WO2025011445A1 - Pyrimidinamine nuak inhibitor, preparation method therefor, and use thereof - Google Patents

Abstract

The present application provides a pyrimidinamine compound. The pyrimidinamine compound is a compound represented by formula I, or a stereoisomer, tautomer, isotope derivative, hydrate, solvate, prodrug, and pharmaceutically acceptable salt thereof. The pyrimidinamine compound according to the present application has excellent NUAK1/NUAK2 kinase inhibitory activity, and can be used for preventing or treating the following diseases by inhibiting NUAK1/NUAK2: neuropsychiatric diseases, metabolic diseases, tumors, fibrotic diseases of the internal organs, and skin fibrosis diseases.

Description

A pyrimidineamine NUAK inhibitor and its preparation method and use

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application No. 202310866021.0 filed on July 13, 2023, the entire contents of which are incorporated herein by reference.

Technical Field

The present invention belongs to the field of small molecule compounds, and specifically relates to a pyrimidineamine NUAK inhibitor and its preparation method and use. The compound can be used to prevent or treat the following diseases by inhibiting NUAK1/NUAK2: neuropsychiatric diseases, metabolic diseases, tumors, visceral fibrosis diseases, skin fibrosis diseases, or used alone to reduce trauma and postoperative scars.

Background Art

Protein kinases are a group of important functional proteins involved in regulating cell metabolism, polarity, growth, division and differentiation. The human genome encodes more than 500 protein kinases, which can transfer the phosphate group on ATP (adenosine triphosphate) to specific serine, threonine or tyrosine residues of substrate proteins to phosphorylate them; most protein kinases are serine/threonine kinases, and there are less than 100 tyrosine kinases. Adenosine monophosphate-activated protein kinase (AMPK) belongs to serine/threonine protein kinases (STKs) and is an important regulator of cellular energy homeostasis in mammals. It regulates sugar and lipid metabolism, cell proliferation and cell polarity by sensing changes in the ratio of AMP (Adenosine monophosphate)/ATP or ADP (adenosine diphosphate)/ATP in cells under metabolic stress (such as hypoxia, heat shock). It can be called a metabolic sensor protein. AMPK is highly conserved in evolution. It is a heterotrimeric protein consisting of an α subunit containing a kinase domain (KD), and β and γ subunits that regulate kinase activity. The γ subunit contains four CBS domains (Cystathionine-β-synthase (CBS) domains) that are responsible for detecting changes in the intracellular AMP/ATP and ADP/ATP ratios (Hardie DG, Trends in Cell Biology.2016,26:190).

AMPK dysfunction can lead to obesity, diabetes, inflammatory diseases, tumors and other diseases, so the body's regulation of AMPK function is critical. First, the activation of AMPK requires the upstream kinase to phosphorylate the threonine (T172) at position 172 of its α subunit. Currently, there are three known AMPK upstream kinases: liver kinase B1 (LKB1), calcium ion/calmodulin-dependent protein kinase 2 (Ca ²⁺ /calmodulin-dependent PK kinase 2, CaMKK2), and transforming growth factor β-activated kinase 1 (TAK1). Corresponding to the activation of AMPK by upstream kinases through phosphorylation of the T172 site, three protein phosphatases are known to inhibit the activity of AMPK by removing the phosphate group on T172, including protein phosphatase 2A (PP2A), protein phosphatase 2C (PP2C), and magnesium/manganese-dependent protein phosphatase 1E (Mg ²⁺ -/Mn ²⁺ -dependent protein phosphatase 1E, PPM1E). When the cell is in a low-energy state (high AMP/ATP or ADP/ATP ratio), the α subunit KD and γ subunit of AMPK are tightly cross-linked, and the β subunit is myristoylated, ensuring that the protein phosphatase cannot access the T172 site and remains activated. When the cell is in a high-energy state, the KD and γ subunits are loosened, and T172 is exposed to the phosphatase, resulting in AMPK inactivation (Steinberg GR, Nature Reviews Drug Discovery. 2019, 18: 527).

In addition to the upstream kinases and protein phosphatases of AMPK that can activate and inactivate AMPK, there is also a class of AMPK-related kinases (ARKs) that participate in regulating the function of AMPK. Currently, there are 12 ARKs (BRSK1, BRSK2, NUAK1, NUAK2, QIK, QSK, SIK, MARK1, MARK2, MARK3, MARK4 and MELK), all of which are serine/threonine protein kinases. The kinase domain of ARKs has a high homology with the α subunit of AMPK. Except for MELK, they can be activated by LKB1, and their kinase activation phosphorylation sites are also equivalent to T172 of AMPK. Functionally, they are also involved in the regulation of cell metabolism, proliferation and polarity. However, unlike AMPK, ARKs cannot be directly regulated by the intracellular AMP/ATP ratio because they do not have regulatory subunits (Bright NJ, Acta Physiologica. 2009, 196: 15).

ARKs are divided into several ARK subfamilies due to their different protein structures and functional characteristics. The NUAK (Nu (novel) and AMPK-related kinase) ARK subfamily contains two members, NUAK1 (originally named ARK5) and NUAK2 (originally named sucrose nonfermenting-like/AMPK related kinase, SNARK). The amino acid sequences of the two are about 55% homologous. According to the amino acid sequences, the estimated molecular weights of NUAK1 and NUAK2 are 76 and 69 kDa, respectively. Very similar, the amino terminus is a kinase domain, and the carboxyl terminus is a ubiquitin-associated domain. It is not clear whether NUAK is also a heterotrimeric structure like AMPKs. NUAK1 and NUAK2 are expressed in most tissues, among which NUAK1 is expressed significantly higher than other parts in organs and tissues such as the brain, skin, muscle, upper digestive tract, and endocrine. The organs and tissues with the highest expression of NUAK2 are the digestive tract, female reproductive system, skin, bone, brain, and endocrine, among which NUAK2 expression shows more tissue specificity. It is worth mentioning that NUAK1 and other ARKs and AMPK proteins are mainly distributed in the cytoplasm, while NUAK2 is mainly distributed in the nucleus, and there are signs that NUAK2 is involved in the expression of genes related to metabolic stress as a transcriptional regulator (Sun X, J of Molecular Endocrinology. 2013, 51: R15).

As serine/threonine protein kinases, NUAK1 and NUAK2 can phosphorylate a variety of protein substrates, including proteins involved in cell signaling, metabolism, cell proliferation, apoptosis, autophagy, and cytoskeletal organization. NUAK1 is known to phosphorylate AMPK, LATS1/2, p53 tumor suppressor protein, and myosin phosphatase target subunit 1 (Mypt1) that regulates actin cytoskeletal organization. NUKA2 can phosphorylate the transcription factor Gli3 of the Hedghog signaling pathway, FoxO1, kinesin light chain 1 (KLC1), and Rho GDP dissociation inhibitor α (Rho GDIα).

The functional regulation of NUAK1 and NUAK2 involves a variety of intracellular signaling systems. In addition to being activated by LKB1 phosphorylation (T211, equivalent to T172 of AMPK) and calcium ion/PKC activation, NUAK1 is the only member of the AMPK-related protein family that can be activated by Akt. Increased NUAK1 activity is also seen in the activation of some growth factors such as insulin-like growth factor 1 (IGF1) signaling pathways. When skeletal muscle cells are in a contractile state, NUAK1 activity is also enhanced. Both NUAK1 and NUAK2 can be activated by LKB1 (T211/NUAK1, T208/NUAK2, equivalent to T172 of AMPK), but NUAK2 can be activated by autophosphorylation. NUAK2 can interact with ubiquitin-specific protease 9 (USP9X) on the X chromosome, which is a deubiquitinating enzyme that can maintain the activity of NUAK2. In different cells, stimuli such as low osmotic pressure, DNA damage, oxidation, and malnutrition can lead to the activation of NUAK2. NUAK2 activation is also seen in the contraction of skeletal muscle cells (Brooks D, Data in Brief. 2022, 43:108482).

Many studies have shown that NUAK plays an important role in the pathogenesis of metabolic diseases, tumors, neurodegenerative diseases, and fibrotic diseases. NUAK1 and NUAK2 homozygous knockout mice basically lead to embryonic death, and NUAK1 heterozygous deletion mice lead to the body and neural tissues (such as cerebral cortex) The human NUAK1 heterozygous gene mutation is associated with autism spectrum disorders (ASD), cognitive deficits, attention deficit/hyperactivity disorder (AD/HD) and schizophrenia. Human NUAK2 gene deletion leads to anencephaly, a severe neural tube defect that causes fetal developmental defects, and its mechanism is related to the loss of YAP function (Bonnard C, J Exp Med. 2020, 217: e20191561). Skeletal muscle cell-specific knockout of the NUAK1 gene can prevent high-fat diet-induced subclinical diabetes; the phenotype of NUAK2 heterozygous deletion mice is similar to a series of clinical manifestations of human type 2 diabetes with obesity, which may be related to the imbalance of cellular autophagy mechanism (Bennison SA, Cellular Signaling. 2022, 100: 110472; Blazejewski SM, Scientific Reports. 2011, 11: 8156).

NUAK1 activation caused by Akt and other protein kinases can promote the survival of tumor cells in an energy-deficient environment and protect tumor cells from apoptosis. NUAK2 also inhibits TNFα and CD95-induced apoptosis through a similar mechanism. In addition, after NUAK1 activation, it promotes the invasion and metastasis of tumor cells by upregulating matrix metalloproteinases (MMP), while NUAK2 participates in the occurrence of tumor metastasis by enhancing the activity of tumor cells (Hou X, Oncogen. 201, 30: 2933; Chen Y, Cell Death and Disease. 2020, 11: 712; Molina E, Cells. 10: 2760; Humbert N, The EMBO Journal. 2010, 29: 376). There is a lot of evidence that NUAK1 and NUAK2 play a role in tumor formation and metastasis. Compared with NUAK1, NUAK2 has a stronger effect in promoting tumor formation and metastasis. NUAK2 inhibitors should be more likely to become new targeted anti-tumor drugs than NUAK1 inhibitors.

