Fused tricyclic PARP1 inhibitor as well as preparation method and application thereof

Abstract

The invention relates to a fused tricyclic PARP1 inhibitor as well as a preparation method and application thereof. The invention particularly relates to a compound capable of inhibiting activity of poly ADP-ribose polymerase, and a pharmaceutical composition and application thereof. The present invention relates in particular to compounds of formula (I) and also to pharmaceutical compositions comprising these compounds and to the use of these compounds for the preparation of medicaments for the prevention and / or treatment of diseases, in particular diseases ameliorated by inhibition of PARP1. # imgabs0 #

Description

A fused tricyclic PARP1 inhibitor, preparation method and use thereof Technical Field

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

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

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

[0004] PARP inhibitors exert their anti-cancer effects by blocking DNA damage repair in highly mutated cancer cells, resulting in "toxic damage" that causes cell death due to homologous recombination repair (HRR) deficiency. Healthy cells contain multiple signaling pathways for DNA repair, so inhibiting only PARP is not very toxic. However, certain tumor cells, due to specific gene mutations such as BRCA, can disrupt other DNA repair pathways, making them dependent on PARP-1 and therefore particularly sensitive to PARP inhibitors. This is why patients with ovarian and breast cancers carrying BRCA mutations are more likely to benefit from PARP inhibitors. PARP2 levels are low, accounting for only 5% to 10% of total PARP activity. Knocking out PARP1 significantly reduces PARP activity compared to knocking out PARP2 (<10%) (Yélamos et al, The EMBO journal 25.18, 2006). Knocking out PARP1 also blocks the inhibitory activity of olaparib on PARP and eliminates the cell proliferation inhibitory effect of olaparib (Murai et al, Cancer research, 2012). These data suggest that PARP1 is the key to determining the efficacy of PARPi. Literature reports indicate that intact PARP2 in the bone marrow is required for mouse survival; PARP2 deficiency leads to reduced RBC, WBC, and BM cell numbers (Farrés et al, Blood, The Journal of the American Society of Hematology 122.1, 2013); and compared to PARP1 knockout, PARP2 knockout reduces T cell and RBC numbers, whereas PARP1 knockout has no significant effect on T cell (Yélamos et al, Blood, The EMBO journal 25.18, 2006) or RBC numbers (Farrés et al, Cell Death & Differentiation 22.7, 2015). Therefore, PARP1 inhibition is the primary source of drug efficacy, while PARP2 inhibition is the primary source of toxicity. The development of highly selective PARP1 / 2 inhibitors could significantly reduce the toxicity caused by PARP2 without significantly reducing drug efficacy.

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

[0006] Summary of the Invention

[0007] The present invention relates to a compound represented by general formula (I), or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, crystal form, solvate, hydrate or prodrug thereof.

[0008] in,

[0009] X 1 Independently selected from -N-, -NR 14 -、-CR 7 -、-CR 7 R 7’ 、-CH2CR 7 R 7’ -、-CR 7 R 7’ -CH2-, O and S;

[0010] X 2 Independently selected from -N-, -NR 15 -、-CR 8 -、-CR 8 R 8’ -, O and S;

[0011] X 3 Independently selected from -N-, -NR 16 -、-CR 9 -, O and S;

[0012] X 4 、X 8 are each independently selected from -N- and -C-;

[0013] is a single bond or a double bond; and X 1 、X 2 、X 3 、X 4 、X 8 Together they constitute a five-membered heteroaryl group, or a partially saturated five-membered or six-membered heterocyclic group; wherein the heteroaryl group or heterocyclic group each independently contains 1, 2 or 3 heteroatoms independently selected from N, O or S;

[0014] R 7 、R 7’ 、R 8 、R 8’ 、R 9 are each independently selected from hydrogen, halogen, hydroxy, cyano, C1-C3 alkoxy, unsubstituted or substituted C3-C6 cycloalkyl or unsubstituted or substituted C1-C6 alkyl; or, R 7 With R 7’ or R 8 With R 8’ Together they constitute a C3-C6 cycloalkyl group; R7 、R 7’ 、R 8 、R 8’ 、R 9 Each is independently preferably hydrogen, halogen or C1-C4 alkyl; R 7 、R 7’ 、R 8 、R 8’ 、R 9 Each independently more preferably is hydrogen, F or methyl; or preferably, R 7 With R 7’ or R 8 With R 8’ Together they form a C3-C4 cycloalkyl group, such as a cyclopropyl or cyclopentyl group;

[0015] R 14 、R 15 、R 16 R is independently selected from hydrogen, unsubstituted or substituted C3-C6 cycloalkyl, unsubstituted or substituted C1-C6 alkyl; 14 、R 15 、R 16 Each is independently preferably hydrogen or C1-C3 alkyl; R 14 、R 15 、R 16 Each independently more preferably is methyl;

[0016] X 5 、X 6 Each independently selected from -N- and -CR 10 -;R 10 R is selected from hydrogen, halogen, cyano, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted C1-C6 alkyl; 10 Preferably, hydrogen, halogen, cyano or C1-C4 alkyl; R 10 More preferably, it is hydrogen, fluorine, chlorine or methyl;

[0017] X 7 -N- or -CR 17 -;R 17 R is selected from hydrogen, halogen, cyano, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted C1-C6 alkyl; 17 Preferably, hydrogen, halogen, cyano or C1-C4 alkyl; R 17 More preferably, it is hydrogen, fluorine, chlorine or methyl;

[0018] R 1 、R 1 '、R 2 、R 3 、R 4 、R 5are each independently selected from hydrogen, unsubstituted or substituted C1-C6 alkyl; or R 4 、R 5 Together with the carbon atoms to which they are attached, they form a C3-C6 cycloalkyl group;

[0019] s and n are each independently selected from 0, 1 and 2;

[0020] Y is N or CH;

[0021] R 6 Selected from:

[0022] Each R 11 independently selected from halogen, cyano, C1-C3 alkoxy, carbonyl, -CONHR 13 , amino, preferably selected from halogen, -CONHR 13 and cyano; more preferably selected from -CONHR 13 ;

[0023] m is 0, 1, 2, or 3;

[0024] R 12 is selected from hydrogen, halogen, cyano, unsubstituted or substituted C1-C4 alkyl;

[0025] R 13 is hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C3-C8 cycloalkyl, unsubstituted or substituted C1-C6 alkoxy, or unsubstituted or substituted 3-8 membered heterocycloalkyl; preferably, R 13 is hydrogen, unsubstituted or halogen-substituted C1-C4 alkyl, C3-C6 cycloalkyl or C1-C6 alkoxy; the heterocycloalkyl group refers to a heterocycloalkyl group containing 1-3 heteroatoms selected from N, O, and S; preferably, R 13 is methyl, ethyl, C2-C3 alkoxy, cyclopropyl, propylene oxide, oxetane or oxolane;

[0026] Among them, R 1 、R 1’ 、R 2 、R 3 、R 4 、R 5 、R 7 、R 7’ 、R 8 、R 8’ 、R 9 、R 10 、R 12 、R 13 、R 14 、R 15 、R 16 、R17 The substitution mentioned herein refers to substitution by one or more selected from C1-C4 alkyl, halogen, hydroxyl, cyano, amino, carboxyl, and C3-C6 cycloalkyl;

[0027] The conditions are:

[0028] When X 5 and X 8 When both are -N-, X 1 、X 2 、X 3 、X 4 At least one is -N-;

[0029] When X 4 and X 5 When both are -N-, X 3 Also -N-;

[0030] When X 3 is oxygen and X 1 、X 2 、X 4 、X 8 -C-, X 7 -CR 17 -, R 1 、R 1’ 、R 17 Not simultaneously hydrogen;

[0031] Preferably, the condition is: when X 8 When it is -N-, X 1 、X 2 、X 3 At least one is -N-.

[0032] Preferably, the condition is: X 5 、X 6 Each independently selected from -CR 10 -, and X 7 -CR 17 -.

[0033] More preferably, the condition is: when X 8 When it is -N-, X 1 、X 2 、X 3 At least one is -N-;

[0034] When X 4 and X 5 When both are -N-, X 3 Also -N-;

[0035] When X 3 is oxygen and X 1 、X 2 、X4 、X 8 -C-, X 7 -CR 17 -, R 1 、R 1’ 、R 17 Not hydrogen at the same time.

[0036] In a preferred embodiment of the present invention, there is provided a compound represented by general formula (II), or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, crystal form, solvate, hydrate or prodrug thereof,

[0037] in,

[0038] X 1 Independently selected from -N-, -NR 14 -、-CR 7 -, O and S;

[0039] X 2 Independently selected from -N-, -NR 15 -and-CR 8 -, O and S;

[0040] X 3 Independently selected from -N-, -NR 16 -and-CR 9 -, O and S;

[0041] X 4 、X 8 are each independently selected from -N- and -C-;

[0042] is a single bond or a double bond; and X 1 、X 2 、X 3 、X 4 、X 8 Together they constitute a five-membered heteroaryl group or a partially saturated five-membered heterocyclic group;

[0043] R 7 、R 8 、R 9 R is independently selected from hydrogen, halogen, unsubstituted or substituted C3-C6 cycloalkyl or unsubstituted or substituted C1-C6 alkyl; 7 、R 8 、R 9 The substitution mentioned in the above is substituted by one or more selected from C1-C4 alkyl, halogen, hydroxyl, cyano, amino, carboxyl, C3-C6 cycloalkyl; R 7 、R 8 、R 9Preferably, hydrogen, halogen or C1-C4 alkyl; R 7 、R 8 、R 9 More preferably, it is hydrogen, F or methyl;

[0044] X 5 、X 6 、X 7 、R 1 、R 1’ 、R 2 、R 3 、R 4 、R 5 、R 6 、R 10 、R 11 、R 12 、R 13 、R 14 、R 15 、R 16 、R 17 , s, n, m, and Y are as described in the general formula (I).

[0045] In a preferred embodiment of the present invention, in the compounds represented by general formula (I) and (II), X 1 、X 2 、X 3 、X 4 、X 8 Together they constitute a partially saturated five-membered heterocyclic group or a partially saturated six-membered heterocyclic group. 4 When it is C, X 3 N or -NR 16 More preferably, when X 4 When C, and X 3 is N.

[0046] In a preferred embodiment of the present invention, in the compounds represented by general formula (I) and (II), X 1 、X 2 、X 3 、X 4 、X 8 Together they constitute a five-membered heteroaryl group.

[0047] In a preferred embodiment of the present invention, in the compounds represented by general formula (I) and (II), X 1 、X 2 、X 3 、X 4 、X 8 Together they constitute a partially saturated five-membered heterocyclic group.

[0048] In a preferred embodiment of the present invention, in the compounds represented by general formula (I) and (II), X1 、X 2 、X 3 、X 4 、X 8 Together they constitute a partially saturated six-membered heterocyclic group.

[0049] In a preferred embodiment of the present invention, in the compounds represented by general formula (I) and (II), X 1 、X 2 、X 3 、X 4 、X 8 At least one of is nitrogen, and X 1 、X 2 、X 3 、X 4 、X 8 Together they constitute a partially saturated five-membered or six-membered heterocyclic group.

[0050] In a preferred embodiment of the present invention, among the compounds represented by general formula (I) and (II),

[0051] X 1 Independently selected from -N-, -NR 14 -、-CR 7 -、-CR 7 R 7’ 、-CH2CR 7 R 7’ -and-CR 7 R 7’ -CH2-;

[0052] X 2 Independently selected from -N-, -NR 15 -、-CR 8 -and-CR 8 R 8’ -;

[0053] X 4 、X 8 are each independently selected from -N- and -C-;

[0054] X 3 Independently selected from -N-, -NR 16 -and-CR 9 -;and

[0055] X 1 、X 2 、X 3 、X 4 、X 8 At least one of is nitrogen, and X 1 、X 2 、X 3 、X4 、X 8 Together they constitute a partially saturated five-membered or six-membered heterocyclic group.

[0056] In a preferred embodiment of the present invention, among the compounds represented by general formula (I) and (II),

[0057] X 1 Independently selected from -CR 7 -、-CR 7 R 7’ 、-CH2CR 7 R 7’ -and-CR 7 R 7’ -CH2-;

[0058] X 2 Independently selected from -CR 8 -and-CR 8 R 8’ -;

[0059] X 4 for -C-;

[0060] X 8 is -N-;

[0061] X 3 Selected from -N- and -NR 16 ;

[0062] And X 1 、X 2 、X 3 、X 4 、X 8 Together they constitute a partially saturated five-membered or six-membered heterocyclic group.

[0063] In the above embodiment, the “partially saturated five-membered or six-membered heterocyclic group” refers to an unsaturated but non-aromatic heterocyclic group.

[0064] Preferably, in the general formula (I) or (II) Selected from the following structures:

[0065] More preferably, it is selected from the following structures:

[0066] Wherein, in the general formula (I) or (II) Preferably selected from the following structures:

[0067] More preferably, it is selected from the following structures:

[0068] More preferably, it is selected from the following structures:

[0069] Among them, R 2 、R 3 、R 4 、R 5 、R 11 、R 12 、R 13 , and m are defined as above.

[0070] More preferably, the compound of formula (I) is selected from the following specific compounds:

[0071] On the other hand, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts, crystal forms, solvates, hydrates or prodrugs, and a pharmaceutically acceptable carrier.

[0072] In certain embodiments of the pharmaceutical composition, the pharmaceutical composition is formulated for intravenous administration, intramuscular administration, oral administration, rectal administration, inhalation administration, nasal administration, topical administration, ophthalmic administration, or ear administration. In other embodiments of the pharmaceutical composition, the pharmaceutical composition is a tablet, pill, capsule, liquid, inhalant, nasal spray solution, suppository, solution, emulsion, ointment, eye drops, or ear drops. In other embodiments of the pharmaceutical composition, it further comprises one or more additional therapeutic agents.

[0073] On the other hand, the present invention provides the use of a compound of formula (I), or its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts, crystal forms, solvates, hydrates or prodrugs, or the pharmaceutical composition thereof in the preparation of a medicament for preventing, treating or ameliorating diseases by inhibiting PARP1.

[0074] On the other hand, the present invention provides a method for preventing, treating or ameliorating diseases by inhibiting PARP1, which comprises administering to an individual in need of such treatment an effective amount of a compound of formula (I), or its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts, crystal forms, solvates, hydrates or prodrugs, or said pharmaceutical composition.

[0075] In some embodiments of the present invention, the disease includes but is not limited to cancer.

[0076] In some embodiments of the present invention, the cancer includes, but is not limited to, a malignant tumor, such as any one of ovarian cancer, breast cancer, fallopian tube cancer, endometrial cancer, peritoneal cancer, gastric cancer, colon cancer, bladder cancer, pancreatic cancer, biliary tract cancer, osteosarcoma, cervical cancer, head and neck tumors, germ cell and embryonal cancers, esophageal cancer, malignant glioma, Ewing sarcoma, pancreatic cancer, melanoma, bile duct cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, lymphoma, and blood cancer.

[0077] In some embodiments of the present invention, the cancer genome is of a type deficient in homologous recombination repair.

[0078] In some embodiments of the invention, the cancer is dependent on a pathway that is deficient in homologous recombination repair of DNA double-strand damage.

[0079] 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.

[0080] In some embodiments of the invention, the cancer comprises one or more cancer cells that lack BRCA1 or BRCA2 or that have a BRCA1 or BRCA2 mutation.

[0081] Terminology

[0082] In the present invention, unless otherwise specified, the terms used in the present invention have the meanings defined below. Terms not clearly defined in the present invention have the general meanings generally understood by those skilled in the art.

[0083] As used herein, the term "halogen" or "halo" refers to fluorine, chlorine, bromine, and iodine.

[0084] As used herein, "heteroaryl" refers to a monocyclic ring system having 5-6 (5-6 members) or 6 (6 members) ring atoms, which contains 1, 2, 3 or 4 heteroatoms independently selected from N, O or S, and the remaining ring atoms are carbon atoms. For example, it contains 1 N heteroatom and optionally further contains 1, 2 or 3 heteroatoms independently selected from N, O or S. When the total number of S and O atoms in the heteroaryl exceeds 1, these S and O heteroatoms are not adjacent to each other. For example, the heteroaryl includes but is not limited to: 5-6 membered monocyclic heteroaryl, that is, a monocyclic aromatic hydrocarbon group having 5 or 6 ring atoms, which contains 1, 2 or 3 heteroatoms independently selected from N, O or S, and the remaining ring atoms are carbon atoms; examples of the heteroaryl include but are not limited to: pyrrolyl, pyrazolyl, imidazolyl, furanyl, thienyl, thiazolyl, isothiazolyl, pyrazinyl, pyridazinyl, pyridinyl or pyrimidinyl.

