Degradation agent of targeted Smurf1 protein, preparation method and application

Abstract

The invention discloses a degradation agent targeting Smurf1 protein as well as a preparation method and application of the degradation agent. The compound as shown in the formula (I) can degrade Smurf1 protein. The compound provided by the invention can be used for preparing a treatment compound for treating diseases caused by Smurf1 protein accumulation, can be used for treating various cancers, and particularly can be used as a treatment compound for treating colorectal cancer. # imgabs0 #

Description

Technical Field

[0001] The present application relates to the field of medicinal chemistry, and specifically to a degradation agent targeting Smurf1 protein, a preparation method and use thereof. Background Art

[0002] HECT ubiquitin ligase Smurf1 is expressed in many organs including bones, cartilage, heart, lungs, nervous system and reproductive organs, and is expressed in all developmental stages from embryo to adult, indicating that Smurf1 plays a comprehensive role in its physiological regulation. Depending on the different tissue cell environments, Smurf1 can selectively regulate the degradation of its key regulatory factors Smad1 / 5, BMP receptors, MEKK2, RhoA, RUNX2, Prickle1, Jun-B, hPEM-2, TRAFs, ING2 and other proteins, and play different roles in pathways such as bone morphogenetic protein (BMP), Wnt and MEKK-JNK. Studies have found that Smurf1 is highly expressed in various tumor tissues such as colorectal cancer, and is closely related to the poor prognosis of tumor patients. Inhibiting Smurf1 expression using siRNA and other technologies can effectively inhibit the proliferation and migration of tumor cells and other malignant behaviors. Given that Smurf1 gene knockout mice survive and no other obvious diseases are observed, the risk of potential side effects of targeted regulation of Smurf1 expression is low, so Smurf1 is an ideal target for tumor treatment. Summary of the invention

[0003] The purpose of the present application is to provide a degradation agent targeting Smurf1 protein, a preparation method and use thereof, which can solve the above-mentioned technical problems.

[0004] The present application provides a compound represented by formula (I):

[0005]

[0006] In some embodiments, the compound is capable of degrading Smurf1 protein.

[0007] The examples of the present application further provide a method for preparing the compound represented by formula (I), comprising the following steps:

[0008] 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione and piperidine-3-carboxylic acid were used to synthesize intermediate 1

[0009] Intermediate 2 was synthesized by combining intermediate 1 with 3-ethynylaniline

[0010] The intermediate 3 was synthesized by reacting 4-(4-aminophenoxy)-N-methylpicolinamide with azidotrimethylsilane.

[0011]

[0012] Intermediate 2 and intermediate 3 are combined to synthesize the compound represented by formula (I).

[0013] The embodiments of the present application further provide a pharmaceutical composition, which comprises the above-mentioned compound or a compound prepared by the above-mentioned method for preparing the compound.

[0014] In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier.

[0015] In some embodiments, the dosage form of the pharmaceutical composition includes at least one of tablets, capsules, powders, granules, injections, suspensions, solutions, creams, suppositories, gels, aerosols, sprays, and powder sprays.

[0016] The embodiments of the present application further provide the use of the above-mentioned compound, its pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate, polymorph, compound prepared by the preparation method of the above-mentioned compound, and the above-mentioned pharmaceutical composition in the preparation of drugs for treating diseases caused by Smurf1 protein accumulation.

[0017] The embodiments of the present application further provide the use of the above-mentioned compounds, their pharmaceutically acceptable salts, enantiomers, stereoisomers, solvates, polymorphs, compounds prepared by the preparation methods of the above-mentioned compounds, and the above-mentioned pharmaceutical compositions in the preparation of drugs for treating cancer.

[0018] In some embodiments, the cancer includes colorectal cancer, gastric cancer, lung cancer, cervical cancer, liver cancer, pancreatic cancer, and breast cancer.

[0019] The embodiments of the present application further provide a degradation agent targeting Smurf1 protein, including the above-mentioned compound, its pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate, polymorph, compound prepared by the preparation method of the above-mentioned compound, and the above-mentioned pharmaceutical composition.

[0020] The beneficial effects of the present application are as follows: the present application provides a compound represented by formula (I), and conducts functional studies and safety evaluations on the compound, and the results show that the compound of the present application can be used as a therapeutic compound for cancer, especially colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 For compound SMART1 1 H-NMR spectrum;

[0023] Figure 2 For compound SMART1 13 C-NMR spectrum;

[0024] Figure 3 The SPR test results of the compounds SMART1 and Smurf1 binding to CRBN protein;

[0025] Figure 4 The immunoprecipitation results of compound SMART1 show that in the presence of compound SMART1, it can bind to Smurf1 and CRBN to form a target protein-PROTAC-E3 ligase ternary complex;

[0026] Figure 5 The results of the experiment on the specific degradation of Smurf1 induced by compound SMART1, wherein, Figure a shows that compound SMART1 can induce the specific degradation of Smurf1, but has no degradation effect on Smurf2 and β-actin, Figure b shows the DC50 test result of compound SMART1 degrading Smurf1, and Figure c shows the DIA test result of compound SMART1 inducing the specific degradation of Smurf1;

[0027] Figure 6 The functional experimental results of compound SMART1, wherein Figure a is the IC50 test results of compound SMART1 on different cells, Figure b is the inhibition results of compound SMART1 on HCT116 cells, and Figure c is the cell cycle test results of compound SMART1 blocking HCT116;

[0028] Figure 7 The pharmacokinetic experimental results of compound SMART1;

[0029] Figure 8 The results of the nude mouse experiment of compound SMART1, wherein Figure a is the body weight of nude mice before the experiment, Figure b is the volume of subcutaneous tumors of nude mice after subcutaneous injection, Figure c is the volume of subcutaneous tumors in the solvent control group and the experimental group, and Figure d is the statistical results of the weight of subcutaneous tumors in the solvent control group and the experimental group;

[0030] Fig. 9 Figure 5 is the safety experiment result of compound SMART1, where a is the setting parameters of the safety test, b is the weight change results of mice in different treatment groups, c is the organ morphology of mice in different treatment groups, d is the HE staining results of organ tissue of mice in different treatment groups, e is the organ weight change of mice in different treatment groups, f is the blood test results of mice in different treatment groups, WBC is white blood cell, RBC is red blood cell, PLT is platelet, NE is neutrophil, LY is lymphocyte, g is the liver and kidney function test results of different treatment groups, where ALT is alanine aminotransferase, GLU is fasting blood glucose, ALB is plasma albumin, TBIL is bilirubin, UREA is urea, and CRE is blood creatinine. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present application. In addition, in the description of the present application, the term "including" means "including but not limited to". The terms first, second, third, etc. are used only as markings, and no numerical requirements or order are imposed. Various embodiments of the present application may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and simplicity, and should not be understood as a hard limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values ​​within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the numbered range, such as 1, 2, 3, 4, 5 and 6, which are applicable regardless of the range. Additionally, whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.

