CN111689991A - Substituted heteroaryl compounds, compositions and uses thereof - Google Patents

Abstract

The invention belongs to the field of medicines, and particularly relates to a substituted heteroaryl compound shown as a formula (I), or a stereoisomer, a tautomer, a nitric oxide, a solvate, a metabolite or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the compound, and application of the compound and the pharmaceutical composition in preparation of medicines for treating proliferative diseases, autoimmune diseases, allergic diseases, inflammatory diseases, transplant rejection, cancers or other diseases of mammals. The compound provided by the invention shows excellent inhibitory activity and optimized kinase selectivity on target kinases. In addition, the compound provided by the invention also has excellent membrane permeability and shows excellent pharmacokinetic properties in an animal body, so the compound provided by the invention has very good development prospect.

Description

Substituted heteroaryl compounds, compositions and uses thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a novel substituted heteroaryl compound, a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the compound, and application of the compound and the pharmaceutical composition in preparation of medicines for treating proliferative diseases, autoimmune diseases, allergic diseases, inflammatory diseases, transplant rejection, cancers or other diseases of mammals. More specifically, the compounds of the present invention can modulate the activity of the TAM kinase family (including Axl, Mer and Tyro-3), and Trk kinase family (tropomyosin receptor kinases including TrkA, TrkB and TrkC), etc., thereby modulating signal transduction inside and outside cells.

Background

The family of protein kinases comprises a large class of structurally related enzymes that control various signal transduction processes within the cell, catalyzing the phosphorylation of target protein substrates. Many diseases are associated with abnormal cellular responses triggered by protein kinase-mediated events. These diseases include benign and malignant proliferative disorders, disorders resulting from inappropriate activation of the immune system, allograft rejection, graft-versus-host disease, autoimmune disorders, inflammatory disorders, bone disorders, metabolic disorders, neurological and neurodegenerative disorders, cancer, cardiovascular disorders, allergy and asthma, alzheimer's disease and hormone-related disorders. Accordingly, protein kinase inhibitors effective in the treatment of diseases have been developed in the medical field.

Kinases can be classified into families by phosphorylated substrates (e.g., protein-tyrosine, protein-serine/threonine, lipids, etc.). Tyrosine phosphorylation is one of the central events that regulates various biological processes such as cell proliferation, migration, differentiation, and survival. Several families of receptor and non-receptor tyrosine kinases control the tyrosine groups that catalyze the transfer of phosphate from ATP to specific cellular protein targets. Currently, motifs generally corresponding to the above-mentioned kinase families have been identified (Hanks et al, FASEB J.,1995,9, 576-596; Knighton et al, Science,1991,253, 407-414; Garcia-Bustos en al EMBO J.,1994,13: 2352-2361). Examples of kinases in the protein kinase family include, but are not limited to, Aurora, Axl, abl, Akt, bcr-abl, Blk, Brk, Btk, c-Met, c-src, c-fms, CDKl, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, cRafl, CSF1R, CSK, EGFR, ErbB2, ErbB3, flatcell 4, Erk, Flt-3, Fak, fes, fgfrfl, FGFR2, FGFR3, FGFR4, FGFR5, Fgr, Fyn, Axl, IGF-1R, INS-R, KDR, Lck, Lyn, MEK, Mer, p38, PDGFR, PIK, PKC, PYK 5, tiros, Tie, hue-2, tyk, tympan, tygene 3, R, and ErbB 5519. wy, R5526, R5548, R.

Cancer (and other hyperproliferative diseases) is characterized by uncontrolled cellular proliferation. The activity of many protein kinases is increased in human tumors compared to normal tissues, and this increased activity may be due to a number of factors, including increased kinase levels, mutations in the expression of co-activators or inhibitory proteins.

Axl is a receptor tyrosine kinase (ligand: growth inhibition specific protein 6, Gas6) which is unique in having two immunoglobulin-like repeats in tandem and two fibronectin type III repeats, a common feature of cell adhesion molecules. Axl kinase belongs to the TAM family of receptor tyrosine kinases, and can activate the tyrosine kinase activity of Axl by combining with ligand Gas6, thereby activating the downstream signal transduction pathway and participating in the processes of growth, migration, aggregation and apoptosis of cells (Rothlin, C.V.; Ghosh, S.; Zuniga, E.I.; Oldstone, M.B.A.; Lemke, G.TAM receptors and emerlepriopic inhibitors of the input immune response. cell 2007,131,1124 and 1136). Axl and Tyro-3 have the most similar gene structures, while Axl and Mer have the most similar amino acid sequences of the tyrosine kinase domain. Like other Receptor Tyrosine Kinases (RTKs), the structure of the TAM family comprises an extracellular domain, a transmembrane domain, and a conserved intracellular kinase domain. The extracellular domain contains two immunoglobulin-like (Ig) domains and two fibronectin domain iii (FN iii) repeats, and is the binding site for endogenous ligands; the conserved amino acid sequence KW (I/L) A (I/L) ES, located in the kinase domain, is a unique structural feature of the TAM family. Members of the TAM family have a Common Ligand, Growth-inhibitory specific protein 6(Gas 6), which binds to all TAM Receptor Tyrosine kinases, of which Axl binds to Gas6 most strongly, 3-10 times stronger than the weakest Mer (Zago' rska A, et al. conversion of TAM Receptor Tyrosine kinase function. Nature Immunology 2014, 15: 920-.

Axl kinase is overexpressed or activated in a variety of cancers, including ovarian cancer, melanoma, renal cell carcinoma, uterine leiomyoma, endometrial cancer, thyroid cancer, gastric cancer, breast cancer, NSCLC, CML, AML, colon cancer, prostate cancer, various lymphomas, and esophageal cancer. Recent studies have shown that overexpression of Axl is particularly severe in cancer cells that develop resistance after chemotherapy and receptor Tyrosine Kinase Inhibitor (TKI) treatment, and is one of the major causes of resistance (Zhang, z.; Lee, j.c.; Lin, l.; et al Activation of the Axl kinase responses to EGFR-targeted therapy in lung cancer. nat. gene.2012, 44, 852-. At present, the emergence of drug resistance in Cancer patients remains a challenge in the course of Cancer therapy, and inhibition of Axl activity can enhance chemotherapy sensitivity and delay the development of drug resistance, and thus, the Axl protooncogene is an attractive and valuable Target for the discovery and development of new therapeutics (fen yrolles C, Spenlinhauer a, Guiet L, et al. Axl Kinase as a Key Target for oncology: Focus on Small Molecule inhibitors. mol Cancer Ther,2014,13(9): 2141-. Based on the implications in a variety of human malignancies, there is a need to design specific and selective inhibitors to treat cancer and other conditions mediated by and/or associated with Axl kinase. The present invention fulfills these needs and provides other related benefits.

The Trk, tropomyosin-receptor-kinase, is the product of the protooncogene Trk, is produced by fusion of tropomyosin and tyrosine-kinase, and is a member of the receptor tyrosine-kinase (RTK) family. Trk receptors are divided into TrkA, TrkB and TrkC, corresponding ligands of the TrkA receptors are NGF, BDNF and NT-3 respectively, another ligand of the TrkB receptors is NT4/5, and the NT-3 can also act on the TrkA and the TrkB. Members of the NTs family signal transduction primarily through autophosphorylation of Trk receptors, producing effects (Huang, e.j.; Reichardt, l.f.trk receptors: rolls in neural signal transduction, annu. rev. biochem.2003,72, 609-.

NTRK gene fusions involving NTRK1, NTRK2, or NTRK3 (encoding neurotrophin receptors TrkA, TrkB, and TrkC, respectively) are oncogenic drivers of various adult and pediatric tumor types. TRK fusion proteins have ligand-independent kinase activity, are capable of activating downstream signaling pathways associated therewith, stimulate cell growth and survival, and cause cancer that is mutually exclusive with other oncogenic drivers in humans. The data indicate that inhibition of TRK fusion proteins (e.g., larotretinib or entretinib) can treat cancer patients who are NTRK fusion positive. (E.Cocco, E.; Scaltrti, M.; Drilon, A., NTRK fusion-positive cameras and TRK inhibitor therapy. Nat Rev Clinoncol.2018, 15(12): 731-

Immunotherapy is a therapeutic method for artificially enhancing or suppressing the immune function of the body to treat diseases in response to a low or high immune state of the body. There are many methods of immunotherapy and are applicable to the treatment of a variety of diseases. Immunotherapy of tumors aims to activate the human immune system, relying on autoimmune functions to kill cancer cells and tumor tissues. Unlike previous surgery, chemotherapy, radiation therapy and targeted therapies, immunotherapy targets not tumor cells and tissues, but the human body's own immune system.

Protein kinase inhibitors have gathered much attention as new immunomodulatory, anti-inflammatory and anti-cancer agents. Therefore, the novel or improved agent for inhibiting protein kinases such as Axl kinase and Trk kinase can be used as an immunomodulator, an antitumor agent, an analgesic, an anti-organ fibrosis drug for organ transplantation, and can be used for the prevention and treatment of autoimmune diseases (e.g., multiple sclerosis, psoriasis, rheumatoid arthritis, asthma, type I diabetes, inflammatory bowel disease, Crohn's disease, polycythemia vera, essential thrombocythemia, myelofibrosis, autoimmune thyroid disease, Alzheimer's disease), diseases involving overactivated inflammatory response (e.g., eczema), allergy, chronic obstructive pulmonary disease, bronchitis, fibrosis, cancer (e.g., gastric cancer, liver cancer, lung cancer, colorectal cancer, prostate cancer, acute myelocytic leukemia, chronic myelocytic leukemia, acute lymphocytic leukemia, Leukemia, multiple myeloma) and other treatments, chronic pain and acute pain, or wherein the pain is associated with cancer, surgery, bone fracture, bone pain due to tumor metastasis, osteoarthritis, silver shoulder arthritis, rheumatoid arthritis, interstitial cystitis, chronic pancreatitis, visceral pain, migraine, chronic low back pain, bladder pain syndrome, or neuropathic pain, among others.

The compounds, compositions and methods described herein directly address these needs and other objectives. In particular, the invention provides a class of compounds that inhibit, modulate and/or modulate the activity of one or more protein kinases, such as TAM kinase (Axl, Mer and Tyro3), and Trk kinase, for the treatment of proliferative diseases, autoimmune diseases, allergic diseases, inflammatory diseases, pain, fibrosis, transplant rejection and complications thereof. Compared with the existing similar compounds, the compound has better pharmacological activity, and particularly shows excellent inhibitory activity and optimized kinase selectivity on target kinases. In addition, the compound also has excellent membrane penetrating property and shows excellent pharmacokinetic property in animal bodies, so the compound has very good development prospect.

Disclosure of Invention

Definition of terms

Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated by the accompanying structural and chemical formulas. The invention is intended to cover alternatives, modifications and equivalents, which may be included within the scope of the invention as defined by the appended claims. One skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described herein. In the event that one or more of the incorporated documents, patents, and similar materials differ or contradict this application (including but not limited to defined terminology, application of terminology, described techniques, and the like), this application controls.

The articles "a," "an," and "the" as used herein are intended to include "at least one" or "one or more" unless otherwise indicated or clearly contradicted by context. Thus, as used herein, the articles refer to one or to more than one (i.e., to at least one) of the objects. For example, "a component" refers to one or more components, i.e., there may be more than one component contemplated for use or use in embodiments of the described embodiments.

"stereoisomers" refers to compounds having the same chemical structure but differing in the arrangement of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis/trans), atropisomers, and the like.

The term "tautomer" or "tautomeric form" refers to structural isomers having different energies that can interconvert by a low energy barrier (lowenergy barrier). If tautomerism is possible (e.g., in solution), then the chemical equilibrium of the tautomer can be reached. For example, proton tautomers (also known as proton transfer tautomers) include interconversions by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers (valenctautomers) include interconversion by recombination of some of the bonding electrons. A specific example of keto-enol tautomerism is the tautomerism of the pentan-2, 4-dione and 4-hydroxypent-3-en-2-one tautomers. Another example of tautomerism is phenol-ketone tautomerism. One specific example of phenol-ketone tautomerism is the tautomerism of pyridin-4-ol and pyridin-4 (1H) -one tautomers. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

The compounds of the invention may be optionally substituted with one or more substituents, as described herein, such as compounds of the general formula of the invention, or as specified in the examples, subclasses, and classes encompassed by the invention.

It is understood that the term "optionally substituted" may be used interchangeably with the term "substituted or unsubstituted". In general, the term "substituted" means that one or more hydrogen atoms in a given structure are replaced with a particular substituent. "optionally" unless otherwise indicated, an optional substituent group may be substituted at each substitutable position of the group. When more than one position in a given formula can be substituted with one or more substituents selected from a particular group, the substituents may be substituted at each position, identically or differently.

The term "optionally substituted with … …" is used interchangeably with the term "unsubstituted or substituted with … …", i.e., the structure is unsubstituted or substituted with one or more substituents described herein, including, but not limited to, H, D, oxo (═ O), F, Cl, Br, -OH, -CN, -NO₂、-NR^cR^d、-C(＝O)R⁹、-OC(＝O)R⁹、-C(＝O)OR^9a、-S(＝O)_0-2R⁹、-OS(＝O)_1-2R⁹、-S(＝O)_1-2OR^9a、-N(R^10a)C(＝O)R¹⁰、-C(＝O)NR^10aR¹⁰、-OC(＝O)NR^10aR¹⁰、-N(R^10a)S(＝O)_1-2R¹⁰、-S(＝O)_1-2NR^10aR¹⁰、-N(R^10a)C(＝O)NR^10aR¹⁰、C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_1-6Haloalkyl, C_1-6Hydroxyalkyl radical, C_1-6Aminoalkyl, cyano-substituted C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylamino radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl, or C_1-9Heteroaryl C_1-6An alkyl group; wherein each C is_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl group, C_1-9Heteroaryl C_1-6Alkyl is independently optionally substituted with 0,1, 2,3 or 4 substituents independently selected from H, D, oxo (═ O), F, Cl, Br, -OH, -NH₂、-CN、-NO₂、C_1-6Alkyl and C_1-6Alkoxy, and the like. Wherein R is^c、R^d、R⁹、R^9a、R¹⁰And R^10aHave the meaning as described in the present invention.

In the various parts of this specification, substituents of the disclosed compounds are disclosed in terms of group type or range. It is specifically intended that the invention includes each and every independent subcombination of the various members of these groups and ranges. For example, the term "C₁-C₆Alkyl "means in particular independently disclosed methyl, ethyl, C₃Alkyl radical, C₄Alkyl radical, C₅Alkyl and C₆An alkyl group.

The term "alkyl" or "alkyl group" as used herein, denotes a saturated, straight or branched chain monovalent hydrocarbon radical containing from 1 to 20 carbon atoms, wherein the alkyl group may be optionally substituted with one or more substituents as described herein. Unless otherwise specified, alkyl groups contain 1-20 carbon atoms. In one embodiment, the alkyl group contains 1 to 12 carbon atoms; in another embodiment, the alkyl group contains 1 to 6 carbon atoms; in yet another embodiment, the alkyl group contains 1 to 4 carbon atoms; in yet another embodiment, the alkyl group contains 1 to 3 carbon atoms. The alkyl group may be optionally substituted with one or more substituents described herein.

Examples of alkyl groups include, but are not limited to, methyl (Me, -CH)₃) Ethyl group (Et, -CH)₂CH₃) N-propyl (n-Pr, -CH)₂CH₂CH₃) Isopropyl group (i-Pr, -CH (CH)₃)₂) N-butyl (n-Bu, -CH)₂CH₂CH₂CH₃) Isobutyl (i-Bu, -CH)₂CH(CH₃)₂) Sec-butyl (s-Bu, -CH (CH)₃)CH₂CH₃) Tert-butyl (t-Bu, -C (CH)₃)₃) N-pentyl (-CH)₂CH₂CH₂CH₂CH₃) 2-pentyl (-CH (CH)₃)CH₂CH₂CH₃) 3-pentyl (-CH (CH)₂CH₃)₂) 2-methyl-2-butyl (-C (CH)₃)₂CH₂CH₃) 3-methyl-2-butyl (-CH (CH)₃)CH(CH₃)₂) 3-methyl-1-butyl (-CH)₂CH₂CH(CH₃)₂) 2-methyl-1-butyl (-CH)₂CH(CH₃)CH₂CH₃) N-hexyl (-CH)₂CH₂CH₂CH₂CH₂CH₃) 2-hexyl (-CH (CH)₃)CH₂CH₂CH₂CH₃) 3-hexyl (-CH (CH)₂CH₃)(CH₂CH₂CH₃) 2-methyl-2-pentyl (-C (CH))₃)₂CH₂CH₂CH₃) 3-methyl-2-pentyl (-CH (CH)₃)CH(CH₃)CH₂CH₃) 4-methyl-2-pentyl (-CH (CH)₃)CH₂CH(CH₃)₂) 3-methyl-3-pentyl (-C (CH)₃)(CH₂CH₃)₂) 2-methyl-3-pentyl (-CH (CH)₂CH₃)CH(CH₃)₂)2, 3-dimethyl-2-butyl (-C (CH)₃)₂CH(CH₃)₂) 3, 3-dimethyl-2-butyl (-CH (CH)₃)C(CH₃)₃) N-heptyl, n-octyl, and the like.

The term "haloalkyl" or "haloalkoxy" means an alkyl or alkoxy group substituted with one or more halogen atoms, examples of which include, but are not limited to, trifluoromethyl (-CF)₃) Trifluoromethoxy (-OCF)₃) Difluoroethyl (-CH)₂CHF₂,-CF₂CH₃,-CHFCH₂F) Trifluoroethyl (-CH)₂CF₃,-CF₂CH₂F,-CFHCHF₂) And the like.

