Deuterated compounds as cdk4 / 6 inhibitors

Abstract

The present application relates to a kind of deuterated compound as CDK4 / 6 inhibitor, its preparation method, pharmaceutical composition containing deuterated compound and its purposes, with better metabolic stability and more excellent pharmacokinetic properties compared with non-deuterated control compound, it is expected to reduce the dosage of clinical use.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to a deuterated compound as a CDK4 / 6 inhibitor, its preparation method, a pharmaceutical composition containing the deuterated compound, and its uses. Background Technology

[0002] Cyclin-dependent kinases (CDKs) are a class of serine / threonine kinases that serve as important intracellular signal transduction molecules. They form CDK-Cyclin complexes with cyclins and participate in cell growth, proliferation, dormancy, or apoptosis.

[0003] Currently, 13 members of the CDK family have been identified, namely CDK1-CDK13, among which CDK1, CDK2, CDK3, CDK4, and CDK6 are involved in regulating cell proliferation. Cyclin is divided into AL, with different CDKs linking to different cyclin subtypes. The Cyclin D family (Cyclin D1, D2, D3) begins to be expressed in the G1 phase, binding to and activating CDK4 and CDK6 to form the CDK4 / 6-Cyclin D complex, which phosphorylates a series of substrates, including retinoblastoma proteins. CDK4 / 6-specific activation is closely related to the proliferation of some tumors; therefore, developing CDK4 / 6 inhibitors has become an effective means of targeted cancer therapy.

[0004] 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentan-1,3'-indole]-5'-yl)-aza-(5-(1-methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (hereinafter referred to as "Control Compound 1") is a highly selective CDK4 / 6 inhibitor. The structural formula of this drug is:

[0005]

[0006] The specific method of its synthesis is described in WO2017 / 092635A1.

[0007] Many drugs have limited use in certain diseases due to poor absorption, distribution, metabolism, and excretion (ADME) properties, which is also the reason why many drug candidates fail to pass clinical trials. Although special formulation processes or prodrug technologies can improve the ADME properties of some drugs in certain cases, the ADME problems of most drug candidates cannot be effectively solved, especially the problem of rapid drug metabolism. As a result, many drugs that could have been highly effective in treating diseases are difficult to develop into drugs because they are metabolized too quickly from the body.

[0008] Therefore, there is still a need for compounds that possess CDK4 / 6 inhibitory activity, better metabolic stability, longer half-life, and better pharmacokinetic properties. Summary of the Invention

[0009] The purpose of this invention is to provide a class of compounds with CDK4 / 6 inhibitory activity, good pharmacokinetic properties, and reduced toxicity.

[0010] The first aspect of the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0011]

[0012] Among them, R 1 -R 29 They are H or D respectively, and R 1 -R 29 At least one of them is D; and

[0013] The compounds of formula (I) do not include the following compounds:

[0014]

[0015] In one implementation, R 1 -R 15 and R 17 -R 29 At least one of them is D; preferably, R 1 -R 15 R 17 and R 19 -R 29 At least one of them is D; preferably, R 1 -R 12 R 17 R 19 -R 29 At least one of them is D; preferably, R 1 -R 3 R8 -R 9 R 17 R 21 R 23 R 27 -R 29 At least one of them is D.

[0016] In one implementation, R 1 -R 3 It is D; and / or R 8 and R 9 It is D; and / or R 13 -R 15 It is D; and / or R 16 It is D; and / or R 17 It is D; and / or R 18 It is D; and / or R 21 and R 23 It is D; and / or R 27 -R 29 It is D.

[0017] In one implementation, R 1 -R 3 It is D; and / or R 8 and R 9 It is D; and / or R 13 -R 15 It is D; and / or R 17 It is D; and / or R 21 and R 23 It is D; and / or R 27 -R 29 It is D.

[0018] In one implementation, R 1 -R 3 It is D; and / or R 8 -R 9 It is D; and / or R 17 It is D; and / or R 21 and R 23 It is D; and / or R 27 -R 29 It is D.

[0019] In one implementation, R 16 Is it H, or R? 13 -R 15 Is it H, or R? 18 It's H.

[0020] In one implementation, where R 16 When it is D, R 1 -R 3 It's D, and R.13 -R 15 It is D; and / or R 1 -R 3 It is D, R 8 -R 9 It's D, and R. 13 -R 15 It is D; and / or R 21 and R 23 It is D; and / or R 27 -R 29 It is D; and / or R 17 It is D and R 27 -R 29 It is D; and / or R 21 It's D, R 23 It's D, and R. 27 -R 29 It is D; and / or R 1 -R 3 It is D, R 21 It is D, R 23 It's D, and R. 27 -R 29 It is D.

[0021] In one embodiment, the compound has the structure shown in formula (II):

[0022]

[0023] Where R 1 R 2 R 3 At least one of them is D, R 13 -R 15 They are H or D, respectively; preferably, R 1 R 2 and R 3 It is D,R 13 -R 15 H or D respectively; more preferably, R 1 R 2 and R 3 It is D,R 13 -R 15 At least one is D.

[0024] In one embodiment, the compound has the structure shown in formula (III):

[0025]

[0026] Among them, R 1 -R 3 R 8 -R 9 R 13-R 15 They are H or D, respectively; preferably, R 1 -R 3 It is D, and / or R 8 -R 9 It is D, and / or R 13 -R 15 It is D; preferably, R 1 -R 3 It is D and R 13 -R 15 It is D; preferably, R 1 -R 3 It is D, R 8 -R 9 It's D, and R. 13 -R 15 It is D.

[0027] In one embodiment, the compound has the structure shown in formula (IV):

[0028]

[0029] Among them, R 1 -R 15 At least one of them is D; preferably, R 1 -R 3 It is D, and / or R 8 -R 9 It is D.

[0030] In one embodiment, the compound has the structure shown in formula (V):

[0031]

[0032] Among them, R 1 -R 3 R 13 -R 16 They are H or D respectively; preferably, R. 1 -R 3 It is D, and / or R 13 -R 15 It is D.

[0033] In one embodiment, the compound has the structure shown in formula (VI):

[0034]

[0035] Among them, R 1 -R 3 R 8 R 9 R 13 -R 16 They are H or D, respectively; preferably, R1 -R 3 It is D, and / or R 13 -R 15 It is D, and / or R 8 and R 9 It is D; preferably, R 16 It is D.

[0036] In one embodiment, the compound has the structure shown in formula (VII):

[0037]

[0038] Among them, R 1 -R 3 R 8 R 9 R 13 -R 26 They are H or D, respectively; preferably, R 1 -R 3 It is D; and / or R 8 and R 9 It is D; and / or R 13 -R 15 It is D; and / or R 16 It is D; and / or R 17 It is D; and / or R 21 and R 23 It is D.

[0039] In a preferred embodiment, the compound is selected from:

[0040]

[0041]

[0042]

[0043]

[0044] Unless otherwise specified, the term "compound of the present invention" refers to the compound of formula (I) and its salts, including pharmaceutically acceptable salts of the compound and all stereoisomers (including but not limited to diastereomers and enantiomers), tautomers, isotopic compounds, their prodrugs, solvates, and hydrates.

[0045] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological potency of the free acid and base of a particular compound without any adverse biological effects. Examples of pharmaceutically acceptable salts include, but are not limited to: (1) acid addition salts, which are salts formed with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, nitric acid, phosphoric acid, etc.; or salts formed with organic acids such as malic acid, fumaric acid, maleic acid, benzoic acid, phenylacetic acid, succinic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, glycolic acid, cinnamic acid, pyruvic acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, acrylic acid, mandelic acid, etc.; or (2) base addition salts, which are salts formed with alkali metals such as lithium, sodium, potassium, etc.; salts formed with alkaline earth metals such as calcium, magnesium, etc.; and salts formed with organic bases such as ammonium, choline, diethanolamine, lysine, ethylenediamine, tert-butylamine, tert-octylamine, tris(hydroxymethyl)aminomethane, N-methylglucosamine, triethanolamine, dehydrorosinamine, etc. Other pharmaceutically acceptable salts are known to those skilled in the art.

[0046] The term "isotopic compound" refers to a compound of general formula (I) of the present invention containing one or more atomic isotopes of natural or non-natural abundance. Non-natural atomic isotopes include, but are not limited to, deuterium. 2 H or D), tritium ( 3 H or T), Iodine-125 ( 125 I), phosphorus-32( 32 P), carbon-13 ( 13 C) or carbon-14 ( 14 C). Unless otherwise specified, any atom in the compounds synthesized in this invention may represent any stable isotope of that atom. Unless otherwise specified, when a position in the structure is defined as H, i.e., hydrogen (H-1), that position contains only the amount of naturally occurring isotope (0.015%).

[0047] “Deuterium” or “D” indicates an isotope of hydrogen containing one proton and one neutron in its nucleus. When a particular position is designated as containing deuterium, it should be understood that the abundance of deuterium at that position is greater than the natural abundance of deuterium (typically 0.015%). Unless otherwise specified, when a position is specifically designated as “D” or “deuterium”, that position should be understood as containing deuterium at an abundance greater than the natural abundance of deuterium.

[0048] The deuteration rate of the compounds synthesized in this invention refers to the ratio of the content of the synthesized isotope to the amount of the naturally occurring isotope. The deuteration rate of each specified deuterium atom in the compounds synthesized in this invention may be at least 3333.3 times (50%), at least 4000 times (60%), at least 4500 times (67.5%), at least 5000 times (75%), at least 5333.3 times (80%), at least 6000 times (90%), at least 6333.3 times (95%), at least 6466.7 times (97%), at least 6566.7 times (98.5%), at least 6600 times (99%), or at least 6633.3 times (99.5%).

[0049] The amount of hydrogen isotopes at a specific position in the compounds synthesized in this invention depends on many factors, including the deuterotopic purity of the deuterated reagent (such as D₂O, D₂, NaBD₄, LiAlD₄, etc.) and the effectiveness of the deuterotope synthesis method. However, as mentioned above, the total amount of such hydrogen isotopes at a specific position will be less than 49.9%. The total amount of hydrogen isotopes at a specific position in the compounds synthesized in this invention will be less than 47.5%, 40%, 32.5%, 25%, 17.5%, 10%, 5%, 3%, 1%, or 0.5%.

[0050] In this article, any atom not designated as deuterium exists at its natural isotopic abundance.

[0051] The term "solvent" refers to the form of the compound of this patented invention that forms a solid or liquid complex by coordinating with solvent molecules. Examples of such forms include hydrates, alcohols, etc.

[0052] The term "prodrug" refers to any pharmaceutical agent that is converted into the parent drug in the body. Prodrugs are often useful because, in some cases, they are easier to administer than the parent drug. For example, they are bioavailable when administered orally, while the parent drug is not. Prodrugs may also improve the solubility of the pharmaceutical composition relative to the parent drug. Prodrugs can be converted into the parent drug via enzymatic methods and metabolic hydrolysis.

[0053] Another aspect of the invention provides a pharmaceutical composition comprising a compound of the invention or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

[0054] The 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. Administration can be local (e.g., transdermal, skin, eye, and mucous membrane delivery, including intranasal, vaginal, and rectal delivery), pulmonary (e.g., by inhalation or blowing of powders or aerosols, including via nebulizers; intratracheal, intranasal), oral, or parenteral administration. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial administration, such as intrathecal or intraventricular administration.

[0055] Certain examples of acceptable carriers or excipients include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. The pharmaceutical composition may also contain: lubricants such as talc, magnesium stearate, and mineral oil; humectants; emulsifiers and suspending agents; preservatives such as methyl benzoate and hydroxypropyl benzoate; sweeteners and flavoring agents. The pharmaceutical compositions of the present invention can be formulated using methods known in the art to provide immediate, sustained, or delayed release of the active ingredient upon administration to a patient.

