Targeting TRIM15 micromolecule conjugate as well as preparation method and application thereof

Abstract

The invention discloses a PROTAC compound for degrading TRIM15 protein. The PROTAC compound has a structure as shown in a formula (I). The molecule can effectively inhibit the RIG-1 signal channel, and has application prospects in preparation of anti-inflammatory or autoimmune disease drugs. Meanwhile, the invention further provides a chemical probe molecule with the structure shown in the formula (III). One end of the molecule is combined with TRIM15, and the other end of the molecule can be combined with tag protein (such as Halog-tag) or other target protein, so that TRIM15 is recruited as E3 ubiquitin ligase to carry out ubiquitination modification on the tag protein (or the target protein). The molecule can be used as an innovative research tool and is used for exploring the biological function of the E3 ligase of TRIM15, the substrate specificity and non-K48 type ubiquitination modification.

Description

Technical Field

[0001] This invention belongs to the fields of medicinal chemistry and chemical biology, specifically relating to a class of novel compounds targeting TRIM15 protein, and their preparation methods and applications as protein degrading agents and / or probes for studying E3 ligase activity. Background Technology

[0002] The tripartite motif (TRIM) protein family comprises over eighty members in the human genome, widely involved in various key life processes such as cell proliferation, differentiation, apoptosis, tumorigenesis, and innate immunity. TRIM15, as a member of this family, possesses an N-terminal Ring domain that endows it with E3 ubiquitin ligase activity. Existing research indicates that TRIM15 plays a crucial role in multiple diseases. On one hand, it is significantly upregulated in various cancers, including melanoma and non-small cell lung cancer, and promotes cancer cell proliferation and metastasis. On the other hand, TRIM15 is also a key driver of osteoarthritis and participates in regulating inflammation and innate immune signaling.

[0003] Specifically, TRIM15 has been identified as a key regulatory protein in the RIG-1-MAVS signaling pathway. Overactivation of this pathway is a contributing factor to various autoimmune diseases, and silencing TRIM15 through genetic means can significantly inhibit the secretion of type I interferon downstream of this signaling pathway, indicating that TRIM15 is a potential important target for anti-inflammatory and therapeutic purposes against autoimmune diseases.

[0004] Given the crucial role of TRIM15 in tumorigenesis and inflammatory signaling pathways, developing small molecule compounds capable of specifically modulating its biological activity has become a key scientific challenge in addressing related disease treatments. However, current intervention strategies targeting TRIM15 are limited to genetic methods such as gene knockout or RNA interference, with no reports of specific small molecule inhibitors or modulators. Therefore, there is an urgent need in this field for two innovative tools: first, small molecules capable of efficiently and specifically degrading TRIM15 protein for the treatment of diseases driven by TRIM15 dysfunction; and second, chemical probes that specifically utilize the E3 ligase activity of TRIM15 for in-depth research into its catalytic mechanisms, substrate recognition, and complex functions in cellular signaling pathways. Summary of the Invention

[0005] The purpose of this invention is to solve the aforementioned technical problems and provide a class of bifunctional compounds targeting the TRIM15 protein based on the same core structural framework, and their applications. Based on newly discovered small molecule ligands for TRIM15, this invention has developed two classes of molecular entities with different functions:

[0006] The first class consists of proteolytic-targeting chimeric (PROTAC) molecules, which can efficiently and specifically degrade TRIM15 protein by recruiting endogenous E3 ubiquitin ligases. These molecules can effectively downregulate TRIM15 protein levels, thereby inhibiting the overactivation of the RIG-1 signaling pathway, and show great promise in the development of novel anti-inflammatory or autoimmune disease treatments.

[0007] The second category consists of TRIM15 recruiting molecules, which can serve as probes for chemical biology research. One end of this molecule specifically binds to TRIM15, while the other end couples with a target protein (such as a fusion protein with a reporter tag like Halo-tag) via a specific target protein-binding ligand (such as a haloalkane). This "guides" the E3 ligase activity of TRIM15 to the target protein, achieving specific ubiquitination modification. These molecules provide innovative tools for studying the E3 ligase activity of TRIM15, substrate specificity, and the biological function of the non-degrading ubiquitination signaling pathway.

[0008] To achieve the above dual objectives, the present invention provides the following technical solution:

[0009] A compound targeting the TRIM15 protein has the structure shown in formula (I):

[0010]

[0011] L is selected from -L1-Linker-E3 Ligand, COOR 11 -CONHR 22 , where R 11 It is H or C1-C5 alkyl, R 22 It is a C1-C5 alkyl group. In a first aspect, namely, in general formula (I), when L is selected from -L1-Linker-E3 Ligand, the present invention provides a PROTAC compound having the structure shown in formula (I-1), its optical isomer, or a pharmaceutically acceptable salt thereof:

[0012]

[0013] In formula (Ⅰ) or formula (I-1):

[0014] Ring A is selected from one or more substituted 4-8 membered aromatic heterocycles, 5-8 membered aromatic rings, 3-10 membered aliphatic heterocycles, and 3-10 membered aliphatic rings; the substituents are selected from: halogen, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxy, carboxyl, ester, amide, hydroxy, and cyano.

[0015] L1 is -CO-

[0016] The linker is a connecting group selected from one or more of the following structures: straight-chain or branched alkyl-(CH2). n1 -、-(CH2O) n2 -、-NR1-、-(CH=CHCH2) n3 -、-(C≡CCH2) n4 - 3-8 membered aliphatic rings, 3-8 membered aliphatic heterocycles, spirocycles, 5-8 membered aromatic rings, 4-8 membered aromatic heterocycles, -(CH2) n5 CONH(CH2) n6 -or any combination thereof;

[0017] Wherein, n1~n6 each independently represent natural numbers from 1 to 30; R1 is H or C1-C10 alkyl; the spirocycle is a structure in which two ring systems are connected by a common atom, wherein the two rings are independently selected from 3-8 membered aliphatic rings or aliphatic heterocycles.

[0018] E3 Ligand is the ligand for E3 ligase.

[0019] As a preferred option, ring A is selected from the following structures:

[0020] Among them, R A Selected from halogens, C1-C3 alkyl groups, C1-C3 haloalkyl groups, C1-C5 alkoxy groups, carboxyl groups, ester groups, amide groups, hydroxyl groups, and cyano groups;

[0021] X is selected from C, CH, and N;

[0022] Y is selected from O and S.

[0023] The ester group is -COORaa or -OCORaa, where Raa is selected from C1 to C3 alkyl groups.

[0024] The amide group is -CONHRaa or -NHCORaa, where Raa is selected from C1 to C3 alkyl groups.

[0025] The preferred A ring is selected from the following structures:

[0026]

[0027] Preferably, the Linker is selected from any one or more of the following units:

[0028] .

[0029] In the above structure, n represents a natural number from 1 to 20; further, n is a natural number from 1 to 10; X1, X2, Y1, and Y2 are each independently selected from N and CH; Z is selected from CH2, O, and C=O.

[0030] Preferably, the linker is selected from -NHCH2(CH2OCH2). n CH2O-, where n is 1, 2, 3, or 4. As a further preferred option, n is 2 or 3.

[0031] As a preferred option, Linker includes, but is not limited to, the following structures:

[0032] .

[0033] Preferably, E3 Ligand is selected from the following structures:

[0034] Where M is C=O or CH2. Preferably, E3 Ligand includes, but is not limited to, the following structures:

[0035] .

[0036] Preferably, the compounds represented by formula (I-1) provided by the present invention include, but are not limited to, the compounds shown in Table 1 below:

[0037] Table 1

[0038]

[0039]

[0040]

[0041] In one aspect of the invention, a pharmaceutical composition is provided comprising a therapeutically effective amount of a compound of formula (I-1), an optical isomer thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0042] In another aspect of the invention, the use of a compound of formula (Ⅰ-1), a pharmaceutically acceptable salt thereof, or the above-described pharmaceutical composition in the preparation of a medicament for: (1) inhibiting the RIG-1 signaling pathway by degrading the TRIM15 protein to treat diseases associated with overactivation of the RIG-1 signaling pathway, such as autoimmune diseases or inflammation; and (2) inducing the degradation of the TRIM15 protein in cells with high TRIM15 protein expression (e.g., human gastric adenocarcinoma, human cervical adenocarcinoma cells) for the preparation of an antitumor drug.

[0043] In a second aspect of the invention, when L is selected from COOR 11 -CONHR 22(where L is represented by L2), this invention provides a small affinity ligand targeting the TRIM15 protein, which specifically binds to the TRIM15 protein. It is a compound of formula (I-2), its optical isomer, or a pharmaceutically acceptable salt thereof:

[0044]

[0045] L2 is selected from COOR 11 -CONHR 22 A ring is the same as that defined in general formula (I) or (I-1) in the first aspect of the present invention.

[0046] In formula (I-2):

[0047] Ring A is selected from one or more substituted 4-8 membered aromatic heterocycles, 5-8 membered aromatic rings, 3-10 membered aliphatic heterocycles, and 3-10 membered aliphatic rings; the substituents are selected from: halogen, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxy, carboxyl, ester, amide, hydroxyl, and cyano.

[0048] As a preferred option, ring A is selected from the following structures:

[0049] in,

[0050] R A Selected from halogens, C1-C3 alkyl groups, C1-C3 haloalkyl groups, C1-C3 alkoxy groups, carboxyl groups, ester groups, amide groups, hydroxyl groups, and cyano groups;

[0051] X is selected from C, CH, and N;

[0052] Y is selected from O and S.

[0053] The preferred L2 is selected from -COOR 11 -CONHR 22 , where R 11 It is H or C1-C5 alkyl, R 22 It is a C1-C5 alkyl group;

[0054] The preferred A ring is selected from the following structures:

[0055]

[0056] Preferably, the small molecules with affinity for TRIM15 protein provided by the present invention include, but are not limited to, the following molecules:

[0057] .

[0058] In another aspect of the invention, the compound of formula (I-2) is provided for use in either of the following purposes: (1) as a control ligand or affinity tool for studying the biological function of the TRIM15 protein; or (2) as an intermediate for preparing a functional molecule targeting the TRIM15 protein, such as a PROTAC compound of formula (I) or a chemical probe of formula (II) below.

[0059] A third aspect of the invention provides a chemical probe (or TRIM15 recruiter) that targets TRIM15, which is capable of recruiting the TRIM15 protein to ubiquitinate a target protein (e.g., a Halo-tag fusion protein).

[0060] To achieve the above objectives, the present invention provides the compound of formula (II), its optical isomer, or a pharmaceutically acceptable salt thereof:

[0061] (II)

[0062] Wherein: the definition of ring A is the same as that in formula (I-1) of the first aspect of the present invention; L1 is -CO; the definition of linker is the same as that in formula (I-1) of the first aspect of the present invention; POI is a chemical group selected from functional groups that can bind to target proteins (such as Halo-tag tags).

[0063] Preferably, POI is a chloroalkane group.

[0064] Preferably, POI is a chemical group selected from the following structures:

[0065] Or C4-C10 haloalkanes.

[0066] Furthermore, the POI is selected from C5-C7 chloroalkanes; the Linker is selected from -NHCH2 (CH2OCH2). n CH2O-, where n is 1, 2, 3, or 4.

[0067] Preferably, the compounds of formula (II) provided by the present invention include, but are not limited to, the compounds shown in Table 2 below:

[0068] Table 2

[0069]

[0070] In another aspect of the invention, the use of compound (II) is provided for either of the following purposes: (1) as a research tool for investigating the catalytic activity and substrate specificity of TRIM15 protein as an E3 ubiquitin ligase; (2) in the preparation of reagents or kits for recruiting TRIM15 protein to ubiquitinate target proteins.

[0071] Definitions and Explanations

[0072] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0073] It should be understood that substitutions and combinations of substitutions as described herein, whether explicitly stated or not, refer to substitutions that conform to the valence of the substituted member. For example, substitution applied to carbon members refers to the tetravalence of C; when applied to nitrogen members, it refers to the trivalence of N; and when generally indicating a positive charge, it refers to the quadratic bond of the nitrogen member. The permissible options for valence are part of the art.

[0074] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0075] The term "pharmaceutically acceptable salt" refers to a salt of the compounds of this invention, prepared by reacting a compound having specific substituents discovered in this invention with a relatively non-toxic acid or base. When the compounds of this invention contain relatively acidic functional groups, base addition salts can be obtained by contacting a neutral form of such compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts or similar salts. When the compounds of this invention contain relatively basic functional groups, acid addition salts can be obtained by contacting a neutral form of such compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, hydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; as well as salts of amino acids (such as arginine) and salts of organic acids such as glucuronic acid. Certain compounds of the present invention contain both basic and acidic functional groups, and thus can be converted into either a base or an acid addition salt.

[0076] The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture thereof.

[0077] The term "isomer" refers to the fact that the compounds of this invention can exist in specific geometric or stereoisomeric forms. This invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this invention.

[0078] Unless otherwise stated, "(D)" or "(+)" indicates right-handed rotation, "(L)" or "(-)" indicates left-handed rotation, and "(DL)" or "(±)" indicates racemic rotation.

[0079] Unless otherwise specified, use wedge-shaped solid line keys ( ) and wedge-shaped dashed key ( ) represents the absolute configuration of a solid center, using a straight solid line key ( ) and straight dashed key ( The relative configuration of the center of a solid is represented by a wavy line ( ). ) indicates a wedge-shaped solid line key ( ) or wedge-shaped dashed key ( ), or use wavy lines ( ) indicates a straight solid line key ( ) and straight dashed key ( ).

[0080] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which can include deuterium and hydrogen variants, provided that the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may or may not be substituted, unless otherwise specified, and the type and number of substituents can be arbitrary on a chemically feasible basis.

[0081] When the listed substituents do not specify which atom they are attached to the substituted group, such substituents can be bonded to any of their atoms. For example, a phenyl group as a substituent can be attached to the substituted group via any carbon atom on the benzene ring. A ring system formed by a substituent being bonded to a central ring (e.g., (As shown) represents the substitution of one of the substituents at any of the substituted positions in the ring system.

[0082] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Thus, for example, if a group is substituted by 0-2 Rs, the group can optionally be substituted by at most two Rs, and the Rs in each case have independent options. Furthermore, combinations of substituents and / or their variants are only permitted if such combinations produce a stable compound.

[0083] Unless otherwise specified, a number range represents all integers including the numbers at both ends of the range. Unless otherwise specified, integers from 0 to 10 represent 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; integers from 1 to 5 represent 1, 2, 3, 4, 5; integers from 1 to 3 represent 1, 2, 3; C 1-3 Alkyl groups represent C1, C2, and C3 alkyl groups; C 1-6 Alkyl groups represent C1, C2, C3, C4, C5, and C6 alkyl groups; C 3-6 Cycloalkyl refers to C3, C4, C5, C6 cycloalkyl, and so on.

[0084] The purpose of this invention is to provide compounds targeting the TRIM15 protein and their applications. Firstly, this invention provides a class of PROTAC molecules with the structure of formula (I), which can specifically induce the degradation of the TRIM15 protein. This molecule can effectively inhibit the RIG-1 signaling pathway and has promising applications in the preparation of drugs for anti-inflammatory or autoimmune diseases. Simultaneously, this invention also provides a class of chemical probe molecules with the structure of formula (III). This molecule binds to TRIM15 at one end and can bind to a tag protein (such as a Halo-tag) or other target protein at the other end, thereby recruiting TRIM15 as an E3 ubiquitin ligase to ubiquitinate the tag protein (or target protein). This molecule can serve as an innovative research tool for exploring the E3 ligase biological function, substrate specificity, and non-K48 ubiquitination modification of TRIM15. Attached Figure Description

[0085] Figure 1 Affinity test for small molecule ligands.

[0086] Figure 2 TRIM15 degradation activity after PROTACs treatment of AGS cells.

[0087] Figure 3 The inhibitory effect of TRIM15 on the RIG-1 signaling pathway.

[0088] Figure 4 The representative compound T24 of formula (III) successfully recruited TRIM15 in HEK-293T cells and significantly enhanced its ubiquitination level of Halo-tagged fusion protein. Detailed Implementation

[0089] The present invention will be described in detail below with reference to embodiments, but this does not imply any adverse limitation on the invention. The present invention has been described in detail, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope thereof.

[0090] In the embodiments, experimental methods without specific conditions are conventional methods and conditions well known in the field, or are operated according to the conditions recommended by the instrument manufacturer.