[0085] As used herein, "heterocyclyl" refers to a fully saturated or partially saturated, non-aromatic monocyclic, bicyclic or tricyclic cyclic group having 3-15 ring atoms (e.g., 4-12 ring atoms, 3-10 ring atoms, 5-10 ring atoms, 4-7 ring atoms, 5-6 ring atoms), for example, a 4- to 7-membered monocyclic, 5- to 6-membered monocyclic ring system containing 1, 2, 3 or 4 heteroatoms independently selected from N, O or S, and the remaining ring atoms being carbon atoms. Nitrogen heteroatoms and sulfur heteroatoms may also be optionally oxidized, for example, sulfur heteroatoms may form -S(O)- or -S(O)2- structures. The heterocyclyl may be a monocyclic group, a bicyclic group, a fused ring group, a spirocyclic group, and a bridged ring group. "5-6 membered heterocyclyl" refers to a heterocyclyl group having 5 or 6 ring atoms, which contains 1, 2, 3 or 4 heteroatoms independently selected from N, O or S, for example, contains 1, 2 or 3 N heteroatoms, which is a monocyclic ring.

[0086] As used herein, "heterocycloalkyl" is a fully saturated heterocyclic group as defined herein, for example, a "3-8 membered heterocycloalkyl" is a saturated heterocyclic ring having 3-8 ring atoms containing 1, 2, 3 or 4 heteroatoms independently selected from N, O or S, for example containing 1 N or O heteroatom.

[0087] As used herein, the terms "optional," "optionally," or "optionally" mean that the subsequently described substitution pattern, event, or circumstance may or may not occur, and that the description includes instances where the substitution pattern occurs as well as instances where the substitution pattern does not occur. For example, "optionally substituted alkyl" includes "unsubstituted alkyl" and "substituted alkyl" as defined herein. It will be understood by those skilled in the art that, for any group containing one or more substituents, the group does not include any substitution pattern that is sterically impractical, chemically incorrect, synthetically infeasible, and / or inherently unstable.

[0088] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the compounds of the present invention and is not biologically or otherwise undesirable. Non-limiting examples of such salts include non-toxic, inorganic or organic base or acid addition salts of the compounds of the present invention. In many cases, the compounds of the present invention are capable of forming acid salts and / or base salts due to the presence of amino and / or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; ammonium, potassium, sodium, calcium, and magnesium salts are particularly preferred. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins, and the like, particularly isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. Pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound (basic or acidic moiety) by conventional chemical methods. Generally, the salts can be prepared by reacting the free acid form of the compound with a stoichiometric amount of an appropriate base (e.g., hydroxide, carbonate, bicarbonate, etc., of Na, Ca, Mg, or K) or by reacting the free base form of the compound with a stoichiometric amount of an appropriate acid. Such reactions are typically carried out in water or an organic solvent, or a mixture of the two. In general, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred where practicable. Other suitable salts can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Company, Easton, Pa., (1985), which is incorporated herein by reference.

[0089] As used herein, the term "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, the like, and combinations thereof, which are well known to those of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, it is contemplated for use in the therapeutic or pharmaceutical composition.

[0090] As used herein, the term "solvate" is intended to include stoichiometric or non-stoichiometric solvent addition forms. If the solvent is water, the solvate formed is a hydrate, and when the solvent is ethanol, the solvate formed is an ethanolate. Hydrates are formed by one or more molecules of water with one molecule of the substance, wherein the water retains its molecular state of HO. Such a combination can form one or more hydrates, such as hemihydrates, monohydrates, and dihydrates.

[0091] As used herein, "prodrug" refers to a chemically modified active or inactive compound that, after administration to a subject, undergoes physiological action in the body (e.g., hydrolysis, necrolysis, etc.) to become a compound of the present invention. The adaptability and technology of making and using prodrugs are well known to those skilled in the art.

[0092] The term "therapeutically effective amount" of the compound of the present invention refers to an amount of the compound of the present invention that can elicit a biological or medical response in an individual or improve symptoms, slow down or delay disease progression, or prevent disease.

[0093] As used herein, the term "subject" refers to an animal. Preferably, the animal is a mammal. Subject also refers to, for example, primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In a preferred embodiment, the subject is a human.

[0094] 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.

[0095] As used herein, the term "treating" any disease or condition refers, in one embodiment, to ameliorating the disease or condition (i.e., arresting or slowing the progression of the disease or at least one of its clinical symptoms). In another embodiment, "treating" refers to improving at least one physical parameter, which may not be perceptible to the patient. In another embodiment, "treating" refers to modulating the disease or condition physically (e.g., stabilizing a perceptible symptom) or physiologically (e.g., stabilizing a physical parameter), or both. Beneficial effects

[0096] The main advantages of the present invention are that 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. DETAILED DESCRIPTION

[0097] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial sources. The starting materials can generally be obtained from commercial sources or easily prepared using methods known to those skilled in the art.

[0098] In each embodiment, the experimental instruments or materials used are as follows:

[0099] 1 H NMR was recorded on a Varian Mercury-300 or Varian Mercury-400 nuclear magnetic resonance spectrometer. 13 C NMR spectra were recorded on a Varian Mercury-400, Varian Mercury-500, or Varian Mercury-600 nuclear magnetic resonance instrument, and chemical shifts are expressed in δ (ppm). Mass spectra were recorded on Finnigan / MAT-95 (EI), Finnigan LCQ / DECA, and Micromass Ultra Q-TOF (ESI) mass spectrometers. Silica gel with a mesh size of 200-300 was used for reversed-phase preparative HPLC separations.

[0100] Abbreviations

[0101] Synthesis of key intermediates

[0102] Intermediate 1a: (R)-6-Fluoro-N-methyl-5-(2-methylpiperazin-1-yl)picolinamide hydrochloride

[0103] Step 1: Synthesis of methyl 5-bromo-6-fluoropicolinate

[0104] Compound 1a-1 (1 g, 4.6 mmol), acetonitrile (30 mL), and silver difluoride (1.76 g, 13.9 mmol) were added sequentially to a 50 mL single-necked flask and stirred overnight. The reaction mixture was filtered, and the filtrate was concentrated and purified via a silica gel column (PE:EA = 5:1) to afford 1a-2 (450 mg, white solid) in a 42% yield. LCMS (ESI): m / z 233.9 [M+H] + ; RT = 1.51 min (3.00 min).

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

[0106] To a 50 mL single-necked flask, 1a-2 (450 mg, 1.9 mmol), (R)-4-Boc-2-methylpiperazine (577 mg, 2.9 mmol), Ruphos Pd G3 (159 mg, 0.19 mmol), cesium carbonate (1.5 g, 4.7 mmol), and dioxane (6 mL) were added sequentially. The mixture was heated at 80°C overnight under nitrogen. The reaction mixture was concentrated and purified on a silica gel column (PE:EA = 2:1) to afford product 1a-3 (300 mg, yellow solid) in a 44% yield. LCMS (ESI): m / z 354.1 [M+H] + ; RT = 1.80 min (3.00 min).

[0107] Step 3: tert-Butyl (R)-4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)-3-methylpiperazine-1-carboxylate

[0108] 1a-3 (100 mg, 0.28 mmol) and methylamine ethanol solution (2 mL) were added to a 50 mL single-necked flask, and the reaction mixture was stirred overnight. Concentration afforded 1a-4 (80 mg, yellow solid) in an 80% yield. LCMS (ESI): m / z 297.1 [M-56+H] + ; RT = 1.70 min (3.00 min).

[0109] Step 4: Synthesis of (R)-6-fluoro-N-methyl-5-(2-methylpiperazin-1-yl)picolinamide hydrochloride

[0110] To a 20 mL single-necked flask, add 1a-4 (80 mg, 0.23 mmol), EA (2 mL), and a 4 M hydrochloric acid solution in dioxane (2 mL). Stir at room temperature for 2 hours. Concentrate to afford 1a (65 mg, yellow oil). Yield: 100%. LCMS (ESI): m / z 253.2 [M+H] +; RT = 0.99 min (3.00 min).

[0111] Intermediate 2a: Synthesis of N-methyl-5-(piperazin-1-yl)pyridineamide hydrochloride

[0112] Step 1: Synthesis of tert-butyl 4-(2-fluoro-6-(methoxycarbonyl)pyridin-3-yl)piperazine-1-carboxylate

[0113] A 50 mL flask was charged with 1a-2 (500 mg, 2.1 mmol), 1-tert-butyloxycarbonylpiperazine (600 mg, 3.2 mmol), Ruphos Pd G3 (180 mg, 0.21 mmol), cesium carbonate (1.7 g, 5.2 mmol), and dioxane (15 mL). The mixture was heated at 80°C overnight under nitrogen. The reaction mixture was concentrated and purified on a silica gel column (PE:EA = 2:1) to afford 2a-1 (640 mg, yellow solid) in an 88% yield. LCMS (ESI): m / z 340.1 [M+H]. + ; RT = 1.74 min (3.00 min).

[0114] Step 2: Synthesis of 5-(4-(tert-Butyloxycarbonyl)piperazin-1-yl)-6-fluoropicolinic acid

[0115] To a dry 50 mL single-necked flask, 2a-1 (320 mg, 0.94 mmol) and THF (8 mL) were added dropwise. An aqueous solution of lithium hydroxide monohydrate (200 mg, 4.7 mmol) (8 mL) was added dropwise and stirred for 2 hours. The reaction mixture was adjusted to pH 6 with 1 M hydrochloric acid. The reaction mixture was concentrated and purified via a reverse phase column (2% to 40% acetonitrile in water) to afford 2a-2 (300 mg, yellow solid) in a 92% yield. LCMS (ESI): m / z 326.1 [M+H] + ; RT = 1.26 min (3.00 min).

[0116] Step 3: Synthesis of tert-butyl 4-(2-fluoro-6-(methylcarbamoyl)pyridin-3-yl)piperazine-1-carboxylate

[0117] Compound 2a-2 (300 mg, 0.92 mmol), 1-hydroxybenzotriazole (149 mg, 1.1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (211 mg, 1.1 mmol), DIEA (237 mg, 1.84 mmol), DMF (6 mL), and methylamine hydrochloride (123 mg, 1.84 mmol) were added to a dry 50 mL single-necked bottle and stirred overnight at room temperature. The reaction mixture was added with water (20 mL) and extracted three times with EA (30 mL). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by reverse phase column chromatography (20% to 70% acetonitrile in water) to afford 2a-3 (250 mg, white solid) in an 80% yield. LCMS (ESI): m / z 283.1 [M-100+H]. + ; RT = 1.66 min (3.00 min).

[0118] Step 4: Synthesis of 6-fluoro-N-methyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0119] To a dry 20 mL single-necked flask, 2a-3 (250 mg, 0.74 mmol), EA (3 mL), and 4 M hydrochloric acid in dioxane (1 mL) were added sequentially. The mixture was stirred at room temperature for 2 h and concentrated to afford product 2a (200 mg, yellow solid). Yield: 100%. LCMS (ESI): m / z 239.1 [M+H] + ; RT = 0.91 min (3.00 min). 1 H NMR (600MHz, CD3OD): 7.98-7.96 (m, 1H), 7.67-7.64 (m, 1H), 3.50-3.48 (m, 4H), 3.45-3.43 (m, 4H), 2.93 (d, J = 3.6Hz, 3H).

[0120] Intermediate 3a: Synthesis of N-methyl-5-(piperazin-1-yl)pyridineamide hydrochloride

[0121] The synthesis method refers to the synthesis of intermediate 1a, except that Boc-piperazine is used instead of (R)-4-Boc-2-methylpiperazine in step 2 of the preparation method of intermediate 1a, and compound 1a-1 is used instead of compound 1a-2 in step 2. LCMS (ESI): m / z 221.2 [M+H] + ; RT = 0.285 min (6.00 min).

[0122] Intermediate 5a: Methyl (6-fluoro-4-oxo-4,5-dihydropyrrolo[1,2-a]quinoxalin-7-yl)methanesulfonate

[0123] Step 1: Synthesis of 1-bromo-2,4-difluoro-3-nitrobenzene

[0124] 5a-1 (25.0 g, 157 mmol) and 100 mL of concentrated sulfuric acid were added to a 250 mL single-necked bottle. N-bromosuccinimide (33.6 g, 188 mmol) was slowly added under ice bath. The reaction was allowed to react overnight at 80°C. The reaction solution was slowly poured into ice water for dilution. The product was extracted twice with EA, dried, concentrated, and then purified by column chromatography (PE) to obtain product 5a-2 (36 g, yellow oil). Yield: 96.6%. 1 H NMR (400MHz, DMSO-d6): δ8.19-8.14(m,1H),7.56-7.51(m,1H).

[0125] Step 2: Synthesis of methyl 1-(4-bromo-3-fluoro-2-nitrophenyl)-1H-pyrrole-2-carboxylate

[0126] 5a-2 (0.5 g, 2.1 mmol), methyl 1H-pyrrole-2-carboxylate (244 mg, 1.89 mmol), and cesium carbonate (1.37 g, 4.2 mmol) were added to a 100 mL single-necked flask containing 15 mL of DMF. The mixture was reacted at room temperature for 5 h. The reaction solution was diluted with water and extracted twice with EA. After drying and concentration, the mixture was purified by column chromatography (PE:EA = 5:1) to afford 5a-3 (4.0 g, yellow solid). Yield: 65.5%, LCMS (ESI): m / z 345.0 [M+H] + ; RT = 1.783 min (2.50 min).

[0127] Step 3: Synthesis of 7-bromo-6-fluoropyrrolo[1,2-a]quinoxalin-4(5H)-one

[0128] 5a-3 (6.5 g, 18.9 mmol) and iron powder (21 g, 37.9 mmol) were added to a single-necked bottle containing 250 mL of acetic acid. The mixture was reacted at 110°C for 3 h. EA was added to the reaction solution and the mixture was directly filtered. The filtrate was concentrated and saturated sodium bicarbonate solution was added. The mixture was extracted three times with EA. The organic phase was washed with water, dried, and concentrated to obtain 5a-4 (650 mg, yellow solid). Yield: 12.2%. 1 H NMR (400MHz, DMSO-d6): δ11.46(s,1H),8.22(m,1H),7.89(d,J=8.0Hz,1H),7.53-7.48(m,1H),7.10(d,J=3.2Hz,1H),6.74-6.73(m,1H).

[0129] Step 4: Synthesis of 6-fluoro-7-(hydroxymethyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one

[0130] To a three-necked flask containing 15 mL of anhydrous dioxane was added 5a-4 (700 mg, 2.49 mmol), XPhos Pd G2 (197 mg, 0.25 mmol), and tri-tert-butyltin methanol (960 mg, 2.99 mmol). The mixture was reacted at 100°C overnight. The reaction solution was quenched with potassium fluoride solution, filtered, and the filtrate was concentrated to afford 5a-5 (430 mg, yellow solid) in a yield of 74.4%. LCMS (ESI): m / z 233.1 [M+H] + ; RT = 0.877 min (2.00 min). 1 H NMR (400MHz, DMSO-d6): δ11.23(s,1H),8.18-8.17(m,1H),7.87(d,J=8.4Hz,1H),7.28-7.24(m,1H),7.08-7.07(s,1H),6.72-6.70(m, 1H),5.36-5.33(m,1H),4.59(d,J=5.6Hz,2H).

[0131] Step 5: Synthesis of methyl (6-fluoro-4-oxo-4,5-dihydropyrrolo[1,2-a]quinoxalin-7-yl)methanesulfonate

[0132] To a 100 mL three-necked flask containing 8 mL of THF was added 5a-5 (150 mg, 0.65 mmol) and triethylamine (164 mg, 1.63 mmol). Methanesulfonyl chloride (89 mg, 0.78 mmol) was added under ice-cooling and the mixture was allowed to react at room temperature for 3 h. The reaction solution was directly concentrated to afford 5a (120 mg, yellow solid). LCMS (ESI): m / z 309.1 [MH] - ; RT = 1.241 min (2.50 min).

[0133] Intermediate 6a: 7-(1-bromoethyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one

[0134] Step 1: Synthesis of methyl 1-(4-bromo-2-nitrophenyl)-1H-pyrrole-2-carboxylate

[0135] 6a-1 (500 mg, 2.27 mmol), methyl 1H-pyrrole-2-carboxylate (341.26 mg, 2.73 mmol), and cesium carbonate (1.48 g, 4.55 mmol) were dissolved in DMF (10 mL). The reaction solution was stirred at 80°C for 16 hours. The reaction solution was diluted with water (20 mL), extracted with EA (20 mL x 3), dried, concentrated, and then column chromatography (PE:EA = 10:1) afforded 6a-2 (383 mg, 1.18 mmol, yield: 51.9%). LCMS (ESI): m / z 325.0 [M]; RT = 1.341 min (2.50 min).

