GSPT1 molecular glue degradation agent compound and application thereof

Abstract

The invention discloses a GSPT1 molecular glue degradation agent compound and application thereof, and particularly provides a compound shown as a formula I or pharmaceutically acceptable salt thereof. The invention aims to solve the technical problem that a high-selectivity and high-efficacy GSPT1 molecular glue degradation agent which can be used by GSPT1 dependent tumor patients is absent clinically at present, and provides a GSPT1 molecular glue degradation agent compound. The compound disclosed by the invention can be combined with an E3 ligase CRBN compound and change the specificity of the compound so as to induce ubiquitination and degradation of a compound related protein GSPT1, is novel in structure and has high selectivity and high degradation efficacy on the GSPT1 protein.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a GSPT1 molecular gel degrading compound and its applications. Background Technology

[0002] Targeted protein degradation technologies, particularly strategies utilizing molecular glue compounds to recruit CRL4CRBN E3 ubiquitin ligases to degrade disease-related proteins, have become a frontier in new drug development. GSPT1 (G1 to S phase transition 1), a key translation termination factor, is overexpressed in a range of malignant tumors such as acute myeloid leukemia (AML) and certain solid tumors. Its overexpression is closely related to the rapid proliferation and survival of tumor cells, making it a highly promising anti-tumor target.

[0003] Although pioneering glutarimide-based molecular gels (such as CC-885) have demonstrated antitumor efficacy in GSPT1 degradation, their clinical application is limited by significant toxic side effects. This is mainly due to their lack of selectivity for GSPT1 degradation, as they simultaneously degrade important transcription factors such as Ikaros (IKZF1) and Aiolos (IKZF3). Subsequent candidate drugs (such as CC-90009) have shown improved selectivity, but their chemical structure and pharmacological properties still have room for optimization, potentially facing challenges in areas such as pharmacokinetics, oral bioavailability, or overcoming drug resistance.

[0004] Therefore, there is an urgent need in this field to develop novel GSPT1 molecular gel degraders with novel structures, high selectivity, high potency, and superior pharmaceutical properties. Providing such new compounds will enable safer and more effective treatment options for patients with GSPT1-dependent tumors, meeting unmet clinical needs. Summary of the Invention

[0005] The technical problem this invention aims to solve is the current lack of highly selective and efficient GSPT1 molecular glue degraders that are available for GSPT1-dependent tumor patients in clinical practice. This invention provides a GSPT1 molecular glue degrader compound and its applications. The compound of this invention has a novel structure, high selectivity for GSPT1 protein, and high GSPT1 degradation efficiency.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] This invention provides a compound of formula I or a pharmaceutically acceptable salt thereof;

[0008] ;

[0009] in,

[0010] Ring A is , or , where X 2 With L 1 connect;

[0011] L 1 For single bond or -NR a -R b - ,pass The end is connected to ring A;

[0012] L 2 C 1-6 Alkylene, -O- or -NR a -;

[0013] Ring B is C 6-12 The aryl group or "a 5-10 membered heteroaryl group selected from one, two, or three of N, O, and S, with one, two, three, or four heteroatoms";

[0014] R 1 and R 2 Independent of H, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups;

[0015] R 3 Halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups;

[0016] n is 0, 1, 2, or 3;

[0017] X 1 For CH or N;

[0018] X 2 For CH or N;

[0019] R a For H or C 1-6 alkyl;

[0020] R b C 1-6 Alkylene.

[0021] In certain preferred embodiments of the present invention, in the compound of Formula I or its pharmaceutically acceptable salt, certain groups may be defined as follows, and other groups may be defined as described in any embodiment of the present invention (hereinafter referred to as "in some embodiments"): L 2C 1-6 Alkylene or -NR a -

[0022] In some implementations, ring B is C. 6-12 Aryl.

[0023] In some implementation schemes, R 1 and R 2 It is a halogen on its own.

[0024] In some implementation schemes, R 3 It is a halogen.

[0025] In some implementations, n is 1.

[0026] In some implementation schemes, R a For H.

[0027] In some implementations, each of the C 1-6 The alkylene group is independently methylene, ethylene, propylene, or butylene, preferably methylene.

[0028] In some implementations, each of the C 6-12 The aryl group is independently phenyl or naphthyl, preferably phenyl.

[0029] In some embodiments, each of the halogens is independently F, Cl, Br or I, preferably F or Br.

[0030] In some implementations, ring A is , , , , , or .

[0031] In some implementations, L 1 It is a single bond or -NH-CH2-.

[0032] In some implementations, L 2 It is either -NH- or -CH2-.

[0033] In some implementation schemes, for , or .

[0034] In some implementations, ring B is a phenyl group.

[0035] In some implementation schemes, for .

[0036] In some implementation schemes, R 1 and R 2 Independently, it is F.

[0037] In some implementation schemes, R 3 It is Br.

[0038] In some embodiments, the compound of formula I is a compound shown in formula I-1 and / or formula I-2:

[0039] , ;

[0040] The definitions of each substituent are independently described in any embodiment of the present invention.

[0041] In some embodiments, the compound of formula I is any of the following compounds:

[0042] , , , .

[0043] The present invention also provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0044] The present invention also provides the use of the compound of Formula I, its pharmaceutically acceptable salt, or the pharmaceutical composition thereof in the preparation of GSPT1 molecular gel degrading agents.

[0045] The present invention also provides the use of the compound of Formula I, its pharmaceutically acceptable salt, or the pharmaceutical composition thereof in the preparation of medicaments for the prevention and / or treatment of diseases associated with GSPT1.

[0046] In some implementations, the GSPT1-related disease is a tumor that highly expresses GSPT1, such as acute myeloid leukemia.

[0047] Unless otherwise specified, the terms used in this invention have the following meanings:

[0048] Those skilled in the art will understand that, according to conventions used in the art, the use of "" in the structural formulas describing the functional groups in this invention is appropriate. The "+" sign indicates that the corresponding group is connected to other segments or groups in the compound through that site. The "-" at the end of a group indicates that the group is connected to other segments in the molecule through that site.

[0049] When the linking groups listed in this invention do not specify their linking direction, the linking direction is the same as the reading order from left to right. Examples are given below. The linker group L1 is -CD-, and -CD- connects ring A and ring B in the same direction as the reading order from left to right to form... and does not constitute Specifically, in this invention, ring A is... express Through N and L in the mother 1 Connected by a single bond, rather than through C to L in the parent compound. 1 Connected by a single key.

