Double E3-PROTAC as well as preparation method and application thereof

Abstract

The invention discloses diE3-PROTAC selected from a compound with a structure as shown in a formula I or pharmaceutically acceptable salt and racemate thereof, n is an integer from 2 to 8, R1 and R2 are respectively and independently selected from X selected from CH2 and C = O, and m is an integer from 2 to 3. The double E3-PROTACs disclosed by the invention have good degradation activity on the BRD4 protein, and the degradation efficiency of the double E3-PROTACs on the BRD4 protein is superior to that of the corresponding traditional PROTACs. The invention also discloses an application of the compound or the pharmaceutically acceptable salt and racemate thereof in preparation of a targeted BRD4 degradation agent. The invention also discloses application of the compound or the pharmaceutically acceptable salt and racemate thereof in preparation of drugs for treating BRD4 protein related diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, and relates to a double E3-PROTAC as well as a preparation method and application thereof. BACKGROUND

[0002] Traditional drug discovery strategies are to find molecules that can directly regulate the activity of related proteins. Most small molecule inhibitors and antibody drugs generally exert their biological functions through "occupation-driven", that is, the drug molecules and the active sites of target proteins bind to compete with endogenous ligands, which makes them need to have good binding affinity and half-life characteristics to exert good drug efficacy. Peptide and antibody drugs have the advantages of high selectivity and high affinity, but their molecular weight is large, which leads to poor membrane permeability and difficulty in acting on intracellular proteins.

[0003] Proteolysis targeting chimerics (PROTAC) is a new direction in the field of drug research and development, which brings dawn to the bottleneck of small molecule drugs and antibody drugs and has attracted widespread attention from researchers. The core of PROTAC is to directly induce target protein degradation through the ubiquitin-proteasome protein degradation pathway in vivo, which is superior to traditional inhibitors and is expected to solve the problems of drug resistance and non-druggable targets. In addition, PROTAC has the advantages of high selectivity and specificity, substoichiometric catalytic activity.

[0004] The classic PROTAC is a bivalent PROTAC, which contains a target protein ligand and a ligand recognizing E3 ligase, and the two ligands are connected by a linker chain. The classic PROTAC has opened up a path for protein degradation, but also faces some limitations, including weak binding affinity, poor drugability (large molecular weight), unpredictable off-target effects and potential toxicity. Therefore, it is necessary to optimize the traditional PROTAC technology to avoid or reduce such harmful shortcomings to better assist clinical medicine.

[0005] In recent years, PROTAC technology has developed rapidly, and as a result, many new technologies have emerged to optimize traditional PROTAC. At present, there have been reports of trivalent PROTACs containing two target protein ligands and one E3 ligase ligand, which have shown stronger drug efficacy than bivalent PROTACs. However, there have been no reports of PROTACs containing two E3 ligase ligands.

[0006] BRD4 is a member of the BET (Bromodomain and extraterminal domain) family, and is a very attractive target in a variety of pathological environments, especially cancer, such as midline carcinoma, acute myeloid leukemia, multiple myeloma, Burkitt's lymphoma and prostate cancer. Small molecule inhibitors targeting BRD4 such as (+)JQ-1, iBET and OTX015 exhibit therapeutic potential in preclinical models of various cancers. However, limited by the defects of small molecule inhibitors, BRD4 inhibitors need to maintain a high dose to ensure the inhibitory effect on BRD4. In this context, scientists hope to apply the PROTAC technology to the degradation of BRD4 protein to achieve remarkable results. SUMMARY

[0007] The purpose of the present application is to provide a kind of double E3-PROTAC, compared with the classic bivalent PROTAC, the double E3-PROTAC has a target protein ligand, two E3 ligase ligands and a connecting chain, by increasing the binding site of PROTAC to improve the efficiency of protein degradation.

[0008] The purpose of the present application is realized by the following technical solutions:

[0009] The double E3-PROTAC is selected from the compound or its pharmaceutically acceptable salt, racemate with the structure as shown in formula I:

[0010]

[0011] Wherein, n is an integer from 2 to 8, R1 and R2 are independently selected from X is selected from CH2, C=O, and m is an integer from 2 to 3; but not including: n=2, R1 is selected from R2 is selected from n=2 or 3, R1 is selected from R2 is selected from

[0012] n=4, R1 is selected from R2 is selected from

[0013] Preferably, n is 2, 3 or 4, R1 and R2 are independently selected from But not including: n=2, R1 is selected from R2 is selected from n=2 or 3, R1 is selected from R2 is selected from n=4, R1 is selected from R2 is selected from

[0014] More preferably, n is 2, 3 or 4, and R1 and R2 are each independently selected from n is 3 or 4, and R1 is selected from R2 is selected from n is 4, and R1 is selected from R2 is selected from

[0015] Specifically, the double E3-PROTAC is selected from a compound as shown in the following structure or a pharmaceutically acceptable salt, racemate thereof:

[0016]

[0017]

[0018] As the most preferred technical solution of the present application, the double E3-PROTAC is selected from a compound as shown in the following structure or a pharmaceutically acceptable salt, racemate thereof:

[0019]

[0020] Another object of the present application is to provide a preparation method of the compound, and the synthetic route is as follows:

[0021]

[0022] wherein, n, R1 and R2 are as described above; Ts represents p-toluenesulfonyl, and PMB represents p-methoxybenzyl;

[0023] comprising the following steps:

[0024] Step (1), compound III ((2,2,5-trimethyl-1,3-dioxane-5-yl)methanol) is reacted with intermediate VI in the presence of an acid binding agent, using anhydrous N,N-dimethylformamide as a reaction solvent, to obtain intermediate VII;

[0025] Step (2), intermediate VII is subjected to hydrolysis reaction under acidic conditions, using a mixed solvent of methanol and water with a volume ratio of 30:1 as a reaction solvent, to obtain intermediate VIII;

[0026] Step (3), intermediate VIII is reacted with compound XI (1-((2-iodoethoxy)methyl)-4-methoxybenzene) in the presence of an acid binding agent, using anhydrous tetrahydrofuran or anhydrous N,N-dimethylformamide as a reaction solvent, to obtain intermediate XII;

[0027] Step (4), intermediate XII is subjected to deprotection reaction in the presence of a strong oxidizing agent, using a mixed solvent of dichloromethane and water with a volume ratio of 10:1 as a reaction solvent, to obtain intermediate XIII;

[0028] Step (5), the intermediate XIII is subjected to oxidation reaction in the presence of the Dess-Martin oxidizing agent to obtain the intermediate XIV, with dichloromethane as the reaction solvent;

[0029] Step (6), the intermediate XIV is subjected to oxidation reaction to obtain the intermediate XV, with a mixed solvent of acetonitrile and water in a volume ratio of 1:1.8-1:2 as the reaction solvent, in the presence of sodium chlorite, sodium dihydrogen phosphate and H2O2.

[0030] Step (7), the compound XV is reacted with R1H and R2H to obtain the intermediate XVI, with dichloromethane, tetrahydrofuran, acetonitrile or N,N-dimethylformamide as the reaction solvent, in the presence of an amide condensing agent and an acid binding agent.

[0031] Step (8), the intermediate XVI is subjected to reduction reaction under the catalysis of a catalyst in a hydrogen atmosphere, with a mixed solvent of methanol and dichloromethane in a volume ratio of 10:1 as the reaction solvent; and the reduction product is reacted with (+)JQ-1 ((S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid) to obtain the compound shown in formula I, with dichloromethane, tetrahydrofuran, acetonitrile or N,N-dimethylformamide as the reaction solvent, in the presence of an amide condensing agent and an acid binding agent.

[0032] In step (1), the molar ratio of the compound III to the intermediate VI is 1:0.9-1:1.1.

[0033] The molar ratio of the compound III to the acid binding agent is 1:3-1:5; and the acid binding agent is selected from NaH, and 60% NaH dispersed in mineral oil is specifically used.

[0034] The reaction temperature is room temperature.

[0035] After the reaction is completed, ice water and saturated NH4Cl aqueous solution are added to quench the reaction, ethyl acetate is used for extraction, organic solvents are removed by drying under reduced pressure, and silica gel column chromatography purification is performed with n-hexane:ethyl acetate = 9:1-3:1 V / V as the eluent to obtain the intermediate VII.

[0036] In step (2), the acid condition is provided by trifluoroacetic acid, and 0.01-0.015 mL of trifluoroacetic acid is added per 1 mL of reaction solvent.

[0037] The molar ratio of the intermediate VII to trifluoroacetic acid is 1:0.4-1:0.6.

[0038] The reaction temperature is room temperature.

[0039] After the reaction, the organic solvent was removed by drying under reduced pressure, and the intermediate VIII was obtained by column chromatography on silica gel using n-hexane: ethyl acetate = 3:1 to 1:3 V / V as eluent,

[0040] In step (3), the molar ratio of the intermediate VIII to the compound XI was 1:3 to 1:5.

[0041] The molar ratio of the intermediate VIII to the acid binding agent was 1:1 to 1:1.5.

[0042] The acid binding agent was selected from NaH, and 60% NaH dispersed in mineral oil was specifically used.

[0043] The reaction temperature was 20 to 66°C.

[0044] After the reaction, the reaction was quenched by adding cold saturated NH4Cl aqueous solution, extracted with ethyl acetate, and the organic solvent was removed by drying under reduced pressure. The intermediate XII was obtained by column chromatography on silica gel using n-hexane: ethyl acetate = 9:1 to 3:1 V / V as eluent.