[0136] Step 2: Synthesis of 7-bromopyrrolo[1,2-a]quinoxalin-4(5H)-one

[0137] 6a-2 (1.5 g, 4.61 mmol) and iron powder (5.15 g, 92.27 mmol) were dissolved in acetic acid (50 mL) and stirred at 110°C for 3 h. The mixture was concentrated to afford a dark brown solid, which was diluted with DCM. The organic phase was washed with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, and concentrated to afford 6a-3 (1.3 g, 4.49 mmol, 100% yield, as a yellow solid). 1 H NMR (400MHz, DMSO-d6): δ11.35(s,1H),8.20(s,1H),8.03(d,J=8.4Hz,1H),7. 45(s,1H),7.37(d,J=8.4Hz,1H),7.06(d,J=3.6Hz,1H),6.71(d,J=2.8Hz,1H).

[0138] Step 3: Synthesis of 7-acetylpyrrolo[1,2-a]quinoxalin-4(5H)-one

[0139] 6a-3 (500 mg, 1.9 mmol), tributyl(1-ethoxyethylene)tin (1.37 g, 3.8 mmol), and PdCl2(PhP3)2 (200 mg, 0.29 mmol) were dissolved in 1,4-dioxane (25 mL) and reacted at 100°C for 16 hours. After cooling to room temperature, 1M aqueous hydrochloric acid (10 mL) was added to the reaction solution and stirred for 10 minutes. Extraction with EA (10 mL × 3), drying, concentration, and column chromatography (THF:PE = 1:1) afforded crude product 6a-4 (220 mg, 0.97 mmol, 51% yield). LCMS (ESI): m / z 227 [M+H] + ; RT = 0.947 (2.50 min).

[0140] Step 4: Synthesis of 7-(1-hydroxyethyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one

[0141] Sodium borohydride (73.58 mg, 1.94 mmol) was slowly added to a solution of 6a-4 (220 mg, 0.97 mmol) in methanol (5 mL) at 0°C and allowed to react for 2 h at room temperature. The solution was quenched with saturated aqueous ammonium chloride (5 mL), extracted with EA, dried, concentrated, and purified by column chromatography (DCM:methanol = 1:1) to afford 6a-5 (200 mg, 0.88 mmol, yield: 90.3%, white solid). LCMS (ESI): m / z 229.1 [MH] - ; RT = 0.877 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ11.23(s,1H),8.15(s,1H),7.98(d,J=8.4Hz,1H),7.33(s,1H),7.16(d,J=8.4Hz,1H) ,7.02(d,J=3.6Hz,1H),6.67(t,J=3.2Hz,1H),5.30(d,J=4.0Hz,1H),4.78-4.75(m,1H),1.35(d,J=6.4Hz,3H).

[0142] Step 5: Synthesis of 7-(1-bromoethyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one

[0143] Phosphorus tribromide (711.55 mg, 2.63 mmol) was slowly added to a solution of 6a-5 (200 mg, 0.87 mmol) in DCM (5 mL) at 0°C and allowed to react for 2 h at room temperature. The reaction solution was quenched with water, extracted with EA, dried, and concentrated to afford the crude product 6a (200 mg, 0.69 mmol, yield: 79.0%), as a white solid. LCMS (ESI): m / z 289.1 [MH] - ; RT = 1.447 (2.50 min).

[0144] Intermediate 7a: 7-(1-bromomethyl)-6-F-pyrazolo[1,2-a]quinoxalin-4(5H)-one

[0145] The synthesis method is similar to that of intermediate 6a, except that 5a-4 prepared in step 3 of intermediate 5a is used instead of 6a-3 used in step 4 of intermediate 6a. LCMS (ESI): m / z 310.0 [M+H] + ; RT = 1.202 min (2.50 min).

[0146] Intermediate 8a: 7-(Bromomethyl)pyrazolo[1,5-a]quinoxalin-4(5H)-one

[0147] Step 1: Synthesis of N-(5-bromo-2-fluorophenyl)-1H-pyrazole-5-carboxamide

[0148] 8a-1 (3.0 g, 15.8 mmol), 1H-pyrazole-5-carboxylic acid (1.95 g, 17.4 mmol), DIEA (6.1 g, 47.4 mmol), and HATU (9.0 g, 23.7 mmol) were added to a single-necked flask containing 30 mL of DMF. The mixture was reacted at room temperature for 16 h. The reaction solution was diluted with water, extracted with EA, washed with saturated sodium chloride solution, dried, concentrated, and purified by column chromatography (PE:EA = 5:1) to afford 8a-2 (2.0 g, yellow solid). Yield: 44.8%. LCMS (ESI): m / z 286.0 [M+H] + ; RT = 1.03 min (2.00 min).

[0149] Step 2: Synthesis of 7-bromopyrazolo[1,5-a]quinoxalin-4(5H)-one

[0150] 8a-2 (2.0 g, 7.07 mmol) was added to a three-necked flask containing 30 mL of DMAC. Sodium hydride (565 mg, 14.1 mmol) was added under ice-cooling. The reaction was incubated at 145°C for 16 h. The reaction solution was quenched with saturated ammonium chloride solution to precipitate a solid, which was filtered and dried to obtain 8a-3 (1.5 g, yellow solid). Yield: 80.7%, LCMS (ESI): m / z 264.0 / 266.0 [M+H] + ; RT = 0.93 min (2.00 min). 1 H NMR (400MHz, DMSO-d6): δ11.50(s,1H),8.03-7.97(m,2H),7.47-7.39(m,2H),7.10(s,1H).

[0151] Step 3: Synthesis of 7-(hydroxymethyl)pyrazolo[1,5-a]quinoxalin-4(5H)-one

[0152] To a single-necked flask containing 10 mL of dioxane was added 8a-3 (500 mg, 1.90 mmol), tributyltin methanol (671 mg, 2.09 mmol), and XPhos Pd G2 (75 mg, 0.095 mmol). The mixture was reacted at 80°C under nitrogen for 16 h. The reaction solution was directly concentrated and then purified by column chromatography (DCM:methanol = 15:1) to afford product 8a-4 (190 mg, yellow solid) in a yield of 45.0%. LCMS (ESI): m / z 214.1 [MH] - ; RT = 0.984 min (2.50 min).

[0153] Step 4: Synthesis of 7-(bromomethyl)pyrazolo[1,5-a]quinoxalin-4(5H)-one

[0154] 8a-4 (80 mg, 0.37 mmol), triphenylphosphine (293 mg, 1.12 mmol), and carbon tetrabromide (248 mg, 0.74 mmol) were added to a 100 mL single-necked flask containing 8 mL of DCM and reacted at room temperature for 2 hours. The reaction solution was directly concentrated to give crude product 8a (80 mg, yellow solid). LCMS (ESI): m / z 278.0 [M+H] + ; RT = 1.405 min (2.50 min).

[0155] Intermediate 9a: 7-(1-bromoethyl)pyrazolo[1,5-a]quinoxalin-4(5H)-one

[0156] The synthesis method is similar to that of intermediate 6a, except that 8a-3 prepared in step 2 of intermediate 8a is used instead of 6a-3 used in step 4 of intermediate 6a. LCMS (ESI): m / z 290.0 [MH] - ; RT = 1.300 min (2.50 min).

[0157] Intermediate 10a: Synthesis of methyl (6-fluoro-4-oxo-4,5-dihydropyrazolo[1,5-a]quinoxalin-7-yl)methanesulfonate

[0158] Step 1: Synthesis of N-(2,6-difluorophenyl)acetamide

[0159] 10a-1 (2.00 g, 15.49 mmol), DCM (20 mL), and acetic anhydride (1.53 mL, 16.27 mmol) were added sequentially to a single-necked flask. The mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure. The residue was diluted with water (20 mL) and the pH was adjusted to 8 with saturated sodium bicarbonate aqueous solution. A white solid precipitated. The filter cake was collected by filtration to obtain 10a-2 (2.60 g, white solid). Yield: 98.07%. LCMS (ESI): m / z 172.2 [M+H] + ; RT = 1.036 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ9.69(s,1H),7.37-7.30(m,1H),7.17-7.13(m,2H),2.08(s,3H).

[0160] Step 2: Synthesis of N-(3-bromo-2,6-difluorophenyl)acetamide

[0161] To a single-necked flask, 10a-2 (1.00 g, 5.84 mmol), concentrated sulfuric acid (10 mL), and N-bromosuccinimide (1.04 g, 5.84 mmol) were added sequentially at room temperature. The mixture was stirred at room temperature and allowed to react for 16 hours. The reaction solution was poured into ice water (50 mL), whereupon a white solid precipitated. The filter cake was collected by filtration under reduced pressure to afford 10a-3 (1.26 g, white solid). Yield: 86.24%. LCMS (ESI): m / z 291.0 [M+H+MeCN]. + ; RT = 1.290 min (2.50 min).

[0162] Step 3: Synthesis of 3-bromo-2,6-difluoroaniline

[0163] To a single-necked flask, 10a-3 (1.00 g, 4.00 mmol), ethanol (6 mL), and concentrated hydrochloric acid (3 mL) were added sequentially at room temperature. The mixture was heated to 70°C and reacted for 2 h. The reaction solution was concentrated under reduced pressure. The residue was adjusted to pH 8 with saturated aqueous sodium bicarbonate solution and extracted with EA. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 10a-4 (650 mg, yellow solid). Yield: 78.14%. LCMS (ESI): m / z 207.9 [M+H+MeCN]. + ; RT = 1.636 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ6.93-6.88(m,1H),6.81-6.76(m,1H),5.54(s,2H).

[0164] Step 4: Synthesis of N-(3-bromo-2,6-difluorophenyl)-1H-pyrazole-5-carboxamide

[0165] To a dry 100 mL three-necked flask at 0°C, 1H-pyrazole-5-carboxylic acid (3.23 g, 28.85 mmol), anhydrous DCM (40 mL), oxalyl chloride (2.28 mL, 26.92 mmol), and DMF (0.10 mL) were added sequentially. The mixture was allowed to react at room temperature for 3 h. The mixture was then slowly added to a mixed solution of 10a-4 (2.00 g, 9.62 mmol) in DCM (30 mL) and pyridine (15 mL) at 0°C. The mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with saturated aqueous ammonium chloride (100 mL) and extracted with DCM (80 mL x 2). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA=5:1) to give 10a-5 (1.80 g, nearly white solid). Yield: 61.97%. LCMS (ESI): m / z 303.9 [M+H] + ; RT = 1.287 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ13.49(s,1H),10.01(s,1H),7.92(d,J=1.6Hz,1H),7.75-7.69(m,1H),7.26-7.21(m,1H),6.77(t,J=2.0Hz,1H).

[0166] Step 5: Synthesis of 7-bromo-6-fluoropyrazolo[1,5-a]quinoxalin-4(5H)-one

[0167] In a dry 50 mL three-necked flask, sodium hydride (60% wt, 185 mg, 4.63 mmol), DMAC (5 mL), and a solution of 10a-5 (700 mg, 2.32 mmol) in DMAC (5 mL) were added sequentially at 0°C. The mixture was heated to 145°C and reacted for 16 hours. The mixture was diluted with saturated aqueous ammonium chloride (50 mL). A precipitate was precipitated, filtered, and the filtrate was extracted with EA (15 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 slurried with EA (10 mL), filtered, and the filter cake was collected to give the crude product 10a-6 (180 mg, yellow solid). LCMS (ESI): m / z 282.0 [M+H] + ; RT = 1.470 min (2.50 min). 1H NMR (400MHz, DMSO-d6): δ12.08 (s, 1H), 8.13 (d, J = 2.0Hz, 1H), 7.89 (dd, J1 = 1.6Hz, J2 = 9.6Hz, 1H), 7.59-7.56 (m, 1H), 7.22 (d, J = 2.0Hz, 1H).

[0168] Step 6: Synthesis of 6-fluoro-7-(hydroxymethyl)pyrazolo[1,5-a]quinoxalin-4(5H)-one

[0169] To a dry 25 mL three-necked flask, 10a-6 (180 mg, 0.64 mmol), 1,4-dioxane (5 mL), and XPhos Pd G2 (50 mg, 0.06 mmol) were added sequentially at room temperature. Under nitrogen, tributyltin carbinol (246 mg, 0.77 mmol) was added. The mixture was heated to 80°C for 16 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was slurried with EA (10 mL), filtered, and the filter cake was collected to afford compound 10a-7 (48 mg, yellow solid) in a 32.25% yield. LCMS (ESI): m / z 234.1 [M+H]+; RT = 0.998 min (2.50 min).

[0170] Step 7: Synthesis of methyl (6-fluoro-4-oxo-4,5-dihydropyrazolo[1,5-a]quinoxalin-7-yl)methanesulfonate

[0171] 10a-7 (48 mg, 0.21 mmol), THF (5 mL), triethylamine (0.09 mL, 0.62 mmol) and methanesulfonyl chloride (0.02 mL, 0.25 mmol) were added sequentially to a 25 mL single-necked flask. The reaction was allowed to react at room temperature for 1 h. The mixture was concentrated under reduced pressure. The mixture was diluted with water (10 mL) and extracted with EA. The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 10a (58 mg, yellow solid). LCMS (ESI): m / z 312.0 [M+H] + ; RT = 1.223 min (2.50 min).

[0172] Intermediate 11a: Synthesis of 7-(bromomethyl)-6-fluoro-1-methyl-1,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-one

[0173] Step 1: Synthesis of 3-bromo-2-fluoro-6-iodobenzoic acid

[0174] 11a-1 (4.0 g, 13.3 mmol) was added to a three-necked flask containing 40 mL of THF at room temperature. Lithium diisopropylamide (7.3 mL, 14.6 mmol) was then added at -78°C. The reaction was allowed to proceed for 1 hour, then the temperature was raised to room temperature and allowed to react overnight. The reaction solution was quenched with saturated ammonium chloride, diluted with water, and the pH was adjusted to 8-9 with dilute sodium hydroxide solution. The solution was extracted twice with EA. The aqueous phase was adjusted to acidic pH with dilute hydrochloric acid and extracted twice with EA. The combined organic phases were washed twice with water and once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to afford 11a-2 (3.2 g, yellow solid). Yield: 69.8%. 1 H NMR (400MHz, CDCl3): δ7.68 (d, J = 8.4Hz, 1H), 7.57-7.53 (m, 1H).

[0175] Step 2: Synthesis of tert-butyl (3-bromo-2-fluoro-6-iodophenyl)carbamate

[0176] To a three-necked flask containing 40 mL of toluene were added 11a-2 (3.0 g, 8.7 mmol), diphenylphosphoryl azide (2.87 g, 10.4 mmol), and triethylamine (1.05 g, 10.4 mmol). The mixture was reacted at 120°C for 1 hour, and then tert-butanol was added. The reaction was continued for 3 hours. The reaction solution was directly concentrated and then purified by column chromatography (PE / EA = 30 / 1) to give 11a-3 (2.9 mg, white solid). LCMS (ESI): m / z 413.8 [MH] - ; RT = 1.684 min (2.50 min).

[0177] Step 3: Synthesis of tert-butyl (3-bromo-2-fluoro-6-(1-methyl-1H-pyrazol-5-yl)phenyl)carbamate

[0178] To a flask containing 15 mL of dioxane and 5 mL of water were added 11a-3 (1.4 g, 3.37 mmol), 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (631 mg, 3.04 mmol), potassium carbonate (1.40 g, 10.1 mmol), and 1,1-bis(diphenylphosphino)diborane iron palladium dichloride (catalytic amount). The reaction was stirred at 70°C for 3 h. The reaction solution was cooled to room temperature, diluted with water, and extracted twice with EA. The combined organic phases were washed twice with water and once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (EA:PE = 1:10) to afford 11a-4 (900 mg, yellow solid) in a yield of 72.3%; LCMS (ESI): m / z 370.0 [M+H]. + ; RT = 1.444 min (2.50 min).

[0179] Step 4: Synthesis of 3-bromo-2-fluoro-6-(1-methyl-1H-pyrazol-5-yl)aniline

[0180] 11a-4 (900 mg, 2.44 mmol) was added to a 50 mL single-necked flask containing 15 mL of methanolic hydrochloric acid. After reaction at room temperature for 3 h, the solvent was removed, the mixture was diluted with water, and the pH was adjusted to alkaline with saturated sodium bicarbonate solution. The mixture was extracted twice with EA. The combined organic phases were washed twice with water and once with saturated brine, dried over anhydrous sodium sulfate, and concentrated to afford 11a-5 (580 mg, yellow solid). Yield: 88.4%; LCMS (ESI): m / z 270.3 / 272.3 [M+H] + ; RT = 1.22 min (2.00 min).

