WO2021047627A1 - 一种可降解蛋白的并环类化合物及其应用 - Google Patents

一种可降解蛋白的并环类化合物及其应用 Download PDF

Info

Publication number
WO2021047627A1
WO2021047627A1 PCT/CN2020/114728 CN2020114728W WO2021047627A1 WO 2021047627 A1 WO2021047627 A1 WO 2021047627A1 CN 2020114728 W CN2020114728 W CN 2020114728W WO 2021047627 A1 WO2021047627 A1 WO 2021047627A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
compound
pharmaceutically acceptable
crbn
acceptable salt
Prior art date
Application number
PCT/CN2020/114728
Other languages
English (en)
French (fr)
Inventor
罗云富
仓勇
彭博
雷茂义
徐雨
陈曙辉
Original Assignee
南京明德新药研发有限公司
上海科技大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京明德新药研发有限公司, 上海科技大学 filed Critical 南京明德新药研发有限公司
Priority to US17/641,759 priority Critical patent/US20220324848A1/en
Priority to CN202080063971.4A priority patent/CN114761400A/zh
Priority to CA3150653A priority patent/CA3150653A1/en
Publication of WO2021047627A1 publication Critical patent/WO2021047627A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • 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
    • A61K31/42—Oxazoles
    • A61K31/423—Oxazoles condensed with carbocyclic 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
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • 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
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4525—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with oxygen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
    • C07D261/20—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
    • C07D307/79—Benzo [b] furans; Hydrogenated benzo [b] furans with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

