WO2021136431A1 - Benzo[d][1,2,3]triazole ether compound - Google Patents

Benzo[d][1,2,3]triazole ether compound Download PDF

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Publication number
WO2021136431A1
WO2021136431A1 PCT/CN2020/141640 CN2020141640W WO2021136431A1 WO 2021136431 A1 WO2021136431 A1 WO 2021136431A1 CN 2020141640 W CN2020141640 W CN 2020141640W WO 2021136431 A1 WO2021136431 A1 WO 2021136431A1
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compound
pharmaceutically acceptable
acceptable salt
cells
synthesis
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PCT/CN2020/141640
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French (fr)
Chinese (zh)
Inventor
张国利
王勇
罗云富
苏胜
陈曙辉
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南京明德新药研发有限公司
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Publication of WO2021136431A1 publication Critical patent/WO2021136431A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4155—1,2-Diazoles non condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/38—Drugs for disorders of the endocrine system of the suprarenal hormones
    • A61P5/44—Glucocorticosteroids; Drugs increasing or potentiating the activity of glucocorticosteroids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C335/00—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C335/04—Derivatives of thiourea
    • C07C335/16—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C335/18—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/16—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms condensed with carbocyclic rings or ring systems
    • C07D249/18—Benzotriazoles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings

Definitions

  • the present invention relates to a series of benzo[d][1,2,3] triazole ether compounds, and specifically relates to compounds represented by formula (I) and pharmaceutically acceptable salts thereof.
  • Rheumatoid Arthritis is a chronic inflammatory, "systemic" autoimmune disease.
  • the joint manifestations of early rheumatoid arthritis are often difficult to distinguish from other types of inflammatory arthritis.
  • Rheumatoid arthritis has more characteristic signs such as joint erosions, rheumatoid nodules and other extra-articular manifestations.
  • Rheumatoid arthritis affects women more than men (3:1), and the age of onset is between 30-55 years old.
  • the pathogenesis of rheumatoid arthritis is very complicated. The main reason is that autoantigens are presented to activated CD4+ T cells by MHC-II positive antigen-presenting cells (APC) to initiate specific immunity. Response: At the same time activated T cells, macrophages, etc. migrate to the synovium, increasing the secretion of various inflammatory cytokines such as TNF ⁇ , IL-1 and IL-6, infiltrating the lubricating membrane joints, leading to corresponding arthritis symptoms.
  • MHC-II positive antigen-presenting cells APC
  • T cells, macrophages, etc. migrate to the synovium, increasing the secretion of various inflammatory cytokines such as TNF ⁇ , IL-1 and IL-6, infiltrating the lubricating membrane joints, leading to corresponding arthritis symptoms.
  • Glucocorticoids are widely used to treat inflammation and immune diseases for decades, including: rheumatoid arthritis, asthma, chronic obstructive pulmonary disease (COPD), osteoarthritis, rheumatic fever, allergic rhinitis, systemic Lupus erythematosus, Crohn’s disease, inflammatory bowel disease, and ulcerative colitis.
  • Glucocorticoid binds to the glucocorticoid receptor (GR), enters the nucleus, affects gene transcription (activation and inhibition), and reduces the production of inflammatory factors.
  • Glucocorticoid receptor is a member of the conserved nuclear receptor superfamily and belongs to nuclear transcription factors. It is widely present in various tissues and cells of the body. Almost all cells are its target cells. Metabolism and immune function play an important regulatory role. GC usually has serious and irreversible side effects, such as: osteoporosis, hyperglycemia, diabetes, hypertension, muscle atrophy, Cushing syndrome, etc., which severely limits the use of GC in chronic diseases.
  • GR ligands which can selectively induce transcriptional inhibition without significant transcriptional activation, reduce the risk of systemic side effects, and maintain anti-inflammatory activity.
  • selective glucocorticoid receptor modulators SGRM
  • Selective glucocorticoid receptor modulators are different from GC. When combined with GR, they trigger complete transcriptional inhibition and only trigger partial transcriptional activation, which can control related side effects while maintaining anti-inflammatory activity.
  • the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
  • Ring A is selected from 5-6 membered heteroaryl and [1,2,4]triazolo[4,3-A]pyridyl, the 5-6 membered heteroaryl and [1,2,4] tri oxazolo [4,3-A] pyridyl optionally substituted with 1, 2 or 3 substituents R a;
  • R 1 is independently selected from H, F, Cl, Br and I;
  • R 2 is selected from C 1-6 alkyl, and the C 1-6 alkyl is optionally substituted with 1, 2 or 3 R b ;
  • n 0, 1, 2 and 3;
  • R a is each independently selected from H, F, Cl and C 1-3 alkyl, the C 1-3 alkyl is optionally substituted with 1, 2 or 3 R;
  • R b is independently selected from H, F, Cl, Br and I;
  • R is independently selected from H, F and Cl
  • the carbon atom with "*" is a chiral carbon atom, which exists in the form of (R) or (S) single enantiomer or rich in one enantiomer;
  • the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
  • Ring A is selected from 5-membered heteroaryl, pyridyl, pyridazinyl, 1H-pyridin-2-onyl and [1,2,4]triazolo[4,3-A]pyridyl, the 5-membered Heteroaryl, pyridyl, pyridazinyl, 1H-pyridin-2-onyl and [1,2,4]triazolo[4,3-A]pyridyl are optionally substituted by 1, 2 or 3 Ra replace;
  • R 1 is independently selected from H, F, Cl, Br and I;
  • R 2 is selected from C 1-6 alkyl, and the C 1-6 alkyl is optionally substituted with 1, 2 or 3 R b ;
  • n 0, 1, 2 and 3;
  • R a is each independently selected from H, F, Cl and C 1-3 alkyl, the C 1-3 alkyl is optionally substituted with 1, 2 or 3 R;
  • R b is independently selected from H, F, Cl, Br and I;
  • R is independently selected from H, F and Cl
  • the carbon atom with "*" is a chiral carbon atom, which exists in the form of (R) or (S) single enantiomer or rich in one enantiomer;
  • the above-mentioned compound or a pharmaceutically acceptable salt thereof is selected from,
  • ring A, R 1 , R 2 and m are as defined in the present invention.
  • R a is independently selected from H, CH 3, CH 2 CH 3, CH (CH 3) 2, CF 3, CHF 2 , and CH 2 F, the other variables are as defined in the present invention .
  • the above-mentioned ring A is selected from pyrazolyl, 1H-pyridin-2-onyl, [1,2,4]triazolo[4,3-A]pyridyl, furyl, thiazole Group, pyridyl and pyridazinyl, the pyrazolyl, 1H-pyridin-2-one, [1,2,4]triazolo[4,3-A]pyridyl, furyl, thiazolyl, pyridyl and pyridazinyl optionally substituted with 1, 2 or 3 substituents R a, the other variables are as defined in the present invention.
  • the above-mentioned ring A is selected from Said 1, 2 or 3 substituents R a, the other variables are as defined in the present invention.
  • the above-mentioned ring A is selected from Other variables are as defined in the present invention.
  • the above-mentioned ring A is selected from Other variables are as defined in the present invention.
  • R 1 is independently selected from H and F, and other variables are as defined in the present invention.
  • the above-mentioned structural unit are independently selected from Other variables are as defined in the present invention.
  • R 2 is selected from C 1-3 alkyl, and the C 1-3 alkyl is optionally substituted with 1, 2 and 3 R b , and other variables are as defined in the present invention.
  • R 2 is selected from CH 3 and The CH 3 and Optionally substituted by 1, 2 and 3 R b , and other variables are as defined in the present invention.
  • R 2 is selected from CH 3 and Other variables are as defined in the present invention.
  • the present invention also provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
  • Ring A is selected from pyrazolyl, 1H-pyridin-2-one and [1,2,4]triazolo[4,3-A]pyridyl, the pyrazolyl, 1H-pyridin-2-one group, and [1,2,4] triazolo [4,3-A] pyridyl optionally substituted with 1, 2 or 3 substituents R a;
  • R 1 is independently selected from H, F, Cl, Br and I;
  • R 2 is selected from C 1-6 alkyl, and the C 1-6 alkyl is optionally substituted with 1, 2 or 3 R b ;
  • n 0, 1, 2 and 3;
  • R a is each independently selected from H, F, Cl and C 1-3 alkyl, the C 1-3 alkyl is optionally substituted with 1, 2 or 3 R;
  • R b is independently selected from H, F, Cl, Br and I;
  • R is independently selected from H, F and Cl
  • the carbon atom with "*" is a chiral carbon atom and exists in the form of (R) or (S) single enantiomer or enriched in one enantiomer.
  • the above-mentioned compound or a pharmaceutically acceptable salt thereof is selected from,
  • ring A, R 1 , R 2 and m are as defined in the present invention.
  • R a is independently selected from H and CH 3, the other variables are as defined in the present invention.
  • the above-mentioned ring A is selected from Said 1, 2 or 3 substituents R a, the other variables are as defined in the present invention.
  • the above-mentioned ring A is selected from Other variables are as defined in the present invention.
  • the above-mentioned ring A is selected from Other variables are as defined in the present invention.
  • R 1 is independently selected from H and F, and other variables are as defined in the present invention.
  • R 2 is selected from C 1-3 alkyl, and the C 1-3 alkyl is optionally substituted with 1, 2 and 3 R b , and other variables are as defined in the present invention.
  • R 2 is selected from CH 3 and The CH 3 and Optionally substituted by 1, 2 and 3 R b , and other variables are as defined in the present invention.
  • R 2 is selected from CH 3 and Other variables are as defined in the present invention.
  • the above-mentioned compound or a pharmaceutically acceptable salt thereof is selected from,
  • T 1 is selected from CH and N;
  • R 1, R 2, R a and m are as defined in the present invention.
  • the above-mentioned compound or a pharmaceutically acceptable salt thereof is selected from,
  • R 1 , R 2 and m are as defined in the present invention.
  • the present invention provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof,
  • the present invention also provides the application of the above-mentioned compound or its pharmaceutically acceptable salt in the preparation of drugs for glucocorticoid receptor-related diseases.
  • the aforementioned glucocorticoid receptor-related disease refers to rheumatoid arthritis.
  • the compound of the present invention has good hMMP1 transcriptional inhibition activity, comparable MMTV transcriptional activation activity, strong anti-inflammatory activity at the cellular level, and moderate CYP3A4 inhibition.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms that are within the scope of reliable medical judgment and are suitable for use in contact with human and animal tissues. , Without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to a salt of the compound of the present invention, which is prepared from a compound with specific substituents discovered in the present invention and a relatively non-toxic acid or base.
  • a base addition salt can be obtained by contacting the compound with a sufficient amount of base in a pure solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salt or similar salts.
  • the acid addition salt can be obtained by contacting the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, the organic acid includes, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid; also include salts of amino acids (such as arginine, etc.) , And salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and
  • the pharmaceutically acceptable salt of the present invention can be synthesized from the parent compound containing acid or base by conventional chemical methods. In general, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of appropriate base or acid in water or organic solvent or a mixture of both.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers Isomers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomers or diastereomer-enriched mixtures, all of these mixtures belong to this Within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All these isomers and their mixtures are included in the scope of the present invention.
  • the compound of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms constituting the compound.
  • compounds can be labeled with radioisotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C).
  • deuterium can be substituted for hydrogen to form deuterated drugs.
  • the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon.
  • deuterated drugs can reduce toxic side effects and increase drug stability. , Enhance the efficacy, extend the biological half-life of drugs and other advantages. All changes in the isotopic composition of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by substituents, and may include deuterium and hydrogen variants, as long as the valence of the specific atom is normal and the substituted compound is stable of.
  • oxygen it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups.
  • optionally substituted means that it can be substituted or unsubstituted. Unless otherwise specified, the type and number of substituents can be arbitrary on the basis that they can be chemically realized.
  • any variable such as R
  • its definition in each case is independent.
  • the group can optionally be substituted with up to two Rs, and R has independent options in each case.
  • combinations of substituents and/or variants thereof are only permitted if such combinations result in stable compounds.
  • linking group When the number of a linking group is 0, such as -(CRR) 0 -, it means that the linking group is a single bond.
  • any one or more sites of the group can be connected to other groups through chemical bonds.
  • the connection method of the chemical bond is not positioned, and there is a H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will correspondingly decrease with the number of chemical bonds connected to become the corresponding valence number ⁇ The group.
  • the chemical bond between the site and other groups can be a straight solid bond Straight dashed key Or wavy line Said.
  • the straight solid bond in -OCH 3 means that it is connected to other groups through the oxygen atom in the group;
  • the straight dashed bond in indicates that the two ends of the nitrogen atom in the group are connected to other groups;
  • the wavy line in indicates that the phenyl group is connected to other groups through the 1 and 2 carbon atoms;
  • C 1-6 alkyl is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 6 carbon atoms.
  • the C 1-6 alkyl group includes C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-4 , C 6 and C 5 alkyl groups, etc.; it may Is monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine).
  • C 1-6 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl) , S-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl, etc.
  • C 1-3 alkyl is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 3 carbon atoms.
  • the C 1-3 alkyl group includes C 1-2 and C 2-3 alkyl groups, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine) .
  • Examples of C 1-3 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.
  • 5-6 membered heteroaryl ring and “5-6 membered heteroaryl group” can be used interchangeably in the present invention.
  • the term “5-6 membered heteroaryl group” means a ring consisting of 5 to 6 ring atoms. It is composed of a monocyclic group with a conjugated ⁇ -electron system, in which 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from O, S and N, and the rest are carbon atoms. Where the nitrogen atom is optionally quaternized, the nitrogen and sulfur heteroatoms may optionally be oxidized (ie NO and S(O) p , p is 1 or 2).
  • the 5-6 membered heteroaryl group can be attached to the rest of the molecule through a heteroatom or a carbon atom.
  • the 5-6 membered heteroaryl group includes 5-membered and 6-membered heteroaryl groups.
  • Examples of the 5-6 membered heteroaryl include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrrolyl, etc.) Azolyl, etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5- Oxazolyl, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl and 4H-1, 2,
  • C n-n+m or C n -C n+m includes any specific case of n to n+m carbons, for example, C 1-12 includes C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , and C 12 , including any range from n to n+m, for example, C 1-12 includes C 1-3 , C 1-6 , C 1-9 , C 3-6 , C 3-9 , C 3-12 , C 6-9 , C 6-12 , and C 9-12, etc.; similarly, from n to n +m member means that the number of atoms in the ring is from n to n+m, for example, 3-12 membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, 9-membered
  • leaving group refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (for example, a nucleophilic substitution reaction).
  • representative leaving groups include triflate; chlorine, bromine, iodine; sulfonate groups, such as mesylate, tosylate, p-bromobenzenesulfonate, p-toluenesulfonic acid Esters, etc.; acyloxy groups, such as acetoxy, trifluoroacetoxy and the like.
  • protecting group includes, but is not limited to, "amino protecting group", “hydroxy protecting group” or “thiol protecting group”.
  • amino protecting group refers to a protecting group suitable for preventing side reactions at the amino nitrogen position.
  • Representative amino protecting groups include, but are not limited to: formyl; acyl, such as alkanoyl (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl, such as tert-butoxycarbonyl (Boc) ; Arylmethyloxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethyloxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-di -(4'-Methoxyphenyl)methyl; silyl groups, such as trimethylsilyl (TMS) and tert-butyldi
  • hydroxy protecting group refers to a protecting group suitable for preventing side reactions of the hydroxyl group.
  • Representative hydroxy protecting groups include but are not limited to: alkyl groups, such as methyl, ethyl, and tert-butyl; acyl groups, such as alkanoyl groups (such as acetyl); arylmethyl groups, such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyl Dimethylsilyl (TBS) and so on.
  • alkyl groups such as methyl, ethyl, and tert-butyl
  • acyl groups such as alkanoyl groups (such as acetyl)
  • arylmethyl groups such as benzyl (Bn), p-methyl Oxybenzyl (P
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and those well known to those skilled in the art Equivalent alternatives, preferred implementations include but are not limited to the embodiments of the present invention.
  • the structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art.
  • the single crystal X-ray diffraction method uses the Bruker D8 venture diffractometer to collect the diffraction intensity data of the cultured single crystal.
  • the light source is CuK ⁇ radiation
  • the scanning method After scanning and collecting relevant data, the direct method (Shelxs97) is further used to analyze the crystal structure to confirm the absolute configuration.
  • the solvent used in the present invention is commercially available.
  • the present invention uses the following abbreviations: THF stands for tetrahydrofuran; DCM stands for dichloromethane; DMF stands for N,N-dimethylformamide; n-BuLi stands for n-butyl lithium; NaH stands for sodium hydrogen; TFA stands for trifluoroacetic acid .
  • HOBt stands for 1-hydroxybenzotriazole
  • EDCI stands for 1-ethyl-(3-dimethylaminopropyl) carbodiimide
  • TBTU stands for O-benzotriazole-N,N,N', N'-tetramethylurea tetrafluoroborate
  • HATU stands for 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate
  • HBTU stands for O- Benzotriazole-tetramethylurea hexafluorophosphate
  • L-Glutamine stands for L-glutamine
  • TR stands for transcriptional inhibition
  • TA stands for transcriptional activation
  • PMA stands for phorbol 12-myristate 13-acetate
  • DPBS stands for and Dulbecco's phosphate buffered solution
  • Trypsin stands for trypsin
  • DMEM stands for modified Eagle's medium
  • aqueous phase was extracted with ethyl acetate (5L ⁇ 2), the organic phases were combined, washed with saturated brine (10L), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure.
  • Petroleum ether (8L) was added to the crude product, stirred at room temperature for 16 hours, and filtered. The filter cake was rinsed with petroleum ether (5 L), and the solid was collected to obtain compound CC-1-4.
  • Tetrahydrofuran 50mL was added to a dry three-necked flask, and after replacing it with nitrogen three times, the temperature of the system was reduced to -15°C, and the compound phenylmagnesium bromide (19.51g, 107.63mmol) was added, and the compound CC-1-2 (10g, 43.05mmol) was added to tetrahydrofuran (150mL), the above system was slowly added (the temperature was maintained at about -5°C), the addition was completed, and the reaction was stirred at 25°C for 12 hours.
  • reaction solution dropped to room temperature, it was poured into water (30 mL) and ammonia (10 mL), and extracted with ethyl acetate (30 mL x 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure.
  • the crude product was separated and purified by preparative HPLC (column type: Xtimate C18 100*30mm*3 ⁇ m; mobile phase: [H 2 O(0.04%HCl)-ACN]; B(ACN)%: 47%-77%, 8min) to obtain WX015 .
  • Experimental example 1 In vitro detection compounds inhibit hMMP1 transcription activity under the luciferase reporter gene screening system
  • the human MMP-1 promoter region (contains two AP-1 binding sites and two PEA3 sites, a total of 249 bp, gene bank catalog #AF023338) was cloned upstream of the luciferase reporter gene.
  • the hMMP-1 promoter reporter gene was constructed and transfected into Hela cells, so that the production of luciferase could be easily detected.
  • the stable recombinant hMMP-1/luciferase cell line was used for the development and verification of this experiment.
  • the cell concentration is 5 ⁇ 10 3 cells/well.
  • the cell plate is placed in a 37°C 5% CO 2 incubator for 18-24 hours.
  • test compound was diluted to 30 mM with DMSO, and stored in aliquots in a refrigerator at -80°C for later use.
  • the final DMSO concentration is 0.1%. PMA needs to be protected from light during use.
  • Negative control 10nM PMA (0.1% DMSO)
  • Test compound the highest concentration is 1000 nM, diluted 4 times, 10 wells in total, repeat.
  • Dexamethasone the highest concentration is 1000nM, diluted 4 times, a total of 10 wells, repeat.
  • the promoter of mouse mammary tumor virus contains a specific binding site that activates GR (GREs).
  • GREs GR
  • a reporter gene luciferase was inserted after the MMTV promoter, and the structure was expressed in a stable manner in the genome of the HeLa cell line.
  • the test compound is used to activate the MMTV promoter, induce the expression of luciferase, and detect its activity by luminescence measurement.
  • the cell plate is placed in a 37°C 5% CO 2 incubator for 18-24 hours.
  • test compound was diluted to 30 mM with DMSO, and stored in aliquots in a refrigerator at -80°C for later use.
  • the final experimental concentration of DMSO is 0.1%.
  • Inoculation of cells Fresh cells were inoculated into a 384 white transparent bottom experimental plate at 4 ⁇ 10 3 cells/30 ⁇ L/well, and cultured in a 37°C 5% CO 2 incubator for 24 hours.
  • Test compound the highest concentration is 1000 nM, diluted 4 times, 10 wells in total, repeat.
  • Dexamethasone the highest concentration is 1000nM, diluted 4 times, a total of 10 wells, repeat.
  • IC 50 is absolute IC 50
  • EC 50 is absolute EC 50
  • Effect represents the maximum effect value.
  • the compound of the present invention shows very good transcription inhibitory activity and equivalent transcription activation activity.
  • mice peripheral blood mononuclear cells
  • PRMI 1640 medium (Invitrogen-11875093, lot 2003787)
  • Fetal Bovine Serum (FBS) (Gibco-10091148, lot 1989478)
  • PBMC cells AllCells, Cat.PB006F-C, lot LP190225B: RPMI 1640 + 10% FBS (Gibico) + 1% PS
  • the compound was diluted from a storage concentration of 10 mM with 100% DMSO to 1 mM, and as the first spot, it was diluted 3 times with 100% DMSO for 8 spots.
  • the second step is 125-fold dilution with serum-free medium. At this time, the DMSO concentration is 0.8%. Then transfer 16.8 ⁇ L of the compound that has been diluted with culture medium to a 100 ⁇ L cell plate, at which time the concentration of DMSO is 0.114%. After the compound is added, the cell plate is placed in a 37°C, 5% CO 2 incubator and incubated for 1 hour.
  • the first step is to dilute LPS with ultrapure water to a storage concentration of 1 mg/mL.
  • the storage concentration of LPS is diluted to 1 ⁇ g/mL with serum-free medium.
  • the third step is 1666.666 times dilution with medium without serum. Then transfer 16.8 ⁇ L of LPS that has been diluted with culture medium to 116.8 ⁇ L of cell plate. At this time, the final concentration of DMSO is 0.1%. After adding LPS, place the cell plate in a 37°C, 5% CO 2 incubator and incubate 18 Hours.
  • Inhibition rate (1-(original value-HPE average)/(ZPE average-HPE average)) ⁇ 100%
  • ZPE 0% inhibition (75pg/mL LPS, 0.1% DMSO)
  • HPE 100% inhibition (without LPS, 0.1% DMSO).
