3H),2.59–2.48(m,2H),2.22–2.04(m,2H),1.62–1.48(m,2H),1.53–1.33(m,4H)ppm.UPLC-MS(ESI)m / z730.3,t R 0.885 min, 97% purity.

[0395]

[0396] The protein degrader PRTB-14 was synthesized and purified using the general synthetic strategy described above to obtain 16 mg of the target product. 1 HNMR(400MHz,DMSO-d6)δ111.07(s,1H),7.75(t,J=3.8Hz,1H),7.72–7.62(m,4H), 7.56(d,J=3.3Hz,1H),7.54–7.48(m,2H),6.37(d,J=3.1Hz,1H),5.78(t,J=9.3Hz,1 H),4.10(td,J=5.3,0.7Hz,2H),3.72–3.65(m,1H),3.20–2.98(m,4H),2.63(s,3H) ,2.59–2.48(m,2H),2.22–2.04(m,2H),1.96(p,J=5.5Hz,2H)ppm.UPLC-MS(ESI)m / z 721.2,t R 1.157 min, 97% purity.

[0397]

[0398] The protein degrader PRTB-15 was synthesized and purified using the general synthesis strategy described above to obtain 18 mg of the target product. 1HNMR(400MHz,DMSO-d6)δ11.07(s,1H),7.75(t,J=3.8Hz,1H),7.73–7.65(m,2H) ,7.69–7.61(m,4H),7.55–7.48(m,2H),5.78(t,J=9.3Hz,1H),4.10(td,J=5.4,1 .2Hz,2H),3.72–3.65(m,1H),3.16(td,J=5.7,3.8Hz,2H),3.15–2.98(m,2H),2. 59–2.48(m,2H),2.22–2.04(m,2H),1.96(p,J=5.5Hz,2H)ppm.UPLC-MS(ESI)m / z 721.2,t R 1.133 min, 97% purity.

[0399]

[0400] The protein degrader PRTB-16 was synthesized and purified using the general synthetic strategy described above to obtain 12 mg of the target product. 1 HNMR(400MHz, DMSO-d6)δ11.08(s,1H),7.76(t,J=4.8Hz,1H),7.69–7.62(m,2H),7. 55–7.48(m,2H),7.02(d,J=0.6Hz,1H),5.78(t,J=9.3Hz,1H),4.13(s,2H),4.03–3. 95(m,1H),3.98(s,3H),3.70–3.55(m,6H),3.40–3.18(m,7H),3.14–2.98(m,2H),2. 63(s,3H),2.59–2.49(m,2H),2.31(s,3H),2.22–2.05(m,2H)ppm.UPLC-MS(ESI)m / z 789.3,t R 0.709 min, 96% purity.

[0401] Example 4: Preparation of compounds PRTA-01 to PRTA-06

[0402]

[0403] Synthetic route 25: Synthesis of compounds 95, 96, and 97

[0404] [Step A] Dissolve tert-butyl((1r,4r)-4-hydroxycyclohexyl)carbamate (5.00 g, 23.22 mmol, 1.0 equiv.) in dry THF (100 mL). Add fractionally of NaH (2.09 g, 52.25 mmol, 60 wt%, 2.25 equiv.) at 0 °C. The reaction mixture is automatically heated to room temperature under an argon atmosphere and stirred for 40 min. Add 2-chloro-4-fluorobenzonitrile (5.42 g, 34.84 mmol, 1.5 equiv.) to the above reaction solution at 0 °C. The reaction mixture is automatically heated to room temperature and stirred for 5 h. TLC monitoring shows no remaining starting material. Pour the reaction mixture into an ice-water mixture. Extract the resulting aqueous phase with EtOAc. Combine the organic phases, wash with saturated brine, and dry with anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, 6.96 g of compound 95 was obtained by chromatographic purification, yielding 85%; MS (ESI) m / z 251.1 ([M+H-Boc)). + ).

[0405] [Step B] At 0°C, TFA (trifluoroacetic acid) (1.85 mL, 28.48 mmol, 4.0 equiv.) was slowly added to a 30 mL solution of dried DCM (1,2-dichloroethane) (30 mL) containing compound 95 (2.50 g, 7.12 mmol, 1.0 equiv.). The reaction mixture was stirred at 0°C for 3 h. TLC monitoring showed no remaining starting material. The reaction solution was diluted with DCM (1,2-dichloroethane), the solvent was removed under reduced pressure, and the residue was dissolved in methanol. After dilution with water, the pH was adjusted to 8 with saturated sodium bicarbonate solution. The aqueous phase was extracted with EtOAc, and the combined organic phases were dried over anhydrous sodium sulfate. After filtration and removal of the solvent under reduced pressure, the mixture was further evaporated under reduced pressure at 45°C and 0.6 mbar for 30 min to obtain 1.33 g of crude product 96. This crude product was used directly in the next step without further purification. MS (ESI) m / z 251.1 ([M+H) + ).

