PCSK9 small-molecule autophagy degrader, preparation method therefor and use thereof

Abstract

The present invention provides a PCSK9 small-molecule autophagy degrader, a preparation method therefor and a use thereof. The PCSK9 small-molecule autophagy degrader is a tetrahydroisoquinoline compound having a chemical structure represented by general formula I or a pharmaceutically acceptable salt or solvate thereof. Such a compound has the effect of degrading PCSK9, and can be used for preparing drugs for preventing and / or treating diseases associated with inhibition of PCSK9 activity.

Description

PCSK9 small molecule autophagy degrading agent, preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, and particularly relates to a PCSK9 small molecule autophagy degrading agent, a preparation method and application thereof. BACKGROUND

[0002] Proprotein convertase subtilisin / kexin type 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 cells by forming a complex with the cell surface low-density lipoprotein receptor (LDLRs), and the low pH condition of the endosome causes the LDL to separate from its receptor, and the LDL is degraded in the lysosome, while the 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 be degraded simultaneously in the cell, thereby inhibiting the ability of LDLR to recycle and clear low-density lipoprotein cholesterol from the blood. Generally speaking, inhibiting the production or secretion of PCSK9 can reduce the level of cholesterol in the blood, and autophagy degrading agents of PCSK9 can be used to treat hypercholesterolemia.

[0003] In general, inhibitors or degrading agents of PCSK9 show good lipid-lowering activity. For example, alirocumab, evolocumab and tafolecimab, three monoclonal antibodies and small interfering RNA Inclisiran are four examples of PCSK9 inhibitors that have been used clinically and can be used to treat cardiovascular and cerebrovascular diseases. According to patents such as CN113876955A, WO2022002160A1, WO2021052472A1, CN113332423A, CN113663075A, WO2021243002A1, WO2021207712A1, WO2021154947A1, WO2021143762A1, CN112083163A, WO2020229718A1, CN113412258A, CN111154760A, WO2020252383 A1, WO2018057409 A1, etc., various PCSK9 drugs have been disclosed for treating or preventing cardiovascular and cerebrovascular diseases, hair loss 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, etc.

[0004] The three currently approved clinical drugs of alirocumab, evolocumab and tafolecimab described above are biological drugs, and the treatment cost is large, the administration route is inconvenient, and the compliance of patients is poor. At present, there is no small molecule PCSK9 drug applied in clinical, therefore, it is of great significance to develop small molecule PCSK9 drugs and apply them to treat or prevent cardiovascular and cerebrovascular diseases, alopecia diseases, vitiligo, keratinization abnormal diseases, scars, pulmonary fibrosis diseases, metabolic syndrome, obesity, diabetes, Alzheimer's disease, anti-rejection, non-alcoholic hepatitis and malignant tumors.

[0005] Autophagy has long been associated with various aspects of regulating immune responses, including pathogen capture, metabolic regulation and cell homeostasis. Current studies have shown that autophagy is related to the occurrence and development of metabolic diseases, malignant tumors, immune disorders and neurodegenerative diseases. Macroautophagy (hereinafter referred to as autophagy) is responsible for degrading cytotoxic misfolded proteins that are recognized and sorted by various selective autophagy receptors. P62 / SQSTM1 / Sequestosome_1 is the main selective autophagy receptor that binds misfolded proteins to cause them to co-aggregate and be delivered to autophagosomes. The transport of misfolded proteins to autophagosomes mediated by P62 requires the oligomerization ability of P62. Through oligomerization, P62 not only packages its cargo, but also delivers the cargo to the autophagosome formation site for autophagic co-degradation. At this time, the unfolded proteins are concentrated together to reduce the volume, making them more easily degraded by autophagy. The PB1 domain of P62 mediates the self-oligomerization of P62. When the autophagosome binds to the lysosome, the unfolded protein-P62 complex delivered to the autophagosome can be degraded by the enzymes of the lysosome. Therefore, designing a P62-mediated degradation method is expected to effectively reduce the content of target proteins. SUMMARY

