CN110790750B - Phthalimide selective androgen receptor degradation agent and preparation method and application thereof - Google Patents

Abstract

The invention discloses a phthalimide chimeric molecule containing 1,2, 3-triazolyl, a preparation method and a selection thereofAn application of sex Androgen Receptor (AR) inhibitor in the field of AR-resistant high-expression cancer belongs to the field of pharmaceutical chemistry. It has the following structural general formula:

the compounds have good combination effect on AR and AR degradation activity, and can be used as further developed candidates or lead compounds for preparing anti-AR high-expression cancer drugs.

Description

Phthalimide selective androgen receptor degradation agent and preparation method and application thereof

Technical Field

The invention relates to the field of medicinal chemistry, in particular to a phthalimide chimeric molecule containing 1,2, 3-triazolyl, a preparation method thereof and application thereof as a selective Androgen Receptor (AR) degradation agent in the field of resisting prostate cancer.

Background

Prostate cancer (PCa) is common in middle-aged and elderly men, and is a common genitourinary malignancy, the worldwide incidence of which is second only to lung cancer, the second highest incidence of cancer, and the mortality of the sixth highest incidence of all male malignancies. The PCa therapeutic drug market is growing at an annual rate of 15-20% worldwide.

Androgen-deprivation therapy of surgical castration or drug castration is the standard of treatment for advanced stage prostate cancer, but after a median time of treatment of 14-30 months, almost all patient lesions develop from androgen-dependent prostate cancer (HDPC) to androgen-independent prostate cancer (HIPC), also known as castration-resistant prostate cancer (CRPC), with a median survival of less than 20 months in patients, resulting in endocrine treatment failure, such that antiandrogen therapy has essentially no effect on such patients, who eventually die from prostate cancer; approximately 50,000 patients will develop metastatic disease each year, which is known as metastatic crpc (mcrpc).

The most major obstacle to the progression of CRPC is the inability of AR signaling antagonists acting through LBD (e.g., bicalutamide, enzalutamide, abiraterone) to inhibit growth driven by N-terminal domain (NTD) dependent constitutively active AR-SV. There is currently no effective treatment for CRPC patients, and although the molecular biological mechanisms involved in their development are not completely understood, a number of studies have shown high AR expression in 80% of advanced CRPC. Degrading agent molecules degrade AR by direct targeting, thereby truly blocking this pathway at the AR level, and may be a potential alternative strategy for treating CRPC.

In 2004, the Ohwen Rous, arone Cheranoo and Alfamil Helichi families achieved the Nobel prize due to the discovery of ubiquitin-mediated protein degradation processes. In 2008, the Crews group reported that nutlin 3a has high binding force (Ki) with E3 ligase Mdm2, and the Ki value is nmol.L^-1Horizontally, the androgen receptor antagonist bicalutamide analogue is covalently linked with nutlin 3a by polyethylene glycol Linker to generate PROTAC molecules (the structure is shown in the specification), the chimera can effectively degrade AR in cells with high androgen expression, and can prevent the degradation of AR after a proteasome inhibitor is added, and the process is proved to occur through a ubiquitin-proteasome pathway.

Mdm 2-based E3 ligase mediates AR targeted degradation of PROTAC.

Disclosure of Invention

The invention aims to provide a novel phthalimide androgen receptor degradation agent and a preparation method thereof; another purpose is to provide the application of the compound as a lead compound for preparing the anti-prostate cancer.

The specific general formula of the phthalimide androgen receptor degrading agent is as follows:

wherein R is C_0-10Straight chain alkyl, phenyl;

m＝1-6；

R₁is- (CH)₂)_a-，-(CH₂CH₂O)_b-，-(CH₂CH₂CH₂O)_c-, a ═ 1 to 10, b ═ 1 to 10, c ═ 1 to 10; w is an O or S atom;

preferred in formula I: r is phenyl or C_1-5A linear alkyl group; m is 1 or 2; w is an O or S atom;

R₁is- (CH)₂)_a-，-(CH₂CH₂O)_b-，-(CH₂CH₂CH₂O)_c-，a＝1-5,b＝1-5，c＝1-2；

The following compounds are preferred:

the invention relates to a triazolyl-containing phthalimide compound which is mainly prepared by the following method:

1. a process for the preparation of a compound of formula II:

the compound of the following general formula II is an intermediate for synthesizing the compound of the general formula I, and R is para-substituted phenyl or straight-chain alkyl; m is 1-6 and has the structure of

The alkyl chain of (A) may be-CH₂-、-CH₂CH₂-、-CH₂CH₂CH₂-and the like; w is an O or S atom;

in a solvent, carrying out electrophilic substitution reaction on commercially available 4-fluoro-2-trifluoromethyl benzonitrile and 5, 5-dimethyl hydantoin under an alkaline condition, wherein the used alkali is one of sodium hydroxide, potassium carbonate, sodium carbonate and the like; the method comprises the following steps of (1) using one or any two of N, N-dimethylformamide, acetonitrile, acetone, dioxane, methanol and ethanol as a solvent, purifying by column chromatography or recrystallization to obtain an intermediate 1, using one or two of ethyl acetate, diethyl ether and N-hexane as a solvent for recrystallization, carrying out nucleophilic substitution reaction on the intermediate 1 and dibromoalkane or phenyl azide with different lengths under an alkaline condition, and using one of sodium hydride, potassium hydride, sodium hydroxide, potassium hydroxide and the like as a base; the solvent is one of tetrahydrofuran, N-dimethylformamide, acetonitrile, dioxane, methanol and ethanol, and finally nucleophilic substitution reaction is carried out on the solvent and sodium azide under the heating condition, the solvent is one of N, N-dimethylformamide, ethyl acetate/water and acetone/water, and the obtained products L-1 and L-2 are purified by column chromatography to obtain pure products.

In a solvent, commercially available methyl 2-aminoisobutyrate hydrochloride undergoes nucleophilic substitution reaction with bromoalkanol and benzyl bromoazide respectively under alkaline conditions to obtain an intermediate 3 or 4 respectively. The alkali is one of potassium carbonate, sodium hydroxide, etc.; the solvent is one of acetonitrile, N-dimethylformamide, acetone, methanol and ethanol; the intermediate 3 or 4 and cyanobenzene isocyanuric acid ester are subjected to cyclization reaction with acetyl under alkaline conditions, the used alkali is one of triethylamine and diisopropylethylamine, and the used solvent is one of tetrahydrofuran, dichloromethane, chloroform, acetone, N-dimethylformamide and the like; the obtained products L-3 and L-4 are purified by column chromatography or recrystallization and the like to obtain pure products. The solvent used for recrystallization is one or a mixture of two of ethyl acetate, diethyl ether, isopropyl ether, ethanol and acetone.

The invention provides a preparation method of the compound, which is synthesized by the following reaction route:

2. a process for the preparation of a compound of formula III:

the compound of the following general formula III is an intermediate for synthesizing the compound of the general formula I, R₁Is C_1-10Straight chain alkyl or C_1-5A polyalkyldiol chain.

