A protac compound targeting degradation of parp7 and preparation method and application thereof

Abstract

The application discloses a PROTAC compound for targeted degradation of PARP7 and a preparation method and application thereof. The application provides a PROTAC compound for targeted degradation of PARP7 with a structure shown in formula (I), a pharmaceutical composition containing a derivative of formula (I) and a hydrate, and isotopic derivatives, chiral isomers, conformers, different salts, prodrugs and preparations of the compounds. The application also provides a preparation method of the PROTAC compound for targeted degradation of PARP7 and application of the compound in treatment of solid tumors and hematological tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a PROTAC compound that targets the degradation of PARP7, its preparation method, and its application. Background Technology

[0002] Poly(ADP-ribose) polymerases (PARPs) are members of a family of seventeen enzymes that regulate fundamental cellular processes including gene expression, protein degradation, and multicellular stress responses. Poly(ADP-ribose) polymerase 7 (PARP7) is an AHR-regulated gene and a key member of the PARP family, involved in a range of cellular processes such as DNA repair and maintenance of genome stability. PARP7 can only transfer a single ADP-ribose (MAR), belonging to the monoPARP family. The PARP catalytic domain of PARP7 contains a zinc finger motif that can confer DNA binding and a WWE domain that mediates protein-protein interactions. Its mediated mono-ADP ribosylation is a reversible post-translational modification involved in various important biological processes, such as immune cell function, transcriptional regulation, protein expression, and DNA repair. PARP7 plays a crucial role in neuronal development, stem cell maintenance, antiviral infection, and cancer. Based on its role in innate immune responses, the role of PARP7 in tumor immunity is also receiving increasing attention. Aberrantly expressed or activated PARP7 can suppress innate immune responses, thereby inhibiting T cell-mediated anti-tumor immunity. In large-scale gene screening, PARP7 has also been identified as an inhibitor of T cell activation. Knocking out PARP7 in melanoma cells enhances the proliferation and growth of co-cultured T cells.

[0003] With the continuous development of drug development technology, targeted protein degradation (TPD) has received considerable attention from medicinal chemists and is considered one of the most promising treatment methods. Protein-targeted degradation chimeras (PROTACs) are an emerging strategy for small molecule drug TPD. A PROTAC is a bifunctional small molecule in which a target protein ligand and an E3 ubiquitin ligase ligand are linked together via a linker arm to form a triplet compound. As a potential therapeutic approach, PROTACs can degrade specific proteins. A PROTAC is a biospecific functional molecule, typically consisting of a compound that binds to a protein target, a small molecule ligand that recruits an E3 ligase, and a linker. Induction by PROTACs leads to selective polyubiquitination of the target protein and subsequent degradation on the proteasome. Compared with traditional small molecule inhibitors, PROTACs have several advantages, including the ability to exert their effects without binding to the active site of the target protein, the ability to degrade drug-resistant targets, and the ability to produce drugs at lower doses. Therefore, they have great potential, especially in the development of anticancer drugs.

[0004] Currently, most reports focus on small molecule structural modifications and prodrug studies of PARP7 inhibitors (RBN-2397), and there are no reports on PROTAC drugs targeting PARP7. Summary of the Invention

[0005] The first objective of this invention is to address the shortcomings of the prior art by providing a PROTAC compound that targets the degradation of PARP7.

[0006] A PROTAC compound that targets the degradation of PARP7, or an optical isomer, racemate, single enantiomer, possible diastereomer, or pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, or solvate thereof, wherein the structure of the derivative is shown in formula (I):

[0007]

[0008] in:

[0009] A represents -O-, -NH-, -NHCO-, -CONH-.

[0010] Linker is selected from straight-chain hydrocarbon groups, oxyheterochains, phenyl groups, heterocyclic groups, or one of the following groups with 1 to 60 carbon atoms:

[0011]

[0012] Where m and n are both natural numbers between 0 and 20. This indicates the site where the linker connects to the aromatic ring of E3 ligase; Indicates the site where the Linker connects to A;

[0013] E3 ligase is selected from one of the following structural segments:

[0014]

[0015] in:

[0016] W is CH2, C=O, NH, or N-alkyl;

[0017] X is either O or S;

[0018] Z is -alkyl, -cycloalkyl, -Cl, -F or -H;

[0019] G is -H, -OH, or -CH2, -heterocyclic, or alkyl;

[0020] R 1 It is a substituent on the aromatic ring, selected from one of -H, -D, -F, -Cl, -Br, -I, -NO2, -CN, -NH2, -OH, -CH3, -CH2F, -CHF2, -CF3, -CH2D, -CHD2 or -CH2CH3.

[0021] Preferably, the chemical structural formula of the PROTAC compound that targets and degrades PARP7 is any one of the following formulas 1 to 52:

[0022]

[0023]

[0024] The second objective of this invention is to provide a method for preparing the aforementioned PROTAC compound that targets and degrades PARP7, employing the following technical solution:

[0025] Option 1

[0026] (1) Intermediate a-1 and compound a-2 were dissolved in N,N-dimethylformamide DMF, N,N-diisopropylethylamine DIPEA was added, and the mixture was stirred at 90°C for 4 h. After post-treatment and purification, compound a-3 was obtained.

[0027] (2) Compound a-3 was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed, the residue obtained by TLC was concentrated under reduced pressure and dissolved in N,N-dimethylformamide DMF. N,N-diisopropylethylamine DIPEA, compound a-4, and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate HATU were added sequentially under ice bath. The mixture was stirred at room temperature for 1 hour and then purified to obtain compound a-5.

[0028] (3) Compound a-5 was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed by TLC, the residue obtained by concentration under reduced pressure was dissolved in EtOH, and Et3N was added. The mixture was stirred at 60°C for 4 hours. After the reaction was completed by TLC, the product was post-processed and purified by column chromatography. The product obtained was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed by TLC, the product was purified by column chromatography to obtain compound (I).

[0029]

[0030] Each Linker is independently selected from one of the following structural segments:

[0031]

[0032] Where m and n are both natural numbers between 0 and 20. This indicates the site where the linker connects to the aromatic ring of E3 ligase.

[0033] Option 2

[0034] (1) Intermediate b-1 and compound b-2 were dissolved in N,N-dimethylformamide DMF, sodium bicarbonate NaHCO3 and potassium iodide KI were added, and the mixture was stirred at 60℃ for 12h. After post-treatment and purification, compound b-3 was obtained.

[0035] (2) Compound b-3 was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed, the residue obtained by TLC was concentrated under reduced pressure and dissolved in N,N-dimethylformamide DMF. N,N-diisopropylethylamine DIPEA, compound a-4, and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate HATU were added sequentially under ice bath. The mixture was stirred at room temperature for 1 hour and then purified to obtain compound b-4.

[0036] (3) Compound b-4 was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed by TLC, the residue obtained by concentration under reduced pressure was dissolved in EtOH, and Et3N was added. The mixture was stirred at 60°C for 4 hours. After the reaction was completed by TLC, the product was post-processed and purified by column chromatography. The product obtained was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed by TLC, the product was purified by column chromatography to obtain compound (I).

[0037]

[0038] Where n is any natural number from 0 to 20.

[0039] Option 3

[0040] (1) Intermediate c-1 and compound c-2 were dissolved in N,N-dimethylformamide DMF, and N,N-diisopropylethylamine DIPEA and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate HATU were added sequentially under ice bath. The mixture was stirred at room temperature for 1 hour and then purified to obtain compound c-3.

[0041] (2) Compound c-3 was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed, the residue obtained by TLC was concentrated under reduced pressure and dissolved in N,N-dimethylformamide DMF. N,N-diisopropylethylamine DIPEA, compound a-4, and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate HATU were added sequentially under ice bath. The mixture was stirred at room temperature for 1 hour and then purified to obtain compound c-3.

[0042] (3) Compound a-5 was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed by TLC, the residue obtained by concentration under reduced pressure was dissolved in EtOH, and Et3N was added. The mixture was stirred at 60°C for 4 hours. After the reaction was completed by TLC, the product was post-processed and purified by column chromatography. The product obtained was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed by TLC, the product was purified by column chromatography to obtain compound (I).

[0043]

[0044] Where n is any natural number from 0 to 20.

[0045] Option 4

[0046] (1) Intermediate d-1 and compound d-2 were dissolved in N,N-dimethylformamide DMF, and N,N-diisopropylethylamine DIPEA and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate HATU were added sequentially under ice bath. The mixture was stirred at room temperature for 1 hour and then purified to obtain compound d-3.

[0047] (2) Compound d-3 was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed, the residue obtained by TLC was concentrated under reduced pressure and dissolved in N,N-dimethylformamide DMF. N,N-diisopropylethylamine DIPEA, compound a-4, and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate HATU were added sequentially under ice bath. The mixture was stirred at room temperature for 1 hour and then purified to obtain compound d-4.

[0048] (3) Compound d-4 was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed by TLC, the residue obtained by concentration under reduced pressure was dissolved in EtOH, and Et3N was added. The mixture was stirred at 60°C for 4 hours. After the reaction was completed by TLC, the product was post-processed and purified by column chromatography. The product was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed by TLC, the product was purified by column chromatography to obtain compound (I).

[0049]

[0050] Where n is any natural number from 0 to 20.

