Protein degradation targeting chimera and methods of making same

Abstract

The application provides a protein degradation targeting chimera and a preparation method thereof, which comprises a target protein ligand, an E3 ligase ligand and a linker, one end of the linker is connected with the target protein ligand, and the other end is connected with the E3 ligase ligand, and the structural formula of the ligase ligand is: the preparation method of the protein degradation targeting chimera provided by the application adopts a new E3 ligase ligand, so that a protein degradation targeting chimera compound with high yield and high purity can be obtained, the protein degradation targeting chimera can effectively degrade various proteins in cells, the type of the protein degradation targeting chimera is expanded, and the protein degradation targeting chimera has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to a protein degradation-targeting chimera and its preparation method. Background Technology

[0002] Targeted protein degradation (TPD) is an emerging therapeutic approach that has attracted significant attention due to its ability to modulate proteins that are difficult to target with traditional small molecules. Protein degradation-targeting chimeras (PROTACs) utilize the ubiquitin-proteasome system (UPS), a natural intracellular protein degradation system, to achieve targeted degradation of a target protein (POI). PROTACs are heterobifunctional molecules composed of three parts: a target protein ligand, an E3 ligase ligand, and a linker connecting the two. Intracellularly, one end of the PROTAC molecule binds to the target protein, and the other end binds to the E3 ligase, forming a target protein-PROTACs-E3 ligase ternary complex. This complex brings the target protein and the E3 ligase closer, prompting the E3 ligase to catalyze the transfer of ubiquitin to the target protein. Subsequently, the ubiquitinated target protein is recognized and degraded by the proteasome, and the physiological function of the target protein disappears with its degradation. Regulating the physiological function of a target protein through degradation opens up an unprecedented new avenue for biomedical research and pharmaceutical development.

[0003] Currently, all PROTAC molecules utilize only a few ligases of this type, primarily VHL, CRBN, MDM2, and IAPs. This is mainly due to the difficulty in developing new, effective E3-binding ligase ligands, and the scarcity of compounds capable of efficient degradation. Summary of the Invention

[0004] In view of this, the present invention aims to propose a protein degradation targeting chimera and its preparation method, so as to provide a novel E3 ligase ligand as part of PROTACs for complete PROTAC chemical synthesis, and to effectively degrade the target protein.

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

[0006] A protein degradation-targeting chimera includes a target protein ligand, an E3 ligase ligand, and a linker. One end of the linker is connected to the target protein ligand, and the other end is connected to the E3 ligase ligand. The structural formula of the ligase ligand is as follows:

[0007]

[0008] Furthermore, the structural formula of the connector is selected from one of the following structural formulas:

[0009]

[0010] Where n is any integer from 1 to 6, m is any integer from 2 to 5, and x is 1 or 2.

[0011] Furthermore, the structural formula of the target protein ligand is selected from one of the following structural formulas:

[0012]

[0013] Furthermore, the structural formula of the protein degradation targeting chimera is selected from one of the following structural formulas:

[0014]

[0015]

[0016]

[0017] The preparation method of the protein degradation targeting chimera as described above includes the following steps:

[0018] (1S,4S)-4-aminocyclohexane-1-carboxylate methyl hydrochloride and Et3N solution were slowly added dropwise to a chloroacetyl chloride solution and stirred overnight at room temperature. The mixture was then washed successively with 0.1M HCl solution, NaHCO3 solution, and brine. The organic phase was dried over anhydrous Na2SO4, and the solvent was removed under vacuum to obtain an oily residue. The residue was dissolved in MeCN, and then (1H-indol-2-yl)methylamine, NaHCO3, and KI were added. The mixture was refluxed for 12 hours, filtered, and the solvent was removed under vacuum. The residue was purified by column chromatography to obtain the first intermediate.

[0019] The first intermediate was dissolved in MeOH and water, LiOH-H2O was added, and the mixture was stirred overnight at room temperature. The pH was adjusted to 7, and the solvent was removed under vacuum to obtain the second intermediate.

[0020] The second intermediate was dissolved in 1,4-dioxane and water, and Na₂CO₃ and Fmoc-Osu were added. The mixture was stirred overnight at room temperature, and the pH was adjusted to 6-7. The solvent was removed under vacuum, the residue was diluted with water, the pH was adjusted to 4, and the mixture was extracted with ethyl acetate. The organic layers were combined, dried over anhydrous Na₂SO₄, filtered, and the solvent was removed under vacuum. The mixture was recrystallized from EtOAc and petroleum ether to give the compound with the following structural formula:

[0021]

[0022] Compared with existing technologies, the protein degradation-targeting chimera and its preparation method described in this invention have the following advantages:

[0023] The protein degradation targeted chimera preparation method described in this invention uses a novel E3 ligase ligand, which can obtain protein degradation targeted chimera compounds with high yield and high purity. It can effectively degrade a variety of intracellular proteins, expand the types of protein degradation targeted chimeras, and has broad application prospects. Attached Figure Description

[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0025] Figure 1 This is a schematic diagram of the degradation experiment results in Application Example 1;

[0026] Figure 2 This is a schematic diagram of the degradation experiment results in Application Example 2;

[0027] Figure 3 This is a schematic diagram of the degradation experiment results in Application Example 3;

[0028] Figure 4 This is a schematic diagram of the degradation experiment results in Application Example 4;

[0029] Figure 5 This is a schematic diagram of the degradation experiment results in Application Example 5. Detailed Implementation

[0030] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0031] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0032] Preparation Example 1

[0033] At 0 °C, a solution of (1S,4S)-4-aminocyclohexane-1-carboxylate hydrochloride (0.7 g, 3.6 mmol, 1 eq.) and Et3N (1.5 mL, 10.8 mmol, 3 eq.) in DCM was slowly added dropwise. The mixture was then stirred overnight at room temperature. The solution was washed successively with 0.1 M HCl solution, NaHCO3 solution, and brine. The organic phase was dried over anhydrous Na2SO4. The solvent was removed under vacuum, giving an oily residue. The residue was dissolved in MeCN, and then (1H-indol-2-yl)methylamine (789 mg, 5.4 mmol, 1.5 eq.), NaHCO3 (908 mg, 10.8 mmol, 3 eq.), and KI (60 mg, 0.36 mmol, 0.1 eq.) were added. The mixture was refluxed for 12 hours. After filtration, the solvent was removed under vacuum, and the residue was purified by column chromatography to obtain the first intermediate (1.2 g).