[0406] [Step C] Compound 4-(4-(tert-butoxycarbonyl)piperazin-1-yl)benzoic acid (4.70 g, 15.33 mmol, 1.05 equiv.), crude product 96 (3.66 g, ~14.60 mmol, 1.00 equiv.), HATU (8.46 g, 21.90 mmol, 1.5 equiv.), and DIPEA (diisopropylethylamine) (7.59 mL, 43.80 mmol, 3.0 equiv.) were dissolved in anhydrous DMF (150 mL). The reaction system was stirred overnight at room temperature under argon protection. TLC showed no residual starting material. The reaction was quenched with water, and the aqueous phase was extracted with EtOAc. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, 6.42 g of compound 97 was obtained by chromatographic purification, yielding 82%; MS (ESI) m / z 439.2 ([M+H-Boc]). + ).

[0407] [Step D] At 0 °C, TFA (trifluoroacetic acid) (0.98 mL, 14.84 mmol, 4.0 equiv.) was slowly added to a 50 mL solution of dried DCM (1,2-dichloroethane) containing compound 97 (2.00 g, 3.71 mmol, 1.0 equiv.). The reaction mixture was stirred at 0 °C for 3 h. TLC monitoring showed no reactants remaining. The reaction mixture was diluted with DCM (1,2-dichloroethane), the solvent was removed under reduced pressure, and the residue was dissolved in methanol. After dilution with water, the pH was adjusted to 8 with saturated sodium bicarbonate solution. The aqueous phase was extracted with EtOAc, and the combined organic phases were dried over anhydrous sodium sulfate. After filtration and removal of the solvent under reduced pressure, the mixture was further evaporated under reduced pressure at 45 °C and 0.6 mbar for 30 min to obtain 1.68 g of crude product 98, which could be used directly in the next step without further purification. 1 H NMR(400MHz, DMSO-d6)δ7.83(d,J=8.8Hz,1H),7.71(d,J=8.9Hz,1H),7.68–7.61 (m,2H),7.16(d,J=2.2Hz,1H),7.09–7.02(m,2H),6.95(dd,J=8.8,2.2Hz,1H),3 .93(tt,J=6.2,3.6Hz,1H),3.75–3.64(m,1H),3.35–3.21(m,4H),3.10–2.95(m, 4H),2.39(p,J=3.2Hz,1H),2.02–1.88(m,2H),1.87–1.63(m,6H)ppm.MS(ESI)m / z 439.2([M+H] + ).

[0408]

[0409] Synthesis of compound 99 via synthetic route 26

[0410] Under an argon atmosphere, CAN (cerium ammonium nitrate) (766 mg, 1.40 mmol, 10.0 equiv.) was added fractionally to a MeCN-H2O (10 mL, 10:1, v / v) solution of compound 90-LB14-PMB (80 mg, 0.14 mmol, 1.0 equiv.). The reaction mixture was stirred at 0 °C for 15 min, followed by stirring at room temperature for 2 h. TLC analysis showed no remaining starting material. A saturated sodium bicarbonate aqueous solution was added, and the aqueous phase was extracted with a DCM-MeOH (5:1, v / v) mixed solvent. The combined organic phases were dried over anhydrous sodium sulfate. After filtration, solvent removal under reduced pressure, and purification by TLC, 45 mg of compound 90-LB14 was obtained, with a yield of 72% and MS (ESI) m / z 339.2 ([M+H-Boc)). + ).

[0411]

[0412] Synthetic route 27: Synthesis of compounds RCH2OTBS Acid and RCH2OTBS Amide

[0413] [Step A] The starting material represented by RCH2OH Ester (1.0 equiv.) or methyl 4-(4-(hydroxymethyl)piperidin-1-yl)benzoate was dissolved in DCM (1,2-dichloroethane) (0.2 M). Imidazole (2.0 equiv.) was added upwards to the solution at 0 °C. The resulting solution was stirred at the same temperature for 15 min, followed by the addition of TBSCl (tert-butyldimethylchlorosilane) (1.2 equiv.) in portions. The resulting reaction mixture was automatically heated to room temperature and stirred for 2 h. TLC analysis showed no remaining starting material. Saturated sodium bicarbonate aqueous solution was added, and the aqueous phase was extracted with DCM (1,2-dichloroethane). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, the compound represented by RCH2OTBS Ester was purified by silica gel column chromatography.