  • Z factor 1-(3 ⁇ (ZPE standard deviation + HPE standard deviation)/(ZPE average value-HPE average value))
  • Model 205 in XLfit statistical software was used for data analysis. Taking the concentration as the abscissa and the inhibition rate as the ordinate, the IC 50 calculation formula is: using a 4-parameter logistic dose-response equation, the concentration and the inhibition rate (%) of the tested compound are plotted, and the required 50% inhibition is determined Compound concentration (IC 50 ).
  • IC 50 is absolute IC 50 . *Test Top concentration 500nM, 3 times dilution, 8 concentration; the rest test Top concentration 1000nM, 3 times dilution, 8 concentration. Top (%) maximum effect value
  • the compound of the present invention shows strong anti-inflammatory activity at the cellular level.
  • the purpose of the research project is to use a 5-in-1 probe substrate of CYP isoenzymes to evaluate the inhibitory effect of the test product on human liver microsomal cytochrome P450 isoenzymes (CYP3A4).
  • HLM human liver microsomes
  • AZD9567 has a moderate inhibitory effect on CYP3A4.
  • Compounds WX002 and WX004 significantly improved the inhibition of CYP3A4.

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Abstract

Disclosed is a series of benzo[d][1,2,3]triazole ether compounds, and specifically disclosed are a compound shown in formula (I) and pharmaceutically acceptable salts thereof.

Description

苯并[d][1,2,3]三唑醚类化合物Benzo[d][1,2,3]triazole ether compounds
本发明主张如下优先权：The present invention claims the following priority:
申请号：CN201911420729.3，申请日：2019年12月31；Application number: CN201911420729.3, application date: December 31, 2019;
申请号：CN202011402983.3，申请日：2020年12月02。Application number: CN202011402983.3, application date: December 02, 2020.
技术领域Technical field
本发明涉及一系列苯并[d][1,2,3]三唑醚类化合物，具体涉及了式(I)所示化合物及其药学上可接受的盐。The present invention relates to a series of benzo[d][1,2,3] triazole ether compounds, and specifically relates to compounds represented by formula (I) and pharmaceutically acceptable salts thereof.
背景技术Background technique
类风湿关节炎(Rheumatoid Arthritis，RA)是一种慢性炎症性、“系统性”自身免疫性疾病，早期类风湿关节炎的关节表现往往很难与其他类型的炎性关节炎区分。类风湿关节炎更具特征性的体征，如关节侵蚀，类风湿结节和其他关节外表现。类风湿性关节炎对女性的影响大于男性(3:1)，发病年龄在30-55岁之间。Rheumatoid Arthritis (RA) is a chronic inflammatory, "systemic" autoimmune disease. The joint manifestations of early rheumatoid arthritis are often difficult to distinguish from other types of inflammatory arthritis. Rheumatoid arthritis has more characteristic signs such as joint erosions, rheumatoid nodules and other extra-articular manifestations. Rheumatoid arthritis affects women more than men (3:1), and the age of onset is between 30-55 years old.
类风湿关节炎的发病机制十分复杂，主要是自身抗原被主要组织相容性复合体Ⅱ(MHC-Ⅱ)型阳性的抗原呈递细胞(APC)呈递给活化的CD4+T细胞，启动特异性免疫应答；同时活化的T细胞、巨噬细胞等向滑膜迁移，使多种炎性细胞因子如TNFα、IL-1和IL-6等分泌增多，浸润滑膜关节，导致相应关节炎症状。The pathogenesis of rheumatoid arthritis is very complicated. The main reason is that autoantigens are presented to activated CD4+ T cells by MHC-Ⅱ positive antigen-presenting cells (APC) to initiate specific immunity. Response: At the same time activated T cells, macrophages, etc. migrate to the synovium, increasing the secretion of various inflammatory cytokines such as TNFα, IL-1 and IL-6, infiltrating the lubricating membrane joints, leading to corresponding arthritis symptoms.
糖皮质激素(GC)广泛用于治疗炎症和免疫疾病长达几十年，包括：类风湿性关节炎、哮喘、慢性阻塞性肺病(COPD)、骨关节炎、风湿热、过敏性鼻炎、系统性红斑狼疮、克罗恩病、炎性肠病以及溃疡性结肠炎。Glucocorticoids (GC) are widely used to treat inflammation and immune diseases for decades, including: rheumatoid arthritis, asthma, chronic obstructive pulmonary disease (COPD), osteoarthritis, rheumatic fever, allergic rhinitis, systemic Lupus erythematosus, Crohn’s disease, inflammatory bowel disease, and ulcerative colitis.
糖皮质激素(GC)结合糖皮质激素受体(GR)，进入细胞核影响基因转录(激活和抑制)，减少炎症因子的产生。糖皮质激素受体是保守的核受体超家族中的一员，属于核转录因子，广泛存在于机体各种组织细胞中，几乎所有细胞都是它的靶细胞，对机体的发育、生长、代谢以及免疫功能等起着重要调节作用。GC通常具有严重且不可逆的副作用，如：骨质疏松、高血糖、糖尿病、高血压、肌肉萎缩、库兴综合征等，严重限制了GC在慢性疾病中使用。Glucocorticoid (GC) binds to the glucocorticoid receptor (GR), enters the nucleus, affects gene transcription (activation and inhibition), and reduces the production of inflammatory factors. Glucocorticoid receptor is a member of the conserved nuclear receptor superfamily and belongs to nuclear transcription factors. It is widely present in various tissues and cells of the body. Almost all cells are its target cells. Metabolism and immune function play an important regulatory role. GC usually has serious and irreversible side effects, such as: osteoporosis, hyperglycemia, diabetes, hypertension, muscle atrophy, Cushing syndrome, etc., which severely limits the use of GC in chronic diseases.
目前已经找到GR配体的实例，可以选择性地诱导转录抑制而没有显著的转录激活，能降低全身性副作用的风险，同时维持抗炎活性，我们称之为选择性糖皮质激素受体调节剂(SGRM)。选择性糖皮质激素受体调节剂(SGRM)与GC不同，在结合GR时，它们在引发完全的转录抑制，只引发部分的转录激活，进而可以在保持抗炎活性的同时控制相关副作用。At present, examples of GR ligands have been found, which can selectively induce transcriptional inhibition without significant transcriptional activation, reduce the risk of systemic side effects, and maintain anti-inflammatory activity. We call them selective glucocorticoid receptor modulators. (SGRM). Selective glucocorticoid receptor modulators (SGRM) are different from GC. When combined with GR, they trigger complete transcriptional inhibition and only trigger partial transcriptional activation, which can control related side effects while maintaining anti-inflammatory activity.
Lena Ropa,et al,J.Med.Chem.2018,61,1785-1799报道了化合物AZD9567，具有抗炎作用的药效，以及溶解度差、血浆清除率高等理化性质，适宜口服或吸入给药。但是数据同时也显示其基因转录的抑制活性不够令人满意，抗炎效果不够理想，同时存在高血糖和骨质疏松等糖皮质激素类药物的 副作用，因此需要开发同时具备良好的基因转录的抑制活性和一般的基因转录激活活性的化合物，在提高抗炎活性的同时降低副作用。Lena Ropa, et al, J. Med. Chem. 2018, 61, 1785-1799 reported that the compound AZD9567 has anti-inflammatory effects, physicochemical properties such as poor solubility and high plasma clearance, and is suitable for oral or inhaled administration. However, the data also shows that its gene transcription inhibitory activity is not satisfactory enough, the anti-inflammatory effect is not ideal, and there are side effects of glucocorticoid drugs such as hyperglycemia and osteoporosis. Therefore, it is necessary to develop a good gene transcription inhibitor. Active and general gene transcription activating compounds can improve anti-inflammatory activity while reducing side effects.
Figure PCTCN2020141640-appb-000001
Figure PCTCN2020141640-appb-000001
发明内容Summary of the invention
本发明提供了式(I)所示化合物或其药学上可接受的盐，The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
Figure PCTCN2020141640-appb-000002
Figure PCTCN2020141640-appb-000002
其中，among them,
环A选自5-6元杂芳基和[1,2,4]三唑并[4,3-A]吡啶基，所述5-6元杂芳基和[1,2,4]三唑并[4,3-A]吡啶基任选被1、2或3个R a取代； Ring A is selected from 5-6 membered heteroaryl and [1,2,4]triazolo[4,3-A]pyridyl, the 5-6 membered heteroaryl and [1,2,4] tri oxazolo [4,3-A] pyridyl optionally substituted with 1, 2 or 3 substituents R a;
R 1分别独立地选自H、F、Cl、Br和I； R 1 is independently selected from H, F, Cl, Br and I;
R 2选自C 1-6烷基，所述C 1-6烷基任选被1、2或3个R b取代； R 2 is selected from C 1-6 alkyl, and the C 1-6 alkyl is optionally substituted with 1, 2 or 3 R b ;
m选自0、1、2和3；m is selected from 0, 1, 2 and 3;
R a分别独立地选自H、F、Cl和C 1-3烷基，所述C 1-3烷基任选被1、2或3个R取代； R a is each independently selected from H, F, Cl and C 1-3 alkyl, the C 1-3 alkyl is optionally substituted with 1, 2 or 3 R;
R b分别独立地选自H、F、Cl、Br和I； R b is independently selected from H, F, Cl, Br and I;
R分别独立地选自H、F和Cl；R is independently selected from H, F and Cl;
带“*”碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在；The carbon atom with "*" is a chiral carbon atom, which exists in the form of (R) or (S) single enantiomer or rich in one enantiomer;
所述“5-6元杂芳基”包含1、2或3个选自N、NH、O、C(＝O)和S的杂原子或杂原子团。The "5-6 membered heteroaryl group" contains 1, 2 or 3 heteroatoms or heteroatom groups selected from N, NH, O, C(=O) and S.
本发明提供了式(I)所示化合物或其药学上可接受的盐，The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
Figure PCTCN2020141640-appb-000003
Figure PCTCN2020141640-appb-000003
其中，among them,
环A选自5元杂芳基、吡啶基、哒嗪基、1H-吡啶-2-酮基和[1,2,4]三唑并[4,3-A]吡啶基，所述5元杂芳基、吡啶基、哒嗪基、1H-吡啶-2-酮基和[1,2,4]三唑并[4,3-A]吡啶基任选被1、2或3个R a取代； Ring A is selected from 5-membered heteroaryl, pyridyl, pyridazinyl, 1H-pyridin-2-onyl and [1,2,4]triazolo[4,3-A]pyridyl, the 5-membered Heteroaryl, pyridyl, pyridazinyl, 1H-pyridin-2-onyl and [1,2,4]triazolo[4,3-A]pyridyl are optionally substituted by 1, 2 or 3 Ra replace;
R 1分别独立地选自H、F、Cl、Br和I； R 1 is independently selected from H, F, Cl, Br and I;
R 2选自C 1-6烷基，所述C 1-6烷基任选被1、2或3个R b取代； R 2 is selected from C 1-6 alkyl, and the C 1-6 alkyl is optionally substituted with 1, 2 or 3 R b ;
m选自0、1、2和3；m is selected from 0, 1, 2 and 3;
R a分别独立地选自H、F、Cl和C 1-3烷基，所述C 1-3烷基任选被1、2或3个R取代； R a is each independently selected from H, F, Cl and C 1-3 alkyl, the C 1-3 alkyl is optionally substituted with 1, 2 or 3 R;
R b分别独立地选自H、F、Cl、Br和I； R b is independently selected from H, F, Cl, Br and I;
R分别独立地选自H、F和Cl；R is independently selected from H, F and Cl;
带“*”碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在；The carbon atom with "*" is a chiral carbon atom, which exists in the form of (R) or (S) single enantiomer or rich in one enantiomer;
所述“5元杂芳基”包含1、2或3个选自N、NH、O、C＝O和S的杂原子或杂原子团。The "5-membered heteroaryl group" contains 1, 2 or 3 heteroatoms or heteroatom groups selected from N, NH, O, C=O and S.
在本发明的一些方案中，上述化合物或其药学上可接受的盐，其选自，In some aspects of the present invention, the above-mentioned compound or a pharmaceutically acceptable salt thereof is selected from,
Figure PCTCN2020141640-appb-000004
Figure PCTCN2020141640-appb-000004
其中，环A、R 1、R 2和m如本发明所定义。 Wherein, ring A, R 1 , R 2 and m are as defined in the present invention.
在本发明的一些方案中，上述R a分别独立地选自H、CH 3、CH 2CH 3、CH(CH 3) 2、CF 3、CHF 2和CH 2F，其他变量如本发明所定义。 In some aspects of the present invention, the above R a is independently selected from H, CH 3, CH 2 CH 3, CH (CH 3) 2, CF 3, CHF 2 , and CH 2 F, the other variables are as defined in the present invention .
在本发明的一些方案中，上述环A选自吡唑基、1H-吡啶-2-酮基、[1,2,4]三唑并[4,3-A]吡啶基、呋喃基、噻唑基、吡啶基和哒嗪基，所述吡唑基、1H-吡啶-2-酮基、[1,2,4]三唑并[4,3-A]吡啶基、呋喃基、噻唑基、吡啶基和哒嗪基任选被1、2或3个R a取代，其他变量如本发明所定义。 In some embodiments of the present invention, the above-mentioned ring A is selected from pyrazolyl, 1H-pyridin-2-onyl, [1,2,4]triazolo[4,3-A]pyridyl, furyl, thiazole Group, pyridyl and pyridazinyl, the pyrazolyl, 1H-pyridin-2-one, [1,2,4]triazolo[4,3-A]pyridyl, furyl, thiazolyl, pyridyl and pyridazinyl optionally substituted with 1, 2 or 3 substituents R a, the other variables are as defined in the present invention.
在本发明的一些方案中，上述环A选自
Figure PCTCN2020141640-appb-000005
Figure PCTCN2020141640-appb-000006
所述
Figure PCTCN2020141640-appb-000007
被1、2或3个R a取代，其他变量如本发明所定义。 In some aspects of the present invention, the above-mentioned ring A is selected from
Figure PCTCN2020141640-appb-000005
Figure PCTCN2020141640-appb-000006
Said
Figure PCTCN2020141640-appb-000007
1, 2 or 3 substituents R a, the other variables are as defined in the present invention.
在本发明的一些方案中，上述环A选自
Figure PCTCN2020141640-appb-000008
Figure PCTCN2020141640-appb-000009
其他变量如本发明所定义。 In some aspects of the present invention, the above-mentioned ring A is selected from
Figure PCTCN2020141640-appb-000008
Figure PCTCN2020141640-appb-000009
Other variables are as defined in the present invention.
在本发明的一些方案中，上述环A选自
Figure PCTCN2020141640-appb-000010
Figure PCTCN2020141640-appb-000011
其他变量如本发明所定义。 In some aspects of the present invention, the above-mentioned ring A is selected from
Figure PCTCN2020141640-appb-000010
Figure PCTCN2020141640-appb-000011
Other variables are as defined in the present invention.
在本发明的一些方案中，上述R 1分别独立地选自H和F，其他变量如本发明所定义。 In some aspects of the present invention, the above-mentioned R 1 is independently selected from H and F, and other variables are as defined in the present invention.
在本发明的一些方案中，上述结构单元
Figure PCTCN2020141640-appb-000012
分别独立地选自
Figure PCTCN2020141640-appb-000013
Figure PCTCN2020141640-appb-000014
其他变量如本发明所定义。 In some aspects of the present invention, the above-mentioned structural unit
Figure PCTCN2020141640-appb-000012
Are independently selected from
Figure PCTCN2020141640-appb-000013
Figure PCTCN2020141640-appb-000014
Other variables are as defined in the present invention.
在本发明的一些方案中，上述R 2选自C 1-3烷基，所述C 1-3烷基任选被1、2和3个R b取代，其他变量如本发明所定义。 In some embodiments of the present invention, the above-mentioned R 2 is selected from C 1-3 alkyl, and the C 1-3 alkyl is optionally substituted with 1, 2 and 3 R b , and other variables are as defined in the present invention.
在本发明的一些方案中，上述R 2选自CH 3和
Figure PCTCN2020141640-appb-000015
所述CH 3和
Figure PCTCN2020141640-appb-000016
任选被1、2和3个R b取代，其他变量如本发明所定义。 In some aspects of the present invention, the above R 2 is selected from CH 3 and
Figure PCTCN2020141640-appb-000015
The CH 3 and
Figure PCTCN2020141640-appb-000016
Optionally substituted by 1, 2 and 3 R b , and other variables are as defined in the present invention.
在本发明的一些方案中，上述R 2选自CH 3和
Figure PCTCN2020141640-appb-000017
其他变量如本发明所定义。 In some aspects of the present invention, the above-mentioned R 2 is selected from CH 3 and
Figure PCTCN2020141640-appb-000017
Other variables are as defined in the present invention.
本发明还提供了式(I)所示化合物或其药学上可接受的盐，The present invention also provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
Figure PCTCN2020141640-appb-000018
Figure PCTCN2020141640-appb-000018
其中，among them,
环A选自吡唑基、1H-吡啶-2-酮基和[1,2,4]三唑并[4,3-A]吡啶基，所述吡唑基、1H-吡啶-2-酮基和[1,2,4]三唑并[4,3-A]吡啶基任选被1、2或3个R a取代； Ring A is selected from pyrazolyl, 1H-pyridin-2-one and [1,2,4]triazolo[4,3-A]pyridyl, the pyrazolyl, 1H-pyridin-2-one group, and [1,2,4] triazolo [4,3-A] pyridyl optionally substituted with 1, 2 or 3 substituents R a;
R 1分别独立地选自H、F、Cl、Br和I； R 1 is independently selected from H, F, Cl, Br and I;
R 2选自C 1-6烷基，所述C 1-6烷基任选被1、2或3个R b取代； R 2 is selected from C 1-6 alkyl, and the C 1-6 alkyl is optionally substituted with 1, 2 or 3 R b ;
m选自0、1、2和3；m is selected from 0, 1, 2 and 3;
R a分别独立地选自H、F、Cl和C 1-3烷基，所述C 1-3烷基任选被1、2或3个R取代； R a is each independently selected from H, F, Cl and C 1-3 alkyl, the C 1-3 alkyl is optionally substituted with 1, 2 or 3 R;
R b分别独立地选自H、F、Cl、Br和I； R b is independently selected from H, F, Cl, Br and I;
R分别独立地选自H、F和Cl；R is independently selected from H, F and Cl;
带“*”碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在。The carbon atom with "*" is a chiral carbon atom and exists in the form of (R) or (S) single enantiomer or enriched in one enantiomer.
在本发明的一些方案中，上述化合物或其药学上可接受的盐，其选自，In some aspects of the present invention, the above-mentioned compound or a pharmaceutically acceptable salt thereof is selected from,
Figure PCTCN2020141640-appb-000019
Figure PCTCN2020141640-appb-000019
其中，环A、R 1、R 2和m如本发明所定义。 Wherein, ring A, R 1 , R 2 and m are as defined in the present invention.
在本发明的一些方案中，上述R a分别独立地选自H和CH 3，其他变量如本发明所定义。 In some aspects of the present invention, the above R a is independently selected from H and CH 3, the other variables are as defined in the present invention.
在本发明的一些方案中，上述环A选自
Figure PCTCN2020141640-appb-000020
所述
Figure PCTCN2020141640-appb-000021
被1、2或3个R a取代，其他变量如本发明所定义。 In some aspects of the present invention, the above-mentioned ring A is selected from
Figure PCTCN2020141640-appb-000020
Said
Figure PCTCN2020141640-appb-000021
1, 2 or 3 substituents R a, the other variables are as defined in the present invention.
在本发明的一些方案中，上述环A选自
Figure PCTCN2020141640-appb-000022
其他变量如本发明所定义。 In some aspects of the present invention, the above-mentioned ring A is selected from
Figure PCTCN2020141640-appb-000022
Other variables are as defined in the present invention.
在本发明的一些方案中，上述环A选自
Figure PCTCN2020141640-appb-000023
其他变量如本发明所定义。 In some aspects of the present invention, the above-mentioned ring A is selected from
Figure PCTCN2020141640-appb-000023
Other variables are as defined in the present invention.
在本发明的一些方案中，上述R 1分别独立地选自H和F，其他变量如本发明所定义。 In some aspects of the present invention, the above-mentioned R 1 is independently selected from H and F, and other variables are as defined in the present invention.
在本发明的一些方案中，上述R 2选自C 1-3烷基，所述C 1-3烷基任选被1、2和3个R b取代，其他变量如本发明所定义。 In some embodiments of the present invention, the above-mentioned R 2 is selected from C 1-3 alkyl, and the C 1-3 alkyl is optionally substituted with 1, 2 and 3 R b , and other variables are as defined in the present invention.
在本发明的一些方案中，上述R 2选自CH 3和
Figure PCTCN2020141640-appb-000024
所述CH 3和
Figure PCTCN2020141640-appb-000025
任选被1、2和3个R b取代，其他变量如本发明所定义。 In some aspects of the present invention, R 2 is selected from CH 3 and
Figure PCTCN2020141640-appb-000024
The CH 3 and
Figure PCTCN2020141640-appb-000025
Optionally substituted by 1, 2 and 3 R b , and other variables are as defined in the present invention.
在本发明的一些方案中，上述R 2选自CH 3和
Figure PCTCN2020141640-appb-000026
其他变量如本发明所定义。 In some aspects of the present invention, the above-mentioned R 2 is selected from CH 3 and
Figure PCTCN2020141640-appb-000026
Other variables are as defined in the present invention.
在本发明的一些方案中，上述化合物或其药学上可接受的盐，其选自，In some aspects of the present invention, the above-mentioned compound or a pharmaceutically acceptable salt thereof is selected from,
Figure PCTCN2020141640-appb-000027
Figure PCTCN2020141640-appb-000027
其中，among them,
T 1选自CH和N； T 1 is selected from CH and N;
R 1、R 2、R a和m如本发明所定义。 R 1, R 2, R a and m are as defined in the present invention.
在本发明的一些方案中，上述化合物或其药学上可接受的盐，其选自，In some aspects of the present invention, the above-mentioned compound or a pharmaceutically acceptable salt thereof is selected from,
Figure PCTCN2020141640-appb-000028
Figure PCTCN2020141640-appb-000028
其中，among them,
R 1、R 2和m如本发明所定义。 R 1 , R 2 and m are as defined in the present invention.
本发明还有一些方案是由上述变量任意组合而来。There are also some schemes of the present invention that come from any combination of the above variables.
本发明提供了下式所示化合物或其药学上可接受的盐，The present invention provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof,
Figure PCTCN2020141640-appb-000029
Figure PCTCN2020141640-appb-000029
Figure PCTCN2020141640-appb-000030
Figure PCTCN2020141640-appb-000030
本发明还提供了上述化合物或其药学上可接受的盐在制备与糖皮质激素受体相关疾病的药物中的应用。The present invention also provides the application of the above-mentioned compound or its pharmaceutically acceptable salt in the preparation of drugs for glucocorticoid receptor-related diseases.
