Compound and application thereof in preparation of medicine for treating systemic lupus erythematosus

Abstract

The invention discloses a compound and application thereof in preparation of a medicine for treating systemic lupus erythematosus. A series of novel small molecule compounds are designed based on DHA by utilizing a PROTAC technology, the novel small molecule compounds have ligands combined with E3 ubiquitin ligase protein and ligands for targeted degradation of protein, the compound shown in the formula (II-1) degrades CARD6 protein through an ubiquitin enzyme system, phenotypic polarization of THP-1 macrophages induced by LPS + IFN-gamma to M1 can be inhibited, CARD6 can be degraded, and the compound can be used for preparing a medicine for preventing and controlling the expression of the compound in the formula (II-1). The inflammation is improved; the compound can effectively improve the renal function of an MRL / lpr model mouse and relieve the renal inflammatory response of the MRL / lpr mouse, can be further developed into a medicine for treating SLE, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of systemic lupus erythematosus, and more particularly to a compound and application thereof in preparing a drug for treating systemic lupus erythematosus. Background Art

[0002] Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease that can affect multiple organs throughout the body and be life-threatening. Dihydroartemisinin (DHA), a first-generation derivative of artemisinin extracted from the Asteraceae plant Artemisia annua, is a widely used clinical drug for the treatment of malaria. In recent years, DHA has also been found to have promising immunomodulatory effects in the treatment of SLE and has entered Phase II clinical trials. However, the target of DHA remains unclear.

[0003] Traditional small molecule inhibitors work by binding to the active site of a target protein, thereby inhibiting its function. For over 100 years, small molecule research and development has successfully combated numerous diseases. However, the technology behind small molecule drug development faces numerous limitations and challenges. For example, small molecule drugs can develop drug resistance, making them incapable of long-term inhibition of target protein activity. Furthermore, small molecule drugs require a sustained concentration in the body to be effective. Furthermore, many targets are considered inaccessible to small molecules, including transcription factors, scaffold proteins, and the undruggable mutant target KRAS.

[0004] Protein degradation targeting chimera (PROTAC) is a new technology that selectively degrades target proteins through the ubiquitin-proteasome system. Traditional small molecule drugs block the function of proteins, while the core of this technology is to try to design small molecule compounds into a new type of drug. By delivering proteins to the proteasome and degrading them, PROTAC can overcome drug resistance and traditional "undruggable" drugs.

[0005] PROTACs have enormous potential for ubiquitination and degradation of undruggable targets. According to incomplete statistics, there are now over 30 targets that can be ubiquitinated and degraded by PROTAC molecules, which are used to treat a variety of diseases, including cancer and neurodegenerative diseases, showing broad therapeutic prospects. However, PROTAC technology has not yet been found to be used in the treatment of SLE.

[0006] PROTAC binds to the target protein and E3 ligase to form a ternary complex. The proteasome recognizes and degrades the polyubiquitinated target protein, and PROTAC is released to participate in another cyclic degradation process. This complex action process makes PROTAC drug development much more difficult than traditional small molecule drugs:

[0007] PROTACs have a larger molecular weight than general small molecule drugs, resulting in poor membrane permeability and bioavailability. Literature reports show that simply changing the length or structure of the linker can have a huge impact on the degradation ability of PROTAC. The design of the linker needs to consider its length, rigidity and chemical stability to ensure effective ternary complex formation. However, since the crystal structure of the POI-Protac-E3 enzyme complex has not yet been resolved, there is no guidance for linker modification. In addition, it was found that the substrates that PROTAC specifically binds to the E3 enzyme receptor protein are accidental, which also affects the effect of the designed PROTAC. Summary of the Invention

[0008] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide a compound and its use in the preparation of a drug for treating systemic lupus erythematosus.

[0009] The first object of the present invention is to provide a use of a degrader of apoptosis enhancing domain protein 6 in the preparation of a drug for systemic lupus erythematosus.

[0010] The second object of the present invention is to provide a use of a degrader of apoptosis enhancing domain protein 6 in the preparation of a drug for renal failure caused by systemic lupus erythematosus.

[0011] The third object of the present invention is to provide a use of a degrader of apoptosis enhancing domain protein 6 in the preparation of a drug for nephritis caused by systemic lupus erythematosus.

[0012] The fourth object of the present invention is to provide a compound.

[0013] The fifth object of the present invention is to provide the use of the compound in the preparation of medicines for systemic lupus erythematosus.

[0014] The sixth object of the present invention is to provide the use of the method in preparing a medicine for treating renal failure caused by systemic lupus erythematosus.

[0015] The seventh object of the present invention is to provide the use of the method in preparing a medicine for nephritis caused by systemic lupus erythematosus.

[0016] The eighth object of the present invention is to provide a method for preparing the compound.

[0017] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0018] The present invention claims protection for the following applications:

[0019] Use of a degrader of apoptosis enhancing domain protein 6 (CARD6) in the preparation of a drug for systemic lupus erythematosus.

[0020] Use of a degrader of apoptosis enhancing domain protein 6 (CARD6) in the preparation of a drug for renal failure caused by systemic lupus erythematosus.

[0021] Use of a degrader of apoptosis enhancing domain protein 6 (CARD6) in the preparation of a drug for nephritis caused by systemic lupus erythematosus.

[0022] Caspase recruitment domain-containing protein 6 (CARD6), gene ID: 84674; protein ID: Q9BX69.

[0023] Preferably, the degradation agent is a compound having the structural formula shown in formula (II-1),

[0024]

[0025] It is further claimed that a compound has the structural formula shown in formula (II-1),

[0026]

[0027] And the following applications:

[0028] The compound is used in preparing medicines for systemic lupus erythematosus.

[0029] The compound is used in preparing medicine for treating renal failure caused by systemic lupus erythematosus.

[0030] The compound is used in preparing medicine for nephritis caused by systemic lupus erythematosus.

[0031] The present invention also claims a method for preparing the compound, comprising the following steps:

[0032] Dihydroartemisinin, a compound represented by the structural formula (I-1), DMAP and DCM are fully mixed to react to obtain a reaction solution 1. EDCI in DCM is added dropwise to the reaction solution 1, and the mixture is fully mixed and reacted until the reaction is completed, and then purified to obtain the product.

[0033]

[0034] Preferably, the purification is: extraction with DCM, retaining the organic phase, and then separation using column chromatography.

[0035] More preferably, the purification is: extracting with DCM three times, combining and retaining the organic phase, and separating by column chromatography,

[0036] Preferably, the molar ratio of dihydroartemisinin, the compound represented by formula (I-1), and DMAP is: (0.8-1.2): (0.8-1.2): (0.8-1.2).

[0037] As a specific example, the molar ratio of dihydroartemisinin, the compound represented by the structural formula (I-1), and DMAP is 1:1:1.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The present invention utilizes PROTAC technology based on DHA to design a series of novel small molecule compounds, which have ligands that bind to E3 ubiquitin ligase proteins and ligands that target protein degradation. Among them, compound A1 degrades CARD6 protein through the ubiquitin proteasome system, can inhibit the polarization of THP-1 macrophages to the M1 phenotype induced by LPS+IFN-γ, and can degrade CARD6 to improve inflammation; it can also effectively improve the renal function of MRL / lpr model mice and alleviate the renal inflammatory response of MRL / lpr mice. It can be further developed into a drug for the treatment of SLE and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the synthetic route of compounds A1, A2, A3 and A6.

[0041] Figure 2 This is the synthetic route for compounds A4, A5, A7 and A8.

[0042] Figure 3 is the H NMR of compound A1 ( 1 H-NMR) spectrum.

[0043] Figure 4 is the H NMR of compound A2 ( 1 H-NMR) spectrum.

[0044] Figure 5 is the H NMR of compound A3 ( 1 H-NMR) spectrum.

[0045] Figure 6 is the H NMR of compound A4 ( 1 H-NMR) spectrum.

[0046] Figure 7 is the H NMR of compound A5 ( 1 H-NMR) spectrum.

[0047] Figure 8 is the H NMR of compound A6 ( 1 H-NMR) spectrum.

[0048] Figure 9 is the H NMR of compound A7 ( 1 H-NMR) spectrum.

[0049] Figure 10 is the H NMR of compound A8 ( 1 H-NMR) spectrum.

[0050] Figure 11 Effects of compounds A1 to A8 on the cell activity of THP-1 macrophages; # P<0.05, ## P<0.01, compared with the blank group.

[0051] Figure 12 Effects of compound A1 and DHA on the activity of THP-1 macrophages CC 50 .

[0052] Figure 13 The inhibitory effect of compound A1 and DHA on the expression of inflammatory factor mRNA in THP-1 macrophages induced by IFN-γ+LPS to M1 phenotype macrophages.

[0053] Figure 14 The inhibitory effect of compound A1 and DHA on the IFN-γ+LPS-induced transformation of THP-1 macrophages into M1 phenotype macrophage inflammatory factor proteins.

