Pyridazine compound and use thereofTechnical Field
The application relates to a novel pyridazine compound or pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the same and application of the pyridazine compound or pharmaceutically acceptable salt thereof as TYK2 inhibitor in preventing or treating related diseases.
Background
TYK2 is one of the members of the JAK family of non-receptor tyrosine kinases. Janus kinases (JAKs) consist of four family members, JAK1, JAK2, JAK3 and TYK 2. When Cytokine receptor (Cytokine Receptor) binds to Cytokine (Cytokine), downstream signaling pathways are activated by phosphorylating STAT proteins, regulating transcription and expression of the relevant genes, and achieving transduction of signals from cell membrane to nucleus. JAK mediated signal transduction pathways play an important role in many functions of cytokine-dependent regulation of cell proliferation, differentiation, apoptosis, and immune response, and are hot targets for the treatment of inflammatory diseases, autoimmune diseases, and cancer.
Recent studies have found that activation of Th17 cells is closely associated with various autoimmune diseases such as Psoriasis (Psoriasis), multiple sclerosis (Multiple Sclerosis, MS), inflammatory bowel disease (Inflammatory Bowel Disease, IBD), and Lupus (Lupus). Under some environmental stimuli, such as trauma or infection, the body expresses self-antigens, thereby activating infiltrating DC cells in the tissue, and activated DC cells cooperatively polarize T cells into Th17 cells by secreting IL-23 and TNF-alpha. Activated Th17 cells further exacerbate inflammatory responses by secreting pro-inflammatory factors such as IL-17A, IL-17F, IL-6 and INF-alpha. Like IL-23, IL-12 can effectively activate Th1 cells to secrete IFNg, thereby causing a variety of systemic autoimmune diseases such as systemic Lupus erythematosus (Systemic Lupus Erythematosus, SLE) and Lupus Nephritis (LN). The type I interferon can enhance the reaction of B cells to antigens and reduce the threshold value of B cell activation, induce the conversion of monocytes to antigen presenting dendritic cells, and further cause diseases such as SLE, SLE and the like. In the serum of SLE patients, ifnα (a type I interferon) protein levels were significantly increased, as were gene expression regulated by type I interferon in Peripheral Blood Mononuclear Cells (PBMCs) and target organs affected by the disease. TYK2 is used as an important signal molecule downstream of cytokines such as IL-23, IL-12, IL-17A, IL-17F, IL-6, INF-alpha and the like, and is involved in regulating and controlling activation of Th17 and Th1 cells. Inhibition of excessive activation of the IL23/IL17/IL 12/INF-alpha-TYK 2 signaling pathway is an effective means of treating Th17 and Th1 cell mediated autoimmune diseases. Antibodies currently targeting IL-17, IL-17R and IL-23R have been approved clinically for the treatment of autoimmune diseases such as psoriasis and inflammatory bowel disease. In clinical trials of systemic lupus erythematosus, anifloumab, which targets type I interferon receptors, also showed significant efficacy.
JAK has 7 structurally homologous domains (JAK Homology Domain, JH), where the JH1 domain is a kinase domain and the JH2 domain is a pseudokinase domain (modulating the activity of JH 1). Currently, TYK2 inhibitors, such as pyroxene PF-06700841, are currently under study to inhibit by binding to the JH1 domain of TYK 2. Because of the high conservation of the JH1 domain of the JAK kinase family, the selectivity of such JH1 TYK2 inhibitors is not very good, and all have strong JAK1/2 inhibition activity. Therefore, the clinical efficacy of the drug is limited by the unavoidable toxicity associated with JAK 1/2. Because the TYK2 JH2 region is largely different from other kinases (except JAK1 JH 2), providing advantages for the design of highly selective TYK2 inhibitors, the development of allosteric inhibitors targeting the TYK2-JH2 domain has attracted great attention from pharmaceutical companies. The TYK2 highly selective inhibitor BMS-986165 developed by BMS showed close therapeutic effects to antibodies in a secondary clinical trial of psoriasis (see reference Phase 2Trial of Selective Tyrosine Kinase 2Inhibition in Psoriasis.N Engl J Med.2018Oct;379 (14): 1313-1321) while patient tolerability was good and no JAK 1/2-related toxic manifestation was observed. BMS-986165 demonstrates that selective targeting TYK2 can exhibit safety over JH type 1 TYK2 inhibitors with increased patient benefit. Compared with the antibody, the TYK2 small molecule inhibitor also has the advantage of convenient administration. In view of the huge autoimmune market and unmet market demand, the development of highly selective TYK2 inhibitors of JH2 type has great market prospects.
Disclosure of Invention
The application provides a compound shown in a formula (I) or pharmaceutically acceptable salt thereof:
wherein,,
R1 selected from C1 -C10 Alkyl, C3 -C10 Cycloalkyl, 3-10 membered heterocyclyl, C6 -C10 Aryl or 5-10 membered heteroaryl, said C1 -C10 Alkyl, C3 -C10 Cycloalkyl, 3-10 membered heterocyclyl, C6 -C10 Aryl or 5-to 10-membered heteroAryl is optionally substituted with R1a Substitution;
each R is1a Independently selected from halogen, OH, CN, =O,Or optionally by R1b Substituted with the following groups: NH (NH)2 、C1 -C6 Alkyl, C3 -C6 Cycloalkyl, 3-6 membered heterocyclyl, C1 -C6 Alkoxy, C3 -C6 Cycloalkyloxy or 3-6 membered heterocyclyloxy;
each R is1b Independently selected from halogen, OH, CN, = O, NH2 、C1 -C6 Alkyl, C3 -C6 Cycloalkyl or 3-6 membered heterocyclyl;
R2 selected from 4-membered heterocyclyl groups, said 4-membered heterocyclyl groups optionally being substituted with R2a Substitution;
each R is2a Independently selected from halogen, CN, OH, =o, or optionally R2b Substituted with the following groups: NH (NH)2 、C1 -C6 Alkyl, C3 -C6 Cycloalkyl, 3-6 membered heterocyclyl, C1 -C6 Alkoxy, C3 -C6 Cycloalkyloxy or 3-6 membered heterocyclyloxy;
each R is2b Independently selected from halogen, OH, CN, = O, NH2 、C1 -C6 Alkyl, C3 -C6 Cycloalkyl or 3-6 membered heterocyclyl;
R3 selected from the group consisting of
n is selected from 0 or 1.
