MTA-cooperative PRMT5 inhibitor compound having tetracyclic fused-ring structure

Abstract

An MTA-cooperative PRMT5 inhibitor of formula (I) having a tetracyclic fused-ring structure, a pharmaceutical composition comprising same, and a preparation method therefor and a use thereof.

Description

MTA-synergistic PRMT5 inhibitor compound with a tetracyclic ring structure Field of the Invention

[0001] The present invention relates to an MTA-synergistic PRMT5 inhibitor compound having a tetracyclic ring structure, a pharmaceutical composition containing the same, and a preparation method and use thereof. Background Art

[0002] Although tremendous progress has been made in tumor treatment over the past decade, such as drugs represented by immune checkpoint inhibitors that have changed the paradigm of tumor treatment, cancer remains a daunting global health challenge and there is still an urgent need to develop new targets and treatments.

[0003] Protein arginine methyltransferases (PRMTs) can transfer the methyl group on S-adenosylmethionine (SAM) to the guanidino nitrogen atom of the protein arginine side chain to generate methylated arginine and S-adenosylhomocysteine ​​(SAH) (Stopa et al. Cell. Mol. Life Sci. 2015, 72, 2041). PRMT-catalyzed protein substrate methylation plays an important role in regulating key cellular processes, including transcription, cell signaling, mRNA translation, DNA damage, receptor trafficking, protein stability, and RNA splicing (Wang et al. J. Med. Chem. 2018, 61, 9429). Based on the catalytic activity and product type of PRMT, PRMTs can be divided into three major categories: type I, type II, and type III. Type I mainly includes PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, and PRMT8, which catalyze the formation of asymmetric dimethylated arginine on substrates; type II includes PRMT5 and PRMT9, which catalyze the formation of symmetric dimethylated arginine on substrates; type III only includes PRMT7, which is responsible for catalyzing the formation of monomethylated arginine on substrates (Shailesh et al. Oncotarget 2018, 9(94), 36705). PRMT5, as a type II arginine methyltransferase, can methylate different substrates, including spliceosomal Sm proteins, nucleolar proteins, p53, histones H2A, H3 and H4, SPT5 transcription elongation factor and MBD2 (Karkhansis, V. et al. Trends Biochem. Sci. 2011, 36, 633). Recent research results indicate that it is an oncogene whose upregulation can lead to tumor cell proliferation and invasiveness in a variety of different cancers, including colorectal cancer, lung cancer, ovarian cancer, prostate cancer, and pancreatic cancer, as well as lymphoma, leukemia, and glioblastoma. Abnormal expression of PRMT5 is associated with a variety of tumors and is an important potential target for the development of anti-tumor drugs (Kim and Ronai, Cell Stress 2020, 4(8), 207).

[0004] The gene encoding methylthioadenosine phosphorylase (MTAP) is located in region 9p21 on chromosome 9p, close to the CDKN2A tumor suppressor gene locus (Marjon et al. Annu. Rev. Cancer Biol. 2021, 5, 372). Loss of CDKN2A is very common in human cancers, and because the two genes are very close, this often leads to co-loss of MTAP (Barbarino et al. J Cell Mol Med. 2020, 24 (10), 5566). MTAP gene loss occurs in approximately 10% of cancers, including pancreatic cancer, lung cancer, and bladder cancer. MTAP loss leads to the accumulation of methylthioadenosine (MTA), which has a similar structure to SAM and sensitizes cells to PRMT5 inhibition by changing the ratio of MTA and SAM (Kryukov et al. Science 2016, 351 (6278), 1218). Elevated MTA selectively competes with SAM for binding to PRMT5 and partially inhibits the methylation activity of PRMT5, thereby reducing the level of symmetric arginine dimethylation in the entire proteome and increasing the sensitivity of cells to regulating methylosome activity (McKinney et al. J. Med. Chem. 2021, 64, 15, 11150).

[0005] Targeting PRMT5 in MTAP-deficient tumor cells is a potential synthetic lethality strategy, providing a promising approach for selectively killing cancer cells with specific genetic mutations (Marjon et al. Annu. Rev. Cancer Biol. 2021, 5, 375). PRMT5 inhibitors can be divided into three categories based on their mechanism of action: SAM-competitive, non-competitive inhibitors, and MTA-synergistic PRMT5 inhibitors (Smith, CR et al. J Med Chem 2022, 65, 1749). MTA-synergistic PRMT5 inhibitors can synergize with MTA to selectively kill MTAP-deficient tumor cells, while having no killing ability against normal cells with wild-type MTAP. Considering that PRMT5 plays a key role in regulating hematopoiesis, this type of inhibitor will have a better therapeutic window than those MTA-non-synergistic inhibitors. Currently, only a few MTA-synergistic PRMT5 inhibitors are in the early clinical stage, so this field still requires more efforts and R&D investment. The compound of the present invention has a tetracyclic ring structure and is an MTA-synergistic PRMT5 inhibitor, which can selectively inhibit the growth of MTAP-deficient cancer cells.

[0006] Summary of the Invention

[0007] In one aspect, the present invention provides a compound of formula (I),

[0008] Its enantiomers, diastereomers, racemates, tautomers, prodrugs, hydrates, solvates or pharmaceutically acceptable salts, wherein:

[0009] X1 is selected from CR1R2, O, NR3 and S, wherein S may be optionally oxidized;

[0010] X2, X3, X4 and X5 are independently selected from CR4 and N;

[0011] X6 and X7 are selected from CR7 and N;

[0012] X8 is selected from C or N;

[0013] X9 is selected from C(=O) or N;

[0014] represents a single bond or a double bond;

[0015] R1 and R2 are independently selected from H, D and C 1-6 Alkyl, or R1 and R2 together with the atoms to which they are attached form a 3- to 6-membered carbocyclic or heterocyclic group;

[0016] R3 is selected from H, C 1-6 Alkyl, C 1-6 acyl and 3- to 6-membered carbocyclyl acyl;

[0017] Each R4 is independently selected from H, halogen, hydroxy, cyano, NR5R6, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy, halogenated C 1-6 Alkoxy, aryl, heteroaryl and heterocyclic groups, wherein the aryl, heteroaryl, C 3-8 Cycloalkyl and heterocycloalkyl are optionally substituted by one or more independently selected from halogen, hydroxy, cyano, NR5R6, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy and halogenated C 1-6 Alkoxy substituents are substituted,

[0018] R5 and R6 are independently selected from H and C 1-6 Alkyl, or R5 and R6 together with the nitrogen atom to which they are attached form a nitrogen-containing heterocyclic group;

[0019] R7 is selected from H, halogen, cyano (CN), C 1-6Alkyl, cyclopropyl and halo C 1-6 alkyl;

[0020] R8 and R9 are independently selected from H and C 1-6 Alkyl, or R8 and R9 together with the atoms to which they are attached form a 3- to 6-membered carbocyclic or heterocyclic group;

[0021] p=2, 3 or 4.

[0022] In another aspect, the present invention provides a compound of formula (II),

[0023] An enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or a pharmaceutically acceptable salt thereof, wherein X1 to X7, R8, R9 and p are as defined above for formula (I).

[0024] In another aspect, the present invention provides a compound of formula (III),

[0025] An enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or a pharmaceutically acceptable salt thereof, wherein X1 to X7, R8, R9 and p are as defined above for formula (I).

[0026] Another aspect of the present invention provides a pharmaceutical composition comprising:

[0027] (1) a therapeutically effective amount of a compound of formula (I), formula (III) or formula (III), its enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof of the present invention as an active ingredient; and

[0028] (2) Pharmaceutically acceptable carrier.

[0029] Another aspect of the present invention provides a method for inhibiting PRMT5 activity in vivo or in vitro, which comprises contacting PRMT5 with an effective amount of at least one compound of formula (I), formula (III) or formula (III), its enantiomers, diastereomers, racemates, tautomers, prodrugs, hydrates, solvates or pharmaceutically acceptable salts thereof.

[0030] Another aspect of the present invention provides a method for inhibiting the growth of MTAP-deficient cancer cells, comprising contacting an effective amount of at least one compound of formula (I), formula (III) or formula (III) of the present invention, its enantiomers, diastereomers, racemates, tautomers, prodrugs, hydrates, solvates or pharmaceutically acceptable salts thereof with the cells.

[0031] Another aspect of the present invention provides the use of a compound of formula (I), formula (III) or formula (III), its enantiomers, diastereomers, racemates, tautomers, prodrugs, hydrates, solvates or pharmaceutically acceptable salts thereof, or a pharmaceutical composition comprising the compound in the preparation of a drug used as a PRMT5 inhibitor, particularly an MTA-synergistic PRMT5 inhibitor.

[0032] Another aspect of the present invention provides the use of a compound of formula (I), formula (III) or formula (III), its enantiomers, diastereomers, racemates, tautomers, prodrugs, hydrates, solvates or pharmaceutically acceptable salts thereof, or a pharmaceutical composition comprising the compound in the preparation of a medicament for treating a disease mediated by PRMT5 or at least partially mediated by PRMT5.

[0033] Another aspect of the present invention provides the use of a compound of formula (I), formula (III) or formula (III), its enantiomers, diastereomers, racemates, tautomers, prodrugs, hydrates, solvates or pharmaceutically acceptable salts thereof, or a pharmaceutical composition comprising the compound in the preparation of a medicament for treating cancer.

[0034] In another aspect of the present invention, provided are compounds of formula (I), formula (III) or formula (III) of the present invention, their enantiomers, diastereomers, racemates, tautomers, prodrugs, hydrates, solvates or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising the compounds, which are used as PRMT5 inhibitors, particularly MTA-synergistic PRMT5 inhibitors.

[0035] Another aspect of the present invention provides a compound of formula (I), formula (III) or formula (III), its enantiomers, diastereomers, racemates, tautomers, prodrugs, hydrates, solvates or pharmaceutically acceptable salts thereof, or a pharmaceutical composition comprising the compound for use in treating diseases mediated by PRMT5 or at least partially mediated by PRMT5.

[0036] Detailed Description of the Invention

[0037] The present invention provides a compound of formula (I),

[0038] Its enantiomers, diastereomers, racemates, tautomers, prodrugs, hydrates, solvates or pharmaceutically acceptable salts, wherein:

[0039] X1 is selected from CR1R2, O, NR3 and S, wherein S may be optionally oxidized;

[0040] X2, X3, X4 and X5 are independently selected from CR4 and N;

[0041] X6 and X7 are selected from CR7 and N;

[0042] X8 is selected from C or N;

[0043] X9 is selected from C(=O) or N;

[0044] represents a single bond or a double bond;

[0045] R1 and R2 are independently selected from H, D and C 1-6 Alkyl, or R1 and R2 together with the atoms to which they are attached form a 3- to 6-membered carbocyclic or heterocyclic group;

[0046] R3 is selected from H, C 1-6 Alkyl, C 1-6 acyl and 3- to 6-membered carbocyclyl acyl;

[0047] Each R4 is independently selected from H, halogen, hydroxy, cyano, NR5R6, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy, halogenated C 1-6 Alkoxy, aryl, heteroaryl and heterocyclic groups, wherein the aryl, heteroaryl, C 3-8 Cycloalkyl and heterocycloalkyl are optionally substituted by one or more independently selected from halogen, hydroxy, cyano, NR5R6, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy and halogenated C 1-6 Alkoxy substituents are substituted,

[0048] R5 and R6 are independently selected from H and C 1-6 Alkyl, or R5 and R6 together with the nitrogen atom to which they are attached form a nitrogen-containing heterocyclic group;

[0049] R7 is selected from H, halogen, cyano (CN), C 1-6 Alkyl, cyclopropyl and halo C 1-6 alkyl;

[0050] R8 and R9 are independently selected from H and C 1-6 Alkyl, or R8 and R9 together with the atoms to which they are attached form a 3- to 6-membered carbocyclic or heterocyclic group;

[0051] p=2, 3 or 4.

[0052] In one embodiment, X1 is selected from CR1R2 and O.

[0053] In one embodiment, X2, X3, X4 and X5 are independently selected from CR4.

[0054] In one embodiment, X6 and X7 are selected from CR7.

[0055] In one embodiment, X8 is C, X9 is N and Represents a double bond.

[0056] In one embodiment, X8 is N, X9 is C(=O) and Indicates a single bond.

[0057] In one embodiment, R1 and R2 are independently selected from H, D and C 1-6 alkyl.

[0058] In one embodiment, each R4 is independently selected from H, halogen, cyano, C 1-6 Alkoxy, halogenated C 1-6 Alkyl and C 3- 8-cycloalkyl.

[0059] In one embodiment, each R4 is independently selected from H and halogen, for example selected from H, F and Cl.

[0060] In one embodiment, R7, R8 and R9 are all H.

[0061] In one embodiment, the compound of formula (I) of the present invention has the structure of formula (II):

[0062] wherein X1 to X7, R8, R9 and p are as defined above for formula (I).

[0063] In one embodiment, the compound of formula (I) of the present invention has the structure of formula (III):

[0064] wherein X1 to X7, R8, R9 and p are as defined above for formula (I).

[0065] In one embodiment, the compound of the present invention is selected from the following compounds:

[0066] In some embodiments, the disease mediated by PRMT5 or mediated at least in part by PRMT5 is cancer.

[0067] In some embodiments, the cancer is a solid tumor or a hematological malignancy (eg, leukemia, lymphoma, or myeloma).

[0068] In some embodiments, the cancer is selected from ovarian cancer, lung cancer (including non-small cell lung cancer), brain tumor (including glioblastoma (GBM)), tenosynovial giant cell tumor, gastrointestinal stromal tumor (GIST), gastric cancer, esophageal cancer, colon cancer, colorectal cancer, pancreatic cancer, prostate cancer, breast cancer, cervical cancer, melanoma, mesothelioma, mesothelial carcinoma, kidney cancer, liver cancer, thyroid cancer, head and neck cancer, urothelial carcinoma, bladder cancer, endometrial cancer, choriocarcinoma, adrenal cancer, sarcoma, leukemia, lymphoma, or myeloma.

[0069] It should be understood that within the scope of the present invention, the various technical features in the above-mentioned embodiments of the present invention and the embodiments specifically described below (such as examples) can be combined with each other to form new or preferred technical solutions.

[0070] the term

[0071] In the present invention, unless otherwise specified, the terms used have the general meanings commonly known to those skilled in the art.

[0072] In the present invention, the halogen is F, Cl, Br or I, preferably F or Cl.

[0073] In the present invention, the term "alkyl" refers to a saturated linear or branched hydrocarbon moiety, for example, the term "C 1-6 The term "alkyl" refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms, including but not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl and hexyl.

[0074] In the present invention, the term "alkoxy" refers to an alkyl group linked through an oxygen atom, such as -O-(C 1-6 For example, the term "C 1-6 The term "alkoxy" refers to a straight or branched chain alkoxy group having 1 to 6 carbon atoms, including but not limited to methoxy, ethoxy, propoxy, isopropoxy, butoxy, and the like.

