HDAC degradation agent with alkane or fatty acid as connecting chain and preparation method and application thereof

Abstract

The present invention provides an HDAC degradation agent with alkanes or fatty acids as connecting chains, and a preparation method and application thereof, relating to the field of medical technology. The HDAC degradation agent provided by the present invention comprises a ubiquitin ligase E3 ligand and vorinostat in its chemical structure, and the ubiquitin ligase E3 ligand and vorinostat are connected by alkanes or fatty acids. The present invention utilizes a protein degradation strategy to design and synthesize a PROTAC molecule with vorinostat as a target protein, which has the potential to solve the serious toxic and side effects of the currently marketed drug vorinostat by significantly reducing the dosage. The preparation method of the HDAC degradation agent provided by the present invention, the raw materials used are simple and easy to obtain, and the method steps of the present invention are simple to operate, the yield is high, and the application of large-scale instruments and equipment is not required, which can not only effectively control the production cost, but also reduce the burden of patient treatment.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an HDAC degradation agent with alkanes or fatty acids as connecting chains, and a preparation method and application thereof. Background Art

[0002] Vorinostat, chemically named "N-hydroxy-N'-phenyl suberamide" or "suberylanilide hydroxamic acid (SAHA)", is the first anti-tumor drug of histone deacetylase (HDAC) inhibitor developed by Merck in the United States. Since vorinostat can selectively induce the late differentiation of tumor cells, thereby inhibiting the proliferation of such cells under appropriate conditions, vorinostat is used to treat tumor patients with continuously proliferating cancer cells. It is also mainly used in clinical practice to treat cutaneous T-cell lymphoma (CTCL: a non-Hodgkin's lymphoma, a T-cell cancer that affects the white blood cells of the skin) that has worsened, persisted and relapsed or has failed to respond to two systemic drug treatments.

[0003] Vorinostat It was approved for marketing in the United States in 2006 and has clear anti-tumor efficacy. However, the drug has obvious toxic side effects. The FDA reported that vorinostat is prone to cause thromboembolism, anemia, hyperglycemia, liver damage and gastrointestinal adverse reactions (FDA (2006) Available at: http: / / www.accessdata.fda.gov / scripts / cder / drugsatfda / index.cfm?fuseaction=Search.DrugDet ails; Krug, LM et al., [J]. Clin. Lung Cancer 2006, 7, 257). The main reason for these serious toxic side effects is the large dose administration to maintain the drug efficacy. Therefore, how to reduce the drug dosage and solve the serious toxicity problem has become the key to ensure that its anti-tumor efficacy can continue.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The first object of the present invention is to provide an HDAC degrading agent to solve at least one of the above problems.

[0006] The second object of the present invention is to provide a method for preparing the above HDAC degrading agent.

[0007] The third object of the present invention is to provide the use of the above HDAC degrading agent in the preparation of tumor therapeutic drugs.

[0008] The fourth object of the present invention is to provide a tumor treatment drug.

[0009] In a first aspect, the present invention provides an HDAC degrader, wherein the chemical structure of the HDAC degrader comprises a ubiquitin ligase E3 ligand and vorinostat, wherein the ubiquitin ligase E3 ligand and vorinostat are connected by an alkane or a fatty acid.

[0010] As a further technical solution, the structure of the HDAC degradation agent with alkane as the connecting chain is as follows:

[0011]

[0012] Wherein, n is a natural number greater than or equal to 1.

[0013] As a further technical solution, the HDAC degradation agent with alkane as the connecting chain includes:

[0014]

[0015] As a further technical solution, the structure of the HDAC degrader with fatty acids as the connecting chain is as follows:

[0016]

[0017] Wherein, n is an integer greater than or equal to 0.

[0018] As a further technical solution, the structure of the HDAC degrader with fatty acids as connecting chains includes:

[0019]

[0020] In a second aspect, the present invention provides a method for preparing the above-mentioned HDAC degradation agent, comprising the following steps:

[0021] Preparation of HDAC degraders with alkanes as connecting chains: a. 2-(2,6-dioxapiperidin-3-yl)-4-fluoroisoindoline-1,3-dione and Condensation reaction to obtain

[0022] b. By Boc deprotection,

[0023] c. A condensation reaction is performed to obtain an HDAC degradation agent with an alkane as a connecting chain; wherein n is a natural number greater than or equal to 1;

[0024] Preparation of HDAC degraders with fatty acids as connecting chains: d. 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione and Condensation reaction to obtain

[0025] e. By Boc deprotection,

[0026] f. A condensation reaction is performed to obtain an HDAC degradation agent with fatty acids as connecting chains; wherein n is an integer greater than or equal to 0.

[0027] As a further technical solution, Boc deprotection is carried out by p-toluenesulfonic acid, or by trifluoroacetic acid (TFA).

[0028] In a third aspect, the present invention provides use of the above HDAC degrader in the preparation of tumor therapeutic drugs.

[0029] As a further technical solution, the tumor includes cutaneous T-cell lymphoma and myeloma.

[0030] In a fourth aspect, the present invention provides a tumor therapeutic drug, which comprises the HDAC degrading agent.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention utilizes a protein degradation strategy to design and synthesize a PROTAC molecule with vorinostat as the target protein, i.e., the HDAC degrader provided by the present invention, which has the potential to solve the serious toxic and side effects of the currently marketed drug vorinostat by significantly reducing the dosage. At the same time, a nitrogen-containing structure is introduced into the PROTAC molecule to improve the membrane permeability of the compound.

[0033] (2) The preparation method of the HDAC degradation agent provided by the present invention uses raw materials that are easy to obtain, and the method steps of the present invention are simple to operate, have a high yield, and do not require the use of large-scale instruments and equipment, which can not only effectively control production costs, but also reduce the burden of patient treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 The immunoblotting analysis results provided for Experimental Example 2 of the present invention. DETAILED DESCRIPTION

[0036] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0037] In view of the serious toxic side effects of the original drug vorinostat due to the large doses administered to maintain the drug efficacy, the present invention provides an HDAC degrader with alkanes or fatty acids as connecting chains, which has the potential to solve the serious toxic side effects of the currently marketed drug vorinostat by significantly reducing the dosage.

[0038] In a first aspect, the present invention provides an HDAC degrader, wherein the chemical structure of the HDAC degrader comprises a ubiquitin ligase E3 ligand and vorinostat, wherein the ubiquitin ligase E3 ligand and vorinostat are connected by an alkane or a fatty acid.

[0039] The present invention utilizes a protein degradation strategy, and PROTAC molecules can be used repeatedly to degrade target proteins, similar to a catalytic reaction, and are administered in catalytic or substoichiometric amounts, thereby overcoming or improving the serious toxic side effects caused by large doses of marketed drugs.

[0040] The inventors have found through research that the HDAC degrading agent provided by the present invention has good degradation ability for HDAC and does not produce side effects at pharmaceutical concentrations.

[0041] Preferably, the structure of the HDAC degrading agent with alkane as the connecting chain is as follows:

[0042]

[0043] Wherein, n is a natural number greater than or equal to 1.

