A degrader targeting smurf1 protein, preparation method and use thereof

Abstract

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

Description

Technical Field

[0001] This application relates to the field of medicinal chemistry, specifically to a degrading agent targeting the Smurf1 protein, its preparation method, and its uses. Background Technology

[0002] The HECT ubiquitin ligase Smurf1 is expressed in many organs, including bone, cartilage, heart, lung, nervous system, and reproductive organs, and is expressed at all developmental stages from embryonic to adulthood, indicating that Smurf1 plays a comprehensive role in physiological regulation. Depending on the tissue and cellular environment, Smurf1 can selectively regulate the degradation of key regulatory factors such as Smad1 / 5, BMP receptor, MEKK2, RhoA, RUNX2, Prickle1, Jun-B, hPEM-2, TRAFs, and ING2, playing 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, including colorectal cancer, and is closely related to poor prognosis in cancer patients. Inhibiting Smurf1 expression using technologies such as siRNA can effectively suppress malignant behaviors such as tumor cell proliferation and migration. Given that Smurf1 gene knockout mice survive and no other obvious diseases have been observed, the risk of potential side effects from targeted regulation of Smurf1 expression is low, making Smurf1 an ideal target for cancer therapy. Summary of the Invention

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

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

[0005]

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

[0007] The embodiments of this application further provide a method for preparing the compound shown in formula (Ⅰ), comprising the following steps:

[0008] Intermediate 1 was synthesized from 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione and piperidine-3-carboxylic acid.

[0009] Intermediate 1 was synthesized from 3-acetyleneaniline to form intermediate 2.

[0010] Intermediate 3 was synthesized from 4-(4-aminophenoxy)-N-methylpyridinamide and azidotrimethylsilane.

[0011]

[0012] Intermediate 2 and intermediate 3 were combined to synthesize the compound shown in formula (I).

[0013] An embodiment of this application further provides a pharmaceutical composition comprising the above-described compound or a compound prepared by the above-described method.

[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 this application further provide the use of the above-described compounds, their pharmaceutically acceptable salts, enantiomers, stereoisomers, solvates, polymorphs, compounds prepared by the methods for preparing the above-described compounds, and the above-described pharmaceutical compositions in the preparation of a medicament for treating conditions caused by the accumulation of Smurf1 protein.

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

[0018] In some embodiments, the cancers include colorectal cancer, stomach cancer, lung cancer, cervical cancer, liver cancer, pancreatic cancer, and breast cancer.

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

[0020] The beneficial effects of this application are as follows: This application provides the compound shown in formula (I) and conducts functional studies and safety evaluations on the compound. The results show that the compound of this application can be used as a therapeutic compound for cancer, especially colorectal cancer. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

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

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

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

[0026] Figure 5 The results show the experimental results of SMART1 inducing specific degradation of Smurf1. Figure a shows that SMART1 can induce specific degradation of Smurf1, but has no degradation effect on Smurf2 and β-actin. Figure b shows the DC50 test results of SMART1 in degrading Smurf1. Figure c shows the DIA test results of SMART1 inducing specific degradation of Smurf1.

[0027] Figure 6 The results of functional experiments on compound SMART1 are shown in Figure a. Figure a shows the IC50 test results of compound SMART1 on different cells, Figure b shows the inhibitory effect of compound SMART1 on HCT116 cells, and Figure c shows the cell cycle assay results of compound SMART1 blocking HCT116 cells.

[0028] Figure 7 These are the pharmacokinetic results of compound SMART1;

[0029] Figure 8 The results of the nude mouse experiment with compound SMART1 are shown in Figure a. The weight of the nude mice before the experiment is shown in Figure b. The volume of the subcutaneous tumors in the nude mice after subcutaneous injection is shown in Figure c. The volume of the subcutaneous tumors in the solvent control group and the experimental group is shown in Figure d. The weight of the subcutaneous tumors in the solvent control group and the experimental group is shown in Figure d.