[0045] In step (4), the strong oxidizing agent was 2,3-dichloro-5,6-dicyano-benzoquinone.

[0046] The molar ratio of the intermediate XII to the strong oxidizing agent was 1:2.2 to 1:2.5.

[0047] The reaction temperature was room temperature.

[0048] After the reaction, the reaction was quenched by adding saturated NaHCO3 aqueous solution, filtered, extracted with dichloromethane, and the organic solvent was removed by drying under reduced pressure. The intermediate XIII was obtained by column chromatography on silica gel using dichloromethane:methanol = 100:1 to 33:1 V / V as eluent.

[0049] In step (5), the molar ratio of the intermediate XIII to the Dess-Martin oxidizing agent was 1:2.5 to 1:3.5.

[0050] The reaction temperature was room temperature.

[0051] After the reaction, the organic solvent was removed by drying under reduced pressure, and the intermediate XIV was obtained by column chromatography on silica gel using dichloromethane:methanol = 100:1 to 4:1 V / V as eluent.

[0052] In step (6), the molar ratio of the intermediate XIV to sodium chlorite was 1:2.6 to 1:2.7; the molar ratio of the intermediate XIV to sodium dihydrogen phosphate was 1:2.6 to 1:2.7; and the molar ratio of the intermediate XIV to H2O2 was 1:20 to 1:28.

[0053] The reaction temperature is room temperature.

[0054] After the reaction, saturated Na2S2O3 aqueous solution and saturated NH4Cl aqueous solution are added, and dichloromethane is used for extraction. The organic phase is dried under reduced pressure to remove the organic solvent, and then column chromatography is performed on silica gel with dichloromethane:methanol:glacial acetic acid = 98:1:1 ~ 89:10:1 V / V as the eluent to obtain the intermediate XV.

[0055] In step (7), (a) when R1 and R2 are the same, the molar ratio of the intermediate XV to the amide condensing agent is 1:6.3; the amide condensing agent is one selected from O-(7-azabenzotriazol-1-yl)-di(dimethylamino)carbonium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-di(dimethylamino)carbonium hexafluorophosphate (HBTU) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) in combination with one selected from N-hydroxy-7-azabenzotriazole (HOAt) or 1-hydroxybenzotriazole (HOBt) at a molar ratio of 1:1.1; the molar ratio of the intermediate XV to the acid binding agent is 1:5; and the molar ratio of the total amount of R1H and R2H to the intermediate XV is 2:1 ~ 2.2:1.

[0056] The reaction temperature is room temperature.

[0057] (b) when R1 and R2 are not the same, including:

[0058] Step (7a), in the presence of an amide condensing agent and an acid binding agent, compound XV is reacted with R1H to obtain the intermediate XVI, using dichloromethane, tetrahydrofuran, acetonitrile or N,N-dimethylformamide as the reaction solvent;

[0059] The molar ratio of the intermediate XV to the amide condensing agent is 1:3.15; the amide condensing agent is one selected from O-(7-azabenzotriazol-1-yl)-di(dimethylamino)carbonium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-di(dimethylamino)carbonium hexafluorophosphate (HBTU) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) in combination with one selected from N-hydroxy-7-azabenzotriazole (HOAt) or 1-hydroxybenzotriazole (HOBt) at a molar ratio of 1:1.1; the molar ratio of the intermediate XV to the acid binding agent is 1:2.5; and the molar ratio of the intermediate XV to R1H is 1:1 ~ 1:1.1.

[0060] The reaction temperature is room temperature.

[0061] Step (7b), an amide condensing agent, an acid binding agent and R2H are added to the reaction solution of step (7a) to obtain the intermediate XVI.

[0062] wherein the molar ratio of the intermediate XV to the amide condensing agent is 1:3.15; the amide condensing agent is one selected from O-(7-azabenzotriazol-l-yl)-bis(dimethylamino)carbonium hexafluorophosphate (HATU), O-(benzotriazol-l-yl)-bis(dimethylamino)carbonium hexafluorophosphate (HBTU) or l-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) in combination with one selected from N-hydroxy-7-azabenzotriazole (HOAt) or l-hydroxybenzotriazole (HOBt) at a molar ratio of 1:1.1; the molar ratio of the intermediate XV to the acid binding agent is 1:2.5; the molar ratio of the intermediate XV to R2H is 1:1 to 1:1.1;

[0063] The temperature of the reaction is room temperature.

[0064] In step (7), after the reaction is completed, the reaction solution is concentrated, and first subjected to silica gel column chromatography with dichloromethane:methanol = 100:1 to 15:1 V / V as the eluent, and then subjected to preparative liquid chromatography with an SP ODS-A chromatographic column (20 x 250 mm), a detection wavelength of 210 nm and 254 nm, and a mobile phase of methanol and water (both containing 0.1% trifluoroacetic acid) with a gradient of methanol:water = 10:90 to 95:5 V / V at a flow rate of 3 mL / min to obtain the intermediate XVI.

[0065] In step (8), the catalyst is selected from 5% Pd / C.

[0066] The mass ratio of the intermediate XVI to the catalyst is 1:1.

[0067] The temperature of the reduction reaction is room temperature.

[0068] After the reduction reaction is completed, the reaction solution is filtered, the filtrate is concentrated and dried to obtain the reduction product.

[0069] The molar ratio of the intermediate XVI to the amide condensing agent is 1:2.6 to 1:2.8; the molar ratio of the intermediate XVI to the acid binding agent is 1:2; and the molar ratio of the intermediate XVI to (+)JQ-1 is 1:1.

[0070] The amide condensing agent is one selected from O-(7-azabenzotriazol-1-yl)-bis(dimethylamino)methylium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-bis(dimethylamino)methylium hexafluorophosphate (HBTU) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) in combination with one selected from N-hydroxy-7-azabenzotriazole (HOAt) or 1-hydroxybenzotriazole (HOBt) at a molar ratio of 1:1.35-1:1.4; and the acid binding agent is selected from N,N-diisopropylethylamine or triethylamine.

[0071] The reaction temperature is room temperature.

[0072] After the reaction is completed, the reaction solution is concentrated, and preparative liquid chromatography is performed, the chromatographic column is SP ODS-A chromatographic column (20*250mm), the detection wavelength is 210nm and 254nm, the mobile phase is methanol and water (both containing 0.1% trifluoroacetic acid), the eluent gradient is methanol:water=10:90-95:5V / V, the flow rate is 3mL / min, and the compound shown in formula I is obtained.

[0073] The degradation activity screening experiment (Western Blot) shows that the double E3-PROTACs have good degradation activity on BRD4 protein, and most of them have better degradation efficiency on BRD4 protein than the corresponding traditional PROTACs, and the double E3-PROTACs expand the development direction of PROTACs technology.

[0074] Another object of the present application is to provide the use of the compound or pharmaceutically acceptable salt, racemate thereof in the preparation of a BRD4 degradation targeting agent.

[0075] Another object of the present application is to provide the use of the compound or pharmaceutically acceptable salt, racemate thereof in the preparation of a BRD4 degradation targeting agent.

[0076] The BRD4 protein related disease is midline carcinoma, acute myeloid leukemia, multiple myeloma, Burkitt's lymphoma, prostate cancer, breast cancer, glioblastoma, and renal cell carcinoma.

[0077] Another object of the present application is to provide a pharmaceutical composition which is a pharmaceutically acceptable dosage form prepared from the compound or pharmaceutically acceptable salt, racemate thereof as an effective ingredient and a pharmaceutically acceptable carrier.

[0078] The present application has the following beneficial effects:

[0079] The double E3-PROTACs of the present application have significant degradation activity on BRD4 protein and are superior to traditional PROTACs. BRIEF DESCRIPTION OF DRAWINGS

[0080] Figure 1 Western Blotting results of the compounds.

[0081] Figure 2 The results of the determination of the gray value of the compounds I-1, I-7, I-13, I-4, I-6, I-10, I-12, I-16, I-18; wherein the numerical value represents the mean ± SD (n = 3, each group), compared with compound XVII, * P<0.05, ** P<0.01, *** P<0.001.

[0082] Figure 3 Western Blotting results of the compounds.

[0083] Figure 4 The results of the determination of the gray value of the compounds I-3, I-9, I-15, I-2, I-5, I-8, I-11, I-14, I-17; wherein the numerical value represents the mean ± SD (n = 3, each group), compared with compound XVII, * P<0.05, ** P<0.01, *** P<0.001. DETAILED DESCRIPTION

[0084] A series of examples are listed below to further illustrate the technical solutions of the present application. These examples are illustrative and should not be construed as limiting the present application.

[0085] Example 1

[0086] Synthetic route of 2,2'-((2-((2-(2-(2-azidoethoxy)ethoxy)methyl)-2-methylpropane-1,3-diyl)bis(oxy))diacetic acid (Intermediate XV-1)

[0087]

[0088] Synthesis of (2,2,5-trimethyl-1,3-dioxan-5-yl)methanol (Intermediate III)

[0089] Dissolve 2-(hydroxymethyl)-2-methylpropane-1,3-diol (60.0 g, 0.50 mol) and p-toluenesulfonic acid (1.72 g, 0.01 mol) in dry acetone (600 ml) and stir at room temperature for 1 day; neutralize the reaction solution with potassium carbonate solid, filter, concentrate, and purify by silica gel column chromatography (eluent: n-hexane: ethyl acetate = 3:1 ~ 1:4 V / V) to obtain a colorless oil (intermediate III, 72.00 g, 0.45 mol, yield 90%, which becomes a white solid at 4°C).