[0181] Step 5: Synthesis of 7-bromo-6-fluoro-1-methyl-1,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-one

[0182] To a 50 mL single-necked flask containing 15 mL of DMF was added 11a-5 (780 mg, 2.9 mmol) and carbonyldiimidazole (1.41 g, 8.70 mmol). The mixture was reacted at 170°C overnight. The reaction solution was diluted with water, and a solid precipitated. After filtration and drying, 11a-6 (650 mg, brown solid) was obtained in a yield of 76.0%. LCMS (ESI): m / z 296.2 [M+H] + ; RT = 0.85 min (2.00 min). 1 H NMR (400MHz, DMSO-d6): δ11.51(s,1H),8.15(s,1H),7.97(d,J=8.8Hz,1H),7.57-7.53(m,1H),4.35(s,3H).

[0183] Step 6: Synthesis of 6-fluoro-7-(hydroxymethyl)-1-methyl-1,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-one

[0184] 11a-6 (200 mg, 0.68 mmol), tributyltin carbinol (239 mg, 0.75 mmol), and XPhos Pd G2 (27 mg, 0.03 mmol) were added to a single-necked bottle containing 10 mL of dioxane. The mixture was reacted at 80°C for 16 hours. The reaction solution was directly concentrated, slurried with EA, and filtered to obtain 11a-7 (140 mg, yellow solid). Yield: 83.8%, LCMS (ESI): m / z 248.2 [M+H] + ; RT = 0.895 min (2.50 min).

[0185] Step 7: Synthesis of 7-(bromomethyl)-6-fluoro-1-methyl-1,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-one

[0186] 11a-7 (70 mg, 0.28 mmol), triphenylphosphine (222 mg, 0.85 mmol), and carbon tetrabromide (188 mg, 0.57 mmol) were added to a 100 mL single-necked flask containing 8 mL of DCM and reacted at room temperature for 2 hours. The reaction solution was directly concentrated to give 11a (50 mg, yellow solid). Crude product, LCMS (ESI): m / z 310.3 [M+H] + ; RT = 0.64 min (2.00 min).

[0187] Intermediate 12a: N-methyl-5-(pyrrolidin-3-yloxy)picolinamide hydrochloride

[0188] Step 1: Synthesis of methyl 5-((1-(tert-butoxycarbonyl)pyrrolidin-3-yl)oxy)picolinate

[0189] Sodium hydride (641 mg, 16 mmol) was added to a dry 250 mL three-necked flask under nitrogen protection. THF (40 mL), 12a-1 (2 g, 10.7 mmol), and 12a-2 (1.66 g, 10.7 mmol) were added in an ice bath and allowed to react at room temperature for 1 hour. The reaction solution was extracted with EA (30 mL × 3). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA = 3:1) to obtain 12a-3 (1 g, yellow oil) in a 29% yield. LCMS (ESI): m / z 323.2 [M+H] + ; RT = 1.59 min (2.50 min).

[0190] Step 2: tert-Butyl 3-((6-(methylcarbamoyl)pyridin-3-yl)oxy)pyrrolidine-1-carboxylate

[0191] To a dry 100 mL single-necked flask, 12a-3 (400 mg, 1.24 mmol) and a 30% ethanolic solution of methylamine (6 mL) were added. The mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure. The residue was diluted with water (10 mL) and extracted with EA (10 mL x 3). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 12a-4 (380 mg, yellow oil). LCMS (ESI): m / z 266 [M+H] + ; RT = 1.560 min (2.50 min). 1H NMR (400MHz, DMSO-d6): δ8.57(d,J=4Hz,1H),8.28(d,J=2.4Hz,1H),7.98(d,J=8.8Hz,1H),7.58-7.56(m,1H),5.18(s,1H ),4.88(d,J=3.2Hz,1H),3.44-3.40(m,3H),3.11(s,1H),2.79(d,J=4.4Hz,3H),1.40(d,J=5.6Hz,9H),1.19-1.56(m,1H).

[0192] Step 3: Synthesis of N-methyl-5-(pyrrolidin-3-yloxy)picolinamide hydrochloride

[0193] In a dry 100 mL single-necked flask, add 12a-4 (380 mg, 1.18 mmol), DCM (6 mL), and a 4.0 M solution of hydrochloric acid in dioxane (6 mL) in an ice bath. Stir at room temperature for 16 hours. The reaction mixture is concentrated under reduced pressure to give 12a (300 mg, crude yellow solid). LCMS (ESI): m / z 220 [M+H] + ; RT = 0.603 min (2.5 min).

[0194] Intermediate 13a: Synthesis of 7-(1-bromoethyl)-6-fluoropyrazolo[1,5-a]quinoxalin-4(5H)-one

[0195] The synthesis method is similar to that of intermediate 6a, except that 10a-6 prepared in step 5 of intermediate 10a is used instead of 6a-3 used in step 4 of intermediate 6a. LCMS (ESI): m / z 310.0 [MH] - ; RT = 1.548 min (2.50 min).

[0196] Intermediate 14a: Synthesis of 7-(1-bromoethyl)-1-methyl-1,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-one

[0197] Step 1: Synthesis of 5-bromo-2-(1-methyl-1H-pyrazol-5-yl)aniline

[0198] Compound 14a-1 (4.0 g, 13.3 mmol), potassium carbonate (3.7 g, 268 mmol), 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborol-2-yl)-1H-pyrazole (2.2 g, 10.7 mmol), and 1,1-bis(diphenylphosphino)diphenylferric palladium chloride (972 mg, 1.34 mmol) were added sequentially to a three-necked flask containing 60 mL of 1,4-dioxane:water (3:1). The mixture was reacted at 50°C under nitrogen for 4 hours. The reaction solution was diluted with water and extracted with EA. The combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated by column chromatography (PE:EA = 3:1) to afford product 14a-2 (2.3 g, yellow solid). Yield: 67.9%. 1 H NMR (400MHz, DMSO-d6): δ7.49(d,J=2.0Hz,1H),6.97(d,J=2.0Hz,1H),6.92(d,J =8.0Hz,1H),6.76-6.73(m,1H),6.26(d,J=2.0Hz,1H),5.21(s,2H),3.63(s,3H).

[0199] Step 2: Synthesis of 7-bromo-1-methyl-1,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-one

[0200] 14a-2 (2.25 g, 8.9 mmol) and carbonyldiimidazole (4.35 g, 26.80 mmol) were added to a single-necked flask containing 30 mL of N-methylpyrrolidone. The reaction was allowed to proceed overnight at 145°C. The reaction solution was diluted with water, and a solid precipitated. The solid was filtered and dried to afford 14a-3 (1.6 g, brownish-white solid). Yield: 45.1%. LCMS (ESI): m / z 280 [M+H]. + ; RT = 1.410 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ11.50 (s, 1H), 8.14-8.10 (m, 2H), 7.64 (d, J = 2Hz, 1H), 7.46-7.44 (m, 1H), 4.34 (s, 3H).

[0201] Step 3: Synthesis of 7-acetyl-1-methyl-1,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-one

[0202] 14a-3 (600 mg, 2.16 mmol), tributyltin (1.56 g, 4.32 mmol), and bistriphenylphosphine palladium dichloride (227 mg, 0.32 mmol) were added to a single-necked bottle containing 10 mL of dioxane. The mixture was reacted at 95°C for 16 hours. The reaction solution was filtered and concentrated, and column chromatography (DCM:MeOH = 20:1) was performed to obtain 14a-4 (300 mg, yellow solid). Yield: 100%, LCMS (ESI): m / z 242.2 [M+H] + ; RT = 1.057 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ11.60(s,1H),8.34(d,J=8.4Hz,1H),8.15(s,1H),8.04(d,J=1.6Hz,1H),7.86-7.84(m,1H),4.41(s,3H),2.65(s,3H).

[0203] Step 4: Synthesis of 7-(1-hydroxyethyl)-1-methyl-1,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-one

[0204] In a single-necked flask, 14a-4 (300 mg, 1.24 mmol), methanol (4 mL), and sodium borohydride (57 mg, 1.50 mmol) were added sequentially in an ice bath. The mixture was stirred at room temperature for 5 hours. The mixture was quenched with saturated ammonium chloride (1 mL), the pH was adjusted to neutral, the mixture was diluted with water, and the mixture was extracted with EA (10 mL x 5). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to afford 14a-5 (50 mg, yellow solid). Yield: 16.5%. LCMS (ESI): m / z 244.1 [M+H] + ; RT = 0.860 min (2.50 min).

[0205] Step 5: Synthesis of 7-(1-bromoethyl)-1-methyl-1,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-one

[0206] To a 100 mL single-necked vial containing 5 mL of DCM was added 14a-5 (50 mg, 0.20 mmol), triphenylphosphine (157 mg, 0.60 mmol), and carbon tetrabromide (133 mg, 0.40 mmol). The mixture was allowed to react at room temperature for 2 hours. The reaction mixture was concentrated to afford 14a (30 mg, yellow solid). Yield: 47.7%. LCMS (ESI): m / z 306.0 [M+H] + ; RT = 1.477 min (2.50 min).

[0207] Intermediate 15a: (1-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[4,3-c]quinolin-7-yl)methyl methanesulfonate

[0208] Step 1: Synthesis of 7-(hydroxymethyl)-1-methyl-1,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-one

[0209] Compound 14a-3 (500 mg, 1.80 mmol), 1,4-dioxane (10 mL) and XPhos Pd G2 (142 mg, 0.18 mmol) were added to a dry 50 mL three-necked flask. Under nitrogen protection, tributyltin methanol (866 mg, 2.70 mmol) was added. The mixture was heated to 80°C for 16 hours, filtered, and the filtrate was concentrated under reduced pressure. The residue was slurried with EA, filtered, and the filter cake was collected to obtain 15a-1 (400 mg, black solid). Yield: 97.05%, LCMS (ESI): m / z 230.1 [M+H] + ; RT = 0.896 min (2.50 min).

[0210] Step 2: Synthesis of (1-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[4,3-c]quinolin-7-yl)methyl methanesulfonate

[0211] 15a-2 (100 mg, 0.44 mmol), THF (5 mL), triethylamine (0.20 mL, 1.31 mmol) and methanesulfonyl chloride (0.06 mL, 0.87 mmol) were added sequentially to a dry single-necked flask. The reaction was allowed to proceed at room temperature for 16 h. The mixture was diluted with water (50 mL) and extracted with EA. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product 15a (56 mg, yellow solid). LCMS (ESI): m / z 308.1 [M+H] + ; RT = 1.083 min (2.50 min).

[0212] Intermediate 16a: 7-(1-bromoethyl)-6-fluoro-1-methyl-1,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-one

[0213] The synthesis method refers to intermediate 14a, except that intermediate 11a-6 is used instead of intermediate 14a-3. LCMS (ESI): m / z 326.0 [M+H] + ; RT = 1.393 min (2.50 min).

[0214] Intermediate 17a: 6-Fluoro-7-(hydroxymethyl)-2-methyl-2,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-one

[0215] The synthesis method is similar to that of intermediate 11a, except that (1-methyl-1H-pyrazol-3-yl)boronic acid is used instead of intermediate 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole. LCMS (ESI): m / z 310.0 [M+H] + ; RT = 1.327 min (2.50 min).

[0216] Intermediate 18a: 7-(1-bromoethyl)-6-fluoro-2-methyl-2,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-one

[0217] Synthesis method: refer to intermediate 16a, LCMS (ESI): m / z 326.1 [M+H] + ; RT = 0.599 min (2.50 min).

[0218] Intermediate 20a: 7-(1-bromoethyl)-2-methyl-2,5-dihydro-4H-pyrazolo[3,4-c]quinolin-4-one

[0219] Step 1: Synthesis of 7-bromo-2-methyl-2,5-dihydro-4H-pyrazolo[3,4-c]quinolin-4-one

[0220] To a dry, single-necked flask, 20a-1 (1.9 g, 6.4 mmol), anhydrous ethanol (30 mL), methylhydrazine sulfate (2.77 g, 19.2 mmol), and glacial acetic acid (0.5 mL) were added. The mixture was reacted at 95°C for 24 hours. The reaction mixture was concentrated under reduced pressure, diluted with water, and the pH was adjusted to 9. The mixture was filtered and the filter cake was dried under vacuum to afford 20a-2 (1.3 g, white solid). Yield: 72.9%. LCMS (ESI): m / z 280.0 [M+H] + ; RT = 1.267 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ11.42(s,1H),8.57(s,1H),7.83(d,J=8.4Hz,1H),7.51(d,J=1.6Hz,1H),7.37-7.34(m,1H),4.13(s,3H).

[0221] Step 2: Synthesis of 7-acetyl-2-methyl-2,5-dihydro-4H-pyrazolo[3,4-c]quinolin-4-one

[0222] To a dry three-necked flask, 20a-2 (400 mg, 1.44 mmol), 1,4-dioxane (10 mL), and bistriphenylphosphine palladium dichloride (98 mg, 0.14 mmol) were added. Under nitrogen, tributyl(1-ethoxyvinyl)tin (779 mg, 2.16 mmol) was added. The mixture was heated to 95°C and reacted for 16 h. The temperature was lowered to 50°C, and hydrochloric acid solution (1.0 M, 4 mL) was slowly added dropwise. The mixture was reacted for 1 h. Saturated potassium fluoride solution (10 mL) was added and stirred for another 1 h. The mixture was filtered. The filtrate was extracted with water and EA (20 mL x 3). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (DCM:methanol = 20:1) to afford 20a-3 (200 mg, yellow solid) in a 57.7% yield. LCMS (ESI): m / z 242.1 [M+H]. + ; RT = 0.877 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ11.50(s,1H),8.77(s,1H),8.00(d,J=8.0Hz,1H),7.93(d,J=1.6Hz,1H),7.80-7.78(m,1H),4.15(s,3H),2.61(s,3H).

[0223] Step 3: Synthesis of 7-(1-hydroxyethyl)-2-methyl-2,5-dihydro-4H-pyrazolo[3,4-c]quinolin-4-one

[0224] In a dry 100 mL single-necked flask, 20a-3 (140 mg, 0.58 mmol), methanol (4 mL), and sodium borohydride (21 mg, 0.58 mmol) were added on an ice bath. The mixture was stirred at room temperature for 3 hours. The mixture was diluted with water and extracted with DCM (15 mL x 4). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product 20a-4 (110 mg, yellow solid). LCMS (ESI): m / z 244.1 [M+H] + ; RT = 1.045 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ11.28(s,1H),8.59(s,1H),7.79(d,J=7.6Hz,1H),7.35(s,1H),7.14( d,J=8.0Hz,1H),5.26(d,J=4.0Hz,1H),4.77-4.74(m,1H),4.12(s,3H),1.34(d,J=6.4Hz,3H).

[0225] Step 4: Synthesis of 7-(1-bromoethyl)-2-methyl-2,5-dihydro-4H-pyrazolo[3,4-c]quinolin-4-one

[0226] 20a-4 (100 mg, 0.41 mmol) and DCM (5 mL) were added sequentially to a dry three-necked flask. Under nitrogen protection, PBr3 (333 mg, 1.23 mmol) was slowly added dropwise at 0°C. The reaction was allowed to react at room temperature for 3 h. The reaction solution was diluted with water (15 mL) and extracted with DCM (15 mL × 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product 20a (100 mg, yellow solid). LCMS (ESI): m / z 306.1 [M+H] + ; RT = 1.054 min (2.50 min).

[0227] Intermediate 21a: 7-(Bromomethyl)-2-methyl-2,5-dihydro-4H-pyrazolo[3,4-c]quinolin-4-one

[0228] The synthesis method was similar to that of intermediate 11a, except that 20a-2 was used instead of 11a-6. LCMS (ESI): m / z 292.0 [M+H] + ; RT = 1.263 min (2.50 min).

[0229] Intermediate 22a: 7-(Bromomethyl)-6-fluorothieno[2,3-c]quinolin-4(5H)-one

[0230] Step 1: Synthesis of methyl 3-bromothiophene-2-carboxylate

[0231] 22a-1 (1 g, 4.83 mmol) was dissolved in methanol (20 mL), and concentrated sulfuric acid (0.5 ml) was added dropwise. The mixture was refluxed at 80°C for 8 h. After cooling to room temperature, the mixture was concentrated, and water (30 mL) was added. The mixture was extracted three times with EA. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and dried in vacuo to afford 22a-2 (950 mg, 4.30 mmol) as a white solid in an 89% yield.