[0056] Compositions can be formulated in unit dosage forms, each dose containing approximately 5 to 1000 mg, more typically approximately 100 to 500 mg of active ingredient.

[0057] The compounds of this invention are CDK4 / 6 inhibitors, and therefore can treat diseases in which the underlying pathology is (at least in part) mediated by CDK4 / 6. These diseases include cancer and other conditions involving impaired cell proliferation, apoptosis, or differentiation.

[0058] Examples of cancers that can be treated with the compounds of this invention include, but are not limited to, cancers such as bladder cancer, breast cancer, colon cancer (e.g., colorectal cancer, such as colonic adenocarcinoma and colonic adenoma), kidney cancer, epidermal cancer, liver cancer, lung cancer (e.g., adenocarcinoma, small cell lung cancer and non-small cell lung cancer), esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer (e.g., exocrine pancreatic cancer), stomach cancer, cervical cancer, thyroid cancer, nasal cancer, head and neck cancer, prostate cancer, and skin cancer (e.g., squamous cell carcinoma). Other examples of cancers that can be treated with the compounds of this invention include lymphoid hematopoietic tumors (e.g., leukemia, acute lymphoblastic leukemia, mantle cell lymphoma, chronic lymphoblastic leukemia, B-cell lymphoma (e.g., diffuse large B-cell lymphoma), T-cell lymphoma, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma, and Burkitt lymphoma); and bone marrow hematopoietic tumors, such as acute and chronic myeloid leukemia, myelodysplastic syndromes, and promyelocytic leukemia. Other cancers include follicular thyroid carcinoma; stromal tumors such as fibrosarcoma or habdomyosarcoma; tumors of the central or peripheral nervous system such as astrocytoma, neuroblastoma, glioma, brain glioma, or schwannoma; melanoma; seminoma; teratoma; osteosarcoma; xeroderma pigmentosum; retinoblastoma; keratoctanthoma; follicular thyroid carcinoma; and Kaposi's sarcoma.

[0059] Another aspect of the present invention provides a method for treating CDK4 / 6-mediated disorders or diseases, comprising administering an effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, to a patient suffering from said cell proliferation disorder via oral or non-oral routes.

[0060] Another aspect of the present invention provides the use of the compound of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a medicament for treating CDK4 / 6-mediated disorders or diseases.

[0061] Another aspect of the present invention provides a compound or a pharmaceutically acceptable salt thereof for treating CDK4 / 6-mediated disorders or diseases.

[0062] Preferably, the aforementioned disorder or disease caused by abnormal cell proliferation in mammals, including humans, refers to cancer in mammals or humans, more preferably to cancer in humans, including malignant solid tumors and malignant non-solid tumors, specifically including but not limited to breast cancer, lung cancer, prostate cancer, leukemia, brain cancer, glioma, and gastric cancer; and / or the cell proliferation disorder is selected from one or more of AIDS, atherosclerosis, and restenosis after vascular stent implantation.

[0063] Another aspect of the present invention provides a method for preparing a compound of formula (VII), comprising the steps of reacting a compound of formula (VIII) with a deuterating reagent in the presence of an acid to obtain a compound of formula (VII).

[0064]

[0065] Among them, R 1 -R 3 R 8 R 9 R 13 -R 26 They are H or D, respectively.

[0066] Preferably, the acid is selected from at least one of deuterated hydrochloric acid, hydrochloric acid, ferric chloride, aluminum chloride, zinc chloride, acetic acid, acetic anhydride, trifluoroacetic acid, trifluoroacetic anhydride, trifluoromethanesulfonic acid, benzoic acid, p-methylbenzoic acid, p-nitrobenzoic acid, p-methoxybenzoic acid, 3,5-dinitrobenzoic acid, diphenylphosphonic acid, phenylphosphine, phosphoric acid, hypophosphoric acid, phenylacetic acid, phenylpropionic acid, trimethylacetic acid, benzenesulfonic acid, p-toluenesulfonic acid, mandelic acid, and salicylic acid; and / or the deuterated reagent is selected from at least one of d6-DMSO, d7-DMF, d6-acetone, CD3OD, D2O, CD3CN, and C6D6; more preferably, the acid is deuterated hydrochloric acid, and / or the deuterated reagent is D2O.

[0067] Preferably, in the above method, R 1 -R 3 It is D; and / or R 8 and R 9 It is D; and / or R 13 -R 15 It is D; and / or R 16 It is D; and / or R 17 It is D; and / or R 21 and R 23 It is D.

[0068] This preparation method achieves deuteration only on the hydrogen atom on the methyl group of the 3H-indole ring of control compound 1, exhibiting extremely high selectivity.

[0069] Another aspect of the present invention provides a compound of formula (IX) or a pharmaceutically acceptable salt thereof.

[0070]

[0071] Among them, R 18 -R 29 They are H or D respectively; X is a leaving group or an amino group; preferably, X is a halogen or an amino group, more preferably fluorine, bromine, chlorine or amino.

[0072] Another aspect of the present invention provides a method for preparing a compound of formula (I), comprising converting a compound of formula (IX) into a compound of formula (I):

[0073]

[0074] Where: R 17 For D; and R 1 -R 16 and R 18 -R 29 H or D, preferred, R 1 -R 3 It is D, and / or R 13 -R 15 It is D, and / or R 16 It is D.

[0075] Another aspect of the present invention provides a method for preparing a compound of formula (IX), comprising the steps of reacting a compound of formula (X) or a salt thereof with a compound of formula (XI) in the presence of an acid to obtain a compound of formula (IX):

[0076]

[0077] Among them, R 18 -R 29 They are H or D, with R being preferred. 18 -R 29 X is hydrogen; X is a leaving group or an amino group; preferably, X is a halogen or an amino group, more preferably fluorine, bromine, chlorine or amino; and / or the salt of the compound of formula (X) is selected from hydrochloride, sulfate, methanesulfonate and p-toluenesulfonate, preferably hydrochloride.

[0078] Preferably, the acid is an organic acid, an inorganic acid, or a Lewis acid; more preferably, the acid is sulfuric acid, hydrochloric acid, phosphoric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, ZnCl2, FeCl3, AlCl3, or SnCl4; more preferably, the acid is sulfuric acid.

[0079] Preferably, the above preparation method further includes the following steps:

[0080] (1) make The reaction with a deuterated reagent yields

[0081] (2) make Transform into Or its salt.

[0082] Preferably, step (1) is performed in the presence of Ir(OMe)(cod)2; the deuterated reagent is selected from at least one of d6-DMSO, d7-DMF, d6-acetone, CD3OD, D2O, CD3CN, and C6D6; preferably, the deuterated reagent is D2O.

[0083] Hydrogen atoms at certain positions on drug molecules are not easily replaced by deuterium due to steric hindrance and other reasons. Compound (IX) is an important intermediate for the synthesis of compound (I). The inventors synthesized R using a specific method. 17 The compound of formula (IX) is D, and the final product is further synthesized using the compound of formula (IX) as an intermediate, achieving the technical effect of deuteration of hydrogen at a specific site of control compound 1.

[0084] The beneficial effects of this invention are:

[0085] 1) Compared with control compound 1, the compounds of the present invention have comparable or superior inhibitory activity against human glioma U87MG cells.

[0086] 2) Compared with control compound 1, the compound of the present invention has better metabolic stability and superior pharmacokinetic properties in human liver microsomes, which is expected to reduce the clinical dosage and thus reduce treatment costs so that more patients can benefit.

[0087] 3) The present invention also provides a method for deuterating hydrogen at a specific position in control compound 1, which has extremely high selectivity and specificity.

[0088] Abbreviations:

[0089] 1 atm: 1 atmosphere

[0090] ADME: Drug absorption, distribution, metabolism, and excretion

[0091] CD: Carbon-deuterium bond

[0092] CH: Carbon-hydrogen bond

[0093] CDK: Cyclin-dependent kinase

[0094] CDCl3: Deuterated chloroform

[0095] DAPI: 4',6-Diamidinyl-2-phenylindole

[0096] DMSO: Dimethyl sulfoxide

[0097] DMSO-d6: Deuterated dimethyl sulfoxide

[0098] DMF: N,N-Dimethylformamide

[0099] DCM: Dichloromethane

[0100] DCl: Deuterated hydrochloric acid

[0101] dtbpy:4,4'-di-tert-butyl-2,2'-bipyridine

[0102] EA: Ethyl acetate

[0103] FBS: Fetal bovine serum

[0104] Grubbs Cat 1st :Di(tricyclohexylphosphine)benzyl ruthenium dichloride

[0105] 1 H-NMR: 1H NMR spectrum

[0106] HPLC: Preparative High-Pressure Liquid Chromatography

[0107] Ir(OMe)(cod)2: Methoxy(cyclooctadiene)iridium(I) dimer LiAlD4: ​​Deuterated lithium aluminum hydride

[0108] MeOH: Methanol

[0109] MEM: Minimum Required Culture Medium

[0110] NaBD4: Sodium deuterated borohydride

[0111] NEAA: Non-essential amino acids

[0112] NBS: N-bromosuccinimide

[0113] Pd / C: Palladium on Carbon

[0114] PE: Petroleum ether

[0115] PBS: Phosphate Buffer

[0116] Pd2(dba)3:tris[dibenzylacetone]dipalladium

[0117] Pd(pph3)4: Tetra(triphenylphosphine)palladium

[0118] Pd(dppf)Cl2:[1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride

[0119] SnCl2: Tin dichloride

[0120] TLC: Thin-layer chromatography

[0121] THF: Tetrahydrofuran

[0122] xant-phos: 4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene Detailed Implementation

[0123] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all chemical raw materials and reagents used in the following examples are commercially available products.

[0124] Example 1 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-(methyl-[D3])-piperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (Compound 1)

[0125]

[0126] Step 1) 6-Nitro-3',6'-dihydro-[3,4'-dipyridine]-1'(2'H)-carboxy-tert-butyl ester

[0127]

[0128] 5-Bromo-2-nitropyridine (20.0 g, 99.05 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxyboronic-2-yl)-5,6-dihydropyridine-1(2H)-carboxy-tert-butyl ester (33.44 g, 108.16 mol), 1,4-dioxane / water (200 mL / 20 mL), potassium carbonate (40.2 g, 291.3 mmol), and Pd(pph3)2Cl2 (0.692 g, 0.985 mmol) were added in portions to a reaction flask. The mixture was heated to 80 °C for 12 hours under nitrogen protection, cooled to room temperature, concentrated, and separated by column chromatography (PE / EA = 1:1 - DCM / MeOH = 20:1) to give 19 g of the title product as a yellow solid. MS (ESI): 306.1 [M+H] + .

[0129] Step 2) 4-(6-aminopyridin-3-yl)piperidine-1-carboxy-tert-butyl ester

[0130]

[0131] 6-Nitro-3',6'-dihydro-[3,4'-dipyridine]-1'(2'H)-carboxyter-butyl ester (1.0 g, 3.27 mmol), ethyl acetate / methanol (6 mL / 6 mL), and Pd / C (0.2 g) were added to a reaction flask. Hydrogen gas was bubbled through the flask (1 atm), and the mixture was heated to 50 °C for 6 hours. The mixture was filtered, concentrated, and 802 mg of the title product was obtained as an off-white solid. MS (ESI): 278.2 [M+H] + .

[0132] Step 3) 5-(1-(methyl-[D3])piperidin-4-yl)pyridin-2-amine

[0133]

[0134] 4-(6-aminopyridin-3-yl)piperidin-1-carboxyter-butyl ester (700.0 mg, 2.53 mmol) was dissolved in tetrahydrofuran (14 mL), and lithium aluminum deuterated hydride (253.4 mg, 6.03 mmol) was slowly added at low temperature. The mixture was then heated to 70 °C and reacted for 3 hours. After the reaction was complete, ice was added to quench the reaction, followed by extraction, concentration, and column chromatography (DCM / MeOH = 20:1-10:1) to give 459 mg of the title compound as a grayish-white solid. MS (ESI): 195.3 [M+H] + .