[0006] In view of the above problems, the present application provides a novel PCSK9 small molecule autophagy degrading agent, which is a tetrahydroisoquinoline compound or a pharmaceutically acceptable salt or solvate thereof. Such compounds have the effect of degrading PCSK9 and can reduce the level of PCSK9, and are expected to be used for preparing a drug for preventing and / or treating diseases related to inhibition of PCSK9 activity. The present application can be achieved by the following technical means:

[0007] In a first aspect, the present application provides a PCSK9 small molecule autophagy degrading agent, which is a tetrahydroisoquinoline compound or a pharmaceutically acceptable salt or solvate thereof according to the general formula I: wherein, X is -CH2- or -C(=O)-; that is, X is methylene or carbonyl; R1 is hydrogen or C1-C 20R1is hydrogen or C1-C6alkyl; preferably, R1is hydrogen or C1-C6alkyl; the linker group A is selected from: C1-C 20 alkyl chain, ether chain, oxa chain, thia chain, aza chain, C2-C 20 alkenyl chain, C2-C 20 alkynyl chain, -C(=O)NH(C1-C 19 alkyl chain, ether chain, oxa chain, thia chain, aza chain, C2-C 19 alkenyl chain, C2-C 19 alkynyl chain), or any substituents on the linker group A are attached to R1and / or any substituents on the linker group A are attached to each other to form a linker fragment having a single ring, multiple ring, spiro ring, or bridged ring structure; R2and R3are independently hydrogen or R8, R9, and R 10 are independently selected from hydrogen, halogen (e.g., fluorine, chlorine), hydroxyl, nitro, C1-C3alkyl (e.g., methyl, ethyl, isopropyl), C1-C3alkoxy (e.g., methoxy), amino, C1-C3alkylamino (e.g., methylamino, dimethylamino), carboxyl, cyano, ester, halogen-substituted C1-C3alkyl (e.g., trifluoromethyl), halogen-substituted C1-C3alkoxy, halogen-substituted C1-C3alkylamino; R5is selected from hydrogen, C1-C3alkyl (e.g., methyl, ethyl, isopropyl), C1-C3alkoxy (e.g., methoxy), halogen, hydroxyl, nitro, amino, C1-C3alkylamino (e.g., methylamino, dimethylamino), carboxyl, cyano, ester; R6is selected from hydrogen, C1-C3alkyl (e.g., methyl, ethyl, isopropyl), C1-C3alkoxy (e.g., methoxy), halogen, hydroxyl, nitro, amino, C1-C3alkylamino (e.g., methylamino, dimethylamino), carboxyl, cyano, ester; R7is an aromatic ring group having 0-3 substituents selected from C1-C3alkyl, C1-C3alkoxy, halogen, hydroxyl, nitro, amino, C1-C3alkylamino, carboxyl, cyano, ester, or an aromatic heterocyclic ring group having 0-3 substituents selected from C1-C3alkyl, C1-C3alkoxy, halogen, hydroxyl, nitro, amino, C1-C3alkylamino, carboxyl, cyano, ester, and having 0-3 (e.g., 2) heteroatoms. The heteroatoms include, but are not limited to, N, O, S, etc.

[0008] Preferably, the linker fragment is selected from the following structures: wherein A1, A2, and A3are independently selected from methylene, O, S, and NH; a, b, c are independently selected from an integer between 0-5 (preferably an integer between 1-5, e.g., 1, 2, 3, 4, 5); or A1, A2, and A3are absent. In some embodiments, the linker fragment Selected from the following structure: Where n and m are independently selected from integers between 1 and 5, for example 1, 2, 3, 4, 5.

[0009] R7 is selected from

[0010] Preferably, R2 is hydrogen, or

[0011] Preferably, R6 is methyl.

[0012] Preferably, the tetrahydroisoquinoline compound of general chemical formula I is selected from the following compounds:

[0013] In a second aspect, the present invention provides a pharmaceutical composition comprising a tetrahydroisoquinoline compound as described above, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.

[0014] Thirdly, the present invention provides the use of the tetrahydroisoquinoline compound as described above, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof, in the preparation of a medicament for the prevention or treatment of diseases related to the inhibition of PCSK9 activity.

