Fused tricyclic compound, pharmaceutical composition thereof, and use thereof

Abstract

Provided are a fused tricyclic compound represented by formula (I), a pharmaceutical composition thereof, and use thereof. The compound has high selectivity for PARP1, has fewer side effects than olaparib, has high clinical application value, and can be used to prepare drugs for preventing and / or treating diseases, in particular diseases ameliorated by the inhibition of PARP1.

Description

Fused tricyclic compounds, pharmaceutical compositions and uses thereof Technical Field

[0001] The present invention relates to the technical field of PARP inhibitor synthesis, and in particular to a class of fused tricyclic compounds, pharmaceutical compositions thereof, and use thereof as PARP1 inhibitors in the preparation of drugs for preventing and / or treating diseases improved 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 PARP-1 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 levels are low, 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 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 fused tricyclic 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), its stereoisomers, geometric isomers, tautomers, and pharmaceutically acceptable salts or hydrates.

[0012] in:

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

[0014] X 2 Selected from N, -C(=O) and CR 8a ;

[0015] X 3 Selected from N, O and CR 8b ;

[0016] X 4 Selected from N and CR 9 ;

[0017] X 5 Selected from N and CR 10 ;

[0018] X 6 Selected from NR 7c and CR 14 R 15 ;

[0019] X 7 、X 8 are each selected from C and N, provided that X 7 and X 8 Not C;X 2 and X 3 Not all N at the same time;

[0020] R 7cis selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted 3 to 8 membered heterocyclyl, substituted or unsubstituted C2-C6 alkynyl; preferably, R 7c is selected from hydrogen, unsubstituted or halogen-substituted C1-C4 alkyl, unsubstituted or halogen-substituted C3-C6 cycloalkyl, C1-C6 alkoxy, 3- to 6-membered heterocyclyl, C2-C6 alkynyl;

[0021] R 8a and R 8b are each independently selected from hydrogen, halogen, cyano, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, preferably, R 8a and R 8b Each is independently selected from hydrogen, fluorine, chlorine, methyl, ethyl, cyclopropyl;

[0022] R 9 and R 10 are each independently selected from hydrogen, halogen, cyano, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; preferably, R 9 and R 10 are each independently selected from hydrogen, halogen, cyano and C1-C4 alkyl; more preferably, R 9 and R 10 are each independently selected from hydrogen, fluorine, chlorine and methyl;

[0023] R 14 and R 15 are each independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl; or R 14 and R 15 Together with the carbon atoms to which they are attached, they form a C3-C6 cycloalkyl group;

[0024] R 1 、R 1’ 、R 2 and R 3 Each is independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl;

[0025] R 4 and R 5 are each independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl; or R 4 and R 5 Together with the carbon atoms to which they are attached, they form a C3-C6 cycloalkyl group;

[0026] s and n are each independently selected from 0, 1 and 2;

[0027] Y is N, C or CH;

[0028] R 6 Selected from:

[0029] R 11 Each independently selected from halogen, cyano, C1-C3 alkoxy, carbonyl, -CONHR 13 and amino, preferably selected from halogen, -CONHR 13 and cyano;

[0030] m is 0, 1, 2 or 3;

[0031] R 12 is selected from hydrogen, cyano, halogen, substituted or unsubstituted C1-C4 alkyl;

[0032] R 13 is selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted 3-8 membered heterocyclyl; preferably, R 13 Selected from hydrogen, unsubstituted or halogen-substituted C1-C4 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy;

[0033] The heterocyclic group refers to a heterocyclic group containing 1-3 heteroatoms selected from N, O, and S; the substitution refers to substitution by one or more selected from C1-C4 alkyl, C1-C6 alkoxy, halogen, hydroxyl, cyano, amino, carboxyl, and C3-C6 cycloalkyl.

[0034] In some embodiments, X 2 N or CR 8a , the definitions of other substituents are the same as above.

[0035] In some embodiments, X 3 N or CR 8b , the definitions of other substituents are the same as above.

[0036] In some embodiments, X 7 is N, and the definitions of other substituents are the same as above.

[0037] In some embodiments, X 8 is N, and the definitions of other substituents are the same as above.

[0038] In some embodiments, X 4 The other substituents are defined as above.

[0039] In some embodiments, X 5 CR 10, R 10 is H, C1-C4 alkyl or halogen, and the definitions of other substituents are the same as above.

[0040] In some embodiments, X 6 NR 7c , the definitions of the other substituents are the same as above, and the compound is not Preferably, R 7c Selected from hydrogen, C1-C4 alkyl which is unsubstituted or substituted by halogen, hydroxy or C3-C6 cycloalkyl, C3-C6 cycloalkyl which is unsubstituted or substituted by halogen, C1-C6 alkoxy, 3 to 6 membered heterocyclyl, C2-C6 alkynyl.

[0041] In some embodiments, in formula (I) Selected from the following structures:

[0042] More preferably, it is selected from the following structures:

[0043] wherein the definitions of the substituents are the same as above; further preferably, R 8a and R 8b Each is independently selected from hydrogen, fluorine, chlorine, methyl, ethyl, and cyano, and the definitions of other substituents are the same as above.

[0044] In some embodiments, in formula (I) Selected from the following structures:

[0045] Among them, R 8a and R 8b Each independently selected from hydrogen, fluorine, chlorine, methyl, ethyl, cyano, R 9 and R 10 are independently selected from hydrogen, fluorine, chlorine, and methyl, and the definitions of the other substituents are the same as above. 7c Selected from hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl, C1-C4 alkoxy.

[0046] In some embodiments, in formula (I) Selected from the following structures:

[0047] Preferably, it is selected from the following structures:

[0048] Among them, each substituent m, R 11 、R 12 、R 13 The definition of is the same as that of the aforementioned formula (I).

[0049] In some embodiments, in formula (I) Selected from the following structures:

[0050] The definitions of the substituents are the same as above.

[0051] Those skilled in the art will understand that are equivalent.

[0052] In some embodiments, there is provided a formula (II), a compound of formula (II), a stereoisomer, a geometric isomer, a tautomer, a pharmaceutically acceptable salt or a hydrate thereof,

[0053] The definitions of the substituents are the same as those in the above formula (I).

[0054] In some embodiments, a compound of formula (IV) or formula (V), its stereoisomers, geometric isomers, tautomers, or pharmaceutically acceptable salts or hydrates are provided.

[0055] The definitions of the substituents are the same as those in the above formula (I).

[0056] In some embodiments, as described in Formula (I), Formula (II), Formula (III), Formula (IV) or Formula (V), X 6 NR 7c , the definitions of other substituents are the same as above.

[0057] In some embodiments, as described in Formula (I), Formula (II), Formula (III), Formula (IV) or Formula (IV), X 6 CR 14 R 15 , the definitions of other substituents are the same as above.

[0058] In another preferred embodiment, as the compound described in formula (II) or formula (IV), R 8a is hydrogen or C1-C2 alkyl; preferably, R 8a is hydrogen or methyl.

[0059] In another preferred embodiment, the compound of formula (III) or formula (V), R 8b is hydrogen or C1-C2 alkyl; preferably, R 8b is hydrogen or methyl.

[0060] Typical compounds of the present invention include, but are not limited to:

[0061] The compounds of formula (I), (II), (III), (IV), and (V) described in the present invention, each specific compound, its stereoisomers, geometric isomers, tautomers, and pharmaceutically acceptable salts and hydrates thereof are collectively referred to as the compounds of the present invention.

[0062] The present invention also provides methods for preparing the compounds of the present invention, intermediates used to prepare the compounds of the present invention, and methods for preparing the intermediates.

[0063] The present invention also provides compositions comprising at least one compound of the present invention.

[0064] Another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds selected from the compounds of the present invention, and optionally a pharmaceutically acceptable carrier.

[0065] In one embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0066] In another embodiment, the present invention provides a combination, in particular a pharmaceutical combination, comprising a therapeutically effective amount of a compound of the present invention, and one or more additional therapeutic agents.

[0067] 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, ophthalmic administration, or otic administration. In other embodiments of the pharmaceutical composition, the pharmaceutical composition is a tablet, pill, capsule, liquid, inhalant, nasal spray solution, suppository, solution, emulsion, ointment, eye drops, or ear drops. In other embodiments of the pharmaceutical composition, it further comprises one or more additional therapeutic agents.

[0068] The pharmaceutical composition of the present invention can be selected to contain at least one pharmaceutically acceptable carrier selected from the following according to the mode of administration and the properties of the dosage form: diluents, adjuvants, excipients, preservatives, fillers, binders, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, temperature-sensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0069] The compounds according to the invention can be used alone, in combination with other compounds according to the invention or in combination with one or more, preferably one or two, other substances, simultaneously or sequentially.

[0070] Another aspect of the present invention provides use of the compound of the present invention or the pharmaceutical composition in the preparation of a PARP1 inhibitor.

[0071] In another aspect, the present invention provides use of the compound of the present invention or the pharmaceutical composition described above in the preparation of a medicament for preventing and / or treating a disease that can be improved by inhibiting PARP1.

[0072] In another aspect, the present invention provides a method for preventing and / or treating diseases that can be improved by inhibiting PARP1, which comprises administering an effective amount of one or more compounds selected from the present invention or the pharmaceutical composition to an individual in need of such treatment.

[0073] In some embodiments of the present invention, the disease improved by inhibiting PARP1 includes but is not limited to cancer.

[0074] In some embodiments of the 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 and embryonal cancers, 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.

[0075] In some embodiments of the present invention, the cancer genome is of a homologous recombination repair-deficient type.

[0076] In some embodiments of the invention, the cancer is dependent on a pathway that is deficient in homologous recombination repair of double-stranded DNA damage.

[0077] 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.

[0078] In some embodiments of the invention, the cancer comprises one or more cancer cells that lack BRCA1 or BRCA2 or have a BRCA1 or BRCA2 mutation.

[0079] Terminology

[0080] 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.

[0081] When the group has a wavy line , the wavy line indicates where the group is attached to the rest of the molecule.

[0082] As used herein, Represents a single bond or a double bond. Those skilled in the art can determine the Whether it represents a single bond or a double bond is within the ability of a person skilled in the art. A person skilled in the art will understand that the relevant rings can be saturated, partially saturated or aromatic.

[0083] 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 and instances where it does not.

[0084] As used herein, "halogen" may be fluorine, chlorine, bromine or iodine. Preferred halogens are fluorine or chlorine.