[0034] At 0 °C, the first intermediate (1.2 g, 3.49 mmol, 1 eq.) was dissolved in MeOH and water, followed by the addition of LiOH-H₂O (1.5 g, 34.9 mmol, 10 eq.). The mixture was then stirred overnight at room temperature. The pH of the mixture was adjusted to 7 at 0 °C, and the solvent was removed under vacuum to obtain the second intermediate. The second intermediate was dissolved in 1,4-dioxane and water at 0 °C, followed by the addition of Na₂CO₃ and Fmoc-OSu (1.75 g, 5.2 mmol, 1.5 eq.). The mixture was stirred overnight at room temperature. The pH of the mixture was adjusted to 6-7 at 0 °C, and the solvent was removed under vacuum. The residue was diluted with water, and the pH of the mixture was adjusted to 4. The mixture was extracted with ethyl acetate, the organic layers were combined, and dried over anhydrous Na₂SO₄. After filtration, the solvent was removed under vacuum. The residue was recrystallized from EtOAc and petroleum ether to give a compound with the following structural formula (1.3 g, yield 65%):

[0035]

[0036] 1H NMR(400MHz,DMSO-d6)δ12.04(s,1H),11.17(s,1H),11.07(s,1H),7.94–7.80(m,3H),7.63 (d,J=7.4Hz,1H),7.50–7.39(m,3H),7.39–7.27(m,3H),7.14–7.01(m,2H),7.01–6.91(m,1 H),6.29(s,1H),6.04(s,1H),4.59(d,J=20.1Hz,2H),4.37–4.20(m,3H),3.91(s,2H),3.82 –3.67(m,1H),2.41–2.34(m,1H),1.90–1.75(m,2H),1.61–1.50(m,4H),1.50–1.38(m,2H).

[0037] HRMS(ESI) for C 33 H 34 N3O5[M+H] + calculated 552.2493, found 552.2491.

[0038] Preparation Example 2

[0039] At 0 °C, 4-methylbenzenesulfonyl chloride (2.17 g, 11.40 mmol) was slowly added to a DCM solution of tert-butyl(3-hydroxypropyl)carbamate (1.00 g, 5.70 mmol), Et3N (1.59 mL, 11.40 mmol), and DMAP (0.35 g, 2.85 mmol). The mixture was then stirred overnight at room temperature. The solution was washed successively with 0.1 M HCl solution, NaHCO3 solution, and brine. The organic layer was dried over anhydrous Na2SO4. After filtration, the solvent was removed under vacuum, and the residue was purified by column chromatography (PE / EA) to give a pale yellow oil (1.03 g, 55% yield). A mixture of a pale yellow oil (1.03 g, 3.13 mmol), 4-amino-3-nitrophenol (0.96 g, 6.26 mmol), and K₂CO₃ (0.87 g, 6.26 mmol) in MeCN was refluxed for 12 hours. After filtration, the solvent was removed under vacuum. The residue was washed with 0.1 M NaOH solution and then extracted with dichloromethane. The organic layer was dried over anhydrous Na₂SO₄. After filtration, the solvent was removed under vacuum, and the residue was purified by column chromatography (PE / EA) to give a compound with the following structural formula (0.39 g, 40% yield):

[0040] 1H NMR(400MHz,Chloroform-d)δ7.54(d,J=3.0Hz,1H),7.06(dd,J=9.1,2.9Hz,1H),6.76(d,J=9.1Hz,1H),5. 89(s,2H),4.71(brs,1H),3.99(t,J=5.9Hz,2H),3.32(q,J=6.5Hz,2H),1.97(p,J=6.4Hz,2H),1.44(s,9H).

[0041] Preparation Example 3

[0042] The difference from Preparation Example 2 is that tert-butyl(3-hydroxypropyl)carbamate was replaced with tert-butyl(4-hydroxybutyl)carbamate, yielding the compound with the following structural formula (yield 20%):

[0043] 1 H NMR(400MHz,Chloroform-d)δ7.55–7.49(m,1H),7.10–7.01(m,1H),6.79–6.71(m,1H),5.88(s,2H),4. 60(brs,1H),3.98–3.88(m,2H),3.24–3.10(m,2H),1.86–1.75(m,2H),1.71–1.61(m,2H),1.44(s,9H).

[0044] Preparation Example 4

[0045] The difference from Preparation Example 2 is that tert-butyl (3-hydroxypropyl)carbamate was replaced with tert-butyl (5-hydroxypentyl)carbamate, yielding the compound with the following structural formula (yield 31%):

[0046] 1 H NMR(400MHz,Chloroform-d)δ7.53(d,J=2.9Hz,1H),7.06(dd,J=9.1,2.9Hz,1H),6.75(d,J=9.2Hz,1H),5.88(s,2H),4 .54(brs,1H),3.92(t,J=6.4Hz,2H),3.15(q,J=6.9,6.4Hz,2H),1.79(p,J=6.4Hz,2H),1.59–1.47(m,4H),1.45(s,9H).

[0047] Preparation Example 5

[0048] The difference from Preparation Example 2 is that tert-butyl (3-hydroxypropyl)carbamate was replaced with tert-butyl (6-hydroxyhexyl)carbamate, yielding the compound with the following structural formula (yield 29%):

[0049] 1 H NMR(400MHz,Chloroform-d)δ7.53(d,J=3.0Hz,1H),7.06(dd,J=9.0,2.9Hz,1H),6.75(d,J=9.2Hz,1H),5. 86(s,2H),4.51(brs,1H),3.92(t,J=6.5Hz,2H),3.19–3.06(m,2H),1.82–1.72(m,2H),1.54–1.36(m,15H).

[0050] Preparation Example 6

[0051] The difference from Preparation Example 2 is that tert-butyl(3-hydroxypropyl)carbamate was replaced with tert-butyl(7-hydroxyheptyl)carbamate, yielding the compound with the following structural formula (yield 22%):

[0052] 1 H NMR(400MHz,Chloroform-d)δ7.53(d,J=2.9Hz,1H),7.06(dd,J=9.1,2.9Hz,1H),6.75(d,J=9.2Hz,1H),5.88(s,2H),4.50(b rs,1H),3.92(t,J=6.5Hz,2H),3.12(q,J=6.4Hz,2H),1.82–1.70(m,2H),1.53–1.45(m,4H),1.44(s,9H),1.40–1.31(m,4H).

[0053] Preparation Example 7

[0054] The difference from Preparation Example 2 is that tert-butyl (3-hydroxypropyl)carbamate was replaced with aminotert-butyl diethylene glycol, yielding the compound with the following structural formula (yield 32%):

[0055] 1H NMR(400MHz,Chloroform-d)δ7.59(d,J=3.0Hz,1H),7.12(dd,J=9.1,2.9Hz,1H),6.76(d,J=9.1Hz,1H),5.88(s, 2H),4.95(brs,1H),4.13–4.07(m,2H),3.84–3.77(m,2H),3.60(t,J=5.2Hz,2H),3.42–3.28(m,2H),1.44(s,9H).

[0056] Preparation Example 8

[0057] The difference from Preparation Example 2 is that tert-butyl (3-hydroxypropyl)carbamate was replaced with aminotert-butyl ester-polyethylene glycol, yielding the compound with the following structural formula (yield 25%):

[0058] 1 H NMR(400MHz,Chloroform-d)δ7.58(d,J=2.9Hz,1H),7.12(dd,J=9.1,2.9Hz,1H),6.76(d,J=9.0Hz,1H),5.89(s,2H),4.99(brs,1H) ,4.11–4.09(m,2H),3.89–3.81(m,2H),3.74–3.67(m,2H),3.67–3.62(m,2H),3.55(t,J=5.2Hz,2H),3.37–3.25(m,2H),1.43(s,9H).