In some embodiments, R1 Selected from C3 -C6 Cycloalkyl, 3-6 membered heterocyclyl, phenyl or 5-6 membered heteroaryl, said C3 -C6 Cycloalkyl, 3-6 membered heterocyclyl, phenyl or 5-6 membered heteroaryl optionally substituted with R1a Substitution;
in some embodiments, R1 Selected from C3 -C6 Cycloalkyl, 3-6 membered heterocyclyl or 5-6 membered heteroaryl, said C3 -C6 Cycloalkyl, 3-6 membered heterocyclyl or 5-6 membered heteroaryl optionally substituted with R1a Substitution;
in some embodiments, R1 Selected from cyclopropyl, 4 membered heterocyclyl or 5-6 membered heteroaryl, optionally substituted with R1a Substitution;
in some embodiments, R1 Selected from cyclopropyl.
In some embodiments, R1 Selected from 4-membered heterocyclyl groups, said 4-membered heterocyclyl groups being selected fromSaid->Optionally by R1a And (3) substitution.
In some embodiments, R1 Selected from 5-6 membered heteroaryl, said 5-6 membered heteroaryl being selected from imidazolyl, pyrazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl, said imidazolyl, pyrazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl optionally being substituted with R1a And (3) substitution.
In some embodiments, R1 Selected from 5-6 membered heteroaryl, said 5-6 membered heteroaryl being selected from pyrazolyl, pyridinyl or pyrazinyl, said pyrazolyl, pyridinyl or pyrazinyl being optionally substituted with R1a And (3) substitution.
In some embodiments, R1 Selected from cyclopropyl group,Pyrazolyl, pyridinyl or pyrazinyl, said cyclopropyl,/->Pyrazolyl, pyridinyl or pyrazinyl are optionally substituted with R1a And (3) substitution.
In some embodiments, R1 Selected from the group consisting ofCyclopropyl or pyrazinyl optionally substituted with R1a And (3) substitution.
In some embodiments, R1a Selected from halogen, OH, CN, =O,Or optionally by R1b Substituted with the following groups: c (C)1 -C6 Alkyl, C3 -C6 Cycloalkyl or 3-6 membered heterocyclyl.
In some embodiments, R1a Selected from OH,Or optionally by R1b Substituted with the following groups: c (C)1 -C6 Alkyl, 4 membered heterocyclyl.
In some embodiments, R1b Independently selected from halogen, OH, CN, = O, NH2 Or C1 -C6 An alkyl group.
In some embodiments, R1a Selected from OH or methyl.
In some embodiments, R1 Selected from cyclopropyl or pyrazinyl.
In some embodiments, n is selected from 0.
In some embodiments, n is selected from 1.
In some embodiments, the building blockSelected from the group consisting of
In some embodiments, the building blockSelected from->
In some embodiments, R2 Selected from 4-membered heterocyclyl containing 1-2 members selected from O, N or S (O)p Wherein p is 0, 1 or 2, said 4 membered heterocyclyl is optionally substituted with R2a And (3) substitution.
In some embodiments, R2 Selected from 4-membered heterocyclic groups selected from azetidinyl, oxetanyl,said azetidinyl, oxetanyl,/->Optionally by R2a And (3) substitution.
In some embodiments, R2a Independently selected from halogen, CN, OH, =o, or optionally R2b Substituted with the following groups: c (C)1 -C6 Alkyl, C3 -C6 Cycloalkyl or 3-6 membered heterocyclyl.
In some embodiments, R2b Independently selected from halogen, OH, CN, =o or C1 -C6 An alkyl group.
In some embodiments, R2a Independently selected from halogen, CN, =o or C1 -C6 An alkyl group.
In some embodiments, R2a Independently selected from methyl.
In some embodiments, R2 Selected from the group consisting of
In some embodiments, R3 Selected from the group consisting of
In some embodiments, R3 Selected from the group consisting of
In some embodiments, the compound of formula (I), or a pharmaceutically acceptable salt thereof, is selected from the following compounds, or pharmaceutically acceptable salts thereof:
the application also provides a pharmaceutical composition which comprises the compound shown in the formula (I) or pharmaceutically acceptable salt thereof and pharmaceutically acceptable auxiliary materials.
Further, the application relates to application of a compound shown in the formula (I) or pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof in preparing a medicament for preventing or treating TYK2 related diseases.
Further, the application relates to the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for preventing or treating TYK 2-related diseases.
Further, the present application relates to the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, as a selective TYK2 inhibitor in the prevention or treatment of a related disease.
Further, the present application relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for preventing or treating TYK 2-related diseases.
The application also relates to a method of treating TYK 2-related disorders, which comprises administering to a patient a therapeutically effective dose of a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein.
Preferred embodiments of the present application wherein said TYK 2-related disease includes, but is not limited to, inflammatory diseases, autoimmune diseases and cancer.
Definition and description of terms
Unless otherwise indicated, the radical and term definitions recited in the specification and claims of the present application, including as examples, exemplary definitions, preferred definitions, definitions recited in tables, definitions of specific compounds in the examples, and the like, may be arbitrarily combined and coupled with each other. Such combinations and combinations of radical definitions and structures of compounds should fall within the scope of the present description.
The term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of non-toxic acids or bases, including salts of inorganic acids and bases, organic acids and bases.
The term "stereoisomers" refers to isomers arising from the spatial arrangement of atoms in a molecule, and includes cis-trans isomers, enantiomers and diastereomers.
The compounds of the application may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms (optical centers) or asymmetric double bonds. Racemates, enantiomers, diastereomers, geometric isomers are all included within the scope of the present application.
The graphic representation of racemates or enantiomerically pure compounds herein is from Maehr, J.chem. Ed.1985, 62:114-120. Unless otherwise indicated, wedge-shaped keys and virtual wedge-shaped keys are usedAnd->) Representing the absolute configuration of a stereogenic center using black real and virtual keys (/ -)>And->) Represents the cis-trans configuration of the alicyclic compound. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, they include the E, Z geometric isomers unless specified otherwise. Likewise, all tautomeric forms are included within the scope of the application.
