E3 ligase FEM1B novel ligand and preparation and application of derivative thereof

Abstract

The invention discloses a novel ligand of E3 ligase FEM1B, and belongs to the field of medicinal chemistry. The molecular structure of the ligand is as shown in a formula 1: # imgabs0 # FEM1B novel ligand has high affinity and specificity to FEM1B in vivo and in vitro, has low toxicity, is obviously lower than that of a reported ligand EN106, has a potential treatment effect on promoting angiogenesis and repairing chronic wound surfaces of diabetes mellitus, can be developed into various derivatives (PROTAC molecules and the like) based on the FEM1B ligand, changes the specificity of a substrate, and can be used for preparing a medicine for treating chronic wound surfaces of diabetes mellitus. The specific pathogenic protein (such as BRD4) in cells is degraded, the treatment effect is further achieved, the toxicity of derivative molecules is small, and absorption and utilization of the human body are facilitated.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular to the preparation and application of a novel ligand and its derivatives that bind to the E3 ligase FEM1B. The ligand molecule and its derivatives can change the substrate specificity of FEM1B, thereby causing the degradation of specific proteins in cells.

[0002] The compounds involved in the present invention have potential anti-inflammatory, anti-oxidative and therapeutic effects on diseases such as cancer. Background Art

[0003] FEM1B (Fem-1 homolog B) protein is a conserved protein in the human genome. It was first discovered in Caenorhabditis elegans and plays an important role in the development and signal transduction of multicellular organisms. In humans, FEM1B is a component of the Cullin2-RING E3 ubiquitin ligase complex. Its main role is the substrate receptor of the complex. It participates in regulating the degradation of multiple proteins (including SLBP, CDK5R1, GLI1, FNIP1, etc.) and has an important influence on processes such as sex determination, cell apoptosis regulation, and reductive stress. FEM1B is expressed in most tissues of the human body and is also located in the cytoplasm and nucleus. The expression level of FEM1B in the bone marrow is significantly higher than that in most tissues, and it is also highly expressed in acute myeloid leukemia.

[0004] The physiological function of FEM1B makes it a potential drug target. Liu Guohui's team can promote angiogenesis and repair chronic diabetic wounds through drug intervention in the FEM1B-FNIP1 axis (PMID: 37521275). In addition, as an E3 ligase, FEM1B also has good application prospects in the field of targeted protein degradation. Daniel Nomura's team developed the first and currently only FEM1B ligand small molecule EN106, and based on it developed it into a degrader targeting proteins such as BRD4 and BCR-ABL. These compounds have the effect of killing tumor cells. (PMID: 34994556 and related patents WO2021183431A1). However, EN106 still has defects such as high toxicity and poor specificity.

[0005] Developing new FEM1B small molecule ligands and using these ligands to invent new targeted protein degraders can provide new options for the treatment of tumors, metabolism and other related diseases. Summary of the invention

[0006] In view of this, the present invention provides, on one hand, a novel ligand of E3 ligase FEM1B having a structural formula as shown in Formula 1, and derivatives thereof, wherein the derivatives include stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs thereof.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a novel ligand for E3 ligase FEM1B, wherein the molecular structure of the ligand is shown in Formula 1:

[0009]

[0010] Wherein: R1 is a substituent at any position on the A ring, selected from hydrogen, halogen, amino, carboxyl, C 1-4 Any one or a combination of two or more of a straight chain, branched chain or cyclic alkyl, -CF3, phenyl, substituted phenyl, or a structure as shown in Formula 2;

[0011]

[0012] R2 is selected from a benzene ring or a benzoheterocyclic ring, wherein the benzene ring includes a benzene ring containing a substituent, and the benzoheterocyclic ring includes a benzoheterocyclic ring containing a substituent;

[0013] Ring A is selected from any one or a combination of two or more of benzene ring, cyclopentane, cyclohexane, piperidine, pyridine, piperazine, morpholine, diazine, triazine, bicyclo[1.1.1]pentane, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, bicyclo[2.2.2]octane, cubane, and adamantane;

[0014] X is selected from any one of nitrogen, oxygen and carbon;

[0015] Y is selected from C 2-6 Alkyl chain, C 2-6 An alkoxy chain, a dipeptide fragment shown in Formula 3, or a combination of any one or more of the structures shown in Formula 4; R3 and R4 in Formula 3 are preferably hydrogen, methyl, isopropyl, or tert-butyl; n in Formula 4 represents any number between 0 and 6, and M represents -N- or -CH-;

[0016]

[0017] Z is selected from Any one of .

[0018] Further, the derivative is as shown in Formula 5, and the derivative includes stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs of the novel ligand;

[0019]

[0020] Wherein: the FEM1B ligand is a compound represented by formula 1;

[0021] LINKER is a linking group selected from the following structures, or any combination of the following structures: C 1-20 Straight and branched alkyl chains, C 1-20 Alkoxy chain, C 7-11 Spiral ring, C 7-11 Azaspiro ring, C 7-11 Oxaspiro ring, any structure shown in Formula 6; in Formula 6, n represents any number between 0 and 8, and M represents -N- or -CH-;

[0022]

[0023] The hinge group is selected from any one of amide, ester, urea, amine, ether, thioether, and triazole;

[0024] POI ligands are small molecules that have direct binding affinity to target proteins, including any one of BRD2 / 3 / 4, FKBP12, K-RAS, SMARCA2, Bruton's tyrosine kinase protein, androgen receptor protein, MYC protein, N-MYC protein, β-catenin or Huntington protein.