在本发明的一些方案中，上述与糖皮质激素受体相关疾病是指类风湿关节炎。In some aspects of the present invention, the aforementioned glucocorticoid receptor-related disease refers to rheumatoid arthritis.
技术效果Technical effect
本发明化合物具有很好的hMMP1转录抑制活性、相当的MMTV转录激活活性，很强的细胞水平的抗炎活性，以及适中的CYP3A4抑制。The compound of the present invention has good hMMP1 transcriptional inhibition activity, comparable MMTV transcriptional activation activity, strong anti-inflammatory activity at the cellular level, and moderate CYP3A4 inhibition.
相关定义Related definitions
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A specific term or phrase should not be considered uncertain or unclear without a special definition, but should be understood in its ordinary meaning. When a trade name appears in this article, it is meant to refer to its corresponding commodity or its active ingredient.
这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and/or dosage forms that are within the scope of reliable medical judgment and are suitable for use in contact with human and animal tissues. , Without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸 氢根，磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。The term "pharmaceutically acceptable salt" refers to a salt of the compound of the present invention, which is prepared from a compound with specific substituents discovered in the present invention and a relatively non-toxic acid or base. When the compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salt or similar salts. When the compound of the present invention contains a relatively basic functional group, the acid addition salt can be obtained by contacting the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, the organic acid includes, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid; also include salts of amino acids (such as arginine, etc.) , And salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups, which can be converted into any base or acid addition salt.
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。The pharmaceutically acceptable salt of the present invention can be synthesized from the parent compound containing acid or base by conventional chemical methods. In general, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of appropriate base or acid in water or organic solvent or a mixture of both.
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers Isomers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomers or diastereomer-enriched mixtures, all of these mixtures belong to this Within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All these isomers and their mixtures are included in the scope of the present invention.
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚( 3H)，碘-125( 125I)或C-14( 14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。 The compound of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms constituting the compound. For example, compounds can be labeled with radioisotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C). For another example, deuterium can be substituted for hydrogen to form deuterated drugs. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs can reduce toxic side effects and increase drug stability. , Enhance the efficacy, extend the biological half-life of drugs and other advantages. All changes in the isotopic composition of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.
术语“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。The term "optional" or "optionally" refers to the event or condition described later that may but not necessarily occur, and the description includes a situation in which the event or condition occurs and a situation in which the event or condition does not occur .
术语“被取代的”是指特定原子上的任意一个或多个氢原子被取代基取代，可以包括重氢和氢的变体，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧(即＝O)时，意味着两个氢原子被取代。氧取代不会发生在芳香基上。术语“任选被取代的”是指可以被取代，也可以不被取代，除非另有规定，取代基的种类和数目在化学上可以实现的基础上可以是任意的。The term "substituted" means that any one or more hydrogen atoms on a specific atom are replaced by substituents, and may include deuterium and hydrogen variants, as long as the valence of the specific atom is normal and the substituted compound is stable of. When the substituent is oxygen (ie =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it can be substituted or unsubstituted. Unless otherwise specified, the type and number of substituents can be arbitrary on the basis that they can be chemically realized.
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被0-2个R所取代，则所述基团可以任选地至多被两个R所取代，并且每种情况下的R都有独立的选项。此外，取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。When any variable (such as R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group can optionally be substituted with up to two Rs, and R has independent options in each case. In addition, combinations of substituents and/or variants thereof are only permitted if such combinations result in stable compounds.
当一个连接基团的数量为0时，比如-(CRR) 0-，表示该连接基团为单键。 When the number of a linking group is 0, such as -(CRR) 0 -, it means that the linking group is a single bond.
当其中一个变量选自单键时，表示其连接的两个基团直接相连，比如A-L-Z中L代表单键时表示该结构实际上是A-Z。When one of the variables is selected from a single bond, it means that the two connected groups are directly connected. For example, when L in A-L-Z represents a single bond, it means that the structure is actually A-Z.
除非另有规定，当某一基团具有一个或多个可连接位点时，该基团的任意一个或多个位点可以通过化学键与其他基团相连。当该化学键的连接方式是不定位的，且可连接位点存在H原子时，则连接化学键时，该位点的H原子的个数会随所连接化学键的个数而对应减少变成相应价数的基团。所述位 点与其他基团连接的化学键可以用直形实线键
Figure PCTCN2020141640-appb-000031
直形虚线键
Figure PCTCN2020141640-appb-000032
或波浪线
Figure PCTCN2020141640-appb-000033
表示。例如-OCH 3中的直形实线键表示通过该基团中的氧原子与其他基团相连；
Figure PCTCN2020141640-appb-000034
中的直形虚线键表示通过该基团中的氮原子的两端与其他基团相连；
Figure PCTCN2020141640-appb-000035
中的波浪线表示通过该苯基基团中的1和2位碳原子与其他基团相连；
Figure PCTCN2020141640-appb-000036
表示该哌啶基上的任意可连接位点可以通过1个化学键与其他基团相连，至少包括
Figure PCTCN2020141640-appb-000037
这4种连接方式，即使-N-上画出了H原子，但是
Figure PCTCN2020141640-appb-000038
仍包括
Figure PCTCN2020141640-appb-000039
这种连接方式的基团，只是在连接1个化学键时，该位点的的H会对应减少1个变成相应的一价哌啶基。 Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of the group can be connected to other groups through chemical bonds. When the connection method of the chemical bond is not positioned, and there is a H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will correspondingly decrease with the number of chemical bonds connected to become the corresponding valence number的组。 The group. The chemical bond between the site and other groups can be a straight solid bond
Figure PCTCN2020141640-appb-000031
Straight dashed key
Figure PCTCN2020141640-appb-000032
Or wavy line
Figure PCTCN2020141640-appb-000033
Said. For example , the straight solid bond in -OCH 3 means that it is connected to other groups through the oxygen atom in the group;
Figure PCTCN2020141640-appb-000034
The straight dashed bond in indicates that the two ends of the nitrogen atom in the group are connected to other groups;
Figure PCTCN2020141640-appb-000035
The wavy line in indicates that the phenyl group is connected to other groups through the 1 and 2 carbon atoms;
Figure PCTCN2020141640-appb-000036
Indicates that any linkable site on the piperidinyl group can be connected to other groups through a chemical bond, including at least
Figure PCTCN2020141640-appb-000037
These four connection methods, even though the H atom is drawn on -N-, but
Figure PCTCN2020141640-appb-000038
Still include
Figure PCTCN2020141640-appb-000039
The group in this connection mode, only when one chemical bond is connected, the H at this position will decrease by one and become the corresponding monovalent piperidinyl group.
除非另有规定，术语“C 1-6烷基”用于表示直链或支链的由1至6个碳原子组成的饱和碳氢基团。所述C 1-6烷基包括C 1-5、C 1-4、C 1-3、C 1-2、C 2-6、C 2-4、C 6和C 5烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C 1-6烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)、丁基(包括n-丁基，异丁基，s-丁基和t-丁基)、戊基(包括n-戊基，异戊基和新戊基)、己基等。 Unless otherwise specified, the term "C 1-6 alkyl" is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 6 carbon atoms. The C 1-6 alkyl group includes C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-4 , C 6 and C 5 alkyl groups, etc.; it may Is monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine). Examples of C 1-6 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl) , S-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl, etc.
除非另有规定，术语“C 1-3烷基”用于表示直链或支链的由1至3个碳原子组成的饱和碳氢基团。所述C 1-3烷基包括C 1-2和C 2-3烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C 1-3烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)等。 Unless otherwise specified, the term "C 1-3 alkyl" is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 3 carbon atoms. The C 1-3 alkyl group includes C 1-2 and C 2-3 alkyl groups, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine) . Examples of C 1-3 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.
除非另有规定，本发明术语“5-6元杂芳环”和“5-6元杂芳基”可以互换使用，术语“5-6元杂芳基”表示由5至6个环原子组成的具有共轭π电子体系的单环基团，其1、2、3或4个环原子为独立选自O、S和N的杂原子，其余为碳原子。其中氮原子任选地被季铵化，氮和硫杂原子可任选被氧化(即NO和S(O) p，p是1或2)。5-6元杂芳基可通过杂原子或碳原子连接到分子的其余部分。所述5-6元杂芳基包括5元和6元杂芳基。所述5-6元杂芳基的实例包括但不限于吡咯基(包括N-吡咯基、2-吡咯基和3-吡咯基等)、吡唑基(包括2-吡唑基和3-吡唑基等)、咪唑基(包括N-咪唑基、2-咪唑基、4-咪唑基和5-咪唑基等)、噁唑基(包括2-噁唑基、4-噁唑基和5-噁唑基等)、三唑基(1H-1,2,3-三唑基、2H-1,2,3-三唑基、1H-1,2,4-三唑基和4H-1,2,4-三唑基等)、四唑基、异噁唑基(3-异噁唑基、4-异噁唑基和5-异噁唑基等)、噻唑基(包括2-噻唑基、4-噻唑基和5-噻唑基等)、呋喃基(包括2-呋喃基和3-呋喃基等)、噻吩基(包括2-噻吩基和3-噻吩基等)、吡啶基(包括2-吡啶基、3-吡啶基和4-吡啶基等)、吡嗪基或嘧啶基(包括2-嘧啶基和4-嘧啶基等)。除非另有规定，C n-n+m或C n-C n+m包括n至n+m个碳的任何一种具体情况，例如C 1-12包括C 1、C 2、C 3、C 4、C 5、C 6、C 7、C 8、C 9、C 10、C 11、和C 12，也 包括n至n+m中的任何一个范围，例如C 1-12包括C 1-3、C 1-6、C 1-9、C 3-6、C 3-9、C 3-12、C 6-9、C 6-12、和C 9-12等；同理，n元至n+m元表示环上原子数为n至n+m个，例如3-12元环包括3元环、4元环、5元环、6元环、7元环、8元环、9元环、10元环、11元环、和12元环，也包括n至n+m中的任何一个范围，例如3-12元环包括3-6元环、3-9元环、5-6元环、5-7元环、6-7元环、6-8元环、和6-10元环等。 Unless otherwise specified, the terms "5-6 membered heteroaryl ring" and "5-6 membered heteroaryl group" can be used interchangeably in the present invention. The term "5-6 membered heteroaryl group" means a ring consisting of 5 to 6 ring atoms. It is composed of a monocyclic group with a conjugated π-electron system, in which 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from O, S and N, and the rest are carbon atoms. Where the nitrogen atom is optionally quaternized, the nitrogen and sulfur heteroatoms may optionally be oxidized (ie NO and S(O) p , p is 1 or 2). The 5-6 membered heteroaryl group can be attached to the rest of the molecule through a heteroatom or a carbon atom. The 5-6 membered heteroaryl group includes 5-membered and 6-membered heteroaryl groups. Examples of the 5-6 membered heteroaryl include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrrolyl, etc.) Azolyl, etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5- Oxazolyl, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl and 4H-1, 2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-isoxazolyl and 5-isoxazolyl, etc.), thiazolyl (including 2-thiazolyl, etc.) , 4-thiazolyl and 5-thiazolyl, etc.), furyl (including 2-furyl and 3-furyl, etc.), thienyl (including 2-thienyl and 3-thienyl, etc.), pyridyl (including 2 -Pyridyl, 3-pyridyl and 4-pyridyl, etc.), pyrazinyl or pyrimidinyl (including 2-pyrimidinyl and 4-pyrimidinyl, etc.). Unless otherwise specified, C n-n+m or C n -C n+m includes any specific case of n to n+m carbons, for example, C 1-12 includes C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , and C 12 , including any range from n to n+m, for example, C 1-12 includes C 1-3 , C 1-6 , C 1-9 , C 3-6 , C 3-9 , C 3-12 , C 6-9 , C 6-12 , and C 9-12, etc.; similarly, from n to n +m member means that the number of atoms in the ring is from n to n+m, for example, 3-12 membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, 9-membered ring , 10-membered ring, 11-membered ring, and 12-membered ring, including any range from n to n+m, for example, 3-12 membered ring includes 3-6 membered ring, 3-9 membered ring, 5-6 membered ring Ring, 5-7 membered ring, 6-7 membered ring, 6-8 membered ring, 6-10 membered ring, etc.
术语“离去基团”是指可以被另一种官能团或原子通过取代反应(例如亲核取代反应)所取代的官能团或原子。例如，代表性的离去基团包括三氟甲磺酸酯；氯、溴、碘；磺酸酯基，如甲磺酸酯、甲苯磺酸酯、对溴苯磺酸酯、对甲苯磺酸酯等；酰氧基，如乙酰氧基、三氟乙酰氧基等等。The term "leaving group" refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (for example, a nucleophilic substitution reaction). For example, representative leaving groups include triflate; chlorine, bromine, iodine; sulfonate groups, such as mesylate, tosylate, p-bromobenzenesulfonate, p-toluenesulfonic acid Esters, etc.; acyloxy groups, such as acetoxy, trifluoroacetoxy and the like.
术语“保护基”包括但不限于“氨基保护基”、“羟基保护基”或“巯基保护基”。术语“氨基保护基”是指适合用于阻止氨基氮位上副反应的保护基团。代表性的氨基保护基包括但不限于：甲酰基；酰基，例如链烷酰基(如乙酰基、三氯乙酰基或三氟乙酰基)；烷氧基羰基，如叔丁氧基羰基(Boc)；芳基甲氧羰基，如苄氧羰基(Cbz)和9-芴甲氧羰基(Fmoc)；芳基甲基，如苄基(Bn)、三苯甲基(Tr)、1,1-二-(4'-甲氧基苯基)甲基；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。术语“羟基保护基”是指适合用于阻止羟基副反应的保护基。代表性羟基保护基包括但不限于：烷基，如甲基、乙基和叔丁基；酰基，例如链烷酰基(如乙酰基)；芳基甲基，如苄基(Bn)，对甲氧基苄基(PMB)、9-芴基甲基(Fm)和二苯基甲基(二苯甲基，DPM)；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。The term "protecting group" includes, but is not limited to, "amino protecting group", "hydroxy protecting group" or "thiol protecting group". The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the amino nitrogen position. Representative amino protecting groups include, but are not limited to: formyl; acyl, such as alkanoyl (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl, such as tert-butoxycarbonyl (Boc) ; Arylmethyloxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethyloxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-di -(4'-Methoxyphenyl)methyl; silyl groups, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS) and so on. The term "hydroxy protecting group" refers to a protecting group suitable for preventing side reactions of the hydroxyl group. Representative hydroxy protecting groups include but are not limited to: alkyl groups, such as methyl, ethyl, and tert-butyl; acyl groups, such as alkanoyl groups (such as acetyl); arylmethyl groups, such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyl Dimethylsilyl (TBS) and so on.
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and those well known to those skilled in the art Equivalent alternatives, preferred implementations include but are not limited to the embodiments of the present invention.
本发明的化合物可以通过本领域技术人员所熟知的常规方法来确认结构，如果本发明涉及化合物的绝对构型，则该绝对构型可以通过本领域常规技术手段予以确证。例如单晶X射线衍射法(SXRD)，把培养出的单晶用Bruker D8 venture衍射仪收集衍射强度数据，光源为CuKα辐射，扫描方式：
Figure PCTCN2020141640-appb-000040
扫描，收集相关数据后，进一步采用直接法(Shelxs97)解析晶体结构，便可以确证绝对构型。 The structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, the single crystal X-ray diffraction method (SXRD) uses the Bruker D8 venture diffractometer to collect the diffraction intensity data of the cultured single crystal. The light source is CuKα radiation, and the scanning method:
Figure PCTCN2020141640-appb-000040
After scanning and collecting relevant data, the direct method (Shelxs97) is further used to analyze the crystal structure to confirm the absolute configuration.
本发明所使用的溶剂可经市售获得。本发明采用下述缩略词：THF代表四氢呋喃；DCM代表二氯甲烷；DMF代表N，N-二甲基甲酰胺；n-BuLi代表正丁基锂；NaH代表钠氢；TFA代表三氟乙酸。HOBt代表1-羟基苯并三唑；EDCI代表1-乙基-(3-二甲基氨基丙基)碳酰二亚胺；TBTU代表O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸；HATU代表2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯；HBTU代表O-苯并三氮唑-四甲基脲六氟磷酸酯；L-Glutamine代表左旋谷酰胺；TR代表转录抑制；TA代表转录激活；PMA代表佛波醇12-十四酸酯13-乙酸酯；DPBS代表和杜氏磷酸缓冲溶液；Trypsin代表胰蛋白酶；DMEM代表改良伊格尔培养基；FBS代表胎牛血清；NEAA代表非必需氨基酸；Sodium Pyruvate代表丙酮酸钠；G418
Figure PCTCN2020141640-appb-000041
代表选择性抗生素(G418硫酸盐)；MMTV代表小鼠乳腺肿瘤病毒；Hygromycin代表潮霉素。 The solvent used in the present invention is commercially available. The present invention uses the following abbreviations: THF stands for tetrahydrofuran; DCM stands for dichloromethane; DMF stands for N,N-dimethylformamide; n-BuLi stands for n-butyl lithium; NaH stands for sodium hydrogen; TFA stands for trifluoroacetic acid . HOBt stands for 1-hydroxybenzotriazole; EDCI stands for 1-ethyl-(3-dimethylaminopropyl) carbodiimide; TBTU stands for O-benzotriazole-N,N,N', N'-tetramethylurea tetrafluoroborate; HATU stands for 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; HBTU stands for O- Benzotriazole-tetramethylurea hexafluorophosphate; L-Glutamine stands for L-glutamine; TR stands for transcriptional inhibition; TA stands for transcriptional activation; PMA stands for phorbol 12-myristate 13-acetate; DPBS stands for and Dulbecco's phosphate buffered solution; Trypsin stands for trypsin; DMEM stands for modified Eagle's medium; FBS stands for fetal bovine serum; NEAA stands for non-essential amino acids; Sodium Pyruvate stands for sodium pyruvate; G418
Figure PCTCN2020141640-appb-000041
Represents selective antibiotic (G418 sulfate); MMTV represents mouse breast tumor virus; Hygromycin represents hygromycin.
具体实施方式Detailed ways
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。The present invention will be described in detail through the following examples, but it is not meant to impose any disadvantageous restriction on the present invention. The present invention has been described in detail herein, and its specific embodiments are also disclosed. For those skilled in the art, various changes and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. Will be obvious.
参考例1：化合物CC-1的合成Reference example 1: Synthesis of compound CC-1
Figure PCTCN2020141640-appb-000042
Figure PCTCN2020141640-appb-000042
步骤1：化合物CC-1-2的合成Step 1: Synthesis of compound CC-1-2
在干燥的三口瓶中加入化合物CC-1-1(10g,52.85mmol)，用二氯甲烷(100mL)溶解后，分批加入N,N’-羰基二咪唑(10.72g,66.11mmol)，加料完毕，氮气置换三次，氮气氛围下于25℃搅拌1小时。然后加入N-甲基-N-甲氧基胺盐酸盐(6.44g,66.06mmol)，随后加入三乙胺(5.35g,52.85mmol)，加料完毕，氮气置换三次，氮气氛围下在25℃搅拌反应11小时。反应完毕，体系用饱和柠檬酸水溶液(100mL)洗涤，分液，收集有机相。有机相用饱和碳酸氢钠溶液(100mL)洗涤，分液，收集有机相。水相用二氯甲烷(100mL×2)萃取，合并有机相，用饱和食盐水(100mL)洗涤，无水硫酸钠干燥，过滤。滤液减压浓缩，得到化合物CC-1-2。Add compound CC-1-1 (10g, 52.85mmol) into a dry three-necked flask, dissolve it with dichloromethane (100mL), add N,N'-carbonyldiimidazole (10.72g, 66.11mmol) in batches, add After completion, nitrogen replacement was performed three times, and stirring was carried out at 25°C for 1 hour under a nitrogen atmosphere. Then N-methyl-N-methoxyamine hydrochloride (6.44g, 66.06mmol) was added, followed by triethylamine (5.35g, 52.85mmol). After the addition was completed, nitrogen was replaced three times, and the temperature was kept at 25°C under a nitrogen atmosphere. The reaction was stirred for 11 hours. After the reaction was completed, the system was washed with saturated citric acid aqueous solution (100 mL), separated, and the organic phase was collected. The organic phase was washed with saturated sodium bicarbonate solution (100 mL), separated, and the organic phase was collected. The aqueous phase was extracted with dichloromethane (100 mL×2), and the organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain compound CC-1-2.
MS–ESI m/z:133.1[M-100+H] +。 MS–ESI m/z: 133.1 [M-100+H] + .
步骤2：化合物CC-1-3的合成Step 2: Synthesis of compound CC-1-3
在干燥的三口瓶中加入化合物1-溴-3,5-二氟苯(270.03g,1.40mol)，降温至-15℃，缓慢滴加异丙基氯化镁的四氢呋喃溶液(2mol/L,699.60mL)。加料完毕，于-15℃搅拌2小时，然后将化合物CC-1-2(130g,559.68mmol)溶于四氢呋喃(1.3L)溶液中，缓慢加入上述反应体系(温度维持在-5℃左右)。加料完毕，将反应液升温至60℃搅拌1小时。反应完成后，待反应液冷却至室温，缓慢加入饱 和氯化铵溶液(1.5L)中，加乙酸乙酯(1L×2)萃取，合并有机相，用无水硫酸钠干燥，过滤。滤液减压浓缩，得到化合物CC-1-3。Add the compound 1-bromo-3,5-difluorobenzene (270.03g, 1.40mol) into a dry three-necked flask, cool to -15℃, and slowly add a solution of isopropylmagnesium chloride in tetrahydrofuran (2mol/L, 699.60mL). ). After the addition is complete, stir at -15°C for 2 hours, then dissolve compound CC-1-2 (130g, 559.68mmol) in a tetrahydrofuran (1.3L) solution, and slowly add the above reaction system (the temperature is maintained at about -5°C). After the addition was completed, the reaction solution was heated to 60°C and stirred for 1 hour. After the reaction is completed, after the reaction solution is cooled to room temperature, slowly add to saturated ammonium chloride solution (1.5L), add ethyl acetate (1L×2) for extraction, combine the organic phases, dry with anhydrous sodium sulfate, and filter. The filtrate was concentrated under reduced pressure to obtain compound CC-1-3.
1H NMR(400MHz,CDCl 3)δ7.47(br d,J＝6.0Hz,2H),7.09-6.95(m,1H),5.54(br d,J＝7.4Hz,1H),5.15(quin,J＝7.4Hz,1H),1.39(s,8H),1.36(d,J＝7.2Hz,3H),1.21(t,J＝7.2Hz,1H)。 1 H NMR (400MHz, CDCl 3 ) δ7.47 (br d, J = 6.0Hz, 2H), 7.09-6.95 (m, 1H), 5.54 (br d, J = 7.4Hz, 1H), 5.15 (quin, J = 7.4 Hz, 1H), 1.39 (s, 8H), 1.36 (d, J = 7.2 Hz, 3H), 1.21 (t, J = 7.2 Hz, 1H).