[0075] In the present invention, the term "cycloalkyl" refers to a saturated cyclic hydrocarbon moiety. 3-8 The term "cycloalkyl" refers to a cyclic alkyl group having 3 to 8 carbon atoms in the ring, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0076] In the present invention, the term "cycloalkoxy" refers to a saturated cycloalkyl group connected through an oxygen atom. 3-8The term "alkylene oxide" refers to a cycloalkyloxy group having 3 to 8 carbon atoms in the ring, including but not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy, and cyclooctyloxy.

[0077] In the present invention, the term "carbocyclic group" refers to a saturated or partially unsaturated cyclic hydrocarbon moiety, such as cycloalkyl and cycloalkenyl.

[0078] The term "aryl" as used herein refers to a carbocyclic hydrocarbon group consisting of one ring or multiple rings such as two fused rings, wherein at least one ring is an aromatic ring. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and the like.

[0079] In the present invention, the term "heterocyclyl" refers to a 3 to 10-membered, for example 3, 4, 5, 6, 7 or 8-membered cyclic group comprising at least one carbon atom and at least one (e.g., 1-3) ring heteroatom selected from N, O, and S, which may be saturated or partially unsaturated, and the S atoms therein may be optionally oxidized. "Heterocyclyl" includes bicyclic structures such as monocyclic, bridged, and spirocyclic rings, for example, 3 to 8-membered heterocyclyls, 3 to 6-membered heterocyclyls, and the like. Examples of heterocyclyls include tetrahydrofuranyl, pyrrolidinyl, oxetanyl, oxetanyl, azetidinyl, aziridine, thiazole, azepanyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, oxetanyl, oxazepanyl, and the like.

[0080] In the present invention, the term "nitrogen-containing heterocyclic group" refers to a 3- to 10-membered, for example, 3-, 4-, 5-, 6-, 7- or 8-membered cyclic group containing at least one nitrogen atom and optionally one or more (e.g., 1-3) additional ring heteroatoms selected from N, O, and S. Examples of "nitrogen-containing heterocyclic groups" include pyrrolidinyl, azetidinyl, aziridine, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, azepanyl, oxazepanyl, and the like.

[0081] In the present invention, the term "heteroaryl" refers to a monocyclic or bicyclic aromatic group having 5 to 10 ring atoms, for example, 5, 6 or 7 ring atoms, which contains at least one (e.g., 1 to 3) ring heteroatoms independently selected from N, O and S in the ring, and the remaining ring atoms are carbon atoms. Examples of heteroaryl groups include imidazolyl, pyridyl, pyrrolyl, thiazolyl, furyl, oxazolyl, isoxazolyl, pyrazolyl, thienyl, pyrimidinyl, 1,2,4-triazolyl, benzoxazolyl, imidazopyridinyl, triazolopyridinyl, benzofuranyl, pyrazolopyrimidinyl, benzodioxolyl, indolyl, quinolinyl, isoquinolinyl, and the like.

[0082] The term "haloalkyl" as used herein refers to an alkyl group as defined herein in which one or more hydrogen atoms, such as 1, 2, 3, 4 or 5 hydrogen atoms, are replaced by halogen atoms, and when more than one hydrogen atom is replaced by a halogen atom, the halogen atoms may be the same or different from one another. Examples of haloalkyl groups include, but are not limited to, -CF3, -CHF2, -CH2CF3, -CH(CF3)2, and the like.

[0083] As used herein, the terms "optional," "optionally," or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted alkyl" includes "unsubstituted alkyl" and "substituted alkyl" as defined herein. It will be understood by those skilled in the art that, for any group containing one or more substituents, the group does not include any substitution pattern that is sterically impractical, chemically incorrect, synthetically infeasible, and / or inherently unstable.

[0084] It will be understood by those skilled in the art that some compounds of formula (I), (II) and (III) may contain one or more chiral centers and therefore exist as two or more stereoisomers. Racemic mixtures of these isomers, individual isomers and mixtures enriched in one enantiomer, as well as diastereomers and mixtures partially enriched in a particular diastereomer when there are two chiral centers are all within the scope of the present invention. It will also be understood by those skilled in the art that the present invention includes all individual stereoisomers (e.g., enantiomers, including atropisomers), racemic mixtures or partially resolved mixtures of compounds of formula (I), (II) and (III), and, where appropriate, individual tautomers thereof.

[0085] "Atropisomerism" is a stereochemical isomerism phenomenon caused by the steric effect that hinders the free rotation of chemical bonds. The resulting resolvable enantiomers are called "atropisomers".

[0086] The racemic mixture can be used in its own form or can be resolved into its individual isomers. Stereochemically pure compounds or mixtures enriched in one or more isomers can be obtained by resolution. Methods for separating isomers are well known (see Allinger NL and Eliel EL, "Topics in Stereochemistry", Volume 6, Wiley Interscience, 1971), including physical methods, such as chromatography using chiral adsorbents. Individual isomers in chiral form can be prepared from chiral precursors. Alternatively, individual isomers can be chemically separated from the mixture by forming diastereomeric salts with chiral acids, fractionating and crystallizing the salts, then freeing one or both of the resolved bases, and optionally repeating this process to obtain one or two isomers that are substantially free of the other isomer, i.e., isomers with an optical purity > 95%. Alternatively, the racemate can be covalently linked to a chiral compound (auxiliary) to provide diastereomers, which can be separated by chromatography or fractional crystallization, followed by chemical removal of the chiral auxiliary to provide the pure enantiomers.

[0087] The term "tautomer" refers to functional group isomers resulting from the rapid shift of an atom between two positions in a molecule. Tautomers can be interconverted, for example, the enol and keto forms are typical tautomers.

[0088] "Pharmaceutically acceptable salt" refers to a salt of a free acid or base of a compound of formula (I) that is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to a subject for treatment. For example, pharmaceutically acceptable salts are acid addition salts, including, for example, addition salts derived from inorganic acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and nitric acid, and organic acids, such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. For a general description of pharmaceutically acceptable salts, see, for example, SM Berge et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977, 66: 1-19, and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, ed. Stahl and Wermuth, Wiley-VCH and VHCA, Zurich, 2002.

[0089] The term "solvate" refers to a solvent addition form containing either stoichiometric or non-stoichiometric amounts of solvent. Some compounds have a tendency to incorporate fixed molar ratios of solvent molecules in their solid state, thereby forming solvates. If the solvent is water, the solvate formed is a hydrate, while 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 form of HO. Such a combination can form one or more hydrates, such as hemihydrates, monohydrates, and dihydrates.

[0090] The term "inhibition" refers to a reduction in the baseline activity of a biological activity or process. The term "inhibit PRMT5 activity" refers to a reduction in PRMT5 activity caused by a direct or indirect response to the presence of a compound of the present invention relative to the activity of PRMT5 in the absence of the compound of the present invention. The reduction in activity may be caused by a direct interaction between a compound of formula (I) as described herein and / or a pharmaceutically acceptable salt thereof and PRMT5, or by an interaction between a compound of formula (I) as described herein and / or a pharmaceutically acceptable salt thereof and one or more other factors that affect PRMT5 activity. For example, the presence of a compound of formula (I) as described herein and / or a pharmaceutically acceptable salt thereof can reduce the activity of PRMT5 by directly binding to PRMT5, can reduce the activity of PRMT5 by directly or indirectly affecting another factor, or can reduce the activity of PRMT5 by directly or indirectly reducing the amount of PRMT5 present in a cell or organism.

[0091] The term "effective amount" as used herein refers to an amount or dosage of a compound of the present invention that is generally sufficient to produce a beneficial therapeutic effect in a patient who needs treatment for a disease or disorder mediated at least in part by PRMT5 activity, such as cancer. The effective amount or dosage of the active ingredient in the present invention can be determined by conventional methods (e.g., modeling, dose escalation studies, or clinical trials) in combination with conventional influencing factors (e.g., the mode or route of administration or administration, the pharmacokinetics of the pharmaceutical ingredient, the severity and course of the disease or disorder, the individual's previous or ongoing treatment, the individual's health status and response to the drug, and the judgment of the attending physician).

[0092] Typical dosage ranges are from about 0.0001 to about 200 mg of active ingredient per kg of individual body weight per day, for example, from about 0.001 to 100 mg / kg / day, or about 0.01 to 35 mg / kg / day, or about 0.1 to 10 mg / kg, taken once a day or in divided dose units (e.g., twice a day, three times a day, four times a day). For a 70 kg person, an exemplary range of suitable dosage is from about 0.05 to about 7 g / day, or about 0.2 to about 5 g / day.

[0093] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0094] In this application, when the name of a compound is inconsistent with its structural formula, the structural formula of the compound shall prevail unless the context indicates that the structure is incorrect and the name of the compound is correct.

[0095] The compounds of formula (I) described herein can be synthesized using commercially available starting materials, by methods known in the art, or by methods disclosed in this patent application, or by analogous methods. DETAILED DESCRIPTION

[0096] The following examples are only intended to further describe the present invention, but are not intended to limit the scope of the present invention.

[0097] Example

[0098] The examples provide the preparation of representative compounds of the present invention and related structural identification data. It must be noted that the following examples are used to illustrate the present invention rather than to limit the present invention.

[0099] 1 H NMR spectra were measured using a Bruker AVANCE NEO 400M, and chemical shifts are expressed in ppm. 1 H NMR notation: s = singlet, d = doublet, t = triplet, m = multiplet, br = broadened, dd = doublet of a doublet, dt = doublet of a triplet. Coupling constants, when given, are given in Hz.

[0100] Mass spectra were obtained using Agilent 6120B and / or Shimadzu LCMS2010 instruments.

[0101] In the following examples, unless otherwise indicated, all temperatures are in degrees Celsius and pressures are at or near atmosphere; unless otherwise indicated, various starting materials and reagents are commercially available or synthesized according to known methods, and commercially available materials and reagents are used directly without further purification.

[0102] Unless otherwise specified in the examples, the solution in the reaction refers to an aqueous solution.

[0103] Compounds are purified using silica gel column chromatography and thin-layer chromatography. Common eluent systems include petroleum ether and ethyl acetate, and dichloromethane and methanol. The volume ratio of the solvent varies depending on the polarity of the compound and can be adjusted by adding a small amount of an acidic or basic reagent, such as acetic acid or triethylamine. Alternatively, purification can be performed using preparative high-performance liquid chromatography.

[0104] The following are some commonly used abbreviations: CD3OD: deuterated methanol. CDCl3: deuterated chloroform. DMSO-d6: deuterated dimethyl sulfoxide. CBr4: carbon tetrabromide. Cs2CO3: cesium carbonate. DCM: dichloromethane. ESI: electrospray ionization. MeCN: acetonitrile. NBS: N-bromosuccinimide. DIAD: diisopropyl azodicarboxylate. DIPEA: N,N-diisopropylethylamine. DMAP: 4-dimethylaminopyridine. HATU: 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate. LDA: lithium diisopropylamide. m-CPBA: m-chloroperbenzoic acid. TsCl: p-toluenesulfonyl chloride. Pd(dppf)Cl2: 1,1'-bis(diphenylphosphino)ferrocenepalladium(II) dichloride. Pd2(dba)3: tris(dibenzylideneacetone)palladiumPd(PPh3)2Cl2: bis(triphenylphosphine)palladium dichloridePd(dtbpf)Cl2: [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichlorideprep-HPLC: preparative high performance liquid chromatographyPPh3: triphenylphosphineTEA: triethylamineTFA: trifluoroacetic acidCAN: ceric ammonium nitrateDess-Martin: Dess-Martin reagentTBAF: tetrabutylammonium fluorideTBDPSCl: tert-butyldiphenylsilyl chlorideSFC: supercritical fluid chromatography

[0105] Example 1:

[0106] Preparation of 1-(1-(aminomethyl)-4-oxo-3,4-dihydrophthalazin-6-yl)-11-fluoro-10-oxo-5,6,7,8-tetrahydro-10H-pyrazolo[5',1':3,4][1,4]diazaoctino[1,2-b]isoquinoline-15-carbonitrile

[0107] first step:

[0108] Compound 2-fluorobenzonitrile (20.0 g, 165 mmol) was dissolved in methanol (80 mL), and acetyl chloride (90 mL, 1.27 mol) was added dropwise at room temperature. The reaction solution was reacted at room temperature for 3 hours. The reaction solution was directly concentrated to obtain a crude product. After beating with petroleum ether, the crude product was filtered and dried to obtain compound 1-1 2-fluorobenzimidic acid methyl ester. LCMS: ESI m / z 154 (M+H) + .

[0109] Step 2:

[0110] Compound 4-acetamidobenzenesulfonyl azide (22.5 g, 93.7 mmol) was dissolved in acetonitrile (300 mL). Diethyl malonate (10.0 g, 62.4 mol) and triethylamine (15.8 g, 156 mmol) were slowly added to the mixture at room temperature. The mixture was allowed to react overnight at room temperature. The reaction solution was directly filtered and concentrated to obtain a residue. The residue was dissolved in dichloromethane (300 mL) and filtered and concentrated again to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-10%) to obtain compound 1-2 as a light yellow oily product, diethyl diazomalonate. LCMS: ESI m / z 187 (M+H) + .

[0111] Step 3:

[0112] Compound 1-1 (methyl 2-fluorobenzimidate) (4.0 g, 26.1 mmol) and compound 1-2 (diethyl diazomalonate) (1.8 g, 9.8 mmol) were dissolved in dichloroethane (10 mL). Dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer (330 mg, 0.522 mmol), silver hexafluoroantimonate (720 mg, 2.09 mmol), and cesium acetate (500 mg, 2.61 mmol) were added sequentially. The reaction system was heated to 60°C under nitrogen protection and allowed to react overnight. After cooling to room temperature, the reaction system was filtered through celite and concentrated to obtain the crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-5%) to obtain compound 1-3. LCMS: ESI m / z 266 (M+H) + .

[0113] Step 4:

[0114] Compound 1-3 (3.7 g, 14.0 mmol) was dissolved in dichloromethane (30 mL) under ice-cooling, and triethylamine (4.23 g, 41.8 mmol) and trifluoromethanesulfonic anhydride (5.12 g, 18.1 mmol) were added. The mixture was reacted under ice-cooling for 30 minutes. The reaction mixture was diluted with water (30 mL) and extracted three times with dichloromethane (30 mL). The combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-30%) to obtain compound 1-4. LCMS: ESI m / z 398 (M+H) + .

[0115] Step 5:

[0116] Compound 1-4 (4.1 g, 8.71 mmol) was dissolved in 1,4-dioxane (40 mL) and water (5 mL). 1-(2-tetrahydropyranyl)-1H-pyrazole-5-boronic acid pinacol ester (2.65 g, 9.51 mmol), potassium carbonate (2.63 g, 19.0 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (0.26 g) were added sequentially. Under nitrogen, the reaction system was heated to 80°C for 2 hours. After cooling to room temperature, the reaction system was quenched with saturated ammonium chloride solution (20 mL) and extracted three times with ethyl acetate (20 mL). The combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-40%) to obtain compound 1-5. LCMS: ESI m / z 400 (M+H) + .