[0050] The term "pharmaceuticalally acceptable salt" refers to a salt prepared from the compounds of the present invention with a relatively non-toxic, pharmaceutically acceptable acid or base. When the compounds of the present invention contain relatively acidic functional groups, a base addition salt can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable base in a pure solution or a suitable inert solvent. When the compounds of the present invention contain relatively basic functional groups, an acid addition salt can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. When the compounds of the present invention contain relatively acidic and relatively basic functional groups, they can be converted into base addition salts or acid addition salts. See Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977), or Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl and Camille G. Wermuth, ed., Wiley-VCH, 2002).

[0051] The term "alkyl" refers to an alkyl group having a specified number of carbon atoms (e.g., C40, C50, C6 ... 1-6 Alkyl groups are straight-chain or branched, saturated monovalent hydrocarbon groups. Alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0052] The term "alkylene" refers to a divalent group that is attached to the rest of the molecule by two single bonds, and the rest of the definition is the same as that of the term "alkyl".

[0053] The term "aryl" refers to an aryl group having a specified number of carbon atoms (e.g., C36, C46, ​​C56, C66). 6-12 Aryl groups are cyclic, unsaturated monovalent hydrocarbon groups, which can be monocyclic or polycyclic (e.g., two or three). When polycyclic, adjacent monocyclic rings share two atoms and one bond, and each ring is aromatic. Aryl groups include, but are not limited to, phenyl and naphthyl groups.

[0054] The term "heteroaryl" refers to a cyclic, unsaturated monovalent group having a specified number of ring atoms (e.g., 5-10), a specified number of heteroatoms (e.g., 1, 2, 3, or 4), and a specified type of heteroatom (1, 2, or 3 of N, O, and S). It can be monocyclic or polycyclic; in the case of polycyclic groups, adjacent monocyclic rings share two atoms and one bond, and each ring is aromatic. Heteroaryl groups are attached to the rest of the molecule via carbon atoms or heteroatoms; they are attached to the rest of the molecule via rings with or without heteroatoms.

[0055] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0056] The term "halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the definition of halogen is the same as that of the term "halogen", and the definition of alkyl is the same as that of the term "alkyl".

[0057] The term "multiple" refers to 2, 3, 4 or 5, preferably 2 or 3.

[0058] The term "alkoxy" refers to the group R. X -O-,R X The definition is the same as the term "alkyl".

[0059] The term "haloalkoxy" refers to an alkoxy group that has been substituted by one or more halogens, where the definition of halogen is the same as that of the term "halogen" and the definition of alkoxy group is the same as that of the term "alkoxy group".

[0060] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0061] The reagents and raw materials used in this invention are all commercially available.

[0062] The positive and progressive effects of this invention are as follows: the compound of this invention can bind to the E3 ligase CRBN complex and change its specificity to induce ubiquitination and degradation of the complex-related protein GSPT1. It has a novel structure and high selectivity and high degradation efficacy for GSPT1 protein. Detailed Implementation

[0063] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0064] The following are some abbreviations used in the experiment and their corresponding full names:

[0065]

[0066] Example 1: N-(3-bromophenyl)-1-[4-(2,6-dioxo-3-piperidinyl)-3,5-difluorophenyl]piperidine-4-carboxamide (Compound 1)

[0067]

[0068] Step 1: Synthesis of Compounds 1-2

[0069] Compound 1-1 (10.4 g, 44.82 mmol) and methyl acrylate (3.92 g, 45.53 mmol, 4.10 mL) were mixed in tetrahydrofuran (100 mL), and sodium methoxide (242.15 mg, 4.48 mmol) was added at 0 °C. The mixture was stirred at 25 °C for 1 hour. The reaction mixture was quenched with saturated ammonium chloride aqueous solution (50 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane:ethyl acetate = 20 / 1 to 5 / 1). Compound 1-2 was given.

[0070] 1 H NMR (400 MHz, DMSO-d6) δ 7.60 (s, 1H), 7.58 (s, 1H), 4.51 (t, J =8.0 Hz, 1H), 3.55 (s, 3H), 2.47 - 2.40 (m, 2H), 2.22 (qd, J = 7.2, 14.0 Hz,1H), 2.13 - 2.02 (m, 1H).

[0071] Step 2: Synthesis of compounds 1-3

[0072] Compounds 1-2 (13 g, 40.87 mmol) were dissolved in acetic acid (100 mL), followed by the addition of sulfuric acid (24.05 g, 245.20 mmol, 13.07 mL). The mixture was stirred at 90 °C for 2 hours under nitrogen protection. After cooling to room temperature, the mixture was slowly poured into ice water and filtered to obtain a filter cake. The filter cake was washed with distilled water (50 mL × 3) and concentrated under reduced pressure using an oil pump to obtain compounds 1-3. m / z = 305.8 [M+H] + .

[0073] 1H NMR (400 MHz, DMSO-d6) δ 11.00 (s, 1H), 7.50 (d, J = 8.4 Hz, 2H), 4.25 (dd, J = 5.2, 12.8 Hz, 1H), 2.90 - 2.74 (m, 1H), 2.55 (br d, J = 3.2 Hz,1H), 2.18 - 2.07 (m, 1H), 2.06 - 1.97 (m, 1H).

[0074] Step 3: Synthesis of compounds 1-5

[0075] Under nitrogen protection, compounds 1-3 (50 mg, 164.43 μmol) and 1-4 (33.51 mg, 180.87 μmol) were dissolved in dioxane (1 mL), followed by the addition of 1,3-bis[2,6-bis(1-propylbutyl)phenyl]-4,5-dichloro-2H-imidazol-1-onthium-2-endosalt; 3-chloropyridine; palladium dichloro(15.99 mg, 16.44 μmol) and Cs₂CO₃(160.72 mg, 493.28 μmol). The mixture was stirred at 100 °C for 2 hours. The mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by preparative thin-layer chromatography on silica gel (petroleum ether: ethyl acetate = 1:2) to give compounds 1-5. m / z = 409.1 [M+H] + .

[0076] Step 4: Synthesis of compounds 1-6

[0077] Compounds 1-5 (35 mg, 85.69 μmol) were dissolved in dichloromethane (1 mL), followed by the addition of trifluoroacetic acid (TFA, 1.54 g, 13.46 mmol, 1 mL). The mixture was stirred at 25 °C for 2 hours. The mixture was concentrated under reduced pressure to obtain trifluoroacetates of compounds 1-6, which were used directly in the next reaction without further purification. m / z = 353.1 [M+H] + .

