Aryl urea PARP1 inhibitor as well as preparation method and application thereof

Abstract

The invention relates to a urea PARP1 inhibitor as well as a preparation method and application thereof. The invention particularly relates to a compound capable of inhibiting activity of poly ADP-ribose polymerase, and a pharmaceutical composition and application thereof. The present invention relates in particular to compounds of formula (I) and also to pharmaceutical compositions comprising these compounds and to the use of these compounds for the preparation of medicaments for the prevention and / or treatment of diseases, in particular diseases ameliorated by inhibition of PARP1.

Description

An aryl urea PARP1 inhibitor, its preparation method and use Technical Field

[0001] The present invention relates to a class of compounds capable of inhibiting poly ADP-ribose polymerase 1 (PARP1) activity and uses thereof, and particularly to a class of aryl urea compounds, a pharmaceutical composition comprising the compounds, and uses thereof in the preparation of drugs for improving diseases, particularly tumor diseases, by inhibiting PARP1. Background Art

[0002] Poly (ADP-ribose) polymerases (PARPs) are an emerging family of enzymes that catalyze the transfer of ADP-ribose to target proteins (poly (ADP-ribosylation)). At least 18 PARP family members are encoded by different genes and share homology in the conserved catalytic domain (Morales et al, Critical Reviews TM in Eukaryotic Gene Expression 24.1, 2014). PARP1, short for poly(ADP-ribose) polymerase 1, is an abundant nuclear protein (Murai et al, Cancer Research 72.21, 2012). PARP1 catalyzes the transfer of ADP-ribose residues from NAD+ to target substrate proteins or nucleic acids, constructing a poly(ADP-ribose) (PAR) chain that is added to downstream target proteins. This post-translational modification is called PARylation. PARPs play an important role in several cellular processes, including cell proliferation and cell death (Murai et al, Cancer Research 72.21, 2012). The main function of PARP is to participate in DNA damage repair. Single-strand breaks (SSBs) are the most common type of damage and can be converted into potentially disruptive and lethal double-strand breaks (DSBs). PARP1 binds to damaged DNA at single-strand breaks (SSBs) and other DNA damage. This event causes a series of conformational changes in the structure of PARP1, thereby activating its catalytic function (Lord et al, Science 355.6330, 2017).

[0003] BRCA1 and BRCA2 proteins are crucial for the repair of double-stranded DNA breaks (DSBs) through a process called homologous recombination repair (HRR), a form of DNA repair that utilizes homologous DNA sequences to guide repair at the site of a DSB (Lord et al, Science 355:6330, 2017). HRR is normally a "conservative" mechanism because it restores the original DNA sequence at the site of DNA damage. When cells are deficient in HRR, whether driven by defects in BRCA1, BRCA2, or other pathway components, non-conservative forms of DNA repair, such as non-homologous end joining (NHEJ), predominate.

[0004] PARP inhibitors exert their anti-cancer effects by blocking DNA damage repair in highly mutated cancer cells, resulting in "toxic damage" that causes cell death due to homologous recombination repair (HRR) deficiency. Healthy cells contain multiple signaling pathways for DNA repair, so inhibiting only PARP is not very toxic. However, certain tumor cells, due to specific gene mutations such as BRCA, can disrupt other DNA repair pathways, making them dependent on PARP1 and therefore particularly sensitive to PARP inhibitors. This is why patients with ovarian and breast cancers carrying BRCA mutations are more likely to benefit from PARP inhibitors. PARP2 is present at low levels, accounting for only 5% to 10% of total PARP activity. Knocking out PARP1 significantly reduces PARP activity compared to knocking out PARP2 (<10%) (Yélamos et al, The EMBO journal 25.18, 2006). Knocking out PARP1 blocks the inhibitory activity of olaparib on PARP and also eliminates the cell proliferation inhibitory effect of olaparib (Murai et al, Cancer research, 2012). These data indicate that PARP1 is the key to determining the efficacy of PARPi. Literature reports indicate that intact PARP2 in the bone marrow is required for mouse survival; PARP2 deficiency leads to reduced RBC, WBC, and BM cell numbers (Farrés et al, Blood, The Journal of the American Society of Hematology 122.1, 2013); and compared to PARP1 knockout, PARP2 knockout reduces T cell and RBC numbers, whereas PARP1 knockout has no significant effect on T cell (Yélamos et al, Blood, The EMBO journal 25.18, 2006) or RBC numbers (Farrés et al, Cell Death & Differentiation 22.7, 2015). Therefore, PARP1 inhibition is the primary source of drug efficacy, while PARP2 inhibition is the primary source of toxicity. The development of highly selective PARP1 / 2 inhibitors could significantly reduce the toxicity caused by PARP2 without significantly reducing drug efficacy.

[0005] In summary, there is an urgent need in this field to develop PARP inhibitors with high efficacy and good safety, especially inhibitors with high selectivity for PARP1.

[0006] Summary of the Invention

[0007] One of the objectives of the present invention is to provide a class of aryl urea compounds that can serve as PARP1 inhibitors.

[0008] A second object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned compound as an active ingredient and an optional pharmaceutically acceptable carrier.

[0009] A third object of the present invention is to provide a use of the above-mentioned compound or a composition comprising the same in the preparation of a PARP1 inhibitor.

[0010] A fourth object of the present invention is to provide a use of the above-mentioned compound or a composition comprising the same in the preparation of a medicament for preventing and / or treating a disease improved by inhibiting PARP1.

[0011] In one aspect, the present invention provides a compound of formula (I), or a stereoisomer, geometric isomer, tautomer, or pharmaceutically acceptable salt, polymorph, solvate, hydrate, or prodrug thereof,

[0012] in,

[0013] is a single bond or a double bond;

[0014] X 1 Selected from -N- and -CR 8 -;

[0015] X 2 Selected from -N- and -CR 9 -;

[0016] X 3 -N- or -CR 10 -;

[0017] R 1 、R 1’ 、R 2 、R 3 Each is independently selected from hydrogen, unsubstituted or substituted C1-C6 alkyl;

[0018] R 4 、R 5 are each independently selected from hydrogen, unsubstituted or substituted C1-C6 alkyl; or R 4 and R 5 Together with the carbon atom to which it is bonded, it forms a C3-C6 cycloalkyl group;

[0019] R 7selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, C3-C6 alkenyl, C3-C6 alkynyl, unsubstituted or substituted C3-C8 cycloalkyl, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted 3 to 8 membered heterocycloalkyl, unsubstituted or substituted 5 to 6 membered heteroaryl, unsubstituted or substituted C6-C 10 aryl;

[0020] R 8 Selected from hydrogen, halogen, cyano, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C3-C6 cycloalkyl, unsubstituted or substituted C1-C6 alkoxy, NH2, -NH-C1-C4 alkyl;

[0021] R 9 Selected from hydrogen, halogen, cyano, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 alkoxy;

[0022] R 10 Selected from hydrogen, halogen, cyano, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted C1-C6 alkyl;

[0023] s and n are each independently selected from 0, 1 and 2;

[0024] Y is N or CH;

[0025] R 6 Selected from:

[0026] Each R 11 Each independently selected from halogen, cyano, C1-C3 alkoxy, carbonyl, -CONHR 13 and amino;

[0027] m is 0, 1, 2, or 3;

[0028] R 12 Selected from hydrogen, cyano, halogen, unsubstituted or substituted C1-C4 alkyl;

[0029] R 13 Selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C3-C8 cycloalkyl, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted 3-8 membered heterocycloalkyl;

[0030] The heterocycloalkyl group refers to a heterocycloalkyl group containing 1 to 3 heteroatoms selected from N, O, and S;

[0031] R 1 、R 1’ 、R 2 、R 3 、R 4 、R 5、R 7 、R 8 、R 9 、R 10 、R 12 、R 13 The substitution mentioned here refers to substitution by one or more selected from deuterium, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C6 alkoxy, halogen, hydroxy, cyano, amino, carboxyl and C3-C6 cycloalkyl.

[0032] In a specific embodiment, the present invention provides a compound of formula (I), or its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts, polymorphs, solvates, hydrates or prodrugs,

[0033] in,

[0034] is a single bond or a double bond;

[0035] X 1 Selected from -N- and -CR 8 -;

[0036] X 2 Selected from -N- and -CR 9 -;

[0037] X 3 -N- or -CR 10 -; preferably, X 1 、X 2 、X 3 Not all N at the same time;

[0038] R 1 、R 1’ 、R 2 、R 3 Each is independently selected from hydrogen, unsubstituted or substituted C1-C6 alkyl;

[0039] R 4 、R 5 are each independently selected from hydrogen, unsubstituted or substituted C1-C6 alkyl; or R 4 and R 5 Together with the carbon atom to which it is bonded, it forms a C3-C6 cycloalkyl group;

[0040] R 7 selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, C3-C6 alkenyl, C3-C6 alkynyl, unsubstituted or substituted C3-C8 cycloalkyl, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted 3 to 8 membered heterocycloalkyl, unsubstituted or substituted 5 to 6 membered heteroaryl, unsubstituted or substituted C6-C10 Aryl; preferably, R 7 is selected from C1-C4 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, 3- to 8-membered heterocycloalkyl, phenyl, and 5- to 6-membered heteroaryl groups which are unsubstituted or substituted by halogen, hydroxy, C1-C4 alkoxy, C2-C4 alkenyl, C2-C4 alkynyl, or C3-C6 cycloalkyl; more preferably, R 7 Selected from C1-C4 alkyl, C3-C6 cycloalkyl which is unsubstituted or substituted by halogen, hydroxy, C1-C4 alkoxy, C2-C4 alkenyl, C2-C4 alkynyl or C3-C6 cycloalkyl;