The compounds of the application may exist in specific geometric or stereoisomeric forms. The present application contemplates all such compounds, including cis and trans isomers, (-) -and (+) -pairs of enantiomers, (R) -and (S) -enantiomers, diastereomers, (D) -isomers, (L) -isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the application. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms, or asymmetric phosphorus atoms may be present in the substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of the present application. The asymmetric atom-containing compounds of the application can be isolated in optically pure or racemic form. Optically pure forms can be resolved from the racemic mixture or synthesized by using chiral starting materials or chiral reagents.
The term "tautomer" refers to a functional group isomer that results from the rapid movement of an atom in a molecule at two positions. The compounds of the present application may exhibit tautomerism. Tautomeric compounds may exist in two or more interconvertible species. Tautomers generally exist in equilibrium and attempts to isolate individual tautomers often result in a mixture whose physicochemical properties are consistent with the mixture of compounds. The location of the equilibrium depends on the chemical nature of the molecule. For example, among many aliphatic aldehydes and ketones such as acetaldehyde, the ketone type predominates; whereas, among phenols, the enol form is dominant. The present application encompasses all tautomeric forms of the compounds.
The term "pharmaceutical composition" means a mixture of one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof with other chemical components, such as physiologically/pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to the organism.
The term "substituted" means that any one or more hydrogen atoms on a particular atom is substituted with a substituent, provided that the valence of the particular atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., =o), meaning that two hydrogen atoms are substituted, oxo does not occur on the aromatic group.
The term "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, ethyl "optionally" substituted with halogen means that ethyl may be unsubstituted (CH2 CH3 ) Monosubstituted (e.g. CH2 CH2 F) Polysubstituted (e.g. CHFCH2 F、CH2 CHF2 Etc.) or fully substituted (CF)2 CF3 ). It will be appreciated by those skilled in the art that for any group comprising one or more substituents, no substitution or pattern of substitution is introduced that is sterically impossible and/or synthetic.
The term "halogen" or "halo" refers to fluorine, chlorine, bromine and iodine.
The term "C1 -C10 Alkyl "is understood to mean a straight-chain or branched saturated monovalent hydrocarbon radical having 1,2, 3,4, 5,6, 7, 8, 9 or 10 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-ethylpropyl, 1, 2-dimethylpropyl, neopentyl, 1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 2, 3-dimethylbutyl, 1, 3-dimethylbutyl, or 1, 2-dimethylbutyl, etc.; preferably, "C1 -C10 Alkyl "may contain" C1 -C6 Alkyl "or" C1 -C3 Alkyl "," C1 -C6 Alkyl "is understood to mean a straight-chain or branched saturated monovalent hydrocarbon radical having 1,2, 3,4, 5,6 carbon atoms," C1 -C3 Alkyl "is understood to mean a straight or branched saturated monovalent hydrocarbon radical having 1,2, 3 carbon atoms.
The term "C1 -C10 Alkoxy "is understood to mean" C1 -C10 Alkyloxy "or" C1 -C10 alkyl-O- ", preferably," C1 -C10 Alkoxy "may contain" C1 -C6 Alkoxy "or" C1 -C3 An alkoxy group.
The term "C3 -C10 Cycloalkyl "is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 10 carbon atoms. Such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl, or a bicyclic hydrocarbon group such as a decalin ring. Preferably, "C1 -C10 Cycloalkyl "may contain" C3 -C6 Cycloalkyl "or" C3 -C4 Cycloalkyl ", the term" C3 -C6 Cycloalkyl "is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 6 carbon atoms, the term" C3 -C4 Cycloalkyl "is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 4 carbon atoms.
The term "C3 -C10 Cycloalkyloxy "can be understood as" C3 -C10 cycloalkyl-O- ", preferably," C3 -C10 Cycloalkyloxy "may comprise" C3 -C6 Cycloalkyl oxy).
The term "3-10 membered heterocyclyl" means a saturated or partially saturated monovalent monocyclic, fused, spiro or bridged ring comprising 1 to 5, preferably 1 to 3 heteroatoms selected from N, O, B and S. In particular, the heterocyclic groups may include, but are not limited to: 4-membered rings such as azetidinyl, oxetanyl; 5-membered rings, such as tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 1,3, 2-dioxaborolan; or a 6 membered ring such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl or trithianyl; or a partially saturated 6-membered ring such as tetrahydropyridinyl; or a 7-membered ring such as diazepanyl. Optionally, the heterocyclyl may be benzo-fused. The heterocyclyl may be bicyclic, such as, but not limited to, a 5,5 membered ring, such as hexahydrocyclopenta [ c ] pyrrol-2 (1H) -yl ring, or a 5,6 membered bicyclic ring, such as hexahydropyrrolo [1,2-a ] pyrazin-2 (1H) -yl ring. The nitrogen atom-containing ring may be partially unsaturated, i.e., it may contain one or more double bonds, such as, but not limited to, 2, 5-dihydro-1H-pyrrolyl, 4H- [1,3,4] thiadiazinyl, 4, 5-dihydro-oxazolyl, or 4H- [1,4] thiazinyl, or it may be benzo-fused, such as, but not limited to, dihydroisoquinolinyl. Optionally, the 3-10 membered heterocyclyl may be a "3-10 membered heterocycloalkyl", meaning a saturated monovalent monocyclic, fused, spiro or bridged ring containing 1-5 heteroatoms; preferably, "3-10 membered heterocycloalkyl" includes 3-7 membered heterocycloalkyl, also 5-6 membered heterocycloalkyl, etc.; according to the application, the heterocyclic group is non-aromatic.
The term "3-6 membered heterocyclyloxy" is understood as "3-6 membered heterocyclyl-O-".
The term "4-membered heterocyclyl" means a saturated or partially saturated monovalent monocyclic ring comprising 1 to 3, preferably 1 to 2 heteroatoms or groups of heteroatoms selected from N, O and S, wherein the heteroatom S can optionally be oxidized (e.g. s=o, SO2 Etc.). In particular, the 4-membered heterocyclic group may include, but is not limited to, for example, azetidinyl, oxetanyl,Etc.; herein, "4-membered heterocyclic group" may include "4-membered heterocycloalkyl group", which is non-aromatic according to the present application.
The term "3-6 membered heterocyclyloxy" refers to "3-6 membered heterocyclyl-O-".