  • the present invention relates to a compound represented by formula (II) and a pharmaceutically acceptable salt thereof or a pharmaceutical composition using the compound as an active ingredient, and the application in the preparation of a degradable protein medicine.
  • Immunomodulator drugs include Thalidomide (Thalidomide), Lenalidomide (Lenalidomide, Lena), and Pomalidomide (Pomalidomide, Poma) use the glutarimide ring structure to insert Cereblon (CRBN) ubiquitin ligase
  • the pocket area recruits the transcription factor Ikaros (IKZF1)/Aiolos (IKZF3), which is dependent on the survival of cancer cells derived from B cells, and promotes its ubiquitination and degradation, thereby producing cytotoxicity.
  • IKZF1 transcription factor Ikaros
  • IKZF3 Aiolos
  • lenalidomide can also target CRBN to mediate the degradation of CK1alpha to treat myelodysplastic syndrome with 5q deletion.
  • CRBN as an important target of anti-tumor and immunomodulator drugs, has been proven in multiple myeloma, chronic lymphocytic leukemia and other hematological malignancies, leprosy nodular erythema and other skin diseases, and systemic lupus erythematosus. Autoimmune diseases have clear curative effects. CRBN plays a very important role in the ubiquitin-proteasome system. There is an urgent need to develop new CRBN modulator drugs to target new substrate proteins for potential treatment of different diseases.
  • the present invention has developed a research and development process for this type of anti-tumor compound, which mainly includes the following steps: 1) Synthesis of compounds containing glutarimide structural fragments Library; 2) screen out cytotoxic compounds; 3) further screen out compounds that rely on CRBN to exert cytotoxicity; 4) use mass spectrometry to find potential compound target proteins; 5) conduct mechanism and preclinical research.
  • Wee1 is an important G2-M checkpoint regulatory protein and a potential target for tumor-targeted drugs. Inhibiting Wee1 in tumor cells damages the G2-M checkpoint, and causes the genome to enter mitosis in a damaged state, leading to tumor cell apoptosis, that is, the catastrophe of mitosis.
  • a series of preclinical studies and clinical studies have proved that Wee1 inhibitors have a good inhibitory effect on tumor cells with missing G1-S checkpoints, and Wee1 can enhance the sensitivity of many DNA damaging agents.
  • the present invention provides a compound represented by formula (II) or a pharmaceutically acceptable salt thereof,
  • E is selected from CH and N;
  • R 1 is selected from C 1-4 alkyl, C 2-3 alkenyl, Cyclopropyl and furanyl, said C 1-4 alkyl and C 2-3 alkenyl group optionally substituted with two, three R a;
  • T 1 is selected from C(R 3 ) and N;
  • R 2 is selected from H, F, Cl, Br, I, CH 3 and CF 3 ;
  • R 3 is selected from H, F, Cl, Br, I and CH 3 , the CH 3 is optionally substituted with 1, 2, 3 halogens;
  • Each R a is selected from halogen, OCH 3 and NH 2;
  • the R 2 is selected from H, Cl and CF 3 , and other variables are as defined in the present invention.
  • the R 2 is Cl, and other variables are as defined in the present invention.
  • the R 3 is selected from H, F, Cl, Br, I and CH 3 , the CH 3 is optionally substituted by 1, 2, 3 F, and other variables are as defined in the present invention .
  • the R 3 is selected from H, F, Cl, Br, I and CH 3 , and other variables are as defined in the present invention.
  • the R 3 is H and CH 3 , and other variables are as defined in the present invention.
  • the variables are as defined in the present invention.
  • the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
  • R 1 is selected from C 2-4 alkyl and The C 2-4 alkyl is optionally substituted with 1, 2, 3 halogens;
  • T 1 is selected from C(R 3 ) and N;
  • R 2 is selected from H, F, Cl, Br, I, CH 3 and CF 3 ;
  • R 3 is selected from H, F, Cl, Br, I, and CH 3 , and the CH 3 is optionally substituted with 1, 2, 3 halogens.
  • the R 2 is selected from H, Cl and CF 3 , and other variables are as defined in the present invention.
  • the R 2 is Cl, and other variables are as defined in the present invention.
  • the R 3 is selected from H, F, Cl, Br, I and CH 3 , the CH 3 is optionally substituted by 1, 2, 3 F, and other variables are as defined in the present invention .
  • the R 3 is selected from H, F, Cl, Br, I and CH 3 , and other variables are as defined in the present invention.
  • the R 3 is CH 3 , and other variables are as defined in the present invention.
  • the R 1 is selected from And C 3-4 alkyl, the C 3-4 alkyl is optionally substituted with 1, 2, 3 F, and other variables are as defined in the present invention.
  • the R 1 is selected from Other variables are as defined in the present invention.
  • the compound or a pharmaceutically acceptable salt thereof is selected from
  • n is selected from 1, 2 and 3; R 2, R 3 and R a are as defined in the present invention.
  • the present invention provides a compound of the following formula or a pharmaceutically acceptable salt thereof, which is selected from
  • the present invention provides a composition comprising a therapeutically effective amount of the above-mentioned compound or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier.
  • the present invention provides the application of the above-mentioned compound or its pharmaceutically acceptable salt or composition in the preparation of a protein degrading medicine.
  • the present invention provides the application of the above-mentioned compound or its pharmaceutically acceptable salt or composition in the preparation of a medicine for degrading Wee1 protein.
  • the present invention provides the application of the above-mentioned compound or its pharmaceutically acceptable salt or composition 4 in the preparation of a drug that holds CRL4 (CRBN) to degrade Wee1 protein.
  • 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 the 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 neutral form of 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 neutral form of 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.
  • enantiomer or “optical isomer” refers to stereoisomers that are mirror images of each other.
  • cis-trans isomer or “geometric isomer” is caused by the inability to freely rotate the double bond or the single bond of the ring-forming carbon atom.
  • diastereomer refers to a stereoisomer in which the molecule has two or more chiral centers and the relationship between the molecules is non-mirror mirror image.
  • wedge-shaped solid line keys And wedge-shaped dashed key Represents the absolute configuration of a solid center, with a straight solid line key And straight dashed key Indicates the relative configuration of the three-dimensional center, using wavy lines Represents a wedge-shaped solid line key Or wedge-shaped dashed key Or use wavy lines Represents a straight solid line key And straight dashed key
  • the compound of the present invention may be specific.
  • tautomer or “tautomeric form” means that at room temperature, the isomers of different functional groups are in dynamic equilibrium and can be transformed into each other quickly. If tautomers are possible (such as in solution), the chemical equilibrium of tautomers can be reached.
  • proton tautomer also called prototropic tautomer
  • proton migration such as keto-enol isomerization and imine-ene Amine isomerization.
  • Valence isomers include some recombination of bonding electrons to carry out mutual transformation.
  • keto-enol tautomerization is the tautomerization between two tautomers of pentane-2,4-dione and 4-hydroxypent-3-en-2-one.
  • the terms “enriched in one isomer”, “enriched in isomers”, “enriched in one enantiomer” or “enriched in enantiomers” refer to one of the isomers or pairs of
  • the content of the enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or 96% or greater, or 97% or greater, or 98% or greater, or 99% or greater, or 99.5% or greater, or 99.6% or greater, or 99.7% or greater, or 99.8% or greater, or greater than or equal 99.9%.
  • the term “isomer excess” or “enantiomeric excess” refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90%, and the content of the other isomer or enantiomer is 10%, the isomer or enantiomer excess (ee value) is 80% .
  • optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If you want to obtain an enantiomer of a compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure The desired enantiomer.
  • the molecule when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), it forms a diastereomeric salt with an appropriate optically active acid or base, and then passes through a conventional method known in the art The diastereoisomers are resolved, and then the pure enantiomers are recovered.
  • the separation of enantiomers and diastereomers is usually accomplished through the use of chromatography, which employs a chiral stationary phase and is optionally combined with chemical derivatization (for example, the formation of amino groups from amines). Formate).
  • 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, prolong 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.
  • 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
  • C 1-4 alkyl is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 4 carbon atoms.
  • the C 1-4 alkyl group includes C 1-2 , C 1-3 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 1-4 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) and so on.
  • C 2-4 alkyl is used to indicate a linear or branched saturated hydrocarbon group composed of 2 to 4 carbon atoms.
  • the C 2-4 alkyl group includes C 2-3 and C 3-4 alkyl groups, etc.; it can be monovalent (such as ethyl), divalent (such as ethylene) or multivalent (such as ethylene) .
  • Examples of C 2-4 alkyl groups include, but are not limited to, ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl and t-butyl) and so on.
  • C 3-4 alkyl is used to indicate a linear or branched saturated hydrocarbon group composed of 3 to 4 carbon atoms.
  • the C 3-4 alkyl group includes C 3 and C 4 alkyl groups, etc.; it can be monovalent (such as propyl), divalent (such as propylene) or multivalent (such as propylene).
  • Examples of C 3-4 alkyl include but are not limited to propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl and t-butyl), etc. .
  • C 2-3 alkenyl is used to mean a linear or branched hydrocarbon group consisting of 2 to 3 carbon atoms containing at least one carbon-carbon double bond, and a carbon-carbon double bond It can be located in any position of the group.
  • the C 2-3 alkenyl group includes C 3 and C 2 alkenyl groups; the C 2-3 alkenyl group may be monovalent, divalent or multivalent. Examples of C 2-3 alkenyl include, but are not limited to, vinyl, propenyl, and the like.
  • halogen or “halogen” by itself or as part of another substituent means a fluorine, chlorine, bromine or iodine atom.
  • haloalkyl is intended to include monohaloalkyl and polyhaloalkyl.
  • halo(C 1 -C 4 )alkyl is intended to include, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, etc. Wait.
  • examples of haloalkyl include, but are not limited to: trifluoromethyl, trichloromethyl, pentafluoroethyl, and pentachloroethyl.
  • 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, an affinity 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 group, such as trimethylsilyl (TMS) and tert-butyldi
  • hydroxyl 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 (
  • 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 compounds of the present invention can have multiple uses or indications, including but not limited to the specific uses or indications listed in this application.
  • the solvent used in the present invention is commercially available.
  • the present invention uses the following abbreviations: aq stands for water; HATU stands for O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate ; EDC stands for N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; m-CPBA stands for 3-chloroperoxybenzoic acid; eq stands for equivalent, equivalent; M stands for mol/L; CDI stands for carbonyl diimidazole; DCM stands for dichloromethane; PE stands for petroleum ether; DIAD stands for diisopropyl azodicarboxylate; DMF stands for N,N-dimethylformamide; DMSO stands for dimethyl sulfoxide Sulfone; EtOAc stands for ethyl acetate; EtOH stands for ethanol; Me
  • the compound of the present invention proved to significantly down-regulate the level of Wee1 protein in MOLT-4 cells.
  • Figure 1 Inhibition rate of WX106 on the growth of different tumor cells
  • Figure 2 The effect of WX106 on colonization of colorectal cancer cell lines
  • Figure 3 The effect of WX106 on the cloning of brain cancer cell lines
  • FIG. 8 WX106 relies on CRBN to inhibit the proliferation of Hep3B cells
  • WX106 relies on CRBN to inhibit the proliferation of Hep3B cells
  • FIG. 11 WX106 relies on CRBN to inhibit the proliferation of SNU182 cells
  • FIG. 12 WX106 promotes the degradation of Wee1 protein in 293T cells
  • FIG. 13 WX106 promotes the degradation of Wee1 protein in MOLT-4 cells
  • FIG. 14 WX106 promotes the degradation of Wee1 protein in MOLT-4 cells
  • FIG. 15 WX106 promotes the degradation of Wee1 protein in U87-MG cells
  • FIG. 16 WX106 promotes the degradation of Wee1 protein in U87-MG cells
  • FIG. 17 WX106 promotes the degradation of Wee1 protein in HCT116 cells
  • FIG. 18 WX106 promotes the degradation of Wee1 protein in LN-229 cells
  • WX106 relies on CRBN to promote the ubiquitination and degradation of Wee1 protein in 293T cells;
  • WX106 relies on CRBN to promote the ubiquitination and degradation of Wee1 protein in U87-MG cells;
  • Figure 21 The effect of WX106 on the stability of Wee1 protein in U87-MG cells
  • FIG. 22 The effect of WX106 on the stability of Wee1 protein in 293T cells
  • FIG. 23 Synergy experiment of WX106 and DNA damaging agent
  • Figure 27 The kinase domain of Wee1 can bind to CRBN;
  • FIG. 28 Degradation of Wee1 by different compounds in MOLT-4 cells
  • FIG. 29 Degradation of Wee1 by different compounds in U87-MG cells.
  • MS--ESI m/z 423.9[M+Na] + .
  • the intermediate WX012-1 (256 mg, 637.75 ⁇ mol) was added to hydrochloric acid/ethyl acetate (4M, 20 mL), and the reaction mixture was stirred and reacted at room temperature for 2 hours. After the completion of the reaction, the reaction solution was concentrated under reduced pressure to remove the solvent, methanol (5 mL) was added to the resulting residue, and the mixture was stirred at room temperature for 0.5 hour. The reaction solution was filtered, the filter cake was washed with methanol (2 mL), the filter cake was collected, and concentrated under reduced pressure to obtain the target compound WX012. MS--ESI m/z: 302.1[M+H] + .
  • the intermediate WX013-2 (1g, 4.34mmol) was dissolved in concentrated sulfuric acid (5mL, purity: 98%), cooled to 0°C, and then potassium nitrate (447.93mg, 4.43mmol) was added to the reaction mixture. The reaction was stirred at 0°C for 1 hour. After the reaction is complete, pour into ice water (100 mL), and extract with 2-methyltetrahydrofuran (60 mL ⁇ 3).
  • Triazole-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphate (182.22mg, 479.24 ⁇ mol) and triethylamine (96.99mg, 958.48 ⁇ mol, 133.41 ⁇ L)
  • the reaction mixture is at room temperature
  • the reaction was stirred for 0.5 hours, and then the hydrochloride salt of intermediate WX013-4 (0.09 g, 319.49 ⁇ mol) was added, and the reaction mixture was stirred and reacted at room temperature for 12 hours.
  • Inhibition rate [(Ac-As)/(Ac-Ab)] ⁇ 100%, (where As represents the absorbance of the experimental well, Ac represents the absorbance of the control well, and Ab represents the absorbance of the blank well).
  • WX106 can inhibit the growth of blood tumor cells and some liver cancer cells by more than 50%.
  • the cells were collected, and protein loading buffer (50mM Tris-HCl, 2% SDS, 0.025% BPB, 1% ⁇ -mercaptoethanol, 10% glycerol) was denatured at 98°C for 10 minutes.
  • the protein was separated by SDS-PAGE and transferred to PVDF membrane (Millipore, IPVH00010), then blocked with 5% skim milk (dissolved in TBST buffer), incubated with the primary antibody overnight at 4°C, and then with the secondary antibody (Anti-rabbit IgG, HRP-linked Antibody, Cell Signaling, 7074S) were incubated at room temperature for 1 hour, and the bands on the membrane were detected by chemiluminescence method (ECL chemiluminescence kit, Biyuntian Biotech, P0018AM).
  • CC885 and different concentrations of WX106 or Poma were added to MOLT-4 cells, and Western blot was used to detect the expression of GSPT1 protein.
  • the degradation of GSPT1 caused by CC885 was reversed.
  • Poma could inhibit CC885's pro-degradation effect on GSPT1.
  • Poma is a compound that binds to CRBN, and Poma has no obvious inhibitory effect on adherent cells. If a compound relies on CRBN to inhibit cell growth, Poma's competitive binding to CRBN will definitely affect the cytotoxicity of this compound.
  • Treat the cells with 1,10,50 ⁇ M Poma and 10 ⁇ M WX106 for 72 hours, use CCK-8 kit to detect cell viability, calculate cell survival rate, survival rate [(As-Ab)/(Ac-Ab)] ⁇ 100 % (Where As represents the absorbance of the experimental wells, Ac represents the absorbance of the control wells, and Ab represents the absorbance of the blank wells), statistics whether Poma's competitive binding will affect the cytotoxicity of WX106 to determine whether the cytotoxicity of WX106 depends on CRBN. This step is applicable to both SUN182 and Hep3B cells.
  • the cell growth competition experiment performed in SNU182 is shown in Figure 6. Poma can competitively inhibit the cytotoxicity caused by WX106.
  • CC885 is the positive control, which is known to rely on CRBN to kill tumor cells.
  • Figure 7 shows a competition experiment conducted in Hep3B. Poma can competitively inhibit the cytotoxicity caused by WX106.
  • sgRNA sequence for CRBN into the Lenti Crispr V2 plasmid, package the virus together with the packaging vectors psPAX2 and pVSVG, infect the cells, screen with puromycin (Invitrogen, A1113803), Western blot to identify the knockout of CRBN, and then perform cell growth testing , Calculate the survival rate.
  • the sgRNA sequence is shown in the table below:
  • WX106 (10 ⁇ M) was added to the wild-type Hep3B cells and Hep3B CRBN KO (knockout) mixed cloned cells respectively, and CCK-8 detected cell proliferation after 72 hours.
  • the experimental results are shown in Figure 8. The results showed that after knocking out CRBN, the sensitivity of Hep3B cells to WX106 was significantly reduced.
  • WX106 (10 ⁇ M) was added to MM.1S cells and MM.1S-p5000 (Poma drug-resistant CRBN low expression cell line) cells, respectively, and CCK-8 was used to detect cell proliferation after 72 hours.
  • the experimental results are shown in Figure 9. In the case of low expression of CRBN, the sensitivity of cells to WX106 is significantly reduced.
  • a stable cell line (pool) of CRBN knockout was established in Hep3B and SNU182 cells, and treated with WX106 (10 ⁇ M) or solvent. The growth of the cells was detected at different time points and the growth curve was drawn. The experimental results are shown in Figure 10 and Figure 11.
  • the cells with normal expression of CRBN are very sensitive to WX106, while the growth rate of the cell lines with low CRBN expression after adding WX106 is not significantly different from that of the solvent group.
  • Compound WX106 relies on CRBN to inhibit the growth of tumor cells.
  • MOLT-4 There are three treatment methods for the selected cell MOLT-4, one is to directly add DMSO as a control, the other is to add 10 ⁇ M WX106, and the third is to add 10 ⁇ M WX106 and 10 ⁇ M MG132 (proteasome inhibitor, APEXBIO, A2585, MG132 can block protein The ubiquitination degradation of ubiquitin), while adding 10 ⁇ M WX106 and 10 ⁇ M MG132 groups for two repetitions, and DMSO control group and 10 ⁇ M WX106 administration group for three repetitions.
  • the treatment time of MOLT-4 is 6 hours.
  • the basic steps for preparing mass spectrometry samples include urea cleavage, reductive alkylation, desalination, and freeze-drying. Afterwards, the prepared samples were subjected to two-hour gradient analysis on the Q Exactive HF mass spectrometer, and subsequent data analysis.
  • Table 1 shows the results of mass spectrometry.
  • the Wee1 signal was visible in the solvent group, and there was no Wee1 signal in the WX106 group. After WX106 and MG132 were added at the same time, the Wee1 signal recovered, suggesting that WX106 promoted the ubiquitination of Wee1 degradation.
  • the cells were seeded in a 6-well plate at a density of about 70%, and compound WX106 of different concentrations (concentrations as shown in the figure) was added, and treated for 6 hours, after which the whole cell sap was collected and the target protein was detected.
  • the antibody used is: (Wee1 (D10D2) Rabbit mAb, Cell Signaling, 13084S).
  • MG132 was used to block the ubiquitination degradation of the protein. This method is also applicable to 293T, MOLT-4, U87-MG, HCT116, LN-229.
  • Figure 12 shows the changes in Wee1 protein abundance of 293T cells treated with different concentrations of WX106 for 6 hours.
  • Figure 13 and Figure 14 show the effect of WX106 on Wee1 protein expression in MOLT-4 cells.
  • the effects of WX106 on the substrates GSPT1 and CK1 ⁇ (Abcam, ab108296) of other compounds with Glutarimide ring structure were simultaneously tested. The results show that WX106 has no significant pro-degradation effect on GSPT1 and CK1 ⁇ .
  • Figure 15 and Figure 16 show that in U87-MG cells, WX106 has a significant pro-degradation effect on Wee1.
  • Figure 17 shows that WX106 has a weak pro-degradation effect on Weel in HCT116 cells.
  • Figure 18 shows the effect of WX106 treatment on Wee1 degradation in LN-229 cells for different times.
  • the 293T cells and 293T CRBN KO cells were treated with DMSO, 1 ⁇ M WX106, 10 ⁇ M WX106 alone or in combination with 10 ⁇ M MG132 for 16 hours, and the whole cell lysate was collected. Western blot was used to detect the expression of Wee1 protein.
  • the experimental results are shown in Figure 19. It can be observed that in wild cells, the effect of WX106 promotes the degradation of Wee1, while the expression of Wee1 in CRBN-deficient cells has no significant change, suggesting that WX106's promotion of Wee1 degradation depends on CRBN.
  • WX106 relies on CRBN to promote the ubiquitination and degradation of Wee1.
  • FIG 21 and Figure 22 were used to detect the effect of WX106 on the stability of Wee1 in U87-MG and 293T cells, respectively. After WX106 treatment, the cells reached the half-life in about 2 hours. In the cell group treated with solvent, the Wee1 protein was obvious. stable.
  • WX106 was combined with DNA damaging agents Temozolomide, Cisplatin, and Gemcitabine to treat U87-MG cells for 72 hours, and CCK-8 was used to detect cell proliferation.
  • the concentration of WX106 is 10 ⁇ M, and the concentration of DNA damaging agent is 0.1 ⁇ M, 1 ⁇ M and 10 ⁇ M.
  • the experimental results are shown in Figure 23.
  • Temozolomide combined with WX106 can significantly inhibit the proliferation of U87-MG cells.
  • Cisplatin, gemcitabine at low concentrations and combined with WX106 can also significantly inhibit the proliferation of U87-MG cells.
  • the plasmid was transfected into 293T cells. After 24 hours, compound WX106 was added to treat the cells for a certain period of time.
  • the cells were lysed with NETN lysis buffer (150mM NaCl, 50mM Tris-HCl pH8.0, 1% NP40), and agarose beads (EZview TM Red anti-HA affinity gel, Sigma, E6779; anti M2 affinity gel, Sigma, A2220) was incubated with the lysis buffer for 2 hours, the beads were washed 4 times with the lysis buffer, and the loading buffer (100mM Tris-HCl, 4% SDS, 0.05% BPB, 2% ⁇ -mercaptoethanol was added) , 20% glycerol), detected by Western blot.
  • NETN lysis buffer 150mM NaCl, 50mM Tris-HCl pH8.0, 1% NP40
  • agarose beads EZview TM Red anti-HA affinity gel, Sigma, E6779; anti M2 affinity gel, Sigma
  • HA CRBN was overexpressed in 293T cells (CRBN was overexpressed in the cell by transfection technology, HA is a polypeptide fused with CRBN.
  • CRBN refers to the endogenous CRBN of the cell
  • FLAG Wee1 in the cell by transfection technology
  • Wee1 is overexpressed in cells
  • FLAG is a polypeptide fusion expression with CRBN
  • Wee1 refers to the endogenous Wee1
  • 24 hours after transfection the cells were treated with 20 ⁇ M MG132 or 10 ⁇ M WX106 1h and 2h before cell collection, respectively for Co. -IP (Co-immunoprecipitation) test.
  • the results show that Wee1 can interact with CRBN.
  • MOLT-4 cells were treated with 10 ⁇ M test compound for 4 hours, and U87-MG cells were treated with 1 ⁇ M test compound for 24 hours.
  • Western blot was used to detect the protein level of Wee1 (Cell Signaling, 13084S), and GAPDH (Santa Cruz, sc-32233) was the internal reference protein.
  • Collect cells lyse the cells with protein loading buffer (1M Tris-HCl, 10% SDS, 0.025% BPB), and perform Western blot analysis after denaturation at 98°C for 10 minutes.
  • the main steps include: 1) Electrophoresis: Separate proteins by SDS-PAGE electrophoresis.
  • PVDF membrane Millipore, IPVH00010
  • BSA Bovine Serum Album, ABCONE, A23088
  • Anti-rabbit secondary antibody Anti-rabbit IgG, HRP-linked Antibody, Cell Signaling, 7074S
  • Anti-mouse secondary antibody HRP-labeled goat anti-mouse IgG, EpiZyme, LF101
  • Electrophoresis buffer Weigh 3.0275g Tris, 14.4135g Glycine, 1g SDS, vortex and mix, and dilute ddH 2 O to 1L.
  • Transfer membrane buffer Weigh 3.03g of Tris, 14.42g of Glycine, 100mL of methanol, vortex and mix well, and dilute the volume of ddH 2 O to 1L.
  • TBST Weigh 6.057g Tris, 8.75g NaCl, 2mL TWeen20, vortex and shake to mix, and dilute ddH 2 O to 1L. Add hydrochloric acid to adjust the pH to 8.0.
  • skimmed milk Weigh 5g of skimmed milk powder, dissolve it in 100mL TBST, vortex and mix well.
  • WX106, WX002, WX003, WX004, WX005 and WX006 all have pro-degradation effects on Wee1; WX106 has the strongest effect.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

