Fused tricyclic compound and pharmaceutical composition and application thereof

Abstract

The invention provides a fused tricyclic compound as shown in a formula (I), and a pharmaceutical composition and application thereof. The compound provided by the invention has high selectivity to PARP1, has fewer side effects than oaparib, has high clinical application value, and can be used for preparing medicines for preventing and / or treating diseases, especially diseases improved by inhibiting PARP1. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of PARP inhibitor synthesis, and in particular to a class of fused tricyclic compounds, pharmaceutical compositions thereof, and use thereof as PARP1 inhibitors in the preparation of drugs for preventing and / or treating diseases improved by inhibiting PARP1. Background Art

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

[0003] BRCA1 and BRCA2 proteins are essential for the repair of double-stranded DNA breaks (DSBs) by a process called homologous recombination repair (HRR), a form of DNA repair that uses homologous DNA sequences to guide repair at DSBs (Lord et al, Science 355.6330, 2017). HRR is normally a "conservative" mechanism because it restores the original DNA sequence at the site of DNA damage. When cells are defective in HRR, whether driven by defects in BRCA1, BRCA2, or other pathway components, non-conservative forms of DNA repair dominate, such as non-homologous end joining (NHEJ).

[0004] PARP inhibitors play an anti-cancer role by blocking DNA damage repair in highly mutated cancer cells, resulting in "toxic damage" that causes cell death due to homologous recombination repair (HRR) deficiency. There are multiple signaling pathways for repairing DNA in healthy cells, so inhibiting PARP alone will not be too toxic to them; but for some tumor cells, because specific gene mutations such as BRCA will destroy other DNA repair pathways, the DNA repair pathway will rely on PARP-1, so it will be particularly sensitive to PARP inhibitors, which is why ovarian cancer and breast cancer patients with BRCA mutations are more likely to benefit from PARP inhibitors. PARP2 content is low, accounting for only 5% to 10% of total PARP activity. Knocking out PARP1 significantly reduced PARP activity compared to knocking out PARP2 (<10%) (Yélamos et al, The EMBO journal 25.18, 2006). Knocking out PARP1 blocked the inhibitory activity of Olaparib on PARP and also eliminated the cell proliferation inhibitory effect of Olaparib (Murai et al, Cancer research, 2012). These data indicate that PARP is the key to determining the efficacy of PARPi. There are corresponding literature reports that intact PARP2 in the bone marrow is required for mouse survival; the loss of PARP2 leads to a decrease in the number of RBCs, WBCs, and BM cells (Farrés et al, Blood, The Journal of the American Society of Hematology 122.1, 2013); compared with the knockout of PARP1, the knockout of PARP2 can reduce the number of T cells and RBCs, while the knockout of PARP1 has no significant effect on the number of T cells (Yélamos et al, Blood, The EMBO journal 25.18, 2006) and RBCs (Farrés et al, Cell Death & Differentiation 22.7, 2015). Therefore, the inhibition of PARP1 is the main source of efficacy, and the inhibition of PARP2 is the main source of toxicity. The development of highly selective PARP1 / 2 inhibitors may significantly reduce the toxicity caused by PARP2 without significantly reducing the efficacy.

[0005] In summary, there is an urgent need to develop PARP inhibitors with high efficacy and good safety, especially inhibitors with high selectivity for PARP1. Summary of the invention

[0006] One of the objectives of the present invention is to provide a class of fused tricyclic compounds which can be used as PARP1 inhibitors.

[0007] The second object of the present invention is to provide a pharmaceutical composition comprising the above compound as an active ingredient and an optional pharmaceutically acceptable carrier.

[0008] The third object of the present invention is to provide a use of the above compound or a composition comprising the same in the preparation of a PARP1 inhibitor.

[0009] A fourth object of the present invention is to provide a use of the above-mentioned compound or a composition comprising the same in the preparation of a drug for preventing and / or treating a disease improved by inhibiting PARP1.

[0010] In one aspect, the present invention provides a compound of formula (I), or a stereoisomer, geometric isomer, tautomer, or a pharmaceutically acceptable salt or hydrate thereof,

[0011]

[0012] in:

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

[0014] X 2 Select from N or CR 8a ;

[0015] X 3 Select from N or CR 8b ;

[0016] X 4 Select from N or CR 9 ;

[0017] X 5 Select from N or CR 10 ;

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

[0019] X 7 , X 8 are each selected from C or N, provided that X 7 and X 8 Not at the same time, it is C;

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

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

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

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

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

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

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

[0027] Y is N, C or CH;

[0028] R 6 Selected from:

[0029]

[0030] R 11Each is independently selected from halogen, cyano, C1-C3 alkoxy, carbonyl, -CONHR 13 and amino, preferably selected from halogen, -CONHR 13 and cyano;

[0031] m is 0, 1, 2 or 3;

[0032] R 12 is selected from hydrogen, cyano, halogen, substituted or unsubstituted C1-C4 alkyl;

[0033] R 13 is selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted 3-8 membered heterocyclic group; wherein the heterocyclic group refers to a heterocyclic group containing 1-3 heteroatoms selected from N, O, S; preferably, R 13 Selected from hydrogen, unsubstituted or halogen-substituted C1-C4 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy;

[0034] The substitution refers to substitution by one or more selected from C1-C4 alkyl, C1-C6 alkoxy, halogen, hydroxyl, cyano, amino, carboxyl and C3-C6 cycloalkyl.

[0035] In some embodiments, X 7 is N, and the definitions of other substituents are the same as above.

[0036] In some embodiments, X 8 is N, and the definitions of other substituents are the same as above.

[0037] In some embodiments, in formula (I) Selected from the following structures:

[0038]

[0039] More preferably, it is selected from the following structures:

[0040]

[0041]

[0042] wherein the definitions of the substituents are the same as above; further preferably, R 8a and R 8b Each is independently selected from hydrogen, fluorine, chlorine, methyl, ethyl and cyano, and the definitions of other substituents are the same as above.

[0043] In some embodiments, in formula (I) Selected from the following structures:

[0044]

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

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

[0047]

[0048]

[0049] Preferably, it is selected from the following structures:

[0050]

[0051] Among them, each substituent m, R 11 , R 12 , R 13 The definition of is the same as that of the above formula (I).

[0052] In some embodiments, in formula (I) Selected from the following structures:

[0053]

[0054] The definitions of the substituents are the same as above.

[0055] Those skilled in the art will understand that are equivalent.

[0056] In some embodiments, a formula (II), a compound of formula (II), or a stereoisomer, geometric isomer, tautomer, or a pharmaceutically acceptable salt or hydrate thereof is provided.

[0057]

[0058] The definitions of the substituents are the same as those in the above formula (I).

[0059] In some embodiments, a compound of formula (IV) or formula (V), or a stereoisomer, geometric isomer, tautomer, or a pharmaceutically acceptable salt or hydrate thereof is provided.

[0060]

[0061] The definitions of the substituents are the same as those in the above formula (I).

[0062] In some embodiments, for compounds described in formula (I), formula (II), formula (III), formula (IV) or formula (V), X 6 NR 7c , the definitions of other substituents are the same as above.

[0063] In some embodiments, for compounds described in formula (I), formula (II), formula (III), formula (IV) or formula (IV), X 6 CR 14 R 15 , the definitions of other substituents are the same as above.

[0064] In another preferred embodiment, in the compound described in formula (II) or formula (IV), R 8a is hydrogen or C1-C2 alkyl; preferably, R 8a It is hydrogen or methyl.

[0065] In another preferred embodiment, in the compound described in formula (III) or formula (V), R 8b is hydrogen or C1-C2 alkyl; preferably, R 8b It is hydrogen or methyl.

[0066] Typical compounds of the present invention include, but are not limited to:

[0067]

[0068]

[0069]

[0070] The terms in the compound are defined as follows:

[0071] "Halogen" may be fluorine, chlorine, bromine or iodine.

[0072] "C1-C6 alkyl" refers to a chain alkyl group having 1 to 6 carbon atoms; specific examples thereof may include methyl, ethyl, propyl, n-propyl, isopropyl, butyl, n-butyl, isobutyl, tert-butyl, 1-methyl-butyl, 1-ethyl-butyl, pentyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 4-methyl-2-pentyl, 3,3-dimethylbutyl, 2-ethylbutyl and the like, but are not limited thereto. The meaning of C1-C4 alkyl is similar.

[0073] "C1-C6 alkoxy" refers to a RO-group, wherein R is a C1-C6 alkyl group as described above. Specific examples of alkoxy include methoxy, ethoxy, n-propoxy, isopropoxy, and the like. The meaning of C1-C3 alkoxy is similar.

[0074] "C3-C8 cycloalkyl" refers to a fully saturated monovalent cyclic hydrocarbon compound group containing 3-8 ring carbon atoms, and specific examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The meaning of C3-C6 cycloalkyl is similar.

[0075] The "3- to 8-membered heterocyclic group" refers to a 3- to 8-membered non-aromatic cyclic group containing 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur in the ring, and specific examples thereof include oxetane, tetrahydroimidazole and tetrahydrofuran.

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

[0077] The compounds of the present invention or their stereoisomers, geometric isomers, tautomers, and pharmaceutically acceptable salts may also exist in the form of polymorphs, solvates (such as hydrates) and prodrugs, and these polymorphs, solvates (such as hydrates) and prodrugs are also included in the scope of the present invention.

