WO2023165373A1 - Micromolecular pcsk9 protein degradation agent, preparation method therefor, and use thereof - Google Patents

Micromolecular pcsk9 protein degradation agent, preparation method therefor, and use thereof Download PDF

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WO2023165373A1
WO2023165373A1 PCT/CN2023/077334 CN2023077334W WO2023165373A1 WO 2023165373 A1 WO2023165373 A1 WO 2023165373A1 CN 2023077334 W CN2023077334 W CN 2023077334W WO 2023165373 A1 WO2023165373 A1 WO 2023165373A1
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chain
membered
pcsk9
alkyl
cycloalkyl
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PCT/CN2023/077334
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French (fr)
Chinese (zh)
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古险峰
欧阳志蓉
郭薇
马牧野
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复旦大学
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    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
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Definitions

  • the invention belongs to the field of medicinal chemistry, and specifically relates to a PCSK9 small-molecule protein degradation agent and a preparation method thereof.
  • This type of compound has the effect of degrading PCSK9 or LC3, and is expected to be used in the preparation of drugs for preventing or treating diseases related to the inhibition of PCSK9 or LC3 activity. drug.
  • Proprotein convertase subtilisin 9 is a serine protease mainly expressed in the liver discovered by Nabil Seidah et al. in 2003.
  • low-density lipoprotein cholesterol (LDL-C) in the blood mainly enters the cell by combining with low-density lipoprotein receptors (LDLRs) on the cell surface to form a complex, and the low pH condition of the endosome causes LDL Separated from its receptor, LDL is degraded in lysosomes, while LDLR is recycled to the cell surface to participate in the next round of LDL-C clearance.
  • LDL-C low-density lipoprotein cholesterol
  • LDLRs low-density lipoprotein receptors
  • PCSK9 binds to LDLRs, causing LDLRs and LDL-C to degrade simultaneously in the cell, thereby inhibiting the ability of LDLR to clear low-density lipoprotein cholesterol (LDL-C) in the blood.
  • inhibiting the production or secretion of PCSK9 can reduce blood cholesterol levels, and PCSK inhibitors can be used to treat hypercholesterolemia.
  • PCSK9 inhibitors of PCSK9 show good lipid-lowering activity
  • two monoclonal antibodies alirocumab and evolocumab and small interfering RNA Inclisiran are three examples of PCSK9 inhibitors that have been used clinically for the treatment of cardiovascular and cerebrovascular diseases .
  • Various PCSK9 inhibitors have been disclosed for the treatment or prevention of cardiovascular and cerebrovascular diseases, alopecia diseases, vitiligo, abnormal keratinization diseases, scars and pulmonary fibrosis diseases, metabolic syndrome, obesity, diabetes, Alzheimer's disease , anti-rejection, non-alcoholic hepatitis and malignant tumors.
  • CN113876955A WO2022002160 A1, WO2021052472 A1, CN113332423A, CN113663075A, WO2021243002 A1, WO2021207712 A1, WO2021154947 A1, WO202114376 2 A1, CN112083163A, WO2020229718 A1, CN113412258A, CN111154760A, WO2020252383A1, WO2018057409 A1.
  • PCSK9 inhibitors used clinically, so small molecule inhibitors of PCSK9 are developed and used for the treatment or prevention of cardiovascular and cerebrovascular diseases, alopecia diseases, vitiligo, abnormal keratinization diseases, scars and pulmonary fibrosis It is of great significance in cancer diseases, metabolic syndrome, obesity, diabetes, Alzheimer's disease, anti-rejection, non-alcoholic hepatitis and malignant tumors.
  • LC3 protein As a marker protein in the autophagy process, LC3 protein not only mediates the autophagy process, but also is related to the formation and fusion of autophagosomes.
  • LC3 protein and autophagy-related proteins is mainly through protein-protein (Protein-protein interaction) interaction, the interface is relatively smooth, and there is no binding pocket for small molecule drugs. Therefore, few small molecule inhibitors of LC3 have been reported so far.
  • the object of the present invention is to provide a tetrahydroisoquinoline compound as a novel PCSK9 small molecule protein degrading agent or LC3 small molecule protein degrading agent, which is used to reduce the level of PCSK9 or LC3, and can be used for the preparation of prevention or treatment and inhibition of PCSK9 or Drugs for diseases associated with LC3 activity.
  • the present invention provides a PCSK9 small molecule protein degradation agent, which is a tetrahydroisoquinoline compound or a pharmaceutically acceptable salt or solvate thereof of the general formula I as follows: :
  • R 1 , R 2 , R 3 and R 4 are independently selected from hydrogen, halogen, hydroxyl, nitro, methoxy, amino, methylamino, dimethylamino, carboxyl, cyano, -CO 2 Me, -CO 2 Et, -CH 3 , -Et, -N 3 , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, wherein the alkyl, alkenyl or alkynyl is optionally replaced by one Or more selected from -O(C 1-6 alkyl), -O(C 3-6 cycloalkyl), -O(C 1-4 alkylene-C 3-6 cycloalkyl), -O(three One-membered to seven-membered heterocyclyl), -O(C 1-4 alkylene-three-membered to seven-membered heterocyclyl), -SH, -S(C 1-6 alkyl), -S(C 3- 6 cycl
  • R 5 is independently selected from hydrogen, methoxy, methyl, halogen, hydroxyl, nitro, amino, methylamino, dimethylamino, carboxyl, cyano, -CO 2 Me, -CO 2 Et, -CH 3 , -Et, -N 3 , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl;
  • R 6 is independently selected from methyl
  • R 7 is independently selected from the following structures:
  • Group B is:
  • R 8 , R 9 and R 10 are independently selected from hydrogen, halogen, hydroxyl, nitro, methoxy, amino, methylamino, dimethylamino, carboxyl, cyano, -CO 2 Me, -CO 2 Et, -CH 3 , -Et, -N 3 ;
  • X is NH, S, O.
  • said R 7 is
  • the group B is R 8 , R 9 and R 10 are independently selected from halogen, hydroxy, methoxy.
  • the compound is selected from the following compounds:
  • the present invention provides a method for preparing the PCSK9 small molecule protein degradation agent as described above, comprising the following steps:
  • R 2 , R 5 , R 8 , and R 10 are as defined above.
  • the present invention provides a pharmaceutical composition, comprising the above-mentioned tetrahydroisoquinoline compound or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.
  • the present invention provides a tetrahydroisoquinoline compound as described above or a pharmaceutically acceptable salt or solvate thereof in the preparation of a drug for preventing or treating a disease related to the inhibition of PCSK9 activity application.
  • the present invention provides a tetrahydroisoquinoline compound as described above or a pharmaceutically acceptable salt or solvate thereof in the preparation of a drug for preventing or treating diseases related to the inhibition of LC3 activity application.
  • the disease is selected from cardiovascular and cerebrovascular diseases, hair loss diseases, vitiligo, abnormal keratinization diseases, scar scar and at least one of pulmonary fibrotic disease, metabolic syndrome, obesity, diabetes, Alzheimer's disease, anti-rejection, nonalcoholic hepatitis, and malignancy.
  • the cardiovascular and cerebrovascular diseases are selected from at least one of cholesterol-related diseases, hyperlipidemia and atherosclerosis;
  • the malignant tumors are selected from leukemia, prostate cancer, thyroid cancer, liver cancer, ovarian cancer, breast cancer Cancer, esophageal cancer, adenocarcinoma, gastric cancer, lung cancer and colorectal cancer.
  • Fig. 1 shows the Western Blot experimental result of the compound prepared according to the embodiments of the present invention
  • FIG. 2 shows the Western Blot experimental results of compounds I-2 and I-4 prepared according to the embodiments of the present invention
  • Fig. 3 shows the qPCR experiment result of the compound 1-3 prepared according to the embodiment of the present invention
  • Figure 4 shows the MTT toxicity test results of compound 1-3 prepared according to the embodiments of the present invention.
  • Fig. 5 shows the Western Blot experiment result of the compound 1-3 prepared according to the embodiment of the present invention in the hyperlipidemia model
  • Fig. 6 shows the Western Blot experiment results when compound I-3 is prepared according to the embodiment of the present invention and used in combination with statins.
  • Fig. 7 shows the Western Blot experimental results of compounds I-1 to I-15 prepared according to the embodiments of the present invention.
  • the present invention provides a novel PCSK9 small-molecule targeted degradation agent, which has a tetrahydroisoquinoline compound of the following general formula I or a pharmaceutically acceptable salt or solvate thereof:
  • R 1 , R 2 , R 3 and R 4 are independently selected from hydrogen, halogen, hydroxyl, nitro, methoxy, amino, methylamino, di Methylamino, carboxyl, cyano, -CO 2 Me, -CO 2 Et, -CH 3 , -Et, -N 3 , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, Wherein said alkyl, alkenyl or alkynyl is optionally replaced by one or more selected from -O(C 1-6 alkyl), -O(C 3-6 cycloalkyl), -O(C 1-4 Alkylene-C 3-6 cycloalkyl), -O (three-membered to seven-membered heterocyclic group), -O (C 1-4 alkylene-three-membered to seven-membered heterocyclic group), -SH, - S (C 1-6 alkyl), -S (C 3-6
  • R 5 is independently selected from hydrogen, methoxy, methyl, halogen, hydroxyl, nitro, amino, methylamino, dimethylamino, carboxyl, cyano, -CO 2 Me, -CO 2 Et, -CH 3 , -Et, -N 3 , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl;
  • R 6 is independently selected from methyl
  • R 7 is independently selected from the following structures:
  • Group B is:
  • R 8 , R 9 and R 10 are independently selected from hydrogen, halogen, hydroxyl, nitro, methoxy, amino, methylamino, dimethylamino, carboxyl, cyano, -CO 2 Me, -CO 2 Et, -CH 3 , -Et, -N 3 ;
  • X is NH, S, O.
  • said R 7 is
  • the group B is R 8 , R 9 and R 10 alone is selected from halogen, hydroxy, methoxy.
  • the compound is selected from the following compounds:
  • R 2 , R 5 , R 8 , and R 10 are as defined above.
  • mn refers to the range from m to n and the subranges and individual point values therein.
  • C1-C20 or “C1-20” covers the range of 1-20 carbon atoms, and should be understood to also cover any sub-ranges therein and each point value, such as C2-C5, C3-C4, C1-C2, C1-C3, C1-C4, C1-C5, C1-C6 etc., and C1, C2, C3, C4, C5, C6, C7, C8, etc.
  • C3-C10 or “C3-10” should also be understood in a similar manner, for example to cover any subranges and point values contained therein, such as C3-C9, C6-C9, C6-C8, C6 -C7, C7-C10, C7-C9, C7-C8, C8-C9, etc. and C3, 4, 5, 6, 7, 8, 9, 10, etc.
  • Other similar expressions herein should also be read in a similar manner.
  • halo or "halogen” or “halo” is understood to mean a fluorine (F), chlorine (Cl), bromine (Br) or iodine (I) atom.
  • alkyl refers to a straight or branched chain saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms attached to the rest of the molecule by a single bond.
  • Alkyl can have 1-20 carbon atoms, that is, “C 1 -C 20 alkyl”, such as C 1-4 alkyl, C 1-3 alkyl, C 1-2 alkyl, C 3 alkyl , C 4 alkyl, C 1-6 alkyl, C 3-6 alkyl.
  • alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2- Methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-di Methylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or their isomers body.
  • Subunit refers to a group obtained by removing a hydrogen atom from a carbon atom containing free valence electrons, and having two attachment sites for attachment to the rest of the molecule.
  • alkylene or “alkylene” refers to a saturated straight or branched chain divalent hydrocarbon group.
  • alkylene when used herein alone or in combination with other groups, refers to a linear or branched saturated divalent hydrocarbon group.
  • C 1-20 alkylene refers to an alkylene group having 1-20 carbon atoms, such as methylene, ethylene, propylene, butylene, pentylene, hexylene, 1- Methylethylene, 2-methylethylene, methylpropylene or ethylpropylene, etc.
  • cycloalkylene refers to a cyclic saturated divalent hydrocarbon group.
  • C 3-6 cycloalkylene refers to a cycloalkylene group having 3-6 carbon atoms, such as cyclopropylidene, cyclobutylene, cyclopentylylene, cyclohexylylene Kei, wait.
  • alkoxyylene refers to "-O-alkylene” or "alkylene-O-”.
  • Examples of "C 1-8 alkoxylidene” include, but are not limited to, -O-methylene, -O-ethylene, -O-propylene, -O-butylene, methylene-O- , Ethylene-O-, Propylene-O-, Butylene-O-, etc.
  • alkenyl refers to a straight or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one double bond. Alkenyl can have 2-20 carbon atoms, namely " C2-20 alkenyl", such as C2-4 alkenyl, C3-4 alkenyl.
  • Non-limiting examples of alkenyl include, but are not limited to, vinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, (E)-but-2-enyl , (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl and the like.
  • alkynyl refers to a straight or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, having at least one triple bond.
  • the alkynyl group may have 2-20 carbon atoms, ie " C2-8alkynyl ", such as C2-4alkynyl , C3-4alkynyl .
  • radicals include, but are not limited to, ethynyl, prop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl, but-3-ynyl, and the like.
  • cyclic hydrocarbon group refers to a saturated or unsaturated non-aromatic cyclic hydrocarbon group composed of carbon atoms and hydrogen atoms, preferably containing 1 or 2 rings.
  • the cyclic hydrocarbon group may be a monocyclic, fused polycyclic, bridged or spiro ring structure.
  • Cycloalkyl can have 3-10 carbon atoms, that is, "C 3-10 cycloalkyl", such as C 3-8 cycloalkyl, C 3-6 cycloalkyl, C 5 cycloalkyl, C 6 cycloalkyl, C 7 cycloalkyl .
  • Non-limiting examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl, spiro[3.3]heptyl, and the like.
  • cycloalkyl refers to a saturated cyclic hydrocarbon group.
  • heterocyclyl or “heterocycloalkyl” refers to a monocyclic ring having, for example, 3-10 (suitably 3-8, more suitably 3-7, especially 4-6) ring atoms Or a bicyclic ring system (three to ten, three to eight, three to seven, four to six), wherein at least one ring atom (eg, 1 or 3) is selected from N, O, heteroatoms of S and P, and the remaining ring atoms are C.
  • the ring system may be saturated (also understood as the corresponding "heterocycloalkyl") or unsaturated (ie have one or more double and/or triple bonds within the ring).
  • the heterocyclic group can be, for example, a four-membered ring, such as azetidinyl, oxetanyl; or a five-membered ring, such as tetrahydrofuranyl, dioxanyl, pyrrolidinyl, imidazolidinyl, pyrazolidine Base, pyrrolinyl, oxopyrrolidinyl, 2-oxoimidazolidin-1-yl; or six-membered rings, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thio Morpholinyl, piperazinyl, 1,1-dioxo-1,2-thiazidin-2-yl or trithianyl; or seven-membered rings such as diaza base ring.
  • the heterocyclyl group may be benzo-fused.
  • the heterocyclyl group may be bicyclic without limitation, for example a five-membered five-membered ring such as a hexahydrocyclopentane[C]pyrrol-2(1H)-yl) ring; or a five-membered six-membered bicyclic ring such as Hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl ring.
  • a heterocycle may be unsaturated, i.e. it may contain one or more double bonds without limitation, for example an unsaturated heterocycle containing a nitrogen atom may be 1,6-dihydropyrimidine , 1,2-dihydropyrimidine, 1,4-dihydropyrimidine, 1,6-dihydropyridine, 1,2-dihydropyridine, 1,4-dihydropyridine, 2,3-dihydro-IH- Pyrrole, 3,4-dihydro-IH-pyrrole, 2,5-dihydro-IH-pyrrolyl, 4H-[1,3,4]thiadiazinyl, 4,5-dihydrooxazolyl or 4H -[1,4]thiazinyl ring, the unsaturated heterocyclic ring containing oxygen atom can be 2H-pyran, 4H-pyran, 2,3-dihydrofuran, the unsaturated heterocyclic ring containing sulfur atom can be Is
  • hydrocarbon chain refers to a chain-like group composed of carbon atoms and hydrogen atoms, which may be straight or branched. said The hydrocarbon chain may be saturated (ie, an alkylene group) or unsaturated, ie, may contain one or more carbon-carbon double or triple bonds.
  • Non-limiting examples of alkylene include, but are not limited to, methylene (-CH 2 -), 1,1-ethylene (-CH(CH 3 )-), 1,2-ethylene (-CH 2 CH 2 -), 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -), 1,7-heptylene (-CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -), etc.
  • the "compound” in the present invention includes all stereoisomers, geometric isomers, tautomers and isotopes.
  • the "compound” of the present invention may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all isomers are included, eg, enantiomers and diastereomers.
  • Compounds containing asymmetric carbon atoms in the present invention can be isolated in optically pure or racemic forms; optically pure forms can be resolved from racemic mixtures, or by using chiral raw materials or chiral reagents synthesis.
  • the "compound” mentioned in the present invention also includes geometric isomers; the geometric isomers may exist as mixtures or separated E or Z structures.
  • the "compound” in the present invention also includes tautomeric forms; tautomeric forms originate from the exchange of a single bond with an adjacent double bond accompanied by the migration of a proton.
  • the "compound” of the present invention also includes atoms of all isotopes, no matter it is an intermediate or a final compound; atoms of isotopes include those with the same number of protons but different mass numbers, for example, isotopes of hydrogen include deuterium and tritium. Also, if desired, for example for specific therapeutic or diagnostic treatments, the compounds of the invention may incorporate isotopes or radioactive isotopes known in the art, eg 3H, 15O, 13C or 15N isotopes.
  • “Pharmaceutically acceptable salt” refers to a pharmaceutically acceptable salt, which can improve the physicochemical or metabolic properties while maintaining the pharmacological activity of its parent compound.
  • Such salts include acid addition salts or base addition salts prepared from pharmaceutically acceptable acids or bases (including organic acids, inorganic acids, organic bases, inorganic bases), or a mixture of both.
  • suitable inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid or similar acids
  • suitable organic acids include fumaric acid, tartaric acid, lactic acid, acetic acid, citric acid, trifluoromethanesulfonic acid, Mandelic acid, salicylic acid or their analogs.
  • the compounds according to the invention may also exist in the form of solvates.
  • hydrates hemihydrate, monohydrate, dihydrate, trihydrate, etc.
  • the compounds of the present invention can be administered in an effective amount by any acceptable means of administration for other similar uses.
  • the compounds of the present invention can be administered orally, parenterally, transdermally, topically, rectally, intraperitoneally or intranasally.
  • the compounds of the invention are usually administered in the form of pharmaceutical compositions.
  • These compositions may be prepared by methods well known in the art of pharmacy and contain at least one active compound.
  • the active ingredient is usually mixed with an excipient, diluted by an excipient or enclosed in a capsule, sachet, paper or other form of container.
  • the excipient can be a solid, semi-solid, or liquid substance, and can be used as a carrier, carrier or medium for the active ingredient.
  • the composition may be a tablet, pill, powder, lozenge, sachet, capsule, elixir, suspension, emulsion, solution, syrup, spray (as a solid or in a liquid medium), ointment, soft and In the form of hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
  • excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, sterile water, syrup and methylcellulose. Additionally, lubricants (such as talc, magnesium stearate, and mineral oil), wetting agents, emulsifying and suspending agents, preservatives (such as methylparaben and propylparaben), sweetening agents, and agents and flavor enhancers.
  • the pharmaceutical composition of the present invention can achieve rapid, sustained or delayed release of the pharmaceutical active ingredient after being administered to the patient through a specific excipient, which is also a method widely used in the field.
  • the active ingredient that is, the compound of the present invention
  • the amount in the pharmaceutical composition and unit dosage form can be changed or adjusted greatly according to the specific application, the activity of the specific compound and the expected concentration.
  • Treatment means any treatment of a disease in a mammal, including: (1) preventing the disease, that is, causing the symptoms of the clinical disease to not develop; (2) inhibiting the disease, that is, preventing the development of clinical symptoms; (3) alleviating the disease, That is to cause the subsidence of clinical symptoms.
  • Embodiment one the preparation of compound I-1
  • the coumarin was dissolved in nitromethane and ammonium acetate was added. After the dissolution was complete, the reaction liquid was moved to 100°C, and after 2 hours of reaction, it was moved to room temperature. Then there is solid precipitation, and when the reaction solution is completely down to room temperature, the precipitated solid is directly filtered, and a small amount of methanol is used to rinse the filter cake, and the filter cake is spin-dried to obtain a yellow solid P1.
  • Step 4 Synthesis of (E)-3-((4-hydroxy-3-methoxyphenethyl)amino)but-2-enoic acid ethyl ester
  • Step 5 Synthesis of ethyl 2-(7-hydroxy-6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)acetate
  • Step 7 2-(2-(tert-butoxycarbonyl)-7-hydroxy-6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)acetic acid Synthesis
  • Step 8 tert-Butyl 7-hydroxy-6-methoxy-1-methyl-1-(2-oxo-2-(thiazol-2-ylamino)ethyl)-3,4-dihydroiso Synthesis of quinoline-2(1H)-carboxylate
  • Step 11 Synthesis of 5-bromo-3-(3-bromo-4-(2-(2-bromoethoxy)ethoxy)-5-methoxybenzylidene)indol-2-one
  • AN-OCH3 was dissolved in anhydrous DMF at room temperature. After reacting for 0.5 hours, 2,2'-dibromodiethyl ether was added and the reaction was continued for 2 hours. After the reaction was completed, the reaction was quenched with ice water, the reaction solution was extracted with EA, and spin-dried An orange-red oily substance can be obtained. Subsequent purification using PE:EA gave AN-OCH3-O5.
  • Step 12 tert-butyl 7-(2-(5-bromo-3-(3-bromo-4-hydroxy-5-methoxybenzylidene)-2-oxoindol-1-yl)ethoxy Base)-6-methoxy-1-methyl-1-(2-oxo-2-(thiazol-2-ylamino)ethyl)-3,4-dihydroisoquinoline-2(1H) -Synthesis of Carboxylate
  • Step 13 2-(7-(2-(2-(5-bromo-3-(3-bromo-4-hydroxy-5-methoxybenzylidene)-2-oxoindol-1-yl Synthesis of )ethoxy)-6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide
  • Embodiment two the preparation of compound 1-2
  • Embodiment three the preparation of compound I-3
  • Embodiment four the preparation of compound I-4
  • Embodiment five the preparation of compound 1-5
  • Embodiment six the preparation of compound I-6
  • Embodiment seven the preparation of compound I-7
  • Embodiment eight the preparation of compound I-8
  • Embodiment nine the preparation of compound I-9
  • Embodiment 10 Preparation of Compound I-10
  • Embodiment 11 Preparation of Compound I-11
  • Embodiment 12 Preparation of Compound I-12
  • Embodiment 13 Preparation of Compound I-13
  • Embodiment 14 Preparation of Compound I-14
  • Embodiment 15 Preparation of Compound I-15
  • Example 16 Determination of the target degradation activity of the compound on PCSK9 at the cell level, the compounds I-1 to I-4 of the present invention were tested by using Western Blot experiments, namely Western blot experiments and cellular immunofluorescence experiments to determine the targeting of the compounds to PCSK9 Degradability.
  • the targeted degradation ability is mainly expressed by two indicators: the maximum degradation degree of PCSK9 protein and the optimal degradation concentration of the compound.
  • the timed quantitative PCR experiment proves that the compound of the present invention has no obvious effect on the expression level of SREBP2-PCSK9-LDLR mRNA, and only degrades PCSK9 at the protein level. And the determination of LDLR and LC3B and other protein levels to support.
  • simvastatin Sigma
  • LDLR antibody was purchased from Abcam
  • PCSK9 and LC3B antibodies were purchased from Cell Signaling Technologies
  • GAPDH antibody was purchased from Bioworld
  • all other reagents were purchased from Sigma Aldrich.
  • the cells were treated with drugs, they were lysed with RIPA (Beyotime) on ice for 30 minutes, and the supernatant was obtained by high-speed centrifugation. After the protein concentration was measured by the BCA method (Beyotime) and balanced to the same concentration, 5X Loading Buffer was added and boiled for 5 minutes to prepare the protein. The samples were used for western blot experiments.
  • Western blotting experiments mainly included separation of proteins by polyacrylamide gel electrophoresis, transfer to PVDF membrane, blocking with 5% skimmed milk powder in TBST solution for 30 minutes, incubation of primary antibody solution at 4°C overnight, incubation of secondary antibody at room temperature for 60 minutes, developing solution (Millipore ) were visualized and recorded using a gel imaging system.
  • Fig. 1 shows the Western Blot experiment result of the compound prepared according to the embodiment of the present invention. It can be seen from Figure 1 that compounds I-1 to I-4 can all reduce PCSK9 at the protein level on the Huh7 cell line, and have an obvious hook-like effect, and the relative effect of the drug I-3 is better. However, the incubation experiment of Huh7 with the linker compounds proved that the linker compounds AN-Br, AN-OCH3 and P8 did not reduce the activity of PCSK9 alone. It shows that the compound of the present invention has better degradation effect on PCSK9 protein.
  • FIG. 2 shows the Western Blot experimental results of compounds I-2 and I-4 prepared according to the embodiments of the present invention.
  • both compounds I-2 and I-4 can produce the effect of reducing LC3 from the protein level to the Huh7 cell line, and have an obvious concentration-dependent effect, indicating that compounds I-2 and I-4 of the present invention It has a good degradation effect on LC3 protein.
  • Primers for real-time quantitative PCR were synthesized by Sangon Inc, TRIZOL was purchased from TAKARA, and other real-time quantitative PCR related reagents were purchased from Yeason.
  • the real-time quantitative PCR results were normalized according to GAPDH.
  • the primer sequences are as follows:
  • LDLR Forward 5'-GACGTGGCGTGAACATCTG-3';
  • PCSK9 Forward, 5'-AGGGGAGGACATCATTGGTG-3';
  • SREBP2 Forward, 5'-CCCTGGGAGACATCGACGA-3';
  • GAPDH Forward, 5'-ATGGGGAAGGTGAAGGTCG-3';
  • Fig. 3 shows the qPCR experimental results of compound I-3 prepared according to the embodiment of the present invention. It can be seen from Figure 3 that compound I-3 has no significant effect on the expression level of SREBP2-PCSK9-LDLR mRNA, and only has an effect on PCSK9 at the protein level.
  • Fig. 4 shows the MTT toxicity test results of compound I-3 prepared according to the embodiment of the present invention. It can be seen from Figure 4 that compound I-3 has no obvious cytotoxicity.
  • Fig. 5 shows the Western Blot experimental results of compound I-3 prepared according to an embodiment of the present invention in a hyperlipidemia model. It can be seen from Figure 5 that PCSK9 increases under the high-fat inflammatory environment simulated by palmitic acid and LPS, and the compound I-3 drug can reduce the PCSK9 content and increase LDLR. Possibility of treating patients with the disease.
  • Fig. 6 shows the Western Blot experimental results when compound I-3 prepared according to the embodiment of the present invention is used in combination with statins.
  • Compound I-3 can produce a synergistic effect with statins—simvastatin, reduce PCSK9 elevation caused by statins, and further increase the protein content of LDLR.
  • statins statins
  • the experimental results show that compound I-3 and statins The possibility of combined use as a therapeutic drug for patients with hyperlipidemia.
  • Fig. 7 shows the Western Blot experimental results of compounds I-1 to I-15 prepared according to the embodiments of the present invention, and compounds I-1, I-8, and I-15 have better PCSK9 degradation effects. It shows that the compound of the present invention has better degradation effect on PCSK9 protein.