Recent studies have shown that NUAK plays a key role in the development of tissue fibrosis by interacting with the transforming growth factor-β (TGF-β) signaling pathway. First, TGF-β can upregulate the gene transcription of NUAK1 and NUAK2 in a variety of epithelial cells such as keratinocytes and human dermal fibroblasts, while the inactivation of MAPK (ERK1/2and p38) signaling inhibits TGF-β-dependent NUAK2 expression; in addition, NUAK2 inhibits the degradation of SMAD3 in cells by binding to the cross-linking domain and MH2 domain on the intracellular signal mediator SMAD3 protein structure of TGF-β. A similar mechanism also exists between NUAK2 and TβRI. The expression of genes encoding profibrotic molecules such as fibronectin (FN), plasminogen activator inhibitor 1 (PAI1), and tissue inhibitor of metalloproteinase-1 (TIMP1) induced by TGF-β depends on the presence of NUAK2, which shows that NUAK2 promotes the occurrence of fibrosis. Studies have also shown that NUAK1 promotes the development of kidney, lung and prostate by upregulating TGF-β and YAP signaling pathways. Liver fibrosis. In animal experiments, inhibition of NUAK1 can reduce scars caused by new trauma and old scar tissue (Gill MK, Nature Communications. 2018.9: 3510). Interestingly, the study found that the expression of the gene encoding FN in keratinocytes with NUAK1 knockout gene was upregulated, and NUAK1 may affect TGF-β signaling through a negative feedback mechanism to inhibit fibrosis (van de Vis RAJ, Cancers. 2021, 13: 3377). In general, inhibition of NUAK may effectively inhibit the fibrosis process of tissues and organs involved.

Since kinases have always been ideal small molecule drug targets in terms of structure and function, and NUAK1 and NUAK2 are involved in the occurrence of many diseases, some NUAK inhibitors are currently in the early stages of research and development, and their effectiveness and safety are still in doubt (Banerjee S, The Biochemical Journal. 2014, 457: 215). Therefore, the development of NUAK selective inhibitors and NUAK1/2 dual-target inhibitors is expected to provide a new direction for innovative treatment of neuropsychiatric diseases (such as Parkinson's disease, Alzheimer's disease), metabolic diseases (such as diabetes, hyperlipidemia, obesity), tumors (such as liver cancer, leukemia, lymphoma, tumor metastasis), visceral fibrosis diseases (such as cirrhosis, renal fibrosis, pulmonary fibrosis, and sequelae of myocarditis) and skin fibrosis diseases (such as scleroderma, keloids, hypertrophic scars, or simply used to reduce trauma and postoperative scars). The development of a new generation of NUAK inhibitors has great potential clinical application value.

Summary of the invention

The purpose of the present invention is to obtain effective NUAK1/NUAK2 inhibitors, which can be used to prepare drugs for preventing or treating the following diseases: neuropsychiatric diseases (such as Parkinson's disease, Alzheimer's disease), metabolic diseases (such as diabetes, hyperlipidemia, obesity), tumors (such as liver cancer, leukemia, lymphoma, tumor metastasis), visceral fibrosis diseases (such as cirrhosis, renal fibrosis, pulmonary fibrosis, and sequelae of myocarditis) and skin fibrosis diseases (such as scleroderma, keloids, hypertrophic scars, or simply used to reduce trauma and post-operative scars).

In order to achieve the above object, in one aspect, the present invention provides a pyrimidine amine compound, which is a compound represented by Formula I as shown below, or its stereoisomers, geometric isomers, tautomers, isotopic derivatives, hydrates, solvates, prodrugs and pharmaceutically acceptable salts:

Wherein, 0 or 1 of X ₁ , X ₂ , and X ₃ is N, and the others are CH. When X ₁ , X ₂ , and X ₃ are CH, H may be substituted by R ₅ or R ₄ ;

A is an optionally substituted 6-10 membered aryl or an optionally substituted 5-10 membered heteroaryl, B is an optionally substituted 3-10 membered cycloalkyl or an optionally substituted 3-10 membered heterocycloalkyl, and the substituents on A or B are selected from one or more of halogen, amino, hydroxy, nitro, cyano, mercapto, optionally substituted C1-8 alkyl, optionally substituted C1-8 alkyloxy, optionally substituted C1-8 alkylthio, and optionally substituted C1-8 alkylamino;

R ₁ and R ₂ are each independently H or optionally substituted C1-8 alkyl;

R ₃ and R ₄ are each independently halogen, amino, hydroxy, nitro, cyano, mercapto, optionally substituted C1-8 alkyl, optionally substituted C1-8 alkyloxy, optionally substituted C1-8 alkylthio, or optionally substituted C1-8 alkylamino;

_R6 is an optionally substituted C1-8 alkyl, an optionally substituted 3-10 membered cycloalkyl, or an optionally substituted 3-10 membered heterocycloalkyl, wherein the substituent on the cycloalkyl or heterocycloalkyl is selected from one or more of halogen, amino, hydroxy, nitro, cyano, mercapto, an optionally substituted C1-8 alkyl, an optionally substituted C1-8 alkyloxy, an optionally substituted C1-8 alkylthio, and an optionally substituted C1-8 alkylamino;

The substituent on the C1-8 alkyl group is selected from one or more of halogen, amino, hydroxyl, nitro, cyano, and mercapto;

n is 0, 1 or 2, m is 0, 1, 2, 3 or 4, and p is 0, 1, 2, 3, 4, 5 or 6.

In one group of embodiments, R ₆ is an optionally substituted 3-10 membered cycloalkyl, preferably an optionally substituted 3-6 membered cycloalkyl, preferably a halogen substituted 3-6 membered cycloalkyl, more preferably R ₆ is a fluorine substituted 3-6 membered cycloalkyl.

In one group of embodiments, _R6 is

In one group of embodiments, _R5 is Preferably, _R5 is

In one group of embodiments, _R5 is substituted at the para or meta position of the pyrimidinyl group.

In one group of embodiments, B is an optionally substituted 3-6 membered heterocycloalkyl, preferably B is an optionally substituted 5-6 membered heterocycloalkyl, preferably B is an optionally substituted piperidinyl or an optionally substituted piperazinyl.

In one group of embodiments, the compound represented by Formula I is Formula I-1 or Formula I-2,

_R7 is selected from halogen, amino, hydroxyl, nitro, cyano, mercapto, optionally substituted C1-8 alkyl, optionally substituted C1-8 alkyloxy, optionally substituted C1-8 alkylthio, optionally substituted C1-8 alkylamino, _R8 is H or optionally substituted C1-8 alkyl; q is 0, 1, 2, 3, or 4, and the substituent on the C1-8 alkyl is selected from one or more of halogen, amino, hydroxyl, nitro, cyano, mercapto; other groups are defined as above. Preferably _R8 is H or methyl.

In one group of embodiments, A is optionally substituted phenyl or optionally substituted 5-membered heteroaryl.

Further, A is one of the following groups which are optionally substituted:

Preferably, it is connected to the amino group at the top and to the B group at the bottom.

In one group of embodiments, X ₁ -X ₃ are all CH.

In one group of embodiments, the substituent on the A ring is a methoxy group, preferably substituted at the ortho position to the amino group.

In one group of embodiments, _R3 is chloro, preferably substituted in the para position relative to the amino group.

In one group of embodiments, the compound represented by formula I is one of the following compounds:

In one group of embodiments, the pharmaceutically acceptable salt is a formate salt.

In one set of embodiments, the isotopic derivative is a deuterated derivative.

On the other hand, the present application also provides a method for preparing the pyrimidine amine compound, which comprises the following steps:

The compound of formula I is prepared from the compound of formula II and the compound of formula III;

or

The compound of formula V is prepared from the compound of formula IV and the compound of formula II, and the compound of formula I is prepared from the compound of formula V and the compound of formula VI;

Optionally, the preparation process includes necessary protection and deprotection steps;

Wherein, Y is a leaving group, preferably Y is a halogen, more preferably Y is Cl, and the definitions of the other groups are as described above.

In one embodiment, the amino group or imino group on the B ring in formula II is first protected, coupled with the compound of formula III, and then the protecting group is removed to prepare the compound of formula I.

In one group of embodiments, the amino or imino group to which _R2 is attached in the compound of formula IV is protected, and the amino or imino group on the B ring in formula II is optionally protected, and then the protected compound of formula IV is coupled with the optionally protected compound of formula II, and the protecting group on the amino or imino group to which _R2 is attached is removed to prepare the compound of formula V, and finally the compound of formula V is reacted with the compound of formula VI, and the protecting group on the B ring is optionally removed to prepare the compound of formula I.

The present application provides a pharmaceutical composition, which uses the pyrimidineamine compound described above in the present application as an active ingredient; the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

The present application provides the use of the pyrimidineamine compounds for preparing NUAK1 or NUAK2 inhibitors.

The present application provides the use of the pyrimidineamine compound for preparing a drug, characterized in that the compound is used to prevent or treat the following diseases by inhibiting NUAK1 or NUAK2: neuropsychiatric diseases, metabolic diseases, tumors, visceral fibrosis diseases, and skin fibrosis diseases.

In one group of embodiments, the disease is Parkinson's disease, Alzheimer's disease, diabetes, hyperlipidemia, obesity, liver cancer, leukemia, lymphoma, tumor metastasis, cirrhosis, renal fibrosis, pulmonary fibrosis, sequelae of myocarditis, scleroderma, keloid, hypertrophic scar, or is used alone to reduce scars after trauma and surgery.

The beneficial effects of the present invention are:

The present invention provides a class of pyrimidine amine compounds. In vitro kinase activity inhibition tests show that the present invention The compound has excellent inhibitory activity on NUAK1/NUAK2 kinases and can be used to prevent or treat the following diseases by inhibiting NUAK1 or NUAK2: neuropsychiatric diseases, metabolic diseases, tumors, visceral fibrosis diseases, and skin fibrosis diseases.