[0091] Example 1. Synthesis of intermediate A3

[0092]

[0093] Step 1: In a dry 200 mL round-bottom flask, add 2.49 g of pyridinemethanol (22.84 mmol), 50 mL of dichloromethane, and 5 g of O-(2,4-dinitrophenyl)hydroxylamine (25.12 mmol) in sequence. Stir at room temperature for 21 h, concentrate under reduced pressure, slurry with dichloromethane and petroleum ether, filter, and obtain 6.45 g of yellow solid A1, which can be used directly in the next step.

[0094] Step 2: In a dry 200 mL round-bottom flask, add intermediate A1 (6.45 g, 20.94 mmol) and 40 mL of DMF. After complete dissolution, place the flask at 0 °C, add K2CO3 (4.05 g, 29.32 mmol), and then slowly add tert-butyl propargyl ester (3.0 mL, 21.99 mmol). Place the reaction system at 25 °C and stir thoroughly for 24 h. After the reaction is complete, filter using a diatomaceous earth pad, add an appropriate amount of DMF to wash the filter cake, add 80 mL of EA to the filtrate to dilute, and wash the organic phase with 40 mL × 3 times of water. Dry with anhydrous sodium sulfate, concentrate under reduced pressure, and the obtained crude product is purified by silica gel column chromatography to obtain 2.33 g of brownish-black oily liquid A2. ESI(M+H) + =249.2. 1 H NMR (400 MHz, Chloroform-d) δ 8.41 (d, J = 7.2 Hz, 1H), 8.28 (s,1H), 8.05 (s, 1H), 6.91 (dd, J = 7.0 Hz, 1H), 4.77 (s, 2H), 1.60 (s, 9H).

[0095] Step 3: Intermediate A2 (1.08 g, 4.35 mmol) was dissolved in 30 mL of anhydrous dichloromethane, and carbon tetrabromide (1.7 g, 5.23 mmol) was added. The mixture was placed under nitrogen protection in an ice bath at 0 °C. PPh3 (2.17 g, 8.27 mmol) was dissolved in 10 mL of DCM solution and added dropwise to the above mixture. The reaction was carried out at room temperature for 2 h. After the reaction was complete, the mixture was slowly added to 40 mL of saturated sodium bicarbonate solution, extracted with 40 mL × 3 dichloromethane, and the organic phases were combined. The mixture was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to give 1.14 g of white solid A3, with a yield of 84%. ESI (M+H) + =311.0. 1H NMR (400 MHz, Chloroform-d) δ 8.46 (d, J = 7.1 Hz, 1H), 8.32 (s, 1H), 8.12 (s, 1H), 6.95 (dd, J = 7.2, 2.1 Hz, 1H), 4.51 (s, 2H), 1.62 (s, 9H).

[0096] Example 2. Synthesis of intermediate A8

[0097]

[0098] Step 1: Dissolve (R)-2-Boc-aminomethylpyrrolidine (2 g, 9.95 mmol) in 20 mL of DMSO, then add L-proline (229 mg, 1.99 mmol), potassium carbonate (2.75 g, 19.90 mmol), iodobenzene (2.23 mL, 19.90 mmol), and CuI (378 mg, 1.99 mmol) sequentially. Under nitrogen protection, react at 90 °C for 4 h. After the reaction is complete, add 40 mL of ethyl acetate to the mixture, wash the organic phase with 40 mL × 3 times of water, dry the organic phase with anhydrous sodium sulfate, concentrate under reduced pressure, and precipitate the crude product by silica gel column chromatography to obtain 646 mg of white solid A4, yield 23%. ESI(M+H) + =277.1. 1 H NMR (400 MHz, Chloroform-d) δ7.26 – 7.20 (m, 2H), 6.72 – 6.67 (m, 3H), 4.68 (s, 1H), 3.86 (s, 1H), 3.49(t, J = 8.4 Hz, 1H), 3.44 – 3.34 (m, 1H), 3.19 – 3.01 (m, 2H), 2.07 – 1.88 (m, 4H), 1.46 (s, 9H).

[0099] Step 2: Add 2 mol / L hydrogen chloride-ethyl acetate solution (11.6 mL, 23.32 mmol) to product A2 (646 mg, 2.33 mmol), stir at room temperature for 4 h, and concentrate under reduced pressure to obtain 496 mg of white product A5. ESI(M+H) + =177.1.

[0100] Step 3: Intermediate A5 (496 mg, 2.33 mmol) was placed in a 50 mL round-bottom flask, 10 mL of dichloromethane was added, and an appropriate amount of DIPEA was added dropwise to neutralize the hydrochloride until dissolved. Then, a catalytic amount of glacial acetic acid (AcOH) and methyl 2-fluoro-5-formylbenzoate (466 mg, 2.56 mmol) were added, and the mixture was stirred at room temperature for 1 h. Sodium triacetoxyborohydride (1.48 g, 6.99 mmol) was added, and stirring continued overnight. After the reaction was complete, 10 mL of water was added, and the mixture was extracted with 10 mL × 3 times dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give 500 mg of white solid (intermediate A6), with a yield of 63%. ESI (M+H) + =343.1. 1 H NMR (400 MHz, Chloroform-d) δ 7.90 (dd, J = 6.9, 2.3 Hz,1H), 7.52 (ddd, J = 8.5, 4.6, 2.4 Hz, 1H), 7.23 – 7.18 (m, 2H), 7.09 (dd, J =10.5, 8.5 Hz, 1H), 6.69 (d, J = 7.3 Hz, 1H), 6.60 (d, J = 6.0 Hz, 2H), 3.93(s, 3H), 3.91 – 3.82 (m, 3H), 3.48 – 3.41 (m, 1H), 3.13 (dd, J = 8.9, 6.7 Hz,1H), 2.84 (dd, J = 11.9, 3.5 Hz, 1H), 2.63 (dd, J = 11.9, 8.3 Hz, 1H), 2.05(s, 2H), 2.01 – 1.97 (m, 3H).

[0101] Step 4: Intermediate A6 (500 mg, 1.46 mmol) was placed in a 25 mL round-bottom flask, and 10 mL of DMF was added to dissolve it completely. Then, potassium carbonate (605 mg, 4.39 mmol) and intermediate A3 (545 mg, 1.75 mmol) were added, and the mixture was reacted at room temperature for 4 h. After the reaction was complete, 30 mL of EA was added for dilution, and the organic phase was washed with 30 mL × 3 times of water. The organic phase was then washed with saturated brine and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to give 330 mg of a colorless oily product (A7), with a yield of 40%. ESI(M+H) + =573.2. 1H NMR (400 MHz, Chloroform-d) δ 8.47 (d, J = 8.0 Hz,1H), 8.32 (s, 1H), 8.05 (s, 1H), 7.94 – 7.90 (m, 1H), 7.55 (ddd, J = 8.5,4.6, 2.4 Hz, 1H), 7.18 – 7.10 (m, 3H), 7.04 (dd, J = 7.1, 1.9 Hz, 1H), 6.64 (tt, J = 7.3, 1.0 Hz, 1H), 6.49 – 6.45 (m, 2H), 3.95 (s, 3H), 3.89 – 3.78 (m,3H), 3.53 (dd, J = 19.3, 13.7 Hz, 2H), 3.32 – 3.25 (m, 1H), 3.05 (td, J =9.5, 6.4 Hz, 1H), 2.70 – 2.63 (m, 1H), 2.36 (dd, J = 13.2, 9.9 Hz, 1H), 2.10– 2.01 (m, 1H), 1.92 – 1.80 (m, 2H), 1.63 (s, 9H), 1.62 – 1.60 (m, 1H).

[0102] Step 5: Add 865 μL of methanol and 2.59 mL of tetrahydrofuran to the obtained product A7 (330 mg, 0.58 mmol), dissolve completely, then add 865 μL of 2 mol / L lithium hydroxide aqueous solution (1.73 mmol), and stir at room temperature for 1 h. After the reaction is complete, adjust the pH to acidic by adding an appropriate amount of 1N hydrochloric acid aqueous solution, extract with 6 mL × 3 dichloromethane, combine the organic phases, dry to anhydrous sodium sulfate, concentrate under reduced pressure, and give 317 mg of white foamy solid A8, yield 98%. ESI(M+H) + =559.1.

[0103] Example 3. Synthesis of Intermediate B1

[0104]

[0105] Step 1: Dissolve 2-(2,6-dioxadiidine-3-yl)-5-hydroxyisodihydroindole-1,3-dione (200 mg, 0.73 mmol) in 5 mL of DMF, add sodium carbonate (387 mg, 3.65 mmol), stir at 100 °C for 15 minutes, then add dropwise a DMF solution of 2-(Boc-amino)bromoethane (179 mg, 0.80 mmol), and incubate overnight at 100 °C. After the reaction is complete, dilute with 15 mL of EA, wash the organic phase with 15 mL × 3 times of water, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and purify the crude product by silica gel column chromatography to give 132 mg of white solid B1-1, yield 43%. ESI(M+H) + =418.1. 1 H NMR (400 MHz, DMSO-d6) δ11.15 (s, 1H), 7.92 – 7.89 (m, 2H), 7.86 (dd, J = 7.9, 1.2 Hz, 1H), 7.13 (t,J = 5.9 Hz, 1H), 5.16 (dd, J = 12.8, 5.4 Hz, 1H), 3.19 (q, J = 6.6 Hz, 2H), 2.89 – 2.84 (m, 1H), 2.65 – 2.52 (m, 4H), 2.10 – 2.01 (m, 1H), 1.38 (s, 9H).

[0106] Step 2: Add 2 mol / L hydrogen chloride-ethyl acetate solution (1.58 mL, 3.16 mmol) to the above product B1-1 (132 mg, 0.32 mmol), stir at room temperature for 2 h, and concentrate under reduced pressure to obtain 103 mg of white solid B1. ESI(M+H) + =318.1.

[0107] Example 4. Synthesis of target compound T1

[0108]

[0109] Step 1: Intermediate A8 (30 mg, 0.054 mmol) was placed in a 25 mL round-bottom flask, and HATU (24.5 mg, 0.064 mmol) and B1 (22.7 mg, 0.064 mmol) were added. Then, 5 mL of dichloromethane was added, followed by dropwise addition of DIPEA (47 μL, 0.269 mmol). The mixture was stirred at room temperature for 1 h. After the reaction was complete, 5 mL of water and 5 mL × 3 mL of dichloromethane were added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give 37 mg of yellow solid T1-1, with a yield of 80%. ESI(M+H) +=858.1. 1 H NMR (400 MHz, Chloroform-d) δ 8.48 (s, 1H), 8.46 (s,1H), 8.30 (s, 1H), 8.10 (dd, 1H), 8.01 (s, 1H), 7.77 (d, 1H), 7.55 – 7.49 (m,1H), 7.38 (d, J = 2.3 Hz, 1H), 7.23 – 7.04 (m, 6H), 6.61 (tt, J = 7.2, 1.1Hz, 1H), 6.47 – 6.43 (m, 2H), 4.95 (dd, J = 12.4, 5.3 Hz, 1H), 4.29 (t, J =5.2 Hz, 2H), 3.95 (q, J = 5.3 Hz, 2H), 3.85 – 3.76 (m, 3H), 3.54 (d, J = 13.0Hz, 2H), 3.27 (t, J = 9.3 Hz, 1H), 3.03 (td, J = 9.4, 6.4 Hz, 1H), 2.87 –2.63 (m, 4H), 2.38 – 2.30 (m, 1H), 2.16 – 2.04 (m, 2H), 1.89 – 1.79 (m, 3H), 1.62 (s, 9H).

[0110] Step 2: Dissolve intermediate T1-1 (37 mg, 0.043 mmol) in 200 μL of dichloromethane, add 100 μL of trifluoroacetic acid, and stir for 20 min. After the reaction is complete, remove the trifluoroacetic acid by rotary evaporation at low temperature. The crude product obtained is purified by preparative thin-layer chromatography to give 13 mg of yellow solid T1, with a yield of 38%. ESI(M+H) + =802.0. 1H NMR (400 MHz, Chloroform-d) δ 8.59(s, 1H), 8.51 (d, J = 7.1 Hz, 1H), 8.44 (s, 1H), 8.14 (d, J = 12.3 Hz, 2H), 7.76 (d, J = 8.3 Hz, 1H), 7.56 – 7.49 (m, 1H), 7.37 (d, J = 2.2 Hz, 1H), 7.25– 7.04 (m, 6H), 6.61 (t, J = 7.2 Hz, 1H), 6.46 (d, J = 8.1 Hz, 2H), 4.95 (dd,J = 12.3, 5.3 Hz, 1H), 4.29 (t, J = 5.3 Hz, 2H), 4.01 – 3.93 (m, 2H), 3.91 –3.76 (m, 3H), 3.66 – 3.54 (m, 2H), 3.28 (t, J = 8.5 Hz, 1H), 3.03 (q, J = 8.4Hz, 1H), 2.93 – 2.67 (m, 4H), 2.43 – 2.34 (m, 1H), 2.17 – 2.02 (m, 2H), 1.96 – 1.78 (m, 2H), 1.75 – 1.62 (m, 1H).

[0111] Example 5. Synthesis of target compound T2

[0112]

[0113] Step 1: Intermediate B2 was synthesized according to the method of Example 3, except that 2-(Boc-amino)bromoethane was replaced with "(4-bromobutyl)carbamate tert-butyl ester". Following the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B2 (24 mg, 0.064 mmol) instead of intermediate A8 (30 mg, 0.054 mmol) to obtain 30 mg T2-1 in 64% yield. ESI(M+H) + =886.2. 1H NMR (400 MHz, Chloroform-d) δ 8.47 (d, J = 8.0 Hz, 1H), 8.40 (s,1H), 8.30 (s, 1H), 8.10 (dd, J = 7.6, 2.4 Hz, 1H), 8.02 (s, 1H), 7.75 (d, J =8.4 Hz, 1H), 7.53 – 7.47 (m, 1H), 7.33 (d, J = 2.3 Hz, 1H), 7.19 – 7.04 (m,5H), 6.84 (dt, J = 12.4, 5.7 Hz, 1H), 6.62 (d, J = 7.3 Hz, 1H), 6.45 (d, J =9.9 Hz, 2H), 4.95 (dd, J = 12.5, 5.3 Hz, 1H), 4.13 (t, J = 6.1 Hz, 2H), 3.82 (dd, J = 13.6, 6.9 Hz, 3H), 3.61 – 3.51 (m, 4H), 3.31 – 3.24 (m, 1H), 3.03(td, J = 9.5, 6.4 Hz, 1H), 2.87 – 2.63 (m, 4H), 2.35 (dd, J = 13.1, 9.8 Hz,1H), 2.17 – 2.05 (m, 2H), 1.97 – 1.81 (m, 7H), 1.62 (s, 9H).

[0114] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T2-1 (30 mg, 0.034 mg) to obtain 20 mg of yellow solid T2, with a yield of 71%. ESI(M+H) + =830.0. 1H NMR (400 MHz, Chloroform-d) δ 8.81 (s, 1H), 8.51 (d, J = 7.1 Hz, 1H), 8.45 (s, 1H), 8.18 (s, 1H), 8.11(d, J = 7.7 Hz, 1H), 7.74 (dd, J = 8.2, 1.7 Hz, 1H), 7.57 – 7.50 (m, 1H), 7.32 (d, J = 2.3 Hz, 1H), 7.19 – 7.05 (m, 5H), 6.88 – 6.79 (m, 1H), 6.62 (t,J = 7.2 Hz, 1H), 6.46 (d, J = 8.1 Hz, 2H), 4.96 (dd, J = 12.3, 5.2 Hz, 1H), 4.15 (d, J = 5.9 Hz, 2H), 3.87 – 3.76 (m, 3H), 3.64 – 3.52 (m, 4H), 3.28 (t,J = 8.4 Hz, 1H), 3.03 (td, J = 9.4, 6.4 Hz, 1H), 2.93 – 2.66 (m, 4H), 2.43 –2.35 (m, 1H), 2.17 – 2.03 (m, 2H), 1.88 (dq, J = 23.7, 7.0, 5.9 Hz, 6H), 1.68(s, 1H).