[0051] Option 5

[0052] (1) Intermediate e-1 and compound e-2 were dissolved in N,N-dimethylformamide DMF, and N,N-diisopropylethylamine DIPEA and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate HATU were added sequentially under ice bath. The mixture was stirred at room temperature for 1 hour and then purified to obtain compound e-3.

[0053] (2) Compound e-3 was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed, the residue obtained by TLC was concentrated under reduced pressure and dissolved in N,N-dimethylformamide DMF. N,N-diisopropylethylamine DIPEA, compound a-4, and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate HATU were added sequentially under ice bath. The mixture was stirred at room temperature for 1 hour and then purified to obtain compound e-5.

[0054] (3) Compound e-5 was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed by TLC, the residue obtained by concentration under reduced pressure was dissolved in EtOH, and Et3N was added. The mixture was stirred at 60°C for 4 hours. After the reaction was completed by TLC, the product was post-processed and purified by column chromatography. The product obtained was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed by TLC, the product was purified by column chromatography to obtain compound (I).

[0055]

[0056] Where n is any natural number from 0 to 20.

[0057] The compound represented by formula (I) of this invention can be prepared by the method described above; however, the conditions of this method, such as reactants, solvents, amounts of the compound used, reaction temperature, and reaction time, are not limited to those explained above. If other small molecule ligands of CRBN or VHL or their derivatives in other E3 ubiquitin ligase complexes are used as starting materials, the route for synthesizing compound (I) is similar to route 1, which will be readily understood by experienced chemists. The compounds of this invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, such combinations being readily performed by those skilled in the art.

[0058] A third objective of this invention is to provide the use of the PROTAC compound described above, which targets the degradation of PARP7, or an optical isomer, racemate, single enantiomer, possible diastereomer, or pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, or solvate thereof, in the preparation of a drug for treating or preventing tumors.

[0059] A fourth objective of this invention is to provide an antitumor drug containing a safe and effective amount of the aforementioned PROTAC compound that targets and degrades PARP7, or an optical isomer, racemate, single enantiomer, possible diastereomer, or pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, or solvate thereof.

[0060] Preferably, the antitumor drug may further include a pharmacologically acceptable salt and a pharmacologically acceptable excipient or carrier.

[0061] Preferably, in the application and the antitumor drug, the tumor includes solid tumors and hematologic malignancies.

[0062] Because 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.

[0063] 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.

[0064] "Pharmaceutically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling 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 (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0065] 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.

[0066] 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: (a) fillers or solubilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0067] 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 opacifying agents, and the release of the active compound or compound in such compositions may be delayed at a specific site within the digestive tract. Examples of encapsulating components that may 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0072] Dosage forms of the compounds of this 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.

[0073] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0074] 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.

[0075] Compared with existing technologies, this invention utilizes high-throughput screening, homologue synthesis, substrate structure design, and fragment assembly strategies to combine the PARP7 inhibitor RBN-2397 with an appropriate linker and E3 ligase, synthesizing novel PROTAC compounds that target and degrade PARP7. This enhances antitumor activity and is groundbreaking compared to simple structural modifications. This invention also provides methods for preparing PROTAC compounds that target and degrade PARP7, their uses, and the inhibitory activity of these compounds on the proliferation of various tumor cell lines. The PROTAC compounds targeting and degrading PARP7 in this invention hold promise as antitumor drug candidates for treating various cancers, such as solid tumors and hematological malignancies. Detailed Implementation

[0076] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Operating methods not specifically specified in the following embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0077] Example 1: Preparation of Compound 1

[0078]

[0079] Step 1: Cesium carbonate (14.0 g, 42.8 mmol, 1.50 equivalent) was added to solutions of 1a (5.00 g, 28.6 mmol, 1.00 equivalent) and 1b (12.8 g, 142.8 mmol, 5.00 equivalent) in 100 mL of anhydrous acetonitrile at room temperature. The resulting solution was stirred at 25 °C for 10 hours. The reactants were then quenched by adding 200 mL of water. The resulting solution was extracted with 3 × 200 mL EtOAc, and the organic layers were combined and dried over anhydrous sodium sulfate. The organic layers were concentrated under vacuum and the crude product was purified by silica gel column chromatography to give 1c (4.20 g, yield 61.7%), the target compound, as a pale yellow liquid.

[0080] Step 2: Add NaOH (3.20 g, 80.5 mmol, 5.00 equivalent) to a solution of 1c (4.2 g, 16.1 mmol, 1.00 equivalent) in MeOH (100 mL) and H₂O (20 mL). Stir the resulting solution at room temperature for 12 hours. Then quench the reaction by adding 200 mL (1 M HCl). Extract the resulting solution with 3 × 500 mL EtOAc, and combine the organic layers and dry them over anhydrous sodium sulfate. Vacuum concentration yielded 1d (3.20 g, 80.6% yield) of the target compound as a pale yellow liquid, which can be used directly in the next reaction without purification.

[0081] Step 3: Potassium carbonate (1.23 g, 11.6 mmol, 2.00 equivalent) was added to a solution of 1e (1.00 g, 5.81 mmol, 1.00 equivalent) and 1f (1.30 g, 6.97 mmol, 1.00 equivalent) in 15 mL of anhydrous NMP at room temperature. The resulting solution was stirred at 25 °C for 10 hours. The reactants were then quenched by adding 100 mL of water. The resulting solution was extracted with 3 × 100 mL EtOAc, and the organic layers were combined and dried over anhydrous sodium sulfate. The organic layers were concentrated under vacuum and the crude product was purified by silica gel column chromatography to give 1 g (1.80 g, 92.2% yield) of the target compound as a white solid.

[0082] Step 4: Add 5 mL of dioxane hydrochloride solution to a solution of 1 g (1.80 g, 2.66 mmol, 1.00 equivalent) of the compound in 5 mL of MeOH at room temperature. Stir the resulting solution at room temperature for 12 hours. After the reaction was confirmed to be complete by TLC, filter the solid and dry it under vacuum to give a white solid compound 1 h (780 mg, yield 62.9%), which can be used directly in the next step without purification.

[0083] Step 5: HATU (609 mg, 1.60 mmol, 1.20 eq) and DIPEA (634 mg, 5.34 mmol, 4.00 eq) were added to a solution of 1d (330 mg, 1.336 mmol, 1.00 eq) in DMF (5 mL) at 0 °C. The resulting solution was stirred at room temperature for 0.5 h, followed by the addition of 1h (391 mg, 130 mmol, 1.10 eq) in portions at 0 °C. The reaction mixture was stirred at room temperature for 9 h. After the reaction was complete as detected by TLC, 50 mL of water was added. The resulting solution was extracted with 3 × 50 mL EtOAc and the organic layers were combined. The organic layer was washed with 25 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:1) to give a white solid compound 1i (509 mg, yield 84.5%) for the next step.

[0084] Step Six: Add NaOH (1.20 g, 29.9 mmol, 5.00 equivalent) to a solution of 1i (2.7 g, 5.99 mmol, 1.00 equivalent) in MeOH (10 mL) and H₂O (5 mL). Stir the resulting solution at room temperature for 12 hours. Then quench the reaction by adding 100 mL (1 M HCl). Extract the resulting solution with 3 × 100 mL ethyl acetate, and combine the organic layers and dry them over anhydrous sodium sulfate. Vacuum concentration yielded 1j (1.60 g, 61.2% yield) as a white solid of the target compound, which can be used directly in the next reaction without purification.

[0085] Step 7: At room temperature, add 1 mL of DIPEA and 1 L of DIPEA (586.5 mg, 2.715 mmol, 1.50 equivalent) to a solution of 1 K (500 mg, 1.81 mmol, 1.00 equivalent) in anhydrous DMF (5 mL). Stir the resulting solution at 90°C for 10 hours. Then quench the reaction mixture by adding 20 mL of water. Extract the resulting solution with 3 × 20 mL EtOAc, and combine the organic layers and dry them over anhydrous sodium sulfate. Concentrate the organic layers under vacuum and purify the crude product by silica gel column chromatography to give 1 M (820 mg, 95.87% yield) of the target compound as a yellow liquid.

[0086] Step 8: Add 1 mL of trifluoroacetic acid to a solution of 1 m (378 mg, 0.88 mmol, 1.00 eq) in DCM (2 mL). Stir the resulting solution at room temperature for 10 hours. After the reaction is complete as detected by TLC, concentrate the reaction solution under vacuum. The resulting 1 n (crude product) can be used directly in the next step without purification. Add HATU (364.8 mg, 1.96 mmol, 1.20 eq) and DIPEA (412.8 mg, 5.34 mmol, 4.00 eq) to a solution of 1 j (350 mg, 0.8 mmol, 1.00 eq) in DMF (5 mL) at 0 °C. Stir the resulting solution at room temperature for 0.5 hours, then add the crude 1 n in portions at room temperature. Stir the reaction mixture at room temperature for 0.5 hours. Then quench the reaction mixture by adding 20 mL of water. Extract the resulting solution with 3 × 10 mL of ethyl acetate, and combine the organic layers and dry them with anhydrous sodium sulfate. The organic layer was concentrated under vacuum and the crude product was purified by silica gel column chromatography to obtain the target compound 1o (240 mg, yield 37.88%), which was a yellow liquid.

