Pyridine amide compound as PARP1 inhibitor, intermediate and preparation method therefor

Abstract

Disclosed are a pyridine amide compound as represented by formula I as a PARP1 inhibitor, an intermediate and a preparation method therefor. The preparation method for the compound as represented by formula (I) solves the problems of difficult access to raw materials of the pyridine amide compound as the PARP1 inhibitor, high technical requirements for the method, etc.

Description

A PARP1 inhibitor pyridine amide compound, an intermediate and its preparation method

[0001] This application claims priority to Chinese patent application 2024107943645, filed on June 19, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of drug synthesis. Specifically, this invention relates to a PARP1 inhibitor pyridine amide compound, an intermediate, and a method for preparing the same. Background Technology

[0003] ADP-ribosylation is an enzymatic reaction that breaks down the substrate nicotinamide adenine dinucleotide (NAD+) into nicotinamide and an ADP ribose group, which is then covalently linked to a receptor protein. ADP ribosylation is a common and reversible post-translational modification of proteins, playing a crucial role in a range of biological processes, including DNA damage repair, cell proliferation and differentiation, metabolism, and stress response.

[0004] Poly(ADP-ribose) polymerase (PARP) is a family of proteins that catalyze the ribosylation of ADP. PARP1 is one of the most widely studied and important members of this family. It is highly expressed in cells and responds rapidly, quickly catalyzing and modifying DNA repair factors and interacting with them to participate in various DNA repair processes. In normal cells, single-strand breaks in DNA can be repaired through base excision. PARP1 uses NAD+ as a substrate, binds to the damage site through its zinc finger domain to change its conformation, and catalyzes the transfer of ADP-ribose groups, ultimately completing single-strand repair. In cancer cells with double-strand breaks (DSB), DNA breaks are mainly repaired through homologous recombination (HR). BRCA1 and BRCA2 are key proteins mediating HR. In cancer cells with BRCA1 / 2 mutations, PARP1 function is also inhibited, causing obstruction of the main DNA repair pathway and ultimately leading to cell death. This is the synthetic lethal effect that has been well-validated in clinical practice. Therefore, PARP1 has become a highly sought-after target for cancer therapy.

[0005] In recent years, four small-molecule PARP inhibitors (PARPi) have been approved by the US FDA for cancer treatment: olaparib, niraparib, rucaparib, and telazoparib. These drugs have demonstrated good clinical efficacy in treating patients with BRCA1 / 2-mutated ovarian and / or breast cancer, and have also shown very promising results in other BRCA-mutated cancers, including prostate and pancreatic cancer.

[0006] Recent studies have suggested that the mechanism of action of PARP1 can be attributed to two distinct but interconnected mechanisms. First, by inhibiting the catalytic activity of PARP1, these PARP1 inhibitors block the synthesis of the PAR chain, thereby inhibiting poly(ADP-ribosylation) and blocking PARP1-mediated DNA damage repair signaling. Second, PARP inhibitors may also induce PARP1 trapping, leading to DNA double-strand breaks and ultimately killing cancer cells. When PARP1 itself undergoes PARylation, it detaches from the DNA damage site due to steric hindrance and charge repulsion of the PAR chain. However, treatment with PARP inhibitors blocks PARP1 self-modification, causing PARP1 to become trapped at the DNA damage site. The long-term presence of the PARP-DNA complex occupies the DNA damage site and interferes with subsequent DNA replication, leading to replication fork arrest and subsequent double-strand DNA damage, ultimately causing cell death. However, PARP1 trapping occurs concurrently with the inhibition of PARP1 catalytic activity. Therefore, the inhibition of PARP1 catalytic activity and PARP1 capture caused by PARP inhibitors are functionally completely different but intrinsically related.

[0007] Based on the synthetic lethal mechanism, cell lines carrying BRCA1 / 2 mutations or homologous recombination deficiency (HRD) are highly sensitive to PARP inhibitors, providing a very broad therapeutic safety window. However, because some normal cells in the body, such as myeloid-derived cells, are often in a state of rapid proliferation, DNA damage is inevitable. Furthermore, many enrolled patients have germline mutations, leading to varying degrees of hematologic toxicity when using approved non-selective PARP1 / 2 inhibitors, including anemia, neutropenia, and thrombocytopenia. Grade III or IV hematologic toxicity often results in dose reduction, discontinuation, or interruption of treatment, severely impacting the therapeutic efficacy of PARP inhibitors.

[0008] On the other hand, there is a potential basis for synergistic effects when chemotherapy drugs and PARP inhibitors are used in combination. However, because chemotherapy drugs inhibit rapidly proliferating cells while also having strong hematologic toxicity, the combined use of the two can lead to an additive effect of hematologic toxicity, which greatly limits the combined use of PARP inhibitors.

[0009] Studies have shown that anemia caused by PARP inhibitors may be mainly due to the inhibition of PARP2 (Farrés J, et al. Cell Death Differ. 2015 Jul; 22(7):1144-57.). Currently marketed PARP inhibitors all have inhibitory activity on both PARP1 and PARP2. If highly selective PARP1 inhibitors can be developed to reduce the inhibition of PARP2, it may be possible to avoid the anemia toxicity caused by existing PARP1 / 2 inhibitors. WO2021013735A1 discloses a series of selective PARP1 inhibitors, such as AZD5305.

[0010] Therefore, developing more highly selective PARP1 inhibitors has significant clinical application value, and there is a demand for such inhibitors in this field. Summary of the Invention

[0011] The purpose of this invention is to provide a pyridine amide compound, its intermediate, and a method for synthesizing the same, in order to solve the problems of scarce raw materials and high technical requirements for PARP1 inhibitor pyridine amide compounds, while also meeting the needs of clinical sample production.

[0012] The present invention solves the above-mentioned technical problems through the following solution.

[0013] This invention provides compounds as shown in Formula I.

[0014] This invention provides a method for preparing the compound shown in Formula I, comprising the following steps: reacting compound 12 with hydrochloric acid to form a salt, thereby obtaining the compound shown in Formula I.

[0015] In some embodiments, during the salt-forming reaction, the hydrochloric acid is added to the system in the form of a dimethyl sulfoxide solution of hydrochloric acid.

[0016] In some embodiments, in the salt-forming reaction, the molar ratio of compound 12 to hydrochloric acid is 1:(1.0-1.5), preferably 1:(1.0-1.05).

[0017] In some embodiments, the salt formation reaction is carried out in an organic solvent, which is one or more of DMSO, DMF and NMP, preferably DMSO.

[0018] In some embodiments, during the salt formation reaction, the mass-to-volume ratio of compound 12 to the organic solvent is (0.05-0.3) g / mL, preferably (0.1-0.2) g / mL, for example 0.14 g / mL.

[0019] In some embodiments, the salt-forming reaction is carried out at a temperature of 10-30°C, preferably 20-40°C.

[0020] In some embodiments, the progress of the salt formation reaction can be detected using conventional monitoring methods in the art (e.g., TLC, HPLC, or NMR), generally with the disappearance of compound 12 or the cessation of the reaction as the reaction endpoint. For example, the reaction time is 0.5-24 hours, preferably 0.5-1 hour.

[0021] In some embodiments, the salt-forming reaction further includes post-processing steps, such as filtration, washing, and drying.

[0022] In some embodiments, the salt-forming reaction specifically includes the following steps: dissolving compound 12 in an organic solvent, adding hydrochloric acid solution (e.g., a dimethyl sulfoxide solution of hydrochloric acid) dropwise at 10-30°C, and after the reaction is complete, raising the temperature to 75-85°C, adding a poor solvent dropwise, cooling to allow crystallization, and obtaining the compound as shown in Formula I after post-treatment filtration, washing, and drying. Preferably, the poor solvent is isopropyl acetate, acetone, or methyl tert-butyl ether, such as isopropyl acetate; the washing in the post-treatment is isopropyl acetate washing.

[0023] In some embodiments, during the salt-forming reaction, the mass-to-volume ratio of compound 12 to the undesirable solvent is (0.03-0.2) g / mL, preferably (0.05-0.1) g / mL, for example 0.077 g / mL.

[0024] In some embodiments, the method for preparing the compound as shown in Formula I further includes a method for preparing compound 12, which specifically includes the following steps: compound 6 undergoes a substitution reaction with compound 11 to obtain compound 12.

[0025] In some embodiments, compound 6 and compound 11 undergo a substitution reaction in the presence of a base and an organic solvent to yield compound 12.

[0026] In some embodiments, the molar ratio of compound 6 to compound 11 in the substitution reaction is 1:(0.9-1.1), preferably 1:(1.05-1.0).

[0027] In some embodiments, the base in the substitution reaction is N,N-diisopropylethylamine, triethylamine, or pyridine, preferably N,N-diisopropylethylamine.

[0028] In some embodiments, in the substitution reaction, the mass ratio of compound 6 to the base is 1:(1-2.5), preferably 1:(1.5-1.9), for example 1:1.88.

[0029] In some embodiments, the organic solvent in the substitution reaction is one or more of DMSO, DMF, or NMP, preferably DMSO.

[0030] In some embodiments, during the substitution reaction, the mass-to-volume ratio of compound 6 to the organic solvent is (0.1-0.5) g / mL, preferably (0.2-0.25) g / mL, for example 0.23 g / mL.

[0031] In some embodiments, the substitution reaction is carried out at a temperature of 60-80°C, preferably 65-75°C.

[0032] In some embodiments, the progress of the substitution reaction can be detected using conventional monitoring methods in the art (e.g., TLC, HPLC, or NMR), generally with the reaction endpoint defined as when HPLC shows compound 11 < 2.00%. For example, the reaction time is 2-5 hours, preferably 2 hours.

[0033] In some embodiments, the substitution reaction further includes post-processing steps, such as crystallization, filtration, stirring, washing, filtration, drying, and recrystallization.

[0034] In some embodiments, the substitution reaction specifically includes the following steps: stirring compound 6, compound 11, a base, and an organic solvent, heating to 65-75°C, adding a crystallization solvent dropwise to induce crystallization at a controlled temperature of 60-75°C, cooling to 20-30°C, stirring, and then filtering, washing, recrystallizing, washing again, and drying to obtain compound 12. Preferably, the crystallization solvent is ethanol; the washing in the post-treatment is anhydrous ethanol; the recrystallization solvent in the post-treatment is DMSO; the recrystallization temperature is 20-30°C; and the washing after recrystallization in the post-treatment is with isopropyl acetate.

[0035] In some embodiments, the method for preparing compound 12 further includes a method for preparing compound 6, specifically including the following steps: compound 5 undergoes a deprotection reaction to obtain compound 6.

[0036] In some embodiments, compound 5 undergoes a deprotection reaction in the presence of an acid reagent and an organic solvent to yield compound 6.

[0037] In some embodiments, the acid reagent in the deprotection reaction is an ethyl hydrogen chloride solution.

[0038] In some embodiments, in the deprotection reaction, the molar ratio of compound 5 to hydrogen chloride is 1:(3-5), preferably 1:4.0.

[0039] In some embodiments, the concentration of the ethyl hydrochloride solution is 4 mol / L during the deprotection reaction.

[0040] In some embodiments, the organic solvent in the deprotection reaction is one or more of dichloromethane, chloroform, or 1,2-dichloroethane, preferably dichloromethane.

[0041] In some embodiments, during the deprotection reaction, the mass-to-volume ratio of compound 5 to the organic solvent is (0.3-1.5) g / mL, preferably (0.5-1) g / mL, for example 0.77 g / mL.

[0042] In some embodiments, the deprotection reaction is carried out at a temperature of 10-30°C, preferably 10-20°C.

[0043] In some embodiments, the progress of the deprotection reaction can be detected using conventional monitoring methods in the art (e.g., TLC, HPLC, or NMR), generally with the reaction endpoint defined as when HPLC shows compound 5 < 1.00%. For example, the reaction time is 0.5-2 hours, preferably 0.5 hours.

[0044] In some embodiments, the deprotection reaction further includes post-processing steps, such as filtration, washing, and drying.

[0045] In some embodiments, the deprotection reaction specifically includes the following steps: dissolving compound 5 in an organic solvent, adding an acid reagent (e.g., ethyl hydrogen chloride solution) dropwise at 10-20°C, raising the temperature to 15-20°C after the addition is complete, and filtering, washing, and drying to obtain compound 6 after the reaction is complete. Preferably, the washing is with dichloromethane.

[0046] In some embodiments, the preparation method of compound 6 further includes a method for preparing compound 5, which specifically includes the following steps: compound 4 undergoes an amide condensation reaction with deuterated methylamine or its salt to obtain compound 5.

[0047] In some embodiments, compound 4 is reacted with the deuterated methylamine or its salt in the presence of a base, a condensing agent, and an organic solvent to undergo an amide condensation reaction to obtain compound 5.

[0048] In some embodiments, the deuterated methylamine salt in the amide condensation reaction is deuterated methylamine hydrochloride.

[0049] In some embodiments, in the amide condensation reaction, the molar ratio of compound 4 to the deuterated methylamine or its salt is 1:(1.0-1.5), preferably 1:1.2.

[0050] In some embodiments, the base in the amide condensation reaction is DIEA.

[0051] In some embodiments, in the amide condensation reaction, the mass ratio of compound 4 to the base is (0.5-1.5):1, preferably (0.6-1):1, for example 0.83:1.

[0052] In some embodiments, the condensation reagent in the amide condensation reaction is HATU or HBTU, preferably HATU.

[0053] In some embodiments, the molar ratio of compound 4 to the condensing agent in the amide condensation reaction is 1:1.2.

[0054] In some embodiments, the organic solvent in the amide condensation reaction is one or more of DMF, DMA, or DMSO, preferably DMF.

[0055] In some embodiments, in the amide condensation reaction, the mass ratio of compound 4 to the organic solvent is (5-15):1, preferably (8-10):1, for example 9.5:1.

[0056] In some embodiments, the reaction temperature in the amide condensation reaction is 10-30°C, preferably 20-30°C.

[0057] In some embodiments, the progress of the amide condensation reaction can be detected using conventional monitoring methods in the art (e.g., TLC, HPLC, or NMR), generally with the reaction endpoint defined as when HPLC shows compound 4 < 1.00%. For example, the reaction time is 0.5-2 hours, preferably 0.5 hours.

[0058] In some embodiments, the amide condensation reaction further includes post-processing steps, such as extraction, washing, and vacuum concentration.

[0059] In some embodiments, the amide condensation reaction specifically includes the following steps: Compound 4, a base (e.g., DIEA), deuterated methylamine hydrochloride, and an organic solvent (e.g., DMF) are stirred; a condensation reagent (e.g., HATU) is slowly added in batches, maintaining the temperature at 20-30°C; after the addition is complete, the reaction is maintained at 20-30°C; after the reaction is complete, post-treatment extraction, washing, and vacuum concentration are performed to obtain compound 5. Preferably, the post-treatment involves ethyl acetate extraction, washing with purified water, washing with saturated sodium bicarbonate solution, washing with saturated sodium chloride solution, and vacuum concentration to obtain compound 5.

[0060] In some embodiments, the method for preparing compound 5 further includes a method for preparing compound 4, which specifically includes the following steps: compound 3 undergoes a hydrolysis reaction to obtain compound 4.

