Tricyclic compounds as MAT2A inhibitors

Abstract

The invention provides a compound as shown in a formula I, or pharmaceutically acceptable salt thereof, a preparation method thereof and application of the compound as an MAT2A inhibitor. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to a tricyclic compound as a MAT2A inhibitor, or a pharmaceutically acceptable salt thereof, a preparation method thereof, a pharmaceutical composition thereof, and use thereof in preparing a compound for treating cancer in a subject suffering from cancer. Background Art

[0002] Methionine adenosyltransferase (MAT) (also known as S-adenosylmethionine synthetase) is a cellular enzyme that catalyzes the synthesis of S-adenosylmethionine (SAM or AdoMet) from methionine and ATP; this catalysis is considered to be the rate-limiting step of the methionine cycle. SAM is an propylamine donor in polyamine biosynthesis and the major methyl donor for DNA methylation, and is involved in gene transcription and cell proliferation as well as the generation of secondary metabolites.

[0003] The three isoforms of human MAT include MAT1 and MAT3, which are expressed in liver tissue, while MAT2A is ubiquitously expressed in human cell types and is the predominant form in human tumors. Crystal structure and mechanistic studies have shown that despite their 85% identity in amino acid sequence, their mechanisms of action differ significantly. MAT2A forms functional homodimers in its purified active form and binds to the regulatory protein MAT2B. MAT2B regulates the activity of MAT2A by increasing the sensitivity of MAT2A to inhibition by the ADOMet product, but does not provide significant rate enhancement. Cellular localization studies have shown that MAT2A is present in both the cytoplasm and the nucleus. Nuclear condensation of MAT2A has been reported to occur during replication and the subsequent G2 phase, satisfying the high methylation requirements of DNA and histone methylation processes in the nucleus during the S phase. The activity of ADOMet in transmethylation reactions has been identified as a rate-limiting factor in the development of lung cancer stem cells, making MAT2A and related enzymes of the methionine cycle targets for anticancer drugs. Methionine for ADOMet production by MAT2A comes from dietary sources or the polyamine cycle, where 5-methylthio-D-ribose 1-phosphate produced by S-methyl-5'-thioadenosine phosphorylase is recycled to methionine. MAT2A protein expression has been reported to be increased in cancers such as colon cancer, hepatocellular carcinoma, gastric cancer, blood and liver cancer. Approximately 15% of human cancers show deletion of the MTAP (S-methyl-5'-thioadenosine phosphorylase) gene and therefore lack the methionine recycling pathway for polyamine synthesis. Loss of MTAP in chr9p21 often includes loss of the CDKN2a tumor suppressor gene locus. Comprehensive genetic lethality studies of MATP- / - cancer cells have shown that these cancer cells have increased sensitivity to inhibition of MAT2A, PRMT5 and PRMT1.

[0004] In hepatocellular carcinoma (HCC), downregulation of MAT1A and upregulation of MAT2A occur, which is called MAT1A:MAT2A switch. The switch accompanied by upregulation of MAT2B leads to lower SAM content, which provides a growth advantage for liver cancer cells. Since MAT2A plays a vital role in promoting the growth of liver cancer cells, it is a target for anti-tumor therapy. Recent studies have shown that silencing by using small interfering RNA substantially inhibits the growth of liver cancer cells and induces apoptosis. See, for example, T. Li et al., J. Cancer 7 (10) (2016) 1317-1327.

[0005] Some MTAP-deficient cancer cell lines are particularly sensitive to inhibition of MAT2A, Marjon et al. (Cell Reports 15(3)(2016) 574–587). MTAP (methylthioadenosine phosphorylase) is an enzyme widely expressed in normal tissues that catalyzes the conversion of methylthioadenosine (MTA) to adenine and 5-methylthioribose-1-phosphate. Adenine is salvaged to produce adenosine monophosphate, and 5-methylthioribose-1-phosphate is converted to methionine and formate. Due to this salvage pathway, MTA can serve as an alternative purine source when de novo purine synthesis is blocked, for example, by antimetabolites such as L-alanosine.

[0006] In other cancers lacking MTAP deletion, including hepatocellular carcinoma and leukemia, MAT2A is dysregulated. J. Cai et al., Cancer Res. 58 (1998) 1444-1450; TS Jani et al., Cell. Res. 19 (2009) 358-369. Silencing MAT2A expression by RNA interference can produce antiproliferative effects in a variety of cancer models, H. Chen et al., Gastroenterology 133 (2007) 207-218; Q. Liu et al. Hepatol. Res. 37 (2007) 376-388.

[0007] Many human and mouse malignant cells lack MTAP activity. MTAP deficiency is not only present in tissue culture cells, but also in primary leukemias, gliomas, melanomas, pancreatic cancer, non-small cell lung cancer (NSCLC), bladder cancer, astrocytomas, osteosarcomas, head and neck cancer, myxoid chondrosarcomas, ovarian cancer, endometrial cancer, breast cancer, soft tissue sarcomas, non-Hodgkin lymphomas and mesothelioma. The gene encoding human MTAP is located in region 9p21 on human chromosome 9p. This region also contains the tumor suppressor genes p16INK4A (also known as CDKN2A) and p15INK4B. These genes encode p16 and p15, which are inhibitors of cyclin D-dependent kinases cdk4 and cdk6, respectively.

[0008] Alternatively, the p16INK4A transcript can be an alternative reading frame (ARF) spliced ​​into a transcript encoding p14ARF. p14ARF binds to MDM2 and prevents degradation of p53 (Pomerantz et al. (1998) Cell 92: 713-723). The 9p21 chromosome region is of interest because it is often homozygous deleted in a variety of cancers, including leukemia, NSLC, pancreatic cancer, glioma, melanoma, and mesothelioma. Deletions usually inactivate more than one gene. For example, Cairns et al. ((1995) Nat. Gen. 11: 210-212) reported that after studying more than 500 primary tumors, almost all of the deletions identified in these tumors involved a 170kb region containing MTAP, p14ARF, and P16INK4A. Carson et al. (WO99 / 67634) reported that there is a correlation between the stage of tumor development and the homozygosity loss of the gene encoding MTAP and the gene encoding p16. For example, it is reported that the loss of the MTAP gene rather than p16INK4A indicates cancer in the early stages of development, while the loss of the genes encoding p16 and MTAP indicates that cancer is in a more advanced stage of cancer development. In some osteosarcoma patients, the MTAP gene is present at diagnosis, but is missing at a later time point (Garcia-Castellano et al., Clin. Cancer Res. 8 (3) 2002 782-787).

[0009] WO2018039972 discloses MAT2A enzyme inhibitors for treating cancer; WO2021158792 mentions MAT2A inhibitors for treating autoimmune diseases or inflammatory diseases; WO2018045071 describes MAT2A enzyme inhibitors for treating diseases or conditions mediated by overexpression of MAT2A; CN202080014106.0 also discloses the compound 3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)-2-phenyl-5-(pyridin-3-ylamino)pyrazolo[1,5-a]pyrimidin-7(4H)-one for treating MTAP-deficient non-small cell lung cancer (NSCLC) in patients in need; CN114874207A discloses MAT2A inhibitors with a 6-in-6 ring core; CN113999232A discloses polycyclic compounds that inhibit MTAP-deficient cancer cells.

[0010] Finding effective MAT2A inhibitors is an important direction in the current research and development of tumor targeted drugs. Summary of the invention SUMMARY OF THE INVENTION

[0012] The present invention meets the significant demand for safe and effective compounds and methods for treating, preventing and controlling cancer while reducing or avoiding the toxicity and / or side effects associated with conventional therapies. To solve the above technical problems, the present invention adopts the following technical solutions:

[0013] In one aspect, the present invention provides a compound as shown in Formula I and its isomers, or a pharmaceutically acceptable salt thereof,

[0014]

[0015] in:

[0016] X is selected from N or O;

[0017] R1 is selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl;

[0018] R2 is selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, -C1-C6 alkylene-R a ;

[0019] R a Selected from 6-10 membered aryl, 5-10 membered heteroaryl, optionally substituted with one or more substituents selected from the following: hydroxyl, halogen, NH2, CN, C1-C6 alkyl, C1-C6 alkoxy;

[0020] or R1, R2 together with the carbon and nitrogen to which they are attached form a 4-6 membered heterocyclic group, wherein the 4- to 6-membered heterocyclic group may be optionally substituted by one or more substituents, each of which is independently selected from the group consisting of halogen, cyano, oxo, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl;

[0021] R3 is selected from 6-10 membered aryl, 5-10 membered heteroaryl, -C1-C6 alkylene-R b The 6-10 membered aryl group and the 5-10 membered heteroaryl group are optionally substituted with one or more substituents selected from the following: hydroxyl, halogen, -NR c R d , -CN, -NHC(O)C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy;

[0022] R b is selected from 6-10 membered aryl, 5-10 membered heteroaryl, optionally substituted with one or more substituents selected from the following: hydroxyl, halogen, -NH2, -CN, C1-C6 alkyl, C1-C6 alkoxy;

[0023] R c , R dare independently selected at each occurrence from hydrogen, C1-C6-alkyl;

[0024] R4 is selected from hydrogen, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 haloalkyl;

[0025] R5 is selected from hydrogen, oxo, halogen, C1-C6-alkyl, C1-C6-alkoxy, C1-C6-haloalkyl.

[0026] As a preferred technical solution, the R1 is selected from hydrogen, methyl, and ethyl.

[0027] As a preferred technical solution, the R2 is selected from hydrogen, difluoroethane, methyl, -methylene-2-pyridine, and ethyl.

[0028] As a preferred technical solution, the R1, R2 together with the carbon and nitrogen to which they are connected form a 5-membered heterocyclic group.

[0029] As a preferred technical solution, the 5-membered heterocyclic group is selected from azacyclopentane.

[0030] As a preferred technical solution, the R3 is selected from

[0031]

[0032] As a preferred technical solution, the R4 is selected from C1-C6-alkyl, halogen, and cyclopropane, preferably methyl, chlorine, and cyclopropane.

[0033] As a preferred technical solution, the R5 is selected from hydrogen, methyl, and oxo.

[0034] The present invention also provides compounds represented by the following formula Ia, Ib and their isomers, or pharmaceutically acceptable salts thereof:

[0035]

[0036] Wherein, R1 is selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl;

[0037] R2 is selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, -C1-C6 alkylene-R a ;

[0038] R a Selected from 6-10 membered aryl, 5-10 membered heteroaryl, optionally substituted with one or more substituents selected from the following: hydroxyl, halogen, NH2, CN, C1-C6 alkyl, C1-C6 alkoxy;

[0039] Or R1, R2 together with the carbon and nitrogen to which they are attached form an azacyclopentane;

[0040] R3 is selected from 6-10 membered aryl, 5-10 membered heteroaryl, -C1-C6 alkylene-R b The 6-10 membered aryl group and the 5-10 membered heteroaryl group are optionally substituted with one or more substituents selected from the following: hydroxyl, halogen, -NR c R d , -CN, -NHC(O)C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy;

[0041] R b is selected from 6-10 membered aryl, 5-10 membered heteroaryl, optionally substituted with one or more substituents selected from the following: hydroxyl, halogen, -NH2, -CN, C1-C6 alkyl, C1-C6 alkoxy;

[0042] R c , R d are independently selected at each occurrence from hydrogen, C1-C6-alkyl;

[0043] R4 is selected from hydrogen, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 haloalkyl;

[0044] R5 is selected from hydrogen, oxo, halogen, C1-C6-alkyl, C1-C6-alkoxy, C1-C6-haloalkyl.

[0045] The present invention also provides the following compounds and their isomers, or pharmaceutically acceptable salts thereof:

[0046]

[0047] The present invention also provides the use of any of the compounds described above and their isomers, or pharmaceutically acceptable salts thereof, in the preparation of a medicament for treating a disease or condition in a subject suffering from the disease or condition, wherein the disease or condition is mediated by overexpression of MAT2A, the disease or condition is preferably cancer, and the cancer is preferably characterized by a reduction or deletion of methylthioadenosine phosphorylase (MTAP) gene expression, a deletion of the MTAP gene, or a reduction in MTAP protein function.

[0048] The inventors have found that this class of compounds is a highly effective MAT2A inhibitor, has extremely strong MAT2A inhibitory activity, and can be used to prepare a method for preventing and / or treating MAT2A inhibition-related indications, including cancers caused by reduced or absent MTAP expression, absent MTAP genes, and reduced MTAP protein function. The present invention is completed based on the above findings. DETAILED DESCRIPTION OF THE INVENTION

[0050] Various aspects and features of the present invention are further described below.

[0051] The compounds according to the present invention may exist in stereoisomeric forms and the present invention therefore includes all stereoisomeric forms.

[0052] The compounds of the present invention have asymmetric centers. The compounds of the present invention containing asymmetrically substituted atoms can be separated into optically active or racemic forms. Those skilled in the art know how to prepare optically active forms, such as by racemic separation or synthesis from optically active starting materials. Unless the specific stereochemistry or isomeric form is specifically stated, the present invention includes all chiral, diastereoisomers and racemates. The methods for preparing the compounds of the present invention and the intermediates thereof are part of the present invention. All stereoisomers of the compounds of the present invention are also part of the present invention.

[0053] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes both occurring and not occurring of said event or circumstance.

[0054] The term "substituted" means that any one or more hydrogen atoms on a particular atom or group are replaced by a substituent, as long as the valence state of the particular atom is normal and the resulting compound is stable after the substitution. When the substituent is =0, it means that two hydrogen atoms are replaced. When multiple hydrogen atoms are replaced by substituents, each substituent is independent of each other and can be the same substituent or different substituents. Unless otherwise specified, the type and number of substituents can be arbitrary on the basis of chemical realization.

[0055] The terms "alkoxy" and "alkylamino" are customary expressions and refer to an alkyl group attached to the remainder of the molecule through an oxygen atom or an amino group, respectively, wherein the alkyl group is as described herein.

[0056] As used herein, the terms "halogen", "halo" and the like represent fluorine, chlorine, bromine or iodine, and particularly represent fluorine, chlorine and bromine, with fluorine and chlorine being particularly preferred.

[0057] As described herein, the term "alkyl" refers to an alkyl group having a specified number of carbon atoms, which is a linear or branched alkyl group, and which may include its subgroups, for example, when "C1-C6 alkyl" is mentioned, it may also include sub-range groups represented by C1-C4 alkyl, C1-C3 alkyl, C2-C6 alkyl, C2-C4 alkyl, etc., and specific groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, etc. It may be divalent, such as methylene, ethylene.

[0058] Represents a single bond or a double bond.

