NAMPT and HDAC double-target protein degradation agent and application thereof

Abstract

The invention discloses a NAMPT and HDAC double-target protein degradation agent or a pharmaceutical salt thereof. The structural general formula of the NAMPT and HDAC double-target protein degradation agent is selected from one of the following structures, according to the invention, preliminary activity testing is carried out on the NAMPT series and the HDAC series. Results show that in all tested compounds, the compound N4 and the compound H4 show obvious degradation effects. The compound provided by the invention can be applied to preparation of a synergistic degradation agent targeting NAMPT and HDAC.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically, it relates to a dual-target protein degrader of NAMPT and HDAC and its application. Background Technology

[0002] Traditional drug development has long followed a "single-target" strategy, designing highly selective molecules to precisely inhibit a single disease-related protein. While this approach has yielded significant results, its efficacy is often limited in complex, multifactorial diseases like cancer due to redundant signaling pathways. Therefore, a "multi-target" strategy capable of simultaneously intervening in multiple key targets has become an important development direction, with protein degradation-targeting chimeras attracting considerable attention due to their ability to target "undruggable" proteins. However, traditional PROTACs, as single heterobifunctional macromolecules, generally suffer from limitations such as large molecular weight and poor membrane permeability, hindering their further development. The design of multi-target degraders faces even greater challenges due to increased molecular complexity. In recent years, the rise of bioorthogonal chemistry has provided innovative solutions to overcome these bottlenecks. This technology enables efficient and specific chemical reactions within living cells without interfering with normal physiological processes. In particular, based on the anti-electron demand Diels-Alder reaction of trans-cyclooctene and tetrazine, the complete PROTAC molecule can be split into two small molecule precursors with good cell permeability, allowing them to self-assemble within the cell, thus cleverly circumventing the physicochemical defects of traditional PROTACs. Summary of the Invention

[0003] The purpose of this invention is to provide a dual-target protein degrader for NAMPT and HDAC.

[0004] Another object of the present invention is to provide the application of the NAMPT and HDAC dual-target protein degrader in the preparation of a treatment for breast cancer.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a dual-target protein degrader of NAMPT and HDAC, or a pharmaceutically acceptable salt thereof, wherein the general structural formula is selected from one of the following structures:

[0007] ;

[0008] General formula H combined with general formula C can serve as a dual-target protein degrader for NAMPT and HDAC, or general formula N combined with general formula C can serve as a dual-target protein degrader for NAMPT and HDAC.

[0009] Alternatively, general formulas N and H can be combined with general formula C as dual-target protein degraders for NAMPT and HDAC;

[0010] in,

[0011] R1 is selected from

[0012] ;

[0013] R4 is selected from C3~C10 cycloalkane groups;

[0014] n1 is selected from positive integers from 1 to 5 (e.g., 1, 2, 3, 4, 5).

[0015] R5 is selected from C3~C10 cycloalkane oxygen groups, ;

[0016] n2 is selected from positive integers from 1 to 5 (e.g., 1, 2, 3, 4, 5).

[0017] R6 is selected from C3~C10 cycloalkane oxygen groups, ;

[0018] R7 is selected from hydrogen and C1~C10 alkyl groups;

[0019] L is selected from -(CH2)n3, -(CH2)n4CONH(CH2)n5-;

[0020] n3 is selected from positive integers from 1 to 20 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0021] n4 is selected from positive integers from 1 to 20 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0022] n5 is selected from positive integers from 1 to 20 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0023] R2 is selected from C3~C10 cycloalkane oxygen groups, ;

[0024] n6 is selected from positive integers from 1 to 10 (e.g., 1, 2, 3, 4, 5, 9, 7, 8, 9).

[0025] R3 is selected from .

[0026] Preferably, in the NAMPT and HDAC dual-target protein degrading agent,

[0027] in,

[0028] R1 is selected from

[0029] ;

[0030] R4 is selected from ;

[0031] n1 is selected from 1, 2, 3, 4, 5;

[0032] R5 is selected from

[0033] ;

[0034] n2 is selected from 1, 2, 3, 4, and 5;

[0035] R6 is selected from

[0036] ;

[0037] L is selected from -(CH2)n3, -(CH2)n4CONH(CH2)n5-;

[0038] n3 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

[0039] n4 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

[0040] n5 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

[0041] R2 is selected from

[0042] ;

[0043] n6 is selected from 1, 2, 3, 4, 5, 9, 7, 8, 9;

[0044] R3 is selected from .