MS–ESI m/z:286.29[M+H] +。 MS–ESI m/z: 286.29 [M+H] + .
步骤3：化合物CC-1-4的合成Step 3: Synthesis of compound CC-1-4
在50L夹套釜中加入异丙醇(3.16kg,52.54mol)和甲苯(12L)，然后向其中分批加入化合物CC-1-3(1.15kg,4.04mol)，搅拌溶解后加入异丙醇铝(412.73g,2.02mol)，在25℃下搅拌1小时，再升温至55℃继续搅拌15小时。反应完毕，将反应液减压浓缩，加乙酸乙酯(5L)溶解，用稀盐酸(1mol/L)将pH调至5-6，分液，收集有机相。水相用乙酸乙酯(5L×2)萃取，合并有机相，用饱和食盐水(10L)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。粗品中加入石油醚(8L)，在室温下搅拌16小时，过滤。滤饼用石油醚(5L)淋洗，收集固体，得到化合物CC-1-4。Add isopropanol (3.16kg, 52.54mol) and toluene (12L) into a 50L jacketed kettle, then add compound CC-1-3 (1.15kg, 4.04mol) to it in batches, stir to dissolve and add isopropanol Aluminum (412.73g, 2.02mol), stirred at 25°C for 1 hour, then heated to 55°C and continued stirring for 15 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, dissolved with ethyl acetate (5L), the pH was adjusted to 5-6 with dilute hydrochloric acid (1mol/L), the layers were separated, and the organic phase was collected. The aqueous phase was extracted with ethyl acetate (5L×2), the organic phases were combined, washed with saturated brine (10L), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Petroleum ether (8L) was added to the crude product, stirred at room temperature for 16 hours, and filtered. The filter cake was rinsed with petroleum ether (5 L), and the solid was collected to obtain compound CC-1-4.
1HNMR(400MHz,CD 3OD)δ6.98(br d,J＝7.0Hz,2H),6.79(br t,J＝9.2Hz,1H),6.55(br d,J＝8.6Hz,1H),4.56(br d,J＝5.4Hz,1H),3.75-3.63(m,1H),1.37(s,9H),1.06(d,J＝6.8Hz,3H)。 1 HNMR (400MHz, CD 3 OD) δ 6.98 (br d, J = 7.0 Hz, 2H), 6.79 (br t, J = 9.2 Hz, 1H), 6.55 (br d, J = 8.6 Hz, 1H), 4.56 (br d, J=5.4 Hz, 1H), 3.75-3.63 (m, 1H), 1.37 (s, 9H), 1.06 (d, J=6.8 Hz, 3H).
MS–ESI m/z:288.30[M+H] +。 MS–ESI m/z: 288.30 [M+H] + .
步骤4：化合物CC-1的合成Step 4: Synthesis of compound CC-1
在干燥的单口瓶中加入化合物CC-1-4(145g,504.70mmol)，加入盐酸/乙酸乙酯(4mol/L，2L)，在25℃下搅拌12小时。反应完毕，将反应液减压浓缩，加乙酸乙酯(1L)溶解，用氢氧化钠水溶液(4mol/L)将pH调至7-8。分液，收集有机相，水相用乙酸乙酯(500mL×2)萃取。有机相合并，用无水硫酸钠干燥，过滤，滤液减压浓缩，得到化合物CC-1。Compound CC-1-4 (145 g, 504.70 mmol) was added to a dry single-neck flask, hydrochloric acid/ethyl acetate (4 mol/L, 2 L) was added, and the mixture was stirred at 25° C. for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, dissolved in ethyl acetate (1L), and the pH was adjusted to 7-8 with an aqueous sodium hydroxide solution (4mol/L). The layers were separated, the organic phase was collected, and the aqueous phase was extracted with ethyl acetate (500 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound CC-1.
1H NMR(400MHz,CD 3OD)δ7.04-6.97(m,2H),6.84(tt,J＝2.4,9.0Hz,1H),4.70(d,J＝4.4Hz,1H),3.28-3.16(m,1H),1.02(d,J＝6.6Hz,3H)。 1 H NMR(400MHz,CD 3 OD)δ7.04-6.97(m,2H), 6.84(tt,J=2.4,9.0Hz,1H), 4.70(d,J=4.4Hz,1H), 3.28-3.16 (m, 1H), 1.02 (d, J=6.6 Hz, 3H).
参照参考例1的合成方法，合成下表中参考例2和参考例8。Refer to the synthesis method of Reference Example 1, and synthesize Reference Example 2 and Reference Example 8 in the following table.
Figure PCTCN2020141640-appb-000043
Figure PCTCN2020141640-appb-000043
参考例3：化合物CD-1的合成Reference example 3: Synthesis of compound CD-1
Figure PCTCN2020141640-appb-000044
Figure PCTCN2020141640-appb-000044
步骤1：化合物CC-1-2的合成Step 1: Synthesis of compound CC-1-2
将化合物CC-1-1(100g,528.5mmol)溶于二氯甲烷(1.0L)，于25℃缓慢加入羰基二咪唑(107.1g,660.6mmol)，搅拌3小时后，加入三乙胺(107.1g,660.7mmol)和N-甲基-N-甲氧基胺盐酸盐(64.4g,660.6mmol)，在25℃下搅拌反应12小时。反应完毕，向反应体系加入水(300mL)和盐酸(1mol/L,100mL)。分液，收集有机相，水相用二氯甲烷萃取(500mL×3)。有机相合并，用无水硫酸钠干燥，过滤，滤液减压浓缩。粗品经柱层析(洗脱剂：石油醚/乙酸乙酯＝2/1–1/1)分离纯化，得到化合物CC-1-2。Compound CC-1-1 (100g, 528.5mmol) was dissolved in dichloromethane (1.0L), carbonyldiimidazole (107.1g, 660.6mmol) was slowly added at 25°C, and after stirring for 3 hours, triethylamine (107.1 g, 660.7 mmol) and N-methyl-N-methoxyamine hydrochloride (64.4 g, 660.6 mmol), the reaction was stirred at 25°C for 12 hours. After the reaction was completed, water (300 mL) and hydrochloric acid (1 mol/L, 100 mL) were added to the reaction system. Separate the liquids, collect the organic phase, and extract the aqueous phase with dichloromethane (500 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate = 2/1-1/1) to obtain compound CC-1-2.
1H NMR(400MHz,DMSO-d 6)δ＝7.05(J,1H)4.35-4.47(m,1H)3.73(s,3H)3.11(s,3H)1.38(s,9H)1.16(J,3H)。 1 H NMR(400MHz,DMSO-d 6 )δ=7.05(J,1H)4.35-4.47(m,1H)3.73(s,3H)3.11(s,3H)1.38(s,9H)1.16(J,3H) ).
步骤2：化合物CD-1-3的合成Step 2: Synthesis of compound CD-1-3
在干燥的三口瓶中加入四氢呋喃(50mL)，用氮气置换3次后，将体系温度降至-15℃，加入化合物苯基溴化镁(19.51g,107.63mmol)，将化合物CC-1-2(10g,43.05mmol)加入四氢呋喃(150mL)中，缓慢加入上述体系(温度维持在-5℃左右)，加料完毕，于25℃搅拌反应12小时。.反应完成后，将反应液缓慢倒入饱和氯化铵溶液(50mL)中，用乙酸乙酯(300mL×3)萃取，合并有机相，用无水硫酸钠干燥，过滤，滤液减压浓缩，得到化合物CD-1-3。Tetrahydrofuran (50mL) was added to a dry three-necked flask, and after replacing it with nitrogen three times, the temperature of the system was reduced to -15°C, and the compound phenylmagnesium bromide (19.51g, 107.63mmol) was added, and the compound CC-1-2 (10g, 43.05mmol) was added to tetrahydrofuran (150mL), the above system was slowly added (the temperature was maintained at about -5°C), the addition was completed, and the reaction was stirred at 25°C for 12 hours. After the completion of the reaction, the reaction solution was slowly poured into saturated ammonium chloride solution (50 mL), extracted with ethyl acetate (300 mL×3), the organic phases were combined, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Compound CD-1-3 was obtained.
步骤3：化合物CD-1-4的合成Step 3: Synthesis of compound CD-1-4
将化合物CD-1-3(9g,36.10mmol)加入甲苯(50mL)中，随后依次加入异丙醇(28.2g,469.3mmol)和异丙醇铝(3.69g,18.05mmol)，氮气保护下25℃搅拌1小时，升温至65℃搅拌反应12小时。反应完毕，反应液减压浓缩得到粗品。粗品用乙酸乙酯(100mL)溶解，缓慢倒入搅拌的稀盐酸(1mol/L, 100mL)中，分液。收集有机相，水相用乙酸乙酯萃取(150mL×3)。有机相合并，用无水硫酸钠干燥，过滤，滤液减压浓缩，得到化合物CD-1-4。Compound CD-1-3 (9g, 36.10mmol) was added to toluene (50mL), and then isopropanol (28.2g, 469.3mmol) and aluminum isopropoxide (3.69g, 18.05mmol) were added in sequence under nitrogen protection. Stir at °C for 1 hour, heat to 65 °C and stir for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in ethyl acetate (100 mL), slowly poured into stirred dilute hydrochloric acid (1 mol/L, 100 mL), and separated. The organic phase was collected, and the aqueous phase was extracted with ethyl acetate (150 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound CD-1-4.
步骤4：化合物CD-1的合成Step 4: Synthesis of compound CD-1
将化合物CD-1-4(8g,31.83mmol)溶于乙酸乙酯(90mL)，加入盐酸/乙酸乙酯(4mol/L,30mL)，在25℃下搅拌12小时。反应完毕，反应液减压浓缩。粗品加甲基叔丁基醚(100mL)，搅拌1小时，过滤，固体真空干燥，得到化合物CD-1。Compound CD-1-4 (8 g, 31.83 mmol) was dissolved in ethyl acetate (90 mL), hydrochloric acid/ethyl acetate (4 mol/L, 30 mL) was added, and the mixture was stirred at 25° C. for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure. The crude product was added with methyl tert-butyl ether (100 mL), stirred for 1 hour, filtered, and the solid was dried in vacuo to obtain compound CD-1.
1H NMR(400MHz,DMSO-d 6)δ＝8.13(3H,br s)7.23-7.36(4H,m)7.19-7.40(1H,m)5.98(1H,d,J＝4.6Hz)4.93(1H,t,J＝3.6Hz)0.89(3H,d,J＝7.2Hz)。 1 H NMR(400MHz,DMSO-d 6 )δ=8.13(3H,br s)7.23-7.36(4H,m)7.19-7.40(1H,m)5.98(1H,d,J=4.6Hz)4.93(1H , t, J = 3.6 Hz) 0.89 (3H, d, J = 7.2 Hz).
参照参考例3的合成方法，合成下表中参考例4。With reference to the synthesis method of Reference Example 3, Reference Example 4 in the following table was synthesized.
Figure PCTCN2020141640-appb-000045
Figure PCTCN2020141640-appb-000045
参考例5：化合物CG-1的合成Reference Example 5: Synthesis of compound CG-1
Figure PCTCN2020141640-appb-000046
Figure PCTCN2020141640-appb-000046
步骤1：化合物CG-1-3的合成Step 1: Synthesis of compound CG-1-3
向预先干燥的单口瓶中加入化合物CG-1-1(2.58g,9.67mmol)，CG-1-2(1g,8.06mmol)，氟化钾(935.99mg,16.11mmol,377.41μL)和N,N-二甲基甲酰胺(10mL)，氮气保护下于25-28℃反应36小时。反应完毕，将反应液缓慢滴加到剧烈搅拌的冰水(40mL)中，有沉淀析出，继续搅拌20分钟，过滤。滤饼用水(20mL)洗涤，真空干燥。所得固体打浆20分钟(石油醚/乙酸乙酯＝7/1，40mL)，过滤，滤饼真空干燥，得到化合物CG-1-3。Compound CG-1-1 (2.58g, 9.67mmol), CG-1-2 (1g, 8.06mmol), potassium fluoride (935.99mg, 16.11mmol, 377.41μL) and N, were added to the pre-dried single-mouth flask. N-dimethylformamide (10 mL) was reacted at 25-28°C for 36 hours under nitrogen protection. After the reaction was completed, the reaction solution was slowly added dropwise to vigorously stirred ice water (40 mL). A precipitate precipitated out. The stirring was continued for 20 minutes and filtered. The filter cake was washed with water (20 mL) and dried in vacuum. The obtained solid was slurried for 20 minutes (petroleum ether/ethyl acetate=7/1, 40 mL), filtered, and the filter cake was vacuum dried to obtain compound CG-1-3.
MS–ESI m/z:372.0[M+H] +。 MS–ESI m/z: 372.0 [M+H] + .
步骤2：化合物CG-1-4的合成Step 2: Synthesis of compound CG-1-4
向预先干燥的单口瓶中加入化合物CG-1-3(1g,2.69mmol)，溶于甲醇(3mL)，然后加入氯化铵(1.44g,26.94mmol)和锌粉(1.76g,26.94mmol)，于15-18℃反应15小时。反应完毕，反应液通过硅藻土过滤，滤液减压浓缩，得到化合物CG-1-4。Add compound CG-1-3 (1g, 2.69mmol) to a pre-dried single-neck bottle, dissolve it in methanol (3mL), then add ammonium chloride (1.44g, 26.94mmol) and zinc powder (1.76g, 26.94mmol) , React at 15-18°C for 15 hours. After the reaction was completed, the reaction solution was filtered through Celite, and the filtrate was concentrated under reduced pressure to obtain compound CG-1-4.
步骤3：化合物CG-1的合成Step 3: Synthesis of compound CG-1
向预先干燥的单口瓶中加入化合物CG-1-4(0.75g,2.20mmol)和醋酸(10mL)，之后加入亚硝酸钠(182.02mg,2.64mmol)的水溶液(1mL)，15-18℃下反应12小时。反应完毕，将反应液减压浓缩。粗产品加氢氧化钠水溶液(1mol/L,10mL)调节至pH>10，用乙酸乙酯(10mL×3)萃取。有机相合并，用饱和食盐水(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。粗品经柱层析(二氯甲烷/甲醇＝100/1-30/1，体积比)分离纯化，得到化合物CG-1。Compound CG-1-4 (0.75g, 2.20mmol) and acetic acid (10mL) were added to a pre-dried single-neck flask, and then an aqueous solution (1mL) of sodium nitrite (182.02mg, 2.64mmol) was added at 15-18°C. React for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure. The crude product was adjusted to pH>10 with aqueous sodium hydroxide solution (1mol/L, 10mL), and extracted with ethyl acetate (10mL×3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified by column chromatography (dichloromethane/methanol=100/1-30/1, volume ratio) to obtain compound CG-1.
1H NMR(400MHz,DMSO-d 6)δ＝8.59(dd,J＝1.0,1.4Hz,1H),8.40(d,J＝3.0Hz,1H),7.87(dd,J＝1.4,8.8Hz,1H),7.83(dd,J＝3.0,9.6Hz,1H),7.69(dd,J＝1.0,8.6Hz,1H),6.60(d,J＝9.6Hz,1H),3.51(s,3H)。 1 H NMR(400MHz,DMSO-d 6 )δ=8.59(dd,J=1.0,1.4Hz,1H), 8.40(d,J=3.0Hz,1H), 7.87(dd,J=1.4,8.8Hz, 1H), 7.83 (dd, J=3.0, 9.6 Hz, 1H), 7.69 (dd, J=1.0, 8.6 Hz, 1H), 6.60 (d, J=9.6 Hz, 1H), 3.51 (s, 3H).
MS–ESI m/z:353.0[M+H] +。 MS–ESI m/z: 353.0 [M+H] + .
参考例6：化合物CH-1的合成Reference Example 6: Synthesis of Compound CH-1
Figure PCTCN2020141640-appb-000047
Figure PCTCN2020141640-appb-000047
步骤1：化合物CH-1-3的合成Step 1: Synthesis of compound CH-1-3
在预先干燥的单口瓶中加入化合物CG-1-1(20g,74.91mmol)和N,N二甲基甲酰胺(200mL)，充分溶解后加入叔丁醇钾(12.61g,112.36mmol)和化合物CH-1-2(9.63g,74.91mmol)，氮气置换三次，在氮气氛围下于30℃反应12小时。反应完毕，向反应液中加入水(100mL)，甲基叔丁基醚(100mL×2)萃取。有机相合并，用饱和食盐水(50mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。粗品通过柱层析(洗脱剂：石油醚/乙酸乙酯＝50/1-5/1，体积比)分离纯化，得到化合物CH-1-3。Add compound CG-1-1 (20g, 74.91mmol) and N,N dimethylformamide (200mL) in a pre-dried single-neck bottle, and after fully dissolving, add potassium tert-butoxide (12.61g, 112.36mmol) and the compound CH-1-2 (9.63 g, 74.91 mmol) was replaced with nitrogen three times and reacted at 30°C for 12 hours under a nitrogen atmosphere. After the reaction was completed, water (100 mL) was added to the reaction solution and extracted with methyl tert-butyl ether (100 mL×2). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate=50/1-5/1, volume ratio) to obtain compound CH-1-3.
步骤2：化合物CH-1-4的合成Step 2: Synthesis of compound CH-1-4
向预先干燥的反应瓶中加入化合物CH-1-3(7g,18.64mmol)和氯化铵(9.97g,186.39mmol)，加入甲醇(60mL)，0℃下向其中分批加入锌粉(12.19g,186.39mmol)。加料完毕，反应液回温至25℃搅拌反应12小时。反应完成，反应液过滤，滤饼用乙酸乙酯(50mL)洗涤，滤液减压浓缩，得到化合物CH-1-4。Compound CH-1-3 (7g, 18.64mmol) and ammonium chloride (9.97g, 186.39mmol) were added to the pre-dried reaction flask, methanol (60mL) was added, and zinc powder (12.19) was added to it in batches at 0°C. g, 186.39 mmol). After the addition is complete, the reaction solution is warmed to 25°C and stirred for 12 hours. After the reaction was completed, the reaction solution was filtered, the filter cake was washed with ethyl acetate (50 mL), and the filtrate was concentrated under reduced pressure to obtain compound CH-1-4.
步骤3：化合物CH-1-5的合成Step 3: Synthesis of compound CH-1-5
向干燥的单口瓶中加入浓盐酸(12M,30.75mL)，缓慢加入化合物CH-1-4(3.0g,8.68mmol)，将亚硝酸钠(2.16g,31.25mmol)溶于水(6mL)中，0℃滴加至上述体系。滴加完毕，于25℃反应14小时。反应完毕后，向反应液中加水(50mL)。反应液过滤，滤饼用水(50mL×2)洗涤，收集所得固体。真空干燥，得到化合物CH-1-5。Add concentrated hydrochloric acid (12M, 30.75mL) to the dry single-neck flask, slowly add compound CH-1-4 (3.0g, 8.68mmol), dissolve sodium nitrite (2.16g, 31.25mmol) in water (6mL) , Add dropwise to the above system at 0°C. After the addition is complete, react at 25°C for 14 hours. After the reaction was completed, water (50 mL) was added to the reaction solution. The reaction solution was filtered, the filter cake was washed with water (50 mL×2), and the resulting solid was collected. Dry under vacuum to obtain compound CH-1-5.
步骤4：化合物CH-1-6的合成Step 4: Synthesis of compound CH-1-6
将化合物CH-1-5(5g,14.02mmol)溶于乙醇(30mL)，于室温下滴加水合肼(2.11g,42.07mmol)，反应在80℃搅拌12小时。反应完毕，反应液减压浓缩，得到粗品。粗品打浆(甲基叔丁基醚/乙酸乙酯＝20:1，100mL)，过滤，收集滤饼，得到化合物CH-1-6。Compound CH-1-5 (5g, 14.02mmol) was dissolved in ethanol (30mL), hydrazine hydrate (2.11g, 42.07mmol) was added dropwise at room temperature, and the reaction was stirred at 80°C for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was slurried (methyl tert-butyl ether/ethyl acetate=20:1, 100 mL), filtered, and the filter cake was collected to obtain compound CH-1-6.
步骤5：化合物CH-1的合成Step 5: Synthesis of compound CH-1
将化合物CH-1-6(0.75g,2.13mmol)溶于二氯甲烷(10mL)，依次加入原甲酸三甲酯(904.09mg,8.52mmol)，三氟乙酸(242.85mg,2.13mmol)，于25℃下搅拌12小时。反应完毕，向反应体系中加入饱和碳酸氢钠水溶液，将pH调至8。分液，收集有机相，水相用二氯甲烷(100mL×2)萃取。有机相合并，用无水硫酸钠干燥，过滤，滤液减压浓缩。粗品(甲基叔丁基醚/乙酸乙酯＝25/1，50mL)打浆，过滤，得到化合物CH-1。Compound CH-1-6 (0.75g, 2.13mmol) was dissolved in dichloromethane (10mL), and trimethyl orthoformate (904.09mg, 8.52mmol) and trifluoroacetic acid (242.85mg, 2.13mmol) were added in sequence. Stir at 25°C for 12 hours. After the reaction was completed, a saturated aqueous sodium bicarbonate solution was added to the reaction system to adjust the pH to 8. Separate the liquids, collect the organic phase, and extract the aqueous phase with dichloromethane (100 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product (methyl tert-butyl ether/ethyl acetate=25/1, 50 mL) was slurried and filtered to obtain compound CH-1.
1H NMR(400MHz,DMSO-d 6)δ9.40(1H,s)9.27(1H,s)8.65(1H,d,J＝1.00Hz)8.05(1H,d,J＝9.60Hz)7.92-7.97(1H,m)7.84-7.90(2H,m)。 1 H NMR(400MHz,DMSO-d 6 )δ9.40(1H,s)9.27(1H,s)8.65(1H,d,J＝1.00Hz)8.05(1H,d,J＝9.60Hz)7.92-7.97 (1H, m) 7.84-7.90 (2H, m).
参考例7：化合物CI-1的合成Reference Example 7: Synthesis of compound CI-1
Figure PCTCN2020141640-appb-000048
Figure PCTCN2020141640-appb-000048
Figure PCTCN2020141640-appb-000049
Figure PCTCN2020141640-appb-000049
步骤1：化合物CI-1-2的合成Step 1: Synthesis of compound CI-1-2
将化合物CG-1-1(2g,7.49mmol)和化合物CI-1-1(727.49mg,7.49mmol)加入至乙醇(15mL)和水(15mL)中，100℃搅拌反应12小时。反应完毕，将反应液减压浓缩，除去乙醇。残余物用乙酸乙酯(30mL×2)萃取，合并有机相，用无水硫酸钠干燥，过滤。滤液减压浓缩，得到化合物CI-1-2。Compound CG-1-1 (2 g, 7.49 mmol) and compound CI-1-1 (727.49 mg, 7.49 mmol) were added to ethanol (15 mL) and water (15 mL), and the reaction was stirred at 100°C for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove ethanol. The residue was extracted with ethyl acetate (30 mL×2), and the organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain compound CI-1-2.
