WO2023001028A1 - 杂芳-3-哌啶二酮类化合物及其应用 - Google Patents

杂芳-3-哌啶二酮类化合物及其应用 Download PDF

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WO2023001028A1
WO2023001028A1 PCT/CN2022/105236 CN2022105236W WO2023001028A1 WO 2023001028 A1 WO2023001028 A1 WO 2023001028A1 CN 2022105236 W CN2022105236 W CN 2022105236W WO 2023001028 A1 WO2023001028 A1 WO 2023001028A1
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compound
room temperature
reaction
stirred
ethyl acetate
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PCT/CN2022/105236
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French (fr)
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雷茂义
徐雨
罗云富
陈曙辉
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南京明德新药研发有限公司
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Priority to CN202280051063.2A priority Critical patent/CN117751112A/zh
Publication of WO2023001028A1 publication Critical patent/WO2023001028A1/zh

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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
    • A61K31/42—Oxazoles
    • A61K31/421—1,3-Oxazoles, e.g. pemoline, trimethadione
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C07D401/02—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 two hetero rings
    • C07D401/04—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 two hetero rings directly linked by a ring-member-to-ring-member bond
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system

Definitions

  • the present invention relates to heteroaryl-3-piperidinedione compounds and applications thereof, in particular to compounds represented by formula (II) and pharmaceutically acceptable salts thereof.
  • Immunomodulatory Drugs including Thalidomide (Thalidomide), Lenalidomide (Lena), and Pomalidomide (Pomalidomide, Poma) use the glutarimide ring structure to insert CRBN ubiquitin
  • the pocket region of the ligase recruits the transcription factor Ikaros (IKZF1)/Aiolos (IKZF3) that B cell-derived cancer cells rely on for survival and promotes its ubiquitination and degradation, thereby producing cytotoxicity.
  • lenalidomide can also target CRBN to mediate the degradation of CK1alpha to treat myelodysplastic syndrome with 5q deletion
  • CC-90009 can target CRBN to mediate GSPT1 (G1 to S Phase Transition 1) degradation to treat acute myeloid leukemia.
  • CRBN has been proven to be effective in multiple myeloma, chronic lymphocytic leukemia and other hematological malignancies, skin diseases such as leprosy erythema nodosum, and systemic lupus erythematosus, etc. Autoimmune diseases have clear curative effects. Methamide drugs have many side effects, and there is an urgent need to develop new CRBN modulator drugs to improve the clinical therapeutic effect.
  • the present invention provides a compound represented by formula (II) or a pharmaceutically acceptable salt thereof,
  • Ring A is selected from benzofuryl, benzisoxazolyl, benzotriazolyl, naphthofuryl, naphthoisoxazolyl and naphthotriazolyl;
  • L 1 is selected from a bond, -C(R a )(R b )-, -N(R c )- and -OCH 2 -;
  • Each R 1 is independently selected from F, Cl, Br, I, C 1-3 alkyl, C 1-3 alkoxy, 5-membered heteroaryl and The C 1-3 alkyl, C 1-3 alkoxy, 5-membered heteroaryl and optionally substituted by 1, 2 or 3 R;
  • n 0, 1, 2 and 3;
  • Each R a and R b are independently selected from H, F, Cl, Br and I;
  • R c is selected from H and CH 3 ;
  • Each R d is independently selected from F, Cl, Br, I, OCH 3 , N(CH 3 ) 2 and morpholinyl.
  • the ring A is selected from benzofuryl, benzisoxazolyl, benzotriazolyl, naphtho[2,3-b]furyl, naphtho[2, 1-b]furyl, naphtho[2,3-d]isoxazolyl, naphtho[1,2-d]isoxazolyl and 1H-naphtho[2,3-d][1,2 ,3] Triazolyl.
  • the ring A is selected from benzofuryl and benzisoxazolyl, and other variables are as defined in the present invention.
  • the ring A is selected from Other variables are as defined herein.
  • the structural unit selected from The # terminal is connected to the phenyl group of formula (II), and other variables are as defined in the present invention.
  • each R 1 is independently selected from F, Cl, Br, I, CH 3 , OCH 3 , OCH 2 CH 3 , thiazolyl and The CH 3 , OCH 3 , OCH 2 CH 3 , thiazolyl and Optionally substituted with 1, 2 or 3 Rd , other variables are as defined herein.
  • each R 1 is independently selected from F, Cl, CH 3 , -OCH 2 CH 2 OCH 3 , -OCH 2 CH 2 N(CH 3 ) 2 , Other variables are as defined herein.
  • the present invention provides a compound represented by formula (II) or a pharmaceutically acceptable salt thereof,
  • Ring A is selected from benzofuryl, benzisoxazolyl, benzotriazolyl, naphthofuryl, naphthoisoxazolyl and naphthotriazolyl;
  • L 1 is selected from -C(R a )(R b )-, -N(R c )- and -OCH 2 -;
  • Each R 1 is independently selected from H, F, Cl, Br, I, C 1-3 alkyl, C 1-3 alkoxy and 5-membered heteroaryl, the C 1-3 alkyl, C 1 -3 alkoxy and 5-membered heteroaryl are optionally substituted by 1, 2 or 3 R;
  • n 0, 1, 2 and 3;
  • R a , R b and R c are independently selected from H, F, Cl, Br and I;
  • R d is selected from F, Cl, Br, I and morpholinyl.
  • the ring A is selected from benzofuryl, benzisoxazolyl, benzotriazolyl, naphtho[2,3-b]furyl, naphtho[2, 1-b]furyl, naphtho[2,3-d]isoxazolyl, naphtho[1,2-d]isoxazolyl and 1H-naphtho[2,3-d][1,2 ,3] Triazolyl.
  • each R1 is independently selected from H, F, Cl, Br, I, CH3 and thiazolyl, and said CH3 and thiazolyl are optionally replaced by 1, 2 or 3 R d is substituted, and other variables are as defined herein.
  • each R 1 is independently selected from H, F, Cl, CH 3 and Other variables are as defined herein.
  • the compound or a pharmaceutically acceptable salt thereof is selected from
  • T1 is selected from CH and N ;
  • R 1 , L 1 and m are as defined in the present invention.
  • the present invention also provides a compound of the following formula or a pharmaceutically acceptable salt thereof
  • the compound or a pharmaceutically acceptable salt thereof is selected from
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms, which are suitable for use in contact with human and animal tissues within the scope of sound medical judgment , without undue toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to the salts of the compounds of the present invention, which are prepared from the compounds with specific substituents found in the present invention and relatively non-toxic acids or bases.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base, either neat solution or in a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the acid, either neat solution or in a suitable inert solvent.
  • Certain specific compounds of the present invention contain basic and acidic functional groups and can thus be converted into either base or acid addition salts.
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing acid groups or bases by conventional chemical methods.
  • such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.
  • the compounds of the invention may exist in particular 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 and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which are subject to the present within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
  • enantiomer or “optical isomer” refer to stereoisomers that are mirror images of each other.
  • cis-trans isomers or “geometric isomers” arise from the inability to rotate freely due to the double bond or the single bond of the carbon atoms forming the ring.
  • diastereoisomer refers to stereoisomers whose molecules have two or more chiral centers and which are not mirror images of the molecules.
  • keys with wedge-shaped solid lines and dotted wedge keys Indicates the absolute configuration of a stereocenter, with a straight solid-line bond and straight dashed keys Indicates the relative configuration of the stereocenter, with a wavy line Indicates wedge-shaped solid-line bond or dotted wedge key or with tilde Indicates a straight solid line key and straight dashed keys
  • tautomer or “tautomeric form” means that isomers with different functional groups are in dynamic equilibrium at room temperature and are rapidly interconvertible. If tautomerism is possible (eg, in solution), then chemical equilibrium of the tautomers can be achieved.
  • proton tautomers also called prototropic tautomers
  • proton tautomers include interconversions via migration of a proton, such as keto-enol isomerization and imine-ene Amine isomerization.
  • Valence isomers (valence tautomers) involve interconversions by recombination of some bonding electrons.
  • keto-enol tautomerization is the interconversion between two tautomers of pentane-2,4-dione and 4-hydroxypent-3-en-2-one.
  • the terms “enriched in an isomer”, “enriched in an isomer”, “enriched in an enantiomer” or “enantiomerically enriched” refer to one of the isomers or enantiomers
  • 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 Greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.
  • the terms “isomer excess” or “enantiomeric excess” refer 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 other isomer or enantiomer is 10%, then the isomer or enantiomeric excess (ee value) is 80% .
  • Optically active (R)- and (S)-isomers as well as D and L-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure desired enantiomer.
  • a diastereoisomeric salt is formed with an appropriate optically active acid or base, and then a diastereomeric salt is formed by a conventional method known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally in combination with chemical derivatization methods (e.g. from amines to amino groups formate).
  • the compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compounds.
  • compounds may be labeled with radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
  • radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
  • heavy hydrogen can be used to replace 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 isotopic composition of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
  • substituted means that any one or more hydrogen atoms on a specified atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence of the specified atom is normal and the substituted compound is stable.
  • Oxygen substitution does not occur on aromatic groups.
  • optionally substituted means that it may or may not be substituted, and unless otherwise specified, the type and number of substituents may be arbitrary on a chemically realizable basis.
  • any variable eg, R
  • its definition is independent at each occurrence.
  • said group may optionally be substituted with up to two R, with independent options for each occurrence of R.
  • substituents and/or variations thereof are permissible only 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.
  • substituent When a substituent is vacant, it means that the substituent does not exist. For example, when X in A-X is vacant, it means that the structure is actually A. When the enumerated substituent does not indicate which atom it is connected to the substituted group, this substituent can be bonded through any atom, for example, pyridyl as a substituent can be connected to any atom on the pyridine ring. The carbon atom is attached to the group being substituted.
  • linking group listed does not indicate its linking direction
  • its linking direction is arbitrary, for example,
  • the connecting group L in the middle is -MW-, at this time -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form It can also be formed by connecting loop A and loop B in the opposite direction to the reading order from left to right
  • any one or more sites of the group can be linked to other groups through chemical bonds.
  • connection method of the chemical bond is not positioned, and there is an H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will decrease correspondingly with the number of chemical bonds connected to become the corresponding valence group.
  • the chemical bonds that the site connects with other groups can use straight solid line bonds Straight dotted key or tilde express.
  • the straight-shaped solid-line bond in -OCH3 indicates that it is connected to other groups through the oxygen atom in the group;
  • the straight dotted line bond indicates that the two ends of the nitrogen atom in the group are connected to other groups;
  • the wavy lines in indicate that the 1 and 2 carbon atoms in the phenyl group are connected to other groups;
  • C n-n+m or C n -C n+m includes any specific instance 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 , also 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, n to n +m means that the number of atoms on the ring is n to n+m, for example, a 3-12-membered ring includes a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-membere
  • C 1-6 alkyl is used to denote a straight or branched chain saturated hydrocarbon group consisting 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, etc.; it can be 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 denote a straight or branched chain saturated hydrocarbon group consisting 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.
  • C 1-3 alkoxy denotes those alkyl groups containing 1 to 3 carbon atoms attached to the rest of the molecule through an oxygen atom.
  • the C 1-3 alkoxy group includes C 1-2 , C 2-3 , C 3 and C 2 alkoxy groups and the like.
  • Examples of C 1-3 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.
  • the terms "5-membered heteroaryl ring” and “5-membered heteroaryl” in the present invention can be used interchangeably.
  • 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from O, S and N, and the rest are carbon atoms.
  • the nitrogen and sulfur heteroatoms may be optionally oxidized (ie, NO and S(O) p , where p is 1 or 2).
  • the 5-6 membered heteroaryl can be attached to the rest of the molecule through a heteroatom or a carbon atom.
  • Examples of the 5-membered heteroaryl group include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl and 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrazolyl, 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, 4- thiazolyl and 5-thiazolyl, etc.
  • furyl including 2-furyl and 3-furyl, etc.
  • thienyl including 2-thienyl and 3-thienyl, etc.
  • 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 the methods well known to those skilled in the art Equivalent alternatives, preferred embodiments include but are not limited to the examples of the present invention.
  • the solvent used in the present invention is commercially available.
  • the structure of the compounds of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention involves the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction (SXRD), the cultured single crystal is collected with a Bruker D8venture diffractometer to collect diffraction intensity data, the light source is CuK ⁇ radiation, and the scanning method is: After scanning and collecting relevant data, the absolute configuration can be confirmed by further analyzing the crystal structure by direct method (Shelxs97).
  • SXRD single crystal X-ray diffraction
  • the cultured single crystal is collected with a Bruker D8venture diffractometer to collect diffraction intensity data
  • the light source is CuK ⁇ radiation
  • the scanning method is: After scanning and collecting relevant data, the absolute configuration can be confirmed by further analyzing the crystal structure by direct method (Shelxs97).
  • Figure 1 Western blot diagram of degraded GSPT1 protein.
  • the compound of the invention has excellent degradation effect on GSPT1 protein, can effectively inhibit tumor cell proliferation, and has significant tumor shrinkage effect.
  • the compound of the present invention has excellent pharmacokinetic properties and has high plasma systemic exposure.
  • the intermediate WX001–3 (520mg, 2.27mmol) was dissolved in ethanol (20mL), then Raney nickel (971.75mg, 2.27mmol, purity: 20%) and hydrochloric acid (4M, 1.70mL) were added ), the reaction mixture was evacuated and replaced several times with hydrogen, and the reaction mixture was stirred and reacted at room temperature under the protection of hydrogen (15psi) for 12 hours. After the reaction was completed, it was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The intermediate WX001–4 was obtained, which was directly used in the next reaction.
  • intermediate WX001–5 (150 mg, 374.20 ⁇ mol) was dissolved in N,N-dimethylformamide (10 mL), followed by potassium tert-butoxide (41.99 mg, 374.20 ⁇ mol). The reaction mixture was stirred at 0°C for 1 hour, acrylamide (26.60 mg, 374.20 ⁇ mol) was added, the reaction mixture was gradually warmed to room temperature and stirred at room temperature for 2 hours.
  • compound WX003-4 (5g, 17.66mmol), tert-butyl carbamate (2.48g, 21.19mmol), 2-di-tert-butylphosphine-2',4',6-triisopropyl Biphenyl (1.05g, 2.47mmol), tris(dibenzylideneacetone)dipalladium (1.13g, 1.24mmol) and potassium phosphate (15.00g, 70.64mmol) were dissolved in toluene (100mL) and water (20mL) , the reaction mixture was warmed to 100 °C and stirred for 12 hours.
  • the compound WX003-10 (21.5g, 88.51mmol) was dissolved in N,N-dimethylformamide (200mL), and triethylamine (26.87g, 265.52mmol, 36.96mL) and morphine were added Phenyl (7.71g, 88.51mmol, 7.79mL), the reaction mixture was heated to 80°C and stirred for 12 hours.
  • compound WX003-11 (8g, 32.11mmol) was dissolved in a mixed solvent of 1,4-dioxane (70mL) and water (14mL), and 3-hydroxyphenylboronic acid (6.64g, 48.17mmol), potassium carbonate (17.75g, 128.45mmol) and 1,1'-bisdiphenylphosphinoferrocene palladium dichloride (1.17g, 1.61mmol), the reaction mixture was heated to 100°C and stirred for 1 hour .
  • the compound WX003-12 (4.3g, 16.39mmol) was dissolved in N,N-dimethylformamide (50mL), and tert-butyl bromoacetate (6.39g, 32.78mmol, 4.84mL) was added and potassium carbonate (6.80g, 49.18mmol), the temperature of the reaction solution was raised to 50°C and the reaction was stirred for 12 hours.
  • the hydrochloride salt of compound WX003–14 (62.99 mg, 176.52 ⁇ mol) was dissolved in N,N–dimethylformamide (1 mL), and then O-(7-azabenzotri Azolazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (111.86mg, 294.20 ⁇ mol) and N,N-diisopropylethylamine (114.07mg, 882.59 ⁇ mol , 153.73 ⁇ L), after stirring at room temperature for 30 minutes, the hydrochloride (60 mg, 147.10 ⁇ mol) of compound WX003-9 was added, and stirring was continued for 1 hour at room temperature.
  • reaction solution was cooled to room temperature, poured into 0.5M dilute hydrochloric acid (100 mL), and extracted with ethyl acetate (50 mL ⁇ 3). The organic phases were combined, washed successively with half-saturated brine (50 mL ⁇ 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent.
  • the compound WX004-4 (700mg, 3.49mmol) was dissolved in acetonitrile (20mL), then potassium iodide (115.84mg, 697.84 ⁇ mol), potassium carbonate (964.49mg, 6.98mmol), 4–(2 - Chloroethyl)morpholine (574.25mg, 3.84mmol), the reaction mixture was warmed to 80°C and stirred for 12 hours. After the reaction was completed, the reaction solution was directly filtered, and the filter cake was rinsed with tetrahydrofuran (20 mL ⁇ 3). The filtrate was collected and concentrated under reduced pressure to remove the solvent.
  • compound WX004-5 (760mg, 2.42mmol) was dissolved in tetrahydrofuran (6mL) and water (2mL), then lithium hydroxide monohydrate (304.92mg, 7.27mmol) was added, and the reaction mixture was stirred at room temperature Reaction 12 Hour. After the reaction is complete, add water (10mL) to the reaction solution, adjust the pH to 7–8 with 6M dilute hydrochloric acid, extract with 2–methyltetrahydrofuran (50mL ⁇ 10), combine the organic phases, dry with anhydrous sodium sulfate, and filter , concentrated under reduced pressure to obtain compound WX004-6.
  • the compound WX004-6 (890mg, 2.97mmol) was dissolved in toluene (10mL), then triethylamine (660.98mg, 6.53mmol, 909.19 ⁇ L), diphenylphosphoryl azide (898.82mg , 3.27mmol, 707.73 ⁇ L), the reaction mixture was stirred at room temperature for 10 minutes, then phenol (1.40g, 14.85mmol, 1.31mL) was added, the reaction mixture was warmed to 100°C and stirred for 1 hour.
  • the compound WX003-4 (5.5g, 19.43mmol) was dissolved in N,N-dimethylformamide (60mL), followed by the addition of zinc cyanide (2.87g, 24.44mmol, 1.55mL) , 2-dicyclohexylphosphine-2,4,6-triisopropylbiphenyl (926.10mg, 1.94mmol), three (dibenzylideneacetone) dipalladium (889.46mg, 971.33 ⁇ mol), the reaction mixture was heated to 80°C and stirred for 12 hours.
  • the organic phases were combined, washed successively with half-saturated brine (50 mL ⁇ 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent.
  • Compound WX005-4 was obtained by preparative HPLC separation (chromatographic column: Waters Xbridge Prep OBD C18 150*40mm*10 ⁇ m; mobile phase: acetonitrile/water; neutral system: NH 4 HCO 3 ).
  • the compound WX004-4 (2g, 9.97mmol) was dissolved in acetonitrile (20mL), followed by the addition of 2-bromoethyl methyl ether (1.39g, 9.97mmol, 936.23 ⁇ L), potassium iodide (165.49mg , 996.92 ⁇ mol), potassium carbonate (2.76g, 19.94mmol), the reaction mixture was heated to 80°C and stirred for 12 hours. After the reaction was completed, the reaction solution was cooled to room temperature, filtered, and the filter cake was rinsed with tetrahydrofuran (20 mL ⁇ 3). The filtrate was collected and concentrated under reduced pressure to remove the solvent.
