Pyridazinone derivatives with parp7 / hdac dual-target inhibitory activity, and preparation method and application thereof

Abstract

The application discloses a pyridazinone derivative with PARP7 / HDAC dual-target inhibition activity, and a preparation method and application thereof. The application provides a pyridazinone derivative with PARP7 / HDAC dual-target inhibition activity shown in a structure of formula (I), a pharmaceutical composition containing the 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 pyridazinone derivative with PARP7 / HDAC dual-target inhibition activity, and application of the compounds in treatment of solid tumors and hematological tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a pyridazinone derivative with PARP7 / HDAC dual-target inhibitory activity and a preparation method and application thereof. BACKGROUND

[0002] Poly(ADP-ribose) polymerase (PARP) is a member of the seventeen enzyme families that regulate basic cellular processes including gene expression, protein degradation, and multicellular stress responses, and Poly(ADP-ribose) polymerases 7 (PARP7) is an important member of the PARP family, which can participate in DNA repair, genome stability maintenance and a series of other cellular processes. PARP7 can only transfer a single ADP-ribose (MAR), and belongs to monoPARP. PARP7 contains a zinc finger motif that confers DNA binding in its PARP catalytic domain, and a WWE domain that mediates protein interactions. The mono-ADP-ribosylation mediated by PARP7 is a reversible post-translational modification involved in a variety of important biological processes, such as immune cell function, transcriptional regulation, protein expression and DNA repair. PARP7 plays an important role in neuronal development, stem cell maintenance, antiviral infection and cancer. Genome-wide association studies identified the 3q25 locus as a susceptibility locus for ovarian cancer, and PARP7 plays a role in this cancer type. Mechanistically, PARP7 can regulate microtubulin stability by ribosylating Tubulin, and inhibit the type I interferon signaling pathway, inhibit the body's anti-tumor immunity, and promote tumor growth and survival. Based on the role of PARP7 in innate immune response, the role of PARP7 in tumor immunity has also been increasingly concerned. Abnormally expressed or activated PARP7 can inhibit T cell-mediated anti-tumor immunity by inhibiting innate immune response. In large-scale gene screening, PARP7 was also identified as an inhibitor of T cell activation. Knocking out PARP7 in melanoma cells can enhance the proliferation and growth of co-cultured T cells.

[0003] Histone deacetylases (HDACs) are involved in the process of tumorigenesis, proliferation, invasion and metastasis by affecting tumor cell proliferation, controlling cell cycle, inducing cancer cell resistance to chemotherapeutic drugs, regulating angiogenesis, regulating the production of proteins responsible for invasion and metastasis. Studies have found that inhibition of HDACs can induce tumor cell cycle arrest, differentiation and apoptosis. Therefore, histone deacetylase inhibitors (HDACi) as anti-tumor drugs have become a current research hotspot. At the same time, based on the synergistic anti-tumor effect of HDACi and other anticancer drugs, the design and synthesis of single molecule fusion drugs have also become the object of scientists' research in recent years.

[0004] It has been reported that the combination of PARP inhibitors and HDAC inhibitors has a synergistic effect, which can promote the accumulation of DNA damage and reduce the response of the homologous recombination pathway in tumor cells, thereby promoting the death of various tumor cells. Tumor cells without homologous recombination repair pathway also have good killing effect. However, the combination of drugs has the disadvantages of complex pharmacokinetics, possible drug interactions and more toxic side effects. A single small molecule with multi-target inhibition activity is expected to avoid these problems. PARP7 is an important member of the PARP family, and its inhibitor may be an excellent tumor treatment drug. It is of great significance to screen new varieties of PARP7 inhibitors. The present application proposes a pyridazinone derivative with PARP7 / HDAC dual-target inhibition activity to cope with the differences of different tumors and the complexity of tumor environment, which can benefit cancer patients more and has far-reaching significance. SUMMARY

[0005] The first object of the present application is to provide a pyridazinone derivative with PARP7 / HDAC dual-target inhibition activity to overcome the shortcomings of the prior art.

[0006] A pyridazinone derivative with PARP7 / HDAC dual-target inhibition activity, or an optical isomer, a racemate, a single enantiomer, a possible diastereoisomer thereof, or a pharmaceutically acceptable salt, a prodrug, a deuterium derivative, a hydrate, a solvate thereof, the structure of the derivative is shown as formula (I):

[0007]

[0008] Among them:

[0009] is a chemical bond or wherein " / " represents the site connected to pyridazinone; represents the site of attachment of L 1 ;

[0010] L 1 is a monocyclic or polycyclic or spirocyclic ring selected from 4-10 membered heterocycloalkyl, including but not limited to one of the following structural fragments:

[0011] wherein represents the site of attachment of L 1 ; represents the site of attachment of L 1 ; 2 ;

[0012] are each independently selected from one of the following structural fragments:

[0013]

[0014] wherein n is any natural number from 4 to 6, represents the site of attachment of L 1 ; 2 ;

[0015] As a preference, the pyridazinone derivative having PARP7 / HDAC dual-target inhibitory activity is any one of compounds 1-30 as shown in the following structures:

[0016]

[0017] wherein n is any natural number from 4 to 6.

[0018] A second object of the present application is to provide a preparation method of the above-mentioned pyridazinone derivative having PARP7 / HDAC dual-target inhibitory activity.

[0019] If is a chemical bond, then compound a-1 can be prepared from a compound containing L I ; 2 fragment by simple acid-amine condensation, and the synthesis method of compound a-2 is shown in WO2020223229, which specifically includes the following steps:

[0020] (1) Intermediate a-1 and compound a-2 are dissolved in EtOH, Et3N is added, and stirred at 60°C for 4h, and then purified after work-up to obtain compound a-3;

[0021] (2) Compound a-3 is dissolved in dichloromethane solution, trifluoroacetic acid solution is added, and stirred at room temperature, and then purified after work-up to obtain compound a-4;

[0022] ​(3) Dissolve compound a-4 in MeOH and DCM, add NaOH in MeOH solution under ice bath stirring, then add hydroxylamine aqueous solution, keep the reaction at 0℃ and stir for 1 h, and then process to obtain compound (I);

[0023] like for Then, using synthetic route two, compound b-5 can be synthesized from L-containing compounds. I With L 2 The fragmented compound is prepared by a simple substitution reaction, specifically including the following steps:

[0024] (1) Compound b-1 and compound b-2 undergo Michael addition under the action of cesium carbonate to obtain compound b-3;

[0025] (2) Compound b-3 was hydrolyzed with sodium hydroxide to give compound b-4;

[0026] (3) Compounds b-4 and b-5 were dissolved in N,N-dimethylformamide (DMF), and N,N-diisopropylethylamine (DIPEA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and 1-hydroxybenzotriazole (HOBt) were added sequentially under ice bath. The mixture was stirred overnight and then purified to obtain compound b-6.

[0027] (4) Compound b-6 was deprotected by trifluoroacetic acid to obtain compound b-7;

[0028] (5) Dissolve compounds b-5 and b-7 obtained in route 1 with EtOH, add Et3N, stir at 60℃ for 4h, and then purify to obtain compound b-8.

[0029] (6) Compound b-8 was dissolved in dichloromethane solution, trifluoroacetic acid solution was added, stirred at room temperature, and then purified by post-treatment to obtain compound b-9;

[0030] (7) Dissolve compound b-9 in MeOH and DCM, add NaOH in MeOH solution under ice bath stirring, then add hydroxylamine aqueous solution, keep the reaction at 0℃ and stir for 1 h, and then process to obtain compound (I);

[0031]

[0032] The compounds of 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 compounds used, reaction temperature, and reaction time, are not limited to those explained above. 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.

[0033] A third objective of this invention is to provide the use of the pyridazinone derivatives having the HDACi pharmacophore, or optical isomers, racemates, single enantiomers, possible diastereomers, or pharmaceutically acceptable salts, prodrugs, deuterated derivatives, hydrates, or solvates thereof, in the preparation of drugs for the treatment or prevention of tumors.

[0034] A fourth objective of this invention is to provide an antitumor drug containing a safe and effective amount of the said pyridazinone derivative having the HDACi pharmacophore, or an optical isomer, racemate, single enantiomer, possible diastereomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, or solvate thereof.

[0035] Preferably, the antitumor drug may further include a pharmacologically acceptable salt and a pharmacologically acceptable excipient or carrier.

[0036] Preferably, in the application and the antitumor drug, the tumor includes solid tumors and hematologic malignancies.

[0037] 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.

[0038] 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.

[0039] "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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0049] 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.

[0050] Compared with existing technologies, the main advantages of this invention include: This invention fuses the structure of pyridazinone with PARP7 inhibitory effects and HDACi design into a single antitumor molecule, providing pyridazinone derivatives with HDACi pharmacophores of the structure shown in formula (I), pharmaceutical compositions containing compounds of formula (I), hydrates, and isotopic derivatives, chiral isomers, allosteres, different salts, prodrugs, and formulations of these compounds. By introducing the polar HDACi pharmacophore, the physicochemical properties, water solubility, and oral absorption and utilization of the original pyridazinone structure can be significantly improved. While retaining PARP7 activity, the affinity activity of HDAC-related targets is increased, thereby enhancing the overall anticancer effect. This invention combines the functional group action element of HDAC with the key structure of pyridazinone in specific PARP7 inhibitor small molecules through high-throughput screening, homologue synthesis, base structure design, and fragment assembly strategies to enhance antitumor activity, which is groundbreaking compared to simple structural modifications. This invention also provides a method for preparing the pyridazinone derivatives with the HDACi pharmacophore, their uses, and the inhibitory activity of these compounds on the proliferation of various tumor cell lines. The pyridazinone derivatives with the HDACi pharmacophore of this invention show promise as anti-tumor drug candidates for treating various cancers, such as solid tumors and hematological malignancies. Detailed Embodiments

[0051] 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.

