Rhein protacs, and preparation method and application thereof

Abstract

The present application relates to rhein PROTACS and a preparation method and application thereof, the present application is applied to the technical field of medicine, can effectively degrade target protein and has an antitumor effect; is applied to the technical field of new raw materials of skin whitening cosmetics, can effectively target degradation target protein and reduce the content of tyrosinase in cells, and has good in vivo and in vitro reducing melanin effect. The present application specifically takes rhein derivatives as ligands, is coupled with E3 ligase through different types and different chain lengths of linkers, so as to design and synthesize a series of rhein PROTACS.

Description

Technical Field

[0001] This invention relates to a method for designing and synthesizing a series of rhein-based PROTACs using rhein as a POI ligand, linalidomide or pomalidomide as an E3 ligand, and selecting aliphatic or PEG chains as the linker, and their applications in skin whitening and anti-tumor effects. Background Technology

[0002] PROTACs are a technology for the chemical degradation of proteins and have become an emerging direction in drug development. A PROTAC consists of three parts: a target protein ligand, an E3 ubiquitin ligase ligand, and an intermediate linker. The principle is that the PROTAC molecule can simultaneously bind to both the target protein and the E3 ubiquitin ligase, forming a ternary complex. This brings the target protein and the E3 ligase closer together, ubiquitinizing the target protein, which is then degraded by the proteasome system (UPS). Even if the drug weakly binds to the target, PROTACs can still locate the target and further analyze it using differential proteomics, amplifying the differences in protein abundance without binding to multiple protein targets. Therefore, the combination of PROTACs and differential proteomics provides a practical and technical possibility for finding antitumor targets for rhein. PROTAC molecules do not need to be highly intercalated to the highly active regions of the target; a transient adhesion effect is sufficient. Degradation of the target molecule can be achieved through weak binding interactions such as specific intermolecular forces and hydrogen bonds. Currently, the unique driving model of protein degradation mediated by PROTACs has great therapeutic potential, so this technology is mainly used in drug development, and some PROTACs have already undergone clinical trials.

[0003] Furthermore, PROTAC drugs have numerous advantages over traditional small molecule drugs, as follows:

[0004] (1) The design of PROTAC drugs is not limited by Lipinski's rule;

[0005] (2) The pharmacological effects of traditional small molecules rely on occupancy-driven occupancy of key sites on target proteins. This does not achieve the desired efficacy and requires maintaining a certain in vivo drug concentration, with relatively high requirements for the binding sites of small molecules. The pharmacological mechanism of PROTAC drugs is the degradation of target proteins to achieve pharmacodynamic effects. Data shows that very high drug concentrations are not required to achieve the desired effect, and the requirements for binding sites are relatively low.

[0006] (3) About 50% of the proteins in the human body have been found to be related to human diseases, of which only about 20% can be bound by traditional small molecules, while PROTAC can theoretically bind to any part of the protein.

[0007] (4) Compared to traditional small molecules, the drug resistance problem of PROTACs can be ignored. Theoretically, PROTACs can be well compatible with mutations in target proteins;

[0008] (5) PROTAC is more durable and has a stronger inhibitory effect.

[0009] Rhein, a component of Polygonaceae plants, is one of the main components of traditional Chinese medicines such as rhubarb, Polygonum multiflorum, and aloe vera. It possesses pharmacological activities including antibacterial, anti-inflammatory, diuretic, lipid-lowering, and anticancer effects. This invention verifies the good inhibitory effect of rhein on tyrosinase through experiments such as tyrosinase inhibition, and also demonstrates its good skin-whitening effects and value through mechanism research. Furthermore, rhein can inhibit tumor cell proliferation, invasion, and metastasis by regulating mitogen-activated protein kinase, phosphatidylinositol 3-kinase, nuclear transcription molecules, and multiple signaling pathways, and promote cell apoptosis. It can also exert anti-tumor effects by combining with radiotherapy and chemotherapy drugs to reduce toxicity and enhance efficacy. Although it has been confirmed that rhein can effectively inhibit various cancers such as liver cancer, breast cancer, and lung cancer, its specific anti-tumor target proteins have not yet been fully elucidated. Summary of the Invention

[0010] Based on the above research background, the first aspect of the present invention is to synthesize a series of rhein PROTACs and a method for preparing the rhein PROTACs.

[0011] A second aspect of the invention relates to providing rhein PROTACs and their application in antitumor activity. The tumors include at least one of liver cancer, breast cancer, lung cancer, tongue cancer, stomach cancer, colorectal cancer, or nervous system cancer.

[0012] A third aspect of the invention relates to providing rhein PROTACs and their whitening effects or applications in medicaments for treating skin diseases. These skin diseases include hyperpigmented skin conditions such as freckles, melasma, nevus of Ota, tar melanosis, acral hyperpigmentation, melanoma, café-au-lait spots, etc.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] The first objective of this invention is discussed below:

[0015] First, a PROTAC compound of rhein represented by the general formula or a pharmacologically or physiologically acceptable salt thereof is provided to synthesize rhein PROTACs with whitening and antitumor activities.

[0016]

[0017] lenalidomide or pomalidomide For the E3 ligand, the Linker is selected from at least one of an adipose chain or a PEG chain, wherein the adipose chain is -(CH2)n-, where n represents a natural number from 3 to 8; and the PEG chain is -(CH2CH2O)n-CH2CH2-, where n represents 2 or 3.

[0018] Preferably, the rhein PROTACs provided by the present invention include, but are not limited to, the compounds shown in Table 1 below:

[0019] Table 1

[0020]

[0021]

[0022]

[0023]

[0024]

[0025] The second objective of this invention is to provide a method for preparing rhein PROTACs.

[0026] A method for preparing general formula rhein PROTACs includes the following steps:

[0027] (1) The rhein PROTACs shown in the above general formula are divided into Series I rhein PROTACs and Series II rhein PROTACs.

[0028] (2) Among them, Series I rhein PROTACs are compounds represented by general formula I, the Linker is selected as an aliphatic chain, and the E3 ligase is lenalidomide or fluorothalidomide.

[0029] General Formula I:

[0030] (3) Series II rhein PROTACs are compounds represented by general formula II. The linker is selected as PEG2 (diethylene glycol) or PEG3 (triethylene glycol), and the E3 ligase is pomalidomide.

[0031] Formula II:

[0032] First, the applicant discovered that rhein itself has a good inhibitory effect on tyrosinase. Therefore, the invention uses rhein as a target protein ligand (POI ligand). Lenalidomide and pomalidomide, which have good water solubility and low molecular weight, were selected as initial E3 ligand small molecules. To facilitate the linker's connection with the E3 ligand, E3 ligands with a certain length of BOC group linking chain were selected to react with lenalidomide.

[0033] The synthesis reaction formula is as follows:

[0034] E3 ligand:

[0035]

[0036] After the E3 ligand is linked to a linker chain of a certain length, it undergoes an amidation condensation reaction with the POI ligand rhein, thereby linking the POI ligand with the E3 ligase ligand to obtain a series of I rhein PROTACS compounds.

[0037] Series I rhein PROTACS compounds are derived from a synthesized E3 ligand intermediate. Rhein was added to DMF (N,N-dimethylformamide) solvent as a raw material, and stirred for a few minutes. Then, DIPEA (N,N-diisopropylethylamine) and HATU (peptide condensation reagent) were added sequentially, and stirred for about 20 minutes. TIC detection (developing solvent: CDCl3:MeOH = 20:1) was performed until the active ester was formed, and then the intermediate was added. The reaction was carried out at room temperature for 12 hours, and TIC detection (developing solvent: dichloromethane:methanol = 20:1) showed that the reaction was complete. The reaction solution was quenched with crushed ice, and extracted thoroughly with DCM (dichloromethane). The organic layer was washed sequentially with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a yellow liquid. This liquid was purified by PTLC to obtain a series of I rhein PROTACs.

[0038] The synthesis equation is as follows:

[0039]

[0040] Series II Synthetic routes for rhein PROTACs

[0041] Using PEG2 and PEG3, D (rhein) and lenalidomide as raw materials, DMF solvent was added and stirred for a few minutes. Then, DIPEA (N,N-diisopropylethylamine) and HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) were added sequentially. The reaction was carried out at room temperature for 24 hours to obtain the intermediate:

[0042] Then, under ice bath conditions, the intermediate was dissolved in DCM (dichloromethane), with anisole added as a protective agent. After stirring thoroughly, TFA (trifluoroacetic acid) was added, and the mixture was allowed to react briefly. The ice bath was then removed, and the reaction was allowed to proceed to room temperature. The reaction was monitored by TLC, and it was completed in approximately 12 hours. After the reaction was stopped, the solution was diluted with water, extracted three times with dichloromethane, and the organic layers were combined. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the intermediate.

[0043] Without purification, it can be used directly in the next reaction. Lenalidomide is added, followed by DMF (N,N-dimethylformamide) solvent. After stirring for a few minutes, DIPEA and HATU are added in sequence. The reaction is carried out at room temperature for 24 hours to obtain a series of II rhein PROTACs.

[0044] The synthesis reaction formula is as follows:

[0045]

[0046] The third objective of this invention is to provide a whitening application of the rhein PROTACs described herein.

[0047] Unless otherwise specified, the equipment, reagents, processes, parameters, etc. involved in this invention are all conventional equipment, reagents, processes, parameters, etc., and no further examples will be provided.

[0048] All ranges listed in this invention include all point values ​​within that range.

[0049] In this invention, "approximately", "about", or "around" refers to the range or value within ±20%.

[0050] In this invention, unless otherwise specified, % refers to mass percentage and ratio refers to mass ratio.

[0051] In this invention, "room temperature" refers to the normal ambient temperature, which can be 10 to 30°C.

[0052] Compared with the prior art, this technical solution has the following advantages:

[0053] This invention uses rhein as the POI ligand, linalidomide and pomalidomide as E3 ligands, and selects PEG and aliphatic chains as linkers to design and synthesize a series of rhein-based PROTACs. It was found that the rhein-based PROTACs series compounds exhibit good antitumor activity and have potential application value in the preparation of antitumor drugs. Simultaneously, the rhein-based PROTACs series compounds also have certain inhibitory tyrosinase activity superior to that of rhein, showing promising application prospects in the preparation of skin whitening agents. Attached Figure Description

[0054] Figure 1 Test for inhibition of tyrosinase activity.

[0055] Figure 2 Cytotoxicity test for B16-F10 cells.

[0056] Figure 3 Haccat cytotoxicity test.