[0117] Step 6:

[0118] Compound 1-5 (4.0 g, 10.0 mmol) was dissolved in tetrahydrofuran (20 mL), methanol (20 mL), and water (5 mL). Sodium hydroxide (2.0 g, 50.0 mmol) was added, and the reaction mixture was heated to 70°C for 2 hours. The reaction mixture was cooled to room temperature, the pH was adjusted to 5 with dilute hydrochloric acid, and extracted three times with ethyl acetate (50 mL). The combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-100%) to obtain compound 1-6. LCMS: ESI m / z 372 (M+H) + .

[0119] Step 7:

[0120] Compound 1-6 (3.6 g, 9.69 mmol) and ammonium chloride (1.04 g, 19.4 mmol) were dissolved in N, N-dimethylformamide (20 mL), and O-(7-azabenzotriazole-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate (5.53 g, 14.5 mmol) and N, N-diisopropylethylamine (3.76 g, 29.1 mmol) were added thereto. The reaction mixture was reacted at room temperature overnight. The reaction solution was poured into water (50 mL) and extracted three times with ethyl acetate (30 mL). The organic phases were combined and washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-80%) to obtain compound 1-7. LCMS: ESI m / z 371 (M+H) + .

[0121] Step 8:

[0122] Compound 1-7 (3.4 g, 9.18 mmol) was dissolved in tetrahydrofuran (20 mL) and methyl N-(triethylammoniumsulfonyl)carbamate (13.5 g, 56.8 mmol) was added at room temperature. The reaction mixture was reacted at room temperature for 1 hour. The reaction mixture was directly filtered and dried to obtain compound 1-8. LCMS: ESI m / z 353 (M+H) + .

[0123] Step 9:

[0124] Compound 1-8 (2.7 g, 7.66 mmol) was dissolved in dichloroethane (30 mL) under an ice bath, and boron tribromide (5.76 g, 23.0 mmol) was added. Under nitrogen, the reaction solution was heated to 70°C for 1 hour. The reaction solution was cooled to room temperature and directly concentrated to obtain a crude product. The crude product was added to ice water (100 mL) and stirred for 10 minutes. The product was filtered and dried to obtain compound 1-9. LCMS: ESI m / z 255 (M+H) + .

[0125] Step 10:

[0126] Compound 1-9 (1.3 g, 5.11 mmol) was dissolved in N,N-dimethylformamide (800 mL), and cesium carbonate (5.0 g, 15.3 mmol) and 1,4-dibromobutane (1.44 g, 6.65 mmol) were added. Under nitrogen protection, the reaction solution was heated to 80 degrees for 3 hours. The reaction solution was cooled to room temperature, diluted with water (300 mL), and extracted three times with ethyl acetate (200 mL). The organic layers were combined, washed with water and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. Purification by silica gel column chromatography (ethyl acetate / petroleum ether = 0-50%) gave compound 1-10. LCMS: ESI m / z 309 (M+H) + .

[0127] Step 11:

[0128] Compound 1-10 (200 mg, 0.649 mmol) was dissolved in N,N-dimethylformamide (5 mL), and N-bromosuccinimide (231 mg, 1.30 mmol) was added. Under nitrogen protection, the reaction solution was heated to 80 degrees and reacted overnight. The reaction solution was cooled to room temperature, diluted with water (20 mL), and extracted three times with ethyl acetate (30 mL). The organic layers were combined, washed with water and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-30%) to obtain compound 1-11. LCMS: ESI m / z 387 (M+H) + .

[0129] Step 12:

[0130] Compound 1-11 (30 mg, 0.08 mmol) and tert-butyl (4-oxo-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydrophthalazine-1-methyl)carbamate (49 mg, 0.12 mmol) were dissolved in dioxane (1 mL) and water (0.2 mL), and 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride (II) (65 mg) and potassium carbonate (12 mg, 0.08 mmol) were added thereto. Under nitrogen protection, the reaction system was heated to 60 degrees for 3 hours. After cooling to room temperature, the mixture was quenched with saturated ammonium chloride solution (5 mL) and extracted three times with ethyl acetate (5 mL). The organic layers were combined, washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-60%) to obtain compound 1-12. LCMS: ESI m / z 582 (M+1) + .

[0131] Step 13:

[0132] Compound 1-12 (15 mg, 0.03 mmol) was dissolved in dichloromethane (1 mL) under ice-cooling, trifluoroacetic acid (0.5 mL) was added, and the reaction mixture was reacted at room temperature for 30 minutes. The reaction solution was directly concentrated and subjected to reverse-phase HPLC to obtain Example 1. 1H NMR(400MHz,MeOD)δ8.24(d,J=8.3Hz,1H),8.21(s,1H),7.99(d,J=0.9Hz,1H),7.8 2-7.79(m,1H),7.71(dd,J=8.3,1.3Hz,1H),7.47(d,J=8.0Hz,1H),7.40(dd,J=11.3 ,8.3Hz,1H),4.90-4.89(m,1H),4.59(dd,J=15.4,7.2Hz,2H),4.46(d,J=16.3Hz,1H ), 4.17(dd,J=14.5,11.6Hz,1H), 3.24(dd,J=14.3,10.2Hz,1H), 2.22–1.83(m,4H). LCMS:ESI m / z 482(M+1) + .

[0133] Example 2:

[0134] Preparation of 1-(1-(aminomethyl)-4-oxo-3,4-dihydrophthalazin-6-yl)-10-oxo-5,6,7,8-tetrahydro-10H-pyrazolo[5',1':3,4][1,4]diazaoctino[1,2-b]isoquinoline-15-carbonitrile

[0135] first step:

[0136] Compound 1,3-dichloroisoquinoline (10 g, 50.5 mmol) was dissolved in tetrahydrofuran (500 mL), cooled to -70°C, and under nitrogen protection, a tetrahydrofuran solution of lithium diisopropylamide (27.8 mL, 2.0 mol / L) was added. The reaction was allowed to react at this temperature for 1 hour, and then methyl chloroformate (6.2 g, 65.6 mmol) was added and the reaction continued for 1 hour. The reaction solution was quenched with dilute hydrochloric acid (1N, 200 mL) and extracted three times with ethyl acetate (200 mL). The combined organic layers were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by silica gel column chromatography (ethyl acetate / petroleum ether = 0-5%) gave compound 2-1. LCMS: ESI m / z 256 (M+H) + .

[0137] Step 2:

[0138] Compound 2-1 (7.0 g, 27.3 mmol) was dissolved in N,N-dimethylformamide (500 mL), sodium methoxide (1.48 g, 27.3 mmol) was added, and the reaction solution was heated to 120 degrees under nitrogen protection for 1 hour. The reaction solution was cooled to room temperature, diluted with water (100 mL), and extracted three times with ethyl acetate (100 mL). The organic layers were combined, washed with water and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. Purification by silica gel column chromatography (ethyl acetate / petroleum ether = 0-5%) gave compound 2-2. LCMS: ESI m / z 252 (M+H) + .

[0139] Step 3:

[0140] Compound 2-2 (2.5 g, 9.93 mmol) was dissolved in 1,4-dioxane (30 mL) and water (5 mL). 1-(2-tetrahydropyranyl)-1H-pyrazole-5-boronic acid pinacol ester (4.14 g, 14.9 mmol), potassium carbonate (4.12 g, 29.8 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (810 mg) were added. Under nitrogen, the reaction solution was heated to 100°C overnight. After cooling to room temperature, the reaction system was quenched with saturated ammonium chloride solution (20 mL) and extracted three times with ethyl acetate (50 mL). The combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-30%) to obtain compound 2-3. LCMS:ESI m / z 368 (M+H) + .

[0141] Step 4:

[0142] Compound 2-3 (3.3 g, 8.71 mmol) was dissolved in tetrahydrofuran (20 mL), methanol (20 mL), and water (5 mL). Sodium hydroxide (1.74 g, 43.5 mmol) was added and the reaction mixture was heated to 70°C under nitrogen for 2 hours. The reaction mixture was cooled to room temperature and the pH was adjusted to 5 with dilute hydrochloric acid (1N). The reaction mixture was diluted with ethyl acetate (20 mL) and extracted three times with ethyl acetate (50 mL). The combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-100%) to obtain compound 2-4. LCMS: ESI m / z 354 (M+H) + .

[0143] Step 5:

[0144] Compound 2-4 (2.8 g, 7.92 mmol) and ammonium chloride (837 mg, 15.8 mmol) were dissolved in N,N-dimethylformamide (30 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.52 g, 11.9 mmol) and N,N-diisopropylethylamine (3.07 g, 23.8 mmol) were added sequentially. The reaction mixture was reacted at room temperature overnight, poured into water (50 mL), and extracted three times with ethyl acetate (50 mL). The organic phases were combined and washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-80%) to obtain compound 2-5. LCMS: ESI m / z 353 (M+H) + .

[0145] Step 6:

[0146] Compound 2-5 (2.4 g, 6.81 mmol) was dissolved in phosphorus oxychloride (15 mL). Under nitrogen, the reaction mixture was heated to 120°C for 1 hour. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude product was dissolved in ethyl acetate (50 mL) and the pH was adjusted to 8-9 with saturated aqueous sodium bicarbonate. The organic phase was collected and the aqueous phase was extracted three times with ethyl acetate (30 mL). The combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-80%) to obtain compound 2-6. LCMS: ESI m / z 251 (M+H) + .

[0147] Step 7:

[0148] Compound 2-6 (1.0 g, 4.00 mmol) was dissolved in N,N-dimethylformamide (10 mL), and sodium thiomethoxide (560 mg, 7.99 mmol) was added. Under nitrogen, the reaction solution was heated to 80°C for 1 hour. The reaction solution was cooled to room temperature, diluted with ethyl acetate (50 mL), and the pH was adjusted to 5 with dilute hydrochloric acid (1 N). The precipitated solid was filtered and dried to obtain compound 2-7. LCMS: ESI m / z 237 (M+H) + .

[0149] Step 8:

[0150] Compound 2-7 (700 mg, 2.96 mmol) was dissolved in N,N-dimethylformamide (150 mL), cesium carbonate (2.90 g, 8.89 mmol) was added thereto, and the reaction solution was heated to 80 degrees under nitrogen protection for 3 hours. The reaction solution was cooled to room temperature, diluted with water (300 mL), and extracted three times with ethyl acetate (100 mL). The organic layers were combined, washed with water and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. Purification by silica gel column chromatography (ethyl acetate / petroleum ether = 0-50%) gave compound 2-8. LCMS: ESI m / z 291 (M+H) + .

[0151] Step 9:

[0152] Compound 2-8 (60 mg, 0.21 mmol) was dissolved in acetic acid (1 mL), and bromine (66 mg, 0.42 mmol) was added. Under nitrogen, the reaction solution was heated to 80°C for 1 hour. The reaction solution was directly concentrated to obtain the crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-30%) to obtain compound 2-9. LCMS: ESI m / z 369 (M+H) + .

[0153] Step 10: According to the synthesis method of step 12 of Example 1, compound 2-9 was converted into compound 2-10. LCMS: ESI m / z 564 (M+H) + .

[0154] Step 11: According to the synthesis method of Step 13 of Example 1, compound 2-10 was converted into Example 2. 1 H NMR (400MHz, MeOD) δ8.49(d,J=8.0Hz,1H),8.25–8.22(m,2H),7.98(d,J=1.3Hz,1H),7.89–7.84(m,1H),7.76–7.67(m,3H),4.97–4.91(m,1H) ),4.61–4.57(m,2H),4.44(d,J=16.3Hz,1H),4.16–4.10(m,1H),3.28– 3.25(m,1H),2.25–2.11(m,2H),2.08–1.97(m,1H),1.93–1.81(m,1H). LCMS:ESI m / z 464(M+H) + .

[0155] Example 3:

[0156] Preparation of 1-(1-(aminomethyl)-4-oxo-3,4-dihydrophthalazin-6-yl)-6,7-dihydro-5H-pyrazolo[5',1':4,5][1,5]oxazocinol[2,3-b]quinoline-14-carbonitrile

[0157] first step:

[0158] The compound 2-hydroxyquinoline-4-carboxylic acid (25.0 g, 132 mmol) was suspended in thionyl chloride (200 mL), and the reaction system was heated to 90 degrees for 2 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure to obtain a white solid. The above white solid was dissolved in tetrahydrofuran (100 mL) and slowly added to ammonia water (150 mL) under an ice bath. The reaction mixture temperature was raised to room temperature and the reaction was continued for 12 hours. Filter, wash the filter cake with ice water, collect the solid, and dry in vacuo to obtain compound 3-1. LCMS: ESI m / z189 (M+H) + .

[0159] Step 2:

[0160] Under ice bath, compound 3-1 (20g, 106mmol) was dissolved in 1.4-dioxane (100mL), and phosphorus oxychloride (100mL) and phosphorus pentachloride (44.3g, 213mmol) were added sequentially. Under nitrogen protection, the reaction was heated to 90 degrees for 2 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure. The resulting crude product was dissolved in ethyl acetate (100mL), placed in an ice bath, and the pH was adjusted to 8 with saturated sodium bicarbonate solution. The organic phase was collected, and the aqueous phase was extracted three times with ethyl acetate (100mL). The combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by silica gel column chromatography (ethyl acetate / petroleum ether = 10-15%) gave compound 3-2. LCMS: ESI m / z 189 (M+H) + . 1 H NMR (400MHz, DMSO) δ8.36(s,1H),8.15–8.11(m,2H),8.03–7.99(m,1H),7.92–7.88(m,1H).

[0161] Step 3:

[0162] Under ice bath, 3-(tetrahydro-2H-pyran-2-yloxy)propan-1-ol (3.82 g, 23.9 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL), and sodium hydride (1.15 g, 47.7 mmol) was added thereto. Under nitrogen protection, the reaction was carried out for 1 hour. Then 2-chloroquinoline-4-carbonitrile (compound 3-2) (3 g, 15.9 mmol) was added and the reaction was carried out at room temperature for 2 hours. The reaction mixture was poured into ice water (50 mL) and extracted three times with ethyl acetate (50 mL). The combined organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. Purification by silica gel column chromatography (ethyl acetate / petroleum ether = 5-7%) gave compound 3-3. LCMS: ESI m / z 313 (M+H) + .

[0163] Step 4:

[0164] Compound 3-3 (3.5 g, 11.2 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL), cooled to minus 70 degrees Celsius, and under nitrogen protection, lithium diisopropylamide tetrahydrofuran solution (11.2 mL, 22.4 mmol) was slowly added dropwise. After the addition was complete, the reaction was continued at this temperature for 1 hour. Iodine (3.41 g, 13.4 mmol) was then dissolved in tetrahydrofuran (5 mL) and slowly added dropwise to the above reaction solution at low temperature. The reaction mixture was slowly restored to room temperature and reacted for 2 hours. The reaction mixture was poured into ice water (50 mL) for quenching, extracted three times with ethyl acetate (100 mL), and the combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. Purified by silica gel column chromatography (ethyl acetate / petroleum ether = 5-10%) to obtain compound 3-4. LCMS: ESI m / z 439 (M+H) + .