  • compound WX005-5 (2.5g, 9.66mmol) was dissolved in tetrahydrofuran (18mL) and water (6mL), then lithium hydroxide monohydrate (1.62g, 38.66mmol) was added, and the reaction mixture was stirred at room temperature. 12 hours. After the reaction was completed, water (20 mL) was added to the reaction solution, the pH was adjusted to 5-6 with 6M dilute hydrochloric acid, and extracted with ethyl acetate (30 mL ⁇ 3). The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain compound WX005-6.
  • the compound WX005-6 (1.9g, 7.77mmol) was dissolved in tert-butanol (30mL), followed by the addition of diphenylphosphoryl azide (3.21g, 11.65mmol, 2.52mL), triethylamine (1.57g, 15.53mmol, 2.16mL), the reaction mixture was warmed to 100°C and stirred for 12 hours. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure to remove the solvent.
  • compound WX005-9 (76mg, 226.34 ⁇ mol) was dissolved in N,N-dimethylformamide (2mL), then compound WX004-2 (70.15mg, 271.61 ⁇ mol), triethylamine (45.81 mg, 452.68 ⁇ mol, 63.01 ⁇ L), the reaction mixture was stirred at room temperature for 12 hours. After the reaction was completed, the reaction solution was poured into water (20 mL), and extracted with ethyl acetate (20 mL ⁇ 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent.
  • the compound WX008-3 (68g, 329.79mmol) was dissolved in dichloromethane (1300mL), the temperature was lowered to -30°C under the protection of nitrogen, and boron tribromide (223.07g, 890.42mmol) was added dropwise , 85.80 mL), the reaction mixture was slowly raised to room temperature and stirred for 1 hour.
  • reaction solution is quenched by pouring it into ice water (2000mL), separating the liquids, collecting the organic phase, extracting the aqueous phase with ethyl acetate (800mL ⁇ 3), combining the organic phases, and successively washing with saturated brine (600mL ⁇ 3 ), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound WX008-4.
  • the compound WX008-5 (3g, 13.62mmol) was dissolved in tetrahydrofuran (40mL), followed by the addition of N-(tert-butoxycarbonyl)ethanolamine (3.29g, 20.43mmol, 3.17mL), azobis Formyldipiperidine (6.87g, 27.25mmol), cooled to 0°C, added dropwise a solution of tributylphosphine (5.51g, 27.25mmol, 6.72mL) dissolved in tetrahydrofuran (10mL), and the reaction mixture returned to room temperature and stirred React for 3 hours.
  • the compound WX008-6 (1.8g, 4.95mmol) was dissolved in tetrahydrofuran (40mL), then simultaneously added acrylamide (352.06mg, 4.95mmol) and potassium tert-butoxide (833.71mg, 7.43mmol) , the reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was poured into water (50 mL), and extracted with ethyl acetate (30 mL ⁇ 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent.
  • compound WX008-9 (10g, 45.66mmol) and compound 2-aminoisobutyric acid (14.13g, 136.98mmol) were added to N,N-dimethylformamide (100mL) and water (10mL ), copper iodide (1.74g, 9.14mmol), copper powder (580.00mg, 9.13mmol), potassium carbonate (31.55g, 228.30mmol), N,N-dimethylglycine (2.35g , 22.83mmol) were slowly added to the reaction solution in sequence, nitrogen was replaced three times, and the reaction mixture was heated to 110°C and stirred for 12 hours.
  • reaction liquid was cooled to room temperature, 100 mL of ice water was added, 6M hydrochloric acid was added to adjust the pH to 4-5, extracted with ethyl acetate (200 mL ⁇ 2), the organic phases were combined, washed with saturated brine (200 mL ⁇ 2), Dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to remove the solvent.
  • the obtained crude product was added to dichloromethane (15mL) and stirred for 1 hour, filtered, the filter cake was washed with dichloromethane (5mL ⁇ 2), the solid was collected, and dried under reduced pressure to obtain compound WX008-10.
  • MS-ESI m/z 242.2 [M+H] + .
  • reaction solution was cooled to room temperature, concentrated under reduced pressure to remove the solvent, added water (20 mL), adjusted the pH to 3-4 with 1M hydrochloric acid, extracted with ethyl acetate (20 mL ⁇ 2), washed with saturated brine (20 mL ⁇ 2 ), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent.
  • the compound WX004-4 (1.96g, 9.77mmol) was dissolved in acetonitrile (30mL), and then N,N-dimethylaminochloroethane hydrochloride (2.81g, 19.54mmol, 936.23 ⁇ L ), potassium iodide (162.18mg, 976.98 ⁇ mol), potassium carbonate (5.40g, 39.08mmol), the reaction mixture was heated to 80°C and stirred for 12 hours. After the reaction was completed, the reaction solution was cooled to room temperature, filtered, and the filter cake was rinsed with tetrahydrofuran (20 mL ⁇ 3). The filtrate was collected and concentrated under reduced pressure to remove the solvent.
  • compound WX009-2 (240mg, 931.27 ⁇ mol) was dissolved in toluene (2.5mL), then triethylamine (207.32mg, 2.05mmol, 285.17 ⁇ L), diphenylphosphoryl azide (281.91 mg, 1.02mmol, 221.98 ⁇ L), the reaction mixture was heated to 100°C and stirred for 10 minutes, then phenol (438.21mg, 4.66mmol, 409.55 ⁇ L) was added, and the reaction mixture was stirred at 100°C for 0.5 hours. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure to remove the solvent.
  • the compound WX003-4 (3g, 10.60mmol), bis-inhalol borate (3.23g, 12.72mmol), potassium acetate (4.16g, 42.39mmol), [1,1-bis (Diphenylphosphine)ferrocene]palladium dichloride dichloromethane (432.67 mg, 529.82 ⁇ mol) was dissolved in dioxane (50 mL), and the reaction mixture was heated to 100° C. and stirred for 5 hours.
  • reaction liquid was lowered to room temperature, filtered directly with diatomaceous earth, the filter cake was rinsed with dichloromethane (30 mL ⁇ 3), and the filtrate was concentrated under reduced pressure to remove the solvent.
  • the compound WX010-2 (1g, 4.54mmol) was dissolved in tetrahydrofuran (20mL), then N-Boc-ethanolamine (1.10g, 6.81mmol, 1.06mL) was added, Pyridine (2.29g, 9.08mmol) was cooled to 0°C, tributylphosphine (1.84g, 9.08mmol, 2.24mL) dissolved in tetrahydrofuran (5mL) was added dropwise, and the reaction mixture was returned to room temperature and stirred for 3 hours. After the reaction was completed, the reaction solution was directly filtered, and the filter cake was rinsed with tetrahydrofuran (5 mL ⁇ 3).
  • the compound WX010-3 (1.1g, 3.03mmol) was dissolved in N,N-dimethylformamide (20mL), then acrylamide (215.15mg, 3.03mmol) and tert-butanol were added simultaneously A mixture of potassium (509.48mg, 4.54mmol), the reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was poured into water (50mL), extracted with ethyl acetate (30mL ⁇ 3), the organic phases were combined, washed successively with half-saturated brine (50mL ⁇ 3), dried over anhydrous sodium sulfate, and filtered , concentrated under reduced pressure to remove the solvent.
  • compound WX011-1 (10g, 60.18mmol) was dissolved in toluene (100mL), then diethyl carbonate (48.75g, 412.68mmol, 50mL) was added, stirred until the system was dissolved, and cooled to 0-5°C , sodium hydrogen (12.03g, 300.90mmol, purity: 60%) was added in batches, and the reaction mixture was heated to 100°C and stirred for 12 hours.
  • the compound WX011-3 (2.55g, 14.31mmol) was dissolved in ethanol (50mL), then hydroxylamine hydrochloride (3.48g, 50.10mmol) was added, and finally sodium ethoxide (3.41g, 50.10mmol) was added, The reaction mixture was warmed to 80°C and stirred for 12 hours. After the reaction was completed, the reaction solution was poured into water (100 mL), adjusted to pH 4-5 with 2M dilute hydrochloric acid, and extracted with ethyl acetate (100 mL ⁇ 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound WX011-4.
  • the compound WX011-5 (1g, 4.52mmol) was dissolved in tetrahydrofuran (20mL), then N-Boc-ethanolamine (1.09g, 6.78mmol, 1.05mL) was added, Pyridine (2.28g, 9.04mmol) was cooled to 0°C under nitrogen protection, tributylphosphine (1.83g, 9.04mmol, 2.23mL) dissolved in 5mL of tetrahydrofuran was added dropwise, and the reaction mixture was returned to room temperature and stirred for 12 hours. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure to remove the solvent.
  • the compound WX011-6 500mg, 1.37mmol was dissolved in tetrahydrofuran (10mL), then simultaneously added acrylamide (97.53mg, 1.37mmol) and potassium tert-butoxide (230.95mg, 2.06mmol) The reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed, water (50 mL) was added to the reaction liquid, and extracted with ethyl acetate (50 mL ⁇ 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent.
  • compound WX003-1 25g, 116.26mmol was dissolved in tetrahydrofuran (250mL), then dimethyl carbonate (31.42g, 348.77mmol, 29.36mL) was added, stirred until the system was dissolved, and cooled to Potassium tert-butoxide (78.27 g, 697.53 mmol) was added in batches at 5-15° C., and the reaction mixture was heated to 80° C. and stirred for 12 hours.
  • the compound WX012-3 (0.5g, 1.76mmol) was dissolved in water (2mL) and toluene (10mL), followed by adding N-Boc-aminomethyltrifluoroborate potassium (1.25g, 5.28 mmol), cesium carbonate (1.72g, 5.28mmol), [(bis(1-adamantyl)-N-butylphosphine)-2-(2-aminobiphenyl)palladium(II) chloride (117.67mg, 175.99 ⁇ mol), the reaction mixture was heated to 105°C and stirred for 12 hours.
  • the compound WX012-4 (600mg, 1.79mmol) was dissolved in tetrahydrofuran (12mL), and acrylamide (127.54mg, 1.79mmol) and potassium tert-butoxide (201.36mg, 1.79mmol) were added simultaneously, and the reaction The mixture was stirred and reacted at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was poured into a mixture solution of water (20 mL) and ethyl acetate (20 mL), and the organic phase was collected after liquid separation, and the aqueous phase was extracted with ethyl acetate (20 mL ⁇ 3).
  • BxPC-3 cells were treated with 10 ⁇ M, 1 ⁇ M and 0.1 ⁇ M of the test compound for 24 hours, respectively.
  • Cells were collected and lysed, denatured at 100°C for 20 min, and subjected to Western blot analysis, using ⁇ -actin as an internal reference protein to detect the protein level of GSPT1.
  • the main steps include: 1) electrophoresis: use Biorad precast gel, and use SDS-PAGE electrophoresis to separate proteins; 2) transfer membrane: use Biorad rapid membrane transfer system to transfer the protein on the gel to PVDF membrane; Put the PVDF membrane with Western blot into the antibody incubation box, add blocking solution to completely cover the PVDF membrane, and incubate at room temperature for 1 hour; 4) Incubate the primary antibody, cover the above PVDF membrane with GSPT1 antibody diluent and incubate overnight on a shaker at 4°C, 5 ) incubate the secondary antibody, wash the membrane three times with Wash buffer, add the secondary antibody diluent (Anti-rabbit IgG, HRP-linked), and incubate on a shaker at room temperature for 1 hour; 5) expose, wash the membrane three times with Wash buffer, and use chemical Luminescence (Clarity Western ECL Substrate) was used to detect bands on the membrane.
  • transfer membrane use Biorad rapid membrane transfer system
  • Protein loading buffer 4 ⁇ Laemmli loaing buffer was diluted 4 times with deionized water, and DTT was added;
  • Electrophoresis solution 10x Tris/Glycine/SDS buffer diluted 10 times with deionized water;
  • Transfer solution prepared by diluting with Trans-Blot Turbo RTA Midi LF PVDF Transfer Kit and methanol;
  • Blocking solution 5% BSA.
  • Wash buffer 0.2% Tween-20 TBS buffer.
  • Both primary and secondary antibody diluents were prepared by diluting with blocking solution
  • Reagent name species Dilution ratio source Anti-GSPT1 R 1:2000 Abcam, ab49878 Anti- ⁇ -actin R 1:2000 CST, #4970 Anti-rabbit IgG, HRP-linked — 1:2000 CST, #7074
  • the compound WX001 of the present invention has obvious degradative effect on GSPT1 protein in BxPC-3 cells.
  • Experimental example 2 Evaluation of target protein degradation in cells HEK293T-LgBiT_GSPT1-nHiBiT
  • This experiment is to detect the degradation effect of the test compound on the target protein GSPT1 in HEK293T-LgBiT cells.
  • DR Degradation rate, DR
  • DR (%) (RLU vehicle control-RLU compound)/(RLU vehicle control-RLU positive control)*100%, the vehicle control is the blank control.
  • the compound of the present invention exhibits excellent target protein degradation in HEK293T-LgBiT_GSPT1-nHiBiT cells.
  • the tumor cell lines were cultured in an incubator at 37 °C, 5% CO 2 or 0% CO 2 according to the above culture conditions. Passage regularly, take cells in logarithmic growth phase for plating
  • Drug addition Take 10 ⁇ L of 10X compound working solution and add it to the cell culture plate. Add 10 ⁇ L of DMSO-cell culture solution mixture to the vehicle control and the blank control.
  • the culture plate was left at room temperature for 10 minutes to stabilize the luminescent signal.
  • IR Inhibition rate
  • IR (%) (RLU vehicle control - RLU compound) / (RLU vehicle control - RLU blank control) * 100%.
  • IR (%) (RLU vehicle control - RLU compound) / (RLU vehicle control - RLU blank control) * 100%.
  • the compound of the present invention exhibits excellent inhibitory effect on cell proliferation in tumor cell lines MV4-11 and MDA-MB-231.
  • Experimental example 4 Evaluation of antiproliferative effects in tumor cell lines NCI-H1581, DMS114, NCI-H69 and NCI-H526
  • Experimental purpose In this experiment, the effect of the test compound on tumor cells NCI-H1581, DMS114, NCI-H69 and The effect of NCI-H526 on the in vitro activity to study the inhibitory effect of the compound on inhibiting cell proliferation.
  • the tumor cell lines were cultured in an incubator at 37 °C, 5% CO 2 or 0% CO 2 according to the above culture conditions. Passage regularly, take cells in logarithmic growth phase for plating
  • Preparation of 1000X compound stock solution Dissolve the compound in DMSO to 10 mM, and then take out a part and dilute to 1000 times of the highest effect concentration. The diluted stock solution is dispensed and ready for use.
  • Drug addition Take 25 ⁇ L of 5X compound working solution and add it to the cell culture plate. Add 25 ⁇ L of DMSO-cell culture medium mixture (DMSO content is 0.5%) to vehicle control and blank control.
  • the culture plate was left at room temperature for 10 minutes to stabilize the luminescent signal.
  • IR Inhibition rate
  • IR (%) (RLU vehicle control - RLU compound) / (RLU vehicle control - RLU blank control) * 100%.
  • IR (%) (RLU vehicle control - RLU compound) / (RLU vehicle control - RLU blank control) * 100%.
  • the compound of the present invention has the inhibitory effect on cell proliferation in tumor cell lines NCI-H1581, DMS114, NCI-H69 and NCI-H526 cell lines
  • the compounds of the present invention exhibit excellent inhibition of cell proliferation in the tumor cell lines NCI-H1581, DMS114, NCI-H69 and NCI-H526.
  • mice C57BL/6N male mice were selected as the test animals, and the drug concentration in the blood plasma of the test compound was quantitatively determined by LC/MS/MS method in order to evaluate the pharmacokinetics of the test drug in mice. academic features.
  • Plasma samples were stored in a -80°C refrigerator before analysis; oral gavage was administered at 0h (before administration) and 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 24h after administration, Blood collection via the jugular vein, about 0.25 mL for each sample, anticoagulated with sodium heparin, placed on wet ice after blood sample collection, and centrifuged to separate plasma within 1 hour (centrifugation conditions: 6000g, 3 minutes, 2-8°C) , Plasma samples were stored in a -80°C freezer before analysis.
  • Cell culture Human triple-negative breast cancer MDA-MB-231 cells (ATCC-HTB-26) were cultured in vitro as a monolayer, the culture conditions were RPMI-1640 medium plus 10% fetal bovine serum, 37°C 5% CO 2 incubator to cultivate. Routine digestion with trypsin-EDTA was performed every two days for passage. When the cell saturation is 80%-90% and the number reaches the requirement, the cells are collected, counted, and inoculated.
  • mice Balb/c nude mice, female, 6 weeks old upon arrival, purchased from Shanghai Bikai Experimental Animal Co., Ltd.
  • the antitumor efficacy of compounds was evaluated by TGI (%) or relative tumor proliferation rate T/C (%).
  • Relative tumor proliferation rate T/C (%) T RTV /C RTV ⁇ 100% (T RTV : average RTV of the treatment group; C RTV : average RTV of the negative control group).
  • the tumor volume at one measurement, T RTV and C RTV take the data of the same day.