[0057] Figure 4 Effects of D and D6 on TYR, MITF, TRP1, and DCT proteins

[0058] Figure 5 right Figure 4 Grayscale analysis of WB images of TYR protein

[0059] Figure 6 right Figure 4 Gray-scale analysis of WB images of MITF protein

[0060] Figure 7 right Figure 5 Grayscale analysis of TRP1 protein WB image

[0061] Figure 8 right Figure 5 Grayscale analysis of DCT protein WB images

[0062] Figure 9 Effects of ND6 on MITF and TYR proteins

[0063] Figure 10 right Figure 9 Gray-scale analysis

[0064] Figure 11 Effects of D and D6 on melanin production in zebrafish

[0065] Figure 12 Zebrafish melanin grayscale analysis Detailed Implementation

[0066] Example 1: Preparation of intermediates s1-s6 (Preparation of E3 ligand)

[0067] Boc-3-aminopropionic acid (1.5 g, 7.8 mmol) and DIPEA (1.23 mmol) were dissolved sequentially in 1 mL of dry DMF solution. The mixture was stirred at room temperature for five minutes, and HATU (0.495 mmol) was added. After monitoring the reaction by TLC for about half an hour, lenalidomide (1314, 5.07 mmol) was added, and the mixture was stirred at room temperature. The reaction was monitored by TLC and was completed in about 12 hours. The reaction was quenched with crushed ice, extracted three times with ethyl acetate, and the organic layers were combined. The mixture was washed successively with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then subjected to PTLC (dichloromethane:methanol = 20:1, V:V) to obtain intermediate S1 (its structural formula is shown in the figure below) (2.00 g, yield 92%).

[0068]

[0069] intermediate s1 / s2 / s3 / s4 / s5 / s6 structure

[0070] n=3s1; n=4s2; n=5s3; n=6s4; n=7s5; n=8s6

[0071] Boc-4-aminobutyric acid (1.5 g, 7.35 mmol) and DIPEA (2.5 g, 14.7 mmol) were dissolved sequentially in 1 mL of dry DMF solution. The mixture was stirred at room temperature for five minutes, and HATU (0.495 mmol) was added. After monitoring the reaction by TLC for about half an hour, lenalidomide (1239 mg, 4.8 mmol) was added, and the mixture was stirred at room temperature. The reaction was monitored by TLC and was completed in about 12 hours. The reaction was quenched with crushed ice, extracted three times with ethyl acetate, and the organic layers were combined. The mixture was washed successively with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then subjected to PTLC (dichloromethane:methanol = 20:1) to give intermediate S2 (1.92 g, 90% yield).

[0072] Boc-5-aminovaleric acid (1.5 g, 6.9 mmol) and DIPEA (2.4 mL, 13.8 mmol) were dissolved sequentially in 1 mL of dry DMF solution. The mixture was stirred at room temperature for five minutes, and HATU (2.1 g, 5.52 mmol) was added. After monitoring the reaction by TLC for about half an hour, lenalidomide (1163 mg, 4.49 mmol) was added, and the mixture was stirred at room temperature. The reaction was monitored by TLC and was completed in about 12 hours. The reaction was quenched with crushed ice, extracted three times with ethyl acetate, and the organic layers were combined. The mixture was washed successively with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then subjected to PTLC (dichloromethane:methanol = 20:1, V:V) to obtain intermediate S3 (1.9 g, 92%).

[0073] The synthesis methods of intermediates s4-s6 are similar to those of s123.

[0074] Intermediate S1 (1.5 g, 3.38 mmol) was dissolved in a mixed solution of DCM / MeOH (V:V = 3:1) (10 mL). TFA (3 mL) was added dropwise under ice bath conditions. After the addition was complete, the mixture was slowly moved to room temperature. The reaction was monitored by TLC and completed in approximately 12 hours. After the reaction was stopped, the solvent was removed by concentration under reduced pressure to obtain intermediate S1-2 (white solid, 1050 mg, 90%), which could be used directly in the next reaction without further purification.

[0075]

[0076] The structure of the intermediate s1-2 / s2-2 / s3-2 / s4-2 / s5-2 / s6-2 is shown in the above structural formula.

[0077] n=3s1-2; n=4s2-2; n=5s3-2; n=6s4-2; n=7s5-2; n=8s6-2

[0078] Intermediate S2 (1.5 g, 3.38 mmol) was dissolved in a mixed solution (10 mL) of DCM / MeOH (dichloromethane / methanol, V:V = 3:1). TFA (trifluoroacetic acid) (3 mL) was added dropwise under ice bath conditions. After the addition was complete, the mixture was slowly moved to room temperature. The reaction was monitored by TLC and completed in approximately 12 hours. After the reaction was stopped, the solvent was removed by concentration under reduced pressure to obtain intermediate S2-2 (white solid, 1050 mg, 90%), which could be used directly in the next reaction without further purification.

[0079] Intermediate S3 (1.5 g, 3.38 mmol) was dissolved in a mixed solution of DCM / MeOH (V:V = 3:1) (10 mL). TFA (3 mL) was added dropwise under ice bath conditions. After the addition was complete, the mixture was slowly moved to room temperature. The reaction was monitored by TLC and completed in approximately 12 hours. After the reaction was stopped, the solvent was removed by concentration under reduced pressure to obtain intermediate S3-2 (white solid, 1050 mg, 90%), which could be used directly in the next reaction without further purification.

[0080] Intermediates s4-2, 5-2, and 6-2 are similar to s1-2.

[0081] Intermediate s2-2: Yield 92%

[0082] Intermediate s3-2: Yield 94%

[0083] Intermediate s4-2: 95% yield

[0084] Intermediate s5-2: Yield 94%

[0085] Intermediate s6-2: Yield 96%

[0086] Example 2 Preparation of final products 3-8

[0087] 40 mg (1 eq) of rhein was placed in a 50 mL round-bottom flask, and EDCl (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) (2 eq), HOBt (p-hydroxybenzonitrile, 2 eq), s1-2-aminopropionic acid-lenalidomide (1.2 eq), triethylamine (4 eq) was added. Finally, 20 mL of dichloromethane was added as the reaction solvent, and the mixture was stirred to dissolve. The amidation reaction was carried out at room temperature for 4 h. A yellow precipitate was formed after the reaction was completed. The amidation reaction solution was extracted with DCM, washed with saturated brine, and purified by PTLC (PE:PA = 20:1) to obtain final product 3 (dark brown solid, yield 38%).

[0088] 40 mg (1 eq) of rhein was placed in a 50 mL round-bottom flask, and EDCl (2 eq), HOBt (2 eq), S2-2-aminobutyric acid-lenalidomide (1.2 eq), and triethylamine (4 eq) were added. Finally, 20 mL of dichloromethane was added as the reaction solvent, and the mixture was stirred to dissolve. The amidation reaction was carried out at room temperature for 4 h. A yellow precipitate was formed after the reaction was completed. The amidation reaction solution was extracted with DCM, washed with saturated brine, and purified by PTLC (PE:PA = 20:1) to obtain the final product 4 (dark brown solid, yield 35%).

[0089] The preparation of final products 5-8 is similar to that of final products 3-4.

[0090] Final product 3: (dark brown solid, yield 38%) 1 H NMR (500MHz, DMSO-d6) δ10.03(s,1H),9.00(s,1H),8.11(s,1H),7.15(s,1H),6.94(d,J=37.8Hz,1H),6.89–6.82(m,1H),6.77(s, 1H), 6.54 (s, 1H), 6.41 (s, 1H), 4.13 (d, J = 13.1Hz, 1H), 3.37 (s, 2H), 1.91 (t, J = 14.8Hz, 2H), 1.74 (d, J = 11.2Hz, 2H), 1.57 (s, 2H). 13C NMR(125MHz,DMSO-d6)δ189.88,182.59,172.51,172.48,172.47,172.44,170.84,169.76 ,167.57,167.55,167.53,162.33,162.00,161.98,138.03,138.00,137.11,137.10,137. 05,137.04,136.07,136.05,134.12,134.09,133.61,133.55,133.54,132.72,132.71,125.51,125.49,125.47,125.45,125.43,125.41,124.30,124.28,124.25,124.23,123.37, 123.32,122.29,122.27,122.23,122.21,120.17,120.14,120.12,120.10,120.07,120.05,120.04,120.04,120.03,118.14,118.13,118.11,118.09,118.07,118.06,117.72,117 .68,117.63,116.07,55.40,55.38,55.35,47.29,47.26,47.23,37.44,37.42,37.18,37.16,31.43,31.41,31.40,31.39,31.37,31.36,31.35,26.54,26.52,26.51,26.49,26.47.

[0091] Final product 4: (dark brown solid, yield 35%) 1 H NMR(500MHz,DMSO-d6)δ11.91(s,2H),11.02(s,1H),9.98(s,1H),9.04(s,1H),8.13(s,1H),7 .84(d,J=4.4Hz,1H),7.82(d,J=5.3Hz,1H),7.81(s,1H),7.73(d,J=7.0Hz,1H),7.46(s,1H), 7.45(s,1H),7.43–7.37(m,1H),5.13(d,J=13.3,5.1Hz,1H),4.49–4.15(m,2H),3.03–2.67(m ,2H),2.61(d,J=18.5Hz,2H),2.46(d,J=7.2Hz,2H),2.38–1.98(m,2H),1.90(p,J=7.3Hz,2H). 13C NMR (125MHz, DMSO-d6) δ191.43,182.57,172.49,172.09,169.63,167.63,167.55,162.36,162.11,138.05,137.43,136.94,133.59,132.85, 132.61,131.92,126.20,124.06,122.94,120.68,120.48,120.10,118 .09,117.29,114.76,56.30,46.73,39.32,34.24,30.81,25.99,24.41.

[0092] Final product 5: (dark brown solid, yield 38%) 1 H NMR (500MHz, DMSO-d6) δ11.90(s,2H),11.02(s,1H),9.98(s,1H),8.99(t,J=5.6Hz,1H),8.09(d,J=7.7Hz,1H) ,7.94(s,1H),7.83(dd,J=6.7,2.1Hz,1H),7.78(d,J=4.8Hz,1H),7.73(d,J=10.1Hz,1H),7.69(d,J=8.4Hz,1H ),7.49–7.45(m,1H),7.39(d,J=8.2Hz,1H),5.13(dd,J=13.3,5.1Hz,1H),4.57–4.20(m,2H),2.69(d,J=12.0H z,2H),2.43(d,J=7.3Hz,2H),2.22–1.91(m,2H),1.67(q,J=7.3Hz,2H),1.64–1.52(m,2H),1.35–1.18(m,2H). 13 C NMR(126MHz,DMSO-d6)δ181.17,181.02,172.93,171.40,171.11,167.92,163.92,162.41,141.83,137.60,133.88,133.78,133.57,133.35,133 .21,132.69,128.66,125.32,124.68,119.43,119.01,117.50,116.18, 116.11,54.14,51.61,46.68,35.87,35.47,31.24,30.84,28.51,22.71.

[0093] Final product 6: (dark brown solid, yield 36%) 1H NMR(500MHz,DMSO-d6)δ11.86(s,2H),11.03(s,1H),9.76(s,1H),8.90(s,1H),8.09(s ,1H),7.86–7.78(m,2H),7.72(s,2H),7.46(s,2H),5.13(dd,J=13.3,5.1Hz,1H),4.35 (d,J=8.9Hz,2H),3.92(s,1H),2.96–2.88(m,1H),2.62(s,1H),2.38(t,J=7.5Hz,2H), 2.03(t,J=8.7Hz,1H),1.75–1.62(m,2H),1.59(t,J=7.4Hz,2H),1.38(t,J=7.9Hz,2H). 13 C NMR (126MHz, DMSO-d6) δ191.52,181.11,172.90,171.36,171.11,167.85,163.84,161.40,161.17,141.85,137.61,133.81,133.54,133.32, 132.64,128.62,125.16,124.55,122.37,119.49,118.93,117.59,117 .46,116.07,51.54,46.47,35.74,31.23,28.65,26.11,24.85,22.65.