[0078] Step 5: Synthesis of Compound 1

[0079] HOBt (8.69 mg, 64.33 μmol), EDCI (12.33 mg, 64.33 μmol), and DIPEA (16.63 mg, 128.66 μmol, 22.41 μL) were added to a DMF (1 mL) solution of compounds 1-6 (20 mg, 42.89 μmol, TFA salt), followed by 3-bromoaniline (7.38 mg, 42.89 μmol, 4.67 μL, 1 eq). The mixture was stirred at 25 °C for 12 h. The reaction solution was filtered to obtain the residue, which was purified by preparative high-performance liquid chromatography (HPLC) (column: Unisil 3-100 C18 Ultra 150×50 mm×3 μm; mobile phase: [water (0.225% formic acid)-acetonitrile]; gradient 40%-70%, 15 min). The collected fraction was lyophilized to obtain compound 1. m / z = 506.2 [M+H] + .

[0080] 1 H NMR (400 MHz, DMSO-d6) δ 10.87 (s, 1H), 10.10 (s, 1H), 7.97 (t, J =2.0 Hz, 1H), 7.50 (td, J = 1.6, 8.0 Hz, 1H), 7.31 - 7.18 (m, 2H), 6.66 (d, J = 12.8 Hz, 2H), 4.05 (dd, J = 5.2, 12.4 Hz, 1H), 3.84 (br d, J = 12.8 Hz, 2H), 2.87 - 2.70 (m, 3H), 2.60 - 2.52 (m, 2H), 2.09 (br dd, J = 3.2, 12.4 Hz,1H), 2.02 - 1.92 (m, 1H), 1.84 (br d, J = 11.6 Hz, 2H), 1.75 - 1.57 (m, 2H).

[0081] Example 2: N-(3-bromophenyl)-4-[4-(2,6-dioxo-3-piperidinyl)-3,5-difluorophenyl]piperazine-1-carboxamide (Compound 2)

[0082]

[0083] Step 1: Synthesis of Compound 2-2

[0084] Under nitrogen protection, 1,3-bis[2,6-bis(1-propylbutyl)phenyl]-4,5-dichloro-2H-imidazol-1-onthium-2-endosalt; 3-chloropyridine; palladium dichloroisocyanurate (15.99 mg, 16.44 μmol, 0.1 eq) and Cs₂CO₃ (160.72 mg, 493.28 μmol) were added to a dioxane (1 mL) solution of compounds 1-3 (50.00 mg, 164.43 μmol) and 2-1 (33.69 mg, 180.87 μmol); 3-chloropyridine; palladium dichloroisocyanurate (15.99 mg, 16.44 μmol, 0.1 eq) and Cs₂CO₃ (160.72 mg, 493.28 μmol). The mixture was stirred at 100 °C for 2 hours. The reaction solution was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by preparative thin-layer chromatography on silica gel (petroleum ether: ethyl acetate = 1:2) to obtain compound 2-2. m / z = 354.1 [M-55] + .

[0085] Step 2: Synthesis of Compounds 2-3

[0086] Trifluoroacetic acid (TFA) (1.54 g, 13.46 mmol, 1 mL) was added to a 1 mL solution of compound 2-2 (45 mg, 109.91 μmol) in dichloromethane. The mixture was stirred at 25 °C for 2 hours. The reaction solution was concentrated under reduced pressure to obtain the trifluoroacetate of compound 2-3, which could be used directly in the next reaction without further purification. m / z = 310.0 [M+H] + .

[0087] Step 3: Synthesis of Compound 2

[0088] To a solution of compounds 2-3 (20 mg, 47.24 μmol, TFA salt) in N,N-dimethylformamide (1 mL), N,N-diisopropylethylamine (18.32 mg, 141.73 μmol, 24.69 μL) was added, followed by phenyl 1-bromo-3-isocyanate (9.36 mg, 47.24 μmol). The reaction mixture was stirred at 25 °C for 12 h. The reaction solution was filtered to obtain a residue, which was purified by preparative high-performance liquid chromatography (HPLC) (column: Unisil 3-100 C18 Ultra 150×50 mm×3 μm; mobile phase: [water (containing 0.225% formic acid)-acetonitrile]; gradient 38%-68%, 15 min). The collected liquid was evaporated to remove the organic phase, and the remaining aqueous phase was lyophilized to finally obtain compound 2. m / z = 507.2 [M+H] + .

[0089] 1H NMR (400 MHz, DMSO-d6) δ 10.88 (s, 1H), 8.78 (s, 1H), 7.80 (t, J =2.0 Hz, 1H), 7.53 - 7.40 (m, 1H), 7.19 (t, J = 8.0 Hz, 1H), 7.13 - 7.07 (m,1H), 6.71 (d, J = 12.4 Hz, 2H), 4.07 (dd, J = 4.8, 12.8 Hz, 1H), 3.63 - 3.48(m, 4H), 3.30 - 3.20 (m, 4H), 2.86 - 2.73 (m, 1H), 2.52 (br s, 1H), 2.15 -2.03 (m, 1H), 2.00 - 1.86 (m, 1H).

[0090] Example 3: N-(3-bromophenyl)-4-[4-(2,6-dioxo-3-piperidinyl)-3,5-difluorophenyl]piperidine-1-carboxamide (Compound 3)

[0091]

[0092] Step 1: Synthesis of Compound 3-2

[0093] Under nitrogen protection, potassium fluoride (34.4 mg, 592 μmol) was added to a mixed solution of compounds 1-3 (60.0 mg, 197 μmol), compound 3-1 (91.5 mg, 296 μmol), and [2-(2-aminophenyl)phenyl]palladium (1+); bis(1-adamantyl)-butylphosphine; methanesulfonate (21.6 mg, 29.6 μmol) in dioxane (2 mL) and water (0.4 mL). The mixture was stirred at 80 °C for 2 hours under nitrogen protection. The reaction mixture was diluted with ethyl acetate (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the residue, which was purified by preparative thin-layer chromatography on silica gel (ethyl acetate: petroleum ether = 1:1) to obtain compound 3-2. m / z = 307.0 [M+H] + .

[0094] Step 2: Synthesis of Compound 3-3

[0095] Under nitrogen protection, palladium on carbon (11.0 mg, 10%) was added to a mixed solution of compound 3-2 (35.0 mg, 86.1 μmol) in tetrahydrofuran (5 mL) and ethanol (5 mL). The suspension was degassed under vacuum and purged several times with hydrogen. The mixture was stirred at 25 °C for 12 h under hydrogen (15 Psi) protection. The reaction mixture was filtered, and the filter cake was washed with tetrahydrofuran (50 mL). The organic phases were combined and concentrated under reduced pressure to give compound 3-3. m / z = 353.0 [M+H-55] + .