Abstract

一种杂芳-3-哌啶二酮类化合物及其应用，具体公开了式(II)所示化合物及其药学上可接受的盐。

Description

杂芳-3-哌啶二酮类化合物及其应用
本发明主张如下优先权：
CN202110815503.4，申请日：2021年07月19日；
CN202111296262.3，申请日：2021年11月03日。
技术领域
本发明涉及杂芳-3-哌啶二酮类化合物及其应用，具体涉及式(II)所示化合物及其药学上可接受的盐。
背景技术
免疫调节剂药物(Immunomodulatory Drugs，IMiDs)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，CC-90009可以靶向CRBN介导GSPT1(G1 to S Phase Transition 1)的降解从而治疗急性髓性白血病。
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。度胺类药物都有较多副作用，当前迫切需要开发新型CRBN调节剂药物，来提高临床治疗效果。
发明内容
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 PCTCN2022105236-appb-000001
其中，
环A选自苯并呋喃基、苯并异噁唑基、苯并三氮唑基、萘并呋喃基、萘并异噁唑基和萘并三氮唑基；
L 1选自键、-C(R a)(R b)-、-N(R c)-和-OCH 2-；
L 2选自-CH 2-、-C 1-6烷基-C(＝O)NH-和-C 1-3烷基-O-；
各R 1分别独立地选自F、Cl、Br、I、C 1-3烷基、C 1-3烷氧基、5元杂芳基和
Figure PCTCN2022105236-appb-000002
所述C 1-3烷基、C 1-3烷氧基、5元杂芳基和
Figure PCTCN2022105236-appb-000003
任选被1、2或3个R d取代；
m选自0、1、2和3；
各R a和R b分别独立地选自H、F、Cl、Br和I；
R c选自H和CH 3；
各R d分别独立地选自F、Cl、Br、I、OCH 3、N(CH 3) 2和吗啉基。
在本发明的一些方案中，所述环A选自苯并呋喃基、苯并异噁唑基、苯并三氮唑基、萘并[2,3-b]呋喃基、萘并[2,1-b]呋喃基、萘并[2,3-d]异噁唑基、萘并[1,2-d]异噁唑基和1H-萘并[2,3-d][1,2,3]三氮唑基。
在本发明的一些方案中，所述结构单元
Figure PCTCN2022105236-appb-000004
选自
Figure PCTCN2022105236-appb-000005
Figure PCTCN2022105236-appb-000006
Figure PCTCN2022105236-appb-000007
其他变量如本发明所定义。
在本发明的一些方案中，所述结构单元
Figure PCTCN2022105236-appb-000008
选自
Figure PCTCN2022105236-appb-000009
Figure PCTCN2022105236-appb-000010
Figure PCTCN2022105236-appb-000011
其他变量如本发明所定义。
在本发明的一些方案中，所述环A选自苯并呋喃基和苯并异噁唑基，其他变量如本发明所定义。
在本发明的一些方案中，所述环A选自
Figure PCTCN2022105236-appb-000012
其他变量如本发明所定义。
在本发明的一些方案中，所述结构单元
Figure PCTCN2022105236-appb-000013
选自
Figure PCTCN2022105236-appb-000014
Figure PCTCN2022105236-appb-000015
#端连接于式(ⅠI)的苯基，其他变量如本发明所定义。
在本发明的一些方案中，所述L 2选自-CH 2-、-(CH 2) 6-C(＝O)NH-和-CH 2CH 2O-，其他变量如本发明所定义
在本发明的一些方案中，所述各R 1分别独立地选自F、Cl、Br、I、CH 3、OCH 3、OCH 2CH 3、噻唑基和
Figure PCTCN2022105236-appb-000016
所述CH 3、OCH 3、OCH 2CH 3、噻唑基和
Figure PCTCN2022105236-appb-000017
任选被1、2或3个R d取代，其他变量如本发明所定义。
在本发明的一些方案中，所述各R 1分别独立地选自F、Cl、CH 3、-OCH 2CH 2OCH 3、-OCH 2CH 2N(CH 3) 2、
Figure PCTCN2022105236-appb-000018
其他变量如本发明所定义。
在本发明的一些方案中，所述结构单元
Figure PCTCN2022105236-appb-000019
选自
Figure PCTCN2022105236-appb-000020
Figure PCTCN2022105236-appb-000021
其他变量如本发明所定义。
在本发明的一些方案中，所述结构单元
Figure PCTCN2022105236-appb-000022
选自
Figure PCTCN2022105236-appb-000023
Figure PCTCN2022105236-appb-000024
其他变量如本发明所定义。
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 PCTCN2022105236-appb-000025
其中，
环A选自苯并呋喃基、苯并异恶唑基、苯并三氮唑基、萘并呋喃基、萘并异恶唑基和萘并三氮唑基；
L 1选自-C(R a)(R b)-、-N(R c)-和-OCH 2-；
L 2选自-CH 2-和-C 1-6烷基-C(＝O)NH-；
各R 1分别独立地选自H、F、Cl、Br、I、C 1-3烷基、C 1-3烷氧基和5元杂芳基，所述C 1-3烷基、C 1-3烷氧基和5元杂芳基任选被1、2或3个R d取代；
m选自0、1、2和3；
各R a、R b和R c分别独立地选自H、F、Cl、Br和I；
R d选自F、Cl、Br、I和吗啉基。
在本发明的一些方案中，所述环A选自苯并呋喃基、苯并异恶唑基、苯并三氮唑基、萘并[2,3-b]呋喃基、萘并[2,1-b]呋喃基、萘并[2,3-d]异恶唑基、萘并[1,2-d]异恶唑基和1H-萘并[2,3-d][1,2,3]三氮唑基。
在本发明的一些方案中，所述结构单元
Figure PCTCN2022105236-appb-000026
选自
Figure PCTCN2022105236-appb-000027
Figure PCTCN2022105236-appb-000028
Figure PCTCN2022105236-appb-000029
其他变量如本发明所定义。
在本发明的一些方案中，所述结构单元
Figure PCTCN2022105236-appb-000030
选自
Figure PCTCN2022105236-appb-000031
Figure PCTCN2022105236-appb-000032
Figure PCTCN2022105236-appb-000033
其他变量如本发明所定义。
在本发明的一些方案中，所述结构单元
Figure PCTCN2022105236-appb-000034
选自
Figure PCTCN2022105236-appb-000035
其他变量如本发明所定义。
在本发明的一些方案中，所述L 2选自-CH 2-和-(CH 2) 6-C(＝O)NH-，其他变量如本发明所定义
在本发明的一些方案中，所述各R 1分别独立地选自H、F、Cl、Br、I、CH 3和噻唑基，所述CH 3和噻唑基任选被1、2或3个R d取代，其他变量如本发明所定义。
在本发明的一些方案中，所述各R 1分别独立地选自H、F、Cl、CH 3和
Figure PCTCN2022105236-appb-000036
其他变量如本发明所定义。
在本发明的一些方案中，所述结构单元
Figure PCTCN2022105236-appb-000037
选自
Figure PCTCN2022105236-appb-000038
其他变量如本发明所定义。
在本发明的一些方案中，所述结构单元
Figure PCTCN2022105236-appb-000039
选自
Figure PCTCN2022105236-appb-000040
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，其化合物选自
Figure PCTCN2022105236-appb-000041
其中，
T 1选自CH和N；
R 1、L 1和m如本发明所定义。
本发明还提供了下式化合物或其药学上可接受的盐
Figure PCTCN2022105236-appb-000042
Figure PCTCN2022105236-appb-000043
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，其化合物选自
Figure PCTCN2022105236-appb-000044
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。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 PCTCN2022105236-appb-000045
和楔形虚线键
Figure PCTCN2022105236-appb-000046
表示一个立体中心的绝对构型，用直形实线键
Figure PCTCN2022105236-appb-000047
和直形虚线键
Figure PCTCN2022105236-appb-000048
表示立体中心的相对构型，用波浪线
Figure PCTCN2022105236-appb-000049
表示楔形实线键
Figure PCTCN2022105236-appb-000050
或楔形虚线键
Figure PCTCN2022105236-appb-000051
或用波浪线
Figure PCTCN2022105236-appb-000052
表示直形实线键
Figure PCTCN2022105236-appb-000053
和直形虚线键
Figure PCTCN2022105236-appb-000054
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，取代基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。当所列举的取代基中没有指明其通过哪一个原子连接到被取代的基团上时，这种取代基可以通过其任何原子相键合，例如，吡啶基作为取代基可以通过吡啶环上任意一个碳原子连接到被取代的基团上。
当所列举的连接基团没有指明其连接方向，其连接方向是任意的，例如，
Figure PCTCN2022105236-appb-000055
中连接基团L为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成
Figure PCTCN2022105236-appb-000056
也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成
Figure PCTCN2022105236-appb-000057
所述连接基团、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。
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 PCTCN2022105236-appb-000058
直形虚线键
Figure PCTCN2022105236-appb-000059
或波浪线
Figure PCTCN2022105236-appb-000060
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 PCTCN2022105236-appb-000061
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 PCTCN2022105236-appb-000062
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 PCTCN2022105236-appb-000063
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 PCTCN2022105236-appb-000064
Figure PCTCN2022105236-appb-000065
这4种连接方式，即使-N-上画出了H原子，但是
Figure PCTCN2022105236-appb-000066
仍包括
Figure PCTCN2022105236-appb-000067
这种连接方式的基团，只是在连接1个化学键时，该位点的H会对应减少1个变成相应的一价哌啶基。
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 1-6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。
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。
除非另有规定，术语“C 1-3烷氧基”表示通过一个氧原子连接到分子的其余部分的那些包含1至3个碳原子的烷基基团。所述C 1-3烷氧基包括C 1-2、C 2-3、C 3和C 2烷氧基等。C 1-3烷氧基的实例包括但不限于甲氧基、乙氧基、丙氧基(包括正丙氧基和异丙氧基)等。
除非另有规定，本发明术语“5元杂芳环”和“5元杂芳基”可以互换使用，术语“5元杂芳基”表示由5至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元杂芳基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)。
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。
本发明所使用的溶剂可经市售获得。
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 D8venture衍射仪收集衍射强度数据，光源为CuKα辐射，扫描方式：
Figure PCTCN2022105236-appb-000068
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。
化合物依据本领域常规命名原则或者使用
Figure PCTCN2022105236-appb-000069
软件命名，市售化合物采用供应商目录名称。
附图说明
图1.降解GSPT1蛋白的Western blot图。
技术效果
本发明化合物对GSPT1蛋白具有优异的降解作用，可有效抑制肿瘤细胞增殖，且具有显著的缩瘤作用。此外，本发明化合物的药代动力学性质优异，具有较高的血浆系统暴露量。
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
Figure PCTCN2022105236-appb-000070
合成路线：
Figure PCTCN2022105236-appb-000071
步骤1：中间体WX001–2的合成
室温和氮气保护下，将WX001–1(1g,4.69mmol)溶于甲苯(50mL)中，随后加入乙氧甲酰基亚甲基三苯基膦(1.96g,5.63mmol)，反应混合物加热至130℃并在130℃下搅拌反应48小时。反应完毕后，冷却至室温，减压浓缩除去溶剂。所得残余物经过柱层析分离(洗脱剂：石油醚/乙酸乙酯＝20/1–10/1，体积比)，得到中间体WX001–2。MS–ESI m/z:282.9[M+H] +,284.9[M+2+H] +. 1H NMR(400MHz,CDCl 3)δ:7.73(d,J＝1.6Hz,1H),7.65(s,1H),7.45–7.41(m,1H),7.39–7.34(m,1H),4.23(q,J＝6.8Hz,2H),3.68(s,2H),1.31(t,J＝7.0Hz,3H)。
步骤2：中间体WX001–3的合成
室温和氮气保护下，将中间体WX001–2(1g,3.53mmol)和氰化锌(497.71mg,4.24mmol)溶于N,N–二甲基甲酰胺(5mL)中，随后加入三(二亚苄基丙酮)二钯(161.72mg,176.61μmol)，2–二环己基膦–2,4,6–三异丙基联苯(168.38mg,353.21μmol)，反应混合物加热至80℃并在80℃下搅拌反应12小时。反应完毕后，冷却至室温，向反应液中加入水(100mL)，然后用乙酸乙酯萃取(50mL×3)，合并有机相，用饱和食盐水(50mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩除去溶剂。所得残余物经过柱层析分离(洗脱剂：先用石油醚/乙酸乙酯＝10/1–6/1，体积比)，得到中间体WX001–3。MS–ESI m/z:229.9[M+H] +. 1H NMR(400MHz,CDCl 3)δ:7.87(s,1H),7.68(s,1H),7.55–7.45(m,2H),4.14(q,J＝7.0Hz,2H),3.64(s,2H),1.22(t,J＝7.2Hz,3H)。
步骤3：中间体WX001–4的合成
室温和氮气保护下，将中间体WX001–3(520mg,2.27mmol)溶于乙醇(20mL)中，随后加入雷尼镍(971.75mg,2.27mmol,纯度：20％)和盐酸(4M,1.70mL)，反应混合物抽真空并用氢气置换几次，反应混合物在室温和氢气(15psi)保护下搅拌反应12小时。反应完毕后，过滤，滤液减压浓缩除去溶剂。得到中间体WX001–4，直接用于下一步反应。 1H NMR(400MHz,D 2O)δ:7.65(s,1H),7.54(s,1H),7.49(d,J＝8.4Hz,1H),7.30(dd,J＝1.2,8.8Hz,1H),4.15(s,2H),4.09(q,J＝7.2Hz,2H),3.74(s,2H),1.13(t,J＝7.0Hz,3H)。
步骤4：中间体WX001–5的合成
0℃和氮气保护下，将中间体WX001–4(250mg,926.88μmol)溶于四氢呋喃(5mL)中，随后加入三乙胺(468.95mg,4.63mmol,645.05μL)，反应混合物在0℃下搅拌反应0.5小时，加入3–氯–4–甲基苯基异氰酸酯(155.34mg,926.88μmol)，反应混合物逐渐升至室温并在室温下搅拌反应2小时。反应完毕后，向反应液中加入水(50mL)，然后用乙酸乙酯萃取(30mL×3)，合并有机相，用食盐水(30mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩除去溶剂。所得残余物经过柱层析分离(洗脱剂：石油醚/乙酸乙酯＝10/1–4/1，体积比)，得到中间体WX001–5。MS–ESI m/z:422.9[M+Na] +,424.9[M+2+Na] +. 1H NMR(400MHz,DMSO_d 6)δ:8.65(s,1H),7.90(s,1H),7.68(d,J＝2.0Hz,1H),7.55–7.49(m,2H),7.28(dd,J＝1.6,8.4Hz,1H),7.18(d,J＝8.4Hz,1H),7.11(dd,J＝2.0,8.4Hz,1H),6.68(t,J＝5.8Hz,1H),4.38(d,J＝6.0Hz,2H),4.10(q,J＝7.0Hz,2H),3.77(s,2H),2.24(s,3H),1.18(t,J＝7.0Hz,3H)。
步骤5：化合物WX001的合成
0℃和氮气保护下，将中间体WX001–5(150mg,374.20μmol)溶于N,N–二甲基甲酰胺(10mL)中，随后加入叔丁醇钾(41.99mg,374.20μmol)。反应混合物在0℃下搅拌反应1小时，加入丙烯酰胺(26.60mg,374.20μmol)，反应混合物逐渐升至室温并在室温下搅拌反应2小时。反应完毕后，加入水(30mL)，然后用乙酸乙酯(20mL×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，减压浓缩除去溶剂。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WX001。MS–ESI m/z:425.9[M+H] +,427.9[M+2+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.91(s,1H),8.65(s,1H),7.90(s,1H),7.67(d,J＝2.4Hz,1H),7.56–7.52(m,2H),7.28(dd,J＝1.6,8.4Hz,1H),7.18(d,J＝8.4Hz,1H),7.11(dd,J＝2.0,8.4Hz,1H),6.67(t,J＝5.8Hz,1H),4.37(d,J＝6.0Hz,2H),4.14(dd,J＝4.8,12.0Hz,1H),2.83–2.71(m,1H),2.63–2.55(m,1H),2.40–2.31(m,1H),2.23(s,3H),2.17–2.07(m,1H)。
实施例2
Figure PCTCN2022105236-appb-000072
合成路线：
Figure PCTCN2022105236-appb-000073
步骤1：化合物WX002–1的合成
室温和氮气保护下，将化合物WX001–4(200mg,741.50μmol)，2-(4-氯苯基)-2,2-二氟乙酸(229.76mg,1.11mmol)和O-(7-氮杂苯并三氮唑-1-基)-N,N,N’,N’-四甲基脲六氟磷酸酯(563.88mg,1.48mmol)溶 于N,N–二甲基甲酰胺(5mL)中，随后加入三乙胺(375.16mg,3.71mmol,516.04μL)，反应混合物在室温下搅拌反应12小时。反应完毕后，向反应混合物中加入水(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，所得残余物经过柱层析分离(洗脱剂：石油醚/乙酸乙酯＝3/1，体积比)，得到化合物WX002–1。MS–ESI m/z:444.0[M+Na] +,446.0[M+Na+2] +. 1H NMR(400MHz,CDCl 3)δ:7.58(s,1H),7.50(d,J＝8.4Hz,2H),7.43–7.33(m,4H),7.14(dd,J＝1.8,8.2Hz,1H),6.72(s,1H),4.52(d,J＝5.6Hz,2H),4.12(q,J＝7.0Hz,2H),3.60(s,2H),1.21(t,J＝7.2Hz,3H)。
步骤2：化合物WX002的合成
0℃和氮气保护下，将化合物WX002–1(0.120g,284.48μmol)溶于N,N–二甲基甲酰胺(10mL)中，加入叔丁醇钾(31.92mg,284.48μmol),反应混合物在0℃下搅拌反应1小时,随后再加入丙烯酰胺(20.22mg,284.48μmol)，反应混合物升温至室温并在室温下搅拌反应2小时。反应完毕后，向反应混合物中加入水(30mL)，用乙酸乙酯(20mL×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WX002。MS–ESI m/z:468.8[M+Na] +,470.8[M+Na+2] +. 1H NMR(400MHz,DMSO_d 6)δ:10.94(s,1H),9.63(t,J＝6.0Hz,1H),7.91(s,1H),7.67–7.57(m,4H),7.53(d,J＝8.4Hz,1H),7.45(s,1H),7.18(dd,J＝1.4,8.2Hz,1H),4.42(d,J＝6.0Hz,2H),4.09(dd,J＝5.2,12.0Hz,1H),2.83–2.72(m,1H),2.68–2.57(m,1H),2.34–2.23(m,1H),2.15–2.06(m,1H)。