[0054] Figure 15 The IC values ​​of compound A1 and DHA for poly(dA:dT)-induced IFN-β mRNA 50 comparison.

[0055] Figure 16 The discovery of CARD6 protein; (A) is the volcano plot of differentially expressed proteins between the blank group and the M1 model group; (B) is the advanced Venn diagram of the differentially downregulated proteins at the intersection of the gradient concentration of compound A1 compared with the M1 group; (C) is the abundance level of 9 differentially downregulated proteins at the intersection.

[0056] Figure 17 Compound A1 can effectively degrade CARD6; (A) Compound A1 degrades CARD6 protein in a concentration-dependent manner; (B) Compound A1 degrades CARD6 protein in a time-dependent manner.

[0057] Figure 18 The degradation of CARD6 protein by compound A1 depends on the formation of a ternary complex; (A) The protein expression level of CARD6 after co-incubation of compound A1 with MG132, carfilzomib, and MLN4924; (B) The protein expression level of CARD6 after co-incubation of compound A1 with DHA and pomalidomide; (C) Co-IP detection of the binding between CRBN and CARD6.

[0058] Figure 19 The binding level of compound A1 to CARD6 was detected by DARTS.

[0059] Figure 20 The effects of compound A1 and DHA on urinary protein in MRL / lpr model mice.

[0060] Figure 21 Effects of compound A1 and DHA on the level of anti-dsDNA IgG in the serum of MRL / lpr model mice ( n=5).

[0061] Figure 22 The effects of compound A1 and DHA on Scr and BUN in the serum of MRL / lpr model mice.

[0062] Figure 23 The effects of compound A1 and DHA on the expression levels of CARD6 and inflammatory factor proteins in the renal tissue of MRL / lpr model mice.

[0063] Figure 24 Effects of compound A1 and DHA on renal pathology in MRL / lpr model mice (63×, HE staining).

[0064] Figure 25 The effects of compound A1 and DHA on M1 phenotypic marker proteins in MRL / lpr model mice. DETAILED DESCRIPTION

[0065] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0066] The THP-1 cells used in the present invention were purchased from ATCC, USA, and female C57BL / 6 mice and MRL / lpr lupus model mice were purchased from Zhuhai Bestone Biotechnology Co., Ltd. and Shanghai Lingchang Biotechnology Co., Ltd., respectively.

[0067] The cell growth medium used in the examples of the present invention is composed of: 1640 basal medium, to which 10% of the total volume of fetal bovine serum and 1% of the total volume of ampicillin / streptomycin are added. The culture medium is stored at 4°C and preheated in a 37°C water bath before use.

[0068] The dihydroartemisinin (DHA) used in the examples of the present invention was purchased from Shanghai Maclean Co., Ltd. with a purity of >99%.

[0069] The organic solvents used in the examples of the present invention, such as petroleum ether, ethyl acetate, dichloromethane, methanol, and ethanol, were purchased from Shanghai Titan Technology Co., Ltd. and were of analytical grade.

[0070] The glassware used in the examples of the present invention, such as round-bottom flask, separatory funnel, reflux condenser, splash guard, adapter, chromatography column, etc., were purchased from Xinweier Glass Instrument Co., Ltd.

[0071] The test tubes, graduated cylinders, thin-layer spotting capillaries, brown bottles, and pressure tips used in the examples of the present invention were all purchased from Beijing Lituo Xingye Biotechnology Co., Ltd.

[0072] 1H NMR spectra were recorded on a Bruker AVANCE III 400 MHz spectrometer using the solvent (CDCl3) peak as the standard. Low-resolution mass spectrometric analysis was performed using an Agilent 6340 or Waters AQUITY UPLC™ / MS.

[0073] All data were analyzed using the statistical software SPSS 25.0, and the measurement data that met the normal distribution were used. If it does not meet the description, use M(P 25 ,P 75 ) were used for description. Two-sample t-tests were performed for measurement data that met the normal distribution and homogeneity of variance. One-way ANOVA was used for measurement data from multiple groups. The nonparametric Mann-Whiteny U test was performed for measurement data that did not meet the normal distribution and homogeneity of variance test. Differences were considered statistically significant when P < 0.05. GraphPad 9.0 software was used to generate bar graphs.

[0074] Compound 1a, Compound 1b, Compound 1c, Compound 1b and Compound 2 refer to the prior art: Chen, D., Lin, S., Zeng, Z., An, J., Yan, W., Gu, Z., Chen, L., and He, B. (2024). Serendipitous discovery of Class I HDAC inhibitors from rational design of molecular glue degraders targeting HDAC. European Journal of Medicinal Chemistry 263, 115926. https: / / doi.org / 10.1016 / j.ejmech.2023.115926;

[0075] Compound 3a, Compound 3b, Compound 3c and Compound 3d refer to the prior art: Xie, S., Sun, Y., Liu, Y., Li, X., Li, X., Zhong, W., Zhan, F., Zhu, J., Yao, H., Yang, D.-H., et al. (2021). Development of Alectinib-Based PROTACs as Novel Potent Degraders of Anaplastic Lymphoma Kinase (ALK). Journal of Medicinal Chemistry 64, 9120-9140.10.1021 / acs.jmedchem.1c00270.

[0076] DMAP, CSA: 1122-58-3, 4-dimethylaminopyridine; DCM, CSA: 75-09-2, dichloromethane; EDCI, CSA: 7084-11-9, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HATU, CSA: 148893-10-1, 2-(7-azobenzotriazole)-tetramethyluronium hexafluorophosphate; DIPEA, CSA: 7087-68-5, N,N-diisopropylethylamine; PE, CSA: 8032-32-4, petroleum ether; EA, CSA: 141-78-6, ethyl acetate; MeOH, CSA: 67-56-1, methanol.

[0077] Example 1 Synthesis of Compounds

[0078] 1. Experimental Methods

[0079] The present invention utilizes PROTAC technology based on DHA to design a series of novel small molecule compounds, which have ligands that bind to E3 ubiquitin ligase proteins and ligands that target protein degradation.

[0080] (1) Synthesis of Compounds A1, A2, A3, and A6

[0081] Synthesis route see Figure 1 Compounds 1a, 1b, 1c and 1d (1 eq) of formula (I) were dissolved in DCM with dihydroartemisinin (28.5 mg, 0.1 mmol, 1 eq) and DMAP (12 mg, 1 eq), respectively, and stirred at 0°C (avoid overheating).

[0082]

[0083] Among them, compound 1a, n=1; compound 1b, n=4; compound 1c, n=6; compound 1d, n=9;

[0084] EDCI was then dissolved in DCM and slowly added dropwise to the above reaction system, and the reaction solution was stirred at room temperature overnight;

[0085] The reaction solution was detected by TLC (volume ratio of PE:EA=1:1 as developing solvent, 365nm ultraviolet light for color development). After the reaction was completed, the reaction solution was poured into water and extracted with DCM (20 mL each time, 3 times in total). The organic phases were combined and concentrated, and then separated by column chromatography (volume ratio of PE:EA=1:1 as eluent) to obtain the final products A6, A1, A2 and A3 corresponding to compounds 1a, 1b, 1c and 1d represented by formula (I) (isolation yield IY=50%). The structural formulas of compounds A1, A2, A3 and A6 are shown in (II).

[0086]

[0087] Among them, for compound A6, n=1; for compound A1, n=4; for compound A2, n=6; and for compound A3, n=9.

[0088] (2) Synthesis of Compounds A4, A5, A7, and A8

[0089] Synthesis route see Figure 2 Compounds 3a, 3b, 3c and 3d (1 eq) of the structural formula (III) were dissolved in DCM to obtain DCM solutions of compounds 3a, 3b, 3c and 3d.

[0090]

[0091] Among them, compound 3a, n=1; compound 3b, n=2; compound 3c, n=3; compound 3d, n=4;

[0092] Compound 2 (0.1 mmol, 1 eq) of formula (IV), HATU (41.8 mg, 0.11 mmol, 1.1 eq), and DIPEA (38.2 μl, 2 eq) were dissolved in DCM and stirred at room temperature for 5 minutes. The mixture was then mixed with DCM solutions of compounds 3a, 3b, 3c, and 3d, respectively, and stirred at room temperature for 2 hours.

[0093]

[0094] The reaction solution was detected by TLC (using DCM:MeOH = 20:1 by volume as the developing solvent and 365 nm UV light for color development). After the reaction was completed, the reaction solution was poured into water and extracted with DCM (20 mL each time, 3 times in total). The organic phases were combined and concentrated, and then separated by column chromatography (using DCM:MeOH = 30:1 by volume as the eluent) to obtain the final products A4, A5, A7 and A8 corresponding to compounds 3a, 3b, 3c and 3d represented by formula (III) (isolation yield = 40%). The structural formulas of compounds A4, A5, A7 and A8 are shown in (V).