[0041] R 8 is selected from hydrogen, halogen, cyano, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C3-C6 cycloalkyl, unsubstituted or substituted C1-C6 alkoxy, NH2, -NH-C1-C4 alkyl; preferably, R 8 is hydrogen, halogen, cyano, NH2, -NH-C1-C4 alkyl, methoxy, C3-C6 cycloalkyl or C1-C4 alkyl; more preferably, R 8 is hydrogen, fluorine, chlorine, methyl, CN, NH2, NHCH3, cyclopropane or methoxy;

[0042] R 9 is selected from hydrogen, halogen, cyano, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 alkoxy; preferably, R 9 is hydrogen, halogen, cyano or C1-C4 alkyl; more preferably, R 9 is hydrogen, fluorine, chlorine or methyl;

[0043] R 10 is selected from hydrogen, halogen, cyano, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted C1-C6 alkyl; preferably, R 10 is hydrogen, halogen, cyano or C1-C4 alkyl; more preferably, R 10 is hydrogen, fluorine, chlorine or methyl;

[0044] s and n are each independently selected from 0, 1 and 2;

[0045] Y is N or CH;

[0046] R 6 Selected from:

[0047] Each R 11 Each independently selected from halogen, cyano, C1-C3 alkoxy, carbonyl, -CONHR 13 and amino, preferably selected from halogen, -CONHR 13 and cyano;

[0048] m is 0, 1, 2, or 3;

[0049] R 12 Selected from hydrogen, cyano, halogen, unsubstituted or substituted C1-C4 alkyl; preferably selected from hydrogen, cyano, halogen, unsubstituted or substituted C1-C4 alkyl by halogen or deuterium;

[0050] R 13 is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C3-C8 cycloalkyl, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted 3-8 membered heterocycloalkyl; preferably, R 13 is selected from hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, which are unsubstituted or substituted by halogen or deuterium;

[0051] The heterocycloalkyl group refers to a heterocycloalkyl group containing 1 to 3 heteroatoms selected from N, O, and S;

[0052] R 1 、R 1’ 、R 2 、R 3 、R 4 、R 5 、R 7 、R 8 、R 9 、R 10 、R 12 、R 13 The substitution mentioned here refers to substitution by one or more selected from deuterium, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C6 alkoxy, halogen, hydroxy, cyano, amino, carboxyl and C3-C6 cycloalkyl.

[0053] In some embodiments, R 1 、R 1’ 、R 2 、R 3 Each is independently selected from hydrogen, C1-C4 alkyl.

[0054] In some embodiments, R 4 、R 5 are each independently selected from hydrogen, unsubstituted or substituted C1-C4 alkyl; or R 4 and R 5 Together with the carbon atom to which it is bonded, it forms a C3-C6 cycloalkyl group.

[0055] In some embodiments, in formula (I) Selected from: wherein the definitions of the substituents are the same as above; preferably, selected from where R10 The definition of is the same as above.

[0056] In some embodiments, in formula (I) Selected from: where R 10 The definition of is the same as above.

[0057] In some embodiments, in formula (I) Selected from the following structures: The definitions of the substituents are the same as above.

[0058] In some embodiments, in formula (I) Selected from: The definitions of the substituents are the same as above.

[0059] Typical compounds of the present invention include, but are not limited to:

[0060] On the other hand, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound as represented by general formula (I), or its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts, crystal forms, solvates, hydrates or prodrugs, and a pharmaceutically acceptable carrier.

[0061] In certain embodiments of the pharmaceutical composition, the pharmaceutical composition is formulated for intravenous administration, intramuscular administration, oral administration, rectal administration, inhalation administration, nasal administration, topical administration, ocular administration, or aural administration.

[0062] In other embodiments of the pharmaceutical composition, the pharmaceutical composition is a tablet, pill, capsule, liquid, inhaler, nasal spray solution, suppository, solution, emulsion, ointment, eye drops, or ear drops.

[0063] In other embodiments of the pharmaceutical composition, it further comprises one or more additional therapeutic agents.

[0064] On the other hand, the present invention provides a compound as represented by general formula (I), or its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts, crystal forms, solvates, hydrates or prodrugs, or the use of said pharmaceutical composition in the preparation of a drug for preventing, treating or improving diseases by inhibiting PARP1.

[0065] On the other hand, the present invention provides a method for preventing, treating or improving diseases by inhibiting PARP1, which comprises administering to an individual in need of such treatment an effective amount of a compound as represented by general formula (I), or its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts, crystal forms, solvates, hydrates or prodrugs, or said pharmaceutical composition.

[0066] In some embodiments of the present invention, the disease includes but is not limited to cancer.

[0067] In some embodiments of the present invention, the cancer genome is of a type deficient in homologous recombination repair.

[0068] In some embodiments of the invention, the cancer is dependent on a pathway that is deficient in homologous recombination repair of DNA double-strand damage.

[0069] In some embodiments of the present invention, the cancer comprises one or more cancer cells that lack the ability to repair DNA double-strand breaks by homologous recombination relative to normal cells.

[0070] In some embodiments of the invention, the cancer comprises one or more cancer cells that lack BRCA1 or BRCA2 or that have a BRCA1 or BRCA2 mutation.

[0071] In some embodiments of the present invention, the cancer includes but is not limited to malignant tumors, such as any one of ovarian cancer, breast cancer, fallopian tube cancer, endometrial cancer, peritoneal cancer, gastric cancer, colon cancer, bladder cancer, pancreatic cancer, biliary tract cancer, osteosarcoma, cervical cancer, head and neck tumors, germ cell cancer, embryonal carcinoma, esophageal cancer, malignant glioma, Ewing sarcoma, pancreatic cancer, melanoma, bile duct cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, lymphoma and blood cancer.

[0072] Terminology

[0073] In the present invention, unless otherwise specified, the terms used in the present invention have the meanings defined below. Terms not clearly defined in the present invention have the general meanings generally understood by those skilled in the art.

[0074] As used herein, the term "halogen" or "halo" refers to fluorine, chlorine, bromine, and iodine.

[0075] As used herein, the terms "optional," "optionally," or "optionally" mean that the subsequently described substitution pattern, event, or circumstance may or may not occur, and that the description includes instances where the substitution pattern occurs as well as instances where the substitution pattern does not occur. For example, "optionally substituted alkyl" includes "unsubstituted alkyl" and "substituted alkyl" as defined herein. It will be understood by those skilled in the art that, for any group containing one or more substituents, the group does not include any substitution pattern that is sterically impractical, chemically incorrect, synthetically infeasible, and / or inherently unstable.

[0076] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the compounds of the present invention and is not biologically or otherwise undesirable. Non-limiting examples of such salts include non-toxic, inorganic or organic base or acid addition salts of the compounds of the present invention. In many cases, the compounds of the present invention are capable of forming acid salts and / or base salts due to the presence of amino and / or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; ammonium, potassium, sodium, calcium, and magnesium salts are particularly preferred. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins, and the like, particularly isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. Pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound (basic or acidic moiety) by conventional chemical methods. Generally, the salts can be prepared by reacting the free acid form of the compound with a stoichiometric amount of an appropriate base (e.g., hydroxide, carbonate, bicarbonate, etc., of Na, Ca, Mg, or K) or by reacting the free base form of the compound with a stoichiometric amount of an appropriate acid. Such reactions are typically carried out in water or an organic solvent, or a mixture of the two. In general, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred where practicable. Other suitable salts can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Company, Easton, Pa., (1985), which is incorporated herein by reference.

[0077] As used herein, the term "solvate" is intended to include stoichiometric or non-stoichiometric solvent addition forms. If the solvent is water, the solvate formed is a hydrate, and when the solvent is ethanol, the solvate formed is an ethanolate. Hydrates are formed by one or more molecules of water with one molecule of the substance, wherein the water retains its molecular state of HO. Such a combination can form one or more hydrates, such as hemihydrates, monohydrates, and dihydrates.

[0078] As used herein, "prodrug" refers to a chemically modified active or inactive compound that, after administration to a subject, undergoes physiological action in the body (e.g., hydrolysis, necrolysis, etc.) to become a compound of the present invention. The adaptability and technology of making and using prodrugs are well known to those skilled in the art.

[0079] The term "therapeutically effective amount" of the compound of the present invention refers to an amount of the compound of the present invention that can elicit a biological or medical response in an individual or improve symptoms, slow down or delay disease progression, or prevent disease.

[0080] As used herein, the term "subject" refers to an animal. Preferably, the animal is a mammal. Subject also refers to, for example, primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In a preferred embodiment, the subject is a human.

[0081] As used herein, the term "inhibit" refers to a reduction or suppression of a particular condition, symptom or disorder or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0082] As used herein, the term "treating" any disease or condition refers, in one embodiment, to ameliorating the disease or condition (i.e., arresting or slowing the progression of the disease or at least one of its clinical symptoms). In another embodiment, "treating" refers to improving at least one physical parameter, which may not be perceptible to the patient. In another embodiment, "treating" refers to modulating the disease or condition physically (e.g., stabilizing a perceptible symptom) or physiologically (e.g., stabilizing a physical parameter), or both. Beneficial effects

[0083] The main advantages of the present invention are that the compounds disclosed herein have high selectivity for PARP1, fewer side effects than olaparib (AZD-2281), and high clinical application value. DETAILED DESCRIPTION

[0084] The present invention will be further described in detail below with reference to specific examples and data. It should be understood that these examples are merely illustrative of the present invention and are intended to illustrate the specific combinations, preparation methods, and functions and effects of the present invention, and are not intended to limit the scope of the invention in any way. The beneficial effects of the pharmaceutical combination of the present invention can also be determined by other test models known to those skilled in the relevant art.