[0085] As used herein, "C1-C6 alkyl" refers to a fully saturated, straight-chain or branched hydrocarbon group having 1 to 6 carbon atoms; specific examples thereof may include methyl, ethyl, propyl, n-propyl, isopropyl, butyl, n-butyl, isobutyl, tert-butyl, 1-methyl-butyl, 1-ethyl-butyl, pentyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 4-methyl-2-pentyl, 3,3-dimethylbutyl, 2-ethyl-butyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, and the like, but are not limited thereto. The meaning of C1-C4 alkyl is similarly deduced.

[0086] As used herein, "C2-C6 alkynyl" refers to a linear or branched monovalent hydrocarbon group having 2 to 6 carbon atoms and containing at least one triple bond. Representative examples include, but are not limited to, ethynyl, propynyl, isopropynyl, butynyl, isobutynyl, pentynyl, isopentenyl, hexynyl, and the like.

[0087] As used herein, "C1-C6 alkoxy" refers to an RO- group, where R is a C1-C6 alkyl group as described above. Specific examples of alkoxy include methoxy, ethoxy, n-propoxy, isopropoxy, and the like. The meaning of C1-C3 alkoxy is analogous.

[0088] As used herein, "C3-C8 cycloalkyl" refers to a fully saturated monovalent cyclic hydrocarbon compound group containing 3-8 ring carbon atoms, and specific examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The meaning of C3-C6 cycloalkyl is similar.

[0089] As used herein, "3-8 membered heterocyclic group" refers to a 3-8 membered non-aromatic cyclic group containing 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur in the ring, and specific examples thereof include oxetane, tetrahydroimidazole and tetrahydrofuran.

[0090] As used herein, "pharmaceutically acceptable salts" refers to salts that retain the biological effects and properties of the compound and are 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 or organic acids, including, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; and organic acids including, 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 also be formed with inorganic or organic bases, including, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are ammonium, potassium, sodium, calcium, and magnesium salts; and organic bases including, for example, primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins, and the like, particularly, for example, 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.

[0091] The compounds of the present invention may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms which may be defined, in terms of absolute stereochemistry, as (R)- or (S)-, or, for amino acids, as (D)- or (L)-. All such possible isomers, as well as their racemic and optically pure forms, whether or not they are specifically described herein, are intended to be included herein.

[0092] "Stereoisomers" refer to compounds made up of the same atoms bonded by the same bonds but having different three-dimensional structures that are not interchangeable. Various stereoisomers and mixtures thereof are contemplated herein, and include "enantiomers," which refer to two stereoisomers whose molecules are non-superimposable mirror images of each other.

[0093] "Tautomer" refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present invention includes tautomers of any of the described compounds.

[0094] The compounds of the present invention or their stereoisomers, geometric isomers, tautomers, and pharmaceutically acceptable salts may also exist in the form of polymorphs, solvates (e.g., hydrates), and prodrugs, and these polymorphs, solvates (e.g., hydrates), and prodrugs are also included in the scope of the present invention.

[0095] 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.

[0096] 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.

[0097] As used herein, the term "therapeutically effective amount" 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, etc.

[0098] 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.

[0099] 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.

[0100] 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

[0101] The compounds of the present invention have high selectivity for PARP1. In specific embodiments, the compounds of the present invention have a selectivity factor of ≥100 for PARP1 / 2, preferably a selectivity factor of ≥200, more preferably a selectivity factor of ≥500, even more preferably ≥800, and most preferably ≥1000. Moreover, the compounds of the present invention have fewer side effects than olaparib (AZD-2281) and have high clinical application value.

[0102] The present invention has been described in detail above, but the above embodiments are merely illustrative in nature and are not intended to limit the present invention. In addition, the present invention is not limited by any theory described in the above prior art or invention summary or the following examples. DETAILED DESCRIPTION

[0103] The present invention will be further described below with reference to the examples. It should be noted that the following examples are provided for illustrative purposes only and do not constitute a limitation on the scope of protection claimed in the present invention.

[0104] 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.

[0105] In each embodiment, the experimental instruments or materials used are as follows:

[0106] 1 H NMR spectra were recorded on a Bruker Advance-400 or -600 NMR spectrometer, 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. Reverse-phase preparative HPLC separations were performed on 200-300 mesh silica gel.

[0107] Abbreviations:

[0108] PE: petroleum ether; EA: ethyl acetate; DIEA: N,N-diisopropylethylamine; DCM: dichloromethane; DMF: N,N-dimethylformamide; 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).

[0109] Example

[0110] Synthesis of key intermediates

[0111] Intermediate 1a: Synthesis of 6-chloro-N-methyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0112] Step 1: Synthesis of 5-bromo-6-chloro-N-methylpicolinamide

[0113] 1a-1 (1.00 g, 3.99 mmol) and a methylamine ethanol solution (33 wt %, 10 mL) were added to a flask in sequence. The reaction was allowed to proceed at room temperature for 16 hours. The reaction solution was concentrated to afford 1a-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) (the time before the bracket is the peak time, and the time in the bracket is the total running time).

[0114] Step 2: Synthesis of tert-butyl 4-(2-chloro-6-(methylcarbamoyl)pyridin-3-yl)piperazine-1-carboxylate

[0115] To a flask were added 1a-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 and the filtrate was concentrated. Purification by silica gel column chromatography (PE:EA = 1:1) afforded 1a-3 (180 mg, yellow oil) in a 20.41% yield. 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).

[0116] Step 3: Synthesis of 6-chloro-N-methyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0117] 1a-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 1a (140 mg, yellow solid). Yield: 94.59%, LCMS (ESI): m / z 255.1 [M+H] + ;RT=0.336min&0.461min(2.50min). 1H-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).

[0118] Intermediate 2a: Synthesis of N-methyl-5-(piperazin-1-yl)pyridineamide hydrochloride

[0119] Step 1: Synthesis of tert-butyl 4-(2-fluoro-6-(methoxycarbonyl)pyridin-3-yl)piperazine-1-carboxylate

[0120] 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).

[0121] Step 2: Synthesis of 5-(4-(tert-Butyloxycarbonyl)piperazin-1-yl)-6-fluoropicolinic acid

[0122] To a dry 50 mL single-necked flask, 2a-1 (320 mg, 0.94 mmol) and tetrahydrofuran (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 by reverse phase column chromatography (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).

[0123] Step 3: Synthesis of tert-butyl 4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazine-1-carboxylate

[0124] 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), N,N-diisopropylethylamine (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 was 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).

[0125] Step 4: Synthesis of 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0126] 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).

[0127] Intermediate 3a: Synthesis of N,6-dimethyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0128] Step 1: Synthesis of 5-bromo-6-methylpicolinic acid

[0129] 3a-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) and adjusted to pH 4 with 3M dilute hydrochloric acid. The filter cake was filtered to obtain 3a-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=8.0Hz,1H),7.78(d,J=8.4Hz,1H),2.67(s,3H).

[0130] Step 2: Synthesis of 5-bromo-N,6-dimethylpicolinamide

[0131] To a dry flask were added 3a-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 tetrahydrofuran (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 ethyl acetate (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 using a preparative plate (PE:EA = 3:2) to afford 3a-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).

[0132] Step 3: Synthesis of tert-butyl 4-(2-methyl-6-(methylcarbamoyl)pyridin-3-yl)piperazine-1-carboxylate

[0133] To a dry flask were added 3a-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 reaction was stirred at 100°C under nitrogen for 16 hours. The reaction mixture was filtered and the filtrate was concentrated. The residue was purified using a preparative plate (PE:EA = 1:1) to afford 3a-4 (160 mg, yellow solid) in a 54.80% yield. LCMS (ESI): m / z 335.1 [M+H]. + .

[0134] Step 4: Synthesis of N,6-dimethyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0135] 3a-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 3a (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).

[0136] Intermediate 4a: Synthesis of N-ethyl-6-fluoro-5-(piperazin-1-yl)picolinamide hydrochloride

[0137] The synthesis method refers to the synthesis of intermediate 2a, except that ethylamine was used instead of methylamine hydrochloride. LCMS (ESI): m / z 253.2 [M+H] + ; RT = 0.527 min (2.5 min).

[0138] Intermediate 5a: Synthesis of N-cyclopropyl-6-fluoro-5-(piperazin-1-yl)picolinamide hydrochloride

[0139] The synthesis method refers to the synthesis of intermediate 2a, except that cyclopropylamine is used instead of methylamine hydrochloride. LCMS (ESI): m / z 264.2 [M+H]+ ; RT = 0.958 min (2.5 min).

[0140] Intermediate 6a: 7-Fluoro-8-(hydroxymethyl)-2,4-dimethyl-4H-imidazo[1,5,4-de]quinoxalin-5(6H)-one

[0141] Step 1: Synthesis of ethyl (4-bromo-3-fluoro-2-nitrophenyl)alaninate

[0142] 6a-1 (5 g, 21 mmol) was dissolved in DMF (25 mL). DL-alanine ethyl ester hydrochloride (2.89 g, 18.9 mmol) and DIEA (8.1 g, 63 mmol) were added sequentially under ice-cooling. The mixture was warmed to room temperature and stirred overnight. Water (100 mL) was added to the reaction solution, which was extracted three times with EA (60 mL). The organic phase was concentrated and purified on a silica gel column (EA:PE = 1:10) to afford 6a-2 (2.6 g, 7.76 mmol) as an orange solid in a 37% yield. 1 H NMR (600MHz, CDCl3) δ7.47 (dd, J=9.2, 7.0Hz, 1H), 6.41 (dd, J=9.4, 1.6Hz, 1H), 4.24 ( q,J=7.0Hz,2H),4.18(t,J=7.0Hz,1H),1.56(d,J=7.0Hz,3H),1.28(t,J=7.0Hz,3H).

[0143] Step 2: Synthesis of ethyl (2-amino-4-bromo-3-fluorophenyl)alaninate

[0144] 6a-2 (600 mg, 1.79 mmol) and ammonium chloride (949 mg, 17.9 mmol) were added to a solution of methanol (20 mL) and water (5 mL). Zinc powder (1.16 g, 17.9 mmol) was added portionwise under an ice bath and stirred at room temperature for 2 hours. The reaction mixture was filtered, the solid was washed with methanol, the filtrate was concentrated and diluted with EA (50 mL), basified with saturated sodium bicarbonate aqueous solution, the layers were separated, the aqueous phase was extracted three times with EA, and the organic phase was concentrated. Crude product 6a-3 (304 mg, 0.996 mmol) was obtained as a brown solid in a yield of 55.6%. LCMS (ESI): m / z 307.0 [M+H] + ; RT = 1.70 min (3.00 min).