[0061] In some embodiments, compound 3 is hydrolyzed in the presence of a base and an organic solvent to yield compound 4.

[0062] In some embodiments, the alkali in the hydrolysis reaction is sodium hydroxide, potassium hydroxide, or lithium hydroxide, preferably lithium hydroxide; in this invention, the alkali is added to the system in the form of an aqueous solution.

[0063] In some embodiments, the molar ratio of compound 3 to the base in the hydrolysis reaction is 1:(1.5-2.5), preferably 1:1.8.

[0064] In some embodiments, the mass-to-volume ratio of the alkali to the water in the hydrolysis reaction is (0.03-0.15) g / mL, preferably (0.05-0.1) g / mL, for example 0.07 g / mL.

[0065] In some embodiments, the organic solvent in the hydrolysis reaction is one or more of dioxane, THF, or acetonitrile, preferably THF.

[0066] In some embodiments, during the hydrolysis reaction, the mass-to-volume ratio of compound 3 to the organic solvent is (0.5-2) g / mL, preferably (0.8-1.5) g / mL, for example 1 g / mL.

[0067] In some embodiments, the hydrolysis reaction is carried out at a temperature of 10-35°C, preferably 20-30°C.

[0068] In some embodiments, the progress of the hydrolysis reaction can be detected using conventional monitoring methods in the art (e.g., TLC, HPLC, or NMR), generally with the reaction endpoint defined as when HPLC shows compound 3 < 1.00%. For example, the reaction time is 0.5-2 hours, preferably 0.5-1 hour.

[0069] In some embodiments, the hydrolysis reaction further includes post-processing steps, such as acidification, filtration, washing, drying, dissolution, washing again, and vacuum concentration.

[0070] In some embodiments, the hydrolysis reaction specifically includes the following steps: dissolving compound 3 in an organic solvent, cooling to 15-20°C, adding lithium hydroxide solution dropwise, controlling the temperature at 15-25°C, maintaining the temperature at 20-30°C after the addition is complete, and after the reaction is complete, undergoing post-treatment acidification, filtration, washing, drying, dissolution, washing again, and vacuum concentration to obtain compound 4. Preferably, the acid used for acidification is hydrochloric acid; the washing after acidification is with purified water; the dissolution is with dichloromethane to dissolve the crude compound 4; and the washing after dissolution is with a 10% sodium chloride aqueous solution.

[0071] In some embodiments, the method for preparing compound 4 further includes a method for preparing compound 3, which specifically includes the following steps: compound 2 undergoes a carbonylation reaction to obtain compound 3.

[0072] In some embodiments, under a CO atmosphere, compound 2 is reacted with methanol in an alkali, a catalyst, and an organic solvent via a carbonylation reaction to yield compound 3.

[0073] In some embodiments, the carbonyl insertion reaction is carried out in a CO atmosphere at a pressure of 0.8-2.0 MPa, preferably (1.2-1.3) MPa.

[0074] In some embodiments, in the carbonylation reaction, the mass-to-volume ratio of compound 2 to methanol is (0.1-1) g / mL, preferably (0.3-0.8) g / mL, for example 0.5 g / mL.

[0075] In some embodiments, the base in the carbonylation reaction is TEA, sodium acetate, or potassium acetate, preferably TEA.

[0076] In some embodiments, in the carbonylation reaction, the mass ratio of compound 2 to the base is (0.8-2):1, preferably (1-1.5):1, for example 1.19:1.

[0077] In some embodiments, the catalyst in the carbonyl insertion reaction is a palladium catalyst, preferably Pd(dppf)Cl2.

[0078] In some embodiments, in the carbonylation reaction, the mass ratio of compound 2 to the catalyst is (11-13):1, preferably (12-12.5):1, for example 12.3:1.

[0079] In some embodiments, in the carbonyl insertion reaction, the organic solvent is one or more of dioxane, THF, and 2-methyltetrahydrofuran, preferably dioxane.

[0080] In some embodiments, during the carbonylation reaction, the mass-to-volume ratio of compound 2 to the organic solvent is (0.1-0.5) g / mL, preferably (0.1-0.3) g / mL, for example 0.2 g / mL.

[0081] In some embodiments, the reaction temperature in the carbonyl insertion reaction is 90-110°C, preferably 100-105°C.

[0082] In some embodiments, the progress of the carbonylation reaction can be detected using conventional monitoring methods in the art (e.g., TLC, HPLC, or NMR), with the reaction endpoint generally defined as when TLC shows that compound 2 has reacted completely. For example, the reaction time is 5-10 hours, preferably 8 hours.

[0083] In some embodiments, the carbonylation reaction further includes post-processing steps, such as concentration, filtration, washing, concentration, dissolution, column chromatography, concentration, crystallization, and drying.

[0084] In some embodiments, the carbonylation reaction specifically includes the following steps: Compound 2 is added to an organic solvent (e.g., dioxane) and methanol under stirring, followed by the sequential addition of a base and a catalyst. After the addition is complete, the mixture is purged with nitrogen three times, and then purged with carbon monoxide twice. Carbon monoxide is introduced to control the pressure, and the temperature is raised to 100-105°C for the reaction. After the reaction is complete, the mixture undergoes post-treatment, concentration, filtration, washing, concentration, dissolution, column chromatography, concentration, crystallization, and drying to obtain Compound 3. Preferably, the concentrated solution is filtered through diatomaceous earth; the washing is performed with ethyl acetate; the dissolution is performed by dissolving the concentrate in dichloromethane; the eluent for column chromatography is PE:EA = 3:1; and the crystallization is performed by crystallizing the crude compound 3 in a mixed solution of tertiary methyl ether and n-hexane (e.g., tertiary methyl ether: n-hexane = 1:1) at a crystallization temperature of 20-30°C.

[0085] In some embodiments, the method for preparing compound 3 further includes a method for preparing compound 2, which specifically includes the following steps: compound 1 undergoes a bromination reaction with a brominating reagent to obtain compound 2.

[0086] In some embodiments, compound 1 undergoes a bromination reaction with the brominating agent in the presence of an organic solvent, wherein the organic solvent is one or more of DMF, DMSO, or DMA, preferably DMF.

[0087] In some embodiments, the brominating agent in the bromination reaction is NBS, liquid bromine, DBH, or NBP, preferably NBS.

[0088] In some embodiments, the molar ratio of compound 1 to the brominating reagent in the bromination reaction is 1:(1.2-2.5), preferably 1:1.8.

[0089] In some embodiments, during the bromination reaction, the mass-to-volume ratio of compound 1 to the organic solvent is (0.15-0.4) g / mL, preferably (0.2-0.3) g / mL, for example 0.24 g / mL.

[0090] In some embodiments, the bromination reaction is carried out at a temperature of 10-30°C, preferably 20-30°C.

[0091] In some embodiments, the progress of the bromination reaction can be monitored using conventional monitoring methods in the art (e.g., TLC, HPLC, or NMR), with the reaction endpoint typically defined as the complete reaction of compound 1 as shown by TLC. For example, the reaction time is 2-5 hours, preferably 2-3 hours.

[0092] In some embodiments, the bromination reaction further includes post-processing steps, such as extraction, washing, and concentration.

[0093] In some embodiments, the bromination reaction specifically includes the following steps: dissolving compound 1 in an organic solvent, cooling to 10-15°C, adding the brominating reagent in batches while controlling the temperature at 10-25°C, reacting at 20-30°C after the addition is complete, and obtaining compound 2 through post-treatment extraction, washing, and concentration. Preferably, the post-treatment extraction is ethyl acetate extraction; the washing is performed sequentially with a 10% (w / w) sodium chloride aqueous solution, a 10% (w / w) sodium bisulfite aqueous solution, and a 10% (w / w) sodium chloride aqueous solution.

[0094] In some embodiments, the method for preparing compound 2 further includes a method for preparing compound 1, which specifically includes the following steps: 2-fluoro-3-bromopyridine undergoes a Buchwald-Hartwig coupling reaction with N-Boc piperazine to obtain compound 1.

[0095] In some embodiments, the 2-fluoro-3-bromopyridine and the N-Boc piperazine undergo a Buchwald-Hartwig coupling reaction in the presence of a base, a phosphorus ligand, a palladium catalyst, and an organic solvent to give compound 1.

[0096] In some embodiments, the molar ratio of 2-fluoro-3-bromopyridine to N-Boc piperazine in the Buchwald-Hartwig coupling reaction is 1:(1.2-2.0), for example 1:1.5.

[0097] In some embodiments, in the Buchwald-Hartwig coupling reaction, the base is cesium carbonate, sodium tert-butoxide, or potassium tert-butoxide, preferably cesium carbonate.

[0098] In some embodiments, in the Buchwald-Hartwig coupling reaction, the mass ratio of the 2-fluoro-3-bromopyridine to the base is (0.1-0.25):1, preferably (0.15-0.2):1, for example 0.18:1.

[0099] In some embodiments, the phosphorus ligand in the Buchwald-Hartwig coupling reaction is Xant-Phos.

[0100] In some embodiments, in the Buchwald-Hartwig coupling reaction, the mass ratio of the 2-fluoro-3-bromopyridine to the phosphorus ligand is (1.5-3):1, preferably (2-2.8):1, for example 2.55:1.

[0101] In some embodiments, the palladium catalyst in the Buchwald-Hartwig coupling reaction is Pd2(dba)3 or Pd(OAc)2, preferably Pd2(dba)3.

[0102] In some embodiments, in the Buchwald-Hartwig coupling reaction, the mass ratio of 2-fluoro-3-bromopyridine to the catalyst is (2.5-4):1, preferably (3-3.5):1, for example 3.2:1.

[0103] In some embodiments, the organic solvent in the Buchwald-Hartwig coupling reaction is one or more of tetrahydrofuran, 1,4-dioxane, or methyl tert-butyl ether, preferably dioxane.

[0104] In some embodiments, in the Buchwald-Hartwig coupling reaction, the mass-to-volume ratio of the 2-fluoro-3-bromopyridine to the organic solvent is (0.05-0.2) g / mL, preferably (0.08-0.15) g / mL, for example 0.1 g / mL.

[0105] In some embodiments, the reaction temperature in the Buchwald-Hartwig coupling reaction is 90-110°C, preferably 100-105°C.

[0106] In some embodiments, the progress of the Buchwald-Hartwig coupling reaction can be detected using conventional monitoring methods in the art (e.g., TLC, HPLC, or NMR), generally with the reaction endpoint defined as the complete reaction of the compound 2-fluoro-3-bromopyridine as indicated by TLC. For example, the reaction time is 5-12 hours, preferably 6 hours.

[0107] In some embodiments, the Buchwald-Hartwig coupling reaction further includes post-processing steps, such as filtration, washing, pulping, filtration, concentration, washing, and concentration.

[0108] In some embodiments, the Buchwald-Hartwig coupling reaction specifically includes the following steps: stirring 2-fluoro-3-bromopyridine, N-Boc piperazine, a base, a phosphorus ligand, and an organic solvent, heating to 100-105°C, and after the reaction is complete, undergoing post-treatment filtration, extraction, washing, drying, and vacuum concentration to obtain compound 1. Preferably, the filtration is diatomaceous earth filtration; the extraction is ethyl acetate extraction; the washing is washing with a saturated ammonium chloride aqueous solution; and the drying is drying with anhydrous sodium sulfate.

[0109] In some embodiments, the method for preparing compound 12 further includes a method for preparing compound 11, which includes the following steps: chlorinating compound 10 in the presence of an acid-binding agent and an organic solvent to obtain compound 11.

[0110] In some embodiments, the chlorinating agent in the chlorination reaction is thionyl chloride.

[0111] In some embodiments, the molar ratio of compound 10 to the chlorinating reagent in the chlorination reaction is 1:(2-5), preferably 1:3.

[0112] In some embodiments, the acid-binding agent in the chlorination reaction is N,N-diisopropylethylamine, triethylamine, or pyridine, preferably N,N-diisopropylethylamine.

[0113] In some embodiments, during the chlorination reaction, the mass ratio of compound 10 to the acid-binding agent is (2.5-4):1, preferably (3-3.5):1, for example 3.39:1.

[0114] In some embodiments, the organic solvent in the chlorination reaction is tetrahydrofuran or dioxane, preferably tetrahydrofuran.

[0115] In some embodiments, during the chlorination reaction, the mass-to-volume ratio of compound 10 to the organic solvent is (0.01-0.1) g / mL, preferably (0.05-0.08) g / mL, for example 0.067 g / mL.

[0116] In some embodiments, the chlorination reaction is carried out at a temperature of 15-30°C, preferably 20-25°C.

[0117] In some embodiments, the progress of the chlorination reaction can be detected using conventional monitoring methods in the art (e.g., TLC, HPLC, or NMR), generally with the reaction endpoint defined as when HPLC shows 10% of the compound < 1.00%. For example, the reaction time is 0.5-2 hours, preferably 1 hour.

[0118] In some embodiments, the chlorination reaction further includes post-processing steps, such as filtration, washing, drying, pulping, filtration, and drying.

[0119] In some embodiments, the chlorination reaction specifically includes the following steps: mixing an organic solvent, compound 10, and an acid-binding agent; cooling the mixture to 15-20°C; adding a chlorinating reagent dropwise while maintaining the temperature at 15-25°C; maintaining the temperature at 20-25°C after the addition is complete; and after the reaction is complete, undergoing post-treatment filtration, washing, drying, pulping, filtration, and drying to obtain compound 11. Preferably, the washing is tetrahydrofuran washing; and the pulping is acetonitrile pulping.

[0120] In some embodiments, the preparation method of compound 11 further includes a preparation method of compound 10, which specifically includes the following steps: under the condition of the presence of an organic solvent, compound 9 is reduced with a reducing agent to obtain compound 10, wherein the reducing agent is lithium aluminum hydride, sodium borohydride or trisec-butyl borohydride.

[0121] In some embodiments, the organic solvent in the reduction reaction is THF or dioxane, preferably THF.

[0122] In some embodiments, in the reduction reaction, the mass ratio of compound 9 to the organic solvent is (0.01-0.05):1, preferably (0.02-0.04):1, for example 0.03:1.

[0123] In some embodiments, the reducing agent in the reduction reaction is lithium aluminum hydride. In this invention, the reducing agent is lithium aluminum hydride added to the reaction system in the form of a lithium aluminum hydride tetrahydrofuran solution.

[0124] In some embodiments, the concentration of the lithium aluminum hydride tetrahydrofuran solution in the reduction reaction is (2-3) M, preferably 2.5 M.

[0125] In some embodiments, in the reduction reaction, the molar ratio of compound 9 to the reducing agent is 1:(1-2), preferably 1:1.2.

[0126] In some embodiments, the reduction reaction is carried out at a temperature of 10-30°C, preferably 10-20°C.

[0127] In some embodiments, the progress of the reduction reaction can be monitored using conventional monitoring methods in the art (e.g., TLC, HPLC, or NMR), with the reaction endpoint typically defined as the complete reaction of compound 9 as indicated by TLC. For example, the reaction time is 12-24 hours, preferably 16 hours.

[0128] In some embodiments, the reduction reaction further includes post-processing steps, such as quenching, separation, extraction, concentration, pulping, filtration, and drying.