[0094] Final product 7: (dark brown solid, yield 34%) 1H NMR(500MHz,DMSO-d6)δ11.92(d,J=5.6Hz,2H),11.02(s,1H),9.94(s,1H),8.96(t,J=5.9Hz,1H),8.15–8.09(m,1H),7.84(d,J=7.8Hz,1H),7.81(dd,J=6.8,2.0Hz,1H),7.80–7.78(m,1H),7.74(d,J=7.4Hz,1H),7.47(d,J=1.7Hz,1H),7.45(d,J=7.6Hz,1H),7.42(d,J=8.4Hz,1H),5.13(dd,J=13.3,5.1Hz,1H),4.47–4.28(m,2H),3.28(t,J=6.8Hz,2H),2.59(d,J=17.8Hz,2H),2.41–2.36(m,2H),2.08–1.89(m,1H),1.62(t,J=7.1Hz,2H),1.56(s,2H),1.36(d,J=3.7Hz,2H),1.35–1.26(m,2H). 13C NMR(125MHz,DMSO-d6)δ189.81,182.83,172.51,172.48,172.47,172.44,172.37,169.85,167.60, 167.58,167.57,167.55,167.53,162.34,161.97,161.95,137.95,137.92,137.11,137.10,137.05 ,137.04,135.60,135.59,135.02,134.99,133.60,133.54,133.21,130.75,130.72,125.48,125.42,124.53,124.52,124.51,124.48,124.46,123.37,123.32,123.30,122.49,122.48,122.43,122.4 2,120.17,120.14,120.12,120.10,120.07,120.05,120.04,119.99,117.04,117.02,117.00,116.96,116.95,115.91,55.25,55.23,55.20,47.34,47.31,47.28,39.66,39.64,39.63,39.61,36.98,3 6.95,36.92,31.47,31.45,31.44,31.42,31.40,31.38,28.71,28.69,28.67,28.65,28.44,28.42,28.40,28.38,28.37,26.92,26.90,26.88,26.86,26.85,26.83,25.47,25.44,25.42,25.41,25.38.

[0095] Final product 8: (dark brown solid, yield 35%) 1H NMR(500MHz,DMSO-d6)δ11.92(d,J=5.8Hz,2H),11.03(s,1H),9.92(s,1H),8.95(d,J=5.8Hz,1H),8.13(d,J=6.8Hz,1H),7.84(d,J=7.7Hz,1H),7.81(d,J=6.3Hz,1H),7.79(s,1H),7.75(s,1H),7.73(s,1H),7.47(s,1H),7.45(d,J=7.4Hz,1H),7.42(d,J=8.3Hz,1H),5.13(dd,J=13.3,5.1Hz,1H),4.47–4.28(m,2H),3.28(q,J=6.6Hz,2H),2.90(d,J=11.1Hz,2H),2.67–2.56(m,2H),2.37(dd,J=9.4,5.8Hz,2H),2.01(dd,J=9.3,3.8Hz,2H),1.60(t,J=7.2Hz,2H),1.54(d,J=6.9Hz,2H),1.34(s,2H),1.31(s,2H). 13C NMR(125MHz,DMSO-d6)δ189.81,182.83,182.79,172.61,172.58,172.57,172.54,172.37,169.80,167.55,1 67.53,167.51,162.34,161.97,161.95,137.95,137.92,137.11,137.10,137.05,137.04,135.60,135.59,13 5.02, 134.99, 133.55, 133.50, 133.21, 130.75, 130.72, 125.50, 125.49, 125.47, 125.43, 124.53, 124.52, 124.51, 124.48, 124.46, 123.33, 123.31, 123.27, 122.49, 122.48, 122.43, 122.42, 120.14, 120.12, 120.10, 120 .07,120.01,119.96,119.91,117.59,117.53,117.04,117.00,116.95,115.91,55.40,55.38,55.36,47.34,47.31,47.28,39.69,39.68,39.66,39.64,36.98,36.95,36.92,31.47,31.45,31.44,31.42,31.41,31.40,31 .38,28.72,28.71,28.69,28.67,28.65,28.31,28.30,28.28,28.26,28.25,27.68,27.66,27.64,27.63,27.61,26.90,26.88,26.86,26.85,26.83,25.47,25.44,25.42,25.41,25.38,24.94,24.93,24.91,24.89,24.87.

[0096] Example 3 Intermediate O1--O3

[0097] Preparation of intermediate O1

[0098] Fluorothalidomide (2 g, 7.241 mmol, 1 eq) was dissolved in 20 mL of DMF with stirring. Then, DIEA (1.872 mL, 14.481 mmol, 2 eq) and tert-butyl [2-(2-aminoethoxy)ethyl]carbamate (2 mL, 9.780 mmol, 1.2 eq) were added, and the mixture was heated to 90 °C and refluxed for 12 h. After the reaction was completed, the mixture was quenched with ice water, extracted with ethyl acetate, and the combined organic phases were washed successively with saturated NaHCO3 and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to obtain O1 (structure shown in the figure) (yellow transparent oil, 1.2451 g).

[0099]

[0100]

[0101] Fluorothalidomide (2 g, 7.241 mmol, 1 eq) was dissolved in 20 mL of DMF with stirring. Then, DIEA (1.872 mL, 14.481 mmol, 2 eq) and tert-butyl 2-(2-(2-aminoethoxy))ethoxy]ethyl]carbamate (2 mL, 8.689 mmol, 1.2 eq) were added, and the mixture was heated to 90 °C and refluxed for 12 h. After the reaction was completed, the mixture was quenched with ice water, extracted with ethyl acetate, and the combined organic phases were washed successively with saturated NaHCO3 and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to obtain O2 (structure shown in the figure) (yellow transparent oil, 1.3533 g).

[0102] Fluorothalidomide (2 g, 7.241 mmol, 1 eq) was dissolved in 20 mL of DMF with stirring. Then, DIEA (1.872 mL, 14.481 mmol, 2 eq) and (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate tert-butyl ester (2 mL, 8.689 mmol, 1.2 eq) were added, and the mixture was heated to 90 °C and refluxed for 12 h. After the reaction was completed, the mixture was quenched with ice water, extracted with ethyl acetate, and the combined organic phases were washed successively with saturated NaHCO3 and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to obtain O3 (structure shown in the figure) (yellow transparent oil, 1.479 g).

[0103] Preparation of final product 9-12

[0104] Weigh out rhein (60 mg, 0.156 mmol) and DIPEA (64.80 mg, 0.468 mmol) and dissolve them sequentially in 1 mL of dry DMF solution. Stir at room temperature for five minutes, add HATU (88.98 mg, 0.234 mmol), and monitor the reaction by TLC for about half an hour. Then add intermediate O2 (80.89 mg, 0.312 mmol) and stir again at room temperature. Monitor the reaction by TLC, and the reaction is complete in about 12 hours. Quench the reaction with crushed ice, extract three times with ethyl acetate, combine the organic layers, wash successively with saturated ammonium chloride solution and saturated brine, dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure, and then perform PTLC (dichloromethane:methanol = 15:1) to obtain final product 9.

[0105] Final product 9 (dark brown solid, yield 35%) 1 H NMR(500MHz,DMSO-d6)δ11.10(s,1H),8.90(s,1H),8.13(s,1H),7.83(d,J=8.5Hz,1H),7.76 (d,J=7.9Hz,1H),7.56(t,J=8.0Hz,1H),7.42(d,J=8.0Hz,1H),7.09(d,J=8.7Hz,1H),7.00(d ,J=7.0Hz,1H),6.54(s,1H),5.04(dd,J=13.0,5.4Hz,1H),2.87(d,J=18.2Hz,2H),2.73(s,1H ),2.65–2.52(m,2H),2.36(s,2H),2.02(s,2H),1.59(s,2H),1.38(s,2H),1.27–0.91(m,2H). 13C NMR(125MHz,DMSO-d6)δ189.81,182.83,172.51,172.47,172.44,170.00,167.57,167.55,166 .19,166.14,164.31,162.34,161.97,161.95,144.22,144.19,137.11,137.10,137.05,137.0 4, 136.27, 136.25, 133.55, 133.50, 133.21, 131.56, 131.52, 131.50, 130.90, 130.84, 124.53, 124.52, 124.51, 124.48, 124.46, 123.33, 123.31, 123.28, 123.26, 120.83, 120.82, 120.78, 12 0.77, 120.50, 120.48, 120.45, 120.43, 120.17, 120.14, 120.12, 120.10, 120.07, 120.01, 119.96, 119.91, 117.04, 117.00, 116.95, 115.91, 113.57, 113.56, 69.66, 69.65, 69.63, 69.62, 69. 38,69.36,69.35,69.33,69.32,69.30,69.28,52.46,52.44,52.41,43.87,43.84,43.81,41.41,41.38,41.35,31.21,31.19,31.17,31.15,31.13,26.60,26.58,26.57,26.55,26.54,26.52.

[0106] Weigh out rhein (60 mg, 0.156 mmol) and DIPEA (64.80 mg, 0.468 mmol) and dissolve them sequentially in 1 mL of dry DMF solution. Stir at room temperature for five minutes, add HATU (88.98 mg, 0.234 mmol), and monitor the reaction by TLC for about half an hour. Then add intermediate O1 (79.74 mg, 0.312 mmol), and stir again at room temperature. Monitor the reaction by TLC, and the reaction is complete in about 12 hours. Quench the reaction with crushed ice, extract three times with ethyl acetate, combine the organic layers, wash successively with saturated ammonium chloride solution and saturated brine, dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure, and then perform PTLC (dichloromethane:methanol = 15:1) to obtain the final product 10.

[0107] Weigh out rhein (60 mg, 0.156 mmol) and DIPEA (64.80 mg, 0.468 mmol) and dissolve them sequentially in 1 mL of dry DMF solution. Stir at room temperature for five minutes, add HATU (88.98 mg, 0.234 mmol), and monitor the reaction by TLC for about half an hour. Then add intermediate O3 (82.19 mg, 0.312 mmol) and stir again at room temperature. Monitor the reaction by TLC, and the reaction is complete in about 12 hours. Quench the reaction with crushed ice, extract three times with ethyl acetate, combine the organic layers, wash successively with saturated ammonium chloride solution and saturated brine, dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure, and then perform PTLC (dichloromethane:methanol = 15:1) to obtain final product 11.