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Abstract

Provided are a micromolecular PCSK9 protein degradation agent and a preparation method therefor. The micromolecular PCSK9 inhibitor is a tetrahydroisoquinoline compound having a chemical structure represented by formula (I), or a pharmaceutically acceptable salt or solvate thereof. The compound has the effect of degrading PCSK9 or degrading LC3, and can be used in preparing a medicament for preventing or treating a PCSK9 or LC3 activity-related disease.

Description

一种PCSK9小分子蛋白降解剂及其制备方法和应用A kind of PCSK9 small molecule protein degradation agent and its preparation method and application 技术领域technical field
本发明属于药物化学领域，具体涉及一种PCSK9小分子蛋白降解剂及其制备方法，这类化合物具有降解PCSK9或降解LC3的作用，有望于制备预防或治疗与抑制PCSK9或LC3活性相关的疾病的药物。The invention belongs to the field of medicinal chemistry, and specifically relates to a PCSK9 small-molecule protein degradation agent and a preparation method thereof. This type of compound has the effect of degrading PCSK9 or LC3, and is expected to be used in the preparation of drugs for preventing or treating diseases related to the inhibition of PCSK9 or LC3 activity. drug.
背景技术Background technique
前蛋白转化酶枯草溶菌素9(PCSK9)是2003年Nabil Seidah等人发现的一种主要在肝脏表达的丝氨酸蛋白酶。正常生理情况下，血液中的低密度脂蛋白胆固醇(LDL-C)主要是通过与细胞表面的低密度脂蛋白受体(LDLRs)结合形成复合物进入细胞，核内体的低pH条件导致LDL与其受体分离，LDL在溶酶体中降解，而LDLR则循环到细胞表面，参与下一轮的LDL-C清除工作。而PCSK9通过与LDLRs结合，使得LDLRs与LDL-C在胞内同时发生降解，进而抑制了LDLR循环清除血中低密度脂蛋白胆固醇(LDL-C)的能力。一般来说，抑制PCSK9的生成或分泌可以降低血中胆固醇水平，PCSK的抑制剂可用于治疗高胆固醇血症。Proprotein convertase subtilisin 9 (PCSK9) is a serine protease mainly expressed in the liver discovered by Nabil Seidah et al. in 2003. Under normal physiological conditions, low-density lipoprotein cholesterol (LDL-C) in the blood mainly enters the cell by combining with low-density lipoprotein receptors (LDLRs) on the cell surface to form a complex, and the low pH condition of the endosome causes LDL Separated from its receptor, LDL is degraded in lysosomes, while LDLR is recycled to the cell surface to participate in the next round of LDL-C clearance. PCSK9 binds to LDLRs, causing LDLRs and LDL-C to degrade simultaneously in the cell, thereby inhibiting the ability of LDLR to clear low-density lipoprotein cholesterol (LDL-C) in the blood. In general, inhibiting the production or secretion of PCSK9 can reduce blood cholesterol levels, and PCSK inhibitors can be used to treat hypercholesterolemia.
一般而言，PCSK9的抑制剂显示出良好的降脂活性，alirocumab和evolocumab两种单克隆抗体和小干扰RNA Inclisiran是已经在临床上使用的三个PCSK9抑制剂实例，用于治疗心脑血管疾病。已经公开了各种PCSK9抑制剂用于治疗或预防心脑血管疾病、脱发性疾病、白癜风、角化异常性疾病、疤痕以及肺纤维化疾病、代谢综合征、肥胖、糖尿病、阿尔茨海默症、抗排斥反应、非酒精性肝炎以及恶性肿瘤。例如CN113876955A、WO2022002160 A1、WO2021052472 A1、CN113332423A、CN113663075A、WO2021243002 A1、WO2021207712 A1、WO2021154947 A1、WO2021143762 A1、CN112083163A、WO2020229718 A1、CN113412258A、CN111154760A、WO2020252383A1、WO2018057409 A1。In general, inhibitors of PCSK9 show good lipid-lowering activity, two monoclonal antibodies alirocumab and evolocumab and small interfering RNA Inclisiran are three examples of PCSK9 inhibitors that have been used clinically for the treatment of cardiovascular and cerebrovascular diseases . Various PCSK9 inhibitors have been disclosed for the treatment or prevention of cardiovascular and cerebrovascular diseases, alopecia diseases, vitiligo, abnormal keratinization diseases, scars and pulmonary fibrosis diseases, metabolic syndrome, obesity, diabetes, Alzheimer's disease , anti-rejection, non-alcoholic hepatitis and malignant tumors. For example CN113876955A, WO2022002160 A1, WO2021052472 A1, CN113332423A, CN113663075A, WO2021243002 A1, WO2021207712 A1, WO2021154947 A1, WO202114376 2 A1, CN112083163A, WO2020229718 A1, CN113412258A, CN111154760A, WO2020252383A1, WO2018057409 A1.
目前获批临床的三种药物均为生物药，治疗成本较大，给药途径不方便，对于患者而言，依从性较差。目前尚无小分子的PCSK9抑制剂应用于临床，因此开发PCSK9的小分子抑制剂并将其用于治疗或预防心脑血管疾病、脱发性疾病、白癜风、角化异常性疾病、疤痕以及肺纤维化疾病、代谢综合征、肥胖、糖尿病、阿尔茨海默症、抗排斥反应、非酒精性肝炎以及恶性肿瘤具有重要意义。The three drugs currently approved for clinical use are all biological drugs, which have high treatment costs, inconvenient administration routes, and poor compliance for patients. At present, there is no small molecule PCSK9 inhibitor used clinically, so small molecule inhibitors of PCSK9 are developed and used for the treatment or prevention of cardiovascular and cerebrovascular diseases, alopecia diseases, vitiligo, abnormal keratinization diseases, scars and pulmonary fibrosis It is of great significance in cancer diseases, metabolic syndrome, obesity, diabetes, Alzheimer's disease, anti-rejection, non-alcoholic hepatitis and malignant tumors.
自噬长期以来一直与调节免疫反应的多个方面有关，包括病原体捕获，代谢调节和 细胞稳态等。目前多项研究表明细胞自噬与代谢性疾病、恶性肿瘤、免疫失调以及神经退行性疾病等的发生发展有关。LC3蛋白作为细胞自噬过程中的标志物蛋白，不仅介导了自噬过程，还与自噬小体的形成和融合等过程有关。但是，LC3蛋白与自噬相关蛋白的作用主要通过蛋白-蛋白Protein-protein interaction)相互作用发挥功能，界面比较平整，缺乏小分子药物的结合口袋。因此，目前很少报道LC3的小分子抑制剂。2021年，中科院上海药物研究所研究员罗成等研究员首次报道了与LC3共价结合的小分子抑制剂。因此开发LC3抑制剂具有重要意义。Autophagy has long been implicated in regulating multiple aspects of the immune response, including pathogen capture, metabolic regulation and Cell homeostasis, etc. At present, many studies have shown that autophagy is related to the occurrence and development of metabolic diseases, malignant tumors, immune disorders and neurodegenerative diseases. As a marker protein in the autophagy process, LC3 protein not only mediates the autophagy process, but also is related to the formation and fusion of autophagosomes. However, the function of LC3 protein and autophagy-related proteins is mainly through protein-protein (Protein-protein interaction) interaction, the interface is relatively smooth, and there is no binding pocket for small molecule drugs. Therefore, few small molecule inhibitors of LC3 have been reported so far. In 2021, Luo Cheng, a researcher at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and other researchers reported for the first time small molecule inhibitors that covalently bind to LC3. Therefore, it is of great significance to develop LC3 inhibitors.
发明内容Contents of the invention
发明要解决的问题：Problems to be solved by the invention:
本发明的目的在于提供一种四氢异喹啉类化合物作为新颖的PCSK9小分子蛋白降解剂或LC3小分子蛋白降解剂，用于降低PCSK9或LC3水平，可用于制备预防或治疗与抑制PCSK9或LC3活性相关的疾病的药物。The object of the present invention is to provide a tetrahydroisoquinoline compound as a novel PCSK9 small molecule protein degrading agent or LC3 small molecule protein degrading agent, which is used to reduce the level of PCSK9 or LC3, and can be used for the preparation of prevention or treatment and inhibition of PCSK9 or Drugs for diseases associated with LC3 activity.
解决问题的手段：Means to solve the problem:
为了解决上述技术问题，第一个方面，本发明提供一种PCSK9小分子蛋白降解剂，为如下式化学结构通式I的四氢异喹啉类化合物或其药学上可接受的盐或溶剂化物：
In order to solve the above-mentioned technical problems, in the first aspect, the present invention provides a PCSK9 small molecule protein degradation agent, which is a tetrahydroisoquinoline compound or a pharmaceutically acceptable salt or solvate thereof of the general formula I as follows: :
其中，in,
连接基团A为：C1-C20的烷基链、醚链、氧杂链、硫杂链、氮杂链、C1-C20烯基链、C1-C20炔基链、-C(＝O)NH(C1-C19的烷基链、醚链、氧杂链、硫杂链、氮杂链、C1-C19烯基、C1-C19炔基)；Linking group A is: C 1 -C 20 alkyl chain, ether chain, oxa chain, thia chain, aza chain, C 1 -C 20 alkenyl chain, C 1 -C 20 alkynyl chain, - C(=O)NH(C 1 -C 19 alkyl chain, ether chain, oxa chain, thia chain, aza chain, C 1 -C 19 alkenyl, C 1 -C 19 alkynyl);
R1、R2、R3以及R4独立地选自氢、卤素、羟基、硝基、甲氧基、氨基、甲氨基、二甲氨基、羧基、氰基、-CO2Me、-CO2Et、-CH3、-Et、-N3、C1-6烷基、C2-6烯基、C2-6炔基，其中所述烷基、烯基或炔基任选地被一个或多个选自-O(C1-6烷基)、-O(C3-6环烃基)、-O(C1-4亚烷基-C3-6环烃基)、-O(三元至七元杂环基)、-O(C1-4亚烷基-三元至七元杂环基)、-SH、-S(C1-6烷基)、-S(C3-6环烃基)、-S(C1-4亚烷基-C3-6环烃基)、-S(三元至七元杂环基)、-S(C1-4亚烷基-三元至七元杂环基)、-NH2、-NH(C1-6烷基)、-N(C1-6烷基)2、-NH(C3-6环烃基)、-N(C3-6环烃基)2、-NH(C1-4亚烷基-C3-6环烃基)、-N(C1-4亚烷基-C3-6环烃基)2、-NH(三 元至七元杂环基)、-N(三元至七元杂环基)2、-NH(C1-4亚烷基-三元至七元杂环基)、-N(C1-4亚烷基-三元至七元杂环基)2所取代；R 1 , R 2 , R 3 and R 4 are independently selected from hydrogen, halogen, hydroxyl, nitro, methoxy, amino, methylamino, dimethylamino, carboxyl, cyano, -CO 2 Me, -CO 2 Et, -CH 3 , -Et, -N 3 , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, wherein the alkyl, alkenyl or alkynyl is optionally replaced by one Or more selected from -O(C 1-6 alkyl), -O(C 3-6 cycloalkyl), -O(C 1-4 alkylene-C 3-6 cycloalkyl), -O(three One-membered to seven-membered heterocyclyl), -O(C 1-4 alkylene-three-membered to seven-membered heterocyclyl), -SH, -S(C 1-6 alkyl), -S(C 3- 6 cycloalkyl), -S (C 1-4 alkylene-C 3-6 cycloalkyl), -S (three-membered to seven-membered heterocyclyl), -S (C 1-4 alkylene-three to seven-membered heterocyclyl), -NH 2 , -NH(C 1-6 alkyl), -N(C 1-6 alkyl) 2 , -NH(C 3-6 cycloalkyl), -N(C 3-6 cycloalkyl) 2 , -NH(C 1-4 alkylene-C 3-6 cycloalkyl), -N(C 1-4 alkylene-C 3-6 cycloalkyl) 2 , -NH( three One-membered to seven-membered heterocyclyl), -N(three- to seven-membered heterocyclyl) 2 , -NH(C 1-4 alkylene-three- to seven-membered heterocyclyl), -N(C 1- 4 alkylene-three-membered to seven-membered heterocyclyl) substituted by 2 ;
R5独立地选自氢、甲氧基、甲基、卤素、羟基、硝基、氨基、甲氨基、二甲氨基、羧基、氰基、-CO2Me、-CO2Et、-CH3、-Et、-N3、C1-6烷基、C2-6烯基、C2-6炔基；R 5 is independently selected from hydrogen, methoxy, methyl, halogen, hydroxyl, nitro, amino, methylamino, dimethylamino, carboxyl, cyano, -CO 2 Me, -CO 2 Et, -CH 3 , -Et, -N 3 , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl;
R6独立地选自甲基；R 6 is independently selected from methyl;
R7独立地选自以下结构：R 7 is independently selected from the following structures:
或者 or
基团B为：
Group B is:
其中R8、R9以及R10独立地选自氢、卤素、羟基、硝基、甲氧基、氨基、甲氨基、二甲氨基、羧基、氰基、-CO2Me、-CO2Et、-CH3、-Et、-N3；X为NH、S、O。Wherein R 8 , R 9 and R 10 are independently selected from hydrogen, halogen, hydroxyl, nitro, methoxy, amino, methylamino, dimethylamino, carboxyl, cyano, -CO 2 Me, -CO 2 Et, -CH 3 , -Et, -N 3 ; X is NH, S, O.
优选地，所述R7为 Preferably, said R 7 is
优选地，所述基团B为R8、R9和R10独立地选自卤素、羟基、甲氧基。Preferably, the group B is R 8 , R 9 and R 10 are independently selected from halogen, hydroxy, methoxy.
优选地，所述化合物选自下述化合物：