[0087] Step 9: Add 1 mL of trifluoroacetic acid to a solution of 1o (230 mg, 0.291 mmol, 1.00 equivalent) in DCM (2 mL). Stir the resulting solution at room temperature for 10 hours. After the reaction is complete as determined by TLC, concentrate the reaction solution under vacuum. The resulting 1p (crude product) can be used directly in the next step without purification. Add 1 mL of Et3N to a solution of 1q (95.5 mg, 0.291 mmol, 1.00 equivalent) in EtOH (5 mL) at room temperature. Stir the resulting solution and add the crude 1p product at room temperature. Then stir the reaction mixture at 60 °C for 12 hours. After the reaction is complete as determined by TLC, add 20 mL of water to quench the reaction mixture. Extract the resulting solution with 3 × 10 mL of ethyl acetate, and combine the organic layers and dry them with anhydrous sodium sulfate. Concentrate the organic layer under vacuum and purify the crude product by silica gel column chromatography to obtain the target compound 1r (165 mg, yield 57.6%) as a yellow solid.

[0088] Step 10: Add 1 mL of trifluoroacetic acid to a solution of 1 r (150 mg, 0.1525 mmol, 1.00 equivalent) in DCM (2 mL). Stir the resulting solution at room temperature for 10 hours. Then quench the reaction mixture by adding 20 mL of water. Extract the resulting solution with 3 × 10 mL of dichloromethane, and combine the organic layers and dry them over anhydrous sodium sulfate. Concentrate the organic layers under vacuum and purify the crude product by silica gel column chromatography to give compound 1 (86 mg, yield 66.07%) as a yellow solid.

[0089] 1H NMR (500MHz, DMSO-d6) δ11.08(s,1H),8.76(s,2H),8.32(t,J=5.6Hz,1H),7.98(s,1H),7.56(dd,J=8.5,7.0Hz,1H),7.08(d,J=8.6Hz ,1H),7.01(d,J=7.0Hz,1H),6.52(t,J=5.9Hz,1H),6.39(dd,J=8.6,4.2Hz,1H),5.30–5.11(m,2H),5.10–4.91(m,1H),4.30–4.09(m, 1H),3.78(dq,J=25.2,4.0Hz,5H),3.68(qt,J=9.6,6.6Hz,2H),3.58–3.45(m,7H),3.29(q,J=6.5Hz,3H),3.22(q,J=6.5Hz,3H),2.87 (ddd,J=16.8,13.7,5.5Hz,1H),2.65–2.55(m,5H),1.54(dq,J=32.2,6.9Hz,5H),1.36(qd,J=8.4,5.6,4.8Hz,5H),1.27–1.03(m,6H).

[0090] Example 2: Preparation of Compound 2

[0091]

[0092] Step 1: Cesium carbonate (28.0 g, 83.7 mmol, 1.50 equivalent) was added to a solution of 2a (10.0 g, 57.1 mmol, 1.00 equivalent) and 2b (25.7 g, 285.7 mmol, 5.0 equivalent) in 200 mL of anhydrous acetonitrile at room temperature. The resulting solution was stirred at 25 °C for 10 hours. The reactants were then quenched by adding 200 mL of water. The resulting solution was extracted with 3 × 200 mL EtOAc, and the organic layers were combined and dried over anhydrous sodium sulfate. The organic layers were concentrated under vacuum and the crude product was purified by silica gel column chromatography to give 2c (7.80 g, yield 52.3%), the target compound, as a pale yellow liquid.

[0093] Step 2: Add NaOH (5.98 g, 149.5 mmol, 5.00 equivalents) to a solution of 2c (7.8 g, 29.9 mmol, 1.00 equivalents) in MeOH (100 mL) and H₂O (30 mL). Stir the resulting solution at room temperature for 12 hours. Then quench the reaction by adding 20 mL (1 M HCl). Extract the resulting solution with 3 × 500 mL EtOAc, and combine the organic layers and dry them with anhydrous sodium sulfate. Concentrate under vacuum to obtain 2d (5.60 g, 75.9% yield) as a colorless liquid of the target compound, which can be used directly in the next reaction without purification.

[0094] Step 3: Potassium carbonate (1.23 g, 11.6 mmol, 2.00 equivalent) was added to a solution of 2e (1.00 g, 5.81 mmol, 1.00 equivalent) and 2f (1.30 g, 6.97 mmol, 1.00 equivalent) in 15 mL of anhydrous NMP at room temperature. The resulting solution was stirred at 25 °C for 10 hours. The reactants were then quenched by adding 100 mL of water. The resulting solution was extracted with 3 × 100 mL EtOAc, and the organic layers were combined and dried over anhydrous sodium sulfate. The organic layers were concentrated under vacuum and the crude product was purified by silica gel column chromatography to give 2 g (1.80 g, 92.2% yield) of the target compound as a white solid.

[0095] Step 4: Add 5 mL of dioxane hydrochloride solution to a solution of 2 g (1.80 g, 2.66 mmol, 1.00 equivalent) of the compound in 5 mL of MeOH at room temperature. Stir the resulting solution at room temperature for 12 hours. After the reaction was confirmed to be complete by TLC, filter the solid and dry it under vacuum to give a white solid compound 2 h (780 mg, yield 62.9%), which can be used directly in the next step without purification.

[0096] Step 5: Add EDCI (4.02 g, 21.06 mmol, 2.60 eq), HOBt (1.42 g, 10.53 mmol, 1.30 eq), and DIPEA (4.18 g, 32.4 mmol, 4.00 eq) to a solution of 2d (2.00 g, 8.10 mmol, 1.00 eq) in 30 mL of DMF at room temperature. Stir the resulting solution at room temperature for 0.5 h, then add 2h (2.62 g, 8.91 mmol, 1.10 eq) in portions at room temperature. Stir the reaction mixture at room temperature for 8 h. After the reaction is complete as detected by TLC, add 300 mL of water. Extract the resulting solution with 3 × 200 mL EtOAc and combine the organic layers. The organic layer was washed with 200 mL of saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:3) to give a white solid compound 2i (3.18 g, yield 87.1%) for the next step of the reaction.

[0097] Step Six: Trifluoroacetic acid (5 mL) was added dropwise to a solution of compound 2i (3.10 g, 6.87 mmol, 1.00 equivalent) in DCM (15 mL) at room temperature. The solution was stirred at room temperature for 10 hours. After the reaction was confirmed to be complete by TLC, the solid was filtered and dried under vacuum to give a white solid compound 2J (2.20 g, yield 91.9%), which could be used directly in the next step without purification.

[0098] Step 7: At room temperature, add 3 mL of Et3N to a solution of 2 J (2.2 g, 6.29 mmol, 1.00 equivalent) in 30 mL of EtOH. Stir the resulting solution at room temperature and add 2 K (2.06 g, 6.29 mmol, 1.00 equivalent of crude product). Then stir the reaction mixture at 60 °C for 12 hours. After the reaction is complete as determined by TLC, 200 mL of water is added to quench the reaction mixture. The resulting solution is extracted with 3 × 200 mL of ethyl acetate, and the organic layers are combined and dried over anhydrous sodium sulfate. The organic layers are concentrated under vacuum and the crude product is purified by silica gel column chromatography to give compound 2l (2.80 g, yield 69.6%) as a yellow solid.

[0099] Step 8: Add 2 L (2.80 g, 4.36 mmol, 1.00 equivalent) of the compound to a solution in DCM (20 mL) and add 6 mL of trifluoroacetic acid. Stir the resulting solution at room temperature for 10 hours. Then quench the reaction mixture by adding 200 mL of water. Extract the resulting solution with 3 × 100 mL of dichloromethane, and combine the organic layers and dry them over anhydrous sodium sulfate. Concentrate the organic layers under vacuum and purify the crude product by silica gel column chromatography to give compound m (1.21 g, yield 54.2%) as a yellow solid.

[0100] Step 9: Add NaOH (473 mg, 11.8 mmol, 5.00 equivalent) to a solution of 2m (1.21 g, 2.36 mmol, 1.00 equivalent) in MeOH (20 mL) and H₂O (5 mL), and stir the resulting solution at room temperature for 12 hours. Then quench the reaction mixture by adding 10 mL (1 M HCl). Extract the resulting solution with 3 × 100 mL of ethyl acetate, and combine the organic layers and dry them over anhydrous sodium sulfate. Vacuum concentration yields 2n ​​(620 mg, 52.6% yield) as a white solid of the target compound, which can be used directly in the next reaction without purification.

[0101] Step 10: At room temperature, add DIPEA (0.2 mL) and 2p (145.2 mg, 0.43 mmol, 1.20 equivalent) to a solution of 2o (100 mg, 0.36 mmol, 1.00 equivalent) in anhydrous NMP (5 mL). Stir the resulting solution at 90°C for 10 hours. Then quench the reaction mixture by adding 50 mL of water. Extract the resulting solution with 3 × 50 mL EtOAc, and combine the organic layers and dry them over anhydrous sodium sulfate. Concentrate the organic layers under vacuum and purify the crude product by silica gel column chromatography to give 2q (169 mg, yield 79.3%) as a yellow liquid containing the target compound.

[0102] Step 11: Add 1 mL of trifluoroacetic acid to the solution of 2q (169 mg, 0.28 mmol, 1.00 equivalent) in DCM (2 mL). Stir the resulting solution at room temperature for 10 hours. After the reaction is complete as detected by TLC, concentrate the reaction solution under vacuum. The resulting 2r (crude product) can be used directly in the next step without purification.

