A compound and uses thereof

Abstract

A CRBN small molecule ligand compound based on a pyridazine-glutarimide core backbone (PDG), its composition, and its application; and a protein degrading agent based on a CRBN small molecule ligand compound based on a pyridazine-glutarimide core backbone (PDG) and its application. Specifically, the CRBN small molecule ligand compound is shown in (I). Wherein, R1 and R2 are selected from one or more of the following: pyridazine core-attached fused rings, H, alkyl, OR, F, CN, CF3, NR2, Ph, 4-Py; Y is selected from one or more of CH, CD, CF, and N; R3 is selected from one or more of OR, NR2, CHR2, and C≡CR; R4 is selected from H or more; R4 is selected from H or alkyl.

Description

A compound and its use Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry technology, and specifically relates to a CRBN small molecule ligand compound based on a pyridazine-glutarimide core skeleton (PDG), a composition thereof, and applications thereof, and also relates to a protein degrader of a CRBN small molecule ligand compound based on a pyridazine-glutarimide core skeleton (PDG), and applications thereof. Background Art

[0002] Cereblon (CRBN) is a substrate receptor protein of the CRL4CRBN isoform E3 ubiquitin ligase complex. Together with the DDB1 (Damage-specific DNA Binding protein 1), the zinc-finger domain protein RBX1, and the Cullin4 (Cul4) scaffold protein, it 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, leading to their degradation via the ubiquitin-proteasome pathway. This in turn affects various physiological activities of the cell, including energy metabolism, membrane potential regulation, and transcription factor degradation.

[0003] In 2010, Ito et al. first demonstrated that thalidomide's direct target is CRBN, a substrate receptor protein for an E3 ubiquitin ligase. Following increasing evidence that immunomodulators exert their anti-tumor effects by targeting CRBN, three articles in the same issue of Science in 2014 [34-36] demonstrated at the molecular level that immunomodulators exert their anti-multiple myeloma effects through a protein ubiquitination and degradation mechanism. The CRL4CRBN (CUL4-Roc1-DDB1-CRBN) E3 ubiquitin ligase is composed of Cullin-4A (Cul4A), regulator of cullins 1 (Roc1), damaged DNA binding protein 1 (DDB1), and CRBN (cereblon). Cul4A has a scaffolding function, while Roc1 has a ring finger domain associated with an E2 ubiquitin-conjugating enzyme. CRBN acts as a substrate receptor, directly binding to specific substrates and mediating their ubiquitination. This ubiquitin ligase marks specific proteins with ubiquitin (Ub). Ubiquitin-tagged proteins are recognized by the proteasome as damaged or defective proteins, which are then hydrolyzed. The interaction of immunomodulators with this E3 ubiquitin ligase activates the activity of the E3 ubiquitin ligase, which is the basis for the cytotoxic and immunomodulatory effects of immunomodulators.

[0004] The ubiquitination-proteasome degradation pathway of proteins is a common and important way of endogenous protein degradation. This pathway requires several consecutive processes to be implemented: 1. ATP provides energy to covalently bind the C-terminus of ubiquitin to the cysteine ​​residue of E1 ubiquitin activating enzyme; 2. E1 ubiquitin activating enzyme transfers ubiquitin to E2 ubiquitin conjugating enzyme, which can transfer ubiquitin to the -NH2 of lysine residues of target proteins; 3. E3 ubiquitin ligase recruits substrate proteins and catalyzes the binding of ubiquitin on E2 ubiquitin conjugating enzyme to substrate proteins; 4. 26S proteasome specifically recognizes substrate proteins marked with ubiquitin and hydrolyzes them.

[0005] After immunomodulators such as thalidomide bind to CRBN, they activate the activity of E3 ubiquitin ligase, prompting E3 ubiquitin ligase to recruit substrate protein IKZF1 / IKZF3 (Ikaros / Aiolos is a zinc finger transcription factor that plays an important role in blood cell differentiation). Then, E3 ubiquitin ligase, substrate protein IKZF1 / IKZF3 and immunomodulator will form a stable ternary complex, thereby transferring ubiquitin to the substrate protein IKZF1 / IKZF3, resulting in the degradation of IKZF1 / IKZF3 by the 26S proteasome. IKZF1 and IKZF3 are essential transcriptional regulatory factors for the proliferation and development of B cells and T cells [John LB, Ward A C. 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 effect on 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 T cell immune response and inhibiting the function of B cells, achieving tumor killing and inhibiting tumor proliferation. Simultaneously, 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 structure of thalidomide, lenalidomide, and pomalidomide, a class of immunomodulators, have emerged.

[0006] PROTAC, Proteolysis Targeting Chimera, protein degradation targeting chimera, is a bifunctional molecule that consists of an E3 ligand structural unit that can bind to a specific E3 ubiquitin ligase, a target protein ligand structural unit that can bind to the target protein, and a linker connecting the two ligands. PROTAC can recruit a specific E3 ubiquitin ligase, bind to a specific target protein to form a [E3 ubiquitin ligase-PROTAC-target protein] ternary complex, induce the target protein to be polyubiquitinated by the recruited E3 ubiquitin ligase, and then cause the degradation of the target protein by the ubiquitin-proteasome pathway.

[0007] Currently, more than 100 proteins have been successfully degraded by PROTACs. 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. Even the transcription factor regulatory protein pirin, epigenetic-related protein PCAF / GCN5, KRAS-G12C, etc., which are considered "undruggable targets", are included.

[0008] In the development of PROTACs, CRBN ligand is the most widely used E3 ubiquitin ligase ligand. By selecting different target protein ligands and CRBN ligands, the designed PROTACs molecules can achieve different effects. However, in reality, there are too few CRBN ligands to choose from, which cannot meet the needs of PROTACs development. In view of the problems of immunomodulator resistance, too few CRBN ligands and lack of structural diversity, the widely used amine CRBN ligands have poor chemical stability, easy to undergo diastereoisomerization in the body, a large number of hydrogen bond donors and acceptors, poor solubility, and poor binding activity, there is an urgent need to develop more CRBN ligands to solve the above problems.

[0009] Summary of the Invention

[0010] In a first aspect, the present invention provides a compound that can bind to CRBN protein, wherein the compound is shown as (I):

[0011] Among them, R 1 and R 2One or more selected from the group consisting of a pyridazine parent nucleus and a ring, 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, in the compound, R 1 and R 2 They may be selected from alkyl groups at the same time.

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

[0015] Furthermore, when R 1 When H, R 2 One or more selected from alkyl, OR, F, CN, CF3, NR2, Ph, 4-Py.

[0016] Furthermore, when R 2 When H, R 1 One or more selected from alkyl, OR, F, CN, CF3, NR2, Ph, 4-Py.

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

[0018] R 3 One or more selected from OR, NR2, CHR2 and C≡CR;

[0019] R 4 is selected from H or alkyl.

[0020] In a second aspect, 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 prepared from the parent compound by addition of a non-toxic acid or base.

[0022] In a third aspect, the present invention provides a protein degrader, which comprises: a target protein ligand (POI ligand) + a linker (Linker) + a CRBN protein ligand; the CRBN protein ligand is the compound described in the first aspect of the present invention, and the structural formula of the protein degrader is (II):

[0023] L of the Linker substructure in Formula II 1 Can be connected to R 2 Connected C, at this time R 2 That is L 1 ; R 3 One or more selected from OR, NR2, CHR2 and C≡CR;

[0024] L of the Linker substructure in Formula II 1 Can be connected to R 3 Connected C, at this time R 3 That is L 1 , R 2 One or more selected from the group consisting of a pyridazine parent nucleus and a ring, H, alkyl, OR, F, CN, CF3, NR2, Ph, and 4-Py.

[0025] In Formula II, R 1 One or more selected from the group consisting of a pyridazine parent nucleus and a ring, H, alkyl, OR, F, CN, CF3, NR2, Ph, and 4-Py.

[0026] R 4 is selected from H or alkyl.

[0027] Y is selected from one or more of CH, CD, CF, CCH3 and N.

[0028] The substructure L of Linker in Formula II 1 、L 3 and L 5 You can choose one or more of the following:

[0029] Among them, R a One or more selected from H, alkyl, SO2(alyl) or SO2(aryl).

[0030] The substructure L of Linker in Formula II 2 、L 4 You can choose one of the following:

[0031] Furthermore, the target protein ligand is a ligand of the target protein that causes related diseases.

[0032] Furthermore, the diseases 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, androgenic 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.

[0033] Furthermore, the target protein ligand, i.e., the POI ligand in Formula II, can be selected from:

[0034] One or more of .

[0035] In a fourth aspect, the present invention provides a pharmaceutical composition comprising a combination of the compound of formula (I) or a pharmaceutically acceptable salt thereof according to the first aspect and a pharmaceutically acceptable diluent or carrier.

[0036] Furthermore, the pharmaceutical composition may also comprise a combination of an enantiomer, diastereomer, stereoisomer or pharmaceutically acceptable salt of the compound of formula (I) and a pharmaceutically acceptable diluent or carrier.

[0037] In a fifth aspect, the present invention provides a pharmaceutical composition for targeted protein degradation, comprising a combination of the protein degrader (II) described in the second aspect of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent or carrier.

[0038] Furthermore, the pharmaceutical composition for targeted protein degradation may also comprise a combination of an enantiomer, diastereomer, stereoisomer or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent or carrier.

[0039] In a sixth aspect, the present invention provides a regulator for regulating a transcriptional regulatory factor, wherein the regulator contains a compound of formula (I) or a pharmaceutically acceptable salt thereof or a protein degrader (II) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof.

[0040] Furthermore, the transcriptional regulatory factor is a regulatory factor necessary for the proliferation and development of B cells and T cells.

[0041] Furthermore, the regulation may be positive regulation and / or negative regulation.

[0042] Furthermore, the transcription factors include but are not limited to IRF4 and Myc.

[0043] In a seventh aspect, the present invention provides a method for treating or preventing pathological conditions or symptoms of diseases 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.

[0044] Furthermore, the pathological conditions or symptoms include, but are not limited to, bone marrow failure, anemia, immune paralysis and infection, fractures and bone pain, high calcium levels and renal failure. DETAILED DESCRIPTION

[0045] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below may be combined with each other as long as they do not conflict with each other.

[0046] The present invention is described below by means of 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 in the scope of protection of the present invention.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0048] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0049] The compound of the CRBN protein of the present invention is shown as (I):

[0050] If it contains a fatty amine, the structure represented includes but is not limited to its free amine form, hydrochloride form, and trifluoroacetate form;

[0051] In some embodiments, R 1 One of the following structures:

[0052] R 2 When it is one of the following structures:

[0053] Then R 3 Select any one of the following structures:

[0054] R 2 When it is one of the following structures:

[0055] R 3 Any of the following structures:

[0056] Y is one of the following structures: CCH3, CD, CF, N;

[0057] R 4 It is one of the following structures: H, CH3, Boc, Cbz, Ph.

[0058] In the above structure, if a fatty amine is contained, the structure represented includes but is not limited to its free amine form, hydrochloride form, and trifluoroacetate form;

[0059] The AB ring is one of the following structures:

[0060] R 1 One of the following structures:

[0061] Y is one of the following structures: CCH3, CD, CF, N; R 4 It is one of the following structures: H, CH3, Boc, Cbz, Ph.

[0062] Table 1 Structures of exemplary compounds that bind to CRBN small molecules

[0063] Table 2 Exemplary compound structures of PROTAC protein degraders

[0064] As used herein, the term "alkyl" includes both branched and straight-chain saturated aliphatic hydrocarbon groups and has a specified number of carbon atoms, generally 1 to about 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.

[0065] The term "stereoisomer" as used herein refers to compounds that have identical chemical constitution but differ with regard to the arrangement of atoms or groups in space, and includes "diastereomers" and "enantiomers".

[0066] As used herein, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral characteristics, and reactivity. Mixtures of diastereomers can be separated by high-resolution analytical procedures such as electrophoresis and crystallization in the presence of a resolving agent or chromatographic method, such as using a chiral HPLC column.

[0067] The term "enantiomers" as used herein refers to two stereoisomers of a compound that are non-superimposable mirror images of each other. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur in chemical reactions or processes without stereoselectivity or stereospecificity.