[0145] Step 3: Synthesis of 7-bromo-8-fluoro-3-methyl-3,4-dihydroquinoxalin-2(1H)-one

[0146] Dissolve 6a-3 in methanol (15 mL) and EA (10 mL), add 4M hydrochloric acid (dioxane solution) (2 mL), and stir at room temperature for 2 hours. The reaction solution is concentrated to give crude product 6a-4 (260 mg, 0.996 mmol). LCMS (ESI): m / z 257.0 [MH] - ; RT = 1.38 min (3.00 min).

[0147] Step 4: Synthesis of 7-bromo-8-fluoro-3-methyl-5-nitroquinoxalin-2(1H)-one

[0148] 6a-4 (260 mg, 1.0 mmol) was dissolved in concentrated sulfuric acid (8 mL, 95% aq.) in an ice-water bath. Potassium nitrate (121 mg, 1.2 mmol) was added and stirred for 2 hours. The reaction solution was slowly added to ice water, and an orange-red solid precipitated. The solid was collected by filtration, washed with water, and then dried under vacuum to obtain 6a-5 (125 mg, 0.42 mmol). The crude product was used directly in the next reaction without purification. LCMS (ESI): m / z 299.9 [MH] - ; RT = 1.33 min (3.00 min).

[0149] Step 5: Synthesis of 5-amino-7-bromo-8-fluoro-3-methyl-3,4-dihydroquinoxalin-2(1H)-one

[0150] 6a-5 (251 mg, 0.831 mmol), ammonium chloride (265 mg, 5 mmol), methanol (10 mL), and water (1 mL) were stirred and zinc powder (325 mg, 5 mmol) was added. The mixture was stirred at room temperature for 3 hours, the reaction mixture was filtered, the solid was washed with methanol, the filtrate was concentrated, diluted with EA (20 mL), and basified with saturated sodium bicarbonate aqueous solution. The aqueous phase was extracted three times with EA, the organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give 6a-6 (130 mg) as a brown solid. LCMS (ESI): m / z 275.9 [M+H] + ; RT = 2.49 min (5.00 min).

[0151] Step 6: Synthesis of 8-bromo-7-fluoro-2,4-dimethyl-4H-imidazo[1,5,4-de]quinoxalin-5(6H)-one

[0152] 6a-6 (130 mg) was dissolved in triethyl orthoacetate (5 mL) and heated to 80°C with stirring under nitrogen for 1 hour. After cooling to room temperature, acetic acid (3 mL) was added and the mixture was heated to 100°C with stirring for 2 hours. The reaction solution was concentrated to remove the solvent, and the resulting crude product was dissolved in DMSO (3 mL) and purified on a reverse phase column (acetonitrile:water (1‰ ammonium bicarbonate = 0%-80%)) to obtain product 6a-7 (30 mg, 0.101 mmol) as a white solid. LCMS (ESI): m / z 298.1 [M+H] + ; RT = 1.25 min (3.00 min). 1 H NMR (600MHz, DMSO) δ11.35(s,1H),7.42(d,J=4.8Hz,1H),5.34(q,J=7.0Hz,1H),2.54(d,J=7.9Hz,3H),1.64(d,J=7.0Hz,3H).

[0153] Step 7: 7-Fluoro-8-(hydroxymethyl)-2,4-dimethyl-4H-imidazo[1,5,4-de]quinoxalin-5(6H)-one

[0154] 6a-7 (30 mg, 0.101 mmol), (tributyltin)methanol (65 mg, 0.202 mmol), and x-phos Pd G2 (16 mg, 0.0202 mmol) were added sequentially to dioxane (5 mL). The mixture was stirred at 110°C under nitrogen for 2 hours. The reaction solution was concentrated and purified on a silica gel column (methanol:DCM = 1:10) to obtain product 6a (15 mg, 0.0602 mmol) as a white solid in a yield of 59.6%. LCMS (ESI): m / z 250.2 [M+H] + ; RT = 0.93 min (3.00 min).

[0155] Intermediate 9a: 8-(Hydroxymethyl)-3-methyl-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0156] Step 1: Synthesis of tert-butyl 2-(5-bromo-7-nitro-1H-indazol-1-yl)propanoate

[0157] 9a-1 (1 g, 4.13 mmol) was dissolved in DMF (20 mL), and 9a-2 (950 mg, 4.54 mmol) and potassium carbonate (1.7 g, 12.4 mmol) were added at room temperature and stirred for 2 hours. The reaction solution was diluted with EA (60 mL), washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified on a silica gel column (EA:PE = 1:5) to obtain 9a-3 (480 mg, 1.30 mmol) as a yellow oily solid. Yield: 31%. 1 H NMR (400MHz, DMSO-d6) δ8.55(d,J=1.8Hz,1H),8.45(s,1H),8.31(d,J=1.8Hz,1H),5.57(q,J=7.1Hz,1H),1.83(d,J=7.1Hz,3H),1.31(s,9H).

[0158] Step 2: Synthesis of 8-bromo-3-methyl-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0159] 9a-3 (1.05 g, 2.96 mmol) was added to acetic acid (10 mL), and iron powder (994 mg, 17.7 mmol) was added. The mixture was stirred at 80°C for 2 hours, filtered through celite, and the filtrate was concentrated and diluted with EA (30 mL). The mixture was washed with saturated aqueous sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified on a silica gel column (EA:PE = 1:3) to give the product 9a-4 (604 mg, 1.86 mmol) as a white solid in a yield of 63%.

[0160] Step 3: Synthesis of 8-(hydroxymethyl)-3-methyl-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0161] 9a-4 (35 mg, 0.130 mmol), (tributyltin)methanol (83 mg, 0.26 mmol), and x-phos Pd G2 (10 mg, 0.013 mmol) were added sequentially to dioxane (10 mL). The mixture was stirred at 110°C under nitrogen for 4 hours. The reaction solution was concentrated and purified on a silica gel column (methanol:DCM = 1:10) to afford product 9a (25 mg, 0.11 mmol) as a white solid in an 83% yield. LCMS (ESI): m / z 216.0 [MH] - .

[0162] Intermediate 10a: 3-ethyl-8-(hydroxymethyl)-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0163] The synthesis method is the same as that of intermediate 9a, except that tert-butyl 2-bromobutyrate is used as the starting material. LCMS (ESI): m / z 232.2 [M+H] + ; RT = 1.11 min (3.00 min).

[0164] Intermediate 11a: 8-(Hydroxymethyl)-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0165] The synthesis method is the same as that of intermediate 9a, except that tert-butyl bromoacetate is used as the starting material. LCMS (ESI): m / z 202.1 [MH] - ; RT = 0.89 min (3.00 min).

[0166] Intermediate 12a: 9-Fluoro-8-(hydroxymethyl)-3-methyl-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0167] Step 1: Synthesis of 5-bromo-6-fluoro-1H-indazole

[0168] 12a-1 (5 g, 24.6 mmol), sodium nitrite (1.7 g, 24.6 mmol), and acetic acid (100 mL) were dissolved in water (2 mL) and reacted at 50°C for 2 hours. The reaction solution was slowly added dropwise to ice water and filtered. The filter cake was washed with water and dried to obtain product 12a-2 (3.8 g, brown solid) in a 72% yield. LCMS (ESI): m / z 213.0 [MH] - ; RT = 1.44 min (3.0 min).

[0169] Step 2: Synthesis of 5-bromo-6-fluoro-7-nitro-1H-indazole

[0170] To 12a-2 (2 g, 9.3 mmol) and concentrated sulfuric acid (20 mL), potassium nitrate (944 mg, 9.3 mmol) was added portionwise under ice-cooling and stirred for 2 hours. The reaction mixture was slowly added dropwise to ice-water and extracted with EA (50 mL). The organic phase was washed with water (30 mL), dried, and concentrated. The residue was purified by silica gel column chromatography (PE:EA = 15:1) to afford 12a-3 (800 mg, yellow solid) in a 33% yield. LCMS (ESI): m / z 258.0 [MH] - ; RT = 1.44 min (3.0 min).

[0171] Step 3: Synthesis of 5-bromo-6-fluoro-1H-indazol-7-amine

[0172] 12a-3 (1.5 g, 5.8 mmol), iron powder (1.9 g, 34.7 mmol), and ammonium chloride (1.8 g, 34.7 mmol) were added to ethanol (30 mL) and water (10 mL). The mixture was reacted at 60°C for 2 hours, filtered, and the filtrate was added with water (30 mL). The mixture was extracted with EA (50 mL), dried, and concentrated to give the crude product 12a-4 (1 g, brown solid) in a 77% yield. LCMS (ESI): m / z 230.0 [M+H] + ; RT = 1.38 min (3.0 min).

[0173] Step 4: Synthesis of tert-butyl 2-(7-amino-5-bromo-6-fluoro-1H-indazol-1-yl)propanoate

[0174] 12a-4 (1.5 g, 6.5 mmol), potassium carbonate (1.8 g, 13.0 mmol), and tert-butyl 2-bromopropionate (2.0 g, 9.8 mmol) were added to DMF (40 mL) and reacted at 60°C for 6 hours. After cooling, water (30 mL) was added, and the mixture was extracted with EA (50 mL). The mixture was dried and concentrated to give the crude product 12a-5 (1 g, brown solid). LCMS (ESI): m / z 302.0 [M-56+H] + ; RT = 1.78 min (3.0 min).

[0175] Step 5: Synthesis of 8-bromo-9-fluoro-3-methyl-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0176] 12a-5 (1 g, 2.8 mmol) and a methanolic hydrochloric acid solution (4 M, 5 mL) were added to methanol (10 mL). The mixture was reacted at 50°C for 2 hours, concentrated, and the residue was purified using a reverse-phase preparative column (2% to 60% acetonitrile (1‰ ammonium bicarbonate)) to give the crude product 12a-6 (140 mg, brown solid) in an 18% yield. LCMS (ESI): m / z 282.0 [MH] - ; RT = 1.41 min (3.0 min).

[0177] Step 6: Synthesis of 9-fluoro-8-(hydroxymethyl)-3-methyl-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0178] 12a-6 (140 mg, 0.49 mmol), (tributyltin)methanol (327 mg, 0.99 mmol), and x-Phos Pd G2 (38 mg, 0.049 mmol) were added to dioxane (10 mL). The reaction was incubated at 100°C under nitrogen for 3 hours. The residue was separated on a silica gel column (DCM:Me = 20:1) to afford intermediate 12a (70 mg, brown solid) in a 60% yield. LCMS (ESI): m / z 234.1 [MH] - ; RT = 1.04 min (3.00 min).