[0085] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial sources. The starting materials can generally be obtained from commercial sources or easily prepared using methods known to those skilled in the art.

[0086] In each embodiment, the experimental instruments or materials used are as follows:

[0087] 1 H NMR was recorded on a Varian Mercury-300 or Varian Mercury-400 nuclear magnetic resonance spectrometer. 13 C NMR spectra were recorded on a Varian Mercury-400, Varian Mercury-500, or Varian Mercury-600 nuclear magnetic resonance instrument, and chemical shifts are expressed in δ (ppm). Mass spectra were recorded on Finnigan / MAT-95 (EI), Finnigan LCQ / DECA, and Micromass Ultra Q-TOF (ESI) mass spectrometers. Silica gel with a mesh size of 200-300 was used for reversed-phase preparative HPLC separations.

[0088] Abbreviations:

[0089] PE: petroleum ether; EA: ethyl acetate; DIEA: N,N-diisopropylethylamine; DCM: dichloromethane; DMF: N,N-dimethylformamide; THF: tetrahydrofuran; XPhos Pd G2: chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II); Ruphos Pd G3: methanesulfonic acid(2-dicyclohexylphosphino-2',6'-diisopropyloxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II).

[0090] Synthesis of key intermediates

[0091] Intermediate 1a: (R)-6-Fluoro-N-methyl-5-(2-methylpiperazin-1-yl)picolinamide hydrochloride

[0092] Step 1: Synthesis of methyl 5-bromo-6-fluoropicolinate

[0093] To a 50 mL single-necked flask, 1a-1 (1 g, 4.6 mmol), acetonitrile (30 mL), and silver difluoride (1.76 g, 13.9 mmol) were added sequentially and stirred at room temperature overnight. The reaction mixture was filtered, the filtrate was concentrated, and the residue was purified on a silica gel column (PE:EA = 5:1) to afford 1a-2 (450 mg, white solid) in a 42% yield. LCMS (ESI): m / z 233.9 [M+H] + ; RT = 1.51 min (3.00 min).

[0094] Step 2: Synthesis of tert-butyl (R)-4-(2-fluoro-6-(methoxycarbonyl)pyridin-3-yl)-3-methylpiperazine-1-carboxylate

[0095] To a 50 mL single-necked flask were added 1a-2 (450 mg, 1.9 mmol), (R)-4-Boc-2-methylpiperazine (577 mg, 2.9 mmol), Ruphos Pd G3 (159 mg, 0.19 mmol), cesium carbonate (1.5 g, 4.7 mmol), and dioxane (6 mL). The mixture was heated at 80°C overnight under nitrogen. The reaction mixture was concentrated, and the residue was purified on a silica gel column (PE:EA = 2:1) to afford product 1a-3 (300 mg, yellow solid) in a 44% yield. LCMS (ESI): m / z 354.1 [M+H] + ; RT = 1.80 min (3.00 min).

[0096] Step 3: (R)-tert-Butyl 4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)-3-methylpiperazine-1-carboxylate

[0097] 1a-3 (100 mg, 0.28 mmol) and methylamine ethanol solution (2 mL) were added to a 50 mL single-necked flask, and the reaction mixture was stirred overnight. Concentration afforded 1a-4 (80 mg, yellow solid) in an 80% yield. LCMS (ESI): m / z 297.1 [M-56+H] + ; RT = 1.70 min (3.00 min).

[0098] Step 4: Synthesis of (R)-6-fluoro-N-methyl-5-(2-methylpiperazin-1-yl)picolinamide hydrochloride

[0099] To a 20 mL single-necked flask, add 1a-4 (80 mg, 0.23 mmol), EA (2 mL), and a 4 M hydrochloric acid solution in dioxane (2 mL). Stir at room temperature for 2 hours. Concentrate to afford 1a (65 mg, yellow oil). Yield: 100%. LCMS (ESI): m / z 253.2 [M+H] + ; RT = 0.99 min (3.00 min).

[0100] Intermediate 2a: Synthesis of N-methyl-5-(piperazin-1-yl)pyridineamide hydrochloride

[0101] Step 1: Synthesis of tert-butyl 4-(2-fluoro-6-(methoxycarbonyl)pyridin-3-yl)piperazine-1-carboxylate

[0102] A 50 mL flask was charged with 1a-2 (500 mg, 2.1 mmol), 1-tert-butyloxycarbonylpiperazine (600 mg, 3.2 mmol), Ruphos Pd G3 (180 mg, 0.21 mmol), cesium carbonate (1.7 g, 5.2 mmol), and dioxane (15 mL). The mixture was heated at 80°C overnight under nitrogen. The reaction mixture was concentrated, and the residue was purified on a silica gel column (PE:EA = 2:1) to afford 2a-1 (640 mg, yellow solid) in an 88% yield. LCMS (ESI): m / z 340.1 [M+H]. + ; RT = 1.74 min (3.00 min).

[0103] Step 2: Synthesis of 5-(4-(tert-Butyloxycarbonyl)piperazin-1-yl)-6-fluoropicolinic acid

[0104] To a dry 50 mL single-necked flask, 2a-1 (320 mg, 0.94 mmol) and THF (8 mL) were added dropwise. An aqueous solution of lithium hydroxide monohydrate (200 mg, 4.7 mmol) (8 mL) was added dropwise and stirred for 2 hours. The reaction mixture was adjusted to pH 6 with 1 M hydrochloric acid. The reaction mixture was concentrated and purified via a reverse phase column (2% to 40% acetonitrile in water) to afford 2a-2 (300 mg, yellow solid) in a 92% yield. LCMS (ESI): m / z 326.1 [M+H] + ; RT = 1.26 min (3.00 min).

[0105] Step 3: Synthesis of tert-butyl 4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazine-1-carboxylate

[0106] To a dry, single-necked flask were added 2a-2 (300 mg, 0.92 mmol), 1-hydroxybenzotriazole (149 mg, 1.1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (211 mg, 1.1 mmol), DIEA (237 mg, 1.84 mmol), DMF (6 mL), and methylamine hydrochloride (123 mg, 1.84 mmol). The mixture was stirred overnight. Water (20 mL) was added to the reaction solution, which was then extracted three times with EA (30 mL). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated. The residue was purified on a reverse-phase column (20% to 70% acetonitrile in water) to afford 2a-3 (250 mg, white solid) in an 80% yield. LCMS (ESI): m / z 283.1 [M-100+H]. + ; RT = 1.66 min (3.00 min).

[0107] Step 4: Synthesis of 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0108] To a dry 20 mL single-necked flask, 2a-3 (250 mg, 0.74 mmol), EA (3 mL), and 4 M hydrochloric acid in dioxane (1 mL) were added sequentially at room temperature. The mixture was stirred at room temperature for 2 hours and concentrated to afford product 2a (200 mg, yellow solid). Yield: 100%. LCMS (ESI): m / z 239.1 [M+H] + ; RT = 0.91 min (3.00 min). 1 H-NMR (600MHz, CD3OD): 7.98-7.96 (m, 1H), 7.67-7.64 (m, 1H), 3.50-3.48 (m, 4H), 3.45-3.43 (m, 4H), 2.93 (d, J = 3.6Hz, 3H).

[0109] Intermediate 3a: N-methyl-5-(pyrrolidin-3-yloxy)picolinamide hydrochloride

[0110] Step 1: Synthesis of methyl 5-((1-(tert-butoxycarbonyl)pyrrolidin-3-yl)oxy)picolinate

[0111] Sodium hydride (641 mg, 16 mmol) was added to a 250 mL flask, and the atmosphere was replaced with nitrogen three times. Under an ice bath, THF (40 mL), 3a-1 (2 g, 10.7 mmol), and 3a-2 (1.66 g, 10.7 mmol) were added and reacted at room temperature for 1 hour. The reaction solution was extracted with EA (30 mL × 3). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA = 3:1) to obtain 3a-3 (1 g, yellow oil) in a 29% yield. LCMS (ESI): m / z 323.2 [M+H] + ; RT = 1.59 min (2.50 min).

[0112] Step 2: tert-Butyl 3-((6-(methylcarbamoyl)pyridin-3-yl)oxy)pyrrolidine-1-carboxylate

[0113] 3a-3 (400 mg, 1.24 mmol) and a 30% ethanolic solution of methylamine (6 mL) were added to a dry 100 mL flask. The mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure. The residue was diluted with water (10 mL) and extracted with EA (10 mL × 3). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 3a-4 (380 mg, yellow oil). LCMS (ESI): m / z 266 [M+H] + ; RT = 1.560 min (2.50 min). 1 H-NMR (400MHz, DMSO-d6): δ8.57(d,J=4Hz,1H),8.28(d,J=2.4Hz,1H),7.98(d,J=8.8Hz,1H),7.58-7.56(m,1H),5.18(s,1 H), 4.88 (d, J = 3.2Hz, 1H), 3.44-3.40 (m, 3H), 3.11 (s, 1H), 2.79 (d, J = 4.4Hz, 3H), 1.40 (d, J = 5.6Hz, 9H), 1.19-1.56 (m, 1H).

[0114] Step 3: Synthesis of N-methyl-5-(pyrrolidin-3-yloxy)picolinamide hydrochloride

[0115] To a single-necked flask at 0°C, 3a-4 (380 mg, 1.18 mmol), DCM (6 mL), and a dioxane hydrochloride solution (4.0 M, 6 mL) were added. The reaction was allowed to proceed at room temperature for 16 hours. The mixture was concentrated under reduced pressure to afford 3a (300 mg, crude yellow solid). LCMS (ESI): m / z 220 [M+H] + ; RT = 0.603 min (2.5 min).