The terms "hydroxyalkyl" and "hydroxyalkoxy" mean an alkyl or alkoxy group, as the case may be, substituted with one or more hydroxy groups, where "hydroxyalkyl" and "hydroxyalkyl" may be used interchangeably, and examples include, but are not limited to, hydroxymethyl (-CH)₂OH), 2-hydroxyethyl (-CH)₂CH₂OH), 1-hydroxyethyl (-CH (OH) CH₃) 2-hydroxypropan-2-yl (-COH (CH)₃)₂) 2-hydroxy-2-methylpropyl (-CH)₂COH(CH₃)₂) 3-hydroxypropyl (-CH)₂CH₂CH₂OH), 2-hydroxypropyl (-CH)₂CH(OH)CH₃) Hydroxy methoxy (-OCH)₂OH), and the like.

The term "cycloalkyl" denotes a monovalent or polyvalent saturated monocyclic, bicyclic or tricyclic ring system containing from 3 to 12 carbon atoms. Bicyclic cycloalkyl groups include spirobicycloalkyl, fused bicycloalkyl and bridged bicycloalkyl groups. In some embodiments, cycloalkyl groups contain 3 to 12 carbon atoms; in other embodiments, cycloalkyl groups contain 3 to 10 carbon atoms; in other embodiments, cycloalkyl groups contain 3 to 8 carbon atoms; in other embodiments, cycloalkyl groups contain 3 to 7 carbon atoms; in other embodiments, cycloalkyl groups contain 3 to 6 carbon atoms; in still other embodiments, cycloalkyl is C₇-C₁₂Cycloalkyl group containing C₇-C₁₂Monocycloalkyl radical, C₇-C₁₂Bicycloalkyl (e.g. C)₇-C₁₂Spirobicycloalkyl radical, C₇-C₁₂Fused bicycloalkyl and C₇-C₁₂Bridged bicycloalkyl) or C₇-C₁₂A tricycloalkyl group. The cycloalkyl groups may be independently unsubstituted or substituted with one or more substituents described herein. The term "monocyclic cycloalkyl" or "monocycloalkyl" denotes a cycloalkyl group of a monocyclic ring system, wherein the cycloalkyl group has the definitions as described above, and the monocyclic cycloalkyl group can independently be unsubstituted or substituted by one or more substituents described herein. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopentyl-1-alkenyl, 1-cyclopentyl-2-alkenyl, 1-cyclopentyl-3-alkenyl, cyclohexyl, 1-cyclohexyl-1-alkenyl, 1-cyclohexyl-2-alkenyl, 1-cyclohexyl-3-alkenyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and the like.

The term "cycloalkylalkyl" includes cycloalkyl-substituted alkyl groups. In some embodiments, cycloalkylalkyl groups refer to "lower cycloalkylalkyl" groups, i.e.Cycloalkyl radicals bound to C_1-6On the alkyl group of (a). In other embodiments, cycloalkylalkyl groups refer to C-containing groups_1-3The "phenylalkylene" of an alkyl group of (a). Specific examples thereof include, but are not limited to, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclopentylethyl, cyclohexylethyl, and the like. The cycloalkyl group on the cycloalkylalkyl group may be further substituted with one or more substituents described herein.

The terms "heterocyclyl" and "heterocycle" are used interchangeably herein and refer to a mono-, bi-or tricyclic ring system containing 3 to 12 ring atoms which is mono-or polyvalent, saturated or partially unsaturated, and which is not aromatic, wherein at least one ring atom is selected from the group consisting of nitrogen, sulfur and oxygen atoms. In some embodiments, heterocyclyl or heterocyclic contains 4-12 ring atoms. In some embodiments, heterocyclyl or heterocyclic contains 5-12 ring atoms. In some embodiments, heterocyclyl or heterocyclic contains 5-8 ring atoms. In some embodiments, heterocyclyl or heterocyclic contains 5-7 ring atoms. Unless otherwise specified, heterocyclyl may be carbon-or nitrogen-based, and-CH₂The group may optionally be replaced by-C (═ O) -, the sulfur atom of the ring may optionally be oxidized to S-oxide, and the nitrogen atom of the ring may optionally be oxidized to N-oxygen compound. The heterocyclic group includes a saturated heterocyclic group (heterocycloalkyl group) and a partially unsaturated heterocyclic group. The heterocyclic group has one or more attachment points to the rest of the molecule. Examples of heterocyclyl groups include, but are not limited to: oxiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrrolinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, tetrahydrofuryl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, 1, 3-dioxolanyl, dithiocyclopentyl, tetrahydropyranyl, dihydropyranyl, 2H-pyranyl, 4H-pyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, dioxanyl, dithianyl, thiaxanyl, homopiperazinyl, homopiperidinyl, oxepanyl, thiepanyl, nitrogen monoheterocycloheptanyl, oxazepinyl, oxazepin

Radicals (e.g. 1, 4-oxaza)

1, 2-oxaza

Alkyl), diazepine

Radicals (e.g. 1, 4-diazepine)

1, 2-diazepines

Basic), dioxa

Radicals (e.g. 1, 4-dioxa)

1, 2-dioxan

Basic), a sulfur aza

Radicals (e.g. 1, 4-thiazepine)

1, 2-thiaza radical

Indolyl), indolinyl, 1,2,3, 4-tetrahydroisoquinolinyl, 1, 3-benzodioxolyl, 2-oxa-5-azabicyclo [2.2.1]Hept-5-yl, 2-azaspiro [4.4]Nonanyl, 1, 6-dioxaspiro [4.4 ]]Nonanyl, 2-azaspiro [4.5 ]]Decyl, 8-azaspiro [4.5 ]]Decyl, 7-azaspiro [4.5 ]]Decyl, 3-azaspiro [5.5 ]]Undecyl, 2-azaspiro [5.5]Undecyl, octahydro-1H-isoindolyl, N-acetyl-L-amino-N-methyl-ethyl,Octahydrocyclopenta [ c ] s]Pyrrolyl, indolinyl, 1,2,3, 4-tetrahydroisoquinolinyl, hexahydrofuro [3,2-b ]]Furyl and dodecahydroisoquinolinyl, and the like. Examples of substitutions of the-CH 2-group in the heterocyclyl by-C (═ O) -include, but are not limited to, 2-oxopyrrolidinyl, oxo-1, 3-thiazolidinyl, 2-piperidinonyl and 3, 5-dioxopiperidinyl. Examples of the sulfur atom in the heterocyclic group being oxidized include, but are not limited to, sulfolane group, 1-dioxothiomorpholinyl group. The heterocyclyl group may be optionally substituted with one or more substituents as described herein.

In yet another embodiment, heterocyclyl is a 4-7 atom heterocyclyl and refers to a monovalent or polyvalent, saturated or partially unsaturated, nonaromatic, monocyclic or bicyclic ring containing 4-7 ring atoms wherein at least one ring atom is selected from the group consisting of nitrogen, sulfur and oxygen atoms. Unless otherwise specified, a heterocyclic group of 4 to 7 atoms may be carbon-based or nitrogen-based, and-CH₂-the group may optionally be replaced by-C (═ O) -. The sulfur atom of the ring may optionally be oxidized to the S-oxide. The nitrogen atom of the ring may optionally be oxidized to an N-oxygen compound. The 4-7 atom heterocyclyl group has one or more attachment points to the rest of the molecule. Among them, examples of the monocyclic heterocyclic group consisting of 4 to 7 atoms include, but are not limited to: azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrrolinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, tetrahydrofuryl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, tetrahydropyranyl, dihydropyranyl, 2H-pyranyl, 4H-pyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, dioxanyl, dithianyl, thiaxanyl, 1, 2-oxazinyl, 1, 2-thiazinyl, hexahydropyridazinyl, homopiperazinyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepanyl, and pyrazolinyl

Radical (1, 4-oxaza)

A base group and a side chain, wherein the base group is 1,2-oxaza

Alkyl), diazepine

Radical (1, 4-diazepine)

1, 2-diazepines

Alkyl) and thiazepine

Radical (1, 4-thiazepine)

1, 2-thiaza radical

Base), etc.; examples of bicyclic heterocyclic groups consisting of 4 to 7 atoms include, but are not limited to: 3-azabicyclo [3,2,0 ] s]Heptane, 3-oxobicyclo [3,2,0 ]]Heptane and the like; examples of the substitution of the-CH 2-group by-C (═ O) -in a heterocyclic group consisting of 4 to 7 atoms include, but are not limited to, 2-oxopyrrolidinyl, oxo-1, 3-thiazolidinyl, 2-piperidonyl and 3, 5-dioxopiperidinyl; examples of the sulfur atom in the heterocyclic group consisting of 4 to 7 atoms being oxidized include, but are not limited to, sulfolane group, 1-dioxotetrahydrothiophene, 1-dioxotetrahydrothiopyran, 1-dioxothiomorpholinyl. Said heterocyclyl group of 4 to 7 atoms may be optionally substituted by one or more substituents as described herein.

The term "heterocyclylalkyl" includes heterocyclyl-substituted alkyl groups in which both heterocyclyl and alkyl have the meanings as described herein, and such examples include, but are not limited to, tetrahydrofuryl methyl, pyrrol-2-ylmethyl, morpholin-4-ylethyl, piperazin-4-ylethyl, piperidin-4-ylethyl, and the like.

The term "aryl" denotes monocyclic, bicyclic and tricyclic carbon ring systems containing 6 to 14 ring atoms, or 6 to 12 ring atoms, or 6 to 10 ring atoms, wherein at least one ring system is aromatic, wherein each ring system contains 3 to 7 atoms in the ring and one or more attachment points to the rest of the molecule. The term "aryl" may be used interchangeably with the term "aromatic ring". Examples of the aryl group may include phenyl, naphthyl and anthracenyl. The aryl group may independently be optionally substituted with one or more substituents described herein.

The term "arylalkyl" or "aralkyl" includes aryl-substituted alkyl groups. In some embodiments, an arylalkyl group refers to a "lower arylalkyl" group, i.e., the aryl group is attached to C_1-6On the alkyl group of (a). In other embodiments, arylalkyl group refers to a C-containing group_1-3The "phenylalkylene" of an alkyl group of (a). Specific examples thereof include, but are not limited to, benzyl, diphenylmethyl, phenethyl, and the like. The aryl group on the arylalkyl group can be further substituted with one or more substituents described herein.

The term "heteroaryl" denotes monocyclic, bicyclic and tricyclic ring systems containing 5 to 12 ring atoms, or 5 to 10 ring atoms, or 5 to 6 ring atoms, wherein at least one ring is aromatic and at least one aromatic ring contains one or more heteroatoms, wherein each ring system contains a ring of 5 to 7 atoms with one or more attachment points to the rest of the molecule. The term "heteroaryl" may be used interchangeably with the terms "heteroaromatic ring" or "heteroaromatic compound". In one embodiment, heteroaryl is a heteroaryl consisting of 5 to 12 atoms containing 1,2,3 or 4 heteroatoms independently selected from O, S and N. In another embodiment, heteroaryl is a heteroaryl consisting of 5 to 10 atoms containing 1,2,3, or 4 heteroatoms independently selected from O, S, and N. In another embodiment, heteroaryl is 5-6 atom composed of 1,2,3, or 4 heteroatoms independently selected from O, S, and N. The heteroaryl group is optionally substituted with one or more substituents described herein.

Examples of heteroaryl groups include, but are not limited to, 2-furyl, 3-furyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, pyridazinyl (e.g., 3-pyridazinyl), 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, tetrazolyl (e.g., 5-tetrazolyl), triazolyl (e.g., 2-triazolyl and 5-triazolyl), and the like, 2-thienyl, 3-thienyl, pyrazolyl (e.g., 2-pyrazolyl), isothiazolyl, 1,2, 3-oxadiazolyl, 1,2, 5-oxadiazolyl, 1,2, 4-oxadiazolyl, 1,2, 3-triazolyl, 1,2, 3-thiadiazolyl, 1,3, 4-thiadiazolyl, 1,2, 5-thiadiazolyl, pyrazinyl, 1,3, 5-triazinyl; the following bicyclic rings are also included, but are in no way limited to these: benzimidazolyl, benzofuranyl, benzothienyl, indolyl (e.g., 2-indolyl), purinyl, quinolyl (e.g., 2-quinolyl, 3-quinolyl, 4-quinolyl), isoquinolyl (e.g., 1-isoquinolyl, 3-isoquinolyl, or 4-isoquinolyl), imidazo [1,2-a ] pyridyl, 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 ] pyridyl, and the like.

The term "heteroarylalkyl" denotes an alkyl group substituted with one or more heteroaryl groups, wherein both the heteroaryl and alkyl groups have the meaning described herein, and such examples include, but are not limited to, pyridine-2-methyl, imidazole-2-methyl, furan-2-ethyl, indole-3-methyl, and the like.

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

As used herein, "pharmaceutically acceptable salts" refer to organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as are: berge et al, descriptive acceptable salts in detail in J. pharmaceutical Sciences,1977,66:1-19. Pharmaceutically acceptable non-toxic acid salts include, but are not limited to, inorganic acid salts formed by reaction with amino groups such as hydrochloric acidSalts, hydrobromides, phosphates, sulphates, perchlorates, and organic acid salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates, or these salts can be obtained by other methods described in the literature, such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, cyclopentylpropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxy-ethanesulfonates, lactobionates, lactates, laurates, malates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, palmitates, pamoates, pectinates, persulfates, 3-phenylpropionates, picrates, pivalates, propionates, stearates, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts obtained with appropriate bases include alkali metals, alkaline earth metals, ammonium and N⁺(C_1-4Alkyl radical)₄A salt. The present invention also contemplates quaternary ammonium salts formed from compounds containing groups of N. Water-soluble or oil-soluble or dispersion products can be obtained by quaternization. Alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Pharmaceutically acceptable salts further include suitable, non-toxic ammonium, quaternary ammonium salts and amine cations resistant to formation of counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C_1-8Sulfonates and aromatic sulfonates.

"solvate" of the present invention refers to an association of one or more solvent molecules with a compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol. The term "hydrate" refers to an association of solvent molecules that is water.

Description of the Compounds of the invention

The invention discloses a novel compound which can be used as an inhibitor of the activity of protein kinases, in particular TAM family kinases (including TYRO3, AXL and MER) and NTRK family kinases (including NTRKA, NTRKB and NTRKC). Compounds that are protein kinase inhibitors are useful for treating diseases associated with inappropriate protein kinase activity, particularly inappropriate TAM kinase, and NTRK kinase activity. Compared with the existing similar compounds, the compound has better pharmacological activity, and particularly shows excellent inhibitory activity and optimized kinase selectivity on target kinases. In addition, the compound also has excellent membrane penetrating property and shows excellent pharmacokinetic property in animal bodies, so the compound has very good development prospect.

The compounds disclosed in the invention can show strong inhibitory activity against one or more protein kinases. In one aspect, the invention relates to a compound having the structure of formula (I):

or a stereoisomer, tautomer, nitroxide, solvate, metabolite, or pharmaceutically acceptable salt thereof;

wherein the content of the first and second substances,

U₁and U₂Are each independently N or-C (R)^a)-；

R¹、R²And R⁴Are each independently H, C_1-6Alkyl radical, C_1-6Haloalkyl, C_1-6Hydroxyalkyl radical, C_1-6Aminoalkyl, cyano-substituted C_1-6Alkyl radical, C_3-10Cycloalkyl radical, C_3-10Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl group, C_1-9Heteroaryl C_1-6An alkyl group; wherein each C is_1-6Alkyl radical, C_1-6Haloalkyl, C_1-6Hydroxyalkyl radical, C_1-6Aminoalkyl, cyano-substituted C_1-6Alkyl radical, C_3-10Cycloalkyl radical, C_3-10Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl and C_1-9Heteroaryl C_1-6Alkyl is independently optionally substituted with 0,1, 2,3 or 4R¹¹Substitution;

each R^a、R³、R⁵、R⁶、R⁷And R⁸Are respectively and independently H, D, F, Cl, Br, -OH, -CN, -NO₂、-NR^cR^d、C_1-6Alkyl radical, C_1-6Haloalkyl, C_1-6Hydroxyalkyl radical, C_1-6Aminoalkyl, cyano-substituted C_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl, or C_1-9Heteroaryl C_1-6An alkyl group; wherein each C is_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl and C_1-9Heteroaryl C_1-6Alkyl is independently optionally substituted with 0,1, 2,3 or 4R¹²Substitution;

or R²And R³Optionally form a heterocyclic ring of 4 to 12 atoms together with the carbon and nitrogen atom to which it is attached, wherein said heterocyclic ring of 4 to 12 atoms is optionally substituted with 0,1, 2,3,4 or 5R¹³Substitution;

each R¹¹、R¹²And R¹³Each independently is H, D, oxo (═ O), F, Cl, Br, -OH, -CN, -NO₂、-NR^cR^d、-C(＝O)R⁹、-OC(＝O)R⁹、-C(＝O)OR^9a、-S(＝O)_0-2R⁹、-OS(＝O)_1-2R⁹、-S(＝O)_1-2OR^9a、-N(R^10a)C(＝O)R¹⁰、-C(＝O)NR^10aR¹⁰、-OC(＝O)NR^10aR¹⁰、-N(R^10a)S(＝O)_1-2R¹⁰、-S(＝O)_1-2NR^10aR¹⁰、-N(R^10a)C(＝O)NR^10aR¹⁰、C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_1-6Haloalkyl, C_1-6Hydroxyalkyl radical, C_1-6Aminoalkyl, cyano-substituted C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylamino radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl, or C_1-9Heteroaryl C_1-6An alkyl group; wherein each C is_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl group, C_1-9Heteroaryl C_1-6Alkyl is independently optionally substituted with 0,1, 2,3 or 4 substituents independently selected from H, D, oxo (═ O), F, Cl, Br, -OH, -NH₂、-CN、-NO₂、C_1-6Alkyl and C_1-6A radical substitution of alkoxy;

each R^c、R^d、R⁹、R^9a、R¹⁰And R^10aAre each independently H, D, C_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl, or C_1-9Heteroaromatic compoundsRadical C_1-6An alkyl group; wherein each C is_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl and C_1-9Heteroaryl C_1-6Alkyl is independently optionally substituted with 0,1, 2,3 or 4 substituents independently selected from H, D, oxo (═ O), F, Cl, Br, -OH, -NH₂、-CN、-NO₂、C_1-6Alkyl and C_1-6A radical substitution of alkoxy; and

n is 0,1, or 2.