实施例3
Figure PCTCN2022105236-appb-000074
合成路线：
Figure PCTCN2022105236-appb-000075
步骤1：化合物WX003–2的合成
室温下，将化合物WX003–1(100g,465.02mmol)溶于氯仿(500mL)和乙酸乙酯(500mL)混合溶剂中，然后加入溴化铜(207.73g,930.04mmol)，反应混合物升温至90℃并搅拌反应20小时。反应完毕后，将反应液降至室温，过滤，滤饼用二氯甲烷(200mL×2)淋洗，收集滤液，得到化合物WX003–2的二氯甲烷溶液，直接用于下一步。
步骤2：化合物WX003–3的合成
氮气保护下，将化合物WX003–2(136.69g,465.03mmol)的二氯甲烷溶液(1.4L)，降温至0℃，缓慢滴加三乙胺(70.58g,697.54mmol,97.09mL)，反应混合物缓慢升至20℃并搅拌反应0.5小时。反应完毕后，向体系内加入水(300mL)，分液，收集有机相，用饱和食盐水(1L)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩除去约一半溶剂，然后加入甲苯(500mL)，继续减压浓缩除去残留的低沸点溶剂(注意不要旋干)，得到化合物WX003–3的甲苯溶液，直接用于下一步。MS–ESI m/z:212.9[M+H] +,215.0[M+H+2] +。
步骤3：化合物WX003–4的合成
室温和氮气保护下，将化合物WX003–3(99g,464.73mmol)的二氯甲烷和甲苯(1L)混合物溶液 中，加入乙氧甲酰基亚甲基三苯基膦(161.90g,464.73mmol)，将反应液升温至130℃并搅拌反应20小时。反应完毕后，将反应液降至室温，减压浓缩得到残余物。向残余物中加入甲基叔丁基醚(800mL)，搅拌30分钟，过滤，滤饼用甲基叔丁基醚(100mL×2)淋洗，滤液减压浓缩得到粗品。粗品经柱层析(洗脱剂：石油醚：乙酸乙酯＝100/1–10/1)分离，得到化合物WX003–4。MS–ESI m/z:283.0[M+H] +,285.0[M+H+2] +. 1H NMR(400MHz,CDCl 3)δ:7.66(d,J＝1.2Hz,1H),7.61(s,1H),7.44(d,J＝8.4Hz,1H),7.38(dd,J＝1.6Hz,8.4Hz,1H),4.20(q,J＝7.0Hz,2H),3.68(d,J＝1.2Hz,2H),1.28(t,J＝7.2Hz,3H)。
步骤4：化合物WX003–5的合成
室温和氮气保护下，将化合物WX003–4(5g,17.66mmol)，氨基甲酸叔丁酯(2.48g,21.19mmol)，2–二叔丁基膦–2’,4’,6–三异丙基联苯(1.05g,2.47mmol)，三(二亚苄基丙酮)二钯(1.13g,1.24mmol)和磷酸钾(15.00g,70.64mmol)溶于甲苯(100mL)和水(20mL)中，反应混合物升温至100℃并搅拌反应12小时。反应完毕后，将反应液降至室温，加入水(50mL)，用乙酸乙酯(100mL×3)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，滤液减压浓缩得到粗品。向粗品中加入正庚烷(100mL)，打浆，过滤，滤饼用正庚烷(50mL×3)洗涤，收集固体，得到化合物WX003–5。 1H NMR(400MHz,CDCl 3)δ:7.77(s,1H),7.57(s,1H),7.44(d,J＝8.4Hz,1H),7.05(dd,J＝1.6Hz,8.4Hz,1H),6.56(s,1H),4.19(q,J＝7.2Hz,2H),3.66(d,J＝0.8Hz,2H),1.54(s,9H),1.27(t,J＝7.2Hz,3H)。
步骤5：化合物WX003–6的合成
室温下，将化合物WX003–5(4.4g,13.78mmol)溶于N,N–二甲基甲酰胺(50mL)中，依次加入叔丁醇钾(2.78g,24.80mmol)和丙烯酰胺(1.18g,16.53mmol)，反应混合物在室温下搅拌反应2小时。反应完毕后，将反应液倒入饱和氯化铵水溶液(200mL)中，用乙酸乙酯(50mL×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，滤液减压浓缩得到粗品。粗品经柱层析(洗脱剂：石油醚：乙酸乙酯＝5/1–1/2)分离，得到化合物WX003–6。
步骤6：化合物WX003–7的合成
室温下，将化合物WX003–6(2.8g,8.13mmol)溶于二氯甲烷(80mL)中，加入盐酸/乙酸乙酯(4M,200mL)，反应混合物在室温下搅拌反应4小时。反应完毕后，将反应液直接减压浓缩除去溶剂。所得残余物加入二氯甲烷(500mL)，用饱和碳酸氢钠水溶液调节pH＝8～9，用二氯甲烷(500mL×3)萃取，合并有机相，用饱和食盐水(200mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，得到化合物WX003–7。MS–ESI m/z:245.2[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.85(s,1H),7.52(s,1H),7.17(d,J＝8.0Hz,1H),6.65(d,J＝1.6Hz,1H),6.52(d,J＝1.4Hz,8.2Hz,1H),5.16(s,2H),3.98(dd,J＝4.8Hz,11.6Hz,1H),2.75–2.64(m,1H),2.58–2.53(m,1H),2.30–2.18(m,1H),2.13–2.03(m,1H)。
步骤7：化合物WX003–8的合成
室温和氮气保护下，将7–(N–叔丁氧羰基氨基)庚酸(100.44mg,409.42μmol)溶于N,N–二甲基甲酰胺(2mL)中，然后加入2–(7–氮杂苯并三氮唑)–N,N,N',N'–四甲基脲六氟磷酸酯(311.35mg,818.85μmol)和N,N–二异丙基乙胺(158.74mg,1.23mmol,213.94μL)，室温下搅拌30分钟，加入化合物WX003–7(100mg,409.42μmol)，反应混合物在室温下继续搅拌1小时。反应完毕后，向反应液中加入水(5mL)和石油醚(3mL)，搅拌10分钟，有固体析出，过滤，用乙酸乙酯(1mL×2)洗涤滤饼，收集滤饼，减压浓缩得到化合物WX003–8。MS–ESI m/z:372.2[M-99] +。
步骤8：化合物WX003–9盐酸盐的合成
室温下，将化合物WX003–8(190mg,402.93μmol)溶于盐酸/乙酸乙酯(3.61mL，4M)中，反应混合物搅拌反应0.5小时。反应完毕后，将反应液过滤，用乙酸乙酯(2mL×3)淋洗滤饼，收集滤饼，得到化合物WX003–9的盐酸盐。MS–ESI m/z:372.2[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.88(s,1H),10.16(s,1H),8.07(s,1H),7.90(s,2H),7.81(s,1H),7.47(d,J＝8.4Hz,1H),7.31(dd,J＝1.2,8.8Hz,1H),4.09(dd,J＝4.6Hz,11.8Hz,1H),2.81–2.65(m,3H),2.60–2.53(m,1H),2.35(t,J＝7.4Hz,2H),2.32–2.23(m,1H),2.15–2.05(m,1H),1.65–1.52(m,4H),1.40–1.26(m,4H)。
步骤9：化合物WX003–11的合成
室温和氮气保护下，将化合物WX003–10(21.5g,88.51mmol)溶于N,N–二甲基甲酰胺(200mL)中，加入三乙胺(26.87g,265.52mmol,36.96mL)和吗啡啉(7.71g,88.51mmol,7.79mL)，反应混合物升温至80℃并搅拌反应12小时。反应完毕后，将反应液降至室温，加入10％食盐水(100mL)，用乙酸乙酯(150mL×3)萃取，合并有机相，用饱和食盐水(200mL×2)洗涤，无水硫酸钠干燥，过滤并减压浓缩得到粗品。粗品经柱层析(洗脱剂：石油醚：乙酸乙酯＝1/0–1/1)分离，得到化合物WX003–11。MS–ESI m/z:248.9[M+H] +,250.9[M+H+2] +. 1H NMR(400MHz,DMSO_d 6)δ:6.92(s,1H),3.69(t,J＝5.0Hz,4H),3.36(t,J＝5.0Hz,4H)。
步骤10：化合物WX003–12的合成
室温和氮气保护下，将化合物WX003–11(8g,32.11mmol)溶于1,4–二氧六环(70mL)和水(14mL)的混合溶剂中，加入3–羟基苯硼酸(6.64g,48.17mmol)，碳酸钾(17.75g,128.45mmol)和1,1'–双二苯基膦二茂铁二氯化钯(1.17g,1.61mmol)，反应混合物升温至100℃并搅拌反应1小时。反应完毕后，将反应液降至室温，用1N盐酸调节pH＝5～6，加入水(100mL)，用乙酸乙酯(150mL×3)萃取，合并有机相，用饱和食盐水(150mL×2)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得到粗品。粗品经柱层析(洗脱剂：石油醚：乙酸乙酯＝1/0–1/1)分离，得到化合物WX003–12。MS–ESI m/z:263.1[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:9.39(s,1H),7.28(d,J＝1.6Hz,1H),7.20(s,1H),7.16(t,J＝7.6Hz,1H),7.01–6.93(m,1H),6.75–6.65(m,1H),3.73(t,J＝4.8Hz,4H),3.42(t,J＝4.8Hz,4H)。
步骤11：化合物WX003–13的合成
室温和氮气保护下，将化合物WX003–12(4.3g,16.39mmol)溶于N,N–二甲基甲酰胺(50mL)中，加入溴乙酸叔丁酯(6.39g,32.78mmol,4.84mL)和碳酸钾(6.80g,49.18mmol)，反应液升温至50℃并搅拌反应12小时。反应完毕后，将反应液降至室温，加入水(50mL)，用乙酸乙酯(50mL×2)萃取，合并有机相，用饱和食盐水(50mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得到残余物。向残余物中加入甲基叔丁基醚(10mL)，搅拌10分钟，过滤，滤饼用甲基叔丁基醚(3mL)淋洗，收集滤饼，得到化合物WX003–13。MS–ESI m/z:377.1[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:7.46(d,J＝7.6Hz,1H),7.38(t,J＝2.0Hz,1H),7.34(s,1H),7.29(t,J＝8.0Hz,1H),6.82(dd,J＝2.4Hz,8.0Hz,1H),4.68(s,2H),3.73(t,J＝4.8Hz,4H),3.44(t,J＝5.0Hz,4H),1.43(s,9H)。
步骤12：化合物WX003–14盐酸盐的合成
室温下，将化合物WX003–13(3.3g,8.77mmol)溶于盐酸/乙酸乙酯(10mL，4M)中，反应混合物在室温下搅拌反应1小时。反应完毕后，将反应液过滤，用乙酸乙酯(5mL×2)淋洗滤饼，收集滤饼，减压 浓缩除去残余溶剂，得到化合物WX003–14的盐酸盐。MS–ESI m/z:321.1[M+H] +。
步骤13：化合物WX003的合成
室温和氮气保护下，将化合物WX003–14的盐酸盐(62.99mg,176.52μmol)溶于N,N–二甲基甲酰胺(1mL)中，然后加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N’,N’-四甲基脲六氟磷酸酯(111.86mg,294.20μmol)和N,N–二异丙基乙胺(114.07mg,882.59μmol,153.73μL)，室温下搅拌30分钟后，加入化合物WX003–9的盐酸盐(60mg,147.10μmol)，室温下继续搅拌1小时。反应完毕后，向反应液中加入水(10mL)，用乙酸乙酯(20mL×4)萃取，合并有机相，用饱和食盐水(20mL×3)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得到残余物。所得残余物经制备HPLC(流动相：乙腈/水；酸性体系：0.04％HCl)分离，得到化合物WX003。MS–ESI m/z:674.3[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.88(s,1H),10.01(s,1H),8.09(t,J＝5.8Hz,1H),8.05(d,J＝1.6Hz,1H),7.81(s,1H),7.49–7.43(m,3H),7.33–7.25(m,3H),6.90–6.85(m,1H),4.49(s,2H),4.08(dd,J＝5.0Hz,11.8Hz,1H),3.73(t,J＝5.0Hz,4H),3.43(t,J＝4.8Hz,4H),3.12(q,J＝6.8Hz,2H),2.78–2.65(m,1H),2.60–2.53(m,1H),2.35–2.25(m,3H),2.15–2.07(m,1H),1.62–1.52(m,2H),1.48–1.40(m,2H),1.32–1.23(m,4H)。
实施例4
Figure PCTCN2022105236-appb-000076
合成路线：
Figure PCTCN2022105236-appb-000077
步骤1：化合物WX004-1的合成
室温和氮气保护下，将化合物WX001-3(3g,13.09mmol)溶于N,N-二甲基甲酰胺(45mL)中，降温至0℃，同时加入丙烯酰胺(930.21mg,13.09mmol)和叔丁醇钾(1.47g,13.09mmol)，反应混合物在0℃下搅拌反应1小时。反应完毕后，将反应液倒入饱和氯化铵(100mL)和乙酸乙酯(50mL)的混合物溶液中，分液后收集有机相，水相用乙酸乙酯萃取(50mL×3)。合并有机相，用半饱和食盐水洗涤(50mL×3)，无水硫酸钠干燥，过滤，减压浓缩除去溶剂。所得残余物经甲醇(10mL)室温搅拌20分钟，有固体析出，过滤，收集固体，减压浓缩得到化合物WX004-1. 1H NMR(400MHz,DMSO_d 6)δ:10.93(s,1H),8.26(d,J＝0.8Hz,1H),8.12(s,1H),7.81(d,J＝8.4Hz,1H),7.57(dd,J＝1.6,8.8Hz,1H),4.20(dd,J＝4.8,12.4Hz,1H),2.80-2.69(m,1H),2.63-2.55(m,1H),2.47-2.34(m,1H),2.15-2.05(m,1H).
步骤2：化合物WX004-2的合成
室温下，将湿钯碳(4g,纯度:10％)加入甲醇(300mL)中，随后加入化合物WX004-1(2.1g,8.26mmol)和浓盐酸(12M,6mL)，氢气置换3次，反应混合物在室温和氢气(15psi)氛围下搅拌反应12小时。反应完毕后，反应液直接用硅藻土过滤，收集滤液，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；中性体系：NH 4HCO 3)，得到化合物WX004-2.MS-ESI m/z:257.1[M-H] -. 1H NMR(400MHz,DMSO_d 6)δ:7.87(s,1H),7.58-7.43(m,2H),7.32-7.20(m,1H),4.21(d,J＝6.0Hz,1H),4.11(dd,J＝4.8,10.0Hz,1H),3.82(s,1H),2.81-2.68(m,1H),2.61-2.54(m,1H),2.39-2.26(m,1H),2.17-2.04(m,1H).
步骤3：化合物WX004-4的合成
室温和氮气保护下，将化合物WX004–3(1g,3.79mmol)溶于N,N-二甲基甲酰胺(10mL)中，随后加入(E)-苯甲醛肟(482.68mg,3.98mmol)，二叔丁基-(2,4,6-三异丙基-3,6-二甲氧基联苯-2-基)膦(229.92mg, 474.36μmol)，氯化烯丙基钯(II)二聚物(69.42mg,189.74μmol)和碳酸铯(1.85g,5.69mmol)，反应混合物升温至90℃并搅拌反应8小时。反应完毕后，反应液冷却至室温，将反应液倒入0.5M稀盐酸(100mL)中，用乙酸乙酯萃取(50mL×3)。合并有机相，依次用半饱和食盐水洗涤(50mL×3)，无水硫酸钠干燥，过滤，减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝1/0–19/1，体积比)，得到化合物WX004-4.MS-ESI m/z:199.1[M-H] -,200.1[M-H+1] -,201.1[M-H+2] -. 1H NMR(400MHz,DMSO_d 6)δ:10.34(s,1H),7.39(d,J＝1.6Hz,1H),7.37(d,J＝1.6Hz,1H),3.82(s,3H),2.22(s,3H).
步骤4：化合物WX004-5的合成
室温和氮气保护下，将化合物WX004-4(700mg,3.49mmol)溶于乙腈(20mL)中，随后加入碘化钾(115.84mg,697.84μmol)，碳酸钾(964.49mg,6.98mmol)，4–(2–氯乙基)吗啡啉(574.25mg,3.84mmol)，反应混合物升温至80℃并搅拌反应12小时。反应完毕后，反应液直接过滤，滤饼用四氢呋喃淋洗(20mL×3)，收集滤液，减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝4/1–1/1，体积比)，得到化合物WX004-5.MS-ESI m/z:314.2[M+H] +,315.2[M+H+1] +,316.2[M+H+2] +. 1H NMR(400MHz,CDCl 3)δ:7.68(d,J＝0.8Hz,1H),7.40(d,J＝0.8Hz,1H),4.18(t,J＝5.6Hz,2H),3.91(s,3H),3.74(t,J＝4.6Hz,4H),2.86(t,J＝5.6Hz,2H),2.61(t,J＝4.4Hz,4H),2.32(s,3H).
步骤5：化合物WX004-6的合成
室温下，将化合物WX004-5(760mg,2.42mmol)溶于四氢呋喃(6mL)和水(2mL)中，随后加入一水合氢氧化锂(304.92mg,7.27mmol)，反应混合物在室温下搅拌反应12小时。反应完毕后，向反应液中加入水(10mL)，用6M稀盐酸调节pH至7–8，用2–甲基四氢呋喃萃取(50mL×10)，合并有机相，用无水硫酸钠干燥，过滤，减压浓缩得到化合物WX004-6. 1H NMR(400MHz,DMSO_d 6)δ:7.51(d,J＝0.8Hz,1H),7.43(d,J＝1.2Hz,1H),4.17(t,J＝5.4Hz,2H),3.57(t,J＝4.6Hz,4H),2.74(t,J＝5.6Hz,2H),2.53-2.51(m,4H),2.25(s,3H).
步骤6：化合物WX004-7的合成
室温和氮气保护下，将化合物WX004-6(890mg,2.97mmol)溶于甲苯(10mL)中，随后加入三乙胺(660.98mg,6.53mmol,909.19μL)，叠氮磷酸二苯酯(898.82mg,3.27mmol,707.73μL)，反应混合物在室温下搅拌10分钟后，加入苯酚(1.40g,14.85mmol,1.31mL)，反应混合物升温至100℃并搅拌反应1小时。反应完毕后，反应液直接减压浓缩除去溶剂，所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝4/1–1/1，体积比，湿法上样)，得到化合物WX004-7.MS-ESI m/z:391.2[M+H] +,392.2[M+H+1] +,393.2[M+H+2] +. 1H NMR(400MHz,CDCl 3)δ:7.45-7.38(m,2H),7.29-7.26(m,1H),7.25-7.16(m,3H),6.93(br s,1H),6.90(d,J＝2.0Hz,1H),4.11(t,J＝5.8Hz,2H),3.73(t,J＝4.8Hz,4H),2.82(t,J＝5.6Hz,2H),2.59(t,J＝4.6Hz,4H),2.23(s,3H).
步骤7：化合物WX004的合成
室温下，将化合物WX004-7(80mg,204.68μmol)和化合物WX004-2(52.86mg,204.68μmol)溶于N,N-二甲基甲酰胺(2mL)，随后加入三乙胺(41.42mg,409.35μmol,56.98μL)，反应混合物在室温下搅拌反应12小时。反应完毕后，将反应液倒入水(10mL)中，用乙酸乙酯萃取(10mL×3)。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；中性体系：NH 4HCO 3)，得到目标化合物WX004。MS-ESI m/z:555.0[M+H] +,556.0[M+H+1] +,557.0[M+H+2] +. 1H NMR(400MHz,DMSO_d 6)δ:10.87(s,1H),8.60(s,1H),7.86(s,1H),7.52-7.47(m,2H),7.24(dd,J＝1.6,8.4Hz,1H),7.11(d,J＝2.0Hz,1H),6.97(d,J＝2.0Hz,1H),6.65(t,J＝5.6Hz,1H),4.32(d,J＝6.0Hz,2H),4.10(dd,J＝4.6,12.2Hz,1H),3.99(t,J＝5.8Hz,2H),3.53(t,J＝4.6Hz,4H),2.78-2.70(m,1H),2.68(t,J＝5.6Hz,2H),2.59-2.50(m,1H),2.44-2.40(m,4H),2.35-2.29(m,1H),2.12-2.08(m,1H),2.06(s,3H).
实施例5
Figure PCTCN2022105236-appb-000078
合成路线：
Figure PCTCN2022105236-appb-000079
步骤1：化合物WX005-1的合成
室温和氮气保护下，将化合物WX003-4(5.5g,19.43mmol)溶于N,N-二甲基甲酰胺(60mL)中，随后依次加入氰化锌(2.87g,24.44mmol,1.55mL)，2-二环己基膦-2,4,6-三异丙基联苯(926.10mg,1.94mmol)，三(二亚苄基丙酮)二钯(889.46mg,971.33μmol)，反应混合物升温至80℃并搅拌反应12小时。反应完毕 后，反应液冷却至室温，加入水(200mL)，加入0.5M氢氧化钠水溶液调节pH＝12～13，用乙酸乙酯萃取(100mL×3)。合并有机相，依次用半饱和食盐水洗涤(50mL×3)，无水硫酸钠干燥，过滤，减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝1/0-9/1，体积比)，得到化合物WX005-1.MS-ESI m/z:230.2[M+H] +. 1H NMR(400MHz,CDCl 3)δ:7.83(t,J＝1.2Hz,1H),7.82-2.79(m,1H),7.67(d,J＝8.4Hz,1H),7.54(dd,J＝1.2,8.0Hz,1H),4.21(q,J＝7.0Hz,2H),3.72(d,J＝1.2Hz,2H),1.28(t,J＝7.2Hz,3H).