[0108] Final product 10 (dark brown solid, yield 39%) 1 H NMR(500MHz,DMSO-d6)δ11.08(s,1H),8.09(s,1H),7.82(s,1H),7.74(s,2H),7.5 2(t,J=8.0Hz,1H),7.40(d,J=8.2Hz,1H),7.12(d,J=8.6Hz,1H),6.95(d,J=7.0Hz ,1H),6.61(t,J=5.9Hz,1H),5.04–4.99(m,1H),4.12(s,4H),3.91(d,J=5.5Hz,1H ),3.63(d,J=5.7Hz,2H),2.90–2.77(m,2H),2.04–2.00(m,1H),1.34–1.18(m,2H). 13 C NMR (125MHz, Common NMR Solvents)δ191.43,182.57,172.08,169.62,167.65,166.70,166.53,162.36,162.11,144.65,137.40,136.94,133.59,132.61,132.3 8,132.22,124.06,120.69,120.68,120.49,120.48,120.10,118.09,114.76,114.15,69.75,69.45,51.34,43.54,40.61,29.89,24.67.

[0109] Final product 11 (dark brown solid, yield 39%) 1H NMR(500MHz,Chloroform-d)δ11.94(s,1H),11.92(s,1H),8.58(s,1H),8.17–8.11(m,1H),7.78(d,J=7.5Hz,1H),7.75(s,1H),7.67(t,J=7.9Hz,1H),7.48(d,J=5.2Hz,1H),7.36(t,J=7.8Hz,1H),7.28(d,J=8.5Hz,1H),6.97(d,J=7.0Hz,1H),6.76(d,J=8.5Hz,1H),6.38(t,J=5.6Hz,1H),4.95–4.82(m,1H),3.71(d,J=3.7Hz,2H),3.68(s,2H),3.66(d,J=4.8Hz,2H),3.65–3.63(m,1H),3.35(q,J=5.4Hz,2H),3.03–2.84(m,2H),2.81–2.70(m,2H),2.22–2.12(m,1H),1.96(s,2H),1.24(s,2H). 13C NMR(125MHz,DMSO-d6)δ189.85,182.83,172.48,170.08,170.05,169.99,169.96,167.48,166 .18,166.16,164.39,164.37,162.38,162.36,162.32,162.30,161.94,144.35,144.29,137.11 ,137.10,137.05,137.04,136.06,136.05,133.51,133.20,131.62,131.60,131.59,131.56,131.54,131.53,130.87,124.55,124.52,124.50,124.44,123.32,123.27,121.03,121.01,120.9 7,120.95,120.51,120.49,120.46,120.45,120.26,120.24,120.23,120.19,120.17,120.00,119.97,119.95,119.92,117.19,115.91,115.90,113.56,70.59,70.58,70.57,69.67,69.65,69 .64,69.62,69.61,69.36,69.34,69.33,69.30,69.28,69.25,69.24,69.22,52.48,52.39,52.37,43.85,43.83,41.34,41.32,31.24,31.22,31.20,26.60,26.58,26.57,26.55,26.54,26.52.

[0110] Example 5 Preparation of intermediate w2-8

[0111] Fluthalidomide (150 mg, 0.543 mmol, 1 eq) was dissolved in DMF, and then DIEA (N,N,diisopropylethylamine, 180 μl, 1.086 mmol, 2 eq) and N-tert-butoxycarbonyl-1,2-ethylenediamine (116 μl, 0.6516 mmol, 1.5 eq) were added sequentially. The mixture was heated to 90 °C and refluxed with stirring. The reaction was monitored by TLC. After 12 h, the reaction was quenched with ice water, and the mixture was filtered to obtain a dark green solid. The solid was washed with pure water to obtain a green solid W-2. The filtrate was extracted with ethyl acetate, and the combined organic phases were washed successively with 5% lemon juice, saturated NaHCO3, and saturated brine. The mixture was dried over anhydrous sodium sulfate and concentrated to obtain a green solid w2 (structural formula shown in the figure).

[0112]

[0113] w2 / w3 / w4 / w5 / w6 / w8 structural

[0114] n=1w2; n=2w3; n=3w4; n=4w5; n=5w6; n=7w8

[0115] Fluthalidomide (150 mg, 0.543 mmol, 1 eq) was dissolved in DMF, and then DIEA (180 μl, 1.086 mmol, 2 eq) and N-Boc-1,3-propanediamine (N-tert-butyloxycarbonyl-1,3-propanediamine) (116 μl, 0.6516 mmol, 1.5 eq) were added sequentially. The mixture was heated to 90 °C and refluxed with stirring. The reaction was monitored by TLC. After 12 h, the reaction was quenched with ice water, and the mixture was filtered to obtain a dark green solid. The solid was washed with pure water to obtain a green solid W-3. The filtrate was extracted with ethyl acetate, and the combined organic phases were washed successively with 5% lemon juice, saturated NaHCO3, and saturated brine. The mixture was dried over anhydrous sodium sulfate and concentrated to obtain a green solid w3 (structural formula shown above).

[0116] Fluthalidomide (150 mg, 0.543 mmol, 1 eq) was dissolved in DMF, and then DIEA (180 μl, 1.086 mmol, 2 eq) and N-tert-butoxycarbonyl-1,4-butanediamine (116 μl, 0.6516 mmol, 1.5 eq) were added sequentially. The mixture was heated to 90 °C and refluxed with stirring. The reaction was monitored by TLC. After 12 h, the reaction was quenched with ice water, and the mixture was filtered to obtain a dark green solid. The solid was washed with pure water to obtain a green solid W-4. The filtrate was extracted with ethyl acetate, and the combined organic phases were washed successively with 5% lemon juice, saturated NaHCO3, and saturated brine. The mixture was dried over anhydrous sodium sulfate and concentrated to obtain a green solid w4 (structural formula shown above).

[0117] Fluthalidomide (150 mg, 0.543 mmol, 1 eq) was dissolved in DMF, and then DIEA (180 μl, 1.086 mmol, 2 eq) and N-(5-aminopentyl)carbamate tert-butyl ester (116 μl, 0.6516 mmol, 1.5 eq) were added successively. The mixture was heated to 90 °C and refluxed with stirring. The reaction was monitored by TLC. After 12 h, the reaction was quenched with ice water, and the mixture was filtered to obtain a dark green solid. The solid was washed with pure water to obtain a green solid W-5. The filtrate was extracted with ethyl acetate, and the combined organic phases were washed successively with 5% lemon juice, saturated NaHCO3, and saturated brine. The mixture was dried over anhydrous sodium sulfate and concentrated to obtain a green solid w5 (structural formula shown above).

[0118] Fluthalidomide (150 mg, 0.543 mmol, 1 eq) was dissolved in DMF, and then DIEA (180 μl, 1.086 mmol, 2 eq) and N-Boc-16-hexanediamine (N-(6-aminohexyl)carbamate tert-butyl ester) (116 μl, 0.6516 mmol, 1.5 eq) were added sequentially. The mixture was heated to 90 °C and refluxed with stirring. The reaction was monitored by TLC. After 12 h, the reaction was quenched with ice water, and the mixture was filtered to obtain a dark green solid. The solid was washed with pure water to obtain a green solid W-6. The filtrate was extracted with ethyl acetate, and the combined organic phases were washed successively with 5% lemon juice, saturated NaHCO3, and saturated brine. The mixture was dried over anhydrous sodium sulfate and concentrated to obtain a green solid w6 (structural formula shown above).

[0119] Fluorothalidomide (150 mg, 0.543 mmol, 1 eq) was dissolved in DMF, and then DIEA (180 μl, 1.086 mmol, 2 eq) and 1-tert-butoxycarbonyl-1,8-diaminooctane (116 μl, 0.6516 mmol, 1.5 eq) were added sequentially. The mixture was heated to 90 °C and refluxed with stirring. The reaction was monitored by TLC. After 12 h, the reaction was quenched with ice water, and the mixture was filtered to obtain a dark green solid. Washing with pure water yielded a green solid W-6. The filtrate was extracted with ethyl acetate, and the combined organic phases were washed successively with 5% lemon juice, saturated NaHCO3, and saturated brine. The mixture was dried over anhydrous sodium sulfate and concentrated to obtain a green solid w8 (structural formula shown above).

[0120] Intermediate w3: (green solid, yield 64%)

[0121] Intermediate w4: (green solid, yield 65%)

[0122] Intermediate w5: (green solid, yield 68%)

[0123] Intermediate w6: (green solid, 70% yield)

[0124] Intermediate w8: (green solid, yield 69%)

[0125] Preparation of final product 12-17

[0126] 40 mg (1 eq) of rhein was placed in a 50 mL round-bottom flask, and EDCL (2 eq), HOBt (2 eq), W2 (1.2 eq), and triethylamine (4 eq) were added. Finally, 20 mL of dichloromethane was added as the reaction solvent, and the mixture was stirred to dissolve. The amidation reaction was carried out at room temperature for 4 h. A yellow precipitate was formed after the reaction was completed. The amidation reaction solution was extracted with DCM, washed with saturated brine, and purified by PTLC (PE:PA = 1:1) to obtain the final product 12 (solid color, yield 37%).

[0127] The preparation of end products 13-17 is similar to that of end product 12.

[0128] Final product 12: (dark brown solid, yield 37%) 1 H NMR(500MHz,DMSO-d6)δ11.11(s,1H),8.98(s,1H),8.12(s,1H),7.95(s,1H),7.82–7.73(m,1H),7.58(s,1H),7.40(s,1H),7.13 (s,1H),7.02(s,1H),6.76(s,1H),5.13–5.00(m,1H),2.86(d,J=19.4Hz,2H),2.66(d,J=63.9Hz,2H),2.02(s,2H),1.84(s,2H). 13 C NMR(125MHz,DMSO-d6)δ189.88,182.52,172.35,169.96,169.93,169.87,169.84,167.60, 167.58,166.18,166.16,164.39,164.37,162.33,162.00,161.98,144.15,144.10,144.08 ,137.11,137.10,137.05,137.04,135.09,135.08,133.66,133.65,133.60,133.59,132.72,132.71,131.41,131.39,131.38,131.35,131.33,131.32,130.55,124.30,124.29,124.2 5,124.23,123.37,123.32,120.84,120.82,120.78,120.76,120.45,120.44,120.42,120.40,120.38,120.37,120.17,120.14,120.12,120.10,120.07,120.05,120.04,120.04,120 .03,117.72,117.68,117.63,116.19,112.36,52.28,52.27,52.25,52.18,44.79,44.77,40.82,40.80,40.79,40.78,30.97,30.95,30.93,26.38,26.36,26.35,26.34,26.33,26.31.