In some embodiments, the first and second substrates are, among others,

R²is H, C_1-4Alkyl radical, C_1-4Haloalkyl, C_1-4Hydroxyalkyl radical, C_1-4Aminoalkyl, cyano-substituted C_1-4Alkyl radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-4Alkyl radical, C_2-7Heterocyclic group, or C_2-7Heterocyclyl radical C_1-4An alkyl group; wherein each C is_1-4Alkyl radical, C_1-4Haloalkyl, C_1-4Hydroxyalkyl radical, C_1-4Aminoalkyl, cyano-substituted C_1-4Alkyl radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-4Alkyl radical, C_2-7Heterocyclyl and C_2-7Heterocyclyl radical C_1-4Alkyl is independently optionally substituted with 0,1, 2,3 or 4R¹²Substitution;

R³is H, D, -CN, C_1-4Alkyl, wherein each of said C_1-4Alkyl is optionally substituted by 0,1, 2,3 or 4R¹¹Substitution;

or R²And R³Optionally form a 5-12 atom heterocycle with the carbon and nitrogen atoms to which it is attached, wherein said 5-12 atom heterocycle is optionally substituted with 0,1, 2,3,4, or 5R¹³And (4) substitution.

In other embodiments, the compounds of the present invention have the structure of formula (II):

wherein the content of the first and second substances,

X¹is O, S, -N (R)^13a)-、-C(＝O)-、-(CH₂)_t1-、-X²-(CH₂)_t1-, or- (CH)₂)_t1-X²-(CH₂)_t2-；

X²Is O, S, -N (R)^13a) -, or-C (═ O) -;

each R^13aEach independently is H, D, oxo (═ O), F, Cl, Br, -OH, -CN, -NO₂Amino group, C_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl group, C_1-9Heteroaryl C_1-6Alkyl, -C (═ O) R⁹、-C(＝O)OR^9a、-S(＝O)_0-2R⁹、-S(＝O)_1-2OR^9a、-S(＝O)_1-2NR^10aR¹⁰or-C (═ O) NR^10aR¹⁰Wherein each of C_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl and C_1-9Heteroaryl C_1-6Alkyl is independently optionally substituted with 0,1, 2,3 or 4 substituents independently selected from H, D, oxo (═ O), F, Cl, Br, -OH, -NH₂、-CN、-NO₂、C_1-6Alkyl and C_1-6A radical substitution of alkoxy;

each t1 and t2 is independently 0,1, 2, or 3; and

m is 0,1, 2,4, or 5.

In some embodiments, wherein X¹Is O, S, -N (R)^13a)-、-C(＝O)-、-(CH₂)_t1-、-X²-(CH₂)_t1-, or- (CH)₂)_t1-X²-(CH₂)_t2-; and X²Is O, S, or-N (R)^13a)-。

In some embodiments, R¹³Each independently is H, D, oxo (═ O), F, Cl, Br, -OH, -NH₂、-CN、-NO₂、C_1-4Alkyl radical, C_2-4Alkenyl radical, C_2-4Alkynyl, C_1-4Haloalkyl, C_1-4Hydroxyalkyl radical, C_1-4Aminoalkyl, cyano-substituted C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkylamino radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-4Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-4Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-4Alkyl radical, C_1-9Heteroaryl, or C_1-9Heteroaryl C_1-4An alkyl group; wherein each C is_1-4Alkyl radical, C_2-4Alkenyl radical, C_2-4Alkynyl, C_1-4Haloalkyl, C_1-4Hydroxyalkyl radical, C_1-4Aminoalkyl, cyano-substituted C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkylamino radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-4Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-4Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-4Alkyl radical, C_1-9Heteroaryl and C_1-9Heteroaryl C_1-4Alkyl is independently optionally substituted with 0,1, 2,3 or 4 substituents independently selected from H, D, oxo (═ O), F, Cl, Br, -OH, -NH₂、-CN、-NO₂、C_1-4Alkyl and C_1-4Alkoxy groups.

In some embodiments, each R is^13aEach independently is H, D, oxo (═ O), F, Cl, Br, -OH, -CN, -NO₂Amino group, C_1-4Alkyl radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-4Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-4Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-4Alkyl radical, C_1-9Heteroaryl, or C_1-9Heteroaryl C_1-4An alkyl group; wherein each C is_1-4Alkyl radical C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-4Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-4Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-4Alkyl radical, C_1-9Heteroaryl and C_1-9Heteroaryl C_1-4Alkyl is independently optionally substituted with 0,1, 2,3 or 4 substituents independently selected from H, D, oxo (═ O), F, Cl, Br, -OH, -NH₂、-CN、-NO₂、C_1-6Alkyl and C_1-6Alkoxy groups.

In some embodiments, each t1 and t2 is independently 0,1, 2, or 3, respectively; and m is 0,1, 2, or 4.

In some embodiments, wherein R¹Is C_1-4Alkyl radical, C_1-4Haloalkyl, C_1-4Hydroxyalkyl radical, C_1-4Aminoalkyl, cyano-substituted C_1-4Alkyl radical, C_3-8Cycloalkyl, phenyl, or C_1-9A heteroaryl group; wherein each C is_1-4Alkyl radical, C_1-4Haloalkyl, C_1-4Hydroxyalkyl radical, C_1-4Aminoalkyl, cyano-substituted C_1-4Alkyl radical, C_3-8Cycloalkyl, phenyl and C_1-9Heteroaryl is independently optionally substituted with 0,1, 2,3 or 4R¹¹And (4) substitution.

In some embodiments, the first and second substrates are, among others,

U₁and U₂Are each independently N or-C (R)^a)-；

Each R^aAnd R⁸Are respectively and independently H, D, F, Cl, Br, -OH, -CN, -NO₂、-NH₂Or C_1-4An alkyl group; wherein each C is_1-4Alkyl is independently optionally substituted with 0,1, 2,3 or 4R¹²Substitution; and

n is 0,1, or 2.

In some embodiments, wherein R⁴H, D, methyl, ethyl, propyl, isopropyl, butyl,C_1-4Haloalkyl, C_1-4Aminoalkyl, cyano-substituted C_1-4Alkyl radical, C_3-6Cycloalkyl radical, C_3-6Cycloalkyl radical C_1-4Alkyl radical, C_2-7Heterocyclic group, or C_2-7Heterocyclyl radical C_1-4An alkyl group.

In some embodiments, wherein R⁵Is H, D, -NR^cR^d、C_1-4Alkyl radical, C_1-4Haloalkyl, C_1-4Hydroxyalkyl radical, C_1-4Aminoalkyl, or cyano-substituted C_1-4An alkyl group.

In some embodiments, wherein R⁶And R⁷Are respectively and independently H, D, F, Cl, Br, -OH, -NR^cR^d、-CN、-NO₂、C_1-4Alkyl radical, C_1-4Haloalkyl, C_1-4Hydroxyalkyl radical, C_1-4Aminoalkyl, or cyano-substituted C_1-4An alkyl group.

In some embodiments, wherein each R is¹¹、R¹²And R¹³Each independently is H, D, oxo (═ O), F, Cl, Br, -OH, -CN, -NO₂、-NR^cR^d、C_1-4Alkyl radical, C_1-4Haloalkyl, C_1-4Hydroxyalkyl radical, C_1-4Aminoalkyl, cyano-substituted C_1-4Alkyl radical, C_1-4Alkoxy, or C_1-4An alkylamino group; wherein each C is_1-4Alkyl radical, C_1-4Haloalkyl, C_1-4Hydroxyalkyl radical, C_1-4Aminoalkyl, cyano-substituted C_1-4Alkyl radical, C_1-4Alkoxy and C_1-4Alkylamino is independently optionally substituted with 0,1, 2,3 or 4 substituents independently selected from H, D, oxo (═ O), F, Cl, Br, -OH, -NH₂、-CN、-NO₂And C_1-4Alkoxy groups.

In some embodiments, wherein each R is^c、R^d、R⁹、R^9a、R¹⁰And R^10aAre each independently H, D, C_1-4Alkyl radical, C_3-6Cycloalkyl, or C_2-7A heterocyclic group; wherein each C is_1-4Alkyl radicalC_3-6Cycloalkyl and C_2-7Heterocyclyl is independently optionally substituted with 0,1, 2,3 or 4 substituents independently selected from H, D, oxo (═ O), F, Cl, Br, -OH, -NH₂、-CN、-NO₂、C_1-6Alkyl and C_1-6Alkoxy groups.

In some embodiments, the compounds of the present invention are compounds having one of the following structures:

or a stereoisomer, tautomer, nitroxide, solvate, metabolite, or pharmaceutically acceptable salt thereof.

Stereoisomers, tautomers, solvates, metabolites or pharmaceutically acceptable salts of the compounds of formula (I) and (II) are included within the scope of the present invention unless otherwise indicated.

The compounds of the present disclosure may contain asymmetric or chiral centers and thus may exist in different stereoisomeric forms. The present invention contemplates that all stereoisomeric forms of the compounds of formula (I) or (II), including but not limited to diastereomers, enantiomers, atropisomers and geometric (or conformational) isomers, and mixtures thereof, such as racemic mixtures, are integral to the invention.

In the structures disclosed herein, when the stereochemistry of any particular chiral atom is not specified, then all stereoisomers of that structure are contemplated as within this invention and are included as disclosed compounds in this invention. When stereochemistry is indicated by a solid wedge (solid wedge) or dashed line representing a particular configuration, then the stereoisomers of the structure are so well-defined and defined.

The compounds of formula (I) or (II) may be present in the form of a salt. In one embodiment, the salt refers to a pharmaceutically acceptable salt. The term "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the mammal being treated therewith. In another embodiment, the salt need not be a pharmaceutically acceptable salt, and may be an intermediate useful in the preparation and/or purification of a compound of formula (I) or (II) and/or in the isolation of an enantiomer of a compound of formula (I) or (II).

In another aspect, the invention relates to intermediates for the preparation of compounds of formula (I) and (II).

In another aspect, the invention relates to methods for the preparation, isolation and purification of compounds of formula (I) and (II).

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention. In one embodiment, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable excipient, diluent or carrier, or a combination thereof. In another embodiment, the pharmaceutical composition may be in a liquid, solid, semi-solid, gel, or spray dosage form.

In some embodiments, the pharmaceutical compositions of the present invention further comprise an additional therapeutic agent.

In another aspect, the invention relates to the use of a compound according to the invention or a pharmaceutical composition according to the invention in the manufacture of a medicament for the prevention or treatment of one or more Axl and/or Trk protein kinase mediated diseases and/or conditions.

In some embodiments, the disease and/or disorder is selected from a proliferative disease, an autoimmune disease, an allergic disease, an inflammatory disease, or transplant rejection.

In some embodiments, the disease and/or disorder is selected from the group consisting of treatment and prevention of TAM kinase, and NTRK kinase-mediated diseases involving signaling pathways. Such diseases include proliferative diseases, autoimmune diseases, allergic diseases, inflammatory diseases, transplant rejection, and complications thereof. In particular, the compounds of the present invention may be used to treat diseases such as cancer, polycythemia vera, essential thrombocythemia, myelofibrosis, myeloid leukemia, acute lymphocytic leukemia, Chronic Myelogenous Leukemia (CML), Chronic Obstructive Pulmonary Disease (COPD), asthma, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, sjogren's syndrome, psoriasis, type I diabetes, respiratory allergic disease, sinusitis, eczema, measles, food allergy, insect venom allergy, inflammatory bowel disease, crohn's disease, rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, organ transplant rejection, tissue transplant rejection, cell transplant rejection, and the like.

Pharmaceutical compositions, formulations and administration of the compounds of the invention

The present invention provides a pharmaceutical composition comprising a compound disclosed herein, or as listed in the examples; and pharmaceutically acceptable adjuvants, diluents, carriers, vehicles or combinations thereof. The amount of compound in the pharmaceutical compositions disclosed herein is that amount which is effective to detect inhibition of a protein kinase in a biological sample or patient.

It will also be appreciated that certain compounds of the invention may be present in free form for use in therapy or, if appropriate, in the form of a pharmaceutically acceptable derivative thereof. Some non-limiting embodiments of pharmaceutically acceptable derivatives include pharmaceutically acceptable salts, esters, salts of such esters, or any additional adduct or derivative that upon administration to a patient in need thereof provides, directly or indirectly, a compound of the present invention or a metabolite or residue thereof.

Various carriers for formulating pharmaceutically acceptable compositions, and well known techniques for their preparation, are disclosed in documents such as Remington, The Science and Practice of Pharmacy,21stedition,2005, ed.D.B.Troy, Lippincott Williams & Wilkins, Philadelphia, and Endencyclopedia of Pharmaceutical Technology, eds.J.Swarbrick and J.C.Boylan,1988-1999, Marcel Dekker, New York, The contents of each of which are incorporated herein by reference. Except insofar as any conventional carrier is incompatible with the disclosed compounds, such as by producing any undesirable biological effect or interacting in a deleterious manner with any other ingredient in a pharmaceutically acceptable composition, its use is contemplated as falling within the scope of the present invention.

The pharmaceutical compositions provided by the present invention may be co-formulated with other active ingredients that do not impair the intended therapeutic effect, or with substances that supplement the intended effect.

Use of the Compounds and compositions of the invention

The present invention provides methods of treating, preventing, or ameliorating one or more symptoms of a disease or disorder mediated or otherwise affected by the behavior of one or more protein kinases, such as TAM family kinases (including Tyro3, AXL, and MER kinases), or NTRK family kinases (NTRKA, NTRKB, and NTRKC), or a disease or disorder mediated or otherwise affected by the behavior of one or more protein kinases, such as TAM family kinases (including Tyro3, AXL, and MER kinases), or NTRK family kinases (NTRKA, NTRKB, and NTRKC), using the compounds and pharmaceutical compositions disclosed herein.

TAM family kinases (including Tyro3, AXL and MER kinases), or NTRK family kinases (NTRKA, NTRKB and NTRKC) may be wild-type and/or mutated to the kinases involved.

In some embodiments, the present invention provides a class of compounds disclosed herein or pharmaceutical compositions comprising a compound disclosed herein for use in treating, preventing or ameliorating one or more symptoms of a disease or disorder mediated or otherwise affected by inappropriate AXL kinase activity or a disease or disorder mediated or otherwise affected by inappropriate AXL kinase activity. In another embodiment, the disease, disorder, or one or more symptoms of a disease or disorder is associated with inappropriate MER kinase behavior. In yet another embodiment, the disease, disorder, or one or more symptoms of the disease or disorder is associated with inappropriate TYRO3 kinase activity.

In some embodiments, the invention provides a class of compounds disclosed herein or pharmaceutical compositions comprising a compound disclosed herein for use in treating, preventing or ameliorating one or more symptoms of a disease or disorder mediated or otherwise affected by inappropriate NTRK-a kinase behavior or a disease or disorder mediated or otherwise affected by inappropriate NTRK-a kinase behavior. In other embodiments, the disease, disorder, or one or more symptoms of the disease or disorder is associated with inappropriate NTRK-B kinase behavior. In some embodiments, the disease, disorder, or one or more symptoms of the disease or disorder is associated with inappropriate NTRK-C kinase behavior.

By "inappropriate AXL kinase activity" is meant AXL kinase activity that occurs in a particular patient that deviates from normal AXL kinase activity. Inappropriate AXL kinase activity may be expressed in the form of, for example, an abnormal increase in activity, or deviations in the time point and control of AXL kinase activity. This inappropriate kinase behavior results, for example, from inappropriate or uncontrolled behavior caused by overexpression or mutation of protein kinases. Accordingly, the present invention provides methods of treating these diseases and disorders.

Consistent with the above description, such diseases or disorders include, but are not limited to: myeloproliferative diseases, such as polycythemia vera (PCV), essential thrombocythemia, Idiopathic Myelofibrosis (IMF); leukemias, e.g., myeloid leukemias including Chronic Myeloid Leukemia (CML), imatinib-resistant CML forms, Acute Myeloid Leukemia (AML) and subtypes of AML, acute megakaryoblastic leukemia (AMKL); lymphoproliferative diseases such as Acute Lymphoblastic Leukemia (ALL) and myeloma; the cancer includes head and neck cancer, prostate cancer, breast cancer, ovarian cancer, melanoma, lung cancer, brain tumor, pancreatic cancer, urothelial cancer, liver cancer, stomach cancer, kidney cancer, etc.; and inflammatory diseases or disorders associated with immune dysfunction, immunodeficiency, immune modulation, autoimmune diseases, tissue transplant rejection, graft-versus-host disease, wound healing, kidney disease, multiple sclerosis, thyroiditis, type I diabetes, sarcoidosis, psoriasis, allergic rhinitis, inflammatory bowel disease including crohn's disease and Ulcerative Colitis (UC), Systemic Lupus Erythematosus (SLE), arthritis, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma and Chronic Obstructive Pulmonary Disease (COPD), and dry eye syndrome (or keratoconjunctivitis sicca (KCS)).

In one aspect, the present invention provides a class of compounds disclosed herein or pharmaceutical compositions comprising a compound disclosed herein for use in the prevention and/or treatment of a proliferative disease, an autoimmune disease, an allergic disease, an inflammatory disease, or transplant rejection in a mammal (including a human).

In another aspect, the present invention provides a method of treating a mammal suffering from or at risk of suffering from a disease disclosed herein, the method comprising administering a condition treating effective amount or a condition preventing effective amount of one or more of the pharmaceutical compositions or compounds disclosed herein.

In particular examples, the proliferative disease is selected from the group consisting of cancer (e.g., colon cancer, glioblastoma, endometrial cancer, liver cancer, lung cancer, melanoma, kidney cancer, thyroid cancer, lymphoma, lymphoproliferative disorders, small cell lung cancer, squamous cell lung cancer, glioma, breast cancer, prostate cancer, ovarian cancer, cervical cancer, and the like), hematological malignancies, such as Acute Myelogenous Leukemia (AML), myelodysplastic syndrome (MDS), myeloproliferative disorders (MPD), Chronic Myelogenous Leukemia (CML), T-cell acute lymphocytic leukemia (T-ALL), B-cell acute lymphocytic leukemia (B-ALL), non-Hodgkin's lymphoma (NHL), B-cell lymphoma, polycythemia vera, essential thrombocythemia, myelofibrosis, multiple myeloma, and the like.