[0032] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art in the field described in this application. Although only preferred methods and materials are described in this application, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail. Unless otherwise specified, "%" is a percentage based on weight.

[0033] PROteolysis Targeting Chimera (PROTAC) is an artificially constructed macromolecular complex tool. One end of it is a ligand that binds to ubiquitin ligase, and the other end is a ligand that allows the target protein in the cell to bind, and the middle is connected by a Linker. PROTAC can induce the target protein to be ubiquitinated and then degraded through the ubiquitin-proteasome pathway. This technology uses the cell's own degradation mechanism to degrade pathogenic proteins. It can be used as a new treatment method for diseases such as cancer, autoimmune diseases and neurological diseases, and has great application prospects. Traditional small molecule compounds inhibit the function of proteins by occupying the active area of ​​the target protein, while protein targets that do not contain protein binding active areas are considered to be "undruggable targets" or "difficult to drug targets", such as transcription factors, scaffold proteins, etc. Unlike traditional small molecule drugs, PROTAC drugs do not need to bind tightly and for a long time to pathogenic targets, and have the advantage of degrading "undruggable" targets. According to existing data, it is estimated that 80% of the current "undruggable" targets can be targeted by PROTAC molecules. In addition, PROTAC technology has a series of advantages such as substrate selectivity, tissue specificity and overcoming drug resistance. The present application obtains PROTAC-type compounds through synthetic screening. The compounds are rationally designed and synthesized using derivatives of Smurf1 inhibitors and CRBN ligands, and are screened using NanoLuc technology. The experimental results show that the ROTAC compounds screened by the present application have good affinity for both the target protein Smurf1 and the E3 ligase CRBN. The compound effectively ubiquitinates the Smurf1 protein and degrades it through the proteasome pathway by forming a ternary complex in the cell, effectively inhibiting tumor growth, indicating that the compound can be used as a degrader of Smurf1 protein, and it can be used as a therapeutic drug for diseases caused by the accumulation of Smurf1 protein. The applicant conducted functional studies and safety evaluations on the compound, confirming that it can be used as a potential drug for the treatment of various cancers, especially colorectal cancer.

[0034] The structure of the compound of the present application is shown in formula (I):

[0035]

[0036] In some embodiments, the compound represented by formula (I) is capable of degrading Smurf1 protein.

[0037] In some embodiments, the compound represented by formula (I) is prepared by the following method:

[0038] 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione and piperidine-3-carboxylic acid were used to synthesize intermediate 1

[0039] Intermediate 2 was synthesized by combining intermediate 1 with 3-ethynylaniline

[0040] The intermediate 3 was synthesized by reacting 4-(4-aminophenoxy)-N-methylpicolinamide with azidotrimethylsilane.

[0041]

[0042] Intermediate 2 and intermediate 3 are combined to synthesize the compound represented by formula (I) (compound SMART1).

[0043] In some embodiments, the preparation route of the above compounds is as follows:

[0044]

[0045] In some embodiments, in the synthesis step of intermediate 1: the molar ratio of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione to piperidine-3-carboxylic acid is 1:(1-2), such as the molar ratio of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione to piperidine-3-carboxylic acid is 1:1.5.

[0046] In some embodiments, in the synthesis step of intermediate 1: the solvent system in which the reactants are located includes DMSO.

[0047] In some embodiments, in the synthesis step of intermediate 1: DIPEA (N,N-diisopropylethylamine) is also present in the reaction system.

[0048] In some embodiments, the molar ratio of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione to DIPEA is 1:(1-3), such as the molar ratio of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione to DIPEA is 1:3.

[0049] In some embodiments, during the synthesis of intermediate 1, the reaction temperature is 85-95°C. For example, the temperature can be any value among 85, 90, 95 or a range consisting of any two values. In a specific embodiment, 90°C can be selected as the synthesis reaction temperature of intermediate 1.

[0050] In some embodiments, the reaction time of intermediate 1 can be 5 to 10 hours. For example, the reaction time can be any value of 5, 6, 7, 8, 9, 10 or a range consisting of any two values. In a specific embodiment, the reaction time can be 6 hours.

[0051] In some embodiments, in the synthesis step of intermediate 2: the molar ratio of intermediate 1 to 3-ethynylaniline is 1:(1-2), such as the molar ratio of intermediate 1 to 3-ethynylaniline is 1:1.2.

[0052] In some embodiments, in the synthesis step of intermediate 2: the solvent system in which the reactants are located includes DMF (dimethylformamide) and DCM (dichloromethane), and the volume ratio of DMF to DCM is 1:(5-10), such as the volume ratio of DMF to DCM is any value of 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or a range consisting of any two values.

[0053] In some embodiments, in the synthesis step of intermediate 2: HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate) and DIPEA are also present in the reaction system.

[0054] In some embodiments, the molar ratio of intermediate 1 to HATU is 1:(1-3), such as the molar ratio of intermediate 1 to HATU is any value of 1:1, 1:1.5, 1:2, 1:2.5, 1:3, or a range consisting of any two values.

[0055] In some embodiments, the molar ratio of intermediate 1 to DIPEA is 1:(1-3), such as the molar ratio of intermediate 1 to DIPEA is any value of 1:1, 1:1.5, 1:2, 1:2.5, 1:3 or a range consisting of any two values.

[0056] In some embodiments, in the synthesis of intermediate 2, the reaction temperature is room temperature.