In a solvent, commercially available 3-hydroxy phthalic anhydride and 3-amino-2, 6-piperidedione hydrochloride react under the condition of heating acetic acid and acetic anhydride, the obtained product is recrystallized in water to obtain an intermediate 10, and then the intermediate is subjected to alkylation reaction with a p-methyl benzene sulfonate alkynyl intermediate with alkyl or polyalkyl glycol chains of different chain lengths under an alkaline condition, wherein the base is one of sodium carbonate and sodium bicarbonate, and the solvent is one of N, N-dimethylformamide, acetonitrile, dioxane, tetrahydrofuran and the like; purifying the obtained product by column chromatography or recrystallization to obtain a pure product, wherein the solvent used for recrystallization is one or a mixture of two of ethyl acetate, diethyl ether, isopropyl ether and the like.

The invention provides a preparation method of the compound, which is synthesized by the following reaction route:

3. process for the preparation of compounds of the general formula I

In organic solvent, compound of formula II and compound of formula III in CuSO₄Sodium ascorbate, CuI/organic base or Cu/CuSO₄1, 3-cycloaddition reaction is carried out under the condition, and the used alkali is diisopropylethylamine and triethylamine; the organic solvent is acetonitrile, tetrahydrofuran/water, tert-butyl alcohol/water, N, N-dimethylformamide/water, methanol/water, etc.; the reaction temperature is between 0 and 120 ℃, usuallyAt room temperature. The obtained product is purified by recrystallization, column chromatography and the like to obtain a pure product. The solvent used for recrystallization is one or a mixture of two of acetonitrile, methanol, ethanol, acetone, ethyl acetate, dichloromethane and chloroform.

The invention provides a preparation method of the compound, which is synthesized by the following reaction route:

the compounds of the invention are targeted proteolytic chimeras (PROTACs) that selectively induce degradation of AR proteins.

The invention has the advantages that: the AR non-steroid antagonist and an E3 ligase complex cereblon protein ligand are connected by a 1,2, 3-triazole Linker to obtain the bifunctional micromolecule. Compared with the prior art, the 1,2, 3-triazole active unit is combined with the E3 ligase ligand phthalimide and the AR ligand to form the targeting chimeric molecule by click chemistry for the first time, the triazole-containing phthalimide compound is synthesized simply, efficiently and environmentally, and in-vitro AR activity combination experiments show that the triazole-containing phthalimide compound has better combination effect and certain degradation effect on AR protein, can be used as an active ingredient for preparing an anti-prostatic cancer antagonistic or degradation drug, and is expected to be well applied to treatment of prostatic cancer, advanced prostatic cancer, castration-resistant prostatic cancer or other hyperandrogenism skin diseases.

Drawings

FIG. 1 is a western blot of AR protein expression in LnCAP cells with the compounds of the present invention;

FIG. 2 is a bar graph showing the effect of the compounds of the present invention on the expression of AR proteins in LnCAP cells.

Detailed Description

The starting materials may be obtained from commercial sources or prepared by methods known in the art or according to the methods described herein.

The compound structure passes through the nucleusMagnetic resonance (¹H-NMR，¹³C-NMR) and mass spectrometry (HRMS).¹H-NMR and¹³C-NMR was measured using a nuclear magnetic resonance apparatus model DPX-400 (Bruker, Germany); the high resolution mass spectrum is measured by a Q-exact mass spectrometer of Saimer Feishale; 200-mesh 300-mesh silica gel (Qingdao ocean chemical production) is used for column chromatography.

EXAMPLE 1 preparation of intermediate L-1

(1) Preparation of intermediate 1

Weighing 508mg of 5, 5-dimethylhydantoin, placing in a bottle shaped like a eggplant, adding 5mL of DMF while stirring to completely dissolve the 5, 5mg of K₂CO₃(1eq), stirring for half an hour at 45 ℃ to fully mix the components, finally slowly adding DMF solution of 4-fluoro-2-trifluoromethylbenzonitrile (300mg), stirring for 5 hours under heating, cooling to room temperature after full reaction, adding 30mL of ethyl acetate for dilution, extracting for 3 times by 20mL of saturated ammonium chloride aqueous solution, extracting for 1 time by saturated salt water, drying an organic layer for 6 hours by anhydrous magnesium sulfate, carrying out suction filtration, concentrating, and finally carrying out column chromatography with 200-300 meshes to obtain 330mg of white solid with the yield of 79%.

Intermediate 1, white solid:¹H NMR(400MHz,DMSO-d₆)δ8.83(s,1H),8.30(d,J＝8.4Hz,1H),8.19(d,J＝1.6Hz,1H),8.04(dd,J＝8.4,1.8Hz,1H),1.43(s,6H).¹³C NMR(101MHz,DMSO-d₆)δ175.72,152.94,136.98,135.98,131.19,130.87,129.93,124.04,123.99,123.60,120.88,115.20,106.55,57.94,24.56.

(2) preparation of intermediate 2

50mg of NaH (3eq) was weighed into a two-neck flask and charged with N₂Replacing air in a two-neck flask for 3-4 times, slowly dropwise adding an intermediate 1(200mg) dissolved in dry DMF (dimethyl formamide) by using a constant-pressure dropping funnel under ice bath, stirring for 30min, slowly dropwise adding dibromopropane (2eq) diluted by 2 times of DMF, stirring for 2h at room temperature, fully reacting, then adding 30mL ethyl acetate for dilution, extracting for 3 times by using 20mL saturated ammonium chloride aqueous solution, extracting for 2 times by using saturated saline solution, drying an organic layer for 5h by using anhydrous magnesium sulfate, performing suction filtration, concentrating, and finally performing column chromatography by using 200-300 meshes to obtain 230mg of colorless liquid, wherein the yield is 85%.

The intermediate 2 is a mixture of two or more,colorless liquid:¹H NMR(400MHz,CDCl₃)δ8.15(s,1H),8.00(dd,J＝8.4,1.8Hz,1H),7.92(d,J＝8.4Hz,1H),3.65(t,J＝6.0Hz,2H),3.57–3.45(m,2H),2.36–2.16(m,2H),1.56(d,J＝2.5Hz,6H).¹³C NMR(101MHz,DMSO-d₆)δ172.67,155.80,139.03,138.78,132.19,130.87,129.93,125.14,123.59,123.60,121.88,114.32,106.55,57.63,44.06,30.02,30.07,23.56.

(3) preparation of intermediate L-1

200mg of intermediate 2 is weighed, completely dissolved in 3mL of acetone solution, and 1mL of water-soluble NaN is slowly dropped into the solution under stirring at room temperature₃(87mg), refluxing for 6h at 80 ℃, evaporating acetone after the reaction is finished, extracting for 3 times by using ethyl acetate, back extracting once by using saturated saline, combining organic layers, drying by using anhydrous magnesium sulfate, performing suction filtration, concentrating under reduced pressure, and purifying by using a column chromatography method to obtain 210mg of a colorless liquid of an intermediate L-1, wherein the yield is 90%.