[0135] Step 4) 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-(methyl-[D3])-piperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine

[0136]

[0137] 5'-(2-chloro-5-fluoropyrimidin-4-yl)-7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indole] (150.0 mg, 0.449 mmol), 5-(1-(methyl-[D3])piperidin-4-yl)pyridin-2-amine (78.4 mg, 0.404 mmol), palladium acetate (10.05 mg, 0.0448 mmol), xant-phos (25.95 mg, 0.0448 mmol), and potassium carbonate (185.9 mg, 1.347 mmol) were added sequentially to a reaction flask, dissolved in toluene (10 mL) and water (2 mL). Nitrogen gas was then introduced, and the mixture was heated to 100 °C for 2 hours. After the reaction was complete, the reaction solution was concentrated, extracted, passed through diatomaceous earth, separated, further concentrated, and then purified by TLC and HPLC. The solution was lyophilized to obtain 90 mg of the title compound as a white solid. MS(ESI): 492.3 [M+H] + . 1H-NMR (400MHz, CDCl3) δ8.45(d,J=3.6Hz,2H),8.35(d,J=8.6Hz,1H),8.29-8.22(m,1H),7.97(s,1H),7.90(d,J=11.0Hz,1H),7.62(d, J=8.7Hz,1H),3.21-3.20(d,J=11.4Hz,2H),2.65-2.53(m,1H),2.40(s,3H),2.37-2.26(m,2H),2.23-1.97(m,8H),1.96-1.87(m,4H).

[0138] Example 2 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-6-[D]-2-amine (Compound 5)

[0139]

[0140] Step 1) 5-Fluoropyrimidine-2,4(1H,3H)-keto-6-[D]

[0141]

[0142] 5-Fluorouracil (5.0 g, 38.4 mmol) and heavy water (40 mL) were added sequentially to a sealed iron container. The mixture was then heated to 190 °C and reacted for 3 hours. After the reaction was complete, the mixture was cooled to room temperature and filtered to obtain 4 g of the title compound as a yellow solid. MS (ESI): 130.2 [MH] + .

[0143] Step 2) 2,4-Dichloro-5-fluoropyrimidine-6-[D]

[0144]

[0145] 5-Fluoropyrimidine-2,4(1H,3H)-one-6-[D] (2.0 g, 15 mmol), N,N-diisopropylethylamine (0.96 g, 7.9 mmol), and phosphorus oxychloride (7 mL) were added sequentially to the reaction flask. The mixture was then heated to 110 °C and reacted for 2 hours. After the reaction was complete, the solvent was evaporated to dryness, ice was added to the reaction flask, and the mixture was extracted, separated, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain 1.3 g of the title compound as a pale yellow oil. MS (ESI): 168.1 [M+H] + .

[0146] Step 3) 5'-(2-chloro-5-fluoropyrimidin-4-yl-6-[D])-7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indole]

[0147]

[0148] Under nitrogen protection, isopropanol (15 mL), 5'-bromo-7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indole] (1.2 g, 4.25 mmol), potassium acetate (0.5 g, 5.1 mmol), and bis-pinacol borate (1.2 g, 4.67 mmol) were added to a reaction flask, and the mixture was stirred. Then, 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride (0.34 g, 0.425 mmol) was added. The mixture was heated to 85 ± 5 °C and reacted for 12 hours. The reaction solution was concentrated under reduced pressure, then extracted with purified water and ethyl acetate. The aqueous phase was discarded after separation. The resulting organic phase was filtered and concentrated under reduced pressure to obtain the intermediate. The intermediate and toluene (20 mL) were transferred to a reaction flask and stirred. Then, 2,4-dichloro-5-fluoropyrimidine-6-[D] (1.2 g, 3.6 mmol) and potassium phosphate (1.94 g, 7.2 mmol) were added and stirred. 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride (0.26 g, 0.36 mmol) was added to the reaction flask, followed by purified water (6 mL). The mixture was heated to 85 ± 5 °C and maintained at this temperature for 3 hours. After the reaction was complete, the mixture was concentrated, extracted, separated, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain 1.0 g of the title compound as a pale yellow oil. MS (ESI): 335.3 [M+H] + .

[0149] Step 4) 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-aza-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-6-[D]-2-amine

[0150]

[0151] 5'-(2-chloro-5-fluoropyrimidin-4-yl-6-[D])-7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indole] (112 mg, 0.334 mmol), 5-(1-methylpiperidin-4-yl)pyridin-2-amine (64.8 mg, 0.334 mmol), palladium acetate (7.5 mg, 0.0334 mmol), xant-phos (19.32 mg, 0.0334 mmol), and potassium carbonate (138 mg, 1 mmol) were added sequentially to a reaction flask in toluene (10 mL) and water (2 mL). Nitrogen gas was then introduced, and the mixture was heated to 100 °C for 2 hours. After the reaction was complete, the reaction solution was concentrated, extracted, filtered through diatomaceous earth, separated, further concentrated, and then purified by TLC and HPLC. The solution was lyophilized to obtain 10 mg of the title compound as a white solid. MS (ESI): 490.3 [M+H]+ . 1 H-NMR (400MHz, CDCl3) δ8.35(d,J=8.4Hz,1H),8.23-8.20(m,2H),7.98(s,1H),7.91(d,J=10.8Hz,1H),7.62(d,J= 7.6Hz,1H),3.15-3.13(m,2H),2.56-2.55(m,1H),2.45(s,3H),2.40(s,3H),2.03-2.24(m,8H),1.91-1.76(m,6H).

[0152] Example 3 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-methylpiperidin-4-yl-3,4-[D2])pyridin-2-yl)pyrimidin-2-amine (Compound 13)

[0153]

[0154] Step 1) 4-(6-aminopyridin-3-yl)piperidine-1-carboxyter-tert-butyl ester-3,4-[D2]

[0155]

[0156] 6-Nitro-3',6'-dihydro-[3,4'-dipyridine]-1'(2'H)-carboxy-tert-butyl ester (200.0 mg, 0.66 mmol), deuterated methanol (5 mL), and Pd / C (20 mg) were added to a reaction flask. Deuterium gas (1 atm) was introduced, and the mixture was heated to 50 °C for 12 hours. The mixture was filtered, concentrated, and 162 mg of the title product was obtained as a gray solid. MS (ESI): 280.3 [M+H] + .

[0157] Step 2) 5-(1-(methylpiperidin-4-yl-3,4-[D2])pyridine-2-amine

[0158]

[0159] 4-(6-aminopyridin-3-yl)piperidin-1-carboxyter-tert-butyl ester-3,4-[D2] (280 mg, 1.00 mmol) was dissolved in tetrahydrofuran (10 mL), and lithium aluminum hydride (91.4 mg, 2.4 mmol) was slowly added at low temperature. The mixture was then heated to 70 °C and reacted for 3 hours. After the reaction was complete, ice was added to quench the reaction, followed by extraction, concentration, and column chromatography (DCM / MeOH = 20:1-10:1) to give 123 mg of the title compound as a grayish-white solid. MS (ESI): 194.3 [M+H] + .

[0160] Step 3) 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-methylpiperidin-4-yl-3,4-[D2])pyridin-2-yl)pyrimidin-2-amine

[0161]

[0162] The following substances were added sequentially to the reaction flask: 5'-(2-chloro-5-fluoropyrimidin-4-yl)-7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indole] (100.0 mg, 0.299 mmol), 5-(1-(methylpiperidin-4-yl-3,4-[D2])pyridine-2-amine (50.5 mg, 0.26 mmol), palladium acetate (6.72 mg, 0.0299 mmol), and xant-phos (17.3 mg). 0.0299 mmol g of potassium carbonate (129.8 mg, 0.897 mmol) and 0.0299 mmol of potassium carbonate were dissolved in toluene (4 mL) and water (1 mL). Nitrogen gas was then introduced, and the mixture was heated to 100 °C for 2 hours. After the reaction was complete, the reaction solution was concentrated, extracted, filtered through diatomaceous earth, separated, further concentrated, and then purified by TLC and HPLC. The solution was lyophilized to obtain 75 mg of the title compound as a white solid. MS (ESI): 491.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.64(brs,1H),8.46(d,J=3.6Hz,1H),8.35(d,J=8.8H z,1H),8.27(d,J=1.6Hz,1H),7.97(s,1H),7.90(d,J=11.2Hz,1H),7.61(dd,J 1 =8.8Hz,J 2 =1.6Hz,1H),3.06-3.03(m,2H),2.40(s,3H),2.38(s,3H),2.23-2.09(m,8H),1.90-1.86(m,5H).

[0163] Example 4 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-methylpiperidin-4-yl)pyridin-2-yl-3,4,6-[D3])pyrimidin-2-amine (Compound 20)

[0164]

[0165] Step 1) Pyridine-[D4]-2-amine

[0166]

[0167] 2-Aminopyridine (3.0 g, 31.9 mmol), Pd / C (600 mg), and heavy water (100 mL) were added sequentially to a sealed iron can. Hydrogen gas was then bubbled through the can for 5 minutes, and the can was heated to 210 °C for 8 hours. After the reaction was complete, dichloromethane and methanol (50 mL) were added to the reaction solution, and the mixture was filtered to remove palladium on carbon. The filtrate was concentrated to give 2.1 g of the title compound as a gray solid. MS (ESI): 99.1 [M+H] + .

[0168] Step 2) 5-Bromopyridine-3,4,6-[D3]-2-amine

[0169]

[0170] Pyridine-[D4]-2-amine (200 mg, 2.02 mmol) was dissolved in DMF (5 mL), and NBS (399 mg, 2.2 mmol) was added to the reaction flask at approximately 10°C. After the addition was complete, the mixture was stirred at room temperature for 2 hours. The reaction was quenched with water, extracted, separated, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain 189 mg of the title compound as a gray solid. MS (ESI): 176.2 [M+H] + .

[0171] Step 3) 6-Amino-3',6'-dihydro-[3,4'-dihydropyridine]-1'(2'H)-tert-butyl carboxylate-2,4,5-[D3]

[0172]

[0173] 5-Bromopyridine-3,4,6-[D3]-2-amine (1.0 g, 5.68 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxyboronic-2-yl)-5,6-dihydropyridine-1(2H)-carboxy-tert-butyl ester (2.15 g, 6.95 mol), 1,4-dioxane / water (6 mL / 1 mL), potassium carbonate (2.175 g, 17.2 mmol), and Pd(pph3)4 (0.65 g, 0.568 mmol) were added fractionally to a reaction flask. The reaction was carried out under nitrogen protection at 100 °C for 1 hour, cooled to room temperature, extracted, concentrated, and separated by column chromatography (PE / EA = 1:1 - DCM / MeOH = 20:1) to give 1.0 g of the title product as a grayish-white solid. MS (ESI): 279.3 [M+H] + .

[0174] Step 4) 5-(1-(methylpiperidin-4-yl)pyridine-3,4,6-[D3]-2-amine

[0175]

[0176] 6-Amino-3',6'-dihydro-[3,4'-dihydropyridine]-1'(2'H)-tert-butyl carboxylate-2,4,5-[D3] (800 mg, 2.85 mmol) was dissolved in tetrahydrofuran (30 mL), and a 2.5 M lithium aluminum hydride solution in tetrahydrofuran (2.86 mL, 7.14 mmol) was slowly added at low temperature. The mixture was then heated to 70 °C and reacted for 4 hours. After the reaction was complete, ice was added to quench the reaction, followed by extraction, concentration, and column chromatography (DCM / MeOH = 20:1-10:1) to give 603 mg of the title compound as a gray solid. MS (ESI): 195.3 [M+H] + .