[0103] Step 12: EDCI (99.32 mg, 0.52 mmol, 2.60 eq), HOBt (35.1 mg, 0.26 mmol, 1.30 eq), and DIPEA (103.2 mg, 0.80 mmol, 4.00 eq) were added to a solution of 2n (100 mg, 0.20 mmol, 1.00 eq) in 3 mL of DMF at room temperature. The resulting solution was stirred at room temperature for 0.5 hours, followed by the addition of 2r (118 mg, 0.24 mmol, 1.20 eq) in portions at room temperature. The reaction mixture was stirred at room temperature for 8 hours. After the reaction was confirmed to be complete by TLC, 50 mL of water was added. The resulting solution was extracted with 3 × 50 mL EtOAc and the organic layers were combined. The organic layer was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give compound 2 as a white solid. 1H NMR (500MHz, DMSO-d6) δ12.44(s,1H),11.09(s,1H),8.78(s,2H),8.43(t,J=5.6Hz,1H),7.91(s,1H),7.57(dd,J=8.6,7.1Hz,1H ),7.13(d,J=8.6Hz,1H),7.03(d,J=7.0Hz,1H),6.59(t,J=5.8Hz,1H),6.26(dq,J=8.1,4.0Hz,1H),5.75(s,1H),5.05(dd,J=12.8 ,5.4Hz,1H),4.15(p,J=6.3Hz,1H),3.79(ddd,J=25.1,6.7,4.0Hz,4H),3.73–3.58(m,5H),3.57–3.50(m,14H),3.46(dd,J=11.9, 6.2Hz,3H),3.38(q,J=5.8Hz,5H),2.88(ddd,J=16.6,13.7,5.4Hz,1H),2.62–2.56(m,3H),2.55–2.51(m,1H),2.06–1.99(m,1H).

[0104] Example 3: Preparation of Compound 3

[0105]

[0106] Refer to the synthesis method of compound 2:

[0107] Step 1: At room temperature, DIPEA (0.2 mL) and 3b (138.2 mg, 0.86 mmol, 1.20 equivalence) were added to a solution of 3a (200 mg, 0.72 mmol, 1.00 equivalence) in anhydrous NMP (5 mL). The solution was stirred at 90 °C for 10 hours. The reactants were then quenched by adding 50 mL of water. The resulting solution was extracted with 3 × 50 mL EtOAc, and the organic layers were combined and dried over anhydrous sodium sulfate. The organic layers were concentrated under vacuum and the crude product was purified by silica gel column chromatography to give 3c (251 mg, yield 83.9%) as a yellow liquid, representing the target compound.

[0108] Step 2: Add 1 mL of trifluoroacetic acid to the solution of 3c (251 mg, 0.60 mmol, 1.00 equivalent) in DCM (5 mL). Stir the resulting solution at room temperature for 10 hours. After the reaction is complete as detected by TLC, concentrate the reaction solution under vacuum. The resulting 3d (crude product) can be used directly in the next step without purification.

[0109] Step 3: EDCI (158 mg, 0.83 mmol, 2.60 eq), HOBt (43 mg, 0.42 mmol, 1.30 eq), and DIPEA (206 mg, 1.6 mmol, 5 eq) were added to a solution of 3e (100 mg, 0.32 mmol, 1.00 eq) in DMF (5 mL) at room temperature. The resulting solution was stirred at room temperature for 0.5 hours, followed by the addition of 3d (120 mg, 0.38 mmol, 1.20 eq) in portions at room temperature. The reaction mixture was stirred at room temperature for 8 hours. After the reaction was confirmed to be complete by TLC, 50 mL of water was added. The resulting solution was extracted with 3 × 50 mL EtOAc and the organic layers were combined. The organic layer was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give 3f (251 mg, yield 83.9%) as a yellow-green liquid, representing the target compound.

[0110] Step 4: Add 1 mL of trifluoroacetic acid to the solution of 3f (251 mg, 0.67 mmol, 1.00 equivalent) in 3 mL of DCM. Stir the resulting solution at room temperature for 10 hours. After the reaction is complete as detected by TLC, concentrate the reaction solution under vacuum. The resulting 3 g (crude product) can be used directly in the next step without purification.

[0111] Step 5: To a solution of 2n (100 mg, 0.20 mmol, 1.00 equivalence) in DMF (5 mL), add EDCI (99.32 mg, 0.52 mmol, 2.60 equivalence), HOBt (35.1 mg, 0.26 mmol, 1.30 eq), and DIPEA (103.2 mg, 0.80 mmol, 4.00 eq). Stir the resulting solution at room temperature for 0.5 hours, then add 3 g (123 mg, 0.24 mmol, 1.20 equivalence) in portions at room temperature. Stir the reaction mixture at room temperature for 8 hours. After the reaction is complete as detected by TLC, add 50 mL of water. Extract the resulting solution with 3 × 50 mL EtOAc and combine the organic layers. Wash the organic layer with 20 mL of saturated brine, dry to anhydrous sodium sulfate, and concentrate under reduced pressure. Purify the crude product by silica gel column chromatography to give a yellow-green solid compound 3. 1H NMR (500MHz, DMSO-d6) δ8.78(s,2H),8.31(t,J=5.6Hz,1H),8.00(t,J=5.6Hz,1H),7.91(s,1H),7.58(dd,J=8.6,7.0Hz,1H),7.24 (q,J=8.5Hz,1H),7.17(d,J=8.7Hz,1H),7.03(d,J=7.0Hz,1H),6.71(t,J=6.1Hz,1H),6.27(dq,J=8.1,4.1Hz,1H),5.06(dd,J=12. 7,5.5Hz,1H),4.15(p,J=6.4Hz,1H),3.74–3.44(m,9H),3.37(q,J=6.7Hz,3H),3.23(dq,J=13.7,6.3Hz,4H),2.89(ddd,J=16.6,13 .6,5.3Hz,1H),2.68–2.53(m,4H),2.03(q,J=7.3Hz,3H),1.48(dp,J=13.0,6.8Hz,6H),1.37–1.22(m,11H),1.16(d,J=6.5Hz,3H).

[0112] Example 4: Preparation of Compound 4

[0113]

[0114] Refer to the synthesis method of compound 2;

[0115] Step 1: At room temperature, DIPEA (0.4 mL) and 4b (150.4 mg, 0.86 mmol, 1.20 equivalence) were added to a solution of 4a (200 mg, 0.72 mmol, 1.00 equivalence) in anhydrous NMP (5 mL). The solution was stirred at 90 °C for 10 hours. The reactants were then quenched by adding 50 mL of water. The resulting solution was extracted with 3 × 50 mL EtOAc, and the organic layers were combined and dried over anhydrous sodium sulfate. The organic layers were concentrated under vacuum and the crude product was purified by silica gel column chromatography to give 4c (259 mg, yield 83.8%) as a yellow liquid, representing the target compound.

[0116] Step 2: Add 1 mL of trifluoroacetic acid to the solution of 4c (259 mg, 0.60 mmol, 1.00 equivalent) in DCM (2 mL). Stir the resulting solution at room temperature for 10 hours. After the reaction is complete as detected by TLC, concentrate the reaction solution under vacuum. The resulting 4d (crude product) can be used directly in the next step without purification.

[0117] Step 3: EDCI (99.32 mg, 0.52 mmol, 2.60 eq), HOBt (35.1 mg, 0.26 mmol, 1.30 eq), and DIPEA (103.2 mg, 0.80 mmol, 4.00 eq) were added to a solution of 2n (100 mg, 0.20 mmol, 1.00 eq) in 30 mL of DMF at room temperature. The resulting solution was stirred at room temperature for 0.5 hours, followed by the addition of 4d (79.2 mg, 0.24 mmol, 1.20 eq) in portions at room temperature. The reaction mixture was stirred at room temperature for 8 hours. After the reaction was confirmed to be complete by TLC, 50 mL of water was added. The resulting solution was extracted with 3 × 50 mL EtOAc and the organic layers were combined. The organic layer was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give a white-yellow-green solid compound 4. 1 H NMR (500MHz, DMSO-d6) δ12.44(s,1H),11.08(s,1H),8.76(s,2H),8.63(t,J=5.5Hz,1H),7.90(s,1H),7.57(dd,J=8.6,7.0Hz ,1H),7.24(d,J=8.7Hz,1H),7.01(d,J=7.0Hz,1H),6.83(t,J=6.2Hz,1H),6.26(dt,J=7.9,4.1Hz,1H),5.04(dd,J=12.8,5.5 Hz,1H),4.13(dq,J=15.7,5.8Hz,2H),3.79(dt,J=24.9,5.4Hz,4H),3.72–3.62(m,2H),3.61–3.45(m,8H),3.43(t,J=5.6Hz, 2H),3.16(d,J=5.2Hz,2H),2.88(ddd,J=16.9,13.7,5.4Hz,1H),2.62–2.54(m,3H),2.07–1.95(m,1H),1.14(d,J=6.4Hz,3H).