MS–ESI m/z:344.9[M+H] +。 MS–ESI m/z: 344.9 [M+H] + .
步骤2：化合物CI-1-3的合成Step 2: Synthesis of compound CI-1-3
将化合物CI-1-2(2.3g,6.68mmol)加入甲醇(25mL)中，搅拌下加入氯化铵(3.58g,66.84mmol)，然后缓慢加入锌粉(4.37g,66.84mmol)，于20℃反应12小时。反应完毕，过滤除去不溶固体，滤液减压浓缩，得到化合物CI-1-3。Compound CI-1-2 (2.3g, 6.68mmol) was added to methanol (25mL), ammonium chloride (3.58g, 66.84mmol) was added with stirring, and then zinc powder (4.37g, 66.84mmol) was slowly added to the mixture. React at °C for 12 hours. After the reaction was completed, the insoluble solid was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain compound CI-1-3.
MS–ESI m/z:315.1[M+H] +。 MS–ESI m/z: 315.1 [M+H] + .
步骤3：化合物CI-1的合成Step 3: Synthesis of compound CI-1
将化合物CI-1-3(2.5g,7.96mmol)加入乙醇(5mL)中，依次加入亚硝酸钠(1.65g,23.88mmol)和浓盐酸(12mol/L,1.99mL)，在15℃下反应12小时。反应完毕，反应液减压浓缩。残余物用乙酸乙酯溶解，饱和碳酸氢钠溶液(30mL×2)洗涤。分液，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。粗产品经制备HPLC(柱型:Agela DuraShell C18 250mm*70mm*10μm；流动相:[H 2O(10mM NH 4HCO 3)-ACN]；B(ACN)％:25％-48％,22min)分离纯化，得到化合物CI-1。 Compound CI-1-3 (2.5g, 7.96mmol) was added to ethanol (5mL), sodium nitrite (1.65g, 23.88mmol) and concentrated hydrochloric acid (12mol/L, 1.99mL) were added successively, and reacted at 15℃ 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure. The residue was dissolved with ethyl acetate and washed with saturated sodium bicarbonate solution (30 mL×2). Liquid separation, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was subjected to preparative HPLC (column type: Agela DuraShell C18 250mm*70mm*10μm; mobile phase: [H 2 O(10mM NH 4 HCO 3 )-ACN]; B(ACN)%: 25%-48%, 22min) After separation and purification, compound CI-1 was obtained.
MS–ESI m/z:325.9[M+H] +。 MS–ESI m/z: 325.9 [M+H] + .
1H NMR(400MHz,CD 3OD)δ8.50(s,1H),7.91(d,J＝7.6Hz,2H),7.81(dd,J＝8.8,1.2Hz,1H),7.40(d,J＝8.8Hz,1H),4.06(s,3H)。 1 H NMR(400MHz,CD 3 OD)δ8.50(s,1H),7.91(d,J=7.6Hz,2H),7.81(dd,J=8.8,1.2Hz,1H),7.40(d,J =8.8 Hz, 1H), 4.06 (s, 3H).
参考例9：化合物CK-1的合成Reference Example 9: Synthesis of Compound CK-1
Figure PCTCN2020141640-appb-000050
Figure PCTCN2020141640-appb-000050
Figure PCTCN2020141640-appb-000051
Figure PCTCN2020141640-appb-000051
步骤1：化合物CK-1-2的合成Step 1: Synthesis of compound CK-1-2
向预先干燥好的单口瓶中加入化合物CK-1-1(4.16g，37.45mmol)，溶于N,N二甲基甲酰胺(50mL)。搅拌均匀后，加入碳酸钾(12.94g,93.63mmol)，加料完毕后置换氮气三次，搅拌0.5小时。分批加入化合物CG-1-1(12g,44.94mmol)，25℃继续搅拌3小时。将反应体系过滤，滤液倒入水(200mL)中，用乙酸乙酯(200mL x3)萃取。有机相合并，无水硫酸钠干燥，过滤，减压浓缩得到化合物CK-1-2。Compound CK-1-1 (4.16 g, 37.45 mmol) was added to the pre-dried single-neck flask, and dissolved in N,N dimethylformamide (50 mL). After stirring uniformly, potassium carbonate (12.94 g, 93.63 mmol) was added, and nitrogen was replaced three times after the addition was completed, and the mixture was stirred for 0.5 hour. Compound CG-1-1 (12 g, 44.94 mmol) was added in batches, and stirring was continued for 3 hours at 25°C. The reaction system was filtered, and the filtrate was poured into water (200 mL), and extracted with ethyl acetate (200 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound CK-1-2.
步骤2：化合物CK-1-3的合成Step 2: Synthesis of compound CK-1-3
向预先干燥好的单口瓶中加入化合物CK-1-2(12.5g,34.90mmol)，溶于甲醇(5mL)，搅拌均匀后，加入氯化铵(3.73g,69.81mmol)。将反应体系降温至0℃后，缓慢加入锌粉(9.13g,139.61mmol)。加料完毕后，置于25℃搅拌4小时。将反应液过滤，滤饼用二氯甲烷(250mL)冲洗后，滤液减压浓缩，得到化合物CK-1-3。Compound CK-1-2 (12.5 g, 34.90 mmol) was added to the pre-dried single-neck flask, dissolved in methanol (5 mL), and after stirring, ammonium chloride (3.73 g, 69.81 mmol) was added. After cooling the reaction system to 0°C, zinc powder (9.13 g, 139.61 mmol) was slowly added. After the addition is complete, place it at 25°C and stir for 4 hours. The reaction solution was filtered, the filter cake was washed with dichloromethane (250 mL), and the filtrate was concentrated under reduced pressure to obtain compound CK-1-3.
步骤3：化合物CK-1的合成Step 3: Synthesis of compound CK-1
向预先干燥好的单口瓶中加入化合物CK-1-3(1.4g,4.27mmol)溶于水(5mL)和盐酸(12M,17.78mL)中。搅拌均匀后，将反应体系降温至0℃后分批加入亚硝酸钠(1.47g,21.33mmol)。加料完毕后，于25℃搅拌2小时。反应液减压浓缩，得到化合物CK-1。Compound CK-1-3 (1.4g, 4.27mmol) was dissolved in water (5mL) and hydrochloric acid (12M, 17.78mL) into the pre-dried single-necked flask. After stirring uniformly, the reaction system was cooled to 0° C. and sodium nitrite (1.47 g, 21.33 mmol) was added in batches. After the addition is complete, stir at 25°C for 2 hours. The reaction solution was concentrated under reduced pressure to obtain compound CK-1.
MS–ESI m/z:340.0[M+H] +。 MS–ESI m/z: 340.0[M+H] + .
1H NMR(400MHz,DMSO-d 6)δ:8.59(s,2H),8.09(s,1H),7.89(dd,J＝8.76,1.2Hz,1H),7.74(d,J＝8.4Hz,1H),4.26(q,J＝7.6Hz,2H),1.47(t,J＝7.2Hz,3H)。 1 H NMR (400MHz, DMSO-d 6 ) δ: 8.59 (s, 2H), 8.09 (s, 1H), 7.89 (dd, J = 8.76, 1.2 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 4.26 (q, J = 7.6 Hz, 2H), 1.47 (t, J = 7.2 Hz, 3H).
实施例1：化合物WX001的合成Example 1: Synthesis of compound WX001
Figure PCTCN2020141640-appb-000052
Figure PCTCN2020141640-appb-000052
Figure PCTCN2020141640-appb-000053
Figure PCTCN2020141640-appb-000053
步骤1：化合物WX001-1的合成Step 1: Synthesis of compound WX001-1
向预先干燥的单口瓶中加入化合物CC-1(87.71mg,468.58μmol)，化合物CG-1(0.15g,425.98μmol)，N,N-二甲基甘氨酸(95.53mg,425.98μmol)，碳酸铯(416.38mg,1.28mmol)，碘化亚铜(40.56mg,212.99μmol)和正丁腈(2mL)。氮气置换三次，氮气氛围下升温至130℃反应12小时。反应完毕，反应液降至室温，加入水(20mL)，用乙酸乙酯(20mL×3)萃取。有机相合并，用饱和食盐水(30mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩至干。粗品经制备TLC(展开剂：二氯甲烷/甲醇＝10/1，体积比)分离纯化，得到化合物WX001-1。Add compound CC-1 (87.71 mg, 468.58 μmol), compound CG-1 (0.15 g, 425.98 μmol), N,N-dimethylglycine (95.53 mg, 425.98 μmol), and cesium carbonate to a pre-dried single-mouth bottle. (416.38 mg, 1.28 mmol), cuprous iodide (40.56 mg, 212.99 μmol) and n-butyronitrile (2 mL). It was replaced with nitrogen three times, and the temperature was raised to 130°C under a nitrogen atmosphere to react for 12 hours. After the reaction was completed, the reaction solution was cooled to room temperature, water (20 mL) was added, and extraction was performed with ethyl acetate (20 mL×3). The organic phases were combined, washed with saturated brine (30 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was separated and purified by preparative TLC (developing solvent: dichloromethane/methanol=10/1, volume ratio) to obtain compound WX001-1.
步骤2：化合物WX001的合成Step 2: Synthesis of compound WX001
向干燥的单口瓶中加入化合物WX001-1(30mg,72.92μmol)，2,2-二氟丙酸(12.04mg,109.38μmol)，HOBt(19.71mg,145.84μmol)，三乙胺(22.14mg,218.76μmol)和N,N-二甲基甲酰胺(0.5mL)，最后加入EDCI(27.96mg,145.84μmol)，于25-30℃反应10小时。反应完毕，粗品通过制备HPLC(柱型:x-charge150mm×25mm×5μm；流动相:[H 2O(0.05％HCl)-ACN]；B(ACN)％:30％-60％,10min)分离纯化，得到化合物WX001。 Add compound WX001-1 (30mg, 72.92μmol), 2,2-difluoropropionic acid (12.04mg, 109.38μmol), HOBt (19.71mg, 145.84μmol), triethylamine (22.14mg, 218.76μmol) and N,N-dimethylformamide (0.5mL). Finally, EDCI (27.96mg, 145.84μmol) was added and reacted at 25-30℃ for 10 hours. After the reaction is complete, the crude product is separated by preparative HPLC (column type: x-charge150mm×25mm×5μm; mobile phase: [H 2 O(0.05% HCl)-ACN]; B(ACN)%: 30%-60%, 10min) Purified to obtain compound WX001.
1H NMR(400MHz,CD 3OD)δ8.79(br d,J＝9.0Hz,1H),8.27(d,J＝2.8Hz,1H),7.91(dd,J＝2.8,9.6Hz,1H),7.73(d,J＝9.0Hz,1H),7.43(dd,J＝2.2,9.0Hz,1H),7.31(d,J＝2.0Hz,1H),7.10(br d,J＝6.0Hz,2H),6.95-6.86(m,1H),6.76(d,J＝9.6Hz,1H),5.38(d,J＝6.6Hz,1H),4.47-4.35(m,1H),3.69(s,3H),1.63(t,J＝19.2Hz,3H),1.42(d,J＝7.0Hz,3H)。 1 H NMR(400MHz,CD 3 OD)δ8.79(br d,J=9.0Hz,1H), 8.27(d,J=2.8Hz,1H),7.91(dd,J=2.8,9.6Hz,1H) ,7.73(d,J=9.0Hz,1H),7.43(dd,J=2.2,9.0Hz,1H),7.31(d,J=2.0Hz,1H),7.10(br d,J=6.0Hz,2H ), 6.95-6.86(m,1H), 6.76(d,J=9.6Hz,1H), 5.38(d,J=6.6Hz,1H), 4.47-4.35(m,1H), 3.69(s,3H) , 1.63 (t, J = 19.2 Hz, 3H), 1.42 (d, J = 7.0 Hz, 3H).
MS–ESI m/z:504.2[M+H] +。 MS–ESI m/z: 504.2 [M+H] + .
参照实施例1中的合成方法，合成下表中各实施例。With reference to the synthesis method in Example 1, the various examples in the following table were synthesized.
Figure PCTCN2020141640-appb-000054
Figure PCTCN2020141640-appb-000054
Figure PCTCN2020141640-appb-000055
Figure PCTCN2020141640-appb-000055
实施例4：化合物WX004的合成Example 4: Synthesis of compound WX004
Figure PCTCN2020141640-appb-000056
Figure PCTCN2020141640-appb-000056
步骤1：化合物WX004-1的合成Step 1: Synthesis of compound WX004-1
将化合物CC-1(30g,160.27mmol)和2,2-二氟丙酸(17.64g,160.27mmol)溶于N,N-二甲基甲酰胺(80mL)，随后依次加入1-羟基苯并三唑(43.31g,320.54mmol)，N,N-二异丙基乙胺(62.14g,480.80mmol)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(61.45g,320.54mmol)，于30℃反应12小时。反应完毕，向反应液中加入水(100mL)，用乙酸乙酯(150mL×3)萃取，收集有机相。有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。粗产品通过柱层析(洗脱剂：石油醚/乙酸乙酯＝80/1-1/1，体积比)分离纯化，得到化合物WX004-1。Compound CC-1 (30g, 160.27mmol) and 2,2-difluoropropionic acid (17.64g, 160.27mmol) were dissolved in N,N-dimethylformamide (80mL), and then 1-hydroxybenzo Triazole (43.31g, 320.54mmol), N,N-Diisopropylethylamine (62.14g, 480.80mmol), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Salt (61.45g, 320.54mmol), react at 30°C for 12 hours. After the reaction was completed, water (100 mL) was added to the reaction solution, extracted with ethyl acetate (150 mL×3), and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate=80/1-1/1, volume ratio) to obtain compound WX004-1.
步骤2：化合物WX004的合成Step 2: Synthesis of compound WX004
将化合物WX004-1(56.69mg,203.01μmol)和化合物CI-1(60mg,184.55μmol)加入正丁腈(1.5mL)中，搅拌下加入碳酸铯(180.39mg,553.66μmol)，N,N-二甲基甘氨酸(28.55mg,276.83μmol)，碘化亚铜(38.66mg,203.01μmol)，氮气置换三次后，氮气保护下升温至130℃搅拌6小时。反应完毕，将反应液倒入水(5mL)中，用乙酸乙酯(5mL×3)萃取。有机相合并，用饱和食盐水(10mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。粗品经制备HPLC(柱型:Phenomenex luna C18 80mm*40mm*3μm；流动相:[H 2O(0.04％HCl)-ACN]；B(ACN)％:35％-60％,7min)分离纯化，得到化合物WX004。 Compound WX004-1 (56.69 mg, 203.01 μmol) and compound CI-1 (60 mg, 184.55 μmol) were added to n-butyronitrile (1.5 mL), and cesium carbonate (180.39 mg, 553.66 μmol) was added with stirring, N, N- Dimethylglycine (28.55mg, 276.83μmol), cuprous iodide (38.66mg, 203.01μmol), replaced with nitrogen three times, heated to 130°C under nitrogen protection and stirred for 6 hours. After the reaction was completed, the reaction solution was poured into water (5 mL), and extracted with ethyl acetate (5 mL×3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified by preparative HPLC (column type: Phenomenex luna C18 80mm*40mm*3μm; mobile phase: [H 2 O(0.04%HCl)-ACN]; B(ACN)%: 35%-60%, 7min), Compound WX004 was obtained.
1H NMR(400MHz,DMSO-d 6)δ8.75(d,J＝8.8Hz,1H),8.49(s,1H),8.03(s,1H),7.78(d,J＝9.2Hz,1H),7.32-7.44(m,2H),7.10-7.22(m,3H),5.37(d,J＝7.2Hz,1H),4.22-4.33(m,1H),3.96(s,3H),1.56(t,J＝19.4Hz,3H),1.34(d,J＝6.8Hz,3H).MS–ESI m/z:477.1[M+H] +。 1 H NMR(400MHz,DMSO-d 6 )δ8.75(d,J=8.8Hz,1H),8.49(s,1H),8.03(s,1H),7.78(d,J=9.2Hz,1H) ,7.32-7.44(m,2H),7.10-7.22(m,3H),5.37(d,J=7.2Hz,1H),4.22-4.33(m,1H),3.96(s,3H),1.56(t , J = 19.4 Hz, 3H), 1.34 (d, J = 6.8 Hz, 3H). MS-ESI m/z: 477.1 [M+H] + .
参照实施例4中的合成方法，合成下表中各实施例。With reference to the synthesis method in Example 4, each of the examples in the following table was synthesized.
Figure PCTCN2020141640-appb-000057
Figure PCTCN2020141640-appb-000057
实施例10：化合物WX010的合成Example 10: Synthesis of compound WX010
Figure PCTCN2020141640-appb-000058
Figure PCTCN2020141640-appb-000058
步骤1：化合物WX010-1的合成Step 1: Synthesis of compound WX010-1
向预先干燥好的单口瓶中加入化合物CC-1(0.5g,2.67mmol)，化合物CK-1(823.52mg,2.43mmol)溶于正丁腈(10mL)中，搅拌均匀后，加入碳酸铯(2.37g,7.28mmol)，碘化亚铜(462.47mg,2.43mmol)，N,N-二甲基甘氨酸(125.20mg,1.21mmol)。加料完毕后，置换氮气三次，置于110℃油浴加热搅拌12小时。将反应体系过滤，滤液倒入水(50mL)中，加入氨水(2mL)，用乙酸乙酯萃取(100mL x 3)。有机相合并，无水硫酸钠干燥，过滤，减压浓缩得到残留物。所得残余物经过柱层析分离(洗脱剂：二氯甲烷/甲醇＝20/1–5/1，体积比)。得到化合物WX010-1。Compound CC-1 (0.5g, 2.67mmol) was added to the pre-dried single-neck flask, compound CK-1 (823.52mg, 2.43mmol) was dissolved in n-butyronitrile (10mL), and after stirring evenly, cesium carbonate ( 2.37g, 7.28mmol), cuprous iodide (462.47mg, 2.43mmol), N,N-dimethylglycine (125.20mg, 1.21mmol). After the addition is complete, the nitrogen is replaced three times, and the mixture is heated and stirred for 12 hours in an oil bath at 110°C. The reaction system was filtered, the filtrate was poured into water (50 mL), ammonia water (2 mL) was added, and the mixture was extracted with ethyl acetate (100 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The obtained residue is separated by column chromatography (eluent: dichloromethane/methanol=20/1-5/1, volume ratio). Compound WX010-1 was obtained.
MS–ESI m/z:399.2[M+H] +. MS–ESI m/z:399.2[M+H] + .
步骤2：化合物WX010的合成Step 2: Synthesis of compound WX010
向预先干燥好的单口瓶中加入2,2-二氟丙酸(99.45mg,903.60μmol)，溶于N,N二甲基甲酰胺(30mL)。搅拌均匀后，加入1-羟基苯并三唑(203.49mg,1.51mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(433.05mg)和N,N-二异丙基乙胺(194.63mg,1.51mmol)，最后加入化合物WX010-1(0.3g,753.00μmol)。加料完毕后，25℃搅拌12小时。将反应液倒入水(50mL)中，乙酸乙酯(100mL x 2)萃取。有机相合并，无水硫酸钠干燥，过滤，减压浓缩得到粗品。粗品经制备HPLC(柱型:Phenomenex luna C18 250mm*50mm*10μm；流动相:[H 2O(0.04％HCl)-ACN]；B(ACN)％:40％-70％,10min)分离纯化，得到WX010。 Add 2,2-difluoropropionic acid (99.45 mg, 903.60 μmol) to the pre-dried single-neck bottle, and dissolve in N,N dimethylformamide (30 mL). After stirring uniformly, add 1-hydroxybenzotriazole (203.49mg, 1.51mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (433.05mg) and N, N-diisopropylethylamine (194.63 mg, 1.51 mmol), and finally compound WX010-1 (0.3 g, 753.00 μmol) was added. After the addition is complete, stir at 25°C for 12 hours. The reaction solution was poured into water (50 mL), and extracted with ethyl acetate (100 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by preparative HPLC (column type: Phenomenex luna C18 250mm*50mm*10μm; mobile phase: [H 2 O(0.04%HCl)-ACN]; B(ACN)%: 40%-70%, 10min). Get WX010.
1H NMR(400MHz,DMSO-d 6)δ8.75(d,J＝9.2Hz,1H),8.54(s,1H),8.05(s,1H),7.79-7.82(d,J＝9.2Hz,1H),7.36-7.41(m,2H),7.14-7.17(m,3H),5.37(d,J＝7.2Hz,1H),4.22-4.31(m,3H),1.57(t,J＝19.2Hz,3H),1.46(t,J＝7.2Hz,3H),1.34(d,J＝6.8Hz,3H).MS–ESI m/z:491.2[M+H] +。 1 H NMR(400MHz,DMSO-d 6 )δ8.75(d,J=9.2Hz,1H),8.54(s,1H),8.05(s,1H),7.79-7.82(d,J=9.2Hz, 1H),7.36-7.41(m,2H),7.14-7.17(m,3H),5.37(d,J=7.2Hz,1H),4.22-4.31(m,3H),1.57(t,J=19.2Hz , 3H), 1.46 (t, J = 7.2 Hz, 3H), 1.34 (d, J = 6.8 Hz, 3H). MS-ESI m/z: 491.2 [M+H] + .
参照实施例10中的合成方法，合成下表中各实施例。With reference to the synthesis method in Example 10, each of the examples in the following table was synthesized.