DETAILED DESCRIPTION

The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

The endpoints and any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

Before describing the present invention in detail, it should be understood that the terms used herein are only intended to describe specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. In order to more fully understand the present invention described herein, the following terms are used and their definitions are as follows. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by those of ordinary skill in the art to which the present invention belongs.

definition

Unless otherwise specified, the following terms used in the present invention have the following definitions.

Features illustrated or described as part of one embodiment or group of embodiments can be used on another embodiment or group of embodiments to yield a still further embodiment.

In the present invention, the ring group It means that there can be n R _{3 s} , m R _{4 s} , and q R _{7 s} connected to any possible position on the ring group, and R ₅ can be connected to any possible position on the ring group.

In the present invention, some substituents The ligation site is indicated.

Unless otherwise specified, the term "optionally substituted" means that the hydrogen on the substituted group is not replaced or one or more substitutable positions of the substituted group are independently substituted by substituents, and the substituents are independently selected from one or more deuterium, halogen, amino, hydroxyl, nitro, cyano, mercapto, oxo, optionally substituted C1-8 alkyl, optionally substituted C1-8 alkyloxy, optionally substituted C1-8 alkylthio, optionally substituted C1-8 One or more alkylamino groups; the substituents on the C1-8 alkyl group are selected from one or more of deuterium, halogen, amino, hydroxyl, nitro, cyano and mercapto; the substituents are selected from "oxo" when two hydrogen atoms at the same substitution position are replaced by oxygen atoms.

The term "aryl" refers to a monocyclic or bicyclic aromatic carbocyclic ring system containing 6 to 10 carbon atoms. Examples of aryl groups include phenyl and naphthyl.

The term "heteroaryl" refers to an aromatic monocyclic or polycyclic ring system containing a 5-10-membered structure, or preferably a 5-8-membered structure, more preferably a 5-6-membered structure, wherein 1, 2, 3, 4 or more ring atoms are heteroatoms and the remaining atoms are carbon, the heteroatoms are independently selected from O, N or S, and the number of heteroatoms is preferably 1, 2, 3 or 4. The heteroaryl group may be an aromatic monocyclic ring containing 1-2 5-6-membered structures selected from N or S, and is preferably an aromatic monocyclic ring containing 1-2 N 5-6-membered structures. Examples of heteroaryl groups include, but are not limited to, furanyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, thiodiazolyl, triazinyl, phthalazinyl, quinolyl, isoquinolyl, pteridinyl, purinyl, indolyl, isoindolyl, indazolyl, benzofuranyl, benzothiophenyl, benzopyridinyl, benzopyrimidinyl, benzo pyrazinyl, benzimidazolyl, benzophthalazinyl, pyrrolo[2,3-b]pyridinyl, imidazo[1,2-a]pyridinyl, pyrazolo[1,5-a]pyridinyl, pyrazolo[1,5-a]pyrimidinyl, imidazo[1,2-b]pyridazinyl, [1,2,4]triazolo[4,3-b]pyridazinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl, [1,2,4]triazolo[1,5-a]pyridinyl, and the like.

The term "cycloalkyl" refers to a saturated monocyclic, bicyclic or tricyclic ring system containing 3-10 carbon atoms, wherein the monocyclic, bicyclic or tricyclic ring does not contain an aromatic ring, including a bridged ring group, a spirocyclic group, a cyclocyclic group, etc. Preferably, it contains 3-10 carbon atoms (C3-10 cycloalkyl), and more preferably 3-8 carbon atoms (C3-8 cycloalkyl), 3-6 carbon atoms (C3-6 cycloalkyl), 4-6 carbon atoms (C4-6 cycloalkyl), 5-6 carbon atoms (C5-6 cycloalkyl). Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylcyclopropyl, 2-ethyl-cyclopentyl, dimethylcyclobutyl, etc.

The term "heterocycloalkyl" refers to a saturated monocyclic, bicyclic or polycyclic hydrocarbon group, preferably containing 3-10 ring atoms, wherein 1, 2, 3 or more ring atoms are selected from N, O or S, and the remaining ring atoms are C, including bridged ring groups, spirocyclic groups, and annular groups. Preferably, it contains 3 to 8 ring atoms (3-8 membered heterocycloalkyl), or 3-6 ring atoms (3-6 membered heterocycloalkyl), or 4-6 ring atoms (4-6 membered heterocycloalkyl), or 5-6 ring atoms (5-6 membered heterocycloalkyl). The number of heteroatoms is preferably 1-4, more preferably 1 to 3 (i.e., 1, 2 or 3). The heterocycloalkyl group may be a 5-6 membered monocyclic heterocycloalkyl group containing 1-2 N. Examples of heterocycloalkyl groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, pyranyl, aziridine, oxirane, thiirane, azetidinyl, oxetanyl, thietanyl, oxhexane, morpholinyl, thiomorpholinyl, dioxanyl, dithioxanyl, oxazolidinyl, thiazolidinyl, pyrazolidinyl, imidazolinidine, and the like.

The term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group, preferably containing 1-8 carbon atoms (C1-8 alkyl) straight or branched groups (the number of carbon atoms is between 1-8, specifically 1, 2, 3, 4, 5, 6, 7 or 8), more preferably containing 1-6 carbon atoms (i.e. C1-6 alkyl, the number of carbon atoms is between 1-6, specifically 1, 2, 3, 4, 5 or 6). Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, n-heptyl, n-octyl, etc.

The terms "alkyloxy", "alkylthio" and "alkylamino" refer to -O-alkyl, -S-alkyl, -NH-alkyl or dialkylamino, respectively, wherein the alkyl is as defined above. Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy, tert-butoxy, etc.; methylthio, ethylthio, propylthio, isopropylthio, butylthio, 1-methylpropylthio, 2-methylpropylthio, tert-butylthio, etc.; methylamino, ethylamino, propylamino, dimethylamino, diethylamino, dipropylamino, methylethylamino, etc.

The term "halogen" refers to F, Cl, Br, I.

The active compound of the present invention is interpreted as including the compound and its stereoisomers, tautomers, isotopic derivatives, hydrates, solvates, prodrugs or pharmaceutically acceptable salts. The stereoisomers, tautomers, isotopic derivatives, hydrates, solvates, prodrugs, isotopic derivatives or pharmaceutically acceptable salts of the compound are obtained by conventional technical means in the art, and exert the same or similar effects in vivo and in vitro with substantially the same mechanism of action as the compound.

The term "stereoisomer" refers to isomers produced by different spatial arrangements of atoms in a molecule, including configurational isomers and conformational isomers, wherein configurational isomers include geometrical isomers (or cis-trans isomers) and optical isomers (including enantiomers and diastereomers). Geometrical isomers may exist in the present compound. Optical isomers refer to substances with exactly the same molecular structure, similar physical and chemical properties, but different optical rotations. The compounds of the present invention may contain asymmetrically substituted in the R or S configuration. Carbon atoms, wherein the terms "R" and "S" are as defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, Pure Appl. Chem. (1976) 45, 13-10. Compounds with asymmetrically substituted carbon atoms (having equal numbers of R and S configurations) are racemic at those carbon atoms. Atoms with an excess of one configuration (relative to the other) result in a higher number of that configuration being present, preferably an excess of about 85%-90%, more preferably an excess of about 95%-99%, and even more preferably an excess of greater than about 99%. Accordingly, the present invention includes racemic mixtures, relative and absolute optical isomers, and mixtures of relative and absolute optical isomers.

The term "tautomer" refers to structural isomers with different energies that can be interconverted through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomers can be achieved. For example, proton tautomers (also known as prototropic tautomers) include interconversions through proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions through the reorganization of some bonding electrons.

The term "isotopic derivative" means that the compounds of the present invention may exist in an isotopically-tagged or enriched form, containing one or more atoms whose atomic mass or mass number is different from the atomic mass or mass number of the atom found in maximum amount in nature. Isotopes may be radioactive or non-radioactive isotopes. Isotopes of atoms such as hydrogen, carbon, phosphorus, sulfur, fluorine, chlorine and iodine include, but are not limited to: ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ O, ³² P, ³⁵ S, ¹⁸ F, ³⁶ Cl and ¹²⁵ I. Compounds containing other isotopes of these and/or other atoms are within the scope of the present invention. Isotopically-labeled compounds of the present invention can be prepared by general methods well known to those of ordinary skill in the art.

The term "hydrate" refers to an association of one or more water molecules with a compound of the present invention.

The term "solvate" refers to an association of one or more solvent molecules with a compound of the present invention.

The term "prodrug" is a designed derivative of an active drug that improves some defined, undesirable physical or biological property. Physical properties are usually related to solubility (too high or insufficient lipid or water solubility) or stability, while problematic biological properties include too rapid metabolism or poor bioavailability, which themselves may be related to physicochemical properties.

The term "pharmaceutically acceptable salt" refers to salts that are suitable for contact with mammalian tissues, especially human tissues, without excessive toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit/risk ratio within the scope of reasonable medical judgment. If the compound is basic, the pharmaceutically acceptable salt comprises a salt prepared from an inorganic acid The pharmaceutically acceptable salts include salts prepared from inorganic bases and/or organic bases.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the relevant governmental regulatory authorities as acceptable for use by humans or livestock.