[0129] In some embodiments, the reduction reaction specifically includes the following steps: compound 9 is dissolved in an organic solvent, cooled to -5 to -5°C under nitrogen protection, and a reducing agent (e.g., a solution of lithium aluminum hydride tetrahydrofuran) is added dropwise. After the addition is complete, the temperature is raised to 10 to 20°C and the reaction is stirred to terminate the reaction. Compound 10 is obtained after post-treatment quenching, separation, extraction, concentration, pulping, filtration, and drying. Preferably, the quenching is performed by adding purified water; the separation is performed by adding a 5% (w / w) NaOH aqueous solution and stirring; the extraction is performed by tetrahydrofuran extraction; and the pulping is performed by pulping with purified water.

[0130] In some embodiments, the preparation method of compound 10 further includes a preparation method of compound 9, which specifically includes the following steps: under a CO atmosphere, compound 8 reacts with methanol in an alkaline, palladium catalyst, and organic solvent via a carbonylation reaction to generate compound 9, wherein the pressure is 0.8-2.0 MPa.

[0131] In some embodiments, the carbonyl insertion reaction is carried out under a CO atmosphere at a pressure of 1 MPa.

[0132] In some embodiments, in the carbonylation reaction, the mass ratio of compound 8 to methanol is (0.3-1):1, preferably (0.5-0.8):1, for example 0.64:1.

[0133] In some embodiments, the base in the carbonylation reaction is TEA, sodium acetate, or potassium acetate, preferably triethylamine.

[0134] In some embodiments, in the carbonylation reaction, the molar ratio of compound 8 to the base is (0.1-0.8):1, preferably (0.2-0.5):1, for example 0.33:1.

[0135] In some embodiments, the palladium catalyst in the carbonyl insertion reaction is [1,1-bis(diphenylphosphine)ferrocene]palladium dichloromethane.

[0136] In some embodiments, in the carbonylation reaction, the molar ratio of compound 8 to the catalyst is (15-21):1, preferably (19-20.5):1, for example 20.2:1.

[0137] In some embodiments, in the carbonyl insertion reaction, the organic solvent is one or more of dioxane, tetrahydrofuran, or 2-methyltetrahydrofuran, preferably dioxane.

[0138] In some embodiments, in the carbonylation reaction, the mass ratio of compound 8 to the organic solvent is (0.1-0.25):1, preferably (0.12-0.2):1, for example 0.16:1.

[0139] In some embodiments, the reaction temperature in the carbonyl insertion reaction is 80-110°C, preferably 80-90°C.

[0140] In some embodiments, the progress of the carbonylation reaction can be monitored using conventional monitoring methods in the art (e.g., TLC, HPLC, or NMR), with the reaction endpoint typically defined as the complete reaction of compound 8 as shown by LCMS. For example, the reaction time is 2-10 hours, preferably 5-8 hours.

[0141] In some embodiments, the carbonylation reaction further includes post-processing steps, such as filtration, pulping, and drying.

[0142] In some embodiments, the carbonyl insertion reaction specifically includes the following steps: adding an organic solvent (e.g., 1,4-dioxane), methanol, compound 8, a base (e.g., TEA), and a palladium catalyst (e.g., [1,1-bis(diphenylphosphine)ferrocene]palladium dichloromethane) sequentially to a high-pressure reactor; stirring the reaction at 80-90°C under a carbon monoxide atmosphere (e.g., 1.0 MPa); and after the reaction is complete, post-treatment filtration, pulping, filtration, and drying yield compound 9. Preferably, the pulping is performed with methanol.

[0143] In some embodiments, the preparation method of compound 9 further includes a preparation method of compound 8, specifically comprising the following steps: compound 7 undergoes a cyclization reaction with n-butyryl chloride to obtain compound 8.

[0144] In some embodiments, compound 7 undergoes a cyclization reaction with n-butyryl chloride in the presence of a base and an organic solvent to yield compound 8.

[0145] In some embodiments, in the cyclization reaction, the molar ratio of compound 7 to n-butyryl chloride is 1:(1-2), preferably 1:1.5.

[0146] In some embodiments, the base in the cyclization reaction is one or more of DMAP, pyridine, or potassium tert-butoxide, preferably DMAP, pyridine, and potassium tert-butoxide.

[0147] In some embodiments, in the cyclization reaction, the molar ratio of compound 7 to DMAP is (9-11):1, preferably (9.5-10.5):1, for example 10:1.

[0148] In some embodiments, in the cyclization reaction, the molar ratio of compound 7 to pyridine is (9-11):1, preferably (9.5-10.5):1, for example 10:1.

[0149] In some embodiments, in the cyclization reaction, the molar ratio of compound 7 to potassium tert-butoxide is 1:(5-7), preferably 1:6.

[0150] In some embodiments, the organic solvent in the cyclization reaction is one or more of toluene, THF, or dioxane, preferably tetrahydrofuran.

[0151] In some embodiments, in the cyclization reaction, the mass ratio of compound 7 to the organic solvent is (0.08-0.2):1, preferably (0.1-0.15):1, for example 0.11:1.

[0152] In some embodiments, the cyclization reaction is carried out at a temperature of 10-70°C, preferably 20-65°C.

[0153] In some embodiments, the progress of the cyclization reaction can be detected using conventional monitoring methods in the art (e.g., TLC, HPLC, or NMR), with the reaction endpoint generally defined as the complete reaction of compound 7 as shown by HPLC. For example, the reaction time is 3-10 hours, preferably 3-5 hours.

[0154] In some embodiments, the cyclization reaction further includes post-processing steps, such as concentration, crystallization, filtration, pulping, filtration and drying.

[0155] In some embodiments, the cyclization reaction specifically includes the following steps: mixing an organic solvent, compound 7, and a base; cooling to 10-20°C under nitrogen protection; adding n-butyryl chloride dropwise; raising the temperature to 20-30°C and stirring the reaction after the addition is complete; and, after post-treatment, concentrating, crystallizing, filtering, pulping, filtering again, and drying to obtain compound 8. Preferably, the crystallization is performed using purified water; and the pulping is performed using purified water.

[0156] In some embodiments, the method for preparing compound 8 further includes a method for preparing compound 7, specifically comprising the following steps: reducing 5-bromo-2-cyano-3-nitropyridine to obtain compound 7.

[0157] In some embodiments, the 5-bromo-2-cyano-3-nitropyridine is reduced with a reducing agent in an organic solvent to yield compound 7.

[0158] In some embodiments, the 5-bromo-2-cyano-3-nitropyridine is added to the system in the form of a 5-bromo-2-cyano-3-nitropyridine acetic acid solution.

[0159] In some embodiments, the reducing agent in the reduction reaction is iron powder.

[0160] In some embodiments, in the reduction reaction, the molar ratio of 5-bromo-2-cyano-3-nitropyridine to the reducing agent is 1:(5-7), preferably 1:5.5.

[0161] In some embodiments, the organic solvent in the reduction reaction is acetic acid.

[0162] In some embodiments, in the reduction reaction, the mass ratio of the 5-bromo-2-cyano-3-nitropyridine to the organic solvent is (0.05-0.15):1, preferably (0.08-0.11):1, for example 0.1:1.

[0163] In some embodiments, the reduction reaction is carried out at a temperature of 10-40°C, preferably 10-30°C.

[0164] In some embodiments, the progress of the reduction reaction can be detected using conventional monitoring methods in the art (e.g., TLC, HPLC, or NMR), generally with the reaction endpoint defined as the complete reaction of the compound 5-bromo-2-cyano-3-nitropyridine as shown by HPLC. For example, the reaction time is 3-5 hours, preferably 3 hours.

[0165] In some embodiments, the reduction reaction further includes post-processing steps, such as filtration, pulping, filtration, pulping, concentration, pulping, filtration, pulping, filtration, and drying.

[0166] In some embodiments, the reduction reaction specifically includes the following steps: adding a reducing agent to an organic solvent at 20-30°C, slowly adding a solution of 5-bromo-2-cyano-3-nitropyridineacetic acid, stirring the reaction at 20-30°C, and after the reaction is complete, the post-treatment involves filtration, pulping, filtration, pulping, concentration, pulping, filtration, pulping, filtration, and drying to obtain compound 7. Preferably, the post-treatment involves filtration, THF pulping, filtration, THF pulping, concentration, purified water pulping, filtration, pulping a mixed solution of dichloromethane and n-hexane, filtration, and drying.

[0167] The present invention also provides a method for preparing compound 12, comprising the following steps: compound 6 undergoes a substitution reaction with compound 11 to obtain compound 12.

[0168] In some embodiments, the compound 12 is prepared as described in any one of the present invention.

[0169] The present invention also provides compound 2,

[0170] This invention also provides a method for preparing compound 2, comprising the following steps: reacting compound 1 with a brominating reagent via a bromination reaction to obtain compound 2.

[0171] In some embodiments, the preparation method of compound 2 is as described in any one of the present invention.

[0172] This invention also provides a method for preparing compound 3, which includes the following steps: compound 2 undergoes a carbonylation reaction to obtain compound 3.

[0173] In some embodiments, the preparation method of compound 3 is as described in any one of the present invention.

[0174] The present invention also provides a method for preparing compound 11, which includes the following steps: compound 10 is subjected to a chlorination reaction to obtain compound 11.

[0175] In some embodiments, the compound 11 is prepared as described in any one of the present invention.

[0176] The present invention also provides the use of compounds of Formula I in the preparation of pharmaceuticals for the treatment or prevention of PARP-mediated diseases, preferably, the diseases being selected from cancer, ischemic diseases and neurodegenerative diseases, more preferably, the pharmaceuticals being used for the treatment or prevention of cancer, for example, cancer lacking the HR-dependent DNADSB repair pathway, cancer having a BRCA1 or BRCA2 defect phenotype, the cancer being selected from breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, hematologic malignancies, gastrointestinal cancers and lung cancer.

[0177] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0178] The reagents and raw materials used in this invention are all commercially available.

[0179] The positive and progressive effects of this invention are as follows:

[0180] (1) To provide a novel method for synthesizing pyridine amide compounds and their intermediates;

[0181] (2) Solve the problems of scarce raw materials and high technical requirements for PARP1 inhibitor pyridine amide compounds. Attached Figure Description

[0182] Figure 1 shows the effect curve of compound 12 of the present invention on PARP1-DNA capture after 2 hours.

[0183] Figure 2 shows the effect curve of compound 12 of the present invention on PARP2-DNA capture after 2 hours.

[0184] Figure 3 shows the time-effect curves of PARP1-DNA capture by compound 12 of the present invention at different time points.

[0185] Figure 4 shows the time-effect curves of PARP2-DNA capture by compound 12 of the present invention at different time points.

[0186] Figure 5 shows the effect of compound 12 of the present invention on tumor volume changes in subcutaneous xenografts of MDA-MB-436 mice.

[0187] Figure 6 shows the induction of DLD-1BRCA2 by compound 12 of the present invention in the first experiment. - / - Apoptosis (ns, no statistically significant difference; *, p<0.05, statistically significant difference; **, p<0.01, statistically significant difference).

[0188] Figure 7 shows how compound 12 of the present invention induced DLD-1BRCA2 in the second experiment. - / -Apoptosis (ns, no statistically significant difference; *, p<0.05, statistically significant difference; **, p<0.01, statistically significant difference).

[0189] Figure 8 shows how compound 12 of the present invention induced DLD-1BRCA2 in the third experiment. - / - Apoptosis (ns, no statistically significant difference; *, p<0.05, statistically significant difference; **, p<0.01, statistically significant difference).

[0190] Figure 9 shows the expression of cleaved Caspase-3 and Caspase-3 proteins in tumor tissue.

[0191] Figure 10 shows the effect of compound 12 and reference AZD5305 on Cleaved Caspase-3 protein in tumor tissue at the same dose (*, p<0.05, no statistically significant difference between other groups by one-way ANOVA).

[0192] Figure 11 shows the effect of compound 12 and reference AZD5305 on Cleaved Caspase-3 protein in tumor tissue at the same dose (*, p<0.05, no statistically significant difference between other groups by one-way ANOVA). Detailed Implementation

[0193] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0194] Method for determining chloride ion content

[0195] Example 1:

[0196] Synthesis of Compound 1

[0197] 25.00 L of dioxane was added to a 100 L glass reactor. While stirring, 2.50 kg of 2-fluoro-3-bromopyridine, 3.97 kg of N-Boc piperazine (1.5 ep), 13.90 kg of cesium carbonate, 0.98 kg of Xant-Phos, and 0.78 kg of Pd2(dba)3 were added sequentially. After the addition was complete, the temperature was raised to 100-105 °C and the reaction was carried out for 6 hours. A sample was then taken; TLC showed that the reactants had reacted completely, indicating the reaction was complete.

[0198] The reaction solution was filtered through diatomaceous earth. The filter cake was washed with 5 x 5 L of ethyl acetate to obtain filtrate 1. The filter cake was then slurried with 18 L of ethyl acetate at 20-30 °C for 1 h and filtered to obtain filtrate 2. Filtrates 1 and 2 were combined and concentrated under reduced pressure at 50 °C to a remaining 18-20 kg. The concentrate was added to a 100 L reactor. The solution was washed twice with 2 x 14 L of saturated ammonium chloride aqueous solution, and the organic phase was separated and concentrated to dryness under reduced pressure at 50 °C. Compound 1 (4.75 kg) was obtained and used directly in the next step.

[0199] 1 H NMR (400MHz, CD3Cl) δ=7.910-7.90(m,1H), δ=7.50-7.47(m,2H), δ=3.39-3.37(m,4H), δ=3.30-3.27(m,4H), δ=1.43(s,9H).

[0200] Example 2:

[0201] Synthesis of Compound 2

[0202] Add 17L of DMF to a 100L glass reactor, then add 4.00kg of Compound 1 while stirring, and cool to 10-15℃. Add 4.56kg of NBS (1.8ep) in batches, controlling the temperature at 10-25℃. After the addition is complete, stir at 20-30℃ for 3 hours. Take a sample, and TLC shows that Compound 1 has reacted completely, indicating the reaction is complete.

[0203] Add 25 L of ethyl acetate and 15 L of 10% sodium chloride aqueous solution to the reaction mixture and stir for 15 min. Allow to stand and separate the phases; extract the aqueous phase with 20 L of ethyl acetate. Combine the organic phases and wash once with 15 L of 10% sodium chloride aqueous solution. Separate the organic phase and wash once with 15 L of 10% sodium bisulfite aqueous solution. Separate the organic phase and wash twice with 2 x 15 L of 10% sodium chloride aqueous solution. Separate the organic phase and concentrate to dryness under reduced pressure at 50 °C. Compound 2, 5.69 kg, is obtained and used directly in the next step.

[0204] 1H NMR (400MHz, CD3Cl) δ = 7.25 (S, 1H), δ = 7.13-7.09 (m, 1H), δ = 3.58-3.55 (t, 4H), δ = 3.02-3.00 (t, 4H), δ = 1.46 (s, 9H).

[0205] Example 3:

[0206] Synthesis of Compound 3

[0207] 25.60 L of dioxane and 10.20 L of methanol were added to a 50 L high-pressure reactor. While stirring, 5.12 kg of compound 2 was added, followed by 4.31 kg of triethylamine and 415 g of Pd(dppf)Cl2. After the addition was complete, the reactor was purged with nitrogen three times, then with carbon monoxide twice. The pressure was maintained at 1.2-1.3 MPa with carbon monoxide, and the temperature was raised to 100-105 °C for 8 hours. Samples were taken, and TLC showed that compound 2 had reacted completely, at which point the reaction was terminated.