[0015] Preferably, the disease is selected from at least one of cardiovascular and cerebrovascular diseases, alopecia, vitiligo, keratosis disorders, scars, pulmonary fibrosis, metabolic syndrome, obesity, diabetes, Alzheimer's disease, anti-rejection reaction, non-alcoholic hepatitis, and malignant tumors.

[0016] 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 at least one of leukemia, prostate cancer, thyroid cancer, liver cancer, ovarian cancer, breast cancer, esophageal cancer, adenocarcinoma, gastric cancer, lung cancer, and colorectal cancer. Attached Figure Description

[0017] Figure 1 shows the Western Blot experimental results of compound I-1 prepared according to an embodiment of the present application; Figure 2 shows the Western Blot experimental results of compound I-2 prepared according to an embodiment of the present application; Figure 3 shows the Western Blot experimental results of compound I-3 prepared according to an embodiment of the present application; Figure 4 shows the Western Blot experimental results of compound I-10 prepared according to an embodiment of the present application; Figure 5 shows the Western Blot experimental results of compound I-6 prepared according to an embodiment of the present application; Figure 6 shows the Western Blot experimental results of compound I-6, PCSK9 ligand P9 and P62 ligand M4 prepared according to an embodiment of the present application; Figure 7 shows the qPCR experimental results of compound I-6 prepared according to an embodiment of the present application; Figure 8 shows the MTT toxicity test determination results of compound I-6 prepared according to an embodiment of the present application; Figure 9 shows the Western Blot experimental results of compound I-6 in a high-fat model prepared according to an embodiment of the present application; Figure 10 shows the Western Blot experimental results of the binding ability of compound I-6 to PCSK9 and P62 protein prepared according to an embodiment of the present application; Figure 11 shows the Western Blot experimental results of compound I-6 in an autophagy and ubiquitination inhibition model prepared according to an embodiment of the present application. DETAILED DESCRIPTION

[0018] The present application is further illustrated by the following examples and the accompanying drawings, which are intended to be illustrative only and not limiting of the present application.

[0019] The following exemplary illustrates the preparation process of the compounds provided by the present application.

[0020] As an example, when X is -C(=O)-, R6 is methyl, and R1 is hydrogen, the synthetic route can be as shown below:

[0021] Synthesis of intermediate compound of formula II:

[0022] Synthesis of intermediate compound of formula III-a:

[0023] Synthesis of target compound I-a using intermediate compound of formula II and compound of formula III-a:

[0024] wherein R2, R3, R5, R7 and the linking group A are defined as above.

[0025] As an example, when X is -CH2-, R6 is methyl, and R1 is hydrogen, the synthetic route can be as follows:

[0026] Synthesis of intermediate formula II compounds:

[0027] Synthesis of intermediate III-b compounds:

[0028] The target compound Ib was synthesized using intermediate compound II and compound III-b:

[0029] R2, R3, R5, R7 and linking group A are as defined above.

[0030] The above synthesis schemes only illustrate some of the preparation methods of the compounds in this invention. Based on the above synthesis schemes, those skilled in the art can use similar methods to synthesize the compounds of this invention, referring to commonly used techniques and existing technologies in the field.

[0031] The expression "mn" used in this paper refers to the range from m to n, the subrange consisting of the individual point values ​​within it, and the individual point values ​​themselves. For example, the expression "C1-C 20 "or "C 1-20 "Covering a range of 1-20 carbon atoms, and should be understood to also include any subranges within this range 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-C..." 10 "or "C 3-10 "It should also be understood in a similar way, for example, it can encompass any subrange and point value contained within it, such as C3-C9, C6-C9, C6-C8, C..." 6- C7, C7-C 10 C7-C9, C7-C8, C8-C9, etc., as well as C3, C4, C5, C6, C7, C8, C9, C 10 Etc. Other similar expressions in this article should also be understood in a similar manner.

[0032] The terms “halogen” or “halogenated” should be understood to refer to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I) atoms.

