Compound for degrading WRN based on hydrophobic label technology and preparation method and application thereof

Abstract

The invention discloses a compound for degrading WRN based on a hydrophobic label technology and a preparation method and application thereof. The structural formula of the compound for targeted degradation of WRN is shown as a general formula (I). Compared with a WRN inhibitor, the compound provided by the invention can degrade WRN protein at a proper dosage. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a new bifunctional small molecule and its preparation method and application, and in particular to a compound for degrading WRN based on hydrophobic tag technology and its preparation method and use. Background Art

[0002] Microsatellite instability-high (MSI-H) is a genomic feature caused by defective mismatch repair (dMMR), with an incidence of approximately 4% in all cancers, but significantly higher in specific cancers such as endometrial cancer (30%), colorectal adenocarcinoma (20%), and gastric adenocarcinoma (20%). Although MSI-H tumors have a high response rate to immune checkpoint inhibitors, a large number of patients still cannot benefit, and new treatment strategies are urgently needed.

[0003] Werner syndrome RecQ helicase (WRN) is a member of the RecQ family, with 3′-5′ exonuclease and helicase activities, and plays a key role in DNA replication, repair, and maintenance of genomic stability. Recent studies have shown that there is a synthetic lethal effect between WRN and MSI-H tumors, and its helicase activity is crucial for resolving the secondary structure of MSI-H-related TA repeat sequences, making it a potential therapeutic target with great potential. Currently, companies such as Roche and Novartis have developed several WRN inhibitors, but only two have entered phase I clinical trials. In addition, studies have found that the non-enzymatic function of WRN regulates key life processes, and long-term inhibition may lead to drug-resistant mutations (such as the decrease in protein level and binding site mutations that occurred during the treatment with HRO-761), suggesting that targeted degradation of WRN may be more effective than simply inhibiting enzyme activity.

[0004] Existing methods for downregulating WRN (such as RNA interference and fusion protein degradation) are complex to operate, while small molecule degraders have significant advantages. Hydrophobic tag (HyT) technology, as a targeted protein degradation strategy, has the characteristics of small molecular weight, avoiding the teratogenic risk of thalidomide derivatives, and no significant "hook effect" compared with PROTACs. This technology binds to the target protein through covalent or high-affinity ligands, and connects hydrophobic tags to simulate the partially denatured state of the protein, thereby inducing degradation. Recent studies have confirmed the successful design of HyT degraders based on N-acyl-N-alkylsulfonamide (NASA) cleavable linkers, and the discovery of covalent WRN inhibitors provides a potential ligand basis for the development of HyT degraders. Summary of the Invention

[0005] Object of the Invention: The first object of the present invention is to provide a new bifunctional small molecule, the second object is to provide a preparation method of the compound, and the third object is to provide a pharmaceutical application of the compound and its pharmaceutical composition.

[0006] Technical solution: A compound for targeted degradation of WRN or a pharmaceutically acceptable salt thereof, and the structural formula of the compound for targeted degradation of WRN is shown as the general formula (I):

[0007]

[0008] Wherein, R is selected from:

[0009]

[0010] L is selected from -NH-C 1～15 linear alkyl, -NH-C 1～7 linear alkyl - O - C 1～7 linear alkyl,

[0011] Wherein R1 is selected from cyclobutyl, cyclopentyl, C 1～6 alkyl;

[0012] R2 is selected from C 1～6 alkyl.

[0013] The compound for targeted degradation of WRN or a pharmaceutically acceptable salt thereof, and L is selected from any one of the following structures:

[0014] L is selected from -NH-C 1～15 linear alkyl, -NH-C 1～7 linear alkyl - O - C 1～7 linear alkyl,

[0015] Wherein m is selected from 1 to 2; R1 is selected from C 1～4 alkyl;

[0016] R2 is selected from C 1～4 alkyl.

[0017] The compound for targeted degradation of WRN or a pharmaceutically acceptable salt thereof, and L is selected from any one of the following structures:

[0018]

[0019] A compound for targeted degradation of WRN or a pharmaceutically acceptable salt thereof, and the structural formula of the compound for targeted degradation of WRN is shown as any one of the following:

[0020]

[0021]

[0022] A method for preparing the compound for targeted degradation of WRN or a pharmaceutically acceptable salt thereof, characterized by comprising the following steps:

[0023]

[0024] The definitions of R and L are the same as above.

[0025] The method for preparing the compound for targeted degradation of WRN or a pharmaceutically acceptable salt thereof is specifically as follows:

[0026] Compound 1-1 and 1-2 are used to prepare compound 1-3 through the Mitsunobu reaction, and the reaction temperature is 0-100 °C;

[0027] Compound 1-3 and 4-chloro-2-(methylthio)-6-(trifluoromethyl)pyrimidine are used to obtain 1-4 through the Suzuki reaction, and the reaction temperature is 25 °C to 150 °C;

[0028] Compound 1-4 is subjected to acid hydrolysis for deprotection to prepare compound I-5, and the reaction temperature is 0-60 °C;

[0029] Compound 1-5 and R-COOH are subjected to a condensation reaction to obtain 1-6, and the reaction temperature is 0-100 °C;

[0030] Compound 1-6 is subjected to an oxidation reaction to obtain 1-7, and the oxidant used is selected from one or a mixture of two of potassium peroxymonosulfate compound, hydrogen peroxide, potassium permanganate, trifluoroperacetic acid, m-chloroperbenzoic acid, ozone, and the reaction temperature is 0-60 °C;

[0031] Compound 1-7 is added with benzyl mercaptan for a substitution reaction to obtain 1-8, and the reaction temperature is 20-120 °C;

[0032] Compound 1-8 is subjected to an oxidation reaction to obtain 1-9, and the oxidant used is selected from chlorine, sulfonyl chloride, and the reaction temperature is -20-40 °C;

[0033] Compound 1-9 and 2-amino-N-(p-tolyl)acetamide are subjected to acylation to obtain compound (I).

[0034] For the compound for targeted degradation of WRN or a pharmaceutically acceptable salt thereof, the pharmaceutically acceptable salt is an acid addition salt formed by the compound and any one of the following acids: hydrogen chloride, hydrogen bromide, sulfuric acid, carbonic acid, oxalic acid, citric acid, malic acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or ferulic acid.

[0035] A pharmaceutical composition comprising the compound for targeted degradation of WRN or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0036] Use of the compound for targeted degradation of WRN or a pharmaceutically acceptable salt thereof, or the said pharmaceutical composition, in the preparation of a medicament for degrading WRN protein.

[0037] Use of the compound for targeted degradation of WRN or a pharmaceutically acceptable salt thereof, or the said pharmaceutical composition, in the preparation of a medicament for treating tumors.

[0038] Preferably, the preparation method comprises the following steps:

[0039] Compound 1-3 is prepared from compound 1-1 and 1-2 through Mitsunobu reaction. The solvent used is one of dichloromethane, ethyl acetate, acetone, tetrahydrofuran, dichloromethane, acetonitrile, toluene, ethylene glycol dimethyl ether, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide or dimethyl sulfoxide, or a mixture of any two of them, preferably tetrahydrofuran; the oxidation reagent used is selected from one of diethyl azodicarboxylate, diisopropyl azodicarboxylate, di-tert-butyl azodicarboxylate, dicyclohexyl azodicarboxylate, or a combination of any two of them, preferably diisopropyl azodicarboxylate. The reaction temperature is 0 - 100 °C, preferably 40 - 60 °C.