[0052] Example 1: Preparation of Compound 1

[0053]

[0054] Step 1: Under nitrogen protection, sodium hydride (24.0 g, 60% purity, 142 mol, 1.20 equivalent) was added dropwise to a solution of 1a (30.0 g, 118.1 mmol, 1.00 equivalent) in 300 mL of DMF. The resulting solution was stirred at room temperature for 1 hour, followed by dropwise addition of [2-(chloromethoxy)ethyl]trimethylsilane (21.6 g, 130 mmol, 1.10 equivalent) at 0 °C. The reaction mixture was stirred at room temperature for 9 hours. After the reaction was confirmed to be complete by TLC, 500 mL of water was added. The resulting solution was extracted with 3 × 500 mL of LEtOAc and the organic layers were combined. The organic layer was washed with 250 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 = 1:4) to give a colorless oily compound 1b (34.0 g, yield 85.4%) for the next step. LCMS:[M+H]+384.70.

[0055] Step 2: At room temperature, potassium hydroxide (14.0 g, 142 mol, 1.20 equivalent) was added in portions to a solution of 1b (32.0 g, 83.3 mmol, 1.00 equivalent) in 300 mL of MeOH. The resulting solution was stirred at room temperature for 10 hours. After the reaction was confirmed to be complete by TLC, the reaction mixture was evaporated to dryness, and then 500 mL of water was added to the crude product. The resulting solution was extracted with 3 × 500 mL of LEtOAc and the organic layers were combined. The organic layer was washed with 250 mL of brine, dried over anhydrous sodium sulfate and subjected to reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:1) to give a yellow oily compound 1c (22.0 g, yield 79.0%) for the next step of the reaction.

[0056] Step 3: 1c (22.0 g, 62.1 mmol, 1.00 equivalent), 1d (35.6 g, 186 mmol, 3.00 equivalent), and CuI (9.46 g, 31.1 mmol, 0.50 equivalent) were added to 250 mL of NMP. The mixture was stirred at 100 °C for 12 hours. After the reaction was confirmed to be complete by TLC, the mixture was cooled and filtered. The reactants were then quenched by adding 1000 mL of water. The resulting solution was extracted with 3 × 1000 mL of LEtOAc. The organic layers were combined and concentrated. The residue was applied to a silica gel column (ethyl acetate: petroleum ether = 1:1). The collected eluates were combined and concentrated to obtain an oily substance, yielding 1e (17.6 g, 87.4% yield) as a pale yellow oil containing the target compound.

[0057] Step 4: TMSI (16.0 g, 80.2 mmol, 1.30 equivalent) was added dropwise to a solution of 1e (20.0 g, 61.7 mmol, 1.00 equivalent) in 200 mL of DMF at 20 °C. The resulting solution was stirred at 85 °C for 10 hours. The reaction mixture was then quenched with 850 mL of water, and the resulting solution was extracted with 3 × 500 mL of LEtOAc. 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 1f (13.0 g, 70.2% yield) as a pale yellow solid.

[0058] Step 5: Add oxaloyl chloride (12.3 g, 96.7 mmol, 3.00 equivalent) dropwise to 1 f (10.0 g, 32.3 mmol, 1.00 equivalent) in 100 mL of DMF at 0–5 °C. 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 × 300 mL of LEtOAc, 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 g (8.4 g, yield 79.4%) of the target compound as a pale yellow liquid.

[0059] Step Six: Triethylamine (308 mg, 3.05 mmol, 4.00 equivalent) and 1 h (12.3 g, 96.7 mmol, 3.00 equivalent) were added to a solution of 1 g (250 mg, 0.762 mmol, 1.00 equivalent) in anhydrous ethanol (5 mL) at 0-5 °C. The resulting solution was stirred at room temperature for 10 h. The reaction mixture was then quenched by adding 20 mL of water. The resulting solution was extracted with 3 × 20 mL of LEtOAc, 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 i (200 mg, yield 58.3%) of the target compound as a white solid.

[0060] Step 7: 1i (200 mg, 0.443 mmol, 1.00 equivalent) was added dropwise to a solution in DCM (3 mL) with 1 mL of trifluoroacetic acid. The resulting solution was stirred at room temperature for 10 hours. The reaction mixture was then quenched by adding 20 mL of water. The resulting solution was extracted with 3 × 10 mL of dichloromethane, 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 1j (70 mg, yield 48.6%) as a pale yellow liquid containing the target compound.

[0061] Step 8: Compound 1j (70 mg, 0.22 mmol, 1.00 equivalent) was placed in a 25 mL round-bottom flask, and DCM solution (1 mL) and MeOH solution (2 mL) were added. Under ice bath conditions, NH2OH aqueous solution (50%, 215 mg, 6.54 mmol, 15.0 equivalent) and NaOH (130.8 mg, 3.27 mmol) were added, and the mixture was stirred for 2 h under ice bath conditions. After the reaction was confirmed to be complete by TLC, the pH was adjusted to 7-8 with HCl aqueous solution (1 M) under stirring. Then, dichloromethane (10 mL) was added for extraction. The aqueous phase was retained and concentrated. The residue was purified by C18 reversed-phase chromatography with H2O / CH3CN (1 / 1) elution to obtain compound 1 (48 mg, yield 68%) as a white solid, the title compound. 1 H NMR (400MHz, DMSO-d6) δ12.43(s,1H),10.34(d,J=1.5Hz,1H),8.67(d,J=1.4Hz,1H),7.85(s,1H),7.08(tt,J=6. 5,3.4Hz,1H),3.35–3.26(m,2H),1.93(t,J=7.4Hz,2H),1.48(h,J=6.9Hz,4H),1.26(qd,J=6.7,4.0,3.3Hz,4H).

[0062] Example 2: Preparation of Compound 2

[0063]

[0064] Step 1: HATU (1.11 g, 2.93 mmol, 1.10 eq) and DIPEA (686 mg, 5.32 mmol, 2.00 eq) were added to a solution of 2a (500 mg, 2.66 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 2b (798 mg, 3.99 mmol, 1.50 eq) in portions at 0 °C. The reaction mixture was stirred at room temperature for 4 h. 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 of LEtOAc 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 colorless oily compound 2c (600 mg, yield 61.0%) for the next step.

[0065] Step 2: Add HCl·dioxane (5 mL) to a solution of compound 2c (600 mg, 1.62 mmol, 1.00 equivalent) in MeOH (5 mL) at room temperature. Stir the resulting solution at room temperature for 12 hours. After TLC detection, the reaction was complete. The reaction solution was evaporated to dryness to give a colorless oily compound 2d (340 mg, yield 85.4%), which can be used directly in the next step without purification.

[0066] Step 3: Triethylamine (277 mg, 2.77 mmol, 3.00 equivalent) and 2d (336 mg, 1.07 mmol, 1.20 equivalent) were added to a solution of 2e (300 mg, 0.914 mmol, 1.00 equivalent) in anhydrous ethanol (5 mL) at 0-5 °C. The resulting solution was stirred at room temperature for 12 hours. The reaction mixture was then quenched by adding 20 mL of water. The resulting solution was extracted with 3 × 20 mL of LEtOAc, 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 2f (320 mg, yield 62.4%) as a white solid, representing the target compound.

[0067] Step 4: 2f (280 mg, 0.498 mmol, 1.00 equivalent) of the compound was added dropwise to a solution in DCM (1 mL) with 1 mL of trifluoroacetic acid. The solution was stirred at room temperature for 4 hours, and then the reaction mixture was quenched by adding 20 mL of water. The resulting solution was extracted with 3 × 10 mL of dichloromethane, 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 (160 mg, yield 74.4%) of the target compound as a pale yellow liquid.

[0068] Step 5: 2 g (140 mg, 0.324 mmol, 1.00 equivalent) of compound was placed in a 25 mL round-bottom flask, and DCM solution (1 mL) and MeOH solution (2 mL) were added. Under ice bath conditions, NH2OH aqueous solution (50%, 321 mg, 4.86 mmol, 15.0 equivalent) and NaOH (129 mg, 3.24 mmol) were added, and the mixture was stirred for 1 h under ice bath conditions. After the reaction was confirmed to be complete by TLC, the pH was adjusted to 7-8 with HCl aqueous solution (1 M) while stirring. Then, dichloromethane (10 mL) was added for extraction. The aqueous phase was retained and concentrated. The residue was purified by C18 reversed-phase chromatography with H2O / CH3CN (1 / 1) elution to obtain compound 2 (70 mg, yield 49.9%) as a white solid, the title compound. 1H NMR(500MHz,DMSO-d6)δ12.49(s,1H),10.32(s,1H),8.65(s,1H),8.02(s,1H),6.40(dq,J=7.0,3.3 Hz,1H),4.38(d,J=13.2Hz,1H),4.09(q,J=5.3Hz,1H),4.05–3.91(m,1H),3.86(d,J=13.7Hz,1H),3. 17(d,J=5.2Hz,2H),3.12–2.99(m,1H),2.60(td,J=13.0,2.7Hz,1H),2.28(t,J=7.5Hz,2H),1.93(t, J=7.4Hz,2H),1.89–1.72(m,2H),1.69–1.51(m,1H),1.47(dd,J=7.3,4.4Hz,4H),1.37–1.14(m,4H).