[0414] [Step B] The compound represented by RCH2OTBS Ester (1.0 equiv.) was dissolved in anhydrous MeOH (0.2 M). Finely ground K2CO3 (3.0 equiv.) was added to the solution in portions at 0 °C. The resulting reaction mixture was automatically heated to room temperature and stirred for 4 h. TLC analysis showed no residue. The mixture was diluted with water, and the pH was adjusted to 5–7 with 3N HCl aqueous solution. The aqueous phase was extracted with EtOAc, and the combined organic phases were dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, the mixture was further evaporated under reduced pressure at 45 °C and 0.6 mbar for 30 min to obtain a series of crude products represented by RCH2OTBS Acid, which could be used directly in the next step without further purification.

[0415] [Step C] Compounds represented by RCH2OTBS Acid (1.0 equiv.), compound 96 (1.05 equiv.), HATU (1.5 equiv.), and DIPEA (diisopropylethylamine) (3.0 equiv.) were dissolved in anhydrous DMF (0.2 M). The resulting reaction system was stirred overnight at room temperature under argon protection. When one of the starting materials was consumed by TLC, the reaction was quenched with water. The aqueous phase was extracted with EtOAc, and the combined organic phases were dried over anhydrous sodium sulfate. After filtration, solvent removal under reduced pressure, and purification by silica gel column chromatography, compounds represented by RCH2OTBS Amide were obtained.

[0416] [Step D] The compound represented by RCH2OTBS Amide (1.0 equiv.) was dissolved in anhydrous THF (0.2 M), and TBAF (tetrabutylamine fluoride) (1.0 M in THF, 1.5 equiv.) was slowly added. The resulting reaction system was stirred at room temperature for 3 h under argon protection. When the starting material was consumed by TLC, the reaction was diluted with water, the aqueous phase was extracted with EtOAc, and the combined organic phases were dried over anhydrous sodium sulfate. After filtration, solvent removal under reduced pressure, and purification by silica gel column chromatography, the compound represented by RCH2OH Amide was obtained.

[0417] [Step E] The compound represented by RCH2OHAmide (1.0 equiv.) was dissolved in anhydrous DCM (1,2-dichloroethane) (0.1 M). DMP (2.0 equiv.) was added in portions at 0 °C. The resulting reaction system was stirred at 0 °C under argon protection for 1 h. When the starting material was consumed by TLC, the reaction was diluted with water, and the reaction was quenched by adding diluted sodium thiosulfate solution and saturated sodium bicarbonate solution. The aqueous phase was extracted with DCM (1,2-dichloroethane), and the combined organic phases were dried over anhydrous sodium sulfate.

[0418] After filtration and solvent removal under reduced pressure, the compound represented by RCHOAmide was purified by silica gel rapid column chromatography.

[0419]

[0420] Following the general synthetic strategy described above, 86 mg of the target product compound 100 was synthesized and purified. 1 H NMR (400MHz, DMSO-d6) δ9.54(d,J=7.5Hz,1H),7.83(d,J=8.8Hz,1H),7.71(d,J=8.9Hz,1H),7. 68–7.61(m,2H),7.16(d,J=2.2Hz,1H),7.09–7.02(m,2H),6.95(dd,J=8.8,2.2Hz,1H),3.93(t t,J=6.2,3.6Hz,1H),3.75–3.64(m,1H),3.45(ddd,J=12.5,8.4,5.8Hz,2H),3.34(ddd,J=12.5 ,8.3,5.8Hz,2H),2.47(dp,J=7.7,5.5Hz,1H),2.02–1.63(m,12H)ppm.MS(ESI)m / z466.2([M+H] + ).

[0421]

[0422] Following the general synthetic strategy described above, 217 mg of the target product compound 101 was synthesized and purified. 1 HNMR(400MHz,DMSO-d6)δ9.54(d,J=7.5Hz,1H),7.93–7.82(m,2H),7.71(d,J=8.9Hz,1H),7.22–7.14(m,2H),6.95(dd,J=8.8,2.2Hz,1H),3.98–3.8 6(m,3H),3.83–3.72(m,1H),3.60(ddd,J=12.3,8.8,6.1Hz,2H),2.47(dp ,J=7.7,5.3Hz,1H),2.02–1.73(m,9H),1.77–1.62(m,5H)ppm.MS(ESI)m / z 468.2([M+H] + ).

