Novel FSP1 degradation agent PROTAC compound as well as preparation method and application thereof

Abstract

The invention relates to a novel FSP1 degradation agent PROTAC compound as well as a preparation method and application thereof, and belongs to the technical field of medicinal chemistry. The novel FSP1 degradation agent PROTAC compound is a compound as shown in a formula I, a pharmaceutically acceptable salt of the compound as shown in the formula I, a hydrate of the compound as shown in the formula I or a solvate of the compound as shown in the formula I. The novel FSP1 degradation agent PROTAC compound provided by the invention has the effect of degrading the activity of ferroptosis inhibitory protein 1 (FSP1).

Description

Technical Field

[0001] The present application relates to the field of pharmaceutical chemistry technology, and in particular to a novel FSP1 degrader PROTAC compound and a preparation method and application thereof. Background Art

[0003] The FSP1 protein pathway is a lipid peroxide metabolism regulation pathway. It has the activity of CoQ10-H2 oxidoreductase and is a good LPO capture antioxidant. It can reduce lipid peroxides formed by cells to normal lipid alcohols. FSP1 regulates lipid peroxide metabolism by maintaining the reducing activity of coenzyme Q10. The FSP1-CoQ10-NADPH pathway exists as an independent parallel system, which cooperates with GPX4 and glutathione to inhibit phospholipid peroxidation and ferroptosis. In the absence of GPX4, FSP1 uses NADPH to regenerate ubiquinol from the oxidized form of ubiquinone and inhibits phospholipid peroxidation and ferroptosis in cells. Studies have found that when GPX4 loses its activity, FSP1 maintains the normal growth of cancer cells. High expression of FSP1 can lead to cell resistance to "ferroptosis", thereby causing resistance to "ferroptosis" inducers. When FSP1 activity is inhibited or the FSP1 gene is knocked out, the GPX4 inhibitor RSL3 shows higher cytotoxicity to a variety of tumor cells.

[0004] At present, the research on ferroptosis inducers is still in its early stages, and there are not many studies on FSP1-targeted drugs. FSP1 inhibitor iFSP1 is expressed in many cancer cells, and treatment with the inhibitor iFSP1 can significantly affect the sensitivity of cell lines to ferroptosis. However, the structure-activity relationship of the FSP1 inhibitor iFSP1 is still unclear, so the design of drugs targeting FSP1 is urgently needed. Summary of the invention

[0005] In view of this, the present application provides a novel FSP1 degrader PROTAC compound and its preparation method and application. The compound has been experimentally proven to have the activity of degrading ferroptosis inhibitory protein 1 (FSP1), and can degrade FSP1, block the metabolism of lipid peroxides in cells, accumulate toxic lipid peroxides in LPO (FSP1-CoQ10-NADPH pathway), and induce ferroptosis in cells. FSP1 is highly expressed in a variety of tumors such as fibrosarcoma, lymphoma, breast cancer, lung cancer, and renal cancer. The ferroptosis induced by the compound can effectively kill tumor cells and provide patients with more optional drugs; it can effectively overcome the defects of the above-mentioned prior art.

[0006] In a first aspect, an embodiment of the present application provides a novel FSP1 degradation agent PROTAC compound, wherein the compound is a compound of Formula I, a pharmaceutically acceptable salt of the compound of Formula I, a hydrate of the compound of Formula I, or a solvate of the compound of Formula I, and the structure of Formula I is shown below:

[0007]

[0008] In Formula I, n is a straight-chain alkyl group of 1 to 2 carbon atoms; R is selected from 1,4-dimethylpiperazine, 1-methyl-4-(1-methylazetidin-3-yl)piperazine, 2,2'-(ethane-1,2-diylbis(oxy))bis(N-methylethane-1-amine), 2,2'-oxybis(N-methylethane-1-amine), N1,N5-dimethylpentane-1,5-diamine, N1,N4-di methylbutane-1,4-diamine, 1-methyl-4-((1-methylazetidin-3-yl)methyl)piperazine, 1-methyl-4-((1-methylazetidin-2-yl)methyl)piperazine, 1-methyl-4-((1-methylpiperidin-4-yl)methyl)piperazine, 1-methyl-4-(4-methylpiperidin-1-yl)piperazine or 1-methyl-4-(1-methylpiperidin-4-yl)piperazine.

[0009] In some embodiments that may include the above embodiments, the compound is selected from N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)4-oxobutanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)3-oxopropanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)3-oxopropanamide, [1,2-a]pyridin-1-yl)-3-(3-(4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)azetidin-1-yl)3-oxopropanamide, N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-N4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethyl)succinamide, N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-N4-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethyl)succinamide -3-yl)-1,3-dioxoisoindol-4-yl)amino)butyl)succinamide, N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-N4-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)hexyl)succinamide, N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-N3-(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)pentyl)malonamide, N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)- ,5]imidazo[1,2-a]pyridin-1-yl)-N3-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)butyl)malonamide, N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-N3-(2-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethyl)malonamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(((4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)methyl)azetidin-1-yl)4-oxobutanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-((4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)methyl)azetidin-1-yl)3-oxopropanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(((4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)methyl)azetidin-1-yl)3-oxopropanamide -yl)methyl)piperidin-1-yl)4-oxobutanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(((1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pyrrolidin-2-yl)methyl)piperazin-1-yl)4-oxobutanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(((1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)methyl)piperazin-1-yl)4-oxobutanamide, -(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(4-(1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pyrrolidin-3-yl)piperazin-1-yl)4-oxobutanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-(((1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)methyl)piperazin-1-yl)3-oxopropanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-(((1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)methyl)piperazin-1-yl)3-oxopropanamide, ]imidazo[1,2-a]pyridin-1-yl)-3-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)piperazin-1-yl)-3-oxopropanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridine-1 -yl)-3-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pyrrolidin-3-yl)piperazin-1-yl)3-oxopropanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-N3-(2-(2-(2,(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethoxyethoxyethyl)malonamide, N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-N4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethoxyethoxyethyl)malonamide 4-yl)amino)ethoxy)ethyl)succinamide or N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-(4-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-3-oxopropanamide.