[0033] The term "alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms, connected to the rest of the molecule by single bonds. Alkyl groups can have 1-20 carbon atoms, i.e., C1-C2. 20 Alkyl groups, such as C1-4 alkyl, C 1-3 alkyl, C 1-2 alkyl, C3alkyl, C4alkyl, 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, t-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-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or isomers thereof. "Alkylene" refers to a radical obtained by removing one hydrogen atom from a carbon atom that contains a free valence electron, having two attachment sites to other parts of the molecule. For example, "alkylene" or "alkylidene" refers to a saturated, straight chain or branched divalent hydrocarbon radical.

[0034] The term "alkylene", used alone or in combination with other radicals, refers to a straight chain or branched saturated divalent hydrocarbon radical. For example, the term "C 1-20 alkylene" refers to an alkylene radical having 1 to 20 carbon atoms, such as methylene, ethylene, propylene, butylene, pentylene, hexylene, 1-methylethylene, 2-methylethylene, methylpropylene, or ethylpropylene, and the like. The term "cycloalkylene" refers to a cyclic saturated divalent hydrocarbon radical. For example, the term "C 3-6 cycloalkylene" refers to a cycloalkylene radical having 3 to 6 carbon atoms, such as cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and the like. The term "alkoxyalkylene" refers to "-O-alkylene" or "alkylene-O-". Examples of "C 1-8 alkoxyalkylene" include, but are not limited to, -O-methylene, -O-ethylene, -O-propylene, -O-butylene, methylene-O-, ethylene-O-, propylene-O-, butylene-O-, and the like.

[0035] The term "alkenyl" refers to a straight chain or branched unsaturated aliphatic hydrocarbon radical having at least one double bond, composed of carbon and hydrogen atoms. Alkenyl groups can have 2 to 20 carbon atoms, i.e., "C 2-20 alkenyl", such as C 2-4 alkenyl, C 3-4 alkenyl. Non-limiting examples of alkenyl groups include, but are not limited to, ethenyl, allyl, (E)-2-methylethenyl, (Z)-2-methylethenyl, (E)-but-2-enyl, (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl, and the like.

[0036] The term "alkynyl" refers to a straight-chain or branched unsaturated aliphatic hydrocarbon group having at least one triple bond, consisting of carbon and hydrogen atoms. The alkynyl group can have 2 to 20 carbon atoms, i.e. "C 2-8 alkynyl", for example C 2-4 alkynyl, C 3-4 alkynyl. Non-limiting examples of alkynyl groups 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.

[0037] The term "cycloalkyl" refers to a saturated or unsaturated non-aromatic cyclic hydrocarbon group consisting of carbon and hydrogen atoms, preferably comprising 1 or 2 rings. The cycloalkyl group can be a monocyclic, fused polycyclic, bridged or spirocyclic structure. The cycloalkyl group can have 3 to 10 carbon atoms, i.e. "C 3-10 cycloalkyl", for example C 3-8 cycloalkyl, C 3-6 cycloalkyl, C5cycloalkyl, C6cycloalkyl, C7cycloalkyl. Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl and spiro[3.3]heptyl, and the like. The term also encompasses cases where a C atom can be substituted by oxo (=0).

[0038] The term "cycloalkyl" refers to a saturated non-aromatic cyclic hydrocarbon group.

[0039] The term "heterocyclyl" or "heterocycloalkyl" refers to a monocyclic or bicyclic ring system (3- to 10-membered, suitably 3- to 8-membered, more suitably 3- to 7-membered, in particular 4- to 6-membered) having, for example, 3 to 10 (suitably 3 to 8, more suitably 3 to 7, in particular 4 to 6) ring atoms, wherein at least one ring atom (e.g. 1 or 3) is a heteroatom selected from N, O, S and P, and the remaining ring atoms are C. The ring system can be saturated (which can also be understood as the corresponding "heterocycloalkyl") or unsaturated (i.e. having one or more double bonds and / or triple bonds within the ring). The "heterocyclyl" or "heterocycloalkyl" is not aromatic. The term also encompasses cases where a C atom can be substituted by oxo (=0) and / or a S atom in the ring can be substituted by 1 or 2 oxo (=0) and / or a P atom in the ring can be substituted by 1 or 2 oxo (=0).