[0057] In some embodiments, the reaction time of intermediate 2 can be 10 to 15 hours. For example, the reaction time can be any value of 10, 11, 12, 13, 14, 15 or a range consisting of any two values. In a specific embodiment, the reaction time can be 12 hours.

[0058] In some embodiments, in the synthesis step of intermediate 3: the molar ratio of 4-(4-aminophenoxy)-N-methylpicolinamide to azidotrimethylsilane (TMSN3) is 1:(1-5), such as the molar ratio of 4-(4-aminophenoxy)-N-methylpicolinamide to azidotrimethylsilane is any value of 1:1, 1:2, 1:3, 1:4, 1:5 or a range consisting of any two values.

[0059] In some embodiments, in the synthesis step of intermediate 3: the solvent system in which the reactants are located includes DMF and ACN (acetonitrile), and the volume ratio of DMF to ACN is 1:(1-5), such as the volume ratio of DMF to ACN is any value of 1:1, 1:2, 1:3, 1:4, 1:5 or a range consisting of any two values.

[0060] In some embodiments, in the synthesis step of intermediate 3: tert-butyl nitrite (t-BuONO) is also present in the reaction system.

[0061] In some embodiments, the molar ratio of 4-(4-aminophenoxy)-N-methylpicolinamide to t-BuONO is 1:(1-5), such as the molar ratio of 4-(4-aminophenoxy)-N-methylpicolinamide to t-BuONO is any value of 1:1, 1:2, 1:3, 1:4, 1:5, or a range consisting of any two values.

[0062] In some embodiments, in the synthesis of intermediate 3, the reaction temperature is 45-55° C. For example, the reaction temperature is any value among 45° C., 50° C., 55° C., or a range consisting of any two values.

[0063] In some embodiments, the reaction time of intermediate 3 can be 5 to 10 hours. For example, the reaction time can be any value of 5, 6, 7, 8, 9, 10 or a range consisting of any two values. In a specific embodiment, the reaction time can be 6 hours.

[0064] In some embodiments, in the synthesis steps of compound SMART1: the molar ratio of intermediate 2 to intermediate 3 is 1:(1-3), such as the molar ratio of intermediate 2 to intermediate 3 is any value of 1:1, 1:2, 1:3 or a range consisting of any two values.

[0065] In some embodiments, in the synthesis step of compound SMART1: the solvent system in which the reactants are located includes methanol. In other embodiments, the solvent system also includes water.

[0066] In some embodiments, in the synthesis step of compound SMART1: CuSO4 is also present in the reaction system. The molar ratio of intermediate 2 to CuSO4 is 1:(1-3), and the molar ratio of intermediate 2 to CuSO4 is any value of 1:1, 1:2, 1:3 or a range consisting of any two values.

[0067] In some embodiments, in the synthesis step of compound SMART1: a sodium citrate solution is also present in the reaction system.

[0068] In some embodiments, the concentration of the sodium citrate solution is 1 mol / L. The molar ratio of the intermediate 2 to the sodium citrate solution is 1:(1-5), and the molar ratio of the intermediate 2 to the sodium citrate solution is any value of 1:1, 1:2, 1:3, 1:4, 1:5 or a range consisting of any two values.

[0069] In some embodiments, in the synthesis step of compound SMART1, the reaction temperature is room temperature.

[0070] In some embodiments, in the synthesis step of compound SMART1, the reaction time can be 10 to 15 hours. For example, the reaction time can be any value of 10, 11, 12, 13, 14, 15 or a range consisting of any two values. In a specific embodiment, the reaction time can be selected as 12 hours.

[0071] Pharmaceutical composition

[0072] The pharmaceutical composition described in the present application includes a compound as shown in formula (I). The mass proportion of the compound shown in formula (I) in the pharmaceutical composition can be 0.001 to 99.9wt%, such as based on the total mass of the pharmaceutical composition, the mass proportion (%) of the compound shown in formula (I) in the pharmaceutical composition is 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, any value or a range consisting of any two values.

[0073] The pharmaceutical composition also includes a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, and such pharmaceutically acceptable material, composition or carrier participates in carrying or transporting the drug from one organ or part of the body to another organ or another part of the body. The mass proportion of the pharmaceutically acceptable carrier in the pharmaceutical composition can be 0.001 to 99.9wt%. For example, based on the total mass of the pharmaceutical composition, the mass proportion (%) of the pharmaceutically acceptable carrier in the pharmaceutical composition is any value of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or a range consisting of any two values.

[0074] The above-mentioned carriers must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

[0075] In some embodiments, pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives and analogs such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl lauryl ester; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; and other nontoxic and compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate, magnesium stearate, and polyoxyethylene-polypropylene oxide copolymers, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0076] Dosage

[0077] In the present application, the pharmaceutical composition can be administered to the subject / patient in any suitable dosage form, and the administration method is not particularly limited. Representative administration methods include but are not limited to: oral administration, intramuscular injection, intravenous injection, intravenous drip, enema, spray, external application, or intraperitoneal injection.

[0078] In the present application, the subject / patient refers to a vertebrate, preferably a mammal, including but not limited to mice, apes, livestock, humans, etc., preferably humans.

[0079] In the present application, examples of dosage forms of the pharmaceutical composition include, but are not limited to, tablets, capsules, powders, granules, injections, suspensions, solutions, creams, suppositories, gels, aerosols, sprays, and powder sprays.

[0080] Example 1: Synthesis of compounds targeting degradation of Smurf1 protein

[0081] Intermediate 1: 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperidine-3-carboxylic acid (1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)

[0082] Synthesis of piperidine-3-carboxylic acid):

[0083]

[0084] 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (1.1 g, 4 mmol, 1 equiv) was dissolved in DMSO (10 mL), and piperidine-3-carboxylic acid (0.775 g, 6 mmol, 1.5 eq uiv) and N,N-diisopropylethylamine (DIPEA) (1.55 g, 12 mmol, 3.0 equiv) were added to a round-bottom flask. The mixture was heated to 90 ° C and stirred for 6 h. The reaction was monitored by TLC until the reaction was complete. 30 mL of water was added to the residue and extracted with ethyl acetate (3×30 mL). The organic layer was then washed with brine and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (DCM: MeOH = 200: 1-50: 1) to give the intermediate 1-(2-(2,6-dioxopiperidin-3-yl))-1,3-dioxoisoindolin-4-yl)piperidine-3-carboxylic acid (1.0 g, yellow solid, yield 65%).