[0165] Step 5:

[0166] Compound 3-4 (2 g, 4.56 mmol) was dissolved in 1.4-dioxane (30 mL) and water (5 mL). 1-(2-tetrahydropyranyl)-1H-pyrazole-5-boronic acid pinacol ester (1.90 g, 6.85 mmol), potassium carbonate (1.89 g, 13.7 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (370 mg, 0.46 mmol) were added sequentially. Under nitrogen, the reaction solution was heated to 80°C for 2 hours. After cooling to room temperature, the mixture was diluted with ethyl acetate (50 mL) and water (20 mL). The organic layer was collected, washed sequentially with water and saturated brine, dried, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 15-20%) to obtain compound 3-5. LCMS: ESI m / z 463 (M+H) +

[0167] Step 6:

[0168] In an ice bath, compound 3-5 (1.6 g, 3.46 mmol) was slowly added to hydrochloric acid / 1.4-dioxane (10 mL, 4 N) and allowed to return to room temperature for 30 minutes. The reaction was diluted with ice water (20 mL) and extracted three times with ethyl acetate (50 mL). The organic layers were combined, washed sequentially with water and saturated brine, dried, filtered, and concentrated to obtain compound 3-6. LCMS: ESI m / z 295 (M+H) + .

[0169] Step 7:

[0170] Compound 3-6 (700 mg, 2.38 mmol) was dissolved in dichloromethane (10 mL) under ice-cooling. Triphenylphosphine (1.25 g, 4.76 mmol) and carbon tetrabromide (1.58 g, 4.76 mmol) were added. The reaction mixture was returned to room temperature and reacted for 2 hours. The mixture was diluted with dichloromethane (50 mL) and water (20 mL). The organic layer was collected, washed with water and saturated brine, dried, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 20-30%) to obtain compound 3-7. LCMS: ESI m / z 357 (M+H) + .

[0171] Step 8:

[0172] Compound 3-7 (200 mg, 0.56 mmol) was dissolved in acetonitrile (100 mL), and cesium carbonate (408 mg, 1.12 mmol) was added. Under nitrogen, the reaction mixture was heated to 60°C for 2 hours. The reaction solution was filtered and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 30-40%) to obtain compound 3-8. LCMS: ESI m / z 277 (M+H) + Step 9: According to the synthesis method of step 11 of Example 1, compound 3-8 was converted into compound 3-9. LCMS: ESI m / z 355 (M+H) + .

[0173] Step 10: According to the synthesis method of step 12 of Example 1, compound 3-9 was converted into compound 3-10. LCMS: ESI m / z 550 (M+H) + .

[0174] Step 11: According to the synthesis method of Step 13 of Example 1, compound 3-10 was converted into Example 3. 1 H NMR (400MHz, MeOD) δ8.21(d,J=8.3Hz,1H),8.13(d,J=7.6Hz,2H),8.02-7.98(m,2H),7.88(d,J=1.2Hz,1H),7.78-7.74(m,1H),7.57(dd,J=8 .3,1.5Hz,1H),4.68-4.58(m,3H),4.30(d,J=16.2Hz,1H),4.15(d,J=16.3Hz,1H),4.07-4.00(m,1H),2.40–2.32(m,1H),2.10–2.02(m,1H). LCMS:ESI m / z 450(M+H) + .

[0175] Example 4:

[0176] 1-(1-(Aminomethyl)-4-oxo-3,4-dihydrophthalazin-6-yl)-5,6,7,8-tetrahydropyrazolo[1',5'-a]azocino[4,3-b]quinoline-14-carbonitrile

[0177] first step:

[0178] Compound 2-chloro-4-cyanoquinoline (6 g, 31.8 mmol) was dissolved in methanol (100 mL), and sodium methoxide (3.44 g, 63.6 mmol) was added. Under nitrogen, the reaction system was heated to 70°C for 2 hours. The mixture was quenched with saturated ammonium chloride solution (20 mL) and extracted three times with ethyl acetate (50 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 10-15%) to obtain compound 4-1. LCMS: ESI m / z 185 (M+1) + .

[0179] Step 2:

[0180] Compound 4-1 (6.2 g, 33.7 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL), cooled to minus 60 degrees Celsius, and lithium diisopropylamide solution (33.7 mL, 67.3 mmol) was slowly added thereto. The reaction solution was reacted at this temperature for 1 hour. Then a tetrahydrofuran solution of iodine (10.3 g, 40.4 mmol) was added thereto, and the reaction mixture was returned to room temperature and stirred for 2 hours. The reaction solution was quenched with ice water (50 mL), extracted three times with ethyl acetate (50 mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 5-10%) to obtain compound 4-2. LCMS: ESI m / z 311 (M+1) + .

[0181] Step 3:

[0182] Compound 4-2 (8 g, 25.8 mmol) was dissolved in 1.4-dioxane (100 mL) and water (10 mL). 1-(2-tetrahydropyranyl)-1H-pyrazole-5-boronic acid pinacol ester (10.7 g, 38.7 mmol), potassium carbonate (8.91 g, 64.5 mmol), and [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium dichloride (0.83 g, 1.29 mmol) were added sequentially. Under nitrogen, the reaction mixture was heated to 80°C for 16 hours. The reaction mixture was diluted with water (20 mL) and extracted three times with ethyl acetate (50 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 10-15%) to obtain compound 4-3. LCMS: ESI m / z 335 (M+1) + .

[0183] Step 4:

[0184] Compound 4-3 (7 g, 20.9 mmol) was dissolved in N,N-dimethylformamide (100 mL), and sodium thiomethoxide (2.93 g, 41.9 mmol) was added. Under nitrogen, the reaction mixture was heated to 90°C for 2 hours. The reaction mixture was then cooled to room temperature and quenched with saturated aqueous sodium hypochlorite. The mixture was extracted three times with ethyl acetate (50 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 15-25%) to obtain compound 4-4. LCMS: ESI m / z 321 (M+1) + .

[0185] Step 5:

[0186] Compound 4-4 (6.1 g, 17.1 mmol) was dissolved in dichloromethane (80 mL) under an ice bath. Triethylamine (7.2 mL, 51.8 mmol) and 4-dimethylaminopyridine (4.19 g, 34.3 mmol) were added under nitrogen. Trifluoromethanesulfonic anhydride (7.25 g, 25.7 mmol) was also added, and the mixture was reacted at the same temperature for 2 hours. The reaction solution was directly concentrated to obtain a crude product, which was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1-10%) to obtain compound 4-5. LCMS: ESI m / z 453 (M+1) + .

[0187] Step 6:

[0188] Compound 4-5 (3.9 g, 7.8 mmol) was dissolved in dioxane (50 mL) and water (10 mL). (E)-4,4,5,5-tetramethyl-2-(4-(tetrahydro-2H-pyran-2-yl)oxy)but-1-en-1-yl)-1,3,2-dioxaborolane (3.3 g, 11.6 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.57 g), and potassium carbonate (3.2 g, 23.3 mmol) were added. Under nitrogen, the reaction mixture was heated to 90°C for 18 hours. After cooling to room temperature, the reaction mixture was directly concentrated to obtain a crude product, which was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1-20%) to obtain compound 4-6. LCMS: ESI m / z 459 (M+1) + .

[0189] Step 7:

[0190] Compound 4-6 (3.1 g, 6.1 mmol) was dissolved in methanol (100 mL), and 10% Pd / C (650 mg) was slowly added. The mixture was reacted under a hydrogen atmosphere for 18 hours. After filtration, the filtrate was concentrated to obtain compound 4-7, which was used directly in the next step. LCMS: ESI m / z 461 (M+1) + .

[0191] Step 8:

[0192] Compound 4-7 (2.5 g, 4.3 mmol) was dissolved in dichloromethane (20 mL) under ice-cooling. A dioxane hydrochloride solution (4 mL, 16 mmol, 4 N) was added, and the reaction system was allowed to react at room temperature for 3 hours. The reaction solution was concentrated to obtain a crude product, which was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1-50%) to obtain compound 4-8. LCMS: ESI m / z 293 (M+1) + .

[0193] Step 9:

[0194] Compound 4-8 (500 mg, 1.5 mmol) was dissolved in dichloromethane (20 mL) under ice-cooling. Carbon tetrabromide (1.02 g, 3.1 mol) and triphenylphosphine (810 mg, 3.1 mmol) were added, and the reaction system was allowed to react at room temperature for 3 hours. The reaction solution was concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 2-50%) to obtain compound 4-9. LCMS: ESI m / z 355 (M+1) + .

[0195] Step 10:

[0196] Compound 4-9 (200 mg, 0.5 mmol) was dissolved in acetonitrile (600 mL), and cesium carbonate (330 mg, 1.0 mmol) was added. Under nitrogen, the reaction solution was heated to 70°C for 5 hours. The reaction solution was filtered and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 2-30%) to obtain compound 4-10. LCMS: ESI m / z 275 (M+1) + .

[0197] Step 11:

[0198] Compound 4-10 (100 mg, 0.3 mmol) was dissolved in acetonitrile (5 mL) under an ice bath. N-bromosuccinimide (80 mg, 0.5 mmol) was added. The reaction mixture was heated to 50°C under nitrogen for 3 hours. The crude product was directly concentrated and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1-25%) to obtain compound 4-11. LCMS: ESI m / z 353 (M+1) + .

[0199] Step 12: According to the synthesis method of Step 12 of Example 1, compound 4-11 was converted into compound 4-12. LCMS: ESI m / z 548 (M+H) + .

[0200] Step 13: According to the synthetic method of Step 13 of Example 1, compound 4-12 was converted into Example 4. 1 H NMR(400MHz,MeOD,containing 0.4eq.HCOOH)δ8.26(d,J=8.5Hz,1H),8.22(s,1H),8.15(d,J=8.4Hz,1H),8.04-7.9 8(m,2H),7.83-7.79(m,2H),7.45(dd,J=8.3,1.5Hz,1H),4.58–4.50(m,1H),4.29(d ,J=16.0Hz,1H),4.12(d,J=16.1Hz,1H),3.75(dd,J=14.6,10.3Hz,1H),3.47–3.45( m,1H),2.73–2.64(m,1H),2.44–2.35(m,1H),2.22–2.13(m,1H),1.92-1.89(m,2H). LCMS:ESI m / z 448(M+H) + .

[0201] Example 5:

[0202] Preparation of 1-(1-(aminomethyl)-4-oxo-3,4-dihydrophthalazin-6-yl)-10-fluoro-5,6,7,8-tetrahydropyrazolo[1',5'-a]azocino[4,3-b]quinoline-14-carbonitrile

[0203] first step:

[0204] Compound 4-chloro-8-fluoroquinoline (13 g, 71.6 mmol) was dissolved in N-methylpyrrolidone (130 mL). Zinc cyanide (12.6 g, 107 mmol) and tetrakis(triphenylphosphine)palladium (4.14 g, 3.58 mmol) were added under nitrogen. The reaction system was heated to 160°C in a microwave reactor for 1 hour. The reaction mixture was diluted with water (100 mL) and extracted three times with ethyl acetate (100 mL). The combined organic phases were washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 15-20%) to obtain compound 5-1. LCMS: ESI m / z 173 (M+H) + .

[0205] Step 2:

[0206] Under ice bath, compound 5-1 (10 g, 58.1 mmol) was dissolved in dichloromethane (100 mL), and m-chloroperbenzoic acid (20.1 g, 116 mmol) was added thereto. The reaction solution was stirred at room temperature overnight. The solution was quenched with saturated aqueous sodium bicarbonate solution (50 mL), the organic layer was collected, and the aqueous phase was extracted three times with dichloromethane (20 mL). The combined organic phases were washed with water and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product of compound 5-2. LCMS: ESI m / z 189 (M+H) + .

[0207] Step 3:

[0208] Under ice bath, compound 5-2 (9 g, 47.8 mmol) was dissolved in chloroform (100 mL), phosphorus oxychloride (36.7 g, 239 mmol) was added thereto, and the reaction solution was heated to 60 degrees under nitrogen protection for 2 hours. Direct concentration gave a crude product, which was dissolved in ethyl acetate (100 mL), and the pH was adjusted to 9 with aqueous sodium hydroxide solution (1N). The organic layer was collected, and the aqueous phase was extracted three times with ethyl acetate (30 mL). The combined organic phases were washed with water and saturated brine in turn, dried over anhydrous sodium sulfate, filtered and concentrated. Purification by silica gel chromatography (ethyl acetate / petroleum ether = 10-15%) gave compound 5-3. LCMS: ESI m / z 207 (M+H) + .

[0209] Step 4:

[0210] Compound 5-3 (8.0 g, 38.7 mmol) was dissolved in methanol (100 mL), and sodium methoxide (10.5 g, 58.1 mmol) was added. The reaction solution was heated to 60 degrees Celsius for 2 hours. The reaction was quenched with saturated ammonium chloride solution (50 mL) and extracted three times with ethyl acetate (100 mL). The combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 10-15%) to obtain compound 5-4. LCMS: ESI m / z 203 (M+H) + .

[0211] Step 5:

[0212] Compound 5-4 (5.3 g, 26.2 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL). Under nitrogen protection, the reaction solution was cooled to -60 degrees Celsius, and lithium diisopropylamide (26.2 mL, 52.4 mmol) was slowly added dropwise thereto. The reaction solution continued to react at this temperature for 1 hour, and then a tetrahydrofuran solution of iodine (7.98 g, 31.5 mmol) was added, and the reaction mixture was returned to room temperature for 2 hours. The reaction solution was poured into ice water to quench, extracted three times with ethyl acetate (50 mL), and the combined organic phase was washed with water and saturated brine in turn, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 5-10%) to obtain compound 5-5. LCMS: ESI m / z 329 (M+H) + .

[0213] Step 6:

[0214] Compound 5-5 (4.5 g, 14.3 mmol) was dissolved in 1.4-dioxane (100 mL) and water (10 mL). 1-(2-tetrahydropyranyl)-1H-pyrazole-5-boronic acid pinacol ester (5.98 g, 21.5 mmol), potassium carbonate (5.94 g, 43.0 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (1.17 g, 1.43 mmol) were added sequentially. The reaction mixture was heated to 80°C under nitrogen for 16 hours. After cooling to room temperature, the reaction was quenched with saturated ammonium chloride solution (50 mL) and extracted three times with ethyl acetate (50 mL). The combined organic phases were washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 30-40%) to obtain compound 5-6. LCMS:ESI m / z 339 (M+H) + .

[0215] Step 7:

[0216] Compound 5-6 (5.4 g, 16.5 mmol) was dissolved in dichloroethane (100 mL) under an ice bath, and trimethylsilyl iodide (4.62 g, 32.9 mmol) was added. Under nitrogen protection, the reaction solution was heated to 60 degrees Celsius for 2 hours. The reaction was quenched with aqueous sodium hydroxide solution (1N) and extracted three times with ethyl acetate (50 mL). The combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 30-35%) to obtain compound 5-7. LCMS: ESI m / z 353 (M+H) + .

[0217] Step 8: Compound 5-7 was converted into compound 5-8 according to the synthesis method of step 5 of Example 4. LCMS: ESI m / z 471 (M+H) + .

[0218] Step 9: According to the synthesis method of step 6 of Example 4, compound 5-8 was converted into compound 5-9. LCMS: ESI m / z 477 (M+H) + .