[0078] As used herein, the term "solvate" is intended to include stoichiometric or non-stoichiometric solvent addition forms. If the solvent is water, the solvate formed is a hydrate, and when the solvent is ethanol, the solvate formed is an ethanolate. Hydrates are formed by one or more molecules of water with one molecule of the substance, wherein the water retains its molecular state of H2O, and such a combination can form one or more hydrates, such as hemihydrates, monohydrates and dihydrates.

[0079] As used herein, "prodrug" refers to an active or inactive compound that is chemically modified and, after administration to an individual, becomes a compound of the present invention through physiological actions in the body (e.g., hydrolysis, neogenesis, etc.). The adaptability and technology of making and using prodrugs are well known to those skilled in the art.

[0080] Another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound selected from the group consisting of formula (I), its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts and hydrates thereof, and optionally a pharmaceutically acceptable carrier.

[0081] In certain embodiments of the pharmaceutical composition, the pharmaceutical composition is formulated for intravenous administration, intramuscular administration, oral administration, rectal administration, inhalation administration, nasal administration, topical administration, eye administration or ear administration. In other embodiments of the pharmaceutical composition, the pharmaceutical composition is a tablet, a pill, a capsule, a liquid, an inhalant, a nasal spray solution, a suppository, a solution, an emulsion, an ointment, an eye drop or an ear drop. In other embodiments of the pharmaceutical composition, it also comprises one or more additional therapeutic agents.

[0082] The pharmaceutical composition of the present invention can be selected to contain at least one pharmaceutically acceptable carrier selected from the following according to the mode of administration and the properties of the dosage form: diluents, adjuvants, excipients, preservatives, fillers, binders, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, temperature-sensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0083] Another aspect of the present invention provides the use of a compound of formula (I), or a stereoisomer, geometric isomer, tautomer thereof, a pharmaceutically acceptable salt or hydrate thereof, or the above pharmaceutical composition in the preparation of a PARP1 inhibitor.

[0084] In another aspect, the present invention provides the use of a compound of formula (I), or a stereoisomer, geometric isomer, tautomer thereof, a pharmaceutically acceptable salt or hydrate thereof, or the above-mentioned pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease improved by inhibiting PARP1.

[0085] In another aspect, the present invention provides a method for preventing and / or treating diseases that are improved by inhibiting PARP1, which comprises administering to an individual in need of such treatment an effective amount of a compound selected from the group consisting of a compound of formula (I), its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts and hydrates thereof, or the pharmaceutical composition.

[0086] In some embodiments of the present invention, the disease improved by inhibiting PARP1 includes but is not limited to cancer.

[0087] In some embodiments of the invention, the cancer includes but is not limited to malignant tumors, such as ovarian cancer, breast cancer, fallopian tube cancer, endometrial cancer, peritoneal cancer, gastric cancer, colon cancer, bladder cancer, pancreatic cancer, biliary tract cancer, osteosarcoma, cervical cancer, head and neck tumors, germ cell and embryonal cancer, esophageal cancer, malignant glioma, Ewing sarcoma, pancreatic cancer, melanoma, bile duct cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, lymphoma and any one of blood cancers.

[0088] In some embodiments of the present invention, the genome of the cancer is of a type deficient in homologous recombination repair.

[0089] In some embodiments of the invention, the cancer is dependent on a pathway that is deficient in homologous recombination repair of double-stranded DNA damage.

[0090] In some embodiments of the present invention, the cancer comprises one or more cancer cells that lack the ability to repair DNA double-strand breaks by homologous recombination relative to normal cells.

[0091] In some embodiments of the invention, the cancer comprises one or more cancer cells that lack BRCA1 or BRCA2 or have a BRCA1 or BRCA2 mutation.

[0092] Terminology

[0093] In the present invention, unless otherwise explicitly stated, the terms used in the present invention have the meanings defined below. Terms not explicitly defined in the present invention have the general meanings generally understood by those skilled in the art.

[0094] The terms "optional," "optional," or "optionally" as used herein mean that the subsequently described substitution pattern, event, or circumstance may or may not occur, and that the description includes instances where the substitution pattern occurs and instances where the substitution pattern does not occur.

[0095] The term "therapeutically effective amount" of the compound of the present invention refers to an amount of the compound of the present invention that can induce a biological or medical response in an individual or improve symptoms, slow down or delay disease progression, or prevent disease, etc.

[0096] As used herein, the term "subject" refers to an animal. Preferably, the animal is a mammal. The subject also refers to, for example, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In a preferred embodiment, the subject is a human.

[0097] As used herein, the term "inhibit" refers to a reduction or suppression of a particular condition, symptom or disorder or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0098] As used herein, the term "treating" any disease or condition refers in one embodiment to ameliorating the disease or condition (i.e., arresting or slowing the development of the disease or at least one of its clinical symptoms). In another embodiment, "treating" refers to improving at least one physical parameter, which may not be perceived by the patient. In another embodiment, "treating" refers to modulating the disease or condition physically (e.g., stabilizing a perceptible symptom) or physiologically (e.g., stabilizing a physical parameter), or both.

[0099] Beneficial effects:

[0100] The compound of the present invention has high selectivity for PARP1, has fewer side effects than olaparib (AZD-2281), and has high clinical application value.

[0101] The present invention has been described in detail above, but the above embodiments are only illustrative in nature and are not intended to limit the present invention. In addition, this article is not limited by any theory described in the above prior art or invention content or the following examples. DETAILED DESCRIPTION

[0102] The present invention is further described below in conjunction with examples. It should be noted that the following examples are provided for illustrative purposes only and do not constitute a limitation on the scope of protection claimed for the present invention.

[0103] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial sources. The raw materials can usually be obtained from commercial sources or easily prepared using methods known to those skilled in the art.

[0104] In each embodiment, the experimental instruments or materials used are as follows:

[0105] 1 H NMR was recorded by Varian Mercury-300 or Varian Mercury-400 NMR spectrometer. 13C NMR was recorded by Varian Mercury-400, Varian Mercury-500 or Varian Mercury-600 NMR instrument, and chemical shift was expressed in δ (ppm); mass spectra were recorded by Finnigan / MAT-95 (EI), Finnigan LCQ / DECA and Micromass Ultra Q-TOF (ESI) mass spectrometers; the silica gel used for reverse phase preparative HPLC separation was 200-300 mesh.

[0106] Abbreviations:

[0107] PE: petroleum ether; EA: ethyl acetate; DIEA: N,N-diisopropylethylamine; DCM: dichloromethane; DMF: N,N-dimethylformamide; XPhos Pd G2: chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II); Ruphos Pd G3: methanesulfonate(2-dicyclohexylphosphino-2',6'-diisopropyloxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II).

[0108] Example

[0109] Synthesis of key intermediates

[0110] Intermediate 1a: Synthesis of 6-chloro-N-methyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0111]

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

[0113] 1a-1 (1.00 g, 3.99 mmol) and ethanol solution of methylamine (33 wt%, 10 mL) were added to a flask in sequence. The mixture was reacted at room temperature for 16 hours. The reaction solution was concentrated to obtain 1a-2 (0.94 g, yellow oil). Yield: 94.37%, LCMS (ESI): m / z 250.9 [M+H] + ; RT = 1.448min (2.50min) (the time before the bracket is the peak time, and the time in the bracket is the total running time).

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

[0115] 1a-2 (620 mg, 2.49 mmol), toluene (15 mL), tert-butyl piperazine-1-carboxylate (370 mg, 1.99 mmol), 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (155 mg, 0.25 mmol), cesium carbonate (2020 mg, 6.21 mmol) and palladium acetate (56 mg, 0.25 mmol) were added to the flask in sequence. The mixture was reacted at 100 °C under nitrogen protection for 16 hours. The reaction solution was filtered and the filtrate was concentrated. Purification by silica gel column chromatography (petroleum ether (PE): ethyl acetate (EA) = 1:1) gave 1a-3 (180 mg, yellow oil), yield: 20.41%, LCMS (ESI): m / z 355.1 [M+H] + ; RT = 1.679 min (2.50 min). 1 H-NMR (400MHz, DMSO-d6): δ8.46(d,J=4.8Hz,1H),7.95(d,J=8.4Hz,1H),7.68(d, J=8.4Hz,1H),3.50(s,4H),3.06-3.04(m,4H),2.80(d,J=4.8Hz,3H),1.43(s,9H).

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

[0117] 1a-3 (180 mg, 0.51 mmol), DCM (2 mL) and dioxane hydrochloride (4.0 M, 2 mL) were added to a dry flask. After stirring at room temperature for 2 hours, the mixture was concentrated to give 1a (140 mg, yellow solid). Yield: 94.59%, LCMS (ESI): m / z 255.1 [M+H] + ;RT=0.336min&0.461min(2.50min). 1 H-NMR (400MHz, DMSO-d6): δ9.40 (s, 2H), 8.50 (d, J = 4.4Hz, 1H), 7.97 (d, J = 8.4Hz, 1H),7.76(d,J=8.4Hz,1H),3.34-3.32(m,4H),3.25(m,4H),2.80(d,J=4.8Hz,3H).

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

[0119]

[0120] Step 1: Synthesis of tert-butyl 4-(2-fluoro-6-(methoxycarbonyl)pyridin-3-yl)piperazine-1-carboxylate

[0121] 1a-2 (500 mg, 2.1 mmol), 1-tert-butyloxycarbonylpiperazine (600 mg, 3.2 mmol), Ruphos Pd G3 (180 mg, 0.21 mmol), cesium carbonate (1.7 g, 5.2 mmol), and dioxane (15 mL) were added to a 50 mL flask and heated at 80 ° C overnight under nitrogen protection. The reaction solution was concentrated and the residue was purified by silica gel column (PE: EA = 2: 1) to obtain 2a-1 (640 mg, yellow solid), yield: 88%. LCMS (ESI): m / z 340.1 [M+H] + ; RT = 1.74min (3.00min).