[0423]

[0424] Synthetic route 28: Synthesis of compounds 102, 103, and 104

[0425] [Step A] 4-fluorobenzonitrile (1.58 g, 13.04 mmol, 1.5 equiv.), 4-(4-bromo-1H-pyrazol-1-yl)piperidine (2.00 g, 8.69 mmol, 1.0 equiv.), and finely ground K₂CO₃ (2.40 g, 17.38 mmol, 2.0 equiv.) were added to dry DMF (100 mL). The resulting reaction mixture was stirred overnight at 60 °C under an argon atmosphere. TLC monitoring showed no remaining starting material. The reaction mixture was diluted with water, and the resulting aqueous phase was extracted with DCM (1,2-dichloroethane). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, chromatographic purification yielded 2.36 g of compound 10⁻², 82% yield, MS (ESI) m / z 331.1 ([M+H)). + ).

[0426] [Step B] Compound 102 (2.20 g, 6.64 mmol, 1.0 equiv.) was added to a 50% aqueous sulfuric acid solution (1 M). The reaction system was heated under reflux for 1 h under an argon atmosphere. TLC monitoring showed no remaining reactants. After cooling, the reaction system was slowly poured into a mixture of saturated sodium bicarbonate aqueous solution and ice. The pH was carefully adjusted to 4–6 with 4N HCl aqueous solution. The aqueous phase was extracted with EtOAc, and the combined organic phases were dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, the mixture was further evaporated under reduced pressure at 45 °C and 0.6 mbar for 30 min to give 2.21 g of crude product compound 103. MS (ESI) m / z 348.0 ([MH)). - ).

[0427] [Step C] Compound 103 (500 mg, ~1.43 mmol, 1.0 equiv.), compound 96 (376 mg, ~1.50 mmol, 1.05 equiv.), HATU (830 mg, 2.14 mmol, 1.5 equiv.), and DIPEA (diisopropylethylamine) (0.75 mL, 4.29 mmol, 3.0 equiv.) were dissolved in anhydrous DMF (20 mL). The reaction system was stirred overnight at room temperature under argon protection. TLC showed no residual starting material. The reaction was quenched with water, and the aqueous phase was extracted with EtOAc. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, solvent removal under reduced pressure, and purification by chromatographic chromatography, 718 mg of compound 104 was obtained, with a yield of 86%. MS (ESI) m / z 582.1 ([M+H)). + ).

[0428]

[0429] Synthesis of compounds 105, 106, and 107 in synthetic route 29

[0430] [Step A] Following the synthetic method for compound 55b, 238 mg of compound 105 was synthesized using the starting material 4-((benzyloxy)methyl)piperidine. Compound 105 (200 mg, 0.35 mmol, 1.0 equiv.) was dissolved in anhydrous MeOH (10 mL), and Pd / C (30 mg, 10 wt% on Carbon) was added. The reaction system was then vigorously stirred for 3 h at room temperature under a hydrogen atmosphere (balloon). TLC showed no residual starting material. After filtration, solvent removal under reduced pressure, and purification by chromatographic chromatography, 65 mg of compound 106 was obtained, with a yield of 61%. MS (ESI) m / z 305.2 ([M+H)). + ).

[0431] [Step B] DMP (116 mg, 0.27 mmol, 1.3 equiv.) was added in portions to a 5 mL solution of anhydrous DCM (1,2-dichloroethane) containing compound 106 (65 mg, 0.21 mmol, 1.0 equiv.) at 0 °C. The reaction mixture was stirred for 1 h at 0 °C under argon protection. When the starting material was consumed by TLC, the reaction was diluted with water, and the reaction was quenched by adding diluted sodium thiosulfate solution and saturated sodium bicarbonate solution. The aqueous phase was extracted with DCM (1,2-dichloroethane), and the combined organic phases were dried over anhydrous sodium sulfate. After filtration, solvent removal under reduced pressure, and purification by chromatographic chromatography, 59 mg of compound 107 was obtained, with a yield of 91%. 1 HNMR(400MHz,DMSO-d6)δ11.07(s,1H),9.54(d,J=7.5Hz,1H),7.51–7.44(m,1H), 6.90(d,J=8.6Hz,1H),3.92(ddd,J=12.3,8.8,6.0Hz,2H),3.68(td,J=6.2,0.7Hz ,1H),3.60(ddd,J=12.3,8.8,6.1Hz,2H),2.59–2.48(m,2H),2.47(dt,J=7.6,5.5 Hz,1H),2.22–2.04(m,2H),1.96–1.83(m,2H),1.83–1.70(m,2H)ppm.MS(ESI)m / z 303.1([M+H] + ).

[0432]

[0433] Following the synthetic method of compound 7, 92 mg of compound 108 was synthesized from the starting material tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate. 1 H NMR (400MHz, DMSO-d6) δ11.07(s,1H),9.23–9.13(m,1H),9.02–8.92(m,1H),7.51–7.44(m,1H),6.89(d,J=8.6Hz,1H), 3.92(s,4H),3.68(td,J=6.2,0.7Hz,1H),3.23(d,J=6.8Hz,2H),2.61–2.48(m,2H),2.22–2.04(m,2H)ppm.MS(ESI)m / z 288.1([M+H] + ).