[0025] Furthermore, the novel ligand of the E3 ligase FEM1B is represented by any structure of L1-L56:

[0026]

[0027]

[0028]

[0029] Furthermore, the structural formula of the derivative is shown in any structure of P1-P9:

[0030]

[0031] Furthermore, the E3 ligase ligand molecule is used in the preparation of a drug for treating a tumor disease. Furthermore, the derivative is used in the preparation of a drug for treating a tumor disease.

[0032] Furthermore, the tumor disease is one or more of breast cancer, pancreatic cancer, colon cancer, prostate cancer, lung cancer, liver cancer, acute myeloid leukemia, and multiple myeloma.

[0033] The beneficial effects of the present invention are:

[0034] 1. The new FEM1B ligand has high affinity and specificity for FEM1B both in vivo and in vitro.

[0035] 2. The new FEM1B ligand has low toxicity, significantly lower than the reported ligand EN106.

[0036] 3. The new FEM1B ligand has potential therapeutic effects, such as promoting angiogenesis and repairing chronic diabetic wounds.

[0037] 4. Based on the FEM1B ligand, a variety of derivatives (PROTAC molecules, etc.) can be developed, which change the substrate specificity, causing the degradation of specific pathogenic proteins (such as BRD4) in the cell, thereby exerting a therapeutic effect.

[0038] 5. Based on the FEM1B ligand, a variety of PROTAC molecules can be developed with low toxicity, which is conducive to human absorption and utilization.

[0039] Other advantages, objectives and features of the present invention will be described in the following description and will be apparent to those skilled in the art to some extent, or those skilled in the art may be taught from the practice of the present invention. The objectives and other advantages of the present invention may be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to make the purpose, technical solution and beneficial effects of the invention clearer, the present invention provides the following drawings for illustration:

[0041] Figure 1 This is a graph showing the experimental results of using fluorescence polarization to determine the ability of a compound to competitively bind to FEM1B;

[0042] Figure 2 This is a diagram showing the experimental results of using microthermophoresis to determine the affinity of the compound to the FEM1B protein;

[0043] Figure 3 This is a graph showing the results of a protein stability experiment using dual fluorescence characterization to determine the ability of a compound to competitively bind to FEM1B in cells;

[0044] Figure 4 This is the result of the CETSA cell thermal shift experiment, which is used to characterize the binding of the compound to intracellular FEM1B;

[0045] Figure 5 This is an experimental result diagram of using Western Blot to detect the effect of PROTAC on specific degradation of BRD4;

[0046] Figure 6This is a graph showing the experimental results of using the Cell-Titer Glo method to detect the toxic effects of compounds on normal cells. DETAILED DESCRIPTION

[0047] The present invention provides the preparation and application of a novel ligand of E3 ligase FEM1B and its derivatives.

[0048] In the present invention, the raw materials can be obtained from commercial sources, or prepared by methods known in the art, or prepared according to the methods described herein. The structures of the compounds were determined by nuclear magnetic resonance (NMR). 1 H-NMR / 13 C-NMR) and / or liquid-mass spectrometry (LC-MS). NMR determination was performed using a Bruker AVANCE IV Dr (600 MHz) nuclear magnetic resonance instrument, and the determination solvents were deuterated chloroform (CDCl3-d), deuterated methanol (MeOD-d4), deuterated dimethyl sulfoxide (DMSO-d6), and TMS was used as the internal standard. LC-MS determination was performed using an Agilent / 1260-6160 single quadrupole liquid-mass spectrometer. The preparative liquid phase model was Agilent 1260 Infinity II. Silica gel column chromatography used 200-300 mesh silica gel from Qingdao Ocean Chemical Plant.

[0049] Embodiment 1:

[0050] Compound N 1 -(2-(4-(2-chloroacetyl)piperazin-1-yl)ethyl)-N 3 Preparation of -(3-hydroxyphenyl)-5-(1-oxoisoindolin-2-yl)isophthalamide (L36), the synthetic route is as follows:

[0051]

[0052] Synthesis of intermediate 1-(4-(2-aminoethyl)piperazin-1-yl)-2-chloroethane-1-one (I-2): I-1 (0.40 g, 1.74 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane (10 mL), triethylamine (0.37 mL, 2.62 mmol, 1.5 equiv) and chloroacetyl chloride (0.15 mL, 1.92 mmol, 1.1 equiv) were added at 0°C, and reacted at room temperature for 2 hours. The mixture was washed with saturated ammonium chloride solution and saturated sodium chloride solution in sequence, extracted with dichloromethane, dried over anhydrous sodium sulfate, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol=25 / 1) to obtain a colorless oily product (0.53 g, yield: 99%). 1HNMR (600MHz, Chloroform-d) δ4.89 (s, 1H), 4.00 (s, 2H), 3.57 (t, J = 5.0 Hz, 2H), 3.46 (t, J = 5.0 Hz, 2H), 3.18 (d, J = 6.8 Hz, 2H), 2.47–2.37 (m, 6H), 1.38 (s, 9H). The above oil (0.53 g, 1.73 mmol, 1.0 equiv) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (1.3 mL, 17.3 mmol, 10.0 equiv) was slowly added dropwise at 0°C, and the reaction was allowed to proceed at room temperature until the reaction was complete. The product I-2 was concentrated by distillation under reduced pressure to obtain a yellow oil, which was used directly in the next step without purification.