[0179] Intermediate 13a: 6-(piperazin-1-yl)nicotinic acid cyanide hydrochloride

[0180] Step 1: Synthesis of tert-butyl 4-(5-cyanopyridin-2-yl)piperazine-1-carboxylate

[0181] 13a-1 (500 mg, 4.09 mmol) and 1-tert-butyloxycarbonylpiperazine (915.2 mg, 4.9 mmol) were added to acetonitrile (10 mL), and DIEA (1.36 mL, 8.19 mmol) was added dropwise. The mixture was reacted at 65°C under nitrogen for 16 hours. The solvent was removed, and the residue was purified by column chromatography (PE:EA = 5:1) to afford 13a-2 (1.10 g, yellow solid) in a 93.22% yield. LCMS (ESI): m / z 289.2 [M+H] + ; RT = 1.728 min (2.50 min).

[0182] Step 2: Synthesis of 6-(piperazin-1-yl)nicotinic acid cyanide hydrochloride

[0183] 13a-2 (1.10 g, 3.81 mmol) was added to DCM (15 mL), and 1,4-dioxane hydrochloride (1.5 mL) was added dropwise under ice-cooling. The mixture was stirred at room temperature for 2 hours and concentrated to give 13a (850 mg, yellow oil). LCMS (ESI): m / z 189.2 [M+H] + ; RT = 0.352 min. 1 H NMR (400MHz, DMSO-d6): δ9.54(s,2H),8.55(d,J=2.4Hz,1H),7.97-7.94(m,1H),7.03(d,J=9.2Hz,1H),3.93-3.91(m,4H),3.16(s,4H).

[0184] Intermediate 14a: 4-(Hydroxymethyl)-8-methyl-6H-2,2a,6,8-tetraazaacenaphthen-7(8H)-one

[0185] Step 1: Synthesis of ethyl 6-bromo-4-((4-methoxybenzyl)amino)pyrazolo[1,5-a]pyridine-3-carboxylate

[0186] Compound 14a-1 (2.0 g, 7.06 mmol), p-anisaldehyde (1.44 g, 10.6 mmol), sodium acetate borohydride (6.0 g, 28.24 mmol), and 100 mL of 1,2-dichloroethane were added to a 250 mL single-necked flask. Under argon, the mixture was heated to 80°C and stirred overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. The residue was extracted with water (100 mL) and ethyl acetate (200 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by a forward column chromatography (PE:EA = 10:1) to afford compound 14a-2 (2.0 g, white solid) in a 70% yield. LCMS (ESI): m / z 404.1 [M+H]. + .

[0187] Step 2: Synthesis of 6-bromo-4-((4-methoxybenzyl)amino)pyrazolo[1,5-a]pyridine-3-carboxylic acid

[0188] 14a-2 (4.0 g, 9.92 mmol), anhydrous tetrahydrofuran (20 mL), anhydrous methanol (20 mL), and 10 mL of distilled water were placed in a 250 mL single-necked flask. Sodium hydroxide (2.0 g, 50.0 mmol) was added and heated to 60°C. Stir for 2 h. After the reaction was complete, the mixture was cooled to room temperature. The organic solvent was removed by concentration, and the pH was adjusted to 5 with 1 M dilute hydrochloric acid. The precipitated white solid was filtered and dried under vacuum. Compound 14a-3 (2.5 g, 6.67 mmol) was obtained in a 67% yield. LCMS (ESI): m / z 274.2 [M+H] + .

[0189] Step 3: Synthesis of 4-bromo-6-(4-methoxybenzyl)-6H-2,2a,6,8-tetraazaanthracen-7(8H)-one

[0190] 14a-3 (500 mg, 1.33 mmol) and triethylamine (400 mg, 4.0 mmol) were added to anhydrous DMF (10 mL). The mixture was cooled to 0°C and diphenylphosphoryl azide (550 mg, 2.0 mmol) was slowly added dropwise. The mixture was stirred for 4 hours. The mixture was heated to 100°C and stirred overnight. The mixture was cooled to room temperature and slowly added dropwise to distilled water. The precipitated gray solid was filtered and dried under vacuum to obtain crude compound 14a-4 (400 mg, white solid) in an 81% yield. LCMS (ESI): m / z 373.0 [M+H] + .

[0191] Step 4: Synthesis of 4-bromo-6-(4-methoxybenzyl)-8-methyl-6H-2,2a,6,8-tetraazaanthracen-7(8H)-one

[0192] 14a-4 (500 mg, 1.34 mmol) and potassium carbonate (555 mg, 4.02 mmol) were added to DMF (10 mL), and iodomethane (286 mg, 2.01 mmol) was added dropwise. The mixture was heated to 60°C and stirred for 4 hours. The mixture was cooled to room temperature and extracted with water (50 mL) and ethyl acetate (50 mL*3). The organic phase was washed with brine and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA=1:1) to obtain 14a-5, 400 mg, with a yield of 77%. LCMS (ESI): m / z 387.0 [M+H] + .

[0193] Step 5: Synthesis of 4-bromo-8-methyl-6H-2,2a,6,8-tetraazaanthracen-7(8H)-one

[0194] 14a-5 (400 mg, 1.04 mmol) was added to TFA (5 mL), stirred at room temperature for 4 hours, and slowly added dropwise to distilled water. The precipitated gray solid was filtered and dried to give 14a-6 (250 mg, white solid) in a 90% yield. LCMS (ESI): m / z 267.1 [M+H] + .

[0195] Step 6: 4-(Hydroxymethyl)-8-methyl-6H-2,2a,6,8-tetraazaacenaphthen-7(8H)-one

[0196] The synthesis method is the same as step 3 of intermediate 9a, LCMS (ESI): m / z 219.1 [M+H] + .

[0197] Intermediate 15a: 8-(Hydroxymethyl)-3,3-dimethyl-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0198] Step 1: Synthesis of tert-butyl (2-(7-bromo-5-chloro-1H-indazol-1-yl)acetate

[0199] 15a-1 (2 g, 8.7 mmol), tert-butyl 2-hydroxyacetate (1.72 g, 13.0 mmol), and triphenylphosphine (4.1 g, 15.65 mmol) were added to toluene (40 mL). DIAD (3.16 g, 15.65 mmol) was added dropwise under nitrogen protection and ice-bath. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated and purified on a silica gel column (EA:PE = 1:4) to obtain product 15a-2 (1.2 g) in a 40% yield. LCMS (ESI): m / z 288.9 / 300.9 [M-55] - .

[0200] Step 2: Synthesis of tert-butyl 2-(7-bromo-5-chloro-1H-indazol-1-yl)-2-methylpropanoate

[0201] 15a-2 (1.2 g, 3.478 mmol) was dissolved in THF (20 mL) and cooled to -78°C in a dry ice acetone bath. LiHMDS (13.91 mmol, 1 M / THF) was slowly added dropwise. The reaction solution was stirred at -78°C for 30 min, and then iodomethane (1.98 g, 13.91 mmol) was added dropwise. Stirring was continued and the temperature was naturally raised to -10°C. The reaction was quenched with saturated aqueous ammonium chloride and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified on a silica gel column (EA:PE = 1:5) to obtain product 15a-3 (1.1 g, 2.95 mmol) in an 85% yield. LCMS (ESI): m / z 316.9 / 318.9 [M-55]. - .

[0202] Step 3: Synthesis of tert-butyl 2-(7-((tert-butoxycarbonyl)amino)-5-chloro-1H-indazol-1-yl)-2-methylpropanoate

[0203] 15a-3 (1 g, 2.68 mmol), tert-butyl carbamate (3.14 g, 26.8 mmol), Pd2(dba)3 (245 mg, 0.268 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (310 mg, 0.536 mmol), and cesium carbonate (2.61 g, 8.04 mmol) were added to dioxane (20 mL) and refluxed at 110°C under nitrogen for 8 hours. The reaction solution was concentrated and purified on a silica gel column (EA:PE = 1:2) to afford 15a-4 (600 mg, 1.46 mmol) in a 54.5% yield. LCMS (ESI): m / z 354 [M-55]. - .

[0204] Step 4: Synthesis of 8-chloro-3,3-dimethyl-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0205] 15a-4 (600 mg, 1.46 mmol) was added to methanol (10 mL), and HCl / 1,4-dioxane (4 mL) was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated and dried under vacuum to give product 15a-5 (320 mg, 1.36 mmol) in a yield of 93.2%. LCMS (ESI): m / z 234.1 [M+H] + .

[0206] Step 5: Synthesis of 8-(hydroxymethyl)-3,3-dimethyl-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0207] Refer to the synthesis method of step 3 of intermediate 9a, LCMS (ESI): m / z 232.1 [M+H] + .

[0208] Intermediate 16a: 8'-(Hydroxymethyl)spiro[cyclopropane-1,3'-pyrazolo[1,5,4-de]quinoxalin]-2'(1'H)-one

[0209] Step 1: Synthesis of 1-(5-bromo-7-nitro-1H-indazol-1-yl)cyclopropane-1-carboxylic acid methylamide

[0210] 16a-1 (500 mg, 2.07 mmol), methyl 2,4-dibromobutyrate (809 mg, 3.11 mmol), and cesium carbonate (2.02 g, 6.21 mmol) were added to 10 mL of anhydrous DMF and stirred at room temperature overnight. The reaction mixture was extracted with water (100 mL) and EA (100 mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by a forward column chromatography (PE:EA = 10:1) to afford compound 16a-2 (80 mg, yellow solid) in a 12% yield. LCMS (ESI): m / z 304.1 [M+H] + .

[0211] Step 2: Synthesis of 8'-bromospiro[cyclopropane-1,3'-pyrazolo[1,5,4-de]quinoxalin]-2'(1'H)-one

[0212] 16a-2 (80 mg, 0.23 mmol) and anhydrous acetic acid (10 mL) were heated to 70°C and stirred for 2 h. After the reaction was complete, the mixture was cooled to room temperature. The organic solvent was removed by concentration, and the sample was then mixed with silica gel and purified by a forward column (dichloromethane:methanol = 10:1). 16a-3 (50 mg, 0.18 mmol, 90% yield) was obtained. LCMS (ESI): m / z 278.0 [MH]-.

[0213] Step 3: Synthesis of 8'-(hydroxymethyl)spiro[cyclopropane-1,3'-pyrazolo[1,5,4-de]quinoxalin]-2'(1'H)-one

[0214] The same synthesis method as intermediate 9a, step 3. LCMS (ESI): m / z 230.1 [M+H] + .