[0095]

[0096] Among them, for compound A4, n=1; for compound A5, n=2; for compound A7, n=3; and for compound A8, n=4.

[0097] (3) Mass spectrometry and NMR detection of compounds

[0098] Mass spectrum of compound A1 ( Figure 3 ) and nuclear magnetic resonance test results are:

[0099] 1H-NMR (400MHz, CDCl3, ppm): 8.04 (s, 1H), 7.49 (t, J = 7.72Hz, 1H), 7.08 (d, J = 7.04Hz, 1H), 6.87 (d, J = 8.52Hz ,1H),6.24(s,1H),5.79(d,J=9.84Hz,1H),5.44(s,1H),4.93-4.89(m,1H),3.27(dt,J1=6.28Hz,J2=6.64Hz ,2H),2.91-2.72(m,3H),2.58-2.53(m,1H),2.44-2.32(m,3H),2.14-2.11(m,1H),2.04(s,1H),2.01(s,1H) ,1.91-1.87(m,1H),1.79-1.24(m,15H),1.05-0.99(m,1H),0.96(d,J=5.88Hz,3H),0.83(d,J=7.08Hz,3H);

[0100] 13C-NMR (400MHz, CDCl3, ppm):172.35,171.01,169.59,168.34,167.74,147.09,136.28,132.64,116.76,111.59,110.84,104.59,91.97,91.64 ,80.27,51.72,49.00,45.40,42.56,37.41,36.36,34.22,31.94,31.54 ,29.10,26.51,26.09,24.72,24.43,22.94,22.14,20.34,15.39,12.26;

[0101] LC-MS: calculated for C 34 H 43 N3O 10 , [M+H] + ,654.59.

[0102] It can be determined that the structural formula of compound A1 is as shown in formula (II-1), and it is named: (3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dio xepino[4,3-i]isochromen-10-yl6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexanoate,

[0103]

[0104] The mass spectrum of compound A2 ( Figure 4 ) and NMR test results are as follows:

[0105] 1 H-NMR (400MHz, CDCl3, ppm): 8.13 (d, J = 3.76Hz, 1H), 7.49 (dd, J1 = 7.44Hz, J2 = 8.2Hz, 1H), 7.08 (d, J = 7.12Hz, 1H), 6 .88(d,J=8.52Hz,1H),6.22(t,J=5.4Hz,1H),5.79(d,J=9.84Hz,1H),5.44(s,1H),4.93-4.89(m,1H),3.28(dt,J1= 6.04Hz, J2=6.8Hz,2H),2.91-2.72(m,3H),2.58-2.53(m,1H),2.41-2.33(m,3H),2.14-2.10(m,1H),2.04(s,1H),2 .01(s,1H),1.91-1.86(m,1H),1.79-1.25(m,19H),1.05-0.99(m,1H),0.96(d,J=6Hz,3H),0.83(d,J=7.08Hz,3H);

[0106] 13 C-NMR (400MHz, CDCl3, ppm): 172.57, 171.23, 169.59, 168.50, 167.75, 147. 12,136.19,132.62,116.74,111.42,109.98,104.54,91.84,91.58,80.25, 51.70,49.98,45.37,42.72,37.36,36.33,34.31,34.21,31.92,31.52,29. 25,28.99,26.82,26.04,24.69,24.60,22.91,22.10,20.30,15.35,12.22;

[0107] LC-MS: calculated for C 36 H 47 N3O 10 , [M+H] + ,682.44.

[0108] It can be determined that the structural formula of compound A2 is as shown in formula (II-2), and it is named: (3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dio xepino[4,3-i]isochromen-10-yl8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octanoate,

[0109]

[0110] Mass spectrum of compound A3 ( Figure 5 ) and NMR test results are as follows:

[0111] 1 H-NMR (400MHz, CDCl3, ppm): 8.03 (s, 1H), 7.52 (t, J = 8.08Hz, 1H), 7.12 (d, J = 7.08Hz, 1H), 6.94 (d, J = 8.52 Hz,1H),6.50(t,J=6.16Hz,1H),5.83(d,J=9.84Hz,1H),5.44(s,1H),4.93-4.89(m,1H),3.67-3.61(m,2H ),2.91-2.68(m,5H),2.59-2.54(m,1H),2.41-2.33(m,1H),2.14-2.10(m,1H),2.04(s,1H),2.01(s,1H), 1.92-1.87(m,1H),1.79-1.24(m,25H),1.05-0.99(m,1H),0.96(d,J=5.88Hz,3H),0.83(d,J=6.56Hz,3H);

[0112] 13C-NMR (400MHz, CDCl3, ppm):172.73,171.04,169.62,168.38,167.78,147.20,1 36.23,132.65,116.78,111.46,109.97,104.58,91.84,91.63,80.30,51.75,49 .01,45.43,42.81,37.40,36.38,34.45,34.25,31.98,31.55,29.52,29.41,29.37,29.28,29.18,27.04,26.10,24.76,24.73,22.96,22.15,20.35,15.40,12.26

[0113] LC-MS: calculated for C 39 H 53 N3O 10 , [M+H] + ,724.54.

[0114] It can be determined that the structural formula of compound A3 is as shown in formula (II-3), and it is named: (3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dio xepino[4,3-i]isochromen-10-yl

[0115] 11-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)unddecanoate,

[0116]

[0117] Mass spectrum of compound A4 ( Figure 6 ) and NMR test results are as follows:

[0118] 1H-NMR(400MHz,CDCl3,ppm):8.43(d,J=13.24Hz,1H),7.51(t,J=7.84Hz,1H),7.10(d,J=7.12Hz,1H),7.00(d,J=8.52Hz,1H),6.43(s,1H),6.34(d,J=4.76Hz,1H),5.77(d,J=9.84Hz,1H),5.44(s,1H),4.95-4.91(m,1H),3.45(s,4H),2.89-2.72(m,5H),2.55-2.32(m,4H),2.13-2.10(m,1H),2.04(s,1H),2.00(s,1H),1.91-1.87(m,1H),1.76-1.25(m,9H),1.04-0.98(m,1H),0.95(d,J=5.72Hz,3H),0.83(d,J=7.08Hz,3H);

[0119] 13 C-NMR(400MHz,CDCl3,ppm):172.26,171.86,171.15,169.54,168.50,167.67,146.92,136.44,132.64,116.99,112.10,110.55,104.64,92.46,91.64,80.27,51.68,49.08,45.35,42.18,39.18,37.41,36.34,34.22,31.87,31.17,30.06,29.83,26.05,24.72,22.90,22.11,20.33,12.18;

[0120] LC-MS:calculated for C 34 H 42 N4O 11 ,[M+H] + ,683.39;

[0121] It can be determined that the structural formula of compound A4 is as shown in formula (V-1), and it is named: (3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dio xepino[4,3-i]isochromen-10-yl4-((2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)amino)-4-oxo butanoate,

[0122]

[0123] Mass spectrum of compound A5 ( Figure 7 ) and NMR test results are as follows:

[0124] 1 H-NMR (400MHz, CDCl3, ppm): 8.18 (d, J = 3.44Hz, 1H), 7.49 (t, J = 7.36Hz, 1H), 7.09 (d, J = 7.08Hz, 1H), 6.89 (d,J=8.56Hz,1H),6.23(s,1H),5.83(s,1H),5.77(d,J=9.88Hz,1H),5.42(d,J=1.96Hz,1H),4.93-4.89( m,1H),3.29(s,4H),2.91-2.70(m,5H),2.60-2.32(m,4H),2.16-2.09(m,1H),2.04(s,1H),2.00(s,1H),1 .91-1.87(m,1H),1.77-1.25(m,13H),1.03-0.98(m,1H),0.95(d,J=5.72Hz,3H),0.83(d,J=7.08Hz,3H);

[0125] 13C-NMR (400MHz, CDCl3, ppm): 171.99, 171.59, 171.34, 169.66, 168.62, 167. 73,147.03,136.31,132.59,116.88,111.62,110.07,104.61,92.37,91.60 ,80.25,51.65,49.03,45.32,42.31,39.14,37.36,36.32,34.19,31.86,31 .54,31.11,30.02,27.11,26.61,26.03,24.69,22.92,22.08,20.31,12.17;

[0126] LC-MS: calculated for C 36 H 46 N4O 11 , [M+H] + ,711.62;

[0127] It can be determined that the structural formula of compound A5 is as shown in formula (V-2), and it is named: (3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dio xepino[4,3-i]isochromen-10-yl4-((4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butyl)amino)-4-oxo butanoate,

[0128]

[0129] Mass spectrum of compound A6 ( Figure 8 ) and NMR test results are as follows:

[0130] 1H-NMR (400MHz, CDCl3, ppm): 8.15 (s, 1H), 7.51 (t, J = 7.76Hz, 1H), 7.11 (d, J = 7.12Hz, 1H), 6.94 (d, J = 8.5 6Hz,1H),6.50(t,J=5.96Hz,1H),5.82(d,J=9.88Hz,1H),5.44(s,1H),4.93-4.89(m,1H),3.67-3.61(m, 2H),2.90-2.71(m,5H),2.59-2.54(m,1H),2.41-2.33(m,1H),2.13-2.10(m,1H),2.04(s,1H),2.00(s,1 H),1.91-1.87(m,1H),1.78-1.25(m,9H),1.05-0.99(m,1H),0.96(d,J=5.84Hz,3H),0.82(d,J=7Hz,3H);

[0131] 13 C-NMR (400MHz, CDCl3, ppm): 171.06, 170.47, 169.36, 168.31, 167.65, 146.44, 136.38, 132.74, 116.58, 112.08, 110.74, 104.65, 92. 48,91.70,80.24,51.70,49.03,45.37,38.14,37.42,36.35,34.25,34.21,31.88,31.55,26.06,24.71,22.89,22.14,20.33,12.24;

[0132] LC-MS: calculated for C 31 H 37 N3O 10 , [M+H] + ,612.20;

[0133] It can be determined that the structural formula of compound A6 is as shown in formula (II-4), and it is named: (3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dio xepino[4,3-i]isochromen-10-yl3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propanoate,

[0134]

[0135] Mass spectrum of compound A7 ( Figure 9 ) and NMR test results are as follows:

[0136] 1 H-NMR (400MHz, CDCl3, ppm): 8.25 (s, 1H), 7.49 (t, J = 7.8Hz, 1H), 7.08 (d, J = 7.08Hz, 1H), 6.88 (d, J = 8.5 2Hz,1H),6.23(t,J=5.16Hz,1H),5.81(s,1H),5.75(d,J=9.84Hz,1H),5.42(s,1H),4.93-4.89(m,1H),3 .29-3.15(m,4H),2.90-2.69(m,5H),2.57-2.32(m,4H),2.14-2.10(m,1H),2.04(s,1H),2.00(s,1H),1. 90-1.85(m,1H),1.76-1.25(m,17H),1.03-0.97(m,1H),0.94(d,J=5.72Hz,3H),0.83(d,J=7.08Hz,3H);

[0137] 13 C-NMR (400MHz, CDCl3, ppm):172.07,171.36,171.11,169.69,168.46,167.75, 147.15,136.29,132.65,116.82,111.58,110.08,104.61,92.37,91.63,80.25 ,51.69,49.04,45.35,42.66,39.56,37.41,36.35,34.22,31.89,31.56,31.14,30.34,29.58,29.12,26.70,26.61,26.07,24.72,22.95,22.11,20.33,12.20;

[0138] LC-MS: calculated for C 38 H 50 N4O 11 ,[M+H] + ,739.47,

[0139] It can be determined that the structural formula of compound A7 is as shown in formula (V-3), and it is named: (3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dio xepino[4,3-i]isochromen-10-yl4-((6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)amino)-4-oxo butanoate,

[0140]

[0141]

[0142] Mass spectrum of compound A8 ( Figure 10 ) and NMR test results are as follows:

[0143] 1 H-NMR (400MHz, CDCl3, ppm): 8.25 (s, 1H), 7.49 (t, J = 7.72Hz, 1H), 7.08 (d, J = 7.08Hz, 1H), 6.88 (d, J = 8 .56Hz,1H),6.23(t,J=5.32Hz,1H),5.78-5.75(m,2H),5.43(d,J=2.04Hz,1H),4.93-4.89(m,1H),3.28 -3.14(m,4H),2.90-2.67(m,5H),2.57-2.33(m,4H),2.15-2.08(m,1H),2.04(s,1H),2.00(s,1H),1.9 1-1.86(m,1H),1.78-1.25(m,21H),1.05-0.98(m,1H),0.95(d,J=5.84Hz,3H),0.84(d,J=7.12Hz,3H);

[0144] 13C-NMR (400MHz, CDCl3, ppm):172.04,171.35,171.24,169.64,168.56,167.76,1 47.15,136.23,132.61,116.80,111.46,109.97,104.59,92.34,91.60,80.24,51 .67,48.99,45.33,42.68,39.75,37.38,36.33,34.20,31.87,31.52,31.06,29.96,29.79,29.56,29.11,26.81,26.77,26.04,24.69,22.92,22.09,20.30,12.16;

[0145] LC-MS: calculated for C 40 H 54 N4O 11 ,[M+H] + ,767.79,

[0146] It can be determined that the structural formula of compound A8 is as shown in formula (V-4), and it is named: (3R,5aS,6R,8aS,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl4-((8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octyl)amino)-4-oxobutanoate,

[0147]

[0148] Example 2 Cytotoxicity of Compounds A1 to A8 on THP-1 Macrophages

[0149] 1. Experimental Methods

[0150] 1. Inoculate THP-1 macrophages and induce them into M0 mononuclear macrophages

[0151] The cell density was adjusted to 5×10 cells per well using RPMI 1640 medium containing 10% fetal bovine serum (V / V). 4 The cells were seeded into a 96-well plate at 100 μL / well, and PMA (100 ng / mL) was added to induce M0 mononuclear macrophages; the cells were cultured at 37° C. and 5% (V / V) CO 2 for 24 hours.

[0152] 2. Medication treatment

[0153] The medium from each well was aspirated, and drug treatment was performed: 100 μL of RPMI 1640 medium supplemented with 10% (v / v) fetal bovine serum was added to each well, diluted to the corresponding concentrations of Compounds A1 to A8 prepared in Example 1 (0.625, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 μM). DHA was used as a positive control. A blank control was prepared with 100 μL of RPMI 1640 medium supplemented with 10% (v / v) fetal bovine serum, without drug. The cells were incubated for 24 hours after drug administration.

[0154] 3. MTT color

[0155] After 24 hours of co-incubation, 10 μL of 5 mg / ml MTT solution was added to each well. The cells were cultured at 37° C. and 5% (V / V) CO 2 for 4 hours, and then DMSO was added until the formazan was completely dissolved under ordinary optical microscope.

[0156] 4. Measurement and calculation

[0157] The absorbance was measured at 490 nm, and the effects of compounds A1 to A8 on the cell activity and survival of THP-1 macrophages were calculated based on the measured OD values, and CC was further calculated. 50 .

[0158] 2. Experimental Results

[0159] Cell viability results Figure 11 The results showed that compared with the blank group (Control), compound A2 prepared in Example 1 significantly reduced the viability of THP-1 macrophages, and the difference was statistically significant (P<0.01), and it had a greater toxicity to THP-1 macrophages; while the remaining 7 compounds had no significant effect on the viability of THP-1 macrophages, and there was no statistical difference (P>0.01). Therefore, the remaining 7 PROTAC compounds were used for subsequent anti-inflammatory activity screening.

[0160] Among them, the compound A1 prepared in Example 1 has lower toxicity to THP-1 macrophages than DHA, and the CC 50 The values ​​were 952.68 μM and 463.12 μM ( Figure 12 ); This shows that A1 is safer and less toxic (only about half of DHA).

[0161] Example 3 Compound Inhibitory Effect on Gene mRNA Expression of Inflammatory Factors in THP-1 Macrophages Polarized to M1 Phenotype

[0162] 1. Experimental Methods

[0163] 1. Inoculate THP-1 macrophages and induce them into M0 mononuclear macrophages

[0164] The cell density was adjusted to 1×10 cells per well using RPMI 1640 medium containing 10% fetal bovine serum (V / V). 6 The cells were seeded into 6-well plates at 100 μl / well and PMA (100 ng / mL) was added for 24 h to induce M0 mononuclear macrophages.

[0165] 2. Induction of M1 phenotype THP-1 macrophages and drug intervention

[0166] After adherence, cells were starved and cultured in serum-free 1640 medium for 24 hours. LPS (100 ng / mL) and IFN-γ (20 ng / mL) were then used to induce M1 monocyte-macrophage cell differentiation. Treatments were then performed: Compounds A1, A3, A4, A5, A6, A7, and A8 prepared in Example 1 at a final concentration of 5 μM were used as the treatment group; M1 monocyte-macrophage cells were treated with corresponding concentrations of DHA as the positive control group; M0 phenotype cells served as the blank control group; and M1 phenotype cells served as the model group. The cells were incubated for 24 hours after dosing.

