Compounds based on the protac strategy, methods of making, pharmaceutical compositions, and uses thereof

Abstract

The application discloses a compound based on a PROTAC strategy and a preparation method, a pharmaceutical composition and application thereof, discloses a compound with AR / AR-V7 protein degradation activity and a preparation method, a pharmaceutical composition and application thereof. The structure of the compound is as shown in the formula I. The compound can effectively degrade AR / AR-V7 proteins in vivo and in vitro, has a significant antitumor effect, has no obvious toxic side effects, and can be used for treating castration-resistant prostate cancer. The preparation method of the compound is simple and easy to operate, and is beneficial to structural expansion.

Description

Technical Field

[0001] This invention relates to a compound based on the PROTAC strategy, its preparation method, pharmaceutical composition, and application, and more particularly to a compound with AR / AR-V7 protein degradation activity, its preparation method, pharmaceutical composition, and application. Background Technology

[0002] The androgen receptor (AR), encoded by eight exons located on Xq11-12, is a member of the steroid and nuclear receptor superfamily. The AR is primarily composed of three functional domains: the N-terminal domain (NTD), the DNA-binding domain (DBD), and the ligand-binding domain (LBD). The NTD is inherently disordered, with its activation domain 1 (AF-1) mediating ligand-independent transcriptional activation. The two transcriptional activation units within AF-1, Tau-1 and Tau-5, are crucial for AR transcriptional activation. In addition to interacting with the LBD, the NTD can also directly interact with coactivators to initiate AR transcription. The DBD, composed of two zinc finger structures, is a highly conserved domain in the AR that assists in binding to DNA response elements, mediating AR transcription. The LBD contains a hidden pocket known as the androgen binding site, which is also the primary site of action for existing AR antagonists. Gene sequencing studies have shown that after a period of anti-androgen treatment, AR will produce point mutants and splicing variants.

[0003] Prostate cancer (PC) has been shown to depend on the abnormal activation of androgen receptor (AR) signaling. Androgen deprivation therapy (ADT) is currently the most effective and widely used clinical strategy for treating prostate cancer, but it inevitably leads to castration-resistant prostate cancer (CRPC) after 1-2 years. Studies have shown that AR remains a key oncogenic driver in CRPC, and anti-androgen drugs that target and block the AR signaling pathway, such as enzalutamide, have become the main means of drug treatment for CRPC.

[0004] However, as the disease progresses, most CRPC patients develop resistance to existing drugs and progress to fatal late metastatic CRPC (mCRPC). The next-generation AR-LBD antagonist darolutamide is effective against several point mutants, including the enzalutamide-resistant F876L. However, the frequency of AR splicing variants after resistance is as high as 70%, with AR-V7 being one of the most widely studied high-frequency splicing variants. Truncated AR-V7 lacks the LBD region but possesses NTD and DBD domains and a unique C-terminus, enabling nuclear transfer without androgen binding and regulating the transcriptional activation of downstream target genes. AR-V7-induced hormone-dependent activation is a crucial mechanism for secondary resistance. Studies have shown that AR-V7 is closely related to the worsening of CRPC and drug resistance; CRPC carrying AR-V7 has a strong invasive ability and poor prognosis, and currently there are no effective treatments available clinically.

[0005]

[0006] Targeting NTDs can simultaneously regulate AR and AR-V7 activity. Currently, the most representative NTD antagonist is the prodrug molecule EPI-506 developed by ESSA. The parent drug molecule EPI-002, generated from the in vivo metabolism of EPI-506, irreversibly covalently binds to the AR-NTD region, thereby inhibiting the interaction between the AR NTD and LBD and preventing activation of the AR / AR-V7 signaling pathway. However, due to insufficient clinical efficacy and metabolic instability, EPI-506 was terminated in a Phase I clinical trial (NCT02606123). Therefore, there is an urgent need to develop new strategies to meet clinical needs. Summary of the Invention

[0007] Objectives of the Invention: The first objective of this invention is to provide a compound with AR / AR-V7 protein degradation activity; the second objective is to provide a method for preparing the compound; the third objective is to provide a pharmaceutical composition comprising the compound; and the fourth objective is to provide a pharmaceutical application of the compound and the pharmaceutical composition thereof.

[0008] Technical solution: The compound based on the PROTAC strategy described in this invention has the structure of Formula I:

[0009]

[0010] in:

[0011] R is selected from

[0012] L is selected from

[0013] X1 and X2 are selected from C or N;

[0014] Z is selected from C(O), C(O)NHR1, NH or does not exist;

[0015] R1 is selected from (CH2) k Or it may not exist;

[0016] Y is selected from 4-6 member heterocycles containing 1-2 nitrogen atoms, (CH2) k NH may not exist;

[0017] m is selected from 0 or 1, n is selected from integers from 2 to 6, p and q are selected from 1 or 2, and k is selected from 1 or 2;

[0018] D is selected from

[0019] Preferably, in the structure:

[0020] The structure satisfies any of the following conditions:

[0021] (1) When X1 is N and X2 is C, Z is C(O), m is 1, p and q are both 2, and Y is selected from piperidinyl or CH2NH;

[0022] (2) When X1 is C and X2 is N, Z is C(O)NH, NH or does not exist, m is 0 or 1, p and q are 1 or 2 and are the same, and Y does not exist;

[0023] (3) When X1 and X2 are both N, Z is C(O)NH(CH2)2, m is 0, p and q are both 2, and Y is piperidinyl.

[0024] Preferably, in the structure:

[0025] Selected from

[0026] Preferably, in the structure:

[0027] D is selected from

[0028] Preferably, the compound described in this invention is selected from any of the following compounds:

[0029]

[0030]

[0031] The pharmaceutically acceptable salts described in this invention are salts formed by the compounds described in this invention with an acid or a base, wherein the acid is selected from hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, or ferulic acid, and the base is selected from alkali metal cation bases, alkaline earth metal cation bases, ammonium cation bases, or choline.

[0032] "Pharmaceutically acceptable salts" refer to salts of compounds prepared by reacting a compound with a relatively non-toxic acid or base, containing specific substituents. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts, or similar salts. When a compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogen phosphates, dihydrogen phosphates, sulfuric acid (forming sulfates or bisulfates), hydroiodic acid, phosphorous acid, etc.); and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid. Acids such as citric acid, tartaric acid, and methanesulfonic acid; organic acid salts also include salts of organic acids such as amino acids (e.g., arginine) and glucuronic acid. Certain compounds contain both basic and acidic functional groups, thus allowing them to be converted into either a base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner, and then the parent compound is separated, thereby regenerating the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as different solubilities in polar solvents.

[0033] Pharmaceutically acceptable salts can be synthesized from parent compounds containing an acid radical or a base using conventional chemical methods. Generally, such salts are prepared by reacting these compounds, in their free acid or base form, with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of both. Non-aqueous media such as ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile are generally preferred.

[0034] The stereoisomers described in this invention are chiral isomers introduced by the chiral carbon in the R group, where the chiral carbon has an R or S configuration. For example: compounds Stereoisomers include:

[0035]

[0036] The method for preparing the compound described in this invention is selected from any of the following methods:

[0037] Method 1:

[0038]

[0039] (a) Compound 1 is substituted with ethyl bromoacetate to form compound 2;

[0040] (b) Compound 2 undergoes deethylation to form compound 3;

[0041] (c) Compound 3 was reacted with a terminal amino linker via an amide condensation reaction to give compound 4;

[0042] (d) Compound 4 reacts with bromopropylene oxide to generate compound 5;

[0043] (e) Compound 5 was given by epoxidative ring-opening in cerium chloride heptahydrate to yield compound 6;

[0044] (f) Compound 6 was deprotected from the Boc protecting group in trifluoroacetic acid to give compound 7;

[0045] (g) Compound 7 undergoes a substitution reaction to produce compound I;

[0046] Reaction conditions: a)ethyl 2-bromoacetate, K2CO3, DMF, 25℃, 12h, 37%; b) KOH, MeOH, H2O, 25℃, 1h, 98%; c) HATU, DMF, 60℃, 12h, 42%-91%; d) epibromohydrin, K2CO3, DMF, 25℃, 12h, 61%-88%; e) CeCl3, MeCN, 70℃, 12h, 85%-95%; f) TFA, DCM, 25℃, 2h, 77%-91%; g) DIPEA, DMF, 90℃, 12h, 13%-47%.

[0047] Method 2:

[0048]

[0049] (h) Compound 1 is substituted with a terminal brominated linker to generate compound 8;

[0050] (i) Compound 8 reacts with bromopropylene oxide to generate compound 9;

[0051] (j) Compound 9 was given by epoxidative ring-opening in cerium chloride heptahydrate to yield compound 10;

[0052] (k) Compound 10 was deprotected from the Boc protecting group in trifluoroacetic acid to give compound 11;

[0053] (l) Compound 11 undergoes a substitution reaction to produce compound I;

[0054] Reaction conditions: h) K₂CO₃, DMF, 25℃, 12h, 30%-50%; i) epibromohydrin, K₂CO₃, DMF, 25℃, 12h, 62%-86%; j) CeCl₃, MeCN, 70℃, 12h, 81%-93%; k) TFA, DCM, 25℃, 2h, 82%-96%; l) DIPEA, DMF, 90℃, 12h, 14%-46%.