[0115] Example 6. Synthesis of target compound T3

[0116]

[0117] Step 1: Intermediate B3 was synthesized according to the method of Example 3, except that 2-(Boc-amino)bromoethane was replaced with "(6-bromohexyl)carbamate tert-butyl ester". Following the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B3 (26 mg, 0.064 mmol) instead of intermediate A8 (30 mg, 0.054 mmol) to obtain 45 mg T3-1 in 92% yield. ESI(M+H) + =914.2. 1H NMR (400 MHz, Chloroform-d) δ 8.50 (s, 1H), 8.47 (d, J = 7.1 Hz,1H), 8.30 (s, 1H), 8.10 (dd, J = 7.7, 2.4 Hz, 1H), 8.01 (s, 1H), 7.75 (d, J =8.3 Hz, 1H), 7.49 (ddd, J = 7.8, 4.9, 2.3 Hz, 1H), 7.31 (d, J = 2.2 Hz, 1H), 7.17 – 7.04 (m, 5H), 6.82 – 6.72 (m, 1H), 6.61 (t, J = 7.3 Hz, 1H), 6.45 (d,J = 8.1 Hz, 2H), 4.95 (dd, J = 12.4, 5.3 Hz, 1H), 4.07 (t, J = 6.4 Hz, 2H), 3.86 – 3.77 (m, 3H), 3.55 (s, 1H), 3.54 – 3.46 (m, 3H), 3.27 (t, J = 8.1 Hz,1H), 3.03 (td, J = 9.5, 6.4 Hz, 1H), 2.79 – 2.62 (m, 4H), 2.35 (dd, J = 13.2,9.9 Hz, 1H), 2.15 – 2.05 (m, 2H), 1.85 (d, J = 6.8 Hz, 5H), 1.70 – 1.66 (m,2H), 1.62 (s, 9H), 1.50 (td, J = 13.0, 11.8, 6.7 Hz, 4H).

[0118] Step 2: Referring to the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T3-1 (45 mg, 0.049 mmol) to obtain 19 mg of yellow solid T3, with a yield of 45%. ESI(M+H) + =858.0. 1H NMR (400 MHz, Chloroform-d) δ 9.13 (s, 1H), 8.51 (d, J = 7.1 Hz, 1H), 8.44 (s, 1H), 8.23(s, 1H), 8.09 (dt, J = 7.0, 3.1 Hz, 1H), 7.75 (d, J 6.45 (d, J = 8.4 Hz, 2H), 4.97 (dd, J = 12.2,5.5 Hz, 1H), 4.08 (t, J = 5.9 Hz, 2H), 3.88 – 3.75 (m, 3H), 3.64 – 3.49 (m,4H), 3.27 (t, J = 8.6 Hz, 1H), 3.06 – 2.97 (m, 1H), 2.93 – 2.63 (m, 4H), 2.38(ddd, J = 13.0, 9.6, 3.2 Hz, 1H), 2.19 – 2.03 (m, 2H), 1.90 – 1.80 (m, 4H), 1.73 – 1.64 (m, 3H), 1.59 – 1.45 (m, 4H).

[0119] Example 7. Synthesis of target compound T4

[0120]

[0121] Step 1: Intermediate B4 was synthesized according to the method of Example 3, except that 2-(Boc-amino)bromoethane was replaced with "(2-(2-bromoethoxy)ethyl)tert-butyl carbamate". Following the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B4 (24 mg, 0.064 mmol) instead of intermediate A8 (30 mg, 0.054 mmol) to obtain 31 mg T4-1 in 64% yield. ESI(M+H) + =902.1. 1H NMR (400 MHz, Chloroform-d) δ 8.63 (s, 1H), 8.47 (d, J= 7.1 Hz, 1H), 8.30 (s, 1H), 8.08 (dd, J = 7.5, 2.3 Hz, 1H), 8.01 (s, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.50 (ddd, J = 7.9, 4.8, 2.4 Hz, 1H), 7.33 (s, 1H), 7.20 – 7.03 (m, 6H), 6.61 (t, J = 7.3 Hz, 1H), 6.45 (d, J = 8.1 Hz, 2H), 4.94(dd, J = 10.7, 5.3 Hz, 1H), 4.24 (d, J = 4.1 Hz, 2H), 3.94 – 3.86 (m, 2H), 3.85 – 3.70 (m, 7H), 3.53 (d, J = 13.7 Hz, 2H), 3.27 (t, J = 9.0 Hz, 1H), 3.03 (dd, J = 9.4, 6.5 Hz, 1H), 2.92 – 2.59 (m, 4H), 2.34 (dd, J = 13.1, 9.9Hz, 1H), 2.15 – 2.04 (m, 2H), 1.90 – 1.80 (m, 2H), 1.69 – 1.63 (m, 1H), 1.62(s, 9H).

[0122] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T4-1 (31 mg, 0.034 mmol) to obtain 12 mg of yellow solid T4, with a yield of 41%. ESI(M+H) + =846.1. 1H NMR (400 MHz, Chloroform-d) δ 10.01 (s, 1H), 8.49 (dd, J = 7.1, 3.0 Hz, 1H), 8.45 (d, J =2.2 Hz, 1H), 8.33 (d, J = 6.5 Hz, 1H), 8.01 (t, J = 8.4 Hz, 1H), 7.76 (dd, J= 8.3, 1.8 Hz, 1H), 7.65 (s, 1H), 7.43 (d, J = 2.6 Hz, 1H), 7.22 – 7.11 (m,4H), 7.07 – 7.00 (m, 1H), 6.95 (s, 1H), 6.61 (s, 1H), 6.45 (s, 3.26 (d, J = 7.9 Hz, 1H), 3.04 – 2.67(m, 5H), 2.40 (t, J = 12.4 Hz, 1H), 2.24 – 2.13 (m, 1H), 2.02 (d, J = 10.3Hz, 1H), 1.89 – 1.76 (m, 2H), 1.72 – 1.51 (m, 1H).

[0123] Example 8. Synthesis of target compound T5

[0124]

[0125] Step 1: Intermediate B5 was synthesized according to the method of Example 3, wherein 2-(Boc-amino)bromoethane was replaced with "(2-(2-(2-bromoethoxy)ethoxy)ethyl)carbamate tert-butyl ester". Referring to the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B5 (26 mg, 0.064 mmol) instead of intermediate A8 (30 mg, 0.054 mmol) to obtain 26 mg T5-1, in 51% yield. ESI(M+H) + =946.2. 1H NMR (400 MHz, Chloroform-d) δ 8.60 (s, 1H), 8.48 (d, J = 7.0 Hz, 1H), 8.30 (s, 1H), 8.08 (dd, J = 7.6, 2.4 Hz, 1H), 8.01(s, 1H), 7.73 (d, J = 8.3 Hz, 1H), 7.52 – 7.46 (m, 1H), 7.33 (d, J = 2.2 Hz, 1H), 7.20 – 7.11 (m, 4H), 7.08 – 7.02 (m, 2H), 6.60 (d, J = 7.3 Hz, 1H), 6.45(d, J = 7.6 Hz, 2H), 4.94 (dd, J = 11.2, 5.3 Hz, 1H), 4.25 – 4.17 (m, 2H), 3.92 – 3.87 (m, 2H), 3.81 (dd, J = 13.5, 8.6 Hz, 3H), 3.75 – 3.72 (m, 2H), 3.71 – 3.67 (m, 6H), 3.52 (d, J = 13.6 Hz, 2H), 3.27 (dd, J = 8.6 Hz, 1H), 3.07 – 2.98 (m, 1H), 2.90 – 2.62 (m, 4H), 2.38 – 2.30 (m, 1H), 2.16 – 2.05(m, 2H), 1.93 – 1.75 (m, 3H), 1.61 (s, 9H).

[0126] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T5-1 (26 mg, 0.027 mmol) to obtain 11 mg of yellow solid T5, with a yield of 48%. ESI(M+H) + =890.1. 1H NMR (400 MHz, Chloroform-d) δ 9.34 (s, 1H), 8.49 (dd, J = 7.0, 2.1 Hz, 1H), 8.42 (d, J =1.0 Hz, 1H), 8.21 (s, 1H), 8.06 (d, J = 8.6 Hz, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.54 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 1.7 Hz, 1H), 7.24 – 7.11 (m, 4H), 7.05 – 6.94 (m, 2H), 6.62 (s, 1H), 6.46 (d, J = 8.0 Hz, 2H), 4.98 (dd, J =12.3, 5.4 Hz, 1H), 4.23 (t, J = 4.7 Hz, 2H), 3.96 – 3.85 (m, 3H), 3.77 (dd, J= 8.2, 4.4 Hz, 4H), 3.70 (s, 6H), 3.66 – 3.50 (m, 2H), 3.33 – 3.23 (m, 1H), 3.03 (q, J = 8.2 Hz, 1H), 2.94 – 2.65 (m, 4H), 2.47 – 2.31 (m, 1H), 2.20 –2.03 (m, 2H), 1.96 – 1.77 (m, 2H), 1.75 – 1.59 (m, 1H).

[0127] Example 9. Synthesis of target compound T6

[0128]

[0129] Step 1: Intermediate B6 was synthesized according to the method of Example 3, wherein 2-(Boc-amino)bromoethane was replaced with "(2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethyl)tert-butyl carbamate". Referring to the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B6 (29 mg, 0.064 mmol) instead of intermediate A8 (30 mg, 0.054 mmol) to obtain 27 mg of T6-1, in a yield of 51%. ESI(M+H) + =990.5. 1H NMR (400 MHz, Chloroform-d) δ 8.58(s, 1H), 8.47 (d, J = 7.1 Hz, 1H), 8.30 (s, 1H), 8.07 – 8.02 (m, 1H), 8.01(s, 1H), 7.73 (d, J = 8.3 Hz, 1H), 7.53 – 7.45 (m, 1H), 7.33 (d, J = 2.3 Hz,1H), 7.23 – 7.03 (m, 6H), 6.61 (t, J = 7.3 Hz, 1H), 6.45 (d, J = 8.1 Hz, 2H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 4.21 (t, J = 4.6 Hz, 2H), 3.91 – 3.85 (m,2H), 3.81 (dd, J = 13.6, 6.1 Hz, 3H), 3.72 – 3.65 (m, 12H), 3.52 (dd, J =13.7, 4.6 Hz, 2H), 3.30 – 3.23 (m, 1H), 3.02 (td, J = 9.4, 6.3 Hz, 1H), 2.91– 2.62 (m, 4H), 2.33 (dd, J = 13.2, 10.0 Hz, 1H), 2.17 – 2.04 (m, 2H), 1.90 –1.76 (m, 2H), 1.71 – 1.63 (m, 1H), 1.61 (s, 9H).

[0130] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T6-1 (27 mg, 0.027 mmol) to obtain 11 mg of yellow solid T6, with a yield of 44%. ESI(M+H) + =934.1. 1H NMR (400 MHz, Chloroform-d) δ 8.93 (s, 1H), 8.50 (d, J = 7.1 Hz, 1H), 8.42 (s, 1H), 8.14(s, 1H), 8.08 (dd, J = 7.5, 2.3 Hz, 1H), 7.73 (d, J = 8.3 Hz, 1H), 7.52 –7.45 (m, 1H), 7.32 (d, J = 2.3 Hz, 1H), 7.26 – 7.02 (m, 6H), 6.61 (t, J = 7.2Hz, 1H), 6.46 (d, J = 8.2 Hz, 2H), 4.95 (dd, J = 12.4, 5.4 Hz, 1H), 4.20 (dd,J = 5.6, 3.6 Hz, 2H), 3.90 – 3.78 (m, 5H), 3.72 – 3.66 (m, 12H), 3.63 – 3.51(m, 2H), 3.28 (t, J = 8.5 Hz, 1H), 3.03 (td, J = 9.4, 6.3 Hz, 1H), 2.92 –2.65 (m, 4H), 2.38 (dd, J = 13.1, 9.6 Hz, 1H), 2.17 – 2.02 (m, 2H), 1.92 –1.80 (m, 2H), 1.76 – 1.62 (m, 1H).

[0131] Example 10. Synthesis of target compound T7

[0132]

[0133] Step 1: Intermediate B7 was synthesized according to the method of Example 3, except that 2-(Boc-amino)bromoethane was replaced with "(14-bromo-3,6,9,12-tetraoxatetradecyl)carbamate tert-butyl ester". Following the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B7 (17 mg, 0.043 mmol) instead of intermediate A8 (20 mg, 0.036 mmol) to obtain 34 mg T7-1 in 92% yield. ESI(M+H) + =1034.5. 1H NMR (400 MHz, Chloroform-d) δ 8.48 (dd, J =7.1, 0.9 Hz, 1H), 8.31 (s, 1H), 8.28 (s, 1H), 8.09 (dd, J = 7.6, 2.3 Hz, 1H), 8.02 (d, J = 1.8 Hz, 1H), 7.76 (d, J = 8.3 Hz, 1H), 7.52 – 7.47 (m, 1H), 7.35 (d, J = 2.2 Hz, 1H), 7.23 – 7.12 (m, 4H), 7.12 – 7.04 (m, 2H), 6.64 – 6.59(m, 1H), 6.48 – 6.44 (m, 2H), 4.95 (dd, 1H), 4.26 – 4.20 (m, 2H), 3.89 – 3.78(m, 5H), 3.71 – 3.64 (m, 16H), 3.53 (d, J = 13.6 Hz, 2H), 3.28 (t, J = 8.1Hz, 1H), 3.07 – 3.00 (m, 1H), 2.92 – 2.62 (m, 4H), 2.38 – 2.31 (m, 1H), 2.16 – 2.04 (m, 2H), 1.91 – 1.77 (m, 2H), 1.68 (s, 1H), 1.62 (s, 9H).

[0134] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T7-1 (34 mg, 0.033 mmol) to obtain 22 mg of yellow solid T7, with a yield of 68%. ESI(M+H) + =978.2. 1H NMR (400 MHz, Chloroform-d) δ 8.86 (s, 1H), 8.50 (d, J = 7.1 Hz, 1H), 8.41 (s, 1H), 8.14(s, 1H), 8.06 (dd, J = 7.4, 2.3 Hz, 1H), 7.73 (d, J = 8.3 Hz, 1H), 7.46 (ddd,J = 8.4, 4.9, 2.3 Hz, 1H), 7.33 (p, J = 5.4, 4.3 Hz, 2H), 7.20 – 7.02 (m,5H), 6.61 (t, J = 7.3 Hz, 1H), 6.47 (d, J = 8.1 Hz, 2H), 4.95 (dd, J = 12.4,5.4 Hz, 1H), 4.21 (dd, J = 5.7, 3.6 Hz, 2H), 3.88 – 3.80 (m, 5H), 3.71 – 3.64(m, 16H), 3.60 – 3.51 (m, 2H), 3.32 – 3.24 (m, 1H), 3.03 (td, J = 9.4, 6.4Hz, 1H), 2.91 – 2.67 (m, 4H), 2.39 (dd, J = 13.2, 9.7 Hz, 1H), 2.17 – 2.03(m, 2H), 1.91 – 1.80 (m, 2H), 1.74 – 1.63 (m, 1H).

[0135] Example 11. Synthesis of intermediate B8

[0136]

[0137] Step 1: Dissolve 100 mg (0.29 mmol) of 5-bromo-2-(2,6-dioxadiidine-3-yl)isodihydroindole-1,3-dione in 2 mL of DMF. Add cuprous iodide (11 mg, 0.058 mmol), bis(triphenylphosphine)palladium(II) dichloride (21 mg, 0.029 mmol), triethylamine (123 μL, 0.87 mmol), and tert-butyl 3-butynedicarbamate (60 mg, 0.35 mmol) sequentially. Under nitrogen protection, react overnight at 90 °C. After the reaction is complete, dilute the reaction solution with 6 mL of ethyl acetate, wash the organic phase with 6 mL × 3 mL of brine, concentrate under reduced pressure, and purify the crude product by silica gel column chromatography to obtain 90 mg of yellow solid B8-1, yield 71%. ESI(M+H) + =426.1. 1H NMR (400 MHz, DMSO-d6) δ 11.15 (s, 1H), 7.92 – 7.89 (m,2H), 7.86 (dd, J = 7.9, 1.2 Hz, 1H), 7.13 (t, J = 5.9 Hz, 1H), 5.16 (dd, J =12.8, 5.4 Hz, 1H), 3.19 (q, J = 6.6 Hz, 2H), 2.89 – 2.84 (m, 1H), 2.65 – 2.52(m, 4H), 2.10 – 2.01 (m, 1H), 1.38 (s, 9H).