Preferably, the compound is selected from the following compounds:

第二个方面，本发明提供一种如上所述的PCSK9小分子蛋白降解剂的制备方法，包括如下步骤：
In a second aspect, the present invention provides a method for preparing the PCSK9 small molecule protein degradation agent as described above, comprising the following steps:
其中，in,
R2、R5、R8、R10如前述所定义的。R 2 , R 5 , R 8 , and R 10 are as defined above.
第三个方面，本发明提供一种药物组合物，包括如上所述的四氢异喹啉类化合物或其药学上可接受的盐或溶剂化物，以及药物上可接受的载体。In a third aspect, the present invention provides a pharmaceutical composition, comprising the above-mentioned tetrahydroisoquinoline compound or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.
第四个方面，本发明提供一种如上所述的四氢异喹啉类化合物或其药学上可接受的盐或溶剂化物在制备用于预防或治疗与抑制PCSK9活性相关的疾病的药物中的应用。In a fourth aspect, the present invention provides a tetrahydroisoquinoline compound as described above or a pharmaceutically acceptable salt or solvate thereof in the preparation of a drug for preventing or treating a disease related to the inhibition of PCSK9 activity application.
第五个方面，本发明提供一种如上所述的四氢异喹啉类化合物或其药学上可接受的盐或溶剂化物在制备用于预防或治疗与抑制LC3活性相关的疾病的药物中的应用。In a fifth aspect, the present invention provides a tetrahydroisoquinoline compound as described above or a pharmaceutically acceptable salt or solvate thereof in the preparation of a drug for preventing or treating diseases related to the inhibition of LC3 activity application.
优选地，所述疾病选自心脑血管疾病、脱发性疾病、白癜风、角化异常性疾病、疤 痕以及肺纤维化疾病、代谢综合征、肥胖、糖尿病、阿尔茨海默症、抗排斥反应、非酒精性肝炎和恶性肿瘤中的至少一种。Preferably, the disease is selected from cardiovascular and cerebrovascular diseases, hair loss diseases, vitiligo, abnormal keratinization diseases, scar scar and at least one of pulmonary fibrotic disease, metabolic syndrome, obesity, diabetes, Alzheimer's disease, anti-rejection, nonalcoholic hepatitis, and malignancy.
优选地，所述心脑血管疾病选自胆固醇相关疾病、高脂血症和动脉粥样硬化中的至少一种；所述恶性肿瘤选自白血病、前列腺癌、甲状腺癌、肝癌、卵巢癌、乳腺癌、食道癌、腺癌、胃癌、肺癌和结直肠癌中的至少一种。Preferably, the cardiovascular and cerebrovascular diseases are selected from at least one of cholesterol-related diseases, hyperlipidemia and atherosclerosis; the malignant tumors are selected from leukemia, prostate cancer, thyroid cancer, liver cancer, ovarian cancer, breast cancer Cancer, esophageal cancer, adenocarcinoma, gastric cancer, lung cancer and colorectal cancer.
附图说明Description of drawings
图1示出了根据本发明实施例制备的化合物的Western Blot实验结果；Fig. 1 shows the Western Blot experimental result of the compound prepared according to the embodiments of the present invention;
图2示出了根据本发明实施例制备的化合物I-2与I-4的Western Blot实验结果；Figure 2 shows the Western Blot experimental results of compounds I-2 and I-4 prepared according to the embodiments of the present invention;
图3示出了根据本发明实施例制备的化合物I-3的qPCR实验结果；Fig. 3 shows the qPCR experiment result of the compound 1-3 prepared according to the embodiment of the present invention;
图4示出了根据本发明实施例制备的化合物I-3的MTT毒性试验测定结果；Figure 4 shows the MTT toxicity test results of compound 1-3 prepared according to the embodiments of the present invention;
图5示出了根据本发明实施例制备的化合物I-3在高脂模型中的Western Blot实验结果；Fig. 5 shows the Western Blot experiment result of the compound 1-3 prepared according to the embodiment of the present invention in the hyperlipidemia model;
图6示出了根据本发明实施例制备化合物I-3与他汀类药物联用时的Western Blot实验结果。Fig. 6 shows the Western Blot experiment results when compound I-3 is prepared according to the embodiment of the present invention and used in combination with statins.
图7示出了根据本发明实施例制备的化合物I-1～I-15的Western Blot实验结果。Fig. 7 shows the Western Blot experimental results of compounds I-1 to I-15 prepared according to the embodiments of the present invention.
具体实施方式Detailed ways
以下结合附图和下述实施方式进一步说明本发明，应理解，附图及下述实施方式仅用于说明本发明，而非限制本发明。在各图中相同或相应的附图标记表示同一部件，并省略重复说明。The present invention will be further described below in conjunction with the drawings and the following embodiments. It should be understood that the drawings and the following embodiments are only used to illustrate the present invention rather than limit the present invention. In each figure, the same or corresponding reference numerals denote the same components, and repeated explanations will be omitted.
本发明提供一种新颖的PCSK9小分子靶向降解剂，其具有如下结构通式I的四氢异喹啉类化合物或其药学上可接受的盐或溶剂化物：
The present invention provides a novel PCSK9 small-molecule targeted degradation agent, which has a tetrahydroisoquinoline compound of the following general formula I or a pharmaceutically acceptable salt or solvate thereof:
其中，in,
连接基团A为：C1-C20的烷基链、醚链、氧杂链、硫杂链、氮杂链、C1-C20烯基链、C1-C20炔基链、-C(＝O)NH(C1-C19的烷基链、醚链、氧杂链、硫杂链、氮杂链、C1-C19烯基、C1-C19炔基)；Linking group A is: C 1 -C 20 alkyl chain, ether chain, oxa chain, thia chain, aza chain, C 1 -C 20 alkenyl chain, C 1 -C 20 alkynyl chain, - C(=O)NH(C 1 -C 19 alkyl chain, ether chain, oxa chain, thia chain, aza chain, C 1 -C 19 alkenyl, C 1 -C 19 alkynyl);
R1、R2、R3以及R4独立地选自氢、卤素、羟基、硝基、甲氧基、氨基、甲氨基、二 甲氨基、羧基、氰基、-CO2Me、-CO2Et、-CH3、-Et、-N3、C1-6烷基、C2-6烯基、C2-6炔基，其中所述烷基、烯基或炔基任选地被一个或多个选自-O(C1-6烷基)、-O(C3-6环烃基)、-O(C1-4亚烷基-C3-6环烃基)、-O(三元至七元杂环基)、-O(C1-4亚烷基-三元至七元杂环基)、-SH、-S(C1-6烷基)、-S(C3-6环烃基)、-S(C1-4亚烷基-C3-6环烃基)、-S(三元至七元杂环基)、-S(C1-4亚烷基-三元至七元杂环基)、-NH2、-NH(C1-6烷基)、-N(C1-6烷基)2、-NH(C3-6环烃基)、-N(C3-6环烃基)2、-NH(C1-4亚烷基-C3-6环烃基)、-N(C1-4亚烷基-C3-6环烃基)2、-NH(三元至七元杂环基)、-N(三元至七元杂环基)2、-NH(C1-4亚烷基-三元至七元杂环基)、-N(C1-4亚烷基-三元至七元杂环基)2所取代；R 1 , R 2 , R 3 and R 4 are independently selected from hydrogen, halogen, hydroxyl, nitro, methoxy, amino, methylamino, di Methylamino, carboxyl, cyano, -CO 2 Me, -CO 2 Et, -CH 3 , -Et, -N 3 , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, Wherein said alkyl, alkenyl or alkynyl is optionally replaced by one or more selected from -O(C 1-6 alkyl), -O(C 3-6 cycloalkyl), -O(C 1-4 Alkylene-C 3-6 cycloalkyl), -O (three-membered to seven-membered heterocyclic group), -O (C 1-4 alkylene-three-membered to seven-membered heterocyclic group), -SH, - S (C 1-6 alkyl), -S (C 3-6 cycloalkyl), -S (C 1-4 alkylene-C 3-6 cycloalkyl), -S (three-membered to seven-membered heterocycle group), -S(C 1-4 alkylene-three- to seven-membered heterocyclic group), -NH 2 , -NH(C 1-6 alkyl), -N(C 1-6 alkyl) 2 , -NH(C 3-6 cycloalkyl), -N(C 3-6 cycloalkyl) 2 , -NH(C 1-4 alkylene-C 3-6 cycloalkyl), -N(C 1-4 Alkylene-C 3-6 cycloalkyl) 2 , -NH (three- to seven-membered heterocyclyl), -N (three- to seven-membered heterocyclyl) 2 , -NH (C 1-4 alkylene -Three-membered to seven-membered heterocyclic group), -N (C 1-4 alkylene-three-membered to seven-membered heterocyclic group) substituted by 2 ;
R5独立地选自氢、甲氧基、甲基、卤素、羟基、硝基、氨基、甲氨基、二甲氨基、羧基、氰基、-CO2Me、-CO2Et、-CH3、-Et、-N3、C1-6烷基、C2-6烯基、C2-6炔基；R 5 is independently selected from hydrogen, methoxy, methyl, halogen, hydroxyl, nitro, amino, methylamino, dimethylamino, carboxyl, cyano, -CO 2 Me, -CO 2 Et, -CH 3 , -Et, -N 3 , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl;
R6独立地选自甲基；R 6 is independently selected from methyl;
R7独立地选自以下结构：R 7 is independently selected from the following structures:
或者 or
基团B为：
Group B is:
其中R8、R9以及R10独立地选自氢、卤素、羟基、硝基、甲氧基、氨基、甲氨基、二甲氨基、羧基、氰基、-CO2Me、-CO2Et、-CH3、-Et、-N3；X为NH、S、O。Wherein R 8 , R 9 and R 10 are independently selected from hydrogen, halogen, hydroxyl, nitro, methoxy, amino, methylamino, dimethylamino, carboxyl, cyano, -CO 2 Me, -CO 2 Et, -CH 3 , -Et, -N 3 ; X is NH, S, O.
在本发明的一个优选实施方式中，所述R7为 In a preferred embodiment of the present invention, said R 7 is
在本发明的一个优选实施方式中，所述基团B为R8、R9和R10独 立地选自卤素、羟基、甲氧基。In a preferred embodiment of the present invention, the group B is R 8 , R 9 and R 10 alone is selected from halogen, hydroxy, methoxy.
在本发明的一个优选实施方式中，所述化合物选自下述化合物：

In a preferred embodiment of the present invention, the compound is selected from the following compounds:

本发明提供的化合物可以通过下述合成方案合成：
The compounds provided by the invention can be synthesized through the following synthetic schemes:
其中，R2、R5、R8、R10如前述所定义的。Wherein, R 2 , R 5 , R 8 , and R 10 are as defined above.
除非另有说明，上述合成方案中所述基团、术语的含义与通式I化合物中的含义相同。Unless otherwise specified, the meanings of the groups and terms in the above synthesis schemes are the same as those in the compounds of general formula I.
上述合成方案只是列举了本发明中部分化合物的制备方法，参考本领域中常用技术手段及现有技术，本领域技术人员在上述合成方案的基础上，可采用类似的方法合成本发明的化合物。The above-mentioned synthesis scheme only lists the preparation methods of some compounds in the present invention. With reference to common technical means and prior art in this field, those skilled in the art can use similar methods to synthesize the compounds of the present invention on the basis of the above-mentioned synthesis scheme.
本文所用的表述m-n指m至n的范围以及由其中的各个点值组成的亚范围以及各个点值。例如，表述“C1-C20”或“C1—20”涵盖1-20个碳原子的范围，并应理解为还涵盖其中的任意亚范围以及每个点值，例如C2-C5、C3-C4、C1-C2、C1-C3、C1-C4、C1-C5、C1-C6 等，以及C1、C2、C3、C4、C5、C6、C7、C8等。例如，表述“C3-C10”或“C3-10”也应当以类似的方式理解，例如可以涵盖包含于其中的任意亚范围和点值，例如C3-C9、C6-C9、C6-C8、C6-C7、C7-C10、C7-C9、C7-C8、C8-C9等以及C3、4、5、6、7、8、9、10等。本文中其他类似的表述也应当以类似的方式理解。The expression mn as used herein refers to the range from m to n and the subranges and individual point values therein. For example, the expression "C1-C20" or "C1-20" covers the range of 1-20 carbon atoms, and should be understood to also cover any sub-ranges therein and each point value, such as C2-C5, C3-C4, C1-C2, C1-C3, C1-C4, C1-C5, C1-C6 etc., and C1, C2, C3, C4, C5, C6, C7, C8, etc. For example, the expression "C3-C10" or "C3-10" should also be understood in a similar manner, for example to cover any subranges and point values contained therein, such as C3-C9, C6-C9, C6-C8, C6 -C7, C7-C10, C7-C9, C7-C8, C8-C9, etc. and C3, 4, 5, 6, 7, 8, 9, 10, etc. Other similar expressions herein should also be read in a similar manner.
术语“卤”或“卤素”或“卤代”应理解为表示氟(F)、氯(Cl)、溴(Br)或碘(I)原子。The term "halo" or "halogen" or "halo" is understood to mean a fluorine (F), chlorine (Cl), bromine (Br) or iodine (I) atom.
术语“烷基”是指由碳原子和氢原子组成的直链或支链的饱和的脂肪烃基团，其通过单键与分子的其余部分连接。“烷基”可以具有1-20个碳原子，即“C1-C20烷基”，例如C1-4烷基、C1-3烷基、C1-2烷基、C3烷基、C4烷基、C1-6烷基、C3-6烷基。烷基的非限制性实例包括但不限于甲基、乙基、丙基、丁基、戊基、己基、异丙基、异丁基、仲丁基、叔丁基、异戊基、2-甲基丁基、1-甲基丁基、1-乙基丙基、1,2-二甲基丙基、新戊基、1,1-二甲基丙基、4-甲基戊基、3-甲基戊基、2-甲基戊基、1-甲基戊基、2-乙基丁基、1-乙基丁基、3,3-二甲基丁基、2,2-二甲基丁基、1,1-二甲基丁基、2,3-二甲基丁基、1,3-二甲基丁基或1,2-二甲基丁基，或者它们的异构体。“亚基”是指在含有自由价电子的碳原子上再去掉一个氢原子而得到的，具有两个与分子其他部分连接的连接位点的基团。例如“亚烷基”或“烷基亚基”指饱和的直链或支链的二价烃基。The term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms attached to the rest of the molecule by a single bond. "Alkyl" can have 1-20 carbon atoms, that is, "C 1 -C 20 alkyl", such as C 1-4 alkyl, C 1-3 alkyl, C 1-2 alkyl, C 3 alkyl , C 4 alkyl, C 1-6 alkyl, C 3-6 alkyl. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2- Methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-di Methylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or their isomers body. "Subunit" refers to a group obtained by removing a hydrogen atom from a carbon atom containing free valence electrons, and having two attachment sites for attachment to the rest of the molecule. For example "alkylene" or "alkylene" refers to a saturated straight or branched chain divalent hydrocarbon group.
术语“亚烷基”，在本文中单独或与其他基团组合使用时，指直链或支链的饱和的二价烃基。例如，术语“C1-20亚烷基”指具有1-20个碳原子的亚烷基，例如亚甲基、亚乙基、亚丙基、亚丁基、亚戊基、亚己基、1-甲基亚乙基、2-甲基亚乙基、甲基亚丙基或乙基亚丙基等。术语“亚环烷基”是指环状的饱和的二价烃基。例如，术语“C3-6亚环烷基”指具有3-6个碳原子的环烷基亚基，例如环丙基亚基、环丁基亚基、环戊基亚基、环己基亚基，等。术语“烷氧基亚基”是指“-O-亚烷基”或“亚烷基-O-”。“C1-8烷氧基亚基”的实例包括但不限于-O-亚甲基、-O-亚乙基、-O-亚丙基、-O-亚丁基、亚甲基-O-、亚乙基-O-、亚丙基-O-、亚丁基-O-，等。The term "alkylene", when used herein alone or in combination with other groups, refers to a linear or branched saturated divalent hydrocarbon group. For example, the term "C 1-20 alkylene" refers to an alkylene group having 1-20 carbon atoms, such as methylene, ethylene, propylene, butylene, pentylene, hexylene, 1- Methylethylene, 2-methylethylene, methylpropylene or ethylpropylene, etc. The term "cycloalkylene" refers to a cyclic saturated divalent hydrocarbon group. For example, the term "C 3-6 cycloalkylene" refers to a cycloalkylene group having 3-6 carbon atoms, such as cyclopropylidene, cyclobutylene, cyclopentylylene, cyclohexylylene Kei, wait. The term "alkoxyylene" refers to "-O-alkylene" or "alkylene-O-". Examples of "C 1-8 alkoxylidene" include, but are not limited to, -O-methylene, -O-ethylene, -O-propylene, -O-butylene, methylene-O- , Ethylene-O-, Propylene-O-, Butylene-O-, etc.
术语“烯基”是指由碳原子和氢原子组成的直链或支链的具有至少一个双键的不饱和脂肪族烃基。烯基可以具有2-20个碳原子，即“C2-20烯基”，例如C2-4烯基、C3-4烯基。烯基的非限制性实例包括但不限于乙烯基、烯丙基、(E)-2-甲基乙烯基、(Z)-2-甲基乙烯基、(E)-丁-2-烯基、(Z)-丁-2-烯基、(E)-丁-1-烯基、(Z)-丁-1-烯基等。The term "alkenyl" refers to a straight or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one double bond. Alkenyl can have 2-20 carbon atoms, namely " C2-20 alkenyl", such as C2-4 alkenyl, C3-4 alkenyl. Non-limiting examples of alkenyl include, but are not limited to, vinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, (E)-but-2-enyl , (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl and the like.
术语“炔基”是指由碳原子和氢原子组成的直链或支链的具有至少一个三键的不饱和脂肪族烃基。炔基可以具有2-20个碳原子，即“C2-8炔基”，例如C2-4炔基、C3-4炔基。炔 基的非限制性实例包括但不限于乙炔基、丙-1-炔基、丙-2-炔基、丁-1-炔基、丁-2-炔基、丁-3-炔基等。The term "alkynyl" refers to a straight or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, having at least one triple bond. The alkynyl group may have 2-20 carbon atoms, ie " C2-8alkynyl ", such as C2-4alkynyl , C3-4alkynyl . alkyne Non-limiting examples of radicals include, but are not limited to, ethynyl, prop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl, but-3-ynyl, and the like.
术语“环烃基”是指由碳原子和氢原子组成的饱和或不饱和的非芳香性的环状烃基，优选包含1或2个环。所述环烃基可以是单环、稠合多环、桥环或螺环结构。环烃基可以具有3-10个碳原子，即“C3-10环烃基”，例如C3-8环烃基、C3-6环烃基、C5环烃基、C6环烃基、C7环烃基。环烃基的非限制性实例包括但不限于环丙基、环丁基、环戊基、环己基、环庚基、双环[2.2.1]庚基和螺[3.3]庚基等。该术语还涵盖这样的情况，其中的C原子可以被氧代(＝O)取代。The term "cyclic hydrocarbon group" refers to a saturated or unsaturated non-aromatic cyclic hydrocarbon group composed of carbon atoms and hydrogen atoms, preferably containing 1 or 2 rings. The cyclic hydrocarbon group may be a monocyclic, fused polycyclic, bridged or spiro ring structure. Cycloalkyl can have 3-10 carbon atoms, that is, "C 3-10 cycloalkyl", such as C 3-8 cycloalkyl, C 3-6 cycloalkyl, C 5 cycloalkyl, C 6 cycloalkyl, C 7 cycloalkyl . Non-limiting examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl, spiro[3.3]heptyl, and the like. The term also covers cases where a C atom may be substituted by oxo (=O).
术语“环烷基”是指饱和的环烃基。The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group.
术语“杂环基”或“杂环烃基”是指具有例如3-10个(适合地具有3-8个，更适合地具有3-7个，特别是4-6个)环原子的单环或双环的环体系(三元至十元、三元至八元、三元至七元、四元至六元)，其中至少一个环原子(例如1或3个)是选自N、O、S和P的杂原子，且其余环原子是C。该环体系可以是饱和(也可以理解为相应的“杂环烷基”)或不饱和的(即在环内具有一个或多个双键和/或三键)。“杂环基”或“杂环烃基”不具备芳香性。该术语还涵盖这样的情况，其中的C原子可以被氧代(＝O)取代和/或环上的S原子可以被1个或2个氧代(＝O)取代和/或环上的P原子可以被1个或2个氧代(＝O)取代。The term "heterocyclyl" or "heterocycloalkyl" refers to a monocyclic ring having, for example, 3-10 (suitably 3-8, more suitably 3-7, especially 4-6) ring atoms Or a bicyclic ring system (three to ten, three to eight, three to seven, four to six), wherein at least one ring atom (eg, 1 or 3) is selected from N, O, heteroatoms of S and P, and the remaining ring atoms are C. The ring system may be saturated (also understood as the corresponding "heterocycloalkyl") or unsaturated (ie have one or more double and/or triple bonds within the ring). "Heterocyclyl" or "heterocycloalkyl" is not aromatic. The term also covers cases where a C atom may be substituted by oxo (=O) and/or a ring S atom may be substituted by 1 or 2 oxo (=O) and/or a ring P Atoms may be substituted with 1 or 2 oxo (=O).
杂环基可以是例如四元环，如氮杂环丁烷基、氧杂环丁烷基；或者五元环，如四氢呋喃基、二噁烷基、吡咯烷基、咪唑烷基、吡唑烷基、吡咯啉基、氧代吡咯烷基、2-氧代咪唑烷-1-基；或者六元环，如四氢吡喃基、哌啶基、吗啉基、二噻烷基、硫代吗啉基、哌嗪基、1,1-二氧代-1,2-噻嗪烷-2-基或三噻烷基；或者七元环，如二氮杂基环。任选地，杂环基可以是苯并稠和的。The heterocyclic group can be, for example, a four-membered ring, such as azetidinyl, oxetanyl; or a five-membered ring, such as tetrahydrofuranyl, dioxanyl, pyrrolidinyl, imidazolidinyl, pyrazolidine Base, pyrrolinyl, oxopyrrolidinyl, 2-oxoimidazolidin-1-yl; or six-membered rings, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thio Morpholinyl, piperazinyl, 1,1-dioxo-1,2-thiazidin-2-yl or trithianyl; or seven-membered rings such as diaza base ring. Optionally, the heterocyclyl group may be benzo-fused.
杂环基可以是双环的，不受其限制，例如五元并五元环，如六氢环戊烷[C]吡咯-2(1H)-基)环；或者五元并六元双环，如六氢吡咯并[1,2-a]吡嗪-2(1H)-基环。The heterocyclyl group may be bicyclic without limitation, for example a five-membered five-membered ring such as a hexahydrocyclopentane[C]pyrrol-2(1H)-yl) ring; or a five-membered six-membered bicyclic ring such as Hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl ring.
如上文所提到的，杂环可以是不饱和的，即其可以包含一个或多个双键，不受其限制，例如包含氮原子的不饱和的杂环可以是1,6-二氢嘧啶、1,2-二氢嘧啶、1,4-二氢嘧啶、1,6-二氢吡啶、1,2-二氢吡啶、1,4-二氢吡啶、2,3-二氢-IH-吡咯、3,4-二氢-IH-吡咯、2,5-二氢-IH-吡咯基、4H-[1,3,4]噻二嗪基、4,5-二氢噁唑基或4H-[1,4]噻嗪基环，包含氧原子的不饱和的杂环可以是2H-吡喃、4H-吡喃、2,3-二氢呋喃，包含硫原子的不饱和的杂环可以是2H-噻喃、4H-噻喃。杂环可以是苯并稠和的，不受其限制，例如二氢异喹啉基环。As mentioned above, a heterocycle may be unsaturated, i.e. it may contain one or more double bonds without limitation, for example an unsaturated heterocycle containing a nitrogen atom may be 1,6-dihydropyrimidine , 1,2-dihydropyrimidine, 1,4-dihydropyrimidine, 1,6-dihydropyridine, 1,2-dihydropyridine, 1,4-dihydropyridine, 2,3-dihydro-IH- Pyrrole, 3,4-dihydro-IH-pyrrole, 2,5-dihydro-IH-pyrrolyl, 4H-[1,3,4]thiadiazinyl, 4,5-dihydrooxazolyl or 4H -[1,4]thiazinyl ring, the unsaturated heterocyclic ring containing oxygen atom can be 2H-pyran, 4H-pyran, 2,3-dihydrofuran, the unsaturated heterocyclic ring containing sulfur atom can be Is 2H-thiopyran, 4H-thiopyran. Heterocycles may be benzo-fused without limitation, eg dihydroisoquinolinyl rings.
术语“烃链”指由碳原子和氢原子构成的链状基团，其可以是直链或支化的。所述 烃链可以是饱和的(即亚烷基)，也可以是不饱和的，即可以包含一个或多个碳碳双键或三键。亚烷基的非限制性实例包括但不限于亚甲基(-CH2-)、1,1-亚乙基(-CH(CH3)-)、1,2-亚乙基(-CH2CH2-)、1,1_亚丙基(-CH(CH2CH3)-)、1,2-亚丙基(-CH2CH(CH3)-)、1,3-亚丙基(-CH2CH2CH2-)、1,4-亚丁基(-CH2CH2CH2CH2-)、1,7-亚庚基(-CH2CH2CH2CH2CH2CH2CH2-)等。The term "hydrocarbon chain" refers to a chain-like group composed of carbon atoms and hydrogen atoms, which may be straight or branched. said The hydrocarbon chain may be saturated (ie, an alkylene group) or unsaturated, ie, may contain one or more carbon-carbon double or triple bonds. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH 2 -), 1,1-ethylene (-CH(CH 3 )-), 1,2-ethylene (-CH 2 CH 2 -), 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -), 1,7-heptylene (-CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -), etc.
本发明所述的“化合物”，包括所有的立体异构体、几何异构体、互变异构体和同位素。The "compound" in the present invention includes all stereoisomers, geometric isomers, tautomers and isotopes.
本发明所述的“化合物”，可以是不对称的，例如，具有一个或多个立体异构体。除非另有说明，所有异构体都包括，如对映异构体和非对映异构体。本发明中含有不对称碳原子的化合物，可以光学活性纯的形式或外消旋形式被分离出来；光学活性纯的形式可以从外消旋混合物拆分，或通过使用手性原料或手性试剂合成。The "compound" of the present invention may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all isomers are included, eg, enantiomers and diastereomers. Compounds containing asymmetric carbon atoms in the present invention can be isolated in optically pure or racemic forms; optically pure forms can be resolved from racemic mixtures, or by using chiral raw materials or chiral reagents synthesis.
本发明所述的“化合物”，还包括几何异构体形式；几何异构体形式可以混合物或分离的E或Z结构形式存在。The "compound" mentioned in the present invention also includes geometric isomers; the geometric isomers may exist as mixtures or separated E or Z structures.
本发明所述的“化合物”，还包括互变异构体形式；互变异构体形式源于一个单键与相邻的双键交换并一起伴随一个质子的迁移。The "compound" in the present invention also includes tautomeric forms; tautomeric forms originate from the exchange of a single bond with an adjacent double bond accompanied by the migration of a proton.
本发明所述的“化合物”，还包括所有同位素的原子，无论是中间体或最后的化合物；同位素的原子包括具有相同的质子数、但有不同质量数的，例如，氢的同位素包括氘和氚。又，如果需要的话，例如为了特别的治疗或者诊断治疗，本发明的化合物可以引进现有技术已知的同位素或放射性同位素、例如3H、15O、13C或15N同位素。The "compound" of the present invention also includes atoms of all isotopes, no matter it is an intermediate or a final compound; atoms of isotopes include those with the same number of protons but different mass numbers, for example, isotopes of hydrogen include deuterium and tritium. Also, if desired, for example for specific therapeutic or diagnostic treatments, the compounds of the invention may incorporate isotopes or radioactive isotopes known in the art, eg 3H, 15O, 13C or 15N isotopes.
“药学上可接受的盐”是指药学上可接受的盐，保持其母体化合物的药理活性的同时可改善理化性质或者代谢性质等。这类盐包括由药学上可接受的酸或者碱(包括有机酸、无机酸、有机碱、无机碱)制备的酸加成盐或碱加成盐，或者两者混合物。在本发明中，合适的无机酸例如盐酸、氢溴酸、硫酸、硝酸、磷酸或类似的酸；合适的有机酸包括富马酸、酒石酸、乳酸、醋酸、柠檬酸、三氟甲磺酸、扁桃酸、水杨酸或其类似物。"Pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt, which can improve the physicochemical or metabolic properties while maintaining the pharmacological activity of its parent compound. Such salts include acid addition salts or base addition salts prepared from pharmaceutically acceptable acids or bases (including organic acids, inorganic acids, organic bases, inorganic bases), or a mixture of both. In the present invention, suitable inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid or similar acids; suitable organic acids include fumaric acid, tartaric acid, lactic acid, acetic acid, citric acid, trifluoromethanesulfonic acid, Mandelic acid, salicylic acid or their analogs.
根据本发明的化合物还可以以溶剂化物形式存在。例如水合物(半水合物、一水合物、二水合物、三水合物等)。The compounds according to the invention may also exist in the form of solvates. For example, hydrates (hemihydrate, monohydrate, dihydrate, trihydrate, etc.).
给药以及药物组合物Administration and pharmaceutical composition
一般来说，本发明的化合物可以在有效量下，通过任何可接受的用于其它类似用途的给药方式进行给药。举例来说，本发明的化合物可以口服、非肠道给药、透皮给药、局部给药、直肠给药、腹腔注射或鼻内给药。 In general, the compounds of the present invention can be administered in an effective amount by any acceptable means of administration for other similar uses. For example, the compounds of the present invention can be administered orally, parenterally, transdermally, topically, rectally, intraperitoneally or intranasally.
当用作药物时，本发明的化合物通常以药物组合物的形式给药。这些组合物可用制药学领域熟知的方法进行制备，这些组合物包含至少一种活性化合物。在配制本发明提供的组合物时，有效成分通常与赋形剂混合，被赋形剂稀释或被以胶囊，小袋，纸或其它形式的容器包裹。当所述赋形剂作为稀释剂时，可以是固体，半固体，或液体物质，可作为有效成分的运载体、载体或媒介。由此，所述组合物可以是药片、药丸、粉末、锭剂、小袋、胶囊、酏剂、混悬剂、乳剂、溶液、糖浆、喷雾(作为固体或在液体介质里)，软膏，软和硬的明胶胶囊，栓剂，无菌注射溶液，和无菌包装粉末的形式。When used as a medicine, the compounds of the invention are usually administered in the form of pharmaceutical compositions. These compositions may be prepared by methods well known in the art of pharmacy and contain at least one active compound. In formulating the compositions provided herein, the active ingredient is usually mixed with an excipient, diluted by an excipient or enclosed in a capsule, sachet, paper or other form of container. When the excipient is used as a diluent, it can be a solid, semi-solid, or liquid substance, and can be used as a carrier, carrier or medium for the active ingredient. Thus, the composition may be a tablet, pill, powder, lozenge, sachet, capsule, elixir, suspension, emulsion, solution, syrup, spray (as a solid or in a liquid medium), ointment, soft and In the form of hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
一些典型的赋形剂包括乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、淀粉、阿拉伯树胶、磷酸钙、无菌水、糖浆和甲基纤维素。另外还可以包括润滑剂(如滑石粉、硬脂酸镁和矿物油)、浸润剂、乳化和悬浊剂、防腐剂(如对羟基苯甲酸甲酯和对羟基苯甲酸丙酯)、甜味剂和增味剂。本发明的药物组合物可以通过具体赋形方式在对病人进行给药之后达到药物活性成分的快速、持续或延时释放，这也是本领域中广泛应用的方法。Some typical excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, sterile water, syrup and methylcellulose. Additionally, lubricants (such as talc, magnesium stearate, and mineral oil), wetting agents, emulsifying and suspending agents, preservatives (such as methylparaben and propylparaben), sweetening agents, and agents and flavor enhancers. The pharmaceutical composition of the present invention can achieve rapid, sustained or delayed release of the pharmaceutical active ingredient after being administered to the patient through a specific excipient, which is also a method widely used in the field.
活性成分，即本发明中的化合物，在药物构成和单位剂型中的数量可根据具体应用情况、特定化合物的活性和预期浓度进行改变或有较大调整。The active ingredient, that is, the compound of the present invention, the amount in the pharmaceutical composition and unit dosage form can be changed or adjusted greatly according to the specific application, the activity of the specific compound and the expected concentration.
“治疗”意味着对哺乳动物体内疾病的任何治疗，包括：(1)防止疾病，即造成临床疾病的症状不发展；(2)抑制疾病，即阻止临床症状的发展；(3)减轻疾病，即造成临床症状的消退。"Treatment" means any treatment of a disease in a mammal, including: (1) preventing the disease, that is, causing the symptoms of the clinical disease to not develop; (2) inhibiting the disease, that is, preventing the development of clinical symptoms; (3) alleviating the disease, That is to cause the subsidence of clinical symptoms.
以下结合具体实施例进一步详细说明本发明。同样应理解，以下实施例只用于对本发明进行进一步说明，不能理解为对本发明保护范围的限制，本领域的技术人员根据本发明的上述内容作出的一些非本质的改进和调整均属于本发明的保护范围。下述示例具体的工艺参数等也仅是合适范围中的一个示例，即本领域技术人员可以通过本文的说明做合适的范围内选择，而并非要限定于下文示例的具体数值。The present invention will be further described in detail below in conjunction with specific examples. It should also be understood that the following examples are only used to further illustrate the present invention, and should not be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art according to the above contents of the present invention all belong to the present invention scope of protection. The specific process parameters and the like in the following examples are only an example of the appropriate range, that is, those skilled in the art can make a selection within the appropriate range through the description herein, and are not limited to the specific values exemplified below.
实施例一：化合物I-1的制备Embodiment one: the preparation of compound I-1
2-(7-(2-(2-(5-溴-3-(3-溴-4-羟基-5-甲氧基亚苄基)-2-氧代吲哚-1-基)乙氧基)-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)-N-(噻唑-2-基)乙酰胺的合成
2-(7-(2-(2-(5-bromo-3-(3-bromo-4-hydroxy-5-methoxybenzylidene)-2-oxoindol-1-yl)ethoxy Synthesis of -6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide
步骤1：2-甲氧基-4-(2-硝基乙烯基)苯酚的合成
Step 1: Synthesis of 2-methoxy-4-(2-nitrovinyl)phenol
室温下，将香豆素溶于硝基甲烷，并加入醋酸胺。溶解完全后将反应液移入100℃条件下，反应2小时后，移至室温。随后有固体析出，反应液完全降至室温时，将析出的固体直接过滤，并使用少量甲醇润洗滤饼，旋干滤饼得黄色固体P1。1H NMR(400MHz,DMSO)δ10.09(s,1H),8.19-8.15(d,J＝13.4Hz,1H),8.05-8.02(d,J＝13.5Hz,1H),7.48(s,1H),7.31-7.29(d,J＝8.1Hz,1H),6.85-6.83(d,J＝8.1Hz,1H),3.82(s,3H).ESI-MS calculated for[M+H]+:196.0，found:196.0.At room temperature, the coumarin was dissolved in nitromethane and ammonium acetate was added. After the dissolution was complete, the reaction liquid was moved to 100°C, and after 2 hours of reaction, it was moved to room temperature. Then there is solid precipitation, and when the reaction solution is completely down to room temperature, the precipitated solid is directly filtered, and a small amount of methanol is used to rinse the filter cake, and the filter cake is spin-dried to obtain a yellow solid P1. 1H NMR (400MHz, DMSO) δ 10.09 (s ,1H),8.19-8.15(d,J=13.4Hz,1H),8.05-8.02(d,J=13.5Hz,1H),7.48(s,1H),7.31-7.29(d,J=8.1Hz, 1H),6.85-6.83(d,J=8.1Hz,1H),3.82(s,3H).ESI-MS calculated for[M+H]+:196.0,found:196.0.
步骤2：2-甲氧基-4-(2-硝基乙基)苯酚的合成
Step 2: Synthesis of 2-methoxy-4-(2-nitroethyl)phenol
将P1溶于THF，并将硼氢化钠溶于THF和EtOH，冰浴下，将溶于THF的P1缓慢滴加入硼氢化钠中，滴加完毕后，将反应液移入室温下反应过夜。随后使用盐酸稀溶液(1M)淬灭反应，并用二氯甲烷萃取，用无水硫酸钠干燥有机相，旋干得黑色粗产物。使用PE：EA柱层析纯化得淡黄色油状物P2。1H NMR(600MHz,DMSO)δ8.84(s,1H),6.85(d,J＝1.6Hz,1H),6.69-6.67(d,J＝8.0Hz,1H),6.63-6.62(dd,J＝8.0,1.7Hz,1H),4.78-4.76(t,J＝7.1Hz,2H),3.74(s,3H),3.12-3.09(t,J＝7.1Hz,2H).ESI-MS calculated for[M-H]+:196.0，found:195.9。P1 was dissolved in THF, and sodium borohydride was dissolved in THF and EtOH. Under an ice bath, P1 dissolved in THF was slowly added dropwise to sodium borohydride. After the dropwise addition, the reaction solution was transferred to room temperature to react overnight. The reaction was subsequently quenched with dilute hydrochloric acid solution (1M), extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate and spin-dried to obtain a black crude product. Purified by PE: EA column chromatography to obtain pale yellow oil P2. 1H NMR (600MHz, DMSO) δ8.84(s, 1H), 6.85(d, J=1.6Hz, 1H), 6.69-6.67(d, J =8.0Hz, 1H), 6.63-6.62(dd, J=8.0, 1.7Hz, 1H), 4.78-4.76(t, J=7.1Hz, 2H), 3.74(s, 3H), 3.12-3.09(t, J＝7.1Hz, 2H). ESI-MS calculated for [M-H]+: 196.0, found: 195.9.
步骤3：4-(2-氨基乙基)-2-甲氧基苯酚的合成
Step 3: Synthesis of 4-(2-aminoethyl)-2-methoxyphenol
将P2溶于无水THF，并置于冰浴下，随后将LiAlH4溶液缓慢地滴加入溶液中。滴加完成后，将反应液移入75℃油浴中回流4小时。随后降至0℃下，并缓慢加入饱和硫酸钠水溶液淬灭反应，滤掉残渣，旋干可得粗产物。将旋干后的粗产物重新溶于二氧六环的盐酸溶液中，并置于-20℃冰箱过夜，可析出灰色固体P3的盐酸盐。1H NMR(600MHz,D2O)δ7.03(s,1H),6.97-6.96(d,J＝8.0Hz,1H),6.88-6.87(d,J＝8.0Hz,1H),3.94(s,3H),3.34-3.31(t,J＝7.1Hz,2H),3.01-2.98(t,J＝7.2Hz,2H).ESI-MS calculated for[M+H]+:168.1，found:168.1。P2 was dissolved in anhydrous THF and placed in an ice bath, then LiAlH4 solution was slowly added dropwise into the solution. After the dropwise addition was completed, the reaction solution was transferred to an oil bath at 75° C. and refluxed for 4 hours. Then it was lowered to 0°C, and the reaction was quenched by slowly adding saturated aqueous sodium sulfate solution, the residue was filtered off, and the crude product was obtained by spinning. The spin-dried crude product was re-dissolved in dioxane hydrochloric acid solution, and placed in a -20°C refrigerator overnight to precipitate the hydrochloride salt of gray solid P3. 1H NMR(600MHz, D2O)δ7.03(s,1H),6.97-6.96(d,J＝8.0Hz,1H),6.88-6.87(d,J＝8.0Hz,1H),3.94(s,3H) ,3.34-3.31(t,J＝7.1Hz,2H), 3.01-2.98(t,J＝7.2Hz,2H).ESI-MS calculated for[M+H]+:168.1,found:168.1.
步骤4：(E)-3-((4-羟基-3-甲氧基苯乙基)氨基)丁-2-烯酸乙酯的合成
Step 4: Synthesis of (E)-3-((4-hydroxy-3-methoxyphenethyl)amino)but-2-enoic acid ethyl ester
将粗产物P3与乙酰乙酸乙酯溶于无水THF中，并加入无水硫酸钠，在氮气保护下反应过夜。反应完成后，过滤除去硫酸钠，旋干得到粗产物。随后用PE：EA过柱，纯化得到黄白色油状化合物P4。ESI-MS calculated for[M+H]+:280.2，found:280.0。The crude product P3 and ethyl acetoacetate were dissolved in anhydrous THF, and anhydrous sodium sulfate was added to react overnight under nitrogen protection. After the reaction was completed, sodium sulfate was removed by filtration, and spin-dried to obtain a crude product. Then, PE:EA was used to pass through the column, and the compound P4 was obtained as a yellow-white oil. ESI-MS calculated for [M+H]+: 280.2, found: 280.0.
步骤5：2-(7-羟基-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)乙酸乙酯的合成
Step 5: Synthesis of ethyl 2-(7-hydroxy-6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)acetate
冰浴下，将五氧化二磷溶于85％的磷酸中，降至室温后，将P4溶于该溶液中，随后将反应液升温至100℃，反应1小时后，将反应液降至0℃，并加入25％的醋酸铵水溶液淬灭反应，二氯甲烷萃取，旋干得紫红色油状物，DCM：MeOH过柱纯化得P5。ESI-MS calculated for[M+H]+:280.2，found:280.2。Under ice bath, dissolve phosphorus pentoxide in 85% phosphoric acid, after cooling down to room temperature, dissolve P4 in the solution, then raise the temperature of the reaction solution to 100°C, and after 1 hour of reaction, reduce the reaction solution to 0 ℃, and added 25% ammonium acetate aqueous solution to quench the reaction, extracted with dichloromethane, and spin-dried to obtain a purple-red oil, which was purified by DCM:MeOH column to obtain P5. ESI-MS calculated for [M+H]+: 280.2, found: 280.2.
步骤6：1-(2-乙氧基-2-氧乙基)-7-羟基-6-甲氧基-1-甲基-3,4-二氢异喹啉-2(1H)-羧酸叔丁酯的合成
Step 6: 1-(2-Ethoxy-2-oxyethyl)-7-hydroxy-6-methoxy-1-methyl-3,4-dihydroisoquinoline-2(1H)-carboxy Synthesis of tert-butyl ester
将P5溶于DCM，并加入DIPEA以及(Boc)2O，反应过夜后，旋干得粗产物，PE：EA过柱的淡黄色油状物P6。1H NMR(600MHz,CDCl3)δ6.81(s,1H),6.52(s,1H),5.51(s,1H),4.13-4.10(q,J＝7.1Hz,1H),3.93-3.89(q,J＝7.1Hz,2H),3.85(s,4H),3.57–3.54(m,1H),2.82–2.76(m,2H),2.65-2.61(ddd,J＝15.0,6.8,3.1Hz,1H),1.68(s,3H),1.50(s,9H),1.09-1.06(t,J＝7.1Hz,3H).ESI-MS calculated for[M+H]+:380.2，found:379.9。Dissolve P5 in DCM, add DIPEA and (Boc)2O, react overnight, spin dry to get the crude product, PE: EA light yellow oil P6 through the column. 1H NMR (600MHz, CDCl3) δ6.81 (s, 1H),6.52(s,1H),5.51(s,1H),4.13-4.10(q,J=7.1Hz,1H),3.93-3.89(q,J=7.1Hz,2H),3.85(s,4H ),3.57–3.54(m,1H),2.82–2.76(m,2H),2.65-2.61(ddd,J＝15.0,6.8,3.1Hz,1H),1.68(s,3H),1.50(s,9H ), 1.09-1.06 (t, J=7.1Hz, 3H). ESI-MS calculated for [M+H]+: 380.2, found: 379.9.
步骤7：2-(2-(叔丁氧基羰基)-7-羟基-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)乙酸的合成
Step 7: 2-(2-(tert-butoxycarbonyl)-7-hydroxy-6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)acetic acid Synthesis
将P6溶于H2O+THF，并加入LiOH，将反应液移至90℃油浴中反应过夜。反应完成后，DCM萃取，无水硫酸钠干燥，旋干得黄色粉末状化合物P7。1H NMR(600MHz,CDCl3)δ6.80(s,1H),6.51(s,1H),4.04-4.01(d,J＝15.5Hz,1H),3.85–3.84(m,4H),3.50–3.49(m,1H),2.84-2.81(d,J＝15.5Hz,1H),2.79-2.75(ddd,J＝14.7,8.3,3.3Hz,1H),2.61-2.58(ddd,J＝15.1,6.5,3.1Hz,1H),1.66(s,3H),1.49(s,9H).ESI-MS calculated for[M-H]-:350.2，found:349.8。Dissolve P6 in H2O+THF, add LiOH, and move the reaction solution to 90°C oil bath to react overnight. After the reaction was completed, DCM was extracted, dried over anhydrous sodium sulfate, and spin-dried to obtain yellow powder compound P7. 1H NMR (600MHz, CDCl3) δ6.80(s, 1H), 6.51(s, 1H), 4.04-4.01(d ,J=15.5Hz,1H),3.85–3.84(m,4H),3.50–3.49(m,1H),2.84-2.81(d,J=15.5Hz,1H),2.79-2.75(ddd,J=14.7 ,8.3,3.3Hz,1H),2.61-2.58(ddd,J＝15.1,6.5,3.1Hz,1H),1.66(s,3H),1.49(s,9H).ESI-MS calculated for[M-H]- :350.2, found: 349.8.
步骤8：叔丁基7-羟基-6-甲氧基-1-甲基-1-(2-氧代-2-(噻唑-2-基氨基)乙基)-3,4-二氢异喹啉-2(1H)-羧酸盐的合成
Step 8: tert-Butyl 7-hydroxy-6-methoxy-1-methyl-1-(2-oxo-2-(thiazol-2-ylamino)ethyl)-3,4-dihydroiso Synthesis of quinoline-2(1H)-carboxylate
室温下，将HATU、DIPEA溶于NMP中，反应1小时后加入P7，随后将反应液移至80℃过夜。反应完成后，DCM萃取，旋干后的粗产物，经PE：EA过柱后得淡黄色粉末 状化合物P8。1H NMR(600MHz,MeOD)δ7.33-7.32(d,J＝3.6Hz,1H),6.99(d,J＝3.6Hz,1H),6.83(s,1H),6.58(s,1H),4.09-4.07(d,J＝14.8Hz,1H),3.92–3.86(m,1H),3.79(s,3H),3.50–3.46(m,1H),3.03-3.00(d,J＝14.8Hz,1H),2.79-2.74(ddd,J＝15.0,8.5,3.5Hz,1H),2.61-2.57(ddd,J＝15.2,6.5,3.2Hz,1H),1.75(s,3H),1.49(s,9H).ESI-MS calculated for[M+H]+:432.2，found:431.7。Dissolve HATU and DIPEA in NMP at room temperature, add P7 after reacting for 1 hour, then move the reaction solution to 80°C overnight. After the reaction was completed, DCM was extracted, the crude product was spin-dried, and the light yellow powder was obtained after passing through the column of PE:EA Compound P8. 1H NMR (600MHz, MeOD) δ7.33-7.32(d, J=3.6Hz, 1H), 6.99(d, J=3.6Hz, 1H), 6.83(s, 1H), 6.58(s, 1H), 4.09-4.07(d, J＝14.8Hz, 1H), 3.92–3.86(m, 1H), 3.79(s, 3H), 3.50–3.46(m, 1H), 3.03-3.00(d, J＝ 14.8Hz, 1H), 2.79-2.74(ddd, J＝15.0, 8.5, 3.5Hz, 1H), 2.61-2.57(ddd, J＝15.2, 6.5, 3.2Hz, 1H), 1.75(s, 3H), 1.49 (s,9H). ESI-MS calculated for [M+H]+: 432.2, found: 431.7.
步骤9：5-溴-3-(3-溴-4-羟基-5-甲氧基亚苄基)吲哚-2-酮的合成
Step 9: Synthesis of 5-bromo-3-(3-bromo-4-hydroxy-5-methoxybenzylidene)indol-2-one
室温下，5-溴氧化吲哚与5-溴香兰素溶于乙酸中，并加入醋酸钠，随后将反应液移至117℃油浴中进行反应，反应过夜后，直接将冰块加至反应液中，可析出淡黄色固体，过滤所得滤渣为粗产品。在75℃条件下，将粗产品溶于THF中，直至澄清透明，随后将溶液降至室温，并缓慢滴加石油醚直至有固体析出。待不再有固体析出后，过滤可得纯净的AN-OCH3。1H NMR(600MHz,DMSO)δ10.72(s,2H),10.35(s,2H),8.52(d,J＝1.4Hz,1H),8.30(d,J＝1.5Hz,1H),7.89(d,J＝1.6Hz,1H),7.84(s,1H),7.80(d,J＝1.4Hz,1H),7.59(s,1H),7.52(d,J＝1.3Hz,1H),7.40(dd,J＝8.3,1.8Hz,1H),7.38(d,J＝1.4Hz,1H),7.33(dd,J＝8.2,1.8Hz,1H),6.85(d,J＝8.3Hz,1H),6.79(d,J＝8.2Hz,1H),3.90(s,3H),3.88(s,3H)。At room temperature, 5-bromooxindole and 5-bromovanillin were dissolved in acetic acid, and sodium acetate was added, and then the reaction solution was moved to an oil bath at 117°C for reaction. After overnight reaction, ice cubes were directly added to In the reaction solution, a light yellow solid can be precipitated, and the filter residue obtained by filtration is a crude product. At 75°C, the crude product was dissolved in THF until it became clear and transparent, then the solution was cooled to room temperature, and petroleum ether was slowly added dropwise until a solid precipitated out. After no solid precipitates out, filter to obtain pure AN-OCH3. 1H NMR (600MHz, DMSO) δ10.72(s, 2H), 10.35(s, 2H), 8.52(d, J=1.4Hz, 1H ),8.30(d,J=1.5Hz,1H),7.89(d,J=1.6Hz,1H),7.84(s,1H),7.80(d,J=1.4Hz,1H),7.59(s,1H ),7.52(d,J=1.3Hz,1H),7.40(dd,J=8.3,1.8Hz,1H),7.38(d,J=1.4Hz,1H),7.33(dd,J=8.2,1.8Hz , 1H), 6.85 (d, J = 8.3Hz, 1H), 6.79 (d, J = 8.2Hz, 1H), 3.90 (s, 3H), 3.88 (s, 3H).
步骤11：5-溴-3-(3-溴-4-(2-(2-溴乙氧基)乙氧基)-5-甲氧基亚苄基)吲哚-2-酮合成
Step 11: Synthesis of 5-bromo-3-(3-bromo-4-(2-(2-bromoethoxy)ethoxy)-5-methoxybenzylidene)indol-2-one
氮气保护，室温条件下，将AN-OCH3溶于无水DMF中，反应0.5小时后，加入2,2'-二溴二乙醚，并继续反应2小时。反应完成后，冰水淬灭反应，EA萃取反应液，旋干 可得橙红色油状物。随后使用PE：EA纯化可得AN-OCH3-O5。1H NMR(600MHz,DMSO)δ10.47(s,2H),10.33(s,1H),8.53(d,J＝1.6Hz,2H),8.35(d,J＝1.6Hz,2H),7.95(d,J＝1.9Hz,2H),7.91(s,2H),7.84(d,J＝1.8Hz,1H),7.68(s,1H),7.54(d,J＝1.3Hz,1H),7.48(dd,J＝8.4,1.9Hz,1H),7.42(dd,J＝8.4,2.0Hz,2H),7.40(s,1H),7.15(d,J＝8.5Hz,1H),7.08(d,J＝8.4Hz,2H),3.95(dd,J＝12.2,6.6Hz,6H),3.91(s,6H),3.88(s,3H),3.76-3.66(m,12H),3.52(dd,J＝10.9,5.2Hz,6H)。Under nitrogen protection, AN-OCH3 was dissolved in anhydrous DMF at room temperature. After reacting for 0.5 hours, 2,2'-dibromodiethyl ether was added and the reaction was continued for 2 hours. After the reaction was completed, the reaction was quenched with ice water, the reaction solution was extracted with EA, and spin-dried An orange-red oily substance can be obtained. Subsequent purification using PE:EA gave AN-OCH3-O5. 1H NMR (600MHz, DMSO) δ10.47(s, 2H), 10.33(s, 1H), 8.53(d, J=1.6Hz, 2H), 8.35 (d,J=1.6Hz,2H),7.95(d,J=1.9Hz,2H),7.91(s,2H),7.84(d,J=1.8Hz,1H),7.68(s,1H),7.54 (d,J=1.3Hz,1H),7.48(dd,J=8.4,1.9Hz,1H),7.42(dd,J=8.4,2.0Hz,2H),7.40(s,1H),7.15(d, J＝8.5Hz, 1H), 7.08(d, J＝8.4Hz, 2H), 3.95(dd, J＝12.2, 6.6Hz, 6H), 3.91(s, 6H), 3.88(s, 3H), 3.76- 3.66 (m, 12H), 3.52 (dd, J=10.9, 5.2Hz, 6H).
步骤12：叔丁基7-(2-(5-溴-3-(3-溴-4-羟基-5-甲氧基亚苄基)-2-氧代吲哚-1-基)乙氧基)-6-甲氧基-1-甲基-1-(2-氧代-2-(噻唑-2-基氨基)乙基)-3,4-二氢异喹啉-2(1H)-羧酸酯的合成
Step 12: tert-butyl 7-(2-(5-bromo-3-(3-bromo-4-hydroxy-5-methoxybenzylidene)-2-oxoindol-1-yl)ethoxy Base)-6-methoxy-1-methyl-1-(2-oxo-2-(thiazol-2-ylamino)ethyl)-3,4-dihydroisoquinoline-2(1H) -Synthesis of Carboxylate
氮气保护，室温条件下，将P8溶于无水DMF中，反应0.5小时后，加入AN-OCH3-O5后，移至75℃条件下反应过夜。反应完成后，冰水淬灭反应，EA萃取反应液，旋干可得橙红色油状物。随后使用DCM：MeOH纯化可得AN-OCH3-O5-P8。ESI-MS calculated for[M+H]+:928.1，found:928.4。Under nitrogen protection, P8 was dissolved in anhydrous DMF at room temperature. After 0.5 hours of reaction, AN-OCH3-O5 was added, and then moved to 75°C for overnight reaction. After the reaction was completed, the reaction was quenched with ice water, the reaction solution was extracted with EA, and spin-dried to obtain an orange-red oil. Subsequent purification using DCM:MeOH afforded AN-OCH3-O5-P8. ESI-MS calculated for [M+H]+: 928.1, found: 928.4.
步骤13:2-(7-(2-(2-(5-溴-3-(3-溴-4-羟基-5-甲氧基亚苄基)-2-氧代吲哚-1-基)乙氧基)-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)-N-(噻唑-2-基)乙酰胺的合成
Step 13: 2-(7-(2-(2-(5-bromo-3-(3-bromo-4-hydroxy-5-methoxybenzylidene)-2-oxoindol-1-yl Synthesis of )ethoxy)-6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide
将AN-OCH3-O5-P8溶于DCM中，并加入TFA，室温下反应0.5小时。旋干反应液DCM：MeOH纯化可得终产物I-1。1H NMR(600MHz,CDCl3)δ8.76(d,J＝1.2Hz,1H), 7.88(d,J＝1.6Hz,2H),7.75(d,J＝1.4Hz,1H),7.73(s,2H),7.56(d,J＝1.7Hz,1H),7.42(s,2H),7.40(d,J＝3.5Hz,3H),7.32(s,1H),7.23(dd,J＝8.4,1.7Hz,2H),7.21(dd,J＝8.3,1.7Hz,1H),7.16(d,J＝1.2Hz,2H),6.92(d,J＝8.4Hz,2H),6.89(s,2H),6.88(s,1H),6.88(s,1H),6.70(s,2H),6.62(s,1H),6.53(s,2H),6.50(s,1H),4.14-4.06(m,4H),4.05(dd,J＝9.1,4.6Hz,2H),4.01(s,3H),3.98(dt,J＝14.7,5.7Hz,5H),3.93(s,6H),3.85-3.78(m,12H),3.77(s,6H),3.75(s,3H),3.30(dd,J＝12.6,6.0Hz,3H),3.23-3.15(m,3H),3.04(d,J＝16.0Hz,2H),2.98(d,J＝16.1Hz,1H),2.88(td,J＝11.0,5.7Hz,3H),2.78-2.72(m,3H),2.70(d,J＝17.3Hz,2H),2.62(d,J＝16.1Hz,1H),1.55(s,5H),1.50(s,3H).13C NMR(151MHz,CDCl3)δ168.72,168.65,168.17,165.93,158.00,148.52,148.46,147.16,146.85,146.13,145.12,142.87,140.56,137.49,137.40,137.34,131.98,131.88,131.01,130.83,127.21,127.17,127.09,127.01,126.26,125.25,125.08,123.29,122.77,121.42,114.39,114.13,113.45,113.08,112.13,111.77,111.54,111.12,110.74,110.71,108.75,107.73,69.61,69.33,69.18,69.10,68.96,56.62,56.53,55.78,55.75,54.80,54.64,46.54,40.45,40.37,38.33,29.67,29.32,29.29,29.17,29.14.ESI-MS calculated for[M+H]+:829.0727，found:829.0706。Dissolve AN-OCH3-O5-P8 in DCM, add TFA, and react at room temperature for 0.5 hours. The reaction liquid was spin-dried to DCM: purified by MeOH to obtain the final product I-1. 1H NMR (600MHz, CDCl3) δ8.76 (d, J=1.2Hz, 1H), 7.88(d, J=1.6Hz, 2H), 7.75(d, J=1.4Hz, 1H), 7.73(s, 2H), 7.56(d, J=1.7Hz, 1H), 7.42(s, 2H), 7.40(d, J=3.5Hz, 3H), 7.32(s, 1H), 7.23(dd, J=8.4, 1.7Hz, 2H), 7.21(dd, J=8.3, 1.7Hz, 1H), 7.16(d ,J=1.2Hz,2H),6.92(d,J=8.4Hz,2H),6.89(s,2H),6.88(s,1H),6.88(s,1H),6.70(s,2H),6.62 (s,1H),6.53(s,2H),6.50(s,1H),4.14-4.06(m,4H),4.05(dd,J=9.1,4.6Hz,2H),4.01(s,3H), 3.98(dt,J=14.7,5.7Hz,5H),3.93(s,6H),3.85-3.78(m,12H),3.77(s,6H),3.75(s,3H),3.30(dd,J= 12.6,6.0Hz,3H),3.23-3.15(m,3H),3.04(d,J=16.0Hz,2H),2.98(d,J=16.1Hz,1H),2.88(td,J=11.0,5.7 Hz, 3H), 2.78-2.72(m, 3H), 2.70(d, J=17.3Hz, 2H), 2.62(d, J=16.1Hz, 1H), 1.55(s, 5H), 1.50(s, 3H ).13C NMR (151MHz, CDCl3) δ168.72, 168.65, 168.17, 165.93, 158.00, 148.52, 148.46, 147.16, 146.85, 146.13, 145.12, 142.87, 140.56, 137.49, 137.4 0,137.34,131.98,131.88,131.01,130.83,127.21 ,127.17,127.09,127.01,126.26,125.25,125.08,123.29,122.77,121.42,114.39,114.13,113.45,113.08,112.13,111.77,111.54,111.12,1 10.74, 110.71, 108.75, 107.73, 69.61, 69.33, 69.18, 69.10 ,68.96,56.62,56.53,55.78,55.75,54.80,54.64,46.54,40.45,40.37,38.33,29.67,29.32,29.29,29.17,29.14.ESI-MS calculated for[M+H]+:829.072 7, found: 829.0706 .