[0122] Step 2: Synthesis of 5-(4-(tert-butyloxycarbonyl)piperazin-1-yl)-6-fluoropicolinic acid

[0123] 2a-1 (320 mg, 0.94 mmol) and tetrahydrofuran (8 mL) were added to a dry 50 mL single-necked flask, and an aqueous solution (8 mL) of lithium hydroxide monohydrate (200 mg, 4.7 mmol) was added dropwise. The mixture was stirred for 2 hours. The pH of the reaction solution was adjusted to 6 with a 1 M hydrochloric acid solution. The reaction solution was concentrated and purified by a reverse phase column (2% to 40% acetonitrile aqueous solution) to obtain 2a-2 (300 mg, yellow solid). The yield was 92%. LCMS (ESI): m / z 326.1 [M+H] + ; RT = 1.26min (3.00min).

[0124] Step 3: Synthesis of tert-butyl 4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazine-1-carboxylate

[0125] 2a-2 (300 mg, 0.92 mmol), 1-hydroxybenzotriazole (149 mg, 1.1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (211 mg, 1.1 mmol), N,N-diisopropylethylamine (DIEA) (237 mg, 1.84 mmol), DMF (6 mL), methylamine hydrochloride (123 mg, 1.84 mmol) were added to a dry single-mouth bottle and stirred overnight. The reaction solution was added with water (20 mL), extracted with EA (30 mL) three times, the organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The residue was purified by reverse phase column (20% to 70% acetonitrile aqueous solution) to obtain 2a-3 (250 mg, white solid), yield: 80%. LCMS (ESI): m / z 283.1 [M-100+H] + ; RT = 1.66 min (3.00 min).

[0126] Step 4: Synthesis of 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0127] 2a-3 (250 mg, 0.74 mmol), EA (3 mL), and 4M hydrochloric acid dioxane solution (1 mL) were added to a dry 20 mL single-necked flask at room temperature, stirred at room temperature for 2 hours, and concentrated to obtain product 2a (200 mg, yellow solid), yield: 100%. LCMS (ESI): m / z 239.1 [M+H] + ; RT = 0.91 min (3.00 min). 1 H-NMR (600MHz, CD3OD): 7.98-7.96 (m, 1H), 7.67-7.64 (m, 1H), 3.50-3.48 (m, 4H), 3.45-3.43 (m, 4H), 2.93 (d, J = 3.6Hz, 3H).

[0128] Intermediate 3a: Synthesis of N,6-dimethyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0129]

[0130] Step 1: Synthesis of 5-bromo-6-methylpicolinic acid

[0131] 3a-1 (500 mg, 2.54 mmol), methanol (6 mL), water (3 mL) and sodium hydroxide (507 mg, 12.69 mmol) were added to a dry flask in sequence. The mixture was reacted at 70 °C for 1 hour. The reaction solution was concentrated under reduced pressure. The mixture was diluted with water (10 mL), adjusted to pH = 4 with 3M dilute hydrochloric acid, filtered, and the filter cake was collected to obtain 3a-2 (300 mg, white solid). Yield: 54.72%, LCMS (ESI): m / z 218.0 [M+H] + ; RT = 1.208min (2.50min). 1 H-NMR (400MHz, DMSO-d6): δ8.12(d,J=8.0Hz,1H),7.78(d,J=8.4Hz,1H),2.67(s,3H).

[0132] Step 2: Synthesis of 5-bromo-N,6-dimethylpicolinamide

[0133] 3a-2 (300 mg, 1.39 mmol), DMF (3 mL), DIEA (0.92 mL, 5.55 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (792 mg, 2.08 mmol) and methylamine tetrahydrofuran solution (2.0 M, 1.39 mL, 2.78 mmol) were added to a dry flask. The reaction was allowed to react at room temperature for 1 hour. The mixture was diluted with water (30 mL) and extracted with ethyl acetate (10 mL×2). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative plate (PE:EA=3:2) to give 3a-3 (215 mg, yellow solid), yield: 67.59%, LCMS (ESI): m / z 231.0 [M+H] + ; RT = 1.427 min (2.50 min). 1 H-NMR (400 MHz, DMSO-d6): δ8.67 (d, J = 4.0 Hz, 1H), 8.18 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 2.82 (d, J = 4.8 Hz, 3H), 2.65 (s, 3H).

[0134] Step 3: Synthesis of tert-butyl 4-(2-methyl-6-(methylcarbamoyl)pyridin-3-yl)piperazine-1-carboxylate

[0135] 3a-3 (200 mg, 0.87 mmol), toluene (8 mL), tert-butyl piperazine-1-carboxylate (179 mg, 0.96 mmol), 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (54 mg, 0.09 mmol), cesium carbonate (711 mg, 2.18 mmol) and palladium acetate (20 mg, 0.09 mmol) were added to a dry flask. The reaction was carried out at 100 °C under nitrogen protection for 16 hours. The reaction solution was filtered and the filtrate was concentrated. The residue was purified by preparative plate (PE: EA = 1: 1) to obtain 3a-4 (160 mg, yellow solid), yield: 54.80%, LCMS (ESI): m / z 335.1 [M+H] +

[0136] Step 4: Synthesis of N,6-dimethyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0137] 3a-4 (160 mg, 0.48 mmol), DCM (2 mL) and hydrochloric acid-dioxane (4.0 M, 2 mL) were added to a dry flask. After reacting at room temperature for 4 hours, the mixture was concentrated to obtain 3a (129 mg, yellow solid). Yield: 99.58%, LCMS (ESI): m / z 235.2 [M+H] + ;RT=0.340 min&0.450 min(2.50 min). 1 H-NMR (400 MHz, DMSO-d6): δ9.44(s,2H),8.56(d,J=4.8 Hz,1H),7.88(d,J=8.0 Hz,1H),7.61(d,J=8.4 Hz,1H),3.25-3.16(m,8H),2.82(d,J=4.4 Hz,3H),2.54(s,3H).

[0138] Intermediate 4a: Synthesis of N-ethyl-6-fluoro-5-(piperazin-1-yl)picolinamide hydrochloride

[0139]

[0140] The synthesis method refers to the synthesis of intermediate 2a, except that ethylamine is used instead of methylamine hydrochloride. LCMS (ESI): m / z 253.2 [M+H] + ; RT = 0.527 min (2.5 min).

[0141] Intermediate 5a: Synthesis of N-cyclopropyl-6-fluoro-5-(piperazin-1-yl)picolinamide hydrochloride

[0142]

[0143] The synthesis method refers to the synthesis of intermediate 2a, except that cyclopropylamine is used instead of methylamine hydrochloride. LCMS (ESI): m / z 264.2 [M+H] + ; RT = 0.958 min (2.5 min).

[0144] Intermediate 6a: 7-Fluoro-8-(hydroxymethyl)-2,4-dimethyl-4H-imidazo[1,5,4-de]quinoxalin-5(6H)-one

[0145]

[0146] Step 1: Synthesis of (4-bromo-3-fluoro-2-nitrophenyl)alanine ethyl ester

[0147] 6a-1 (5 g, 21 mmol) was dissolved in DMF (25 mL), and DL-alanine ethyl ester hydrochloride (2.89 g, 18.9 mmol) and DIEA (8.1 g, 63 mmol) were added in turn under ice bath, and the mixture was heated to room temperature and stirred overnight. Water (100 mL) was added to the reaction solution, and EA (60 mL) was extracted three times, and the organic phase was concentrated and purified by silica gel column (EA:PE=1:10) to obtain 6a-2 (2.6 g, 7.76 mmol), an orange solid, with a yield of 37%. 1 H NMR (600MHz, CDCl3) δ7.47 (dd, J=9.2, 7.0Hz, 1H), 6.41 (dd, J=9.4, 1.6Hz, 1H), 4.24 ( q,J=7.0Hz,2H),4.18(t,J=7.0Hz,1H),1.56(d,J=7.0Hz,3H),1.28(t,J=7.0Hz,3H).

[0148] Step 2: Synthesis of (2-amino-4-bromo-3-fluorophenyl)alanine ethyl ester

[0149] 6a-2 (600 mg, 1.79 mmol) and ammonium chloride (949 mg, 17.9 mmol) were added to a solution of methanol (20 mL) and water (5 mL). Zinc powder (1.16 g, 17.9 mmol) was added in batches under an ice bath and stirred at room temperature for 2 hours. The reaction solution was filtered, the solid was washed with methanol, the filtrate was concentrated and diluted with EA (50 mL), alkalized with saturated sodium bicarbonate aqueous solution, separated, the aqueous phase was extracted three times with EA, and the organic phase was concentrated. The crude product 6a-3 (304 mg, 0.996 mmol) was obtained as a brown solid with a yield of 55.6%. LCMS (ESI): m / z 307.0 [M+H] + ; RT = 1.70min (3.00min).

[0150] Step 3: Synthesis of 7-bromo-8-fluoro-3-methyl-3,4-dihydroquinoxalin-2(1H)-one

[0151] Dissolve 6a-3 in methanol (15 mL), EA (10 mL), add 4M hydrochloric acid gas (dioxane solution) (2 mL), and stir at room temperature for 2 hours. The reaction solution is concentrated to give crude product 6a-4 (260 mg, 0.996 mmol), LCMS (ESI): m / z 257.0 [MH] - ; RT = 1.38 min (3.00 min).