[0044] When n=2, the HDAC degradation agent with alkane as the connecting chain is:

[0045]

[0046] When n=3, the HDAC degradation agent with alkane as the connecting chain is:

[0047]

[0048] When n=4, the HDAC degradation agent with alkane as the connecting chain is:

[0049]

[0050] Preferably, the structure of the HDAC degrader with fatty acids as connecting chains is as follows:

[0051]

[0052] Wherein, n is an integer greater than or equal to 0.

[0053] When n=1, the HDAC degrader with fatty acids as the connecting chain is:

[0054] When n=2, the HDAC degrader with fatty acids as the connecting chain is:

[0055]

[0056] When n=0, the HDAC degrader with fatty acids as the connecting chain is:

[0057]

[0058] In a second aspect, the present invention provides a method for preparing the above-mentioned HDAC degradation agent, comprising the following steps:

[0059] Preparation of HDAC degraders with alkanes as connecting chains: a. 2-(2,6-dioxapiperidin-3-yl)-4-fluoroisoindoline-1,3-dione and Condensation reaction to obtain

[0060] b. By Boc deprotection,

[0061] c. A condensation reaction is performed to obtain an HDAC degradation agent with an alkane as a connecting chain; wherein n is a natural number greater than or equal to 1;

[0062] Preparation of HDAC degraders with fatty acids as connecting chains: d. 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione and Condensation reaction to obtain

[0063] e. By Boc deprotection,

[0064] f. A condensation reaction is performed to obtain an HDAC degradation agent with fatty acids as connecting chains; wherein n is a natural number greater than or equal to 0.

[0065] The inventors have found through research that the condensation reaction in step a or step d has the advantages of high yield, mild reaction conditions, and short reaction time.

[0066] The preparation method of the HDAC degradation agent provided by the present invention uses raw materials that are easy to obtain, and the method steps of the present invention are simple to operate, with high yield, and does not require the use of large-scale instruments and equipment, which can not only effectively control production costs, but also reduce the burden of patient treatment.

[0067] As a further technical solution, Boc deprotection is carried out by p-toluenesulfonic acid, or Boc deprotection is carried out by TFA.

[0068] In a third aspect, the present invention provides use of the above HDAC degrader in the preparation of tumor therapeutic drugs.

[0069] The HDAC degrading agent provided by the present invention has good degradation ability for HDAC and does not produce side effects at a medicinal concentration, and can be used for the preparation of tumor therapeutic drugs.

[0070] As a further technical solution, the tumor includes cutaneous T-cell lymphoma and myeloma.

[0071] In a fourth aspect, the present invention provides a tumor therapeutic drug, which comprises the HDAC degrading agent.

[0072] Since the tumor therapeutic drug contains an HDAC degrader, it has all the functions of an HDAC degrader and has a certain therapeutic effect on tumors including cutaneous T-cell lymphoma and myeloma.

[0073] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0074] Example 1 Preparation of tert-butyl (2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-ylamino)butyl)carbamate (ii-a)

[0075] The reaction formula is as follows:

[0076]

[0077] At 25.0°C, 2-(2,6-dioxapiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (21.0 g, 76.0 mmol, 1.00 eq) and N,N-diisopropylethylamine (DIPEA) (29.4 g, 228 mmol, 39.7 mL, 3.00 eq) were dissolved in N-methylpyrrolidone (NMP) (945 mL), and compound ia (15.7 g, 83.6 mmol, 1.10 eq) was added to the above solution under stirring. Stir at 90.0°C (oil bath) for 12 hours. TLC (petroleum ether / ethyl acetate = 1 / 1). 3.00 L of water was added to the mixture to remove NMP, and then extracted with ethyl acetate (500 ml × 2). Saturated brine (100 ml × 3) was added to the organic phase. The organic solvent was added to sodium sulfate and dried, and then the organic solvent was rotary evaporated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 0 / 1), washed again with saturated saline solution (100 ml x 5), and then rotary evaporated to obtain compound ii-a (10.9 g, 23.8 mmol, 31.3% yield) as a green foam.

[0078] Characterization of the product: 1 H NMR: (400MHz, DMSO-d6) δ11.13(s,1H),7.61(dd,J=8.4,7.3Hz,1H),7.14(d,J=8 .6Hz,1H),7.06(d,J=7.0Hz,1H),6.87(t,J=5.5Hz,1H),6.59(t,J=5.9Hz,1H),5 .09(dd,J=12.8,5.4Hz,1H),3.34(q,J=6.8Hz,2H),3.00-2.89(m,3H),2.66-2.5 7(m,2H),2.11-2.05(m,1H),1.61-1.56(m,2H),1.52-1.47(m,2H),1.41(s,9H).

[0079] LCMS: 445.2[M+H], 467.3[M+Na], 889.4[2M+H].

[0080] Example 2 Preparation of 4-((4-aminobutyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (iii-a)

[0081] The reaction formula is as follows:

[0082]

[0083] Compound ii-a (10.9 g, 24.5 mmol, 1.00 eq) was dissolved in ACN (150 mL) at 25.0 °C, stirred and p-toluenesulfonic acid (TsOH) (8.45 g, 49.0 mmol, 2.00 eq) was added, and stirred at 40.0 °C for 3.5 hours to form a light yellow solution. The reaction solution was rotary evaporated to remove most of the solvent, and then filtered to collect the filter cake. A mixture of dichloromethane (100 mL) and methanol (5.00 mL) was slurried at 40.0 °C, filtered and rotary evaporated to obtain compound iii-a (8.80 g, 17.0 mmol, 69.4% yield, TsOH) as a light yellow solid.

[0084] Characterization of the product: 1 H NMR: (400MHz, MeOD-d4) δ7.72(d,J=8.2Hz,2H),7.57(dd,J=8.5,7.2Hz,1H),7.24(d,J=8.0Hz,2H),7.08-7.06(m,2H),5.07(dd,J=1 2.5,5.5Hz,1H),3.38(t,J=6.4Hz,2H),2.99(t,J=6.9Hz,2H),2.92-2.67(m,3H),2.37(s,3H),2.14-2.09(m,1H),1.82-1.70(m,4H).

[0085] 13 C NMR: (101MHz, MeOD-d4) δ173.34,170.29,169.38,167.91,146.70,142.14,140.38,135.96,132.55,128 .49(2C),125.57(2C),116.67,110.57,109.78,53.65,41.46,39.5,30.81,25.97,25.64,22.24,19.56.

[0086] LCMS: 345.2[M+H], 367.1[M+Na].

