PARP7 inhibitor for cancer immunotherapy

Abstract

The invention discloses a PARP7 inhibitor for cancer immunotherapy, and belongs to the field of chemical medicines. The invention provides an azacyclo-substituted pyrimidine compound with a structure as shown in a general formula (I), the compound can be used as a PARP7 inhibitor, the activity or selectivity of the compound is remarkably improved compared with that of existing active compounds RBN-2397 and S-XY-05, and the compound can be used for treating and / or preventing PARP7 regulated diseases, such as hyperproliferative diseases, autoimmune or inflammatory diseases and the like. In addition, the compound or the salt thereof of the present invention has little side effects, and has good pharmacometabolism and pharmacokinetics.

Description

Technical Field

[0001] This invention belongs to the field of chemical medicine, and specifically relates to a PARP7 inhibitor for cancer immunotherapy. Background Technology

[0002] Studies have shown that most members of the poly(ADP-ribose) polymerase (PARP) family in the human body exhibit monoADP-ribotransferase activity. The MonoPARP protein family is closely associated with the development of cancer, inflammation, and neurodegenerative diseases. PARP7, a member of the monoPARP protein family, is a novel negative regulator of nucleic acid sensors in cells and is overexpressed in various tumor cells. PARP7 regulates multiple cellular processes and biological functions, including innate immune regulation, coronavirus infection, and the progression of ischemic stroke. The cGAS STING pathway in tumor immunotherapy is widely recognized. However, in various tumors, PARP7 inhibits the phosphorylation of TANK-binding kinase 1 (TBK1), thereby suppressing its activation of the cGAS STING signaling pathway. Since cancer cells can use PARP7 to suppress interferon signaling, allowing them to "hide" outside the immune system, many cancer cells depend on PARP7 for survival. Research has found that inhibiting PARP7 can restore intracellular interferon signaling, restore the body's innate and adaptive immunity, and thus inhibit cancer cell growth. In cancer models such as lung cancer and colorectal cancer, PARP7 inhibitors have shown durable tumor growth inhibition.

[0003] Currently, no PARP7 inhibitors have been approved for marketing. RBN-2397, developed by Ribon, is the first compound with strong inhibitory activity and selectivity against PARP7 and is currently in Phase I clinical trials (NCT04053673). However, RBN-2397 has a high clearance rate in vivo, resulting in low in vivo exposure and oral bioavailability. Furthermore, the inhibitory activity of RBN-2397 as a monotherapy is limited, and it currently needs to be combined with CYP450 inhibitors to reduce its clearance rate in order to exert its effect. Summary of the Invention

[0004] [Technical Issues]

[0005] This invention provides a PARP7 inhibitor with superior inhibitory activity compared to existing active compounds, offering a better treatment or alleviation method for PARP7-mediated diseases such as hyperproliferative disorders, autoimmune diseases, or inflammatory diseases.

[0006] [Technical Solution]

[0007] This invention provides a class of novel compounds that can be used as PARP7 antagonists, including novel compounds with PARP7 inhibitory activity and / or good pharmacodynamic / pharmacokinetic properties, as well as pharmaceutically acceptable salts thereof, and their use in treating or alleviating PARP7-mediated diseases such as hyperproliferative disorders, autoimmune or inflammatory diseases.

[0008] Detailed plan content:

[0009] One object of the present invention is to provide nitrogen-heterocyclic substituted pyrimidine compounds of the structure shown in general formula (I), or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope-labeled compounds, or prodrugs thereof:

[0010]

[0011] In the formula:

[0012] A is selected from:

[0013]

[0014] L is selected from:

[0015]

[0016] Furthermore, A is either A1 or A2, and L is either L1 or L2;

[0017] Alternatively, A can be A3 or A4, and L can be any of L1, L2 or L3.

[0018] In this invention, Indicates the connection site.

[0019] Preferably, the present invention specifically provides the following nitrogen-substituted pyrimidine compounds:

[0020]

[0021] The present invention relates to compounds of general formula (I) or pharmaceutically acceptable salts thereof, wherein the pharmaceutically acceptable salt is an inorganic salt or an organic salt, wherein the inorganic salt is selected from hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, nitrate, phosphate, and acid phosphate; and wherein the organic salt is selected from acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, sulfonate, benzenesulfonate, and salicylate.

[0022] On the other hand, the present invention provides the use of compounds of the above general formula (I) or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope labels or prodrugs thereof in the preparation of drugs for use as inhibitors of the PARP7 signaling pathway.

[0023] In another aspect, the present invention also provides the use of compounds of the above general formula (I) or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope labels or prodrugs thereof in the preparation of medicaments for treating or alleviating PARP7-mediated diseases.

[0024] PARP7-mediated diseases include: hyperproliferative disorders, autoimmune or inflammatory diseases, etc.

[0025] PARP7-mediated diseases include cancers such as breast cancer, central nervous system cancer, endometrial cancer, kidney cancer, colorectal cancer, lung cancer, esophageal cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, head and neck cancer (upper respiratory and digestive tract cancers), urethral cancer, colon cancer, leukemia, lymphoma, liver cancer (e.g., hepatocellular carcinoma), bladder cancer, bone cancer, glioma, breast cancer, cervical cancer, colon cancer, endometrial cancer, epithelial cancer, esophageal cancer, Ewing's sarcoma, pancreatic cancer, gallbladder cancer, stomach cancer, gastrointestinal tumors, head and neck cancer (upper respiratory and digestive tract cancers), intestinal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, and uterine cancer.

[0026] PARP7-mediated diseases also include cardiology, virology, neurodegeneration, inflammation, and pain.

[0027] Furthermore, the present invention also provides the use of compounds of the above general formula (I) or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope labels or prodrugs in medicaments for treating acute, subacute or chronic liver diseases.

[0028] The present invention also provides a method for preparing compounds of general formula (I).

[0029] The present invention also provides pharmaceutical compositions comprising the above-described compound of general formula (I) of the present invention or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label or prodrug, and pharmaceutical excipients.

[0030] The pharmaceutical excipients include pharmaceutically acceptable carriers, excipients, or diluents.

[0031] Pharmaceutically acceptable carriers include microspheres, nanoparticles, and liposomes.

[0032] In one embodiment of the present invention, the dosage form of the pharmaceutical composition includes injection, lyophilized powder for injection, suspension, implant, embolization, capsule, tablet, pill and oral liquid.