实施例二：化合物I-2的制备Embodiment two: the preparation of compound 1-2
2-(7-(2-(2-(3,5-二溴-4-羟基亚苄基)-5-溴代-2-氧代吲哚-1-基)乙氧基)-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)-N-(噻唑-2-基)乙酰胺的合成
2-(7-(2-(2-(3,5-dibromo-4-hydroxybenzylidene)-5-bromo-2-oxoindol-1-yl)ethoxy)-6- Synthesis of Methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide
参照实施例一I-1的合成路线。I-2：1H NMR(600MHz,DMSO)δ8.77(s,10H),7.82(s,2H),7.77(d,J＝1.8Hz,4H),7.58(s,4H),7.48(s,1H),7.48(s,4H),7.27(dd,J＝8.4,1.7Hz,1H),7.24(d,J＝3.5Hz,5H),7.15(dd,J＝8.3,1.8Hz,4H),7.03(d,J＝8.4Hz,1H),6.93(d,J＝8.3Hz,4H),6.82(s,1H),6.81(s,4H),6.73(s,1H),6.72(s,4H),4.03(dd,J＝10.2,5.2Hz,5H),3.98(dd,J＝10.2,5.2Hz,5H),3.93(dd,J＝9.6,5.6Hz,8H),3.90(d,J＝6.0Hz,2H),3.73(t,J＝4.5Hz,10H),3.71–3.66(m,25H),3.50(d,J＝2.8Hz,2H),3.41(s,8H),3.19(s,10H),2.95(d,J＝5.7Hz,1H),2.92(d,J＝6.6Hz,4H),2.85(s,4H),2.82(s,1H),1.64(s,15H).13C NMR(151MHz,DMSO)δ168.54,165.57,157.04,148.39,146.85,139.09,138.08,137.87,134.79,128.39, 127.04,124.38,119.74,114.90,114.07,113.00,112.10,110.29,109.90,68.81,68.17,68.08,57.40,55.54,43.83,37.58,29.10,28.78,25.91,22.17,14.04.ESI-MS calculated for[M+H]+:876.9725，found:876.9695。Refer to the synthetic route of Example 1-1. I-2: 1H NMR (600MHz, DMSO) δ8.77(s, 10H), 7.82(s, 2H), 7.77(d, J=1.8Hz, 4H), 7.58(s, 4H), 7.48(s, 1H),7.48(s,4H),7.27(dd,J=8.4,1.7Hz,1H),7.24(d,J=3.5Hz,5H),7.15(dd,J=8.3,1.8Hz,4H), 7.03(d,J=8.4Hz,1H),6.93(d,J=8.3Hz,4H),6.82(s,1H),6.81(s,4H),6.73(s,1H),6.72(s,4H ),4.03(dd,J=10.2,5.2Hz,5H),3.98(dd,J=10.2,5.2Hz,5H),3.93(dd,J=9.6,5.6Hz,8H),3.90(d,J= 6.0Hz, 2H), 3.73(t, J=4.5Hz, 10H), 3.71–3.66(m, 25H), 3.50(d, J=2.8Hz, 2H), 3.41(s, 8H), 3.19(s, 10H), 2.95(d, J=5.7Hz, 1H), 2.92(d, J=6.6Hz, 4H), 2.85(s, 4H), 2.82(s, 1H), 1.64(s, 15H).13C NMR (151MHz,DMSO)δ168.54,165.57,157.04,148.39,146.85,139.09,138.08,137.87,134.79,128.39, 127.04, 124.38, 119.74, 114.90, 114.07, 113.00, 112.10, 110.29, 109.90, 68.81, 68.17, 68.08, 57.40, 55.54, 43.83, 37.58, 29.10, 28.78, 25.9 1,22.17,14.04.ESI-MS calculated for [M+ H]+: 876.9725, found: 876.9695.
实施例三：化合物I-3的制备Embodiment three: the preparation of compound I-3
2-(7-(4-(5-溴-3-(3-溴-4-羟基-5-甲氧基亚苄基)-2-氧代吲哚-1-基)丁氧基)-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)-N-(噻唑-2-基)乙酰胺的合成
2-(7-(4-(5-bromo-3-(3-bromo-4-hydroxy-5-methoxybenzylidene)-2-oxoindol-1-yl)butoxy)- Synthesis of 6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide
参照实施例一I-1的合成路线。I-3：1H NMR(600MHz,CDCl3)δ8.78(s,1H),7.91(d,J＝1.6Hz,2H),7.76(s,1H),7.75(s,2H),7.60(d,J＝1.6Hz,1H),7.43(s,2H),7.40(d,J＝3.5Hz,3H),7.35(dd,J＝8.4,1.7Hz,2H),7.34(s,1H),7.33–7.31(m,1H),7.17(d,J＝1.2Hz,2H),6.88(t,J＝3.4Hz,3H),6.80(d,J＝8.3Hz,2H),6.76(d,J＝8.3Hz,1H),6.69(s,2H),6.63(s,1H),6.52(s,2H),6.51(s,1H),4.05–3.99(m,9H),3.94(s,6H),3.91–3.83(m,6H),3.78(s,6H),3.77(s,3H),3.32–3.27(m,3H),3.19(dd,J＝12.8,5.2Hz,3H),3.02(dd,J＝23.2,16.0Hz,3H),2.91–2.85(m,3H),2.78–2.68(m,5H),2.65(d,J＝16.0Hz,1H),1.93–1.85(m,12H),1.56(s,6H),1.52(s,3H).13C NMR(151MHz,CDCl3)δ168.66,168.57,167.87,165.63,157.91,148.35,148.26,147.06,146.78,146.05,145.01,142.33,139.97,137.53,137.36,137.24,131.93,131.80,131.75,130.92,130.78,127.12,127.06,126.93,126.73,126.63,126.41,125.47,125.01,123.24,122.90,121.63,114.31,114.06,113.40,112.97,111.94,111.24,110.89,110.61,110.05,109.63,108.66,107.62,68.75,56.55,56.43,55.67,54.72,54.60,46.54,46.45,39.62,38.25,29.57,29.53,29.20,29.06,26.35,26.28,24.13,24.06.ESI-MS calculated for[M+H]+:813.0778，found:813.0775。Refer to the synthetic route of Example 1-1. I-3: 1H NMR (600MHz, CDCl3) δ8.78(s, 1H), 7.91(d, J=1.6Hz, 2H), 7.76(s, 1H), 7.75(s, 2H), 7.60(d, J＝1.6Hz, 1H), 7.43(s, 2H), 7.40(d, J＝3.5Hz, 3H), 7.35(dd, J＝8.4, 1.7Hz, 2H), 7.34(s, 1H), 7.33– 7.31(m, 1H), 7.17(d, J=1.2Hz, 2H), 6.88(t, J=3.4Hz, 3H), 6.80(d, J=8.3Hz, 2H), 6.76(d, J=8.3 Hz,1H),6.69(s,2H),6.63(s,1H),6.52(s,2H),6.51(s,1H),4.05–3.99(m,9H),3.94(s,6H),3.91 –3.83(m,6H),3.78(s,6H),3.77(s,3H),3.32–3.27(m,3H),3.19(dd,J＝12.8,5.2Hz,3H),3.02(dd,J ＝23.2,16.0Hz,3H),2.91–2.85(m,3H),2.78–2.68(m,5H),2.65(d,J＝16.0Hz,1H),1.93–1.85(m,12H),1.56( s,6H),1.52(s,3H).13C NMR(151MHz,CDCl3)δ168.66,168.57,167.87,165.63,157.91,148.35,148.26,147.06,146.78,146.05,145.01,142.33,139 .97, 137.53, 137.36, 137.24 ,131.93,131.80,131.75,130.92,130.78,127.12,127.06,126.93,126.73,126.63,126.41,125.47,125.01,123.24,122.90,121.63,114.31,1 14.06, 113.40, 112.97, 111.94, 111.24, 110.89, 110.61, 110.05 . 13,24.06.ESI-MS calculated for [M +H]+: 813.0778, found: 813.0775.
实施例四：化合物I-4的制备Embodiment four: the preparation of compound I-4
2-(7-(4-(3-(3,5-二溴-4-羟基苄叉)-5-溴代-2-氧代吲哚-1-基)丁氧基)-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)-N-(噻唑-2-基)乙酰胺的合成
2-(7-(4-(3-(3,5-dibromo-4-hydroxybenzylidene)-5-bromo-2-oxoindol-1-yl)butoxy)-6-methyl Synthesis of Oxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide
参照实施例一I-1的合成路线。I-4：1H NMR(600MHz,DMSO)δ8.80(s,6H),7.86(s,1H),7.83(s,1H),7.80(d,J＝1.8Hz,2H),7.60(s,2H),7.48(s,1H),7.48(d,J＝3.5Hz,2H),7.36(dd,J＝8.3,1.5Hz,1H),7.24(d,J＝3.5Hz,3H),7.22(dd,J＝8.3,1.8Hz,2H),7.02(d,J＝8.4Hz,1H),6.93(d,J＝8.3Hz,2H),6.83(s,2H),6.83(s,1H),6.72(s,3H),4.02–3.96(m,4H),3.91(d,J＝9.6Hz,4H),3.81(d,J＝8.6Hz,7H),3.71(s,6H),3.70(s,3H),3.51(s,2H),3.22(s,1H),3.17(t,J＝17.1Hz,6H),2.95–2.89(m,3H),2.84(d,J＝16.8Hz,3H),1.72(d,J＝4.9Hz,12H),1.63(s,9H).13C NMR(151MHz,DMSO)δ168.51,165.54,157.06,148.54,146.88,139.14,137.94,137.85,137.79,128.54,127.06,124.40,124.34,119.86,114.96,114.03,113.00,112.01,110.64,109.42,69.86,68.39,55.54,43.91,37.58,31.37,31.23,29.90,29.09,28.78,26.19,24.19,22.17,14.04.ESI-MS calculated for[M+H]+:860.9776，found:860.9756。Refer to the synthetic route of Example 1-1. I-4: 1H NMR (600MHz, DMSO) δ8.80(s,6H),7.86(s,1H),7.83(s,1H),7.80(d,J＝1.8Hz,2H),7.60(s, 2H),7.48(s,1H),7.48(d,J=3.5Hz,2H),7.36(dd,J=8.3,1.5Hz,1H),7.24(d,J=3.5Hz,3H),7.22( dd,J=8.3,1.8Hz,2H),7.02(d,J=8.4Hz,1H),6.93(d,J=8.3Hz,2H),6.83(s,2H),6.83(s,1H), 6.72(s,3H),4.02–3.96(m,4H),3.91(d,J=9.6Hz,4H),3.81(d,J=8.6Hz,7H),3.71(s,6H),3.70(s ,3H),3.51(s,2H),3.22(s,1H),3.17(t,J＝17.1Hz,6H),2.95–2.89(m,3H),2.84(d,J＝16.8Hz,3H) ,1.72(d,J＝4.9Hz,12H),1.63(s,9H).13C NMR(151MHz,DMSO)δ168.51,165.54,157.06,148.54,146.88,139.14,137.94,137.85,137.79,128.54,127. 06,124.40 ,124.34,119.86,114.96,114.03,113.00,112.01,110.64,109.42,69.86,68.39,55.54,43.91,37.58,31.37,31.23,29.90,29.09,28.78,26.1 9,24.19,22.17,14.04.ESI-MS calculated for [M+H]+: 860.9776, found: 860.9756.
实施例五：化合物I-5的制备Embodiment five: the preparation of compound 1-5
2-(7-(2-(2-(3,5-二溴-4-羟基亚苄基)-5-碘代-2-氧代吲哚-1-基)乙氧基)-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)-N-(噻唑-2-基)乙酰胺的合成
2-(7-(2-(2-(3,5-dibromo-4-hydroxybenzylidene)-5-iodo-2-oxoindol-1-yl)ethoxy)-6- Synthesis of Methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide
参照实施例一I-1的合成路线。I-5：1H NMR(600MHz,DMSO)δ8.83(d,J＝52.6Hz,2H),8.08(s,1H),7.93(s,1H),7.81(s,2H),7.57(s,1H),7.49(d,J＝8.1Hz,1H),7.45(s,3H),7.38(d,J＝8.1Hz,1H),7.21(s,2H),6.89(d,J＝8.2Hz,1H),6.81(d,J＝11.9Hz,3H),6.69(s,1H),6.67(s,1H),3.96(s,2H),3.90(s,2H),3.84–3.74(m,4H),3.73(s,1H),3.69(s,7H),3.25(s,2H),3.17(s,1H),3.13(s,3H),2.89(d,J＝16.5Hz,2H),2.80(d,J＝15.5Hz,2H),1.69(s,8H), 1.59(s,6H).13C NMR(151MHz,DMSO)δ168.63,167.69,165.34,157.12,148.36,148.28,146.73,140.95,139.03,138.94,138.23,137.77,134.90,132.86,128.91,128.65,125.37,124.67,117.38,115.06,114.81,113.85,112.02,110.82,110.76,109.98,83.94,68.39,68.21,56.83,55.51,44.33,37.62,26.51,26.22,26.11,24.19,23.95.ESI-MS calculated for[M+H]+:924.9588，found:924.9581。Refer to the synthetic route of Example 1-1. I-5: 1H NMR (600MHz, DMSO) δ8.83(d, J=52.6Hz, 2H), 8.08(s, 1H), 7.93(s, 1H), 7.81(s, 2H), 7.57(s, 1H), 7.49(d, J=8.1Hz, 1H), 7.45(s, 3H), 7.38(d, J=8.1Hz, 1H), 7.21(s, 2H), 6.89(d, J=8.2Hz, 1H), 6.81(d, J=11.9Hz, 3H), 6.69(s, 1H), 6.67(s, 1H), 3.96(s, 2H), 3.90(s, 2H), 3.84–3.74(m, 4H ),3.73(s,1H),3.69(s,7H),3.25(s,2H),3.17(s,1H),3.13(s,3H),2.89(d,J＝16.5Hz,2H),2.80 (d,J=15.5Hz,2H),1.69(s,8H), 1.59(s,6H).13C NMR(151MHz,DMSO)δ168.63,167.69,165.34,157.12,148.36,148.28,146.73,140.95,139.03,138.94,138.23,137.77,134.90,132. 86,128.91,128.65,125.37,124.67, 117.38, 115.06, 114.81, 113.85, 112.02, 110.82, 110.76, 109.98, 83.94, 68.39, 68.21, 56.83, 55.51, 44.33, 37.62, 26.51, 26.22, 26.11, 24.19 ,23.95.ESI-MS calculated for[M+H] +: 924.9588, found: 924.9581.
实施例六：化合物I-6的制备Embodiment six: the preparation of compound I-6
2-(7-(4-(3-(3,5-二溴-4-羟基苄叉)-5-碘代-2-氧代吲哚-1-基)丁氧基)-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)-N-(噻唑-2-基)乙酰胺的合成
2-(7-(4-(3-(3,5-dibromo-4-hydroxybenzylidene)-5-iodo-2-oxoindol-1-yl)butoxy)-6-methyl Synthesis of Oxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide
参照实施例一I-1的合成路线。I-6：1H NMR(600MHz,DMSO)δ8.83(d,J＝52.6Hz,2H),8.08(s,1H),7.93(s,1H),7.81(s,2H),7.57(s,1H),7.49(d,J＝8.1Hz,1H),7.45(s,3H),7.38(d,J＝8.1Hz,1H),7.21(s,2H),6.89(d,J＝8.2Hz,1H),6.81(d,J＝11.9Hz,3H),6.69(s,1H),6.67(s,1H),3.96(s,2H),3.90(s,2H),3.84–3.74(m,4H),3.73(s,1H),3.69(s,7H),3.25(s,2H),3.17(s,1H),3.13(s,3H),2.89(d,J＝16.5Hz,2H),2.80(d,J＝15.5Hz,2H),1.69(s,8H),1.59(s,6H).13C NMR(151MHz,DMSO)δ168.63,167.69,165.34,157.12,148.36,148.28,146.73,140.95,139.03,138.94,138.23,137.77,134.90,132.86,128.91,128.65,125.37,124.67,117.38,115.06,114.81,113.85,112.02,110.82,110.76,109.98,83.94,68.39,68.21,56.83,55.51,44.33,37.62,26.51,26.22,26.11,24.19,23.95.ESI-MS calculated for[M+H]+:908.9639，found:908.9644。Refer to the synthetic route of Example 1-1. I-6: 1H NMR (600MHz, DMSO) δ8.83(d, J=52.6Hz, 2H), 8.08(s, 1H), 7.93(s, 1H), 7.81(s, 2H), 7.57(s, 1H), 7.49(d, J=8.1Hz, 1H), 7.45(s, 3H), 7.38(d, J=8.1Hz, 1H), 7.21(s, 2H), 6.89(d, J=8.2Hz, 1H), 6.81(d, J=11.9Hz, 3H), 6.69(s, 1H), 6.67(s, 1H), 3.96(s, 2H), 3.90(s, 2H), 3.84–3.74(m, 4H ),3.73(s,1H),3.69(s,7H),3.25(s,2H),3.17(s,1H),3.13(s,3H),2.89(d,J＝16.5Hz,2H),2.80 (d,J＝15.5Hz,2H),1.69(s,8H),1.59(s,6H).13C NMR(151MHz,DMSO)δ168.63,167.69,165.34,157.12,148.36,148.28,146.73,140.95,139.03, 138.94,138.23,137.77,134.90,132.86,128.91,128.65,125.37,124.67,117.38,115.06,114.81,113.85,112.02,110.82,110.76,109.98,83 .94,68.39,68.21,56.83,55.51,44.33,37.62,26.51, 26.22, 26.11, 24.19, 23.95. ESI-MS calculated for [M+H]+: 908.9639, found: 908.9644.
实施例七：化合物I-7的制备Embodiment seven: the preparation of compound I-7
2-(7-((5-(5-溴-3-(3-溴-4-羟基-5-甲氧基亚苄基)-2-氧代吲哚-1-基)戊基)氧基)-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)-N-(噻唑-2-基)乙酰胺的合成
2-(7-((5-(5-bromo-3-(3-bromo-4-hydroxy-5-methoxybenzylidene)-2-oxoindol-1-yl)pentyl)oxy Synthesis of -6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide
参照实施例一I-1的合成路线。I-7：ESI-MS calculated for[M+H]+:826.1，found:826.5。Refer to the synthetic route of Example 1-1. I-7: ESI-MS calculated for [M+H]+: 826.1, found: 826.5.
实施例八：化合物I-8的制备Embodiment eight: the preparation of compound I-8
2-(7-((8-(5-溴-3-(3-溴-4-羟基-5-甲氧基亚苄基)-2-氧代吲哚-1-基)辛基)氧基)-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)-N-(噻唑-2-基)乙酰胺的合成
2-(7-((8-(5-bromo-3-(3-bromo-4-hydroxy-5-methoxybenzylidene)-2-oxoindol-1-yl)octyl)oxy Synthesis of -6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide
参照实施例一I-1的合成路线。I-8：ESI-MS calculated for[M+H]+:869.1405，found:869.1403。Refer to the synthetic route of Example 1-1. I-8: ESI-MS calculated for [M+H]+: 869.1405, found: 869.1403.
实施例九：化合物I-9的制备Embodiment nine: the preparation of compound I-9
2-(7-(2-(2-(2-(2-(5-溴-3-(3-溴-4-羟基-5-甲氧基苄叉)-2-氧代吲哚-1-基)乙氧基)乙氧基)乙氧基)-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)-N-(噻唑-2-基)乙酰胺的合成
2-(7-(2-(2-(2-(2-(5-bromo-3-(3-bromo-4-hydroxy-5-methoxybenzylidene)-2-oxoindole-1 -yl)ethoxy)ethoxy)ethoxy)-6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazole Synthesis of -2-yl)acetamide
参照实施例一I-1的合成路线。I-9：1H NMR(600MHz,DMSO+CDCl3)δ8.38(dd,J＝66.8,15.5Hz,6H),7.94(d,J＝15.5Hz,1H),7.75(d,J＝11.3Hz,2H),7.60(d,J＝10.2Hz,2H),7.52(d,J＝11.4Hz,1H),7.39(d,J＝2.2Hz,3H),7.19-7.14(m,3H),7.12(d,J＝3.2Hz,3H),7.04(d,J＝7.5Hz,1H),6.94(d,J＝8.3Hz,2H),6.83(s,3H),6.58(s,3H),3.93-3.81(m,12H),3.74(s,9H),3.71-3.63(m,15H),3.63-3.60(m,6H),3.50(d,J＝5.1Hz,12H),3.48-3.44(m,12H),3.11-3.05(m,3H),3.02(d,J＝6.1Hz,3H),2.96(d,J＝15.1Hz,3H),2.78(dd,J＝14.7,6.7Hz,3H),2.75-2.67(m,3H),2.67-2.59(m,3H),1.39(s,9H).13C NMR(151MHz,DMSO)δ169.24,165.52,157.35,149.84,147.39,146.18,140.29,137.88,137.61,137.51,132.97,128.75,127.09,126.50,119.32,113.21,112.85,112.18,111.25,109.80,72.26,69.94,69.88,69.78,69.01,68.12,68.01,59.82,55.37,54.76,45.93,37.88,29.03,28.77,26.41,20.83,14.15.ESI-MS calculated for[M+H]+:917.1252，found:917.1261。Refer to the synthetic route of Example 1-1. I-9: 1H NMR (600MHz, DMSO+CDCl3) δ8.38 (dd, J = 66.8, 15.5Hz, 6H), 7.94 (d, J = 15.5Hz, 1H), 7.75 (d, J = 11.3Hz, 2H), 7.60(d, J=10.2Hz, 2H), 7.52(d, J=11.4Hz, 1H), 7.39(d, J=2.2Hz, 3H), 7.19-7.14(m, 3H), 7.12( d,J=3.2Hz,3H),7.04(d,J=7.5Hz,1H),6.94(d,J=8.3Hz,2H),6.83(s,3H),6.58(s,3H),3.93- 3.81(m,12H),3.74(s,9H),3.71-3.63(m,15H),3.63-3.60(m,6H),3.50(d,J＝5.1Hz,12H),3.48-3.44(m, 12H), 3.11-3.05(m, 3H), 3.02(d, J=6.1Hz, 3H), 2.96(d, J=15.1Hz, 3H), 2.78(dd, J=14.7, 6.7Hz, 3H), 2.75-2.67(m,3H),2.67-2.59(m,3H),1.39(s,9H).13C NMR(151MHz,DMSO)δ169.24,165.52,157.35,149.84,147.39,146.18,140.29,137.88,137.61 , 137.51, 132.97, 128.75, 127.09, 126.50, 119.32, 113.21, 112.85, 112.18, 111.25, 109.80, 72.26, 69.94, 69.88, 69.78, 69.01, 68.12, 68.01, 59 .82,55.37,54.76,45.93,37.88,29.03,28.77, 26.41, 20.83, 14.15. ESI-MS calculated for [M+H]+: 917.1252, found: 917.1261.
实施例十：化合物I-10的制备Embodiment 10: Preparation of Compound I-10
2-(7-((5-(5-溴-3-(3,5-二溴-4-羟基亚苄基)-2-氧代吲哚-1-基)戊基)氧基)-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)-N-(噻唑-2-基)乙酰胺的合成
2-(7-((5-(5-bromo-3-(3,5-dibromo-4-hydroxybenzylidene)-2-oxoindol-1-yl)pentyl)oxy)- Synthesis of 6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide
参照实施例一I-1的合成路线。1H NMR(600MHz,DMSO)δ8.77(s,2H),7.90(s,1H),7.81(s,2H),7.78(s,1H),7.57(s,1H),7.45(s,1H),7.39(s,2H),7.32(d,J＝8.3Hz,1H),7.20(d,J＝8.1Hz,1H),7.12(s,2H),6.99(d,J＝8.2Hz,1H),6.90(d,J＝8.1Hz,1H),6.81(s,2H),6.58(s,2H),3.86(dd,J＝12.9,5.0Hz,4H),3.80-3.71(m,4H),3.65(s,6H),3.11(d,J＝11.8Hz,2H),3.04(d,J＝5.8Hz,2H),2.98(s,1H),2.96(s,1H),2.83(s,1H),2.81(s,1H),2.71(s,2H),2.65(d,J＝ 15.2Hz,2H),1.69(s,4H),1.66–1.60(m,4H),1.44(s,6H),1.42-1.36(m,4H).13C NMR(151MHz,DMSO)δ169.16,167.97,166.09,165.52,165.42,157.32,147.54,146.42,140.40,139.52,139.28,137.69,137.61,135.07,132.60,128.84,128.66,126.84,126.61,124.39,122.59,119.64,116.81,115.23,115.13,114.52,113.94,113.25,112.87,112.68,112.20,111.18,111.14,111.02,110.09,109.34,72.34,68.43,60.30,55.42,54.99,48.66,45.79,37.84,31.35,29.09,28.80,28.76,28.70,28.58,27.44,27.19,23.15,22.98,22.16,14.02.I-10：ESI-MS calculated for[M+H]+:874.9933，found:874.9936。Refer to the synthetic route of Example 1-1. 1H NMR(600MHz,DMSO)δ8.77(s,2H),7.90(s,1H),7.81(s,2H),7.78(s,1H),7.57(s,1H),7.45(s,1H) ,7.39(s,2H),7.32(d,J=8.3Hz,1H),7.20(d,J=8.1Hz,1H),7.12(s,2H),6.99(d,J=8.2Hz,1H) ,6.90(d,J=8.1Hz,1H),6.81(s,2H),6.58(s,2H),3.86(dd,J=12.9,5.0Hz,4H),3.80-3.71(m,4H), 3.65(s,6H),3.11(d,J=11.8Hz,2H),3.04(d,J=5.8Hz,2H),2.98(s,1H),2.96(s,1H),2.83(s,1H ),2.81(s,1H),2.71(s,2H),2.65(d,J＝ 15.2Hz,2H),1.69(s,4H),1.66–1.60(m,4H),1.44(s,6H),1.42-1.36(m,4H).13C NMR(151MHz,DMSO)δ169.16,167.97,166.09 ,165.52,165.42,157.32,147.54,146.42,140.40,139.52,139.28,137.69,137.61,135.07,132.60,128.84,128.66,126.84,126.61,124.39,1 22.59, 119.64, 116.81, 115.23, 115.13, 114.52, 113.94, 113.25 ,112.87,112.68,112.20,111.18,111.14,111.02,110.09,109.34,72.34,68.43,60.30,55.42,54.99,48.66,45.79,37.84,31.35,29.09,28.8 0,28.76,28.70,28.58,27.44,27.19,23.15 , 22.98, 22.16, 14.02. I-10: ESI-MS calculated for [M+H]+: 874.9933, found: 874.9936.
实施例十一：化合物I-11的制备Embodiment 11: Preparation of Compound I-11
2-(7-((8-(5-溴-3-(3,5-二溴-4-羟基亚苄基)-2-氧代吲哚-1-基)辛基)氧基)-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)-N-(噻唑-2-基)乙酰胺的合成
2-(7-((8-(5-bromo-3-(3,5-dibromo-4-hydroxybenzylidene)-2-oxoindol-1-yl)octyl)oxy)- Synthesis of 6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide
参照实施例一I-1的合成路线。I-11：ESI-MS calculated for[M+H]+:917.0403，found:917.0406。Refer to the synthetic route of Example 1-1. I-11: ESI-MS calculated for [M+H]+: 917.0403, found: 917.0406.
实施例十二：化合物I-12的制备Embodiment 12: Preparation of Compound I-12
2-(7-(2-(2-(2-(2-(5-溴-3-(3,5-二溴-4-羟基亚苄基)-2-氧吲哚-1-基)乙氧基)乙氧基)-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)-N-(噻唑-2-基)乙酰胺的合成
2-(7-(2-(2-(2-(2-(5-bromo-3-(3,5-dibromo-4-hydroxybenzylidene)-2-oxindol-1-yl) Ethoxy)ethoxy)-6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide Synthesis
参照实施例一I-1的合成路线。I-12：1H NMR(600MHz,DMSO)δ8.80(s,2H),8.55(s,1H),7.89(d,J＝6.5Hz,2H),7.81(s,1H),7.77(s,2H),7.58(s,2H),7.46(s,1H),7.42(s,3H),7.31(t,J＝9.1Hz,2H),7.19(d,J＝8.2Hz,1H),7.15(s,2H),7.03(d,J＝8.3Hz,1H),6.94(d,J＝ 8.1Hz,1H),6.89(s,1H),6.83(s,2H),6.63(d,J＝8.2Hz,3H),6.59(d,J＝8.2Hz,1H),4.00(dd,J＝14.9,9.5Hz,6H),3.93-3.87(m,6H),3.68(s,3H),3.67(s,6H),3.65-3.60(m,12H),3.52(d,J＝8.8Hz,12H),3.45(s,12H),3.23(d,J＝4.8Hz,3H),3.13(s,3H),3.06(d,J＝15.1Hz,3H),2.99(dd,J＝15.0,8.1Hz,3H),2.84-2.75(m,3H),2.70(d,J＝16.0Hz,3H),1.53(s,3H),1.52(s,6H).13C NMR(151MHz,DMSO)δ170.44,169.46,168.45,166.39,166.02,165.61,157.71,157.67,149.86,148.08,146.81,141.18,139.79,139.66,138.48,138.37,138.07,135.40,135.02,134.14,131.85,131.65,129.31,128.85,127.33,126.58,126.44,124.58,122.95,120.02,117.60,115.57,115.46,115.42,114.86,113.87,113.35,113.27,113.14,112.49,111.89,111.33,111.21,111.10,110.32,104.80,70.29,70.23,70.17,70.13,69.44,69.39,69.36,68.52,68.46,68.28,68.20,56.19,56.08,56.01,55.80,55.36,49.08,45.63,45.48,42.62,38.16,38.09,28.30,28.23,28.15,28.05.ESI-MS calculated for[M+H]+:965.0250，found:965.0251。Refer to the synthetic route of Example 1-1. I-12: 1H NMR (600MHz, DMSO) δ8.80(s, 2H), 8.55(s, 1H), 7.89(d, J=6.5Hz, 2H), 7.81(s, 1H), 7.77(s, 2H), 7.58(s, 2H), 7.46(s, 1H), 7.42(s, 3H), 7.31(t, J=9.1Hz, 2H), 7.19(d, J=8.2Hz, 1H), 7.15( s, 2H), 7.03(d, J=8.3Hz, 1H), 6.94(d, J= 8.1Hz,1H),6.89(s,1H),6.83(s,2H),6.63(d,J=8.2Hz,3H),6.59(d,J=8.2Hz,1H),4.00(dd,J= 14.9,9.5Hz,6H),3.93-3.87(m,6H),3.68(s,3H),3.67(s,6H),3.65-3.60(m,12H),3.52(d,J＝8.8Hz,12H ),3.45(s,12H),3.23(d,J=4.8Hz,3H),3.13(s,3H),3.06(d,J=15.1Hz,3H),2.99(dd,J=15.0,8.1Hz ,3H),2.84-2.75(m,3H),2.70(d,J＝16.0Hz,3H),1.53(s,3H),1.52(s,6H).13C NMR(151MHz,DMSO)δ170.44,169.46, 168.45, 166.39, 166.02, 165.61, 157.71, 157.67, 149.86, 148.08, 146.81, 141.18, 139.79, 139.66, 138.48, 138.37, 138.07, 135.40, 135.02, 13 4.14, 131.85, 131.65, 129.31, 128.85, 127.33, 126.58, 126.44, 124.58, 122.95, 120.02, 117.60, 115.57, 115.46, 115.42, 114.86, 113.87, 113.35, 113.27, 113.14, 112.49, 111.89, 111.33, 111.21, 111.10, 11 0.32, 104.80, 70.29, 70.23, 70.17, 70.13, 69.44, 69.39, ES I-MS calculated for[M+H] +: 965.0250, found: 965.0251.
实施例十三：化合物I-13的制备Embodiment 13: Preparation of Compound I-13
2-(7-((5-(3,5-二溴-4-羟基亚苄基)-5-碘代-2-氧代吲哚-1-基)戊基)氧基)-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)-N-(噻唑-2-基)乙酰胺的合成
2-(7-((5-(3,5-dibromo-4-hydroxybenzylidene)-5-iodo-2-oxoindol-1-yl)pentyl)oxy)-6- Synthesis of Methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide
参照实施例一I-1的合成路线。I-13：1H NMR(600MHz,DMSO)δ8.78(s,2H),8.12(s,1H),7.92(s,2H),7.82(s,1H),7.81(s,2H),7.57(s,1H),7.56(s,2H),7.48(d,J＝8.6Hz,1H),7.44(s,1H),7.39(s,2H),7.37(d,J＝8.1Hz,2H),7.11(s,3H),6.88(d,J＝8.1Hz,1H),6.80(d,J＝11.3Hz,5H),6.58(s,3H),3.86(dd,J＝12.9,5.7Hz,6H),3.76(s,6H),3.65(s,9H),3.12-3.08(m,3H),3.04(d,J＝5.6Hz,3H),2.97(d,J＝15.0Hz,3H),2.80(d,J＝14.9Hz,3H),2.73(d,J＝15.4Hz,3H),2.65(d,J＝15.7Hz,3H),1.69(s,6H),1.66-1.60(m,6H),1.44(s,9H),1.40(d,J＝6.9Hz,6H).13C NMR(151MHz,DMSO)δ170.44,169.46,168.45,166.39,166.02,165.61,157.71,157.67,149.86,148.08,146.81,141.18,139.79,139.66,138.48,138.37,138.07,135.40,135.02,134.14,131.85,131.65,129.31,128.85,127.33,126.58,126.44,124.58,122.95,120.02,117.60,115.57,115.46,115.42,114.86,113.87,113.35,113.27,113.14,112.49,111.89,111.33,111.21,111.10,110.32,104.80,70.29,70.23,70.17,70.13,69.44,69.39,69.36,68.52,68.46,68.28,68.20,56.19,56.08,56.01,55.80,55.36,49.08,45.63,45.48,42.62,38.16,38.09,28.30,28.23, 28.15,28.05.ESI-MS calculated for[M+H]+:922.9795，found:922.9787。Refer to the synthetic route of Example 1 I-1. I-13: 1H NMR (600MHz, DMSO) δ8.78(s, 2H), 8.12(s, 1H), 7.92(s, 2H), 7.82(s, 1H), 7.81(s, 2H), 7.57( s,1H),7.56(s,2H),7.48(d,J=8.6Hz,1H),7.44(s,1H),7.39(s,2H),7.37(d,J=8.1Hz,2H), 7.11(s,3H),6.88(d,J=8.1Hz,1H),6.80(d,J=11.3Hz,5H),6.58(s,3H),3.86(dd,J=12.9,5.7Hz,6H ),3.76(s,6H),3.65(s,9H),3.12-3.08(m,3H),3.04(d,J＝5.6Hz,3H),2.97(d,J＝15.0Hz,3H),2.80 (d, J=14.9Hz, 3H), 2.73(d, J=15.4Hz, 3H), 2.65(d, J=15.7Hz, 3H), 1.69(s, 6H), 1.66-1.60(m, 6H) ,1.44(s,9H),1.40(d,J=6.9Hz,6H).13C NMR(151MHz,DMSO)δ170.44,169.46,168.45,166.39,166.02,165.61,157.71,157.67,149.86,148.08,146.81 ,141.18 ,139.79,139.66,138.48,138.37,138.07,135.40,135.02,134.14,131.85,131.65,129.31,128.85,127.33,126.58,126.44,124.58,122.95,1 20.02, 117.60, 115.57, 115.46, 115.42, 114.86, 113.87, 113.35 . 28,68.20,56.19,56.08,56.01,55.80,55.36 ,49.08,45.63,45.48,42.62,38.16,38.09,28.30,28.23, 28.15, 28.05. ESI-MS calculated for [M+H]+: 922.9795, found: 922.9787.
实施例十四：化合物I-14的制备Embodiment 14: Preparation of Compound I-14
2-(7-((8-(3,5-二溴-4-羟基亚苄基)-5-碘代-2-氧代吲哚-1-基)辛基)氧基)-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)-N-(噻唑-2-基)乙酰胺的合成2-(7-((8-(3,5-dibromo-4-hydroxybenzylidene)-5-iodo-2-oxoindol-1-yl)octyl)oxy)-6- Synthesis of Methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide
参照实施例一I-1的合成路线。I-14：ESI-MS calculated for[M+H]+:965.0265，found:965.0271。Refer to the synthetic route of Example 1-1. I-14: ESI-MS calculated for [M+H]+: 965.0265, found: 965.0271.
实施例十五：化合物I-15的制备Embodiment 15: Preparation of Compound I-15
2-(7-(2-(2-(2-(3-(3,5-二溴-4-羟基亚苄基)-5-碘-2-氧代吲哚-1-基)乙氧基)乙氧基)-6-甲氧基-1-甲基-1,2,3,4-四氢异喹啉-1-基)-N-(噻唑-2-基)乙酰胺的合成
2-(7-(2-(2-(2-(3-(3,5-dibromo-4-hydroxybenzylidene)-5-iodo-2-oxoindol-1-yl)ethoxy Synthesis of (yl)ethoxy)-6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide
参照实施例一I-1的合成路线。I-15：ESI-MS calculated for[M+H]+:1013.0113，found:1013.0123Refer to the synthetic route of Example 1-1. I-15: ESI-MS calculated for [M+H]+: 1013.0113, found: 1013.0123
实施例十六：细胞水平测定化合物对PCSK9的靶向降解活性试验，对本发明的化合物I-1～I-4采用Western Blot实验即蛋白印迹实验和细胞免疫荧光实验来测定化合物对PCSK9的靶向降解能力。该靶向降解能力主要用PCSK9蛋白最大降解程度以及化合物最佳降解浓度两个指标来表示。同时采用定时定量PCR实验证明本发明化合物对SREBP2-PCSK9-LDLR mRNA表达水平没有明显影响，仅在蛋白水平对PCSK9有降解作用。并对LDLR以及LC3B等蛋白水平的测定来进行佐证。Example 16: Determination of the target degradation activity of the compound on PCSK9 at the cell level, the compounds I-1 to I-4 of the present invention were tested by using Western Blot experiments, namely Western blot experiments and cellular immunofluorescence experiments to determine the targeting of the compounds to PCSK9 Degradability. The targeted degradation ability is mainly expressed by two indicators: the maximum degradation degree of PCSK9 protein and the optimal degradation concentration of the compound. At the same time, the timed quantitative PCR experiment proves that the compound of the present invention has no obvious effect on the expression level of SREBP2-PCSK9-LDLR mRNA, and only degrades PCSK9 at the protein level. And the determination of LDLR and LC3B and other protein levels to support.
实验方法：experimental method:
细胞系和试剂Cell Lines and Reagents
Huh7细胞(ATCC)在补充有10％胎牛血清(BI)和1％青霉素和链霉素(Sigma)的高糖DMEM培养基(Hyclone)中培养，培养箱环境为37℃，95％的空气和5％二氧化碳。除棕榈酸溶液外，其他药物溶液均以1000倍实际使用浓度储存于DMSO或PBS中。油酸和(Sigma)棕榈酸(Sigma)加热溶解后，溶于含15％FAF-BSA(Yeason)的高糖DMEM中，过滤除菌后，配成含5mM的油酸:棕榈酸＝2:1或棕榈酸溶液并储存。 Huh7 cells (ATCC) were cultured in high-glucose DMEM medium (Hyclone) supplemented with 10% fetal bovine serum (BI) and 1% penicillin and streptomycin (Sigma), the incubator environment was 37 °C, 95% air and 5% carbon dioxide. Except palmitic acid solution, other drug solutions were stored in DMSO or PBS at 1000 times the actual concentration. After heating and dissolving oleic acid and (Sigma) palmitic acid (Sigma), dissolve in high-sugar DMEM containing 15% FAF-BSA (Yeason), filter and sterilize, and make oleic acid containing 5mM:palmitic acid=2: 1 or palmitic acid solution and store.
细胞生长到约70％密度时，直接给药或者造模后给药。造模使用500μM棕榈酸和1μg/mL LPS共同处理或500μM油酸:棕榈酸＝2:1处理24小时，他汀组则另外再孵育4小时辛伐他汀(Sigma)后，与化合物共孵育。When the cell grows to about 70% density, it is administered directly or after modeling. Modeling was co-treated with 500 μM palmitic acid and 1 μg/mL LPS or 500 μM oleic acid:palmitic acid=2:1 for 24 hours, and the statin group was incubated with simvastatin (Sigma) for another 4 hours, and co-incubated with the compound.
LDLR抗体购自Abcam，PCSK9、LC3B抗体购自Cell Signaling Technologies，GAPDH抗体购自Bioworld，所有其他试剂均从Sigma Aldrich购得。LDLR antibody was purchased from Abcam, PCSK9 and LC3B antibodies were purchased from Cell Signaling Technologies, GAPDH antibody was purchased from Bioworld, and all other reagents were purchased from Sigma Aldrich.
实验方法：experimental method:
细胞经过药物处理后，RIPA(Beyotime)冰上裂解30分钟，高速离心取上清，经BCA法(Beyotime)测定蛋白浓度并平衡至同一浓度后，加入5X Loading Buffer，煮沸5分钟制成蛋白上样液用于蛋白印迹实验。蛋白印迹实验主要包括聚丙烯酰胺凝胶电泳分离蛋白，PVDF膜转印，含5％脱脂奶粉的TBST溶液封闭30分钟，一抗溶液4℃孵育过夜，二抗室温孵育60分钟，显影液(Millipore)显影并使用凝胶成像系统记录。After the cells were treated with drugs, they were lysed with RIPA (Beyotime) on ice for 30 minutes, and the supernatant was obtained by high-speed centrifugation. After the protein concentration was measured by the BCA method (Beyotime) and balanced to the same concentration, 5X Loading Buffer was added and boiled for 5 minutes to prepare the protein. The samples were used for western blot experiments. Western blotting experiments mainly included separation of proteins by polyacrylamide gel electrophoresis, transfer to PVDF membrane, blocking with 5% skimmed milk powder in TBST solution for 30 minutes, incubation of primary antibody solution at 4°C overnight, incubation of secondary antibody at room temperature for 60 minutes, developing solution (Millipore ) were visualized and recorded using a gel imaging system.
图1示出了根据本发明实施例制备的化合物的Western Blot实验结果。从图1可以看出，化合物I-1～I-4均能够对Huh7细胞系产生从蛋白水平降低PCSK9的效果，并且具有明显勾状效应，其中I-3药物相对作用效果更好。而Huh7与接头化合物孵育实验证明其接头化合物AN-Br、AN-OCH3以及P8并没有单独降低PCSK9活性。说明本发明化合物对PCSK9蛋白有较好的降解作用。Fig. 1 shows the Western Blot experiment result of the compound prepared according to the embodiment of the present invention. It can be seen from Figure 1 that compounds I-1 to I-4 can all reduce PCSK9 at the protein level on the Huh7 cell line, and have an obvious hook-like effect, and the relative effect of the drug I-3 is better. However, the incubation experiment of Huh7 with the linker compounds proved that the linker compounds AN-Br, AN-OCH3 and P8 did not reduce the activity of PCSK9 alone. It shows that the compound of the present invention has better degradation effect on PCSK9 protein.
图2示出了根据本发明实施例制备的化合物I-2与I-4的Western Blot实验结果。从图2可以看出，化合物I-2与I-4均能够对Huh7细胞系产生从蛋白水平降低LC3的效果，并且具有明显的浓度依赖作用，表明本发明化合物的I-2与I-4对LC3蛋白有较好的降解作用。Figure 2 shows the Western Blot experimental results of compounds I-2 and I-4 prepared according to the embodiments of the present invention. As can be seen from Figure 2, both compounds I-2 and I-4 can produce the effect of reducing LC3 from the protein level to the Huh7 cell line, and have an obvious concentration-dependent effect, indicating that compounds I-2 and I-4 of the present invention It has a good degradation effect on LC3 protein.
实验方法：experimental method:
定时定量PCR实验：Timed quantitative PCR experiment:
用于实时定量PCR的引物由Sangon Inc合成，TRIZOL购自TAKARA，其余实时定量PCR相关试剂均购自Yeason。实时定量PCR结果根据GAPDH进行均一化处理。引物序列如下：Primers for real-time quantitative PCR were synthesized by Sangon Inc, TRIZOL was purchased from TAKARA, and other real-time quantitative PCR related reagents were purchased from Yeason. The real-time quantitative PCR results were normalized according to GAPDH. The primer sequences are as follows:
LDLR：正向5'-GACGTGGCGTGAACATCTG-3'；LDLR: Forward 5'-GACGTGGCGTGAACATCTG-3';
反向，5'-CTGGCAGGCAATGCTTTGG-3'；Reverse, 5'-CTGGCAGGCAATGCTTTGG-3';
PCSK9：正向，5'-AGGGGAGGACATCATTGGTG-3'；PCSK9: Forward, 5'-AGGGGAGGACATCATTGGTG-3';
反向，5-CAGGTTGGGGGTCAGTACC-3；reverse, 5-CAGGTTGGGGGTCAGTACC-3;
SREBP2：正向，5'-CCCTGGGAGACATCGACGA-3'； SREBP2: Forward, 5'-CCCTGGGAGACATCGACGA-3';
反向，5'-CGTTGCACTGAAGGGTCCA-3'；Reverse, 5'-CGTTGCACTGAAGGGTCCA-3';
GAPDH：正向，5'-ATGGGGAAGGTGAAGGTCG-3'；GAPDH: Forward, 5'-ATGGGGAAGGTGAAGGTCG-3';
反向，5'-GGGGTCATTGATGGCAACAATA-3'。Reverse, 5'-GGGGTCATTGATGGCAACAATA-3'.
图3示出了根据本发明实施例制备的化合物I-3的qPCR实验结果。从图3可以看出，化合物I-3对SREBP2-PCSK9-LDLR mRNA表达水平没有明显影响，仅在蛋白水平对PCSK9有作用。Fig. 3 shows the qPCR experimental results of compound I-3 prepared according to the embodiment of the present invention. It can be seen from Figure 3 that compound I-3 has no significant effect on the expression level of SREBP2-PCSK9-LDLR mRNA, and only has an effect on PCSK9 at the protein level.
图4示出了根据本发明实施例制备的化合物I-3的MTT毒性试验测定结果。从图4可以看出，化合物I-3无明显细胞毒性。Fig. 4 shows the MTT toxicity test results of compound I-3 prepared according to the embodiment of the present invention. It can be seen from Figure 4 that compound I-3 has no obvious cytotoxicity.
图5示出了根据本发明实施例制备的化合物I-3在高脂模型中的Western Blot实验结果。从图5可以看出，在棕榈酸、LPS模拟的高脂炎症环境下，PCSK9升高，化合物I-3药物能够降低PCSK9含量并提高LDLR，实验结果表明了化合物I-3药物作为高脂血症患者治疗药物的可能性。Fig. 5 shows the Western Blot experimental results of compound I-3 prepared according to an embodiment of the present invention in a hyperlipidemia model. It can be seen from Figure 5 that PCSK9 increases under the high-fat inflammatory environment simulated by palmitic acid and LPS, and the compound I-3 drug can reduce the PCSK9 content and increase LDLR. Possibility of treating patients with the disease.
图6示出了根据本发明实施例制备的化合物I-3与他汀类药物联用时的Western Blot实验结果。化合物I-3药物能够与他汀类药物—辛伐他汀产生协同效果，降低由他汀类药物引起的PCSK9升高，并进一步提高LDLR的蛋白含量，实验结果表明了化合物I-3药物和他汀类药物联合使用作为高脂血症患者治疗药物的可能性。Fig. 6 shows the Western Blot experimental results when compound I-3 prepared according to the embodiment of the present invention is used in combination with statins. Compound I-3 can produce a synergistic effect with statins—simvastatin, reduce PCSK9 elevation caused by statins, and further increase the protein content of LDLR. The experimental results show that compound I-3 and statins The possibility of combined use as a therapeutic drug for patients with hyperlipidemia.
图7示出了根据本发明实施例制备的化合物I-1～I-15的Western Blot实验结果，化合物I-1、I-8、I-15的PCSK9降解效果更优。说明本发明化合物对PCSK9蛋白有较好的降解作用。Fig. 7 shows the Western Blot experimental results of compounds I-1 to I-15 prepared according to the embodiments of the present invention, and compounds I-1, I-8, and I-15 have better PCSK9 degradation effects. It shows that the compound of the present invention has better degradation effect on PCSK9 protein.
以上的具体实施方式对本发明的目的、技术方案和有益效果进行了进一步详细说明，应当理解的是，以上仅为本发明的一种具体实施方式而已，并不限于本发明的保护范围，在不脱离本发明的基本特征的宗旨下，本发明可体现为多种形式，因此本发明中的实施形态是用于说明而非限制，由于本发明的范围由权利要求限定而非由说明书限定，而且落在权利要求界定的范围，或其界定的范围的等价范围内的所有变化都应理解为包括在权利要求书中。凡在本发明的精神和原则之内的，所做出的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。 The above specific embodiments have further described the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above is only a specific embodiment of the present invention, and is not limited to the protection scope of the present invention. The present invention can be embodied in various forms under the purpose of departing from the basic characteristics of the present invention, so the embodiments in the present invention are for illustration rather than limitation, because the scope of the present invention is defined by the claims rather than by the description, and All changes that fall within the range defined in the claims, or within the range equivalent to the range defined in the claims, should be construed as being included in the claims. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (8)

