CN118440056A - Compounds based on CRBN ligands that induce IRAK4 degradation, and preparation methods and applications thereof - Google Patents

Abstract

The present invention discloses a compound based on CRBN ligand-induced IRAK4 degradation or a pharmaceutically acceptable salt, hydrate, stereoisomer, tautomer, prodrug thereof, the structure of which is shown in formula (I) or formula (II); wherein: X is n is an integer of 3-11, m is an integer of 2-10, h is an integer of 1-5; R is k is an integer of 1-5. The present invention also discloses a preparation method and a pharmaceutical composition of the compound, and the use of the compound in the preparation of a drug for treating or preventing a disease associated with IRAK4. The compound of the present invention has excellent IRAK4 protein degradation ability and high selectivity for IRAK4 target, thereby exerting a stronger anti-inflammatory effect than existing IRAK4 inhibitors, and can be used to prepare anti-inflammatory drugs.

Description

Compounds based on CRBN ligands that induce IRAK4 degradation, and preparation methods and applications thereof

Technical Field

The present invention relates to the field of drug compound synthesis, and in particular to a compound based on CRBN ligand-induced IRAK4 degradation, and a preparation method and application thereof.

Background technique

Interleukin 1 receptor (IL-1R)-associated kinase 4 (IRAK4) is a serine/threonine kinase located at the intersection of Toll-like receptor (TLR) and IL-1R signaling, and plays an important role in TLR/IL-1R-mediated myeloid differentiation factor 88 (MyD88)-dependent signaling pathways. Dysregulation of IRAK4 has been implicated in a variety of inflammatory diseases, such as sepsis, psoriasis, systemic lupus erythematosus, and rheumatoid arthritis. Therefore, targeting IRAK4 is a potential therapeutic strategy for the treatment of autoimmune and inflammatory diseases.

IRAK4 consists of an N-terminal death domain and an activation domain. Upon binding to TLR ligands, IRAKs and MyD88 form a signaling body called "myddosome". The formation of myddosome brings the kinase domains of IRAK4 into close proximity and activation, thereby mediating inflammatory signal transduction downstream of TLR. Given the important role of IRAK4 in the TLR signaling pathway, several IRAK4 small molecule inhibitors, including PF-06650833, BAY-1830839 and Edecesertib, have entered clinical trials.

However, both the kinase function and scaffold function of IRAK4 can mediate TLR signaling pathway transduction. Although the above-mentioned small molecule compounds can inhibit the kinase activity of IRAK4, they are likely to enhance the scaffold function of IRAK4 and obtain a more stable myddosome complex, which makes the effect of IRAK4 small molecule inhibitors very limited.

The recently developed proteolysis targeting chimera (PROTAC) technology can simultaneously regulate the catalytic and non-enzymatic functions of the target protein, thus providing a potential strategy to overcome the limitations of IRAK4 inhibitors. PROTAC is a heterobifunctional molecule that can induce ubiquitination of the target protein and promote its degradation through the ubiquitin-proteasome system. Compared with small molecule inhibitors, the potential advantage of PROTAC is that it can remove all functions of the target protein, thereby completely blocking IRAK4-mediated inflammatory factor signal transduction. However, only a few PROTACs are currently designed based on the non-enzymatic functions of the target protein, such as Aurora-A14 and focal adhesion kinase (FAK), as well as other non-kinase targets.

Currently, there are multiple IRAK4 degraders used to treat IRAK4-related diseases, such as the compound KT-474 used to treat inflammation-related diseases. KT-474 has entered Phase 2 clinical trials for the treatment of suppurative hidradenitis (NCT06028230) and atopic dermatitis (NCT06058156). The discovery of these drugs confirmed the role of degrading IRAK4 in blocking its non-enzymatic function.

However, despite the reported role of IRAK4 in the TLR signaling pathway, no IRAK4 degraders have been found for the treatment of other TLR-mediated inflammatory diseases such as acute lung injury (ALI) and psoriasis.

Summary of the invention

The present invention provides a compound based on CRBN ligand-induced IRAK4 degradation. The compound or its pharmaceutical composition has excellent IRAK4 protein degradation ability and high selectivity for IRAK4 target, thereby exerting a stronger anti-inflammatory effect than existing IRAK4 inhibitors, and can be used to prepare anti-inflammatory drugs.

The technical solution of the present invention is as follows:

A compound based on CRBN ligand-induced IRAK4 degradation, or a pharmaceutically acceptable salt, hydrate, stereoisomer, tautomer, or prodrug thereof, the structure of which is shown in formula (I) or formula (II);

in:

X is n is an integer from 3 to 11, m is an integer from 2 to 10, and h is an integer from 1 to 5;

R is k is an integer from 1 to 5.

The present invention uses a connecting chain to connect an IRAK4 small molecule inhibitor and a Cereblon protein ligand in an E3 ubiquitin ligase complex to prepare a protein degradation targeting complex (PROTACs) bifunctional small molecule, which can selectively induce IRAK4 protein degradation by ubiquitination labeling of IRAK4 and has good anti-inflammatory activity.

Preferably, n is an integer of 5 to 11; m is an integer of 2 to 8; h is an integer of 1 to 4; and k is an integer of 1 to 5. The preferred compound has good inducing IRAK4 degradation and anti-inflammatory activity.

Most preferably, n is 5, 7, 9 or 11; m is 2, 5 or 8; h is an integer of 1-4, and k is 3 or 4. The preferred compounds have better inducing IRAK4 degradation and anti-inflammatory activity.

Preferably, R is

Preferably, the compound is selected from:

The compounds of the present invention also include stereoisomers of the compounds of formula (I) and (II). All stereoisomers of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers and their mixtures (such as racemates), are included in the scope of the present invention.

The compounds of the present invention also include tautomers of the compounds represented by formula (I) and (II). "Tautomers" or "tautomeric forms" refer to structural isomers of different energies that can be mutually converted via a low energy barrier.

The compounds described in the present invention also include prodrugs of derivatives of the compounds shown in formula (I) and (II). The derivatives of the compounds shown in formula (I) and (II) may themselves have weak activity or even no activity, but after administration, they are converted into corresponding biologically active forms under physiological conditions (for example, by metabolism, solvent decomposition or other means).

The compounds described in the present invention also include pharmaceutically acceptable salts of the compounds represented by formula (I) and (II), including addition salts formed with the following acids: hydrochloric acid, hydroquinone, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, theanine disulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid or benzoic acid; and acid salts of hydrochloric acid, hydroquinone, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, trifluoroacetic acid, maleic acid, benzenesulfonic acid or sulphuric acid.

The present invention also discloses a method for preparing the compound, comprising: reacting a compound of formula (a) or formula (b) with a compound of formula (c) or formula (d) to obtain a compound of formula (I) or formula (II);

in:

Z is n is an integer from 3 to 11, m is an integer from 2 to 10, and h is an integer from 1 to 5;

Y is k is an integer from 1 to 5.