[0053] Synthesis of intermediate 5-((2-cyanobenzyl)amino)isophthalic acid dimethyl ester (I-5): I-3 (1.00 g, 4.78 mmol, 1.0 equiv) was dissolved in acetonitrile (20 mL), and I-4 (0.94 g, 4.78 mmol, 1.0 equiv) and potassium carbonate (0.99 g, 7.17 mmol, 1.5 equiv) were added, and refluxed for 24 hours. The mixture was cooled to room temperature and poured into ice water (35 mL), and a solid was precipitated, which was filtered and dried to obtain a white solid I-5 (1.55 g, yield: 99%).

[0054] Synthesis of intermediate 5-(1-oxoisoindolin-2-yl)isophthalic acid (I-6): Disperse I-5 (1.55 g, 4.78 mmol) in 6.0 M sodium hydroxide aqueous solution (20 mL) and reflux for 24 hours. Cool to 0 ° C, acidify with 6.0 M hydrochloric acid aqueous solution, precipitate solid, filter, and dry the filter cake. Disperse the above solid in glacial acetic acid (35 mL) and reflux for 24 hours. Cool to room temperature, filter, rinse the filter cake with a large amount of ether, and dry the filter cake to obtain a white solid I-6 (1.27 g, yield: 90%). 1 H NMR (600MHz, DMSO-d6) δ13.17(s,2H),8.72(s,2H),8.26(s,1H),7.81(d,J=7.6Hz,1H),7.74–7.64(m,2H),7.60–7.49(m,1H),5.14(s,2H).

[0055] Synthesis of intermediate 3-((3-benzyloxyphenyl)carbamoyl)-5-(1-oxoisoindolin-2-yl)benzoic acid (I-7): I-6 (0.30 g, 1.01 mmol, 1.0 equiv) was dissolved in anhydrous acetonitrile (10 mL) and anhydrous DMF (2 mL), and 3-benzyloxyaniline (0.22 g, 1.11 mmol, 1.1 equiv), HATU (0.46 g, 1.21 mmol, 1.2 equiv), and DIPEA (0.35 ml, 2.02 mmol, 2.0 equiv) were added, and the mixture was reacted at room temperature for 8 hours. The mixture was concentrated by distillation under reduced pressure, diluted with water, and adjusted to pH = 2 with 1.0 M hydrochloric acid aqueous solution. The solid was precipitated, filtered, and the filter cake was dried to obtain a brown solid I-7 (0.32 g, yield: 67%). 1 H NMR(600MHz,DMSO-d6)δ10.46(s,1H),8.77(s,1H),8.40(s,1H),8.32(t,J= 1.5Hz,1H),7.82(dd,J=7.6,1.1Hz,1H),7.73–7.68(m,2H),7.60(t,J=2.2H z,1H),7.59–7.55(m,1H),7.49–7.44(m,2H),7.43–7.38(m,3H),7.36–7.31 (m,1H),7.27(t,J=8.1Hz,1H),6.80–6.77(m,1H),5.14(s,2H),5.11(s,2H).

[0056] Intermediate N 1 -(3-Benzyloxyphenyl)-N 3 -Synthesis of (2-(4-(2-chloroacetyl)piperazin-1-yl)ethyl)-5-(1-oxoisoindolin-2-yl)isophthalamide (I-8): I-7 (0.28 g, 0.58 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane (5 mL) and anhydrous DMF (2 mL), and I-2 (0.12 g, 0.58 mmol, 1.0 equiv), HATU (0.33 g, 0.87 mmol, 1.5 equiv), and DIPEA (0.25 mL, 1.45 mmol, 2.5 equiv) were added and reacted at room temperature for 8 hours. The product was washed successively with saturated ammonium chloride solution, saturated sodium bicarbonate solution and saturated sodium chloride solution, extracted with dichloromethane and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (dichloromethane / methanol=20 / 1) to obtain yellow oily I-8 (0.12 g, yield: 31%).

[0057] Compound N 1 -(2-(4-(2-chloroacetyl)piperazin-1-yl)ethyl)-N 3Synthesis of -(3-hydroxyphenyl)-5-(1-oxoisoindolin-2-yl)isophthalamide (L36): I-8 (0.12 g, 0.18 mmol) was dissolved in toluene (2 mL), trifluoroacetic acid (2 mL) was added, and the reaction was carried out at room temperature until the reaction was complete. The mixture was concentrated by distillation under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol=15 / 1) to obtain a white solid L36 (57 mg, yield: 57%). 1 HNMR(600MHz,DMSO-d6)δ10.32(s,1H),9.45(s,1H),8.65(t,J=5.7Hz,1H),8.54(t,J= 2.0Hz,2H),8.16(t,J=1.6Hz,1H),7.84(d,J=7.4Hz,1H),7.74–7.69(m,2H),7.61–7.53 (m,1H),7.37(t,J=2.2Hz,1H),7.20–7.11(m,2H),6.57–6.50(m,1H),5.16(s,2H),4.3 7(s,2H),3.52–3.42(m,6H),2.55(t,J=6.9Hz,2H),2.50(p,4H),2.44(t,J=5.2Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ166.97,165.58,165.04,164.52,157.57,141.09,140.02,139.64,136.27,135.59,132.62,132.07,129 .31,128.35,123.46,123.42,121.74,120.67,111.14,111.01,107.51,56.70,52.75,52.31,50.63,45.39,41.85,41.67,36.92.