[0138] Step 2: Add 2 mol / L hydrogen chloride-ethyl acetate solution (1.05 mL, 2.11 mmol) to the above product B8-1 (90 mg, 0.21 mmol), and stir at room temperature for 2 h to obtain intermediate B8. ESI(M+H) + =326.0.

[0139] Example 12. Synthesis of target compound T8

[0140]

[0141] Step 1: Following the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B8 (35 mg, 0.107 mmol) instead of intermediate A8 (50 mg, 0.089 mmol) to obtain 40 mg of T8-1, with a yield of 52%. ESI(M+H) + =866.2. 1HNMR (400 MHz, Chloroform-d) δ 8.47 (d, J = 7.1 Hz, 1H), 8.41 (s, 1H), 8.30(s, 1H), 8.13 (dd, J = 7.6, 2.4 Hz, 1H), 8.02 (s, 1H), 7.85 (d, J = 1.2 Hz, 1H), 7.81 – 7.71 (m, 2H), 7.52 (ddd, J = 7.8, 4.9, 2.3 Hz, 1H), 7.22 – 7.02(m, 5H), 6.62 (t, J = 7.3 Hz, 1H), 6.46 (d, J = 8.1 Hz, 2H), 4.97 (dd, J =12.3, 5.3 Hz, 1H), 3.89 – 3.70 (m, 5H), 3.55 (dd, J = 13.7, 4.0 Hz, 2H), 3.28(t, J = 8.0 Hz, 1H), 3.07 – 2.98 (m, 1H), 2.91 – 2.82 (m, 3H), 2.79 – 2.61(m, 3H), 2.41 – 2.31 (m, 1H), 2.18 – 2.05 (m, 2H), 1.92 – 1.83 (m, 2H), 1.68– 1.64 (m, 1H), 1.62 (s, 9H).

[0142] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T8-1 (40 mg, 0.046 mmol) to obtain 13 mg of yellow solid T8, with a yield of 35%. ESI(M+H) + =810.1. 1H NMR (400 MHz, Chloroform-d) δ 8.64 (s, 1H), 8.51 (d, J = 7.1 Hz, 1H), 8.45 (s, 1H), 8.16(s, 2H), 7.84 (s, 1H), 7.79 – 7.70 (m, 2H), 7.58 – 7.50 (m, 1H), 7.19 – 7.07(m, 5H), 6.62 (t, J = 7.3 Hz, 1H), 6.47 (d, J = 8.1 Hz, 2H), 4.98 (dd, J =12.3, 5.3 Hz, 1H), 3.88 – 3.74 (m, 5H), 3.60 (dd, J = 17.7, 13.9 Hz, 2H), 3.29 (t, J = 8.5 Hz, 1H), 3.03 (td, J = 9.4, 6.4 Hz, 1H), 2.92 – 2.68 (m,6H), 2.44 – 2.33 (m, 1H), 2.19 – 2.06 (m, 2H), 1.92 – 1.82 (m, 2H), 1.73 –1.65 (m, 1H).

[0143] Example 13. Synthesis of target compound T9

[0144]

[0145] Step 1: Intermediate B9 was synthesized according to the method of Example 11, except that tert-butyl 3-butynedicarbamate was replaced with "5-hexyn-1-ylcarbamate". Following the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B9 (38 mg, 0.098 mmol) instead of intermediate A8 (50 mg, 0.089 mmol) to obtain 55 mg T9-1 in 69% yield. ESI(M+H) + =894.2. 1H NMR (400 MHz, Chloroform-d) δ 8.47 (d, J = 7.1 Hz, 1H), 8.42 (s,1H), 8.30 (s, 1H), 8.11 (dd, J = 7.6, 2.3 Hz, 1H), 8.01 (s, 1H), 7.85 (s,1H), 7.81 – 7.69 (m, 2H), 7.50 (ddd, J = 8.0, 5.0, 2.4 Hz, 1H), 7.19 – 7.01(m, 4H), 6.81 (dt, J = 12.4, 5.6 Hz, 1H), 6.61 (t, J = 7.3 Hz, 1H), 6.45 (d,J = 8.1 Hz, 2H), 5.00 – 4.91 (m, 1H), 3.82 (dd, J = 13.5, 8.1 Hz, 3H), 3.55(t, J = 9.3 Hz, 4H), 3.27 (t, J = 8.4 Hz, 1H), 3.02 (td, J = 9.5, 6.4 Hz,1H), 2.95 – 2.82 (m, 2H), 2.79 – 2.61 (m, 4H), 2.53 (t, J = 6.7 Hz, 2H), 2.35(dd, J = 13.2, 9.9 Hz, 1H), 2.17 – 2.03 (m, 2H), 1.87 – 1.82 (m, 3H), 1.76 –1.70 (m, 2H).

[0146] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T9-1 (39 mg, 0.044 mmol) to obtain 23 mg of yellow solid T9, with a yield of 64%. ESI(M+H) + =838.2. 1H NMR (400 MHz, Chloroform-d) δ 10.13 (s, 1H), 8.38 (s, 1H), 8.26 (s, 1H), 8.01 (s, 1H), 7.95 (dd, J = 7.7, 2.3 Hz, 1H), 7.71 (s, 1H), 7.68 – 7.56 (m, 2H), 7.40 (ddd, J =7.8, 5.0, 2.1 Hz, 1H), 7.05 – 6.92 (m, 4H), 6.89 (dd, J = 11.6, 5.8 Hz, 1H), 6.49 (t, J = 7.2 Hz, 1H), 6.34 (d, J = 8.1 Hz, 2H), 4.90 – 4.76 (m, 1H), 3.76– 3.64 (m, 3H), 3.50 – 3.40 (m, 4H), 3.16 (t, J = 8.4 Hz, 1H), 2.91 (td, J =9.5, 6.3 Hz, 1H), 2.78 – 2.70 (m, 2H), 2.69 – 2.47 (m, 4H), 2.43 (t, J = 6.7Hz, 2H), 2.32 – 2.21 (m, 1H), 2.05 – 1.91 (m, 2H), 1.76 – 1.69 (m, 3H), 1.67– 1.60 (m, 2H).

[0147] Example 14. Synthesis of target compound T10

[0148]

[0149] Step 1: Intermediate B10 was synthesized according to the method in Example 11, except that tert-butyl 3-butynedicarbamate was replaced with tert-butyl octyl-7-ynthin-1-carbamate. Intermediate A8 (50 mg, 0.089 mmol), TCFH (30 mg, 0.107 mmol), and 2 mL of DMF were added to a 25 mL round-bottom flask, followed by N-methylimidazole (35 μL, 0.448 mmol) and intermediate B10 (41 mg, 0.098 mmol). The reaction was carried out at room temperature for 1 h. After the reaction was complete, 6 mL of ethyl acetate was added for dilution, and the organic phase was washed with 6 mL × 3 mL of water. The solution was concentrated under reduced pressure and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography to give 59 mg of T10-1, with a yield of 71%. ESI(M+H) + =922.2. 1H NMR (400 MHz, Chloroform-d) δ 8.48 (dd, J = 7.1, 0.8Hz, 1H), 8.31 (s, 1H), 8.25 (s, 1H), 8.12 (dd, J = 7.6, 2.4 Hz, 1H), 8.02 (d,J = 1.7 Hz, 1H), 7.85 (s, 1H), 7.81 – 7.70 (m, 2H), 7.49 (ddd, J = 7.8, 4.9,2.4 Hz, 1H), 7.18 – 7.04 (m, 4H), 6.76 (dt, J = 12.3, 5.6 Hz, 1H), 6.62 (t, J= 7.2 Hz, 1H), 6.49 – 6.40 (m, 2H), 4.97 (dd, J = 12.4, 5.3 Hz, 1H), 3.82 (dd, J = 13.5, 8.8 Hz, 3H), 3.58 – 3.46 (m, 4H), 3.32 – 3.23 (m, 1H), 3.03(td, J = 9.5, 6.3 Hz, 1H), 2.95 – 2.61 (m, 4H), 2.47 (t, J = 6.9 Hz, 2H), 2.35 (dd, J = 13.2, 9.9 Hz, 1H), 2.18 – 2.02 (m, 2H), 1.93 – 1.80 (m, 2H),1.73 – 1.63 (m, 5H), 1.62 (s, 9H), 1.56 – 1.43 (m, 4H).

[0150] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T10-1 (59 mg, 0.063 mmol) to obtain 29 mg of yellow solid T10, with a yield of 68%. ESI(M+H) + =866.2. 1H NMR (400 MHz, Chloroform-d) δ 8.94 (s, 1H), 8.53 (d, J = 7.1 Hz, 1H), 8.45 (s, 1H), 8.16(s, 1H), 8.12 (d, J = 9.9 Hz, 1H), 7.84 (s, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.70 (d, J = 9.1 Hz, 1H), 7.55 – 7.48 (m, 1H), 7.17 – 7.05 (m, 4H), 6.83 –6.74 (m, 1H), 6.61 (t, J = 7.3 Hz, 1H), 6.45 (d, J = 8.1 Hz, 2H), 4.98 (dd, J= 12.3, 5.2 Hz, 1H), 3.88 – 3.76 (m, 3H), 3.63 – 3.47 (m, 4H), 3.27 (t, J =8.5 Hz, 1H), 3.07 – 2.98 (m, 1H), 2.93 – 2.63 (m, 4H), 2.46 (t, J = 6.9 Hz, 2H), 2.37 (dd, J = 13.1, 9.7 Hz, 1H), 2.18 – 2.11 (m, 1H), 2.06 (t, J = 8.9Hz, 1H), 1.93 – 1.80 (m, 2H), 1.65 (dd, J = 13.4, 5.9 Hz, 5H), 1.48 (dq, J =23.4, 8.1 Hz, 4H).

[0151] Example 15. Synthesis of target compound T11

[0152]

[0153] Step 1: Intermediate B11 was synthesized according to the method of Example 11, wherein tert-butyl 3-butynedicarbamate was replaced with "(2-(prop-2-yn-1-yloxy)ethyl)tert-butyl carbamate". Referring to the synthesis method of the target compound in Example 4, intermediate B10 (35 mg, 0.098 mmol) was used instead of intermediate B1 (50 mg, 0.089 mmol) to obtain 49 mg T11-1, with a yield of 61%. ESI(M+H) + =896.2. 1H NMR (400 MHz, Chloroform-d) δ 8.47 (d, J =7.0 Hz, 1H), 8.38 (d, J = 4.2 Hz, 1H), 8.30 (s, 1H), 8.09 (s, 1H), 8.02 (d, J= 1.8 Hz, 1H), 7.88 (s, 1H), 7.80 – 7.73 (m, 2H), 7.50 (ddd, J = 8.0, 4.8,2.3 Hz, 1H), 7.18 – 7.04 (m, 5H), 6.62 (t, J = 7.3 Hz, 1H), 6.46 (d, J = 8.1Hz, 2H), 4.96 (dd, J = 12.5, 5.3, 1.8 Hz, 1H), 4.46 (s, 2H), 3.86 – 3.74 (m,7H), 3.53 (dd, J = 13.7, 2.0 Hz, 2H), 3.27 (t, J = 8.0 Hz, 1H), 3.03 (td, J =9.4, 6.3 Hz, 1H), 2.93 – 2.63 (m, 4H), 2.34 (dd, J = 13.2, 9.9 Hz, 1H), 2.18– 2.05 (m, 2H), 1.90 – 1.81 (m, 2H), 1.74 – 1.72 (m, 1H), 1.62 (s, 9H).

[0154] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T11-1 (49 mg, 0.054 mmol) to obtain 27 mg of yellow solid T11, with a yield of 59%. ESI(M+H) + =840.1. 1H NMR (400 MHz, Chloroform-d) δ 9.14 (s, 1H), 8.51 (d, J = 7.1 Hz, 1H), 8.44 (s, 1H), 8.19(s, 1H), 8.10 (dt, J = 7.6, 2.3 Hz, 1H), 7.88 (s, 1H), 7.79 – 7.70 (m, 2H), 7.53 (ddd, J = 7.8, 4.8, 2.3 Hz, 1H), 7.21 – 7.02 (m, 5H), 6.61 (t, J = 7.2Hz, 1H), 6.45 (d, J = 7.3 Hz, 2H), 4.98 (dd, J = 11.6, 5.2 Hz, 1H), 4.50 –4.41 (m, 2H), 3.87 – 3.73 (m, 7H), 3.64 – 3.49 (m, 2H), 3.27 (t, J = 8.4 Hz,1H), 3.07 – 2.97 (m, 1H), 2.93 – 2.61 (m, 4H), 2.42 – 2.32 (m, 1H), 2.20 –2.00 (m, 2H), 1.92 – 1.77 (m, 2H), 1.76 – 1.59 (m, 1H).

[0155] Example 16. Synthesis of target compound T12

[0156]

[0157] Step 1: Intermediate B12 was synthesized according to the method of Example 11, wherein tert-butyl 3-butynedicarbamate was replaced with "(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)tert-butyl carbamate". Referring to the synthesis method of the target compound in Example 4, intermediate B12 (42 mg, 0.098 mmol) was used instead of intermediate B1 (50 mg, 0.089 mmol) to obtain 39 mg T12-1, with a yield of 46%. ESI(M+H) + =940.2. 1H NMR (400 MHz, Chloroform-d) δ 8.48(d, J = 7.3 Hz, 2H), 8.30 (s, 1H), 8.09 (dd, J = 7.6, 2.4 Hz, 1H), 8.02 (s,1H), 7.88 (d, J = 1.1 Hz, 1H), 7.82 – 7.73 (m, 2H), 7.49 (ddd, J = 7.9, 4.8,2.3 Hz, 1H), 7.22 – 7.03 (m, 5H), 6.61 (t, J = 7.3 Hz, 1H), 6.50 – 6.42 (m,2H), 5.01 – 4.93 (m, 1H), 4.46 (s, 2H), 3.85 – 3.76 (m, 5H), 3.75 – 3.69 (m,6H), 3.53 (d, J = 13.7 Hz, 2H), 3.27 (t, J = 8.9 Hz, 1H), 3.03 (td, J = 9.5,6.3 Hz, 1H), 2.94 – 2.60 (m, 4H), 2.34 (dd, J = 13.2, 9.9 Hz, 1H), 2.17 –2.05 (m, 2H), 1.90 – 1.81 (m, 2H), 1.76 – 1.73 (m, 1H), 1.62 (s, 9H).

[0158] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T12-1 (39 mg, 0.041 mmol) to obtain 17 mg of yellow solid T12, with a yield of 46%. ESI(M+H) + =884.2. 1H NMR (400 MHz, Chloroform-d) δ 9.30 (s, 1H), 8.50 (dd, J = 7.1, 2.3, 0.8 Hz, 1H), 8.43 (d, J= 0.8 Hz, 1H), 8.21 (s, 1H), 8.08 (dt, J = 7.7, 2.8 Hz, 1H), 7.89 (s, 1H), 7.82 – 7.73 (m, 2H), 7.55 (ddd, J = 8.4, 4.9, 2.4 Hz, 1H), 7.22 (d, J = 4.9Hz, 1H), 7.17 – 7.10 (m, 2H), 7.08 – 6.99 (m, 2H), 6.64 – 6.57 (m, 1H), 6.45(dd, J = 7.8, 2.7 Hz, 2H), 5.03 – 4.96 (m, 1H), 4.47 (s, 2H), 3.90 – 3.82 (m,1H), 3.82 – 3.77 (m, 4H), 3.75 – 3.70 (m, 6H), 3.62 (dd, J = 14.7, 3.3 Hz, 1H), 3.54 (t, J = 13.2 Hz, 1H), 3.31 – 3.22 (m, 1H), 3.02 (td, J = 9.4, 6.4Hz, 1H), 2.93 – 2.64 (m, 4H), 2.38 (ddd, J = 12.4, 9.8, 2.3 Hz, 1H), 2.18 –2.11 (m, 1H), 2.10 – 2.02 (m, 1H), 1.90 – 1.78 (m, 2H), 1.69 (q, J = 9.3, 8.1Hz, 1H).