The present invention also provides a pharmaceutical composition, comprising a compound as shown in formula (I) or formula (II) or a pharmaceutically acceptable salt, hydrate, stereoisomer, tautomer, prodrug thereof, and a pharmaceutically acceptable excipient.

The pharmaceutical composition is added with pharmaceutically acceptable excipients to prepare a clinically acceptable dosage form, which is an injection, tablet or capsule.

The present invention also provides an IRAK4 degrader, the active ingredient of which is a compound represented by formula (I) or (II) or a pharmaceutically acceptable salt, hydrate, stereoisomer, tautomer, or prodrug thereof.

The present invention also provides the use of a compound represented by formula (I) or formula (II) or a pharmaceutically acceptable salt, hydrate, stereoisomer, tautomer, or prodrug thereof in the preparation of a drug for treating or preventing diseases associated with IRAK4.

Diseases associated with IRAK4 include cancer, neurodegenerative disorders, viral diseases, autoimmune diseases, inflammatory diseases, genetic disorders, hormone-related diseases, metabolic disorders, diseases associated with organ transplantation, immunodeficiency disorders, destructive bone diseases, proliferative disorders, infectious diseases, conditions associated with cell death, thrombin-induced platelet aggregation, liver diseases, pathological immune conditions involving T cell activation, cardiovascular disorders or CNS disorders.

Further, diseases associated with IRAK4 include sepsis, acute lung injury, epidermal hyperplasia, psoriasis, prostatic hyperplasia, lymphoma, breast cancer, follicular carcinoma, undifferentiated tumor, papillary tumor, melanoma, ABC-DLBCL, Hodgkin's lymphoma, primary cutaneous T-cell lymphoma, chronic lymphocytic leukemia, smoldering indolent multiple myeloma, leukemia, diffuse large B-cell lymphoma DLBCL, chronic lymphocytic leukemia CLL, chronic lymphocytic lymphoma, primary effusion lymphoma, Burkitt's lymphoma leukemia, acute lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, multiple myeloma, Alzheimer's disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, diabetes treatment, metabolic syndrome, obesity; autoimmune blood disorders such as hemolytic anemia, aplastic anemia, pure red cell anemia and idiopathic thrombocytopenia, systemic lupus erythematosus, rheumatoid arthritis, hidradenitis suppurativa, atopic dermatitis, COPD, lung disease, acid-induced lung damage, atopic dermatitis, asthma, allergies, bronchiolitis, bronchitis, chronic transplant rejection, colitis, conjunctivitis, cystitis, hepatitis, hidradenitis suppurativa, alcoholic fatty liver disease, non-alcoholic fatty liver disease, inflammatory bowel disease, oophoritis, acute and chronic gout, chronic gouty arthritis, rheumatoid arthritis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing the degradation effects of IRAK4 degraders LC-MI-1, LC-MI-3, LC-MI-6, LC-MI-8, LC-MI-9 and compound JH-I-25 on IRAK4 in cell test example 1.

FIG2 shows the therapeutic effects of LC-MI-3 and the control compound JH-I-25 on LPS-induced acute lung injury in mice in Animal Test Example 1;

Among them, (A, B) are the results of enzyme-linked immunosorbent assay (ELISA) of IL-6 and TNFα in mouse bronchoalveolar lavage fluid; (C, D) are the results of QPCR experiment of il6 and tnfa mRNA in mouse lung tissue; (E, F) are representative images of H&E staining and F4/80 immunohistochemistry of mouse lung sections and their quantitative statistical results; (G) Western blot results of mouse lung tissue and its quantitative analysis.

FIG3 shows the therapeutic effects of LC-MI-3 and the control compound JH-I-25 on LPS-induced sepsis in mice in animal level test example 2;

Among them, (A) is a schematic diagram of the experimental process; (B) is a representative picture of the mouse back skin on the sixth day of modeling; (C) is the daily scoring of the peeling and redness level of the mouse back skin; (D) is a representative picture of H&E staining of the mouse back skin; (E) is the Baker pathology score result for the representative picture in (D); (F) is the back skin thickness measured by the mouse on the sixth day; (G) is the statistical result of the weight of the mouse spleen obtained on the sixth day; (H) is the statistical result of the weight of the mice during the experiment.

Detailed ways

The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be pointed out that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on the present invention.

The structure of the compound was determined by nuclear magnetic resonance (1H-NMR) and high resolution mass spectrometry (HRMS), the NMR measurement was performed using an ACF-400BRUK nuclear magnetic resonance spectrometer, and the measurement solvent was deuterated dimethyl sulfoxide (DMSO-D6). Column chromatography used 200-300 mesh silica gel.

Embodiment 1:

Preparation: N-(4-(4-(3-((3-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)prop-2-yn-1-yl)oxy)propanoyl)piperazin-1-yl)-2-methoxyphenyl)-6-(1H-pyrazol-5-yl)picolinamide (LC-MI-3)

3-((3-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)prop-2-yn-1-yl)oxy)propionic acid (52 mg), N-(2-methoxy-4-(piperazine-1-yl)phenyl)-6-(1H-pyrazol-5-yl)picolinamide (50 mg), HATU (75 mg), DIPEA (85 mg) were added to 2 mL of DMF and stirred at room temperature for 45 min. 50 mL of water was added to quench the mixture, and the mixture was extracted with dichloromethane (20 mL × 3). The organic phases were combined, washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, and concentrated in vacuo. The product LC-MI-3 was obtained by purification by silica gel chromatography.

The mass spectrometry and nuclear magnetic resonance characterization of the product are as follows: ¹ H NMR (400 MHz, DMSO-d ₆ )δ13.23(s,1H),11.14(s,1H),10.58(s,1H),8.21(s,2H),8.14–8.00(m,2H),7.93(d,J＝9.6Hz,4H),6.99(s,1H),6.75(s,1H),6.56(dd,J＝8.9,2.5Hz ,1H),5.16(dd,J＝12.9,5.4Hz,1H),4.47(s,2H),3.98(s,3H),3.83(t,J＝6.4Hz,2H),3.68–3.57(m,5H),3.17(d,J＝24.0Hz,5H),2.72(t,J＝6.5Hz,2H),2 .63–2.51(m,2H); ¹³ C NMR (101MHz, DMSO) δ173.20,170.20,169.14,166.90,166.81,138.05,132.24,131.01,128.70,126.24,124.26,91.36,84.58,66.54,58.52,49.6 0,45.32,41.39,40.61,40.40,40.19,39.98,39.77,39.56,39.35,33.04,31.37,22.39.LC-MS m/z:745.50(M+H) ⁺ calcd for C ₃₉ H ₃₆ N ₈ O ₈ ,744.27

Embodiment 2:

Preparation of N-(4-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidine-4-carbonyl)piperazin-1-yl)-2-methoxyphenyl)-6-(1H-pyrazole-5-methyl)picolinamide (LC-MI-2)

1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidine-4-carboxylic acid (52 mg), N-(2-methoxy-4-(piperazin-1-yl)phenyl)-6-(1H-pyrazol-5-yl)picolinamide (50 mg), HATU (75 mg), DIPEA (85 mg) were added to 2 mL of DMF and stirred at room temperature for 45 min. 50 mL of water was added to quench the mixture, and the mixture was extracted with dichloromethane (20 mL × 3). The organic phases were combined, washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, and concentrated in vacuo. The product LC-MI-2 was obtained by purification by silica gel column chromatography.