[0232] Step 2: Synthesis of (2-(methoxycarbonyl)thiophen-3-yl)boronic acid

[0233] To a 50 mL dry three-necked flask were added 22a-2 (700 mg, 3.17 mmol), pinacol diboronate (1.61 g, 6.34 mmol), Pd(dppf)Cl2 (695 mg, 0.76 mmol), potassium acetate (932 mg, 9.51 mmol), and dioxane (20 mL) in sequence. The mixture was stirred at 100°C under nitrogen for 8 hours. The reaction mixture was filtered through celite, and the filtrate was concentrated and purified on a reverse-phase column (acetonitrile:water (1‰ NH4HCO3)) to afford 22a-3 (40 mg, 0.215 mmol) as a white solid in a 6.8% yield. LCMS (ESI): m / z 186.9 [M+H] + ; RT = 2.61 min (5.00 min).

[0234] Step 3: Synthesis of methyl 3-(4-bromo-2-((tert-butoxycarbonyl)amino)-3-fluorophenyl)thiophene-2-carboxylate

[0235] To a 25 mL single-necked flask were added 22a-3 (40 mg, 0.215 mmol), 11a-3 (98 mg, 0.236 mmol), Pd(ppf)Cl2 (16 mg, 0.0215 mmol), K2CO3 (89 mg, 0.645 mmol), dioxane (8 mL), and water (2 mL) in sequence. The mixture was stirred at 70°C under nitrogen for 1 hour. The reaction mixture was diluted with EA, washed twice with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified on a silica gel column (EA:PE = 1:5) to afford 22a-4 (71 mg, 0.165 mmol) as a white solid in a 76.7% yield. LCMS (ESI): m / z 331.9 [M+H-100]. + ; RT = 1.85 min (3.00 min).

[0236] Step 4: Synthesis of 7-bromo-6-fluorothieno[2,3-c]quinolin-4(5-hydrogen)-one

[0237] 22a-4 (71 mg, 0.165 mmol) was dissolved in methanol (4 mL) at room temperature. HCl / dioxane (2 mL, 4 M) was added. The reaction mixture was stirred at 45°C for 1 hour. The reaction mixture was concentrated and dried under vacuum to afford 22a-5 (49 mg, 0.148 mmol) as a white solid in a 90% yield. LCMS (ESI): m / z 299.9 [M+H] + ; RT = 1.46 min (3.00 min)

[0238] Step 5: Synthesis of 6-fluoro-7-(hydroxymethyl)thieno[2,3-c]quinolin-4(5-hydrogen)-one

[0239] To a 25 mL single-necked flask were added 22a-5 (49 mg, 0.148 mmol), (tributyltin)methanol (71 mg, 0.222 mmol), X-phos Pd G2 (11.6 mg, 0.0148 mmol), and dioxane (5 mL). The mixture was stirred at 100°C under nitrogen for 4 hours, concentrated, and purified on a silica gel column (EA:PE = 1:5) to afford 22a-6 (31 mg, 0.124 mmol) as a white solid in an 83.8% yield. LCMS (ESI): m / z 250.1 [M+H] + ; RT = 0.95 min (3.00 min).

[0240] Step 6: 7-(Bromomethyl)-6-fluorothieno[2,3-c]quinolin-4(5H)-one

[0241] To a 25 mL single-necked flask was added 22a-6 (31 mg, 0.124 mmol) and DCM (5 mL) at room temperature, and phosphorus tribromide (168 mg, 0.622 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 2 hours and then concentrated to give crude product 22a (50 mg, 100%). LCMS (ESI): m / z 311.9 [M+H] + ; RT = 1.41 min (3.00 min).

[0242] Intermediate 23a: 7-(Bromomethyl)-6-fluorothieno[3,4-c]quinolin-4(5H)-one

[0243] The synthesis method refers to intermediate 22a, except that 4-bromothiophene-3-carboxylic acid was used instead of 22a-1. LCMS (ESI): m / z 311.9 [M+H] + ; RT = 1.57 min (3.00 min).

[0244] Intermediate 24a: 6-(piperazin-1-yl)nicotinate hydrochloride

[0245] Step 1: Synthesis of tert-butyl 4-(5-cyanopyridin-2-yl)piperazine-1-carboxylate

[0246] To a 100 mL three-necked flask, 24a-1 (500 mg, 4.09 mmol), 1-tert-butyloxycarbonylpiperazine (915.24 mg, 4.91 mmol), and acetonitrile (10 mL) were added sequentially. DIEA (1.36 mL, 8.19 mmol) was added dropwise. The mixture was reacted at 65°C under nitrogen atmosphere for 16 hours. The solvent was removed, and the residue was purified by column chromatography (PE:EA = 5:1) to afford 24a-2 (1.10 g, yellow solid). Yield: 93.22%. LCMS (ESI): m / z 289.2 [M+H] + ; RT = 1.728 min (2.50 min).

[0247] Step 2: Synthesis of 6-(piperazin-1-yl)nicotinate hydrochloride

[0248] To a dry 100 mL single-necked flask, 24a-2 (1.10 g, 3.81 mmol) and DCM (15 mL) were added sequentially. 1,4-Dioxane hydrochloride (1.5 mL) was added dropwise under ice-cooling. The mixture was stirred at room temperature for 2 hours. The solvent was removed to afford 24a (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).

[0249] Intermediate 25a: 1-(2,4-difluorophenyl)piperazine

[0250] Step 1: Synthesis of tert-butyl 4-(2,4-difluorophenyl)piperazine-1-carboxylate

[0251] To a dry 100 mL three-necked flask were added 25a-1 (2.00 g, 8.33 mmol), 1-tert-butyloxycarbonylpiperazine (2.33 g, 12.50 mmol), cesium carbonate (6.79 g, 20.83 mmol), 1,4-dioxane (50 mL), and Ruphos Pd G3 (698 mg, 0.83 mmol). The mixture was heated to 100°C under nitrogen for 16 hours. Filtering and removal of the solvent followed by column chromatography (PE:EA = 20:1) afforded 25a-2 (720 mg, black solid) in a 25.00% yield. LCMS (ESI): m / z 243.1 [M+H-56]. + ; RT = 1.624 min (2.50 min).

[0252] Step 2: Synthesis of 1-(2,4-difluorophenyl)piperazine

[0253] To a dry 100 mL single-necked flask, 25a-2 (1.10 g, 3.81 mmol) and DCM (15 mL) were added sequentially. 1,4-Dioxane hydrochloride (1.5 mL) was added dropwise in an ice bath, and the mixture was stirred at room temperature for 2 h. The solvent was removed, and the mixture was extracted with water and EA. The aqueous phase was adjusted to a weak base with saturated sodium bicarbonate and extracted with EA. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to afford crude 25a (180 mg, black oil). LCMS (ESI): m / z 199.1 [M+H] + ; RT = 0.672 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ7.20-7.14(m,1H),7.07-6.95(m,2H),2.91-2.82(m,8H).

[0254] Intermediate 26a: 7-(Bromomethyl)-2-methyl-2,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-one

[0255] The synthesis method was similar to that of intermediate 11a, except that 5-bromo-2-iodoaniline was used instead of 11a-3. LCMS (ESI): m / z 292.0 [M+H] + ; RT = 1.234 min (2.50 min).

[0256] Intermediate 27a: 7-(Hydroxymethyl)-2-methyloxazolo[4,5-c]quinolin-4(5H)-one

[0257] Step 1: Synthesis of methyl (4-iodo-3-(2-methyloxazole-4-carboxamide)benzoate

[0258] To a 25 mL dry single-necked flask were added 27a-1 (340 mg, 2.68 mmol), methyl 3-amino-4-iodobenzoate (741 mg, 2.68 mmol), EA (20 mL), and T3P (5.12 g, 8.04 mmol, 50% EA solution) in sequence. The mixture was stirred at 80°C under nitrogen for 16 h. After cooling to room temperature, water (20 mL) was added and stirred for 2 minutes. The layers were then separated. The aqueous phase was extracted with EA, and the organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and purified on a silica gel column (EA:PE = 3:1) to afford 27a-2 (420 mg, 1.09 mmol) as a pale yellow solid in a yield of 40.6%. LCMS (ESI): m / z 387.0 [M+H]. +; RT = 1.74 min (3.0 min).

[0259] Step 2: Synthesis of methyl 3-(N-(tert-butoxycarbonyl)-2-methyloxazole-4-carboxamido)-4-iodobenzoate

[0260] To a 25 mL dry, single-necked flask were added 27a-2 (420 mg, 1.09 mmol), DCM (15 mL), di-tert-butyl dicarbonate (356 mg, 1.63 mmol), and DMAP (199 mg, 1.63 mmol) in sequence and stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure and purified on a silica gel column (EA:PE = 1:5) to afford 27a-3 (350 mg, 0.72 mmol) as a white solid in a 66% yield. LCMS (ESI): m / z 387.0 [M+H-100]. + ; RT = 1.80 min (3.00 min).

[0261] Step 3: Synthesis of methyl 2-methyl-4-oxo-4,5-dihydrooxazolo[4,5-c]quinoline-7-carboxylate

[0262] 27a-3 (320 mg, 0.658 mmol), Pd(OAc)2 (30 mg, 0.132 mmol), triphenylphosphine (34.6 mg, 0.132 mmol) and potassium carbonate (182 mg, 1.316 mmol) were added to DMF (5 mL) under nitrogen protection. The reaction solution was stirred at 100°C under microwave conditions for 1.5 h. The reaction solution was filtered and washed with EA. The filtrate was concentrated and purified by reverse phase column (acetonitrile: water (1‰ HCOOH)). After lyophilization, the product 27a-4 (140 mg, 0.534 mmol) was obtained as an off-white solid in a yield of 82.5%.

[0263] LCMS (ESI): m / z 259.1 [M+H] + ; RT = 1.28 min (3.00 min)

[0264] Step 4: Synthesis of 7-(hydroxymethyl)-2-methyloxazolo[4,5-c]quinolin-4(5H)-one

[0265] 27a-4 (140 mg, 0.534 mmol) was dissolved in THF (10 mL), and LiAlH4 (1.08 mL, 1 M / THF) was added dropwise in an ice bath. Stirring was continued for 2 hours. The reaction solution was quenched with methanol, the pH was adjusted to weak acidity with trifluoroacetic acid, and concentrated under reduced pressure. The residue was dissolved in dimethyl sulfoxide (5 mL) and purified on a reverse phase column (acetonitrile:water (1‰ NH4HCO3)) to afford 27a-5 (75 mg, 0.326 mmol) as an off-white solid in a 60% yield. LCMS (ESI): m / z 231.1 [M+H] + ; RT = 1.07 min (3.00 min).

[0266] Step 5: Synthesis of 7-(bromomethyl)-2-methyloxazolo[4,5-c]quinolin-4(5-hydro)-one

[0267] In a dry 25 mL single-necked flask, 27a-5 (40 mg, 0.174 mmol) and DCM (5 mL) were added sequentially at room temperature, followed by the dropwise addition of phosphorus tribromide (235 mg, 0.869 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated to obtain an oil (60 mg, crude product). LCMS (ESI): m / z 292.9 [M+H] + ; RT = 1.39 min (3.00 min).

[0268] Intermediate 28a: 7-(Chloromethyl)-2-methylthiazolo[4,5-c]quinolin-4(5H)-one

[0269] Step 1: Synthesis of ethyl 5-bromo-2-methylthiazole-4-carboxylate

[0270] 28a-1 (2.0 g, 11.7 mmol), N-bromosuccinimide (4.16 g, 23.4 mmol), and anhydrous acetonitrile (20 mL) were added to a 50 mL single-necked vial. The mixture was heated to 90°C under argon and stirred for 16 hours. The mixture was cooled to room temperature, concentrated under reduced pressure, and filtered through a forward phase column (PE:EA = 5:1) to afford 28a-2 (1.3 g, yellow solid) in a 45% yield. LCMS (ESI): m / z 250.1 [M+H]+; RT = 1.30 min (3.0 min).

[0271] Step 2: Synthesis of methyl 2-methyl-4-oxo-4,5-dihydrothiazo[4,5-c]quinoline-7-carboxylate

[0272] 28a-2 (100 mg, 0.4 mmol), 2-amino-4-methoxycarbonylphenylboronic acid pinacol ester (166 mg, 0.6 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (29 mg, 0.04 mmol), potassium carbonate (166 mg, 1.2 mmol), dioxane (5 mL), and water (1 mL) were added to a single-necked flask. The mixture was heated to 80°C under argon and stirred for 16 h. The mixture was cooled to room temperature, and water was added. The mixture was extracted with EA. The organic phase was dried over anhydrous sodium sulfate, and the residue was purified by a forward column chromatography (DCM:methanol = 10:1) to afford 28a-3 (50 mg, white solid) in a 45% yield. LCMS (ESI): m / z 275.1 [M+H]+; RT = 1.03 min (3.0 min).

[0273] Step 3: Synthesis of 7-(hydroxymethyl)-2-methylthiazolo[4,5-c]quinolin-4(5H)-one

[0274] 28a-3 (50 mg, 0.18 mmol) and anhydrous THF (5 mL) were added to a 50 mL single-necked flask, cooled to 0°C, and a 1 M solution of lithium aluminum tetrahydride (0.5 mL, 0.5 mmol) was slowly added dropwise. The mixture was stirred for 2 h, quenched with methanol, and 2 mL of trifluoroacetic acid was added dropwise. The mixture was stirred for 10 min, concentrated, and the residue was purified by reverse-phase column chromatography (1% to 30% acetonitrile / 0.1% formic acid in water) to afford 28a-4 (30 mg, white solid) in a 45% yield. LCMS (ESI): m / z 247.1 [M+H]+; RT = 0.54 min (3.0 min).

[0275] Step 4: Synthesis of 7-(chloromethyl)-2-methylthiazolo[4,5-c]quinolin-4(5H)-one

[0276] 28a-4 (30 mg, 0.12 mmol) and anhydrous DCM (5 mL) were added to a 50 mL single-necked flask, cooled to 0°C, and thionyl chloride (1 mL) was slowly added dropwise. The mixture was stirred for 2 hours and concentrated under reduced pressure to afford crude product 28a (30 mg, white solid) in a 100% yield. LCMS (ESI): m / z 265.1 [M+H] + ; RT = 1.41 min (3.0 min).

[0277] Intermediate 31a: 6-Fluoro-7-(bromomethyl)-[1,2,3]triazolo[1,5-a]quinoxalin-4(5H)-one

[0278] Step 1: Synthesis of 6-bromo-5-fluoro-3-oxo-3,4-dihydroquinoxaline-2-carbaldehyde

[0279] 31a-1 (500 mg, 1.9 mmol), tin dioxide (328 mg, 2.9 mmol), and dioxane (10 mL) were added sequentially to a dry 50 mL single-necked flask at room temperature. The mixture was heated to 100°C and stirred overnight. The reaction mixture was filtered and the filtrate was concentrated to give 31a-2 (600 mg, oil). LCMS (ESI): m / z 271.2 [M+H] + ; RT = 1.11 min (3.00 min).

[0280] Step 2: Synthesis of 7-bromo-6-fluoro-[1,2,3]triazolo[1,5-a]quinoxalin-4(5H)-one

[0281] To a dry, single-necked flask, 31a-2 (700 mg, 2.6 mmol), 4-methylbenzenesulfonylhydrazide (579 mg, 3.1 mmol), and methanol (10 mL) were added sequentially. The mixture was stirred at room temperature for 4 hours. The filtrate was concentrated, and the residue was purified using a reverse-phase preparative column (1% to 50% acetonitrile) to afford the product 31a-3 (600 mg, red solid) in a 71% yield. LCMS (ESI): m / z 282.8 [M+H] + ; RT = 1.617 min (3.00 min).

[0282] Step 3: Synthesis of 6-fluoro-7-(hydroxymethyl)-[1,2,3]triazolo[1,5-a]quinoxalin-4(5H)-one

[0283] To a dry 50 mL single-necked flask was added 31a-3 (200 mg, 0.71 mmol), (tributyltin)methanol (273 mg, 0.85 mmol), x-phos Pd G2 (56 mg, 0.071 mmol), and dioxane (10 mL) at room temperature. The mixture was heated at 80°C overnight under nitrogen. The reaction mixture was concentrated and purified on a silica gel column (DCM:methanol = 30:1) to afford 31a-4 (100 mg, white solid) in a 60% yield. LCMS (ESI): m / z 234.9 [M+H]. + ; RT = 1.317 min (3.00 min).