[0116] Intermediates 4a, 5a:

[0117] The synthesis method is the same as that of intermediate 2a, except that ethylamine and cyclopropylamine are used instead of methylamine hydrochloride. 4a: LCMS (ESI): m / z 253.2 [M+H] + ; RT=0.527min (2.5min). 5a: LCMS(ESI):m / z 264.2[M+H] + ; RT = 0.958 min (2.5 min).

[0118] Intermediate 6a: 1-(4-(bromomethyl)-3-fluoropyridin-2-yl)-3-ethylurea

[0119] Step 1: Synthesis of methyl 2-(3-ethylureido)-3-fluoroisonicotinate

[0120] 6a-1 (500 mg, 2.65 mmol), 6a-2 (350 mg, 4.00 mmol), tris(dibenzylideneacetone)dipalladium (121 mg, 0.132 mmol), 2-(di-tert-butylphosphino)-1,1'-binaphthyl (105 mg, 0.265 mmol), and potassium phosphate (1.3 g, 4.00 mmol) were added to 10 mL of ethylene glycol dimethyl ether and stirred at 80°C overnight under argon. The mixture was cooled to room temperature and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (PE:EA = 1:1) to give 6a-3 (300 mg, white solid) in 47% yield. LCMS (ESI): m / z 242.1 [M+H] + ; RT = 1.36 min (3.0 min).

[0121] Step 2: Synthesis of 1-ethyl-3-(3-fluoro-4-(hydroxymethyl)pyridin-2-yl)urea

[0122] 6a-3 (100 mg, 0.41 mmol) and anhydrous THF (5 mL) were added to a 50 mL single-necked bottle and cooled to 0°C. A 1 M solution of lithium aluminum tetrahydride (0.8 mL, 0.82 mmol) was slowly added dropwise and stirred for 2 hours. The mixture was quenched with methanol and 2 mL of trifluoroacetic acid was added dropwise. The mixture was stirred for 10 minutes and concentrated. The residue was purified by reverse-phase column chromatography (1% to 30% acetonitrile / 0.1% aqueous ammonium bicarbonate) to afford 6a-4 (50 mg, white solid) in a 57% yield. LCMS (ESI): m / z 214.1 [M+H] + ; RT = 1.10 min (3.0 min).

[0123] Step 3: Synthesis of 1-(4-(bromomethyl)-3-fluoropyridin-2-yl)-3-ethylurea

[0124] 6a-4 (50 mg, 0.23 mmol) and DCM (5 mL) were added to a 50 mL single-necked flask, cooled to 0°C, and phosphorus tribromide (0.5 mL) was slowly added dropwise. The mixture was stirred for 2 hours and concentrated under reduced pressure to obtain crude product 6a (64 mg, white solid). Yield: 100%. LCMS (ESI): m / z 276.1 [M+H] + ; RT = 1.45 min (3.0 min).

[0125] Intermediate 7a: 1-(4-(bromomethyl)-3-fluoropyridin-2-yl)-3-cyclopropylurea

[0126] The synthesis method is the same as 6a, except that cyclopropyl urea is used instead of ethyl urea (6a-2) as the starting material. LCMS (ESI): m / z 288.1 [M+H] + ; RT = 1.47 min (3.0 min).

[0127] Intermediate 8a: 6-chloro-N-methyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0128] Step 1: Synthesis of 5-bromo-6-chloro-N-methylpicolinamide

[0129] 8a-1 (1.00 g, 3.99 mmol) and a 33% wt ethanol solution of methylamine (10 mL) were added to a flask. The mixture was allowed to react at room temperature for 16 hours. The reaction solution was concentrated to afford 8a-2 (0.94 g, yellow oil) in a 94.37% yield. LCMS (ESI): m / z 250.9 [M+H] + ; RT = 1.448 min (2.50 min).

[0130] Step 2: Synthesis of tert-butyl 4-(2-chloro-6-(methylcarbamoyl)pyridin-3-yl)piperazine-1-carboxylate

[0131] To a flask were added 8a-2 (620 mg, 2.49 mmol), toluene (15 mL), tert-butyl piperazine-1-carboxylate (370 mg, 1.99 mmol), 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (155 mg, 0.25 mmol), cesium carbonate (2020 mg, 6.21 mmol), and palladium acetate (56 mg, 0.25 mmol). The mixture was reacted at 100°C under nitrogen atmosphere for 16 hours. The reaction mixture was filtered, the filtrate was concentrated, and the residue was purified by silica gel column chromatography (PE:EA = 1:1) to afford 8a-3 (180 mg, yellow oil) in a yield of 20.41%. LCMS (ESI): m / z 355.1 [M+H] + ; RT = 1.679 min (2.50 min). 1 H-NMR (400MHz, DMSO-d6): δ8.46(d,J=4.8Hz,1H),7.95(d,J=8.4Hz,1H),7.68(d, J=8.4Hz,1H),3.50(s,4H),3.06-3.04(m,4H),2.80(d,J=4.8Hz,3H),1.43(s,9H).

[0132] Step 3: Synthesis of 6-chloro-N-methyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0133] 8a-3 (180 mg, 0.51 mmol), DCM (2 mL) and dioxane hydrochloride (4.0 M, 2 mL) were added to a dry flask. The mixture was stirred at room temperature for 2 hours and then concentrated to afford 8a (140 mg, yellow solid). Yield: 94.59%, LCMS (ESI): m / z 255.1 [M+H] + ;RT=0.336min&0.461min(2.50min). 1 H-NMR (400MHz, DMSO-d6): δ9.40 (s, 2H), 8.50 (d, J = 4.4Hz, 1H), 7.97 (d, J = 8.4Hz, 1H), 7.76 (d, J = 8.4Hz, 1H), 3.34-3.32 (m, 4H), 3.25 (m, 4H), 2.80 (d, J = 4.8Hz, 3H).

[0134] Intermediate 9a: N,6-dimethyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0135] Step 1: Synthesis of 5-bromo-6-methylpicolinic acid

[0136] 9a-1 (500 mg, 2.54 mmol), methanol (6 mL), water (3 mL), and sodium hydroxide (507 mg, 12.69 mmol) were added to a dry flask in sequence. The mixture was reacted at 70°C for 1 hour. The reaction solution was concentrated under reduced pressure. The mixture was diluted with water (10 mL), the pH was adjusted to 4 with 3M dilute hydrochloric acid, and the filter cake was collected by filtration to obtain 9a-2 (300 mg, white solid). Yield: 54.72%. LCMS (ESI): m / z 218.0 [M+H] + ; RT = 1.208 min (2.50 min). 1 H-NMR (400MHz, DMSO-d6): δ8.12 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 2.67 (s, 3H).

[0137] Step 2: Synthesis of 5-bromo-N,6-dimethylpicolinamide

[0138] To a dry flask were added 9a-2 (300 mg, 1.39 mmol), DMF (3 mL), DIEA (0.92 mL, 5.55 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (792 mg, 2.08 mmol), and a 2.0 M solution of methylamine in THF (1.39 mL, 2.78 mmol). The reaction was allowed to react at room temperature for 1 hour. The mixture was diluted with water (30 mL) and extracted with EA (10 mL x 2). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative chromatography (PE:EA = 3:2) to afford 9a-3 (215 mg, yellow solid) in a 67.59% yield. LCMS (ESI): m / z 231.0 [M+H]. + ; RT = 1.427 min (2.50 min). 1 H-NMR (400MHz, DMSO-d6): δ8.67(d,J=4.0Hz,1H), 8.18(d,J=8.4Hz,1H), 7.75(d,J=8.4Hz,1H), 2.82(d,J=4.8Hz,3H), 2.65(s,3H).

[0139] Step 3: Synthesis of tert-butyl 4-(2-methyl-6-(methylcarbamoyl)pyridin-3-yl)piperazine-1-carboxylate

[0140] To a dry flask were added 9a-3 (200 mg, 0.87 mmol), toluene (8 mL), tert-butyl piperazine-1-carboxylate (179 mg, 0.96 mmol), 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (54 mg, 0.09 mmol), cesium carbonate (711 mg, 2.18 mmol), and palladium acetate (20 mg, 0.09 mmol). The mixture was reacted at 100°C under nitrogen atmosphere for 16 hours. The reaction mixture was filtered and the filtrate was concentrated. The residue was purified by preparative chromatography (PE:EA = 1:1) to afford 9a-4 (160 mg, yellow solid) in a 54.80% yield. LCMS (ESI): m / z 335.1 [M+H] + .

[0141] Step 4: Synthesis of N,6-dimethyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0142] 9a-4 (160 mg, 0.48 mmol), DCM (2 mL) and hydrochloric acid-dioxane (4.0 M, 2 mL) were added to a dry flask. The mixture was reacted at room temperature for 4 hours and then concentrated to afford 9a (129 mg, yellow solid). Yield: 99.58%, LCMS (ESI): m / z 235.2 [M+H]+ ;RT=0.340min&0.450min(2.50min). 1 H-NMR (400MHz, DMSO-d6): δ9.44(s,2H),8.56(d,J=4.8Hz,1H),7.88(d,J=8.0Hz, 1H), 7.61 (d, J = 8.4Hz, 1H), 3.25-3.16 (m, 8H), 2.82 (d, J = 4.4Hz, 3H), 2.54 (s, 3H).