In another aspect, the present invention provides a method of treating and/or preventing a mammal susceptible to or suffering from an autoimmune disease, said method comprising administering a therapeutically effective amount or a prophylactically effective amount of one or more of the presently disclosed pharmaceutical compositions or compounds.

In a particular embodiment, the autoimmune disease is selected from COPD, asthma, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, sjogren's syndrome, psoriasis, type I diabetes and inflammatory bowel disease.

In another aspect, the present invention provides a method of treating and/or preventing a mammal susceptible to or suffering from an allergic disease, said method comprising administering a therapeutically effective amount or a prophylactically effective amount of one or more of the pharmaceutical compositions or compounds disclosed herein. In a particular embodiment, the allergic disease is selected from respiratory allergic disease, sinusitis, eczema and measles, food allergy and insect venom allergy.

In a particular embodiment, the allergic disease is selected from respiratory allergic disease, sinusitis, eczema and measles, food allergy and insect venom allergy.

In another aspect, the present invention provides a method of treating and/or preventing a mammal susceptible to or suffering from an inflammatory disease, comprising administering a therapeutically effective amount or a prophylactically effective amount of one or more of the pharmaceutical compositions or compounds disclosed herein.

In another aspect, the invention provides a class of compounds disclosed herein for use as medicaments, in particular for use as medicaments for the treatment and/or prevention of the diseases described herein. Also provided is the use of a compound disclosed herein for the manufacture of a medicament for the treatment and/or prevention of a disease described herein.

Combination therapy

The compounds of the present invention may be administered as the sole active agent or may be administered in combination with other therapeutic agents, including other compounds that have the same or similar therapeutic activity and are identified as safe and effective for such combination administration.

In one aspect, the invention provides a method of treating, preventing or ameliorating a disease or condition comprising administering a safe and effective amount of a combination comprising a compound of the disclosure and one or more therapeutically active agents. In one embodiment, the combination comprises one or two additional therapeutic agents.

Examples of other therapeutic agents include, but are not limited to: anti-cancer agents, including chemotherapeutic agents and antiproliferative agents; an anti-inflammatory agent; and an immunomodulator or immunosuppressant.

In another aspect, the invention provides products comprising a compound of the invention and at least one other therapeutic agent, formulated for simultaneous, separate or sequential administration in therapy. In some embodiments, the treatment is a treatment for a disease or condition mediated by one or more protein kinase, such as AXL kinase, or NTRK kinase activity. The products provided by the combined preparation include compositions comprising the disclosed compounds and other therapeutic agents in the same pharmaceutical composition, or in different forms, e.g., kits.

In another aspect, the present invention provides a pharmaceutical composition comprising a compound disclosed herein and one or more additional therapeutic agents. In one embodiment, the pharmaceutical composition may comprise a pharmaceutically acceptable excipient as described above.

In another aspect, the invention provides a kit comprising two or more separate pharmaceutical compositions, wherein at least one pharmaceutical composition comprises a compound disclosed herein. In one embodiment, the kit comprises means for separately holding the compositions, such as a container, a separate bottle, or a separate foil box. An example of such a kit is a blister pack, which is commonly used for packaging tablets, capsules and the like.

The compounds disclosed herein may be administered as a single active ingredient or as, for example, an adjuvant, co-administered with other therapeutic agents.

In some embodiments, the additional therapeutic agent comprises a chemotherapeutic agent and/or an antiproliferative agent. Known chemotherapeutic agents include, but are not limited to, other therapeutic or anti-cancer drugs that may be used in combination with the compounds of the present invention, surgery, radiation therapy (a few examples are gamma radiation, neutron beam radiation, electron beam radiation, proton therapy, brachytherapy and systemic radioisotopes), endocrine therapy, taxanes (taxol, docetaxel, etc.), platinum derivatives (cisplatin), carboplatin (carboplatin), biological response modifiers (interferons, interleukins), tumor necrosis factor (TNF, TRAIL receptor targets), hyperthermia and cryotherapy, agents to mitigate any adverse effects (e.g., anti-emetics), and other approved chemotherapeutic drugs including, but not limited to, alkylating drugs (mechlorethamine), chlorambucil (chlorembucil), cyclophosphamide (cyclophoramide), malan (melphala), and the like, Ifosfamide (ifosfamide)), antimetabolites (methotrexate), pemetrexed (pemetrexed), etc., purine and pyrimidine antagonists (6-mercaptopurine (6-mertepurine), 5-fluorouracil (5-fluorouricil), cytarabine (cytarabine), gemcitabine (gemcitabine), spindle inhibitors (vinblastine), vincristine (vincristine), vinorelbine (vinorelbine), podophyllotoxin (etoposide), irinotecan (irinotecan), topotecan (topotecan), antibiotics (doxorubicin), bleomycin (bleomycin), mitomycin (mitomycin)), nitrosoureas (carmustine, loxastine (loxastine), cell cycle inhibitors (cyclin P), mitosin (CDK-S), and (CDK-S (caspase (CDK-NP), and (CDK-S (CEM), and (CDK-E (CEM-D), and (CEM) Leuprolide (leuprolide), flutamide (flutamide), megestrol (megestrol), dexamethasone (dexamethasone), and the like). Anti-angiogenic agents (avastin, etc.). Mabs (belimumab), brentuximab, cetuximab (cetuximab), gemtuzumab (gemtuzumab), ipilimumab (ipilimumab), ofatumumab, panitumumab (panitumumab), ranibizumab (ranibizumab), rituximab (rituximab), tositumomab (tositumomab), trastuzumab (trastuzumab). Kinase inhibitors (imatinib), sunitinib (sunitinib), sorafenib (sorafenib), erlotinib (erlotinib), gefitinib (gefitinib), dasatinib (dasatinib), nilotinib (nilotinib), lapatinib (lapatinib), crizotinib (crizotinib), ruxotinib (ruxolitinib), vemurafenib (vemurafenib), vandetanib (vandetanib), pazopanib (pazopanib), and the like). Drugs inhibit or activate cancer pathways such as mTOR, the HIF (hypoxia inducible factor) pathway, and others.

The compounds disclosed herein may also be combined with other therapeutic procedures to enhance efficacy. For example, hormone therapy or special radiation therapy is administered. The compounds disclosed herein are particularly useful as radiosensitizers, particularly for the treatment of tumors that are weakly sensitive to those radiation treatments.

"combination" means a fixed combination or a kit of parts for the combined administration in the form of a single dosage unit, wherein a compound disclosed in the invention and a combination partner may be administered separately at the same time or may be administered separately within certain time intervals, in particular such that the combination partners show a cooperative, e.g. synergistic, effect. The terms "co-administration" or the like as used herein are intended to encompass administration of the selected combination partner to a single individual in need thereof (e.g., a patient), and are intended to encompass treatment regimens in which the substances are not necessarily administered by the same route of administration or simultaneously.

Method of treatment

In one embodiment, the presently disclosed methods of treatment comprise administering to a patient in need thereof a safe and effective amount of a compound of the present invention or a pharmaceutical composition comprising a compound of the present invention. Various embodiments disclosed herein include methods of treating a disease referred to herein by administering to a patient in need thereof a safe and effective amount of a disclosed compound or a pharmaceutical composition comprising a disclosed compound.

In embodiments thereof, the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered once or several times at different time intervals over a specified period of time according to a dosing regimen. For example, once, twice, three times or four times daily. In one embodiment, the administration is once daily. In yet another embodiment, the administration is twice daily. The administration may be carried out until the desired therapeutic effect is achieved or the desired therapeutic effect is maintained indefinitely. Suitable dosing regimens for the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds depend on the pharmacokinetic properties of the compound, such as dilution, distribution and half-life, which can be determined by the skilled person. In addition, suitable dosing regimens for the compounds or pharmaceutical compositions comprising the disclosed compounds, including the duration of the regimen, will depend on the condition being treated, the severity of the condition being treated, the age and physical condition of the patient being treated, the medical history of the patient being treated, the nature of concurrent therapy, the desired therapeutic effect, and other factors within the knowledge and experience of the skilled artisan. Such a skilled artisan will also appreciate that adjustments to the subject's response to the dosage regimen, or the need for changes in the subject's patient over time, may be required.

The compounds disclosed herein may be administered simultaneously, or before or after, one or more other therapeutic agents. The compounds of the invention may be administered separately from the other therapeutic agents, by the same or different routes of administration, or together with them in pharmaceutical compositions.

General synthetic schemes

To illustrate the invention, the following examples are set forth. It is to be understood that the invention is not limited to these embodiments, but is provided as a means of practicing the invention.

In general, the compounds of the invention can be prepared by the methods described herein, wherein the substituents are as defined in formula (I) or (II), unless otherwise indicated. The following reaction schemes and examples serve to further illustrate the context of the invention.

The examples described below, unless otherwise indicated, are all temperatures set forth in degrees Celsius. Reagents were purchased from commercial suppliers such as Aldrich Chemical Company, Arco Chemical Company and Alfa Chemical Company and were used without further purification unless otherwise indicated. General reagents were purchased from Shantou Wen Longdan chemical plant, Guangzhou chemical reagent plant, Fuchen chemical reagent plant of Tianjin City, Wuhan Xin Huayuan scientific and technological development Co., Ltd, and Qingdao Kayakeli.

The anhydrous tetrahydrofuran, dioxane, toluene and ether are obtained through reflux drying of metal sodium. The anhydrous dichloromethane and chloroform are obtained by calcium hydride reflux drying. Ethyl acetate, petroleum ether, N-hexane, N, N-dimethylacetamide and N, N-dimethylformamide were used as they were previously dried over anhydrous sodium sulfate.

The following reactions are generally carried out under positive pressure of nitrogen or argon or by sleeving a dry tube over an anhydrous solvent (unless otherwise indicated), the reaction vial being stoppered with a suitable rubber stopper and the substrate being injected by syringe. The glassware was dried.

The column chromatography is performed using a silica gel column. Silica gel (300 and 400 meshes) was purchased from Qingdao oceanic chemical plants.

¹H NMR spectra were recorded using a Bruker 300MHz, 400MHz or 600MHz NMR spectrometer.¹H NMR Spectrum in CDC1₃、DMSO-d₆、CD₃OD or acetone-d₆As solvent (in ppm) withTMS (0ppm) or chloroform (7.26ppm) was used as reference standard. When multiple peaks occur, the following abbreviations will be used: s (singlets), d (doublets), t (triplets), m (multiplets), br (broadcasters), br (broadendigles), dd (doublets of doublets), dt (doublets of triplets). Coupling constants are expressed in hertz (Hz).

Low resolution Mass Spectrometry (MS) data were determined using Agilent 6120 quadrupole HPLC-M (column model: Zorbax SB-C18,2.1 × 30mm,3.5 microns, 6min, flow rate of 0.6 mL/min. mobile phase: 5% -95% (CH with 0.1% formic acid)₃CN) in (H containing 0.1% formic acid)₂O) by electrospray ionization (ESI) at 210nm/254nm, with UV detection.

Pure compounds were detected by UV at 210nm/254nm using Agilent 1260pre-HPLC or Calesep pump 250pre-HPLC (column model: NOVASEP 50/80mm DAC).

Typical synthetic procedures for preparing the disclosed compounds are shown in schemes 1-2 below. Unless otherwise stated, each R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、U₁、U₂And n each have the definitions as described herein; PG (Picture experts group)¹And PG²Is a protecting group.

Synthesis scheme 1:

having the formula(6)The compounds of the invention of the structures shown can be prepared by general synthetic methods as described in scheme 1, with reference to the examples for specific procedures. In FIG. 1, boron ester derivatives are prepared under basic conditions(1)In the presence of a suitable base (e.g., cesium carbonate, potassium carbonate, sodium carbonate, etc.), and a suitable Pd catalyst (e.g., Pd (OAc))₂、Pd(dppf)₂Cl₂Or Pd₂(dba)₃Etc.) with a substituted heteroaryl compound(2)By coupling reaction to obtainTo the compound(3). Removal of protective group PG¹Then obtaining the aromatic amine derivative(4). Carboxylic acid derivatives(5)With compounds in the presence of condensing agents (e.g. EDCI or HATU)(4)Condensing to obtain target kinase inhibitor(6)。

Carboxylic acid derivatives(5)Can be obtained by synthetic methods described in the literature (see, for example, "Practical synthesis of bicyclic pyrazol-5-one derivatives." Xuejin Feng, Michael A. Xi, Yanjun Wu, Xialogang Wang, Ning Xi Tetrahedron Lett.2017,58, 46-49; Facile synthesis of bicyclic1-arylpyrazol-5-ones. "Wu, Y.; Wang, K.; Li, Z.; Bai, X.; Xi, N.tetrahedron Lett.2014,55, 142-.

Synthesis scheme 2:

having the formula(6)The compounds of the invention of the structures shown can also be prepared by general synthetic methods as depicted in scheme 2, with reference to the examples for specific procedures. In FIG. 2, aryl or heteroaryl compounds (A)7)With compounds in the presence of condensing agents (e.g. EDCI or HATU)(5)Condensed to obtain a compound(8). Under alkaline conditions, boron ester derivatives(10)In the presence of a suitable base (e.g., cesium carbonate, potassium carbonate, sodium carbonate, etc.), and a suitable Pd catalyst (e.g., Pd (OAc))₂、Pd(dppf)₂Cl₂Or Pd₂(dba)₃Etc.) with a substituted heteroaryl compound(8)Carrying out coupling reaction to obtain a compound(9). Under alkaline conditions, boron ester derivatives(9)In the presence of a suitable base (e.g., cesium carbonate, potassium carbonate, sodium carbonate, etc.), and a suitable Pd catalyst (e.g., Pd (OAc))₂、Pd(dppf)₂Cl₂Or Pd₂(dba)₃Etc.) with a substituted heteroaryl derivative(2)Carrying out coupling reaction to obtain the target kinase inhibitor(6)。

Examples

Example 1N- (4- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -2-oxo-1-phenyl-1, 2,4,5,6, 7-hexahydropyrazolo [1,5-a ] pyridine-3-carboxamide

Step 1) N- (4-bromophenyl) -2-oxo-1-phenyl-1, 2,4,5,6, 7-hexahydropyrazolo [1,5-a ] pyridine-3-carboxamide

2-oxo-1-phenyl-1, 2,4,5,6, 7-hexahydropyrazolo [1,5-a ] is contained]A mixture of pyridine-3-carboxylic acid (650mg, 65%, 1.636mmol), triethanolamine (0.7mL, 5mmol), 4-bromoaniline (282mg, 1.639mmol) and HATU (622mg, 1.636mmol) in dichloromethane (20mL) was stirred at room temperature for 23 h. Water (20mL) was added to the reaction solution to dilute, and extracted with dichloromethane (30mL × 2), the organic phases were combined and concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (EtOAc/MeOH (v/v) ═ 100/1) to give the title compound (386.8mg, 57.35%) as a yellow solid. MS (ESI, pos.ion) M/z:414.2[ M + H ]]⁺。¹H NMR(400MHz,CDCl₃)(ppm):10.61(s,1H),7.54(dd,J＝16.0,8.3Hz,4H),7.45(t,J＝7.4Hz,1H),7.42-7.31(m,4H),3.55(t,J＝5.9Hz,2H),3.38(t,J＝6.4Hz,2H),2.12-2.02(m,2H),1.96-1.86(m,2H)。

Step 2) 2-oxo-1-phenyl-N- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) -1,2,4,5,6, 7-hexahydropyrazolo [1,5-a ] pyridine-3-carboxamide

To a solution containing N- (4-bromophenyl) -2-oxo-1-phenyl-1, 2,4,5,6, 7-hexahydropyrazolo [1,5-a ]]To a solution of pyridine-3-carboxamide (386mg, 0.936mmol), bis-pinacolato diboron (268mg, 1.056mmol) and 1,1' -bis (diphenylphosphino) ferrocene dichloropalladium (II) (104mg, 0.142mmol) in 1, 4-dioxane (8mL) was added potassium acetate (235mg, 2.395 mmol). The reaction solution was heated to reflux under nitrogen for 7.5 hours, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (PE/EtOAc (v/v) ═ 2/1) to give the title compound as a white solid (368.5mg, 85.70%). MS (ESI, pos. ion) M/z 460.2[ M + H ]]⁺。¹H NMR(400MHz,CDCl₃)(ppm):10.67(s,1H),7.75(d,J＝8.4Hz,2H),7.68(d,J＝8.5Hz,2H),7.53(t,J＝7.6Hz,2H),7.44(t,J＝7.4Hz,2H),7.35(d,J＝7.3Hz,2H),3.55(t,J＝5.9Hz,2H),3.40(t,J＝6.4Hz,2H),2.07(dt,J＝11.6,5.8Hz,3H),1.91(m,3H),1.33(s,13H)。

Step 3) N- (4- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -2-oxo-1-phenyl-1, 2,4,5,6, 7-hexahydropyrazolo [1,5-a ] pyridine-3-carboxamide

To a solution containing 2-oxo-1-phenyl-N- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl]-1,2,4,5,6, 7-hexahydropyrazolo [1,5-a]Pyridine-3-carboxamide (368mg, 0.801mmol) and 5-bromo-7-methyl-7H-pyrrolo [2,3-d]To a solution of pyrimidin-4-amine (182mg, 0.801mmol) in 1, 4-dioxane (8mL) was added 1,1' -bis (diphenylphosphino) ferrocene dichloropalladium (II) dichloromethane complex (134mg, 0.161mmol) and cesium carbonate (522mg, 1.602 mmol). The reaction solution was heated to 110 ℃ under nitrogen protection, stirred for 15 hours, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (DCM/MeOH (v/v) ═ 10/1) to give a yellow solid, which was further purified by anion separation column to give the title compound (14.7mg, 3.46%) as a white solid. MS (ESI, pos. ion) M/z 480.4[ M + H ]]⁺。HRMS(ESI⁺) Calculated value C₂₇H₂₆N₇O₂[M+H]⁺480.21, found 480.2155.¹H NMR(400MHz,CDCl₃)(ppm):10.66(s,1H),8.33(s,1H),7.75(d,J＝8.5Hz,2H),7.55(t,J＝7.6Hz,2H),7.45(t,J＝7.5Hz,1H),7.39(dd,J＝14.3,7.9Hz,4H),5.11(s,2H),3.83(s,3H),3.57(t,J＝5.9Hz,2H),3.42(t,J＝6.4Hz,3H),2.13-2.05(m,3H),1.97-1.89(m,3H)。

Example 2N- (5- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) pyridin-2-yl) -2-oxo-1-phenyl-2, 4,6, 7-tetrahydro-1H-pyrazolo [5,1-c ] [1,4] oxazine-3-carboxamide

Step 1) 2-oxo-1-phenyl-2, 4,6, 7-tetrahydro-1H-pyrazolo [5,1-c ] [1,4] oxazine-3-carbonyl chloride

To a solution of 2-oxo-1-phenyl-2, 4,6, 7-tetrahydro-1H-pyrazolo [5,1-c ] [1,4] oxazine-3-carboxylic acid (480mg, 1.845mmol) in tetrahydrofuran (10mL) was added oxalyl chloride (0.6mL, 7 mmol). The reaction mixture was heated to 60 ℃ and stirred for 4h, and concentrated under reduced pressure to give the title compound (515mg, 100.2%) as a yellow solid.