[0030] Figure 9 Figure 1 shows the safety test results for compound SMART1. Figure a shows the safety test setup parameters; Figure b shows the weight changes of mice in different treatment groups; Figure c shows the organ morphology of mice in different treatment groups; Figure d shows the HE staining results of organ tissues of mice in different treatment groups; Figure e shows the organ weight changes of mice in different treatment groups; Figure f shows the blood test results of mice in different treatment groups, where WBC is white blood cells, RBC is red blood cells, PLT is platelets, NE is neutrophils, and LY is lymphocytes; Figure g shows 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 serum creatinine. Detailed Implementation

[0031] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. In addition, in the description of this application, the term "comprising" means "including but not limited to". The terms first, second, third, etc. are used only as illustrative purposes and do not impose numerical requirements or establish an order. Various embodiments of this 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 conciseness and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values ​​within that 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., and single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range. Additionally, whenever a numerical range is specified in this document, it means that any referenced number (fraction or integer) within the range is included.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art described herein. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this application. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail. Unless otherwise stated, “%” means percentage based on weight.

[0033] PROteolysis Targeting Chimera (PROTAC) is an artificially constructed macromolecular complex tool. One end of it has a ligand that binds to a ubiquitin ligase, and the other end has a ligand that allows the intracellular target protein to bind, connected by a linker. PROTAC induces ubiquitination of the target protein, which is then degraded via the ubiquitin-proteasome pathway. This technology utilizes the cell's own degradation mechanisms to degrade pathogenic proteins, and has great potential as a novel treatment for diseases such as cancer, autoimmune diseases, and neurological disorders. Traditional small molecule compounds inhibit protein function by occupying the active region of the target protein. Protein targets that do not contain protein-binding active regions are considered "undruggable targets" or "difficult-to-drug targets," such as transcription factors and scaffold proteins. Unlike traditional small molecule drugs, PROTAC drugs do not require tight, long-term binding to the pathogenic target, giving them the advantage of degrading "undruggable" targets. Based on current data, it is estimated that 80% of currently "undruggable" targets can be targeted by PROTAC molecules. Furthermore, PROTAC technology offers a range of advantages, including substrate selectivity, tissue specificity, and the ability to overcome drug resistance. This application obtained a PROTAC-type compound through synthetic screening. This compound was rationally designed and synthesized using a derivative of a Smurf1 inhibitor and a CRBN ligand, and then screened using NanoLuc technology. Experimental results show that the PROTAC-type compound obtained in this application exhibits good affinity for both the target protein Smurf1 and the E3 ligase CRBN. This compound effectively ubiquitinates Smurf1 protein and degrades it via the proteasome pathway by forming a ternary complex within the cell, effectively inhibiting tumor growth. This indicates that the compound can act as a Smurf1 protein degrader and may serve as a therapeutic agent for diseases caused by Smurf1 protein accumulation. The applicant conducted functional studies and safety evaluations of this compound, confirming its potential as a treatment for various cancers, especially colorectal cancer.

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

[0035]

[0036] In some embodiments, the compound shown in formula (I) can degrade the Smurf1 protein.

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

[0038] Intermediate 1 was synthesized from 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione and piperidine-3-carboxylic acid.

[0039] Intermediate 1 was synthesized from 3-acetyleneaniline to form intermediate 2.

[0040] Intermediate 3 was synthesized from 4-(4-aminophenoxy)-N-methylpyridinamide and azidotrimethylsilane.

[0041]

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

[0043] In some embodiments, the preparation route of the above-mentioned 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 1:1.5.

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

[0047] In some embodiments, during the synthesis 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 1:3.

[0049] In some embodiments, the reaction temperature in the synthesis of intermediate 1 is 85-95°C. The temperature can be any value among 85, 90, and 95 or a range 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, or 10, or a range of any two values. In a specific embodiment, the reaction time can be selected as 6 hours.

[0051] In some embodiments, in the synthesis step of intermediate 2, the molar ratio of intermediate 1 to 3-acetylene aniline is 1:(1-2), such as the molar ratio of intermediate 1 to 3-acetylene aniline being 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 any value or any combination of two values ​​in the volume ratio of DMF to DCM of 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.

[0053] In some embodiments, during the synthesis of intermediate 2, HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea 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 to 3), such as any value or a range of any two values ​​among 1:1, 1:1.5, 1:2, 1:2.5, and 1:3.