[0059] Preparation Example 9

[0060] The difference from Preparation Example 2 is that tert-butyl (3-hydroxypropyl)carbamate was replaced with aminotert-butyl tetraethylene glycol, yielding the compound with the following structural formula (yield 32%):

[0061] 1 H NMR(400MHz,Chloroform-d)δ7.57(d,J=2.9Hz,1H),7.11(dd,J=9.1,2.9Hz,1H),6.76(d,J=9.0Hz,1H),5.93(s,2H),5.05(b rs,1H),4.12–4.09(m,2H),3.88–3.82(m,2H),3.75–3.60(m,8H),3.54(t,J=5.2Hz,2H),3.31(q,J=5.5Hz,2H),1.44(s,9H).

[0062] Preparation Example 10

[0063] The difference from Preparation Example 2 is that tert-butyl (3-hydroxypropyl)carbamate was replaced with aminotert-butyl ester-polyethylene glycol, yielding the compound with the following structural formula (yield 33%):

[0064] 1 H NMR(400MHz,Chloroform-d)δ7.57(d,J=3.0Hz,1H),7.11(dd,J=9.1,2.9Hz,1H),6.76(d,J=9.0Hz,1H),5.92(s,2H),5.03(b rs,1H),4.12–4.08(m,2H),3.88–3.81(m,2H),3.74–3.59(m,12H),3.53(t,J=5.2Hz,2H),3.31(q,J=5.8Hz,2H),1.44(s,9H).

[0065] Preparation Example 11

[0066] A mixture of the compound obtained in Preparation Example 2 (0.39 g, 1.25 mmol) and 10% Pd / C (wet) (78 mg) in MeOH was degassed under vacuum and then flushed with H2. The mixture was stirred overnight at room temperature. After filtration, the solvent was removed under vacuum. The crude product was ready for the next step without purification. At 0 °C, DIPEA (96 ml, 0.55 mmol) was slowly added to a solution of the compound obtained in Preparation Example 1 (120 mg, 0.22 mmol), the crude product (80 mg, 0.28 mmol), and HBTU (107 mg, 0.28 mmol) in MeCN. The mixture was stirred overnight at room temperature. The solvent was removed under vacuum. The residue was dissolved in dichloromethane and washed with brine. The organic layer was dried over anhydrous Na2SO4. After filtration, the solvent was removed under vacuum. The residue was dissolved in HOAc, and the mixture was stirred at 70 °C for 1 hour. After vacuum evaporation of the solvent, the residue was purified by flash column chromatography to give a compound with the following structural formula (119 mg, yield 68%):

[0067] 1H NMR (400MHz, Chloroform-d) δ9.38 (s, 1H), 7.65 (d, J = 7.7Hz, 2H), 7.54–7.41 (m, 2H), 7.39–7. 26(m,4H),7.23–7.20(m,1H),7.15–6.99(m,4H),6.98–6.91(m,1H),6.82–6.73(m,1H),6.31( s,1H),6.18(s,1H),4.95–4.84(m,1H),4.49–4.33(m,3H),4.16–3.74(m,7H),3.33–3.15(m,2 H),2.90–2.77(m,1H),1.96–1.78(m,4H),1.78–1.64(m,2H),1.61–1.47(m,4H),1.42(s,9H).

[0068] Preparation Example 12

[0069] The difference from Preparation Example 11 is that the compound obtained in Preparation Example 2 was replaced with the compound obtained in Preparation Example 3, resulting in a compound with the following structural formula (yield 57%):

[0070]

[0071] 1 H NMR(400MHz,Chloroform-d)δ9.43(s,1H),7.71–7.62(m,2H),7.55–7.41(m,2H) ),7.40–7.27(m,4H),7.24–7.21(m,1H),7.20–6.87(m,5H),6.85–6.78(m,1H),6 .19(s,1H),4.81–4.67(m,1H),4.64–4.27(m,4H),4.19–4.05(m,1H),4.00–3.7 3(m,5H),3.21–3.04(m,2H),2.90–2.74(m,1H),1.90–1.44(m,12H),1.43(s,9H)

[0072] Preparation Example 13

[0073] The difference from Preparation Example 11 is that the compound obtained in Preparation Example 2 was replaced with the compound obtained in Preparation Example 4, resulting in a compound with the following structural formula (yield 58%):

[0074]

[0075] 1H NMR(400MHz,Chloroform-d)δ9.41(s,1H),7.65(d,J=7.7Hz,2H),7.53–7.42(m,2H),7.41 –7.26(m,4H),7.24–7.15(m,2H),7.14–6.95(m,4H),6.82(d,J=9.0Hz,1H),6.20(s,1H),4. 76–4.54(m,2H),4.53–4.23(m,3H),4.19–4.04(m,1H),4.03–3.72(m,5H),3.20–2.99(m,2H ),2.92–2.78(m,1H),1.96–1.81(m,2H),1.81–1.65(m,4H),1.64–1.45(m,8H),1.43(s,9H)

[0076] Preparation Example 14

[0077] The difference from Preparation Example 11 is that the compound obtained in Preparation Example 2 was replaced with the compound obtained in Preparation Example 5, resulting in a compound with the following structural formula (yield 56%):

[0078]

[0079] 1 H NMR(400MHz,Chloroform-d)δ9.37(s,1H),7.66(d,J=7.7Hz,2H),7.52–7.43(m,2H),7.42–7.37 (m,1H),7.36–7.25(m,4H),7.14–6.96(m,5H),6.84(d,J=8.8Hz,1H),6.18(s,1H),4.63–4.59(m, 1H),4.39(d,J=6.1Hz,2H),4.17–4.06(m,1H),4.04–3.76(m,6H),3.13–3.01(m,2H),2.88–2.77 (m,1H),1.97–1.82(m,2H),1.76–1.64(m,4H),1.63–1.46(m,6H),1.44(s,9H),1.37–1.27(m,4H)

[0080] Preparation Example 15

[0081] The difference from Preparation Example 11 is that the compound obtained in Preparation Example 2 was replaced with the compound obtained in Preparation Example 6, resulting in a compound with the following structural formula (yield 60%):

[0082]

[0083] 1 H NMR(400MHz,Chloroform-d)δ7.65(d,J=7.5Hz,2H),7.53–7.42(m,2H),7.41–7.30(m, 2H),7.30–7.25(m,3H),7.23–7.16(m,1H),7.15–6.95(m,4H),6.88–6.81(m,1H),6.34 (s,1H),6.19(s,1H),4.64–4.57(m,2H),4.38(d,J=6.3Hz,2H),4.17–3.75(m,6H),3.1 4–3.00(m,2H),2.87–2.79(m,1H),1.94–1.44(m,12H),1.43(s,9H),1.36–1.23(m,6H)

[0084] Preparation Example 16

[0085] The difference from Preparation Example 11 is that the compound obtained in Preparation Example 2 was replaced with the compound obtained in Preparation Example 7, resulting in a compound with the following structural formula (yield 62%):

[0086]

[0087] 1 H NMR(400MHz,Chloroform-d)δ7.70(d,J=7.4Hz,2H),7.53–7.40(m,3H),7.40–7.30(m,4H),7. 22–6.98(m,5H),6.96–6.85(m,1H),6.40(s,1H),6.20(s,1H),5.00(brs,1H),4.67–4.59(m,1H ),4.59–4.41(m,2H),4.38–4.28(m,1H),4.22–4.04(m,4H),4.01–3.76(m,5H),3.66–3.52(m, 3H),3.41–3.28(m,2H),2.96–2.85(m,1H),1.86–1.68(m,4H),1.68–1.51(m,4H),1.44(s,9H).