[0068] As used herein, the terms "pharmaceutically acceptable salts" and "salts of compounds" are interchangeable and refer to derivatives of the disclosed compounds wherein the parent compound is formed by the preparation of non-toxic acid or base addition salts; examples of pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition 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, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates, and arylsulfonates, as appropriate.

[0069] Example

[0070] Example 1 Synthesis of Compound 1

[0071] [Step A] In a round-bottom flask, the raw materials 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%) were added successively, and 50 mL of a mixed solution of isopropanol and water (2:1, v / v) was injected and stirred. The reaction system was stirred at 80°C (oil bath) under argon atmosphere for 9 h. TLC confirmed the absence of residual starting material. After cooling the reaction system to room temperature, the product was diluted with 40 mL of water and extracted with EtOAc. The combined organic phases were washed with saturated brine and dried. The solvent was removed by filtration and purified by chromatography to afford 2.68 g of compound 44 in a 68% yield. MS (ESI) m / z 412.2 was obtained.

[0072] [Step B] To a round-bottom flask, compound 44 (1.00 g, 2.43 mmol) was added, along with 20 mL of anhydrous ethanol and 200 mg of Pd / C (20 wt%, 10% on carbon). The mixture was stirred under a hydrogen balloon for 8 h. TLC analysis revealed no residual starting material, and LC-MS analysis revealed no unreacted intermediate. The reaction mixture was filtered and dried under reduced pressure to afford 612 mg of crude compound 45, which was used in the next step without purification. MS (ESI) revealed m / z 234.1.

[0073] [Step C] To a solution of crude product 45 (612 mg, ~2.62 mmol, ~1.0 equiv.) in acetone (15 mL) were added PMBCl (360 μL, 2.62 mmol, 1.0 equiv.), KCO (724 mg, 5.24 mmol, 2.0 equiv.), and TBAI (193 mg, 0.52 mmol, 0.2 equiv.), respectively. The reaction mixture was stirred for 12 h. TLC confirmed the absence of residual starting material. The mixture was diluted with 20 mL of water and extracted with EtOAc. The combined organic phases were washed with saturated brine and dried. The solvent was removed by filtration and purified by chromatography to afford 515 mg of compound 46 in a two-step yield of 60%. MS (ESI) m / z 354.1.

[0074] [Step D] To a solution of lithium diisopropylamide (LDA) in anhydrous THF (tetrahydrofuran) (~0.68 mmol, 7.0 mL, ~1.2 equiv.) at -78°C under argon was added trimethylsilyldiazomethane (360 μL, 1.90 Min hexanes, 0.68 mmol, 1.2 equiv.) dropwise. The reaction was stirred at the same temperature for 30 min. Compound 46 (200 mg, 0.57 mmol, 1.0 equiv.) in anhydrous THF (tetrahydrofuran) (5 mL) was then added dropwise. The reaction was stirred at the same temperature for 1 h, then warmed to room temperature and heated to reflux for 3 h. TLC analysis indicated no residual starting material. After cooling the reaction to room temperature, the mixture was diluted with 10 mL of ice water and extracted with EtOAc. The combined organic phases were washed with saturated brine and dried. After filtration and removal of the solvent under reduced pressure, the product was purified by chromatography to give 103 mg of compound 47 with a yield of 52%. MS (ESI) m / z was 350.1.

[0075] [Step E] To a solution of compound 47 (60 mg, 0.17 mmol, 1.0 equiv.) in MeCN-H₂O (20 mL, 10:1, v / v) at 0°C under argon was added CAN (ceric ammonium nitrate) (941 mg, 1.72 mmol, 10.0 equiv.) portionwise. The reaction mixture was stirred at 0°C for 15 min and then at room temperature for 2 h. TLC analysis indicated no residual starting material. Saturated aqueous sodium bicarbonate (10 mL) was added, and the mixture was extracted with EtOAc. The combined organic phases were washed with saturated brine and dried. Filtration, removal of the solvent under reduced pressure, and purification by chromatography afforded 34 mg of compound 1 in 85% yield (see Synthetic Scheme 1).

[0076] UPLC-MS (ESI) m / z 230.1, t R 4.516min. 1 H NMR(400MHz,Chloroform-d)δ7.69(dd,J=

[0077] 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.

[0078] Synthesis route 1 Synthesis route of compound 1

[0079] Example 2 Preparation of Compounds 2-43

[0080] [Step A]:

[0081] Negishi reaction (Rt.2):

[0082] Celite (10 wt% of Zn) was added to a round-bottom flask and heated with a heat gun (400°C) under high vacuum for 15 min. After cooling to room temperature, zinc powder (200 mesh, 2.0 equiv.) was added, followed by the addition of dry DMA (N,N-dimethylacetamide) (1 M) and 1,2-dibromoethane (0.12 equiv.). The reaction system was heated at 70°C for 15 min, cooled to room temperature, and trimethylsilyl chloride (TMSCl) (0.12 equiv.) was added dropwise. The mixture was stirred at room temperature for 1 h, followed by the addition of a dry DMA (0.4 M) solution of tert-butyl3-iodoazetidine-1-carboxylate (1-Boc-3-iodoazetidine) (1.5 equiv.) while maintaining the reaction temperature below 40°C. The reaction system was stirred at 40°C for 2 h after the addition. After cooling to room temperature and allowing to stand for 30 minutes, 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, a 0.4 M solution of 3,6-dichloropyridazine (1.0 equiv.) in dry DMA was added. The reaction mixture was stirred at 70°C for 2 hours. TLC confirmed the absence of residual starting material. After cooling to room temperature, the reaction was quenched by adding ammonium chloride solution and extracted with EtOAc. The combined organic phases were washed with brine and dried. Filtration, the solvent was removed under reduced pressure, and the Negishi coupling product was purified by silica gel flash column chromatography.

[0083] Suzuki reaction (Rt.3):

[0084] A round-bottom flask was charged with 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%). The mixture was purged with argon and then charged with dry DME (0.2 M) and pre-deoxygenated aqueous sodium bicarbonate (50% by volume of DME). The reaction system was stirred at 80°C for 8 h. TLC confirmed the absence of residual starting material. The mixture was cooled to room temperature, diluted with water, extracted with EtOAc (3×), and the combined organic phases were washed with saturated brine and dried. The solvent was removed by filtration and then purified by silica gel flash column chromatography to obtain the Suzuki coupling product.

[0085] [Step B](Rt.2~6)

[0086] A round-bottom flask was charged with a 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%). The atmosphere was replaced with argon, and the flask was tightly capped 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 system was stirred at 80°C under an argon atmosphere for 9 h. TLC confirmed the absence of residual starting material. After cooling the reaction system to room temperature, it was diluted with water and extracted with EtOAc. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was removed by filtration and then purified by silica gel flash column chromatography to obtain the Suzuki coupling product.

[0087] [Step C](Rt.2~6)

[0088] A bis-benzyl-protected pyridinedione derivative was dissolved in MeOH-THF (0.1M, 1:1, v / v), and 10% Pd / C (20 wt%) was added. After argon replacement, the atmosphere was replaced with hydrogen while stirring. The reaction system was vigorously stirred at room temperature for 8-16 hours. TLC analysis indicated no residual starting material, and LC-MS analysis indicated no unreacted intermediate. The reaction solution was filtered through a silica gel cake, the solvent was removed under reduced pressure, and then purified by silica gel flash column chromatography to obtain the glutarimide derivative.

[0089] [Step D](Rt.2, 3, 5)

[0090] The glutarimide derivative obtained in the previous step was dissolved in DCM (1,2-dichloroethane) (0.2 M). HCl (4.0 M, ~6 equiv.) in dioxane was slowly added dropwise at 0°C. The reaction system was stirred at room temperature for 2 hours. TLC confirmed the absence of residual starting material. An equal volume of diethyl ether was added to the reaction solution, and the mixture was stirred for 0.5 hours. The resulting solid was filtered and washed with PE. Residual solvent was removed under vacuum to obtain the secondary amine hydrochloride as a white solid.

[0091] Synthesis Route 2 Synthesis Route of Compounds 2 to 8

[0092] According to the synthetic route of synthetic route Rt.2, 3,6-dichloropyridazine (200 mg, 1.34 mmol) was used as the equivalent standard raw material in the Step A-Negishi reaction to obtain 268 mg of Negishi coupling product 48 with a yield of 74%. MS (ESI) m / z 214.0 ([M+H–t-Bu] + ).

[0093] In Step B, compound 48 (80 mg, 0.30 mmol) was used as the equivalent standard raw material to obtain 103 mg of Suzuki coupling product 49 with a yield of 66%. MS (ESI) m / z 469.2 ([M+H–t-Bu] + ).

[0094] In Step C, compound 49 (100 mg, 0.19 mmol) was used as the equivalent standard raw material to obtain 31 mg of product 50 with a yield of 47%. MS (ESI) m / z 291.1 ([M+H–t-Bu] + ).

[0095] In Step D, compound 50 (31 mg, 0.09 mmol) was used as the equivalent standard raw material to obtain 23 mg of the target product 2 with a yield of 92%.

[0096] UPLC-MS (ESI) m / z 247.1, t R 0.972min. 1 H NMR(400MHz,D2O)δ7.67(dd,J=8.3,0.6

[0097] 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.

[0098] Following the above operation and the synthetic route of Rt.3, 3,6-dichloropyridazine (200 mg, 1.34 mmol) was used as the equivalent standard raw material in the Step A-Suzuki reaction to obtain 301 mg of Suzuki coupling product 51 with a yield of 76%. MS (ESI) m / z 240.1 ([M+H–t-Bu] + ).

[0099] In Step B, compound 51 (250 mg, 0.85 mmol) was used as the equivalent standard raw material to obtain 224 mg of Suzuki coupling product 52 with a yield of 48%. MS (ESI) m / z 495.2 ([M+H–t-Bu] + ).

[0100] In Step C, compound 52 (200 mg, 0.36 mmol) was used as the equivalent standard raw material to obtain 55 mg of product 53 with a yield of 40%. MS (ESI) m / z 319.1 ([M+H–t-Bu] + ).

[0101] In Step D, compound 53 (55 mg, 0.15 mmol) was used as the equivalent standard raw material to obtain 43 mg of the target product 3 with a yield of 95%. UPLC-MS (ESI) m / z 275.2, t R 0.941min. 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.

[0102] (2) Aromatic nucleophilic

[0103] Aromatic nucleophilic substitution reaction (Rt.4, 5):

[0104] To a stirring solution of 3,6-dichloropyridazine (1.0 equiv.) in dry NMP (N-methylpyrrolidone) (0.2 M) were added a secondary amine (1.0 equiv.) and K2CO3 (3.0 equiv.). After purging with argon, the reaction system was stirred at 110°C for 8 h. TLC confirmed the absence of residual starting material. 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. Filtration, removal of the solvent under reduced pressure, and purification by chromatography afforded the aromatic nucleophilic substitution product.

[0105] Following the above operation and the synthetic route of Rt.4, in Step A - aromatic nucleophilic substitution, 3,6-dichloropyridazine (200 mg, 1.34 mmol) was used as the equivalent standard raw material and 3-(benzyloxy)azetidine hydrochloride (268 mg, 1.47 mmol, 1.1 equiv.) was used as the reactant to obtain 303 mg of 3-(3-(benzyloxy)azetidin-1-yl)-6-chloropyridazine (54a) in an 82% yield. MS (ESI) m / z 276.1.

[0106] In Step B, compound 54a (250 mg, 0.91 mmol) was used as the equivalent standard raw material to obtain 289 mg of product 55a with a yield of 60%. MS (ESI) m / z 531.6.

[0107] In Step C, compound 55a (200 mg, 0.36 mmol) was used as the equivalent standard raw material to obtain 43 mg of the target product 4 with a yield of 44%. UPLC-MS (ESI) m / z 263.1, t R 2.689min. 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.

[0108] Following the above procedures, according to the synthetic route of Rt.4, in Step A, aromatic nucleophilic substitution, 3,6-dichloropyridazine (200 mg, 1.34 mmol) was used as the equivalent standard raw 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 obtain 346 mg of product 54b in 85% yield, MS (ESI) m / z 304.1; and (S)-2-((benzyloxy)methyl)pyrrolidine (256 mg, 1.34 mmol, 1.0 equiv.) was used as the reactant to obtain 334 mg of product 54c in 82% yield, MS (ESI) m / z 304.1.