[0129] Final product 13: (dark brown solid, yield 38%) 1H NMR(500MHz,DMSO-d6)δ11.10(s,1H),8.92(s,1H),8.03(d,J=79.0Hz,1H),7.81(s, 1H),7.74(s,1H),7.56(t,J=7.9Hz,1H),7.39(s,1H),7.11(d,J=8.3Hz,1H),6.99(d ,J=6.9Hz,1H),6.58(d,J=6.2Hz,1H),5.04(dd,J=12.8,5.4Hz,1H),2.97–2.67(m,2 H),2.67–2.52(m,2H),2.03(dt,J=12.1,5.5Hz,1H),1.63(s,2H),1.40–0.65(m,1H). 13 C NMR(125MHz,DMSO-d6)δ189.88,182.59,172.35,170.06,170.03,169.97,169.94,167.60,16 7.58,167.57,166.18,166.16,164.39,164.37,162.33,162.00,161.98,144.12,144.06,137. 11,137.10,137.05,137.04,135.09,135.08,133.61,133.60,133.55,133.54,132.72,132.71,131.41,131.39,131.38,131.35,131.33,131.32,130.55,124.30,124.29,124.25,124.23, 123.37,123.32,120.84,120.82,120.78,120.76,120.53,120.52,120.50,120.47,120.46,120.44,120.17,120.14,120.12,120.10,120.07,120.05,120.04,120.04,120.03,117.72,117 .68,117.63,116.07,112.58,52.27,52.18,42.82,42.80,42.79,42.77,38.40,38.38,38.37,38.35,31.19,31.17,31.15,28.36,28.34,28.32,26.38,26.36,26.35,26.34,26.33,26.31.

[0130] Final product 14: (dark brown solid, yield 39%) 1H NMR(500MHz,Chloroform-d)δ8.99(s,1H),8.27(t,J=6.8Hz,1H),7.93(d,J=1.4Hz,1H),7.79(dd,J=7.5,1.5Hz,1H),7.74(t,J=5.9Hz,1H),7.71–7.67(m,1H),7.66(d,J=2.4Hz,1H),7.53(t,J=7.5Hz,1H),7.46(dd,J=7.5,1.6Hz,1H),7.34(dd,J=7.5,1.5Hz,1H),7.13(dd,J=7.5,1.6Hz,1H),5.50(t,J=7.0Hz,1H),3.40(qd,J=6.9,1.9Hz,2H),3.28(ddtd,J=48.7,12.7,7.0,5.9Hz,2H),2.73–2.51(m,2H),2.32–2.00(m,2H),1.76–1.61(m,2H),1.61–1.48(m,2H). 13C NMR(125MHz,DMSO-d6)δ189.88,182.59,172.35,170.15,170.12,170.06,170.03,167.60,167.58 ,167.57,166.18,166.16,164.30,164.29,162.33,162.00,161.98,144.12,144.06,137.11,137. 10, 137.05, 137.04, 136.83, 136.82, 133.61, 133.60, 133.55, 133.54, 132.80, 132.79, 131.48, 131.46, 131.45, 131.42, 131.40, 131.39, 130.40, 124.52, 124.50, 124.47, 124.45, 123.36, 123.32, 123.30, 120.84, 120.82, 120.78, 120.76, 120.57, 120.55, 120.54, 120.50, 120.17, 120.14, 120.12, 120.10, 120.07, 120.05, 120.04, 120.03, 117.04, 117.00, 116.95, 115.94, 112.58, 52.22, 52.1 3,43.40,43.38,43.37,43.35,39.73,39.71,39.69,39.67,31.19,31.17,31.15,26.86,26.84,26.83,26.81,26.79,26.74,26.72,26.70,26.69,26.67,26.60,26.58,26.57,26.55,26.54,26.53.

[0131] Final product 15: (dark brown solid, yield 36%) 1H NMR(500MHz,Chloroform-d)δ8.99(s,1H),8.27(t,J=6.8Hz,1H),7.93(d,J=1.4Hz,1H),7.79(dd,J=7.5,1.5Hz,1H),7.74(t,J=5.9Hz,1H),7.71–7.67(m,1H),7.66(d,J=2.4Hz,1H),7.53(t,J=7.5Hz,1H),7.46(dd,J=7.5,1.6Hz,1H),7.34(dd,J=7.5,1.5Hz,1H),7.13(dd,J=7.5,1.6Hz,1H),5.50(t,J=7.0Hz,1H),3.40(qd,J=6.9,1.9Hz,2H),3.28(ddtd,J=48.7,12.7,7.0,5.9Hz,2H),2.73–2.51(m,2H),2.32–2.00(m,2H),1.76–1.61(m,2H),1.61–1.48(m,2H). 13C NMR (125MHz, DMSO-d6) δ189.81,182.59,172.40,170.15,170.12,170.06,170.03,167.60,167.58,167. 57,166.18,166.16,164.31,164.30,162.33,162.00,161.98,144.12,144.06,137.11,137.10,137.05, 137.04, 136.83, 136.82, 133.61, 133.60, 133.55, 133.54, 132.93, 132.92, 131.60, 131.59, 131.57, 131.54, 131.52, 131.51, 130.40, 124.52, 124.50, 124.47, 124.45, 123.36, 123.32, 123.30, 120.84, 120.82, 120.78, 120.76, 120.57, 120.55, 120.54, 120.50, 120.48, 120.17, 120.14, 120.12, 120.10, 120.07, 120.05, 120.04, 120.03, 117.04, 117.00, 116.95, 115.94, 112.58, 52.22, 52.20, 52.13, 43.54, 43.52, 43.51 ,43.49,39.80,39.78,39.76,39.74,31.19,31.17,31.15,28.91,28.89,28.87,28.86,28.84,28.73,28.71,28.69,28.68,28.65,26.60,26.58,26.57,26.55,26.54,26.53,25.02,25.00,24.98,24.96,24.95.

[0132] Final product 16: (dark brown solid, yield 35%) 1H NMR(500MHz,Chloroform-d)δ8.99(s,1H),8.18(t,J=6.8Hz,1H),7.92(d,J=1.5Hz,1H),7.83–7.79(m,1H),7.79–7.76(m,1H),7.72–7.67(m,1H),7.67(d,J=3.8Hz,1H),7.50(t,J=7.4Hz,1H),7.43(dd,J=7.5,1.6Hz,1H),7.34(dd,J=7.4,1.6Hz,1H),7.13(dd,J=7.3,1.7Hz,1H),5.50(t,J=7.0Hz,1H),3.50–3.29(m,2H),3.29–3.04(m,2H),2.84–2.50(m,2H),2.35–2.02(m,2H),1.80–1.65(m,2H),1.62(qd,J=7.1,2.0Hz,2H),1.49–1.33(m,2H),1.33–1.32(m,2H),1.32–1.11(m,2H). 13CNMR(125MHz,DMSO-d6)δ189.81,182.83,182.79,172.40,170.15,170.12,170.06,170.03,167.60,167.58 ,167.57,166.18,166.16,164.31,164.30,162.34,161.97,161.95,144.02,137.11,137.10,137.05,137.0 4,135.60,135.59,133.60,133.54,133.21,131.59,131.57,131.52,131.51,130.87,124.53,124.52,124.51,124.48,124.46,123.37,123.32,123.30,120.83,120.82,120.78,120.76,120.57,120.55,120.54,120 .50,120.17,120.14,120.12,120.10,120.07,120.05,120.04,120.03,119.99,117.04,117.00,116.95,115.91,113.64,52.48,52.39,43.55,43.53,43.52,43.50,39.66,39.64,39.63,39.61,31.19,31.17,31.15, 29.13,29.11,29.09,29.07,29.05,28.92,28.91,28.89,28.87,28.85,28.72,28.70,28.68,28.67,28.65,27.35,27.33,27.31,27.30,27.28,26.60,26.58,26.57,26.55,26.52,24.98,24.96,24.94,24.93,24.91.

[0133] Final product 17: (dark brown solid, yield 33%) 1 H NMR(500MHz,DMSO-d6)δ11.10(s,1H),8.88

[0134] (s,1H),8.11(s,1H),7.81(s,1H),7.74(s,1H),7.70(d,J=16.2Hz,1H),7.59–7.50(m,1H),7.39(d,J=7.9Hz,1H),7.05(d,J=8.4Hz,1H),6.98(d,J=7.0Hz,1H),6.54–6.44(m,1H),5.04(dd,J=12.8,5.4Hz,1H),3.29–3.26(m,2H),2.87(dt,J=13.7,9.9Hz,2H),2.56(s,1H),2.06–1.95(m,1H),1.55(q,J=7.2Hz,4H),1.32(s,8H).13C NMR(126MHz,DMSO)δ172.72,170.58,168.91,167.07,164.96,161.88,153.55,147.34,146.85,142.31,138.72,136.71,134.73,132.62,131.91,126.37,124.77,124.26,123.69,120.38,118.63,117.60,116.55,111.69,109.44,49.68,42.31,31.83,29.21,29.12,26.82,26.73,24.96,22.62,18.96,17.95. 13C NMR (125MHz, DMSO-d6) δ189.81,182.83,182.79,172.48,170.06,170.03,169.97,169.94,167.55,167.5 3,167.51,166.17,166.16,164.31,164.30,162.34,161.97,161.95,144.02,137.11,137.10,137.05,13 7.04, 135.60, 135.59, 133.55, 133.50, 133.21, 131.58, 131.56, 131.55, 131.50, 130.87, 124.53, 124.52, 124.51, 124.48, 124.46, 123.33, 123.31, 123.27, 123.26, 120.84, 120.82, 120.78, 120.76, 120.55, 120 .50,120.17,120.14,120.12,120.10,120.07,120.01,119.96,119.91,117.04,117.00,116.95,115.91,113.64,52.48,52.46,52.39,43.53,43.51,43.49,43.47,39.70,39.68,39.66,39.64,31.19,31.17,31. 15,29.11,29.09,29.07,29.05,28.90,28.88,28.86,28.84,28.82,28.80,28.72,28.70,28.68,28.67,27.36,27.34,27.32,27.30,27.29,26.60,26.58,26.57,26.55,26.52,24.96,24.94,24.92,24.90,24.89.

[0135] Example 6: Preparation of intermediates N7-N16 and NJ7-NJ16

[0136] (1) Preparation of intermediate N7

[0137] 2 g (0.011 mol, 1 equivalent) of potassium phthalimide was weighed into a 50 mL round-bottom flask. 20 mL of acetone was added, and TBAB (0.32 g, 0.001 mol, 0.1 equivalent) and 1,2-dibromoethane (3.9 mL, 0.033 mol, 3 equivalent) were added sequentially under stirring. The mixture was stirred and refluxed at 70 °C for 2 h. TIC analysis (PE:EtOAc = 6:1 as the developing solvent) showed that the reaction was complete. Post-treatment: The mixture was cooled, quenched with ice water, and repeatedly extracted with DCM. Vacuum distillation yielded a yellow liquid, which was purified by column chromatography (PE:EtOAc = 10:1) to obtain intermediate N7-NN2 (structural formula shown in the figure) (white solid, yield 86.8%).

[0138]

[0139] Structure of intermediates N7-NN2 to N7-NN6

[0140] The synthesis methods of intermediates N7-NN3 to N1-NN16 are similar to those of N7-NN2.