[0085] 1 HNMR (400MHz, DMSO-d6) δ = 12.33 (s, 1H), 11.07 (s, 1H), 7.69 (dd, J = 8.2, 7.3Hz, 1H),7.36(t,J=8.0Hz,2H),5.11(dd,J=12.7,5.4Hz,1H),3.80(d,J=11.6Hz,1H) ,3.56(d,J=11.9Hz,1H),3.08-2.80(m,3H),2.60(d,J=8.1Hz,3H),2.11-1.97(m ,2H),1.82(d,J=12.8Hz,1H),1.75-1.62(m,1H),1.55(dd,J=13.3,11.4Hz,1H).

[0086] HRMS:m / zcalcdforC 19 H 19 N3O6[M+H]+:386.13,found:386.20.

[0087] Intermediate 2: Synthesis of 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-N-(3-ethynylphenyl)piperidine-3-carboxamide:

[0088]

[0089] In a 25 mL round bottom flask, the intermediate 1-(2-(2,6-dioxopiperidin-3-yl))-1,3-dioxoisoindolin-4-yl)piperidine-3-carboxylic acid (1) (385 mg, 1 mmol, 1.0 equiv) was added to DMF (2 mL) and DCM (10 mL), followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (570 mg, 1.5 mmol, 1.5 equiv) and DIPEA (194 mg, 1.5 mmol, 1.5 equiv). The reaction mixture was stirred at room temperature for 30 min, then 3-ethynylaniline (1.2 mmol, 1.2 equiv) was added and stirred at room temperature for 12 h. The reaction was monitored by TLC until the reaction was complete. The residue was added with 30 mL of water and extracted with ethyl acetate (3×30 mL). The organic layer was then washed with brine and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (DCM:MeOH=200:1-50:1) to obtain intermediate 2 as a light red solid (330 mg, yield 68%).

[0090] 1 HNMR (400MHz, DMSO-d6) δ=11.06(s,1H),10.09(s,1H),7.80(s,1H),7.71(d,J=8.0Hz,1H),7.58(d,J=8.2Hz ,1H),7.41(d,J=8.5Hz,1H),7.35(d,J=7.2Hz,2H),7.15(d,J=7.6Hz,1H),5.13-5.09(m,1H),4.16(s,1H),3 .85(d,J=11.5Hz,1H),3.71(d,J=11.7Hz,1H),2.89(d,J=7.2Hz,3H),2.78(d,J=9.7Hz,1H),2.59(d,J=17.0 Hz,2H),2.04(d,J=7.4Hz,3H),1.82(s,2H).HRMS:m / zcalcdforC27H24N4O5[M+H]+:485.17,found:485.23.

[0091] Intermediate 3: Synthesis of 4-(4-azidophenoxy)-N-methylpicolinamide

[0092]

[0093] In a 25 mL round-bottom flask, 4-(4-aminophenoxy)-N-methylpicolinamide (120 mg, 0.5 mmol, 1.0 equiv) was added to DMF (2 mL) and CH3CN (6 mL), and t-BuONO (152.0 mg, 1.5 mmol, 3.0 equiv) was added. The reaction mixture was stirred at room temperature for 30 min, and then TMSN3 (170.0 mg, 1.5 mmol, 3.0 equiv) was added and stirred at 50 ° C for 6 h. The reaction was monitored by TLC until the reaction was complete. The residue was concentrated by rotary evaporation, 30 mL of water was added, and extracted with ethyl acetate (3×30 mL). The organic layer was then washed with brine and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (PE: EA = 4: 1 to 2: 1) to give 4-(4-azidophenoxy)-N-methylpicolinamide as a yellow solid (Intermediate 3) (108 mg, yield 80%).

[0094] 1 HNMR (400MHz, CDCl3-d) δ = 8.45 (d, J = 5.7Hz, 1H), 8.18 (s, 1H), 7.74 (d, J = 2.5Hz, 1H), 7.12 (s, 4H), 7.04 (dd ,J=5.7,2.6Hz,1H),3.03(d,J=5.0Hz,3H).HRMS:m / zcalcdforC13H12N5O2[M+H]+:270.10,found:270.16.

[0095] Synthesis of compound SMART1:

[0096]

[0097] In a 25 mL round-bottom flask, 4-(4-azidophenoxy)-N-methylpicolinamide (135 mg, 0.5 mmol, 1.0 equiv) and 1-(2-(2,6-dioxopiperidin-3-yl))-1,3-dioxoisoindol-4-yl)-N-(3-ethynylphenyl)piperidine-3-carboxamide (242 mg, 0.5 mmol, 1.0 equiv) were added to 10 mL of methanol, followed by 1 N CuSO4 solution (1.0 mL, 2.0 equiv) and 1 N sodium citrate solution (2 mL, 4.0 equiv). The reaction mixture was stirred at room temperature for 12 h. The reaction was monitored by TLC until the reaction was complete. The residue was concentrated by rotary evaporation, and the crude product was purified by silica gel column chromatography (DCM: MeOH = 200: 1 to 100: 1) to obtain compound SMART1 as a yellow solid (220 mg, yield 69%).

[0098] 1HNMR(400MHz, DMSO-d6)δ=11.08(s,1H),10.17(s,1H),9.31(s,1H),8.80(s,1H),8.57(d,J=4.8Hz,1H),8.33(s ,1H),8.12(d,J=8.0Hz,2H),7.70(t,J=7.4Hz,1H),7.66-7.56(m,2H),7.51(d,J=7.5Hz,3H),7.43(t,J=8.2Hz, 2H),7.35(d,J=6.5Hz,1H),7.26(s,1H),5.13(d,J=7.9Hz,1H),3.90(d,J=11.4Hz,1H),3.73(d,J=9.3Hz,1H),3 .09(t,J=11.0Hz,1H),2.86(dd,J=10.9,8.4Hz,6H),2.60(d,J=8.0Hz,2H),2.08(s,2H),1.92-1.58(m,3H).13CN MR(101MHz,DMSO-d6)δ=173.23,172.73,170.48,167.55,166.82,165.59,164. 15,153.77,153.12,151.08,150.33,147.80,140.27,136.23,134.57,134.16, 131.11,129.85,124.71,122.79,122.71,120.92,120.31,119.55,116.97,116.51,115.03,109.85,53.90,49.30,43.87,31.44,27.99,26.49,24.90,22.56.