[0045] Most preferably, the structure of the NAMPT and HDAC dual-target protein degrader is selected from one of the following structures:

[0046] .

[0047] In a second aspect, the present invention provides the use of the NAMPT and HDAC dual-target protein degrader in the preparation of a medicament for treating breast cancer.

[0048] A third aspect of the present invention provides the application of the NAMPT and HDAC dual-target protein degrader in the preparation of the NAMPT and HDAC dual-target protein degrader.

[0049] In a fourth aspect, the present invention provides the application of compound N4 in combination with compound C2 in the preparation of a dual-target protein degrader for NAMPT and HDAC.

[0050] In a fifth aspect, the present invention provides the application of compound H4 in combination with compound C2 in the preparation of a dual-target protein degrader for NAMPT and HDAC.

[0051] In a sixth aspect, the present invention provides the use of compound H4 and compound N4 in combination with compound C2 in the preparation of a dual-target protein degrader for NAMPT and HDAC.

[0052] In a seventh aspect, the present invention provides the use of compound H4 and compound N4 in combination with compound C2, or compound N4 in combination with compound C2, or compound H4 in combination with compound C2 in the preparation of a medicament for treating breast cancer.

[0053] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0054] This invention provides a bioorthogonal intracellular self-assembly strategy with a PROTAC degrader that synergistically degrades NAMPT and HDAC. Preliminary activity tests were conducted on the NAMPT series (compounds N1-N5) and the HDAC series (compounds H1-H4). Results showed that compounds N4 and H4 exhibited significant degradation effects among all tested compounds. Therefore, compounds N4 and H4 were selected for subsequent experiments. MDA-MB-231 cells were co-treated with compounds N4 and H4 for 24 h, followed by treatment with compound C2 for 48 h. The results showed that compounds N4 and H4 synergistically induced the degradation of NAMPT and HDAC proteins, accompanied by the characteristic "hook effect" of PROTAC. The degradation activity of NAMPT and HDAC was very strong, DC… 50 The values ​​were 25.4 nM and 9.42 nM, respectively. In summary, compounds N4 and H4 function as highly efficient and synergistic degraders targeting NAMPT and HDAC. Therefore, the compounds provided by this invention can be used to prepare synergistic degraders targeting NAMPT and HDAC.

[0055] This invention provides a bioorthogonal intracellular self-assembly strategy for the dual degradation of NAMPT and HDAC1. Utilizing cell-permeable precursor compounds N4, H4, and C2, two heterobifunctional degradative agents are formed in situ via an intracellular click reaction. Experimental results show that this dual-target degradative agent exhibits highly efficient dual-target degradation activity in MDA-MB-231 cells. The optimal single-target combination (compounds N4+C2 and H4+C2) achieves the half-maximal effective concentration (DC) for the degradation of their respective target proteins NAMPT and HDAC1. 50The concentrations reached 0.321 μM and 0.347 μM, respectively, demonstrating their high degradation activity. More importantly, when compounds N4, H4, and C2 were used in combination, they could simultaneously induce the degradation of both proteins in the same cell, achieving half-maximal effective concentrations (DCs) for the target proteins NAMPT and HDAC1. 50 The molecular weights (MW) reached 25.4 nM and 9.42 nM, achieving dual-target knockdown and exhibiting superior degradation effects compared to single-target degradation. This invention breaks down traditional large bifunctional PROTAC molecules into multiple small, cell-permeable precursors (such as compounds N4, H4, and C2). These small precursor molecules more easily enter cells and assemble into their active forms on demand within the cell via bioorthogonal reactions (click chemistry). This fundamentally solves the drug-likeness bottleneck caused by the large molecular weight and poor membrane permeability of traditional PROTAC molecules. This invention not only establishes a highly efficient and controllable method for dual-target protein degradation but also conceptually expands the scope of PROTAC technology, paving a practical path for developing multi-target degradative agents using bioorthogonal chemistry.

[0056] This invention employs an innovative strategy, combining small-molecule precursors targeting NAMPT and HDAC with small-molecule precursors that recruit CRBN E3 ligases. These precursors then self-assemble within cells to form two classes of PROTAC degraders targeting NAMPT and HDAC, respectively. This invention enables the simultaneous and synergistic degradation of NAMPT and HDAC within the cell. Attached Figure Description

[0057] Figure 1 A schematic diagram showing the results of screening compounds with degradation activity.