[0219] Step 10: Compound 5-9 was converted into compound 5-10 according to the synthesis method of step 7 of Example 4. LCMS: ESI m / z 479 (M+H) + .

[0220] Step 11: According to the synthesis method of Step 8 of Example 4, compound 5-10 was converted into compound 5-11. LCMS: ESI m / z 311 (M+H) + .

[0221] Step 12: According to the synthesis method of step 9 of Example 4, compound 5-11 was converted into compound 5-12. LCMS: ESI m / z 373 (M+H) + .

[0222] Step 13: According to the synthesis method of step 10 of Example 4, compound 5-12 was converted into compound 5-13. LCMS: ESI m / z 293 (M+H) + .

[0223] Step 14: According to the synthesis method of step 11 of Example 4, compound 5-13 was converted into compound 5-14. LCMS: ESI m / z 371 (M+H) + .

[0224] Step 15: According to the synthesis method of Step 12 of Example 1, compound 5-14 was converted into compound 5-15. LCMS: ESI m / z 566 (M+H) + .

[0225] Step 16: According to the synthetic method of Step 13 of Example 1, compound 5-15 was converted into Example 5. 1 H NMR(400MHz,MeOD,containing 0.6eq.HCOOH)δ8.22(s,1H),8.15(d,J=8.3Hz,1H),7.88–7.82(m,2H),7. 79-7.72(m,2H),7.44-7.42(m,1H),4.53(dd,J=14.9Hz,6.1Hz,1H),4.46( d,J=16.2Hz,1H),4.30(d,J=16.2Hz,1H),3.80–3.72(m,1H),3.55-3.49(m ,1H),2.73-2.65(m,1H),2.45-2.36(m,1H),2.20–2.12(m,1H),1.96–1.87 -1.88(m,2H). LCMS:ESI m / z 466(M+H) + .

[0226] Intermediate 1:

[0227] Preparation of 2-amino-3-cyclopropylbenzoic acid

[0228] first step:

[0229] Compound 2-amino-3-bromobenzoic acid methyl ester (3.0 g, 13.0 mmol) and cyclopropylboronic acid (1.7 g, 19.71 mmol) were dissolved in toluene (15 mL) and water (5 mL). Potassium phosphate (8.3 g, 39.1 mmol), tricyclohexylphosphine (370 mg, 1.3 mmol) and palladium acetate (290 mg, 1.3 mmol) were then added to the system. Under nitrogen protection, the reaction mixture was heated to 100° C. and reacted for 12 hours. Cooled to room temperature, the reaction mixture was diluted with water (10 mL) and extracted three times with ethyl acetate (30 mL). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product, which was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 30-50%) to give 2-amino-3-cyclopropylbenzoic acid methyl ester (2.0 g, yield: 80.3%) as a pale yellow solid. LCMS:ESI m / z 192 (M+H) + .

[0230] Step 2:

[0231] The compound 2-amino-3-cyclopropylbenzoic acid methyl ester (2.0 g, 10.5 mmol) was dissolved in methanol (15 mL) and water (5 mL), lithium hydroxide (500 mg, 20.9 mmol) was added thereto, and the reaction solution was heated to 50 ° C for 12 hours. After cooling to room temperature, the reaction solution was diluted with water (10 mL), and then the pH was adjusted to 5-6 and extracted three times with ethyl acetate (30 mL). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give a pale yellow solid crude product 2-amino-3-cyclopropylbenzoic acid (1.8 g, yield: 97.1%), which can be used directly in the next reaction. LCMS: ESI m / z 178 (M+H) + .

[0232] Intermediate 2:

[0233] Preparation of 2-amino-3-cyanobenzoic acid

[0234] first step:

[0235] The compound methyl 2-amino-3-bromobenzoate (3.0 g, 13.0 mmol) was dissolved in N,N-dimethylformamide (20 mL), and then zinc cyanide (1.5 g, 13.1 mmol) and 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride (II) (950 mg, 1.3 mmol) were added thereto. The reaction mixture was heated to 120°C under nitrogen protection for 12 hours. After cooling to room temperature, the reaction mixture was diluted with water (20 mL) and extracted three times with ethyl acetate (50 mL). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 30-50%) to obtain a light yellow solid product methyl 2-amino-3-cyanobenzoate (2.0 g, yield: 86.7%). LCMS: ESI m / z 177 (M+H) + .

[0236] Step 2:

[0237] The compound 2-amino-3-cyanobenzoic acid methyl ester (2.0 g, 11.4 mmol) was dissolved in methanol (15 mL) and water (5 mL), lithium hydroxide (540 mg, 22.7 mmol) was added thereto, and the reaction solution was heated to 50 ° C for 12 hours. After cooling to room temperature, the reaction solution was diluted with water (10 mL), and then the pH was adjusted to 5-6 and extracted three times with ethyl acetate (30 mL). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give a pale yellow solid crude product 2-amino-3-cyanobenzoic acid (1.76 g, yield: 95.6%), which can be used directly in the next reaction. LCMS: ESI m / z 163 (M+H) + .

[0238] Intermediate 3:

[0239] Synthesis of 6,7,8,9-tetrahydropyrazolo[1,5-a]azocine-5(4H)-one

[0240] first step:

[0241] 6-Chloro-1-hexene (330 g, 2.78 mol) was dissolved in dichloromethane (3 L). Meta-chloroperbenzoic acid (624 g, 3.6 mol) was added portionwise at room temperature and stirred overnight. The mixture was filtered and the filtrate washed sequentially with sodium thiosulfate solution, sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. This was purified by vacuum distillation (50°C fraction) to obtain 2-(4-chlorobutyl)oxirane (340 g, 90% yield), a colorless, transparent liquid. 1 H NMR(400MHz, CDCl3)δ3.56(t,J=6.6Hz,2H),2.96–2.88(m,1H),2.76(t,J=4.5Hz, 1H), 2.48 (dd, J=5.0, 2.7Hz, 1H), 1.84 (dd, J=13.4, 6.7Hz, 2H), 1.68–1.47 (m, 4H).

[0242] Step 2:

[0243] The compound 1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (243 g, 1.6 mol) was dissolved in ultra-dry tetrahydrofuran (2.3 L), cooled to -60 ° C, and n-butyl lithium (1 L, 1.6 mol / L) was slowly added dropwise thereto. The reaction mixture was stirred at this temperature for 2 hours. Then, the compound 2-(4-chlorobutyl) oxirane (235 g, 1.75 mol) was dissolved in ultra-dry tetrahydrofuran (200 mL) and stirred at -60 ° C. The reaction mixture was added dropwise to the reaction system. After the addition was completed, the reaction system was naturally warmed to room temperature and stirred overnight. The reaction was quenched with saturated ammonium chloride solution at zero degrees Celsius, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product, which was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10-30%) to give a yellow oily liquid product 6-chloro-1-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)hexan-2-ol (250 g, yield: 55%). 1 H NMR (400MHz, DMSO) δ7.35(s,1H),6.11(d,J=1.3Hz,1H),5.40(ddd,J=15.5,9.7,2.3Hz,1H),4.71(t,J=5.4Hz,1H),3.86(d,J=10.6Hz,1H),3.75–3.54( m,4H),2.75(d,J=6.1Hz,2H),2.35–2.21(m,1H),1.97(s,1H),1.84–1.77(m ,1H),1.68(dd,J=6.3,3.0Hz,3H),1.57–1.46(m,3H),1.38(s,3H).LCMS:ESI m / z 287(M+1) + .

[0244] Step 3:

[0245] The compound 6-chloro-1-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)hexan-2-ol (300 g, 1.05 mol) was dissolved in ethanol (1.5 L). Concentrated hydrochloric acid (500 mL) was added dropwise and stirred at room temperature overnight. The pH was adjusted to a weak base with saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, 6-chloro-1-(1H-pyrazol-5-yl)hexan-2-ol (230 g, yield: 108%). This crude product was used directly in the next reaction. LCMS: ESI m / z 203 (M+H) + .

[0246] Step 4:

[0247] The compound 6-chloro-1-(1H-pyrazol-5-yl)hexan-2-ol (202 g, 1 mol) was dissolved in dichloromethane (2 L). Imidazole (136 g, 2 mol) was added at room temperature, followed by portionwise addition of tert-butyldimethylsilyl chloride (151 g, 1 mol). The mixture was stirred at room temperature overnight. The reaction mixture was washed sequentially with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5-20%) to obtain the product 5-(2-((tert-butyldimethylsilyl)oxy)-6-chlorohexyl)-1H-pyrazole (200 g, yield: 63%) as a yellow oily liquid. LCMS: ESI m / z 317 (M+H) + .

[0248] Step 5:

[0249] The compound 5-(2-((tert-butyldimethylsilyl)oxy)-6-chlorohexyl)-1H-pyrazole (50 g, 160 mmol) was dissolved in dimethyl sulfoxide (300 mL). The mixture was added dropwise to a solution of potassium hydroxide (9 g, 160 mol) and potassium carbonate (22 g, 160 mmol) in dimethyl sulfoxide (150 mL) at 20° C. The mixture was reacted at room temperature for 5 hours. The reaction system was poured into saturated NH 4 Cl solution at 0° C., and the pH was adjusted to weak alkaline with saturated sodium bicarbonate. The mixture was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5-20%) to obtain a yellow oily liquid product 5-((tert-butyldimethylsilyl)oxy)-4,5,6,7,8,9-hexahydropyrazolo[1,5-a]azocine (15.0 g, yield: 33%). LCMS:ESI m / z 281(M+H) + .

[0250] Step 6:

[0251] The compound 5-((tert-butyldimethylsilyl)oxy)-4,5,6,7,8,9-hexahydropyrazolo[1,5-a]azocine (28 g, 100 mmol) was dissolved in dichloromethane (100 mL). A solution of hydrogen chloride in dioxane (150 mL, 4 N) was added dropwise. The mixture was stirred at room temperature overnight, concentrated, and then dissolved in dichloromethane (100 mL). The mixture was washed with sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product 5-hydroxy-4,5,6,7,8,9-hexahydropyrazolo[1,5-a]azocine (20.0 g, yield: 120%). LCMS: ESI m / z 167 (M+H) + .

[0252] Step 7:

[0253] The compound oxalyl chloride (46 g, 360 mmol) was dissolved in ultra-dry dichloromethane (350 mL), and a solution of dimethyl sulfoxide (26 g 330 mmol) in dichloromethane (100 mL) was added dropwise thereto at -60 ° C. After the addition was complete, the mixture was stirred at -60 ° C for 30 minutes. At this temperature, a solution of the compound 5-hydroxy-4,5,6,7,8,9-hexahydropyrazolo[1,5-a]azocine (50 g, 300 mmol) in dichloromethane (300 mL) was added dropwise to the reaction mixture. After the addition was complete, the reaction was continued for 30 minutes, and then triethylamine (152 g, 150 mmol) was added dropwise thereto, and the reaction was continued at this temperature for 30 minutes. The reaction was quenched with water at -10°C, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product, which was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5-20%) to give a yellow oily liquid product 6,7,8,9-tetrahydropyrazolo[1,5-a]azocine-5(4H)-one (30 g, yield: 60%). 1 H NMR(400MHz, CDCl3) δ7.49(d,J=1.5Hz,1H),6.13(d,J=1.3Hz,1H),4.26–4.18(m,2H),3.77 (s,2H),2.36–2.28(m,2H),1.93(ddd,J=8.1,7.1,4.1Hz,2H),1.76(dt,J=8.2,6.1Hz,2H). LCMS:ESI m / z 165(M+H) + .

[0254] Alternative synthesis of intermediate 3

[0255] Preparation of 6,7,8,9-tetrahydropyrazolo[1,5-a]azocine-5(4H)-one

[0256] first step:

[0257] Under ice, 6-chloro-1-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)hexan-2-ol (25.0 g, 87.2 mmol) was dissolved in dichloromethane (250 mL). Imidazole (8.9 g, 130.8 mmol) and tert-butyldiphenylsilyl chloride (26.4 g, 95.9 mmol) were then added sequentially. The reaction mixture was reacted at room temperature under nitrogen for 12 hours. The reaction mixture was diluted with water (200 mL) and extracted three times with dichloromethane (150 mL). The combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to afford the crude product, 5-(2-((tert-butyldiphenylsilyl)oxy)-6-chlorohexyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (40 g, yield: 83.0%), as a yellow oil. LCMS:ESI m / z 525 (M+H) + .

[0258] Step 2:

[0259] Under ice bath, compound 5-(2-((tert-butyldiphenylsilyl)oxy)-6-chlorohexyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (32.0 g, 61.0 mmol) was dissolved in dioxane (100 mL), and then dioxane hydrochloride solution (200 mL, 4N) was slowly added thereto. The reaction mixture was reacted at room temperature for 4 hours under nitrogen protection. The pH of the mixture was adjusted to 9 with saturated sodium bicarbonate solution, and the organic phase was collected, filtered, dried, and concentrated to give a brown oily crude product 5-(2-((tert-butyldiphenylsilyl)oxy)-6-chlorohexyl)-1H-pyrazole (14 g, yield: 52.1%). LCMS: ESI m / z 441 (M+H) + .

[0260] Step 3:

[0261] Under ice, the compound 5-(2-((tert-butyldiphenylsilyl)oxy)-6-chlorohexyl)-1H-pyrazole (13.0 g, 29.5 mmol) was dissolved in N,N-dimethylformamide (50 mL). Sodium hydride (2.36 g, 58.9 mmol, 60%) was then slowly added to the solution. The mixture was reacted under nitrogen for 2 hours. The reaction system was quenched with saturated ammonium chloride solution (50 mL) and extracted three times with ethyl acetate (100 mL). The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 0-40%) to obtain 5-((tert-butyldiphenylsilyl)oxy)-4,5,6,7,8,9-hexahydropyrazolo[1,5-a]azocine (7.0 g, yield: 55.8%) as a colorless oil. LCMS:ESI m / z 405 (M+H) + .

[0262] Step 4:

[0263] On an ice bath, the compound 5-((tert-butyldiphenylsilyl)oxy)-4,5,6,7,8,9-hexahydropyrazolo[1,5-a]azepine (7.0 g, 17.3 mmol) was dissolved in tetrahydrofuran (70 mL). Under nitrogen, tetrabutylammonium fluoride (26 mL, 26.0 mmol, 1 mmol / mL tetrahydrofuran solution) was added. The reaction system was allowed to react at room temperature for 3 hours. The reaction mixture was diluted with water (50 mL) and extracted three times with ethyl acetate (50 mL). The combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by silica gel column chromatography (ethyl acetate:petroleum ether = 0-60%) gave 5-hydroxy-4,5,6,7,8,9-hexahydropyrazolo[1,5-a]azepine (1.4 g, yield: 46.3%) as a yellow oil. LCMS:ESI m / z167(M+H) + .