[0096] Step 3: Synthesis of Compounds 3-4

[0097] A hydrochloric acid / dioxane solution (2 M, 4.5 mL) was added to a dichloromethane (1.5 mL) solution of compound 3-3 (35.0 mg, 85.7 μmol). The mixture was stirred at 25 °C for 12 hours. The reaction mixture was concentrated under reduced pressure to give the hydrochloride salt of compound 3-4. m / z = 309.2 [M+H] + .

[0098] Step 4: Synthesis of Compound 3

[0099] To a mixed solution of compounds 3-4 (25.0 mg, 72.5 μmol, hydrochloride) and N,N-diisopropylethylamine (46.9 mg, 363 μmol) in N,N-dimethylformamide (1 mL), 1-bromo-3-isocyanate (17.2 mg, 87.0 μmol) was added. The mixture was stirred at 25 °C for 2 hours. After filtration, the filtrate was purified by preparative high-performance liquid chromatography (HPLC) (column: Unisil 3-100 C18 Ultra 150×50 mm×3 μm; mobile phase: [water (containing 0.225% formic acid)-acetonitrile]; gradient 39%-69%, 15 min) to obtain compound 3. m / z = 508.2 [M+H] + .

[0100] 1H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 8.69 (s, 1H), 7.82 (s, 1H), 7.50 - 7.43 (m, 1H), 7.19 (t, J = 8.0 Hz, 1H), 7.13 - 7.03 (m, 3H), 4.27 (d,J = 13.2 Hz, 2H), 4.20 (dd, J = 5.2, 12.8 Hz, 1H), 2.92 - 2.75 (m, 4H), 2.54 (d, J = 3.6 Hz, 1H), 2.19 - 2.10 (m, 1H), 2.05 - 1.93 (m, 1H), 1.82 (d, J =11.6 Hz, 2H), 1.66 - 1.50 (m, 2H).

[0101] Example 4: N-(3-bromophenyl)-4-[4-(2,6-dioxo-3-piperidinyl)-3,5-difluorophenyl]cyclohexyl-3-ene-1-carboxamide (Compound 4)

[0102]

[0103] Step 1: Synthesis of Compound 4-2

[0104] To a mixed solution of compound 4-1 (54.73 mg, 177.58 μmol), compound 1-3 (50 mg, 164.43 μmol), and potassium fluoride (28.66 mg, 493.28 μmol) in dioxane (1 mL) and water (0.2 mL), [2-(2-aminophenyl)phenyl]palladium (1+); bis(1-adamantyl)-butylphosphine; methanesulfonate (17.96 mg, 24.66 μmol) was added. The mixture was stirred at 80 °C for 2 hours under nitrogen protection. The reaction mixture was filtered, and the residue was purified by preparative thin-layer chromatography on silica gel (petroleum ether: ethyl acetate = 2:1) to give product compound 4-2.

[0105] Step 2: Synthesis of Compound 4-3

[0106] Trifluoroacetic acid (3 mL) was added to a 1 mL solution of compound 4-2 (60 mg, 147.99 μmol) in dichloromethane. The mixture was stirred at 25 °C for 1 hour under nitrogen protection. The reaction solution was concentrated to obtain compound 4-3, which could be used directly in the next reaction without purification. m / z = 350.2 [M+H] + .

[0107] Step 3: Synthesis of Compound 4

[0108] At 0 °C, N,N-diisopropylethylamine (46.25 mg, 357.83 μmol, 62.33 μL) and HOBt (14.51 mg, 107.35 μmol) were added to a solution of compound 4-3 (25 mg, 71.57 μmol) in N,N-dimethylformamide (1 mL), followed by EDCI (20.58 mg, 107.35 μmol), and the mixture was stirred at 0 °C for 30 min. Then, 3-bromoaniline (14.77 mg, 85.88 μmol, 9.35 μL) was added to the mixture, and the mixture was stirred at 25 °C for 12 h. The reaction mixture was filtered, and the residue after concentration of the green filtrate was purified by preparative high-performance liquid chromatography (HPLC) (column: Unisil 3-100C18 Ultra 150×50 mm×3 μm; mobile phase: [water (containing 0.225% formic acid)-acetonitrile]; gradient 44%-74%, 15 min). The purified residue was then freeze-dried to give compound 4. m / z = 503.2 [M+H] + , 505.2 [M+3] + .

[0109] 1 H NMR (400 MHz, DMSO-d6) δ 10.96 (s, 1H), 10.14 (s, 1H), 7.99 (s,1H), 7.51 (d, J = 8.0 Hz, 1H), 7.23 (m, 4H), 6.39 (s, 1H), 4.21 (m, 1H), 2.81(m, 1H), 2.58 (m, 2H), 2.41 (m, 3H), 2.08 (m, 4H), 1.73 (m, 1H).

[0110] Example 5: 1,4-cis-N-(3-bromophenyl)-4-[4-(2,6-dioxo-3-piperidinyl)-3,5-difluorophenyl]cyclohexaneformamide (compound 5A or 5B) and 1,4-trans-N-(3-bromophenyl)-4-[4-(2,6-dioxo-3-piperidinyl)-3,5-difluorophenyl]cyclohexaneformamide (compound 5B or 5A)

[0111]

[0112] Step 1: Synthesis of Compound 5-1

[0113] Under nitrogen protection, palladium on carbon (12 mg, 10%) was added to a mixed solution of compound 4-2 (45 mg, 110.99 μmol) in tetrahydrofuran (5 mL) and ethanol (5 mL). The suspension was degassed under vacuum and purged several times with hydrogen. The mixture was stirred at 25 °C for 12 h under a hydrogen (15 Psi) atmosphere. The reaction mixture was filtered, and the filter cake was washed with tetrahydrofuran (50 mL). The organic phases were combined and concentrated under reduced pressure to give compound 5-1, which could be used directly for the next reaction without purification. m / z = 352.1 [M-55] + .

[0114] Step 2: Synthesis of Compound 5-2

[0115] To a solution of compound 5-1 (40 mg, 98.17 μmol) in dichloromethane (1 mL), 3 mL of trifluoroacetic acid was added. The mixture was stirred at 25 °C for 1 hour under nitrogen protection. After concentration, the resulting product, compound 5-2, was used directly in the next reaction without purification. m / z = 352.1 [M+H] + .