[0033] [Beneficial Effects]

[0034] The compounds of this invention can serve as PARP7 inhibitors, exhibiting significantly improved activity or selectivity compared to existing inhibitors (RBN-2397, S-XY-05), and can be used for the treatment and / or prevention of PARP7-regulated diseases, such as hyperproliferative disorders, autoimmune diseases, or inflammatory diseases. Furthermore, the compounds of this invention, or their salts, have virtually no side effects and exhibit favorable drug metabolism and pharmacokinetics. Therefore, the compounds designed in this invention have excellent prospects for development and application. Detailed Implementation

[0035] The technical solution of the present invention will be described in detail below with reference to the embodiments.

[0036] In this invention, “administering” or “giving” an individual compound means providing the compound of this invention to an individual in need of treatment.

[0037] The term "pharmaceutically acceptable salt" refers to a salt that, within reasonable medical judgment, is suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and has a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art.

[0038] Unless otherwise specified, all stereoisomers, such as enantiomers and diastereomers, are contemplated. Compounds of the present invention containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic form. Methods for preparing optically active forms from optically inactive raw materials are known in the art, for example by resolving racemic mixtures or by stereoselective synthesis. Geometric isomers of alkenes, C=N double bonds, etc., may also be present in the compounds described herein, and all such stable isomers are covered in this invention. Cis and trans geometric isomers of the compounds of the present invention can be isolated in the form of mixtures of isomers or as separate isomers.

[0039] The compounds of this invention also include tautomeric forms. Tautomeric forms arise from the exchange of single bonds with adjacent double bonds and the accompanying proton migration. Tautomeric forms include proton transfer tautomers in isoprotonated states having the same empirical formula and total charge. Exemplary proton transfer tautomers include keto-enol pairs, amide-imine pairs, lactam-lactamimide pairs, enamine-imine pairs, and cyclic forms in which protons can occupy two or more positions in a heterocyclic system, such as 1H- and 3H-imidazolium, 1H-, 2H- and 4H-1,2,4-triazoles, 1H- and 2H-isoindole, and 1H- and 2H-pyrazoles. Tautomeric forms can be in equilibrium or spatially locked to one form through appropriate substitution. Examples of tautomeric forms, pyridazine-3(2H)-one and pyridazine-3-ol, are described below:

[0040]

[0041] Unless otherwise specified, the term “compound” as used herein is intended to include all stereoisomers, geometric isomers, tautomers and isotopes of the structure described.

[0042] All compounds and their pharmaceutically acceptable salts can be found together with other substances, such as water and solvents (e.g., in hydrate and solvate form) or can be separated.

[0043] The present invention also provides pharmaceutical compositions comprising the above-described compound of general formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0044] The compounds of the present invention or pharmaceutically acceptable salts thereof can be formulated into solid dosage forms for oral administration, including, but not limited to, capsules, tablets, pills, powders, granules, etc. In these solid dosage forms, the compound of general formula (I) of the present invention is mixed as the active ingredient with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate. Or it may be mixed with the following components: (1) fillers or solubilizers, such as starch, lactose, sucrose, glucose, mannitol and silica; (2) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, gum arabic; (3) humectants, such as glycerin; (4) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginic acid, certain silicates and sodium carbonate; (5) slowing agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glyceryl monostearate; (8) adsorbents, such as kaolin; (9) lubricants, such as talc, calcium stearate, solid polyethylene glycol, sodium dodecyl sulfate, etc., or mixtures thereof. Buffers may also be included in capsules, tablets and pills.

[0045] The solid dosage forms, such as tablets, sugar pills, capsules, pellets, and granules, can be coated or microencapsulated with coating and shell materials such as enteric coatings and other materials known in the art. They may contain opaque agents, and the release of the active ingredient from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active ingredient may also be formed into microcapsules with one or more of the excipients described above.

[0046] The compounds of the present invention or pharmaceutically acceptable salts thereof can be formulated into liquid dosage forms for oral administration, including, but not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, tinctures, etc. In addition to the compound of general formula (I) or its pharmaceutically acceptable salt as the active ingredient, the liquid dosage form may contain inert diluents conventionally used in the art, such as water and other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn oil, olive oil, castor oil, sesame oil, etc., or mixtures thereof. In addition to these inert diluents, the liquid dosage forms of the present invention may also include conventional adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0047] The suspending agent includes, for example, ethoxylated octadecyl alcohol, polyoxyethylene sorbitol, and dehydrated sorbitol, microcrystalline cellulose, agar, or mixtures thereof.

[0048] The compounds of this invention and their pharmaceutically acceptable salts can be formulated into dosage forms for parenteral injection, including, but not limited to, physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions and dispersions. Suitable carriers, diluents, solvents, and excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0049] The compounds of this invention or pharmaceutically acceptable salts thereof can be formulated into dosage forms for topical administration, including ointments, powders, suppositories, drops, sprays, and inhalers. The compounds of general formula (I) of this invention or pharmaceutically acceptable salts thereof, as active ingredients, are mixed under sterile conditions with a physiologically acceptable carrier and optionally with preservatives, buffers, and propellants, if necessary.

[0050] The pharmaceutical compositions of the present invention comprise a compound of general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier, excipient, and diluent. In preparing the pharmaceutical compositions, the compound of general formula (I) or a pharmaceutically acceptable salt thereof is typically mixed with a pharmaceutically acceptable carrier, excipient, or diluent. The content of the compound of general formula (I) or a pharmaceutically acceptable salt thereof can be 0.01-1000 mg, for example 0.05-800 mg, 0.1-500 mg, 0.01-300 mg, 0.01-200 mg, 0.05-150 mg, 0.05-50 mg, etc.

[0051] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. The technical solutions of the present invention will be described in detail below with reference to the embodiments.

[0052] The following examples are illustrative and not limiting of the synthesis of compounds of general formula (I). All temperatures are in degrees Celsius. Unless otherwise stated, all evaporations were performed under reduced pressure. Unless otherwise stated, reagents were purchased from commercial suppliers and used without further purification. The structures of the final products, intermediates, and starting materials were confirmed by standard analytical methods, such as elemental analysis and spectroscopic characterization, such as MS and NMR. Abbreviations used are conventional abbreviations in the art.

