Novel fsp1 degrader protac compounds, preparation method and application thereof

Abstract

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

Description

Novel PROTAC-type FSP1 degrading agents, their preparation methods and applications Technical Field

[0001] This application relates to the field of medicinal chemistry, and in particular to a novel FSP1 degrading agent PROTAC-type compound, its preparation method, and its application. Background Technology

[0002] The FSP1 protein pathway is a lipid peroxide metabolism regulatory pathway with CoQ10-H2 oxidoreductase activity. It is a good LPO-scavenging antioxidant, reducing cellular lipid peroxides to normal lipid alcohols. FSP1 regulates lipid peroxide metabolism by maintaining the reductive activity of coenzyme Q10. The FSP1-CoQ10-NADPH pathway exists as an independent parallel system, cooperating with GPX4 and glutathione to inhibit phospholipid peroxidation and ferroptosis. In the absence of GPX4, FSP1 uses NADPH to regenerate ubiquinone from its oxidized form, ubiquinone, and inhibits phospholipid peroxidation and ferroptosis in cells. Studies have found that when GPX4 is inactive, FSP1 maintains normal cancer cell growth. High FSP1 expression leads to cellular resistance to ferroptosis, thus causing resistance to ferroptosis inducers. When FSP1 activity is inhibited or the FSP1 gene is knocked out, the GPX4 inhibitor RSL3 exhibits higher cytotoxicity against various tumor cell lines.

[0003] Research on ferroptosis inducers is still in its early stages, and there are few drugs targeting FSP1. The FSP1 inhibitor iFSP1 is expressed in various cancer cells, and treatment with iFSP1 significantly affects the sensitivity of cell lines to ferroptosis. However, the structure-activity relationship of the FSP1 inhibitor iFSP1 is unclear, therefore, the design of drugs targeting FSP1 is urgently needed. Summary of the Invention

[0004] In view of this, this application provides a novel PROTAC-type compound that degrades FSP1, along with its preparation method and application. This compound has been experimentally demonstrated to have the activity of degrading ferroptosis inhibitor protein 1 (FSP1). By degrading FSP1, it blocks intracellular lipid peroxide metabolism, leading to the accumulation of toxic lipid peroxides in the LPO (FSP1-CoQ10-NADPH pathway) and inducing ferroptosis. FSP1 is highly expressed in various tumors such as fibrosarcoma, lymphoma, breast cancer, lung cancer, and renal cell carcinoma. The ferroptosis induced by this compound can effectively kill tumor cells, providing patients with more drug options and effectively overcoming the shortcomings of the existing technologies.

[0005] The first aspect of this application provides a novel FSP1 degrading agent, a PROTAC-type compound, wherein the compound is a compound of Formula I, a pharmaceutically acceptable salt of a compound of Formula I, a hydrate of a compound of Formula I, or a solvate of a compound of Formula I, and the structure of Formula I is shown below:

[0006]

[0007] In Formula I, n is a straight-chain alkyl group with 1-2 carbon atoms; R is selected from 1,4-dimethylpiperazine, 1-methyl-4-(1-methylazacyclobutane-3-yl)piperazine, 2,2'-(ethane-1,2-diylbis(oxy))bis(N-methylethane-1-amine), 2,2'-oxybis(N-methylethyl-1-amine), N1,N5-dimethylpentane-1,5-diamine, N1,N4-di Methylbutane-1,4-diamine, 1-methyl-4-((1-methylazacyclobutane-3-yl)methyl)piperazine, 1-methyl-4-((1-methylazacyclobutane-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.

[0008] 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-oxobutyramide, 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-oxopropionamide, 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-dioxoisoindole-4-yl)piperazin-1-yl)azacyclobutane-1-yl)3-oxopropionamide, 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-dioxoisoindole-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-dioxoisoindole-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-dioxoisoindole-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-dioxoisoindole-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-dioxoisoindole-4-yl)amino)pentyl)malonamide, 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-dioxoisoindole-4-yl)amino)pentyl)malonamide, N1-(2,4-dicyano-3-(p-tolyl)benzo[4 [,5]imidazo[1,2-a]pyridin-1-yl)-N3-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-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-dioxoisoindole-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)azacyclobutane-1-yl)4-oxobutyramide, 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)azacyclobutane-1-yl)3-oxopropionamide, 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)piperazin-1 - (2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(((1-(2-(2-(2,6-dioxopirin-3-yl)-1,3-dioxoisoindol-4-yl)pyrrolidine-2-yl)methyl)piperazin-1-yl)4-oxobutyramide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-4-(((1-(2-(2-(2,6-dioxopirin-3-yl)-1,3-dioxoisoindol-4-yl)piperazin-1-yl)4-oxobutyramide, 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)pyrrolidine-3-yl)piperazin-1-yl)4-oxobutyramide, 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-oxopropionamide, 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-oxopropionamide, N-(2,4-dicyano-3-(p-tolyl)benzo[4,5] Imidazolo[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-oxopropionamide, 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)pyrrolidine-3-yl)piperazin-1-yl)3-oxopropionamide, N-(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-dioxoisoindole-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-dioxoisoindole- 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-oxopropionamide.