[0090] Synthesis of 2-(2-azidoethoxy)ethan-1-ol (intermediate V-1)

[0091] Prepare a 40% solution of diethyl azodicarboxylate (DEAD, 74.19 mL, 0.47 mol) in toluene and reserve. In a reaction flask, dissolve diethylene glycol (compound IV-1, 50.00 g, 44.72 mL, 0.47 mol) and triphenylphosphine (PPh3, 123.60 g, 0.47 mol) in tetrahydrofuran (THF, 1.8 L). At 0°C, add the 40% solution of DEAD in toluene and diphenylphosphoryl azide (DPPA, 101.54 mL, 0.47 mol) to the reaction flask sequentially, slowly raise to room temperature, and stir overnight; concentrate the reaction solution by rotary evaporation, and purify by silica gel column chromatography (eluent: n-hexane: ethyl acetate = 3:1 ~ 1:4 V / V) to obtain a colorless oil (intermediate V-1, 15.4 g, 0.12 mol, yield 25%). This step can only be purified to 60% purity, which can be used for the next reaction.

[0092] Synthesis of 2-(2-azidoethoxy)ethyl 4-methylbenzenesulfonate (intermediate VI-1)

[0093] Dissolve 2-(2-azidoethoxy)ethan-1-ol (intermediate V-1, 32.75 g, 0.25 mol) in 500 mL of dichloromethane, add triethylamine (41.60 ml, 0.30 mol), slowly add p-toluenesulfonyl chloride (47.70 g, 0.25 mol) and 4-dimethylaminopyridine (DMAP, 1.53 g, 12.5 mmol) at 0°C, slowly raise to room temperature, and stir overnight; filter the reaction solution, wash the filtrate with saturated brine, concentrate by rotary evaporation, and purify by silica gel column chromatography (eluent: n-hexane: ethyl acetate = 4:1 ~ 2:3 V / V) to obtain a colorless oil (intermediate VI-1, 64.10 g, 0.23 mol, yield 90%).

[0094] Synthesis of 5-((2-(2-azidoethoxy)ethoxy)methyl)-2,2,5-trimethyl-1,3-dioxane (intermediate VII-1)

[0095] (2,2,5-trimethyl-1,3-dioxan-5-yl)methanol (Intermediate III, 10.17 g, 63.54 mmol) was dissolved in 70 mL of dry N,N-dimethylformamide (DMF), 60% NaH (dispersed in mineral oil, 8.47 g, 211.76 mmol) was added slowly at 0 °C, stirred for 30 min, 2-(2-azidoethoxy)ethyl 4-methylbenzenesulfonate (Intermediate VI-1, 15.09 g, 52.94 mmol) was added, stirred at room temperature overnight; the reaction was quenched by the addition of ice water and saturated aqueous NH4Cl solution, extracted with ethyl acetate for 3 times, the organic phase was combined, washed with saturated brine, dried over anhydrous magnesium sulfate, concentrated by rotary evaporation, purified by silica gel column chromatography (eluent: n-hexane: ethyl acetate = 9:1 ~ 3:1 V / V) to give colorless oil (Intermediate VII-1, 10.41 g, 38.12 mmol, 60% yield).

[0096] Synthesis of (2-((2-(2-azidoethoxy)ethoxy)methyl)-2-methylpropane-1,3-diol (Intermediate VIII-1)

[0097] Intermediate VII-1 (5.50 g, 20.15 mmol) was dissolved in 52 mL of a mixture solvent of methanol and water (30:1 V / V), trifluoroacetic acid (TFA, 0.77 mL, 10.07 mmol) was added dropwise at 0 °C, stirred at room temperature overnight, TLC detection showed that the starting material was consumed, directly concentrated, purified by silica gel column chromatography (eluent: n-hexane: ethyl acetate = 3:1 ~ 1:3 V / V) to give colorless oil (Intermediate VIII-1, 4.23 g, 18.14 mmol, 90% yield).

[0098] Synthesis of 2-((4-methoxybenzyl)oxy)ethan-1-ol (Intermediate X)

[0099] Dissolve ethylene glycol (15.00 g, 13.48 mL, 241.66 mmol) in 125.7 mL of tetrahydrofuran, add 60% NaH (4.83 g, 120.83 mmol) at 0 °C, stir at room temperature for 1 hour; add 4-methoxybenzyl chloride (PMBCl, 10.92 mL, 80.55 mmol), tetrabutylammonium chloride (TBAC, 2.24 g, 8.06 mmol) and potassium iodide (KI, 1.34 g, 8.06 mmol) successively, heat to reflux at 66 °C for 10 hours, cool to room temperature, add ice water and saturated aqueous NH4Cl solution to quench the reaction, extract with ethyl acetate for 3 times, combine the organic phases, wash the organic phase with saturated brine, dry over anhydrous magnesium sulfate, concentrate by rotary evaporation, purify by silica gel column chromatography (eluent: n-hexane: ethyl acetate = 9:1 ~ 3:2 V / V) to give colorless oil (intermediate X, 11.00 g, 60.41 mmol, yield 75%).

[0100] Synthesis of 1-((2-iodoethoxy)methyl)-4-methoxybenzene (intermediate XI)

[0101] In a reaction flask, dissolve triphenylphosphine (25.81 g, 98.40 mmol) and imidazole (6.70 g, 98.4 mmol) in 526.5 mL of dichloromethane, cool to 0 °C, add iodine (24.97 g, 98.40 mmol), stir at room temperature for 10 minutes, add a solution of 2-((4-methoxybenzyl)oxy)ethan-1-ol (intermediate X, 13.90 g, 76.30 mmol) in dichloromethane (183.1 mL), stir at room temperature overnight; TLC detection shows that the starting material is consumed, add saturated aqueous Na2S2O3 solution and saturated aqueous NaHCO3 solution to quench the reaction, separate the phases, extract the aqueous phase with ethyl acetate for 3 times, combine the organic phases, wash the organic phase with saturated brine, dry over anhydrous magnesium sulfate, concentrate by rotary evaporation, purify by silica gel column chromatography (eluent: n-hexane: ethyl acetate = 1:0 ~ 9:1 V / V) to give pale yellow oil (intermediate XI, 17.82 g, 61.04 mmol, yield 80%).

[0102] Synthesis of 14-azido-7-((2-((4-methoxybenzyl)oxy)ethoxy)methyl)-1-(4- methoxyphenyl)-7-methyl-2,5,9,12-tetraoxatetradecane (intermediate XII-1)

[0103] Intermediate VIII-1 (1.10 g, 4.72 mmol) was dissolved in 23.6 mL of anhydrous tetrahydrofuran, 60% NaH (1.13 g, 28.32 mmol) was added slowly at 0 °C, stirred for 30 min; intermediate XI (5.51 g, 18.88 mmol) was added, stirred at 66 °C overnight; the reaction was quenched by adding ice water and saturated aqueous NH4Cl solution, extracted with ethyl acetate for 3 times, the organic phase was combined, washed with saturated brine, dried over anhydrous magnesium sulfate, concentrated by rotary evaporation, purified by silica gel column chromatography (eluent: n-hexane: ethyl acetate = 9:1 ~ 3:1 V / V) to give a light green oil (intermediate XII-1, 1.24 g, 2.22 mmol, yield 47%).

[0104] Synthesis of 2,2'-((2-((2-(2-azidoethoxy)ethoxy)methyl)-2-methylpropane-1,3- diyl)bis(oxy))bis(ethan-1-ol) (intermediate XIII-1)

[0105] Intermediate XII-1 (1.21 g, 2.16 mmol) was dissolved in 23.76 mL of a mixed solvent of dichloromethane and water (10:1 V / V), 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 1.08 g, 4.75 mmol) was added at 0 °C, stirred at room temperature overnight; the reaction was quenched by adding saturated aqueous NaHCO3 solution, filtered, extracted with dichloromethane for 3 times, the organic phase was combined, washed with saturated brine, dried over anhydrous magnesium sulfate, concentrated by rotary evaporation; purified by silica gel column chromatography (eluent: dichloromethane:methanol = 100:1 ~ 33:1 V / V) to give a colorless oil (intermediate XIII-1, 0.62 g, 1.94 mmol, yield 90%).

[0106] Synthesis of 2,2'-((2-((2-(2-azidoethoxy)ethoxy)methyl)-2-methylpropane-1,3- diyl)bis(oxy))bis(acetic acid) (intermediate XV-1)

[0107] Intermediate XII-1 (300.0 mg, 0.94 mmol) was dissolved in 9.35 mL of dichloromethane, Dess-Martin oxidant (DMP, 1.19 g, 2.80 mmol) was added at 0 °C, stirred at room temperature for 3 hours; the reaction was directly concentrated, purified by silica gel column chromatography (eluent: dichloromethane:methanol = 100:1 ~ 4:1 V / V) to give intermediate XIV-1 crude, which could be directly used for the next step, yield 60%.