[0159] Example 17. Synthesis of target compound T13

[0160]

[0161] Step 1: Intermediate B13 was synthesized according to the method of Example 11, wherein tert-butyl 3-butynedicarbamate was replaced with "(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl)tert-butyl carbamate". Referring to the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B13 (47 mg, 0.098 mmol) instead of intermediate A8 (50 mg, 0.089 mmol) to obtain 40 mg T13-1, yield 45%. ESI(M+H)+ =984.2. 1 H NMR (400 MHz, Chloroform-d)δ 8.48 (d, J = 7.1 Hz, 1H), 8.31 (d, J = 4.5 Hz, 2H), 8.10 (dd, J = 7.6, 2.4Hz, 1H), 8.02 (s, 1H), 7.88 (s, 1H), 7.84 – 7.74 (m, 2H), 7.49 (ddd, J = 7.8,4.8, 2.4 Hz, 1H), 7.20 – 7.05 (m, 5H), 6.62 (td, J = 7.3, 1.1 Hz, 1H), 6.48 –6.42 (m, 2H), 4.97 (dd, J = 12.3, 5.3 Hz, 1H), 4.44 (s, 2H), 3.83 – 3.68 (m,15H), 3.53 (d, J = 13.7 Hz, 2H), 3.28 (t, J = 8.9 Hz, 1H), 3.03 (td, J = 9.5,6.3 Hz, 1H), 2.94 – 2.63 (m, 4H), 2.34 (dd, J = 13.2, 9.9 Hz, 1H), 2.12 –1.97 (m, 2H), 1.89 – 1.80 (m, 2H), 1.69 – 1.66 (m, 1H), 1.62 (s, 9H).

[0162] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T13-1 (39 mg, 0.041 mmol) to obtain 21 mg of yellow solid T13, with a yield of 57%. ESI(M+H) + =928.2. 1H NMR (400 MHz, Chloroform-d) δ 8.91 (s, 1H), 8.50 (d, J = 7.1 Hz, 1H), 8.42 (s, 1H), 8.13(s, 1H), 8.08 (dd, J = 7.5, 2.4 Hz, 1H), 7.85 (s, 1H), 7.80 – 7.71 (m, 2H), 7.47 (ddd, J = 8.4, 4.8, 2.4 Hz, 1H), 7.28 (d, J = 11.5 Hz, 1H), 7.18 – 7.00(m, 4H), 6.61 (t, J = 7.3 Hz, 1H), 6.46 (d, J = 7.7 Hz, 2H), 4.98 (dd, J =12.3, 5.3 Hz, 1H), 4.43 (s, 2H), 3.83 – 3.69 (m, 15H), 3.62 – 3.53 (m, 2H), 3.31 – 3.24 (m, 1H), 3.03 (td, J = 9.5, 6.4 Hz, 1H), 2.94 – 2.65 (m, 4H), 2.38 (dd, J = 13.1, 9.7 Hz, 1H), 2.18 – 2.02 (m, 2H), 1.95 – 1.80 (m, 2H), 1.78 – 1.58 (m, 1H).

[0163] Example 18. Synthesis of intermediate B14

[0164]

[0165] Step 1: In a 50 mL round-bottom flask, 2-(2,6-dioxadiazin-3-yl)-5-fluoroisoindoline-1,3-dione (1.5 g, 5.43 mmol), piperazine-1-carboxylic acid tert-butyl ester (1.12 g, 5.98 mmol), 10 mL DMSO, and DIPEA (2.8 mL, 16.30 mmol) were added sequentially. The reaction was carried out at 100 °C for 1 h. After the reaction was complete, 30 mL of ethyl acetate was added for dilution, and the organic phase was washed with 30 mL × 3 mL of water. The mixture was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to give 1.2 g of yellow solid B14-1, with a yield of 50%. ESI(M+H) + =443.0. 1H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1H), 7.69 (d, J = 8.4Hz, 1H), 7.34 (d, J = 2.2 Hz, 1H), 7.24 (dd, J = 8.6, 2.3 Hz, 1H), 5.08 (dd,J = 13.0, 5.4 Hz, 1H), 3.46 (s, 8H), 2.93 – 2.83 (m, 1H), 2.63 – 2.52 (m,2H), 2.06 – 1.98 (m, 1H), 1.42 (s, 9H).

[0166] Step 2: Add 13.5 mL of 2 mol / L hydrogen chloride-ethyl acetate solution (27.15 mmol) to the above product B14-1 (1.2 g, 2.72 mmol), stir at room temperature for 2 h, concentrate under reduced pressure, add 15 mL of saturated sodium bicarbonate solution to liberate, extract with 15 mL × 3 dichloromethane, combine the organic phases, concentrate under reduced pressure, and give 956 mg of yellow solid B14. ESI(M+H) + =343.0.

[0167] Example 19. Synthesis of intermediate B15

[0168]

[0169] Step 1: In a dry 25 mL round-bottom flask, add intermediate B14 (100 mg, 0.29 mmol), dissolve completely in 2 mL of LDM, then add DIPEA (203 μL, 1.17 mmol) and tert-butyl (2-(2-bromoethoxy)ethyl)carbamate (109 mg, 0.35 mmol). Stir overnight at 60 °C. After the reaction is complete, dilute with 6 mL of ethyl acetate, wash the organic phase with 6 mL × 3 mL of water, concentrate under reduced pressure, and purify the crude product by silica gel column chromatography to give 106 mg of yellow solid B15-1, yield 69%. ESI (M+H) + =530.1. 1H NMR (400 MHz, Chloroform-d) δ 8.06 (s, 1H), 7.70 (d, 1H), 7.28 (d,J = 2.2 Hz, 1H), 7.05 (dd, J = 8.6, 2.4 Hz, 1H), 5.21 (s, 1H), 4.93 (dd, 1H),3.63 (t, J = 5.5 Hz, 2H), 3.54 (t, J = 5.2 Hz, 2H), 3.46 (t, J = 5.2 Hz, 4H),3.32 (dd, J = 5.6 Hz, 2H), 2.92 – 2.72 (m, 3H), 2.69 – 2.61 (m, 6H), 2.17 –2.09 (m, 1H), 1.43 (s, 9H).

[0170] Step 2: Add 1 mL of 2 mol / L hydrogen chloride-ethyl acetate solution (2.00 mmol) to the above product B15-1 (106 mg, 0.20 mmol), stir at room temperature for 2 h, and concentrate under reduced pressure to obtain compound B15. ESI(M+H) + =430.1.

[0171] Example 20. Synthesis of target compound T14

[0172]

[0173] Step 1: Following the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B14 (27 mg, 0.078 mmol) instead of intermediate A8 (40 mg, mmol) to obtain 16 mg T14-1, with a yield of 25%. ESI(M+H) + =883.2. 1H NMR(400 MHz, Chloroform-d) δ 8.48 (d, J = 7.0 Hz, 1H), 8.39 (s, 1H), 8.31 (s,1H), 8.06 (s, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.47 (ddd, 1H), 7.42 (dd, J =6.5, 2.2 Hz, 1H), 7.27 (d, J = 2.3 Hz, 1H), 7.17 – 7.03 (m, 5H), 6.63 (t, J =7.3 Hz, 1H), 6.47 (d, J = 8.6 Hz, 2H), 4.94 (dd, J = 12.3, 5.4 Hz, 1H), 3.97(s, 2H), 3.81 (dd, J = 18.7, 13.7 Hz, 3H), 3.63 – 3.50 (m, 4H), 3.45 (s, 2H), 3.31 (q, J = 8.2, 6.9 Hz, 3H), 3.05 (td, J = 9.5, 6.3 Hz, 1H), 2.91 – 2.65(m, 4H), 2.36 (dd, J = 13.1, 9.7 Hz, 1H), 2.18 – 2.05 (m, 2H), 1.89 (dt, J =14.2, 8.7 Hz, 2H), 1.75 – 1.73 (m, 1H), 1.62 (s, 9H).

[0174] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T14-1 (16 mg, 0.018 mmol) to obtain 7 mg of yellow solid T14, with a yield of 46%. ESI(M+H) + =827.2. 1H NMR (400 MHz, Chloroform-d) δ 8.51 (d, J = 7.1 Hz, 1H), 8.48 (s, 1H), 8.43 (s, 1H), 8.17(s, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.48 (d, J = 6.8 Hz, 2H), 7.29 (d, J = 2.3Hz, 1H), 7.18 – 7.11 (m, 3H), 7.04 (ddd, J = 12.6, 7.8, 2.1 Hz, 2H), 6.62 (d,J = 7.2 Hz, 1H), 6.48 (d, J = 8.7 Hz, 2H), 4.95 (dd, J = 12.3, 5.4 Hz, 1H),3.97 (s, 2H), 3.89 – 3.79 (m, 3H), 3.68 – 3.53 (m, 4H), 3.46 (s, 2H), 3.37 –3.30 (m, 3H), 3.09 – 3.02 (m, 1H), 2.91 – 2.71 (m, 4H), 2.47 – 2.37 (m, 1H), 2.16 – 2.05 (m, 2H), 1.94 – 1.85 (m, 2H), 1.78 – 1.69 (m, 1H).

[0175] Example 21. Synthesis of target compound T15

[0176]

[0177] Step 1: Following the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B15 (46 mg, 0.098 mmol) instead of intermediate A8 (50 mg, 0.089 mmol) to obtain 43 mg T15-1, with a yield of 49%. ESI(M+H) + =971.3. 1HNMR (400 MHz, Chloroform-d) δ 8.47 (d, J = 7.1 Hz, 1H), 8.30 (s, 1H), 8.24(s, 1H), 8.11 (dd, J = 7.6, 2.4 Hz, 1H), 8.01 (s, 1H), 7.67 (dd, J = 8.5, 1.7Hz, 1H), 7.54 – 7.49 (m, 1H), 7.22 (d, J = 2.3 Hz, 1H), 7.18 – 6.99 (m, 6H), 6.62 (t, J = 7.3 Hz, 1H), 6.46 (d, 2H), 4.93 (dd, J = 12.2, 5.3 Hz, 1H), 3.82(dd, J = 13.6, 10.3 Hz, 3H), 3.74 – 3.64 (m, 6H), 3.54 (dd, J = 13.7, 2.7 Hz, 2H), 3.40 (s, 4H), 3.28 (t, J = 8.9 Hz, 1H), 3.03 (td, J = 9.5, 6.4 Hz, 1H),2.93 – 2.63 (m, 10H), 2.35 (dd, J = 13.2, 9.9 Hz, 1H), 2.16 – 2.03 (m, 2H),1.84 (dt, J = 12.0, 7.1 Hz, 2H), 1.72 – 1.68 (m, 1H), 1.62 (s, 9H).

[0178] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T15-1 (43 mg, 0.044 mmol) to obtain 22 mg of yellow solid T15, with a yield of 55%. ESI(M+H) + =914.2. 1H NMR (400 MHz, Chloroform-d) δ 10.40 (s, 1H), 8.26 (d, J = 7.1 Hz, 1H), 8.10 (s, 1H), 7.85(s, 1H), 7.83 – 7.77 (m, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.29 (d, J = 5.4 Hz,1H), 7.15 (d, J = 10.3 Hz, 1H), 7.00 – 6.76 (m, 7H), 6.35 (t, J = 7.2 Hz,1H), 6.21 (d, J = 8.1 Hz, 2H), 4.73 – 4.67 (m, 1H), 3.59 (d, J = 13.8 Hz, 3H), 3.46 (d, J = 10.3 Hz, 6H), 3.36 (d, J = 12.9 Hz, 2H), 3.16 (t, J = 5.1Hz, 4H), 3.04 (t, J = 8.5 Hz, 1H), 2.78 (td, J = 9.4, 6.2 Hz, 1H), 2.54 –2.34 (m, 10H), 2.14 (dd, J = 13.2, 9.8 Hz, 1H), 1.89 – 1.81 (m, 2H), 1.63 (h,J = 6.8, 6.3 Hz, 2H), 1.45 (q, J = 10.9, 8.7 Hz, 1H).

[0179] Example 22. Synthesis of target compound T16

[0180]

[0181] Step 1: Following the method of Example 19, intermediate B16 was synthesized, wherein (2-(2-bromoethoxy)ethyl)carbamate tert-butyl ester was replaced with "(2-(2-(2-bromoethoxy)ethoxy)ethyl)carbamate tert-butyl ester" to obtain compound B16. Referring to the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B16 (51 mg, 0.098 mmol) instead of intermediate A8 (50 mg, 0.089 mmol) to obtain 67 mg of T16-1, with a yield of 74%. ESI(M+H) + =1014.2. 1H NMR (400MHz, Chloroform-d) δ 8.47 (d, J = 7.1 Hz, 2H), 8.30 (s, 1H), 8.07 (dd, J =7.6, 2.4 Hz, 1H), 8.02 (d, J = 1.7 Hz, 1H), 7.66 (dd, J = 8.5, 1.2 Hz, 1H), 7.49 (dt, J = 7.6, 4.1 Hz, 1H), 7.25 (d, J = 2.1 Hz, 1H), 7.23 – 6.99 (m,6H), 6.61 (t, J = 7.3 Hz, 1H), 6.45 (d, J = 8.1 Hz, 2H), 4.92 (dd, J = 12.3,5.3 Hz, 1H), 3.81 (dd, J = 13.6, 5.4 Hz, 3H), 3.70 – 3.64 (m, 10H), 3.52 (dd,J = 13.7, 4.6 Hz, 2H), 3.41 (t, J = 5.1 Hz, 4H), 3.27 (t, J = 8.0 Hz, 1H), 3.07 – 2.98 (m, 1H), 2.87 – 2.64 (m, 10H), 2.36 – 2.31 (m, 1H), 2.13 – 2.06(m, 2H), 1.88 – 1.80 (m, 2H), 1.70 – 1.65 (m, 1H), 1.62 (s, 9H).

[0182] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T16-1 (67 mg, 0.066 mmol) to obtain 23 mg of yellow solid T16, with a yield of 36%. ESI(M+H) + =958.2. 1H NMR (400 MHz, Chloroform-d) δ 8.97 (s, 1H), 8.45 (d, J = 7.0 Hz, 1H), 8.31 (s, 1H), 8.11(s, 1H), 8.04 (dd, J = 7.4, 2.3 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.46 –7.37 (m, 2H), 7.20 (s, 1H), 7.17 – 7.10 (m, 2H), 7.04 (dd, J = 11.1, 8.4 Hz,1H), 6.99 – 6.90 (m, 2H), 6.60 (t, J = 7.3 Hz, 1H), 6.46 (d, J = 8.1 Hz, 2H), 4.93 (dd, J = 12.3, 5.4 Hz, 1H), 3.81 (dd, J = 16.0, 10.8 Hz, 3H), 3.73 –3.62 (m, 10H), 3.61 – 3.47 (m, 2H), 3.41 (t, J = 5.2 Hz, 4H), 3.31 – 3.24 (m,1H), 3.02 (td, J = 9.4, 6.3 Hz, 1H), 2.88 – 2.63 (m, 10H), 2.37 (dd, J =13.1, 9.6 Hz, 1H), 2.09 (ddd, J = 17.0, 9.9, 6.4 Hz, 2H), 1.92 – 1.78 (m,2H), 1.77 – 1.65 (m, 1H).

[0183] Example 23. Synthesis of target compound T17

[0184]

[0185] Step 1: Following the method of Example 19, intermediate B17 was synthesized, wherein (2-(2-bromoethoxy)ethyl)carbamate tert-butyl ester was replaced with "(2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethyl)carbamate tert-butyl ester" to obtain compound B17. Referring to the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B17 (55 mg, 0.098 mmol) instead of intermediate A8 (50 mg, 0.089 mmol) to obtain 54 mg of T17-1, with a yield of 57%. ESI(M+H) + =1058.2. 1H NMR(400 MHz, Chloroform-d) δ 8.51 (s, 1H), 8.47 (d, J = 7.9 Hz, 1H), 8.30 (s,1H), 8.07 – 7.98 (m, 2H), 7.65 (d, J = 8.5 Hz, 1H), 7.49 (ddd, J = 7.9, 4.8,2.3 Hz, 1H), 7.28 (s, 1H), 7.24 (d, J = 2.3 Hz, 1H), 7.16 – 7.01 (m, 5H), 6.61 (t, J = 7.3 Hz, 1H), 6.45 (d, J = 8.1 Hz, 2H), 4.92 (dd, J = 12.3, 5.3Hz, 1H), 3.81 (dd, J = 13.7, 4.5 Hz, 3H), 3.70 – 3.63 (m, 14H), 3.52 (dd, J =13.7, 4.5 Hz, 2H), 3.42 (t, J = 5.1 Hz, 4H), 3.30 – 3.24 (m, 1H), 3.02 (td, J= 9.5, 6.3 Hz, 1H), 2.86 – 2.64 (m, 10H), 2.34 (d, J = 3.2 Hz, 1H), 2.11 –2.04 (m, 2H), 1.89 – 1.80 (m, 2H), 1.70 – 1.64 (m, 1H), 1.62 (s, 9H).