[0040] The heterocyclyl can be, for example, a four-membered ring, such as azetidinyl, oxetanyl; or a five-membered ring, such as tetrahydrofuranyl, dioxanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, oxopyrrolidinyl, 2-oxoimidazolidin-1-yl; or a six-membered ring, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, 1,1-dioxo-1,2-thiazinanyl or trithianyl; or a seven-membered ring, such as diazepinyl; or a nine-membered ring, such as diazabicyclo[4.2.1]nonanyl; or a ten-membered ring, such as diazabicyclononanyl. The heterocyclic group may optionally be benzofused.

[0041] Heterocyclic groups can be bicyclic, without restriction, such as five-membered fused five-membered rings, such as hexahydrocyclopentane[C]pyrrole-2(1H)-yl ring; or five-membered fused six-membered bicyclic rings, such as hexahydropyrrolo[1,2-a]pyrazine-2(1H)-yl ring.

[0042] As mentioned above, heterocycles can be unsaturated, meaning they can contain one or more double bonds without limitation. For example, unsaturated heterocycles containing nitrogen atoms can 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-pyrroleyl, 4H-[1,3,4]thiadiazinyl, 4,5-dihydrooxazolyl, or 4H-[1,4]thiazinyl rings. Unsaturated heterocycles containing oxygen atoms can be 2H-pyran, 4H-pyran, or 2,3-dihydrofuran. Unsaturated heterocycles containing sulfur atoms can be 2H-thiaran or 4H-thiaran. Heterocycles can be benzofused without limitation, such as dihydroisoquinoline rings.

[0043] The term "aryl" or "aryl ring group" refers to a monocyclic all-carbon aromatic ring (i.e., monocyclic aryl) or a polycyclic aromatic ring system (i.e., polycyclic aryl) having a conjugated π-electron system, having 6 to 14 ring atoms (i.e., 6 to 14 membered aryl). In some embodiments, the aryl group may be an aryl group having 6 to 10 ring atoms (i.e., C6-C). 10 Aryl). The monocyclic aryl group, such as phenyl. The polycyclic aryl group, non-limiting examples include, but are not limited to, naphthyl, anthracene, phenanthrene, etc.

[0044] The term "heteroaryl" or "aromatic heterocyclic group" refers to a monocyclic heteroaryl ring (i.e., monocyclic heteroaryl) or polycyclic heteroaryl ring system (i.e., polycyclic heteroaryl) having a conjugated π-electron system, containing at least one (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur (the nitrogen may optionally be oxidized, i.e., to form nitrogen oxides; the sulfur may optionally be oxidized, i.e., to form sulfoxides or sulfones), having 5 to 14 ring atoms (i.e., 5 to 14 membered heteroaryl groups). Non-limiting examples include, but are not limited to: pyridine, thiophene, triazole, oxazole, benzofuran, and isoindoline.

[0045] The term "hydrocarbon chain" refers to a chain-like group composed of carbon and hydrogen atoms, which can be straight-chain or branched. The hydrocarbon chain can be saturated (i.e., alkylene), or it can be unsaturated, i.e., it can contain one or more carbon-carbon double or triple bonds. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylidene (-CH(CH3)-), 1,2-ethylidene (-CH2CH2-), 1,1-propylidene (-CH(CH2CH3)-), 1,2-propylidene (-CH2CH(CH3)-), 1,3-propylidene (-CH2CH2CH2-), 1,4-butylidene (-CH2CH2CH2CH2-), 1,7-heptamethylene (-CH2CH2CH2CH2CH2CH2CH2-), and the like.

[0046] The term "compound" as used herein includes all stereoisomers, geometric isomers, tautomers, and isotopologues.

[0047] The term "compound" as used herein includes all stereoisomers, geometric isomers, tautomers, and isotopologues.

[0048] The term "compound" as used herein includes all stereoisomers, geometric isomers, tautomers, and isotopologues.