[0108] Synthesis of 2,2'-((2-((2-(2-azidoethoxy)ethoxy)methyl)-2-methylpropane-1,3- diyl)bis(oxy))bis(acetic acid) (intermediate XV-1)

[0109] Intermediate XIV-1 (200.0 mg, 0.94 mmol) was dissolved in 5.68 mL of acetonitrile to give solution 1, which was used later. Sodium dihydrogen phosphate (NaH2PO4, 302.9 mg, 2.52 mmol) was dissolved in 2.27 mL of water to give solution 2, which was used later. Solution 2 was added to solution 1, and then 1.45 mL of 50% hydrogen peroxide (H2O2, 21.32 mmol) was added to give a reaction solution; sodium chlorite (NaClO2, 251.0 mg, 2.52 mmol) was dissolved in 7.57 mL of water at 0 °C, and then added dropwise to the reaction solution. After the dropwise addition was completed, the reaction solution was slowly warmed to room temperature and stirred overnight. The reaction was quenched by adding saturated aqueous Na2S2O3 and saturated aqueous NH4Cl, and extracted with dichloromethane for 3-5 times. The organic phase was combined and dried over anhydrous magnesium sulfate, and concentrated by rotary evaporation. Purification by silica gel column chromatography (eluent: pure dichloromethane to dichloromethane:methanol:glacial acetic acid = 98:1:1 to 89:10:1 V / V) gave intermediate XV-1 (229.6 mg, 0.66 mmol, 70% yield) as a colorless oil.

[0110] Example 2

[0111] Synthesis of 1-azido-11-((carboxymethoxy)methyl)-11-methyl-3,6,9,13-tetraoxapentadecanoic acid (Intermediate XV-2)

[0112]

[0113] The preparation method of intermediate XV-1 in Example 1 was referred to, and tetraethylene glycol was used to replace diethylene glycol (compound IV-1) at an equimolar amount, and other conditions were unchanged. Purification by silica gel column chromatography (eluent: pure dichloromethane to dichloromethane:methanol:glacial acetic acid = 98:1:1 to 89:10:1 V / V) gave intermediate XV-2 as a colorless oil.

[0114] Example 3

[0115] Synthesis of 1-azido-14-((carboxymethoxy)methyl)-14-methyl-3,6,9,12,16-pentaoxaoctadecanoic acid (Intermediate XV-3)

[0116]

[0117] The preparation method of intermediate XV-1 in Example 1 was referred to, and tetraethylene glycol was used to replace diethylene glycol (compound IV-1) at an equimolar amount, and other conditions were unchanged. Purification by silica gel column chromatography (eluent: pure dichloromethane to dichloromethane:methanol:glacial acetic acid = 98:1:1 to 89:10:1 V / V) gave intermediate XV-2 as a colorless oil.

[0118] Example 4

[0119] 2,2'-((2-((2-(2-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)acetamido)ethoxy)ethoxy)methyl)- 2-methylpropane-l,3-diyl)bis(oxy))bis(N-(2-((2-(2,6-dioxopiperidin-3-yl)- 1,3-dioxoisoindolin-4-yl)amino)ethyl)acetamide) (Compound I-1) Synthesis

[0120]

[0121] 2,2'-((2-((2-(2-(2-azidoethoxy)ethoxy)methyl)-2-methylpropane-l,3-diyl)bis(oxy))bis(N-(2-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)ethyl)acetamide) (Intermediate XVI-1) Synthesis

[0122] Into a reaction vial, was added Intermediate XV-1 (34.9 mg, 0.1 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-l-yl)urem hexafluorophosphate (HATU, 114.1 mg, 0.30 mmol), N-hydroxy-7-azabenzotriazole (HOAt, 44.9 mg, 0.33 mmol), DMF (1 mL) and N,N-diisopropylethylamine (DIEA, 84 μL, 0.5 mmol) and stirred for 5 min; 4-((2-aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-l,3-dione (69.5 mg, 0.22 mmol) was added and stirred at room temperature for 2 h; the reaction mixture was directly concentrated, purified by silica gel column chromatography (eluent: dichloromethane:methanol = 100:1 ~ 15:1 V / V) and then by preparative liquid chromatography (column: SPODS-A column (20 x 250 mm), detection wavelength: 210 nm and 254 nm, mobile phase: water and methanol (both containing 1% trifluoroacetic acid), flow rate: 3 mL / min, eluent gradient: methanol:water = 10:90 ~ 95:5 V / V) to give a yellow solid (Intermediate XVI-1, 75.6 mg, 0.08 mmol, 80% yield).

[0123] Synthesis of Compound I-1

[0124] Intermediate XVI-1 (20.0 mg, 0.021 mmol) was dissolved in 2.1 mL of a mixture of methanol and dichloromethane (10:1 V / V) and 20 mg of 5% Pd / C was added. The reaction bottle was sealed, the air in the bottle was pumped out with a vacuum pump and hydrogen was filled into the bottle. The reaction was carried out at room temperature. The reaction progress was monitored by TLC or LCMS (liquid chromatography-mass spectrometry). After 5 hours of reaction, the starting material was consumed as detected by TLC or LCMS. The reaction solution was filtered, the filtrate was concentrated, and was lyophilized to give a crude product, which was used as is. In another reaction bottle, (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid (8.5 mg, 0.021 mmol), HATU (8.8 mg, 0.023 mmol), HOAt (4.3 mg, 0.032 mmol), DMF (0.21 mL), and N,N-diisopropylethylamine (DIEA, 7 μL, 0.042 mmol) were added in sequence. The mixture was stirred for 5 minutes. The reaction solution was added to the crude product used as is. The mixture was stirred at room temperature for 2 hours. The reaction solution was directly concentrated and purified by preparative liquid chromatography. The preparative liquid chromatography conditions were as follows: column: SP ODS-A column (20 x 250 mm), detection wavelength: 210 nm and 254 nm, flow rate: 3 mL / min, mobile phase: methanol and water (both containing 0.1% trifluoroacetic acid), eluent gradient: methanol:water = 10:90 to 95:5 V / V, to give a yellow solid (compound I-1, 20.5 mg, 0.016 mmol, 75% yield).

[0125] HR-MS: C 62 H 68 ClN 13 O 15 S for [M+H] + , calculated 1302.4440, found 1302.4434.

[0126] Example 5

[0127] Synthesis of (2R,4S)-1-((2R)-15-azido-2-(tert-butyl)-8-((2-((2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)amino)-2-oxoethoxy)methyl)-8-methyl-4-oxo-6,10,13-trioxa-3-azapentadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (Intermediate XVI-2)

[0128]

[0129] Synthesis of (2R,4S)-1-((2R)-15-azido-2-(tert-butyl)-8-((2-((2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)amino)-2-oxoethoxy)methyl)-8-methyl-4-oxo-6,10,13-trioxa-3-azapentadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (Intermediate XVI-2)

[0130] To a reaction vial was added successively intermediate XV-1 (34.9 mg, 0.1 mmol), HATU (57.0 mg, 0.15 mmol), HOAt (22.5 mg, 0.165 mmol), DMF (1 mL) and DIEA (42 μL, 0.25 mmol), stirred for 5 min; added 4-((2-aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (34.8 mg, 0.11 mmol), stirred at room temperature for 2 h; added again HATU (57.0 mg, 0.15 mmol), HOAt (22.5 mg, 0.165 mmol), DIEA (42 μL, 0.25 mmol) and (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (51.4 mg, 0.11 mmol), stirred at room temperature for 2 h; the reaction mixture was directly concentrated, purified by silica gel column chromatography (eluent: dichloromethane:methanol = 100:1 ~ 15:1 V / V) and preparative liquid chromatography, the conditions of preparative liquid chromatography were as follows: column: SP ODS-A column (20 x 250 mm), detection wavelength: 210 nm and 254 nm, mobile phase: methanol and water (both containing 0.1% trifluoroacetic acid), eluent gradient: methanol:water = 10:90 ~ 95:5 V / V, flow rate: 3 mL / min, to give a light yellow solid (intermediate XVI-2, 31.8 mg, 0.03 mmol, 30% yield).

[0131] Synthesis of compound I-2

[0132] Refer to the preparation method of compound I-1, replace intermediate XVI-1 with equimolar amount of intermediate XVI-2, and other conditions remain unchanged to give compound I-2, a light yellow solid, yield 75%.

[0133] HR-MS: C 69 H 82 ClN 13 O 14 S2 for [M+H] + , calculated 1416.5307, found 1416.5289.

[0134] Example 6

[0135] Synthesis of (2S,4R)-1-((17S)-17-(tert-butyl)-1-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-11-((2-(((R)-1-((2R,4S)-4- hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1- oxobutan-2-yl)amino)-2-oxoethoxy)methyl)-11-methyl-2,15-dioxo-6,9,13-trioxa-3,16- diazaoctadec-18-yl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (Compound I-3)

[0136]

[0137] Synthesis of (2S,4R)-1-((2S,14R)-8-((2-(2-azidoethoxy)ethoxy)methyl)-2-(tert-butyl)-14- ((2R,4S)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)- 8,15,15-trimethyl-4,12-dioxo-6,10-dioxa-3,13-diazahexadecyl)-4-hydroxy-N-(4-(4- methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (Intermediate XVI-3)

[0138] To a reaction flask was added Intermediate XV-1 (34.9 mg, 0.1 mmol), HATU (114.1 mg, 0.30 mmol), HOAt (44.9 mg, 0.33 mmol), DMF (1 mL) and DIEA (84 μί, 0.5 mmol) successively, stirred for 5 min; (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4- hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (102.7 mg, 0.22 mmol) was added, stirred at room temperature for 2 h; the reaction mixture was directly concentrated, purified by silica gel column chromatography (eluent: dichloromethane:methanol = 100:1 ~ 15:1 V / V) first, then by preparative liquid chromatography, the conditions of preparative liquid chromatography were as follows: column: SP ODS-A column (20 x 250 mm), detection wavelength: 210 nm and 254 nm, mobile phase: methanol and water (both containing 0.1% trifluoroacetic acid), eluent gradient: methanol:water = 10:90 ~ 95:5 V / V, flow rate: 3 mL / min, to give a white solid (Intermediate XVI-3, 93.9 mg, 0.08 mmol, 80% yield).