Figure PCTCN2020141640-appb-000059
Figure PCTCN2020141640-appb-000059
实施例12：化合物WX012的合成Example 12: Synthesis of compound WX012
Figure PCTCN2020141640-appb-000060
Figure PCTCN2020141640-appb-000060
步骤1：化合物WX012-2的合成Step 1: Synthesis of compound WX012-2
将化合物WX012-1(500mg,2.52mmol)和呋喃-3-硼酸嚬哪醇酯(734.91mg,3.79mmol)加入N,N-二甲基甲酰胺(5mL)中，随后加入醋酸铜(917.23mg,5.05mmol)和吡啶(798.91mg,10.10mmol)。反应于氧气氛围下升温至100℃搅拌12小时。反应完毕，将反应液倒入水(20mL)中，加乙酸乙酯萃取(10mL×3)。有机相合并后加半饱和食盐水(20mL×2)洗，饱和食盐水(30mL)洗，无水硫酸钠干燥，过滤，滤液45℃减压浓缩。残余物通过柱层析(洗脱剂：石油醚/乙酸乙酯＝99/1–9/1，体积比)分离纯化，得到WX012-2。Compound WX012-1 (500mg, 2.52mmol) and furan-3-boronic acid lanacol ester (734.91mg, 3.79mmol) were added to N,N-dimethylformamide (5mL), followed by copper acetate (917.23mg , 5.05 mmol) and pyridine (798.91 mg, 10.10 mmol). The reaction was heated to 100°C under an oxygen atmosphere and stirred for 12 hours. After the reaction was completed, the reaction solution was poured into water (20 mL), and ethyl acetate was added for extraction (10 mL×3). The organic phases were combined and washed with half-saturated brine (20 mL×2), washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure at 45°C. The residue was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate=99/1-9/1, volume ratio) to obtain WX012-2.
MS–ESI m/z:266.0[M+H] +。 MS–ESI m/z: 266.0 [M+H] + .
步骤2：化合物WX012的合成Step 2: Synthesis of compound WX012
将化合物WX004-1(453.62mg,1.62mmol)和化合物WX012-2(390mg,1.48mmol)加入正丁腈(6mL)中，随后加入碳酸铯(1.44g,4.43mmol)，N,N-二甲基甘氨酸(228.44mg,2.22mmol)和碘化亚铜(309.39mg,1.62mmol)。氮气置换三次，氮气保护下升温至130℃搅拌6小时。反应完毕，反应液倒入水(15mL)和氨水(10mL)中，加乙酸乙酯萃取(15mL×3)。有机相合并，无水硫酸钠干燥，过滤，滤液减压浓缩。粗品先经过制备HPLC(柱型:Phenomenex Gemini-NX 150×30mm×5μm；流动相: [H 2O(10mM NH 4HCO 3)-ACN]；B(CAN)％:30％-60％,8min)分离，再经过手性SFC(柱型:DAICEL CHIRALPAK AD(250mm×30mm×10μm)；流动相:[A:CO 2；B:0.1％NH 3H 2O MeOH]；B％:24％-24％,min)分离纯化，得到WX012。 Compound WX004-1 (453.62mg, 1.62mmol) and compound WX012-2 (390mg, 1.48mmol) were added to n-butyronitrile (6mL), followed by cesium carbonate (1.44g, 4.43mmol), N,N-dimethyl Glycine (228.44 mg, 2.22 mmol) and cuprous iodide (309.39 mg, 1.62 mmol). Replace with nitrogen three times, and heat up to 130°C under nitrogen protection and stir for 6 hours. After the reaction was completed, the reaction solution was poured into water (15 mL) and ammonia (10 mL), and extracted with ethyl acetate (15 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was first subjected to preparative HPLC (column type: Phenomenex Gemini-NX 150×30mm×5μm; mobile phase: [H 2 O(10mM NH 4 HCO 3 )-ACN]; B(CAN)%: 30%-60%, 8min ) Separation, and then go through chiral SFC (column type: DAICEL CHIRALPAK AD (250mm×30mm×10μm); mobile phase: [A: CO 2 ; B: 0.1% NH 3 H 2 O MeOH]; B%: 24%- 24%, min) was separated and purified to obtain WX012.
1H NMR(400MHz,DMSO-d 6)δ＝8.77(d,J＝9.2Hz,1H),8.59(s,1H),7.97(t,J＝1.8Hz,1H),7.90(d,J＝9.2Hz,1H),7.44(d,J＝2.0Hz,1H),7.42(d,J＝2.0Hz,1H),7.39(d,J＝2.4Hz,1H),7.21(dd,J＝2.0,0.8Hz,1H),7.12-7.19(m,3H),5.38(d,J＝7.2Hz,1H),4.22-4.35(m,1H),1.57(t,J＝19.4Hz,3H),1.34(d,J＝6.4Hz,3H).MS–ESI m/z:463.3[M+H] +。 1 H NMR (400MHz, DMSO-d 6 )δ=8.77(d,J=9.2Hz,1H), 8.59(s,1H), 7.97(t,J=1.8Hz,1H), 7.90(d,J= 9.2Hz, 1H), 7.44 (d, J = 2.0 Hz, 1H), 7.42 (d, J = 2.0 Hz, 1H), 7.39 (d, J = 2.4 Hz, 1H), 7.21 (dd, J = 2.0, 0.8Hz,1H),7.12-7.19(m,3H),5.38(d,J=7.2Hz,1H),4.22-4.35(m,1H),1.57(t,J=19.4Hz,3H),1.34( d, J=6.4 Hz, 3H). MS-ESI m/z: 463.3 [M+H] + .
实施例13：化合物WX013的合成Example 13: Synthesis of compound WX013
Figure PCTCN2020141640-appb-000061
Figure PCTCN2020141640-appb-000061
步骤1：化合物WX013-2的合成Step 1: Synthesis of compound WX013-2
向干燥的单口瓶中加入化合物WX013-1(50g,204.07mmol)和N,N-二甲基甲酰胺(500mL)，0℃下分批加入氢化钠(9.79g,244.88mmol,60％含量)。搅拌0.5小时，加入2-(三甲基硅)乙氧基甲基氯(51.03g,306.10mmol)，置于25℃反应12小时。反应完毕，向反应液中加入饱和氯化铵水溶液(500mL)，乙酸乙酯(500mL×2)萃取。有机相合并，半饱和食盐水(200mL×2)洗，饱和食盐水(200mL)洗，无水硫酸钠干燥，过滤，滤液减压浓缩。粗品经柱层析(洗脱剂：石油醚/乙酸乙酯＝5/1-1/1，体积比)纯化，得到WX013-2。Add compound WX013-1 (50g, 204.07mmol) and N,N-dimethylformamide (500mL) to a dry single-neck bottle, add sodium hydride (9.79g, 244.88mmol, 60% content) in batches at 0°C . After stirring for 0.5 hours, 2-(trimethylsilyl)ethoxymethyl chloride (51.03 g, 306.10 mmol) was added, and the mixture was placed at 25° C. to react for 12 hours. After the reaction was completed, saturated ammonium chloride aqueous solution (500 mL) was added to the reaction solution, and ethyl acetate (500 mL×2) was extracted. The organic phases were combined, washed with half-saturated brine (200 mL×2), washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product is purified by column chromatography (eluent: petroleum ether/ethyl acetate = 5/1-1/1, volume ratio) to obtain WX013-2.
MS–ESI m/z:376.0[M+H] +。 MS–ESI m/z: 376.0 [M+H] + .
步骤2：化合物WX013-3的合成Step 2: Synthesis of compound WX013-3
在干燥的单口瓶中加入化合物WX004-1(7g,25.07mmol)和正丁腈(75mL)，充分溶解之后依次 加入化合物WX013-2(7.84g,20.89mmol,1.00eq)，碳酸铯(20.42g,62.68mmol,3eq)和N,N-二甲基甘氨酸(3.23g,31.34mmol,1.5eq)，氮气置换三次后加入碘化亚铜(1.99g,10.45mmol,0.5eq)，再氮气置换三次后，120℃搅拌反应12小时。反应完毕，向反应液中加入氨水(50mL)和水(50mL)，乙酸乙酯(100mL×2)萃取。有机相合并，无水硫酸钠干燥，过滤，滤液减压浓缩。粗品经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝3/1-1/1，体积比)纯化，得到目标化合物WX013-3。Add compound WX004-1 (7g, 25.07mmol) and n-butyronitrile (75mL) into a dry single-necked flask, and then add compound WX013-2 (7.84g, 20.89mmol, 1.00eq), cesium carbonate (20.42g, 62.68mmol, 3eq) and N,N-dimethylglycine (3.23g, 31.34mmol, 1.5eq), after nitrogen replacement three times, add cuprous iodide (1.99g, 10.45mmol, 0.5eq), and then nitrogen replacement three times , The reaction was stirred at 120°C for 12 hours. After the reaction was completed, ammonia water (50 mL) and water (50 mL) were added to the reaction solution, and extracted with ethyl acetate (100 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate=3/1-1/1, volume ratio) to obtain the target compound WX013-3.
MS–ESI m/z:527.1[M+H] +。 MS–ESI m/z: 527.1 [M+H] + .
步骤3：化合物WX013-4的合成Step 3: Synthesis of compound WX013-4
向干燥的单口瓶中加入化合物WX013-3(6.6g,12.53mmol)和盐酸乙酸乙酯溶液(4mol/L,32.69mL)，保持25℃反应12小时。反应完毕，将反应液减压浓缩。残余物中加入饱和碳酸氢钠水溶液(50mL)，乙酸乙酯(50mL×2)萃取。有机相合并，无水硫酸钠干燥，过滤，滤液减压浓缩。粗品经柱层析(洗脱剂：石油醚/乙酸乙酯＝19/1–7/3，体积比)分离纯化，得到WX013-4。Compound WX013-3 (6.6 g, 12.53 mmol) and hydrochloric acid ethyl acetate solution (4 mol/L, 32.69 mL) were added to the dry single-neck flask, and the reaction was maintained at 25° C. for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure. Saturated aqueous sodium bicarbonate solution (50 mL) was added to the residue, and extracted with ethyl acetate (50 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate = 19/1-7/3, volume ratio) to obtain WX013-4.
1H NMR(400MHz,DMSO-d 6)δ＝8.72(d,J＝8.8Hz,1H),7.71-7.97(m,1H),7.08-7.20(m,4H),5.34(d,J＝7.2Hz,1H),4.15-4.34(m,1H),1.56(t,J＝19.4Hz,3H),1.32(d,J＝6.4Hz,3H)。 1 H NMR(400MHz,DMSO-d 6 )δ=8.72(d,J=8.8Hz,1H),7.71-7.97(m,1H),7.08-7.20(m,4H),5.34(d,J=7.2 Hz, 1H), 4.15-4.34 (m, 1H), 1.56 (t, J=19.4 Hz, 3H), 1.32 (d, J=6.4 Hz, 3H).
MS–ESI m/z:397.2[M+H] +。 MS–ESI m/z: 397.2 [M+H] + .
步骤4：化合物WX013的合成Step 4: Synthesis of compound WX013
将化合物WX013-4(300mg,756.93μmol)和4-溴噻唑(248.31mg,1.51mmol)加入N,N-二甲基甲酰胺(3mL)中，加入N,N-二甲基-1,2-环己二胺(107.67mg,756.93μmol)，碳酸铯(739.87mg,2.27mmol)和碘化亚铜(144.16mg,756.93μmol)。氮气置换三次后，反应液升温至100℃搅拌反应12小时。反应完毕，将反应液倒入水(15mL)中，加乙酸乙酯(10mL×3)萃取。有机相合并，加半饱和食盐水(30mL×2)洗，饱和食盐水(30mL)洗，无水硫酸钠干燥，过滤，滤液减压浓缩。粗品先经过柱层析(洗脱剂：石油醚/乙酸乙酯＝19/1–17/3，体积比)分离纯化，再进行SFC手性拆分(柱型:DAICEL CHIRALPAK AD(250mm×30mm×10μm)；流动相:[A:CO 2；B:0.1％NH 3H 2O EtOH]；B％:35％-35％,10min)纯化，得到化合物WX013。 Compound WX013-4 (300mg, 756.93μmol) and 4-bromothiazole (248.31mg, 1.51mmol) were added to N,N-dimethylformamide (3mL), and N,N-dimethyl-1,2 -Cyclohexanediamine (107.67 mg, 756.93 μmol), cesium carbonate (739.87 mg, 2.27 mmol) and cuprous iodide (144.16 mg, 756.93 μmol). After replacing with nitrogen three times, the reaction solution was heated to 100°C and stirred for 12 hours. After the reaction was completed, the reaction solution was poured into water (15 mL), and ethyl acetate (10 mL×3) was added for extraction. The organic phases were combined, washed with half-saturated brine (30 mL×2), washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate = 19/1–17/3, volume ratio), and then subjected to SFC chiral resolution (column type: DAICEL CHIRALPAK AD (250mm×30mm) ×10 μm); mobile phase: [A: CO 2 ; B: 0.1% NH 3 H 2 O EtOH]; B%: 35%-35%, 10 min) to obtain compound WX013.
1H NMR(400MHz,DMSO-d 6)δ＝9.38(d,J＝2.0Hz,1H),8.78(d,J＝8.8Hz,1H),8.23(d,J＝2.0Hz,1H),8.14(dd,J＝8.4,1.6Hz,1H),7.45(s,1H),7.41-7.45(m,1H),7.11-7.20(m,3H),5.39(d,J＝7.2Hz,1H),4.19-4.36(m,1H),1.56(t,J＝19.6Hz,3H),1.34(d,J＝6.8Hz,3H)。 1 H NMR(400MHz,DMSO-d 6 )δ=9.38(d,J=2.0Hz,1H), 8.78(d,J=8.8Hz,1H), 8.23(d,J=2.0Hz,1H), 8.14 (dd,J=8.4,1.6Hz,1H),7.45(s,1H),7.41-7.45(m,1H),7.11-7.20(m,3H),5.39(d,J=7.2Hz,1H), 4.19-4.36 (m, 1H), 1.56 (t, J = 19.6 Hz, 3H), 1.34 (d, J = 6.8 Hz, 3H).
MS–ESI m/z:480.1[M+H] +。 MS–ESI m/z: 480.1 [M+H] + .
参照实施例13中的合成方法，用片段2替换WX004-1，合成下表中各实施例。Referring to the synthesis method in Example 13, replace WX004-1 with fragment 2 to synthesize the examples in the following table.
Figure PCTCN2020141640-appb-000062
Figure PCTCN2020141640-appb-000062
实施例15：化合物WX015的合成Example 15: Synthesis of compound WX015
Figure PCTCN2020141640-appb-000063
Figure PCTCN2020141640-appb-000063
步骤1：化合物WX015-1的合成Step 1: Synthesis of compound WX015-1
在预先干燥好的单口瓶中加入化合物CG-1-1(10g,37.45mmol)和吡啶-2-胺(3.52g,37.45mmol)。用N,N-二甲基甲酰胺(100mL)溶解后，加入叔丁醇钾(1.0M,56.18mL)，升温至60℃搅拌反应16小时。将反应液降温至室温后，加入到水(300mL)中，用乙酸乙酯(100mL x3)萃取。有机相合并，用半饱和食盐水(100mL x3)洗涤，无水硫酸钠干燥，过滤，减压浓缩至干。粗品经快速柱层析(石油醚:乙酸乙酯＝20:1至5:1，体积比)分离纯化得到WX015-1。Compound CG-1-1 (10g, 37.45mmol) and pyridin-2-amine (3.52g, 37.45mmol) were added to the pre-dried single-necked flask. After dissolving with N,N-dimethylformamide (100 mL), potassium tert-butoxide (1.0M, 56.18 mL) was added, and the temperature was raised to 60° C. and the reaction was stirred for 16 hours. After the reaction solution was cooled to room temperature, it was added to water (300 mL), and extracted with ethyl acetate (100 mL x 3). The organic phases were combined, washed with half-saturated brine (100 mL x3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to dryness. The crude product was separated and purified by flash column chromatography (petroleum ether: ethyl acetate = 20:1 to 5:1, volume ratio) to obtain WX015-1.
1H NMR(400MHz,DMSO-d 6)δ9.69(s,1H),8.28(d,J＝2.0Hz,1H),8.16(br d,J＝3.6Hz,1H),8.04-7.98(m,1H),7.92(dd,J＝1.9,9.0Hz,1H),7.73-7.66(m,1H),7.08(d,J＝8.4Hz,1H),6.94(dd,J＝5.3,6.8Hz,1H)。 1 H NMR(400MHz,DMSO-d 6 )δ9.69(s,1H), 8.28(d,J=2.0Hz,1H), 8.16(br d,J=3.6Hz,1H), 8.04-7.98(m ,1H),7.92(dd,J=1.9,9.0Hz,1H),7.73-7.66(m,1H),7.08(d,J=8.4Hz,1H),6.94(dd,J=5.3,6.8Hz, 1H).
MS–ESI m/z:342.0[M+H] +。 MS–ESI m/z: 342.0[M+H] + .
步骤2：化合物WX015-2的合成Step 2: Synthesis of compound WX015-2
向预先干燥的反应瓶中加入化合物WX015-1(0.5g,1.47mmol)和氯化铵(392.05mg,7.33mmol)，加入甲醇(10mL)，然后分批加入锌粉(479.25mg,7.33mmol)。加料完毕，反应液在25℃搅拌反应2小时。将反应液通过硅藻土过滤，滤饼用甲醇(20mL)淋洗。滤液减压浓缩，得到WX015-2。Add compound WX015-1 (0.5g, 1.47mmol) and ammonium chloride (392.05mg, 7.33mmol) to the pre-dried reaction flask, add methanol (10mL), and then add zinc powder (479.25mg, 7.33mmol) in batches . After the addition was completed, the reaction solution was stirred and reacted at 25°C for 2 hours. The reaction solution was filtered through Celite, and the filter cake was rinsed with methanol (20 mL). The filtrate was concentrated under reduced pressure to obtain WX015-2.
1H NMR(400MHz,DMSO-d 6)δ＝8.03(dd,J＝1.4,5.0Hz,1H),7.86(s,1H),7.48(ddd,J＝1.9,7.0, 8.4Hz,1H),7.15(d,J＝8.4Hz,1H),7.08(d,J＝2.0Hz,1H),6.84(dd,J＝2.2,8.3Hz,1H),6.68-6.59(m,2H),5.05(s,2H)。 1 H NMR(400MHz,DMSO-d 6 )δ=8.03(dd,J=1.4,5.0Hz,1H),7.86(s,1H),7.48(ddd,J=1.9,7.0, 8.4Hz,1H), 7.15 (d, J = 8.4 Hz, 1H), 7.08 (d, J = 2.0 Hz, 1H), 6.84 (dd, J = 2.2, 8.3 Hz, 1H), 6.68-6.59 (m, 2H), 5.05 (s ,2H).
步骤3：化合物WX015-3的合成Step 3: Synthesis of compound WX015-3
向干燥的单口瓶中加入浓盐酸(4mL)，后加入化合物WX015-2(0.4g,1.29mmol)，降温至0℃，将亚硝酸钠(266.12mg,3.86mmol)用水(2mL)溶解后缓慢滴入反应液中。滴毕，升温至25℃反应14小时。将反应液过滤，滤饼用水(10mL)洗涤。收集所得固体，真空干燥，得到WX015-3。Add concentrated hydrochloric acid (4mL) to the dry single-neck flask, then add compound WX015-2 (0.4g, 1.29mmol), cool to 0℃, dissolve sodium nitrite (266.12mg, 3.86mmol) in water (2mL) and slowly Drop into the reaction solution. After dropping, the temperature was raised to 25°C for 14 hours. The reaction solution was filtered, and the filter cake was washed with water (10 mL). The obtained solid was collected and dried under vacuum to obtain WX015-3.
1H NMR(400MHz,DMSO-d 6)δ＝8.73-8.67(m,1H),8.64(d,J＝0.8Hz,1H),8.38(d,J＝8.6Hz,1H),8.27-8.21(m,1H),8.19-8.12(m,1H),7.97(dd,J＝1.3,8.8Hz,1H),7.55(dd,J＝5.0,6.6Hz,1H)。 1 H NMR(400MHz, DMSO-d 6 )δ=8.73-8.67(m,1H), 8.64(d,J=0.8Hz,1H), 8.38(d,J=8.6Hz,1H), 8.27-8.21( m, 1H), 8.19-8.12 (m, 1H), 7.97 (dd, J=1.3, 8.8 Hz, 1H), 7.55 (dd, J=5.0, 6.6 Hz, 1H).
步骤4：化合物WX015的合成Step 4: Synthesis of compound WX015
在预先干燥的反应瓶中加入化合物WX004-1(50mg,179.06μmol)，化合物WX015-3(57.68mg,179.06μmol)和正丁腈(2mL)，然后加入碳酸铯(175.03mg,537.19μmol)，N,N-二甲基甘氨酸(18.47mg,179.06μmol)，最后加入碘化亚铜(17.05mg,89.53μmol)。反应在氮气氛围下于120℃搅拌16小时。反应液降至室温后，倒入水(30mL)和氨水(10mL)，乙酸乙酯(30mL x3)萃取。有机相合并，用饱和食盐水(50mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩至干。粗品经制备HPLC(柱型:Xtimate C18 100*30mm*3μm；流动相:[H 2O(0.04％HCl)-ACN]；B(ACN)％:47％-77％,8min)分离纯化得到WX015。 Add compound WX004-1 (50 mg, 179.06 μmol), compound WX015-3 (57.68 mg, 179.06 μmol) and n-butyronitrile (2 mL) into the pre-dried reaction flask, and then add cesium carbonate (175.03 mg, 537.19 μmol), N , N-Dimethylglycine (18.47mg, 179.06μmol), and finally copper iodide (17.05mg, 89.53μmol) was added. The reaction was stirred at 120°C for 16 hours under a nitrogen atmosphere. After the reaction solution dropped to room temperature, it was poured into water (30 mL) and ammonia (10 mL), and extracted with ethyl acetate (30 mL x 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The crude product was separated and purified by preparative HPLC (column type: Xtimate C18 100*30mm*3μm; mobile phase: [H 2 O(0.04%HCl)-ACN]; B(ACN)%: 47%-77%, 8min) to obtain WX015 .
1H NMR(400MHz,DMSO-d 6)δ＝8.78-8.71(m,1H),8.64(br d,J＝4.0Hz,1H),8.56(d,J＝9.2Hz,1H),8.23(d,J＝8.4Hz,1H),8.08-8.02(m,1H),7.47-7.41(m,2H),7.31(d,J＝2.0Hz,1H),7.10(br d,J＝6.4Hz,2H),6.91-6.85(m,1H),5.37(d,J＝6.6Hz,1H),4.44-4.35(m,1H),1.61(t,J＝19.2Hz,3H),1.41(d,J＝6.8Hz,3H)。 1 H NMR(400MHz,DMSO-d 6 )δ=8.78-8.71(m,1H), 8.64(br d,J=4.0Hz,1H), 8.56(d,J=9.2Hz,1H), 8.23(d ,J=8.4Hz,1H),8.08-8.02(m,1H),7.47-7.41(m,2H),7.31(d,J=2.0Hz,1H),7.10(br d,J=6.4Hz,2H ),6.91-6.85(m,1H),5.37(d,J=6.6Hz,1H),4.44-4.35(m,1H),1.61(t,J=19.2Hz,3H),1.41(d,J= 6.8Hz, 3H).
MS–ESI m/z:474.2[M+H] +。 MS–ESI m/z: 474.2 [M+H] + .