[0208] The reaction solution was removed and concentrated under reduced pressure at 50°C to a remaining 18-22 kg. The concentrate was filtered through diatomaceous earth, and the filter cake was washed with 10 L of ethyl acetate. The filtrate was collected and concentrated to dryness under reduced pressure at 50°C. The concentrate was dissolved in 2.5 L of dichloromethane and loaded into a glass column pre-loaded with 9 kg of 100-200 mesh silica gel. Eluent (PE:EA = 3:1) was added, and column chromatography was initiated. After TLC detection, the eluent was concentrated to dryness to obtain 3.07 kg of crude product. The crude product was dissolved in 9 L of methyl ether and added to a 50 L reactor. 9 L of n-hexane was added, and the mixture was stirred at 20-30°C for 3 h. The mixture was filtered, and the filter cake was dissolved in 4 L of methyl ether and added to a 20 L reactor. 4 L of n-hexane was added, and the mixture was stirred at 20-30°C for 4 h. The mixture was filtered again, and the filter cake was washed with 3 L of n-hexane. The filter cake was then dried in a forced-air oven at 50°C. A total of 1.63 kg of compound 3 was obtained, with a yield of 35%.

[0209] 1 H NMR (400MHz, MeOD) δ = 7.9310-7.9075 (m, 1H), δ = 7.5026-7.4567 (m, 1H), δ = 3.898 4(s,3H), δ=3.5999-3.5769(m,4H), δ=3.3138-3.2075(m,4H), δ=1.4739(s,9H).

[0210] Example 4:

[0211] Synthesis of Compound 4

[0212] Add 2.20 L of tetrahydrofuran and 2.20 kg of compound 3 to a 20 L glass reactor, stir, and cool to 15-20 °C. Add lithium hydroxide solution (0.31 kg of lithium hydroxide dissolved in 4.40 L of purified water) dropwise, controlling the temperature at 15-25 °C. After the addition is complete, maintain the temperature at 20-30 °C for 0.5 h, then take a sample; HPLC shows that compound 3 < 1.00%, indicating the reaction is complete. Add HCl solution (1.71 kg of concentrated hydrochloric acid dissolved in 1.45 L of purified water) dropwise, controlling the temperature at 20-30 °C with stirring; after the addition is complete, maintain the temperature at 20-30 °C with stirring for 1 h, filter, wash the filter cake with 4.40 L of purified water, and vacuum the filter cake at 50 °C for 48 h; weigh 2.17 kg of crude compound 4, yield 102%.

[0213] 63.00L of dichloromethane and 2.10kg of compound 4 were added to a 100L glass reactor and stirred at 20-30℃ until dissolved. The mixture was then washed with 20.00L of 10% sodium chloride aqueous solution, and the organic layer was separated. The mixture was concentrated under reduced pressure to obtain 2.02kg of compound 4, with a yield of 96%.

[0214] 1 H NMR (400MHz, DMSO) δ = 13.0362 (s, 1H), δ = 7.9177-7.8980 (m, 1H), δ = 7.5676-7.52 08(m,1H), δ=3.4887-3.3646(m,4H), δ=3.1777-3.1530(m,4H), δ=1.4255(s,9H).

[0215] Example 5:

[0216] Synthesis of Compound 5

[0217] Add 18.50 kg DMF, 1.95 kg compound 4, 2.34 kg DIEA, and 507 g deuterated methylamine hydrochloride sequentially to a 100 L glass reactor; stir, and slowly add 2.73 kg HATU in batches, controlling the temperature at 20-30 °C. After the addition is complete, maintain the temperature at 20-30 °C for 0.5 h and take a sample; HPLC showed that compound 4 < 1.00%, indicating the reaction was complete. Add 40.00 L purified water and 29.00 L ethyl acetate to the reaction solution for extraction; separate the organic layer for later use, and extract the aqueous layer with 20.00 L ethyl acetate; separate the aqueous layer and discard it, combine the two organic layers, and wash twice with 2 × 20.00 L purified water; separate the organic layer, wash once with 20.00 L saturated sodium bicarbonate solution, and then wash once with 20.00 L saturated sodium chloride solution. The organic layer was separated and concentrated under reduced pressure at 45-50℃ until no distillate was obtained, yielding compound 5 1.77 kg, yield: 86%.

[0218] 1 H NMR (400MHz, DMSO) δ = 8.4062 (s, 1H), δ = 7.8689-7.8496 (m, 1H), δ = 7.6118-7.565 2(m,1H), δ=3.4916-3.3477(m,4H), δ=3.1286-3.1040(m,4H), δ=1.4278(s,9H).

[0219] Example 6:

[0220] Synthesis of Compound 6

[0221] Add 6.50 L of dichloromethane, 1.60 kg of compound 5, and 160 g of activated carbon to a 20 L glass reactor. Stir at 20-30 °C for 0.5-1 h, filter, and reserve the filtrate. Add 11.20 L of ethyl hydrogen chloride solution (4 mol / L) to a 50 L glass reactor, cool to 10-15 °C, and stir. Add the above filtrate dropwise, maintaining the temperature at 10-20 °C. After the addition is complete, maintain the temperature at 15-20 °C for 0.5 h and take a sample. HPLC shows that compound 5 < 1.00%, indicating the reaction is complete. Filter, wash the filter cake with 3.00 L of dichloromethane, and dry the filter cake under vacuum at 40 °C for 16 h to obtain 1.35 kg of compound 6, with a yield of 104%.

[0222] 1 H NMR (400MHz, MeOD) δ = 7.9516-7.9287 (m, 1H), δ = 7.6741-7.6283 (m, 1H), δ = 3.5367-3.5112 (m, 4H), δ = 3.4694-3.4444 (m, 4H); 13 C NMR (400MHz, MeOD) δ: 47.7607-47.7180, 44.6403, 26.0427, 25.8357, 25.6250, 25.4195.

[0223] Example 7:

[0224] Synthesis of Compound 7

[0225] Add acetic acid (30 kg) to the reactor, and then add 7.14 kg (127.85 mol) of reduced iron powder while stirring at 20-30°C. Next, slowly add a solution of 5-bromo-2-cyano-3-nitropyridine (5.3 kg, 23.24 mol) in acetic acid (25 kg) (dropwise for approximately 6 hours). Stir the reaction at 20-30°C for 3 hours, then stop the reaction. Centrifuge the mixture, and then slurry the filter cake with THF for 3 hours before centrifuging again. Repeat the centrifugation process with 32 kg of THF. Combine the filtrates and concentrate under reduced pressure at 50°C. The concentrate was slurried with purified water (40 kg) at 60-70℃ for 1 h, then centrifuged at 20-30℃. The filter cake was slurried with a mixed solution of dichloromethane (6.9 kg) and n-hexane (3.7 kg) at 20-30℃ for 1 h, centrifuged, and dried with forced air at 45-55℃ to obtain a light yellow compound 7 (3.8 kg, yield 84%).

[0226] 1 H NMR (400MHz, DMSO-d6): δ7.93(d,1H),7.45(d,1H),6.54(s,2H).

[0227] Example 8:

[0228] Synthesis of Compound 8

[0229] Add tetrahydrofuran (33.5 kg), compound 7 (3.8 kg, 19.20 mol), DMAP (0.23 kg, 1.92 mol), and pyridine (2.40 kg, 1.92 mol) to the reactor. Under nitrogen protection, lower the temperature to 10-20℃. Add n-butyryl chloride (3.10 kg, 29.10 mol) dropwise. After the addition is complete, raise the temperature to 20-30℃ and stir for 2 hours. Add potassium tert-butoxide (12.70 kg, 113.60 mol) in portions, and stir at 55-65℃ for 3 hours to stop the reaction. Concentrate the reaction solution directly in the reactor under reduced pressure at 50-60℃ until almost no distillate is distilled off. Add 20 kg of purified water and continue concentrating until no distillate is distilled off. Add 18 kg of purified water and lower the temperature to 20-30℃. Centrifuge and filter, then slurry the filter cake with 19 kg of purified water at 10-20℃ for 1 h, centrifuge and filter again, and dry the filter cake with forced air at 45-55℃ to obtain compound 8 (4.3 kg, yield 84%).

[0230] 1 H NMR (400MHz, DMSO-d6) δ = 10.93 (s, 1H), δ = 8.54-8.33 (m, 1H), δ = 8.32-8.28 (m, 1H), δ = 4.31-4.28 (m, 2H), δ = 1.05-1.02 (m, 3H).

[0231] Example 9:

[0232] Synthesis of Compound 9

[0233] 1,4-Dioxane (21.7 kg) and methanol (5.5 kg) were added to a high-pressure reactor, followed by compound 8 (3.5 kg, 13.10 mol), triethylamine (3.9 kg, 39.20 mol), and [1,1-bis(diphenylphosphine)ferrocene]palladium dichloromethane (0.47 kg, 0.65 mol). After addition, the reaction mixture was stirred at 80-90 °C for 5-8 h under a carbon monoxide atmosphere at 1.0 MPa. LC-MS showed complete consumption of the starting materials. After cooling, the reaction mixture was filtered. The filter cake was slurried with methanol (10.50 kg) at 10-20 °C for 1 h, filtered, and dried by forced air at 45-55 °C to obtain compound 9 (2.7 kg, yield 84%).

[0234] 1 H NMR (400MHz, DMSO-d6) δ = 11.02 (H-15, s, 1H), δ = 8.35-8.34 (m, 1H), δ = 7.59-7 .58(m,1H), δ=4.67-4.66(m,2H), δ=3.91-3.90(m,3H); δ=1.06-1.02(m,3H).

[0235] Example 10:

[0236] Synthesis of Compound 10

[0237] Tetrahydrofuran (52.5 kg) and Compound 9 (1.5 kg, 6.07 mol) were added to the reactor, and the mixture was cooled to -5 to -5°C under nitrogen protection. Lithium aluminum hydride solution (2.5 M / THF, 2.7 kg, 7.28 mol) was added dropwise, and the mixture was heated to 10-20°C and stirred for 16 h to stop the reaction. The reaction solution was cooled to 5-15°C, and purified water (2.0 kg) was slowly added dropwise to quench the reaction. Then, 5% NaOH aqueous solution (15.0 kg) was added, and the mixture was stirred and separated at 20-30°C. The aqueous phase was extracted six times with tetrahydrofuran (6 × 12.5 kg). The organic phases were combined and concentrated under reduced pressure at 50°C. The concentrate was filtered, and the filter cake was slurried with purified water (3.0 kg) at 10-20°C for 1 h, filtered, and dried by forced air at 45-55°C to obtain Compound 10 (0.87 kg, yield 65.41%).

[0238] 1 H NMR (400MHz, DMSO) δ = 11.0182 (H-15, s, 1H), δ = 8.3393-8.3352 (m, 1H), δ = 7.5855-7.5838 (m, 1H), δ=6.3741(s,2H), δ=5.5126-5.4871(m,1H), δ=4.6573-4.6466(m,2H), δ=1.0580-1.0214(m,3H); 13 C NMR (400MHz, DMSO) δ: 162.2565, 147.4702, 140.8701, 139.0095, 133.2015, 129.6908, 119.8844, 107.3262, 60.3807, 17.2246, 12.1835.

[0239] Example 11:

[0240] Synthesis of Compound 11

[0241] Add 15.00 L of tetrahydrofuran, 1.00 kg of compound 10, and 295 g of N,N-diisopropylethylamine to a 100 L glass reactor. Stir and cool to 15-20 °C. Add 1.63 kg of thionyl chloride dropwise, controlling the temperature at 15-25 °C. After the addition is complete, maintain the reaction temperature at 20-25 °C for 1 h and take a sample. HPLC showed that compound 10 < 1.00%, indicating the reaction was complete. Filter, wash the filter cake with 2.00 L of tetrahydrofuran, dry the filter cake at 40 °C for 16 h, weigh it, and obtain 1.37 kg of crude compound 11.

[0242] 13.00 L of acetonitrile and 1.37 kg of compound 11 were added to a 20 L glass reactor and stirred at 20-30 °C for 3 h. The mixture was filtered, and the filter cake was dried at 40 °C for 16 h. The cake was then weighed to obtain 1.23 kg of compound 11, with a yield of 98%.

[0243] 1 H NMR (400MHz, DMSO) δ = 11.3723 (s, 1H), δ = 8.5037-8.4995 (m, 1H), δ = 7.7668-7.7628 (m, 1H), δ = 6.5683 (m, 3H), δ = 4.9629 (s, 2H), δ = 2.6225-2.5568 (m 2H), δ=1.0733-1.0367(m 3H).

[0244] Example 12:

[0245] Synthesis of Compound 12

[0246] Add 5.50 L of dimethyl sulfoxide, 1.29 kg of compound 6, 2.42 kg of N,N-diisopropylethylamine, and 1.10 kg of compound 11 sequentially to a 50 L glass reactor. Stir and heat to 65-75 °C for 2 h. Take a sample; HPLC showed that compound 11 < 2.00%, indicating the reaction was complete. Add 8.80 kg of anhydrous ethanol dropwise to the reactor, maintaining the temperature at 60-75 °C. After the addition is complete, cool to 20-30 °C and stir for 16 h. Filter, and wash the filter cake with 2.20 L of anhydrous ethanol. Filter the mixture, add the filter cake and 8.80L of dimethyl sulfoxide to a 50L glass reactor, heat to 90-100℃ and stir for 1 hour, then cool to 20-30℃ and stir for 1 hour. Filter the mixture, wash the filter cake with 4.40L of isopropyl acetate, filter the mixture, and dry the filter cake at 50-55℃ with forced air for 40 hours. Stop drying, remove the cake and weigh it to obtain 1.48kg of crude compound 12.

[0247] The crude product was added to a 50L glass reactor along with 5.92L of dimethyl sulfoxide. The mixture was heated to 85-95℃ and stirred for 1.5h, then cooled to 20-30℃ and stirred for 1h. The mixture was filtered, and the filter cake was washed with 5.92L of isopropyl acetate. After filtration, the filter cake was dried under vacuum at 50-55℃ for 15h. The cake was then weighed to obtain compound 12 1.29kg, with a yield of 73%.

[0248] 1H NMR (400MHz, DMSO) δ = 10.8994 (s, 1H), δ = 8.3778 (s, 1H), δ = 8.3219-8.3181 (d, 1H), δ = 7.8559-7.8332 (m, 1H), δ = 7.5916-7.5445 (m, 1H) , δ = 7.5341-7.5308 (m, 1H), δ = 6.3355 (s, 1H), δ = 3.6465 (s, 2H), δ = 3.1766 (s, 4H), δ = 2.5739-2.5128 (m, 4H), δ = 1.0095-0.9730 (m, 3H).