[0139] Synthesis of compound I-3

[0140] The compound I-3 was prepared according to the procedure described for the preparation of compound I-1 by replacing intermediate XVI-1 with intermediate XVI-3 in an equimolar amount, and otherwise as described. Compound I-3 was obtained as a white solid in 75% yield.

[0141] HR-MS: C 76 H 96 ClN 13 O 13 S3 for [M+H] + , calculated 1530.6174, found 1530.6148.

[0142] Example 7

[0143] Synthesis of 2,2'-((2-((2-(2-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)acetamido)ethoxy)ethoxy)methyl)- 2-methylpropane-l,3-diyl)bis(oxy))bis(N-(3-((2-(2,6-dioxopiperidin-3-yl)-l- oxoisoindolin-4-yl)amino)propyl)acetamide) (compound I-4)

[0144]

[0145] Synthesis of 2,2'-((2-((2-(2-(2-azidoethoxy)ethoxy)methyl)-2-methylpropane-l,3- diyl)bis(oxy))bis(N-(3-((2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-4-yl)amino)propyl) acetamide) (intermediate XVI-4)

[0146] To a reaction vial was added successively intermediate XV-1 (34.9 mg, 0.1 mmol), HATU (114.1 mg, 0.30 mmol), HOAt (44.9 mg, 0.33 mmol), DMF (1 mL) and DIEA (84 μL, 0.5 mmol), stirred for 5 min; added 3-(4-((3-aminopropyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (69.5 mg, 0.22 mmol), stirred at room temperature for 2 h; the reaction mixture was directly concentrated, purified by silica gel column chromatography (eluent: dichloromethane:methanol = 100:1 ~ 15:1 V / V) and preparative liquid chromatography, the conditions of preparative liquid chromatography were as follows: column: SP ODS-A column (20 x 250 mm), detection wavelength: 210 nm and 254 nm, mobile phase: methanol and water (both containing 0.1% trifluoroacetic acid), eluent gradient: methanol:water = 10:90 ~ 95:5 V / V, flow rate: 3 mL / min, to give a light yellow solid (intermediate XVI-4, 75.6 mg, 0.08 mmol, 80% yield).

[0147] Synthesis of compound I-4

[0148] Refer to the preparation method of compound I-1, replace intermediate XVI-1 with equimolar amount of intermediate XVI-4, and other conditions remain unchanged to obtain compound I-4, a light yellow solid, in a yield of 75%.

[0149] HR-MS: C 64 H 76 ClN 13 O 13 S for [M+H] + , calculated 1302.5168, found 1302.5153.

[0150] Example 8

[0151] Synthesis of (2S,4R)-1-((17S)-17-(tert-butyl)-1-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-11-((2-((3-((2-(2,6- dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)propyl)amino)-2-oxoethoxy)methyl)- 11-methyl-2,15-dioxo-6,9,13-trioxa-3,16-diazaoctadecan-18-yl)-4-hydroxy-N-(4-(4- methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (compound I-5)

[0152] Synthesis of (2S,4R)-1-((17S)-17-(tert-butyl)-1-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-11-((2-((3-((2-(2,6- dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)propyl)amino)-2-oxoethoxy)methyl)- 11-methyl-2,15-dioxo-6,9,13-trioxa-3,16-diazaoctadecan-18-yl)-4-hydroxy-N-(4-(4- methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (compound I-5)

[0153] Synthesis of (2S,4R)-1-((2S)-8-((2-(2-azidoethoxy)ethoxy)methyl)-2-(tert-butyl)-16-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)amino)-8-methyl-4,12-dioxo-6,10-dioxa-3,13-diazahexadecyl)-4-hydroxy-N-(4-(4-methylthiazo-5-yl)benzyl)pyrrolidine-2-carboxamide (intermediate XVI-5)

[0154] Intermediate XV-1 (34.9 mg, 0.1 mmol), HATU (57.0 mg, 0.15 mmol), HOAt (22.5 mg, 0.165 mmol), DMF (1 mL), and DIEA (42 μL, 0.25 mmol) were added sequentially to the reaction flask, and the mixture was stirred for 5 minutes. Then, 3-(4-((3-aminopropyl)amino)-1-oxoisoindololin-2-yl)piperidin-2,6-dione (34.8 mg, 0.11 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Finally, HATU (57.0 mg, 0.15 mmol) and HOAt were added. t (22.5 mg, 0.165 mmol), DIEA (42 μL, 0.25 mmol), and (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (51.4 mg, 0.11 mmol) were stirred at room temperature for 2 hours. The reaction solution was directly concentrated, purified by silica gel column chromatography (eluent: dichloromethane:methanol = 100:1 to 15:1 V / V), and then purified by preparative liquid chromatography. The preparative liquid chromatography conditions were: column: SP An ODS-A column (20×250 mm) was used, with detection wavelengths of 210 nm and 254 nm. The mobile phase consisted of methanol and water (both containing 0.1% trifluoroacetic acid), and the eluent gradient was methanol:water = 10:90 to 95:5 V / V. The flow rate was 3 mL / min, yielding a white solid (intermediate XVI-5, 31.8 mg, 0.03 mmol, yield 30%).

[0155] Synthesis of compound I-5

[0156] Following the preparation method of compound I-1, intermediate XVI-1 was replaced with an equimolar amount of intermediate XVI-5, with other conditions remaining unchanged, to obtain compound I-5, a white solid with a yield of 75%.

[0157] HR-MS:C 70 H 86 ClN 13 O 13S2 for [M+H] + Calculated 1416.5671, found 1416.5612.

[0158] Example 9

[0159] 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][l,2,4]triazolo[4,3- a][l,4]diazepin-6-yl)-N-(8-((2-((2-(2-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin- 4-yl)amino)ethyl)amino)-2-oxoethoxy)methyl)-l6-((2-(2,6-dioxopiperidin-3-yl)-l- oxoisoindolin-4-yl)amino)-8-methyl- 12-oxo-3,6,10-trioxa- 13-azahexadecyl)acetamide (Compound I-6)

[0160]

[0161] 2-(3-(2-(2-azidoethoxy)ethoxy)-2-((2-((2-(2-(2-(2,6-dioxopiperidin-3-yl)-l,3- dioxoisoindolin-4-yl)amino)ethyl)amino)-2-oxoethoxy)methyl)-2-methylpropoxy)-N-(3- ((2-(2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolin-4-yl)amino)propyl)acetamide (Intermediate XVI-6)

[0162] To a reaction vial was added successively intermediate XV-1 (34.9 mg, 0.1 mmol), HATU (57.0 mg, 0.15 mmol), HOAt (22.5 mg, 0.165 mmol), DMF (1 mL) and DIEA (42 μL, 0.25 mmol), stirred for 5 min; 3-(4-((3-aminopropyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (34.8 mg, 0.11 mmol) was added, stirred at room temperature for 2 h; HATU (57.0 mg, 0.15 mmol), HOAt (22.5 mg, 0.165 mmol), DIEA (42 μL, 0.25 mmol) and 4-((2-aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (34.8 mg, 0.11 mmol) were added, stirred at room temperature for 2 h; the reaction mixture was directly concentrated, purified by silica gel column chromatography (eluent: dichloromethane:methanol = 100:1 ~ 15:1 V / V) and preparative liquid chromatography, the conditions of preparative liquid chromatography were as follows: column: SP ODS-A column (20 x 250 mm), detection wavelength: 210 nm and 254 nm, mobile phase: methanol and water (both containing 0.1% trifluoroacetic acid), eluent gradient: methanol:water = 10:90 ~ 95:5 V / V, flow rate: 3 mL / min, to give a yellow solid (intermediate XVI-6, 28.4 mg, 0.03 mmol, 30% yield).

[0163] Synthesis of compound I-6

[0164] Refer to the preparation method of compound I-1, replace intermediate XVI-1 with equimolar amount of intermediate XVI-6, and other conditions remain unchanged, to give a yellow solid in 75% yield.

[0165] HR-MS: C 63 H 72 ClN 13 O 14 S for [M+H] + , calculated 1302.4804, found 1302.4773.

[0166] Example 10

[0167] 1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetylamino)-N-(2-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin- 4-yl)amino)ethyl)-11-((2-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino) ethyl)amino)-2-oxoethoxy)methyl)-11-methyl-3,6,9,13-tetraoxapentadecan-15-amide (Compound I-7)

[0168]

[0169] The compound I-7 was obtained by referring to the preparation method of the compound I-1, replacing the intermediate XV-1 with the intermediate XV-2 in an equimolar amount, and other conditions were unchanged, to obtain the compound I-7, yellow solid, yield 75%.

[0170] HR-MS: C 64 H 72 ClN 13 O 16 S for [M+H] + , calculated 1346.4702, found 1346.4692.

[0171] Example 11

[0172] (2S,4R)-1-((2OS)-20-(tert-butyl)-1-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-14-((2-((2-(2-(2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)amino)-2-oxoethoxy)methyl)-14-methyl- 2,18-dioxo-6,9,12,16-tetraoxa-3,19-diazapentacosa-21-oyl)-4-hydroxy-N-(4-(4- methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (Compound I-8)

[0173]

[0174] The compound I-8 was obtained by referring to the preparation method of the compound I-2, replacing the intermediate XV-1 with the intermediate XV-2 in an equimolar amount, and other conditions were unchanged, to obtain the compound I-8, yellowish solid, yield 75%.

