Kif18a inhibitor compounds, pharmaceutical compositions, and methods of making and using the same

Abstract

The present application provides a compound shown in formula (I), a pharmaceutical composition and a preparation method and application thereof. The compound has good KIF18A inhibition effect, can be combined with microtubules to form a complex to regulate KIF18A protein, is used for treating KIF18A-mediated diseases and / or diseases, such as tumor diseases, and is used for preparing medicines for such diseases or diseases.

Description

[0001] This application claims priority to the following patent applications:

[0002] The prior application filed with the China National Intellectual Property Administration on August 5, 2022, with patent application number 202210944271.7 and title "KIF18A inhibitor compound, pharmaceutical composition and preparation method and application thereof";

[0003] The prior application filed with the China National Intellectual Property Administration on November 22, 2022, with patent application number 202211469122.6 and title "KIF18A inhibitor compound, pharmaceutical composition and preparation method and application thereof";

[0004] The full text of the prior application is incorporated herein by reference. Technical Field

[0005] This invention belongs to the pharmaceutical field, specifically relating to a KIF18A inhibitor compound, a pharmaceutical composition, its preparation method, and its application. Background Technology

[0006] Cancer is one of the most serious diseases affecting human health, with mortality and morbidity rates often ranking among the highest of all diseases. Although the quality of life for some patients has been greatly improved with the continuous development and progress of medical technology and drug research, there are still many unmet clinical needs in the search for effective treatments or cures for different cancers, and new targets will provide new possibilities for future cancer drug development.

[0007] Cancer cells exhibit unregulated cell proliferation due to damage or loss of one or more genes that regulate the cell cycle. Various kinases and kinesins have been identified as playing key roles in the regulation and progression of cell cycle and mitosis in both normally dividing cells and cancer cells.

[0008] Kinesin molecules are kinetic proteins that use intracellular microtubules as their orbital pathways; also known as molecular motors, they convert ATP energy into mechanical energy and are closely related to eukaryotic cell division, mitosis, meiosis, tissue and organ growth and development, neuronal development, and signal transduction. Kinesin members share a relatively conserved motor domain. Based on the location of the motor domain in the molecule, the kinesin family is broadly divided into three categories: N-type kinesins, where the amino (-NH2) terminal region of the polypeptide chain contains a motor domain; M-type kinesins, where the middle region contains a motor domain; and C-type kinesins, where the carboxyl (-COOH) terminal region contains a motor domain.

[0009] Chromosomal instability is a hallmark of cancer, caused by errors in chromosome segregation during mitosis. Targeting chromosomal instability is an emerging therapeutic strategy in drug development. KIF18A, a member of the N-type Kinesin-8 kinesin family, has been shown to play a role in maintaining the integrity of the bipolar spindle and promoting the survival of chromosomally unstable cancer cells. Mitosis is an effective intervention point, and many antimitotic drugs are used clinically to treat human cancers. The most widely used microtubule inhibitors both stabilize microtubules and prevent microtubule assembly. Currently, antimitotic drugs have limitations due to their narrow therapeutic window, and these issues necessitate the development of new targets to address them.

[0010] Although tubulin inhibitors are widely used as standard treatments for various human cancer types, these drugs can cause collateral damage to normal cells, including myelosuppression and neurotoxicity. Since KIF18A may not be essential in normal diploid somatic cells (KIF18A knockout mice are viable but have reproductive defects, suggesting that KIF18A is not a necessary gene for normal somatic cell division), targeting KIF18A could potentially significantly reduce its toxicity, thus improving the therapeutic safety window of tubulin-targeting drugs in clinical practice.

[0011] KIF18A protein is highly expressed in various tumors, including colorectal cancer, breast cancer, lung cancer, pancreatic cancer, prostate cancer, bladder cancer, head and neck cancer, cervical cancer, and ovarian cancer. KIF18A plays a crucial role in the occurrence, development, and metastasis of breast cancer, and its high expression predicts poor prognosis. KIF18A is essential for the proliferation of chromosomally unstable cells derived from triple-negative breast cancer or colorectal cancer, but is not required in diploid cells. Knockout of the KIF18A gene leads to infertility in male mice, but female mice are unaffected. KIF18A mRNA expression is significantly associated with higher tumor grade and larger tumors in breast cancer patients, and KIF18A is an independent predictor of lymph node metastasis in breast cancer, with a risk factor of 3.2. Furthermore, inhibiting KIF18A expression not only affects its key function in cell mitosis but also reduces cancer cell migration by stabilizing leading-edge microtubules, ultimately leading to inactivation of the PI3K-AKT signaling pathway and inducing apoptosis.

[0012] Therefore, the development of KIF18A protein inhibitors may be a new breakthrough in cancer drugs. Summary of the Invention

[0013] To address the aforementioned technical problems, the present invention provides a compound of formula (I), its racemic mixture, stereoisomer, tautomer, isotope label, solvate, polymorph, pharmaceutically acceptable salt, or prodrug compound:

[0014]

[0015] Among them, X1, X2, and X3 may be the same or different, and are independently selected from N or CR0; R0 is selected from H, halogen, cyano, and C. 1-12 Alkyl, Halogenated C 1-12 Alkyl, cyano C 1-12 Alkyl, C 1-12 Alkoxy, halogenated C 1-12 Alkoxy, cyano C 1-12 Alkoxy;

[0016] A is selected from unsubstituted or arbitrarily assigned to one, two or more R. a The following groups are substituted: C 1-12 Alkyl, Halogenated C 1-12 Alkyl, cyano C 1-12 Alkyl, C 1-12 Alkoxy, halogenated C 1-12 Alkoxy, cyano C 1-12 Alkoxy, C 3-12 cycloalkyl, C 3-12 Cycloalkyloxy, C 3-12 Cycloalkylthio groups, 3-14 membered heterocyclic groups; each R a They may be the same or different, and are independently selected from halogens, cyano groups, and C. 1-12 Alkyl, Halogenated C 1-12 Alkyl, cyano C 1-12 Alkyl, C 1-12 Alkoxy, halogenated C 1-12 Alkoxy, cyano C 1-12 Alkoxy;

[0017] Y1, Y2, and Y3 may be the same or different, and are independently selected from N or CH;

[0018] M is selected from unsubstituted or optionally by one, two or more Rs. b The following groups are substituted: C 3-12 cycloalkyl, C 3-12 Cycloalkenyl, 3-14 membered heterocyclic groups; each R b They may be the same or different, and are independently selected from halogens, cyano groups, and C. 1-12 Alkyl, Halogenated C 1-12 Alkyl, cyano C 1-12 Alkyl, C 1-12 Alkoxy, cyano C 1-12 Alkoxy;

[0019] E is selected from unsubstituted or optionally by one, two or more R. c The following groups are substituted: -NH-S(=O)2-R c1-S(=O)2-NH-R c2 -S(=O)(=NH)-R c3 -N(R) c4 (R) c5 ), 3-14 membered heterocyclic groups; each R c They may be the same or different, and are independently selected from OH, halogen, cyano, and C groups. 1-12 Alkyl, C 1-12 Alkoxy, halogenated C 1-12 Alkyl, Halogenated C 1-12 Alkoxy, cyano C 1-12 Alkyl, cyano C 1-12 Alkoxy, -N(R) c6 (R) c7 );

[0020] R c1 R c2 R c3 R c4 R c5 R c6 R c7 Whether the two are the same or different, they are selected independently from H and C. 1-12 Alkyl, hydroxyl C 1-12 Alkyl, Halogenated C 1-12 Alkyl, Halogenated C 1-12 Alkoxy, cyano C 1-12 Alkyl, cyano C 1-12 Alkoxy, C 3-12 Cycloalkyl, 3-14 membered heterocyclic groups, C 1-12 Alkoxy-C 1-12 Alkyl, hydroxyl C 1-12 Alkyl-C 3-12 Cycloalkyl.

[0021] According to some implementation schemes, X2 and X3 are not both N.

[0022] According to some implementation schemes, when X1 and X2 are N or CR0, X3 is CR0, and R0 is selected from H, halogen, CN, and C. 1-6 Alkyl group; when X1 is N or CH, X2 is CH, X3 is N or CR0, and R0 is selected from H, halogen, CN, C. 1-6 Alkyl group; when X1 is N or CR0 and X2 is N, X3 is N or CR0; R0 is selected from H, halogen, CN, C. 1-6 alkyl.

[0023] According to some implementation schemes, A is selected from C. 1-6 Alkyl, C 3-8 cycloalkyl, C 3-8 Cycloalkyloxy, 3-8 membered heterocyclic group, halogenated 3-8 membered heterocyclic group, halogenated C3-8 Cycloalkyl.

[0024] According to some implementation schemes, A is selected from cyclopropyl, isopropyl,

[0025] According to some implementation schemes, when Y1 is CH, Y2 is CH, and Y3 is N or CH; when Y1 is CH, Y2 is N, and Y3 is N or CH; when Y1 is N, Y2 is CH, and Y3 is N or CH.

[0026] According to some implementation schemes, M is selected from C. 3-12 cycloalkyl, C 3-12 Cycloalkenyl, 3-12 membered heterocyclic group. Preferably, M is selected from C 3-12 Fused cycloalkyl groups, C 3-12 Fused ring cycloalkenyl, 3-12 membered fused ring heterocyclic group, C 3-12 Spirocycloalkyl, C 3-12 Spirocyclic cycloalkenyl, 3-12 membered spirocyclic heterocyclic, C 3-12 Bridged cycloalkyl, C 3-12 Bridged ring cycloalkenyl or 3-12 membered bridged ring heterocyclic group.

[0027] According to some implementation schemes, M is selected from...

[0028] According to some implementation schemes, E is selected from -NH-S(=O)2-R c1 -S(=O)2-NH-R c2 -S(=O)(=NH)-R c3 -N(R) c4 (R) c5 C 1-6 Alkyl-substituted 3-6 membered heterocyclic groups,

[0029] R c1 R c2 R c3 R c4 R c5 Whether the two are the same or different, they are independently selected from H, hydroxyl C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, cyano C 1-6 Alkyl, C 1-6 Alkyl-NH-C 1-6 Alkyl-, (C 1-6 (alkyl)2N-C 1-6 Alkyl, C 3-6 cycloalkyl-NH-C 1-6 Alkyl-, C 1-6 Alkoxy-C 1-6 Alkyl-, C3-6 cycloalkyl, 3-6 membered heterocyclic groups; or

[0030] R c1 R c2 R c3 R c4 R c5 Whether the two are the same or different, they are independently selected from H, hydroxyl C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, cyano C 1-6 Alkyl, C 1-6 Alkyl-NH-C 1-6 Alkyl-, (C 1-6 (alkyl)2N-C 1-6 Alkyl, C 3-6 cycloalkyl-NH-C 1-6 Alkyl-, C 1-6 Alkoxy-C 1-6 Alkyl-, C 3-6 Cycloalkyl, 3-6 membered heterocyclic groups, hydroxyl-C 3-6 Cycloalkyl, hydroxy C 1-6 Alkyl-C 3-6 Cycloalkyl.

[0031] According to some implementation schemes, E is selected from...

[0032] According to some embodiments, the compound represented by formula (I) has the following structure:

[0033]

[0034] Among them, A, E, M, X2, X3, Y1, Y2, and Y3 have the definitions described above.

[0035] According to some embodiments, the compound represented by formula (I) has the structure represented by formula (II), preferably the structure represented by formula (III), and more preferably the structure represented by formula (IV):

[0036]

[0037] Among them, A, E, X1, X2, X3, R c1 It has the definition described above; It represents a carbon-carbon single bond or a carbon-carbon double bond.

[0038] According to some embodiments, the compound shown in formula (I) is selected from the following structures:

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045] The present invention also provides a method for preparing the compound shown in formula (I), comprising the following steps:

[0046]

[0047] (1) Compound a reacts with compound b to give compound c;

[0048] (2) Compound c reacts with EH to give the compound shown in formula (I);

[0049] Among them, A, E, M, X1, X2, X3, Y1, Y2, and Y3 independently have the definitions described above; X is selected from halogens, such as Cl, Br, and I.

[0050] The present invention also provides a pharmaceutical composition comprising at least one of the following: a compound of formula (I), a racemic mixture, a stereoisomer, a tautomer, an isotope label, a solvate, a polymorph, a pharmaceutically acceptable salt, or a prodrug compound thereof, in a therapeutically effective amount.

[0051] According to embodiments of the present invention, the pharmaceutical composition further includes one or more pharmaceutically acceptable excipients.

[0052] According to embodiments of the present invention, the pharmaceutical composition may further contain one or more additional therapeutic agents.

[0053] The present invention also provides a method for treating tumor diseases, comprising administering to a patient a preventive or therapeutically effective amount of at least one of the following: a compound of formula (I), its racemic mixture, stereoisomer, tautomer, isotope label, solvate, polymorph, pharmaceutically acceptable salt, or prodrug compound.

[0054] The present invention also provides a method for treating tumor diseases, comprising administering to a patient a preventive or therapeutically effective amount of the above-described pharmaceutical composition.

[0055] The cancers mentioned include colorectal cancer, breast cancer, lung cancer, pancreatic cancer, prostate cancer, bladder cancer, head and neck cancer, cervical cancer, and ovarian cancer.

[0056] In some implementations, the patient includes mammals, preferably humans.

[0057] The present invention also provides at least one of the following: a compound of formula (I) for treating tumor diseases, a racemic mixture, a stereoisomer, a tautomer, an isotope label, a solvate, a polymorph, a pharmaceutically acceptable salt, or a prodrug compound thereof, or a pharmaceutical composition thereof.

[0058] The present invention also provides the use of at least one of the compounds of formula (I), racemates, stereoisomers, tautomers, isotope labels, solvates, polymorphs, pharmaceutically acceptable salts or prodrug compounds thereof in the preparation of pharmaceuticals.

[0059] According to embodiments of the present invention, the use may be in the preparation of medicaments for treating KIF18A-mediated conditions and / or diseases, such as in the preparation of KIF18A inhibitor medicaments.

[0060] According to embodiments of the present invention, the disease is, for example, cancer, including colorectal cancer, breast cancer, lung cancer, pancreatic cancer, prostate cancer, bladder cancer, head and neck cancer, cervical cancer, or ovarian cancer.

[0061] Beneficial effects

[0062] The compounds of the present invention have good KIF18A inhibitory activity. These compounds can regulate KIF18A protein alone or by forming a binding complex with microtubules, for the treatment of KIF18A-mediated conditions and / or diseases, such as tumor diseases, and for the preparation of medicaments for such conditions or diseases.

[0063] Terminology Definitions and Explanations

[0064] Unless otherwise stated, the definitions of groups and terms recorded in this application specification and claims, including definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, and definitions of specific compounds in the examples, can be arbitrarily combined and combined with each other. Such combinations and combinations of group definitions and compound structures should be understood as being within the scope of this application specification and / or claims.

