Pyrimidine PLK1 inhibitor as well as preparation method and application thereof

Abstract

The invention provides a novel PLK1 inhibitor which can be used for preventing and / or treating diseases related to PLK1, such as tumors, has higher PLK1 inhibition activity and tumor cell killing activity, improves physicochemical properties such as solubility and permeability, and has higher bioavailability.

Description

[0001] This application claims priority to an earlier application filed on August 23, 2024 with the China National Intellectual Property Administration, application number 202411163027.2, entitled "A pyrimidine PLK1 inhibitor and its preparation method and use", the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This invention relates to the field of biochemistry, specifically to a pyrimidine PLK1 inhibitor, its preparation method, and its uses. Background Technology

[0003] PLK1 belongs to the Polo-like kinase (PLKs) family (including PLK1 / 2 / 3 / 4) and is a highly conserved Ser / Thr protein kinase that plays an important role in normal cell cycle function, centrosome maturation, and cytoplasmic segregation. PLK1 regulates the G2 / M transition by phosphorylating downstream cyclin proteins such as WEE1 / CDK1; PLK1 inhibition leads to mitotic arrest in the G2 phase and induces apoptosis (Sci Signal. 2018 Aug 14; 11(543):eaar4195). In addition, PLK1 also has regulatory effects on cellular functions other than mitosis, such as PLK1 repairing damaged DNA and promoting tumor cell proliferation (Mol Cell. 2021 Mar 4; 81(5):1084-1099.e6.). PLK1 overexpression is found in most malignant tumors, including colorectal cancer, breast cancer, lung cancer, prostate cancer, leukemia and pancreatic cancer, and mediates poor prognosis (Genes 2019, 10, 208; World J Gastroenterol 2005; 11(36): 5644-5650). In addition, PLK1 is highly associated with multiple tumor-related targets and pathways. RAS mutations cause mitotic stress, making tumor cells more dependent on PLK1 for mitosis. Interference with mitosis can promote synthetic lethality in tumor cells. PLK1 knockout or inhibition of PLK1 kinase activity can arrest mitosis in RAS mutant cells at the G2 / M phase (Cell 137, 835–848, May 29, 2009). PLK1 can directly phosphorylate p53 protein, mediating p53 instability, or indirectly promote the phosphorylation of TOPORS and GTSE1, degrading p53 and causing it to lose its anti-tumor effect. PLK1 can directly stabilize or promote the expression of the oncogenic protein Myc, promoting tumor development and progression. PLK1 can also inhibit PTEN phosphorylation and activate the PI3K signaling pathway.

[0004] Therefore, small molecule enzymes targeting PLK1 have broad therapeutic effects on PLK1-related tumors.

[0005] PCM-075 is currently the most advanced PLK1 inhibitor in clinical trials. It has shown significant anti-tumor activity in preclinical models and clinical studies, whether used alone or in combination. However, its solubility, permeability, and bioavailability are relatively low in preclinical studies, and its anti-tumor activity needs further improvement. Patent application WO2009040399A discloses a class of PLK1 inhibitors, among which compound A1C3M2 shows some PLK1 inhibitory activity, but its ability to kill tumor cells is weak, and its low solubility further limits its in vivo application. Therefore, this therapeutic area still needs molecules with higher activity, better physicochemical properties, and higher bioavailability.

[0006] Summary of the Invention

[0007] This invention provides a novel PLK1 inhibitor with higher PLK1 inhibitory activity and tumor cell killing activity, and improved physicochemical properties such as solubility and permeability, resulting in higher bioavailability. It can be used to prevent and / or treat PLK1-related diseases, such as tumors.

[0008] According to one aspect of the invention, the invention provides compounds of formula (I) or their deuterated derivatives or pharmaceutically acceptable salts thereof:

[0009]

[0010] in:

[0011] -XY- is selected from -CH2-CH2-, -CH=CH-, -N=CH-, and -CH=N-.

[0012] n is 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0013] R1 is selected from H and C. 1-4 Straight-chain or branched alkyl groups, C 3-7 Cycloalkyl groups, 3-7 membered heterocyclic alkyl groups containing 1-3 independently selected N, O, S cyclic heteroatoms connected to a proximal group via a C or N ring, wherein the alkyl group, cycloalkyl group, or heterocyclic alkyl group is optionally surrounded by one or more independently selected F, Cl, Br, -OR4, CN, -NR4R5, =O, C 1-4 Substituted with straight-chain or branched alkyl groups;

[0014] R2 is independently selected from F, Cl, Br, OH, CN, =O, C 1-4 Straight-chain or branched alkyl groups, C 3-7Cycloalkyl groups, 3-7 membered heterocyclic alkyl groups containing 1-3 independently selected N, O, S cyclic heteroatoms connected to a proximal group via a C or N ring, wherein the alkyl group, cycloalkyl group, or heterocyclic alkyl group is optionally surrounded by one or more independently selected F, Cl, Br, -OR4, CN, -NR4R5, =O, C 1-4 Substituted with straight-chain or branched alkyl groups;

[0015] R1 and R2, together with the atoms on the piperazine ring to which they are jointly attached, can form a 3-7 membered heterocyclic alkyl group containing 1-3 independent cyclic heteroatoms selected from N, O, and S, wherein the heterocyclic alkyl group is optionally surrounded by one or more independent atoms selected from F, Cl, Br, -OR4, CN, -NR4R5, C 1-4 Straight-chain or branched alkyl groups, C 2-4 Straight-chain or branched alkenyl, C 2-4 Substitution of straight-chain or branched alkynyl groups;

[0016] Two R2 atoms, together with the atoms on the piperazine ring they are connected to, can form 3-7 membered heterocyclic alkyl groups containing 1-3 independent cyclic heteroatoms selected from N, O, and S. 3-7 Cycloalkyl group, wherein the heterocycloalkyl group is optionally selected by one or more elements independently selected from F, Cl, Br, -OR4, CN, -NR4R5, C 1-4 Straight-chain or branched alkyl groups, C 2-4 Straight-chain or branched alkenyl, C 2-4 Substitution of straight-chain or branched alkynyl groups;

[0017] R3 is selected from hydrogen, C 1-4 Straight-chain or branched alkyl groups, C 3-7 Cycloalkyl groups, 3-7 membered heterocyclic alkyl groups containing 1-3 cyclic heteroatoms independently selected from N, O, and S, wherein the alkyl group, cycloalkyl group, or heterocyclic alkyl group is optionally surrounded by one or more elements independently selected from F, Cl, Br, -OR4, CN, -NR4R5, and C. 1-4 Straight-chain or branched alkyl groups, C 2-4 Straight-chain or branched alkenyl, C 2-4 Straight-chain or branched alkynyl groups, C 3-7 The alkyl group is replaced by a 3-7 membered heterocyclic alkyl group containing 1-3 independent cyclic heteroatoms selected from N, O, and S, connected to a proximal group via a C or N on the ring.

[0018] R4 and R5 are independently selected from H, OH, CN, NH2, and C. 1-4 Straight-chain or branched alkyl groups, C 2-4 Straight-chain or branched alkenyl, C 2-4 Straight-chain or branched alkynyl groups, C 1-4 Straight-chain or branched alkoxy groups, C 3-7Cycloalkyl, 3-7 membered heterocyclic alkyl groups containing 1-3 independent cyclic heteroatoms selected from N, O, S, connected by a C or N atom on the ring to a proximal group, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, or heterocyclic alkyl group is optionally surrounded by one or more elements selected from F, Cl, Br, OH, CN, NH2, C 1-4 Substituted with straight-chain or branched alkyl groups;

[0019] In a preferred embodiment

[0020] -XY- is selected from -CH2-CH2-, -CH=CH-, and -N=CH-;

[0021] n is 0, 1, 2, 3 or 4;

[0022] R1 is selected from H and C. 1-4 Straight-chain or branched alkyl groups, C 3-7 Cycloalkyl group, wherein the alkyl group, or cycloalkyl group, is optionally composed of one or more elements independently selected from F, Cl, Br, -OR4, CN, -NR4R5, =O, C. 1-4 Substituted with straight-chain or branched alkyl groups;

[0023] R2 is independently selected from F, Cl, Br, OH, CN, =O, C 1-4 Straight-chain or branched alkyl groups, C 3-7 Cycloalkyl group, wherein the alkyl group, or cycloalkyl group, is optionally composed of one or more elements independently selected from F, Cl, Br, -OR4, CN, -NR4R5, =O, C. 1-4 Substituted with straight-chain or branched alkyl groups;