[0069] Example 3: Preparation of Compound 3

[0070]

[0071] Step 1: HATU (2.09 g, 5.52 mmol, 1.10 equivalence) and DIPEA (1.792 g, 15.06 mmol, 3.00 eq) were added to a solution of 3b (1.15 g, 5.02 mmol, 1.00 equivalence) in DMF (5 mL) at 0 °C. The resulting solution was stirred at room temperature for 0.5 h, followed by the addition of 3a (0.908 g, 5.02 mmol, 1.00 equivalence) in portions at 0 °C. The reaction mixture was stirred at room temperature for 4 h. 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 of LEtOAc 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 colorless oily compound 3c (820 mg, yield 45.9%) for the next step.

[0072] Step 2: Add HCl·dioxane (5 mL) to a solution of compound 3c (800 mg, 2.24 mmol, 1.00 equivalent) in MeOH (5 mL) at room temperature. Stir the resulting solution at room temperature for 12 hours. After TLC detection, the reaction was complete. The reaction solution was evaporated to dryness to obtain a colorless oily compound 3d (470 mg, yield 71.8%), which can be used directly in the next step without purification.

[0073] Step 3: Triethylamine (277 mg, 2.77 mmol, 5.00 equivalent) and 3d (470 mg, 1.60 mmol, 1.76 equivalent) were added to a solution of 3e (300 mg, 0.914 mmol, 1.00 equivalent) in anhydrous ethanol (5 mL) at 0-5 °C. The resulting solution was stirred at room temperature for 12 hours. The reaction mixture was then quenched by adding 20 mL of water. The resulting solution was extracted with 3 × 20 mL of LEtOAc, 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 3f (320 mg, yield 64.0%) as a white solid, representing the target compound.

[0074] Step 4: 3f (320 mg, 0.58 mmol, 1.00 equivalent) of the compound was added dropwise to a solution in DCM (5 mL). The solution was stirred at room temperature for 10 hours. The reaction mixture was then quenched by adding 20 mL of water. The resulting solution was extracted with 3 × 10 mL DCM, and the organic layers were combined and dried over anhydrous sodium sulfate. The organic layers were concentrated under vacuum and purified by silica gel column chromatography to give 3 g (95 mg, yield 38.5%) of the target compound as a pale yellow liquid.

[0075] Step 5: 3 g (95 mg, 0.227 mmol, 1.00 equivalent) of compound was placed in a 25 mL round-bottom flask, and DCM solution (1 mL) and MeOH solution (2 mL) were added. Under ice bath conditions, NH2OH aqueous solution (50%, 224.7 mg, 3.41 mmol, 15.0 equivalent) and NaOH (90.8 mg, 2.27 mmol, 10.0 equivalent) were added, and the mixture was stirred for 1 h under ice bath conditions. After the reaction was confirmed to be complete by TLC, the pH was adjusted to 7-8 with HCl aqueous solution (1 M) under stirring. Then, dichloromethane (10 mL) was added for extraction. The aqueous phase was retained and concentrated. The residue was purified by C18 reversed-phase chromatography with H2O / CH3CN (1 / 1) elution to obtain compound 3 (40 mg, yield 42.1%) as a white solid, the title compound. 1H NMR(500MHz,DMSO-d6)δ10.32(s,1H),8.64(s,1H),7.95(s,1H),7.81(t,J=5.6Hz,1H) ,3.71–3.55(m,2H),3.30(s,2H),3.16(t,J=12.5Hz,2H),3.00(q,J=6.6Hz,2H),2.42–2 .31(m,1H),1.92(t,J=7.4Hz,2H),1.72(dd,J=13.6,3.8Hz,2H),1.62(qd,J=11.9,3.9 Hz,2H),1.46(p,J=7.5Hz,2H),1.36(p,J=7.2Hz,2H),1.20(qd,J=9.7,9.0,6.0Hz,2H).

[0076] Example 4: Preparation of Compound 4

[0077]

[0078] Step 1: Cesium carbonate (14.0 g, 42.8 mmol, 1.50 equivalent) was added to a solution of 4a (5.00 g, 28.6 mmol, 1.00 equivalent) and 4b (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 of LEtOAc, 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 (4.20 g, yield 61.7%) as a pale yellow liquid, representing the target compound.

[0079] Step 2: NaOH (3.20 g, 80.5 mmol, 5.00 equivalent) was added to a solution of 4c (4.2 g, 16.1 mmol, 1.00 equivalent) in MeOH (100 mL) and H₂O (20 mL). The resulting solution was stirred at room temperature for 12 hours. The reaction mixture was then quenched by adding 200 mL (1 M HCl). The resulting solution was extracted with 3 × 500 mL of LEtOAc, and the organic layers were combined and dried over anhydrous sodium sulfate. Vacuum concentration yielded the target compound 4d (3.20 g, 80.6% yield) as a pale yellow liquid, which could be used directly in the next reaction without purification.

[0080] Step 3: Potassium carbonate (1.23 g, 11.6 mmol, 2.00 equivalent) was added to a solution of 4e (1.00 g, 5.81 mmol, 1.00 equivalent) and 4f (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 of LEtOAc, 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 4 g (1.80 g, 92.2% yield) of the target compound as a white solid.

[0081] Step 4: Add 5 mL of dioxane hydrochloride solution to a solution of 4 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 4 h (780 mg, yield 62.9%), which can be used directly in the next step without purification.

[0082] Step 5: HATU (609 mg, 1.60 mmol, 1.20 equivalence) and DIPEA (634 mg, 5.34 mmol, 4.00 eq) were added to a solution of 4d (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 4h (391 mg, 130 mmol, 1.10 equivalence) in portions at 0 °C. The reaction mixture was stirred at room temperature for 9 h. 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 of LEtOAc 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 4i (509 mg, yield 84.5%) for the next step.

[0083] Step Six: Add 1 mL of trifluoroacetic acid to a solution of compound 4i (400 mg, 0.887 mmol, 1.00 equivalent) in DCM (3 mL) at room temperature. Stir the resulting solution at room temperature for 12 hours. After the reaction is complete as detected by TLC, evaporate the reaction solution to dryness to obtain a colorless oily compound 4j (300 mg, crude product), which can be used directly in the next step without purification.

[0084] Step 7: At room temperature, triethylamine (1 mL) and 4j (300 mg, 0.84 mmol, 1.10 equivalent) were added to a solution of 4k (250 mg, 0.762 mmol, 1.00 equivalent) in anhydrous ethanol (5 mL). The solution was stirred at 65°C for 12 hours. The reaction mixture was then quenched by adding 20 mL of water. The resulting solution was extracted with 3 × 20 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 4l (180 mg, yield 36.7%) of the target compound as a white solid.

[0085] Step 8: Add 1 mL of trifluoroacetic acid to a solution of 4 L (180 mg, 0.28 mmol, 1.00 equivalent) in 2 mL of DCM. 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 4 M (52 mg, yield 36.2%) as a colorless liquid.

[0086] Step 9: Compound 4m (52 ​​mg, 0.101 mmol) was placed in a 25 mL round-bottom flask, and DCM solution (1 mL) and MeOH solution (2 mL) were added. Under ice bath conditions, NH2OH aqueous solution (50%, 100.3 mg, 1.50 mmol) and NaOH (40.5 mg, 1.01 mmol) were added, and the mixture was stirred for 1 h under ice bath conditions. While stirring, the pH was adjusted to 7–8 with HCl aqueous solution (1 M). After concentration, the residue was purified by C18 reversed-phase chromatography eluting with H2O / CH3CN (1 / 1) to obtain compound 4 (32 mg, yield 61.5%) as a white solid, the title compound. 1 H NMR(500MHz,DMSO-d6)δ12.43(s,1H),11.08(s,1H),9.02(s,1H),8.69(s,2H),7. 90(s,1H),6.26(dt,J=8.6,4.1Hz,1H),4.12(dt,J=20.3,6.4Hz,1H),3.78(dt,J= 24.7,5.2Hz,4H),3.74–3.62(m,2H),3.53(d,J=5.3Hz,4H),3.48(d,J=5.6Hz,2H) ,3.16(d,J=3.2Hz,1H),2.58(t,J=6.5Hz,2H),2.54(s,1H),1.14(d,J=6.5Hz,3H) 13C NMR(126MHz,DMSO-d6)δ168.9,161.3,157.9,157.1,146.1,128.6,125.9, 123.8,114.9,73.4,67.0,48.5,44.6,43.6,43.2,40.7,40.4,32.7,17.6.

[0087] Example 5: Preparation of Compound 5

[0088]

[0089] Following the synthetic method of compound 4, the following steps were performed: First, a substitution reaction was conducted to obtain a yellow solid compound 5c (4.00 g, yield 85.2%); second, a dioxane hydrochloride solution was used to remove the tert-butyloxycarbonyl group to obtain a crude yellow solid compound 5d (2.10 g, yield 76.9%); third, compound 5d and compound 5e were subjected to acid-ammonia condensation to obtain a white solid compound 5f (1.05 g, yield 57.6%); fourth, a protecting group was removed by TFA to obtain a colorless oily compound 5g (300 mg, crude); fifth, a nucleophilic substitution reaction was conducted to obtain a colorless oily compound 5h (220 mg, yield 46.9%); sixth, a protecting group was removed by TFA to obtain a white solid compound 5i (105 mg, yield 66.7%); seventh, a light yellow solid compound 5 (44 mg, yield 46.3%) was obtained by amine exchange and C18 reversed-phase chromatography. 1 H NMR (400MHz, DMSO-d6) δ12.47(s,1H),11.02(s,1H),8.91(s,1H),8.51(d,J=2.4Hz,1H), 7.92(s,1H),7.87(dd,J=9.0,2.5Hz,1H),6.84(d,J=9.0Hz,1H),6.30(dt,J=8.6,4.2Hz, 1H), 4.14 (dp, J=10.1, 6.1Hz, 1H), 3.67 (q, J=6.3Hz, 2H), 3.61 (dd, J=6.6, 3.3Hz, 2H), 3. 54(t,J=5.1Hz,6H),3.48(d,J=5.6Hz,2H),2.58(t,J=6.5Hz,2H),1.14(d,J=6.5Hz,3H). 13 C NMR(126MHz,DMSO-d6)δ168.9,159.5,157.9,147.2,146.1,136.2,128.6,125 .9,123.8,117.1,105.8,73.4,66.9,48.5,44.4,44.2,44.0,40.6,32.7,17.6.