[0010] In some embodiments that may include the above embodiments, R is selected from any of the following structures:

[0011]

[0012] The second aspect of the present application also provides a method for preparing the novel FSP1 degrader PROTAC compound, comprising the following steps:

[0013] iFSP and compound 1 were dissolved in DMF, and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate and N,N-diisopropylethylamine were added to react for 3-5 hours. After the reaction, the mixture was extracted with dichloromethane and water. The organic layer was washed with saturated sodium chloride solution, and the organic layer was dried by spin drying. The sample was mixed with 200-300 mesh silica gel, and the mixture was passed through a column with a volume ratio of dichloromethane:methanol of 50 mL:1 mL to obtain an intermediate compound. The intermediate compound was dissolved in a volume ratio of methanol:water of 5 mL:1 mL. To the mixed solution, lithium hydroxide was added, and the reaction was carried out at room temperature for 3-5 hours. After the reaction was completed, it was extracted with ethyl acetate, and concentrated hydrochloric acid was added to the organic layer to adjust the pH to less than 1, and a solid was precipitated to obtain compound 2; Compound 2 and compound 3 were dissolved in 20 mL of DMF, and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate and N,N-diisopropylethylamine (ie, HATU and DIPEA) were added to react for 5-8 hours. After the reaction was completed, post-treatment was performed to obtain the target compound. The reaction formula is shown below:

[0014]

[0015] In some embodiments that may include the above embodiments, the specific process of the post-treatment is: after the reaction is completed, it is concentrated under reduced pressure, and the target compound is obtained after separation by preparing a thin layer plate.

[0016] The third aspect of the embodiments of the present application also provides a pharmaceutical composition, which contains the above-mentioned novel FSP1 degrader PROTAC compound and optionally a pharmaceutically acceptable additive or excipient.

[0017] The fourth aspect of the embodiments of the present application also provides the use of the above-mentioned novel FSP1 degrader PROTAC compound in the preparation of ferroptosis inducers.

[0018] The fifth aspect of the embodiments of the present application also provides the use of the above-mentioned novel FSP1 degrader PROTAC compound in the preparation of anti-tumor drugs.

[0019] In some embodiments that may include the above embodiments, the tumor is a ferroptotic tumor, and the ferroptotic tumor is selected from fibrosarcoma, lymphoma, breast cancer, lung cancer or kidney cancer.

[0020] In some embodiments that may include the above embodiments, the compound induces ferroptosis by degrading FSP1, thereby inhibiting the survival of tumor cells and achieving an anti-tumor effect.

[0021] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0022] The novel FSP1 degrader PROTAC compounds of the embodiments of the present application have significant inhibitory effects on the activity of ferroptosis inhibitory protein 1 (FSP1). The compounds have been experimentally proven to have significant inhibitory effects on the activity of ferroptosis inhibitory protein 1 (FSP1). They can inhibit FSP1, block the metabolism of lipid peroxides in cells, accumulate toxic lipid peroxides in LPO (FSP1-CoQ10-NADPH pathway), and induce ferroptosis in cells. FSP1 is highly expressed in a variety of tumors such as fibrosarcoma, lymphoma, breast cancer, lung cancer, and renal cancer. The ferroptosis induced by the compounds effectively kills tumor cells, giving patients more optional drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 Western blotting experiments verify the degradation level of FSP1 degrader Example 7 (2307) at different concentrations, where the abscissa is the concentration of 2307, the ordinate is the protein level, and the EC50 value is 131.7 nM;

[0025] Figure 2 This is a diagram of the cell degradation activity of the novel FSP1 degrader PROTAC compounds of some embodiments of the present application (Example 1 (2301), Example 2 (2302), Example 3 (2303), Example 4 (2304), Example 5 (2305), Example 6 (2306), Example 7 (2307), Example 8 (2308), Example 9 (2309), Example 10 (2310), Example 11 (2311), Example 12 (2312), Example 13 (2313), Example 14 (2314), Example 15 (2315), Example 16 (2316), Example 17 (2317), Example 18 (2318), Example 19 (2319) and Example 20 (2320)). The degradation level of the FSP1 degrader was verified by Western blotting experiment, wherein the abscissa is the compound name and the ordinate is the protein level. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0027] In the following examples and comparative examples, unless otherwise specified, all raw materials can be obtained by commercial purchase or conventional methods.

[0028] The term "substituted" in the examples of the present application means that one or more hydrogen atoms in the group are independently replaced by a corresponding number of substituent groups.

[0029] In the examples of the present application, the additives or excipients are substances other than the active ingredients, which have been reasonably evaluated in terms of safety and are included in the pharmaceutical preparations, and may be gum arabic, syrup, lanolin, starch, magnesium chloride, cyclodextrin, sebacic acid, dextrin, medicinal calcium sulfate, glycerol, mannitol, sorbitol, inositol, thiol, tromethamine, phenol, m-cresol, benzyl alcohol, parabens, methyl paraben, tert-butyl alcohol, benzalkonium chloride, chlorobutanol, thimerosal, etc.

[0030] The derivatives of the compounds of general formula I in the examples of the present application are used as prodrugs, which means that they themselves may have weak activity or even no activity, but after administration, they are converted into corresponding biologically active forms under physiological conditions (for example, by metabolism, solvent decomposition or other means).

[0031] The chemical structures of the examples of the present application were confirmed by 1H-NMR and 13C-NMR. Flash column chromatography was performed on silica gel H (10-40 μM).