[0024] R3 is selected from hydrogen, C 1-4 Straight-chain or branched alkyl groups, C 3-7 Cycloalkyl, wherein the alkyl group, or cycloalkyl group, is optionally selected independently from one or more elements chosen from F, Cl, Br, -OR4, CN, -NR4R5, C. 1-4 Straight-chain or branched alkyl groups, C 2-4 Straight-chain or branched alkenyl, C 2-4 Straight-chain or branched alkynyl groups, C 3-7 Substituted with cycloalkyl groups;

[0025] R4 and R5 are independently selected from H, OH, CN, NH2, and C. 1-4 Straight-chain or branched alkyl groups, C 2-4 Straight-chain or branched alkenyl, C 2-4 Straight-chain or branched alkynyl groups, C 1-4 Straight-chain or branched alkoxy groups, C 3-7 Cycloalkyl, wherein the alkyl, alkenyl, alkynyl, alkoxy, or cycloalkyl group is optionally surrounded by one or more elements selected from F, Cl, Br, OH, CN, NH2, C. 1-4Substituted with straight-chain or branched alkyl groups;

[0026] In a preferred embodiment

[0027] -XY- is selected from -CH2-CH2-, -CH=CH-, and -N=CH-;

[0028] n is 0;

[0029] R1 is selected from C 1-4 Straight-chain or branched alkyl groups, wherein the alkyl group is optionally composed of one or more elements independently selected from F, Cl, Br, -OR4, CN, -NR4R5, C. 1-4 Substituted with straight-chain or branched alkyl groups;

[0030] R3 is selected from hydrogen, C 1-4 Straight-chain or branched alkyl groups, wherein the alkyl group is optionally composed of one or more elements independently selected from F, Cl, Br, -OR4, CN, -NR4R5, C. 1-4 Substituted with straight-chain or branched alkyl groups;

[0031] R4 and R5 are independently selected from H;

[0032] In a preferred embodiment

[0033] -XY- is -CH2-CH2-;

[0034] n is 0;

[0035] R1 is selected from C 1-4 Straight-chain or branched alkyl groups;

[0036] R3 is selected from hydrogen, C 1-4 Straight-chain or branched alkyl groups, wherein the alkyl group is optionally composed of one or more elements independently selected from F, Cl, Br, -OR4, CN, -NR4R5, C. 1-4 Substituted with straight-chain or branched alkyl groups;

[0037] R4 and R5 are independently selected from H;

[0038] In a preferred embodiment, the compounds of the present invention are selected from the following compounds or their deuterated derivatives or pharmaceutically acceptable salts:

[0039]

[0040] In this invention, although substituents are disclosed as groups or ranges, the groups or ranges of this invention specifically refer to each particular group covered therein. For example, the term "C" 1-4"Straight-chain or branched alkyl" specifically refers to independently disclosed methyl (i.e., C1 alkyl), ethyl (i.e., C2 alkyl), straight-chain or branched propyl (i.e., C3 alkyl), and straight-chain or branched butyl (i.e., C4 alkyl).

[0041] In this invention, alkenyl groups include cis and trans.

[0042] In this invention, "C" 2-4 Examples of "straight-chain or branched alkenyl" include, but are not limited to, vinyl, allyl, propen-2-yl, buten-1-yl, buten-2-yl, etc.

[0043] In this invention, "C" 2-4 Examples of "straight-chain or branched alkynyl" include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-2-yl, butyn-3-yl, etc.

[0044] In this invention, "C" 3-7 Examples of "cycloalkyl" include cyclopropane, cyclobutane, cyclopentane, cyclohexane, and cycloheptane.

[0045] In this invention, examples of "3-7 membered heterocyclic alkyl groups containing 1-3 independently selected cyclic heteroatoms from N, O, and S" include, but are not limited to, ethylene oxide, cyclothioethane, aziridine, oxacyclobutane, N-heterocyclic butane, α-lactam ring, β-lactam ring, β-lactone, tetrahydrofuran, thiocyclopentane, pyrrolidine, pyrrolidone, pyrrolidone, dioxolane, oxazolidinone, oxazolidinone, isoxazolidinone, thiazolinone, isotoxazolidinone, thiazolinone, isothiazolidinone, thiazolinone, imidazolinone, imidazolinone, pyrazolone, tetrahydropyran, dihydropyran, pyran, piperidine, piperidinone, 1,4-dioxane, morpholine, morpholinone, piperazine, aziridine, hexane, thiocycloheptane, 1,4-oxazane, and 1,4-thiazolinone.

[0046] The compounds of this invention can be asymmetric, for example, having one or more stereocenters. All stereoisomers, such as enantiomers and diastereomers, are included within the scope of this invention unless otherwise specified. In this invention, compounds containing asymmetrically substituted carbon atoms can be isolated in either optically active or racemic form. Various methods for preparing the optically active form are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis.

[0047] This invention also includes pharmaceutically acceptable salts of the said compounds. The compounds of this invention can be prepared into pharmaceutically acceptable salts by reacting with non-toxic inorganic or organic acids. Inorganic acids include, for example, hydrobromic acid, hydroiodic acid, phosphoric acid, sulfuric acid, nitric acid, hydrogen sulfate, boric acid, hemisulfate, etc.; organic acids include, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, heptanoic acid, undecanoic acid, palmitic acid, stearic acid, oleic acid, oxalic acid, malonic acid, adipic acid, lactic acid, malic acid, maleic acid, tartaric acid, citric acid, succinic acid, ascorbic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphorsulfonic acid, citric acid, fumaric acid, gluconic acid, and various amino acids, etc.

[0048] The present invention also includes hydrates and solvates of the said compounds.

[0049] The present invention also includes all forms of the compounds in which the atoms are various isotopes. Isotopes include all atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include deuterium.

[0050] The present invention also includes prodrugs of the said compounds. A "prodrug" refers to a compound obtained by structural modification that has no or low activity in vitro but releases the compound through enzymatic or non-enzymatic conversion in vivo to exert its pharmacological effect.

[0051] Those skilled in the art will understand that the compounds of the present invention can be prepared by various methods disclosed in the literature. The compounds of the present invention can be prepared by reaction in a suitable solvent, and those skilled in the art of organic synthesis can readily select a suitable solvent that does not substantially react with the reactants, intermediates, or products. The reaction can be carried out in one solvent or a mixture of more than one solvent. The compounds of the present invention can be prepared by reaction at a suitable temperature, for example, between the solidification temperature and the boiling point of the solvent. Methods for preparing the compounds of the present invention involve the protection and deprotection of various chemical groups, and those skilled in the art of organic synthesis can readily determine whether protection and deprotection of chemical groups are necessary and to select suitable protecting groups. The reaction for preparing the compounds of the present invention can be monitored using any method known in the art, such as nuclear magnetic resonance spectroscopy, infrared spectroscopy, mass spectrometry, chromatography, etc.

[0052] The compounds of the present invention can be prepared, for example, by the following method:

[0053] Method I:

[0054]

[0055] The definitions of the groups in each formula are as described above.

[0056] The synthesis of Ib can be carried out in a solvent in the presence of a base, a ligand, and a catalyst. The solvent can be selected from 1,4-dioxane, tetrahydrofuran, water, methanol, toluene, xylene, dimethyl sulfoxide, N,N-dimethylformamide, or any mixture thereof; the base can be selected from potassium phosphate, potassium carbonate, sodium carbonate, cesium carbonate, sodium bicarbonate, etc.; the ligand can be selected from triphenylphosphine, dppp, dppf, etc.; and the catalyst can be selected from palladium acetate, tetratriphenylphosphine palladium, Pd2(dba)3, Pd(dppf)Cl2, Pd(PPh3)2Cl2, etc.

[0057] The synthesis of compound I can be carried out in a solvent in the presence of a base, a ligand, and a catalyst. The solvent can be selected from tetrahydrofuran, 1,4-dioxane, tert-butanol, toluene, xylene, DME, dimethyl sulfoxide, N,N-dimethylformamide, or any mixture thereof; the base can be selected from potassium carbonate, sodium carbonate, cesium carbonate, lithium bis(trimethylsilylamine), potassium tert-butoxide, sodium tert-butoxide, etc.; the ligand can be selected from BINAP, Davephos, Xphos, Xantphos, etc.; and the catalyst can be selected from Pd2(dba)3, Pd(OAc)2, etc.

[0058] Method II:

[0059]

[0060] The definitions of the groups in each formula are as described above.

[0061] The synthesis of compound II-b can be carried out in a solvent in the presence of a base, a ligand, and a catalyst. The solvent can be selected from tetrahydrofuran, 1,4-dioxane, tert-butanol, toluene, xylene, DME, dimethyl sulfoxide, N,N-dimethylformamide, or any mixture thereof; the base can be selected from potassium carbonate, sodium carbonate, cesium carbonate, lithium bis(trimethylsilylamine), potassium tert-butoxide, sodium tert-butoxide, etc.; the ligand can be selected from BINAP, Davephos, Xphos, Xantphos, etc.; and the catalyst can be selected from Pd2(dba)3, Pd(OAc)2, etc.