[0059] The terms "heterocycle", "heterocyclic" or "heterocyclyl" are used interchangeably and refer to substituted and unsubstituted 3 to 7-membered monocyclic groups, 7 to 11-membered bicyclic groups, and 10 to 15-membered tricyclic groups having at least one heteroatom (O, S or N) in at least one ring, the heteroatom-containing ring preferably having 1, 2 or 3 heteroatoms selected from O, S and N. Each ring of such heteroatom-containing groups may contain one or two oxygen or sulfur atoms and / or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less, and further provided that the ring contains at least one carbon atom. Nitrogen and sulfur atoms may be optionally oxidized, and nitrogen atoms may be optionally quaternized. The fused rings completing the bicyclic and tricyclic groups may contain only carbon atoms, and may be saturated, partially saturated or fully unsaturated. The heterocyclic group may be attached to any available nitrogen or carbon atom. As used herein, the terms "heterocycle," "heterocycloalkyl," "heterocyclo," "heterocyclic," and "heterocyclyl" include "heteroaryl" groups, as defined below.

[0060] "Aryl" when used alone or as part of another term refers to a carbocyclic aromatic radical, whether fused or not, having the specified number of carbon atoms, or if no carbon number is specified, up to 14 carbon atoms, such as C6-C14-aryl. Specific aryl groups are phenyl, naphthyl, biphenyl, phenanthrenyl, tetraphenylene, and the like.

[0061] In addition to the heteroaryl groups described below, exemplary monocyclic heterocyclic groups include azetidinyl, pyrrolidinyl, oxetanyl, imidazolinyl, oxazolidinyl, isoxazolinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, azepinyl, 1-pyridonyl, 4-piperidonyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxolane, and tetrahydro-1,1-dioxythienyl, etc. Exemplary bicyclic heterocyclic groups include quinuclidinyl.

[0062] The term "heteroaryl" refers to substituted and unsubstituted aromatic 5- or 6-membered monocyclic groups, 9- or 10-membered bicyclic groups, and 11- to 14-membered tricyclic groups, which have at least one heteroatom (O, S or N) in at least one ring, and the heteroatom-containing ring preferably has 1, 2 or 3 heteroatoms selected from O, S and N. Each ring of the heteroaryl containing heteroatoms can contain one or two oxygen or sulfur atoms and / or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less, and each ring has at least one carbon atom. The condensed rings completing the bicyclic and tricyclic groups can contain only carbon atoms and can be saturated, partially saturated or unsaturated. Nitrogen and sulfur atoms can be optionally oxidized, and nitrogen atoms can be optionally quaternized. As a bicyclic or tricyclic heteroaryl, it must include at least one completely aromatic ring, but the other one or more condensed rings can be aromatic or non-aromatic. Heteroaryl can be attached to any available nitrogen or carbon atom of any ring. If the other ring is cycloalkyl or heterocyclic it is additionally optionally substituted with =0 (oxo) where valence permits.

[0063] Unless otherwise indicated, when referring to a specifically named aryl (e.g., phenyl), heterocyclyl (e.g., pyrrolidinyl, piperidinyl, and morpholinyl), or heteroaryl (e.g., tetrazolyl, imidazolyl, pyrazolyl, triazolyl, thiazolyl, and furanyl) group, the reference is intended to include rings optionally having 0 to 3, preferably 0 to 2, substituents selected from the substituents listed above for arylheterocyclyl and / or heteroaryl groups.

[0064] As described herein, the term "pharmaceutically acceptable salt" means that the salt is not only physiologically acceptable to the subject, but also refers to a synthetic substance with pharmaceutical use value, such as a salt formed as an intermediate when performing chiral separation. Although the salt of this intermediate cannot be directly administered to the subject, the salt can play a role in obtaining the final product of the present invention. Specifically including acid (organic acid and inorganic acid) addition salts or base addition salts (including organic bases and inorganic bases).

[0065] As described herein, the term "disease" refers to a physical state of the subject, which is related to the disease described in the present invention, for example, peripheral arterial disease and neurodegeneration-related diseases described in the present invention.

[0066] The cancer treatments of the present invention include standard treatments such as surgery, radiation therapy, chemotherapy and hormone therapy.

[0067] "Cancer" or "malignancy" refers to any of a variety of diseases characterized by uncontrolled abnormal proliferation of cells, the ability of affected cells to spread locally or via the bloodstream and lymphatic system to other sites in the body (i.e., metastasis), and any of a number of characteristic structural and / or molecular features. "Cancer cell" refers to a cell that is undergoing an early, intermediate, or advanced stage of multistep neoplastic progression. Cancers include mesothelioma, neuroblastoma, rectal cancer, colon cancer, familial adenomatous polyposis cancer and hereditary nonpolyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, gastric cancer, adenocarcinoma, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal parenchymal cancer, ovarian cancer, cervical cancer, uterine corpus cancer, endometrial cancer, choriocarcinoma, pancreatic cancer, prostate cancer, bladder cancer, testicular cancer, breast cancer, urinary tract cancer, melanoma, brain tumors, head and neck cancer, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hepatocellular carcinoma, gallbladder cancer, bronchial tumor, advanced solid tumors, small cell lung cancer, metastatic non-small cell lung cancer, multiple myeloma, basal cell tumor, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma and plasma cell tumor, lymphoma, pancreatic ductal adenocarcinoma.

[0068] The compound of the present invention or the pharmaceutical composition containing the same can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, intravenous drip, subcutaneous injection, nasal cavity, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0069] The dosage form can be a liquid dosage form, a solid dosage form or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o / w types, w / o types and multiple emulsions), suspensions, injections (including water injections, powder injections and infusions), eye drops, nasal drops, lotions and liniments, etc.; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), sprays, etc.; semisolid dosage forms can be ointments, gels, pastes, etc.

[0070] In order to achieve the purpose of medication and enhance the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known administration method.

[0071] The compound or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs. When the compound of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0072] Beneficial technical effects

[0073] The inventors found that the compounds of the present invention have good MAT2A inhibitory activity, and the IC50 is less than that of the positive control drug AG270. The present invention provides a class of MAT2A inhibitor compounds with novel structure and strong activity, which have good application prospects in preventing and / or treating indications related to MAT2A inhibition, such as reduction or loss of MTAP expression, loss of MTAP gene, reduction of MTAP protein function, etc. DETAILED DESCRIPTION

[0074] The following embodiments are helpful for those skilled in the art to better understand the technical solution of the present invention, but the protection scope of the present invention includes but is not limited to them.

[0075] For all the following examples, standard procedures and methods known to those skilled in the art can be used. Unless otherwise indicated, all temperatures are expressed in ° C (Celsius). The structures of the compounds are determined by nuclear magnetic resonance spectroscopy (NMR) and / or mass spectroscopy (MS).

[0076] The structure of the compound of the present invention is determined by nuclear magnetic resonance (NMR) or / and liquid-mass spectrometry (LC-MS). The NMR chemical shift (δ) is in parts per million (ppm). The nuclear magnetic resonance is measured using a Brukeravance-400 nuclear magnetic spectrometer, the solvents are deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD) and deuterated chloroform (CDCl3), and the internal standard is tetramethylsilane (TMS).

[0077] Liquid phase mass spectrometry LC-MS measurement The liquid phase part uses ACQUITY UPLC ultra-high pressure liquid chromatography, and the mass spectrometry part uses XevoG2-SQtof mass spectrometer.

[0078] The starting materials in the examples of the present invention are known and can be purchased commercially, or can be synthesized using or according to methods known in the art.

[0079] Example 1: Synthesis of 5-chloro-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one

[0080] Step 1: Synthesis of 2,4,6-trichloronicotinic acid

[0081] Add a mixture of 2,4,6-trichloropyridine (10g, 54.9mmol, 1.0eq) and tetrahydrofuran (100mL) to a three-necked flask, replace nitrogen, cool to -78°C, slowly add n-butyl lithium (26.4mL, 65.9mmol, 1.2eq), stir for 1 hour at -78°C, then add excess dry ice to the reaction flask, and then stir for 2 hours. After the reaction is completed, add dilute hydrochloric acid to quench the reaction, then add ethyl acetate to extract, combine the organic phases, dry with saturated brine and anhydrous sodium sulfate, respectively, and concentrate to obtain a crude brown oil 2,4,6-trichloronicotinic acid (11.9g, yield 95.9%). LCMS (TOF MSES+) m / z [M+H] + :225.9.

[0082] Step 2: Synthesis of 2,4,6-trichlorononanamide

[0083] 2,4,6-trichloronicotinic acid (11.9 g, 52.6 mmol, 1.0 eq) and N,N-dimethylformamide (192 mg, 2.6 mmol, 0.05 eq) were added to dichloromethane (100 mL), and then oxalyl chloride (20.0 g, 157.8 mmol, 3.0 eq) was added dropwise and reacted for 2 hours. The reaction solution was then concentrated and dichloromethane was added. Ammonia water (11.8 mL, 157.8 mmol, 3.0 eq) was slowly added under ice-water bath conditions and reacted for 1 hour. After the reaction was completed, the reaction solution was directly concentrated, ethyl acetate and water were added to extract the liquid, the organic phase was combined and concentrated, and ethyl acetate / petroleum ether was used to slurry to obtain a yellow solid 2,4,6-trichlorononanamide (6.6 g, yield 56.0%). LCMS (TOF MS ES+) m / z [M+H] + :224.9. 1 H NMR (400MHz, DMSO) δ8.20 (s, 1H), 8.07 (s, 1H), 8.00 (d, J = 1.2Hz, 1H).

[0084] Step 3: Synthesis of 2,6-dichloro-4-(phenylamino)nicotinamide

[0085] Dissolve aniline (2.99 g, 32.2 mmol, 1.1 eq) in tetrahydrofuran (30 mL), add lithium bis(trimethylsilyl)amide (35.1 mL, 35.1 mmol, 1.2 eq) dropwise under ice bath, and stir for 30 minutes. Then add 2,4,6-trichlorononanamide (6.6 g, 29.3 mmol, 1.0 eq) and stir for 30 minutes. After the reaction is completed, add saturated ammonium chloride solution to quench, then add ethyl acetate to extract, combine and concentrate the organic phase, separate and purify by normal phase, and obtain yellow solid 2,6-dichloro-4-(phenylamino)nicotinamide (4.1 g, yield 49.8%). LCMS (TOF MS ES+) m / z [M+H] + :282.0. 1 H NMR (400MHz, DMSO) δ8.61(s,1H),8.19(s,1H),7.86-7.82(m,1H),7.47-7.38(m,2H),7.29-7.18(m,3H),6.67(s,1H).

[0086] Step 4: Synthesis of 5,7-dichloro-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione

[0087] 2,6-dichloro-4-(phenylamino)nicotinamide (4.0 g, 14.2 mmol, 1.0 eq) was added to tetrahydrofuran (50 mL), sodium hydride (1.7 g, 42.5 mmol, 3.0 eq) was added under ice bath, stirred for 30 minutes, and then N,N'-carbonyldiimidazole (4.6 g, 28.3 mmol, 2.0 eq) was added, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, ice water was added to quench, and then dilute hydrochloric acid was added to adjust the pH to 2-3, and then ethyl acetate was added for extraction, and the organic phase was combined and concentrated to obtain a yellow solid 5,7-dichloro-1-phenylpyridine [4,3-d] pyrimidine-2,4 (1H, 3H) -dione (2.4 g, yield 55.0%). LCMS (TOF MS ES+) m / z [M+H] + :307.9. 1 H NMR (400MHz, DMSO) δ12.05(s,1H),7.73–7.54(m,3H),7.49–7.40(m,2H),6.24(s,1H).

[0088] Step 5: Synthesis of tert-butyl (2-hydroxyethyl)carbamate

[0089] Dissolve ethanolamine (5.0 g, 82 mmol, 1.0 eq) and triethylamine (16.6 g, 164 mmol, 1.0 eq) in methanol (50 mL), add di-tert-butyl dicarbonate (19.7 g, 90 mmol, 1.1 eq) under ice bath, and stir at room temperature for 1 hour. After the reaction is completed, concentrate the reaction solution to obtain colorless oily (2-hydroxyethyl) carbamic acid tert-butyl ester (13.2 g). LCMS (TOF MS ES+) m / z [M+H] + :162.1. 1 H NMR (400MHz, DMSO) δ6.63 (t, J = 5.8 Hz, 1H), 4.54 (t, J = 5.6 Hz, 1H), 3.40–3.31 (m, 2H), 2.97 (q, J = 6.2 Hz, 2H), 1.37 (s, 9H).

[0090] Step 6: Synthesis of tert-butyl {2-[(7-chloro-2,4-dioxo-1-phenyl-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]ethyl}carbamate

[0091] Dissolve tert-butyl (2-hydroxyethyl)carbamate (1.15 g, 7.14 mmol, 1.1 eq) in tetrahydrofuran (40 mL), add sodium hydride (519 mg, 12.9 mmol, 2.0 eq) under ice bath, and stir for 30 minutes. Then add 5,7-dichloro-1-phenylpyridinium [4,3-d] pyrimidine-2,4 (1H, 3H) -dione (2.0 g, 6.49 mmol, 1.0 eq) and stir for 1 hour. After the reaction is completed, add saturated ammonium chloride solution to quench, extract the aqueous phase with ethyl acetate, combine and concentrate the organic phase, and separate and purify in the forward direction to obtain a yellow solid {2- [(7-chloro-2,4-dioxo-1-phenyl-1,2,3,4-tetrahydropyrido [4,3-d] pyrimidin-5-yl) oxy] ethyl} carbamate (2.4 g, yield 85.6%). LCMS (TOF MS ES+) m / z [M+H] + :433.1. 1 H NMR (400MHz, DMSO) δ11.67(s,1H),7.59(dd,J=13.3,6.9Hz,3H),7.42(d,J=7.4Hz,2H) ,6.83(s,1H),5.83(d,J=1.9Hz,1H),4.38(d,J=6.3Hz,2H),3.34(s,2H),1.38(s,9H).

[0092] Step 7: Synthesis of 5-(2-aminoethoxy)-7-chloro-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride

[0093] Add tert-butyl {2-[(7-chloro-2,4-dioxo-1-phenyl-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]ethyl}carbamate (2.4 g, 5.55 mmol, 1.0 eq) to hydrogen chloride / 1,4-dioxane solution (20 mL) and 1,4-dioxane (30 mL) and stir for 16 hours. After the reaction is completed, filter, wash the filter cake with methyl tert-butyl ether, and dry the filter cake to obtain a white solid 5-(2-aminoethoxy)-7-chloro-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (1.8 g, yield 88.1%). LCMS (TOF MS ES+) m / z [M+H] + :333.0. 1 H NMR (400MHz, D2O) δ7.67–7.57(m,3H),7.37–7.29(m,2H),6.32(d,J=1.0Hz,1H),4.70(s,2H),3.51–3.44(m,2H).