式(II)所示化合物及其药学上可接受的盐或以该化合物为活性成分的药物组合物，以及在制备降解蛋白药物中的应用。

Description

一种可降解蛋白的并环类化合物及其应用
本申请主张如下优先权：
CN201910865744.2，申请日2019-09-12。
技术领域
本发明涉及式(II)所示化合物及其药学上可接受的盐或以该化合物为活性成分的药物组合物，以及在制备降解蛋白药物中的应用。
背景技术
免疫调节剂药物包括沙利度胺(Thalidomide)，来那度胺(Lenalidomide,Lena)，和泊马度胺(Pomalidomide,Poma)利用戊二酰亚胺环结构插入Cereblon(CRBN)泛素连接酶的口袋区域，招募B细胞来源的癌细胞生存所依赖的转录因子Ikaros(IKZF1)/Aiolos(IKZF3)并促进其泛素化降解，进而产生细胞毒作用。除了介导IKZF1/3的泛素化降解，来那度胺还可以靶向CRBN介导CK1alpha的降解从而治疗5q缺失的骨髓增生异常综合症。
CRBN作为抗肿瘤和免疫调节剂药物的重要靶点，已被证实在多发性骨髓瘤、慢性淋巴细胞白血病等多种血液性恶性肿瘤、麻风结节性红斑等皮肤病、和系统性红斑狼疮等自免疫性疾病具有明确的疗效。CRBN在泛素-蛋白酶体系统中具有非常重要的作用。当前迫切需要开发新型CRBN调节剂药物，来靶向新的底物蛋白，用于不同种疾病的潜在治疗。
发明内容
本发明为了发现更多靶向CRBN的类似“分子胶水”，开发了这类抗肿瘤化合物的研发流程，主要包括以下几个步骤：1)合成含有戊二酰亚胺(glutarimide)结构片段的化合物库；2)筛选出有细胞毒性的化合物；3)进一步筛选出依赖CRBN而发挥细胞毒性的化合物；4)利用质谱技术找到潜在的化合物靶标的蛋白；5)进行机制及临床前的研究。
根据上述流程，评估了大量化合物。发现化合物WX106能够促进Wee1激酶的泛素化和蛋白酶体介导的降解。Wee1是重要的G2-M检查点调控蛋白，是一种有潜力的肿瘤靶向药物的作用靶点。在肿瘤细胞中抑制Wee1，损害了G2-M检查点，会导致基因组在损伤的状态下进入有丝分裂，导致肿瘤细胞凋亡，即有丝分裂灾难。一系列的临床前研究和临床研究证明了Wee1抑制剂对G1-S检查点缺失的肿瘤细胞有很好的抑制作用，并且Wee1能够增强很多DNA损伤剂的敏感性。
通过定向合成的化合物库，结合表型筛选和基于蛋白降解的质谱分析，高效发现新的分子胶水化合物，靶向新底物蛋白，泛素化新底物蛋白并通过蛋白酶体进行降解，用于多种疾病的潜在治疗。
本发明提供了式(ⅠI)所示的化合物或其药学上可接受的盐，
Figure PCTCN2020114728-appb-000001
其中，
E选自CH和N；
R 1选自C 1-4烷基、C 2-3烯基、
Figure PCTCN2020114728-appb-000002
环丙基和呋喃基，所述C 1-4烷基和C 2-3烯基任选被1、2、3个R a取代；
T 1选自C(R 3)和N；
R 2选自H、F、Cl、Br、I、CH 3和CF 3；
R 3选自H、F、Cl、Br、I和CH 3，所述CH 3任选被1、2、3个卤素取代；
各R a选自卤素、OCH 3和NH 2；
在本发明的一些方案中，所述R 2选自H、Cl和CF 3，其他变量如本发明所定义。
在本发明的一些方案中，所述R 2为Cl，其他变量如本发明所定义。
在本发明的一些方案中，所述R 3选自H、F、Cl、Br、I和CH 3，所述CH 3任选被1、2、3个F取代，其他变量如本发明所定义。
在本发明的一些方案中，所述R 3选自H、F、Cl、Br、I和CH 3，其他变量如本发明所定义。
在本发明的一些方案中，所述R 3为H和CH 3，其他变量如本发明所定义。
在本发明的一些方案中，所述R 1选自
Figure PCTCN2020114728-appb-000003
CH＝CH 2、CH 3、CH 2CH 3和CH 2CH 2CH 3，所述CH 3、CH 2CH 3和CH 2CH 2CH 3任选被1、2、3个R a取代，其他变量如本发明所定义。
在本发明的一些方案中，所述R 1选自
Figure PCTCN2020114728-appb-000004
Figure PCTCN2020114728-appb-000005
-CH＝CH 2、-CH 2NH 2、-CH 2CH 3、-CH 2CH 2OCH 3和-CH 2CH 2CH 3，其他变量如本发明所定义。
本发明提供了式(Ⅰ)所示的化合物或其药学上可接受的盐，
Figure PCTCN2020114728-appb-000006
其中，
R 1选自C 2-4烷基和
Figure PCTCN2020114728-appb-000007
所述C 2-4烷基任选被1、2、3个卤素取代；
T 1选自C(R 3)和N；
R 2选自H、F、Cl、Br、I、CH 3和CF 3；
R 3选自H、F、Cl、Br、I和CH 3，所述CH 3任选被1、2、3个卤素取代。
在本发明的一些方案中，所述R 2选自H、Cl和CF 3，其他变量如本发明所定义。
在本发明的一些方案中，所述R 2为Cl，其他变量如本发明所定义。
在本发明的一些方案中，所述R 3选自H、F、Cl、Br、I和CH 3，所述CH 3任选被1、2、3个F取代，其他变量如本发明所定义。
在本发明的一些方案中，所述R 3选自H、F、Cl、Br、I和CH 3，其他变量如本发明所定义。
在本发明的一些方案中，所述R 3为CH 3，其他变量如本发明所定义。
在本发明的一些方案中，所述R 1选自
Figure PCTCN2020114728-appb-000008
和C 3-4烷基，所述C 3-4烷基任选被1、2、3个F取代，其他变量如本发明所定义。
在本发明的一些方案中，所述R 1选自
Figure PCTCN2020114728-appb-000009
Figure PCTCN2020114728-appb-000010
其他变量如本发明所定义。
在本发明的一些方案中，所述化合物或其药学上可接受的盐，其选自
Figure PCTCN2020114728-appb-000011
其中，m选自1、2和3；R 2、R 3和R a如本发明所定义。
本发明提供下式化合物或其药学上可接受的盐，其选自
Figure PCTCN2020114728-appb-000012
本发明还有一些方案有上述变量任意组合而来。
本发明提供了一种组合物，包括治疗有效量的上述的化合物或其药学上可接受的盐作为活性成分以及药学上可接受的载体。
本发明提供了上述化合物或其药学上可接受的盐或组合物在制备降解蛋白药物中的应用。
本发明提供了上述化合物或其药学上可接受的盐或组合物在制备降解Wee1蛋白药物中的应用。
本发明提供了上述化合物或其药学上可接受的盐或组合物4在制备挟持CRL4(CRBN)降解Wee1蛋白药物中的应用。
定义和说明
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。
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。
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的中性形式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的中性形式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。
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。
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。
除非另有说明，术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。
除非另有说明，术语“顺反异构体”或者“几何异构体”系由因双键或者成环碳原子单键不能自由旋转而引起。
除非另有说明，术语“非对映异构体”是指分子具有两个或多个手性中心，并且分子间为非镜像的关系的立体异构体。
除非另有说明，“(D)”或者“(+)”表示右旋，“(L)”或者“(-)”表示左旋，“(DL)”或者“(±)”表示外消旋。
除非另有说明，用楔形实线键
Figure PCTCN2020114728-appb-000013
和楔形虚线键
Figure PCTCN2020114728-appb-000014
表示一个立体中心的绝对构型，用直形实线键
Figure PCTCN2020114728-appb-000015
和直形虚线键
Figure PCTCN2020114728-appb-000016
表示立体中心的相对构型，用波浪线
Figure PCTCN2020114728-appb-000017
表示楔形实线键
Figure PCTCN2020114728-appb-000018
或楔形虚线键
Figure PCTCN2020114728-appb-000019
或用波浪线
Figure PCTCN2020114728-appb-000020
表示直形实线键
Figure PCTCN2020114728-appb-000021
和直形虚线键
Figure PCTCN2020114728-appb-000022
本发明的化合物可以存在特定的。除非另有说明，术语“互变异构体”或“互变异构体形式”是指在室温下，不同官能团异构体处于动态平衡，并能很快的相互转化。若互变异构体是可能的(如在溶液中)，则可以达到互变异构体的化学平衡。例如，质子互变异构体(proton tautomer)(也称质子转移互变异构体(prototropic tautomer))包括通过质子迁移来进行的互相转化，如酮-烯醇异构化和亚胺-烯胺异构化。价键异构体(valence tautomer)包括一些成键电子的重组来进行的相互转化。其中酮-烯醇互变异构化的具体实例是戊烷-2,4-二酮与4-羟基戊-3-烯-2-酮两个互变异构体之间的互变。
除非另有说明，术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100％，并且，该异构体或对映体的含量大于等于60％，或者大于等于70％，或者大于等于80％，或者大于等于90％，或者大于等于95％，或者大于等于96％，或者大于等于97％，或者大于等于98％，或者大于等于99％，或者大于等于99.5％，或者大于等于99.6％，或者大于等于99.7％，或者大于等于99.8％，或者大于等于99.9％。
除非另有说明，术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如，其中一种异构体或对映体的含量为90％，另一种异构体或对映体的含量为10％，则异构体或对映体过量(ee值)为80％。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外， 对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。
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。
“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。
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。
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。
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。
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。
当一个取代基为空缺时，表示该取代基是不存在的，比如A-X中X为空缺时表示该结构实际上是A。
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。
除非另有规定，术语“C 1-4烷基”用于表示直链或支链的由1至4个碳原子组成的饱和碳氢基团。所述C 1-4烷基包括C 1-2、C 1-3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 1-4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)等。
除非另有规定，术语“C 2-4烷基”用于表示直链或支链的由2至4个碳原子组成的饱和碳氢基团。所述C 2-4烷基包括C 2-3和C 3-4烷基等；其可以是一价(如乙基)、二价(如亚乙基)或者多价(如次乙基)。C 2-4烷基的实例包括但不限于乙基(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)等。
除非另有规定，术语“C 3-4烷基”用于表示直链或支链的由3至4个碳原子组成的饱和碳氢基团。所述C 3-4烷基包括C 3和C 4烷基等；其可以是一价(如丙基)、二价(如亚丙基)或者多价(如次丙基)。C 3-4烷基的实例包括但不限于丙基(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)等。
除非另有规定，“C 2-3烯基”用于表示直链或支链的包含至少一个碳-碳双键的由2至3个碳原子组成的碳氢基团，碳-碳双键可以位于该基团的任何位置上。所述C 2-3烯基包括C 3和C 2烯基；所述C 2-3烯基可以是一价、二价或者多价。C 2-3烯基的实例包括但不限于乙烯基、丙烯基等。
除非另有规定，术语“卤代素”或“卤素”本身或作为另一取代基的一部分表示氟、氯、溴或碘原子。此外，术语“卤代烷基”意在包括单卤代烷基和多卤代烷基。例如，术语“卤代(C 1-C 4)烷基”意在包括但不仅限于三氟甲基、2，2，2-三氟乙基、4-氯丁基和3-溴丙基等等。除非另有规定，卤代烷基的实例包括但不仅限于：三氟甲基、三氯甲基、五氟乙基，和五氯乙基。
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(例如亲和取代反应)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。
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。
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。
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 PCTCN2020114728-appb-000023
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。
本发明的化合物可以有多种用途或适应症，包括但不限于本申请所列举的具体用途或适应症。
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：aq代表水；HATU代表O-(7-氮杂苯并三唑-1-基)-N，N，N'，N'-四甲基脲六氟磷酸盐；EDC代表N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐；m-CPBA代表3-氯过氧苯甲酸；eq代表当量、等量；M代表mol/L；CDI代表羰基二咪唑；DCM代表二氯甲烷；PE代表石油醚；DIAD代表偶氮二羧酸二异丙酯；DMF代表N，N-二甲基甲酰胺；DMSO代表二甲亚砜；EtOAc代表乙酸乙酯；EtOH代表乙醇；MeOH代表甲醇；CBz代表苄氧羰基，是一种胺保护基团；BOC代表叔丁氧羰基是一种胺保护基团；HOAc代表乙酸；ACN乙腈BH 3代表氰基硼氢化钠；r.t.代表室温；O/N代表过夜；THF代表四氢呋喃；Boc 2O代表二-叔丁基二碳酸酯；三氟乙酸代表三氟乙酸；DIPEA代表二异丙基乙基胺；SOCl 2代表氯化亚砜；CS 2代表二硫化碳；TsOH代表对甲苯磺酸；NFSI代表N-氟-N-(苯磺酰基)苯磺酰胺；NCS代表1-氯吡咯烷-2,5-二酮；n-Bu 4NF代表氟化四丁基铵；iPrOH代表2-丙醇；mp代表熔点；LDA代表二异丙基胺基锂。BSA代表牛血清蛋白；CC885的结构为
Figure PCTCN2020114728-appb-000024
来那度胺的结构为
Figure PCTCN2020114728-appb-000025
化合物依据本领域常规命名原则或者使用
Figure PCTCN2020114728-appb-000026
软件命名，市售化合物采用供应商目录名称。技术效果