[0062] The synthesis of compound I-1 can be carried out in a solvent in the presence of formamide or formamidinium. The solvent may be selected from formic acid, tetrahydrofuran, 1,4-dioxane, tert-butanol, isopropanol, n-butanol, DME, dimethyl sulfoxide, N,N-dimethylformamide, or any mixture thereof.

[0063] Method III:

[0064]

[0065] The definitions of the groups in each formula are as described above.

[0066] The synthesis of compound III-b can be carried out in a solvent in the presence of a base, a ligand, and a catalyst. The solvent can be selected from tetrahydrofuran, 1,4-dioxane, tert-butanol, toluene, xylene, DME, dimethyl sulfoxide, N,N-dimethylformamide, or any mixture thereof; the base can be selected from potassium carbonate, sodium carbonate, cesium carbonate, lithium bis(trimethylsilylamine), potassium tert-butoxide, sodium tert-butoxide, etc.; the ligand can be selected from BINAP, Davephos, Xphos, Xantphos, etc.; and the catalyst can be selected from Pd2(dba)3, Pd(OAc)2, etc.

[0067] The synthesis of compound I-2 can be carried out in a solvent in the presence of hydrazine. The solvent may be selected from formic acid, tetrahydrofuran, 1,4-dioxane, methanol, ethanol, n-propanol, isopropanol, n-butanol, tert-butanol, DME, dimethyl sulfoxide, N,N-dimethylformamide, or any mixture thereof.

[0068] The compounds of the present invention are capable of inhibiting PLK1. Therefore, according to another aspect of the present invention, the present invention provides a method for inhibiting PLK1 using the compounds of the present invention.

[0069] According to another aspect of the invention, the invention provides a method for preventing and / or treating PLK1-related diseases, wherein an individual in need is given an effective amount of a compound of the invention or a pharmaceutical composition containing a compound of the invention for prevention and / or treatment. PLK1-related diseases include any disease directly and / or indirectly related to the expression and / or activity of PLK1, such as diseases associated with PLK1 overexpression; diseases that are prevented and / or treated by modulating, for example, PLK1 activity.

[0070] PLK1-related diseases include solid tumors and hematologic malignancies. Examples of solid tumors include, but are not limited to, colorectal cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, gastric cancer, head and neck cancer, ovarian cancer, uterine cancer, glioma, liver cancer, esophageal cancer, bladder cancer, kidney cancer, lymphoma, melanoma, and osteosarcoma. Examples of hematologic malignancies include, but are not limited to, leukemia and myelodysplastic syndrome.

[0071] According to another aspect of the invention, the invention provides the use of the compounds of the invention in the preparation of PLK1 inhibitors.

[0072] According to another aspect of the invention, the invention provides the use of the compounds of the invention in the preparation of medicaments for the prevention and / or treatment of PLK1-related diseases.

[0073] When using the compounds of the present invention for the prevention and / or treatment of PLK1-related diseases, the compounds of the present invention may be administered in the form of a pharmaceutical composition. Therefore, according to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising the compounds of the present invention and a pharmaceutically acceptable carrier.

[0074] Those skilled in the art will understand that the pharmaceutical compositions of the present invention can be prepared by various methods disclosed in the literature. The compounds or pharmaceutical compositions of the present invention can be administered via a variety of routes, depending on whether local or systemic treatment is required and the area to be treated. For example, they can be administered orally, parenterally (e.g., intravenously, arterially, subcutaneously, intraperitoneally, intramuscularly, or by infusion), intracranially (e.g., intrathecal or intraventricular), transdermally, through the eyes, nose, vagina, rectum, or lungs (e.g., by inhalation or blowing in powder or aerosol).

[0075] For oral administration, the pharmaceutical compositions of the present invention are typically provided in the form of tablets, capsules, or solutions. Tablets may contain the compounds of the present invention or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers. These carriers include, but are not limited to, diluents, disintegrants, binders, lubricants, colorants, or preservatives. Capsules include hard capsules and soft capsules. For parenteral administration, the pharmaceutical compositions of the present invention may be administered by intravenous, intramuscular, or subcutaneous injection. They are typically provided as sterile aqueous solutions or suspensions or lyophilized powders, adjusted to a suitable pH and isotonicity.

[0076] The effective amount of the compounds of this invention can be determined based on the specific therapeutic use, the method of administration, and the individual's condition, such as the patient's. Those skilled in the art are capable of determining the effective amount of the compounds of this invention. Typical dosage ranges are, for example, from 1 μg / kg / day to 1000 mg / kg / day.

[0077] When used to prevent and / or treat PLK1-related diseases, the compounds of this invention can be used in combination with one or more other drugs. Other drugs include immune checkpoint inhibitors, targeted therapies, and chemotherapy drugs, etc. Detailed Implementation

[0078] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by this invention.

[0079] Preparation Example 1. Synthesis of common intermediate common int-1

[0080]

[0081] Synthesis of 1-(4-methoxy-3-nitrophenyl)-4-methylpiperazine (2):

[0082] Compounds 4-bromo-1-methoxy-2-nitrobenzene (500.0 mg, 2.155 mmol), N-methylpiperazine (280.6 mg, 2.80 mmol), 2-dicyclohexylphosphine-2′,6′-dimethoxy-biphenyl (88.5 mg, 0.215 mmol), cesium carbonate (1404.2 mg, 4.31 mmol), and tris(dibenzylacetone)palladium (98.7 mg, 0.11 mmol) were dissolved in 1,4-dioxane (8 mL). The mixture was purged with nitrogen three times, heated to 90 °C, and stirred for 6 hours. The reaction mixture was analyzed by LC-MS to confirm complete reaction. The reaction solution was diluted with water (15 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:1) to obtain a yellow oil (230.0 mg). LC-MS:[M+H] + :252.1.

[0083] Synthesis of 2-methoxy-5-(4-methylpiperazin-1-yl)aniline (common int-1):

[0084] Compound 1-(4-methoxy-3-nitrophenyl)-4-methylpiperazine (180.0 mg, 0.72 mmol) and Pd / C (80.0 mg, 10% w / w) were suspended in methanol (5 mL), purged three times with hydrogen, and then stirred at room temperature for 4 hours. After the reaction was complete as monitored by LC-MS, the reaction solution was filtered through diatomaceous earth. The filter cake was washed three times with ethyl acetate, and the filtrate was evaporated to dryness to give a yellow solid (150.0 mg). LC-MS: [M+H] + :222.2.

[0085] Preparation Example 2. Synthesis of the common intermediate common int-2

[0086]

[0087] Synthesis of 4-((2,2-diethoxyethyl)amino)-6-oxo-3,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (2):

[0088] 15.00 g (70.35 mmol) of tert-butyl 2,4-dipiperidinone-1-carboxylate and 9.37 g (70.35 mmol) of aminoacetaldehyde diethanoloxide were dissolved in toluene (30 mL). The mixture was stirred at 75 °C for 3 hours. TLC showed complete reaction of the starting material. The solution was concentrated to obtain a pale yellow oil (20.60 g), which was used directly in the next reaction without purification. LC-MS: [M+H-Boc] + :229.1.

[0089] Synthesis of 1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (3):

[0090] 20.60 g (62.73 mmol) of compound 4-((2,2-diethoxyethyl)amino)-6-oxo-3,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (10 mL) was added. The mixture was stirred at room temperature for 3 hours, concentrated, and then methyl tert-butyl ether (30 mL) was added. The mixture was stirred and filtered. The filter cake was slurried with methyl tert-butyl ether (30 mL) and cyclohexane (30 mL) and filtered. The concentrate yielded a yellow solid compound (3.20 g). LC-MS: [M+H] + :137.1.

[0091] Synthesis of 4-oxo-6,7-dihydro-1H-pyrrolo[3,2-c]pyridine-1,5(4H)-dicarboxylic acid ditert-butyl ester (4):

[0092] Compound 1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (3.20 g, 23.50 mmol), triethylamine (9.50 g, 93.88 mmol), and 4-dimethylaminopyridine (287.1 mg, 2.35 mmol) were dissolved in acetonitrile (50 mL), and di-tert-butyl dicarbonate (19.00 g, 87.06 mmol) was slowly added dropwise. The reaction was carried out at room temperature for 16 hours under nitrogen protection. After concentration, 50 mL of water was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was then concentrated and purified by column chromatography (petroleum ether:ethyl acetate = 5:1) to give a white solid compound (3.20 g). 1 H NMR (400MHz, DMSO) δ7.24 (d, J = 3.6 Hz, 1H), 6.49 (d, J = 4.0 Hz, 1H), 3.95 (t, J = 6.4 Hz, 2H), 3.15 (t, J = 6.4 Hz, 2H), 1.56 (s, 9H), 1.46 (s, 9H).