[0099] HRMS: m / z calcd for C 40 H 36 N9O7[M+H]+:754.27,found:754.32.

[0100] Example 2: Binding assay of compound SMART1 to Smurf1 / CRBN

[0101] Although the design process of compound SMART1 used the ligands of Smurf1 and CRBN, the changes in functional groups introduced during the synthesis of PROTAC may lead to large changes in spatial conformation and affect its binding to the target protein. In order to evaluate the binding of compound SMART1 to Smurf1 / CRBN, we used SPR technology to determine the affinity of the compound to the full-length Smurf1 (GST-Smurf1) and CRBN-TBD (GST-TBD) domain proteins.

[0102] (1) Expression and purification of GST-Smurf1 and GST-TBD

[0103] The GST-Smurf1 plasmid and the GST-TBD plasmid were transformed into the competent BL21 (DE3) strain, respectively, and cultured in a 37°C incubator overnight; a single clone was picked and cultured in LB liquid medium containing ampicillin (100 μg / mL) for 12 h (37°C, 220 rpm); the culture volume of the culture medium containing the expression bacteria was expanded to 2 L at a ratio of 1:50, and the culture was continued for about 2 h, and the OD (600 nm) value reached 0.6; ITPG (final concentration 100 μM) was added to the culture medium containing the expression bacteria, and the culture was transferred to a temperature-controlled shaker (25°C, 160 rpm) and continued for 12 h to induce protein expression; the expression bacteria were collected by low-temperature centrifuge at 4000 rpm × 10 min, and the supernatant was discarded and the bacteria were frozen at -80°C; the bacteria were lysed by adding 100 mL of lysis buffer (350 mM NaCl, 20 mM Tris-HCl, 5 mM MgCl2, 1 mM The cells were resuspended in EDTA, 20% glycerol, 0.1% NP40, 1 μM PMSF, 1 μM DTT, pH = 8.0), and lysozyme (100 μg / mL) was added for lysis on ice for 10 min, and further ultrasonic lysis (120 W, 3s+3s, 10 min); the lysate was centrifuged at high speed (13000 g × 10 min), and the supernatant was filtered through a 0.45 μm filter; the lysate was balanced with BeyoGold TMGST-tag Purification Resin: Take 1 mL of Resin and add 5 mL of lysis buffer, mix by inversion for 5 minutes, centrifuge at 1000 rpm for 1 minute, discard the supernatant, and repeat twice; Binding: Add the filtered lysis buffer supernatant to the balanced Resin, bind at 4°C for 4 hours; Washing: Centrifuge (1000 rpm×2 min), discard the supernatant, and wash with lysis buffer 3 times, 5 minutes each time; Elution: Incubate the washed Resin with the eluent (20 mM Tris-HCl, 350 mM NaCl, 20 mM reduced glutathione GSH, pH=8.0) for 10 minutes, centrifuge at 1000 rpm for 1 minute, and the supernatant is the eluent containing the GST-Smurf1 target protein; The above eluent is further replaced with the buffer (PBS, 350 mM NaCl) by molecular sieve Superdex200, and the purity of the obtained protein is identified by Coomassie Brilliant Blue staining; The obtained protein can be immediately used for SPR affinity determination.

[0104] (2) SPR affinity determination (taking compound SMART1 binding to GST-Smurf1 as an example)

[0105] Pre-experimental preparation: Biacore T200, CM5 chip (catalog number: 29-1049-88); amino coupling kit (catalog number: BR-1000-50); coupling buffer: 10mM sodium acetate pH4.0 (catalog number: BR-1003-49); 10xPBS-P+ (catalog number: 28-9950-84); analytical grade DMSO, deionized water; GST, GST-Smurf1 protein (concentration greater than 0.5mg / mL); compound SMART1 (DMSO dissolved, 80μM); other consumables: capless 1.5mL EP tube (catalog number: BR-1002-87), rubber bottle cap type 2 (catalog number: BR-1004-11), 96-well plate (catalog number: BR-1005-03), 96-well plate sealing film (catalog number: 28-9758-16), purchased from Cytiva.

[0106] Protein ligand coupling: CM5 was selected for the chip, and the four channels FC1 and 2 were set separately (channels 1 and 2 were used in pairs, channel 1 was used as a reference to couple GST protein, and channel 2 was coupled to GST-Smurf1). The chip was first activated with EDC / NHS, and the ligand was fixed by amino direct coupling. The ligand protein was diluted to 50 μg / mL with sodium acetate at pH 4.0, coated on the chip surface to the target coupling amount, and then blocked with ethanolamine.

[0107] Running buffer and sample configuration:

[0108] Preparation of running buffer and solvent calibration curve The running buffer for small molecule samples is 1×PBS-P+ containing 5% DMSO (the DMSO content can be adjusted according to the solubility of the sample, and the maximum is not more than 10%). Take 105mL of 10×PBS-P+ and dilute it to 1L with deionized water to make 1.05×PBS-P+. According to Table 1, add DMSO to prepare 5% DMSO running buffer and 4.5% and 5.8% solvent calibration stock solutions.

[0109] Table 1

[0110] 4.5% DMSO 5.8% DMSO 5.0%DMSO running buffer 1.05x PBS-P+ 9.5mL 9.5mL 950mL 100% DMSO 0.45mL 0.58mL 50mL Final volume ~10mL ~10mL 1000mL

[0111] Prepare a 5% DMSO concentration calibration curve by mixing 4.5% and 5.8% stock solutions according to Table 2

[0112] Table 2

[0113] Buffer / Vial 1 2 3 4 5 6 7 8 4.5% DMSO 0 200 400 600 800 1000 1200 1400 5.8% DMSO 1400 1200 1000 800 600 400 200 0

[0114] Small molecule sample preparation: dilute 1mM small molecule stock solution 20 times with 1×PBS-P+ buffer without DMSO to obtain 1000μL of 50μM small molecule in 1×PBS-P+ containing 5% DMSO, and then use the prepared Running Buffer containing 5% DMSO to dilute the analyte downward by two times into 6 concentration gradients (200μL each), namely 4μM, 2μM, 1μM, 0.5μM, 0.25μM, 0.125μM, 0.0625μM, 0.0315μM. Set a repeat concentration at intervals and add a 0 concentration.