[0141] N7-NN3 (white solid, yield 80.9%)

[0142] N7-NN4 (white solid, yield 80.5%)

[0143] N7-NN5 (white solid, yield 82.7%)

[0144] N7-NN6 (white solid, yield 78.7%)

[0145] (2) Preparation of intermediate N8

[0146] At room temperature, 1 g (0.004 mol, 1 equivalent) of N7-NN2 was weighed into a 25 ml round-bottom flask. DMF (15 ml) was added and stirred until homogeneous. Then, K2CO3 (1.7 g, 0.012 mol, 3 equivalents) and KI (0.2 g, 0.0012 mmol, 0.3 equivalents) were added. The mixture was heated to 75°C, and γ-aminobutyric acid (0.82 g, 0.008 mol, 2 equivalents) was slowly added. TIC (developing solvent PE:EA = 7:1) analysis showed no N1-NN2 starting material after 24 h of reaction. Post-treatment: The reaction was quenched with ice water, resulting in the precipitation of a yellow solid. The mixture was stirred at room temperature for 30 min and extracted thoroughly with EtOAc. The organic layer was washed sequentially with saturated brine, dried over anhydrous sodium sulfate, and subjected to reduced pressure with concentrated acid to obtain a colorless, transparent liquid, N8-J7 (yellow oily liquid, yield 85.1%). n1 = 1, n2 = 1 NJ7

[0147] The synthesis methods of intermediates N8-J8 to N8-J16 are similar to those of N8-J7.

[0148] N8-J8: Yellow oily liquid, yield 85.6%

[0149] N8-J9: Yellow oily liquid, yield 85.3%

[0150] N8-J10: Yellow oily liquid, yield 83.2%

[0151] N8-J11: Yellow oily liquid, yield 82.3%

[0152] N8-J12: Yellow oily liquid, yield 81.3%

[0153] N8-J13: Yellow oily liquid, yield 80.7%

[0154] N8-J14: Yellow oily liquid, yield 80.7%

[0155] N8-J15: Yellow oily liquid, yield 84.7%

[0156] N8-J16: Yellow oily liquid, yield 86.7%

[0157] (4) Preparation of intermediates NJ7~NJ18

[0158] Weigh 50 mg of N8-NJ7 (0.0094 mmol, 1 equivalent) into a 25 ml round-bottom flask, add 20 ml of DMF and stir well. Then add 30 ml of 80% hydrazine hydrate and stir at room temperature for 12 h. A white solid appears. Post-treatment: Quench the reaction with ice water, extract thoroughly with EtOAc, wash the organic layer sequentially with saturated brine, dry with anhydrous sodium sulfate, and obtain a colorless transparent liquid NJ7 (yellow oily liquid, yield 92.5%) under reduced pressure and concentrated acid.

[0159]

[0160] Structure of NJ7~NJ16

[0161] The synthesis methods of intermediates NJ8 to NJ16 are similar to those of NJ7.

[0162] NJ8: Yellow oily liquid, yield 94.5%

[0163] NJ9: Yellow oily liquid, yield 95.6%

[0164] NJ10: Yellow oily liquid, yield 97.8%

[0165] NJ11: Yellow oily liquid, yield 94.4%

[0166] NJ12: Yellow oily liquid, yield 94.7%

[0167] NJ13: Yellow oily liquid, yield 94.3%

[0168] NJ14: Yellow oily liquid, yield 94.9%

[0169] NJ15: Yellow oily liquid, yield 94.5%

[0170] NJ16: Yellow oily liquid, yield 95.5%

[0171] Preparation of final product 18-27

[0172] Weigh rhein (50 mg, 0.176 mmol, 1 equivalent) into a 25 mL flask, add DMF (1 mL) solvent, stir for a few minutes, then add DIPEA (36 μl, 0.21 mmol, 3 equivalents) and HATU (33 mg, 0.084 mmol, 1.2 equivalents) sequentially. Stir for about 60 min, and monitor with TIC (developing solvent: CDCl3:MeOH = 20:1). After the formation of the active ester, add NJ7-6 (40 mg, 0.1 mmol, 1.5 equivalents). React at room temperature for 12 h, and monitor with TIC (developing solvent: dichloromethane:methanol = 20:1) to confirm complete reaction of the starting material. Quench the reaction solution with crushed ice, extract thoroughly with DCM, wash the organic layer sequentially with saturated ammonium chloride solution and saturated brine, dry with anhydrous sodium sulfate, concentrate under reduced pressure to obtain a yellow liquid, and purify by PTIC to obtain product 18 (dark brown solid, yield 52.7%).

[0173] Final product 18 (dark brown solid, yield 55%) 1H NMR(500MHz,Chloroform-d)δ9.30(s,1H),8.72(s,1H),8.50(t,J=7.0Hz,1H),7.92(d,J=1.5Hz,1H),7.88(dd,J=7.5,1.5Hz,1H),7.79(dd,J=7.4,1.6Hz,1H),7.78–7.74(m,1H),7.71–7.67(m,1H),7.67(d,J=3.9Hz,1H),7.37(t,J=7.5Hz,1H),7.34(dd,J=7.4,1.5Hz,1H),4.49(t,J=7.0Hz,1H),4.32(d,J=6.6Hz,2H),3.73(p,J=6.3Hz,1H),3.46–3.22(m,2H),2.84–2.70(m,2H),2.70–2.59(m,2H),2.59–2.42(m,2H),2.32(td,J=7.1,2.1Hz,2H),2.10–1.93(m,2H),1.93–1.84(m,2H),1.84–1.70(m,2H). 13C NMR (125MHz, DMSO-d6) δ189.81,182.83,172.58,171.96,169.84,169.80,169.77,167.57,167.54,167.53,167 .51,162.34,161.97,161.94,138.05,137.99,137.11,137.10,137.05,137.04,135.60,135.59,135.01,135.0 0,133.56,133.55,133.50,133.49,133.21,133.20,130.73,125.49,125.48,125.45,125.43,125.42,124.53,124.53,124.51,124.48,124.46,123.33,123.27,122.51,122.47,122.45,122.44,122.40,120.17,120.14,120 .12,120.10,120.07,120.02,119.97,119.96,119.95,119.91,117.62,117.59,117.56,117.54,117.51,117.04,117.00,116.95,115.91,55.41,55.39,55.38,55.37,55.35,48.19,48.17,48.16,48.14,48.13,48.11,47.34 ,47.31,47.28,46.35,46.33,46.31,46.29,46.28,46.26,38.12,38.10,38.09,38.07,35.22,35.19,35.16,31.44,31.43,31.42,31.40,28.13,28.11,28.09,26.90,26.88,26.86,26.85,26.83,24.79,24.77,24.76,24.74.

[0174] Final product 19 (dark brown solid, yield 52%) 1H NMR(500MHz,Chloroform-d)δ9.30(s,1H),8.72(s,1H),8.18(t,J=6.8Hz,1H),7.92(d,J=1.4Hz,1H),7.88(dd,J=7.5,1.5Hz,1H),7.79(dd,J=7.4,1.6Hz,1H),7.78–7.73(m,1H),7.72–7.67(m,1H),7.67(d,J=3.9Hz,1H),7.37(t,J=7.5Hz,1H),7.34(dd,J=7.4,1.5Hz,1H),4.49(t,J=7.0Hz,1H),4.32(d,J=6.6Hz,2H),3.49(p,J=6.2Hz,1H),3.40(qd,J=7.0,1.9Hz,2H),2.88–2.74(m,2H),2.74–2.65(m,2H),2.59(qt,J=12.4,7.0Hz,2H),2.32(td,J=7.1,2.1Hz,2H),2.09–1.90(m,2H),1.89–1.73(m,2H),1.61–1.53(m,2H),1.53–1.45(m,2H). 13C NMR (125MHz, DMSO-d6) δ189.81,182.83,172.58,171.96,169.84,169.80,169.77,167.55,167.53,167.51,167.49,167 .46,162.34,161.97,161.94,138.05,137.99,137.13,137.12,137.10,137.07,137.06,135.28,135.26,135.01,135.00 ,133.56,133.55,133.50,133.49,133.21,133.20,130.73,125.55,125.53,125.50,125.48,125.47,124.53,124.53,124.51,124.48,124.46,123.33,123.31,123.28,123.25,122.26,122.22,122.21,122.19,122.15,120.17,120.14,120. 12,120.10,120.07,119.97,119.96,119.95,119.91,117.62,117.59,117.56,117.54,117.51,117.04,117.00,116.95,115.91,55.40,55.39,55.38,55.37,55.35,48.19,48.16,48.14,48.13,48.11,47.90,47.88,47.86,47.84,47.83,47. 81,47.34,47.31,47.28,39.73,39.71,39.69,39.67,35.22,35.19,35.16,31.44,31.43,31.42,31.40,27.03,27.01,26.99,26.97,26.95,26.62,26.60,26.59,26.57,26.55,26.13,26.11,26.09,26.07,26.05,24.79,24.77,24.76,24.74.

[0175] Final product 20 (dark brown solid, yield 51%) 1H NMR(500MHz,Chloroform-d)δ9.30(s,1H),8.72(s,1H),8.12(t,J=6.8Hz,1H),7.92(d,J=1.5Hz,1H),7.88(dd,J=7.5,1.5Hz,1H),7.79(dd ,J=7.4,1.6Hz,1H),7.78–7.75(m,1H),7.68(d,J=7.5Hz,1H),7.67–7.63(m,1H),7.37(t,J=7.5Hz,1H),7.34(dd,J=7.5,1.5Hz,1H),4.49( t,J=7.0Hz,1H),4.32(d,J=6.6Hz,2H),3.50(p,J=6.3Hz,1H),3.38(p,J=7.0Hz,2H),2.90–2.75(m,2H),2.74–2.65(m,2H),2.59(qt,J=12. 4,7.0Hz,2H),2.32(td,J=7.1,2.1Hz,2H),2.09–1.91(m,2H),1.90–1.74(m,2H),1.69–1.57(m,2H),1.57–1.47(m,2H),1.47–1.25(m,2H). 13 C NMR (125MHz, DMSO-d6) δ189.80,183.20,173.24,171.95,171.07,168.36,167. 55,162.48,161.89,138.48,137.09,135.90,133.68,133.46,133.33,132.61,1 24.90, 124.60, 124.50, 123.52, 121.67, 120.21, 115.82, 114.85, 113.78, 54.76, 48.01, 47.90, 47.29, 39.89, 36.25, 31.60, 29.00, 27.82, 25.70, 25.42, 25.04.

[0176] Final product 21 (dark brown solid, yield 53%) 1H NMR(500MHz,Chloroform-d)δ9.30(s,1H),8.72(s,1H),8.18(t,J=6.8Hz,1H),7.96(dd,J=7.5,1.5Hz,1H),7.91(d,J=1.4Hz,1H),7.79(t,J=1.4H z,1H),7.78(t,J=1.4Hz,1H),7.74–7.68(m,1H),7.67(d,J=6.8Hz,1H),7 .37(t,J=7.5Hz,1H),7.33(dd,J=7.4,1.5Hz,1H),4.49(t,J=7.0Hz,1H),4 .32(d,J=6.6Hz,2H),3.65(p,J=6.3Hz,1H),3.32(ddq,J=74.9,12.5,7.0 Hz,2H),2.94–2.75(m,2H),2.75–2.66(m,2H),2.59(qt,J=12.4,7.0Hz,2 H),2.32(td,J=7.1,2.1Hz,2H),2.07–1.91(m,2H),1.90–1.80(m,2H),1. 80–1.65(m,2H),1.65–1.53(m,2H),1.47–1.33(m,2H),1.33–1.23(m,2H). 13 C NMR(125MHz,DMSO-d6)δ189.80,183.20,173.24,171.95,171.07,168.36,167.47 ,162.48,161.87,138.47,137.09,135.90,133.68,133.46,133.33,132.61,124. 90,124.60,124.52,123.52,121.67,120.21,115.82,114.89,114.10,54.51,47.90,47.73,47.29,39.75,36.25,31.67,28.88,27.57,27.53,27.18,26.01,25.70.