[0093] Synthesis of 4-oxo-1,4,6,7-tetrahydro-5H-pyrrolo[3,2-c]pyridine-5-carboxylic acid tert-butyl ester (5):

[0094] 3.20 g (9.51 mmol) of compound 4-oxo-6,7-dihydro-1H-pyrrolo[3,2-c]pyridine-1,5(4H)-dicarboxylic acid di-tert-butyl ester was dissolved in methanol (15 mL), and ammonia (5 mL) was added. The reaction was carried out at 75 °C for 5 hours. The mixture was concentrated, extracted with 15 mL of water and ethyl acetate (15 mL × 3), and the organic phases were combined and dried over anhydrous sodium sulfate. The mixture was then concentrated and purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to give a white solid compound (1.9 g). 1 H NMR (400MHz, DMSO) δ11.35 (s, 1H), 6.77–6.70 (m, 1H), 6.31 (t, J = 2.4Hz, 1H), 3.91 (t, J = 6.0Hz, 2H), 2.83 (t, J = 6.4Hz, 2H), 1.45 (s, 9H).

[0095] Synthesis of tert-butyl 1-methyl-4-oxo-1,4,6,7-tetrahydro-5H-pyrrolo[3,2-c]pyridine-5-carboxylic acid (6):

[0096] Compound 4-oxo-1,4,6,7-tetrahydro-5H-pyrrolo[3,2-c]pyridine-5-carboxylic acid tert-butyl ester (1.90 g, 8.04 mmol) and cesium carbonate (3.90 g, 11.97 mmol) were added to N,N-dimethylformamide (15 mL), followed by slow dropwise addition of iodomethane (1.70 g, 11.98 mmol). The reaction was carried out at room temperature for 2 hours. After extraction with 15 mL of water and ethyl acetate (15 mL × 3), the organic phases were combined and dried over anhydrous sodium sulfate. The mixture was then concentrated and purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to give a white solid compound (1.6 g). 1 H NMR (400MHz, DMSO) δ6.75 (d, J = 2.8 Hz, 1H), 6.32 (d, J = 3.2 Hz, 1H), 3.92 (t, J = 6.4 Hz, 2H), 3.53 (d, J = 3.2 Hz, 3H), 2.83 (t, J = 6.0 Hz, 2H), 1.45 (s, 9H).

[0097] Synthesis of tert-butyl 2-bromo-1-methyl-4-oxo-1,4,6,7-tetrahydro-5H-pyrrolo[3,2-c]pyridine-5-carboxylic acid (7):

[0098] 1-Methyl-4-oxo-1,4,6,7-tetrahydro-5H-pyrrolo[3,2-c]pyridine-5-carboxylic acid tert-butyl ester (1.60 g, 6.39 mmol) was dissolved in anhydrous tetrahydrofuran (18 mL) and methanol (6 mL). N-bromosuccinimide (1.16 g, 6.52 mmol) was slowly added under nitrogen protection at -65 °C for 4 hours. The mixture was concentrated, extracted with 15 mL of water and ethyl acetate (15 mL × 3), and the organic phases were combined and dried over anhydrous sodium sulfate. The mixture was then purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to give a white solid compound (1.3 g). 1 H NMR (400MHz, DMSO) δ6.49 (s, 1H), 3.93 (t, J = 6.4Hz, 2H), 3.49 (s, 3H), 2.88 (t, J = 6.4Hz, 2H), 1.45 (s, 9H).

[0099] Synthesis of 1-methyl-4-oxo-2-boronate pinacol ester-1,4,6,7-tetrahydro-5H-pyrrolo[3,2-c]pyridine-5-carboxylic acid tert-butyl ester (8):

[0100] Compounds 2-bromo-1-methyl-4-oxo-1,4,6,7-tetrahydro-5H-pyrrolo[3,2-c]pyridine-5-carboxylic acid tert-butyl ester (400.0 mg, 1.215 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium(II) dichloride (87.8 mg, 0.12 mmol), pinacol diboronate (401.4 mg, 1.58 mmol), and potassium acetate (358.0 mg, 3.65 mmol) were dissolved in 1,4-dioxane (5.0 mL). The reaction system was subjected to nitrogen protection at 80 °C for 0.5 h. After the reaction was complete, excess solvent was removed under vacuum to give a black solid (459.1 mg). LC-MS: [M+H-tBu] + :321.2.

[0101] Synthesis of 2-(2-chloro-5-fluoropyrimidin-4-yl)-1-methyl-4-oxo-1,4,6,7-tetrahydro-5H-pyrrolo[3,2-c]pyridine-5-carboxylic acid tert-butyl ester (common int-2):

[0102] 1-Methyl-4-oxo-2-borate pinacol ester-1,4,6,7-tetrahydro-5H-pyrrolo[3,2-c]pyridine-5-carboxylic acid tert-butyl ester (459.1 mg, 1.22 mmol), 2,4-dichloro-5-fluoropyrimidine (243.6 mg, 1.46 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium(II) dichloride (87.8 mg, 0.12 mmol), and potassium phosphate (774.4 mg, 3.65 mmol) were dissolved in a mixed solution of 1,4-dioxane and water (5.5 mL, 1,4-dioxane:water = 10:1). The reaction system was subjected to nitrogen protection at 90 °C for 3 hours. After the reaction was complete, water (10.0 mL) was added to quench the reaction. The mixture was extracted with ethyl acetate (10 mL × 3), the organic phases were combined, washed with saturated brine, and finally dried over anhydrous sodium sulfate. The solution was filtered and concentrated to obtain the crude product. Separation by column chromatography (dichloromethane:methanol = 10:1) yielded a yellow solid (65.0 mg). LC-MS: [M+H-tBu] + :325.1,327.1.

[0103] Preparation Example 3. Synthesis of common intermediates common int-3, common int-4, and common int-5

[0104]

[0105] Synthesis of 2-chloro-4-(1-ethoxyvinyl)-5-fluoropyrimidine (2):

[0106] Compounds 2,4-dichloro-5-fluoropyrimidine (4.00 g, 23.96 mmol), tributyl(1-ethoxyvinyl)tin (9.50 g, 26.30 mmol), and bis(triphenylphosphine)palladium dichloride (0.84 g, 1.20 mmol) were dissolved in N,N-dimethylformamide (50 mL) and reacted at 90 °C under nitrogen protection for 3 hours. The reaction was quenched with ice water, extracted with ethyl acetate (20 mL × 3), and the organic phases were combined and dried over anhydrous sodium sulfate. The mixture was purified by column chromatography (petroleum ether:ethyl acetate = 10:1) to give a colorless oily compound (3.40 g). 1 H NMR (400MHz, DMSO) δ8.88(d,J=3.2Hz,1H),5.19(d,J=2.8Hz,1H),4.85(d,J=2.8Hz,1H),3.93(q,J=6.8Hz,2H),1.32(t,J=7.2Hz,3H); LC-MS:[M+H] + Synthesis of 2-bromo-1-(2-chloro-5-fluoropyrimidin-4-yl)ethane-1-one (common int-3):

[0107] 2-Chloro-4-(1-ethoxyvinyl)-5-fluoropyrimidine (500.0 mg, 2.47 mmol) was dissolved in tetrahydrofuran (4.5 mL) and water (1.8 mL). N-bromosuccinimide (571.3 mg, 3.21 mmol) was added at 0 °C, and the reaction was carried out at room temperature for 15 minutes. The mixture was extracted with ethyl acetate (10 mL × 3), and the organic phases were combined and dried over anhydrous sodium sulfate. The resulting light yellow colorless oily compound (350.0 mg) was directly used in the next step of the reaction.

[0108] Synthesis of 2-(2-chloro-5-fluoropyrimidin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (commonint-4):

[0109] Compounds 2-bromo-1-(2-chloro-5-fluoropyrimidin-4-yl)ethane-1-one (1.6 g, 6.31 mmol), 2,4-piperidinedione (0.86 g, 7.60 mmol), and ammonium acetate (0.97 g, 12.58 mmol) were dissolved in ethanol and reacted at room temperature for 20 hours. The mixture was then concentrated and purified by column chromatography (dichloromethane:methanol = 10:1) to give a yellow solid compound (360.0 mg). 1 H NMR (400MHz, DMSO) δ12.36 (s, 1H), 8.73 (d, J = 3.2Hz, 1H), 7.28 (s, 1H), 7.14 (s, 1H), 3.41 (m, 2H), 2.89 (t, J = 6.8Hz, 2H); LC-MS: [M+H] + :267.0,269.0.