The term "C6 -C10 Aryl "is understood to mean preferably a mono-, bi-or tricyclic hydrocarbon ring of monovalent aromatic or partly aromatic nature having 6 to 10 carbon atoms. In particular having 6 carbon atoms ("C)6 Aryl "), such as phenyl; or a ring having 9 carbon atoms ("C)9 Aryl "), e.g. indanyl or indenyl, or a ring having 10 carbon atoms (" C "10 Aryl "), such as tetrahydronaphthyl, dihydronaphthyl or naphthyl.
The term "5-10 membered heteroaryl" is understood to include monovalent monocyclic, bicyclic or tricyclic aromatic ring systems having 5 to 10 ring atoms and containing 1 to 5 heteroatoms independently selected from N, O and S. "5-6 membered heteroaryl" means a heteroatom having 5 or 6 ring atoms and which contains 1 to 4, preferably 1 to 3, heteroatoms each independently selected from N, O and S. In particular, heteroaryl is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl and the like, and their benzo derivatives, such as benzofuranyl, benzothienyl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazole, indazolyl, indolyl, isoindolyl and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and the like, and their benzo derivatives, such as quinolinyl, quinazolinyl, isoquinolinyl, and the like.
The term "treating" means administering a compound or formulation of the application to prevent, ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:
(i) Preventing the occurrence of a disease or disease state in a mammal, particularly when such mammal is susceptible to the disease state, but has not been diagnosed as having the disease state;
(ii) Inhibiting a disease or disease state, i.e., inhibiting its progression;
(iii) The disease or condition is alleviated, even if the disease or condition subsides.
The term "therapeutically effective amount" means an amount of a compound of the application that (i) treats or prevents a particular disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the compound of the present application that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one of ordinary skill in the art based on his own knowledge and disclosure.
The term "adjuvant" refers to a pharmaceutically acceptable inert ingredient. Examples of the category of the term "excipient" include, without limitation, binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, and the like. Excipients can enhance the handling characteristics of the pharmaceutical formulation, i.e., by increasing flowability and/or tackiness, making the formulation more suitable for direct compression. Typical examples of "pharmaceutically acceptable carriers" suitable for use in the above formulations are: saccharides, starches, cellulose and derivatives thereof, and the like.
The term "pharmaceutically acceptable excipients" refers to those excipients which do not significantly stimulate the organism and which do not impair the biological activity and properties of the active compound. Suitable excipients are well known to the person skilled in the art, such as carbohydrates, waxes, water soluble and/or water swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like.
The words "comprise", "comprising" or "includes" and variations thereof such as include or comprise are to be interpreted in an open, non-exclusive sense, i.e. "including but not limited to".
The compounds of the present application may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by combining with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present application.
The application also includes isotopically-labeled compounds of the application which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic weight or mass number different from the atomic weight or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as, respectively2 H、3 H、11 C、13 C、14 C、13 N、15 N、15 O、17 O、18 O、31 P、32 P、35 S、18 F、123 I、125 I and36 cl, and the like.
Certain isotopically-labeled compounds of the application (e.g., with3 H is H14 C-labeled) can be used in compound and/or substrate tissue distribution analysis. Tritiation (i.e3 H) And carbon-14 (i.e14 C) Isotopes are particularly preferred for their ease of preparation and detectability. Positron emitting isotopes, such as15 O、13 N、11 C and C18 F can be used in Positron Emission Tomography (PET) studies to determine substrate occupancy. Isotopically-labeled compounds of the present application can generally be prepared by following procedures analogous to those disclosed in the schemes and/or examples below by substituting an isotopically-labeled reagent for an non-isotopically-labeled reagent.
In addition, the use of heavier isotopes (such as deuterium (i.e.2 H) Substitution may provide certain therapeutic advantages resulting from higher metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements), and thus may be preferred in certain circumstances, where deuterium substitution may be partial or complete, partial deuterium substitution meaning that at least one hydrogen is substituted with at least one deuterium.
The pharmaceutical compositions of the present application may be prepared by combining the compounds of the present application with suitable pharmaceutically acceptable excipients, for example, in solid, semi-solid, liquid or gaseous formulations such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres, aerosols and the like.
Typical routes of administration of the compounds of the application or pharmaceutically acceptable salts thereof or pharmaceutical compositions thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.
The pharmaceutical compositions of the present application may be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, freeze-drying, and the like.
In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical compositions may be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of the present application to be formulated into tablets, pills, troches, dragees, capsules, liquids, gels, slurries, suspensions and the like for oral administration to a patient.
The solid oral compositions may be prepared by conventional mixing, filling or tabletting methods. For example, it can be obtained by the following method: the active compound is mixed with solid auxiliary materials, the resulting mixture is optionally milled, if desired with other suitable auxiliary materials, and the mixture is then processed to granules, giving a tablet or dragee core. Suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, and the like.
The pharmaceutical compositions may also be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms.
In all methods of administration of the compounds of formula I described herein, the dosage administered is from 0.01 to 100mg/kg body weight, preferably from 0.05 to 50mg/kg body weight, more preferably from 0.1 to 30mg/kg body weight, either alone or in divided doses.
The chemical reactions of the embodiments of the present application are accomplished in a suitable solvent that is compatible with the chemical changes of the present application and the reagents and materials required therefor. In order to obtain the compounds of the present application, it is sometimes necessary for a person skilled in the art to modify or select the synthesis steps or reaction schemes on the basis of the embodiments already present.
Detailed Description
The following examples illustrate the technical aspects of the application in detail, but the scope of the application is not limited thereto.
The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) and/or Mass Spectrometry (MS). The NMR shift is in the unit of 10-6 (ppm). The solvent for NMR measurement wasDeuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol and the like, and the internal standard is Tetramethylsilane (TMS); IC (integrated circuit)50 "means half inhibition concentration" means concentration at which half of the maximum inhibition effect is achieved.