The mass spectrum and nuclear magnetic resonance characterizations of the product are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ13.24 (s, 1H), 11.09 (s, 1H), 10.61 (s, 1H), 8.27 (d, J = 8.7 Hz, 1H), 8.20 (d, J = 7.8 Hz, 1H), 8.14–8.01 (m, 2H), 7.96–7.92 (m, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.34 (d, J = 2.2 Hz, 1H), 7.25 (dd, J = 8.8, 2.2 Hz, 1H), 7.02– 6.97(m,1H),6.79(s,1H),6.60(dd,J＝8.8,2.4Hz,1H),5.08(dd,J＝12.9,5.4Hz,1H),4.08(d,J＝12.9Hz,2H),4.00(s,2H),3.88(s,1H),3.73(s,2H),3. 64(s,3H),3.21(s,3H),3.18–3.02(m,5H),2.64–2.53(m,2H),1.81–1.57(m,4H). ¹³ C NMR (101MHz, DMSO) δ173.29,172.75,170.58,168.08,167.44,155.30,134.52,125.49,118.10,108.31,49.21,47.18,45.12,41.53,39.56,39.35 ,37.26,31.45,27.93,22.67,22.53.LC-MS m/z:746.30(M+H) ⁺ calcd for C ₃₉ H ₃₆ N ₈ O ₈ ,745.30

Embodiment 3:

Preparation of N-(4-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)azetidine-3-carbonyl)piperazin-1-yl)-2-methoxyphenyl)-6-(1H-pyrazole-5-methyl)picolinamide (LC-MI-1)

1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)azetidine-3-carboxylic acid (48 mg), N-(2-methoxy-4-(piperazine-1-yl)phenyl)-6-(1H-pyrazol-5-yl)picolinamide (50 mg), HATU (75 mg), DIPEA (85 mg) were added to 2 mL of DMF and stirred at room temperature for 45 min. 50 mL of water was added to quench the mixture, and the mixture was extracted with dichloromethane (20 mL × 3). The organic phases were combined, washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, and concentrated in vacuo. The product LC-MI-1 was obtained by purification by silica gel chromatography.

The mass spectrum and nuclear magnetic resonance characterizations of the product are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ13.23 (s, 1H), 11.08 (s, 1H), 10.58 (s, 1H), 8.17 (s, 2H), 8.10 (t, J = 7.6 Hz, 1H), 8.04 (dd, J = 7.3, 1.4 Hz, 1H), 7.90 (s, 1H), 7.66 (d, J = 8.2 Hz, 1H), 6.99 (d, J = 2.2 Hz, 1H), 6.85 (d, J = 2.1 Hz, 1H), 6.79 (d, J = 2.5 Hz, 1H ),6.71(dd,J＝8.4,2.1Hz,1H),6.60(dd,J＝8.8,2.5Hz,1H),5.06(dd,J＝12.9,5.4Hz,1H),4.27(t,J＝8.5Hz,2H),4.22–4.14(m,2H),4.04–3.93(m,5H),3.68 (t,J＝5.1Hz,2H),3.51(t,J＝5.1Hz,2H),3.24–3.15(m,5H),2.63–2.51(m,2H). ¹³ C NMR (101MHz, DMSO) δ173.29,170.58,169.79,167.91,167.64,155.45,134.26,125.30,117.69,105.05,100.97,54.11,49.19,40.58,40.37,40.1 6,39.95,39.74,39.53,39.32,31.69,31.43.LC-MS m/z:718.31(M+H) ⁺ calcd for C ₃₇ H ₃₅ N ₉ O ₇ ,717.27

Embodiment 4:

Preparation of N-(4-(4-(3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy(ethoxy)ethoxy(propoxy)piperazin-1-yl)-2-methoxyphenyl)-6-(1H-pyrazole-5-methyl)picolinamide (LC-MI-4)

3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)ethoxy)glycolic acid (58 mg), N-(2-methoxy-4-(piperazin-1-yl)phenyl)-6-(1H-pyrazol-5-yl)picolinamide (50 mg), HATU (75 mg), DIPEA (85 mg) were added to 2 mL of DMF and stirred at room temperature for 45 min. 50 mL of water was added to quench the mixture, and the mixture was extracted with dichloromethane (20 mL×3). The organic phases were combined, washed with saturated brine (50 mL×3), dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel chromatography to obtain the product LC-MI-4.

The mass spectrum and nuclear magnetic resonance characterizations of the product are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ13.24 (s, 1H), 11.13 (s, 1H), 10.56 (s, 1H), 8.17 (s, 2H), 8.10 (t, J = 7.6 Hz, 1H), 8.06–8.00 (m, 1H), 7.90 (s, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.44 (d, J = 2.3 Hz, 1H), 7.35 (dd, J = 8.3, 2.3 Hz, 1H), 6.99 (d, J = 2.3 Hz, 1H). z,1H),6.75(d,J＝2.6Hz,1H),6.56(dd,J＝8.9,2.5Hz,1H),5.12(dd,J＝12.9,5.4Hz,1H),4.32–4.25(m,2H),3.96(s,3H),3.81–3.74(m,2H),3.70–3.51(m ,12H),3.17(t,J＝5.0Hz,2H),3.12(t,J＝5.2Hz,2H),2.66–2.51(m,4H). ¹³ C NMR (101MHz, DMSO) δ173.26,170.43,169.34,167.33,167.26,164.37,134.36,125.72,123.50,121.33,109.33,100.83,70.41,70.16,69.10,68. 87,67.32,49.58,49.41,49.18,45.32,41.32,40.58,40.37,40.17,39.96,39.75,39.54,39.33,33.23,31.41,22.52.LC-MS m/z:795.43(M+H) ⁺ calcdfor C ₄₀ H ₄₂ N ₈ O ₁₀ ,794.30

Embodiment 5:

Preparation of N-(4-(4-(3-(2-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy(ethoxy)ethoxy(propoxy)piperazin-1-yl)-2-methoxyphenyl)-6-(1H-pyrazole-5-methyl)picolinamide (LC-MI-5)

3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy(ethoxy)ethoxy(methoxy)propionic acid (64 mg), N-(2-methoxy-4-(piperazine-1-yl)phenyl)-6-(1H-pyrazol-5-yl)picolinamide (50 mg), HATU (75 mg), DIPEA (85 mg) were added to 2 mL of DMF and stirred at room temperature for 45 min. 50 mL of water was added to quench the mixture, and the mixture was extracted with dichloromethane (20 mL×3). The organic phases were combined, washed with saturated brine (50 mL×3), dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel chromatography to obtain the product LC-MI-5.