[0167] 3. Collect total cell RNA

[0168] RNA extraction kit (Chengdu Fuji Biotechnology Co., Ltd., catalog number: RE-03113) was used. The specific method is as follows:

[0169] The supernatant was aspirated and 1 ml of PBS was added to each well of the 6-well plate to wash. Buffer CRL1 was added to lyse the cells and the plate was centrifuged at 12,000 rpm for 2 min before passing through a DNA column. The filtrate was collected and 1.6x Buffer CRL2 was added. The mixture was mixed well and passed through an RNA column. After centrifugation, 500 μL of Buffer RW1 was added for protein removal and two washes with 700 μL of RW2 were added for desalting. After centrifugation of the empty tube to remove residual ethanol, 30 μL of 65°C preheated enzyme-free water was added to each tube. After centrifugation, RNA was collected and the 260 / 280 absorbance ratio was measured using a UV spectrophotometer to calculate the RNA concentration.

[0170] 4. Reverse transcription of RNA into cDNA

[0171] Reaction system: RNA 1 mg, 5× PrimeScript RT Master Mix (TAKARA) 4 μl, add enzyme-free water to a total volume of 20 μl; after gentle mixing, reverse transcription was performed at 37°C for 15 min and 85°C for 5 s.

[0172] 5. Real-time fluorescence quantitative PCR detection of each sample

[0173] The mRNA levels of inflammatory factor genes TNF-α, IL-1β and IL-6 in each sample.

[0174] Reaction system (10 μl): cDNA template 1 μl, qPCR Master Mix (Promega) 5 μl, forward and reverse primers 0.4 μl, and enzyme-free water 3.2 μl were mixed and incubated at 95°C for 30 seconds for 1 cycle. PCR amplification program: 95°C for 5 seconds for 40 cycles; annealing: 60°C for 30-34 seconds.

[0175] Table 1 Forward and reverse primers for each target RNA

[0176]

[0177]

[0178] 6. Calculation

[0179] According to the CT value of each sample, (—△△Ct) The RNA level of each sample relative to the control group was calculated.

[0180] 2. Experimental Results

[0181] The results are as follows Figure 13 The results showed that when treated with 5 μM of DHA, the mRNA levels of TNF-α, IL-1β and IL-6 in the model group were significantly increased compared with the blank group. After treatment with DHA and the 7 compounds prepared in Example 1, the gene expression levels of these inflammatory factors decreased; among them, the compound A1 prepared in Example 1 had the most significant effect and could effectively reduce the mRNA levels of inflammatory factors TNF-α, IL-1β and IL-6.

[0182] Example 4 Inhibitory Effect of Compounds on Protein Levels of Inflammatory Factors Polarized to M1 Phenotype in THP-1 Macrophages

[0183] 1. Experimental Methods

[0184] 1. Inoculate THP-1 macrophages and induce them into M0 mononuclear macrophages

[0185] The THP-1 cell density was adjusted to 1×10 cells per well using RPMI 1640 medium containing 10% fetal bovine serum (V / V). 6 The cells were seeded into 6-well plates at 100 μl / well and PMA (100 ng / mL) was added for 24 h to induce M0 mononuclear macrophages.

[0186] 2. Induction of M1 phenotype THP-1 macrophages and drug intervention

[0187] After adherence, the cells were starved for 24 hours after being replaced with serum-free 1640 medium. LPS (100 ng / mL) and IFN-γ (20 ng / mL) were then used to induce M1 monocyte-macrophage cell differentiation. The cells were then treated with various concentrations (0.625, 1.25, 2.5, 5, and 10 μM) of compounds A1, A3, A4, A5, A6, A7, and A8 prepared in Example 1 and incubated with M1 monocyte-macrophage cells. DHA at corresponding concentrations was used in the positive control group. M0 phenotype cells served as a blank control group, and M1 phenotype cells served as a model group. The cells were incubated for 24 hours after administration.

[0188] 3. Extraction of total cell protein

[0189] Cells from each group were collected and washed twice with 4°C pre-cooled PBS. 100 μl of RIPA mixed lysis buffer (RIPA: phosphatase inhibitor: protease inhibitor = 1000:10:1) was added to each well. Cells were scraped with a cell scraper and collected in a 1.5 ml centrifuge tube. The cells were centrifuged at 12000 rpm at 4°C for 15 min, and the supernatant was transferred to a new centrifuge tube.

[0190] 4. Detect sample protein concentration

[0191] The protein concentration of the samples was determined using the BCA protein concentration assay kit (Shanghai Biotech Co., Ltd., P0012S). The specific method is as follows:

[0192] The protein standard was diluted with PBS to a gradient concentration of 0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml. 20 μl of the diluted protein standard and protein samples extracted from each group of cells were added to a 96-well plate, and 200 μl of BCA working solution (volume ratio of A solution: B solution is 50:1) was added to each well. The plates were incubated at 37°C for 30 min. The absorbance was measured at 562 nm by a microplate reader, and the protein concentration in the samples was calculated based on the standard curve.

[0193] Finally, the protein sample extracted from each group of cells was diluted to a concentration of 2 mg / mL, placed at 100°C for denaturation for 5 minutes, cooled, and stored in a -20°C refrigerator for later use.

[0194] 5. Western blotting analysis

[0195] Prepare a 10% SDS-polyacrylamide gel by adding various reagents in sequence. After injecting the separating gel, quickly add anhydrous ethanol to flatten it. Once solidified, pour off the anhydrous ethanol, blot dry with filter paper, and then inject the stacking gel. Insert the electrophoresis comb. Load 10 μL of protein sample per well. After setting the marker wells, connect the power supply and run electrophoresis at 80V for 25 minutes, then switch to 120V for 80 minutes.

[0196] 6. Transfer

[0197] Remove the separation gel and soak it in transfer buffer. Cut the target protein according to the marker and cut a 0.22 μm PVDF membrane according to the size of the gel. After activation in methanol for 2 minutes, soak it in transfer buffer. Prepare a "sandwich" transfer cassette in the order of the black side of the cassette at the bottom, sponge, wet filter paper, gel, PVDF membrane, wet filter paper, sponge, and white side as the top. Fill the electrophoresis tank with transfer buffer, cover the transfer cassette and the top with ice, turn on the power, and transfer under the constant current of 300 mA. Transfer for large molecules (>70 kDa) for 120 minutes and for small molecules (<70 kDa) for 50 minutes.

[0198] 7. Protein Blocking: Remove the PVDF membrane and place it in blocking solution. Soak it in a shaking plate at room temperature for 1 hour to block nonspecific antigens. After blocking, wash it three times with TBST on a shaking plate at room temperature for 5 minutes each time.

[0199] 8. Antibody Incubation: Incubate with primary antibody at 4°C overnight. Wash the membrane three times on a shaker at low speed, then incubate with secondary antibody at room temperature for 1 hour before washing.

[0200] 9. Development: Turn on the automatic imaging chemiluminescence instrument, cool the instrument to -30℃, immerse the membrane in ECL luminescent solution (solution A and solution B), turn on white light to focus, then develop and record.

[0201] 2. Experimental Results

[0202] The results are as follows Figure 14 Protein-level studies revealed that compounds A1, A4, and A8 prepared in Example 1 exhibited significant inhibitory effects, effectively reducing the expression levels of CD86, TNF-α, and IL-1β. Further experiments demonstrated that compound A1 prepared in Example 1 reduced the expression levels of CD86, TNF-α, and IL-1β proteins in a concentration-dependent manner. Therefore, compound A1 prepared in Example 1 was selected for subsequent experiments.

[0203] Example 5 Effects of Compounds on IFN-β Levels in THP-1 Cells Stimulated by Double-Stranded DNA Poly(dA:dT)

[0204] 1. Experimental Methods

[0205] THP-1 cells were induced to M0 monocyte-macrophage cells using PMA (100 ng / mL) according to the method of Example 2. After adherence, the cells were starved for 24 hours by replacing the serum-free 1640 medium and then transfected with 2 μg / mL poly(dA:dT) to induce IFN-β production in THP-1 monocyte-macrophage cells.

[0206] Compound A1 prepared in Example 1 was co-incubated with poly(dA:dT)-transfected THP-1 mononuclear macrophages at different concentrations (0.3125, 0.625, 1.25, 2.5, 5, 10, 20, and 40 μM) for 24 h, and then the expression of IFN-β was detected;

[0207] The same concentration of DHA was co-incubated with THP-1 mononuclear macrophages transfected with poly(dA:dT) for 24 h as a positive control to detect the expression of IFN-β.

[0208] According to the method of Example 3, total cellular RNA was reverse transcribed into cDNA, and each sample was detected by real-time fluorescence quantitative PCR and calculated. The specific primers are shown in Table 2.

[0209] Table 2

[0210]

[0211] 2. Experimental Results

[0212] See the results Figure 15 The compound A1 prepared in Example 1 exerted an inhibitory effect on IFN-β gene level that was 4 times that of DHA, and the IC 50 The values ​​were 0.61 and 2.5 μM, respectively.

[0213] Based on Examples 1 to 4, it was found that compared with DNA, the compound A1 prepared in Example 1 can improve the anti-inflammatory activity and has lower toxicity.