[0110] Synthesis of 1-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2-(2-chloro-5-fluoropyrimidin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (common int-5):

[0111] Compound 2-(2-chloro-5-fluoropyrimidin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (320.0 mg, 1.20 mmol) and cesium carbonate (1173.0 mg, 3.60 mmol) were dissolved in N,N-dimethylformamide (5 mL), followed by the addition of tert-butyl-(2-iodoethoxy)dimethylsilane (446.5 mg, 1.56 mmol). The reaction was carried out at room temperature for 4 hours. After extraction with 15 mL of water and ethyl acetate (10 mL × 3), the organic phases were combined and dried over anhydrous sodium sulfate. The concentrate was then purified by pTLC (dichloromethane:methanol = 15:1) to give a white solid compound (190.0 mg).1 HNMR(400MHz,DMSO)δ8.78(d,J=3.6Hz,1H),7.32(s,1H),7.21(d,J=4.2Hz,1H),4.58(t,J=5.2Hz,2H),3 .88(t,J=5.2Hz,2H),3.47–3.41(m,2H),2.96(t,J=6.8Hz,2H),0.73(s,9H),-0.15(s,6H); LC-MS:[M+H] + :425.1,427.1.

[0112] Preparation Example 4. Synthesis of the common intermediate common int-6

[0113]

[0114] Synthesis of ethyl 2-amino-5-(2-chloro-5-fluoropyrimidin-4-yl)-1H-pyrrole-3-carboxylate (1):

[0115] Ethyl 3-amino-3-iminopropionic acid hydrochloride (36.5 g, 219.07 mmol) was dissolved in ethanol (500.0 mL), and the solution was cooled to 0 °C. Sodium ethoxide (74.9 g, 220.13 mmol, 20 wt% in EtOH) was added, and the mixture was stirred for 30 minutes. Then, a solution of 2-bromo-1-(2-chloro-5-fluoropyrimidin-4-yl)ethane-1-one (27.7 g, 109.29 mmol) in ethanol (277.5 mL) was slowly added dropwise. The mixture was then allowed to return to room temperature and reacted for 16 hours. After the reaction was complete, the solution was concentrated under reduced pressure, diluted with water, extracted three times with ethyl acetate and washed twice with brine. The resulting organic phase was dried over anhydrous sodium sulfate and concentrated under vacuum. The crude product was purified by silica gel chromatography (petroleum ether:ethyl acetate = 5:1) to give a yellow solid (1.8 g). LC-MS: [M+H] + :285.0.

[0116] Synthesis of ethyl 2-amino-5-(2-chloro-5-fluoropyrimidin-4-yl)-1-methyl-1H-pyrrole-3-carboxylate (2):

[0117] Ethyl 2-amino-5-(2-chloro-5-fluoropyrimidin-4-yl)-1H-pyrrole-3-carboxylic acid (1.8 g, 6.32 mmol), benzyltriethylammonium chloride (1.4 g, 6.15 mol), and dimethyl sulfate (0.4 g, 3.17 mmol) were dissolved in dichloromethane solution (50 mL). The solution was cooled to 0 °C, and sodium hydroxide (1.52 g, 19.00 mmol, 50 wt% in H2O) was added. The reaction was carried out at room temperature for 16 hours. After the reaction was complete, citric acid was added to adjust the pH to 5–6, and the solution was diluted with water. The mixture was extracted three times with ethyl acetate and washed twice with brine. The resulting organic phase was dried over anhydrous sodium sulfate and concentrated under vacuum. The crude product was purified by silica gel chromatography (petroleum ether:ethyl acetate = 5:1) to give a white solid (570.0 mg). LC-MS: [M+H] + :299.1.

[0118] Synthesis of ethyl 2-(bis(tert-butoxycarbonyl)amino)-5-(2-chloro-5-fluoropyrimidin-4-yl)-1-methyl-1H-pyrrole-3-carboxylic acid (common int-6):

[0119] Ethyl 2-amino-5-(2-chloro-5-fluoropyrimidin-4-yl)-1-methyl-1H-pyrrole-3-carboxylate (570.0 mg, 1.91 mmol), di-tert-butyl dicarbonate (832.9 mg, 3.82 mmol), triethylamine (289.1 mg, 2.86 mmol), and 4-dimethylaminopyridine (23.3 mg, 0.19 mmol) were dissolved in dichloromethane (10 mL), and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was concentrated under vacuum, and the crude product was purified by silica gel chromatography (petroleum ether:ethyl acetate = 10:1) to give a white solid (500.0 mg). LC-MS: [M+H] + :499.1.

[0120] Example 1. Synthesis of compound DP01

[0121]

[0122] Synthesis of tert-butyl 2-(5-fluoro-2-((2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1-methyl-4-oxo-1,4,6,7-tetrahydro-5H-pyrrolo[3,2-c]pyridine-5-carboxylic acid (1):

[0123] 2-Methoxy-5-(4-methylpiperazin-1-yl)aniline (41.5 mg, 0.19 mmol), 2-(2-chloro-5-fluoropyrimidin-4-yl)-1-methyl-4-oxo-1,4,6,7-tetrahydro-5H-pyrrolo[3,2-c]pyridine-5-carboxylic acid tert-butyl ester (65.0 mg, 0.17 mmol), tris(dibenzylacetone)palladium (15.6 mg, 0.017 mmol), 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (21.2 mg, 0.034 mmol), and cesium carbonate (111.5 mg, 0.34 mmol) were dissolved in 1,4-dioxane (2 mL). The reaction system was subjected to nitrogen protection at 100 °C for 2 hours. After the reaction was complete, an aqueous solution (10 mL) was added to quench the reaction. The mixture was extracted with ethyl acetate (10 mL × 3), and the organic phases were combined. The mixture was then washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Separation by column chromatography (dichloromethane:methanol = 10:1) yielded a yellow solid (90.0 mg). LC-MS: [M+H] + :566.3.

[0124] Synthesis of 2-(5-fluoro-2-((2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1-methyl-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (DP01):

[0125] 90.0 mg (0.16 mmol) of 2-(5-fluoro-2-((2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1-methyl-4-oxo-1,4,6,7-tetrahydro-5H-pyrrolo[3,2-c]pyridine-5-carboxylic acid tert-butyl ester was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (0.3 mL) was added to the solution. The reaction system was reacted at room temperature for 1 hour. After the reaction was complete, the solution was concentrated to obtain the crude product. Separation by column chromatography (dichloromethane:methanol = 10:1) yielded a yellow solid (60.0 mg). 1 HNMR (400MHz, CDCl3) δ8.29(d,J=3.2Hz,1H),8.14(d,J=2.8Hz,1H),7.55(s,1H),7.38(d,J=3.6Hz,1H),6.81(d,J=8.8Hz,1H),6.54(dd,J=8.8,3.2Hz, 1H),5.46(s,1H),3.93(s,3H),3.86(s,3H),3.67–3.63(m,2H),3.23–3.10( m,4H),2.93(t,J=6.8Hz,2H),2.70–2.54(m,4H),2.38(s,3H); LC-MS:[M+H]+ :466.2.

[0126] Example 2. Synthesis of compound DPO2

[0127]

[0128] Synthesis of 2-(5-fluoro-2-((2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1-methyl-1,5-dihydro-4H-pyrrolo[3,2-c]pyridin-4-one (DP02):

[0129] 2-(5-fluoro-2-((2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1-methyl-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (30.0 mg, 0.064 mmol) was dissolved in N,N-dimethylformamide (1 mL), and 60% sodium hydroxide (25.6 mg, 0.64 mmol) was slowly added under ice bath conditions. The reaction system was stirred at room temperature for 48 hours. After the reaction was complete, the mixture was concentrated to obtain the crude product. After the reaction was complete, an aqueous solution (10 mL) was added to quench the reaction, and the mixture was extracted three times with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. A yellow solid (1.5 mg) was obtained by preparative separation. 1 H NMR (400MHz, MeOD) δ8.44(d,J=3.2Hz,1H),7.99(d,J=2.8Hz,1H),7.48(d,J=3.2Hz,1H),7.26(d,J=7.2Hz,1H),6.93(d,J=8.8Hz,1H),6.7 6(d,J=7.6Hz,1H),6.67(dd,J=8.8,3.2Hz,1H),4.03(s,3H),3.87(s,3H),3.15–3.08(m,4H),2.69–2.54(m,4H),2.34(s,3H).LC-MS:[M+H] + :464.2.