[0264] Step 5:

[0265] Under ice, 5-hydroxy-4,5,6,7,8,9-hexahydropyrazolo[1,5-a]azepine (1.4 g, 8.4 mmol) was dissolved in dichloromethane (20 mL). Dess-Martin reagent (5.4 g, 12.6 mmol) was slowly added, and the reaction system was allowed to react at room temperature for 12 hours. The reaction mixture was diluted with water (30 mL) and extracted three times with dichloromethane (30 mL). The combined organic phases were washed with saturated sodium bisulfite solution, saturated sodium bicarbonate solution, water, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by silica gel column chromatography (ethyl acetate:petroleum ether = 0-50%) afforded 6,7,8,9-tetrahydropyrazolo[1,5-a]azepine-5(4H)-one as a colorless oil (900 mg, yield: 61.8%). LCMS: ESI m / z 165 (M+H) + .

[0266] Intermediate 4:

[0267] Preparation of 14-chloro-12-cyclopropyl-5,6,7,8-tetrahydropyrazolo[1',5':1,2]azocino[4,3-b]quinoline

[0268] first step:

[0269] Under ice-cooling, compound 6,7,8,9-tetrahydropyrazolo[1,5-a]azocine-5(4H)-one (1.0 g, 6.1 mmol) was dissolved in acetonitrile (10 mL). 2-Amino-5-bromobenzoic acid (1.45 g, 6.7 mmol) was added, followed by the slow addition of phosphorus oxychloride (10 mL). Under nitrogen protection, the reaction mixture was heated to 85°C for 2 hours. The mixture was cooled to room temperature and concentrated to obtain a crude product. The reaction was quenched with saturated sodium bicarbonate solution (50 mL) and extracted three times with ethyl acetate (50 mL). The organic layers were combined, washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification by silica gel column chromatography (ethyl acetate:petroleum ether = 0-30%) gave 12-bromo-14-chloro-5,6,7,8-tetrahydropyrazolo[1',5':1,2]azocine[4,3-b]quinoline (1.8 g, yield: 77.4%) as a yellow solid. LCMS: ESI m / z 362 (M+H) + .

[0270] Step 2:

[0271] Dissolve 12-bromo-14-chloro-5,6,7,8-tetrahydropyrazolo[1',5':1,2]azocine[4,3-b]quinoline (1.7 g, 4.7 mmol) and cyclopropylboronic acid (440 mg, 5.2 mmol) in toluene (20 mL) and water (4 mL). Add tricyclohexylphosphine (260 mg, 0.94 mmol), potassium phosphate (1.99 g, 9.4 mmol), and palladium acetate (110 mg, 0.47 mmol) sequentially. Heat the reaction mixture to 100°C under nitrogen for 12 hours. Cool to room temperature, pour the reaction mixture into cold water, and extract three times with ethyl acetate (20 mL). Combine the organic layers, wash sequentially with water and saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude product. Purification by silica gel column chromatography (ethyl acetate / petroleum ether = 0-20%) gave 14-chloro-12-cyclopropyl-5,6,7,8-tetrahydropyrazolo[1',5':1,2]azocino[4,3-b]quinoline (1.22 g, yield: 76.4%) as a pale yellow solid. LCMS: ESI m / z 324 (M+H) + .

[0272] Intermediate 5:

[0273] Preparation of 5,6,7,8-tetrahydropyrazolo[1',5':1,2]azocinolo[4,3-b][1,8]naphthyridine-14-carbonitrile

[0274] first step:

[0275] Under ice bath, compound 6,7,8,9-tetrahydropyrazolo[1,5-a]azocine-5(4H)-one (1.5 g, 9.1 mmol) and 2-aminonicotinic acid (1.3 g, 9.1 mmol) were dissolved in acetonitrile (20 mL), and phosphorus oxychloride (20 mL) was slowly added. Under nitrogen protection, the reaction mixture was heated to 85°C for 2 hours. After cooling to room temperature, it was concentrated and dissolved in ethyl acetate. The pH was adjusted to 8-9 with saturated sodium bicarbonate solution. The organic phase was collected and the aqueous phase was extracted three times with ethyl acetate (10 mL). The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification by silica gel column chromatography (ethyl acetate:petroleum ether = 0-40%) gave 14-chloro-5,6,7,8-tetrahydropyrazolo[1',5':1,2]azocine[4,3-b][1,8]naphthyridine (1.1 g, yield: 40.2%) as a white solid. LCMS: ESI m / z 285 (M+H) + .

[0276] Step 2:

[0277] Dissolve the compound 14-chloro-5,6,7,8-tetrahydropyrazolo[1',5':1,2]azocine[4,3-b][1,8]naphthyridine (540 mg, 1.9 mmol) in N,N-dimethylacetamide (10 mL). Add zinc cyanide (334 mg, 2.8 mmol), zinc powder (12.4 mg, 0.19 mmol), 1,1'-bis(diphenylphosphino)ferrocene (105 mg, 0.19 mmol), and tris(dibenzylideneacetone)palladium (174 mg, 0.19 mmol) sequentially. Heat the reaction mixture to 120°C under nitrogen for 2 hours. Cool to room temperature, pour the reaction mixture into cold water, and extract three times with ethyl acetate (15 mL). Combine the organic layers, wash sequentially with water and saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude product. Purification by silica gel column chromatography (ethyl acetate:petroleum ether = 0-80%) gave a brown solid product, 5,6,7,8-tetrahydropyrazolo[1',5':1,2]azocine[4,3-b][1,8]naphthyridine-14-carbonitrile (430 mg, yield: 78.2%). LCMS: ESI m / z 276 (M+H) + .

[0278] Intermediate 6:

[0279] Preparation of 10,12-difluoro-5,6,7,8-tetrahydropyrazolo[1',5':1,2]azocino[4,3-b]quinoline-14-carbonitrile

[0280] first step:

[0281] Under ice-cooling, the compound 6-chloro-1-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)hexan-2-ol (11.6 g, 40.5 mmol) was dissolved in dichloromethane (120 mL), and Dess-Martin periodinane (25.7 g, 60.67 mmol) was added. The reaction system was reacted under ice-cooling for 2 hours. The reaction mixture was diluted with water (50 mL), and the dichloromethane phase was collected and then washed with saturated sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product 6-chloro-1-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)hexan-2-one (9.6 g, yield 79.2%) as a colorless oil. LCMS: ESI m / z 285 (M+H) + .

[0282] Step 2:

[0283] Under ice-cooling, the compound 6-chloro-1-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)hexan-2-one (3.0 g, 10.53 mmol) was dissolved in dioxane (10 mL), and a solution of hydrogen chloride in dioxane (20 mL) was added. The reaction mixture was reacted under ice-cooling for 2 hours, directly concentrated, then dissolved in dichloromethane, washed with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product 6-chloro-1-(1H-pyrazol-5-yl)hexan-2-one (1.8 g, yield: 80.9%) as a colorless oil. LCMS: ESI m / z 201 (M+H) + .

[0284] Step 3:

[0285] Under ice bath, compound 6-chloro-1-(1H-pyrazol-5-yl)hexan-2-one (5.0 g, 24.9 mmol) and 2-amino-3,5-difluorobenzoic acid (3.76 g, 27.4 mmol) were dissolved in acetonitrile (25 mL), and then phosphorus oxychloride (25 mL) was slowly added thereto. Under nitrogen protection, the reaction mixture was heated to 60 ° C for 12 hours. The mixture was cooled to room temperature and concentrated to obtain a crude product. After concentration, it was dissolved in ethyl acetate and the pH was adjusted to 8-9 with saturated sodium bicarbonate solution. The organic phase was collected, and the aqueous phase was extracted three times with ethyl acetate (50 mL). The organic phases were combined, washed with water and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. Purification by silica gel column chromatography (ethyl acetate:petroleum ether = 0-40%) gave 4-chloro-2-(4-chlorobutyl)-6,8-difluoro-3-(1H-pyrazol-5-yl)quinoline (4.6 g, yield: 49.2%) as a white solid. LCMS: ESI m / z 356 (M+H) + .

[0286] Step 4:

[0287] Under ice, the compound 4-chloro-2-(4-chlorobutyl)-6,8-difluoro-3-(1H-pyrazol-5-yl)quinoline (2.3 g, 6.5 mmol) was dissolved in acetonitrile (600 mL). Cesium carbonate (4.2 g, 12.9 mmol) was added, and the reaction mixture was heated to 70°C under nitrogen for 2 hours. The mixture was cooled to room temperature, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 0-40%) to obtain 14-chloro-10,12-difluoro-5,6,7,8-tetrahydropyrazolo[1',5':1,2]azocino[4,3-b]quinoline (1.2 g, yield: 55.2%) as a yellow solid. LCMS: ESI m / z 320 (M+H) + .

[0288] Step 5:

[0289] Dissolve 14-chloro-10,12-difluoro-5,6,7,8-tetrahydropyrazolo[1',5':1,2]azocine[4,3-b]quinoline (300 mg, 0.9 mmol) in N-methylpyrrolidone (10 mL). Add zinc cyanide (130 mg, 1.1 mmol), 2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl (40 mg, 0.09 mmol), and allylpalladium(II) chloride dimer (410 mg, 1.60 mmol) sequentially. Under nitrogen, heat the reaction mixture to 160°C for 4 hours. Cool to room temperature, pour the reaction mixture into cold water, and extract three times with ethyl acetate (15 mL). Combine the organic layers, wash sequentially with water and saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude product. Purification by silica gel column chromatography (ethyl acetate:petroleum ether = 0-30%) gave 10,12-difluoro-5,6,7,8-tetrahydropyrazolo[1',5':1,2]azocine[4,3-b]quinoline-14-carbonitrile (230 mg, yield: 75.1%) as a yellow solid. LCMS: ESI m / z 311 (M+H) + .

[0290] Intermediate 7:

[0291] Preparation of 6,7,8,9-tetrahydro-5H-pyrazolo[1',5':1,2]azanonacyclo[4,3-b]quinoline-15-carbonitrile

[0292] first step:

[0293] Lithium chloride (3.96 g, 93.3 mmol) was suspended in tetrahydrofuran (400 mL), and copper chloride (6.3 g, 46.7 mmol) was added. The mixture was reacted at room temperature under nitrogen for 1 hour. The mixture was then cooled to 0°C in an ice bath, and a solution of 1-bromo-4-chlorobutane (40.0 g, 233.3 mmol) and allylmagnesium bromide (315 mL, 315 mmol) in tetrahydrofuran was added. The reaction was continued at room temperature for 2 hours. The mixture was cooled to 0°C, quenched with 10% sulfuric acid solution (300 mL), and extracted three times with methyl tert-butyl ether (450 mL). The organic layers were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by silica gel column chromatography (petroleum ether = 100%) gave a colorless oily crude product 7-chlorohept-1-ene (30 g, yield: 92.1%). The crude product contained the starting material 1-bromo-4-chlorobutane. 1H NMR (400MHz, DMSO) δ5.85–5.72(m,1H),5.06–4.89(m,2H),3.62(t,J=6.6Hz,2H),1.95-1.89(m,2H),1.87-1.82(m,2H),1.42–1.33(m,4H).

[0294] Step 2:

[0295] Under an ice bath, dissolve 7-chlorohept-1-ene (34.0 g, 256.4 mmol) in dichloromethane (350 mL). Slowly add m-chloroperbenzoic acid (76.0 g, 440.4 mmol). The mixture is reacted at 0°C for 12 hours under nitrogen. The reaction mixture is filtered, and the pH is adjusted to 7-8 with saturated sodium bicarbonate solution (500 mL). The dichloromethane layer is collected, and the aqueous phase is extracted three times with dichloromethane (300 mL). The combined organic phases are washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to afford crude 2-(5-chloropentyl)oxirane (25.0 g, yield: 62.3%) as a colorless oil. This crude product can be used directly in the next reaction.

[0296] Step 3:

[0297] Dissolve 1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (10.0 g, 65.7 mmol) in tetrahydrofuran (100 mL). Under nitrogen, cool to -70°C in a dry ice acetone bath. Slowly add n-butyl lithium (39.4 mL, 98.6 mmol) and allow to react at this temperature for 1 hour. Then, add 2-(5-chloropentyl)oxirane (25.0 g, 164.3 mmol) dropwise to the reaction mixture at this temperature. Continue to react at -70°C for 1 hour. The reaction system was heated to 0°C and quenched with hydrochloric acid solution (50 mL, 1 N). The organic phase was collected, and the aqueous phase was extracted three times with ethyl acetate (100 mL). The organic phases were combined, washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 0-30%) to obtain 7-chloro-1-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)heptan-2-ol (2.2 g, yield: 8.9%) as a colorless oil. LCMS: ESI m / z 301 (M+H) + .

[0298] Step 4:

[0299] Under ice-cooling, the compound 7-chloro-1-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)heptan-2-ol (2.2 g, 7.3 mmol) was dissolved in dichloromethane (30 mL), and Dess-Martin reagent (4.65 g, 10.9 mmol) was added. The mixture was reacted at room temperature under nitrogen for 2 hours. The mixture was then placed in an ice-cooling bath, diluted with water, and the organic phase was collected. The aqueous phase was extracted three times with dichloromethane (30 mL). The organic layers were combined and washed sequentially with saturated sodium bicarbonate solution, sodium bisulfite solution, water, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification by silica gel column chromatography (ethyl acetate:petroleum ether = 1:5) gave 7-chloro-1-[2-(3,4,5,6-tetrahydro-2H-pyran-2-yl)pyrazol-3-yl]heptan-2-one (1.1 g, yield: 45.3%) as a pale yellow oil. LCMS: ESI m / z 299 (M+H) + .

[0300] Step 5:

[0301] Under ice, 7-chloro-1-[2-(3,4,5,6-tetrahydro-2H-pyran-2-yl)pyrazol-3-yl]heptane-2-one (1.1 g, 3.7 mmol) was dissolved in dichloromethane (10 mL), and dioxane hydrochloride (20 mL, 4 N) was added. The reaction mixture was allowed to react at room temperature for 2 hours. The mixture was diluted with ethyl acetate and the pH was adjusted to 10 with saturated sodium bicarbonate solution. The organic layer was collected and washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by silica gel column chromatography (ethyl acetate / petroleum ether = 1:1) afforded 7-chloro-1-(1H-pyrazol-5-yl)heptane-2-one (560 mg, yield: 63.8%) as a pale yellow oil. LCMS: ESI m / z 215 (M+H) + .

[0302] Step 6:

[0303] Under ice, 7-chloro-1-(1H-pyrazol-5-yl)heptan-2-one (520 mg, 2.4 mmol) and 2-aminobenzoic acid (0.4 g, 2.9 mmol) were dissolved in acetonitrile (8 mL). Phosphorus oxychloride (8 mL) was added, and the reaction mixture was heated to 80°C under nitrogen for 12 hours. After cooling to room temperature, the mixture was concentrated to yield a crude product, which was then dissolved in ethyl acetate (20 mL) and the pH adjusted to 7-8 with saturated sodium bicarbonate solution. The organic phase was collected, and the aqueous phase was extracted three times with ethyl acetate (10 mL). The combined organic layers were washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to yield the crude product. Purification by silica gel column chromatography (ethyl acetate:petroleum ether = 0-50%) afforded 4-chloro-2-(5-chloropentyl)-3-(1H-pyrazol-5-yl)quinoline (150 mg, yield: 17.6%) as a white solid. LCMS:ESI m / z 334 (M+H) + .