[0215] Intermediate 17a: 9'-Fluoro-8'-(hydroxymethyl)spiro[cyclopropane-1,3'-pyrazolo[1,5,4-de]quinoxalin]-2'(1'H)-one

[0216] Refer to the synthesis method of intermediate 16a. LCMS (ESI): m / z 248.1 [M+H] + .

[0217] Intermediate 18a: 3-cyclopropyl-8-(hydroxymethyl)-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0218] Refer to the synthesis method of intermediate 16a. LCMS (ESI): m / z 244.1 [M+H] + .

[0219] Intermediate 19a: 4-cyclopropyl-8-(hydroxymethyl)-4H-imidazo[1,5,4-de]quinoxalin-5(6H)-one

[0220] Step 1: Synthesis of methyl 2-[(4-bromo-2,6-dinitrophenyl)amino]-2-cyclopropylacetate

[0221] 19a-1 (3.7 g, 0.0131 mol) was dissolved in DMF (20 mL), and 19a-2 (2.39 g, 0.0144 mol) and DIEA (5.08 g, 0.0393 mol) were added sequentially, and stirred at 40°C overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified on a silica gel column (PE:EA = 10:1) to obtain 19a-3 (3.3 g, yield: 60.3%, yellow solid). LCMS (ESI): m / z 374.0 [M+H] + .

[0222] Step 2: Synthesis of 5-amino-7-bromo-3-cyclopropyl-1H-quinoxalin-2-one

[0223] 19a-3 (800 mg, 2.1382 mmol), NH4Cl (1.143 g, 21.381 mmol), and Fe powder (1.194 g, 21.381 mmol) were added to MeOH (50 mL) and H2O (10 mL) and stirred at 80°C for 16 hours. The reaction solution was filtered, the solid was washed with methanol, the filtrate was concentrated, and then diluted with ethyl acetate. The solution was basified with saturated sodium bicarbonate aqueous solution. The layers were separated, the aqueous phase was extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The reaction solution was concentrated to give the crude product 19a-4 (670 mg, yield: 100%), as a brown solid. LCMS (ESI): m / z 280.0 [M+H] + .

[0224] Step 3: Synthesis of 5-amino-7-bromo-3-cyclopropyl-3,4-dihydro-1H-quinoxalin-2-one

[0225] 19a-4 (670 mg, 2.3918 mmol), NH4Cl (1.279 g, 21.918 mmol), and Fe (1.564 g, 21.918 mmol) were added to MeOH (50 mL) and H2O (10 mL), and the reaction solution was stirred at 25°C for 2 hours. The reaction solution was filtered, the solid was washed with methanol, the filtrate was concentrated, and then diluted with ethyl acetate. The solution was basified with saturated sodium bicarbonate aqueous solution, and the layers were separated. The aqueous phase was extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The reaction solution was concentrated to give the crude product 19a-5 (600 mg, yield: 80%, brown solid). LCMS (ESI): m / z 282.0 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ10.12(s,1H),6.37(d,J=2.2Hz,1H),6.23(d,J=2.2Hz,1H),5 .13-5.10(m,3H),3.14(dd,J=2.0,8.0Hz,1H),1.06-0.88(m,1H),0.51-0.15(m,4H).

[0226] Step 4: Synthesis of 8-bromo-4-cyclopropyl-4H-imidazo[1,5,4-de]quinoxalin-5(6H)-one

[0227] 19a-5 (2.2 g, 2.1266 mmol) was dissolved in trimethyl orthoformate (20 mL), and formic acid (0.5 mL) was added. The reaction mixture was stirred at 100°C for 2 hours. The reaction mixture was concentrated to obtain a crude product, which was then slurried through EA / PE (1 / 10, 10 mL) to obtain product 19a-6 (1.8 g, yield: 70.5%, yellow solid). LCMS (ESI): m / z 292.0 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ11.01(s,1H),8.40(s,1H),7.46(d,J=1.2Hz,1H),6.73 (t,J=1.2Hz,1H),4.69(d,J=9.0Hz,1H),1.60-1.23(m,1H),0.81-0.41(m,4H).

[0228] Step 5: Synthesis of 4-cyclopropyl-8-(hydroxymethyl)-4H-imidazo[1,5,4-de]quinoxalin-5(6H)-one

[0229] Refer to the synthesis method of intermediate 9a step 3. LCMS (ESI): m / z 244.2 [M+H] + .

[0230] Intermediate 20a: 8-(Hydroxymethyl)-4-methyl-6-hydrogen-2H,4H-oxazolo[3,4,5-de]quinoxaline-2,5-dione

[0231] Step 1: Synthesis of 2-amino-5-bromo-3-nitrophenol

[0232] Compound 20a-1 (2.5 g, 16.23 mmol) was dissolved in 1,4-dioxane / acetic acid (20 mL / 10 mL). Bromine (2.49 g, 24.35 mmol) was slowly added dropwise under an ice bath. After completion of the addition, the mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated aqueous sodium sulfite solution and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated. The crude product was purified on a silica gel column (EA:PE = 1:2) to afford product 20a-2 (2.5 g, 10.7 mmol) in a yield of 66%. LCMS (ESI): m / z 231 / 233 [MH] - .

[0233] Step 2: Synthesis of 6-bromo-4-nitrobenzo[d]oxazol-2(3H)-one

[0234] Compound 20a-2 (1 g, 4.31 mmol) and N,N'-carbonyldiimidazole (1.05 g, 6.47 mmol) were dissolved in tetrahydrofuran (20 mL) and stirred at 70°C for 2 h. The reaction solution was concentrated to remove the solvent, and water (30 mL) was added to the residue. The mixture was extracted with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate. After concentration, the crude product was purified on a silica gel column (EA:PE = 1:4) to afford product 20a-3 (800 mg, 3.09 mmol) in a yield of 71.6%. LCMS (ESI): m / z 257 / 259 [MH] - .

[0235] Step 3: Synthesis of methyl 2-(6-bromo-4-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)propanoate

[0236] Compound 20a-3 (500 mg, 1.94 mmol), methyl 2-hydroxypropionate (302 mg, 2.91 mmol), and triphenylphosphine (914 mg, 3.49 mmol) were added to toluene (40 mL). DIAD (705 mg, 3.49 mmol) was added dropwise under nitrogen and ice-cooled. The mixture was allowed to warm to room temperature and stirred for 4 hours. The reaction solution was concentrated and purified on a silica gel column (EA:PE = 1:4) to afford product 20a-4 (311 mg, 0.901 mmol) as a yellow solid in a 46% yield. LCMS (ESI): m / z 345 / 347 [M+H]. + .

[0237] Step 4: Synthesis of 8-bromo-4-methyl-6-hydrogen-2H,4H-oxazolo[3,4,5-de]quinoxaline-2,5-dione

[0238] Compound 20a-4 (341 mg, 0.991 mmol) and iron powder (444 mg, 7.93 mmol) were added to acetic acid (10 mL), and the reaction mixture was allowed to react at 70°C for 2 hours. The reaction mixture was concentrated and purified on a silica gel column (methanol:DCM = 1:10) to afford product 20a-5 (195 mg, 0.689 mmol) as a white solid in a yield of 69.5%. LCMS (ESI): m / z 281 / 283 [MH] - .

[0239] Step 5: Synthesis of 8-(hydroxymethyl)-4-methyl-6-hydrogen-2H,4H-oxazolo[3,4,5-de]quinoxaline-2,5-dione

[0240] Compound 20a-5 (100 mg, 0.353 mmol), (tributyltin)methanol (175 mg, 0.530 mmol), and x-phos Pd G2 (28 mg, 0.0353 mmol) were added to dioxane (15 mL) and stirred at 110°C under nitrogen for 8 hours. The reaction solution was concentrated and purified on a silica gel column to afford product 20a (68 mg, 0.289 mmol) as a white solid in an 82% yield. LCMS (ESI): m / z 235.0 [M+H] + .

[0241] Intermediate 21a: 8-(Hydroxymethyl)-4-methyl-4H-imidazo[1,5,4-de]quinoxaline-2,5(1H,6H)-dione

[0242] Step 1: Synthesis of 8-bromo-4-methyl-4H-imidazo[1,5,4-de]quinoxaline-2,5(1H,6H)-dione

[0243] To a solution of 21a-1 (80 mg, 0.189 mmol) in dichloromethane (10 mL) were added triphosgene (28 mg, 0.094 mmol) and pyridine (45 mg, 0.567 mmol) and allowed to react at room temperature for 2 h. The reaction solution was concentrated and purified by column chromatography (dichloromethane:methanol = 20:1) to afford 21a-2 (13.5 mg, white solid) in a 16% yield. LCMS (ESI): m / z 281.9 [M+H] + .

[0244] Step 2: Synthesis of 8-(hydroxymethyl)-4-methyl-4H-imidazo[1,5,4-de]quinoxaline-2,5(1H,6H)-dione

[0245] Refer to the synthesis method of intermediate 9a step 3. LCMS (ESI): m / z 234.0 [M+H] +.

[0246] Intermediates 22a-40a: Refer to the synthesis method of intermediate 14a, except that 14a-1 or iodomethane is replaced by the raw material intermediates in Table 1 below.

[0247] Table 1

[0248] Intermediate 41a: 4-cyclobutyl-8-(hydroxymethyl)-4H-imidazo[1,5,4-de]quinoxalin-5(6H)-one

[0249] Refer to the synthesis of intermediate 19a, except that methyl 2-amino-2-cyclobutylacetate was used as the starting material. LCMS (ESI): m / z 258.1 [M+H] + .

[0250] Synthesis of compound 1

[0251] 6a (15 mg, 0.06 mmol) was dissolved in DCM (8 mL), and phosphorus tribromide (0.5 mL) was added dropwise at room temperature. Stirring was continued for 2 h after the addition was complete. The reaction solution was concentrated and dried under vacuum to afford crude product 6a-1. The crude product 6a-1 was dissolved in acetonitrile (5 mL), and DIEA (23 mg, 0.18 mmol) and compound 2a (16 mg, 0.06 mmol) were added sequentially. The mixture was stirred at 80°C for 2 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in DMSO (3 mL). The product was purified by reverse phase column chromatography (acetonitrile:water (1‰ ammonium bicarbonate) = 0% to 80%) to afford compound 1 (10 mg, 0.0213 mmol) as a white solid in a yield of 35.4%. LCMS (ESI): m / z 470.3 [M+H]. + ; RT = 1.24 min (3.00 min). 1 H NMR (400MHz, DMSO-d6) δ11.19(s,1H),8.40(d,J=4.8Hz,1H),7.85(t,J=7.6Hz,1H),7.61-7.51(m,1H),7.10(d,J=4.8Hz,1H ),5.33(q,J=6.8Hz,1H),3.64(s,2H),3.15(s,4H),2.77(t,J=3.8Hz,3H),2.58(s,4H),2.53(s,3H),1.65(d,J=7.0Hz,3H).