参照实施例15中的合成方法，用片段2替换吡啶-2-胺，合成下表中各实施例。Referring to the synthesis method in Example 15, substituting fragment 2 for pyridin-2-amine, each example in the following table was synthesized.
Figure PCTCN2020141640-appb-000064
Figure PCTCN2020141640-appb-000064
各实施例中目标化合物的 1HNMR和MS数据 1 HNMR and MS data of the target compound in each example
Figure PCTCN2020141640-appb-000065
Figure PCTCN2020141640-appb-000065
Figure PCTCN2020141640-appb-000066
Figure PCTCN2020141640-appb-000066
Figure PCTCN2020141640-appb-000067
Figure PCTCN2020141640-appb-000067
实验例1：体外检测化合物在荧光素酶报告基因筛选体系下抑制hMMP1转录活性Experimental example 1: In vitro detection compounds inhibit hMMP1 transcription activity under the luciferase reporter gene screening system
实验目的：Purpose:
人类MMP-1启动子区(含两个AP-1结合位点和两个PEA3位点，共249bp，基因库目录#AF023338)克隆到荧光素酶报告基因的上游。构建hMMP-1启动子报告基因并转染到Hela细胞中，从而可以容易地检测荧光素酶的产生。稳定重组细胞hMMP-1/荧光素酶的细胞株被用于本实验的开发和验证。培养基及试剂：The human MMP-1 promoter region (contains two AP-1 binding sites and two PEA3 sites, a total of 249 bp, gene bank catalog #AF023338) was cloned upstream of the luciferase reporter gene. The hMMP-1 promoter reporter gene was constructed and transfected into Hela cells, so that the production of luciferase could be easily detected. The stable recombinant hMMP-1/luciferase cell line was used for the development and verification of this experiment. Medium and reagents:
1.常规细胞培养基1. Conventional cell culture medium
DMEM 90％,FBS 10％,1mM NAA,1mM Sodium Pyruvate,4mM L-Glutamine,300μg/mL G418,4℃储存。DMEM 90%, FBS 10%, 1mM NAA, 1mM Sodium Pyruvate, 4mM L-Glutamine, 300μg/mL G418, stored at 4℃.
2.冷冻液2. Freezing fluid
DMEM 75％,FBS 20％,DMSO 5％。使用前配制。DMEM 75%, FBS 20%, DMSO 5%. Prepare before use.
3.实验细胞培养基3. Experimental cell culture medium
DMEM 97％,活性炭处理血清(Charcoal stripped serum)3％。DMEM 97%, activated charcoal treated serum (Charcoal stripped serum) 3%.
4.Bright-Glo试剂盒4.Bright-Glo kit
将一瓶Bright Glo Buffer全部转移到棕色的荧光底物瓶中，倒置混合直至底物完全溶解，适量分装，-80℃储存。Transfer a bottle of Bright Glo Buffer to the brown fluorescent substrate bottle, mix it upside down until the substrate is completely dissolved, aliquot the appropriate amount and store at -80°C.
方法method
冷冻细胞制备细胞悬浮液Frozen cells to prepare cell suspension
1.细胞解冻1. Thaw cells
1)冻存细胞快速解冻，置于37℃水浴中不断搅拌直至完全溶解。1) The frozen cells are quickly thawed and placed in a 37°C water bath with constant stirring until completely dissolved.
2)将上述细胞加入到15mL离心管中(内含有5mL预热的细胞培养基)，然后1000转，离心5分钟。2) Add the above-mentioned cells to a 15mL centrifuge tube (containing 5mL of pre-warmed cell culture medium), then centrifuge at 1000 rpm for 5 minutes.
3)弃上清液，加入5mL预热细胞培养基重悬细胞。3) Discard the supernatant and add 5 mL of pre-warmed cell culture medium to resuspend the cells.
4)将细胞悬浮液转移至60mM细胞培养皿中，置于37℃5％CO 2培养箱中培养。. 4) Transfer the cell suspension to a 60mM cell culture dish and place it in a 37°C 5% CO 2 incubator for culture. .
2.传代培养2. Subculture
1)当细胞生长达到80-90％时，进行细胞传代培养。pGL6.0-TA-hMMP-1 HeLa细胞通常每周传代两次，1:3或1:6稀释传代。1) When the cell growth reaches 80-90%, the cells are subcultured. pGL6.0-TA-hMMP-1 HeLa cells are usually passaged twice a week, diluted 1:3 or 1:6.
2)小心吸出所有培养基，用适量DPBS轻轻冲洗细胞层，然后吸出。2) Carefully aspirate all the medium, gently rinse the cell layer with an appropriate amount of DPBS, and then aspirate.
3)加适量的0.05％Typsine EDTA置于CO 2培养箱孵育在3-5分钟消化细胞。 3) Add an appropriate amount of 0.05% Typsine EDTA in a CO 2 incubator and incubate for 3-5 minutes to digest the cells.
4)用适量的预热的细胞培养基重悬细胞，并稀释传代培养。4) Resuspend the cells with an appropriate amount of pre-warmed cell culture medium, and dilute and subculture.
3.每隔一天换一次培养基3. Change the medium every other day
1)轻轻吸掉所有培养基。1) Gently aspirate all the medium.
2)加入新鲜细胞培养基(37℃预热)(100mm皿加10mL或150mm皿加入20mL)。2) Add fresh cell culture medium (preheated at 37°C) (100mm dish with 10mL or 150mm dish with 20mL).
4.冻存细胞4. Freeze cells
1)重复传代培养步骤1-4。1) Repeat the subculture steps 1-4.
2)细胞1000转离心5分钟。2) Centrifuge the cells at 1000 rpm for 5 minutes.
3)吸去上清液，用冻存液重新悬浮，计数并稀释到(2-3)×10 6个/mL。每个细胞冷冻管中加入1mL悬浮细胞。 3) Aspirate the supernatant, resuspend with cryopreservation solution, count and dilute to (2-3)×10 6 /mL. Add 1 mL of suspended cells to each cell freezing tube.
4)把冻存管放入冻存盒中，然后将冻存盒转移到-80℃冰箱中并过夜。4) Put the cryopreservation tube into the freezer box, and then transfer the freezer box to -80°C refrigerator and overnight.
5)将冻存管转移到到液氮中(-196℃)。5) Transfer the cryotube to liquid nitrogen (-196°C).
5.接种细胞5. Seed cells
1)小心吸出所有培养基，用适量DPBS轻轻冲洗细胞层，然后吸出。1) Carefully aspirate all the medium, gently rinse the cell layer with an appropriate amount of DPBS, and then aspirate.
2)加适量的0.05％Typsine EDTA置于CO 2培养箱孵育在3-5分钟消化细胞。 2) Add an appropriate amount of 0.05% Typsine EDTA in a CO 2 incubator and incubate for 3-5 minutes to digest the cells.
3)用适量的细胞培养基重新悬浮细胞。3) Resuspend the cells with an appropriate amount of cell culture medium.
4)计算所需的细胞量。细胞浓度为5×10 3个/孔。 4) Calculate the amount of cells required. The cell concentration is 5×10 3 cells/well.
5)用适当的细胞培养液稀释细胞悬浮液。5) Dilute the cell suspension with an appropriate cell culture medium.
6)细胞悬浮液分装到一次性无菌加样槽中。6) Dispense the cell suspension into a disposable aseptic sample tank.
7)30μL每孔接种到384孔板中。7) Inoculate 30 μL per well into a 384-well plate.
8)细胞板放置在37℃5％CO 2培养箱培养18-24小时。 8) The cell plate is placed in a 37°C 5% CO 2 incubator for 18-24 hours.
化合物配制Compound formulation
1.PMA:1.PMA:
PMA用DMSO稀释溶解到10mM，避光分装保存在-80℃冰箱中备用。Dilute PMA with DMSO and dissolve it to 10mM, store in aliquots protected from light in a refrigerator at -80℃ for later use.
2.地塞米松(Dexamethasone):2.Dexamethasone (Dexamethasone):
用DMSO稀释溶解到30mM，避光分装保存在-80℃冰箱中备用。Dilute and dissolve with DMSO to 30mM, and store in -80℃ refrigerator for later use, protected from light.
3. 10倍浓度化合物配制:3. Preparation of 10 times concentration compound:
试验化合物用DMSO稀释到30mM，分装保存在-80℃冰箱中备用。The test compound was diluted to 30 mM with DMSO, and stored in aliquots in a refrigerator at -80°C for later use.
用DMSO稀释化合物到1mM,0.25mM,0.0625mM,0.015625mM,0.0039mM,0.0009765mM,0.000244mM,0.000061mM,0.00001526mM和0.0000038125mM，然后用含有100nM的PMA无血清 培养基稀释100倍，最后得到10×浓度化合物实验板。Dilute the compound with DMSO to 1 mM, 0.25 mM, 0.0625 mM, 0.015625 mM, 0.0039 mM, 0.0009765 mM, 0.000244 mM, 0.000061 mM, 0.00001526 mM and 0.0000038 125 mM, then dilute 100 times with PMA serum-free medium containing 100 nM, and finally get 10× concentration compound experiment plate.
最终DMSO浓度为0.1％。PMA使用中需要避光。The final DMSO concentration is 0.1%. PMA needs to be protected from light during use.
hMMP1 GR转录抑制活性试验hMMP1 GR transcription inhibitory activity test
1)接种细胞：新鲜细胞以5×10 3个/30μL/孔接种到384白色实验板中，在37℃5％CO 2培养箱中培养24小时。 1) Inoculation of cells: Fresh cells were inoculated into a 384 white experiment plate at 5×10 3 cells/30 μL/well, and cultured in a 37°C 5% CO 2 incubator for 24 hours.
2)化合物配制：在实验开始前配置好化合物板，准备好10×浓度的参照化合物(地塞米松)和受试化合物，最后得到10×浓度化合物实验板。2) Compound preparation: prepare the compound board before the start of the experiment, prepare a 10× concentration of the reference compound (dexamethasone) and test compound, and finally obtain a 10× concentration compound test board.
3)加入化合物：用Bravo转移3.3μL 10×化合物并加入到细胞板中。在37℃5％CO 2培养箱中培养18小时。 3) Add compound: Transfer 3.3 μL of 10× compound with Bravo and add to the cell plate. Incubate in a 37°C 5% CO 2 incubator for 18 hours.
4)30μL Bright-Glo荧光检测试剂转入到细胞板中。4) 30μL Bright-Glo fluorescence detection reagent is transferred to the cell plate.
5)离心孵育2分钟5) Centrifuge and incubate for 2 minutes
6)用Envision读板机测定荧光数值。6) Measure the fluorescence value with Envision plate reader.
数据处理及分析Data processing and analysis
阳性对照：10nM PMA+1000nM地塞米松(0.1％DMSO)Positive control: 10nM PMA+1000nM dexamethasone (0.1% DMSO)
阴性对照：10nM PMA(0.1％DMSO)Negative control: 10nM PMA (0.1% DMSO)
试验化合物：最高浓度1000nM，4倍稀释，共10个孔，重复。Test compound: the highest concentration is 1000 nM, diluted 4 times, 10 wells in total, repeat.
地塞米松：最高浓度1000nM，4倍稀释，共10个孔，重复。Dexamethasone: the highest concentration is 1000nM, diluted 4 times, a total of 10 wells, repeat.
利用作图软件GraphPad Prism5制作受试化合物浓度曲线，计算IC 50值。实验结果见表1。 Produced using mapping software GraphPad Prism5 concentration of test compound curve, 50 value IC. The experimental results are shown in Table 1.
实验例2：体外检测化合物在荧光素酶报告基因筛选体系下激活MMTV转录活性Experimental Example 2: In vitro detection compounds activate MMTV transcription activity under the luciferase reporter gene screening system
实验目的：Purpose:
小鼠乳腺肿瘤病毒(MMTV)的启动子包含激活GR(GREs)的特异性结合位点。为了确定化合物的活性，在MMTV启动子后面插入了一个报告基因(荧光素酶)，并且该结构在HeLa细胞系的基因组中以稳定的方式表达。采用待测化合物激活MMTV启动子，诱导荧光素酶的表达，通过发光测量来检测其活性。The promoter of mouse mammary tumor virus (MMTV) contains a specific binding site that activates GR (GREs). In order to determine the activity of the compound, a reporter gene (luciferase) was inserted after the MMTV promoter, and the structure was expressed in a stable manner in the genome of the HeLa cell line. The test compound is used to activate the MMTV promoter, induce the expression of luciferase, and detect its activity by luminescence measurement.
培养基及试剂：Medium and reagents:
1.常规细胞培养基1. Conventional cell culture medium
DMEM 90％,FBS 10％,Hygromycin 100μg/mLDMEM 90%, FBS 10%, Hygromycin 100μg/mL
2.冷冻液2. Freezing fluid
DMEM 75％,FBS 20％,DMSO 5％。DMEM 75%, FBS 20%, DMSO 5%.
3.实验细胞培养基3. Experimental cell culture medium
DMEM 97％,活性炭处理血清(Charcoal stripped serum)3％。DMEM 97%, activated charcoal treated serum (Charcoal stripped serum) 3%.
4.Bright-Glo试剂盒4.Bright-Glo kit
将一瓶Bright Glo Buffer全部转移到棕色的荧光底物瓶中，倒置混合直至底物完全溶解，适量分装，-80℃储存。Transfer a bottle of Bright Glo Buffer to the brown fluorescent substrate bottle, mix it upside down until the substrate is completely dissolved, aliquot the appropriate amount and store at -80°C.
方法method
冷冻细胞制备细胞悬浮液Frozen cells to prepare cell suspension
1.细胞解冻1. Thaw cells
1)将冻存细胞置于37℃水浴中不断搅拌直至完全解冻。1) Place the frozen cells in a 37°C water bath and keep stirring until they are completely thawed.
2)将上述细胞加入到15mL离心管中(内含有5mL预热的细胞培养基)，然后1000转，离心5分钟。2) Add the above-mentioned cells to a 15mL centrifuge tube (containing 5mL of pre-warmed cell culture medium), then centrifuge at 1000 rpm for 5 minutes.
3)弃上清液，加入5mL预热细胞培养基重悬细胞。3) Discard the supernatant and add 5 mL of pre-warmed cell culture medium to resuspend the cells.
4)将细胞悬浮液转移至60mm细胞培养皿中，置于37℃5％CO 2培养箱中培养。. 4) Transfer the cell suspension to a 60mm cell culture dish and place it in a 37°C 5% CO 2 incubator for culture. .
2.传代培养2. Subculture
1)当细胞生长达到80-90％时，进行细胞传代培养。通常每周传代两次，1:3或1:6稀释传代。1) When the cell growth reaches 80-90%, the cells are subcultured. Usually passaged twice a week, diluted 1:3 or 1:6.
2)小心吸出所有培养基，用适量DPBS轻轻冲洗细胞层，然后吸出。2) Carefully aspirate all the medium, gently rinse the cell layer with an appropriate amount of DPBS, and then aspirate.
3)加适量的0.05％Typsine EDTA置于CO 2培养箱孵育在3-5分钟消化细胞。 3) Add an appropriate amount of 0.05% Typsine EDTA in a CO 2 incubator and incubate for 3-5 minutes to digest the cells.
4)用适量的预热的细胞培养基重悬细胞，并稀释传代培养。4) Resuspend the cells with an appropriate amount of pre-warmed cell culture medium, and dilute and subculture.
3.每隔一天换一次培养基3. Change the medium every other day
1)轻轻吸掉所有培养基。1) Gently aspirate all the medium.
2)加入新鲜细胞培养基(37℃预热)(100mm皿加10mL或150mm皿加入20mL)。2) Add fresh cell culture medium (preheated at 37°C) (100mm dish with 10mL or 150mm dish with 20mL).
4.冻存细胞4. Freeze cells
1)重复传代培养步骤1-4。1) Repeat the subculture steps 1-4.
2)细胞1000转离心5分钟。2) Centrifuge the cells at 1000 rpm for 5 minutes.
3)吸去上清液，用冻存液重新悬浮，计数并稀释到(2-3)×10 6个/mL。每个细胞冷冻管中加入1mL悬浮细胞。 3) Aspirate the supernatant, resuspend with cryopreservation solution, count and dilute to (2-3)×10 6 /mL. Add 1 mL of suspended cells to each cell freezing tube.
4)把冻存管放入冻存盒中，然后将冻存盒转移到-80℃冰箱中并过夜。4) Put the cryopreservation tube into the freezer box, and then transfer the freezer box to -80°C refrigerator and overnight.
5)将冻存管转移到到液氮中(-196℃)。5) Transfer the cryotube to liquid nitrogen (-196°C).
5.接种细胞5. Seed cells
1)小心吸出所有培养基，用适量DPBS轻轻冲洗细胞层，然后吸出。1) Carefully aspirate all the medium, gently rinse the cell layer with an appropriate amount of DPBS, and then aspirate.
2)加适量的0.05％Typsine EDTA置于CO 2培养箱孵育在3-5分钟消化细胞。 2) Add an appropriate amount of 0.05% Typsine EDTA in a CO 2 incubator and incubate for 3-5 minutes to digest the cells.
3)用适量的细胞培养基重新悬浮细胞。3) Resuspend the cells with an appropriate amount of cell culture medium.
4)计算所需的细胞量，细胞浓度为4×10 3个/孔。 4) Calculate the amount of cells required, and the cell concentration is 4×10 3 cells/well.
5)用适当的细胞培养液稀释细胞悬浮液。5) Dilute the cell suspension with an appropriate cell culture medium.
6)细胞悬浮液分装到一次性无菌加样槽中。6) Dispense the cell suspension into a disposable aseptic sample tank.
7)30μL每孔接种到384孔板中。7) Inoculate 30 μL per well into a 384-well plate.
8)细胞板放置在37℃5％CO 2培养箱培养18-24小时。 8) The cell plate is placed in a 37°C 5% CO 2 incubator for 18-24 hours.
化合物配制Compound formulation
1.地塞米松(Dexamethasone):1. Dexamethasone (Dexamethasone):
用DMSO稀释溶解到30mM，避光分装保存在-80℃冰箱中备用。Dilute and dissolve with DMSO to 30mM, and store in -80℃ refrigerator for later use, protected from light.
2. 4倍浓度化合物配制:2. Preparation of 4 times concentration compound:
试验化合物用DMSO稀释到30mM，分装保存在-80℃冰箱中备用。The test compound was diluted to 30 mM with DMSO, and stored in aliquots in a refrigerator at -80°C for later use.
用DMSO稀释化合物到1mM,0.25mM,0.0625mM,0.015625mM,0.0039mM,0.0009765mM,0.000244mM,0.000061mM,0.00001526mM和0.0000038125mM，然后用含活性炭处理血清培养基稀释25倍，最后得到4×浓度化合物实验板，使用前配制。Dilute the compound with DMSO to 1 mM, 0.25 mM, 0.0625 mM, 0.015625 mM, 0.0039 mM, 0.0009765 mM, 0.000244 mM, 0.000061 mM, 0.00001526 mM and 0.0000038 125 mM, and then use activated charcoal to treat the serum medium and dilute it by 25 times to obtain 4× Concentration compound experiment board, prepared before use.
最终DMSO实验浓度为0.1％。The final experimental concentration of DMSO is 0.1%.
MMTV_GR转录激活活性试验MMTV_GR transcription activation activity test
1)接种细胞：新鲜细胞以4×10 3个/30μL/孔接种到384白色透明底实验板中，在37℃5％CO 2培养箱中培养24小时。 1) Inoculation of cells: Fresh cells were inoculated into a 384 white transparent bottom experimental plate at 4×10 3 cells/30 μL/well, and cultured in a 37°C 5% CO 2 incubator for 24 hours.
2)化合物配制：在实验开始前配置好化合物板，准备好4×浓度的参照化合物(地塞米松)和受试化合物，最后得到4×浓度化合物实验板。2) Compound preparation: prepare a compound board before the start of the experiment, prepare a 4× concentration of the reference compound (dexamethasone) and the test compound, and finally obtain a 4× concentration compound test board.
3)加入化合物：用Bravo转移10μL 4×化合物并加入到细胞板中。在37℃5％CO 2培养箱中培养18小时。 3) Add compound: Transfer 10 μL of 4× compound with Bravo and add to the cell plate. Incubate in a 37°C 5% CO 2 incubator for 18 hours.
4)30μL Bright-Glo荧光检测试剂转入到细胞板中。4) 30μL Bright-Glo fluorescence detection reagent is transferred to the cell plate.
5)离心孵育2分钟5) Centrifuge and incubate for 2 minutes
6)用Envision读板机测定荧光数值。6) Measure the fluorescence value with Envision plate reader.
数据处理及分析Data processing and analysis
阳性对照：1000nM地塞米松(0.1％DMSO)Positive control: 1000nM dexamethasone (0.1% DMSO)
阴性对照：0.1％DMSONegative control: 0.1% DMSO
试验化合物：最高浓度1000nM，4倍稀释，共10个孔，重复。Test compound: the highest concentration is 1000 nM, diluted 4 times, 10 wells in total, repeat.
地塞米松：最高浓度1000nM，4倍稀释，共10个孔，重复。Dexamethasone: the highest concentration is 1000nM, diluted 4 times, a total of 10 wells, repeat.
利用作图软件GraphPad Prism5制作受试化合物浓度曲线，计算IC 50值。 Produced using mapping software GraphPad Prism5 concentration of test compound curve, 50 value IC.
实验结果见表1。The experimental results are shown in Table 1.
表1本发明化合物体外筛选测试结果Table 1 In vitro screening test results of the compounds of the invention
Figure PCTCN2020141640-appb-000068
Figure PCTCN2020141640-appb-000068
Figure PCTCN2020141640-appb-000069
Figure PCTCN2020141640-appb-000069
注：IC 50为绝对IC 50；EC 50为绝对EC 50；Effect代表最大效应值。 Note: IC 50 is absolute IC 50 ; EC 50 is absolute EC 50 ; Effect represents the maximum effect value.
结论：本发明化合物显示了很好的转录抑制活性及相当的转录激活活性。Conclusion: The compound of the present invention shows very good transcription inhibitory activity and equivalent transcription activation activity.
实验例3：体外检测化合物对人外周血单核细胞TNF-α的抑制活性Experimental example 3: In vitro detection of the compound's inhibitory activity on human peripheral blood mononuclear cells TNF-α
实验目的：根据人外周血单核细胞(hPBMC)中TNF-α的水平来表达测试化合物细胞水平的抗炎活性。Experimental purpose: according to the level of TNF-α in human peripheral blood mononuclear cells (hPBMC) to express the anti-inflammatory activity of the test compound at the cellular level.