[0175] HR-MS: C71 H 86 ClN 13 O 15 S2 for[M+H] + ,calculated1460.5569,found 1460.5438.

[0176] Example 12

[0177] (2S,4R)-1-((S)-20-tert-butyl)-1-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-14-((2-(S)-1-((2S,4R)-4- hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3- dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)methyl)-14-methyl-2,18-dioxo-6,9,12,16- tetraoxa-3,19-diazahenicosan-21-yl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine- 2-carboxamide (Compound I-9)

[0178]

[0179] The compound I-9 was prepared according to the method for preparing the reference compound I-3, using intermediate XV-2 instead of intermediate XV-1 in an equimolar amount, and without other changes, to obtain the compound I-9 as a white solid with a yield of 75%

[0180] HR-MS: C 78 H 100 ClN 13 O 14 S3 for[M+H] + ,calculated1574.6436,found 1574.6432.

[0181] Example 13

[0182] 1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetylamino)-N-(3-((2-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin- 4-yl)amino)propyl)-11-((2-((3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4- yl)amino)propyl)propyl)-2-oxoethoxy)methyl)-11-methyl-3,6,9,13-tetraoxapentadecan- 15-amide (Compound I-10)

[0183]

[0184] The compound I-10 was prepared according to the method for preparing the reference compound I-4, using intermediate XV-2 instead of intermediate XV-1 in an equimolar amount, and without modifying the other conditions. It was obtained in the form of a light yellow solid in a yield of 75%.

[0185] 1 H NMR (500 MHz, DMSO-d6) δ 10.88 (s, 2H), 7.62 (t, J = 6.0 Hz, 2H), 7.36 (d, J = 8.5 Hz, 2H), 7.33-7.27 (m, 2H), 7.14 (t, J = 7.7 Hz, 2H), 6.81 (d, J = 7.4 Hz, 2H), 6.61 (d, J = 8.1 Hz, 2H), 4.98 (dd, J = 13.2, 5.2 Hz, 2H), 4.10 (d, J = 17.1 Hz, 2H), 4.01 (d, J = 17.1 Hz, 2H), 3.72 (s, 4H), 3.39 (s, 5H), 3.37-3.29 (m, 5H), 3.23-3.13 (m, 9H), 3.13-3.05 (m, 6H), 3.00 (t, J = 7.0 Hz, 4H), 2.79 (m, 2H), 2.48 (s, 5H), 2.38 (m, 6H), 2.28 (s, 3H), 2.17 (m, 2H), 1.90 (m, 2H), 1.61 (m, 4H), 1.49 (s, 3H), 0.77 (s, 3H). HR-MS: C 66 H 80 ClN 13 O 14 S for [M+H] + , calculated 1346.5430, found 1346.5403.

[0186] Example 14

[0187] Synthesis of (2S,4R)-1-((2OS)-20-(tert-butyl)-1-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-14-((2-((3-((2-(2,6- dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)propyl)amino)-2-oxoethoxy)methyl)- 14-methyl-2,18-dioxo-6,9,12,16-tetraoxa-3,19-diazacosa-21-yl)-4-hydroxy-N-(4-(4- methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (Compound I-11)

[0188]

[0189] Refer to the preparation method of Compound I-5, replace intermediate XV-1 with intermediate XV-2 in equal molar amount, and the rest is the same, to obtain Compound I-11, white solid, yield 75%.

[0190] HR-MS: C 72 H 90 ClN 13 O 14 S2 for[M+H] + , calculated 1482.5752, found 1482.5738.

[0191] Example 15

[0192] 1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetamido)-N-(2-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin- 4-yl)amino)ethyl)-11-((2-((3-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino) propyl)amino)-2-oxoethoxy)methyl)-11-methyl-3,6,9,13-tetraoxapentacosa-15-amide (Compound I-12) Synthesis

[0193]

[0194] Refer to the preparation method of Compound I-6, replace intermediate XV-1 with intermediate XV-2 in equal molar amount, and the rest is the same, to obtain Compound I-12, yellow solid, yield 75%.

[0195] HR-MS: C65 H 76 ClN 13 O 15 S for[M+H] + , calculated 1346.5066, found 1346.5045.

[0196] Example 16

[0197] 1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetamino)-N-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin- 4-yl)amino)ethyl)-14-((2-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)amino)-2-oxoethoxy)methyl)-14-methyl-3,6,9,12,16- pentaoxaoctadecan-18-amide (Compound I-13)

[0198]

[0199] The compound I-13 was prepared according to the method for preparing the reference compound I-1, replacing the intermediate XV-1 with the intermediate XV-3 in an equimolar amount, and other conditions were unchanged, to obtain the compound I-13, a yellow solid, in a yield of 75%.

[0200] HR-MS: C 66 H 76 ClN 13 O 17 S for[M+H] + , calculated 1390.4964, found 1390.4943.

[0201] Example 17

[0202] Synthesis of (2R,4S)-1-((23R)-23-(tert-butyl)-1-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-17-((2-((2-((2-(2-(2,6- dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)amino)-2-oxoethoxy) methyl)-17-methyl-2,21-dioxo-6,9,12,15,19-pentaoxa-3,22-diazatetracosan-24-yl)-4- hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (Compound I-14)

[0203]

[0204] The compound I-14 was prepared according to the preparation method of the reference compound I-2, using intermediate XV-3 instead of intermediate XV-1 in an equimolar amount, and without other changes, to give the compound I-14 as a light yellow solid in a yield of 75%.

[0205] HR-MS: C 73 H 90 ClN 13 O 16 S2 for [M+H] + , calculated 1504.5831, found 1504.5810.

[0206] Example 18

[0207] Synthesis of (2S,4R)-1-((23S)-23-tert-butyl)-1-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-17-((2-(R)-1-((2R,4S)-4- hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1- oxobutan-2-yl)amino)-2-oxoethoxy)methyl)-17-methyl-2,21-dioxo-6,9,12,15,19- pentaoxa-3,22-diazatetracosan-24-yl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine- 2-carboxamide (Compound I-15)

[0208]

[0209] The compound I-15 was obtained in white solid with 75% yield according to the preparation method of the compound I-3, using intermediate XV-3 instead of intermediate XV-1 in equimolar amount, and other conditions unchanged.

[0210] HR-MS: C 80 H 104 ClN 13 O 15 S3 for[M+H] + , calculated 1618.6698, found 1618.6686.

[0211] Example 19

[0212] 1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetamido)-N-(3-((2-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4- yl)amino)propyl)-14-(((3-((2-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)propyl) amino)-2-oxoethoxy)methyl)-14-methyl-3,6,9,12,16-pentaoxaoctadecan-18-amide (Compound 16)

[0213]

[0214] The compound I-16 was obtained in pale yellow solid with 75% yield according to the preparation method of the compound I-4, using intermediate XV-3 instead of intermediate XV-1 in equimolar amount, and other conditions unchanged.

[0215] HR-MS: C 68 H 84 ClN 13 O 15 S for[M+H] + , calculated 1390.5692, found 1390.5686.

[0216] Example 20

[0217] Synthesis of (2S,4R)-1-((23S)-23-(tert-butyl)-1-((S)-4-(4-chlorophenyl)-2,3,9- trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-17-((2-((3-((2-(2,6- dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)propyl)amino)-2-oxoethoxy)methyl)- 17-methyl-2,21-dioxo-6,9,12,15,19-pentaoxa-3,22-diazatetracosan-24-oyl)-4- hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (Compound I-17)

[0218]

[0219] Refer to the preparation method of Compound I-5, replace intermediate XV-1 with intermediate XV-3 in equimolar amount, and the rest is the same, to obtain Compound I-17, white solid, yield 75%.

[0220] HR-MS: C 74 H 94 ClN 13 O 15 S2 for [M+H] + , calculated 1526.6015, found 1526.6008.

[0221] Example 21

[0222] 1-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)acetamido)-N-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin- 4-yl)amino)ethyl)-14-((2-((3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino) propyl)amino)-2-oxoethoxy)methyl)-14-methyl-3,6,9,12,16-pentaoxaoctadecan-18- amide (Compound I-18) Synthesis

[0223]

[0224] Refer to the preparation method of Compound I-6, replace intermediate XV-1 with intermediate XV-3 in equimolar amount, and the rest is the same, to obtain Compound I-18, yellow solid, yield 75%.

[0225] HR-MS: C67 H 80 ClN 13 O 16 S for[M+H] + , calculated 1390.5328, found 1390.5309.

[0226] Example 22

[0227] Synthesis route of 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2- f][l,2,4]triazolo[4,3-a][l,4]diazepin-6-yl)-N-(l-((2-(2,6-dioxopiperidin-3-yl)-l,3- dioxoisoindolin-4-yl)amino)-4-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)acetamide (Compound XVII)

[0228]

[0229] Synthesis of tert-butyl (l-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)amino)-4-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)carbamate (Intermediate XIX-1)

[0230] To the reaction bottle, 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaoctadecan-16-oic acid (51.4 mg, 0.16 mmol), HATU (62.0 mg, 0.16 mmol), HOAt (22.2 mg, 0.16 mmol), DMF (1 mL) and DIEA (85 μL, 0.49 mmol) were added in turn, stirred for 5 min; 4-((2-aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-l,3-dione (50.0 mg, 0.16 mmol) was added, stirred at room temperature for 2 h; after the reaction was completed, saturated brine was added, extracted with dichloromethane for 3 times, the organic phases were combined and concentrated; purified by silica gel column chromatography (eluent dichloromethane:methanol = 100:1 ~ 15:1 V / V) to obtain a yellow solid (Intermediate XIX-1, 77.4 mg, 0.13 mmol, 80% yield).