[0058] Example 2

[0059] Compound N 1 -(3-(4-(2-chloroacetyl)piperazin-1-yl)propyl)-N 3 -(3-Hydroxyphenyl)adamantane-1,3-dicarboxamide (L45), the synthetic route is as follows:

[0060]

[0061] Synthesis of intermediate I-9: (1) Dissolve tert-butyl N-[3-(piperazin-1-yl)propyl]carbamate (0.30 g, 1.23 mmol, 1.0 equiv) in anhydrous dichloromethane (10 mL), add triethylamine (0.26 mL, 1.85 mmol, 1.5 equiv) and chloroacetyl chloride (0.10 mL, 1.29 mmol, 1.1 equiv) at 0°C, and react at room temperature for 2 hours. Wash with saturated ammonium chloride solution and saturated sodium chloride solution in sequence, extract with dichloromethane, dry with anhydrous sodium sulfate, and purify the crude product by silica gel column chromatography (dichloromethane / methanol=25 / 1) to obtain a colorless oily product (0.25 g, yield: 64%). 1 HNMR (600MHz, Chloroform-d) δ5.16 (s, 1H), 4.05 (s, 2H), 3.66–3.59 (m, 2H), 3.55–3.48 (m, 2H), 3.19 (d, J=6.3Hz, 2H), 2.48 (t, J=5.1Hz, 2H), 2.42 (t, J=6.7Hz, 4H), 1.66 (t, J=6.7Hz, 2H), 1.42 (s, 9H). (2) The above product (0.22g, 0.69mmol, 1.0equiv) was dissolved in dichloromethane (5mL), trifluoroacetic acid (0.53mL, 6.90mmol, 10.0equiv) was slowly added dropwise at 0°C, and the reaction was allowed to proceed at room temperature until the reaction was complete. The product I-9 was concentrated by distillation under reduced pressure to obtain a yellow oily product, which was used directly in the next step without purification.

[0062] Synthesis of L45: 1,3-adamantanedicarboxylic acid (150 mg, 0.67 mmol, 1.0 equiv) was dissolved in anhydrous DMF (3 mL), and 3-aminophenol (80 mg, 0.74 mmol, 1.1 equiv), T4P (48-52% in Ethyl Acetate) (0.61 mL, 1.01 mmol, 1.5 equiv), and DIPEA (0.23 mL, 1.34 mmol, 2.0 equiv) were added. After reacting at room temperature for 2 hours, I-9 (0.67 mmol, 1.0 equiv), T4P (48-52% in Ethyl Acetate) (0.61 mL, 1.01 mmol, 1.5 equiv), and DIPEA (0.23 mL, 1.34 mmol, 2.0 equiv) were added and reacted at room temperature for 1 hour. The reaction mixture was washed successively with saturated ammonium chloride solution, saturated sodium bicarbonate solution and saturated sodium chloride solution, extracted with ethyl acetate and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 15 / 1) to give a white solid L45 (17 mg, yield: 5%). 1H NMR(600MHz, Methanol-d4)δ7.12(t,J=2.2Hz,1H),7.09(t,J=8.1Hz,1H),6.96–6.92(m,1H) ,6.56–6.53(m,1H),4.21(s,2H),3.64–3.60(m,2H),3.59–3.56(m,2H),3.25(t,J=6.8Hz,2H) ,2.53(t,J=5.1Hz,2H),2.48(t,J=5.1Hz,2H),2.43(t,J=7.1Hz,2H),2.21(p,J=3.1Hz,2H),2 .03(s,2H),1.96(q,J=13.4Hz,4H),1.91–1.82(m,4H),1.80–1.75(m,2H),1.75–1.69(m,2H). 13 C NMR(151MHz,Methanol-d4)δ179.94,178.23,167.54,158.79,140.69,130.33,113.67,112.46,109.77,5 7.24,54.18,53.68,46.98,43.21,43.07,42.41,41.87,41.55,39.37,39.17,39.07,36.43,29.82,26.99.

[0063] Example 3

[0064] Derivatives (1R,3S,5R,7S)-N 1 -(3-(4-(2-chloroacetyl)piperazin-1-yl)propyl)-N 3 Preparation of -(3-(2-(5-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-yl)acetamido)pentanamide)ethoxy)-5-hydroxyphenyl)adamantane-1,3-dicarboxamide (P7), the synthetic route is as follows:

[0065]