[0143] Intermediate 25a-4: methyl 2-chloro-3-fluoro-6-methylisonicotinate

[0144] Step 1: Synthesis of methyl 5-fluoro-2-methylisonicotinate

[0145] Under argon, 25a-1 (8.5 g, 36.32 mmol), trimethylboroxane (3.5 M, 20.74 mL, 72.64 mmol), potassium carbonate (10.1 g, 72.64 mmol), and 1,1-bis(diphenylphosphino)diphenylferric palladium chloride (2.6 g, 3.63 mmol) were added to 1,4-dioxane (100 mL). The mixture was heated to 90°C under nitrogen for 10 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA = 100:1-10:1) to afford 25a-2 (4.46 g, white solid) in a 72.59% yield. LCMS (ESI): m / z 170.1 [M+H]. + ; RT = 1.067 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ8.67(d,J=2.4Hz,1H),7.72(d,J=6.0Hz,1H),3.95(s,3H),2.57-2.56(m,3H).

[0146] Step 2: Synthesis of 5-fluoro-4-(methoxycarbonyl)-2-methylpyridine 1-oxide

[0147] At 0°C under argon, m-chloroperbenzoic acid (13.7 g, 79.11 mmol) was slowly added portionwise to 25a-2 (4.46 g, 26.37 mmol) dissolved in DCM (100 mL). After the addition was complete, the temperature was raised to 50°C and the reaction mixture was allowed to react for 4 h. The reaction mixture was cooled in an ice bath for 30 minutes. A large amount of solid precipitated and was filtered. The filter cake was washed with DCM, and the filtrate was collected and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA = 10:1 to 1:1) to afford 25a-3 (4.40 g, white solid) in a 90.13% yield. LCMS (ESI): m / z 186.1 [M+H] + ; RT = 0.857 min (2.50 min). 1 H-NMR (400MHz, DMSO-d6): δ8.68(d,J=6.4Hz,1H),7.98(d,J=9.2Hz,1H),3.87(s,3H),2.34(s,3H).

[0148] Step 3: Synthesis of methyl 2-chloro-3-fluoro-6-methylisonicotinate

[0149] To 25a-3 (4.4 g, 23.76 mmol) dissolved in 1,2-dichloroethane (100 mL) at 0°C under argon was slowly added phosphorus oxychloride (10.9 g, 71.29 mmol) and a drop of DMF. After the addition was complete, the temperature was raised to 80°C and the reaction was allowed to react for 4 h. The reaction solution was cooled in an ice bath for 30 min, resulting in the precipitation of a large amount of solid. The solid was filtered, and the filter cake was washed with DCM. The filtrate was collected and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA = 10:1 to 1:1) to afford 25a-4 (3.2 g, white solid) in a 66.1% yield. LCMS (ESI): m / z 204.0 [M+H] + ; RT = 1.245 min (2.50 min). 1 H-NMR (400MHz, DMSO-d6): δ7.70 (d, J = 4.4Hz, 1H), 3.91 (s, 3H), 2.51-2.49 (m, 3H).

[0150] Intermediate 26a-3: methyl 2-chloro-3-fluoro-6-methoxyisonicotinate

[0151] Step 1: Synthesis of 5-fluoro-2-methoxy-4-(methoxycarbonyl)pyridine 1-oxide

[0152] 26a-1 (200 mg, 1.08 mmol) was dissolved in DCM (5 mL), and m-chloroperbenzoic acid (744 mg, 4.32 mmol) was added at 0°C. The mixture was stirred at room temperature overnight. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The product was purified by column chromatography (PE:EA=3:1) to give 26a-2 (152 mg, white solid). Yield: 70.0%. LCMS (ESI): m / z 202.1 [M+H] + ; RT = 0.738 min (2.50 min). 1 H NMR (400MHz, CDCl3) δ8.28 (d, J = 5.3Hz, 1H), 7.42 (d, J = 7.0Hz, 1H), 4.11 (s, 3H), 3.98 (s, 3H).

[0153] Step 2: Synthesis of methyl 2-chloro-3-fluoro-6-methoxyisonicotinate

[0154] 26a-2 (2.5 g, 12.43 mmol) was dissolved in phosphorus oxychloride (20 mL) and reacted at 105°C for 16 hours. The reaction solution was cooled to room temperature and concentrated in vacuo. The residue was added to ice water and extracted with DCM. The organic phase was collected and concentrated. The residue was purified by column chromatography (EA:PE = 1:10) to afford 26a-3 (2 g, 9.11 mmol, white solid) in a 73% yield. LCMS (ESI): m / z 220.1 [M+H] + ; RT = 1.195 min (2.50 min). 1 H NMR (400MHz, CDCl3): δ7.19 (d, J=3.6Hz, 1H), 3.91 (s, 3H), 3.88 (s, 3H).

[0155] Intermediates 10a-18a, 21a-67a: The synthesis method is the same as 6a, except that the starting materials 6a-1 or 6a-2 are replaced by the raw material intermediates in the following table:

[0156] Intermediate 19a: 1-(2,4-difluorophenyl)piperazine

[0157] Step 1: Synthesis of tert-butyl 4-(2,4-difluorophenyl)piperazine-1-carboxylate

[0158] To a dry flask were added 19a-1 (2.00 g, 8.33 mmol), 1-tert-butyloxycarbonylpiperazine (2.33 g, 12.50 mmol), cesium carbonate (6.79 g, 20.83 mmol), 1,4-dioxane (50 mL), and Ruphos Pd G3(II) (698 mg, 0.83 mmol). The mixture was heated to 100°C under nitrogen and allowed to react for 16 hours. The mixture was filtered, the filtrate was evaporated to dryness, and the residue was purified by column chromatography (PE:EA = 20:1) to afford 19a-2 (720 mg, black solid) in a 25.00% yield. LCMS (ESI): m / z 243.1 [M+H-56]. + ; RT = 1.624 min (2.50 min).

[0159] Step 2: Synthesis of 1-(2,4-difluorophenyl)piperazine

[0160] 19a-2 (1.10 g, 3.81 mmol) and DCM (15 mL) were added to a flask, and 1,4-dioxane hydrochloride (1.5 mL) was added dropwise under ice-cooling. The mixture was stirred at room temperature for 2 hours. The mixture was dried by spin-drying, and water and EA were added. The aqueous phase was adjusted to a weak alkaline state with saturated sodium bicarbonate and extracted with EA. The organic phase was washed twice with saturated brine, dried over anhydrous sodium sulfate, and dried by spin-drying to obtain crude product 19a (180 mg, black oil). LCMS (ESI): m / z 199.1 [M+H] + ; RT = 0.672 min (2.50 min). 1 H-NMR (400MHz, DMSO-d6): δ7.20-7.14(m,1H),7.07-6.95(m,2H),2.91-2.82(m,8H).

[0161] Intermediate 20a: (6-(3-ethylureido)-5-fluoropyrimidin-4-yl)methyl methanesulfonate

[0162] Step 1: Synthesis of 1-(6-chloro-5-fluoropyrimidin-4-yl)-3-ethylurea

[0163] 20a-1 (1.00 g, 5.99 mmol), 1-ethylurea (0.53 g, 5.99 mmol), potassium carbonate (1.66 g, 11.98 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (0.35 g, 0.60 mmol), and tris(dibenzylideneacetone)dipalladium (0.55 g, 0.60 mmol) were added to 1,4-dioxane (40 mL). The mixture was reacted at 90°C under nitrogen for 15 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA = 5:1) to obtain 20a-2 (0.86 g, white solid) in a yield of 65.68%. LCMS (ESI): m / z 219.0 [M+H]. + ; RT = 1.219 min (2.50 min).

[0164] Step 2: Synthesis of 1-ethyl-3-(5-fluoro-6-(hydroxymethyl)pyrimidin-4-yl)urea

[0165] 20a-2 (660 mg, 3.02 mmol), 1,4-dioxane (25 mL), tributyltinmethanol (145 mg, 4.53 mmol), and XPhos Pd G2 (474 ​​mg, 0.60 mmol) were added to 1,4-dioxane (25 mL) and reacted at 80°C under nitrogen for 16 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (100% EA) to obtain 20a-3 (371 mg, yellow solid) in a yield of 49.02%. LCMS (ESI): m / z 215.1 [M+H] + ; RT = 0.827 min (2.50 min).

[0166] Step 3: Synthesis of (6-(3-ethylureido)-5-fluoropyrimidin-4-yl)methyl methanesulfonate

[0167] 20a-3 (337 mg, 1.57 mmol) and triethylamine (0.66 mL, 4.72 mmol) were added to THF (20 mL), and methanesulfonyl chloride (0.15 mL, 1.89 mmol) was added dropwise. The reaction was allowed to react at room temperature for 2 hours. The reaction solution was diluted with water (30 mL) and extracted with EA (20 mL × 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product 20a (436 mg, yellow solid). LCMS (ESI): m / z 293.0 [M+H] + ; RT = 1.077 min (2.50 min).

[0168] Intermediates 43a-44a, 48a-49a, 54a, 59a-60a: Synthesized by the same method as 20a, except that the starting material 20a-1 or ethyl urea was replaced by the intermediates listed in the table below.

[0169] Intermediate 24a: 1-(4-(bromomethyl)-3-fluoropyridin-2-yl)-3-(thiazol-2-yl)urea

[0170] Step 1: Synthesis of methyl 2-(diphenylmethylene)amino)-3-fluoroisonicotinate

[0171] 24a-4-1 (2.00 g, 10.6 mmol), diphenylmethane (1.9 g, 10.6 mmol), cesium carbonate (6.9 g, 21.2 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (613 mg, 1.06 mmol) and tris(dibenzylideneacetone)dipalladium (970 mg, 1.06 mmol) were added to anhydrous toluene (40 mL). The mixture was heated to 80 ° C under nitrogen protection and reacted for 16 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography to give compound 24a-4-2 (3.1 g, yellow oil). LCMS (ESI): m / z 335.1 [M+H] + ; RT = 1.617 min (2.50 min).