[0118] Example 5: Preparation of Compound 5

[0119]

[0120] Refer to the synthesis method of compound 2;

[0121] Step 1: At room temperature, DIPEA (0.4 mL) and 4b (162.4 mg, 0.86 mmol, 1.20 equivalence) were added to a solution of 4a (200 mg, 0.72 mmol, 1.00 equivalence) in anhydrous NMP (5 mL). The solution was stirred at 90 °C for 10 hours. The reactants were then quenched by adding 50 mL of water. The resulting solution was extracted with 3 × 50 mL EtOAc, and the organic layers were combined and dried over anhydrous sodium sulfate. The organic layers were concentrated under vacuum and the crude product was purified by silica gel column chromatography to give 4c (263 mg, yield 82.4%) as a yellow liquid, representing the target compound.

[0122] Step 2: Add 1 mL of trifluoroacetic acid to the solution of 5c (263 mg, 0.59 mmol, 1.00 equivalent) in 3 mL of DCM. Stir the resulting solution at room temperature for 10 hours. After the reaction is complete as detected by TLC, concentrate the reaction solution under vacuum. The resulting 5d (crude product) can be used directly in the next step without purification.

[0123] Step 3: EDCI (99.32 mg, 0.52 mmol, 2.60 eq), HOBt (35.1 mg, 0.26 mmol, 1.30 eq), and DIPEA (103.2 mg, 0.80 mmol, 4.00 eq) were added to a solution of 2n (100 mg, 0.20 mmol, 1.00 eq) in 5 mL of DMF at room temperature. The resulting solution was stirred at room temperature for 0.5 hours, followed by the addition of 5d (82.5 mg, 0.24 mmol, 1.20 eq) in portions at room temperature. The reaction mixture was stirred at room temperature for 8 hours. After the reaction was confirmed to be complete by TLC, 50 mL of water was added. The resulting solution was extracted with 3 × 50 mL EtOAc and the organic layers were combined. The organic layer was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give a yellow-green solid compound 5. 1H NMR (500MHz, DMSO-d6) δ12.44(s,1H),11.08(s,1H),8.77(s,2H),8.38(t,J=5.6Hz,1H),7.91(s,1H),7.56(dd,J=8. 6,7.0Hz,1H),7.11(d,J=8.5Hz,1H),7.01(d,J=7.0Hz,1H),6.56(t,J=6.0Hz,1H),6.26(dd,J=8.6,4.3Hz,1H),5.04 (dd,J=12.7,5.5Hz,1H),4.14(t,J=7.1Hz,1H),3.79(dt,J=25.1,5.2Hz,4H),3.55–3.46(m,5H),3.27(q,J=5.9Hz,2 H),3.13(qd,J=7.3,4.1Hz,7H),2.92–2.82(m,1H),2.06–1.99(m,1H),1.60(q,J=7.6Hz,4H),1.15(d,J=6.4Hz,3H).

[0124] Example 6: Preparation of Compound 6

[0125]

[0126] Refer to the synthesis method of compound 2;

[0127] Step 1: At room temperature, DIPEA (0.4 mL) and 6b (174.5 mg, 0.86 mmol, 1.20 equivalence) were added to a solution of 6a (200 mg, 0.72 mmol, 1.00 equivalence) in anhydrous NMP (5 mL). The solution was stirred at 90 °C for 10 hours. The reactants were then quenched by adding 50 mL of water. The resulting solution was extracted with 3 × 50 mL EtOAc, and the organic layers were combined and dried over anhydrous sodium sulfate. The organic layers were concentrated under vacuum and the crude product was purified by silica gel column chromatography to give 6c (281 mg, yield 85.2%) as a yellow liquid, representing the target compound.

[0128] Step 2: Add 1 mL of trifluoroacetic acid to a solution of 6c (281 mg, 0.61 mmol, 1.00 equivalent) in 3 mL of DCM. Stir the resulting solution at room temperature for 10 hours. After the reaction is complete as detected by TLC, concentrate the reaction solution under vacuum. The resulting 6d (crude product) can be used directly in the next step without purification.

[0129] Step 3: EDCI (99.32 mg, 0.52 mmol, 2.60 eq), HOBt (35.1 mg, 0.26 mmol, 1.30 eq), and DIPEA (103.2 mg, 0.80 mmol, 4.00 eq) were added to a solution of 2n (100 mg, 0.20 mmol, 1.00 eq) in 30 mL of DMF at room temperature. The resulting solution was stirred at room temperature for 0.5 hours, followed by the addition of 6d (86 mg, 0.24 mmol, 1.20 eq) in portions at room temperature. The reaction mixture was stirred at room temperature for 8 hours. After the reaction was confirmed to be complete by TLC, 50 mL of water was added. The resulting solution was extracted with 3 × 50 mL EtOAc and the organic layers were combined. The organic layer was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give a yellow-green solid compound 6. 1 H NMR (500MHz, DMSO-d6) δ11.09(s,1H),8.76(d,J=0.9Hz,2H),8.33(t,J=5.3Hz,1H),7.95(d,J=37.8Hz,1H),7.57(dd,J=8.6,7.0Hz,1H),7.0 9(d,J=8.7Hz,1H),7.01(d,J=7.0Hz,1H),6.33(ddd,J=65.2,8.2,3.9Hz,1H),5.04(dd,J=12.8,5.4Hz,1H),3.87–3.73(m,4H),3.68(td,J=9. 8,5.2Hz,2H),3.49(t,J=5.3Hz,2H),3.30(q,J=6.7Hz,2H),3.24(dt,J=10.0,5.0Hz,2H),3.17(d,J=5.3Hz,4H),2.88(ddd,J=16.8,13.8,5.4 Hz,1H),2.64–2.56(m,3H),2.02(dtd,J=11.1,5.6,2.8Hz,1H),1.58(dp,J=27.1,7.3Hz,4H),1.43–1.35(m,2H),1.16(dd,J=6.5,4.3Hz,3H).

[0130] Example 7: Preparation of Compound 7

[0131]

[0132] Refer to the synthesis method of compound 2;

[0133] Step 1: At room temperature, DIPEA (0.4 mL) and 7b (186.6 mg, 0.86 mmol, 1.20 equivalent) were added to a solution of 7a (200 mg, 0.72 mmol, 1.00 equivalent) in anhydrous NMP (5 mL). The solution was stirred at 90 °C for 10 hours. The reactants were then quenched by adding 50 mL of water. The resulting solution was extracted with 3 × 50 mL EtOAc, and the organic layers were combined and dried over anhydrous sodium sulfate. The organic layers were concentrated under vacuum and the crude product was purified by silica gel column chromatography to give 7c (271 mg, yield 79.7%) as a yellow liquid, representing the target compound.

[0134] Step 2: Add 1 mL of trifluoroacetic acid to the solution of 7c (271 mg, 0.57 mmol, 1.00 equivalent) in 3 mL of DCM. Stir the resulting solution at room temperature for 10 hours. After the reaction is complete as detected by TLC, concentrate the reaction solution under vacuum. The resulting 7d (crude product) can be used directly in the next step without purification.

[0135] Step 3: EDCI (99.32 mg, 0.52 mmol, 2.60 eq), HOBt (35.1 mg, 0.26 mmol, 1.30 eq), and DIPEA (103.2 mg, 0.80 mmol, 4.00 eq) were added to a solution of 2n (100 mg, 0.20 mmol, 1.00 eq) in 5 mL of DMF at room temperature. The resulting solution was stirred at room temperature for 0.5 hours, followed by the addition of 7d (89.2 mg, 0.24 mmol, 1.20 eq) in portions at room temperature. The reaction mixture was stirred at room temperature for 8 hours. After the reaction was confirmed to be complete by TLC, 50 mL of water was added. The resulting solution was extracted with 3 × 50 mL EtOAc and the organic layers were combined. The organic layer was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give a yellow-green solid compound 7. 1H NMR (500MHz, DMSO-d6) δ11.08(s,1H),8.77(s,2H),8.33(t,J=5.6Hz,1H),7.98(s,1H),7.56(dd,J=8.6,7.0Hz,1H),7.07(d,J=8.6H z,1H),7.00(d,J=7.0Hz,1H),6.53(dt,J=23.0,6.5Hz,2H),6.39(dq,J=8.2,4.1Hz,1H),5.26–5.10(m,2H),5.04(dd,J=12.7,5.5Hz ,1H),4.19(p,J=6.5Hz,1H),3.87–3.61(m,7H),3.50(dt,J=16.6,5.2Hz,7H),3.27(q,J=6.7Hz,2H),3.21(q,J=6.6Hz,2H),2.87(dd d,J=16.8,13.8,5.4Hz,1H),2.64–2.51(m,4H),2.02(dtd,J=13.0,5.2,2.2Hz,1H),1.53(dt,J=35.7,7.0Hz,5H),1.18–1.14(m,3H).

[0136] Example 8: Preparation of Compound 8

[0137]

[0138] Refer to the synthesis method of compound 2;

[0139] Step 1: At room temperature, DIPEA (0.4 mL) and 8b (198.7 mg, 0.86 mmol, 1.20 equivalence) were added to a solution of 8a (200 mg, 0.72 mmol, 1.00 equivalence) in anhydrous NMP (5 mL). The solution was stirred at 90°C for 10 hours. The reactants were then quenched by adding 50 mL of water. The resulting solution was extracted with 3 × 50 mL EtOAc, and the organic layers were combined and dried over anhydrous sodium sulfate. The organic layers were concentrated under vacuum and the crude product was purified by silica gel column chromatography to give 8c (293 mg, yield 83.7%) as a yellow liquid, representing the target compound.