[0284] Step 4: Synthesis of 6-fluoro-7-(bromomethyl)-[1,2,3]triazolo[1,5-a]quinoxalin-4(5H)-one

[0285] To a dry 50 mL single-necked flask, 31a-4 (20 mg, 0.085 mol) and dioxane (3 mL) were added sequentially at room temperature. Phosphorus tribromide (69 mg, 0.25 mmol) was then added dropwise under ice-cooling. The mixture was stirred at room temperature for 2 hours, and the filtrate was concentrated to give the crude product (40 mg, oil). LCMS (ESI): m / z 296.9 [M+H] + ; RT = 1.45 min (3.00 min).

[0286] Intermediate 32a: 7-(1-bromoethyl)-6-fluoro-[1,2,3]triazolo[1,5-a]quinoxalin-4(5H)-one

[0287] The synthesis method was similar to that of 31a, except that tributyl(1-ethoxyethylene)tin and PdCl2(PPh3)2 were used in the Stille coupling reaction. LCMS (ESI): m / z 310.9 [M+H]+; RT = 1.55 min (3.00 min).

[0288] Intermediate 33a: 8-(Chloromethyl)imidazo[1,2-c]quinazolin-5(6H)-one

[0289] Step 1: Synthesis of tert-butyl 2-bromo-1H-imidazole-1-carboxylate

[0290] 33a-1 (200 mg, 1.36 mmol), di-tert-butyl dicarbonate (445 mg, 2.04 mmol), triethylamine (412 mg, 4.08 mmol), and DMF (5 mL) were added to a 50 mL single-necked vial and stirred at room temperature under argon for 2 h. The reaction mixture was extracted with water and EA, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by a forward column chromatography (PE:EA = 5:1) to afford 33a-2 (250 mg, white solid) in a 74% yield. LCMS (ESI): m / z 146.9 [M+H-100]. + ; RT = 1.58 min (3.0 min).

[0291] Step 2: Synthesis of methyl 5-carbonyl-5,6-dihydroimidazo[1,2-c]quinazoline-8-carboxylate

[0292] 33a-2 (100 mg, 0.4 mmol), methyl 3-amino-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (166 mg, 0.6 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (29 mg, 0.04 mmol), potassium carbonate (166 mg, 1.2 mmol), dioxane (5 mL), and water (1 mL) were added to a 50 mL single-necked vial and stirred at 80°C under argon for 16 hours. The mixture was cooled to room temperature and extracted with EA. The organic phase was dried over anhydrous sodium sulfate, and the residue was purified by a forward column chromatography (DCM:methanol = 10:1) to afford 33a-3 (50 mg, white solid) in a 45% yield. LCMS (ESI): m / z 244.1 [M+H]. + ; RT = 1.22 min (3.0 min).

[0293] Step 3: Synthesis of 8-(hydroxymethyl)imidazo[1,2-c]quinazolin-5(6H)-one

[0294] 33a-3 (50 mg, 0.20 mmol) and anhydrous THF (5 mL) were added to a 50 mL single-necked bottle and cooled to 0°C. A 1 M solution of lithium aluminum tetrahydride (0.5 mL, 0.5 mmol) was slowly added dropwise, and the mixture was stirred for 2 h. The mixture was quenched with methanol, and 2 mL of trifluoroacetic acid was added dropwise. The mixture was stirred for 10 min and concentrated. The residue was purified by reverse-phase column chromatography (1% to 30% acetonitrile / 0.1% aqueous ammonium bicarbonate) to afford 33a-4 (20 mg, white solid) in a 47% yield. LCMS (ESI): m / z 216.1 [M+H]. + ; RT = 0.99 min (3.0 min).

[0295] Step 4: Synthesis of 8-(chloromethyl)imidazo[1,2-c]quinazolin-5(6H)-one

[0296] 33a-4 (20 mg, 0.09 mmol) and DCM (5 mL) were added to a single-necked bottle, cooled to 0°C, and thionyl chloride (1 mL) was slowly added dropwise. The mixture was stirred for 2 hours and concentrated under reduced pressure to obtain crude product 33a (20 mg, white solid). LCMS (ESI): m / z 234.1 [M+H] + ; RT = 1.28 min (3.0 min).

[0297] Intermediate 34a: Synthesis of N-ethyl-6-fluoro-5-(piperazin-1-yl)picolinamide hydrochloride

[0298] The synthesis method refers to the synthesis of intermediate 2a, except that ethylamine was used instead of methylamine hydrochloride in the condensation step. LCMS (ESI): m / z 253.2 [M+H] + ; RT = 0.527 min (2.5 min).

[0299] Intermediate 35a: (R)-6-Fluoro-5-(piperazin-1-yl)-N-(tetrahydrofuran-3-yl)picolinamide hydrochloride

[0300] The synthesis method refers to the synthesis of intermediate 2a, except that (R)-tetrahydrofuran-3-amine is used instead of methylamine hydrochloride in the condensation step. LCMS (ESI): m / z 395.2 [M+H] + ; RT = 1.582 min (2.50 min).

[0301] Intermediate 36a: 6-chloro-N-methyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0302] Step 1: Synthesis of 5-bromo-6-chloro-N-methylpicolinamide

[0303] To a dry 100 mL single-necked flask, 36a-1 (1.00 g, 3.99 mmol) and a 33% wt ethanolic solution of methylamine (10 mL) were added sequentially. The reaction was allowed to react at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure to afford 36a-2 (0.94 g, yellow oil) in a 94.37% yield. LCMS (ESI): m / z 250.9 [M+H]+; RT = 1.448 min (2.50 min).

[0304] Step 2: Synthesis of tert-butyl 4-(2-chloro-6-(methylcarbamoyl)pyridin-3-yl)piperazine-1-carboxylate

[0305] To a dry 50 mL single-necked flask were added 36a-2 (620 mg, 2.49 mmol), toluene (15 mL), tert-butyl piperazine-1-carboxylate (370 mg, 1.99 mmol), 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (155 mg, 0.25 mmol), cesium carbonate (2020 mg, 6.21 mmol), and palladium acetate (56 mg, 0.25 mmol). The mixture was heated to 100°C under nitrogen and allowed to react for 16 hours. The reaction mixture was filtered, the filtrate collected, and concentrated under reduced pressure. Purification by silica gel column chromatography (PE:EA = 1:1) afforded 36a-3 (180 mg, yellow oil) in a 20.41% yield. LCMS (ESI): m / z 355.1 [M+H]+; RT = 1.679 min (2.50 min).1 H NMR (400MHz, DMSO-d6): δ8.46(d,J=4.8Hz,1H),7.95(d,J=8.4Hz,1H),7.68(d,J =8.4Hz,1H),3.50(s,4H),3.06-3.04(m,4H),2.80(d,J=4.8Hz,3H),1.43(s,9H).

[0306] Step 3: Synthesis of 6-chloro-N-methyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0307] To a dry flask were added 36a-3 (180 mg, 0.51 mmol), DCM (2 mL), and hydrochloric acid-dioxane (4.0 M, 2 mL). The mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure to afford 36a (140 mg, yellow solid) in a 94.59% yield. LCMS (ESI): m / z 255.1 [M+H]+; RT = 0.336 min & 0.461 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ9.40 (s, 2H), 8.50 (d, J = 4.4Hz, 1H), 7.97 (d, J = 8.4Hz, 1 H),7.76(d,J=8.4Hz,1H),3.34-3.32(m,4H),3.25(m,4H),2.80(d,J=4.8Hz,3H).

[0308] Intermediate 37a: N,6-dimethyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0309] Step 1: Synthesis of 5-bromo-6-methylpicolinic acid

[0310] To a dry flask were added 37a-1 (500 mg, 2.54 mmol), methanol (6 mL), water (3 mL), and sodium hydroxide (507 mg, 12.69 mmol) in sequence. The mixture was reacted at 70°C for 1 hour. The reaction solution was concentrated under reduced pressure. The mixture was diluted with water (10 mL) and adjusted to pH 4 with 3M dilute hydrochloric acid. The filter cake was filtered to obtain 37a-2 (300 mg, white solid). Yield: 54.72%. LCMS (ESI): m / z 218.0 [M+H] + ; RT = 1.208 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ8.12(d,J=8.0Hz,1H),7.78(d,J=8.4Hz,1H),2.67(s,3H).

[0311] Step 2: Synthesis of 5-bromo-N,6-dimethylpicolinamide

[0312] To a dry flask were added 37a-2 (300 mg, 1.39 mmol), DMF (3 mL), DIEA (0.92 mL, 5.55 mmol), HATU (792 mg, 2.08 mmol), and a 2.0 M solution of methylamine in THF (1.39 mL, 2.78 mmol). The reaction was allowed to react at room temperature for 1 h. The mixture was diluted with water (30 mL) and extracted with EA (10 mL x 2). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure. The residue was purified using a preparative plate (PE:EA = 3:2) to afford 37a-3 (215 mg, yellow solid) in a 67.59% yield. LCMS (ESI): m / z 231.0 [M+H]+; RT = 1.427 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ8.67(d,J=4.0Hz,1H), 8.18(d,J=8.4Hz,1H), 7.75(d,J=8.4Hz,1H), 2.82(d,J=4.8Hz,3H), 2.65(s,3H).

[0313] Step 3: Synthesis of tert-butyl 4-(2-methyl-6-(methylcarbamoyl)pyridin-3-yl)piperazine-1-carboxylate

[0314] To a dry flask were added 37a-3 (200 mg, 0.87 mmol), toluene (8 mL), tert-butyl piperazine-1-carboxylate (179 mg, 0.96 mmol), 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (54 mg, 0.09 mmol), cesium carbonate (711 mg, 2.18 mmol), and palladium acetate (20 mg, 0.09 mmol). The mixture was heated to 100°C under nitrogen for 16 hours. The reaction mixture was filtered, the filtrate collected, and concentrated under reduced pressure. The residue was purified using a preparative plate (PE:EA = 1:1) to afford 37a-4 (160 mg, yellow solid) in a 54.80% yield. LCMS (ESI): m / z 335.1 [M+H]. + ; RT = 1.623 min (2.50 min). 1 H NMR (400MHz, DMSO-d6): δ8.44(d,J=4.8Hz,1H),7.80(d,J=8.4Hz,1H),7.49(d,J=8.4Hz ,1H),3.49(s,4H),2.89-2.87(m,4H),2.81(d,J=4.8Hz,3H),2.51(s,3H),1.43(s,9H).

[0315] Step 4: Synthesis of N,6-dimethyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0316] 37a-4 (160 mg, 0.48 mmol), DCM (2 mL), and hydrochloric acid-dioxane (4.0 M, 2 mL) were added to a dry flask. The reaction was allowed to react at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure to afford 37a (129 mg, yellow solid) in a 99.58% yield. LCMS (ESI): m / z 235.2 [M+H] + ;RT=0.340min&0.450min(2.50min). 1 H NMR (400MHz, DMSO-d6): δ9.44(s,2H),8.56(d,J=4.8Hz,1H),7.88(d,J=8.0Hz,1 H),7.61(d,J=8.4Hz,1H),3.25-3.16(m,8H),2.82(d,J=4.4Hz,3H),2.54(s,3H).

[0317] Intermediate 38a: Synthesis of N-cyclopropyl-6-fluoro-5-(piperazin-1-yl)picolinamide hydrochloride

[0318] The synthesis method refers to the synthesis of intermediate 2a, except that cyclopropylamine is used instead of methylamine hydrochloride in the condensation step. LCMS (ESI): m / z 264.2 [M+H] + ; RT = 0.958 min (2.5 min).

[0319] Intermediate 40a: 8-(Bromomethyl)-2-methylimidazo[1,2-c]quinazolin-5(6H)-one

[0320] The synthesis method refers to the synthesis of intermediate 33a, except that 2-bromo-4-methyl-1H-imidazole is used as the starting material in step 1 and phosphorus tribromide is used as the reaction reagent in step 4. LCMS (ESI): m / z 292.0 [M+H] + ; RT = 1.25 min (3.00 min).

[0321] Intermediate 41a: Synthesis of 8-(bromomethyl)-7-fluoro-dihydroimidazo[1,2-c]quinazolin-5(3H)-one

[0322] Step 1: Synthesis of 3-bromo-6-(4,5-dihydro-1H-imidazol-2-yl)-2-fluoroaniline

[0323] Compound 41a-1 (500 mg, 2.33 mmol) was dissolved in methanol (20 mL), and ethylenediamine dihydrochloride (3.1 g, 23.3 mmol) and sodium carbonate (3.7 g, 35 mmol) were added sequentially. The mixture was heated at reflux for 72 hours. The mixture was concentrated, water (20 mL) was added, and the mixture was filtered. The solid was dissolved in DMSO (5 mL) and purified on a reverse phase column (acetonitrile:water) to afford 41a-2 (130 mg, 0.504 mmol) as a white solid in a yield of 21.6%. LCMS (ESI): m / z 260.0 [M+H] + ; RT = 0.75 min (3.00 min).

[0324] Step 2: Synthesis of 8-bromo-7-fluoro-2,6-dihydroimidazo[1,2-c]quinazolin-5(3H)-one

[0325] 41a-2 (50 mg, 0.194 mmol) was dissolved in DMF (5 mL), and N,N'-carbonyldiimidazole (94 mg, 0.582 mmol) and DMAP (24 mg, 0.194 mmol) were added sequentially. The mixture was stirred at 100°C for 3 h. After the reaction solution was cooled to room temperature, the solid was collected by filtration, washed with EA, and dried in vacuo to obtain 41a-3 (41 mg, 0.144 mmol) as a white solid in a yield of 74.2%. LCMS (ESI): m / z 286.0 [M+H] + ; RT = 1.22 min (3.00 min).

[0326] Step 3: Synthesis of 7-fluoro-8-(hydroxymethyl)-2,6-dihydroimidazo[1,2-c]quinazolin-5(3H)-one

[0327] 41a-3 (41 mg, 0.144 mmol), (tributyltin)methanol (69 mg, 0.216 mmol), and x-phos Pd G2 (11.3 mg, 0.0144 mmol) were added sequentially to dioxane (5 mL). The mixture was stirred at 100°C under nitrogen for 6 hours, concentrated, and purified on a silica gel column (methanol:DCM=1:10) to afford 41a-4 (29 mg, 0.123 mmol) as a white solid in an 85.7% yield. LCMS (ESI): m / z 236.1 [M+H]+; RT = 0.91 min (3.00 min).

[0328] Step 4: Synthesis of 8-(bromomethyl)-7-fluoro-dihydroimidazo[1,2-c]quinazolin-5(3H)-one

[0329] 41a-4 (29 mg, 0.123 mmol) was dissolved in DCM (8 mL), and phosphorus tribromide (0.5 mL) was added dropwise at room temperature. The mixture was stirred for 2 hours. The reaction solution was concentrated to remove excess phosphorus tribromide and dried to obtain crude product 41a (35 mg). LCMS (ESI): m / z 298.2 [M+H] + ; RT = 1.23 min (3.00 min).

[0330] Intermediate 42a: Synthesis of 8-(bromomethyl)-2,6-dihydroimidazo[1,2-c]quinazolin-5(3H)-one

[0331] The synthesis method is the same as 41a, except that 2-amino-4-bromobenzonitrile is used as the starting material. LCMS (ESI): m / z 280.0 [M+H] + ; RT = 0.580 min (2.50 min).

[0332] Intermediate 43a: Synthesis of 8-(bromomethyl)-7-methyl-dihydroimidazo[1,2-c]quinazolin-5(3H)-one

[0333] The synthesis method is the same as 41a, except that 2-amino-4-bromo-3-methylbenzonitrile is used as the starting material. LCMS (ESI): m / z 294.1 [M+H] + ; RT = 1.30 min (3.00 min).

[0334] Intermediate 44a: Synthesis of N-cyclopropyl-6-methyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0335] The synthesis method is the same as 37a, except that cyclopropylamine is used as the starting material. LCMS (ESI): m / z 261.1 [M+H] + ; RT = 1.05 min (3.0 min).

[0336] Intermediates 45a: 8-(Bromomethyl)-7-chloro-dihydroimidazo[1,2-c]quinazolin-5(3H)-one and 46a: 8-(Bromomethyl)-9-chloro-2,6-dihydroimidazo[1,2-c]quinazolin-5(3H)-one

[0337] Step 1: Synthesis of 5-bromo-2-(4,5-dihydro-1H-imidazol-2-yl)aniline

[0338] To a dry 100 mL single-necked flask, 45a-1 (5 g, 25.5 mmol), phosphorus pentasulfide (487 mg, 2.55 mmol), and ethylenediamine (10 mL) were added sequentially at room temperature. The mixture was reacted at 100°C for 4 hours. Water (100 mL) was added to the reaction solution, filtered, and the filter cake was washed with water and dried to obtain 45a-2 (1.5 g, light yellow solid). Yield: 82%. LCMS (ESI): m / z 240.1 [M+H]. + ; RT = 0.98 min (3.00 min).