步骤2：化合物WX005-2的合成
室温和氮气保护下，将化合物WX005-1(3.1g,13.52mmol)溶于N,N-二甲基甲酰胺(50mL)中，降温至0℃加入丙烯酰胺(961.22mg,13.52mmol)和叔丁醇钾(1.67g,14.88mmol)，反应混合物0℃搅拌反应0.5小时。反应完毕后，将反应液倒入水(100mL)中，用乙酸乙酯萃取(50mL×3)，合并有机相，依次用半饱和食盐水洗涤(50mL×3)，无水硫酸钠干燥，过滤，减压浓缩除去溶剂。所得残余物经甲醇(10mL)室温搅拌30分钟，有固体析出，过滤，收集固体，真空干燥得到化合物WX005-2. 1H NMR(400MHz,DMSO_d 6)δ：10.95(s,1H),8.26-8.15(m,2H),7.81(d,J＝8.4Hz,1H),7.67(dd,J＝1.2,8.4Hz,1H),4.22(dd,J＝4.6,12.6Hz,1H),2.83-2.70(m,1H),2.66-2.55(m,1H),2.42-2.26(m,1H),2.17-2.06(m,1H).
步骤3：化合物WX005-3的合成
室温下，将湿钯碳(0.3g,纯度:10％)加入四氢呋喃(30mL)中，随后加入化合物WX005-2(750mg,2.95mmol)，二碳酸二叔丁酯(772.60mg,3.54mmol,813.26μL)，氢气置换3次，反应混合物在室温和氢气环境下(15psi)搅拌反应12小时。反应完毕后，反应液直接用硅藻土过滤，收集滤液，减压浓缩除去溶剂。所得粗品经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝4/1-3/2，体积比)，得到化合物WX005-3.MS-ESI m/z:357.1[M-H] -.
步骤4：化合物WX005-4盐酸盐的合成
室温下，将化合物WX005-3(700mg,1.95mmol)溶于盐酸/乙酸乙酯(4M,14.00mL)中，反应混合物在室温下搅拌反应10分钟，有固体析出。反应完毕后，反应液直接过滤，收集滤饼，减压干燥，得到化合物WX005-4的盐酸盐.MS-ESI m/z:257.0[M-H] -. 1H NMR(400MHz,DMSO_d 6)δ：10.91(s,1H),8.45(s,3H),7.95(s,1H),7.74(s,1H),7.62(d,J＝8.0Hz,1H),7.37(dd,J＝1.2,8.0Hz,1H),4.20-4.08(m,3H),2.83-2.70(m,1H),2.63-2.53(m,1H),2.41-2.27(m,1H),2.16-2.05(m,1H).经制备HPLC分离(色谱柱：Waters Xbridge Prep OBD C18 150*40mm*10μm；流动相：乙腈/水；中性体系：NH 4HCO 3)，得到化合物WX005-4。
步骤5：化合物WX005-5的合成
室温和氮气保护下，将化合物WX004-4(2g,9.97mmol)溶于乙腈(20mL)中，随后加入2-溴乙基甲基醚(1.39g,9.97mmol,936.23μL)，碘化钾(165.49mg,996.92μmol)，碳酸钾(2.76g,19.94mmol)，反应混合物升温至80℃并搅拌反应12小时。反应完毕后，反应液冷却至室温，过滤，滤饼用四氢呋喃淋洗(20mL×3)，收集滤液，减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝1/0-19/1，体积比)，得到化合物WX005-5. 1H NMR(400MHz,CDCl 3)δ:7.68(d,J＝1.6Hz,1H),7.40(d,J＝0.8Hz,1H),4.22-4.16(m,2H),3.91(s,3H),3.82-3.77(m,2H),3.47(s,3H),2.35(s,3H).
步骤6：化合物WX005-6的合成
室温下，将化合物WX005-5(2.5g,9.66mmol)溶于四氢呋喃(18mL)和水(6mL)中，随后加入一水合氢氧化锂(1.62g,38.66mmol)，反应混合物在室温下搅拌反应12小时。反应完毕后，向反应液中加 入水(20mL)，用6M稀盐酸调节pH至5～6，用乙酸乙酯萃取(30mL×3)。合并有机相，用无水硫酸钠干燥，过滤，减压浓缩除去溶剂，得到化合物WX005-6. 1H NMR(400MHz,DMSO_d 6)δ:13.17(br s,1H),7.53(d,J＝0.8Hz,1H),7.41(d,J＝0.8Hz,1H),4.22-4.16(m,2H),3.73-3.67(m,2H),3.33(s,3H),2.26(s,3H).
步骤7：化合物WX005-7的合成
室温和氮气保护下，将化合物WX005-6(1.9g,7.77mmol)溶于叔丁醇(30mL)中，随后加入叠氮磷酸二苯酯(3.21g,11.65mmol,2.52mL)，三乙胺(1.57g,15.53mmol,2.16mL)，反应混合物升温至100℃并搅拌反应12小时。反应完毕后，反应液直接减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝1/0-19/1，体积比)，得到化合物WX005-7.MS-ESI m/z:260.1[M-55] +,261.1[M+H-55] +,262.1[M+2-55] +. 1H NMR(400MHz,CDCl 3)δ:7.01(br s,1H),6.90(d,J＝2.0Hz,1H),6.40(br s,1H),4.14-4.08(m,2H),3.79-3.74(m,2H),3.46(s,3H),2.23(s,3H),1.52(s,9H).
步骤8：化合物WX005-8盐酸盐的合成
室温下，将化合物WX005-7(1.65g,5.22mmol)溶于盐酸/乙酸乙酯(4M,30mL)中，反应混合物在室温下搅拌反应0.5小时，有固体析出。反应完毕后，反应液直接过滤，收集滤饼，真空干燥，得到化合物WX005-8的盐酸盐.MS-ESI m/z:214.1[M-H] -. 1H NMR(400MHz,DMSO_d 6)δ:6.80(d,J＝1.6Hz,1H),6.72(d,J＝1.6Hz,1H),4.11-4.04(m,2H),3.71-3.65(m,2H),3.33(s,3H),2.14(s,3H).
步骤9：化合物WX005-9的合成
室温和氮气保护下，将化合物WX005-8的盐酸盐(200mg,793.22μmol)溶于二氯甲烷(5mL)中，随后加入三乙胺(234.59mg,2.32mmol,322.68μL)，降温至0℃滴加氯甲酸苯酯(174.23mg,1.11mmol,139.38μL)，反应混合物在室温下搅拌反应0.5小时。反应完毕后，向反应液中加入水(20mL)，用二氯甲烷萃取(10mL×3)。合并有机相，用无水硫酸钠干燥，过滤，减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝1/0-4/1，体积比)，得到化合物WX005-9.MS-ESI m/z:336.2[M+H] +,337.2[M+H+1] +,338.2[M+H+2] +. 1H NMR(400MHz,CDCl 3)δ:7.44-7.37(m,2H),7.26-7.22(m,1H),7.21-7.15(m,3H),6.93(d,J＝2.0Hz,1H),6.89(br s,1H),4.13-4.08(m,2H),3.78-3.72(m,2H),3.45(s,3H),2.25(s,3H).
步骤10：化合物WX005的合成
室温下，将化合物WX005-9(80mg,238.25μmol)溶于N,N-二甲基甲酰胺(2mL)中，随后依次加入化合物WX005-4的盐酸盐(98.54mg,334.34μmol)，三乙胺(72.32mg,714.75μmol,99.48μL)，反应混合物在室温下搅拌反应12小时。反应完毕后，将反应液倒入水(20mL)中，用乙酸乙酯萃取(20mL×3)，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：HCl)，得到目标化合物WX005。MS-ESI m/z:500.2[M+H] +,501.1[M+H+1] +,502.1[M+H+2] +. 1H NMR(400MHz,DMSO_d 6)δ:10.89(s,1H),8.67(s,1H),7.86(s,1H),7.53(d,J＝8.0Hz,1H),7.47(s,1H),7.20(dd,J＝1.0,8.2Hz,1H),7.17(d,J＝2.0Hz,1H),6.99(d,J＝2.0Hz,1H),6.73(t,J＝6.0Hz,1H),4.39(d,J＝5.6Hz,2H),4.12(dd,J＝4.8,12.0Hz,1H),4.06-4.01(m,2H),3.70-3.64(m,2H),3.31(s,3H),2.80-2.69(m,1H),2.62-2.53(m,1H),2.32-2.25(m,1H),2.15-2.07(m,4H).
实施例6
Figure PCTCN2022105236-appb-000080
合成路线：
Figure PCTCN2022105236-appb-000081
化合物WX006的合成
室温下，将化合物WX004-7(50mg,127.92μmol)和化合物WX005-4的盐酸盐(36.34mg,140.72μmol)溶于N,N-二甲基甲酰胺(1mL)中，随后加入三乙胺(25.89mg,255.85μmol,35.61μL)，反应混合物在室温下搅拌反应12小时。反应完毕后，向反应液中加入水(10mL)，用乙酸乙酯萃取(20mL×3)。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；中性体系：NH 4HCO 3)，得到目标化合物WX006.MS-ESI m/z:555.3[M+H] +,556.2[M+H+1] +,557.3[M+H+2] +. 1H NMR(400MHz,DMSO_d 6)δ:10.89(s,1H),8.66(s,1H),7.86(s,1H),7.53(d,J＝8.0Hz,1H),7.47(s,1H),7.19(dd,J＝1.2,8.4Hz,1H),7.15(d,J＝2.0Hz,1H),7.01(d,J＝1.6Hz,1H),6.74(t,J＝5.8Hz,1H),4.38(d,J＝6.0Hz,2H),4.12(dd,J＝5.0,11.8Hz,1H),4.03(t,J＝5.6Hz,2H),3.57(t,J＝4.6Hz,4H),2.80-2.68(m,3H),2.60-2.54(m,1H),2.47-2.43(m,4H),2.32-2.25(m,1H),2.15-2.05(m,4H).
实施例7
Figure PCTCN2022105236-appb-000082
合成路线：
Figure PCTCN2022105236-appb-000083
化合物WX007的合成
室温下，将化合物WX005-9(76mg,226.34μmol)溶于N,N-二甲基甲酰胺(2mL)中，随后依次加入化合物WX004-2(70.15mg,271.61μmol)，三乙胺(45.81mg,452.68μmol,63.01μL)，反应混合物在室温下搅拌反应12小时。反应完毕后，将反应液倒入水(20mL)中，用乙酸乙酯萃取(20mL×3)。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：HCl)，得到目标化合物WX007.MS-ESI m/z:500.2[M+H] +,501.2[M+H+1] +,502.2[M+H+2] +. 1H NMR(400MHz,DMSO_d 6)δ:10.89(s,1H),8.63(s,1H),7.89(s,1H),7.53(d,J＝8.8Hz,1H),7.51(d,J＝1.2Hz,1H),7.27(dd,J＝1.6,8.4Hz,1H),7.17(d,J＝2.0Hz,1H),6.98(d,J＝1.8Hz,1H),6.67(t,J＝5.8Hz,1H),4.36(d,J＝5.6Hz,2H),4.13(dd,J＝4.8,12.0Hz,1H),4.07-4.00(m,2H),3.70-3.64(m,2H),3.31(s,3H),2.81-2.70(m,1H),2.61-2.58(m,1H),2.40-2.33(m,1H),2.17-2.11(m,1H),2.10(s,3H).
实施例8
Figure PCTCN2022105236-appb-000084
合成路线：
Figure PCTCN2022105236-appb-000085
步骤1：化合物WX008-2的合成
室温下，将硫酸(1.22kg,12.19mol,663.00mL,纯度:98％)滴加到冰水(221mL)中，之后加入化合物WX008-1(170g,1.37mol)，降温至0℃，滴加4-氯乙酰乙酸乙酯(247.93g,1.51mol)，反应混合物在室温下搅拌反应12小时。反应完毕后，将反应液倒入800mL冰水中淬灭，有固体析出，过滤，收集滤饼，滤饼减压真空干燥，得到化合物WX008-2.MS-ESI m/z:225.1[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:7.40(d,J＝9.2Hz,1H),7.31(d,J＝3.2Hz,1H),7.29-7.21(m,1H),6.68(s,1H),5.06(s,2H),3.84(s,3H).
步骤2：化合物WX008-3的合成
室温下，将氢氧化钠(45.45g,1.14mol)溶于2M氢氧化钠的水(568.125mL)溶液中，之后加入化合物WX008-2(250g,1.11mol)，反应混合物升温至80℃搅拌反应12小时。反应完毕后，反应液冷却至室温，加入水(1000mL)，用乙酸乙酯(600mL)萃取，有机相丢弃，水相用12M浓盐酸调节pH为5-6，有固体析出，用乙酸乙酯(600mL×3)萃取，合并有机相，依次用饱和食盐水(400mL×3)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得到化合物WX008-3.MS-ESI m/z:207.1[M+H] +.
步骤3：化合物WX008-4的合成
室温和氮气保护下，将化合物WX008-3(68g,329.79mmol)溶于二氯甲烷(1300mL)中，氮气保护下降温至-30℃，逐滴滴加三溴化硼(223.07g,890.42mmol,85.80mL)，反应混合物缓慢升至室温搅拌反应1小时。反应完毕后，反应液倒入冰水(2000mL)中淬灭，分液，收集有机相，水相用乙酸乙酯(800mL×3)萃取，合并有机相，依次用饱和食盐水(600mL×3)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得到化合物WX008-4.MS-ESI m/z:193.1[M+H] +.
步骤4：化合物WX008-5的合成
室温和氮气保护下，将化合物WX008-4(126g,655.68mmol)溶于乙醇(410mL)中，加入浓硫酸(12.86g,131.14mmol,6.99mL,纯度:98％)，反应混合物升温至80℃搅拌反应12小时。反应完毕后，反应混合物减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝1/0-9/1，体积比)，得到化合物WX008-5.MS-ESI m/z:221.1[M+H] +. 1H NMR(400MHz,CDCl 3)δ:7.59(s,1H),7.31(d,J＝8.4Hz,1H),6.98(d,J＝2.4Hz,1H),6.81(dd,J＝2.6,8.6Hz,1H),5.70(s,1H),4.20(q,J＝7.0Hz,2H),3.64(d,J＝1.2Hz,2H),1.27(t,J＝7.2Hz,3H).
步骤5：化合物WX008-6的合成
室温和氮气保护下，将化合物WX008-5(3g,13.62mmol)溶于四氢呋喃(40mL)中，随后加入N-(叔丁氧羰基)乙醇胺(3.29g,20.43mmol,3.17mL)，偶氮二甲酰二哌啶(6.87g,27.25mmol)，降温至0℃，滴加溶于四氢呋喃(10mL)的三丁基膦(5.51g,27.25mmol,6.72mL)溶液，反应混合物恢复至室温并搅拌反应3小时。反应完毕后，反应液直接过滤，滤饼用四氢呋喃淋洗(5mL×3)，收集滤液，减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝1/0-9/1，体积比)，得到化合物WX008-6.MS-ESI m/z:264.2[M+H-100] +. 1H NMR(400MHz,CDCl 3)δ:7.61(s,1H),7.37(d,J＝8.8Hz,1H),7.02(d,J＝2.8Hz,1H),6.91(dd,J＝2.6,9.0Hz,1H),5.04(br s,1H),4.20(q,J＝7.2Hz,2H),4.06(t,J＝5.2Hz,2H),3.66(d,J＝1.2Hz,2H),3.61-3.52(m,2H),1.47(s,9H),1.29(t,J＝7.2Hz,3H).
步骤6：化合物WX008-7的合成
室温和氮气保护下，将化合物WX008-6(1.8g,4.95mmol)溶于四氢呋喃(40mL)中，随后同时加入丙烯酰胺(352.06mg,4.95mmol)和叔丁醇钾(833.71mg,7.43mmol)，反应混合物在室温下搅拌反应1小时。反应完毕后，将反应液倒入水(50mL)中，用乙酸乙酯萃取(30mL×3)。合并有机相，用无水硫酸钠干燥，过滤，减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝9/1-7/3，体积比)。得到化合物WX008-7. 1H NMR(400MHz,CDCl 3)δ:8.09(br s,1H),7.56(s,1H),7.40(d,J＝9.2Hz,1H),6.97-6.95(m,1H),6.95-6.91(m,1H),5.02(br s,1H),4.06(t,J＝5.0Hz,2H),3.97(t,J＝7.6Hz,1H),3.62-3.49(m,2H),2.88-2.69(m,2H),2.42-2.32(m,2H),1.47(s,9H).
步骤7：化合物WX008-8盐酸盐的合成
室温下，将化合物WX008-7(600mg,1.54mmol)溶于盐酸/乙酸乙酯(4M,20mL)中，反应混合物在室温下搅拌反应1小时，有固体析出。反应完毕后，反应液直接过滤，收集固体，减压干燥得到化合物WX008-8的盐酸盐.MS-ESI m/z:287.2[M-H] -. 1H NMR(400MHz,DMSO_d 6)δ:10.89(s,1H),8.25(br s,3H),7.88(s,1H),7.51(d,J＝9.2Hz,1H),7.16(d,J＝2.8Hz,1H),6.98(dd,J＝2.4,8.8Hz,1H),4.23-4.16(m,2H),4.13(dd,J＝4.8,12.0Hz,1H),3.25-3.16(m,2H),2.81-2.70(m,1H),2.63-2.55(m,1H),2.39-2.27(m,1H),2.16-2.05(m,1H).
步骤8：化合物WX008-10的合成
室温和氮气保护下，将化合物WX008-9(10g,45.66mmol)和化合物2-氨基异丁酸(14.13g,136.98mmol)加入到N,N-二甲基甲酰胺(100mL)和水(10mL)的混合溶液中，将碘化亚铜(1.74g,9.14mmol)，铜粉(580.00mg,9.13mmol)，碳酸钾(31.55g,228.30mmol)，N,N-二甲基甘氨酸(2.35g,22.83mmol)依次慢慢的加入反应液中，置换氮气三次，反应混合物升温至110℃搅拌反应12小时。反应完毕后，反应液冷却到室温，加入100mL冰水，6M盐酸调节pH至4-5，用乙酸乙酯萃取(200mL×2)，合并有机相，用饱和食盐水洗涤(200mL×2)，无水硫酸钠干燥，过滤，减压浓缩除去溶剂。所得粗品加入到二氯甲烷(15mL)中搅拌1小时，过滤，滤饼用二氯甲烷洗涤(5mL×2)，收集固体，减压干燥得到化合物WX008-10.MS-ESI m/z:242.2[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:12.53(s,1H),7.59(t,J＝8.8Hz,1H),6.96(s,1H),6.33(d,J＝8.8Hz,1H),6.15(d,J＝14.4Hz,1H),1.45(s,6H).