[0304] Step 7:

[0305] The compound 4-chloro-2-(5-chloropentyl)-3-(1H-pyrazol-5-yl)quinoline (150 mg, 0.45 mmol) was dissolved in acetonitrile (150 mL) and cesium carbonate (292 mg, 0.9 mmol) was added. Under nitrogen, the reaction mixture was heated to 70°C for 2 hours. After cooling to room temperature, filtration, and concentration, the crude product was obtained. Purification by silica gel column chromatography (ethyl acetate:petroleum ether = 0-40%) afforded 15-chloro-6,7,8,9-tetrahydro-5H-pyrazolo[1',5':1,2]azanonacyclo[4,3-b]quinoline (70 mg, yield: 49.8%) as a yellow solid. LCMS: ESI m / z 298 (M+H) + .

[0306] Step 8:

[0307] Compound 15-chloro-6,7,8,9-tetrahydro-5H-pyrazolo[1',5':1,2]azanonacyclo[4,3-b]quinoline (50 mg, 0.17 mmol) was dissolved in N,N-dimethylacetamide (3 mL). Zinc cyanide (21 mg, 0.18 mmol), zinc powder (2 mg, 0.02 mmol), 1,1'-bis(diphenylphosphino)ferrocene (10 mg, 0.02 mmol), and tris(dibenzylideneacetone)dipalladium (2 mg) were added sequentially. Under nitrogen, the reaction mixture was heated to 120°C for 12 hours. After cooling to room temperature, the reaction solution was poured into cold water and extracted three times with ethyl acetate (10 mL). The organic layers were combined, washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the product. Purification by silica gel column chromatography (ethyl acetate:petroleum ether = 0-80%) gave a brown solid product, 6,7,8,9-tetrahydro-5H-pyrazolo[1',5':1,2]azepano[4,3-b]quinoline-15-carbonitrile (30 mg, yield: 49.6%). LCMS: ESI m / z 289 (M+H) + .

[0308] Examples 4A and 4B:

[0309] Preparation of Compound 4A and Compound 4B

[0310] first step:

[0311] Under ice bath, compound 6,7,8,9-tetrahydropyrazolo[1,5-a]azocine-5(4H)-one (8.8 g, 53.6 mmol) and 2-aminobenzoic acid (8.08 g, 58.6 mmol) were dissolved in acetonitrile (50 mL), and phosphorus oxychloride (50 mL) was slowly added thereto. The reaction mixture was slowly heated to 60°C under nitrogen protection for 12 hours, cooled to room temperature, and concentrated under reduced pressure. The concentrated crude product was dissolved in ethyl acetate, and the pH was adjusted to 8-9 with saturated sodium bicarbonate solution under ice bath. The organic phase was collected, and the aqueous phase was extracted three times with acetic acid (30 mL). The organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product, which was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 0-50%) to give compound 6-1 (650 mg, yield: 57.9%). LCMS:ESI m / z 284 (M+H) + .

[0312] Step 2:

[0313] Compound 6-1 (13.4 g, 47.2 mmol) was dissolved in N,N-dimethylformamide (200 mL) at room temperature. Zinc cyanide (6.65 g, 56.7 mmol), 2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl (4.01 g, 9.45 mmol), and allylpalladium(II) chloride dimer (1.72 g, 4.72 mmol) were added. The reaction mixture was heated to 160°C under nitrogen for 12 hours. After cooling to room temperature, the mixture was diluted with water (200 mL) and extracted three times with ethyl acetate (200 mL). The organic phases were combined, washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 0-40%) to obtain compound 4-10 (9.6 g, yield: 74.1%). LCMS:ESI m / z275(M+H) + .

[0314] Step 3:

[0315] Compound 4-10 (9.0 g, 29.53 mmol) was dissolved in acetonitrile (150 mL) and N-bromosuccinimide (5.78 g, 32.48 mmol) was added at room temperature. Under nitrogen, the reaction mixture was heated to 50°C for 3 hours and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 5-20%) to obtain compound 4-11 (10.0 g, yield: 86.3%). LCMS: ESI m / z 353 (M+H) + .

[0316] Step 4:

[0317] Compound 4-11 (10.2 g, 25.9 mmol) was dissolved in dioxane (250 mL) and water (50 mL). Tert-butyl (4-oxo-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydrophthalazin-1-methyl)carbamate (15.6 g, 38.9 mmol, synthesized according to WO2021050915), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex (1.9 g, 2.60 mmol), and potassium carbonate (10.8 g, 77.9 mmol) were then added. The reaction mixture was heated to 65°C under nitrogen for 18 hours. After cooling to room temperature, the mixture was filtered and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate:petroleum ether=50-100%) to obtain compound 6-4 (3.5 g, yield 22.1%). LCMS: ESI m / z 548 (M+H) + .

[0318] Step 5:

[0319] In an ice bath, compound 6-4 (7.7 g, 12.7 mmol) was dissolved in dichloromethane (50 mL), and trifluoroacetic acid (30 mL) was slowly added thereto. The reaction was carried out at room temperature for 30 minutes, and the crude product was directly concentrated to obtain the crude product, which was purified by reverse phase preparative method to obtain compound 4 (5.6 g, yield: 96.8%). 1 H NMR(400MHz,MeOD)8.26(d,J=8.5Hz,1H),8.23(s,1H),8.13(d,J=8.3Hz,1H),8.0 5–7.97(m,2H),7.88(d,J=1.3Hz,1H),7.80–7.77(m,1H),7.40(dd,J=8.4,1.5Hz, 1H),4.54–4.50(m,2H),4.33(d,J=16.3Hz,1H),3.77-3.71(m,1H),3.49-3.44(m, 1H),2.71–2.65(m,1H),2.42-2.37(m,1H),2.18-2.14(m,1H),1.98-1.87(m,2H). LCMS:ESI m / z 448 (M+H) + .

[0320] Step 6:

[0321] Compound 4 was separated by SFC (Thar350 preparative SFC, Column: ChiralPak AD, 300×50 mm, ID 10 μm, Mobile phase: A for CO2 and B for Ethanol (0.1% NH3H2O), Gradient: B 55%, Flow rate: 200 mL / min, Back pressure: 100 bar, Column temperature: 38°C, Wavelength: 220 nm, Cycle-time: ~9 min) to give compound 4A (retention time: 5.5 minutes) and compound 4B (retention time: 8.9 minutes).

[0322] Examples 5A and 5B:

[0323] Preparation of Compound 5A and Compound 5B

[0324] Compound 5 prepared in Example 5 was separated by SFC (Waters Thar 80preparative SFC, Column: ChiralPak C-IG, 250×30mm ID, 5μm, Mobile phase: A for CO2 and B for MEOH (0.1% 2mol / L NH3 in MeOH), Gradient: B 50%, Flow rate: 60mL / min, Back pressure: 100bar, Column temperature: 35℃, Wavelength: 220nm, Run time: 30min, Cycle-time: ~20min) to obtain compound 5A (retention time: 10.0min) and compound 5B (retention time: 17.5min).

[0325] Example 7:

[0326] Preparation of 1-(4-(aminomethyl)-8-cyano-1-oxo-1,2-dihydrophthalazin-6-yl)-5,6,7-8-tetrahydropyrazolo[1',5':1,2]azocino[4,3-b]quinoline-14-carbonitrile

[0327] first step:

[0328] Compound 4-11 (55 mg, 0.16 mmol) was dissolved in 1,4-dioxane (3 mL) and water (0.6 mL), and (4-(bis(tert-butoxycarbonyl(amino)-8-cyano-1-oxo-1,2-dihydronaphthyridin-6-yl)boronic acid (70 mg, 0.16 mmol, synthesized with reference to WO2023098439), potassium carbonate (65 mg, 0.47 mmol) and [1,1'-bis(diphenylphosphino)bis(1,1'-diphenylphosphino)]-1,4-dioxane were added in sequence under nitrogen protection. [Fe] Palladium (II) dichloromethane complex (12.9 mg) was added, and the reaction system was heated to 65°C overnight. After cooling to room temperature, the reaction system was quenched with saturated ammonium chloride solution (5 mL) and extracted three times with ethyl acetate (5 mL). The combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 0-80%) to obtain compound 7-1 (40 mg, yield: 37.7%). LCMS: ESI m / z 673 (M+H) + .

[0329] Step 2:

[0330] Compound 7-1 (40 mg, 0.06 mmol) was dissolved in dichloromethane (1 mL) under ice-cooling, and trifluoroacetic acid (0.2 mL) was added. The mixture was allowed to react at room temperature for 30 minutes. The crude product was directly concentrated and purified by reverse phase preparative method to obtain compound 7 (9 mg, yield: 32.0%). LCMS: ESI m / z 473 (M+H) + .

[0331] Examples 8A and 8B:

[0332] Preparation of compounds 8A and 8B

[0333] first step:

[0334] Under ice bath, compound 6,7,8,9-tetrahydropyrazolo[1,5-a]azocine-5(4H)-one (10.0 g, 60.89 mmol) and 2-amino-3,5-difluorobenzoic acid (11.6 g, 66.98 mmol) were dissolved in acetonitrile (50 mL), and phosphorus oxychloride (50 mL) was slowly added thereto. The reaction mixture was slowly heated to 60° C. under nitrogen protection for 12 hours, cooled to room temperature, and concentrated under reduced pressure. The concentrated crude product was dissolved in ethyl acetate, and the pH was adjusted to 8-9 with saturated sodium bicarbonate solution under ice bath. The organic phase was collected, and the aqueous phase was extracted three times with acetic acid (100 mL). The organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product, which was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 0-50%) to give compound 8-1 (10.0 g, yield: 48.8%). LCMS:ESI m / z 320 (M+H) + .

[0335] Step 2:

[0336] At room temperature, compound 8-1 (10.0 g, 31.27 mmol) was dissolved in N,N-dimethylacetamide (100 mL), and zinc cyanide (4.41 g, 37.53 mmol), 2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl (1.73 g, 3.12 mmol) and allylpalladium (II) chloride dimer (1.43 g, 1.56 mmol) were added thereto. Under nitrogen protection, the reaction mixture was heated to 120 ° C for 12 hours. The mixture was cooled to room temperature, diluted with water (200 mL), and extracted three times with ethyl acetate (200 mL). The organic phases were combined, washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The product was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 0-50%) to obtain compound 8-2 (3.0 g, yield: 24.7%) and a crude product of compound 8-2 containing some impurities (7.0 g, purity: ~60%). LCMS: ESI m / z 311 (M+H) + .

[0337] Step 3:

[0338] Compound 8-2 (3.0 g, 9.67 mmol) was dissolved in acetonitrile (30 mL), and N-bromosuccinimide (1.72 g, 9.67 mmol) was added at room temperature. Under nitrogen, the reaction mixture was heated to 60°C for 1 hour, cooled to room temperature, diluted with water (50 mL), and extracted three times with ethyl acetate (100 mL). The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 0-50%) to obtain compound 8-3 (2.9 g, yield: 61.7%). LCMS: ESI m / z 389 (M+H) + .

[0339] Step 4:

[0340] Compound 8-3 (2.9 g, 7.45 mmol) was dissolved in dioxane (30 mL) and water (10 mL). Tert-butyl (4-oxo-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydrophthalazin-1-methyl)carbamate (3.59 g, 8.94 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex (610 mg, 0.75 mmol), and potassium carbonate (3.09 g, 22.35 mmol) were added. The reaction mixture was heated to 70°C under nitrogen for 24 hours. The mixture was cooled to room temperature, diluted with water (50 mL), and extracted three times with ethyl acetate (50 mL). The organic phases were combined and washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography (methanol:dichloromethane = 0-20%) to obtain compound 8-4 (720 mg, yield 15.7%). LCMS: ESI m / z 584 (M+H) + .

[0341] Step 5:

[0342] Compound 8-4 (720 mg, 1.23 mmol) was dissolved in dichloromethane (10 mL) under an ice bath, and trifluoroacetic acid (5 mL) was slowly added. The reaction was allowed to react at room temperature for 1 hour. The mixture was diluted with dichloromethane (5 mL), and the pH was adjusted to 8-9 with saturated sodium bicarbonate. The organic phase was collected, and the aqueous phase was extracted three times with dichloromethane (5 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was purified by reverse phase preparative method to obtain compound 8 (400 mg, yield: 65.7%). 1 HNMR(400MHz,MeOD)δ8.22(s,1H),8.15(d,J=8.3Hz,1H),7.88(d,J=1.2Hz, 1H),7.75–7.68(m,1H),7.54–7.49(m,1H),7.42(dd,J=8.3,1.5Hz,1H),4.55 –4.45(m,2H),4.34(d,J=16.2Hz,1H),3.80–3.71(m,1H),3.55–3.45(m,1H), 2.72–2.63(m,1H),2.43–2.35(m,1H),2.20–2.12(m,1H),1.96–1.84(m,2H). LCMS:ESI m / z 484 (M+H) + .

[0343] Step 6:

[0344] Compound 8 was separated by SFC (WATERS 150 preparative SFC, Column: ChiralPak AD, 250×30mm ID, 10μm, Mobile phase: A for CO2 and B for Isopropanol (0.1% NH3H2O), Gradient: B 40%, Flow rate: 120mL / min, Back pressure: 100bar, Column temperature: 38℃, Wavelength: 220nm, Cycle time: ~9min) to obtain compound 8A (retention time: 4.2 minutes) and compound 8B (retention time: 7.5 minutes).

[0345] The following compounds were synthesized from the corresponding intermediates according to the synthetic methods of Examples 4A and 4B (the corresponding starting materials are marked in the starting material column):

[0346] Example 16:

[0347] 1-(4-(Aminomethyl)-1-oxo-1,2-dihydrophthalazin-6-yl)-10-(trifluoromethyl)-5,6,7,8-tetrahydropyrazolo[1',5':1,2]azocino[4,3-b]quinoline-14-carbonitrile

[0348] first step:

[0349] The compound 2-amino-3-(trifluoromethyl)benzoic acid (1.0 g, 4.8 mmol) was dissolved in phosphorus oxychloride (10 mL), and 6,7,8,9-tetrahydropyrazolo[1,5-a]azocine-5(4H)-one (1.5 g, 4.8 mmol) was added thereto. The reaction solution was heated to 60°C under nitrogen protection for 5 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure, and the pH was adjusted to 7-8. The organic phase was collected, and the aqueous phase was extracted three times with acetic acid (20 mL). The organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 10-30%) to obtain compound 16-1 (600 mg, yield: 35%). LC-MS (ESI) m / z: 352 (M+H) + .

[0350] Step 2::

[0351] Compound 16-1 (600 mg, 1.7 mmol) was dissolved in N-methylpyrrolidone (10 mL), and 2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl (144 mg, 0.34 mmol), allylpalladium(II) chloride dimer (63 mg, 0.17 mmol), and zinc cyanide (200 mg, 1.7 mmol) were added. The resulting reaction mixture was heated to 150°C under nitrogen for 5 hours. After cooling to room temperature, the mixture was diluted with water (10 mL) and extracted three times with ethyl acetate (20 mL). The organic phases were combined, washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 10-30%) to obtain compound 16-2 (350 mg, yield: 60%). LC-MS (ESI) m / z: 343 (M+H) + .