[0055] Method 3:

[0056]

[0057] (m) Compound 12 is substituted with morpholine to generate compound 13;

[0058] (n) Compound 13 reacts with carbon tetrabromide to give hydroxybrominated product 14;

[0059] (o) Compound 1 was reacted with a terminal brominated linker to give compound 15;

[0060] (p) Compound 15 is substituted with compound 14 to give compound 16;

[0061] (q) Compound 16 was deprotected from the Boc protecting group in trifluoroacetic acid to give compound 17;

[0062] (r) Compound 17 undergoes a substitution reaction to produce compound I;

[0063] Reaction conditions: m) Morpholine, DMF, 100℃, 16h, 90%; n) CBr4, PPh3, 25℃, 3h, 70%; o) K2CO3, DMF, 25℃, 12h, 31%-57%; p) K2CO3, DMF, 90℃, 6h, 72%-88%; q) TFA, DCM, 25℃, 2h, 80%-95%; r) DIPEA, DMF, 90℃, 12h, 11%-49%.

[0064] Method 4:

[0065]

[0066] (s) Compound 3 was amide-condensed with a terminal amino linker to give compound 18;

[0067] (t) Compound 18 and Compound 14 were substituted to give Compound 19;

[0068] (u) Compound 19 was deprotected from the Boc protecting group in trifluoroacetic acid to give compound 20;

[0069] (v) Compound 20 undergoes a substitution reaction to produce compound I;

[0070] Reaction conditions: s) HATU, DMF, 60℃, 12h, 42%-82%; t) K2CO3, DMF, 90℃, 6h, 64%-86%; u) TFA, DCM, 25℃, 2h, 78%-94%; v) DIPEA, DMF, 90℃, 12h, 12%-46%.

[0071] The definitions of L and D are as described above.

[0072] The pharmaceutically acceptable salt of the compound is obtained by salting the compound I prepared by the above method with the corresponding acid or base.

[0073] The pharmaceutical compositions of the present invention comprise the compounds of the present invention or their pharmaceutically acceptable salts and pharmaceutically acceptable carriers.

[0074] Preferably, the formulation of the drug combination is selected from tablets, capsules, powders, pills, granules, injections, oral liquids, syrups, inhalers, ointments, patches, or suppositories.

[0075] "Pharmaceutically acceptable carriers" are excipients widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods to facilitate the dissolution of the active ingredient at a desired rate after administration to a subject, or to promote the effective absorption of the active ingredient after administration to a subject. The pharmaceutical excipients may be inert fillers or provide a function, such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient. The pharmaceutical excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0076] The pharmaceutical compositions described in this invention can be prepared using any method known to those skilled in the art, based on the disclosure. For example, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0077] The pharmaceutical compositions of this invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical compositions of this invention can also be controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0078] The compounds or pharmaceutical compositions thereof described in this invention are used in the preparation of drugs for AR / AR-V7 protein degrading agents.

[0079] Preferably, the drug is a drug for treating castration-resistant prostate cancer.

[0080] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0081] The compounds designed in this invention can effectively degrade AR / AR-V7 protein both in vitro and in vivo, exhibiting significant anti-tumor activity (in vitro molecular-level degradation activity DC). 50 The optimal value is below 50 nM, with an IC50 value indicating inhibitory activity at the cellular level. 50 The optimal concentration is below 50 nM, which significantly inhibits the volume and weight of tumors in vivo, and has no obvious toxic side effects. It can be used to treat castration-resistant prostate cancer. The compound preparation method is simple and easy to implement, which is conducive to structural expansion. Attached Figure Description

[0082] Figure 1 The results show the degradation activity of compounds NP-1 to NP-24 on AR / AR-V7 protein in 22Rv1 cells;

[0083] Figure 2 Results of the antiproliferative activity of compound NP-18 against tumor cells; A: 22Rv1 cells, B: LnCaP cells;

[0084] Figure 3Results of the antitumor activity of compound NP-18 in tumor-bearing mice; A-C: Changes in tumor volume and mass 21 days after administration; D: Changes in mouse body weight during administration; E: Immunofluorescence staining detection of AR / AR-V7 protein content in tumor tissue sections 21 days after administration. Detailed Implementation

[0085] The technical solution of the present invention will be further described below with reference to the embodiments.

[0086] 1 H-NMR was measured using a Bruker AV300 (300MHz) NMR spectrometer (TMS as internal standard), and mass spectrometry was performed using a Shimadzu GC / MS-QP2010 mass spectrometer (EI-MS) and an Agilent 1100LC-MSD-Trap / SL mass spectrometer (ESI-MS).

[0087] Column chromatography used 100-200 mesh, 200-300 mesh, or 300-400 mesh silica gel (Qingdao Ocean Chemical Plant), with petroleum ether-ethyl acetate or chloroform-methanol systems as eluents. Thin-layer chromatography (TLC) was performed using GF254 TLC plates (Yantai Jiangyou Silica Gel Development Co., Ltd.); the TLC developing system was a petroleum ether-ethyl acetate system or a chloroform-methanol system, with a small amount of acetic acid added if necessary; TLC was visualized using a ZF7 three-way UV analyzer (Henan Gongyi Yuhua Instrument Co., Ltd.). The purity of some compounds was determined using Shimadzu HPLC at 254 nm, with a methanol / water system as the mobile phase.

[0088] This invention provides a method for synthesizing an AR / AR-V7 degrader targeting NTDs or a pharmaceutically acceptable salt thereof. The preparation method includes routes 1, 2, 3, and 4.

[0089] Example 1

[0090] Route 1:

[0091]

[0092] Reaction conditions: a)ethyl 2-bromoacetate, K2CO3, DMF, 25℃, 12h, 37%; b) KOH, MeOH, H2O, 25℃, 1h, 98%; c) HATU, DMF, 60℃, 12h, 42%-91%; d) epibromohydrin, K2CO3, DMF, 25℃, 12h, 61%-88%; e) CeCl3, MeCN, 70℃, 12h, 85%-95%; f) TFA, DCM, 25℃, 2h, 77%-91%; g) DIPEA, DMF, 90℃, 12h, 13%-47%.

[0093] (1) Synthesis of ethyl acetate 2-(4-(2-(4-hydroxyphenyl)prop-2-yl)phenoxy)

[0094]

[0095] Bisphenol A (1 g, 4.4 mmol), ethyl bromoacetate (0.7 g, 4.4 mmol), and potassium carbonate (0.6 g, 13.2 mmol) were dissolved in 10 mL of DMF and reacted overnight at room temperature. After the reaction was complete, the solution was poured into water, and the pH of the reaction mixture was adjusted to 7 by adding 10% dilute hydrochloric acid solution. The solution was then extracted with ethyl acetate, and the organic phase was washed three times with saturated brine. After drying with anhydrous sodium sulfate, the solution was subjected to column chromatography (DCM:MeOH = 400:1-500:1) to give a white solid 2 (0.5 g, 37%). 1 HNMR (300MHz, CDCl3) δ7.20-6.98(m,4H),6.83-6.67(m,4H),4.59(s,2H),4.27(q,J=7.1Hz,2H),1.62(s,6H),1.30(t,J=7.1Hz,3H).

[0096] (2) Synthesis of 2-(4-(2-(4-hydroxyphenyl)prop-2-yl)phenoxy)acetic acid (3)

[0097]

[0098] Compound 2 (508 mg, 1.6 mmol) was dissolved in 10 mL of methanol solution, and then 10 mL of water was added. Potassium hydroxide (1.2 g, 21.4 mmol) was added under an ice-water bath. The reaction was allowed to proceed at room temperature for 0.5 h. The pH was adjusted to 2 by adding 10% dilute hydrochloric acid solution. The mixture was then filtered to obtain a white solid 3 (450 mg, 98.2%). 1 H NMR (300MHz, DMSO-d6) δ9.17(s,1H),7.13-7.05(m,2H),7.02-6.95(m,2H),6.85-6.73(m,2H),6.70-6.59(m,2H),4.61(s,2H),1.55(s,6H).

[0099] (3) Synthesis of tert-butyl (2-(4-(2-(4-hydroxyphenyl)prop-2-yl)phenoxy)acetyl)carbamate (5a)

[0100]

[0101] Compound 3 (450 mg, 1.6 mmol), HATU (896 mg, 2.4 mmol), and DIPEA (0.41 mL, 4.7 mmol) were dissolved in 10 mL of DMF and reacted at room temperature for 0.5 h. Then, tert-butyl (2-aminoethyl)carbamate (384 mg, 2.4 mmol) was added and the reaction was continued for 2 h. The reaction was then checked for completeness. The reaction solution was poured into water to adjust to neutral and extracted with ethyl acetate. The organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, and subjected to column chromatography (DCM:MeOH = 100:1-200:1) to obtain a brown oily substance 5a (603 mg, 88.0%). 1 HNMR (300MHz, CDCl3) δ7.80 (s, 1H), 7.39 (t, J = 5.7Hz, 1H), 7.18-7.09 (m, 2H), 7.08-6.99 (m, 2H), 6.84-6.74 (m,4H),5.17(t,J=6.0Hz,1H),4.45(s,2H),3.44-3.32(m,2H),3.16-3.05(m,2H),1.60(s,6H),1.42(s,9H).

[0102] Synthesis of (4) tert-butyl carbamate (6a)

[0103]

[0104] Compound 5a (500 mg, 1.2 mmol) and potassium carbonate (485 mg, 3.5 mmol) were dissolved in 10 mL of DMF, and epichlorohydrin (176 mg, 1.3 mmol) was added. The mixture was reacted at 50 °C under nitrogen protection for 6 h. After the reaction was completed, the reaction solution was poured into water and extracted with ethyl acetate. The organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain a colorless oily substance 6a (480 mg, 82.6%), which was added directly without purification.