[0172] Step 2: Synthesis of methyl 2-amino-3-fluoroisonicotinate

[0173] 24a-4-2 (3.1 g, 9.3 mmol) was added to a solution of hydrochloric acid / dioxane (4 M, 30 mL), and water (0.5 mL) was added. The reaction was allowed to react at room temperature for 16 h. Water (50 mL) was added, and the mixture was extracted with PE (50 mL × 2). The aqueous phase was adjusted to pH 8-9 and extracted with EA (40 mL × 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to afford 24a-4 (1.3 g, yellow solid). Yield: 82.4%. LCMS (ESI): m / z 171.1 [M+H]. + ; RT = 0.910 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ7.84 (d, J=5.2Hz, 1H), 6.80-6.78 (m, 1H), 6.55 (s, 2H), 3.86 (s, 3H).

[0174] Step 3: Synthesis of 4-nitrophenylthiazol-2-ylcarbamate

[0175] 24a-1 (200 mg, 2.0 mmol) and pyridine (237 mg, 3.0 mmol) were added to DCM (15 mL), and 24a-2 (484 mg, 2.4 mmol) was added dropwise at 0°C. The mixture was allowed to react at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure, diluted with water, and filtered. The solid was dried under vacuum to give the crude product 24a-3 (450 mg, white solid), which was used directly in the next step.

[0176] Step 4: Synthesis of methyl 3-fluoro-2-(3-(thiazol-2-yl)ureido)isonicotinate

[0177] 24a-3 (51 mg, 0.19 mmol) and 24a-4 (30 mg, 0.18 mmol) were added to pyridine (3 mL) and stirred at room temperature for 1.5 hours. The residue was diluted with water (10 mL), the filter cake was collected by filtration, and washed with EA to give 24a-4 (50 mg, yellow oil), LCMS (ESI): m / z 297.0 [M+H] + ; RT = 1.390 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ12.12(s,1H),10.16(s,1H),8.34(d,J=4.8Hz,1H), 7.52-7.50(m,1H),7.44(d,J=3.6Hz,1H),7.22(d,J=3.2Hz,1H),3.92(s,3H).

[0178] Step 5: Synthesis of 1-(3-fluoro-4-(hydroxymethyl)pyridin-2-yl)-3-(thiazol-2-yl)urea

[0179] Under ice bath, 24a-4 (70 mg, 0.24 mmol), THF (3 mL), and a 2.5 M THF solution of lithium aluminum tetrahydride (0.2 mL) were added to a dry 25 mL three-necked flask and reacted at room temperature for 2 hours. The reaction solution was quenched with methanol (0.05 mL), 15% sodium hydroxide (0.05 mL), and methanol (0.05 mL), filtered, and concentrated under reduced pressure to give 24a-5 (70 mg, crude yellow solid). LCMS (ESI): m / z 269.1 [M+H-HCl]. + ; RT = 1.057 min (2.5 min).

[0180] Step 6: Synthesis of 1-(4-(bromomethyl)-3-fluoropyridin-2-yl)-3-(thiazol-2-yl)urea

[0181] 24a-5 (40 mg, 0.15 mmol) was added to a 100 mL single-necked flask containing 3 mL of dioxane. Phosphine tribromide (162 mg, 0.6 mmol) was added dropwise under ice-cooling and allowed to react overnight at room temperature. The reaction solution was directly concentrated to dryness to obtain 24a (50 mg, yellow solid). LCMS (ESI): m / z 331.0 [M+H-HCl] + ; RT = 1.508 min (2.5 min).

[0182] Intermediates 30a, 35a, 47a, 65a:

[0183] The synthesis method is the same as 8a, except that the raw material intermediates in the following table are used instead of 8a-1 or methylamine as the starting materials:

[0184] Intermediates 45a, 46a, 66a:

[0185] The synthesis method is the same as 9a, except that the raw material intermediates in the following table are used instead of methylamine in step 2:

[0186] Intermediate 52a: 6-(piperazin-1-yl)nicotinonitrile

[0187] The synthesis method was the same as that of 19a, except that 2-bromo-5-cyanopyridine was used instead of 19a-1 as the starting material. LCMS (ESI): m / z 189.1 [M+H] + .

[0188] Intermediate 53a: Methyl ((3-fluoro-2-(3-(prop-2-yn-1-yl)ureido)pyridin-4-yl)methanesulfonate

[0189] Step 1: Synthesis of methyl 2-(diphenylmethylene)amino)-3-fluoroisonicotinate

[0190] 53a-1 (3.00 g, 15.83 mmol), benzophenone imine (2.87 g, 15.83 mmol), cesium carbonate (10.32 g, 31.66 mmol), tris(dibenzylideneacetone)dipalladium (1.45 g, 1.58 mmol), and 4,5-bis(diphenylphosphino-9,9-dimethylxanthene) (1.83 g, 3.16 mmol) were added to toluene (50 mL). The mixture was reacted at 80°C under nitrogen for 10 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA = 5:1) to obtain 53a-2 (3.9 g, yellow oily liquid) in a yield of 73.71%. LCMS (ESI): m / z 335.0 [M+H].+ .

[0191] Step 2: Synthesis of methyl 2-amino-3-fluoroisonicotinate

[0192] 53a-2 (3.9 g, 11.66 mmol) was added to a 4 M dioxane hydrochloride solution (20 mL). The reaction was allowed to react at room temperature for 2 hours. The reaction solution was concentrated and diluted with EA. The organic phase was poured into an ice-cold saturated aqueous sodium bicarbonate solution and extracted with EA. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA = 1:1) to afford 53a-3 (1.1 g, white solid). Yield: 55.43%. LCMS (ESI): m / z 171.1 [M+H]. + .

[0193] Step 3: Synthesis of methyl 3-fluoro-2-((phenoxycarbonyl)amino)isonicotinate

[0194] To a THF solution (15 mL) of 53a-3 (910 mg, 5.35 mmol) was added phenyl chlorocarbonate (837 mg, 5.35 mmol), and pyridine (1.69 g, 21.40 mmol) was slowly added under ice-cooling conditions. The reaction was allowed to react at room temperature for 10 hours. The reaction solution was poured into water and extracted with EA. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA=1:1) to give the crude product 53a-4 (880 mg, yellow solid). LCMS (ESI): m / z 291.0 [M+H] +

[0195] Step 4: Synthesis of methyl 3-fluoro-2-(3-(prop-2-yn-1-yl)ureido)isonicotinate

[0196] 53a-4 (780 mg, 2.69 mmol), THF (15 mL), and propargylamine (1.48 mg, 26.87 mmol) were added sequentially to a microwave tube and microwaved for 0.5 h. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography (PE:EA=1:1) to give the crude product 53a-5 (290 mg, white solid). LCMS (ESI): m / z 252.0 [M+H] + .

[0197] Step 5: Synthesis of 1-(3-fluoro-4-(hydroxymethyl)pyridin-2-yl)-3-(prop-2-yn-1-yl)urea

[0198] To a solution of 53a-5 (290 mg, 1.15 mmol) in THF (5 mL) was slowly added dropwise a solution of lithium aluminum tetrahydride-THF (2.50 M, 0.92 mL, 2.30 mmol) at 0°C. The mixture was reacted at 0°C for 2 hours. Methanol (10 mL) was added under ice-cooling, stirred for 0.5 hours, and concentrated under reduced pressure. The residue was purified by column chromatography (DCM:methanol = 10:1) to afford 53a-6 (110 mg, light yellow solid) in a yield of 42.69%. 1 H-NMR (400MHz, DMSO-d6): δ9.25 (s, 1H), 9.16 (d, J = 5.2Hz, 1H), 8.05 (d, J = 5.2Hz, 1H),7.13(t,J=4.8Hz,1H),5.53(t,J=5.6Hz,2H),4.04-4.02(m,2H),3.13(s,1H).

[0199] Step 6: Synthesis of methyl ((3-fluoro-2-(3-(prop-2-yn-1-yl)ureido)pyridin-4-yl)methanesulfonate

[0200] To a solution of 53a-6 (100 mg, 0.43 mmol) in THF (10 mL) at 0°C were added triethylamine (0.18 mL, 1.29 mmol) and methanesulfonyl chloride (0.05 mL, 0.65 mmol). The mixture was allowed to react at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to afford 53a (130 mg, crude product, yellow solid). LCMS (ESI): m / z 302.0 [M+H] + .

[0201] Intermediates 57a, 58a, 61a, 62a:

[0202] The synthesis method is the same as 53a, except that the starting material 53a-3 is replaced by the raw material intermediates in the following table:

[0203] Synthesis of compound 1

[0204] Intermediate 6a (64 mg, 0.23 mmol), intermediate 2a (70 mg, 0.25 mmol), DIEA (148 mg, 1.15 mmol), and anhydrous acetonitrile (10 mL) were added to a 50 mL single-necked bottle and stirred at 70°C for 16 hours. The mixture was concentrated under reduced pressure, and the crude product was purified by reverse-phase column chromatography (1% to 50% acetonitrile / 0.1% aqueous ammonium bicarbonate) to afford compound 1 (50 mg, white solid) in a 50% yield. LCMS (ESI): m / z 434.1 [M+H] + ; RT = 1.43 min (3.0 min). 1H NMR (400MHz, DMSO-d6) δ9.04 (d, J=1.0Hz, 1H), 8.95-8.92 (m, 1H), 8.41 (q, J= 4.6Hz,1H),8.02(d,J=5.2Hz,1H),7.85(dd,J=8.0,1.2Hz,1H),7.56(dd,J=10 .6,8.2Hz,1H),7.08(t,J=4.8Hz,1H),3.63(s,2H),3.30-3.20(m,2H),3.20- 3.15(m,4H),2.78(d,J=4.8Hz,3H),2.65-2.55(m,4H),1.12(t,J=7.2Hz,3H).