[0109] In Step B, compound 54b (250 mg, 0.82 mmol) was used as the equivalent standard raw material to obtain 267 mg of product 55b with a yield of 58%, MS (ESI) m / z 559.3; compound 54c (250 mg, 0.82 mmol) was used as the equivalent standard raw material to obtain 276 mg of product 55c with a yield of 60%, MS (ESI) m / z 559.3.

[0110] In Step C, compound 55b (200 mg, 0.36 mmol) was used as the equivalent standard raw material to obtain 54 mg of the target product 5 with a yield of 52%. UPLC-MS (ESI) m / z 291.2, t R 2.323min. 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.

[0111] Compound 55c (200 mg, 0.36 mmol) was used as the equivalent standard raw material to obtain 51 mg of the target product 6 with a yield of 49%. UPLC-MS (ESI) m / z 291.2, t R 2.323min. 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.

[0112] Following the above procedures, according to the synthetic route of Rt.5, in Step A, aromatic nucleophilic substitution, 3,6-dichloropyridazine (200 mg, 1.34 mmol) was used as the equivalent standard starting material and tert-butyl piperazine-1-carboxylate (250 mg, 1.34 mmol, 1.0 equiv.) was used as the reactant to afford 344 mg of product 54d in 86% yield. MS (ESI) m / z 199.1 ([M+H–Boc] + ).

[0113] In Step B, compound 54d (250 mg, 0.84 mmol) was used as the equivalent standard raw material to obtain 255 mg of product 55d with a yield of 55%. MS (ESI) m / z 454.2 ([M+H–Boc] + ).

[0114] In Step C, compound 55d (200 mg, 0.36 mmol) was used as the equivalent standard raw material to obtain 70 mg of product 56 with a yield of 52%. MS (ESI) m / z 276.4 ([M+H–Boc] + ).

[0115] In Step D, compound 56 (70 mg, 0.13 mmol) was used as the equivalent standard raw material to obtain 34 mg of the target product 7 with a yield of 88%.

[0116] UPLC-MS (ESI) m / z 276.1, t R 0.982min. 1 H NMR(400MHz,D2O)δ7.97(d,J=1.3Hz,

[0117] 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.

[0118] Following the above operation and the synthetic route of Rt. 6, 133 mg of Suzuki coupling product 57 was obtained in Step B using 3-chloro-6-methoxypyridazine (100 mg, 0.69 mmol) as the equivalent standard raw material. The yield was 48%. MS (ESI) m / z 400.2.

[0119] In Step C, compound 57 (100 mg, 0.25 mmol) was used as the equivalent standard raw material to obtain 18 mg of the target product 8 with a yield of 32%.

[0120] UPLC-MS (ESI) m / z 222.1, t R 3.567min. 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.

[0121] According to the method of synthetic route 3-Eq.1, compounds CMP-F, CMP-CN, and CMP-CF3 were synthesized, starting material (1.0 equiv.), MeONa (1.5 equiv.).

[0122] Synthesis route 3 Synthesis reaction formula of 4-substituted pyridazine derivatives

[0123] Compounds CMP-Et, CMP-IP, CMP-Cp, CCP-Cp, and CMP-NC were synthesized according to the method in Synthesis Route 3-Eq. 2. 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 Na₂S₂O₃ (1.2 equiv.), respectively. After purging with argon, the reaction was allowed to proceed at 60°C under an argon atmosphere for 6-12 h. TLC confirmed the absence of residual starting material. After cooling to room temperature, Na₂CO₃ was added to neutralize the reaction. The reaction was quenched with saturated sodium thiosulfate solution, extracted with EtOAc, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. Filtration, removal of the solvent under reduced pressure, and purification by silica gel flash column chromatography yielded the desired compound.

[0124] Compounds CMP-Ph, CMP-Py, CCP-Ph, and CCP-Py were synthesized according to the method in Synthesis Route 3-Eq. To a dry, argon-filled, branched reaction tube were added the 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%). After purging with argon three times, the reaction was then filled with deoxygenated ultrapure water (4:1, v / v). After purging with argon, the reaction was continued at 70°C under an argon atmosphere for 10-18 h. TLC analysis revealed no residual starting material. After cooling to room temperature, the reaction mixture was diluted with EtOAc. The resulting filtrate was filtered, diluted with water, and extracted with EtOAc. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the desired compound was purified by silica gel flash column chromatography.

[0125] [Rt.7]

[0126] According to synthetic route 4-Rt.7, in the first step, a 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 to an oven-dried, argon-filled, open-end reaction tube. The mixture was then evacuated with an oil pump for 10 minutes, after which the atmosphere was replaced with argon. Dry toluene (0.1 M) was added, and the resulting mixture was stirred at room temperature for 10 minutes before being heated at 100°C under argon for 8-15 hours. TLC analysis indicated no residual starting material. After cooling the reaction system to room temperature, the solvent was removed under reduced pressure, and the mixture was dissolved in DCM (1,2-dichloroethane), filtered, and the solvent removed under reduced pressure. The mixture was then purified by silica gel flash column chromatography to yield the intermediate CbzNN-PM-R and a series of compounds.

[0127] Synthesis Route 4 Synthesis Route of Compounds 9-15, 19, and 20

[0128] Table 3 Intermediate CbzNN-PM-R series of compounds

[0129] In the second step, NaI (3.0 equiv.) was added to a stirring solution of CbzNN-PM-R (1.0 equiv.) in dry MeCN. The suspension was placed in an ice-water bath at 0°C. Trimethylsilyl chloride (3.0 equiv.) was slowly added dropwise to the reaction system. After addition, the reaction system was brought to room temperature and stirred for 6-14 hours. TLC confirmed the absence of residual starting material. The reaction was quenched by adding sodium bicarbonate solution and sodium thiosulfate solution and stirred for another 30 minutes. The mixture was then extracted with DCM-MeOH (5-10:1, v / v). The organic phases were combined and dried. The mixture was filtered and the solvent removed under reduced pressure to yield the crude 3(2H)-pyridazinone derivative. To the crude 3(2H)-pyridazinone derivative in a round-bottom flask at 0°C, add anhydrous toluene (0.2 M). POCl3 (1.5 equiv.) is slowly added, followed by the dropwise addition of DIPEA (3.0 equiv.). The reaction system is stirred at room temperature for 15 minutes and then at 100°C for 4-8 hours. TLC analysis indicates no residual starting material. After the reaction system is cooled to room temperature, all volatile components are removed under reduced pressure. The resulting residue is dissolved in DCM (1,2-dichloroethane). The DCM phase is quickly washed with ice water (<1 minute). The resulting organic phase is washed with icy saturated brine and dried over anhydrous sodium sulfate. The product is filtered, the solvent is removed under reduced pressure, and then purified by flash column chromatography on silica gel to yield the 3-chloropyridazine derivative.

[0130] Table 43-Chloropyridazine derivatives

[0131] In the third step, according to Step B in Synthesis Route 2, a series of intermediate CbzNN-P(Py)-R compounds were synthesized using the 3-chloropyridazine derivative obtained in the second step as an equivalent standard raw material.

[0132] Table 5 Intermediate CbzNN-P(Py)-R series of compounds

[0133] In the fourth step, according to the operations of Steps C and D in Synthesis Route 2, the CbzNN-P(Py)-R intermediate obtained in the third step was used as an equivalent reference raw material to complete the synthesis of target products 9-15, 19, and 20.

[0134] Table 6 Synthesis of products 9 to 15, 19, and 20

[0135] Synthesis Route 5 Synthesis route of compounds 21-24, 31, and 32

[0136] [Rt.8]

[0137] In the synthetic route 5-Rt.8, the first step is carried out according to Step B in synthetic route 2, using 3,6-dichloropyridazine derivatives as equivalent standard raw materials to synthesize a series of intermediate C-(R)P-Py compounds.

[0138] Table 7 Intermediate C-(R)P-Py series of compounds

[0139] In the second step, benzyl piperazine-1-carboxylate (1.5 equiv.) and Cs2CO3 (3.0 equiv.) were added to a stirring solution of C-(R)P-Py (1.0 equiv.) in dry NMP (N-methylpyrrolidone). The suspension was stirred at 110°C under an argon atmosphere for 8-15 hours. TLC analysis indicated no residual starting material. After cooling to room temperature, the reaction was diluted with water and quenched with saturated sodium bicarbonate and sodium thiosulfate solutions. Stirring was continued for 30 minutes. The reaction was then extracted 4× with DCM-MeOH (5-10:1, v / v). The organic phases were combined and dried. Filtration, the solvent was removed under reduced pressure, and the mixture was purified by silica gel flash column chromatography to yield the CbzNN-(R)P-Py series.

[0140] Table 8 CbzNN-(R)P-Py series compounds

[0141] In the third step, according to Step C and D in Synthesis 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.

[0142] Table 9 Synthesis of target products 21-24, 31, and 32

[0143] Synthesis Route 6 Synthesis route of compounds 16 and 28

[0144] A round-bottom flask was charged with KH (2.24 g, 30 wt%, 16.76 mmol, 2.0 equiv.), and anhydrous n-hexane (5 mL) was added, stirred, allowed to stand, and the supernatant was discarded. This was repeated twice. The residual solvent was removed by an oil pump and the gas was replaced with argon. Dry THF (tetrahydrofuran) (20 mL) was injected, and the mixture was placed in an ice-water bath at 0°C. A solution of PMBOH (p-methoxybenzyl alcohol) (1.50 g, 10.89 mmol, 1.3 equiv.) in THF (tetrahydrofuran) (5 mL) was added dropwise with stirring. Subsequently, 18-C-6 (111 mg, 0.42 mmol, 0.05 equiv.) was added, and the reaction system was stirred at room temperature for 2 h. A solution of 3,6-dichloro-4-methoxypyridazine (1.50 g, 8.38 mmol, 1.0 equiv.) in dry THF (20 mL) was added dropwise to the reaction mixture at 0°C. The reaction was stirred at room temperature for 6 h. TLC analysis indicated no residual 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. The residue was filtered, the solvent removed under reduced pressure, and purified by chromatography to afford 1.08 g of compound 58 in a 46% yield.

[0145] MS (ESI) m / z 281.0. 1 H NMR(400MHz,Chloroform-d)δ7.37(d,J=8.2Hz,2H),7.17–

[0146] 6.77(m,3H),5.32(s,2H),3.98(s,3H),3.78(s,3H)ppm.

[0147] [Synthetic Route 6Rt.9]:

[0148] In the first step, referring to the first step of synthetic route 4-Rt.7, compound 58 (1.00 g, 3.56 mmol) was used as the equivalent standard raw material to synthesize 1.07 g of compound 59 with a yield of 65%, MS (ESI) m / z 465.2.

[0149] 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. With stirring, 1.0 equiv. of CAN (ceric ammonium nitrate) was added every 1 h (a total of 9.13 g, 17.22 mmol, 8.0 equiv.). TLC analysis indicated no residual starting material. The reaction was quenched by addition of 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. Filtration and removal of the solvent under reduced pressure afforded 511 mg of crude compound 60, which was used directly in the next step without further purification. MS (ESI) m / z 345.1.

[0150] In the third step, referring to the third step of synthetic route 4-Rt.7, crude product 60 (511 mg, ~1.48 mmol) was used as the equivalent standard raw material to synthesize 258 mg of compound 61 with a two-step yield of 33%, MS (ESI) m / z 363.1.

[0151] In the fourth step, referring to the fourth step of synthetic route 4-Rt.7, compound 61 (300 mg, 0.83 mmol) was used as the equivalent standard raw material to synthesize 312 mg of compound 62 with a yield of 61%, MS (ESI) m / z 618.3.

[0152] In the fifth and sixth steps, referring to the fifth and sixth steps of synthetic route 4-Rt.7, compound 62 (300 mg, 0.83 mmol) was used as the equivalent standard raw material to synthesize 52 mg of target compound 16 with a yield of 38%.

[0153] UPLC-MS (ESI) m / z 306.2, t R 0.911min. 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.

[0154] [Synthetic Route 6Rt.10]

[0155] In the first step, referring to the fourth step of synthetic route 4-Rt.7, compound 58 (1.00 g, 3.56 mmol) was used as the equivalent standard raw material to synthesize 1.03 g of compound 64 with a yield of 54%. MS (ESI) m / z 536.2.