[0065] Unless otherwise stated, the numerical ranges described in this specification and claims are equivalent to describing at least each specific integer value therein. For example, the numerical range "1-12" is equivalent to describing each integer value in the numerical range "1-12", namely 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.

[0066] Term "C" 1-12 "alkyl" should be understood to refer to straight-chain and branched alkyl groups having 1 to 12 carbon atoms, "C 1-8"Alkyl" refers to straight-chain and branched alkyl groups having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. 1-6 "Alkyl" means a straight-chain or branched alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or their isomers.

[0067] Term "C" 3-12 "Cycloalkyl" should be understood to refer to saturated monovalent monocyclic, bicyclic (such as fused ring, bridged ring, spiro ring) hydrocarbon rings or tricyclic alkanes, having 3 to 12 carbon atoms, preferably "C". 3-10 "Cycloalkyl", more preferably "C" 3-8 cycloalkyl. The term "C" 3-12 "Cycloalkyl" should be understood to refer to a saturated monovalent monocyclic, bicyclic (e.g., bridged, spirocyclic) hydrocarbon ring or tricyclic alkane having 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. The C... 3-12 Cycloalkyl groups can be monocyclic hydrocarbon groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or cyclodecyl; or bicyclic hydrocarbon groups, such as borneolyl, indolyl, hexahydroindolyl, tetrahydronaphthyl, decahydronaphthyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.1]heptenyl, 6,6-dimethylbicyclo[3.1.1]heptyl, 2,6,6-trimethylbicyclo[3.1.1]heptyl, bicyclo[2.2.2]octyl, 2,7-diazaspiro[3,5]nonyl, 2,6-diazaspiro[3,4]octyl; or tricyclic hydrocarbon groups, such as adamantyl.

[0068] Term "C" 3-12 "Cycloalkenyl" should be understood as referring to a monovalent monocyclic, bicyclic (such as fused ring, bridged ring, spiro ring) hydrocarbon ring or tricyclic alkene containing a carbon-carbon double bond, having 3 to 12 carbon atoms, preferably "C". 3-10 "Cycloalkenyl", more preferably "C" 3-8 "Cycloalkenyl". The term "C" 3-12 "Cycloalkenyl" should be understood as representing a monovalent monocyclic, bicyclic (such as bridged or spirocyclic) hydrocarbon ring, or tricyclic alkene containing a carbon-carbon double bond, having 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. The C... 3-12Cycloalkenyl groups can be monocyclic hydrocarbon groups, such as cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononenyl or cyclodecenyl, or bicyclic hydrocarbon groups such as spiro[2.5]oct-5-enyl, spiro[3.5]non-6-enyl, spiro[4.5]dec-7-enyl.

[0069] Unless otherwise defined, the term "3-14 membered heterocyclic group" refers to a saturated or unsaturated non-aromatic ring or ring system, for example, a 4-, 5-, 6-, or 7-membered monocyclic ring, a 7-, 8-, 9-, 10-, 11-, or 12-membered bicyclic ring (such as a fused ring, bridged ring, or spirocyclic ring), or a 10-, 11-, 12-, 13-, or 14-membered tricyclic ring system, and contains at least one, for example, 1, 2, 3, 4, 5, or more heteroatoms selected from O, S, and N, wherein N and S may optionally be oxidized to various oxidation states to form nitrides, -S(O)-, or -S(O)2- states. Preferably, the heterocyclic group may be selected from "3-10 membered heterocyclic groups". The term "3-10 membered heterocyclic group" means a saturated or unsaturated non-aromatic ring or ring system containing at least one heteroatom selected from O, S, and N. The heterocyclic group can be connected to the rest of the molecule via any one of the carbon atoms or a nitrogen atom (if present). The heterocyclic group can include fused or bridged rings and spirocyclic rings. Specifically, the heterocyclic group can include, but is not limited to: 4-membered rings, such as azirrobutyl or oxobutyl; 5-membered rings, such as tetrahydrofuranyl, dioxacyclopentenyl, pyrrolyl, imidazoalkyl, pyrazolealkyl, or pyrrololinyl; or 6-membered rings, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, piperazineyl, or trithiaalkyl; or 7-membered rings, such as diazacycloheptyl. Optionally, the heterocyclic group can be benzofused. The heterocyclic group can be bicyclic, such as, but not limited to, a 5,5-membered ring, like a hexahydrocyclopentano[c]pyrrole-2(1H)-yl ring, or a 5,6-membered bicyclic ring, like a hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl ring. The heterocyclic group can be partially unsaturated, meaning it can contain one or more double bonds, such as, but not limited to, dihydrofuranyl, dihydropyranyl, 2,5-dihydro-1H-pyrroleyl, 4H-[1,3,4]thiadiazinyl, 1,2,3,5-tetrahydrooxazolyl, or 4H-[1,4]thiazinyl, or it can be benzofused, such as, but not limited to, dihydroisoquinolinyl. When the 3-14-membered heterocyclic group is linked to other groups to form the compounds of the present invention, the carbon atom on the 3-14-membered heterocyclic group can be linked to other groups, or the heterocyclic atom on the 3-14-membered heterocyclic ring can be linked to other groups. For example, when the 3-14 membered heterocyclic group is selected from piperazine, the nitrogen atom on the piperazine group can be attached to other groups. Or when the 3-14 membered heterocyclic group is selected from piperidinium, the nitrogen atom on the piperidinium ring and the carbon atom at its para position can be attached to other groups.

[0070] The term "spirocycle" refers to a ring system in which two rings share a single ring atom.

[0071] The term "fused ring" refers to a ring system in which two rings share two cyclic atoms.

[0072] The term "bridged ring" refers to a ring system in which two rings share three or more cyclic atoms.

[0073] The term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0074] "Halogenation" refers to the replacement of a substance by one or more halogens.

[0075] Those skilled in the art will understand that the compounds shown in formula (I) can exist in the form of various pharmaceutically acceptable salts. If these compounds have a basic center, they can form acid addition salts; if these compounds have an acidic center, they can form base addition salts; if these compounds contain both an acidic center (e.g., a carboxyl group) and a basic center (e.g., an amino group), they can also form internal salts.

[0076] The compounds of the present invention may exist as solvates (such as hydrates), wherein the compounds of the present invention contain a polar solvent, particularly, for example, water, methanol, or ethanol, as a structural element of the lattice of the compound. The amount of the polar solvent, particularly water, may be stoichiometric or non-stoichiometric.

[0077] Depending on their molecular structure, the compounds of the present invention can be chiral, and therefore may exist in various enantiomeric forms. Thus, these compounds can exist in racemic or optically active forms. The compounds of the present invention encompass isomers of each chiral carbon in the R or S configuration, or mixtures thereof, and racemates. The compounds of the present invention or their intermediates can be isolated as enantiomeric compounds by chemical or physical methods known to those skilled in the art, or used in this form for synthesis. In the case of racemic amines, diastereomers are obtained from the mixture by reaction with an optically active resolving agent. Examples of suitable resolving agents are optically active acids, such as tartaric acid in both R and S forms, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, suitable N-protected amino acids (e.g., N-benzoylproline or N-benzenesulfonylproline), or various optically active camphorsulfonic acids. Chromatographic enantiomeric separation can also be advantageously performed using optically active resolving agents (e.g., dinitrobenzoylphenylglycine immobilized on silica gel, cellulose triacetate or other carbohydrate derivatives, or chiral derivatized isobutylene ester polymers). Suitable eluents for this purpose are aqueous or alcoholic solvent mixtures, such as hexane / isopropanol / acetonitrile.

[0078] The corresponding stable isomers can be separated using known methods, such as extraction, filtration, or column chromatography.

[0079] The term "patient" refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, or primates, with humans being the most preferred.

[0080] The term “therapeutic effective amount” refers to the amount of an active compound or drug that researchers, veterinarians, physicians, or other clinicians are searching for in tissues, systems, animals, individuals, or humans to elicit a biological or medical response. It includes one or more of the following: (1) prevention of disease: e.g., prevention of disease, disorder, or condition in individuals susceptible to disease, disorder, or symptom but not yet experiencing or exhibiting the pathology or symptoms of the disease; (2) suppression of disease: e.g., suppression of disease, disorder, or symptom in individuals experiencing or exhibiting the pathology or symptoms of the disease, disorder, or symptom (i.e., prevention of further development of the pathology and / or symptoms); (3) relief of disease: e.g., relief of disease, disorder, or symptom in individuals experiencing or exhibiting the pathology or symptoms of the disease, disorder, or symptom (i.e., reversal of the pathology and / or symptoms). Detailed Implementation

[0081] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0082] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0083] Example 1

[0084] N-[2-(4,4-difluoropiperidin-1-yl)-6-methylpyrimidin-4-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (Compound 1)

[0085]

[0086]

[0087] The first step was the synthesis of methyl 4-bromo-2-(spiro[2.5]oct-5-en-6-yl)benzoate (compounds 1-3):

[0088] Under nitrogen protection, at room temperature, the [1,1-bis(diphenylphosphine)ferrocene]palladium dichloromethane complex (167.25 mg, 0.205 mmol, 0.1 eq) was added to a solution of methyl 4-bromo-2-iodobenzoate (compound 1-1, 700 mg, 2.053 mmol, 1 eq), sodium carbonate (326.41 mg, 3.079 mmol, 1.5 eq), and 1,4,5,5-tetramethyl-2-{spiro[2.5]oct-5-en-6-yl}-1,3,2-dioxabopentane (compound 1-2, 480.74 mg, 2.053 mmol, 1 eq) in 1,4-dioxane (8 mL) and water (1.6 mL). The reaction mixture was heated to 80 °C and stirred for 1 hour. The reaction solution was cooled to room temperature, filtered, and the filter cake was washed with ethyl acetate (3 × 5 mL). The filtrate was diluted with water (30 mL), and the mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined and backwashed with saturated brine (2 × 40 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase column chromatography under the following conditions: column size, C18; mobile phase: acetonitrile and water, gradient 10% to 70%; elution time: 10 min; yielding methyl 4-bromo-2-(spiro[2.5]oct-5-en-6-yl)benzoate (compounds 1-3, 320 mg, 48.52%).

[0089] MS(ESI, m / z): 320.95 [M+H] + RT(min):1.492

[0090] The second step is the synthesis of 4-bromo-2-{spiro[2,5]oct-5-en-6-yl}benzoic acid (compounds 1-4):

[0091] At room temperature, lithium hydroxide (111.84 mg, 4.670 mmol, 5 eq) was added to a solution of methyl 4-bromo-2-(spiro[2.5]oct-5-en-6-yl)benzoate (compounds 1-3, 300 mg, 0.934 mmol, 1 eq) in methanol (2 mL), tetrahydrofuran (2 mL), and water (2 mL). The reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with water (15 mL), and the mixture was extracted with ethyl acetate (1 × 10 mL). The aqueous phase was collected and acidified to pH 4–5 with dilute hydrochloric acid at 1 mol / L. The aqueous phase was extracted with ethyl acetate (3 × 15 mL). The organic phase was collected and backwashed with saturated brine (2 × 20 mL), and dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure to give 4-bromo-2-{spiro[2.5]oct-5-en-6-yl}benzoic acid (compounds 1-4, 250 mg, 87.14%).

[0092] MS(ESI,m / z):306.90[M+H]+,RT(min):1.323

[0093] Step 3: Synthesis of 4-bromo-N-(2-(4,4-difluoropiperidin-1-yl)-6-methylpyrimidin-4-yl)-2-(spiro[2.5]oct-5-en-6-yl)benzamide (compounds 1-6)

[0094] Under nitrogen protection, at room temperature, 1-methyl-1H-imidazolium (106.91 mg, 1.304 mmol, 4 eq) was added to a solution of 4-bromo-2-{spiro[2.5]oct-5-en-6-yl}benzoic acid (compounds 1-4, 100 mg, 0.326 mmol, 1 eq), 2-(4,4-difluoropiperidin-1-yl)-6-methylpyrimidin-4-amine (compounds 1-5, 89.16 mg, 0.391 mmol, 1.2 eq), and tetramethylchloroformamidine hexafluorophosphate (182.68 mg, 0.652 mmol, 2 eq) in N,N-dimethylformamide (3 mL). The reaction mixture was heated to 80 °C and stirred overnight. The reaction mixture was cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, backwashed with water (3 × 20 mL), and dried over anhydrous sodium sulfate. Filtration, concentration of the filtrate under reduced pressure, and purification of the residue by preparative chromatography (ethyl acetate: petroleum ether = 1:5) to give 4-bromo-N-(2-(4,4-difluoropiperidin-1-yl)-6-methylpyrimidin-4-yl)-2-(spiro[2.5]oct-5-en-6-yl)benzamide (compound 1-6, 40 mg, 21.77%).

[0095] MS(ESI, m / z): 517.30 [M+H] + RT(min): 1.477

[0096] Step 4: Synthesis of N-[2-(4,4-difluoropiperidin-1-yl)-6-methylpyrimidin-4-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2,5]oct-5-en-6-yl}benzamide (compound 1)

[0097] Under nitrogen protection, at room temperature, palladium acetate (1.74 mg, 0.008 mmol, 0.1 eq) and 2-di-tert-butylphospho-2',4',6'-triisopropylbiphenyl (6.57 mg, 0.015 mmol, 0.2 eq) were added to a solution of 4-bromo-N-[2-(4,4-difluoropiperidin-1-yl)-6-methylpyrimidin-4-yl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (compounds 1-6, 40 mg, 0.077 mmol, 1 eq), 2-hydroxyethanesulfonamide (compounds 1-7, 19.35 mg, 0.154 mmol, 2 eq) and sodium tert-butoxide (22.29 mg, 0.231 mmol, 3 eq) in N,N-dimethylformamide (2 mL). The reaction mixture was heated to 140 °C and stirred for 1 hour. The reaction solution was cooled to room temperature, filtered, and the filter cake was washed with ethyl acetate (3 × 3 mL). The filtrate was diluted with ethyl acetate (15 mL), the mixture was washed with water (3 × 10 mL), and the organic phase was dried over anhydrous sodium sulfate. The mixture was filtered, concentrated under reduced pressure, and the crude product was purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: column specifications: Kinetex EVO C18 Column, 30*150, 5 μm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60 mL / min; elution gradient: 20% B to 75% B; elution time: 8 min, yielding N-[2-(4,4-difluoropiperidin-1-yl)-6-methylpyrimidin-4-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (compound 1, 15.5 mg, 32.18%).

[0098] MS(ESI, m / z): 561.90 [M+H] + RT(min):1.766

[0099] 1 H NMR (400MHz, DMSO-d6) δ10.08(s,2H),7.49(d,J=8.4Hz,1H),7.21-7.14(m,2H),7.05(d,J=2.3Hz,1H),5.73(s,1H),4.95(s,1H),3 .83(s,4H),3.76(t,J=6.6Hz,2H),3.29(d,J=6.7Hz,2H),2.29(s,5H),2.03-1.84(m,6H),1.41(t,J=6.0Hz,2H),0.31-0.18(m,4H).