[0434]

[0435] Synthesis of compound PRTA-01 via synthetic route 30

[0436] Compounds 107 (22 mg, 0.07 mmol, 1.0 equiv.) and 98 (38 mg, 0.09 mmol, 1.2 equiv.) were dissolved in 3 mL of anhydrous DCE (1,2-dichloroethane) containing 10 v% AcOH (0.30 mL). The mixture was stirred for 15 min, then NaBH(OAc)3 (18 mg, 0.09 mmol, 1.2 equiv.) was added, and stirring continued for 3 h. When the starting material was consumed by TLC, the reaction was diluted with water, and the reaction was quenched with diluted sodium thiosulfate solution and saturated sodium bicarbonate solution. The aqueous phase was extracted with DCM (1,2-dichloroethane), and the combined organic phases were dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, 36 mg of compound PRTA-01 was obtained by preparative HPLC purification, with a yield of 68%. 1H NMR (400MHz, DMSO-d6) δ11.07(s,1H),7.83(d,J=8.8Hz,1H),7.71(d,J=8.9Hz,1H),7.68–7. 62(m,2H),7.51–7.44(m,1H),7.16(d,J=2.2Hz,1H),7.09–7.02(m,2H),6.98–6.86(m,2H),4. 02–3.89(m,3H),3.82(ddd,J=12.4,8.1,6.9Hz,2H),3.75–3.65(m,2H),3.21(ddd,J=7.5,5.7 ,3.0Hz,5H),2.65–2.45(m,9H),2.22–2.04(m,2H),2.02–1.63(m,16H)ppm.UPLC-MS(ESI)m / z 725.3,t R 0.831 min, 97% purity.

[0437]

[0438] Synthetic route 31 Synthesis of compound PRTA-02

[0439] Compounds 7 (35 mg, 0.11 mmol, 1.0 equiv.) and 101 (63 mg, 0.13 mmol, 1.2 equiv.) were dissolved in 3 mL of anhydrous DCE containing 10 v% AcOH (0.30 mL) at room temperature. The mixture was stirred for 15 min, then NaBH(OAc)3 (31 mg, 0.15 mmol, 1.3 equiv.) was added, and stirring continued for 2 h. When the starting material was consumed by TLC, the reaction was diluted with water, and the reaction was quenched with diluted sodium thiosulfate solution and saturated sodium bicarbonate solution. The aqueous phase was extracted with DCM (1,2-dichloroethane), and the combined organic phases were dried over anhydrous sodium sulfate. After filtration, solvent removal under reduced pressure, and purification by preparative HPLC, 51 mg of compound PRTA-02 was obtained, with a yield of 62%. 1H NMR (400MHz, DMSO-d6) δ11.07(s,1H),7.93–7.82(m,2H),7.71(d,J=8.8Hz,1H),7.47(dd,J=8.5,0.6Hz,1H),7.23 –7.14(m,2H),6.98–6.88(m,2H),4.02–3.92(m,2H),3.96–3.89(m,1H),3.88–3.77(m,2H),3.81–3.72(m,1H),3.6 8(td,J=6.2,0.6Hz,1H),3.65–3.51(m,4H),2.73–2.64(m,2H),2.67–2.61(m,2H),2.64–2.57(m,1H),2.53(ddd,J =8.2,7.4,0.9Hz,2H),2.49(dd,J=11.5,4.9Hz,1H),2.22–2.04(m,2H),2.02–1.62(m,13H)ppm.UPLC-MS(ESI)m / z 727.3,t R 0.685 min, 98% purity.

[0440]

[0441] Synthesis of compound PRTA-03 via synthetic route 32

[0442] The protein degrader PRTA-03 was synthesized and purified from compounds 101 and 40 using the same method as PRTA-02 to obtain 44 mg of the target product. 1 HNMR(400MHz,DMSO-d6)δ10.64(s,1H),7.93–7.82(m,2H),7.71(d,J=8.8Hz,1H),7.23–7.14(m, 2H),7.06(d,J=8.0Hz,1H),7.00(d,J=8.0Hz,1H),6.95(dd,J=8.8,2.2Hz,1H),4.02–3.95(m,1H ),3.99–3.89(m,4H),3.84(d,J=7.8Hz,1H),3.84–3.78(m,1H),3.82–3.72(m,1H),3.66–3.51(m ,4H),2.75–2.57(m,7H),2.49(dd,J=11.4,4.9Hz,1H),2.02–1.62(m,13H)ppm.UPLC-MS(ESI)m / z 728.3,t R 0.602 min, 97% purity.