[0090] Example 6: Preparation of Compound 6

[0091]

[0092] Step 1: NaOH (1.20 g, 29.9 mmol, 5.00 equivalent) was added to a solution of 6a (2.7 g, 5.99 mmol, 1.00 equivalent) in MeOH (10 mL) and H₂O (5 mL). The solution was stirred at room temperature for 12 hours. The reaction mixture was then quenched by adding 100 mL (1 M HCl). The resulting solution was extracted with 3 × 100 mL of ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate. Vacuum concentration yielded 6b (1.60 g, 61.2% yield) as a white solid, which could be used directly in the next reaction without further purification.

[0093] Step 2: HATU (626 mg, 1.65 mmol, 1.20 equivalence) and DIPEA (490 mg, 4.12 mmol, 3.00 eq) were added to a solution of 6b (600 mg, 1.37 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 6c (535 g, 2.75 mmol, 2.00 equivalence) in portions at 0 °C. The reaction mixture was stirred at room temperature for 1 h. 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 of ethyl acetate, and the organic layers were combined. The organic layers were 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 6d (720 mg, yield 90.7%) for the next step.

[0094] Step 3: Add 5 mL of trifluoroacetic acid to a solution of compound 6d (700 mg, 1.21 mmol, 1.00 equivalent) in DCM (5 mL) at room temperature. Stir the resulting solution at room temperature for 12 hours. After TLC detection, the reaction was complete. The reaction solution was evaporated to dryness to obtain a colorless oily compound 6e (600 mg, crude product), which can be used directly in the next step without purification.

[0095] Step 4: At room temperature, triethylamine (1 mL) and 6e (600 mg, crude product) were added to a solution of 6f (360 mg, 1.10 mmol, 1.00 equivalent) in anhydrous ethanol (5 mL). The resulting solution was stirred at 60 °C for 4 hours. The reactants were then quenched by adding 20 mL of water. The resulting solution was extracted with 3 × 20 mL of LEtOAc, 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 6 g (420 mg, yield 49.6%) of the target compound as a colorless liquid.

[0096] Step 5: 6 g (10.0 g, 32.3 mmol, 1.00 equivalent) of the compound was added dropwise to a solution in DCM (5 mL) with 2 mL of trifluoroacetic acid. The solution was stirred at room temperature for 4 hours. The reaction mixture was then quenched by adding 50 mL of water. The resulting solution was extracted with 3 × 50 mL DCM, 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 the target compound as a white crystalline solid (260 mg, yield 78.3%) after 6 hours.

[0097] Step 6: Compound 6h (180 mg, 0.28 mmol) was placed in a 25 mL round-bottom flask, and DCM solution (2.5 mL) and MeOH solution (5 mL) were added. Under ice bath conditions, NH2OH aqueous solution (50%, 278 mg, 8.43 mmol, 15.0 equivalent) and NaOH (112 mg, 2.81 mmol, 10.0 equivalent) were added, and the mixture was stirred for 1 h under ice bath conditions. While stirring, the pH was adjusted to 7-8 with HCl aqueous solution (1 M). After concentration, the residue was purified by C18 reversed-phase chromatography eluting with H2O / CH3CN (1 / 1) to give the title compound 6 (115 mg, yield 63.5%) as a white solid. 1H NMR (500MHz, DMSO-d6) δ12.44(s,1H),10.32(s,1H),8.77(d,J=1.4Hz,2H),8.65(d,J=2.0Hz,1H),8.32(t,J=5.5Hz,1H),7.91 (s,1H),6.27(dt,J=8.7,4.3Hz,1H),4.15(p,J=6.5Hz,1H),4.09(qd,J=5.3,1.5Hz,1H),3.79(dt,J=25.2,5.1Hz,4H),3.68(tt ,J=10.2,5.2Hz,2H),3.52(t,J=5.2Hz,4H),3.48(d,J=5.5Hz,2H),3.32(s,1H),3.21(q,J=6.7Hz,2H),3.16(dd,J=5.2,1.2Hz, 1H), 2.59 (t, J=6.4Hz, 2H), 1.93 (t, J=7.4Hz, 2H), 1.48 (p, J=7.2Hz, 4H), 1.27 (dh, J=12.2, 6.8, 5.2Hz, 4H), 1.19–1.09 (m, 3H). 13 C NMR(126MHz,DMSO-d6)δ169.1,168.9,163.4,161.3,157.9,157.5,146.1,128.6,125.9,1 16.6,73.4,67.0,48.5,44.6,43.6,43.2,40.7,32.7,32.2,29.1,28.3,26.2,25.1,17.6.

[0098] Example 7: Preparation of Compound 7

[0099]

[0100] Following the synthetic method of compound 6, the first step involved acid-ammonia condensation to obtain a white solid compound 7c (500 mg, 85% yield); the second step involved the removal of a tert-butyloxycarbonyl group from trifluoroacetic acid to obtain a crude colorless oily liquid compound 7d (460 mg, 100% yield); the third step involved nucleophilic substitution to obtain a colorless oily compound 7f (410 mg, 63.7% yield); the sixth step involved the removal of a protecting group by TFA to obtain a white solid compound 7g (160 mg, 48.3% yield); and the seventh step involved purification by amine exchange and C18 reversed-phase chromatography to obtain a pale yellow solid compound 7 (105 mg, 65.6% yield). 1HNMR(400MHz,DMSO-d6)δ12.45,10.34,8.77,8.74–8.58(m),8.34(t,J=5.6Hz),7. 91,6.28(dq,J=8.3,4.0Hz),4.15(p,J=6.3Hz),3.88–3.73(m),3.67(q,J=6.3Hz), 3.53(d,J=5.2Hz),3.48(d,J=5.6Hz),3.20(q,J=6.7Hz),2.58(t,J=6.5Hz),1.94( t, J=7.4Hz), 1.49 (h, J=6.9Hz), 1.26 (ddt, J=15.2, 9.8, 5.9Hz), 1.14 (d, J=6.5Hz). 13 C NMR (126MHz, DMSO-d6) δ 169.1, 168.9, 163.4, 161.3, 157.9, 157.5, 146.1, 128.6, 125.9, 123.8, 73.4, 67.0, 48.5, 44.6, 43.6, 43.2, 40.7, 32.7, 32.2, 28.9, 26.1, 24.9, 17.6. Example 8: Preparation of Compound 8

[0101]

[0102] Following the synthetic method of compound 6, the first step involved acid-ammonia condensation to obtain a white solid compound 8c (550 mg, 83% yield); the second step involved the removal of the tert-butyloxycarbonyl group from trifluoroacetic acid to obtain a crude colorless oily liquid compound 8d (720 mg, 100% yield); the third step involved nucleophilic substitution to obtain a colorless oily compound 8f (390 mg, 52.5% yield); the sixth step involved the removal of the protecting group from TFA to obtain a white solid compound 8g (180 mg, 55.9% yield); and the seventh step involved purification by amine exchange and C18 reversed-phase chromatography to obtain a pale yellow solid compound 8 (93 mg, 51.7% yield). 1H NMR(500MHz,DMSO-d6)δ8.78(s,2H),8.36(t,J=5.6Hz,1H),7.91(s,1H),6.26(d,J=7 .2Hz,1H),4.15(p,J=6.7Hz,1H),3.79(ddd,J=25.2,6.9,4.1Hz,4H),3.73–3.63(m,2H ),3.53(t,J=5.3Hz,4H),3.48(d,J=5.6Hz,2H),3.21(q,J=6.3Hz,2H),3.17(s,1H),2 .59(t,J=6.5Hz,2H),1.97(t,J=7.0Hz,2H),1.58–1.41(m,4H),1.15(d,J=6.5Hz,3H). 13 C NMR(126MHz,DMSO-d6)δ169.0,168.9,163.4,161.3,157.9,157.5,146.1,128.6,126.0,1 23.8,116.6,73.4,67.0,48.5,44.6,43.6,43.2,40.7,38.7,32.7,32.0,28.8,22.8,17.6.

[0103] Example 9: Preparation of Compound 9

[0104]

[0105] Following the synthetic method of compound 6, the first step involved acid-ammonia condensation to obtain a white solid compound 9c (900 mg, yield 88%); the second step involved the removal of a tert-butyloxycarbonyl group from trifluoroacetic acid to obtain a crude colorless oily liquid compound 9d (940 mg, yield 100%); the third step involved nucleophilic substitution to obtain a colorless oily compound 9f (623 mg, yield 49.8%); the sixth step involved the removal of a protecting group from trifluoroacetic acid to obtain a white solid compound 9g (289 mg, yield 48.8%); and the seventh step involved purification by amine exchange and C18 reversed-phase chromatography to obtain a pale red solid compound 9 (193 mg, yield 66.8%). 1H NMR(500MHz,DMSO-d6)δ8.78(s,2H),8.36(t,J=5.6Hz,1H),7.91(s,1H),6.26(d,J=7 .2Hz,1H),4.15(p,J=6.7Hz,1H),3.79(ddd,J=25.2,6.9,4.1Hz,4H),3.73–3.63(m,2H ),3.53(t,J=5.3Hz,4H),3.48(d,J=5.6Hz,2H),3.21(q,J=6.3Hz,2H),3.17(s,1H),2 .59(t,J=6.5Hz,2H),1.97(t,J=7.0Hz,2H),1.58–1.41(m,4H),1.15(d,J=6.5Hz,3H). 13 C NMR(126MHz,DMSO-d6)δ169.0,168.9,163.4,161.3,157.9,157.5,146.1,128.6,126.0,1 23.8,116.6,73.4,67.0,48.5,44.6,43.6,43.2,40.7,38.7,32.7,32.0,28.8,22.8,17.6.