[0100] Example 2

[0101] N-[2-(4,4-difluoropiperidin-1-yl)-6-methylpyrimidin-4-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2,5]octane-6-yl}benzamide (compound 2)

[0102]

[0103] Platinum dioxide (0.40 mg, 0.002 mmol, 0.10 eq) was added to a 1 mL solution of N-[2-(4,4-difluoropiperidin-1-yl)-6-methylpyrimidin-4-yl]-4-(2-hydroxyethanesulfonylamino)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (10 mg, 0.018 mmol, 1 eq) in ethyl acetate at room temperature and under a hydrogen atmosphere. The pressure was increased to 4 MPa, and the mixture was stirred at room temperature for 5 days. The reaction solution was filtered, and the filter cake was washed with ethyl acetate (3 × 3 mL). The filtrate was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (HPLC) under the following conditions (Column specifications: XBridge Shield RP18 OBD Column, 30*150mm, 5μm, n; Mobile phase A: water (10mmol / L) ammonium bicarbonate; Mobile phase B: acetonitrile; Flow rate: 60mL / min; Elution gradient: 38%B to 70%B in 10min, 70%B; Detection wavelength: UV 220nm; Retention time (min): 8.82), yielding N-[2-(4,4-difluoropiperidin-1-yl)-6-methylpyrimidin-4-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]octane-6-yl}benzamide (0.84mg, 8.24%).

[0104] MS(ES,m / z):564.40[M+H] + RT(min):1.816

[0105] 1 H NMR: (400MHz, CDCl3) δ7.86(s,1H),7.47-7.43(m,2H),7.28(s,1H),7.20-7.17(m ,1H),6.78(s,1H),4.14(t,J=5.2Hz,2H),3.93(s,4H),3.37-3.26(m,2H),3.05(t ,J=12.0Hz,1H),2.41(s,3H),2.01-1.94(m,4H),1.93-1.79(m,4H),1.64-1.62(m ,2H),1.26(s,1H),0.92(d,J=13.2Hz,2H),0.33-0.30(m,2H),0.28-0.24(m,2H).

[0106] Example 3

[0107] N-[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (compound 3)

[0108]

[0109] Step 1: Synthesis of 2-(4,4-difluoropiperidin-1-yl)-6-nitropyridine (compound 3-3):

[0110] Under nitrogen protection, at room temperature, 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (392.76 mg, 0.63 mmol, 0.1 eq) and palladium acetate (283.22 mg, 1.26 mmol, 0.2 eq) were added to a toluene (10 mL) solution of 2-chloro-6-nitropyridine (1 g, 6.31 mmol, 1 eq), 4,4-difluoropiperidine hydrochloride (1.15 g, 9.46 mmol, 1.5 eq), and cesium carbonate (6.17 g, 18.92 mmol, 3 eq). The reaction mixture was heated to 100 °C and stirred for 16 hours. The reaction mixture was cooled to room temperature, diluted with water (50 mL), and extracted with ethyl acetate (3 × 50 mL). The organic phases were combined, backwashed with saturated brine (1 × 60 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in petroleum ether / ethyl acetate (10:1) to give 2-(4,4-difluoropiperidin-1-yl)-6-nitropyridine (980 mg, 63.88%).

[0111] MS(ESI,m / z):244.00[M+H] + RT(min):1.298

[0112] The second step is the synthesis of 6-(4,4-difluoropiperidin-1-yl)pyridine-2-amine (compounds 3-4):

[0113] Under nitrogen protection, iron powder (1.01 g, 18.09 mmol, 5 eq) and ammonium chloride (967.69 mg, 18.09 mmol, 5 eq) were added to a mixed solution of 2-(4,4-difluoropiperidin-1-yl)-6-nitropyridine (880 mg, 3.62 mmol, 1 eq) in 10 mL of ethanol and 1 mL of water at room temperature. The reaction solution was heated to 60 °C and stirred for one hour. The reaction was stopped by liquid chromatography-mass monitoring. The solution was cooled to room temperature and diluted with water (30 mL). The mixture was filtered, and the filter cake was washed with ethyl acetate (3 × 20 mL). The filtrate was extracted with ethyl acetate (3 × 50 mL). The organic phases were combined, backwashed with saturated brine (1 × 60 mL), and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give 6-(4,4-difluoropiperidin-1-yl)pyridine-2-amine (600 mg, 77.77%).

[0114] MS(ESI, m / z): 214.05 [M+H] + RT(min): 0.740

[0115] The third step is the synthesis of 4-bromo-N-[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (compound 3-5):

[0116] Under nitrogen protection, at room temperature, N,N,N',N'-tetramethylchloroformamide hexafluorophosphate (236.30 mg, 0.41 mmol, 2 eq), N-methylimidazolium (172.87 mg, 1.03 mmol, 5 eq), and 4-bromo-2-{spiro[2.5]oct-5-en-6-yl}benzoic acid (253.54 mg, 0.41 mmol, 2 eq) were added to a solution of 6-(4,4-difluoropiperidin-1-yl)pyridine-2-amine (90 mg, 0.21 mmol, 1 eq) in N,N-dimethylformamide (1 mL). The reaction mixture was heated to 80 °C and stirred for one hour. The reaction mixture was cooled to room temperature and diluted with water (10 mL). The reaction mixture was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, backwashed with saturated brine (3 × 5 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in petroleum ether / ethyl acetate (5:1) to give 4-bromo-N-[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (90 mg, 42.44%).

[0117] MS(ESI, m / z): 501.95 [M+H] + RT(min):1.497

[0118] The fourth step is the synthesis of N-[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2,5]oct-5-en-6-yl}benzamide (compound 3):

[0119] Under nitrogen protection, at room temperature, a solution of 4-bromo-N-[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]-2-{spiro[2,5]oct-5-en-6-yl}benzamide (75 mg, 0.15 mmol, 1 eq) in 1,4-dioxane (2 mL) was prepared, and then dicyclohexyl(3-isopropoxy-2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphonane (15.97 mg, 0.03 mmol, 0.2 eq) and (methanesulfonic acid {dicyclohexyl(3-isopropoxy-2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphonane}(2'-methylamino-1,1'-biphenyl) were added. 2-hydroxyethyl sulfonamide (2-hydroxyethyl)palladium(II) (13.71 mg, 0.015 mmol, 0.1 eq), cesium carbonate (145.92 mg, 0.45 mmol, 3 eq), and 2-hydroxyethyl sulfonamide (37.36 mg, 0.30 mmol, 2 eq) were added. The reaction mixture was heated to 120 °C and stirred for 1 hour. The reaction mixture was then cooled to room temperature. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, backwashed with saturated brine (1 × 20 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) under the following conditions (Kinetex column). EVO prep C18, 30*150, 5μm; mobile phase A: water (10mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 35%B to 65%B in 8min, 65%B; wavelength: 220nm; RT (min): 7.32), yielding N-[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (22.9mg, 28.01%).

[0120] MS(ESI,m / z):547.00[M+H] + RT(min):1.706

[0121] 1H NMR(400MHz,DMSO-d6)δ9.98(s,1H),9.68(s,1H),7.61–7.50(m,2H),7.41( s,1H),7.21–7.14(m,1H),7.05(d,J=2.2Hz,1H),6.66(d,J=8.3Hz,1H),5.78 (s,1H),4.95(s,1H),3.76(t,J=6.6Hz,2H),3.66(s,4H),3.29(d,J=6.7Hz,2 H),2.27(s,2H),1.96(d,J=15.8Hz,6H),1.41(s,2H),0.24(d,J=2.6Hz,4H).

[0122] Example 4

[0123] N-[3-(4,4-difluoropiperidin-1-yl)phenyl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (compound 4)

[0124]

[0125] Step 1: Synthesis of 4,4-difluoro-1-(3-nitrophenyl)piperidine (compound 4-2):

[0126] Under nitrogen protection, at room temperature, 4,4-difluoropiperidine hydrochloride (2.31 g, 19.041 mmol, 1.5 eq), cesium carbonate (12.41 g, 38.082 mmol, 3 eq), 1,1-binaphthyl-2,2-bis(diphenylphosphine) (1580.93 mg, 2.539 mmol, 0.2 eq), and palladium acetate (285.00 mg, 1.269 mmol, 0.1 eq) were added to a 20 mL solution of 3-nitrochlorobenzene (2 g, 12.694 mmol, 1 eq) in toluene. The reaction mixture was heated to 90 °C and stirred for 1 hour. The reaction mixture was cooled to room temperature and quenched with water (80 mL). The reaction mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic phases were backwashed with saturated brine (1 × 80 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in petroleum ether / ethyl acetate (10:1) to give 4,4-difluoro-1-(3-nitrophenyl)piperidine (2.92 g, 94.96%).

[0127] MS(ESI,m / z):243.00[M+H] + RT(min):1.242

[0128] The second step is the synthesis of 3-(4,4-difluoropiperidin-1-yl)aniline (compound 4-3):

[0129] Under nitrogen protection, iron powder (576.37 mg, 10.321 mmol, 5.00 eq) and ammonium chloride (552.07 mg, 10.32 mmol, 5 eq) were added to a solution of 4,4-difluoro-1-(3-nitrophenyl)piperidine (500 mg, 2.06 mmol, 1 eq) in ethanol (5 mL) and water (1 mL) at room temperature. The reaction mixture was heated to 60 °C and stirred for 1 hour. The reaction mixture was cooled to room temperature, and the reaction mixture was extracted with ethyl acetate (3 × 40 mL). The organic phases were combined, backwashed with saturated brine (1 × 40 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in petroleum ether / ethyl acetate (10:1) to give 3-(4,4-difluoropiperidin-1-yl)aniline (400 mg, 91.32%).

[0130] MS(ESI, m / z): 213.30 [M+H] + RT(min): 0.590

[0131] The third step is the synthesis of 4-bromo-N-[3-(4,4-difluoropiperidin-1-yl)phenyl]-2-{spiro[2,5]oct-5-en-6-yl}benzamide (compound 4-4):

[0132] Under nitrogen protection, 4-bromo-2-{spiro[2.5]oct-5-en-6-yl}benzoic acid (17.37 mg, 0.056 mmol, 0.6 eq), tetramethylchlorourea hexafluorophosphate (2 mg, 0.007 mmol, 0.08 eq), and N-methylimidazole (5 mg, 0.061 mmol, 0.65 eq) were added to a solution of 3-(4,4-difluoropiperidin-1-yl)aniline (20 mg, 0.094 mmol, 1 eq) in N,N-dimethylformamide (1 mL) at room temperature. The reaction mixture was heated to 80 °C and stirred for 12 hours. The reaction mixture was cooled to room temperature and diluted with water (10 mL). The reaction mixture was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, backwashed with saturated saline solution (1 × 10 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in petroleum ether / ethyl acetate (10:1) to give 4-bromo-N-[3-(4,4-difluoropiperidin-1-yl)phenyl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (25 mg, 53.19%).

[0133] MS(ESI,m / z):501.00[M+H] + RT(min):1.281

[0134] The fourth step is the synthesis of N-[3-(4,4-difluoropiperidin-1-yl)phenyl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2,5]oct-5-en-6-yl}benzamide (compound 4):

[0135] Under nitrogen protection, at room temperature, 2-hydroxyethanesulfonamide (7.50 mg, 0.06 mmol, 1.2 eq), palladium acetate (1.1 mg, 0.005 mmol, 0.1 eq), 2-di-tert-butylphospho-2',4',6'-triisopropylbiphenyl (4.25 mg, 0.01 mmol, 0.2 eq), and sodium tert-butoxide (14.4 mg, 0.15 mmol, 3 eq) were added to a solution of 4-bromo-N-[3-(4,4-difluoropiperidin-1-yl)phenyl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (25 mg, 0.05 mmol, 1 eq) in N,N-dimethylformamide (0.5 mL), at room temperature. The reaction mixture was heated to 140 °C and stirred for 1 hour. The reaction mixture was cooled to room temperature and diluted with water (10 mL). The reaction mixture was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, backwashed with saturated saline solution (1×10 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (HPLC) under the following conditions (column specifications: Kinetex EVO C18 column, 30*150, 5 μm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60 mL / min; elution gradient: 35% B to 60% B in 8 min, 60% B; detection wavelength: 220 nm; RT1 (min): 7.48). N-[3-(4,4-difluoropiperidin-1-yl)phenyl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (1.3 mg, 23.73%) was obtained.

[0136] MS(ESI, m / z): 545.90 [M+H] + RT(min): 1.603

[0137] 1H NMR (400MHz, DMSO-d6) δ9.78 (s, 1H), 7.40 (d, J = 8.2Hz, 1H), 7.35 (s, 1H), 7.1 4(d,J=8.2Hz,3H),7.06(s,1H),6.72(d,J=7.6Hz,1H),5.71(s,1H),4.96(s, 1H),3.75(t,J=6.7Hz,2H),3.29(s,6H),2.31(s,2H),2.12–2.00(m,4H),1.9 2(s,2H),1.37(t,J=6.0Hz,2H),0.22(d,J=3.0Hz,2H),0.19(d,J=3.0Hz,2H).

[0138] Example 5

[0139] N-[3-(4,4-difluoropiperidin-1-yl)-5-methylphenyl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (compound 5)

[0140]

[0141] Step 1: Synthesis of 4,4-difluoro-1-(3-methyl-5-nitrophenyl)piperidine (compound 5-2):

[0142] Under nitrogen protection, at room temperature, tris(dibenzylacetone)dipalladium (0.53 g, 0.583 mmol, 0.1 eq), and 4,5-bis(diphenylphosphonic acid)-9,9-dimethylethane (0.67 g, 1.166 mmol, 0.2 eq) were added to a solution of 1,4-dioxane (10 mL) containing 1-chloro-3-methyl-5-nitrobenzene (1.0 g, 5.828 mmol, 1.0 eq), 4,4-difluoropiperidine hydrochloride (1.10 g, 6.994 mmol, 1.2 eq), and sodium tert-butoxide (1.68 g, 17.484 mmol, 3.0 eq). The reaction mixture was heated to 110 °C and stirred for 16 hours. The reaction mixture was cooled to room temperature and diluted with water (40 mL). The reaction mixture was extracted with ethyl acetate (3 × 60 mL). The organic phases were combined, backwashed with saturated brine (1 × 80 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in petroleum ether / ethyl acetate (9:1) to give 4,4-difluoro-1-(3-methyl-5-nitrophenyl)piperidine (1.1 g, 73.65%).

[0143] MS(ESI, m / z): 257.30 [M+H] + ,RT(min):1.205.