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

[0056] In some embodiments, the reaction temperature in the synthesis of intermediate 2 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 or a range of any two values ​​among 10, 11, 12, 13, 14, and 15. In a specific embodiment, the reaction time can be selected as 12 hours.

[0058] In some embodiments, in the synthesis step of intermediate 3, the molar ratio of 4-(4-aminophenoxy)-N-methylpyridine amide to tris(azido)silane (TMSN3) is 1:(1-5), such as any value or a range of any two values ​​among 1:1, 1:2, 1:3, 1:4, and 1:5.

[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 to 5), such as any value or any combination of two values ​​of the volume ratio of DMF to ACN of 1:1, 1:2, 1:3, 1:4, 1:5.

[0060] In some embodiments, during the synthesis 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-methylpyridine amide to t-BuONO is 1:(1 to 5), such as any value or any range of two values ​​among 1:1, 1:2, 1:3, 1:4, and 1:5.

[0062] In some embodiments, the reaction temperature during the synthesis of intermediate 3 is 45–55°C. For example, the reaction temperature can be any value or a range of any two of 45°C, 50°C, and 55°C.

[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, or 10, or a range of any two values. In a specific embodiment, the reaction time can be selected as 6 hours.

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

[0065] In some embodiments, the solvent system in which the reactants are synthesized includes methanol, and in other embodiments, the solvent system also includes water.

[0066] In some embodiments, during the synthesis 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 or a range of any two values ​​from 1:1, 1:2, 1:3.

[0067] In some embodiments, during the synthesis 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 to mol / L. The molar ratio of intermediate 2 to sodium citrate solution is 1:(1 to 5), which is any value or a range of any two values ​​from 1:1, 1:2, 1:3, 1:4, 1:5.

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

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

[0071] Pharmaceutical Composition

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

[0073] The pharmaceutical composition also includes a pharmaceutically acceptable carrier. A "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, which participates in the transport or delivery of the drug from one organ or part of the body to another organ or part of the body. The pharmaceutically acceptable carrier may constitute 0.001 to 99.9 wt% of the pharmaceutical composition. Specifically, based on the total mass of the pharmaceutical composition, the percentage of the pharmaceutically acceptable carrier (%) may be any value or a range of any two values ​​from 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99.

[0074] The aforementioned carrier must be "acceptable," meaning it is compatible with other ingredients in the formulation and does not harm 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 carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth gum 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; alginate; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer; and other non-toxic and compatible substances used in pharmaceutical preparations. Wetting agents, emulsifiers and lubricants, such as sodium dodecyl sulfonate, magnesium stearate, and polyoxyethylene-polypropylene copolymers, as well as colorants, release agents, coating agents, sweeteners, flavorings and fragrances, preservatives and antioxidants may also be present in the composition.

[0076] Administration method

[0077] In this application, the pharmaceutical composition can be administered to the subject / patient in any suitable dosage form, and there are no particular restrictions on the method of administration. Representative methods of administration include, but are not limited to, oral, intramuscular, intravenous, intravenous drip, enema, spray, external application, or intraperitoneal injection.

[0078] In this application, the subject / patient refers to a vertebrate, preferably a mammal, which includes, but is not limited to, rodents, apes, livestock, and humans, and is preferably a human.

[0079] Examples of dosage forms for pharmaceutical compositions in this application include, but are not limited to, tablets, capsules, powders, granules, injections, suspensions, solutions, creams, suppositories, gels, aerosols, sprays, and powder inhalers.