[0088] Preparation Example 17

[0089] The difference from Preparation Example 11 is that the compound obtained in Preparation Example 2 was replaced with the compound obtained in Preparation Example 8, resulting in a compound with the following structural formula (yield 58%):

[0090]

[0091] 1H NMR(400MHz,Chloroform-d)δ7.70(d,J=7.3Hz,2H),7.59–7.42(m,3H),7.42–7.27(m,4H),7.24– 6.99(m,5H),6.96–6.86(m,1H),6.20(s,1H),5.11–4.94(m,1H),4.68–4.42(m,3H),4.38–4.28(m ,1H),4.26–4.06(m,3H),3.96–3.79(m,4H),3.77–3.67(m,2H),3.67–3.59(m,2H),3.55(t,J=5.1 Hz,2H),3.36–3.27(m,2H),2.98–2.86(m,1H),1.90–1.70(m,4H),1.69–1.52(m,4H),1.43(s,9H).

[0092] HRMS(ESI) for C 50 H 59 N6O8[M+H] + calculated 871.4389, found 871.4389.

[0093] Preparation Example 18

[0094] The difference from Preparation Example 11 is that the compound obtained in Preparation Example 2 was replaced with the compound obtained in Preparation Example 9, resulting in a compound with the following structural formula (yield 55%):

[0095]

[0096] 1H NMR(400MHz,Chloroform-d)δ7.66(d,J=7.8Hz,2H),7.48(d,J=9.0Hz,2H),7.41–7.27(m,4H),7.24–7.15(m,2H), 7.15–6.95(m,5H),6.91–6.80(m,1H),6.19(s,1H),5.27(s,1H),5.11(t,J=5.8Hz,1H),4.61(s,1H),4.39(d,J=6.3 Hz,2H),4.14–4.02(m,3H),4.02–3.96(m,1H),3.92(s,2H),3.89–3.77(m,3H),3.73–3.52(m,10H),3.48(t,J=5.2H z,2H),3.30–3.19(m,2H),2.94–2.79(m,1H),2.00–1.84(m,2H),1.82–1.67(m,2H),1.65–1.45(m,4H),1.42(s,9H)

[0097] Preparation Example 19

[0098] The difference from Preparation Example 11 is that the compound obtained in Preparation Example 2 was replaced with the compound obtained in Preparation Example 10, resulting in a compound with the following structural formula (yield 51%):

[0099]

[0100] 1 H NMR(400MHz,Chloroform-d)δ7.73–7.61(m,2H),7.51–7.44(m,2H),7.43–7.37(m,1H),7.35–7.27(m,3H),7.24–7.16(m,2H ),7.15–7.05(m,2H),7.05–7.01(m,2H),6.82(d,J=8.8Hz,1H),6.19(s,1H),5.16–5.06(m,1H),4.66–4.53(m,1H),4.44–4. 35(m,3H),4.11–4.04(m,2H),3.94(s,2H),3.84–3.77(m,2H),3.71–3.66(m,2H),3.66–3.58(m,6H),3.58–3.49(m,5H),3.4 7–3.37(m,2H),3.26–3.15(m,2H),2.95–2.82(m,1H),1.97–1.83(m,2H),1.83–1.70(m,2H),1.63–1.49(m,4H),1.42(s,9H)

[0101] Preparation Example 20

[0102] Trifluoroacetic acid was slowly added dropwise to a solution of the compound obtained in Preparation Example 11 (60 mg, 0.07 mmol) in DCM at 0 °C. The mixture was stirred at 0 °C for 1 hour. The solvent was then removed under vacuum, and the residue was dissolved in DMF, followed by the addition of JQ1-COOH (32 mg, 0.08 mmol), HATU (42 mg, 0.11 mmol), and DIPEA (61 μL, 0.35 mmol). The reaction mixture was stirred overnight at room temperature. The mixture was diluted with EtOAc and washed with brine. The organic phase was dried over Na2SO4. After removing the solvent under vacuum, the residue was purified by column chromatography to give an intermediate. Piperidine was slowly added dropwise to the intermediate in a solution of DCM and MeCN to remove the Fmoc group. After stirring at room temperature for 2 hours, the solvent was removed under vacuum, and the residue was purified by HPLC to give a compound with the following structural formula (yield 30%):

[0103]

[0104] 1 H NMR(400MHz,Chloroform-d)δ10.35(s,1H),7.57–7.49(m,2H),7.31(d,J=8.7Hz,3H),7.25–7.21(m,3 H),7.07–6.99(m,2H),6.76(dd,J=8.8,2.4Hz,1H),6.37–6.32(m,1H),4.71(dd,J=9.3,4.6Hz,1H),4. 22(s,1H),4.00(s,1H),3.91(s,2H),3.82–3.72(m,2H),3.44–3.29(m,3H),3.27(s,2H),2.87–2.74(m ,1H),2.36(s,3H),2.28(s,3H),2.03–1.85(m,4H),1.76–1.66(m,4H),1.59(s,3H),1.57–1.49(m,2H).

[0105] HRMS(ESI) for C 46 H 50 ClN 10 O3S[M+H] + calculated 857.3477, found 857.3486

[0106] Preparation Example 21

[0107] The difference from Preparation Example 20 is that the compound obtained in Preparation Example 11 was replaced with the compound obtained in Preparation Example 12, resulting in a compound with the following structural formula (yield 35%):

[0108]

[0109] 1 H NMR (400MHz, Chloroform-d) δ10.30 (s, 1H), 7.51–7.45 (m, 2H), 7.37 (d, J = 8.5Hz, 3H), 7.29–7.27 (m, 1H), 7.25–7. 24(m,1H),7.22–7.16(m,2H),7.03–6.95(m,2H),6.86(brs,1H),6.76–6.69(m,1H),6.28(s,1H),4.66(t,J=6.9Hz, 1H),4.00–3.91(m,1H),3.88–3.75(m,4H),3.63–3.53(m,1H),3.43–3.34(m,1H),3.33–3.22(m,2H),3.17(s,2H),2 .92–2.81(m,1H),2.59(s,3H),2.34(s,3H),2.02–1.89(m,2H),1.86–1.62(m,8H),1.61(s,3H),1.57–1.46(m,2H).

[0110] HRMS(ESI) for C 47 H 52 ClN 10 O3S[M+H] + calculated 871.3628, found 871.3623.