[0094] Step 8: Synthesis of 5-chloro-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one

[0095] 5-(2-Aminoethoxy)-7-chloro-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (200 mg, 0.54 mmol, 1.0 eq) and 1,8-diazabicyclo[5.4.0]undec-7-ene (247 mg, 1.62 mmol, 3.0 eq) were dissolved in N,N-dimethylformamide (5 mL), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (422 mg, 0.81 mmol, 1.5 eq) was added under ice bath and stirred for 1 hour. After the reaction, water and ethyl acetate were added for extraction, and the organic phases were combined and concentrated to obtain a white solid 5-chloro-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one (19 mg, yield 11.2%). LCMS (TOF MS ES+) m / z [M+H] + :315.0. 1 H NMR (400MHz, DMSO) δ8.67(t,J=6.2Hz,1H),7.64–7.49(m,3H),7.33–7.26(m,2H),5.90(s,1H),4.64(t,J=5.0Hz,2H),4.09(q,J=5.4Hz,2H).

[0096] Example 2: Synthesis of (S)-5-chloro-9-ethyl-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-naphthalene-2(8H)-one

[0097] Step 1: Synthesis of tert-butyl (S)-(1-hydroxybutan-2-yl)carbamate

[0098] (S)-2-aminobutan-1-ol (2.0 g, 22.44 mmol, 1.0 eq) and triethylamine (4.54 g, 44.88 mmol, 2.0 eq) were dissolved in methanol (20 mL), di-tert-butyl dicarbonate (5.14 g, 23.56 mmol, 1.05 eq) was added under ice bath, and stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to obtain yellow oily (S)-tert-butyl (1-hydroxybutan-2-yl)carbamate (4.1 g). 1H NMR(400MHz,DMSO-d6)δ6.39(d,J=7.8Hz,1H),4.50(t,J=5.4Hz,1H),3.34–3.17(m,3H ),1.57–1.47(m,1H),1.37(s,9H),1.23(dt,J=14.3,7.2Hz,1H),0.81(t,J=7.4Hz,3H).

[0099] Step 2: Synthesis of 5,7-dichloro-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione

[0100] Dissolve (S)-tert-butyl (1-hydroxybutan-2-yl)carbamate (677 g, 3.58 mmol, 1.1 eq) in tetrahydrofuran (10 mL), add sodium hydride (260 mg, 6.52 mmol, 2.0 eq) under ice bath, stir for 30 minutes, then add 5,7-dichloro-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (1.0 g, 3.26 mmol, 1.0 eq), stir for 1 hour. After the reaction was completed, saturated ammonium chloride solution was added to quench, the aqueous phase was extracted with ethyl acetate, the organic phases were combined and concentrated, and the mixture was separated and purified in the forward direction to obtain yellow solid (S)-{1-[(7-chloro-2,4-dioxo-1-phenyl-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]butan-2-yl}carbamic acid tert-butyl ester (1.11 g, yield 85.6%). LCMS (TOF MS ES+) m / z[M+H]+: 461.15.

[0101] Step 3: Synthesis of (S)-5-(2-aminobutoxy)-7-chloro-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride

[0102] (S)-{1-[(7-chloro-2,4-dioxo-1-phenyl-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]butan-2-yl}carbamic acid tert-butyl ester (700 g, 1.52 mmol, 1.0 eq) was added to a hydrogen chloride-1,4-dioxane solution (5.7 mL) and 1,4-dioxane (5 mL) and stirred for 16 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with methyl tert-butyl ether, and the filter cake was dried to obtain a white solid (S)-5-(2-aminobutoxy)-7-chloro-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (535 mg, yield 97.81%). LCMS(TOF MS ES+)m / z[M+H]+:361.1H NMR (400MHz, DMSO-d6) δ11.80(s,1H),8.21(s,3H),7.68–7.54(m,3H),7.44(m,J=5.5,1.7Hz,2H),5.91(s,1H),4.58(dd,J =11.5, 4.8Hz, 1H), 4.46 (dd, J = 11.5, 5.7Hz, 1H), 3.48 (t, J = 6.0Hz, 1H), 1.81 (m, J = 28.2, 7.1Hz, 2H), 1.00 (t, J = 7.5Hz, 3H).

[0103] Step 4: Synthesis of (S)-5-chloro-9-ethyl-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-naphthalen-2(8H)-one

[0104] (S)-5-(2-Aminobutoxy)-7-chloro-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (150 mg, 0.38 mmol, 1.0 eq) and 1,8-diazabicyclo[5.4.0]undec-7-ene (171 mg, 1.13 mmol, 3.0 eq) were dissolved in N,N-dimethylformamide (2.5 mL), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (294 mg, 0.57 mmol, 1.5 eq) was added under ice bath and stirred for 1 hour. After the reaction, water and ethyl acetate were added for extraction, and the organic phases were combined and concentrated, and the mixture was purified to obtain a white solid (S)-5-chloro-9-ethyl-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-naphthalene-2(8H)-one (6.3 mg, yield 5.0%). LCMS(TOF MS ES+)m / z[M+H]+:343.08.1H NMR (400MHz, DMSO-d6) δ8.13(d,J=9.0Hz,1H),7.65–7.56(m,2H),7.59–7.49(m,1H),7.37(d,J=8.0Hz,1H),7.29(d,J=7.6Hz,1H),5.94(s,1 H), 4.94 (d, J = 10.6Hz, 1H), 4.78 (d, J = 8.3Hz, 1H), 4.36 (dd, J = 10.8, 3.1Hz, 1H), 2.03–1.95 (m, 1H), 1.90–1.81 (m, 1H), 1.05 (t, J = 7.4Hz, 3H).

[0105] Example 3: Synthesis of 5-methyl-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one

[0106] Step 1: Synthesis of 5-methyl-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one

[0107] 5-Chloro-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one (30 g, 0.096 mmol, 1.0 eq), methylboric acid (11.4 mg, 0.19 mmol), tetrakis(triphenylphosphine)palladium (11 mg, 0.0096 mmol, 0.1 eq) and potassium carbonate (39.6 mg, 0.29 mmol, 3.0 eq) were dissolved in a mixed solution of 1,4-dioxane (1 mL) and water (0.2 mL). After nitrogen was replaced, the mixture was heated to 120° C. and stirred for 4 hours to prepare and purify 5-methyl-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one (18 mg, yield 64.29%) as a white solid. LCMS(TOF MS ES+)m / z[M+H]+:295.1H NMR(400MHz, DMSO-d6)δ8.67(t,J=6.1Hz,1H),7.65–7.45(m,3H),7.32–7.21( m, 2H), 5.83 (s, 1H), 4.64 (t, J = 4.9Hz, 2H), 4.08 (q, J = 5.4Hz, 2H), 2.24 (s, 3H).

[0108] Example 4: Synthesis of 5-cyclopropyl-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacyclohepta[de]naphthalene-2(8H)-one

[0109] Step 1: Synthesis of 2,6-dichloro-4-[(2-methylpyridin-3-yl)amino]nicotinamide

[0110] Dissolve 3-amino-2-methylpyridine (2.11 g, 19.6 mmol, 1.1 eq) in tetrahydrofuran (50 mL), add lithium bis(trimethylsilyl)amide (35.6 mL, 35.6 mmol, 2.0 eq) dropwise under ice bath, and stir for 30 minutes. Then add 2,4,6-trichlorononanamide (4.0 g, 17.8 mmol, 1.0 eq) and stir for 30 minutes. After the reaction is completed, add saturated ammonium chloride solution to quench, then add ethyl acetate to extract, combine and concentrate the organic phase, separate and purify by normal phase, and obtain yellow solid 2,6-dichloro-4-[(2-methylpyridin-3-yl)amino]nicotinamide (2.9 g, yield 54.8%). LCMS (TOF MS ES+) m / z [M+H] + :297.0. 1H NMR(400MHz, DMSO)δ8.48(s,1H),8.41(m,J=4.8,1.6Hz,1H),8.31–8.16(m,1H),7.95–7.6 9(m,1H),7.61(m,J=7.9,1.6Hz,1H),7.31(m,J=7.9,4.8Hz,1H),6.14(s,1H),2.35(s,3H).

[0111] Step 2: Synthesis of 5,7-dichloro-1-(2-methylpyridin-3-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione

[0112] 2,6-dichloro-4-[(2-methylpyridin-3-yl)amino]nicotinamide (2.85 g, 9.6 mmol, 1.0 eq) was added to tetrahydrofuran (50 mL), sodium hydride (768 mg, 19.2 mmol, 2.0 eq) was added under ice bath, stirred for 30 minutes, and then N,N'-carbonyldiimidazole (2.33 g, 14.4 mmol, 1.5 eq) was added, and the reaction was allowed to react at room temperature for 2 hours. After the reaction was completed, ice water was added to quench the reaction, and then saturated ammonium chloride solution was added to quench the reaction, and then ethyl acetate was added to extract, and the organic phase was combined and concentrated to obtain a yellow solid 5,7-dichloro-1-(2-methylpyridin-3-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (2.8 g, yield 90.5%). LCMS (TOF MS ES+) m / z [M+H] + :322.0. 1 H NMR (400MHz, DMSO) δ12.20(s,1H),8.74(dd,J=5.1,1.6Hz,1H),8.07–7.97(m,1H),7.67–7.63(m,1H),6.57(s,1H),2.39(s,3H).

[0113] Step 3: Synthesis of tert-butyl (2-{[7-chloro-1-(2-methylpyridin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl]oxy}ethyl)carbamate

[0114] Dissolve tert-butyl (2-hydroxyethyl)carbamate (1.54 g, 9.53 mmol, 1.1 eq) in tetrahydrofuran (40 mL), add sodium hydride (693 mg, 12.9 mmol, 17.3 eq) under ice bath, and stir for 30 minutes. Then add 5,7-dichloro-1-phenylpyridino[4,3-d]pyrimidine-2,4(1H,3H)-dione (2.8 g, 8.67 mmol, 1.0 eq) and stir for 1 hour. After the reaction is completed, add saturated ammonium chloride solution to quench, extract the aqueous phase with ethyl acetate, combine and concentrate the organic phases, and separate and purify in the forward direction to obtain a yellow solid (tert-butyl 2-{[7-chloro-1-(2-methylpyridin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydropyridino[4,3-d]pyrimidin-5-yl]oxy}ethyl)carbamate (1.1 g, yield 28.3%). LCMS (TOF MS ES+) m / z [M+H] + :448.2. 1 H NMR (400MHz, DMSO) δ11.82(s,1H),8.64(dd,J=4.8,1.6Hz,1H),7.84(dd,J=8.0,1.6Hz,1H),7.49(dd,J=8.0,4.8Hz ,1H),6.85(t,J=5.7Hz,1H),5.90(s,1H),4.38(M,J=11.0,5.5Hz,2H),3.41–3.34(m,2H),2.27(s,3H),1.38(s,9H).

[0115] Step 4: Synthesis of 5-(2-aminoethoxy)-7-chloro-1-(2-methylpyridin-3-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride

[0116] Tert-butyl (2-{[7-chloro-1-(2-methylpyridin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl]oxy}ethyl)carbamate (1.1 g, 2.46 mmol, 1.0 eq) was added to a hydrogen chloride / 1,4-dioxane solution (20 mL) and 1,4-dioxane (20 mL) and stirred for 16 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with methyl tert-butyl ether, and the filter cake was dried to obtain a white solid 5-(2-aminoethoxy)-7-chloro-1-(2-methylpyridin-3-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (1.0 g, yield 96.7%). LCMS (TOF MS ES+) m / z [M+H] + :348.0. 1H NMR (400MHz, DMSO) δ11.99(s,1H),8.84(dd,J=5.4,1.6Hz,1H),8.28(d,J=7.7Hz,4H),7.8 3(dd,J=8.1,5.3Hz,1H),6.34(s,1H),4.69–4.54(m,2H),3.66–3.56(m,2H),2.45(s,3H).

[0117] Step 5: Synthesis of 5-chloro-3-(2-methylpyridin-3-yl)-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacyclohepta[de]naphthalen-2(8H)-one

[0118] 5-(2-Aminoethoxy)-7-chloro-1-(2-methylpyridin-3-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (200 mg, 0.47 mmol, 1.0 eq) and 1,8-diazabicyclo[5.4.0]undec-7-ene (253 mg, 1.66 mmol, 3.5 eq) were dissolved in N,N-dimethylformamide (8 mL), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (366 mg, 0.71 mmol, 1.5 eq) was added under ice bath and stirred for 1 hour. After the reaction, water and ethyl acetate were added for extraction, and the organic phases were combined and concentrated, and the white solid 5-chloro-3-(2-methylpyridin-3-yl)-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane[de]naphthalene-2(8H)-one (8 mg, yield 5.2%) was obtained. LCMS (TOF MS ES+) m / z [M+H] + :330.0. 1 H NMR(400MHz,DMSO)δ8.75(t,J=6.1Hz,1H),8.61(dd,J=4.8,1.6Hz,1H),7.70(dt,J=8.2,2.0Hz,1H), 7.46 (dd, J = 7.9, 4.8 Hz, 1H), 5.96 (s, 1H), 4.65 (q, J = 6.3 Hz, 2H), 4.12 (q, J = 6.7 Hz, 2H), 2.21 (s, 3H).

[0119] Example 5: Synthesis of 5-chloro-10-methyl-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one

[0120] Step 1: Synthesis of tert-butyl {2-[(7-chloro-2,4-dioxo-1-phenyl-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]ethyl}(methyl)carbamate

[0121] Dissolve tert-butyl (2-hydroxyethyl)(methyl)carbamate (439.6 mg, 2.51 mmol, 1.1 eq) in tetrahydrofuran (15 mL), add sodium hydride (182 mg, 4.56 mmol, 2.0 eq) under ice bath, stir for 30 minutes, then add 5,7-dichloro-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (700 g, 2.28 mmol, 1.0 eq), stir for 1 hour. After the reaction was completed, saturated ammonium chloride solution was added to quench, the aqueous phase was extracted with ethyl acetate, the organic phases were combined and concentrated, and the normal phase was separated and purified, dichloromethane: methanol = 13:1, to obtain a white solid {2-[(7-chloro-2,4-dioxo-1-phenyl-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]ethyl}(methyl)carbamic acid tert-butyl ester (550 mg, yield 48.67%). LCMS (TOFMS ES+) m / z [M+H] + :447.13.1H NMR (400MHz, DMSO-d6) δ11.69(d,J=8.0Hz,1H),7.67–7.52(m,3H),7.41(d,J=7.5Hz,2H),5.83(s, 1H), 4.47 (d, J = 15.9Hz, 2H), 3.55 (d, J = 5.8Hz, 2H), 2.96 (d, J = 17.9Hz, 3H), 1.36 (d, J = 15.8Hz, 9H).