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

[0081] Intermediate 1: 1-(2-(2,6-dioxopiperidin-3-yl))-1,3-dioxoisoindololin-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-dioxadiazin-3-yl)-4-fluoroisoindoline-1,3-dione (1.1 g, 4 mmol, 1 equiv) was dissolved in DMSO (10 mL). 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 complete. The residue was extracted with 30 mL of water and 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 to 50:1) to give the intermediate 1-(2-(2,6-dioxopiperidin-3-yl))-1,3-dioxoisoindoline-4-yl)piperidin-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 (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, intermediate 1-(2-(2,6-dioxopiperidin-3-yl))-1,3-dioxoisoindoline-4-yl)piperidin-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'-tetramethylurea 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-acetyleneaniline (1.2 mmol, 1.2 equiv) was added and stirred at room temperature for 12 h. The reaction was monitored by TLC until complete. The residue was extracted with ethyl acetate (3 × 30 mL) by adding 30 mL of water. The organic layer was then washed with brine and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (DC M:MeOH = 200:1 to 50:1) to give intermediate 2, a pale 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-methylpyridine amide (120 mg, 0.5 mmol, 1.0 equiv) was added to DMF (2 mL) and CH3CN (6 mL), followed by t-BuONO (152.0 mg, 1.5 mmol, 3.0 equiv). The reaction mixture was stirred at room temperature for 30 min, then TMSN3 (170.0 mg, 1.5 mmol, 3.0 equiv) was added and the mixture was stirred at 50 °C for 6 h. The reaction was monitored by TLC until complete. The residue was concentrated by rotary evaporation, and 30 mL of water was added. The residue was 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-methylpyridine carboxamide 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-methylpyridinecarboxamide (135 mg, 0.5 mmol, 1.0 equiv) and 1-(2-(2,6-dioxopiperidin-3-yl))-1,3-dioxoisoindol-4-yl)-N-(3-ethynylphenyl)piperidin-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 complete. The residue was concentrated by rotary evaporation, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 200:1–100:1) to give 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 determination of compound SMART1 with Smurf1 / CRBN

[0101] Although the design process of compound SMART1 used ligands for Smurf1 and CRBN, the functional group changes introduced during the synthesis of PROTAC may lead to significant spatial conformational changes, affecting its binding to target proteins. To evaluate the binding of compound SMART1 to Smurf1 / CRBN, we used SPR technology to determine the affinity of the compound for full-length Smurf1 (GST-Smurf1) and CRBN-TBD (GST-TBD) domain proteins, respectively.

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

[0103] GST-Smurf1 and GST-TBD plasmids were transformed into competent cells of BL21(DE3) strain and cultured overnight at 37°C. Single colonies were picked and cultured in LB liquid medium containing ampicillin (100 μg / mL) for 12 h (37°C, 220 rpm). The culture medium containing the expression bacteria was expanded to 2 L at a ratio of 1:50 and cultured for about 2 h until 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 medium was transferred to a temperature-controlled shaker (25°C, 160 rpm) and cultured for another 12 h to induce protein expression. The expression bacteria were collected by centrifuging at 4000 rpm for 10 min, the supernatant was discarded, and the cells were frozen at -80°C. Cell lysis was performed by adding 100 mL of lysis buffer (350 mM NaCl, 20 mM Tris-HCl, 5 mM MgCl2, 1 mM... Bacterial cells were resuspended in EDTA, 20% glycerol, 0.1% NP40, 1 μM PMSF, 1 μM MTT, pH 8.0. Lysozyme (100 μg / mL) was added and the cells were lysed on ice for 10 min, followed by sonication (120 W, 3 s + 3 s, 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 then equilibrated with BeyoGold. TMGST-tag Purification Resin: Add 1 mL of Resin to 5 mL of lysis buffer, invert and mix for 5 min, centrifuge at 1000 rpm for 1 min, discard the supernatant, and repeat twice; Binding: Add the supernatant of the filtered lysis buffer to the equilibrated Resin, and bind at 4℃ for 4 h; Washing: Centrifuge (1000 rpm × 2 min), discard the supernatant, and wash 3 times with lysis buffer, 5 min each time; Elution: Incubate the washed Resin with elution buffer (20 mM Tris-HCl, 350 mM NaCl, 20 mM reduced glutathione GSH, pH = 8.0) for 10 min, centrifuge at 1000 rpm for 1 min, and the supernatant is the elution buffer containing the GST-Smurf1 target protein; The above elution buffer is further processed through Superdex 200 molecular sieve replacement buffer (PBS, 350 mM NaCl), 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 determination of affinity (taking the binding of compound SMART1 to GST-Smurf1 as an example)