[0140] Step 2: Add 1 mL of trifluoroacetic acid to the solution of 8c (293 mg, 0.60 mmol, 1.00 equivalent) in 3 mL of DCM. Stir the resulting solution at room temperature for 10 hours. After the reaction is complete as detected by TLC, concentrate the reaction solution under vacuum. The resulting 8d (crude product) can be used directly in the next step without purification.

[0141] Step 3: EDCI (99.32 mg, 0.52 mmol, 2.60 eq), HOBt (35.1 mg, 0.26 mmol, 1.30 eq), and DIPEA (103.2 mg, 0.80 mmol, 4.00 eq) were added to a solution of 2n (100 mg, 0.20 mmol, 1.00 eq) in 30 mL of DMF at room temperature. The resulting solution was stirred at room temperature for 0.5 hours, followed by the addition of 8d (92.6 mg, 0.24 mmol, 1.20 eq) in portions at room temperature. The reaction mixture was stirred at room temperature for 8 hours. After the reaction was confirmed to be complete by TLC, 50 mL of water was added. The resulting solution was extracted with 3 × 50 mL EtOAc and the organic layers were combined. The organic layer was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give a yellow-green solid compound 8. 1 H NMR (500MHz, DMSO-d6) δ12.44(s,1H),11.09(s,1H),8.77(s,2H),7.91(s,1H),7.57(dd,J=8.6,7.0Hz,1H),7.08(d,J=8.6Hz,1H),7.01(d, J=7.0Hz,1H),6.52(t,J=6.0Hz,1H),6.27(dq,J=8.1,4.0Hz,1H),5.05(dd,J=12.7,5.4Hz,1H),3.86–3.79(m,2H),3.76(dd,J=6.8,4.1Hz,2 H),3.71–3.64(m,2H),3.53(t,J=5.5Hz,4H),3.49(d,J=5.6Hz,2H),3.29(q,J=6.6Hz,2H),3.22(q,J=6.6Hz,2H),2.62–2.56(m,3H),2.53( d,J=4.6Hz,1H),2.02(ddq,J=10.5,5.4,2.4Hz,1H),1.57(t,J=7.0Hz,2H),1.50(q,J=6.3Hz,2H),1.40–1.31(m,6H),1.15(d,J=6.5Hz,3H).

[0142] Example 9: Preparation of Compound 9

[0143]

[0144] Refer to the synthesis method of compound 2;

[0145] Step 1: At room temperature, DIPEA (0.4 mL) and 9b (210.8 mg, 0.86 mmol, 1.20 equivalence) were added to a solution of 9a (200 mg, 0.72 mmol, 1.00 equivalence) in anhydrous NMP (5 mL). The solution was stirred at 90°C for 10 hours. The reactants were then quenched by adding 50 mL of water. The resulting solution was extracted with 3 × 50 mL EtOAc, and the organic layers were combined and dried over anhydrous sodium sulfate. The organic layers were concentrated under vacuum and the crude product was purified by silica gel column chromatography to give 9c (304 mg, yield 84.4%) as a yellow liquid, representing the target compound.

[0146] Step 2: Add 1 mL of trifluoroacetic acid to a solution of 9c (304 mg, 0.60 mmol, 1.00 equivalent) in 3 mL of DCM. Stir the resulting solution at room temperature for 10 hours. After the reaction is complete as detected by TLC, concentrate the reaction solution under vacuum. The resulting 9d (crude product) can be used directly in the next step without purification.

[0147] Step 3: EDCI (99.32 mg, 0.52 mmol, 2.60 eq), HOBt (35.1 mg, 0.26 mmol, 1.30 eq), and DIPEA (103.2 mg, 0.80 mmol, 4.00 eq) were added to a solution of 2n (100 mg, 0.20 mmol, 1.00 eq) in 30 mL of DMF at room temperature. The resulting solution was stirred at room temperature for 0.5 hours, followed by the addition of 9d (96 mg, 0.24 mmol, 1.20 eq) in portions at room temperature. The reaction mixture was stirred at room temperature for 8 hours. After the reaction was confirmed to be complete by TLC, 50 mL of water was added. The resulting solution was extracted with 3 × 50 mL EtOAc and the organic layers were combined. The organic layer was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give a yellow-green solid compound 9. 1H NMR (500MHz, DMSO-d6) δ12.44(s,1H),11.09(s,1H),8.77(s,2H),8.32(t,J=5.6Hz,1H),7.91(s,1H),7.57(dd,J=8.6,7.0Hz,1H),7. 08(d,J=8.6Hz,1H),7.01(d,J=7.0Hz,1H),6.52(t,J=6.0Hz,1H),6.27(dt,J=8.6,4.2Hz,1H),5.75(s,2H),5.04(dd,J=12.8,5.5Hz, 1H),4.15(p,J=6.5Hz,1H),3.79(dt,J=24.9,5.2Hz,4H),3.56–3.46(m,6H),3.29(q,J=6.6Hz,2H),3.22(q,J=6.6Hz,2H),2.88(ddd, J=16.8,13.8,5.4Hz,1H),2.62–2.56(m,3H),2.53(s,1H),2.06–2.00(m,1H),1.53(ddt,J=33.6,12.4,6.8Hz,5H),1.20–1.09(m,6H).

[0148] Example 10: Preparation of Compound 10

[0149]

[0150] Refer to the synthesis method of compound 2;

[0151] Step 1: At room temperature, DIPEA (0.4 mL) and 10b (252.3 mg, 0.86 mmol, 1.20 equivalent) were added to a solution of 10a (200 mg, 0.72 mmol, 1.00 equivalent) in anhydrous NMP (5 mL). The solution was stirred at 90 °C for 10 hours. The reactants were then quenched by adding 50 mL of water. The resulting solution was extracted with 3 × 50 mL EtOAc, and the organic layers were combined and dried over anhydrous sodium sulfate. The organic layers were concentrated under vacuum and the crude product was purified by silica gel column chromatography to give 10c (336 mg, yield 85.3%) as a yellow liquid, representing the target compound.

[0152] Step 2: Add 1 mL of trifluoroacetic acid to a solution of 10 c (336 mg, 0.61 mmol, 1.00 equivalent) in 3 mL of DCM. Stir the resulting solution at room temperature for 10 hours. After the reaction is complete as detected by TLC, concentrate the reaction solution under vacuum. The resulting 10 d (crude product) can be used directly in the next step without purification.

[0153] Step 3: EDCI (99.32 mg, 0.52 mmol, 2.60 eq), HOBt (35.1 mg, 0.26 mmol, 1.30 eq), and DIPEA (103.2 mg, 0.80 mmol, 4.00 eq) were added to a solution of 2n (100 mg, 0.20 mmol, 1.00 eq) in 30 mL of DMF at room temperature. The resulting solution was stirred at room temperature for 0.5 hours, followed by the addition of 10d (107.5 mg, 0.24 mmol, 1.20 eq) in portions at room temperature. The reaction mixture was stirred at room temperature for 8 hours. After the reaction was confirmed to be complete by TLC, 50 mL of water was added. The resulting solution was extracted with 3 × 50 mL EtOAc and the organic layers were combined. The organic layer was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give a yellow-green solid compound 10. 1 H NMR (500MHz, DMSO-d6) δ12.44(s,1H),11.08(s,1H),8.77(s,2H),8.42(t,J=5.6Hz,1H),7.90(s,1H),7.57(dd,J=8.6,7.1Hz,1H),7.12 (d,J=8.6Hz,1H),7.03(d,J=7.0Hz,1H),6.58(t,J=5.8Hz,1H),6.27(dt,J=8.6,4.1Hz,1H),5.05(dd,J=12.7,5.5Hz,1H),3.78(dt,J=2 4.9,5.0Hz,4H),3.71–3.64(m,2H),3.60(t,J=5.4Hz,2H),3.56–3.53(m,2H),3.51(d,J=3.8Hz,8H),3.49(t,J=5.4Hz,3H),3.45(q,J=5 .5Hz,2H),3.37(q,J=5.9Hz,2H),2.88(ddd,J=16.7,13.7,5.4Hz,1H),2.58(p,J=4.9Hz,3H),2.05–1.99(m,1H),1.15(d,J=6.5Hz,3H).

[0154] Example 11: Preparation of Compound 11

[0155]

[0156] Refer to the synthesis method of compound 2;

[0157] Step 1: At room temperature, DIPEA (0.4 mL) and 11b (214.2 mg, 0.86 mmol, 1.20 equivalent) were added to a solution of 11a (200 mg, 0.72 mmol, 1.00 equivalent) in anhydrous NMP (5 mL). The solution was stirred at 90 °C for 10 hours. The reactants were then quenched by adding 50 mL of water. The resulting solution was extracted with 3 × 50 mL EtOAc, and the organic layers were combined and dried over anhydrous sodium sulfate. The organic layers were concentrated under vacuum and the crude product was purified by silica gel column chromatography to give 11c (288 mg, yield 79.5%) as a yellow liquid, representing the target compound.

[0158] Step 2: Add 1 mL of trifluoroacetic acid to the solution of 11c (288 mg, 0.57 mmol, 1.00 equivalent) in DCM (3 mL). Stir the resulting solution at room temperature for 10 hours. After the reaction is complete as detected by TLC, concentrate the reaction solution under vacuum. The resulting 11d (crude product) can be used directly in the next step without purification.