[0105] Synthesis of (5) tert-butyl carbamate (7a)

[0106]

[0107] Compound 6a (480 mg, 1.0 mmol) was dissolved in 10 mL of acetonitrile, and CeCl3·7H2O (1 g, 2.7 mmol) was added and refluxed overnight. The reaction solution was directly prepared into sand and then subjected to column chromatography (DCM:MeOH = 100:1-200:1) to obtain a white solid 7a (450 mg, 87.2%). 1 H NMR (300MHz, CDCl3) δ7.24-7.05(m,5H),6.85-6.76(m,4H),4.94(s,1H),4.45(s,2H),4.25-4.14(m,1H),4.10 -4.00(m,2H),3.80-3.64(m,2H),3.48-3.37(m,2H),3.33-3.22(m,2H),2.91(s,1H),1.63(s,6H),1.42(s,9H).

[0108] (6) Synthesis of N-(2-aminoethyl)-2-(4-(2-(4-(3-chloro-2-hydroxypropoxy)phenyl)prop-2-yl)phenoxy)acetamide (8a)

[0109]

[0110] Compound 7a (450 mg, 1.1 mmol) was dissolved in 10 mL of dichloromethane, and trifluoroacetic acid (1 mL, 13.1 mmol) was added under an ice-water bath. The reaction was carried out at room temperature for 1 h, and the reaction solution was evaporated to dryness and added directly to the solution to obtain a brown oily substance 8a (400 mg, 86.4%).

[0111] (7) Synthesis of 2-(4-(2-(4-(3-chloro-2-hydroxypropoxy)phenyl)prop-2-yl)phenoxy)-N-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)ethyl)acetamide (NP-1)

[0112]

[0113] Compound 8a (200 mg, 0.5 mmol) and 2-(2,6-dioxadiazin-3-yl)-5-fluoroisoindoline-1,3-dione (138 mg, 0.5 mmol) were dissolved in 10 mL of DMF, and DIPEA (0.35 mL, 2 mmol) was added. The reaction was carried out at 100 °C for 6 h. The reaction was monitored by TLC until it was complete. The reaction solution was poured into water and extracted with ethyl acetate. The organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain a crude yellow oil. Thin-layer chromatography was used to purify the crude product to obtain a yellow solid NP-1 (50 mg, 15.4%). 1HNMR (400MHz, CDCl3) δ7.53 (d, J = 8.3Hz, 1H), 7.16-7.08 (m, 4H), 7.02 (t, J = 6.3Hz, 1H), 6. 88(d,J=2.2Hz,1H),6.84-6.75(m,4H),6.68(dd,J=2.2,8.3Hz,1H),5.38(t,J=5.1Hz,1H) ,4.96-4.86(m,1H),4.49(s,2H),4.22-4.15(m,1H),4.09-4.01(m,2H),3.82-3.67(m,2H) ,3.66-3.58(m,2H),3.38-3.31(m,2H),2.90-2.62(m,3H),2.12-2.04(m,1H),1.61(s,6H). 13 C NMR (101MHz, CDCl3) δ171.3,170.2,168.9,167.9,167.4,156.1,154.9,153.5,144.9,134.5,128.1,127.8,125.5,1 18.3,116.5,114.1,114.0,105.9,69.9,68.4,67.3,49.0,46.0,44.1,41.8,38.2,31.4,30.9,22.7.HRMS(ESI):m / z calcd forC 35 H 37 ClN4NaO8 + [M+Na] + ,699.2192;found,699.2191.

[0114] (8) Synthesis of 2-(4-(2-(4-(3-chloro-2-hydroxypropoxy)phenyl)propyl-2-yl)phenoxy)-N-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)propyl)acetamide (NP-2)

[0115]

[0116] The synthesis method of compound NP-2 is the same as that of compound NP-1 described above. The final product is a yellow solid NP-2 (58 mg, 22.6% yield). 1H NMR (400MHz, CDCl3) δ8.82 (s, 1H), 7.49 (d, J = 8.4Hz, 1H), 7.22-7.07 (m, 4H), 6.96-6.87 (m, 2H) ,6.85-6.78(m,4H),6.70(dd,J=2.2,8.4Hz,1H),5.49(t,J=5.8Hz,1H),4.98-4.89(m,1H),4.5 2(s,2H),4.26-4.17(m,1H),4.12-3.99(m,2H),3.85-3.68(m,2H),3.52-3.42(m,2H),3.26-3. 17(m,2H),2.97(s,1H),2.90-2.67(m,3H),2.17-2.06(m,1H),1.88-1.77(m,2H),1.64(s,6H). 13 C NMR (101MHz, CDCl3) δ171.5,169.4,169.1,168.1,167.5,156.1,154.9,153.7,144.8,143.6,134.6,128.1,127.8,125.4, 117.9,116.7,114.1,114.0,105.7,69.9,68.5,67.3,49.0,46.0,41.8,40.2,36.3,31.4,30.9,28.5,22.8.HRMS(ESI):m / z calcd for C 36 H 39 ClN4NaO8 + [M+Na] + ,713.2349; found,713.2347.

[0117] (9) Synthesis of 2-(4-(2-(4-(3-chloro-2-hydroxypropoxy)phenyl)prop-2-yl)phenoxy)-N-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)butyl)acetamide (NP-3)

[0118]

[0119] The synthesis method of compound NP-3 is the same as that of compound NP-1 described above. The final product is a yellow solid NP-3 (49 mg, 13.5% yield). 1H NMR (300MHz, CDCl3) δ8.78 (s, 1H), 7.48 (d, J = 8.3Hz, 1H), 7.21-7.07 (m, 4H), 6.88-6.7 1(m,6H),6.64(dd,J=2.2,8.3Hz,1H),5.12(t,J=5.3Hz,1H),4.97-4.87(m,1H),4.48( s,2H),4.23-4.15(m,1H),4.10-3.99(m,2H),3.80-3.67(m,2H),3.43-3.35(m,2H),3. 21-3.12(m,2H),2.87-2.70(m,4H),2.13-2.05(m,1H),1.69-1.63(m,4H),1.62(s,6H). 13 CNMR (75MHz, CDCl3) δ171.6,169.2,168.9,168.1,167.5,156.1,155.0,153.7,144.7,143.6,134.5,128.1,127.8,125.4,117 .7,116.2,114.1,114.0,105.8,69.9,68.5,67.4,49.0,46.0,43.0,41.8,38.6,31.5,31.0,27.4,25.8,22.8.HRMS(ESI):m / z calcd forC 37 H 41 ClN4NaO8 + [M+Na] + ,727.2505; found,727.2509.

[0120] (10) Synthesis of 2-(4-(2-(4-(3-chloro-2-hydroxypropoxy)phenyl)prop-2-yl)phenoxy)-N-(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)pentyl)acetamide (NP-4)

[0121]

[0122] The synthesis method of compound NP-4 is the same as that of compound NP-1 described above. The final product is a yellow solid NP-4 (62 mg, 21.7% yield). 1H NMR (400MHz, CDCl3) δ8.67 (s, 1H), 7.51 (d, J = 8.3Hz, 1H), 7.19-7.09 (m, 4H), 6.86 (d, J = 2.1Hz, 1H), 6.83-6.77(m,4H),6.71(t,J=6.2Hz,1H),6.65(dd,J=2.2,8.4Hz,1H),4.97(t,J=5.2Hz,1H),4.95- 4.89(m,1H),4.47(s,2H),4.23-4.15(m,1H),4.09-3.97(m,2H),3.82-3.63(m,2H),3.42-3.33(m,2 H),3.20-3.07(m,2H),2.92-2.65(m,4H),2.14-2.04(m,1H),1.70-1.55(m,10H),1.46-1.35(m,2H). 13 C NMR (101MHz, CDCl3) δ171.5,169.0,168.8,168.0,156.1,155.0,153.8,144.7,143.6,134.5,128.1,127.8,125.5,117.8,11 6.2,114.1,114.0,105.8,69.9,68.4,67.4,49.0,46.0,43.2,41.8,38.5,31.5,31.0,29.3,28.2,23.9,22.8.HRMS(ESI):m / z calcd for C 38 H 43 ClN4NaO8 + [M+Na] + ,741.2662; found,741.2678.

[0123] (11) Synthesis of 2-(4-(2-(4-(3-chloro-2-hydroxypropoxy)phenyl)prop-2-yl)phenoxy)-N-(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)pentyl)acetamide (NP-5)

[0124]

[0125] The synthesis method of compound NP-5 is the same as that of compound NP-1 described above. The final product is a yellow solid NP-5 (36 mg, 13.5% yield). 1H NMR (300MHz, CDCl3) δ8.49 (s, 1H), 7.55 (d, J = 8.3Hz, 1H), 7.19-7.07 (m, 4H), 6.90 (d, J = 2.1Hz, 1 H),6.85-6.77(m,4H),6.73-6.62(m,2H),4.97-4.87(m,1H),4.82(t,J=5.3Hz,1H),4.46(s,2H), 4.24-4.15(m,1H),4.10-4.00(m,2H),3.80-3.67(m,2H),3.40-3.30(m,2H),3.22-3.11(m,2H), 2.89-2.81(m,2H),2.78-2.70(m,1H),2.15-2.03(m,1H),1.67-1.53(m,10H),1.45-1.31(m,4H). 13 C NMR (75MHz, CDCl3) δ171.4,168.7,168.5,168.0,167.5,156.1,155.0,153.8,144.7,143.6,134.6,128.1,127.8,125.5,117.9,11 6.2,114.1,114.0,106.0,69.9,68.4,67.4,49.0,46.0,43.2,41.8,38.7,31.5,31.0,29.4,28.7,26.3,26.2,22.8.HRMS(ESI):m / z calcd for C 39 H 45 ClN4NaO8 + [M+Na] + ,755.2818; found,755.2812.