[0053] Example 1: (S)-5-((1-(3-oxo-3-(1-(5-(trifluoromethyl)pyrimidin-2-yl)-5,6-2H-1,2,4-triazine-4(1H)-yl)propoxy)propane-2-yl)amino)-4-(trifluoromethyl)pyridazine-3(2H)-one (I)

[0054]

[0055] Step 1: Preparation of tert-butyl-(2-((5-(trifluoromethyl)pyrimidin-2-yl)amino)ethyl)carbamate (I-3)

[0056] N,N-diisopropylethylamine (DIEA) (55.0 mL, 121 mmol) was added to a DMF (200 mL) solution of 2-chloro-5-(trifluoromethyl)pyrimidine (starting material I-1, 20.0 g, 110 mmol), followed by the addition of tert-butyl (2-aminoethyl)carbamate (19.3 g, 340 mmol). The reaction mixture was shaken at room temperature for 2 h. LC / MS indicated the reaction was complete. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give intermediate I-3 (25.2 g, 75% yield). MS-ESI (m / z): 307.1 [M+l] + .

[0057] Step 2: Preparation of tert-butyl-(2-(1-(5-(trifluoromethyl)pyrimidin-2-yl)hydrazyl)ethyl)carbamate (I-4)

[0058] 25.2 g (82.3 mmol) of tert-butyl-(2-((5-(trifluoromethyl)pyrimidin-2-yl)amino)ethyl)carbamate was suspended in 100 mL (600 mmol) of 6N hydrochloric acid aqueous solution. The solution was cooled to 0 °C, and 20 mL of water solution containing 8.51 g (123 mmol) of sodium nitrite was slowly added. The mixture was stirred at 0 °C for 15 minutes, then at room temperature for 30 minutes. Stannous chloride dihydrate (37.2 g (165 mmol)) was then added at 0 °C, and the mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was neutralized with 1N sodium hydroxide and extracted with ethyl acetate (150 mL x 3). The combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. Further purification by column chromatography yielded a white solid intermediate I-4 (19.3 g, yield: 73%).

[0059] MS-ESI (m / z): 322.2 [M+l] + .

[0060] Step 3: Preparation of 2-(1-(5-(trifluoromethyl)pyrimidin-2-yl)hydrazino)ethyl-1-amine (I-5)

[0061] Trifluoroacetic acid (TFA) (4.91 mL, 66 mmol) was added to a solution of tert-butyl-(2-(1-(5-(trifluoromethyl)pyrimidin-2-yl)hydrazino)ethyl)carbamate (19.3 g, 60 mmol) in dichloromethane (200 mL), and the reaction mixture was stirred at room temperature for 2 h. LC / MS analysis showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure to give a yellow solid, which was used without further purification.

[0062] MS-ESI (m / z): 222.1 [M+l] + .

[0063] Step 4: Preparation of 1-(5-(trifluoromethyl)pyrimidin-2-yl)-1,4,5,6-tetrahydro-1,2,4-triazine (I-6)

[0064] Trimethoxymethane (14.4 g, 136 mmol) was added to a methanol (100 mL) solution of the above-mentioned 2-(1-(5-(trifluoromethyl)pyrimidin-2-yl)hydrazino)ethyl-1-amine reaction mixture, and the mixture was stirred under reflux at 65 °C for 1 hour. LC / MS analysis showed that the reaction was complete. The reaction mixture was cooled at room temperature and concentrated under reduced pressure to give the residue. Further purification by column chromatography gave a white solid intermediate I-6 (11.0 g, yield: 70%).

[0065] MS-ESI (m / z): 232.1 [M+l] + .

[0066] Step 5: Preparation of 4,5-dibromo-2-((2-(trimethylsilyl)ethoxy)methyl)pyridazine-3(2H)-one (I-8)

[0067] Sodium hydride (3.97 g, 165 mmol) was added dropwise to a solution of 4,5-dibromo-2,3-dihydropyridazin-3-one (starting material I-7, 35.0 g, 138 mmol) in 300 mL of DMF at 0 °C under nitrogen atmosphere. The resulting solution was stirred at room temperature for 1 hour, followed by dropwise addition of [2-(chloromethoxy)ethyl]trimethylsilane (25.3 g, 152 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was then quenched by adding 1.2 L of water. The resulting solution was extracted with 3 × 1.5 L of EtOAc and the organic layers were combined. The organic layers were washed with 3 × 5 L of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give intermediate I-8 (42.3 g, yield: 79%) as a yellow solid.

[0068] MS-ESI (m / z): 383.0 [M+l] + .

[0069] Step 6: Preparation of 4-bromo-5-chloro-2-((2-(trimethylsilyl)ethoxy)methyl)pyridazine-3(2H)-one (I-9)

[0070] Lithium chloride (4.7 g, 110 mmol) was added to a solution of 4,5-dibromo-2-((2-(trimethylsilyl)ethoxy)methyl)pyridazin-3(2H)-one (42.3 g, 110 mmol) in NMP (400 mL), and the resulting solution was stirred at 95 °C for 4 hours. LC / MS analysis showed that the reaction was complete. The solution was diluted with 2 L of water, extracted with 3 × 3 L of EtOAc, and the organic layers were combined. The organic layers were washed with 3 × 10 L of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Further column chromatography purification gave a green solid intermediate I-9 (22.4 g, yield: 60%).

[0071] MS-ESI (m / z): 338.9 [M+l] + .

[0072] Step 7: Preparation of 4-chloro-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)pyridin-2(1H)-one (I-11)

[0073] CuI (2.5 g, 13.2 mol) was added to a solution of 4-bromo-5-chloro-2-((2-(trimethylsilyl)ethoxy)methyl)pyridazine-3(2H)-one (intermediate I-9, 22.4 g, 66 mmol) in NMP (300 mL) at room temperature, followed by the dropwise addition of methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (starting material I-10, 38.0 g, 198 mmol). The solution was stirred at 80 °C for 2 hours. LC / MS analysis showed that the reaction was complete. The reactants were then quenched with 1 L of water and extracted with 3 × 1.2 L of EtOAc. The organic layers were combined and washed with 3 × 3 L of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Further column chromatography purification gave green solid intermediate I-11 (16.4 g, yield: 76%).

[0074] MS-ESI (m / z): 328.1 [M+l] + .

[0075] Step 8: Preparation of (S)-5-((1-hydroxypropane-2-yl)amino)-4-(trifluoromethyl)-2-((2-(trimethylsilyl)ethoxy)methyl)pyridazine-3(2H)-one (I-13)

[0076] A solution of 4-chloro-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)pyridin-2(1H)-one (16.4 g, 50 mmol), TEA (6.9 mL, 50 mmol), and (S)-2-aminoprop-1-ol (3.76 g, 50 mmol) in EtOH (200 mL) was stirred at 60 °C for 1 hour. LC / MS analysis showed that the reaction was complete. The solvent was concentrated under vacuum, and the residue was further purified by column chromatography to give a green solid intermediate I-13 (10.4 g, yield: 58%). MS-ESI (m / z): 368.2 [M+l] + .