[0111] Preparation Example 22

[0112] The difference from Preparation Example 20 is that the compound obtained in Preparation Example 11 was replaced with the compound obtained in Preparation Example 13, resulting in a compound with the following structural formula (yield 34%):

[0113]

[0114] 1H NMR(400MHz,Chloroform-d)δ11.35(brs,1H),10.32(s,1H),7.57–7.46(m,2H),7.37(d,J=8.7Hz,2H),7.28(d,J=8.8Hz,3H),7. 24–7.18(m,1H),7.06–6.96(m,2H),6.76(dd,J=8.8,2.4Hz,1H),6.30(d,J=2.1Hz,1H),4.67(t,J=7.0Hz,1H),4.02–3.97(m,1H), 3.88–3.81(m,4H),3.63–3.53(m,1H),3.49(s,1H),3.45–3.36(m,1H),3.31–3.23(m,2H),3.20(s,2H),2.94–2.84(m,1H),2.60( s,3H),2.34(s,3H),2.04–1.91(m,2H),1.86–1.79(m,2H),1.77–1.65(m,4H),1.61(s,3H),1.59–1.49(m,4H),1.49–1.38(m,2H).

[0115] HRMS(ESI) for C 48 H 54 ClN 10 O3S[M+H] + calculated 885.3790,found 885.3800

[0116] Preparation Example 23

[0117] The difference from Preparation Example 20 is that the compound obtained in Preparation Example 11 was replaced with the compound obtained in Preparation Example 14, resulting in a compound with the following structural formula (yield 29%):

[0118]

[0119] 1H NMR(400MHz,Chloroform-d)δ10.19(s,1H),7.54–7.46(m,2H),7.43–7.34(m,3H),7.30(d,J=8.4Hz,3H),7.24–7.19(m,1H ),7.05–6.98(m,2H),6.93–6.85(m,2H),6.78(d,J=8.3Hz,1H),6.32(s,1H),4.65(t,J=6.9Hz,1H),4.03–3.99(m,1H),3.92 –3.84(m,4H),3.55(dd,J=14.4,7.0Hz,1H),3.39(dd,J=14.6,6.3Hz,1H),3.30–3.21(m,4H),2.96–2.87(m,1H),2.61(s,3 H),2.35(s,3H),2.04–1.93(m,2H),1.89–1.81(m,2H),1.78–1.68(m,4H),1.54(dd,J=14.1,9.0Hz,4H),1.45–1.32(m,4H).

[0120] HRMS(ESI) for C 49 H 56 ClN 10 O3S[M+H] + calculated 899.3941, found 899.3940

[0121] Preparation Example 24

[0122] The difference from Preparation Example 20 is that the compound obtained in Preparation Example 11 was replaced with the compound obtained in Preparation Example 15, resulting in a compound with the following structural formula (yield 31%):

[0123]

[0124] 1H NMR(400MHz,Chloroform-d)δ10.30(s,1H),7.57–7.46(m,2H),7.43–7.34(m,3H),7.30(d,J=8.8Hz,2H),7.25–7.18(m,1H),7.07–6.9 7(m,2H),6.95–6.89(m,1H),6.79(dd,J=8.8,2.4Hz,1H),6.33–6.28(m,1H),4.64(t,J=7.0Hz,1H),4.03–3.98(m,1H),3.91–3.82(m,4 H),3.55(dd,J=14.6,7.3Hz,1H),3.49(s,1H),3.37(dd,J=14.6,6.5Hz,1H),3.32–3.18(m,4H),2.96–2.85(m,1H),2.62(s,3H),2.35( s,3H),2.05–1.91(m,2H),1.89–1.80(m,2H),1.78–1.66(m,4H),1.61(s,3H),1.58–1.44(m,4H),1.43–1.34(m,2H),1.33–1.26(m,4H).

[0125] HRMS(ESI) for C 50 H 58 ClN 10 O3S[M+H] + calculated 913.4097, found 913.4094

[0126] Preparation Example 25

[0127] The difference from Preparation Example 20 is that the compound obtained in Preparation Example 11 was replaced with the compound obtained in Preparation Example 16, resulting in a compound with the following structural formula (yield 30%):

[0128]

[0129] 1H NMR(400MHz,Chloroform-d)δ10.22(s,1H),7.58(d,J=8.3Hz,1H),7.54–7.47(m,1H),7.41(d,J=8.5Hz,2H),7.34–7.27( m,3H),7.25–7.20(m,2H),7.08–6.95(m,2H),6.95–6.79(m,2H),6.32(d,J=1.8Hz,1H),4.68(t,J=7.1Hz,1H),4.17(t,J=4 .5Hz,2H),4.05(s,1H),3.89(s,2H),3.84–3.72(m,2H),3.68–3.53(m,3H),3.53–3.39(m,5H),3.25(s,2H),3.00–2.86(m ,2H),2.61(s,3H),2.39(s,3H),2.09–1.93(m,2H),1.90–1.80(m,2H),1.79–1.70(m,2H),1.66(s,3H),1.63–1.53(m,2H).

[0130] 13 C NMR (101MHz, CDCl3) δ170.71,170.30,163.99,158.53,155.87,155.10,150.0 1,136.97,136.60,136.33,131.76,131.27,130.97,130.71,129.90,128.72,1 28.35,121.40,120.08,119.39,111.25,100.99,69.82,69.68,68.90,54.18,51.90,47.09,44.17,39.34,38.85,36.62,29.23,26.60,14.38,13.09,11.68.

[0131] HRMS(ESI) for C 47 H 52 ClN 10 O4S[M+H] + calculated 887.3577, found 887.3580

[0132] Preparation Example 26

[0133] The difference from Preparation Example 20 is that the compound obtained in Preparation Example 11 was replaced with the compound obtained in Preparation Example 17, resulting in a compound with the following structural formula (yield 28%):

[0134]

[0135] 1 1H NMR (400 MHz, Chloroform-d) δ 11.74 (brs, 1H), 10.25 (s, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.52–7.47 (m, 1H), 7.43–7.36 (m, 2H), 7.31 (d, J = 8.8 Hz, 2H), 7.21 (dd, J = 7.0, 3.7 Hz, 2H), 7.08–6.96 (m, 3H), 6.81 (dd, J = 8.8, 2.5 Hz, 1H), 6.31 (d, J = 2.1 Hz, 1H), 4.71 (t, J = 6.9 Hz, 1H), 4.22–4.09 (m, 2H), 4.09–3.99 (m, 1H), 3.87 (s, 2H), 3.83 (t, J = 5.0 Hz, 2H), 3.75–3.63 (m, 4H), 3.63–3.54 (m, 2H), 3.54–3.42 (m, 4H), 3.25 (s, 2H), 3.02–2.93 (m, 1H), 2.64 (s, 3H), 2.39 (s, 3H), 2.12–1.97 (m, 2H), 1.94–1.83 (m, 2H), 1.81–1.70 (m, 2H), 1.65 (s, 3H), 1.63–1.53 (m, 2H).

[0136] 13 13C NMR (101 MHz, CDCl3) δ 170.79, 170.45, 164.03, 155.85, 155.04, 149.90, 136.97, 136.90, 136.62, 136.49, 131.83, 131.19, 131.01, 130.69, 129.90, 128.72, 128.33, 121.36, 120.05, 119.35, 111.25, 100.97, 70.65, 70.18, 69.82, 69.43, 68.30, 54.17, 51.91, 47.08, 44.32, 39.44, 38.74, 36.43, 29.22, 26.70, 26.61, 14.37, 13.08, 11.74.