[0443]

[0444] Synthesis of compound PRTA-04 via synthetic route 33

[0445] The protein degrader PRTA-04 was synthesized and purified from compounds 101 and 9 using the same method as PRTA-02 to obtain 53 mg of the target product. 1 HNMR(400MHz,DMSO-d6)δ11.13(s,1H),7.93–7.82(m,2H),7.71(d,J=8.8Hz,1H),7.23–7.14 (m,2H),6.95(dd,J=8.8,2.3Hz,1H),6.67(d,J=0.6Hz,1H),4.11(t,J=6.1Hz,1H),4.02–3.9 2(m,2H),3.96–3.89(m,1H),3.88–3.77(m,2H),3.81–3.71(m,1H),3.67–3.57(m,2H),3.61– 3.52(m,2H),2.73–2.45(m,8H),2.23–2.04(m,2H),2.02–1.62(m,14H)ppm.UPLC-MS(ESI)m / z 741.3,t R 0.698 min, 96% purity.

[0446]

[0447] Synthetic route 34 Synthesis of compound PRTA-05

[0448] The protein degrader PRTA-05 was synthesized and purified from compounds 101 and 108 using the same method as PRTA-01 to obtain 34 mg of the target product. 1H NMR (400MHz, DMSO-d6) δ11.07(s,1H),7.83(d,J=8.8Hz,1H),7.71(d,J=8.9Hz,1H),7.68–7.61(m,2H),7.51–7.44(m,1H ),7.16(d,J=2.2Hz,1H),7.09–7.02(m,2H),6.95(dd,J=8.8,2.2Hz,1H),6.89(d,J=8.6Hz,1H),3.98–3.90(m,1H),3.93( s,2H),3.87(s,2H),3.75–3.64(m,2H),3.42(ddd,J=12.3,8.3,6.1Hz,2H),3.09(ddd,J=12.3,8.2,6.2Hz,2H),2.60(dd ,J=11.2,3.7Hz,1H),2.59–2.48(m,3H),2.22–2.04(m,2H),2.02–1.88(m,2H),1.88–1.63(m,11H)ppm.UPLC-MS(ESI)m / z 737.3,t R 0.672 min, 98% purity.

[0449]

[0450] Synthetic route 35 Synthesis of compound PRTA-06

[0451] [Step A] In a round-bottom flask, add Pd(PPh3)2Cl2 (8 mg, 0.01 mmol, 0.1 equiv.) and CuI (2 mg, 0.13 mmol, 0.12 equiv.). After purging with argon three times, add dry DMF (1.0 mL), compound 104 (63 mg, 0.11 mmol, 1.0 equiv.), compound 87 (54 mg, 0.16 mmol, 1.5 equiv.), and Et3N (1.0 mL). After purging with argon again, stir the reaction mixture overnight at 70 °C under an argon atmosphere. TLC showed no remaining starting material. Dilute the reaction mixture with water, extract the aqueous phase with DCM (1,2-dichloroethane), and dry the combined organic phases with anhydrous sodium sulfate. After filtration, solvent removal under reduced pressure, and purification by TLC, 62 mg of compound 109 was obtained, yielding 68%. MS (ESI) m / z 837.3 ([M+H-Boc)). + ).

[0452] [Step B] Under an argon atmosphere, at 0°C, CAN (cerium ammonium nitrate) (192 mg, 0.35 mmol, 5.0 equiv.) was added in portions to a MeCN-H2O (5 mL, 10:1, v / v) solution of compound 109 (62 mg, 0.07 mmol, 1.0 equiv.). The reaction mixture was stirred at 0°C for 10 min, followed by stirring at room temperature for 1 h. TLC analysis showed no remaining starting material. A saturated sodium bicarbonate aqueous solution was added, and the aqueous phase was extracted with a DCM-MeOH (5:1, v / v) mixed solvent. The combined organic phases were dried over anhydrous sodium sulfate. After filtration and solvent removal under reduced pressure, 21 mg of compound PRTA-06 was obtained by preparative HPLC, with a yield of 40%. 1 H NMR (400MHz, DMSO-d6) δ11.07(s,1H),7.87(d,J=1.8Hz,1H),7.83(d,J=8.8Hz,1H) ,7.74–7.65(m,3H),7.68–7.61(m,3H),7.16(d,J=2.2Hz,1H),7.09–7.02(m,2H),6 .95(dd,J=8.8,2.2Hz,1H),4.47(pd,J=3.1,0.7Hz,1H),3.93(tt,J=6.2,3.6Hz,1H ),3.75–3.56(m,6H),2.59–2.48(m,2H),2.22–1.63(m,15H)ppm.UPLC-MS(ESI)m / z 717.3,t R 0.713 min, 98% purity.