[0040] Compound 1-4 is obtained from compound 1-3 through Suzuki reaction. The solvent used is selected from one of tetrahydrofuran, acetonitrile, N-methylpyrrolidone, N,N-dimethylacetamide, dimethyl sulfoxide, N,N-dimethylformamide, 1,4-dioxane or water, or a mixture of any two of them, preferably a mixed solvent of 1,4-dioxane and water at a ratio of 5:1; the base used is selected from potassium bicarbonate, potassium carbonate, sodium bicarbonate, sodium carbonate, potassium phosphate, sodium acetate, triethylamine or N,N-diisopropylethylamine, preferably potassium phosphate; the catalyst used is selected from tetrakis(triphenylphosphine)palladium (Pd(PPh3)4), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (Pd(dppf)Cl2), palladium acetate (Pd(OAc)2), tris(dibenzylideneacetone)dipalladium (Pd2(dba)3) or bis(triphenylphosphine)dichloropalladium (Pd(PPh3)2Cl2), preferably Pd(dppf)Cl2; the reaction temperature is 25 °C to 150 °C, preferably 60 - 100 °C.

[0041] Compound I-5 is prepared by acid hydrolysis and deprotection of compound 1-4. The acid used is hydrogen chloride, trifluoroacetic acid, trifluoromethanesulfonic acid, sulfuric acid, hydrobromic acid or phosphoric acid, preferably hydrogen chloride; the reaction solvent used is one of ethyl acetate, tetrahydrofuran, dioxane, methanol, ethanol or ether, or a mixture of any two of them, preferably ethyl acetate; the reaction temperature is 0 - 60 °C, preferably 20 - 30 °C.

[0042] Compound 1-6 was obtained by the condensation reaction of compounds 1-5. The solvents used were tetrahydrofuran, acetonitrile, N-methylpyrrolidone, N,N-dimethylacetamide, dimethyl sulfoxide, N,N-dimethylformamide, 1,4-dioxane, or a mixture of any two of them. The condensing agents used were N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), 1-hydroxybenzotriazole (HOBt), propylphosphonic anhydride (T3P), and cyanuric chloride, preferably N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU). The bases used were selected from triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium hydride, potassium tert-butoxide, sodium methoxide, or sodium ethoxide, preferably N,N-diisopropylethylamine. The reaction temperature was 0-100°C, preferably 15-35°C.

[0043] Compound 1-7 was obtained by the oxidation reaction of compound 1-6. The solvents used were dichloromethane, ethyl acetate, acetone, tetrahydrofuran, dichloromethane, acetonitrile, toluene, ethylene glycol dimethyl ether, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, or dimethyl sulfoxide, preferably dichloromethane. The oxidizing agents used were selected from potassium peroxymonosulfate compound salt, hydrogen peroxide, potassium permanganate, trifluoroperacetic acid, m-chloroperbenzoic acid, ozone, or a mixture of any two of them, preferably m-chloroperbenzoic acid. The reaction temperature was 0-60°C, preferably 20-30°C.

[0044] Compound 1-8 was obtained by the substitution reaction of compound 1-7. The bases used were selected from triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium hydride, potassium tert-butoxide, sodium methoxide, or sodium ethoxide, preferably triethylamine. The solvents used were dichloromethane, ethyl acetate, acetone, tetrahydrofuran, dichloromethane, acetonitrile, toluene, ethylene glycol dimethyl ether, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, or dimethyl sulfoxide, preferably acetonitrile. The reaction temperature was 20-120°C, preferably 40-80°C.

[0045] Compound 1-9 is obtained from compounds 1-8 through an oxidation reaction. The oxidizing agent used is selected from chlorine gas, sulfonyl chloride, preferably sulfonyl chloride; the solvent is selected from dichloromethane, ethyl acetate, acetone, tetrahydrofuran, dichloromethane, acetonitrile, toluene, ethylene glycol dimethyl ether, 1,4-dioxane, water, or a mixture of any two of them, preferably a mixture of dichloromethane and water at a ratio of 5:1; the acid used is selected from hydrogen chloride, trifluoroacetic acid, trifluoromethanesulfonic acid, sulfuric acid, hydrobromic acid, or phosphoric acid, acetic acid, strongly acidic cation exchange resin, or a mixture of any two of them; the reaction temperature is -20 - 40°C, preferably 0 - 20°C.

[0046] Compound 1-H (i.e., general formula I) is obtained by acylating compound 1-9. The base used is selected from triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium hydride, potassium tert-butoxide, sodium methoxide, or sodium ethoxide, preferably triethylamine; the solvent used is selected from dichloromethane, ethyl acetate, acetone, tetrahydrofuran, dichloromethane, acetonitrile, toluene, ethylene glycol dimethyl ether, 1,4-dioxane, water, or a mixture of any two of them, preferably ethyl acetate.

[0047] Among them, the definitions of R and L are as described above.

[0048] The above-prepared compound 1-H is formed into a salt with a pharmaceutically acceptable acid to obtain the pharmaceutically acceptable salt described herein.

[0049] The pharmaceutical composition of the present invention comprises the compound of the present invention or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be excipients widely used in the field of drug production. The excipients are mainly used to provide a safe, stable, and functional pharmaceutical composition, and can also provide methods to enable the active ingredient to dissolve at a desired rate after the subject receives the administration, or to promote the effective absorption of the active ingredient after the subject receives the administration of the composition. The pharmaceutical excipients can be inert fillers or provide certain functions, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipients can include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesion agents, glidants, wetting agents, gelling agents, absorption retardants, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0050] The pharmaceutical composition of the present invention can be prepared by any method known to those skilled in the art according to the disclosed content. For example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding, or freeze-drying processes.

[0051] The pharmaceutical composition of the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be in a controlled-release or sustained-release dosage form (such as liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, cachets, soft capsules and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, injectable solutions, dry powder preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, injectable suspensions and injectable emulsions. Examples of other suitable preparations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalants; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

[0052] The compound or its pharmaceutically acceptable salt or its pharmaceutical composition of the present invention is used in the preparation of a drug for degrading WRN protein. Preferably, the drug is a drug for treating tumors. More preferably, the drug is a drug for treating colorectal cancer, gastric cancer, prostate cancer, endometrial cancer, adrenocortical cancer, uterine cancer, cervical cancer, esophageal cancer, breast cancer, kidney cancer or ovarian cancer.

[0053] Hydrates, solvates or crystals of the compound of general formula (I) of the present invention and their applications in the preparation of anti-tumor drugs are also within the protection scope of the present invention.