[0144] The second step is the synthesis of 3-(4,4-difluoropiperidin-1-yl)-5-methylaniline (compound 5-3):

[0145] Under nitrogen protection, iron powder (653.79 mg, 11.706 mmol, 6.0 eq) and ammonium chloride (313.11 mg, 5.853 mmol, 3.0 eq) were added to a solution of 4,4-difluoro-1-(3-methyl-5-nitrophenyl)piperidine (500 mg, 1.951 mmol, 1.0 eq) in ethanol (5 mL) and water (1 mL) at room temperature. The reaction mixture was heated to 60 °C and stirred for 1 hour. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with ethyl acetate (3 × 10 mL). The filtrate was collected and diluted with water (40 mL), and extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, backwashed with saturated brine (1 × 40 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography in petroleum ether / ethyl acetate (9:1) to give 3-(4,4-difluoropiperidin-1-yl)-5-methylaniline (410 mg, 92.87%).

[0146] MS(ESI,m / z):227.00[M+H] + ,RT(min):0.681.

[0147] The third step is the synthesis of 4-bromo-N-[3-(4,4-difluoropiperidin-1-yl)-5-methylphenyl]-2-{spiro[2,5]oct-5-en-6-yl}benzamide (compound 5-4):

[0148] Under nitrogen protection, at room temperature, tetramethylchloroformamide salt of hexafluorophosphate (36.54 mg, 0.130 mmol, 2.0 eq) and 1-methylimidazole (53.46 mg, 0.650 mmol, 10.0 eq) were added to a solution of 4-bromo-2-{spiro[2.5]oct-5-en-6-yl}benzoic acid (20 mg, 0.065 mmol, 1.0 eq), 3-(4,4-difluoropiperidin-1-yl)-5-methylaniline (17.68 mg, 0.078 mmol, 1.2 eq) in N,N-dimethylformamide (1 mL). The reaction mixture was heated to 80 °C and stirred for 2 hours. The reaction mixture was cooled to room temperature and diluted with water (10 mL). The mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, backwashed with saturated brine (1 × 20 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was subjected to silica gel column chromatography in petroleum ether / ethyl acetate (9:1) to give 4-bromo-N-[3-(4,4-difluoropiperidin-1-yl)-5-methylphenyl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (30 mg, 89.40%).

[0149] MS(ESI, m / z): 514.95 [M+H] + ,RT(min):1.319.

[0150] The fourth step is the synthesis of N-[3-(4,4-difluoropiperidin-1-yl)-5-methylphenyl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2,5]oct-5-en-6-yl}benzamide (compound 5):

[0151] Under nitrogen protection, at room temperature, palladium acetate (1.09 mg, 0.005 mmol, 0.1 eq) and 2-di-tert-butylphospho-2',4',6'-triisopropylbiphenyl (4.12 mg, 0.010 mmol, 0.2 eq) were added to a solution of 4-bromo-N-[3-(4,4-difluoropiperidin-1-yl)-5-methylphenyl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (25 mg, 0.049 mmol, 1.0 eq), 2-hydroxyethanesulfonamide (6.07 mg, 0.049 mmol, 1.0 eq), and sodium tert-butoxide (13.98 mg, 0.147 mmol, 3.0 eq) in N,N-dimethylformamide (1 mL). The reaction mixture was monitored to complete. The reaction mixture was cooled to room temperature and diluted with water (10 mL). The mixture was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, backwashed with saturated brine (1×10 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) under the following conditions (column: XBridgePrep OBD C18 column, 30*150 mm, 5 μm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 42% B to 65% B, 65% B over 8 min; wavelength: 220 nm; retention time (min): 7.62) to obtain N-[3-(4,4-difluoropiperidin-1-yl)-5-methylphenyl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (3.12 mg, 11.49%).

[0152] MS(ESI, m / z): 559.90 [M+H] + , RT(min):1.715.

[0153] 1 H NMR: (400MHz, DMSO-d6) δ9.73 (s, 2H), 7.41 (d, J = 8.3 Hz, 1H), 7.17 (dd, J = 8.3, 2.2H z,1H),7.13(s,1H),7.09(d,J=2.2Hz,1H),6.98(s,1H),6.56(s,1H),5.71(s,1H),4 .97(s,1H),3.76(t,J=6.6Hz,2H),3.29–3.26(m,6H),2.33(s,2H),2.22(s,3H),2.0 3(s,4H),1.92(s,2H),1.38(t,J=6.0Hz,2H),0.24–0.23(m,2H),0.19–0.17(m,2H).

[0154] Example 6

[0155] N-(6-(4,4-difluoropiperidin-1-yl)pyrazin-2-yl)-4-((2-hydroxyethyl)sulfonamide)-2-(spiro[2.5]oct-5-en-6-yl)benzamide (compound 6)

[0156]

[0157]

[0158] The first step is the synthesis of 6-(4,4-difluoropiperidin-1-yl)pyrazin-2-amine (compound 6-2):

[0159] Under nitrogen protection, potassium carbonate (480 mg, 3.468 mmol, 3 eq) and 4,4-difluoropiperidine hydrochloride (272 mg, 1.734 mmol, 1.5 eq) were added to a solution of 6-bromopyrazin-2-amine (200 mg, 1.156 mmol, 1 eq) in N,N-dimethylacetamide (2 mL) at room temperature. The reaction mixture was heated to 120 °C and stirred for 16 hours. The reaction mixture was cooled to room temperature and diluted with water (20 mL). The mixture was extracted with ethyl acetate (3 × 30 mL), the organic phases were combined, backwashed with saturated brine (2 × 30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in petroleum ether / ethyl acetate (1:1) to give 6-(4,4-difluoropiperidine-1-yl)pyrazin-2-amine (180 mg, 72.82%).

[0160] MS(ESI, m / z): 215.35 [M+H] + ,RT(min):0.649.

[0161] The second step is the synthesis of 4-bromo-N-(6-(4,4-difluoropiperidin-1-yl)pyrazin-2-yl)-2-(spiro[2.5]oct-5-en-6-yl)benzamide (compound 6-3):

[0162] Under nitrogen protection, at room temperature, N,N,N',N'-tetramethylchloromethane hexafluorophosphate (182.68 mg, 0.652 mmol, 4 eq) and N-methylimidazolium (133.64 mg, 1.630 mmol, 10 eq) were added to a solution of 6-(4,4-difluoropiperidin-1-yl)pyrazin-2-amine (41.84 mg, 0.196 mmol, 1.2 eq), 4-bromo-2-(spiro[2.5]oct-5-en-6-yl)benzoic acid (50 mg, 0.163 mmol, 1.00 eq) in dichloromethane (1 mL). The reaction mixture was heated to 80 °C and stirred for 5 hours. The reaction mixture was cooled to room temperature, diluted with water (10 mL), extracted with ethyl acetate (2 × 30 mL), the organic phases were combined, backwashed with saturated brine (1 × 30 mL), and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in petroleum ether / ethyl acetate (1:1) to give 4-bromo-N-(6-(4,4-difluoropiperidin-1-yl)pyrazin-2-yl)-2-(spiro[2.5]oct-5-en-6-yl)benzamide (40 mg, 48.82%).

[0163] MS(ESI, m / z): 503.30 [M+H] + ,RT(min):1.395.

[0164] The third step was the synthesis of N-(6-(4,4-difluoropiperidin-1-yl)pyrazin-2-yl)-4-((2-hydroxyethyl)sulfonamide)-2-(spiro[2.5]oct-5-en-6-yl)benzamide (compound 6):

[0165] Under nitrogen protection, at room temperature, 4-bromo-N-(6-(4,4-difluoropiperidin-1-yl)pyrazin-2-yl)-2-(spiro[2.5]oct-5-en-6-yl)benzamide (40 mg, 0.079 mmol, 1 eq), cesium carbonate (77.67 mg, 0.237 mmol, 3 eq) and 2-hydroxyethane-1-sulfonamide (11.93 mg, 0.095 mmol, 1.2 eq) in N,N-dimethylformamide (1 mL) was added to a solution of methanesulfonic acid {bicyclohexyl(3-isopropoxy-2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphonane}(2'-methylamino-1,1'-biphenyl-2-yl)palladium(II) (7.30 mg, 0.008 mmol, 0.1 eq), dicyclohexyl(2-yl)phosphonate (2'-methylamino-1,1'-biphenyl-2-yl)palladium(II) (7.30 mg, 0.008 mmol, 0.1 eq) in N,N-dimethylformamide (1 mL). Cyclohexyl(3-isopropoxy-2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphonane (8.50 mg, 0.016 mmol, 0.2 eq). The reaction mixture was heated to 100 °C and stirred for 1 hour. The reaction mixture was cooled to room temperature, diluted with water (10 mL), extracted with ethyl acetate (3 × 30 mL), the organic phases were combined, backwashed with saturated brine (2 × 30 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative high performance liquid chromatography to give N-(6-(4,4-difluoropiperidin-1-yl)pyrazin-2-yl)-4-((2-hydroxyethyl)sulfonamide)-2-(spiro[2.5]oct-5-en-6-yl)benzamide (9.67 mg, 21.93%).

[0166] MS(ESI, m / z): 548.35 [M+H] + ,RT(min):1.369.

[0167] 1 H NMR(400MHz,DMSO-d6)δ10.08(s,1H),9.98(s,1H),8.58(s,1H),8.13(s,1H), 7.51(d,J=8.3Hz,1H),7.18(dd,J=8.4,2.2Hz,1H),7.06(d,J=2.2Hz,1H),5.73 (s,1H),4.95(s,1H),3.76(t,J=6.6Hz,2H),3.71(s,4H),3.31(s,2H),2.29(s ,2H),1.99(s,4H),1.95(s,2H),1.40(t,J=5.9Hz,2H),0.23(d,J=12.1Hz,4H).

[0168] Example 7

[0169] N-[2-(4,4-difluoropiperidin-1-yl)pyridin-4-yl]-4-((2-hydroxyethyl)sulfonamide)-2-(spiro[2.5]oct-5-en-6-ylbenzamide (compound 8)

[0170]

[0171] Step 1: Synthesis of 2-(4,4-difluoropiperidin-1-yl)pyridine-4-amine (compound 8-2):

[0172] Under nitrogen protection, N,N-diisopropylethylamine (1508.02 mg, 11.667 mmol, 3 eq) and 4,4-difluoropiperidine hydrochloride (706.66 mg, 5.833 mmol, 1.5 eq) were added to a solution of 2-chloropyridine-4-amine (500 mg, 3.889 mmol, 1 eq) in N-methylpyrrolidone (5 mL) at room temperature. The reaction mixture was microwaved at 160 °C for 2 hours. The reaction solution was cooled to room temperature and quenched with water (5 mL). The reaction mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic phases were backwashed with saturated brine (3 × 30 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in petroleum ether / ethyl acetate (5:1) to give 2-(4,4-difluoropiperidin-1-yl)pyridine-4-amine (300 mg, 36.17%).

[0173] MS:(ESI,m / z):214.05[M+H] + RT(min): 0.820

[0174] The second step is the synthesis of 4-bromo-N-(2-(4,4-difluoropiperidin-1-yl)pyridin-4-yl)-2-spiro[2.5]oct-5-en-6-yl)benzamide (compound 8-3):

[0175] Under nitrogen protection, at room temperature, N,N,N',N'-tetramethylchloromethane hexafluorophosphate (526.34 mg, 1.876 mmol, 4 eq), 4-bromo-2-(spiro[2.5]oct-5-en-6-ylbenzoic acid (172.87 mg, 0.563 mmol, 1.2 eq), and N-methylimidazolium (385.05 mg, 4.690 mmol, 10 eq) were added to a solution of 2-(4,4-difluoropiperidin-1-yl)pyridine-4-amine (100 mg, 0.469 mmol, 1 eq) in dichloromethane (2 mL). The reaction solution... The mixture was heated to 80°C and stirred for 2 hours. The reaction mixture was cooled to room temperature and quenched with water (10 mL). The reaction mixture was extracted with dichloromethane (3 × 20 mL). The organic phases were combined, backwashed with saturated brine (1 × 20 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in petroleum ether / ethyl acetate (10:1) to give 4-bromo-N-(2-(4,4-difluoropiperidin-1-yl)pyridin-4-yl)-2-spiro[2.5]oct-5-en-6-yl)benzamide (78 mg, 30.43%).

[0176] MS:(ESI,m / z):502.35[M+H] + RT(min):1.368

[0177] The third step is the synthesis of N-[2-(4,4-difluoropiperidin-1-yl)pyridin-4-yl]-4-((2-hydroxyethyl)sulfonamide)-2-(spiro[2.5]oct-5-en-6-ylbenzamide (compound 8):

[0178] Under nitrogen protection, at room temperature, dicyclohexyl(3-isopropoxy-2′,4′,6′-triisopropyl-[1,1′-biphenyl]-2-yl)phosphine was added to a 1,4-dioxane (1 mL) solution of 4-bromo-N-(2-(4,4-difluoropiperidin-1-yl)pyridin-4-yl)-2-spiro[2,5]oct-5-en-6-yl)benzamide (68 mg, 0.124 mmol, 1 eq), 2-hydroxyethane-1-sulfonamide (23.35 mg, 0.186 mmol, 1.5 eq), and cesium carbonate (121.59 mg, 0.372 mmol, 3 eq). (6.65 mg, 0.012 mmol, 0.1 eq), (methanesulfonic acid {bicyclohexyl(3-isopropoxy-2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphonane}(2'-methylamino-1,1'-biphenyl-2-yl)palladium(II) (22.85 mg, 0.025 mmol, 0.2 eq). The reaction mixture was heated to 100 °C and stirred for 1 hour. The reaction mixture was cooled to room temperature and quenched with water (3 mL). The reaction mixture was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, backwashed with saturated brine (1 × 20 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography under the following conditions: column specification: XBridge Prep OBD C18, 30*150mm, 5μm; Mobile phase A: water (10mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60ml / min; elution gradient: 46%B to 73%B in 8min; detection wavelength: 254nm; retention time (min): 6.72. N-[2-(4,4-difluoropiperidin-1-yl)pyridin-4-yl]-4-((2-hydroxyethyl)sulfonamide)-2-(spiro[2.5]oct-5-en-6-ylbenzamide (11.18mg, 16.44%) was obtained.

[0179] MS:(ESI,m / z):547.00[M+H] + RT(min):1.611

[0180] 1 H NMR: (400MHz, DMSO-d6) δ10.15(s,1H),9.99(s,1H),8.01(d,J=5.6Hz,1H),7.44(d,J=8.4Hz,1H), 7.25(s,1H),7.18(dd,J=8.3,2.2Hz,1H),7.10(d,J=2.1Hz,1H),6.94(d,J=5.4Hz,1H),5.69(s,1H) ,4.98(s,1H),3.76(s,2H),3.62(t,J=5.8Hz,4H),3.28(d,J=6.6Hz,2H),2.32(s,2H),1.98(dt,J= 14.6,8.4Hz,4H),1.90(s,2H),1.37(t,J=6.0Hz,2H),0.22(d,J=3.6Hz,2H),0.17(d,J=3.8Hz,2H).