[0252] Synthesis of compounds 4-9 and 11-82

[0253] According to the synthesis method of compound 1, intermediates 10a and 2a were replaced with the intermediates in Table 2 below to synthesize compounds 4-9 and 11-82.

[0254] Table 2

[0255] Synthesis of compounds 4-1 and 4-2

[0256] Compound 4 (45 mg) was separated by chiral column, column type 250×25mm10μm, mobile phase A:B (10:1, A: ethanol (+0.2% 7.0mol / L ammonia methanol); B: acetonitrile (+0.2% 7.0mol / L ammonia methanol)), to obtain compound 4-1 (15.98mg) and compound 4-2 (17.69mg);

[0257] Compound 4-1: peak time 3.662 min; ee%>99. LCMS (ESI): m / z 438.3 [M+H] + ; RT = 0.467 min (6.00 min). 1 H-NMR (400MHz, DMSO-d6): δ11.01(s,1H),8.41-8.37(m,1H),8.02(s,1H),7.83(d,J=7.2Hz,1H),7.58-7.53(m,1H),7.19(s,1H),6. 72(s,1H),5.34-5.31(m,1H),3.55(s,2H),3.15(s,4H),2.75(d,J=3.6Hz,3H),2.67(d,J=2.8Hz,4H),1.75(dd,J=2.8Hz,6.4Hz,3H).

[0258] Compound 4-2: peak time 7.369 min; ee%>94. LCMS (ESI): m / z 438.3 [M+H] + ; RT = 1.770 min (6.00 min). 1H-NMR (400MHz, DMSO-d6): δ11.04(s,1H),8.40-8.38(m,1H),8.03(s,1H),7.84(d,J=8.0Hz,1H),7.55(dd,J=8.0Hz,10.4Hz,1H), 7.19(s,1H),6.73(s,1H),5.36-5.31(m,1H),3.56(s,2H),3.15(s,4H),2.76(d,J=4.8Hz,3H),2.54(s,4H),1.75(d,J=6.8Hz,3H).

[0259] Compounds 5-1 / 5-2, 6-1 / 6-2, 7-1 / 7-2, 11-1 / 11-2, 43-1 / 43-2, 45-1 / 45-2, 47-1 / 47-2, and 49-1 / 49-2 were obtained by chiral separation of compounds 5, 6, 7, 11, 43, 45, 47, and 49, respectively. 250×25mm, 10μm, mobile phase: methanol (+0.1% 7.0 mol / L ammonia methanol), ee%>99. See Table 3 below. Compounds 43, 45, 47, and 49 were prepared according to the synthetic method of compound 1, replacing intermediates 10a and 2a with 18a and 1a (compound 43), 18a and 3a (compound 45), 19a and 1a (compound 47), and 19a and 3a (compound 49), respectively. The resulting crude products were directly subjected to the above chiral resolution.

[0260] Table 3

[0261] Comparative Example

[0262] Synthesis of compound B1

[0263] Step 1: Synthesis of 6-bromo-3-iodo-4-nitro-1H-indazole

[0264] DMF (40 mL), B1-1 (3.0 g, 12.4 mmol) and N-iodosuccinimide (3.08 g, 13.7 mmol) were added to a 100 mL single-necked flask and reacted at 60° C. for 2 h. The reaction solution was added with water and extracted with ethyl acetate. The mixture was washed with saturated sodium chloride solution and concentrated, and then column chromatography (PE / EA=3 / 1) was performed to obtain B1-2 (3.9 g, yellow solid). Yield: 85.4%. LCMS (ESI): m / z 367.7 [MH] - ; RT = 1.384 min (2.50 min).

[0265] Step 2: Synthesis of 6-bromo-3-iodo-1-methyl-4-nitro-1H-indazole

[0266] To a 100 mL single-necked flask were added 25 mL of DMF, B1-2 (4.0 g, 10.9 mmol), sodium hydride (1.1 g, 27.2 mmol), and iodomethane (2.3 g, 16.3 mmol). The mixture was reacted at room temperature for 2 hours. The reaction solution was quenched with saturated aqueous ammonium chloride solution, extracted with EA, washed with saturated sodium chloride solution, dried, concentrated, and purified by column chromatography (PE / EA = 5 / 1) to give B1-3 (2.8 g, yellow solid). Yield: 67.5%. LCMS (ESI): m / z 383.8 [M+H] + ; RT = 1.593 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ8.53(d,J=1.2Hz,1H), 8.02(d,J=1.2Hz,1H), 4.14(s,3H).

[0267] Step 3: Synthesis of N-(6-bromo-1-methyl-4-nitro-1H-indazol-3-yl)-1,1-diphenylmethaneimine

[0268] To a 100 mL single-necked flask containing 30 mL of toluene were added B1-3 (2.0 g, 5.25 mmol), benzophenone imine (855 mg, 4.72 mmol), cesium carbonate (5.1 g, 15.7 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (152 mg, 0.26 mmol) and tris(dibenzylideneacetone)dipalladium (240 mg, 0.26 mmol). The mixture was reacted at 80°C overnight, filtered, and the filtrate was concentrated and subjected to column chromatography (PE / EA=10 / 1) to give B1-4 (1.3 g, yellow solid). Yield: 57.1%, LCMS (ESI): m / z 435.0 [M+H] + ; RT = 1.534 min (2.50 min).

[0269] Step 4: Synthesis of 6-bromo-1-methyl-4-nitro-1H-indazol-3-amine

[0270] B1-4 (1.1 g, 2.5 mmol) was added to a 100 mL single-necked bottle containing 8 mL of ethyl acetate hydrochloride. The mixture was reacted at room temperature for 2 hours. The reaction solution was filtered, washed with ethyl acetate, and dried to obtain B1-5 (600 mg, yellow solid). Yield: 87.7%. LCMS (ESI): m / z 272.9 [M+H] + ; RT = 1.180 min (2.50 min).

[0271] Step 5: Synthesis of 6-bromo-N-ethyl-1-methyl-4-nitro-1H-indazol-3-amine

[0272] To a 50 mL single-necked flask containing 15 mL of methanol and 15 mL of dioxane were added B1-5 (500 mg, 1.93 mmol), acetaldehyde (127 mg, 2.89 mmol) and acetic acid (two drops). The mixture was reacted at 60°C for 1 h, followed by the addition of sodium cyanoborohydride (363 mg, 5.78 mmol). The reaction solution was added with water, extracted with EA, washed with saturated sodium chloride solution, dried, concentrated, and purified by column chromatography (PE / EA = 3 / 1) to give B1-6 (500 mg, yellow solid). Yield: 87.1%. LCMS (ESI): m / z 301.0 [M+H] + ; RT = 1.690 min (2.50 min).

[0273] Step 6: Synthesis of 6-bromo-N3-ethyl-1-methyl-1H-indazole-3,4-diamine

[0274] To a 100 mL single-necked flask containing 10 mL of methanol and 1 mL of water were added B1-6 (670 mg, 2.25 mmol) and ammonium chloride (962 mg, 18.0 mmol). Zinc powder (1.18 g, 18.0 mmol) was added under ice bath. The mixture was reacted at room temperature for 2 h, filtered, and the filtrate was concentrated and diluted with water. The mixture was extracted with EA, and the organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated to give B1-7 (840 mg, yellow solid). LCMS (ESI): m / z 271.0 [M+H] + ; RT = 1.179 min (2.50 min).

[0275] Step 7: Synthesis of 7-bromo-3-ethyl-1-methyl-1,5-dihydropyrazolo[3,4,5-d]quinazolin-4(3H)-one

[0276] To a 100 mL single-necked flask containing 15 mL of N-methylpyrrolidone, add B1-7 (560 mg, 2.09 mmol) and carbonyldiimidazole (1.69 g, 10.4 mmol). The reaction was incubated at 140°C for 2 h. The reaction solution was diluted with water, and the precipitate was filtered and dried to give B1-8 (380 mg, yellow solid). Yield: 61.9%. LCMS (ESI): m / z 297.1 [M+H]. + ; RT = 0.890 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ10.51(s,1H),7.06(s,1H),6.16(s,1H),3.82-3.77(m,5H),1.24-1.20(m,3H).

[0277] Step 8: Synthesis of 3-ethyl-7-(hydroxymethyl)-1-methyl-1,5-dihydropyrazolo[3,4,5-d]quinazolin-4(3H)-one

[0278] To a 50 mL single-necked flask, 10 mL of dioxane, B1-8 (200 mg, 0.68 mmol), tributyltin carbinol (262 mg, 0.82 mmol), and XPhos Pd G2 (27 mg, 0.034 mmol) were added. The mixture was reacted at 80°C for 16 hours. The reaction solution was concentrated and subjected to column chromatography (PE / EA = 1 / 1) to afford B1-9 (140 mg, yellow solid). Yield: 83.6%, LCMS (ESI): m / z 247.2 [M+H] + ; RT = 0.561 min (2.50 min).

[0279] Step 9: Synthesis of 7-(bromomethyl)-3-ethyl-1-methyl-1,5-dihydropyrazolo[3,4,5-d]quinazolin-4(3H)-one

[0280] B1-9 (70 mg, 0.28 mmol), triphenylphosphine (223 mg, 0.85 mmol), and carbon tetrabromide (189 mg, 0.57 mmol) were added to a 50 mL single-necked bottle containing 8 mL of DCM and reacted at room temperature for 2 hours. The reaction solution was concentrated to give B1-10 (70 mg, yellow solid). LCMS (ESI): m / z 3B1.0 [M+H] + ; RT = 1.438 min (2.50 min).

[0281] Step 10: Synthesis of 5-(4-((3-ethyl-1-methyl-4-oxo-1,3,4,5-tetrahydropyrazolo[3,4,5-d]quinazolin-7-yl)methyl)piperazin-1-yl)-6-fluoro-N-methylpicolinamide

[0282] To a 50 mL single-necked flask containing 5 mL of acetonitrile was added B1-10 (80 mg, 0.26 mmol), intermediate 2a (71 mg, 0.26 mmol), and DIEA (166 mg, 1.29 mmol). The mixture was reacted at 70°C for 2 hours. The reaction solution was purified by Pre-HPLC (formic acid) to obtain B1 (37.72 mg, white solid) in a yield of 31.4%. LCMS (ESI): m / z 467.4 [M+H] + ; RT = 3.215 min (6.00 min). 1H NMR (400MHz, DMSO-d6): δ10.31(s,1H),8.40(d,J=4.8Hz,1H),7.86-7.83(m,1H),7.59-7.54(m,1H),6.70(s,1H), 6.13(s,1H),3.82-3.77(m,5H),3.51(s,2H),3.17(s,4H),2.77(d,J=4.8Hz,3H),2.55(s,4H),1.24-1.20(m,3H).