[0054] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The compound designed by the present invention can significantly inhibit the proliferation of tumor cells, and the inhibition activity reaches the micromolar concentration level. It has the dual functions of targeted inhibition and degradation, so as to improve the drug resistance and toxicity of anti-tumor drugs. Description of the Drawings

[0055] Figure 1 It is a result diagram of the effect of the compound on the expression of WRN in HCT-116 cells;

[0056] Figure 2 It is a result diagram of the effect of the compound on the cell viability of HCT-116 cells. Detailed Embodiments

[0057] In the examples, the NMR spectral data of the end products and intermediates were measured on a 300 MHz or 400 MHz nuclear magnetic resonance spectrometer of Bruker company, using DMSO-d6 or CDCl3-d3 as the solvent and TMS as the internal standard; the high-resolution mass spectrometry (HRMS) was measured on a mass spectrometer of model Q-TOF 6520 of Agilent company.

[0058] The reagents used in the synthesis, purification and separation of the compounds are as follows: (1) Column chromatography silica gel: 200 or 300 mesh silica gel was purchased from Qingdao Ocean Chemical Industry; (2) HSGF254 type TLC thin layer chromatography plate: purchased from Yantai Chemical Industry Research Institute; (3) Conventional solvents such as petroleum ether, dichloromethane, ethyl acetate, methanol, etc. used in the column chromatography elution system and chemical reagents required for the reaction are commercially available chemical pure or analytical pure products except as otherwise specified.

[0059] Example 1:

[0060] Step 1: Synthesis of tert-butyl (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxymethyl)carbamate. Under a nitrogen atmosphere, an anhydrous tetrahydrofuran solution of compound 1-1, tert-butyl (2-hydroxyethyl)carbamate and PPh3 was mixed with DIAD for 5 minutes. The reaction was stirred at 50 °C until the starting materials completely disappeared (monitored by thin layer chromatography). After quenching the reaction with water at room temperature, the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography to obtain compound 1-3. 1 H NMR(300MHz, Chloroform-d)δ7.86-7.69(m, 2H), 6.95-6.84(m, 2H), 4.05(t, J = 5.1Hz, 2H), 3.55(q, J = 5.4Hz, 2H), 1.46(s, 9H), 1.35(s, 12H).

[0061] Step 2: tert-Butyl ((4-(2-(methylthio)-6-(trifluoromethyl)pyrimidin-4-yl)phenoxy)methyl)carbamate. 4-Chloro-2-(methylthio)-6-(trifluoromethyl)pyrimidine, compound 1-3, Pd(PPh3)2Cl2 and K3PO4 were added to a two-necked flask. After adding a 5:1 mixture of 1,4-dioxane and water, the flask was degassed under a nitrogen atmosphere. After stirring at 100 °C for 2 - 5 hours, the reaction mixture was cooled to room temperature and extracted with ethyl acetate. After concentrating the organic layer, it was purified by column chromatography to obtain compound 1-4. 11H NMR (300 MHz, Chloroform-d) δ 8.19 - 8.09 (m, 2H), 7.59 (s, 1H), 7.04 - 7.00 (m, 2H), 4.12 (t, J = 5.1 Hz, 2H), 3.60 (q, J = 5.5 Hz, 2H), 2.68 (s, 3H), 1.49 (s, 9H).

[0062] Step 3: (4-(2-(Methylthio)-6-(trifluoromethyl)pyrimidin-4-yl)phenoxy)methanamine. Dissolve compound 1-4 in anhydrous dichloromethane, and then add trifluoroacetic acid; the mixture is stirred at room temperature for 0.5 - 1.5 hours, and then the solution is concentrated under reduced pressure to obtain compound 1-5. The crude product enters the next step without further purification. 1 1H NMR (300 MHz, DMSO-d6) δ 8.49 (s, 2H), 8.34 (d, J = 8.6 Hz, 2H), 8.15 (s, 1H), 7.16 (d, J = 8.7 Hz, 2H), 4.34 (t, J = 5.0 Hz, 2H), 3.24 (d, J = 5.2 Hz, 2H), 2.63 (s, 3H).

[0063] Step 4: 2-((3R,5R,7R)-Adamantan-1-yl)-N-((4-(2-(methylthio)-6-(trifluoromethyl)pyrimidin-4-yl)phenoxy)methyl)acetamide. Dissolve 2-((3r,5r,7r)-adamantan-1-yl)acetic acid in N,N-dimethylformamide (DMF), and then add HATU and diisopropylethylamine (DIPEA). After stirring at room temperature for 30 minutes under nitrogen protection, add compound 1-5 to the mixture and continue stirring overnight. The reaction mixture is poured into water and extracted with ethyl acetate. The organic layer is washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain compound 1-6. 1 1H NMR (300 MHz, Chloroform-d) δ 8.06 (d, J = 8.9 Hz, 2H), 7.52 (s, 1H), 6.96 (d, J = 8.9 Hz, 2H), 6.01 (t, J = 5.9 Hz, 1H), 4.10 (dd, J = 6.1, 4.2 Hz, 2H), 3.67 (q, J = 5.4 Hz, 2H), 2.62 (s, 3H), 1.95 (s, 2H), 1.89 (q, J = 3.1 Hz, 3H), 1.71 - 1.48 (m, 12H).

[0064] Step 5: 2-((3R,5R,7R)-Adamantan-1-yl)-N-((4-(2-(methylsulfonyl)-6-(trifluoromethyl)pyrimidin-4-yl)phenoxy)methyl)acetamide. M-CPBA was added to the anhydrous dichloromethane solution of Compound 1-6 at room temperature and stirred. After the reaction was completed, saturated sodium bicarbonate solution and saturated sodium thiosulfate solution were added to the product, and the layers were separated. The organic layer was collected, washed with saturated sodium thiosulfate solution and saturated sodium bicarbonate solution, dried over sodium sulfate, and concentrated under reduced pressure to obtain Compound 1-7. 1 H NMR(300MHz, Chloroform-d)δ8.33 - 8.18(m, 2H), 8.10(s, 1H), 7.15 - 6.98(m, 2H), 5.86(s, 1H), 4.18(t, J = 5.1Hz, 2H), 3.73(q, J = 5.5Hz, 2H), 3.48(s, 3H), 1.99(s, 2H), 1.94(s, 3H), 1.71 - 1.53(m, 12H).

[0065] Step 6: 2-((3R,5R,7R)-Adamantan-1-yl)-N-((4-(2-(benzylthio)-6-(trifluoromethyl)pyrimidin-4-yl)phenoxy)methyl)acetamide. In a round-bottom flask equipped with a stir bar, acetonitrile, benzyl mercaptan, triethylamine, and Compound 1-7 were mixed. The reaction mixture was stirred at 80 °C for 0.5 - 1 hour. After the reaction was completed, the volatiles were removed under vacuum. The resulting residue was purified by column chromatography to obtain Compound 1-8. 1 H NMR(300MHz, Chloroform-d)δ8.11 - 7.95(m, 1H), 7.54(d, J = 1.7Hz, 1H), 7.51 - 7.43(m, 1H), 7.29(ddd, J = 13.0, 7.9, 6.3Hz, 2H), 7.03 - 6.91(m, 1H), 4.48(s, 1H), 4.11(d, J = 5.1Hz, 1H), 3.69(q, J = 5.4Hz, 1H), 1.98(s, 1H), 1.93(q, J = 3.1Hz, 2H), 1.73 - 1.53(m, 6H).