EXAMPLE 1 Synthesis of Compound 001
6- (cyclopropanecarboxamide) -4- ((2-methoxy-3- (1- (1-methylazetidin-3-yl) -1H-1,2, 4-triazol-3-yl) phenyl) amino) -N-methylpyridazine-3-carboxamide
Synthetic route and specific synthetic steps:
the first step: synthesis of 3- (2-methoxy-3-nitrophenyl) -1- (thietane-1, 1-dioxide-3-yl) -1H-1,2, 4-triazole 001-b
001-a (300 mg,1.36 mmol), tert-butyl 3-iodoazetidine-1-carboxylate (1.20 g,4.10 mmol) was added to a solution of cesium carbonate (1.3 g,4.10 mmol) in DMF (50.0 mL), degassed three times under nitrogen and the reaction heated to 100deg.C and stirred for 18 hours. After completion of the reaction, the reaction mixture was added to 100mL of water, extracted twice with ethyl acetate (50.0 mL), and the organic phase was backwashed with brine, dried and concentrated. The crude product was isolated and purified by silica gel column chromatography to give 001-b (273 mg, yield 53%).
LCMS:Rt:1.60min；MS m/z(ESI):376.2[M+H].
And a second step of: synthesis of tert-butyl 3- (3- (3-amino-2-methoxyphenyl) -1H-1,2, 4-triazol-1-yl) azetidin-1-carboxylate 001-c
Pd/C (109 mg,40% mmol) was added to a solvent of 001-b (2793 mg,0.73 mmol) in methanol (8.0 mL) at room temperature, degassed three times under hydrogen, and the reaction was heated to 30℃and stirred for 18 hours. After completion of the reaction, the reaction mixture was dried by filtration and purified by column chromatography on silica gel to give 001-c (205 mg, yield 81%).
LCMS:Rt:1.43min；MS m/z(ESI):346.2[M+H].
And a third step of: synthesis of 4- ((3- (1- (1- (1- (tert-butoxycarbonyl) azetidin-3-yl) -1H-1,2, 4-triazol-3-yl) -2-methoxyphenyl) amino) -6-chloropyridazine-3-carboxylic acid 001-d
001-c (175 mg,0.51 mmol), lithium 4, 6-dichloropyridazine-3-carboxylate (201 mg,4.10 mmol) was added to a solution of zinc acetate (188 mg,1.01 mmol) in isopropanol/water (15 mL/5 mL), degassed three times under nitrogen, the reaction was heated to 85℃and stirred for 18 hours, then the reaction was concentrated under reduced pressure, the crude product was prepared in reverse phase with acetonitrile/water (0.1% ammonia) as the mobile phase, and purified to give 001-d (110 mg, yield 44%).
LCMS:Rt:1.59min；MS m/z(ESI):502.0[M+H].
Fourth step: synthesis of tert-butyl 3- (3- (3- ((6-chloro-3- (methylcarbamoyl) pyridazin-4-yl) amino) -2-methoxyphenyl) -1H-1,2, 4-triazol-1-yl) 1-carboxylic acid azetidine 001-e
001-d (102 mg,0.204 mmol), EDCI (59 mg,0.306 mmol), HOBT (19 mg,0.143 mmol), NMI (8 mg,0.102 mmol), methylamine (70 mg,1.02 mmol) were dissolved in acetonitrile (1 mL) and methylpyrrolidone (1 mL), and the reaction was heated to 35℃and stirred under argon for 4 hours. After completion of the reaction, the reaction mixture was concentrated, and the crude product was prepared in reverse phase, acetonitrile/water (0.1% aqueous ammonia) as a mobile phase, and purified to give 001-e (41 mg, yield: 39%).
LCMS:Rt:2.06min；MS m/z(ESI):515.0[M+H].
Fifth step: synthesis of tert-butyl 3- (3- (3- ((6- (cyclopropanecarboxamide) -3- (methylcarbamoyl) pyridazin-4-yl) amino) -2-methoxyphenyl) -1H-1,2, 4-triazole-1-azetidine-1-carboxylate 001-f
001-e (28 mg,0.055 mmol), cyclopropanecarboxamide (46 mg,0.550 mmol), tris (dibenzylideneacetone) dipalladium (Pd) at room temperature2 (dba)3 5mg, 0.006mmol), 4, 5-bis (diphenylphosphine) -9, 9-dimethylxanthene (XantPhos, 3mg, 0.006mmol), cesium carbonate (36 mg,0.110 mmol) was added to dioxane (5.0 mL), the reaction was heated to 110℃and stirred under argon for 18 hours. Concentrating the reaction solution under reduced pressure after the reaction is completed, preparing the crude product by reverse phase, and preparing the crude product by reverse phaseNitrile/water (0.1% aqueous ammonia) was used as a mobile phase, and purification gave 001-f (12 mg, yield: 38%).
LCMS:Rt:2.010min；MS m/z(ESI):564.1[M+H].
Sixth step: synthesis of 6- (cyclopropanecarboxamide) -4- ((2-2-methoxy-3- (1- (1-methylazetidin-3-yl) -1H-1,2, 4-triazol-3-yl) phenyl) amino) -N-methylpyridazine-3-carboxamide 001
Trifluoroacetic acid (0.5 mL) was added to a solution of 001-f (8 mg,0.014 mmol) in dichloromethane (3.0 mL) at room temperature, and the reaction was stirred at 25℃under argon for 2 hours. After the reaction was concentrated under reduced pressure, the concentrated solution was dissolved in methanol (1.0 mL), paraformaldehyde (6 mg,0.18 mmol) was added, and sodium cyanoborohydride (11 mg,0.18 mmol) was stirred at 25℃under argon for 18 hours. After completion of the reaction was detected, the reaction solution was filtered and the filtrate was concentrated under reduced pressure, and the crude product was prepared by reverse phase preparative liquid chromatography (acetonitrile/water (0.1% aqueous ammonia) as a mobile phase) and purified to give 001 (3.1 mg, yield: 30%).
LCMS:Rt:5.811min；MS m/z(ESI):478.2[M+H].
1 H NMR(400MHz,DMSO-d6)δ11.34(s,1H),11.01(s,1H),9.18(d,J＝4.8Hz,1H),8.72(s,1H),8.17(s,1H)7.68(d,J＝8.0Hz,1H)，7.54-7.52(m,1H),7.29(t,J＝8.0Hz,1H),5.15-5.12(m,1H),3.76-3.72(m,5H),3.46(t,J＝6.8Hz,2H),2.86(d,J＝4.8Hz,3H),2.34(s,3H),2.10-2.07(m,1H),0.83-0.81(m,3H).