  1. 一种PCSK9小分子蛋白降解剂，其特征在于，为如下式化学结构通式I的四氢异喹啉类化合物或其药学上可接受的盐或溶剂化物：
    A PCSK9 small molecule protein degradation agent is characterized in that, it is a tetrahydroisoquinoline compound or a pharmaceutically acceptable salt or solvate thereof of the following formula chemical structure general formula I:
    其中，in,
    连接基团A为：C1-C20的烷基链、醚链、氧杂链、硫杂链、氮杂链、C1-C20烯基链、C1-C20炔基链、-C(＝O)NH(C1-C19的烷基链、醚链、氧杂链、硫杂链、氮杂链、C1-C19烯基、C1-C19炔基)；Linking group A is: C 1 -C 20 alkyl chain, ether chain, oxa chain, thia chain, aza chain, C 1 -C 20 alkenyl chain, C 1 -C 20 alkynyl chain, - C(=O)NH(C 1 -C 19 alkyl chain, ether chain, oxa chain, thia chain, aza chain, C 1 -C 19 alkenyl, C 1 -C 19 alkynyl);
    R1、R2、R3以及R4独立地选自氢、卤素、羟基、硝基、甲氧基、氨基、甲氨基、二甲氨基、羧基、氰基、-CO2Me、-CO2Et、-CH3、-Et、-N3、C1-6烷基、C2-6烯基、C2-6炔基，其中所述烷基、烯基或炔基任选地被一个或多个选自-O(C1-6烷基)、-O(C3-6环烃基)、-O(C1-4亚烷基-C3-6环烃基)、-O(三元至七元杂环基)、-O(C1-4亚烷基-三元至七元杂环基)、-SH、-S(C1-6烷基)、-S(C3-6环烃基)、-S(C1-4亚烷基-C3-6环烃基)、-S(三元至七元杂环基)、-S(C1-4亚烷基-三元至七元杂环基)、-NH2、-NH(C1-6烷基)、-N(C1-6烷基)2、-NH(C3-6环烃基)、-N(C3-6环烃基)2、-NH(C1-4亚烷基-C3-6环烃基)、-N(C1-4亚烷基-C3-6环烃基)2、-NH(三元至七元杂环基)、-N(三元至七元杂环基)2、-NH(C1-4亚烷基-三元至七元杂环基)、-N(C1-4亚烷基-三元至七元杂环基)2所取代；R 1 , R 2 , R 3 and R 4 are independently selected from hydrogen, halogen, hydroxyl, nitro, methoxy, amino, methylamino, dimethylamino, carboxyl, cyano, -CO 2 Me, -CO 2 Et, -CH 3 , -Et, -N 3 , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, wherein the alkyl, alkenyl or alkynyl is optionally replaced by one Or more selected from -O(C 1-6 alkyl), -O(C 3-6 cycloalkyl), -O(C 1-4 alkylene-C 3-6 cycloalkyl), -O(three One-membered to seven-membered heterocyclyl), -O(C 1-4 alkylene-three-membered to seven-membered heterocyclyl), -SH, -S(C 1-6 alkyl), -S(C 3- 6 cycloalkyl), -S (C 1-4 alkylene-C 3-6 cycloalkyl), -S (three-membered to seven-membered heterocyclyl), -S (C 1-4 alkylene-three to seven-membered heterocyclyl), -NH 2 , -NH(C 1-6 alkyl), -N(C 1-6 alkyl) 2 , -NH(C 3-6 cycloalkyl), -N(C 3-6 cycloalkyl) 2 , -NH(C 1-4 alkylene-C 3-6 cycloalkyl), -N(C 1-4 alkylene-C 3-6 cycloalkyl) 2 , -NH( Three-membered to seven-membered heterocyclic group), -N(three-membered to seven-membered heterocyclic group) 2 , -NH(C 1-4 alkylene-three- to seven-membered heterocyclic group), -N(C 1 -4 alkylene-three-membered to seven-membered heterocyclic group) substituted by 2 ;
    R5独立地选自氢、甲氧基、甲基、卤素、羟基、硝基、氨基、甲氨基、二甲氨基、羧基、氰基、-CO2Me、-CO2Et、-CH3、-Et、-N3、C1-6烷基、C2-6烯基、C2-6炔基；R 5 is independently selected from hydrogen, methoxy, methyl, halogen, hydroxyl, nitro, amino, methylamino, dimethylamino, carboxyl, cyano, -CO 2 Me, -CO 2 Et, -CH 3 , -Et, -N 3 , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl;
    R6独立地选自甲基；R 6 is independently selected from methyl;
    R7独立地选自以下结构：
    或者     R 7 is independently selected from the following structures:
    or
    基团B为：
    Group B is:
    其中R8、R9以及R10独立地选自氢、卤素、羟基、硝基、甲氧基、氨基、甲氨基、二甲氨基、羧基、氰基、-CO2Me、-CO2Et、-CH3、-Et、-N3；X为NH、S、O。Wherein R 8 , R 9 and R 10 are independently selected from hydrogen, halogen, hydroxyl, nitro, methoxy, amino, methylamino, dimethylamino, carboxyl, cyano, -CO 2 Me, -CO 2 Et, -CH 3 , -Et, -N 3 ; X is NH, S, O.
  2. 根据权利要求1所述的PCSK9小分子蛋白降解剂，其特征在于，所述R7为 PCSK9 small molecule protein degradation agent according to claim 1, is characterized in that, described R 7 is
  3. 根据权利要求1或2所述的PCSK9小分子蛋白降解剂，其特征在于，所述基团B为R8、R9和R10独立地选自卤素、羟基、甲氧基。PCSK9 small molecule protein degradation agent according to claim 1 or 2, is characterized in that, described group B is R 8 , R 9 and R 10 are independently selected from halogen, hydroxy, methoxy.
  4. 根据权利要求1-3任一项所述的PCSK9小分子蛋白降解剂，其特征在于，所述化合物选自下述化合物：