[0032] Example 1

[0033] The preparation process of N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)4-oxobutanamide is as follows:

[0034] 1-amino-3-(p-tolyl)benzo[4,5]imidazo[1,2]pyridine-2,4-dicarbonitrile (iFSP) and 4-methoxy-4-oxobutyric acid were dissolved in DMF, and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate and N,N-diisopropylethylamine were added to react for 3-5 hours. After the reaction, the mixture was extracted with dichloromethane and water, and the organic layer was washed with saturated sodium chloride solution. The organic layer was dried by spin drying, and the sample was mixed with 200-300 mesh silica gel. The mixed solution with a volume ratio of dichloromethane:methanol of 50 mL:1 mL was passed through a column to obtain 4-(2,4-dicyano-3-(m-tolyl)benzene 4-((2,4-dicyano-3-(m-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)amino)-4-oxobutyric acid methyl ester; 4-(2,4-dicyano-3-(m-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)amino)-4-oxobutyric acid methyl ester is dissolved in a mixed solution of methanol: water in a volume ratio of 5mL:1mL, lithium hydroxide is added, and the reaction is carried out at room temperature for 3-5h. After the reaction is completed, it is extracted with ethyl acetate, and concentrated hydrochloric acid is added to the organic layer to adjust the pH to less than 1, and a solid is precipitated to obtain 4-((2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)amino)-4-oxobutyric acid.

[0035] 4-((2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)amino)-4-oxobutanoic acid, 2-(2,6-dioxopiperidin-3-yl)-4-(4-methylpiperazine-1-yl)isoindole-1,3-dione and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate were dissolved in 5 mL of N,N-dimethylformamide, and N,N-diisopropylethylamine was added to react at room temperature for 5 h. After the reaction was completed, the mixture was extracted three times with dichloromethane and water, and the organic layer was taken and extracted with a saturated sodium chloride solution. The organic layer was concentrated in vacuo, mixed with 200-300 mesh silica gel, and purified with a mixed solution of dichloromethane:methanol in a volume ratio of 50 mL:1 mL to obtain the target compound with a yield of 95%.

[0036] The structure of the target compound is shown below:

[0037]

[0038] 1H NMR(400MHz, DMSO-d6)δ9.01(s,3H),7.71(td,J=7.9,5.4Hz,1H),7.56–7.46(m,2H),7. 43(d,J=8.0Hz,1H),7.38(dd,J=7.0,3.1Hz,3H),7.36–7.33(m,1H),7.33–7.29(m,1H), 5.10(dd,J=12.9,5.3Hz,1H),3.67(s,4H),2.79(d,J=26.0Hz,2H),2.41(d,J=7.0Hz,4H ),2.33(d,J=6.6Hz,1H),2.03(d,J=12.8Hz,1H),1.34–1.26(m,2H),1.26–1.21(m,5H).

[0039] Example 2

[0040] N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazine-1-yl)3-oxopropanamide. The synthetic route of this embodiment is the same as that of Example 1.

[0041] The structure of the target compound is shown below:

[0042]

[0043] 1H NMR (400MHz, DMSO-d6) δ8.69(d,J=8.2Hz,1H),7.72(d,J=8.1Hz,1H),7.67(dd ,J=8.5,7.2Hz,1H),7.44(dd,J=8.2,1.9Hz,3H),7.36(d,J=2.7Hz,2H),7.34( d,J=1.9Hz,2H),7.29–7.25(m,2H),5.09(dd,J=12.9,5.4Hz,1H),3.79–3.68( m,5H),3.62(s,3H),2.60(s,1H),2.41(s,4H),2.05–1.96(m,1H),1.23(s,2H).

[0044] Example 3

[0045] N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-(3-(4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazine-1-yl)azetidine-1-yl)3-oxopropanamide. The synthetic route of this embodiment is the same as that of Example 1.

[0046] The structure of the target compound is shown below:

[0047]

[0048] 1H NMR(400MHz,DMSO-d6)δ8.71(dd,J=12.2,8.2Hz,1H),7.75–7.66(m,2H),7.49–7.40(m ,4H),7.38–7.34(m,3H),7.34–7.27(m,2H),5.08(dd,J=12.8,5.5Hz,1H),4.29(s,1H) ,4.14(s,1H),3.94(t,J=8.6Hz,1H),3.79(s,1H),3.22(q,J=7.2,5.8Hz,6H),2.58(d, J=17.1Hz,2H),2.41(s,4H),2.07–1.98(m,1H),1.36–1.28(m,1H),1.27–1.21(m,3H).

[0049] Example 4

[0050] N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridine-1-yl)-N4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethyl)succinamide. The synthesis route of this embodiment is the same as that of Example 1.

[0051] The structure of the target compound is shown below:

[0052]

[0053] 1H NMR (500MHz, DMSO-d6) δ8.94(d,J=8.2Hz,1H),8.21(t,J=5.6Hz,1H),7.93(d,J=8.1Hz,1H),7.53(ddd,J=11.9,5.9,2 .5Hz,2H),7.29(d,J=1.7Hz,4H),7.07(d,J=8.6Hz,1H),6.98(d,J=7.1Hz,1H),6.55(t,J=5.8Hz,1H),5.03(dd,J=12. 7,5.4Hz,1H),3.56(t,J=5.5Hz,3H),3.50(s,4H),3.48(dd,J=6.0,3.5Hz,3H),3.20(q,J=5.8Hz,2H),3.08(t,J=7.0H z,2H),2.84(q,J=6.9Hz,2H),2.60–2.54(m,2H),2.42(s,3H),2.01(ddd,J=12.9,5.7,3.3Hz,1H),0.88–0.78(m,3H).

[0054] Example 5

[0055] N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridine-1-yl)-N4-(4-((2-(2,6-dioxopiperidine-3-yl)-1,3-dioxoisoindol-4-yl)amino)butyl)succinamide. The synthesis route of this embodiment is the same as that of Example 1.

[0056] The structure of the target compound is shown below:

[0057]

[0058] 1H NMR(400MHz,DMSO-d6)δ11.08(s,1H),8.63(s,1H),7.85(s,1H),7.69(s,1H),7.55(s,1H ),7.41(d,J=7.9Hz,3H),7.34(d,J=8.0Hz,2H),7.25(s,1H),7.08(s,1H),6.99(s,1H),6 .52(s,1H),5.04(s,1H),3.26(s,2H),2.88(s,3H),2.67(t,J=7.6Hz,2H),2.55(s,1H),2 .43(s,2H),2.41(s,3H),2.01(s,1H),1.54(s,2H),1.45(s,2H),1.24(d,J=10.2Hz,2H).