EXAMPLE 2 Synthesis of Compound 002
6- (cyclopropanecarboxamide) -4- ((2-methoxy-3- (1- (oxetan-3-yl) -1H-1,2, 4-triazol-3-yl) phenyl) amino) -N-methylpyridazine-3-carboxamide
Synthetic route and specific synthetic steps:
the first step: synthesis of 3- (2-methoxy-3-nitrophenyl) -1- (oxetan-3-yl) -1H-1,2, 4-triazole 002-b
002-a (5.0 g,22.7 mmol), 3-iodo-oxetane (12.5 g,68.1 mmol) and cesium carbonate (22.2 g,68.1 mmol) were added to DMF (40 mL), heated to 100deg.C for 16h, after LCMS detects the disappearance of starting material, the reaction solution was cooled to room temperature and filtered, and the filtrate was purified by reverse phase column chromatography to give 002-b (3.18 g, yield 51%).
LCMS:Rt:1.29min；MS m/z(ESI):277.1[M+H].
1 H NMR(400MHz,CDCl3 )δ8.43(s,1H),8.22(dd,J＝8.0Hz,J＝1.6Hz,1H),7.86(dd,J＝8.0Hz,J＝1.6Hz,1H),7.33(t,J＝8.0Hz,1H),5.66-5.62(m,1H),5.20-5.11(m,4H),3.98(s,3H).
And a second step of: synthesis of 2-methoxy-3- (1- (oxetan-3-yl) -1H-1,2, 4-triazol-3-yl) aniline 002-c
002-b (3.18 g,11.5 mmol) and palladium on carbon (1.2 g, 40%) were added to methanol (150 mL) and the mixture was then heated to 30deg.C under hydrogen and stirred for 5 hours, and LCMS detected the disappearance of starting material. The reaction solution was filtered and concentrated to give 002-c (2.6 g, yield 93%, purity 88%).
LCMS:Rt:1.20min；MS m/z(ESI):247.2[M+H].
And a third step of: synthesis of 6-chloro-4- ((2-methoxy-3- (1- (oxetan-3-yl) -1H-1,2, 4-triazol-3-yl) phenyl) amino) pyridazine-3-carboxylic acid 002-e
002-c (2.6 g,10.6 mmol), lithium 4, 6-dichloropyridazine-3-carboxylate (4.2 g,21.2 mmol) and zinc acetate (3.9 g,21.2 mmol) were added to a mixed solvent of isopropanol (90 mL) and water (30 mL), and the reaction was heated to 85℃and stirred for 18 hours. After the completion of the reaction, the reaction mixture was cooled to room temperature, concentrated under reduced pressure, and the obtained solid was dissolved in DMF and filtered, followed by purification by reverse phase column chromatography (0.01% TFA) to give 002-e (2.1 g, yield 50%).
LCMS:Rt:1.61min；MS m/z(ESI):403.1[M+H].
1 H NMR(400MHz,DMSO-d6)δ11.24(s,1H),8.74(s,1H),7.80(d,J＝8.0Hz,1H),7.64(d,J＝8.0Hz,1H),7.34(t,J＝8.0Hz,1H),7.19(s,1H),5.80-5.76(m,1H),5.00-4.94(m,4H),3.75(s,3H).
Fourth step: synthesis of 6-chloro-4- ((2-methoxy-3- (1- (oxetan-3-yl) -1H-1,2, 4-triazol-3-yl) phenyl) amino) -N-methylpyridazine-3-carboxamide 002-f
002-e (2.1 g,5.2 mmol), methylamine hydrochloride (1.8 g,26.0 mmol), EDCI (1.2 g,6.2 mmol), HOBt (1.2 g,6.2 mmol) and N-methylimidazole (298 mg,3.6 mmol) were added to a mixed solvent of N-methylpyrrolidone (15 mL) and acetonitrile (30 mL), and the reaction solution was heated to 30℃and stirred for 3 hours. After the completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated and purified by reverse phase column chromatography (0.1% TFA) to give 1.1g of a crude product. The crude product was further purified by normal phase column chromatography to give 002-f (235 mg, yield 11.0%).
LCMS:Rt:1.57min；MS m/z(ESI):416.1[M+H].
1 H NMR(400MHz,CD3 OD)δ11.20(s,1H),9.41(d,J＝4.8Hz,1H),8.74(s,1H),7.77(dd,J＝7.6Hz，J＝1.6Hz，1H),7.64(dd,J＝8.0Hz,J＝1.6Hz，1H),7.32(t,J＝8.0Hz,1H),7.23(s,1H),5.80-5.76(m,1H),5.00-4.95(m,4H),3.76(s,3H),2.87(d,J＝4.8Hz,3H).
Fifth step: synthesis of 6- (cyclopropanecarboxamide) -4- ((2-methoxy-3- (1- (oxetan-3-yl) -1H-1,2, 4-triazol-3-yl) phenyl) amino) -N-methylpyridazine-3-carboxamide 002
Pd was taken at room temperature2 (dba)3 (22.0 mg,0.024 mmol), xantPhos (14.0 mg,0.024 mmol), cesium carbonate (156.0 mg,0.48 mmol), cyclopropylamide (41.0 mg,0.48 mmol) and 002-f (100.0 mg,0.24 mmol) were added to dioxane (15 mL). Heated to 110 c and stirred under argon for 18 hours. After the completion of the reaction, the reaction mixture was cooled to room temperature, filtered, concentrated, and purified by DMF (0.1% FA) to give 002 (50.6 mg, yield 45%).
LCMS:Rt:7.14min；MS m/z(ESI):465.3[M+H].
1 H NMR(400MHz,DMSO-d6)δ11.33(s,1H),11.01(s,1H),9.17(q,J＝4.4Hz，1H),8.73(s,1H),8.17(s,1H),7.70(dd,J＝8.0Hz，J＝1.6Hz，1H),7.54(dd,J＝8.0Hz,J＝1.2Hz，1H),7.30(t,J＝8.0Hz,1H),5.81-5.74(m,1H),4.97(d,J＝6.8Hz,4H),3.76(s,3H),2.87(d,J＝4.8Hz,3H),2.11-2.05(m,1H),0.83-0.82(m,4H).