[0058] Figure 2 This is a schematic diagram showing the experimental results of treating MDA-MB-231 cells with compounds N4 and H4 for 24 hours, followed by treatment with compound C2 for 48 hours. Detailed Implementation

[0059] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0060] All reagents and raw materials used in the following examples are commercially available or can be prepared according to literature methods. Experimental methods without specific conditions are performed under standard conditions or as recommended by the manufacturer.

[0061]

[0062] Example 1

[0063] Preparation method of compound 3a:

[0064] Compound 1 (prepared according to the literature: Acta Pharm. Sin. B 12 (2022) 2859-2868.) (250 mg, 0.639 mmol, 1 eq) was dissolved in 8 mL of N,N-dimethylformamide. Compound 2a (213 mg, 0.767 mmol, 1.2 eq) and N,N-diisopropylethylamine (334 μL, 1.917 mmol, 3 eq) were added, and the mixture was heated to 60 °C and reacted for 8 h, monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, diluted with ethyl acetate, washed with saturated sodium chloride solution, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 100:6) to give 153 mg of compound 3a as a white solid, with a yield of 40.69%.

[0065] Preparation method of compound N1:

[0066] Compound 3a (132 mg, 0.224 mmol) was dissolved in 4 mL of dichloromethane, and trifluoroacetic acid (2 mL) was added dropwise. The reaction was carried out at room temperature for 1 h, monitored by TLC. After the reaction was complete, the solution was concentrated under reduced pressure, redissolved in N,N-dimethylformamide (8 mL), and (E)-cyclooctyl-4-en-1-yl(4-nitrophenyl) carbonate (50 mg, 0.172 mmol) and N,N-diisopropylethylamine (90 μL, 0.468 mmol) were added. The reaction was carried out at room temperature for 16 h, monitored by TLC. After the reaction was complete, the solution was diluted with ethyl acetate, washed with saturated sodium chloride solution, and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 100:8) to give 52 mg of compound N1 as a white solid, with a yield of 47.27%.

[0067] Example 2

[0068] Preparation method of compound 3b:

[0069] The synthesis method was the same as that for compound 3a, except that compound 2a was replaced with compound 2b, yielding 775 mg of compound 3b as a gray solid, with a yield of 68.83%.

[0070] Preparation method of compound 4b:

[0071] Compound 3b (650 mg, 1.174 mmol, 1 eq) and lithium hydroxide (141 mg, 5.870 mmol, 5 eq) were dissolved in a mixed solvent consisting of 6 mL methanol, 4 mL tetrahydrofuran, and 2 mL water. The reaction was carried out at room temperature for 18 h, monitored by TLC. After the reaction was complete, the mixture was concentrated under reduced pressure, diluted with water, and the pH was adjusted to weakly acidic with saturated citric acid solution. The mixture was filtered, and the filter cake was dried to give 600 mg of compound 4b as a white solid, with a yield of 97.24%.

[0072] Preparation method of compound 5b:

[0073] Compound 4b (350 mg, 0.666 mmol, 1 eq) was dissolved in 8 mL of dichloromethane, and tert-butyl(3-aminopropyl)carbamate (139 mg, 0.799 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDCI (192 mg, 0.999 mmol, 1.5 eq), 1-hydroxybenzotriazole HOBT (135 mg, 0.999 mmol, 1.5 eq), and N,N-diisopropylethylamine DIPEA (348 μL, 1.998 mmol, 3 eq) were added. The reaction was carried out at room temperature for 24 h, and the reaction was monitored by TLC. After the reaction was complete, the mixture was diluted with dichloromethane, washed with saturated sodium bicarbonate solution, and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 100:6) to give 297 mg of compound 5b as a white solid, with a yield of 65.42%.

[0074] Preparation method of compound N2:

[0075] The synthesis method was the same as that for compound N1, except that compound 3a was replaced with compound 5b to obtain 53 mg of compound N2, a white solid, with a yield of 42.06%.

[0076] Example 3

[0077] Preparation method of compound 3c:

[0078] The synthesis method was the same as that for compound 3a, except that compound 2a was replaced with compound 2c, yielding 612 mg of compound 3c, a white solid, in a yield of 63.03%.

[0079] Preparation method of compound 4c:

[0080] The synthesis method was the same as that for compound 4b, except that compound 3b was replaced with compound 3c, yielding 360 mg of compound 4c, a white solid, with a yield of 83.92%.

[0081] Preparation method of compound 5c:

[0082] The synthesis method was the same as that for compound 5b, except that compound 4b was replaced with compound 4c, yielding 278 mg of compound 5c, a white solid, in a yield of 48.10%.