Intermediate L-1, colorless liquid:¹H NMR(400MHz,CDCl₃)δ8.00–7.93(m,1H),7.92(s,1H),7.79(d,J＝8.2Hz,1H),3.78(dd,J＝17.0,8.7Hz,2H),3.48(t,J＝6.2Hz,2H),2.14(d,J＝5.8Hz,2H),1.68(s,J＝64.4Hz,6H).¹³C NMR(101MHz,CDCl₃)δ178.64,175.09,137.12,135.19,133.82,132.83,132.16,130.11,127.08,126.94,126.02,123.29,120.57,114.89,109.89,49.11,41.71,27.47,23.04.

EXAMPLE 2 preparation of intermediate L-2

Weighing p-methylaniline, adding into a flask, sequentially adding 1mL of concentrated hydrochloric acid and 1mL of water, stirring at room temperature for half an hour, then carrying out ice bath to below zero ℃, and quickly adding NaNO dissolved by 1mL of ice water below the liquid level of the system₂(1.1eq) solution, stirring for 30min, cooling to-4 deg.C in a salt bath, and adding dropwise NaN dissolved in 1.5mL of water₃(3.0eq) solution is stirred for 4-8 h until TLC monitoring reaction is complete, a neutralization system of sodium carbonate is slowly added under the condition of ice bath stirring, then ethyl acetate is used for extraction, saturated salt solution is used for washing for 2 times, low-temperature spin drying is carried out, a phenylazide product is obtained, then NBS and AIBN are taken, and the mixture is subjected to CCl reaction₄Refluxing to obtain benzyl bromide azide intermediate, and finally weighing intermediate 1, and intermediate L-1180mg of L-2 was obtained as a tan solid in 65% yield.

Intermediate L-2, tan solid:¹H NMR(400MHz,DMSO-d₆)δ8.33(d,J＝8.4Hz,1H),8.26(s,1H),8.10(d,J＝8.3Hz,1H),7.51(d,J＝8.3Hz,2H),7.10(d,J＝8.3Hz,2H),4.62(s,2H),1.41(s,6H).¹³C NMR(101MHz,DMSO-d₆)δ174.55,153.06,138.34,136.83,135.97,134.86,131.51,131.19,130.87,130.55,129.99,129.38,126.31,124.07,124.02,123.59,120.87,118.98,115.16,106.73,106.71,61.92,41.91,22.62.

EXAMPLE 3 preparation of intermediates L-3 and L-4

Methyl 2-aminoisobutyrate hydrochloride (200mg) was weighed to react with bromopropanol (1.2eq) to give intermediate 3, which was then reacted with less toxic phenylisothiocyanato to give 3-a in high yield, followed by reaction of 3-a with TsCl (1.5eq) and DABCO (2eq) to give intermediate 3-b, and finally with NaN₃(3eq) refluxing for 6h at 80 ℃, evaporating acetone after the reaction is finished, extracting for 3 times by using ethyl acetate, performing back extraction once by using saturated saline solution, combining organic layers, drying by using anhydrous magnesium sulfate, performing suction filtration, performing reduced pressure concentration, and purifying by using a column chromatography method to obtain 210mg of ligand L-3 yellow solid with the yield of 90%.

Intermediate 3-a, colorless oily liquid:¹H NMR(400MHz,CDCl₃)δ7.96(d,J＝8.3Hz,1H),7.90(d,J＝1.4Hz,1H),7.78(dd,J＝8.3,1.8Hz,1H),3.96–3.87(m,2H),3.75(s,2H),2.30(s,1H),2.06(dt,J＝12.2,6.0Hz,2H),1.61(s,6H).¹³C NMR(101MHz,DMSO-d₆)δ182.68,175.73,138.23,135.26,132.96,130.64,123.67,117.98,116.93,105.07,66.74,59.56,44.25,33.14,24.53.

intermediate 3-b, a colorless oil,¹H NMR(400MHz,CDCl₃)δ7.94(t,J＝8.0Hz,1H),7.88(d,J＝12.3Hz,1H),7.77(t,J＝11.1Hz,3H),7.34(t,J＝9.8Hz,2H),4.30–4.11(m,6H),3.81(dd,J＝21.2,13.3Hz,2H),2.44(d,J＝8.8Hz,3H),2.25(dt,J＝15.2,6.0Hz,2H),2.04(p,J＝6.0Hz,2H),1.58(d,J＝12.5Hz,6H).¹³C NMR(101MHz,CDCl₃)δ171.71,79.63,74.58,70.62,70.52,70.46,69.25,69.09,62.96,58.39,48.82,42.33,36.73,32.76,28.61.

the intermediate L-3, a yellow solid,¹H NMR(400MHz,CDCl₃)δ8.00–7.93(m,1H),7.92(s,1H),7.79(d,J＝8.2Hz,1H),3.78(dd,J＝17.0,8.7Hz,2H),3.48(t,J＝6.2Hz,2H),2.14(d,J＝5.8Hz,2H),1.68(s,J＝64.4Hz,6H).¹³C NMR(101MHz,CDCl₃)δ178.64,175.09,137.12,135.19,133.82,133.49,133.16,132.83,132.16,130.11,127.08,127.04,126.99,126.94,126.02,123.29,120.57,117.84,114.89,109.89,65.22,49.11,41.71,27.47,23.04.

methyl 2-aminoisobutyrate hydrochloride (200mg) was weighed to react with p-benzyl bromoazide (1.2eq) in acetonitrile as solvent and potassium carbonate as base to give intermediate 4, which was then reacted with less toxic phenylisothiocyanic acid ester to give intermediate L-4 at 140mg with 75% yield.

Intermediate L-4, tan solid:¹H NMR(400MHz,DMSO-d₆)δ8.33(d,J＝8.4Hz,1H),8.26(s,1H),8.10(d,J＝8.3Hz,1H),7.51(d,J＝8.3Hz,2H),7.10(d,J＝8.3Hz,2H),4.62(s,2H),1.41(s,6H).¹³C NMR(101MHz,DMSO-d₆)δ174.55,153.06,138.34,136.83,135.97,134.86,131.51,131.19,130.87,130.55,129.99,129.38,126.31,124.07,124.02,123.59,120.87,118.98,115.16,106.73,106.71,61.92,41.91,22.62.

example 4 Synthesis of intermediate 10

Weighing 3-hydroxyphthalic anhydride, completely dissolving the 3-hydroxyphthalic anhydride by using acetic acid as a solvent, then adding sodium acetate (2eq) and 3-amino-2, 6-piperidinedione hydrochloride (1eq), stirring for 10 hours at 110 ℃, fully and completely reacting, evaporating the acetic acid solvent under reduced pressure, adding water, stirring for half an hour, precipitating a gray solid, filtering by suction, and drying to obtain an intermediate 10 with the yield of 85%.

Intermediate 10, off white solid:¹H NMR(400MHz,DMSO-d₆)δ11.19(s,1H),11.09(s,1H),7.66(dd,J＝8.3,7.3Hz,1H),7.32(d,J＝7.1Hz,1H),7.25(d,J＝8.2Hz,1H),5.08(dd,J＝12.9,5.4Hz,1H),2.89(ddd,J＝17.3,14.0,5.4Hz,1H),2.56(dd,J＝20.8,10.8Hz,2H),2.09–1.96(m,1H).¹³C NMR(101MHz,DMSO-d₆)δ173.71,171.58,169.97,168.61,156.58,136.46,134.21,122.40,115.74,112.53,52.77,29.68,24.11.