[0249] Example 13:

[0250] Synthesis of the target compound

[0251] Add 6.00 L of dimethyl sulfoxide and 1.20 kg of compound 12 to a 50 L glass reactor. Maintain the temperature at 20-30 °C and stir. Add dimethyl sulfoxide hydrochloride solution (267 g concentrated hydrochloric acid dissolved in 2.40 L of dimethyl sulfoxide) dropwise. After the addition is complete, maintain the temperature at 20-40 °C and stir until the system is clear. Add 120 g of activated carbon and heat to 65-70 °C, stirring for 0.5-1 h. Filter the solution, collect the filtrate, heat to 75-85 °C and stir. Add 6.00 L of isopropyl acetate dropwise, maintaining the temperature at 75-85 °C. After the addition is complete, control the cooling rate, lowering the temperature to 55-60 °C over 1.5-2.5 h. Add 9.60 L of isopropyl acetate dropwise, completing the addition over 20-40 min. After the addition is complete, cool to 20-30 °C and stir for 12-15 h. The filter cake was filtered, washed with 6.40 L of isopropyl acetate, filtered dry, and vacuum dried at 50 °C for 24 h to obtain 1.16 kg of the target compound, with a yield of 90% (the chloride ion content was determined to be 7.3% by the chloride ion content determination method, which corresponds to a hydrochloric acid to compound 12 ratio of 1:1).

[0252] 1 H NMR (400MHz, DMSO) δ = 11.87 (s, 1H), δ = 11.22 (s, 1H), δ = 8.71 (d, 1H), δ = 8.42 (s, 1H), δ = 7.87-7.89 (dd, 1H), δ = 7.74-7.76 (d, 1H), δ = 7.67-7.7 1(dd,1H),δ=6.42-6.45(s,2H),δ=4.54-4.58(s,2H),δ=3.68-3.70(s ,2H), δ=3.34-3.44(m,6H), δ=2.57-2.51(q,2H), δ=1.03-0.97(t,3H).

[0253] Experimental Example 1: Inhibitory effect of compound 12 on PARP1 / PARP2 enzyme activity

[0254] PARP1 / PARP2 enzyme activities were detected using a chemiluminescence method. First, histone (Active Motif, 81126) was incubated in 384-well plates for 2 hours. Then, different dilutions of the example compounds and either PARP1 working solution (Abcam, ab279663) or PARP2 working solution (BPS, 80502) were added. Max control wells contained only PARP1 or PARP2 working solution, while Min control wells contained only assay buffer. The plates were incubated at room temperature for 15 minutes, followed by the addition of biotin-labeled substrate NAD. + (BPS, 80610) After incubation at room temperature for 2 hours, the ADP riboyl group of the substrate remained biotinylated on the histone after enzyme catalysis. Streptavidin-HRP solution (Abcam, AB7403) was added to develop biotin color, and the values ​​were read on an EnSight (PE) instrument. The inhibition rate was calculated using the fluorescence values ​​of the Max and Min wells. A dose-response curve was fitted using GraphPad Prism 5 software to obtain the IC50 of each compound on the enzyme activity. 50 value.

[0255] The results are shown in Table 1 below. Compound 12 exhibited dose-dependent inhibition of PARP1 enzyme, with significant inhibitory activity reaching picomolar concentration levels, similar to the reference compounds AZD5305 and Olaparib. Regarding PARP2 selectivity, compound 12 showed similar selectivity to the reference AZD5305. Compared to the currently marketed PARP1 inhibitor Olaparib, compound 12 significantly improved PARP2 selectivity while maintaining inhibition of PARP1 enzyme activity, with a 30-fold increase in selectivity.

[0256] Table 1 shows the inhibitory effects of compounds on PARP1 / PARP2 enzyme activity.

[0257] Experimental Example 2: Compound induces the DNA capture ability of PARP1 / PARP2

[0258] The DNA capture capacity of PARP1 / PARP2 was detected using HTRF (homogeneous time-resolved fluorescence). First, PARP1 (BPS, 80501) or PARP2 (BPS, 80502) was labeled with Mab anti-GST-Tb crypate (Cisbio, 61GSTTLA). The DNA-labeled probe (Generay) was damaged, and different concentrations of the example compounds were added. 50 μM AZD2281 was added to the Max control wells, and medium buffer was added to the Min wells. After incubation at room temperature for 1 hour, the substrate NAD was added. + (Sigma, 10127965001) After 10 minutes of incubation, PARP exhibits enzymatic activity, causing it to be released from the damaged DNA. At this stage, only a fluorescence signal with an emission wavelength of 615 nm can be detected. When PARP is inhibited, it is induced to bind to the damaged DNA, causing energy transfer. Two emission wavelengths can then be detected: one is the fluorescence signal emitted by the damaged DNA probe itself at 615 nm, and the other is the fluorescence signal emitted at 665 nm due to energy transfer after PARP binds to the damaged DNA. The fluorescence ratio of 665 nm to 615 nm represents the amount of PARP-captured DNA complex. The capture capacity induced by each compound was calculated using the fluorescence values ​​of the Max and Min wells. The dose-response curve was fitted using the analysis software GraphPad Prism5 to obtain the EC5 value of each compound inducing the DNA capture capacity of the PARP enzyme. 50 Value (required concentration to achieve 50% capture).

[0259] The results are shown in Table 2 below. Compound 12 induced PARP to capture DNA at single-digit nanomolar concentrations, demonstrating significant capture ability similar to the reference compound AZD53305 and superior to Olaparib. Regarding PARP2 selectivity, compound 12 achieved approximately 100-fold selectivity, outperforming the reference AZD5305. Compared to the currently marketed PARP1 compound Olaparib, compound 12 significantly improved activity against PARP1 and selectivity against PARP2, with a 3-fold increase in activity and a 333-fold increase in selectivity.

[0260] Table 2. DNA capture ability of PARP induced by the compounds

[0261] Experimental Example 3: Inhibitory effect of the compound on PARP1 / PARP2 enzyme activity at the cellular level.

[0262] The inhibitory effects of the compounds on PARP1 / PARP2 enzyme activity at the cellular level were detected using high-content imaging. A549 WT, PARP1-KO, and PARP2-KO cell lines were self-established. After cell resuscitation and stabilization, a sufficient number of cells were collected, and 100 μL of cell suspension was seeded into 96-well plates. The next day, serially diluted concentrations of the example compounds were added. Max control wells received only buffer, while Min control wells received 500 nM AZD5305 (A549 WT and PARP2-KO cell lines) or 1 μM AZD2281 (PARP1-KO cell line). Incubation was performed for 1.5 hours (A549 WT and PARP2-KO cell lines) or 2 hours (PARP1-KO cell line). Remove the supernatant and incubate with 0.4 mM H2O2 for 10 minutes (A549 WT and PARP2-KO cell lines) or 1.5 mM H2O2 for 15 minutes (PARP1-KO cell line) to induce significant DNA damage. Remove the supernatant, fix with 4% paraformaldehyde for 20 minutes, then treat with 0.5% Triton X-100 for 20 minutes to increase cell membrane permeability. Incubate with 3% BSA for 1 hour to avoid nonspecific binding. Add the primary antibody Poly(ADP-ribose) monoclonal antibody (CST, 83732S) diluted 1:500 and incubate overnight. Then add the secondary antibody Goat anti-Rabbit IgG, Alexa Fluor diluted 1:500. TM Incubate at 488 (Invitrogen, A-11034) for 1 hour. Add 50 μL DAI (Invitrogen, R37606) to stain cell nuclei for 30 minutes. Wash twice with PBS, then incubate in OPERETTA CLASSES. TM Cells were photographed on a PE (polyepidermal imager) in non-confocal mode under a 20x water objective, with five regions photographed for each well. For data analysis, DAPI-stained nuclei were used to distinguish between the nucleus and cytoplasm, and the average intensity of Alexa 488 in the nuclei of each well was calculated. The inhibition rate was calculated using the fluorescence values ​​from the Max and Min wells, and dose-response curves were fitted using GraphPad Prism 5 software to determine the IC50 of each compound on enzyme activity. 50 value.

[0263] The results are shown in Table 3. Compound 12 showed dose-dependent inhibition of enzyme activity in both A549 WT and PARP2-KO cell lines, with significant inhibitory activity. Even at single-digit nanomolar concentrations, the inhibitory activity was similar, indicating that the cellular enzyme activity mainly originates from PARP1. Compound 12 showed similar inhibitory activity against PARP1 compared to the reference AZD5305. Compound 12 exhibited relatively weak inhibitory activity in the A549PARP1-KO cell line. No enzyme activity inhibition was detected at the highest concentration of 40 μM, indicating that after PARP1 knockout, compound 12 did not significantly inhibit PARylation at the cellular level, and its inhibitory effect on enzymes other than PARP1 was not significant. Compared to the reference AZD5305, compound 12 showed a selectivity fold greater than 3-6 times, demonstrating better selectivity.

[0264] Table 3 shows the inhibitory effects of compounds on PARP1 / PARP2 enzyme-mediated parylation activity at the cellular level.

[0265] Experimental Example 4: Test of the anti-proliferative activity of the compound against tumor cells

[0266] The anti-proliferative assay of the compounds against tumor cells was performed using the most widely used ATP concentration detection method. Both MDA-MB-231 and MDA-MB-436 cell lines were derived from ATCC, DLD-1 human colorectal adenocarcinoma epithelial cells, and DLD-1 BRCA2 cells. - / - Cells were obtained from Horizon. Cells were resuscitated and, once stabilized, collected with a viable cell count greater than 90%. 450-500 cells were seeded into 384-well plates. The next day, serially diluted compounds from the example were added. Max wells received only buffer, and Min wells received 50 μM AZD2281. Incubation was performed for 7 days. On the 8th day, [the following was added]... Reagent (Promega, G7573) was incubated at room temperature for 30 minutes, and values ​​were read on Envision (PE). Inhibition rates were calculated using the fluorescence values ​​of the Max and Min wells. Dose-response curves were fitted using GraphPad Prism 5 software to determine the IC50 of each compound on enzyme activity. 50 value.

[0267] The results are shown in Table 4 below. In both the BRCA1-mutated MDA-MB-436 human triple-negative breast cancer cell line and the BRCA1-normal MDA-MB-231 human triple-negative breast cancer cell line, compound 12 exhibited dose-dependent antiproliferative activity against the BRCA1-mutated MDA-MB-436 cell line. Compound 12 inhibited the proliferation of this cancer cell line at single-digit nanomolar concentrations, similar to the reference AZD5305. For the BRCA1-normal MDA-MB-231 cell line, compound 12 showed no inhibitory activity against cell proliferation at the highest concentration of 10 μM, consistent with the reference AZD5305. This indicates that compound 12 showed significant selectivity in both BRCA1-mutated and BRCA1-normal cell lines, with a selection factor reaching several thousand times. Compared to the marketed PARPi Olaparib, compound 12 showed a 154-fold increase in antiproliferative activity against the BRCA1-mutated MDA-MB-436 cell line and a 471-fold increase in selectivity for PARP2, significantly improving both PARP1 inhibitory activity and PARP2 selectivity.

[0268] As shown in Table 5, in the BRCA2-mutated DLD-1 BRCA2 - / - In the BRCA2-normal DLD-1 human colorectal adenocarcinoma epithelial cell line, all the compounds in the examples exhibited dose-dependent antiproliferative activity against BRCA2-mutant cell lines. Compound 12 showed inhibitory activity against cancer cell proliferation at single-digit nanomolar concentrations, similar to the reference AZD5305. For the BRCA2-normal DLD-1 cell line, compound 12 showed no inhibitory activity against cell proliferation at the highest concentration of 10 μM, consistent with the reference AZD5305, indicating that compound 12 showed significant selectivity in both BRCA2-mutant and BRCA2-normal cell lines, with a selectivity fold of several thousand times.

[0269] The antiproliferative activity of the compounds in BRCA1-mutated and normal, and BRCA2-mutated and normal cancer cell lines, as demonstrated by the examples, indicates that the compounds possess strong antiproliferative activity against target cells and are targeted and safe for clinical treatment.

[0270] Table 4. Antiproliferative activity of compounds against BRCA1-mutated and BRCA1-normal cancer cells.

[0271] Table 5. Antiproliferative activity of compounds against BRCA2-mutated and BRCA2-normal cancer cells.

[0272] By combining enzymatic activities at both the molecular and cellular levels, inducing PARP's ability to capture damaged DNA and its anti-proliferative activity against cancer cells, compound 12 exhibits excellent selective inhibitory activity against PARP1 and anti-proliferative capacity against target cells. It is significantly superior to the marketed PARPi Olaparib in both PARP1 activity and PARP2 selectivity. Compared with the reference compound AZD5305, it shows a significant improvement in PARP1 selectivity at the cellular level.

[0273] Example 5: Selectivity of compounds for PARP family members PARP3, 5a, 6, 7, and 11

[0274] The activities of PARP family enzymes were detected using a chemiluminescence method. First, histone (Active Motif, 81126) was incubated in a 384-well plate for 2 hours.

[0275] Different dilutions of the example compounds and PARP3 working solution (BPS, 80503) were added. Max control wells were added only with the respective PARP working solutions, and Min control wells were added only with the assay buffer. The mixtures were incubated at room temperature for 15 minutes, followed by the addition of the biotin-labeled substrate NAD. + (BPS, 80610), PARP3 activating DNA (Generay), incubated at room temperature for 2 hours. After the substrate is catalyzed by the enzyme, the ADP riboyl group binds to the histone and still carries the biotin label. Striptigin-HRP solution (Abcam, ab7403) is added to make biotin develop color, and the value is read on the EnSight (PE) instrument.

[0276] Different dilutions of the example compounds and PARP5a working solution (BPS, 80504) were added. Max control wells were added only with the respective PARP working solution, and Min control wells were added only with assay buffer. The mixtures were incubated at room temperature for 15 minutes, followed by the addition of the biotin-labeled substrate NAD. + (BPS, 80610), incubated at room temperature for 2 hours. After the substrate is catalyzed by the enzyme, the ADP riboyl group binds to the histone and still carries the biotin label. Striptigin-HRP solution (Abcam, ab7403) is added to make biotin develop color, and the value is read on the EnSight (PE) instrument.

[0277] Different dilutions of the example compounds and PARP6 working solution (BPS, 80506) were added. Max control wells were added only with the respective PARP working solutions, and Min control wells were added only with the assay buffer. The mixtures were incubated at room temperature for 15 minutes, followed by the addition of the biotin-labeled substrate NAD. +(BPS, 80610), incubated at room temperature for 2 hours. After the substrate is catalyzed by the enzyme, the ADP riboyl group binds to the histone and still carries the biotin label. Striptigin-HRP solution (Abcam, ab7403) is added to make biotin develop color, and the value is read on the EnSight (PE) instrument.

[0278] The PARP7 Chemiluminescent assay kit (BPS, 79729) was used. Different dilutions of the example compounds and PARP7 working solution (BPS, 80527) were added. The Max control wells were added only with each PARP working solution, and the Min control wells were added only with the assay buffer. The mixture was incubated at room temperature for 15 minutes. The substrate mixture provided by the assay kit (BPS, 78371) was then added, and the mixture was incubated at room temperature for 1 hour. After the substrate was catalyzed by the enzyme, the ADP riboyl group was bound to histone and still carried the biotin label. Treptavidin-HRP solution (BPS, 80611) was added to develop the biotin color, and the values ​​were read on the EnSight (PE) instrument.