[0077] Step 9: Preparation of methyl-(S)-3-(2-((6-oxo-5-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1,6-dihydropyridin-4-yl)amino)propoxy)propionate (I-15)

[0078] A solution of 5-[[(S)-1-hydroxypropyl-2-yl]amino]-4-(trifluoromethyl)-2-((2-(trimethylsilyl)ethoxy)methyl)-2,3-dihydropyridazin-3-one (10.4 g, 28 mmol), methyl propionate (24.37 g, 283 mmol), and Cs₂CO₃ (9.2 g, 28 mmol) in MeCN (200 mL) was stirred at 25 °C for 4 hours. LC / MS analysis showed that the reaction was complete. The solvent was concentrated under vacuum, and the residue was further purified by column chromatography to give a green solid intermediate I-15 (6.2 g, yield: 48%).

[0079] MS-ESI (m / z): 354.2 [M+l] + .

[0080] Step 10: Preparation of methyl-(S)-3-(2-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)amino)propoxy)propionate (I-16)

[0081] A solution of methyl-(S)-3-(2-((6-oxo-5-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1,6-dihydropyridin-4-yl)amino)propoxy)propionate (6.2 g, 14 mmol) and TFA (20 mL) in DCM (40 mL) was stirred at 25 °C for 0.5 h. LC / MS analysis showed that the reaction was complete. The resulting mixture was concentrated under vacuum to give a white solid intermediate I-16 (3.9 g, yield: 90%).

[0082] MS-ESI (m / z): 324.1 [M+l] + .

[0083] Step 11: Preparation of (S)-3-(2-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)amino)propoxy)propionic acid (I-17)

[0084] A solution of methyl-(S)-3-(2-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)amino)propoxy)propionate (3.9 g, 12 mmol), LiOH·H₂O (2.5 g, 60 mmol) in MeOH (15 mL) and H₂O (50 mL) was stirred at 25 °C for 0.5 h. LC / MS analysis showed that the reaction was complete. The pH of the solution was adjusted to 6 with TFA. The resulting mixture was concentrated under vacuum and the residue was purified by C18 reversed-phase chromatography eluting with H₂O / MeCN (6:1) to give a yellow oily intermediate I-17 (3.3 g, yield: 90%).

[0085] MS-ESI (m / z): 324.1 [M+l] + .

[0086] Step 12: Preparation of (S)-5-((1-(3-oxo-3-(1-(5-(trifluoromethyl)-2-pyrimidinyl)-5,6-2H-1,2,4-triazin-1(4H)-yl)propoxy)2-propyl)amino)-4-(trifluoromethyl)o-diazabenzyl-3(2H)-one (I)

[0087] A solution of (S)-3-(2-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)amino)propoxy)propionic acid (1.5 g, 5 mmol), 1-(5-(trifluoromethyl)pyrimidin-2-yl)-1,4,5,6-tetrahydro-1,2,4-triazine (0.7 g, 3.3 mmol), HATU (2.1 g, 6 mmol), and DIEA (2.1 g, 16 mmol) in DMF (25 mL) was stirred at room temperature for 4 hours. LC / MS analysis showed that the reaction was complete. The reactants were then quenched by adding 100 mL of water. The resulting solution was extracted with 3 × 60 mL EtOAc and concentrated under reduced pressure. The residue was further purified by column chromatography to give (S)-5-((1-(3-oxo-3-(1-(5-(trifluoromethyl)pyrimidin-2-yl)-5,6-2H-1,2,4-triazin-4(1H)-yl)propoxy)propane-2-yl)amino)-4-(trifluoromethyl)pyridazin-3(2H)-one (I) (1.6 g, yield: 58%)

[0088] MS-ESI (m / z): 523.2 [M+l] + .

[0089] 1H NMR(400MHz,DMSO-d6)δ12.31(s,1H),8.69(s,2H),7.97(s,1H),7.94(s,1H),7.2 7(s,1H),4.58(t,J=5.3Hz,2H),4.26(dt,J=10.4,5.1Hz,1H),3.95(dt,J=10.6,5. 5Hz,1H),3.64(qt,J=11.4,7.2Hz,2H),3.53-3.39(m,1H),3.35(dd,J=11.7,5.9H z,1H),3.27(dd,J=11.7,5.7Hz,1H),2.68(t,J=7.3Hz,2H),1.22(d,J=6.5Hz,3H).

[0090] Example 2: (S)-5-((1-(3-oxo-3-(4-(5-(trifluoromethyl)-2-pyrimidinyl)-5,6-dihydro-1,2,4-triazin-1(4H)-yl)propoxy)2-propyl)amino)-4-(trifluoromethyl)pyridazin-3(2H)-one (II)

[0091]

[0092] Step 1: Preparation of 2-((5-(trifluoromethyl)pyrimidin-2-yl)amino)ethyl-1-ol (II-2)

[0093] DIEA (55.0 mL, 121 mmol) was added to a DMF (200 mL) solution of 2-chloro-5-(trifluoromethyl)pyrimidine (starting material I-1, 20.0 g, 110 mmol), followed by the addition of 2-aminoethyl-1-ol (20.1 g, 340 mmol). The reaction mixture was shaken at room temperature for 2 h. LC / MS indicated that the reaction was complete. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give intermediate II-2 (17.5 g, 77% yield).

[0094] MS-ESI (m / z): 208.1 [M+l] + .

[0095] Step 2: Preparation of 2-((5-(trifluoromethyl)pyrimidin-2-yl)amino)ethoxy-methanesulfonate (II-3)

[0096] Intermediate II-2 (17.5 g, 84 mmol) was dissolved in DCM (200 mL). Triethylamine (19 mL, 135 mmol) and methanesulfonyl chloride (11.6 g, 101 mmol) were added sequentially under ice bath conditions. After the addition was complete, the reaction was carried out at room temperature for 3 hours. LC / MS analysis showed that the reaction was complete. The reaction was then quenched with water (100 mL), extracted with DCM (200 mL x 3), and the organic phases were combined. The mixture was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude intermediate II-3 (24.0 g, yield: 95%), which was used directly in the next step without purification.

[0097] MS-ESI (m / z): 301.0 [M+l] + .

[0098] Step 3: Preparation of N-(2-hydrazinoethyl)-5-(trifluoromethyl)pyrimidine-2-amine (II-4)

[0099] Intermediate II-3 (24.0 g, 80 mmol) was dissolved in ethanol (300 mL), and 80% hydrazine hydrate (7.0 g, 112 mmol) was added. The mixture was heated to 60 °C and reacted for 5 hours. LC / MS analysis showed that the reaction was complete. The mixture was then concentrated under reduced pressure to obtain intermediate II-4 (15.2 g, yield: 89%), which was used directly in the next step without purification.