[0066] Synthesis of intermediate I-10: tert-butyl 4-(3-aminopropyl)piperazine-1-carboxylate (2.0 g, 8.2 mmol, 1.2 equiv) was dissolved in anhydrous DMF (30 mL), and 1,3-adamantanedicarboxylic acid (1.5 g, 6.8 mmol, 1.0 equiv), HATU (2.9 g, 7.5 mmol, 1.1 equiv), and DIPEA (3.0 mL, 17.1 mmol, 2.5 equiv) were added, and the mixture was reacted at room temperature for 1 hour. Then, 5-aminobenzene-1,3-diol hydrochloride (1.1 g, 6.8 mmol, 1.0 equiv), HATU (2.9 g, 7.5 mmol, 1.1 equiv), and DIPEA (3.0 mL, 17.1 mmol, 2.5 equiv) were added, and the mixture was reacted at room temperature for 4 hours. Add 2.0M sodium hydroxide aqueous solution (30mL), stir for 10 minutes, and then adjust the pH to weak acidity with 1.0M hydrochloric acid aqueous solution. Wash with saturated ammonium chloride solution and saturated sodium chloride solution in sequence, extract with ethyl acetate, dry with anhydrous sodium sulfate, and purify the crude product by silica gel column chromatography (dichloromethane / methanol=10 / 1) to obtain a yellow solid product (2.8g, yield: 61%). 1 HNMR(400MHz,Methanol-d4)δ6.57(d,J=2.2Hz,2H),6.06(t,J=2.2Hz,1H),3.65(s,3H),3.28(d,J=6.5Hz,2H ),3.08(s,3H),2.96(t,J=7.6Hz,2H),2.24–2.16(m,2H),2.04–1.83(m,14H),1.79–1.72(m,2H),1.47(s,9H).

[0067] Synthesis of intermediate I-11: I-10 (0.9 g, 1.7 mmol, 1.0 equiv) was dissolved in acetone (20 mL), and (2-bromoethyl) benzyl carbamate (0.5 g, 1.9 mmol, 1.1 equiv), Cs2CO3 (0.7 g, 2.0 mmol, 1.2 equiv), KI (28 mg, 0.2 mmol, 0.1 equiv) were added, and refluxed for 24 hours. The mixture was concentrated by distillation under reduced pressure, washed with water and saturated sodium chloride solution in turn, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 15 / 1) to obtain a white solid product (0.3 g, yield: 26%). 1H NMR(400MHz,Chloroform-d)δ7.50(s,1H),7.36–7.27(m,7H),6.98(s,1H),6.4 1(d,J=2.3Hz,1H),6.17(d,J=2.2Hz,1H),5.46(t,J=6.0Hz,1H),5.08(s,2H),3. 92(t,J=5.2Hz,2H),3.56–3.47(m,6H),3.31(q,J=5.6Hz,2H),2.50–2.41(m,6H ),2.19(s,2H),2.00(s,2H),1.90–1.82(m,6H),1.75–1.66(m,6H),1.45(s,9H).

[0068] Synthesis of intermediate I-12: I-11 (0.3 g, 0.4 mmol) was dissolved in methanol (15 mL), 10% Pd / C (32 mg) was added, hydrogen was replaced, and the reaction was carried out at room temperature overnight. The mixture was filtered and the filtrate was concentrated by distillation under reduced pressure to obtain a white solid product (0.2 g, yield: 77%). 1 H NMR (400MHz, Methanol-d4) δ6.73(p,J=1.9Hz,2H),6.17(t,J=2.2Hz,1H),3.95(t,J=5.3Hz,2H),3.43(t,J=5.0Hz,4H),3.23(t,J=6.8 Hz,2H),2.98(t,J=5.3Hz,2H),2.43–2.36(m,6H),2.23–2.18(m,2H),2.02(s,2H),1.99–1.81(m,8H),1.81–1.66(m,4H),1.45(s,9H).

[0069] Synthesis of intermediate I-13: (+)-JQ-1 carboxylic acid (88 mg, 0.2 mmol, 1.0 equiv) was dissolved in anhydrous DMF (2 mL), and tert-butyl 5-aminopentanoate (40 mg, 0.2 mmol, 1.0 equiv), HATU (125 mg, 0.3 mmol, 1.5 equiv), and DIPEA (115 mL, 0.7 mmol, 3.0 equiv) were added, and reacted at room temperature for 1 hour. The mixture was washed with saturated ammonium chloride solution and saturated sodium chloride solution in sequence, extracted with ethyl acetate, and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 15 / 1) to obtain a colorless oily product (100 mg, yield: 83%). 1HNMR(400MHz,Chloroform-d)δ7.37(d,J=8.6Hz,2H),7.32–7.27(m,2H),6.83(t,J=5. 7Hz,1H),4.61(t,J=7.0Hz,1H),3.57–3.48(m,1H),3.39–3.27(m,2H),3.26–3.16(m,1 H), 2.64 (s, 3H), 2.37 (s, 3H), 2.19 (t, J = 6.9 Hz, 2H), 1.64 (s, 3H), 1.61–1.49 (m, 4H), 1.41 (s, 9H). The above-obtained oil (140 mg, 0.3 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (0.5 mL) was added dropwise, and the reaction was carried out at room temperature until the reaction was complete. The yellow oil product I-13 was obtained by distillation and concentration under reduced pressure, and was used directly in the next step without purification.

[0070] Synthesis of intermediate I-14: I-13 (0.1 mmol, 1.0 equiv) was dissolved in anhydrous DMF (2 mL), and I-12 (75 mg, 0.1 mmol, 1.0 equiv), HATU (62 mg, 0.2 mmol, 1.3 equiv), and DIPEA (65 mL, 0.4 mmol, 3.0 equiv) were added, and the mixture was reacted at room temperature for 1 hour. A 2.0 M aqueous sodium hydroxide solution (2 mL) was added, and the mixture was stirred for 10 minutes, and the pH was adjusted to a weak acidity with a 1.0 M aqueous hydrochloric acid solution. The mixture was washed with a saturated ammonium chloride solution and a saturated sodium chloride solution, extracted with ethyl acetate, and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to obtain a white solid (108 mg, yield: 80%). 1 H NMR(400MHz, Methanol-d4)δ7.45–7.37(m,4H),6.78–6.74(m,1H),6.69–6.66(m,1H),6.14–6.10( m,1H),4.69–4.59(m,1H),3.98–3.90(m,2H),3.52(t,J=4.8Hz,6H),3.43–3.36(m,1H),3.29–3.22( m,4H),2.73–2.67(m,7H),2.63(t,J=7.4Hz,2H),2.41(s,3H),2.26(t,J=7.2Hz,2H),2.16(q,J=3. 0Hz,2H),2.00(s,2H),1.96–1.73(m,11H),1.68(d,J=27.1Hz,7H),1.62–1.52(m,2H),1.45(s,9H).