[0122] Step 2: Synthesis of {7-chloro-5-[2-(methylamino)ethoxy]-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride

[0123] Add tert-butyl {2-[(7-chloro-2,4-dioxo-1-phenyl-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]ethyl}(methyl)carbamate (550 g, 1.79 mmol, 1.0 eq) to a hydrogen chloride / 1,4-dioxane solution (8 mL) and tetrahydrofuran (8 mL) and stir for 16 hours. After the reaction is completed, filter, wash the filter cake with ether, and dry the filter cake to obtain a white solid 7-chloro-5-[2-(methylamino)ethoxy]-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (481 mg, yield 100%). LCMS (TOF MS ES+) m / z [M+H] + :347.08.1 H NMR(400MHz,DMSO-d6)δ11.80(s,1H),9.06(s,2H),7.69–7.54(m,3H),7.47–7.3 9(m,2H),5.90(s,1H),4.66(t,J=5.1Hz,2H),3.37(s,2H),2.76(d,J=3.8Hz,3H).

[0124] Step 3: Synthesis of 5-chloro-10-methyl-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one

[0125] 7-Chloro-5-[2-(methylamino)ethoxy]-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (100 mg, 0.26 mmol, 1.0 eq) and 1,8-diazabicyclo[5.4.0]undec-7-ene (118.5 mg, 0.78 mmol, 3.0 eq) were dissolved in N,N-dimethylformamide (2 mL), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (204 mg, 0.39 mmol, 1.5 eq) was added under ice bath and stirred for 1 hour. After the reaction, water and ethyl acetate were added for extraction, and the organic phases were combined and concentrated to obtain a white solid 5-chloro-10-methyl-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one (5.9 mg, yield 2.89%). LCMS (TOF MS ES+) m / z [M+H] + :329.07. 1 H NMR (400MHz, DMSO-d6) δ7.69-7.49(m,3H),7.35-7.22(m,2H),5.85(s,1H),4.76-4.58(m,2H),4.04-3.90(m,2H),3.28(s,3H).

[0126] Example 6: 5-Chloro-10-(2,2-difluoroethyl)-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacyclohepta[de]naphthalen-2(8H)-one

[0127] Step 1: Synthesis of tert-butyl (2-(benzyloxy)ethyl)carbamate

[0128] Dissolve 2-(Benzyloxy)ethane-1-amine (10g, 66.2mmol, 1.0eq) in methanol (100mL), then add triethylamine (20.1g, 198.7mmol, 3.0eq), slowly add di-tert-butyl dicarbonate (21.7g, 99.3mmol, 1.5eq) under ice bath conditions, and then stir for 2 hours. After the reaction is completed, add ethyl acetate and water for extraction, collect the organic layer, dry it with saturated brine and anhydrous sodium sulfate, and concentrate to obtain white solid [2-(benzyloxy)ethyl]carbamic acid tert-butyl ester (11.9g, yield 71.7%). LCMS (TOF MS ES+) m / z [M+H] + :252.

[0129] Step 2: Synthesis of tert-butyl [2-(benzyloxy)ethyl](2,2-difluoroethyl)carbamate

[0130] Add tert-butyl [2-(benzyloxy)ethyl]carbamate (11.9 g, 47.4 mmol, 1.0 eq) to ultra-dry tetrahydrofuran (100 mL), then add sodium hydrogen (3.4 g, 142.2 mmol, 3.0 eq) under ice bath conditions, stir for 10 minutes, add 1,1-difluoro-2-iodoethane (9.1 g, 47.4 mmol, 1.0 eq), and then react at room temperature for 3 hours. After the reaction is completed, add saturated ammonium chloride solution under ice bath conditions to quench the reaction solution, then add ethyl acetate to extract the liquid, collect the organic layer, dry it with saturated brine and anhydrous sodium sulfate, combine and concentrate the organic phase to obtain a crude brown oil [tert-butyl [2-(benzyloxy)ethyl)(2,2-difluoroethyl)carbamate (12.2 g, yield 81.9%). LCMS (TOF MS ES+) m / z [M+H] + :316.

[0131] Step 3: Synthesis of tert-butyl (2,2-difluoroethyl)(2-hydroxyethyl)carbamate

[0132] Dissolve [2-(benzyloxy)ethyl](2,2-difluoroethyl)carbamic acid tert-butyl ester (12.2g, 38.7mmol, 1.0eq) in methanol (50mL), then add palladium carbon (1.22g), and stir at room temperature for 16 hours under hydrogen protection. After the reaction is completed, filter the reaction liquid, wash the filter cake with methanol, concentrate the filtrate, and separate and purify with normal phase to obtain a transparent oily substance (2,2-difluoroethyl)(2-hydroxyethyl)carbamic acid tert-butyl ester (7.9g, yield 90.8%). LCMS (TOF MS ES+) m / z [M+H] + :226.

[0133] Step 4: Synthesis of tert-butyl {2-[(7-chloro-2,4-dioxo-1-phenyl-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]ethyl}(2,2-difluoroethyl)carbamate

[0134] Dissolve tert-butyl (2,2-difluoroethyl)(2-hydroxyethyl)carbamate (7.9 g, 35.1 mmol, 1.0 eq) in ultra-dry tetrahydrofuran (40 mL), add sodium hydride (1.69 g, 70.2 mmol, 2.0 eq) under ice bath, stir for 30 minutes, then add 5,7-dichloro-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (10.7 g, 35.1 mmol, 1.0 eq), stir for 1 hour. After the reaction was completed, saturated ammonium chloride solution was added to quench, the aqueous phase was extracted with ethyl acetate, the organic phases were combined and concentrated, and the forward separation and purification were performed to obtain a yellow solid {2-[(7-chloro-2,4-dioxo-1-phenyl-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]ethyl}(2,2-difluoroethyl)carbamic acid tert-butyl ester (9.6 g, yield 55.0%). LCMS (TOF MSES+) m / z [M+H] + :497.

[0135] Step 5: Synthesis of 7-chloro-5-{2-[(2,2-difluoroethyl)amino]ethoxy}-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride

[0136] tert-Butyl {2-[(7-chloro-2,4-dioxo-1-phenyl-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]ethyl}(2,2-difluoroethyl)carbamate (9.6 g, 19.4 mmol, 1.0 eq) was added to a hydrogen chloride / 1,4-dioxane solution (20 mL) and 1,4-dioxane (30 mL) and stirred for 16 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with methyl tert-butyl ether, and the filter cake was dried to obtain a white solid 7-chloro-5-{2-[(2,2-difluoroethyl)amino]ethoxy}-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (7.6 g, yield 90.5%). LCMS (TOF MS ES+) m / z [M+H] + :432.

[0137] Step 6: Synthesis of 5-chloro-10-(2,2-difluoroethyl)-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacyclohepta[de]naphthalen-2(8H)-one

[0138] 7-Chloro-5-{2-[(2,2-difluoroethyl)amino]ethoxy}-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (200 mg, 0.46 mmol, 1.0 eq) and 1,8-diazabicyclo[5.4.0]undec-7-ene (212 mg, 1.39 mmol, 3.0 eq) were dissolved in N,N-dimethylformamide (5 mL), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (362 mg, 0.70 mmol, 1.5 eq) was added under ice bath and stirred for 1 hour. After the reaction, water and ethyl acetate were added for extraction, and the organic phases were combined and concentrated to obtain a white solid 5-chloro-10-(2,2-difluoroethyl)-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane[de]naphthalene-2(8H)-one (25 mg, yield 14.3%). LCMS (TOF MS ES+) m / z [M+H] + :379.

[0139] Example 7: Synthesis of 5-cyclopropyl-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacyclohepta[de]naphthalene-2(8H)-one

[0140] Step 1: Synthesis of 4-(Benzo[d]thiazol-6-amino)-2,6-dichloronicotinamide

[0141] Dissolve benzo[d]thiazol-6-amine (2.0 g, 13.4 mmol, 0.75 eq) in tetrahydrofuran (30 mL), add lithium bis(trimethylsilyl)amide (26.8 mL, 26.8 mmol, 1.5 eq) dropwise under ice bath, and stir for 30 minutes. Then add 2,4,6-trichlorononanamide (4.0 g, 17.87 mmol, 1.0 eq) and stir for 60 minutes. After the reaction is completed, add saturated ammonium chloride solution to quench, then add ethyl acetate to extract, combine and concentrate the organic phase, and separate and purify with normal phase to obtain yellow solid 4-(benzo[d]thiazol-6-amino)-2,6-dichloronicotinamide (1.5 g, yield 25%). LCMS (TOF MS ES+) m / z [M+H] + :338.9.0. 1 H NMR (400MHz, DMSO-d6) δ9.36 (d, J = 0.7Hz, 1H), 8.80 (s, 1H), 8.22 (s, 1H), 8.14–8.03 (m, 2H), 7.86 (s, 1H), 7.41 (dd, J = 8.7, 2.2Hz, 1H), 6.76 (s, 1H).

[0142] Step 2: Synthesis of 1-(Benzo[d]thiazol-6-yl)-5,7-dichloropyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione

[0143] 4-(Benzo[d]thiazole-6-amino)-2,6-dichloronicotinamide (1.6 g, 4.74 mmol, 1.0 eq) was added to tetrahydrofuran (20 mL), sodium hydride (378 mg, 9.47 mmol, 2.0 eq) was added under ice bath, stirred for 30 minutes, and then N,N'-carbonyldiimidazole (1.15 g, 7.1 mmol, 1.5 eq) was added, and the reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, a saturated ammonium chloride solution was added to quench, and then ethyl acetate was added for extraction, the organic phases were combined and concentrated, and normal phase separation was performed, and dichloromethane:methanol = 20:1 to obtain a yellow solid 1-(benzo[d]thiazole-6-yl)-5,7-dichloropyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (1.28 g, yield 74.41%). LCMS (TOF MS ES+) m / z [M+H] + :364.9. 1 H NMR (400MHz, DMSO-d6) δ12.12(s,1H),9.59(s,1H),8.33(d,J=8.7Hz,1H),8.30(d,J=2.0Hz,1H),7.64-7.57(m,1H),6.47(s,1H).

[0144] Step 3: Synthesis of tert-butyl 2-{[1-(benzo[d]thiazol-6-yl)-7-chloro-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl]oxy}carbamate

[0145] Dissolve tert-butyl (2-hydroxyethyl)carbamate (602 g, 3.71 mmol, 1.1 eq) in tetrahydrofuran (15 mL), add sodium hydride (270 mg, 6.76 mmol, 2.0 eq) under ice bath, stir for 30 minutes, then add 1-(benzo[d]thiazol-6-yl)-5,7-dichloropyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (1.23 g, 3.38 mmol, 1.0 eq), stir for 2 hours. After the reaction was completed, saturated ammonium chloride solution was added to quench, the aqueous phase was extracted with ethyl acetate, the organic phases were combined and concentrated, and the yellow solid 2-{[1-(benzo[d]thiazol-6-yl)-7-chloro-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl]oxy}carbamic acid tert-butyl ester (835 mg, yield 45.62%) was obtained. LCMS (TOF MS ES+) m / z [M+H] + :490.08.1 H NMR (400MHz, DMSO-d6) δ11.74(s,1H),9.56(s,1H),8.40–8.16(m,2H),7.58(dd,J=8.7,2.0Hz,1H ), 6.86 (t, J = 5.8Hz, 1H), 5.97 (s, 1H), 4.38 (t, J = 6.1Hz, 2H), 3.34 (d, J = 6.9Hz, 2H), 1.38 (s, 9H).

[0146] Step 4: Synthesis of 5-(2-aminoethoxy)-1-(benzo[d]thiazol-6-yl)-7-chloropyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride

[0147] 2-{[1-(Benzo[d]thiazol-6-yl)-7-chloro-2,4-dioxo-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl]oxy}carbamic acid tert-butyl ester (830 g, 1.69 mmol, 1.0 eq) was added to a solution of hydrogen chloride / 1,4-dioxane (8 mL) and tetrahydrofuran (8 mL) and stirred for 16 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with ether, and the filter cake was dried to obtain a white solid 5-(2-aminoethoxy)-1-(benzo[d]thiazol-6-yl)-7-chloropyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (730 mg). LCMS (TOF MS ES+) m / z [M+H] + :390.

[0148] Step 5: Synthesis of 3-(Benzo[d]thiazol-6-yl)-5-chloro-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacyclohepta[d]naphthalen-2(8H)-one

[0149] 5-(2-Aminoethoxy)-1-(benzo[d]thiazol-6-yl)-7-chloropyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (240 mg, 0.56 mmol, 1.0 eq) and 1,8-diazabicyclo[5.4.0]undec-7-ene (256 mg, 1.68 mmol, 3.0 eq) were dissolved in N,N-dimethylformamide (2 mL). 1H-Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (440 mg, 0.84 mmol, 1.5 eq) was added under ice-cooling and stirred for 1 hour. After the reaction, water and ethyl acetate were added for extraction, and the organic phases were combined and concentrated to obtain a white solid 3-(benzo[d]thiazol-6-yl)-5-chloro-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacyclohepta[d]naphthalene-2(8H)-one (5.9 mg, yield 2.89%). LCMS (TOF MS ES+) m / z [M+H] + :372. 1 H NMR (400MHz, DMSO-d6) δ9.54(s,1H),8.73(t,J=6.1Hz,1H),8.27(d,J=8.6Hz,1H),8.18(d,J=2. 0Hz, 1H), 7.45 (dd, J = 8.6, 2.1Hz, 1H), 6.04 (s, 1H), 4.66 (t, J = 5.1Hz, 2H), 4.12 (d, J = 5.4Hz, 2H).

[0150] Example 8: Synthesis of 5-cyclopropyl-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacyclohepta[de]naphthalene-2(8H)-one

[0151] Step 1: Synthesis of 5-cyclopropyl-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacyclohepta[de]naphthalen-2(8H)-one

[0152] 5-Chloro-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one (40 mg, 0.127 mmol, 1.0 eq), cyclopropaneboric acid (21.9 mg, 0.254 mmol, 2.0 eq), tetrakis(triphenylphosphine)palladium (14.7 mg, 0.012 mmol, 0.1 eq) and potassium carbonate (52.7 mg, 0.382 mmol, 3.0 eq) were added to 1,4-dioxane (2 mL) and water (0.4 mL), the system was replaced with nitrogen, and the reaction was carried out at 120°C for 3 hours. After the reaction, water and dichloromethane were added for extraction, and the organic phases were combined and concentrated to obtain a white solid 5-cyclopropyl-3-phenyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane[de]naphthalene-2(8H)-one (17 mg, yield 41.7%). LCMS (TOF MS ES+) m / z [M+H] + :321.1. 1 H NMR (400MHz, DMSO) δ8.59(t,J=6.1Hz,1H),7.62–7.48(m,3H),7.30–7.23(m,2H),5.90(s,1H),4.52(t,J=5.0Hz ,2H),4.07(q,J=5.4Hz,2H),1.85(m,J=8.2,4.7Hz,1H),0.94(m,J=4.8,2.6Hz,2H),0.87(m,J=8.4,3.2Hz,2H).