The mass spectrum and nuclear magnetic resonance characterization of the product are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ13.24 (s, 1H), 11.13 (s, 1H), 10.59 (s, 1H), 8.25 (s, 1H), 8.19 (s, 1H), 8.10 (t, J = 7.6 Hz, 1H), 8.06–8.00 (m, 1H), 7.93 (s, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.43 (d, J = 2.3 Hz, 1H), 7.35 (dd, J = 8. 3,2.3Hz,1H),6.99(s,1H),6.76(s,1H),6.57(dd,J＝8.9,2.5Hz,1H),5.12(dd,J＝12.9,5.4Hz,1H),4.33–4.26(m,2H),3.99(s,3H),3.80–3.74(m,2H),3 .70–3.47(m,16H),3.21–3.09(m,4H),2.66–2.53(m,4H). ¹³ C NMR (101MHz, DMSO) δ173.24,170.40,169.36,167.32,167.25,164.37,134.34,125.70,123.49,121.30,109.32,100.83,70.42,70.28,70.23,70. 17,69.11,68.87,67.31,49.62,49.43,49.17,45.34,41.33,40.59,40.38,40.17,39.96,39.75,39.54,39.33,33.26,31.42,22.54.LC-MS m/z:839.55(M+H) ⁺ calcd for C ₄₂ H ₄₆ N ₈ O ₁₁ ,838.33

Embodiment 6:

Preparation of N-(4-(4-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexanoyl)piperazin-1-yl)-2-methoxyphenyl)-6-(1H-pyrazol-5-yl)picolinamide (LC-MI-6)

6-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexanoic acid (52 mg), N-(2-methoxy-4-(piperazine-1-yl)phenyl)-6-(1H-pyrazol-5-yl)picolinamide (50 mg), HATU (75 mg), DIPEA (85 mg) were added to 2 mL of DMF and stirred at room temperature for 45 min. 50 mL of water was added to quench the mixture, and the mixture was extracted with dichloromethane (20 mL × 3). The organic phases were combined, washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, and concentrated in vacuo. The product LC-MI-6 was obtained by purification by silica gel column chromatography.

The mass spectrum and nuclear magnetic resonance characterizations of the product are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ13.23 (s, 1H), 11.11 (s, 1H), 10.60 (s, 1H), 8.25 (s, 1H), 8.19 (s, 1H), 8.14–8.00 (m, 2H), 7.93 (s, 1H), 7.58 (dd, J=8.6, 7.1 Hz, 1H), 7.10 (d, J=8.6 Hz, 1H), 7.02 (d, J=7.0 Hz, 1H), 6.99 (s, 1H), 6.76 (s, 1H), 6.61– 6.50(m,2H),5.06(dd,J＝12.9,5.4Hz,1H),3.99(s,2H),3.61(q,J＝5.5Hz,4H),3.36–3.26(m,2H),3.14(d,J＝15.4Hz,4H),2.96–2.81(m,1H),2.65–2. 52(m,2H),2.38(t,J＝7.4Hz,2H),1.59(dp,J＝14.7,7.2Hz,4H),1.46–1.16(m,4H). ¹³ C NMR (101MHz, DMSO) δ173.29,171.07,170.58,169.42,167.78,146.90,136.75,132.66,117.65,110.85,109.47,49.01,45.23,42.24,41.34,40.6 0,40.40,40.19,39.98,39.77,39.56,39.35,32.61,31.45,29.03,26.56,25.00,22.63.LC-MS m/z:748.37(M+H) ⁺ calcd for C ₃₉ H ₄₁ N ₉ O ₇ ,747.31

Embodiment 7:

Preparation of N-(4-(4-(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octanoyl)piperazin-1-yl)-2-methoxyphenyl)-6-(1H-pyrazol-5-yl)picolinamide (LC-MI-7)

8-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octanoic acid (54 mg), N-(2-methoxy-4-(piperazine-1-yl)phenyl)-6-(1H-pyrazol-5-yl)picolinamide (50 mg), HATU (75 mg), DIPEA (85 mg) were added to 2 mL of DMF and stirred at room temperature for 45 min. 50 mL of water was added to quench the mixture, and the mixture was extracted with dichloromethane (20 mL × 3). The organic phases were combined, washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, and concentrated in vacuo. The product LC-MI-7 was obtained by purification by silica gel column chromatography.

The mass spectrum and nuclear magnetic resonance characterizations of the product are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ13.24 (s, 1H), 11.11 (s, 1H), 10.60 (s, 1H), 8.26 (s, 1H), 8.18 (s, 1H), 8.10 (t, J = 7.6 Hz, 1H), 8.04 (d, J = 8.5 Hz, 1H), 7.93 (s, 1H), 7.58 (dd, J = 8.6, 7.0 Hz, 1H), 7.09 (d, J = 8.6 Hz, 1H), 7.01 (dd, J = 9.8, 3.6 Hz, 2H), 6.76 (s, 1H), 6. .58(dd,J＝8.8,2.5Hz,1H),6.52(t,J＝6.0Hz,1H),5.06(dd,J＝12.9,5.4Hz,1H),3.98(s,3H),3.60(q,J＝5.8,5.1Hz,4H),3.29(q,J＝6.7Hz,2H),3.15(d,J ＝18.0Hz,3H),2.65–2.52(m,2H),2.35(t,J＝7.4Hz,2H),1.62–1.48(m,5H),1.39–1.17(m,8H). ¹³ C NMR(101MHz,DMSO)δ173.29,171.15,170.57,169.44,167.78,146.90,136.74,132.65,117.64,110.84,109.47,49.01,45.24,42.29,41.32,40.60,40.39,40.30,40.18,39.97,39.76,39.56,39.35,32.68,31.45,29.21,29.12,29.07,26.72,25.22,22.63.LC-MSm/z:776.70(M+H) ⁺ calcd for C ₄₁ H ₄₅ N ₉ O ₇ ,775.34

Embodiment 8:

Preparation of N-(4-(4-(12-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)dodecanoyl)piperazin-1-yl)-2-methoxyphenyl)-6-(1H-pyrazol-5-yl)picolinamide (LC-MI-8)

12-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)dodecanoic acid (63 mg), N-(2-methoxy-4-(piperazine-1-yl)phenyl)-6-(1H-pyrazol-5-yl)picolinamide (50 mg), HATU (75 mg), DIPEA (85 mg) were added to 2 mL of DMF and stirred at room temperature for 45 min. 50 mL of water was added to quench the mixture, and the mixture was extracted with dichloromethane (20 mL × 3). The organic phases were combined, washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, and concentrated in vacuo. The product LC-MI-8 was obtained by purification by silica gel column chromatography.