[0186] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T17-1 (54 mg, 0.051 mmol) to obtain 13 mg of yellow solid T17, with a yield of 25%. ESI(M+H) + =1002.2. 1H NMR (400 MHz, Chloroform-d) δ 8.67 (s, 1H), 8.47 (d, J = 7.1 Hz, 1H), 8.38 (s, 1H), 8.10(s, 1H), 8.05 (dd, J = 7.5, 2.3 Hz, 1H), 7.63 (d, J = 8.5 Hz, 1H), 7.46 (ddd,J = 8.0, 4.8, 2.3 Hz, 1H), 7.29 (s, 1H), 7.24 (d, J = 2.2 Hz, 1H), 7.14 (t, J= 7.7 Hz, 2H), 7.05 (dd, J = 11.3, 8.4 Hz, 1H), 7.00 (dd, J = 8.7, 2.4 Hz, 2H), 6.62 (t, J = 7.2 Hz, 1H), 6.48 (d, J = 8.1 Hz, 2H), 4.93 (dd, J = 12.2,5.3 Hz, 1H), 3.81 (d, J = 13.6 Hz, 3H), 3.71 – 3.62 (m, 14H), 3.60 – 3.52 (m,2H), 3.44 (d, J = 5.4 Hz, 4H), 3.29 (t, J = 8.4 Hz, 1H), 3.04 (td, J = 9.3,6.2 Hz, 1H), 2.90 – 2.69 (m, 10H), 2.40 (dd, J = 13.2, 9.7 Hz, 1H), 2.14 –2.06 (m, 2H), 1.91 – 1.82 (m, 2H), 1.76 – 1.68 (m, 1H).

[0187] Example 24. Synthesis of intermediate B18

[0188]

[0189] Step 1: Add 5 mL of dichloromethane to intermediate B14 (100 mg, 0.29 mmol), followed by acetic acid (2.3 μL, 0.058 mmol) and tert-butyl 3-formylazetane-1-carboxylic acid (56 mg, 0.32 mmol). Stir at room temperature for 1 h, then add sodium triacetoxyborohydride (186 mg, 0.88 mmol) and continue the reaction for another 4 h until complete. Add 10 mL of water and extract with 10 mL × 3 times dichloromethane. Combine the organic phases, concentrate under reduced pressure, and purify the crude product by silica gel column chromatography to obtain 65 mg of yellow solid B18-1, yield 44%. ESI (M+H)+ =512.1. 1 H NMR (400 MHz, Chloroform-d) δ 8.28 (s, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.27 (d, J = 2.3 Hz, 1H), 7.04 (dd, J = 8.6, 2.4Hz, 1H), 4.95 (d, J = 5.3 Hz, 1H), 4.03 (t, J = 8.3 Hz, 2H), 3.61 (dd, J =8.6, 5.3 Hz, 2H), 3.40 (t, J = 5.1 Hz, 4H), 2.92 – 2.71 (m, 4H), 2.64 (d, J =7.4 Hz, 2H), 2.57 (t, J = 5.1 Hz, 4H), 2.16 – 2.08 (m, 1H), 1.43 (s, 9H).

[0190] Step 2: Add 2 mol / L hydrogen chloride-ethyl acetate solution (0.64 mL, 1.27 mmol) to the above product B18-1 (65 mg, 0.13 mmol), stir at room temperature for 2 h, and concentrate under reduced pressure to obtain 67 mg of yellow solid B18. ESI(M+H) + =412.1.

[0191] Example 25. Synthesis of target compound T18

[0192]

[0193] Step 1: Following the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B18 (44 mg, 0.098 mmol) instead of intermediate A8 (50 mg, 0.089 mmol) to obtain 34 mg T18-1, with a yield of 40%. ESI(M+H) + =952.2. 1HNMR (400 MHz, Chloroform-d) δ 8.48 (dd, J = 7.1, 0.9 Hz, 1H), 8.31 (s, 1H), 8.21 (s, 1H), 8.05 (d, J = 1.7 Hz, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.55 (dd, J= 6.5, 2.2 Hz, 1H), 7.45 (dt, J = 7.5, 3.1 Hz, 1H), 7.26 (s, 1H), 7.15 (dd, J= 8.5, 7.2 Hz, 2H), 7.10 – 7.00 (m, 3H), 6.63 (t, J = 7.3 Hz, 1H), 6.50 –6.42 (m, 2H), 4.93 (dd, J = 12.4, 5.3 Hz, 1H), 4.30 (dd, J = 10.2, 8.4 Hz,1H), 4.14 (t, J = 8.6 Hz, 1H), 3.90 – 3.72 (m, 5H), 3.53 (dd, J = 19.5, 13.6Hz, 2H), 3.38 (t, J = 5.1 Hz, 4H), 3.31 – 3.23 (m, 1H), 3.09 – 2.99 (m, 1H), 2.93 – 2.71 (m, 4H), 2.65 (dt, J = 9.0, 3.1 Hz, 3H), 2.56 (q, J = 5.0 Hz,4H), 2.39 – 2.32 (m, 1H), 2.15 – 2.03 (m, 2H), 1.92 – 1.83 (m, 2H), 1.73 –1.69 (m, 1H), 1.63 (s, 9H).

[0194] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T18-1 (34 mg, 0.036 mmol) to obtain 23 mg of yellow solid T18, with a yield of 72%. ESI(M+H) + =896.2. 1H NMR (400 MHz, Chloroform-d) δ 8.72 (s, 1H), 8.49 (d, J = 7.0 Hz, 1H), 8.40 (s, 1H), 8.08 (d, J = 4.8 Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.56 (q, J = 5.2, 3.8 Hz, 1H), 7.41 – 7.34 (m, 1H), 7.25 (s, 1H), 7.16 (ddd, J = 9.0, 7.3, 2.0 Hz, 2H), 7.05– 6.96 (m, 3H), 6.67 – 6.59 (m, 1H), 6.49 (dd, J = 8.2, 4.5 Hz, 2H), 4.98 –4.88 (m, 1H), 4.32 (t, J = 9.3 Hz, 1H), 4.17 (t, J = 8.6 Hz, 1H), 3.92 – 3.81(m, 5H), 3.64 – 3.51 (m, 2H), 3.43 (d, J = 5.0 Hz, 4H), 3.31 (t, J = 8.5 Hz,1H), 3.06 (td, J = 10.0, 9.5, 4.8 Hz, 1H), 2.96 (t, J = 7.2 Hz, 1H), 2.90 –2.60 (m, 10H), 2.40 (ddd, J = 12.9, 9.6, 3.0 Hz, 1H), 2.17 – 2.06 (m, 2H), 1.97 – 1.83 (m, 2H), 1.81 – 1.66 (m, 1H).

[0195] Example 26. Synthesis of target compound T19

[0196]

[0197] Step 1: Following the method of Example 24, intermediate B19 was synthesized, except that 3-formylazetane-1-carboxylic acid tert-butyl ester was replaced with "1-Boc-3-pyrrolidinecarboxaldehyde" to obtain compound B19. Referring to the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B19 (45 mg, 0.098 mmol) instead of intermediate A8 (50 mg, 0.089 mmol) to obtain 30 mg T19-1, with a yield of 35%. ESI(M+H) + =966.3. 1H NMR (400 MHz, Chloroform-d) δ 8.47 (d, J= 7.1 Hz, 1H), 8.38 (d, J = 5.7 Hz, 1H), 8.31 (d, J = 1.8 Hz, 1H), 8.05 (s,1H), 7.70 – 7.64 (m, 1H), 7.44 – 7.38 (m, 2H), 7.28 (d, J = 2.3 Hz, 1H), 7.17– 7.01 (m, 5H), 6.63 (dd, J = 7.3, 2.7 Hz, 1H), 6.46 (t, J = 7.2 Hz, 2H), 4.93 (dt, J = 12.2, 4.8 Hz, 1H), 3.81 (t, J = 12.5 Hz, 3H), 3.58 – 3.48 (m,2H), 3.44 – 3.37 (m, 3H), 3.34 – 3.26 (m, 4H), 3.13 – 2.99 (m, 2H), 2.91 –2.66 (m, 4H), 2.65 – 2.56 (m, 3H), 2.55 – 2.42 (m, 4H), 2.39 – 2.28 (m, 2H), 2.15 – 2.02 (m, 3H), 1.89 – 1.76 (m, 4H), 1.62 (s, 9H).

[0198] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T19-1 (30 mg, 0.031 mmol) to obtain 14 mg of yellow solid T19, with a yield of 50%. ESI(M+H) + =910.2. 1H NMR (400 MHz, Chloroform-d) δ 8.64 (s, 1H), 8.48 (d, J = 7.0 Hz, 1H), 8.42 – 8.36 (m, 1H), 8.23 ​​– 7.98 (m, 1H), 7.66 (dd, J = 12.1, 8.5 Hz, 1H), 7.52 – 7.42 (m, 1H), 7.40 – 7.30 (m, 1H), 7.27 (d, J = 2.6 Hz, 1H), 7.18 – 7.12 (m, 2H), 7.09 –6.94 (m, 3H), 6.62 (t, J = 7.3 Hz, 1H), 6.48 (d, J = 8.1 Hz, 2H), 4.94 (dt, J= 12.0, 5.5 Hz, 1H), 3.89 – 3.78 (m, 3H), 3.70 – 3.58 (m, 2H), 3.45 (q, J =5.5, 5.0 Hz, 3H), 3.35 – 3.28 (m, 4H), 3.18 – 3.01 (m, 2H), 2.92 – 2.35 (m,13H), 2.17 – 1.97 (m, 3H), 1.95 – 1.83 (m, 2H), 1.80 – 1.64 (m, 2H).

[0199] Example 27. Synthesis of target compound T20

[0200]

[0201] Step 1: Following the method of Example 24, intermediate B20 was synthesized, wherein 3-formylazetane-1-carboxylic acid tert-butyl ester was replaced with "1-tert-butoxycarbonylpiperidine-4-carboxaldehyde" to obtain compound B20. Referring to the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B20 (47 mg, 0.098 mmol) instead of intermediate A8 (50 mg, 0.089 mmol) to obtain 34 mg T20-1, with a yield of 39%. ESI(M+H) + =980.1. 1H NMR (400 MHz, Chloroform-d) δ 8.47 (d,J = 6.9 Hz, 1H), 8.31 (s, 1H), 8.29 (s, 1H), 8.04 (s, 1H), 7.68 (d, J = 8.5Hz, 1H), 7.43 – 7.35 (m, 2H), 7.27 (d, J = 2.4 Hz, 1H), 7.18 – 7.11 (m, 2H), 7.09 – 7.00 (m, 3H), 6.63 (t, J = 7.3 Hz, 1H), 6.47 (dd, J = 7.5, 1.5 Hz,2H), 4.97 – 4.89 (m, 1H), 4.77 (d, J = 13.1 Hz, 1H), 3.88 – 3.75 (m, 3H), 3.59 – 3.44 (m, 3H), 3.40 (t, J = 5.0 Hz, 4H), 3.32 – 3.25 (m, 1H), 3.08 –3.00 (m, 1H), 2.91 – 2.62 (m, 5H), 2.55 (t, J = 5.2 Hz, 4H), 2.35 (dd, J =13.1, 9.9 Hz, 1H), 2.29 – 2.17 (m, 2H), 2.15 – 2.03 (m, 2H), 1.97 – 1.64 (m,9H), 1.62 (s, 9H).

[0202] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T20-1 (34 mg, 0.034 mmol) to obtain 23 mg of yellow solid T20, with a yield of 72%. ESI(M+H) + =924.1. 1H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 8.82 (d, J = 7.1 Hz, 1H), 8.37 (s, 1H), 8.10 (s,1H), 7.69 (d, J = 8.3 Hz, 1H), 7.50 (t, J = 7.1 Hz, 1H), 7.36 (d, J = 6.5 Hz, 2H), 7.27 (t, J = 8.9 Hz, 2H), 7.14 (d, J = 7.2 Hz, 1H), 7.04 (td, J = 7.7,3.5 Hz, 2H), 6.51 (t, J = 7.2 Hz, 1H), 6.40 (d, J = 7.5 Hz, 2H), 5.08 (dd, J= 12.8, 5.4 Hz, 1H), 4.51 (d, J = 12.6 Hz, 1H), 3.96 – 3.62 (m, 5H), 3.61 –3.28 (m, 10H), 3.19 (t, J = 8.7 Hz, 2H), 3.01 – 2.73 (m, 5H), 2.62 – 2.53 (m,3H), 2.29 (t, J = 11.6 Hz, 1H), 2.07 – 1.92 (m, 3H), 1.90 – 1.71 (m, 4H), 1.64 – 1.50 (m, 2H).

[0203] Example 28. Synthesis of intermediate B21

[0204]

[0205] Step 1: Add 4-(Boc-amino)cyclohexanecarboxylic acid (78 mg, 0.32 mmol) and HATU (133 mg, 0.35 mmol) to a dry 25 mL round-bottom flask. Dissolve completely in 3 mL of DCM. Then add intermediate B14 (100 mg, 0.29 mmol) and DIPEA (254 μL, 1.46 mmol). Stir at room temperature for 1 h. After the reaction is complete, add 9 mL of water to the system and extract with 9 mL × 3 DCM. Combine the organic phases, dry to anhydrous sodium sulfate, concentrate under reduced pressure, and purify the crude product by silica gel column chromatography to obtain 89 mg of yellow solid B21-1, yield 54%. ESI(M+H) + =568.1. 1H NMR (400 MHz, Chloroform-d) δ 8.28 (s,1H), 7.72 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 2.1 Hz, 1H), 7.06 (dd, J = 8.7,2.4 Hz, 1H), 4.94 (dd, J = 12.1, 5.3 Hz, 1H), 4.88 – 4.35 (m, 1H), 3.74 (d, J= 40.1 Hz, 5H), 3.48 – 3.40 (m, 4H), 2.94 – 2.69 (m, 3H), 2.50 (dd, J = 62.4,9.2 Hz, 1H), 2.16 – 2.07 (m, 2H), 1.88 – 1.70 (m, 4H), 1.68 – 1.56 (m, 3H), 1.43 (s, 9H).

[0206] Step 2: Add 2 mol / L hydrogen chloride-ethyl acetate solution (mL, mmol) to the above product B21-1 (mg, mmol), stir at room temperature for 2 h, and concentrate under reduced pressure to obtain a yellow solid B21. ESI(M+H) + =468.1.

[0207] Example 29. Synthesis of target compound T21

[0208]

[0209] Step 1: Following the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B21 (39 mg, 0.078 mmol) instead of intermediate A8 (40 mg, 0.071 mmol) to obtain 32 mg T21-1, with a yield of 44%. ESI(M+H) + =1008.2. 1HNMR (400 MHz, Chloroform-d) δ 8.47 (d, J = 7.1 Hz, 1H), 8.35 (s, 1H), 8.30(s, 1H), 8.09 (ddd, J = 14.0, 7.6, 2.4 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H),7.72 (d, J = 8.5 Hz, 1H), 7.52 – 7.46 (m, 1H), 7.29 (d, J = 2.3 Hz, 1H), 7.19– 6.89 (m, 6H), 6.64 – 6.59 (m, 1H), 6.46 (dt, J = 8.6, 1.3 Hz, 2H), 4.98 –4.90 (m, 1H), 4.18 (d, J = 121.0 Hz, 1H), 3.86 – 3.77 (m, 5H), 3.75 – 3.68(m, 2H), 3.57 – 3.50 (m, 2H), 3.49 – 3.41 (m, 4H), 3.28 (t, J = 8.6 Hz, 1H), 3.08 – 3.00 (m, 1H), 2.94 – 2.57 (m, 5H), 2.35 (ddd, J = 12.8, 9.8, 2.5 Hz, 1H), 2.30 – 2.22 (m, 1H), 2.15 – 2.05 (m, 2H), 2.04 – 1.98 (m, 1H), 1.87 –1.71 (m, 9H), 1.62 (s, 9H).