[0115] Multi-cycle kinetic assay:

[0116] Place the prepared solvent calibration solution (solvent calibration is performed once at the beginning and once at the end of the test), concentration gradient small molecules, and glycine-HCl regeneration solution on the sample rack, set up the surface test-regeneration process of the sample, and run the program.

[0117] Result analysis:

[0118] Open Biacore T200 Evaluation Software, find the saved result file, click Binding to reference in the Plot on the left, check whether each point is consistent or less than 20% of the corresponding response value in the binding level, and then check whether the response value of each point in the binding level is obviously concentration-dependent.

[0119] Click on solvent correction to perform solvent correction analysis. The solvent correction curve is generally required to fall within -500 to +1000RU, and the two vertical lines fall within the correction curve range. 2 Less than 2. If it exceeds this range by a large amount, it is mostly due to inaccurate configuration of DMSO concentration.

[0120] Click Kinetics / Affinity in the middle of the top, and click Surface bound in the drop-down bar. In the pop-up window, select appropriate, at least 5 continuous concentrations for fitting. For unnecessary concentrations, remove the check mark in front of the concentration in the sample concentration table. Select FC=2-1corr in Curve.

[0121] Affinity analysis, select Steady State Affinity as the Model, click Fit in the upper left corner to fit the data, and click Finish in the lower right corner to complete. After fitting, the affinity of the compound to GST-Smurf1 is KD = 400.9nM, and the affinity to CRBN-TBD is KD = 21.06μM (the results are shown in Figure 3 shown).

[0122] Example 3: Compound SMART1 mediates the formation of a ternary complex between Smurf1 and CRBN

[0123] An important factor for whether the compound SMART1 can mediate the ubiquitination of the substrate is to induce the formation of the target protein (Smurf1)-PROTAC-E3 ligase (CRBN) ternary complex. In order to detect whether our synthesized compound SMART1 can induce the formation of the Smurf1-PROTAC-CRBN ternary complex in cells, we used immunocoprecipitation and NanoBRET experiments for determination and analysis.

[0124] The co-immunoprecipitation experiment was performed as follows:

[0125] 1. Cell treatment: HEK293T cells were cultured in T25 culture flasks to 80% confluence, the proteasome inhibitor MG132 was added to the cells for 1 h, and then different concentrations of compounds were added to the cultured cells;

[0126] 2. Cell collection: After 4 hours, discard the culture medium, rinse the cells once with pre-cooled PBS, collect the cells with a cell scraper, then wash once with PBS, centrifuge at 3000 rpm for 3 minutes, and discard the supernatant;

[0127] 3. Cell lysis: add 600 μL HEPES lysis buffer (with protease inhibitors and phosphatase inhibitors) to resuspend the cells, lyse on ice for 10 min, then continue to ultrasonically disrupt the cells in an ice bath (30w, 3min, on for 1s, off for 1.5s), centrifuge at 4°C, 12000rpm for 10min, and collect the supernatant;

[0128] 4. Pre-clearing: Add 20 μL protein-A / G-agarose beads to the above supernatant, incubate on a rotating mixer at 4°C for 3 h, centrifuge at 1000 rpm for 1 min, and collect the supernatant;

[0129] 5. Antigen-antibody binding: Take 40 μL of the supernatant as the total cell lysate, add 1 μg of Flag antibody to the remaining supernatant, and incubate on a rotating mixer at 4°C for 3 h;

[0130] 6. Antibody Protein A / G binding: Add 40 μL Protein-A / G-agarose beads and continue incubation on a rotating mixer at 4°C for 8 hours;

[0131] 7. Washing: Centrifuge at 1000 rpm for 3 min at 4°C, discard the supernatant, add 1 mL of cell lysis buffer, resuspend and wash in a rotating mixer at 4°C for 10 min, repeat 3 times;

[0132] 8. Sample preparation and detection: Aspirate the supernatant from the washed agarose beads, add 40 μL 2× loading buffer (100 mM Tris-HCl, pH 6.8, 4% (W / V) SDS, 0.2% (W / V) bromophenol blue, 20% (V / V) glycerol, 2% (W / V) β-ME, 0.2 mM PMSF), mix well and boil in 100°C boiling water for 15 min, and detect by immunoblotting.

[0133] The NanoBRET assay is as follows:

[0134] 1. Build Smurf1- -CRBN carrier;

[0135] 2. Transfection cell preparation: HEK293T cells were cultured in 6-well plates to 80% confluence;

[0136] 3. Cell transfection: 2 μg Smurf1- Fusion vector DNA + 0.2 μg -CRBN fusion vector DNA was routinely transfected into cells and cultured for 24 h;

[0137] 4. Preparation of test cells: collect transfected cells by trypsin digestion, wash once with PBS, and use test medium (Opti- I low serum medium, no phenol red + 4% FBS) and adjust the cell density to 2 × 10 5 cells / mL;

[0138] 5. Split the cells into two parts and add NanoBRET TM 618 Ligand or solvent DMSO:

[0139] Experimental sample (+ligand): add 1 μL of 0.1 mM NanoBRET TM 618 ligand (final concentration 100 nM).

[0140] No receptor control (–ligand): Add 1 μl DMSO per ml of cells (final concentration 0.1% DMSO).