步骤9：化合物WX008-11的合成
室温下，将化合物WX008-10(4g,16.58mmol)，异硫氰酸甲酯(1.82g,24.87mmol,1.70mL)溶于乙醇(80mL)中，缓慢加入三乙胺(5.71g,56.37mmol,7.85mL)，反应混合物升温至80℃并搅拌反应12小时。反应完成，反应液冷却到室温，减压浓缩除去溶剂，加入水(20mL)，1M的盐酸调节pH至3-4，用乙酸乙酯萃取(20mL×2)，饱和食盐水洗涤(20mL×2)，无水硫酸钠干燥，过滤，减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝1/0-2/3，体积比)，得到化合物WX008-11.MS-ESI m/z:297.1[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:13.44(s,1H),7.99(t,J＝8.2Hz,1H),7.43(dd,J＝1.6,11.2Hz,1H),7.30(dd,J＝1.6,8.4Hz,1H),3.22(s,3H),1.38(s,6H).
步骤10：化合物WX008的合成
室温下，将化合物WX008-8的盐酸盐(100mg,307.92μmol)溶于N,N-二甲基甲酰胺(2mL)中，随后加入化合物WX008-11(109.49mg,369.50μmol)，最后加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N’,N’-四甲基脲六氟磷酸酯(234.16mg,615.84μmol)和三乙胺(124.63mg,1.23mmol,171.43μL)，反应混合物在室温下搅拌反应5小时。反应完毕后，将反应液倒入水(20mL)中，用乙酸乙酯萃取(20mL×3)。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：HCl)，得到目标化合物WX008。MS-ESI m/z:567.2[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.87(s,1H),8.72(t,J＝4.8Hz,1H),7.85(s,1H),7.73(t,J＝8.2Hz,1H),7.47(d,J＝8.8Hz,1H),7.41(dd,J＝1.8,10.6Hz,1H),7.27(dd,J＝1.8,8.2Hz,1H),7.16(d,J＝2.4Hz,1H),6.94(dd,J＝2.6,9.0Hz,1H),4.19-4.06(m,3H),3.66(q,J＝5.6Hz,2H),3.22(s,3H),2.81-2.65(m,1H),2.61-2.54(m,1H),2.37-2.29(m,1H),2.14-2.06(m,1H),1.37(s,6H).
实施例9
Figure PCTCN2022105236-appb-000086
合成路线：
Figure PCTCN2022105236-appb-000087
步骤1：化合物WX009-1的合成
室温和氮气保护下，将化合物WX004-4(1.96g,9.77mmol)溶于乙腈(30mL)中，随后加入N,N-二甲氨基氯乙烷盐酸盐(2.81g,19.54mmol,936.23μL)，碘化钾(162.18mg,976.98μmol)，碳酸钾(5.40g,39.08mmol)，反应混合物升温至80℃并搅拌反应12小时。反应完毕后，反应液冷却至室温，过滤，滤饼用四氢呋喃淋洗(20mL×3)，收集滤液，减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝4/1-2/3～二氯甲烷/甲醇＝1/0-19/1，体积比)，得到化合物WX009-1.MS-ESI m/z:272.2[M+H] +,273.2[M+H+1] +,274.2[M+H+2] +.
步骤2：化合物WX009-2的合成
室温下，将化合物WX009-1(0.8g,2.94mmol)溶于四氢呋喃(9mL)和水(3mL)中，随后加入一水合氢氧化锂(494.12mg,11.78mmol)，反应混合物在室温下搅拌反应2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；中性体系：NH 4HCO 3)，得到化合物WX009-2.MS-ESI m/z:256.1[M-H] -,257.0[M-H+1] -,258.1[M-H+2] -. 1H NMR(400MHz,DMSO_d 6)δ:7.51(d,J＝1.2Hz,1H),7.41(d,J＝0.8Hz,1H),4.18(t,J＝5.6Hz,2H),2.82(t,J＝5.4Hz,2H),2.34(s,6H),2.24(s,3H).步骤3：化合物WX009-3的合成
室温和氮气保护下，将化合物WX009-2(240mg,931.27μmol)溶于甲苯(2.5mL)中，随后加入三乙胺(207.32mg,2.05mmol,285.17μL)，叠氮磷酸二苯酯(281.91mg,1.02mmol,221.98μL)，反应混合物升温至100℃搅拌10分钟后，加入苯酚(438.21mg,4.66mmol,409.55μL)，反应混合物在100℃下继续搅拌反应0.5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；中性体系：NH 4HCO 3)，得到化合物WX009-3.MS-ESI m/z:349.2[M+H] +,350.2[M+H+1] +,351.2[M+H+2] +. 1H NMR(400MHz,DMSO_d 6)δ:10.31(s,1H),7.48-7.40(m,2H),7.29-7.25(m,1H),7.25-7.21(m,2H),7.20-7.15(m,2H),4.02(t,J＝5.6Hz,2H),2.70(t,J＝5.6Hz,2H),2.26(s,6H),2.14(s,3H).
步骤4：化合物WX009的合成
室温下，将化合物WX009-3(15mg,43.00μmol)溶于N,N-二甲基甲酰胺(0.4mL)中，随后依次加入化合物WX005-4(12.22mg,47.30μmol)，三乙胺(13.05mg,129.01μmol,17.96μL)，反应混合物在室温 下搅拌反应12小时。反应完毕后，反应液直接经制备HPLC分离(流动相：乙腈/水；中性体系：NH 4HCO 3)，得到目标化合物WX009.MS-ESI m/z:513.2[M+H] +,514.3[M+H+1] +,515.2[M+H+2] +. 1H NMR(400MHz,DMSO_d 6)δ:10.89(s,1H),8.67(s,1H),7.87(s,1H),7.53(d,J＝8.0Hz,1H),7.47(s,1H),7.19(d,J＝8.4Hz,1H),7.17(d,J＝2.0Hz,1H),6.99(d,J＝1.6Hz,1H),6.74(t,J＝5.8Hz,1H),4.38(d,J＝6.0Hz,2H),4.12(dd,J＝4.8,12.0Hz,1H),3.99(t,J＝5.6Hz,2H),2.79-2.69(m,1H),2.65(t,J＝5.8Hz,2H),2.61-2.56(m,1H),2.32-2.26(m,1H),2.22(s,6H),2.15-2.06(m,4H).
实施例10
Figure PCTCN2022105236-appb-000088
合成路线：
Figure PCTCN2022105236-appb-000089
步骤1：化合物WX010-1的合成
室温和氮气保护下，将化合物WX003-4(3g,10.60mmol)，双联嚬哪醇硼酸酯(3.23g,12.72mmol)，醋酸钾(4.16g,42.39mmol)，[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷(432.67mg,529.82μmol)溶于二氧六环(50mL)中，反应混合物升温至100℃并搅拌反应5小时。反应完毕后，将反应液降至室温，直接用硅藻土过滤，滤饼用二氯甲烷(30mL×3)淋洗，滤液减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝30/1，体积比)，得到化合物WX010-1.
步骤2：化合物WX010-2的合成
室温下，将化合物WX010-1(3.1g,9.39mmol)和碳酸氢钠(1.58g,18.78mmol,730.29μL)溶于四氢呋喃(40mL)和水(20mL)的混合溶剂中，降温至0℃，滴加双氧水(7.15g,63.06mmol,6.06mL,纯度:30％)，反应混合物在0℃下搅拌反应2小时。反应完毕后，向反应液加入15％的亚硫酸水溶液(50mL)淬灭，并搅拌10分钟，用1N盐酸调节pH至5～6，用乙酸乙酯萃取(50mL×3)，有机相用饱和食盐水(50mL)洗涤，无水硫酸钠干燥，过滤，旋干。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝6/1，体积比)，得到化合物WX010-2.
步骤3：化合物WX010-3的合成
室温和氮气保护下，将化合物WX010-2(1g,4.54mmol)溶于四氢呋喃(20mL)中，随后加入N-Boc-乙醇胺(1.10g,6.81mmol,1.06mL)，偶氮二甲酰二哌啶(2.29g,9.08mmol)，降温至0℃，滴加溶于四氢呋喃(5mL)的三丁基磷(1.84g,9.08mmol,2.24mL)，反应混合物恢复至室温搅拌反应3小时。反应完毕后，反应液直接过滤，滤饼用四氢呋喃淋洗(5mL×3)，收集滤液，减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝1/0-9/1，体积比)，得到化合物WX010-3.MS-ESI m/z:308.1[M-55] +. 1H NMR(400MHz,CDCl 3)δ:7.54(s,1H),7.44(d,J＝8.4Hz,1H),7.00(d,J＝2.0Hz,1H),6.89(dd,J＝2.2,8.6Hz,1H),5.03(br s,1H),4.19(q,J＝7.0Hz,2H),4.06(t,J＝5.0Hz,2H),3.66(d,J＝1.2Hz,2H),3.62-3.51(m,2H),1.47(s,9H),1.28(t,J＝7.0Hz,3H).
步骤4：化合物WX010-4的合成
室温和氮气保护下，将化合物WX010-3(1.1g,3.03mmol)溶于N,N-二甲基甲酰胺(20mL)中，随后同时加入丙烯酰胺(215.15mg,3.03mmol)和叔丁醇钾(509.48mg,4.54mmol)的混合物，反应混合物在室温下搅拌反应1小时。反应完毕后，将反应液倒入水(50mL)中，用乙酸乙酯萃取(30mL×3)，合并有机相，依次用半饱和食盐水洗涤(50mL×3)，无水硫酸钠干燥，过滤，减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝4/1-1/1，体积比)，得到化合物WX010-4. 1H NMR(400MHz,CDCl 3)δ:8.17(br s,1H),7.49(s,1H),7.36(d,J＝8.8Hz,1H),7.02(d,J＝2.4Hz,1H),6.89(dd,J＝2.2,8.6Hz,1H),5.03(br s,1H),4.06(t,J＝5.0Hz,2H),3.97(t,J＝7.4Hz,1H),3.62-3.50(m,2H),2.85-2.65(m,2H),2.42-2.29(m,2H),1.46(s,9H).
步骤5：化合物WX010-5盐酸盐的合成
室温下，将化合物WX010-4(650mg,1.67mmol)溶于盐酸/乙酸乙酯(4M,20mL)中，反应混合物在室温下搅拌反应15分钟，有固体析出。反应完毕后，反应液直接过滤，收集滤饼，减压干燥，得到化合物WX010-5的盐酸盐.MS-ESI m/z:287.0[M-H] -. 1H NMR(400MHz,DMSO_d 6)δ:10.89(s,1H),8.23(br s,3H),7.81(s,1H),7.49(d,J＝8.8Hz,1H),7.25(d,J＝2.0Hz,1H),6.94(dd,J＝2.2,8.6Hz,1H),4.23(t,J＝5.0Hz,2H),4.10(dd,J＝4.8,12.0Hz,1H),3.27-3.17(m,2H),2.81-2.66(m,1H),2.62-2.52(m,1H),2.37-2.23(m,1H),2.16-2.05(m,1H).
步骤6：化合物WX010的合成
室温下，将化合物WX010-5的盐酸盐(70mg,215.54μmol)溶于N,N-二甲基甲酰胺(2mL)中，随后加入化合物WX008-11(70.26mg,237.10μmol)，最后加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N’,N’-四甲基脲六氟磷酸酯(163.91mg,431.09μmol)，三乙胺(87.24mg,862.18μmol,120.01μL)，反应混合物在室温下搅拌反应12小时。反应完毕后，将反应液倒入水中(20mL)，用乙酸乙酯萃取(20mL×3)，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：HCl)，得到目标化合物WX010.MS-ESI m/z:567.0[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:10.88(s,1H),8.72(t,J＝4.8Hz,1H),7.78(s,1H),7.72(t,J＝8.0Hz,1H),7.46(d,J＝8.4Hz,1H),7.41(dd,J＝1.6,10.8Hz,1H),7.26(dd,J＝1.8,8.2Hz,1H),7.23(d,J＝2.0Hz,1H),6.91(dd,J＝2.2,8.6Hz,1H),4.17(t,J＝5.6Hz,2H),4.08(dd,J＝4.8,11.6Hz,1H),3.70-3.62(m,2H),3.22(s,3H),2.77-2.69(m,1H),2.61-2.56(m,1H),2.32-2.23(m,1H),2.16-2.06(m,1H),1.37(s,6H).
实施例11
Figure PCTCN2022105236-appb-000090
合成路线：
Figure PCTCN2022105236-appb-000091
步骤1：化合物WX011-2的合成
室温下，将化合物WX011-1(10g,60.18mmol)溶于甲苯(100mL)中，随后加入碳酸二乙酯(48.75g,412.68mmol,50mL)，搅拌至体系溶清，降温至0～5℃，分批加入钠氢(12.03g,300.90mmol,纯度:60％)，反应混合物升温至100℃搅拌反应12小时。反应完毕后，将反应液倒入水(500mL)中，用甲基叔丁基醚萃取(100mL)，有机相丢弃，水相用6M稀盐酸调节pH至5～6，用乙酸乙酯/四氢呋喃(3:1)萃取(500mL×3)。合并有机相，用无水硫酸钠干燥，过滤，减压浓缩除去溶剂。所得残余物经甲基叔丁基醚(50mL)室温搅拌30分钟，有固体析出，过滤，收集固体，减压干燥得到化合物WX011-2.MS-ESI m/z:193.1[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:12.35(br s,1H),7.71(d,J＝8.8Hz,1H),6.97-6.89(m,2H),5.44(s,1H),3.85(s,3H).
步骤2：化合物WX011-3的合成
室温和氮气保护下，将化合物WX011-2(3g,15.61mmol)溶于二氯甲烷(60mL)中，氮气保护降温至-30℃滴加三溴化硼(11.73g,46.83mmol,4.51mL)，反应混合物恢复至室温搅拌反应12小时。反应完毕后，将反应液缓慢倒入冰水中(200mL)，减压浓缩除去溶剂，用(乙酸乙酯/四氢呋喃＝3/1)萃取(200mL×3)。合并有机相，用饱和食盐水洗涤(50mL×2)，无水硫酸钠干燥，过滤，减压浓缩除去溶剂。所得残余物经甲基叔丁基醚(20mL)室温搅拌30分钟，有固体析出，过滤，收集固体，减压干燥得到化合物WX011-3.MS-ESI m/z:179.0[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:12.22(s,1H),10.51(s,1H),7.63(d,J＝8.4Hz,1H),6.76(dd,J＝2.2,8.6Hz,1H),6.66(d,J＝2.0Hz,1H),5.38(s,1H).
步骤3：化合物WX011-4的合成
室温和氮气保护下，将化合物WX011-3(2.55g,14.31mmol)溶于乙醇(50mL)中，随后加入盐酸羟胺(3.48g,50.10mmol)，最后加入乙醇钠(3.41g,50.10mmol)，反应混合物升温至80℃搅拌反应12小时。反应完毕后，将反应液倒入水(100mL)中，用2M稀盐酸调节pH至4～5，用乙酸乙酯萃取(100mL×3)。合并有机相，用无水硫酸钠干燥，过滤，减压浓缩得到化合物WX011-4.MS-ESI m/z:194.1[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:12.77(s,1H),10.34(s,1H),7.58(d,J＝8.4Hz,1H),6.93(d,J＝1.6Hz,1H),6.85(dd,J＝2.0,8.4Hz,1H),3.97(s,2H).
步骤4：化合物WX011-5的合成
室温和氮气保护下，将化合物WX011-4(2.7g,13.98mmol)溶于乙醇(30mL)中，随后加入浓硫酸(920.00mg,9.19mmol,0.5mL,纯度：98％)，反应混合物升温至60℃搅拌反应16小时。反应完毕后，反应液直接减压浓缩除去溶剂，所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝1/0-4/1，体积比)，得到化合物WX011-5.MS-ESI m/z:222.1[M+H] +. 1H NMR(400MHz,CDCl 3)δ:7.53(d,J＝8.8Hz,1H),6.94(d,J＝2.0Hz,1H),6.84(dd,J＝2.0,8.4Hz,1H),5.94(s,1H),4.23(q,J＝7.0Hz,2H),3.99(s,2H),1.28(t,J＝7.2Hz,3H).
步骤5：化合物WX011-6的合成
室温和氮气保护下，将化合物WX011-5(1g,4.52mmol)溶于四氢呋喃(20mL)中，随后加入N-Boc-乙醇胺(1.09g,6.78mmol,1.05mL)，偶氮二甲酰二哌啶(2.28g,9.04mmol)，氮气保护降温至0℃，滴加溶于5mL四氢呋喃的三丁基膦(1.83g,9.04mmol,2.23mL)，反应混合物恢复至室温搅拌反应12小时。反应完毕后，反应液直接减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝1/0-9/1，体积比)，得到化合物WX011-6.MS-ESI m/z:365.2[M+H] +. 1H NMR(400MHz,CDCl 3)δ:7.56(d,J＝8.8Hz,1H),6.99(d,J＝2.0Hz,1H),6.93(dd,J＝2.0,8.8Hz,1H),5.01(br s,1H),4.21(q,J＝7.2Hz,2H),4.09(t,J＝5.0Hz,2H),3.98(s,2H),3.64-3.55(m,2H),1.46(s,9H),1.26(t,J＝7.2Hz,3H).
步骤6：化合物WX011-7的合成
室温和氮气保护下，将化合物WX011-6(500mg,1.37mmol)溶于四氢呋喃(10mL)中，随后同时加入丙烯酰胺(97.53mg,1.37mmol)和叔丁醇钾(230.95mg,2.06mmol)的混合物，反应混合物在室温下搅拌反应1小时。反应完毕后，向反应液加入水(50mL)，用乙酸乙酯萃取(50mL×3)。合并有机相，用无水硫酸钠干燥，过滤，减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝4/1-1/1， 体积比)，得到化合物WX011-7. 1H NMR(400MHz,CDCl 3)δ:8.11(br s,1H),7.57(d,J＝8.8Hz,1H),7.01(d,J＝2.0Hz,1H),6.95(dd,J＝2.0,8.8Hz,1H),5.00(br s,1H),4.28(dd,J＝5.2,8.4Hz,1H),4.10(t,J＝5.2Hz,2H),3.66-3.51(m,2H),3.09-2.96(m,1H),2.81-2.71(m,1H),2.66-2.55(m,1H),2.49-2.40(m,1H),1.47(s,9H).
步骤7：化合物WX011-8盐酸盐的合成
室温下，将化合物WX011-7(240mg,616.33μmol)溶于盐酸/乙酸乙酯(4M,4.99mL)中，反应混合物在室温下搅拌反应15分钟，有固体析出。反应完毕后，反应液直接过滤，收集滤饼，减压干燥得到化合物WX011-8的盐酸盐.MS-ESI m/z:290.2[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:11.08(s,1H),8.27(br s,3H),7.75(d,J＝8.8Hz,1H),7.38(d,J＝2.0Hz,1H),7.04(dd,J＝2.0,8.8Hz,1H),4.54(dd,J＝5.0,11.8Hz,1H),4.31(t,J＝5.0Hz,2H),3.30-3.20(m,2H),2.82-2.71(m,1H),2.65-2.56(m,1H),2.49-2.41(m,1H),2.24-2.14(m,1H).