[0181] Example 8

[0182] N-(2-(3,3-difluoroazacyclobutane-1-yl)-6-methylpyrimidin-4-yl)-4-((2-hydroxyethyl)sulfonamide)-2-(spiro[2.5]oct-5-en-6-yl)benzamide (compound 29)

[0183]

[0184]

[0185] The first step is the synthesis of 2-(3,3-difluoroazacyclobutan-1-yl)-6-methylpyrimidin-4-amine (compound 29-3):

[0186] Under nitrogen protection, 2-chloro-6-methylpyrimidin-4-amine (1.00 g, 6.965 mmol, 1 eq), N,N-diisopropylethylamine (2.70 g, 20.895 mmol, 3 eq), and 3,3-difluoroazacyclobutane hydrochloride (1.35 g, 10.447 mmol, 1.5 eq) in N-methylpyrrolidone (20 mL) were stirred at 140 °C for 16 h. The reaction mixture was cooled to room temperature and diluted with water (50 mL). The mixture was extracted with ethyl acetate (3 × 50 mL), the organic phases were combined, backwashed with water (3 × 50 mL) and saturated brine (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase column chromatography under the following conditions: C18 column, mobile phase: water and acetonitrile, 10% to 30% gradient for 10 min, detector: UV 254 nm, yielding 2-(3,3-difluorozacyclobutane-1-yl)-6-methylpyrimidin-4-amine (910 mg, 65.26%).

[0187] MS(ESI,m / z):201.25[M+H] + ,RT(min):0.627.

[0188] The second step was the synthesis of 4-bromo-N-(2-(3,3-difluoroazacyclobutan-1-yl)-6-methylpyrimidin-4-yl)-2-(spiro[2.5]oct-5-en-6-yl)benzamide (compound 29-4):

[0189] Under nitrogen protection, at room temperature, N,N,N',N'-tetramethylchloromethane hexafluorophosphate (182.68 mg, 0.652 mmol, 4 eq) and N-methylimidazolium (133.64 mg, 1.630 mmol, 10 eq) were added to a solution of 2-(3,3-difluoroazacyclobutan-1-yl)-6-methylpyrimidin-4-amine (39.1 mg, 0.196 mmol, 1.20 eq), 4-bromo-2-(spiro[2.5]oct-5-en-6-yl)benzoic acid (50 mg, 0.163 mmol, 1.00 eq) in dichloromethane (1 mL). The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was extracted with dichloromethane (3 × 10 mL). The organic phases were combined, backwashed with saturated brine (2 × 10 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in petroleum ether / ethyl acetate (5:1) to give 4-bromo-N-(2-(3,3-difluoroazacyclobutane-1-yl)-6-methylpyrimidin-4-yl)-2-(spiro[2.5]oct-5-en-6-yl)benzamide (60 mg, 75.33%).

[0190] MS(ESI, m / z): 489.25 [M+H] + ,RT(min):1.455.

[0191] The third step was the synthesis of N-(2-(3,3-difluoroazacyclobutane-1-yl)-6-methylpyrimidin-4-yl)-4-((2-hydroxyethyl)sulfonamide)-2-(spiro[2.5]oct-5-en-6-yl)benzamide (compound 29):

[0192] Under nitrogen protection, at room temperature, 4-bromo-N-(2-(3,3-difluoroazacyclobutane-1-yl)-6-methylpyrimidin-4-yl)-2-(spiro[2,5]oct-5-en-6-yl)benzamide (40 mg, 0.082 mmol, 1 eq), cesium carbonate (79.9 mg, 0.246 mmol, 3 eq), and 2-hydroxyethane-1-sulfonamide (12.27 mg, 0.098 mmol, 1.2 eq) in N,N-dimethylformamide (1 mL) was added to a solution of methanesulfonic acid {bicyclohexyl(3-isopropoxy-2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphonane} (2'-methyl) Amino-1,1'-biphenyl-2-yl)palladium(II) (7.51 mg, 0.008 mmol, 0.1 eq), dicyclohexyl(3-isopropoxy-2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphonane (8.74 mg, 0.016 mmol, 0.2 eq). The reaction solution was heated to 100 °C and stirred for 1 hour. The reaction solution was cooled to room temperature and extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, backwashed with saturated brine (2 × 10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography (HPLC) under the following conditions (column specification: Xselect). CSH C18 OBD Column 30*150mm 5μm,n; Mobile phase A: water (10mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60mL / min; elution gradient: 35%B to 65%B in 10min, 80%B; detection wavelength: UV 220nm; retention time (min): 9.2). N-(2-(3,3-difluoroazacyclobutane-1-yl)-6-methylpyrimidin-4-yl)-4-((2-hydroxyethyl)sulfonamide)-2-(spiro[2.5]oct-5-en-6-yl)benzamide (16.63mg, 37.60%) was obtained.

[0193] MS(ESI, m / z): 534.45 [M+H] + ,RT(min):1.456.

[0194] 1H NMR (400MHz, DMSO-d6) δ10.33(s,1H),9.98(s,1H),7.46(d,J=8.4Hz,1H),7.37(s ,1H),7.15(dd,J=8.4,2.2Hz,1H),7.05(d,J=2.3Hz,1H),5.71(s,1H),4.95(s,1H ),4.38(t,J=12.5Hz,4H),3.75(t,J=6.6Hz,2H),3.31(s,2H),2.31(s,3H),2.29- 2.24(m,2H),1.94(d,J=3.6Hz,2H),1.41(t,J=5.9Hz,2H),0.24(d,J=12.7Hz,4H).

[0195] Example 9

[0196] N-[6-(4,4-difluoropiperidin-1-yl)-4-methylpyridin-2-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (compound 40)

[0197]

[0198] The first step is the synthesis of 6-(4,4-difluoropiperidin-1-yl)-4-methylpyridin-2-amine (compound 40-2):

[0199] Under nitrogen protection, sodium tert-butoxide (2.02 g, 21.042 mmol, 6 eq), 2-dicyclohexylphospho-2,6-diisopropoxy-1,1-biphenyl (163.63 mg, 0.351 mmol, 0.1 eq), 1-{[2',6'-bis(prop-2-yloxy)-[1,1'-biphenyl]-2-yl]dicyclohexyl-λ5-phosphono}-1-chloro-2H,3H,4H-benzo[c]1-aza-2-palladiumcyclohexane (255.52 mg, 0.351 mmol, 0.1 eq) and 4,4-difluoropiperidine hydrochloride (849.51 mg, 7.014 mmol, 2 eq) were added to a tetrahydrofuran (10.0 mL) solution of 6-chloro-4-methylpyridin-2-amine (500 mg, 3.51 mmol, 1 eq) at room temperature. The reaction mixture was heated to 100°C and stirred for one hour. The desired product was observed in the liquid chromatography-mass spectrometry (LC-MS). The reaction mixture was cooled to room temperature and quenched with water (20 mL). The reaction mixture was extracted with ethyl acetate (3 × 40 mL). The combined organic phases were backwashed with saturated brine (1 × 50 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in petroleum ether / ethyl acetate (5:1) to give 6-(4,4-difluoropiperidin-1-yl)-4-methylpyridin-2-amine (110 mg, 13.80%).

[0200] MS(ESI, m / z): 228.40 [M+H] + RT(min): 0.584

[0201] The second step is the synthesis of 4-bromo-N-[6-(4,4-difluoropiperidin-1-yl)-4-methylpyridin-2-yl]-2-{spiro[2,5]oct-5-en-6-yl}benzamide (compound 40-3):

[0202] Under nitrogen protection, tetramethylchloromethane hexafluorophosphate (80.38 mg, 0.288 mmol, 4 eq) and N-methylimidazolium (58.8 mg, 0.720 mmol, 10 eq) were added to a solution of 4-bromo-2-{spiro[2.5]oct-5-en-6-yl}benzoic acid (22 mg, 0.072 mmol, 1.00 eq) in dichloromethane (2 mL) and stirred for 2 minutes. Then, 6-(4,4-difluoropiperidin-1-yl)-4-methylpyridin-2-amine (24.41 mg, 0.108 mmol, 1.50 eq) was added at room temperature. The reaction mixture was heated to 80 °C and reacted for 1 hour. The reaction mixture was cooled to room temperature and quenched with water (10 mL). The reaction mixture was extracted with ethyl acetate (3 × 10 mL). The combined organic phases were backwashed with saturated brine (1 × 10 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in petroleum ether / ethyl acetate (5:1) to give 4-bromo-N-[6-(4,4-difluoropiperidin-1-yl)-4-methylpyridin-2-yl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (35 mg, 94.63%).

[0203] MS(ESI, m / z): 516.15 [M+H] + RT(min):1.322

[0204] The third step was the synthesis of N-[6-(4,4-difluoropiperidin-1-yl)-4-methylpyridin-2-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2,5]oct-5-en-6-yl}benzamide (compound 40):

[0205] Under nitrogen protection, at room temperature, dicyclohexyl(3-isopropoxy-2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphonane (5.18 mg,0.010 mmol) was added to a 1,4-dioxane (1 mL) solution of 4-bromo-N-[6-(4,4-difluoropiperidin-1-yl)-4-methylpyridin-2-yl]-2-{spiro[2,5]oct-5-en-6-yl}benzamide (25 mg, 0.048 mmol, 1 eq) at room temperature. 0.2 eq), methanesulfonic acid {biscyclohexyl[3-isopropoxy-2',4',6'-triisopropyl-(1,1'-biphenyl)-2-yl]phosphonane}(2'-methylamino-1,1'-biphenyl-2-yl)palladium(II) (4.45 mg, 0.005 mmol, 0.1 eq), cesium carbonate (47.32 mg, 0.144 mmol, 3 eq), and 2-hydroxyethanesulfonamide (12.12 mg, 0.096 mmol, 2 eq). The reaction mixture was heated to 120 °C. After reacting for one hour, the reaction mixture was cooled to room temperature and quenched with water (10 mL). The reaction mixture was extracted with ethyl acetate (3 × 10 mL). The combined organic phases were backwashed with saturated brine (1 × 10 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) under the following conditions (column: XBridge Shield RP18 OBD column, 30*150mm, 5μm; mobile phase A: water (10mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 40%B to 73%B, 73%B over 10 min; wavelength: 220 nm; RT1 (min): 8.27). N-[6-(4,4-difluoropiperidin-1-yl)-4-methylpyridin-2-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (7.98 mg, 29.40%) was obtained.

[0206] MS(ESI, m / z): 561.15 [M+H] + RT(min):1.847

[0207] 1H NMR (400MHz, DMSO-d6) δ9.97 (s, 1H), 9.55 (s, 1H), 7.54 (d, J = 8.3Hz, 1H), 7.31 ( s,1H),7.17(d,J=8.8Hz,1H),7.04(s,1H),6.51(d,J=5.6Hz,1H),5.79(s,1H), 4.94(s,1H),3.76(t,J=6.6Hz,2H),3.65(s,4H),3.23(s,2H),2.37(s,2H),2.2 5(d,J=10.3Hz,3H),1.99(s,2H),1.93(s,4H),1.44–1.38(m,2H),0.25(s,4H).

[0208] Example 10

[0209] N-[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]-5-((2-hydroxyethyl)sulfonamide)-3-{spiro[2.5]oct-5-en-6-yl}pyridin-2-carboxamide (compound 41)

[0210]

[0211] The first step is the synthesis of methyl 3-chloro-5-nitropyridine-2-carboxylate (compound 41-2):

[0212] At room temperature, thionyl chloride (10 mL) was added to 3-chloro-5-nitropyridine-2-onitrile (3.9 g, 21.248 mmol, 1 eq) in methanol (10 mL). After the addition was complete, the mixture was stirred at 50 °C for 16 hours. The reaction mixture was cooled to room temperature and quenched with water (100 mL). The reaction mixture was extracted with ethyl acetate (3 × 100 mL). The combined organic phases were backwashed with saturated brine (1 × 100 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative chromatography using petroleum ether / ethyl acetate (10:1) to give methyl 3-chloro-5-nitropyridine-2-carboxylate (1.5 g, 32.60%).

[0213] MS(ESI, m / z): 216.90 [M+H] + , RT(min):0.828.

[0214] The second step is the synthesis of methyl 5-nitro-3-{spiro[2.5]oct-5-en-6-yl}pyridine-2-carboxylate (compound 41-3):

[0215] Under nitrogen protection, at room temperature, 1,1'-bis(diphenylphosphino)ferrocene-palladium(II) chloride (II) (31.25 mg, 0.043 mmol, 0.1 eq) and sodium carbonate (146.81 mg, 1.386 mmol, 3.0 eq) were added to a solution of methyl 3-chloro-5-nitropyridine-2-carboxylate (100 mg, 0.462 mmol, 1.00 eq) in 1,4-dioxane (1.7 mL) and water (0.4 mL). The reaction mixture was heated to 80 °C and stirred for 1 hour. The reaction mixture was quenched with water at room temperature. The reaction mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, backwashed with saturated brine (1 × 20 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative chromatography using petroleum ether / ethyl acetate (8:1) to give methyl 5-nitro-3-{spiro[2.5]oct-5-en-6-yl}pyridine-2-carboxylate (50 mg, 37.56%).

[0216] MS(ESI,m / z):289.00[M+H] + ,RT(min):1.110.

[0217] The third step is the synthesis of 5-nitro-3-{spiro[2.5]oct-5-en-6-yl}pyridine-2-carboxylic acid (compound 41-4):

[0218] Lithium hydroxide (33.23 mg, 1.390 mmol, 10.0 eq) was added to methyl 5-nitro-3-{spiro[2.5]oct-5-en-6-yl}pyridine-2-carboxylic acid (40 mg, 0.139 mmol, 1.0 eq) in methanol (2 mL) and water (0.4 mL) at room temperature. The reaction mixture was heated to 60 °C and stirred for 1 hour. The reaction mixture was cooled to room temperature and quenched with water. The reaction mixture was acidified to pH 6 with 1 mol / L hydrochloric acid. The reaction mixture was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, backwashed with saturated brine (1 × 30 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure to give 5-nitro-3-{spiro[2.5]oct-5-en-6-yl}pyridine-2-carboxylic acid (37 mg, 97.23%).

[0219] MS(ESI, m / z): 274.90 [MH] + ,RT(min):0.672.

[0220] The fourth step is the synthesis of N-[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]-5-nitro-3-{spiro[2.5]oct-5-en-6-yl}pyridine-2-carboxamide (compound 41-5):

[0221] Under nitrogen protection, N-methylimidazole (89.81 mg, 1.090 mmol, 10.0 eq) was added to dichloromethane (1 mL) containing 5-nitro-3-{spiro[2.5]oct-5-en-6-yl}pyridine-2-carboxylic acid (30 mg, 0.109 mmol, 1.0 eq), 6-(4,4-difluoropiperidin-1-yl)pyridine-2-amine (25.66 mg, 0.120 mmol, 1.1 eq), and tetramethylchloroformamide hexafluorophosphate (92.07 mg, 0.327 mmol, 3.0 eq). After the addition was complete, the mixture was stirred at 80 °C for 2 hours. The reaction mixture was quenched with water at room temperature. The reaction mixture was extracted with ethyl acetate (3 × 20 mL). The combined organic phases were backwashed with saturated brine (1 × 20 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by preparative chromatography using petroleum ether / ethyl acetate (3:2) to give N-[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]-5-nitro-3-{spiro[2.5]oct-5-en-6-yl}pyridin-2-carboxamide (30 mg, 58.42%).