The mass spectrum and nuclear magnetic resonance characterizations of the product are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ13.23 (s, 1H), 11.10 (s, 1H), 10.60 (s, 1H), 8.26 (d, J = 8.2 Hz, 1H), 8.19 (s, 1H), 8.09 (t, J = 7.5 Hz, 1H), 8.03 (d, J = 6.9 Hz, 1H), 7.93 (s, 1H), 7.57 (dd, J = 8.6, 7.1 Hz, 1H), 7.07 (d, J = 8.6 Hz, 1H), 7.04-6.96 (m, 2H), 6.77 (s, 1H), 6.58 (dd, J = 8.9,2.6Hz,1H),6.50(t,J＝5.9Hz,1H),5.05(dd,J＝12.9,5.4Hz,1H),3.98(d,J＝6.3Hz,2H),3.60(q,J＝5.4,5.0Hz,4H),3.27(q,J＝6.7Hz,2H),3.15(d,J＝ 17.8Hz, 5H), 2.64–2.52 (m, 1H), 2.34 (t, J = 7.4Hz, 2H), 1.53 (dt, J = 21.4, 7.4Hz, 5H), 1.39–1.13 (m, 16H). ¹³ C NMR (101MHz, DMSO) δ173.28,171.16,170.56,169.42,167.77,146.89,136.72,132.64,110.82,109.46,49.01,45.26,42.30,41.32,40.61,40.40 ,40.19,39.98,39.77,39.56,39.36,32.70,31.45,29.46,29.42,29.35,29.24,29.13,26.78,25.29,22.63.LC-MS m/z:832.75(M+H) ⁺ calcd for C ₄₅ H ₅₃ N ₉ O ₇ ,831.41

Embodiment 9:

Preparation of N-(4-(4-(2-)((2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)amino)-2-oxoethyl)piperazin-1-yl)-2-methoxyphenyl)-6-(1H-pyrazol-5-yl)picolinamide (LC-MI-9)

2-(4-(4-(6-(1H-pyrazol-5-yl)pyridinylamino)-3-methoxyphenyl)piperazin-1-yl)acetic acid (60 mg), 4-((2-aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione (44 mg), HATU (79 mg), DIPEA (89 mg) were added to 2 mL of DMF and stirred at room temperature for 45 min. 50 mL of water was added to quench the mixture, and the mixture was extracted with dichloromethane (20 mL×3). The organic phases were combined, washed with saturated brine (50 mL×3), dried over anhydrous sodium sulfate, and concentrated in vacuo. The product LC-MI-9 was obtained by purification by silica gel column chromatography.

The mass spectrum and nuclear magnetic resonance characterization of the product are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ13.23 (s, 1H), 11.10 (s, 1H), 10.58 (s, 1H), 8.19 (s, 1H), 8.14–8.00 (m, 4H), 7.93 (s, 1H), 7.60 (dd, J=8.6, 7.1 Hz, 1H), 7.23 (d, J=8.6 Hz, 1H), 7.05 (d, J=7.0 Hz, 1H), 6.99(s,1H),6.76–6.69(m,2H),6.53(dd,J＝8.9,2.5Hz,1H),5.06(dd,J＝12.9,5.4Hz,1H),3.97(s,3H),3.51–3.40(m,2H),3.18(s,4H),2.99(s,2H), 2.65–2.51(m,6H),1.38–1.17(m,4H). ¹³ C NMR (101MHz, DMSO) δ173.26,170.55,169.19,167.78,146.87,136.71,132.69,117.75,111.05,109.69,53.27,49.01,48.85,41.86,40.60,40.39 ,40.19,39.98,39.77,39.56,39.35,38.19,31.44,29.47,22.65.LC-MS m/z:735.44(M+H) ⁺ calcd for C ₃₇ H ₃₈ N ₁₀ O ₇ ,734.29

Embodiment 10:

Preparation of N-(4-(4-(2-((5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)pentyl)amino)-2-oxoethyl)piperazin-1-yl)-2-methoxyphenyl)-6-(1H-pyrazol-5-yl)picolinamide (LC-MI-10)

2-(4-(4-(6-(1H-pyrazol-5-yl)pyridinylamino)-3-methoxyphenyl)piperazin-1-yl)acetic acid (60 mg), 4-((5-aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione (50 mg), HATU (79 mg), DIPEA (89 mg) were added to 2 mL of DMF and stirred at room temperature for 45 min. 50 mL of water was added to quench the mixture, and the mixture was extracted with dichloromethane (20 mL×3). The organic phases were combined, washed with saturated brine (50 mL×3), dried over anhydrous sodium sulfate, and concentrated in vacuo. The product LC-MI-10 was obtained by purification by silica gel column chromatography.

The mass spectrum and nuclear magnetic resonance characterization of the product are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ13.23 (s, 1H), 11.10 (s, 1H), 10.59 (s, 1H), 8.20 (d, J = 15.0 Hz, 2H), 8.10 (t, J = 7.6 Hz, 1H), 8.09–8.00 (m, 1H), 7.93 (s, 1H), 7.78 (t, J = 5.7 Hz, 1H), 7.63–7.54 (m, 1H), 7.10 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.99 (m, 1H). s,1H),6.73(s,1H),6.54(dt,J＝11.8,4.4Hz,2H),5.05(dd,J＝12.9,5.4Hz,1H),3.98(s,3H),3.30(dd,J＝13.4,6.9Hz,3H),3.20(d,J＝5.7Hz,5H),3.13(q ,J＝6.6Hz,2H),2.98(s,2H),2.95–2.81(m,2H),2.63–2.51(m,4H),1.66–1.28(m,6H). ¹³ C NMR (101MHz, DMSO) δ173.28,170.56,169.43,169.34,167.77,146.89,136.76,132.65,117.67,110.87,109.49,53.29,49.01,48.89,42.26,40.6 0,40.39,40.19,39.98,39.77,39.56,39.35,38.53,31.44,29.40,28.82,24.15,22.63.LC-MS m/z:776.34(M+H) ⁺ calcd forC ₄₀ H ₄₄ N ₁₀ O ₇ ,777.43

Embodiment 11:

Preparation of N-(4-(4-(2-)((8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octyl)amino)-2-oxoethyl)piperazin-1-yl)-2-methoxyphenyl)-6-(1H-pyrazol-5-yl)picolinamide (LC-MI-11)

2-(4-(4-(6-(1H-pyrazol-5-yl)pyridinylamino)-3-methoxyphenyl)piperazin-1-yl)acetic acid (60 mg), 4-((8-aminooctyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione (55 mg), HATU (79 mg), DIPEA (89 mg) were added to 2 mL of DMF and stirred at room temperature for 45 min. 50 mL of water was added to quench the mixture, and the mixture was extracted with dichloromethane (20 mL × 3). The organic phases were combined, washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, and concentrated in vacuo. The product LC-MI-11 was obtained by purification by silica gel column chromatography.