Step 2) N- (5-bromopyridin-2-yl) -2-oxo-1-phenyl-2, 4,6, 7-tetrahydro-1H-pyrazolo [5,1-c ] [1,4] oxazine-3-carboxamide

After stirring a solution of 5-bromopyridin-2-amine (320mg, 1.849mmol) and triethylamine (0.8mL, 6mmol) in methylene chloride (20mL) at room temperature for 10 minutes, 2-oxo-1-phenyl-2, 4,6, 7-tetrahydro-1H-pyrazolo [5,1-c ] was added to the mixture][1,4]Oxazine-3-carbonyl chloride (515mg, 1.848 mmol). The reaction solution was stirred at room temperature for 24 hours, diluted dropwise with water (20mL), and extracted with dichloromethane (50 mL). The organic phase was separated and concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (DCM/MeOH (v/v) ═ 100/1), and the collected yellow solid was recrystallized from a mixed solution of dichloromethane/methanol/petroleum ether (6mL/0.5mL/12mL) to give the title compound (380mg, 49.53%) as a white solid. MS (ESI, pos. ion) M/z 415.0[ M + H ]]⁺。¹H NMR(400MHz,CDCl₃)(ppm):10.58(s,1H),8.13(s,1H),7.99(d,J＝8.8Hz,1H),7.57(dd,J＝8.8,2.0Hz,1H),7.37(t,J＝7.5Hz,2H),7.30(d,J＝6.8Hz,1H),7.21(d,J＝7.6Hz,2H),5.04(s,2H),3.97(t,J＝4.8Hz,2H),3.50(t,J＝4.7Hz,2H)。

Step 3) 2-oxo-1-phenyl-N- (5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2-yl) -2,4,6, 7-tetrahydro-1H-pyrazolo [5,1-c ] [1,4] oxazine-3-carboxamide

To a solution of 1, 4-dioxane (10mL) containing N- (5-bromopyridin-2-yl) -2-oxo-1-phenyl-2, 4,6, 7-tetrahydro-1H-pyrazolo [5,1-c ] [1,4] oxazine-3-carboxamide (380mg, 0.915mmol), bis-pinacolato diboron (260mg, 1.024mmol) and tetrakis (triphenylphosphine) palladium (0) (151mg, 0.130mmol) was added potassium acetate (278mg, 2.833 mmol). The reaction mixture was heated to reflux under nitrogen for 5 hours, and concentrated under reduced pressure to give the title compound (425mg, 100.4%) as a white solid.

Step 4) N- (5- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) pyridin-2-yl) -2-oxo-1-phenyl-2, 4,6, 7-tetrahydro-1H-pyrazolo [5,1-c ] [1,4] oxazine-3-carboxamide

To a compound containing 2-oxo-1-phenyl-N- (5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2-yl) -2,4,6, 7-tetrahydro-1H-pyrazolo [5, 1-c)][1,4]Oxazine-3-carboxamide (425mg, 0.919mmol) and 5-bromo-7-methyl-7H-pyrrolo [2,3-d]To a mixture of pyrimidin-4-amine (235mg, 1.035mmol), 1, 4-dioxane (10mL) and water (5mL) were added tetrakis (triphenylphosphine) palladium (0) (215mg, 0.186mmol) and potassium carbonate (390mg, 2.794 mmol). The reaction solution was heated to 105 ℃ under nitrogen protection, stirred for 20 hours, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (DCM/MeOH (v/v) ═ 50/1) to give a yellow solid, which was further purified by anion separation column to give the title compound (73mg, 16.46%) as a white solid. MS (ESI, pos. ion) M/z 483.1[ M + H ]]⁺。HRMS(ESI⁺) Calculated value C₂₅H₂₂N₈O₃([M+H]⁺) 483.18, found 483.1886.¹H NMR(400MHz,DMSO-d₆)(ppm):10.77(s,1H),8.37(d,J＝1.7Hz,1H),8.27(d,J＝8.5Hz,1H),8.16(s,1H),7.84(dd,J＝8.5,2.1Hz,1H),7.64-7.58(m,2H),7.56-7.50(m,3H),7.37(s,1H),6.18(s,2H),5.13(s,2H),4.12-4.08(m,2H),3.74(s,3H),3.70(t,J＝4.5Hz,2H)。¹³C NMR(151MHz,CDCl₃)(ppm):163.48,161.98,156.86,155.43,152.09,151.22,151.04,147.73,137.79,132.95,129.63,128.80,126.57,125.68,124.08,113.97,112.34,101.16,98.26,47.39,31.24,23.76,22.58,19.69。

Example 3N- (4- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

Step 1) N- (4-bromophenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] is added]A solution of pyrazole-3-carboxylic acid (700mg, 2.87mmol, 100%), 4-bromoaniline (490mg, 2.87mmol) and HATU (1.15g, 2.87mmol) in dichloromethane (20mL) was stirred at room temperature for 4 hours. The reaction was quenched with water (30mL), extracted with dichloromethane (50mL x 3), and combined withThe organic phase was washed with brine (10mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the title compound as a white solid (1020mg, 89.4%). MS (ESI, pos.ion) M/z 398.1[ M + H ]]⁺。

Step 2) 2-oxo-1-phenyl-N- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) -2,4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

To a solution containing N- (4-bromophenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ]]Pyrazole-3-carboxamide (1020mg, 2.56mmol), Bipinolaoupinodiboron (733mg, 2.88mmol) and Pd (dppf) Cl₂(II) (285mg, 0.39mmol) in 1, 4-dioxane (20mL) was added potassium acetate (630mg, 6.42 mmol). The reaction solution was heated to reflux under nitrogen for 7 hours, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (DCM/MeOH (v/v) ═ 20/1) to give the title compound as a white solid (900mg, 78.9%). MS (ESI, pos.ion) M/z 446.3[ M + H ]]⁺。

Step 3) N- (4- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

To a solution containing 2-oxo-1-phenyl-N- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) -2,4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] c]Pyrazole-3-carboxamide (600mg, 1.34mmol) and 5-bromo-7-methyl-7H-pyrrolo [2,3-d]Pd (dppf) Cl was added to a mixture of pyrimidine-4-amine (343mg, 1.51mmol), 1, 4-dioxane (20mL) and water (2mL)₂(II) (200mg, 0.269mmol) and cesium carbonate (876mg, 2.69 mmol). The reaction solution was heated to reflux under nitrogen for 7 hours, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (MeOH/DCM (v/v) ═ 1/20) to give the title compound as a white solid (120mg, 19.13%).

MS(ESI,pos.ion)m/z:466.1[M+H]⁺。HRMS(ESI⁺) Calculated value C₂₆H₂₃N₇O₂[M+H]⁺466.1913, found 466.1983.¹H NMR(400MHz,CDCl₃)(ppm):10.27(s,1H),8.36(s,1H),7.79(d,J＝8.4Hz,2H),7.54(d,J＝7.6Hz,2H),7.45(d,J＝8.4Hz,4H),6.94(s,1H),5.14(s,2H),3.86(s,3H),3.75(s,2H),3.36(s,2H),2.56(s,2H),1.28(s,2H)。¹³C NMR(101MHz,DMSO-d₆)(ppm):165.82,161.80,160.73,157.62,151.98,151.61,150.89,137.81,134.86,129.99,129.79,129.39,127.77,125.57,124.59,123.87,119.91,115.22,100.35,98.84,97.02,49.94,31.18,26.01,22.26。

Example 4N- (4- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -2-oxo-1-phenyl-2, 4,6, 7-tetrahydro-1H-pyrazolo [5,1-c ] [1,4] oxazine-3-carboxamide

Step 1) (tert-butyl 4- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) carbamate

To a mixture of 1, 4-dioxane (200mL) containing tert-butyl (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -phenyl) carbamate (16.88g, 52.88mmol), 5-bromo-7-methyl-pyrrolo [2,3-d ] pyrimidin-4-amine (10g, 44.04mmol) and water (50mL) was added tetrakis (triphenylphosphine) palladium (0) (5.2g, 4.50mmol) and cesium carbonate (36g, 110 mmol). The reaction solution was heated to 105 ℃ under nitrogen and stirred for 24.5 hours, water (200mL) and ethyl acetate (100mL) were added and stirred for 10min, the liquid was separated, the aqueous phase was extracted with ethyl acetate (250mL × 3), the organic phases were combined and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE/EtOAc (v/v) ═ 1/1) to give the title compound (11.63g, 77.8%) as a yellow solid.

Step 2)5- (4-aminophenyl) -7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-4-amine

A solution of tert-butyl (4- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) carbamate (4.5g, 13mmol) and concentrated hydrochloric acid (13mL, 12M) in methanol (75mL) was heated to 60 ℃ and stirred for 5 hours. The reaction mixture was concentrated under reduced pressure, methylene chloride (100mL) was added, and the mixture was basified with a saturated sodium bicarbonate solution (100 mL). The separated aqueous phase was extracted with dichloromethane (200mL), the organic phases were combined and washed with water (50mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the title compound as a yellow solid (2.58g, 81%).

Step 3) N- (4- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -2-oxo-1-phenyl-2, 4,6, 7-tetrahydro-1H-pyrazolo [5,1-c ] [1,4] oxazine-3-carboxamide

To a solution containing 2-oxo-1-phenyl-2, 4,6, 7-tetrahydro-1H-pyrazolo [5,1-c ]][1,4]Oxazine-3-carboxylic acid (100mg, 0.384mmol) and 5- (4-aminophenyl) -7-methyl-7H-pyrrolo [2,3-d]To a solution of pyrimidin-4-amine (90mg, 0.376mmol) in N, N-dimethylformamide (4mL) were added EDCI (101mg, 0.516mmol) and HOAT (112mg, 0.806 mmol). The reaction mixture was heated to 55 ℃ and stirred for 6 hours, and concentrated under reduced pressure. The residue was washed with water (10mL) to give the title compound as a brown solid (65.6mg, 32.0%). MS (ESI, pos.ion) M/z 482.1[ M + H ]]⁺。HRMS(ESI⁺) Calculated value C₂₆H₂₄N₇O₃[M+H]⁺482.19, found 482.1928.¹H NMR(400MHz,DMSO-d₆)(ppm):10.48(s,1H),8.12(s,1H),7.74(d,J＝8.6Hz,2H),7.64-7.57(m,2H),7.56-7.48(m,6H),7.15(s,2H),6.64(s,1H),5.13(s,2H),4.10(t,J＝4.8Hz,2H),3.73-3.68(m,5H)。¹³C NMR(151MHz,DMSO-d₆)(ppm):162.07,161.09,151.56,149.67,138.83,137.15,132.36,129.98,129.74,129.63,129.32,127.63,126.95,121.18,119.54,102.86,98.57,96.04,63.56,63.23,45.70,30.18。

Example 5N- (4- (4-amino-7-cyclopropyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

Step 1) 4-chloro-7-cyclopropyl-5-iodo-7H-pyrrolo [2,3-d ] pyrimidine

To a solution containing 4-chloro-5-iodo-7H-pyrrolo [2,3-d]To a solution of pyrimidine (5.00g, 17.89mmol) in DCE (300ml) were added cyclopropylboronic acid (3.07g, 35.78mmol), copper acetate (3.57g, 17.89mmol), sodium carbonate (3.79g, 35.78mmol) and 2, 2' -bipyridine (2.79g, 17.89 mmol). The reaction was heated to reflux and stirred for 1 hour, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (EtOAc/PE (v/v) ═ 1/10) to give a white solidTitle compound as a solid (2.30g, 40.2%). MS (ESI, pos. ion) M/z 320.0[ M + H ]]⁺。¹H NMR(400MHz,DMSO-d₆)(ppm):8.64(s,1H),7.93(s,1H),3.65-3.55(m,1H),1.07(dd,J＝10.2,3.2Hz,4H)。

Step 2) (tert-butyl 4- (4-chloro-7-cyclopropyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) carbamate

To a solution containing 4-chloro-7-cyclopropyl-5-iodo-7H-pyrrolo [2,3-d]To a mixture of pyrimidine (2.30g, 7.20mmol) in 1, 4-dioxane (75mL) and water (15mL) was added tert-butyl (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) carbamate (2.30g, 7.20mmol), 1' -bis (diphenylphosphino) ferrocene dichloropalladium (II) (0.53g, 0.72mmol) and sodium carbonate (2.29g, 21.60 mmol). The reaction was heated to reflux and stirred for 8 hours, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (EtOAc/PE (v/v) ═ 1/3) to give the title compound (2.40g, 86.6%) as a white solid. MS (ESI, pos.ion) M/z 385.2[ M + H ]]⁺。¹H NMR(400MHz,DMSO-d₆)(ppm):9.42(s,1H),8.66(s,1H),7.70(s,1H),7.51(d,J＝8.4Hz,2H),7.39(d,J＝8.5Hz,2H),3.70-3.60(m,1H),1.14(s,2H),1.10(d,J＝2.4Hz,2H),1.07(s,9H)。

Step 3)5- (4-aminophenyl) -7-cyclopropyl-7H-pyrrolo [2,3-d ] pyrimidin-4-amine

In an autoclave, the mixture of (4- (4-chloro-7-cyclopropyl-7H-pyrrolo [2, 3-d)]Pyrimidin-5-yl) phenyl) carbamic acid tert-butyl ester (1.50g, 3.90mmol) in 1, 4-dioxane (10ml) was added aqueous ammonia (50ml, 25% aqueous solution). The reaction was heated to 130 ℃ and stirred for 24 hours, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (MeOH/DCM (v/v) ═ 1/30) to give the title compound (0.70g, 68.0%) as a yellow solid. MS (ESI, pos. ion) M/z 266.2[ M + H ]]⁺。¹H NMR(400MHz,DMSO-d₆)(ppm):8.12(s,1H),7.10(d,J＝8.2Hz,2H),7.03(s,1H),6.65(d,J＝8.2Hz,2H),5.98(s,2H),5.19(s,2H),3.58-3.49(m,1H),1.04-0.97(m,4H)。

Step 4) N- (4- (4-amino-7-cyclopropyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

To a solution containing 5- (4-aminophenyl) -7-cyclopropyl-7H-pyrrolo [2,3-d]To a solution of pyrimidin-4-amine (0.25g, 0.94mmol) in dichloromethane (30ml) was added 2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ]]Pyrazole-3-carboxylic acid (0.23g, 0.94mmol), EDCI (0.36g, 1.89mmol) and HOAT (0.26g, 1.89 mmol). The reaction solution was heated to reflux and stirred overnight, quenched with water (50ml), and extracted with dichloromethane (200ml x 3). The organic phases were combined, washed with saturated sodium bicarbonate solution (50mL) and brine (100mL x 2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (MeOH/DCM (v/v) ═ 1/30) to give the title compound (0.30g, 65.0%) as a yellow solid. MS (ESI, pos. ion) M/z 492.2[ M + H ]]⁺。HRMS(ESI⁺) Calculated value C₂₈H₂₆N₇O₂[M+H]⁺492.2148, found 492.2140.¹H NMR(400MHz,DMSO-d₆)(ppm):10.26(s,1H),8.18(s,1H),7.72(d,J＝8.4Hz,2H),7.54(dd,J＝14.5,7.2Hz,4H),7.42(d,J＝8.3Hz,3H),7.25(s,1H),6.17(s,2H),3.81(t,J＝6.9Hz,2H),3.59-3.48(m,1H),3.18(t,J＝7.3Hz,2H),2.47-2.38(m,2H),1.04(dd,J＝7.9,6.1Hz,4H)。¹³C NMR(151MHz,DMSO-d₆)(ppm):165.79,161.79,160.73,157.30,151.85,151.60,151.57,151.55,137.87,134.83,129.81,129.74,129.47,129.34,127.78,123.84,123.02,121.21,119.81,115.27,100.76,96.98,60.23,49.95,27.12,26.02,22.26,6.50。

Example 6N- (5- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) pyridin-2-yl) -2-oxo-1-phenyl-1, 2,4,5,6, 7-hexahydropyrazolo [1,5-a ] pyridine-3-carboxamide

Step 1) N- (5-bromopyridin-2-yl) -2-oxo-1-phenyl-1, 2,4,5,6, 7-hexahydropyrazolo [1,5-a ] pyridine-3-carboxamide

To 2-oxo-1-phenyl-4, 5,6, 7-tetrahydropyrazolo [1,5-a ]]To a mixture of pyridine-3-carboxylic acid (1.6g, 6.2mmol) in DCM (50mL) was added 1-hydroxy 7-azabenzotriazole (170mg, 1.22mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimideImine hydrobromide (1.6g, 8.2mmol) and 5-bromopyridin-2-amine (1.2g, 6.7 mmol). The mixture was heated to 45 ℃ and stirred for 21 h. TLC showed no starting material present, then water (20mL) was added dropwise to the mixture and the resulting mixture was extracted twice with DCM (50mL) and concentrated in vacuo to give a yellow solid. The residue was purified by flash column chromatography (PE: EtOAc ═ 1:1-0:1) to give N- (5-bromo-2-pyridinyl) -2-oxo-1-phenyl-4, 5,6, 7-tetrahydropyrazolo [1,5-a ] N- (5-bromo-2-pyridinyl)]Pyridine-3-carboxamide is a yellow solid (1.35g, 3.27mmol, yield 53%). MS (ESI, pos. ion) M/z 412.9[ M + H ]]⁺。

Step 2) 2-oxo-1-phenyl-N- (5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2-yl) -1,2,4,5,6, 7-hexahydropyrazolo [1,5-a ] pyridine-3-carboxamide

To N- (5-bromo-2-pyridyl) -2-oxo-1-phenyl-4, 5,6, 7-tetrahydropyrazolo [1,5-a ]]Pyridine-3-carboxamide (1.35g, 3.27mmol), 4,4,5, 5-tetramethyl-2- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1,3, 2-dioxaborolan (0.95g, 3.7mmol) and Pd (dppf)₂Cl₂To the suspension (0.4g, 0.5mmol) was added a solution of KOAc (0.8g, 8mmol) in 1, 4-dioxane (30 mL). The reaction mixture is stirred under N₂Stirring under reflux for 5h under atmosphere. TLC showed no starting material present and concentrated in vacuo to give the title compound (1.5g, 3.27mmol, 100.0% yield).