As used herein, the term "treatment" refers to any administration of a therapeutic agent according to a therapeutic regimen that achieves the desired effect, i.e., partially or completely alleviates, improves, alleviates, inhibits, delays the onset, reduces the severity, and/or reduces the incidence of one or more symptoms or features of a specific disease, disorder, and/or condition; in some embodiments, the administration of a therapeutic agent according to a therapeutic regimen is associated with the achievement of the desired effect. This treatment can be directed to subjects who do not show relevant diseases, disorders, and/or conditions and/or to subjects who only show early signs of diseases, disorders, and/or conditions. Alternatively or additionally, this treatment can be directed to subjects who show one or more determined signs of relevant diseases, disorders, and/or conditions. In some embodiments, treatment can be directed to subjects who have been diagnosed with relevant diseases, disorders, and/or conditions. In some embodiments, treatment can be directed to subjects known to have one or more susceptibility factors that are statistically associated with an increased risk of developing relevant diseases, disorders, and/or conditions.

According to the present invention, the medicine prepared in the pharmaceutical use described in the present application may contain, in addition to the pyrimidineamine compound of the present invention as an active ingredient, other agents that can be used to prevent or treat related diseases as another active ingredient. When the medicine contains multiple active ingredients, each active ingredient can be administered simultaneously, sequentially or separately according to the judgment of the physician.

Hereinafter, the effects of the specific compounds of the present invention will be described in detail through examples.

Example

Example 1 General method for synthesizing compound 518 (TDM-181118)

Step 1: Compound 518c

Tert-butyl (S)-4-(4-((4-(4-(2,2-difluorocyclopropane-1-carboxamido)phenyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)piperidine-1-carboxylate

To a mixture of compound 518a (20 mg, 0.065 mmol), compound 518b (26 mg, 0.097 mmol), palladium acetate, xantphos (37 mg, 0.065 mmmol) and cesium carbonate (42 mg, 0.129 mmmol) was added dioxane (2 mL). The mixture was degassed under vacuum, replaced with argon several times, heated to 100 ° C and stirred for 2 hours. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (10% methanol in dichloromethane/dichloromethane=0-27/73) to obtain a yellow solid product (compound 518c, 17.3 mg, yield 49.3%). LCMS[M+1] ⁺ =540.

Step 2: Compound 518

(S)-2,2-Difluoro-N-(4-(2-((1-(piperidin-4-yl)-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)phenyl)cyclopropane-1-carboxamide

A 4M hydrochloric acid dioxane solution (0.07 mL) was added to a solution of compound 518c (17.3 mg, 0.028 mmol) in methanol (0.5 mL) and dichloromethane (2 mL), and the mixture was stirred at room temperature overnight. The reactant was concentrated under reduced pressure, and the residue was purified by preparative HPLC (formic acid) to give a white solid product (compound 518, TDM-181118, 13.5 mg, yield 33%). LCMS [M+1] ⁺ = 440.

¹ H NMR (400MHz, DMSO) δ10.76(s,1H),9.47(s,1H),8.45(d,J＝5.1Hz,1H),8.35(s,1H),8.13(d,J＝8.7Hz, 2H),7.98(s,1H),7.77(d,J＝8.5Hz,2H),7.62 (s,1H),7.24(d,J＝5.2Hz,1H),4.33(s,1H),3.21(d,J＝12.5Hz,2H),2.93-2.75(m,3H),2.13 -1.87( m,6H).

N-(4-(2-((1-(piperidin-4-yl)-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)phenyl)cyclopropanecarboxamide (TDM-181119) was synthesized as a yellow solid (18.1 mg, yield 22.5%) by a similar method to Example 1.

Example 2 General method for the synthesis of compound 522 (TDM-181122)

Step 1: Compound 522c

2-Chloro-4-(3-nitrophenyl)pyrimidine

To a solution of compound 522a (149 mg, 1 mmol), compound 522b (200 mg, 1.2 mmol) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1 mmol) in 1,2-dimethoxyethane (5 mL) was added a solution of sodium bicarbonate (158 mg, 1.88 mmol) in water (0.5 mL). The reaction solution was replaced with argon several times, heated to 80°C and stirred for 7 hours. The reaction was detected to be complete. Post-treatment: The reaction solution was concentrated to dryness, and the crude product was purified by column chromatography (10% MeOH in DCM/DCM = 0 to 1/99) to obtain the target compound (compound 522c, 137 mg, yield 58.1%) as a yellow solid, LCMS [M+1] ⁺ = 236.

Step 2: Compound 522d

3-(2-Chloropyrimidin-4-yl)aniline

Iron powder (162 mg, 2.91 mmol) and ammonium chloride (155 mg, 2.91 mmol) were added to a solution of compound 522c (137 mg, 0.58 mmol) in tetrahydrofuran (14 mL) and water (14 mL). The reaction solution was heated to 65°C and stirred for 3 hours. The reaction was detected to be complete. Post-treatment: The reaction solution was cooled to room temperature, filtered, the filtrate was concentrated, pulled dry, and sodium bicarbonate aqueous solution and ethyl acetate (20 mL) were added and extracted three times. The organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and pulled dry to obtain the target compound (compound 522d, 117 mg, yield 99%) as a yellow solid, LCMS [M+1] ⁺ = 206.

Step 3: Compound 522f

N-(3-(2-chloropyrimidin-4-yl)phenyl)cyclopropanecarboxamide

To a solution of compound 522e (75 mg, 0.88 mmol) in N,N-dimethylformamide (5 mL) were added 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (333 mg, 0.88 mmol), N,N-diisopropylethylamine (151 mg, 1.17 mmol) and compound 522d (120 mg, 0.58 mmol). The reaction solution was heated to 50°C and stirred for two hours. The reaction was detected to be complete. Post-treatment: The reaction solution was poured into water, ethyl acetate (3*100 mL) was added for extraction, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and pulled dry. The crude product was passed through a column [eluent: petroleum ether/ethyl acetate = 0-30%] to obtain the target compound (compound 522f, 122.6 mg, yield 77%) as a yellow solid, LCMS [M+1] ⁺ = 274.

Step 4: Compound 522h

tert-Butyl-4-(4-(4-(3-(cyclopropanecarboxamido)phenyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)piperidine-1-carboxylate

Into a 100-mL three-necked flask were added compound 522f (122.6 mg, 0.45 mmol), compound 522g (143 mg, 0.54 mmol), palladium acetate (20 mg, 0.09 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthene (104 mg, 0.18 mmol), cesium carbonate (293 mg, 0.9 mmol), and 1,4-dioxane (10 mL). The reaction solution was replaced with argon several times, heated to 100°C and stirred for 2 hours. The reaction was detected to be complete. Post-treatment: the reaction solution was concentrated to dryness, ethyl acetate (100 mL) and water (100 mL) were added for extraction, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and dried, and the crude product was passed through a column [eluent: (dichloromethane: methanol = 10:1)/DCM = 0-30%] to give the target compound as a yellow solid (compound 522h, 181 mg, yield 80%), LCMS [M+1] ⁺ = 504.

Step 5: Compound 2

N-(3-(2-(1-(piperidin-4-yl)-1H-pyrazol-4-ylamino)pyrimidin-4-ylphenyl)cyclopropane Formamide

To a solution of compound 522h (181 mg, 0.36 mmol) in dichloromethane (5 mL) and methanol (1 mL) was added a solution of hydrochloric acid-dioxane (1.35 mL, 5.4 mmol), and the reaction solution was stirred at room temperature for 1 hour. The reaction was detected to be complete. Post-treatment: The reaction solution was concentrated to dryness, and the crude product was purified to obtain the target compound (compound 522, TDM-181122, 46 mg, yield 31.7%) as a yellow solid, LCMS [M+1] ⁺ = 404.

¹ H NMR (400MHz, DMSO) δ10.46 (s, 1H), 9.54 (s, 1H), 8.72 (s, 1H), 8.48 (d, J = 5.1Hz, 1H ),8.35(s,1H),8.16(s,1H),7.75(d,J＝7.6Hz,1H),7.57(s,2H),7.46(t,J＝7.9Hz ,1H),7.21(d,J＝5.1Hz,1H),4.41(s,1H),3.23(d,J＝12.5Hz,2H),2.81(t,J＝11.0 Hz, 2H), 2.01 (dd, J = 25.5, 11.1Hz, 4H), 1.89-1.78 (m, 1H), 0.85 (d, J = 7.7Hz, 4H).

Example 3 General method for the synthesis of compound 525 (TDM-181125)

Step 1: Compound 525

(S)-2,2-Difluoro-N-(4-(2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-ylphenyl)cyclopropane-1-carboxamide

To a solution of compound 525a (50 mg, 0.161 mmol) and compound 525b (53.6 mg, 0.242 mmol) in n-butanol (5 mL) was added p-toluenesulfonic acid monohydrate (61 mg, 0.322 mmol). The mixture was heated to 110° C. and stirred for 3 h. The mixture was then added to water (60 mL), extracted with ethyl acetate (30 mL*3), the organic layers were combined, washed with brine and dried over sodium sulfate, the filtrate was concentrated under reduced pressure, and the residue was purified by preparative HPLC (formic acid) to give a white solid product (compound 525, TDM-181125, 27.5 mg, 34.5% yield). LCMS [M+1] ⁺ = 495.

¹ H NMR (400MHz, DMSO) δ10.67(s,1H),8.41(d,J＝5.2Hz,1H),8.12(d,J＝8.8Hz,2H),7.92(s,1H),7.89( d,J＝8.7Hz,1H),7.74(d,J＝8.8Hz,2H),7.28(d, J＝5.3Hz,1H),6.66(d,J＝2.5Hz,1H),6.54(dd,J＝8.8,2.5Hz,1H),3.83(s,3H),3.18–3.08(m,4 H), 2.85 (ddd, J＝13.6, 10.8, 8.1Hz, 1H), 2.52 (d, J＝1.9Hz, 4H), 2.25 (s, 3H), 2.12-1.93 (m, 2H).