培养基及试剂：Medium and reagents:
PRMI 1640培养基(Invitrogen-11875093,lot 2003787)PRMI 1640 medium (Invitrogen-11875093, lot 2003787)
双抗(PS)(Invitrogen-15140122,lot 2019317)Double antibody (PS) (Invitrogen-15140122, lot 2019317)
胎牛血清(FBS)(Gibco-10091148,lot 1989478)Fetal Bovine Serum (FBS) (Gibco-10091148, lot 1989478)
台盼蓝(Gibco-15250061,lot 1311086)Trypan Blue (Gibco-15250061, lot 1311086)
TNF-αElisa试剂盒Set A(BD-555212,lot 7171693),TNF-αElisa Kit Set A (BD-555212, lot 7171693),
TNF-a Elisa试剂盒Set B(BD-550534,lot 9095783)TNF-a Elisa Kit Set B (BD-550534, lot 9095783)
PBMC细胞(AllCells,Cat.PB006F-C，lot LP190225B):RPMI 1640+10％FBS(Gibico)+1％PSPBMC cells (AllCells, Cat.PB006F-C, lot LP190225B): RPMI 1640 + 10% FBS (Gibico) + 1% PS
方法method
实验步骤：Experimental steps:
1.PBMC实验1. PBMC experiment
1)复苏细胞1) Resuscitate cells
a)在37℃水浴锅中连续搅拌，快速解冻冷冻细胞。a) Stir continuously in a 37°C water bath to quickly thaw frozen cells.
b)在50mL离心管中加入25毫升新鲜的预热解冻培养基。然后轻轻一点一滴地加入细胞。b) Add 25 ml of fresh preheated thawing medium to a 50 ml centrifuge tube. Then add the cells little by little.
c)将细胞以2000rpm离心10分钟(升速9，降速0)。c) Centrifuge the cells at 2000 rpm for 10 minutes (increasing speed 9, slowing down 0).
d)丢弃上清液，将细胞颗粒重新悬浮于25mL新鲜预热的RPMI 1640培养基中。d) Discard the supernatant, and resuspend the cell pellet in 25 mL of freshly pre-warmed RPMI 1640 medium.
2)在96孔板中种细胞2) Seed cells in 96-well plates
a)解冻复苏重悬细胞后，计数细胞数，计算实验所需细胞总数。a) After thawing, resuscitating and resuspending the cells, count the number of cells and calculate the total number of cells required for the experiment.
b)用适当体积的细胞培养基稀释细胞悬液。b) Dilute the cell suspension with an appropriate volume of cell culture medium.
c)将细胞悬液倒入无菌的一次性容器中。c) Pour the cell suspension into a sterile disposable container.
d)转移100000细胞/(100μl)的细胞悬液到96孔板每个孔中。d) Transfer 100,000 cells/(100μl) of cell suspension to each well of 96-well plate.
e)将细胞板放入加湿的空气中，5％CO 2培养箱，37℃，放置2小时。 e) Put the cell plate in humidified air, 5% CO 2 incubator, 37°C, and place it for 2 hours.
2.化合物剂量梯度稀释2. Compound dose gradient dilution
第一步将化合物从储藏浓度10mM用100％DMSO稀释到1mM，作为第一个点用100％DMSO3倍稀释8个点。第二步用不含有血清的培养基125倍稀释，此时DMSO的浓度是0.8％。然后转16.8μL已经用培养基稀释好的化合物到100μL的细胞板里，此时DMSO的浓度是0.114％。加好化合物后将细胞板放入37℃，5％CO 2培养箱中孵育1个小时。 In the first step, the compound was diluted from a storage concentration of 10 mM with 100% DMSO to 1 mM, and as the first spot, it was diluted 3 times with 100% DMSO for 8 spots. The second step is 125-fold dilution with serum-free medium. At this time, the DMSO concentration is 0.8%. Then transfer 16.8 μL of the compound that has been diluted with culture medium to a 100 μL cell plate, at which time the concentration of DMSO is 0.114%. After the compound is added, the cell plate is placed in a 37°C, 5% CO 2 incubator and incubated for 1 hour.
3.LPS稀释(20191125)3. LPS dilution (20191125)
第一步将LPS用超纯水稀释到储藏浓度1mg/mL。第二步储藏浓度的LPS用不含血清的培养基稀释到1μg/mL。第三步用不含有血清的培养基1666.666倍稀释。然后转16.8μL已经用培养基稀释好的LPS到116.8μL的细胞板里，此时DMSO终浓度是0.1％，加好LPS后将细胞板放入37℃，5％CO 2培养箱中孵育18个小时。 The first step is to dilute LPS with ultrapure water to a storage concentration of 1 mg/mL. In the second step, the storage concentration of LPS is diluted to 1 μg/mL with serum-free medium. The third step is 1666.666 times dilution with medium without serum. Then transfer 16.8 μL of LPS that has been diluted with culture medium to 116.8 μL of cell plate. At this time, the final concentration of DMSO is 0.1%. After adding LPS, place the cell plate in a 37°C, 5% CO 2 incubator and incubate 18 Hours.
ELISA实验ELISA experiment
第一天：first day:
1.将TNF-α抗体在包被液中稀释至1×，然后每孔100μL加到96孔高结合性能的板子中，板子用膜封住放到4℃18个小时。1. Dilute the TNF-α antibody to 1× in the coating solution, then add 100μL per well to a 96-well high-binding plate, seal the plate with a membrane and place it at 4℃ for 18 hours.
2.配制2000毫升清洗缓冲液至1×备用。2. Prepare 2000 ml of washing buffer to 1× for use.
第二天：the next day:
3.包被的板子过夜后，将包被液倒掉，用清洗缓冲液每孔300μL/孔清洗3遍。3. After the coated plate is overnight, pour out the coating solution, and wash with 300μL/well of washing buffer 3 times per well.
4.板子清洗过后加每孔200μL的封闭缓冲液，板子用膜封住。放到25度孵育箱中孵育一个小时。4. After the plate is cleaned, add 200μL of blocking buffer per well, and seal the plate with a membrane. Place it in the 25°C incubator and incubate for one hour.
5.将孵育18个小时的细胞板子放到离心机中离心，温度：25℃，转速：2000转，时间：10分钟，升速：9，降速：1。离心后取每孔100μL细胞上清到3599细胞板中，后放到4℃冰箱备用。5. Put the cell plate incubated for 18 hours in a centrifuge, temperature: 25°C, rotation speed: 2000 rpm, time: 10 minutes, speed up: 9, speed down: 1. After centrifugation, 100μL of cell supernatant from each well was transferred to a 3599 cell plate, and then placed in a refrigerator at 4°C for later use.
6.将细胞上清用封闭缓冲液稀释40倍放到4度冰箱待用，后配制标准品也放置4℃冰箱备用。6. Dilute the cell supernatant 40 times with blocking buffer and put it in a refrigerator at 4°C for later use. After preparing the standard, place it in a refrigerator at 4°C for later use.
7.封闭完成后，将封闭液倒掉，用清洗缓冲液每孔300μL清洗3遍。7. After the blocking is completed, pour out the blocking solution, and wash 3 times with 300 μL of washing buffer per well.
8.将稀释好的细胞上清样品以及标准品加到ELISA板子中，板子用膜封住。后放到25℃孵育箱中孵育两个小时。8. Add the diluted cell supernatant samples and standards to the ELISA plate, and seal the plate with a membrane. Then put it in an incubator at 25°C and incubate for two hours.
9.将板中液体倒掉，用清洗缓冲液每孔300μL清洗5遍。9. Pour out the liquid in the plate, and wash 5 times with 300 μL of washing buffer per well.
10.配制抗体，并每孔加入100μL，用封板膜封板。后放到25℃孵育箱中孵育一个小时。10. Prepare the antibody, add 100μL to each well, and seal the plate with a sealing film. Then put it in an incubator at 25°C and incubate for one hour.
11.将板中液体倒掉，用清洗缓冲液每孔300μL清洗7遍。11. Pour out the liquid in the plate, and wash 7 times with 300μL of washing buffer per well.
12.配制显色液，每孔加100μL。后避光放到25℃孵育箱中孵育半个小时。12. Prepare color developing solution, add 100μL to each hole. After dark, put it in an incubator at 25°C and incubate for half an hour.
13.每孔加50μL终止液，离心，温度：25℃，转速：1000转，时间：1分钟，升速：9，降速：9。13. Add 50μL stop solution to each well, centrifuge, temperature: 25℃, rotation speed: 1000 rpm, time: 1 minute, increase speed: 9, decrease speed: 9.
14.离心后30分钟内在Envision上读数，设置为吸收光450减去吸收光570的值为最终的原始数据使用值。14. Read on Envision within 30 minutes after centrifugation, and set the value of absorption light 450 minus absorption light 570 as the final raw data usage value.
数据处理data processing
根据原始数据计算抑制率，抑制率计算公式为：Calculate the inhibition rate based on the original data. The calculation formula for the inhibition rate is:
抑制率＝(1-(原始值-HPE平均值)/(ZPE平均值-HPE平均值))×100％Inhibition rate=(1-(original value-HPE average)/(ZPE average-HPE average))×100%
其中ZPE为：0％抑制(75pg/mL LPS,0.1％DMSO)，HPE为：100％抑制(不含LPS，0.1％DMSO)。Among them, ZPE is: 0% inhibition (75pg/mL LPS, 0.1% DMSO), and HPE is: 100% inhibition (without LPS, 0.1% DMSO).
S/B:ZPE平均值/HPE平均值S/B: ZPE average/HPE average
Z-factor:Z factor＝1-(3×(ZPE标准差+HPE标准差)/(ZPE平均值–HPE平均值))Z-factor: Z factor = 1-(3×(ZPE standard deviation + HPE standard deviation)/(ZPE average value-HPE average value))
用XLfit统计软件中的Model 205进行数据分析。以浓度为横坐标，抑制率为纵坐标，IC 50的计算公式为：用4参数logistic剂量响应方程，绘制了被测化合物的浓度和抑制率(％)，并确定了50％抑制所需的化合物浓度(IC 50)。 Model 205 in XLfit statistical software was used for data analysis. Taking the concentration as the abscissa and the inhibition rate as the ordinate, the IC 50 calculation formula is: using a 4-parameter logistic dose-response equation, the concentration and the inhibition rate (%) of the tested compound are plotted, and the required 50% inhibition is determined Compound concentration (IC 50 ).
将标准曲线的原始值输入到GraphPad Prism中，绘制出标准曲线，计算化合物的IC 50及最大效应值。测试结果见表2。 Input the original value of the standard curve into GraphPad Prism, draw the standard curve, and calculate the IC 50 and maximum effect value of the compound. The test results are shown in Table 2.
表2本发明化合物对人外周血单核细胞TNF-α的抑制活性测试结果Table 2 Test results of the inhibitory activity of the compounds of the present invention on human peripheral blood mononuclear cells TNF-α
Figure PCTCN2020141640-appb-000070
Figure PCTCN2020141640-appb-000070
注：IC 50均为绝对IC 50。*测试Top浓度500nM，3倍稀释，8浓度；其余测试Top浓度1000nM，3倍稀释，8浓度。Top(％)最大效应值 Note: IC 50 is absolute IC 50 . *Test Top concentration 500nM, 3 times dilution, 8 concentration; the rest test Top concentration 1000nM, 3 times dilution, 8 concentration. Top (%) maximum effect value
结论：本发明化合物显示出很强的细胞水平的抗炎活性。Conclusion: The compound of the present invention shows strong anti-inflammatory activity at the cellular level.
实验例4：人肝微粒体CYP抑制实验Experimental example 4: CYP inhibition experiment of human liver microsomes
研究项目的目的是采用CYP同工酶的5合1探针底物来评价供试品对人肝微粒体细胞色素P450同工酶(CYP3A4)的抑制性。The purpose of the research project is to use a 5-in-1 probe substrate of CYP isoenzymes to evaluate the inhibitory effect of the test product on human liver microsomal cytochrome P450 isoenzymes (CYP3A4).
混合人肝微粒体(HLM)购自Corning Inc.(Steuben,New York,USA)或者其他的供应商，使用前都储存在低于-70℃条件下。Mixed human liver microsomes (HLM) were purchased from Corning Inc. (Steuben, New York, USA) or other suppliers, and stored at a temperature below -70°C before use.
将稀释好的系列浓度的供试品工作液加入到含有人肝微粒体、探针底物和循环体系的辅助因子的孵育体系中，不含供试品而含有溶剂的对照作为酶活性对照(100％)。探针底物生成的代谢产物在样品中的浓度采用液相色谱-串联质谱(LC-MS/MS)方法进行测定。使用SigmaPlot(V.11)对供试品平均百分比活性对浓度作非线性回归分析。通过三参数或四参数反曲对数方程来计算IC 50值。测试结果如表3： Add the diluted working solution of the test substance to the incubation system containing human liver microsomes, the probe substrate and the cofactors of the circulating system. The control without the test substance but containing the solvent is used as the enzyme activity control ( 100%). The concentration of the metabolites generated by the probe substrate in the sample was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Use SigmaPlot (V.11) to make a non-linear regression analysis of the average percentage activity versus concentration of the test product. The IC 50 value is calculated by a three-parameter or four-parameter inverse logarithmic equation. The test results are shown in Table 3:
表3本发明化合物体外检测对CYP同工酶的抑制Table 3 Inhibition of CYP isoenzymes in vitro by the compounds of the present invention
Figure PCTCN2020141640-appb-000071
Figure PCTCN2020141640-appb-000071
结论：AZD9567对CYP3A4有中强度抑制。化合物WX002、WX004明显改善了对CYP3A4的抑制。Conclusion: AZD9567 has a moderate inhibitory effect on CYP3A4. Compounds WX002 and WX004 significantly improved the inhibition of CYP3A4.

Claims (15)

  1. 式(I)所示化合物或其药学上可接受的盐，The compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
    Figure PCTCN2020141640-appb-100001
    Figure PCTCN2020141640-appb-100001
    其中，among them,
    环A选自5元杂芳基、吡啶基、哒嗪基、1H-吡啶-2-酮基和[1,2,4]三唑并[4,3-A]吡啶基，所述5元杂芳基、吡啶基、哒嗪基、1H-吡啶-2-酮基和[1,2,4]三唑并[4,3-A]吡啶基任选被1、2或3个R a取代； Ring A is selected from 5-membered heteroaryl, pyridyl, pyridazinyl, 1H-pyridin-2-onyl and [1,2,4]triazolo[4,3-A]pyridyl, the 5-membered Heteroaryl, pyridyl, pyridazinyl, 1H-pyridin-2-onyl and [1,2,4]triazolo[4,3-A]pyridyl are optionally substituted by 1, 2 or 3 Ra replace;
    R 1分别独立地选自H、F、Cl、Br和I； R 1 is independently selected from H, F, Cl, Br and I;
    R 2选自C 1-6烷基，所述C 1-6烷基任选被1、2或3个R b取代； R 2 is selected from C 1-6 alkyl, and the C 1-6 alkyl is optionally substituted with 1, 2 or 3 R b ;
    m选自0、1、2和3；m is selected from 0, 1, 2 and 3;
    R a分别独立地选自H、F、Cl和C 1-3烷基，所述C 1-3烷基任选被1、2或3个R取代； R a is each independently selected from H, F, Cl and C 1-3 alkyl, the C 1-3 alkyl is optionally substituted with 1, 2 or 3 R;
    R b分别独立地选自H、F、Cl、Br和I； R b is independently selected from H, F, Cl, Br and I;
    R分别独立地选自H、F和Cl；R is independently selected from H, F and Cl;
    带“*”碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在；The carbon atom with "*" is a chiral carbon atom, which exists in the form of (R) or (S) single enantiomer or rich in one enantiomer;
    所述“5元杂芳基”包含1、2或3个选自N、NH、O、C＝O和S的杂原子或杂原子团。The "5-membered heteroaryl group" contains 1, 2 or 3 heteroatoms or heteroatom groups selected from N, NH, O, C=O and S.
  2. 根据权利要求1所述化合物或其药学上可接受的盐，其选自，The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which is selected from,
    Figure PCTCN2020141640-appb-100002
    Figure PCTCN2020141640-appb-100002
    其中，环A、R 1、R 2和m如权利要求1所定义。 Wherein, ring A, R 1 , R 2 and m are as defined in claim 1.
  3. 根据权利要求1或2所述化合物或其药学上可接受的盐，其中，R a分别独立地选自H、CH 3、CH 2CH 3、CH(CH 3) 2、CF 3、CHF 2和CH 2F。 The compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2, wherein, R a is independently selected from H, CH 3, CH 2 CH 3, CH (CH 3) 2, CF 3, CHF 2 , and CH 2 F.
  4. 根据权利要求1或2所述化合物或其药学上可接受的盐，其中，环A选自吡唑基、1H-吡啶-2-酮基、[1,2,4]三唑并[4,3-A]吡啶基、呋喃基、噻唑基、吡啶基和哒嗪基，所述吡唑基、1H-吡啶-2-酮基、[1,2,4]三唑并[4,3-A]吡啶基、呋喃基、噻唑基、吡啶基和哒嗪基任选被1、2或3个R a取代。 The compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein ring A is selected from pyrazolyl, 1H-pyridin-2-onyl, [1,2,4]triazolo[4, 3-A]pyridyl, furyl, thiazolyl, pyridyl and pyridazinyl, the pyrazolyl, 1H-pyridin-2-onyl, [1,2,4]triazolo[4,3- A] pyridinyl, furanyl, thiazolyl, pyridyl and pyridazinyl optionally substituted with 1, 2 or 3 R a.
  5. 根据权利要求4所述化合物或其药学上可接受的盐，其中，环A选自
    Figure PCTCN2020141640-appb-100003
    Figure PCTCN2020141640-appb-100004
    所述
    Figure PCTCN2020141640-appb-100005
    任选被1、2或3个R a取代。     The compound or a pharmaceutically acceptable salt thereof according to claim 4, wherein ring A is selected from
    Figure PCTCN2020141640-appb-100003
    Figure PCTCN2020141640-appb-100004
    Said
    Figure PCTCN2020141640-appb-100005
    Optionally substituted with 1,2 or 3 substituents R a.
  6. 根据权利要求5所述化合物或其药学上可接受的盐，其中，环A选自
    Figure PCTCN2020141640-appb-100006
    Figure PCTCN2020141640-appb-100007
    The compound or a pharmaceutically acceptable salt thereof according to claim 5, wherein ring A is selected from
    Figure PCTCN2020141640-appb-100006
    Figure PCTCN2020141640-appb-100007
  7. 根据权利要求1或2所述化合物或其药学上可接受的盐，其中，R 1分别独立地选自H和F。 The compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein R 1 is independently selected from H and F, respectively.
  8. 根据权利要求7所述化合物或其药学上可接受的盐，其中，结构单元
    Figure PCTCN2020141640-appb-100008
    分别独立地选自
    Figure PCTCN2020141640-appb-100009
    The compound or a pharmaceutically acceptable salt thereof according to claim 7, wherein the structural unit
    Figure PCTCN2020141640-appb-100008
    Are independently selected from
    Figure PCTCN2020141640-appb-100009
  9. 根据权利要求1或2所述化合物或其药学上可接受的盐，其中，R 2选自C 1-3烷基，所述C 1-3烷基任选被1、2和3个R b取代。 The compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein R 2 is selected from a C 1-3 alkyl group, and the C 1-3 alkyl group is optionally substituted by 1, 2 and 3 R b replace.
  10. 根据权利要求9所述化合物或其药学上可接受的盐，其中，R 2选自CH 3和
    Figure PCTCN2020141640-appb-100010
    所述CH 3
    Figure PCTCN2020141640-appb-100011
    任选被1、2和3个R b取代。     The compound or a pharmaceutically acceptable salt thereof according to claim 9, wherein R 2 is selected from CH 3 and
    Figure PCTCN2020141640-appb-100010
    The CH 3
    Figure PCTCN2020141640-appb-100011
    Optionally substituted with 1, 2 and 3 R b .
  11. 根据权利要求10所述化合物或其药学上可接受的盐，其中，R 2选自CH 3和
    Figure PCTCN2020141640-appb-100012
    The compound or a pharmaceutically acceptable salt thereof according to claim 10, wherein R 2 is selected from CH 3 and
    Figure PCTCN2020141640-appb-100012
  12. 根据权利要求1～11任意一项所述化合物或其药学上可接受的盐，其选自，The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, which is selected from,
    Figure PCTCN2020141640-appb-100013
    Figure PCTCN2020141640-appb-100013
    其中，among them,
    T 1选自CH和N； T 1 is selected from CH and N;
    R 1、R 2、R a和m如权利要求1～11任意一项所定义。 R 1, R 2, R a and m are as claimed in any one of 1 to 11 as defined in claim.
  13. 下式所示化合物或其药学上可接受的盐，The compound represented by the following formula or a pharmaceutically acceptable salt thereof,
    Figure PCTCN2020141640-appb-100014
    Figure PCTCN2020141640-appb-100014
    Figure PCTCN2020141640-appb-100015
    Figure PCTCN2020141640-appb-100015
  14. 根据权利要求1～13任意一项所述的化合物或其药学上可接受的盐在制备与糖皮质激素受体相关疾病的药物中的应用。The use of the compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof in the preparation of drugs for glucocorticoid receptor-related diseases.
  15. 根据权利要求14所述的应用，所述与糖皮质激素受体相关疾病是指类风湿关节炎。The use according to claim 14, wherein the disease related to glucocorticoid receptor refers to rheumatoid arthritis.
PCT/CN2020/141640 2019-12-31 2020-12-30 Benzo[d][1,2,3]triazole ether compound WO2021136431A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114478273A (en) * 2022-01-19 2022-05-13 汉瑞药业(荆门)有限公司 Preparation method of metahydroxylamine bitartrate

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012104659A1 (en) * 2011-02-04 2012-08-09 University Of Nottingham Novel ether linked compounds and improved treatments for cardiac and cardiovascular disease
CN105461711A (en) * 2014-06-17 2016-04-06 南京明德新药研发股份有限公司 Pyrido[1,2-a]pyrimidone analogs as PI3K inhibitors
WO2017215506A1 (en) * 2016-06-13 2017-12-21 南京明德新药研发股份有限公司 Benzotriazole-derived α and β unsaturated amide compound used as tgf-βr1 inhibitor

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012104659A1 (en) * 2011-02-04 2012-08-09 University Of Nottingham Novel ether linked compounds and improved treatments for cardiac and cardiovascular disease
CN105461711A (en) * 2014-06-17 2016-04-06 南京明德新药研发股份有限公司 Pyrido[1,2-a]pyrimidone analogs as PI3K inhibitors
WO2017215506A1 (en) * 2016-06-13 2017-12-21 南京明德新药研发股份有限公司 Benzotriazole-derived α and β unsaturated amide compound used as tgf-βr1 inhibitor

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114478273A (en) * 2022-01-19 2022-05-13 汉瑞药业(荆门)有限公司 Preparation method of metahydroxylamine bitartrate

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