[0279] The PARP11 Chemiluminescent assay kit (BPS, 80561) was used. Different dilutions of the example compounds and PARP11 working solution (BPS, 80511) were added. The Max control wells were added only with the PARP working solution, and the Min control wells were added only with the assay buffer. The mixture was incubated at room temperature for 15 minutes. The substrate mixture provided by the assay kit (BPS, 78371) was then added, and the mixture was incubated at room temperature for 1 hour. After the substrate was catalyzed by the enzyme, the ADP riboyl group was bound to histone and still carried the biotin label. Treptavidin-HRP solution (BPS, 80611) was added to develop the biotin color, and the values ​​were read on the EnSight (PE) instrument.

[0280] The inhibition rate was calculated using the fluorescence values ​​of the Max and Min wells. Dose-response curves were fitted using the analysis software GraphPad Prism 5 to determine the IC50 of each compound on enzyme activity. 50 value.

[0281] The results are shown in Table 6 below. Compound 12 showed weaker inhibitory effects on all PARP enzymes than on PARP1 enzyme activity. The values ​​in parentheses in Table 6 represent the ratios to PARP1 enzyme activity. Compared with the marketed PARPi Olaparib, Compound 12 showed significantly improved selectivity for all PARP enzymes except for slightly weaker selectivity for PARP11. Compared with the reference AZD5305, Compound 12 showed significantly improved selectivity for all tested PARP enzymes.

[0282] Compound 12 is a PARP1-specific selective inhibitor, exhibiting superior selectivity to AZD5305 for PARP3, PARP5a, PARP6, PARP7, and PARP11, with a particularly significant advantage in selectivity for PARP11; and superior selectivity to Olaparib for PARP3, PARP5a, PARP6, and PARP7, with a particularly significant advantage in selectivity for PARP3.

[0283] Table 6. Inhibition of PARP3 / PARP5a / PARP6 / PARP7 / PARP11 enzyme activity by compounds (nM)

[0284] Effect of Compound 6 on DNA Capture Induced by PARP1 / 2 in FP Experiment

[0285] To further explore the effects of compound 12 on PARP1-DNA and PARP2-DNA capture, and the differences between them, fluorescence polarization (FP) was used for further evaluation. PARP1 / 2 enzymes bind to fluorescently labeled DNA to form a larger complex, resulting in slower rotation of the fluorescently labeled DNA and thus emitting a higher polarization value. This effect was observed after the addition of NAD+. + Subsequently, PARP1 / 2 enzymes undergo self-ribosylation and accumulate negative charges. When the negative charge accumulates to a certain level, it causes the fluorescently labeled DNA to dissociate, resulting in faster rotation of the fluorescently labeled DNA and a decrease in polarization intensity. Adding a PARP inhibitor affects PARP-DNA capture, and the degree of this effect can be detected by changes in polarization intensity. First, 25 nL of different concentrations of the compound were transferred to 384-well plates at 1000-fold final concentrations. 25 nL of 100% DMSO was added to the Min and Max wells, respectively. Then, 5 μL of enzyme solution containing 10 nM PARP1 (BPS, 80501) and 1 nM FAM-PARP1-DNA (Generay, customized) or 10 nM PARP2 (BPS, 80502) and 1 nM FAM-PARP2-DNA (Generay, customized) was added. The plates were centrifuged at 1000 rpm for 1 minute, then incubated at room temperature for 30 minutes. Finally, 5 μL of 1 mM NAD+ substrate was added. + (MCE,HY-B0445), add 5 μL of 1 mM NAD substrate to the min well. + Add Assay buffer to the Max well, centrifuge at 1000 rpm for 1 minute, react at 25°C, and test at the following different time points:

[0286] For PARP1 capture test readings (Envision) at 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 30 hours, 48 ​​hours and 54 hours.

[0287] For PARP2 capture test plate reader readings at 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours and 48 hours.

[0288] The induced capture capacity of the compounds was calculated using the fluorescence values ​​of the Max and Min wells. Dose-response curves were fitted using the analysis software GraphPad Prism 5 to determine the EC5 values ​​of each compound's ability to induce PARP enzyme DNA capture. 50 value.

[0289] The results are shown in Table 7 and Figures 1-2. Taking the 2-hour time point as an example, compound 12 showed a 48-fold increase in its ability to induce PARP1-DNA capture compared to the marketed PARPi Olaparib, and similar activity to the reference AZD5305. Compound 12 showed almost no ability to capture PARP2-DNA, similar to the reference AZD5305; while the marketed PARPi Olaparib showed similar capture of PARP2-DNA as it did of PARP1-DNA, with no selectivity.

[0290] This indicates that compound 12 has a much higher selectivity for PARP1 than PARP2, and is expected to significantly reduce the blood toxicity caused by PARP2.

[0291] As shown in Table 7 and Figures 3-4, further comparisons were made of the ability of compound 12 and the reference AZD5305 to capture PARP1-DNA. Over time, the activity of compound 12 was maintained for more than 54 hours, and at 54 hours, its activity was 14 times stronger than that of the reference AZD5305. This suggests better anti-tumor proliferation activity.

[0292] Table 7 Effects of compounds on PARP1 / 2-DNA capture

[0293] Experimental Example 7: Antitumor effect of compound on a mouse model of human breast cancer cell MDA-MB-436 xenograft.

[0294] Compound 12 was further validated in vivo using a mouse subcutaneous xenograft model of the BRCA1-mutated MDA-MB-436 cell line. Human breast cancer MDA-MB-436 cells (ATCC, HTB-130) were cultured in vitro as a monolayer under the following conditions: L-15 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin solution, and 0.01 mg / mL bovine insulin, in a CO2-free incubator at 37°C. Cells were passaged twice a week using trypsin-EDTA digestion. When cell saturation reached 80%-90%, cells were harvested, counted, and 0.2 mL of 1×10⁻⁶ cells was added. 7 One MDA-MB-436 cell was subcutaneously seeded into the right posterior dorsal region of each mouse (PBS:Matrigel = 1:1). The average tumor volume reached 152 mm. 3 The mice were started to be administered drugs at a certain time, with the day designated as Day 0. Tumor volume and mouse weight were monitored twice a week for 28 days.

[0295] The tumor diameter was measured using vernier calipers. The formula for calculating tumor volume is: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. The tumor-suppressing efficacy of the compound is evaluated using the relative tumor shrinkage rate Reg%. Reg% reflects the rate at which the tumor volume shrinks after treatment. Reg% = (V0 - V t ) / V0×100%, where V0 is the tumor volume measured at the time of group administration (i.e., d0), V t The tumor volume at a particular measurement.

[0296] The compound response criteria in mouse models are derived from mRECIST (Revised Solid Tumor Response Criteria) (Gao et al., 2015), and are defined as follows:

[0297] CR (complete remission): Best Response <-95% and Best Avg Response <-40%;

[0298] Partial remission (PR): Best Response < -50% and Best Avg Response < -20%;

[0299] SD (Stable Disease): Best Response <35% and Best Avg Response <30%;

[0300] PD (Disease Progression): Other Categories;

[0301] ORR% equals the sum of the percentages of complete remission (CR) and partial remission (PR).

[0302] Statistical analysis was performed using Prism software, including the mean (mean) and standard error (SEM) of tumor volume at each time point for each group. For statistical analysis of TV, the raw TV data from each measurement were used to compare differences between groups. Two-way ANOVA was used to include both drug administration and time factors in the analysis, and Tukey's multiple comparisons test was applied. A p-value < 0.05 was considered statistically significant.

[0303] As shown in Figure 5, at the same dose, both compound 12 and the reference AZD5305 promoted tumor regression. Compound 12 caused a greater degree of tumor regression than the reference AZD5305, with an ORR of 60%, while the reference ORR was 20%.

[0304] This indicates that in the human breast cancer MDA-MB-436 mouse subcutaneous xenograft model, compound 12 exhibits superior antitumor growth activity compared to the reference AZD5305.

[0305] Experimental Example 8: The effect of compound 8 on promoting apoptosis in BRCA2-deficient cell lines

[0306] Mechanism analysis of the antiproliferative effect of the compound on BRCA-mutated tumors, and flow cytometry analysis of the compound's effect on apoptosis in BRCA2-mutated cell lines. BRCA2-deficient human colon cancer DLD-1 cells (ATCC, HTB-130) were cultured adherently in RPMI 1640 medium supplemented with 1% fetal bovine serum and 100 μg / mL hygromycin B. Once the cells entered the logarithmic growth phase, they were harvested and placed in 6-well plates (600,000 cells per well) for overnight incubation at 37°C / 5% CO2. Different concentrations of the compound were added to each well for incubation. After 7 days, the cells were harvested and placed in 96-well plates. 195 μL of Annexin V-FITC binding buffer was added to each well to resuspend the cells, followed by 5 μL of Annexin V-FITC (beyotime, C1052). The mixture was gently mixed and incubated at room temperature for 30 minutes. The cells were washed twice with PBS, centrifuged at 300g for 5 minutes, and 500 μL of PBS was added to each well for reselection. Finally, 5 μL of [the compound name is missing here, likely a continuation of the previous sentence] was added to each well. Gently mix with PI (beyotime, C1052), stain at room temperature for 30 minutes, wash once with PBS, centrifuge at 300g for 5 minutes, resuspend cells in 500μL PBS, and transfer 300μL of cells to a flow cytometry tube for analysis (BD Bioscience, FACSVerse). Analyze the data collected by the flow cytometer using FlowJo software.

[0307] Apoptosis Analysis: Drag the apoptotic sample into FlowJo and double-click the raw data to open the graph window. Select FSC-A for the X-axis and SSC-A for the Y-axis. Analyze the apoptotic cells using the cell population shown in the above figure. Cell debris is not analyzed. In the SSC / FSC graph, double-click "Analyzed Cell Population," select FITC for the X-axis to represent Annexin V-FITC, and PerCP for the Y-axis to represent PI. Use the quadrature gate tool to delineate live and apoptotic cells. Annexin V positive cells are apoptotic, Annexin V positive and PI negative cells are early apoptotic cells, and Annexin V positive and PI positive cells are late apoptotic cells. Use PRISM for plotting and analysis. Statistical analysis uses two-way ANOVA to include different compounds and concentrations in the analysis. Tukey's multiple comparisons test is applied, and p < 0.05 is considered statistically significant.

[0308] The results are shown in Figures 6-8. Three replicate experiments were conducted, and compound 12 induced apoptosis in significantly higher proportions of cells at different concentrations compared to the reference AZD5305, with some concentrations showing statistically significant differences. This suggests that compound 12 is superior to AZD5305 in inducing apoptosis in BRCA2-deficient human colon cancer DLD-1 cells.

[0309] Experimental Example 9: The effect of compound 9 on promoting apoptosis in BRCA1-mutated human breast cancer xenografts

[0310] To further validate the pro-apoptotic effects of the compounds in vivo, the changes in cleaved caspase-3 in subcutaneous xenograft tissue of MDA-MB-436 mice were detected by Western blotting. After 10 days of treatment with different compounds, animals were euthanized 0.25 hours and 24 hours after the last administration, and tumor tissue was collected for analysis. The flash-frozen tumor tissue was placed on dry ice, and 350 μL of complete cell lysis buffer (containing 1% protease inhibitor and phosphatase inhibitor) was added. The tissue was lysed using a tissue grinder for 5 minutes, and the lysate was placed on ice for 30 minutes. The tissue was centrifuged at 12,000 rpm and 4°C for 10 minutes, and the supernatant was transferred to a new 1.5 mL centrifuge tube. Protein quantification was performed using a BCA quantitative kit. Based on the quantification results, the sample protein concentration was adjusted to 2 μg / μL, and LDS loading buffer (4X) and sample reducing agent (10X) were added. The sample was heated at 100°C for 10 minutes. For Western blotting, load 10 μL of protein into each well of an SDS-PAGE gel and incubate at 80 V for 30 minutes; then perform electrophoresis at 120 V for 90 minutes. Transfer the membrane using an iBlot2 transfer kit and transfer apparatus for 7 minutes. Cut the membrane to the molecular weight of the protein to be detected. Wash the membrane three times with 1xTBST for 5 minutes each time. Add the primary antibodies Cleaved Caspase-3 (Asp175)(5A1E) Rabbit mAb (CST, 9664), Caspase-3 Antibody (CST, 9662), and β-Actin Antibody (CST, 4967) and incubate overnight at 4°C. Wash the membrane three times with 1xTBST for 10 minutes each time. Add the secondary antibody Goat anti-Rabbit IgG-HRP (Thermo Fisher, 31462) and incubate at room temperature for 1 hour. Wash the membrane three times with 1xTBST for 10 minutes each time. Add West HRP substrate from the Femto ultrasensitive chemiluminescence kit was used for chemiluminescence assay, which was detected and photographed on a Tanon 5200 Multi analyzer. Quantitative analysis was performed using Alpha View software to perform relative quantification of the density intensity of the immunoblot spectral bands. β-Actin, a housekeeping protein, was used to check the consistency of sample loading in the immunoblot assay. The density intensity of the cleaved caspase-3 band was standardized by comparing it with the density intensity of the total caspase-3 band. Then, the relative density intensity of cleaved caspase-3 in the Vehicle control group was set to 1, and the relative expression levels of cleaved caspase-3 in each treatment group were converted and plotted.The values ​​were analyzed graphically using Prism software, including the mean and standard error (SEM) of the relative expression levels of cleaved caspase-3 at each time point for each group. Differences between groups were compared, and one-way ANOVA was used for comparisons among multiple groups. For unequal variances (significant differences in F-values), the Games-Howell test was applied. If no significant difference was found in the F-values, Tukey's multiple comparisons test was used. A p-value < 0.05 was considered statistically significant.

[0311] The results, as shown in Figures 9 and 10-11, indicate that compound 12 promoted the increase of cleaved caspase-3 in subcutaneous xenograft tissue of MDA-MB-436 mice, with a greater increase than that of the reference AZD5305 at 0.25 hours and 24 hours after the last administration. This suggests that in vivo, compound 12 has a better effect on promoting tumor cell apoptosis than the reference AZD5305 in BRCA1-mutant MDA-MB-436 tumors, consistent with its better tumor-suppressive effect.

[0312] In summary, the compounds of this invention are highly selective PARP1 inhibitors, exhibiting superior tumor growth inhibition compared to AZD5305 in the BRCA1 MDA-MB-436 human breast cancer tumor model. This advantage is further confirmed by comparisons of their promotion of PARP1-DNA capture and induction of apoptosis. Compared to marketed PARP inhibitors, they significantly improve selectivity for PARP2, and compared to the reference AZD5305, they significantly improve selectivity for other PARP family members. This suggests that compounds of this invention, such as compound 12, hold promise as safer and more effective highly selective PARP1 inhibitors, providing higher quality treatment options for clinical patients.

Claims

1. A compound as shown in Formula I, 2. A method for preparing a compound as shown in Formula I, characterized in that, It includes the following steps: Compound 12 undergoes a salt-forming reaction with hydrochloric acid to obtain the compound shown in Formula I.