[0059] Example 6

[0060] N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridine-1-yl)-N4-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)hexyl)succinamide. The synthesis route of this embodiment is the same as that of Example 1.

[0061] The structure of the target compound is shown below:

[0062]

[0063] 1H NMR (500MHz, DMSO-d6) δ11.08(s,1H),8.98(d,J=8.2Hz,1H),8.14(t,J=5.7Hz,1H),7.94(d,J=8.1Hz,1H),7.65–7.6 0(m,1H),7.57–7.52(m,2H),7.28(s,4H),7.00(d,J=7.0Hz,1H),6.96(d,J=8.6Hz,1H),6.40(t,J=5.9Hz,1H),5.03(d d,J=12.7,5.4Hz,1H),4.47–4.26(m,2H),3.09(t,J=6.8Hz,2H),3.04(q,J=6.6Hz,4H),2.83(t,J=6.9Hz,2H),2.41( s,3H),2.18(t,J=7.4Hz,1H),2.04–1.99(m,1H),1.49–1.45(m,2H),1.34(d,J=7.8Hz,4H),1.30(s,1H),1.26(s,2H).

[0064] Example 7

[0065] N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridine-1-yl)-N3-(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)pentyl)malonamide. The synthesis route of this embodiment is the same as that of Example 1.

[0066] The structure of the target compound (labeled as 2307) is shown below:

[0067]

[0068] 1H NMR (400MHz, DMSO-d6) δ11.09(s,1H),8.68(d,J=8.2Hz,1H),7.75(d,J=8.1Hz,1H),7.55(q,J=7.8,6.8Hz,1H),7 .45(d,J=8.1Hz,3H),7.37(d,J=7.9Hz,2H),7.28(t,J=7.7Hz,1H),7.05–6.95(m,2H),6.46(t,J=5.9Hz,1H),5.04 (dd,J=12.9,5.4Hz,1H),3.60(pd,J=6.6,3.8Hz,1H),3.22–3.07(m,5H),2.95–2.80(m,2H),2.71(d,J=17.2Hz,2H ), 2.56 (s, 1H), 2.42 (s, 3H), 2.03 (ddt, J = 13.1, 5.9, 3.4Hz, 1H), 1.48 (dp, J = 14.7, 7.1Hz, 4H), 1.36–1.29 (m, 2H).

[0069] Example 8

[0070] N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridine-1-yl)-N3-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)butyl)malonamide. The synthetic route of this embodiment is the same as that of Example 1.

[0071] The structure of the target compound is shown below:

[0072]

[0073] 1H NMR(400MHz,DMSO-d6)δ11.08(s,1H),7.66(s,1H),7.57(s,2H),7.42(d, J=7.9Hz,2H),7.36(s,2H),7.22(s,1H),7.12(s,1H),6.98(s,2H),6.52(s ,2H),5.07(s,1H),3.56(s,1H),3.50(s,3H),3.14(s,3H),2.97(s,1H),2. 41(s,3H),2.00(d,J=15.2Hz,2H),1.60(s,2H),1.26(s,2H),1.23(s,2H).

[0074] Example 9

[0075] N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-N3-(2-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethyl)malonamide. The synthetic route of this embodiment is the same as that of Example 1.

[0076] The structure of the target compound is shown below:

[0077]

[0078] 1H NMR (400MHz, DMSO-d6) δ11.06(s,1H),9.57(s,1H),8.60(s,1H),7.70(d,J=7.8Hz,1H),7.61–7.52(m,1H),7.42(d,J=8.0Hz,3H),7.35(d,J= 7.9Hz,2H),7.23(dt,J=16.5,8.0Hz,2H),7.18–7.11(m,1H),5.07(d,J=8.6Hz,1H),4.63(s,1H),3.88(d,J=7.4Hz,1H),3.60–3.44(m,2H),3 .22(d,J=13.0Hz,2H),2.98–2.76(m,2H),2.74–2.66(m,2H),2.65–2.57(m,2H),2.57–2.52(m,1H),2.44(d,J=9.9Hz,1H),2.41(s,3H),2.36 (d,J=5.9Hz,1H),2.27(d,J=7.0Hz,1H),2.20–2.10(m,1H),1.98(dd,J=11.5,6.2Hz,2H),1.90–1.72(m,2H),1.38–1.25(m,2H),1.23(s,2H).

[0079] Example 10

[0080] N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(((4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazine-1-yl)methyl)azetidine-1-yl)4-oxobutanamide. The synthetic route of this embodiment is the same as that of Example 1.

[0081] The structure of the target compound is shown below:

[0082]

[0083] 1H NMR (400MHz, DMSO-d6) δ11.09(s,1H),9.80(s,1H),7.73(t,J=8.1Hz,1H),7.63(t,J=7.8Hz,1H),7 .46–7.40(m,3H),7.40–7.33(m,3H),7.33–7.26(m,2H),7.26–7.20(m,1H),5.20–4.94(m,1H),4.26 (t,J=8.4Hz,1H),3.93(s,4H),3.62–3.55(m,2H),2.96–2.83(m,5H),2.69(d,J=7.3Hz,1H),2.60( d,J=18.1Hz,2H),2.38(d,J=15.2Hz,4H),2.30–2.17(m,3H),2.09–1.96(m,1H),1.31–1.21(m,3H).

[0084] Embodiment 11

[0085] N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-((4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazine-1-yl)methyl)azetidine-1-yl)3-oxopropanamide. The synthetic route of this embodiment is the same as that of Example 1.

[0086] The structure of the target compound is shown below:

[0087]

[0088] 1H NMR(400MHz,DMSO-d6)δ9.90(s,2H),7.73(s,1H),7.57(s,1H),7.49(s,1H),7.42(d,J=7.3 Hz,3H),7.39–7.29(m,4H),7.23(s,1H),7.14(s,2H),4.54(s,1H),4.02(s,2H),3.60(s,1H) ,3.56(d,J=7.2Hz,1H),3.44(d,J=2.5Hz,4H),3.27(s,3H),2.96(s,1H),2.89(d,J=9.8Hz,2 H), 2.60 (s, 1H), 2.40 (s, 3H), 2.37 (s, 1H), 2.28 (s, 2H), 2.18 (s, 1H), 1.23 (d, J = 9.3Hz, 2H).