[0141] 6. Incubate the plate at 37°C, 5% CO2 for at least 4-6 hours to overnight (18-24 hours);

[0142] 7. Add compound at ten times higher concentration than the final concentration in the assay medium (e.g., if the final concentration is 1 μM, add 10 μM);

[0143] 8. In Opti- Preparation of NanoBRET in Low Serum Medium (without Phenol Red) TM Nano- 5X solution of Substrate;

[0144] 9. Add the substrate to the cells and shake the plate to mix for 30 seconds;

[0145] 10. Within 10 minutes after adding the substrate, use the NanoBRET TM PPI detection method compatible luminescence detector (CellTiter- 2.0Assay, Promega) to measure the donor emission signal (460 nm) and the acceptor emission signal (618 nm);

[0146] 11.NanoBRET TM Calculation: Divide the acceptor emission value (e.g. 618 nm) by the donor emission value (e.g. 460 nm) for each sample to obtain the raw NanoBRET TM ratio;

[0147] 12. Multiply each raw BRET value by 1000 to obtain the raw NanoBRET TMUnits (usually decimal values) are converted to milliBRET units (mBU; integer);

[0148] 13. Determine the Average NanoBRET for Each Group of Samples TM Ratio: Samples include NanoBRET TM The experimental samples with 618 ligand and the control samples without receptor. The background or bleed-through caused by the donor should also be considered, so the mean value of the experimental samples should be subtracted from the mean value of the control samples without receptor to obtain the corrected NanoBRET TM ratio.

[0149] The results are as follows Figure 4 As shown in Figure 2, the presence of compound SMART1 can effectively promote the binding of Smurf1 to CRBN in a concentration-dependent manner, while the two end ligands of compound SMART1, SMART1-L Neither Poma nor Pomalidomide can induce Smurf1 to bind to CRBN ( Figure 4 NanoBRET experiments also confirmed that SMART1 promoted the binding of Smurf1 to CRBN in a concentration-dependent manner (Figure a). Figure 4 (as shown in Figure b).

[0150] Example 4: Concentration dependence experiment of biological activity evaluation of compound SMART1 at the level of protein immunoblotting experiment

[0151] 1. Cell plating: Take HCT116 cells in the logarithmic growth phase and prepare 10 4 Cell suspension of 100 cells / mL was inoculated into 12-well plates at 1 mL / well and placed in a 37°C incubator to adhere overnight.

[0152] 2. Compound treatment: Prepare medium containing different concentrations of compound SMART1 (10μM, 3μM, 1μM, 0.3μM, 0.1μM, 0.03μM, 0.01μM, 0.003μM, 0.001μM, 0.0003μM, 0.0001μM), add the compound to the cells by replacing the medium, add DMSO to the control group, and continue culturing for 12h;

[0153] 3. Western Blot sample preparation and detection: discard the culture medium, wash the cells once with PBS, add 120 μL 1× Loading Buffer to the treated cells, transfer to ice, place on a shaker for 10 min to fully lyse, collect the samples and place in a boiling water bath for 20 min, mix well and use directly for Western Blot detection;

[0154] 4. The development results were scanned in grayscale using Image J to obtain the grayscale value of the target protein involved;

[0155] 5. Obtained gray values ​​and performed relative statistics, and used GraphPad to fit the DC of compound-induced degradation of target protein 50 .

[0156] Time-dependent experiments

[0157] 1. Cell plating: Take HCT116 cells in the logarithmic growth phase and prepare 10 4 Cell suspension of 100 cells / mL was inoculated into 12-well plates at 1 mL / well and placed in a 37°C incubator to adhere overnight.

[0158] 2. Compound treatment: Add compounds to cells at a final concentration of 0.1 μM for different treatment times;

[0159] 3. Discard the culture medium, wash the cells once with PBS, and prepare and detect Western Blot samples.

[0160] The results are as follows Figure 5 As shown, the compound SMART1 (SMART-1) can specifically and efficiently induce Smurf1 degradation in the nM concentration range, DC 50 =1.303nM, but did not affect the expression of Smurf2, a member of the same family ( Figure 5 ), DIA quantitative mass spectrometry analysis further confirmed that compound SMART1 specifically induced the degradation of Smurf1 ( Figure 5 (as shown in Figure c).

[0161] Example 4: Evaluation of the biological activity of compound SMART1 in inhibiting cell proliferation in different tumor cells

[0162] 1. Cell plating: Take cells in the logarithmic growth phase (colorectal cancer cell HCT116, gastric cancer cell MGC-803, lung cancer cell H1299, cervical cancer cell HeLa, liver cancer cell Hep3B, LM3, pancreatic cancer cell SW1990, breast cancer cell MDA-MB-231), prepare 10 4 Cell suspension of 100 μL / well was inoculated into 96-well plates and placed in a 37°C incubator to adhere overnight.

[0163] 2. Compound treatment: Compound treatment: Prepare culture medium containing different concentrations of compound SMART1 (10μM, 3μM, 1μM, 0.3μM, 0.1μM, 0.03μM, 0.01μM, 0.003μM, 0.001μM, 0.0003μM, 0.0001μM), add the compound to the cells by replacing the culture medium, add DMSO to the control group, and continue culturing;

[0164] 3. Set different time points (24h, 48h, 72h, 96h), prepare culture medium containing 10% CCK-8 reagent (Beijing Langbolide), add CCK-8 reagent to the cultured cells by changing the culture medium, and incubate in a 37°C incubator for 1h;

[0165] 4. Measure the OD (450nm) value with an enzyme-labeled instrument;

[0166] 5. Use GraphPad to fit the IC of the compound to inhibit cell growth 50 .

[0167] The results are as follows Figure 6 As shown in Figure a, compound SMART1 has growth inhibitory effects in multiple tumor cell lines, and the effect is most significant in HCT116, IC 50 =22.04 nM. The clone formation experiment further confirmed that SMART1 significantly inhibited the clone formation of HCT116 cells ( Figure 6 (as shown in Figure b); cell cycle experimental analysis found that HCT116 treated with SMART1 was blocked in the G2-M phase.