The mass spectrum and nuclear magnetic resonance characterization of the product are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ13.24 (s, 1H), 11.10 (s, 1H), 10.59 (s, 1H), 8.22 (dd, J = 16.6, 8.2 Hz, 2H), 8.10 (t, J = 7.6 Hz, 1H), 8.04 (d, J = 7.6 Hz, 1H), 7.94 (s, 1H), 7.75 (t, J = 6.0 Hz, 1H), 7.61–7.53 (m, 1H), 7.07 (d ,J＝8.6Hz,1H),7.04–6.96(m,2H),6.73(s,1H),6.59–6.47(m,2H),5.06(dd,J＝12.9,5.4Hz,1H),3.98(s,3H),3.31–3.18(m,6H),3.10(q,J＝6.7Hz,2H) ,2.98(s,2H),2.64–2.55(m,6H),1.61–1.18(m,14H). ¹³ C NMR(101MHz,DMSO)δ173.29,170.57,169.43,169.29,167.77,146.88,136.73,132.63,117.59,110.84,109.46,61.74,53.28,49.01,48.91,42.30,40.59,40.38,40.17,39.96,39.75,39.55,39.34,38.65,31.44,29.65,29.20,29.14,26.81,26.75,22.63.LC-MSm/z:819.62(M+H) ⁺ calcd for C ₄₃ H ₅₀ N ₁₀ O ₇ ,818.39

Embodiment 12:

Preparation of N-(4-(4-(2-((2-(2-)((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy(ethyl)amino)-2-oxoethyl)piperazin-1-yl)-2-methoxyphenyl)-6-(1H-pyrazol-5-yl)picolinamide (LC-MI-12)

2-(4-(4-(6-(1H-pyrazol-5-yl)pyridinylamino)-3-methoxyphenyl)piperazin-1-yl)acetic acid (60 mg), 4-(2-(2-aminoethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione (50 mg), HATU (79 mg), DIPEA (89 mg) were added to 2 mL of DMF and stirred at room temperature for 45 min. 50 mL of water was added to quench the mixture, and the mixture was extracted with dichloromethane (20 mL×3). The organic phases were combined, washed with saturated brine (50 mL×3), dried over anhydrous sodium sulfate, and concentrated in vacuo. The product LC-MI-12 was obtained by purification by silica gel chromatography.

The mass spectrum and nuclear magnetic resonance characterization of the product are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ13.23 (s, 1H), 11.11 (s, 1H), 10.58 (s, 1H), 8.22 (d, J = 11.2 Hz, 2H), 8.10 (t, J = 7.6 Hz, 1H), 8.04 (d, J = 8.5 Hz, 1H), 7.93 (s, 1H), 7.79 (s, 1H), 7.58 (t, 1H), 7.14 (d, J = 8.6 Hz, 1H), 7.04 (d, J = 7.0 Hz, 1H), 6.99 (s, 1H), 6.70(s,1H),6.61(t,J＝5.7Hz,1H),6.51(dd,J＝8.9,2.5Hz,1H),5.06(dd,J＝12.9,5.4Hz,1H),4.00–3.93(m,3H),3.64(t,J＝5.4Hz,2H),3.50(dt,J＝13. 9,5.7Hz,4H),3.32(q,J＝6.0Hz,3H),3.18(s,5H),3.00(s,2H),2.64–2.52(m,6H). ¹³ C NMR (101MHz, DMSO) δ173.27,170.53,169.45,167.74,146.86,136.73,132.53,117.89,111.21,109.73,69.33,69.05,53.21,49.03,48.89,42.23 ,40.59,40.39,40.18,39.97,39.76,39.55,39.34,38.52,31.44,22.62.LC-MS m/z:779.56(M+H) ⁺ calcd for C ₃₉ H ₄₂ N ₁₀ O _8, 778.32

Embodiment 13:

Preparation of N-(4-(4-(2-((2-(2-(2-(2-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy(ethyl)amino)-2-oxoethyl)piperazin-1-yl)-2-methoxyphenyl)-6-(1H-pyrazol-5-yl)picolinamide (LC-MI-13)

2-(4-(4-(6-(1H-pyrazol-5-yl)pyridinylamino)-3-methoxyphenyl)piperazin-1-yl)acetic acid (60 mg), 4-((2-(2-(2-aminoethoxy)ethoxy)(ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione (55 mg), HATU (79 mg), and DIPEA (89 mg) were added to 2 mL of DMF and stirred at room temperature for 45 min. 50 mL of water was added to quench the mixture, and the mixture was extracted with dichloromethane (20 mL×3). The organic phases were combined, washed with saturated brine (50 mL×3), dried over anhydrous sodium sulfate, and concentrated in vacuo. The product LC-MI-13 was purified by silica gel chromatography.

The mass spectrum and nuclear magnetic resonance characterizations of the product are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ13.23 (s, 1H), 11.10 (s, 1H), 10.58 (s, 1H), 8.20 (d, J = 9.3 Hz, 2H), 8.10 (t, J = 7.5 Hz, 1H), 8.04 (d, J = 6.9 Hz, 1H), 7.94 (s, 1H), 7.75 (t, J = 5.9 Hz, 1H), 7.56 (dd, J = 8.6, 7.1 Hz, 1H), 7.10 (d, J = 8.6 Hz, 1H), 7.03 (d, J = 7.0 Hz, 1H), 6. .99(s,1H),6.71(s,1H),6.60(t,J＝5.8Hz,1H),6.53(dd,J＝8.9,2.5Hz,1H),5.06(dd,J＝12.9,5.4Hz,1H),3.92(d,J＝43.5Hz,3H),3.66–3.51(m,6H),3. 45(p,J＝5.7Hz,5H),3.37–3.24(m,3H),3.19(t,J＝4.8Hz,4H),2.99(s,2H),2.64–2.52(m,6H). ¹³ C NMR (101MHz, DMSO) δ173.27,170.55,169.57,169.42,167.74,146.81,136.66,132.53,117.83,111.15,109.71,70.28,70.01,69.53,69.34,53.2 3,49.03,48.96,42.14,40.60,40.39,40.18,39.97,39.76,39.55,39.35,38.58,31.45,22.61.LC-MS m/z:823.62(M+H) ⁺ calcd for C ₄₁ H ₄₆ N ₁₀ O ₉ ,822.34

Embodiment 14:

Preparation of (4-(4-(17-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxo-6,9,12,15-tetraoxo-3-azaheptadecanyl)piperazin-1-yl)-2-methoxyphenyl)-6-(1H-pyrazol-5-yl)picolinamide (LC-MI-14)

2-(4-(4-(6-(1H-pyrazol-5-yl)pyridinylamino)-3-methoxyphenyl)piperazin-1-yl)acetic acid (60 mg), 4-((14-amino-3,6,9,12-tetraoxy-tradecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione (67 mg), HATU (79 mg), DIPEA (89 mg) were added to 2 mL of DMF and stirred at room temperature for 45 min. 50 mL of water was added to quench the mixture, and the mixture was extracted with dichloromethane (20 mL×3). The organic phases were combined, washed with saturated brine (50 mL×3), dried over anhydrous sodium sulfate, and concentrated in vacuo. The product LC-MI-14 was obtained by purification by silica gel chromatography.