[0100] MS-ESI (m / z): 222.1 [M+l] + .

[0101] Step 4: Preparation of 4-(5-(trifluoromethyl)pyrimidin-2-yl)-1,4,5,6-tetrahydro-1,2,4-triazine (II-5)

[0102] A methanol (4 mL) solution of intermediate II-4 (15.2 g, 69 mmol) was added to a reaction mixture of TMO (35.7 g, 337 mmol), and the mixture was stirred under reflux at 65 °C for 1 hour. LC / MS analysis showed that the reaction was complete. The reaction mixture was cooled at room temperature and concentrated under reduced pressure to obtain a residue. Further purification by column chromatography gave intermediate II-5 (9.5 g, yield: 60%) as a white solid.

[0103] MS-ESI (m / z): 232.1 [M+l] + .

[0104] Step 5: Preparation of (S)-5-((1-(3-oxo-3-(4-(5-(trifluoromethyl)-2-pyrimidinyl)-5,6-dihydro-1,2,4-triazine-1(4H)-yl)propoxy)2-propyl)amino)-4-(trifluoromethyl)pyridazine-3(2H)-one (II)

[0105] A solution of (S)-3-(2-((6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl)amino)propoxy)propionic acid (1.6 g, 6 mmol), 4-(5-(trifluoromethyl)pyrimidin-2-yl)-1,4,5,6-tetrahydro-1,2,4-triazine (0.8 g, 3.3 mmol), HATU (2.1 g, 6 mmol), and DIEA (2.1 g, 16 mmol) in DMF (25 mL) was stirred at room temperature for 4 hours. LC / MS analysis showed that the reaction was complete. The reactants were quenched by adding 100 mL of water. The resulting solution was extracted with 3 × 60 mL EtOAc and concentrated under reduced pressure. The residue was further purified by column chromatography to give (S)-5-((1-(3-oxo-3-(4-(5-(trifluoromethyl)-2-pyrimidinyl)-5,6-dihydro-1,2,4-triazin-1(4H)-yl)propoxy)-2-propyl)amino)-4-(trifluoromethyl)pyridazin-3(2H)-one (II) (1.5 g, yield: 56%)

[0106] MS-ESI (m / z): 232.1 [M+l] + .

[0107] 1 H NMR(400MHz,DMSO-d6)δ12.25(s,1H),8.68(s,2H),7.97(s,1H),7.95(s,1H),7.19(s,1H),4.44-4.30(m,2H),3.87-3 .60(m,5H),3.54(dd,J=11.3,5.7Hz,1H),3.26(dd,J=11.2,5.7Hz,1H),2.77(t,J=7.1Hz,2H),1.23(d,J=6.8Hz,3H).

[0108] Example 3: (S)-2-(1-oxo-1-(1-(5-(trifluoromethyl)-2-pyrimidinyl)-5,6-dihydro-1,2,4-triazine-1(4H)-yl)2-propane)-2H-indazole-7-carboxamide (III)

[0109]

[0110] Step 1: Preparation of 1H-indazole-7-carboxamide (III-6)

[0111] 1H-Indazole-7-carboxylic acid (III-5) (10 g, 62 mmol) was dissolved in 100 mL of THF. DMF (1.2 mL, 15 mmol) was then added to the solution as a catalyst. The mixture was cooled in an ice bath, and then thionyl chloride (10 mL, 135 mmol) was added dropwise. The resulting mixture was stirred at 50 °C for 3 h until TLC indicated completion. After cooling to room temperature, the solvent and thionyl chloride were removed under reduced pressure. Then, ammonia (24 mL, 617 mmol) was slowly added at -10 °C, followed by 40 mL of THF. After reacting at -10 °C for 2 h, LC / MS analysis showed completion. The solvent was removed under reduced pressure. The residue was added to 30 mL of water and stirred for 30 min. The precipitate was collected by filtration. The precipitate was dried under vacuum to give a white solid (8.3 g, yield: 83%).

[0112] MS-ESI (m / z): 162.1 [M+l] + .

[0113] Step 2: Preparation of ethyl-2-(7-carbamoyl-2H-indazole-2-yl)propionate (III-8)

[0114] Ethyl bromopropionate (III-7) (10.3 g, 57 mmol) and 1H-indazole-7-carboxamide (III-6) (8.3 g, 52 mmol) were dissolved in 200 mL of DMF. Cesium carbonate (50.3 g, 154 mmol) was then added. The resulting mixture was stirred at 90 °C for 3 h until LC / MS analysis showed the reaction was complete. After cooling to room temperature, 500 mL of water was added, and the mixture was extracted with ethyl acetate (700 mL × 3). The organic phase was washed with saturated brine (2500 mL × 3), dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the residue. Further purification by column chromatography gave a white solid (III-8) (9.7 g, yield: 72%). MS-ESI (m / z): 262.2 [M+l] + .

[0115] Step 3: Preparation of 2-(7-carbamoyl-2H-indazole-2-yl)propionic acid (III-9)

[0116] Ethyl-2-(7-carbamoyl-2H-indazole-2-yl)propionate (III-8) (9.7 g, 37 mmol) was dissolved in a mixed solvent (MeOH / H₂O = 3:1, 20 mL), and 5N NaOH (30 mL) was added. The mixture was stirred at room temperature for 1 h until LC / MS analysis showed that the reaction was complete. The reaction mixture was then diluted with water (50 mL) and 2N HCl solution was added to pH = 5. The mixture was stirred at room temperature for 15 min, and a white precipitate formed. The precipitate was collected by filtration, washed with water (20 mL), and dried under vacuum to give a white solid (III-9) (8.1 g, 93%).

[0117] MS-ESI (m / z): 234.1 [M+l] + .

[0118] Step 4: Preparation of 2-(1-oxo-1-(1-(5-(trifluoromethyl)-2-pyrimidinyl)-5,6-dihydro-1,2,4-triazine-1(4H)-yl)2-propane)-2H-indazole-7-carboxamide (III-10)

[0119] A solution of 2-(7-carbamoyl-2H-indazole-2-yl)propionic acid (III-9) (4.1 g, 17 mmol), 1-(5-(trifluoromethyl)pyrimidin-2-yl)-1,4,5,6-tetrahydro-1,2,4-triazine (I-6) (2.2 g, 10 mmol), HATU (6.6 g, 18 mmol), and DIEA (6 g, 47 mmol) in DMF (100 mL) was stirred at room temperature for 4 hours. LC / MS analysis showed that the reaction was complete. The reactants were quenched by adding 300 mL of water. The resulting solution was extracted with 3 × 500 mL EtOAc and concentrated under reduced pressure. The residue was further purified by column chromatography to give a pale white solid (III-10) (2.2 g, yield: 52%). MS-ESI (m / z): 447.2 [M+l] + .

