A wrn degrader and methods of making and using the same

Abstract

The application discloses a WRN degrading agent, a preparation method and application thereof. The application provides triazole derivatives with structures of formula (I), (II) and (III), a pharmaceutical composition containing the compounds and pharmaceutically acceptable salts thereof. The application evaluates the effect of the triazole derivatives by improving the inhibition and degradation ability of the drugs on WRN and the growth inhibition effect on MSI cells, and the application can degrade WRN protein while inhibiting the activity of WRN, has good in-vitro activity and stability, and has the feasibility of further developing drugs for treating MSI cancer patients.

Description

Technical Field

[0001] This invention relates to the fields of medicinal chemistry and pharmacotherapeutic science, specifically to a WRN degrading agent, its preparation method, and its uses. Background Technology

[0002] Cells in any organism possess multiple DNA repair mechanisms to maintain the stability and integrity of the genome, which is crucial for survival. Mismatch repair (MMR) is a repair mechanism that recognizes DNA molecules containing mismatched bases and restores the nucleotide sequence to normal. Mutations in MMR-related genes can lead to abnormal MMR function, preventing the recognition and repair of mismatched bases, resulting in DNA disorder and creating opportunities for tumor development. Therefore, the MMR system is an important defense mechanism in the body for maintaining stability and ensuring safety, maintaining the integrity and stability of genomic DNA.

[0003] Abnormal DNA mismatch repair function leads to high mutation and instability in the genome, manifested as frequent insertions and deletions of repetitive DNA sequences (microsatellites) throughout the genome. This phenomenon is known as microsatellite instability (MSI). Since the MMR system plays a role in maintaining the integrity and stability of the genome, MSI caused by MMR defects can serve as a characteristic of tumor genetic instability. Detecting MSI plays an important role in early tumor diagnosis, assessing tumor progression, determining tumor sensitivity to chemotherapy, and predicting potential cancer populations.

[0004] Synthetic lethality refers to the genetic interaction between two genes, where the simultaneous inactivation of two functional genes leads to cell death, while abnormal expression of only one gene does not cause significant changes in cell viability. Developing drugs that precisely target tumors and selectively kill tumor cells based on the theory of synthetic lethality is currently a hot topic in cancer research. Patients can improve treatment outcomes, reduce side effects, and achieve better survival rates through innovative therapeutic drugs.

[0005] WRN (Werner syndrome protein) is an important member of the RecQ helicase family. Numerous studies have found that WRN plays a crucial regulatory role in vital activities such as DNA damage repair, telomere maintenance, and autophagy, maintaining genome stability. Current research has shown that in DNA MMR-deficient MSI tumor cells, knocking out the WRN gene or depleting the WRN protein can induce a synthetic lethal effect, leading to tumor cell death. Therefore, WRN may be a novel synthetic lethal target for MSI malignancies.

[0006] Currently, the development of WRN inhibitors is still in its early stages, and no drugs have been marketed. Only three inhibitors have entered clinical trials: Novartis' HRO761, Roche's VVD133214, and GlaxoSmithKline's NCTO6710847. Based on publicly available data, existing WRN inhibitors still have some shortcomings, such as poor efficacy and the emergence of drug resistance.

[0007] Targeted protein degradation has emerged as an effective solution to target drug resistance, effectively mitigating or even preventing resistance by degrading the target protein. For a protein to perform its normal physiological function, its domains need to be in appropriate relative positions to ensure good stability and interaction with the substrate. In recent years, researchers have discovered that small molecules can act on multiple domains of a protein, causing misalignment of the protein structure, resulting in an unstable conformation and ultimately its degradation by the proteasome. Currently, no small-molecule degraders targeting WRN have been reported. Summary of the Invention

[0008] Purpose of the invention: To address the problems existing in the prior art, this invention provides a novel triazole derivative WRN degrader. This type of triazole derivative effectively solves the problem that there are currently no marketed WRN inhibitors and WRN protein degraders. Compared with clinical WRN inhibitors, this invention not only enhances the inhibitory activity of drugs against WRN but also has the function of degrading WRN. Therefore, this invention is expected to solve the problem of poor efficacy of WRN inhibitors during treatment.

[0009] The present invention also provides a method for preparing the WRN degrading agent, a pharmaceutical composition thereof, and its application.

[0010] Technical solution: To achieve the above objectives, the present invention provides a triazole derivative or a pharmaceutically acceptable salt thereof with WRN degradation activity, the structure of which is shown in general formula (I).

[0011]

[0012] in:

[0013] R1 is selected from substituted or unsubstituted C. 1-8 Alkyl, substituted or unsubstituted 5-6 membered monocyclic aryl, substituted or unsubstituted 5-6 membered monocyclic heterocyclic, or substituted or unsubstituted 5-6 membered monocyclic heteroaryl; the substituted C 1-8 Alkyl, 5-6 membered monocyclic aryl, 5-6 membered monocyclic heterocyclic or 5-6 membered monocyclic heteroaryl are optionally substituted by 0-3 independent substituents selected from -OH, halogen, -CN or NH2; the 5-6 membered monocyclic heterocyclic or 5-6 membered monocyclic heteroaryl contains 1-2 heteroatoms independently selected from N, O or S;

[0014] R2 is selected from hydrogen or substituted or unsubstituted 5-6 membered monocyclic aryl, substituted or unsubstituted 5-6 membered monocyclic heteroaryl, or substituted or unsubstituted 5-6 membered fused heteroaryl; the substituted 5-6 membered monocyclic aryl, 5-6 membered monocyclic heteroaryl, or 5-6 membered fused heteroaryl are optionally substituted by 0-3 independent substituents selected from halogen, -CN, CF3, or SF5; the 5-6 membered monocyclic heteroaryl or 5-6 membered fused heteroaryl contains at least one N atom;

[0015] R3 is selected from hydrogen or substituted or unsubstituted 5-6 membered monocyclic heteroaryl or substituted or unsubstituted 4-10 membered fused heterocyclic group; the substituted 5-6 membered monocyclic heterocyclic group or 4-10 membered fused heterocyclic group is optionally substituted by 0-3 independent substituents selected from -OH, halogen, -CN or NH2; the 5-6 membered monocyclic heteroaryl group contains 1-3 independent heteroatoms selected from N, O or S;

[0016] R4 is selected from hydrogen or substituted or unsubstituted 5-6 membered monocyclic heteroaryl, substituted or unsubstituted 3-6 membered cycloalkyl, substituted or unsubstituted 4-10 membered heterocyclic or substituted or unsubstituted 4-10 membered fused heterocyclic; wherein the substituted 5-6 membered monocyclic heteroaryl, 3-6 membered cycloalkyl, 4-10 membered heterocyclic or 4-10 membered fused heterocyclic is optionally surrounded by 0-3 independent elements selected from -OH, halogen, methyl, C 1-6 Alkyl groups or -CN substituents may be used; the 5-6 member monocyclic heteroaryl, 4-10 member heterocyclic or 4-10 member fused heterocyclic groups contain 1-3 heteroatoms independently selected from N, O or S.

[0017] The present invention relates to a triazole derivative or a pharmaceutically acceptable salt thereof having WRN degradation activity, the structure of which is shown in general formula (II).

[0018]

[0019] in:

[0020] X, Y, and Z are each independently selected from CH or N, and X, Y, and Z contain at least one N atom;

[0021] R3 is selected from hydrogen or substituted or unsubstituted 5-6 membered monocyclic heteroaryl or substituted or unsubstituted 4-10 membered fused heterocyclic group; the substituted 5-6 membered monocyclic heterocyclic group or 4-10 membered fused heterocyclic group is optionally substituted by 0-3 independent substituents selected from -OH, halogen, -CN or NH2; the 5-6 membered monocyclic heteroaryl group contains 1-3 independent heteroatoms selected from N, O or S;

[0022] R5 is selected from H, halogen, CN, C. 1-6 Alkyl, C 1-6Haloalkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, C 3-6 An alkoxy or 4-10 membered heterocyclic group, wherein the 4-10 membered heterocyclic group comprises 1-3 heteroatoms independently selected from N, O or S;

[0023] Ring A and the benzene ring attached thereto form a fused ring, wherein ring A is selected from substituted or unsubstituted 5-7-membered aryl or substituted or unsubstituted 5-7-membered heterocyclic groups; the substituted 5-7-membered aryl or 5-7-membered heterocyclic groups are optionally surrounded by 0-3 independent groups selected from -OH, halogen, C 1-6 Alkyl or -CN substituents; the 5-7 aryl or 5-7 heterocyclic group contains 0-2 heteroatoms independently selected from N, O or S;

[0024] m is selected from 0, 1, 2 or 3.

[0025] The present invention relates to a triazole derivative or a pharmaceutically acceptable salt thereof having WRN degradation activity, the structure of which is shown in general formula (III).