[0205] Synthesis of compounds 2-9, 12-13, 17, 19-24, 31-35, 40-46, 48-152

[0206] According to the method described in compound 1, intermediates 6a and 2a were replaced by the intermediates in the table below to synthesize compounds 2-9, 12-13, 17, 19-24, 31-35, 40-46, and 48-152. The structural formulas of the compounds in each example are shown in the previous table.

[0207] Synthesis of compound 47

[0208] Step 1: Synthesis of methyl 3-fluoro-2-(3-(2-hydroxyethyl)ureido)isonicotinate

[0209] The synthesis method is the same as that of intermediate 6a-3, except that hydroxyethyl urea is used as the starting material to obtain 47-2 as a white solid in a yield of 41%. LCMS (ESI): m / z 258.2 [M+H] + .

[0210] Step 2: Synthesis of methyl 2-(3-(2-((tert-butyldimethylsilyl)oxy)ethyl)ureido)-3-fluoroisonicotinate

[0211] 47-2 (250 mg, 1.59 mmol), tert-butyldimethylsilyl chloride (175 mg, 1.17 mmol), and imidazole (132 mg, 1.946 mmol) were added to DMF (15 mL) and stirred at room temperature for 3 hours. The reaction solution was diluted with EA (50 mL) and washed with saturated aqueous sodium chloride. The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified on a silica gel column (EA:PE = 1:4) to obtain 47-3 (322 mg, 0.868 mmol) as a white solid in an 89% yield. LCMS (ESI): m / z 372.2 [M+H] + .

[0212] Step 3: Synthesis of 1-(2-((tert-butyldimethylsilyl)oxy)ethyl)-3-(3-fluoro-4-(hydroxymethyl)pyridin-2-yl)urea

[0213] The synthesis method is the same as that of 6a-4, except that 47-3 is used as the starting material to obtain 47-4 as a white solid in a yield of 49%. LCMS (ESI): m / z 344.2 [M+H] + .

[0214] Step 4: Synthesis of (2-(3-(2-((tert-butyldimethylsilyl)oxy)ethyl)ureido)-3-fluoropyridin-4-yl)methyl methanesulfonate

[0215] 47-4 (50 mg, 0.146 mmol) was dissolved in DCM (5 mL). Triethylamine (61 mg, 0.584 mmol) and methanesulfonyl chloride (33 mg, 0.292 mmol) were added sequentially at room temperature. After the addition was complete, the mixture was stirred for 1 hour. The reaction solution was diluted with DCM (15 mL), washed with saturated sodium bicarbonate aqueous solution, dried over anhydrous sodium sulfate, concentrated, and dried to obtain crude product 47-5 (61 mg, 0.146 mmol), which was used directly in the next reaction without purification. LCMS (ESI): m / z 422.2 [M+H] + .

[0216] Step 5: Synthesis of 5-(4-((2-(3-(2-((tert-butyldimethylsilyl)oxy)ethyl)ureido)-3-fluoropyridin-4-yl)methyl)piperazin-1-yl)-6-fluoro-N-methylpicolinamide

[0217] 47-5 (61 mg, 0.146 mmol) was dissolved in acetonitrile (5 mL), and DIEA (94 mg, 0.73 mmol) and intermediate 2a (40 mg, 0.146 mmol) were added sequentially. The reaction solution was heated to 70°C and stirred for 4 hours. The mixture was concentrated and purified on a silica gel column (EA:PE = 3:1) to afford product 47-6 (36 mg, 0.639 mmol) as a white solid in a 44% yield. LCMS (ESI): m / z 564.3 [M+H] + .

[0218] Step 6: Synthesis of 6-fluoro-5-(4-((3-fluoro-2-(3-(2-hydroxyethyl)ureido)pyridin-4-yl)methyl)piperazin-1-yl)-N-methylpicolinamide

[0219] 47-6 (36 mg, 0.639 mmol) was dissolved in methanol (5 mL), and HCl / methanol (0.5 mL, 4 M) was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated and purified by reverse phase column chromatography (acetonitrile:water = 0%-50%). The product 47 (14 mg, 0.0312 mmol) was obtained as a white solid in a yield of 48.8%. LCMS (ESI): m / z 450.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δ9.07(m,2H),8.40(q,J=4.4Hz,1H),8.01(d,J=5.1Hz,1H),7.90-7.80(m,1H),7.56(dd,J=10.6,8.2Hz,1H) ,7.08(t,J=4.9Hz,1H),4.77(s,1H),3.63(s,2H),3.49(s,2H),3.30-3.26(m,2H),3.17(m,4H),2.76(d,J=4.8Hz,3H),2.59(m,4H).

[0220] Biological Examples

[0221] Experimental Example 1: Evaluation of the PARP1 / 2 inhibitory activity of compounds

[0222] The PARP1 / 2 inhibitory activity of the disclosed compounds was tested in an assay using histones as substrates.

[0223] Experimental purpose: According to the established experimental method, the IC50 value of the compound of the present application for inhibition of PARP1 / 2 enzyme activity was detected, with AZD-2281 (Olaparib) as the positive control compound.

[0224] Reagents: Recombinant human PARP1 protein (Abcam, cat. ab279663); recombinant human PARP2 protein (BPS, cat. 80502); recombinant histone H1 (Active Motif, cat. 81126); NAD+, Biotin-Labeled (BPS, cat. 80610); SuperBlock (TBS) Blocking Buffer (Thermo Scientific TM ,cat.37535);Streptavidin(HRP)(Abcam,cat.ab7403); Peroxidase Chemiluminescent Substrate Kit (Seracare, cat.5430-0040); 20xPBS (CST, cat.9808S); 20xPBST (CST, cat.9809S); AZD2281 (Selleck, cat.S1060)

[0225] Experimental method 1: PARP1 inhibitory activity

[0226] 1. Compound configuration:

[0227] The compounds were diluted with DMSO to a solution with a final concentration of 1000 times in a 384-well plate and used for later use.

[0228] 2. Coating microplate:

[0229] 1) Dilute histones in PBS, add 25 μL of the histone mixture to each well, and incubate for 2 hours. 2) Wash each well five times with PBST. Remove the solution on a clean paper towel. 3) Add 75 μL of blocking buffer to each well and incubate at room temperature for 1 hour. 4) Wash each well five times with PBST. Remove the solution on a clean paper towel.

[0230] 3. Ribosylation reaction:

[0231] 1) Transfer 25 nL of the reserved compound at a 1000x final concentration to a 384-well plate. Add 25 nL of 100% DMSO to each of the Min and Max control wells. 2) Prepare a PARP1 solution at a 2.5x final concentration in 1× Assay buffer. 3) Add 10 μL of enzyme solution to each of the compound and Max control wells; add 10 μL of 1× Assay buffer to the Min control well. 4) Centrifuge at 1000 rpm for 60 seconds and incubate at room temperature for 15 minutes. 5) Prepare a substrate solution at a 1.67x final concentration in 1× Assay buffer. Add 500 μM NAD+ to the substrate solution and add 15 μL of substrate solution to each well to initiate the reaction. 6) Centrifuge at 1000 rpm for 60 seconds and incubate at room temperature for 2 hours. 7) Wash each well five times with PBST. Drain the solution on a clean paper towel.

[0232] 4. Detection:

[0233] 1) Prepare Streptavidin-HRP solution, add 25 μL to each well, centrifuge at 1000 rpm for 60 seconds, and incubate at room temperature for 30 minutes. 2) Wash each well five times with PBST. Drain the solution on a clean paper towel. 3) Add 50 μL of ELISA Chemiluminescent Substrate to each well. 4) Centrifuge at 1000 rpm for 60 seconds. Read the plate using EnSight after 5 minutes.

[0234] 5. Data Analysis:

[0235] Inhibition rate % = (maximum signal - compound signal) / (maximum signal - minimum signal) × 100, where "minimum signal" is the mean value of the negative control wells, and "maximum signal" is the mean value of the positive control wells.

[0236] Fitting the dose-effect curve: With the log value of the concentration as the X-axis and the percentage inhibition rate as the Y-axis, the log (inhibitor) vs. response-variable slope of the analysis software GraphPadPrism5 was used to fit the dose-effect curve to obtain the inhibition IC of the compound of the present disclosure on the enzyme activity. 50 The fitting formula is: Y = bottom + (top - bottom) / (1 + 10^((logIC 50 -X)*HillSlope)).

[0237] Experimental method 2: PARP2 inhibitory activity

[0238] The PARP2 inhibitory activity assay was performed in the same manner as in assay 1, except that recombinant human PARP2 solution was used instead of the PARP1 solution used in step 2 (2) of "3. Ribosylation Reaction," and 500 μM NAD+ was not added in the fifth step of the glycosylation reaction.

[0239] Table 1. IC values ​​of the compounds of the present disclosure for inhibition of PARP1 / 2 enzymes 50

[0240] Note: “ / ” means not tested.