[0120] Step 5: Preparation of (S)-2-(1-oxo-1-(1-(5-(trifluoromethyl)-2-pyrimidinyl)-5,6-dihydro-1,2,4-triazine-1(4H)-yl)2-propane)-2H-indazole-7-carboxamide (III)

[0121] (S)-2-(1-oxo-1-(1-(5-(trifluoromethyl)-2-pyrimidinyl)-5,6-dihydro-1,2,4-triazin-1(4H)-yl)2-propane)-2H-indazole-7-carboxamide (III-10) was obtained by HPLC chiral resolution (1.1 g, yield: 47%).

[0122] MS-ESI (m / z): 447.2 [M+l] + .

[0123] 1 H NMR(400MHz, DMSO-d6)δ8.68(s,2H),8.31(d,J=1.6Hz,1H),8.13(dt,J=7.8,1.4Hz,1H),8.00-7.94(m,2H),7.6 6(s,2H),7.54-7.47(m,1H),5.21(q,J=6.9Hz,1H),4.52-4.36(m,2H),4.33-4.19(m,2H),1.61(d,J=6.6Hz,3H).

[0124] Example 4: (S)-2-(1-oxo-1-(4-(5-(trifluoromethyl)-2-pyrimidinyl)-5,6-dihydro-1,2,4-triazine-1(4H)-yl)2-propane)-2H-indazole-7-carboxamide (IV)

[0125]

[0126] Step 1: Preparation of 2-(1-oxo-1-(4-(5-(trifluoromethyl)-2-pyrimidinyl)-5,6-dihydro-1,2,4-triazine-1(4H)-yl)2-propane)-2H-indazole-7-carboxamide (III-11)

[0127] A solution of 2-(7-carbamoyl-2H-indazole-2-yl)propionic acid (III-9) (4.1 g, 17 mmol), 4-(5-(trifluoromethyl)pyrimidin-2-yl)-1,4,5,6-tetrahydro-1,2,4-triazine (II-5) (2.2 g, 10 mmol), HATU (6.6 g, 18 mmol), and DIEA (6 g, 47 mmol) in DMF (100 mL) was stirred at room temperature for 4 hours. LC / MS analysis showed that the reaction was complete. The reactants were quenched by adding 300 mL of water. The resulting solution was extracted with 3 × 500 mL EtOAc and concentrated under reduced pressure. The residue was further purified by column chromatography to give a white solid (III-11) (2.3 g, yield: 54%). MS-ESI (m / z): 447.2 [M+l] + .

[0128] Step 2: Preparation of (S)-2-(1-oxo-1-(4-(5-(trifluoromethyl)-2-pyrimidinyl)-5,6-dihydro-1,2,4-triazine-1(4H)-yl)2-propane)-2H-indazole-7-carboxamide (IV)

[0129] (S)-2-(1-oxo-1-(4-(5-(trifluoromethyl)-2-pyrimidinyl)-5,6-dihydro-1,2,4-triazin-1(4H)-yl)2-propane)-2H-indazole-7-carboxamide (III-11) was obtained by chiral resolution by HPLC.

[0130] MS-ESI (m / z): 447.2 [M+l] + .

[0131] 1 H NMR (400MHz, DMSO-d6) δ8.69(s,2H),8.35-8.29(m,1H),8.11(dt,J=7.9,1.4Hz,1H),8.05-7.95(m,2H),7.79(d,J=7.9Hz,1H) ,7.69(d,J=7.9Hz,1H),7.54-7.46(m,1H),5.20(q,J=6.7Hz,1H),4.48-4.31(m,2H),3.88-3.71(m,2H),1.63(d,J=6.7Hz,3H).

[0132] Example 5: (S)-2-(1-O-1-(4-(5-(trifluoromethyl)-2-pyrimidinyl)-1-piperazinyl)-2-propane)-2H-indazole-4-carboxamide (V)

[0133]

[0134] Step 1: Preparation of tert-butyl 4-(5-(trifluoromethyl)pyrimidin-2-yl)piperazine-1-carboxylate (III-3)

[0135] A solution of 2-chloro-5-(trifluoromethyl)pyrimidine (10.29 g, 56 mmol), piperazine-1-carboxylic acid tert-butyl ester (III-2) (10.0 g, 54 mmol), and K₂CO₃ (14.8 g, 107 mmol) in NMP (80 mL) was stirred at 80 °C for 1 hour, followed by the addition of 200 mL H₂O. The solid was collected by filtration to give a white solid (III-3) (16.4 g, yield: 92%). MS-ESI (m / z): 333.2 [M+l] + .

[0136] Step 2: Preparation of 2-(piperazin-1-yl)-5-(trifluoromethyl)pyrimidine (III-4)

[0137] A solution of tert-butyl 4-(5-(trifluoromethyl)pyrimidin-2-yl)piperazine-1-carboxylate (16.4 g, 49 mmol) in HCl / dioxane (30 mL / 4 M) was stirred at room temperature for 1 hour. The solution was collected by filtration as a pale yellow solid (III-4) (10.9 g, yield: 95%). MS-ESI (m / z): 233.1 [M+l] +

[0138] Step 3: Preparation of 1H-indazole-4-carboxamide (III-13)

[0139] The synthesis was performed following the method described in Example 3 for compound (III-6), yielding a pale yellow solid (III-13) (7.9 g, yield: 81%). MS-ESI (m / z): 162.1 [M+l] +

[0140] Step 4: Preparation of ethyl-2-(4-carbamoyl-2H-indazole-2-yl)propionate (III-14)

[0141] The synthesis was performed following the method described in Example 3 for compound (III-8), yielding a white solid (III-14) (8.4 g, yield: 76%). MS-ESI (m / z): 262.1 [M+l] + .

[0142] Step 5: Preparation of 2-(4-carbamoyl-2H-indazole-2-yl)propionic acid (III-15)

[0143] The synthesis was performed following the method described in Example 3 for compound (III-9), yielding a white solid (III-15) (6.5 g, yield: 58%). MS-ESI (m / z): 234.1 [M+1]. + .