[0156] 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 with stirring at 0°C. The reaction system was stirred at room temperature for 2-3 h. TLC analysis indicated no residual starting material. All volatile components were removed under reduced pressure to yield 780 mg of crude product 65, which was used directly in the next step. MS (ESI) m / z 416.2 was obtained.

[0157] In the third step, referring to the third step of synthetic route 4-Rt.7, crude product 65 (780 mg, ~1.87 mmol) was used as the equivalent standard raw material to synthesize 381 mg of compound 66 with a two-step yield of 47%, MS (ESI) m / z 434.1.

[0158] In the fourth step, referring to the first step of synthetic route 4-Rt.7, compound 66 (300 mg, 0.69 mmol) was used as the equivalent standard raw material to synthesize 378 mg of compound 67 with a yield of 74% and MS (ESI) m / z 618.3.

[0159] In the fifth and sixth steps, referring to the fifth and sixth steps of the synthetic route 4-Rt.7, compound 67 (300 mg, 0.83 mmol) was used as the equivalent standard raw material to synthesize 62 mg of the target compound 16 with a yield of 45%. UPLC-MS (ESI) m / z 306.2, t R 0.921min. 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.

[0160] To a solution of 3,6-dichloropyridazin-4-amine (1.0 g, 6.10 mmol, 1.0 equiv.) in anhydrous THF (tetrahydrofuran) (30 mL) were added Boc2O (5.32 g, 24.40 mmol, 4.0 equiv.) and DMAP (4-dimethylaminopyridine) (74 mg, 0.61 mmol, 0.1 equiv.). The reaction system was stirred under reflux for 18 h. TLC analysis revealed no residual starting material. After cooling the reaction system to room temperature, the solvent was removed under reduced pressure and purified by silica gel flash column chromatography to afford 1.91 g of compound CCP-NDB in 86% yield. MS (ESI) m / z 364.2 was obtained.

[0161] According to the first step of Rt.11 in synthetic route 7 and referring to the first step of synthetic route 4-Rt.7, compound CCP-NDB (1.50 g, 4.12 mmol) was used as the equivalent standard raw material to synthesize 1.40 g of compound CbzNN-CP-NDB with a yield of 62% and MS (ESI) m / z 548.2.

[0162] 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 in dioxane (5 mL) was added to the solution with stirring at 0°C. The reaction system was stirred at room temperature for 2-4 hours. TLC analysis indicated no residual starting material. All volatile components were removed under reduced pressure. The resulting residue was dispersed in water and EtOAc, and the pH was adjusted to 8 with sodium bicarbonate. The aqueous phase was extracted with EtOAc (3 x 40 mL). The combined organic phases were washed with saturated sodium chloride solution and dried. The product was filtered, the solvent was removed under reduced pressure, and the product was purified by chromatography to yield 678 mg of compound CbzNN-CP-N in 89% yield. MS (ESI) m / z 348.1 was obtained.

[0163] In the third step, referring to the fourth step of synthetic route 4-Rt.7, compound CbzNN-CP-N (600 mg, 1.72 mmol) was used as the equivalent standard raw material to synthesize 582 mg of compound CbzNN-P(Py)-N with a yield of 56% and MS (ESI) m / z 536.2.

[0164] In the fourth step, according to Steps C and D in Synthesis Route 2, the compound CbzNN-P(Py)-N (500 mg, 0.83 mmol) obtained in Step 3 was used as the equivalent standard raw material to obtain 81 mg of the target product 17 with a yield of 30%.

[0165] Synthesis Route 7 Synthesis route of compounds 17 and 29

[0166] 17: UPLC-MS (ESI) m / z 291.2, t R 0.758min. 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.

[0167] According to the first step of Rt.12 in synthetic route 7 and referring to the fourth step of synthetic route 4-Rt.7, compound CCP-NDB (1.5 g, 4.12 mmol) was used as the equivalent standard raw material to synthesize 1.86 g of compound NDB-CP-Py with a yield of 73%. MS (ESI) m / z 619.2.

[0168] In the second step, referring to the second step of Rt.11 in synthetic route 7, compound NDB-CP-Py (1.5 g, 2.42 mmol) was used as the equivalent standard raw material to synthesize 913 mg of compound N-CP-Py with a yield of 90% and MS (ESI) m / z 419.1.

[0169] In the third step, referring to the first step of synthetic route 4-Rt.7, compound N-CP-Py (800 mg, 1.91 mmol) was used as the equivalent standard raw material to synthesize 610 mg of compound N-(CbzNN)P-Py with a yield of 53% and MS (ESI) m / z 603.2.

[0170] In the fourth step, following the procedures of Steps C and D in Synthesis Route 2, the compound N-(CbzNN)P-Py (600 mg, 1.00 mmol) obtained in Step 3 was used as the equivalent standard raw material to obtain 75 mg of the target product 29 with a yield of 23%. UPLC-MS (ESI) m / z 291.2, t R 0.788min. 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.

[0171] According to the synthesis route Rt.13 in Synthesis Route 8, referring to the synthesis route 4-Rt.7 steps 1, 2, 3, and 4, and the method of Step C in Synthesis Route 2, compound CMP-NC (2.00 g, 6.22 mmol) was used as the starting material. After five steps, 138 mg of compound BocNN-GP-NH was obtained, MS (ESI) m / z 419.2.

[0172] In the sixth step, BocNN-GP-NH (100 mg, 0.24 mmol, 1.0 equiv.) was dissolved in DCE (5 mL). 37 wt% aqueous HCHO (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. The reaction mixture was warmed to room temperature and stirred for an additional 12 h. TLC indicated no residual starting material. The reaction was quenched by addition of saturated sodium bicarbonate solution. The aqueous phase was extracted with DCM-MeOH (5:1, v / v) (3 × 5 mL), and the combined organic phases were dried. Filtration and removal of the solvent under reduced pressure afforded the crude product, BocNN-GP-NM, which was used directly in the next step without further purification. MS (ESI) revealed m / z 433.2.

[0173] In the seventh step, referring to the method of Step D in synthetic route 2, the crude product obtained in the sixth step was used as the equivalent standard raw material to obtain 37 mg of the target product 18 with a yield of 38%.

[0174] Synthesis route 8 Synthesis route of compound 18

[0175] 18: UPLC-MS (ESI) m / z [M+2H] 2+ 167.1,t R 0.781min. 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.

[0176] According to the first step of Rt.14 in synthetic route 9, refer to the fourth step of synthetic route 4-Rt.7; the second and third steps of Rt.14 in synthetic route 9, refer to the second and third steps of synthetic route 4-Rt.7; the fourth step of Rt.14 in synthetic route 9, refer to the first step of synthetic route 4-Rt.7; the fifth to seventh steps of Rt.14 in synthetic route 9, refer to 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.

[0177] 30: UPLC-MS (ESI) m / z [M+2H] 2+ 167.1,t R 0.741min. 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.

[0178] Synthesis route 9 Synthesis route of compound 30

[0179] Referring to the synthesis 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 equivalent standard raw materials, respectively.

[0180] 25: UPLC-MS (ESI) m / z 294.1, t R 0.915min. 1 H 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.

[0181] 26: UPLC-MS (ESI) m / z 301.1, tR 0.932min. 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.

[0182] 27: UPLC-MS (ESI) m / z 344.1, t R 0.942min. 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.

[0183] Referring to the synthetic route 2-R t.5, compounds 33, 34, 35, 36, and 37 were synthesized using 3,6-dichlor o-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 equivalent standard raw materials, respectively.

[0184] 33: UPLC-MS (ESI) m / z 304.2, t R 1.103min. 1 H 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.

[0185] 34: UPLC-MS (ESI) m / z 316.2, t R 1.116min. 1H 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.

[0186] 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.

[0187] 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(dd d,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.

[0188] 37: UPLC-MS (ESI) m / z 328.1, t R 1.120min. 1 H 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.

[0189] According to the synthetic route Rt.15 in synthetic route 10,

[0190] In the first step, compound 56 (200 mg, 0.53 mmol, 1.0 equiv.) was dissolved in dry DCM (1,2-dichloroethane) (10 mL). 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. The reaction was stirred at room temperature for 5 h. TLC indicated no residual starting material. The reaction was quenched by addition of saturated sodium bicarbonate solution. The aqueous phase was extracted with DCM-MeOH (10:1, v / v) (3 × 10 mL). The combined organic phases were dried. The product was filtered, the solvent was removed under reduced pressure, and the product was purified by silica gel flash column chromatography to afford 192 mg of compound 69 in a 76% yield. MS (ESI) m / z 476.2 was obtained.

[0191] 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. The reaction was stirred at the same temperature for 1 h. (A) At the same temperature, a solution of NFSI (91 mg, 0.29 mmol, 1.6 equiv.) in dry THF (2.0 mL) was added dropwise to the reaction solution. The reaction was stirred at the same temperature for 2-5 h. LC-MS analysis indicated no residual starting material. The reaction was quenched by addition of saturated ammonium chloride solution. After warming 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 removal of the solvent under reduced pressure, the product was purified by chromatography to yield 61 mg of compound 70 in a 69% yield. MS (ESI) m / z 494.2. (B) Alternatively, AcOD (17 μL, 0.29 mmol, 1.6 equiv.) was added dropwise to the reaction mixture at the same temperature, and the reaction was stirred for 2–5 h. LC-MS analysis revealed no residual starting material. The reaction mixture was warmed to room temperature and diluted with water. 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 removal of the solvent under reduced pressure, the product was purified by chromatography to yield 75 mg of compound 71 in an 88% yield. MS (ESI) m / z 477.2.

[0192] In the third step, referring to the method of Step D in synthetic route 2, compound 70 (50 mg, 0.10 mmol) or 71 (60 mg, 0.13 mmol) obtained in the second step was used as the equivalent standard raw material to obtain 24 mg and 23 mg of target products 38 and 39, respectively, with yields of 72% and 59%.

[0193] 38: UPLC-MS (ESI) m / z 294.1, t R 0.965min. 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.

[0194] 39: UPLC-MS (ESI) m / z 277.2, t R 0.977min. 1 H 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.

[0195] Synthesis Route 10 Synthesis route of compounds 38 and 39

[0196] According to the synthetic route Rt.16 in synthetic route 11, in the first step, to a stirred solution of uracil (500 mg, 4.46 mmol, 1.0 equiv.) in DME (20 mL) were added Boc2O (2.92 g, 13.38 mmol, 3.0 equiv.) and DMAP (4-dimethylaminopyridine) (163 mg, 1.34 mmol, 0.3 equiv.). The reaction system was heated under reflux overnight. TLC showed no residual starting material. After the reaction system was cooled to room temperature, all volatile components were removed under reduced pressure to obtain the crude product N1Boc-N3Boc-PM, which was used directly in the next step without further purification. MS (ESI) m / z 257.1 ([M+Ht-Bu] + ).

[0197] 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. The solvent was removed by filtration and then purified by chromatography to obtain 786 mg of compound N1H-N3Boc-PM in 83% yield. MS (ESI) m / z 213.1 was obtained.

[0198] Synthesis route 11 Synthesis route of compound N1H-N3Boc-PM

[0199] Following the synthetic route in Scheme 12, in the first step, N₁H-N₃Boc-PM (100 mg, 0.47 mmol, 1.0 equiv.), chiral prolinamide cat-1 (31 mg, 0.09 mmol, 0.3 equiv.), and 3-chloro-6-iodopyridazine (170 mg, 0.71 mmol, 1.5 equiv.) were added to an argon-filled reaction tube. The atmosphere was replaced with argon, and deoxygenated ultrapure water (2.5 mL) was added. CuI (9 mg, 0.05 mmol, 0.1 equiv.) and K₂CO₃ (130 mg, 0.94 mmol, 2.0 equiv.) were added. The atmosphere was replaced with argon, and the reaction system was stirred at 100°C for 24 h. TLC analysis confirmed the absence of residual starting material. After the reaction system was cooled 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 removal of the solvent under reduced pressure, the product was purified by chromatography to give 89 mg of compound 72 with a yield of 58%. MS (ESI) m / z was 325.1.