[0152] Step 4: Synthesis of 7-bromo-8-fluoro-3-methyl-5-nitroquinoxalin-2(1H)-one

[0153] 6a-4 (260 mg, 1.0 mmol) was dissolved in concentrated sulfuric acid (8 mL, 95% aq.) in an ice-water bath, potassium nitrate (121 mg, 1.2 mmol) was added, and stirred for 2 hours. The reaction solution was slowly added to ice water, and an orange-red solid precipitated. The solid was collected by filtration, washed with water, and then vacuum dried to obtain 6a-5 (125 mg, 0.42 mmol). The crude product was used directly in the next step without purification. LCMS (ESI): m / z 299.9 [MH] - ; RT = 1.33min (3.00min).

[0154] Step 5: Synthesis of 5-amino-7-bromo-8-fluoro-3-methyl-3,4-dihydroquinoxalin-2(1H)-one

[0155] 6a-5 (251 mg, 0.831 mmol), ammonium chloride (265 mg, 5 mmol), methanol (10 mL) and water (1 mL) were stirred and zinc powder (325 mg, 5 mmol) was added. The mixture was stirred at room temperature for 3 hours, the reaction solution was filtered, the solid was washed with methanol, the filtrate was concentrated, diluted with EA (20 mL), alkalized with saturated sodium bicarbonate aqueous solution, the aqueous phase was extracted with EA three times, the organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain 6a-6 (130 mg) as a brown solid. LCMS (ESI): m / z 275.9 [M+H] + ; RT = 2.49 min (5.00 min).

[0156] Step 6: Synthesis of 8-bromo-7-fluoro-2,4-dimethyl-4H-imidazo[1,5,4-de]quinoxalin-5(6H)-one

[0157] 6a-6 (130 mg) was dissolved in triethyl orthoacetate (5 mL), heated to 80°C and stirred for 1 h under nitrogen protection, and then cooled to room temperature and acetic acid (3 mL) was added and continued to heat to 100°C and stirred for 2 h. The reaction solution was concentrated to remove the solvent, and the resulting crude product was dissolved in DMSO (3 mL) and purified by reverse phase column (acetonitrile: water (1‰ ammonium bicarbonate = 0%-80%)) to obtain product 6a-7 (30 mg, 0.101 mmol) as a white solid. LCMS (ESI): m / z 298.1 [M+H] + ; RT = 1.25min (3.00min). 1 H NMR (600MHz, DMSO) δ11.35(s,1H),7.42(d,J=4.8Hz,1H),5.34(q,J=7.0Hz,1H),2.54(d,J=7.9Hz,3H),1.64(d,J=7.0Hz,3H).

[0158] Step 7: 7-Fluoro-8-(hydroxymethyl)-2,4-dimethyl-4H-imidazo[1,5,4-de]quinoxalin-5(6H)-one

[0159] 6a-7 (30 mg, 0.101 mmol), (tributyltin)methanol (65 mg, 0.202 mmol) and x-phos Pd G2 (16 mg, 0.0202 mmol) were added to dioxane (5 mL) in sequence, and stirred at 110°C for 2 hours under nitrogen protection. The reaction solution was concentrated and purified by silica gel column (methanol: DCM = 1: 10) to obtain product 6a (15 mg, 0.0602 mmol) as a white solid. Yield: 59.6%. LCMS (ESI): m / z 250.2 [M+H] + ; RT = 0.93min (3.00min).

[0160] Intermediate 7a: 4-ethyl-7-fluoro-8-(hydroxymethyl)-2-methyl-4H-imidazo[1,5,4-de]quinoxalin-5(6H)-one

[0161]

[0162] The synthesis method refers to intermediate 6a, except that DL-2-aminobutyric acid methyl ester hydrochloride is used instead of DL-alanine ethyl ester hydrochloride, LCMS (ESI): m / z 264.2 [M+H] + ; RT = 1.01 min (3.00 min).

[0163] Intermediate 8a: 9-Fluoro-8-(hydroxymethyl)-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0164]

[0165] Step 1: Synthesis of tert-butyl 2-(5-bromo-7-nitro-1H-indazol-1-yl)acetate

[0166] 8a-1 (1 g, 4.13 mmol) was dissolved in DMF (20 mL), tert-butyl bromoacetate (967 mg, 4.96 mmol) and potassium carbonate (1.72 g, 12.4 mmol) were added at room temperature, stirred overnight at room temperature, diluted with EA (60 mL), washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column (EA: PE = 1: 10) to obtain 8a-2 (480 mg, 1.30 mmol), a yellow oily solid, yield: 31%. 1H NMR (400MHz, DMSO-d6) δ8.61(d,J=1.8Hz,1H),8.47(s,1H),8.40(d,J=1.8Hz,1H),5.45(s,2H),1.44(s,9H).

[0167] Step 2: Synthesis of tert-butyl 2-(7-amino-5-bromo-1H-indazol-1-yl)acetate

[0168] 8a-2 (1.05 g, 2.96 mmol) was added to acetic acid (10 mL), and iron powder (994 mg, 17.7 mmol) was added. The mixture was stirred at 50 °C for 2 hours, filtered through diatomaceous earth, and the filtrate was concentrated. The mixture was diluted with EA (30 mL), washed with saturated aqueous sodium bicarbonate solution, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column (EA:PE=1:3) to obtain the product 8a-3 (604 mg, 1.86 mmol) as a white solid. The yield was 63%. 1 HNMR (600MHz, DMSO-d6) δ7.90(s,1H),7.21(d,J=1.7Hz,1H),6.73(d,J=1.7Hz,1H),5.39(s,2H),5.36(s,2H),1.36(s,9H).

[0169] Step 3: Synthesis of tert-butyl 2-(7-amino-5-bromo-6-fluoro-1H-indazol-1-yl)acetate

[0170] 1-Chloromethyl-4-fluoro-1,4-diazabicyclo[2.2.2]octane di(tetrafluoroborate) salt (653 mg, 1.85 mmol) was dissolved in DMF (4 mL), and the solution was added dropwise to a solution of 8a-3 (400 mg, 1.23 mmol) in anhydrous tetrahydrofuran (40 mL) at room temperature. The mixture was stirred at room temperature for 6 hours. The reaction solution was diluted with water (30 mL), extracted three times with EA (40 mL), and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column (EA:PE=1:10) to obtain 8a-4 (121 mg, 0.352 mmol) as a light yellow solid. Yield: 29%. 1 H NMR (400MHz, DMSO-d6) δ7.94 (s, 1H), 7.37 (d, J = 5.9Hz, 1H), 5.40 (s, 2H), 5.31 (s, 2H), 1.35 (s, 9H).

[0171] Step 4: Synthesis of 8-bromo-9-fluoro-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one 6

[0172] 8a-4 (61 mg, 0.178 mmol) was dissolved in methanol (5 mL), 4M hydrochloric acid gas (dioxane solution) (2 mL) was added, and the mixture was stirred at 70°C for 4 hours. The reaction solution was concentrated, and the crude product was dissolved in EA (10 mL), washed with saturated sodium bicarbonate aqueous solution, dried over anhydrous sodium sulfate, and concentrated to give 8a-5 (35 mg, 0.130 mmol) as a white solid with a yield of 73%. LCMS (ESI): m / z 268.0 [MH] - ; RT = 1.35min (3.00min).

[0173] Step 5: Synthesis of 9-fluoro-8-(hydroxymethyl)-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0174] 8a-5 (35 mg, 0.130 mmol), (tributyltin)methanol (83 mg, 0.26 mmol) and x-phos Pd G2 (10 mg, 0.013 mmol) were added to dioxane (10 mL) in sequence, and stirred at 110 ° C for 4 hours under nitrogen protection. The reaction solution was concentrated and purified by silica gel column (methanol: DCM = 1: 10) to obtain product 8a (25 mg, 0.11 mmol) as a white solid. Yield: 87%. LCMS (ESI): m / z 220.1 [MH] - ; RT = 0.94 min (3.00 min).

[0175] Intermediate 9a: 8-(Hydroxymethyl)-3-methyl-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0176]

[0177] Step 1: Synthesis of tert-butyl 2-(5-bromo-7-nitro-1H-indazol-1-yl)propanoate

[0178] 9a-1 (1 g, 4.13 mmol) was dissolved in DMF (20 mL), 9a-2 (950 mg, 4.54 mmol) and potassium carbonate (1.7 g, 12.4 mmol) were added at room temperature, and stirred for 2 hours. The reaction solution was diluted with EA (60 mL), washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column (EA: PE = 1: 5) to obtain 9a-3 (480 mg, 1.30 mmol) as a yellow oily solid. Yield: 31%. 1H NMR (400MHz, DMSO-d6) δ8.55(d,J=1.8Hz,1H),8.45(s,1H),8.31(d,J=1.8Hz,1H),5.57(q,J=7.1Hz,1H),1.83(d,J=7.1Hz,3H),1.31(s,9H).

[0179] The method of step 2 and step 3 is the same as the experimental method of intermediates 8a-5 and 8a to obtain 9a as a white solid with a yield of 83%. LCMS (ESI): m / z 216.0 [MH] - ; RT = 1.04min (3.00min).

[0180] Intermediate 10a: 3-ethyl-8-(hydroxymethyl)-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0181]

[0182] The synthesis method is the same as that of intermediate 9a, except that tert-butyl 2-bromobutyrate is used as the starting material. LCMS (ESI): m / z 232.2 [M+H] + ; RT = 1.11 min (3.00 min).

[0183] Intermediate 11a: 8-(Hydroxymethyl)-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0184]

[0185] The synthesis method is the same as that of intermediate 9a, except that tert-butyl bromoacetate is used as the starting material. LCMS (ESI): m / z 202.1 [MH] - ; RT = 0.89 min (3.00 min).