[0144] Step 6: Preparation of 2-(1-O-1-(4-(5-(trifluoromethyl)-2-pyrimidinyl)-1-piperazinyl)-2-propane)-2H-indazole-4-carboxamide (III-16)

[0145] The synthesis was performed following the method described in Example 3 for compound (III-10), yielding a pale white solid (III-15) (1.6 g, yield: 43%). MS-ESI (m / z): 447.2 [M+l] + .

[0146] Step 7: Preparation of (S)-2-(1-oxo-1-(4-(5-(trifluoromethyl)-2-pyrimidinyl)-1-piperazinyl)-2-propane)-2H-indazole-4-carboxamide (V)

[0147] The synthesis was performed according to the method for compound (III) in Example 3, yielding (S)-2-(1-oxo-1-(4-(5-(trifluoromethyl)-2-pyrimidinyl)-1-piperazinyl)-2-propane)-2H-indazole-4-carboxamide (V) (0.7 g, yield: 41%).

[0148] MS-ESI (m / z): 447.2 [M+l] + .

[0149] 1 H NMR(400MHz,DMSO-d6)δ8.63(s,2H),8.45(s,1H),7.96-7.89(m,1H),7.75-7.67(m,1H),7.61-7.52(m,3 H),5.16(q,J=6.6Hz,1H),3.74(ddd,J=10.7,7.1,4.5Hz,2H),3.65-3.45(m,6H),1.63(d,J=6.7Hz,3H).

[0150] Example 6: (S)-2-(1-oxo-1-(4-(5-(trifluoromethyl)-2-pyrimidinyl)-1-piperazinyl)-2-propane)-2H-benzo[d][1,2,3]triazole-4-carboxamide (VI)

[0151]

[0152] Step 1: Preparation of 1H-benzo[d][1,2,3]triazole-4-carboxamide (III-18)

[0153] The synthesis was performed following the method described in Example 3 for compound (III-6), yielding a pale yellow solid (III-18) (6.9 g, yield: 79%). MS-ESI (m / z): 163.0 [M+l] + .

[0154] Step 2: Preparation of ethyl-2-(4-carbamoyl-2H-benzo[d][1,2,3]triazol-2-yl)propionate (III-19)

[0155] The synthesis was performed following the method described in Example 3 for compound (III-8), yielding a white solid (III-19) (8.1 g, yield: 71%). MS-ESI (m / z): 263.2 [M+l] + .

[0156] Step 3: Preparation of 2-(4-carbamoyl-2H-benzo[d][1,2,3]triazol-2-yl)propionic acid (III-20)

[0157] The synthesis was performed following the method described in Example 3 for compound (III-9), yielding a white solid (III-20) (6.3 g, yield: 54%). MS-ESI (m / z): 235.1 [M+l] + .

[0158] Step 4: Preparation of 2-(1-oxo-1-(4-(5-(trifluoromethyl)-2-pyrimidinyl)-1-piperazinyl)-2-propane)-2H-benzo[d][1,2,3]triazole-4-carboxamide (III-21)

[0159] The synthesis was performed following the method described in Example 3 for compound (III-10), yielding a pale white solid (III-21) (1.3 g, yield: 41%). MS-ESI (m / z): 449.2 [M+l] + .

[0160] Step 5: Preparation of (S)-2-(1-oxo-1-(4-(5-(trifluoromethyl)-2-pyrimidinyl)-1-piperazinyl)-2-propane)-2H-benzo[d][1,2,3]triazole-4-carboxamide (VI)

[0161] The synthesis was performed according to the method for compound (III) in Example 3, yielding (S)-2-(1-oxo-1-(4-(5-(trifluoromethyl)-2-pyrimidinyl)-1-piperazinyl)-2-propane)-2H-benzo[d][1,2,3]triazole-4-carboxamide (VI) (0.8 g, yield: 62%).

[0162] MS-ESI (m / z): 449.2 [M+l] + .

[0163] 1 ¹H NMR (400MHz, DMSO-d⁶) δ 8.61 (s, 2H), 8.08 (dd, J = 7.7, 1.1Hz, 1H), 7.97 (dd, J = 7.8, 1.2Hz, 1H), 7.88 (s, 2H), 7.63 (t, J = 7.9Hz, 1H), 5.41 (q, J = 6.8Hz, 1H), 3.78 (ddd, J = 11.6, 6.8, 4.4Hz, 2H), 3.65 (ddd, J = 12.3, 6.6, 4.5Hz, 2H), 3.59–3.44 (m, 4H), 1.73 (d, J = 6.7Hz, 3H). Example 7: Inhibitory activity of the compound against PARP7.

[0164] Experimental materials: PARP7 Chemiluminescent Assay Kit, BPS Bioscience; DMSO, Sinopharm, Nivo, PerkinElmer.

[0165] Experimental methods:

[0166] (1) Preparation of solutions and buffer solutions

[0167] Preparation of 10X PBS: Weigh 720 mg KH3PO4, 45 g NaCl and 5.311 g Na2HPO4·12H2O and dissolve them in 500 mL of deionized water. Adjust the pH of the system to 7.4, sterilize at 121 °C for 30 minutes, cool and store at 4 °C for later use.

[0168] Preparation of 1X PBS: Dilute 10X PBS with deionized water 10 times, that is, add 1 part of 10X PBS to 9 parts of deionized water for dilution.

[0169] Wash buffer preparation: 1X PBS contains 0.05% Tween-20.

[0170] Preparation of 1X PARP buffer: (Prepare fresh for immediate use) Dilute 10X PARP buffer 10 times with deionized water and place on ice for later use.

[0171] (2) Preparation of working solution of test compound

[0172] According to the testing requirements, the test compounds (including Examples 1-6, and control compounds RBN-2397 and S-XY-05) were diluted to the required concentration with 100% DMSO, and then diluted 10 times with 1X PARP buffer to prepare a 10X working solution of the test compounds.

[0173] (3) Experimental steps:

[0174] a. Thaw the 5X histone mixture on ice the day before the experiment;

[0175] b. Preparation of 1X histone mixture: Use 1X PBS to prepare 1X histone mixture from 5X histone mixture; take 25 μL of 1X histone mixture into each well and incubate overnight at 4C.

[0176] c. Add 100 μL of blocking buffer to each well into the test plate and incubate at 25°C for 90 minutes;

[0177] d. After incubation, shake off the liquid in the test plate and wash the plate three times.

[0178] e. Take 2.5 μL of the working solution of the test compound in each well and add it to the test plate according to the experimental layout diagram; add the corresponding volume of 1X PARP buffer containing 10% DMSO to the positive control well, and add the corresponding volume of 1X PARP buffer to the blank control well.