Step 3) N- (5- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) pyridin-2-yl) -2-oxo-1-phenyl-1, 2,4,5,6, 7-hexahydropyrrolo [1,5-a ] pyridine-3-carboxamide

To 2-oxo-1-phenyl-N- [5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2-pyridine]-4,5,6, 7-tetrahydropyrazolo [1,5-a]Pyridine-3-carboxamide (1.5g, 3.3mmol) and 5-bromo-7-methyl-pyrrolo [2,3-d]Pyrimidin-4-amine (1.0g, 4.4mmol), H₂O (5mL) in 1, 4-dioxane (10mL) was added 1,1' -bis (diphenylphosphino) ferrocene-palladium (II) dichloride dichloromethane complex (0.66g, 0.79mmol) and Cs₂CO₃(2.6g, 8.0 mmol). Mixing the mixture in N₂Stirred overnight at 105 ℃ under atmosphere and concentrated in vacuo. The residue was purified by flash column chromatography (DCM: MeOH ═ 50:1-10:1),a yellow solid (240mg) was obtained. Purifying the solid with anion separation column to obtain N- (5- (4-amino-7-methyl-7H-pyrrolo [2, 3-d)]Pyrimidin-5-yl) pyridin-2-yl) -2-oxo-1-phenyl-1, 2,4,5,6, 7-hexahydropyrazolo [1,5-a]Pyridine-3-carboxamide was a white solid (6.9mg, 0.014mmol, yield 0.44%). MS (ESI, pos. ion) M/z 481.4[ M + H ]]+。¹H NMR(600MHz,DMSO)11.84(s,1H),8.62(d,J＝8.3Hz,1H),8.48(s,1H),8.15(dd,J＝7.9,1.4Hz,1H),7.51–7.48(m,1H),7.26(d,J＝8.7Hz,2H),7.25–7.21(m,1H),6.92(d,J＝8.7Hz,2H),5.41(s,2H),3.72(s,4H),2.55(s,3H)。¹³C NMR(151MHz,CDCl₃)163.48,161.98,156.86,155.43,152.09,151.22,151.04,147.73,137.79,132.95,129.63,128.80,126.57,125.68,124.08,113.97,112.34,101.16,98.26,47.39,31.24,23.76,22.58,19.69。

Example 7N- (4- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) -3-fluorophenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

Step 1) N- (4-bromo-3-fluorophenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

To 4-bromo-3-fluoroaniline (200mg, 1.0mmol) in CH₂Cl₂(6mL) to the solution was added 2-oxo-1-phenyl-5, 6-dihydro-4H-pyrrolo [1,2-b ]]Pyrazole-3-carboxylic acid (274mg, 1.1mmol), EDCI (240mg, 12mmol) and HOAT (28mg, 0.2 mmol). After stirring at reflux overnight, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate 1/1) to give the title compound as a yellow solid (260mg, 61%). MS (ESI, pos. ion) M/z 416.0(M + 1).¹H NMR(400MHz,CDCl₃)10.31(s,1H),7.84-7.81(dd,J＝10.9,2.3Hz,1H),7.56-7.52(t,J＝7.8Hz,2H),7.47-7.39(m,4H),7.19-7.17(dd,J＝8.6,1.9Hz,1H),3.76-3.73(t,J＝6.9Hz,2H),3.34-3.31(t,J＝7.4Hz,2H),2.59-2.52(m,2H)。

Step 2) N- (3-fluoro-4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

To N- (4-bromo-3-fluorophenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ]]To a solution of pyrazole-3-carboxamide (260mg, 0.62mmol) in dioxane (6mL) was added 4,4,5, 5-tetramethyl-2- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1,3, 2-dioxaborolan (178mg, 0.69mmol), Pd (dppf) Cl₂.CH₂Cl₂(52mg, 0.063mmol) and potassium acetate (161mg, 1.6 mmol). The reaction was degassed with nitrogen and after stirring the mixture at reflux overnight, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate 2/1) to give the title compound as a yellow solid (210mg, 73%). MS (ESI, pos.ion) M/z 464.2(M + 1).

Step 3) N- (4- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) -3-fluorophenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

To N- (3-fluoro-4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] c]Dioxane (6mL)/H of pyrazole-3-carboxamide (210mg, 0.45mmol)₂O (1mL) solution was added with 5-bromo-7-methyl-pyrrolo [2,3-d]Pyrimidin-4-amine (108mg, 0.45mmol), Pd (dppf) Cl₂.CH₂Cl₂(37mg, 0.045mmol) and cesium carbonate (301mg, 0.91 mmol). The reaction was degassed with nitrogen and after stirring the mixture at reflux overnight, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (dichloromethane/methanol-500/1) to give the title compound as a yellow solid (46mg, 21%). MS (ESI, pos. ion) M/z 484.2(M + 1).¹HNMR(400MHz,DMSO)10.38(s,1H),8.15(s,1H),7.88-7.85(d,J＝12.8Hz,1H),7.60-7.52(m,4H),7.45-7.41(t,J＝7.3Hz,1H),7.36-7.35(m,2H),7.30(s,1H),6.03(s,2H),3.85-2.81(t,J＝6.9Hz,2H),3.75(s,3H),3.20-3.17(t,J＝6.8Hz,2H),2.46-2.43(m,2H)。

Example 8N- (4- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) -2-fluorophenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

Step 1) 2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carbonyl chloride

To 2-oxo-1-phenyl-5, 6-dihydro-4H-pyrrolo [1,2-b ]]Pyrazole-3-carboxylic acid (200mg, 0.82mmol) in CH₂Cl₂To the solution (5mL) were added DMF (1mg, 0.014mmol) and oxalyl chloride (0.1mL, 1 mmol). After stirring at room temperature for 4 hours, the reaction mixture was concentrated in vacuo and used in the next step without further purification.

Step 2) N- (4-bromo-2-fluorophenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

To 4-bromo-2-fluoroaniline (175mg, 0.9mmol) in CH₂Cl₂To the solution (3mL) was added triethylamine (0.25mL, 1.8mmol) and the reaction mixture was stirred at 0 ℃. Adding 2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] to the reaction mixture]Pyrazole-3-carbonyl chloride (215mg, 0.82mmol) in CH₂Cl₂(2mL) of the solution. After stirring at room temperature overnight, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate-1/1) to give the title compound as a yellow solid (0.15g, 44%, two steps). MS (ESI, pos. ion) M/z 416.0(M + 1).¹H NMR(400MHz,CDCl₃)10.47(s,1H),8.46-8.42(t,J＝8.6Hz,1H),7.55-7.51(t,J＝7.8Hz,2H),7.45-7.43(d,J＝7.6Hz,2H),7.41-7.37(t,J＝7.4Hz,1H),7.30-7.25(m,2H),3.76-3.73(t,J＝6.9Hz,2H),3.35-3.31(t,J＝7.4Hz,2H),2.59-2.51(m,2H)。

Step 3) N- (2-fluoro-4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

To N- (4-bromo-2-fluorophenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ]]To a solution of pyrazole-3-carboxamide (150mg, 0.36mmol) in dioxane (6mL) was added 4,4,5, 5-tetramethyl-2- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1,3, 2-dioxaborolan (187mg, 0.72mmol), Pd (dppf) Cl₂.CH₂Cl₂(30mg, 0.036mmol) and potassium acetate (93 m)g, 0.9 mmol). The reaction was degassed with nitrogen. After the mixture was stirred at reflux overnight, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate 2/1) to give the title compound as a yellow solid (135mg, 81%). MS (ESI, pos. ion) M/z 464.3(M + 1).¹HNMR(400MHz,CDCl₃)10.59-10.58(d,J＝2.3Hz,1H),8.57-8.53(t,J＝7.8Hz,1H),7.59-7.54(dd,J＝11.6,6.5Hz,2H),7.52-7.50(d,J＝8.0Hz,2H),7.45-7.44(d,J＝7.6Hz,2H),7.40-7.36(t,J＝7.3Hz,1H),3.76-3.72(t,J＝6.9Hz,2H),3.36-3.32(t,J＝7.4Hz,2H),2.58-2.51(m,2H),1.35(s,12H)。

Step 4) N- (4- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) -2-fluorophenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

To N- (2-fluoro-4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] c]Dioxane (6mL)/H of pyrazole-3-carboxamide (135mg, 0.29mmol)₂Adding 5-bromo-7-methyl-pyrrolo [2,3-d ] into O (1mL) solution]Pyrimidin-4-amine (70mg, 0.29mmol), Pd (dppf) Cl₂.CH₂Cl₂(24mg, 0.029mmol) and cesium carbonate (194mg, 0.58 mmol). The reaction was degassed with nitrogen. After the mixture was stirred at reflux overnight, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (dichloromethane/methanol-500/1) to give the title compound as a yellow solid (46mg, 33%). MS (ESI, pos. ion) M/z 484.2(M + 1).¹HNMR(400MHz,DMSO)10.51-10.50(d,J＝1.8Hz,1H),8.53-8.49(t,J＝8.4Hz,1H),8.16(s,1H),7.59-7.52(m,4H),7.45-7.41(t,J＝7.0Hz,1H),7.35-7.32(m,2H),7.27-7.25(d,J＝8.5Hz,1H),6.18(s,2H),3.84-3.81(t,J＝6.8Hz,2H),3.74(s,3H),3.20-3.17(t,J＝7.2Hz,2H),2.48-2.41(m,2H)。

Example 9N- (4- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) -2-fluorophenyl) -1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazole-4-carboxamide

Step 1)1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazole-4-carbonyl chloride

To 1, 5-dimethyl-3-oxo-2-phenyl-pyrazole-4-carboxylic acid (300mg, 1.3mmol) in CH₂Cl₂To the solution (6mL) were added DMF (1mg, 0.014mmol) and oxalyl chloride (0.2mL,2 mmol). After stirring at room temperature for 4 hours, the reaction mixture was concentrated in vacuo and used in the next step without further purification.

Step 2) N- (4-bromo-2-fluorophenyl) -1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazole-4-carboxamide

To 4-bromo-2-fluoroaniline (274mg, 1.4mmol) in CH₂Cl₂Triethylamine (0.36mL, 2.6mmol) was added to the solution (4mL) and the reaction mixture was stirred at 0 ℃. To the reaction mixture was added 1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazole-4-carbonyl chloride (322mg, 1.3mmol) in CH₂Cl₂(2mL) of the solution. After stirring at room temperature overnight, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate-1/1) to give the title compound as a yellow solid (0.44g, 85%, two steps). MS (ESI, pos. ion) M/z:404.0(M + 1).¹H NMR(400MHz,CDCl3)10.98(s,1H),8.46-8.42(t,J＝8.7Hz,1H),7.58-7.54(t,J＝7.6Hz,2H),7.49-7.46(t,J＝7.4Hz,1H),7.39-3.37(d,J＝7.4Hz,2H),7.28-7.25(m,2H),3.37(s,3H),2.80(s,3H)。

Step 3) N- (2-fluoro-4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) -1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazole-4-carboxamide

To a solution of N- (4-bromo-2-fluorophenyl) -1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazole-4-carboxamide (440mg, 1.1mmol) in dioxane (6mL) was added 4,4,5, 5-tetramethyl-2- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1,3, 2-dioxaborolan (564mg,2.2mmol), Pd (dppf) Cl₂.CH₂Cl₂(90mg,0.11mmol) and potassium acetate (281mg, 2.7 mmol). The reaction was degassed with nitrogen. After the mixture was stirred at reflux overnight, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate 2/1) to give the title compound as a yellow solid (410mg, 83%)。MS(ESI,pos.ion)m/z:452.3(M+1)。¹H NMR(600MHz,CDCl₃)11.10(s,1H),8.57-8.54(t,J＝7.8Hz,1H),7.58-7.53(m,4H),7.48-7.45(t,J＝7.5Hz,1H),7.39-7.37(d,J＝7.4Hz,2H),3.37(s,3H),2.81(s,3H),1.35(s,12H)。

Step 4) N- (4- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) -2-fluorophenyl) -1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazole-4-carboxamide

To a dioxane (8mL)/H of N- (2-fluoro-4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) -1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazole-4-carboxamide (410mg, 0.91mmol)₂O (1.5mL) solution was added with 5-bromo-7-methyl-pyrrolo [2,3-d]Pyrimidin-4-amine (217mg, 0.91mmol), Pd (dppf) Cl₂.CH₂Cl₂(75mg, 0.091mmol) and cesium carbonate (604mg, 1.8 mmol). The reaction was degassed with nitrogen. After the mixture was stirred at reflux overnight, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (dichloromethane/methanol-500/1) to give the title compound as a yellow solid (78mg, 18%). MS (ESI, pos. ion) M/z 472.1(M + 1).¹H NMR(400MHz,DMSO)11.05(s,1H),8.54-8.50(t,J＝8.4Hz,1H),8.16(s,1H),7.62-7.59(t,J＝7.5Hz,2H),7.55-7.51(m,1H),7.46-7.44(d,J＝7.6Hz,2H),7.35-7.30(m,2H),7.26-7.24(d,J＝8.3Hz,1H),6.17(s,2H),3.74(s,3H),3.38(s,3H),2.73(s,3H)。

Example 10N- (4- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazole-4-carboxamide

Step 1) N- (4-bromophenyl) -1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazole-4-carboxamide

To 4-bromoaniline (500mg, 2.9mmol) in CH₂Cl₂To the solution (15mL) was added 1, 5-dimethyl-3-oxo-2-phenyl-pyrazole-4-carboxylic acid (739mg, 3.2mmol), EDCI (675mg, 3.5mmol) and HOAT (80mg, 0.58 mmol). After stirring at reflux overnight, the reaction mixture was concentrated in vacuo. Residue ofPurification by silica gel column chromatography (petroleum ether/ethyl acetate 1/1) gave the title compound as a yellow solid (340mg, 31%). MS (ESI, pos.ion) M/z:386.0(M + 1).¹H NMR(400MHz,CDCl₃)10.74(s,1H),7.59-7.55(m,4H),7.50-7.47(t,J＝7.4Hz,1H),7.43-7.41(d,J＝8.8Hz,2H),7.38-7.36(d,J＝7.7Hz,2H),3.37(s,3H),2.80(s,3H)。

Step 2)1, 5-dimethyl-3-oxo-2-phenyl-N- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) -2, 3-dihydro-1H-pyrazole-4-carboxamide

To a solution of N- (4-bromophenyl) -1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazole-4-carboxamide (340mg, 0.88mmol) in dioxane (6mL) was added 4,4,5, 5-tetramethyl-2- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1,3, 2-dioxaborolan (250mg, 0.97mmol), Pd (dppf) Cl₂.CH₂Cl₂(73mg, 0.088mmol) and potassium acetate (227mg, 2.2 mmol). The reaction was degassed with nitrogen. After the mixture was stirred at reflux overnight, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate 1/1) to give the title compound as a yellow solid (280mg, 73%). MS (ESI, pos. ion) M/z:434.3(M + 1).¹H NMR(400MHz,CDCl3)10.81(s,1H),7.79-7.77(d,J＝8.3Hz,2H),7.71-7.69(d,J＝8.3Hz,2H),7.58-7.54(t,J＝7.7Hz,2H),7.49-7.46(t,J＝7.4Hz,1H),7.38-7.37(d,J＝7.5Hz,2H),3.36(s,3H),2.81(s,3H),1.35(s,12H)。

Step 3) N- (4- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazole-4-carboxamide

To a solution of 1, 5-dimethyl-3-oxo-2-phenyl-N- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) -2, 3-dihydro-1H-pyrazole-4-carboxamide (280mg, 0.65mmol) in dioxane (6mL)/H₂Adding 5-bromo-7-methyl-pyrrolo [2,3-d ] into O (1mL) solution]Pyrimidin-4-amine (154mg, 0.64mmol), Pd (dppf) Cl₂.CH₂Cl₂(53mg, 0.064mmol) and cesium carbonate (430mg, 1.3 mmol). The reaction was degassed with nitrogen. After the mixture was stirred at reflux overnight, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (II)Methyl chloride/methanol-500/1) to give the title compound as a yellow solid (68mg, 23%). MS (ESI, pos. ion) M/z 454.3(M + 1).¹H NMR(400MHz,DMSO)10.82(s,1H),8.15(s,1H),7.71-7.69(d,J＝8.5Hz,2H),7.62-7.58(t,J＝7.6Hz,2H),7.54-7.50(t,J＝7.4Hz,1H),7.46-7.44(d,J＝7.4Hz,2H),7.41-7.39(d,J＝8.4Hz,2H),7.28(s,1H),6.06(s,2H),3.74(s,3H),3.37(s,3H),2.72(s,3H)。