本发明化合物证明了对MOLT-4细胞内Wee1蛋白水平明显下调作用。
附图说明
图1：WX106对不同肿瘤细胞生长的抑制率；
图2：WX106对结直肠癌细胞系克隆形成的影响；
图3：WX106对脑癌胞系克隆形成的影响；
图4：热位移实验结果；
图5：WX106与CC885竞争性结合CRBN实验；
图6：SNU182中，WX106与Poma竞争性结合CRBN实验；
图7：Hep3B细胞中，WX106与Poma竞争性结合CRBN实验；
图8：WX106依赖CRBN抑制Hep3B细胞增殖实验；
图9：WX106依赖CRBN抑制MM.1S细胞增殖实验；
图10：WX106依赖CRBN抑制Hep3B细胞增殖实验；
图11：WX106依赖CRBN抑制SNU182细胞增殖实验；
图12：WX106促进293T细胞中Wee1蛋白的降解实验；
图13：WX106促进MOLT-4细胞中Wee1蛋白的降解实验；
图14：WX106促进MOLT-4细胞中Wee1蛋白的降解实验；
图15：WX106促进U87-MG细胞中Wee1蛋白的降解实验；
图16：WX106促进U87-MG细胞中Wee1蛋白的降解实验；
图17：WX106促进HCT116细胞中Wee1蛋白的降解实验；
图18：WX106促进LN-229细胞中Wee1蛋白的降解实验；
图19：WX106依赖CRBN促进293T细胞中Wee1蛋白的泛素化降解实验；
图20：WX106依赖CRBN促进U87-MG细胞中Wee1蛋白的泛素化降解实验；
图21：WX106对U87-MG细胞中Wee1蛋白稳定性的影响；
图22：WX106对293T细胞中Wee1蛋白稳定性的影响；
图23：WX106与DNA损伤剂的协同作用实验；
图24：WX106增强Wee1与CRBN复合体的形成实验1；
图25：WX106增强Wee1与CRBN复合体的形成实验2；
图26：WX106增强Wee1与CRBN复合体的形成实验3；
图27：Wee1的激酶结构域能够与CRBN的结合；
图28：不同化合物在MOLT-4细胞中对Wee1的降解作用；
图29：不同化合物在U87-MG细胞中对Wee1的降解作用。
具体实施方式
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。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。
实施例1：WX106
Figure PCTCN2020114728-appb-000027
Figure PCTCN2020114728-appb-000028
步骤1：中间体WX001–2的合成
室温和氮气保护下，将WX001–1(100.00g,465.02mmol)溶于三氯甲烷(500mL)和乙酸乙酯(500mL)的混合溶液中，随后加入溴化铜(207.73g,930.04mmol)，反应混合物加热至100℃并在100℃下搅拌反应14小时。反应完毕后，反应液冷却至室温，过滤，滤液减压浓缩除去溶剂。往残渣中加水(200mL)，并用二氯甲烷(200mL×3)萃取。合并有机相，用饱和食盐水(300mL×2)洗涤，无水硫酸钠干燥，过滤。得到中间体WX001–2溶解在二氯甲烷(465.02mmol,600mL)中，直接用于下一步反应。
步骤2：中间体WX001–3的合成
0℃和氮气保护下，向中间体WX001–2的二氯甲烷溶液(465.02mmol,600mL)中加入三乙胺(47.06g,465.02mmol,64.73mL)，反应混合物升至室温并在室温下搅拌反应0.5小时。反应完毕后，向反应液中加入水(300mL)，用二氯甲烷(200mL×3)萃取。合并有机相，用饱和食盐水(400mL×2)洗涤，无水硫酸钠干燥，过滤。得到中间体WX001–3溶解在二氯甲烷(465.02mmol,1200mL)中，直接用于下一步反应。
步骤3：中间体WX001–4的合成
室温和氮气保护下，向中间体WX001–3的二氯甲烷溶液(465.02mmol,1200mL)中加入甲苯(2000mL)，随后加入乙基(三苯基膦)乙酸酯(194.40g,558.02mmol)，反应混合物加热至130℃并在130℃下搅拌反应60小时。反应完毕后，冷却至室温，减压除去溶剂。所得残余物经过柱层析分离(洗脱剂：石油醚/乙酸乙酯＝1/0–70/1，体积比)，得到中间体WX001–4。 1H NMR(400MHz,CDCl 3)δ:7.71(d,J＝2.0Hz,1H),7.64(s,1H),7.40(d,J＝8.6Hz,1H),7.35(d,J＝8.8Hz,1H),4.21(q,J＝7.2Hz,2H),3.66(d,J＝0.8Hz,2H),1.29(t,J＝7.2Hz,3H)。
步骤4：中间体WX001–5的合成
室温和氮气保护下，将中间体WX001–4(10.00g,35.32mmol)和二苯甲酮亚胺(7.04g,38.85mmol,6.52mL)加入到二氧六环(100mL)中，随后依次加入三(二亚苄基丙酮)二钯(1.62g,1.77mmol)，4,5–双二 苯基膦–9,9–二甲基氧杂蒽(2.04g,3.53mmol)和碳酸铯(17.26g,52.98mmol)，反应混合物加热至80℃并在80℃下搅拌反应3小时。反应完毕后，冷却至室温，加入水(150mL)，用乙酸乙酯(100mL×3)萃取。合并有机相，用饱和食盐水(100mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压除去溶剂，所得残余物经过柱层析分离(洗脱剂：石油醚/乙酸乙酯＝1/0–10/1，体积比)，得到中间体WX001–5。MS–ESI m/z:384.4[M+H] +. 1H NMR(400MHz,CDCl 3)δ:7.76(d,J＝6.8Hz,2H),7.56(s,1H),7.53–7.46(m,1H),7.44–7.39(m,2H),7.26–7.19(m,4H),7.15–7.10(m,2H),6.94(d,J＝2.0Hz,1H),6.66(dd,J＝2.2,8.6Hz,1H),4.16(q,J＝7.2Hz,2H),3.56(s,2H),1.27(t,J＝7.2Hz,3H)。
步骤5：中间体WX001–6的合成
0℃和氮气保护下，将中间体WX001–5(15.83g,19.03mmol,纯度：46.10％)加入到N,N–二甲基甲酰胺(100mL)中，随后加入叔丁醇钾(2.14g,19.03mmol)，然后将丙烯酰胺(1.35g,19.03mmol)加入到上述反应液中，反应混合物在0℃和氮气保护下搅拌反应1小时。反应完毕后，加入水(50mL)，用乙酸乙酯(30mL×3)萃取。合并有机相，用饱和食盐水(20mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压除去溶剂，所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝10/1–1/2，体积比)，得到中间体WX001–6。 1H NMR(400MHz,DMSO_d 6)δ:10.87(s,1H),7.79(s,1H),7.68(d,J＝6.8Hz,2H),7.56–7.50(m,1H),7.49–7.45(m,2H),7.37–7.27(m,4H),7.15(dd,J＝3.0,6.6Hz,2H),6.86(d,J＝2.0Hz,1H),6.69(dd,J＝2.2,8.6Hz,1H),3.99(dd,J＝5.0,11.8Hz,1H),2.70–2.63(m,1H),2.49–2.40(m,1H),2.13–2.00(m,1H),1.98–1.90(m,1H)。
步骤6：中间体WX001–7的合成
室温和氮气保护下，将中间体WX001–6(1.55g,2.71mmol,纯度：71.39％)加入到盐酸/乙酸乙酯(4M,29.62mL)中，反应混合物在室温下搅拌反应12小时。反应完毕后，减压除去溶剂，所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝10/1–0/1，体积比)，得到中间体WX001–7。MS–ESI m/z:245.1[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.97(s,1H),10.23(br s,2H),8.04(s,1H),7.71(d,J＝8.8Hz,1H),7.56(d,J＝2.0Hz,1H),7.33(dd,J＝2.0,8.8Hz,1H),4.19(dd,J＝4.6,12.2Hz,1H),2.87–2.75(m,1H),2.68–2.57(m,1H),2.39–2.23(m,1H),2.22–2.08(m,1H)。
步骤7：化合物WX106的合成
室温和氮气保护下，将中间体WX001–7(1.88g,6.00mmol,纯度：77.97％)和3–氯–4–甲基–苯甲酸(1.02g,6.00mmol)溶解于N,N–二甲基甲酰胺(10mL)中，加入2–(7–氧化苯并三氮唑)–N,N,N',N'–四甲基脲六氟磷酸酯(3.42g,9.00mmol)和三乙胺(607.29mg,6.00mmol,835.34μL)，反应混合物在室温下搅拌反应3小时。反应完毕后，加入水(50mL)，用乙酸乙酯(30mL×3)萃取。合并有机相，用饱和食盐水(30mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压除去溶剂，所得残余物经柱层析分离(洗脱剂：石 油醚/乙酸乙酯＝10/1–1/2，体积比)后，再经制备HPLC分离(流动相：乙腈/水；酸性体系：0.05％HCl)，得到目标化合物WX106。MS–ESI m/z:397.1[M+H] +,399.0[M+2+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.96(s,1H),10.33(s,1H),8.05(d,J＝1.2Hz,1H),8.00(d,J＝2.0Hz,1H),7.92(s,1H),7.87(dd,J＝1.6,8.0Hz,1H),7.67(dd,J＝2.0,8.8Hz,1H),7.57(d,J＝8.8Hz,1H),7.52(d,J＝8.0Hz,1H),4.14(dd,J＝4.8,12.0Hz,1H),2.85–2.75(m,1H),2.68–2.57(m,1H),2.42(s,3H),2.36–2.25(m,1H),2.19–2.10(m,1H)。
实施例2：WX002
Figure PCTCN2020114728-appb-000029
室温和氮气保护下，将中间体WX001–7(150.00mg,614.14μmol)和2–(三氟甲基)异烟酸(117.37mg,614.14μmol)溶于N,N–二甲基甲酰胺(3mL)中，随后加入O–(7–氮杂苯并三唑–1–基)–N,N,N,N–四甲基脲六氟磷酸盐(350.27mg,921.21μmol)和三乙胺(186.43mg,1.84mmol,256.44μL)，反应混合物在室温下搅拌反应15小时。反应完毕后，加入水(30mL)稀释，用乙酸乙酯(30mL×3)萃取。合并有机相，用饱和食盐水(40mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压除去溶剂。所得残余物经过制备HPLC(流动相：乙腈/水；酸性体系：0.05％HCl)分离，得到目标化合物WX002。MS–ESI m/z:418.0[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.94(s,1H),10.75(s,1H),9.00(d,J＝5.2Hz,1H),8.39(s,1H),8.22(d,J＝4.8Hz,1H),7.99(d,J＝1.6Hz,1H),7.94(s,1H),7.67(dd,J＝1.8,9.0Hz,1H),7.61(d,J＝8.8Hz,1H),4.15(dd,J＝4.8,12.0Hz,1H),2.82–2.74(m,1H),2.69–2.61(m,1H),2.35–2.28(m,1H),2.20–2.11(m,1H)。
实施例3：WX003
Figure PCTCN2020114728-appb-000030
Figure PCTCN2020114728-appb-000031
室温和氮气保护下，将中间体WX001–7(0.15g,534.37μmol,盐酸盐)和对甲基苯甲酸(87.30mg,641.24μmol)加入到N,N–二甲基甲酰胺(10mL)中，加入O–(7–氮杂苯并三唑–1–基)–N,N,N,N–四甲基脲六氟磷酸盐(304.77mg,801.55μmol)和三乙胺(162.22mg,1.60mmol,223.13μL)，反应混合物在室温下搅拌反应12小时。反应完毕后，反应混合物加入水(50mL)稀释，用乙酸乙酯(30mL×3)萃取。合并有机相，用饱和食盐水(50mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压除去溶剂。所得残余物经制备HPLC分离(流动相：乙腈/水；酸性体系：0.05％HCl)，得到目标化合物WX003。MS–ESI m/z:363.2[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.96(s,1H),10.21(s,1H),8.02(d,J＝2.0H,1H),7.91(s,2H),7.89(s,1H),7.66(dd,J＝2.0,8.8Hz,1H),7.56(d,J＝8.8Hz,1H),7.34(d,J＝8.0Hz,2H),4.14(dd,J＝4.8,12.0Hz,1H),2.86–2.74(m,1H),2.70–2.55(m,1H),2.40(s,3H),2.35–2.24(m,1H),2.20–2.10(m,1H)。
实施例4：WX004
Figure PCTCN2020114728-appb-000032
室温和氮气保护下，将中间体WX001–7(0.15g,534.37μmol,盐酸盐)和3–氯苯甲酸(100.40mg,641.24μmol)加入到N,N–二甲基甲酰胺(10mL)中，加入O–(7–氮杂苯并三唑–1–基)–N,N,N,N–四甲基脲六氟磷酸盐(304.77mg,801.55μmol)和三乙胺(162.22mg,1.60mmol,223.13μL)，反应混合物在室温下搅拌反应2小时。反应完毕后，反应混合物加入水(50mL)稀释，用乙酸乙酯(30mL×3)萃取。合并有机相，用饱和食盐水(50mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压除去溶剂。所得残余物经制备HPLC分离(流动相：乙腈/水；酸性体系：0.05％HCl)，得到目标化合物WX004。MS–ESI m/z:383.1[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.96(s,1H),10.41(s,1H),8.04(t,J＝1.6H,1H),8.00(d,J＝1.6Hz,1H),7.94(d,J＝7.6Hz,1H),7.93(s,1H),7.73–7.63(m,2H),7.61–7.54(m,2H),4.15(dd,J＝5.0,12.2Hz, 1H),2.88–2.74(m,1H),2.69–2.55(m,1H),2.37–2.26(m,1H),2.20–2.10(m,1H)。
实施例5：WX005
Figure PCTCN2020114728-appb-000033
室温和氮气保护下，将中间体WX001–7(0.15g,534.37μmol,盐酸盐)和苯甲酸(78.31mg,641.24μmol)加入到N,N–二甲基甲酰胺(10mL)中，加入O–(7–氮杂苯并三唑–1–基)–N,N,N,N–四甲基脲六氟磷酸盐(304.77mg,801.55μmol)和三乙胺(162.22mg,1.60mmol,223.13μL)，反应混合物在室温下搅拌反应2小时。反应完毕后，反应混合物加入水(50mL)稀释，用乙酸乙酯(30mL×3)萃取。合并有机相，用饱和食盐水(50mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压除去溶剂。所得残余物经制备HPLC分离(流动相：乙腈/水；酸性体系：0.05％HCl)，得到目标化合物WX005。MS–ESI m/z:349.1[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.96(s,1H),10.31(s,1H),8.03(d,J＝2.0Hz,1H),7.98(dd,J＝1.6,6.8Hz,2H),7.92(s,1H),7.67(dd,J＝2.0,8.8Hz,1H),7.63–7.49(m,4H),4.15(dd,J＝4.8,12.0Hz,1H),2.88–2.74(m,1H),2.71–2.56(m,1H),2.37–2.26(m,1H),2.20–2.10(m,1H)。