[0210] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T21-1 (32 mg, 0.032 mmol) to obtain 18 mg of yellow solid T21, with a yield of 60%. ESI(M+H) + =952.2. 1H NMR (400 MHz, Chloroform-d) δ 8.61 – 8.46 (m, 2H), 8.42 (d, J = 14.0 Hz, 1H), 8.13 (d, J =11.3 Hz, 1H), 7.94 – 7.75 (m, 1H), 7.72 (dd, J = 8.6, 2.9 Hz, 1H), 7.30 –7.26 (m, 2H), 7.20 – 6.83 (m, 6H), 6.65 – 6.60 (m, 1H), 6.50 (dd, J = 25.2,8.0 Hz, 2H), 4.95 (dd, J = 12.4, 5.3 Hz, 1H), 4.40 (d, 1H), 3.84 (dd, J =14.6, 10.6 Hz, 5H), 3.73 (d, J = 18.4 Hz, 2H), 3.61 (d, J = 15.1 Hz, 2H), 3.45 (s, 4H), 3.37 – 3.26 (m, 1H), 3.13 – 3.00 (m, 1H), 2.92 – 2.68 (m, 5H), 2.52 – 2.39 (m, 1H), 2.25 (d, J = 11.7 Hz, 1H), 2.14 – 1.72 (m, 12H).

[0211] Example 30. Synthesis of target compound T22

[0212]

[0213] Step 1: Following the method of Example 18, intermediate B22 was synthesized, wherein piperazine-1-carboxylic acid tert-butyl ester was replaced with "2,7-diazaspiro[3.5]nonane-7-carboxylic acid tert-butyl ester" to obtain compound B22. Referring to the synthesis method of the target compound in Example 4, intermediate B1 was replaced with intermediate B22 (41 mg, 0.098 mmol) instead of intermediate A8 (50 mg, 0.089 mmol) to obtain 27 mg T22-1, with a yield of 33%. ESI(M+H) + =923.2. 1H NMR (400 MHz, Chloroform-d) δ 8.47 (d, J= 7.1 Hz, 1H), 8.31 (s, 1H), 8.16 (s, 1H), 8.06 (s, 1H), 7.65 (d, J = 8.2 Hz,1H), 7.46 – 7.34 (m, 2H), 7.17 – 7.03 (m, 4H), 6.79 (d, J = 2.2 Hz, 1H), 6.63(t, J = 7.3 Hz, 1H), 6.53 (dd, J = 8.4, 2.2 Hz, 1H), 6.47 (d, J = 8.3 Hz, 2H), 4.93 (dd, J = 12.2, 5.3 Hz, 1H), 3.90 – 3.62 (m, 9H), 3.58 – 3.48 (m,2H), 3.37 – 3.21 (m, 3H), 3.09 – 3.01 (m, 1H), 2.91 – 2.65 (m, 4H), 2.40 –2.32 (m, 1H), 2.15 – 2.04 (m, 2H), 1.99 – 1.85 (m, 4H), 1.72 – 1.66 (m, 3H), 1.63 (s, 9H).

[0214] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T22-1 (27 mg, 0.029 mmol) to obtain 13 mg of yellow solid T22, with a yield of 52%. ESI(M+H) + =867.1. 1H NMR (400 MHz, Chloroform-d) δ 8.54 (s, 1H), 8.51 (d, J = 7.1 Hz, 1H), 8.45 (s, 1H), 8.15(s, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.43 (d, J = 5.5 Hz, 2H), 7.18 – 7.04 (m,4H), 6.77 (d, J = 2.1 Hz, 1H), 6.63 (t, J = 7.3 Hz, 1H), 6.52 – 6.46 (m, 3H), 4.93 (dd, J = 12.3, 5.3 Hz, 1H), 3.90 – 3.72 (m, 9H), 3.58 (d, J = 24.3 Hz,2H), 3.34 – 3.20 (m, 3H), 3.06 (dd, J = 9.6, 6.6 Hz, 1H), 2.91 – 2.68 (m,4H), 2.40 (dd, J = 13.1, 9.7 Hz, 1H), 2.15 – 2.02 (m, 2H), 1.97 – 1.84 (m, 4H), 1.82 – 1.65 (m, 3H).

[0215] Example 31. Synthesis of target compound T23

[0216]

[0217] Step 1: Following the synthetic method of the target compound in Example 4, intermediate B1 was replaced with compound "2-(2-((6-chlorohexyl)oxy)ethoxy)ethylamine hydrochloride" (10 mg, 0.038 mmol) instead of intermediate A8 (20 mg, 0.036 mmol) to obtain 20 mg of compound T23-1, with a yield of 74%. ESI(M+H) + =764.1.

[0218] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T23-1 (20 mg, 0.026 mmol) to obtain 9.6 mg of yellow solid T23, with a yield of 52%. ESI(M+H) + =708.1. 1H NMR (400 MHz, Chloroform-d) δ 8.51 (d, J = 7.1 Hz, 1H), 8.44 (s, 1H), 8.17 – 8.10 (m, 2H), 7.47 (ddd, J = 7.9, 4.9, 2.4 Hz, 1H), 7.18 – 7.13 (m, 2H), 7.12 – 7.05 (m,2H), 6.63 (t, J = 7.3 Hz, 1H), 6.48 (d, J = 8.0 Hz, 2H), 3.85 (t, J = 13.9Hz, 3H), 3.71 (d, J = 2.3 Hz, 4H), 3.66 (td, J = 6.8, 4.1 Hz, 3H), 3.62 –3.53 (m, 4H), 3.48 (dt, J = 13.8, 6.7 Hz, 4H), 3.33 – 3.27 (m, 1H), 3.05 (td,J = 9.4, 6.4 Hz, 1H), 2.70 (dd, J = 13.1, 3.2 Hz, 1H), 2.41 (dd, J = 13.3,9.8 Hz, 1H), 2.11 – 2.03 (m, 1H), 1.88 (td, J = 10.2, 9.2, 4.5 Hz, 2H), 1.78– 1.70 (m, 3H), 1.58 (p, J = 6.7 Hz, 3H), 1.46 – 1.40 (m, 2H).

[0219] Example 32. Synthesis of target compound T24

[0220]

[0221] Step 1: Following the synthetic method of the target compound in Example 4, intermediate B1 was replaced with compound "2-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)ethylamine hydrochloride" (12 mg, 0.039 mmol) instead of intermediate A8 (20 mg, 0.036 mmol) to obtain 23 mg of compound T24-1, with a yield of 79%. ESI(M+H) + =808.1.

[0222] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T24-1 (23 mg, 0.029 mmol) to obtain 11.4 mg of yellow liquid T24, with a yield of 52%. ESI(M+H) + =752.1.1 H NMR (400 MHz, Chloroform-d) δ 8.46 (d, J = 37.7 Hz, 2H), 8.23 ​​(d, J = 34.7 Hz, 2H), 7.40 (d, J = 7.5 Hz, 1H), 7.17 (t, J = 7.7 Hz, 2H), 7.05 (t, J = 9.8 Hz, 1H), 6.97(d, J = 6.5 Hz, 1H), 6.71 (t, J = 7.2 Hz, 1H), 6.59 (d, J = 7.9 Hz, 2H), 3.83(q, J = 16.7, 13.2 Hz, 4H), 3.68 (t, J = 11.3 Hz, 10H), 3.58 (s, 3H), 3.45(dt, J = 24.1, 7.7 Hz, 5H), 3.08 (t, J = 8.5 Hz, 1H), 2.76 (d, J = 12.9 Hz,1H), 2.52 (d, J = 11.3 Hz, 1H), 2.08 (s, 1H), 1.92 (d, J = 16.9 Hz, 2H), 1.78 (d, J = 8.7 Hz, 1H), 1.71 (q, J = 7.0 Hz, 2H), 1.60 – 1.50 (m, 2H), 1.43 –1.32 (m, 4H).

[0223] Example 33. Synthesis of target compound T25

[0224]

[0225] Step 1: Following the synthetic method of the target compound in Example 4, intermediate B1 was replaced with compound "18-chloro-3,6,9,12-tetraoxo-octadecane-1-amine hydrochloride" (14 mg, 0.04 mmol) instead of intermediate A8 (20 mg, 0.036 mmol) to obtain 23 mg of compound T25-1, with a yield of 74%. ESI(M+H) + =852.1.

[0226] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate T25-1 (23 mg, 0.027 mmol) to obtain 13 mg of yellow liquid T25, with a yield of 60%. ESI(M+H) + =796.1. 1H NMR (400 MHz, Chloroform-d) δ 8.45 (d, J = 36.6 Hz, 2H), 8.18 (s, 2H), 7.49 – 7.31 (m, 2H), 7.19 (d, J = 7.7 Hz, 2H), 7.11 – 6.92 (m, 2H), 6.83 – 6.61 (m, 3H), 3.96 –3.37 (m, 26H), 3.11 (d, J = 8.5 Hz, 1H), 2.84 (d, J = 12.8 Hz, 1H), 2.63 (s,1H), 2.13 – 1.79 (m, 4H), 1.72 (p, J = 6.8 Hz, 2H), 1.54 (s, 2H), 1.39 (t, J= 7.8 Hz, 2H), 1.31 (d, J = 7.4 Hz, 2H).

[0227] Example 34. Synthesis of small molecule ligand A9

[0228]

[0229] Step 1: Following the synthesis method of the target compound in Example 4, intermediate B1 was replaced with methylamine (3 mg, 0.098 mmol) instead of intermediate A8 (50 mg, 0.089 mmol) to obtain 30 mg of compound A9-1, with a yield of 58%. ESI(M+H) + =572.1.

[0230] Step 2: Following the synthesis method of the target compound in Example 4, intermediate T1-1 was replaced with intermediate A9-1 (30 mg, 0.087 mmol) to obtain 16 mg of white solid A9, with a yield of 59%. ESI(M+H) + =516.1. 1H NMR (400 MHz, Chloroform-d) δ 8.50 (d, J = 26.8 Hz, 2H), 8.16 (d, J = 10.0 Hz, 2H), 7.53 –7.44 (m, 1H), 7.20 – 7.04 (m, 4H), 6.82 (dd, J = 13.3, 5.4 Hz, 1H), 6.64 (t,J = 7.2 Hz, 1H), 6.49 (d, J = 8.0 Hz, 2H), 3.84 (dd, J = 14.0, 10.7 Hz, 3H), 3.59 (dd, J = 21.4, 13.9 Hz, 2H), 3.31 (t, J = 8.4 Hz, 1H), 3.05 (d, J = 4.5Hz, 4H), 2.69 (dd, J = 13.3, 3.3 Hz, 1H), 2.41 (dd, J = 13.2, 9.6 Hz, 1H), 2.11 – 2.03 (m, 1H), 1.97 – 1.82 (m, 2H), 1.76 – 1.64 (m, 1H).

[0231] Example 35. Evaluation of Bioactivity

[0232] I. Affinity and activity testing of small molecule ligands

[0233] Microscale Thermophoresis (MST)

[0234] Drug (A9) was dissolved in DMSO to prepare a 10 mM stock solution, which was then diluted with MST buffer (50 mM Tris pH 8.0, 80 mM NaCl, 0.05% TWEEN 20, 0.3 mM TCEP) to a working solution of 500 μM–0.15 nM. EGFP-labeled TRIM15-SPRY purified protein samples were diluted to 400 nM with MST buffer, and then an equal volume of protein sample was mixed with the compound working solution. After incubation at room temperature for 15 minutes, the sample was added to a Monolith NT.115 capillary tube and processed using the Monolith NT.115 instrument. Data were analyzed using the manufacturer's MO affinity analysis software (v2.3) (NanoTemper). The ordinate Fnorm (normalized fluorescence) represents the fluorescence intensity of unbound molecules as a function of bound state, representing fluorescence intensity; the abscissa Ligand Concentration represents ligand concentration. The results showed that the small molecule ligand had good affinity, with a Kd value of 164 nM ± 83 nM and a signal-to-noise ratio of 8.6 (above 5 is considered a good signal-to-noise ratio).

[0235] II. Western blot analysis of the degradation effect of compounds on TRIM15

[0236] Using DMSO as a negative control, three concentration gradients were set up, and Western blot was used to determine the degradation of TRIM15 protein in AGS cell line by the compounds.

[0237] Experimental materials:

[0238] Cell line: AGS cells

[0239] Culture medium: RPMI-1640 Medium

[0240] Drug preparation method: Dissolve the drug in DMSO to prepare a 10 mM stock solution, and dilute it according to a certain ratio to obtain the corresponding concentration.

[0241] 1. In vitro culture and drug administration of AGS cells

[0242] (1) In vitro culture of AGS cells:

[0243] The selected AGS was placed in a solution containing 5% CO. 2 The cells were cultured in a 37°C constant temperature incubator under the following conditions: RPMI-1640 Medium + 10% Hyclone serum. When the cell density reached 70-90%, the cells were passaged for future experiments.

[0244] (2) Cell seeding: Digest the cells and add the cell suspension to a 15 mL centrifuge tube. Centrifuge at 800 rpm for 4 min, resuspend the cells in 2 mL of culture medium and count them. Seed the cell suspension in a 24-well plate with 100,000 cells per well and leave for 12 h until all cells adhere to the plate.

[0245] (3) Cell drug delivery: Dissolve compounds (T1~T22) in DMSO, add 1 μL of the compound to each well, and the final concentrations are 0.5 μM, 1 μM and 5 μM, respectively. Incubate at 37°C in a 5% CO2 atmosphere. 2 The cells were incubated in a cell culture incubator for 12 h, with the DMSO group serving as a blank control.

[0246] 2. Cell lysis

[0247] At the end of the reaction time, collect the cells and wash them once with PBS. Add the appropriate volume of 4% SDS to lyse the cells according to the cell quantity, and sonicate until the cells are no longer viscous. Centrifuge at 12,000 rpm at room temperature for 30 min. Transfer the supernatant to a new EP tube for protein quantification.

[0248] 3. Protein quantification

[0249] Take 2 mg / mL BSA standard and dilute it 2 / 3 times to prepare the concentrations used for the standard curve, namely 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.0625 mg / mL. For each sample, 200 μL of the mixture of solution A and solution B from the BCA quantitative kit is required. Take the corresponding volume of solution A and solution B (volume ratio 50:1) and mix well. Add 10 μL of BSA at different concentrations and the sample to a 96-well plate, then add 200 μL of the mixed solution A and solution B, gently pat to mix, and incubate at 37℃ in the dark for 30 min. After the reaction, measure the absorbance at 562 nm and calculate the sample protein concentration using the standard curve.

[0250] 4. Western Blot

[0251] (1) Protein sample preparation

[0252] Take 40 μg of protein and add a certain amount of 6×Loading Buffer according to the volume to make the final concentration 2×Loading Buffer; heat at 95℃ for 10 min to denature, and after cooling, centrifuge and mix well for Western Blot experiment. Freeze the remaining sample at -80℃.

[0253] (2) Making adhesive

[0254] a. Separating gel: Select the concentration of the separating gel to be prepared according to the molecular weight of the target protein. First, set up the gel clamp: clamp the two glass plates of different thicknesses and keep the ground flat; add the separating gel reagent to the centrifuge tube in sequence and vortex to mix; after mixing, add the separating gel between the two glass plates, then add an appropriate amount of water on the top layer of the gel surface, let it stand at room temperature, and wait for it to solidify.

[0255] b. Concentrated Gel: Discard the water on the top of the separating gel and blot the remaining liquid with filter paper. Prepare the concentrated gel and vortex it to mix well. After mixing, add the concentrated gel between the two glass plates, insert the gel casting comb, and let it stand at room temperature until it solidifies.

[0256] (3) Electrophoresis

[0257] a. Preparation: Assemble the prepared Tricine SDS-PAGE gel with the electrophoresis apparatus. Add a certain amount of 1×Tricine buffer to the inner electrophoresis tank and add 1×Running buffer to the outer tank. Let it stand for several minutes and check for leakage.

[0258] b. Sample loading: Vortex the sample to mix well, take a certain amount of sample (8-10 μL) and load it. After loading, turn on the electrophoresis apparatus, first adjust to constant voltage mode, electrophoresis at 80 V for 20 min, and confirm that the current is normal. After the sample enters the separating gel, adjust the voltage to 150 V. When the sample reaches the appropriate position, stop electrophoresis.