[0089] Example 12

[0090] N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(((4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazine-1-yl)methyl)piperidin-1-yl)4-oxobutanamide. The synthetic route of this embodiment is the same as that of Example 1.

[0091] The structure of the target compound is shown below:

[0092]

[0093] 1H NMR (400MHz, DMSO-d6) δ11.06(s,1H),9.44(s,1H),8.60(s,1H),7.68(d,J=8.0Hz,1H),7.56(t,J=8.3Hz,1H),7.46–7.37(m,3H),7.34(d ,J=8.0Hz,2H),7.22(dd,J=15.2,7.3Hz,2H),7.15(dd,J=12.1,6.4Hz,1H),5.05(s,1H),4.60(s,1H),3.87(s,1H),3.50(d,J=32.5Hz,2H ),3.21(d,J=10.4Hz,2H),2.85(dd,J=18.3,4.6Hz,2H),2.66(d,J=5.8Hz,2H),2.63–2.57(m,2H),2.54(s,1H),2.44(d,J=8.0Hz,1H),2. 40(s,3H),2.38–2.29(m,3H),2.29–2.22(m,1H),2.21–2.06(m,2H),1.98(dd,J=10.1,5.0Hz,2H),1.89–1.66(m,2H),1.38–1.20(m,3H).

[0094] Example 13

[0095] N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(((1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pyrrolidin-2-yl)methyl)piperazine-1-yl)4-oxobutanamide. The synthetic route of this embodiment is the same as that of Example 1.

[0096] The structure of the target compound is shown below:

[0097]

[0098] 1H NMR (400MHz, DMSO-d6) δ11.06(s,1H),9.57(s,1H),8.60(s,1H),7.70(d,J=7.8Hz,1H),7.61–7.52(m,1H),7.42(d,J=8.0Hz,3H),7.35(d,J= 7.9Hz,2H),7.23(dt,J=16.5,8.0Hz,2H),7.18–7.11(m,1H),5.07(d,J=8.6Hz,1H),4.63(s,1H),3.88(d,J=7.4Hz,1H),3.60–3.44(m,2H),3 .22(d,J=13.0Hz,2H),2.98–2.76(m,2H),2.74–2.66(m,2H),2.65–2.57(m,2H),2.57–2.52(m,1H),2.44(d,J=9.9Hz,1H),2.41(s,3H),2.36 (d,J=5.9Hz,1H),2.27(d,J=7.0Hz,1H),2.20–2.10(m,1H),1.98(dd,J=11.5,6.2Hz,2H),1.90–1.72(m,2H),1.38–1.25(m,2H),1.23(s,2H).

[0099] Embodiment 14

[0100] N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(4-(1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)piperazine-1-yl)4-oxobutanamide, the synthetic route of the application example is the same as that of Example 1.

[0101] The structure of the target compound is shown below:

[0102]

[0103] 1H NMR(500MHz,Chloroform-d)δ9.14(s,1H),8.20(dd,J=7.5,1.6Hz,1H),7.81(dd,J=7.4,1.5Hz,1H),7.71(s,1H),7.55–7.47(m,4H) ,7.41(td,J=7.8,2.1Hz,2H),7.35–7.29(m,3H),5.29(t,J=6.9Hz,1H),4.26(ddt,J=27.1,12.4,7.1Hz,2H),3.67(dt,J=12.6,7.2Hz ,1H),3.63–3.56(m,5H),3.01–2.79(m,1H),2.66(t,J=5.2Hz,4H),2.61–2.54(m,2H),2.50(td,J=5.0,1.3Hz,2H),2.46(td,J=4.9,1 .3Hz,2H),2.40(q,J=7.0Hz,1H),2.33(s,3H),2.13–2.00(m,2H),1.95(dq,J=13.9,7.0Hz,1H),1.78(ddt,J=32.1,13.3,6.9Hz,2H).

[0104] Embodiment 15

[0105] N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(((1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pyrrolidin-3-yl)methyl)piperazine-1-yl)4-oxobutanamide. The synthetic route of this embodiment is the same as that of Example 1.

[0106] The structure of the target compound is shown below:

[0107]

[0108] 1H NMR (400MHz, DMSO-d6) δ11.08(s,1H),9.60(s,1H),8.62(d,J=8.2Hz,1H),7.74–7.60(m,2H),7.42(dd,J= 13.0,8.1Hz,3H),7.33(dd,J=7.7,3.6Hz,3H),7.30–7.21(m,2H),5.08(dd,J=12.8,5.4Hz,1H),3.76(d,J= 11.9Hz,3H),3.55(d,J=7.2Hz,1H),3.44(s,2H),2.94–2.79(m,5H),2.70(dd,J=15.2,5.7Hz,5H),2.62–2 .52(m,2H),2.39(s,3H),2.28(dd,J=12.0,5.7Hz,1H),2.07–1.90(m,3H),1.71(s,2H),1.38–1.20(m,2H).

[0109] Example 16

[0110] N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(((1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)methyl)piperazine-1-yl)4-oxobutanamide. The synthetic route of this embodiment is the same as that of Example 1.

[0111] The structure of the target compound is shown below:

[0112]

[0113] 1H NMR(400MHz, DMSO-d6)δ9.49(s,1H),7.76–7.59(m,3H),7.48–7.39(m,3H),7.33(dd,J=13.0, 5.6Hz,3H),7.30–7.21(m,3H),5.08(dd,J=13.0,5.5Hz,1H),3.67(s,4H),2.86(p,J=8.6Hz,4H ),2.76–2.64(m,4H),2.58(d,J=17.8Hz,2H),2.40(s,4H),2.30–2.24(m,2H),2.01(dd,J=14. 1,7.2Hz,2H),1.82(s,4H),1.37(d,J=10.6Hz,1H),1.35–1.31(m,1H),1.24(d,J=10.0Hz,3H).

[0114] Embodiment 17

[0115] N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(4-(1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pyrrolidin-3-yl)piperazine-1-yl)4-oxobutanamide. The synthetic route of this embodiment is the same as that of Example 1.