[0222] MS(ESI, m / z): 470.25 [M+H] + ,RT(min):1.379.

[0223] Step 5: Synthesis of 5-amino-N-[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]-3-{spiro[2,5]oct-5-en-6-yl}pyridin-2-carboxamide (compound 41-6):

[0224] Iron powder (10.71 mg, 0.192 mmol, 3.0 eq) and ammonium chloride (20.51 mg, 0.384 mmol, 6.0 eq) were added to N-[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]-5-nitro-3-{spiro[2.5]oct-5-en-6-yl}pyridine-2-carboxamide (30 mg, 0.064 mmol, 1.0 eq) in ethanol (1 mL) and water (0.2 mL) at room temperature. The reaction mixture was heated to 60 °C and stirred for 1 hour. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with ethyl acetate (3 × 10 mL). The filtrate was concentrated under reduced pressure. The resulting residue was purified by preparative chromatography using petroleum ether / ethyl acetate (5:1) to give 5-amino-N-[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]-3-{spiro[2.5]oct-5-en-6-yl}pyridin-2-carboxamide (30 mg, 90.80%).

[0225] MS(ESI, m / z): 440.10 [M+H] + ,RT(min):1.055.

[0226] Step 6: Synthesis of 2-[(6-{[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]carbamoyl}-5-{spiro[2.5]oct-5-en-6-yl}pyridin-3-yl)aminosulfonyl]ethyl acetate (compounds 41-8):

[0227] At 0 °C, ethyl 2-chlorosulfonyl acetate (11.2 mg, 0.061 mmol, 1.1 eq) and potassium carbonate (15.1 mg, 0.110 mmol, 2.0 eq) were added to a solution of 5-amino-N-[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]-3-{spiro[2.5]oct-5-en-6-yl}pyridine-2-carboxamide (30 mg, 0.055 mmol, 1.0 eq) in acetonitrile (1 mL). The reaction was carried out at room temperature for 1 hour, and the reaction solution was quenched with water at room temperature. The reaction mixture was extracted with ethyl acetate (3 × 20 mL). The combined organic phases were backwashed with saturated brine (1 × 20 mL) and dried over sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative chromatography using petroleum ether / ethyl acetate (5:1) to give ethyl acetate 2-[(6-{[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]carbamoyl}-5-{spiro[2.5]oct-5-en-6-yl}pyridin-3-yl)aminosulfonyl] (20 mg, 62.11%).

[0228] MS(ESI, m / z): 590.10 [M+H] + ,RT(min):1.196.

[0229] Step 7: Synthesis of N-[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]-5-((2-hydroxyethyl)sulfonamide)-3-{spiro[2.5]oct-5-en-6-yl}pyridine-2-carboxamide (compound 41):

[0230] Under nitrogen protection, lithium aluminum hydride (0.02 mL, 2.5 mol / L, 1.2 eq) was added dropwise to a tetrahydrofuran solution (20 mg, 0.034 mmol, 1 eq) of ethyl 2-[(6-{[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]carbamoyl}-5-{spiro[2.5]oct-5-en-6-yl}pyridin-3-yl)aminosulfonyl]acetate (20 mg, 0.034 mmol, 1 eq) at -40 °C. After the addition was complete, the system was stirred at room temperature for 1 hour. The desired product was observed in the liquid chromatography-mass spectrometry (LC-MS). The reaction solution was quenched with water at 0°C. The mixture was extracted with ethyl acetate (3 × 20 mL), the organic phases were combined, backwashed with saturated brine (1 × 30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography under the following conditions (column specifications: XBridge Shield RP18 OBD column, 30*150 mm, 5 μm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 28% B to 63% B, 63% B within 8 min; wavelength: 220 nm; retention time (min): 7.93). N-[6-(4,4-difluoropiperidin-1-yl)pyridin-2-yl]-5-((2-hydroxyethyl)sulfonamide)-3-{spiro[2.5]oct-5-en-6-yl}pyridin-2-carboxamide (3.3 mg, 17.77%) was obtained.

[0231] MS(ESI, m / z): 547.95 [M+H] + ,RT(min):1.487.

[0232] 1 H NMR: (400MHz, DMSO-d6) δ10.38(s,1H),10.11(s,1H),8.37(s,1H),7.60(t,J=8.0 Hz,1H),7.48(d,J=7.8Hz,1H),7.43(d,J=2.5Hz,1H),6.69(d,J=8.3Hz,1H),5.62 (s,1H),4.96(s,1H),3.78(t,J=6.2Hz,2H),3.68(s,4H),3.37(d,J=12.5Hz,2H), 2.30(s,2H),1.99(d,J=15.1Hz,6H),1.50(d,J=6.0Hz,2H),0.33(d,J=3.4Hz,4H).

[0233] Example 11

[0234] N-[6-(4,4-difluoropiperidin-1-yl)-5-fluoropyridin-2-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (compound 42)

[0235]

[0236] The first step is the synthesis of 6-(4,4-difluoropiperidin-1-yl)-5-fluoropyridine-2-amine (compound 42-2):

[0237] Under nitrogen protection, N,N-diisopropylethylamine (609.01 mg, 4.713 mmol, 3 eq) and 4,4-difluoropiperidine hydrochloride (495.04 mg, 3.142 mmol, 2 eq) were added to a solution of 6-bromo-5-fluoropyridine-2-amine (300 mg, 1.571 mmol, 1 eq) in N-methylpyrrolidone (3 mL) at room temperature. The mixture was heated to 200 °C and stirred in a microwave for one hour. The reaction solution was cooled to room temperature and extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, backwashed with water (3 × 10 mL) and saturated brine (3 × 10 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure to give 6-(4,4-difluoropiperidine-1-yl)-5-fluoropyridine-2-amine (150 mg, 41.32%).

[0238] MS(ESI, m / z): 232.05 [M+H] + RT(min):1.210

[0239] The second step is the synthesis of 4-bromo-N-[6-(4,4-difluoropiperidin-1-yl)-5-fluoropyridin-2-yl]-2-{spiro[2,5]oct-5-en-6-yl}benzamide (compound 42-3):

[0240] At room temperature, N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (199.01 mg, 0.710 mmol, 4 eq) and N-methylimidazolium (145.59 mg, 1.775 mmol, 10 eq) were added to a solution of 4-bromo-2-(spiro[2.5]oct-5-en-6-yl)benzoic acid (55 mg, 0.177 mmol, 1 eq) and 6-(4,4-difluoropiperidin-1-yl)-5-fluoropyridine-2-amine (82 mg, 0.355 mmol, 2 eq) in dichloromethane (1.5 mL). The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was extracted with dichloromethane (3 × 10 mL). The organic phases were combined, backwashed with saturated brine (1 × 10 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography with petroleum ether / ethyl acetate (10:1) to give 4-bromo-N-[6-(4,4-difluoropiperidin-1-yl)-5-fluoropyridin-2-yl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (55 mg, 59.60%).

[0241] MS(ESI, m / z): 520.11 [M+H] + RT(min):1.559

[0242] The third step was the synthesis of N-[6-(4,4-difluoropiperidin-1-yl)-5-fluoropyridin-2-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (compound 42):

[0243] Under nitrogen protection, at room temperature, cesium carbonate (103.31 mg, 0.318 mmol, 3 eq), 2-hydroxyethane-1-sulfonamide (20 mg, 0.159 mmol, 1.5 eq), and dicyclohexyl(3-isopropoxy-2', 4', 6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphonane (11.3 mg, 0.021 mmol, 0.2 eq) were added to a 1,4-dioxane (2 mL) solution of 4-bromo-N-[6-(4,4-difluoropiperidin-1-yl)-5-fluoropyridin-2-yl]-2-{spiro[2,5]oct-5-en-6-yl}benzamide (55 mg, 0.106 mmol, 1 eq) at room temperature. q), (methanesulfonic acid {bicyclohexyl(3-isopropoxy-2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphonane}(2'-methylamino-1,1'-biphenyl-2-yl)palladium(II) (9.71 mg, 0.011 mmol, 0.1 eq). The reaction solution was heated to 100 °C and stirred for 1 hour. The reaction mixture was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, backwashed with saturated brine (1 × 20 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography under the following conditions (wavelength: 254 nm / 220 nm, column: XSelect). CSH Fluoro Phenyl 5μm, 30mm*150mm, mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile, flow rate: 60mL / min, gradient: 45% B to 65% B over 10min, retention time (min): 10.08). N-[6-(4,4-difluoropiperidin-1-yl)-5-fluoropyridin-2-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (6.03mg, 10.10%) was obtained.

[0244] MS(ESI, m / z): 565.40 [M+H] + RT(min):1.666

[0245] 1H NMR(400MHz, CDCl3)δ8.78(s,1H),7.86(d,J=8.4Hz,1H),7.81–7.75(m,1H),7.33(dd,J=12.2,8.5 Hz,1H),7.22(dd,J=8.4,2.3Hz,1H),7.13(d,J=2.3Hz,1H),6.90(s,1H),6.01–5.95(m,1H),4.13( t,J=5.2Hz,2H),3.63(t,J=5.8Hz,4H),3.35(t,J=5.2Hz,2H),2.47(s,1H),2.33(q,J=5.2Hz,2H), 2.12(q,J=3.9,3.3Hz,2H),2.05(dt,J=13.6,5.7Hz,4H),1.50(t,J=6.0Hz,2H),0.38–0.24(m,4H).

[0246] Example 12

[0247] N-[2-(4,4-difluoropiperidin-1-yl)-3-fluoropyridin-4-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (compound 43)

[0248]

[0249]

[0250] The first step is the synthesis of 2-(4,4-difluoropiperidin-1-yl)-3-fluoropyridine-4-amine (compound 43-2):

[0251] Under nitrogen protection, 4,4-difluoropiperidine-4-amine (100 mg, 0.68 mmol, 1 eq) and diisopropylethylamine (265 mg, 2.05 mmol, 3 eq) were added to a solution of 2-chloro-3-fluoropyridine-4-amine (100 mg, 0.68 mmol, 1 eq) in N-methylpyrrolidone (1 mL) at room temperature. The reaction mixture was heated to 200 °C and microwaved for 3 hours. The reaction mixture was cooled to room temperature, quenched with water, and extracted with ethyl acetate (3 × 50 mL). The organic phases were combined, backwashed with water (3 × 40 mL) and saturated brine (2 × 60 mL), and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give 2-(4,4-difluoropiperidine-1-yl)-3-fluoropyridine-4-amine (38 mg, 2.41%).

[0252] MS(ESI, m / z): 232.05 [M+H] + ,RT(min):0.556.

[0253] The second step is the synthesis of 4-bromo-N-[2-(4,4-difluoropiperidin-1-yl)-3-fluoropyridin-4-yl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (compound 43-3):

[0254] Under nitrogen protection, at room temperature, 4-bromo-2-{spiro[2.5]oct-5-en-6-yl}benzoic acid (47.83 mg, 0.156 mmol, 1.2 eq), tetramethylchloromethanediamine hexafluorophosphate (47.83 mg, 0.156 mmol, 1.2 eq), and N-methylimidazolium (106.53 mg, 1.300 mmol, 10 eq) were added to a solution of 2-(4,4-difluoropiperidin-1-yl)-3-fluoropyridine-4-amine (30 mg, 0.130 mmol, 1.2 eq) in dichloromethane (5 mL). The reaction mixture was heated to 80 °C and stirred for 2 hours. The reaction mixture was cooled to room temperature, quenched with water, and the reaction mixture was extracted with dichloromethane (3 × 30 mL). The organic phases were combined, backwashed with saturated brine (1 × 30 mL), and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in petroleum ether / ethyl acetate (3:1) to give 4-bromo-N-[2-(4,4-difluoropiperidin-1-yl)-3-fluoropyridin-4-yl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (45 mg, 66.65%).

[0255] MS(ESI, m / z): 520.11 [M+H] + RT(min):1.773

[0256] The third step was the synthesis of N-[2-(4,4-difluoropiperidin-1-yl)-3-fluoropyridin-4-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (compound 43):

[0257] Under nitrogen protection, at room temperature, 2-hydroxyethanesulfonamide (19.24 mg, 0.154 mmol, 2 eq), palladium acetate (1.73 mg, 0.008 mmol, 0.1 eq), 2-di-tert-butylphospho-2',4',6'-triisopropylbiphenyl (6.53 mg, 0.015 mmol, 0.2 eq), and sodium tert-butoxide (22.16 mg, 0.231 mmol, 3 eq) were added to a solution of 4-bromo-N-[2-(4,4-difluoropiperidin-1-yl)-3-fluoropyridin-4-yl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (40 mg, 0.077 mmol, 1 eq) in N,N-dimethylformamide (5 mL), at room temperature. The reaction solution was heated to 140 °C and stirred for 1 hour. The reaction mixture was cooled to room temperature and quenched with water. The reaction mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, backwashed with saturated brine (1 × 30 mL), and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: column specifications: Xselect CSH C18 OBD, 30*150 mm, 5 μm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 60 mL / min; elution gradient: 65% B to 90% B in 8 min, 90% B; detection wavelength: 220 nm; retention time (min): 7.78. N-[2-(4,4-difluoropiperidin-1-yl)-3-fluoropyridin-4-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (2.3 mg, 5.25%) was obtained.

[0258] MS(ESI, m / z): 564.95 [M+H] + ,RT(min):1.613.

[0259] 1H NMR (400MHz, DMSO-d6) δ10.13-9.79 (s, 2H), 8.00-7.87 (d, J = 5.5Hz, 1H), 7.65-7.53 (s, 1H), 7.54-7 .42(d,J=8.4Hz,1H),7.28-7.10(d,J=8.5Hz,1H),7.10-6.94(s,1H),5.78-5.67(s,1H),5.05-4.86 (s,1H),3.85-3.65(t,J=6.6Hz,2H),3.58-3.32(m,4H),3.29-3.19(m,2H),2.39-2.26(s,2H),2.24 -1.98(m,4H),1.98-1.76(s,2H),1.46-1.29(m,2H),0.37-0.22(d,J=3.5Hz,2H),0.22-0.06(m,2H).