3. The method for preparing the compound as shown in Formula I according to claim 2, characterized in that, The salt-forming reaction satisfies one or more of the following conditions: 1) In the salt-forming reaction, the hydrochloric acid is added to the system in the form of a dimethyl sulfoxide solution of hydrochloric acid; 2) In the salt-forming reaction, the molar ratio of compound 12 to hydrochloric acid is 1:(1.0-1.5), preferably 1:(1.0-1.05); 3) The salt formation reaction is carried out in an organic solvent, for example, one or more of DMSO, DMF and NMP, preferably DMSO; 4) In the salt-forming reaction, the mass-to-volume ratio of compound 12 to the organic solvent is (0.05-0.3) g / mL. The preferred concentration is (0.1-0.2) g / mL, for example, 0.14 g / mL; 5) In the salt formation reaction, the reaction temperature is 10-30℃, preferably 20-40℃; 6) The reaction time is 0.5-24 hours, preferably 0.5-1 hour; and 7) The salt-forming reaction also includes post-processing steps, such as filtration, washing, and drying; Preferably, the salt-forming reaction specifically includes the following steps: Compound 12 is dissolved in an organic solvent, and hydrochloric acid solution is added dropwise at 10-30°C. After the reaction is complete, the temperature is raised to 75-85°C, and a poor solvent is added dropwise. The mixture is then cooled to crystallize. After post-treatment, filtration, washing, and drying, the compound shown in Formula I is obtained. Preferably, the hydrochloric acid solution is a dimethyl sulfoxide solution of hydrochloric acid, and the poor solvent is isopropyl acetate, acetone, or methyl tert-butyl ether, such as isopropyl acetate. The mass-to-volume ratio of compound 12 to the poor solvent is (0.03-0.2) g / mL, preferably (0.05-0.1) g / mL, for example, 0.077 g / mL. The washing in the post-treatment is with isopropyl acetate.

4. The method for preparing the compound as shown in Formula I according to claim 2, characterized in that, The method for preparing the compound shown in Formula I further includes a method for preparing compound 12, which specifically includes the following steps: compound 6 undergoes a substitution reaction with compound 11 to obtain compound 12.

5. The method for preparing the compound as shown in Formula I according to claim 4, characterized in that, Compound 6 and compound 11 undergo a substitution reaction in the presence of a base and an organic solvent to give compound 12; Preferably, the substitution reaction of compound 6 with compound 11 satisfies one or more of the following conditions: 1) In the substitution reaction, the molar ratio of compound 6 to compound 11 is 1:(0.9-1.1), preferably 1:(1.05-1.0); 2) In the substitution reaction, the base is N,N-diisopropylethylamine, triethylamine, or pyridine, preferably N,N-diisopropylethylamine; 3) In the substitution reaction, the mass ratio of compound 6 to the base is 1:(1-2.5), preferably 1:(1.5-1.9), for example 1:1.88; 4) In the substitution reaction, the organic solvent is one or more of DMSO, DMF, or NMP, preferably DMSO; 5) In the substitution reaction, the mass-to-volume ratio of compound 6 to the organic solvent is (0.1-0.5) g / mL, preferably (0.2-0.25) g / mL, for example 0.23 g / mL; 6) In the substitution reaction, the reaction temperature is 60-80℃, preferably 65-75℃; 7) The reaction time is 2-5 hours, preferably 2 hours; and 8) The substitution reaction also includes post-processing steps, such as crystallization, filtration, stirring, washing, filtration, drying and recrystallization; More preferably, the substitution reaction specifically includes the following steps: Compound 6, Compound 11, a base, and an organic solvent were stirred and heated to 65-75°C for reaction. After the reaction was completed, a crystallization solvent was added dropwise to induce crystallization, with the temperature controlled at 60-75°C. After the addition was complete, the temperature was lowered to 20-30°C and stirred. After post-treatment, filtration, washing, recrystallization, washing, and drying, Compound 12 was obtained. Preferably, the crystallization solvent was ethanol; the washing in the post-treatment was anhydrous ethanol; the recrystallization solvent in the post-treatment was DMSO; the recrystallization temperature was 20-30°C; and the washing after recrystallization in the post-treatment was with isopropyl acetate.

6. The method for preparing the compound as shown in Formula I according to claim 4, characterized in that, The preparation method of compound 12 further includes a preparation method of compound 6, specifically including the following steps: compound 5 undergoes a deprotection reaction to obtain compound 6.

7. The method for preparing the compound as shown in Formula I according to claim 6, characterized in that, Compound 5 was deprotected in the presence of an acid reagent and an organic solvent to give compound 6. Preferably, the deprotection reaction satisfies one or more of the following conditions: 1) In the deprotection reaction, the acid reagent is an ethyl hydrogen chloride solution; 2) In the deprotection reaction, the molar ratio of compound 5 to hydrogen chloride is 1:(3-5), preferably 1:4.0; 3) In the deprotection reaction, the concentration of the ethyl hydrogen chloride solution is 4 mol / L; 4) In the deprotection reaction, the organic solvent is one or more of dichloromethane, chloroform, or 1,2-dichloroethane, preferably dichloromethane; 5) In the deprotection reaction, the mass-to-volume ratio of compound 5 to the organic solvent is (0.3-1.5) g / mL. Preferably, it is (0.5-1) g / mL, for example, 0.77 g / mL; 6) In the deprotection reaction, the reaction temperature is 10-30℃, preferably 10-20℃; 7) The reaction time is 0.5-2 hours, preferably 0.5 hours; and 8) The deprotection reaction also includes post-processing steps, such as filtration, washing, and drying; More preferably, the deprotection reaction specifically includes the following steps: Compound 5 is dissolved in an organic solvent, and an acid reagent is added dropwise at 10-20°C. After the addition is complete, the temperature is raised to 15-20°C to react. After the reaction is completed, the mixture is filtered, washed, and dried to obtain compound 6. Preferably, the washing is performed with dichloromethane.

8. The method for preparing the compound as shown in Formula I according to claim 6, characterized in that, The preparation method of compound 6 further includes a preparation method of compound 5, which specifically includes the following steps: compound 4 undergoes an amide condensation reaction with deuterated methylamine or its salt to obtain compound 5.

9. The method for preparing the compound as shown in Formula I according to claim 8, characterized in that, Compound 4 reacts with the deuterated methylamine or its salt in the presence of a base, a condensing agent, and an organic solvent in an amide condensation reaction to yield compound 5; Preferably, the amide condensation reaction satisfies one or more of the following conditions: 1) In the amide condensation reaction, the deuterated methylamine salt is deuterated methylamine hydrochloride; 2) In the amide condensation reaction, the molar ratio of compound 4 to the deuterated methylamine or its salt is 1:(1.0-1.5), preferably 1:1.2; 3) In the amide condensation reaction, the base is DIEA; 4) In the amide condensation reaction, the mass ratio of compound 4 to the base is (0.5-1.5):1, preferably (0.6-1):1, for example 0.83:1; 5) In the amide condensation reaction, the condensation reagent is HATU or HBTU, preferably HATU; 6) In the amide condensation reaction, the molar ratio of compound 4 to the condensing reagent is 1:1.2; 7) In the amide condensation reaction, the organic solvent is one or more of DMF, DMA or DMSO, preferably DMF; 8) In the amide condensation reaction, the mass ratio of compound 4 to the organic solvent is (5-15):1, preferably (8-10):1, for example 9.5:1; 9) In the amide condensation reaction, the reaction temperature is 10-30℃, preferably 20-30℃; 10) The reaction time is 0.5-2 hours, preferably 0.5 hours; and 11) The amide condensation reaction further includes post-processing steps, such as extraction, washing, and vacuum concentration; More preferably, the amide condensation reaction specifically includes the following steps: Compound 4, a base, deuterated methylamine hydrochloride, and an organic solvent are stirred, and a condensation reagent is slowly added in batches while maintaining the temperature at 20-30°C. After the addition is complete, the mixture is kept at 20-30°C for further reaction. After the reaction is complete, compound 5 is obtained by post-treatment extraction, washing, and vacuum concentration. Preferably, the post-treatment is ethyl acetate extraction, washing with purified water, washing with saturated sodium bicarbonate solution, washing with saturated sodium chloride solution, and vacuum concentration to obtain compound 5.

10. The method for preparing the compound of formula I as described in claim 8, characterized in that, The method for preparing compound 5 further includes a method for preparing compound 4, which specifically includes the following steps: compound 3 undergoes a hydrolysis reaction to obtain compound 4.

11. The method for preparing the compound of formula I as described in claim 10, characterized in that, Compound 3 was hydrolyzed in the presence of a base and an organic solvent to give compound 4. Preferably, the hydrolysis reaction satisfies one or more of the following conditions: 1) In the hydrolysis reaction, the base is sodium hydroxide, potassium hydroxide or lithium hydroxide, preferably lithium hydroxide; 2) In the hydrolysis reaction, the alkali is added to the system in the form of an aqueous alkali solution; 3) In the hydrolysis reaction, the molar ratio of compound 3 to the base is 1:(1.5-2.5), preferably 1:1.8; 4) In the hydrolysis reaction, the mass-to-volume ratio of the alkali to the water is (0.03-0.15) g / mL, preferably (0.05-0.1) g / mL, for example 0.07 g / mL; 5) In the hydrolysis reaction, the organic solvent is one or more of dioxane, THF, or acetonitrile, preferably THF; 6) In the hydrolysis reaction, the mass-to-volume ratio of compound 3 to the organic solvent is (0.5-2) g / mL, preferably (0.8-1.5) g / mL, for example 1 g / mL; 7) In the hydrolysis reaction, the reaction temperature is 10-35℃, preferably 20-30℃; 8) The reaction time is 0.5-2 hours, preferably 0.5-1 hour; and 9) The hydrolysis reaction also includes post-processing steps, such as acidification, filtration, washing, drying, dissolution, washing and vacuum concentration; More preferably, the hydrolysis reaction specifically includes the following steps: Compound 3 was dissolved in an organic solvent, cooled to 15-20°C, and lithium hydroxide solution was added dropwise while maintaining the temperature at 15-25°C. After the addition was complete, the mixture was kept at 20-30°C for further reaction. After the reaction was completed, the mixture underwent post-treatment acidification, filtration, washing, drying, dissolution, washing, and vacuum concentration to obtain compound 4. Preferably, the acid used for acidification was hydrochloric acid; the washing after acidification was done with purified water; the dissolution was done with dichloromethane to dissolve the crude compound 4; and the washing after dissolution was done with a 10% sodium chloride aqueous solution.

12. The method for preparing the compound as shown in Formula I according to claim 10, characterized in that, The method for preparing compound 4 further includes a method for preparing compound 3, which specifically includes the following steps: compound 2 undergoes a carbonylation reaction to obtain compound 3.

13. The method for preparing the compound as shown in Formula I according to claim 12, characterized in that, Under a CO atmosphere, compound 2 reacts with methanol in an alkaline, catalytic, and organic solvent reaction to yield compound 3; Preferably, compound 2 undergoes a carbonylation reaction satisfying one or more of the following conditions: 1) In the carbonyl insertion reaction, the pressure is 0.8-2.0 MPa under a CO atmosphere, preferably (1.2-1.3) MPa; 2) In the carbonylation reaction, the mass-to-volume ratio of compound 2 to methanol is (0.1-1) g / mL, preferably (0.3-0.8) g / mL, for example 0.5 g / mL; 3) In the carbonylation reaction, the base is TEA, sodium acetate or potassium acetate, preferably TEA; 4) In the carbonylation reaction, the mass ratio of compound 2 to the base is (0.8-2):1, preferably (1-1.5):1, for example 1.19:1; 5) In the carbonyl insertion reaction, the catalyst is a palladium catalyst, preferably Pd(dppf)Cl2; 6) In the carbonylation reaction, the mass ratio of compound 2 to the catalyst is (11-13):1, preferably (12-12.5):1, for example 12.3:1; 7) The organic solvent is one or more of dioxane, THF, and 2-methyltetrahydrofuran, preferably dioxane; 8) In the carbonylation reaction, the mass-to-volume ratio of compound 2 to the organic solvent is (0.1-0.5) g / mL, preferably (0.1-0.3) g / mL, for example 0.2 g / mL; 9) In the carbonyl insertion reaction, the reaction temperature is 90-110℃, preferably 100-105℃; 10) The reaction time is 5-10 hours, preferably 8 hours; and 11) The carbonyl insertion reaction also includes post-processing steps, such as concentration, filtration, washing, concentration, dissolution, column chromatography, concentration, crystallization and drying; More preferably, the carbonyl insertion reaction specifically includes the following steps: Compound 2 was added under stirring with an organic solvent and methanol, followed by the addition of alkali and catalyst. After the addition was complete, the mixture was purged with nitrogen three times and then with carbon monoxide twice. The pressure was controlled by introducing carbon monoxide, and the temperature was raised to 100-105°C for reaction. After the reaction was completed, the mixture underwent post-treatment, concentration, filtration, washing, concentration, dissolution, column chromatography, concentration, crystallization, and drying to obtain compound 3. Preferably, the concentrated solution was filtered through diatomaceous earth; the washing was performed with ethyl acetate; the dissolution was performed by dissolving the concentrate in dichloromethane; the eluent for column chromatography was PE:EA = 3:1; the crystallization was performed by crystallizing the crude compound 3 in a mixed solution of methyl ether and n-hexane, wherein the volume ratio of methyl ether to n-hexane in the mixed solution was 1:1, and the crystallization temperature was 20-30°C.

14. The method for preparing the compound of formula I as described in claim 12, characterized in that, The preparation method of compound 3 further includes a preparation method of compound 2, which specifically includes the following steps: compound 1 undergoes a bromination reaction with a brominating reagent to obtain compound 2.

15. The method for preparing the compound of formula I as described in claim 14, characterized in that, The bromination reaction satisfies one or more of the following conditions: 1) The compound 1 and the brominating agent undergo a bromination reaction in the presence of an organic solvent, for example, the organic solvent is one or more of DMF, DMSO or DMA, preferably DMF; 2) In the bromination reaction, the brominating agent is one or more of NBS, liquid bromine, DBH or NBP, preferably NBS; 3) In the bromination reaction, the molar ratio of compound 1 to the brominating reagent is 1:(1.2-2.5), preferably 1:1.8; 4) In the bromination reaction, the mass-to-volume ratio of compound 1 to the organic solvent is (0.15-0.4) g / mL, preferably (0.2-0.3) g / mL, for example 0.24 g / mL; 5) In the bromination reaction, the reaction temperature is 10-30℃, preferably 20-30℃; 6) The reaction time is 2-5 hours, preferably 2-3 hours; and 7) The bromination reaction also includes post-processing steps, such as extraction, washing, and concentration; Preferably, the bromination reaction specifically includes the following steps: Compound 1 is dissolved in an organic solvent, cooled to 10-15°C, and a brominating reagent is added in batches while maintaining the temperature at 10-25°C. After the addition is complete, the reaction is carried out at 20-30°C. After the reaction is completed, compound 2 is obtained by post-treatment extraction, washing, and concentration. Preferably, the extraction in the post-treatment is ethyl acetate extraction. The washing is performed sequentially with a 10% sodium chloride aqueous solution, a 10% sodium bisulfite aqueous solution, and a 10% sodium chloride aqueous solution.

16. The method for preparing the compound of formula I as described in claim 14, characterized in that, The method for preparing compound 2 further includes a method for preparing compound 1, which specifically comprises the following steps: 2-fluoro-3-bromopyridine undergoes a Buchwald-Hartwig coupling reaction with N-Boc piperazine to obtain compound 1.