[0186] Intermediate 12a: 9-Fluoro-8-(hydroxymethyl)-3-methyl-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0187]

[0188] Step 1: Synthesis of 5-bromo-6-fluoro-1H-indazole

[0189] 12a-1 (5 g, 24.6 mmol), sodium nitrite (1.7 g, 24.6 mmol) and acetic acid (100 mL) were dissolved in water (2 mL), reacted at 50 ° C for 2 hours, and the reaction solution was slowly added dropwise to ice water, filtered, and the filter cake was washed with water and dried to obtain the product 12a-2 (3.8 g, brown solid), yield: 72%. LCMS (ESI): m / z 213.0 [MH] - ; RT = 1.44min (3.0min).

[0190] Step 2: Synthesis of 5-bromo-6-fluoro-7-nitro-1H-indazole

[0191] 12a-2 (2 g, 9.3 mmol) and concentrated sulfuric acid (20 mL) were added with potassium nitrate (944 mg, 9.3 mmol) in batches under ice bath and stirred for 2 hours. The reaction solution was slowly added dropwise to ice water, extracted three times with EA (50 mL), the organic phase was washed twice with water (30 mL), dried, concentrated, and the residue was passed through a silica gel column (PE: EA = 15: 1) to obtain 12a-3 (800 mg, yellow solid), yield: 33%. LCMS (ESI): m / z 258.0 [MH] - ; RT = 1.44min (3.0min).

[0192] Step 3: Synthesis of 5-bromo-6-fluoro-1H-indazol-7-amine

[0193] 12a-3 (1.5 g, 5.8 mmol), iron powder (1.9 g, 34.7 mmol), ammonium chloride (1.8 g, 34.7 mmol) were added to ethanol (30 mL) and water (10 mL), reacted at 60 ° C for 2 hours, filtered, the filtrate was added with water (30 mL), extracted twice with EA (50 mL), dried, and concentrated to give the crude product 12a-4 (1 g, brown solid), yield: 77%. LCMS (ESI): m / z 230.0 [M+H] + ; RT = 1.38min (3.0min).

[0194] Step 4: Synthesis of tert-butyl 2-(7-amino-5-bromo-6-fluoro-1H-indazol-1-yl)propanoate

[0195] 12a-4 (1.5 g, 6.5 mmol), potassium carbonate (1.8 g, 13.0 mmol), tert-butyl 2-bromopropionate (2.0 g, 9.8 mmol) were added to N,N-dimethylformamide (40 mL), and the reaction solution was reacted at 60°C for 6 hours, and then water (30 mL) was added after cooling, and EA (50 mL) was used for extraction twice, dried, and concentrated to obtain the crude product 12a-5 (1 g, brown solid). LCMS (ESI): m / z 302.0 [M-56+H] + ; RT = 1.78 min (3.0 min).

[0196] Step 5: Synthesis of 8-bromo-9-fluoro-3-methyl-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0197] 12a-5 (1 g, 2.8 mmol) and methanol solution of hydrochloric acid (4 M, 5 mL) were added to methanol (10 mL), reacted at 50 ° C for 2 hours, concentrated, and the residue was purified by reverse phase preparative column (2% to 60% acetonitrile (1‰ ammonium bicarbonate)) to obtain crude product 12a-6 (140 mg, brown solid), yield: 18%. LCMS (ESI): m / z 282.0 [MH] - ; RT = 1.41min (3.0min).

[0198] Step 6: Synthesis of 9-fluoro-8-(hydroxymethyl)-3-methyl-1H-pyrazolo[1,5,4-de]quinoxalin-2(3H)-one

[0199] 12a-6 (140 mg, 0.49 mmol), (tributyltin)methanol (327 mg, 0.99 mmol), x-Phos Pd G2 (38 mg, 0.049 mmol) were added to dioxane (10 mL), and the mixture was reacted at 100 °C for 3 hours under nitrogen protection. The residue was separated by silica gel column (DCM:Me=20:1) to obtain intermediate 12a (70 mg, brown solid), yield: 60%. LCMS (ESI): m / z 234.1 [MH] - ; RT = 1.04min (3.00min).

[0200] Intermediate 13a: 6-(piperazine-1-yl) nicotinic acid cyanide hydrochloride

[0201]

[0202] Step 1: Synthesis of tert-butyl 4-(5-cyanopyridin-2-yl)piperazine-1-carboxylate

[0203] 13a-1 (500 mg, 4.09 mmol) and 1-tert-butyloxycarbonylpiperazine (915.2 mg, 4.9 mmol) were added to acetonitrile (10 mL), and DIEA (1.36 mL, 8.19 mmol) was added dropwise. The mixture was reacted at 65°C under nitrogen protection for 16 hours. The solvent was removed and the residue was subjected to column chromatography (PE:EA=5:1) to obtain 13a-2 (1.10 g, yellow solid). Yield: 93.22%, LCMS (ESI): m / z 289.2 [M+H] + ; RT = 1.728min (2.50min).

[0204] Step 2: Synthesis of 6-(piperazin-1-yl)nicotinic acid cyanide hydrochloride

[0205] 13a-2 (1.10 g, 3.81 mmol) was added to DCM (15 mL), and 1,4-dioxane hydrochloride (1.5 mL) was added dropwise under ice bath. The mixture was stirred at room temperature for 2 hours and concentrated to give 13a (850 mg, yellow oil). LCMS (ESI): m / z 189.2 [M+H] + ; RT = 0.352 min. 1 H NMR (400MHz, DMSO-d6): δ9.54(s,2H),8.55(d,J=2.4Hz,1H),7.97-7.94(m,1H),7.03(d,J=9.2Hz,1H),3.93-3.91(m,4H),3.16(s,4H).

[0206] Synthesis of compound 1

[0207]

[0208] 6a (15 mg, 0.06 mmol) was dissolved in DCM (8 mL), and phosphorus tribromide (0.5 mL) was added dropwise at room temperature. After the addition was completed, the mixture was stirred for 2 h. The reaction solution was concentrated and dried under vacuum. The crude product was dissolved in acetonitrile (5 mL), and DIEA (23 mg, 0.18 mmol) and compound 2a (16 mg, 0.06 mmol) were added in sequence. The mixture was stirred at 80 ° C for 2 h. The reaction solution was concentrated under reduced pressure. The crude product was dissolved in DMSO (3 mL) and purified by reverse phase column (acetonitrile: water (1‰ ammonium bicarbonate) = 0%-80%) to obtain compound 1 (10 mg, 0.0213 mmol) as a white solid. The yield was 35.4%. LCMS (ESI): m / z 470.3 [M+H] + ; RT = 1.24min (3.00min). 1H NMR (400MHz, DMSO-d6) δ11.19(s,1H),8.40(d,J=4.8Hz,1H),7.85(t,J=7.6Hz,1H),7.61-7.51(m,1H),7.10(d,J=4.8Hz,1H ),5.33(q,J=6.8Hz,1H),3.64(s,2H),3.15(s,4H),2.77(t,J=3.8Hz,3H),2.58(s,4H),2.53(s,3H),1.65(d,J=7.0Hz,3H).

[0209] Synthesis of Compound 2-Compound 9, 12, 16, 25, 26, 35

[0210] According to the method described in compound 1, intermediates 6a and 2a were replaced by the intermediates in the following table to synthesize compounds 2-9, 12, 16, 25, 26, and 35.

[0211]

[0212]

[0213] Synthesis of Compounds 4-1 and 4-2

[0214]

[0215] Compound 4 (45 mg) was separated by chiral column, column type IH, 250×25mm10μm, mobile phase A:B (10:1, A: ethanol (+0.2% 7.0mol / L ammonia methanol); B: acetonitrile (+0.2% 7.0mol / L ammonia methanol)), compound 4-1 (15.98mg) and compound 4-2 (17.69mg) were obtained;

[0216] Compound 4-1: Peak time 3.662 min; ee%>99. LCMS (ESI): m / z 438.3 [M+H] + ; RT = 0.467min (6.00min). 1H-NMR (400MHz, DMSO-d6): δ11.01(s,1H),8.41-8.37(m,1H),8.02(s,1H),7.83(d,J=7.2Hz,1H),7.58-7.53(m,1H),7.19(s,1H),6.72 (s,1H),5.34-5.31(m,1H),3.55(s,2H),3.15(s,4H),2.75(d,J=3.6Hz,3H),2.67(d,J=2.8Hz,4H),1.75(dd,J1=2.8Hz,J2=6.4Hz,3H).

[0217] Compound 4-2: Peak time 7.369 min; ee%>94. LCMS (ESI): m / z 438.3 [M+H] + ; RT = 1.770min (6.00min). 1 H-NMR (400MHz, DMSO-d6): δ11.04(s,1H),8.40-8.38(m,1H),8.03(s,1H),7.84(d,J=8.0Hz,1H),7.55(dd,J1=8.0Hz,J2=10.4Hz,1H ),7.19(s,1H),6.73(s,1H),5.36-5.31(m,1H),3.56(s,2H),3.15(s,4H),2.76(d,J=4.8Hz,3H),2.54(s,4H),1.75(d,J=6.8Hz,3H).

[0218] Compounds 5-1, 5-2, 6-1, 6-2, 7-1, and 7-2 were obtained by chiral separation of compounds 5, 6, and 7, respectively. IH, 250×25mm 10μm, mobile phase: methanol (+0.1% 7.0mol / L ammonia methanol), ee%>99, as shown in the following table.