[0339] Step 2: Synthesis of 3-bromo-2-chloro-6-(4,5-dihydro-1H-imidazol-2-yl)aniline

[0340] To a dry 50 mL single-necked flask were added 45a-2 (500 mg, 2.09 mmol), N-chlorosuccinimide (335 mg, 2.5 mmol), p-toluenesulfonic acid (40 mg, 0.21 mmol), and DMF (10 mL). The mixture was reacted at 90°C for 6 hours. Water (30 mL) was added to the reaction solution, and the mixture was extracted with EA (40 mL). The organic phase was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. The filtrate was concentrated, and the residue was purified on a silica gel column (PE:EA = 3:1) to obtain a mixture of 45a-3 and 45a-4 (100 mg, yellow solid) in a yield of 17%. LCMS (ESI): m / z 274.0 [M+H]. + ;RT=1.19&1.21min(3.00min).

[0341] Step 3: Synthesis of 8-bromo-7-chloro-2,6-dihydroimidazo[1,2-c]quinazolin-5(3H)-one

[0342] A mixture of 45a-3 and 45a-4 (100 mg, 0.37 mmol), N,N'-carbonyldiimidazole (118 mg, 0.73 mmol), DMAP (45 mg, 0.37 mmol), and DMF (3 mL) were added sequentially to a single-necked flask and heated to 100°C for 2 hours. The reaction mixture was added with water (10 mL), filtered, and the filter cake was washed with water and dried to afford a mixture of 45a-5 and 45a-6 (90 mg, yellow solid) in an 82% yield. LCMS (ESI): m / z 299.9 [M+H] + ;RT=1.29&1.32min(3.00min).

[0343] Step 4: Synthesis of 7-chloro-8-(hydroxymethyl)-2,6-dihydroimidazo[1,2-c]quinazolin-5(3H)-one

[0344] A mixture of 45a-5 and 45a-6 (90 mg, 0.3 mmol), (tributyltin)methanol (192 mg, 0.6 mmol), x-Phos Pd G2 (23 mg, 0.03 mmol), and dioxane (5 mL) were added sequentially to a dry flask. The mixture was reacted at 100°C under nitrogen protection for 6 hours. The reaction was concentrated and the residue was purified by reverse phase preparative column (1% to 50% acetonitrile). 45a-7 (15 mg, yellow solid) and 45a-8 (25 mg, yellow solid) were dried.

[0345] 45a-7:LCMS(ESI):m / z 252.1[M+H] + ; RT = 0.95 min (3.00 min). 1 H NMR (600MHz, DMSO-d6): δ7.80(d,J=8.0Hz,1H),7.30(d,J=8.0Hz,1H),3.96(m,2H),3.87(m,2H).

[0346] Step 5: Synthesis of 8-(bromomethyl)-7-chloro-dihydroimidazo[1,2-c]quinazolin-5(3H)-one

[0347] 45a-7 (15 mg, 0.06 mmol) and dioxane (3 mL) were added to a single-necked flask, and phosphorus tribromide (48 mg, 0.18 mmol) was added dropwise in an ice bath. The mixture was stirred at room temperature for 2 hours and concentrated to give crude product 45a (20 mg, yellow solid). LCMS (ESI): m / z 314.0 & 316.0 [M+H] + ; RT = 1.28 min (3.00 min).

[0348] Step 6: Synthesis of 8-(bromomethyl)-9-chloro-2,6-dihydroimidazo[1,2-c]quinazolin-5(3H)-one

[0349] 45a-8 (25 mg, 0.1 mmol) and dioxane (3 mL) were added to a single-necked flask, and phosphorus tribromide (81 mg, 0.3 mmol) was added dropwise in an ice bath. The mixture was stirred at room temperature for 2 hours and concentrated to afford 46a (30 mg, yellow solid). LCMS (ESI): m / z 314.0 & 316.0 [M+H] + ; RT = 1.32 min (3.00 min).

[0350] Intermediate 47a: Synthesis of 8-(bromomethyl)-7-fluoro-2,2-dimethyl-2,6-dihydroimidazo[1,2-c]quinazolin-5(3H)-one

[0351] Step 1: Synthesis of 2-((7-bromo-2-chloro-8-fluoroquinazolin-4-yl)amino)-2-methylpropan-1-ol

[0352] 47a-1 (300 mg, 1.01 mmol) was dissolved in THF (5 mL), and 47a-2 (108 mg, 1.21 mmol) and triethylamine (306 mg, 3.03 mmol) were added sequentially. The mixture was stirred at room temperature for 2 h. The reaction solution was diluted with water (15 mL) and extracted three times with EA (20 mL). The organic phase was washed with brine, dried over anhydrous sodium sulfate, and concentrated to afford 47a-3 (320 mg, 0.92 mmol) as a white solid in a 91% yield. LCMS (ESI): m / z 349.9 [M+H] + ; RT = 1.62 min (3.0 min).

[0353] Step 2: Synthesis of 8-bromo-5-chloro-7-fluoro-2,2-dimethyl-2,3-dihydroimidazo[1,2-c]quinazoline

[0354] 47a-3 (320 mg, 0.92 mmol) was dissolved in toluene (10 mL) and phosphorus oxychloride (1 mL) was added dropwise in an ice bath. The reaction was allowed to proceed at room temperature for 3 hours. The pH was adjusted to 7-8 with aqueous sodium carbonate. The product was extracted with EA, dried over anhydrous sodium sulfate, concentrated, and purified on a silica gel column (PE:EA = 1:1) to afford 47a-4 (215 mg, 0.651 mmol) as a white solid in a 70.8% yield. LCMS (ESI): m / z 331.9 [M+H]. + ; RT = 1.62 min (3.0 min).

[0355] Step 3: Synthesis of 8-bromo-7-fluoro-2,2-dimethyl-2,6-dihydroimidazo[1,2-c]quinazolin-5(3H)-one

[0356] 47a-4 (215 mg, 0.651 mmol) was added to acetic acid (5 mL) and heated to 110°C for 2 hours. The reaction mixture was concentrated to remove the acetic acid, and the residue was added with water (15 mL). The pH was adjusted to 7-8 with saturated sodium bicarbonate solution and extracted three times with EA (20 mL). The organic phase was washed with brine, dried over anhydrous sodium sulfate, and concentrated to afford 47a-5 (180 mg, 0.577 mmol) as a white solid in a 93.8% yield. LCMS (ESI): m / z 314.0 [M+H] + ; RT = 1.32 min (3.0 min).

[0357] Step 4: 7-Fluoro-8-(hydroxymethyl)-2,2-dimethyl-2,6-dihydroimidazo[1,2-c]quinazolin-5(3H)-one

[0358] 47a-5 (80 mg, 0.256 mmol), (tributyltin)methanol (165 mg, 0.513 mmol), and X-Phos Pd G2 (20 mg, 0.0256 mmol) were added to anhydrous dioxane (5 mL). The mixture was heated to 110°C under argon and stirred for 2 hours. The mixture was concentrated and the residue was purified by column chromatography (DCM:methanol = 10:1) to afford 47a-6 (48 mg, 0.154 mmol) as a white solid in a 60% yield. LCMS (ESI): m / z 264.2 [M+H] + ; RT = 1.02 min (3.0 min).

[0359] Step 5: Synthesis of 8-(bromomethyl)-7-fluoro-2,2-dimethyl-2,6-dihydroimidazo[1,2-c]quinazolin-5(3H)-one

[0360] 47a-6 (48 mg, 0.154 mmol) was dissolved in DCM (10 mL), and phosphorus tribromide (0.5 mL) was added dropwise at room temperature. After the addition was complete, stirring was continued for 2 hours. The reaction solution was concentrated to remove the solvent and excess phosphorus tribromide, and the crude product 47a (60 mg) was dried in vacuo. LCMS (ESI): m / z 326.2 [M+H] + ; RT = 1.38 min (3.0 min)

[0361] Intermediates 48a-56a, 59a-60a: The synthesis method is the same as 41a or 47a, except that the raw material intermediates in the table below are used instead of ethylenediamine or 47a-2 as the starting material

[0362] Intermediate 57a: 9-(Chloromethyl)-3,3,8-trifluoro-2,3,4,7-tetrahydro-6-pyrimido[1,2-c]quinazolin-6-one

[0363] Compound 57a-1 (20 mg, 0.07 mmol, synthesized the same way as 47a-6, except that 3-amino-2,2-difluoropropan-1-ol was used instead of 47a-2) and anhydrous DCM (10 mL) were added to a 50 mL single-necked flask. The mixture was cooled to 0°C and thionyl chloride (1 mL) was slowly added dropwise. The mixture was stirred for 2 hours and concentrated under reduced pressure to obtain crude compound 57a (25 mg, white solid). Yield: 100%. LCMS (ESI): m / z 304.1 [M+H]. + ; RT = 1.50 min (3.0 min).

[0364] Intermediate 58a: 9-(Chloromethyl)-8-fluoro-4-methyl-2,3,4,7-tetrahydro-6-pyrimido[1,2-c]quinazolin-6-one

[0365] The synthesis method is the same as 57a, except that 4-amino-2-butanol is used instead of 47a-2. LCMS (ESI): m / z 282.1 [M+H] + ; RT = 1.28 min (3.0 min).

[0366] Intermediate 61a: N-ethyl-6-methyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0367] The synthesis method was the same as 37a, except that an ethyl group was used instead of a methyl group. LCMS (ESI): m / z 249.3 [M+H]+; RT = 1.0 min (3.00 min).

[0368] Intermediate 62a: 6-Chloro-N-ethyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0369] The synthesis method was the same as 36a, except that an ethyl group was used instead of a methyl group. LCMS (ESI): m / z 269.3 [M+H]+; RT = 1.2 min (3.00 min).

[0370] Intermediate 63a: 6-chloro-N-cyclopropyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0371] The synthesis method was the same as 36a, except that a cyclopropyl group was used instead of a methyl group. LCMS (ESI): m / z 281.3 [M+H]+; RT = 1.3 min (3.00 min).

[0372] Intermediate 64a: 8'-(1-chloroethyl)-7'-fluoro-3'-H-spiro[cyclopropane-1,2'-imidazo[1,2-c]quinazoline]-5'(6'H)-one

[0373] Step 1: 8'-Acetyl-7'-fluoro-3'-H-spiro[cyclopropane-1,2'-imidazo[1,2-c]quinazolin]-5'(6'H)-one

[0374] Compound 64a-1 (50 mg, 0.16 mmol), 64a-2 (87 mg, 0.24 mmol), and triphenylphosphine palladium dichloride (11 mg, 0.016 mmol) were added to 5 mL of anhydrous dioxane and stirred at 100°C for 6 h. After concentration, the residue was added with 5N dilute hydrochloric acid (10 mL) and stirred at room temperature for 1 h. The mixture was then neutralized with saturated aqueous sodium bicarbonate until alkaline. The mixture was extracted with EA, and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated, and purified by normal phase column chromatography (PE:EA = 3:1) to afford compound 64a-3 (20 mg, white solid) in a 46% yield. LCMS (ESI): m / z 274.1 [M+H]+; RT = 1.21 min (3.0 min).

[0375] Step 2: 7'-Fluoro-8'-(1-hydroxyethyl)-3'-H-spiro[cyclopropane-1,2'-imidazo[1,2-c]quinazoline]-5'(6'H)-one

[0376] 64a-3 (50 mg, 0.18 mmol) was dissolved in anhydrous methanol (10 mL), sodium borohydride (69 mg, 1.8 mmol) was added, and the mixture was stirred at 0°C for 2 h. The mixture was concentrated under reduced pressure, extracted with ethyl acetate (30 mL*3) and water (20 mL), and the organic phase was dried over anhydrous sodium sulfate and concentrated to afford compound 64a-4 (30 mg, white solid) in a 60% yield. LCMS (ESI): m / z 276.2 [M+H] + ; RT = 1.09 min (3.0 min).

[0377] Step 3: 8'-(1-chloroethyl)-7'-fluoro-3'-H-spiro[cyclopropane-1,2'-imidazo[1,2-c]quinazoline]-5'(6'H)-one

[0378] Compound 64a-4 (20 mg, 0.073 mmol) was dissolved in anhydrous dichloromethane (10 mL), cooled to 0°C, and thionyl chloride (1 mL) was slowly added dropwise. The mixture was stirred for 2 hours and concentrated under reduced pressure to obtain crude product 64a (20 mg, white solid). Yield: 100%. LCMS (ESI): m / z 294.1 [M+H] + ; RT = 1.38 min (3.0 min).

[0379] Intermediate 65a: (2R)-8-(1-bromoethyl)-7-fluoro-2-methyl-2,6-dihydroimidazo[1,2-c]quinazolin-5(3H)-one

[0380] The synthesis method is the same as 64a, except that (R)-8-bromo-7-fluoro-2-methyl-2,6-dihydroimidazo[1,2-c]quinazolin-5(3H)-one is used instead of 64a-1. LCMS (ESI): m / z 326.0 [M+H] + ; RT = 1.37 min (3.0 min).

[0381] Intermediate 66a: (R)-(2-methyl-5-oxo-2,3,5,6-tetrahydroimidazo[1,2-c]quinazolin-8-yl)methyl methanesulfonate

[0382] To a 50 mL single-necked flask, 66a-1 (100 mg, 0.43 mmol), THF (10 mL), triethylamine (0.18 mL, 1.29 mmol), and methanesulfonyl chloride (0.05 mL, 0.65 mmol) were added sequentially at 0°C. The reaction was allowed to proceed at room temperature for 2 h. The reaction solution was concentrated to give 66a (120 mg, crude product, yellow solid). LCMS (ESI): m / z 310.0 [M+H] + ; RT = 0.807 min (2.50 min).

[0383] Intermediate 67a: 9-(Bromomethyl)-2,3,4,7-tetrahydro-6-pyrimido[1,2-c]quinazolin-6-one

[0384] The synthesis method is the same as 45a, except that 2-amino-4-bromobenzonitrile and propylenediamine are used as starting materials. LCMS (ESI): m / z 296.0 [M+H] + ; RT = 0.715 min (2.50 min).

[0385] Intermediate 68a: N-methyl-5-(piperazin-1-yl)picolinamide hydrochloride

[0386] The synthesis method refers to the synthesis of intermediate 2a, except that step 1 is omitted and 5-bromopyridine-2-carboxylic acid methyl ester and 4-Boc piperazine are used for coupling reaction. LCMS (ESI): m / z 221.2 [M+H] + ; RT = 0.285 min (6.00 min).

[0387] Intermediate 69a: 2-Chloro-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide hydrochloride

[0388] The synthesis method refers to the synthesis of intermediate 36a, except that N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester was used as the starting material. LCMS (ESI): m / z 252.2 [M+H] + ; RT = 0.95 min (3.00 min).

[0389] Intermediate 70a: 6-chloro-N-methyl-5-(piperidin-4-yl)picolinamide hydrochloride

[0390] 69a (240 mg, 0.85 mmol) and platinum dioxide (30 mg) were added to methanol (5 mL) in sequence at room temperature. The mixture was stirred at room temperature under hydrogen for 4 hours. The reaction solution was filtered and the filtrate was concentrated to give 70a (130 mg, white solid). LCMS (ESI): m / z 254.3 [M+H] + ; RT = 0.97 min (3.00 min).

[0391] Intermediate 71a: (R)-6-chloro-N-methyl-5-(3-methylpiperazin-1-yl)picolinamide

[0392] The synthesis method refers to the synthesis of intermediate 36a, except that (S)-1-N-Boc-2-methylpiperazine was used as the starting material. LCMS (ESI): m / z 269.2 [M+H] + ; RT = 0.91 min (3.0 min).

[0393] Intermediate 72a: 6-chloro-N-(2,2-difluoroethyl)-5-(piperazin-1-yl)picolinamide

[0394] The synthesis method refers to the synthesis of intermediate 36a, except that 2,2-difluoroethane-1-amine was used as the starting material. LCMS (ESI): m / z 305.2 [M+H] + ; RT = 1.02 min (3.0 min).

[0395] Intermediate 73a: 6-(difluoromethyl)-N-methyl-5-(piperazin-1-yl)picolinamide

[0396] The synthesis method refers to the synthesis of intermediate 36a, except that 5-bromo-6-(difluoromethyl)-N-methylpicolinamide was used as the starting material. LCMS (ESI): m / z 271.3 [M+H] + ; RT = 1.54 min (3.0 min).