[0177] Step 5) 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-methylpiperidin-4-yl)pyridin-2-yl-3,4,6-[D3])pyrimidin-2-amine

[0178]

[0179] Add 5'-(2-chloro-5-fluoropyrimidin-4-yl)-7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indole] (257 mg, 0.769 mmol), 5-(1-(methylpiperidin-4-yl)pyridine-3,4,6-[D3]-2-amine (150 mg, 0.769 mmol), Pd2(dba)3 (70 mg, 0.077 mmol), and xant-phos (8) to the reaction flask in sequence. 8.9 mg (0.154 mmol) and cesium carbonate (750 mg, 2.3 mmol) were dissolved in 1,4-dioxane (8 mL), then nitrogen gas was introduced, and the mixture was microwaved to 100 °C for 1 hour. After the reaction was complete, the reaction solution was concentrated, extracted, filtered through diatomaceous earth, separated, further concentrated, and then purified by TLC and HPLC. The solution was lyophilized to obtain 94 mg of the title compound as a white solid. MS (ESI): 492.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.44(d,J=3.6Hz,1H),8.00(s,1H),7.98(s,1H),7.61(dd,J 1 =9.2Hz, J 2 =3.6Hz,1H),3.45-3.44(m,2H),2.69(s,3H),2.65-2.64(m,3H),2.40(s,3H),2.39-2.34(m,2H),2.19-1.86(m,10H).

[0180] Example 5 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl-6'-[D])-N-(5-(1-methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (Compound 21)

[0181]

[0182] Step 1) 4-Bromo-2-fluoro-3,6-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)amine

[0183]

[0184] 4-Bromo-2-fluoroaniline (10 g, 53 mmol), 4,4,5,5-tetramethyl-1,3,2-dioxaborane (40 g, 159 mmol), dtbpy (620 mg, 2.12 mmol), and Ir(OMe)(cod)₂ (700 mg, 1.06 mmol) were added sequentially to a reaction flask. Tetrahydrofuran (100 mL) was then added to dissolve the compounds. Nitrogen gas was purged, and the mixture was heated to 85 °C for 12 hours. After the reaction was complete, the mixture was filtered, concentrated, extracted, dried over anhydrous sodium sulfate, filtered again, concentrated, and separated by column chromatography to obtain 3.1 g of the title compound as a white solid. MS (ESI): 442.1 [M+H] + .

[0185] Step 2) 4-Bromo-2-fluorophenyl-3,6-[D2]-amine

[0186]

[0187] 4-Bromo-2-fluoro-3,6-bis(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)amine (5.34 g, 14.8 mmol), Ir(OMe)(cod)2 (0.39 g, 0.6 mmol), heavy water (10 mL), and tetrahydrofuran (40 mL) were added sequentially to a sealed iron container. The reaction mixture was then heated to 145 °C and reacted for 5 hours. After the reaction was complete, the mixture was cooled, extracted with ethyl acetate, concentrated, and separated by column chromatography to obtain 2.85 g of the title compound as a pale yellow oil. MS (ESI): 192.1 [M+H] + . 1 H-NMR (400MHz, DMSO-d6) δ7.30 (s, 1H), 5.30 (brs, 2H).

[0188] Step 3) (4-bromo-2-fluorobenzene-3,6-[D2])hydrazine hydrochloride

[0189]

[0190] 4-Bromo-2-fluorobenzyl-3,6-[D2]-amine (850 mg, 4.45 mmol) was dissolved in 8 mL of hydrochloric acid under nitrogen protection. The reaction solution was then cooled to -5 °C, and sodium nitrite (370 mg, 4.67 mmol) dissolved in 4 mL of water was slowly added dropwise. After maintaining the reaction at -5 °C for 30 minutes, SnCl2 (2.1 g, 8.9 mmol) dissolved in 1 mL of HCl was slowly added dropwise to the reaction solution. The reaction was then carried out at 0 °C for 3 hours. After the reaction was complete, the mixture was filtered, extracted, and concentrated to give 936 mg of the title compound as a white solid. MS (ESI): 192.1 [M+H] + .

[0191] Step 4) 5'-Bromo-7'-Fluoro-2'-Methylspiro[cyclopentane-1,3'-indole]-5'-yl-6'-[D]

[0192]

[0193] (4-Bromo-2-fluorobenzene-3,6-[D2])hydrazine hydrochloride (936 mg, 3.89 mmol), cyclopentyl ethyl ketone (500 mg, 4.27 mmol), and triethylamine (800 mg, 7.7 mmol), along with 15 mL of methanol, were added sequentially to the reaction flask. The reaction mixture was then maintained at 35 °C for 3 hours. After cooling to -5 °C, concentrated sulfuric acid (1.6 g, 15.56 mmol) was slowly added dropwise. The reaction was continued at 35 °C for another 12 hours. Upon completion of the reaction, the pH was adjusted to 8 with ammonia, and the mixture was extracted, concentrated, and separated by column chromatography to obtain 439 mg of the title compound as a yellow oil. MS (ESI): 283.1 [M+H] + .

[0194] Step 5) 5'-(2-chloro-5-fluoropyrimidin-4-yl)-7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-6'-[D]

[0195]

[0196] Under nitrogen protection, isopropanol (10 mL), 5'-bromo-7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl-6'-[D] (239 mg, 0.84 mmol), potassium acetate (99.6 mg, 1 mmol), and dipinacolborate (258 mg, 1 mmol) were added to a reaction flask. The mixture was stirred, and then 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride (61.9 mg, 0.084 mmol) was added. The temperature was raised to 85 ± 5 °C and the reaction was maintained for 12 hours. The reaction solution was concentrated under reduced pressure, then extracted with purified water and ethyl acetate. The liquid was separated, and the aqueous phase was discarded. The obtained organic phase was filtered and concentrated under reduced pressure to obtain the intermediate. The intermediate and 1,4-dioxane (10 mL) were transferred to a reaction flask and stirred. Then, 2,4-dichloro-5-fluoropyrimidine (141.5 mg, 0.84 mmol) and potassium phosphate (449 mg, 2.1 mmol) were added, and the mixture was stirred. Tetra(triphenylphosphine)palladium (89.8 mg, 0.07 mmol) was added to the reaction flask, followed by purified water (3.3 mL). The mixture was then microwaved at 100 °C for 1 hour. After the reaction was complete, the mixture was concentrated, extracted, separated, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain 163 mg of the title compound as a yellow solid. MS (ESI): 335.1 [M+H] + .

[0197] Step 6) 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl-6'-[D])-aza-(5-(1-methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine

[0198]

[0199] The following substances were added sequentially to the reaction flask: 5'-(2-chloro-5-fluoropyrimidin-4-yl)-7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-6'-[D] (163 mg, 0.49 mmol), 5-(1-(methylpiperidin-4-yl)pyridin-2-amine (112 mg, 0.58 mmol), Pd2(dba)3 (44.6 mg, 0.05 mmol), and xant-phos (56.4 mg, 0.4 mmol). 0.1 mmol of cesium carbonate (476 mg, 1.47 mmol) and 1,4-dioxane (10 mL) were dissolved in 1,4-dioxane. Nitrogen gas was then introduced, and the mixture was heated to 100 °C and microwaved for 1 hour. After the reaction was complete, the reaction solution was concentrated, extracted, filtered through diatomaceous earth, separated, further concentrated, and then purified by TLC and HPLC. The solution was lyophilized to obtain 116 mg of the title compound as a pale yellow solid. MS (ESI): 490.3 [M+H] + . 1H-NMR (400MHz, CDCl3) δ8.45(d,J=3.6Hz,1H),8.35(s,1H),8.33(s,1H),8.24(d,J=2.4Hz,1H),7.98(s,1H),7.62(dd,J 1 =8.8Hz,J 2 =2.4Hz,1H),3.16-3.13(m,2H),2.59-2.53(m,1H),2.45(s,3H),2.40(s,3H),2.27-2.08(m,8H),1.96-1.87(m,6H).

[0200] Example 6 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl-4'-[D])-aza-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (Compound 25)

[0201]

[0202] Step 1) (5-bromo-2-fluorophenyl) tert-butyl carbamate

[0203]

[0204] 5-Bromo-2-fluoroaniline (1.89 g, 10 mmol) was added sequentially to the reaction flask and suspended in water (10 mL). The addition of di-tert-butyl dicarbonate (2.16 g, 12 mmol) resulted in heating and bubbling. The reaction mixture was further diluted with water (50 mL) and then heated at 80 °C for 8 hours. Upon cooling to room temperature, water (100 mL) was added, and the precipitate was collected by filtration and thoroughly washed with water. The solid residue was extracted into dichloromethane (100 mL), dried over magnesium sulfate, filtered, and concentrated to dryness to give 2.0 g of the title compound as a pale yellow solid. MS (ESI): 289.1 [M+H] + .

[0205] Step 2 (3-amino-4-fluorophenyl)boronic acid

[0206]

[0207] To a reaction flask, tert-butyl (5-bromo-2-fluorophenyl)carbamate (2 g, 6.92 mmol), 1,4-dioxane (10 mL), bis(pinacol)diboron (2.74 g, 10.38 mmol), and potassium acetate (1.36 g, 13.84 mmol) were added sequentially. Nitrogen gas was bubbled through the mixture for 5 minutes, followed by the addition of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.56 g, 0.069 mmol) in combination with dichloromethane. The reaction mixture was heated to 100 °C for 12 hours. Upon cooling, the reaction mixture was partitioned between ethyl acetate (80 mL) and water (40 mL). The organic phase was washed with brine (20 mL), dried over magnesium sulfate, filtered, and concentrated. The residue was dissolved in 10 mL of chloroform, and then 1 mL of trifluoroacetic acid was added. The mixture was reacted at room temperature for 5 hours. The solution was then concentrated to dryness. The crude residue was partitioned between chloroform (50 mL) and water (50 mL). The aqueous phase was washed with 10% methanol in chloroform (50 mL), the pH was adjusted to 7 by adding sodium bicarbonate, and then washed with dichloromethane. The aqueous phase was concentrated to dryness to give a solid residue, which was washed with dichloromethane, dissolved in methanol, filtered, and concentrated to give 1.18 g of the title compound as a white solid. MS (ESI): 156.3 [M+H] + .

[0208] Step 3) 2-Fluorophenyl-5-[D]amine

[0209]

[0210] (3-Amino-4-fluorophenyl)boric acid (1.18 g, 7.54 mmol), Ir(OMe)(cod)₂ (131 mg, 0.226 mmol), heavy water (5 mL), and tetrahydrofuran (20 mL) were added sequentially to a sealed iron container. The reaction mixture was then heated to 145 °C and reacted for 5 hours. After the reaction was complete, the mixture was cooled, extracted with ethyl acetate, concentrated, and separated by column chromatography to obtain 900 mg of the title compound as a pale yellow oil. MS (ESI): 113.3 [M+H] + .

[0211] Step 4) 4-Bromo-2-fluorophenyl-5-[D]amine

[0212]

[0213] 2-Fluorophenyl-5-[D]-amine (400 mg, 3.57 mmol) was dissolved in THF (5 mL), and NBS (690 mg, 3.89 mmol) was added to the reaction flask at approximately 10 °C. After the addition was complete, the mixture was stirred at room temperature for 5 hours. The reaction was quenched with water, extracted, separated, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain 438 mg of the title compound as a white solid. MS (ESI): 191.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ7.17 (d, J = 10.8Hz, 1H), 6.70 (m, 1H), 3.73 (brs, 2H).