[0026]

[0027] in:

[0028] R3 is selected from hydrogen or a substituted 5-6 membered monocyclic heteroaryl or a 4-10 membered fused heterocyclic group; the substituted 5-6 membered monocyclic heterocyclic group or the 4-10 membered fused heterocyclic group is optionally substituted by 0-3 independent substituents selected from -OH, halogen, -CN or NH2. The 5-6 membered monocyclic heteroaryl group contains 1-3 independent heteroatoms selected from N, O or S.

[0029] R4 is selected from hydrogen or substituted or unsubstituted 5-6 membered monocyclic heteroaryl, substituted or unsubstituted 3-6 membered cycloalkyl, substituted or unsubstituted 4-10 membered heterocyclic or substituted or unsubstituted 4-10 membered fused heterocyclic; wherein the substituted 5-6 membered monocyclic heteroaryl, 3-6 membered cycloalkyl, 4-10 membered heterocyclic or 4-10 membered fused heterocyclic is optionally surrounded by 0-3 independent elements selected from -OH, halogen, methyl, C 1-6 Alkyl groups or -CN substituents may be used; the 5-6 member monocyclic heteroaryl, 4-10 member heterocyclic or 4-10 member fused heterocyclic groups contain 1-3 heteroatoms independently selected from N, O or S.

[0030] The triazole derivatives or pharmaceutically acceptable salts thereof with WRN degradation activity described in this invention are selected from any one of the following:

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040] The method for preparing a triazole derivative or a pharmaceutically acceptable salt thereof with WRN degradation activity according to the present invention comprises the following steps:

[0041] (1) Bromination of compound a1-1 yields compound a1;

[0042] (2) Compound a2-1 undergoes chlorination under the action of sulfonyl chloride to give compound a2-2;

[0043] (3) Compound a2-2 reacts with N-Boc-piperazine in the presence of triethylamine to give compound a2-3;

[0044] (4) Compound a2-3 reacts with a1 under the action of phosphoric acid to give compound a2;

[0045] (5) Compound a3-1 undergoes acylation under alkaline conditions to give compound a3-2;

[0046] (6) Compound a3-2 reacts with a2 under alkaline conditions to give compound a3;

[0047] (7) Compound a4-1 loses its protecting group under acidic conditions to obtain compound a4-2;

[0048] (8) Compound a4-2 undergoes Suzuki coupling with a3 under the action of a catalyst to obtain compound a4;

[0049] (9) Compound a4 was reacted with a condensing agent to obtain compound a5-2;

[0050] (10) Compound a5-2 was deprotected under acidic conditions to obtain compound a5;

[0051] (11) Compound a5 was reacted with a condensing agent to obtain the target compound;

[0052] The reaction formula is shown below:

[0053]

[0054] As a preferred option, (1) compound a1-1 is brominated by reacting sodium bromate and sodium bromide in sulfuric acid solution to obtain compound a1;

[0055] (2) Compound a2-1 undergoes chlorination under the action of sulfonyl chloride to give compound a2-2;

[0056] (3) Compound a2-2 reacts with N-Boc-piperazine in the presence of triethylamine to give compound a2-3;

[0057] (4) Compound a2-3 reacts with a1 under the action of phosphoric acid to give compound a2;

[0058] (5) Compound a3-1 reacts with bromoacetyl bromide under alkaline conditions to give compound a3-2;

[0059] (6) Compound a3-2 reacts with a2 under alkaline conditions to give compound a3;

[0060] (7) Compound a4-1 loses its protecting group under acidic conditions to obtain compound a4-2;

[0061] (8) Compound a4-2 undergoes Suzuki coupling with a3 under the action of a catalyst to obtain compound a4;

[0062] (9) Compound a4 was reacted with amide condensing agent HATU to obtain compound a5-2;

[0063] (10) Compound a5-2 was deprotected by the Boc protecting group under acidic conditions to obtain compound a5;

[0064] (11) Compound a5 was reacted with the amide condensing agent EDCI to obtain the target compound. The use of the triazole derivatives or pharmaceutically acceptable salts thereof with WRN degradation activity described in this invention in the preparation of reagents with WRN inhibitory and WRN protein degradation functions.

[0065] The use of the triazole derivatives or pharmaceutically acceptable salts thereof with WRN degradation activity as described in this invention in the preparation of medicaments for the treatment or prevention of tumors.

[0066] The tumor is a microsatellite unstable tumor, including one or more of colorectal cancer, endometrial cancer, gastric cancer, liver cancer, and other cancers with microsatellite instability.

[0067] Furthermore, since the compounds of the present invention have the activity of inhibiting the proliferation of various tumor cell lines, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate various diseases, including various cancers.

[0068] The WRN degrading agent of the present invention comprises the triazole derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0069] The pharmaceutical composition for treating or preventing tumors according to the present invention contains a therapeutically effective amount of the triazole derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0070] The pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhaler, ointment, suppository or patch.

[0071] The "safe and effective amount" described in this invention refers to an amount of compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1–2000 mg of the compound of this invention per dose, more preferably, 5–1000 mg of the compound of this invention per dose. Preferably, "one dose" refers to one capsule or tablet.

[0072] Here, "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g.), wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0073] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and local administration.

[0074] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (1) fillers or solubilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (2) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (3) humectants, such as glycerin; (4) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (5) slowing agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glyceryl monostearate; (8) adsorbents, such as kaolin; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in the dosage forms of capsules, tablets, and pills.

[0075] Solid dosage forms, such as tablets, sugar pills, capsules, pellets, and granules, can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opaque agents, and the release of the active compound or compound in such compositions can be delayed at a specific site within the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0076] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0077] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0078] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances.

[0079] The composition for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous water, dispersion, suspension or emulsion, and sterile powder for reconstitution into a sterile injectable solution or dispersion. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0080] The dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or, if necessary, propellants.

[0081] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0082] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–5000 mg, preferably 5–2000 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0083] This invention provides a method for preparing a class of triazole derivatives with WRN degradation activity, as well as the method and its uses. This invention provides triazole derivatives with structures of formulas (I), (II), and (III), pharmaceutical compositions comprising the compounds, and pharmaceutically acceptable salts thereof. The efficacy of the triazole derivatives was evaluated by enhancing their ability to inhibit and degrade WRN and their growth-inhibiting effect on MSI cells. These derivatives not only enhance WRN inhibitory activity but also degrade WRN proteins, exhibiting good in vitro activity and stability, and possessing the feasibility for further development for the treatment of MSI cancer patients.

[0084] This invention modifies the structure of WRN inhibitors by introducing structural units capable of degrading WRN. This allows the inhibitors to degrade WRN while simultaneously inhibiting it, thus addressing the problems of drug resistance and poor activity in current WRN inhibitors.

[0085] This invention, through specific structural design and fragment assembly, constructs a triazole derivative that not only exhibits WRN inhibitory activity but also significantly enhances WRN degradation activity, demonstrating excellent biological activity and drug-like properties. The compound constructed through specific structural modification and fragment introduction can act as a WRN inhibitor, exhibiting superior WRN inhibitory activity compared to the existing compound HRO761, and also significantly superior WRN protein degradation activity.

[0086] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0087] 1. This invention combines and fuses WRN inhibitors with specific fragment structures into a single antitumor molecule through homologous synthesis, base structure design, and fragment assembly strategies. This molecule exhibits both WRN inhibitory activity and excellent WRN degradation activity. The combination of these two elements significantly enhances antitumor activity, representing a groundbreaking approach compared to simple structural modifications. The compounds of formula (I) and / or formula (II) and / or (III) constructed in this invention, or their pharmaceutical compositions, as well as different salts and formulations of these compounds, synergistically enhance the inhibitory effect of compounds on MSI tumors.

[0088] 2. This invention also provides a method for preparing the WRN degrading agents, their uses, and the inhibitory activity of these compounds on the proliferation of various tumor cell lines. The triazole derivatives with WRN-degrading activity described in this invention show promise as candidate drugs for treating MSI cancers.

[0089] (3) The compounds of the present invention are easy to prepare and inexpensive, and have simple structure, ingenious design, cheap and readily available raw materials, safe and environmentally friendly synthesis process, and easy to scale up production. Detailed Implementation

[0090] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below with reference to specific embodiments and comparative examples. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0091] Unless otherwise specified, the equipment used in this embodiment, comparative example and experimental example are all conventional experimental equipment, and the materials and reagents used are all commercially available unless otherwise specified. The experimental methods are also conventional experimental methods unless otherwise specified.

[0092] The technical solution of the present invention will be further described below with reference to the embodiments. The known starting materials of the present invention can be synthesized by or according to methods known in the art, or can be purchased from companies such as Leyan, Bid Pharmaceutical, Aladdin, and Anaiji.