EXAMPLE 5 preparation of intermediate 6

Potassium tert-butoxide 64mg (1.2eq) was weighed out and dissolved in dry THF solution, N₂Adding polyethylene glycol chains of different lengths 200mg (1eq) under the environment, stirring for 30min under an ice salt bath, then dropwise adding a bromopropyne solution diluted by one time of THF by using a constant-pressure dropping funnel, stirring overnight at room temperature, fully reacting, finally removing insoluble substances by using a suction filtering funnel, removing THF at a low temperature by spinning, concentrating under reduced pressure, and purifying by using column chromatography to obtain a yellow liquid 6 with the yield of 38%.

EXAMPLE 6 Synthesis of intermediate III-1

200mg of intermediate 10 are weighed out and completely dissolved in DMF solution, and 56mg of K are added₂CO₃(1.2eq) fully stirring, finally slowly dropwise adding a DMF solution of alkynyl p-methylbenzenesulfonate with an alkyl chain or a polyethylene glycol chain, stirring for 6 hours at 85 ℃, detecting by TLC until the reaction is complete, cooling to room temperature, adding 30mL of dichloromethane diluted solution, extracting for 3 times by using 20mL of saturated ammonium chloride aqueous solution, extracting twice by using saturated saline solution, drying an organic layer for 5 hours by using anhydrous sodium sulfate, carrying out suction filtration, adding a small amount of ethyl acetate for recrystallization, and separating out white solid to obtain the target compound III-1.

Intermediate III-1, a white solid,¹H NMR(400MHz,DMSO-d₆)δ11.10(s,1H),7.85–7.77(m,1H),7.54(d,J＝8.5Hz,1H),7.47(d,J＝7.2Hz,1H),5.08(dd,J＝12.8,5.4Hz,1H),4.32(t,J＝6.6Hz,2H),2.97–2.85(m,2H),2.70(td,J＝6.5,2.6Hz,2H),2.66–2.51(m,2H),2.14–1.94(m,1H).¹³C NMR(101MHz,DMSO-d₆)δ173.71,169.97,169.74,168.61,155.71,136.92,133.20,118.92,118.87,116.95,84.40,70.14,68.24,52.77,29.68,28.29,24.11,18.81.

example 7: preparation of 4- (3- (4- (4- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dione-isoindolin-4-yl) oxy) ethyl) -1H-1,2, 3-triazol-1-yl) benzyl) -4, 4-dimethyl-2, 5-dioxoimidazolidin-1-yl) -2- (trifluoromethyl) benzonitrile (I-1) as shown in the general formula (I).

120mg of alkynyl III-1 ligand is weighed, 5mL of tetrahydrofuran is added, 2eq of azide L-4 ligand 297mg are added after ultrasonic dissolution, and 41mg of CuSO is added₄And 45mg VcNa, and finally 1.5mL of H₂And O, stirring for 7-11 h at room temperature until the reaction is complete, performing suction filtration by using diatomite to remove metal insoluble substances, performing rotary evaporation at 39 ℃ to remove most tetrahydrofuran, adding 40mL of ethyl acetate to dilute the system, extracting for three times by using 24mL of saturated saline solution, adding anhydrous magnesium sulfate to an organic layer, drying, performing suction filtration and concentration, and performing column chromatography to obtain a white solid 170mg with the yield of 80%. Yellow solid, m.p. 158.2-159.5 ℃,¹H NMR(400MHz,CDCl₃)δ8.94(s,1H),8.74(s,1H),8.13(s,1H),8.01(d,J＝8.6Hz,1H),7.94(d,J＝8.4Hz,1H),7.80(d,J＝8.4Hz,2H),7.71(t,J＝7.9Hz,1H),7.56(d,J＝8.4Hz,2H),7.50(d,J＝7.2Hz,1H),7.24(d,J＝8.6Hz,1H),4.97(dd,J＝12.0,5.2Hz,1H),4.75(d,J＝15.7Hz,1H),4.55(d,J＝15.8Hz,1H),4.52–4.43(m,1H),4.26(dd,J＝13.8,6.6Hz,1H),3.40(d,J＝4.8Hz,2H),2.84(dd,J＝26.3,13.9Hz,2H),2.74(dd,J＝28.1,12.3Hz,2H),2.21–2.09(m,1H),1.67(s,2H),1.49(d,J＝27.4Hz,6H).¹³C NMR(101MHz,CDCl₃)δ173.71,172.68,169.97,169.74,168.61,155.71,155.21,152.26,138.65,138.61,136.92,134.79,133.20,132.75,132.32,132.13,131.76,123.67,122.79,118.92,118.87,116.95,116.93,116.67,115.34,113.05,68.74,64.49,52.77,39.74,29.68,27.72,24.53,24.11.ESI-HRMS:m/z cacld.For C₃₇H₂₉F₃N₈O₇[M+H]+:755.2111,found 755.2132.

example 8: preparation of 4- (3- (4- (4- (3- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dione-isoindolin-4-yl) oxy) propyl) -1H-1,2, 3-triazol-1-yl) benzyl) -4, 4-dimethyl-2, 5-dioxoimidazolidin-1-yl) -2- (trifluoromethyl) benzonitrile (I-2).

The synthesis procedure is as in example 7, yellow brown solid, m.p. 156.5-157.9 ℃,¹H NMR(400MHz,CDCl₃)δ8.37(s,1H),8.18(s,1H),8.07–8.01(m,1H),7.99–7.91(m,2H),7.75(d,J＝8.4Hz,2H),7.70–7.64(m,2H),7.52(d,J＝8.4Hz,2H),7.45(d,J＝7.3Hz,1H),7.24(d,J＝8.5Hz,1H),4.95(dd,J＝11.9,5.3Hz,1H),4.68(d,J＝7.1Hz,2H),4.32–4.22(m,3H),3.09(t,J＝6.9Hz,3H),2.91–2.78(m,2H),2.79–2.66(m,2H),2.36(dd,J＝12.7,6.4Hz,2H),2.16–2.07(m,1H),1.70(s,2H),1.47(d,J＝4.8Hz,6H).¹³C NMR(101MHz,CDCl₃)δ173.71,172.68,169.97,169.74,168.61,155.71,155.21,149.66,138.65,138.61,136.92,134.79,133.20,132.75,132.32,132.13,131.76,123.67,122.90,122.79,118.92,118.87,116.95,116.93,116.67,113.05,69.99,64.49,52.77,39.74,29.68,25.79,24.53,24.11,23.95.ESI-HRMS:m/z cacld.For C₃₈H₃₁F₃N₈O₇[M+H]+:769.2268,found 769.2261.

example 9: preparation of 4- (3- (4- (4- ((3- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dione-isoindolin-4-yl) oxy) propoxy) methyl) -1H-1,2, 3-triazol-1-yl) benzyl) -4, 4-dimethyl-2, 5-dioxoimidazolidin-1-yl) -2- (trifluoromethyl) benzonitrile (I-3).