[0214] Step 5) (4-bromo-2-fluorophenyl-5-[D])hydrazine hydrochloride

[0215]

[0216] 4-Bromo-2-fluorophenyl-5-[D]-amine (950 mg, 5 mmol) was dissolved in 14 mL of hydrochloric acid under nitrogen protection. The reaction solution was then cooled to -5 °C, and sodium nitrite (2.26 g, 10 mmol) dissolved in 7 mL of water was slowly added dropwise. After maintaining the reaction at -5 °C for 30 minutes, SnCl₂·H₂O (1.1 g, 6 mmol) dissolved in 3 mL of HCl was slowly added dropwise to the reaction solution. The reaction was then carried out at 0 °C for 3 hours. After the reaction was complete, the mixture was filtered, extracted, and concentrated to give 730 mg of the title compound as a yellow solid. MS (ESI): 206.1 [M+H] + .

[0217] Step 6) 5'-Bromo-7'-Fluoro-2'-Methylspiro[cyclopentane-1,3'-indole]-5'-yl-4'-[D]

[0218]

[0219] (4-Bromo-2-fluorophenyl-5-[D])hydrazine hydrochloride (730 mg, 3.54 mmol), cyclopentyl ethyl ketone (383 mg, 3.54 mmol), and triethylamine (716 mg, 7.07 mmol), along with 12 mL of methanol, were added sequentially to the reaction flask. The reaction mixture was then maintained at 35 °C for 3 hours. After cooling to -5 °C, concentrated sulfuric acid (1.42 g, 14.5 mmol) was slowly added dropwise. The reaction was continued at 40 °C for 12 hours. Upon completion of the reaction, the pH was adjusted to 8 with ammonia, and the mixture was extracted, concentrated, and separated by column chromatography to obtain 540 mg of the title compound as a yellow oil. MS (ESI): 283.1 [M+H] + .

[0220] Step 7) 5'-(2-chloro-5-fluoropyrimidin-4-yl)-7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-4'-[D]

[0221]

[0222] Under nitrogen protection, isopropanol (10 mL), 5'-bromo-7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl-4'-[D] (540 mg, 1.91 mmol), potassium acetate (225.2 mg, 2.30 mmol), and dipinacolborate (730 mg, 2.87 mmol) were added to a reaction flask. The mixture was stirred, and then 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride (134 mg, 0.18 mmol) was added. The mixture was heated to 85 ± 5 °C and reacted for 12 hours. The reaction solution was concentrated under reduced pressure, then extracted with purified water and ethyl acetate. The aqueous phase was discarded after separation. The resulting organic phase was filtered and concentrated under reduced pressure to obtain the intermediate. The intermediate and 1,4-dioxane (8 mL) were transferred to a reaction flask and stirred. Then, 2,4-dichloro-5-fluoropyrimidine (281 mg, 1.67 mmol) and potassium phosphate (886.5 mg, 4.18 mmol) were added, and the mixture was stirred. Tetra(triphenylphosphine)palladium (161 mg, 0.14 mmol) was added to the reaction flask, followed by purified water (2 mL). The mixture was then microwaved at 100 °C for 1 hour. After the reaction was complete, the mixture was concentrated, extracted, separated, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain 157 mg of the title compound as a yellow solid. MS (ESI): 335.1 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.55 (d, J = 3.2 Hz, 1H), 7.94 (d, J = 10.4 Hz, 1H), 2.44 (s, 3H), 2.21-2.06 (m, 6H), 1.97-1.88 (m, 2H).

[0223] Step 8) 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl-4'-[D])-N-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine)

[0224]

[0225] 5'-(2-chloro-5-fluoropyrimidin-4-yl)-7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-4'-[D] (112 mg, 0.58 mmol), 5-(1-(methylpiperidin-4-yl)pyridin-2-amine (63 mg, 0.19 mmol), Pd2(dba)3 (18 mg, 0.02 mmol), xant-phos (22 mg, 0.04 mmol), and cesium carbonate (184 mg, 0.57 mmol) dissolved in 1,4-dioxane (5 mL) were added sequentially to a reaction flask. Nitrogen gas was then introduced, and the mixture was microwaved at 100 °C for 1 hour. After the reaction was complete, the reaction solution was concentrated, extracted, filtered through diatomaceous earth, separated, further concentrated, and then purified by TLC and HPLC. The solution was lyophilized to obtain 89 mg of the title compound as a pale yellow solid. MS (ESI): 490.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.44(d,J=3.6Hz,1H),8.35(d,J=8.4Hz,1H),8.24(d,J=2.0Hz,1H),8.16(s,1H),7.91(d,J=11.2Hz,1H),7.63(dd,J 1 =8.8Hz,J 2 =2.4Hz,1H),3.13-3.10(m,2H),2.58-2.53(m,1H),2.44(s,3H),2.40(s,3H),2.26-2.08(m,8H),2.03-1.90(m,6H).

[0226] Example 7 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl-3,4-[D2])-aza-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (Compound 27)

[0227]

[0228] Step 1) Ethyl 2-acetyl-2-(2-propen-1-yl)-4-pentenoate

[0229]

[0230] Ethyl acetoacetate (13.2 g, 100 mmol), potassium carbonate (42 g, 304 mmol), and 60 mL of DMF were added sequentially to the reaction flask. Then, 3-bromopropene (30 g, 248 mmol) was slowly added dropwise. Under nitrogen protection, the mixture was heated to 85 °C and reacted for 12 hours. The reaction solution was poured into ice water, extracted with ethyl acetate, and the organic phase was washed three times with brine (3 x 100 mL). The solution was concentrated, and column chromatography was used to obtain 7 g of the title compound as an anhydrous oil. MS (ESI): 211.3 [M+H] + .

[0231] Step 2) 3-(2-Propylene-1-yl)-5-Hexen-2-one

[0232]

[0233] Ethyl 2-acetyl-2-(2-propen-1-yl)-4-pentenoate (7.0 g, 35 mmol) was dissolved in 20 mL of DMF and added to a reaction flask. LiCl (2.8 g, 60 mmol) was added, and the reaction mixture was heated to 150 °C for 20 hours after purging with nitrogen. After the reaction was complete, the mixture was cooled to room temperature, ice water was added, and the solution was extracted, separated, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain 2.4 g of the title compound as a colorless oil. MS (ESI): 139.3 [M+H] + .

[0234] Step 3) 1-(3-cyclopenten-1-yl)ethyl ketone

[0235]

[0236] 3-(2-propen-1-yl)-5-hexen-2-one (2.4 g, 17 mmol) and Grubbscat were added sequentially to the reaction flask. 1st (720 mg, 0.87 mmol) and 20 mL of DCM were reacted under nitrogen atmosphere at room temperature for 15 hours. After the reaction was complete, the mixture was filtered, concentrated, and separated by column chromatography to obtain 450 mg of the title compound as a colorless oil. MS (ESI): 111.3 [M+H] + .

[0237] Step 4) 1-(cyclopentyl-3,4-[D2]) ethyl ketone

[0238]

[0239] 1-(3-cyclopenten-1-yl)acetone (450 mg, 4.5 mmol), cesium carbonate (70 mg, 0.21 mmol), palladium on carbon (20 mg), and 5 mL of deuterated methanol (CH3OD) were added sequentially to a reaction flask. Deuterium gas (1 atm) was then introduced, and the mixture was heated to 40 °C for 12 hours. After the reaction was complete, the mixture was filtered and concentrated to obtain 300 mg of the title compound as a yellow oil. MS (ESI): 115.1 [M+H] + .

[0240] Step 5) 5'-Bromo-7'-Fluoro-2'-Methylspiro[cyclopentane-1,3'-indole]-3,4-[D2]

[0241]

[0242] 4-Bromo-2-fluorophenylhydrazine hydrochloride (600 mg, 2.91 mmol), 1-(cyclopentyl-3,4-[D2]) acetone (300 mg, 2.71 mmol), and triethylamine (600 mg, 5.9 mmol), along with 12 mL of methanol, were added sequentially to a reaction flask. The reaction mixture was then maintained at 35 °C for 3 hours. After cooling to -10 °C, concentrated sulfuric acid (1.2 g, 12.2 mmol) was slowly added dropwise. The reaction was continued at 40 °C for 12 hours. Upon completion of the reaction, the pH was adjusted to 8 with ammonia, and the mixture was extracted, concentrated, and separated by column chromatography to obtain 130 mg of the title compound as a yellow oil. MS (ESI): 284.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ7.24 (d, J = 3.6 Hz, 1H), 7.27 (d, J = 8.8 Hz, 1H), 2.32 (s, 3H), 2.06-2.01 (m, 4H), 1.83-1.80 (m, 2H).

[0243] Step 6) 5'-(2-chloro-5-fluoropyrimidin-4-yl)-7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-3,4-[D2]

[0244]

[0245] Under nitrogen protection, isopropanol (10 mL), 5'-bromo-7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl-3,4-[D2] (130 mg, 0.46 mmol), potassium acetate (58 mg, 0.59 mmol), and dipinacol borate (152 mg, 0.59 mmol) were added to a reaction flask. The mixture was stirred, and then 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride (134 mg, 0.18 mmol) was added. The temperature was raised to 85 ± 5 °C and the reaction was maintained for 12 hours. The reaction solution was concentrated under reduced pressure, then extracted with purified water and ethyl acetate. The liquid was separated, and the aqueous phase was discarded. The obtained organic phase was filtered and concentrated under reduced pressure to obtain the intermediate. The intermediate and 1,4-dioxane (8 mL) were transferred to a reaction flask and stirred. Then, 2,4-dichloro-5-fluoropyrimidine (106 mg, 0.63 mmol) and potassium phosphate (267 mg, 1.26 mmol) were added and stirred. Tetra(triphenylphosphine)palladium (48.5 mg, 0.042 mmol) was added to the reaction flask, followed by purified water (2 mL). The mixture was microwaved at 100 °C for 1 hour. After the reaction was complete, the mixture was concentrated, extracted, separated, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain 87 mg of the title compound as a yellow solid. MS (ESI): 336.1 [M+H] + .

[0246] Step 7) 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl-3,4-[D2])-aza-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine)

[0247]

[0248] Add 5'-(2-chloro-5-fluoropyrimidin-4-yl)-7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-3,4-[D2] (70 mg, 0.208 mmol), 5-(1-(methylpiperidin-4-yl)pyridin-2-amine (50 mg, 0.25 mmol), Pd2(dba)3 (19.2 mg, 0.021 mmol), and xant-phos (24 mg, 0.25 mmol) sequentially to the reaction flask. 0.042 mg (mg, 0.042 mmol) and cesium carbonate (202.8 mg, 0.624 mmol) were dissolved in 1,4-dioxane (8 mL), and then the mixture was purged with nitrogen and microwaved at 100 °C for 1 hour. After the reaction was complete, the reaction solution was concentrated, extracted, filtered through diatomaceous earth, separated, further concentrated, and then purified by TLC and HPLC. The solution was lyophilized to obtain 25 mg of the title compound as a pale yellow solid. MS (ESI): 491.3 [M+H] + . 1H-NMR (400MHz, CDCl3) δ8.45(d,J=3.6Hz,1H),8.35(d,J=8.4Hz,1H),8.29(s,1H ),8.24(d,J=2.0Hz,1H),7.91(d,J=0.8Hz,1H),7.88(d,J=0.8Hz,1H),7.62(dd,J 1 =8.8Hz,J 2 =2.4Hz,1H),3.12-3.09(m,2H),2.59-2.51(m,1H),2.42(s,3H),2.40(s,3H),2.23-2.07(m,7H),1.97-1.88(m,5H).