[0009] In some embodiments that may include the above embodiments, R is selected from any of the following structures:

[0010]

[0011] The second aspect of this application also provides a method for preparing the above-mentioned novel FSP1 degrading agent PROTAC-type compound, comprising the following steps:

[0012] iFSP and compound 1 were dissolved in DMF, and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate and N,N-diisopropylethylamine were added. The reaction was carried out for 3-5 hours. After the reaction, the mixture was extracted with dichloromethane and water. The organic layer was then washed with saturated sodium chloride solution, evaporated to dryness, mixed with 200-300 mesh silica gel, and passed through a column chromatography with a dichloromethane:methanol volume ratio of 50 mL:1 mL to obtain the intermediate compound. The intermediate compound was then dissolved in a methanol:water volume ratio of 5 mL:1 mL. Lithium hydroxide was added to the mixture, and the reaction was carried out at room temperature for 3-5 hours. After the reaction was completed, the mixture was extracted with ethyl acetate. The organic layer was taken and the pH was adjusted to less than 1 with concentrated hydrochloric acid, and a solid precipitated to obtain compound 2. Compound 2 and compound 3 were dissolved in 20 mL of LMF, and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea and N,N-diisopropylethylamine (i.e., HATU and DIPEA) were added to carry out the reaction for 5-8 hours. After the reaction was completed, post-treatment was performed to obtain the target compound. The reaction formula is shown below:

[0013]

[0014] In some embodiments that may include the above embodiments, the post-processing process is as follows: after the reaction is completed, the mixture is concentrated under reduced pressure, and the target compound is obtained after separation by thin-layer plate preparation.

[0015] A third aspect of this application also provides a pharmaceutical composition comprising the aforementioned novel FSP1 degrading agent PROTAC-type compound and optionally pharmaceutically acceptable adjuvants or excipients.

[0016] The fourth aspect of this application also provides the application of the aforementioned novel FSP1 degrading agent PROTAC-type compounds in the preparation of ferroptosis inducers.

[0017] The fifth aspect of this application also provides the application of the aforementioned novel FSP1 degrading agent PROTAC compounds in the preparation of antitumor drugs.

[0018] In some embodiments that may include the above embodiments, the tumor is a ferroptotic tumor, which is selected from fibrosarcoma, lymphoma, breast cancer, lung cancer, or kidney cancer.

[0019] In some embodiments that may include the above examples, the compound induces ferroptosis by degrading FSP1, thereby inhibiting the survival of tumor cells and achieving an anti-tumor effect.

[0020] Compared with the prior art, the embodiments of this application have the following beneficial effects:

[0021] The novel PROTAC-type compounds, FSP1 degraders described in this application, exhibit significant inhibitory activity against ferroptosis inhibitor protein 1 (FSP1). Experimentally demonstrated, these compounds significantly inhibit FSP1 activity, blocking intracellular lipid peroxide metabolism by inhibiting FSP1, leading to the accumulation of toxic lipid peroxides in the LPO (FSP1-CoQ10-NADPH pathway) and inducing ferroptosis. FSP1 is highly expressed in various tumors, including fibrosarcoma, lymphoma, breast cancer, lung cancer, and renal cell carcinoma. The ferroptosis induced by these compounds effectively kills tumor cells, providing patients with more drug options. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 shows the degradation level of FSP1 degrader Example 7 (2307) at different concentrations as verified by Western blotting experiments. The horizontal axis represents the concentration of 2307, the vertical axis represents the protein level, and the EC50 value is 131.7 nM.

[0024] Figure 2 shows the cellular degradation activity of the novel FSP1 degrading agent PROTAC compounds in some embodiments of this 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 degrading agent was verified by Western blotting. The horizontal axis represents the compound name, and the vertical axis represents the protein level. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0026] In the following examples and comparative examples, unless otherwise specified, all raw materials can be obtained by commercial purchase or conventional methods.