[0116] The structure of the target compound is shown below:

[0117]

[0118] 1H NMR(400MHz,DMSO-d6)δ11.05(s,1H),9.94(s,2H),7.67(dd,J=26.3,8.0Hz,1H),7.59–7 .45(m,2H),7.42(dt,J=8.2,3.2Hz,2H),7.36–7.33(m,1H),7.30–7.22(m,2H),7.16–7.0 2(m,3H),5.06(dd,J=12.9,5.5Hz,1H),2.93–2.79(m,2H),2.69(dd,J=13.8,5.8Hz,4H), 2.58(d,J=17.8Hz,3H),2.40(s,4H),2.15–1.94(m,3H),1.69(s,1H),1.31–1.21(m,3H).

[0119] Embodiment 18

[0120] N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-(((1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)methyl)piperazine-1-yl)3-oxopropanamide. The synthetic route of this embodiment is the same as that of Example 1.

[0121] The structure of the target compound is shown below:

[0122]

[0123] 1H NMR(500MHz,Chloroform-d)δ9.13(s,1H),8.12(dd,J=7.5,1.4Hz,1H),7.81(dd,J=7.4,1.5Hz,1H),7.71(s,1 H),7.55–7.51(m,1H),7.50–7.44(m,3H),7.41(ddd,J=11.9,7.3,2.1Hz,2H),7.34–7.27(m,3H),5.28(t,J=6. 9Hz,1H),4.35–4.08(m,2H),3.66–3.56(m,6H),3.07(s,2H),2.96–2.85(m,1H),2.65–2.54(m,4H),2.33(d,J= 1.2Hz,3H),2.21(t,J=5.4Hz,4H),2.13–2.03(m,1H),1.86–1.76(m,1H),1.66–1.57(m,1H),1.51–1.40(m,3H).

[0124] Embodiment 19

[0125] N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)piperazine-1-yl)-3-oxopropanamide. The synthetic route of this embodiment is the same as that of Example 1.

[0126] The structure of the target compound is shown below:

[0127]

[0128] 1H NMR(400MHz,DMSO-d6)δ11.08(s,1H),9.60(s,2H),8.66(d,J=8.2Hz,1H),7.77–7.6 1(m,2H),7.51–7.40(m,3H),7.33(ddd,J=15.7,8.1,3.4Hz,4H),5.09(dd,J=12.8,5 .5Hz,1H),3.73(d,J=13.3Hz,2H),3.66–3.55(m,1H),2.92–2.79(m,3H),2.64–2.52 (m,1H),2.41(s,3H),2.07–1.98(m,1H),1.79(d,J=95.4Hz,2H),1.35–1.22(m,3H).

[0129] Embodiment 20

[0130] N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pyrrolidin-3-yl)piperazine-1-yl)3-oxopropanamide. The synthetic route of this embodiment is the same as that of Example 1.

[0131] The structure of the target compound is shown below:

[0132]

[0133] 1H NMR (500MHz, DMSO-d6) δ11.09(s,1H),8.67(d,J=8.2Hz,1H),7.73(d,J=8.1Hz,1H),7.66( td,J=7.5,6.6,4.2Hz,1H),7.51–7.45(m,1H),7.44–7.41(m,2H),7.38–7.32(m,3H),7.32 –7.25(m,3H),3.70(d,J=11.7Hz,4H),3.59(t,J=3.3Hz,4H),3.50(s,3H),3.19–3.00(m,2 H), 2.82 (d, J = 12.1Hz, 2H), 2.68 (s, 1H), 2.58 (td, J = 18.5, 17.7, 4.9Hz, 2H), 2.41 (s, 4H).

[0134] Embodiment 21

[0135] O-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pyrrolidin-3-yl)methyl)piperazine-1-yl)-3-oxopropanamide, the synthetic route of the application example is the same as that of Example 1.

[0136] The structure of the target compound is shown below:

[0137]

[0138] 1H NMR(500MHz,Chloroform-d)δ8.76(s,1H),7.75(ddd,J=8.6,7.5,1.5Hz,2H),7.66(s,1H),7.54–7.44(m,3H),7.39(t,J=7.4Hz,1H),7.27 (dtd,J=7.4,4.0,1.5Hz,3H),7.17(ddd,J=28.9,7.6,2.1Hz,2H),5.35–5.26(m,1H),4.33(d,J=12.4Hz,1H),4.05–3.89(m,2H),3.74–3.61 (m,2H),3.54(td,J=12.3,3.7Hz,1H),3.41–3.26(m,2H),3.05(ddd,J=12.5,3.7,1.5Hz,1H),2.95–2.80(m,2H),2.75(ddd,J=12.7,3.6,1. 3Hz,1H),2.60–2.43(m,3H),2.28(d,J=1.3Hz,3H),2.23–2.13(m,2H),2.11–1.98(m,2H),1.88–1.67(m,3H),1.26(dq,J=13.7,7.0Hz,1H).

[0139] Embodiment 22

[0140] N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pyrrolidin-2-yl)methyl)piperazine-1-yl)-3-oxopropanamide, the synthetic route of the application example is the same as that of Example 1.

[0141] The structure of the target compound is shown below:

[0142]

[0143] 1H NMR(500MHz,Chloroform-d)δ7.81(ddd,J=7.6,3.8,1.5Hz,2H),7.57–7.42(m,4H),7.36–7.20(m,5H),5.28(t,J =6.9Hz,1H),4.48(td,J=12.3,3.3Hz,1H),4.14(d,J=12.4Hz,1H),4.00–3.87(m,2H),3.69(d,J=12.2Hz,1H),3.6 1–3.41(m,4H),3.37(ddd,J=12.6,3.3,1.4Hz,1H),3.05(td,J=12.3,3.7Hz,1H),2.96–2.81(m,2H),2.65–2.53( m,2H),2.43(dd,J=12.4,7.1Hz,1H),2.35–2.26(m,4H),2.19–2.02(m,4H),1.84–1.72(m,1H),1.55–1.43(m,1H).