步骤8：化合物WX011的合成
室温下，将化合物WX011-8的盐酸盐(70mg,214.89μmol)溶于N,N-二甲基甲酰胺(2mL)中，随后加入化合物WX008-11(70.04mg,236.38μmol)，最后加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N’,N’-四甲基脲六氟磷酸酯(163.42mg,429.78μmol)和三乙胺(86.98mg,859.56μmol,119.64μL)，反应混合物在室温下搅拌反应12小时。反应完毕后，反应液直接过滤，滤液经制备HPLC分离(流动相：乙腈/水；酸性体系：HCl)，得到目标化合物WX011。MS-ESI m/z:567.9[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:11.08(s,1H),8.75(t,J＝6.2Hz,1H),7.76-7.68(m,2H),7.41(dd,J＝1.4,11.0Hz,1H),7.36(d,J＝1.6Hz,1H),7.27(dd,J＝1.8,8.2Hz,1H),7.00(dd,J＝2.0,8.8Hz,1H),4.52(dd,J＝5.2,12.0Hz,1H),4.24(t,J＝5.4Hz,2H),3.70(q,J＝5.8Hz,2H),3.22(s,3H),2.81-2.71(m,1H),2.64-2.55(m,1H),2.47-2.40(m,1H),2.25-2.14(m,1H),1.37(s,6H).
实施例12
Figure PCTCN2022105236-appb-000092
合成路线：
Figure PCTCN2022105236-appb-000093
步骤1：化合物WX012-1的合成
室温和氮气保护下，将化合物WX003-1(25g,116.26mmol)溶于四氢呋喃(250mL)中，随后加入碳酸二甲酯(31.42g,348.77mmol,29.36mL)，搅拌至体系溶清，降温至5～15℃分批加入叔丁醇钾(78.27g,697.53mmol)，反应混合物升温至80℃搅拌反应12小时。反应完毕后，将反应液倒入水(500mL)中，用甲基叔丁基醚萃取(100mL)，有机相丢弃，水相用6M盐酸调节pH至5～6，有固体析出，过滤，收集固体，减压干燥得到化合物WX012-1.MS-ESI m/z:241.0[M+H] +,243.1[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:12.71(s,1H),7.72(d,J＝8.4Hz,1H),7.67(d,J＝2.0Hz,1H),7.52(dd,J＝1.8,8.6Hz,1H),5.61(s,1H).
步骤2：化合物WX012-2的合成
室温下，将化合物WX012-1(24g,99.57mmol)溶于乙醇(300mL)中，随后加入盐酸羟胺(24.22g,348.49mmol)，最后加入醋酸钠(28.59g,348.49mmol)，反应混合物升温至80℃搅拌反应12小时。反应完毕后，将反应液倒入水(1000mL)中，用6M稀盐酸调节pH至4～5，减压浓缩除去部分乙醇，用乙酸乙酯萃取(500mL×3)。合并有机相，用无水硫酸钠干燥，过滤，减压浓缩得到化合物WX012-2.MS-ESI m/z:256.0[M+H] +,258.0[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:12.65(s,1H),8.11(d,J＝1.2Hz,1H),7.82(d,J＝8.4Hz,1H),7.59(dd,J＝1.4,8.6Hz,1H),4.12(s,2H).
步骤3：化合物WX012-3的合成
室温和氮气保护下，将化合物WX012-2(24g,93.73mmol)溶于乙醇(200mL)中，随后加入浓硫酸(5.52g,55.15mmol,3mL,纯度:98％)，反应混合物升温至60℃搅拌反应12小时。反应完毕后，反应液减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝1/0-17/3，体积比)，得到化合物WX012-3.MS-ESI m/z:284.0[M+H] +,286.0[M+H] +. 1H NMR(400MHz,CDCl 3)δ:7.78(d,J＝1.2Hz,1H),7.59(d,J＝8.4Hz,1H),7.46(dd,J＝1.6,8.4Hz,1H),4.22(q,J＝7.0Hz,2H),4.03(s,2H),1.27(t,J＝7.2Hz,3H).
步骤4：化合物WX012-4的合成
室温和氮气保护下，将化合物WX012-3(0.5g,1.76mmol)溶于水(2mL)和甲苯(10mL)中，随后依次加入N-Boc-氨基甲基三氟硼酸钾(1.25g,5.28mmol)，碳酸铯(1.72g,5.28mmol)，[(二(1-金刚烷基)-N-丁基膦)-2-(2-氨基联苯)氯化钯(II)(117.67mg,175.99μmol)，反应混合物升温至105℃搅拌反应12小时。反应完毕后，将反应液倒入水(20mL)中，用乙酸乙酯萃取(30mL×3)。合并有机相，用无水硫酸钠干燥，过滤，减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝1/0-41/9，体积比)，得到化合物WX012-4.MS-ESI m/z:335.2[M+H] +. 1H NMR(400MHz,CDCl 3)δ:7.66(d,J＝8.0Hz,1H),7.50(s,1H),7.27(d,J＝7.6Hz,1H),5.01(s,1H),4.48(d,J＝6.0Hz,2H),4.22(q,J＝7.2Hz,2H),4.03(s,2H),1.48(s,9H),1.26(t,J＝7.0Hz,3H).
步骤5：化合物WX012-5的合成
室温和氮气保护下，将化合物WX012-4(600mg,1.79mmol)溶于四氢呋喃(12mL)中，同时加入丙烯酰胺(127.54mg,1.79mmol)和叔丁醇钾(201.36mg,1.79mmol)，反应混合物在室温下搅拌反应0.5小时。反应完毕后，将反应液倒入水(20mL)和乙酸乙酯(20mL)的混合物溶液中，分液后收集有机相，水相用乙酸乙酯萃取(20mL×3)。合并有机相，依次用饱和食盐水洗涤(50mL)，无水硫酸钠干燥，过滤，减压浓缩除去溶剂。所得残余物经柱层析分离(洗脱剂：石油醚/乙酸乙酯＝4/1-1/1，体积比)，得到化合物WX012-5. 1H NMR(400MHz,CDCl 3)δ:8.14(s,1H),7.66(d,J＝8.0Hz,1H),7.53(s,1H),7.26(d,J＝7.6Hz 1H),5.03(s,1H),4.48(d,J＝5.6Hz,2H),4.32(dd,J＝5.2,8.8Hz,1H),3.07-2.95(m,1H),2.82-2.71(m,1H),2.68-2.56(m,1H),2.51-2.40(m,1H),1.48(s,9H).
步骤6：化合物WX012-6盐酸盐的合成
室温下，将化合物WX012-5(390mg,1.09mmol)溶于盐酸/乙酸乙酯(4M,15mL)中，反应混合物在室温下搅拌反应15分钟。反应完毕后，反应液直接过滤，收集滤饼，减压干燥得到化合物WX012-6的盐酸盐.MS-ESI m/z:260.2[M+H] +. 1H NMR(400MHz,DMSO_d 6)δ:11.11(s,1H),8.55(br s,3H),7.93-7.88(m,2H),7.52(d,J＝8.4Hz,1H),4.63(dd,J＝5.2,12.0Hz,1H),4.26-4.17(m,2H),2.85-2.73(m,1H),2.66-2.53(m,2H),2.26-2.16(m,1H).
步骤7：化合物WX012的合成
室温下，将化合物WX004-7(50mg,127.92μmol)和化合物WX012-6的盐酸盐(41.61mg,140.71μmol)溶于N,N-二甲基甲酰胺(1mL)中，随后加入三乙胺(38.83mg,383.76μmol,53.41μL)，反应混合物在室温下搅拌反应12小时。反应完毕后，反应液直接过滤。所得滤液经制备HPLC分离(流动相：乙腈/水；中性体系：NH 4HCO 3)，得到目标化合物WX012.MS-ESI m/z:556.0[M+H] +,557.0[M+H+1] +,558.0[M+H+2] +. 1H NMR(400MHz,DMSO_d 6)δ:11.10(s,1H),8.77(s,1H),7.80(d,J＝8.8Hz,1H),7.61(s,1H),7.33(dd,J＝1.2,9.2Hz,1H),7.16(d,J＝2.0Hz,1H),7.02(d,J＝2.0Hz,1H),6.86(t,J＝6.2Hz,1H),4.58(dd,J＝5.0,12.2Hz,1H),4.45(d,J＝5.6Hz,2H),4.03(t,J＝5.6Hz,2H),3.59-3.55(m,4H),2.83-2.73(m,1H),2.71(t,J＝5.6Hz,2H),2.65-2.56(m,1H),2.48-2.44(m,4H),2.24-2.15(m,2H),2.10(s,3H).
实施例13
Figure PCTCN2022105236-appb-000094
合成路线：
Figure PCTCN2022105236-appb-000095
化合物WX013的合成
室温下，将化合物WX005-9(50mg,148.91μmol)和化合物WX012-6的盐酸盐(48.44mg,163.80μmol)溶于N,N-二甲基甲酰胺(1mL)中，随后加入三乙胺(45.20mg,446.72μmol,62.18μL)，反应混合物在室温下搅拌反应2小时。反应完毕后，反应液直接过滤，所得滤液经制备HPLC分离(流动相：乙腈/水；酸性体系：HCl)，得到目标化合物WX013.MS-ESI m/z:501.0[M+H] +,501.9[M+H+1] +,503.0[M+H+2] +. 1H NMR(400MHz,DMSO_d 6)δ:11.10(s,1H),8.78(s,1H),7.80(d,J＝8.4Hz,1H),7.61(s,1H),7.33(d,J＝8.4Hz,1H),7.18(d,J＝1.6Hz,1H),7.00(d,J＝1.6Hz,1H),6.86(t,J＝5.8Hz,1H),4.58(dd,J＝4.8,12.0Hz,1H),4.45(d,J＝6.0Hz,2H),4.03(t,J＝4.6Hz,2H),3.67(t,J＝4.6Hz,2H),3.32(s,3H),2.83-2.72(m,1H),2.65-2.52(m,2H),2.23-2.15(m,1H),2.11(s,3H).
实施例14
Figure PCTCN2022105236-appb-000096
合成路线：
Figure PCTCN2022105236-appb-000097
化合物WX014的合成
室温下，将化合物WX009-3(30mg,86.00μmol)和化合物WX012-6的盐酸盐(27.98mg,94.60μmol)溶于N,N-二甲基甲酰胺(1mL)中，随后加入三乙胺(26.11mg,258.01μmol,35.91μL)，反应混合物在室温下搅拌反应2小时。反应完毕后，反应液直接过滤，收集滤液，所得滤液经制备HPLC分离(流动相：乙腈/水；中性体系：NH 4HCO 3)，得到目标化合物WX014.MS-ESI m/z:514.0[M+H] +,515.0[M+H+1] +,516.0[M+H+2] +. 1H NMR(400MHz,DMSO_d 6)δ:11.10(s,1H),8.77(s,1H),7.80(d,J＝8.0Hz,1H),7.61(s,1H),7.33(d,J＝8.4Hz,1H),7.17(s,1H),7.00(s,1H),6.85(t,J＝5.8Hz,1H),4.58(dd,J＝4.8,12.0Hz,1H),4.45(d,J＝6.0Hz,2H),3.99(t,J＝5.6Hz,2H),2.85-2.72(m,1H),2.70-2.60(m,4H),2.22(s,6H),2.19-2.15(m,1H),2.10(s,3H).
实施例15和实施例16
Figure PCTCN2022105236-appb-000098
合成路线：
Figure PCTCN2022105236-appb-000099
化合物WX015或WX016的合成
化合物WX005(100mg,200.02μmol)经过超临界流体色谱(分离条件，色谱柱：DAICEL CHIRALPAK AD(250mm*30mm,10μm)；流动相：A:CO 2；B:IPA；B％:55％-55％，8min)分离。收集保留时间为1.711min的样品，再经制备HPLC分离(流动相：乙腈/水；酸性体系：HCl)，得到目标化合物WX015(ee％:98.68％)。收集保留时间为2.135min的样品，再经制备HPLC分离(流动相：乙腈/水；酸性体系：HCl)，得到目标化合物WX016(ee％:100.0％)。SFC分析方法，色谱柱：Chiralpak AD-3,50×4.6mm I.D.,3μm；流动相:A:CO 2；B:IPA(0.1％IPAm,v/v)。
化合物WX015.MS–ESI m/z:500.1[M+H] +,501.2[M+H+1] +,502.2[M+H+2] +. 1H NMR(400MHz,DMSO_d 6)δ:10.89(s,1H),8.67(s,1H),7.86(s,1H),7.53(d,J＝4.0Hz,1H),7.47(s,1H),7.20(dd,J＝1.2,8.4Hz,1H),7.17 (d,J＝2.0Hz,1H),6.99(d,J＝1.6Hz,1H),6.73(t,J＝6.0Hz,1H),4.39(d,J＝6.0Hz,2H),4.12(dd,J＝4.8,12.0Hz,1H),4.06–4.01(m,2H),3.70–3.65(m,2H),3.32(s,3H),2.81–2.69(m,1H),2.61–2.53(m,1H),2.38–2.27(m,1H),2.16–2.05(m,4H).
化合物WX016.MS–ESI m/z:500.2[M+H] +,501.1[M+H+1] +,502.1[M+H+2] +. 1H NMR(400MHz,DMSO_d 6)δ:10.90(s,1H),8.68(s,1H),7.87(s,1H),7.53(d,J＝8.0Hz,1H),7.48(s,1H),7.23–7.15(m,2H),6.99(s,1H),6.74(t,J＝5.8Hz,1H),4.38(d,J＝5.6Hz,2H),4.12(dd,J＝4.8,12.0Hz,1H),4.06–4.00(m,2H),3.71–3.63(m,2H),3.32(s,3H),2.82–2.68(m,1H),2.61–2.54(m,1H),2.37–2.25(m,1H),2.15–2.05(m,4H).
生物实验
实验例1：降解GSPT1蛋白的作用
分别用10μM、1μM和0.1μM的待测化合物处理BxPC-3细胞24小时。收集并裂解细胞，100℃变性20min之后进行Western blot分析，以β-actin为内参蛋白检测GSPT1的蛋白水平。主要步骤包括：1)电泳：采用Biorad预制胶，用SDS-PAGE电泳法分离蛋白；2)转膜：使用Biorad快速转膜体系，将凝胶上蛋白转移至PVDF膜；3)封闭：将带有蛋白印迹的PVDF膜放入抗体孵育盒中，加入封闭液完全覆盖PVDF膜，室温孵育1小时；4)孵育一抗，用GSPT1抗体稀释液覆盖上述PVDF膜于4℃摇床上孵育过夜，5)孵育二抗，用Wash buffer洗膜三遍，加入二抗稀释液(Anti-rabbit IgG,HRP-linked)，室温摇床上孵育1小时；5)曝光，用Wash buffer洗膜三遍，用化学发光法(Clarity Western ECL Substrate)检测膜上的条带。
仪器，实验材料及缓冲液配方
(1)细胞
详细信息见表1。
表1.细胞
细胞名称 类别 细胞来源 货号
BxPC-3 人胰腺癌肿瘤细胞 ATCC CRL-1687
(2)试剂
详细信息见表2。
表2.试剂
Figure PCTCN2022105236-appb-000100
Figure PCTCN2022105236-appb-000101
蛋白上样缓冲液：4×Laemmli loaing buffer用去离子水稀释4倍，并加入DTT；
电泳液：10x Tris/Glycine/SDS buffer用去离子水10倍稀释；
转膜液：用Trans-Blot Turbo RTA Midi LF PVDF Transfer Kit外加甲醇稀释配制；
封闭液：5％BSA。
Wash buffer：0.2％Tween-20 TBS buffer。
一二抗稀释液均用封闭液稀释配制
(3)抗体
详细信息见表3。
表3.抗体
试剂名称 种属 稀释比 来源
Anti-GSPT1 R 1:2000 Abcam，ab49878
Anti-β-actin R 1:2000 CST，#4970
Anti-rabbit IgG,HRP-linked — 1:2000 CST，#7074
(4)仪器
Biorad PowerPac Basic Power Supply电泳仪
Biorad
Figure PCTCN2022105236-appb-000102
Tetra Cell小型垂直电泳槽
BioRad Trans-Blot Turbo快速转印仪
ChemiDoc Imaging System成像仪
具体筛选结果见图1。
结论：本发明化合物WX001对BxPC-3细胞内GSPT1蛋白有明显的促降解作用。
实验例2：在细胞HEK293T-LgBiT_GSPT1-nHiBiT中的靶蛋白降解作用评估
实验目的：本实验通过检测受试化合物在细胞HEK293T-LgBiT中对靶蛋白GSPT1的降解作用。
实验材料：
1.细胞和培养基
细胞：HEK293T-LgBiT_GSPT1-nHiBiT polyclone
培养基：DMEM+10％FBS+2mM GlutaMax+1mM sodium pyruvate+1X penicillin/streptomycin
阳性对照：1000nM；阴性对照:0.1％DMSO
2.试剂与耗材
详细信息见表4。
表4.试剂与耗材
Figure PCTCN2022105236-appb-000103
3.仪器
详细信息见表5。
表5.仪器
仪器名称 生产商 仪器型号
ECHO移液器 Labcyte Echo 550
Bravo自动液体工作站 Agilent 16050-101
Envision读板器 Perkin Elmer 2104
自动加样器 Thermofisher Multidrop Combi
细胞计数器 Thermo Countess II FL
实验方案：
第1天
1.化合物的准备
(1).将待测化合物的粉末用DMSO溶解到10mM作为储存浓度，用移液器手动吸取9μL 10mM或者3mM的待测化合物，至LDV板的第1列和第13列；
(2).用multidrop Combi加6μL DMSO至第2-12列以及14-24列；
(3).用Bravo将待测化合物进行3倍稀释化(3μL+6μL)，从第1-11列及第13-23列；
(4).按照板布局，用Echo将25nL化合物溶液(LDV板第1-24列)转移到实验板；
(5).用Echo将25nL的1mM阳性对照的溶液转移到测定板中作为100％降解对照(即LC,HPE),25nL DMSO转移到测定板作为0％对照(即HC,ZPE)。
2.细胞铺板
(1).弃去细胞培养基，DPBS清洗一次，胰酶消化细胞，细胞计数，制备细胞悬液4×10 5cells/mL；
(2).用MultiDrop Combi中速加入25μL/孔的细胞悬液至含有待测化合物的实验板；
(3).将含有细胞的实验板放回培养箱中37℃、5％CO 2培养16～18小时。
第2天
(1).用MultiDrop Combi高速往实验板中加入25μL/孔检测试剂(NanoGlo裂解溶液+底物+LgBit蛋白)至测定板，震荡10min；
(2).离心2000rpm×1min除去气泡；
(3).Envision,US Luminescence检测方法读板。
3.数据分析
用下列公式来计算检测化合物的降解率(Degradation rate，DR)：DR(％)＝(RLU溶媒对照-RLU化合物)/(RLU溶媒对照-RLU阳性对照)*100％,溶媒对照即空白对照.在Excel中计算不同浓度化合物的降解率，然后用XLFit软件作抑制曲线图和计算相关参数，包括最小降解率，最大降解率及DC 50。
测试结果见表6。
表6 本发明化合物在细胞HEK293T-LgBiT_GSPT1-nHiBiT中的靶蛋白降解作用
化合物 DC 50(nM) 最大降解率(％)
WX005 2.51 100.32％
WX006 1.94 99.92％
WX012 1.18 99.85％
WX013 1.07 100.36％
结论：本发明化合物在细胞HEK293T-LgBiT_GSPT1-nHiBiT中展现出优异的靶蛋白降解作用。
实验例3：在肿瘤细胞系MV4-11和MDA-MB-231中的抗增殖作用评估
实验目的：本实验通过检测受试化合物对肿瘤细胞MV4-11和MDA-MB-231体外活性的影响从而研究化合物抑制细胞增殖的抑制作用。
实验材料：
1.细胞系及培养方法
详细信息见表7。
表7.细胞系及培养方法
细胞系 肿瘤类型 生长特点 培养方法
MV4-11 白血病 悬浮 RPMI1640+10％FBS
MDA-MB-231 乳腺癌 贴壁 Leibovitz's L-15+10％FBS(0％CO 2)
2.培养基及试剂
详细信息见表8。
表8.培养基及试剂
培养基及试剂 生产商 货号
RPMI 1640 GIBCO 22400-089
Leibovitz's L-15 SIGMA L1518
Dulbecco's PBS Hyclone SH30256.01
FBS Hyclone SY30087.03
Antibiotic-antimycotic GIBCO 15240-062
0.25％Trypsin GIBCO 25200072
DMSO SIGMA D2650
3.多孔板
Greiner
Figure PCTCN2022105236-appb-000104
96-孔板，平底黑板(带盖及透明底),#655090.