Example 11N- (4- (4-amino-7-isopropyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -2-oxo-1-phenyl-1, 2,4,5,6, 7-hexahydropyrazolo [1,5-a ] pyridine-3-carboxamide

To 5- (4-aminophenyl) -7-isopropyl-7H-pyrrolo [2,3-d]Pyrimidine-4-amine (100mg, 0.37mmol) in CH₂Cl₂(2mL) to the solution was added 2-oxo-1-phenyl-4, 5,6, 7-tetrahydropyrazolo [1,5-a ]]Pyridine-3-carboxylic acid (116mg, 0.45mmol), EDCI (110mg, 0.56mmol) and HOAT (10mg,0.072 mmol). After stirring at reflux for 8h, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (dichloromethane/methanol ═ 100/0) to give the title compound as a yellow solid (97mg, 51%). MS (ESI, pos.ion) M/z:508.4(M + 1).¹H NMR(400MHz,DMSO)10.68(s,1H),8.13(s,1H),7.71-7.69(d,J＝8.4Hz,2H),7.61-7.57(t,J＝7.5Hz,2H),7.53-7.51(d,J＝7.2Hz,1H),7.48-7.46(d,J＝7.4Hz,2H),7.43-7.41(m,3H),6.05(s,2H),5.02-4.92(m,1H),3.59-3.56(t,J＝5.7Hz,2H),3.24-3.21(t,J＝6.2Hz,2H),1.99-1.98(m,2H),1.84-1.81(m,2H),1.47-1.45(d,J＝6.7Hz,6H)。

Example 12N- (4- (4-amino-7-isopropyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

Step 1) 4-chloro-5-iodo-7-isopropyl-7H-pyrrolo [2,3-d ] pyrimidine

To 4-chloro-5-iodo-7H-pyrrolo [2,3-d]Pyrimidines (2.0g, 7)0mmol) in THF (35mL) was added potassium tert-butoxide (1.23g, 10.7mmol) and the reaction mixture was stirred for 0.5 h. To the reaction mixture was added 2-iodopropane (0.9mL, 9.0 mmol). After stirring overnight at 70 ℃, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate 3/1) to give the title compound as a yellow solid (1.42g, 63%). MS (ESI, pos.ion) M/z:322.1(M + 1).¹H NMR(400MHz,CDCl3)8.61(s,1H),7.47(s,1H),5.18-5.08(m,1H),1.54-1.52(d,J＝6.8Hz,6H)。

Step 2)4- (4-chloro-7-isopropyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) aniline

To 4-chloro-5-iodo-7-isopropyl-7H-pyrrolo [2,3-d]Dioxane (20mL)/H of pyrimidine (1.31g, 4.07mmol)₂To a solution of O (5mL) was added 4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) aniline (0.91g, 4.1mmol), Pd (dppf) Cl₂.CH₂Cl₂(0.34g, 0.41mmol) and sodium carbonate (0.87g,8.2 mmol). The reaction was degassed with nitrogen. After the mixture was stirred at 90 ℃ overnight, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate 5/1) to give the title compound as a yellow solid (0.68g, 58%). MS (ESI, pos. ion) M/z 287.2(M + 1).¹H NMR(400MHz,DMSO)8.60(s,1H),7.80(s,1H),7.19-7.17(d,J＝8.3Hz,2H),6.63-6.61(d,J＝8.3Hz,2H),5.14(s,2H),5.11-5.06(m,1H),1.52-1.50(d,J＝6.7Hz,6H)。

Step 3)5- (4-aminophenyl) -7-isopropyl-7H-pyrrolo [2,3-d ] pyrimidin-4-amine

To 4- (4-chloro-7-isopropyl-7H-pyrrolo [2, 3-d)]To a solution of pyrimidin-5-yl) aniline (0.68g, 2.4mmol) in dioxane (20mL) was added ammonium hydroxide (20 mL). After the mixture was stirred in a closed tube at 130 ℃ for 24 hours, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (dichloromethane/methanol-100/1) to give the title compound as a yellow solid (0.30g, 47%). MS (ESI, pos. ion) M/z 268.2(M + 1).¹H NMR(400MHz,DMSO)8.09(s,1H),7.23(s,1H),7.13-7.11(d,J＝8.2Hz,2H),6.67-6.65(d,J＝8.2Hz,2H),5.97(s,2H),5.16(s,2H),4.99-4.89(m,1H),1.45-1.43(d,J＝6.7Hz,6H)。

Step 4) N- (4- (4-amino-7-isopropyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

To 5- (4-aminophenyl) -7-isopropyl-7H-pyrrolo [2,3-d]Pyrimidine-4-amine (100mg, 0.37mmol) in CH₂Cl₂(2mL) to the solution was added 2-oxo-1-phenyl-5, 6-dihydro-4H-pyrrolo [1,2-b ]]Pyrazole-3-carboxylic acid (110mg, 0.45mmol), EDCI (110mg, 0.56mmol) and HOAT (10mg,0.072 mmol). After the mixture was stirred at reflux for 9h, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (dichloromethane/methanol ═ 100/0) to give the title compound as a yellow solid (94mg, 51%). MS (ESI, pos.ion) M/z 494.2(M + 1).¹H NMR(400MHz,DMSO)10.26(s,1H),8.13(s,1H),7.74-7.72(d,J＝8.3Hz,2H),7.59-7.51(m,4H),7.44-7.41(m,4H),6.06(s,2H),5.00-4.92(td,J＝13.3,6.6Hz,1H),3.82-3.79(t,J＝6.8Hz,2H),3.20-3.17(t,J＝5.8Hz,3H),2.46-2.40(m,2H),1.47-1.46(d,J＝6.7Hz,6H)。

Example 13N- (4- (4-amino-7- (cyclopropylmethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

Step 1) 4-chloro-7- (cyclopropylmethyl) -5-iodo-7H-pyrrolo [2,3-d ] pyrimidine

4-chloro-5-iodo-7H-pyrrolo [2,3-d]A mixture of pyrimidine (12.00g, 42.94mmol), KOH (4.82g, 85.9mmol) in DMSO (60mL) was stirred at 53 ℃. 2-bromoethanol (12mL) was then added dropwise to the mixture and the reaction mixture was held for 24 hours. The mixture was poured into water (150mL) and filtered under vacuum to give the title compound as a yellow oil (11.53g, 83.00%). MS (ESI, pos. ion) M/z 323.90[ M + H ]]⁺。¹H NMR(400MHz,DMSO)8.63(s,1H),7.98(s,1H),4.93(t,J＝5.4Hz,1H),4.31(t,J＝5.4Hz,2H),3.75(q,J＝5.4Hz,2H)。

Step 2) (4- (4-chloro-7- (cyclopropylmethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) carbamic acid tert-butyl ester

Reacting (4- (4-chloro-7- (cyclopropylmethyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-5-yl) phenyl) carbamic acid tert-butyl ester (2.66g, 7.97mmol), N- [4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl]Tert-butyl carbamate (3.03g,9.49mmol), H₂O(7.5mL)，Pd(dppf)Cl₂(0.85g, 0.79mmol) and Na₂CO₃(2.12g, 20.0mmol) of a mixture of 1, 4-dioxane (35mL) was stirred at 93 ℃ for 12h and then concentrated in vacuo. The residue was purified by flash column chromatography (PE: EA ═ 5:1) to give the title compound as a yellow solid (1.37mg, 43.1%). MS (ESI, pos. ion) M/z 399.10[ M + H ]]⁺。

Step 3) (4- (4-amino-7- (cyclopropylmethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) carbamic acid tert-butyl ester

Reacting (4- (4-amino-7- (cyclopropylmethyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-5-yl) phenyl) carbamic acid tert-butyl ester (1.37g, 3.43mmol) and NH₄A mixture of OH (28%, 40mL) in 1, 4-dioxane (10mL) was stirred at 120 ℃ for 37h, then concentrated in vacuo. The residue was purified by flash column chromatography (DCM: MeOH ═ 30:1) to give the title compound as a yellow solid (675.6mg, 51.8%). MS (ESI, pos. ion) M/z 280.20[ M + H ]]⁺。

Step 4)5- (4-aminophenyl) -7- (cyclopropylmethyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-amine

Reacting 5- (4-aminophenyl) -7- (cyclopropylmethyl) -7H-pyrrolo [2,3-d]A mixture of pyrimidin-4-amine (675.6mg, 1.78mmol) and HCl (37%, 5mL) in methanol (10mL) was stirred at 60 ℃ for 5h, then concentrated in vacuo. Vacuum filtration gave the title compound as a yellow solid (312.5mg, 62.85%). MS (ESI, pos. ion) M/z 280.20[ M + H ]]⁺。

Step 5) N- (4- (4-amino-7- (cyclopropylmethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -2-oxo-1-phenyl-2, 4,5, 6-tetrahydro-1H-pyrrolo [1,2-b ] pyrazole-3-carboxamide

2-oxo-1-phenyl-5, 6-dihydro-4H-pyrrolo [1, 2-b)]Pyrazole-3-carboxylic acid (139.6mg, 0.57mmol), 5- (4-aminophenyl) -7- (cyclopropylmethyl) pyrrolo [2,3-d]Pyrimidin-4-amine (159.9mg, 0.5725mmol), HOAT (24.3mg, 0.18mmol) and EDCI (198.5mg, 1.03 mmol)l) mixture of DCM (10mL) was stirred at 45 ℃ for 12 h. The mixture was concentrated in vacuo to give the title compound as a yellow solid (103.5g, 35.76%).¹H NMR(400MHz,DMSO)10.26(s,1H),8.14(s,1H),7.73(d,J＝8.4Hz,2H),7.63–7.49(m,4H),7.46–7.33(m,4H),6.09(s,2H),4.02(d,J＝7.1Hz,2H),3.80(t,J＝6.8Hz,2H),3.18(dd,J＝9.5,4.9Hz,2H),2.47–2.38(m,2H),0.92–0.77(m,1H),0.57–0.46(m,2H),0.43(d,J＝4.0Hz,2H)。¹³C NMR(151MHz,DMSO)165.80,161.80,160.73,157.70,151.98,150.57,137.78,134.84,130.06,129.80,129.41,127.77,123.84,123.57,119.87,115.24,100.33,97.01,49.95,48.51,26.02,22.26,12.08,4.13。MS(ESI,pos.ion)m/z:503.20[M+H]⁺。

Examples 14 to 17 and example 20 were prepared by a method similar to example 2 and examples 18 to 19 were prepared by a method similar to example 10, see in particular table E:

TABLE E

Biological assay

The LC/MS/MS system for analysis included an Agilent 1200 series vacuum degassing furnace, a binary injection pump, an orifice plate autosampler, a column oven, an Agilent G6430 three-stage quadrupole mass spectrometer with an electrospray ionization (ESI) source. The quantitative analysis was performed in MRM mode, with the parameters of the MRM transition as shown in table a:

TABLE A

Multiple reaction detection scan	490.2→383.1
		Fragmentation voltage	230V
Capillary voltage	55V
		Temperature of drying gas	350℃
Atomizer	0.276MPa
		Dry air flow rate	10L/min

Analysis 5. mu.L of sample was injected using an Agilent XDB-C18, 2.1X 30mm, 3.5. mu.M column. Analysis conditions were as follows: the mobile phase was 0.1% aqueous formic acid (A) and 0.1% methanolic formic acid (B). The flow rate was 0.4 mL/min. Mobile phase gradients are shown in table B:

TABLE B

Time of day	Gradient of mobile phase B
		0.5min	5％
1.0min	95％
		2.2min	95％
2.3min	5％
		5.0min	Terminate

Also used for the analysis was an Agilent 6330 series LC/MS spectrometer equipped with a G1312A binary syringe pump, a G1367A auto sampler and a G1314C UV detector; the LC/MS/MS spectrometer uses an ESI radiation source. The appropriate cation model treatment and MRM conversion for each analyte was performed using standard solutions for optimal analysis. During the analysis a Capcell MP-C18 column was used, with the specifications: 100X 4.6mm I.D., 5. mu.M (Phenomenex, Torrance, California, USA). The mobile phase was 5mM ammonium acetate, 0.1% aqueous methanol (a): 5mM ammonium acetate, 0.1% methanolic acetonitrile solution (B) (70:30, v/v); the flow rate is 0.6 mL/min; the column temperature was kept at room temperature; 20 μ L of sample was injected.

Example A stability in human and rat liver microsomes

Human or rat liver microsomes were incubated in duplicate wells in polypropylene tubes. A typical incubation mixture comprises human or rat liver microsomes (0.5mg protein/mL), the compound of interest (5. mu.M) and a total volume of 200. mu.L of NADPH (1.0mM) potassium phosphate buffer (PBS, 100mM, pH 7.4), and the test compound is dissolved in DMSO and diluted with PBS to give a final DMSO solution concentration of 0.05%. And incubated in a water bath at 37 ℃ in air communication, and after 3 minutes of pre-incubation, protein was added to the mixture and the reaction was started. At various time points (0, 5, 10, 15, 30 and 60 minutes), the reaction was stopped by the addition of the same volume of ice-cold acetonitrile. The samples were stored at-80 ℃ until LC/MS/MS analysis.

The concentration of compound in the human or rat liver microsome incubation mixture was determined by the method of LC/MS. The linear range of concentration ranges is determined for each test compound.

Parallel incubation experiments were incubated at 37 ℃ using denatured microsomes as negative controls, and reactions were terminated at different time points (0, 15 and 60 min).

Verapamil (1 μm) was used as a positive control, incubated at 37 ℃ and the reaction was terminated at different time points (0, 5, 10, 15, 30 and 60 min). Positive and negative control samples were included in each assay method to ensure integrity of the microsomal incubation system.

In addition, stability data for the compounds of the invention in human or rat liver microsomes can also be obtained from the following assays. Human or rat liver microsomes were incubated in duplicate wells in polypropylene tubes. A typical incubation mixture comprises human or rat liver microsomes (final concentration: 0.5mg protein/mL), the test compound (final concentration: 1.5. mu.M) and a total volume of 30. mu.L of potassium phosphate buffer (containing 1.0mM EDTA, 100mM, pH 7.4). The test compound was dissolved in DMSO and diluted with potassium phosphate buffer solution to make the final concentration of DMSO 0.2%. After a pre-incubation time of 10 minutes, 15. mu.L of NADPH (final concentration: 2mM) was added for the enzymatic reaction, and the whole assay was carried out in an incubation tube at 37 ℃. At various time points (0, 15, 30 and 60 minutes), the reaction was stopped by the addition of 135. mu.L acetonitrile (containing IS). The supernatant was collected by centrifugation at 4000rpm for 10 minutes to remove protein and analyzed by LC-MS/MS. In the above assay, ketanserin (1 μ M) was selected as a positive control, incubated at 37 ℃ and the reaction terminated at different time points (0, 15, 30 and 60 min). Positive and negative control samples were included in each assay method to ensure integrity of the microsomal incubation system.

Data analysis

For each reaction, the concentration of compound (in percent) in human or rat liver microsome incubations was plotted as a percentage of the zero time point to infer intrinsic hepatic clearance CL in vivo_int(see, e.g., Naritomi Y, Terashita S, Kimura S, Suzuki A, Kagayama A, Sugiyama Y.prediction of humancosmetic clearings from in vivo and experimental experiments and in vitro strategies with lipid microorganisms from animals and humans. drug Metabolism and distribution 2001,29: 1316-.

The compounds of the invention exhibit suitable stability when incubated in human and rat liver microsomes.

TABLE 1 hepatic microparticle stability in humans and rats

Example B pharmacokinetics of mice, rats, dogs and monkeys after oral or intravenous administration of a quantitative amount of a compound of the present invention Evaluation of

Pharmacokinetic studies of the compounds of the invention in mice, rats, dogs or monkeys were evaluated. The compounds of the invention are administered as aqueous solutions or 2% HPMC + 1% Tween-80 aqueous solution, 5% DMSO + 5% saline solution, 4% MC or capsules. For intravenous administration, animals are given a dose of 1 or 2 mg/kg. For oral doses (p.o.), rats and mice were 5 or 10mg/kg, and dogs and monkeys were 10 mg/kg. Blood (0.3mL) was taken at time points of 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 12 and 24 hours and centrifuged at 3,000 or 4,000rpm for 10 minutes. The plasma solutions were collected and stored at-20 ℃ or-70 ℃ until the LC/MS/MS analysis described above was performed.

When the compound provided by the invention is administrated by intravenous injection or oral administration, the compound shows good pharmacokinetic property, good absorption and ideal half-life (T)_1/2) And higher oral bioavailability (F).

TABLE 2 rat PK data

TABLE 3 mouse PK data

TABLE 4 dog PK data

TABLE 5 monkey PK data

Example C kinase Activity assay

The utility of the disclosed compounds as protein kinase inhibitors can be evaluated by the following experiments.

Kinase assays by detecting incorporation of gamma-³³Myelin Basic Protein (MBP) of P-ATP. Mu.g/ml MBP (Sigma # M-1891) Tris buffered saline (TBS; 50mM Tris pH 8.0, 138mM NaCl, 2.7mM KCl) was prepared and coated in high binding white 384 well plates (Greiner) at 60. mu.L per well. Incubate at 4 ℃ for 24 hours. The plate was then washed 3 times with 100. mu.L TBS. Kinase reaction in a total volume of 34. mu.L of kinase buffer (prepared as required, e.g., 5mM hepes pH 7.6, 15mM NaCl, 0.01% bovine serum albumin (Sigma # I-5506), 10mM MgCl₂1mM DTT, 0.02% TritonX-100). Compounds were dissolved in DMSO and added to each well at a final concentration of 1% compound in DMSO solution. The assay for each compound was performed in at least two trials. For example, the final concentration of the enzyme is 10nM or 20 nM. Addition of unlabeled ATP (10. mu.M) and gamma-³³P-labelled ATP (2 × 10 per well 10)⁶cpm, 3000Ci/mmol), the reaction was initiated with shaking at room temperature for 1 hour, the 384 well plates were washed with 7 × PBS, and 50. mu.L of scintillation fluid per well was added.