The mass spectrum and nuclear magnetic resonance characterization of the product are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ13.23 (s, 1H), 11.10 (s, 1H), 10.59 (s, 1H), 8.26–8.16 (m, 1H), 8.13–8.00 (m, 2H), 7.93 (s, 1H), 7.76 (t, J = 5.8 Hz, 1H), 7.58 (ddd, J = 13.9, 8.6, 7.1 Hz, 1H), 7.18–6.96 (m, 3H ),6.73(s,1H),6.58(tt,J＝9.0,4.6Hz,3H),5.06(dt,J＝13.0,4.9Hz,1H),4.03–3.92(m,2H),3.67–3.38(m,17H),3.33–3.16(m,6H),3.00(s,2H),2. 95–2.82(m,5H),2.59(dt,J＝17.9,6.9Hz,5H). ¹³ C NMR (101MHz, DMSO) δ173.27,170.53,169.57,169.39,167.75,146.84,136.65,132.53,117.85,111.12,109.69,70.34,70.30,70.26,70.22,70.16 ,70.00,69.44,69.32,69.23,53.24,49.02,42.14,40.59,40.38,40.17,39.97,39.76,39.55,39.34,38.56,31.45,22.62.LC-MSm/z:911.33(M+H) ⁺ calcd for C ₄₅ H ₅₄ N ₁₀ O ₁₁ ,910.40

Cell test example 1: Degradation of IRAK4 by LC-MI-3

1.1 Cell culture and treatment

BaF3 cells stably transfected with IRAK4 (BaF3-TEL-IRAK4) were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody, and plated into 6-well plates at a concentration of 10 ⁶ cells/well and left to stand overnight. Then, the control compound JH-I-25 (a kinase inhibitor of IRAK4, reference DOI: 10.1074/jbc.RA118.005428) or the specified IRAK4 degrader was treated at 100 nM for 12 hours. The cells were collected and lysed, and the degradation ability of IRAK4 was determined by Western blot after protein quantification.

1.2 Experimental Results

As shown in Figure 1, treatment of Baf3 cells stably expressing IRAK4 with JH-I-25 or five candidate IRAK4 degraders resulted in different degrees of IRAK4 degradation. Among the IRAK4 degraders, LC-MI-3 had the best degradation effect.

Biological test example 1: Reversal effect of MI-3 on acute lung injury (ALI) in mice

1.1 Establishment of acute lung injury model:

C57BL/6 mice were randomly divided into 4 groups (n=8): control group (Con), LPS-induced ALI group (LPS), LPS+20mg/kg LC-MI-3 group and LPS+20mg/kg JH-I-25 group. Mice were intragastrically gavaged with LC-MI-3 and JH-I-25 solutions at 200μL/20g body weight, and the control group and LPS group were given the same volume of vehicle (5% DMSO, 30% PEG-400 and 65% saline). After the mice were treated with vehicle, LC-MI-3 or JH-I-25 for 1h, 5mg/kg LPS was injected into the trachea to establish the ALI model. Mice were killed 6 hours later to collect bronchoalveolar lavage fluid (BALF) and lung tissue samples.

1.2 Determination of inflammatory factors in bronchoalveolar lavage fluid

The collected BALF was centrifuged at 1000 rpm for 10 min at 4°C, and the supernatant was used for the determination of inflammatory factor (TNF-α and IL-6) concentrations. The expression levels of the two inflammatory factors were determined by TNF-α (Cat. No. 88-7324; ThermoFisher) and IL-6 (Cat. No. 88–7064; Thermo Fisher Scientific) ELISA kits according to the manufacturer's instructions.

1.3 qPCR evaluation of transcriptional levels of inflammatory factors in lung tissue

Total RNA from lung tissue was extracted using AG RNAex Pro RNA Extraction Reagent (Cat. No. AG21101; Accurate Biotechnology) and SteadyPure Universal RNA Extraction Kit (Cat. No. AG21024; Accurate Biotechnology). RNA concentration was then determined and quantitatively reverse transcribed into cDNA using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific) and Evo M-MLV Mix Kit with gDNACleanfor qPCR (Cat. No. AG11728; Accurate Biotechnology) according to the manufacturer's instructions. After reverse transcription, qPCR amplification was performed using the SYBR Green Premix Pro Taq HS qPCR Kit (Cat. No. AG11733; Accurate Biotechnology) on the Bio-Rad CFX Connect Real-Time system, and the test results were quantitatively analyzed using the 2 ^-ΔΔCt method for the relative expression of inflammatory factors.

All primer sequences used for qPCR are as follows:

Mouse TNFα:

F: 5’-TGATCCGCGACGTGGAA-3’ (SEQ ID NO.1)

R: 5’-ACCGCCTGGAGTTCTGGAA-3’ (SEQ ID NO.2)

Mouse IL-6:

F: 5’-GAGGATACCACTCCCAACAGACC-3’ (SEQ ID NO.3)

R: 5’-AAGTGCATCATCGTTGTTCATACA-3’ (SEQ ID NO.4)

Mouse GAPDH:

F: 5’-GGAGCGAGATCCCTCCAAAAT-3’ (SEQ ID NO.5)

R: 5’-GGCTGTTGTCATACTTCTCATGG-3’ (SEQ ID NO.6)

1.4 Immunohistochemical analysis

Mouse lung tissue was fixed in 4% paraformaldehyde for 48 h, dehydrated with alcohol and embedded in paraffin, the tissue was cut into 5 μm sections and then dewaxed and rehydrated, and then stained with hematoxylin and eosin (HE). Another section was soaked in citric acid buffer (pH 6.0) to repair antigens. Then endogenous peroxidase was blocked with 3% hydrogen peroxide and incubated with 5% BSA for 1 h, incubated with F4/80 primary antibody on a shaker at 4 ° C for 12 h, and then incubated with secondary antibody for 15 min and developed with DAB colorimetric agent. The obtained immunohistochemical sections were photographed using an EVOS M7000 microscope (Thermo Fisher) and quantitatively analyzed using ImageJ software.

1.5 Western blot analysis

The obtained lung tissue samples were separated into proteins using tissue lysis buffer, and the protein concentration of the samples was determined and normalized using the BCA method. The obtained samples were separated by SDS-PAGE and transferred to a membrane. The protein band images were taken on the Vilber Fusion FX system using NcmECL Ultra chemiluminescent reagent, and the grayscale values of the bands were determined using ImageJ software.

1.6 Statistical analysis

All data are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism8 software (GraphPad software). Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Dunnett's test for multiple comparisons. When P < 0.05, the obtained data were considered statistically significant.