[0106] Example 10: Preparation of Compound 10

[0107]

[0108] Following the synthetic method of compound 6, the first step involved hydrolysis to obtain 10b (440 mg, 75.9% yield); the second step involved acid-ammonia condensation to obtain a white solid compound 10d; the third step involved the removal of the tert-butyloxycarbonyl group from trifluoroacetic acid to obtain a colorless oily liquid (460 mg, 100% crude product yield); the fourth step involved nucleophilic substitution to obtain a colorless oily compound 10f (320 mg, 54.5% yield); the fifth step involved the removal of the protecting group from TFA to obtain a white solid compound 10g (200 mg, 75.3% yield); and the sixth step involved purification by amine exchange and C18 reversed-phase chromatography to obtain a pale yellow solid compound 10 (70 mg, 41.2% yield). 1H NMR (400MHz, DMSO-d6) δ12.47(s,1H),10.35(d,J=1.5Hz,1H),8.64(dd,J=30.9,2.0Hz,2H),8.25(t,J=5.6Hz,1H),8.0 6–7.84(m,2H),6.85(d,J=9.0Hz,1H),6.30(dt,J=8.6,4.2Hz,1H),4.17(s,1H),4.13(dt,J=10.4,5.2Hz,1H),3.68(q, J=6.2Hz,2H),3.62(dd,J=6.7,3.3Hz,2H),3.55(p,J=5.3Hz,6H),3.49(d,J=5.6Hz,2H),3.19(dd,J=15.4,6.0Hz,3H), 2.59(t,J=6.5Hz,2H), 1.94(t,J=7.3Hz,2H), 1.49(p,J=7.1Hz,4H), 1.26(dq,J=9.6,5.9Hz,4H), 1.15(d,J=6.4Hz,3H).

[0109] Example 11: Preparation of Compound 11

[0110]

[0111] Step 1: At room temperature, 11a (2.9 g, 11.1 mmol) was placed in a solution of DCM (30 mL) and trifluoroacetic acid (5 mL) was added. The reaction mixture was stirred at room temperature in the dark for 6 h. The solvent was removed under reduced pressure to obtain intermediate 11b (crude product), which was used directly in the next stage without further purification.

[0112] Step 2: Add Et3N (10 mL), EtOH (100 mL), and intermediate 11c (3.7 g, 11.1 mmol) to the crude intermediate 11b. Stir the reaction mixture at 60 °C for 12 h. Monitor the reaction by TLC. Quench the mixture with cold water (200 mL) at room temperature and extract with EtOAc (300 mL × 3). Wash the combined organic layers with brine (100 mL × 2), dry with Na2SO4, and concentrate under vacuum. Purify the crude product by column chromatography (DCM / MEOH = 20:1, v / v) to give intermediate 11d (2.7 g, 54% yield), a colorless oil.

[0113] Step 3: Add 5 mL of trifluoroacetic acid to a solution of compound 11d (2.7 g, 5.95 mmol) in DCM (30 mL) at room temperature. Stir the resulting solution at room temperature for 12 hours. Stir at room temperature for 2 hours until TLC (DCM / MeOH = 20:1) shows the reaction is complete. Quench with cold water (100 mL) at room temperature and extract with DCM (200 mL × 2). Wash the combined organic layers with brine (100 mL × 2), dry with Na2SO4, and concentrate under vacuum. Obtain colorless oily compound 11e (1.8 g, 94% yield) which can be used directly in the next step without purification.

[0114] Step 4: Add NaOH (1.1 g, 27.8 mmol, 5.00 equivalent) to a solution of 11e (1.8 g, 5.6 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 mixture by adding 50 mL (1 M HCl). Extract the resulting solution with 3 × 100 mL of LEtOAc, and combine the organic layers and dry them over anhydrous sodium sulfate. Concentrate under vacuum to obtain 11f (1.50 g, 87.3% yield) as a pale yellow liquid, which can be used directly in the next reaction without purification.

[0115] Step 5: Et3N (3.8 g, 37.61 mmol, 2.00 equivalent) was added to a solution of 11 g (3.00 g, 18.80 mmol, 1.00 equivalent) and 11 h (4.20 g, 22.57 mmol, 1.20 equivalent) in 50 mL of anhydrous THF at room temperature. The resulting solution was stirred at 0 °C for 1 h. The reactants were then quenched by adding 100 mL of water. The resulting solution was extracted with 3 × 100 mL of LEtOAc, 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 the target compound 11i (1.80 g, 92.2% yield) as a yellow solid.

[0116] Step Six: Add 15 mL of dioxane hydrochloride solution to a solution of compound 11i (5.3 g, 17.1 mmol, 1.00 equivalent) in MeOH (30 mL) 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 obtain a yellow solid compound 11j (3.7 g, yield 88.2%), which can be used directly in the next step without purification.

[0117] Step 7: At room temperature, DIPEA (1669.8 mg, 12.92 mmol, 1.00 equivalence) and EDCI (1610.28 mg, 8.40 mmol, 2.60 equivalence) and HOBt (567.50 mg, 4.20 mmol, 1.30 equivalence) were added to a solution of 11f (1.00 g, 3.23 mmol, 1.00 equivalence) and 11j (872.9 mg, 3.55 mmol, 1.10 equivalence) in 15 mL of anhydrous DMF. 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 of LEtOAc, 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 the target compound 11k (920.0 mg, 57% yield) as a white solid.

[0118] Step 8: At room temperature, iron powder (791 mg, 9.20 mmol, 5.00 equivalent) and ammonium chloride (492.1 mg, 9.20 mmol, 5.00 equivalent) were added to a solution of 11k (920.0 mg, 1.84 mmol, 1.00 equivalent) in anhydrous ethanol (15 mL) and water (5 mL). The reaction mixture was then stirred at 60 °C for 10 hours. The solution was filtered through diatomaceous earth and then 100 mL of water was added. The resulting solution was extracted with 3 × 100 mL of LEtOAc, 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 the target compound 11i (420 mg, yield 48.2%) as a white solid.

[0119] Step 9: Add DIPEA (166.4 mg, 1.29 mmol, 1.00 equivalence) and EDCI (214.3 mg, 1.12 mmol, 2.00 equivalence) and HOBt (75.46 mg, 0.56 mmol, 1.30 equivalence) to a solution of 11i (200.0 mg, 0.43 mmol, 1.00 equivalence) and 11m (88.9 mg, 0.51 mmol, 1.20 equivalence) in 15 mL of anhydrous DMF at 0 °C. Stir the reaction mixture at room temperature for 9 hours. After the reaction is complete as detected by TLC, add 50 mL of water. Extract the resulting solution with 3 × 50 mL of LEtOAc and combine the organic layers. 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 11n (140.0 mg, yield 52.3%) for the next step of the reaction.

[0120] Step 10: Compound 11n (140.0 mg, 0.22 mmol) was placed in a 25 mL round-bottom flask, and DCM solution (2.5 mL) and MeOH solution (5 mL) were added. Under ice bath conditions, NH2OH aqueous solution (50%, 221.3 mg, 3.35 mmol, 15.0 equivalent) and NaOH (134.2 mg, 3.35 mmol, 15.0 equivalent) were added, and the mixture was stirred for 1 h under ice bath conditions. While stirring, the pH was adjusted to 7–8 with HCl aqueous solution (1 M). After concentration, the residue was purified by C18 reversed-phase chromatography eluting with H2O / CH3CN (1 / 1) to obtain the title compound 11 (115 mg, yield 63.5%) as a white solid. 1 H NMR(500MHz,DMSO-d6)δ12.44(s,1H),10.33(s,1H),9.80(s,1H),8.66(s,1H),8.54(s,2 H),7.91(s,1H),6.26(dd,J=8.5,4.2Hz,1H),4.14(p,J=6.3Hz,1H),3.74–3.58(m,6H),3 .49(dd,J=9.4,5.3Hz,6H),2.58(t,J=6.5Hz,2H),2.27(t,J=7.4Hz,2H),1.95(t,J=7.3H z,2H),1.53(dp,J=29.9,7.5Hz,4H),1.26(tt,J=9.6,6.5Hz,2H),1.15(d,J=6.4Hz,3H). 13 C NMR (126MHz, DMSO-d6) δ171.2, 169.0, 168.8, 157.9, 150.1, 146.1, 128.6, 124.8, 124. 6,73.4,67.0,48.5,44.6,43.9,43.5,40.7,35.7,32.7,32.1,28.2,24.9,24.7,17.6.

[0121] Example 12: Preparation of Compound 12

[0122]

[0123] Following the synthetic method of compound 11, 12c (160 mg, yield 75.9%) was prepared by acid-ammonia condensation reaction in the first step, and compound 12 (70 mg, yield 43.8%) was prepared by amino-ester exchange and C18 reversed-phase chromatography in the second step. 1H NMR(500MHz,DMSO-d6)δ12.44(s,1H),10.33(s,1H),9.80(s,1H),8.54(s,3H),7.91(s ,1H),6.27(dq,J=8.1,4.0Hz,1H),4.15(p,J=6.5Hz,1H),3.76–3.58(m,6H),3.50(dt, J=9.6,5.2Hz,6H),2.58(t,J=6.5Hz,2H),2.27(t,J=7.4Hz,2H),1.93(t,J=7.4Hz,2H) ,1.52(dp,J=37.7,7.0Hz,4H), 1.26(qd,J=9.7,7.8,4.8Hz,4H), 1.15(d,J=6.5Hz,3H). 13 CNMR(126MHz,DMSO-d6)δ171.3,169.1,168.8,158.0,157.9,150.0,146.1,128.6,124.8, 73.4,68.0,48.5,44.6,43.9,43.5,40.7,35.8,32.7,32.2,28.4,28.4,25.0,24.9,17.6.