[0249] Example 8 5-Fluoro-4-(7'-Fluoro-2'-(Methyl-[D3])spiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (Compound 32)

[0250]

[0251] Step 1) 5-Fluoro-4-(7'-Fluoro-2'-(Methyl-[D3])spiro[cyclopentane-1,3'-indole]-5'-yl)-aza-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine)

[0252]

[0253] 5-fluoro-4-(7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-methylpiperidin-4-yl)pyridin-2-yl)pyrimidine-2-amino (230 mg, 0.47 mmol), deuterated hydrochloric acid (87 mg, 0.47 mmol), and 10 mL of heavy water were added sequentially to the reaction flask. The mixture was then heated to 95 °C and reacted for 3 hours. After the reaction was complete, the mixture was cooled to room temperature, and the pH was adjusted to 8 with deuterated sodium hydroxide. The mixture was extracted with DCM, concentrated, and recrystallized from DCM and deuterated methanol to give 163 mg of the title compound as a pale yellow solid. MS (ESI): 492.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.44(d,J=3.6Hz,1H),8.34(d,J=8.8Hz,1H),8.23(d,J=2.0Hz,1H),8.14(s,1H),7.98(s,1H),7.91(dd,J 1 =11.2Hz,J 2=1.2Hz, 1H), 7.61(dd, J 1 =8.8Hz,J 2 =2.4Hz,1H),3.04-3.01(m,2H),2.53-2.49(m,1H),2.37(s,3H),2.22-2.07(m,8H),1.91-1.70(m,6H).

[0254] Example 9 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-(methyl-[D3])-piperidin-4-yl)pyridin-2-yl-3,4,6-[D3])pyrimidin-2-amine (Compound 2)

[0255]

[0256] The title compound was synthesized following steps similar to those in Examples 1 and 4.

[0257] MS(ESI): 495.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.44(d,J=3.6Hz,1H),8.05(s,1H),7.98(s,1H),7.91(d,J=11.2Hz,1H),3.34- 3.21(m,2H),2.65–2.60(m,1H),2.52–2.50(m,2H),2.40(s,3H),2.24–2.11(m,6H),2.09–1.88(m,6H).

[0258] Example 10 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-(methyl-[D])piperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (Compound 3)

[0259]

[0260] The title compound was synthesized following a similar procedure to that in Example 1. MS (ESI): 490.4 [M+H] + .

[0261] Example 11 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-(methyl-[D2])piperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (Compound 4)

[0262]

[0263] The title compound was synthesized following a similar procedure to that in Example 1. MS (ESI): 491.4 [M+H] + .

[0264] Example 12 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-methylpiperidin-4-yl-3,4-[D2])pyridin-2-yl)pyrimidin-6-[D]-2-amine (Compound 6)

[0265]

[0266] The title compound was synthesized following steps similar to those in Examples 2 and 3.

[0267] MS(ESI): 492.3 [M+H] + . 1 H-NMR (400MHz, DMSO-d6) δ9.99 (s, 1H), 8.19 (d, J = 2.4Hz, 1H), 8.12 (d, J = 8.8Hz, 1H), 8.01 (s, 1H), 7.84 (d, J = 11.6Hz, 1H), 7.66 (dd, J 1 =8.4Hz, J 2 =2.4Hz,1H),2.88-2.85(m,2H),2.34(s,3H),2.20(s,3H),2.10-2.05(m,6H),2.08-1.96(m,2H),1.75-1.72(m,5H).

[0268] Example 13 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-methylpiperidin-4-yl)pyridin-2-yl-3,4,6-[D3])pyrimidin-6-[D]-2-amine (Compound 7)

[0269]

[0270] The title compound was synthesized following steps similar to those in Examples 2 and 4.

[0271] MS(ESI): 493.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.01-7.98(m,2H),7.91(d,J=10.8Hz,1H),3.20-3.17(m,2H) ,2.62-2.55(m,1H),2.48(s,3H),2.41(s,3H),2.33-2.03(m,8H),2.08-1.86(m,6H).

[0272] Example 14 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-methyl-[D3])piperidin-4-yl)pyridin-2-yl)pyrimidin-6-[D]-2-amine (Compound 8)

[0273]

[0274] The title compound was synthesized following steps similar to those in Examples 1 and 2.

[0275] MS(ESI): 493.2 [M+H] + . 1 H-NMR (400MHz, DMSO-d6) δ9.99(s,1H),8.20(d,J=2.4Hz,1H),8.13(d,J=8.8Hz,1H),8.01(s,1H),7.85(d,J=12.0Hz,1H),7.66(dd,J 1 =8.4Hz, J 2 =2.0Hz,1H),2.89-2.85(m,2H),2.75-2.71(m,1H),2.34(s,3H),2.10-1.96(m,8H),1.76-1.65(m,6H).

[0276] Example 15 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-methyl-[D3])piperidin-4-yl-3,4-[D2])pyridin-2-yl-3,4,6-[D3])pyrimidin-6-[D]-2-amine (Compound 9)

[0277]

[0278] The title compound was synthesized following steps similar to those in Examples 1-4.

[0279] MS(ESI): 498.3 [M+H] + . 1 H-NMR(400MHz, CDCl3)δ8.02(s,1H),7.98(s,1H),7.91(dd,J 1 =10.8Hz, J 2 =0.8Hz,1H),3.17-3.14(m,2H),2.41(s,3H),2.26-2.08(m,8H),1.97-1.88(m,7H).

[0280] Example 16 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-methyl-[D3])piperidin-4-yl-3,4-[D2])pyridin-2-yl)pyrimidin-6-[D]-2-amine (Compound 10)

[0281]

[0282] The title compound was synthesized following steps similar to those in Examples 1-3.

[0283] MS(ESI): 495.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ9.99 (s, 1H), 8.19 (d, J = 2.4Hz, 1H), 8.12 (d, J = 8.8Hz, 1H), 8.01 (s, 1H), 7.84 (d, J = 11.6Hz, 1H), 7.65 (dd, J 1 =8.4Hz, J 2 =2.4Hz,1H),2.89-2.86(m,2H),2.34(s,3H),2.08-2.05(m,6H),2.01-1.96(m,2H),1.75-1.72(m,5H).

[0284] Example 17 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-methylpiperidin-4-yl-3,4-[D2])pyridin-2-yl-3,4,6-[D3])pyrimidin-6-[D]-2-amine (Compound 11)

[0285]

[0286] The title compound was synthesized following steps similar to those in Examples 2-4.

[0287] MS(ESI): 495.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ7.98 (s, 1H), 7.95 (s, 1H), 7.91 (d, J = 10.8Hz, 1H), 3.15 -3.12(m,2H),2.45(s,3H),2.41(s,3H),2.23-2.09(m,7H),1.91-1.88(m,6H).

[0288] Example 18 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-aza-(5-(1-methyl-[D3])piperidin-4-yl)pyridin-2-yl-3,4-[D2]-6-[D])pyrimidin-6-[D]-2-amine (Compound 12)

[0289]

[0290] The title compound was synthesized following steps similar to those in Examples 1, 2, and 4.

[0291] MS(ESI): 496.0 [M+H] + . 1 H-NMR(400MHz,DMSO-d6)δ10.00(s,1H),8.18(s,1H),8.01(s,1H),7.84(d,J=11 .6Hz,1H),2.91-2.88(m,2H),2.34(s,3H),2.10-1.99(m,9H),1.77-1.65(m,6H).

[0292] Example 19 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-methyl-[D3])piperidin-4-yl-3,4-[D2])pyridin-2-yl)pyrimidin-2-amine (Compound 14)

[0293]

[0294] The title compound was synthesized following steps similar to those in Examples 1 and 3.

[0295] MS(ESI): 494.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.51(brs,1H),8.46(d,J=3.6Hz,1H),8.35(d,J=8.8H z,1H),8.26(d,J=2.0Hz,1H),7.97(s,1H),7.90(d,J=11.2Hz,1H),7.61(dd,J 1 =8.4Hz, J 2 =2.0Hz,1H),3.09-3.07(m,2H),2.40(s,3H),2.18-2.08(m,8H),1.90-1.87(m,5H).

[0296] Example 20 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-methylpiperidin-4-yl-3,4-[D2])pyridin-2-yl-3,4,6-[D3])pyrimidin-2-amine (Compound 15)

[0297]

[0298] The title compound was synthesized following steps similar to those in Examples 3 and 4.

[0299] MS(ESI): 494.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.43(d,J=3.6Hz,1H),7.98(s,1H),7.95(s,1H),7.91(d,J=10.8Hz ,1H),3.23-3.21(m,2H),2.51(s,3H),2.41(s,3H),2.33-2.20(m,2H),2.19-1.82(m,11H).

[0300] Example 21 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-methyl-[D3])piperidin-4-yl-3,4-[D2])pyridin-2-yl-3,4,6-[D3])pyrimidin-2-amine (Compound 16)

[0301]

[0302] The title compound was synthesized following steps similar to those in Examples 1, 3, and 4.

[0303] MS(ESI): 497.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.43 (d, J = 3.6 Hz, 1H), 7.98 (s, 1H), 7.95 (s, 1H), 7.91 (dd, J 1 =10.8Hz, J 2 =1.2Hz,1H),3.17-3.15(m,2H),2.41(s,3H),2.27-2.19(m,2H),2.16-1.87(m,11H).

[0304] Example 22 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-methylpiperidin-4-yl-2,2,6,6-[D4])pyridin-2-yl)pyrimidin-2-amine (Compound 17)

[0305]

[0306] The title compound was synthesized following a similar procedure to that in Example 3. MS (ESI): 493.3 [M+H] + .

[0307] Example 23 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-methylpiperidin-4-yl-4-[D])pyridin-2-yl)pyrimidin-2-amine (Compound 18)

[0308]

[0309] The title compound was synthesized following a similar procedure to that in Example 3. MS (ESI): 490.3 [M+H] + .

[0310] Example 24 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-methylpiperidin-4-yl-2,2,3,3,4,5,5,6,6-[D9])pyridin-2-yl)pyrimidin-2-amine (Compound 19)

[0311]

[0312] The title compound was synthesized following a similar procedure to that in Example 3. MS (ESI): 498.2 [M+H] + .

[0313] Example 25 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl-6'-[D])-aza-(5-(1-(methyl-[D3])piperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (Compound 22)

[0314]

[0315] The title compound was synthesized following steps similar to those in Examples 1 and 5.

[0316] MS(ESI): 493.3 [M+H] + . 1H-NMR (400MHz, CDCl3) δ8.43(d,J=3.6Hz,1H),8.36(d,J=8.8Hz,1H),8.21(d,J=2.4Hz,1H),8.02(s,1H),7.98(s,1 H),7.65(d,J=8.8Hz,1H),3.28-3.27(m,2H),2.62-2.53(m,3H),2.40(s,3H),2.26-1.90(m,8H),1.88-1.72(m,4H).

[0317] Example 26 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl-6'-[D])-aza-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-6-[D]-2-amine (Compound 23)

[0318]

[0319] The title compound was synthesized following steps similar to those in Examples 2 and 5.

[0320] MS(ESI): 491.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.34(d,J=8.4Hz,1H),8.24(s,1H),8.23(d,J=8.0Hz,1H),7.98(s,1H),7.61(dd,J 1 =8.4Hz, J 2 =2.4Hz,1H),3.08-3.05(m,2H),2.53-2.50(m,1H),2.40(s,3H),2.39(s,3H),2.22-2.08(m,8H),1.91-1.84(m,6H).

[0321] Example 27 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl-6'-[D])-N-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl-3,4,6-[D3])pyrimidin-2-amine (Compound 24)

[0322]

[0323] The title compound was synthesized following steps similar to those in Examples 4 and 5.

[0324] MS(ESI): 493.3 [M+H] + . 1H-NMR (400MHz, CDCl3) δ8.44 (d, J = 3.6Hz, 1H), 8.01 (s, 1H), 7.98 (s, 1H), 3.14-3.11 (m, 2H),2.57-2.53(m,1H),2.44(s,3H),2.40(s,3H),2.24-2.07(m,8H),1.96-1.88(m,6H).