EXAMPLE 3 Synthesis of Compound 003
4- ((2-methoxy-3- (1- (oxy-ethane-3-yl) -1H-1,2, 4-triazol-3-yl) phenyl) amino) -N-methyl-6- (pyrazin-2-ylamino) pyridazine-3-carboxamide
Synthetic route and specific synthetic steps:
002-f (25.0 mg,0.06 mmol) pyrazin-2-amine (28.5 mg,0.30 mmol), pd2 (dba)3 (5.5 mg, 0.006mmol), t-BuBrettPhos (2.9 mg, 0.006mmol), cesium carbonate (58.7 mg,0.18 mmol) was added to 1, 4-dioxane (1.0 mL), and the reaction was heated to 130℃and stirred under argon for 3 hours. The reaction solution was filtered, and the filtrate was concentrated and purified under reduced pressure to give 003 (4.1 mg, yield: 14%).
LCMS:Rt:5.927min；MS m/z(ESI):475.1[M+H].
1 H NMR(400MHz,DMSO-d6)δ11.07(s,1H),10.50(s,1H),9.19(d,J＝4.8Hz,1H),8.99(d,J＝1.2Hz,1H),8.73(s,1H),8.23-8.22(m,1H),8.14(d,J＝2.8Hz,1H),8.04(s,1H),7.71-7.66(m,2H),7.35(t,J＝8.0Hz,1H),5.82-5.75(m,1H),5.00-4.97(m,4H),3.79(s,3H),2.87(d,J＝4.8Hz,3H).
EXAMPLE 4 Synthesis of Compound 004
6- (cyclopropanecarboxamide) -4- ((2-methoxy-3- (1- (thietane-1, 1-dioxide-3-yl) -1H-1,2, 4-triazol-3-yl) phenyl) amino) -N-methylpyridazine-3-carboxamide
Synthetic route and specific synthetic steps:
the first step: synthesis of 3- (2-methoxy-3-nitrophenyl) -1- (thietane-1, 1-dioxide-3-yl) -1H-1,2, 4-triazole 004-b
004-a (300.0 mg,1.36 mmol), thietane-1, 1-dioxide-3-yl-methylsulfonate (544.0 mg,2.72 mmol) and cesium carbonate (1.33 g,4.08 mmol) were added to DMF (10 mL) and heated to 60℃for 16h. After the completion of the reaction, the reaction mixture was cooled to room temperature and then filtered, and the filtrate was purified by reverse phase column chromatography to give 004-b (250 mg, yield 56%).
LCMS:Rt:1.347min；MS m/z(ESI):325.1[M+H].
1 H NMR(400MHz,DMSO-d6)δ8.89(s,1H),8.23(dd,J1 ＝8.0Hz,J2 ＝2.0Hz,1H),7.98(dd,J1 ＝8.0Hz,J2 ＝2.0Hz,1H),7.46(t,J＝8.0Hz,1H),5.59-5.55(m,1H),4.92-4.86(m,2H),4.80-4.74(m,2H)3.87(s,3H).
And a second step of: synthesis of 2-methoxy-3- (1-thietane-1, 1-dioxide-3-yl) -1H-1,2, 4-triazole-3-yl) aniline 004-c
004-b (230 mg,0.710 mmol) and palladium on carbon (40 mg, 40%) were added to methanol (10.0 mL), and the mixture was then heated to 30deg.C under hydrogen atmosphere and stirred for 16 hours. The reaction solution was filtered and concentrated to give 004-c (190 mg, yield 91%).
LCMS:Rt:0.887min；MS m/z(ESI):295.1[M+H].
And a third step of: synthesis of 6-chloro-4- ((2-methoxy-3- (1-thietane-1, 1-dioxide-3-yl) -1H-1,2, 4-triazol-3-yl) phenyl) amino) pyridazine-3-carboxylic acid 004-d
004-c, lithium 4, 6-dichloropyridazine-3-carboxylate (236 mg,1.29 mmol) and zinc acetate (237 mg,1.29 mmol) were added to a mixed solvent of isopropyl alcohol (9.0 mL) and water (3.0 mL), and the reaction solution was heated to 85℃and stirred for 22 hours. After the completion of the reaction, the reaction mixture was cooled to room temperature, concentrated under reduced pressure, and the obtained solid was dissolved in DMF and filtered, followed by purification by reverse phase column chromatography (0.01% TFA) to give 004-d (255 mg, yield 88%).
LCMS:Rt:1.327min；MS m/z(ESI):451.0[M+H].
1 H NMR(400MHz,DMSO-d6)δ8.84(s,1H),7.81(d,J＝8.0Hz，1H),7.64(d,J＝7.6Hz,1H),7.68(s,1H),7.34(t,J＝8.0Hz,2H),5.58-5.53(m,1H),4.92-4.86(m,2H),4.79-4.74(m,2H),3.75(s,3H).
Fourth step: synthesis of 6-chloro-4- ((2-methoxy-3- (1- (1-thietane-1, 1-dioxide-3-yl) -1H-1,2, 4-triazol-3-yl) phenyl) amino) -N-methylpyridazine-3-carboxamide 004-e
004-d (50.0 mg,0.111 mmol), methylamine hydrochloride (37.0 mg,0.56 mmol), EDCI (25.0 mg,0.133 mmol), HOBt (18.0 mg,0.13 mmol) and N-methylimidazole (6.4 mg,0.07 mmol) were added to a mixed solvent of N-methylpyrrolidone (2.0 mL) and acetonitrile (5.0 mL), and the reaction solution was heated to 30℃and stirred for 18 hours. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated and purified by reverse phase column chromatography (0.1% tfa) to give 004-e (15.0 mg, yield 29%).
LCMS:Rt:1.337min；MS m/z(ESI):464.1[M+H].
1 H NMR(400MHz,DMSO-d6)δ11.18(s,1H),9.41(d,J＝4.8Hz，1H),8.83(s,1H),7.77(dd,J1 ＝8.0Hz,J2 ＝1.6Hz,1H),7.65(dd,J1 ＝8.0Hz,J2 ＝1.2Hz,1H),7.32(t,J＝8.0Hz,1H),7.23(s,1H),5.57-5.53(m,1H),4.91-4.85(m,2H),4.79-4.74(m,2H),3.76(s,3H),2.87(d,J＝4.4Hz,3H).