[0116] Step 3: Synthesis of Compound 5A and Compound 5B

[0117] At 0 °C, N,N-diisopropylethylamine (55.18 mg, 426.93 μmol, 74.36 μL) and HOBt (17.31 mg, 128.08 μmol) were added to a 1 mL solution of N,N-dimethylformamide containing compound 5-2 (30 mg, 85.39 μmol), followed by the addition of EDCI (24.55 mg, 128.08 μmol), and the mixture was stirred at 0 °C for 30 min. Then, 3-bromoaniline (17.63 mg, 102.46 μmol, 11.16 μL) was added to the mixture, and the mixture was stirred at 25 °C for 12 h. The reaction mixture was filtered to obtain a residue, which was purified by preparative high performance liquid chromatography (column: Unisil 3-100 C18 Ultra 150×50 mm×3 μm; mobile phase: [water (containing 0.225% formic acid)-acetonitrile]; gradient 45%-75%, 15.0 min). After lyophilization, compounds 5A and 5B were obtained, respectively.

[0118] Compound 5A: HPLC retention time RT = 2.926 min, m / z = 505.2 [M+H] + 507.2 [M+3] + .

[0119] 1H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 9.52 (s, 1H), 7.99 (s, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.23 (m, 2H), 6.97 (d, J = 10.4 Hz, 2H), 4.13 (m,1H), 2.81 (m, 1H), 2.68 (m, 2H), 2.58 (m, 1H), 2.13 (m, 1H), 2.01 (m, 3H), 1.85 (m, 2H), 1.68 (m, 4H).

[0120] Compound 5B: HPLC retention time RT = 2.965 min, m / z = 505.2 [M+H] + 507.2 [M+3] + .

[0121] 1 H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 10.07 (s, 1H), 8.00 (s,1H), 7.51 (d, J = 8.0 Hz, 1H), 7.23 (m, 2H), 7.04 (d, J = 10.0 Hz, 2H), 4.19(m, 1H), 2.81 (m, 1H), 2.60 (m, 2H), 2.42 (m, 1H), 2.13 (m, 1H), 1.95 (m,5H), 1.53 (m, 4H).

[0122] Example 6: N-(3-bromophenyl)-4-[4-(2,6-dioxo-3-piperidinyl)-3,5-difluorophenyl]-1,2,3,6-tetrahydropyridine-1-carboxamide (Compound 6)

[0123]

[0124] Step 1: Synthesis of Compound 6-1

[0125] Trifluoroacetic acid (768 mg, 6.73 mmol) was added to a mixed solution of compound 3-2 (15.0 mg, 36.9 μmol) in dichloromethane (1.5 mL). The mixture was stirred at 25 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure to give the trifluoroacetate of compound 6-1. m / z = 307.2 [M+H] + .

[0126] Step 2: Synthesis of Compound 6

[0127] To a solution of compound 6-1 (15.0 mg, 35.7 μmol, trifluoroacetate) and N,N-diisopropylethylamine (23.1 mg, 178 μmol) in N,N-dimethylformamide (1 mL), phenyl 1-bromo-3-isocyanate (7.07 mg, 35.7 μmol) was added. The mixture was stirred at 25 °C for 2 h. The reaction mixture was filtered, and the filtrate was purified by preparative high-performance liquid chromatography (HPLC) (column: Unisil 3-100 C18 Ultra 150 × 50 mm × 3 μm; mobile phase: [water (containing 0.225% formic acid) - acetonitrile]; gradient 39%-69%, 15 min) to obtain the crude product. The crude product was further purified by preparative high-performance liquid chromatography (HPLC) (column: Unisil 3-100 C18 Ultra 150×50 mm×3 μm; mobile phase: [water (containing 0.225% formic acid)-acetonitrile]; gradient 39%-69%, 15 min). Finally, it was purified by preparative HPLC (column: Waters Xbridge 150×25 mm×5 μm; mobile phase: [water (10 mM NH4HCO3)-acetonitrile]; gradient 40%-70%, 9 min) to obtain compound 6. m / z = 506.2 [M+H] + .

[0128] 1 H NMR (400 MHz, DMSO-d6) δ 10.97 (s, 1H), 8.74 (s, 1H), 7.81 (t, J =2.0 Hz, 1H), 7.51 - 7.44 (m, 1H), 7.29 - 7.18 (m, 3H), 7.15 - 7.08 (m, 1H),6.41 (s, 1H), 4.23 (dd, J = 5.2, 12.4 Hz, 1H), 4.16 (d, J = 1.6 Hz, 2H), 3.66(t, J = 5.6 Hz, 2H), 3.38 (s, 1H), 2.86 - 2.76 (m, 1H), 2.55 (d, J = 3.2 Hz,1H), 2.20 - 2.07 (m, 1H), 2.06 - 1.96 (m, 1H).

[0129] Example 7: 2-(3-bromophenyl)-N-[1-[4-(2,6-dioxo-3-piperidinyl)-3,5-difluorophenyl]-4-piperidinyl]acetamide (Compound 7)

[0130]

[0131] Step 1: Synthesis of Compound 7-3

[0132] Compound 7-1 (800 mg, 3.99 mmol), compound 7-2 (1.27 g, 3.99 mmol), Xantphos (231.13 mg, 399.45 μmol), Pd(OAc)2 (44.84 mg, 199.72 μmol), and Cs2CO3 (3.90 g, 11.98 mmol) were added to toluene (20 mL). The solution was subjected to a vacuum-nitrogen purging cycle three times, followed by stirring at 70 °C for 12 hours under nitrogen protection. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1) to give compound 7-3. m / z = 391.1 [M+H] + .

[0133] 1 H NMR (400 MHz, DMSO-d6) δ 6.81-6.87(m, 1H), 6.79-6.81 (m, 1H), 6.71-6.79 (m, 1H), 3.68-3.74 (m, 2H), 3.46-3.48 (m, 1H), 3.35-3.37 (m, 2H), 2.79-2.86 (m, 2H), 1.71-1.77 (m, 2H), 1.37 (s, 9H).

[0134] Step 2: Synthesis of Compounds 7-5

[0135] Compounds 7-3 (1.3 g, 3.32 mmol), 7-4 (1.53 g, 3.65 mmol), Pd(dppf)Cl2 (121.56 mg, 166.13 μmol), and Na2CO3 (704.34 mg, 6.65 mmol) were dissolved in a mixed solvent of dioxane (20 mL) and water (5 mL). The solution was subjected to a vacuum-nitrogen purging cycle three times, followed by stirring at 90 °C for 12 hours under nitrogen protection. The reaction mixture was poured into water (20 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with saturated brine (45 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. This residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 50:1 ~ 8:1) to give compound 7-5. m / z = 602.3 [M+H]+ .