[0283] Synthesis of compound B2

[0284] Step 1: Synthesis of N-(6-bromo-1-methyl-4-nitro-1H-indazol-3-yl)-2,2,2-trifluoroacetamide

[0285] B1-5 (1.8 g, 6.67 mmol), DIEA (2.58 g, 20.0 mmol), and trifluoroacetic anhydride (2.10 g, 10.0 mmol) were added to a 100 mL single-necked flask containing 20 mL of DCM. The mixture was reacted at room temperature for 2 hours. Water was added to the reaction solution, extracted with EA, washed with saturated sodium chloride solution, and dried and concentrated to obtain B2-1 (2.3 g, yellow solid). LCMS (ESI): m / z 368.9 [M+H] + ; RT = 1.352 min (2.50 min).

[0286] Step 2: Synthesis of N-(6-bromo-1-methyl-4-nitro-1H-indazol-3-yl)-2,2,2-trifluoro-N-methylacetamide

[0287] B2-1 (2.3 g, 6.28 mmol), cesium carbonate (4.08 g, 12.6 mmol), and iodomethane (1.34 g, 9.42 mmol) were added to a 100 mL single-necked flask containing 25 mL of DMF. The mixture was reacted at 30°C for 2 hours. Water was added to the reaction solution, extracted with EA, washed with saturated sodium chloride solution, and dried and concentrated to obtain B2-2 (2.3 g, yellow solid). LCMS (ESI): m / z 380.9 [M+H] + ; RT = 1.519 min (2.50 min).

[0288] Step 3: Synthesis of 6-bromo-N,1-dimethyl-4-nitro-1H-indazol-3-amine

[0289] B2-2 (2.6 g, 6.8 mmol) and aqueous sodium hydroxide solution (2 mL, 6N) were added to a single-necked flask containing 30 mL of methanol. The mixture was reacted at room temperature for 30 minutes. The reaction solution was filtered, and the filter cake was washed with water and methanol, respectively. After drying, the product B2-3 (1.5 g, black solid) was obtained. Yield: 77.3%, LCMS (ESI): m / z 287.0 [M+H] + ; RT = 1.495 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ8.25(s,1H),7.83(s,1H),5.58-5.54(m,1H),3.88(s,3H),2.87(d,J=5.2Hz,3H).

[0290] Step 4: Synthesis of 6-bromo-N,1-dimethyl-1H-indazole-3,4-diamine

[0291] To a 100 mL single-necked flask containing 30 mL of methanol and 3 mL of water, add B2-3 (1.4 g, 4.93 mmol) and ammonium chloride (2.1 g, 39.4 mmol). Zinc powder (2.58 g, 39.4 mmol) was added at 0°C and allowed to react at room temperature for 2 hours. The reaction solution was filtered, the filtrate was concentrated to dryness, diluted with water, and extracted with EA. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain B2-4 (840 mg, brown solid). LCMS (ESI): m / z 255.0 [M+H] + ; RT = 0.558 min (2.50 min).

[0292] Step 5: Synthesis of 7-bromo-1,3-dimethyl-1,5-dihydropyrazolo[3,4,5-d]quinazolin-4(3H)-one

[0293] To a 50 mL single-necked flask containing 15 mL of N-methylpyrrolidone, add B2-4 (800 mg, 3.15 mmol) and carbonyldiimidazole (2.55 g, 1.57 mmol). The mixture was reacted at 140°C for 2 h. The mixture was diluted with water to precipitate, which was filtered and dried to give B2-5 (700 mg, gray solid). Yield: 79.3%. LCMS (ESI): m / z 281.0 [M+H]. + ; RT = 1.098 min (2.50 min). 1 HNMR (400MHz, DMSO-d6): δ10.52(s,1H),7.05(s,1H),6.16(s,1H),3.77(s,3H),3.22(s,3H).

[0294] Step 6: Synthesis of 7-(hydroxymethyl)-1,3-dimethyl-1,5-dihydropyrazolo[3,4,5-d]quinazolin-4(3H)-one

[0295] To a 50 mL single-necked bottle containing 10 mL of dioxane was added B2-5 (300 mg, 1.07 mmol), tributyltin carbinol (413 mg, 1.29 mmol), and XPhos Pd G2 (42 mg, 0.05 mmol). The mixture was reacted at 80°C for 16 hours. The reaction solution was concentrated and purified by column chromatography (PE / EA = 1 / 1) to afford B2-6 (220 mg, black solid). Yield: 88.5%, LCMS (ESI): m / z 233.1 [M+H] + ; RT = 0.924 min (2.50 min).

[0296] Step 7: Synthesis of 7-(bromomethyl)-1,3-dimethyl-1,5-dihydropyrazolo[3,4,5-d]quinazolin-4(3H)-one

[0297] To a 50 mL single-necked flask containing 8 mL of dichloromethane, add B2-6 (35 mg, 0.15 mmol), triphenylphosphine (B19 mg, 0.45 mmol), and carbon tetrabromide (100 mg, 0.30 mmol) and react at room temperature for 2 hours. The reaction solution was concentrated to obtain B2-7 (35 mg, yellow solid). LCMS (ESI): m / z 297.0 [M+H] + ; RT = 1.039 min (2.50 min).

[0298] Step 8: Synthesis of 5-(4-((1,3-dimethyl-4-oxo-1,3,4,5-tetrahydropyrazolo[3,4,5-d]quinazolin-7-yl)methyl)piperazin-1-yl)-6-fluoro-N-methylpicolinamide

[0299] To a 25 mL single-necked flask containing 5 mL of acetonitrile was added B2-7 (35 mg, 0.24 mmol), intermediate 2a (35 mg, 0.24 mmol), and N,N-diisopropylethylamine (77 mg, 1.19 mmol). The mixture was reacted at 70°C for 2 hours. The reaction solution was purified by Pre-HPLC (formic acid) to afford B2 (14.35 mg, white solid) in a yield of 27.7%. LCMS (ESI): m / z 453.4 [M+H] + ; RT = 3.166 min (6.00 min). 1H NMR (400MHz, DMSO-d6): δ10.35(s,1H),8.41-8.38(m,1H),7.85-7.83(m,1H),7.58-7.54(m,1H),6.70(s, 1H), 6.13 (s, 1H), 3.76 (s, 3H), 3.51 (s, 2H), 3.24 (s, 3H), 3.17 (s, 4H), 2.76 (d, J = 4.8Hz, 3H), 2.54 (s, 4H).

[0300] Synthesis of compound B3

[0301] Step 1: Synthesis of 7-bromo-6-fluoro-1,3-dimethyl-1,5-dihydropyrazolo[3,4,5-de]quinazolin-4(3H)-one

[0302] To a 100 mL single-necked flask containing 10 mL of acetic acid, B2-5 (200 mg, 0.71 mmol) and N-fluorobisbenzenesulfonamide (293 mg, 0.93 mmol) were added. The reaction was incubated at 100°C for 16 hours. After concentration, the product was purified by Pre-HPLC (ammonium bicarbonate) to afford B3-1 (50 mg, reddish-brown solid) in a yield of 23.5%. LCMS (ESI): m / z 299.0 [M+H] + ; RT = 1.403 min (2.50 min). 1 HNMR (400MHz, DMSO-d6): δ7.19 (d, J = 3.6Hz, 1H), 3.77 (s, 3H), 3.22 (s, 3H).

[0303] Step 2: 6-Fluoro-7-(hydroxymethyl)-1,3-dimethyl-1,5-dihydropyrazolo[3,4,5-de]quinazolin-4(3H)-one

[0304] To a 50 mL single-necked bottle containing 5 mL of dioxane was added B3-1 (10 mg, 0.03 mmol), tributyltin carbinol (13 mg, 0.04 mmol), and XPhos Pd G2 (11 mg, 0.014 mmol). The mixture was reacted at 80°C for 16 hours. The reaction solution was concentrated and slurried with pure EA to obtain B3-2 (6 mg, brown solid). Yield: 71.5%, LCMS (ESI): m / z 251.1 [M+H] + ; RT = 0.896 min (2.50 min).

[0305] Step 3: 7-(Bromomethyl)-6-fluoro-1,3-dimethyl-1,5-dihydropyrazolo[3,4,5-de]quinazolin-4(3H)-one

[0306] To a 50 mL single-necked flask containing 8 mL of dichloromethane, add B3-2 (11 mg, 0.044 mmol), triphenylphosphine (35 mg, 0.13 mmol), and carbon tetrabromide (29 mg, 0.088 mmol) and react at room temperature for 2 hours. The reaction solution was concentrated to obtain B3-3 (11 mg, yellow solid). Crude product, LCMS (ESI): m / z 313.0 [M+H] + ; RT = 1.370 min (2.50 min).

[0307] Step 4: Synthesis of 5-(4-(((6-fluoro-1,3-dimethyl-4-oxo-1,3,4,5-tetrahydropyrazolo[3,4,5-de]quinazolin-7-yl)methyl)piperazin-1-yl)-N,6-dimethylpicolinamide

[0308] To a 25 mL single-necked vial containing 5 mL of acetonitrile was added B3-3 (30 mg, 0.1 mmol), intermediate 3a (47 mg, 0.15 mmol), and DIEA (77 mg, 0.6 mmol). The mixture was reacted at 70°C for 1.5 hours. The reaction solution was purified by Pre-HPLC (formic acid) to afford B3 (32 mg, white solid) in a yield of 71.3%. LCMS (ESI): m / z 467.8 [M+H] + ; RT = 3.362 min (6.00 min). 1 H NMR (400MHz, DMSO-d6): δ10.68(s,1H),8.55-8.51(m,1H),7.76(d,J=8.4Hz,1H),7.45(d,J=8.4Hz,1H),6.75(d,J =3.2Hz,1H),3.73(s,3H),3.61(s,2H),3.20(s,3H),2.92(s,4H),2.78(d,J=4.8Hz,3H),2.61(s,4H),2.44(s,3H).

[0309] Biological Experiment Examples

[0310] Experimental Example 1: Evaluation of the PARP1 / 2 inhibitory activity of compounds

[0311] The PARP1 / 2 inhibitory activity of the compounds of the present invention was tested in an assay using histone as a substrate.