[0177] Final product 22 (dark brown solid, yield 55%) 1H NMR (500MHz, Chloroform-d) δ9.30 (s, 1H), 8.72 (s, 1H), 8.18 (t, J = 6.8Hz, 1H), 7. 96(dd,J=7.5,1.5Hz,1H),7.91(d,J=1.4Hz,1H),7.80(dd,J=7.5,1.5Hz,1H),7.7 7(dd,J=7.5,1.5Hz,1H),7.72–7.67(m,1H),7.66(d,J=3.9Hz,1H),7.37(t,J=7.5 Hz,1H),7.33(dd,J=7.4,1.5Hz,1H),4.49(t,J=7.0Hz,1H),4.32(d,J=6.6Hz,2H) ,3.65(p,J=6.3Hz,1H),3.33(ddq,J=69.4,12.5,7.0Hz,2H),2.97–2.75(m,2H),2 .75–2.67(m,2H),2.59(qt,J=12.4,7.0Hz,2H),2.32(td,J=7.1,2.1Hz,2H),1.99 (qq,J=12.5,7.1Hz,2H),1.91–1.80(m,2H),1.80–1.66(m,2H),1.63(pd,J=7.1,2 .0Hz,2H),1.51–1.34(m,2H),1.33(td,J=3.2,2.8,1.2Hz,2H),1.32–1.16(m,2H). 13 C NMR(125MHz,DMSO-d6)δ189.80,183.20,173.24,171.95,171.07,168.36,167.47, 162.48,162.13,138.47,136.99,135.90,133.68,133.43,133.12,132.61,124.90, 124.60,124.50,123.52,121.67,120.21,115.82,114.89,113.50,54.51,47.90,47.73,47.29,39.74,36.25,31.67,29.15,28.88,27.23,26.53,26.29,26.01,25.70.

[0178] Final product 23 (dark brown solid, yield 52.8%) 1H NMR(500MHz,Chloroform-d)δ9.30(s,1H),8.72(s,1H),8.18(t,J=6.8Hz,1H), 7.96(dd,J=7.5,1.5Hz,1H),7.91(d,J=1.5Hz,1H),7.80(dd,J=3.1,1.5Hz,1H), 7.79(dd,J=3.1,1.5Hz,1H),7.68(d,J=7.4Hz,1H),7.67(d,J=3.5Hz,1H),7.37( t,J=7.5Hz,1H),7.33(dd,J=7.5,1.5Hz,1H),4.49(t,J=7.0Hz,1H),4.32(d,J=6 .6Hz,2H),3.65(p,J=6.3Hz,1H),3.33(ddq,J=68.9,12.3,7.0Hz,2H),2.92–2.7 5(m,2H),2.75–2.67(m,2H),2.59(qt,J=12.4,7.0Hz,2H),2.32(td,J=7.1,2.1H z,2H),2.11–1.92(m,2H),1.91–1.80(m,2H),1.80–1.65(m,2H),1.65–1.54(m,2 H),1.46–1.31(m,2H),1.31–1.28(m,2H),1.28–1.27(m,2H),1.27–1.16(m,2H). 13 C NMR(125MHz,DMSO-d6)δ189.80,183.04,173.24,171.95,171.07,168.36,167.36,16 2.49,162.13,138.47,136.96,135.90,133.68,133.43,133.12,132.61,125.21,124 .60,124.53,123.52,121.67,120.21,115.82,114.89,113.50,54.51,47.90,47.73,47.29,39.75,36.10,31.67,28.88,28.57,28.53,27.23,26.65,26.53,26.01,25.70.

[0179] Final product 24 (dark brown solid, yield 53.2%) 1H NMR(500MHz,Chloroform-d)δ9.30(s,1H),8.72(s,1H),8.14(t,J=6.8Hz,1H),7.9 6(dd,J=7.5,1.5Hz,1H),7.91(d,J=1.5Hz,1H),7.80(dd,J=3.1,1.5Hz,1H),7.79( dd,J=3.1,1.5Hz,1H),7.68(d,J=7.4Hz,1H),7.67(d,J=3.5Hz,1H),7.37(t,J=7.5 Hz,1H),7.33(dd,J=7.5,1.5Hz,1H),4.49(t,J=7.0Hz,1H),4.32(d,J=6.6Hz,2H),3 .65(p,J=6.3Hz,1H),3.31(ddq,J=80.4,12.3,7.0Hz,2H),2.97–2.75(m,2H),2.75 –2.66(m,2H),2.59(qt,J=12.4,7.0Hz,2H),2.32(td,J=7.1,2.1Hz,2H),2.08–1.9 2(m,2H),1.91–1.80(m,2H),1.80–1.65(m,2H),1.65–1.56(m,2H),1.46–1.32(m,2 H),1.32–1.28(m,2H),1.28–1.26(m,2H),1.26(d,J=2.9Hz,2H),1.25–1.09(m,2H). 13 C NMR (125MHz, DMSO-d6) δ189.80,183.04,173.24,171.95,171.07,168.36,167.23,162 .49,162.13,138.15,136.96,135.90,133.68,133.43,133.12,132.61,125.21,124.60 ,124.53,123.52,121.67,120.21,115.82,114.89,113.50,54.51,47.90,47.73,47.29,39.77,36.12,31.67,29.58,28.86,28.56,28.53,27.23,27.07,26.53,25.70,25.45.

[0180] Final product 25 (dark brown solid, yield 52%) 1H NMR(500MHz,Chloroform-d)δ9.30(s,1H),8.72(s,1H),8.14(t,J=6.8Hz,1H),7.96( dd,J=7.5,1.5Hz,1H),7.91(d,J=1.5Hz,1H),7.80(dd,J=3.1,1.5Hz,1H),7.79(dd,J= 3.1,1.5Hz,1H),7.72–7.67(m,1H),7.67(d,J=3.5Hz,1H),7.37(t,J=7.5Hz,1H),7.33 (dd,J=7.5,1.5Hz,1H),4.49(t,J=7.0Hz,1H),4.32(d,J=6.6Hz,2H),3.65(p,J=6.3Hz ,1H),3.31(ddq,J=80.4,12.3,7.0Hz,2H),2.91–2.75(m,2H),2.75–2.66(m,2H),2.59 (qt,J=12.4,7.0Hz,2H),2.32(td,J=7.1,2.1Hz,2H),2.09–1.92(m,2H),1.91–1.80(m ,2H),1.80–1.67(m,2H),1.63(pd,J=7.1,1.7Hz,2H),1.48–1.32(m,2H),1.32–1.28(m ,2H),1.28–1.26(m,2H),1.26(t,J=1.4Hz,2H),1.26–1.25(m,2H),1.25–1.12(m,2H). 13 C NMR (125MHz, DMSO-d6) δ189.80,183.04,173.29,171.95,170.84,168.36,167.23,162.4 9,162.13,138.15,136.96,135.99,133.68,133.43,133.12,132.61,125.21,124.60,12 4.53, 123.52, 122.03, 120.21, 115.82, 114.89, 113.16, 54.51, 47.90, 47.73, 47.29, 39.77, 36.12, 31.67, 29.13, 29.09, 28.86, 28.56, 28.53, 27.23, 27.07, 26.53, 25.70, 25.45.

[0181] Final product 26 (dark brown solid, yield 54.3%) 1H NMR (500MHz, Chloroform-d) δ9.30 (s, 1H), 8.72 (s, 1H), 8.14 (t, J = 6.8Hz, 1H), 7.96 (dd ,J=7.5,1.5Hz,1H),7.91(d,J=1.5Hz,1H),7.80(dd,J=3.1,1.5Hz,1H),7.79(dd,J=3.1, 1.5Hz,1H),7.68(d,J=7.4Hz,1H),7.67(d,J=3.5Hz,1H),7.37(t,J=7.5Hz,1H),7.33(d d,J=7.5,1.6Hz,1H),4.49(t,J=7.0Hz,1H),4.32(d,J=6.6Hz,2H),3.65(p,J=6.3Hz,1H) ,3.31(ddq,J=80.4,12.3,7.0Hz,2H),2.95–2.75(m,2H),2.75–2.66(m,2H),2.65–2.48 (m,2H),2.32(td,J=7.1,2.1Hz,2H),2.08–1.92(m,2H),1.90–1.80(m,2H),1.80–1.66(m ,2H),1.62(dtd,J=14.0,7.0,5.4Hz,2H),1.49–1.32(m,2H),1.32–1.28(m,2H),1.28–1 .26(m,2H),1.26–1.26(m,2H),1.26–1.25(m,2H),1.25–1.24(m,2H),1.24–1.17(m,2H). 13 C NMR (125MHz, DMSO-d6) δ189.80,183.04,173.29,171.95,170.84,168.36,167.37,162.49 ,162.12,138.15,136.97,135.99,133.68,133.43,133.12,132.61,125.21,124.60,124.5 3,123.52,122.03,120.21,115.82,114.89,113.11,54.51,47.90,47.73,47.29,39.77,36.12,31.67,29.24,29.13,29.09,28.86,28.57,28.53,27.23,27.07,26.53,25.70,25.45.

[0182] Final product 27 (dark brown solid, yield 55.2%) 1H NMR(500MHz,Chloroform-d)δ9.30(s,1H),8.72(s,1H),8.14(t,J=6.8Hz,1H),7.96(dd,J= 7.5,1.5Hz,1H),7.91(d,J=1.5Hz,1H),7.80(dd,J=3.1,1.5Hz,1H),7.79(dd,J=3.1,1.5Hz, 1H),7.68(d,J=7.4Hz,1H),7.67(d,J=3.5Hz,1H),7.37(t,J=7.5Hz,1H),7.33(dd,J=7.5,1 .6Hz,1H),4.49(t,J=7.0Hz,1H),4.32(d,J=6.6Hz,2H),3.65(p,J=6.3Hz,1H),3.31(ddq,J= 80.4,12.3,7.0Hz,2H),2.94–2.75(m,2H),2.75–2.64(m,2H),2.64–2.49(m,2H),2.32(td, J=7.1,2.1Hz,2H),2.10–1.91(m,2H),1.91–1.80(m,2H),1.80–1.66(m,2H),1.63(pd,J=7.1 ,1.7Hz,2H),1.47–1.32(m,2H),1.32–1.28(m,2H),1.28–1.27(m,2H),1.26(dd,J=2.5,1.0 Hz,2H),1.25(t,J=1.7Hz,2H),1.25–1.24(m,2H),1.23(d,J=2.0Hz,2H),1.23–1.16(m,2H). 13 C NMR(125MHz,DMSO-d6)δ189.80,183.04,173.29,171.95,170.84,168.19,167.37,162.49,1 62.12,138.03,136.97,135.99,133.68,133.43,133.12,132.61,125.21,124.60,124.53,1 23.52,122.03,120.21,115.81,114.89,113.11,54.51,47.90,47.73,47.29,39.79,36.12,31.67,29.28,29.23,29.11,29.08,28.86,28.57,28.53,27.23,26.84,26.45,26.01,25.70.