[0126] (12) Synthesis of 5-(4-(1-(2-(4-(2-(4-(3-chloro-2-hydroxypropoxy)phenyl)propyl-2-yl)phenoxy)acetyl)piperidin-4-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (NP-6)

[0127]

[0128] The synthesis method of compound NP-6 is the same as that of compound NP-1 described above. The final product is a yellow solid NP-6 (77 mg, 25.7% yield). 1H NMR (400MHz, CDCl3) δ8.70 (s, 1H), 7.68 (d, J = 8.4Hz, 1H), 7.27 (s, 1H), 7.16-7.08 (m, 4H), 7.03 ( dd,J=2.3,8.6Hz,1H),6.86-6.76(m,4H),4.97-4.88(m,1H),4.71-4.55(m,3H),4.21-4.13(m,1 H),4.10-3.99(m,3H),3.79-3.64(m,2H),3.44-3.34(m,4H),3.05(t,J=12.6Hz,1H),1.53-1.33 (m,8H),2.59-2.49(m,1H),2.16-2.05(m,1H),1.92-1.83(m,2H),1.6(s,6H),1.92-1.83(m,2H). 13 C NMR (75MHz, CDCl3) δ171.5,168.7,168.0,167.3,166.5,156.1,155.7,155.4,144.0,143.7,134.2,127.9,125.4,119.6,118 .0,114.0,113.9,108.7,69.8,68.5,67.8,61.4,49.2,48.5,47.7,46.0,44.7,41.8,31.0,28.7,27.8,22.7.HRMS(ESI):m / z calcd for C 42 H 48 ClN5NaO8 + [M+Na] + ,808.3084;found,808.3084.

[0129] (13) Synthesis of 2-(4-(2-(4-(3-chloro-2-hydroxypropoxy)phenyl)prop-2-yl)phenoxy)-N-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)azacyclobutane-3-yl)acetamide (NP-7)

[0130]

[0131] The synthesis method of compound NP-7 is the same as that of compound NP-1 mentioned above. The final product is a yellow solid NP-7 (89 mg, 31.6% yield). 1H NMR (400MHz, CDCl3) δ8.29 (s, 1H), 7.65 (d, J = 8.3Hz, 1H), 7.18 (s, 1H), 7.17-7.10 (m, 4H), 6.83(d,J=3.2Hz,1H),6.82-6.74(m,4H),6.54(dd,J=2.2,8.3Hz,1H),5.07-4.96(m,1H), 4.96-4.87(m,1H),4.49(s,2H),4.44-4.35(m,2H),4.25-4.15(m,1H),4.10-4.00(m,2H), 3.96-3.86(m,2H),3.81-3.67(m,2H),2.90-2.63(m,4H),2.16-2.06(m,1H),1.63(s,6H). 13 CNMR(101MHz, CDCl3)δ171.0,168.5,168.3,167.7,167.4,156.1,154.9,154.6,145.0,143.5,134.3,134.3,128.2,127.8,125 .4,118.9,114.7,114.2,114.0,105.4,69.9,68.4,67.4,58.7,49.1,46.0,41.8,39.9,31.4,31.0,22.7.HRMS(ESI):m / zcalcd for C 36 H 37 ClN4NaO8 + [M+Na] + ,711.2192;found,711.2162.

[0132] (14) Synthesis of 2-(4-(2-(4-(3-chloro-2-hydroxypropoxy)phenyl)prop-2-yl)phenoxy)-N-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-4-yl)acetamide (NP-8)

[0133]

[0134] The synthesis method of compound NP-8 is the same as that of compound NP-1 described above. The final product is a yellow solid NP-8 (59 mg, 23.3% yield). 1H NMR (400MHz, CDCl3) δ8.68 (s, 1H), 7.69 (d, J = 8.5Hz, 1H), 7.28 (s, 1H), 7.19-7.10 (m, 4H), 7.0 6(dd,J=2.4,8.6Hz,1H),6.85-6.78(m,4H),6.59(d,J=8.2Hz,1H),4.99-4.91(m,1H),4.48(s, 2H),4.25-4.13(m,2H),4.11-4.01(m,2H),3.95-3.86(m,2H),3.82-3.69(m,2H),3.19-3.07( m,2H),3.01(d,J=5.8Hz,1H),2.91-2.70(m,3H),2.15-2.03(m,3H),1.64(s,6H),1.55(m,2H). 13 C NMR (101MHz, CDCl3) δ171.4,168.6,168.0,168.0,167.3,156.2,155.0,154.9,144.8,143.6,134.4,128.1,127.8,125.5, 119.2,118.1,114.2,114.0,108.8,69.9,68.5,67.4,49.2,47.0,46.2,46.0,41.8,31.4,31.1,31.0,22.7.HRMS(ESI):m / z calcd for C 38 H 41 ClN4NaO8 + [M+Na] + ,739.2505; found,739.2501.

[0135] (15) Synthesis of 5-(((1-(2-(4-(2-(4-(3-chloro-2-hydroxypropoxy)phenyl)prop-2-yl)phenoxy)acetyl)piperidin-4-yl)methyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (NP-9)

[0136]

[0137] The synthesis method of compound NP-9 is the same as that of compound NP-1 described above. The final product is a yellow solid NP-9 (46 mg, 11.2% yield). 1H NMR (400MHz, CDCl3) δ8.32(s,1H),7.58(d,J=8.3Hz,1H),7.17-7.07(m,4H),6.92(d,J=2.1Hz,1H ),6.87-6.76(m,4H),6.71(dd,J=2.2,8.3Hz,1H),4.96-4.88(m,1H),4.69-4.57(m,3H),4.23-4.1 5(m,1H),4.11-3.99(m,3H),3.81-3.65(m,2H),3.11-2.99(m,3H),2.89-2.71(m,3H),2.65-2.53 (m,1H),2.15-2.07(m,1H),1.94-1.85(m,1H),1.85-1.77(m,2H),1.62(s,6H),1.24-1.13(m,3H). 13 C NMR (101MHz, CDCl3) δ171.3,168.7,167.9,167.4,166.5,156.1,155.7,153.6,144.1,143.7,134.7,127.8,125.6,118.4, 116.5,114.0,113.9,106.0,69.9,68.4,67.8,49.1,48.8,46.0,42.2,41.8,35.7,31.5,31.0,29.7,22.8.HRMS(ESI):m / z calcd for C 39 H 43 ClN4NaO8 + [M+Na] + ,753.2662;found,753.2661.

[0138] (16) Synthesis of 2-(4-(2-(4-(3-chloro-2-hydroxypropoxy)phenyl)propyl-2-yl)phenoxy)-N-(3-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)propyl)acetamide (NP-10)

[0139]

[0140] The synthesis method of compound NP-10 is the same as that of compound NP-1 described above. The final product is a yellow solid NP-10 (53 mg, 22.8% yield). 1HNMR (400MHz, CDCl3) δ8.85 (s, 1H), 7.48-7.41 (m, 1H), 7.19-7.10 (m, 4H), 7.07 (d, J = 7.1Hz, 1H) ,6.93(t,J=6.2Hz,1H),6.89-6.76(m,5H),6.45(t,J=6.0Hz,1H),4.95-4.86(m,1H),4.48(s,2H ),4.25-4.15(m,1H),4.09-4.01(m,2H),3.81-3.66(m,2H),3.52-3.42(m,2H),3.35-3.26(m,2H ),3.12(d,J=5.8Hz,1H),2.85-2.62(m,3H),2.07-2.00(m,1H),1.92-1.82(m,1H),1.63(s,6H). 13 C NMR (101MHz, CDCl3) δ171.7,169.4,169.1,168.8,167.7,156.2,155.0,146.6,144.7,143.5,136.2,132.6,128.1,127.8, 116.5,114.1,114.0,111.6,110.1,69.8,68.5,67.4,48.9,46.0,41.8,39.8,36.5,31.4,31.0,29.1,22.7.HRMS(ESI):m / z calcd for C 36 H 40 ClN4O8 + [M+H] + ,691.2529;found,691.2497.

[0141] (17) Synthesis of 2-(4-(2-(4-(3-chloro-2-hydroxypropoxy)phenyl)prop-2-yl)phenoxy)-N-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)azacyclobutane-3-yl)acetamide (NP-11)

[0142]

[0143] The synthesis method of compound NP-11 is the same as that of compound NP-1 described above. The final product was a yellow solid NP-11 (64 mg, 24.8% yield). 1H NMR (400MHz, DMSO-d6) δ11.08(s,1H),8.77(d,J=7.0Hz,1H),7.58(t,J=7.7Hz,1H),7.15( s,1H),7.14-7.05(m,4H),6.87-6.76(m,4H),5.53(d,J=5.2Hz,1H),5.12-5.00(m,1H),4.7 3-4.61(m,1H),4.53-4.44(m,3H),4.16-4.03(m,2H),4.03-3.96(m,1H),3.96-3.88(m,2H) ,3.80-3.60(m,2H),2.94-2.80(m,1H),2.62-2.51(m,2H),2.06-1.93(m,1H),1.57(s,6H). 13 C NMR(101MHz,DMSO-d6)δ173.3,170.5,168.4,167.7,167.0,156.6,156.0,148.3,143.8,143.2,135.5,133.7,127.9, 127.9,120.6,114.6,114.3,112.4,110.7,69.3,69.1,67.4,61.0,49.1,47.3,41.7,31.4,31.2,22.6.HRMS(ESI):m / z calcdfor C 36 H 38 ClN4O8 + [M+H] + ,689.2373; found,689.2355.