IC inhibited by the test method₅₀And/or suppression constant K_i。IC₅₀Defined as the concentration of compound that inhibits 50% of the enzyme activity under the conditions tested. Dilution factor using 1/2logA curve containing 10 concentration points was made and IC was estimated₅₀Values (e.g., a typical curve is made by the concentration of compound at 3. mu.M, 1. mu.M, 0.3. mu.M, 0.1. mu.M, 0.03. mu.M, 0.01. mu.M, 0.003. mu.M, 0.001. mu.M, 0.0003. mu.M, 0. mu.M), or at 10. mu.M, 3. mu.M, 1. mu.M, 0.3. mu.M, 0.1. mu.M, 0.03. mu.M, 0.01. mu.M, 0.003. mu.M, 0.001. mu.M, 0. mu.M).

AXL(h)

AXL (h) pH 7.0 at 8mM MOPS, 0.2mM EDTA, 250. mu. M KKSRGDYMTMQIG, 10mM magnesium acetate and [ gamma-³³P-ATP](specific activity and concentration determined as required) in the presence of conditions. The reaction was started after the addition of the MgATP mixture. After incubation at room temperature for 40 minutes, a phosphoric acid solution was added thereto to a concentration of 0.5% to terminate the reaction. 10 μ L of the reaction solution was distributed in a spot form on a P30 filter, and washed 4 times with 0.425% phosphoric acid solution and 1 time with methanol for 4 minutes. After drying, the measurement was carried out with a scintillation counter.

TrkA(h)

TrkA (h) at 8mM MOPS pH 7.0, 0.2mM EDTA, 250. mu. M KKKSPGEYVNIEFG, 10mM magnesium acetate and [ gamma-³³P-ATP](specific activity and concentration determined as required) in the presence of conditions. The reaction was started after the addition of the MgATP mixture. After incubation at room temperature for 40 minutes, a phosphoric acid solution was added thereto to a concentration of 0.5% to terminate the reaction. 10 μ L of the reaction solution was distributed in a spot form on a P30 filter, and washed 4 times with 0.425% phosphoric acid solution and 1 time with methanol for 4 minutes. After drying, the measurement was carried out with a scintillation counter.

TrkB(h)

TrkB (h) at 8mM MOPS pH 7.0, 0.2mM EDTA, 0.1mg/mL poly (Glu, Tyr)4:1,10mM magnesium acetate and [ gamma-³³P-ATP](specific activity and concentration determined as required) in the presence of conditions. The reaction was started after the addition of the MgATP mixture. After incubation at room temperature for 40 minutes, a phosphoric acid solution was added thereto to a concentration of 0.5% to terminate the reaction. mu.L of the reaction solution was distributed in a spot form on the A filter, and washed 4 times with 0.425% phosphoric acid solution and 1 time with methanol for 4 minutes. After drying, the measurement was carried out with a scintillation counter.

TrkC(h)

TrkB (h) at 8mM MOPS pH 7.0, 0.2mM EDTA, 500. mu. M GEEPLYWSFPAKKK,10mM magnesium acetate and [ gamma-³³P-ATP](specific activity and concentration determined as required) in the presence of conditions. The reaction was started after the addition of the MgATP mixture. After incubation at room temperature for 40 minutes, a phosphoric acid solution was added thereto to a concentration of 0.5% to terminate the reaction. mu.L of the reaction solution was distributed in a spot form on the A filter, and washed 4 times with 0.425% phosphoric acid solution and 1 time with methanol for 4 minutes. After drying, the measurement was carried out with a scintillation counter.

Table 6 results of axl (h), trka (h), TrkB and TrkC kinase assays for compounds provided herein

As can be seen from table 6, the compounds of the present invention showed better axl (h), trka (h), TrkB and TrkC kinase inhibitory activity in kinase assays.

Example D cell Activity assay

The method is completed by detecting the inhibition effect of two compounds on the growth of Ba/F3 AXL cell line and Ba/F3 partial cell line. Harvesting cells in logarithmic growth phase, counting the cells by adopting a platelet counter, and detecting the cell viability by using a trypan blue exclusion method to ensure that the cell viability is over 90 percent; adjusting the cell concentration; add 90 μ Ι _ of cell suspension to 96-well plates, respectively; cells in 96-well plates were incubated at 37 ℃ with 5% CO₂And cultured overnight under 95% humidity conditions. Preparing 10 times of drug solution, wherein the highest concentration is 100 mu M, the concentration is 9, the dilution is 3.16 times, 10 mu L of drug solution is added into each hole of a 96-hole plate inoculated with cells, and three multiple holes are arranged at each drug concentration; the cells in the dosed 96-well plate were placed at 37 ℃ in 5% CO₂95% humidity barThe culture was continued for 72 hours under the conditions, after which the CTG analysis was performed. The CTG reagents were thawed and the cell plates were equilibrated to room temperature for 30 minutes, an equal volume of CTG solution was added to each well, the cells were lysed by shaking on an orbital shaker for 5 minutes, the cell plates were placed at room temperature for 20 minutes to stabilize the luminescence signal, and the luminescence values were read. Data were analyzed using GraphPad Prism 5.0 software, fitted to the data using non-linear sigmoidal regression to derive a dose-effect curve, and IC was calculated therefrom₅₀Value, cell survival (%) ═ Lum (Lum)_{Drug to be tested}-Lum_{Culture fluid control})/(Lum_{Cell controls}-Lum_{Culture fluid control})×100％。

TABLE 7 inhibitory Effect of the Compounds provided by the present invention on the growth of two strains of Ba/F3 cells

Example E cytochrome P450 enzyme Induction assay

The cytochrome P450 induction assay can identify the potential of a test compound to induce CYP1a2, CYP2B6 or CYP3a4 in human hepatocytes.

Experimental System

Inducer and substrate:

the appropriate CYP enzyme positive control (inducer), negative control (flumazenil is commonly used), and probe substrate (used only for enzyme activity assay, and not for mRNA analysis) were selected. Common CYP enzyme kits are shown in table 8 below, and other types of kits may be selected by reference for purposes.

TABLE 8 common CYP enzyme kits

Hepatocyte donor:

evaluation of induction capacity was performed using hepatocytes of three donors.

Cytotoxicity test:

in the induction experiment, the influence of the test drug on the activity of the liver cells should be detected in parallel, and CCK-8, LDH, MTT, neutral red and the like can be adopted as the method.

Experimental procedure

Hepatocyte recovery/passage, plating:

recovering the cryopreserved hepatic cells or subculturing the hepatic cells meeting the requirement, and inoculating the cryopreserved hepatic cells on a proper cell culture plate according to a certain density. And (3) operating according to the culture requirements of the required culture model (such as monolayer cells, sandwich culture, 3D culture and the like) to meet the cell growth requirements.

And (3) incubation of the inducer in corresponding holes, adding a dosing solution which is prepared by using an incubation solution and contains a positive/negative control drug or a test drug and is preheated, continuously incubating for 2-3 days, changing the solution every day, and performing multiple-hole parallel operation on each compound. Samples may be taken at various time points during the last day of the test drug incubation and analyzed for actual concentrations, if desired.

Substrate incubation and CCK-8 incubation:

adding the prepared and preheated substrate working solution into the culture plate after the last administration for incubation for a certain time. The cytotoxic assay plate was incubated with CCK-8 for a period of time.

Cytotoxicity and enzyme activity assays:

the measurement of absorbance was performed using a microplate reader or a spectrophotometer to calculate cytotoxicity. Substrate working solution incubation followed by sample treatment was analyzed to calculate enzyme activity.

Extraction of mRNA, reverse transcription and fluorescent quantitative PCR:

the procedures of the above experimental procedures were performed according to the relevant kit instructions.

Data processing

Respectively calculating the induction times of mRNA level, the induction times of enzyme activity and the induction activities of the mRNA level and the enzyme activity relative to a positive control drug in an enzyme induction experiment; the cytotoxicity assay calculated the ratio of cell viability to the blank after each compound administration.

Fold induction calculation of mRNA levels:

the Ct value of the target gene is subtracted from the Ct value of the internal reference (such as 18s) to obtain the delta Ct value of the target gene, and then the delta Ct value is involved in the calculation of the relative expression difference (induction multiple) of the mRNA, namely the following formula is used for judging the induction condition of the mRNA expression level.

Δ Ct-target gene-Ct 18s

Δ Δ Ct-control/sample- Δ Ct-vehicle control relative expression difference 2- Δ Δ Ct.

Relative enzyme activity calculation:

specific metabolites of each CYP sub-enzyme probe substrate were detected using a confirmed assay, and enzyme activity induction was calculated by comparing the differences in metabolites of the test group and the vehicle control group. Relative positive control activity was calculated according to the following formula:

relative positive control activity (%) × (test group sample activity-vehicle group sample activity)/(positive control group sample activity-vehicle group sample activity) × 100.

Evaluation criteria:

usually, the enzyme activity induction factor of the positive control drug is 2 times or more, the induction factor of the mRNA level is 4 times or more, the experimental system is considered to be normal, and the standard can be properly reduced when the capability of inducing individual sub-enzymes of some cells is weaker. The tested drug group shows that the cell activity is less than 70 percent, and the liver cell toxicity is considered. The test drug is considered to be at an induction risk of not less than 2 times the induction factor of the mRNA level and not less than 20% of the induction factor of the mRNA level relative to the positive control drug. Meanwhile, in order to reduce false negative, if any of the mRNA level induction factor ≧ 4 times, the enzymatic activity induction factor ≧ 2 times, and the enzymatic activity induction factor ≧ 20% relative to the positive control drug is satisfied, it is considered that there is an induction risk.

The results of the enzyme induction experiments for some compounds of the invention at a concentration of 10 μ M are shown in Table 9:

TABLE 9 results of enzyme induction experiments of the compounds of the present invention

Finally, it should be noted that there are other ways of implementing the invention. Accordingly, the embodiments of the present invention will be described by way of illustration, but not limitation to the description of the present invention, and modifications made within the scope of the present invention or equivalents added to the claims are possible. All publications or patents cited herein are incorporated by reference.

Claims (10)

1. A compound having the structure shown in formula (I):

or a stereoisomer, tautomer, nitroxide, solvate, or pharmaceutically acceptable salt thereof;

wherein the content of the first and second substances,

U₁and U₂Are each independently N or-C (R)^a)-；

R¹、R²And R⁴Are each independently H, C_1-6Alkyl radical, C_1-6Haloalkyl, C_1-6Hydroxyalkyl radical, C_1-6Aminoalkyl, cyano-substituted C_1-6Alkyl radical, C_3-10Cycloalkyl radical, C_3-10Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl group, C_1-9Heteroaryl C_1-6An alkyl group; wherein each C is_1-6Alkyl radical, C_1-6Haloalkyl, C_1-6Hydroxyalkyl radical, C_1-6Aminoalkyl, cyano-substituted C_1-6Alkyl radical, C_3-10Cycloalkyl radical, C_3-10Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl and C_1-9Heteroaryl C_1-6Alkyl is independently optionally substituted with 0,1, 2,3 or 4R¹¹Substitution;

each R^a、R³、R⁵、R⁶、R⁷And R⁸Are respectively and independently H, D, F, Cl, Br, -OH, -CN, -NO₂、-NR^cR^d、C_1-6Alkyl radical, C_1-6HalogenatedAlkyl radical, C_1-6Hydroxyalkyl radical, C_1-6Aminoalkyl, cyano-substituted C_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl, or C_1-9Heteroaryl C_1-6An alkyl group; wherein each C is_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl and C_1-9Heteroaryl C_1-6Alkyl is independently optionally substituted with 0,1, 2,3 or 4R¹²Substitution;

or R²And R³Optionally form a heterocyclic ring of 4 to 12 atoms together with the carbon and nitrogen atom to which it is attached, wherein said heterocyclic ring of 4 to 12 atoms is optionally substituted with 0,1, 2,3,4 or 5R¹³Substitution;

each R¹¹、R¹²And R¹³Each independently is H, D, oxo (═ O), F, Cl, Br, -OH, -CN, -NO₂、-NR^cR^d、-C(＝O)R⁹、-OC(＝O)R⁹、-C(＝O)OR^9a、-S(＝O)_0-2R⁹、-OS(＝O)_1-2R⁹、-S(＝O)_1-2OR^9a、-N(R^10a)C(＝O)R¹⁰、-C(＝O)NR^10aR¹⁰、-OC(＝O)NR^10aR¹⁰、-N(R^10a)S(＝O)_1-2R¹⁰、-S(＝O)_1-2NR^10aR¹⁰、-N(R^10a)C(＝O)NR^10aR¹⁰、C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_1-6Haloalkyl, C_1-6Hydroxyalkyl radical, C_1-6Aminoalkyl, cyano-substituted C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylamino radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl, or C_1-9Heteroaryl C_1-6An alkyl group; wherein each C is_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl group, C_1-9Heteroaryl C_1-6Alkyl is independently optionally substituted with 0,1, 2,3 or 4 substituents independently selected from H, D, oxo (═ O), F, Cl, Br, -OH, -NH₂、-CN、-NO₂、C_1-6Alkyl and C_1-6A radical substitution of alkoxy;

each R^c、R^d、R⁹、R^9a、R¹⁰And R^10aAre each independently H, D, C_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl, or C_1-9Heteroaryl C_1-6An alkyl group; wherein each C is_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl and C_1-9Heteroaryl C_1-6Alkyl is independently optionally substituted with 0,1, 2,3 or 4 substituents independently selected from H, D, oxo (═ O), F, Cl, Br, -OH, -NH₂、-CN、-NO₂、C_1-6Alkyl and C_1-6A radical substitution of alkoxy; and

n is 0,1, or 2.

2. The compound of claim 1, having the structure of formula (II):

wherein the content of the first and second substances,

X¹is O, S, -N (R)^13a)-、-C(＝O)-、-(CH₂)_t1-、-X²-(CH₂)_t1-, or- (CH)₂)_t1-X²-(CH₂)_t2-；

X²Is O, S, -N (R)^13a) -, or-C (═ O) -;

each R^13aEach independently is H, D, oxo (═ O), F, Cl, Br, -OH, -CN, -NO₂Amino group, C_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl group, C_1-9Heteroaryl C_1-6Alkyl, -C (═ O) R⁹、-C(＝O)OR^9a、-S(＝O)_0-2R⁹、-S(＝O)_1-2OR^9a、-S(＝O)_{1- 2}NR^10aR¹⁰or-C (═ O) NR^10aR¹⁰Wherein each of C_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_3-8Cycloalkyl radical C_1-6Alkyl radical, C_2-7Heterocyclic group, C_2-7Heterocyclyl radical C_1-6Alkyl radical, C_6-12Aryl radical, C_6-12Aryl radical C_1-6Alkyl radical, C_1-9Heteroaryl and C_1-9Heteroaryl C_1-6Alkyl is independently optionally substituted with 0,1, 2,3 or 4 substituents independently selected from H, D, oxo (═ O), F, Cl, Br, -OH, -NH₂、-CN、-NO₂、C_1-6Alkyl and C_1-6A radical substitution of alkoxy;

each t1 and t2 is independently 0,1, 2, or 3; and

m is 0,1, 2,4, or 5.

3. The compound according to claim 1 or 2, wherein,

R¹is C_1-4Alkyl radical, C_1-4Haloalkyl, C_1-4Hydroxyalkyl radical, C_1-4Aminoalkyl, cyano-substituted C_1-4Alkyl radical, C_3-8Cycloalkyl, phenyl, or C_1-9A heteroaryl group; wherein each C is_1-4Alkyl radical, C_1-4Haloalkyl, C_1-4Hydroxyalkyl radical, C_1-4Aminoalkyl, cyano-substituted C_1-4Alkyl radical, C_3-8Cycloalkyl, phenyl and C_1-9Heteroaryl is independently optionally substituted with 0,1, 2,3 or 4R¹¹And (4) substitution.

4. The compound according to claim 1 or 2, wherein,

R⁴is H, D, methyl, ethyl, propyl, isopropyl, butyl, C_1-4Haloalkyl, C_1-4Aminoalkyl, cyano-substituted C_1-4Alkyl radical, C_3-6Cycloalkyl radical, C_3-6Cycloalkyl radical C_1-4Alkyl radical, C_2-7Heterocyclic group, or C_2-7Heterocyclyl radical C_1-4An alkyl group.

5. The compound of claim 1, which is a compound having one of the following structures:

or a stereoisomer, tautomer, nitroxide, solvate, or pharmaceutically acceptable salt thereof.

6. A pharmaceutical composition comprising a compound of any one of claims 1-5, or a stereoisomer, a tautomer, a nitric oxide, a solvate, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant, diluent, or carrier, or a combination thereof.

7. The pharmaceutical composition of claim 6, further comprising an additional therapeutic agent.

8. Use of a compound according to any one of claims 1 to 5 or a pharmaceutical composition according to any one of claims 6 to 7 in the manufacture of a medicament for the prevention or treatment of one or more diseases and/or conditions mediated by Axl and/or Trk protein kinases.

9. The use according to claim 8, the disease and/or disorder being selected from a proliferative disease, an autoimmune disease, an allergic disease, an inflammatory disease, or transplant rejection.

10. The use according to claim 9, the disease and/or disorder being selected from cancer, polycythemia vera, essential thrombocythemia, acute myeloid leukemia, acute lymphocytic leukemia, myelofibrosis, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic obstructive pulmonary disease, asthma, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, sjogren's syndrome, psoriasis, type I diabetes, respiratory allergic disease, sinusitis, eczema, measles, food allergy, insect venom allergy, inflammatory bowel disease, crohn's disease, rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, organ transplant rejection, tissue transplant rejection or cell transplant rejection.