[0259] (4) Transfer and sealing

[0260] a. Transfer: Add a sponge, thick filter paper, and thin filter paper to the transfer clamp in sequence; Take the gel: Use a peeling spatula to remove the concentrated gel clumps from the edges and carefully remove the separating gel, placing it in the center of the filter paper; Apply the membrane: Apply the PVDF membrane, pre-activated with methanol, to the gel from one side, removing any air bubbles; then add thin filter paper, thick filter paper, and sponge in sequence, and clamp the transfer clamp in place, assembling the transfer clamp. Add an appropriate amount of transfer solution to the transfer tank, add an appropriate amount of water to the outer layer of the transfer tank, and place an ice pack inside to keep the transfer tank in ice water to prevent the large amount of heat released during the transfer process from affecting the transfer effect.

[0261] b. Blocking: After the transfer is complete, open the transfer clamp, remove the gel and filter paper, and observe the PVDF membrane (if the transfer is successful, you can see that the protein marker has been completely transferred to the PVDF membrane). Then, cut the PVDF membrane according to the molecular weight of the target protein and place the membrane in 5% BSA at room temperature for 60 min.

[0262] (5) Incubate antibodies

[0263] a. Incubation with primary antibody: After blocking, recover BSA and wash away residual BSA with TBST buffer, then add an appropriate amount of primary antibody (dilute with antibody diluent according to the recommended dilution ratio in the antibody instructions), and incubate at 4 ℃ on a horizontal shaker for 8-12 h.

[0264] b. Incubation of secondary antibody: The primary antibody is recovered and washed three times with TBST (15 min, 5 min, 5 min), and the secondary antibody is added (generally prepared at a ratio of secondary antibody to 5% BSA = 1:5000). The mixture is then incubated on a horizontal shaker at room temperature for 60 min.

[0265] (6) Development

[0266] a. Membrane washing: Discard the secondary antibody and wash the membrane three times with TBST (15 min, 5 min, 5 min).

[0267] b. Exposure: Prepare ECL developer solution: Solution A: Solution B = 1:1, prepare fresh before use. Place the PVDF membrane on filter paper to remove residual TBST buffer. Use tweezers to pick up the strip and place it in the center of the exposure plate. Add ECL developer solution to evenly cover the strip. Finally, use an AI800 ultra-sensitive multi-functional imager to acquire the chemiluminescence signal.

[0268] The degradation activity of the molecules in the examples is detailed in Table 3, where “+” indicates degradation effect (degradation rate of 1~20% (inclusive) is marked as “+”, 20~40% (inclusive) is marked as “++”, 40~60% (inclusive) is marked as “+++”, 60~80% (inclusive) is marked as “++++”, and 80~100% (inclusive) is marked as “+++++”), and “-” indicates that no degradation effect was observed.

[0269] Table 3 TRIM15 degradation activity after PROTACs treatment of AGS cells

[0270]

[0271] Experimental results: Using GAPDH (an enzyme in glycolysis whose expression level is generally constant in the same cell or tissue and is rarely affected by external inducers) protein as an internal control, normalized analysis revealed that the compound of this invention has a certain degradation effect on TRIM15 in AGS cells (results are shown in Appendix). Figure 2 Among them, compound T6 exhibits the best degradation activity.

[0272] III. Inhibitory effect of TRIM15 degradation on RIG-1 signaling pathway

[0273] Using DMSO as a negative control, the expression of RIG-I downstream related genes in HeLa cell lines treated with compound (T6) was determined by quantitative real-time polymerase chain reaction.

[0274] Experimental materials:

[0275] Cell line: HeLa cells

[0276] Culture medium: Dulbecco's Modified Eagle Medium

[0277] Drug preparation method: Dissolve the drug in DMSO to prepare a 10 mM stock solution, and dilute it according to a certain ratio to obtain the corresponding concentration.

[0278] 1. In vitro culture and drug administration of HeLa cells

[0279] (1) In vitro culture of HeLa cells:

[0280] The selected HeLa cells were cultured in a 37°C constant temperature incubator containing 5% CO2 under the following conditions: Dulbecco's Modified Eagle Medium + 10% Hyclone serum. The cells were passaged when the cell density reached 70-90% for use in subsequent experiments.

[0281] (2) Cell seeding: Digest the cells and add the cell suspension to a 15 mL centrifuge tube. Centrifuge at 800 rpm for 4 min. Resuspend the cells in 2 mL of culture medium and count them. Seed the cell suspension in a 12-well plate with 250,000-300,000 cells per well and leave for 12 h until all cells adhere to the plate.

[0282] (3) Cell administration: The compound was dissolved in DMSO to prepare an appropriate concentration. 1 μL of the compound was added to each well, with a final concentration of 2 μM. After administration, the cells were incubated at 37°C in a cell culture incubator containing 5% CO2 for 12 h. The DMSO group was used as a blank control.

[0283] (4) Cell transfection: using Opti-MEM respectively TM Dilute the corresponding amount of Lipofectamine TM 3000 (3 μL / well) and Poly (I:C) (5 mg / ml), Poly (I:C) was added to Lipofectamine TM Vortex to mix thoroughly and let stand for 10 min. Add 100 μL of the mixture to each well and incubate the cells at 37°C with 5% CO2. 2Transfect in a cell culture incubator for 9-12 hours.

[0284] 2. Cell lysis

[0285] At the end of the treatment period, the cells were washed once with pre-cooled 1×PBS, lysed with 1 mL of Trizol solution, and transferred to 1.5 mL Eppendorf tubes.

[0286] 3. RNA extraction

[0287] (1) RNA extraction

[0288] Add 200 μL of CHCl3 to each sample tube and vortex thoroughly for at least 15 seconds to form a homogeneous suspension. Centrifuge at 12000 rpm and 4°C for 15 minutes. After centrifugation, the liquid will separate into three layers, from top to bottom: a colorless aqueous phase (where RNA is distributed), an intermediate layer, and a lower red organic phase. Carefully aspirate 200-400 μL of the upper aqueous phase into a new Eppendorf tube.

[0289] (2) Precipitate RNA

[0290] Add the same volume of isopropanol to the Eppendorf tube, gently invert it to mix thoroughly, let stand for 5-8 minutes, and centrifuge at 12000 rpm and 4°C for 10 minutes.

[0291] (3) Cleaning RNA

[0292] Centrifuge and discard the supernatant. Add 1 mL of 75% ethanol (DEPC water: anhydrous ethanol = 1:3) to wash the precipitate twice. Finally, leave the container open at room temperature to evaporate any remaining liquid.

[0293] (4) RNA quantification

[0294] Dissolve the RNA in different volumes of DECP water (20-30 μL) according to the total amount of RNA, ensure complete dissolution, determine the concentration using ThermoScientific NanoDrop, and store at -80℃, avoiding repeated freeze-thaw cycles.

[0295] 4. Reverse transcription

[0296] (1) Preparation system

[0297] Using TransScript ® Uni One-Step gDNA Removal and cDNA Synthesis SuperMix Kit. Prepare 0.2 mL eight-tube sets, add 2 μg RNA, 10 μL 2xTS Uni ReactionMix, and 1 μL TransScript to each sample.® Uni RT / RI Enzyme Mix, 1 μL Anchored Oligo(dT), Primer (0.5 pg / ul), 1 μL gDNA Remover, add RNase-free Water to a total volume of 20 μL, and mix thoroughly.

[0298] (2) Reverse transcription

[0299] Place the sample into the C1000 Touch™ Thermal Cycler instrument and set the program as follows: Lid: 105℃, Volume: 20μL, 1. 50℃, 30 min. 2. 85℃, 5 s. 3. 4℃, ∞, run.

[0300] 5. Quantitative Real-time Polymerase Chain Reaction

[0301] (1) Preparation system

[0302] Prepare 0.1 mL eight-tube strips. Add 10 μL TB green, 1.25 μL front primer, 1.25 μL back primer, and 100 ng cDNA to each sample. Add DEPC water to a final volume of 20 μL. Set up a duplicate set of wells and mix thoroughly.

[0303] (2) Running the instrument

[0304] Power on the device, select the corresponding program, place the eight-tube array into the detection area, and begin detection. Select Sample editor, RelQuant, name the genes and samples in sequence, select the positive control, and generate replicates.

[0305] (3) Analyze the data

[0306] Select Analysis to analyze Tm calling and Advanced relative quantification, calculate the results, and export the results. Experimental data were analyzed using GraphPad Prism 8 software, and a two-tailed Student's t-test was performed on the two groups of data. All statistical data are presented as mean ± standard error (SEM), n≥3; a p-value less than 0.05 indicates statistical significance.

[0307] Experimental results showed that in HeLa (human cervical cancer cell line) cells, under poly(I:C) stimulation (a synthetic double-stranded RNA used to mimic the process of viral infection), PROTAC significantly inhibited the mRNA expression of downstream genes of the RIG-I signaling pathway, including IFNB (interferon β), TNFα (tumor necrosis factor-α), IFIT1 (interferon-induced protein 1), IFIT2 (interferon-induced protein 2), and IFIT3 (interferon-induced protein 3). However, in TRIM15 knockout HeLa cells (HeLa-Trim15-KO), PROTAC had no significant effect on downstream genes of RIG-I, indicating that the effect of PROTAC is dependent on TRIM15.

[0308] IV. Immunoprecipitation detection of protein ubiquitination modification level

[0309] (1) Cell culture and transfection

[0310] HEK-293T cells were cultured in a 5% CO2 solution. 2 The cells were cultured in a 37°C constant temperature incubator under the following conditions: DMEM (Dulbecco's Modified Eagle Medium) + 10% Hyclone serum. Transfection was performed when the cell density reached 50-60%.

[0311] (2) Cell transfection:

[0312] Plasmid transfection was performed using JetPRIME transfection reagent. For example, in a 10cm culture dish, 500 μL of buffer, 10 μg of plasmid, and 20 μL of JetPRIME were added to a 1.5ml centrifuge tube. The mixture was vortexed, allowed to stand for 10 min, and then slowly added to the cell culture medium. After 6 h, the cell culture medium was replaced, and 10 μM of compound T24 was administered simultaneously. Cells were cultured for another 24 h before collection.

[0313] (3) Cell lysis and quantification: Collect cells that overexpress the target protein, wash them once with PBS, add an appropriate volume of 4% SDS lysis buffer, sonicate until no longer viscous, centrifuge at 12000 rpm for 30 min at room temperature, and transfer the supernatant to another EP tube; use the BCA kit to determine the protein concentration and take an appropriate amount of protein to add to Loading to prepare an Input sample.

[0314] (4) Calculate by adding 20 μL of HA / Flag beads to each sample group. Take an appropriate amount of beads, wash them once with 500mM NaCl washing solution, and then aliquot them into EP tubes. Take 1 mg of protein lysis buffer for each sample group and add it to the beads. Add 500mM NaCl washing solution to make up the volume to 1 mL, mix well, and incubate overnight at 4℃.

[0315] (5) Centrifuge at 6,000 rpm and 4℃ for 60 s, discard the supernatant, add 1 mL of 500mM NaCl washing solution to wash the beads, and wash on a shaker for 3 min; repeat the above operation 5 times, add 40 μL of 2× Loading buffer to elute the protein, place in a 95℃ metal bath for 10 min to heat and denature the protein, and perform Western blot experiment after the sample cools down.

[0316] Experimental results showed that in HEK-293T (human embryonic kidney cells) cells, simultaneous overexpression of the Myc-tagged TRIM15 protein (TRIM15-Myc) and the HA-tagged EGFP-Halotag fusion protein / Flag-tagged P53-Halotag fusion protein, followed by administration of compound T24, significantly enhanced the ubiquitination (UB) level of the fusion proteins.

Claims

1. A compound targeting the TRIM15 protein, characterized in that, It has the structure shown in equation (Ⅰ): ； Or it may be an optical isomer of the structure shown in formula (Ⅰ) or a pharmaceutically acceptable salt thereof; In formula (Ⅰ): L is selected from -L1-Linker-E3 Ligand, COOR 11 -CONHR 22 , where R 11 It is H or C1-C5 alkyl, R 22 It is a C1-C5 alkyl group; Ring A is selected from 4-8 membered aromatic heterocycles, 5-8 membered aromatic rings, 3-10 membered aliphatic heterocycles, and 3-10 membered aliphatic rings substituted with one or more substituents; the substituents are selected from: halogen, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxy, carboxyl, hydroxyl, cyano, ester, and amide. L1 is -CO-; Linker is a connecting group, composed of one or more of the following structures: straight-chain or branched alkyl-(CH2). n1 -、-(CH2O) n2 -、-NR1-、-(CH=CHCH2) n3 -、-(C≡CCH2) n4 - 3-8 membered aliphatic rings, 3-8 membered aliphatic heterocycles, spirocycles, 5-8 membered aromatic rings, 4-8 membered aromatic heterocycles, -(CH2) n5 CONH(CH2) n6 -; Wherein, n1~n6 each independently represent natural numbers from 1 to 30; R1 is H or C1-C10 alkyl; the spirocycle is a structure in which two ring systems are connected by a common atom, wherein the two rings are each independently selected from 3-8 membered aliphatic rings or aliphatic heterocycles; E3 Ligand is an E3 ligase ligand; The ester group is -COORaa or -OCORaa, and Raa is selected from C1~C3 alkyl groups; The amide group is -CONHRaa or -NHCORaa, where Raa is selected from C1 to C3 alkyl groups.

2. The compound according to claim 1, characterized in that, Ring A is selected from the following structures: ; in, R A The group is selected from halogens, C1-C3 alkyl groups, C1-C3 haloalkyl groups, C1-C5 alkoxy groups, carboxyl groups, hydroxyl groups, ester groups, amide groups, and cyano groups; the ester group is -COORaa or -OCORaa, and Raa is selected from C1-C3 alkyl groups; the amide group is -CONHRaa or -NHCORaa, and Raa is selected from C1-C3 alkyl groups. X is selected from C, CH, and N; Y is selected from O and S; Linker is selected from the following structures: ； In the above structure, n represents a natural number from 1 to 20; X1, X2, Y1, and Y2 are each independently selected from N and CH; Z is selected from CH2, O, and C=O. E3 Ligand is selected from the following structures: ; where M is C=O or CH2.

3. The compound according to claim 1 or 2, characterized in that, Ring A has the following structure: ； Linker is selected from the following structures: ； E3 Ligand is selected from the following structures: 。 4. The compound according to claim 1, characterized in that, Selected from the following compounds: Or it may be an optical isomer of the structure shown or a pharmaceutically acceptable salt thereof.

5. A pharmaceutical composition, characterized in that, It comprises the compound or prodrug as described in any one of claims 1 to 4.

6. Use of the compound according to any one of claims 1-4 or the pharmaceutical composition according to claim 5 in any of the following applications: When L is selected from -L1-Linker-E3 Ligand, it is applied in one of the following ways: (1) Application in the preparation of drugs that inhibit the RIG-1 signaling pathway by degrading TRIM15 protein; (2) As a research tool probe, it is used to explore the biological function of TRIM15 protein; The L is selected from COOR 11 -CONHR 22 , where R 11 It is H or C1-C5 alkyl, R 22 When it is a C1-C5 alkyl group, its application is as a TRIM15 targeting ligand in the preparation of bifunctional compounds targeting the TRIM15 protein.

7. A compound, characterized in that, Having the structure shown in Formula (II) or being an optical isomer of the structure shown in Formula (II) or a pharmaceutically acceptable salt thereof: ； Wherein, the A-ring is the same as the A-ring structure defined in claim 1, 2 or 3; L1 is -CO-; Linker as defined in claim 1, 2 or 3; POI is a chemical group selected from the following structures: Or C4-C10 haloalkanes.

8. The compound according to claim 7, characterized in that, POI is selected from C5-C7 chloroalkanes; Linker is selected from -NHCH2(CH2OCH2). n CH2O-, where n is 1, 2, 3, or 4.

9. The compound according to claim 7, characterized in that, Selected from the following compounds: 。 10. A pharmaceutical composition, characterized in that... It comprises the compound of any one of claims 7 to 9, its isomers, its pharmaceutically acceptable salts or prodrugs, and one or more pharmaceutically acceptable carriers, diluents or excipients.

11. The use of the compound according to any one of claims 7 to 9, or the pharmaceutical composition according to claim 10, in any of the following uses: (1) the use of the compound in the preparation of a research tool probe for studying the activity of TRIM15 protein E3 ubiquitin ligase; (2) the use in the preparation of a kit for recruiting TRIM15 protein ubiquitination target substrates.

Images