[0130] Example 3. Synthesis of compound DPO3

[0131]

[0132] Synthesis of 1-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2-(5-fluoro-2-((2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (1):

[0133] Compounds 1-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2-(2-chloro-5-fluoropyrimidin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (60.0 mg, 0.14 mmol), 2-methoxy-5-(4-methylpiperazin-1-yl)aniline (31.2 mg, 0.14 mmol), cesium carbonate (138.0 mg, 0.42 mmol), 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (17.6 mg, 0.028 mmol), and palladium acetate (3.2 mg, 0.014 mmol) were dissolved in 1,4-dioxane (1 mL) and reacted at 100 °C under nitrogen protection for 6 hours. The solution was concentrated and purified by column chromatography (dichloromethane:methanol-acetic acid = 10:1) to give a yellow solid compound (35.0 mg). LC-MS:[M+H] + :610.2.

[0134] Synthesis of 2-(5-fluoro-2-((2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1-(2-hydroxyethyl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (DP03):

[0135] The compound 1-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2-(5-fluoro-2-((2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (30.0 mg, 0.049 mmol) was dissolved in tetrahydrofuran (0.5 mL), and 1 M tetrabutylammonium fluoride (0.07 mL, 0.07 mmol) was added. The mixture was reacted at room temperature for 1 hour, and the resulting product was purified to obtain a white solid compound (7.3 mg). 1 H NMR (400MHz, DMSO) δ8.43(d,J=4.0Hz,1H),8.23(s,1H),7.38(d,J=2.8Hz,1H),7.1 9(s,1H),7.06(d,J=4.0Hz,1H),6.92(d,J=8.8Hz,1H),6.67(dd,J=8.8,2.8Hz,1H) ,4.69(t,J=5.2Hz,1H),4.38(t,J=4.8Hz,2H),3.73(s,3H),3.42–3.36(m,4H),3.0 4–2.99(m,4H),2.90(t,J=6.8Hz,2H),2.47–2.43(m,4H),2.21(s,3H); LC-MS:[M+H] + :496.3.

[0136] Example 4. Synthesis of compound DPO4

[0137]

[0138] Synthesis of ethyl 2-(bis(tert-butoxycarbonyl)amino)-5-(5-fluoro-2-((2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1-methyl-1H-pyrrole-3-carboxylic acid (1):

[0139] Ethyl 2-(bis(tert-butoxycarbonyl)amino)-5-(2-chloro-5-fluoropyrimidin-4-yl)-1-methyl-1H-pyrrole-3-carboxylate (50.0 mg, 0.10 mmol), 2-methoxy-5-(4-methylpiperazin-1-yl)aniline (26.6 mg, 0.12 mmol), tris(dibenzylacetone)palladium (9.18 mg, 0.010 mmol), 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (12.4 mg, 0.020 mmol), and cesium carbonate (65.2 mg, 0.20 mmol) were dissolved in 1,4-dioxane (3 mL). The mixture was stirred at 100 °C under an argon atmosphere for 6 hours. After the reaction was complete, the reaction solution was concentrated under vacuum, and the crude product was purified by silica gel chromatography (dichloromethane:methanol = 10:1) to obtain 40.0 mg. LC-MS:[M+H] + :684.3.

[0140] Synthesis of 6-(5-fluoro-2-((2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-7-methyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (DP04):

[0141] Ethyl 2-(bis(tert-butoxycarbonyl)amino)-5-(5-fluoro-2-((2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1-methyl-1H-pyrrole-3-carboxylic acid (40.0 mg, 0.0585 mmol) was dissolved in a mixed solution of formamide (6.7 mL), N,N-dimethylformamide (2.5 mL), and formic acid (1.0 mL). The mixture was stirred at 200 °C for 1 hour. The reaction solution was diluted with water and extracted three times with ethyl acetate. The combined organic phases were washed twice with brine. The resulting organic phase was dried over anhydrous sodium sulfate and concentrated under vacuum. The crude product was then used to prepare a white solid (1.0 mg). 1H NMR (400MHz, DMSO) δ12.15(s,1H),9.58(s,1H),8.55(d,J=3.4,1H),8.49(s ,1H),8.04(d,J=3.9,1H),7.49(d,J=2.8,1H),7.18(d,J=3.4,1H),6.98(d,J =8.9,1H),6.75(dd,J=8.9,2.9,1H),3.90(s,3H),3.76(s,3H),3.65(d,J=1 3.5,4H),3.51(d,J=11.9,2H),3.21–3.14(m,2H),2.86(s,3H).LC-MS:[M+H] + :465.2.

[0142] Example 5. Synthesis of compound DP05

[0143]

[0144] Synthesis of 2-(5-fluoro-2-((2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (DP05):

[0145] Compounds 2-(2-chloro-5-fluoropyrimidin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (40.0 mg, 0.15 mmol), 2-methoxy-5-(4-methylpiperazin-1-yl)aniline (33.2 mg, 0.15 mmol), tris(dibenzylindeneacetone)palladium (27.5 mg, 0.03 mmol), 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (37.3 mg, 0.06 mmol), and cesium carbonate (97.7 mg, 0.30 mmol) were dissolved in 1,4-dioxane (5 mL). The mixture was stirred at 100 °C for 32 hours under an argon atmosphere. After the reaction was complete, the reaction solution was concentrated under vacuum to obtain a crude product, which was then separated to yield a yellow solid (5.1 mg). 1H NMR (400MHz, DMSO) δ12.06(s,1H),8.52(d,J=3.3Hz,1H),8.13(d,J=2.8Hz,1H),7.80(s,1H),7.21(s,1H),7.09–7.04(m,1H),6.92(d,J=8.9 Hz,1H),6.53(dd,J=8.8,2.8Hz,1H),3.83(s,3H),3.42(d,J=2.2Hz,6H),3.07–3.03(m,4H),2.88(t,J=6.8Hz,2H),2.23(s,3H).LC-MS:[M+H] + :452.2.

[0146] Example 6. Test of the inhibitory activity of the compound against PLK1

[0147] The test compound was serially diluted 3-fold with DMSO in a 384-well dilution plate. 25 nL of the diluted compound was transferred to each well using ECHO, ensuring the DMSO concentration was 0.5%. 2.5 μL of 2×PLK1 enzyme solution was transferred to each well, centrifuged at 1000 rpm for 1 min, and incubated at 25°C for 10 min. 2.5 μL of a 2×ATP & PLK1 mixture was transferred to each well, centrifuged at 1000 rpm for 1 min, and incubated at 25°C for 60 min. 4 μL of ADP-Glo ​​Reagent was transferred to each well, centrifuged at 1000 rpm for 1 min, and incubated at 25°C for 40 min. 8 μL of ADP-Glo ​​Detection reagent was transferred to each well, centrifuged at 1000 rpm for 1 min, and incubated at 25°C for 40 min. The RLU (Relative Luminescence Unit) signal was read using a BMG microplate reader. The signal intensity was used to characterize the kinase activity. The IC50 of the positive drug was calculated using the nonlinear fitting formula (1) in GraphPad software. 50 :

[0148] Y=Bottom + (Top-Bottom) / (1+10^((LogIC50-X) ×HillSlope)) (1)

[0149] Where X represents the compound concentration log value, and Y represents the inhibition rate (%inhibition).

[0150] The results show that the compound of the present invention has good PLK1 inhibitory activity, IC50 50 Less than 1 nM, preferably less than 0.1 nM, IC50 of representative compounds 50 As shown in Table 1 below.

[0151] Example 7. HCT116 cell proliferation inhibition activity test

[0152] HCT116 cells were revived using complete culture medium (M'5A Medium + 10% FBS + 1% P / S). After approximately two passages, cell lines in good growth condition were selected. Cells in the logarithmic growth phase were collected by digestion and centrifugation and counted. After resuspending the cells, the cell suspension was seeded into 384-well plates, with 40 μL of cell suspension added to each well, and cultured overnight. The test compound was prepared as a stock solution using DMSO. Nine concentration gradients were obtained by stepwise 3-fold dilution with DMSO, starting at 1 μM. The compound was added to each well, and the culture plate was incubated at 37°C, 100% relative humidity, and 5% CO2 for 3 days. CellCounting-Lite 2.0 Luminescent Cell Viability Assay was added to each well, shaken for 2 min, and then incubated at room temperature for 30 min. The fluorescence value (RLU) of each well was then read in BMG. The cell proliferation inhibition rate data were processed using the following formula:

[0153] Inhibition Rate(Inh%)=100-(RLUCpd-RLUMin) / (RLUMax-RLUMin)*100%;

[0154] Wherein, RLUCpd represents the average fluorescence value of each well containing a compound, RLUMax represents the average fluorescence value of the DMSO well, and RLUMin represents the average fluorescence value of the blank well.