[0124] Example 13: Preparation of Compound 13

[0125]

[0126] Step 1: CS₂CO₃ (11.2 g, 34.5 mmol, 1.50 equivalent) was added to a solution of 13a (3.0 g, 23.00 mmol, 1.00 equivalent) and 13b (5.8 g, 27.58 mmol, 1.20 equivalent) in 50 mL of anhydrous DMF at room temperature. The resulting solution was stirred at 60 °C for 3 hours. The reactants were then quenched by adding 100 mL of water. The resulting solution was extracted with 3 × 100 mL of LEtOAc, 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 13c (2.80 g, yield 47.2%) as a grayish-white solid.

[0127] Step 2: At room temperature, K₂CO₃ (3.8 g, 37.61 mmol, 2.00 equivalent) was added to a solution of 13c (2.8 g, 10.82 mmol, 1.00 equivalent) in 50 mL of anhydrous NMP. The solution was stirred at 60 °C for 12 hours. The reactants were then quenched by adding 100 mL of water. The resulting solution was extracted with 3 × 100 mL of LEtOAc, 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 the target compound 13e (2.30 g, yield 52.2%) as a yellow solid.

[0128] Step 3: Add 10 mL of dioxane hydrochloride solution to a solution of compound 13e (2.3 g, 5.63 mmol, 1.00 equivalent) in MeOH (20 mL) at room temperature. Stir the resulting solution at room temperature for 12 hours. After TLC detection, the reaction was complete. Filter the solid and dry it under vacuum to obtain a yellow solid compound 13f (1.7 g, yield 88.3%), which can be used directly in the next step without purification.

[0129] Step 4: At room temperature, DIPEA (668.7 mg, 5.17 mmol, 4.00 equivalent) and EDCI (644.1 mg, 3.36 mmol, 2.60 equivalent) and HOBt (227.0 g, 1.68 mmol, 1.30 equivalent) were added to a solution of 13 f (533.8 mg, 1.55 mmol, 1.00 equivalent) and 13 g (400 mg, 1.29 mmol, 1.00 equivalent) in 15 mL of anhydrous DMF. The resulting solution was stirred at 25 °C for 10 h. The reactants were then quenched by adding 100 mL of water. The resulting solution was extracted with 3 × 100 mL of LEtOAc, 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 the target compound as a white solid at 13 h (270 mg, yield 31.3%).

[0130] Step 5: Compound 13h (270 mg, 0.45 mmol) was placed in a 25 mL round-bottom flask, and DCM solution (2.5 mL) and MeOH solution (5 mL) were added. Under ice bath conditions, NH2OH aqueous solution (50%, 445 mg, 6.75 mmol, 15.0 equivalent) and NaOH (270 mg, 6.75 mmol, 15.0 equivalent) were added, and the mixture was stirred for 1 h under ice bath conditions. While stirring, the pH was adjusted to 7-8 with HCl aqueous solution (1 M). After concentration, the residue was purified by C18 reversed-phase chromatography eluting with H2O / CH3CN (1 / 1) to obtain the title compound 13 (120 mg, yield 44.5%) as a white solid. 1 H NMR(500MHz,DMSO-d6)δ12.44(s,1H),10.33(s,1H),9.80(s,1H),8.66(s,1H),8.54(s,2 H),7.91(s,1H),6.26(dd,J=8.5,4.2Hz,1H),4.14(p,J=6.3Hz,1H),3.74–3.58(m,6H),3 .49(dd,J=9.4,5.3Hz,6H),2.58(t,J=6.5Hz,2H),2.27(t,J=7.4Hz,2H),1.95(t,J=7.3H z,2H),1.53(dp,J=29.9,7.5Hz,4H),1.26(tt,J=9.6,6.5Hz,2H),1.15(d,J=6.4Hz,3H). 13 C NMR (126MHz, DMSO-d6) δ169.0,168.8,157.9,157.1,146.1,145.7,145.6,128.6,125.9,1 23.8,73.4,69.2,67.0,48.5,44.7,44.3,43.9,40.7,32.8,32.2,28.5,25.0,24.9,17.6.

[0131] Example 14: Preparation of Compound 14

[0132]

[0133] Following the synthetic method of compound 17, the first step involved a substitution reaction to obtain 14c (3.1 g, yield 66.2%); the second step involved a substitution reaction to obtain a white solid compound 14e (3.4 g, yield 74.2%); the third step involved the removal of the tert-butyloxycarbonyl group from dioxane hydrochloride to obtain a crude white solid 14f (2.8 g, yield 95.9%); the fourth step involved an acid-ammonia condensation reaction to obtain a colorless solid compound 14h (140 mg, yield 22.1%); and the sixth step involved purification by amine exchange and C18 reversed-phase chromatography to obtain a light white solid compound 14 (70 mg, yield 50.2%). 1 H NMR(500MHz,DMSO-d6)δ12.45(s,1H),10.76(s,1H),9.08(s,1H),8.27(s,2H),7.90(s,1H),7 .58(d,J=7.9Hz,2H),7.51–7.44(m,2H),6.47(d,J=15.8Hz,1H),6.26(dt,J=8.6,4.2Hz,1H), 5.13(s,2H),4.14(dd,J=10.5,4.4Hz,1H),3.72–3.65(m,2H),3.63(q,J=5.8,4.6Hz,2H),3.5 8(dd,J=6.9,3.7Hz,2H),3.49(q,J=5.4Hz,6H),2.57(t,J=6.5Hz,2H),1.14(d,J=6.4Hz,3H). 13 C NMR (126MHz, DMSO-d6) δ168.9,157.9,157.2,146.2,146.1,145.2,137.9,134.6,130.7,128.6, 128.4,127.6,126.0,123.8,119.3,73.4,70.6,67.0,48.5,44.7,44.3,43.9,40.7,32.8,17.6.

[0134] Example 15: Preparation of Compound 15

[0135]

[0136] Following the synthetic method of compound 13, the first step involved a substitution reaction to obtain 15c (3.2 g, yield 61.2%); the second step involved a substitution reaction to obtain a white solid compound 15e (2.5 g, yield 51.2%); the third step involved the removal of the tert-butyloxycarbonyl group from dioxane hydrochloride to obtain a crude white solid 15f (1.8 g, yield 96.9%); the fourth step involved an acid-ammonia condensation reaction to obtain a colorless solid compound 15h (110 mg, yield 22.3%); and the fifth step involved purification by amine exchange and C18 reversed-phase chromatography to obtain a light white solid compound 15 (70 mg, yield 50.2%). 1 H NMR(500MHz,DMSO-d6)δ8.19(s,2H),7.90(s,1H),6.25(dt,J=8.1,4.0Hz,1H),4.13( p,J=6.4Hz,1H),3.94(t,J=6.5Hz,2H),3.73–3.64(m,2H),3.64–3.54(m,4H),3.49(q ,J=5.7Hz,6H),2.58(t,J=6.5Hz,2H),1.95(t,J=7.3Hz,2H),1.65(p,J=6.7Hz,2H),1 .50(p,J=7.5Hz,2H),1.37(p,J=7.1Hz,2H),1.32–1.23(m,2H),1.15(d,J=6.5Hz,3H). 13 C NMR(126MHz,DMSO-d6)δ169.1,168.9,157.9,157.1,146.1,145.8,145.6,128.6,126.0,123 .8,73.4,69.3,67.0,48.6,44.7,44.4,43.9,40.7,32.8,32.2,28.6,28.3,25.1,25.1,17.6.

[0137] Example 16: Preparation of Compound 16

[0138]

[0139] Following the synthetic method of compound 13, the first step involved a substitution reaction to obtain 16c (3.2 g, yield 59.4%); the second step involved a substitution reaction to obtain a white solid compound 16e (2.5 g, yield 51.1%); the third step involved the removal of the tert-butyloxycarbonyl group from dioxane hydrochloride to obtain a crude white solid 16f (1.8 g, yield 94.0%); the fourth step involved an acid-ammonia condensation reaction to obtain a colorless solid compound 16h (165 mg, yield 27.4%); and the fifth step involved purification by amine exchange and C18 reversed-phase chromatography to obtain a light white solid compound 16 (83 mg, yield 50.3%). 1H NMR(500MHz,DMSO-d6)δ12.45(s,1H),11.22(s,1H),9.06(s,1H),8.27(s,2H), 7.90(s,1H),7.82–7.69(m,2H),7.56–7.42(m,2H),6.26(dq,J=7.9,4.0Hz,1H), 5.16(s,2H),4.14(p,J=6.4Hz,1H),3.72–3.64(m,2H),3.60(ddd,J=24.6,6.7, 3.8Hz, 4H), 3.49 (q, J = 5.4Hz, 6H), 2.57 (t, J = 6.5Hz, 2H), 1.14 (d, J = 6.5Hz, 3H). 13 C NMR (126MHz, DMSO-d6) δ168.8,157.9,157.2,146.2,146.1,145.2,139.7,132.4, 128.6,127.6,127.1,73.4,70.4,67.0,48.5,44.6,44.2,43.8,40.7,32.7,17.6.