实施例6：WX006
Figure PCTCN2020114728-appb-000034
室温和氮气保护下，将中间体WX001–7(0.100g,402.96μmol,纯度：98.42％)和丁酸(35.50mg,402.96μmol)加入到N,N–二甲基甲酰胺(5mL)中，加入O–(7–氮杂苯并三唑–1–基)–N,N,N,N–四甲基脲六氟磷酸盐(229.82mg,604.43μmol)和三乙胺(122.33mg,1.21mmol,168.26μL)，反应混合物在室温下搅 拌反应3小时。反应完毕后，反应混合物加入水(20mL)稀释，用乙酸乙酯(15mL×3)萃取。合并有机相，用饱和食盐水(15mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压除去溶剂。所得残余物经制备HPLC分离(流动相：乙腈/水；酸性体系：0.05％HCl)，得到目标化合物WX006。MS–ESI m/z:315.2[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.95(s,1H),9.91(s,1H),7.88(s,1H),7.87(d,J＝1.6Hz,1H),7.49(d,J＝8.8Hz,1H),7.45(dd,J＝1.8,8.6Hz,1H),4.11(dd,J＝4.8,12.0Hz,1H),2.87–2.74(m,1H),2.70–2.55(m,1H),2.28(t,J＝7.2Hz,2H),2.26–2.21(m,1H),2.17–2.09(m,1H),1.68–1.55(m,2H),0.92(t,J＝7.4Hz,3H)。
实施例7：WX007
Figure PCTCN2020114728-appb-000035
室温和氮气保护下，将中间体WX001–7(0.150g,614.14μmol)和呋喃–2–甲酸(137.67mg,1.23mmol)溶于N,N–二甲基甲酰胺(6mL)中，依次加入O–(7–氮杂苯并三唑–1–基)–N,N,N,N–四甲基脲六氟磷酸盐(350.27mg,921.20μmol)和三乙胺(186.43mg,1.84mmol,256.44μL)，反应混合物在室温下搅拌反应12小时。反应完毕后，反应混合物加入水(50mL)稀释，用乙酸乙酯(30mL×3)萃取。合并有机相，用饱和食盐水(30mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压除去溶剂，所得残余物经制备HPLC分离(流动相：乙腈/水；酸性体系：0.05％HCl)，得到目标化合物WX007。MS–ESI m/z:339.0[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.94(s,1H),10.22(s,1H),7.97-7.92(m,2H),7.90(s,1H),7.66(dd,J＝2.2,9.0Hz,1H),7.55(d,J＝8.8Hz,1H),7.32(d,J＝3.2Hz,1H),6.70(dd,J＝2.0,3.2Hz,1H),4.13(dd,J＝5.0,12.2Hz,1H),2.85–2.74(m,1H),2.70–2.57(m,1H),2.36–2.24(m,1H),2.19–2.10(m,1H)。
实施例8：WX008
Figure PCTCN2020114728-appb-000036
Figure PCTCN2020114728-appb-000037
室温和氮气保护下，将中间体WX001–7(0.150g,614.14μmol)和环丙基甲酸(63.44mg,736.96μmol)溶于N,N–二甲基甲酰胺(10mL)中，依次加入O–(7–氮杂苯并三唑–1–基)–N,N,N,N–四甲基脲六氟磷酸盐(350.27mg,921.20μmol)和三乙胺(186.43mg,1.84mmol,256.44μL)，反应混合物在室温下搅拌反应12小时。反应完毕后，反应混合物加入水(50mL)稀释，用乙酸乙酯(40mL×3)萃取。合并有机相，用饱和食盐水(30mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压除去溶剂，所得残余物经制备HPLC分离(流动相：乙腈/水；酸性体系：0.05％HCl)，得到目标化合物WX008。MS–ESI m/z:313.0[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.93(s,1H),10.23(s,1H),7.88(s,1H),7.87(s,1H),7.53–7.47(m,1H),7.46–7.41(m,1H),4.10(dd,J＝4.8,12.0Hz,1H),2.78–2.73(m,1H),2.61(s,1H),2.27–2.17(m,1H),2.16–2.13(m,1H),1.78–1.78(m,1H),0.80–077(m,4H)。
实施例9：WX009
Figure PCTCN2020114728-appb-000038
室温和氮气保护下，将中间体WX001–7(152.00mg,541.49μmol,盐酸盐)和丙烯酸(39.02mg,541.49μmol,37.16μL)溶解于N,N–二甲基甲酰胺(5mL)中，依次加入O–(7–氮杂苯并三唑–1–基)–N,N,N,N–四甲基脲六氟磷酸盐(308.84mg,812.24μmol)和三乙胺(164.38mg,1.62mmol,226.11μL)，反应混合物在室温下搅拌反应15小时。反应完毕后，加入水(30mL)稀释，用乙酸乙酯(30mL×3)萃取。合并有机相，用饱和食盐水(40mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压除去溶剂，所得残余物经过制备HPLC分离(流动相：乙腈/水；酸性体系：0.05％HCl)，得到目标化合物WX009。MS–ESI m/z:299.0[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.95(s,1H),10.20(s,1H),7.94(s,1H),7.90(s,1H),7.53(br s,2H),6.44(dd,J＝10.2,17.0Hz,1H),6.25(dd,J＝2.0,17.2Hz,1H),5.74(dd,J＝2.0,10.0Hz,1H),4.12(dd,J＝5.2,12.0Hz,1H),2.83–2.74(m,1H),2.65–2.55(m,1H),2.29–2.20(m,1H),2.17–2.07(m,1H)。
实施例10：WX010
Figure PCTCN2020114728-appb-000039
室温和氮气保护下，将中间体WX001–7(0.150g,614.14μmol)和丙酸(45.49mg,614.14μmol,45.82μL)溶于N,N–二甲基甲酰胺(3mL)中，依次加入O–(7–氮杂苯并三唑–1–基)–N,N,N,N–四甲基脲六氟磷酸盐(350.27mg,921.21μmol)和三乙胺(186.43mg,1.84mmol,256.44μL)，反应混合物在室温下搅拌反应15小时。反应完毕后，反应混合物加入水(30mL)稀释，用乙酸乙酯(30mL×3)萃取。合并有机相，用饱和食盐水(40mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压除去溶剂，所得残余物经制备HPLC分离(流动相：乙腈/水；酸性体系：0.05％HCl)，得到目标化合物WX010。MS–ESI m/z:301.0[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.92(s,1H),9.88(s,1H),7.88-7.84(m,2H),7.51–7.40(m,2H),4.10(dd,J＝4.8,12.0Hz,1H),2.83–2.72(m,1H),2.63–2.55(m,1H),2.31(q,J＝7.5Hz,2H),2.28–2.19(m,1H),2.16–2.08(m,1H),1.09(t,J＝7.6Hz,3H)。
实施例11：WX011
Figure PCTCN2020114728-appb-000040
室温和氮气保护下，将中间体WX001–7(0.100g,402.96μmol)和3–甲氧基丙酸(41.95mg,402.96μmol,37.79μL)溶于N,N–二甲基甲酰胺(5mL)中，依次加入O–(7–氮杂苯并三唑–1–基)–N,N,N,N–四甲基脲六氟磷酸盐(229.82mg,604.43μmol)和三乙胺(122.33mg,1.21mmol,168.26μL)，反应混合物在室温下搅拌反应3小时。反应完毕后，反应混合物加入水(20mL)稀释，用乙酸乙酯(10mL×3)萃取。合并有机相，用饱和食盐水(10mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压除去溶剂。所得残余 物经制备HPLC分离(流动相：乙腈/水；酸性体系：0.05％HCl)，得到目标化合物WX011。MS–ESI m/z:331.2[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.91(s,1H),9.98(s,1H),7.89-7.87(m,2H),7.50(d,J＝8.8Hz,1H),7.44(dd,J＝2.0,8.8Hz,1H),4.11(dd,J＝5.0,12.2Hz,1H),3.62(t,J＝6.2Hz,2H),3.24(s,3H),2.84–2.73(m,1H),2.69–2.58(m,1H),2.54(q,J＝6.2Hz,2H),2.35–2.20(m,1H),2.15–2.09(m,1H)。
实施例12：WX012
Figure PCTCN2020114728-appb-000041
步骤1：中间体WX012-1的合成
室温和氮气保护下，将中间体WX001-7(202mg,719.61μmol,盐酸盐)和N–(叔丁氧羰基)甘氨酸(126.06mg,719.61μmol)溶于N,N–二甲基甲酰胺(10mL)中，随后依次加入O–(7–氮杂苯并三唑–1–基)–N,N,N,N–四甲基脲六氟磷酸盐(410.43mg,1.08mol)和三乙胺(218.45mg,2.16mmol,300.49μL)，反应混合物在室温下搅拌反应14小时。反应完毕后，向反应液中加入水(50mL)稀释，用乙酸乙酯(50mL×3)萃取。合并有机相，用饱和食盐水(100mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压除去溶剂，所得残余物经过板分离(洗脱剂：石油醚/乙酸乙酯＝1/2，体积比)，得到中间体WX012-1。MS–ESI m/z:423.9[M+Na] +. 1H NMR(400MHz,DMSO_d 6)δ:10.92(s,1H),9.94(s,1H),7.98–7.81(m,2H),7.51(d,J＝8.8Hz,1H),7.47–7.41(m,1H),7.02(t,J＝5.8Hz,1H),4.11(dd,J＝5.0,11.8Hz,1H),3.72(d,J＝6.4Hz,2H),2.83–2.74(m,1H),2.62–2.56(m,1H),2.34–2.21(m,1H),2.16–2.10(m,1H),1.39(s,9H)。
步骤2：化合物WX012的合成
室温和氮气保护下，将中间体WX012-1(256mg,637.75μmol)加入到盐酸/乙酸乙酯(4M,20mL)中，反应混合物在室温下搅拌反应2小时。反应完毕后，反应液减压浓缩除去溶剂，所得残余物中加入甲醇(5mL)，在室温下搅拌0.5小时。反应液过滤，滤饼用甲醇(2mL)洗涤，收集滤饼，减压浓缩得到目标化合物WX012。MS–ESI m/z:302.1[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.94(s,1H),10.64(br s,1H),8.03(br s,2H),7.92(s,1H),7.86(br s,1H),7.59–7.54(m,1H),7.51–7.43(m,1H),4.13(dd,J＝4.2,11.8Hz,1H),3.75(s,2H),2.84–2.73(m,1H),2.68–2.56(m,1H),2.34–2.19(m,1H),2.18–2.08(m,1H)。
实施例13：WX013
Figure PCTCN2020114728-appb-000042
步骤1：中间体WX013–2的合成
室温下，将中间体WX013–1(5g,24.37mmol)和丙烯酰胺(1.73g,24.37mmol)溶于四氢呋喃(150mL)中，随后缓慢滴加叔丁醇钾的四氢呋喃溶液(1M,24.37mL)，反应混合物在室温下搅拌反应12小时。反应完毕后，倒入冰水(200mL)中，用2–甲基四氢呋喃(150mL×3)萃取。合并有机相，用饱和食盐水(150mL×3)洗涤，无水硫酸钠干燥，过滤，减压浓缩除去溶剂。残留物经过柱层析分离(中性氧化铝：200–300目，洗脱剂：石油醚/乙酸乙酯＝1/0–0/1，体积比)，得到中间体WX013–2。 1H NMR(400MHz,DMSO_d 6)δ:11.12(s,1H),7.87(d,J＝8.0Hz,1H),7.77(d,J＝8.4Hz,1H),7.70–7.63(m,1H),7.40(t,J＝7.2Hz,1H),4.62(dd,J＝5.2,11.6Hz,1H),2.85–2.74(m,1H),2.65–2.52(m,2H),2.24–2.18(m,1H)。
步骤2：中间体WX013–3的合成
室温和氮气保护下，将中间体WX013–2(1g,4.34mmol)溶于浓硫酸(5mL,纯度：98％)，冷却至0℃，随后加入硝酸钾(447.93mg,4.43mmol)，反应混合物在0℃下搅拌反应1小时。反应完毕后，倒入冰水(100mL)中，用2–甲基四氢呋喃(60mL×3)萃取。合并有机相，用水(80mL×3)洗涤，饱和食盐水(80mL×3)洗涤，无水硫酸钠干燥，过滤，减压浓缩除去溶剂，得到中间体WX013–3，直接用于下一步反应。
步骤3：中间体WX013–4的合成
室温和氮气保护下，将上述中间体WX013–3(0.7g,2.54mmol)溶于乙醇(14mL)中，加入氯化亚锡二水合物(4.02g,17.80mmol)，反应混合物加热至50℃并在50℃下搅拌反应12小时。反应完毕后，冷却至室温，反应液减压浓缩除去溶剂，加入水(100mL)稀释，用饱和碳酸氢钠水溶液调节pH值至约7，用2–甲基四氢呋喃(150mL×3)萃取。合并有机相，用无水硫酸钠干燥，过滤，减压浓缩除去溶剂，所 得残余物经制备HPLC分离(流动相：乙腈/水；酸性体系：0.05％HCl)，得到中间体WX013–4的盐酸盐。
步骤4：化合物WX013的合成
室温和氮气保护下，将3–氯–4–甲基苯甲酸(54.50mg,319.49μmol)溶于N,N–二甲基甲酰胺(3mL)，依次加入O–(7–氮杂苯并三唑–1–基)–N,N,N,N–四甲基脲六氟磷酸盐(182.22mg,479.24μmol)和三乙胺(96.99mg,958.48μmol,133.41μL)，反应混合物在室温下搅拌反应0.5小时，再加入中间体WX013–4的盐酸盐(0.09g,319.49μmol)，反应混合物在室温下搅拌反应12小时。反应完毕后，反应液直接减压浓缩除去溶剂，所得残余物经制备HPLC分离(流动相：乙腈/水；酸性体系：0.05％HCl)，得到目标化合物WX013。MS–ESI m/z:398.0[M+H] +. 1H NMR(400MHz,MeOD)δ:8.22(d,J＝2.0Hz,1H),7.99(d,J＝2.0Hz,1H),7.84(dd,J＝2.0,9.2Hz,1H),7.81(dd,J＝1.8,7.8Hz,1H),7.65(d,J＝8.8Hz,1H),7.46(d,J＝8.0Hz,1H),4.53(dd,J＝5.0,11.0Hz,1H),2.86–2.80(m,2H),2.65–2.53(m,1H),2.46(s,3H),2.41–2.35(m,1H)。
实验例1：细胞实验
将不同的细胞种入96孔板中(贴壁细胞1000/孔的密度，悬浮细胞10000/孔的密度)，加入10μM WX106作用72小时，加入CCK-8试剂(美仑生物公司，MA0218-L)反应2小时后用酶标仪(SpectraMax i3)进行读板，并计算抑制率。抑制率＝[(Ac-As)/(Ac-Ab)]×100％，(其中As表示实验孔吸光度，Ac表示对照孔吸光度，Ab表示空白孔吸光度)。