[0155] In Excel, the inhibition rates corresponding to different concentrations of the compound were calculated. Then, the inhibition rate curves were fitted using GraphPad Prism software, and the IC50 was calculated. 50 IC50 value, representing the IC50 of a compound 50 As shown in Table 1 below.

[0156] The results showed that the compound of the present invention has good inhibitory activity against HCT116 tumor cells, IC50. 50 Less than 20 nM, preferably less than 10 nM, IC50 of representative compounds 50 As shown in Table 1 below.

[0157] Example 8. Solubility Test

[0158] (1) Solution preparation: Prepare a 10 mM stock solution of the compound to be tested using DMSO. Test solution (buffer) preparation: Weigh 7.098 g Na2HPO4 and dissolve it in 500 mL of ultrapure water by sonication; weigh 3.40 g KH2PO4 and dissolve it in 250 mL of ultrapure water by sonication. After mixing the above buffer solutions, adjust the pH to 7.40.

[0159] (2) Experimental Operation

[0160] a) Add 30 μL of the compound stock solution to the wells of a 96-well solubility plate, creating a double-well configuration.

[0161] b) Add 970 μL of test solution to the well of the above compound.

[0162] c) Place the PTFE-encapsulated stainless steel rod stirrer into each hole, and then seal the holes with PTDE / SIL silicone plugs;

[0163] d) Transfer the solubility sample plate to a Thermomixer Comfort plate shaker and shake at 1100 rpm and 25°C for 2 hours.

[0164] e) After 2 hours, use a magnet to remove the stirring rod;

[0165] f) Transfer 200 μL of sample to a filter plate, filter under vacuum, and collect the filtrate;

[0166] g) Add 10 μL of the filtered sample and 10 μL of DMSO to 980 μL of methanol, and further dilute with methanol:water (1:1) 5 times as the filtered sample for LC-MS / MS analysis;

[0167] h) For standard samples, dilute the 10 mM DMSO stock solution to 300 μM;

[0168] i) Add 10 μL of 300 μM DMSO sample and 10 μL of buffer to 980 μL of methanol, and then dilute 5 times with methanol:water (1:1) as a standard sample for LC-MS / MS analysis.

[0169] (3) Data Analysis

[0170] Calculate solubility using Microsoft Excel.

[0171]

[0172] Where DF represents the dilution factor, Area is the area under the curve, and std is the standard.

[0173] The results showed that, compared with the clinical compound PCM-075 and the patented molecule WO2009040399-A1C3M2, the solubility of the compounds of the present invention was significantly improved. The solubility of representative compounds is shown in Table 1 below.

[0174] Table 1. Inhibitory activity and solubility tests of representative compounds against PLK1 and HCT116

[0175] Compound numbering <![CDATA[PLK1 IC 50 (nM)]]> <![CDATA[HCT116 IC 50 (nM)]]> Solubility (μM) pH 7.4 DP01 0.06 6.8 301.1 WO2009040399-A1C3M2 0.5 46.1 15.7 PCM-075 0.66 49.8 22.1

[0176] The compounds of this invention have a strong inhibitory effect on PLK1 kinase activity and HCT116 cell proliferation. Compared with the patented molecule WO2009040399-A1C3M2 and the clinical molecule PCM-075, they not only improve the inhibitory activity against PLK1 and HCT116, but also significantly improve solubility.

[0177] Example 9: Permeability Test

[0178] (1) MDCK-MDR1 cell culture: MDR1 cells were seeded at a density of 20,000 cells / well in a 24-well Transwell plate insertion system and cultured for 1-2 days until a cell monolayer was formed.

[0179] (2) Experimental Procedure: Test compounds DP01, PCM-075, and reference compounds (Metoprolol, Atenolol, Digoxin) were diluted to 10 μM from 10 mM stock solutions with buffer (HBSS containing BSA) and applied to the top or bottom of cell monolayers. The permeability of the test compounds from A to B (Papp(AB)) or from B to A (Papp(BA)) was repeatedly measured by incubation at 37°C, 5% CO2, and 95% relative humidity for 120 min. Furthermore, the efflux ratio of each compound was determined. Test and reference compounds were analyzed by LC-MS / MS, and quantification was performed based on the peak area ratio of analyte / internal standard (IS, Imipramine).

[0180] (3) Data Analysis:

[0181] The formula for calculating the apparent permeability coefficient Papp (cm / s) is: Papp=(dCr / dt)x Vr / (Ax C0)

[0182] The formula for calculating the Efflux ratio is: Efflux Ratio = Papp(BA) / Papp(AB).

[0183] Where dCr / dt represents the cumulative change in compound concentration in the receiving chamber over time (unit: seconds); Vr represents the solution volume in the receiving chamber (0.6 mL on the top side and 0.9 mL on the base side); and A represents the transport surface area, i.e., the surface area of ​​a monolayer of cells is 0.84 cm². 2 C0 represents the initial concentration in the donor chamber.

[0184] (4) Classification criteria:

[0185] Permeability <![CDATA[P app (10 -6 cm / s)]]> high >10 middle 1～10 Low ≤1

[0186] Table 2. MDCK-MDR1 cell permeability results

[0187] Compound numbering <![CDATA[P app(A-B) (10 -6 cm / s)]]> <![CDATA[P app(B-A) (10 -6 cm / s)]]> Efflux Ratio Metoprolol 35.0 35.9 1.02 Atenolol 0.18 0.37 2.01 Digoxin 1.06 23.85 22.44 DP01 12.01 59.73 4.97 PCM-075 3.52 33.37 9.48

[0188] The compounds of this invention exhibit high permeability in MDCK-MDR1 cells, with increased permeability and reduced efflux ratio compared to PCM-075.

[0189] Example 10: Mouse PK Test

[0190] (1) Experimental animals: Two groups of female Balb / c nude mice, three mice in each group, SPF grade, provided by Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0191] (2) Drug preparation: On the day of administration, take a certain amount of compounds DP01 and PC-M075 from the examples and prepare them using a solvent:

[0192] Prepare a 2 mg / mL solution by mixing 5% DMSO, 10% Solutol, and 85% Saline.

[0193] (3) Dosage and administration: The dosage is 20 mg / kg, and the administration volume is 10 mL / kg. Weigh the animal before administration and calculate the dosage based on its body weight. Administer orally via gavage. Fast the animal overnight (10-14 hours) before administration, and feed it 4 hours after administration.

[0194] (4) Blood collection and processing: Blood samples were collected at 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, and 24h after drug administration. Blood was collected via the cheek, approximately 0.05mL per sample, anticoagulated with EDTA-2K. After collection, the samples were placed on wet ice and centrifuged within 1 hour to separate the plasma (centrifugation conditions: 6000g, 3 minutes, 2-8℃). Plasma samples were stored at -80℃ before analysis.

[0195] (5) Plasma sample analysis: After all samples are completely thawed, mix well for 10–30 seconds, and centrifuge at 4000 rpm and 4°C for 0.5 minutes. Transfer 15 μL of plasma sample (15 μL of blank plasma for both blank sample and internal standard blank sample) to a 96-well plate, and add 150 μL of internal standard (50% methanol-acetonitrile solution (concentration: 100 ng / mL)) solution (150 μL of 50% methanol-acetonitrile solution for blank sample). Vortex the sample for 5 minutes, centrifuge at 4000 rpm and 4°C for 5 minutes, transfer 100 μL to 100 μL of water, mix well, and analyze by LC-MS / MS.

[0196] (6) Results analysis: Pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.2.0 based on blood drug concentration data at different time points.

[0197] Table 3. Pharmacokinetic parameters of the compounds of the present invention in Balb / c nude mice.

[0198]

[0199] The compounds of this invention exhibit high blood concentrations, exposure levels, and bioavailability in Balb / c nude mice, demonstrating significant pharmacokinetic advantages over PCM075.