[0137] HRMS (ESI) for C 49 H 56 ClN 10 O5S [M + H] + calculated 931.3839, found 931.3841

[0138] Preparation Example 27

[0139] The difference from Preparation Example 20 is that the compound obtained in Preparation Example 11 was replaced with the compound obtained in Preparation Example 18, resulting in a compound with the following structural formula (yield 32%):

[0140]

[0141] 1 H NMR(400MHz,Chloroform-d)δ11.71(brs,1H),10.34(s,1H),7.58–7.45(m,3H),7.40(d,J=8.5Hz,2H),7.30(d,J=8.7Hz,2H),7.24 –7.18(m,1H),7.07–6.97(m,3H),6.80(d,J=8.3Hz,1H),6.30(d,J=2.0Hz,1H),4.69(t,J=7.0Hz,1H),4.09(t,J=5.0Hz,2H),4.04–3 .98(m,1H),3.85(s,2H),3.83–3.78(m,2H),3.75–3.56(m,9H),3.56–3.49(m,3H),3.48(s,2H),3.43–3.33(m,2H),3.22(s,2H),2.9 7–2.86(m,1H),2.63(s,3H),2.37(s,3H),2.04–1.95(m,2H),1.91–1.80(m,2H),1.76–1.67(m,2H),1.63(s,3H),1.61–1.52(m,2H).

[0142] 13 C NMR (101MHz, CDCl3) δ170.87,170.68,164.04,155.80,155.09,149.90,137.04,136.86,13 6.63,136.51,131.95,131.15,130.90,130.51,129.89,128.72,128.33,121.34,120.05,1 19.34,118.77,111.21,100.94,96.65,70.77,70.63,70.30,69.79,69.73,68.28,54.29,51.87,50.19,47.05,44.32,39.54,38.76,36.41,29.21,26.72,26.59,14.36,13.07,11.75.

[0143] HRMS(ESI) for C 51H 60 ClN 10 O6S[M+H] + calculated 975.4107, found 975.4111

[0144] Preparation Example 28

[0145] The difference from Preparation Example 20 is that the compound obtained in Preparation Example 11 was replaced with the compound obtained in Preparation Example 19, resulting in a compound with the following structural formula (yield 34%):

[0146]

[0147] 1 H NMR(400MHz,Chloroform-d)δ11.47(brs,1H),10.32(s,1H),7.55(d,J=7.8Hz,1H),7.49(dt,J=7.4,2.8Hz,1H),7.42–7.36(m,2H),7.31(d,J =8.8Hz,2H),7.25–7.16(m,2H),7.06–6.92(m,3H),6.80(dd,J=8.7,2.4Hz,1H),6.29(d,J=2.1Hz,1H),4.67(t,J=7.0Hz,1H),4.13–3.96(m,4 H),3.84(s,2H),3.80(t,J=4.8Hz,2H),3.73–3.67(m,2H),3.67–3.61( m,6H),3.61–3.57(m,3H),3.57–3.50(m,4H),3.50–3.42(m,4H),3.22( s,2H),2.97–2.87(m,1H),2.62(s,3H),2.36(s,3H),2.05–1.93(m,2H) ,1.89–1.80(m,2H),1.75–1.66(m,2H),1.62(s,3H),1.60–1.51(m,2H).

[0148] HRMS(ESI) for C 53 H 63 ClN 10 O7SNa[M+Na] + calculated 1041.4183, found 1041.4176

[0149] Preparation Example 29

[0150] The difference from Preparation Example 2 is that tert-butyl(3-hydroxypropyl)carbamate was replaced with tert-butyl-4-(2-hydroxyethyl)piperazine-1-carboxylic acid ester, yielding the compound with the following structural formula (yield 45%):

[0151] 1 H NMR(400MHz,Chloroform-d)δ7.57(d,J=3.0Hz,1H),7.09(dd,J=9.1,2.9Hz,1H),6.76(d,J=9.2Hz,1H),5.91( s,2H),4.07(t,J=5.6Hz,2H),3.46(t,J=5.1Hz,4H),2.80(t,J=5.6Hz,2H),2.51(t,J=5.1Hz,4H),1.46(s,9H)

[0152] Preparation Example 30

[0153] The difference from Preparation Example 29 is that tert-butyl-4-(2-hydroxyethyl)piperazine-1-carboxylate was replaced with tert-butyl-4-(3-hydroxypropyl)piperazine-1-carboxylate, yielding the compound with the following structural formula (yield 36%):

[0154] 1 H NMR(400MHz,Chloroform-d)δ7.55(d,J=3.0Hz,1H),7.06(dd,J=9.0,2.9Hz,1H),6.75(d,J=9.2Hz,1H),5.88(s,2H),3.99 (t,J=6.3Hz,2H),3.44(t,J=5.2Hz,4H),2.52(t,J=7.3Hz,2H),2.41(t,J=5.1Hz,4H),1.96(p,J=6.5Hz,2H),1.46(s,9H).

[0155] Preparation Example 31

[0156] The difference from Preparation Example 11 is that the compound obtained in Preparation Example 2 was replaced with the compound obtained in Preparation Example 29, resulting in a compound with the following structural formula (yield 51%):

[0157]

[0158] 1H NMR(400MHz,Chloroform-d)δ9.14(s,1H),7.66(d,J=7.7Hz,2H),7.53–7.42(m,2H),7.42–7.33(m,1H ),7.33–7.26(m,3H),7.24–7.22(m,1H),7.20–6.96(m,5H),6.88–6.75(m,1H),6.18(s,1H),4.63–4.54 (m,1H),4.45–4.31(m,3H),4.14–4.00(m,3H),3.99–3.94(m,1H),3.86(s,2H),3.49–3.37(m,4H),2.90 –2.82(m,1H),2.82–2.69(m,2H),2.55–2.42(m,4H),1.93–1.65(m,4H),1.64–1.48(m,4H),1.46(s,9H)

[0159] Preparation Example 32

[0160] The difference from Preparation Example 31 is that the compound obtained in Preparation Example 29 was replaced with the compound obtained in Preparation Example 30, resulting in a compound with the following structural formula (yield 54%):

[0161]

[0162] 1 H NMR(400MHz,Chloroform-d)δ7.66(d,J=7.8Hz,2H),7.52–7.41(m,2H),7.37–7.26(m, 4H),7.24–7.22(m,1H),7.17–6.97(m,5H),6.88–6.78(m,1H),6.17(s,1H),4.61–4.56 (m,1H),4.44–4.27(m,3H),4.02–3.79(m,6H),3.45–3.37(m,4H),2.91–2.75(m,1H),2 .53–2.43(m,2H),2.41–2.34(m,4H),1.99–1.73(m,5H),1.65–1.50(m,5H),1.46(s,9H)

[0163] Preparation Example 33

[0164] The difference from Preparation Example 20 is that the compound obtained in Preparation Example 11 was replaced with the compound obtained in Preparation Example 31, resulting in a compound with the following structural formula (yield 30%):

[0165] 1H NMR(400MHz,Chloroform-d)δ10.38(s,1H),7.64–7.57(m,1H),7.53–7.46(m,1H),7.43–7.37(m,3H),7.35–7.28(m,3H), 7.24–7.19(m,1H),7.05–6.97(m,3H),6.85–6.78(m,1H),6.31(s,1H),5.29(s,1H),4.81(t,J=6.7Hz,1H),4.06–3.96(m, 3H),3.85(s,2H),3.78–3.60(m,4H),3.60–3.48(m,3H),3.24(s,2H),2.98–2.88(m,1H),2.78–2.70(m,2H),2.63(s,3H), 2.59–2.41(m,4H),2.37(s,3H),2.08–1.96(m,2H),1.90–1.83(m,2H),1.79–1.72(m,2H),1.64(s,3H),1.60–1.51(m,2H).