[0260] Example 13

[0261] N-[2-(4,4-difluorocyclohexyl)-6-methylpyrimidin-4-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2,5]oct-5-en-6-yl}benzamide (compound 44)

[0262]

[0263] The first step is the synthesis of 2-(4,4-difluorocyclohexyl-1-en-1-yl)-6-methylpyrimidin-4-amine (compound 44-2):

[0264] Under nitrogen protection, at room temperature, 4,4-difluorocyclohexene-1-boranoic acid pinacol ester (1631.54 mg, 6.686 mmol, 1.2 eq), potassium phosphate (2365.56 mg, 11.144 mmol, 2 eq), and 1,1-bis(diphenylphosphino)ferrocene palladium dichloride (454.69 mg, 0.557 mmol, 0.1 eq) were added to a 20 mL 4 / 1 v / v solution of 2-chloro-4-amino-6-methylpyrimidine (800 mg, 5.572 mmol, 1 eq) of 1,4-dioxane and water. The reaction mixture was heated to 80 °C and stirred for 4 h. The reaction mixture was cooled to room temperature and extracted with ethyl acetate (3 × 50 mL). The combined organic phases were backwashed with saturated brine (1 × 50 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in ethyl acetate / petroleum ether (1:1) to give 2-(4,4-difluorocyclohex-1-en-1-yl)-6-methylpyrimidin-4-amine (684.5 mg, 89.975%).

[0265] MS:(ESI,m / z):226.10[M+H] + ,RT(min):0.685.

[0266] The second step is the synthesis of 2-(4,4-difluorocyclohexane-1-yl)-6-methylpyrimidin-4-amine (compound 44-3):

[0267] Under nitrogen protection, palladium on carbon (68 mg, 0.21 mmol, 0.69 eq) was added to a 15 mL ethanol solution of 2-(4,4-difluorocyclohexyl-1-en-1-yl)-6-methylpyrimidin-4-amine (684.5 mg, 3.039 mmol, 1 eq) at room temperature, and hydrogen gas was introduced to replace the hydrogen gas. The reaction solution was allowed to react at room temperature for 6 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give 2-(4,4-difluorocyclohexyl)-6-methylpyrimidin-4-amine (620 mg, 90%).

[0268] MS:(ESI,m / z):228.20[M+H] + ,RT(min):0.737.

[0269] The third step is the synthesis of 4-bromo-N-[2-(4,4-difluorocyclohexyl)-6-methylpyrimidin-4-yl]-2-{spiro[2,5]oct-5-en-6-yl}benzamide (compound 44-4):

[0270] Under nitrogen protection, tetramethylchloroformamidine hexafluorophosphate (182.68 mg, 0.652 mmol, 4 eq) and N-methylimidazole (133.64 mg, 1.630 mmol, 10 equiv) were added to a solution of 2-(4,4-difluorocyclohexyl)-6-methylpyrimidin-4-amine (55.49 mg, 0.244 mmol, 1.5 eq) and 4-bromo-2-{spiro[2.5]oct-5-en-6-yl}benzoic acid (50 mg, 0.163 mmol, 1.00 eq) in dichloromethane (5.00 mL) and N-methylimidazole (133.64 mg, 1.630 mmol, 10 equiv) at room temperature. The reaction solution was heated to 60 °C and stirred for 1 hour. The reaction solution was cooled to room temperature and extracted with dichloromethane (3 x 10 mL). The organic phases were combined and backwashed with saturated brine (1 x 30 mL). The solution was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography with ethyl acetate / petroleum ether (5:1) to give 4-bromo-N-[2-(4,4-difluorocyclohexyl)-6-methylpyrimidin-4-yl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (48 mg, 57.10%).

[0271] MS:(ESI,m / z):516.30[M+H] + ,RT(min):1.517.

[0272] The fourth step is the synthesis of N-[2-(4,4-difluorocyclohexyl)-6-methylpyrimidin-4-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2,5]oct-5-en-6-yl}benzamide (compound 44):

[0273] Under nitrogen protection, at room temperature, a solution of 4-bromo-N-[2-(4,4-difluorocyclohexyl)-6-methylpyrimidin-4-yl]-2-{spiro[2,5]oct-5-en-6-yl}benzamide (40 mg, 0.077 mmol, 1 eq) and 2-hydroxyethanesulfonamide (11.63 mg, 0.092 mmol, 1.2 eq) in N,N-dimethylformamide (1 mL) was added, along with (methanesulfonic acid {bicycloethyl(3-isopropoxy-2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphonane}(2'-methylamino-1,1'-biphenyl-2-yl)palladium(II) (7.11 mg, 0.008 mmol, 0.1 eq), bicycloethyl(3-isopropoxy-2',4',6'-triisopropyl-[ 1,1'-Biphenyl]-2-yl)phosphonane (8.28 mg, 0.015 mmol, 0.2 eq) and cesium carbonate (75.71 mg, 0.232 mmol, 3.00 eq). The reaction mixture was heated to 100 °C and stirred for 1 hour. The reaction mixture was cooled to room temperature, and the mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, backwashed with saturated sodium chloride solution (1 × 30 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative high performance liquid chromatography to obtain N-[2-(4,4-difluorocyclohexyl)-6-methylpyrimidin-4-yl]-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (13.02 mg, 29.92%).

[0274] MS:(ESI,m / z):561.55[M+H] + ,RT(min):1.712.

[0275] 1H NMR: (400MHz, DMSO-d6) δ10.50(s,1H),10.02(s,1H),7.81(s,1H),7.51(d,J=8.4Hz,1H),7. 18(dd,J=8.5,2.2Hz,1H),7.07(d,J=2.3Hz,1H),5.72(s,1H),4.90(s,1H),3.76(t,J=6.6Hz, 2H),3.32-3.31(m,2H),2.87(d,J=12.3Hz,1H),2.43(s,3H),2.29(s,2H),2.08(d,J=10.2Hz ,2H),2.01-1.74(m,8H),1.39(t,J=6.0Hz,2H),0.22(d,J=3.9Hz,2H),0.17(d,J=3.8Hz,2H).

[0276] Example 14

[0277] 4-((2-hydroxyethyl)sulfonamide)-N-[6-methyl-2-(pyrrolidone-1-yl)pyrimidin-4-yl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (Compound 45)

[0278]

[0279] The first step is the synthesis of 6-methyl-2-(pyrrolidone-1-yl)pyrimidin-4-amine (compound 45-2):

[0280] Under nitrogen protection at room temperature, N,N-diisopropylethylamine (1.82 mL, 10.449 mmol, 3 eq) was added to a solution of 2-chloro-6-methylpyrimidin-4-amine (500 mg, 3.483 mmol, 1 eq) and pyrrolidine (297.23 mg, 4.180 mmol, 1.2 eq) in N-methylpyrrolidone (5 mL). The reaction mixture was heated to 160 °C and stirred for 1 hour. The reaction mixture was cooled to room temperature and quenched with water (30 mL). The reaction mixture was extracted with ethyl acetate (3 × 20 mL). The combined organic phases were backwashed with saturated sodium chloride solution (3 × 20 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in dichloromethane / methanol (10:1) to give 6-methyl-2-(pyrrolidone-1-yl)pyrimidine-4-amine (380 mg, 61.22%).

[0281] MS:(ESI,m / z):179.15[M+H] + RT(min): 0.471

[0282] The second step is the synthesis of 4-bromo-N-[6-methyl-2-(pyrrolidin-1-yl)pyrimidin-4-yl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (compound 45-3):

[0283] Under nitrogen protection, at room temperature, N-methylimidazolium (187.10 mg, 2.280 mmol, 10 eq) was added to a 2 mL solution of 6-methyl-2-(pyrrolidone-1-yl)pyrimidin-4-amine (48.74 mg, 0.274 mmol, 1.2 eq), N,N,N',N'-tetramethylchloromethanemidazone hexafluorophosphate (255.75 mg, 0.912 mmol, 4 eq), and 4-bromo-2-(spiro[2.5]oct-5-en-6-yl)benzoic acid (70 mg, 0.228 mmol, 1.00 eq). The reaction mixture was heated to 100 °C and stirred for 16 hours. The reaction mixture was cooled to room temperature and quenched with water (20 mL). The reaction mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic phases were backwashed with saturated sodium chloride solution (3 × 20 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography in petroleum ether / ethyl acetate (10:1) to give 4-bromo-N-[6-methyl-2-(pyrrolidone-1-yl)pyrimidin-4-yl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (70 mg, 65.72%).

[0284] MS:(ESI,m / z):467.35[M+H] + RT(min):1.563

[0285] Step 3: Synthesis of 4-((2-hydroxyethyl)sulfonamide)-N-[6-methyl-2-(pyrrolidone-1-yl)pyrimidin-4-yl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (compound 45):

[0286] Under nitrogen protection, at room temperature, dicyclohexyl(3-isopropoxy-2′,4′,6′-triisopropyl-[1,1′-biphenyl]-2-yl)phosphonane (11.44 mg,0.021 mmol,0.2 eq) and ( ) were added to a solution of 4-bromo-N-[6-methyl-2-(pyrrolidone-1-yl)pyrimidin-4-yl]-2-{spiro[2,5]oct-5-en-6-yl}benzamide (50 mg, 0.107 mmol, 1 eq), 2-hydroxyethanesulfonamide (16.06 mg, 0.128 mmol, 1.2 eq), and cesium carbonate (104.56 mg, 0.321 mmol, 3 eq) in N,N-dimethylformamide (2 mL). Mesylate {bicyclohexyl(3-isopropoxy-2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphonane}(2'-methylamino-1,1'-biphenyl-2-yl)palladium(II) (9.83 mg, 0.011 mmol, 0.1 eq). The reaction mixture was heated to 100 °C and stirred for 1 hour. The reaction mixture was cooled to room temperature and quenched with water (10 mL). The reaction mixture was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, backwashed with saturated brine (3 × 10 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) under the following conditions: column specification: Kinetex. 5 μm EVO C18, 30 mm * 150 mm; Mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; Flow rate: 60 mL / min; Elution gradient: 30% B to 65% B in 8 min; Detection wavelength: 254 nm / 220 nm; Retention time (min): 7.18. 4-((2-hydroxyethyl)sulfonamide)-N-[6-methyl-2-(pyrrolidone-1-yl)pyrimidin-4-yl]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (25.8 mg, 46.81%) was obtained.

[0287] MS:(ESI,m / z):512.00[M+H] + RT(min):1.572

[0288] 1H NMR: (400MHz, DMSO-d6) δ9.96 (s, 1H), 9.85 (s, 1H), 7.51 (d, J = 8.4Hz, 1H), 7.23–7.09(m,2H),7.05(d,J=2.3Hz,1H),5.84–5.69(m,1H),4.95(s,1H), 3.76(t,J=6.6Hz,2H),3.42(s,4H),3.29(d,J=6.6Hz,2H),2.26(s,5H),1. 98(s,2H),1.94–1.78(m,4H),1.43(t,J=6.0Hz,2H),0.26(d,J=5.7Hz,4H).

[0289] Example 15

[0290] N-(2-(3-azabicyclo[3.1.0]hexane-3-yl-6-methylpyrimidin-4-yl)-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (compound 46)

[0291]

[0292] The first step was the synthesis of 2-(3-azabicyclo[3.1.0]hex-3-yl-6-methylpyrimidin-4-amine (compound 46-2):

[0293] Under nitrogen protection, N,N-diisopropylethylamine (1350.36 mg, 10.449 mmol, 3 eq) and 3-azabicyclo[3.1.0]hexane (434.29 mg, 5.224 mmol, 1.5 eq) were added to a solution of 2-chloro-6-methylpyrimidin-4-amine (500 mg, 3.483 mmol, 1 eq) in N-methylpyrrolidone (5 mL, 51.850 mmol, 14.89 eq) at room temperature. The reaction mixture was microwaved at 160 °C for 2 hours. The reaction solution was cooled to room temperature and quenched with water (20 mL). The reaction mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic phases were backwashed with saturated brine (2 × 20 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography in petroleum ether / ethyl acetate (5:1) to give 2-(3-azabicyclo[3.1.0]hex-3-yl-6-methylpyrimidin-4-amine (230 mg, 34.71%).

[0294] MS:(ESI,m / z):191.10[M+H] + RT(min): 0.608

[0295] The second step was the synthesis of N-(2-(3-azabicyclo[3.1.0]hexane-3-yl-6-methylpyrimidin-4-yl)-4-bromo-2-{spiro[2.5]oct-5-en-6-yl}benzamide (compound 46-3):

[0296] Under nitrogen protection, at room temperature, N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (438.42 mg, 1.56 mmol, 4 eq) and N-methylimidazolium (320.73 mg, 3.900 mmol, 10 eq) were added to a solution of 4-bromo-2-(spiro[2.5]oct-5-en-6-ylbenzoic acid (30 mg, 0.099 mmol, 1.00 eq), 2-(3-azabicyclo[3.1.0]hex-3-yl-6-methylpyrimidin-4-amine (27.87 mg, 0.150 mmol, 1.5 eq) in dichloromethane (2 mL). The liquid was heated to 80°C and stirred for 2 hours. The reaction mixture was quenched with water (3 mL) at room temperature. The reaction mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, backwashed with saturated brine (3 × 30 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography with petroleum ether / ethyl acetate (5:1) to give N-(2-(3-azabicyclo[3.1.0]hexane-3-yl-6-methylpyrimidin-4-yl)-4-bromo-2-(spiro[2.5]oct-5-en-6-ylbenzamide) (27 mg, 57.61%).

[0297] MS:(ESI,m / z):478.90[M+H] + RT(min):1.114

[0298] The third step was the synthesis of N-(2-(3-azabicyclo[3.1.0]hexane-3-yl-6-methylpyrimidin-4-yl)-4-((2-hydroxyethyl)sulfonamide)-2-(spiro[2.5]oct-5-en-6-ylbenzamide (compound 46):

[0299] Under nitrogen protection, at room temperature, dicyclohexyl(3-isopropoxy-2′,4′,6′-triisopropyl-[1,1′-biphenyl]-2-yl)phosphonane (6.02 mg,0.011 mmol,0.2 eq) and (2.5) octyl-5-en-6-ylbenzamide (27 mg, 0.056 mmol, 1 eq), 2-hydroxyethanesulfonamide (10.57 mg, 0.084 mmol, 1.5 eq) were added to a solution of N,N-dimethylformamide (1 mL) containing N-(2-(3-azabicyclo[3.1.0]hexane-3-yl-6-methylpyrimidin-4-yl)-4-bromo-2-(spiro[2.5]octyl-5-en-6-ylbenzamide (27 mg, 0.056 mmol, 1 eq), 2-hydroxyethanesulfonamide (10.57 mg, 0.084 mmol, 1.5 eq) and N,N-dimethylformamide (1 mL) were added. 4',6'-Triisopropyl-[1,1'-biphenyl]-2-yl)phosphonane}(2'-methylamino-1,1'-biphenyl-2-yl)palladium(II) (5.17 mg, 0.006 mmol, 0.1 eq), cesium carbonate (55.05 mg, 0.168 mmol, 3 eq). The reaction mixture was heated to 100 °C and stirred for 1 hour. The reaction mixture was cooled to room temperature, quenched with water, and extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, backwashed with saturated brine (3 × 30 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) under the following conditions: column specification: XBridge. Prep OBD C18, 30*150mm, 5μm; Mobile phase A: water (10mmol / L ammonium bicarbonate), Mobile phase B: acetonitrile; Flow rate: 60mL / min; Elution gradient: 46%B to 73%B in 8min; Detection wavelength: 220nm; Retention time (min): 6.72. N-(2-(3-azabicyclo[3.1.0]hexane-3-yl-6-methylpyrimidin-4-yl)-4-((2-hydroxyethyl)sulfonamide)-2-{spiro[2.5]oct-5-en-6-yl}benzamide (6.25mg, 21.00%) was obtained.