[0219]

[0220] Other compounds were prepared by referring to the synthetic methods of similar compounds and intermediates mentioned above.

[0221]

[0222] Comparative Example

[0223] Synthesis of compound B1

[0224]

[0225] Step 1: Synthesis of 6-bromo-3-iodo-4-nitro-1H-indazole

[0226] DMF (40 mL), B1-1 (3.0 g, 12.4 mmol) and N-iodosuccinimide (3.08 g, 13.7 mmol) were added to a 100 mL single-mouth bottle, and the mixture was reacted at 60 ° C for 2 hours. Water was added to the reaction solution, extracted with ethyl acetate, washed with saturated sodium chloride solution, concentrated and column chromatography (PE / EA=3 / 1) was performed to obtain B1-2 (3.9 g, yellow solid), yield: 85.4%, LCMS (ESI): m / z 367.7 [MH] - ; RT = 1.384min (2.50min).

[0227] Step 2: Synthesis of 6-bromo-3-iodo-1-methyl-4-nitro-1H-indazole

[0228] 25 mL DMF, B1-2 (4.0 g, 10.9 mmol), sodium hydride (1.1 g, 27.2 mmol) and iodomethane (2.3 g, 16.3 mmol) were added to a 100 mL single-mouth bottle, and the reaction was carried out at room temperature for 2 hours. The reaction solution was quenched with saturated aqueous ammonium chloride solution, extracted with EA, washed with saturated sodium chloride solution, dried and concentrated, and then column chromatography (PE / EA=5 / 1) was performed to obtain B1-3 (2.8 g, yellow solid), yield: 67.5%, LCMS (ESI): m / z 383.8 [M+H] + ; RT = 1.593min (2.50min). 1 H NMR (400MHz, DMSO-d6): δ8.53(d,J=1.2Hz,1H), 8.02(d,J=1.2Hz,1H), 4.14(s,3H).

[0229] Step 3: Synthesis of N-(6-bromo-1-methyl-4-nitro-1H-indazol-3-yl)-1,1-diphenylmethaneimine

[0230] In a 100 mL single-mouth bottle containing 30 mL of toluene, B1-3 (2.0 g, 5.25 mmol), benzophenone imine (855 mg, 4.72 mmol), cesium carbonate (5.1 g, 15.7 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (152 mg, 0.26 mmol) and tris(dibenzylideneacetone)dipalladium (240 mg, 0.26 mmol) were added, and the mixture was reacted at 80°C overnight. The reaction solution was filtered, and the filtrate was concentrated and then subjected to column chromatography (PE / EA=10 / 1) to obtain B1-4 (1.3 g, yellow solid). Yield: 57.1%, LCMS (ESI): m / z 435.0 [M+H] + ; RT = 1.534min (2.50min).

[0231] Step 4: Synthesis of 6-bromo-1-methyl-4-nitro-1H-indazol-3-amine

[0232] B1-4 (1.1 g, 2.5 mmol) was added to a 100 mL single-mouth bottle containing 8 mL of hydrochloric acid ethyl acetate, and the mixture was reacted at room temperature for 2 hours. The reaction solution was filtered, washed with ethyl acetate, and dried to obtain B1-5 (600 mg, yellow solid). Yield: 87.7%, LCMS (ESI): m / z 272.9 [M+H] + ; RT = 1.180min (2.50min).

[0233] Step 5: Synthesis of 6-bromo-N-ethyl-1-methyl-4-nitro-1H-indazol-3-amine

[0234] B1-5 (500 mg, 1.93 mmol), acetaldehyde (127 mg, 2.89 mmol) and acetic acid (two drops) were added to a 50 mL single-mouth bottle containing 15 mL methanol and 15 mL dioxane. After reacting at 60 ° C for 1 h, sodium cyanoborohydride (363 mg, 5.78 mmol) was added. Water was added to the reaction solution, extracted with EA, washed with saturated sodium chloride solution, dried and concentrated, and then column chromatography (PE / EA=3 / 1) was performed to obtain B1-6 (500 mg, yellow solid). Yield: 87.1%, LCMS (ESI): m / z 301.0 [M+H] + ; RT = 1.690 min (2.50 min).

[0235] Step 6: Synthesis of 6-bromo-N3-ethyl-1-methyl-1H-indazole-3,4-diamine

[0236] In a 100 mL single-mouth bottle containing 10 mL of methanol and 1 mL of water, B1-6 (670 mg, 2.25 mmol) and ammonium chloride (962 mg, 18.0 mmol) were added, and zinc powder (1.18 g, 18.0 mmol) was added under ice bath. The reaction was carried out at room temperature for 2 h. The reaction solution was filtered, the filtrate was concentrated and diluted with water, extracted with EA, the organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain B1-7 (840 mg, yellow solid). LCMS (ESI): m / z 271.0 [M+H] + ; RT = 1.179 min (2.50 min).

[0237] Step 7: Synthesis of 7-bromo-3-ethyl-1-methyl-1,5-dihydropyrazolo[3,4,5-d]quinazolin-4(3H)-one

[0238] Add B1-7 (560 mg, 2.09 mmol) and carbonyldiimidazole (1.69 g, 10.4 mmol) to a 100 mL single-mouth bottle containing 15 mL N-methylpyrrolidone, react at 140°C for 2 h, dilute the reaction solution with water, precipitate, filter and dry to obtain B1-8 (380 mg, yellow solid), yield: 61.9%. LCMS (ESI): m / z 297.1 [M+H] + ; RT = 0.890 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ10.51(s,1H),7.06(s,1H),6.16(s,1H),3.82-3.77(m,5H),1.24-1.20(m,3H).

[0239] Step 8: Synthesis of 3-ethyl-7-(hydroxymethyl)-1-methyl-1,5-dihydropyrazolo[3,4,5-d]quinazolin-4(3H)-one

[0240] 10 mL of dioxane, B1-8 (200 mg, 0.68 mmol), tributyltin methanol (262 mg, 0.82 mmol) and XPhos Pd G2 (27 mg, 0.034 mmol) were added to a 50 mL single-mouth bottle, and the mixture was reacted at 80°C for 16 hours. The reaction solution was concentrated and subjected to column chromatography (PE / EA=1 / 1) to obtain B1-9 (140 mg, yellow solid). Yield: 83.6%, LCMS (ESI): m / z 247.2 [M+H] + ; RT = 0.561 min (2.50 min).

[0241] Step 9: Synthesis of 7-(bromomethyl)-3-ethyl-1-methyl-1,5-dihydropyrazolo[3,4,5-d]quinazolin-4(3H)-one

[0242] Add B1-9 (70 mg, 0.28 mmol), triphenylphosphine (223 mg, 0.85 mmol) and carbon tetrabromide (189 mg, 0.57 mmol) to a 50 mL single-mouth bottle containing 8 mL of DCM and react at room temperature for 2 hours. The reaction solution was concentrated to obtain B1-10 (70 mg, yellow solid). LCMS (ESI): m / z 3B1.0 [M+H] + ; RT = 1.438min (2.50min).

[0243] Step 10: Synthesis of 5-(4-((3-ethyl-1-methyl-4-oxo-1,3,4,5-tetrahydropyrazolo[3,4,5-d]quinazolin-7-yl)methyl)piperazin-1-yl)-6-fluoro-N-methylpicolinamide

[0244] B1-10 (80 mg, 0.26 mmol), intermediate 2a (71 mg, 0.26 mmol) and DIEA (166 mg, 1.29 mmol) were added to a 50 mL single-mouth bottle containing 5 mL of acetonitrile, and the mixture was reacted at 70°C for 2 hours. The reaction solution was purified by Pre-HPLC (formic acid) to obtain B1 (37.72 mg, white solid), with a yield of 31.4%. LCMS (ESI): m / z 467.4 [M+H] + ; RT = 3.215min (6.00min). 1 H NMR (400MHz, DMSO-d6): δ10.31(s,1H),8.40(d,J=4.8Hz,1H),7.86-7.83(m,1H),7.59-7.54(m,1H),6.70(s,1H), 6.13(s,1H),3.82-3.77(m,5H),3.51(s,2H),3.17(s,4H),2.77(d,J=4.8Hz,3H),2.55(s,4H),1.24-1.20(m,3H).

[0245] Synthesis of compound B2

[0246]

[0247]

[0248] Step 1: Synthesis of N-(6-bromo-1-methyl-4-nitro-1H-indazol-3-yl)-2,2,2-trifluoroacetamide

[0249] Add B1-5 (1.8 g, 6.67 mmol), DIEA (2.58 g, 20.0 mmol) and trifluoroacetic anhydride (2.10 g, 10.0 mmol) to a 100 mL single-mouth bottle containing 20 mL of DCM, and react at room temperature for 2 hours. Add water to the reaction solution, extract with EA, wash with saturated sodium chloride solution, dry and concentrate to obtain B2-1 (2.3 g, yellow solid). LCMS (ESI): m / z 368.9 [M+H] + ; RT = 1.352min (2.50min).

[0250] Step 2: Synthesis of N-(6-bromo-1-methyl-4-nitro-1H-indazol-3-yl)-2,2,2-trifluoro-N-methylacetamide

[0251] Add B2-1 (2.3 g, 6.28 mmol), cesium carbonate (4.08 g, 12.6 mmol) and methyl iodide (1.34 g, 9.42 mmol) to a 100 mL single-mouth bottle containing 25 mL of DMF, and react at 30°C for 2 hours. Add water to the reaction solution, extract twice with EA, wash with saturated sodium chloride solution, dry and concentrate to obtain B2-2 (2.3 g, yellow solid). LCMS (ESI): m / z 380.9 [M+H] + ; RT = 1.519 min (2.50 min).