[0144] Example 2

[0145] Route 2:

[0146]

[0147] Reaction conditions: h) K₂CO₃, DMF, 25℃, 12h, 30%-50%; i) epibromohydrin, K₂CO₃, DMF, 25℃, 12h, 62%-86%; j) CeCl₃, MeCN, 70℃, 12h, 81%-93%; k) TFA, DCM, 25℃, 2h, 82%-96%; l) DIPEA, DMF, 90℃, 12h, 14%-46%.

[0148] Synthesis of (1) tert-butyl (2-(4-(2-(4-hydroxyphenyl)prop-2-yl)phenoxy)ethyl)carbamate (11a)

[0149]

[0150] Bisphenol A (1 g, 4.4 mmol), tert-butyl (2-bromoethyl)carbamate (1.0 g, 4.4 mmol), and potassium carbonate (0.6 g, 13.2 mmol) were dissolved in 10 mL of DMF and reacted overnight at room temperature. After the reaction was complete, the solution was poured into water, and the pH of the reaction mixture was adjusted to 7 by adding 10% dilute hydrochloric acid solution. The solution was then extracted with ethyl acetate, and the organic phase was washed three times with saturated brine. After drying with anhydrous sodium sulfate, the solution was subjected to column chromatography (DCM:MeOH = 300:1-400:1) to give a white solid 2 (653 mg, 40%). 1 H NMR (300MHz, CDCl3) δ7.21-7.04(m,4H),6.85-6.68(m,4H),5.62(s,1H),5.0 3(s,1H),3.98(t,J=5.1Hz,2H),3.56-3.46(m,2H),1.63(s,6H),1.45(s,9H).

[0151] (2) Synthesis of tert-butyl (2-(4-(2-(4-hydroxyphenyl)prop-2-yl)phenoxy)ethyl)carbamate (12a)

[0152]

[0153] Compound 11a (500 mg, 1.4 mmol) and potassium carbonate (559 mg, 4.1 mmol) were dissolved in 10 mL of DMF, and epichlorohydrin (206 mg, 1.5 mmol) was added. The mixture was reacted at 50 °C under nitrogen protection for 6 h. After the reaction was completed, the reaction solution was poured into water and extracted with ethyl acetate. The organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain a colorless oily substance 12a (490 mg, 85%), which was added directly without purification.

[0154] Synthesis of (3) tert-butyl carbamate (13a)

[0155]

[0156] Compound 12a (490 mg, 1.2 mmol) was dissolved in 5 mL of acetonitrile, and CeCl3·7H2O (854 mg, 2.3 mmol) was added and refluxed overnight. The reaction solution was directly prepared into sand and then subjected to column chromatography (DCM:MeOH = 100:1-200:1) to obtain a white solid 13a (466 mg, 87%). 1H NMR (300MHz, CDCl3) δ7.17-7.10(m,4H),6.84-6.77(m,4H),5.14-5.04(m,1H),4.25-4.15(m,1H),4.12-4.02(m ,2H),4.02-3.95(m,2H),3.82-3.68(m,2H),3.56-3.47(m,2H),3.01(d,J=5.7Hz,1H),1.64(s,6H),1.46(s,9H).

[0157] (4) Synthesis of 3-(4-(2-(4-(2-aminoethoxy)phenyl)prop-2-yl)phenoxy)-2-chloro-1-propanol (14a)

[0158]

[0159] Compound 13a (450 mg, 1.0 mmol) was dissolved in 5 mL of dichloromethane, and trifluoroacetic acid (1 mL, 13.1 mmol) was added under ice-water bath. The reaction was carried out at room temperature for 1 h, and the reaction solution was evaporated to dryness and added directly to the solution to obtain a brown oily substance 14a (336 mg, 95%).

[0160] (5) Synthesis of 4-((2-(4-(2-(4-(2-chloro-3-hydroxypropoxy)phenyl)prop-2-yl)phenoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (NP-12)

[0161]

[0162] Compound 14a (450 mg, 1.2 mmol) and 2-(2,6-dioxadiazin-3-yl)-4-fluoroisoindoline-1,3-dione (331 mg, 1.2 mmol) were dissolved in 10 mL of DMF, and DIPEA (1.1 mL, 6 mmol) was added. The reaction was carried out at 100 °C for 6 h. The reaction was monitored by TLC until it was complete. The reaction solution was poured into water and extracted with ethyl acetate. The organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain a crude yellow oil. Thin-layer chromatography was used to purify the crude product to obtain a yellow solid NP-12 (128 mg, 17.2%). 1H NMR (400MHz, CDCl3) δ8.22 (s, 1H), 7.51 (dd, J = 7.1, 8.5Hz, 1H), 7.15-7.09 (m, 5 H),6.98(d,J=8.5Hz,1H),6.85-6.79(m,4H),6.58(t,J=6.0Hz,1H),4.96-4.85 (m,1H),4.23-4.17(m,1H),4.15(t,J=5.4Hz,2H),4.09-4.02(m,2H),3.80-3.7 1(m,2H),3.70-3.66(m,2H),2.90-2.59(m,4H),2.15-2.02(m,1H),1.63(s,6H). 13 C NMR (75MHz, CDCl3) δ171.6,169.4,168.7,167.7,156.2,156.1,146.7,143.9,143.7,136.1,132.5,127.8,11 6.8,114.0,114.0,111.9,110.5,69.9,68.4,66.3,48.9,46.0,42.0,41.8,31.4,31.0,22.7.HRMS(ESI):m / z calcd for C 33 H 34 ClN3NaO7 + [M+Na] + ,642.1978; found,642.1968.

[0163] (6) Synthesis of 4-((3-(4-(2-(4-(3-chloro-2-hydroxypropoxy)phenyl)propyl-2-yl)phenoxy)propyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (NP-13)

[0164]

[0165] The synthesis method of compound NP-13 is the same as that of compound NP-12 mentioned above. The final product is a yellow solid NP-13 (34 mg, 22.6% yield). 1H NMR (400MHz, CDCl3) δ8.60 (s, 1H), 7.44 (dd, J = 7.1, 8.5Hz, 1H), 7.19-7.10 (m ,4H),7.07(d,J=7.0Hz,1H),6.91(d,J=8.6Hz,1H),6.88-6.75(m,4H),6.46( s,1H),4.95-4.86(m,1H),4.23-4.16(m,1H),4.10-4.01(m,4H),3.79-3.66( m,2H),3.53-3.44(m,2H),2.87-2.69(m,3H),2.17-2.03(m,3H),1.63(s,6H). 13 C NMR (75MHz, CDCl3) δ171.6,169.4,168.7,167.7,156.5,156.1,146.9,143.9,143.3,136.2,132.5,127.9,127.8,1 16.6,113.9,113.9,111.5,110.0,69.9,68.5,65.4,48.9,46.0,41.7,40.1,31.4,31.0,29.1,22.8.HRMS(ESI):m / z calcd for C 34 H 36 ClN3NaO7 + [M+Na] + ,656.2134; found,656.2130.

[0166] (7) Synthesis of 4-((4-(4-(2-(4-(3-chloro-2-hydroxypropoxy)phenyl)prop-2-yl)phenoxy)butyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (NP-14)

[0167]

[0168] The synthesis method of compound NP-14 is the same as that of compound NP-12 mentioned above. The final product is a yellow solid NP-14 (112 mg, 35.8% yield). 1H NMR (400MHz, CDCl3) δ8.10 (s, 1H), 7.51 (dd, J = 7.1, 8.5Hz, 1H), 7.21-7.08 (m, 5H), 6.92(d,J=8.5Hz,1H),6.87-6.77(m,4H),6.32(t,J=5.7Hz,1H),5.02-4.83(m,1H), 4.27-4.17(m,1H),4.14-4.04(m,2H),4.03-3.98(m,2H),3.83-3.69(m,2H),3.42- 3.35(m,2H),2.96-2.72(m,3H),2.20-2.11(m,1H),1.95-1.84(m,4H),1.66(s,6H). 13 C NMR (75MHz, CDCl3) δ171.2,169.5,168.4,167.7,156.7,156.0,146.9,144.0,143.1,136.2,132.5,127.9,127.8,116. 7,113.9,113.8,111.5,110.0,69.9,68.4,67.2,48.9,46.0,42.3,41.7,31.4,31.0,26.7,26.1,22.8.HRMS(ESI):m / z calcd for C 35 H 38 ClN3NaO7 + [M+Na] + ,670.2291; found,670.2284.

[0169] (8) Synthesis of 4-((6-(4-(2-(4-(3-chloro-2-hydroxypropoxy)phenyl)prop-2-yl)phenoxy)hexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (NP-15)

[0170]

[0171] The synthesis method of compound NP-15 is the same as that of compound NP-12 mentioned above. The final product is a yellow solid NP-15 (59 mg, 29.7% yield). 1H NMR (300MHz, CDCl3) δ8.33 (s, 1H), 7.48 (dd, J = 7.1, 8.5Hz, 1H), 7.16-7.07 (m, 5H), 6.8 8(d,J=8.5Hz,1H),6.84-6.77(m,4H),4.97-4.83(m,1H),4.23-4.16(m,1H),4.11-4.00 (m,2H),3.97-3.90(m,2H),3.83-3.64(m,2H),3.32-3.23(m,2H),2.89-2.69(m,3H),2. 14-2.08(m,1H),1.82-1.75(m,2H),1.73-1.66(m,2H),1.63(s,6H),1.55-1.45(m,4H). 13 CNMR(101MHz, CDCl3)δ171.4,169.5,168.6,167.7,156.9,156.0,147.0,144.0,142.9,136.2,132.5,127.9,127.7,116.7,11 3.9,113.8,111.4,109.8,69.9,68.4,67.6,48.9,46.0,42.6,41.7,31.4,31.0,29.7,29.2,26.8,25.9,22.8.HRMS(ESI):m / z calcdfor C 37 H 42 ClN3NaO7 + [M+Na] + ,698.2604; found,698.2598.