[0105] Preparation before the experiment: Biacore T200, CM5 chip (catalog number: 29-1049-88); amino-coupling kit (catalog number: BR-1000-50); coupling buffer: 10mM sodium acetate pH 4.0 (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 (dissolved in DMSO, 80μM); other consumables: 1.5mL EP tubes without caps (catalog number: BR-1002-87), rubber 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: The CM5 chip was selected, and four channels FC1 and FC2 were configured separately (channels 1 and 2 were used in pairs; channel 1 served as the reference for coupling GST protein, and channel 2 was coupled to GST-Smurf1). The chip was first activated with EDC / NHS, and ligands were immobilized using the direct amino-coupling method. The ligand protein was diluted to 50 μg / mL with sodium acetate at pH 4.0 and coated onto the chip surface to the target coupling amount, followed by blocking with ethanolamine.

[0107] Run buffer and sample preparation:

[0108] Prepare the running buffer and solvent correction curve. For small molecule samples, use 1×PBS-P+ containing 5% DMSO (the DMSO content can be adjusted according to the sample solubility, but should not exceed 10%). Take 105 mL of 10×PBS-P+ and dilute it with deionized water to 1 L to prepare 1.05×PBS-P+. Then, according to Table 1, add DMSO to prepare 5% DMSO running buffer and 4.5% and 5.8% solvent correction 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% mother liquor 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] For small molecule sample preparation, dilute 1 mM of the small molecule stock solution 20-fold with 1×PBS-P+ buffer without DMSO to obtain 1000 μL of the small molecule in 50 μM 1×PBS-P+ containing 5% DMSO. Then, use a prepared Running Buffer containing 5% DMSO to perform down-dilute six concentration gradients (200 μL each): 4 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM, 0.125 μM, 0.0625 μM, and 0.0315 μM. A repeat concentration is set at intervals, followed by a zero concentration.

[0115] Multi-cycle dynamics detection:

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

[0117] Results analysis:

[0118] Open the Biacore T200 Evaluation Software and locate the saved results file. Click "Binding to reference" in the left-hand Plot section to check if the responses at each point are consistent or less than 20% of the corresponding response values ​​at the binding level. Then, check if there is a significant concentration dependence in the response values ​​at each point of the binding level.

[0119] Click "solvent correction" to perform solvent correction analysis. The solvent correction curve should generally fall within the range of -500 to +1000 RU, with both vertical lines falling within the correction curve range. The fitted Chi... 2 Less than 2. If it exceeds this range significantly, it is usually due to inaccurate DMSO concentration preparation.

[0120] Click on Kinetics / Affinity in the top center, and then click on Surface bound in the drop-down menu. In the pop-up window, select appropriate values, at least 5 consecutive concentrations, for fitting. Unnecessary concentrations can be unchecked in the sample concentration table. Select FC = 2 - 1corr for the Curve.

[0121] For 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 the process. The fitted data showed that the affinity KD for the compound with GST-Smurf1 was 400.9 nM, and the affinity KD for CRBN-TBD was 21.06 μM (results are shown below). Figure 3 (As shown).

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

[0123] A key factor in whether compound SMART1 can mediate substrate ubiquitination is the induction of the formation of the target protein (Smurf1)-PROTAC-E3 ligase (CRBN) ternary complex. To detect whether our synthesized compound SMART1 can induce the formation of the Smurf1-PROTAC-CRBN ternary complex in cells, we performed immunoprecipitation and NanoBRET assays.

[0124] The immunoprecipitation experiment is as follows:

[0125] 1. Cell treatment: HEK293T cells were cultured in T25 flasks until 80% confluence, and the cells were treated with the proteasome inhibitor MG132 for 1 hour. Then, different concentrations of the compound were added to the cultured cells.

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

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

[0128] 4. Pre-cleaning: Add 20 μL of protein-A / G-agarose beads to the supernatant above, incubate on a rotary 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 total cell lysate, add 1 μg of Flag antibody to the remaining supernatant, and incubate on a rotary mixer at 4°C for 3 h;

[0130] 6. Antibody Protein A / G binding: Add 40 μL of Protein-A / G-agarose beads and incubate for 8 h at 4°C on a rotary mixer;

[0131] 7. Washing: Centrifuge at 1000 rpm for 3 min at 4℃, discard the supernatant, add 1 mL of cell lysis buffer to resuspend, and wash at 4℃ using a rotary mixer for 10 min. Repeat 3 times.