[0252] Step 3: Synthesis of 6-bromo-N,1-dimethyl-4-nitro-1H-indazol-3-amine

[0253] B2-2 (2.6 g, 6.8 mmol) and aqueous sodium hydroxide solution (2 mL, 6N) were added to a single-mouth bottle containing 30 mL of methanol, and the reaction was carried out at room temperature for 30 minutes. The reaction solution was filtered, and the filter cake was washed with water and methanol respectively. After drying, the product B2-3 (1.5 g, black solid) was obtained. The yield: 77.3%, LCMS (ESI): m / z 287.0 [M+H] + ; RT = 1.495min (2.50min). 1 H NMR (400MHz, DMSO-d6): δ8.25(s,1H),7.83(s,1H),5.58-5.54(m,1H),3.88(s,3H),2.87(d,J=5.2Hz,3H).

[0254] Step 4: Synthesis of 6-bromo-N,1-dimethyl-1H-indazole-3,4-diamine

[0255] Add B2-3 (1.4 g, 4.93 mmol) and ammonium chloride (2.1 g, 39.4 mmol) to a 100 mL single-mouth bottle containing 30 mL of methanol and 3 mL of water, and add zinc powder (2.58 g, 39.4 mmol) at 0°C. React at room temperature for 2 hours, filter the reaction solution, concentrate the filtrate to dryness, dilute with water, extract with EA, wash the organic phase with water and saturated brine, dry with anhydrous sodium sulfate, and concentrate to obtain B2-4 (840 mg, brown solid). LCMS (ESI): m / z 255.0 [M+H] + ; RT = 0.558 min (2.50 min).

[0256] Step 5: Synthesis of 7-bromo-1,3-dimethyl-1,5-dihydropyrazolo[3,4,5-d]quinazolin-4(3H)-one

[0257] B2-4 (800 mg, 3.15 mmol) and carbonyldiimidazole (2.55 g, 1.57 mmol) were added to a 50 mL single-mouth bottle containing 15 mL N-methylpyrrolidone, and the mixture was reacted at 140°C for 2 h. The reaction solution was diluted with water, and the precipitate was filtered and dried to obtain B2-5 (700 mg, gray solid), with a yield of 79.3%. LCMS (ESI): m / z 281.0 [M+H] + ; RT = 1.098min (2.50min). 1 H NMR (400MHz, DMSO-d6): δ10.52(s,1H),7.05(s,1H),6.16(s,1H),3.77(s,3H),3.22(s,3H).

[0258] Step 6: Synthesis of 7-(hydroxymethyl)-1,3-dimethyl-1,5-dihydropyrazolo[3,4,5-d]quinazolin-4(3H)-one

[0259] Add B2-5 (300 mg, 1.07 mmol), tributyltin methanol (413 mg, 1.29 mmol) and XPhos Pd G2 (42 mg, 0.05 mmol) to a 50 mL single-mouth bottle containing 10 mL of dioxane, react at 80 ° C for 16 hours, concentrate the reaction solution, and column chromatography (PE / EA = 1 / 1) to obtain B2-6 (220 mg, black solid). Yield: 88.5%, LCMS (ESI): m / z 233.1 [M+H] + ; RT = 0.924min (2.50min).

[0260] Step 7: Synthesis of 7-(bromomethyl)-1,3-dimethyl-1,5-dihydropyrazolo[3,4,5-d]quinazolin-4(3H)-one

[0261] Add B2-6 (35 mg, 0.15 mmol), triphenylphosphine (B19 mg, 0.45 mmol) and carbon tetrabromide (100 mg, 0.30 mmol) to a 50 mL single-mouth bottle containing 8 mL of dichloromethane and react at room temperature for 2 hours. The reaction solution was concentrated to obtain B2-7 (35 mg, yellow solid). LCMS (ESI): m / z 297.0 [M+H] + ; RT = 1.039min (2.50min).

[0262] Step 8: Synthesis of 5-(4-((1,3-dimethyl-4-oxo-1,3,4,5-tetrahydropyrazolo[3,4,5-d]quinazolin-7-yl)methyl)piperazin-1-yl)-6-fluoro-N-methylpicolinamide

[0263] B2-7 (35 mg, 0.24 mmol), intermediate 2a (35 mg, 0.24 mmol) and N,N-diisopropylethylamine (77 mg, 1.19 mmol) were added to a 25 mL single-mouth bottle containing 5 mL of acetonitrile, and the mixture was reacted at 70°C for 2 hours. The reaction solution was purified by Pre-HPLC (formic acid) to obtain B2 (14.35 mg, white solid), with a yield of 27.7%. LCMS (ESI): m / z 453.4 [M+H] + ; RT = 3.166min (6.00min). 1 H NMR (400MHz, DMSO-d6): δ10.35(s,1H),8.41-8.38(m,1H),7.85-7.83(m,1H),7.58-7.54(m,1H),6.70(s, 1H), 6.13 (s, 1H), 3.76 (s, 3H), 3.51 (s, 2H), 3.24 (s, 3H), 3.17 (s, 4H), 2.76 (d, J = 4.8Hz, 3H), 2.54 (s, 4H).

[0264] Synthesis of compound B3

[0265]

[0266] Step 1: Synthesis of 7-bromo-6-fluoro-1,3-dimethyl-1,5-dihydropyrazolo[3,4,5-de]quinazolin-4(3H)-one

[0267] B2-5 (200 mg, 0.71 mmol) and N-fluorobisbenzenesulfonamide (293 mg, 0.93 mmol) were added to a 100 mL single-mouth bottle containing 10 mL of acetic acid, and the mixture was reacted at 100°C for 16 hours. After concentration, B3-1 (50 mg, reddish brown solid) was prepared using Pre-HPLC (ammonium bicarbonate). The yield was 23.5%. LCMS (ESI): m / z 299.0 [M+H] + ; RT = 1.403min (2.50min). 1 H NMR (400MHz, DMSO-d6): δ7.19 (d, J = 3.6Hz, 1H), 3.77 (s, 3H), 3.22 (s, 3H).

[0268] Step 2: 6-Fluoro-7-(hydroxymethyl)-1,3-dimethyl-1,5-dihydropyrazolo[3,4,5-de]quinazolin-4(3H)-one

[0269] Add B3-1 (10 mg, 0.03 mmol), tributyltin methanol (13 mg, 0.04 mmol) and XPhos Pd G2 (11 mg, 0.014 mmol) to a 50 mL single-mouth bottle containing 5 mL of dioxane, and react at 80 ° C for 16 hours. After the reaction solution is concentrated, it is slurried with pure EA to obtain B3-2 (6 mg, brown solid). Yield: 71.5%, LCMS (ESI): m / z 251.1 [M+H] + ; RT = 0.896 min (2.50 min).

[0270] Step 3: 7-(Bromomethyl)-6-fluoro-1,3-dimethyl-1,5-dihydropyrazolo[3,4,5-de]quinazolin-4(3H)-one

[0271] Add B3-2 (11 mg, 0.044 mmol), triphenylphosphine (35 mg, 0.13 mmol) and carbon tetrabromide (29 mg, 0.088 mmol) to a 50 mL single-mouth bottle containing 8 mL of dichloromethane and react at room temperature for 2 hours. The reaction solution was concentrated to obtain B3-3 (11 mg, yellow solid). Crude product, LCMS (ESI): m / z 313.0 [M+H] + ; RT = 1.370min (2.50min).

[0272] Step 4: Synthesis of 5-(4-(((6-fluoro-1,3-dimethyl-4-oxo-1,3,4,5-tetrahydropyrazolo[3,4,5-de]quinazolin-7-yl)methyl)piperazin-1-yl)-N,6-dimethylpicolinamide

[0273] B3-3 (30 mg, 0.1 mmol), intermediate 3a (47 mg, 0.15 mmol) and DIEA (77 mg, 0.6 mmol) were added to a 25 mL single-mouth bottle containing 5 mL of acetonitrile, and the mixture was reacted at 70°C for 1.5 hours. The reaction solution was prepared by Pre-HPLC (formic acid) to obtain B3 (32 mg, white solid), with a yield of 71.3%. LCMS (ESI): m / z 467.8 [M+H] + ; RT = 3.362min (6.00min). 1H NMR (400MHz, DMSO-d6): δ10.68(s,1H),8.55-8.51(m,1H),7.76(d,J=8.4Hz,1H),7.45(d,J=8.4Hz,1H),6.75(d,J =3.2Hz,1H),3.73(s,3H),3.61(s,2H),3.20(s,3H),2.92(s,4H),2.78(d,J=4.8Hz,3H),2.61(s,4H),2.44(s,3H).

[0274] Biological Experiment Examples

[0275] Experimental Example 1: Evaluation of PARP1 / 2 Inhibitory Activity of Compounds

[0276] The PARP1 / 2 inhibitory activity of the compounds of the present invention was tested in an assay using histone as a substrate.

[0277] Experimental purpose: According to the established experimental method, detect the inhibitory IC of the compounds of the present invention on PARP1 / 2 enzyme activity 50 AZD-2281 (Olaparib) was used as a positive control compound.