[0172] (9) Synthesis of 5-((4-(4-(2-(4-(3-chloro-2-hydroxypropoxy)phenyl)prop-2-yl)phenoxy)butyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (NP-16)

[0173]

[0174] The synthesis method of compound NP-16 is the same as that of compound NP-12 mentioned above. The final product is a yellow solid NP-16 (83 mg, 28.8% yield). 1H NMR (400MHz, CDCl3) δ8.20 (s, 1H), 7.58 (d, J=8.3Hz, 1H), 7.19-7.09 (m, 4H), 6.93 (d, J= 2.2Hz,1H),6.85-6.76(m,4H),6.69(dd,J=2.2,8.3Hz,1H),4.97-4.88(m,1H),4.72-4.6 5(m,1H),4.24-4.15(m,1H),4.12-4.03(m,2H),4.10-3.95(m,2H),3.80-3.67(m,2H),3 .32-3.25(m,2H),2.96-2.61(m,4H),2.14-2.07(m,1H),1.93-1.80(m,4H),1.64(s,6H). 13 C NMR (101MHz, CDCl3) δ171.2,168.5,167.9,167.4,156.6,156.0,153.6,144.0,143.3,134.7,127.9,127.8,125.6,118 .3,116.3,113.9,113.8,106.1,69.9,68.4,67.2,49.1,46.0,43.2,41.7,31.4,31.0,26.7,25.8,22.8.HRMS(ESI):m / z calcd forC 35 H 38 ClN3NaO7 + [M+Na] + ,670.2291; found,670.2293.

[0175] (10) Synthesis of 5-((4-(4-(2-(4-(3-chloro-2-hydroxypropoxy)phenyl)propyl-2-yl)phenoxy)butyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (NP-17)

[0176]

[0177] The synthesis method of compound NP-17 is the same as that of compound NP-12 mentioned above. The final product is a yellow solid NP-17 (94 mg, 25.3% yield). 1H NMR (400MHz, CDCl3) δ8.22(s,1H),7.58(d,J=8.3Hz,1H),7.17-7.10(m,4H),6.93(d,J=2.1Hz,1H),6.8 4-6.76(m,4H),6.70(dd,J=2.2,8.3Hz,1H),4.97-4.87(m,1H),4.60-4.54(m,1H),4.24-4.14(m,1H),4 .09-4.02(m,2H),3.97-3.90(m,2H),3.79-3.69(m,2H),3.26-3.17(m,2H),2.92-2.69(m,3H),2.66(d, J=6.0Hz,1H),2.15-2.07(m,1H),1.83-1.74(m,2H),1.72-1.66(m,2H),1.63(s,6H),1.55-1.43(m,4H). 13 C NMR (101MHz, CDCl3) δ171.2,168.5,168.0,167.5,156.8,156.0,153.7,144.0,142.9,134.7,127.9,127.7,125.6,118.2,11 6.3,113.9,113.8,106.1,69.9,68.4,67.6,49.1,46.0,43.5,41.7,31.5,31.0,29.2,29.0,26.7,25.9,22.8.HRMS(ESI):m / z calcd for C 37 H 42 ClN3NaO7 + [M+Na] + ,698.2604;found,698.2561.

[0178] Example 3

[0179] Route 3:

[0180]

[0181] Reaction conditions: m) Morpholine, DMF, 100℃, 16h, 90%; n) CBr4, PPh3, 25℃, 3h, 70%; o) K2CO3, DMF, 25℃, 12h, 31%-57%; p) K2CO3, DMF, 90℃, 6h, 72%-88%; q) TFA, DCM, 25℃, 2h, 80%-95%; r) DIPEA, DMF, 90℃, 12h, 11%-49%.

[0182] Synthesis of (1) (6-morpholinylpyridin-2-yl)methanol (16)

[0183]

[0184] Compound 15 (1 g, 5.3 mmol) was dissolved in 2 mL of morpholine and reacted at 100 °C for 16 h. The reaction was monitored by TLC until it ended. The reaction solution was poured into water and extracted with ethyl acetate. The organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain crude product 16 (926 mg, 90%), which was added directly without purification.

[0185] (2) Synthesis of 4-(6-(bromomethyl)pyridin-2-yl)morpholine (17)

[0186]

[0187] Compound 16 (926 mg, 4.8 mmol) and triphenylphosphine (1.3 g, 4.8 mmol) were dissolved in 10 mL of anhydrous dichloromethane. Carbon tetrabromide (1.6 g, 4.8 mmol) was slowly added under an ice-water bath. After the addition was complete, the reaction was carried out at 25 °C for 3 h. The reaction was stopped by TCL monitoring. The mixture was washed with water and extracted with dichloromethane. Column chromatography (DCM:MeOH = 400:1-500:1) gave a white solid 17 (864 mg, 70%). 1 H NMR (300MHz, DMSO-d6) δ7.59-7.51(m,1H),6.82(d,J=7.2Hz,1H),6.76(d,J=8.5Hz,1H),4.50(s,2H),3.76-3.64(m,4H),3.44(m,4H).

[0188] Synthesis of (3) tert-butyl carbamate (20a)

[0189]

[0190] Compound 11a (500 mg, 1.4 mmol) and potassium carbonate (559 mg, 4.1 mmol) were dissolved in 10 mL of DMF. Compound 17 (346 mg, 1.4 mmol) was added and the mixture was reacted at 90 °C under nitrogen protection for 6 h. After the reaction was completed, the reaction solution was poured into water and extracted with ethyl acetate. The organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness. The solution was then subjected to column chromatography (DCM:MeOH = 200:1-300:1) to obtain a brown solid 20a (500 mg, 67.8%). 1H NMR (300MHz, CDCl3) δ7.51(dd,J=7.4,8.4Hz,1H),7.16-7.11(m,4H),6.92-6.84(m,3H),6.81-6.77(m,2H),6.54(d ,J=8.4Hz,1H),5.01(s,3H),3.99(t,J=5.1Hz,2H),3.87-3.76(m,4H),3.56-3.47(m,6H),1.63(s,6H),1.45(s,9H).

[0191] (4) Synthesis of 2-(4-(2-(4-((6-morpholinylpyridin-2-yl)methoxy)phenyl)prop-2-yl)phenoxy)ethyl-1-amine (21a)

[0192]

[0193] Compound 20a (500 mg, 0.9 mmol) was dissolved in 10 mL of dichloromethane, and trifluoroacetic acid (2 mL, 26.2 mmol) was added under ice-water bath. The reaction was carried out at room temperature for 1 h, and the reaction solution was evaporated to dryness and added directly to the solution to obtain brown oily substance 21a (380 mg, 94.5%).

[0194] (5) Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-((2-(4-(2-(4-((6-morpholinylpyridin-2-yl)methoxy)phenyl)prop-2-yl)phenoxy)ethyl)amino)isoindoline-1,3-dione (NP-18)

[0195]

[0196] Compound 21a (300 mg, 0.7 mmol) and 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (194 mg, 0.7 mmol) were dissolved in 10 mL of DMF, and DIPEA (0.37 mL, 2.1 mmol) was added. The reaction was carried out at 100 °C for 6 h. The reaction was monitored by TLC until it was complete. The reaction solution was poured into water and extracted with ethyl acetate. The organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain a crude yellow oil. Thin-layer chromatography was used to purify the crude product to obtain a yellow solid NP-18 (86 mg, 18.3%). 1H NMR (300MHz, CDCl3) δ8.38 (s, 1H), 7.50 (dd, J = 7.3, 8.4Hz, 2H), 7.18-7.07 (m, 5H), 6.98(d,J=8.5Hz,1H),6.93-6.76(m,5H),6.59(t,J=5.9Hz,1H),6.53(d,J=8.5Hz,1 H),5.01(s,2H),4.95-4.86(m,1H),4.14(t,J=5.4Hz,2H),3.88-3.77(m,4H),3.74- 3.63(m,2H),3.56-3.43(m,4H),2.94-2.58(m,3H),2.15-2.04(m,1H),1.62(s,6H). 13 C NMR (75MHz, CDCl3) δ171.0,169.3,168.3,167.6,156.5,156.2,146.7,143.8,143.2,138.3,136.9,136.1,132.5,127.9,12 7.7,116.8,114.2,113.9,112.0,111.0,110.5,105.6,66.8,66.3,48.9,45.6,42.0,41.7,31.4,31.0,22.8.HRMS(ESI):m / z calcd for C 40 H 42 N5O7 + [M+H] + ,704.3079; found,704.3073.

[0197] (6) Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-((4-(4-(2-(4-((6-morpholinylpyridin-2-yl)methoxy)phenyl)prop-2-yl)phenoxy)methyl)piperidin-1-yl)isoindoline-1,3-dione (NP-19)

[0198]

[0199] The synthesis method of compound NP-19 is the same as that of compound NP-18 mentioned above. The final product is a yellow solid NP-19 (64 mg, 24.8% yield). 1H NMR (400MHz, CDCl3) δ8.51 (s, 1H), 7.62-7.49 (m, 2H), 7.39 (d, J = 7.1Hz, 1H) ,7.22-7.13(m,5H),6.93-6.87(m,3H),6.85-6.80(m,2H),6.56(d,J=8.4Hz, 1H),5.04(s,2H),5.02-4.96(m,1H),3.89-3.78(m,8H),3.55-3.52(m,4H), 3.01-2.66(m,5H),2.17-2.08(m,1H),2.07-1.95(m,3H),1.71-1.61(m,8H). 13 C NMR (101MHz, CDCl3) δ171.3,168.4,167.5,166.7,159.1,156.8,156.5,155.8,150.9,143.4,143.2,138.3,135.5,134.1,127.8,127.7,123 .7,117.3,115.4,114.2,113.8,111.0,105.6,72.3,70.9,66.8,51.7,51.4,49.1,45.6,41.7,35.7,31.4,31.1,29.2,22.7.HRMS(ESI):m / z calcd for C 44 H 48 N5O7 + [M+H] + ,758.3548;found,758.3536.