1.7 Experimental Results

FIG2 shows the experimental data measured from samples obtained from the above four groups of mice.

(1) The results in Figure 2 (A) and (B) showed that LPS could induce the protein expression of IL-6 and TNFα in BALF, while LC-MI-3 could inhibit the expression of these two inflammatory factors.

(2) The results in Figure 2 (C) and (D) showed that LPS could induce the transcriptional expression of IL-6 and TNFα in lung tissue, while LC-MI-3 could inhibit the expression of these two inflammatory factors.

(3) Figure 2 (E) shows the HE staining and pathological scores of lung tissues of the four groups of mice. The results showed that compared with the control group, the edema and lung injury in the LPS group were the most severe, while both MI-3 and JH-I-25 treatments could reverse LPS-induced edema and lung injury, among which LC-MI-3 treatment had the best effect.

(4) Figure 2 (F) shows the F4/80 immunohistochemistry and pathological scores of lung tissues of the four groups of mice. The results showed that compared with the control group, the inflammatory cell infiltration in the LPS group was the most serious, while both LC-MI-3 and JH-I-25 treatments could reverse the LPS-induced inflammatory cell infiltration, among which LC-MI-3 treatment had the best effect.

(5) Figure 2 (G) shows the western blot bands and grayscale value statistical analysis of lung tissues of four groups of mice. The results showed that compared with the control group, both NFκB and MAPK signaling pathways were activated in the LPS group, but JH-I-25 treatment could only block LPS-induced MAPK signaling pathway activation, while LC-MI-3 could simultaneously block LPS-induced NFκB and MAPK signaling pathway activation.

Biological test example 2: The therapeutic effect of LC-MI-3 on psoriasis in mice

2.1 Establishment of psoriasis model

As shown in Figure 3 (A), C57BL/6 mice were randomly divided into 4 groups (n=8): imiquimod (IMQ) group, IMQ+JH-I-25 group, IMQ+LC-MI-3 group and IMQ+dexamethasone (Dex) group. IMQ cream was applied to the shaved back of mice at 3.125 mg/mouse/day to establish a psoriasis model. For the IMQ group, mice were gavaged with vehicle (5% DMSO, 30% PEG-400, 65% saline) every day. For the IMQ+JH-I-25 group and IMQ+LC-MI-3 group, mice were gavaged with 20 mg/kg JH-I-25 or LC-MI-3 every day. For the Dex group, mice were gavaged with 3 mg/kg Dex every day. The severity of psoriasis was monitored every day by recording skin thickness, erythema and desquamation. Mice were killed and photographed on the 6th day, and the back skin and spleen of mice were collected. The back skin was stained with HE according to the immunohistochemical analysis method in 1.4 of Biological Test Example 1.

2.2 Experimental Results

FIG3 shows the experimental data measured from samples obtained from the above four groups of mice.

(1) Figure 3 (B) shows representative images of the back skin of mice in each group on the sixth day of model establishment. The results showed that IMQ induced keratinocyte proliferation, epidermal hyperplasia and redness and swelling in the back skin of mice, while JH-I-25, LC-MI-3 and Dex could reverse these symptoms, among which LC-MI-3 and Dex had better efficacy, and the efficacy of the two was comparable.

(2) Figure 3 (C) shows the skin thickness curves of the back skin of each group of mice over 6 days. The results showed that IMQ induced thickening of the mouse skin, while JH-I-25, LC-MI-3 and Dex could reverse the skin thickening caused by IMQ, among which LC-MI-3 and Dex had better efficacy, and the efficacy of the two was comparable.

(3) Figure 3 (D)-(E) shows the HE staining sections and pathological scores of the back skin of each group of mice within 6 days. The results show that IMQ induces keratinocyte proliferation and epidermal hyperplasia in the back skin of mice, and JH-I-25, LC-MI-3 and Dex can reverse these symptoms caused by IMQ, among which LC-MI-3 and Dex have better efficacy, and the efficacy of the two is comparable. In addition, Figure 3 (F) shows the back epidermal thickness measured in HE sections, and the results also confirm that MI-3 can better inhibit IMQ-induced epidermal hyperplasia.

(4) Figure 3 (G) shows the spleen weights of mice in each group on day 6. The results showed that IMQ induced an abnormal increase in the spleen weight of mice, and JH-I-25, LC-MI-3, and Dex could reverse this effect of IMQ, among which LC-MI-3 and Dex had better efficacy, and the efficacy of the two was comparable.

(5) Figure 3 (H) shows the weight changes of mice in each group during the experiment. The results showed that IMQ and the three drugs had no significant effect on the weight of mice, indicating that the above drugs had no toxic effects on mice.

The embodiments described above provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements and equivalent substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims (9)

1. A compound for inducing IRAK4 degradation based on CRBN ligand, or pharmaceutically acceptable salts, hydrates, stereoisomers, tautomers, prodrugs thereof, wherein the structure of the compound is shown as formula (i) or formula (ii);

Wherein:

X is N is an integer of 3-11, m is an integer of 2-10, and h is an integer of 1-5;

R is K is an integer of 1 to 5.

2. A compound based on CRBN ligand-induced IRAK4 degradation according to claim 1, wherein n is an integer from 5 to 11; m is an integer from 2 to 8; h is an integer of 1 to 4; k is an integer of 1 to 5.

3. A compound based on CRBN ligand-induced IRAK4 degradation according to claim 2, wherein n is 5, 7, 9 or 11; m is 2, 5 or 8; h is an integer from 1 to 4, and k is 3 or 4.

4. A compound according to claim 1, wherein R is

5. A compound for inducing IRAK4 degradation based on CRBN ligand according to claim 1, wherein said compound is selected from the group consisting of:

6. a pharmaceutical composition comprising a compound of formula (i) or formula (ii) as defined in claim 1 or a pharmaceutically acceptable salt, hydrate, stereoisomer, tautomer, prodrug thereof, and a pharmaceutically acceptable excipient.

7. An IRAK4 degrading agent, characterized in that the active ingredient is a compound shown in a formula (i) or a formula (ii) as set forth in claim 1 or pharmaceutically acceptable salts, hydrates, stereoisomers, tautomers and prodrugs thereof.

8. Use of a compound of formula (i) or (ii) as defined in claim 1 or a pharmaceutically acceptable salt, hydrate, stereoisomer, tautomer, prodrug thereof for the preparation of a medicament for the treatment or prophylaxis of a disease associated with IRAK 4.

9. The use according to claim 8, wherein the diseases associated with IRAK4 comprise cancer, neurodegenerative disorders, viral diseases, autoimmune diseases, inflammatory diseases, genetic disorders, hormone-related diseases, metabolic disorders, diseases associated with organ transplantation, immunodeficiency disorders, destructive bone diseases, proliferative disorders, infectious diseases, conditions associated with cell death, thrombin-induced platelet aggregation, liver diseases, pathological immune conditions involving T cell activation, cardiovascular disorders or CNS disorders.