[0093] Example 1

[0094] Synthesis of intermediate a1

[0095]

[0096] Step 1: Add water (300 mL), 98% concentrated sulfuric acid (150 g), 3-amino-1,2,4-triazole a1-1 (50 g), and sodium bromide (150 g) to a reaction vessel, and heat the system to 55°C. Dissolve sodium bromate (45 g) in water (150 mL) and add it dropwise to the reaction vessel while maintaining the temperature at 55°C. After the addition is complete, maintain the temperature for 20 hours. After the reaction is complete, cool the temperature to 20°C–30°C, quench with saturated sodium sulfite, and adjust the pH to 6.0 with 20% sodium hydroxide solution, then filter. Add n-butanol to the filtrate for extraction (100 mL * 3), and concentrate under reduced pressure until no fraction remains. Add ethyl acetate to dissolve, filter with silica gel, and concentrate the ethyl acetate until no fraction remains. Add 1,4-dioxane (50 mL), heat to 80°C, stir, cool to room temperature, filter, and vacuum dry the filter cake for 12 hours to obtain a white solid a1 (54 g, yield 55.8%). LC-MS (ESI): m / z: 162.95 [M+H] + .

[0097] Example 2

[0098] Preparation of intermediate a2

[0099]

[0100] Step 1: Dissolve ethyl propionyl acetate a2-1 (50 g, 346.8 mmol) in dichloromethane (350 mL), add SO2Cl2 (67.5 g, 500.2 mmol) at 0 °C, and react at room temperature for 2 hours until the reaction is complete. Add 300 mL of water to the reaction system, adjust the pH to 6 with 20% sodium hydroxide solution, extract with dichloromethane (100 mL * 3), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate to obtain a pale yellow oily substance a2-2 (100 g). No further purification is required; proceed directly to the next step of the reaction. LCMS ESI-MS m / z: 179.04 [M+H] + .

[0101] Step 2: Dissolve intermediate a2-2 (50 g, 280.8 mmol) and triethylamine (92.6 g, 915.1 mmol) in 250 mL of acetonitrile and stir at room temperature for 2.5 hours. Dissolve N-Boc-piperazine (57 g, 306.0 mmol) in 250 mL of acetonitrile and add it dropwise to the above reaction solution. React at 60 °C for 16 hours until the reaction is complete. After cooling to room temperature, filter under vacuum, wash the filter cake with a small amount of acetonitrile, evaporate the filtrate to dryness, add 500 mL of water, extract with ethyl acetate (100 mL * 3), dry with anhydrous sodium sulfate, evaporate the solvent under reduced pressure, and purify the residue by column chromatography (petroleum ether / ethyl acetate = 10 / 1) to give a pale yellow solid a2-3 (37 g, yield 40.14%). LC-MS (ESI): m / z: 329.20 [M+H]+ .

[0102] Step 3: Dissolve intermediate a2-3 (22.2 g, 67.6 mmol) and 5-bromo-1-H-3-amino-1,2,4-triazole a1 (11 g, 67.6 mmol) from the previous step in 200 mL of ethanol. Slowly add phosphoric acid (7.8 g, 67.6 mmol). After the addition is complete, heat to 80 °C and react for 12 hours. Cool to room temperature and remove the solvent by vacuum evaporation. Pour the reaction solution into 100 mL of ice water, adjust the pH to approximately 8 with saturated sodium bicarbonate solution, extract with dichloromethane (100 mL * 3), dry with anhydrous sodium sulfate, concentrate, and separate the crude product by column chromatography (dichloromethane / methanol = 40 / 1) to obtain a yellow solid a2 (4.4 g, yield 15.3%). LC-MS (ESI): m / z: 427.10 [M+H] + .

[0103] Example 3

[0104] Preparation of intermediate a3

[0105]

[0106] Step 1: Dissolve 10 g (51.3 mmol) of 3-chloro-4-aminotrifluorotoluene a3-1 and 6.25 g (51.2 mmol) of 4-dimethylaminopyridine in 100 mL of anhydrous dichloromethane. Cool the reaction mixture to 0 °C and slowly add 12.4 g (61.4 mmol) of bromoacetyl bromide dropwise. Stir at 25 °C for 2 hours. After the reaction is complete, add 30 mL of water and 30 mL of dichloromethane, extract and separate the layers. Evaporate the solvent from the organic layer under reduced pressure and perform column chromatography (petroleum ether / ethyl acetate = 5 / 1 gradient elution) to give a pale yellow solid a3-2 (13.78 g, yield 85.60%). LC-MS (ESI): m / z: 315.93 [M+H] + .

[0107] Step 2: Intermediate a3-2 (1.8 g, 5.7 mmol) was dissolved in N,N-dimethylformamide (20 mL), and intermediate a2 (2.1 g, 4.9 mmol) and N,N-diisopropylethylamine (1.68 g, 13.0 mmol) were added. The reaction was carried out at 50 °C for 3 hours. After the reaction was complete, water (200 mL) and ethyl acetate (100 mL) were added to the reaction system, and the mixture was extracted and separated. The organic phase was washed once with saturated sodium chloride aqueous solution and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and column chromatography (petroleum ether / ethyl acetate = 1 / 1) was performed to obtain intermediate a3 (1 g, yield 26.46%). LC-MS (ESI): m / z: 662.10 [M+H] + .

[0108] Example 4

[0109] Preparation of intermediate a4

[0110]

[0111] Step 1: Dissolve 50 g (161.7 mmol) of 1,2,3,6-tetrahydro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)pyridine a4-1 in ethyl acetate (500 mL), and slowly add dropwise 200 mL (800 mmol) of 4N HCl in ethyl acetate. Stir at room temperature for 2 hours. Distill off the solvent under reduced pressure, and slurry the ethyl acetate at room temperature (30 mL) to give a pale yellow solid a4-2 (28 g, yield 70.52%). LC-MS (ESI): m / z: 210.16 [M+H] + .

[0112] Step 2: Under nitrogen protection, intermediates a4-2 (2 g, 8.1 mmol), a3 (3.59 g, 5.4 mmol), and potassium carbonate (2.23 g, 16.2 mmol) from the previous step were dissolved in 120 mL of dioxane. 40 mL of water was added, along with the catalyst Pd(dppf)C12·DCM (0.16 g, 0.2 mmol). The mixture was heated to 85 °C and reacted for 12 hours, after which the reaction was stopped. 100 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (30 mL x 3). The extract was dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (dichloromethane / methanol = 10 / 1) to obtain a white solid a4 (1.23 g, 35% yield). LC-MS (ESI): m / z: 665.25 [M+H] + .

[0113] Example 5

[0114] Preparation of intermediate a5

[0115]

[0116] Step 1: Pyrazolo[1,5-a]pyrimidine-3-carboxylic acid a5-1 (0.3 g, 1.8 mmol), a4 (1 g, 1.5 mmol), and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU) (0.684 g, 1.8 mmol) were dissolved in N,N-dimethylformamide (15 mL), and N,N-diisopropylethylamine (0.58 g, 4.5 mmol) was added dropwise. The mixture was stirred at room temperature for 3 hours until the reaction was complete. The reaction solution was then diluted with water (150 mL) and extracted with ethyl acetate (50 mL * 3). The organic phase was washed once with saturated sodium chloride aqueous solution and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. The crude product was separated by column chromatography (dichloromethane / methanol = 20 / 1) to obtain a white solid a5-2 (0.68 g, yield 56%). LC-MS(ESI): m / z: 810.28 [M+H] + .

[0117] Step 2: Dissolve a5-2 (0.5 g, 1.8 mmol) from the previous step in dichloromethane (5 mL), then add trifluoroacetic acid (1 mL). Stir the mixture at 25 °C for 3 hours. After the reaction is complete, concentrate the reaction solution by vacuum distillation, add isopropyl ether (2 mL), and stir at room temperature. Filter to obtain a pale yellow solid a5 (0.46 g, yield 91%). LC-MS (ESI): m / z: 710.23 [M+H] + .

[0118] Example 6

[0119] Preparation of target molecules L1-L35

[0120]

[0121] 5-Hydroxy-6-methylpyrimidine-4-carboxylic acid b1 (36 mg, 0.23 mmol) was dissolved in N,N-dimethylformamide (3 mL). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (61 mg, 0.32 mmol) and N-hydroxy-7-azabenzotriazole (HOAT) (15 mg, 0.11 mmol) were added. After stirring at 25 °C for 30 minutes, intermediate a5 (150 mg, 0.21 mmol) and N,N-diisopropylethylamine (190 mg, 1.47 mmol) were added. The mixture was stirred at 25 °C for 2 hours until the reaction was complete. Water (30 mL) was added, and a solid precipitated. The solid was filtered, dried, and separated by column chromatography (dichloromethane / methanol = 20 / 1) to give a pale yellow solid L1 (60 mg, 35% yield). LC-MS(ESI): m / z: 846.25 [M+H] + . 1H NMR(300MHz,Chloroform-d)δ9.20(s,1H),8.75(d,J=6.9Hz,1H),8.62(d,J=7.2Hz,2H ),8.43(d,J=10.8Hz,2H),7.62(s,1H),7.51(d,J=8.7Hz,1H),6.99(s,2H),5.65(s,1H ),5.23(s,2H),4.80(d,J=12.9Hz,1H),4.50(s,2H),3.81(s,3H),3.51(s,1H),3.08(d ,J=45.9Hz,4H),2.83(s,2H),2.60(s,3H),1.40(d,J=17.7Hz,2H),1.34-1.24(m,3H).