[0352] Step 3:

[0353] Under ice bath, compound 16-2 (300 mg, 0.88 mmol) was dissolved in acetonitrile (5 mL), and iodine (249 mg, 0.87 mmol) and cerium ammonium nitrate (694 mg, 1.3 mmol) were added to the mixture. Under nitrogen protection, the reaction mixture was heated to 60 ° C for 3 hours. The reaction solution was diluted with water (20 mL) and extracted three times with ethyl acetate (20 mL). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 10-30%) to obtain compound 16-3 (150 mg, yield: 36.6%). LC-MS (ESI) m / z: 469 (M+H) + .

[0354] Step 4:

[0355] Compound 16-3 (150 mg, 0.32 mmol) and tert-butyl (4-oxo-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydrophthalazin-1-methyl)carbamate (192 mg, 0.48 mmol) were dissolved in 1,4-dioxane (5 mL) and water (1 mL). Potassium carbonate (133 mg, 0.96 mmol) and methanesulfonic acid [n-butyldi(1-adamantyl)phosphine] (2-amino-1,1'-biphenyl-2-yl) palladium (II) (23 mg) were added. Under nitrogen protection, the reaction mixture was heated to 65° C. and reacted overnight. The reaction mixture was diluted with water (10 ml) and extracted three times with ethyl acetate (20 ml). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 0-80%) to obtain compound 16-4 (80 mg, yield: 40.5%). LC-MS (ESI) m / z: 616 (M+H) + .

[0356] Step 5:

[0357] Under ice bath, compound 16-4 (80 mg, 0.13 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (1 mL) was added thereto. The reaction mixture was reacted at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in dichloromethane (5 mL). The pH was adjusted to 8-9 with saturated sodium bicarbonate solution. The dichloromethane layer was collected, and the aqueous phase was extracted three times with dichloromethane (10 mL). The organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product, which was purified by reverse phase preparation to give compound 16-5 (60 mg, yield: 89.5%). 1 H NMR (400MHz, MeOD) δ8.35(d,J=7.2Hz,1H),8.29–8.21(m,2H),8.14(d,J=8.3Hz,1H),7.96(d,J=1.3Hz,1H),7.91–7.85(m,1H),7.42–7.36(m ,1H),4.59–4.43(m,3H),3.78–3.70(m,1H),3.55–3.49(m,1H),2.75– 2.66(m,1H),2.44–2.37(m,1H),2.22–2.12(m,1H),2.00–1.87(m,2H). LCMS:ESI m / z 516(M+H) + .

[0358] The following compounds were synthesized from the corresponding starting materials according to the synthesis method of Example 16 (the corresponding starting materials are marked in the starting material column):

[0359] PRMT5 enzyme activity test method

[0360] Test compounds and positive compounds:

[0361] All compounds were prepared into 10mM or 20mM stock solutions in DMSO.

[0362] Positive compound: MRTX9768 (CAS#2629314-68-5; MCE, cat#HY-138684)

[0363] Experimental conditions:

[0364] The positive compound MRTX9768 was serially diluted 4-fold starting from 200 μM in DMSO, for a total of 10 concentrations

[0365] The test compound was serially diluted 3-fold starting from 200 μM in DMSO for a total of 10 concentrations

[0366] Add 1 μL of serially diluted compound to 65.67 μL of assay buffer to obtain 3X working solutions of test compound and positive compound.

[0367] Prepare 3X positive control (3 μM MRTX9768) and 3X negative control (1.5% DMSO)

[0368] Seal the dilution plate and shake the plate on a plate shaker for 15 minutes.

[0369] Experimental materials and reagents:

[0370] Consumables and instruments:

[0371] Experimental methods:

[0372] Add 4 μL of 3X serial dilutions of compound to a 384-well assay plate (6008280, PerkinElmer)

[0373] Prepare 3X PRMT5 enzyme working solution

[0374] Add 4 μL of 3X PRMT5 enzyme working solution to a 384-well assay plate.

[0375] Seal the test plate, centrifuge the 384-well test plate at 1000 rpm for 1 minute, and incubate in a 25°C incubator for 30 minutes.

[0376] Prepare 3X PRMT5 enzyme substrate working solution

[0377] Add 4 μL of 3X PRMT5 enzyme substrate working solution to the 384-well assay plate to start the reaction.

[0378] Seal the test plate, centrifuge the 384-well test plate at 1000 rpm for 1 minute, and incubate in a 25°C incubator for 90 minutes.

[0379] Prepare 4X detection solution using Protin-A-Eu, anti-histone H4 (R3 dimethyl symmetry) antibody and streptavidin-d2

[0380] Add 4 μL of 4X detection solution to a 384-well assay plate

[0381] Seal the test plate, centrifuge the 384-well test plate at 1000 rpm for 1 minute, and incubate in a 25°C incubator for 30 minutes.

[0382] Read on a BMG plate reader at wavelengths of 665 nm and 615 nm

[0383] Data processing:

[0384] The formula for calculating the inhibition rate % is as follows:

[0385] Average RR of positive control

[0386] Average RR of negative control

[0387] Determination of the antiproliferative effects of compounds on HCT116 cells

[0388] The anti-cell proliferation effect of the compounds was determined using HCT116 WT and HCT116 MTAP(- / -) cell lines. The cells were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin and placed in a 37°C, 5% CO2 incubator. After washing the cells twice with PBS, the cells were trypsinized and centrifuged for cell counting. The cell viability for the experiment reached more than 90% before the experiment could be performed. HCT116 WT and HCT116 MTAP(- / -) cells were plated into 384-well cell culture plates, with 100 cells per well and 40 μL of culture medium.

[0389] The compound was serially diluted 4-fold using DMSO, starting from 10 mM, for a total of 10 concentrations. 40 nL of the DMSO serially diluted compound was transferred to a 384-well cell culture plate using an Echo550. The 384-well plate was placed in a cell culture incubator (37°C, 5% CO2) for 10 days.

[0390] After 10 days of culture, 40 μL of CTG detection reagent (Promega, cat# G7573) was added to the 384-well microplate and mixed on a shaker for 10 minutes to induce cell lysis. The 384-well microplate was then incubated in the dark at 37°C, 5% CO₂, and 4% CO₂ for 30 minutes. The cells were read using an Envision multi-functional microplate reader.

[0391] The inhibitory rate of the drug on tumor cell growth was calculated as follows:

[0392] Tumor cell growth inhibition rate % = 100-(Signal cmpd -Signal Ave_BL ) / (Signal Ave_VC -Signal Ave_BL )X100

[0393] In-cell lysine symmetric dimethylation assay (SDMA In Cell Western)

[0394] All compounds were prepared as 10 mM or 20 mM stock solutions in DMSO. Test compounds were serially diluted 4-fold in DMSO starting at 5 mM for a total of 10 concentrations. The compound plate was centrifuged at 1000 rpm for 1 min.

[0395] HCT116 MTAP(- / -) cells or HCT116 WT cells were cultured in RPMI 1640 medium supplemented with 10% FBS (v / v) and 100 units / mL of penicillin-streptomycin mixture at 37°C and 5% CO2. Thirty microliters of cells were added to a poly-D-lysine-coated 384-well plate, with 500 cells per well. The 384-well plate was incubated at 37°C and 5% CO2 for 24 hours. Compounds were serially diluted, and 60 nL of compound from the 384-well dilution plate was added to the 384-well plate. The 384-well plate was incubated at 37°C and 5% CO2 for 96 hours.

[0396] Remove the culture medium from the 384-well cell plate, add 50 μL of 4% paraformaldehyde to each well, and incubate at room temperature for 20 minutes to fix the cells. Aspirate the paraformaldehyde and wash four times with PBS (PBST) wash buffer containing 0.1% Tween 20. Add 30 μL of ice-cold methanol to each well and place the 384-well cell plate at -20°C for 10 minutes. Aspirate the methanol and wash the cell plate four times with 100 μL of PBST wash buffer per well. Add 30 μL of Odyssey blocking buffer containing 0.05% Tween 20 to each cell plate and incubate with shaking at room temperature for 2 hours. Remove the blocking buffer and add 30 μL of Symmetric Di-Methyl Arginine Motif[sdme-RG]MultiMab to each well. TM Rabbit primary antibody (1:500 dilution in blocking buffer) was added and incubated overnight (16 hours) at 4°C. The primary antibody was removed by aspiration and the cell plate was washed 4 times with 100 μL of washing buffer per well, soaking for 5 minutes each time. 30 μL of goat anti-rabbit IgG ( The cells were then incubated with secondary antibodies (1:800 dilution for 800CW and 1:10,000 dilution for DRAQ5) for 2 hours at room temperature. The cell plates were washed four times with 100 μL of wash buffer per well. The cell plates were dried at room temperature and the integrated intensity at 700 nm and 800 nm was measured and imaged using a LI-COR Odyssey.

[0397] The ratio for each well was determined by the formula: SDMA 800 nm value / DRAQ5 700 nm value. The average ratio of the control wells treated with DMSO alone (minimum inhibition) was calculated and used to determine the percent inhibition for each test well in the cell plate. The percent inhibition was determined and the IC was generated using three replicate wells per concentration of compound. 50 Inhibition rate % = 100 - (single test sample ratio / minimum inhibition ratio) * 100.

[0398] The experimental results of the compounds of the present invention obtained through the above experiments are shown in Table 1 below.

[0399] Table 1. Compound activity data

[0400] The experimental results showed that the compounds of the present invention exhibited very strong PRMT5 enzyme inhibitory activity and showed better cell proliferation inhibition activity in MTAP-deficient cells than in wild-type cells. This also shows that the compounds of the present invention have the potential to selectively kill MTAP-deficient cancer cells while having no side effects on normal cells, indicating that the compounds of the present invention have a good therapeutic safety window.

Claims

1. A compound of formula (I), Its enantiomers, diastereomers, racemates, tautomers, prodrugs, hydrates, solvates or pharmaceutically acceptable salts, wherein: X1 is selected from CR1R2, O, NR3 and S, wherein S may be optionally oxidized; X2, X3, X4 and X5 are independently selected from CR4 and N; X6 and X7 are selected from CR7 and N; X8 is selected from C or N; X9 is selected from C(=O) or N; represents a single bond or a double bond; R1 and R2 are independently selected from H, D and C 1-6 Alkyl, or R1 and R2 together with the atoms to which they are attached form a 3- to 6-membered carbocyclic or heterocyclic group; R3 is selected from H, C 1-6 Alkyl, C 1-6 acyl and 3- to 6-membered carbocyclyl acyl; Each R4 is independently selected from H, halogen, hydroxy, cyano, NR5R6, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy, halogenated C 1-6 Alkoxy, aryl, heteroaryl and heterocyclic groups, wherein the aryl, heteroaryl, C 3-8 Cycloalkyl and heterocycloalkyl are optionally substituted by one or more independently selected from halogen, hydroxy, cyano, NR5R6, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy and halogenated C 1-6 Alkoxy substituents are substituted, R5 and R6 are independently selected from H and C 1-6 Alkyl, or R5 and R6 together with the nitrogen atom to which they are attached form a nitrogen-containing heterocyclic group; R7 is selected from H, halogen, cyano, C 1-6 Alkyl, cyclopropyl and halo C 1-6 alkyl; R8 and R9 are independently selected from H and C 1-6 Alkyl, or R8 and R9 together with the atoms to which they are attached form a 3- to 6-membered carbocyclic or heterocyclic group; p=2, 3 or 4.

2. The compound according to claim 1, its enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof, wherein X1 is selected from CR1R2 and O.

3. The compound according to claim 1 or 2, its enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof, wherein X2, X3, X4 and X5 are independently selected from CR4.

4. The compound according to any one of claims 1 to 3, its enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof, wherein X6 and X7 are selected from CR7.

5. A compound according to any one of claims 1 to 4, its enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof, wherein X8 is C, X9 is N and Represents a double bond.

6. The compound according to any one of claims 1 to 4, its enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof, wherein X8 is N, X9 is C(=O) and Indicates a single bond.

7. The compound according to any one of claims 1 to 6, its enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof, wherein X1 is CR1R2 and R1 and R2 are independently selected from H, D and C 1-6 alkyl.

8. The compound according to any one of claims 1 to 7, its enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof, wherein R4 is independently selected from H, halogen, cyano, C 1-6 Alkoxy, halogenated C 1-6 Alkyl and C 3-8 Cycloalkyl is, for example, selected from H and halogen, for example selected from H, F and Cl.

9. The compound according to any one of claims 1 to 8, its enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof, wherein R7, R8 and R9 are all H.

10. The compound according to claim 1, its enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof, wherein the compound has the structure of formula (II): wherein X1 to X7, R8, R9 and p are as defined in claim 1.

11. The compound according to claim 1, its enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof, wherein the compound has the structure of formula (III): wherein X1 to X7, R8, R9 and p are as defined in claim 1.

12. The compound according to claim 1, its enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof, wherein the compound is selected from the following compounds:

13. A pharmaceutical composition comprising: (1) a therapeutically effective amount of a compound according to any one of claims 1 to 12, its enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or pharmaceutically acceptable salt as an active ingredient; and (2) Pharmaceutically acceptable carrier.

14. Use of the compound according to any one of claims 1 to 12, its enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 13 in the preparation of a medicament for use as a PRMT5 inhibitor, particularly an MTA-synergistic PRMT5 inhibitor.

15. Use of the compound of any one of claims 1 to 12, its enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 13 in the preparation of a medicament for treating a disease mediated by or at least partially mediated by PRMT5.

16. The use of claim 15, wherein the disease mediated by PRMT5 or at least in part by PRMT5 is cancer.

17. The use of claim 16, wherein the cancer is a solid tumor or a hematological malignancy, such as leukemia, lymphoma or myeloma.

18. The method of claim 16, wherein the cancer is selected from the group consisting of ovarian cancer, lung cancer (including non-small cell lung cancer), brain tumor (including glioblastoma (GBM)), giant cell tumor of the tendon sheath, gastrointestinal stromal tumor (GIST), gastric cancer, esophageal cancer, colon cancer, colorectal cancer, pancreatic cancer, prostate cancer, breast cancer, cervical cancer, melanoma, mesothelioma, mesothelial carcinoma, kidney cancer, liver cancer, thyroid cancer, head and neck cancer, urothelial carcinoma, bladder cancer, endometrial cancer, choriocarcinoma, adrenal cancer, sarcoma, leukemia, lymphoma, or myeloma.

19. A method for inhibiting PRMT5 activity in vivo or in vitro, comprising contacting PRMT5 with an effective amount of at least one compound according to any one of claims 1 to 12, its enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate or pharmaceutically acceptable salt thereof.

20. A method for inhibiting the growth of MTAP-deficient cancer cells, comprising contacting the cells with an effective amount of at least one compound according to any one of claims 1 to 12, its enantiomer, diastereomer, racemate, tautomer, prodrug, hydrate, solvate, or pharmaceutically acceptable salt.

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