[0200] In the second step, referring to the first step of the synthetic route 4-Rt.7, compound 72 (70 mg, 0.22 mmol) was used as the equivalent standard raw material to synthesize 64 mg of compound 73 with a yield of 63%. MS (ESI) m / z 419.2 ([M+Ht-Bu] + ).

[0201] In steps 3 and 4, the same procedures as Steps C and D in Synthesis Route 2 were used, using compound 73 (50 mg, 1.00 mmol) obtained in step 2 as the equivalent standard raw material to obtain 24 mg of the target product 40 with a yield of 72%. UPLC-MS (ESI) m / z 277.1, t R 0.768min. 1H 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.

[0202] Synthesis Route 12 Synthesis Route of Compound 40

[0203] Following the synthetic route in Scheme 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). KCO (2.26 g, 16.36 mmol, 2.0 equiv.) was added to this solution at 0°C. A solution of tert-buty1piperazine-1-carboxylate (1.98 g, 10.63 mmol, 1.3 equiv.) in dry DMF (20 mL) was then slowly added dropwise to the reaction mixture at the same temperature. The reaction mixture was allowed to warm to room temperature and stirred for 3 h. TLC confirmed the absence of residual starting material. The reaction mixture 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 removal of the solvent under reduced pressure, the product was purified by chromatography to give 2.45 g of compound 74 with a yield of 90%. MS (ESI) m / z was 333.1.

[0204] In the second step, according to Step B in Synthesis Scheme 2, compound 74 (1.00 g, 3.00 mmol) was used as the equivalent standard raw material to synthesize 1.08 g of compound 75 with a yield of 61% and MS (ESI) m / z 588.2.

[0205] In the third step, 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 the solution at 0°C. The reaction system was stirred at room temperature for 10 min and then refluxed 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 by vacuum evaporation. The product was then purified by silica gel flash column chromatography to afford 344 mg of compound 76 in a 77% yield. MS (ESI) m / z 584.3 was obtained. (B) Alternatively, a sealed reaction tube was used to add dimethylamine methanol solution (0.58 mL, 2.0 M in HCl) to the solution at 0°C. MeOH, 1.15 mmol, 1.5 equiv.), the reaction system was stirred at room temperature for 10 min, then sealed and heated with stirring at 100°C for 4 h. The reaction system was cooled to room temperature, the pressure was released, and TLC detected that no starting material remained. 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 mixture was purified by chromatography to obtain 269 mg of compound 77 in a yield of 59%. MS (ESI) m / z 597.3.

[0206] In steps 4 and 5, the reaction was carried out according to steps C and D in route 2, using compound 76 (250 mg, 0.43 mmol) obtained in step 2 as the equivalent standard raw material to obtain 51 mg of the target product 41 with a yield of 35%; or 77 (200 mg, 0.34 mmol) as the equivalent standard raw material to obtain 31 mg of the target product 42 with a yield of 26%.

[0207] 41: UPLC-MS (ESI) m / z 306.2, t R 0.682min. 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.

[0208] 42: UPLC-MS (ESI) m / z 319.2, t R 0.595min. 1H 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.

[0209] Synthesis Route 13 Synthesis route of compounds 41 and 42

[0210] To a solution of compound 56 (100 mg, 0.27 mmol, 1.0 equiv.) in dry THF (tetrahydrofuran) (3 mL) was slowly added dropwise TMSCHN2 (trimethylsilyl)diazomethane (0.35 mL, 2.0 M in hexanes, 0.71 mmol, 3.0 equiv.). The reaction system was stirred at room temperature for 12 h. TLC analysis indicated no residual starting material. 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. The product was filtered, the solvent was removed under reduced pressure, and the product was purified by silica gel flash column chromatography to afford 78 mg of compound 82 in a 75% yield. MS (ESI) m / z 334.1 ([M+Ht-Bu] + ).

[0211] Following the operation in Step D of Synthesis Scheme 2, compound 82 (50 mg, 0.13 mmol) obtained in the previous step was used as the equivalent standard raw material to obtain 33 mg of the target product 43 with a yield of 80%.

[0212] UPLC-MS (ESI) m / z 290.2, t R 1.021min. 1 H NMR (400MHz, Methanol-d4) δ7.47 (dd, J=

[0213] 8.6,0.6Hz,1H),6.95(d,J=8.3Hz,1H),4.03(td,J=6.5,0.7Hz,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.

[0214] Synthesis Route 14 Synthesis Route of Compound 43

[0215] Example 3 Preparation of Compounds PRTB-01 to PRTB-16

[0216] Synthesis of compound JQ1 acid in Synthetic Route 15

[0217] To a solution of JQ1 (1.00 g, 2.19 mmol, 1.0 equiv.) in dry DCM (1,2-dichloroethane) (20 mL) was slowly added TFA (trifluoroacetic acid) (8 mL) dropwise at 0°C, and the reaction system was stirred at room temperature for 6 h. TLC showed no residual starting material. The reaction solution was concentrated under reduced pressure to obtain a residue, which was dissolved in EtOAc, washed with saturated brine, and dried over anhydrous sodium sulfate. The residue was filtered, the solvent was removed under reduced pressure, and then purified by silica gel flash column chromatography to obtain 805 mg of compound JQ1 acid in a yield of 92%. MS (ESI) m / z 399.1 ([M+H] + ).

[0218] Synthesis of compound JQ1-LB1 in Synthetic Route 16

[0219] Compound 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 system was stirred at room temperature overnight under argon. TLC confirmed the absence of residual starting material. The reaction was quenched with water, the aqueous phase was 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 then purified by silica gel flash column chromatography to obtain 72 mg of compound JQ1-LB1 in an 84% yield. MS (ESI) m / z 472.0 ([M+H-Boc] + ).

[0220] Referring to the synthesis method of compound JQ1-LB1, tert-butyl(5-aminopentyl)carbamate was substituted for tert-butyl(4-aminobutyl)carbamate to obtain 68 mg of compound JQ1-LB2LB13 with a yield of 78%. MS (ESI) m / z 486.0 ([M+H-Boc] + ).

[0221] Referring to the synthesis method of compound JQ1-LB1, tert-butyl(6-aminohexyl)carbamate was substituted for tert-butyl(4-aminobutyl)carbamate to obtain 65 mg of compound JQ1-LB3 with a yield of 73%. MS (ESI) m / z 500.1 ([M+H-Boc] + ).

[0222] Synthetic Route 17 Compound 7-LB4

[0223] 4-((tert-butoxycarbonyl)amino)butanoic acid id (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. A solution of compound 7 (70 mg, 0.22 mmol, 1.0 equiv.) in anhydrous DCM (1,2-dichloroethane) (2 mL) and TEA (187 μL, 1.35 mmol, 6.0 equiv.) were added to the above reaction solution, and the reaction system was stirred at room temperature for 3 h. TLC confirmed the absence of residual starting material. The reaction was quenched by addition of saturated sodium bicarbonate solution at 0°C. The resulting aqueous phase was extracted with a DCM-MeOH (5:1) mixture, and the combined organic phases were dried over anhydrous sodium sulfate. After filtration and removal of the solvent under reduced pressure, the product was purified by silica gel flash column chromatography to give 68 mg of compound 7-LB4 with a yield of 66%. MS (ESI) m / z 361.4 ([M+H-Boc] + ).

[0224] Referring to the synthesis method of compound 7-LB4, 5-((tert-butoxycarbonyl)amino)pentanoic acid was substituted for 4-((tert-butoxycarbonyl)amino)butanoic acid to obtain 77 mg of compound 7-LB5 with a yield of 72%. MS (ESI) m / z 375.4 ([M+H-Boc] + ).

[0225] Referring to the synthesis method of compound 7-LB4, 2-(2-((tert-butoxycarbonyl)amino)ethoxy)acetic acid was substituted for 4-((tert-butoxycarbonyl)amino)butanoic acid to obtain 70 mg of compound 7-LB6 with a yield of 65%. MS (ESI) m / z 377.4 ([M+H-Boc] + ).

[0226] Referring to the synthesis method of compound 7-LB4, 2-(2-((tert-butoxycarbonyl)amino)ethoxy)acetic acid was used instead of 4-((tert-butoxycarbonyl)amino)butanoic acid to obtain 67 mg of compound 7-LB7 with a yield of 61%. MS (ESI) m / z 391.4 ([M+H-Boc] + ).

[0227] Referring to the synthesis method of compound 7-LB6, compound 40 was substituted for compound 7 to obtain 75 mg of compound 40-LB8 with a yield of 70%. MS (ESI) m / z 377.4 ([M+H-Boc] + ).

[0228] Referring to the synthesis method of compound 7-LB6, compound 9 was substituted for compound 7 to obtain 76 mg of compound 9-LB9 with a yield of 69%. MS (ESI) m / z 391.4 ([M+H-Boc] + ).

[0229] Referring to the synthesis method of compound 7-LB6, compound 35 was substituted for compound 7 to obtain 74 mg of compound 35-LB10 with a yield of 62%. MS (ESI) m / z 431.5 ([M+H-Boc] + ).

[0230] Referring to the synthesis method of compound 7-LB6, compound 36 was substituted for compound 7 to obtain 84 mg of compound 36-LB11 with a yield of 71%. MS (ESI) m / z 427.5 ([M+H-Boc] + ).

[0231] Referring to the synthesis method of compound 7-LB6, compound 41 was substituted for compound 7 to obtain 74 mg of compound 41-LB16 with a yield of 65%. MS (ESI) m / z 407.4 ([M+H-Boc] + ).

[0232] Synthesis Route 18: Synthesis of Compounds 55a-OH, 55a-LB12, and 4-LB12

[0233] [Step A] Following the synthesis of compound 55a, 3-((tert-butyldimethylsilyl)oxy)azetidine was substituted for 3-(benzyloxy)azetidine to obtain 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). To this solution was added dropwise a solution of tetrabutylammonium fluoride (TBAF) in THF (tetrahydrofuran) (2.16 mL, 2.16 mmol, 1.0 M in THF, 1.5 equiv.). The reaction mixture was stirred at room temperature for 4 h. TLC confirmed the absence of residual starting material. The mixture was quenched with saturated sodium bicarbonate solution. The resulting 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 product was purified by chromatography to give 594 mg of compound 55a-OH with a yield of 93%. MS (ESI) m / z 441.5 ([M+H] + ).

[0234] [Step B] To a solution of compound 55a-OH (400 mg, 0.91 mmol, 1.0 equiv.) in anhydrous DMF (5 mL) was added NaH (54 mg, 1.36 mmol, 60 wt%, 1.5 equiv.) at 0°C. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was placed in an ice-water bath, and a solution of tert-butyl(5-bromopentyl)carbamate (266 mg, 1.00 mmol, 1.1 equiv.) in anhydrous DMF (2 mL) was added dropwise. The reaction system was stirred at room temperature for another 5 h. TLC analysis indicated no residual starting material. The reaction was quenched by addition of saturated ammonium chloride solution. The resulting aqueous phase was extracted with EtOAc, and the combined organic phases were dried over anhydrous sodium sulfate. The product was filtered, the solvent removed under reduced pressure, and then purified by silica gel flash column chromatography to afford 330 mg of compound 55a-LB12 in a 58% yield. MS (ESI) m / z 526.7 ([M+H-Boc] + ).

[0235] [Step C] Compound 55a-LB12 (300 mg, 0.48 mmol, 1.0 equiv.) was dissolved in a mixture of MeOH and THF (4:1, 5 mL). Pd / C (60 mg, 10 wt% on carbon) was added and the reaction system was stirred overnight at room temperature under a hydrogen atmosphere (balloon). TLC analysis indicated no residual starting material. The product was filtered, the solvent was removed under reduced pressure, and purified by chromatography to afford 173 mg of compound 4-LB12 in 81% yield. MS (ESI) m / z 348.4 ([M+H-Boc] + ).

[0236] Synthesis Route 19: Synthesis of Compounds 83, 84, 85, 86, and 87

[0237] [Step A] Following the synthesis of compound 44, 1-(6-chloropyridazin-3-yl)ethan-1-one was replaced with 6-chloropyridazine-3-carbaldehyde to yield 1.42 g of compound 44-Aldehyde. Compound 44-Aldehyde (1.00 g, 2.52 mmol, 1.0 equiv.) was dissolved in a 5:2 MeOH-THF mixture (14 mL). Pd / C (100 mg, 10 wt% on carbon) was added, and the reaction system was stirred overnight at room temperature under a hydrogen atmosphere (balloon). TLC analysis revealed no residual starting material. The product was filtered, the solvent removed under reduced pressure, and purified by chromatography to yield 427 mg of compound 83 in 77% yield. MS (ESI) m / z 220.2 ([M+H] + ).