[0071] Synthesis of compound P7: (1) I-14 (108 mg, 0.1 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (0.5 mL) was added dropwise, and the reaction was carried out at room temperature until the reaction was complete. The product was concentrated by distillation under reduced pressure to obtain a yellow oily product, which was used directly in the next step without purification. (2) The above product was dissolved in anhydrous dichloromethane (3 mL), triethylamine (69 μL, 0.5 mmol, 5.0 equiv) and chloroacetyl chloride (8 μL, 0.1 mmol, 1.0 equiv) were added at 0°C, and the reaction was carried out at room temperature for 30 minutes. The product was concentrated by distillation under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to obtain a white solid (51 mg, yield: 48%). 1 H NMR (600MHz, Methanol-d4) δ7.45–7.41(m,2H),7.40(d,J=8.8Hz,2H),6.74(t,J=2.0Hz,1H),6.67(t,J=2.0Hz,1H),6.13(t,J=2.2Hz,1H) ,4.63(dd,J=8.7,5.6Hz,1H),4.20(s,2H),3.94(td,J=5.2,2.9Hz,2H),3.63–3.58(m,2H),3.58–3.55(m,2H),3.54–3.48(m,2H),3.39(dd, J=15.0,8.7Hz,1H),3.30–3.20(m,5H),2.67(s,3H),2.54(t,J=5.1Hz,2H),2.48(t,J=5.1Hz,2H),2.45–2.40(m,5H),2.26(t,J=7.3Hz,2H ),2.16(t,J=3.3Hz,2H),1.99(s,2H),1.94–1.84(m,6H),1.80(d,J=12.2Hz,2H),1.74–1.67(m,6H),1.66–1.63(m,3H),1.61–1.55(m,2H). 13C NMR(151MHz,Methanol-d4)δ179.88,178.07,176.14,172.64,167.44,166.17,161.27,159.66,1 56.97,152.11,141.37,138.08,137.93,133.47,133.19,131.99,131.91,131.32,129.78,102.38 ,100.08,99.15,67.41,57.23,55.24,54.12,53.64,46.85,43.27,42.96,42.38,41.91,41.50,40.05,39.33,39.15,39.02,38.85,36.55,36.38,29.91,29.78,26.87,24.34,14.44,12.97,11.65.

[0072] Similarly, according to the steps of Examples 1-3, compounds L1-L56 or their derivatives P1-P9 are prepared.

[0073] Example 4: Fluorescence polarization was used to determine the ability of compounds to competitively bind to FEM1B.

[0074] The method mainly includes the following steps: (1) Prepare a buffer solution (40 mM HEPES, 150 mM NaCl, 0.05% Tween20, 1 mM DTT, pH = 7.5). (2) Use the buffer solution to prepare 500 nM FEM1B protein for use, and prepare a 40 nM FNIP1 substrate peptide solution with a FITC fluorescent group at the N-terminus for use, and prepare a series of gradient dilutions of compounds for use (up to 2.5 mM, containing 8% DMSO). (3) Add 10 μL of 500 nM FEM1B to a 384-well plate, and then add 5 μL of the diluted compound to each well, and incubate at room temperature for 2 hours. (4) Next, add 5 μL of 40 nM FITC-FINP1 fluorescent peptide to the mixture and incubate at room temperature for 1 hour. (5) Use an ELISA reader to detect fluorescence. If the compound binds to the target protein, it will release free FITC peptides, resulting in a weakening of the fluorescence polarization signal. By plotting the fluorescence polarization values ​​corresponding to different compound concentrations, the inhibition constant (K) of the small molecule can be calculated. i ) or IC 50 value.

[0075] The results are attached Figure 1 As shown, the IC of the example compound L36 of the present invention is 50 The IC value of the positive control compound EN106 was 1.9 μM under the same assay conditions. 50The IC value of the compound P7 of the present invention is 6.6 μM, so L36 is superior to EN106 in the ability to bind to FEM1B. 50 It is 0.81 μM, indicating that after the ligand is transformed into PROTAC, it still has a strong FEM1B affinity.

[0076] Example 5: Determination of the affinity of the compounds for FEM1B protein using microthermophoresis.

[0077] The main steps include: dilute the His dye 1:50, dilute the FEM1B protein to 1.8μM with PBS buffer, and then take 50μL of the diluted protein and incubate with 50μL of the diluted dye at room temperature for 30 minutes. The incubated protein was centrifuged at 4°C for 10 minutes, and then 90μL of the supernatant was taken and diluted 3 times with 180μL PBS buffer, and the final protein concentration was 300nM. Next, the small molecule stock solution with the highest concentration of 10mM was diluted 25 times with PBS buffer, and the other concentration gradients were diluted twice with PBS buffer containing 4% DMSO. Take 4μL of FEM1B protein solution and incubate with 4μL of small molecules of different concentrations at room temperature for 1 hour. Use a special capillary to draw the sample, perform experimental detection with an MST instrument, fit the relationship curve between fluorescence intensity and small molecule concentration, and obtain the dissociation constant (K) of the interaction. d ), and related kinetic information.