[0087] Example 3N 1 -(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-ylamino)butyl)amino)-2-oxoethoxy)phenyl)-N-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-ylamino)butyl)amino)-2-oxoethoxy)phenyl)- 8 Preparation of -Hydroxyoctanediamine (III)

[0088] The reaction formula is as follows:

[0089]

[0090] Compound iv (3.00 g, 8.87 mmol, 1.00 eq) was dissolved in 12.0 mL of N,N-dimethylformamide, and then N,N-diisopropylethylamine (9.17 g, 70.9 mmol, 12.3 mL, 8.00 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (4.05 g, 10.6 mmol, 1.20 eq) were added, and finally compound iii-a (3.21 g, 9.31 mmol, 1.05 eq) was added and reacted at 15 °C for 1 hour. The product was purified by preparative HPLC with a chromatographic column of Phenomenex luna C18 250*50mm*10um; mobile phase: [water(TFA)-ACN]; B%: 17%-47%, 30 min, to obtain compound III (1.55 g, 2.25 mmol, 25.3% yield) as a yellow powder.

[0091] Characterization of the product: 1 H NMR (400MHz, MeOD-d4) δ7.55(dd,J=8.5,7.2Hz,1H),7.32(d,J=8.9Hz,2H),7.07-7.03(m,2H),6.73(d,J=8.9Hz,2H),5.06(dd,J=12.5,5.4Hz,1H),4.3 3(s,2H),3.38-3.34(m,4H),2.91-2.82(m,1H),2.76-2.67(m,2H),2.31(t, J=7.4Hz,2H),2.11(t,J=7.2Hz,3H),1.69-1.61(m,8H),1.37-1.35(m,4H).

[0092] 13 C NMR(101MHz,MeOD-d4)δ173.24,172.81,170.24,169.53,169.35,167.94(2C),153.93,146.82,135.86,132.53,130.32,121.99(2C),11 6.66,114.80(2C),110.42,109.69,74.92,48.81,41.67,38.28,36.24,32.04,30.80,28.43,28.36,26.28,26.19,25.34,24.82,22.41.

[0093] LCMS: 665.2 [M+H].

[0094] Example 4 Preparation of tert-butyl (5-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-amino)pentyl)carbamate (ii-b)

[0095] The reaction formula is as follows:

[0096]

[0097] At 25.0°C, 2-(2,6-dioxapiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (500 mg, 1.81 mmol, 1.00 eq) was dissolved in NMP (15 mL), DIPEA (702 mg, 5.43 mmol, 946 μL, 3.00 eq) was added, and compound ib (402 mg, 1.99 mmol, 1.10 eq) was added to the above solution under stirring. Stir at 90.0°C (oil bath) for 12 hours. Then, the mixture was diluted with 40 mL of water and quenched, and extracted with ethyl acetate (150 mL × 2); the organic layers were combined and washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the organic solvent was rotary evaporated to obtain a crude product. The concentrate was purified by column chromatography (petroleum ether / ethyl acetate = 5 / 1 to 1 / 1) and then rotary evaporated to obtain compound ii-b (706 mg, 1.54 mmol, 85.1% yield) as a yellow-green liquid.

[0098] Characterization of the product: 1 H NMR(400MHz,MeOD-d4)δ10.71(brs,1H),7.47(t,J=7.8Hz,1H),6.99-6.94(m,2H),6.49(brs,1H),6.36(brs,1H),5.05(dd,J=12.6,5.4Hz,1 H),3.25(t,J=7.0Hz,2H),3.04(t,J=6.4Hz,2H),2.91-2.65(m,3H),2.12-2.08(m,1H),1.67-1.60(m,2H),1.54-1.47(m,2H),1.42(s,11H).

[0099] 13 C NMR(101MHz,MeOD-d4))δ173.32,170.19,169.38,167.93,157.10,146.75,135.87,132.42,116.59,1 10.40,109.49,78.46,60.18,42.00,39.84,30.91,29.34,28.65,27.50,23.80,22.44,19.58,13.16.

[0100] LCMS: 459.5 [M+H] + .

[0101] Example 5 Preparation of 4-((5-aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (iii-b)

[0102] The reaction formula is as follows:

[0103]

[0104] Compound ii-b (830 mg, 1.81 mmol, 1.00 eq) was dissolved in ACN (15 mL) at 25.0 °C, stirred and TsOH (623 mg, 3.62 mmol, 2.00 eq) was added, and stirred at 40.0 °C for 3.5 hours to form a light yellow solution. The reaction solution was rotary evaporated to remove most of the solvent, and then filtered to collect the filter cake. A mixture of dichloromethane (100 mL) and methanol (5.00 mL) was slurried at 40.0 °C, filtered and rotary evaporated to obtain compound iii-b (609 mg, 1.70 mmol, 93.9% yield) as a yellow solid.

[0105] Characterization of the product: 1 H NMR(500MHz,MeOD-d4)δ7.70(d,J=8.2Hz,2H),7.55(dd,J=8.5,7.2Hz,1H),7.23(d,J=8.0Hz,2H),7.05-7.03(m,2H),5.05(dd,J=112.5,5.5 Hz,1H),3.33(t,J=7.0Hz,2H),2.94-2.82(m,3H),2.77-2.69(m,2H),2.36(s,3H),2.13-2.08(m,1H),1.73-1.66(m,4H),1.52-1.46(m,2H).

[0106] 13 C NMR(126MHz,MeOD-d4)δ174.63,171.66,170.79,169.25,148.12,143.47,141.75,137.29,133.92,129.84( 2C),126.94(2C),118.02,111.90,111.14,50.18,43.09,40.63,32.19,29.76,28.30,24.77,23.79,21.30.

[0107] LCMS: 359.4 [M+H] + .

[0108] Example 6N 1 -(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-ylamino)pentyl)amino)-2-oxoethoxy)phenyl)-N 8 Preparation of -Hydroxyoctanamide (IV)

[0109] The reaction formula is as follows:

[0110]

[0111] Compound iv (670 mg, 1.98 mmol, 1.00 eq) and N-hydroxy-7-azabenzotriazole (HOAt) (404 mg, 2.97 mmol, 1.50 eq) were dissolved in 10 mL of dimethyl sulfoxide (DMSO), and N-methylmorpholine (NMM) (604 mg, 5.94 mmol, 657 μL, 3.00 eq) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (569 mg, 2.97 mmol, 1.50 eq) were added and stirred for 0.5 hours; compound iii-b (710 mg, 1.98 mmol, 1.00 eq) was added. The reaction solution was stirred at 25°C for 12 hours. Then, 40 mL of water was added to quench, and the mixture was extracted with ethyl acetate (250 mL × 2); the organic layers were combined, washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the organic solvent was rotary evaporated to obtain a crude product. The concentrate was separated by column chromatography (methanol / ethyl acetate = 1 / 50 to 1 / 10) to obtain compound IV (204 mg, 0.30 mmol, 15.2% yield) as a yellow solid.

[0112] Characterization of the product: 1 H NMR (400MHz, MeOD-d4) δ10.84(s,1H),9.55(s,1H),8.77(d,J=3.8Hz,1H),8.46(d,J=8.2Hz,1H ),7.75(s,1H),7.65(s,1H),7.55(t,J=7.7Hz,1H),7.35(d,J=8.6Hz,2H),7.06-7.02(m,2H),6. 76(d,J=8.7Hz,2H),4.34-4.31(m,3H),3.34(d,J=9.1Hz,4H),2.93-2.70(m,3H),2.33(t,J=7.3 Hz,2H),2.13(t,J=7.1Hz,3H),1.79-1.63(m,9H),1.49(q,J=7.4Hz,4H),1.28(t,J=7.1Hz,1H).