[0183] Example 8: Preparation of ND6 (Negative Control)

[0184] Take 40 mg (1 eq) of rhein into a 50 mL round-bottom flask, add EDCL (2 eq), HOBt (2 eq), 6-aminohexanoic acid-3-(4-amino-1-oxoisoindol-2-yl)-1-methylpiperidin-2,6-dione (1.2 eq), triethylamine (4 eq), and finally add 20 mL of dichloromethane as the reaction solvent. Stir to dissolve and carry out the amidation reaction at room temperature for 4 h. When the reaction is complete, a yellow precipitate is formed. Wash the amidation reaction solution with DCM, pure water, and ethanol to obtain the product ND6.

[0185] The final product was ND6 (dark brown solid, yield 64.2%). .1 H NMR (500MHz, Pyridine-d5) δ13.17(s,1H),9.66(d,J=5.8Hz,1H),8.28(d,J=1.7Hz,1H),7.87(d,J=7.4Hz,1H),7.64(t,J=7.9 Hz,1H),7.48(dd,J=8.6,7.1Hz,1H),7.40–7.36(m,1H),7.19(d,J=3.7Hz,1H),6.91(d,J=8.5Hz,1H),6.61(t,J=5.7Hz,1H),5. 55(dd,J=12.8,5.3Hz,1H),3.70(q,J=6.7Hz,2H),3.13(dq,J=11.7,6.0,5.3Hz,3H),3.08(d,J=5.3Hz,1H),3.02–2.95(m,1H) ,2.95–2.73(m,2H),2.41–2.04(m,2H),1.72(q,J=7.4Hz,2H),1.53–1.47(m,2H),1.40(d,J=7.4Hz,2H),1.34(d,J=7.4Hz,2H). 13C NMR(126MHz,pyridine-d5)δ173.61,171.25,170.62,168.77,167.92,166.18 ,164.11,163.11,159.96,147.18,144.21,138.09,136.67,136.31,135.48,13 4.73,134.38,133.01,125.22,120.44,119.07,117.42,116.71,111.42,110. 64,57.93,50.65,43.06,40.91,32.51,30.34,29.75,27.90,26.60,23.74.13C NMR (126MHz, DMSO) δ171.91,171.43,170.69,167.90,155.62,134.60,132.66,128.57,125.43,11 7.98,77.32,53.13,51.04,46.72,41.79,35.74,31.40,29.33,28.28,26.59,26.01,24.89,21.88.

[0186] Example 9: This invention provides an assay (IC50) for the antitumor activity of rhein PROTACs compounds.

[0187] The tumor cell proliferation inhibition ability of the rhein PROTACs compounds of this invention was tested using the conventional CCK-8 assay. When the tumor cells grew to 80%–90% confluence in the culture dish, the cells were digested, centrifuged, and resuspended in fresh DMEM / 1640 complete medium. Cell counts were performed under a microscope, and the cell suspension was then diluted to 50 cells / μL. A ring of sterile PBS was placed around the outermost edge of each well in a 96-well plate (100 μL per well), and the remaining wells were each coated with 100 μL of the diluted cell suspension. The plates were then incubated at 37°C in a 5% CO2 incubator. After 24 hours, the culture medium in the 96-well plate was aspirated, and the drug was serially diluted with fresh complete culture medium. The diluted solutions were then added sequentially to the 96-well plate. After 48 hours of incubation, the drug-containing culture medium was removed, and CCK-8 solution diluted with the culture medium (100 μL culture medium + 10 μL CCK-8) was added. The plate was incubated at 37°C in a 5% CO2 incubator for 30–40 min. The absorbance was measured at OD 450 using a microplate reader, and the inhibition rate was calculated based on the absorbance at OD 450. The half-maximal inhibitory concentration (IC50) was calculated using SPSS software.

[0188] The calculation formula is as follows:

[0189] Inhibition rate = [(Ac-As) / (Ac-Ab)] × 100%

[0190] As: Absorbance of experimental wells (including cells, culture medium, CCK-8 solution and drug solution);

[0191] Ac: Absorbance of control wells (containing cells, culture medium, and CCK-8 solution, but excluding drugs);

[0192] Ab: Absorbance of blank wells (containing culture medium and CCK-8 solution, but excluding cells and drugs).

[0193] Experimental Results: To further evaluate the in vitro antitumor activity of the target compounds, three tumor cell lines—HeLa cells (cervical cancer cells), MCF-7 cells (human breast cancer cells), and A549 cells (human lung cancer cells)—were selected as test tumor lines, with rhein D serving as the control group. The test results are shown in Table 1. Overall, the antitumor activity of the rhein PROTACs was superior to that of the rhein derivative itself. (Combined with IC50 analysis...) 50 Based on the compound structure, DO-2 was selected as the drug group for further proteomics research and analysis.

[0194] Table 1. In vitro antitumor activity (IC50) of D and D1–D18

[0195]

[0196]

[0197] This invention provides the detection of the skin-whitening activity of rhein PROTACs compounds.

[0198] Example 10: Test for Inhibition of Tyrosinase Activity

[0199] This invention tests the inhibition of tyrosinase activity by rhein and its PROTACs derivatives, using arbutin as a positive control, and evaluates the inhibition rate of tyrosinase activity by measuring OD values.

[0200] Experimental Procedure: Prepare tyrosinase solution (400 μ / mL). Weigh 0.01972 g of L-DOPA and dissolve it in 10 mL of 1×PBS (concentration 10 mM). Prepare sample solutions with final concentrations of 250 μM, 125 μM, 62.5 μM, and 31.25 μM using 0.01 M PBS, with arbutin as a positive control. Add 100 μL of sample solution to a 2 mL centrifuge tube, then add 150 μL of L-DOPA, and finally add 50 μL of tyrosinase. Mix well and add 200 μL to a 96-well plate. Measure OD475 using a microplate reader. Use arbutin as a positive control and 0.01 M PBS as a blank control. Perform three replicates for each concentration. The formula for calculating the tyrosinase inhibition rate is as follows:

[0201] Tyrosinase inhibition rate (%) = 100 × (A blank group - A treatment group) / A blank group

[0202] Experimental results: Figure 1 It can be seen that 250 μM D inhibited tyrosinase by 13%, while D6 inhibited tyrosinase by 32%, which was significantly higher than that of D.

[0203] Example 11 Cytotoxicity Test

[0204] 100 μL of B16-F10 cells (Procell Life Science & Technology Co., Ltd., WuHan, China) were added to a 96-well plate (density 5 × 10⁻⁶). 5 Cells were incubated at 37°C in a 5% incubator for 12 hours, followed by the addition of rhein (D) (final concentrations of 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM), with 1% DMSO as a negative control. After 48 hours, the cell supernatant was removed, and 10% CCK-8 solution (Beyotime Biotechnology, Shanghai, China) was added. OD450 was measured after 30 minutes.

[0205] Cell viability (%) = (A with drug - A blank) / (A0 with drug - A blank)

[0206] Adosed is the absorbance of a well containing cells, CCK-8 solution, and drug solution; Ablank is the absorbance of a well containing culture medium and CCK-8 solution but without cells; A0dosed is the absorbance of a well containing cells and CCK-8 solution but without drug.

[0207] Following cytotoxicity testing, the analysis revealed final concentrations of rhein (D) of 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM. Figure 2 , Figure 3 It can be seen that when the drug concentration is below 25 μM, D and D6 have weak killing effects on B16-F10 cells and Haccat cells. When the drug concentration is above 25 μM, the cytotoxicity of D and D6 is smaller, and the cytotoxicity of D6 is smaller than that of D.

[0208] Example 12 Protein Blotting

[0209] This invention first detects the expression of melanin-related proteins in cells after administration of drugs D, D6, and ND6 (negative control). Figure 4 , 5As shown in Figures 6, 7, and 8, to demonstrate that tyrosinase is degraded via ubiquitination, Western blot was used to explore the process by which D6 induces tyrosinase degradation through the ubiquitin-proteasome system (UPS). ND6-mediated downregulation of tyrosinase is irreversible (e.g., ...). Figure 9 , Figure 10 As shown in the figure, this indicates that D6-induced tyrosinase degradation depends on ubiquitination and the proteasome cascade. These results demonstrate that D6 can induce tyrosinase to form a ternary chain with the E3 ligase, ultimately leading to tyrosinase ubiquitination and degradation.

[0210] Example 13 Zebrafish Experiment

[0211] Zebrafish have optically transparent bodies, rapid external development, and short growth cycles. More importantly, their tyrosinase is highly homologous to human tyrosinase, and the presence of melanin on their surface allows for easy observation of the pigmentation process without complex experimental procedures. Due to these advantages, they are commonly used as animal models for pigmentation research.

[0212] Three hours post-fertilization (hpf) in zebrafish embryos, the embryos were treated with rhein (D) at concentrations of 3.125 μM, 6.25 μM, and 12.5 μM. Arbutin was selected as a positive inhibitor, and embryos treated with DMSO served as a negative control.

[0213] Approximately 3 hours after fertilization, 20 zebrafish embryos were transferred into single wells of a 6-well plate containing 10 mL of embryonic fluid. These embryos were incubated with specified concentrations of rhein (D) and arbutin, then cultured at a constant temperature of 28 ± 0.5 °C, with the medium changed every 24 hours. Body pigmentation was assessed after 72 hours. For better observation, the embryos were anesthetized with 0.4% (w / v) tricaine and photographed under a microscope (Olympus, Japan). Figure 11 ), and used ImageJ to calculate the melanin content in zebrafish skin (e.g., Figure 12 It was found that the ability of 12.5 μM to inhibit melanin production was comparable to that of arbutin at the same concentration.

[0214] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. Rhein (PROTAC) compounds or their pharmacologically or physiologically acceptable salts, wherein, The rhein PROTAC is selected from at least one of the following compounds:

2. The use of the rhein PROTAC compound according to claim 1 or its pharmacologically or physiologically acceptable salt in the preparation of an antitumor drug, wherein the antitumor drug is an anticancer drug that inhibits tyrosinase activity.

3. The application according to claim 2, characterized in that: The tumor is at least one of breast cancer, lung cancer, and cervical cancer.

4. The use of the rhein PROTAC compound according to claim 1 or a pharmacologically or physiologically acceptable salt thereof in the preparation of skin whitening cosmetics or medicaments for treating skin diseases, wherein the medicament for treating skin diseases is a drug that treats by inhibiting tyrosinase activity.

5. A method for preparing a PROTAC compound, comprising the following steps: in, 6. A method for preparing a PROTAC compound, characterized in that, Includes the following steps: Where n = 2, 3, 4, 5,

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