[0066] Step 7: 4-(4-((2-((3R,5R,7R)-adamantan-1-yl)acetamido)methoxy)phenyl)-6-(trifluoromethyl)pyrimidine-2-sulfonyl chloride. Sulfuryl chloride was added to a vigorously stirred solution of Compound 1-8 and strongly acidic C-type cation exchange resin in dichloromethane / water (10:1). The mixture was stirred for 0.5 - 3 hours at room temperature in the dark. After completion of the reaction, the mixture was filtered, dried over anhydrous sodium sulfate, and then the volatiles were removed under vacuum to obtain 1-9 sulfonyl chloride. The crude 1-9 was directly carried into the next step without further purification.

[0067] Step 8: 2-((3R,5R,7R)-adamantan-1-yl)-N-((4-(2-(N-(2-oxo-2-(p-toluidino)ethyl)sulfamoyl)-6-(trifluoromethyl)pyrimidin-4-yl)phenoxy)methyl)acetamide. 1-9 was added to a solution of 2-amino-N-(p-tolyl)acetamide in ethyl acetate with stirring in the dark. The mixture was stirred overnight at room temperature, and then the volatiles were removed under vacuum. The resulting residue was purified by silica gel column chromatography to obtain the target compound 1-H-1. 1 H NMR (300 MHz, DMSO-d6) δ 9.94 (s, 1H), 8.67 (s, 2H), 8.39 (d, J = 8.6 Hz, 2H), 7.98 (t, J = 5.6 Hz, 1H), 7.30 (d, J = 8.1 Hz, 2H), 7.04 (t, J = 9.0 Hz, 4H), 4.18 - 4.04 (m, 4H), 3.45 (q, J = 5.3 Hz, 2H), 2.22 (s, 3H), 1.86 (s, 5H), 1.62 (d, J = 12.5 Hz, 3H), 1.55 (d, J = 3.0 Hz, 9H). 13 C NMR (101 MHz, DMSO-d6) δ 170.30, 167.25, 166.20, 166.10, 162.62, 136.09, 132.21, 130.31, 129.02, 126.19, 119.04, 115.04, 114.03, 66.75, 50.00, 46.38, 32.19, 28.06, 20.42.

[0068] Example 2: The tert-butyl (2-hydroxyethyl)carbamate in Step 1 of Example 1 was replaced with tert-butyl (4-hydroxybutyl)carbamate, and the remaining steps were kept unchanged to obtain the target compound 1-H-2. 11H NMR (300 MHz, DMSO-d6) δ 9.94 (s, 1H), 8.67 (d, J = 4.2 Hz, 2H), 8.37 (d, J = 8.9 Hz, 2H), 7.75 (t, J = 5.6 Hz, 1H), 7.29 (d, J = 8.4 Hz, 2H), 7.02 (dd, J = 8.8, 3.2 Hz, 4H), 4.18 - 3.94 (m, 4H), 3.09 (q, J = 6.6 Hz, 2H), 2.21 (s, 3H), 1.89 (s, 3H), 1.82 (s, 2H), 1.79 - 1.69 (m, 2H), 1.69 - 1.48 (m, 14H). 13 13C NMR (101 MHz, DMSO-d6) δ 170.26, 166.64, 136.56, 129.45, 119.48, 115.46, 50.57, 46.83, 42.62, 38.41, 36.92, 32.62, 28.51, 26.60, 26.32, 20.86.

[0069] Example 3: Replace the tert-butyl (2-hydroxyethyl) carbamate in Step 1 of Example 1 with tert-butyl (5-hydroxypentyl) carbamate, and keep the remaining steps unchanged to obtain the target compound 1-H-3. 1 1H NMR (400 MHz, Methanol-d4) δ 8.35 (s, 1H), 8.26 (d, J = 9.0 Hz, 2H), 7.28 (d, J = 8.5 Hz, 2H), 7.03 (d, J = 8.4 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 4.17 (s, 2H), 4.03 (t, J = 6.3 Hz, 2H), 3.22 (t, J = 6.6 Hz, 2H), 2.27 (s, 3H), 1.94 (s, 5H), 1.84 (p, J = 6.5 Hz, 2H), 1.74 (d, J = 12.1 Hz, 3H), 1.70 - 1.63 (m, 9H), 1.57 (tdd, J = 13.1, 11.1, 9.0, 5.7 Hz, 4H). 13 13C NMR (101 MHz, Methanol-d4) δ 173.80, 168.45, 164.80, 136.74, 134.91, 131.23, 130.15, 127.60, 121.12, 116.16, 114.65, 69.26, 51.92, 47.86, 47.86, 43.77, 40.14, 37.90, 33.79, 30.26, 30.16, 29.86, 24.62, 20.93.

[0070] Example 4: Replace tert-butyl (2-hydroxyethyl) carbamate in Step 1 of Example 1 with tert-butyl (6-hydroxyhexyl) carbamate, and keep the remaining steps unchanged to obtain the target compound 1-H-4. 1 H NMR (300 MHz, DMSO-d6) δ 9.95 (s, 1H), 8.68 (s, 1H), 8.65 (s, 1H), 8.37 (d, J = 8.8 Hz, 2H), 7.67 (t, J = 5.4 Hz, 1H), 7.39 - 7.21 (m, 2H), 7.01 (d, J = 8.0 Hz, 4H), 4.35 - 3.78 (m, 4H), 3.03 (q, J = 6.2 Hz, 2H), 2.20 (s, 3H), 1.88 (s, 3H), 1.80 (s, 2H), 1.78 - 1.68 (m, 2H), 1.58 (d, J = 19.0 Hz, 13H), 1.47 - 1.33 (m, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 169.73, 166.14, 155.55, 132.16, 130.29, 129.00, 119.04, 67.89, 50.14, 42.18, 38.22, 36.50, 32.17, 29.18, 28.57, 28.09, 26.25.25.21.20.41.

[0071] Example 5: Replace tert-butyl (2-hydroxyethyl) carbamate in Step 1 of Example 1 with tert-butyl (7-hydroxyheptyl) carbamate, and keep the remaining steps unchanged to obtain the target compound 1-H-5. 1 H NMR (300 MHz, Methanol-d4) δ 8.35 (s, 1H), 8.26 (d, J = 8.9 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 8.3 Hz, 2H), 6.95 (d, J = 8.9 Hz, 2H), 4.18 (s, 2H), 4.02 (t, J = 6.4 Hz, 2H), 3.18 (td, J = 6.6, 2.3 Hz, 2H), 2.27 (s, 3H), 1.94 (d, J = 5.3 Hz, 5H), 1.84 - 1.77 (m, 2H), 1.75 - 1.61 (m, 12H), 1.58 - 1.39 (m, 8H). 13C NMR (101 MHz, Methanol-d4) δ 173.75, 169.58, 168.45, 164.84, 136.74, 134.90, 131.23, 130.14, 121.12, 116.16, 114.60, 69.34, 51.92, 47.86, 43.77, 37.91, 33.78, 30.44, 30.16, 30.13, 30.07, 27.97, 27.05, 20.93.