[0312] Experimental purpose: According to the established experimental method, the IC of the compounds of the present invention on the inhibition of PARP1 / 2 enzyme activity was detected. 50 AZD-2281 (Olaparib) was used as a positive control compound.

[0313] Experimental reagents:

[0314] 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); 20×PBS (CST, cat.9808S); 20×PBST (CST, cat.9809S); AZD2281 (Selleck, cat.S1060)

[0315] Experimental method 1: PARP1 inhibitory activity

[0316] 1. Compound preparation: Dilute the compound with DMSO to a solution with a final concentration of 1000 times in a 384-well plate and set aside.

[0317] 2. Coating microplate:

[0318] 1) Dilute Histone with 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.

[0319] 3. Ribosylation reaction:

[0320] 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.

[0321] 4. Detection:

[0322] 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.

[0323] 5. Data Analysis

[0324] The inhibition rate was calculated using the following formula: inhibition rate % = (maximum signal - compound signal) / (maximum signal - minimum signal) × 100, where "minimum signal" is the mean value of the negative control wells without enzyme addition, and "maximum signal" is the mean value of the positive control wells without compound addition.

[0325] 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 invention on the enzyme activity. 50 The fitting formula is: Y = bottom + (top - bottom) / (1 + 10^((logIC 50 -X)×HillSlope)), where "top" and "bottom" are the maximum inhibition rate and the minimum inhibition rate, respectively; "HillSlope" is the absolute value of the maximum slope of the curve.

[0326] Experimental method 2: PARP2 inhibitory activity

[0327] 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.

[0328] Table 4. IC values ​​of the compounds of the present invention for inhibition of PARP1 / 2 enzyme 50

[0329] Note: “ / ” means not tested.

[0330] As shown in Table 4, the compounds of the present invention exhibit high selectivity for PARP1, with good selectivity for PARP1, with a selectivity factor of ≥100. These compounds can reduce the toxicity caused by PARP2 without significantly reducing efficacy. Furthermore, the compounds of the present invention exhibit significantly better selectivity for PARP1 than comparative compounds B1, B2, B3, and AZD-2281.

[0331] Experimental Example 2: MDA-MB-436 cell proliferation inhibition test

[0332] 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.

[0333] Table 5. MDA-MB-436 cell proliferation inhibitory activity of the compounds of the present invention

[0334] The experimental results show that the compound of the present invention has significant proliferation inhibitory activity on MDA-MB-436 cells.

[0335] Experimental Example 3: Preliminary pharmacokinetic test

[0336] 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).

[0337] The animals were fasted for 12 hours before the experiment and allowed to drink water freely. They were fed 4 hours after the administration.

[0338] 2. Blood collection time and sample processing

[0339] Intravenous and oral administration: 0.25h, 0.5h, 1.0h, 2.0h, 3.0h, 4.0h, 6.0h, 8.0h and 24h after administration.

[0340] 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.

[0341] 3. Sample testing and data analysis

[0342] 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).

[0343] 4. Experimental Results

[0344] Table 6. Pharmacokinetic parameters of the compounds of the present invention in mouse plasma

[0345] Note: iv: intravenous injection; po: oral administration.

[0346] 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.

[0347] Experimental Example 4: Testing of blood-brain barrier properties in rats

[0348] The compound of the present invention was prepared into an aqueous solution containing 5% DMSO and 95% (containing 20% ​​hydroxypropyl-β-cyclodextrin) and administered intravenously to three male Wistar Han rats at 1 mg / kg. Four hours later, carotid artery blood samples were collected and brain tissue was collected after cerebral perfusion. Drug concentrations were determined by LC-MS / MS. The results are shown in the table below:

[0349] Table 7. Brain permeability of the compounds of the present invention in rat blood-brain barrier

[0350] Experimental Example 5: PDE3A enzyme activity test

[0351] Phosphodiesterase 3A (PDE3A) is primarily distributed in the heart, vascular smooth muscle, and platelets, and regulates myocardial contractility, vascular smooth muscle contraction, and platelet aggregation. If a compound has a strong inhibitory effect on PDE3A, it may cause side effects such as increased cardiac contractility, decreased blood pressure, arrhythmias, and thrombocytopenia (References: Bowes, J.; Brown, A.J.; Hamon, J.; Jarolimek, W.; Sridhar, A.; Waldron, G.; Whitebread, S. Reducing safety-related drug attrition: the use of in vitro pharmacological profiling. Nat Rev Drug Discov 2012, 11(12), 909-922.)

[0352] Experimental Methods: Various concentrations of compound, PDE3A protein (ICE, G13020203013) (0.2 nM), and FAM-cAMP (BPS, 60200) (200 nM) were added to a 384-well plate (Corning, 4514) and allowed to react at room temperature for 60 minutes. 15 μL of the binding reagent mixture (IMAP FP IPP Explorer Kit, Molecular Devices, R8124) was added to each well and incubated at room temperature for 60 minutes. FP signals (ex 490 nm / em 520 nm) were read using a Microplate Reader (BMG, PHERAstar FSX).

[0353] Table 8. Inhibitory effects of the compounds of the present invention on PDE3A

[0354] The experimental results show that some of the compounds of the present invention do not have a strong inhibitory effect on PDE3A.

[0355] In summary, the compounds of the present invention have high selectivity for PARP1. The inventors have also unexpectedly discovered that the compounds of the present invention exhibit high brain penetrance (brain / blood ratio greater than 10%) in a rat blood-brain barrier assay. Furthermore, the compounds of the present invention have good physical and chemical stability, good bioavailability (e.g., low clearance), and good drugability. Therefore, the compounds of the present invention have fewer side effects than olaparib (AZD-2281) and have high clinical application value.

[0356] The above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced with equivalents, without departing from the spirit and substance of the claims of the present invention; and such modifications or replacements remain within the scope of the claims of the present invention.

Claims

1. A compound of formula (I), its stereoisomers, geometric isomers, tautomers, or pharmaceutically acceptable salts or hydrates thereof, in: is a single bond or a double bond; X 2 Selected from N, -C(=O) and CR 8a ; X 3 Selected from N, O and CR 8b ; X 4 Selected from N and CR 9 ; X 5 Selected from N and CR 10 ; X 6 Selected from NR 7c and CR 14 R 15 ; X 7 , X 8 are each selected from C or N, provided that X 7 and X 8 Not C, X at the same time 2 and X 3 Not all are N; R 7c is selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted 3 to 8 membered heterocyclyl, substituted or unsubstituted C2-C6 alkynyl; preferably, R 7c is selected from hydrogen, unsubstituted or halogen, hydroxy or C3-C6 cycloalkyl substituted C1-C4 alkyl, unsubstituted or halogen substituted C3-C6 cycloalkyl, C1-C6 alkoxy, 3 to 6 membered heterocyclyl, C2-C6 alkynyl; R 8a and R 8b are each independently selected from hydrogen, halogen, cyano, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, preferably, R 8a and R 8b Each is independently selected from hydrogen, fluorine, chlorine, methyl, ethyl, cyclopropyl; R 9 and R 10 are each independently selected from hydrogen, halogen, cyano, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; preferably, R 9 and R 10 are each independently selected from hydrogen, halogen, cyano and C1-C4 alkyl; more preferably, R 9 and R 10 are each independently selected from hydrogen, fluorine, chlorine and methyl; R 14 and R 15 are each independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl; or R 14 and R 15 Together with the carbon atoms to which they are attached, they form a C3-C6 cycloalkyl group; R 1 , R 1’ , R 2 and R 3 Each is independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl; R 4 and R 5 are each independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl; or R 4 and R 5 Together with the carbon atoms to which they are attached, they form a C3-C6 cycloalkyl group; s and n are each independently selected from 0, 1 and 2; Y is N, C or CH; R 6 Selected from: R 11 Each is independently selected from halogen, cyano, C1-C3 alkoxy, carbonyl, -CONHR 13 and amino, preferably selected from halogen, -CONHR 13 and cyano; m is 0, 1, 2 or 3; R 12 is selected from hydrogen, cyano, halogen, substituted or unsubstituted C1-C4 alkyl; R 13 is selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted 3-8 membered heterocyclyl; preferably, R 13 Selected from hydrogen, unsubstituted or halogen-substituted C1-C4 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy; The heterocyclic group refers to a heterocyclic group containing 1-3 heteroatoms selected from N, O, and S; the substitution refers to substitution by one or more selected from C1-C4 alkyl, C1-C6 alkoxy, halogen, hydroxyl, cyano, amino, carboxyl, and C3-C6 cycloalkyl.

2. The compound according to claim 1, its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts or hydrates thereof, wherein: In formula (I) Selected from the following structures: The definitions of the substituents are the same as those in claim 1.

3. The compound according to claim 2, its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts or hydrates thereof, wherein: In formula (I) Selected from the following structures: Where R 8a and R 8b are each independently selected from hydrogen, fluorine, chlorine, methyl, ethyl, cyano, R 9 and R 10 Each is independently selected from hydrogen, fluorine, chlorine, and methyl. The definitions of other substituents are the same as those in claim 1.

4. The compound according to any one of claims 1 to 3, its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts or hydrates thereof, wherein: In formula (I) Selected from the following structures: The definitions of the substituents are the same as those in claim 1.

5. The compound according to claim 4, its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts or hydrates thereof, wherein: In formula (I) Selected from the following structures: The definitions of the substituents are the same as those in claim 1.

6. The compound according to any one of claims 1 to 5, its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts or hydrates thereof, wherein: The compound of formula I is selected from the following compounds:

7. A pharmaceutical composition comprising a compound as claimed in any one of claims 1 to 6, one or more of its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts and hydrates thereof, and optionally a pharmaceutically acceptable carrier.

8. Use of the compound according to any one of claims 1 to 6, its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts or hydrates thereof, or the pharmaceutical composition according to claim 7 in the preparation of a PARP1 inhibitor.

9. Use of the compound according to any one of claims 1 to 6, its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts or hydrates thereof, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for preventing and / or treating a disease improved by inhibiting PARP1; Preferably, the disease improved by inhibiting PARP1 includes cancer.

10. The use according to claim 9, wherein The cancer includes malignancies such as any 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 and embryonal cancers, 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; Preferably, the genome of the cancer is of the type deficient in homologous recombination repair; Preferably, the cancer is dependent on a pathway where double-strand DNA damage is repaired but homologous recombination is deficient; Preferably, 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; Preferably, the cancer comprises one or more cancer cells that lack BRCA1 or BRCA2, or that have a BRCA1 or BRCA2 mutation.

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