[0136] 1 H NMR (400 MHz, DMSO-d6) δ 7.58 (d, J = 8.0 Hz, 1H), 7.40-7.44 (m,2H), 7.35-7.39 (m, 2H), 7.32-7.34 (m, 4H), 7.25-7.30 (m, 1H), 6.80-6.89 (m,1H), 6.63-6.72 (m, 2H), 6.52 (d, J = 8.0 Hz, 1H), 5.35 (s, 2H), 5.33 (s, 2H),3.70-3.82 (m, 2H), 3.41-3.52 (m, 1H), 2.77-2.91 (m, 2H), 1.70-1.85 (m, 2H),1.40-1.50 (m, 2H), 1.38 (s, 9H).

[0137] Step 3: Synthesis of compounds 7-6

[0138] The fixed-bed reactor (FLR1, 5 mL volume) was completely filled with 1.7 g of granular catalyst Pd(OH)2 / Al2O3. The hydrogen back pressure regulating valve was set to 1 MPa, and the hydrogen flow rate was controlled at (60,000 eq, 10,200 sccm). A mixture of compound 7-5 (0.2 g, 332.40 μmol) in N,N-dimethylformamide (8 mL) and tetrahydrofuran (8 mL) was pumped via pump 1 (S1, P1, flow rate 0.4 mL / min) to flow reactor 1 (FLR1, stainless steel, fixed bed, inner diameter 6.350 (1 / 4") mm, volume 5.000 mL, temperature 50.0 °C). The reaction mixture was continuously collected from the reactor outlet into a container. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 ~ 1:1) to give compound 7-6. m / z = 424.2 [M+H] + .

[0139] 1H NMR (400 MHz, DMSO-d6) δ 10.87 (s, 1H), 6.84 (br d, J = 8.0 Hz,1H), 6.63 (s, 1H), 6.60 (s, 1H), 3.97-4.12 (m, 2H), 3.67-3.73 (m, 2H), 3.40-3.49 (m, 2H), 2.76-2.85 (m, 2H), 2.04-2.15 (m, 1H), 1.90-1.98 (m, 1H), 1.71-1.80 (m, 2H), 1.41-1.45 (m, 1H), 1.38 (s, 9H), 1.31-1.36 (m, 1H).

[0140] Step 4: Synthesis of Compound 7-7

[0141] A mixture of compound 7-6 (90 mg, 212.54 μmol) and hydrochloric acid / dioxane solution (2 M, 5 mL) was stirred at 25 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to give the hydrochloride salt of compound 7-7 directly for use in the next step without purification. m / z = 324.1 [M+H] + .

[0142] Step 5: Synthesis of Compound 7

[0143] N,N-diisopropylethylamine (30.1 mg, 232 μmol) was added to a mixed solution of 2-(3-bromophenyl)acetic acid (10.0 mg, 46.5 μmol) and compound 7-7 (16.7 mg, 46.5 μmol, hydrochloride) in N,N-dimethylformamide (1 mL). The mixture was stirred at 25 °C for 0.5 h. Subsequently, HOBt (8.17 mg, 60.5 μmol, 1.3 eq) and EDCI (11.6 mg, 60.5 μmol) were added to the mixture, and the reaction mixture was stirred at 25 °C for another 2 h. The pH of the mixture was adjusted to 4 with formic acid. After filtration, the filtrate was purified by preparative high-performance liquid chromatography (HPLC) (column: Unisil 3-100 C18 Ultra 150×50 mm×3 μm; mobile phase: [water (containing 0.225% formic acid)-acetonitrile]; gradient 37%-67%, 15 min) to obtain product compound 7. m / z = 522.2 [M+H) + .

[0144] 1H NMR (400 MHz, DMSO-d6) δ 10.87 (s, 1H), 8.08 (d, J = 7.6 Hz, 1H), 7.55 - 7.37 (m, 2H), 7.33 - 7.21 (m, 2H), 6.71 - 6.58 (m, 2H), 4.04 (dd, J =4.8, 12.4 Hz, 1H), 3.80 - 3.66 (m, 3H), 3.39 (s, 2H), 2.89 (t, J = 11.2 Hz,2H), 2.82 - 2.70 (m, 1H), 2.54 (s, 1H), 2.16 - 2.02 (m, 1H), 1.99 - 1.89 (m,1H), 1.75 (d, J = 10.1 Hz, 2H), 1.46 - 1.38 (m, 2H).

[0145] Example 8: 2-(3-bromophenyl)-N-[[1-[4-(2,6-dioxo-3-piperidinyl)-3,5-difluorophenyl]-4-piperidinyl]methyl]acetamide (Compound 8)

[0146]

[0147] Step 1: Synthesis of Compound 8-2

[0148] Under nitrogen protection, Cs₂CO₃ (145 mg, 446 μmol), 1,3-bis[2,6-bis(1-propylbutyl)phenyl]-4,5-dichloro-2H-imidazol-1-onthium-2-endosalt, 3-chloropyridine, palladium dichloro(14.5 mg, 14.9 μmol), and compound 8-1 (35.1 mg, 164 μmol) were added to a 2 mL solution of dioxane (45.0 mg, 148 μmol) of compound 1-3; 3-chloropyridine; palladium dichloro(14.5 mg, 14.9 μmol); and compound 8-1 (35.1 mg, 164 μmol). The mixture was stirred at 100 °C for 2 hours. The reaction mixture was filtered, and the filtrate was purified by preparative thin-layer chromatography on silica gel (petroleum ether: ethyl acetate = 1:1) to give product compound 8-2.

[0149] Step 2: Synthesis of Compound 8-3

[0150] Compound 8-2 (40.0 mg, 91.4 μmol) was added to a hydrochloric acid / dioxane solution (2 M, 2.17 mL), and the mixture was stirred at 25 °C for 12 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give the hydrochloride salt of compound 8-3. m / z = 338.0 [M+H] + .

[0151] Step 3: Synthesis of Compound 8

[0152] To a mixture of compound 8-3 (15.0 mg, 40.1 μmol, hydrochloride), 2-(3-bromophenyl)acetic acid (9.53 mg, 44.3 μmol), and N,N-diisopropylethylamine (26.0 mg, 201 μmol) in N,N-dimethylformamide (1 mL), HOBt (7.08 mg, 52.4 μmol) and EDCI (10.0 mg, 52.4 μmol) were added. The mixture was stirred at 25 °C for 2 h. The pH of the reaction mixture was adjusted to 4 with formic acid, filtered, and the filtrate was purified by preparative high-performance liquid chromatography (HPLC) (column: Unisil 3-100 C18 Ultra 150×50 mm×3 μm; mobile phase: [water (containing 0.225% formic acid)-acetonitrile]; gradient 37%-67%, 15 min) to give compound 8. m / z = 536.3 [M+H] + .