[0132] 8. Sample preparation and detection: Remove the supernatant from the washed agarose beads, add 40 μL of 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, boil in 100℃ water for 15 min, and then perform immunoblotting detection.

[0133] The NanoBRET experiment is as follows:

[0134] 1. Construct Smurf1- -CRBN vector;

[0135] 2. Cell transfection preparation: Culture HEK293T cells in 6-well plates until 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 another 24 hours;

[0137] 4. Cell preparation for detection: Collect transfected cells by trypsin digestion, wash once with PBS, and then use detection medium (Opti-). I. Adjust the cell density to 2 × 10⁶ cells / year using low serum-free medium (phenol red-free + 4% FBS). 5 cells / mL;

[0138] 5. Divide the cells into two parts and add as described below. NanoBRET TM 618 ligand or solvent DMSO:

[0139] Experimental sample (+ligand): Add 1 μL of 0.1 mM solution per milliliter of cells. NanoBRET TM 618 ligand (final concentration 100 nM).

[0140] Receptor-free control (–ligand): Add 1 μl DMSO per milliliter of cells (final concentration 0.1% DMSO).

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

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

[0143] 8. In Opti- NanoBRET was prepared in low serum medium (phenol red-free). TM Nano- 5X solution of Substrate;

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

[0145] 10. Within 10 minutes of adding the substrate, use with NanoBRET. TM PPI detection method compatible luminescence detector (CellTiter-) The 2.0 Assay (Promega) measures 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) of each sample by the donor emission value (e.g., 460 nm) to obtain the original NanoBRET. TM ratio;

[0147] 12. Multiply each original BRET value by 1000, that is, multiply the original NanoBRET value by 1000. TMConvert units (usually decimal values) to milliBRET units (mBU; integers);

[0148] 13. Determine the mean NanoBRET for each group of samples. TM Ratio: The sample includes samples containing NanoBRET TM Experimental samples with the 618 ligand and receptor-free control samples. Background or osmosis caused by the donor should also be considered; therefore, the average value of the experimental samples should be subtracted from the average value of the receptor-free control to obtain the corrected NanoBRET. TM ratio.

[0149] The results are as follows Figure 4 As shown, the presence of compound SMART1 effectively promotes the binding of Smurf1 to CRBN in a concentration-dependent manner, while the two ligands at both ends of compound SMART1, SMART1-L... Neither Poma nor Pomalidomide could induce Smurf1 to bind to CRBN. Figure 4 (As shown in Figure a) The NanoBRET experiment also confirmed that SMART1 promotes the binding of Smurf1 to CRBN in a concentration-dependent manner. Figure 4 (As shown in Figure b).

[0150] Example 4: Concentration-dependent assay for evaluating the bioactivity of compound SMART1 at the level of Western blotting.

[0151] 1. Cell plating: HCT116 cells in the logarithmic growth phase were used to prepare 10-1 cells. 4 The cell suspension was seeded at 1 mL / well into a 12-well plate and incubated overnight at 37°C for cell adhesion.

[0152] 2. Compound treatment: Culture media containing different concentrations of compound SMART1 (10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM, 0.003 μM, 0.001 μM, 0.0003 μM, 0.0001 μM) were prepared. The compounds were added to the cells by changing the culture medium. DMSO was added to the control group. The cells were cultured for another 12 h.

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

[0154] 4. The development results were obtained by grayscale scanning using Image J to obtain the grayscale values ​​of the internal components and the target protein;

[0155] 5. The acquired grayscale values ​​were statistically analyzed, and the DC values ​​of the target protein induced by the fitted compound were used in GraphPad. 50 .

[0156] Time-dependent experiments

[0157] 1. Cell plating: HCT116 cells in the logarithmic growth phase were used to prepare 10-1 cells. 4 The cell suspension was seeded at 1 mL / well into a 12-well plate and incubated overnight at 37°C for cell adhesion.

[0158] 2. Compound treatment: The compound was added to the cells at a final concentration of 0.1 μM and treated for different durations.