[0144] Embodiment 23

[0145] N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridine-1-yl)-N3-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethoxyethoxyethyl)malonamide. The synthetic route of this embodiment is the same as that of Example 1.

[0146] The structure of the target compound is shown below:

[0147]

[0148] 1H NMR (500MHz, DMSO-d6) δ8.69(dd,J=8.2,2.6Hz,1H),8.17(s,1H),7.72(dd,J=8.1,3.2Hz,1H),7.59–7.49(m,2H),7.43(dd,J= 8.2,2.0Hz,2H),7.37–7.33(m,2H),7.26(td,J=7.9,3.1Hz,1H),7.10–6.95(m,3H),6.55(dt,J=14.1,5.8Hz,1H),3.61(ddq,J =9.6,5.7,3.0,2.4Hz,2H),3.55(t,J=5.5Hz,2H),3.51–3.47(m,4H),3.46(d,J=2.7Hz,3H),3.43–3.41(m,3H),3.31(s,3H),3 .29–3.24(m,3H),2.41(s,4H),2.26(td,J=7.7,6.4,3.3Hz,1H),2.01(ddt,J=13.7,10.3,5.6Hz,1H),1.24(d,J=13.4Hz,3H).

[0149] Embodiment 24

[0150] N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridine-1-yl)-N4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethyl)succinamide. The synthetic route of this embodiment is the same as that of Example 1.

[0151] The structure of the target compound is shown below:

[0152]

[0153] 1H NMR(500MHz,DMSO-d6)δ13.01(s,1H),8.96(dd,J=8.3,4.4Hz,1H),8.21(td,J=5.7,2.7Hz,1H),7.96–7.90( m,1H),7.64–7.59(m,1H),7.54(ddt,J=8.3,6.2,4.3Hz,2H),7.29(t,J=1.2Hz,4H),7.09–6.96(m,2H),6.54( q,J=6.0Hz,1H),3.50(dq,J=5.4,3.0,2.3Hz,3H),3.44(s,2H),3.40(td,J=5.9,1.8Hz,3H),3.23(qd,J=5.8, 3.4Hz, 2H), 3.09 (td, J=6.9, 4.2Hz, 2H), 2.85 (q, J=7.0Hz, 2H), 2.42 (s, 3H), 2.27–2.20 (m, 2H), 1.23 (s, 5H).

[0154] Embodiment 25

[0155] N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(3-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)azetidine-1-yl)4-oxobutanamide, the synthetic route of the application example is the same as that of Example 1.

[0156] The structure of the target compound is shown below:

[0157]

[0158] 1H NMR(500MHz,Chloroform-d)δ9.15(s,1H),8.04(dd,J=7.4,1.5Hz,1H),7.81(dd,J=7.5,1.5Hz,1H),7.71(s,1H),7.69–7.61(m,2H),7.53(td,J=7 .4,1.6Hz,1H),7.45(t,J=7.5Hz,1H),7.37–7.30(m,3H),7.25(ddd,J=13 .9,7.5,2.0Hz,2H),5.32(t,J=6.8Hz,1H),4.05(dd,J=11.3,7.1Hz,1H),3 .97(dd,J=11.2,6.9Hz,1H),3.82(dd,J=11.2,6.9Hz,1H),3.72(dd,J=11.2,7.0Hz,1H),2.98(t,J=5.1Hz,4H),2.93–2.84(m,1H),2.75(p,J=7.1Hz ,1H),2.67(t,J=5.1Hz,4H),2.63–2.54(m,2H),2.50(td,J=4.7,1.2Hz,2 H), 2.46 (td, J = 4.8, 1.3Hz, 2H), 2.33 (d, J = 1.4Hz, 3H), 2.12–2.03 (m, 1H).

[0159] Embodiment 26

[0160] N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-(4-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazine-1-yl)methyl)piperidin-1-yl)-3-oxopropanamide. The synthetic route of this embodiment is the same as that of Example 1.

[0161] The structure of the target compound is shown below:

[0162]

[0163] 1H NMR (500MHz, DMSO-d6) δ11.09(s,1H),8.67(d,J=8.3Hz,1H),7.73(d,J=8.0Hz,1H),7.47(d,J=7.6Hz,1H) ,7.44(d,J=7.9Hz,3H),7.36(d,J=7.9Hz,4H),7.28(t,J=7.8Hz,2H),4.59–4.38(m,2H),4.04(d,J=12.5Hz ,1H),3.55(s,2H),3.51(s,1H),3.44(s,3H),3.07(t,J=12.5Hz,2H),2.94–2.81(m,3H),2.65–2.55(m,3H) ,2.41(s,5H),2.31–2.15(m,3H),2.02(ddd,J=12.7,7.7,4.9Hz,1H),1.71(t,J=16.6Hz,2H),1.22(s,3H).

[0164] Application Examples

[0165] active:

[0166] Cell activity: Western blotting was used for testing. The testing process is as follows:

[0167] 1. Sample preparation: First, proteins need to be extracted from cells or tissues. Usually, protein lysis buffer is used to lyse cells or tissues, and then the supernatant is obtained as a sample by centrifugation.

[0168] 2. Electrophoresis: The extracted protein samples are subjected to polyacrylamide gel electrophoresis to separate proteins of different molecular weights.

[0169] 3. Membrane transfer: Transfer the proteins on the gel after electrophoresis to a solid support using a polyvinylidene fluoride membrane.

[0170] 4. Blocking: Place the transferred membrane in blocking solution and use skim milk to prevent nonspecific binding.

[0171] 5. Primary antibody incubation: Add specific antibodies to bind to the target protein, usually incubating overnight at 4°C to ensure sufficient binding.

[0172] 6. Secondary antibody incubation: Incubate with a secondary antibody corresponding to the species of the primary antibody, usually at room temperature for 1 hour.

[0173] 7. Chemiluminescent detection: Use chemiluminescent reagents to detect bound antibodies, and use chemiluminescent imaging systems for image acquisition and analysis.

[0174] When the internal reference remains the same, the protein expression of the target gene decreases with the increase of drug concentration. Therefore, Example 7 (Compound 2307) exhibits a significant inhibitory effect in breast cancer cells MDA-MB-231, with an EC50 value of 131.7 nM.