[0083] Preparation method of compound N3:

[0084] The synthesis method was the same as that for compound N1, except that compound 3a was replaced with compound 5c to obtain 38 mg of compound N3, a white solid, with a yield of 33.63%.

[0085] Example 4

[0086] Preparation method of compound N4:

[0087] Compound 5b (140 mg, 0.205 mmol) was dissolved in 4 mL of dichloromethane, and trifluoroacetic acid (2 mL) was added dropwise. The reaction was carried out at room temperature for 1 h, and monitored by TLC. After the reaction was complete, the solution was concentrated under reduced pressure, redissolved in dichloromethane (6 mL), and 2-(4-(6-methyl-1,2,4,5-tetraazine-3-yl)phenyl)acetic acid (52 mg, 0.226 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (59 mg, 0.308 mmol), 1-hydroxybenzotriazole (42 mg, 0.308 mmol), and N,N-diisopropylethylamine (107 μL, 0.615 mmol) were added. The reaction was carried out at room temperature for 16 h, and monitored by TLC. After the reaction was complete, the mixture was diluted with dichloromethane, washed with saturated sodium bicarbonate solution, and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 100:8) to give 70 mg of compound N4 as a red solid, with a yield of 42.94%.

[0088] Example 5

[0089] Preparation method of compound N5:

[0090] The synthesis method was the same as that for compound N4, except that compound 5b was replaced with compound 5c to obtain 53 mg of compound N5, a red solid, with a yield of 55.79%.

[0091]

[0092] Example 6

[0093] Preparation method of compound 8a:

[0094] 4-Aminomethylbenzoate hydrochloride, i.e., compound 6 (1.196 g, 5.933 mmol, 1.2 eq), was dissolved in dichloromethane (40 mL). Compound 7a (1 g, 4.944 mmol, 1 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.422 g, 7.416 mmol, 1.5 eq), 1-hydroxybenzotriazole (1.002 g, 7.416 mmol, 1.5 eq), and N,N-diisopropylethylamine (2.583 mL, 14.832 mmol, 3 eq) were added. The reaction was carried out at room temperature for 18 h, and the reaction was monitored by TLC. After the reaction was complete, the mixture was diluted with dichloromethane, washed with saturated sodium bicarbonate solution, and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 100:8) to give 1.334 g of compound 8a as a white solid, with a yield of 77.20%.

[0095] Preparation method of compound 9a:

[0096] Compound 8a (1.334 g, 3.818 mmol, 1 eq) and lithium hydroxide (457 mg, 19.09 mmol, 5 eq) were dissolved in a mixed solvent consisting of 6 mL methanol, 4 mL tetrahydrofuran, and 2 mL water. The reaction was carried out at room temperature for 18 h, monitored by TLC. After the reaction was complete, the solution was concentrated under reduced pressure, diluted with water, and the pH was adjusted to weakly acidic with saturated citric acid solution. The solution was filtered, and the filter cake was dried to give 939 mg of compound 9a as a white solid, with a yield of 73.30%.

[0097] Preparation method of compound 10a:

[0098] Compound 9a (400 mg, 1.193 mmol, 1 eq) was dissolved in dichloromethane (15 mL), and o-phenylenediamine (142 mg, 1.312 mmol, 1.1 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (343 mg, 1.790 mmol, 1.5 eq), 1-hydroxybenzotriazole (242 mg, 1.790 mmol, 1.5 eq), and N,N-diisopropylethylamine (623 μL, 3.579 mmol, 3 eq) were added. The reaction was carried out at room temperature for 18 h, and monitored by TLC. After the reaction was complete, the mixture was diluted with dichloromethane, washed with saturated sodium bicarbonate solution, and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 100:5) to give 212 mg of compound 10a as a white solid, with a yield of 41.73%.

[0099] Preparation method of compound H1:

[0100] Compound 10a (100 mg, 0.235 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (1 mL) was added dropwise. The reaction was carried out at room temperature for 1 h, and monitored by TLC. After the reaction was complete, the solution was concentrated under reduced pressure and dissolved in N,N-dimethylformamide (6 mL). (E)-cyclooctyl-4-en-1-yl(3-aminopropyl)carbamate hydrochloride (68 mg, 0.259 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate HATU (107 mg, 0.228 mmol), and N,N-diisopropylethylamine (123 μL, 0.705 mmol) were added. The reaction was carried out at room temperature for 16 h, and monitored by TLC. After the reaction was complete, the mixture was diluted with ethyl acetate, washed with saturated sodium bicarbonate solution, and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 100:8) to give 81 mg of compound H1 as a white solid, with a yield of 59.56%.