[0325] Example 28 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl-4',6'-[D2])-aza-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (Compound 26)

[0326]

[0327] The title compound was synthesized following steps similar to those in Example 6.

[0328] MS(ESI): 491.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.43(d,J=3.6Hz,1H),8.32(d,J=8.4Hz,1H),8.24(d,J=2.4Hz,1H),8.15(s,1H),7.60(dd,J 1 =8.8Hz,J 2 =2.4Hz,1H),3.21-3.17(m,2H),2.58-2.53(m,1H),2.48(s,3H),2.43(s,3H),2.29-2.01(m,8H),1.98-1.86(m,6H).

[0329] Example 29 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl-3,4-[D2])-N-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl-3,4,6-[D3])pyrimidin-2-amine (Compound 28)

[0330]

[0331] The title compound was synthesized following steps similar to those in Examples 4 and 7.

[0332] MS(ESI): 494.3 [M+H] + . 1H-NMR (400MHz, CDCl3) δ8.40(d,J=3.6Hz,1H),8.10(s,1H),7.98(s,1H),7.91(d,J=10.8Hz,1H),3. 17-3.14(m,2H),2.57-2.56(m,1H),2.46(s,3H),2.40(s,3H),2.29-2.00(m,7H),1.97-1.88(m,5H).

[0333] Example 30 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl-3,4-[D2])-aza-(5-(1-(methyl-[D3])piperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (Compound 29)

[0334]

[0335] The title compound was synthesized following steps similar to those in Examples 1 and 7.

[0336] MS(ESI): 494.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.44(d,J=3.6Hz,1H),8.36(d,J=8.8Hz,1H),8.22(d,J=2.0Hz,1H),8.06(s,1H),7.98(s,1H),7.91(dd,J 1 =10.8Hz, J 2 =1.2Hz, 1H), 7.66(dd, J 1 =8.8Hz,J 2 =2.0Hz,1H),3.27-3.26(m,2H),2.63-2.60(m,1H),2.40(s,3H),2.19-2.07(m,7H),1.97-1.86(m,5H).

[0337] Example 31 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl-3,4-[D2])-aza-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-6-[D]-2-amine (Compound 30)

[0338]

[0339] The title compound was synthesized following steps similar to those in Examples 2 and 7.

[0340] MS(ESI): 492.3 [M+H] + .1 H-NMR (400MHz, CDCl3) δ8.34(d,J=3.6Hz,1H),8.22(d,J=2.0Hz,1H),8.14(s,1H),7.98(s,1H),7.91(d,J=10.8Hz,1H),7.63(dd,J 1 =8.8Hz,J 2 =2.4Hz,1H),3.20-3.17(m,2H),2.62-2.54(m,1H),2.47(s,3H),2.40(s,3H),2.30-2.01(m,7H),1.98-1.86(m,5H).

[0341] Example 32 5-Fluoro-4-(7'-Fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5'-yl-3,4-[D2])-aza-(5-(1-(methylpiperidin-4-yl-3,4-[D2])pyridin-2-yl-3,4,6-[D3])pyrimidin-2-amine (Compound 31)

[0342]

[0343] The title compound was synthesized following steps similar to those in Examples 1, 3, 4, and 7.

[0344] MS(ESI): 499.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.44(d,J=3.6Hz,1H),8.06(s,1H),7.98(s,1H),7.91(dd,J 1 =10.8Hz, J 2 =1.2Hz,1H),3.28-3.25(m,2H),2.40(s,3H),2.19-2.09(m,6H),1.96-1.86(m,5H).

[0345] Example 33 5-Fluoro-4-(7'-Fluoro-2'-(Methyl-D3)spiro[cyclopentane-1,3'-indole]-5'-yl-2,2,3,3,4,4,5,5-[D8])-nitro-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (Compound 33)

[0346]

[0347] The title compound was synthesized following similar steps to those in Examples 7 and 8. MS (ESI): 500.3 [M+H] + .

[0348] Example 34 5-Fluoro-4-(7'-Fluoro-2'-(Methyl-[D3])spiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-6-[D]-2-amine (Compound 34)

[0349]

[0350] The title compound was synthesized following steps similar to those in Examples 2 and 8.

[0351] MS(ESI): 493.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.34(d,J=8.8Hz,1H),8.24(s,1H),8.23(s,1H),7.98(s,1H),7.91(dd,J 1 =10.8Hz, J 2 =0.8Hz, 1H), 7.61(dd, J 1 =8.8Hz,J 2 =2.4Hz,1H),3.05-3.02(m,2H),2.54-2.50(m,1H),2.38(s,3H),2.22-2.08(m,8H),1.91-1.83(m,6H).

[0352] Example 35 5-Fluoro-4-(7'-Fluoro-2'-(Methyl-[D3])spiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-(methyl-[D3])piperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (Compound 35)

[0353]

[0354] The title compound was synthesized following steps similar to those in Examples 1 and 8.

[0355] MS(ESI): 495.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.44(d,J=3.6Hz,1H),8.34(d,J=8.4Hz,1H),8.23(d,J=2.4Hz,1H),8.13(s,1H),7.98(s,1H),7.91(d,J=11.2Hz,1H),7.62(dd,J 1 =8.4Hz, J 2=2.0Hz,1H),3.07-3.04(m,2H),2.57-2.51(m,1H),2.20-2.08(m,8H),1.91-1.88(m,6H).

[0356] Example 36 5-Fluoro-4-(7'-Fluoro-2'-(Methyl-[D3])spiro[cyclopentane-1,3'-indole]-5'-yl)-nitro-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl-3,4,6-[D3])pyrimidine-2-amine (Compound 36)

[0357]

[0358] The title compound was synthesized following steps similar to those in Examples 4 and 8.

[0359] MS(ESI): 495.1 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.45 (d, J = 3.6 Hz, 1H), 8.43 (s, 1H), 7.98 (s, 1H), 7.91 (dd, J 1 =11.2Hz,J 2 =1.2Hz,1H),3.04-3.01(m,2H),2.53-2.49(m,1H),2.37(s,1H),2.21-2.08(m,8H),1.91-1.82(m,6H).

[0360] Example 37 5-Fluoro-4-(7'-Fluoro-2'-(Methyl-[D3])spiro[cyclopentane-1,3'-indole]-5'-yl-6'-[D])-nitro-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (Compound 37)

[0361]

[0362] The title compound was synthesized following steps similar to those in Examples 5 and 8.

[0363] MS(ESI): 493.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.44(d,J=4.0Hz,1H),8.34(d,J=8.4Hz,1H),8.23(d,J=2.0Hz,1H),8.13(s,1H),7.98(s,1H),7.62(dd,J 1 =8.8Hz,J 2=2.4Hz,1H),3.08-3.05(m,2H),2.55-2.49(m,1H),2.40(s,3H),2.20-2.07(m,8H),1.91-1.88(m,6H).

[0364] Example 38 5-Fluoro-4-(7'-Fluoro-2'-(Methyl-[D3])spiro[cyclopentane-1,3'-indole]-5'-yl-6'-[D])-aza-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-6-[D]-2-amine (Compound 38)

[0365]

[0366] The title compound was synthesized following steps similar to those in Examples 4, 5, and 8.

[0367] MS(ESI): 494.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ12.87(brs,1H),8.65(d,J=8.8Hz,1H),8.23(s,1H),8.02(s,1H),8.00(s ,1H),3.68-3.65(m,2H),2.98-2.92(m,3H),2.87(s,3H),2.75-2.66(m,2H),2.40-1.89(m,10H).

[0368] Example 39 5-Fluoro-4-(7'-Fluoro-2'-(Methyl-[D3])spiro[cyclopentan-1,3'-indole]-5'-yl)-nitro-(5-(1-(methyl-[D3])piperidin-4-yl-3,4-[D2])pyridin-2-yl-3,4,6-[D3])pyrimidin-2-amine (Compound 39)

[0369]

[0370] The title compound was synthesized following steps similar to those in Examples 1, 3, 4, and 8.

[0371] MS(ESI): 500.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.44(d,J=3.6Hz,1H),8.06(s,1H),7.98(s,1H),7.91(dd,J 1 =10.8Hz, J 2=1.2Hz,1H),3.17-3.15(m,2H),2.30-2.20(m,1H),2.19-2.03(m,8H),1.93-1.87(m,4H).

[0372] Example 40 5-Fluoro-4-(7'-Fluoro-2'-(Methyl-[D3])spiro[cyclopentane-1,3'-indole]-5'-yl-3,4-[D2])-aza-(5-(1-(methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (Compound 40)

[0373]

[0374] The title compound was synthesized following steps similar to those in Examples 7 and 8.

[0375] MS(ESI): 494.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.44(d,J=3.6Hz,1H),8.34(d,J=8.4Hz,1H),8.23(s, 1H),8.21(s,1H),8.14(s,1H),7.98(s,1H),7.91(d,J=11.2Hz,1H),7.62(dd,J 1 =8.4Hz, J 2 =2.0Hz,1H),3.08-3.05(m,2H),2.55-2.51(m,1H),2.40(s,3H),2.18-2.07(m,6H),1.91-1.89(m,6H).

[0376] Example 41 5-Fluoro-4-(7'-Fluoro-2'-(Methyl-[D3])spiro[cyclopentane-1,3'-indole]-5'-yl-3,4-[D2])-aza-(5-(1-(methyl-[D3])piperidin-4-yl-3,4--[D2])pyridin-2-yl-3,4,6-[D3])pyrimidin-2-amine (Compound 41)

[0377]

[0378] The title compound was synthesized following steps similar to those in Examples 1, 3, 4, 7, and 8.

[0379] MS(ESI): 502.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.43 (d, J = 3.6 Hz, 1H), 8.07 (s, 1H), 7.98 (s, 1H), 7.91 (dd, J1 =10.8Hz, J 2 =2.8Hz,1H),3.06-3.03(m,2H),2.20-2.07(m,6H),1.91-1.86(m,5H).

[0380] Example 42 5-Fluoro-4-(7'-Fluoro-2'-(Methyl-[D3])spiro[cyclopentan-1,3'-indole]-5'-yl-3,4-[D2])-aza-(5-(1-(Methyl-[D3])piperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (Compound 42)

[0381]

[0382] The title compound was synthesized following steps similar to those in Examples 1, 7, and 8.

[0383] MS(ESI): 497.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.43(d,J=3.6Hz,1H),8.34(d,J=7.6Hz,1H),8.21(d,J=2.4Hz,1H),8.02(s,1H),7.98(s,1H),7.91(dd,J 1 =11.2Hz,J 2 =1.6Hz, 1H), 7.63(dd, J 1 =8.4Hz, J 2 =2.4Hz,1H),3.09-3.07(m,2H),2.54-2.51(m,1H),2.18-2.07(m,6H),1.89-1.88(m,6H).

[0384] Example 43 5-Fluoro-4-(7'-Fluoro-2'-(Methyl-[D3])spiro[cyclopentan-1,3'-indole]-5'-yl-3,4-[D2])-aza-(5-(1-(1-(methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-6-[D]-2-amine) (Compound 43)

[0385]

[0386] The title compound was synthesized following steps similar to those in Examples 2, 7, and 8.

[0387] MS(ESI): 495.3 [M+H] + . 1H-NMR (400MHz, CDCl3) δ8.37(d,J=8.8Hz,1H),8.23(d,J=2.0Hz,1H),8.07(s,1H),7.98(s,1H),7.91(d,J=11.2Hz,1 H),7.66(d,J=8.4Hz,1H),3.33-3.31(m,2H),2.62(s,3H),2.52-2.49(m,2H),2.27-2.07(m,7H),1.99-1.87(m,4H).