[0027] In the embodiments of this application, the term "substituted" refers to one or more hydrogen atoms in a group being independently replaced by a corresponding number of substituent groups.

[0028] In the embodiments of this application, the excipients or additives are substances, other than the active ingredient, that have undergone reasonable safety assessments and are included in the pharmaceutical formulation. These may include gum arabic, syrup, lanolin, starch, magnesium chloride, cyclodextrin, sebacic acid, dextrin, pharmaceutical-grade calcium sulfate, glycerin, mannitol, sorbitol, inositol, thiols, tromethamine, phenol, m-cresol, benzyl alcohol, parabens, methylparaben, tert-butanol, benzalkonium chloride, chlorobutanol, thimerosal, etc.

[0029] In the embodiments of this application, the derivatives of the compound of general formula I are used as prodrugs, indicating that they may have weak or even no activity on their own, but after administration, they are converted into the corresponding biologically active form under physiological conditions (e.g., through metabolism, solvation or other means).

[0030] The chemical structures of all embodiments in this application were confirmed by 1H-NMR and 13C-NMR. Rapid column chromatography was performed on silica gel H (10-40 μM).

[0031] Example 1

[0032] 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-oxobutyramide is as follows:

[0033] 1-Amino-3-(p-Tolyl)benzo[4,5]imidazo[1,2]pyridine-2,4-dianitrile (iFSP) and 4-methoxy-4-oxobutyric acid were dissolved in DMF. N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate and N,N-diisopropylethylamine were added, and the reaction was carried out for 3-5 hours. After the reaction, the mixture was extracted with dichloromethane and water. The organic layer was then washed with saturated sodium chloride solution, evaporated to dryness, mixed with 200-300 mesh silica gel, and passed through a column chromatography with a dichloromethane:methanol volume ratio of 50 mL:1 mL to obtain 4-(2,4-dicyano-3-(m-tolyl)benzene) Methyl 4-(2,4-dicyano-3-(m-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)amino)-4-oxobutyrate was prepared by dissolving methyl 4-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)amino)-4-oxobutyrate in a mixture of methanol and water at a volume ratio of 5 mL:1 mL. Lithium hydroxide was added, and the mixture was reacted at room temperature for 3-5 h. After the reaction was completed, the mixture was extracted with ethyl acetate. The organic layer was taken and the pH was adjusted to less than 1 with concentrated hydrochloric acid. A solid was precipitated to obtain 4-((2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)amino)-4-oxobutyric acid.

[0034] 4-((2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)amino)-4-oxobutyric acid, 2-(2,6-dioxadiazin-3-yl)-4-(4-methylpiperazin-1-yl)isoindol-1,3-dione, and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea were dissolved in 5 mL of N,N-dimethylformamide, and then N,N-diisopropylethylamine was added. The reaction was carried out at room temperature for 5 h. After the reaction was completed, the mixture was extracted three times with dichloromethane and water. The organic layer was collected and then extracted again with saturated sodium chloride solution. The mixture was concentrated under vacuum and purified with a 50 mL:1 mL mixture of dichloromethane and methanol mixed with 200-300 mesh silica gel to obtain the target compound with a yield of 95%.

[0035] The structure of the target compound is shown below:

[0036]

[0037] 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).

[0038] Example 2

[0039] 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-oxopropionamide, the synthetic route of this example is the same as that of Example 1.

[0040] The structure of the target compound is shown below:

[0041]

[0042] 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).

[0043] Example 3

[0044] 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)azacyclobutane-1-yl)3-oxopropionamide, the synthetic route in this example is the same as in Example 1.

[0045] The structure of the target compound is shown below:

[0046]

[0047] 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).

[0048] Example 4

[0049] 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, the synthetic route in this example is the same as that in Example 1.

[0050] The structure of the target compound is shown below:

[0051]

[0052] 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).

[0053] Example 5

[0054] 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-dioxoisoindole-4-yl)amino)butyl)butanediamide, the synthetic route of this example is the same as that of Example 1.

[0055] The structure of the target compound is shown below:

[0056]

[0057] 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).

[0058] Example 6

[0059] 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-dioxoisoindole-4-yl)amino)hexyl)succinamide, the synthetic route of this example is the same as that of Example 1.

[0060] The structure of the target compound is shown below:

[0061]

[0062] 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).

[0063] Example 7

[0064] 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-dioxoisoindole-4-yl)amino)pentyl)malonamide, the synthetic route of this example is the same as that of Example 1.

[0065] The structure of the target compound (labeled 2307) is shown below:

[0066]

[0067] 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).