Fifth step: synthesis of 6- (cyclopropanecarboxamide) -4- ((2-methoxy-3- (1- (oxetan-3-yl) -1H-1,2, 4-triazol-3-yl) phenyl) amino) -N-methylpyridazine-3-carboxamide 004
Pd was taken at room temperature2 (dba)3 (7.0 mg, 0.0070 mmol), xantPhos (4.0 mg, 0.0070 mmol), DBU (20.0 mg,0.130 mmol), cyclopropylamide (28.0 mg,0.325 mmol) and 004-e (30.0 mg,0.065 mmol) were added to dioxane (5.0 mL). Heated to 110 c and stirred under argon for 18 hours. The reaction solution was cooled to room temperature, filtered, concentrated, and purified by DMF (0.1% FA) to give 004 (12.5 mg, yield 37%). LCMS, rt 5.667min; MS m/z(ESI):513.1[M+H].
1 H NMR(400MHz,DMSO-d6)δ11.34(s,1H),11.03(s,1H),9.17(d,J＝4.4Hz，1H),8.82(s,1H),8.16(s,1H),7.71(dd,J＝7.6Hz，J＝1.2Hz，1H),7.55(dd,J＝7.6Hz,J＝1.6Hz，1H),7.30(t,J＝8.0Hz,1H),5.57-5.53(m,1H),4.91-4.85(m,2H),4.79-4.74(m,2H),3.76(s,3H),2.86(d,J＝4.8Hz,3H),2.10-2.07(m,1H),0.83-0.82(m,4H).
Biological Activity and related Property test cases
Test example 1: inhibition of STAT3 phosphorylation in Jurkat cells
Experimental principle: after Jurkat cells were co-incubated with the compound and the stimulator, fluorescence energy transfer was detected by Homogeneous Time Resolved Fluorescence (HTRF) method using Cisbio corporation STAT3 phosphorylation assay kit, reflecting inhibition of phosphorylation.
Experimental instrument:
| instrument for measuring and controlling the intensity of light | Branding | Model number |
| Biological safety cabinet | Thermo Scientific | 1300Series A2 |
| Centrifugal machine | Eppendorf | 5702 |
| CO2 Incubator | Thermo Scientific | 1300SERIES A2 |
| Cell counter | Invitrogen | C10281 |
| Envision | Perkin Elmer | 2014 |
Experimental materials:
the experimental method comprises the following steps:
planting 9 μl/well Jurkat cells in 384 well plates, with HBSS as solution and cell density of 100000/well, adding 60 nL/well of test compound, and 5% CO at 37deg.C2 Is incubated for 5 minutes in the incubator. Then 3. Mu.L/well of stimulator IFN was added at a final concentration of 250ng/mL and incubated for 15 minutes. The extent of STAT3 phosphorylation was detected using the pSTAT3 kit from Cisbio corporation, and finally the fluorescence signals at 665nm and 615nm were read on an Envision microplate reader to calculate the median inhibitory concentration (IC50 ) See table 1.
TABLE 1 inhibition of STAT3 phosphorylation in Jurkat cells by the Compounds of the application
| Examples compound numbering | Jurkat pStat3 IC50 (nM) |
| 001 | 15.9 |
| 002 | 191.0 |
| 003 | 5.7 |
| 004 | 40.0 |
Test example 2 determination of the metabolic stability of the Compounds of the application in liver microsomes
The metabolic stability of the compounds of the application in liver microsomes was determined using the following test methods.
1. Test material and instrument
1. Liver microsome source: human liver microsome (Corning 452117) CD-1 mouse liver microsome (XENOTECH M1000)
2.Na2 HPO4 (Tianjin city light complex fine chemical engineering institute 20180130)
3.KH2 PO4 (Tianjin city light complex fine chemical engineering institute 20180920)
4.MgCl2 (Tianjin city light complex fine chemical engineering institute 20191216)
5.NADPH(Solarbio 1216C022)
6. Positive control compound verapamil (Sigma MKBV 4993V)
7.AB Sciex Triple Quad 4000 liquid chromatography-mass spectrometer
2. Test procedure
Preparation of 1.100mM Phosphate Buffer (PBS): 7.098g of Na is weighed2 HPO4 500mL of pure water was added for ultrasonic dissolution as solution A. 3.400g KH was weighed out2 PO4 250mL of pure water was added for ultrasonic dissolution as solution B. Solution A was placed on a stirrer and solution B was slowly added until the pH reached 7.4 to prepare 100mM PBS buffer.
2. Preparation of the reaction System
The reaction system was prepared according to the following table
3. The reaction was pre-incubated in a 37℃water bath for 10 minutes. To the reaction system, 40. Mu.L of 10mM NADPH solution (NADPH was dissolved in 100mM phosphate buffer) was added, and the final concentration of NADPH was 1mM. As a negative control, 40. Mu.L of phosphate buffer was used instead of NADPH solution. The effect of the negative control is to exclude the effect of the chemical stability of the compound itself.
4. To the reaction system, 4. Mu.L of 100. Mu.M of the compound of the present application and verapamil as a positive control compound were added to initiate the reaction, and the final concentration of the compound was 1. Mu.M.
5. After 0.5, 15, 30, 45 and 60 minutes, the vortex shaker was thoroughly mixed, 50 μl of each incubation sample was removed and the reaction was quenched with 4 times glacial acetonitrile containing an internal standard. The sample was centrifuged at3,220 g for 45 minutes. After centrifugation, 90. Mu.L of the supernatant was transferred to a sample plate, and 90. Mu.L of ultrapure water was added and mixed for LC-MS/MS analysis.
All data were calculated by Microsoft Excel software. The peak area was detected by extracting the ion spectrum. The in vitro half-life (t1/2 )。
Half-life in vitro (t)1/2 ) Calculated by slope:
in vitro t1/2 ＝0.693/k
the in vitro intrinsic clearance (in. Mu.L/min/mg protein) was calculated using the following formula:
in vitro CLint ＝k×volume of incubation(μL)/amount of proteins(mg)
t calculated by the above formula1/2 And CLint The values are shown in Table 2.
TABLE 2 half-life and intrinsic clearance values in liver microsomes of compounds of the application