[0166] HRMS(ESI) for C 49 H 55 ClN 11 O3S[M+H] + calculated 912.3893,found 912.3897

[0167] Preparation Example 34

[0168] The difference from Preparation Example 33 is that the compound obtained in Preparation Example 31 was replaced with the compound obtained in Preparation Example 32, resulting in a compound with the following structural formula (yield 28%):

[0169] 1H NMR(400MHz, Methanol-d4)δ7.49–7.36(m,6H),7.22(d,J=8.0Hz,1H),7.09–7.04(m,1H),7.03–6.95(m,1H),6.95– 6.85(m,2H),6.30(s,1H),4.73–4.66(m,1H),4.64–4.59(m,1H),4.11(t,J=6.2Hz,2H),3.98–3.92(m,1H),3.91(s, 2H),3.84–3.71(m,2H),3.68–3.61(m,2H),3.61–3.51(m,2H),3.29(s,2H),3.05–2.94(m,1H),2.70(s,3H),2.67–2 .60(m,4H),2.58–2.48(m,2H),2.45(s,3H),2.12–1.98(m,4H),1.96–1.86(m,2H),1.70(s,3H),1.69–1.64(m,4H).

[0170] HRMS(ESI) for C 50 H 56 ClN 11 O3SNa[M+Na] + calculated 948.3869, found 948.3867

[0171] Preparation Example 35

[0172] Trifluoroacetic acid was slowly added dropwise to a solution of the compound obtained in Preparation Example 11 (60 mg, 0.07 mmol) in DCM at 0 °C. The mixture was stirred at 0 °C for 1 hour. The solvent was then removed under vacuum, and the residue was dissolved in DMF, followed by the addition of 2-(4-(1,2-diphenylbut-1-en-1-yl)phenoxy)acetic acid (32 mg, 0.08 mmol), HATU (42 mg, 0.11 mmol), and DIPEA (61 μL, 0.35 mmol). The reaction mixture was stirred overnight at room temperature. The mixture was diluted with EtOAc and washed with brine. The organic phase was dried over Na2SO4. After removing the solvent under vacuum, the residue was purified by column chromatography to give an intermediate. Piperidine was slowly added dropwise to a solution of the intermediate in DCM and MeCN to remove the Fmoc group. After stirring at room temperature for 2 hours, the solvent was removed under vacuum, and the residue was purified by HPLC to give a compound with the following structural formula (yield 31%):

[0173]

[0174] 1H NMR(400MHz,Chloroform-d)δ10.00(d,J=28.9Hz,1H),9.69(d,J=21.5Hz,1H),7.56–7.46(m, 3H),7.23–6.93(m,15H),6.91–6.83(m,4H),6.39–6.34(m,1H),4.54–4.46(m,2H),4.07–3.96 (m,2H),3.96–3.85(m,3H),3.64–3.48(m,2H),3.26(s,2H),2.97–2.84(m,1H),2.46(q,J=6.4 ,5.9Hz,2H),2.09–1.95(m,4H),1.94–1.79(m,5H),1.69–1.57(m,2H),0.92(t,J=7.4Hz,3H).

[0175] HRMS(ESI) for C 51 H 55 N6O4[M+H] + calculated 815.4279, found 815.4276

[0176] Preparation Example 36

[0177] The difference from Preparation Example 35 is that the compound obtained in Preparation Example 11 was replaced with the compound obtained in Preparation Example 16, resulting in a compound with the following structural formula (yield 31%):

[0178]

[0179] 1H NMR(400MHz,Chloroform-d)δ9.94(s,1H),7.44–7.35(m,2H),7.29–7.16(m,3H),7.09(d,J=7.0Hz,2H),7.05–6.93(m, 6H),6.93–6.83(m,4H),6.81–6.61(m,5H),6.37(d,J=8.5Hz,1H),6.21(s,1H),4.35(s,1H),4.18(s,1H),3.94–3.84(m, 3H),3.76(s,2H),3.65–3.54(m,2H),3.52–3.47(m,1H),3.47–3.40(m,2H),3.40–3.33(m,2H),3.14(s,2H),2.77–2.72( m,1H),2.38–2.26(m,2H),1.95–1.79(m,2H),1.78–1.57(m,4H),1.52–1.38(m,2H),1.13(s,2H),0.78(t,J=7.4Hz,3H).

[0180] HRMS(ESI) for C 52 H 57 N6O5[M+H] + calculated 845.4385, found 845.4389

[0181] Preparation Example 37

[0182] Trifluoroacetic acid was slowly added dropwise to a solution of the compound obtained in Preparation Example 11 (50 mg, 0.06 mmol) in DCM at 0 °C. The mixture was stirred at 0 °C for 1 hour. The solvent was then removed under vacuum, and the residue was dissolved in DMF, followed by the addition of 2-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)acetic acid (23 mg, 0.06 mmol), HATU (38 mg, 0.10 mmol), and DIPEA (55 μL, 0.32 mmol). The reaction mixture was stirred overnight at room temperature. The mixture was diluted with EtOAc and washed with brine. The organic phase was dried over Na2SO4. After removing the solvent under vacuum, the residue was purified by column chromatography to give an intermediate. Piperidine was slowly added dropwise to a solution of the intermediate in DCM and MeCN to remove the Fmoc group. After stirring at room temperature for 2 hours, the solvent was removed under vacuum, and the residue was purified by HPLC to obtain a compound with the following structural formula (yield 34%):

[0183]

[0184] 1H NMR(400MHz, Methanol-d4)δ7.56–7.49(m,1H),7.33–7.24(m,2H),7.20–7.14(m,2H),7.14–7.07( m,1H),6.99(s,1H),6.92–6.77(m,6H),6.18(s,1H),4.02(t,J=5.8Hz,2H),3.85–3.81(m,1H),3.8 0(s,2H),3.43(t,J=6.5Hz,2H),3.19(s,2H),3.01(s,2H),2.98–2.90(m,4H),2.81–2.70(m,1H),2 .63–2.56(m,4H),1.97(p,J=6.1Hz,2H),1.91–1.78(m,2H),1.78–1.66(m,2H),1.57–1.48(m,4H).