[0153] Example 9: 5-Chloro-8-methyl-3-(3-methylpyridin-2-yl)-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacyclohepta[de]naphthalen-2(8H)-one

[0154] Step 1: Synthesis of 2,6-dichloro-4-[(3-methylpyridin-2-yl)amino]nicotinamide

[0155] Dissolve 2-amino-3-methylpyridine (3.48 g, 32.2 mmol, 1.1 eq) in tetrahydrofuran (30 mL), add lithium bis(trimethylsilyl)amide (35.1 mL, 35.1 mmol, 1.2 eq) dropwise under ice bath, and stir for 30 minutes. Then add 2,4,6-trichlorononanamide (6.6 g, 29.3 mmol, 1.0 eq) and stir for 30 minutes. After the reaction is completed, add saturated ammonium chloride solution to quench, then add ethyl acetate to extract, combine and concentrate the organic phase, and separate and purify by normal phase to obtain yellow solid 2,6-dichloro-4-[(3-methylpyridin-2-yl)amino]nicotinamide (4.5 g, yield 51.6%). LCMS (TOF MS ES+) m / z [M+H]+ :297.0.

[0156] Step 2: Synthesis of 5,7-dichloro-1-(3-methylpyridin-2-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione

[0157] 2,6-dichloro-4-(phenylamino)nicotinamide (3.25 g, 10.93 mmol, 1.0 eq) was added to tetrahydrofuran (50 mL), sodium hydride (874.6 mg, 21.8 mmol, 2.0 eq) was added under ice bath, stirred for 30 minutes, and then N,N'-carbonyldiimidazole (2.65 g, 16.4 mmol, 1.5 eq) was added, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, ice water was added to quench, and then dilute hydrochloric acid was added to adjust the pH to 2-3, and then ethyl acetate was added for extraction, and the organic phase was combined and concentrated to obtain a yellow solid 5,7-dichloro-1-phenylpyridine [4,3-d] pyrimidine-2,4 (1H, 3H) -dione (1.74 g, yield 49.29%). LCMS (TOF MS ES+) m / z [M+H] + :323.

[0158] Step 3: Synthesis of tert-butyl (2-hydroxypropyl)carbamate

[0159] Dissolve tert-butyl (2-hydroxypropyl)carbamate (3.0 g, 17.32 mmol, 1.0 eq) in methanol (20 mL), add sodium borohydride (720 mg, 19.05 mmol, 1.1 eq) under ice bath, and stir at room temperature for 1 hour. After the reaction is completed, concentrate the reaction solution to obtain colorless oily tert-butyl (2-hydroxypropyl)carbamate (2.74 g, yield 90.36%). LCMS (TOF MS ES+) m / z [M+H] + :175.12.

[0160] Step 4: Synthesis of tert-butyl {2-[(7-chloro-1-(3-methylpyridin-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl)oxy]propyl}carbamate

[0161] Dissolve tert-butyl (2-hydroxypropyl)carbamate (1.19 g, 6.81 mmol, 1.1 eq) in tetrahydrofuran (40 mL), add sodium hydride (297.1 mg, 12.38 mmol, 2.0 eq) under ice bath, stir for 30 minutes, then add 5,7-dichloro-1-(3-methylpyridin-2-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (2.0 g, 6.19 mmol, 1.0 eq), stir for 1 hour. After the reaction was completed, saturated ammonium chloride solution was added to quench, the aqueous phase was extracted with ethyl acetate, the organic phases were combined and concentrated, and the forward separation and purification were performed to obtain a yellow solid {2-[(7-chloro-1-(3-methylpyridin-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl)oxy]propyl}carbamic acid tert-butyl ester (1.63 g, yield 57.12%). LCMS (TOF MS ES+) m / z [M+H] + :462.15.

[0162] Step 5: Synthesis of 5-[(1-aminopropan-2-yl)oxy]-7-chloro-1-(3-methylpyridin-2-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride

[0163] 2-[(7-chloro-1-(3-methylpyridin-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl)oxy]propyl}carbamic acid tert-butyl ester (1.2 g, 2.6 mmol, 1.0 eq) was added to a hydrogen chloride / 1,4-dioxane solution (15 mL) and 1,4-dioxane (20 mL) and stirred for 14 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with methyl tert-butyl ether, and the filter cake was dried to obtain a white solid 5-[(1-aminopropan-2-yl)oxy]-7-chloro-1-(3-methylpyridin-2-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (0.883 g, yield 94.05%). LCMS (TOF MS ES+) m / z [M+H] + :362.09.

[0164] Step 6: Synthesis of 5-chloro-8-methyl-3-(3-methylpyridin-2-yl)-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacyclohepta[de]naphthalen-2(8H)-one

[0165] 5-[(1-aminopropan-2-yl)oxy]-7-chloro-1-(3-methylpyridin-2-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (350 mg, 0.97 mmol, 1.0 eq) and 1,8-diazabicyclo[5.4.0]undec-7-ene (313.6 mg, 2.91 mmol, 3.0 eq) were dissolved in N,N-dimethylformamide (5 mL), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (756.6 mg, 1.45 mmol, 1.5 eq) was added under ice-cooling and stirred for 1 hour. After the reaction, water and ethyl acetate were added for extraction, and the organic phases were combined and concentrated to obtain a white solid 5-chloro-8-methyl-3-(3-methylpyridin-2-yl)-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane[de]naphthalene-2(8H)-one (38.6 mg, yield 11.6%). LCMS (TOF MS ES+) m / z [M+H] + :344.1.

[0166] Example 10: 5-Chloro-3-(3-methylpyridin-2-yl)-9,10-dihydro-1,3,6,7,10-pentaazacycloheptadiene-2,8(3H,7H)-dione

[0167] Step 1: Synthesis of tert-butyl (2-amino-2-oxoethyl)carbamate

[0168] 2-Aminoacetamide (1.66 g, 22.44 mmol, 1.0 eq) and triethylamine (4.54 g, 44.88 mmol, 2.0 eq) were dissolved in methanol (20 mL), di-tert-butyl dicarbonate (5.14 g, 23.56 mmol, 1.05 eq) was added under ice bath, and stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to obtain yellow oily (2-amino-2-oxoethyl)carbamic acid tert-butyl ester (3.9 g, crude). LCMS (TOF MS ES + )m / z[M+H] + :175.1.

[0169] Step 2: Synthesis of tert-butyl {2-[(7-chloro-1-(3-methylpyridin-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl)amino]-2-oxoethyl}carbamate

[0170] Dissolve tert-butyl (2-amino-2-oxoethyl)carbamate (623 mg, 3.58 mmol, 1.1 eq) in tetrahydrofuran (10 mL), add sodium hydride (260 mg, 6.52 mmol, 2.0 eq) under ice bath, stir for 30 minutes, then add 5,7-dichloro-1-(3-methylpyridin-2-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (1.05 g, 3.26 mmol, 1.0 eq), stir for 1 hour. After the reaction was completed, saturated ammonium chloride solution was added to quench, the aqueous phase was extracted with ethyl acetate, the organic phases were combined and concentrated, and the mixture was separated and purified in the forward direction to obtain a yellow solid {2-[(7-chloro-1-(3-methylpyridin-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl)amino]-2-oxoethyl}carbamic acid tert-butyl ester (1.01 g, yield 67.3%). LCMS (TOFMS ES+) m / z [M+H] + :461.1.

[0171] Step 3: Synthesis of 2-amino-N-[7-chloro-1-(3-methylpyridin-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl]acetamide hydrochloride

[0172] Tert-butyl {2-[(7-chloro-1-(3-methylpyridin-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl)amino]-2-oxoethyl}carbamate (699 mg, 1.52 mmol, 1.0 eq) was added to a hydrogen chloride / 1,4-dioxane solution (5.7 mL) and 1,4-dioxane (5 mL) and stirred for 16 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with methyl tert-butyl ether, and the filter cake was dried to obtain a white solid 2-amino-N-[7-chloro-1-(3-methylpyridin-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl]acetamide hydrochloride (507 mg, yield 92.91%). LCMS (TOF MSES+) m / z [M+H] + :361.0.

[0173] Step 4: Synthesis of 5-chloro-3-(3-methylpyridin-2-yl)-9,10-dihydro-1,3,6,7,10-pentaazacycloheptadiene-2,8(3H,7H)-dione

[0174] 2-Amino-N-[7-chloro-1-(3-methylpyridin-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl]acetamide hydrochloride (150 mg, 0.38 mmol, 1.0 eq) and 1,8-diazabicyclo[5.4.0]undec-7-ene (171 mg, 1.13 mmol, 3.0 eq) were dissolved in N,N-dimethylformamide (2.5 mL), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (294 mg, 0.57 mmol, 1.5 eq) was added under ice bath and stirred for 1 hour. After the reaction, water and ethyl acetate were added for extraction, and the organic phases were combined and concentrated to obtain a white solid 5-chloro-3-(3-methylpyridin-2-yl)-9,10-dihydro-1,3,6,7,10-pentaazacycloheptadiene-2,8(3H,7H)-dione (14.2 mg, yield 11.3%). LCMS (TOF MS ES+) m / z [M+H] + :343.06.

[0175] Example 11: 5-Chloro-3-phenyl-10-(pyridin-2-ylmethyl)-9,10-dihydro-3H-7-ethanedioic acid-1,3,6,10-tetraazacycloheptane-2(8H)-one

[0176] Step 1: Synthesis of 2-(chloromethyl)pyridine

[0177] Pyridinemethanol (1.5 g, 13.75 mmol, 1.0 eq) and dichlorothionyl (1.64 g, 13.75 mmol, 1.0 eq) were condensed and refluxed at 90 ° C for 3 hours. After the reaction was completed, the reaction solution was concentrated, extracted with ethyl acetate and water for 3 times, and the organic phases were combined and concentrated to obtain 2-(chloromethyl)pyridine (1.50 g, 85.71% yield). LCMS (TOF MS ES+) m / z[M+H]+: 128.0235.

[0178] Step 2: Synthesis of tert-butyl [2-(benzyloxy)ethyl](pyridin-2-ylmethyl)carbamate

[0179] 2-(Chloromethyl)pyridine (1.50 g, 11.76 mmol, 1.1 eq) was dissolved in tetrahydrofuran (40 mL), sodium hydride (513 mg, 21.38 mmol, 2.0 eq) was added under ice bath, and stirred for 30 minutes. Subsequently, [2-ethyl (benzyloxy)] tert-butylcarbamic acid (2.69 g, 10.69 mmol, 1.0 eq) was added and stirred for 1 hour. After the reaction was completed, saturated ammonium chloride solution was added to quench, the aqueous phase was extracted with ethyl acetate, the organic phase was combined and concentrated, and the positive separation and purification were performed to obtain [2-(benzyloxy) ethyl] (pyridin-2-ylmethyl) carbamic acid tert-butyl ester (3.15 g, yield 86.07%). LCMS (TOF MS ES+) m / z[M+H]+: 343.1919.

[0180] Step 3 Synthesis of tert-butyl (2-hydroxyethyl) (pyridin-2-ylmethyl) carbamate

[0181] Dissolve tert-butyl [2-(benzyloxy)ethyl](pyridine-2-methylene)carbamate (3.00 g, 8.76 mmol, 1.0 eq) in methanol (40 mL), then add 10% palladium carbon catalyst (300 mg) and stir for 3 hours under hydrogen atmosphere. After the reaction, the catalyst is filtered through diatomaceous earth and washed twice with methanol, combined and concentrated to obtain tert-butyl (2-hydroxyethyl) (pyridin-2-ylmethyl)carbamate (2.00 g, yield 90.50%). LCMS (TOF MS ES+) m / z[M+H]+: 253.1520.

[0182] Step 4: Synthesis of tert-butyl {2-[(7-chloro-2,4-dioxo-1-phenyl-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]ethyl}(pyridin-2-ylmethyl)carbamate

[0183] Dissolve tert-butyl (2-hydroxyethyl)(pyridine-2-methylene)carbamate (1.15 g, 3.73 mmol, 1.0 eq) in tetrahydrofuran (40 mL), add sodium hydride (179 mg, 7.46 mmol, 2.0 eq) under ice bath, and stir for 30 minutes. Then add 5,7-dichloro-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (942 mg, 3.73 mmol, 1. 0eq), stirred for 1 hour. After the reaction was completed, saturated ammonium chloride solution was added to quench, the aqueous phase was extracted with ethyl acetate, the organic phase was combined and concentrated, and the forward separation and purification were performed to obtain a yellow solid {2-[(7-chloro-2,4-dioxo-1-phenyl-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]ethyl}(pyridin-2-ylmethyl)carbamic acid tert-butyl ester (1.64g, yield 83.67%). LCMS (TOF MS ES+) m / z[M+H]+: 524.1631.

[0184] Step 5: Synthesis of 7-chloro-1-phenyl-5-{2-[(pyridin-2-ylmethyl)amino]ethoxy}-pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride

[0185] Tert-butyl {2-[(7-chloro-2,4-dioxo-1-phenyl-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]ethyl}(pyridin-2-methylene)carbamate (2.4 g, 4.58 mmol, 1.0 eq) was added to a hydrogen chloride / 1,4-dioxane solution (20 mL) and 1,4-dioxane (30 mL) and stirred for 16 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with methyl tert-butyl ether, and the filter cake was dried to obtain a white solid 7-chloro-1-phenyl-5-{2-[(pyridin-2-ylmethyl)amino]ethoxy}-pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (1.7 g, yield 87.63%). LCMS (TOF MS ES+)m / z[M+H]+:424.1157.

[0186] Step 6: Synthesis of 5-chloro-3-phenyl-10-(pyridin-2-ylmethyl)-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one

[0187] 7-Chloro-1-phenyl-5-{2-[(pyridin-2-ylmethyl)amino]ethoxy}-pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (229 mg, 0.54 mmol, 1.0 eq) and 1,8-diazabicyclo[5.4.0]undec-7-ene (247 mg, 1.62 mmol, 3.0 eq) were dissolved in N,N-dimethylformamide (5 mL), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (422 mg, 0.81 mmol, 1.5 eq) was added under ice-cooling and stirred for 1 hour. After the reaction, water and ethyl acetate were added for extraction, and the organic phases were combined and concentrated, and the white solid 5-chloro-3-phenyl-10-(pyridin-2-ylmethyl)-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one (21 mg, yield 11.2%) was obtained. LCMS (TOF MS ES+) m / z[M+H]+: 406.1028.

[0188] Example 12: 3-(5-amino-2-fluorophenyl)-5-chloro-10-methyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacyclohepta[de]naphthalen-2(8H)-one

[0189] Step 1: Synthesis of 2,6-dichloro-4-[(2-fluoro-5-nitrophenyl)amino]nicotinamide

[0190] Dissolve 2-fluoro-5-nitroaniline (3.0 g, 19.23 mmol, 1.1 eq) in tetrahydrofuran (30 mL), add lithium bis(trimethylsilyl)amide (38.5 mL, 38.46 mmol, 2.0 eq) dropwise under ice bath, and stir for 30 minutes. Then add 2,4,6-trichlorononanamide (3.91 g, 17.48 mmol, 1.0 eq) and stir for 30 minutes. After the reaction is completed, add saturated ammonium chloride solution to quench, then add ethyl acetate to extract, combine and concentrate the organic phase, and separate and purify by normal phase to obtain yellow solid 2,6-dichloro-4-[(2-fluoro-5-nitrophenyl)amino]nicotinamide (4.1 g, yield 62%). LCMS (TOF MS ES+) m / z [M+H] + :344.98.