对18种肿瘤细胞系，使用10μM的WX106处理72小时，计算WX106对细胞生长的抑制率。实验结果如图1所示。
结论：WX106对血液肿瘤细胞和一些肝癌细胞有大于50％的生长抑制作用。
实验例2：克隆形成实验
检测化合物对细胞形成单克隆能力的影响。将细胞以低密度种植在6孔板上(约500个细胞每孔)，加入10μM WX106处理，待长成肉眼可见的克隆之时，用结晶紫(碧云天生物公司，C0121)染色，对克隆数进行统计。每种处理方式至少3个重复。实验结果如图2和3所示。
结论：10μM WX106抑制一些结直肠癌细胞和脑瘤细胞系的克隆形成。
实验例3：热位移实验
利用小分子化合物与蛋白结合可以提高蛋白热稳定性的原理。收集3×10 7个细胞，用磷酸缓冲液(PBS)清洗2次。在预冷的PBS中加入100×蛋白酶抑制剂(Protease Inhibitor Cocktail，APEXBIO公司，K1007)，悬浮细胞，每个1.5EP管分500μL细胞悬液。将EP管置于液氮速冻5min，迅速转移至26℃水浴锅，待液体融化一半时转移至4℃，直至完全融化。此冻融程序进行3次，使得细胞中的蛋白完全析出。冻融产物4℃离心20min，吸取上清混合，再分装至1.5mL EP管中，分别加入DMSO和 WX106，使WX106在溶液中的浓度为100μM。室温缓慢摇晃孵育30min，使药物与蛋白充分结合。将溶液分装至200μL PCR管，设置PCR温度(44，47，50，53，56，59℃)，每个温度3min。待所有温度热激结束后，分别收集每个温度下的DMSO和WX106处理样品，4℃离心20min，进行BCA蛋白(Thermo)浓度定量，同时进行Western blot检测CRBN(Anti-CRBN antibody，sigma公司，HPA045910)蛋白含量变化。蛋白含量变化如图4所示。分别在293T细胞裂解物中加入DMSO或WX106(100μM)，室温孵育30分钟，然后在特定温度下进行热激。检测裂解物中CRBN的蛋白表达变化。在56℃和59℃可明显观察到CRBN的蛋白丰度增多。
结论：WX106能够与CRBN结合而使其稳定。
实验例4：蛋白降解竞争实验
原理：已知化合物CC885能够挟持CRBN促进GSPT1(abcam,Anti-eRF3/GSPT1 antibody(ab49878))的泛素化降解，如果一个化合物能够结合CRBN，且本身没有明显的促GSPT1降解的作用，它将会竞争性抑制CC885对GSPT1的促降解作用。用0.1，1，10μM的WX106与10nM的CC885共同处理MOLT-4细胞4小时，用Western blot检测GSPT1的蛋白水平，Poma(泊马度胺，Pomalidomide，Selleck，S1567)在实验中作为阳性对照。收集细胞，蛋白上样缓冲液(50mM Tris-HCl，2％SDS，0.025％BPB,1％β-巯基乙醇，10％甘油)，98℃变性10min。用SDS-PAGE法分离蛋白并转移到PVDF膜(Millipore，IPVH00010)中，再用5％脱脂牛奶(溶于TBST缓冲液)封闭后，在4℃条件下与一抗过夜孵育，再与二抗(Anti-rabbit IgG,HRP-linked Antibody，Cell Signaling，7074S)室温孵育1小时，用化学发光法(ECL化学发光试剂盒，碧云天生物公司，P0018AM)检测膜上的条带。
如图5所示，在MOLT-4细胞中分别加入CC885和不同浓度的WX106或Poma，Western blot检测GSPT1蛋白表达。随着WX106浓度的增加，CC885引起的GSPT1的降解被逆转，Poma作为阳性对照，能够抑制CC885对GSPT1的促降解作用。
结论：WX106可以竞争性地结合CRBN。
实验例5：细胞生长竞争实验
原理：已知Poma是结合CRBN的化合物，且Poma对贴壁细胞没有明显的抑制作用，如果一个化合物依赖于CRBN抑制细胞生长，Poma竞争性结合CRBN一定会影响这个化合物的细胞毒作用。用1，10，50μM的Poma与10μM的WX106处理细胞72小时，用CCK-8试剂盒检测细胞活力，计算细胞生存率，生存率＝[(As-Ab)/(Ac-Ab)]×100％(其中As表示实验孔吸光度，Ac表示对照孔吸光度，Ab表示空白孔吸光度)，统计Poma竞争性结合是否会影响WX106的细胞毒作用，以确定WX106的细胞毒作用是否依赖于CRBN。该步骤适用于SUN182与Hep3B二种细胞。
在SNU182中进行的细胞生长竞争实验如图6所示，Poma能够竞争性地抑制WX106引起的细胞毒作用。在这个实验中CC885是阳性对照，已知它是依赖于CRBN而杀伤肿瘤细胞的。
图7为在Hep3B中进行的竞争实验，Poma能够竞争性地抑制WX106引起的细胞毒作用。
结论：WX106的细胞毒作用依赖于CRBN。
实验例6：比较野生型细胞与CRBN缺失或低表达细胞株对WX106的敏感性的差异
将针对CRBN的sgRNA序列插入lenti Crispr V2质粒，与包装载体psPAX2和pVSVG共同包装病毒，感染细胞，用puromycin(Invitrogen，A1113803)进行筛选，Western blot鉴定CRBN的敲除情况，之后进行细胞的生长检测，计算生存率。sgRNA序列见下表：
基因名称 序列
CRBN#1-oligo1 5'-CACCGGTCCTGCTGATCTCCTTCGC-3'
CRBN#1-oligo2 5'-AAACGCGAAGGAGATCAGCAGGACC-3'
CRBN#2-oligo1 5’-CACCGATAGTACCTAGGTGCTGATA-3’
CRBN#2-oligo2 5’-AAACTATCAGCACCTAGGTACTATC-3’
CRBN#3-oligo1 5’-CACCGCGCACCATACTGACTTCTTG-3’
CRBN#3-oligo2 5’-AAACCAAGAAGTCAGTATGGTGCGC-3’
在野生型Hep3B细胞和Hep3B CRBN KO(knockout)混合克隆细胞中分别加入WX106(10μM)，72h后CCK-8检测细胞增殖，实验结果如图8所示。结果显示敲除CRBN后，Hep3B细胞对WX106的敏感性显著降低。
在MM.1S细胞和MM.1S-p5000(Poma耐药的CRBN低表达细胞株)细胞中分别加入WX106(10μM)，72h后CCK-8检测细胞增殖，实验结果如图9所示。在CRBN低表达的情况下，细胞对WX106的敏感性显著降低。
分别在Hep3B和SNU182细胞中建立了CRBN敲除的稳定细胞系(pool)，用WX106(10μM)或溶剂处理，在不同时间点检测细胞的生长情况，并绘制生长曲线，实验结果如图10和图11中。CRBN正常表达的细胞对WX106非常敏感，而CRBN低表达的细胞株加入WX106后细胞的生长速度与加入溶剂组相比无显著差别。
结论：化合物WX106依赖于CRBN抑制肿瘤细胞的生长。
实验例7：非标记定量质谱进行蛋白组学分析
选用的细胞MOLT-4，有三种处理方式，一是直接加DMSO作为对照，二是加10μM WX106，三是同时加10μM WX106和10μM MG132(蛋白酶体抑制剂，APEXBIO，A2585，MG132可阻断蛋白的泛素化降解)，同时加10μM WX106和10μM MG132组做两个重复，DMSO的对照组和10μM WX106的给药组做三个重复。MOLT-4的药物处理时间为6小时。制备质谱样本，基本步骤包括尿素裂解，还原烷基化，脱盐，冻干。之后制备的样本在Q Exactive HF质谱仪上进行两个小时梯度分析，及后续数据分析。
表1为质谱的结果，在两种细胞株中，溶剂组可见Wee1的信号，WX106组无Wee1的信号，同时加入WX106和MG132后，Wee1的信号回复，提示，WX106促进了Wee1的泛素化降解。
表1
Figure PCTCN2020114728-appb-000043
实验例8：药物对蛋白降解作用的检测
将细胞以70％左右密度种于6孔板，加入不同浓度(浓度如附图所示)的化合物WX106，处理6小时，之后收取全细胞液，对目的蛋白进行检测。使用的抗体为：(Wee1(D10D2)Rabbit mAb，Cell Signaling，13084S)。为了研究药物导致的蛋白降解是否通过泛素化途径，用MG132来阻断蛋白的泛素化降解。此方式也适用于293T、MOLT-4、U87-MG、HCT116、LN-229。
图12为不同浓度的WX106处理293T细胞6个小时后，Wee1蛋白丰度的变化。图13和图14为在MOLT-4细胞中WX106对Wee1蛋白表达的影响。在图13中同时检测了WX106对其它Glutarimide(戊二酰亚胺)环结构的化合物的底物GSPT1和CK1α(Abcam，ab108296)的影响，结果表明WX106对GSPT1和CK1α没有显著的促降解作用。图15和图16显示了在U87-MG细胞中，WX106对Wee1有明显的促降解作用。图17显示WX106对HCT116细胞中的Wee1有弱的促降解作用。图18显示了在LN-229细胞中，WX106处理不同时间对Wee1降解作用的影响。
结论：以上结果表明WX106能够促进Wee1蛋白的降解。
分别用DMSO，1μM WX106，10μM WX106单独或联合10μM MG132处理293T细胞和293T CRBN KO细胞16h，收取全细胞裂解液，Western blot检测Wee1蛋白表达，实验结果如图19所示。可以观察到在野生细胞中，WX106的作用促进了Wee1的降解，而CRBN缺失细胞中Wee1的表达无显著的变化，提示WX106促Wee1的降解作用依赖于CRBN。野生型细胞中，当加入MG132时，WX106对Wee1的促降解作用不存在，提示WX106影响了Wee1的泛素化降解。分别用10μM WX106或WX106联合10μM MG132处理U87-MG细胞和U87-MG CRBN KO细胞16h，Western blot检测Wee1蛋白表达，实验结果如图20所示，观察到了与图19类似的结果。
结论：WX106依赖于CRBN促进Wee1的泛素化降解。
实验例9：药物对蛋白稳定性的影响
将细胞以70％左右密度种于6孔板，用10μM WX106或溶剂处理细胞，同时加入100μg/mL放线菌酮Cycloheximide(CHX)(APEXBIO,A8244)阻止蛋白的生物合成，在不同的时间间隔收样，最后检测WX106对Wee1稳定性的影响。
图21和图22分别在U87-MG和293T细胞中检测WX106对Wee1稳定性的影响，当WX106处理后，细胞在2个小时左右达到了半衰期，而用溶剂处理的细胞组中，Wee1蛋白明显稳定。
结论：WX106影响了Wee1蛋白的稳定性。
实验例10：协同用药试验
WX106分别与DNA损伤剂替莫唑胺(Temozolomide),顺铂(Cisplatin),吉西他滨(Gemcitabine)联合处理U87-MG细胞72小时，CCK-8检测细胞的增殖水平。其中WX106浓度为10μM，DNA损伤剂浓度为0.1μM，1μM和10μM。实验结果如图23所示。
结论：替莫唑胺与WX106联用能显著抑制U87-MG细胞的增殖。顺铂，吉西他滨低浓度和与WX106联用也能显著抑制U87-MG细胞的增殖。
实验例11：免疫共沉淀
将质粒转染入293T细胞，24小时后，加入化合物WX106处理细胞一定时间，用NETN裂解液(150mM NaCl，50mM pH8.0的Tris-HCl，1％NP40)裂解细胞，用琼脂糖珠(EZview TM Red抗-HA亲和凝胶，Sigma，E6779；抗
Figure PCTCN2020114728-appb-000044
M2亲和凝胶，Sigma，A2220)与裂解液孵育2小时，用裂解液洗珠子4次，加入上样缓冲液(100mM Tris-HCl，4％SDS，0.05％BPB,2％β-巯基乙醇，20％甘油)，用Western blot进行检测。
图24中，在293T细胞中过表达HA CRBN(通过转染技术在细胞内过表达CRBN，HA是一段多肽与CRBN融合表达。CRBN指细胞内源的CRBN)和FLAG Wee1(通过转染技术在细胞内过表达Wee1，FLAG是一段多肽与CRBN融合表达，Wee1指细胞内源的Wee1)，转染后24小时，分别在收集细胞前1h和2h用20μM的MG132或10μM WX106处理细胞，进行Co-IP(免疫共沉淀)试验。结果表明，Wee1能够与CRBN发生相互作用，在加入WX106的情况下，Wee1与CRBN形成的复合体明显增加。在图25中，用不同标签的Wee1进行Co-IP实验，结果与图24类似。图26中用过表达的CRBN与内源的Wee1进行免疫共沉淀，可见CRBN能够与Wee1发生相互作用，并且在加入WX106的情况下，Wee1与CRBN形成的复合体明显增加。在图27中，分别克隆不同的Wee1片段，与CRBN进行Co-IP。结果表明Wee1的激酶结构域能够与CRBN发生相互作用。
结论：WX106促进了Wee1与CRBN复合体的形成。
实验例12：降解Wee1蛋白的化合物检测
分别用10μM的待测化合物处理MOLT-4细胞4小时，和1μM的待测化合物处理U87-MG细胞24小时。用Western blot检测Wee1(Cell Signaling，13084S)的蛋白水平，GAPDH(Santa Cruz,sc-32233)为内参蛋白。收集细胞，蛋白上样缓冲液(1M Tris-HCl，10％SDS，0.025％BPB)裂解细胞，98℃变性10min之后进行Western blot分析。主要步骤包括：1)电泳：用SDS-PAGE电泳法分离蛋白，电泳过程中先将电压调至80v约30min，之后将电压调至120v，直至溴酚蓝跑出胶。2)转膜，使用湿转的方法，将凝胶与甲醇激活的PVDF膜贴紧，上下两层包裹滤纸后放入凝胶支架转印夹，并固定到转印槽，恒流250mA，90min；3)封闭，将带有蛋白印迹的PVDF膜放入抗体孵育盒中，加入5％脱脂牛 奶完全覆盖PVDF膜，室温摇床上孵育1小时；4)孵育一抗，用5％BSA配制一抗(1:1000)，并在4℃摇床上孵育过夜，5)孵育二抗，用TBST洗膜三遍，加入二抗(用5％脱脂牛奶配制)，(根据一抗的种属来源，采用不同的二抗，稀释范围1:3000-1:10000)，室温摇床上孵育一个小时；5)曝光，用TBST洗膜三遍，用化学发光法检测膜上的条带。
仪器，实验材料及缓冲液配方
Biorad PowerPac Basic Power Supply电泳仪
Biorad
Figure PCTCN2020114728-appb-000045
Tetra Cell小型垂直电泳槽
BioRad Mini Trans-Blot Electrophoretic Transfer Cell小型Trans-Blot转印槽
ChemiDoc Imaging System成像仪
凝胶制备试剂盒：PAGE凝胶制备试剂盒(10％)，EpiZyme，PG112
PVDF膜：Millipore，IPVH00010
脱脂奶粉：上海生工/BBI，A600669
BSA：Bovine Serum Albumin，ABCONE，A23088
抗兔源二抗：Anti-rabbit IgG,HRP-linked Antibody，Cell Signaling，7074S
抗鼠源二抗：HRP标记山羊抗小鼠IgG，EpiZyme，LF101
ECL化学发光试剂盒：碧云天生物公司，P0018AM
Figure PCTCN2020114728-appb-000046
电泳缓冲液：称取Tris 3.0275g，Glycine 14.4135g，SDS 1g，漩涡震荡混匀，ddH 2O定容至1L。
转膜缓冲液：称取Tris 3.03g，Glycine 14.42g，100mL甲醇，漩涡震荡混匀，ddH 2O定容至1L。
TBST：称取Tris 6.057g，NaCl 8.75g，2mL TWeen20，漩涡震荡混匀，ddH 2O定容至1L。加盐酸调pH值至8.0。
5％脱脂牛奶:称取5g脱脂奶粉，溶于100mL TBST中漩涡震荡混匀。
5％BSA:称取5g BSA，溶于100mL TBST中，漩涡震荡混匀。
细胞来源
细胞 来源
293T Thermo Fisherscientific
Other cells ATCC(American Type Culture Collection)
具体筛选结果见图28与图29。
结论：WX106，WX002，WX003，WX004，WX005和WX006对Wee1均有促降解作用；WX106 的作用效果最强。