Example 4 General method for synthesizing compound 533 (TDM-181133)

Step 1: Compound 533c

2-Chloro-4-(2-nitrophenyl)pyrimidine

Sodium bicarbonate (315.88 mg, 3.76 mmol) was added to a solution of compound 533a (297.96 mg, 2 mmol) and compound 533b (400 mg, 2.4 mmol) in dimethoxymethane (15 mL)/water (1.5 mL). Tetrakis(triphenylphosphine)palladium (231.12 mg, 0.2 mmol) was added to the mixture, and the mixture was stirred at 80°C for 7 hours. The mixture was diluted with water and extracted with ethyl acetate (50 mL×3). The combined organic layer was washed with water (3×50 mL) and brine (3×50 mL), the organic layer was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was purified by flash column chromatography (TLC: petroleum ether/acetic acid) Ethyl ester = 3/1, petroleum ether:ethyl acetate = 80:20 when passing through the column) to give the target compound as a white solid (compound 533c, 269.5 mg, 57.2% yield), LCMS [M+1] ⁺ = 236.1.

Step 2: Compound 533d

2-(2-Chloropyrimidin-4-yl)aniline

Iron powder (318.35 mg, 5.7 mmol), ammonium chloride (304.9 mg, 5.7 mmol) were added to a mixture of a tetrahydrofuran solution (40 mL) of compound 533c (269.5 mg, 1.14 mmol) and water (40 mL), and the mixture was stirred at 65°C for 5 hours, diluted with water and extracted with ethyl acetate (50 mL×3). The combined organic layer was washed with water (3×50 mL) and brine (3×50 mL), separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the target compound (compound 533d, 140 mg, 59.8% yield) was obtained by flash column chromatography (TLC: petroleum ether/ethyl acetate=5/1, petroleum ether:ethyl acetate=71:19 when passing through the column), LCMS [M+1] ⁺ =206.1.

Step 3: Compound 533f

N-(2-(2-chloropyrimidin-4-yl)phenyl)cyclopropanecarboxamide

To a pyridine solution (7 mL) of compound 533d (120 mg, 0.58 mmol) was added compound 533e (50.28 mg, 0.58 mmol), and phosphorus oxychloride (134.32 mg, 0.88 mmol) was added under ice bath. The mixture was stirred for 2 hours. The mixture was diluted with water and extracted with ethyl acetate (50 mL×3). The combined organic layers were washed with water (3×50 mL) and brine (3×50 mL). The organic layer was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by flash column chromatography (TLC: petroleum ether/ethyl acetate=3/1, petroleum ether:ethyl acetate=85:15) to give the target compound (compound 533f, 92.8 mg, 58.07% yield) as a white solid. LCMS [M+1] ⁺ =274.1.

Step 4: Compound 533h

Tert-butyl 4-(4-((4-(2-(cyclopropanecarboxamido)phenyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)piperidine-1-carboxylate

Compound 533f (72.8 mg, 0.27 mmol) was added with compound 533g (86.3 mg, 0.32 mmol) and cesium carbonate (175.9 mg, 0.54 mmol). Palladium acetate (12.1 mg, 0.05 mmol) was added to 1,4-dioxane solution (10 mL) under argon protection and filtered. The mixture was then stirred at 100 ° C for 2 hours, the mixture was diluted with water and extracted with ethyl acetate (50 mL × 3). The combined organic layer was washed with water (3 × 50 mL) and brine (3 × 50 mL), the organic layer was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the mixture was purified by flash column chromatography (TLC: dichloromethane/methanol = 10/1, When passed through a column, dichloromethane:methanol=70:30) to obtain the target compound as a white solid (compound 533h, 92.1 mg, 68.2% yield), LCMS [M+1] ⁺ =504.3.

Step 5: Compound 533

N-(2-(2-((1-(piperidin-4-yl)-1H-pyrazol-4-yl)amino)pyrimidin-4-ylphenyl)cyclopropanecarboxamide

To a solution (8 mL) of compound 533h (72.1 mg, 0.14 mmol) in dichloromethane/methanol 10:1 was added 4 mol/L dioxane hydrochloride (0.6 mL), and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC to give the target compound as a white solid (compound 533, TDM-181133, 23.4 mg, 40.6% yield), LCMS [M+1] ⁺ = 404.1.

¹ H NMR(400MHz,DMSO)δ11.45(s,1H),9.68(s,1H),8.54(d,J＝5.2Hz,1H),8.29(s,1H),8 .14(d,J＝7.7Hz,1H),7.95(s,1H),7.84-7.69(m,1H),7.57(s,1H),7.51-7.39(m,1H) ,7.23(dd,J＝11.0,4.2Hz,1H),7.10(d,J＝5.2Hz,1H),4.33(d,J＝10.6Hz,2H),3.25(d ,J＝12.4Hz,2H),2.86(t,J＝11.4Hz,2H),2.01(dd,J＝33.8,10.8Hz,5H),0.75(s,4H).

Example 5 General method for the synthesis of compound 541 (TDM-181141)

Step 1: Compound 541c

4-(5-((tert-Butoxycarbonyl)amino)thiazol-2-yl)-3,6-dihydropyridine-1(2H)-carboxylic acid Benzyl ester

To a mixture of compound 541a (610 mg, 2.185 mmol), compound 541b (900 mg, 2.622 mmol), potassium phosphate (1.39 g, 0.219 mmol) and 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride (160 mg, 6.555 mmol), dioxane (90 mL) and water (10 mL) were added, the mixture was degassed under vacuum and purged with argon several times, then heated to 90 degrees and stirred for 2.5 hours. The mixture was added with water (300 mL), extracted with ethyl acetate (80 mL*3), the organic layers were combined, washed with brine, the filtrate was concentrated under reduced pressure, and purified by silica gel chromatography (ethyl acetate/petroleum ether=0-40/60) to obtain a yellow solid product (compound 541c, 780 mg, 85.9% yield). LCMS[M+1] ⁺ =416.1.

Step 2: Compound 541d

Benzyl 4-(5-aminothiazol-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate

Zinc bromide (1.68 g, 7.466 mmol) was added to a solution of compound 541c (517 mg, 1.244 mmol) in dichloromethane (20 mL) and methanol (2 mL) under argon atmosphere, and the mixture was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (ethyl acetate/petroleum ether=0-55/45) to obtain a brown solid product (compound 541d, 410 mg, crude product). LCMS [M+1] ⁺ = 316.

Step 3: Compound 541f

Benzyl 4-(5-((4-(4-(cyclopropanecarboxamido)phenyl)pyrimidin-2-yl)amino)thiazol-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate

To a mixture of compound 541e (100 mg, 0.315 mmol), compound 541d (230 mg, 0.731 mmol), tridibenzylideneacetone dipalladium (67 mg, 0.073 mmol), xphos (69.6 mg, 0.146 mmol) and cesium carbonate (238 mg, 0.73 mmol), dioxane (5 mL) was added. The mixture was degassed under vacuum, replaced with argon several times, heated to 100 degrees and stirred for 3 hours. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (10% methanol in dichloromethane/dichloromethane=0-60/40) to obtain a black solid product (compound 541f, 185 mg, crude product). LCMS[M+1] ⁺ =533.

Step 4: Compound 541

N-(4-(2-((2-(piperidin-4-yl)thiazol-5-yl)amino)pyrimidin-4-yl)phenyl)cyclopropanecarboxamide

Compound 541f (100 mg, 0.181 mmol) was dissolved in tetrahydrofuran (2 mL) and 2,2,2-trifluoro Palladium hydroxide on carbon was added to the solution in ethanol (10 mL), the mixture was degassed under vacuum, replaced with hydrogen several times, the mixture was heated to 40 degrees and stirred overnight. The mixture was then filtered and the filtrate was concentrated under reduced pressure, and the residue was purified by preparative HPLC (methanol) to obtain a yellow solid product (compound 541, TDM-181141, 6.1 mg, 8% yield). LCMS [M+1] ⁺ = 421.

¹ H NMR (400MHz, DMSO) δ10.74(s,1H),10.53(s,1H),8.53(d,J＝5.3Hz,1H),8.40(s,1 H),8.19(d,J＝8.3Hz,2H),7.80(d,J＝8.8Hz,2H),7.40(t,J＝2.6Hz,2H),3.17(d,J＝ 12.6Hz,3H),3.10(d,J＝11.3Hz,1H),2.80(t,J＝11.2Hz,2H),2.05(d,J＝11.6Hz,2H ), 1.84 (dt, J＝12.4, 6.3Hz, 1H), 1.74 (dd, J＝21.3, 11.7Hz, 2H), 0.89-0.78 (m, 4H).

Example 6: General method for the synthesis of compound 548 (TDM-181148)

Step 1: Compound 548c

tert-Butyl-4-(4-((4-(4-(cyanomethyl)carbamoyl)-1H-pyrazol-1-yl)pyrimidin-2-yl)amino)-3-methoxyphenyl)piperidine-1-carboxylate

To a mixture of compound 548a (70 mg, 0.266 mmol), compound 548b (98 mg, 0.319 mmol), palladium acetate (12 mg, 0.053 mmol), xantphos (61.6 mg, 0.106 mmol) and cesium carbonate (173 mg, 0.532 mmol) was added dioxane (5 mL). The mixture was degassed under vacuum, replaced with argon several times, heated to 100 degrees and stirred for 2 hours. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (10% methanol in dichloromethane/dichloromethane = 0-14/86) to obtain a yellow solid product (compound 548c, 81 mg, 57.2% yield). LCMS [M+1] ⁺ = 533.

Step 2: Compound 548

N-(Cyanomethyl)-1-(2-((2-methoxy-4-(piperidin-4-yl)phenyl)amino)pyrimidin-4-yl)-1H-pyrazole-4-carboxamide

To a solution of compound 548c (73 mg, 0.137 mmol) in dichloromethane (5 mL) and methanol (0.5 mL) was added zinc bromide (247 mg, 1.096 mmol) under argon atmosphere, and the mixture was stirred at room temperature. The mixture was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC (formic acid) to give the desired product (Compound 548, TDM-181148, 6 mg, 10.1% yield) as a colorless oil. LCMS [M+1] ⁺ =433.