[0200] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The compound represented by formula (I) or its deuterated derivatives or their pharmaceutically acceptable salts: in: -XY- is selected from -CH2-CH2-, -CH=CH-, -N=CH-, and -CH=N-. n is 0, 1, 2, 3, 4, 5, 6, 7 or 8; R1 is selected from H and C. 1-4 Straight-chain or branched alkyl groups, C 3-7 Cycloalkyl groups, 3-7 membered heterocyclic alkyl groups containing 1-3 independently selected N, O, S cyclic heteroatoms connected to a proximal group via a C or N ring, wherein the alkyl group, cycloalkyl group, or heterocyclic alkyl group is optionally surrounded by one or more independently selected F, Cl, Br, -OR4, CN, -NR4R5, =O, C 1-4 Substituted with straight-chain or branched alkyl groups; R2 is independently selected from F, Cl, Br, OH, CN, =O, C 1-4 Straight-chain or branched alkyl groups, C 3-7 Cycloalkyl groups, 3-7 membered heterocyclic alkyl groups containing 1-3 independently selected N, O, S cyclic heteroatoms connected to a proximal group via a C or N ring, wherein the alkyl group, cycloalkyl group, or heterocyclic alkyl group is optionally surrounded by one or more independently selected F, Cl, Br, -OR4, CN, -NR4R5, =O, C 1-4 Substituted with straight-chain or branched alkyl groups; R1 and R2, together with the atoms on the piperazine ring to which they are jointly attached, can form a 3-7 membered heterocyclic alkyl group containing 1-3 independent cyclic heteroatoms selected from N, O, and S, wherein the heterocyclic alkyl group is optionally surrounded by one or more independent atoms selected from F, Cl, Br, -OR4, CN, -NR4R5, C 1-4 Straight-chain or branched alkyl groups, C 2-4 Straight-chain or branched alkenyl, C 2-4 Substitution of straight-chain or branched alkynyl groups; Two R2 atoms, together with the atoms on the piperazine ring they are connected to, can form 3-7 membered heterocyclic alkyl groups containing 1-3 independent cyclic heteroatoms selected from N, O, and S. 3-7 Cycloalkyl group, wherein the heterocycloalkyl group is optionally selected by one or more elements independently selected from F, Cl, Br, -OR4, CN, -NR4R5, C 1-4 Straight-chain or branched alkyl groups, C 2-4 Straight-chain or branched alkenyl, C 2-4 Substitution of straight-chain or branched alkynyl groups; R3 is selected from hydrogen, C 1-4 Straight-chain or branched alkyl groups, C 3-7 Cycloalkyl groups, 3-7 membered heterocyclic alkyl groups containing 1-3 cyclic heteroatoms independently selected from N, O, and S, wherein the alkyl group, cycloalkyl group, or heterocyclic alkyl group is optionally surrounded by one or more elements independently selected from F, Cl, Br, -OR4, CN, -NR4R5, and C. 1-4 Straight-chain or branched alkyl groups, C 2-4 Straight-chain or branched alkenyl, C 2-4 Straight-chain or branched alkynyl groups, C 3-7 The alkyl group is replaced by a 3-7 membered heterocyclic alkyl group containing 1-3 independent cyclic heteroatoms selected from N, O, and S, connected to a proximal group via a C or N on the ring. R4 and R5 are independently selected from H, OH, CN, NH2, and C. 1-4 Straight-chain or branched alkyl groups, C 2-4 Straight-chain or branched alkenyl, C 2-4 Straight-chain or branched alkynyl groups, C 1-4 Straight-chain or branched alkoxy groups, C 3-7 Cycloalkyl, 3-7 membered heterocyclic alkyl groups containing 1-3 independent cyclic heteroatoms selected from N, O, S, connected by a C or N atom on the ring to a proximal group, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, or heterocyclic alkyl group is optionally surrounded by one or more elements selected from F, Cl, Br, OH, CN, NH2, C 1-4 The groups are replaced by straight-chain or branched alkyl groups.

2. The compound of claim 1 or its deuterated derivative or a pharmaceutically acceptable salt thereof, wherein, -XY- is selected from -CH2-CH2-, -CH=CH-, and -N=CH-; n is 0, 1, 2, 3 or 4; R1 is selected from H and C. 1-4 Straight-chain or branched alkyl groups, C 3-7 Cycloalkyl group, wherein the alkyl group, or cycloalkyl group, is optionally composed of one or more elements independently selected from F, Cl, Br, -OR4, CN, -NR4R5, =O, C. 1-4 Substituted with straight-chain or branched alkyl groups; R2 is independently selected from F, Cl, Br, OH, CN, =O, C 1-4 Straight-chain or branched alkyl groups, C 3-7 Cycloalkyl group, wherein the alkyl group, or cycloalkyl group, is optionally composed of one or more elements independently selected from F, Cl, Br, -OR4, CN, -NR4R5, =O, C. 1-4 Substituted with straight-chain or branched alkyl groups; R3 is selected from hydrogen, C 1-4 Straight-chain or branched alkyl groups, C 3-7 Cycloalkyl, wherein the alkyl group, or cycloalkyl group, is optionally selected independently from one or more elements chosen from F, Cl, Br, -OR4, CN, -NR4R5, C. 1-4 Straight-chain or branched alkyl groups, C 2-4 Straight-chain or branched alkenyl, C 2-4 Straight-chain or branched alkynyl groups, C 3-7 Substituted with cycloalkyl groups; R4 and R5 are independently selected from H, OH, CN, NH2, and C. 1-4 Straight-chain or branched alkyl groups, C 2-4 Straight-chain or branched alkenyl, C 2-4 Straight-chain or branched alkynyl groups, C 1-4 Straight-chain or branched alkoxy groups, C 3-7 Cycloalkyl, wherein the alkyl, alkenyl, alkynyl, alkoxy, or cycloalkyl group is optionally surrounded by one or more elements selected from F, Cl, Br, OH, CN, NH2, C. 1-4 The groups are replaced by straight-chain or branched alkyl groups.

3. The compound of claim 1 or its deuterated form or a pharmaceutically acceptable salt thereof, wherein, -XY- is selected from -CH2-CH2-, -CH=CH-, and -N=CH-; n is 0; R1 is selected from C 1-4 Straight-chain or branched alkyl groups, wherein the alkyl group is optionally composed of one or more elements independently selected from F, Cl, Br, -OR4, CN, -NR4R5, C. 1-4 Substituted with straight-chain or branched alkyl groups; R3 is selected from hydrogen, C 1-4 Straight-chain or branched alkyl groups, wherein the alkyl group is optionally composed of one or more elements independently selected from F, Cl, Br, -OR4, CN, -NR4R5, C. 1-4 Substituted with straight-chain or branched alkyl groups; R4 and R5 are independently selected from H.

4. The compound of claim 1 or its deuterated derivative or a pharmaceutically acceptable salt thereof, wherein, -XY- is -CH2-CH2-; n is 0; R1 is selected from C 1-4 Straight-chain or branched alkyl groups; R3 is selected from hydrogen, C 1-4 Straight-chain or branched alkyl groups, wherein the alkyl group is optionally composed of one or more elements independently selected from F, Cl, Br, -OR4, CN, -NR4R5, C. 1-4 Substituted with straight-chain or branched alkyl groups; R4 and R5 are independently selected from H.

5. The compound of claim 1 or its deuterated derivative or a pharmaceutically acceptable salt thereof, wherein, The compound is selected from the following structures:

6. A pharmaceutical composition comprising the compound as described in any one of claims 1-5 and a pharmaceutically acceptable carrier.

7. A method for preventing and / or treating PLK1-related disease, wherein an individual in need is given a preventive and / or therapeutically effective amount of the compound as claimed in any one of claims 1-5 or the pharmaceutical composition as claimed in claim 6. Preferably, the PLK1-related diseases include solid tumors, hematologic malignancies, etc. Examples of solid tumors include, but are not limited to, colorectal cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, gastric cancer, head and neck cancer, ovarian cancer, uterine cancer, glioma, liver cancer, esophageal cancer, bladder cancer, kidney cancer, lymphoma, melanoma, osteosarcoma, etc.; examples of hematologic malignancies include, but are not limited to, leukemia, myelodysplastic syndrome, etc. PLK1-related diseases include solid tumors and hematologic malignancies. Examples of solid tumors include, but are not limited to, colorectal cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, gastric cancer, head and neck cancer, ovarian cancer, uterine cancer, glioma, liver cancer, esophageal cancer, bladder cancer, kidney cancer, lymphoma, melanoma, and osteosarcoma. Examples of hematologic malignancies include, but are not limited to, leukemia and myelodysplastic syndrome.

8. Use of the compound according to any one of claims 1-5 in the preparation of a PLK1 inhibitor.

9. Use of the compound of any one of claims 1-5 or the pharmaceutical composition of claim 6 in the preparation of a medicament for the prevention and / or treatment of PLK1-related diseases; Preferably, the PLK1-related diseases include solid tumors, hematologic malignancies, etc. Examples of solid tumors include, but are not limited to, colorectal cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, gastric cancer, head and neck cancer, ovarian cancer, uterine cancer, glioma, liver cancer, esophageal cancer, bladder cancer, kidney cancer, lymphoma, melanoma, osteosarcoma, etc.; examples of hematologic malignancies include, but are not limited to, leukemia, myelodysplastic syndrome, etc.

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