[0072] Example 6: Replace the tert-butyl (2-hydroxyethyl) carbamate in Step 1 of Example 1 with tert-butyl (8-hydroxyoctyl) carbamate, and keep the remaining steps unchanged to obtain the target compound 1-H-6. 1 H NMR (400 MHz, Methanol-d4) δ 8.35 (s, 1H), 8.26 (d, J = 9.0 Hz, 2H), 7.28 (d, J = 8.5 Hz, 2H), 7.02 (d, J = 8.4 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 4.18 (s, 2H), 4.02 (t, J = 6.4 Hz, 2H), 3.18 (td, J = 6.8, 4.8 Hz, 2H), 2.27 (s, 3H), 1.94 (d, J = 7.2 Hz, 5H), 1.85 - 1.77 (m, 2H), 1.74 (d, J = 12.1 Hz, 3H), 1.70 - 1.61 (m, 9H), 1.52 (q, J = 8.8, 7.9 Hz, 4H), 1.40 (p, J = 5.0 Hz, 6H). 13 C NMR (101 MHz, Methanol-d4) δ 173.70, 168.41, 164.80, 134.84, 131.20, 130.14, 127.44, 121.08, 116.12, 114.59, 69.33, 51.90, 47.86, 43.73, 40.23, 37.88, 33.76, 30.47, 30.41, 30.31, 30.19, 30.13, 27.98, 27.03, 20.95.

[0073] Example 7: Replace the tert-butyl (2-hydroxyethyl) carbamate in Step 1 of Example 1 with tert-butyl (10-hydroxydecyl) carbamate, and keep the remaining steps unchanged to obtain the target compound 1-H-7. 11H NMR (300 MHz, Methanol-d4) δ 8.34 (s, 1H), 8.25 (d, J = 8.9 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 4.18 (s, 2H), 4.01 (t, J = 6.4 Hz, 2H), 3.21 - 3.11 (m, 2H), 2.27 (s, 3H), 1.94 (d, J = 7.0 Hz, 5H), 1.82 - 1.76 (m, 2H), 1.75 - 1.61 (m, 12H), 1.50 (q, J = 6.8 Hz, 4H), 1.36 (d, J = 6.3 Hz, 10H). 13 13C NMR (101 MHz, Methanol-d4) δ 173.74, 169.62, 168.46, 164.88, 134.90, 131.25, 130.15, 127.58, 121.12, 116.18, 69.39, 51.93, 43.77, 40.25, 37.92, 33.79, 30.63, 30.60, 30.50, 30.45, 30.35, 30.22, 30.18, 28.05, 27.10, 20.93.

[0074] Example 8: Replace the tert-butyl (2-hydroxyethyl) carbamate in Step 1 of Example 1 with tert-butyl (12-hydroxydodecyl) carbamate, and keep the remaining steps unchanged to obtain the target compound 1-H-8. 1 1H NMR (400 MHz, Methanol-d4) δ 8.35 (d, J = 1.3 Hz, 1H), 8.30 - 8.21 (m, 2H), 7.35 - 7.21 (m, 2H), 7.02 (d, J = 8.2 Hz, 2H), 6.95 (d, J = 8.7 Hz, 2H), 4.18 (s, 2H), 4.02 (t, J = 6.4 Hz, 2H), 3.16 (t, J = 6.9 Hz, 2H), 2.27 (s, 3H), 1.94 (d, J = 10.2 Hz, 5H), 1.85 - 1.71 (m, 5H), 1.66 (dd, J = 16.3, 2.5 Hz, 9H), 1.50 (dp, J = 15.4, 6.3, 5.2 Hz, 4H), 1.34 (d, J = 10.5 Hz, 14H). 1313C NMR (101 MHz, Methanol-d4) δ 173.70, 169.56, 168.43, 164.85, 136.74, 131.22, 130.14, 127.51, 121.11, 116.17, 69.38, 51.92, 47.87, 43.76, 40.25, 37.91, 33.77, 30.70, 30.67, 30.65, 30.50, 30.45, 30.38, 30.21, 30.17, 28.06, 27.10, 20.94.

[0075] Example 9: Replace the tert-butyl (2-hydroxyethyl) carbamate in Step 1 of Example 1 with tert-butyl (5-hydroxypentyl) carbamate, and replace 2-((3r,5r,7r)-adamantan-1-yl) acetic acid in Step 4 with 2-((2R,5S)-2-isopropyl-5-methylcyclohexyloxy) acetic acid, and keep the remaining steps unchanged to obtain the target compound 1-H-9. 1 1H NMR (400 MHz, Methanol-d4) δ 8.29 (s, 1H), 8.21 (d, J = 9.0 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 8.5 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 4.18 (s, 2H), 4.04 (d, J = 15.2 Hz, 1H), 3.98 (t, J = 6.3 Hz, 2H), 3.88 (d, J = 15.1 Hz, 1H), 3.30 (td, J = 6.8, 1.9 Hz, 2H), 3.18 (td, J = 10.6, 4.1 Hz, 1H), 2.24 (s, 3H), 2.19 (qd, J = 7.0, 2.8 Hz, 1H), 2.06 (ddd, J = 12.1, 5.7, 3.8 Hz, 1H), 1.81 (p, J = 6.6 Hz, 2H), 1.68 - 1.56 (m, 4H), 1.56 - 1.44 (m, 3H), 1.36 - 1.25 (m, 2H), 1.02 - 0.96 (m, 1H), 0.92 (s, 3H), 0.90 (s, 3H), 0.89 - 0.86 (m, 1H), 0.79 (d, J = 6.9 Hz, 3H). 1313C NMR (151 MHz, Methanol-d4) δ 171.70, 168.10, 167.00, 166.32, 163.38, 156.52, 156.28, 135.35, 133.51, 129.85, 128.80, 126.13, 119.75, 119.54, 114.83, 113.23, 113.21, 80.42, 67.87, 67.41, 47.87, 46.57, 39.89, 38.38, 34.21, 31.27, 28.86, 28.45, 25.69, 23.04, 23.02, 21.31, 20.00, 19.61, 15.35.

[0076] Example 10: Replace the tert-butyl(2-hydroxyethyl)carbamate in Step 1 of Example 1 with tert-butyl(5-hydroxypentyl)carbamate, and replace 2-((3r,5r,7r)-adamantan-1-yl)acetic acid in Step 4 with (S)-2-(4-isobutylphenyl)propanoic acid, and keep the remaining steps unchanged to obtain the target compound 1-H-10. 1 1H NMR (300 MHz, Methanol-d4) δ 8.37 (s, 1H), 8.27 (d, J = 8.9 Hz, 2H), 7.26 (t, J = 7.8 Hz, 4H), 7.08 (d, J = 8.1 Hz, 2H), 7.02 (d, J = 8.2 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 4.18 (s, 2H), 3.97 (t, J = 6.4 Hz, 2H), 3.61 (q, J = 7.0 Hz, 1H), 3.20 (dp, J = 20.2, 6.7 Hz, 2H), 2.42 (d, J = 7.2 Hz, 2H), 2.26 (s, 3H), 1.79 (dp, J = 10.4, 6.6 Hz, 3H), 1.62 - 1.50 (m, 2H), 1.44 (d, J = 7.1 Hz, 5H), 0.86 (d, J = 6.6 Hz, 6H). 13 13C NMR (151 MHz, Methanol-d4) δ 175.89, 168.24, 167.07, 166.39, 163.43, 156.61, 156.37, 140.05, 139.12, 135.33, 133.56, 129.84, 128.84, 128.75, 126.71, 126.24, 121.39, 119.77, 114.81, 113.24, 113.22, 67.86, 46.47, 45.87, 44.61, 38.76, 30.01, 28.64, 28.37, 22.88, 21.27, 19.50, 17.37.