[0159] Step 3: EDCI (99.32 mg, 0.52 mmol, 2.60 eq), HOBt (35.1 mg, 0.26 mmol, 1.30 eq), and DIPEA (103.2 mg, 0.80 mmol, 4.00 eq) were added to a solution of 2n (100 mg, 0.20 mmol, 1.00 eq) in 5 mL of DMF at room temperature. The resulting solution was stirred at room temperature for 0.5 hours, followed by the addition of 11d (97 mg, 0.24 mmol, 1.20 eq) in portions at room temperature. The reaction mixture was stirred at room temperature for 8 hours. After the reaction was confirmed to be complete by TLC, 50 mL of water was added. The resulting solution was extracted with 3 × 50 mL EtOAc and the organic layers were combined. The organic layer was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give a yellow-green solid compound 11. 1H NMR (500MHz, DMSO-d6) δ12.44(s,1H),11.08(s,1H),8.77(s,2H),8.40(t,J=5.6Hz,1H),7.91(s,1H),7.56(dd,J=8.6,7.1Hz,1H),7.11( d,J=8.6Hz,1H),7.02(d,J=7.0Hz,1H),6.58(t,J=5.8Hz,1H),6.26(dq,J=8.0,4.0Hz,1H),5.05(dd,J=12.8,5.4Hz,1H),3.77(dt,J=25. 5,5.5Hz,4H),3.71–3.64(m,2H),3.62(t,J=5.5Hz,2H),3.57(dp,J=5.0,2.6Hz,4H),3.52(q,J=3.9Hz,5H),3.48(d,J=5.6Hz,2H),3.44( q,J=5.6Hz,2H),3.38(q,J=5.9Hz,2H),2.94–2.81(m,1H),2.67–2.54(m,3H),2.02(ddd,J=12.9,5.7,3.2Hz,1H),1.15(d,J=6.6Hz,3H).

[0160] Example 12: Preparation of Compound 12

[0161]

[0162] Refer to the synthesis method of compound 2;

[0163] Step 1: At room temperature, DIPEA (0.4 mL) and 12b (176.3 mg, 0.86 mmol, 1.20 equivalence) were added to a solution of 12a (200 mg, 0.72 mmol, 1.00 equivalence) in anhydrous NMP (5 mL). The solution was stirred at 90 °C for 10 hours. The reactants were then quenched by adding 50 mL of water. The resulting solution was extracted with 3 × 50 mL EtOAc, and the organic layers were combined and dried over anhydrous sodium sulfate. The organic layers were concentrated under vacuum and the crude product was purified by silica gel column chromatography to give 12c (247 mg, yield 74.6%) as a yellow liquid, representing the target compound.

[0164] Step 2: Add 1 mL of trifluoroacetic acid to the solution of 12c (247 mg, 0.53 mmol, 1.00 equivalent) in DCM (3 mL). Stir the resulting solution at room temperature for 10 hours. After the reaction is complete as detected by TLC, concentrate the reaction solution under vacuum. The resulting 12d (crude product) can be used directly in the next step without purification.

[0165] Step 3: EDCI (99.32 mg, 0.52 mmol, 2.60 eq), HOBt (35.1 mg, 0.26 mmol, 1.30 eq), and DIPEA (103.2 mg, 0.80 mmol, 4.00 eq) were added to a solution of 2n (100 mg, 0.20 mmol, 1.00 eq) in 30 mL of DMF at room temperature. The resulting solution was stirred at room temperature for 0.5 hours, followed by the addition of 12d (86.4 mg, 0.24 mmol, 1.20 eq) in portions at room temperature. The reaction mixture was stirred at room temperature for 8 hours. After the reaction was confirmed to be complete by TLC, 50 mL of water was added. The resulting solution was extracted with 3 × 50 mL EtOAc and the organic layers were combined. The organic layer was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give compound 12 as a white solid. 1 H NMR (500MHz, DMSO-d6) δ12.44(s,1H),11.09(s,1H),8.75(s,2H),7.91(s,1H),7.55(dd,J=8.6,7.1Hz,1H),7.13(d,J=8.6Hz,1H),7 .01(d,J=7.0Hz,1H),6.59(t,J=5.8Hz,1H),6.27(dq,J=8.1,4.1Hz,1H),5.02(dd,J=12.9,5.4Hz,1H),3.83–3.73(m,4H),3.71–3.6 6(m,2H),3.64(t,J=5.5Hz,3H),3.57(t,J=5.9Hz,3H),3.52(t,J=5.3Hz,4H),3.47(dd,J=9.2,5.4Hz,4H),3.40(q,J=6.2Hz,2H),2. 87(ddd,J=17.0,13.9,5.4Hz,1H),2.68–2.56(m,3H),2.56–2.51(m,1H),2.01(dtt,J=14.7,6.0,3.1Hz,1H),1.15(d,J=6.5Hz,3H).

[0166] Example 13: Preparation of Compound 13

[0167]

[0168] Refer to the synthesis method of compound 2;

[0169] Step 1: To a solution of 13a (200 mg, 0.43 mmol, 1.00 eq) in DMF (5 mL), add EDCI (213.54 mg, 1.12 mmol, 2.60 eq), HOBt (75.47 mg, 0.56 mmol, 1.30 eq), and DIPEA (221.88 mg, 1.72 mmol, 4.00 eq). Stir the resulting solution at room temperature for 0.5 hours, then add 13b (126.42 mg, 0.52 mmol, 1.20 eq) in portions at room temperature. Stir the reaction mixture at room temperature for 8 hours. After the reaction is complete as detected by TLC, add 50 mL of water. Extract the resulting solution with 3 × 50 mL EtOAc and combine the organic layers. The organic layer was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain the target compound, which was a yellow liquid with 13c (173 mg, yield 61.3%).

[0170] Step 2: Add 1 mL of trifluoroacetic acid to a solution of 13c (173 mg, 0.26 mmol, 1.00 equivalent) in 3 mL of DCM. Stir the resulting solution at room temperature for 10 hours. After the reaction is complete as detected by TLC, concentrate the reaction solution under vacuum. The resulting 13d (crude product) can be used directly in the next step without purification.

[0171] Step 3: EDCI (99.32 mg, 0.52 mmol, 2.60 eq), HOBt (35.1 mg, 0.26 mmol, 1.30 eq), and DIPEA (103.2 mg, 0.80 mmol, 4.00 eq) were added to a solution of 2n (100 mg, 0.20 mmol, 1.00 eq) in DMF (5 mL) at room temperature. The resulting solution was stirred at room temperature for 0.5 hours, followed by the addition of 13d (133.68 mg, 0.24 mmol, 1.20 eq) in portions at room temperature. The reaction mixture was stirred at room temperature for 8 hours. After the reaction was confirmed to be complete by TLC, 50 mL of water was added. The resulting solution was extracted with 3 × 50 mL EtOAc and the organic layers were combined. The organic layer was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give compound 13 as a white solid. 1H NMR (500MHz, DMSO-d6) δ12.44(s,1H),8.77(s,2H),8.31(t,J=5.6Hz,1H),7.98(d,J=8.6Hz,1H),7.91(s,1H),6. 27(dq,J=8.0,3.9Hz,1H),4.16(t,J=6.4Hz,2H),3.82(t,J=5.3Hz,2H),3.77(dd,J=7.0,4.0Hz,2H),3.71–3.64(m ,2H),3.61(s,3H),3.56–3.50(m,4H),3.49(d,J=5.6Hz,2H),3.21(q,J=6.6Hz,2H),2.59(t,J=6.5Hz,2H),2.19(d dq,J=21.5,14.2,7.5Hz,2H),1.48(td,J=8.3,4.4Hz,4H),1.33–1.25(m,4H),1.15(d,J=6.4Hz,3H),0.92(s,9H).

[0172] Example 14: Preparation of Compound 14

[0173]

[0174] Refer to the synthesis method of compound 2;

[0175] Step 1: To a solution of 14a (200 mg, 0.43 mmol, 1.00 eq) in DMF (5 mL), add EDCI (213.54 mg, 1.12 mmol, 2.60 eq), HOBt (75.47 mg, 0.56 mmol, 1.30 eq), and DIPEA (221.88 mg, 1.72 mmol, 4.00 eq). Stir the resulting solution at room temperature for 0.5 hours, then add 14b (155.32 mg, 0.52 mmol, 1.20 eq) in portions at room temperature. Stir the reaction mixture at room temperature for 8 hours. After the reaction is complete as detected by TLC, add 50 mL of water. Extract the resulting solution with 3 × 50 mL EtOAc and combine the organic layers. The organic layer was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain the target compound as a yellow liquid at 14c (192 mg, yield 62.7%).

[0176] Step 2: Add 1 mL of trifluoroacetic acid to a solution of 14c (192 mg, 0.27 mmol, 1.00 equivalent) in 3 mL of DCM. Stir the resulting solution at room temperature for 10 hours. After the reaction is complete as detected by TLC, concentrate the reaction solution under vacuum. The resulting 14d (crude product) can be used directly in the next step without purification.