[0300] MS:(ESI,m / z):524.00[M+H] + RT(min):1.633

[0301] 1H NMR: (400MHz, DMSO-d6) δ10.00(s,1H),9.89(s,1H),7.50(d,J=8.3Hz,1H),7.17(d d,J=8.5,2.3Hz,2H),7.04(d,J=2.2Hz,1H),5.74(s,1H),4.95(s,1H),3.75(d,J=7. 3Hz,4H),3.35(m,4H),2.26(s,2H),2.24(s,3H)1.97(s,2H),1.63–1.57(m,2H),1.4 2(t,J=6.0Hz,2H),0.73–0.66(m,1H),0.24(d,J=7.6Hz,4H),0.07(q,J=4.3Hz,1H).

[0302] Example 16

[0303] N-[6-(4,4-difluoropiperidin-1-yl)-4-methylpyridin-2-yl]-4-[(1-hydroxy-2-methylpropyl-2-yl)amino]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (compound 47)

[0304]

[0305] The first step was the synthesis of N-[6-(4,4-difluoropiperidin-1-yl)-4-methylpyridin-2-yl]-4-[(1-hydroxy-2-methylpropyl-2-yl)amino]-2-{spiro[2,5]oct-5-en-6-yl}benzamide (compound 47):

[0306] Under nitrogen protection, at room temperature, 2-amino-2-methyl-1-propanol (10.36 mg, 0.116 mmol, 1.2 eq) and (2-amino-1,1'-biphenyl-2-yl)palladium(II) (5.18 mg, 0.097 mmol, 1 eq) were added sequentially to a solution of 4-bromo-N-[6-(4,4-difluoropiperidin-1-yl)-4-methylpyridin-2-yl]-2-{spiro[2,5]oct-5-en-6-yl}benzamide (50 mg, 0.097 mmol, 1 eq) in 1,4-dioxane (3 mL). 0 mmol (0.1 eq), dicyclohexyl(3-isopropyl-2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphonane (17.79 mg, 0.019 mmol, 0.2 eq) and cesium carbonate (94.64 mg, 0.290 mmol, 3.00 eq) were added. The reaction solution was heated to 60 °C and stirred for 1 hour. The reaction solution was cooled to room temperature, quenched with water (10 mL), and extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, backwashed with saturated brine (2 × 10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography under the following conditions: column specification X Select. CSH Fluoro Phenyl 5μm, 30mm*150mm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 60ml / min; elution gradient: 25%B to 50%B in 8min; detection wavelength: 254nm / 220nm; retention time (min): 7.25, yielding N-[6-(4,4-difluoropiperidin-1-yl)-4-methylpyridin-2-yl]-4-[(1-hydroxy-2-methylpropyl-2-yl)amino]-2-{spiro[2.5]oct-5-en-6-yl}benzamide (3.02mg, 5.78%).

[0307] MS:(ESI,m / z):525.30[M+H] + RT(min):2.302

[0308] 1H NMR (400MHz, CDCl3) δ8.74(s,1H),8.36(s,1H),7.88(d,J=8.7Hz,1H),7.56(s,1H),6.93(d,J=9.0Hz,1H),6.75(s,1H),6.28( s,1H),5.97(s,1H),3.92(s,2H),3.69(s,4H),2.30(s,5H),2.13(s,2H),1.98(s,4H),1.52(s,2H),1.36(s,6H),0.31(s,4H).

[0309] Example 17

[0310] N-[6-(4,4-difluoropiperidin-1-yl)-4-methylpyridin-2-yl]-4-{[1-(hydroxymethyl)cyclopropyl]amino}-2-{spiro[2,5]oct-5-en-6-yl}benzamide (compound 48)

[0311]

[0312] The first step was the synthesis of N-[6-(4,4-difluoropiperidin-1-yl)-4-methylpyridin-2-yl]-4-{[1-(hydroxymethyl)cyclopropyl]amino}-2-{spiro[2,5]oct-5-en-6-yl}benzamide (compound 48):

[0313] Under nitrogen protection, at room temperature, (1-aminocyclopropyl)methanol (10.12 mg, 0.116 mmol, 1.2 eq) and (2-methylamino-1,1'-biphenyl-2-yl)palladium(II) (5.18 mg, 0.010 mmol, 1 eq) were added to a solution of 4-bromo-N-[6-(4,4-difluoropiperidin-1-yl)-4-methylpyridin-2-yl]-2-{spiro[2,5]oct-5-en-6-yl}benzamide (50 mg, 0.097 mmol, 1 eq) in 1,4-dioxane (3 mL). 0.1 mmol (eq), dicyclohexyl(3-isopropyl-2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphonane (17.79 mg, 0.019 mmol, 0.2 eq) and cesium carbonate (22.11 mg, 0.291 mmol, 3 eq) were added. The reaction solution was heated to 60 °C and stirred for 1 hour. The reaction solution was cooled to room temperature, quenched with water (10 mL), and extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, backwashed with saturated brine (1 × 10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography under the following conditions: column specification: XBridge BEH Shield RP18, 30mm*150mm, 5μm; Mobile phase A: water (10mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; Flow rate: 60ml / min; Elution gradient: 53% B to 72% B in 10min; Detection wavelength: 254nm / 220nm; Retention time (min): 8.47. N-[6-(4,4-difluoropiperidin-1-yl)-4-methylpyridin-2-yl]-4-{[1-(hydroxymethyl)cyclopropyl]amino}-2-{spiro[2.5]oct-5-en-6-yl}benzamide (2.30mg, 4.20%) was obtained.

[0314] MS:(ESI,m / z):523.50[M+H] + RT(min): 3.147

[0315] 1H NMR (400MHz, CDCl3) δ8.83(s,1H),7.86(d,J=8.5Hz,1H),7.61(s,1H),6.75(dd,J=8.6,2.4Hz,1H),6.47(d,J=2.5Hz,1H),6.27(s,1H),5.94(s,1H), 4.68(s,1H),3.72(s,4H),3.68(s,2H),2.31(s,5H),2.14(s,2H),2.02(d, J=14.5Hz,4H),1.25(s,2H),1.20(s,1H),0.89(s,4H),0.51–0.15(m,4H).

[0316] Biological evaluation

[0317] Test Example 1

[0318] Test Name: Imaging-Based Nuclear Counting Analysis (NCA) in OVCAR-3 Cells

[0319] Day 0: Compound dilution and treatment

[0320] a) The final test concentrations of AM-5308 were: 10000, 3333.3, 1111.1, 370.3, 123.4, 41.1, 13.7, 4.5, 1.5, and 0.5 nM.

[0321] b) The test compounds were finally tested at the following concentrations: 10000, 3333.3, 1111.1, 370.3, 123.4, 41.1, 13.7, 4.5, 1.5, and 0.5 nM.

[0322] c) The cells were cultured for 4 days in an incubator at 37°C and 5% CO2.

[0323] d) The DMSO concentration is 0.1%.

[0324] On day 1, cells were seeded into 384-well cell culture plates.

[0325] a) When the cell confluence reaches 80%-90%, process the cells.

[0326] b) Resuspend the cells in the culture medium, and then count and dilute the cells at the desired density.

[0327] c) Add 30 μL / well of cell suspension containing appropriate cells to a 384-well plate: 600 cells / well.

[0328] Test on day 4

[0329] a) Add 30 μL of 8% fixative (final concentration 4%) and incubate the plate at room temperature for 30 minutes.

[0330] b) Centrifuge plate, 1000 RPM, 30 s.

[0331] c) Wash twice with 60 μl / well of PBS.

[0332] d) After fixation, the cells were permeabilized and stained in 60 μL of wash buffer containing 2 μg / mL Hoechst 33342 DNA dye (1% BSA, 0.2% Triton X-100, 1X PBS).

[0333] e) Seal the plate and incubate it at room temperature in the dark for 1 hour.

[0334] f) Wash 3 times with PBS.

[0335] g) Add 50 μL PBS per well and scan the plate using HCS.

[0336] h) Data Acquisition and Detection

[0337] Data Analysis

[0338] Inhibition rate (%) = 100 - (Compound well reading - Low reading control well reading) / (High reading control well reading - Low reading control well reading) * 100

[0339] High reading control wells: cells with 30 nL DMSO; Low reading control wells: 10 μM AM-5308 wells.

[0340] Calculate IC using GraphPad Prism 8 software 50 (nM) and plot the effect-dose curve of the compound.

[0341] Table 1 Cell activity data of the compounds in this application

[0342]

[0343]

[0344] Test Example 2

[0345] Test Name: ADP-Glo TM kinase assay

[0346] Operating steps:

[0347] 1) Prepare 1× reaction buffer.

[0348] 2) Transfer 100 nmL of the diluted compound stock solution to each well of the reaction plate using an Echo 655. The final concentration of DMSO is 1%.

[0349] 3) Seal the reaction plate with a sealing film and centrifuge at 1000g for 1 minute.

[0350] 4) Prepare 2× enzyme solution using 1× reaction buffer.

[0351] 5) Add 5 μL of 2× enzyme solution to each well of the reaction plate. Seal the plate with sealing film, centrifuge at 1000g for 1 minute, and incubate at room temperature for 15 minutes.

[0352] 6) Prepare a 2×ATP solution using 1× reaction buffer.

[0353] 7) Add 5 μL of 2×ATP solution to the reaction plate, centrifuge at 1000g for 1 minute to start the reaction.

[0354] 8) React at room temperature for 60 minutes.

[0355] 9) Add 10 μL of ADP Glo reagent. Centrifuge at 1000g for 1 minute and incubate at room temperature for 60 minutes.

[0356] 10) Add 20 μL of kinase detection reagent. Centrifuge at 1000g for 1 minute and incubate at room temperature for 60 minutes.

[0357] 11) Centrifuge at 1000g for 1 minute.

[0358] 12) Read the light emission signal on Envision 2104.

[0359] Data Analysis:

[0360] The inhibition percentage is calculated as follows:

[0361] % Inhibition rate = 100 - (Signal) cmpd -Signal Ave_PC ) / (Signal Ave_VC -Signal Ave_PC )×100

[0362] Signal cmpd : The average value of the compounds tested on the reaction plate.

[0363] Signal Ave_PC : The average value of the positive control on the reaction plate.

[0364] Signal Ave_VC : The average value of the negative control on the reaction plate.

[0365] Computing IC 50 And the dose-response curve of the fitted compound:

[0366] Using GraphPad 8.0, the IC of the compound is obtained using a nonlinear fitting formula. 50.

[0367] 3) Quality Control

[0368] Z factor > 0.5; S / B > 2.

[0369] Table 2 Enzyme activity data of the compounds in this application

[0370]

[0371] The embodiments of the technical solution of the present invention have been described above by way of example. It should be understood that the protection scope of the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the protection scope of the claims of this application.

Claims

1. The compound represented by formula (I), its racemate, stereoisomer, tautomer, or pharmaceutically acceptable salt: （I） in, X1, X2, and X3 may be the same or different, and are independently selected from N or CR0; R0 is selected from H, halogens, and C. 1-6 Alkyl or halogenated C 1-6 alkyl; A is selected from , or ; Y1, Y2, and Y3 may be the same or different, and are independently selected from N or CH; M is selected from ; E is selected from .

2. The compound according to claim 1, wherein its racemic, stereoisomer, tautomer, or pharmaceutically acceptable salt, wherein, X2 and X3 are not both N.

3. The compound according to claim 1, wherein its racemic, stereoisomer, tautomer, or pharmaceutically acceptable salt, wherein, When X1 is N or CH, and X2 is CH, X3 is N or CR0, and R0 is selected from H, halogen, or C. 1-6 alkyl; When X1 is N or CR0, and X2 is N, X3 is N or CR0; R0 is selected from H, halogen, or C. 1-6 alkyl.

4. The compound according to claim 1, wherein its racemic, stereoisomer, tautomer, or pharmaceutically acceptable salt, wherein, When X1 and X2 are N, X3 is CR0, and R0 is selected from H or C. 1-6 alkyl; When X1 is N or CH, X2 is CH, X3 is N or CR0, and R0 is selected from H or C. 1-6 alkyl.

5. The compound according to claim 1, wherein its racemic, stereoisomer, tautomer, or pharmaceutically acceptable salt, wherein, When Y1 is N, Y2 is CH, and Y3 is either N or CH.

6. The compound according to claim 1, wherein its racemic, stereoisomer, tautomer, or pharmaceutically acceptable salt, wherein, The compound is selected from the following structures: 、 、 、 、 ； Wherein, A, E, M, X2, X3, Y1, Y2, and Y3 have the definitions described in claim 1.

7. The compound according to claim 1, wherein its racemic, stereoisomer, tautomer, or pharmaceutically acceptable salt, wherein, The compound has the structure shown in formula (III): (III) Wherein, A and E have the definitions described in claim 1; It represents a carbon-carbon double bond.

8. For the following compounds, their racemic, stereoisomer, tautomer, or pharmaceutically acceptable salt: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 9. A method for preparing the racemic, stereoisomer, tautomer, or pharmaceutically acceptable salt of the compound according to any one of claims 1-7, wherein, Includes the following steps: (1) Compound a reacts with compound b to give compound c; (2) Compound c reacts with EH to give the compound shown in formula (I); Wherein, A, E, M, X1, X2, X3, Y1, Y2, and Y3 independently have the definition described in any one of claims 1-7; X is selected from Cl, Br, and I.

10. A pharmaceutical composition comprising a therapeutically effective amount of at least one of the compounds of any one of claims 1-8, a racemic mixture, a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof.

11. Use of at least one of the compounds of any one of claims 1-8, their racemic, stereoisomers, tautomers, or pharmaceutically acceptable salts in the preparation of a medicament for treating KIF18A-mediated symptoms and / or diseases.

12. The use according to claim 11, wherein, The intended use is in the preparation of KIF18A inhibitors.

13. The use according to claim 11, wherein, The disease in question is cancer.

14. The use according to claim 13, wherein, The cancers mentioned include colorectal cancer, breast cancer, lung cancer, pancreatic cancer, prostate cancer, bladder cancer, head and neck cancer, cervical cancer, or ovarian cancer.

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