[0113] 13 C NMR(101MHz,MeOD-d4)δ174.65,174.15,174.06,171.72,170.84,170.74,169.30,155.28,148.19,137.24,133.85,132.33,131.70,129.85,123 .30,118.00,116.18,111.75,110.94,76.28,66.64,43.24,39.83,37.64 ,33.42,32.20,31.69,29.86,26.75,26.23,25.12,23.79,20.24,14.46.

[0114] LCMS: 679.5 [M+H] + ,701.6[M+Na] + .

[0115] Example 7 Preparation of tert-butyl (2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-amino)hexyl)carbamate (ii-c)

[0116] The reaction formula is as follows:

[0117]

[0118] The compound 2-(2,6-dioxapiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (1.00 g, 3.62 mmol, 1.00 eq) was dissolved in NMP (10 mL), and DIPEA (1.40 g, 10.86 mmol, 1.89 mL, 3.00 eq) was added; then compound ic (861 mg, 3.98 mmol, 1.10 eq) was added. The reaction solution was stirred at 90 ° C for 12 hours. Then, it was diluted with 35 mL of water and extracted with ethyl acetate (250 mL × 1); the organic layers were combined and washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the organic solvent was rotary evaporated to obtain a crude product. The concentrate was purified by column chromatography (petroleum ether / ethyl acetate = 5 / 1 to 1 / 1) to obtain compound ii-c (1.54 g, 3.26 mmol, 90.1% yield) as a yellow-green liquid.

[0119] Characterization of the product: 1H NMR(400MHz,MeOD-d4))δ7.51(m,1H),7.01-6.98(m,2H),5.05(dd,J=12.6,5.4Hz,1H),3.30- 3.26(m,2H),3.02(t,J=6.7Hz,2H),2.92-2.65(m,4H),1.68-1.61(m,2H),1.49-1.36(m,15H).

[0120] 13 C NMR(101MHz,MeOD-d4))δ174.67,171.59,170.75,169.29,148.18,137.21,133.83,117.94,111.70,110 .88,79.77,61.52,43.32,41.23,32.22,30.89,30.24,28.80,27.65,27.56,23.78,20.88,18.53,14.47.

[0121] LCMS: 473.1[M+H] + .

[0122] Example 8 Preparation of 4-((6-aminohexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (iii-c)

[0123] The reaction formula is as follows:

[0124]

[0125] Compound ii-c (747 mg, 1.58 mmol, 1.00 eq) was dissolved in a mixed solution of dichloromethane (DCM) (4 mL) and trifluoroacetic acid (TFA) (2 mL); the reaction solution was stirred at 25°C for 4 hours and concentrated in vacuo; ether (10 mL) was added and stirred at 25°C for 12 hours; compound iii-c (535 mg, 1.44 mmol, 91.1% yield) was obtained by vacuum filtration and filter cake drying as a yellow solid.

[0126] Characterization of the product: 1H NMR (400MHz, MeOD-d4) δ7.53(t,J=7.8Hz,1H),7.02(d,J=7.7Hz,2H),5.05(dd,J=12.5,5.4Hz,1H),3.32(d,J=6.5 Hz,2H),2.92(t,J=7.5Hz,2H),2.87-2.65(m,3H),2.13-2.09(m,1H),1.68(d,J=6.8Hz,4H),1.47(d,J=3.2Hz,4H).

[0127] 13 C NMR(101MHz,MeOD-d4)δ174.66,171.69,170.79,169.28,148.20,137.25,133.87,117 .98,111.78,110.96,50.15,43.19,40.62,32.20,30.02,28.46,27.42,27.14,23.77.

[0128] LCMS: 373.4 [M+H] + .

[0129] Example 9N 1 -(4-((6-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-ylamino)hexyl)amino)-4-oxobutyl)phenyl)-N-(4-((6-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-ylamino)hexyl)amino)-4-oxobutyl)phenyl)- 8 Preparation of -Hydroxyoctanediamine (V)

[0130] The reaction formula is as follows:

[0131]

[0132] Compound iv (465 mg, 1.38 mmol, 1.00 eq) and HOAt (282 mg, 2.07 mmol, 1.50 eq) were dissolved in 10 mL of DMSO, and NMM (419 mg, 4.14 mmol, 455 μL, 3.00 eq) and EDCI (397 mg, 2.07 mmol, 1.50 eq) were added and stirred for 0.5 hours; compound iii-c (514 mg, 1.38 mmol, 1.00 eq) was added. The reaction solution was stirred at 25 °C for 12 hours. Then 40 mL of water was added to quench the mixture, and the mixture was extracted with ethyl acetate (250 mL × 2); the organic layers were combined and washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the organic solvent was rotary evaporated to obtain a crude product. The concentrate was separated by column chromatography (methanol / ethyl acetate = 1 / 30 to 1 / 10) to obtain compound V (243 mg, 0.345 mmol, 25.0% yield) as a yellow solid;

[0133] Characterization of the product: 1 H NMR(400MHz,MeOD-d4)δ7.72(dd,J=5.7,3.3Hz,2H),7.61(dd,J=5.7,3.3Hz,2H),7. 53(t,J=7.8Hz,1H),7.30(d,J=8.9Hz,2H),7.03-7.01(m,2H),6.71(d,J=8.9Hz,2H), ,4.31-4.29(m,5H),3.25(t,J=6.9Hz,2H),2.89-2.65(m,3H),2.30(t,J=7.5Hz,2H), 2.10((t,J=7.4Hz,3H),1.76-1.70(m,4H),1.48-1.43(m,6H),0.98(t,J=7.4Hz,6H).

[0134] 13 C NMR(101MHz,MeOD-d4)δ174.68,174.19,171.70,170.78,169.35,169.32, 155.30,148.28,137.24,133.90,133.56,132.35,131.72,129.86,123.31 ,118.02,116.18,111.72,110.95,76.27,66.66,43.31,40.01,37.65,33. 43,32.20,31.71,30.20,29.86,27.63,26.77,26.25,23.80,20.25,14.04.

[0135] LCMS: 705.8 [M+H] + ,727.6[M+Na] + .

[0136] Example 10 Preparation of tert-butyl (2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-ylamino)-4-oxobutyl)carbamate (va)

[0137] The reaction formula is as follows:

[0138]

[0139] Compound 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione (500 mg, 1.93 mmol, 1.00 eq) and compound va (392 mg, 1.93 mmol, 1.00 eq) were dissolved in DMSO (6 mL), and HOAt (395 mg, 2.90 mmol, 1.50 eq), NMM (586 mg, 5.79 mmol, 637 μL, 3.00 eq), 1EDCI (556 mg, 2.90 mmol, 1.50 eq) were added. The reaction solution was stirred at 25 °C for 12 hours. Then 15 mL of water was added to quench, and the mixture was extracted with ethyl acetate (150 mL × 3); the organic layers were combined and washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the organic solvent was rotary evaporated to obtain a crude product. The concentrate was separated by column chromatography (petroleum ether / ethyl acetate=1 / 5-0 / 1) to obtain compound vi-a (707 mg, 1.59 mmol 82.4% yield) as a white solid.