[0214] Example 6: Label-free quantitative proteomics assessment of CARD6 protein degradation

[0215] 1. Experimental Methods

[0216] 1. Inoculation of THP-1 macrophages

[0217] The THP-1 cell density was adjusted to 1×10 cells per well using RPMI 1640 medium containing 10% fetal bovine serum (V / V). 6 The cells were seeded into 10 cm dishes, 10 mL / dish, and PMA (100 ng / mL) was added for 24 h to induce M0 mononuclear macrophages.

[0218] 2. Induction of M1 phenotype THP-1 macrophages and drug intervention

[0219] After adherence, the cells were replaced with serum-free 1640 medium and starved for 24 h. LPS (100 ng / mL) and IFN-γ (20 ng / mL) were used to induce M1 mononuclear macrophages.

[0220] Treatments were performed: Compound A1 prepared in Example 1 at different concentrations (0.625, 1.25, 2.5, 5, and 10 μM) was administered to M1 monocytes and macrophages as the administration group; M0 served as the blank control group (control); and M1 served as the model group. The cells were incubated for 24 hours after administration.

[0221] 3. Lyse cells and determine protein concentration

[0222] The samples incubated for 24 hours were lysed using lysis buffer (8 M urea, 100 mM NH4HCO3, pH = 8) and ultrasonically lysed on ice for 5 minutes; after centrifugation at 16000g for 15 minutes at 4°C, the supernatant was collected and the protein concentration was determined using the BCA protein quantification kit.

[0223] 4. Reduction and alkylation of protein samples

[0224] The collected supernatant was reduced with 20 mM DTT at 55°C for 45 min, cooled to room temperature, and alkylated with 40 mM IAA for 30 min. Pre-cooled acetone was added and vortexed to mix. The cell sample was precipitated at -20°C for 2 h, and then centrifuged at 16,000 g for 15 min at 4°C to collect the precipitate.

[0225] 5. Protein digestion

[0226] The precipitate was dissolved in 8 M urea, 100 mM TEAB, pH = 8.5, and trypsin (100 μg protein: 2.5 μg trypsin) and 30 μL CaCl2 (1 mM) were added. After mixing, the mixture was digested at 37°C and 300 rpm on a shaker for 18 hours.

[0227] 6. Sample desalting and enrichment

[0228] Add 0.1% formic acid aqueous solution to the activated Oasis HLB extraction column, add the sample to the extraction column for desalting, collect the eluate in a 1.5 mL centrifuge tube, and spin dry it at 60°C using a vacuum concentrator.

[0229] 7. LC-MS / MS detection

[0230] The samples were reconstituted with 0.1% formic acid and 1% acetonitrile and centrifuged at 16,000 g for 1 h at 4°C. The supernatant was collected and analyzed by LC-MS / MS using an Ultimate 3000RSLC nanosystem coupled to an Orbitrap Fusion Lumos hybrid mass spectrometer (Thermo Scientific).

[0231] 8. Data Analysis

[0232] Proteome Discovery (version 2.4) was used to search the mass spectrometry raw data library, and peptides were matched and proteins were identified using the Sequest HT algorithm. The standard database was derived from the UniProtKB human FASTA database (Homo_sapiens_uniprot-reviewed; version January 2023).

[0233] 2. Experimental Results

[0234] The results are as follows Figure 16 The results showed that compared with the Control group, there were 249 differentially upregulated proteins and 192 differentially downregulated proteins in the M1 inflammation model group ( Figure 16 After treatment with compound A1 prepared in Example 1, compared with the M1 inflammation model group, there were 9 cross-linked differentially down-regulated proteins at each concentration in the A1 group ( Figure 16 B), among which CARD6 (apoptosis enhancing domain protein 6) is the most significantly degraded protein ( Figure 16 C).

[0235] Example 7 Compound A1 can effectively degrade CARD6 protein

[0236] 1. Experimental Methods

[0237] 1. Induce M0 monocytes and macrophages

[0238] The THP-1 cell density was adjusted to 5×10 cells per well using RPMI 1640 medium containing 10% fetal bovine serum (V / V). 5 The cells were seeded into 6-well plates at 2 mL / well and PMA (100 ng / mL) was added for 24 h to induce M0 mononuclear macrophages.

[0239] 2. Induction of M1 phenotype THP-1 macrophages and drug intervention

[0240] After attachment, the cells were starved for 24 hours by replacing the serum-free 1640 medium. LPS (100 ng / mL) and IFN-γ (20 ng / mL) were used to induce M1 mononuclear macrophages, and the following treatments were performed:

[0241] M1 mononuclear macrophages were added with different concentrations (0.625, 1.25, 2.5, 5, 10 μM) of compound A1 and incubated for 1 to 24 hours as the drug-treated group; M1 mononuclear macrophages were added with DMSO and incubated for 1 to 24 hours as the blank control.

[0242] 3. Western blot analysis

[0243] Western blot analysis was performed according to the method of Example 4 to detect CARD6 protein. 2. Experimental Results

[0244] 2. Experimental Methods

[0245] Western blot analysis showed that A1 degraded CARD6 protein in a concentration-dependent manner between 0.625 and 10 μM ( Figure 17 A in the figure); and CARD6 protein began to degrade at 1 hour and degraded in a time-dependent manner within 24 hours ( Figure 17 B in the);

[0246] Example 8 Compound A1 Degradation of CARD6 Protein Depends on the Formation of a Ternary Complex

[0247] 1. Experimental Methods

[0248] 1. Induce M0 mononuclear macrophages into M0 mononuclear macrophages

[0249] The THP-1 cell density was adjusted to 5×10 cells per well using RPMI 1640 medium containing 10% fetal bovine serum (V / V). 5 The cells were seeded into 6-well plates at 2 mL / well and PMA (100 ng / mL) was added for 24 h to induce M0 mononuclear macrophages.

[0250] 2. Induction of M1 phenotype THP-1 macrophages and drug intervention

[0251] After attachment, the cells were starved for 24 hours by replacing the serum-free 1640 medium. LPS (100 ng / mL) and IFN-γ (20 ng / mL) were used to induce M1 mononuclear macrophages, and the following treatments were performed:

[0252] (1) M1 mononuclear macrophages were pre-incubated with 10 μM of compound A1 prepared in Example 1 for 4 h, and then the proteasome inhibitor MG132 (5 μM), carfilzomib (0.4 μM), and NAE inhibitor MLN4924 (1 μM) were added and incubated for 4 h.

[0253] (2) M1 monocytes were pre-incubated with 10 μM of Compound A1 for 4 h, and then administered with one to three of the following: Compound A1 (10 μM) prepared in Example 1, DHA (10 μM), and Pomalidomide (10 μM), a ligand of CRBN E3 ligase.

[0254] 3. Western blot analysis

[0255] Western blot analysis was performed according to the method of Example 4 to detect CARD6 protein.

[0256] 2. Experimental Results

[0257] Compound A1 prepared in Example 1 was intervened by adding proteasome inhibitor MG132, carfilzomib, and NAE inhibitor MLN4924, and its CARD6 degradation was inhibited ( Figure 18 A in

[0258] When the compound A1 or DHA prepared in Example 1 was co-treated with the ligand of CRBN E3 ligase, pomalidomide, the degradation effect of A1 or DHA on CARD6 protein was weakened by pomalidomide ( Figure 18 B) in.

[0259] Example 9 Co-IP detection of the effect of compound A1 on the binding of CRBN and CARD6

[0260] 1. Experimental Methods

[0261] (1) Cell plating

[0262] 293T cells (3×10 5 The cells were inoculated into 10 cm culture dishes, 10 mL / dish, and incubated with DMEM (Gibico, USA, catalog number: C11995500BT) complete medium for 24 h.

[0263] (2) Transfection

[0264] Overexpression: 30 minutes before transfection, fresh DMEM complete medium was replaced. 30 μL polyJet (SignaGen, USA, catalog number: s1100688) was added to 250 μL DMEM blank medium to prepare transfection solution. His-CRBN plasmid (5 μg, pCMV-CRBN(human)-Myc-6×His-Zeo, Miaoling Biotechnology, P58926) and HA-CARD6 plasmid (5 μg, pCMV-3×HA-CARD6(1-949aa)-Neo, Miaoling Biotechnology, P38960) were added to 250 μL DMEM blank medium to prepare DNA solution. After 5 minutes, the transfection solution was added to the DNA solution, mixed, and incubated for 15 minutes to obtain the transfection mixture. 500 μL of the transfection mixture was added to each dish for transfection.

[0265] (3) Administration

[0266] 24 hours after transfection, the medium of the negative control group and the overexpression group was replaced with 10 mL of DMEM complete medium containing 10 μM DHA solution or the solution of compound A1 prepared in Example 1, respectively.