4.细胞活性实验所用试剂及仪器
(1)Promega CellTiter-Glo发光法细胞活性检测试剂盒(Promega-G7573).
(2)2104
Figure PCTCN2022105236-appb-000105
读板器，PerkinElmer.
实验方案：
1.细胞培养
将肿瘤细胞系按上述培养条件在37℃，5％CO 2或0％CO 2的培养箱中进行培养。定期传代，取处于对数生长期的细胞用于铺板
2.细胞铺板
(1).用台盼兰进行细胞染色并计数活细胞。
(2).将细胞浓度调整至合适浓度，见表9。
表9.细胞系及浓度
Cell line Density(per 96-well)
MDA-MB-231 5000
MV4-11 6000
(3).在培养板中每孔加入90μL细胞悬液，在空白对照空中加入不含细胞的培养液。
(4).将培养板在37℃,5％CO 2或0％CO 2，及100％相对湿度的培养箱中培养过夜。
3.化合物存储板制备
制备400X化合物存储板：将化合物用DMSO从最高浓度梯度稀释至最低浓度。每次现用现配。
4.10X化合物工作液的配制及化合物处理细胞
(1).10X化合物工作液的配制：加入化合物或DMSO后用排枪吹打混匀在V形底的96孔板中加入78μL细胞培养液，从400X化合物存储板中吸取2μL化合物加入96孔板的细胞培养液中。在溶媒对照和空白对照中加入2μL DMSO。加入化合物或DMSO后用排枪吹打混匀。
(2).加药：取10μL的10X化合物工作液加入到细胞培养板中。在溶媒对照和空白对照中加入10μL DMSO-细胞培养液混合液。
(3).将96孔细胞板放回培养箱中培养3天。
5.CellTiter-Glo发光法细胞活性检测
以下步骤按照Promega CellTiter-Glo发光法细胞活性检测试剂盒(Promega-G7573)的说明书来进行。
(1).将CellTiter-Glo缓冲液融化并放置至室温。
(2).将CellTiter-Glo底物放置至室温。
(3).在一瓶CellTiter-Glo底物中加入10mL CellTiter-Glo缓冲液以溶解底物，从而配制CellTiter-Glo工作液。
(4).缓慢涡旋震荡使充分溶解。
(5).取出细胞培养板放置30分钟使其平衡至室温。
(6).在每孔中加入50μL(等于每孔中细胞培养液一半体积)的CellTiter-Glo工作液。用铝箔纸包裹细胞板以避光。
(7).将培养板在轨道摇床上振摇2分钟以诱导细胞裂解。
(8).培养板在室温放置10分钟以稳定发光信号。
(9).在2104EnVision读板器上检测发光信号。
6.数据分析
用下列公式来计算检测化合物的抑制率(Inhibition rate，IR)：IR(％)＝(RLU溶媒对照–RLU化合物)/(RLU溶媒对照–RLU空白对照)*100％.在Excel中计算不同浓度化合物的抑制率，然后用GraphPad Prism软件作抑制曲线图和计算相关参数，包括最小抑制率，最大抑制率及IC 50。
实验结果：测试结果见表10。
表10 本发明化合物在MV4-11与MDA-MB-231细胞系中的细胞增殖抑制作用
Figure PCTCN2022105236-appb-000106
“/”代表未检测。
结论：
本发明化合物在肿瘤细胞系MV4-11与MDA-MB-231中展现出优异细胞增殖的抑制作用。
实验例4：在肿瘤细胞系NCI-H1581、DMS114、NCI-H69和NCI-H526中的抗增殖作用评估实验目的：本实验通过检测受试化合物对肿瘤细胞NCI-H1581、DMS114、NCI-H69和NCI-H526体外活性的影响从而研究化合物抑制细胞增殖的抑制作用。
实验材料：
1.细胞系及培养方法
详细信息见表11。
表11.细胞系及培养方法
细胞系 肿瘤类型 生长特点 培养方法
NCI-H1581 非小细胞肺癌 贴壁 RPMI 1640+10％FBS(5％CO 2)
DMS114 小细胞肺癌 贴壁 RPMI 1640+10％FBS(5％CO 2)
NCI-H69 小细胞肺癌 悬浮 RPMI 1640+10％FBS(5％CO 2)
NCI-H526 小细胞肺癌 悬浮 RPMI 1640+10％FBS(5％CO 2)
2.培养基及试剂
详细信息见表12。
表12.培养基及试剂
培养基及试剂 生产商 货号
RPMI 1640 GIBCO 22400-089
Leibovitz's L-15 SIGMA L1518
Dulbecco's PBS GIBCO 2160-051
FBS GIBCO 10091-148
Antibiotic-antimycotic GIBCO 15240-062
0.25％Trypsin GIBCO 25200072
DMSO SIGMA D2650
3.多孔板
Corning Costar 96-孔板，平底白板(带盖及透明底),#3599.
4.细胞活性实验所用试剂及仪器
(3)Promega CellTiter-Glo发光法细胞活性检测试剂盒(Promega-G7573).
(4)2104
Figure PCTCN2022105236-appb-000107
读板器，PerkinElmer.
实验方案：
1.细胞培养
将肿瘤细胞系按上述培养条件在37℃，5％CO 2或0％CO 2的培养箱中进行培养。定期传代，取处于对数生长期的细胞用于铺板
2.细胞铺板
(1).用台盼兰进行细胞染色并计数活细胞。
(2).将细胞浓度调整至合适浓度，见表13。
表13.细胞系及浓度
Cell line Density(per 96-well)
NCI-H1581 12000
DMS114 5000
NCI-H69 20000
NCI-H526 10000
(3).在培养板中每孔加入100μL细胞悬液，在空白对照空中加入不含细胞的培养液。
(4).将培养板在37℃,5％CO 2或0％CO 2，及100％相对湿度的培养箱中培养过夜。
3.化合物储液配制
制备1000X化合物储液：将化合物用DMSO溶解至10mM，再取出部分稀释至最高作用浓度的1000倍。稀释好的储液分装备用。
4. 5X化合物工作液的配制及化合物处理细胞。每次现用现配。
(1).5X化合物工作液的配制：在V形底的96孔板中加入119.4μL细胞培养液，后续梯度孔中加入96μL DMSO-细胞培养液混合液(DMSO含量为0.5％)，从1000X化合物储液中吸取0.6μL化合物加入96孔板的细胞培养液中，用排枪吹打混匀。依次用排枪吸取24μL化合物-细胞培养液混合液至后续孔中，每次吹打混匀。
(2).加药：取25μL的5X化合物工作液加入到细胞培养板中。在溶媒对照和空白对照中加入25μL DMSO-细胞培养液混合液(DMSO含量为0.5％)。
(3).将96孔细胞板放回培养箱中培养3天。
5.CellTiter-Glo发光法细胞活性检测
以下步骤按照Promega CellTiter-Glo发光法细胞活性检测试剂盒(Promega-G7573)的说明书来进行。
(1).将CellTiter-Glo缓冲液融化并放置至室温。
(2).将CellTiter-Glo底物放置至室温。
(3).在一瓶CellTiter-Glo底物中加入10mL CellTiter-Glo缓冲液以溶解底物，从而配制CellTiter-Glo工作液。
(4).缓慢涡旋震荡使充分溶解。
(5).取出细胞培养板放置30分钟使其平衡至室温。
(6).在每孔中加入50μL(等于每孔中细胞培养液一半体积)的CellTiter-Glo工作液。用铝箔纸包裹细胞板以避光。
(7).将培养板在轨道摇床上振摇2分钟以诱导细胞裂解。
(8).培养板在室温放置10分钟以稳定发光信号。
(9).在2104 EnVision读板器上检测发光信号。
6.数据分析
用下列公式来计算检测化合物的抑制率(Inhibition rate，IR)：IR(％)＝(RLU溶媒对照–RLU化合物)/(RLU溶媒对照–RLU空白对照)*100％.在Excel中计算不同浓度化合物的抑制率，然后用GraphPad Prism软件作抑制曲线图和计算相关参数，包括最小抑制率，最大抑制率及IC 50。
实验结果：测试结果见表14。
表14本发明化合物在肿瘤细胞系NCI-H1581、DMS114、NCI-H69和NCI-H526细胞系中的细胞增殖抑制作用
Figure PCTCN2022105236-appb-000108
结论：
本发明化合物在肿瘤细胞系NCI-H1581、DMS114、NCI-H69和NCI-H526中展现出优异细胞增殖的抑制作用。
实验例5：化合物的小鼠药代动力学评价
实验目的：
本研究受试动物选用C57BL/6N雄性小鼠，应用LC/MS/MS法定量测定小鼠静脉注射或口服给予测试化合物血浆中的药物浓度，以评价受试药物在小鼠体内的药代动力学特征。
实验材料：
C57BL/6N小鼠(雄性,20-30g,7～10周龄，北京维通利华)。
实验操作：
将测试化合物的澄清或悬浮溶液经尾静脉注射到C57BL/6N小鼠体内(过夜禁食)，或灌胃给予到C57BL/6N小鼠体内(过夜禁食)。静脉注射给药于0h(给药前)和给药后0.083，0.25，0.5，1，2，4，6，8，24h，经脸颊采血，每个样品采集约0.05mL，肝素钠抗凝，血浆样本采集后放置湿冰上，并于1小时之内离心分离血浆(离心条件：6000g，3分钟，2-8℃)，血液样本在分析前存放时则放于-80℃冰箱内；口服灌胃给药于0h(给药前)和给药后0.083，0.25，0.5，1，2，4，6，8，24h，经脸颊采血，每个样品采集约0.05mL，肝素钠抗凝，血浆样本采集后放置湿冰上，并于1小时之内离心分离血浆(离心条件：6000g，3分钟，2-8℃)，血液样本在分析前存放时则放于-80℃冰箱内。通过不同时间点的血药浓度数据，运用Phoenix WinNonlin8.2.0计算药代动力学参数，提供AUC 0-t、AUC 0-∞、MRT 0-∞、C max、T max、和T 1/2等参数及其平均值和标准差。
测试结果见表15。
表15 本发明化合物在小鼠中的药代动力学参数
Figure PCTCN2022105236-appb-000109
结论：本发明化合物的口服血浆系统暴露量(AUC 0-inf)较高。在啮齿动物小鼠中，其药代动力学性质较优。
实验例6：化合物的大鼠药代动力学评价
实验目的：
本研究受试动物选用SD雄性大鼠，应用LC/MS/MS法定量测定大鼠静脉注射或口服给予测试化合物不同时间点的血浆中的药物浓度，以评价受试药物在大鼠体内的药代动力学特征。
实验材料：
Sprague Dawley(SD)大鼠(雄性,200-300g,7～10周龄，北京维通利华)。
实验操作：
将测试化合物的澄清或悬浮溶液经尾静脉注射到SD大鼠体内(过夜禁食)，或灌胃给予到SD大鼠体内(过夜禁食)。静脉注射给药于0h(给药前)和给药后0.083，0.25，0.5，1，2，4，6，8，24h，经颈静脉采血，每个样品采集约0.25mL，肝素钠抗凝，血液样本采集后放置湿冰上，并于1小时之内离心分离血浆(离心条件：6000g，3分钟，2-8℃)。血浆样本在分析前存放时则放于-80℃冰箱内；口服灌胃给药于0h(给药前)和给药后0.083，0.25，0.5，1，2，4，6，8，24h，经颈静脉采血，每个样品采集约0.25mL，肝素钠抗凝，血液样本采集后放置湿冰上，并于1小时之内离心分离血浆(离心条件：6000g，3分钟，2-8℃)，血浆样本在分析前存放时则放于-80℃冰箱内。通过不同时间点的血药浓度数据，运用Phoenix WinNonlin8.2.0计算药代动力学参数，提供AUC 0-t、AUC 0-∞、MRT 0-∞、C max、T max、和T 1/2等参数及其平均值和标准差。
测试结果见表16。
表16 本发明化合物在大鼠中的药代动力学参数
Figure PCTCN2022105236-appb-000110
结论：本发明化合物的口服血浆系统暴露量(AUC 0-inf)较高。在啮齿动物大鼠中，其药代动力学性质较优。
实验例7：化合物在人三阴性乳腺癌MDA-MB-231细胞皮下异种移植肿瘤Balb/c裸小鼠模型的体内药效学研究
细胞培养：人三阴性乳腺癌MDA-MB-231细胞(ATCC-HTB-26)体外单层培养，培养条件为RPMI-1640培养基中加10％胎牛血清，37℃ 5％CO 2孵箱培养。每两天一次用胰酶-EDTA进行常规消化处理传代。当细胞饱和度为80％-90％，数量到达要求时，收取细胞，计数，接种。
实验动物：Balb/c裸小鼠,雌性，到货时为6周龄，购于上海必凯实验动物有限公司。
实验方案：
细胞接种：将0.2mL(5×10 6个)MDA-MB-231细胞(加基质胶，体积比为1:1)皮下接种于每只小鼠的右后背，肿瘤平均体积达到约110mm 3时开始分组给药。七天为一个给药周期，每天给药一次，给药间隔24小时，将实验化合物口服给药，一共给药四个周期。受试化合物WX005给药剂量分别为10mg/kg、30mg/kg和100mg/kg，WX013给药剂量分别为30mg/kg。每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计 算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用TGI(％)或相对肿瘤增殖率T/C(％)评价。相对肿瘤增殖率T/C(％)＝T RTV/C RTV×100％(T RTV：治疗组RTV平均值；C RTV：阴性对照组RTV平均值)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV＝V t/V 0，其中V 0是分组给药时(即D0)测量所得肿瘤体积，V t为某一次测量时的肿瘤体积，T RTV与C RTV取同一天数据。
实验结果：
测试结果见表17。
表17 本发明化合物在人三阴性乳腺癌MDA-MB-231细胞皮下异种移植肿瘤Balb/c裸小鼠模型测试结果
Figure PCTCN2022105236-appb-000111
溶媒：1％DMSO/20％PEG400/0.2％Tween80/78.8％H 2O。
TGI：Tumor Growth Inhibition(肿瘤增长抑制率)。TGI(％)＝[1–(某处理组给药结束时平均瘤体积—该处理组给药时平均瘤体积)/(溶剂对照组结束治疗时平均瘤体积—溶剂对照组开始治疗时平均瘤体积)]×100％。
结论：
本发明化合物WX005和WX013在人三阴性乳腺癌MDA-MB-231异种移植瘤模型上的体内药效展示了显著的缩瘤作用。

Claims (14)

  1. 式(ⅠI)所示的化合物或其药学上可接受的盐，
    Figure PCTCN2022105236-appb-100001
    其中，
    环A选自苯并呋喃基、苯并异噁唑基、苯并三氮唑基、萘并呋喃基、萘并异噁唑基和萘并三氮唑基；
    L 1选自键、-C(R a)(R b)-、-N(R c)-和-OCH 2-；
    L 2选自-CH 2-、-C 1-6烷基-C(＝O)NH-和-C 1-3烷基-O-；
    各R 1分别独立地选自F、Cl、Br、I、C 1-3烷基、C 1-3烷氧基、5元杂芳基和
    Figure PCTCN2022105236-appb-100002
    所述C 1-3烷基、C 1-3烷氧基、5元杂芳基和
    Figure PCTCN2022105236-appb-100003
    任选被1、2或3个R d取代；
    m选自0、1、2和3；
    各R a和R b分别独立地选自H、F、Cl、Br和I；
    R c选自H和CH 3；
    各R d分别独立地选自F、Cl、Br、I、OCH 3、N(CH 3) 2和吗啉基。
  2. 根据权利要求1所述化合物或其药学上可接受的盐，其中，结构单元
    Figure PCTCN2022105236-appb-100004
    选自
    Figure PCTCN2022105236-appb-100005
    Figure PCTCN2022105236-appb-100006
  3. 根据权利要求2所述化合物或其药学上可接受的盐，其中，结构单元
    Figure PCTCN2022105236-appb-100007
    选自
    Figure PCTCN2022105236-appb-100008
  4. 根据权利要求1所述化合物或其药学上可接受的盐，其中，环A选自苯并呋喃基和苯并异噁唑基。
  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 PCTCN2022105236-appb-100009
    Figure PCTCN2022105236-appb-100010
  6. 根据权利要求1～3任意一项所述化合物或其药学上可接受的盐，其中，结构单元
    Figure PCTCN2022105236-appb-100011
    选自
    Figure PCTCN2022105236-appb-100012
    #端连接于式(ⅠI)的苯基。
  7. 根据权利要求1～3任意一项所述化合物或其药学上可接受的盐，其中，L 2选自-CH 2-、-(CH 2) 6-C(＝O)NH-和-CH 2CH 2O-。
  8. 根据权利要求1～3任意一项所述化合物或其药学上可接受的盐，其中，各R 1分别独立地选自F、Cl、Br、I、CH 3、OCH 3、OCH 2CH 3、噻唑基和
    Figure PCTCN2022105236-appb-100013
    所述CH 3、OCH 3、OCH 2CH 3、噻唑基和
    Figure PCTCN2022105236-appb-100014
    任选被1、2或3个R d取代。
  9. 根据权利要求8所述化合物或其药学上可接受的盐，其中，各R 1分别独立地选自F、Cl、CH 3、-OCH 2CH 2OCH 3、-OCH 2CH 2N(CH 3) 2、
    Figure PCTCN2022105236-appb-100015
  10. 根据权利要求1～3任意一项所述化合物或其药学上可接受的盐，其中，结构单元
    Figure PCTCN2022105236-appb-100016
    选自
    Figure PCTCN2022105236-appb-100017
    Figure PCTCN2022105236-appb-100018
  11. 根据权利要求1～3任意一项所述化合物或其药学上可接受的盐，其中，结构单元
    Figure PCTCN2022105236-appb-100019
    选自
    Figure PCTCN2022105236-appb-100020
  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，其化合物选自
    Figure PCTCN2022105236-appb-100021
    其中，
    T 1选自CH和N；
    R 1、L 1和m如权利要求1～11任意一项所定义。
  13. 下式化合物或其药学上可接受的盐
    Figure PCTCN2022105236-appb-100022
    Figure PCTCN2022105236-appb-100023
  14. 根据权利要求13所述化合物或其药学上可接受的盐，其化合物选自
    Figure PCTCN2022105236-appb-100024
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