[0140] Characterization of the product: 1 H NMR (400MHz, MeOD-d4) δ7.76(d,J=7.9Hz,1H),7.64(d,J=7.4Hz,1H),7.52(t,J=7.7Hz,1H),7.25(t,J=7.7Hz,1H),7.12(d,J=7.4Hz,1H),6. 91(d,J=7.8Hz,1H),5.18(dd,J=13.3,5.1Hz,1H),4.50(d,J=2.0Hz,2H),3.14(t,J=6.8Hz,2H),2.66(s,6H),1.89-1.82(m,2H),1.42(s,9H).

[0141] 13C NMR(101MHz,MeOD-d4)δ174.66,174.03,172.07,171.09,158.69,136.23,134.62,133.89,130.04 ,127.62,121.34,113.22,79.99,53.56,40.62,40.40,34.49,32.35,28.78,27.25,24.20,24.13.

[0142] LCMS: 445.6 [M+H] + .

[0143] Example 11 Preparation of (4-amino-N-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)butyramide (vii-a)

[0144] The reaction formula is as follows:

[0145]

[0146] Compound vi-a (707 mg, 1.59 mmol, 1.00 eq) was dissolved in a mixed solution of DCM (10 mL) and TFA (5 mL); the reaction solution was stirred at 25° C. for 4 hours and concentrated in vacuo; ether (15 mL) was added and stirred at 25° C. for 12 hours; the mixture was filtered off under reduced pressure and the filter cake was dried to obtain the following compound vii-a (472 mg, 1.37 mmol, 86.2% yield) as a white solid;

[0147] Characterization of the product: 1 H NMR (400MHz, MeOD-d4) δ10.72(s,1H),9.93(s,1H),7.78(d,J=7.9Hz,1H),7.63(d,J=7.4Hz,1H),7.50(t,J=7.7Hz,1H),5.15(dd ,J=13.3,5.0Hz,1H),4.47(d,J=2.9Hz,2H),3.04(t,J=7.4Hz,2H),2.62(t,J=7.0Hz,2H),2.46-2.41(m,1H),2.18-1.99(m,5H).

[0148] 13 C NMR(101MHz,MeOD-d4)δ174.65,172.99,172.20,171.02,135.94,134.46,133. 85,130.08,127.54,121.33,43.57,40.33,33.76,32.29,31.17,24.11,21.60.

[0149] LCMS: 345.4 [M+H] + .

[0150] Example 12N 1 -(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-4-oxobutyl)amino)-2-oxoethoxy)phenyl)-N- ... 8 Preparation of -Hydroxyoctanamide (VI)

[0151] The reaction formula is as follows:

[0152]

[0153] Compound vii-a (300 mg, 0.87 mmol, 1.00 eq) and compound iv (294 mg, 0.87 mmol, 1.00 eq) were dissolved in DMF (6 mL), and DIPEA (900 mg, 6.96 mmol, 1.21 mL, 8.00 eq) and HATU (395 mg, 1.04 mmol, 1.20 eq) were added; the reaction solution was stirred at 25 ° C for 12 hours; then diluted with 20 mL of water to quench, extracted with ethyl acetate (175 mL × 3); the organic layers were combined, washed with saturated brine (100 mL × 3), dried over anhydrous sodium sulfate, filtered, and the organic solvent was rotary evaporated to obtain a crude product. The concentrate was separated by column chromatography (ethyl acetate / methanol = 30 / 1 to 20 / 1) to obtain compound VI (144 mg, 0.22 mmol, 25.3% yield) as a white solid.

[0154] Characterization of the product: 1 H NMR (400MHz, MeOD-d4) δ8.73(d,J=4.3Hz,1H),8.43(d,J=8.3Hz,1H),7.74(d,J=8.1Hz,1H),7.64(d ,J=7.4Hz,1H),7.51(d,J=8.2Hz,1H),7.29(d,J=8.8Hz,2H),6.71(d,J=8.8Hz,2H),4.49(s,2H),4. 31-4.07(m,3H),3.35(t,J=6.7Hz,2H),2.94-2.85(m,1H),2.78-2.74(m,1H),2.51-2.42(m,3H),2. 28(t,J=7.4Hz,2H),2.20-2.08(m,3H),1.97-1.87(m,2H),1.75-1.40(m,7H),1.24(t,J=7.1Hz,1H).

[0155] 13C NMR(101MHz,MeOD-d4)δ174.65,174.22,173.74,172.06,171.07,171.00,155.32,152.15,134.55,133.90,132.33,131.65,130.05,129.93, 129.85,137.55,123.38,122.14,116.18,76.33,66.65,39.36,37.61, 34.43,33.40,32.35,31.70,29.83,29.75,26.74,26.38,24.09,20.25.

[0156] LCMS: 665.5 [M+H] + .

[0157] Example 13 Preparation of tert-butyl (2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-ylamino)-5-oxopentyl)carbamate (vi-b)

[0158] The reaction formula is as follows:

[0159]

[0160] Compound 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione (2.00 g, 7.72 mmol, 1.00 eq) and compound vb (1.84 g, 8.49 mmol, 1.10 eq) were dissolved in ACN (30 mL), and pyridine (1.83 g, 23.16 mmol, 1.87 mL, 3.00 eq) was added; then phosphorus oxychloride (POCl3) (1.42 g, 9.26 mmol, 863 μL, 1.20 eq) was added under ice bath. The reaction solution was stirred at 25 ° C for 3 hours. Then 25 mL of water was slowly added dropwise to quench, and extracted with ethyl acetate (200 mL × 3); the organic layers were combined and washed with saturated brine (150 mL × 3), dried over anhydrous sodium sulfate, filtered, and the organic solvent was rotary evaporated to obtain a crude product. The concentrate was separated by column chromatography (petroleum ether / ethyl acetate = 1 / 10 to 0 / 1) to obtain compound vi-b (1.06 g, 2.31 mmol, 30.0% yield) as a white solid.

[0161] Characterization of the product: 1H NMR (400MHz, DMSO-d6) δ11.03(s,1H),9.78(s,1H),7.82(d,J=6.6Hz,1H),7.52-7.47(m,2H),6.83(s,1H),5.15(dd,J=13.2,5.0Hz,1H), 4.36(q,J=17.5Hz,2H),2.96-2.88(m,3H),2.61(d,J=17.0Hz,1H),2.37-2.17(m,3H),2.04-1.99(m,1H),1.61-1.40(m,4H),1.38(s,9H).