[0068] Example 8

[0069] N1-(2,4-dicyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-N3-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)butyl)malonamide, the synthetic route of this example is the same as that of Example 1.

[0070] The structure of the target compound is shown below:

[0071]

[0072] 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).

[0073] Example 9

[0074] 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-dioxoisoindole-4-yl)amino)ethoxy)ethyl)malonamide, the synthetic route of this example is the same as that of Example 1.

[0075] The structure of the target compound is shown below:

[0076]

[0077] 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).

[0078] Example 10

[0079] 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)azacyclobutane-1-yl)4-oxobutanamide, the synthetic route in this example is the same as that in Example 1.

[0080] The structure of the target compound is shown below:

[0081]

[0082] 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).

[0083] Example 11

[0084] 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)azacyclobutane-1-yl)3-oxopropionamide, the synthetic route of this example is the same as that of Example 1.

[0085] The structure of the target compound is shown below:

[0086]

[0087] 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).

[0088] Example 12

[0089] 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)piperidin-1-yl)4-oxobutyramide, the synthetic route in this example is the same as in Example 1.

[0090] The structure of the target compound is shown below:

[0091]

[0092] 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).

[0093] Example 13

[0094] 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)pyrrolidine-2-yl)methyl)piperazin-1-yl)4-oxobutyramide, the synthetic route in this example is the same as in Example 1.

[0095] The structure of the target compound is shown below:

[0096]

[0097] 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).

[0098] Example 14

[0099] 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)piperazin-1-yl)4-oxobutyramide, the synthetic route of the application examples is the same as that of Example 1.

[0100] The structure of the target compound is shown below:

[0101]

[0102] 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).

[0103] Example 15

[0104] 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)pyrrolidine-3-yl)methyl)piperazin-1-yl)4-oxobutyramide, the synthetic route in this example is the same as in Example 1.

[0105] The structure of the target compound is shown below:

[0106]

[0107] 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).

[0108] Example 16

[0109] 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-oxobutyramide, the synthetic route in this example is the same as in Example 1.

[0110] The structure of the target compound is shown below:

[0111]

[0112] 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).

[0113] Example 17

[0114] 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)pyrrolidine-3-yl)piperazin-1-yl)4-oxobutyramide, the synthetic route in this example is the same as in Example 1.

[0115] The structure of the target compound is shown below:

[0116]

[0117] 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).

[0118] Example 18

[0119] 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-oxopropionamide, the synthetic route of this example is the same as that of Example 1.

[0120] The structure of the target compound is shown below:

[0121]

[0122] 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).

[0123] Example 19

[0124] 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)piperazin-1-yl)-3-oxopropionamide, the synthetic route in this example is the same as in Example 1.

[0125] The structure of the target compound is shown below:

[0126]

[0127] 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).

[0128] Example 20

[0129] 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)pyrrolidine-3-yl)piperazin-1-yl)3-oxopropionamide, the synthetic route in this example is the same as in Example 1.

[0130] The structure of the target compound is shown below:

[0131]

[0132] 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).

[0133] Example 21

[0134] 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)pyrrolidine-3-yl)methyl)piperazin-1-yl)-3-oxopropionamide, the synthetic route of the application examples is the same as that of Example 1.

[0135] The structure of the target compound is shown below:

[0136]

[0137] 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).

[0138] Example 22

[0139] 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)pyrrolidine-2-yl)methyl)piperazin-1-yl)-3-oxopropionamide, the synthetic route of the application examples is the same as that of Example 1.

[0140] The structure of the target compound is shown below:

[0141]

[0142] 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).

[0143] Example 23

[0144] 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)ethoxyethoxyethyl)malonamide, the synthetic route of this example is the same as that of Example 1.

[0145] The structure of the target compound is shown below:

[0146]

[0147] 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).

[0148] Example 24

[0149] 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-dioxoisoindole-4-yl)amino)ethoxy)ethyl)succinamide, the synthetic route in this example is the same as that in Example 1.

[0150] The structure of the target compound is shown below:

[0151]

[0152] 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).

[0153] Example 25

[0154] 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)azacyclobutane-1-yl)4-oxobutanamide, the synthetic route of the application examples is the same as that of Example 1.

[0155] The structure of the target compound is shown below:

[0156]

[0157] 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).

[0158] Example 26

[0159] 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-oxopropionamide, the synthetic route in this example is the same as in Example 1.

[0160] The structure of the target compound is shown below:

[0161]

[0162] 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).