[0153] 1 H NMR (400 MHz, DMSO-d6) δ 10.86 (s, 1H), 8.12 (t, J = 5.6 Hz, 1H), 7.47 (s, 1H), 7.45 - 7.38 (m, 1H), 7.28 - 7.22 (m, 2H), 6.61 (d, J = 12.8 Hz,2H), 4.03 (dd, J = 5.2, 12.4 Hz, 1H), 3.75 (d, J = 12.8 Hz, 2H), 3.43 (s,2H), 2.97 (t, J = 6.0 Hz, 2H), 2.84 - 2.63 (m, 3H), 2.54 - 2.51 (m, 1H), 2.16- 2.02 (m, 1H), 2.00 - 1.88 (m, 1H), 1.66 (d, J = 13.2 Hz, 2H), 1.61 - 1.49 (m, 1H), 1.22 - 1.04 (m, 2H).

[0154] Example 9: 2-(3-bromophenyl)-N-[1-[4-(2,6-dioxo-3-piperidinyl)-3,5-difluorophenyl]-3-piperidinyl]acetamide (Compound 9)

[0155]

[0156] Step 1: Synthesis of Compound 9-2

[0157] To a solution of dioxane (2 mL) of compounds 1-3 (45.0 mg, 148 μmol), Cs₂CO₃ (145 mg, 446 μmol), 1,3-bis[2,6-bis(1-propylbutyl)phenyl]-4,5-dichloro-2H-imidazol-1-onthium-2-endosalt, 3-chloropyridine, palladium dichloroisocyanurate (14.5 mg, 14.9 μmol), and compound 9-1 (32.8 mg, 164 μmol) were added. The mixture was stirred at 100 °C for 2 hours. The reaction mixture was filtered, and the filtrate was purified by preparative silica gel thin-layer chromatography (petroleum ether: ethyl acetate = 1:1) to give compound 9-2. m / z = 424.1 [M+H] + .

[0158] Step 2: Synthesis of Compound 9-3

[0159] Compound 9-2 (45.0 mg, 106 μmol) was added to a hydrochloric acid / dioxane solution (2 M, 2.0 mL), and the mixture was stirred at 25 °C for 12 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give the hydrochloride salt of compound 9-3. m / z = 324.0 [M+H] + .

[0160] Step 3: Synthesis of Compound 9

[0161] To a mixed solution of compound 9-3 (15.0 mg, 41.7 μmol, hydrochloride), 2-(3-bromophenyl)acetic acid (9.90 mg, 46.1 μmol), and N,N-diisopropylethylamine (27.1 mg, 209 μmol) in N,N-dimethylformamide (1 mL), HOBt (7.35 mg, 54.4 μmol) and EDCI (10.4 mg, 54.4 μmol) were added. The mixture was stirred at 25 °C for 2 h. The pH of the reaction mixture was adjusted to 4 with formic acid, filtered, and the filtrate was purified by preparative high-performance liquid chromatography (HPLC) (column: Unisil 3-100 C18 Ultra 150×50 mm×3 μm; mobile phase: [water (containing 0.225% formic acid)-acetonitrile]; gradient 37%-67%, 15 min) to give compound 9. m / z = 522.2 [M+H] + .

[0162] 1H NMR (400 MHz, DMSO-d6) δ 10.87 (s, 1H), 8.16 (d, J = 7.2 Hz, 1H), 7.47 (s, 1H), 7.45 - 7.39 (m, 1H), 7.31 - 7.20 (m, 2H), 6.59 (d, J = 12.8 Hz,2H), 4.04 (dd, J = 5.2, 12.4 Hz, 1H), 3.73 - 3.64 (m, 1H), 3.63 - 3.51 (m,2H), 3.44 (s, 2H), 3.00 - 2.88 (m, 1H), 2.82 - 2.70 (m, 2H), 2.49 - 2.45 (m,1H), 2.14 - 2.01 (m, 1H), 1.99 - 1.89 (m, 1H), 1.84 - 1.66 (m, 2H), 1.57 -1.40 (m, 2H).

[0163] Example 10: 2-(3-bromophenyl)-N-({1-[4-(2,6-dioxo-3-piperidinyl)-3,5-difluorophenyl]-3-piperidinyl}methyl)acetamide (Compound 10)

[0164]

[0165] Step 1: Synthesis of Compound 10-2

[0166] Under nitrogen protection, 1,3-bis[2,6-bis(1-propylbutyl)phenyl]-4,5-dichloro-2H-imidazol-1-onthium-2-endosalt; 3-chloropyridine; palladium dichloroisocyanurate (31.99 mg, 32.89 μmol) and Cs₂CO₃ (321.44 mg, 986.56 μmol) were added to a 4 mL solution of dioxane (4 mL) of compounds 1-3 (100.00 mg, 328.85 μmol) and 10-1 (77.52 mg, 361.74 μmol). The mixture was stirred at 100 °C for 2 hours. The reaction mixture was filtered, concentrated under reduced pressure, and the residue was purified by preparative silica gel thin-layer chromatography (petroleum ether: ethyl acetate = 1:1) to obtain compound 10-2. m / z = 438.2 [M+H] + .

[0167] Step 2: Synthesis of compound 10⁻³

[0168] Compound 10⁻² (120 mg, 274.30 μmol) was added to a hydrochloric acid / dioxane solution (2 M, 4 mL), and the mixture was stirred at 25 °C for 2 hours. The reaction mixture was concentrated under reduced pressure to give the residue compound 10⁻³, which was used directly in the next reaction without further purification. m / z = 338.0 [M+H] + .

[0169] Step 3: Synthesis of Compound 10

[0170] HOBt (7.54 mg, 55.80 μmol), EDCI (10.70 mg, 55.80 μmol), and N,N-diisopropylethylamine (14.42 mg, 111.61 μmol, 19.43 μL) were added to a solution of 2-(3-bromophenyl)acetic acid (8.00 mg, 37.20 μmol) in N,N-dimethylformamide (0.5 mL). Compound 10⁻³ (15.30 mg, 40.92 μmol, hydrochloride) was then added, and the mixture was stirred at 25 °C for 12 hours.

[0171] The reaction mixture was filtered to obtain a residue, which was then purified by preparative high-performance liquid chromatography (HPLC) (column: Unisil 3-100C18 Ultra 150×50 mm×3 μm; mobile phase: [water (containing 0.225% formic acid)-acetonitrile]; gradient 39%-69%, 15 min). The collected liquid was concentrated under reduced pressure to remove the organic phase, and the remaining aqueous phase was freeze-dried to obtain compound 10. m / z = 534.3 [M+H] + .