[0122] Following the synthetic route of intermediate L1, and using similar starting materials / compounds, the following target intermediate was synthesized.

[0123] Compound L2 was obtained by replacing b1 with 3-hydroxy-2-pyridinecarboxylic acid using the same synthetic method as compound L1.

[0124] Using the same synthetic method as compound L1, replacing b1 with 2-methyl-3-hydroxyquinoline-4-carboxylic acid yields compound L3.

[0125] Using the same synthetic method as compound L1, b1 was replaced with 4-hydroxyfurano[2,3-c]pyridine-5-carboxylic acid to obtain compound L4.

[0126] Using the same synthetic method as compound L1, replacing b1 with 1-methylpyrazole-4-carboxylic acid yields compound L5.

[0127] Using the same synthetic method as compound L1, replacing b1 with thiazol-5-carboxylic acid yields compound L6.

[0128] Using the same synthetic method as compound L1, b1 was replaced with 7-hydroxy-2,3-dihydrofurano[3,2-c]pyridine-6-carboxylic acid to obtain compound L7.

[0129] Using the same synthetic method as compound L1, compound L8 was obtained by replacing the benzene ring with pyridine.

[0130] Using the synthetic method of compound L1, 3-chloro-4-aminotrifluorotoluene was replaced with 2-chloro-4-trifluoromethoxyaniline to obtain compound L9.

[0131] Using the synthetic method of compound L1, replacing a5-1 with 4,5,6,7-tetrahydropyrazol[1,5-a]pyridine-2-carboxylic acid, we obtained compound L10.

[0132] Using the synthetic method of compound L1, by replacing a5-1 with pyrazolo[1,5-a]pyrimidine-2-carboxylic acid, compound L11 was obtained.

[0133] Using the synthetic method of compound L1, b1 was replaced with 3-hydroxy-2-pyridinecarboxylic acid, and intermediate a5-1 was replaced with pyrazolo[1,5-a]pyrimidine-2-carboxylic acid to obtain compound L12.

[0134] Using the synthetic method of compound L1, b1 was replaced with 7-hydroxy-2,3-dihydrofurano[3,2-c]pyridine-6-carboxylic acid, and intermediate a5-1 was replaced with pyrazolo[1,5-a]pyrimidine-2-carboxylic acid to obtain compound L13.

[0135] Using the synthetic method of compound L1, b1 was replaced with 4-hydroxyfurano[2,3-c]pyridine-5-carboxylic acid, and a5-1 was replaced with 4-hydroxy-2,3-dihydrofurano[2,3-c]pyridine-5-carboxylic acid to obtain compound L14.

[0136] Using the synthetic method of compound L1, b1 was replaced with 7-hydroxyfurano[3,2-c]pyridine-6-carboxylic acid, and a5-1 was replaced with 4-hydroxy-2,3-dihydrofurano[2,3-c]pyridine-5-carboxylic acid to obtain compound L15.

[0137] Using the synthetic method of compound L1, b1 was replaced with 7-hydroxy-2,3-dihydrofurano[3,2-c]pyridine-6-carboxylic acid, and a5-1 was replaced with 4-hydroxy-2,3-dihydrofurano[2,3-c]pyridine-5-carboxylic acid to obtain compound L16.

[0138] Using the synthetic method of compound L1, by replacing b1 with acetic acid and a5-1 with 4-hydroxy-2,3-dihydrofurano[2,3-c]pyridine-5-carboxylic acid, compound L17 was obtained.

[0139] Using the synthetic method of compound L1, replacing a5-1 with acetic acid yields compound L18.

[0140] Using the synthetic method of compound L1, replacing a5-1 with cyclopropane carboxylic acid yields compound L19.

[0141] Using the synthetic method of compound L1, by replacing a5-1 with imidazo[1,2-a]pyrazine-2-carboxylic acid and b1 with 3-hydroxy-2-pyridinecarboxylic acid, compound L20 was obtained.

[0142] Using the synthetic method of compound L1, by replacing a5-1 with 4-fluoropyrazolo[1,5-a]pyridine-3-carboxylic acid and replacing b1 with 3-hydroxy-2-pyridinecarboxylic acid, compound L21 was obtained.

[0143] Using the synthetic method of compound L1, replacing a5-1 with 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-2-carboxylic acid and replacing b1 with 3-hydroxy-2-pyridinecarboxylic acid, we obtained compound L22.

[0144] Using the synthetic method of compound L1, by replacing a5-1 with pyrazolo[1,5-a]pyrimidine-6-carboxylic acid and b1 with 3-hydroxy-2-pyridinecarboxylic acid, compound L23 was obtained.

[0145] Using the synthetic method of compound L1, the benzene ring was replaced with pyridine, and b1 was replaced with 3-hydroxy-2-pyridinecarboxylic acid to obtain compound L24.

[0146] Using the synthetic method of compound L1, replacing a4-1 with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)cyclohex-3-en-1-amine and replacing b1 with 3-hydroxy-2-pyridinecarboxylic acid, we obtained compound L25.

[0147] Using the synthetic method of compound L1, by replacing a5-1 with pyrazine 2-carboxylate and b1 with 3-hydroxy-2-pyridinecarboxylic acid, compound L26 was obtained.

[0148] Using the synthetic method of compound L1, by replacing a5-1 with benzo[d]thiazole-2-carboxylic acid and b1 with 3-hydroxy-2-pyridinecarboxylic acid, compound L27 was obtained.

[0149] Using the synthetic method of compound L1, by replacing a5-1 with thiazol-5-carboxylic acid and b1 with 3-hydroxy-2-pyridinecarboxylic acid, compound L28 was obtained.

[0150] Using the synthetic method of compound L1, replacing a5-1 with 1-ethyl-1H-pyrazole-3-carboxylic acid and replacing b1 with 3-hydroxy-2-pyridinecarboxylic acid, compound L29 was obtained.

[0151] Using the synthetic method of compound L1, by replacing a5-1 with pyrimidine-4-carboxylic acid and b1 with 3-hydroxy-2-pyridinecarboxylic acid, compound L30 was obtained.

[0152] Using the synthetic method of compound L1, by replacing a5-1 with pyrazolo[1,5-a]pyridine-3-carboxylic acid and b1 with 3-hydroxy-2-pyridinecarboxylic acid, compound L31 was obtained.

[0153] Using the synthetic method of compound L1, replacing a5-1 with 5-ethyl-1-methyl-1H-pyrazole-3-carboxylic acid and replacing b1 with 3-hydroxy-2-pyridinecarboxylic acid, we obtained compound L32.

[0154] Using the synthetic method of compound L1, replacing a5-1 with 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-2-carboxylic acid and replacing b1 with 3-hydroxy-2-pyridinecarboxylic acid, we obtained compound L33.

[0155] Using the synthetic method of compound L1, replacing a5-1 with pyrazolo[1,5-a]pyridine-2-carboxylic acid and replacing b1 with 3-hydroxy-2-pyridinecarboxylic acid, we obtained compound L34.

[0156] Using the synthetic method of compound L1, replacing a5-1 with pyrazolo[1,5-a]pyrimidine-6-carboxylic acid and replacing b1 with 3-hydroxy-2-pyridinecarboxylic acid, we obtained compound L35.

[0157] Using the synthetic method of compound L1, by replacing a5-1 with piperidinecarboxylic acid and b1 with 3-hydroxy-2-pyridinecarboxylic acid, compound L36 was obtained.

[0158] Using the synthetic method of compound L1, by replacing a5-1 with pyrazolo[1,5-a]pyrimidine-2-carboxylic acid, b1 with 4-hydroxyfurano[2,3-c]pyrimidine-5-carboxylic acid, and 3-chloro-4-aminotrifluorotoluene with (4-amino-3-chlorophenyl)sulfur pentafluoride, compound L37 was obtained.

[0159] Using the synthetic method of compound L1, by replacing a5-1 with pyrazolo[1,5-a]pyrimidine-2-carboxylic acid, b1 with 3-hydroxy-2-pyridinecarboxylic acid, and 3-chloro-4-aminotrifluorotoluene with (4-amino-3-chlorophenyl)sulfur pentafluoride, compound L38 was obtained.