[0159] 3. Discard the culture medium, wash the cells once with PBS, and prepare and detect them using Western blotting.

[0160] The results are as follows Figure 5 As shown, compound SMART1 (SMART-1) can specifically and efficiently induce the degradation of Smurf1 in the nM concentration range, DC 50 =1.303 nM, without affecting the expression of Smurf2, a member of the same family ( Figure 5 As shown in Figures a and b), DIA quantitative mass spectrometry analysis further confirmed that compound SMART1 specifically induces the degradation of Smurf1. Figure 5 (As shown in Figure c).

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

[0162] 1. Cell Plating: Cells in the logarithmic growth phase (colorectal cancer cells HCT116, gastric cancer cells MGC-803, lung cancer cells H1299, cervical cancer cells HeLa, liver cancer cells Hep3B, LM3, pancreatic cancer cells SW1990, breast cancer cells MDA-MB-231) were plated in 10-1 saturates. 4 The cell suspension was seeded at 100 μL / well into a 96-well plate and incubated overnight at 37°C for cell adhesion.

[0163] 2. Compound treatment: Prepare culture media containing different concentrations of compound SMART1 (10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM, 0.003 μM, 0.001 μM, 0.0003 μM, 0.0001 μM). Add the compound to the cells by changing the culture medium. DMSO was added to the control group. Continue culturing.

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

[0165] 4. Measure the OD (450nm) value using an enzyme-linked immunosorbent assay (ELISA) reader;

[0166] 5. Using GraphPad to fit the IC50 of compounds that inhibit cell growth 50 .

[0167] The results are as follows Figure 6 As shown in Figure a, compound SMART1 exhibits growth-inhibiting effects in various tumor cell lines, with the most significant effect observed in HCT116, at an IC50 value of [missing value]. 50 =22.04 nM. Clonogenic assays further confirmed that SMART1 significantly inhibited clonogenicity in HCT116 cells. Figure 6 (As shown in Figure b); Cell cycle experiments revealed that SMART1-treated HCT116 cells were blocked in the G2-M phase.

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

[0169] To evaluate the pharmacokinetic properties of the compound in vivo and to explore the administration route and dosage for subsequent pharmacodynamic experiments, pharmacokinetic experiments were conducted using Kunming mice. Nine mice were randomly numbered and weighed, and then randomly divided into three groups using a parallel design: intravenous administration, oral administration, and intraperitoneal administration. Whole blood samples of approximately 20-30 μL were collected at 2 min, 5 min, 15 min, 30 min, 60 min, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration (intravenous) to determine the concentration of the compound. Following 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 values ​​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 values ​​were 27231.88±3442.81 (ng / mL), 171.49±42.19 (ng / mL), and 766.27, respectively. The values ​​were ±446.64 (ng / mL) and 3027.14±208.50 (ng / mL), respectively. The time to peak concentration (Tmax) was (0.03±0.00) h, (0.83±0.29) h, (0.50±0.00) h, and (0.03±0.00) h, respectively. The half-life (t1 / 2) was (1.80±0.32) h, (8.48±6.69) h, (2.10±0.90) h, and (1.21±0.16) h, respectively. The mean residence time (MRTlast) was (0.70±0.22) h, (3.20±1.58) h, (2.48±0.82) h, and (0.56±0.13) h, respectively. The results showed that the bioavailability of the compound administered by gavage in Kunming mice was 2.29%, and the bioavailability of the compound administered by intraperitoneal administration in Kunming mice 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 tumor formation in nude mice

[0172] 1. Cell Culture: Cell state is crucial for tumorigenesis experiments. Culture cells in the logarithmic growth phase (when cells reach approximately 80-90% confluence) and replace the culture medium with fresh one the night before collecting the cells.

[0173] 2. Cell collection and preparation: Digest cells with trypsin, stop digestion with complete culture medium, collect cells by centrifugation (800 rpm, 3 min), wash twice with pre-cooled PBS to remove serum from the 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 cell digestion, the cells should be subcutaneously injected into nude mice as soon as possible (generally within half an hour). Each nude mouse should be inoculated with 0.1 mL of cells. Tumors will form approximately one week later, with a tumor volume of about 100 mm. 3 The mice with tumors were grouped.