[0101] Example 7

[0102] Preparation method of compound 8b:

[0103] The synthesis method was the same as that for compound 8a, except that compound 7a was replaced with compound 7b, to obtain 1.185 g of compound 8b, a white solid, with a yield of 94.05%.

[0104] Preparation method of compound 9b:

[0105] The synthesis method was the same as that for compound 9a, except that compound 8a was replaced with compound 8b, yielding 999 mg of compound 9b as a white solid with a yield of 93.89%.

[0106] Preparation method of compound 10b:

[0107] The synthesis method was the same as that for compound 10a, except that compound 9a was replaced with compound 9b, yielding 235 mg of compound 10b, a white solid, in 38.78% yield.

[0108] Preparation method of compound H2:

[0109] Compound 10b (114 mg, 0.224 mmol) was dissolved in dichloromethane (4 mL), and trifluoroacetic acid (2 mL) was added dropwise. The reaction was carried out at room temperature for 1 h, monitored by TLC. After the reaction was complete, the solution was concentrated under reduced pressure, redissolved in N,N-dimethylformamide (8 mL), and (E)-cyclooctyl-4-en-1-yl(4-nitrophenyl) carbonate (50 mg, 0.172 mmol) and N,N-diisopropylethylamine (150 μL, 0.86 mmol) were added. The reaction was carried out at room temperature for 16 h, monitored by TLC. After the reaction was complete, the solution was diluted with ethyl acetate, washed with saturated sodium chloride solution, and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 100:8) to give 60 mg of compound H2 as a white solid, with a yield of 61.86%.

[0110] Example 8

[0111] Preparation method of compound 8c:

[0112] The synthesis method was the same as that for compound 8a, except that compound 7a was replaced with compound 7c, to obtain 1.199 g of compound 8c, a white solid, with a yield of 98.12%.

[0113] Preparation method of compound 9c:

[0114] The synthesis method was the same as that for compound 9a, except that compound 8a was replaced with compound 8c, yielding 1.144 g of compound 9c, a white solid, with a yield of 98.28%.

[0115] Preparation method of compound 10c:

[0116] The synthesis method was the same as that for compound 10a, except that compound 9a was replaced with compound 9c, yielding 264 mg of compound 10c, a white solid, with a yield of 43.93%.

[0117] Preparation method of compound H3:

[0118] The synthesis method was the same as that for compound H2, except that compound 10b was replaced with compound 10c, yielding 44 mg of compound H3, a white solid, with a yield of 43.14%.

[0119] Example 9

[0120] Preparation method of compound H4:

[0121] Compound 10c (152 mg, 0.282 mmol) was dissolved in dichloromethane (4 mL), and trifluoroacetic acid (2 mL) was added dropwise. The reaction was carried out at room temperature for 1 h, and monitored by TLC. After the reaction was complete, the solution was concentrated under reduced pressure, redissolved in dichloromethane (6 mL), and 2-(4-(6-methyl-1,2,4,5-tetraazine-3-yl)phenyl)acetic acid (50 mg, 0.217 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (63 mg, 0.326 mmol), 1-hydroxybenzotriazole (44 mg, 0.326 mmol), and N,N-diisopropylethylamine (113 μL, 0.651 mmol) were added. The reaction was carried out at room temperature for 16 h, and monitored by TLC. After the reaction was complete, the mixture was diluted with dichloromethane, washed with saturated sodium bicarbonate solution, and the organic phase was collected. It was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 100:8) to give 46 mg of compound H4 as a red solid, with a yield of 32.62%.

[0122]

[0123] Example 10

[0124] Preparation method of compound 12:

[0125] Compound 11 (400 mg, 1.459 mmol) and tert-butyl 6-bromohexanoate (440 mg, 1.751 mmol) were dissolved in N,N-dimethylformamide (15 mL), potassium carbonate (605 mg, 4.377 mmol) and potassium iodide (121 mg, 0.730 mmol) were added, and the mixture was heated to 110 °C and reacted for 18 h, monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, diluted with ethyl acetate, washed with saturated sodium chloride solution, and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 100:3) to give 425 mg of compound 12 as a yellow oil, with a yield of 65.59%.