[0267] (4) Antibody preincubation

[0268] 2 μL Goat anti-mouse IgG HRP (Qisong Biotechnology, Catalog No.: QSab6555), 2 μL HA-Tag Mouse mAb (Shanghai Abimat Biopharmaceutical Co., Ltd., Catalog No.: 26D11), and 2 μL His-Tag (2A8) Antibody (Shanghai Abimat Biopharmaceutical Co., Ltd., Catalog No.: M20001) were aspirated and pre-incubated with 40 μL Beads, 400 μL IP buffer was added, and the mixture was incubated at 4°C overnight to obtain the antibody pre-incubation solution.

[0269] (5) Cell lysis

[0270] Cell lysate was prepared with a volume ratio of Western and IP cell lysis buffer (Beyotime, cat. no.: P0013): phosphatase inhibitor cocktail C (Beyotime, cat. no.: P1091) = 50:1.

[0271] After 24 hours of incubation with drug administration, the cells were washed twice with pre-cooled PBS after removing the supernatant. Cell lysis buffer was added to the culture dish at 400 μL / dish and lysed on ice for 0.5 hours.

[0272] (6) Pre-cleaning

[0273] After lysis, centrifuge at 12000g for 15 min at 4°C, collect the supernatant, add 20 μl of Beads, rotate on a drum for 1 h, centrifuge at 1000g for 1 min at 4°C, and let stand on ice for 1 min.

[0274] (7) Protein quantification

[0275] Quantification was performed using a BCA protein quantification kit (the method was the same as in Example 4). The total protein concentration was adjusted to 2 mg / mL. 40 μL was added to 8 μL of 5× dual-color SDS-PAGE protein loading buffer and placed in a 100°C metal bath and boiled for 10 min for protein denaturation. This was used as the unincubated protein sample for subsequent Western blot analysis. The remaining protein sample was used for subsequent analysis.

[0276] (8) Washing the antibody and beads mixture:

[0277] After overnight incubation, place the antibody pre-incubation solution in (4) on ice, centrifuge and remove the supernatant, add 1 ml of pre-cooled IP buffer, invert 8-10 times, centrifuge at 1000 g for 1 min at 4°C and remove the supernatant, repeat twice, and leave about 100 μL to obtain the beads mixture.

[0278] (9) Incubation

[0279] The remaining protein sample was added to the washed antibody and beads mixture and incubated on a roller at 4°C overnight.

[0280] (10) Washing

[0281] Place the mixture on ice after overnight incubation, centrifuge to remove the supernatant, add 1 ml of pre-cooled IP Buffer, invert 8-10 times, centrifuge at 1000 g for 1 min at 4°C, repeat 3 times, and analyze by Western blot.

[0282] Aspirate the supernatant with a sample-loading pipette tip and retain the beads.

[0283] (11) Add 40 μL of 5× dual-color SDS-PAGE protein loading buffer, place in a 100°C metal bath and boil for 10 min to denature the protein, and perform Western blot analysis (according to the method of Example 4) together with the unincubated protein sample.

[0284] 2. Experimental Results

[0285] When CRBN-His and CARD6-HA plasmids were co-transfected into cells, CRBN-His was used as bait protein to precipitate CARD6-HA. Only in the compound A1 group prepared in Example 1, CRBN-His and CARD6-HA were bound ( Figure 18 C).

[0286] Examples 8 and 9 show that the degradation effect of compound A1 prepared in Example 1 on CARD6 depends on the formation of a ternary complex with CRBNE3 ligase, and CRBN binds to the CARD6 protein as a substrate receptor, promoting degradation induced by the ubiquitin-proteasome system.

[0287] Example 10 DARTS Experiment Compound A1 Targets CARD6

[0288] 1. Experimental Methods

[0289] (1) THP-1 cell inoculation and transfection

[0290] According to the method of Example 9, THP-1 cells were seeded in a 10 cm dish, induced to adhere to the wall with PMA for 24 h, and then transfected with the pCMV-3×HA-CARD6(1-949aa)-Neo plasmid. After culturing for 24 h, the cells were collected.

[0291] (2) Cracking

[0292] Wash the cells 2-3 times with pre-chilled PBS, add 300 μL / dish of Western and IP cell lysis buffer (Biyuntian, Cat. No.: P0013), gently scrape the cells with a cell scraper, place them in a pre-chilled centrifuge tube, invert them several times, and place them on ice for 10 min to lyse.

[0293] (3) Protein quantification

[0294] After centrifugation at 12000g for 10 min at 4°C, the supernatant was collected and quantified using a BCA protein quantification kit (the method is the same as in Example 4), and diluted to 2 μg / μL with 1× TNC buffer (ECOTOP SCIENTIFIC, catalog number: ED-9214-500ml2);

[0295] (4) Combination

[0296] The protein samples were divided into groups, with 100 μg in each group.

[0297] Control group: protein samples were added with an equal volume of DMSO;

[0298] A1 drug group: 2.5, 5, and 10 μM drug solutions were added to the protein samples, respectively.

[0299] All groups were mixed and then combined at low temperature overnight.

[0300] (5) Enzymatic hydrolysis

[0301] Prepare 100 ng / mL protease stock solution (SIGMA, Catalog No.: P5147-1G) and dilute it 1:1000 with TNC buffer (ECOTOPSCIENTIFIC, Catalog No.: ED-9214-500ml). Add protease to the sample that has been cold-bound overnight and incubate at 37°C for 15 minutes.

[0302] (6) Termination reaction

[0303] The reaction was terminated by adding 5× Loading buffer (Ford Biotechnology, Catalog No.: FD002). The protein sample was denatured by boiling in a metal bath at 100°C for 10 min and then subjected to Western blot analysis.

[0304] 2. Experimental Results

[0305] The results are as follows Figure 19 As shown, the CARD6 protein was hydrolyzed by Pronase. After the CARD6 protein was co-treated with the compound A1 prepared in Example 1, the enzymatic hydrolysis of CARD6 by Pronase was significantly reduced, and the stability of the CARD6 protein was increased, indicating that the compound A1 prepared in Example 1 directly targeted the CARD6 protein.

[0306] Effects of Example 11 Compounds on MRL / lpr Model Mice

[0307] 1. Experimental Grouping and Sample Collection

[0308] After one week of adaptive feeding, 9-week-old female C57BL / 6 mice and MRL / lpr lupus model mice were randomly divided into four groups:

[0309] DHA group: MRL / lpr lupus model mice were given DHA by intraperitoneal injection starting from the 10th week of age, once a day at 0.2 mL / 10 g body weight;

[0310] Group A1: MRL / lpr lupus model mice were intraperitoneally injected with compound A1 prepared in Example 1 at a rate of 0.2 mL / 10 g body weight once daily starting from the 10th week of age;

[0311] MRL / lpr model group (MRL / lpr): MRL / lpr lupus model mice were injected with normal saline intraperitoneally starting from the 10th week of age, once a day at a dose of 0.2 mL / 10 g body weight;

[0312] Blank control group (control): C57BL / 6 mice were intraperitoneally injected with normal saline starting from the 10th week of age, once a day at a dose of 0.2 mL / 10 g body weight.

[0313] The mice were injected continuously for 4 weeks, and urine was collected for 24 hours every week using metabolic cages without food or water.

[0314] At the end of the fourth week, mice were fasted for 12 hours, anesthetized with 1% sodium pentobarbital solution, and blood was collected. The blood was allowed to stand at room temperature for 2-4 hours, then centrifuged at 3500 rpm and 4°C for 15 minutes. The supernatant was then extracted for analysis. The kidneys were quickly extracted, weighed, and half of the right kidney was cut longitudinally and fixed in a centrifuge tube containing 4% paraformaldehyde. The remaining kidney was washed with saline and stored frozen at -80°C until use.

[0315] 2. Urinary protein levels in MRL / lpr model mice

[0316] 1. Experimental methods

[0317] Detection of urine protein level using CBB method

[0318] 2. Experimental results

[0319] The results are as follows Figure 20 Compared with the control group, the urine protein in the MRL / lpr model group increased in the tenth week, peaked in the 11th week, and continued until the 14th week. Compared with the model group, the urine protein in the DHA and A1 groups decreased significantly, with statistically significant differences. Furthermore, the urine protein in the A1 group was significantly lower than that in the DHA group.

[0320] This indicates that both DHA and A1 can effectively reduce nephritis caused by systemic lupus erythematosus, and A1 is more effective.

[0321] Effect of Compound A1 on Anti-dsDNA IgG Antibody Levels in Serum of MRL / lpr Mice

[0322] 1. Experimental methods

[0323] The content of anti-dsDNA IgG antibodies in serum was detected and calculated according to the instructions (Jiangsu Enzyme Immunity Industrial Co., Ltd., product number: MM-45766M1).