[0175] 4. Intraperitoneal administration: Compound SMART1 was dissolved in DMSO to prepare a 10 mg / mL compound solution. A solvent was prepared according to a castor oil:PBS ratio of 1:8. One part of the compound solution was added to nine parts of the solvent to prepare a clear solution of DMSO:castor oil:PBS = 1:1:8, with a compound concentration of 1 mg / mL. Mice were administered the solution intraperitoneally at a standard dose of 25 mg / kg. The control group received an equal volume of the solvent intraperitoneally.

[0176] 5. Administer the medication daily, while simultaneously measuring mouse body weight, recording the volume of the subcutaneous tumor, and summarizing the curves showing changes in mouse body weight and the volume of the subcutaneous tumor.

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

[0178] The results are as follows Figure 8 As shown, the results revealed that the compound SMART1 significantly inhibited the subcutaneous tumorigenicity of HCT116 cells in nude mice.

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

[0180] Six- to eight-week-old female CD-1 mice were treated intraperitoneally with different concentrations of compound SMART1 (dissolved in 10% DMSO, 10% castor oil, and 80% PBS). Mouse weight was measured daily. At the end of the experiment, mice were euthanized, and blood and major organs were collected for analysis. Toxicity studies included hematological, biochemical, and histological analyses.

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

[0182] In the above embodiments, the descriptions of each embodiment have different focuses. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0183] The above provides a detailed description of a Smurf1 protein degrader, its preparation method, and its uses, as provided in the embodiments of this application. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A compound represented by Formula (I): ###0001### (I) comprising the steps of: synthesizing an intermediate 2; synthesizing an intermediate 3; and synthesizing a compound represented by Formula (I) by combining the intermediate 2 with the intermediate 3. （Ⅰ）。 2. The process for the preparation of a compound according to claim 1, characterized in that, 2. The method of claim 1, wherein the compound is represented by Formula (I): ###0002### (I) comprising the steps of: synthesizing an intermediate 2; synthesizing an intermediate 3; and synthesizing a compound represented by Formula (I) by combining the intermediate 2 with the intermediate 3. Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione with piperidine-3-carboxylic acid to intermediate 1 ; Intermediate 1 was synthesized from 3-aminobenzonitrile and intermediate 2 was synthesized from intermediate 1 and 3-ethynylaniline ; Synthesis of 4-(4-aminophenoxy)-N-methylpicolinamide from intermediate 3 with azidotrimethylsilane ; 3. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound represented by Formula (I): ###0003### (I) or a pharmaceutically acceptable salt thereof.

3. A pharmaceutical composition, characterized by, 4. The pharmaceutical composition of claim 3, wherein the pharmaceutically acceptable carrier comprises at least one of a tablet, a capsule, a powder, a granule, a cream, a suppository, and a gel.

4. The pharmaceutical composition of claim 3, wherein, 5. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition is at least one of an injection and a spray.

5. The pharmaceutical composition of claim 3, wherein, 6. The use of a compound represented by Formula (I): ###0004### (I) or a pharmaceutically acceptable salt thereof, a pharmaceutical composition of any one of claims 3 to 5 in the manufacture of a medicament for treating a condition caused by accumulation of Smurf 1 protein.

6. The pharmaceutical composition of claim 3, wherein, 7. The use of a compound represented by Formula (I): ###0005### (I) or a pharmaceutically acceptable salt thereof, a pharmaceutical composition of any one of claims 3 to 5 in the manufacture of a medicament for treating a cancer, wherein the cancer comprises colorectal cancer, gastric cancer, lung cancer, cervical cancer, liver cancer, pancreatic cancer, and breast cancer.

8. The use of a compound represented by Formula (I): ###0006### (I) or a pharmaceutically acceptable salt thereof, a pharmaceutical composition of any one of claims 3 to 5 in the manufacture of a medicament for treating a cancer, wherein the cancer comprises colorectal cancer, gastric cancer, lung cancer, cervical cancer, liver cancer, pancreatic cancer, and breast cancer. ​ 9. A degrader targeting Smurf1 protein, characterized in that, ​

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