[0140] Example 17: Preparation of Compound 17

[0141]

[0142] Step 1: NaH (60% purity) (574.2 mg, 14.35 mmol, 1.50 equivalent) was added to a solution of 17a (2.4 g, 9.57 mmol, 1.00 equivalent) mmol in 50 mL of anhydrous DMF at 0 °C. After stirring the resulting solution at 0 °C for 1 hour, 17b (2.23 g, 11.48 mmol, 1.00 equivalent) was slowly added, and the reaction was stirred at 0 °C for 2 hours. The reactants were then quenched by adding 100 mL of water. The resulting solution was extracted with 3 × 100 mL of LEtOAc, 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 17c (2.4 g, 77.3% yield) as a colorless oily liquid.

[0143] Step 2: At room temperature, Na₂CO₃ (1.58 g, 14.90 mmol, 1.00 equivalent) and DCM (0.5 mL) were added to a solution of 17c (2.4 g, 7.45 mmol, 1.00 equivalent) and 17d (1.77 g, 11.18 mmol, 1.20 equivalent) in 50 mL of anhydrous DMF and 10 mL of H₂O. Oxygen was then purged with nitrogen for 15 min, followed by the addition of (dppf)PdCl₂ (272.7 mg, 0.37 mmol, 0.05 equivalent), and the solution was stirred at 60 °C for 12 h. The reactants were then quenched with 100 mL of water. The resulting solution was extracted with 3 × 100 mL of LEtOAc, 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 the target compound 17e (950 mg, yield 41.2%) as a yellow solid.

[0144] Step 3: At room temperature, K₂CO₃ (851.3 mg, 6.17 mmol, 2.00 equivalent) was added to a solution of 17e (950 mg, 3.08 mmol, 1.00 equivalent) and 14d (861.2 mg, 4.63 mmol, 1.20 equivalent) in 10 mL of anhydrous NMP. The resulting solution was stirred at 100 °C for 12 hours. The reactants were then quenched by adding 100 mL of water. The resulting solution was extracted with 3 × 100 mL of LEtOAc, 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 17f (980 mg, yield 70.2%) as a yellow solid, representing the target compound.

[0145] Step 4: Add 5 mL of dioxane hydrochloride solution to a solution of compound 17f (980 mg, 7.49 mmol, 1.00 equivalent) in MeOH (10 mL) at room temperature. Stir the resulting solution at room temperature for 10 hours. After TLC detection, the reaction was complete. Filter the solid and dry it under vacuum to give 17 g (805 mg, yield 98.3%) of yellow solid compound, which can be used directly in the next step without purification.

[0146] Step 4: At room temperature, DIPEA (668.7 mg, 5.17 mmol, 1.10 equivalents), EDCI (643.0 mg, 3.35 mmol, 2.60 equivalents), and HOBt (227.0 g, 1.68 mmol, 1.30 equivalents) were added to a solution of 17 g (560.4 mg, 1.42 mmol, 1.10 equivalents) and 17 h (400 mg, 1.29 mmol, 1.00 equivalents) in 15 mL of anhydrous DMF. The resulting solution was stirred at 25 °C for 10 h. The reactants were then quenched by adding 100 mL of water. The resulting solution was extracted with 3 × 100 mL of LEtOAc, 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 the target compound as a white solid (17 i, 223.0 mg, yield 31.3%).

[0147] Step 5: Compound 17i (220 mg, 0.34 mmol) was placed in a 25 mL round-bottom flask, and DCM solution (2.5 mL) and MeOH solution (5 mL) were added. Under ice bath conditions, NH2OH aqueous solution (50%, 445 mg, 338.3 mg, 15.0 equivalent) and NaOH (205.0 mg, 5.13 mmol, 15.0 equivalent) were added, and the mixture was stirred for 1 h under ice bath conditions. While stirring, the pH was adjusted to 7-8 with HCl aqueous solution (1 M). After concentration, the residue was purified by C18 reversed-phase chromatography eluting with H2O / CH3CN (1 / 1) to obtain the title compound 17 (110.2 mg, yield 54.4%) as a white solid. 1 H NMR(500MHz,DMSO-d6)δ12.45(s,1H),10.34(s,1H),8.62(s,2H),8.12(s,1H),7.91(s,1H),7.83(d,J =0.8Hz,1H),6.27(dq,J=8.2,4.0Hz,1H),4.15(p,J=6.0Hz,1H),4.08(t,J=7.0Hz,2H),3.74(t,J=5.2H z,2H),3.68(q,J=6.7Hz,4H),3.52(q,J=4.9Hz,4H),3.49(d,J=5.5Hz,2H),2.59(t,J=6.5Hz,2H),1.9 4(t,J=7.4Hz,2H),1.84–1.73(m,2H),1.52(p,J=7.5Hz,2H),1.26–1.19(m,2H),1.15(d,J=6.5Hz,3H). 13C NMR (126MHz, DMSO-d6) δ168.9,168.8,159.8,157.9,154.4,146.1,135.3,128.6,126.0 ,116.0,115.8,73.4,67.03,48.5,44.7,43.7,43.3,40.7,32.8,32.1,29.6,25.6,24.6.

[0148] Example 18: Preparation of Compound 18

[0149]

[0150] Following the synthetic method of compound 17, the following steps were performed: 18c (2.1 g, 85.2% yield) was obtained via a substitution reaction in the first step; 18e (970.2 mg, 48.2% yield) was obtained via a Suzuki coupling reaction in the second step; 18f (1050.1 mg, 74.2% yield) was obtained via a substitution reaction in the third step; 18f (560 mg, 75.9% yield) was obtained via the removal of the tert-butyloxycarbonyl group from dioxane hydrochloride in the fourth step; 18i (140 mg, 33.1% yield) was obtained via an acid-ammonia condensation reaction in the fifth step; and 18i (70 mg, 50.2% yield) was obtained via an amino ester exchange reaction and purification by C18 reversed-phase chromatography in the sixth step. 1 H NMR(500MHz,DMSO-d6)δ12.44(s,1H),10.34(s,1H),8.62(s,2H),8.18–8.06(m,1H),7.91(s,1H),7.83(d, J=0.7Hz,1H),6.27(dq,J=8.1,4.0Hz,1H),4.19–4.11(m,1H),4.08(t,J=7.1Hz,2H),3.73(q,J=3.7,2.3Hz, 2H),3.68(q,J=6.9Hz,4H),3.52(q,J=4.8Hz,4H),3.49(d,J=5.6Hz,2H),2.59(t,J=6.5Hz,2H),1.93(t,J=7 .4Hz,2H),1.77(p,J=6.7Hz,2H),1.47(p,J=7.2Hz,2H),1.24(dq,J=8.3,4.3Hz,4H),1.15(d,J=6.5Hz,3H). 13C NMR (126MHz, DMSO-d6) δ168.9,159.8,157.9,154.4,146.1,135.3,128.6,126.0,116.0,115 .8,73.4,67.0,51.4,48.5,44.7,43.7,43.3,40.7,32.8,32.2,29.7,28.1,25.7,25.0,17.6.

[0151] Example 19: Preparation of Compound 19

[0152]

[0153] Step 1: Potassium carbonate (1.23 g, 11.6 mmol, 2.00 equivalent) was added to a solution of 19a (1.00 g, 5.81 mmol, 1.00 equivalent) and 19b (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 reaction mixture was then quenched by adding 100 mL of water. The resulting solution was extracted with 3 × 100 mL of LEtOAc, 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 19c (1.53 g, yield 81.6%) as a white solid, representing the target compound.

[0154] Step 2: Add 5 mL of dioxane hydrochloride solution to a solution of compound 19c (1.53 g, 4.75 mmol, 1.00 equivalent) in MeOH (5 mL) at room temperature. Stir the resulting solution at room temperature for 12 hours. After TLC detection, the reaction was complete. Filter the solid and dry it under vacuum to give a white solid compound 19d (1.03 mg, yield 97.8%), which can be used directly in the next step without purification.

[0155] Step 3: DIPEA (668.7 mg, 5.17 mmol, 1.00 equivalent) and EDCI (644.1 mg, 3.36 mmol, 2.60 equivalent) and HOBt (227.0 g, 1.68 mmol, 1.30 equivalent) were added to a solution of 19d (286.6 mg, 1.29 mmol, 1.00 equivalent) and 19e (400 mg, 1.29 mmol, 1.00 equivalent) in anhydrous DMF (5 mL) 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 of LEtOAc, 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 19f (190.0 mg, yield 28.7%) as a white solid.

[0156] Step 4: Compound 19f (190 mg, 0.37 mmol) was placed in a 25 mL round-bottom flask, and DCM solution (2.5 mL) and MeOH solution (5 mL) were added. Under ice bath conditions, NH2OH aqueous solution (50%, 366.5 mg, 5.55 mmol, 15.0 equivalent) and NaOH (222.1 mg, 5.55 mmol, 15.0 equivalent) were added, and the mixture was stirred for 1 h under ice bath conditions. While stirring, the pH was adjusted to 7–8 with HCl aqueous solution (1 M). After concentration, the residue was purified by C18 reversed-phase chromatography eluting with H2O / CH3CN (1 / 1) to obtain the title compound 13 (90.0 mg, yield 47.3%) as a white solid. 1 H NMR (500MHz, DMSO-d6) δ8.56(d,J=1.3Hz,1H),8.22(d,J=1.4Hz,1H),7.91(s,1H),6.27(dq,J=8.1,4.0Hz,1H),4.15(p,J=6.3Hz,1H ),3.75–3.62(m,6H),3.57(dd,J=6.7,3.9Hz,4H),3.48(d,J=5.6Hz,2H),2.59(t,J=6.4Hz,2H),2.54(s,1H),1.15(d,J=6.5Hz,3H). 13 C NMR(126MHz,DMSO-d6)δ170.0,161.3,157.9,154.8,146.1,141.1,132.9,130 .0,128.6,125.9,123.8,73.5,66.9,48.5,44.3,43.8,43.5,40.4,32.7,17.6.