[0177] Step 3: EDCI (99.32 mg, 0.52 mmol, 2.60 eq), HOBt (35.1 mg, 0.26 mmol, 1.30 eq), and DIPEA (103.2 mg, 0.80 mmol, 4.00 eq) were added to a solution of 2n (100 mg, 0.20 mmol, 1.00 eq) in 30 mL of DMF at room temperature. The resulting solution was stirred at room temperature for 0.5 hours, followed by the addition of 14d (147.12 mg, 0.24 mmol, 1.20 eq) in portions at room temperature. The reaction mixture was stirred at room temperature for 8 hours. After the reaction was confirmed to be complete by TLC, 50 mL of water was added. The resulting solution was extracted with 3 × 50 mL EtOAc and the organic layers were combined. The organic layer was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to give compound 14 as a white solid. 1 H NMR (500MHz, DMSO-d6) δ12.45(s,1H),8.78(s,2H),8.31(t,J=5.6Hz,1H),7.97(d,J=8.7Hz,1H),7.91(s,1H) ,6.27(dq,J=7.9,3.9Hz,1H),4.16(dd,J=8.2,6.0Hz,2H),3.82(t,J=5.3Hz,2H),3.77(dt,J=5.5,3.0Hz,2H) ,3.71–3.65(m,2H),3.61(s,3H),3.54(d,J=5.5Hz,4H),3.49(d,J=5.6Hz,2H),3.21(q,J=6.7Hz,2H),2.59(t ,J=6.5Hz,2H),2.25–2.05(m,2H),1.65–1.33(m,5H),1.32–1.20(m,14H),1.15(d,J=6.5Hz,4H),0.92(s,9H).

[0178] Example 15: Test of the inhibitory activity of the compound against PARP7 enzyme

[0179] 1. Experimental materials and instruments

[0180] Experimental materials: PARP7 (BPS, Cat. No. 80527), histone (Active Motif, Cat. No. 81167), SuperSignal ELISA Femto Substrate (THERMO PIERCE, Cat. No. 37074), Biotin-NAD+ (R&D, Cat. No. 6573), Strep-HRP (Thermo Pierce, Cat. No. 21127)

[0181] Instrument: SpectraMax Paradigm multi-functional microplate detector;

[0182] 2. Experimental Methods

[0183] Prepare histone-coated 384-well plates, adding 25 μL of histone solution to each well and incubating overnight at 4°C. Prepare PBST buffer, blocking buffer, and detection buffer. Wash the histone-coated 384-well plates three times with PBST buffer. Block the reaction with 50 μL of blocking buffer over 1 hour at room temperature. Wash the plates three times with PBST buffer. Prepare a 2000x compound in the source plate. Transfer 50 nmL of the compound from the source plate to a 96-well intermediate plate with 19.95 μL of detection buffer. Incubate at 1000 rpm for 1 min. Transfer 5 μL of DMSO / compound to each well. Enzyme reaction: Incubate the enzyme mixture at 25°C for 10 min. Add 10 μL of enzyme mixture (desired minimum control), incubate the compound at room temperature for 10 min, and add 10 μL of detection buffer. Add 10 μL of 2.5x biotin-NAD+ to each well and incubate at 25°C for 90 min. Wash the plates three times with PBST buffer.

[0184] Probe: Add 25 μL of Stre-HRP. Incubate at room temperature for 1 h, then wash the plate three times with PBS buffer. Add 25 μL of the quantitative enhancer mixture. Incubate for 10 min. Add 2.5 μL of quantitative red stop solution and shake the plate for 10–30 s to stop peroxidase activity.

[0185] Data processing:

[0186] Use equation (1) to fit the data in Excel to obtain the suppression value.

[0187] Equation (1): Inh%=(Max-Signal) / (Max-Min)*100.

[0188] Use equation (2) to fit the data in XL-Fit to obtain IC. 50 value.

[0189] Equation (2): Y = Bottom + (Top - Bottom) / (1 + (IC) 50 / X)*HillSlope)

[0190] Where Y is the inhibition percentage and X is the compound concentration.

[0191] 3. Experimental Results

[0192] The inhibitory effects of some target compounds on PARP7 enzyme were determined using the experimental methods described above. The results are shown in Table 1.

[0193] Table 1. Half-maximal inhibitory concentrations of the target compounds against PARP7 enzyme.

[0194]

[0195] Conclusion: The representative compound of this invention can effectively inhibit PARP7 enzyme activity.

[0196] Example 16: Test of the inhibitory activity of some compounds against tumor cells

[0197] 1. Cell lines and cell culture.

[0198] Human lung cancer adenocarcinoma cells (NCI-H1373) were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. Cells were cultured in modified RPMI medium supplemented with 10% (vol / vol) fetal bovine serum, 50 μg / mL penicillin, and 50 μg / mL streptomycin. All cell lines were incubated at 37°C in a humidified atmosphere containing 5% CO2.

[0199] 2. Cell viability assay.

[0200] Cells were seeded at the previously optimized seeding density into two 384-well white-walled tissue culture plates and incubated overnight in a 5% CO2 incubator. Cell viability was assessed 5 days after drug treatment using a CellTiter-Glo luminescence activity assay (Promega). Cell viability was measured immediately after drug administration (day 0) and after 6 days of incubation using Cell TiterGlo (CTG, Promega, G7573). Relative viability was calculated by normalizing the raw luminescence counts to those of cells treated with DMSO control (Ctrl.). The half-maximal inhibitory concentration (IC50) was calculated using Graphpad / Prism 8 software, and dose-response curves were fitted.

[0201] Table 2. Half-maximal inhibitory concentrations (IC50) of the target compounds against tumor cells.

[0202] Compd. <![CDATA[IC 50 (μM)]]> 1 +++ 2 +++ 3 +++ 4 +++ 5 +++ 6 ++ 7 ++ 8 ++ 9 ++ 10 ++ 11 ++ 12 +++ 13 +++ 14 +++ RBN-2397 ++++

[0203] Note: + indicates compound IC.50 >50.0μM

[0204] ++ indicates that the compound is 50.0 μM > IC50. 50 >10.0μM

[0205] +++ indicates that the compound is more than 10.0 μM at IC50. 50 >1.0μM

[0206] ++++ indicates compound IC 50 <1.0μM

[0207] Conclusion: The representative compound of this invention can effectively inhibit human lung cancer adenocarcinoma cells.

Claims

1. A PROTAC compound targeting degradation of PARP7, or a pharmaceutically acceptable salt thereof, characterized in that, The structure of the compound is any one of the following formulas 1 to 14: 。 2. A method of preparing the PROTAC compounds 1-12 of claim 1 for targeted degradation of PARP7, characterized in that, The method includes the following steps: (1) Intermediate a-1 and compound a-2 were dissolved in N,N-dimethylformamide DMF, N,N-diisopropylethylamine DIPEA was added, and the mixture was stirred at 90°C for 4 h. After post-treatment and purification, compound a-3 was obtained. (2) Compound a-3 was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed, the residue obtained by TLC was concentrated under reduced pressure and dissolved in N,N-dimethylformamide DMF. N,N-diisopropylethylamine DIPEA, compound a-4, and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate HATU were added sequentially under ice bath. The mixture was stirred at room temperature for 1 hour and then purified to obtain compound a-5. (3) Compound a-5 was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed by TLC, the residue obtained by concentration under reduced pressure was dissolved in EtOH, and Et3N was added. The mixture was stirred with compound a-6 at 60℃ for 4 hours. After the reaction was completed by TLC, the product was post-processed and purified by column chromatography. The product was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed by TLC, the product was purified by column chromatography to obtain compound (I). Each Linker is independently selected from one of the following structural segments: wherein, represents the site of attachment of the Linker to the aromatic ring of the E3 ligase.

3. A method of preparing the PROTAC compound 13-14 of claim 1 for targeted degradation of PARP7, characterized in that, The method includes the following steps: (1) Intermediate d-1 and compound d-2 were dissolved in N,N-dimethylformamide DMF, and N,N-diisopropylethylamine DIPEA and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate HATU were added sequentially under ice bath. The mixture was stirred at room temperature for 1 hour and then purified to obtain compound d-3. (2) Compound d-3 was dissolved in dichloromethane solution, and trifluoroacetic acid solution was added. The mixture was stirred at room temperature. After the reaction was completed, the residue obtained by TLC was concentrated under reduced pressure and dissolved in N,N-dimethylformamide DMF. N,N-diisopropylethylamine DIPEA, compound a-4, and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate HATU were added in sequence under ice bath. The mixture was stirred at room temperature for 1 hour and then purified to obtain compound d-4. (3) Compound d-4 was dissolved in dichloromethane solution, trifluoroacetic acid solution was added, and the mixture was stirred at room temperature. After the reaction was completed by TLC, the residue obtained by concentration under reduced pressure was dissolved in EtOH, Et3N was added, and the mixture was stirred with compound a-6 at 60℃ for 4 h. After the reaction was completed by TLC, the mixture was post-processed and purified by column chromatography. The product obtained was dissolved in dichloromethane solution, trifluoroacetic acid solution was added, and the mixture was stirred at room temperature. After the reaction was completed by TLC, the product was purified by column chromatography to obtain compound (I). 。 4. The use of the PROTAC compound that targets and degrades PARP7 according to claim 1, or a pharmaceutically acceptable salt thereof, in the preparation of a drug for treating or preventing tumors, characterized in that... The tumor in question is adenocarcinoma of human lung cancer.

5. An antitumor drug comprising a safe and effective amount of the PROTAC compound that targets and degrades PARP7 as described in claim 1, or a pharmaceutically acceptable salt thereof; the antitumor drug further comprising a pharmacologically acceptable excipient or carrier.

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