[0162] 13 C NMR(101MHz,DMSO-d6)δ182.35,180.74,180.55,177.31,175.24,174.02,173.29,168.26,144.54,143 .27,142.15,138.16,138.11,134.70,128.47,86.84,84.16,44.92,40.70,38.59,37.76,32.14,31.93.

[0163] LCMS: 459.4 [M+H] + .

[0164] Example 14 Preparation of 5-amino-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)pentanamide (vii-b)

[0165] The reaction formula is as follows:

[0166]

[0167] Compound vi-b (1.06 g, 2.31 mmol, 1.00 eq) was dissolved in a mixed solution of DCM (10 mL) and TFA (5 mL); the reaction solution was stirred at 25° C. for 4 hours and concentrated in vacuo; ether (10 mL) was added and stirred at 25° C. for 12 hours; the mixture was filtered off under reduced pressure and the filter cake was dried to obtain the following compound vii-b (802 mg, 2.24 mmol, 97.0% yield) as a white solid;

[0168] Characterization of the product: 1H NMR (400MHz, MeOD-d4) δ10.76(s,1H),9.89(s,1H),7.76(d,J=7.8Hz,1H),7.64(d,J=7.4Hz,1H),7.51(t,J=7.7Hz,1H),5.16(dd,J=13.2,4.9Hz ,1H),4.47(s,2H),2.98-2.93(m,2H),2.88(dd,J=13.3,4.9Hz,1H),2.7 6(d,J=16.0Hz,1H),2.54-2.40(m,3H),2.18-2.16(m,1H),1.76(s,4H);

[0169] 13 C NMR(101MHz,MeOD-d4)δ174.65,172.98,172.20,171.02,135.93,134.46,133.85, 130.08,127.54,121.32,53.61,43.57,40.33,33.76,32.29,31.17,24.11,21.59;

[0170] LCMS: 359.6 [M+H] + .

[0171] Example 15 (N 1 -(4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-5-oxopentyl)amino)-2-oxoethoxy)phenyl)-N 8 Preparation of -Hydroxyoctanediamine (VII)

[0172] The reaction formula is as follows:

[0173]

[0174] Compound vii-b (330 mg, 0.92 mmol, 1.00 eq) and compound iv (311 mg, 0.92 mmol, 1.00 eq) were dissolved in DMF (10 mL), and DIPEA (951 mg, 7.36 mmol, 1.28 mL, 8.00 eq) and HATU (418 mg, 1.10 mmol, 1.20 eq) were added; the reaction solution was stirred at 25 ° C for 12.5 hours; then diluted with 25 mL of water to quench, extracted with ethyl acetate (200 mL × 3); the organic layers were combined, washed with saturated brine (125 mL × 3), dried over anhydrous sodium sulfate, filtered, and the organic solvent was rotary evaporated to obtain a crude product. The concentrate was separated by column chromatography (ethyl acetate / methanol = 30 / 1 to 10 / 1) to obtain compound VII (125 mg, 0.18 mmol, 19.6% yield) as a white solid.

[0175] Characterization of the product: 1 H NMR(400MHz,MeOD-d4)δ10.63(brs,1H),9.78(brs,1H),9.47(brs,1H),9.05(brs,1H),8.63( brs,1H),7.63(d,J=7.9Hz,1H),7.55(d,J=7.5Hz,1H),7.41(t,J=7.7Hz,1H),7.20(d,J=8.8H z,2H),6.61(d,J=8.8Hz,2H),4.51(s,1H),4.39-4.38(m,2H),4.24(s,2H),2.84-2.64(m,2H) ,2.41-2.36(m,3H),2.20(t,J=7.4Hz,2H),2.12-1.91(m,5H),1.67-1.46(m,9H),1.19(s,3H).

[0176] 13 C NMR(101MHz,MeOD-d4)δ174.73,174.32,172.11,171.11,155.32,136.40,134.54,133.91,131.64,130.87,130.79,130.08,127.87,123.45,12 1.48,116.21,39.78,37.64,36.70,33.37,33.04,32.35,30.73,30.57, 30.30,29.80,29.73,28.10,26.76,26.20,24.08,24.00,23.71,14.44.

[0177] LCMS: 679.1[M+H]+ ,701.1[M+Na] + .

[0178] Example 16 Preparation of tert-butyl (2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-ylamino)-3-oxopropyl)carbamate (vi-c)

[0179] The reaction formula is as follows:

[0180]

[0181] Compound 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione (22.0 g, 84.8 mmol, 1.00 eq), compound vc (19.2 g, 101 mmol, 1.20 eq), 4-dimethylaminopyridine (DMAP) (12.4 g, 101 mmol, 1.20 eq), and EDCI (19.5 g, 101 mmol, 1.20 eq) were dissolved in DMF (154 mL) at 25 °C. Stir at 40.0 °C for 3.5 hours. The solution was first subjected to rotary evaporation, then quenched by adding 300 mL of water, extracted with ethyl acetate (50 mL × 2), the organic layers were combined, washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the organic solvent was rotary evaporated to obtain a crude product. The concentrate was purified by column chromatography (dichloromethane / methanol=100 / 1 to 0 / 1) to obtain compound vi-c (26.7 g, 60.8 mmol, 71.7% yield) as a white solid.

[0182] Characterization of the product: 1 H NMR(400MHz,DMSO-d4)δ11.03(s,1H),9.87(s,1H),7.81(dd,J=7.1,1.5Hz,1H) ,7.53-7.47(m,2H),6.91(t,J=5.2Hz,1H),5.15(dd,J=13.3,5.1Hz,1H),4.37(q ,J=17.6Hz,2H),3.26-3.22(m,2H),2.98-2.89(m,1H),2.62(d,J=16.6Hz,1H), 2.54-2.53(m,2H)2.35(qd,J=13.3,4.4Hz,1H),2.05-1.99(m,1H),1.37(s,9H).

[0183] LCMS: 429.2 [MH], 859.4 [2M-H].

[0184] Example 17 Preparation of 3-amino-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)propanamide (vii-c)

[0185] The reaction formula is as follows:

[0186]

[0187] At 25.0°C, compound vi-c (12.0 g, 27.8 mmol, 1.00 eq) was dissolved in ACN (360 mL) and stirred, TsOH (12.0 g, 69.6 mmol, 2.50 eq) was added, heated to 40.0°C, and the suspension was stirred for 36 hours. The reaction suspension was filtered at 40.0°C and a white filter cake was collected. The filter cake was suspended in a mixture of DCM (150 mL) and MeOH (50 mL), stirred at 40.0°C for 30 min, and then filtered at 40.0°C to collect the filter cake. This operation was repeated three times. The filter cake was rotary evaporated to obtain compound vii-c (8.80 g, 17.5 mmol, 62.8% yield, TsOH) as a white solid.