¹ H NMR (400MHz, DMSO) δ9.46 (s, 1H), 9.08 (s, 1H), 8.57 (d, J = 5.4Hz, 1H), 8.48 (s, 1H), 8. 40(s,1H),8.28(d,J＝6.2Hz,1H),7.98(d,J＝8.2Hz,1H),7.28(d,J＝5.4Hz,1H),6.96( s,1H),6.88(d,J＝8.2Hz,1H),4.34(d,J＝4.9Hz,2H),3.87(s,3H),3.67(s,1H),3.32( d,J＝11.6Hz,2H),2.92(t,J＝11.5Hz,2H),2.80(t,J＝11.5Hz,1H),1.98–1.76(m,4H).

2,2-Difluoro-N-(4-(2-methoxy-4-piperidin-4-phenyl)aminopyrimidin-4-ylpyridin-2-yl)cyclopropane-1-carboxamide (TDM181149) was synthesized as a yellow solid (12.3 mg, 13.4% yield) using a similar method to Example 6; 2,2-difluoro-N-(4-(2-methoxy-4-methylpiperidin-4-phenyl)amino)pyrimidin-4-ylpyridin-2-yl)cyclopropane-1-carboxamide (TDM-181150) was synthesized as a yellow solid (30.4 mg, 32.4% yield).

Example 7 General method for the synthesis of compound 551 (TDM-181151)

Step 1: Compound 551b

Di-tert-butyl (4-bromopyridin-2-yl) iminodicarbonate

To a solution of compound 551a (1 g, 5.78 mmol) in tetrahydrofuran (25 mL) was added LiHMDS (11.56 mL, 11.56 mmol), and stirred for 30 minutes, then di-tert-butyl dicarbonate (2.52 g, 11.56 mol) was added, and stirred at room temperature overnight. The mixture was treated with an aqueous solution of ammonium chloride, extracted with ethyl acetate (50 mL*3), the organic layers were combined, washed with brine, dried over sodium sulfate, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (ethyl acetate/petroleum ether=0-5/95) to give a yellow oily product (compound 551b, 1.69 g, 78.3% yield). LCMS[M+1] ⁺ =373.

Step 2: Compound 551d

To a mixture of compound 551b (1.49 g, 3.922 mmol), compound 551c (1.52 g, 5.987 mmol), potassium acetate (784 mg, 7.984 mmol) and 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride (292 mg, 0.399 mmol) was added dioxane (40 mL). The mixture was degassed under vacuum, replaced with argon several times, heated to 90 degrees and stirred overnight. The mixture was then used directly in the next reaction. LCMS [M+1] ⁺ = 339.1.

Step 3: Compound 551f

Water (7 mL) and dioxane (70 mL) were added to a mixture of compound 551d (3.992 mmol), compound 551e (714 mg, 4.79 mmol), cesium carbonate (2.6 g, 7.984 mmol), and 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride (292 mg, 0.399 mmol). The mixture was degassed under vacuum, replaced with argon several times, heated to 100 degrees and stirred for 4 hours. Water was added to the mixture and extracted with ethyl acetate (60 mL*3), the organic layers were combined, washed with brine, dried with sodium sulfate, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (ethyl acetate/petroleum ether=0-14/86) to obtain a white solid product (compound 551f, 789 mg, 48.6% yield). LCMS[M+1] ⁺ =407.

Step 4: Compound 551g

4-(2-Chloropyrimidin-4-yl)pyridin-2-amine

Trifluoroacetic acid (4.43 g, 38.875 mmol) was added to a solution of compound 551f (790 mg, 1.944 mmol) in dichloromethane (30 mL). The mixture was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure, the residue was added with water and neutralized with aqueous sodium carbonate solution, extracted with ethyl acetate (90 mL*3), the organic layers were combined, washed with brine, dried over sodium sulfate, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (ethyl acetate/dichloromethane=0-70/30) to obtain a colored solid product (compound 551 g, 398 mg, 99% yield). LCMS[M+1] ⁺ =207.

Step 5: Compound 551i

N-(4-(2-chloropyrimidin-4-yl)pyridin-2-yl)-2,2-difluorocyclopropane-1-carboxamide

Phosphorus oxychloride (591 mg, 3.852 mmol) was added to a solution of compound 551g (398 mg, 1.926 mmol) and compound 551h (470 mg, 3.852 mmol) in pyridine (20 mL) under argon atmosphere, and the mixture was stirred at room temperature for 30 minutes. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (ethyl acetate/petroleum ether=0-35/65) to obtain a white solid product (compound 551i, 500 mg, 85.6% yield). LCMS [M+1] ⁺ = 311.

Step 6: Compound 551h

Tert-butyl 4-(4-((4-(2-(2,2-difluorocyclopropane-1-carboxamido)pyridin-4-yl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)piperidine-1-carboxylate

To a mixture of compound 551i (80 mg, 0.257 mmol), compound 551j (82.3 mg, 0.309 mmol), palladium acetate (11.6 mg, 0.051 mmol), xantphos (59 mg, 0.103 mmol) and cesium carbonate (167 mg, 0.514 mmol) was added dioxane (5 mL). The mixture was degassed under vacuum, replaced with argon several times, heated to 100 degrees and stirred for 2 hours. The mixture was heated under reduced pressure. The mixture was concentrated under reduced pressure and the residue was purified by silica gel chromatography (10% methanol in dichloromethane/dichloromethane=0-55/45) to give a yellow solid product (Compound 551k, 102.8 mg, 74% yield). LCMS [M+1] ⁺ =541.

Step 7: Compound 551

2,2-Difluoro-N-(4-(2-((1-(piperidin-4-yl)-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)pyridin-2-yl)cyclopropane-1-carboxamide

To a solution of compound 551k (103 mg, 0.191 mmol) in dichloromethane (10 mL) and methanol (2 mL) was added a solution of dioxane hydrochloride (0.76 mL). The mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC (formic acid) to give a yellow solid product (compound 551, TDM-181151, 45 mg, 53.5% yield). LCMS [M+1] ⁺ = 541

¹ H NMR (400MHz, DMSO) δ11.23(s,1H),9.72(s,1H),8.95(s,1H),8.59(d,J＝5.0Hz,1H),8.52(d,J＝5.2Hz, 1H),8.35(s,1H),8.21(s,1H),7.79(d,J=4. 7Hz,1H),7.54(s,1H),7.35(s,1H),4.35(s,1H),3.20-3.13(m,2H),3.11 -3.01(m,1H),2.73(s,2H),2.09(d,J＝9.0Hz,2H),1.98(d,J＝8.3Hz,4H).

Example 8 General method for the synthesis of compound 562 (TDM-181162)

Step 1: Compound 562c

tert-Butyl (3-(2-chloropyrimidin-4-yl)phenyl)carbamate

Water (3 mL) and dioxane (18 mL) were added to a mixture of compound 562a (280 mg, 1.88 mmol), compound 562b (500 mg, 1.566 mmol), cesium carbonate (1.02 g, 3.132 mmol), and 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride (114.6 mg, 0.157 mmol). The mixture was heated to 100°C and stirred for 3 h. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (ethyl acetate/petroleum ether=0-25/75) to obtain a white solid product (compound 562c, 351.4 mg, 73.4% yield). LCMS[M+1] ⁺ =306.

Step 2: Compound 562e

4-(5-((4-(3-((tert-butoxycarbonyl)amino)phenyl)pyrimidin-2-yl)amino)thiol Benzyl oxazol-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate

To a mixture of compound 562c (300 mg, 0.981 mmol), compound 562d (619 mg, 1.962 mmol), trisdibenzylideneacetone dipalladium (90 mg, 0.098 mmol), xphos (93 mg, 0.1962 mmol, and cesium carbonate (640 mg, 1.962 mmol) was added dioxane (20 mL). The mixture was degassed under vacuum, replaced with argon several times, heated to 100 degrees and stirred. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (ethyl acetate/petroleum ether=0~73/27) to give a brown solid product (compound 562e, 500 mg, 87.2% yield). LCMS [M+1] ⁺ =585.

Step 3: Compound 562f

Benzyl 4-(5-((4-(3-aminophenyl)pyrimidin-2-yl)amino)thiazol-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate

To a solution of compound 562e (500 mg, 0.852 mmol) in dichloromethane (20 mL) was added a solution of dioxane hydrochloride (2.13 mL). The mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (10% methanol (NH ₃ ) in dichloromethane/dichloromethane=0-45/55) to give a brown solid product (compound 562f, 364 mg, 88.2% yield). LCMS [M+1] ⁺ =485.

Step 4: Compound 562h

Benzyl (S)-4-(5-((4-(3-(2,2-difluorocyclopropane-1-carboxamido)phenyl)pyrimidin-2-yl)amino)thiazol-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate

Phosphorus oxychloride (230 mg, 1.500 mmol) was added to a solution of compound 562f (365 mg, 0.75 mmol) and compound 562g (137 mg, 1.125 mmol) in dry pyridine (10 mL) at 0°C under argon atmosphere, and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (10% methanol in dichloromethane/dichloromethane = 0/100 to 50/50) to give a yellow solid product (compound 562h, 100 mg, 22.7% yield). LCMS [M+1] ⁺ = 589.1.

Step 5: Compound 562i

(S)-2,2-Difluoro-N-(3-(2-((2-(1,2,3,6-tetrahydropyridin-4-yl)thiazol-5-yl)amino)pyrimidin-4-yl)phenyl)cyclopropane-1-carboxamide

To a solution of compound 562h (70 mg, 0.119 mmol) in HBr/CH ₃ COOH (5 mL) was added at 0°C, and the mixture was stirred at 0°C for 0.5 h. The mixture was concentrated under reduced pressure, and the residue was used directly in the next reaction. LCMS [M+1] ⁺ =455.