[0077] Example 11: Replace the tert-butyl (2-hydroxyethyl) carbamate in Step 1 of Example 1 with tert-butyl (5-hydroxypentyl) carbamate, and replace 2-((3r,5r,7r)-adamantan-1-yl) acetic acid in Step 4 with 9H-fluorene-9-carboxylic acid, and keep the remaining steps unchanged to obtain the target compound 1-H-11. 1 H NMR (600 MHz, DMSO-d6) δ 8.65 (s, 1H), 8.38 (d, J = 8.9 Hz, 2H), 7.87 (d, J = 7.5 Hz, 2H), 7.51 (d, J = 7.5 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.31 (td, J = 5.0, 4.0, 2.3 Hz, 4H), 7.04 (dd, J = 16.4, 8.5 Hz, 4H), 4.80 (s, 1H), 4.08 - 4.05 (m, 4H), 3.17 (d, J = 6.6 Hz, 2H), 2.21 (s, 3H), 1.78 (p, J = 6.7 Hz, 2H), 1.60 - 1.53 (m, 2H), 1.49 (p, J = 7.4 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 169.71, 167.68, 166.48, 163.19, 155.85, 155.61, 143.38, 141.69, 136.44, 132.54, 130.66, 129.34, 127.95, 127.51, 126.37, 125.09, 121.70, 120.43, 119.87, 119.43, 115.39, 114.33, 68.28, 60.09, 46.73, 38.96, 29.23, 28.58, 23.26, 20.74.

[0078] Example 12: Replace the tert-butyl (2-hydroxyethyl) carbamate in Step 1 of Example 1 with tert-butyl (5-hydroxypentyl) carbamate, and replace 2-((3r,5r,7r)-adamantan-1-yl) acetic acid in Step 4 with 2-((1R,3R,5S,7R)-3,5-dimethyladamantan-1-yl) acetic acid, and keep the remaining steps unchanged to obtain the target compound 1-H-12. 11H NMR (300 MHz, Methanol-d4) δ 8.36 (s, 1H), 8.26 (d, J = 8.9 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 7.03 (d, J = 8.3 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 4.17 (s, 2H), 4.04 (t, J = 6.3 Hz, 2H), 3.22 (t, J = 6.4 Hz, 2H), 2.27 (s, 3H), 2.05 (p, J = 3.1 Hz, 1H), 1.98 (s, 2H), 1.84 (p, J = 6.5 Hz, 2H), 1.58 (dp, J = 11.5, 6.3, 5.3 Hz, 4H), 1.47 (d, J = 2.9 Hz, 2H), 1.37 - 1.18 (m, 10H), 0.81 (s, 6H). 13 13C NMR (151 MHz, Methanol-d4) δ 172.43, 168.21, 167.07, 166.36, 163.42, 156.59, 156.35, 135.32, 133.56, 129.85, 128.77, 126.25, 119.78, 119.56, 114.83, 113.25, 113.24, 67.93, 50.68, 49.85, 48.72, 46.49, 46.49, 42.74, 40.88, 38.75, 34.02, 30.85, 29.80, 29.73, 28.84, 28.49, 23.23, 19.53.

[0079] Example 13: Replace the tert-butyl (2-hydroxyethyl) carbamate in Step 1 of Example 1 with tert-butyl (5-hydroxypentyl) carbamate, and replace 2-((3r,5r,7r)-adamantan-1-yl) acetic acid in Step 4 with (3s,5s,7s)-3,5,7-trifluoroadamantane-1-carboxylic acid, and keep the rest of the steps unchanged to obtain the target compound 1-H-13. 11H NMR (300 MHz, Methanol-d4) δ 8.36 (s, 1H), 8.27 (d, J = 7.2 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 7.03 (d, J = 8.2 Hz, 2H), 6.97 (d, J = 7.5 Hz, 2H), 4.17 (s, 2H), 4.08 - 3.99 (m, 2H), 3.26 (t, J = 6.8 Hz, 2H), 2.27 (s, 3H), 2.16 (p, J = 4.5 Hz, 3H), 2.05 (dq, J = 9.4, 5.2, 4.8 Hz, 3H), 1.98 - 1.92 (m, 6H), 1.84 (p, J = 6.6 Hz, 2H), 1.61 (q, J = 7.8, 6.9 Hz, 2H), 1.57 - 1.48 (m, 2H). 13 13C NMR (151 MHz, Methanol-d4) δ 174.14, 168.22, 167.08, 166.35, 163.41, 156.59, 156.35, 135.32, 133.58, 129.86, 128.76, 126.26, 119.78, 114.82, 113.26, 113.25, 67.77, 46.47, 45.84, 45.72, 45.59, 41.81, 41.69, 39.15, 28.62, 28.43, 22.93, 19.51.

[0080] Example 14: Replace the tert-butyl (2-hydroxyethyl) carbamate in Step 1 of Example 1 with tert-butyl (5-hydroxypentyl) carbamate, and replace 2-((3r,5r,7r)-adamantan-1-yl) acetic acid in Step 4 with 2,2-diphenylacetic acid, and keep the remaining steps unchanged to obtain the target compound 1-H-14. 1 1H NMR (300 MHz, Methanol-d4) δ 8.37 (s, 1H), 8.27 (d, J = 8.9 Hz, 2H), 7.30 (d, J = 1.4 Hz, 2H), 7.28 (s, 10H), 7.28 - 7.24 (m, 5H), 7.02 (d, J = 8.3 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 4.17 (s, 2H), 3.99 (t, J = 6.2 Hz, 2H), 3.30 (d, J = 6.5 Hz, 1H), 2.26 (s, 3H), 1.77 (p, J = 6.6 Hz, 2H), 1.56 (p, J = 6.9 Hz, 2H), 1.43 - 1.35 (m, 2H). 1313C NMR (151 MHz, Methanol-d4) δ 168.24, 163.41, 143.53, 133.56, 130.27, 129.85, 128.76, 127.51, 126.56, 126.27, 119.77, 114.82, 67.83, 46.47, 39.65, 39.52, 28.51, 28.50, 28.36, 23.05, 19.51.

[0081] Example 15: Replace the tert-butyl (2-hydroxyethyl) carbamate in Step 1 of Example 1 with tert-butyl (5-hydroxypentyl) carbamate, and replace 2-((3r,5r,7r)-adamantan-1-yl) acetic acid in Step 4 with 2,2,2-triphenylacetic acid, and keep the remaining steps unchanged to obtain the target compound 1-H-15. 1 1H NMR (300 MHz, Methanol-d4) δ 8.36 (s, 1H), 8.26 (d, J = 8.9 Hz, 2H), 7.34 - 7.20 (m, 13H), 7.02 (d, J = 8.2 Hz, 2H), 6.93 (d, J = 9.0 Hz, 2H), 4.97 (s, 1H), 4.17 (s, 2H), 3.97 (t, J = 6.3 Hz, 2H), 3.28 (t, J = 6.8 Hz, 2H), 2.25 (s, 3H), 1.85 - 1.73 (m, 2H), 1.59 (q, J = 6.9 Hz, 2H), 1.54 - 1.44 (m, 2H). 13 13C NMR (151 MHz, Methanol-d4) δ 173.29, 168.24, 167.08, 166.37, 163.42, 139.83, 135.31, 133.58, 129.84, 128.76, 128.47, 128.04, 126.63, 126.24, 119.78, 114.83, 113.26, 113.25, 67.85, 57.86, 46.47, 46.47, 38.99, 28.66, 28.37, 22.99, 19.51.