Claims (14)

  1. 式(ⅠI)所示的化合物或其药学上可接受的盐，
    Figure PCTCN2020114728-appb-100001
    其中，
    E选自CH和N；
    R 1选自C 1-4烷基、C 2-3烯基、
    Figure PCTCN2020114728-appb-100002
    环丙基和呋喃基，所述C 1-4烷基和C 2-3烯基任选被1、2、3个R a取代；
    T 1选自C(R 3)和N；
    R 2选自H、F、Cl、Br、I、CH 3和CF 3；
    R 3选自H、F、Cl、Br、I和CH 3，所述CH 3任选被1、2、3个卤素取代；
    各R a选自卤素、OCH 3和NH 2。
  2. 根据权利要求1所述的化合物或其药学上可接受的盐，R 2选自H、Cl和CF 3。
  3. 根据权利要求2所述的化合物或其药学上可接受的盐，R 2为Cl。
  4. 根据权利要求1～3任意一项所述的化合物或其药学上可接受的盐，R 3选自H、F、Cl、Br、I和CH 3，所述CH 3任选被1、2、3个F取代。
  5. 根据权利要求4所述的化合物或其药学上可接受的盐，R 3选自H、F、Cl、Br、I和CH 3。
  6. 根据权利要求5所述的化合物或其药学上可接受的盐，R 3为H和CH 3。
  7. 根据权利要求1所述的化合物或其药学上可接受的盐，R 1选自
    Figure PCTCN2020114728-appb-100003
    Figure PCTCN2020114728-appb-100004
    CH＝CH 2、CH 3、CH 2CH 3和CH 2CH 2CH 3，所述CH 3、CH 2CH 3和CH 2CH 2CH 3任选被1、2、3个R a取代。
  8. 根据权利要求7所述的化合物或其药学上可接受的盐，R 1选自
    Figure PCTCN2020114728-appb-100005
    Figure PCTCN2020114728-appb-100006
    -CH＝CH 2、-CH 2NH 2、-CH 2CH 3、-CH 2CH 2OCH 3和-CH 2CH 2CH 3。
  9. 根据权利要求1～6任意一项所述的化合物或其药学上可接受的盐，其选自
    Figure PCTCN2020114728-appb-100007
    其中，
    m选自1、2和3；
    R a如权利要求1所定义；
    R 2如权利要求1～3任意一项所定义；
    R 3如权利要求1、4～6任意一项所定义。
  10. 下式化合物或其药学上可接受的盐，其选自
    Figure PCTCN2020114728-appb-100008
    Figure PCTCN2020114728-appb-100009
  11. 一种组合物，包括治疗有效量的根据权利要求1～10任意一项所述的化合物或其药学上可接受的盐作为活性成分以及药学上可接受的载体。
  12. 根据权利要求1～10任意一项所述的化合物或其药学上可接受的盐或根据权利要求11所述的组合物在制备降解蛋白药物中的应用。
  13. 根据权利要求1～10任意一项所述的化合物或其药学上可接受的盐或根据权利要求11所述的组合物在制备降解Wee1蛋白药物中的应用。
  14. 根据权利要求1～10任意一项所述的化合物或其药学上可接受的盐或根据权利要求11所述的组合物在制备挟持CRL4降解Wee1蛋白药物中的应用。
PCT/CN2020/114728 2019-09-12 2020-09-11 一种可降解蛋白的并环类化合物及其应用 WO2021047627A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US17/641,759 US20220324848A1 (en) 2019-09-12 2020-09-11 Fused cyclic compound capable of degrading protein and use thereof
CN202080063971.4A CN114761400A (zh) 2019-09-12 2020-09-11 一种可降解蛋白的并环类化合物及其应用
CA3150653A CA3150653A1 (en) 2019-09-12 2020-09-11 FUSED CYCLIC COMPOUND CAPABLE OF DEGRADING A PROTEIN AND ITS USE