[0388] Example 44 5-Fluoro-4-(7'-Fluoro-2'-(Methyl-[D3])spiro[cyclopentan-1,3'-indole]-5'-yl-3,4-[D2])-aza-(5-(1-(methyl-[D3])piperidin-4-yl)pyridin-2-yl)pyrimidin-6-[D]-2-amine (Compound 44)

[0389]

[0390] The title compound was synthesized following steps similar to those in Examples 1, 2, 7, and 8.

[0391] MS(ESI): 498.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.39(d,J=8.8Hz,1H),8.22(d,J=3.6Hz,1H),8.05(s,1H),7.98(s,1H),7.91(dd,J 1 =11.2Hz,J 2 =0.8Hz,1H),7.70(d,J=8.4Hz,1H),3.59-3.54(m,2H),2.84-2.75(m,3H),2.55-2.40(m,2H),2.55-2.40(m,2H),2.22-1.87(m,5H).

[0392] Example 45 5-Fluoro-4-(7'-Fluoro-2'-(Methyl-[D3])spiro[cyclopentane-1,3'-indole]-5'-yl-4'-[D])-aza-(5-(1-(methyl-[D3])piperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (Compound 45)

[0393]

[0394] The title compound was synthesized following steps similar to those in Examples 1, 6, and 8.

[0395] MS(ESI): 496.3 [M+H] +. 1 H-NMR (400MHz, CDCl3) δ8.43(d,J=3.6Hz,1H),8.34(d,J=8.8Hz,1H),8.22(d,J=2.4Hz,1H),8.10(s,1H),7.91(d,J=11.2Hz,1H),7.62(dd,J 1 =8.8Hz,J 2 =2.4Hz,1H),3.05-3.02(m,2H),2.54-2.48(m,1H),2.20-2.09(m,8H),1.91-1.80(m,6H).

[0396] Example 46 5-Fluoro-4-(7'-Fluoro-2'-(Methyl-[D3])spiro[cyclopentane-1,3'-indole]-5'-yl-4'-[D])-aza-(5-(1-(methyl-[D3])piperidin-4-yl)pyridin-2-yl)pyrimidin-6-[D]-2-amine (Compound 46)

[0397]

[0398] The title compound was synthesized following steps similar to those in Examples 1, 2, 6, and 8.

[0399] MS(ESI): 497.3 [M+H] + . 1 H-NMR (400MHz, CDCl3) δ8.34(d,J=8.8Hz,1H),8.22(d,J=2.0Hz,1H),8.14(s,1H),7.91(d,J=10.8Hz,1H),7.62(dd,J 1 =8.4Hz, J 2 =2.4Hz,1H),3.06-3.03(m,2H),2.53-2.43(m,1H),2.18-2.08(m,8H),1.90-1.87(m,6H).

[0400] The beneficial effects of the present invention will be illustrated below through experimental examples.

[0401] Experimental Example 1: Determination of the inhibitory effect of the compound of the present invention on the proliferation of human glioma U87MG cells.

[0402] 1.1 Experimental Materials

[0403] Human glioma cells U87 MG were purchased from Nanjing Kebai Biotechnology Co., Ltd.; control compound 1 was prepared according to the preparation method in patent WO2017 / 092635A1; cell detection equipment was an In Cell Analyzer 2200 (GE Healthcare); reagents or consumables used in the experiment are shown in the table below:

[0404] Table 1

[0405]

[0406]

[0407] 1.2 Experimental Preparation

[0408] 1.2.1 Preparation of U87 MG medium: MEM (low glucose) + 10% FBS + 1% penicillin / streptomycin + 1% sodium pyruvate + 1% MEM NEAA

[0409] 1.2.2 Preparation of solutions of control compound 1 and test compound:

[0410] (1) Preparation of control compound 1 solution

[0411] a. Add 15 μL of 1.25 mM control compound 1 solution to well B1 of a 96-well plate. Add 20 μL of DMSO to each well from B2 to B11. Take 5 μL of the solution from well B1 and add it to well B2. Mix well and dilute sequentially to well B11 to obtain control compound 1 solutions diluted 3 times.

[0412] b. Add 95.2 μL of culture medium to wells C2-C11 of a 96-well plate, and add 4.8 μL of diluted solution from wells B2-B11 to wells C2-C11 (well C1 concentration 60 μM), and mix well.

[0413] (2) Preparation of the test compound solution

[0414] a. Take 15 μL of the 1.25 mM test compound solution and add it to well D1 of a 96-well plate. Add 20 μL of DMSO to each well from D2 to D11. Take 5 μL of the solution from well D1 and add it to well D2. Mix well and dilute sequentially up to D11 to obtain test compound solutions diluted 3 times.

[0415] b. Add 95.2 μL of culture medium to wells E2-E11 of a 96-well plate, and add 4.8 μL of the diluted solution from D2-D11 to wells E2-E11 (well E1 concentration 60 μM), and mix well.

[0416] 1.3 Experimental Procedure

[0417] 1.3.1 U87 MG cells were seeded at 4000 cells / 100 μL / well into 96-well black transparent bottom cell plates and cultured overnight at 37°C;

[0418] 1.3.2 The diluted control compound 1 solution and the test compound solution were added at 20 μL / well to the cell-inoculated culture plate and incubated at 37°C for 72 h.

[0419] 1.3.3 Fixation: Remove the 96-well plate, remove the culture medium, and add 50 μL of neutral formaldehyde fixative (formaldehyde:PBS = 1:9) to each well at room temperature. Fix for 10-30 min at room temperature in the dark.

[0420] 1.3.4 Wash twice with 1×PBS (50 μL / well);

[0421] 1.3.5 Permeation treatment: 0.2% Triton TM X-100 (50 μL / well) permeation treatment for 5-10 min;

[0422] 1.3.6 Wash twice with 1×PBS (50 μL / well);

[0423] 1.3.7 Staining: DAPI (50 μL / well) staining (PBS:DAPI = 5000:1), incubate at room temperature in the dark for 20 min;

[0424] 1.3.8 Wash 3 times with 1×PBS (50 μL / well), then add PBS (100 μL / well);

[0425] 1.3.9 In-Cell Analyzer scan to analyze the number of cells per well;

[0426] 1.3.10 Data Processing:

[0427] The inhibition rate of each compound at each concentration point was calculated using the following formula, and the IC50 was obtained by curve fitting using Graphpad Prism 6.0 software. 50 value.

[0428]

[0429] 1.4 Experimental Results

[0430] Table 2. Results of the inhibitory activity of the tested compounds on U87MG cells.

[0431]

[0432]

[0433] As can be seen from the data in Table 2, the deuterated test compound and the undeuterated control compound have similar inhibitory activity against U87MG cells. The above experimental results prove that the compound of the present invention maintains the cellular activity of the drug after deuteration modification relative to control compound 1.

[0434] Test Example 2: Stability of the compound of the present invention in human liver microsomes

[0435] 2.1 Experimental Materials

[0436] Human liver microsomes were purchased from Corning and stored at -80°C; reduced nicotinamide adenine dinucleotide phosphate (NADPH) was purchased from Chem-impex International; control compound 1 was prepared according to the preparation method in patent WO2017 / 092635A1; the reference standards were testosterone, diclofenac and propafenone.

[0437] 2.2 Experimental Conditions

[0438] - Test concentration: 1 μM (DMSO 0.01%);

[0439] - Buffer solution: 100mM phosphate buffer, pH 7.4;

[0440] - Microsomal protein concentration: 0.5 mg / ml;

[0441] - Cofactors: NADPH and MgCl2;

[0442] -NADPH concentration: 1 nM.

[0443] 2.3 Experimental Procedure

[0444] 2.3.1 Prepare eight 96-well incubation plates, named T0, T5, T10, T20, T30, T60, Blank60, and NCF60, respectively. The reaction time points for the first six incubation plates are 0, 5, 10, 20, 30, and 60 minutes, respectively. Do not add the test sample, control compound 1, or control to the Blank 60 plate, and take a sample after 60 minutes of incubation. In the NCF60 plate, use potassium phosphate buffer instead of NADPH working solution for 60 minutes of incubation.

[0445] 2.3.2 Add 5 μL of the test sample, control compound 1 or control working solution and 445 μL of microparticle working solution to the T0, T5, T10, T20, T30, T60 and NCF60 plates respectively. Add only microparticle working solution to the Blank60 plate. Then place the above incubation plates in a 37°C water bath for pre-incubation for about 10 minutes.

[0446] 2.3.3 After the pre-incubation, except for the NCF60 plate and T0 plate, add 44 μL of NADPH working solution to each sample well to start the reaction, and add 50 μL of potassium phosphate buffer to each well of the NCF60 plate.

[0447] 2.3.4 After incubation for appropriate times (5, 10, 20, 30 and 60 minutes), add 180 μL of stop solution (acetonitrile solution containing 200 ng / mL tolbutamide and 200 ng / mL labetalol) to each sample well to terminate the reaction.

[0448] 2.3.5 Preparation of T0 plate: First, add 180 μL of stop solution (acetonitrile solution containing 200 ng / mL tolbutamide and 200 ng / mL labetalol) to the T0 plate, and then add 6 μL of NADPH working solution.

[0449] 2.3.6 Shake all sample plates well and centrifuge at 4000 rpm for 20 minutes. Then, take 80 μL of the supernatant from each well and dilute it into 240 μL of pure water for LC-MS / MS analysis.

[0450] 2.3.7 Data Analysis:

[0451] The concentrations of the test sample, reference compound 1, and reference standard in the sample were determined using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The slope value (Ke) was determined by linear regression of the remaining percentage of the sample against the natural logarithm of the incubation time curve. The in vitro half-life (in vitro Tday) was also determined. 1 / 2 Determined by the slope value:

[0452] 2.4 Experimental results on the metabolic stability of the compounds of this invention in human liver microsomes

[0453] Test the half-life (T) of control compound 1 1 / 2 The average half-life was 23.9 minutes. Within the same batch of tested samples, compounds with a half-life 1.4 to 10 minutes shorter than control compound 1 were defined as C; compounds with a half-life within ±1 minute of control compound 1 were defined as B; compounds with a half-life 1.4 to 10 minutes longer than control compound 1 were defined as A; and compounds with a half-life 15 to 30 minutes longer than control compound 1 were defined as A... + .

[0454] Table 3. Results of metabolic stability assays of the test compounds in human liver microsomes.

[0455]

[0456]

[0457] As can be seen from the data in Table 3, compounds 1, 2, 9, 12-15, 21, 22, 24, 25, 27-32, and 35-44 exhibited better metabolic stability in human liver microsomes than control compound 1. In particular, compound 35 had a half-life in human liver microsomes that was 15-30 minutes longer than control compound 1, and its metabolic stability in human liver microsomes was significantly improved compared to control compound 1. This indicates that some compounds in this invention have a longer duration of action and may have better clinical application value.

[0458] The half-lives of compounds 7 and 10, which served as control compounds, were shorter than those of control compound 1. This indicates that the effect of deuteration on the metabolic properties of compounds is unpredictable. Deuteration at certain sites may not only fail to prolong the half-life but may actually shorten it and degrade its pharmacokinetic properties.

[0459] The present invention has been described through the above embodiments. However, it should be understood that the above embodiments are for illustrative purposes only and are not intended to limit the invention to the scope of the described embodiments. Furthermore, those skilled in the art will understand that the present invention is not limited to the above embodiments, and many more variations and modifications can be made based on the teachings of the present invention, all of which fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A compound or a pharmaceutically acceptable salt thereof: The compound is selected from: , , , , , , , , , , , , , , , , , , , , , , , , , and .

2. A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

3. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2, in the preparation of a medicament for treating human glioma.

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