[0231] Synthesis of 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-N-(2-((2-(2,6-dioxopiperidin-3- yl)-l,3-dioxoisoindolin-4-yl)amino)ethyl)acetamide (Intermediate XX-1)

[0232] Intermediate XIX-1 (43.0 mg, 0.071 mmol) was dissolved in 0.318 mL of dichloromethane, 0.106 mL of trifluoroacetic acid (TFA) was added dropwise, stirred at room temperature overnight, TLC detection at this time the raw material was consumed, direct concentration; silica gel column chromatography (eluent dichloromethane:methanol = 100:1 ~ 10:1 V / V) purification, to get yellow oil (intermediate XX-1, 32.3 mg, 0.064 mmol, 90% yield).

[0233] Synthesis of compound XVII

[0234] To the reaction bottle was added (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid (21.8 mg, 0.054 mmol), HATU (20.7 mg, 0.054 mmol), HOAt (7.4 mg, 0.054 mmol), DMF (0.3 mL) and DIEA (36 μL, 0.216 mmol) in turn, stirred for 5 min; intermediate XX-1 (27.3 mg, 0.054 mmol) was added, stirred at room temperature for 2 h; after the reaction was completed, the reaction solution was directly concentrated, purified by preparative liquid chromatography, and the conditions of preparative liquid chromatography were as follows: column: SP ODS-A column (20 x 250 mm), detection wavelength: 210 nm and 254 nm, mobile phase was methanol and water (both containing 0.1% trifluoroacetic acid), flow rate: 3 mL / min, eluent gradient was methanol: water = 10:90 ~ 95:5 V / V, to get yellow solid (compound XVII, 38.4 mg, 0.043 mmol, 80% yield).

[0235] Example 23

[0236] Synthesis of 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepin-6-yl)-N-(15-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)- 11-oxo-3,6,9-trioxa-12-azapentadecyl)acetamide (compound XVIII)

[0237]

[0238] The compound XVIII was prepared according to the preparation method of the reference compound XVII, by replacing 4-((2-aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione with 3-(4-((3-aminopropyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione in an equimolar amount, and by keeping other conditions unchanged, to obtain the compound XVIII, a light yellow solid, in a yield of 80%.

[0239] Example 24

[0240] The protein degradation activity of the compounds I-1 to I-18 was detected by Western Blot.

[0241] Cell strain: Hela cells.

[0242] Tested compounds: the compounds I-1 to I-18, the compound XVII (traditional PROTACs), the compound XVIII (traditional PROTACs), and (+)JQ-1.

[0243] Drug preparation: the compounds I-1 to I-18, the two control compounds, and (+)JQ-1 were respectively dissolved in DMSO to prepare a stock solution with a concentration of 20 mmol / L, and then DMEM complete culture medium was used to prepare a drug solution with a concentration of 1 μmol / L, which was ready for use.

[0244] Cell culture: the Hela cells in a good growth state were selected for digestion, transferred into 9 cm cell culture dishes, and cultured in a 37℃, 5% CO2 incubator for 24 h, and then the drug solution was given, and the cell culture dishes were further cultured in the incubator for 12 h. DMSO and DMEM complete culture medium were mixed in a volume ratio of 1:19, and an equal volume of the mixture was used to replace the drug solution, to serve as a DMSO group.

[0245] Extraction of cell protein: the old culture medium was discarded, the cells were rinsed with 2 mL pre-cooled PBS for 2-3 times, and the PBS was discarded; 100 μL of prepared lysis solution (1 mL lysis solution + 10 μL PMSF) was added to the cell culture dish, the lysis solution was evenly spread, and lysis was performed on ice for 20 min; after the lysis was completed, a clean spatula was used to quickly scrape the cells to one side of the culture dish, and then a syringe was used to transfer the cell fragments and lysis solution to a 1.5 mL centrifuge tube; the centrifuge was pre-cooled to 4℃, and centrifugation was performed at 4℃ and 12000 rpm for 15 min; after the centrifugation was completed, the supernatant was transferred to a 1.5 mL centrifuge tube, and stored on ice, to be used for BCA protein quantification experiments. An appropriate amount of the supernatant was taken, and about 1 / 4 of the volume of the supernatant was added to the loading buffer, which was subjected to a metal bath at 95℃ for 10 min, cooled to room temperature, and stored in a -80℃ refrigerator, for standby use.

[0246] BCA method for determining the concentration of cell protein: BCA reagent A and reagent B are prepared into BCA working solution according to the volume ratio of 50:1, and are used. Respectively 0, 1, 2, 4, 8, 12, 16, 20 μL of protein standard solution (0.5 mg / mL) is added to the 96-well plate, then PBS is added to make the total volume 20 μL, then 200 μL of BCA working solution is added, and it is placed in a 37°C incubator for 30 min. The absorbance of the sample at 562 nm is detected by the enzyme-labeled instrument, and the standard curve is drawn according to the absorbance value of the standard protein. Take 10 μL of the protein sample to be tested, dilute it to 20 μL with water, add 200 μL of BCA working solution, and place it in a 37°C incubator for 30 min. The absorbance of the sample at 562 nm is detected by the enzyme-labeled instrument, and then the concentration of the protein to be tested is calculated according to the standard curve.

[0247] Western blot detection of BRD4 expression in Hela cells:

[0248] (1) Gel preparation: After aligning the clean glass plate, clamp it tightly to assemble the electrophoresis tank, then vertically place it on the shelf to prepare for pouring the glue. According to the molecular weight of the target protein, prepare 6% separation glue, pour the separation glue into the glass plate of the electrophoresis tank, add anhydrous ethanol above the glue, and the liquid level is flat with the short glass plate. Room temperature for 30 min. When a clear interface appears between the separation glue and the anhydrous ethanol, it means that the separation glue has been polymerized. Slowly pour off the anhydrous ethanol. Pour the prepared concentrated glue onto the separation glue, and the liquid level is flat with the short glass plate. Then quickly insert the sample lane comb into the liquid surface to avoid air bubbles. Room temperature until the glue solidifies.

[0249] (2) Sample loading: Place the prepared polymerized glue into the electrophoresis tank, then add electrophoresis buffer to the inner tank of the electrophoresis tank until it is immersed. Add the same electrophoresis buffer to the outer tank, and the liquid level is about one-third of the height of the electrophoresis tank. Carefully pull out the comb vertically to avoid damaging the sample lane. Slowly add an appropriate amount of protein sample and standard protein Marker to the bottom of each gel well with a pipette.

[0250] (3) Electrophoresis: Cover the electrophoresis instrument, connect the electrophoresis instrument and the power supply device. Electrophoresis at 80V for 30 min. When the sample reaches the separation glue, adjust the voltage to 120V and continue electrophoresis. Refer to the Marker position to determine whether to end the electrophoresis.

[0251] (4) Membrane transfer: Take out the gel and soak it in the transfer solution, separate the glass plates, cut off the concentrated glue part, and soak the remaining part in the transfer solution. Use tweezers to hold the PVDF, soak it in methanol for activation, and use it. Add double-layer filter paper, PVDF membrane, gel, double-layer filter paper to the transfer tank in turn, and lay them flat and in alignment. Add the transfer solution and set the conditions, and transfer the membrane in an ice water bath.

[0252] (5) Antibody incubation: After the PVDF membrane is transferred, wash the membrane with the washing solution for 10 min three times, then add an appropriate amount of 5% blocking solution, and block at room temperature for 1 h; after blocking, discard the blocking solution. Dilute the target protein primary antibody, and prepare it for use. Place the PVDF membrane in the primary antibody dilution solution and incubate at 4°C overnight; after the primary antibody incubation is complete, recover the primary antibody dilution solution and wash the PVDF membrane with PBST buffer three times for 10 min each time. Dilute the target protein secondary antibody, and prepare it for use. Place the PVDF membrane in the secondary antibody dilution solution and incubate at room temperature on a shaker for 1 h, then wash the membrane with TBST buffer three times for 10 min each time. Complete the antibody incubation.

[0253] (6) Development and analysis: Prepare the development working solution (A solution: B solution = 1:1) and prepare it for use. Take out the PVDF membrane, evenly add the development working solution to the surface of the membrane, and finally expose it to light using a gel imaging system and save the exposure image. Scan the gray scale of each group of protein bands using ImageJ software, use the β-actin internal reference protein as the reference gray scale, and compare the expression levels of each group of proteins.

[0254] Each treatment is repeated three times, and the experimental results are expressed as the average value.

[0255] The protein degradation activity results of compounds I-1 to I-18 are shown in Figures 1-4 It can be observed that in Hela cells, most of the compounds have good degradation effect on BRD4 protein, and the degradation efficiency is better than that of the corresponding traditional PROTACs.