[0324] 2. Experimental results

[0325] The results are as follows Figure 21 The results showed that compared with the control group, the anti-dsDNA IgG antibody levels in the serum of MRL / lpr model mice were significantly increased, and the difference was statistically significant (P < 0.01). After treatment with compound A1 prepared in Example 1, the anti-dsDNA IgG antibody levels in the serum of mice in group A1 were significantly decreased compared with those in the MRL / lpr model group (P < 0.01). This shows that compound A1 prepared in the example can reduce the production of anti-dsDNA IgG antibody levels in MRL / lpr model mice.

[0326] Effects of Compound A1 on Serum Creatinine and Urea Nitrogen Levels in MRL / lpr Mice

[0327] 1. Experimental methods

[0328] The serum creatinine (Scr) and urea nitrogen (BUN) levels were determined and calculated according to the manufacturer's instructions.

[0329] 2. Experimental results

[0330] The results are as follows Figure 22The results showed that compared with the Control group, the Scr and BUN levels in the serum of the MRL / lpr model group mice were significantly increased (P<0.01); compared with the model group mice, the Scr and BUN levels in the serum of the DHA group and A1 group mice were significantly decreased (P<0.01), and the Scr and BUN levels of the A1 group mice were significantly lower than those of the DHA group.

[0331] 5. Effect of Compound A1 on Serum Inflammatory Factor Levels in MRL / lpr Mice

[0332] 1. Experimental methods

[0333] The levels of inflammatory factors TNF-α (Biolegend, catalog number: 575209), IL-1β (Biolegend, catalog number: 579409), and IL-6 (Biolegend, catalog number: 575709) in serum were detected and calculated according to the instructions.

[0334] 2. Experimental results

[0335] The results are as follows Figure 23 Results showed that compared with the control group, the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the serum of MRL / lpr model mice were significantly increased (P<0.01). Compared with the MRL / lpr model group, the expression levels of TNF-α, IL-1β, and IL-6 in mice in group A1 were significantly decreased after drug intervention (P<0.01).

[0336] 6. Renal Histopathological Observation

[0337] 1. Experimental methods

[0338] The kidneys collected by dissection were cleaned with physiological saline, and after absorbing excess liquid with filter paper, the right kidney of the rat was cut into two parts longitudinally. One part was fixed in 4% paraformaldehyde, and then H&E staining was used to observe and analyze the pathological morphology of the kidney.

[0339] The specific operation of H&E staining is as follows:

[0340] (1) Part of the fixed kidney was taken and cut into 4 μm thick paraffin sections after a series of operations including dehydration, clearing, wax immersion, and embedding;

[0341] (2) Dewaxing: Soak in xylene three times for 15 min each time, then soak in anhydrous alcohol, 95% alcohol, 85% alcohol, and 70% alcohol for 5 min each, and then wash with ultrapure water for 5 min.

[0342] (3) H&E staining: add 10% hematoxylin for staining for 15 minutes; differentiate with 0.5% hydrochloric acid ethanol for 1 minute, rinse with 1% ammonia water for 1 minute, rinse with ultrapure water for 5 minutes after each reagent reaction, and finally stain the cytoplasm with 1% eosin solution;

[0343] (4) Dehydration and transparency: Soak in gradient ethanol (70%-80%-95% anhydrous ethanol)-xylene-xylene in sequence to dehydrate and transparentize, and shake on a shaker for 2 minutes;

[0344] (5) Sealing: After sealing and drying, observe and photograph the slides under a microscope.

[0345] 2. Experimental results

[0346] The results are as follows Figure 24 The results showed that in the control group, the glomerular structure was clearly defined and the renal tubules were arranged in an orderly manner. In the model group, however, the glomerular structure was blurred, mesangial matrix proliferation was observed (red arrows), the renal tubules were disordered, and a large number of inflammatory cells infiltrated the renal interstitium (black arrows). Compared with the model group, the glomerular morphology and structure of the DHA and A1 groups were restored, and mesangial proliferation and inflammatory infiltration were reduced, indicating that both A1 and DHA can alleviate renal damage in MRL / lpr mice, with compound A1 being more effective.

[0347] 6. Western blot detection of protein expression levels

[0348] 1. Experimental methods

[0349] (1) Total protein extraction from kidney tissue

[0350] 15 mg of collected kidney tissue was weighed into a 2 mL homogenization centrifuge tube, and lysis buffer (1000 RIPA: 10 phosphatase inhibitor: 1 protease inhibitor) was prepared according to the ratio. 150 μL of lysis buffer was added to each tube, and the tube was placed in an automatic homogenizer for homogenization. The tube was lysed on ice for 30 min, vortexed every 3 to 5 min, and centrifuged at 4°C and 14,000 g / min for 10 min. The supernatant was collected and the protein concentration was determined using a BCA kit.

[0351] (2) Western blot detection

[0352] SDS-polyacrylamide gel electrophoresis: Prepare a 10% SDS-polyacrylamide gel. After injecting the separating gel, quickly add anhydrous ethanol to flatten it. After solidification, pour off the anhydrous ethanol, blot dry with filter paper, and then inject the stacking gel. Insert the electrophoresis comb. Load 10 μL of protein sample per well. After setting the marker wells, connect the power supply and run the electrophoresis at 80V for 25 minutes, then switch to 120V for 80 minutes.

[0353] Transfer: Remove the separation gel and soak it in transfer buffer. Cut the target protein according to the marker and cut a 0.22μm PVDF membrane according to the size of the gel. After activation in methanol for 2 minutes, soak it in transfer buffer. Prepare a "sandwich" transfer clip with the black side of the clip at the bottom, and the sponge, wet filter paper, gel, PVDF membrane, wet filter paper, sponge, and white side as the top layer in this order. Fill the electrophoresis tank with transfer buffer, cover the transfer box and the top with ice, turn on the power, and transfer under the constant current of 300mA. Transfer for large molecules (>70kDa) for 120min and for small molecules (<70kDa) for 50min.

[0354] Protein blocking: Remove the PVDF membrane and place it in blocking solution on a shaker at room temperature for 1 hour to block nonspecific antigens. After blocking, wash with TBST three times for 5 minutes each on a shaker at room temperature.

[0355] Incubation with antibodies: Incubate with primary antibodies against CARD6 (Abcam, Catalog No. ab227189), iNOS (Abcam, Catalog No. ab178945), CD86 (Abcam, Catalog No. ab239075), TNF-α (Abcam, Catalog No. ab183218), and IL-1β (Abcam, Catalog No. ab2105) overnight at 4°C. Wash the membrane three times on a rocking platform and incubate with secondary antibodies against Goat anti-rabbit IgG (H+L) (Catalog No. FDR007) or Goat anti-mouse IgG (H+L) (Catalog No. FDM007) at room temperature for 1 hour, then wash the membrane.

[0356] Development: Turn on the automatic imaging chemiluminescence instrument, cool the instrument to -30℃, immerse the membrane in ECL luminescent solution (A / B solution), turn on white light to focus, then develop and record.

[0357] 2. Experimental results

[0358] The results are as follows Figure 25 The results showed that compared with the Control group, the expression level of CARD6 protein in the kidneys of mice in the MRL / lpr model group was increased, accompanied by the upregulation of the expression levels of M1 phenotype marker proteins iNOS, CD86, TNF-α, and IL-1β; compared with the model group, the expression levels of the above proteins in the kidneys of mice in the DHA group and A1 group were significantly decreased, indicating that both A1 and DHA inhibited the expression level of M1 phenotype marker proteins in the kidneys of MRL / lpr mice by degrading CARD6 protein, thereby exerting an anti-inflammatory effect.

Claims

1. Use of a degrader of apoptosis-enhancing domain protein 6 in the preparation of a drug for systemic lupus erythematosus.

2. Use of a degrader of apoptosis-enhancing domain protein 6 in the preparation of a drug for renal failure caused by systemic lupus erythematosus.

3. Use of a degrader of apoptosis enhancing domain protein 6 in the preparation of a drug for nephritis caused by systemic lupus erythematosus.

4. The use according to any one of claims 1 to 3, characterized in that The degradation agent is a compound having a structural formula as shown in formula (II-1), 5. A compound, characterized in that Its structural formula is shown in formula (II-1), 6. Use of the compound according to claim 5 in the preparation of medicines for systemic lupus erythematosus.

7. Use of the compound according to claim 5 in the preparation of a medicament for renal failure caused by systemic lupus erythematosus.

8. Use of the compound according to claim 5 in the preparation of a medicament for nephritis caused by systemic lupus erythematosus.

9. The method for preparing the compound according to claim 5, characterized in that: The following steps are involved: Dihydroartemisinin, a compound represented by the structural formula (I-1), DMAP and DCM were fully mixed to react to obtain a reaction solution 1. EDCI in DCM was added dropwise to the reaction solution 1, and the mixture was fully mixed and reacted until the reaction was completed, and then purified to obtain the product.

10. The preparation method according to claim 9, characterized in that The purification comprises: extracting with DCM, retaining the organic phase, and separating by column chromatography.

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