[0126] Preparation method of compound C1:

[0127] Compound 12 (190 mg, 0.427 mmol) was dissolved in dichloromethane (4 mL), and trifluoroacetic acid (2 mL) was added dropwise. The reaction was carried out at room temperature for 1 h, monitored by TLC. After the reaction was complete, the solution was concentrated under reduced pressure, redissolved in dichloromethane (6 mL), and methyltetraazine hydrochloride (122 mg, 0.512 mmol), HATU (195 mg, 0.512 mmol), and N,N-diisopropylethylamine (223 μL, 1.281 mmol) were added. The reaction was carried out at room temperature for 16 h, monitored by TLC. After the reaction was complete, the solution was diluted with dichloromethane, washed with saturated sodium bicarbonate solution, and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 100:8) to give 219 mg of compound C1 as a red solid, with a yield of 89.75%.

[0128] Example 11

[0129] Preparation method of compound 13:

[0130] The synthesis method was the same as that of compound 12, except that tert-butyl 6-bromohexanoate was replaced with N-Boc-6-bromohexylamine, yielding 297 g of compound 13, a yellow oil with a yield of 42.19%.

[0131] Preparation method of compound C2:

[0132] Compound 13 (106 mg, 0.224 mmol) was dissolved in dichloromethane (4 mL), and trifluoroacetic acid (2 mL) was added dropwise. The reaction was carried out at room temperature for 1 h, monitored by TLC. After the reaction was complete, the solution was concentrated under reduced pressure, redissolved in N,N-dimethylformamide (8 mL), and (E)-cyclooctyl-4-en-1-yl(4-nitrophenyl) carbonate (50 mg, 0.172 mmol) and N,N-diisopropylethylamine (150 μL, 0.86 mmol) were added. The reaction was carried out at room temperature for 16 h, monitored by TLC. After the reaction was complete, the solution was diluted with ethyl acetate, washed with saturated sodium chloride solution, and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 100:6) to give 47 mg of compound C2 as a white solid, in a yield of 52.22%.

[0133] The NMR and MS data of the compounds of this invention are detailed in Table 1.

[0134] Table 1

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141] Example 1

[0142] Instruments, materials, reagents, cells, and related pretreatments:

[0143] Instruments: 4℃, -20℃ and -80℃ refrigerators, clean bench, 37℃ constant temperature incubator, low and high speed centrifuges, 2.5μL, 10 μL, 20 μL, 100 μL, 200 μL and 1000 μL micro-adjustable pipettes, metal bath, vertical electrophoresis tank, rapid transfer instrument, LI-COR Odyssey infrared imaging system.

[0144] Materials: Corning 6-well cell culture plates, 1.5 mL, 5 mL, 15 mL and 50 mL centrifuge tubes, 10 μL, 200 μL and 1 mL sterile pipette tips, ultrapure water, electrophoresis buffer, rapid transfer buffer, TBST, PBS, 10% fetal bovine serum, 1% penicillin-streptomycin, DMEM basal medium, DMSO, lysis buffer, protein loading buffer.

[0145] Preparation of the test drug: The test drug was dissolved in DMSO to prepare a 10mM stock solution, which was then stored in a -20℃ refrigerator for later use.

[0146] Cells: Human breast cancer cells MDA-MB-231 cells.

[0147] MDA-MB-231 cell culture: MDA-MB-231 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were cultured at 37°C and 5% CO2.

[0148] Antibodies: Anti-Visfatin antibody [EPR21980] (1:1000 for WB, Abcamab236874), HDAC1 (D5C6U) Rabbit Monoclonal Antibody (1:1000 for WB, CST #34589), HRP Conjugated GAPDH Recombinant Rabbit Monoclonal Antibody [JF81-04] (1:50000 for WB, HUABIO ET1702-66), HRP-conjugated Goat Anti-Rabbit IgG (H+L) (1:10000 for WB, Proteintech SA00001-2).