[0238] [Step B] Referring to the synthesis method of compound 46, compound 83 was used as the equivalent standard raw material (400 mg, 1.82 mmol, 1.0 equiv.) to obtain 530 mg of compound 84 with a yield of 86%. MS (ESI) m / z 340.4 ([M+H] + ).

[0239] [Step C] To a suspension of NaH (14 mg, 0.35 mmol, 60 wt%, 1.2 equiv.) in anhydrous THF (tetrahydrofuran) (1 mL) at 0°C was added a solution of tert-butyl 2-(diethoxyphosphoryl)acetate (82 mg, 0.32 mmol, 1.1 equiv.) in anhydrous THF (1 mL) dropwise. 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 mixture at 0°C, and the reaction mixture was stirred at the same temperature for 3 h. A solution of compound 84 (100 mg, 0.29 mmol, 1.0 equiv.) in anhydrous THF (1 mL) was added dropwise at the same temperature. After addition, the reaction system was stirred at the same temperature for 15 min. NaH (14 mg, 0.35 mmol, 60 wt%, 1.2 equiv.) was then added, and the mixture was allowed to warm to room temperature and stirred for 1 h. TLC showed no residual starting material. The reaction was quenched by adding water. 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 removal of the solvent under reduced pressure, the product was purified by silica gel flash column chromatography to afford 97 mg of compound 85 in a 76% yield. MS (ESI) m / z 380.4 ([M+Ht-Bu] + ).

[0240] [Step D] To a solution of compound 85 (90 mg, 0.21 mmol, 1.0 equiv.) in anhydrous DCM (1,2-dichloroethane) (3 mL) at 0°C was added TFA (trifluoroacetic acid) (2 mL) dropwise. The reaction system was allowed to warm to room temperature and stirred for 4 h. TLC analysis indicated no residual starting material. All volatile components were removed under reduced pressure to afford 46 mg of crude product 86, which was used directly in the next step without purification.

[0241] [Step E] To a solution of lithium diisopropylamide (LDA) in tetrahydrofuran (tetrahydrofuran) (1 mL) (~1.20 mmol, ~1.20 equiv.) was added TMSCHN2 (trimethylsilyl)diazomethane (1.90 M in hexanes, 0.18 mL, 0.35 mmol, 1.2 equiv.) dropwise at -78°C. The reaction was stirred at the same temperature for 30 min. Compound 84 (100 mg, 0.29 mmol, 1.0 equiv.) in anhydrous THF (1 mL) was added dropwise at -78°C. The reaction system was stirred at -78°C for 1 h and then heated to reflux for 2 h. TLC confirmed the absence of residual starting material. The reaction was cooled to room temperature and quenched with water. The resulting aqueous phase was extracted with a mixture of DCM (1,2-dichloroethane) and MeOH (5:1). The combined organic phases were dried over anhydrous sodium sulfate. After filtration and removal of the solvent under reduced pressure, the mixture was purified by silica gel flash column chromatography to give 61 mg of compound 87 with a yield of 63%. MS (ESI) m / z 336.4 ([M+H] + ).

[0242] Synthesis Route 20: Synthesis of Compound 88

[0243] Compound 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 dry MeCN (10 mL). The reaction mixture was stirred under reflux overnight. TLC confirmed the absence of residual starting material. The mixture was diluted with MTBE (methyl tert-butyl ether) and filtered. The solvent was removed under reduced pressure and purified by silica gel flash column chromatography to afford 104 mg of compound 88 in a 50% yield. MS (ESI) m / z 205.1 ([M+H-Boc] + ).

[0244] Referring to the synthesis method of compound 88, 4-bromo-1H-pyrazole was substituted for 3-bromo-1H-pyrazole to obtain 187 mg of compound 89 with a yield of 90%. MS (ESI) m / z 205.1 ([M+H-Boc] + ).

[0245] Synthesis of Compounds 90-LB14-PMB and 90-LB14 in Synthetic Route 20

[0246] [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 dry THF (tetrahydrofuran) (2 mL). The reaction mixture was purged with argon three times and stirred at 50°C under argon for 3 h. TLC analysis revealed no residual starting material. The reaction mixture was cooled to room temperature and diluted with MTBE (methyl tert-butyl ether). The reaction mixture was filtered, the solvent was removed by vacuum evacuation, and the product was purified by chromatography to afford 86 mg of compound 90-LB14-PMB in a 64% yield. MS (ESI) m / z 459.5 ([M+H-Boc] + ).

[0247] [Step B] To a solution of compound 90-LB14-PMB (80 mg, 0.14 mmol, 1.0 equiv.) in MeCN-H2O (10 mL, 10:1, v / v) at 0°C under argon atmosphere was added CAN (ceric ammonium nitrate) (766 mg, 1.40 mmol, 10.0 equiv.) portionwise. The reaction mixture was stirred at 0°C for 15 min and then at room temperature for 2 h. TLC analysis indicated no residual starting material. Saturated aqueous sodium bicarbonate was added, and the aqueous phase was extracted with a mixed solvent of DCM (1,2-dichloroethane) and MeOH (5:1, v / v). The combined organic phases were dried over anhydrous sodium sulfate. The product was filtered, the solvent was removed by vacuum evaporation, and the product was purified by preparative TLC to afford 45 mg of compound 90-LB14 in a 72% yield. MS (ESI) m / z 339.2 ([M+H-Boc] + ).

[0248] Referring to the synthesis method of compound 90-LB14, 42 mg of compound 91-LB15 was obtained with a yield of 68%. MS (ESI) m / z 339.2 ([M+H-Boc] + ).

[0249] Synthesis of compound 92 in Synthetic Route 21

[0250] To dry DMF (2 mL) at 0°C was added NaH (644 mg, 16.11 mmol, 60 wt%, 1.2 equiv.). Tert-butyl 2-hydroxyacetate (1.77 g, 13.43 mmol, 1.0 equiv.) was then 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 one portion. The resulting mixture was allowed to warm to room temperature under an argon atmosphere and stirred overnight. TLC confirmed the absence of residual starting material. The reaction was quenched by careful addition of ice water. The aqueous phase was extracted with EtOAc, and the combined organic phases were washed with saturated brine. The organic phases were filtered, the solvent was removed under reduced pressure, and the product was purified by chromatography to afford 2.06 g of compound 92 in a 63% yield. MS (ESI) m / z 189.6 ([M+Ht-Bu] + ).

[0251] Synthesis of Compounds 93 and 94 in Synthetic Route 22

[0252] Referring to the synthesis method of compound 8, compound 92 was used to synthesize compound 93. 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 to the solution at 0°C. The reaction system was automatically warmed to room temperature under an argon atmosphere and stirred for 4 hours. TLC showed that no starting material remained. After removing the solvent under reduced pressure, 842 mg of crude product compound 94 was obtained, which was used directly in the next step without further purification. MS (ESI) m / z 266.2 ([M+H] + ).

[0253] Synthesis Route 23 Synthesis of protein degraders PRTB-01, PRTB-02, PRTB-03, and PRTB-13

[0254] Protein degraders PRTB-01, PRTB-02, PRTB-03, and PRTB-13 were synthesized according to the synthetic strategy in the figure above.

[0255] [Step A] Dissolve JQ1-LB1, JQ1-LB2, LB13, 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 warmed to room temperature under an argon atmosphere and stirred for 4-6 hours. LC-MS analysis indicates no residual starting material. Remove the solvent under reduced pressure and dissolve in methanol. Adjust the pH to 8 with saturated sodium bicarbonate solution. Extract with a DCM-MeOH (5:1, v / v) mixed solvent. Combine the organic phases and dry them over anhydrous sodium sulfate. Filter, remove the solvent under reduced pressure, and continue to dry under reduced pressure at 45°C and 0.6 mbar for 30 minutes to obtain the crude product, JQ1-LBx-NH2, which is used directly in the next step.

[0256] [Step B] 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.) were dissolved in dry DMF (0.2 M), and then HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate) (1.5 equiv.) and DIPEA (diisopropylethylamine) (3.0 equiv.) were added sequentially. The reaction system was stirred at room temperature overnight under argon protection. When one of the starting materials was consumed as determined by LC-MS, the reaction was quenched with water. The aqueous phase was extracted with a DCM-MeOH (5:1, v / v) mixture, and the combined organic phases were dried over anhydrous sodium sulfate. Filtration and removal of the solvent under reduced pressure were followed by purification by preparative HPLC to yield PRTB-01, PRTB-02, PRTB-03, and PRTB-13, respectively.

[0257] The protein degrader PRTB-01 was synthesized and purified according to 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 0.842min, 96% purity.

[0258] The protein degrader PRTB-02 was synthesized and purified according to 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 0.855min, 98% purity.

[0259] The protein degrader PRTB-03 was synthesized and purified according to 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 0.851min, 96% purity.

[0260] 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 0.780min, 98% purity.

[0261] Synthesis Route 24: Synthesis of Protein Degraders PRTB-04-PRTB-12, PRTB-14-PRTB-16

[0262] Protein degraders PRTB-04~PRTB-12, PRTB-14~PRTB-16 were synthesized according to the synthesis strategy in the above figure.

[0263] [Step A] 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 were dissolved in dry DCM (1,2-dichloroethane) (0.2 M). TFA (trifluoroacetic acid) (1 / 2 volume of DCM) was slowly added to the solution at 0°C. The reaction system was automatically warmed to room temperature under an argon atmosphere and stirred for 4 hours. LC-MS analysis showed no residual starting material. The solvent was removed under reduced pressure and the product was dissolved in methanol. Saturated sodium bicarbonate solution was added to adjust the pH to 8, and then the product was extracted with a DCM-MeOH (5:1, v / v) mixed solvent. The combined organic phases were dried over anhydrous sodium sulfate. The residue was filtered, the solvent was removed by rotary evaporation under reduced pressure, and the mixture was dried under reduced pressure at 45°C and 0.6 mbar for 30 min to obtain the crude product u-LBz-NH2, which was used in the next step without further purification.

[0264] [Step B] JQ1-Acid (1.0 equiv.) and one of the crude u-LBz-NH2 products obtained in the previous step (1.0 equiv.) were dissolved in dry DMF (0.2 M). HATU (1.5 equiv.) and DIPEA (diisopropylethylamine) (3.0 equiv.) were then added sequentially. The reaction system was stirred at room temperature under argon overnight. When one of the starting materials was consumed, as determined by LC-MS, the reaction was quenched with water. The aqueous phase was extracted with a DCM-MeOH (5:1, v / v) mixture, and the combined organic phases were dried over anhydrous sodium sulfate. Filtration and removal of the solvent under reduced pressure were followed by purification by preparative HPLC to yield 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.

[0265] The protein degrader PRTB-04 was synthesized and purified according to 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 0.671min, 98% purity.

[0266] The protein degrader PRTB-05 was synthesized and purified according to 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 0.685min, 98% purity.

[0267] The protein degrader PRTB-06 was synthesized and purified according to 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 0.601min, 98% purity.

[0268] The protein degrader PRTB-07 was synthesized and purified according to 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 0.618min, 98% purity.

[0269] The protein degrader PRTB-08 was synthesized and purified according to 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 0.511min, 99% purity.

[0270] The protein degrader PRTB-09 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)δ11.13(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 0.624min, 98% purity.

[0271] The protein degrader PRTB-10 was synthesized and purified according to 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 0.701min, 96% purity.

[0272] The protein degrader PRTB-11 was synthesized and purified according to 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 0.695min, 96% purity.

[0273] The protein degrader PRTB-12 was synthesized and purified according to the general synthesis 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 0.885min, 97% purity.

[0274] The protein degrader PRTB-14 was synthesized and purified according to the general synthesis 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.

[0275] The protein degrader PRTB-15 was synthesized and purified according to 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 1.133min, 97% purity.

[0276] The protein degrader PRTB-16 was synthesized and purified according to the general synthesis 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 0.709min, 96% purity.