[0191] Step 2: Synthesis of 5,7-dichloro-1-(2-fluoro-5-nitrophenyl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione

[0192] 2,6-dichloro-4-[(2-fluoro-5-nitrophenyl)amino]nicotinamide (4.1 g, 11.92 mmol, 1.0 eq) was added to tetrahydrofuran (50 mL), sodium hydride (953.54 mg, 23.84 mmol, 2.0 eq) was added under ice bath, stirred for 30 minutes, and then N,N'-carbonyldiimidazole (2.89 g, 17.88 mmol, 1.5 eq) was added, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, ice water was added to quench, and then dilute hydrochloric acid was added to adjust the pH to 2-3, and then ethyl acetate was added for extraction, and the organic phase was combined and concentrated to obtain yellow solid 5,7-dichloro-1-(2-fluoro-5-nitrophenyl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (2.4 g, 54.55% yield). LCMS (TOF MS ES+) m / z [M+H] + :370.9.

[0193] Step 3: Synthesis of tert-butyl (2-{[7-chloro-1-(2-fluoro-5-nitrophenyl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl]oxy}ethyl)(methyl)carbamate

[0194] Dissolve tert-butyl (2-hydroxyethyl)(methyl)carbamate (1.14 g, 7.13 mmol, 1.1 eq) in tetrahydrofuran (40 mL), add sodium hydride (519 mg, 12.9 mmol, 2.0 eq) under ice bath, stir for 30 minutes, then add 5,7-dichloro-1-(2-fluoro-5-nitrophenyl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (2.4 g, 6.48 mmol, 1.0 eq), stir for 1 hour. After the reaction was completed, saturated ammonium chloride solution was added to quench, the aqueous phase was extracted with ethyl acetate, the organic phases were combined and concentrated, and the mixture was separated and purified in the forward direction to obtain a yellow solid (2-{[7-chloro-1-(2-fluoro-5-nitrophenyl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl]oxy}ethyl)(methyl)carbamic acid tert-butyl ester (2.4 g, yield 67.79%). LCMS (TOF MS ES+) m / z [M+H] + :510.1.

[0195] Step 4: Synthesis of 7-chloro-1-(2-fluoro-5-nitrophenyl)-5-[2-(methylamino)ethoxy]pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride

[0196] Tert-butyl (2-{[7-chloro-1-(2-fluoro-5-nitrophenyl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl]oxy}ethyl)(methyl)carbamate (2.4 g, 4.71 mmol, 1.0 eq) was added to a hydrogen chloride / 1,4-dioxane solution (8 mL) and 1,4-dioxane (8 mL) and stirred for 16 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with methyl tert-butyl ether, and the filter cake was dried to obtain a white solid 7-chloro-1-(2-fluoro-5-nitrophenyl)-5-[2-(methylamino)ethoxy]pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (1.8 g, yield 86.1%). LCMS (TOF MS ES+) m / z [M+H] + :409.03.

[0197] Step 5: Synthesis of 5-chloro-3-(2-fluoro-5-nitrophenyl)-10-methyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacyclohepta[de]naphthalen-2(8H)-one

[0198] 7-Chloro-1-(2-fluoro-5-nitrophenyl)-5-[2-(methylamino)ethoxy]pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (200 mg, 0.44 mmol, 1.0 eq) and 1,8-diazabicyclo[5.4.0]undec-7-ene (200 mg, 1.32 mmol, 3.0 eq) were dissolved in N,N-dimethylformamide (2 mL), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (343 mg, 0.66 mmol, 1.5 eq) was added under ice bath and stirred for 1 hour. After the reaction, water and ethyl acetate were added for extraction, and the organic phases were combined and concentrated to obtain a white solid 5-chloro-3-(2-fluoro-5-nitrophenyl)-10-methyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane[de]naphthalene-2(8H)-one (25 mg, yield 14.53%). LCMS (TOF MS ES+) m / z [M+H] + :392.04.

[0199] Step 6: Synthesis of 3-(5-amino-2-fluorophenyl)-5-chloro-10-methyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacyclohepta[de]naphthalen-2(8H)-one

[0200] 5-Chloro-3-(2-fluoro-5-nitrophenyl)-10-methyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptan[de]naphthalen-2(8H)-one (25 mg, 0.064 mmol, 1.0 eq), iron powder (35 mg, 0.64 mmol, 10.0 eq) and ammonium chloride (34 mg, 0.64 mmol, 10.0 eq) were dissolved in methanol (1 mL) and water (1 mL), stirred at room temperature for 2 hours, filtered, concentrated, extracted with ethyl acetate and water, and the organic phases were combined and concentrated to obtain 3-(5-amino-2-fluorophenyl)-5-chloro-10-methyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptan[de]naphthalen-2(8H)-one (10 mg, yield 43.49%) as a white solid. LCMS (TOF MS ES+) m / z [M+H] + :362.07.

[0201] Example 13: 5-Chloro-3-[5-(dimethylamino)-2-fluorophenyl]-10-methyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-naphthalen-2(8H)-one

[0202] 3-(5-Amino-2-fluorophenyl)-5-chloro-10-methyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacyclohepta[de]naphthalen-2(8H)-one (155 mg, 0.43 mmol, 1.0 eq) and potassium carbonate (178 mg, 1.29 mmol, 3.0 eq) were added to N,N-dimethylformamide (2.0 mL), and iodomethane (122 mg, 0.86 mmol, 2.0 eq) was added dropwise to the reaction system under stirring, and then placed in a sealed tube and reacted at 60°C for 2 hours. After the reaction was completed, potassium carbonate was removed by filtration, and the filtrate was purified by preparative purification to obtain a white solid 5-chloro-3-[5-(dimethylamino)-2-fluorophenyl]-10-methyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-naphthalene-2(8H)-one (35.6 mg, yield 21.32%). LCMS (TOF MS ES+) m / z [M+H] + :390.11.

[0203] Example 14: N-[3-(5-chloro-10-methyl-2-oxo-2,8,9,10-tetrahydro-3H-7-oxo-1,3,6,10-tetraazacycloheptyl[de]naphthalen-3-yl)-4-fluorophenyl]acetamide

[0204] 3-(5-Amino-2-fluorophenyl)-5-chloro-10-methyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacyclohepta[de]naphthalen-2(8H)-one (155 mg, 0.43 mmol, 1.0 eq) and triethylamine (130 mg, 1.29 mmol, 3.0 eq) were added to dichloromethane (2.0 mL), and acetyl chloride (51 mg, 0.65 mmol, 1.5 eq) was slowly added dropwise to the reaction system at 0°C, and then the mixture was heated to room temperature and stirred for 2 hours. After the reaction, the mixture was diluted with dichloromethane (50 mL), and then the organic phase was washed with water (50 mL x 3) and saturated brine (50 mL). The organic phase was dried, concentrated, and purified by preparative purification to obtain a white solid N-[3-(5-chloro-10-methyl-2-oxo-2,8,9,10-tetrahydro-3H-7-oxo-1,3,6,10-tetraazacycloheptane[de]naphthalen-3-yl)-4-fluorophenyl]acetamide (29.50 mg, yield 17.05%). LCMS (TOF MS ES+) m / z [M+H] + :404.08.

[0205] Example 15: 5-Chloro-3-(4-methoxybenzyl)-10-methyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one

[0206] Step 1: Synthesis of 2,6-dichloro-4-[(4-methoxybenzyl)amino]nicotinamide

[0207] Dissolve (4-methoxyphenyl)methylamine (5.00g, 36.5mmol, 1.1eq) in tetrahydrofuran (50mL), add lithium bis(trimethylsilyl)amide (39.8mL, 39.8mmol, 1.2eq) dropwise under ice bath, and stir for 30 minutes. Then add 2,4,6-trichlorononanamide (7.43g, 33.2mmol, 1.0eq) and stir for 30 minutes. After the reaction is completed, add saturated ammonium chloride solution to quench, then add ethyl acetate to extract, combine and concentrate the organic phase, and purify by normal phase separation to obtain yellow solid 2,6-dichloro-4-[(4-methoxybenzyl)amino]nicotinamide (5.64g, yield 52.3%). LCMS (TOF MS ES+) m / z [M+H] + :326.2.

[0208] Step 2: Synthesis of 5,7-dichloro-1-(4-methoxybenzyl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione

[0209] 2,6-dichloro-4-[(4-methoxybenzyl)amino]nicotinamide (5.62 g, 17.3 mmol, 1.0 eq) was added to tetrahydrofuran (50 mL), sodium hydride (1.24 g, 51.9 mmol, 3.0 eq) was added under ice bath, stirred for 30 minutes, and then N,N'-carbonyldiimidazole (5.61 g, 34.6 mmol, 2.0 eq) was added, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, ice water was added to quench, and then dilute hydrochloric acid was added to adjust the pH to 2-3, and then ethyl acetate was added for extraction, and the organic phase was combined and concentrated to obtain a yellow solid 5,7-dichloro-1-(4-methoxybenzyl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (3.27 g, yield 53.9%). LCMS (TOF MS ES+) m / z [M+H] + :352.2.

[0210] Step 3: Synthesis of tert-butyl {2-[(7-chloro-1-(4-methoxybenzyl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl)oxy]ethyl}(methyl)carbamate

[0211] Dissolve tert-butyl (2-hydroxyethyl)carbamate (1.64 g, 10.2 mmol, 1.1 eq) in tetrahydrofuran (55 mL), add sodium hydride (446 mg, 18.6 mmol, 2.0 eq) under ice bath, stir for 30 minutes, then add 5,7-dichloro-1-(4-methoxybenzyl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (3.25 g, 9.3 mmol, 1.0 eq), stir for 1 hour. After the reaction was completed, saturated ammonium chloride solution was added to quench, the aqueous phase was extracted with ethyl acetate, the organic phases were combined and concentrated, and the mixture was separated and purified in the forward direction to obtain a yellow solid {2-[(7-chloro-1-(4-methoxybenzyl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl)oxy]ethyl}(methyl)carbamic acid tert-butyl ester (4.08 g, yield 89.5%). LCMS (TOF MS ES+) m / z [M+H] + :491.1.

[0212] Step 4: Synthesis of 7-chloro-1-(4-methoxybenzyl)-5-[2-(methylamino)ethoxy]pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione

[0213] Add tert-butyl {2-[(7-chloro-1-(4-methoxybenzyl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl)oxy]ethyl}(methyl)carbamate (4.07 g, 8.30 mmol, 1.0 eq) to a solution of hydrogen chloride (40 mL) and 1,4-dioxane (60 mL) and stir for 16 hours. After the reaction is completed, filter, wash the filter cake with methyl tert-butyl ether, and dry the filter cake to obtain a white solid 7-chloro-1-(4-methoxybenzyl)-5-[2-(methylamino)ethoxy]pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (2.77 g, yield 85.7%). LCMS (TOF MS ES+) m / z [M+H] + :391.1.

[0214] Step 5: Synthesis of 5-chloro-3-(4-methoxybenzyl)-10-methyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one

[0215] 7-Chloro-1-(4-methoxybenzyl)-5-[2-(methylamino)ethoxy]pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (500 mg, 1.28 mmol, 1.0 eq) and 1,8-diazabicyclo[5.4.0]undec-7-ene (584 mg, 3.85 mmol, 3.0 eq) were dissolved in N,N-dimethylformamide (8 mL). 1H-Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (1.07 g, 1.92 mmol, 1.5 eq) was added under ice-cooling and stirred for 1 hour. After the reaction, water and ethyl acetate were added for extraction, and the organic phases were combined and concentrated to obtain a white solid 5-chloro-3-(4-methoxybenzyl)-10-methyl-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one (57 mg, yield 12.01%). LCMS (TOF MSES+) m / z [M+H] + :373.1.

[0216] Example 16: 5-Chloro-10-methyl-(3-thiazol-2-yl)-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane[de]naphthalen-2(8H)-one

[0217] Step 1: Synthesis of 2,6-dichloro-4-(thiazol-2-ylamino)nicotinamide

[0218] Dissolve 2-aminothiazole (3.22 g, 32.2 mmol, 1.1 eq) in tetrahydrofuran (30 mL), add lithium bis(trimethylsilyl)amide (35.1 mL, 35.1 mmol, 1.2 eq) dropwise under ice bath, and stir for 30 minutes. Then add 2,4,6-trichloronicotinamide (6.6 g, 29.3 mmol, 1.0 eq) and stir for 30 minutes. After the reaction is completed, add saturated ammonium chloride solution to quench, then add ethyl acetate to extract, combine and concentrate the organic phase, and purify by normal phase separation to obtain yellow solid 2,6-dichloro-4-(thiazol-2-ylamino)nicotinamide (3.86 g, yield 45.8%). LCMS (TOF MS ES+) m / z [M+H] + :288.9.

[0219] Step 2: Synthesis of 5,7-dichloro-1-(thiazol-2-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione

[0220] 2,6-dichloro-4-(thiazol-2-ylamino)nicotinamide (4.09 g, 14.2 mmol, 1.0 eq) was added to tetrahydrofuran (50 mL), sodium hydride (1.70 g, 42.5 mmol, 3.0 eq) was added under ice bath, stirred for 30 minutes, and then N,N'-carbonyldiimidazole (4.60 g, 28.3 mmol, 2.0 eq) was added, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, ice water was added to quench, and then dilute hydrochloric acid was added to adjust the pH to 2-3, and then ethyl acetate was added for extraction, and the organic phase was combined and concentrated to obtain a yellow solid 5,7-dichloro-1-(thiazol-2-yl)pyridine [4,3-d] pyrimidine-2,4 (1H, 3H) -dione (2.27 g, yield 51.0%). LCMS (TOF MS ES+) m / z [M+H] + :314.9.

[0221] Step 3: Synthesis of tert-butyl {2-[(7-chloro-2,4-dioxo-1-(thiazol-2-yl)-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]ethyl}carbamate

[0222] Dissolve tert-butyl (2-hydroxyethyl)carbamate (1.28 g, 7.95 mmol, 1.1 eq) in tetrahydrofuran (40 mL), add sodium hydride (347 mg, 14.46 mmol, 2.0 eq) under ice-cooling, and stir for 30 minutes. Then add 5,7-dichloro-1-(thiazol-2-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (2.27 g, 7.23 mmol, 1.0 eq), and stir for 1 hour. After the reaction was completed, saturated ammonium chloride solution was added to quench, the aqueous phase was extracted with ethyl acetate, the organic phases were combined and concentrated, and the forward separation and purification were performed to obtain a yellow solid {2-[(7-chloro-2,4-dioxo-1-(thiazol-2-yl)-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]ethyl}carbamic acid tert-butyl ester (2.62 g, 80.2% yield). LCMS (TOF MS ES+) m / z [M+H] + :454.0.