[0149] Changes in NAMPT and HDAC protein levels after treatment of MDA-MB-231 cells with the compounds prepared in this invention:

[0150] MDA-MB-231 cells were fed at a concentration of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / well in 6-well plates and cultured at 37°C and 5% CO2 for 24 h. After washing once with PBS, cells were treated with serially diluted compounds prepared in this invention for 24 h. After treatment, the culture medium was aspirated, and cells were washed three times with PBS. Then, serially diluted CRBN ligands (compounds C1 and C2 prepared in this invention) were added, and incubation continued for another 48 h. The culture medium was discarded, and cells were washed three times with pre-chilled PBS. Subsequently, 80 μL of pre-chilled lysis buffer (containing a mixture of 1% protease inhibitor and phosphatase inhibitor) was added to each well, and cells were lysed on ice for 30 min. Cell lysates were collected using cell scraping and centrifuged at 15,000 rpm for 15 min at 4°C. The resulting supernatant was collected as the total protein extract. Protein loading buffer was added, and the protein sample was boiled at 100°C for 5 min to denature the protein sample. Denatured proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane using a semi-dry method. After transfer, the membrane was blocked with 5% BSA (prepared in TBST) for 2 hours at room temperature. The membrane was then incubated overnight at 4°C with the corresponding primary antibody, followed by washing three times with TBST for 10 min each time. Subsequently, the membrane was incubated with HRP-conjugated secondary antibody at room temperature for 1 hour. After thorough washing with TBST, protein bands were observed using a LI-COR Odyssey infrared imaging system. Using GAPDH as an internal control, the grayscale values ​​of the target protein bands were analyzed using ImageJ software to calculate the relative expression levels.

[0151] Experimental results are as follows Figure 1 As shown, Figure 1 The diagram illustrates the results of screening compounds with degradation activity. As can be seen from the figure, preliminary activity tests were conducted on the NAMPT series (compounds N1-N5) and the HDAC series (compounds H1-H4). Among all tested compounds, compounds N4 and H4 showed significant degradation effects, while the degradation effects of other compounds were not obvious. Therefore, compounds N4 and H4 were selected for subsequent experiments in this invention.

[0152] Precursor compounds N1–N3 were paired with CRBN ligand compound C1, or precursor compounds N4–N5 were paired with compound C2. Similarly, precursor compounds H1–H3 were paired with compound C1, or precursor compound H4 was paired with compound C2 for assembly. Results showed that compound N4+C2 exhibited the strongest NAMPT degradation, particularly in DCs within MDA-MB-231 cells. 50 The concentration was 0.321 μM. Similarly, compound H4+C2 exhibited the best HDAC1 degradation activity in the same cell line DC. 50 The concentration was 0.347 μM. Both proteins exhibited a significant hook effect at higher concentrations during degradation. Therefore, this invention selected a combination of compounds N4, H4, and C2 to further evaluate the dual-target degradation efficacy.

[0153] Example 2

[0154] Changes in NAMPT and HDAC protein levels after co-treatment of MDA-MB-231 cells with compounds N4, H4, and C2:

[0155] MDA-MB-231 cells were fed at a concentration of 1.5 × 10⁻⁶. 5Cells were seeded at a density of 100 cells / well in 6-well plates and cultured at 37°C and 5% CO2 for 24 h. After washing once with PBS, cells were co-treated with serially diluted compounds N4 and H4 for 24 h. After treatment, the culture medium was aspirated, and cells were washed three times with PBS. Then, serially diluted compound C2 was added, and incubation continued for another 48 h. The culture medium was discarded, and cells were washed three times with pre-chilled PBS. Subsequently, 80 μL of pre-chilled lysis buffer (containing a mixture of 1% protease inhibitor and phosphatase inhibitor) was added to each well, and cells were lysed on ice for 30 min. Cell lysates were collected using cell scrapings and centrifuged at 15,000 rpm for 15 min at 4°C. The resulting supernatant was collected as the total protein extract. Protein loading buffer was added, and the protein sample was boiled at 100°C for 5 min to denature the protein sample. Denatured proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes using a semi-dry method. After transfer, the membrane was blocked with 5% BSA (prepared in TBST) for 2 hours at room temperature. The membrane was then incubated overnight at 4°C with the corresponding primary antibody, followed by washing three times with TBST for 10 min each time. Subsequently, the membrane was incubated with HRP-conjugated secondary antibody at room temperature for 1 hour. After thorough washing with TBST, protein bands were observed using a LI-COR Odyssey infrared imaging system. Using GAPDH as an internal control, the grayscale values ​​of the target protein bands were analyzed using ImageJ software to calculate the relative expression levels.