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179] Example 7

[0180] Preparation of intermediate c1

[0181]

[0182] Step 1: Dissolve intermediate a3 (1 g, 1.5 mmol) from the previous step in dichloromethane (10 mL), then add trifluoroacetic acid (2 mL). Stir the mixture at 25 °C for 3 hours. After the reaction is complete, concentrate the reaction solution by vacuum distillation, add isopropyl ether (4 mL), and slurry at room temperature. Filter to obtain intermediate c1-1 (0.8 g, yield 94%). LC-MS (ESI): m / z: 562.05 [M+H] + .

[0183] 3-Hydroxy-2-pyridinecarboxylic acid (104 mg, 0.75 mmol) was dissolved in N,N-dimethylformamide (2 mL). EDCI (180 mg, 0.94 mmol) and HOAT (141 mg, 0.31 mmol) were added, and the mixture was stirred at 25 °C for 30 minutes. Then, intermediate C1-1 (350 mg, 0.63 mmol) and N,N-diisopropylethylamine (240 mg, 1.86 mmol) were added, and the mixture was stirred at 25 °C for 2 hours until the reaction was complete. Water (30 mL) was added, and a solid precipitated. The solid was filtered, dried, and separated by column chromatography (dichloromethane / methanol = 20 / 1) to give a pale yellow solid C1 (170 mg, 40% yield). LC-MS (ESI): m / z: 683.170 [M+H] + .

[0184] Example 8

[0185] Preparation of target molecules L39-L45

[0186]

[0187] Step 1: Piperazine (0.5 g, 5.8 mmol) was dissolved in DMSO (5 mL), and triethylamine (1.61 mL, 11.6 mmol) was added. The mixture was stirred at room temperature for 5 minutes, and intermediate C1 (1.3 g, 1.9 mmol) and potassium acetate (1.14 g, 11.6 mmol) were added. The reaction was heated at 120 °C for 16 hours. After the reaction was completed, the mixture was cooled to room temperature, quenched with water (2 mL), and concentrated under reduced pressure. The crude product was separated by column chromatography (dichloromethane / methanol = 10 / 1) to give C2 (0.26 g, yield 20%). LC-MS (ESI): m / z: 689.23 [M+H] + .

[0188] Step 2: Pyrazolo[1,5-a]pyrimidine-2-carboxylic acid (57 mg, 0.35 mmol) was dissolved in N,N-dimethylformamide (5 mL), then HATU (0.17 g, 0.87 mmol), N,N-diisopropylethylamine (0.11 g, 0.87 mmol), and intermediate C2 (0.2 g, 0.3 mmol) were added. The mixture was stirred at 25 °C for 3 hours. After the reaction was complete, water (20 mL) was added, and the mixture was filtered. The filter cake was dried and separated by column chromatography (dichloromethane / methanol = 20 / 1) to give a pale yellow solid L39 (97 mg, yield 40%). LC-MS (ESI): m / z: 834.25 [M+H] + . 1 H NMR(300MHz,Chloroform-d)δ9.27(s,1H),9.12-9.10(m,1H),8.49–8.46(m,1H),8.18-8.16 (m,1H),7.77(d,J=2.4Hz,1H),7.69(d,J=7.2Hz,1H),7.60-7.58(m,1H),7.50-7.47(m,1H),7 .32–7.29(m,1H),6.94(t,J=6.9Hz,1H),6.89(s,1H),4.77(s,1H),4.00-3.95(m,2H),3.88- 3.74(m,8H),3.73-3.71(m,2H),3.62-3.58(m,4H),2.69-2.52(m,H),1.14(d,J=15.6Hz,3H).

[0189] Following the synthetic route of compound L39, and using similar starting materials / compounds, the following target compounds were synthesized.

[0190] Using the synthetic method of L39, pyrazolo[1,5-a]pyrimidine-3-carboxylic acid was replaced with pyrazolo[1,5-a]pyrimidine-2-carboxylic acid to obtain compound L40.

[0191] Using the synthetic method of L39, pyrazolo[1,5-a]pyrimidine-3-carboxylic acid was replaced with pyrazine 2-carboxylic acid to obtain compound L41.

[0192] Using the synthetic method of L39, pyrazolo[1,5-a]pyrimidine-3-carboxylic acid was replaced with pyrazine 2-carboxylic acid, and 3-hydroxy-2-pyridinecarboxylic acid was replaced with 5-hydroxy-6-methylpyrimidine-4-carboxylic acid to obtain compound L42.

[0193] Using the synthetic method of L39, 3-chloro-4-aminotrifluorotoluene was replaced with (4-amino-3-chlorophenyl)sulfur pentafluoride to form compound L43.

[0194] Using the L39 synthetic method, pyrazolo[1,5-a]pyrimidine-3-carboxylic acid was replaced with pyrazine 2-carboxylic acid, and piperazine was replaced with cyclohexene amino group to obtain compound L44.

[0195] Using the L39 synthetic method, pyrazolo[1,5-a]pyrimidine-3-carboxylic acid was replaced with cyclopropionic acid, and piperazine was replaced with cyclohexene amino group to obtain compound L45.

[0196]

[0197]

[0198]

[0199]

[0200] Preparation of intermediate d1

[0201]

[0202] Step 1: Ethyl propionyl ethyl d1-1 (9.8 g, 67.6 mmol) and 5-bromo-1-H-3-amino-1,2,4-triazole a1 (11 g, 67.6 mmol) were dissolved in 100 mL of acetic acid. The mixture was heated to 90 °C and reacted for 12 hours. After cooling to room temperature, the solvent was removed by vacuum evaporation. Ethyl acetate (30 mL) was added, and the mixture was stirred at room temperature and filtered to obtain a pale yellow solid d1-2 (12.1 g, yield 73.3%). LC-MS (ESI): m / z: 242.98 [M+H] + .

[0203] Step 2: Dissolve intermediates d1-2 (10 g, 41.3 mmol) and a3-2 (14.4 g, 45.4 mmol) from the previous step in N,N-dimethylformamide (100 mL), add N,N-diisopropylethylamine (16 g, 124 mmol), heat to 50 °C and react for 3 hours. Cool to room temperature, add 500 mL of water, extract with ethyl acetate (150 mL * 3), dry to anhydrous sodium sulfate, concentrate, and separate the crude product by column chromatography (petroleum ether / ethyl acetate = 2 / 1) to obtain a white solid d1 (3.23 g, yield 67%). LC-MS (ESI): m / z: 477.98 [M+H] + .

[0204] Example 10

[0205] Preparation of intermediate d2

[0206]

[0207] Step 1: Under nitrogen protection, intermediate d1 (10 g, 20.9 mmol), 1,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester (5.26 g, 25.1 mmol), and potassium carbonate (5.8 g, 41.8 mmol) were dissolved in 100 mL of dioxane. 33 mL of water was added, along with catalyst Pd(dppf)C12·DCM (1.7 g, 2 mmol). The mixture was heated to 100 °C and reacted for 8 hours. After the reaction was complete, 100 mL of water was added to the reaction solution, and the mixture was filtered. The filter cake was dried, and 20 mL of isopropyl ether was added and stirred at room temperature. The mixture was then filtered to obtain crude product d2-1 (7.03 g, 70% yield). LC-MS (ESI): m / z: 481.12 [M+H] + .

[0208] Step 2: Pyrazolo[1,5-a]pyrimidine-2-carboxylic acid (2.04 g, 12.5 mmol) was dissolved in N,N-dimethylformamide (20 mL), followed by the addition of HATU (5.94 g, 15.6 mmol), N,N-diisopropylethylamine (4.03 g, 31.2 mmol), and intermediate d2-1 (5 g, 10.4 mmol). The mixture was stirred at 25 °C for 3 hours. After the reaction was complete, water (200 mL) was added, and the mixture was filtered. The filter cake was dried and separated by column chromatography (dichloromethane / methanol = 20 / 1) to obtain d2-2 (4.1 g, yield 63%). LC-MS (ESI): m / z: 626.15 [M+H] + .

[0209] Step 3: Dissolve the intermediate d2-2 (4 g, 6.4 mmol) from the previous step in N,N-dimethylformamide (40 mL), add N-bromosuccinimide (232 mg, 6.4 mmol), heat to 40 °C under nitrogen protection, and react for 4 hours. Monitor the reaction by TLC until the starting material is completely reacted. Add 250 mL of water; a solid precipitates. Filter and dry to obtain d2 (3.8 g, yield 84%). LC-MS (ESI): m / z: 704.06 [M+H] + .