Step 6: Compound 562

(S)-2,2-Difluoro-N-(3-(2-((2-(piperidin-4-yl)thiazol-5-yl)amino)pyrimidin-4-yl)phenyl)cyclopropane-1-carboxamide

Pd/C was added to a solution of compound 562i (0.119 mmol) in methanol (7 mL), the mixture was degassed under vacuum, replaced with hydrogen several times, and the mixture was stirred at room temperature for 5 hours. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (formic acid) to give a yellow solid product (compound 562, TDM-181162, 11 mg, 20.2% yield). LCMS [M+1] ⁺ = 457.

¹ H NMR (400MHz, DMSO) δ10.82 (s, 1H), 10.75 (s, 1H), 8.69 (s, 1H), 8.59 (d, J = 5.2Hz, 1H),8.39(s,1H),7.88(d,J＝7.6Hz,1H),7.62(d,J＝8.2Hz,1H),7.52(t,J＝7.9Hz ,1H),7.46–7.30(m,2H),3.18(d,J＝10.7Hz,2H),3.14–3.06(m,1H),2.95–2.87( m,1H),2.82(dd,J＝20.5,8.6Hz,2H),2.16–1.92(m,4H),1.80(t,J＝12.0Hz,2H).

Example 9: General method for the synthesis of compound 576 (TDM-181176)

Step 1: Compound 576c

tert-Butyl (4-(2-chloropyrimidin-4-yl)benzyl)carbamate

Water (4 mL) and dioxane (24 mL) were added to a mixture of compound 576a (212 mg, 1.439 mmol), compound 576b (500 mg, 1.5 mmol), 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride (105 mg, 0.144 mmol) and potassium carbonate (398 mg, 8.889 mmol), and the mixture was degassed under vacuum, purged several times, and then heated to 80 ° C and stirred for 3 hours. The mixture was added with water and extracted with ethyl acetate, concentrated under reduced pressure, and the residue was purified by silica gel chromatography (ethyl acetate/petroleum ether=0-25/75) to obtain a yellow solid product (compound 576c, 362 mg, 78.7% yield). LCMS[M+1] ⁺ =320.

Step 2: Compound 576d

(4-(2-Chloropyrimidin-4-yl)phenyl)methanamine

Trifluoroacetic acid (1 mL) was added to a solution of compound 576c (262 mg, 0.819 mmol) in dichloromethane (10 mL) and methanol (1 mL), and the mixture was stirred at room temperature for 4 hours. The mixture was concentrated under reduced pressure, the residue was added with water, neutralized with aqueous sodium carbonate solution and extracted with ethyl acetate, the organic layers were combined, washed with brine, dried over sodium sulfate, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (10% methanol in dichloromethane/dichloromethane=0-40/60) to give a white solid product (compound 576c). Product 576d, 130 mg, 72.2% yield). LCMS [M+1] ⁺ =220.

Step 3: Compound 576f

(S)-N-(4-(2-chloropyrimidin-4-yl)benzyl)-2,2-difluorocyclopropane-1-carboxamide

To a solution of compound 576d (130 mg, 0.592 mmol), compound 576e (108 mg, 0.888 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (358 mg, 0.888 mmol) in N,N-dimethylformamide (10 mL) was added N,N-diisopropylethylamine (115 mg, 0.888 mmol), and the mixture was stirred at 35° C. for 1 hour. The mixture was added with water (80 mL) and extracted with ethyl acetate (30 mL*3), the organic layers were combined, washed with brine (50 mL*2), dried over sodium sulfate, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (10% methanol in dichloromethane/dichloromethane=0-45/55) to give a white solid product (compound 576f, 143 mg, 74.6% yield). LCMS [M+1]+=324.

Step 4: Compound 576h

Benzyl (S)-4-(4-((4-(4-(2,2-difluorocyclopropane-1-carboxamido)methyl)phenyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)piperidine-1-carboxylate

To a solution of compound 576f (143 mg, 0.442 mmol) and compound 576g (159 mg, 0.530 mmol) in n-butanol (10 mL) was added p-toluenesulfonic acid monohydrate (168 mg, 0.884 mmol). The mixture was heated to 100° C. and stirred. Water (70 mL) was added to the mixture and extracted with ethyl acetate (30 mL*3). The organic layers were combined and washed with brine. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel chromatography (10% methanol in dichloromethane/dichloromethane=0-35/65) to give a white solid product (compound 576h, 153 mg, 48.3% yield. LCMS[M+1] ⁺ =588.

Step 5: Compound 576

(S)-2,2-Difluoro-N-(4-(2-((1-(piperidin-4-yl)-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)benzyl)cyclopropane-1-carboxamide

Compound 576h (110 mg, 0.187 mmol) was dissolved in HBr-AcOH (5 mL) at 0°C, and the mixture was stirred at 0°C for 30 minutes. The mixture was concentrated under reduced pressure at 45°C, and the residue was purified by preparative HPLC (formic acid) to give a yellow solid product (compound 576, TDM-181176, 5.2 mg, 6.1% yield). LCMS [M+1] ⁺ = 454.

¹ H NMR (400MHz, DMSO) δ9.51 (s, 1H), 8.92 (t, J = 5.7Hz, 1H), 8.48 (d, J = 5.1Hz, 1H), 8.34 (s, 1H), 8.11 (d, J＝8.2Hz,2H) ,7.98(s,1H),7.62(s,1H),7.43(d,J＝8.1Hz,2H),7.27(d,J＝5.2Hz,1H),4.43(dd,J＝16.7,4.9Hz, 2H),4.34(dd,J ＝18.9,7.6Hz,1H),3.21(d,J＝12.4Hz,2H),2.82(t,J＝11.7Hz,2H),2.72–2.62(m,1H),2.52(s,1H),2.06 (d,J＝11.1Hz,2H),2.00–1.80(m,4H).

Example 10: General method for the synthesis of compound 580 (TDM-181180)

Step 1: Compound 580c

tert-Butyl (4-(2,5-dichloropyrimidin-4-yl)benzyl)carbamate

In a 50 mL flask, compound 580a (275 mg, 1.5 mmol), compound 580b (500 mg, 1.5 mmol), potassium carbonate (414 mg, 3 mmol), [1,1'-bis(diphenylphosphino)dihydrochloride] [ferrocene] palladium dichloride (110 mg, 0.15 mmol) was dissolved in (1,4-dioxane: water) = (6: 1) solution (25 mL), and the mixture was stirred at 80 ° C for 3 hours in Ar. The reaction was detected by TLC and LCMS. The product was detected by LCMS [M + 1-56] ⁺ = 298.0. The mixture was added to water (30 mL) and extracted with ethyl acetate (2 * 30 mL), and the organic layers were combined. The combined organic phases were washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The organic layer was concentrated and purified by flash chromatography (12g column, 25 minutes), eluted with PE / EA = 0 ~ 100%, and a yellow solid (compound 580c, 462 mg, yield 87%) was obtained as the product. LCMS [M + 1-56] ⁺ = 298.0.

Step 2: Compound 580e

Benzyl 4-(4-((4-(4-)(((tert-butoxycarbonyl)amino)methyl)phenyl)-5-chloropyrimidin-2-yl)amino)-1H-pyrazol-1-yl)piperidine-1-carboxylate

Take a 50mL flask, compound 580c (200mg, 0.56mmol), compound 580d (167mg, 0.56mmol), palladium acetate (28.5mg, 0.11mmol), 4,5-bis (diphenylphosphine) -9,9- dimethylxanthene (130mg, 0.22mmol) and cesium carbonate (364mg, 1.12mmol) are dissolved in ultra-dry 1,4-dioxane (20mL), and the mixture is stirred in argon at 100 degrees Celsius for 2 hours. The reaction is detected by TLC and LCMS. The product is confirmed by LCMS [M + 1-56] ⁺ = 562.2. The mixture is added with water (30mL), extracted with ethyl acetate (2 * 30mL), and the organic layer is combined. The combined organic phase is washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The organic layer was concentrated and purified by flash chromatography (12 g column, 25 minutes), eluting with PE/EA=0-100% to obtain the product as a yellow solid (Compound 580e, 250 mg, yield 71%). LCMS [M+1-56] ⁺ =562.2.

Step 3: Compound 580f

Benzyl 4-(4-((4-(4-aminomethyl)phenyl)-5-chloropyrimidin-2-yl)amino)-1H-pyrazol-1-yl)piperidine-1-carboxylate

Compound 580e (200 mg, 0.324 mmol) was dissolved in dichloromethane (20 mL) at 0°C, trifluoroacetic acid (2 mL) was added, and the mixture was stirred at room temperature for 1 hour. The reaction was detected by TLC and LCMS. Pour into water (30 mL) and adjust to pH>8 with potassium carbonate (50%). Water (30 ml) and ethyl acetate (3*30 ml) were added to extract the mixture, and the organic layers were combined. The combined organic phases were washed with brine, dried over anhydrous sodium sulfate and the organic layer was concentrated under reduced pressure and purified by flash chromatography (12 g column, 30 minutes), eluted with DCM/(DCM:MeOH=10:1)=0~90%, and a yellow oily compound (compound 580f, 126 mg, yield 75%) was obtained as the target product. LCMS[M+1] ⁺ =518.2.

Step 4: Compound 580h

Benzyl (S)-4-(4-((5-chloro-4-(4-)((2,2-difluorocyclopropane-1-carboxamido)methyl)phenyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)piperidine-1-carboxylate

Take a 50mL flask, dissolve compound 580f (126mg, 0.24mmol), compound 580g (43.9mg, 0.36mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (137mg, 0.36mmol) in N,N-dimethylformamide (15mL), add N,N-diisopropylethylamine (46mg, 0.36mmol), and stir the mixture at room temperature for 1 hour. The reaction was detected by TLC and LCMS. Water (30mL) was added to the mixture, extracted with ethyl acetate (3*30mL), and the organic layers were combined. The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The organic layer was concentrated and purified by flash chromatography (12 g column, 30 min) using DCM/(DCM:MeOH=10:1)=0-30% as eluent to give a yellow oily compound (compound 580h, 130 mg, yield 87%) as the target product. LCMS [M+1] ⁺ =622.3.