[0078] The results are attached Figure 2 As shown in the MST test, the dissociation constant K between the example compound P7 of the present invention and FEM1B is d It is 0.71 μM, indicating that its affinity is strong and is consistent with the value measured by the FP method.

[0079] Example 6: Protein stability assay using dual fluorescence characterization to determine the ability of compounds to competitively bind to FEM1B in cells.

[0080] The method mainly includes the following steps: (1) constructing a pCDH-EGFP-FNIP1-IRES-mCherry recombinant plasmid for visually judging the degradation of FNIP1 in vivo. Constructing a pLVX-2xFlag-FEM1B recombinant plasmid for overexpressing FEM1B protein in cells. (2) Cultivating 293T cells in a 10 cm culture dish to a cell density of 70-80%. (3) Transient plasmid transfection: prepare two 1.5mL EP tubes, add 500ul serum-free and anti-antibody-free DMEM to each tube, and mark them as tube A and tube B; add 5μg pCDH-EGFP-FNIP1-IRES-mCherry and 5μg pLVX-2xFlag-FEM1B to tube A, and add 30ul PEI to tube B; let the two tubes stand at room temperature for 5 minutes; add the PEI diluted mixture in tube B to the plasmid mixture in tube A, pipette and mix, and let stand at room temperature for 15 minutes; add the mixture dropwise to 293T cells and shake gently; after 6 hours, aspirate the original culture medium and replace it with 5ml fresh culture medium. (4) 24 hours after transfection, plate the cells, digest the 10cm cells transfected with plasmid, centrifuge, add 3ml DMEM to resuspend, take 20ul cell suspension for cell counting, and dilute the cells according to 400,000 cells per well of 12-well plate and plate them. (5) 12 hours after cell plating, add the compound at a working concentration of 10μM after the cells adhere to the wall, and place in a carbon dioxide incubator at 37℃ for 12 hours. (6) 48 hours after transfection, collect samples for flow cytometry: discard the DMEM in each well, wash twice with PBS, and discard the PBS; add 100ul of phenol red-free trypsin to each well to digest the cells, and then add 500ul of PBS containing 5% FBS to terminate; collect the digested cells into a 1.5ml EP tube, centrifuge at 800rpm for 3min; discard the supernatant, take 300ul of PBS containing 2% FBS to resuspend the cells; transfer the resuspended cells to a flow tube for flow cytometry detection. (7) Cell flow cytometry: set the number of collected cells to 10,000 total cells, set the voltage and fluorescence compensation, and the flow rate to low speed; circle the living cell group, single cell group, and fluorescent cell group for cell analysis. (8) Flowjo data analysis: Use SSC-A and FSC-A to circle the live cell population; use FSC-A and FSC-H to circle the single cell population; finally use EGFP-A and mCherry-A to analyze the fluorescent cell population; use Mean to calculate the average fluorescence value of EGFP and mCherry; export the data. (9) GraphPad Prism data processing: Calculate the EGFP / mCherry ratio as the vertical axis, compare the ratio between the DMSO group and the drug-treated group, and measure the activity of the compound.

[0081] The results are attached Figure 3As shown in the figure, compared with cells treated with DMSO, after adding the example compound L36 of the present invention, the EGFP / mCherry ratio increased significantly, indicating that it can competitively bind to FEM1B in cells and inhibit the degradation of EGFP-FNIP1. L36 is almost the same as EN106 at the cellular level.

[0082] Example 7: CETSA cell thermal migration experiment.

[0083] The main steps include the following: cells were treated with DMSO or designated compounds with fresh medium at 37°C for 3 hours. Subsequently, cells were collected, washed once with binding buffer (cold PBS containing DMSO or designated compounds and a mixture of protease inhibitors), and resuspended in 1.5 mL of binding buffer. Next, 100 μL of the cell suspension was transferred to a PCR tube, heated at the designated temperature for 3 minutes in a Bio-Rad C1000 thermal cycler, and then incubated at 25°C for 3 minutes. After three rapid freeze-thaw cycles in liquid nitrogen, the lysate was transferred to a 1.5 mL Eppendorf tube and centrifuged at 15,000 g for 20 minutes at 4°C. The resulting supernatant was transferred to a new Eppendorf tube, mixed with 5× loading buffer, boiled for 15 minutes, and then subjected to immunoblotting analysis.

[0084] The results are attached Figure 4 As shown, compared with cells treated with DMSO, after the addition of the example compound L36 of the present invention, the thermal stability of the intracellular FEM1B protein in the range of 55-58°C was enhanced, while the intracellular reference protein β-actin had no obvious change. This result suggests that L36 can bind to FEM1B in cells.

[0085] Example 8: Use Western Blot to detect the effect of PROTAC in specifically degrading BRD4.