[0156] Experimental results are as follows Figure 2 As shown, Figure 2 This diagram illustrates the results of treating MDA-MB-231 cells with compounds N4 and H4 for 24 hours, followed by treatment with compound C2 for 48 hours. The diagram shows that compounds N4 and C2 react intracellularly to generate NAMPT degraders; compounds H4 and C2 react intracellularly to generate HDAC degraders. When all three components are administered, dual-target degradation is achieved, with better results than dual-target degradation induced by the two components mentioned above. The combination of N4 / C2, synergistically with H4 / C2, induces the degradation of NAMPT and HDAC proteins, accompanied by a characteristic "hook effect." The degradative activity of NAMPT and HDAC is very strong, DC... 50 The values ​​were 25.4 nM and 9.42 nM, respectively. In summary, compounds N4, H4, and C2 function as highly efficient and synergistic degraders targeting NAMPT and HDAC.

[0157] As shown in the figure, the combination of compounds N4, H4, and C2 induces the simultaneous degradation of NAMPT and HDAC1 proteins in MDA-MB-231 cells. The degradation activity of NAMPT and HDAC1 is very strong, significantly better than single-target degradation. NAMPT's DC... 50 The molecular weight (MnM) is 25.5 nM, and that of HDAC1 is 9.42 nM. In summary, the combination of compounds N4, H4, and C2 functions as a highly efficient and synergistic degrader targeting NAMPT and HDAC.

[0158] In this invention, among the synthesized compounds, the first step is single-target degradation: compounds N4 and C2 exhibit the best degradation activity for NAMPT after undergoing a bioorthogonal reaction in cells, while compounds H4 and C2 exhibit the best degradation activity for HDAC1 after undergoing a bioorthogonal reaction in cells; then, compounds N4 and H4 are co-incubated, followed by incubation of compound C2, to achieve dual-target degradation of NAMPT and HDAC1, which is superior to the degradation effect of single-target degradation.

[0159] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A dual-targeting protein degrader of NAMPT and HDAC or a pharmaceutically acceptable salt thereof, characterized in that, The general structural formula is selected from one of the following structures: ； General formula H combined with general formula C can serve as a dual-target protein degrader for NAMPT and HDAC, or general formula N combined with general formula C can serve as a dual-target protein degrader for NAMPT and HDAC. Alternatively, general formulas N and H can be combined with general formula C as dual-target protein degraders for NAMPT and HDAC; in, R1 is selected from ； R4 is selected from C3~C10 cycloalkane groups; n1 is selected from positive integers from 1 to 5; R5is selected from C3-C10cycloalkyloxy, ; n2 is selected from positive integers from 1 to 5; R6is selected from C3-C10cycloalkyloxy, ; R7 is selected from hydrogen and C1~C10 alkyl groups; L is selected from -(CH2)n3, -(CH2)n4CONH(CH2)n5-; n3 is selected from positive integers from 1 to 20; n4 is selected from positive integers from 1 to 20; n5 is selected from positive integers from 1 to 20; R2is selected from C3-C10cycloalkyloxy, ; n6 is selected from positive integers from 1 to 10; R3is selected from .

2. The NAMPT and HDAC dual-target protein degrader or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, In the NAMPT and HDAC dual-target protein degrading agent R1 is selected from ； R4is selected from ; n1 is selected from 1, 2, 3, 4, 5; R5 is selected from ； n2 is selected from 1, 2, 3, 4, and 5; R6 is selected from ； L is selected from -(CH2)n3, -(CH2)n4CONH(CH2)n5-; n3 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; n4 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; n5 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; R2 is selected from ； n6 is selected from 1, 2, 3, 4, 5, 9, 7, 8, 9; R3is selected from .

3. The NAMPT and HDAC dual-target protein degrader or its pharmaceutical salt according to claim 2, characterized in that, The structure of the NAMPT and HDAC dual-target protein degrader is selected from one of the following structures: 。 4. The use of the NAMPT and HDAC dual-target protein degrader as described in any one of claims 1 to 3 in the preparation of a medicament for treating breast cancer.

5. The use of the NAMPT and HDAC dual-target protein degrader according to any one of claims 1 to 3 in the preparation of the NAMPT and HDAC dual-target protein degrader.

6. The use of compound N4 of claim 3 in combination with compound C2 in the preparation of a dual-target protein degrader for NAMPT and HDAC.

7. The use of compound H4 as described in claim 3 in combination with compound C2 in the preparation of a dual-target protein degrader for NAMPT and HDAC.

8. The use of compound H4 and compound N4 as described in claim 3 in combination with compound C2 in the preparation of a dual-target protein degrader for NAMPT and HDAC.

9. The use of compound H4 and compound N4 as described in claim 3 in combination with compound C2, or compound N4 in combination with compound C2, or compound H4 in combination with compound C2, in the preparation of a medicament for treating breast cancer.

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