[0277] Example 4 Preparation of Compounds PRTA-01 to PRTA-06

[0278] Synthesis of Compounds 95, 96, and 97 in Synthetic Route 25

[0279] [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 NaH (2.09 g, 52.25 mmol, 60 wt%, 2.25 equiv.) portionwise at 0°C. The reaction system was allowed to warm 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 reaction mixture at 0°C. The reaction system was allowed to warm to room temperature and stirred for 5 h. TLC confirmed the absence of residual starting material. The reaction mixture was poured into ice-water, and the resulting aqueous phase was extracted with EtOAc. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After filtration and removal of the solvent under reduced pressure, the mixture was purified by chromatography to give 6.96 g of compound 95 with a yield of 85%. MS (ESI) m / z 251.1 ([M+H-Boc] + ).

[0280] [Step B] To a solution of compound 95 (2.50 g, 7.12 mmol, 1.0 equiv.) in dry DCM (1,2-dichloroethane) (30 mL) was slowly added TFA (1.85 mL, 28.48 mmol, 4.0 equiv.) at 0°C. The reaction system was stirred at 0°C for 3 h. TLC confirmed the absence of residual 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. The reaction solution was diluted with water and 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. Filtration, removal of the solvent under reduced pressure, and continued vacuum drying at 45°C and 0.6 mbar for 30 min to obtain 1.33 g of crude product 96, which was used directly in the next step without further purification. MS (ESI) m / z 251.1 ([M+H] + ).

[0281] [Step C] Dissolve 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.) in anhydrous DMF (150 mL). Stir the reaction under argon at room temperature overnight. TLC analysis indicated 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 removal of the solvent under reduced pressure, the product was purified by chromatography to give 6.42 g of compound 97 with a yield of 82%. MS (ESI) m / z 439.2 ([M+H-Boc] + ).

[0282] [Step D] To a solution of compound 97 (2.00 g, 3.71 mmol, 1.0 equiv.) in dry DCM (1,2-dichloroethane) (50 mL) was slowly added TFA (0.98 mL, 14.84 mmol, 4.0 equiv.) at 0°C. The reaction was stirred at 0°C for 3 h. TLC confirmed the absence of starting material. The reaction was diluted with DCM (1,2-dichloroethane), the solvent removed under reduced pressure, and the residue dissolved in methanol. The reaction was diluted with water and the pH 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. The reaction mixture was filtered, the solvent removed under reduced pressure, and the mixture was dried under reduced pressure at 45°C and 0.6 mbar for 30 min to afford 1.68 g of crude product 98, which was used 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] + ).

[0283] Synthesis of Compound 99 in Synthetic Route 26

[0284] To a solution of compound 90-LB14-PMB (80 mg, 0.14 mmol, 1.0 equiv.) in MeCN-H2O (10 mL, 10:1, v / v) at 0°C under argon atmosphere was added CAN (ceric ammonium nitrate) (766 mg, 1.40 mmol, 10.0 equiv.) portionwise. The reaction mixture was stirred at 0°C for 15 min and then at room temperature for 2 h. TLC analysis indicated no residual starting material. Saturated aqueous sodium bicarbonate was added, and the aqueous phase was extracted with a DCM-MeOH (5:1, v / v) solvent mixture. The combined organic phases were dried over anhydrous sodium sulfate. The product was filtered, the solvent removed under reduced pressure, and purified by preparative TLC to afford 45 mg of compound 90-LB14 in a 72% yield. MS (ESI) m / z 339.2 ([M+H-Boc] + ).

[0285] Synthesis of RCH2OTBS Acid and RCH2OTBS Amide

[0286] [Step A] The starting material 99 represented by RCH2OH Ester (1.0 equiv.) or methyl4-(4-(hydroxymethyl)piperidin-1-yl)benzoate was dissolved in DCM (1,2-dichloroethane) (0.2 M). Imidazole (2.0 equiv.) was added to the solution at 0°C and stirred at the same temperature for 15 minutes. Tert-butyldimethylsilyl chloride (TBSCl) (1.2 equiv.) was then added portionwise. The reaction mixture was allowed to warm to room temperature and stirred for 2 hours. TLC confirmed the absence of residual starting material. Saturated aqueous sodium bicarbonate 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. The mixture was filtered, the solvent removed under reduced pressure, and then purified by silica gel flash column chromatography to yield the compound represented by RCH2OTBS Ester.

[0287] [Step B] Dissolve the compound represented by RCH2OTBS Ester (1.0 equiv.) in anhydrous MeOH (0.2 M). Add finely ground K2CO3 (3.0 equiv.) portionwise to the solution at 0°C. The resulting reaction mixture is allowed to warm to room temperature and stirred for 4 hours. TLC confirms the absence of residual starting material. Dilute with water, then adjust the pH to 5-7 with 3N aqueous HCl. Extract the aqueous phase with EtOAc, and dry the combined organic phases over anhydrous sodium sulfate. Filter, remove the solvent in a vacuum spin-drying cycle, and continue to spin-dry at 45°C and 0.6 mbar for 30 minutes to obtain a series of crude products represented by RCH2OTBS Acid, which are used in the next step without further purification.

[0288] [Step C] Dissolve the compound represented by RCH2OTBS Acid (1.0 equiv.), compound 96 (1.05 equiv.), HATU (1.5 equiv.), and DIPEA (diisopropylethylamine) (3.0 equiv.) in anhydrous DMF (0.2 M). Stir the resulting reaction system at room temperature overnight under argon. Once one of the starting materials has been consumed, as determined by TLC, quench the reaction with water. The aqueous phase is extracted with EtOAc, and the combined organic phases are dried over anhydrous sodium sulfate. Filtration, removal of the solvent under reduced pressure, and purification by silica gel flash column chromatography yield the compound represented by RCH2OTBS Amide.

[0289] [Step D] Dissolve the compound represented by RCH2OTBS Amide (1.0 equiv.) in anhydrous THF (0.2 M). Slowly add TBAF (tetrabutylammonium fluoride) (1.0 M in THF, 1.5 equiv.). Stir the resulting reaction mixture at room temperature under argon for 3 h. Once the starting material is consumed as determined by TLC, dilute the reaction with water. The aqueous phase is extracted with EtOAc, and the combined organic phases are dried over anhydrous sodium sulfate. Filtrate, remove the solvent under reduced pressure, and purify by silica gel flash column chromatography to yield the compound represented by RCH2OH Amide.

[0290] [Step E] Dissolve the compound represented by RCH2OHAmide (1.0 equiv.) in anhydrous DCM (1,2-dichloroethane) (0.1 M). Add DMP (2.0 equiv.) portionwise at 0°C. Stir the resulting reaction system at 0°C under argon for 1 hour. When the starting material is consumed as determined by TLC, dilute the reaction with water and quench with dilute sodium thiosulfate solution and saturated sodium bicarbonate solution. Extract the aqueous phase with DCM (1,2-dichloroethane), and dry the combined organic phases over anhydrous sodium sulfate.

[0291] After filtration and removal of the solvent under reduced pressure, the product was purified by silica gel flash column chromatography to obtain a compound represented by RCHOAmide.

[0292] According to the above general synthetic strategy, 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] + ).

[0293] According to the above general synthetic strategy, 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] + ).

[0294] Synthesis Route 28: Synthesis of Compounds 102, 103, and 104

[0295] [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 at 60°C under argon atmosphere overnight. TLC monitoring confirmed that no starting material remained. The reaction system 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. Filtration, removal of the solvent under reduced pressure, and purification by chromatography afforded 2.36 g of compound 102 in 82% yield. MS (ESI) m / z 331.1 ([M+H] + ).

[0296] [Step B] Compound 102 (2.20 g, 6.64 mmol, 1.0 equiv.) was added to a 50% aqueous solution of sulfuric acid (1 M). The reaction system was heated to reflux under argon atmosphere for 1 h. TLC monitoring showed that no starting material remained. The reaction system was cooled and slowly poured into a mixture of saturated aqueous sodium bicarbonate and ice. The pH was carefully adjusted to 4-6 with a 4N aqueous solution of HCl. The aqueous phase was extracted with EtOAc, and the combined organic phases were dried over anhydrous sodium sulfate. The solvent was removed by filtration and then dried under reduced pressure at 45°C and 0.6 mbar for 30 min to obtain 2.21 g of crude product Compound 103. MS (ESI) m / z 348.0 ([MH] - ).

[0297] [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 at room temperature overnight under argon protection. TLC showed that no starting material remained. The reaction was quenched with water, the aqueous phase was extracted with EtOAc, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure and purified by chromatography to obtain 718 mg of compound 104 in an 86% yield. MS (ESI) m / z 582.1 ([M+H] + ).

[0298] Synthesis Route 29: Synthesis of Compounds 105, 106, and 107

[0299] [Step A] Following the synthesis of 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). Pd / C (30 mg, 10 wt% on carbon) was added, and the reaction system was vigorously stirred at room temperature under a hydrogen atmosphere (balloon) for 3 h. TLC analysis revealed no residual starting material. The product was filtered, the solvent was removed under reduced pressure, and then purified by chromatography to afford 65 mg of compound 106 in a 61% yield. MS (ESI) m / z 305.2 ([M+H] + ).

[0300] [Step B] To a solution of compound 106 (65 mg, 0.21 mmol, 1.0 equiv.) in anhydrous DCM (1,2-dichloroethane) (5 mL) was added portionwise DMP (116 mg, 0.27 mmol, 1.3 equiv.) at 0°C. The resulting reaction system was stirred at 0°C under argon for 1 h. When the starting material was consumed as determined by TLC, the reaction was diluted with water and quenched with dilute 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. Filtration, removal of the solvent under reduced pressure, and purification by chromatography afforded 59 mg of compound 107 in a 91% yield. 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] + ).

[0301] According to the synthesis method of compound 7, 92 mg of compound 108 was synthesized using 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] + ).

[0302] Synthesis of compound PRTA-01 in Synthetic Route 30

[0303] Compounds 107 (22 mg, 0.07 mmol, 1.0 equiv.) and 98 (38 mg, 0.09 mmol, 1.2 equiv.) were dissolved in anhydrous DCE (1,2-dichloroethane) (3 mL) containing 10% AcOH (0.30 mL) at room temperature. The mixture was stirred for 15 minutes, then NaBH(OAc) (18 mg, 0.09 mmol, 1.2 equiv.) was added and stirring continued for 3 hours. When the starting material was consumed as determined by TLC, the reaction was diluted with water and quenched with dilute 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. The residue was filtered, the solvent was removed by vacuum evaporation, and then purified by preparative HPLC to afford 36 mg of compound PRTA-01 in a 68% yield. 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 0.831min, 97% purity.

[0304] Synthesis of compound PRTA-02 in Synthetic Route 31

[0305] Compounds 7 (35 mg, 0.11 mmol, 1.0 equiv.) and 101 (63 mg, 0.13 mmol, 1.2 equiv.) were dissolved in anhydrous DCE (3 mL) containing 10% AcOH (0.30 mL) at room temperature. The mixture was stirred for 15 minutes, then NaBH(OAc)3 (31 mg, 0.15 mmol, 1.3 equiv.) was added and stirring continued for 2 hours. When the starting material was consumed as determined by TLC, the reaction was diluted with water and quenched with dilute 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. The residue was filtered, the solvent removed under reduced pressure, and purified by preparative HPLC to afford 51 mg of compound PRTA-02 in a 62% yield. 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 0.685min, 98% purity.

[0306] Synthesis of compound PRTA-03 in Synthetic Route 32

[0307] The protein degrader PRTA-03 was synthesized and purified from compounds 101 and 40 according to the synthesis method of 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 0.602min, 97% purity.

[0308] Synthesis of compound PRTA-04 in Synthetic Route 33

[0309] The protein degrader PRTA-04 was synthesized and purified from compounds 101 and 9 according to the synthesis method of 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 0.698min, 96% purity.

[0310] Synthesis of compound PRTA-05 in Synthetic Route 34

[0311] The protein degrader PRTA-05 was synthesized and purified from compounds 101 and 108 according to the synthesis method of 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 0.672min, 98% purity.