Step 5: Compound 580

(S)-N-(4-(5-chloro-2-((1-(piperidin-4-yl)-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)benzyl)-2,2-difluorocyclopropane-1-carboxamide

Take a 25 mL flask, add compound 580h (40 mg, 0.06 mmol) at 0°C, cool for a while, then dropwise add hydrobromic acid in acetic acid (2 ml), and stir the mixture at 0°C for 0.5 h. The reaction is detected by TLC and LCMS. The crude product is prepared and purified by HPLC to obtain a white solid product (compound 580, TDM-181180, 12 mg, yield 37.5%), LCMS [M+1] ⁺ = 488.2.

¹ H NMR(401MHz,DMSO)δ9.78(s,1H),8.93(s,1H),8.54(s,1H),8.31(s,1H),7 .88(s,1H),7.80(s,2H),7.58(s,1H),7.43(d,J＝7.8Hz,2H),4.48–4.36(m, 2H),4.29(s,1H),3.20(d,J＝10.9Hz,2H),2.80(t,J＝11.8Hz,2H),2.68(td, J＝6.4,3.0Hz,1H),2.52(s,1H),2.01(t,J＝11.3Hz,2H),1.96-1.78(m,4H).

Test example: Enzyme activity inhibition assay of NUAK1/NUAK2 kinase inhibitors

The effect of the pyrimidine amine small molecules involved in this application on the inhibitory activity of NUAK1 and NUAK2 kinases adopts a kinase activity test method based on P81 filter paper combined with hot spot technology (Nat Biotechnol. 2011, 29: 1039), which is briefly described as follows:

The test buffer system contained 20 mM Hepes (pH 7.5), 10 mM MgCl ₂ , 1 mM EGTA, 0.01% Brij 35 (L23 polyoxyethylene lauryl ether), 0.02 mg/ml BSA (bovine serum albumin), 0.1mM Na ₃ VO ₄ , 2mM DTT, 1% DMSO.

Test steps:

1. Prepare 20 μM substrate solution with freshly prepared test buffer. The substrate is CHKtide polypeptide fragment (Sanchez Y. Science. 1997, 277: 1497-1501).

2. Add kinase and mix gently (the final concentration of NUAK1 is 10nM; the final concentration of NUAK2 is 50nM);

3. Add the test compound dissolved in 100% DMSO to the above kinase reaction mixture using sonication pipetting technology (Echo550; nanoliter) and incubate at room temperature for 5 minutes;

4. Add ³³ P-labeled ATP (the ratio of unlabeled ATP/labeled ATP is 25:1 or 2.5:1);

5. Incubate at room temperature for 2 hours;

6. Detect kinase activity by P81 filter paper combined with hot spot technology.

The IC ₅₀ of the test compound against NUAK1/NUAK2 was calculated according to the above detection. See Table 1 for specific results.

_IC50 calculations were performed using the formula derived from a sigmoidal dose-response curve (variable slope):

Y = Bottom + (Top-Bottom) / (1 + 10^((LogEC50-X) * slope, where X is the Log value of the compound concentration, Y is the response (inhibition rate of kinase activity), and Y rises from the bottom to the top along the S-shaped curve as the concentration increases.

Table 1 IC ₅₀ (nM) of the test compounds of this application against NUAK1/NUAK2

From the results in Table 1, it can be seen that the pyrimidine amine compounds of the present application have excellent inhibitory activity against NUAK1/NUAK2, and are dual-target small molecule kinase inhibitors. The IC ₅₀ of the compounds against NUAK1/NUAK2 can reach several nM or tens of nM. Therefore, the above experiments have proved that the pyrimidine amine compounds of the present application can be used as NUAK1/NUAK2 inhibitors.

The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims (28)

A pyrimidine amine compound, characterized in that the pyrimidine amine compound is a compound represented by formula I, or a stereoisomer, tautomer, isotope derivative, hydrate, solvate, prodrug, and pharmaceutically acceptable salt thereof;
Wherein, 0 or 1 of X ₁ , X ₂ , and X ₃ is N, and the others are CH. When X ₁ , X ₂ , and X ₃ are CH, H may be substituted by R ₅ or R ₄ ; A is an optionally substituted 6-10 membered aryl or an optionally substituted 5-10 membered heteroaryl, B is an optionally substituted 3-10 membered cycloalkyl or an optionally substituted 3-10 membered heterocycloalkyl, and the substituents on A or B are selected from one or more of halogen, amino, hydroxy, nitro, cyano, mercapto, optionally substituted C1-8 alkyl, optionally substituted C1-8 alkyloxy, optionally substituted C1-8 alkylthio, and optionally substituted C1-8 alkylamino; R ₁ and R ₂ are each independently H or optionally substituted C1-8 alkyl; R ₃ and R ₄ are each independently halogen, amino, hydroxy, nitro, cyano, mercapto, optionally substituted C1-8 alkyl, optionally substituted C1-8 alkyloxy, optionally substituted C1-8 alkylthio, or optionally substituted C1-8 alkylamino; _R6 is an optionally substituted C1-8 alkyl, an optionally substituted 3-10 membered cycloalkyl, or an optionally substituted 3-10 membered heterocycloalkyl, wherein the substituent on the cycloalkyl or heterocycloalkyl is selected from one or more of halogen, amino, hydroxy, nitro, cyano, mercapto, an optionally substituted C1-8 alkyl, an optionally substituted C1-8 alkyloxy, an optionally substituted C1-8 alkylthio, and an optionally substituted C1-8 alkylamino; The substituent on the C1-8 alkyl group is selected from one or more of halogen, amino, hydroxyl, nitro, cyano, and mercapto; n is 0, 1 or 2, m is 0, 1, 2, 3 or 4, and p is 0, 1, 2, 3, 4, 5 or 6. The compound according to claim 1, characterized in that R ₆ is an optionally substituted 3-10 membered cycloalkyl. The compound according to claim 2, characterized in that R ₆ is an optionally substituted 3-6 membered cycloalkyl. The compound according to claim 3, characterized in that _R6 is a 3-6 membered cycloalkyl substituted with halogen. The compound according to claim 4, characterized in that _R6 is a fluorine-substituted 3-6 membered cycloalkyl group. The compound according to claim 3, characterized in that R ₆ is The compound according to claim 6, characterized in that R ₅ is The compound according to claim 7, characterized in that R ₅ is The compound according to any one of claims 1 to 8, characterized in that R ₅ is substituted at the para position or meta position of the pyrimidine group. The compound according to any one of claims 1 to 9, characterized in that B is an optionally substituted 3-6 membered heterocycloalkyl. The compound according to claim 10, characterized in that B is an optionally substituted 5-6 membered heterocycloalkyl. The compound according to claim 11, characterized in that B is an optionally substituted piperidinyl or an optionally substituted piperazinyl. The compound according to claim 12, characterized in that the compound represented by formula I is formula I-1 or formula I-2,
_R7 is selected from halogen, amino, hydroxyl, nitro, cyano, mercapto, optionally substituted C1-8 alkyl, optionally substituted C1-8 alkyloxy, optionally substituted C1-8 alkylthio, optionally substituted C1-8 alkylamino, _R8 is H or optionally substituted C1-8 alkyl; q is 0, 1, 2, 3, or 4, and the substituents on the C1-8 alkyl are selected from one or more of halogen, amino, hydroxyl, nitro, cyano, and mercapto. The compound according to claim 13, characterized in that R ₈ is H or methyl. The compound according to any one of claims 1 to 14, characterized in that A is an optionally substituted phenyl group or an optionally substituted 5-membered heteroaryl group. The compound according to claim 15, characterized in that A is one of the following groups which are optionally substituted:
The compound according to any one of claims 1 to 16, characterized in that X ₁ to X ₃ are all CH. The compound according to any one of claims 1 to 17, characterized in that the substituent on ring A is a methoxy group, preferably substituted at the ortho position of the amino group. The compound according to any one of claims 1 to 18, characterized in that R ₃ is chlorine, preferably substituted at the para position of the amino group. The compound according to any one of claims 1 to 19, characterized in that the compound represented by formula I is one of the following compounds:

According to any one of claims 1-20, the pharmaceutically acceptable salt is a formate salt. According to any one of claims 1 to 21, the isotope derivative is a deuterated compound. The method for preparing the compound according to any one of claims 1 to 22, comprising the following steps: The compound of formula I is prepared from the compound of formula II and the compound of formula III; or The compound of formula V is prepared from the compound of formula IV and the compound of formula II, and the compound of formula I is prepared from the compound of formula V and the compound of formula VI; Optionally, the preparation process includes necessary protection and deprotection steps;
Wherein, Y is a leaving group, preferably Y is a halogen, more preferably Y is Cl, and the definitions of other groups are the same as those in claims 1-22. A pharmaceutical composition, characterized in that the compound according to any one of claims 1 to 22 is used as an active ingredient. The pharmaceutical composition according to claim 24, characterized in that it comprises a pharmaceutically acceptable carrier or excipient. Use of the compound according to any one of claims 1 to 22 for preparing a NUAK1 or NUAK2 inhibitor. The use of the compound according to any one of claims 1 to 22 for preparing a drug, characterized in that the compound is used to prevent or treat the following diseases by inhibiting NUAK1 or NUAK2: neuropsychiatric diseases, metabolic diseases, tumors, visceral fibrosis diseases, and skin fibrosis diseases. The use according to claim 27 is characterized in that the disease is Parkinson's disease, Alzheimer's disease, diabetes, hyperlipidemia, obesity, liver cancer, leukemia, lymphoma, tumor metastasis, cirrhosis, renal fibrosis, pulmonary fibrosis, sequelae of myocarditis, scleroderma, keloid, hypertrophic scar, or is used alone to reduce trauma and postoperative scars.