[0278] Experimental reagents:

[0279] Recombinant human PARP1 protein (Abcam, cat. ab279663); Recombinant human PARP2 protein (BPS, cat. 80502); Recombinant histone H1 (Active Motif, cat. 81126); NAD+, Biotin-Labeled (BPS, cat. 80610); SuperBlock (TBS) Blocking Buffer (Thermo Scientific TM ,cat.37535);Streptavidin(HRP)(Abcam,cat.ab7403); Peroxidase ChemiluminescentSubstrate Kit (Seracare, cat.5430-0040); 20×PBS (CST, cat.9808S); 20×PBST (CST, cat.9809S); AZD2281 (Selleck, cat.S1060)

[0280] Experimental method 1: PARP1 inhibitory activity

[0281] 1. Compound preparation: dilute the compound with DMSO to a solution with a final concentration of 1000 times in a 384-well plate for later use.

[0282] 2. Coating microplate:

[0283] 1) Dilute Histone with PBS, add 25 μL of histone mixture to each well, and incubate for 2 hours. 2) Wash each well with PBST solution 5 times. Remove the solution on a clean paper towel. 3) Add 75 μL of blocking buffer to each well and incubate at room temperature for 1 hour. 4) Wash each well with PBST solution 5 times. Remove the solution on a clean paper towel.

[0284] 3. Ribosylation reaction:

[0285] 1) Transfer 25nL of the reserved 1000-fold final concentration compound to the 384 reaction plate, and add 25nL of 100% DMSO to the Min control well and the Max control well. 2) Prepare a 2.5-fold final concentration PARP1 solution with 1×Assay buffer. 3) Add 10μL of enzyme solution to the compound well and the Max control well; add 10μL of 1×Assay buffer to the Min control well. 4) Centrifuge at 1000rpm for 60 seconds and incubate at room temperature for 15 minutes. 5) Prepare a 1.67-fold final concentration substrate solution with 1×Assay buffer, add 500μM NAD+ to the substrate solution, add 15μL of substrate solution to each well, and start the reaction. 6) Centrifuge at 1000rpm for 60 seconds and incubate at room temperature for 2h. 7) Wash each well 5 times with PBST solution. Remove the solution on a clean paper towel.

[0286] 4. Detection:

[0287] 1) Prepare Streptavidin-HRP solution, add 25μL to each well, centrifuge at 1000rpm for 60 seconds, and incubate at room temperature for 30 minutes. 2) Wash each well 5 times with PBST solution. Remove the solution on a clean paper towel. 3) Add 50μL ELISA Chemiluminescent Substrate to each well. 4) Centrifuge at 1000rpm for 60 seconds, and read with EnSight after 5 minutes.

[0288] 5. Data Analysis

[0289] The inhibition rate was calculated using the following formula: inhibition rate % = (maximum signal - compound signal) / (maximum signal - minimum signal) × 100, where "minimum signal" is the mean of the negative control wells and "maximum signal" is the mean of the positive control wells.

[0290] Fitting dose-effect curve: With the log value of concentration as the X-axis and the percentage inhibition rate as the Y-axis, the log (inhibitor) vs. response-variable slope of the analysis software GraphPadPrism5 was used to fit the dose-effect curve, thereby obtaining the inhibitory IC50 value of the compound of the present invention on the enzyme activity. The fitting formula is: Y = bottom + (top - bottom) / (1 + 10^((logIC50-X)*HillSlope)).

[0291] Experimental method 2: PARP2 inhibition activity

[0292] The PARP2 inhibitory activity experimental method is the same as experimental method 1, except that recombinant human PARP2 solution is used instead of the PARP1 solution used in step 2 "2)" of "3. Ribosylation reaction", and 500 μM NAD+ is not added in the fifth step of the glycosylation reaction.

[0293] Table 1. IC values ​​of the inhibitory effects of the compounds of the present invention on PARP1 / 2 enzymes 50

[0294]

[0295]

[0296] Note: “ / ” means not detected.

[0297] Experimental Example 2: MDA-MB-436 Cell Proliferation Inhibition Test

[0298] Human breast cancer MDA-MB-436 (purchased from ATCC) cells were cultured in DMEM medium (supplemented with 10% fetal bovine serum and 1% double antibody) at 37°C and 5% carbon dioxide. Cells in the logarithmic growth phase were taken, digested and a certain concentration of cell suspension was prepared. The cell suspension was inoculated into a 96-well plate, 100 μL of cell suspension was added to each well of the 96-well plate, and different concentrations of compounds were added after incubation overnight. The plates were placed in a cell culture incubator and incubated for 7 days. After the culture was completed, 50 μL of CellTiter-Glo reagent was added to each well, mixed with a microplate shaker for 2 minutes, and placed at room temperature for 60 minutes. The multimode microplate reader reads the fluorescence value according to the formula: [(1-(RLU compound -RLU blank ) / (RLU control -RLU blank )) × 100%] to calculate the cell proliferation inhibition rate. GraphPad Prism 6.0 software was used to fit and obtain the IC 50 value.

[0299] Table 2. MDA-MB-436 cell proliferation inhibitory activity of the compounds of the present invention

[0300]

[0301]

[0302] The experimental results show that the compound of the present invention has significant proliferation inhibition activity on MDA-MB-436 cells.

[0303] Experimental Example 3: Preliminary pharmacokinetic testing

[0304] 1. Take healthy ICR mice and administer the test compound by intravenous injection (1 mg / kg) and oral gavage (5 mg / kg). For each administration route, 9 male mice, weighing 30-35 g, were randomly divided into 3 groups, 3 mice in each group.

[0305] The animals were fasted for 12 hours before the experiment and allowed to drink water freely. They were fed 4 hours after the administration.

[0306] 2. Blood collection time and sample processing

[0307] Intravenous and oral administration: 0.25h, 0.5h, 1.0h, 2.0h, 3.0h, 4.0h, 6.0h, 8.0h and 24h after administration.

[0308] Blood was collected continuously, and 3 animals were collected at each time point. Plasma collection and processing: 30-40 μL of venous blood was collected from the mouse retroorbital venous plexus at the above set time points, placed in an EDTA-K2 test tube, centrifuged at 3500 rpm for 10 minutes, and plasma was separated and frozen in a -20°C refrigerator.

[0309] 3. Sample testing and data analysis

[0310] The concentration of the compound in mouse plasma was determined by LC / MS / MS, and the pharmacokinetic parameters after administration were calculated using the non-compartmental model using Phoenix 8.3 software (Pharsight, USA).

[0311] 4. Experimental Results

[0312] Table 3. Pharmacokinetic parameters of the compounds of the present invention in mouse plasma

[0313]

[0314] Note: iv: intravenous injection; po: oral administration.

[0315] The experimental results show that the pharmacokinetics of the compound of the present invention in mice after oral administration show a long half-life (T 1 / 2), lower clearance (CL) and higher bioavailability.

[0316] Experimental Example 4: Testing of blood-brain barrier properties in rats

[0317] The compound of the present invention was prepared into an aqueous solution containing 5% DMSO and 95% (containing 20% ​​hydroxypropyl-β-cyclodextrin), and intravenously injected into 3 Wistar Han rats (male) at 1 mg / kg. After 4 hours, carotid artery blood samples were collected and brain tissues were collected after brain perfusion. The drug concentration was determined by LC-MS / MS method, and the results are shown in the following table:

[0318] Table 4. Brain permeability of the compounds of the present invention in the rat blood-brain barrier

[0319]

[0320]

[0321] As shown in Table 1, the compounds of the present invention have high selectivity for PARP1, can reduce the toxicity produced by PARP2 without significantly reducing the efficacy, and have good selectivity for PARP1 / 2. As shown in Table 2, the compounds of the present invention have strong proliferation inhibitory activity on human breast cancer cells (MDA-MB-436). The inventors also unexpectedly found that the compounds of the present invention showed high brain permeability (brain / blood ratio greater than 10%) in the rat blood-brain barrier test, and the compounds of the present invention have good physical and chemical stability, good bioavailability (such as low clearance rate) and good drugability. Therefore, the compounds of the present invention have fewer side effects than olaparib (AZD-2281) and have high clinical application value.

[0322] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents, without departing from the spirit and essence of the claims of the present invention; and these modifications or replacements are still within the scope defined by the claims of the present invention.

Claims

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

2. The compound according to claim 1, or its stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt or hydrate, wherein: In formula (I) Selected from the following structures: The definitions of the substituents are the same as those in claim 1.

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

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

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

6. The compound according to any one of claims 1 to 5, or its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts or hydrates thereof, wherein: The compound of formula I is selected from the following compounds: 。 7. A pharmaceutical composition comprising a compound selected from any one of claims 1 to 6, one or more of its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts and hydrates thereof, and optionally a pharmaceutically acceptable carrier.

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

9. Use of the compound according to any one of claims 1 to 6, or its stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt or hydrate, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for preventing and / or treating a disease improved by inhibiting PARP1; Preferably, the disease improved by inhibiting PARP1 includes cancer.

10. The use according to claim 9, wherein The cancer includes malignancies such as any of ovarian cancer, breast cancer, fallopian tube cancer, endometrial cancer, peritoneal cancer, gastric cancer, colon cancer, bladder cancer, pancreatic cancer, biliary tract cancer, osteosarcoma, cervical cancer, head and neck tumors, germ cell and embryonal cancers, esophageal cancer, malignant glioma, Ewing sarcoma, pancreatic cancer, melanoma, bile duct cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, lymphoma, and blood cancer; Preferably, the genome of the cancer is of the type deficient in homologous recombination repair; Preferably, the cancer is dependent on a pathway where double-strand DNA damage is repaired but homologous recombination is deficient; Preferably, the cancer comprises one or more cancer cells that lack the ability to repair DNA double-strand breaks by homologous recombination relative to normal cells; Preferably, the cancer comprises one or more cancer cells that lack BRCA1 or BRCA2, or that have a BRCA1 or BRCA2 mutation.

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