[0453] Example 5: Binding activity of the compound to the E3 ubiquitin ligase CRBN

[0454] The binding activity of the compound disclosed in this patent to the E3 ubiquitin ligase CRBN is determined by the competitive binding of the compound to the CRBN protein by thalidomide labeled with XL665, thereby preventing fluorescence resonance energy transfer (FRET).

[0455] CRBN binding activity assay kit (CEREBLON BINDING KITS (PE, 64BDCRBNPEG)).

[0456] The detection is based on homogeneous time-resolved fluorescence (HTRF) technology. When the donor and acceptor are close, the donor can transfer energy to the acceptor, exciting it and causing it to emit 665nm emission light.

[0457] The donor used in this kit is a europium-labeled GST antibody (GST Eu Cryptate Antibody), and the receptor is thalidomide-redreagent labeled with XL665. The donor binds to the GST-tagged CRBN protein. The compound disclosed in this patent competitively binds to the CRBN protein, and the binding activity of the compound is determined by the fluorescence value emitted by the receptor at 665 nm. The stronger the binding affinity of the compound, the weaker the signal.

[0458] The donor and receptor were diluted 50-fold using the PROTAC binding buffer 1 provided in the kit. The CRBN protein was diluted 45-fold. The 8 mM compound solution disclosed in this patent was diluted 10-fold using the 1X diluent provided in the kit, and then serially diluted 5-fold, for a total of 7 dilutions.

[0459] Add 5 μl of the compound, 5 μl of diluted CRBN protein, and 10 μl of donor-receptor mixture to the wells of a white 384-well plate (PE, Part number: 6008280). Incubate at room temperature for 3 hours.

[0460] Calculate the ratio of the acceptor to the donor emitted signal for each individual pore.

[0461] Ratio = (665nm signal value / 620nm signal value) * 10 4

[0462] Calculate the coefficient of variation (CV) as follows: (Standard deviation (SD) / Ratio) * 100

[0463] Using GraphPad Prism, calculate IC based on compound concentration, ratio, and coefficient of variation. 50 value.

[0464] Table 10 shows the binding activities of compounds 1-18 to the E3 ubiquitin ligase CRBN.

[0465]

[0466]

[0467]

[0468] Table 11 shows the binding activities of compounds 19-34 to the E3 ubiquitin ligase CRBN.

[0469]

[0470]

[0471] Table 12 shows the binding activities of compounds 35-43 to the E3 ubiquitin ligase CRBN.

[0472]

[0473]

[0474] Example 6: Evaluation of the effect of protein degrading compounds on the reduction of expression levels of bromine domain proteins (BRD).

[0475] In this experiment, human chronic myeloid monocytic leukemia MV-4-11 cells were cultured in IMDM medium containing 10% fetal bovine serum (Gibco, USA) at 37°C, 5% CO2, and 95% humidity. When the cell density reached 80%, a medium containing the compound was used to seed the cells into 6-well plates at a density of 1 x 102. 6 / ml, to achieve final compound concentrations of 0.1, 1, 10, and 20 μM. After 24 hours of treatment, cells were collected, washed twice with PBS, and RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors was added to the cells. After lysis and centrifugation, total protein extract was obtained, and the protein concentration in the extract was determined by the BCA method.

[0476] Protein electrophoresis was performed using SDS-PAGE, followed by constant current transfer at 200 mA for 150 min to transfer proteins onto PVDF membranes. The PVDF membranes were then placed in 5% skim milk and blocked at room temperature for 1 h. BRD4 (#13440, CST) (1:1000) and C-MYC (ab32072, 1:1000) were added, and the membranes were incubated overnight at 4°C. The membranes were washed three times with TBST for 10 min each time, then incubated with secondary antibody (1:5000, absin) at room temperature for 60 min. The membranes were washed three times with TBST for 10 min each time, and ECL chemiluminescence buffer was added for exposure. GAPDH protein was used as an internal control for each sample. Protein profiles were analyzed using ImageJ software for grayscale value analysis. The formula used was: Grayscale correction value = (Target protein grayscale value / Corresponding internal control grayscale value > 10). 3 The grayscale correction value for each sample was calculated. This value was then compared with the grayscale correction value of the control group to calculate the degradation rate. Finally, the DC of the compound was obtained by nonlinear curve fitting using Prism with logarithmic concentration-inhibition rate. 50 and D max value.