[0163] Application examples

[0164] active:

[0165] Cell viability was tested using Western blotting, and the testing procedure is as follows:

[0166] 1. Sample preparation: The first step is to extract proteins from cells or tissues. Typically, protein lysis buffer is used to lyse cells or tissues, and the supernatant is obtained by centrifugation as the sample.

[0167] 2. Electrophoresis: The extracted protein samples are subjected to polyacrylamide gel electrophoresis to separate proteins of different molecular weights.

[0168] 3. Transfer: The proteins on the gel after electrophoresis are transferred to a solid support, using a polyvinylidene fluoride membrane.

[0169] 4. Blocking: Place the transferred membrane into a blocking solution using skim milk to prevent nonspecific binding.

[0170] 5. Primary antibody incubation: Add specific antibodies to bind to the target protein, usually incubated overnight at 4°C to ensure adequate binding.

[0171] 6. Secondary antibody incubation: Incubate with a secondary antibody corresponding to the species of the primary antibody, usually for 1 hour at room temperature.

[0172] 7. Chemiluminescence detection: Chemiluminescence reagents are used to detect the bound antibodies, and images are acquired and analyzed using a chemiluminescence imaging system.

[0173] With the internal control kept constant, the protein expression level of the target gene decreased with increasing drug concentration. Therefore, Example 7 (compound 2307) showed a significant inhibitory effect on breast cancer cells MDA-MB-231, with an EC50 value of 131.7 nM.

[0174] As shown in Figure 1, the extracellular inhibitory effect of Compound 2307 on FSP1 in Example 7: Compound 2307 showed a significant inhibitory effect on FSP1.

[0175] Figure 2 shows the extracellular inhibitory effects of some examples on FSP1. Examples 1 (2301), 2 (2302), 3 (2303), 4 (2304), 5 (2305), 6 (2306), 7 (2307), 8 (2308), 9 (2309), 10 (2310), 11 (2311), 12 (2312), 13 (2313), 14 (2314), 15 (2315), 16 (2316), 17 (2317), 18 (2318), 19 (2319), and 20 (2320) demonstrate the extracellular inhibitory effects on FSP1.

[0176] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A PROTAC-type compound that degrades FSP1, 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-oxobutyramide, 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-di... Cyano-3-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyridin-1-yl)-N3-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-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-dioxoisoindole-4-yl)amino)ethoxy)ethyl)malonamide, N ... [4,5]imidazo[1,2-a]pyridin-1-yl)-4-(((4-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)piperazin-1-yl)methyl)piperidin-1-yl)4-oxobutyramide, 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-dioxoisoindole-4-yl)piperidin-4-yl)methyl)piperazin-1-yl)4-oxobutyramide, 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-dioxoisoindole-4-yl)piperidin-4-yl)piperazin-1-yl)-3-oxopropionamide, 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-dioxoisoindole-4-yl)pyrrolidine-3-yl)piperazin-1-yl)3-oxopropionamide.

2. A method for preparing the PROTAC-type compound that degrades FSP1 according to claim 1, characterized in that, The process includes the following steps: iFSP and compound 1 are dissolved in DMF, N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate and N,N-diisopropylethylamine are added, and the reaction is carried out for 3-5 hours. After the reaction is completed, the mixture is extracted with dichloromethane and water, the organic layer is washed with saturated sodium chloride solution, the organic layer is dried by rotary evaporation, mixed with 200-300 mesh silica gel, and filtered through a column to obtain the intermediate compound. The intermediate compound was dissolved in a methanol:water mixture of 5 mL:1 mL, lithium hydroxide was added, and the reaction was carried out at room temperature for 3-5 h. After the reaction was completed, the mixture was extracted with ethyl acetate, and the organic layer was taken. The pH was adjusted to less than 1 with concentrated hydrochloric acid, and a solid precipitated to obtain compound 2. Compounds 2 and 3 were dissolved in 20 mL of LDM, and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate and N,N-diisopropylethylamine were added. The reaction was carried out for 5-8 h. After the reaction was completed, post-treatment was performed to obtain the target compound. The reaction formula is shown below: 。 3. The method for preparing the FSP1 degrading agent PROTAC-type compound according to claim 2, characterized in that, The specific post-processing procedure is as follows: after the reaction is completed, the mixture is concentrated under reduced pressure, and the target compound is obtained after separation by thin-layer plate preparation.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the FSP1 degrading agent PROTAC class compound as described in claim 1, and optionally pharmaceutically acceptable adjuvants or excipients.

5. The application of the PROTAC-type compounds, which are FSP1 degraders according to claim 1, in the preparation of ferroptosis inducers.

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