The synthesis procedure was as in example 7, yellow brown solid, m.p.:215.2-216.1 ℃,¹H NMR(400MHz,DMSO-d₆)δ11.10(s,1H),8.77(s,1H),8.34(d,J＝8.4Hz,1H),8.26(d,J＝1.5Hz,1H),8.11(d,J＝8.4Hz,1H),7.82(d,J＝8.4Hz,1H),7.79(dd,J＝8.4,7.4Hz,1H),7.64(d,J＝8.5Hz,2H),7.50(d,J＝8.5Hz,1H),7.41(d,J＝7.2Hz,1H),5.05(dd,J＝12.7,5.4Hz,1H),4.69(s,2H),4.62(s,2H),4.27(t,J＝6.1Hz,2H),3.71(t,J＝6.2Hz,2H),2.95–2.81(m,1H),2.58(d,J＝17.8Hz,1H),2.28–2.15(m,1H),2.03(dd,J＝11.5,5.5Hz,2H),1.44(s,6H).¹³C NMR(101MHz,DMSO-d₆)δ174.62,172.80,169.90,166.80,155.80,153.16,145.15,138.37,137.05,136.79,136.02,133.10,130.15,128.94,124.22,121.99,119.87,116.14,115.23,65.76,63.21,62.04,48.69,28.75,22.60,21.94.ESI-HRMS:m/z cacld.For C₃₉H₃₃F₃N₈O₈[M+H]+:799.2373,found 799.2329.

example 10: preparation of 4- (3- (4- (4- ((2- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dione-isoindolin-4-yl) oxy) ethoxy) methyl) -1H-1,2, 3-triazol-1-yl) benzyl) -4, 4-dimethyl-2, 5-dioxoimidazolidin-1-yl) -2- (trifluoromethyl) benzonitrile (I-4).

The synthesis procedure was as in example 7, yellow brown solid, m.p. 226.5-227.9 ℃,¹H NMR(400MHz,DMSO-d₆)δ11.10(s,1H),8.76(s,1H),8.34(d,J＝8.4Hz,1H),8.26(s,1H),8.11(d,J＝8.3Hz,1H),7.86(d,J＝8.4Hz,2H),7.82–7.74(m,1H),7.65(d,J＝8.5Hz,2H),7.52(d,J＝8.5Hz,1H),7.44(t,J＝7.0Hz,1H),5.07(dd,J＝12.6,5.4Hz,1H),4.70(s,2H),4.63(s,2H),4.38–4.30(m,2H),3.85–3.79(m,2H),3.70(dd,J＝5.9,3.2Hz,2H),3.65(dd,J＝5.7,3.2Hz,2H),2.93–2.80(m,1H),2.63–2.51(m,2H),2.31–2.22(m,2H),2.08–1.99(m,1H),1.42(d,J＝15.1Hz,6H).¹³C NMR(101MHz,DMSO-d₆)δ174.62,172.80,169.90,166.80,155.80,153.16,145.15,138.37,137.05,136.79,136.02,133.10,130.15,128.94,124.22,121.99,119.87,116.14,115.23,65.76,63.21,62.04,48.69,28.75,22.60,21.94.ESI-HRMS:m/z cacld.For C₄₀H₃₅F₃N₈O₉[M+H]+:829.2479,found 829.2470.

example 11: preparation of 4- (3- (4- ((2- (2- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dione-isoindolin-4-yl) oxy) ethoxy) methyl) -1H-1,2, 3-triazol-1-yl) benzyl) -4, 4-dimethyl-2, 5-dioxoimidazolidin-1-yl) -2- (trifluoromethyl) benzonitrile (I-5).

The synthesis procedure was as in example 7, yellow brown solid, m.p.:214.1-215.6 ℃,¹H NMR(400MHz,CDCl₃)δ8.47(s,1H),8.19(d,J＝1.5Hz,1H),8.10–8.02(m,2H),7.94(d,J＝8.4Hz,1H),7.74(d,J＝8.3Hz,2H),7.69–7.62(m,1H),7.53(d,J＝8.3Hz,2H),7.44(d,J＝7.2Hz,1H),7.25(d,J＝8.5Hz,1H),4.93(dd,J＝11.7,5.4Hz,1H),4.77(s,2H),4.67(s,2H),4.37–4.30(m,2H),3.97–3.90(m,2H),3.79(dd,J＝9.1,4.2Hz,2H),3.76(d,J＝14.2Hz,3H),3.69(dd,J＝10.0,5.1Hz,5H),2.85(dd,J＝12.1,8.4Hz,1H),2.81–2.68(m,2H),2.27–2.18(m,1H),2.10(dd,J＝12.8,7.9Hz,1H),1.46(s,6H).¹³C NMR(101MHz,CDCl₃)δ174.32,171.13,168.53,167.01,165.82,156.54,153.38,136.64,136.20,135.39,133.74,133.44,128.07,123.08,123.03,119.11,117.08,115.92,114.98,108.44,77.25,66.45,66.08,64.37,62.12,49.13,47.81,37.62,31.40,29.87,29.20,23.37,23.34,22.64.ESI-HRMS:m/z cacld.For C₄₂H₃₉F₃N₈O₁₀[M+H]+:873.2741,found 873.2751.

example 12: preparation of 4- (3- (4- (4- (13- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dione-isoindolin-4-yl) oxy) -2,5,8, 11-ethoxy) -1H-1,2, 3-triazol-1-yl) benzyl) -4, 4-dimethyl-2, 5-dioxoimidazolidin-1-yl) -2- (trifluoromethyl) benzonitrile (I-6).

The synthesis procedure was as in example 7, yellow brown solid, m.p. 236.3-237.5 ℃,¹H NMR(400MHz,CDCl₃)δ8.43(s,1H),8.19(d,J＝1.5Hz,1H),8.11–8.02(m,1H),7.95(d,J＝8.5Hz,1H),7.74(d,J＝8.5Hz,1H),7.66(dd,J＝8.3,7.4Hz,1H),7.54(d,J＝8.5Hz,1H),7.44(d,J＝7.2Hz,1H),7.25(s,1H),4.93(dd,J＝12.0,5.3Hz,1H),4.79(d,J＝12.9Hz,1H),4.68(s,1H),4.35–4.28(m,1H),3.96–3.88(m,1H),3.79–3.72(m,2H),3.71–3.64(m,3H),2.90–2.79(m,1H),2.73(dd,J＝33.7,18.4,11.7Hz,1H),2.34(t,J＝6.7Hz,1H),2.15–2.05(m,1H),1.87(dt,J＝12.7,6.2Hz,2H),1.46(s,6H).¹³C NMR(101MHz,DMSO-d₆)δ174.62,172.80,169.90,166.80,155.80,153.16,145.15,138.37,137.05,136.79,136.02,133.10,130.15,128.94,124.22,121.99,119.87,116.14,115.23,65.76,63.21,62.04,48.69,28.75,22.60,21.94.ESI-HRMS:m/z cacld.For C₄₄H₄₃F₃N₈O₁₁[M+H]+:917.3003,found917.3014.

example 14: preparation of 4- (3- (3- (4- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dione-isoindolin-4-yl) oxy) ethyl) -1H-1,2, 3-triazol-1-yl) propyl) -4, 4-dimethyl-2, 5-dioxoimidazolidin-1-yl) -2- (trifluoromethyl) benzonitrile (I-7).