[0200] (7) Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(3-(4-(2-(4-((6-morpholinylpyridin-2-yl)methoxy)phenyl)prop-2-yl)phenoxy)azacyclobutane-1-yl)isoindoline-1,3-dione (NP-20)

[0201]

[0202] The synthesis method of compound NP-20 is the same as that of compound NP-18 mentioned above. The final product is a yellow solid NP-20 (124 mg, 33.9% yield). 1H NMR (400MHz, CDCl3) δ8.60 (s, 1H), 7.56-7.44 (m, 2H), 7.22 (d, J = 7.0Hz, 1H), 7.19-7.12 ( m,4H),3.94-3.87(m,3H),6.74-6.66(m,2H),6.64(d,J=8.4Hz,1H),6.56(d,J=8.4Hz,1H ),5.07-5.00(m,3H),4.99-4.90(m,1H),4.76-4.65(m,2H),4.27(dd,J=4.1,9.9Hz,2H), 3.89-3.79(m,4H),3.58-3.49(m,4H),2.92-2.66(m,3H),2.16-2.05(m,1H),1.65(s,6H). 13 C NMR (101MHz, CDCl3) δ171.4,168.5,167.6,166.9,159.1,156.6,155.7,154.3,147.6,144.2,143.1,138.3,134.9,133.8,128.1,1 27.7,119.5,114.2,114.0,113.0,111.1,111.0,105.6,70.9,66.8,66.0,61.0,49.0,45.6,41.7,31.4,31.1,22.7.HRMS(ESI):m / z calcd for C 41 H 42 N5O7 + [M+H] + ,716.3079;found,716.3071.

[0203] Example 4

[0204] Route 4:

[0205]

[0206] Reaction conditions: s) HATU, DMF, 60℃, 12h, 42%-82%; t) K2CO3, DMF, 90℃, 6h, 64%-86%; u) TFA, DCM, 25℃, 2h, 78%-94%; v) DIPEA, DMF, 90℃, 12h, 12%-46%.

[0207] Synthesis of (1) tert-butyl carbamate (24a)

[0208]

[0209] Compound 5b (500 mg, 1.1 mmol) and potassium carbonate (456 mg, 3.3 mmol) were dissolved in 10 mL of DMF. 17 (414 mg, 1.5 mmol) was added and the mixture was reacted at 90 °C under nitrogen protection for 6 h. After the reaction was completed, the reaction solution was poured into water and extracted with ethyl acetate. The organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness. Column chromatography (DCM:MeOH = 200:1-300:1) was performed to give a white solid 24a (500 mg, 73.5%). 1 H NMR (300MHz, CDCl3) δ7.54-7.46(m,1H),7.25-7.06(m,5H),6.91-6.79(m,5H),6.54(d,J=8.4Hz,1H),5.04-4.91(m,3H), 4.46(s,2H),3.90-3.71(m,4H),5.44-3.46(m,4H),3.43-3.33(m,2H),3.18-3.08(m,2H),1.71-1.57(m,8H),1.43(s,9H).

[0210] (2) Synthesis of N-(2-aminoethyl)-2-(4-(2-(4-((6-morpholinylpyridin-2-yl)methoxy)phenyl)prop-2-yl)phenoxy)acetamide (25a)

[0211]

[0212] Compound 24a (500 mg, 0.8 mmol) was dissolved in 10 mL of dichloromethane, and trifluoroacetic acid (2 mL, 26.2 mmol) was added under ice-water bath conditions. The mixture was reacted at room temperature for 1 h, and the reaction solution was evaporated to dryness and added directly to the solution to obtain a brown oily substance 25a (350 mg, 86.8%).

[0213] (3) Synthesis of N-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)propyl)-2-(4-(2-(4-((6-morpholinylpyridin-2-yl)methoxy)phenyl)propyl-2-yl)phenoxy)acetamide (NP-21)

[0214]

[0215] Compound 25a (300 mg, 0.6 mmol) and 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (160 mg, 0.6 mmol) were dissolved in 10 mL of DMF, and DIPEA (0.32 mL, 1.8 mmol) was added. The reaction was carried out at 100 °C for 6 h. The reaction was monitored by TLC until it was complete. The reaction solution was poured into water and extracted with ethyl acetate. The organic phase was washed three times with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain a crude yellow oil. Thin-layer chromatography was used to purify the crude product to obtain a yellow solid NP-21 (57 mg, 12.3%). 1 H NMR(400MHz, CDCl3)δ8.79(s,1H),7.53-7.39(m,2H),7.17-7.02(m,5H),6.92-6.7 4(m,7H),6.53(d,J=8.5Hz,1H),6.44(t,J=5.9Hz,1H),5.00(s,2H),4.93-4.85(m, 1H),4.46(s,2H),3.85-3.77(m,4H),3.52-3.47(m,4H),3.47-3.41(m,2H),3.34-3 .25(m,2H),2.86-2.60(m,3H),2.06-2.01(m,1H),1.91-1.81(m,2H),1.61(s,6H). 13 C NMR (101MHz, CDCl3) δ171.6,169.4,169.0,168.7,167.6,159.1,156.6,155.7,155.0,146.6,144.8,142.9,138.3,136.2,132.6,128.1,127 .7,116.5,114.3,114.1,111.5,111.0,110.2,105.6,70.9,67.4,66.8,48.9,45.6,41.8,39.8,36.5,31.4,31.0,29.2,22.7.HRMS(ESI):m / z calcd for C 43 H 47 N6O8 + [M+H] + ,775.3450; found,775.3418.

[0216] (4) Synthesis of N-(2-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)aminoethoxy)ethoxy)ethyl)-2-(4-(2-(4-((6-morpholinylpyridin-2-yl)methoxy)phenyl)prop-2-yl)phenoxy)acetamide (NP-22)

[0217]

[0218] The synthesis method for compound NP-22 is the same as that for compound NP-20 described above. The final product was a yellow solid NP-22 (128 mg, 33.5% yield). 1 H NMR(300MHz, CDCl3)δ8.91(s,1H),7.53-7.40(m,2H),7.18-7.04(m,6H),6.90-6.8 2(m,4H),6.82-6.76(m,2H),6.53(d,J=8.4Hz,1H),6.46(t,J=5.6Hz,1H),5.01(s, 2H),4.94-4.85(m,1H),4.45(s,2H),3.84-3.77(m,4H),3.71-3.45(m,19H),3.45- 3.38(m,2H),2.87-2.72(m,2H),2.72-2.64(m,1H),2.11-2.02(m,1H),1.60(s,6H). 13 C NMR (101MHz, CDCl3) δ171.5,169.3,168.7,168.6,167.7,158.9,156.6,155 .6,155.1,146.8,144.6,143.0,138.4,136.0,132.5,128.0,127.7,116.8,1 14.2,114.2,111.6,110.9,110.3,105.7,70.7,70.6,70.6,70.3,69.7,69.5 ,67.5,66.7,48.9,45.6,42.4,41.7,38.8,31.4,31.0,22.8.HRMS(ESI):m / z calcd for C 48 H 56 N6O 11 + [M+H] + ,915.3899;found,915.3889.

[0219] (5) Synthesis of N-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperidin-4-yl)-2-(4-(2-(4-((6-morpholinylpyridin-2-yl)methoxy)phenyl)prop-2-yl)phenoxy)acetamide (NP-23)

[0220]

[0221] The synthesis method of compound NP-23 is the same as that of compound NP-20 mentioned above. The final product is a yellow solid NP-23 (113 mg, 31.2% yield). 1 H NMR (300MHz, CDCl3) δ8.38 (s, 1H), 7.62-7.45 (m, 2H), 7.39 (d, J = 7.1Hz, 1H), 7.21-7.05 (m ,5H),6.94-6.74(m,5H),6.59(d,J=8.3Hz,1H),6.53(d,J=8.4Hz,1H),5.05-4.90(m,3H), 4.47(s,2H),4.17-4.00(m,1H),3.89-3.77(m,4H),3.75-3.62(m,2H),3.55-3.43(m,4H), 3.09-2.95(m,2H),2.92-2.66(m,3H),2.21-2.02(m,3H),1.85-1.72(m,2H),1.63(s,6H). 13 C NMR (101MHz, CDCl3) δ171.4,168.5,167.9,167.4,166.8,159.1,156.6,155 .7,155.0,150.4,144.8,143.0,138.3,135.7,134.1,128.1,127.7,123.8,1 17.7,115.8,114.2,114.1,111.0,105.6,77.5,77.1,76.8,70.9,67.4,66.8 ,50.5,49.2,45.8,45.6,41.8,32.1,31.4,31.0,29.7,22.7.HRMS(ESI):m / z calcd for C 45 H 49 N6O8 + [M+H] + ,801.3606;found,801.3602.