[0175] like Figure 1 As shown, Example 7 (Compound 2307) has an inhibitory effect on FSP1 in vitro: Example 7 (Compound 2307) exhibits a significant inhibitory effect on FSP1.

[0176] like Figure 2 As shown, some of the examples have extracellular effects on FSP1 inhibitors. Example 1 (2301), Example 2 (2302), Example 3 (2303), Example 4 (2304), Example 5 (2305), Example 6 (2306), Example 7 (2307), Example 8 (2308), Example 9 (2309), Example 10 (2310), Example 11 (2311), Example 12 (2312), Example 13 (2313), Example 14 (2314), Example 15 (2315), Example 16 (2316), Example 17 (2317), Example 18 (2318), Example 19 (2319) and Example 20 (2320) have extracellular effects on FSP1 inhibitors.

[0177] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A novel FSP1 degrader PROTAC compound, characterized in that: The compound is a compound of Formula I, a pharmaceutically acceptable salt of the compound of Formula I, a hydrate of the compound of Formula I, or a solvate of the compound of Formula I, and the structure of Formula I is shown below: In Formula I, n is a straight-chain alkyl group of 1 to 2 carbon atoms; R is selected from 1,4-dimethylpiperazine, 1-methyl-4-(1-methylazetidin-3-yl)piperazine, 2,2'-(ethane-1,2-diylbis(oxy))bis(N-methylethane-1-amine), 2,2'-oxybis(N-methylethane-1-amine), N1,N5-dimethylpentane-1,5-diamine, N1,N4-di methylbutane-1,4-diamine, 1-methyl-4-((1-methylazetidin-3-yl)methyl)piperazine, 1-methyl-4-((1-methylazetidin-2-yl)methyl)piperazine, 1-methyl-4-((1-methylpiperidin-4-yl)methyl)piperazine, 1-methyl-4-(4-methylpiperidin-1-yl)piperazine or 1-methyl-4-(1-methylpiperidin-4-yl)piperazine.

2. The novel FSP1 degrader PROTAC compound according to claim 1, characterized in that The compound is selected from N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)4-oxobutanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)3-oxopropanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)- )-3-(3-(4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)azetidin-1-yl)3-oxopropanamide, N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-N4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethyl)succinamide, N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-N4-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3 -dioxoisoindol-4-yl)amino)butyl)succinamide, N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-N4-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)hexyl)succinamide, N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-N3-(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)pentyl)malonamide, N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl) oxazolo[1,2-a]pyridin-1-yl)-N3-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)butyl)malonamide, N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-N3-(2-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethyl)malonamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(((4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)methyl)azetidin-1-yl)4-oxobutanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-((4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)methyl)azetidin-1-yl)3-oxopropanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(((4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)methyl)azetidin-1-yl)3-oxopropanamide -yl)methyl)piperidin-1-yl)4-oxobutanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(((1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pyrrolidin-2-yl)methyl)piperazin-1-yl)4-oxobutanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(((1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)methyl)piperazin-1-yl)4-oxobutanamide, -(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(4-(1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pyrrolidin-3-yl)piperazin-1-yl)4-oxobutanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-(((1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)methyl)piperazin-1-yl)3-oxopropanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-(((1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)methyl)piperazin-1-yl)3-oxopropanamide, ]imidazo[1,2-a]pyridin-1-yl)-3-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperidin-4-yl)piperazin-1-yl)-3-oxopropanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridine-1 -yl)-3-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)pyrrolidin-3-yl)piperazin-1-yl)3-oxopropanamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-N3-(2-(2-(2,(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethoxyethoxyethyl)malonamide, N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-N4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)ethoxy)ethoxyethoxyethyl)malonamide 4-yl)amino)ethoxy)ethyl)succinamide or N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-3-(4-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)-3-oxopropanamide.

3. The novel FSP1 degrader PROTAC compound according to claim 1, characterized in that The R is selected from any of the following structures:

4. A method for preparing a novel FSP1 degrader PROTAC compound according to any one of claims 1 to 3, characterized in that: The following steps are involved: iFSP and compound 1 were dissolved in DMF, and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate and N,N-diisopropylethylamine were added to react for 3-5 hours. After the reaction, the mixture was extracted with dichloromethane and water. The organic layer was washed with saturated sodium chloride solution, and the organic layer was dried by spin drying. The sample was mixed with 200-300 mesh silica gel, and the mixture was passed through a column with a volume ratio of dichloromethane:methanol of 50 mL:1 mL to obtain an intermediate compound. The intermediate compound was dissolved in methanol:water of 50 mL:1 mL. mL:1mL of the mixed solution, add lithium hydroxide, react at room temperature for 3-5h, after the reaction is completed, extract with ethyl acetate, take the organic layer and add concentrated hydrochloric acid to adjust the pH to less than 1, precipitate solid to obtain compound 2; compound 2 and compound 3 are dissolved in 20mL DMF, add N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate and N,N-diisopropylethylamine, react for 5-8h, after the reaction is completed, post-process to obtain the target compound, the reaction formula is as follows:

5. The method for preparing the novel FSP1 degrader PROTAC compound according to claim 4, characterized in that: The specific process of the post-treatment is: after the reaction is completed, the reaction is concentrated under reduced pressure, and a thin layer plate is prepared for separation to obtain the target compound.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the novel FSP1 degrader PROTAC compound according to any one of claims 1 to 3 and optionally a pharmaceutically acceptable additive or excipient.

7. Use of the novel FSP1 degrader PROTAC compound according to any one of claims 1 to 3 in the preparation of a ferroptosis inducer.

8. Use of the novel FSP1 degrader PROTAC compound according to any one of claims 1 to 3 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that: The tumor is a ferroptotic tumor, and the ferroptotic tumor is selected from fibrosarcoma, lymphoma, breast cancer, lung cancer or kidney cancer.

10. The use according to claim 8, characterized in that: The compound induces ferroptosis by degrading FSP1, thereby inhibiting the survival of tumor cells and achieving an anti-tumor effect.

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