[0241] Example 2: MDA-MB-436 cell proliferation inhibition test

[0242] Human breast cancer MDA-MB-436 (purchased from ATCC) cells were cultured in DMEM medium (supplemented with 10% fetal bovine serum and 1% double antibody) at 37°C and 5% carbon dioxide. Cells in the logarithmic growth phase were taken, digested, and a cell suspension of a certain concentration was prepared. The cell suspension was inoculated into a 96-well plate, and 100 μL of cell suspension was added to each well of the 96-well plate. After incubation overnight, different concentrations of compounds were added and placed in a cell culture incubator for 7 days. After the culture was completed, 50 μL of CellTiter-Glo reagent was added to each well, mixed with a microplate shaker for 2 minutes, and placed at room temperature for 60 minutes. The fluorescence value is read by the multimode microplate reader according to the formula: [(1-(RLU compound -RLU blank ) / (RLU control -RLU blank )) × 100%] to calculate the cell proliferation inhibition rate. GraphPad Prism 6.0 software was used to fit the IC 50 value.

[0243] Table 2. Inhibitory activity of the compounds disclosed herein on MDA-MB-436 cell proliferation

[0244] The experimental results show that the compound of the present invention has significant proliferation inhibitory activity on MDA-MB-436 cells.

[0245] Example 3: Evaluation of bidirectional permeability using the MDR1-MDCK II cell model

[0246] MDR1-MDCK Ⅱ cells were cultured at 3.3×10 5Cells were seeded into 96-well plates at a concentration of 10 cells / mL and grown for 4-7 days to form a monolayer of confluent cells. The test compound was added at a concentration of 2 μM to the dosing end wells on the apical or basolateral side of the monolayer cells and incubated at 37.0°C in a 5.0% CO2 incubator for 2.5 hours. The integrity of the cell monolayer was determined by the fluorescein exclusion assay. The buffer was removed from the apical and basolateral sides, and the concentration of the test compound was determined using LC-MS / MS. The concentration data was used to calculate the apparent permeability coefficient for transport from the apical side to the basolateral side and from the basolateral side to the apical side of the monolayer cells, and to calculate the efflux rate. Calculation formula: Efflux Ratio = Papp(BA) / Papp(AB)

[0247] Table 3. Bidirectional permeability of compounds of the present disclosure into MDR1-MDCK II cells

[0248] The experimental results show that the compound of the present invention has higher cell permeability and lower efflux rate in MDR1-MDCK Ⅱ cells.

[0249] Experimental Example 4: Preliminary pharmacokinetic test

[0250] 1. Healthy ICR mice were randomly divided into 3 groups of 3 mice each, with 9 male mice weighing 30-35 g for each administration route, and the test compound was administered intravenously (1 mg / kg) or orally (5 mg / kg).

[0251] The animals were fasted for 12 hours before the experiment and allowed to drink water freely. They were fed 4 hours after the administration.

[0252] 2. Blood collection time and sample processing

[0253] Intravenous and oral administration: 0.25h, 0.5h, 1.0h, 2.0h, 3.0h, 4.0h, 6.0h, 8.0h and 24h after administration.

[0254] Blood was collected continuously, with 3 animals collected at each time point. Plasma collection and processing: 30-40 μL of venous blood was collected from the retroorbital venous plexus of mice at the above-set time points, placed in EDTA-K2 tubes, centrifuged at 3500 rpm for 10 minutes, and plasma was separated and frozen in a -20°C refrigerator.

[0255] 3. Sample testing and data analysis

[0256] The concentration of the compound in mouse plasma was determined by LC / MS / MS, and the pharmacokinetic parameters after administration were calculated using a non-compartmental model using Phoenix 8.3 software (Pharsight, USA).

[0257] 4. Experimental Results

[0258] Table 4. Pharmacokinetic parameters of the compounds of the present disclosure in mouse plasma

[0259] Note: “ / ”: not tested; iv: intravenous injection (1 mg / kg); po: oral administration (5 mg / kg).

[0260] The experimental results show that the pharmacokinetics of the compound of the present invention in mice after oral administration show a long half-life (T 1 / 2 ), lower clearance (CL) and higher bioavailability.

[0261] As can be seen from Table 1, the compounds of the present disclosure have high selectivity for PARP1 and may reduce the toxicity caused by PARP2 without significantly reducing the efficacy. As can be seen from Table 2, the compounds of the present disclosure have strong proliferation inhibitory activity on human breast cancer cells (MDA-MB-436). The inventors also unexpectedly found that the compounds of the present disclosure have high cell permeability and low efflux rate in MDR1-MDCKⅡ cells, and the compounds of the present disclosure have good physical and chemical stability, good bioavailability (such as low clearance rate) and good drugability. Therefore, the compounds of the present disclosure have fewer side effects than olaparib (AZD-2281) and have high clinical application value.

Claims

1. A compound represented by the general formula (I), or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, crystal form, solvate, hydrate or prodrug thereof, in, is a single bond or a double bond; X 1 Selected from -N- and -CR 8 -; X 2 Selected from -N- and -CR 9 -; X 3 -N- or -CR 10 -; R 1 , R 1’ , R 2 , R 3 Each is independently selected from hydrogen, unsubstituted or substituted C1-C6 alkyl; R 4 , R 5 are each independently selected from hydrogen, unsubstituted or substituted C1-C6 alkyl; or R 4 and R 5 Together with the carbon atom to which it is bonded, it forms a C3-C6 cycloalkyl group; R 7 is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, C3-C6 alkenyl, C3-C6 alkynyl, unsubstituted or substituted C3-C8 cycloalkyl, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted 3- to 8-membered heterocycloalkyl, unsubstituted or substituted 5- to 6-membered heteroaryl, unsubstituted or substituted C6-C 10 Aryl; preferably, R 7 is selected from C1-C4 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, 3- to 8-membered heterocycloalkyl, phenyl, 5- to 6-membered heteroaryl, which are unsubstituted or substituted by halogen, hydroxy, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 alkoxy or C3-C6 cycloalkyl; more preferably, R 7 Selected from C1-C4 alkyl, C3-C6 cycloalkyl which is unsubstituted or substituted by halogen, hydroxy, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 alkoxy or C3-C6 cycloalkyl; R 8 is selected from hydrogen, halogen, cyano, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C3-C6 cycloalkyl, unsubstituted or substituted C1-C6 alkoxy, NH2, -NH-C1-C4 alkyl; preferably, R 8 is hydrogen, halogen, methoxy, cyano, NH2, -NH-C1-C4 alkyl, C3-C6 cycloalkyl or C1-C4 alkyl; more preferably, R 8 is hydrogen, fluorine, chlorine, methyl, CN, NH2, NHCH3, cyclopropane or methoxy; R 9 is selected from hydrogen, halogen, cyano, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 alkoxy; preferably, R 9 is hydrogen, halogen, cyano or C1-C4 alkyl; more preferably, R 9 is hydrogen, fluorine, chlorine or methyl; R 10 is selected from hydrogen, halogen, cyano, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted C1-C6 alkyl; preferably, R 10 is hydrogen, halogen, cyano or C1-C4 alkyl; more preferably, R 10 is hydrogen, fluorine, chlorine or methyl; s and n are each independently selected from 0, 1 and 2; Y is N or CH; R 6 Selected from: Each R 11 Each is independently selected from halogen, cyano, C1-C3 alkoxy, carbonyl, -CONHR 13 and amino, excellent Preferably selected from halogen, -CONHR 13 and cyano; m is 0, 1, 2, or 3; R 12 is selected from hydrogen, cyano, halogen, unsubstituted or substituted C1-C4 alkyl; R 13 is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C3-C8 cycloalkyl, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted 3-8 membered heterocycloalkyl; preferably, R 13 Selected from hydrogen, unsubstituted, or halogen- or deuterium-substituted C1-C4 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy; The heterocycloalkyl group refers to a heterocycloalkyl group containing 1 to 3 heteroatoms selected from N, O, and S; The substitution refers to substitution by one or more selected from deuterium, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C6 alkoxy, halogen, hydroxyl, cyano, amino, carboxyl and C3-C6 cycloalkyl.

2. The compound according to claim 1, or its stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, crystal form, solvate, hydrate or prodrug, wherein: In the general formula (I) Selected from:

3. The compound according to claim 2, or its stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, crystal form, solvate, hydrate or prodrug, wherein: In the general formula (I) Selected from:

4. The compound according to claim 1, or its stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, crystal form, solvate, hydrate or prodrug, wherein: In the general formula (I) Selected from the following structures:

5. The compound according to claim 1, or its stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, crystal form, solvate, hydrate or prodrug, wherein: In the general formula (I) Selected from the following structures:

6. The compound according to any one of claims 1 to 5, or its stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, crystal form, solvate, hydrate or prodrug, wherein: The compound represented by the general formula (I) is selected from the following compounds:

7. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 6, or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, crystalline form, solvate, hydrate or prodrug thereof, and a pharmaceutically acceptable carrier.

8. Use of the compound according to any one of claims 1 to 6, or its stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, crystal form, solvate, hydrate or prodrug, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for preventing, treating or ameliorating a disease by inhibiting PARP1.

9. The use according to claim 8, wherein The disease is cancer comprising one or more cancer cells of a type that lacks BRCA1 or BRCA2 or has a BRCA1 or BRCA2 mutation.

10. The use according to claim 8, wherein The disease is selected from any one of ovarian cancer, breast cancer, fallopian tube cancer, endometrial cancer, peritoneal cancer, gastric cancer, colon cancer, bladder cancer, pancreatic cancer, biliary tract cancer, osteosarcoma, cervical cancer, head and neck tumors, germ cell cancer, embryonal carcinoma, esophageal cancer, malignant glioma, Ewing sarcoma, pancreatic cancer, melanoma, bile duct cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, lymphoma and blood cancer.

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