[0188] Characterization of the product: 1 H NMR (400MHz, DMSO-d6) δ11.05 (s, 1H), 10.07 (s, 1H), 7.86 (dd, J = 7.2, 1.6Hz, 1H), 7.7 6(brs,3H),7.57-7.53(m,2H),7.50(d,J=8.1Hz,2H),7.13(d,J=7.9Hz,2H),5.18(dd, J=13.3,5.1Hz,1H),4.40(q,J=17.6Hz,2H),3.13(q,J=6.1Hz,2H),3.00-2.90(m,1H) ,2.77(t,J=6.7Hz,2H),2.63(d,J=16.9Hz,1H),2.37-2.27(m,4H),2.07-2.03(m,1H).

[0189] 13 C NMR(101MHz,DMSO-d6)δ173.38,171.56,169.06,168.28,145.72,138.43,134.13,133.83,133.20,1 29.18,128.65(2C),125.96(2C),125.72,119.78,51.97,46.91,35.48,33.30,31.69,23.15,21.27.

[0190] LCMS:331.1[M+H],661.3[2M+H].

[0191] Example 18 (N 1 -(4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-3-oxopropyl)amino)-2-oxoethoxy)phenyl)-N 8 Preparation of -Hydroxyoctanamide (VIII)

[0192] The reaction formula is as follows:

[0193]

[0194] Compound iv (1.70 g, 5.02 mmol, 1.00 eq) and compound vii-c (1.66 g, 5.02 mmol, 1.00 eq) were dissolved in 5 mL of DMF, and then DIPEA (5.19 g, 40.2 mmol, 7.00 mL, 8.00 eq) and HATU (2.29 g, 6.03 mmol, 1.20 eq) were added, and the mixture was reacted at 15°C for 1 hour. The mixture was purified by preparative high performance liquid chromatography, using a Phenomenex Luna C18 200*40mm*10um column; mobile phase: [water(FA)-ACN]; B%: 8%-38%, 10 min, to obtain compound VIII (1.60 g, 2.39 mmol, 47.5% yield) as a white powder.

[0195] Characterization of the product: 1 H NMR: (400MHz, MeOD-d4) δ7.77-7.72(m,1H),7.68-7.65(m,1H),7.53(t,J=7.7Hz,1H),7.33-7.31(m ,2H),6.75-6.72(m,2H),5.18(dd,J=5.2,13.3Hz,1H),4.54-4.48(m,2H),4.33(s,2H),3.65(t,J=6. 2Hz,2H),2.96-2.87(m,1H),2.81-2.75(m,1H),2.70(t,J=6.4Hz,2H),2.50(qt,J=4.6,13.3Hz,1H) ,2.31(t,J=7.5Hz,2H),2.22-2.16(m,1H),2.07-2.03(m,2H),1.67-1.54(m,4H),1.36-1.31(m,4H).

[0196] 13C NMR: (101MHz, MeOD-d4) δ173.22,172.87,170.82,170.60,170.60,170.52,169.71,153.95,135.23,133.10,132.56,130.31,128.63,1 26.49,122.04(2C),120.14,114.81(2C),74.97,52.21,36.23,35.42,35.33,35.20,32.00,30.95,28.38,28.34,25.32,24.72,22.72.

[0197] LCMS: 651.2 [M+H].

[0198] Experimental Example 1 Anti-cell proliferation effect of the product in the tumor cell line MM.1S

[0199] Experimental Materials

[0200] Table 1. Cell lines and culture methods

[0201]

[0202] Culture medium

[0203] Table 2. Culture media and reagents

[0204] Culture medium and reagents Manufacturer RPMI1640 GIBCO Antibiotic-Antimycotic Invitrogen Dulbecco's PBS Corning FBS Corning DMSO Sigma 0.25%Trypsin GIBCO

[0205] Experimental methods

[0206] Cell culture. Tumor cell lines were cultured in an incubator at 37°C and 5% CO2 according to the culture conditions shown in Table 2. Cells were subcultured regularly and cells in the logarithmic growth phase were used for plating.

[0207] Plate the cells. Stain the cells with trypan blue and count the live cells. Adjust the cell concentration to an appropriate density (3000 per well). Add 90 μL of cell suspension to each well of the culture plate, and add 90 μL of culture medium without cells to the blank control. Incubate the culture plate in an incubator at 37°C, 5% CO2, and 100% relative humidity overnight.

[0208] Preparation of working solution and treatment of cells with compounds. Add 78 μL of cell culture medium to a V-bottom 96-well plate, and draw 2 μL of compound from the 400X compound storage plate and add it to the cell culture medium in the 96-well plate. Add 2 μL of DMSO to the vehicle control and blank control. Mix by pipetting with a dispenser after adding compound or DMSO. Take 10 μL of 10X compound working solution and add it to the cell culture plate. Add 10 μL of DMSO-cell culture medium mixture to the vehicle control and blank control. The final DMSO concentration is 0.25%. Return the 96-well cell plate to the incubator and culture for 3 days.

[0209] CellTiter-Glo luminescent cell activity detection was performed according to the instructions of the Promega CellTiter-Glo luminescent cell activity detection kit (Promega-G7573).

[0210] Data analysis

[0211] The inhibition rate (IR) of the test compound was calculated using the following formula: IR (%) = (1-(RLU compound-RLU blank control) / (RLU vehicle control-RLU blank control))*100%. The inhibition rates of different concentrations of the compound were calculated in Excel, and then the inhibition curves were plotted and related parameters, including the minimum inhibition rate, maximum inhibition rate and IC50, were calculated using GraphPad Prism software.

[0212] Experimental Results

[0213] Table 3. IC50 values ​​of target products and Vorinostat in MM.1S cells

[0214]

[0215] Among them, compound VI has a slight inhibitory effect on cell proliferation in the MM.1S cell line, with a relative IC50 of 3.281 μM, but the maximum inhibition rate is less than 50% (23.10%); compound IV has a significant inhibitory effect on cell proliferation in the MM.1S cell line, with an absolute IC50 and relative IC50 of 2.333 μM and 0.481 μM, respectively, and a maximum inhibition rate of 78.14%; compound V has an inhibitory effect on cell proliferation in the MM.1S cell line, with an absolute IC50 and relative IC50 of 24.611 μM and 1.730 μM, respectively, and a maximum inhibition rate of 60. 03%; Compound VIII has a slight inhibitory effect on cell proliferation in the MM.1S cell line, with a relative IC50 of 6.702 μM, but the maximum inhibition rate is less than 50% (28.72%); Compound VII has a slight inhibitory effect on cell proliferation in the MM.1S cell line, with a relative IC50 of 8.816 μM, and a maximum inhibition rate of less than 50% (40.73%); Compound III has a significant inhibitory effect on cell proliferation in the MM.1S cell line, with an absolute IC50 and relative IC50 of 6.181 μM and 1.176 μM, respectively, and a maximum inhibition rate of 79.70%;

[0216] The reference drug Vorinostat significantly inhibited cell proliferation in the MM.1S cell line, with absolute IC50 and relative IC50 of 0.927μM and 0.944μM, respectively, and the maximum inhibition rate reached 100%;

[0217] The experimental results of the 6 compounds on the inhibition of cell proliferation in the MM.1S tumor cell line showed that all of them had a certain degree of inhibition of cell proliferation. Among them, the HDAC degraders with alkanes as the connecting chain had a better inhibitory effect on cell proliferation in the MM.1S cell line than the HDAC degraders with fatty acids as the connecting chain.