[0210] Example 11

[0211] Preparation of intermediate e1

[0212]

[0213] Step 1: Under nitrogen protection, bromophenol e1-2 (3.84 g, 22.2 mmol) was dissolved in 39 mL of anhydrous N,N-dimethylformamide. The mixture was cooled to 0 °C, and potassium carbonate (3.68 g, 26.6 mmol) and 4-chloro-5-methoxy-6-methylpyrimidine e1-1 (3.53 g, 22.2 mmol) were added. The mixture was heated to 80 °C, and after 2 hours, the reaction was monitored by TLC to indicate completion. 400 mL of water was added, and the mixture was extracted with ethyl acetate (100 mL * 3). The extract was washed with saturated brine, dried over anhydrous sodium sulfate, and then distilled under reduced pressure. The crude product was separated by column chromatography (petroleum ether / ethyl acetate = 20 / 1) to obtain e1-3 (3.52 g, yield 54%). LC-MS (ESI): m / z: 295.00 [M+H] + .

[0214] Step 2: Under nitrogen protection, intermediate e1-3 (3.52 g, 12.0 mmol) from the previous step was dissolved in 40 mL of dichloromethane. The mixture was cooled to -78 °C, and 5 mL of boron tribromide was slowly added dropwise. After stirring for 1 hour, the mixture was heated to room temperature and the reaction continued for 7 hours until completion. The reaction was quenched dropwise with methanol at -78 °C, and then extracted with 100 mL of water and 30 mL of dichloromethane. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, and then distilled under reduced pressure. No further purification was required to obtain a white solid e1-4 (1.6 g). LC-MS (ESI): m / z: 280.98 [M+H] + .

[0215] Step 3: Under nitrogen protection, intermediate e1-4 (1.4 g, 5.0 mmol) was dissolved in 14 mL of dioxane. Pinaryl diboronate (2.53 g, 10.0 mmol), potassium acetate (1.47 g, 15 mmol), and Pd(dppf)Cl2 (0.36 g, 0.5 mmol) were added. The reaction was carried out at 90 °C for 9 hours until completion. 30 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL * 3). The extract was washed with saturated brine, dried over anhydrous sodium sulfate, and then distilled under reduced pressure. Column chromatography (petroleum ether / ethyl acetate 3:1) was performed to give a white solid e1 (0.92 g, yield 56%). LC-MS (ESI): m / z: 328.16 [M+H] + .

[0216] Example 12

[0217] Preparation of target molecules L46-L51

[0218]

[0219] Under nitrogen protection, intermediates e1 (0.1 g, 20.9 mmol), d2 (5.26 g, 25.1 mmol), and potassium carbonate (5.8 g, 41.8 mmol) were dissolved in 100 mL of dioxane, 33 mL of water was added, and catalyst Pd(dppf)C12 (1.7 g, 2.0 mmol) was added. The mixture was heated to 100 °C and reacted for 8 hours. After the reaction was complete, 100 mL of water was added to the reaction solution, and the mixture was filtered. The filter cake was dried and separated by column chromatography (dichloromethane / methanol = 20 / 1) to obtain the target molecule L46 (4.1 g, yield 36%). LC-MS (ESI): m / z: 626.15 [M+H] +. 1H NMR(300MHz,Chloroform-d)δ9.27(s,1H),9.23-9.14(m,1H),8.72(m,J=7.2,1.2Hz,1H),8.29(s,1H),8.25-8.14 (m,1H),8.04(s,1H),7.77(d,J=1.5Hz,1H),7.69(d,J=7.5Hz,1H),7.61-7.54(m,3H),7.24(d,J=6.6Hz,1H),7.13- 7.08(m,2H),7.07-7.05(m,1H),7.03-7.00(m,1H),6.10-6.04(m,1H),5.26(s,2H),4.25-4.20(m,1H),4.09(m,J=4 .8,1.2Hz,1H),4.00-3.94(m,1H),3.90-3.86(m,1H),3.42–3.32(m,2H),2.60-2.51(m,2H),1.11(t,J=7.8Hz,3H).

[0220] Following the synthetic route of compound L46, and using similar starting materials / compounds, the following target intermediate was synthesized.

[0221] Using the synthetic method of L46, replacing d1-1 with 2-chloro-3-methoxypyridine yielded compound L47.

[0222] Using the synthetic method of L46, pyrazolo[1,5-a]pyrimidine-2-carboxylic acid was replaced with pyrazolo[1,5-a]pyrimidine-3-carboxylic acid to obtain compound L48.

[0223] Using the synthetic method of L46, e1-1 was replaced with 2-chloro-3-methoxypyridine, and pyrazolo[1,5-a]pyrimidine-2-carboxylic acid was replaced with pyrazolo[1,5-a]pyrimidine-3-carboxylic acid to obtain compound L49.

[0224] Using the L46 synthetic method, pyrazolo[1,5-a]pyrimidine-2-carboxylic acid was replaced with pyrazine 2-carboxylic acid to obtain compound L50.

[0225] Using the L46 synthetic method, e1-1 was replaced with 2-chloro-3-methoxypyridine, pyrazolo[1,5-a]pyrimidine-2-carboxylic acid was replaced with pyrazolo[1,5-a]pyrimidine-3-carboxylic acid, and 3-chloro-4-aminotrifluorotoluene was replaced with (4-amino-3-chlorophenyl)sulfur pentafluoride to obtain compound L51.

[0226]

[0227]

[0228]

[0229] Example 14

[0230] Preparation of intermediate f3

[0231]

[0232] Step 1: Under nitrogen protection, tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate f3-1 (169 mg, 0.85 mmol) and trifluoroacetic acid (172 mg, 1.7 mmol) were dissolved in DMSO (2 mL). Starting material d2 (400 mg, 0.57 mmol) was added, and the mixture was heated to 85 °C and reacted for 12 hours. The reaction was then stopped. 20 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL * 3). The extract was dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography (dichloromethane / methanol = 20 / 1) to obtain solid f3-2 (0.21 g, yield 45%). LC-MS (ESI): m / z: 822.27 [M+H] + .

[0233] Step 2: Dissolve intermediate f3-2 (0.2 g, 0.24 mmol) from the previous step in dichloromethane (2 mL), then add trifluoroacetic acid (0.4 mL). Stir the mixture at 25 °C for 3 hours. After the reaction is complete, concentrate the reaction solution by vacuum distillation, add isopropyl ether (3 mL), and stir at room temperature. Filter to obtain f3 (0.16 g, yield 94%). LC-MS (ESI): m / z: 722.23 [M+H] + .

[0234] Example 15

[0235] Preparation of target compounds L52-L56

[0236]

[0237] 3-Hydroxy-2-pyridinecarboxylic acid (36.2 mg, 0.26 mmol) was dissolved in N,N-dimethylformamide (2 mL), and EDCI (63.3 mg, 0.33 mmol) and HOAT (15 mg, 0.11 mmol) were added. The solution was incubated at 25°C.

[0238] After stirring for 30 minutes, intermediate f3 (160 mg, 0.22 mmol) and DIEPA (86 mg, 0.66 mmol) were added, and the mixture was stirred at 25°C for 2 hours until the reaction was complete. Water (10 mL) was added, and a solid precipitated. The solid was filtered, dried, and separated by column chromatography (methane / methanol = 20 / 1) to obtain the target product L52 (60 mg, yield 32%). LC-MS (ESI): m / z: 843.24 [M+H] + . 1 HNMR (300MHz, Chloroform-d)

[0239] δ11.27(s,1H),9.13-9.10(m,1H),8.51-8.46(m,1H),8.22-8.16(m,1H),7.77(d,J

[0240] =1.5Hz,1H),7.69(d,J=7.2Hz,1H),7.62-7.58(m,1H),7.52-7.47(m,1H),

[0241] 7.33-7.29(m,1H),6.94(t,J=6.9Hz,1H),6.89(s,1H),5.70-5.65(m,1H),4.77(s,

[0242] 2H),4.33-4.24(m,1H),4.03-3.91(m,2H),3.87(s,2H),3.75(s,2H),3.70(d,J=

[0243] 1.5Hz,4H),3.36-3.27(m,2H),2.63-2.52(m,2H),1.14(t,J=7.5Hz,3H).

[0244] Following the synthetic route of compound L52, and using similar starting materials / compounds, the following target intermediate was synthesized.

[0245] Using the synthetic method of L52, 3-hydroxy-2-pyridinecarboxylic acid was replaced with 5-hydroxy-6-methylpyrimidine-4-carboxylic acid to obtain compound L53.

[0246] Using the synthetic method of L52, 2,6-diazaspiro[3.3]heptane was replaced with 1,4-diazacycloheptane, and 3-hydroxy-2-pyridinecarboxylic acid was replaced with 5-hydroxy-6-methylpyrimidine-4-carboxylic acid to obtain compound L54.

[0247] Using the synthetic method of L52, 2,6-diazaspiro[3.3]heptane was replaced with 1,4-diazacycloheptane to obtain compound L55.