[0223] Step 4: Synthesis of 7-chloro-5-[2-(methylamino)ethoxy]-1-(thiazol-2-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride

[0224] Tert-butyl {(2-((7-chloro-2,4-dioxo-1-(thiazol-2-yl)-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy)ethyl)}carbamate (2.62 g, 5.78 mmol, 1.0 eq) was added to a hydrogen chloride / 1,4-dioxane solution (20 mL) and 1,4-dioxane (30 mL) and stirred for 16 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with methyl tert-butyl ether, and the filter cake was dried to obtain a white solid 7-chloro-5-[2-(methylamino)ethoxy]-1-(thiazol-2-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (1.91 g, yield 85.0%). LCMS (TOF MS ES+) m / z [M+H] + :390.0.

[0225] Step 5: Synthesis of 5-chloro-10-methyl-3-(thiazol-2-yl)-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane[de]naphthalen-2(8H)-one

[0226] 7-Chloro-5-(2-(methylamino)ethoxy)-1-(thiazol-2-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (200 mg, 0.51 mmol, 1.0 eq) and 1,8-diazabicyclo[5.4.0]undec-7-ene (233 mg, 1.53 mmol, 3.0 eq) were dissolved in N,N-dimethylformamide (5 mL), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (395 mg, 0.76 mmol, 1.5 eq) was added under ice-cooling and stirred for 1 hour. After the reaction, water and ethyl acetate were added for extraction, and the organic phases were combined and concentrated to obtain a white solid 5-chloro-10-methyl-(3-thiazol-2-yl)-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane [de] naphthalene-2(8H)-one (25.6 mg, yield 15.0%). LCMS (TOFMS ES+) m / z [M+H] + :336.0.

[0227] Example 17: 5-Chloro-10-methyl-3-(1-methyl-1H-imidazol-2-yl)-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane[de]naphthalen-2(8H)-one

[0228] Step 1: Synthesis of 2,6-dichloro-4-[(1-methyl-1H-imidazol-2-yl)amino)nicotinamide

[0229] 1-Methyl-1H-imidazole-2-amine (2.21 g, 22.4 mmol, 1.0 eq) was dissolved in tetrahydrofuran (100 mL), and after replacing nitrogen, it was cooled to 0°C, and lithium bis(trimethylsilyl)amide (23.40 mL, 26.88 mmol, 1.2 eq) was slowly added. At 0°C, it was stirred for 1 hour, and then 2,4,6-trichloronicotinamide (5.0 g, 22.4 mmol, 1.0 eq) was added to the reaction bottle, and then stirred for 2 hours. After the reaction was completed, dilute hydrochloric acid was added to quench the reaction, and then ethyl acetate was added for extraction. The organic phases were combined, dried with saturated brine and anhydrous sodium sulfate, respectively, and concentrated. The normal phase separation and purification gave 2,6-dichloro-4-[(1-methyl-1H-imidazole-2-yl)amino]nicotinamide (3.6 g, yield 56.9%). LCMS (TOF MS ES+) m / z [M+H] + :286.4.

[0230] Step 2: Synthesis of 5,7-dichloro-1-(1-methyl-1H-imidazol-2-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione

[0231] 2,6-dichloro-4-[(1-methyl-1H-imidazol-2-yl)amino]nicotinamide (3.61 g, 12.7 mmol, 1.0 eq) was dissolved in tetrahydrofuran (30 mL), followed by the addition of sodium hydride (917.7 mg, 38.2 mmol, 3.0 eq) and N,N-dicarbonyl imidazole (5.22 g, 31.75 mmol, 2.5 eq) and the reaction was continued for 3 hours. After the reaction was complete, the reaction solution was quenched with water and then extracted with ethyl acetate. The organic phases were combined and concentrated, and the normal phase separation and purification were performed to obtain 5,7-dichloro-1-(1-methyl-1H-imidazol-2-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (2.21 g, yield 56.0%). LCMS (TOF MS ES+) m / z [M+H] + :312.9.

[0232] Step 3: Synthesis of tert-butyl {2-[(7-chloro-1-(1-methyl-1H-imidazol-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]ethyl}(methyl)carbamate

[0233] 5,7-Dichloro-1-(1-methyl-1H-imidazol-2-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (2.21 g, 7.1 mmol, 1.0 eq) and sodium hydride (511.00 mg, 21.3 mmol, 3.0 eq) were dissolved in tetrahydrofuran (30 mL), and then tert-butyl (2-hydroxyethyl)carbamate (1.4 g, 8.5 mmol, 1.2 eq) was added dropwise and reacted at room temperature for 5 hours. After the reaction was completed, the mother liquor was concentrated and purified by normal phase separation to obtain solid {2-[(7-chloro-1-(1-methyl-1H-imidazol-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]ethyl}(methyl)carbamic acid tert-butyl ester (1.60 g, yield 49.8%). LCMS (TOF MS ES+) m / z [M+H] + :451.0.

[0234] Step 4: Synthesis of 7-chloro-1-(1-methyl-1H-imidazol-2-yl)-5-[2-(methylamino)ethoxy]pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione

[0235] Tert-butyl {2-[(7-chloro-1-(1-methyl-1H-imidazol-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]ethyl}(methyl)carbamate (1.60 g, 3.5 mmol, 1.0 eq) was dissolved in 1,4-dioxane (10.0 mL), and concentrated hydrochloric acid was added and stirred for 2 hours at room temperature. After the reaction was completed, the reaction solution was extracted with ethyl acetate and concentrated to obtain a colorless oily substance 7-chloro-1-(1-methyl-1H-imidazol-2-yl)-5-[2-(methylamino)ethoxy]pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (1.22 g, yield 99.7%). LCMS (TOF MS ES+) m / z [M+H] + :351.1.

[0236] Step 5: 5-Chloro-10-methyl-3-(1-methyl-1H-imidazol-2-yl)-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane[de]naphthalen-2(8H)-one

[0237] 7-Chloro-1-(1-methyl-1H-imidazol-2-yl)-5-[2-(methylamino)ethoxy]pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (198.0 mg, 0.51 mmol, 1.0 eq) was added to N,N-dimethylformamide (5 mL), followed by 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate (395 mg, 0.76 mmol, 1.5 eq) and 1,8-diazabicyclo[5.4.0]undec-7-ene (233 mg, 1.53 mmol, 3.0 eq), and the mixture was reacted at room temperature for 5 hours. After the reaction, the solid 5-chloro-10-methyl-3-(1-methyl-1H-imidazol-2-yl)-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane[de]naphthalen-2(8H)-one (15.6 mg, yield 9.2%) was obtained by preparation and purification. LCMS (TOF MS ES+) m / z [M+H] + :333.9.

[0238] Example 18: Synthesis of 5-chloro-10-methyl-3-(1-methyl-1H-pyrazol-4-yl)-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one

[0239] Step 1: Synthesis of 2,6-dichloro-4-[(1-methyl-1H-pyrazol-4-yl)amino]nicotinamide

[0240] 1-Methyl-1H-pyrazol-4-amine (3.54 g, 36.5 mmol, 1.1 eq) was dissolved in tetrahydrofuran (50 mL), and lithium bis(trimethylsilyl)amide (39.8 mL, 39.8 mmol, 1.2 eq) was added dropwise under ice bath, and stirred for 30 minutes. Then 2,4,6-trichlorononanamide (7.43 g, 33.2 mmol, 1.0 eq) was added and stirred for 30 minutes. After the reaction was completed, saturated ammonium chloride solution was added to quench, and then ethyl acetate was added for extraction, the organic phases were combined and concentrated, and the normal phase was separated and purified to obtain a yellow solid 2,6-dichloro-4-[(1-methyl-1H-pyrazol-4-yl)amino]nicotinamide (5.87 g, yield 62.0%). LCMS (TOF MS ES+) m / z [M+H] + :286.0.

[0241] Step 2: Synthesis of 5,7-dichloro-1-(1-methyl-1H-pyrazol-4-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione

[0242] 2,6-dichloro-4-[(1-methyl-1H-pyrazol-4-yl)amino]nicotinamide (5.50 g, 19.3 mmol, 1.0 eq) was added to tetrahydrofuran (50 mL), sodium hydride (1.39 g, 57.9 mmol, 3.0 eq) was added under ice bath, stirred for 30 minutes, and then N,N'-carbonyldiimidazole (6.3 g, 38.6 mmol, 2.0 eq) was added, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, ice water was added to quench, and then dilute hydrochloric acid was added to adjust the pH to 2-3, and then ethyl acetate was added for extraction, and the organic phase was combined and concentrated to obtain a yellow solid 5,7-dichloro-1-(1-methyl-1H-pyrazol-4-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (3.08 g, yield 51.3%). LCMS (TOF MS ES+) m / z [M+H] + :312.0.

[0243] Step 3: Synthesis of tert-butyl {2-[(7-chloro-1-(1-methyl-1H-pyrazol-4-yl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl)oxy]ethyl}(methyl)carbamate

[0244] Dissolve tert-butyl (2-hydroxyethyl)carbamate (1.96 g, 10.4 mmol, 1.1 eq) in tetrahydrofuran (55 mL), add sodium hydride (454 mg, 18.9 mmol, 2.0 eq) under ice-cooling, and stir for 30 minutes. Then add 5,7-dichloro-1-(1-methyl-1H-pyrazol-4-yl)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (2.95 g, 9.5 mmol, 1.0 eq) and stir for 1 hour. After the reaction was completed, saturated ammonium chloride solution was added to quench, the aqueous phase was extracted with ethyl acetate, the organic phases were combined and concentrated, and the mixture was separated and purified in the forward direction to obtain a yellow solid {2-[(7-chloro-1-(1-methyl-1H-pyrazol-4-yl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl)oxy]ethyl}(methyl)carbamic acid tert-butyl ester (3.78 g, yield 88.4%). LCMS (TOF MS ES+) m / z [M+H] + :451.1.

[0245] Step 4: Synthesis of 7-chloro-1-(1-methyl-1H-pyrazol-4-yl)-5-[2-(methylamino)ethoxy]pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione

[0246] tert-Butyl {2-[(7-chloro-1-(1-methyl-1H-pyrazol-4-yl)-2,4-dioxo-1,2,3,4-tetrahydropyridin[4,3-d]pyrimidin-5-yl)oxy]ethyl}(methyl)carbamate (3.71 g, 8.24 mmol, 1.0 eq) was added to a solution of hydrogen chloride (40 mL) and 1,4-dioxane (60 mL) and stirred for 16 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with methyl tert-butyl ether, and the filter cake was dried to obtain a white solid 7-chloro-1-(1-methyl-1H-pyrazol-4-yl)-5-[2-(methylamino)ethoxy]pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (2.54 g, yield 88.1%). LCMS (TOF MS ES+) m / z [M+H] + :351.1.

[0247] Step 5: Synthesis of 5-chloro-10-methyl-3-(1-methyl-1H-pyrazol-4-yl)-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one

[0248] 7-Chloro-1-(1-methyl-1H-pyrazol-4-yl)-5-[2-(methylamino)ethoxy]pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (179 mg, 0.51 mmol, 1.0 eq) and 1,8-diazabicyclo[5.4.0]undec-7-ene (233 mg, 1.53 mmol, 3.0 eq) were dissolved in N,N-dimethylformamide (10 mL). 1H-Benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (395 mg, 0.76 mmol, 1.5 eq) was added under ice-cooling and stirred for 1 hour. After the reaction, water and ethyl acetate were added for extraction, and the organic phases were combined and concentrated to obtain a white solid 5-chloro-10-methyl-3-(1-methyl-1H-pyrazol-4-yl)-9,10-dihydro-3H-7-oxa-1,3,6,10-tetraazacycloheptane-2-(8H)-one (20.3 mg, yield 12.0%). LCMS (TOF MS ES+) m / z [M+H] + :333.1.

[0249] Example 19: (S)-5-chloro-3-phenyl-8a,9,10,11-tetrahydro-8H-7-oxa-1,3,6,11a-tetraazanaphtho[1,8-ef]azur-2(3H)-one

[0250] Step 1: Synthesis of tert-butyl (S)-2-(hydroxymethyl)pyrrolidine-1-carboxylate

[0251] Dissolve L-prolinol (2.0 g, 19.8 mmol, 1.0 eq) and triethylamine (4.0 g, 39.6 mmol, 2.0 eq) in methanol (50 mL), add di-tert-butyl dicarbonate (4.8 g, 21.8 mmol, 1.1 eq) under ice bath, and stir at room temperature for 1 hour. After the reaction is completed, concentrate the reaction solution to obtain a colorless oily substance (S)-2-(hydroxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (3.98 g, yield 100%). LCMS (TOF MS ES+) m / z [M+H] + :202.1.

[0252] Step 2: Synthesis of (S)-tert-butyl 2-{[(7-chloro-2,4-dioxo-1-phenyl-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]methyl}pyrrolidine-1-carboxylate

[0253] Dissolve (S)-tert-butyl 2-(hydroxymethyl)pyrrolidine-1-carboxylate (1.44 g, 7.14 mmol, 1.1 eq) in tetrahydrofuran (40 mL), add sodium hydride (519 mg, 12.9 mmol, 2.0 eq) under ice bath, stir for 30 minutes, then add 5,7-dichloro-1-phenylpyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (2.0 g, 6.49 mmol, 1.0 eq), stir for 1 hour. After the reaction was completed, saturated ammonium chloride solution was added to quench, the aqueous phase was extracted with ethyl acetate, the organic phases were combined and concentrated, and the positive separation and purification were performed to obtain a yellow solid (S)-2-{[(7-chloro-2,4-dioxo-1-phenyl-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]methyl}pyrrolidine-1-carboxylic acid tert-butyl ester (2.49 g, yield 81.2%). LCMS (TOF MS ES+) m / z [M+H] + :473.1.

[0254] Step 3: Synthesis of (S)-7-chloro-1-phenyl-5-(pyrrolidin-2-ylmethoxy)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride

[0255] (S)-2-{[(7-chloro-2,4-dioxo-1-phenyl-1,2,3,4-tetrahydropyrido[4,3-d]pyrimidin-5-yl)oxy]methyl}pyrrolidine-1-carboxylic acid tert-butyl ester (2.4 g, 5.07 mmol, 1.0 eq) was added to a hydrogen chloride / 1,4-dioxane solution (20 mL) and 1,4-dioxane (20 mL) and stirred for 16 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with methyl tert-butyl ether, and the filter cake was dried to obtain a white solid (S)-7-chloro-1-phenyl-5-(pyrrolidin-2-ylmethoxy)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (1.78 g, yield 85.6%). LCMS (TOF MS ES+) m / z [M+H] + :373.0.