[0157] Example 20: Preparation of Compound 20

[0158]

[0159] Following the synthetic method of compound 19, the first step involved a substitution reaction to obtain 20c (810 mg, yield 82.3%); the second step involved the removal of the tert-butyloxycarbonyl group from dioxane hydrochloride to obtain a crude white solid 20d (460 mg, yield 78.4%); the third step involved an acid-ammonia condensation reaction to obtain a colorless solid compound 20f (160 mg, yield 37.9%); and the fifth step involved purification by amine exchange and C18 reversed-phase chromatography to obtain a pale yellow solid compound 20 (77 mg, yield 48.1%). 1H NMR(500MHz,DMSO-d6)δ8.66(d,J=8.2Hz,2H),7.95–7.82(m,1H),6.26(tt, J=8.7,4.2Hz,1H),4.13(tp,J=13.0,6.6,5.9Hz,1H),3.72–3.56(m,5H),3.5 5–3.49(m,2H),3.49–3.35(m,12H),3.36–3.19(m,2H),2.45(dt,J=13.1,6. 4Hz,2H),1.92(tt,J=7.0,3.3Hz,3H),1.87–1.76(m,1H),1.18–1.10(m,3H). 13 C NMR (126MHz, DMSO-d6) δ168.90,168.88,168.7,162.8,160.20,160.19,160. 0,157.0,146.22,146.18,128.73,128.69,126.0,123.8,114.3,73.6,73.5, 66.93,66.89,66.8,55.2,54.9,53.9,48.64,48.59,48.4,46.6,45.9,45.8,45.3,44.5,34.5,34.4,34.0,33.82,33.79,33.7,33.2,17.60,17.58,17.55.

[0160] Example 21: Preparation of compound 21

[0161]

[0162] Following the synthetic method of compound 19, the first step involved a substitution reaction to obtain 21c (3.5 g, yield 75.9%). The second step involved the removal of the tert-butyloxycarbonyl group from dioxane hydrochloride to obtain a crude white solid 21d (2.2 g, yield 88.2%). The third step involved an acid-ammonia condensation reaction to obtain a colorless solid compound 21f (320 mg, yield 37.3%). The fifth step involved purification by amine exchange and C18 reversed-phase chromatography to obtain a light white solid compound 18 (142 mg, yield 44.4%). 1H NMR(500MHz,DMSO-d6)δ8.65(s,2H),7.90(d,J=4.1Hz,1H),6.26(dt,J=8.9,4 .5Hz,1H),4.12(h,J=6.1Hz,1H),3.79–3.72(m,2H),3.71–3.50(m,5H),3.45– 3.42(m,2H),3.39(dt,J=10.7,4.0Hz,3H),3.22(dd,J=12.3,4.7Hz,1H),3.07 –2.89(m,2H),2.54(s,1H),2.45(td,J=6.4,1.9Hz,2H),1.13(d,J=6.5Hz,3H). 13 C NMR(126MHz,DMSO-d6)δ168.9,160.14,157.95,156.73,146.13,128.68,128.63,125,90,125.8 0,115.07,73.46,73.38,66.9,66.8,50.5,50.3,49.9,49.2,48.5,41.6,40.4,34.3,17.6,17.5.

[0163] Example 9: Test of the inhibitory activity of the compound against PARP7 enzyme

[0164] 1. Experimental materials and instruments

[0165] 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)

[0166] Instrument: SpectraMax Paradigm multi-functional microplate detector;

[0167] 2. Experimental Methods

[0168] 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 nmol of the compound from the source plate to a 96-well intermediate plate using 19.95 μL of detection buffer. Incubate at 1000 rpm for 1 min. Transfer 5 μL of LDMSO / 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.

[0169] 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.

[0170] Data processing:

[0171] Use equation (1) to fit the data in Excel to obtain the suppression value.

[0172] Equation (1): Inh%=(Max-Signal) / (Max-Min)*100.

[0173] Use equation (2) to fit the data in XL-Fit to obtain IC. 50 value.

[0174] Equation (2): Y = Bottom + (Top - Bottom) / (1 + (IC) 50 / X)*HillSlope)

[0175] Where Y is the inhibition percentage and X is the compound concentration.

[0176] 3. Experimental Results

[0177] The inhibitory effects of some target compounds on PARP7 enzyme were determined using the experimental methods described above, and the results are shown in Table 1.

[0178] Table 1. Half-maximal inhibitory concentrations of the target compounds against PARP7 enzyme.

[0179]

[0180] Note: N / D – not measured

[0181] Conclusion: The representative compound of this invention can effectively inhibit PARP7 enzyme activity.

[0182] Example 10: Test of the inhibitory activity of the compound against HDAC1 and HDAC6 enzymes

[0183] 1. Experimental materials and instruments

[0184] Experimental materials: HDAC1 (BPS, Cat. No. 50051); Tris buffer; HDAC6 (BPS, Cat. No. 50006); 384-well plate (Perkin Elmer, Cat. No. 6007279); DMSO (Coolaber);

[0185] Instrument: SpectraMax Paradigm multi-functional microplate detector;

[0186] 2. Experimental Methods

[0187] Prepare 1x assay buffer: Prepare 1x assay buffer (modified Tris buffer); Serial dilution: Transfer the compound to the assay plate in 100% DMSO via Echo, with DMSO content not exceeding 1%; Prepare enzyme solution: Prepare enzyme solution in 1x assay buffer; Prepare substrate solution: Add trypsin and acetylated peptide substrate to 1x assay buffer to prepare substrate solution; Transfer 15 μL of enzyme solution to the assay plate or transfer 15 μL of 1x assay buffer for low control; Incubate at room temperature for 15 minutes; Add 10 μL of substrate solution to each well to start the reaction; Read the plate on Paradigm with excitation wavelength of 355 nm and emission wavelength of 460 nm; Curve fitting: Fit the data in Excel using Equation (1) to obtain the inhibition value Equation (1): Inh% = (Max-Signal) / (Max-Min)*100 Fit the data in XL-Fit using Equation (2) to obtain IC50. 50 Value equation (2): Y = bottom + (top - bottom) / (1 + (IC) 50 / X)*HillSlope). Where Y is the inhibition percentage and X is the compound concentration.

[0188] 3. Experimental Results

[0189] The inhibitory effects of some target compounds on HDAC1 and HDAC6 enzymes were determined using the experimental methods described above. The results are shown in Table 2.

[0190] Table 2. Half-maximal inhibitory concentrations (IC50) of the target compounds against HDAC1 and HDAC6 enzymes.

[0191] Compound No. HDAC1 IC 50 (nM) HDAC6 IC 50 (nM) 1 49 6.7 2 328 72 3 N / D N / D 4 31 71 5 134 36 6 63 8.7 7 99 39 8 929 342 9 >1250 >1250 10 48 7.2 11 62.2 6.9 12 96.5 30.5 13 169.2 22.4 14 35.2 6.4 15 34.1 11.7 16 375.0 9.4 17 2.2 5.4 18 11.0 8.5 19 954.0 819.0 20 72.0 166.0 21 80.2 214.9 SAHA 15 6 RBN-2397 >10000 >10000

[0192] Note: N / D – not determined.

[0193] Conclusion: The representative compounds of this invention can effectively inhibit the activity of HDAC1 and HDAC6 enzymes.

[0194] Example 24: Test of the inhibitory activity of some compounds against tumor cells

[0195] 1. Cell lines and cell culture.

[0196] Human lung adenocarcinoma NCI-H1373 cells, NCI-H2066 cells, human myeloid monocytic leukemia cells MV-4-11, and human histiocytic lymphoma cells U937 were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. NCI-H1373 and CT26 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.

[0197] 2. Cell viability assay.

[0198] 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 using a CellTiter-Glo luminescence activity assay (Promega) 6 days after drug treatment. Cell viability was measured immediately after drug administration (day 0) and after 6 days of incubation using CellTiterGlo (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 / Prism8 software, and dose-response curves were fitted.

[0199] Table 3. Half-maximal inhibitory concentrations of the target compounds against tumor cells.

[0200]

[0201] Conclusion: The representative compounds of this invention can effectively inhibit various tumors, including hematologic malignancies and solid tumors.

Claims

1. A pyridazinone derivative with dual PARP7 / HDAC inhibitory activity, characterized in that, The structure of the derivative is shown in formula (I): ； in: For chemical bonds or ;in" "express The site where pyridazinone is attached; "express With L 1 The site of connection; L 1 It is selected from one of the following structural segments: , , , , , , ; in" " indicates L 1 and The site of connection; " indicates L 1 With L 2 The site of connection; Each of the following structural segments is selected independently: ； Where n is any natural number from 4 to 6, " indicates L 1 With L 2 The site of connection.

2. The pyridazinone derivative with dual PARP7 / HDAC inhibitory activity according to claim 1, characterized in that, The chemical structural formula of the pyridazinone derivative with dual PARP7 / HDAC inhibitory activity is any one of the following formulas 1 to 30: 。 3. The use of the pyridazinone derivative with PARP7 / HDAC dual-target inhibitory activity as described in any one of claims 1-2 in the preparation of antitumor drugs.

4. The application according to claim 3, characterized in that... The tumors include solid tumors and hematologic tumors.

5. An antitumor drug comprising a safe and effective amount of a pyridazinone derivative as described in any one of claims 1-2, having dual PARP7 / HDAC inhibitory activity.

6. The antitumor drug according to claim 5, characterized in that... It also includes pharmacologically acceptable salts and pharmacologically acceptable excipients or carriers.

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