[0248]

[0249]

[0250]

[0251] Example 16

[0252] Examples of tests for the biological activity and related properties of compounds

[0253] Experimental Methods: 60 μL of the compound prepared in this invention was added to a 384-well plate and serially diluted 1:3 in DMSO. 0.15 μL of the diluted compound solution was transferred to each of the 384 wells, with two replicates per column. 5 μL of WRN (WRN protein fragments 517AA-1238AA) was added to each well, and the plate was centrifuged at 1000 rpm for 1 min. The plate was incubated at 25°C for 10 min. 5 μL of dsDNA working solution (WRN protein fluorescent substrate) was added as the reaction substrate, and the plate was centrifuged at 1000 rpm for 1 min. 5 μL of ATP working solution was added to initiate the reaction, and the plate was centrifuged at 1000 rpm for 1 min. The plate was incubated at 25°C for 20 min. Fluorescence signals at Ex620 nm and Em685 nm were read using BMG.

[0254] Data calculation: Calculation formula: Compound percentage inhibition rate (%inh) = 100 * (average high control - compound well) / (average high control - average low control).

[0255] Reliability testing of low-control and high-control data:

[0256] High control: DMSO and enzyme

[0257] Low control: DMSO and assay buffer

[0258] S / B = Average high control / Average low control

[0259] CV% (low control) = 100 * (SD low control / average low control)

[0260] CV% (high control) = 100 * (SD high control / average high control)

[0261] Z' = 1 - 3 * (SD low control + SD high control) / (mean high control - mean low control)

[0262] IC analysis of compounds using XLfit 5.5.0 50 Fitting nonlinear regression equations

[0263] Y=Bottom+(Top-Bottom) / (1+10^((LogIC50 -X)*HillSlope))

[0264] X: Logarithm of compound concentration

[0265] Y: Percentage inhibition rate (%inh)

[0266] Top and Bottom: Stable numbers in the same units as Y.

[0267] logIC 50 Same logarithmic unit as X

[0268] HillSlope: slope or gradient.

[0269] Experimental results:

[0270] The half-maximal inhibitory concentrations (IC50) of some compounds against WRN were tested using the experimental methods described above, and the results are shown in Table 1.

[0271] Example 17

[0272] The degradation activity of the compound on WRN protein was investigated. Details are as follows:

[0273] Experimental methods

[0274] Day 1: HCT116 cells (7*10) 5 Seeds were sown into 6-well plates at a concentration of 2 mL / well and incubated overnight.

[0275] Day 2: Add 1 μM of the compound and DMSO to the control group; incubate for 24 hours.

[0276] Day 3: Cell collection. Add lysis buffer to sample tubes, grind thoroughly, place samples on ice for 30 minutes, then centrifuge at 13000 rpm for 20 minutes at 4°C. Transfer the supernatant to 1.5 mL EP tubes and aliquot. Determine the protein concentration of each sample using the BCA method, and adjust the concentration of all samples to the same value using lysis buffer. Add loading buffer and incubate in a 95°C water bath for 5 minutes. Add the prepared reagents sequentially to the plate, run JESS (Multifunctional Automated Protein Expression Quantitative Analysis System) to read the data, and calculate the WRN protein degradation rate by observing changes in protein fluorescence.

[0277] The degradation activity of some compounds on WRN was determined using the experimental method described above. The results are shown in Table 1. (A > 80%; 80% ≥ B > 60%; 60% ≥ C > 40%; D ≤ 40%)

[0278] Table 1. Inhibitory and degradation activities of some compounds against WRN

[0279]

[0280]

[0281]

[0282] Conclusion: The compounds constructed in this invention exhibit excellent WRN inhibitory activity, and some compounds show superior activity compared to the positive control compound HRO761. Furthermore, this invention represents the first time that compounds have been proposed to effectively degrade WRN protein, and some compounds demonstrate significantly better WRN protein degradation activity than the positive control compound HRO761.

[0283] Example 18

[0284] Assay for antiproliferative activity against MSI-H tumor cells

[0285] Principle: Microsatellite unstable MSI-H tumor cells are sensitive to WRN inhibitors in their cell proliferation; microsatellite stable MSS tumor cells are insensitive to WRN inhibitors in their proliferation. Testing the activity of both demonstrates the molecule of this invention's inhibitory and synthetic lethal effects on WRN at the cellular level, thereby targeting and killing MSI-H cells.

[0286] Experimental methods

[0287] Day 0: HCT116 cells (500 cells / well) were seeded into 96-well plates and incubated at 37°C with CO2.

[0288] Day 1: Add compound (10 μM, 3-fold dilution, n=2), incubate at 37°C with CO2.

[0289] Day 6: Add Cell Counting-lite 2.0 fluorescence cell viability assay reagent to each well, shake well for 2 minutes, and incubate at room temperature for 30 minutes. Read the luminescence on the BMG.

[0290] Experimental results

[0291] The antiproliferative effects of some compounds against MSI cytotoxicity were determined using the experimental methods described above, and the results are shown in the table below.

[0292] Table 2. Half-maximal inhibitory concentrations of some compounds against MSI-H cells (HCT116).

[0293]

[0294]

[0295] Conclusion: The compounds of this invention can effectively inhibit the proliferation of MSI cells, and some compounds have significantly better activity than the positive compound HRO761.

[0296] Example 19

[0297] Liver microsomal stability assay of the compound. Details are as follows:

[0298] Experimental methods

[0299] Prepare 0.5587 mg / mL liver microsomes (LM solution) in PBS (100 mM, pH 7.4). Add 358 μL of 0.5587 mg / mL LM solution to a 96-well plate (culture plate). Add 40 μL of 10 mM NADPH PBS solution or 40 μL of PBS (negative control) to a culture dish, centrifuge at 800 rpm for 10 s, and incubate at 37 °C for 10 min. Add 2 μL of 2 μM test compound to the culture dish to start the reaction. At 0.5, 15, 30, 45, and 60 minutes, transfer 50 μL of culture medium from the incubation plate to a sample plate containing 200 μL of cold methanol and IS*, respectively. Centrifuge the sample plate at 3220 g for 40 min. Transfer 100 μL of the supernatant to an analytical plate containing an appropriate volume of water for LC-MS / MS analysis.

[0300] *IS: 35 ng / mL ketoprofen, 7.5 ng / mL carbamazepine, 5 ng / mL diphenhydramine, 10 ng / mL tolbutamide.

[0301] Data Computing

[0302] The gradient k(ln(remaining percentage of compound) vs. incubation time) of the straight line was calculated using Microsoft Excel.

[0303] Calculate in vitro t using the following formula. 1 / 2 and in vitro Clint:

[0304] In vitro t 1 / 2 (min)=0.693 / k

[0305] In vitro Clint (μL / min / mg protein) = Incubation volume (μL) / Protein incubation amount (mg) * 0.693 / t 1 / 2 .

[0306] The stability of some compounds on rat liver microsomes was determined using the experimental methods described above, and the results are shown in Table 3.

[0307] Table 3. Results of in vitro liver microsomal stability experiments of the compounds in rats.

[0308]

[0309]

[0310] Results: This indicates that the compounds of the present invention have good metabolic stability.

[0311] Example 20

[0312] The target selectivity experiment of the compound is as follows:

[0313] Experimental Method: Add 40 μL of the compound to each well of a 384-well dilution plate. Continuously dilute the compound 1:3 in DMSO. Transfer 0.1 μL of the diluted compound solution from each row to a 384-well plate, with two replicates per column. Add 5 μL of enzyme working solution (RecQ1, BLM, RecQ4, or RecQ5) to each well of the 384-well plate, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 10 min. Add 5 μL of the fluorescent substrate dsDNA working solution for each enzyme to be tested as the reaction substrate, centrifuge at 1000 rpm for 1 min. Add 5 μL of ATP working solution to start the reaction, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 60 min. Add 5 μL of ADP-Glo ​​Reagent to each well of the 384-well plate, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 40 min. Add 10 μL of detection solution to a 384-well plate, centrifuge at 1000 rpm for 1 min, incubate at 25 °C for 40 min, and read the luminescence signal using BMG.

[0314] Compound pore inhibition rate (%inh) = 100 * (high control - compound pores) / (high control - low control)

[0315] Reliability testing of low-control and high-control data analysis:

[0316] High control: DMSO and enzyme

[0317] Low control: DMSO and assay buffer

[0318] S / B = Average high control / Average low control

[0319] CV% (low control) = 100 * (SD low control / average low control)

[0320] CV% (high control) = 100 * (SD high control / average high control)

[0321] Z' = 1 - 3 * (SD low control + SD high control) / (mean high control - mean low control)

[0322] Using XLfit 5.5.0 to analyze the compound IC of the nonlinear regression equation 50 Perform fitting

[0323] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50-X)*HillSlope))

[0324] X: Log of compound concentration

[0325] Y: Average inhibition rate (%inh)

[0326] TOP and Bottom: Same units as Y

[0327] logIC 50 Same logarithmic unit as X

[0328] HillSlope: slope or gradient.