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910865744 2019-09-12
CN201910865744.2 2019-09-12

Publications (1)

Publication Number Publication Date
WO2021047627A1 true WO2021047627A1 (zh) 2021-03-18

Family

ID=74866096

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/114728 WO2021047627A1 (zh) 2019-09-12 2020-09-11 一种可降解蛋白的并环类化合物及其应用

Country Status (4)

Country Link
US (1) US20220324848A1 (zh)
CN (1) CN114761400A (zh)
CA (1) CA3150653A1 (zh)
WO (1) WO2021047627A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023001028A1 (zh) * 2021-07-19 2023-01-26 南京明德新药研发有限公司 杂芳-3-哌啶二酮类化合物及其应用
WO2023036175A1 (zh) * 2021-09-08 2023-03-16 南京明德新药研发有限公司 戊二酰亚胺类化合物与其应用
EP4043455A4 (en) * 2019-09-12 2023-09-20 Medshine Discovery Inc. BICYCLIC COMPOUND AS A CRBN PROTEIN REGULATOR

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115380026B (zh) * 2020-03-17 2023-11-07 南京明德新药研发有限公司 蛋白降解调节剂与其使用方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015200795A1 (en) * 2014-06-27 2015-12-30 Celgene Corporation Compositions and methods for inducing conformational changes in cereblon other e3 ubiquitin ligases
WO2016057503A1 (en) * 2014-10-07 2016-04-14 Celgene Corporation Use of biomarkers for predicting clinical sensitivity to cancer treatment
CN106458993A (zh) * 2014-04-14 2017-02-22 阿尔维纳斯股份有限公司 基于酰亚胺的蛋白水解调节剂和相关使用方法
WO2017120446A1 (en) * 2016-01-08 2017-07-13 Celgene Corporation Methods for treating cancer and the use of biomarkers as a predictor of clinical sensitivity to therapies
CN108366992A (zh) * 2015-11-02 2018-08-03 耶鲁大学 蛋白水解靶向嵌合体化合物及其制备和应用方法
WO2018208123A1 (ko) * 2017-05-12 2018-11-15 한국화학연구원 신규한 피페리딘-2,6-디온 유도체 및 이의 용도

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017197046A1 (en) * 2016-05-10 2017-11-16 C4 Therapeutics, Inc. C3-carbon linked glutarimide degronimers for target protein degradation

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106458993A (zh) * 2014-04-14 2017-02-22 阿尔维纳斯股份有限公司 基于酰亚胺的蛋白水解调节剂和相关使用方法
WO2015200795A1 (en) * 2014-06-27 2015-12-30 Celgene Corporation Compositions and methods for inducing conformational changes in cereblon other e3 ubiquitin ligases
WO2016057503A1 (en) * 2014-10-07 2016-04-14 Celgene Corporation Use of biomarkers for predicting clinical sensitivity to cancer treatment
CN108366992A (zh) * 2015-11-02 2018-08-03 耶鲁大学 蛋白水解靶向嵌合体化合物及其制备和应用方法
WO2017120446A1 (en) * 2016-01-08 2017-07-13 Celgene Corporation Methods for treating cancer and the use of biomarkers as a predictor of clinical sensitivity to therapies
WO2018208123A1 (ko) * 2017-05-12 2018-11-15 한국화학연구원 신규한 피페리딘-2,6-디온 유도체 및 이의 용도

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4043455A4 (en) * 2019-09-12 2023-09-20 Medshine Discovery Inc. BICYCLIC COMPOUND AS A CRBN PROTEIN REGULATOR
WO2023001028A1 (zh) * 2021-07-19 2023-01-26 南京明德新药研发有限公司 杂芳-3-哌啶二酮类化合物及其应用
WO2023036175A1 (zh) * 2021-09-08 2023-03-16 南京明德新药研发有限公司 戊二酰亚胺类化合物与其应用

Also Published As

Publication number Publication date
CA3150653A1 (en) 2021-03-18
CN114761400A (zh) 2022-07-15
US20220324848A1 (en) 2022-10-13

Similar Documents

Publication Publication Date Title
WO2021047627A1 (zh) 一种可降解蛋白的并环类化合物及其应用
JP6948659B1 (ja) ピリダジニルチアアゾールカルボキシアミド化合物
CN110267958A (zh) 制备吡唑并[1,5-a]嘧啶及其盐的方法
JP2023109922A (ja) 癌を治療する方法
US20230357272A1 (en) Octahydropyrazinodiazanaphthyridine dione compounds
CN112204029A (zh) 治疗性化合物
WO2017201069A1 (en) Oxoindoline derivatives as protein function modulators
WO2017053706A1 (en) Tead transcription factor autopalmitoylation inhibitors
WO2021088938A1 (zh) 四氢吡啶并嘧啶类抑制剂及其制备方法和应用
TW202227389A (zh) 新穎化合物
JP7481435B2 (ja) Ｃｒａｃ阻害剤としての２ｈ－ベンゾピラン誘導体
WO2021098859A1 (zh) 氮杂七元环类抑制剂及其制备方法和应用
TWI807697B (zh) 呋喃稠環取代的戊二醯亞胺類化合物
CN112055714A (zh) 二甲基氧膦类化合物
WO2022014639A1 (ja) Ｅｇｆｒ阻害剤
AU2020410900B2 (en) Compound used as RET kinase inhibitor and application thereof
CN109096219B (zh) 一种新型抗pd-l1化合物、其应用及含其的组合物
CN111315750B (zh) 作为mTORC1/2双激酶抑制剂的吡啶并嘧啶类化合物
CA3177298C (en) Compounds containing benzosultam as erk inhibitors
WO2019242642A1 (zh) 一种otr抑制剂的可药用盐、晶型及制备方法
WO2023221975A1 (zh) 包含蛋白降解剂类生物活性化合物的抗体药物偶联物及其制备方法和用途
CN113286594B (zh) 吡啶并嘧啶类化合物在制备治疗鼻咽癌药物中的应用
CN115340527B (zh) 一种bcl-xl抑制剂及其制备方法和用途
WO2023036217A1 (zh) 丙烯酰胺类化合物及其应用
TW202340146A (zh) 抗體結合的化學降解誘導物及其方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20862675

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3150653

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20862675

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 10/08/2022)

122 Ep: pct application non-entry in european phase

Ref document number: 20862675

Country of ref document: EP

Kind code of ref document: A1