[0256] Compounds I-1, I-7, I-13, I-4, I-6, I-10, I-12, I-16 and I-18 have good degradation activity on BRD4 protein; compared with compound XVII and compound XVIII (traditional PROTACs), compounds I-6, I-10, I-12, I-16 and I-18 have significant differences, indicating that their degradation activity is significantly better than the control, and the strongest degradation activity is compound I-10, which has a protein degradation rate of 80% at a drug concentration of 1 μM and a drug time of 12 hours. While the traditional PROTACs have a protein degradation rate of only 10% under the same drug concentration and time. Figure 1 and Figure 2

[0257] Compounds I-8, I-14 and I-17 have good degradation activity on BRD4 protein. Compared with compound XVII and compound XVIII, compounds I-14 and I-17 have better degradation activity, and compound I-14 has significant differences, indicating that the degradation activity of compound I-14 is significantly better than the control, and the protein degradation rate reaches 50% at a drug concentration of 1 μM and a drug time of 12 hours.​Figure 3 and Figure 4 ).

[0258] In summary, it is shown that the dual E3-PROTACs have better degradation activity on BRD4 protein, and are superior to the classic bivalent PROTACs.

Claims

1. A compound with the structure shown in Formula I, or a pharmaceutically acceptable salt or racemate thereof: in, n is an integer from 2 to 8, and R1 and R2 are independently selected from... X is selected from CH2 and C=O, and m is an integer from 2 to 3.

2. The compound according to claim 1, or its pharmaceutically acceptable salt or racemate, characterized in that: n is 2, 3, or 4, and R1 and R2 are independently selected from... But not including: n=2, R1 is selected from R2 is selected from n = 2 or 3, R1 is selected from R2 is selected from n=4, R1 is selected from R2 is selected from 3. The compound according to claim 2, or its pharmaceutically acceptable salt or racemate, characterized in that: n is 2, 3, or 4, and R1 and R2 are independently selected from... n is 3 or 4, and R1 is selected from... R2 is selected from n is 4, and R1 is selected from... R2 is selected from 4. Compounds with the following structures, or their pharmaceutically acceptable salts or racemates:

5. Compounds with the following structures or their pharmaceutically acceptable salts or racemates:

6. A method for preparing the compound according to claim 1, characterized in that: The synthesis route is as follows: Wherein, n, R1, and R2 are as described in claim 1; Includes the following steps: Step (1): Using anhydrous N,N-dimethylformamide as the reaction solvent, in the presence of an acid-binding agent, compound III reacts with intermediate VI to obtain intermediate VII; Step (2): Using a mixed solvent of methanol and water in a volume ratio of 30:1 as the reaction solvent, intermediate VII is hydrolyzed under acidic conditions to obtain intermediate VIII. Step (3): Using anhydrous tetrahydrofuran or anhydrous N,N-dimethylformamide as the reaction solvent, intermediate VIII reacts with compound XI in the presence of an acid-binding agent to obtain intermediate XII; Step (4): Using a mixed solvent of dichloromethane and water in a volume ratio of 10:1 as the reaction solvent, intermediate XII undergoes a deprotection reaction in the presence of a strong oxidizing agent to obtain intermediate XIII; Step (5): Using dichloromethane as the reaction solvent, intermediate XIII undergoes an oxidation reaction in the presence of Des Martin oxidant to obtain intermediate XIV; Step (6): Using a mixed solvent of acetonitrile and water in a volume ratio of 1:1.8 to 1:2 as the reaction solvent, intermediate XIV is oxidized under the conditions of sodium chlorite, sodium dihydrogen phosphate and H2O2 to obtain intermediate XV. Step (7): Using dichloromethane, tetrahydrofuran, acetonitrile or N,N-dimethylformamide as the reaction solvent, in the presence of amide condensing agent and acid-binding agent, compound XV reacts with R1H and R2H to obtain intermediate XVI. Step (8): Using a mixed solvent of methanol and dichloromethane in a volume ratio of 10:1 as the reaction solvent, the intermediate XVI is reduced by a catalyst under a hydrogen atmosphere; then, using dichloromethane, tetrahydrofuran, acetonitrile, or N,N-dimethylformamide as the reaction solvent, in the presence of an amide condensing agent and an acid-binding agent, the reduction product is reacted with (+)JQ-1((S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazaphen-6-yl)acetic acid) to obtain the compound shown in Formula I.

7. The method for preparing the compound according to claim 6, characterized in that: In step (1), the molar ratio of compound III to intermediate VI is 1:0.9 to 1:1.1; the molar ratio of compound III to acid-binding agent is 1:3 to 1:5; the acid-binding agent is selected from NaH; the reaction temperature is room temperature; In step (2), the acidic conditions are provided by trifluoroacetic acid, and 0.01 to 0.015 mL of trifluoroacetic acid is added for every 1 mL of reaction solvent; the reaction temperature is room temperature; In step (3), the molar ratio of intermediate VIII to compound XI is 1:3 to 1:5; the molar ratio of intermediate VIII to acid-binding agent is 1:1 to 1:1.5; the acid-binding agent is selected from NaH; the reaction temperature is 20 to 66°C. In step (4), the strong oxidizing agent is 2,3-dichloro-5,6-dicyanobenzoquinone; the molar ratio of intermediate XII to the strong oxidizing agent is 1:2.2 to 1:2.5; and the reaction temperature is room temperature. In step (5), the molar ratio of intermediate XIII to Desmond oxidant is 1:2.5 to 1:3.5; the reaction temperature is room temperature; In step (6), the molar ratio of intermediate XIV to sodium chlorite is 1:2.6 to 1:2.7; the molar ratio of intermediate XIV to sodium dihydrogen phosphate is 1:2.6 to 1:2.7; the molar ratio of intermediate XIV to H2O2 is 1:20 to 1:28; and the reaction temperature is room temperature. In step (7), (a) when R1 and R2 are the same, the molar ratio of intermediate XV to amide condensing agent is 1:6.3; the amide condensing agent is a combination of one selected from O-(7-azabenzotriazole-1-yl)-di(dimethylamino)carbomonium hexafluorophosphate, O-(benzotriazole-1-yl)-di(dimethylamino)carbomonium hexafluorophosphate, or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) and one selected from N-hydroxy-7-azabenzotriazole or 1-hydroxybenzotriazole in a molar ratio of 1:1.1; the molar ratio of intermediate XV to acid-binding agent is 1:5; the total amount of R1H and R2H and the molar ratio of intermediate XV are 2:1 to 2.2:1; the reaction temperature is room temperature. (b) When R1 and R2 are not the same, including: Step (7a): Using dichloromethane, tetrahydrofuran, acetonitrile or N,N-dimethylformamide as the reaction solvent, in the presence of an amide condensing agent and an acid-binding agent, compound XV reacts with R1H to obtain intermediate XVI. The molar ratio of intermediate XV to amide condensing agent is 1:3.15; the amide condensing agent is a combination of one selected from O-(7-azabenzotriazol-1-yl)-di(dimethylamino)carbomony hexafluorophosphate, O-(benzotriazol-1-yl)-di(dimethylamino)carbomony hexafluorophosphate, or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and one selected from N-hydroxy-7-azabenzotriazole or 1-hydroxybenzotriazole in a molar ratio of 1:1.1; the molar ratio of intermediate XV to acid-binding agent is 1:2.5; the molar ratio of intermediate XV to R1H is 1:1 to 1:1.1; the reaction temperature is room temperature; Step (7b): Add amide condensing agent, acid-binding agent, and R2H to the reaction solution of step (7a) to obtain intermediate XVI; The molar ratio of intermediate XV to amide condensing agent is 1:3.15; the amide condensing agent is a combination of one selected from O-(7-azabenzotriazole-1-yl)-di(dimethylamino)carbomonium hexafluorophosphate, O-(benzotriazole-1-yl)-di(dimethylamino)carbomonium hexafluorophosphate, or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and one selected from N-hydroxy-7-azabenzotriazole or 1-hydroxybenzotriazole in a molar ratio of 1:1.1; the molar ratio of intermediate XV to acid-binding agent is 1:2.5; the molar ratio of intermediate XV to R2H is 1:1 to 1:1.1; and the reaction temperature is room temperature. In step (8), the catalyst is selected from 5% Pd / C; the mass ratio of intermediate XVI to catalyst is 1:1; the reduction reaction temperature is room temperature; The molar ratio of intermediate XVI to amide condensing agent is 1:2.6 to 1:2.8; the molar ratio of intermediate XVI to acid-binding agent is 1:2; and the molar ratio of intermediate XVI to (+)JQ-1 is 1. The amide condensing agent is selected from one of O-(7-azabenzotriazole-1-yl)-di(dimethylamino)carbomony hexafluorophosphate, O-(benzotriazole-1-yl)-di(dimethylamino)carbomony hexafluorophosphate, or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and is combined with N-hydroxy-7-azabenzotriazole or 1-hydroxybenzotriazole in a molar ratio of 1:1.35 to 1:1.4; the acid-binding agent is selected from N,N-diisopropylethylamine or triethylamine.

8. The use of the compound of any one of claims 1-5 or its pharmaceutically acceptable salt or racemate in the preparation of a BRD4-targeting degrader.

9. Use of the compound of any one of claims 1-5 or its pharmaceutically acceptable salt or racemate in the preparation of a medicament for treating BRD4 protein-related diseases; wherein the BRD4 protein-related diseases are midline carcinoma, acute myeloid leukemia, multiple myeloma, Burkitt lymphoma, prostate cancer, breast cancer, glioblastoma, and renal cell carcinoma.

10. A pharmaceutical composition, characterized in that: It uses the compound described in any one of claims 1-5 or its pharmaceutically acceptable salt or racemate as the active ingredient, and is formulated into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier.

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