[0082] Example 16: Replace tert-butyl (2-hydroxyethyl) carbamate in Step 1 of Example 1 with tert-butyl (2-(2-hydroxyethoxy)ethyl) carbamate, and keep the remaining steps unchanged to obtain the target compound 1-H-16. 1H NMR (600 MHz, Methanol-d4) δ 8.26 (s, 1H), 8.18 (d, J = 8.9 Hz, 2H), 7.19 (d, J = 8.5 Hz, 2H), 6.92 (dd, J = 14.9, 8.6 Hz, 4H), 4.12 - 4.08 (m, 2H), 4.08 (s, 2H), 3.78 - 3.73 (m, 2H), 3.54 (t, J = 5.6 Hz, 2H), 3.31 (t, J = 5.6 Hz, 2H), 2.17 (s, 3H), 1.83 (d, J = 4.6 Hz, 5H), 1.62 (d, J = 11.6 Hz, 3H), 1.53 (d, J = 3.0 Hz, 9H). 13 C NMR (151 MHz, Methanol-d4) δ 172.43, 168.01, 166.94, 166.26, 163.01, 156.51, 135.22, 133.46, 129.76, 128.67, 126.44, 119.66, 114.80, 113.21, 113.19, 69.40, 68.86, 67.53, 50.38, 46.39, 42.20, 42.20, 38.72, 36.39, 32.28.28.65.19.42.

[0083] Example 17: Replace tert-butyl (2-hydroxyethyl) carbamate in Step 1 of Example 1 with tert-butyl 4-(2-hydroxyethyl)piperidine-1-carboxylate, and keep the remaining steps unchanged to obtain the target compound 1-H-17. 1 H NMR (600 MHz, Methanol-d4) δ 8.27 (s, 1H), 8.18 (d, J = 8.6 Hz, 2H), 7.16 (d, J = 8.1 Hz, 2H), 6.92 (d, J = 8.1 Hz, 2H), 6.89 (d, J = 8.5 Hz, 2H), 4.07 (d, J = 6.5 Hz, 1H), 4.05 (s, 2H), 4.02 (t, J = 6.3 Hz, 2H), 4.01 - 3.95 (m, 3H), 2.16 (s, 3H), 1.86 (s, 3H), 1.79 - 1.71 (m, 4H), 1.69 (t, J = 6.1 Hz, 3H), 1.65 (d, J = 12.4 Hz, 4H), 1.58 (d, J = 10.2 Hz, 10H). 13C NMR (151 MHz, Methanol-d4) δ 168.11, 163.19, 129.76, 128.63, 126.27, 119.67, 113.15, 65.53, 46.35, 45.52, 42.47, 41.61, 36.36, 34.98, 33.31, 32.82, 32.44, 31.65, 28.70, 19.39.

[0084] Example 18: Replace the tert-butyl (2-hydroxyethyl) carbamate in Step 1 of Example 1 with tert-butyl 2-hydroxy-7-azaspiro[3.5]nonane-7-carboxylate, and keep the remaining steps unchanged to obtain the target compound 1-H-18. 1 H NMR (400 MHz, Methanol-d4) δ 8.35 (s, 1H), 8.25 (d, J = 8.6 Hz, 2H), 7.27 (d, J = 8.2 Hz, 2H), 7.02 (d, J = 8.2 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 4.84 - 4.74 (m, 1H), 4.17 (s, 2H), 3.61 - 3.49 (m, 4H), 2.55 - 2.46 (m, 2H), 2.27 (s, 3H), 2.21 (d, J = 5.7 Hz, 2H), 2.00 - 1.90 (m, 5H), 1.71 (d, J = 23.4 Hz, 15H), 1.63 - 1.58 (m, 1H). 13 C NMR (151 MHz, Methanol-d4) δ 170.61, 168.15, 167.04, 166.35, 161.84, 156.60, 156.36, 135.31, 133.55, 129.88, 128.77, 126.37, 119.79, 115.37, 113.28, 113.26, 68.12, 46.50, 44.44, 44.13, 42.57, 39.45, 39.26, 38.48, 37.17, 36.48, 33.42, 28.81, 19.56.

[0085] Example 19: Replace the tert-butyl (2-hydroxyethyl) carbamate in Step 1 of Example 1 with tert-butyl trans-(4-hydroxymethyl)cyclohexylcarbamate, and keep the remaining steps unchanged to obtain the target compound 1-H-19. 11H NMR (600 MHz, Methanol-d4) δ 8.34 (s, 1H), 8.25 (d, J = 8.9 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 8.1 Hz, 2H), 6.96 (d, J = 8.9 Hz, 2H), 4.15 (s, 2H), 3.85 (d, J = 6.3 Hz, 2H), 3.66 (tt, J = 11.2, 3.6 Hz, 1H), 2.26 (s, 3H), 1.96 (qd, J = 5.9, 4.0, 2.9 Hz, 8H), 1.91 (s, 2H), 1.83 - 1.70 (m, 5H), 1.70 - 1.61 (m, 11H). 13 13C NMR (151 MHz, Methanol-d4) δ 171.56, 168.23, 167.06, 166.37, 163.50, 135.33, 133.55, 129.85, 128.75, 126.28, 119.76, 114.80, 113.26, 113.24, 72.84, 50.44, 48.43, 46.47, 42.38, 36.85, 36.55, 32.42, 32.42, 31.82, 28.81, 28.12, 19.52.

[0086] Example 20: Replace the tert-butyl (2-hydroxyethyl) carbamate in Step 1 of Example 1 with tert-butyl cis-(4-hydroxymethyl)cyclohexylcarbamate, and keep the remaining steps unchanged to obtain the target compound 1-H-20. 1 1H NMR (600 MHz, Methanol-d4) δ 8.31 (s, 1H), 8.23 (d, J = 9.0 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 8.2 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 4.15 (s, 2H), 3.93 (d, J = 6.7 Hz, 3H), 2.24 (s, 3H), 1.97 (s, 2H), 1.94 (t, J = 3.3 Hz, 4H), 1.72 (q, J = 4.9 Hz, 8H), 1.65 (d, J = 2.9 Hz, 12H). 1313C NMR (151 MHz, Methanol-d4) δ 171.86, 168.17, 167.05, 166.33, 163.50, 156.57, 156.32, 135.30, 133.56, 129.85, 128.75, 126.24, 119.79, 114.82, 113.25, 113.23, 71.48, 50.24, 46.50, 45.89, 42.39, 36.55, 35.18, 32.49, 28.81, 28.47, 24.32, 19.54.