[0185] HRMS(ESI) for C 52 H 57 N6O5[M+H] + calculated 786.4198, found 786.4200

[0186] Preparation Example 38

[0187] The difference from Preparation Example 37 is that the compound obtained in Preparation Example 11 was replaced with the compound obtained in Preparation Example 16, resulting in a compound with the following structural formula (yield 30%):

[0188]

[0189] 1 H NMR(400MHz, Methanol-d4)δ7.58(d,J=8.3Hz,1H),7.38(d,J=7.7Hz,1H),7.32–7.17(m,4H),7. 05(s,1H),6.98(t,J=7.4Hz,1H),6.94–6.82(m,4H),6.28(s,1H),4.20–4.14(m,2H),3.96–3.84( m,5H),3.70(t,J=5.2Hz,2H),3.52(t,J=5.2Hz,2H),3.30(s,2H),3.10(s,2H),3.07–2.98(m,4H) ,2.92–2.82(m,1H),2.72–2.65(m,4H),2.02–1.90(m,2H),1.88–1.79(m,2H),1.72–1.58(m,4H).

[0190] HRMS(ESI) for C 44 H 54 N 11 O5[M+H] + calculated 816.4304, found 816.4309

[0191] Preparation Example 39

[0192] The difference from Preparation Example 26 is that JQ1-COOH was replaced with 2-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)acetic acid, yielding a compound with the following structural formula (yield 29%):

[0193]

[0194] 1 H NMR(400MHz, Methanol-d4)δ7.75–7.69(m,1H),7.44–7.35(m,2H),7.35–7.18(m,3H),7.02–6.87(m,5H ),6.82(dd,J=8.7,2.4Hz,1H),6.29(s,1H),4.14(t,J=4.5Hz,2H),3.96–3.84(m,5H),3.79–3.73(m,2H) ,3.73–3.66(m,2H),3.62(t,J=5.2Hz,2H),3.47(t,J=5.2Hz,2H),3.30(s,2H),3.20–3.12(m,4H),3.10 (s,2H),3.01–2.91(m,1H),2.75–2.68(m,4H),2.08–1.96(m,2H),1.94–1.85(m,2H),1.72–1.62(m,4H).

[0195] HRMS(ESI) for C 46 H 58 N 11 O6[M+H] + calculated 860.4566, found 860.4565

[0196] Preparation Example 40

[0197] The difference from Preparation Example 27 is that JQ1-COOH was replaced with 2-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)acetic acid, yielding a compound with the following structural formula (yield 27%):

[0198]

[0199] 1 H NMR (400MHz, Methanol-d4) δ7.77(d,J=8.0Hz,1H),7.48(s,1H),7.39(d,J=7.9Hz,2H),7.29–7.18(m,2H), 7.02–6.88(m,5H),6.83(dd,J=8.7,2.4Hz,1H),6.30(s,1H),4.14–4.08(m,2H),3.92(s,2H),3.82(t,J=4.4 Hz,2H),3.73–3.61(m,9H),3.58(t,J=5.3Hz,2H),3.44(t,J=5.1Hz,2H),3.30(s,2H),3.23–3.14(m,4H),3 .10(s,2H),3.01–2.95(m,1H),2.77–2.68(m,4H),2.10–1.97(m,2H),1.95–1.86(m,2H),1.73–1.63(m,4H).

[0200] HRMS(ESI) for C 48 H 62 N 11 O7[M+H] + calculated 904.4828,found 904.4823.HRMS(ESI)for C48H61N11O7Na[M+Na] + calculated 926.4648, found 926.4641

[0201] Preparation Example 41

[0202] The difference from Preparation Example 20 is that JQ1-COOH was replaced with 2-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)acetic acid, yielding a compound with the following structural formula (yield 33%):

[0203]

[0204] 1H NMR(400MHz, Methanol-d4)δ7.84–7.77(m,1H),7.53(s,1H),7.46–7.36(m,2H),7.30–7.18(m,2H),7.05(s, 1H),7.02–6.84(m,5H),6.30(s,1H),4.11(t,J=6.1Hz,2H),3.91(d,J=10.2Hz,3H),3.74–3.69(m,2H),3.68 –3.63(m,2H),3.35(s,2H),3.30(s,2H),3.26–3.18(m,4H),3.04–2.95(m,1H),2.80–2.72(m,4H),2.65(t,J =7.3Hz,2H),2.62–2.57(m,2H),2.56–2.50(m,2H),2.11–1.99(m,4H),1.96–1.87(m,2H),1.72–1.64(m,4H).

[0205] HRMS(ESI) for C 47 H 59 N 12 O4[M+H] + calculated 855.4777,found 855.4780

[0206] Application Example 1

[0207] The compounds obtained in Preparation Example 25 and Preparation Example 34 were tested for their degradation effect on the target protein by immunoblotting.

[0208] The detection method is as follows:

[0209] U2OS cells were treated with DMSO solvent or compounds of varying concentrations for 8 hours. BRD2, BRD4(long), and BRD4(short) proteins, and the control β-actin were detected by Western blotting. The results are shown below. Figure 1 As shown.

[0210] Application Example 2

[0211] The degradation effect of the compound obtained in Preparation Example 35 on the target protein was detected by immunoblotting.

[0212] The detection method is as follows:

[0213] MCF-7 cells were treated with DMSO solvent or different concentrations of compounds for 18 hours. ERα protein and control β-actin were detected by Western blotting. The results are as follows: Figure 2 As shown.

[0214] Application Example 3

[0215] The degradation effect of the compound obtained in Preparation Example 39 on the target protein was detected by immunoblotting.

[0216] The detection method is as follows:

[0217] U2OS cells were treated with DMSO solvent or compounds of varying concentrations for 8 hours. SMARCA2 protein and control β-actin were detected by Western blotting. Results are shown below. Figure 3 As shown.

[0218] Application Example 4

[0219] The compounds prepared in Examples 20-28 were tested for their degradation effect on target proteins using immunoblotting experiments.

[0220] The detection method is as follows:

[0221] U2OS cells were treated with DMSO solvent or a compound at a concentration of 500 nM for 8 hours. BRD4 (long), BRD4 (short) proteins and control β-actin were detected by Western blotting. The results are as follows: Figure 4 As shown.

[0222] Application Example 5

[0223] The compounds obtained in Preparation Examples 33 and 34 were tested for their degradation effect on the target protein by immunoblotting.

[0224] The detection method is as follows:

[0225] U2OS cells were treated with DMSO solvent or a compound at a concentration of 500 nM for 8 hours. BRD4 (long), BRD4 (short) proteins and control β-actin were detected by Western blotting. The results are as follows: Figure 5 As shown.

[0226] from Figures 1-5 The test results show that the protein degradation targeting chimera prepared in this invention can effectively degrade BRD2, BRD4(long), BRD4(short), ERα, and SMARCA2 proteins. Further research can be conducted on the degradation of other proteins, including but not limited to EGFR, WDR5, SMURF1, PI3K, KRAS, and TGF-TGF-β. The protein degradation targeting chimera prepared in this invention has broad application prospects in the field of protein degradation.

Claims

1. A protein degradation-targeting chimera, characterized in that: The structural formula of the protein degradation-targeting chimera is selected from one of the following structural formulas: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。

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