17. The method for preparing the compound of formula I as described in claim 16, characterized in that, The 2-fluoro-3-bromopyridine and the N-Boc piperazine underwent a Buchwald-Hartwig coupling reaction in the presence of a base, a phosphorus ligand, a palladium catalyst, and an organic solvent to give compound 1. Preferably, the Buchwald-Hartwig coupling reaction satisfies one or more of the following conditions: 1) In the Buchwald-Hartwig coupling reaction, the molar ratio of 2-fluoro-3-bromopyridine to N-Boc piperazine is 1:(1.2-2.0), for example 1:1.5; 2) In the Buchwald-Hartwig coupling reaction, the base is cesium carbonate, sodium tert-butoxide, or potassium tert-butoxide, preferably cesium carbonate; 3) The mass ratio of the 2-fluoro-3-bromopyridine to the base is (0.1-0.25):1, preferably (0.15-0.2):1, for example 0.18:

1. 4) In the Buchwald-Hartwig coupling reaction, the phosphorus ligand is Xant-Phos; 5) In the Buchwald-Hartwig coupling reaction, the mass ratio of the 2-fluoro-3-bromopyridine to the phosphorus ligand is (1.5-3):1, preferably (2-2.8):1, for example 2.55:1; 6) In the Buchwald-Hartwig coupling reaction, the palladium catalyst is Pd2(dba)3 or Pd(OAc)2, preferably Pd2(dba)3; 7) In the Buchwald-Hartwig coupling reaction, the mass ratio of 2-fluoro-3-bromopyridine to the catalyst is (2.5-4):1, preferably (3-3.5):1, for example 3.2:1; 8) In the Buchwald-Hartwig coupling reaction, the organic solvent is one or more of tetrahydrofuran, 1,4-dioxane or methyl tert-butyl ether, preferably dioxane; 9) In the Buchwald-Hartwig coupling reaction, the mass-to-volume ratio of the 2-fluoro-3-bromopyridine to the organic solvent is (0.05-0.2) g / mL, preferably (0.08-0.15) g / mL, for example 0.1 g / mL; 10) In the Buchwald-Hartwig coupling reaction, the reaction temperature is 90-110℃, preferably 100-105℃; 11) The reaction time is 5-12 hours, preferably 6 hours; and 12) The Buchwald-Hartwig coupling reaction also includes post-processing steps, such as filtration, washing, pulping, filtration, concentration, washing and concentration. More preferably, the Buchwald-Hartwig coupling reaction specifically includes the following steps: 2-Fluoro-3-bromopyridine, N-Boc piperazine, a base, a phosphorus ligand, and an organic solvent are stirred and heated to 100-105°C for reaction. After the reaction is completed, the mixture is subjected to post-treatment filtration, extraction, washing, drying, and vacuum concentration to obtain compound 1. Preferably, the filtration is diatomaceous earth filtration; the extraction is ethyl acetate extraction; the washing is washing with saturated ammonium chloride aqueous solution; and the drying is drying with anhydrous sodium sulfate.

18. The method for preparing the compound of formula I as described in claim 4, characterized in that, The method for preparing compound 12 further includes a method for preparing compound 11, which includes the following steps: in the presence of an acid-binding agent and an organic solvent, compound 10 is subjected to a chlorination reaction to obtain compound 11.

19. The method for preparing the compound of formula I as described in claim 18, characterized in that, The chlorinating reagent satisfies one or more of the following conditions: 1) In the chlorination reaction, the chlorinating reagent is thionyl chloride; 2) In the chlorination reaction, the molar ratio of compound 10 to the chlorination reagent is 1:(2-5), preferably 1:3; 3) In the chlorination reaction, the acid-binding agent is N,N-diisopropylethylamine, triethylamine, or pyridine, preferably N,N-diisopropylethylamine; 4) In the chlorination reaction, the mass ratio of compound 10 to the acid-binding agent is (2.5-4):1, preferably (3-3.5):1, for example 3.39:1; 5) In the chlorination reaction, the organic solvent is tetrahydrofuran or dioxane, preferably tetrahydrofuran; 6) In the chlorination reaction, the mass-to-volume ratio of compound 10 to the organic solvent is (0.01-0.1) g / mL, preferably (0.05-0.08) g / mL, for example 0.067 g / mL; 7) In the chlorination reaction, the reaction temperature is 15-30℃, preferably 20-25℃; 8) The reaction time is 0.5-2 hours, preferably 1 hour; and 9) The chlorination reaction also includes post-processing steps, such as filtration, washing, drying, pulping, filtration and drying; Preferably, the chlorination reaction specifically includes the following steps: The organic solvent, compound 10, and acid-binding agent are stirred and mixed, and the temperature is lowered to 15-20°C. A chlorinating reagent is added dropwise while controlling the temperature at 15-25°C. After the addition is complete, the reaction is maintained at 20-25°C. After the reaction is completed, compound 11 is obtained by post-processing, filtration, washing, drying, pulping, filtration, and drying. Preferably, the washing is tetrahydrofuran washing, and the pulping is acetonitrile pulping.

20. The method for preparing the compound of formula I as described in claim 18, characterized in that, The preparation method of compound 11 further includes a preparation method of compound 10, which specifically includes the following steps: under the condition of the presence of an organic solvent, compound 9 is reduced with a reducing agent to obtain compound 10, wherein the reducing agent is lithium aluminum hydride, sodium borohydride or trisec-butyl borohydride.

21. The method for preparing the compound as shown in Formula I according to claim 20, characterized in that, The reduction reaction satisfies one or more of the following conditions: 1) In the reduction reaction, the organic solvent is THF or dioxane, preferably THF; 2) In the reduction reaction, the mass ratio of compound 9 to the organic solvent is (0.01-0.05):1, preferably (0.02-0.04):1, for example 0.03:1; 3) In the reduction reaction, the reducing agent is lithium aluminum hydride, for example, the reducing agent is lithium aluminum hydride added to the reaction system in the form of lithium aluminum hydride tetrahydrofuran solution; 4) In the reduction reaction, the concentration of the lithium aluminum hydride tetrahydrofuran solution is (2-3) M, preferably 2.5 M; 5) In the reduction reaction, the molar ratio of compound 9 to the reducing reagent is 1:(1-2), preferably 1:1.2; 6) In the reduction reaction, the reaction temperature is 10-30℃, preferably 10-20℃; 7) The reaction time is 12-24 hours, preferably 16 hours; and 8) The reduction reaction also includes post-processing steps, such as quenching, separation, extraction, concentration, pulping, filtration and drying; Preferably, the reduction reaction specifically includes the following steps: Compound 9 is dissolved in an organic solvent, cooled to -5 to -5°C under nitrogen protection, and a reducing agent (e.g., a solution of lithium aluminum hydride and tetrahydrofuran) is added dropwise. After the addition is complete, the temperature is raised to 10 to 20°C and the reaction is stirred to stop the reaction. After post-treatment quenching, separation, extraction, concentration, pulping, filtration, and drying, compound 10 is obtained. Preferably, the quenching is performed by adding purified water; the separation is performed by adding a 5% (w / w) NaOH aqueous solution and stirring; the extraction is performed by tetrahydrofuran extraction; and the pulping is performed by pulping with purified water.

22. The method for preparing the compound as shown in Formula I according to claim 20, characterized in that, The preparation method of compound 10 further includes a preparation method of compound 9, which specifically includes the following steps: under a CO atmosphere, compound 8 and methanol undergo an intercalation reaction in an alkali, a palladium catalyst, and an organic solvent to generate compound 9, wherein the pressure is 0.8-2.0 MPa.

23. The method for preparing the compound as shown in Formula I according to claim 22, characterized in that, The carbonyl insertion reaction satisfies one or more of the following conditions: 1) In the carbonyl insertion reaction, the pressure is 1 MPa under a CO atmosphere; 2) In the carbonylation reaction, the mass ratio of compound 8 to methanol is (0.3-1):1, preferably (0.5-0.8):1, for example 0.64:1; 3) In the carbonylation reaction, the base is TEA, sodium acetate or potassium acetate, preferably triethylamine; 4) In the carbonylation reaction, the molar ratio of compound 8 to the base is (0.1-0.8):1, preferably (0.2-0.5):1, for example 0.33:1; 5) In the carbonyl insertion reaction, the palladium catalyst is [1,1-bis(diphenylphosphine)ferrocene]palladium dichloromethane; 6) In the carbonylation reaction, the molar ratio of compound 8 to the catalyst is (15-21):1, preferably (19-20.5):1, for example 20.2:1; 7) In the carbonylation reaction, the organic solvent is one or more of dioxane, tetrahydrofuran or 2-methyltetrahydrofuran, preferably dioxane; 8) In the carbonylation reaction, the mass ratio of compound 8 to the organic solvent is (0.1-0.25):1, preferably (0.12-0.2):1, for example 0.16:1; 9) In the carbonyl insertion reaction, the reaction temperature is 80-110℃, preferably 80-90℃; 10) The reaction time is 2-10 hours, preferably 5-8 hours; and 11) The carbonylation reaction also includes post-processing steps, such as filtration, pulping, filtration and drying; Preferably, the carbonyl insertion reaction specifically includes the following steps: Organic solvent, methanol, compound 8, alkali and palladium catalyst are added sequentially to a high-pressure reactor. The reaction is carried out under a carbon monoxide atmosphere at 80-90°C with stirring. After the reaction is completed, compound 9 is obtained by post-treatment filtration, pulping, filtration and drying. Preferably, the pulping is methanol pulping.

24. The method for preparing the compound as shown in Formula I according to claim 22, characterized in that, The preparation method of compound 9 further includes a preparation method of compound 8, which specifically includes the following steps: compound 7 undergoes a cyclization reaction with n-butyryl chloride to obtain compound 8.

25. The method for preparing the compound as shown in Formula I according to claim 24, characterized in that, Compound 7 undergoes a cyclization reaction with n-butyryl chloride in the presence of a base and an organic solvent to give compound 8. Preferably, the cyclization reaction satisfies one or more of the following conditions: 1) In the cyclization reaction, the molar ratio of compound 7 to n-butyryl chloride is 1:(1-2), preferably 1:1.5; 2) In the cyclization reaction, the base is one or more of DMAP, pyridine, or potassium tert-butoxide, preferably DMAP, pyridine, and potassium tert-butoxide; 3) In the cyclization reaction, the molar ratio of compound 7 to DMAP is (9-11):1, preferably (9.5-10.5):1, for example 10:1; 4) In the cyclization reaction, the molar ratio of compound 7 to pyridine is (9-11):1, preferably (9.5-10.5):1, for example 10:1; 5) In the cyclization reaction, the molar ratio of compound 7 to potassium tert-butoxide is 1:(5-7), preferably 1:6; 6) In the cyclization reaction, the organic solvent is one or more of toluene, THF, or dioxane, preferably tetrahydrofuran; 7) In the cyclization reaction, the mass ratio of compound 7 to the organic solvent is (0.08-0.2):1, preferably (0.1-0.15):1, for example 0.11:1; 8) In the cyclization reaction, the reaction temperature is 10-70℃, preferably 20-65℃; 9) The reaction time is 3-10 hours, preferably 3-5 hours; and 10) The cyclization reaction also includes post-processing steps, such as concentration, crystallization, filtration, pulping, filtration and drying; More preferably, the cyclization reaction specifically includes the following steps: The organic solvent, compound 7, and alkali are mixed, and the mixture is cooled to 10-20°C under nitrogen protection. Butyryl chloride is added dropwise, and the mixture is heated to 20-30°C and stirred to react. After the reaction is completed, the mixture is concentrated, crystallized, filtered, pulped, filtered again, and dried to obtain compound 8. Preferably, the crystallization is performed using purified water, and the pulping is performed using purified water.

26. The method for preparing the compound of formula I as described in claim 24, characterized in that, The method for preparing compound 8 further includes a method for preparing compound 7, which specifically includes the following steps: reducing 5-bromo-2-cyano-3-nitropyridine to obtain compound 7.

27. The method for preparing the compound as shown in Formula I according to claim 26, characterized in that, The 5-bromo-2-cyano-3-nitropyridine was reduced with a reducing agent in an organic solvent to give compound 7; Preferably, the reduction reaction satisfies one or more of the following conditions: 1) The 5-bromo-2-cyano-3-nitropyridine was added to the system in the form of a 5-bromo-2-cyano-3-nitropyridine acetic acid solution; 2) In the reduction reaction, the reducing agent is iron powder; 3) In the reduction reaction, the molar ratio of 5-bromo-2-cyano-3-nitropyridine to the reducing agent is 1:(5-7), preferably 1:5.5; 4) In the reduction reaction, the organic solvent is acetic acid; 5) In the reduction reaction, the mass ratio of 5-bromo-2-cyano-3-nitropyridine to the organic solvent is (0.05-0.15):1, preferably (0.08-0.11):1, for example 0.1:1; 6) In the reduction reaction, the reaction temperature is 10-40℃, preferably 10-30℃; 7) The reaction time is 3-5 hours, preferably 3 hours; and 8) The reduction reaction also includes post-processing steps, such as filtration, pulping, filtration, pulping, concentration, pulping, filtration, pulping, filtration and drying; More preferably, the reduction reaction specifically includes the following steps: In an organic solvent, a reducing agent is added at 20-30°C, and a solution of 5-bromo-2-cyano-3-nitropyridineacetic acid is slowly added dropwise. The reaction is stirred at 20-30°C. After the reaction is completed, the post-treatment involves filtration, pulping, filtration, pulping, concentration, pulping, filtration, pulping, filtration, and drying to obtain compound 7. Preferably, the post-treatment involves filtration, THF pulping, filtration, THF pulping, concentration, purified water pulping, filtration, pulping with a mixed solution of dichloromethane and n-hexane, filtration, and drying.

28. A method for preparing compound 12, characterized in that, It includes the following steps: compound 6 undergoes a substitution reaction with compound 11 to obtain compound 12. Preferably, the method for preparing compound 12 is as described in any one of claims 5-27.

29. A compound 2, 30. A method for preparing compound 2, characterized in that, It includes the following steps: Compound 1 undergoes a bromination reaction with a brominating reagent to obtain Compound 2. Preferably, the method for preparing compound 2 is as described in any one of claims 15-17.

31. A method for preparing compound 3, characterized in that, It includes the following steps: Compound 2 undergoes a carbonylation reaction to obtain Compound 3. Preferably, the method for preparing compound 3 is as described in any one of claims 13-17.

32. A method for preparing compound 11, characterized in that, It includes the following steps: compound 10 is subjected to a chlorination reaction to obtain compound 11. Preferably, the method for preparing compound 11 is as described in any one of claims 19-27.

33. The use of the compound of Formula I as claimed in claim 1 in the preparation of a medicament for treating or preventing PARP-mediated diseases, preferably, the diseases being selected from cancer, ischemic diseases, and neurodegenerative diseases; more preferably, the medicament is used to treat or prevent cancer, for example, cancer lacking the HR-dependent DNA DSB repair pathway, cancer having a BRCA1 or BRCA2 defect phenotype, and cancer being selected from breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, hematologic malignancies, gastrointestinal cancer, and lung cancer.

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