[0218] Experimental Example 2: Immunoblotting analysis of the degradation effect of compounds on HDAC6 in MDA-MB-231 cells

[0219] Experimental Materials

[0220] Table 4. Cell lines and culture methods

[0221]

[0222] Table 5. Reagents and consumables

[0223]

[0224]

[0225] 1X TBST: 50mL 20X TBS plus 949mL ultrapure water, then add 1mL Tween 20 and mix well.

[0226] Electrophoresis buffer: Add 50 mL 20X MES SDS electrophoresis buffer to 950 mL ultrapure water and mix well.

[0227] Table 6. Antibodies

[0228] Serial number Antibody supplier 1 HDAC6 Abcam 4 Goat anti-rabbit IgG-HRP (goat anti-rabbit secondary antibody) LI-COR 5 Goat anti-mouse IgG-HRP (goat anti-mouse secondary antibody) LI-COR

[0229] Experimental methods

[0230] Cell culture: MDA-MB-231 cells were routinely subcultured in a carbon dioxide incubator at 37°C, 0% CO2, and 100% relative humidity, and the cells were collected, counted, and plated when they grew to the exponential phase.

[0231] Compound preparation: Use DMSO to dilute the compound from high concentration to low concentration, and prepare it immediately before adding the drug.

[0232] Cell plating and compound treatment: Collect cells, count with a cell counter after trypan blue staining; adjust the cell concentration to 0.16M / mL, and plate 1.9mL of the mixed cell suspension into a 6-well plate; 20X compound preparation: take 147μL of culture medium into a 1.5mL centrifuge tube, add 3μL of 1000X compound respectively, and vortex to mix; take 294μL of culture medium into a 1.5mL centrifuge tube, add 6μL DMSO, vortex to mix, as a control; compound treatment: take 100μL of 20X compound into a 6-well plate and shake, add 100μL DMSO-culture medium to each well of the control group and shake, the final DMSO concentration is 0.1%; place the 6-well plate in a carbon dioxide incubator at 37°C, 0% CO2, and 100% relative humidity for 24h.

[0233] Cell collection: Collect the cell pellet and store it in a -80℃ refrigerator for long-term storage.

[0234] Immunoblot analysis

[0235] Protein extraction and protein quantification. Take out the cell sample from the -80℃ refrigerator; add 40 / 120μL RIPA lysis buffer (containing 1% protease inhibitor and 1% phosphatase inhibitor); place the cells on ice for 30 minutes, vortex and shake once every 5 minutes; centrifuge at 12,000rpm and 4℃ for 10 minutes, take the supernatant into a new 1.5mL centrifuge tube; use the BCA quantitative kit for protein quantification; prepare protein loading buffer, adjust the protein concentration to 1.6μg / μL according to the quantitative results, add LDS loading buffer (4X) and sample reducing agent (10X), heat the sample at 100℃ for 10 minutes; store the sample in a -80℃ refrigerator.

[0236] Immunoblotting. Sample loading: Thaw the sample and add the protein sample Novex 4-12% Bis-TrisGel (20 wells), 25 μL per well; electrophoresis: 1X MES electrophoresis buffer, 80V, 30min, 120V, 90min; transfer: using 2. Transfer membrane with transfer kit and transfer instrument, P3 program runs for 7 minutes; Block: The membrane is placed in blocking solution (10mL Odyssey blocking solution) and incubated at room temperature for 1 hour; Incubate with primary antibody: Add 10mL primary antibody of corresponding dilution and incubate at 4℃ overnight; Wash the membrane with 10mL 1XTBST, 6 times, and change the solution every 5 minutes; Incubate with secondary antibody: Add 10mL secondary antibody of corresponding dilution and incubate at room temperature for 1 hour; Wash the membrane with 10mL 1XTBST, 3 times, and change the solution every 5 minutes; Wash the membrane with 10mL 1XTBS, 3 times, and change the solution every 5 minutes; Detect near-infrared fluorescence signals with Odyssey Clx. Image Studio software was used to perform relative quantification of the density intensity of immunoblot luminescence bands.

[0237] Data analysis

[0238] Based on the spectral density intensity obtained by the software, the expression ratio of the target protein relative to the internal reference is calculated, and then the standardized expression fold of the target protein in the experimental group is calculated based on the blank control group. The standardized expression fold of the protein in each group is presented in the GraphPad Prism graph.

[0239] Experimental Results

[0240] The expression of HDAC6 and Acetylated α-tubulin in MDA-MB-231 cells was analyzed by immunoblotting to evaluate the pharmacodynamic response of the compound in MDA-MB-231 cells. Figure 1 shown.

[0241] The experimental results showed that compared with the control group, the protein expression level of HDAC6 in MDA-MB-231 cells was significantly reduced after being treated with Compound 1 (Compound VI) at a concentration of 1000nM for 24h; there was no degradation effect on the HDAC6 protein in MDA-MB-231 cells after being treated with Compound 2 (Compound IV) or 3 (Compound V) for 24h; the protein expression level of HDAC6 in MDA-MB-231 cells was significantly low after being treated with Compound 4 (Compound VIII) for 24h; the protein expression level of HDAC6 in MDA-MB-231 cells was significantly low after being treated with Compound 5 (Compound VII) for 24h, and the protein degradation effect was more significant under treatment with a concentration of 1000nM; the protein expression level of HDAC6 in MDA-MB-231 cells was significantly low after being treated with Compound 6 (Compound III) for 24h, and the protein degradation effect was more significant under treatment with a concentration of 1000nM.

[0242] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention 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 invention.

Claims

1. A HDAC degradation agent, characterized in that The chemical structure of the HDAC degrader comprises a ubiquitin ligase E3 ligand and vorinostat, wherein the ubiquitin ligase E3 ligand and vorinostat are connected by alkanes or fatty acids; The HDAC degradation agent with alkane as the connecting chain is: The structure of the HDAC degrading agent with fatty acids as connecting chains is:

2. The method for preparing the HDAC degradation agent according to claim 1, characterized in that: The following steps are involved: Preparation of HDAC degraders with alkanes as connecting chains: a. and Condensation reaction to obtain b. By Boc deprotection, c. A condensation reaction is performed to obtain an HDAC degrading agent with an alkane as a connecting chain; Preparation of HDAC degraders with fatty acids as connecting chains: d. and Condensation reaction to obtain e. By Boc deprotection, f. A condensation reaction is performed to obtain an HDAC degrading agent with fatty acids as connecting chains.

3. The preparation method according to claim 2, characterized in that: Boc deprotection was performed by p-toluenesulfonic acid or Boc deprotection was performed by trifluoroacetic acid.

4. Use of the HDAC degrader according to claim 1 in the preparation of tumor therapeutic drugs; The tumor is myeloma.

5. A tumor treatment drug, characterized in that: The tumor treatment drug includes the HDAC degrader according to claim 1.

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