The synthesis procedure was the same as in example 7, white solid, m.p. 185.1-186.8 ℃,¹H NMR(400MHz,DMSO-d₆)δ11.11(s,1H),8.32(d,J＝8.4Hz,1H),8.19(d,J＝1.8Hz,1H),8.04(dd,J＝8.4,1.8Hz,1H),7.96(s,1H),7.80(dd,J＝8.4,7.3Hz,1H),7.51(d,J＝8.5Hz,1H),7.45(d,J＝7.2Hz,1H),5.09(dd,J＝12.9,5.4Hz,1H),4.43(t,J＝7.2Hz,2H),4.33–4.20(m,2H),3.43–3.35(m,2H),2.96–2.88(m,1H),2.84(d,J＝7.6Hz,2H),2.66–2.52(m,2H),2.17(dt,J＝12.8,6.4Hz,2H),2.13–2.06(m,2H),2.08–2.00(m,1H),1.45(s,6H).¹³C NMR(101MHz,DMSO-d₆)δ173.71,172.68,169.97,169.74,168.61,155.81,155.71,150.80,138.65,136.92,133.20,132.75,132.32,131.76,123.67,120.49,118.92,118.87,116.95,116.93,116.67,113.05,69.99,64.60,52.77,48.05,36.61,29.68,25.79,24.53,24.11,23.95,23.91.ESI-HRMS:m/z cacld.For C₃₃H₂₉F₃N₈O₇[M+H]+:707.2111,found 707.2121.

example 15: preparation of 4- (3- (4- (4- (3- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dione-isoindolin-4-yl) oxy) propyl) -1H-1,2, 3-triazol-1-yl) butyl) -4, 4-dimethyl-2, 5-dioxoimidazolidin-1-yl) -2- (trifluoromethyl) benzonitrile (I-8).

The synthesis procedure was the same as in example 7, white solid, m.p. 174.3-175.6 ℃,¹H NMR(400MHz,CDCl₃)δ8.68(s,1H),8.13(s,1H),8.00(d,J＝8.3Hz,1H),7.92(d,J＝8.4Hz,1H),7.67(t,J＝7.9Hz,1H),7.52(s,1H),7.45(d,J＝7.2Hz,1H),7.22(d,J＝8.5Hz,1H),4.96(dd,J＝11.9,5.2Hz,1H),4.38(t,J＝6.8Hz,2H),4.32–4.16(m,2H),3.37(t,J＝7.3Hz,2H),3.00(dd,J＝13.8,7.0Hz,2H),2.85(dd,J＝22.0,9.3Hz,2H),2.76(t,J＝7.4Hz,2H),2.29(dd,J＝12.8,6.4Hz,2H),2.15(dd,J＝15.7,7.9Hz,1H),2.01–1.92(m,2H),1.70(dt,J＝14.8,7.4Hz,2H),1.49(s,6H).¹³C NMR(101MHz,CDCl₃)δ173.71,172.68,169.97,169.74,168.61,155.81,155.71,150.80,138.65,136.92,133.20,132.75,132.32,131.76,123.67,120.49,118.92,118.87,116.95,116.93,116.67,113.05,69.99,64.60,52.77,51.79,40.72,29.68,27.72,25.79,24.88,24.53,24.11,23.95.ESI-HRMS:m/z cacld.For C₃₅H₃₃F₃N₈O₇[M+H]+:735.2424,found 735.2421.

example 16: preparation of 4- (3- (3- (4- ((3- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dione-isoindolin-4-yl) oxy) propoxy) methyl) -1H-1,2, 3-triazol-1-yl) propyl) -4, 4-dimethyl-2, 5-dioxoimidazolidin-1-yl) -2- (trifluoromethyl) benzonitrile (I-9).

The synthesis procedure was the same as in example 7, white solid, m.p. 196.4-197.5 ℃,¹H NMR(400MHz,DMSO-d₆)δ11.11(s,1H),8.31(d,J＝8.4Hz,1H),8.19(d,J＝1.7Hz,1H),8.13(s,1H),8.04(dd,J＝8.4,1.8Hz,1H),7.80(dd,J＝8.4,7.4Hz,1H),7.50(d,J＝8.5Hz,1H),7.44(d,J＝7.2Hz,1H),5.09(dd,J＝12.8,5.4Hz,1H),4.53(s,2H),4.45(t,J＝7.2Hz,2H),4.26(t,J＝6.2Hz,2H),3.65(t,J＝6.2Hz,2H),3.39(t,J＝7.3Hz,2H),2.97–2.80(m,1H),2.65–2.52(m,2H),2.19(dd,J＝13.7,6.6Hz,2H),2.08–2.03(m,1H),2.02–2.00(m,1H),1.46(d,J＝5.3Hz,6H).¹³C NMR(101MHz,CDCl₃)δ174.32,171.13,168.53,167.01,165.82,156.54,153.38,136.64,136.20,135.39,133.74,133.44,128.07,123.08,123.03,119.11,117.08,115.92,114.98,108.44,77.25,66.45,66.08,64.37,62.12,49.13,47.81,37.62,31.40,29.87,29.20,23.37,23.34,22.64.ESI-HRMS:m/z cacld.For C₃₅H₃₃F₃N₈O₈[M+H]+:751.2373,found751.2365.

example 17, 4- (3- (4- (4- ((2- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dione-isoindolin-4-yl) oxy) ethoxy) methyl) -1H-1,2, 3-triazol-1-yl) butyl) -4, 4-dimethyl-2, 5-dioxoimidazolidin-1-yl) -2- (trifluoromethyl) benzonitrile (I-10) is prepared.

The synthesis procedure was the same as in example 7, white solid, m.p.:197.2-198.6 ℃,¹H NMR(400MHz,CDCl₃)δ8.48(s,1H),8.13(d,J＝1.8Hz,1H),7.99(dd,J＝8.5,1.9Hz,1H),7.92(d,J＝8.4Hz,1H),7.67(dd,J＝8.4,7.4Hz,1H),7.59(s,1H),7.46(d,J＝7.2Hz,1H),7.29–7.23(m,2H),4.95(dd,J＝12.1,5.3Hz,1H),4.67(s,2H),4.40(t,J＝6.9Hz,2H),4.35(t,J＝4.6Hz,2H),3.95–3.90(m,2H),3.80(dd,J＝5.7,2.8Hz,2H),3.74–3.69(m,2H),3.48(s,1H),3.40–3.33(m,2H),2.85(dd,J＝18.3,16.0Hz,1H),2.80–2.69(m,2H),2.16–2.08(m,1H),2.02–1.94(m,2H),1.71(t,J＝7.3Hz,2H),1.50(s,6H).¹³C NMR(101MHz,DMSO-d₆)δ174.62,172.80,169.90,166.80,155.80,153.16,145.15,138.37,137.05,136.79,136.02,133.10,130.15,128.94,124.22,121.99,119.87,116.14,115.23,65.76,63.21,62.04,48.69,28.75,22.60,21.94.ESI-HRMS:m/z cacld.For C₃₇H₃₇F₃N₈O₉[M+H]+:795.2636,found 795.2616.

example 18 preparation of 4- (3- (4- (4- ((2- (2- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dione-isoindolin-4-yl) oxy) ethoxy) methyl) -1H-1,2, 3-triazol-1-yl) butyl) -4, 4-dimethyl-2, 5-dioxoimidazolidin-1-yl) -2- (trifluoromethyl) benzonitrile (I-11).