[0477] Table 13 summarizes the evaluation of the effects of protein degraders PRTB-01-16 on reducing BRD protein expression. Among them, DC... 50 The protein degrader concentration at which the BRD protein is induced to degrade to 50% level is represented by a level grade A (DC). 50<1μM), B(DC) 50 1~10μM), C(DC) 50 >10μM). Where D max The percentage of total BRD protein that can be induced by the protein degrading agent is categorized as A(D). max >85%), B(D) max 85-50%), C(DC) 50 <50%

[0478] Table 13 Evaluation of the effect of protein degraders PRTB-01-16 on reducing BRD protein expression.

[0479]

[0480]

[0481]

[0482]

[0483] Example 7: Evaluation of the effect of protein-degrading compounds on the reduction of androgen receptor (AR) expression.

[0484] In this experiment, the human prostate cancer cell line LNCaP was cultured in RPMI-1640 medium containing 10% fetal bovine serum (Gibco, USA) at 37°C, 5% CO2, and 95% humidity. When the cell density reached 80%, the cells were seeded into 6-well plates at 5 × 10⁶ cells per well. 5 Cells were cultured for 24 hours, and the culture medium was replaced with one containing the compound to achieve final compound concentrations of 0.01, 0.1, 1, and 10 μM. After 24 hours of treatment, the supernatant was removed, and the cells were washed twice with PBS. RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors was added to the cells. After lysis and centrifugation, the total protein extract was obtained, and the protein concentration in the extract was determined by the BCA method.

[0485] Protein electrophoresis was performed using SDS-PAGE, followed by electroporation at a constant current of 200 mA for 90 min to transfer the protein onto a PVDF membrane. The PVDF membrane was placed in a solution of 5% skim milk and blocked at room temperature for 1 h. Anti-Androgen Receptor antibody [EPR1535(2)](ab133273) (1:10000) was added and incubated overnight at 4 °C. The membrane was washed three times with TBST for 10 min each time. Secondary antibody (1:5000, absin) was added and incubated at room temperature for 60 min. The membrane was washed three times with TBST for 10 min each time. ECL luminescent solution was added and the membrane was exposed. Each sample was simultaneously tested with α-tubulin protein as an internal control. The protein map was analyzed by grayscale value using ImageJ software. The formula used was: Grayscale correction value = (target protein grayscale value / corresponding internal control grayscale value > 10). 3 The grayscale correction value for each sample was calculated. This value was then compared with the grayscale correction value of the control group to calculate the degradation rate. Finally, the DC of the compound was obtained by nonlinear curve fitting using Prism with logarithmic concentration-inhibition rate. 50 and D max value.

[0486] Table 14 summarizes the evaluation of the effects of protein degraders PRTA-01 to 06 on reducing AR protein expression. Among them, DC... 50 The protein degrader concentration at which AR protein is induced to degrade to 50% level is represented by level A (DC). 50 <1μM), B(DC) 50 1~10μM), C(DC) 50 >10μM). Where D max The percentage of total AR protein that can be induced by the protein degrading agent to the maximum extent of BRD protein degradation is graded as A(D). max >85%), B(D) max 85-50%), C(D) max <50%).

[0487] Table 14 Evaluation of the effect of protein degraders PRTA-01-06 on reducing AR protein expression levels

[0488]

Claims

1. A protein degrading agent or a pharmaceutically acceptable salt thereof, characterized by, The specific structure of the protein degrading agent is selected from any of the following: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 2. A pharmaceutical composition for targeting the degradation of a protein, said pharmaceutical composition comprising a combination of the protein degrading agent of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent or carrier.

3. A regulator for modulating transcriptional regulatory factors, said regulator comprising the protein degrader of claim 1 or a pharmaceutically acceptable salt thereof.

4. The use of the protein degrading agent as described in claim 1 in the preparation of a medicament for treating or preventing the pathological condition or symptoms of a disease caused by BRD protein expression; wherein the specific structure of the protein degrading agent is selected from any of the following: 、 、 、 、 、 、 、 、 、 、 or .

5. The use as described in claim 4, wherein the diseases include chronic myeloid leukemia, acute myeloid leukemia, T-cell acute lymphoblastic leukemia, Alzheimer's disease, gout, autoimmune diseases, acne, synovial sarcoma, lung cancer, multiple myeloma, lymphoma, tumor metastasis, cervical cancer, neuroblastoma, hepatocellular carcinoma, colorectal cancer, pancreatic cancer, malignant rhabdomyosarcoma, and oral squamous cell carcinoma.

6. The use of the protein degrading agent as described in claim 1 in the preparation of a medicament for treating or preventing the pathological conditions or symptoms of diseases caused by AR protein expression; wherein the specific structure of the protein degrading agent is selected from any of the following: 、 、 、 or .

7. The use as described in claim 6, wherein the disease includes prostate cancer, breast cancer, androgenetic alopecia, and acne.

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