    The PCSK9 small molecule protein degradation agent according to any one of claims 1-3, wherein the compound is selected from the following compounds:

  5. 一种如权利要求1-4任一项所述的PCSK9小分子蛋白降解剂的制备方法，其特征在于，包括如下步骤：
    A preparation method of the PCSK9 small molecule protein degradation agent as described in any one of claims 1-4, is characterized in that, comprises the steps:
    其中，in,
    R2、R5、R8、R10如权利要求1所定义的。R 2 , R 5 , R 8 , R 10 are as defined in claim 1 .
  6. 一种药物组合物，其特征在于，包括如权利要求1-4任一项所述的PCSK9小分子蛋白降解剂，以及药物上可接受的载体。A pharmaceutical composition, characterized in that it comprises the PCSK9 small molecule protein degradation agent according to any one of claims 1-4, and a pharmaceutically acceptable carrier.
  7. 如权利要求1-4任一项所述的PCSK9小分子蛋白降解剂在制备用于预防或治疗与抑制PCSK9活性相关的疾病的药物中的应用。Application of the PCSK9 small molecule protein degrading agent according to any one of claims 1-4 in the preparation of medicines for preventing or treating diseases related to the inhibition of PCSK9 activity.
  8. 如权利要求1-4任一项所述的PCSK9小分子蛋白降解剂在制备用于预防或治疗与抑制 The PCSK9 small molecule protein degradation agent as described in any one of claims 1-4 is used for prevention or treatment and inhibition in preparation
PCT/CN2023/077334 2022-03-02 2023-02-21 Micromolecular pcsk9 protein degradation agent, preparation method therefor, and use thereof WO2023165373A1 (en)