[0312] Synthesis of compound PRTA-06 in Synthetic Route 35

[0313] [Step A] A round-bottom flask was charged with Pd(PPh3)2Cl2 (8 mg, 0.01 mmol, 0.1 equiv.) and CuI (2 mg, 0.13 mmol, 0.12 equiv.). After purging the argon atmosphere three times, 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) were added sequentially. After purging the argon atmosphere again, the reaction system was stirred at 70°C under an argon atmosphere overnight. TLC analysis confirmed that no starting material remained. The reaction system was diluted with water, and the resulting aqueous phase was extracted with DCM (1,2-dichloroethane). The combined organic phases were dried over anhydrous sodium sulfate. The mixture was filtered, the solvent was removed by vacuum evaporation, and the mixture was purified by TLC preparative plate to obtain 62 mg of compound 109 in a 68% yield. MS (ESI) m / z 837.3 ([M+H-Boc] + ).

[0314] [Step B] To a solution of compound 109 (62 mg, 0.07 mmol, 1.0 equiv.) in MeCN-H₂O (5 mL, 10:1, v / v) at 0°C under argon was added CAN (ceric ammonium nitrate) (192 mg, 0.35 mmol, 5.0 equiv.) portionwise. The reaction mixture was stirred at 0°C for 10 min and then at room temperature for 1 h. TLC confirmed the absence of residual starting material. Saturated aqueous sodium bicarbonate was added, and the aqueous phase was extracted with a DCM-MeOH (5:1, v / v) mixture. The combined organic phases were dried over anhydrous sodium sulfate. Filtration and removal of the solvent under reduced pressure were followed by preparative HPLC to yield 21 mg of compound PRTA-06 in a 40% yield. 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 0.713min, 98% purity.

[0315] Example 5 Binding Activity of Compounds to E3 Ubiquitin Ligase CRBN

[0316] The binding activity of the compound disclosed in this patent and the E3 ubiquitin ligase CRBN is determined by the competitive binding of the compound disclosed in this patent with thalidomide labeled with XL665 to the CRBN protein, thereby preventing the occurrence of fluorescence resonance energy transfer (FRET), thereby judging the binding force of the compound to the CRBN protein.

[0317] CRBN binding activity test kit (CEREBLON BINDING KITS (PE, 64BD CRBN PEG).

[0318] The detection is based on homogeneous time-resolved fluorescence (HTRF) technology. When the donor (Donor) and acceptor (Acceptor) are close to each other, the donor can transfer energy to the acceptor, causing it to be excited and emit light at 665nm.

[0319] This kit uses a europium-labeled GST antibody (GST Eu Cryptate Antibody) as the donor and XL665-labeled thalidomide (Thalidomide-Red Reagent) as the receptor. The donor binds to the GST-tagged CRBN protein, and the compounds disclosed in this patent compete with thalidomide for binding to the CRBN protein. The binding activity of the compound is determined by the fluorescence emitted by the receptor at 665 nm. Stronger binding compounds produce weaker signals.

[0320] The donor and receptor were each diluted 50-fold using the PROTAC binding buffer 1 provided in the kit. The CRBN protein was diluted 45-fold. An 8 mM solution of the compound disclosed in this patent was diluted 10-fold using the 1X diluent provided in the kit. The diluent was then serially diluted 5-fold, for a total of seven dilutions.

[0321] 5 μl of compound, 5 μl of diluted CRBN protein, and 10 μl of equal volumes of donor-receptor mixture were sequentially added to the wells of a white 384-well plate (PE, Part number: 6008280) and incubated at room temperature for 3 hours.

[0322] The ratio of the acceptor and donor emission signals was calculated for each individual well.

[0323] Ratio=665nm signal value / 620nm signal value*10 4

[0324] Calculate the coefficient of variation (CV) = standard deviation (SD) / ratio mean * 100

[0325] IC was calculated based on compound concentration, Ratio, and coefficient of variation using GraphPad Prism. 50 value.

[0326] Table 10 Binding activity of compounds 1-18 to E3 ubiquitin ligase CRBN

[0327] Table 11 Binding activity of compounds 19-34 to E3 ubiquitin ligase CRBN

[0328] Table 12 Binding activity of compounds 35-43 to E3 ubiquitin ligase CRBN

[0329] Example 6 Evaluation of the Effect of Protein Degrader Compounds on Reducing Bromodomain Protein (BRD) Expression

[0330] The human chronic myelomonocytic leukemia MV-4-11 cells selected for this experiment were cultured in IMDM medium containing 10% fetal bovine serum (Gibco, USA) in an incubator at 37°C, 5% CO2, and 95% humidity. When the growth density reached 80%, the cells were seeded into 6-well plates using medium containing the compound at a cell density of 1x10 6 / ml, with final compound concentrations of 0.1, 1, 10, and 20 μM. After 24 hours of treatment, cells were harvested and washed twice with PBS. RIPA lysis buffer containing protease and phosphatase inhibitors was added to the cells. Total protein extracts were obtained after lysis and centrifugation, and the protein concentration in the extracts was determined by the BCA assay.

[0331] Protein electrophoresis was performed using SDS-PAGE, and then the protein was transferred to a PVDF membrane using a constant current electrophoresis at 200 mA for 150 minutes. The PVDF membrane was placed in 5% skim milk and blocked at room temperature for 1 hour. BRD4 (#13440, CST) (1:1000) and C-MYC (ab32072, 1:1000) were added and incubated at 4°C overnight. The membrane was washed with TBST 3 times for 10 minutes each time. The secondary antibody (1:5000, absin) was incubated at room temperature for 60 minutes. The membrane was washed with TBST 3 times for 10 minutes each time. ECL luminescent solution was added and exposed. Each sample was simultaneously detected using GAPDH protein as an internal reference. The protein map was analyzed for grayscale values ​​using the analysis software ImageJ. The formula used was: grayscale correction value = (grayscale value of target protein / corresponding grayscale value of internal reference>10 3 , calculate the grayscale correction value of each sample. Then compare it with the grayscale correction value of the control group to calculate the degradation rate. Then use Prism to perform nonlinear curve fitting with logarithmic concentration-inhibition rate to obtain the DC of the compound. 50 and D max value.

[0332] Table 13 summarizes the evaluation of the protein degradation agents PRTB-01 to 16 on the reduction of BRD protein expression. 50 represents the concentration of protein degrader when the protein degrader induces BRD protein degradation to 50%, and the level is graded as A (DC 50 <1μΜ), B(DC 50 1~10μΜ), C(DC 50 >10 μM). Where D max It represents the ratio of the maximum extent of BRD protein degradation induced by protein degraders to the total amount of BRD protein, and the level is graded as A (D max >85%)、B(D max 85~50%)、C(DC 50 <50%)

[0333] Table 13 Evaluation of the effect of protein degraders PRTB-01 to 16 on reducing BRD protein expression

[0334] Example 7 Evaluation of the Effect of Protein Degrader Compounds on Reducing androgen Receptor (AR) Expression

[0335] The human prostate cancer cell line LNCaP cells selected in this experiment were cultured in RPMI-1640 medium containing 10% fetal bovine serum (Gibco, USA) in an incubator at 37°C, 5% CO2, and 95% humidity. When the growth density reached 80%, the cells were seeded into 6-well plates at 5×10 cells per well. 5 After 24 hours of culture, the medium containing the compound was replaced to a final concentration 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 and phosphatase inhibitors was added to the cells. After lysis and centrifugation, a total protein extract was obtained. The protein concentration in the extract was determined by BCA assay.

[0336] Protein electrophoresis was performed using SDS-PAGE, and then the protein was transferred to a PVDF membrane using a constant current electrophoresis at 200 mA for 90 minutes. The PVDF membrane was placed in 5% skim milk and blocked at room temperature for 1 hour. Anti-Androgen Receptor antibody [EPR1535(2)] (ab133273) (1:10000) was added and incubated at 4°C overnight. The membrane was washed with TBST 3 times for 10 minutes each time. The secondary antibody (1:5000, absin) was incubated at room temperature for 60 minutes. The membrane was washed with TBST 3 times for 10 minutes each time. ECL luminescent solution was added and exposed. Each sample was simultaneously detected using α-tubulin protein as an internal reference. The protein spectrum was analyzed by grayscale value analysis using the analysis software ImageJ. The formula used was: grayscale correction value = (target protein grayscale value / corresponding internal reference grayscale value > 10 3 , calculate the grayscale correction value of each sample. Then compare it with the grayscale correction value of the control group to calculate the degradation rate. Then use Prism to perform nonlinear curve fitting with logarithmic concentration-inhibition rate to obtain the DC of the compound. 50 and D max value.

[0337] Table 14 summarizes the evaluation of the protein degradation agents PRTA-01 to PRTA-06 on the reduction of AR protein expression. 50 Represents the concentration of protein degrader when the protein degrader induces AR protein degradation to 50%, and the level is graded as A (DC 50<1μΜ), B(DC 50 1~10μΜ), C(DC 50 >10 μM). Where D max It represents the ratio of the maximum extent of BRD protein degradation induced by protein degraders to the total amount of AR protein, and the level is graded as A (D max >85%)、B(D max 85~50%)、C(D max <50%).

[0338] Table 14 Evaluation of the effect of protein degradation agents PRTA-01 to PRTA-06 on reducing AR protein expression

Claims

1. A compound that can bind to CRBN protein, the compound is as shown in (I): in, R 1 and R 2 One or more selected from the group consisting of a pyridazine nucleus and a ring, H, alkyl, OR, F, CN, CF3, NR2, Ph, and 4-Py; Y is selected from one or more of CH, CD, CF and N; R 3 One or more selected from OR, NR2, CHR2 and C≡CR; R 4 Selected from H or alkyl.

2. The compound binding to CRBN protein according to claim 1, characterized in that The fused ring may be selected from fused ring, cyclopentane, cyclohexane, benzene and / or pyrazine.

3. The compound binding to CRBN protein according to claim 1, characterized in that In the compound, R 1 and R 2 They may be selected from alkyl groups at the same time.

4. The compound binding to CRBN protein according to claim 1, characterized in that In the compound, R 1 and R 2 It cannot be selected from H or one or more of alkyl, OR, F, CN, CF3, NR2, Ph, 4-Py at the same time.

5. A pharmaceutically acceptable salt of the compound (I) that binds to the CRBN protein as claimed in claim 1, wherein the pharmaceutically acceptable salt refers to a salt prepared by adding a non-toxic acid or base to the parent compound.

6. A protein degrader, comprising: a target protein ligand (POI ligand) + a linker (Linker) + a CRBN protein ligand; the CRBN protein ligand is compound (I), and the structural formula of the protein degrader is (II): 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 One or more selected from OR, NR2, CHR2 and C≡CR; 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 One or more selected from the group consisting of a pyridazine nucleus and a ring, H, alkyl, OR, F, CN, CF3, NR2, Ph, and 4-Py. In Formula II, R 1 One or more selected from the group consisting of a pyridazine nucleus and a ring, H, alkyl, OR, F, CN, CF3, NR2, Ph, and 4-Py. R 4 Selected from H or alkyl. Y is selected from one or more of CH, CD, CF, CCH3 and N.

7. The protein degradation agent according to claim 6, characterized in that The Linker substructure L 1 , L 3 and L 5 You can choose one or more of the following: Ra=H, alkyl, SO2(alyl), SO2(aryl) Among them, R a Selected from one or more of H, alkyl, SO2(alyl) or SO2(aryl).

8. The protein degradation agent according to claim 6, characterized in that The Linker substructure L 2 , L 4 You can choose one or more of the following:

9. The protein degradation agent according to claim 6, characterized in that The target protein ligand, i.e., the POI ligand in formula II, can be selected from the following: One or more of .

10. A pharmaceutical composition comprising a combination of the compound of formula (I) or a pharmaceutically acceptable salt thereof according to the first aspect and a pharmaceutically acceptable diluent or carrier.

11. A pharmaceutical composition for targeted protein degradation, comprising a combination of the protein degrading agent (II) or a pharmaceutically acceptable salt thereof according to the second aspect of the present invention and a pharmaceutically acceptable diluent or carrier.

12. The pharmaceutical composition according to claim 9 or 10, characterized in that The pharmaceutical composition may also comprise a combination of an enantiomer, a diastereomer, a stereoisomer or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent or carrier.

13. A regulator for regulating a transcriptional regulatory factor, the regulator comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof or a protein degrading agent (II) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer or tautomer thereof.

14. A method for treating or preventing pathological conditions or symptoms of diseases 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.

Images