[0086] The main steps include: transfer the suspended cells (such as THP-1) to an EP tube, add lysis buffer (containing protease and phosphatase inhibitors) after centrifugation and washing, lyse on ice for 10 minutes, and centrifuge at 4°C. Take the supernatant for protein quantification, use an enzyme reader to measure the absorbance, and draw a standard curve. Take 40μL of protein supernatant, add 5X loading buffer, boil and centrifuge. Prepare separation gel and concentrated gel, fill and seal the gel. Prepare concentrated gel and insert the comb. Prepare samples, add 1X running gel buffer to the electrophoresis tank, load 10μg sample, electrophoresis: 80V, 20min; 120V, 1 hour. Soak the PVDF membrane in methanol, place it in the electrophoresis tank, and use an ice box or ice bag to transfer the membrane at 300mA for 120min. Take the membrane and prepare 5% skim milk for blocking for 2 hours. Incubate with primary antibody (BRD4 or BRD31:1000, β-Actin 1:6000) at 4°C overnight. Wash the membrane 3 times, 10 minutes each time. Incubate with secondary antibody (BRD4 rabbit anti-1:3000, β-Actin mouse anti-1:6000) at room temperature for 90 minutes. Wash the membrane 3 times, 10 minutes each time. Finally, perform color development detection.

[0087] The results are attached Figure 5 As shown in the figure, after treating cells with P7, intracellular BRD4 decreased in a concentration-dependent manner and was almost completely degraded at a concentration of 10 μM, while BRD3 and internal reference proteins did not change significantly. This result suggests that P7 can recruit FEM1B and specifically degrade BRD4 in cells.

[0088] Example 9: The Cell-Titer Glo method was used to detect the toxic effects of the compounds on normal cells.

[0089] The method mainly includes the following steps: using a white 96-well plate, adding 190ul of cell suspension to each well according to 1000 cells per well, and standing overnight. The next day, the ligand or derivative was first diluted three times with DMSO, then diluted 50 times with blank medium, and finally 10μL of the 50-fold diluted small molecule was added to the wells of the pre-laid plate, and the final dilution multiple was 1000 times, and incubated for 48 hours. After adding the ligand / derivative of the present invention, 100μL DMEM was discarded, 40uL of CellTiter-Glo reagent was added to each well, and the shaker was placed in a shaker at 150rpm for 10 minutes, and the chemiluminescent signal was finally detected using an enzyme reader.

[0090] The results are attached Figure 6 As shown, after normal cells were treated with L36 or P7, almost no cell death occurred even at higher concentrations, suggesting that this type of compound has low toxicity.

[0091] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A novel ligand for E3 ligase FEM1B, characterized in that: The molecular structure of the ligand is shown in Formula 1: Wherein: R1 is a substituent at any position on the A ring, selected from hydrogen, halogen, amino, carboxyl, C 1-4 Any one or a combination of two or more of a straight chain, branched chain or cyclic alkyl, -CF3, phenyl, substituted phenyl, or a structure as shown in Formula 2; R2 is selected from a benzene ring or a benzoheterocycle, wherein the benzene ring is a benzene ring containing a substituent, and the benzoheterocycle is a benzoheterocycle containing a substituent; Ring A is selected from any one or a combination of two or more of benzene ring, cyclopentane, cyclohexane, piperidine, pyridine, piperazine, morpholine, diazine, triazine, bicyclo[1.1.1]pentane, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, bicyclo[2.2.2]octane, cubane, and adamantane; X is selected from any one of nitrogen, oxygen and carbon; Y is selected from C 2-6 Alkyl chain, C 2-6 An alkoxy chain, a dipeptide fragment shown in Formula 3, or a combination of any one or more of the structures shown in Formula 4; R3 and R4 in Formula 3 are preferably hydrogen, methyl, isopropyl, or tert-butyl; n in Formula 4 represents any number between 0 and 6, and M represents -N- or -CH-; Z is selected from Any one of .

2. The derivative prepared from the novel ligand of E3 ligase FEM1B according to claim 1, characterized in that: The derivative is as shown in Formula 5, and the derivative includes stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs of the novel ligand; Wherein: the FEM1B ligand is a compound represented by formula 1; LINKER is a linking group selected from the following structures, or any combination of the following structures: C 1-20 Straight and branched alkyl chains, C 1-20 Alkoxy chain, C 7-11 Spiral ring, C 7-11 Azaspiro ring, C 7-11 Oxaspiro ring, any structure shown in Formula 6; in Formula 6, n represents any number between 0 and 8, and M represents -N- or -CH-; The hinge group is selected from any one of amide, ester, urea, amine, ether, thioether, and triazole; POI ligands are small molecules that have direct binding affinity to target proteins, and the target protein is any one of BRD2 / 3 / 4, FKBP12, K-RAS, SMARCA2, Bruton's tyrosine kinase protein, androgen receptor protein, MYC protein, N-MYC protein, β-catenin or Huntington protein.

3. The novel ligand of E3 ligase FEM1B according to claim 1, characterized in that: The structural formula of the novel ligand is specifically any one of the structures L1-L56:

4. The derivative according to claim 2, characterized in that: The specific structure of the derivative is shown in any structure of P1-P9:

5. Use of the novel ligand of E3 ligase FEM1B according to claim 1 in the preparation of a drug for treating tumor diseases.

6. Use of the derivative according to claim 2 for preparing a drug for treating tumor diseases.

7. The use according to claim 5 or 6, characterized in that: The tumor disease is one or more of breast cancer, pancreatic cancer, colon cancer, prostate cancer, lung cancer, liver cancer, acute myeloid leukemia, and multiple myeloma.

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