[0087] Example 21

[0088] Activity test

[0089] Western blot to detect the effect of the compound on the expression of WRN in HCT-116 cells

[0090] 1. Experimental method

[0091] After the HCT-116 cells were treated with the compound, the culture medium was discarded, and the cells were washed 2 - 3 times with PBS. Protease inhibitor and RIPA lysis buffer were added successively to completely lyse the cells, and then centrifugation was carried out (4°C, 12,000 g, 10 min). The supernatant was the obtained total protein solution. Referring to the instructions of the kit, 7.5% separating gel and 5% stacking gel were prepared according to the different molecular weights of the proteins to be measured, and the SDS-PAGE gel was poured. After adding an appropriate amount of pre-cooled 1× electrophoresis buffer, the previously bio-labeled samples or total protein extracts in the cells were added to the wells (pre-stained protein Marker and samples). Electrophoresis was carried out at a constant voltage of 90 V for about 30 min. After the samples entered the separating gel, the voltage was adjusted to 120 V and electrophoresis was continued. When the target band reached the appropriate position (referring to the position of the pre-stained protein Marker), electrophoresis was terminated. Electrophoresis was terminated when the bromophenol blue just ran out, and transfer was carried out. The band of the target protein was stripped out, a PVDF membrane was attached, and transferred to the PVDF membrane by electrophoresis. Then it was blocked with 5% skim milk on a shaker for 1 h. The primary antibody was added and incubated overnight at 4°C, and then washed three times with TBST, with each washing time being 10 min. The secondary antibody was added and incubated at room temperature for 2 h, and then washed three times with TBST, with each washing time being 10 min. The ECL mixed solution was prepared in a ratio of ECLA:ECLB = 1:1 in the darkroom in advance. Then the treated PVDF membrane was placed face up in the exposure cassette, the prepared ECL mixed solution was added, and after reacting for 1 - 2 min, the reaction solution was discarded. The exposure conditions were adjusted according to the luminescence intensity of the developing reagent, and exposure was started. H3B-219 was used as the positive drug for detection with the compound of the present invention.

[0092] 2. Experimental results

[0093] As Figure 1 shown, when the compound disclosed in the present invention is administered at concentrations of 1 and 10 μM for 24 h, it exhibits a certain degree of degradation effect on WRN.

[0094] Example 22

[0095] Digest and count the HCT-116 cells in the logarithmic growth phase, adjust the cell density to 5×10 3 cells / well, and inoculate them into a 96-well plate. Add 100 μL of cell suspension to each well. Place the 96-well plate in an incubator at 37 °C and 5% CO2 for 24 hours to allow the cells to adhere. Prepare target compound solutions and 5-FU solutions with different concentrations, and set 3 replicates for each concentration. Aspirate the culture medium in the 96-well plate, and add 100 μL of culture medium containing different concentrations of the compound to each well. Set a positive control group (5-FU) and a negative control group (0.1% DMSO). Place the 96-well plate in an incubator at 37 °C and 5% CO2 and continue to culture for 48 hours. Add 20 μL of MTT solution (5 mg / mL) to each well and continue to culture for 4 hours. Carefully aspirate the supernatant, add 150 μL of DMSO to each well, and shake for 10 minutes to fully dissolve the crystals. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance (OD value) of each well at a wavelength of 570 nm. Calculate the cell survival rate: Cell survival rate (%) = (OD value of the experimental group - OD value of the blank group) / (OD value of the negative control group - OD value of the blank group) × 100%. Use software such as GraphPad Prism to plot the cell survival rate curve and calculate the IC 50 value (the concentration of the compound required to inhibit 50% of cell proliferation), and the results are as Figure 2 shown.

Claims

1. A compound or a pharmaceutically acceptable salt thereof that targets the degradation of WRN, characterized in that, The structural formula of the compound for targeted degradation of WRN is shown as general formula (I): Wherein, R is selected from: L is selected from -NH-C 1～15 a straight-chain alkyl group, -NH-C 1～7 a straight-chain alkyl group -O-C 1～7 a straight-chain alkyl group, wherein R1 is selected from cyclobutyl, cyclopentyl, C 1～6 alkyl; R2 is selected from C 1～6 alkyl groups.

2. The compound or a pharmaceutically acceptable salt thereof for targeted degradation of WRN according to claim 1, wherein L is selected from any one of the following structures: L is selected from -NH-C 1～15 linear alkyl, -NH-C 1～7 linear alkyl -O-C 1～7 linear alkyl, where m is selected from 1 to 2; R1 is selected from C 1～4 alkyl; R2 is selected from C 1～4 alkyl groups.

3. The compound or its pharmaceutically acceptable salt that targets and degrades WRN according to claim 1, characterized in that, L is selected from any one of the following structures:

4. A compound capable of targeting and degrading WRN or a pharmaceutically acceptable salt thereof, characterized in that, The structural formula of the compound for targeted degradation of WRN is shown as any one of the following:

5. A method for preparing the compound targeting the degradation of WRN or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, Comprising the following steps: The definitions of R and L are the same as those in claim 1.

6. The preparation method of the compound for targeted degradation of WRN or its pharmaceutically acceptable salt according to claim 5, characterized in that, Specifically: Compound 1-1 and 1-2 are used to prepare compound 1-3 through Mitsunobu reaction, and the reaction temperature is 0-100 °C; Compound 1-3 and 4-chloro-2-(methylthio)-6-(trifluoromethyl)pyrimidine are used to obtain 1-4 through Suzuki reaction, and the reaction temperature is 25 °C to 150 °C; Compound 1-4 is deprotected by acid hydrolysis to prepare compound I-5, and the reaction temperature is 0-60 °C; Compound 1-5 and R-COOH are condensed to obtain 1-6, and the reaction temperature is 0-100 °C; Compound 1-6 is oxidized to obtain 1-7, and the oxidant used is selected from one or a mixture of two of potassium peroxymonosulfate complex salt, hydrogen peroxide, potassium permanganate, trifluoroperacetic acid, m-chloroperbenzoic acid, ozone, and the reaction temperature is 0-60 °C; Compound 1-7 is added with benzyl mercaptan and undergoes substitution reaction to obtain 1-8, and the reaction temperature is 20-120 °C; Compound 1-8 is oxidized to obtain 1-9, and the oxidant used is selected from chlorine, sulfonyl chloride, and the reaction temperature is -20-40 °C; Compound 1-9 and 2-amino-N-(p-tolyl)acetamide are acylated to obtain compound (I).

7. The compound or its pharmaceutically acceptable salt for targeted degradation of WRN according to claim 1, characterized in that, The pharmaceutically acceptable salt is an acid addition salt formed by the compound and any one of the following acids: hydrogen chloride, hydrogen bromide, sulfuric acid, carbonic acid, oxalic acid, citric acid, malic acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or ferulic acid.

8. A pharmaceutical composition, characterized in that, Comprising the compound for targeted degradation of WRN or its pharmaceutically acceptable salt as claimed in claim 1 and a pharmaceutically acceptable carrier.

9. Use of the compound for targeted degradation of WRN or its pharmaceutically acceptable salt as claimed in claim 1 or the pharmaceutical composition as claimed in claim 8 in the preparation of a medicament for WRN protein degrading agent.

10. Use of the compound for targeted degradation of WRN or its pharmaceutically acceptable salt as claimed in claim 1 or the pharmaceutical composition as claimed in claim 8 in the preparation of a medicament for treating tumors.

Images