The synthesis procedure was the same as in example 7, white solid, m.p. 203.2-204.5 deg.C,¹H NMR(400MHz,CDCl₃)δ8.85(d,J＝18.9Hz,1H),8.13(d,J＝1.3Hz,1H),8.03–7.89(m,1H),7.67(dt,J＝17.0,6.1Hz,2H),7.55(s,1H),7.45(d,J＝7.2Hz,1H),7.38(dd,J＝8.7,2.2Hz,1H),7.26(s,1H),4.96(dd,J＝11.1,4.4Hz,1H),4.65(d,J＝13.1Hz,2H),4.34(dd,J＝9.2,5.5Hz,3H),3.97–3.90(m,2H),3.78(dd,J＝5.5,3.6Hz,4H),3.66(d,J＝9.4Hz,6H),3.37(d,J＝6.4Hz,2H),2.84(dd,J＝16.0,12.1Hz,2H),2.76(dt,J＝13.5,9.9Hz,2H),2.15–2.08(m,2H),1.99(dd,J＝13.6,6.6Hz,2H),1.91(s,1H),1.69(dt,J＝16.1,6.7Hz,2H),1.52(d,J＝17.4Hz,6H).¹³C NMR(101MHz,CDCl₃)δ174.32,171.13,168.53,167.01,165.82,156.54,153.38,136.64,136.20,135.39,133.74,133.44,128.07,123.08,123.03,119.11,117.08,115.92,114.98,108.44,77.25,66.45,66.08,64.37,62.12,49.13,47.81,37.62,31.40,29.87,29.20,23.37,23.34,22.64.ESI-HRMS:m/z cacld.For C₃₉H₄₁F₃N₈O₁₀[M+H]+:839.2898,found 839.2887.

example 19 preparation of 4- (3- (4- (4- (13- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dione-isoindolin-4-yl) oxy) -2,5,8, 11-ethoxy) -1H-1,2, 3-triazol-1-yl) butyl) -4, 4-dimethyl-2, 5-dioxoimidazolidin-1-yl) -2- (trifluoromethyl) benzonitrile (I-12).

The synthesis procedure was the same as in example 7, white solid, m.p. 216.3-217.7 ℃,¹H NMR(400MHz,CDCl₃)δ8.98(s,1H),8.14(s,1H),8.01(d,J＝8.3Hz,1H),7.93(d,J＝8.4Hz,1H),7.67(t,J＝7.8Hz,1H),7.59(s,1H),7.45(d,J＝7.2Hz,1H),7.27(d,J＝8.5Hz,1H),4.97(dd,J＝7.2,3.4Hz,1H),4.67(s,2H),4.36(t,J＝6.1Hz,5H),3.94(d,J＝4.4Hz,2H),3.84–3.56(m,12H),2.91–2.81(m,1H),2.81(s,2H),2.15–2.06(m,1H),2.01–1.91(m,2H),1.73(d,J＝6.8Hz,2H),1.51(s,6H),1.43–1.36(m,2H).¹³C NMR(101MHz,CDCl₃)δ174.59,171.40,168.45,167.01,165.61,156.38,152.88,145.25,136.52,136.45,135.34,133.73,133.30,128.04,123.35,123.04,122.99,122.61,120.62,119.46,117.25,116.09,115.06,108.15,77.32,71.12,70.52,70.48,69.76,69.31,69.25,64.55,61.91,49.91,49.11,40.00,31.39,29.72,28.79,23.83,23.43,22.62.ESI-HRMS:m/z cacld.For C₄₁H₄₅F₃N₈O₁₁[M+H]+:883.3160,found 883.3158.

example 20 preparation of 4- (3- (5- (4- ((2- (2- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dione-isoindolin-4-yl) oxy) ethoxy) methyl) -1H-1,2, 3-triazol-1-yl) pentyl) -4, 4-dimethyl-2, 5-dioxoimidazolidin-1-yl) -2- (trifluoromethyl) benzonitrile (I-13).

The synthesis procedure was the same as in example 7, white solid, m.p. 206.4-207.7 ℃,¹H NMR(400MHz,CDCl₃)δ8.64(s,1H),8.14(s,2H),8.00(d,J＝8.4Hz,2H),7.92(d,J＝8.4Hz,2H),7.70–7.64(m,2H),7.58(s,2H),7.46(d,J＝7.2Hz,2H),7.26(s,2H),4.95(dd,J＝11.7,5.5Hz,1H),4.68(d,J＝4.4Hz,3H),4.35(dd,J＝12.2,5.8Hz,6H),3.96–3.91(m,3H),3.78(dd,J＝10.6,5.6Hz,3H),3.67(dd,J＝6.4,4.1Hz,10H),3.36–3.28(m,2H),2.92–2.81(m,1H),2.80–2.71(m,2H),2.16–2.08(m,1H),2.01–1.94(m,2H),1.73(d,J＝6.8Hz,2H),1.56–1.48(s,6H).¹³C NMR(101MHz,DMSO-d₆)δ174.62,172.80,169.90,166.80,155.80,153.16,145.15,138.37,137.05,136.79,136.02,133.10,130.15,128.94,124.22,121.99,119.87,116.14,115.23,65.76,63.21,62.04,48.69,28.75,22.60,21.94.ESI-HRMS:m/z cacld.For C₄₀H₄₃F₃N₈O₁₀[M+H]+:853.3054,found 853.3051.

example 21, 4- (3- (3- (4- ((2- (2- (2- ((2- (2, 6-dioxopiperidin-3-yl) -1, 3-dione-isoindolin-4-yl) oxy) ethoxy) methyl) -1H-1,2, 3-triazol-1-yl) propyl) -4, 4-dimethyl-5-oxo-2-thioimidazolidin-1-yl) -2 (trifluoromethyl) benzonitrile (I-14) was prepared.