[0179] f. After the enzyme is completely dissolved, dilute the enzyme stock solution to 6 ng / μL with 1X PARP buffer;

[0180] g. Add 10 portions of enzyme solution per well to the test plate, and add the corresponding volume of 1X PARP buffer to the blank control wells. The enzyme quantity is now 60 ng per well. Note: This step must be performed on ice.

[0181] h. Add 12.5 μL of master mixture (12.5 μL master mixture includes 1.25 μL of 10X PARP buffer, 1.25 μL of Opti-PARP 10X Assay mixture and 10 μL of water) to each well of the test plate; seal the test plate and incubate at 25°C for 60 minutes.

[0182] i. After incubation, shake off the liquid in the test plate and wash the plate three times.

[0183] j. Dilute the Streptavidin-HRP in the kit 50 times with Blocking buffer solution, add 25 μL to each well of the test plate, and incubate at 25°C for 30 minutes;

[0184] k. After incubation, shake off the liquid in the test plate and wash the plate three times.

[0185] 1. Mix ELISA ECL Substrate A and ELISA ECL Substrate B in the 1:1 kit, add 50 μL of the mixture to each well of the test plate, and immediately perform luminescence detection using Nivo and read the luminescence value (RLU).

[0186] m. Enzyme activity calculation: %Enzyme Activity = (RLU(Sample) - RLU(Blank)) / (RLU(Pos.Ctrl) - RLU(Blank)) x 100%; Enzyme inhibition rate = 1 - %Enzyme Activity, IC50 analysis was performed using PrismGraphPad software. 50 The fitting results are shown in Table 1 below.

[0187] Table 1. Number of enzyme inhibitory activities of the test compounds against PARP7

[0188]

[0189]

[0190] Example 8: Antiproliferative activity of the compound against tumor cells

[0191] Cell lines: NCI-H1373 (purchased from the Cell Bank of the Chinese Academy of Sciences), CFPAC-1 (purchased from the Cell Bank of the Chinese Academy of Sciences)

[0192] Experimental procedure:

[0193] a. A group of cancer cell lines cultured to the logarithmic growth phase were plated into 96-well plates at a density of 3000 / well in a medium containing fetal bovine serum;

[0194] b. Cells were treated with the test compound or medium (DMSO) after 24 hours, and day 0 plates were collected for analysis;

[0195] c. After application, the 96-well plate was placed in a 37°C, 5% CO2 incubator and incubated for 6 days. 20 μL of 1.0% MTT thiazolyl blue solution was added to each well.

[0196] d. Continue to place in a constant temperature incubator. After 4 hours, use aspirate to remove the supernatant culture medium, add 150 μL of DMSO to each well, and mix on a decolorizing shaker until the crystals dissolve.

[0197] e. Measure the absorbance at 570 nm using a multi-functional microplate reader. Repeat the experiment twice for each combination of cell line, sample, and sample concentration.

[0198] Using Graphpad Prism5 software, ICs are analyzed according to the following formula. 50 Value calculation.

[0199]

[0200] The drug group refers to the group treated with the test compound according to the above method; the blank control group refers to the group with only culture medium added, without cells and the test compound; the positive control group refers to the group with cells and culture medium added, with 0.5% DMSO added, without the test compound.

[0201]

[0202] Xm: Logarithm of the maximum designed concentration; i: Logarithm of each concentration ratio; ΣP: Sum of growth inhibition rates of each group; 0.5: Empirical constant.

[0203] Calculate IC based on the inhibition rate obtained from the above formula. 50 Values. The specific results are shown in Table 2 below.

[0204] Table 2. Data on the antiproliferative activity of the tested compounds against tumor cells.

[0205]

[0206] As can be seen from Tables 1 and 2, under the same test batch, Examples 1-6 of the present invention have better PARP7 enzyme inhibitory activity and more significant anti-proliferative activity against tumor cells compared to RBN-2397 and S-XY-05, and have higher drug potential.

[0207] Although the invention has been described through specific embodiments above, it should not be construed as being limited thereto; rather, the invention encompasses the general aspects previously disclosed. Various modifications and embodiments are possible without departing from the spirit and scope of the invention.

Claims

1. A nitrogen-heterocyclic substituted pyrimidine compound having the structure shown in general formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug thereof: In the formula, A is selected from: L is selected from: Furthermore, A is either A1 or A2, and L is either L1 or L2; Alternatively, A can be A3 or A4, and L can be any of L1, L2 or L3.

2. The nitrogen-heterocyclic substituted pyrimidine compound according to claim 1, or its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug, wherein the nitrogen-heterocyclic substituted pyrimidine compound is selected from:

3. The nitrogen-substituted pyrimidine compound according to claim 1 or 2, characterized in that, The pharmaceutically acceptable salt is a salt formed by the compound and an acid selected from hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, or ferulic acid.

4. A pharmaceutical composition, characterized in that, The compound comprises a nitrogen-heterocyclic substituted pyrimidine compound or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, prodrug, and pharmaceutical excipient thereof, as described in any one of claims 1 to 3.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical excipients include pharmaceutically acceptable carriers, excipients, or diluents.

6. Use of any nitrogen-heterocyclic substituted pyrimidine compound of claims 1 to 3, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug in the preparation of a drug for inhibiting the PARP7 signaling pathway.

7. Use of any nitrogen-heterocyclic substituted pyrimidine compound of claims 1 to 3, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug in the preparation of a medicament for treating or alleviating PARP7-mediated diseases.

8. The use according to claim 7, characterized in that, PARP7-mediated diseases include hyperproliferative disorders, autoimmune diseases, or inflammatory diseases.

9. The use according to claim 7, characterized in that, PARP7-mediated diseases include cancers such as breast cancer, central nervous system cancer, endometrial cancer, kidney cancer, colorectal cancer, lung cancer, esophageal cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, head and neck cancer, urethral cancer, colon cancer, leukemia, lymphoma, liver cancer, bladder cancer, bone cancer, glioma, breast cancer, cervical cancer, colon cancer, endometrial cancer, epithelial cancer, esophageal cancer, Ewing's sarcoma, pancreatic cancer, gallbladder cancer, stomach cancer, gastrointestinal tumors, head and neck cancer, intestinal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, liver cancer, lung cancer, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, and uterine cancer.

10. The use according to claim 7, characterized in that, PARP7-mediated diseases include heart disease, viral infections, neurodegeneration, inflammation, or pain.

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