[0397] Intermediate 74a: N-methyl-5-(((2R,3S)-2-methylazetidin-3-yl)oxy)picolinamide

[0398] The synthesis method is the same as that of intermediate 12a, except that (2R,3R)-tert-butyl 3-hydroxy-2-methylazetidine-1-carboxylate is used as the starting material. LCMS (ESI): m / z 222.2 [M+H] + ; RT = 0.44 min (3.0 min).

[0399] Synthesis of compound 4

[0400] To a 25 mL single-necked flask containing 5 mL of acetonitrile were added 5a (150 mg, 0.48 mmol), 3a (124 mg, 0.48 mmol), and DIEA (187 mg, 1.45 mmol). The mixture was reacted at 70°C for 2 h. The reaction solution was purified by Pre-HPLC (formic acid) to give compound 4 (40 mg, white solid) in a yield of 19%.

[0401] LCMS (ESI): m / z 435.05 [M+H] + ; RT = 3.198 min (6.00 min).

[0402] 1 H NMR (400MHz, DMSO-d6): δ11.30(s,1H),8.39(d,J=4.8Hz,1H),8.26(s,1H),8.19(s,1H),7.89(d,J=8.4Hz,1H),7.83(d,J=8.8Hz,1H),7.39(d, J=8.4Hz,1H),7.25(s,1H),7.08(d,J=3.2Hz,1H),6.73-6.71(m,1H),3. 66(s,2H),3.34-3.23(m,4H),2.78(d,J=4.8Hz,3H),2.64-2.54(m,4H).

[0403] Synthesis of Compound 5-Compound 204

[0404] According to the method described in compound 4, intermediates 5a and 3a were replaced by the intermediates in the table below to synthesize compounds 5 to 204. The structural formulas of the compounds in the examples can be found in the previous table.

[0405] The present invention will be further described in detail below with reference to specific examples and data. It should be understood that these examples are merely illustrative of the present invention and are intended to illustrate the specific combinations, preparation methods, and functions and effects of the present invention, and are not intended to limit the scope of the invention in any way. The beneficial effects of the pharmaceutical combination of the present invention can also be determined by other test models known to those skilled in the relevant art.

[0406] Biological Examples

[0407] Experimental Example 1: Evaluation of the PARP1 / 2 inhibitory activity of compounds

[0408] The PARP1 / 2 inhibitory activity of the disclosed compounds was tested in an assay using Histone as a substrate.

[0409] Experimental purpose: According to the established experimental method, the IC50 value of the compound of the present application for inhibition of PARP1 / 2 enzyme activity was detected, with AZD-2281 (Olaparib) as the positive control compound.

[0410] Experimental reagents:

[0411] Recombinant human PARP1 protein (Abcam, cat. ab279663); recombinant human PARP2 protein (BPS, cat. 80502); recombinant histone H1 (Active Motif, cat. 81126); NAD+, Biotin-Labeled (BPS, cat. 80610); SuperBlock (TBS) Blocking Buffer (Thermo Scientific TM ,cat.37535);Streptavidin(HRP)(Abcam,cat.ab7403); Peroxidase Chemiluminescent Substrate Kit (Seracare, cat.5430-0040); 20xPBS (CST, cat.9808S); 20xPBST (CST, cat.9809S); AZD2281 (Selleck, cat.S1060)

[0412] Experimental method 1: PARP1 inhibitory activity

[0413] 1. Compound preparation: Dilute the compound with DMSO to a solution with a final concentration of 1000 times in a 384-well plate and set aside.

[0414] 2. Coating microplate:

[0415] 1) Dilute Histone with PBS, add 25 μL of Histone mixture to each well, and incubate for 2 hours. 2) Wash each well five times with PBST. Remove the solution on a clean paper towel. 3) Add 75 μL of Blocking Buffer to each well and incubate at room temperature for 1 hour. 4) Wash each well five times with PBST. Remove the solution on a clean paper towel.

[0416] 3. Ribosylation reaction:

[0417] 1) Transfer 25 nL of the reserved compound at a 1000x final concentration to a 384-well plate. Add 25 nL of 100% DMSO to each of the Min and Max control wells. 2) Prepare a PARP1 solution at a 2.5x final concentration in 1x Assay buffer. 3) Add 10 μL of enzyme solution to each of the compound and Max control wells; add 10 μL of 1x Assay buffer to the Min control well. 4) Centrifuge at 1000 rpm for 60 seconds and incubate at room temperature for 15 minutes. 5) Prepare a substrate solution at a 1.67x final concentration in 1x Assay buffer. Add 500 μM NAD+ to the substrate solution and add 15 μL of substrate solution to each well to initiate the reaction. 6) Centrifuge at 1000 rpm for 60 seconds and incubate at room temperature for 2 hours. 7) Wash each well five times with PBST. Drain the solution on a clean paper towel.

[0418] 4. Detection:

[0419] 1) Prepare Streptavidin-HRP solution, add 25 μL to each well, centrifuge at 1000 rpm for 60 seconds, and incubate at room temperature for 30 minutes. 2) Wash each well five times with PBST. Drain the solution on a clean paper towel. 3) Add 50 μL of ELISA Chemiluminescent Substrate to each well. 4) Centrifuge at 1000 rpm for 60 seconds. Read the plate using EnSight after 5 minutes.

[0420] 5. Data analysis,

[0421] The inhibition rate was calculated using the following formula: inhibition rate % = (maximum signal - compound signal) / (maximum signal - minimum signal) × 100, where "minimum signal" is the mean value of the negative control wells and "maximum signal" is the mean value of the positive control wells.

[0422] Fitting the dose-effect curve: With the log value of the concentration as the X-axis and the percentage inhibition rate as the Y-axis, the log (inhibitor) vs. response-variable slope of the analysis software GraphPadPrism5 was used to fit the dose-effect curve to obtain the inhibition IC of the compound of the present disclosure on the enzyme activity. 50 The fitting formula is: Y = bottom + (top - bottom) / (1 + 10^((logIC50-X)*HillSlope)).

[0423] Experimental method 2: PARP2 inhibitory activity

[0424] The PARP2 inhibitory activity assay was performed in the same manner as in assay 1, except that recombinant human PARP2 solution was used instead of the PARP1 solution used in step 2 (2) of "3. Ribosylation Reaction," and 500 μM NAD+ was not added in the fifth step of the glycosylation reaction.

[0425] Table 1. IC50 values ​​of the compounds disclosed herein for inhibition of PARP1 / 2 enzymes Note: " / " means the data has not been tested.

[0426] The results in Table 1 indicate that the compounds of the present invention have high selectivity for PARP1 and may reduce the toxicity of PARP2 without significantly reducing efficacy. The inventors have also unexpectedly discovered that the compounds disclosed herein have excellent physical and chemical stability, good bioavailability (e.g., low clearance), and good drugability. Therefore, the compounds of the present invention have fewer side effects than olaparib (AZD-2281) and have high clinical application value.

[0427] Example 2: MDA-MB-436 cell proliferation inhibition test

[0428] Human breast cancer MDA-MB-436 (purchased from ATCC) cells were cultured in DMEM medium (supplemented with 10% fetal bovine serum and 1% double antibody) at 37°C and 5% carbon dioxide. Cells in the logarithmic growth phase were taken, digested, and a cell suspension of a certain concentration was prepared. The cell suspension was inoculated into a 96-well plate, and 100 μL of cell suspension was added to each well of the 96-well plate. After incubation overnight, different concentrations of compounds were added and placed in a cell culture incubator for 7 days. After the culture was completed, 50 μL of CellTiter-Glo reagent was added to each well, mixed with a microplate shaker for 2 minutes, and placed at room temperature for 60 minutes. The fluorescence value is read by the multimode microplate reader according to the formula: [(1-(RLU compound -RLU blank) / (RLU control -RLU blank )) × 100%] to calculate the cell proliferation inhibition rate. GraphPad Prism 6.0 software was used to fit the IC 50 value.

[0429] Table 2. Inhibitory activity of the compounds disclosed herein on MDA-MB-436 cell proliferation

[0430] The experimental results show that the compound of the present invention has significant proliferation inhibitory activity on MDA-MB-436 cells.

[0431] Example 3: Evaluation of bidirectional permeability using the MDR1-MDCKⅡ cell model

[0432] MDR1-MDCKⅡ cells were cultured at 3.3 x 10 5 Cells were seeded into 96-well plates at a concentration of 10 cells / mL and grown for 4-7 days to form a monolayer of confluent cells. The test compound was added at a concentration of 2 μM to the dosing end wells on the apical or basolateral side of the monolayer cells and incubated at 37.0°C in a 5.0% CO2 incubator for 2.5 hours. The integrity of the cell monolayer was determined by the fluorescein exclusion assay. The buffer was removed from the apical and basolateral sides, and the concentration of the test compound was determined using LC-MS / MS. The concentration data was used to calculate the apparent permeability coefficient of transport from the apical side to the basolateral side and from the basolateral side to the apical side of the monolayer cells, and to calculate the efflux rate. Calculation formula: Efflux Ratio = Papp (BA) / Papp (AB)

[0433] Table 3. Bidirectional permeability of compounds of the present disclosure to MDR1-MDCKII cells

[0434] The experimental results show that the compound of the present invention has higher cell permeability and lower efflux rate in MDR1-MDCKⅡ cells.

[0435] Experimental Example 4: Preliminary pharmacokinetic test

[0436] 1. Healthy ICR mice were randomly divided into 3 groups of 3 mice each, with 9 male mice weighing 30-35 g for each administration route, and the test compound was administered intravenously (1 mg / kg) or orally (5 mg / kg).

[0437] The animals were fasted for 12 hours before the experiment and allowed to drink water freely. They were fed 4 hours after the administration.

[0438] 2. Blood collection time and sample processing

[0439] Intravenous and oral administration: 0.25h, 0.5h, 1.0h, 2.0h, 3.0h, 4.0h, 6.0h, 8.0h and 24h after administration.

[0440] Blood was collected continuously, with 3 animals collected at each time point. Plasma collection and processing: 30-40 μL of venous blood was collected from the retroorbital venous plexus of mice at the above-set time points, placed in EDTA-K2 tubes, centrifuged at 3500 rpm for 10 minutes, and plasma was separated and frozen in a -20°C refrigerator.

[0441] 3. Sample testing and data analysis

[0442] The concentration of the compound in mouse plasma was determined by LC / MS / MS, and the pharmacokinetic parameters after administration were calculated using a non-compartmental model using Phoenix 8.3 software (Pharsight, USA).

[0443] 4. Experimental Results

[0444] Table 4. Pharmacokinetic parameters of the compounds of the present invention in mouse plasma Note: iv: intravenous injection (1 mg / kg); po: oral administration (5 mg / kg).

[0445] 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.

[0446] As shown in Table 1, the compounds of the present invention have high activity against PARP1 and high selectivity for PARP2, and can reduce the toxicity caused by PARP2 without significantly reducing the efficacy. As shown in Table 2, the compounds of the present invention have strong proliferation inhibitory activity against human breast cancer cells (MDA-MB-436). The inventors also unexpectedly found that the compounds of the present invention have high cell permeability and low efflux rate in MDR1-MDCKⅡ cells, 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.

Claims

1. A compound represented by general formula (I), or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt, crystal form, solvate, hydrate or prodrug thereof, in, X 1 Independently selected from -N-, -NR 14 -、-CR 7 -、-CR 7 R 7’ 、-CH2CR 7 R 7’ -、-CR 7 R 7’ -CH2-, O and S; X 2 Independently selected from -N-, -NR 15 -、-CR 8 、-CR 8 R 8’ -, O and S; X 3 Independently selected from -N-, -NR 16 -、-CR 9 -, O and S; X 4 、X 8 are each independently selected from -N- and -C-; is a single bond or a double bond; and X 1 、X 2 、X 3 、X 4 、X 8 Together they constitute a five-membered heteroaryl, or a partially saturated five-membered or six-membered heterocyclic group; wherein the heteroaryl or heterocyclic group each independently contains 1, 2 or 3 heteroatoms independently selected from N, O or S; R 7 、R 7’ 、R 8 、R 8’ 、R 9 are each independently selected from hydrogen, halogen, hydroxy, cyano, C1-C3 alkoxy, unsubstituted or substituted C3-C6 cycloalkyl or unsubstituted or substituted C1-C6 alkyl; or, R 7 With R 7’ or R 8 With R 8’ Together they constitute a C3-C6 cycloalkyl group; R 7 、R 7’ 、R 8 、R 8’ 、R 9 Each is independently preferably hydrogen, halogen or C1-C4 alkyl; R 7 、R 7’ 、R 8 、R 8’ 、R 9 are each independently more preferably hydrogen, F or methyl; or preferably, R 7 With R 7’ or R 8 With R 8’ Together they form a C3-C4 cycloalkyl group, such as a cyclopropyl or cyclopentyl group; R 14 、R 15 、R 16 R is independently selected from hydrogen, unsubstituted or substituted C3-C6 cycloalkyl, unsubstituted or substituted C1-C6 alkyl; 14 、R 15 、R 16 Each is independently preferably hydrogen or C1-C3 alkyl; R 14 、R 15 、R 16 Each independently more preferably is methyl; X 5 、X 6 Each independently selected from -N- and -CR 10 -;R 10 R is selected from hydrogen, halogen, cyano, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted C1-C6 alkyl; 10 Preferably, hydrogen, halogen, cyano or C1-C4 alkyl; R 10 More preferably, it is hydrogen, fluorine, chlorine or methyl; X 7 -N- or -CR 17 -;R 17 R is selected from hydrogen, halogen, cyano, unsubstituted or substituted C1-C6 alkoxy, unsubstituted or substituted C1-C6 alkyl; 17 Preferably, hydrogen, halogen, cyano, or C1-C4 alkyl; R 17 More preferably, it is hydrogen or fluorine, chlorine, or methyl; R 1 、R 1’ 、R 2 、R 3 、R 4 、R 5 are each independently selected from hydrogen, unsubstituted or substituted C1-C6 alkyl; or R 4 、R 5 Together with the carbon atoms to which they are attached, they form a C3-C6 cycloalkyl group; s and n are each independently selected from 0, 1 and 2; Y is N or CH; R 6 Selected from: Each R 11 independently selected from halogen, cyano, C1-C3 alkoxy, carbonyl, -CONHR 13 , amino, preferably selected from halogen, -CONHR 13 and cyano; more preferably selected from -CONHR 13 ; m is 0, 1, 2, or 3; R 12 Selected from hydrogen, cyano, halogen, unsubstituted or substituted C1-C4 alkyl; R 13 is hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C3-C8 cycloalkyl, unsubstituted or substituted C1-C6 alkoxy, or unsubstituted or substituted 3-8 membered heterocycloalkyl; preferably, R 13 is hydrogen, unsubstituted or halogen-substituted C1-C4 alkyl, C3-C6 cycloalkyl, or C1-C6 alkoxy; the heterocycloalkyl group refers to a heterocycloalkyl group containing 1-3 heteroatoms selected from N, O, and S; preferably, R 13 is methyl, ethyl, C2-C3 alkoxy, cyclopropyl, propylene oxide, oxetane or oxolane; Among them, R 1 、R 1’ 、R 2 、R 3 、R 4 、R 5 、R 7 、R 7’ 、R 8 、R 8’ 、R 9 、R 10 、R 12 、R 13 、R 14 、R 15 、R 16 、R 17 The substitution mentioned herein refers to substitution by one or more selected from C1-C4 alkyl, C1-C6 alkoxy, halogen, hydroxy, cyano, amino, carboxyl, and C3-C6 cycloalkyl; The conditions are: When X 5 and X 8 When both are -N-, X 1 、X 2 、X 3 、X 4 At least one is -N-; When X 4 and X 5 When both are -N-, X 3 Also -N-; When X 3 is oxygen and X 1 、X 2 、X 4 、X 8 -C-, X 7 -CR 17 -, R 1 、R 1’ 、R 17 Not simultaneously hydrogen; Preferably, the conditions are: When X 8 When it is -N-, X 1 、X 2 、X 3 At least one is -N-.

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

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

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

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

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

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

9. The use according to claim 8, wherein The disease is cancer, and the genome of the cancer is of a type deficient in homologous recombination repair, Alternatively, the cancer is dependent on a pathway that repairs double-strand DNA damage but lacks homologous recombination, Alternatively, 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, Alternatively, the cancer comprises one or more cancer cells that lack BRCA1 or BRCA2 or that have a BRCA1 or BRCA2 mutation.

10. The use according to claim 9, wherein The cancer includes, but is not limited to, 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, glioblastoma, Ewing sarcoma, pancreatic cancer, melanoma, bile duct cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, lymphoma, and blood cancer.

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