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Citations (3)

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CN105777633A (en) * 2015-01-09 2016-07-20 成都贝斯凯瑞生物科技有限公司 Tetrahydroisoquinoline derivative and application thereof
US20190119236A1 (en) * 2016-02-23 2019-04-25 Portola Pharmaceuticals, Inc. Compounds for binding proprotein convertase subtilisin/kexin type 9 (pcsk9)
US20200369657A1 (en) * 2016-09-20 2020-11-26 Merck Sharp & Dohme Corp. Substituted 1-methyl-1,2,3,4-tetrahydroisoquinoline molecules as pcsk9 allosteric binders

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Publication number Priority date Publication date Assignee Title
US9877957B2 (en) * 2014-09-05 2018-01-30 Merck Sharp & Dohme Corp. Tetrahydroisoquinoline derivatives useful as inhibitors of diacylglyceride O-acyltransferase 2

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105777633A (en) * 2015-01-09 2016-07-20 成都贝斯凯瑞生物科技有限公司 Tetrahydroisoquinoline derivative and application thereof
US20190119236A1 (en) * 2016-02-23 2019-04-25 Portola Pharmaceuticals, Inc. Compounds for binding proprotein convertase subtilisin/kexin type 9 (pcsk9)
US20200369657A1 (en) * 2016-09-20 2020-11-26 Merck Sharp & Dohme Corp. Substituted 1-methyl-1,2,3,4-tetrahydroisoquinoline molecules as pcsk9 allosteric binders

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