[0222] (6) Synthesis of N-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazin-1-yl)ethyl)-2-(4-(2-(4-((6-morpholinylpyridin-2-yl)methoxy)phenyl)prop-2-yl)phenoxy)acetamide (NP-24)

[0223]

[0224] The synthesis method of compound NP-24 is the same as that of compound NP-20 mentioned above. The final product is a yellow solid NP-24 (101 mg, 31.2% yield).1 H NMR (400MHz, CDCl3) δ8.72(s,1H),7.57(t,J=7.8Hz,1H),7.49(t,J=7.9Hz,1H),7.39(d,J=7. 2Hz,1H),7.18-7.07(m,6H),6.89-6.78(m,5H),6.52(d,J=8.4Hz,1H),4.99(s,2H),4.97-4.90 (m,1H),4.49(s,2H),3.85-3.77(m,4H),3.54-3.48(m,4H),3.48-3.43(m,2H),3.34-3.21(m, 4H),2.88-2.70(m,3H),2.68-2.62(m,4H),2.60-2.54(m,2H),2.11-2.05(m,1H),1.60(s,6H). 13 C NMR (101MHz, CDCl3) δ171.3,168.4,168.4,167.3,166.7,159.1,156.6,1 55.7,155.1,150.2,144.7,142.9,138.2,135.7,134.2,128.0,127.6,12 3.4,117.5,115.9,114.2,114.1,110.9,105.6,70.9,67.5,66.8,56.3,5 2.7,51.0,49.2,45.6,41.7,35.4,31.4,31.0,29.7,22.7.HRMS(ESI):m / z calcd forC 46 H 52 N7O8 + [M+H] + ,830.3872;found,830.3863.

[0225] Example 5: Western blot analysis of the effect of the compound on AR / AR-V7 protein expression in 22Rv1 cells

[0226] 1. Experimental Methods

[0227] After culturing 22Rv1 / LnCaP cells in 6-well plates for 24 h, the old culture medium was discarded, and culture medium containing the compound sample was added for another 24 h. The culture medium was then discarded, and the cells were washed twice with PBS. Cells were collected in 2 mL EP tubes, and 30–100 μL of RIPA lysis buffer (containing 1% PMSF) was added according to the cell count. The cells were dispersed and lysed on ice for 30 min, vortexing every 10 min. After lysis, the cells were centrifuged at 12000 rpm, 4 °C for 15 min, and the supernatant was collected. The protein was quantified using a BCA protein quantification kit, diluted to the same concentration, and loading buffer was added. The diluted protein was placed in boiling water for 10 min to denature it. An equal volume of protein was added to the sample wells of a pre-prepared gel, concentrated at 60 V for 30 min, and separated by electrophoresis at 120 V until bromophenol blue was detected. Cut a PVDF membrane to the same size as the gel, pre-activate it by soaking it in methanol and ultrapure water for 1 min each, then place the PVDF membrane on top of the gel, with a layer of sponge and a layer of filter paper on top and bottom. Transfer the protein from the gel to the PVDF membrane at a constant current of 300 mA for 75 min. After transfer, place the PVDF membrane in TBST solution containing 10% skim milk powder and block at room temperature for 2 h. Incubate with primary antibody: 4°C for 12 h. After completion, wash the membrane three times with TBST solution for 10 min each time. Incubate at room temperature for 1 h. After completion, wash the membrane three times with TBST solution for 10 min each time. Perform imaging analysis using the Bio-Rad gel imaging system.

[0228] 2. Experimental Results

[0229] like Figure 1 As shown, the previously reported AR / AR-V7 NTD antagonist EPI-001 had no significant effect on the content of AR and AR-V7 proteins, while the compounds NP-1 to NP-24 of this invention exhibited varying degrees of degradation activity against AR and AR-V7 at 1 μM and 5 μM, especially compound NP-18, which almost completely degraded AR and AR-V7 at a concentration of 1 μM (degradation rates of 90.1 ± 0.03% and 81.8 ± 0.09%, respectively). Further testing of the degradation efficacy of the preferred compound NP-18 against the target protein in 22Rv1 yielded AR-FLDC. 50 =0.018±0.013μM, AR-V7 DC 50 =0.026±0.013μM ( Figure 2 A) AR-FL DC in LnCaP 50 =0.14±0.08μM ( Figure 2 B).

[0230] Example 6: Detection of the antiproliferative activity of the compound against 22Rv1 cells using the CCK-8 assay

[0231] 1. Experimental Methods

[0232] 22Rv1 / LnCaP cells in good logarithmic growth phase were seeded in 96-well plates with cell suspension (100 μL / well). The plates were pre-cultured in an incubator (37℃, 5% CO2) for 12-24 hours. The culture medium was then removed from each well, and 200 μL of the test drug dissolved in the medium was added to each well. After 4 days of drug administration, the culture medium was discarded, and 200 μL of fresh culture medium and 20 μL of CCK-8 solution were added to each well. The 96-well plates were incubated for 2 hours. The absorbance was measured at 450 nm using a microplate reader, and cell survival curves were plotted to determine the IC50 of the compound on the proliferation of 22Rv1 / LnCaP cells. 50 .

[0233] 2. Experimental Results

[0234] As shown in Table 1, NP-18, a compound with good degradation activity against AR / AR-V7, also exhibits excellent anti-proliferative activity, with an IC50 value in the 22Rv1 cell line. 50 The value reached 0.038±0.018, which was better than the positive control EPI-002.

[0235] Table 1. Antiproliferative activity IC50 of the compounds in 22Rv1 cells 50 result

[0236]

[0237] Example 7: Drug efficacy experiment in nude mice bearing human prostate cancer cells 22Rv1

[0238] 1. Experimental Methods

[0239] Logarithmic growth phase human prostate cancer (22Rv1) cells were cultured in complete culture medium. Before passage, cells were digested with 0.25% trypsin-EDTA, centrifuged at 1000 rpm for 5 min, and collected. Cell density was adjusted with complete culture medium, and cells were passaged at a ratio of 1:3. Cell inoculation: Fifteen qualified male BALB / c-nu mice were selected, and tumor cells were subcutaneously inoculated into their backs at a number of 2 × 10⁶ cells. 6 Each mouse was selected as a group. After tumor growth, once the diameter reached 5-7 mm, 15 mice with uniformly grown tumors were randomly and evenly divided into 3 groups of 5 animals each, based on tumor volume and body weight. Grouping and drug administration details are shown in Table 2. During the drug administration observation period, the diet and mental state of all animals were recorded daily. Mice were weighed every 2 days, and the major diameter (a) and minor diameter (b) of the tumor were measured every 2 days using calipers. The tumor volume (TV) was calculated using the following formula: TV = 1 / 2 × a × b 2Where a and b represent the length and width of the tumor, respectively. At the end of the observation period, the tumor was removed. Before euthanasia, a photograph was taken, the tumor was weighed, and the tissue was fixed and preserved for immunohistochemical detection of AR and AR-V7 protein expression.

[0240] Table 2.22 Summary of Rv1 tumor-bearing nude mouse efficacy test groups and dosage designs

[0241]

[0242] 2. Experimental Results

[0243] like Figure 3 As shown, after 21 days of administration of compound NP-18, tumor growth in the treated group was significantly slowed down. Figure 3 The results (A-3C) indicate that compound NP-18 has an inhibitory effect on the growth of 22Rv1 cell xenograft tumors, and is superior to the positive control EPI-002. Furthermore, compound NP-18 has minimal impact on mouse body weight during the dosing cycle, suggesting that NP-18 has better safety than EPI-002. Figure 3 D). Furthermore, NP-18 exerts its anti-tumor effect in vivo by effectively degrading AR / AR-V7. Figure 3 E).

Claims

1. A compound based on the PROTAC strategy, characterized in that, It has the structure of Formula I: I， in: R is selected from ; L is selected from or n is an integer selected from 2 to 6; D is selected from or .

2. A compound based on the PROTAC strategy, characterized in that, Compounds selected from any of the following: 。 3. A pharmaceutically acceptable salt, characterized in that, The compound of claim 1 is formed by reacting with an acid or a base, wherein the acid is selected from hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, or ferulic acid, and the base is selected from alkali metal cationic bases, alkaline earth metal cationic bases, ammonium cationic bases, or choline.

4. A method for preparing the compound according to claim 1, characterized in that, Choose from any of the following methods: Method 3: ； (m) Compound 12 is substituted with morpholine to generate compound 13; (n) Compound 13 reacts with carbon tetrabromide to give hydroxybromoproduct 14; (o) Compound 1 was substituted with a terminal bromolinker to give compound 15; (p) Compound 15 is substituted with compound 14 to give compound 16; (q) Compound 16 was deprotected from its Boc protecting group in trifluoroacetic acid to give compound 17; (r) Compound 17 undergoes a substitution reaction to produce compound I; Method 4: ； (s) Compound 3 was amide condensed with a terminal amino linker to give compound 18; (t) Compound 18 and compound 14 undergo a substitution reaction to give compound 19; (u) Compound 19 was deprotected from its Boc protecting group in trifluoroacetic acid to give compound 20; (v) Compound 20 undergoes a substitution reaction to produce compound I; Wherein, L and D are defined as described in claim 1; The pharmaceutically acceptable salt of the compound is obtained by salting the compound I prepared by the above method with the corresponding acid or base.

5. A pharmaceutical composition, characterized in that, It comprises the compound of claim 1 or 2 or the pharmaceutically acceptable salt of claim 3 and a pharmaceutically acceptable carrier.

6. Use of a compound according to claim 1 or 2 or a pharmaceutical composition according to claim 5 in the preparation of a medicament for degrading AR / AR-V7 protein.

7. The application according to claim 6, characterized in that, The drug in question is for the treatment of castration-resistant prostate cancer.

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