[0172] 1H NMR (400 MHz, DMSO-d6) δ 10.86 (s, 1H), 8.14 (br t, J = 5.6 Hz,1H), 7.48 (s, 1H), 7.41 (td, J = 2.0, 7.2 Hz, 1H), 7.30 - 7.22 (m, 2H), 6.54(d, J = 12.8 Hz, 2H), 4.04 (br dd, J = 5.2, 12.4 Hz, 1H), 3.66 - 3.54 (m,2H), 3.44 (s, 2H), 3.09 - 2.96 (m, 2H), 2.84 - 2.67 (m, 2H), 2.52 (br s, 1H),2.46 (br s, 1H), 2.15 - 2.01 (m, 1H), 1.99 - 1.89 (m, 1H), 1.76 - 1.60 (m,3H), 1.45 (br d, J = 12.0 Hz, 1H), 1.16 - 1.03 (m, 1H).

[0173] Experimental Example 1. Evaluation of the degradation activity of compounds on GSPT1 protein using the HiBiT assay.

[0174] 1. Experimental procedures;

[0175] 1.1 Preparation of the 293A-GSPT1-HiBiT cell line:

[0176] a) Dilute 293A-GSPT1-HiBiT cells to a density of 2.22 × 10⁻⁶ using DMEM medium (containing 10% FBS and 1% P / S) for passage and cell growth. 5 / mL.

[0177] b) In 96-well opaque cell culture plates (PerkinElmer; catalog number 6005680), seed cells at a density of 20,000 cells / 100 μL per well. After seeding, incubate the culture plates in a 5% CO2 environment for 12–15 hours.

[0178] 1.2 Adding compounds:

[0179] The test compound was serially diluted 5-fold using DMSO to establish 10 concentrations: 5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, 0.0128 nM, and 0 nM. The diluted compound was added to 293A-GSPT1-HiBiT cells, with 0.1% DMSO as a control. The cells were centrifuged at 800 rpm for 2 min, and the mixture was thoroughly mixed before treatment for 6 h.

[0180] 1.3 Cell lysis detection

[0181] a) Calculate the amount of Nano-Glo® HiBiT lysis reagent (Promega; catalog number N3040) required to complete the experiment. Take a new test tube, dilute LgBiTProtein (1:100) and Nano-Glo® HiBiT Lytic Substrate (1:50) with an appropriate volume of room temperature Nano-Glo® HiBiT Lytic Buffer, and mix by inverting.

[0182] b) Remove the culture plate containing 293A-GSPT1-HiBiT cells from the incubator and allow it to equilibrate to room temperature. Add an equal volume of Nano-Glo® HiBiT lysis reagent to each well of culture medium and mix well. Column 1 contains only HiBiT lysis reagent with substrate but no LgBiT as a blank control. For best results, place the plate on a shaker (300-600 rpm) for 10 minutes or mix the samples by pipetting.

[0183] c) Wait 10 minutes to allow the LgBiT and HiBiT in the lysis buffer to reach equilibrium. Use Varioskan LUX (Thermo) to measure the luminescence signal, with a detection time set to 1 second / well.

[0184] 2. Data Processing

[0185] a) The raw data are the detected chemiluminescence values ​​(RLU). After subtracting the RLU values ​​of the BLANK control group, the average chemiluminescence value (RLU) of the DMSO control group is used as the baseline level of the target protein. The degradation fraction and degradation percentage are calculated according to the following formula:

[0186] Degradation fraction = (Mean RLU of DMSO control group - Mean RLU of BLANK blank group) / (RLU of treatment group - Mean RLU of BLANK blank group)

[0187] Degradation percentage = (1 - Degradation fraction) × 100%

[0188] b) Using GraphPad software, the compound concentration and degradation percentage were analyzed, and nonlinear regression fitting (dose-response curve) was performed to obtain the DC50 value.

[0189] 3. Experimental Results

[0190]

[0191] 4. Conclusion: The compounds obtained in this invention have good GSPT1 protein degradation activity at the cellular level, and some compounds have high Dmax values ​​for GSPT1 protein degradation.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof; ； in, Ring A is , or , where X 2 With L 1 connect; L 1 For single bond or -NR a -R b - ,pass The end is connected to ring A; L 2 C 1-6 Alkylene, -O- or -NR a -; Ring B is C 6-12 The aryl group or "a 5-10 membered heteroaryl group selected from one, two, or three of N, O, and S, with one, two, three, or four heteroatoms"; R 1 and R 2 Independent of H, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups; R 3 Halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups; n is 0, 1, 2, or 3; X 1 For CH or N; X 2 For CH or N; R a For H or C 1-6 alkyl; R b C 1-6 Alkylene.

2. The compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or more of the following conditions: (1) L 2 C 1-6 Alkylene or -NR a -; (2) Ring B is C 6-12 Aryl; (3) R 1 and R 2 Halogens are independent of each other; (4) R 3 It is a halogen; (5) n is 1; (6) R a For H.

3. The compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or more of the following conditions: (1) Each of the C's 1-6 The alkylene group is independently methylene, ethylene, propylene, or butylene; (2) Each of the C's 6-12 The aryl group can be phenyl or naphthyl independently; (3) Each of the halogens is independently F, Cl, Br or I.

4. The compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or more of the following conditions: (1) Each of the C's 1-6 The alkylene group is independently methylene; (2) Each of the C's 6-12 The aryl group is independently a phenyl group; (3) Each of the halogens is independently F or Br.

5. The compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or more of the following conditions: (1) Ring A is , , , , , or ; (2) L 1 It is a single bond or -NH-CH2-; (3) L 2 It is -NH- or -CH2-; (4) Ring B is phenyl; (5) R 1 and R 2 Independently defined as F; (6) R 3 It is Br.

6. The compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or two of the following conditions: (1) for , or ; (2) for .

7. The compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compound of formula I is the compound shown in formula I-1 and / or formula I-2: ， ； Among them, R 1 R 2 R 3 n, ring A, ring B, L 1 and L 2 The definition is independent of any one of claims 1-6.

8. The compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compound of formula I is any one of the following compounds: ， ， ， 。 9. A pharmaceutical composition comprising a compound of formula I as described in any one of claims 1-8 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

10. Use of a compound of formula I as claimed in any one of claims 1-8, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as claimed in claim 9, wherein the use is selected from: (1) Preparation of GSPT1 molecular adhesive degrading agent; (2) Prepare drugs for the prevention and / or treatment of diseases related to GSPT1.

11. The use as described in claim 10, characterized in that, The disease associated with GSPT1 is acute myeloid leukemia.

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