The synthesis procedure is as in example 7, red brown solid, m.p. 221.3-222.6 ℃,¹H NMR(400MHz,CDCl₃)δ8.67(s,1H),7.95(t,J＝6.9Hz,1H),7.88(d,J＝1.5Hz,1H),7.80–7.73(m,1H),7.67(dd,J＝8.7,7.1Hz,2H),7.46(t,J＝6.0Hz,1H),7.25(s,1H),4.96(dd,J＝11.3,4.9Hz,1H),4.70(s,2H),4.47(t,J＝6.6Hz,2H),4.37–4.31(m,3H),3.94(dd,J＝10.8,6.0Hz,3H),3.81–3.75(m,5H),3.74–3.64(m,8H),2.90–2.81(m,2H),2.80–2.70(m,2H),2.55–2.44(m,2H),2.12(dd,J＝7.4,4.9Hz,2H),1.53(s,6H).¹³C NMR(101MHz,CDCl₃)δ174.32,171.13,168.53,167.01,165.82,156.54,153.38,136.64,136.20,135.39,133.74,133.44,128.07,123.08,123.03,119.11,117.08,115.92,114.98,108.44,77.25,66.45,66.08,64.37,62.12,49.13,47.81,37.62,31.40,29.87,29.20,23.37,23.34,22.64.ESI-HRMS:m/z cacld.For C₃₈H₃₉F₃N₈O₉S[M+H]+:841.2513,found 841.2516.

example 22 AR protein binding level Activity assay of Compounds of the invention

The compounds of the invention were tested for in vitro AR affinity by Fluorescence Polarization (FP) to verify the binding interaction between the chimeric molecule and the AR protein. The primary sieving concentration of the compound of the present invention was 10 μm.

Preparing complete Buffer solution by using 1ml Buffer solution and 2 mu L1M DTT, then diluting the compound to be tested to 4X final concentration, controlling DMSO not to exceed 1%, firstly preparing 4X fluoromone by using fresh complete Buffer solution^TMTracer solution, then 5. mu.L of 4X Fluormone^TMTracer solution was added to 384 well assay plates followed by 4X AR solution with complete AR buffer, 5 μ L of 4X AR-LBD solution was added to assay plates, then diluted test compound was added to 384 well plates 10 μ L per well, mixed well and the plates were covered to protect the test agent from light. Setting 1X Fluormone^TM Tracer，1X AR-LBD\Fluormone^TMTracer and 1X AR-LBD \ Fluormone^TMTracer \ R1881 and solvent control group, each group 3 multiple wells, at least 3h incubation under dark at room temperature, finally placing the test board in a multifunctional microplate reader, measuring the fluorescence polarization value of each well at 485nm excitation wavelength and 535nm emission wavelength, taking the average value of three independent experiments, and analyzing the data by GrapHpad-prism software.

The fluorescence polarization inhibitory activity of the test compound at a concentration of 10. mu.M was calculated according to the following formula:

mP_100％100% inhibition, mP_0％Inhibition 0%, mpcound — the polarization value of the compound.

mP and corresponding concentrations of chimeric molecules at different concentrations IC was calculated using the formula GraphPad Prism fitted curves₅₀。

TABLE 1 binding Activity of preferred Compounds for AR

Example 23 degradation of Androgen Receptor (AR) by Compounds of the invention

The degradation test of the compound synthesized by the invention on Androgen Receptor (AR) is verified by extracting protein from prostate cancer cells LNCaP with high expression of androgen receptor through Western Blot experiment. The experimental result shows that the chimeric molecule has better degradation activity on androgen receptor.

(I) Experimental method

(1) Preparing a BCA standard curve: and adding PBS into 5mg/ml of standard protein to dilute the standard protein into standard protein with different concentrations. And (3) mixing solution A of the BCA working solution: mixing the solution B according to the volume ratio of 50: 1. In a 96-well plate, 19. mu.l of PBS, 1. mu.l of standard protein, and 200. mu.l of working solution were sequentially added, and incubated at 37 ℃ for 20 min. After the incubation, the OD value was measured at a wavelength of 562nm using a microplate reader. The absorbance values were substituted into the standard curve to calculate the protein concentration.

Cell plating: LNCaP which grows well in logarithmic phase is taken to prepare single cell suspension, and the single cell suspension is evenly inoculated in a six-hole plate in equal cell number.

Adding medicine: stock solutions of the preferred compounds of the present invention were diluted to 10. mu.M each, and 2ml of each of the drug-containing medium and the control medium was added to the corresponding six-well plate.

Collecting cells: after 24h of drug action, the primary medium and the cells collected by digestion were collected by centrifugation and washed 3 times with PBS to obtain a white cell pellet in a 1.5ml EP tube.

Cracking: and (3) adding the prepared RIPA lysate according to the cell number, then placing the cell on ice for lysis, and lightly blowing and beating an EP tube to ensure that the cell can be fully lysed. After full cracking, centrifuging at 12000rpm at 4 deg.C for 10-15min with a low temperature high speed centrifuge, transferring the supernatant to 1.5ml EP tube with a 1ml pipette, and discarding the bottom solid.

Protein quantification: mu.l of the sample was taken for protein quantification and the concentration of protein in each sample was calculated from the standard curve.

Protein denaturation: the collected protein lysate was added with 1/5 volume Loading Buffer and completely denatured by boiling in 100 ℃ boiling water in a water bath for 10 min. Storing at-20 ℃ in dark for later use.

Preparing glue, loading, electrophoresis, membrane transferring and dyeing: and (3) taking out the NC membrane after the electrophoresis membrane conversion is finished, washing the NC membrane once by using PBS, placing the NC membrane into ponceau red dye solution for protein dyeing, and judging whether the protein is smoothly converted into the membrane and the amount of the protein on the membrane according to the dyeing condition. The desired proteins of different molecular weights were cut according to the pre-stained protein Maker and then washed 3 times with TBST.

Sealing milk, sealing primary antibody, sealing secondary antibody, exposing, taking out NC membrane, washing with TBST for 3 times, each time for about 10 min. Primary antibody was diluted according to the instructions, AR and internal control GAPDH used in this experiment were diluted 1000-fold with TBST and incubated overnight at 4 ℃. The goat anti-rabbit and goat anti-mouse secondary antibodies used were both 5000-fold and 10000-fold diluted with TBST. And (3) adding the diluted secondary antibody to a corresponding NC membrane after uniformly mixing in a vortex mode, and incubating for 2h at room temperature. Uniformly mixing the solution A and the solution B of the ECL luminous liquid according to the size of the strip in a ratio of 1:1, adding the mixture on a film for reaction for 2min, and placing the film in a dark box for exposure.

Data processing: after scanning the films, the films were subjected to gray scale analysis using Image J software, and their data were statistically analyzed using GrapHpad-prism software.

(II) Experimental data and results

The AR degradation activity profile of preferred compounds of the invention is shown in FIGS. 1 and 2.

From the above biological activity evaluation, at a concentration of 10 μ M, all of the triazolyl-containing phthalimide compounds showed the ability to competitively bind to the AR-LBD ligand binding pocket with the fluorescence-labeled androgen fluorone, indicating that the compounds could inhibit the transcription activity of AR by competitively binding to AR with DHT, and some of the compounds also showed significant AR degradation activity. The compound of the invention is used as an active ingredient for preparing a new anticancer drug, and has potential application value.

Claims (1)

1. The phthalimide compound containing 1,2, 3-triazolyl is characterized in that: the compound has the following structural formula:

I-2。

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