[0329] The selectivity of some compounds was determined using the experimental method described above, and the results are shown in Table 5.

[0330] Table 4. Target selectivity experiments of compounds

[0331]

[0332] Results: The representative compound of this invention did not have an inhibitory effect on other RecQ proteins, but had good selectivity for WRN.

[0333] The above results indicate that the compound designed in this invention has a good anti-proliferative effect on MSI-H tumor cells, and can achieve a good tumor suppression effect by simultaneously inhibiting and degrading WRN.

Claims

1. A triazole derivative or a pharmaceutically acceptable salt thereof having WRN-degrading activity, characterized in that, The structure of the compound is shown in general formula (I). in: R1 is selected from substituted or unsubstituted C. 1-8 Alkyl, substituted or unsubstituted 5-6 membered monocyclic aryl, substituted or unsubstituted 5-6 membered monocyclic heterocyclic, or substituted or unsubstituted 5-6 membered monocyclic heteroaryl; the substituted C 1-8 Alkyl, 5-6 membered monocyclic aryl, 5-6 membered monocyclic heterocyclic or 5-6 membered monocyclic heteroaryl are optionally substituted by 0-3 independent substituents selected from -OH, halogen, -CN or NH2; the 5-6 membered monocyclic heterocyclic or 5-6 membered monocyclic heteroaryl contains 1-2 heteroatoms independently selected from N, O or S; R2 is selected from hydrogen or substituted or unsubstituted 5-6 membered monocyclic aryl, substituted or unsubstituted 5-6 membered monocyclic heteroaryl, or substituted or unsubstituted 5-6 membered fused heteroaryl; the substituted 5-6 membered monocyclic aryl, 5-6 membered monocyclic heteroaryl, or 5-6 membered fused heteroaryl are optionally substituted by 0-3 independent substituents selected from halogen, -CN, CF3, or SF5; the 5-6 membered monocyclic heteroaryl or 5-6 membered fused heteroaryl contains at least one N atom; R3 is selected from hydrogen or substituted or unsubstituted 5-6 membered monocyclic heteroaryl or substituted or unsubstituted 4-10 membered fused heterocyclic group; the substituted 5-6 membered monocyclic heterocyclic group or 4-10 membered fused heterocyclic group is optionally substituted by 0-3 independent substituents selected from -OH, halogen, -CN or NH2; the 5-6 membered monocyclic heteroaryl group contains 1-3 independent heteroatoms selected from N, O or S; R4 is selected from hydrogen or substituted or unsubstituted 5-6 membered monocyclic heteroaryl, substituted or unsubstituted 3-6 membered cycloalkyl, substituted or unsubstituted 4-10 membered heterocyclic or substituted or unsubstituted 4-10 membered fused heterocyclic; wherein the substituted 5-6 membered monocyclic heteroaryl, 3-6 membered cycloalkyl, 4-10 membered heterocyclic or 4-10 membered fused heterocyclic is optionally surrounded by 0-3 independent elements selected from -OH, halogen, methyl, C 1-6 Alkyl groups or -CN substituents may be used; the 5-6 member monocyclic heteroaryl, 4-10 member heterocyclic or 4-10 member fused heterocyclic groups contain 1-3 heteroatoms independently selected from N, O or S.

2. A triazole derivative or a pharmaceutically acceptable salt thereof having WRN-degrading activity, characterized in that, The structure of the compound is shown in general formula (II). in: X, Y, and Z are each independently selected from CH or N, and X, Y, and Z contain at least one N atom; R3 is selected from hydrogen or substituted or unsubstituted 5-6 membered monocyclic heteroaryl or substituted or unsubstituted 4-10 membered fused heterocyclic group; the substituted 5-6 membered monocyclic heterocyclic group or 4-10 membered fused heterocyclic group is optionally substituted by 0-3 independent substituents selected from -OH, halogen, -CN or NH2; the 5-6 membered monocyclic heteroaryl group contains 1-3 independent heteroatoms selected from N, O or S; R5 is selected from H, halogen, CN, C. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, C 3-6 An alkoxy or 4-10 membered heterocyclic group, wherein the 4-10 membered heterocyclic group comprises 1-3 heteroatoms independently selected from N, O or S; Ring A and the benzene ring attached thereto form a fused ring, wherein ring A is selected from substituted or unsubstituted 5-7-membered aryl or substituted or unsubstituted 5-7-membered heterocyclic groups; the substituted 5-7-membered aryl or 5-7-membered heterocyclic groups are optionally surrounded by 0-3 independent groups selected from -OH, halogen, C 1-6 Alkyl or -CN substituents; the 5-7 aryl or 5-7 heterocyclic group contains 0-2 heteroatoms independently selected from N, O or S; m is selected from 0, 1, 2 or 3.

3. A triazole derivative or a pharmaceutically acceptable salt thereof having WRN-degrading activity, characterized in that, The structure of the compound is shown in preferred general formula (III). in: X, Y, and Z are each independently selected from CH or N, and X, Y, and Z contain at least one N atom; R3 is selected from hydrogen or substituted or unsubstituted 5-6 membered monocyclic heteroaryl or substituted or unsubstituted 4-10 membered fused heterocyclic group; the substituted 5-6 membered monocyclic heterocyclic group or 4-10 membered fused heterocyclic group is optionally substituted by 0-3 independent substituents selected from -OH, halogen, -CN or NH2; the 5-6 membered monocyclic heteroaryl group contains 1-3 independent heteroatoms selected from N, O or S; R4 is selected from hydrogen or substituted or unsubstituted 5-6 membered monocyclic heteroaryl, substituted or unsubstituted 3-6 membered cycloalkyl, substituted or unsubstituted 4-10 membered heterocyclic or substituted or unsubstituted 4-10 membered fused heterocyclic; wherein the substituted 5-6 membered monocyclic heteroaryl, 3-6 membered cycloalkyl, 4-10 membered heterocyclic or 4-10 membered fused heterocyclic is optionally surrounded by 0-3 independent elements selected from -OH, halogen, methyl, C 1-6 Alkyl groups or -CN substituents may be used; the 5-6 member monocyclic heteroaryl, 4-10 member heterocyclic or 4-10 member fused heterocyclic groups contain 1-3 heteroatoms independently selected from N, O or S.

4. The triazole derivative or a pharmaceutically acceptable salt thereof having WRN-degrading activity according to any one of claims 1-3, characterized in that, The compound is selected from any one of the following:

5. A method for preparing a triazole derivative or a pharmaceutically acceptable salt thereof with WRN degradation activity as described in claim 1, characterized in that, The steps include the following: (1) Bromination of compound a1-1 yields compound a1; (2) Compound a2-1 undergoes chlorination under the action of sulfonyl chloride to give compound a2-2; (3) Compound a2-2 reacts with N-Boc-piperazine in the presence of triethylamine to give compound a2-3; (4) Compound a2-3 reacts with a1 under the action of phosphoric acid to give compound a2; (5) Compound a3-1 undergoes acylation under alkaline conditions to give compound a3-2; (6) Compound a3-2 reacts with a2 under alkaline conditions to give compound a3; (7) Compound a4-1 loses its protecting group under acidic conditions to obtain compound a4-2; (8) Compound a4-2 undergoes Suzuki coupling with a3 under the action of a catalyst to obtain compound a4; (9) Compound a4 was reacted with a condensing agent to obtain compound a5-2; (10) Compound a5-2 was deprotected under acidic conditions to obtain compound a5; (11) Compound a5 was reacted with a condensing agent to obtain the target compound; The reaction formula is shown below:

6. Use of a triazole derivative or a pharmaceutically acceptable salt thereof having WRN degradation activity as described in any one of claims 1-3 in the preparation of a reagent having WRN inhibition and WRN protein degradation functions.

7. Use of a triazole derivative or a pharmaceutically acceptable salt thereof having WRN-degrading activity as described in any one of claims 1-3 in the preparation of a medicament for treating or preventing tumors.

8. The use according to claim 7, characterized in that, The tumor is a microsatellite unstable tumor, including one or more of colorectal cancer, endometrial cancer, gastric cancer, and liver cancer.

9. A WRN inhibitor and WRN protein degradation reagent, characterized in that, It includes any of the triazole derivatives or pharmaceutically acceptable salts thereof as described in claims 1-3, and pharmaceutically acceptable carriers or excipients.

10. A pharmaceutical composition for treating or preventing tumors, characterized in that, The pharmaceutical composition contains a therapeutically effective amount of any of the triazole derivatives of claims 1-3 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

11. The pharmaceutical composition according to claim 10, wherein the pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhaler, ointment, suppository or patch.

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