[0256] Step 4: Synthesis of (S)-5-chloro-3-phenyl-8a,9,10,11-tetrahydro-8H-7-oxa-1,3,6,11a-tetraazanaphtho[1,8-ef]azur-2(3H)-one

[0257] (S)-7-Chloro-1-phenyl-5-(pyrrolidin-2-ylmethoxy)pyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride (200 mg, 0.49 mmol, 1.0 eq) and 1,8-diazabicyclo[5.4.0]undec-7-ene (223 mg, 1.47 mmol, 3.0 eq) were dissolved in N,N-dimethylformamide (6 mL), 1-propylphosphonic anhydride (234 mg, 0.74 mmol, 1.5 eq) was added under ice-cooling and stirred for 1 hour. After the reaction, water and ethyl acetate were added for extraction, and the organic phases were combined and concentrated to obtain a white solid (S)-5-chloro-3-phenyl-8a,9,10,11-tetrahydro-8H-7-oxa-1,3,6,11a-tetraazanaphtho[1,8-ef]azur-2(3H)-one (23.9 mg, yield 13.8%). LCMS (TOF MS ES+) m / z [M+H] + :355.0.

[0258] Experimental Example 1: Test of the binding ability of the compound of the present invention to mat2a protein

[0259] Experimental purpose: To detect the binding ability of the compound to MAT2A protein by CETSA experimental method

[0260] Background principle: The CETSA experiment is a molecular detection method to measure the affinity between drugs and target proteins. The principle is that after the drug binds to the target protein, its structure becomes more stable. Use candidate drugs to treat cell or tissue samples. If the candidate drug is an inhibitor of MAT2A, then the candidate drug can bind to MAT2A, making the MAT2A protein more stable. After heating the sample, the MAT2A protein in the sample will be more easily detected by specific antibodies, and the MAT2A protein will be easier to detect through Western blot experiments; conversely, the stability of the MAT2A protein will be worse after heating, and the amount of protein detected will be lower. In this way, the binding ability of the drug to the target protein is evaluated, which is used for the screening of MAT2A protein inhibitors.

[0261] Specific experimental process:

[0262] Take HCT116 cells in the logarithmic growth phase (cell viability>90%), wash the cells 3 times with PBS, centrifuge at 2000g for 2min; lyse on ice for 30min with cell lysis solution containing protease inhibitor PMSF; use BCA kit to measure the concentration of protein samples. Incubate the samples with candidate drugs, control drugs and control reagents for 30min, heat each group of samples at about 10 set temperature points; restore room temperature, centrifuge the samples at 20000g, collect the supernatant; use sample buffer to heat and denature the protein samples at 100℃ for 10min. After the samples return to room temperature, perform western blot detection on the samples; the protein loading amount is controlled at 20ug. After determining the mutation temperature, the compound concentration gradient is generally set to 9 points, the samples are incubated, and the same operation is performed as above for western blot detection. Protein electrophoresis: the voltage of the concentrated gel is set to 60v, and the voltage of the separation gel is set to 120v; after the electrophoresis is completed, start electrotransfer. The conditions of electroporation were set at 250 mA for 2 hours; 5% BSA was blocked for 1 hour; specific antibody 1 was added and incubated overnight on a shaker at 4°C; TBST was washed 4 times, 2.5 minutes each time; 2 antibodies were incubated on a shaker at room temperature for 1 hour; TBST was washed 4 times, 2.5 minutes each time; ECL was used for development to detect the expression of TYK2 protein in different groups and at various temperature points. Western blot bands were converted and processed by image J and GraphPad software, and EC50 was calculated.

[0263] EC50 is the concentration for 50% of maximal effect, which refers to the drug concentration that can cause 50% of individuals to be effective. AG270 (Compound 153 reported in CN109890822A) of Angios Pharmaceuticals Co., Ltd. was used as a positive reference compound.

[0264] The EC50 values ​​of each compound are classified according to the following description:

[0265] “+” indicates that the EC50 value is greater than 100 μM;

[0266] “++” indicates that the EC50 value is less than 100 μM and greater than 10 μM;

[0267] "+++" indicates that the EC50 value is less than 10 μM.

[0268] Table 1: EC50 results

[0269] Compound No. EC50 1 ++ 2 +++ 3 ++ 4 +++ 5 ++ 6 +++ 7 ++ 8 ++ 9 +++ 10 ++ 11 +++ 12 ++ 13 ++ 14 +++ 15 ++ 16 +++ 17 ++ 18 +++ 19 ++ Positive drug AG270 ++

[0270] EC50 experimental data show that the compounds of the present invention have good binding ability with MAT2A protein. Compared with the positive control drug AG270 (prepared according to the preparation method of compound 153 reported in CN109890822A), the compounds of the present invention have comparable or even stronger binding ability with MAT2A protein.

[0271] Experimental Example 2: Effect of the compounds of the present invention on HCT116 MTAP - / - Cell proliferation assay

[0272] Experimental purpose: The purpose of this test case is to test the effect of compounds on HCT116 MTAP - / - Cell proliferation effect.

[0273] Background: Methionine adenosyltransferase 2A (MAT2A) is considered a synthetic lethal target in cancers that lack the methylthioadenosine phosphorylase (MTAP) gene. The MTAP gene is adjacent to the CDKN2A tumor suppressor and is co-deleted with CDKN2A in approximately 15% of cancers. Therefore, the effects of compounds on HCT116 MTAP were tested. - / - The inhibition rate of cell proliferation is used for screening of MAT2A protein inhibitors.

[0274] Specific experimental process:

[0275] Construct HCT116 MTAP knockout cells and screen monoclones. - / - and WT cells, inoculated in 96-well plates, 90uL per well, 1000 cells / well, and incubated overnight at 37°C. On the second day, 10μl of compounds of different concentrations (DMSO final concentration was 1%) were added and incubated at 37°C for 10 days. On the 10th day, the old culture medium was removed, 110ul of culture medium (the ratio of culture medium to CCK8 was 100:10) was added, and incubated at 37°C for 1-4h. The absorbance was detected at 450nM, and the IC50 was calculated by GraphPad software. The compounds were screened by comparing with positive drugs.

[0276] IC50 (half maximal inhibitory concentration) refers to the half inhibitory concentration of the measured antagonist. It indicates the half amount of a drug or substance (inhibitor) in inhibiting a certain biological process (or certain substances contained in this process, such as enzymes, cell receptors or microorganisms). AG270 from Angios Pharmaceuticals Co., Ltd. was used as a positive reference compound.

[0277] The test results are shown in Table 2 below, where the IC50 values ​​of each compound are classified according to the following description:

[0278] +++ less than 100 nM, ++ between 100 nM and 10 μM, + greater than 10 μM.

[0279] The test results are shown in Table 2 below:

[0280] Table 2: IC50 experimental data

[0281]

[0282] The results show that the compounds of the present invention can inhibit HCT116 MTAP-null cells, and the IC50 value reaches nM level, which is equivalent to or less than the positive control drug AG270. This strong inhibitory effect has important therapeutic significance for the treatment of symptoms or diseases related to MAT2A inhibition.

[0283] Experimental Example 3: Determination of the effect of the compounds of the present invention on the function of MAT2A protease

[0284] Experimental purpose: The purpose of this test case is to test the ability of the compound to inhibit the function of MAT2A protease.

[0285] Background Principle: The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) plays an important role in metabolism and epigenetics because it is the main producer of the universal methyl donor s-adenosylmethionine (SAM). ATP and L-Met generate SAM and phosphate groups under the action of MAT2A. Therefore, after compound incubation, the ability of the compound to inhibit the enzyme function of MAT2A is evaluated by detecting the amount of SAM generated, which is used for the screening of MAT2A protein inhibitors.

[0286] Specific experimental process:

[0287] MAT2A protein expression: Full-length MAT2A was cloned into the pET24N vector with an n-terminal (His) 6x tag and a tobacco etch virus (TEV) protease cleavage site. The constructed vector was transformed into Escherichia coli BL21 (DE3), and the cells were shaken to an OD of 0.6, and 1mM IPTG was added and cultured at 18°C ​​for 16h. The cells were collected, ultrasonically disrupted, and the supernatant was taken by centrifugation. The protein was purified by Ni-NTA, and the protein concentration and purity were determined after dialysis. SAM assay: Reaction system: add 91-x ul of 50mM TrisHCl pH 7.5, 1.5uL (10 / 3M) of KCl, 1.5uL (1M) of MgCl2, 1uL (100mM) of ATP, 1uL (80mM) of L-Met, 1uL (30Mm PH7.67) of EDTA, 1uL 5% BSA, 2uL of the above DMSO-dissolved drugs, x uL of MAT2A protein. The experimental groups were prepared with different drug concentrations, 2uL (100x) was taken out and added to the reaction system, reacted at 37°C for 18h, and solvent control group and blank control group were set up respectively. The reaction was terminated, 40uL of the system was taken out, and 4uL 10% SDS was added to quench the reaction. IC50 was calculated by GraphPad software, and the compounds were screened by comparing with positive drugs.

[0288] The test results are shown in Table 3 below, where the IC50 values ​​of each compound are classified according to the following description:

[0289] +++ is less than 10 nM, ++ is between 10 nM and 1 μM, and + is greater than 1 μM.

[0290] The test results are shown in Table 3 below:

[0291] Table 3: IC50 experimental data

[0292] Compound No. IC50 1 ++ 2 +++ 3 ++ 4 ++ 5 ++ 6 ++ 7 +++ 8 + 9 ++ 10 +++ 11 ++ 12 ++ 13 ++ 14 ++ 15 ++ 16 +++ 17 ++ 18 +++ 19 ++ AG270 ++

[0293] The results show that the compounds of the present invention have a very strong inhibitory effect on the protease function of MAT2A, and the IC50 values ​​all reach the nM level, which is lower than the positive control drug AG270. This strong inhibitory effect has important therapeutic significance for the treatment of symptoms or diseases related to MAT2A inhibition.

[0294] The above examples are only representative. It can be seen from the above examples that the compounds of the present invention are ideal and highly effective MAT2A inhibitors, and can be expected to be used for treating or preventing symptoms or diseases related to MAT2A inhibition.

[0295] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, and they should be covered by the protection scope of the present invention.

Claims

1. A compound represented by formula (I) and its isomers, or a pharmaceutically acceptable salt thereof, in: X is selected from N or O; R1 is selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl; R2 is selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, -C1-C6 alkylene-R a ; R a is selected from 6-10 membered aryl, 5-10 membered heteroaryl, optionally substituted with one or more substituents selected from the following: hydroxyl, halogen, NH2, CN, C1-C6 alkyl, C1-C6 alkoxy; or R1, R2 together with the carbon and nitrogen to which they are attached form a 4-6 membered heterocyclic group, wherein the 4- to 6-membered heterocyclic group may be optionally substituted by one or more substituents, each of which is independently selected from the group consisting of halogen, cyano, oxo, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl; R3 is selected from 6-10 membered aryl, 5-10 membered heteroaryl, -C1-C6 alkylene-R b The 6-10 membered aryl group and the 5-10 membered heteroaryl group are optionally substituted with one or more substituents selected from the following: hydroxyl, halogen, -NR c R d , -CN, -NHC(O)C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy; R b is selected from 6-10 membered aryl, 5-10 membered heteroaryl, optionally substituted with one or more substituents selected from the following: hydroxyl, halogen, - NH2, -CN, C1-C6 alkyl, C1-C6 alkoxy; R c , R d are independently selected at each occurrence from hydrogen, C1-C6-alkyl; R4 is selected from hydrogen, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 haloalkyl; R5 is selected from hydrogen, oxo, halogen, C1-C6-alkyl, C1-C6-alkoxy, C1-C6-haloalkyl.

2. The compound according to claim 1, its isomer, or its pharmaceutically acceptable salt, characterized in that: The R1 is selected from hydrogen, methyl and ethyl.

3. The compound according to claim 1-2 and its isomers, or pharmaceutically acceptable salts thereof, characterized in that: The R2 is selected from hydrogen, difluoroethane, methyl, -methylene-2-pyridine, and ethyl.

4. The compound according to claim 1-3 and its isomers, or pharmaceutically acceptable salts thereof, characterized in that: The R1, R2 together with the carbon and nitrogen to which they are attached form a 5-membered heterocyclic group, and the 5-membered heterocyclic group is preferably selected from azacyclopentane.

5. The compound according to claim 1-4 and its isomers, or pharmaceutically acceptable salts thereof, characterized in that: The R3 is selected from 6. The compound according to claim 1-5 and its isomers, or pharmaceutically acceptable salts thereof, characterized in that: The R4 is selected from C1-C6 alkyl, halogen, cyclopropane, preferably methyl, chlorine, cyclopropane.

7. The compound according to claim 1-6 and its isomers, or pharmaceutically acceptable salts thereof, characterized in that: The R5 is selected from hydrogen, methyl, and oxo.

8. A compound represented by the following formula (Ia), (Ib) and its isomers, or a pharmaceutically acceptable salt thereof, R1 is selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl; R2 is selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, -C1-C6 alkylene-R a ; R a Selected from 6-10 membered aryl, 5-10 membered heteroaryl, optionally substituted with one or more substituents selected from the following: hydroxyl, halogen, NH2, CN, C1-C6 alkyl, C1-C6 alkoxy; Or R1, R2 together with the carbon and nitrogen to which they are attached form an azacyclopentane; R3 is selected from 6-10 membered aryl, 5-10 membered heteroaryl, -C1-C6 alkylene-R b The 6-10 membered aryl group and the 5-10 membered heteroaryl group are optionally substituted with one or more substituents selected from the following: hydroxyl, halogen, -NR c R d , -CN, -NHC(O)C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkoxy; R b is selected from 6-10 membered aryl, 5-10 membered heteroaryl, optionally substituted with one or more substituents selected from the following: hydroxyl, halogen, -NH2, -CN, C1-C6 alkyl, C1-C6 alkoxy; R c , R d are independently selected at each occurrence from hydrogen, C1-C6-alkyl; R4 is selected from hydrogen, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 haloalkyl; R5 is selected from hydrogen, oxo, halogen, C1-C6-alkyl, C1-C6-alkoxy, C1-C6-haloalkyl.

9. A compound and its isomers, or a pharmaceutically acceptable salt thereof, wherein the compound is:

10. Use of a compound according to any one of claims 1 to 9 and its isomers, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating a disease or condition in a subject suffering from the disease or condition, wherein the disease or condition is mediated by overexpression of MAT2A, the disease or condition is preferably cancer, and the cancer is preferably characterized by a reduction or deletion of methylthioadenosine phosphorylase (MTAP) gene expression, a deletion of the MTAP gene, or a reduction in MTAP protein function.

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