[0168] Example 5: Pharmacokinetic study of the compound in mice

[0169] In order to evaluate the pharmacokinetic properties of the compound in vivo and explore the administration method and dosage of subsequent pharmacodynamic experiments, this application selected Kunming mice for pharmacokinetic experiments. After 9 mice were randomly numbered and weighed, they were randomly divided into 3 groups using a parallel design and given intravenous, oral, and intraperitoneal administration. About 20-30 μL of whole blood was collected at 2min, 5min, 15min, 30min, 60min, 2h, 4h, 6h, 8h, 12h, and 24h after administration (intravenously) to detect the concentration of the compound. After IV (intravenous administration, 5 mg / kg), PO (oral administration, 25 mg / kg), IP (intraperitoneal injection, 5 mg / kg), and IV (2 mg / kg) administration, the AUCall were 5588.60±1284.74 (h*ng / mL), 537.92±76.54 (h*ng / mL), 1897.90±710.43 (h*ng / mL), and 725.54±103.83 (h*ng / mL), respectively, and the Cmax (peak drug concentration) were 27231.88±3442.81 (ng / mL), 171.49±42.19 (ng / mL), and 766.27 ±446.64 (ng / mL), 3027.14±208.50 (ng / mL), Tmax (peak time) was (0.03±0.00) h, (0.83±0.29) h, (0.50±0.00) h, (0.03±0.00) h, t1 / 2 (drug half-life) was (1.80±0.32) h, (8.48±6.69) h, (2.10±0.90) h, (1.21±0.16) h, MRTlast (mean residence time) was (0.70±0.22) h, (3.20±1.58) h, (2.48±0.82) h, (0.56±0.13) h. The results showed that the bioavailability of the compound in Kunming mice after oral administration was 2.29%, and the bioavailability of the compound in Kunming mice after intraperitoneal administration was 34.84%.

[0170] Based on the above results, intraperitoneal injection was selected as the administration method for the next pharmacodynamic study.

[0171] Example 6: Growth inhibition experiment of compound SMART1 on subcutaneous tumors in nude mice

[0172] 1. Cell culture: Cell status is the key to tumorigenesis experiments. Culture cells in the logarithmic growth phase (cell confluence is about 80-90%) and replace fresh culture medium the night before collecting cells.

[0173] 2. Cell collection and preparation: Trypsinize cells, terminate digestion with complete medium, collect cells by centrifugation (800rpm, 3min), wash twice with pre-cooled PBS to remove serum from cells, adjust the final cell concentration to 5×10^7 cells / mL after cell counting, and place the cell suspension on ice to reduce cell metabolism;

[0174] 3. Subcutaneous injection for tumor formation: After the cells are digested, they should be inoculated into the subcutaneous tissue of nude mice as soon as possible (generally within half an hour). Each nude mouse is inoculated with 0.1 mL of cells. Tumors will form after about one week, and the tumor volume is about 100 mm 3 , tumor-forming mice were divided into groups;

[0175] 4. Intraperitoneal injection: Compound SMART1 was dissolved in DMSO to prepare a 10 mg / mL compound solution, and the solvent was prepared according to the ratio of castor oil: PBS = 1:8. One part of the compound solution was added to 9 parts of the solvent to finally prepare DMSO: castor oil: PBS = 1:1:8, and the compound was 1 mg / mL of the clear solution; mice were administered intraperitoneally according to the 25 mg / Kg standard, and the control group was intraperitoneally injected with an equal volume of solvent;

[0176] 5. Administer the drug daily, measure the weight of the mice, record the volume of the subcutaneous tumor, and summarize the change curve of the weight of the mice and the change curve of the volume of the subcutaneous tumor of the mice;

[0177] 6. Sacrifice the mice, remove the subcutaneous tumors and weigh them.

[0178] The results are as follows Figure 8 As shown, the results showed that compound SMART1 significantly inhibited the subcutaneous tumor-forming ability of HCT116 cells in nude mice.

[0179] Example 7: Safety evaluation study of compound SMART1 in CD1 mice

[0180] 6-8 weeks old CD-1 female mice were intraperitoneally administered with different concentrations of compound SMART1 (the compound was dissolved in 10% DMSO, 10% castor oil, and 80% PBS). The body weight of mice was measured every day. At the end of the experiment, the mice were euthanized, and blood and major organs were collected for analysis. Toxicity studies included hematological, biochemical indices, and histological analysis.

[0181] The results are as follows Fig. 9 As shown, the results showed that compared with the control group mice, the CD-1 mice treated with different doses of compound SMART1 had no obvious abnormalities in body weight, blood biochemistry, blood phase, pathological sections and other indicators, indicating that compound SMART1 has good safety in disease prevention.

[0182] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0183] The above is a detailed introduction to a degradation agent targeting Smurf1 protein, a preparation method and a use provided in the examples of the present application. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above examples is only used to help understand the method of the present application and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present application, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. The compound represented by formula (I):

2. The compound according to claim 1, characterized in that The compound is capable of degrading Smurf1 protein.

3. The method for preparing the compound according to claim 1, characterized in that ,, including the following steps: 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione and piperidine-3-carboxylic acid were used to synthesize intermediate 1 Intermediate 2 was synthesized by combining intermediate 1 with 3-ethynylaniline The intermediate 3 was synthesized by reacting 4-(4-aminophenoxy)-N-methylpicolinamide with azidotrimethylsilane. Intermediate 2 and intermediate 3 are combined to synthesize the compound represented by formula (I).

4. A pharmaceutical composition, characterized in that The invention comprises a compound as claimed in any one of claims 1 to 2 or a compound prepared by the method for preparing a compound as claimed in claim 3.

5. The pharmaceutical composition according to claim 3, characterized in that Includes a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 3, characterized in that The dosage form of the pharmaceutical composition includes at least one of tablets, capsules, powders, granules, injections, suspensions, solutions, creams, suppositories, gels, aerosols, sprays and powder sprays.

7. Use of the compound described in any one of claims 1 and 2, its pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate, polymorph, the compound prepared by the preparation method of the compound described in claim 3, and the pharmaceutical composition described in any one of claims 4 to 6 in the preparation of a drug for treating a disease caused by accumulation of Smurf1 protein.

8. Use of the compound described in any one of claims 1 to 2, its pharmaceutically acceptable salts, enantiomers, stereoisomers, solvates, polymorphs, the compound prepared by the method for preparing the compound described in claim 3, and the pharmaceutical composition described in any one of claims 4 to 6 in the preparation of drugs for treating cancer.

9. The use according to claim 8, characterized in that The cancers include colorectal cancer, gastric cancer, lung cancer, cervical cancer, liver cancer, pancreatic cancer, and breast cancer.

10. A degradation agent targeting Smurf1 protein, characterized in that: Including the compound as described in any one of claims 1 and 2, its pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate, polymorph, the compound prepared by the preparation method of the compound as described in claim 3, and the pharmaceutical composition as described in any one of claims 4 to 6.

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