[0049] The term "compound" as used herein includes all stereoisomers, geometric isomers, tautomers, and isotopologues.

[0050] The term "compound" as used herein includes all stereoisomers, geometric isomers, tautomers, and isotopologues. 3 H, 15 O, 13 C or 15 N isotopes.

[0051] "Pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt which can improve physicochemical properties or metabolic properties, etc. while maintaining the pharmacological activity of the 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 mixtures thereof. In the present application, suitable inorganic acids are, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid or the like; suitable organic acids include fumaric acid, tartaric acid, lactic acid, acetic acid, citric acid, trifluoromethanesulfonic acid, mandelic acid, salicylic acid or the like.

[0052] The compounds according to the present application can also exist in the form of solvates. For example, hydrates (hemihydrates, monohydrates, dihydrates, trihydrates, etc.).

[0053] Dosing

[0054] In general, the compounds of the present application can be administered in an effective amount by any of the accepted modes of administration for agents that serve similar utilities. For example, the compounds of the present application can be administered orally, parenterally, transdermally, topically, rectally, intraperitoneally, or intranasally.

[0055] Pharmaceutical compositions

[0056] The present application also provides a pharmaceutical composition comprising the PCSK9 small molecule autophagy degrader described above, and a pharmaceutically acceptable carrier.

[0057] When used as a medicine, the compounds of the present application are usually administered in the form of a pharmaceutical composition. These compositions can be prepared using methods well known in the art of pharmacy, and they contain at least one active compound. The amount of active ingredient, i.e. the compound of the present application, in the pharmaceutical composition will vary according to the particular application, activity and intended concentration of the compound.

[0058] In formulating the compositions provided by the present application, the active ingredient is typically combined with an excipient (carrier), diluted by an excipient, or enclosed within such a carrier. The excipient can be a solid, a semi-solid, or a liquid. As used herein, the term "excipient" includes any ingredient other than the compound of the present application. When the excipient serves as a diluent, carrier or vehicle, it can be solid, semi-solid, or liquid. In this manner, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[0059] Some typical excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, sterile water, syrups, and cellulose. Other adjuvants such as lubricants and flushing agents, emulsifiers and suspending agents, preservatives, sweeteners and flavoring agents can also be included. The pharmaceutical compositions of the present application can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing well-known methods in the art of pharmaceutical formulation.

[0060] The present application also provides use of the PCSK9 small molecule autophagy degrading agent or the pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease associated with inhibition of PCSK9 activity.

[0061] "Treatment" means any treatment of a disease in a mammal, including: (1) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop; (2) inhibiting the disease, i.e., arresting the development of clinical symptoms; and (3) relieving the disease, i.e., causing the regression of clinical symptoms.

[0062] The disease is selected from at least one 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.

[0063] Preferably, the cardiovascular and cerebrovascular diseases are selected from at least one of cholesterol-related diseases, hyperlipidemia, and atherosclerosis; and the malignant tumors are selected from at least one of leukemia, prostate cancer, thyroid cancer, liver cancer, ovarian cancer, breast cancer, esophageal cancer, adenocarcinoma, gastric cancer, lung cancer, and colorectal cancer.

[0064] The present application will be further described in detail by the following specific examples. It should also be understood that the following examples are only used to further illustrate the present application, and should not be understood as limiting the scope of the present application. Some non-essential improvements and adjustments made by those skilled in the art based on the above description of the present application are within the scope of the present application. The following examples are only one example in the appropriate range, i.e. those skilled in the art can make appropriate selection within the range according to the description herein, and are not limited to the specific values of the following examples.

[0065] In the present application, the ratio of the eluent refers to the volume ratio.

[0066] Example 1: Preparation of compound I-1

[0067] Synthesis of 3-(benzyloxy)-N-(8-((6-methoxy-1-methyl-1-(2-oxo-2-(thiazol-2- ylamino)ethyl)-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)octyl)benzamide

[0068] Step 1: Synthesis of 2-methoxy-4-(2-nitrovinyl)phenol (P1)

[0069] In 350 ml of nitromethane, ammonium acetate (16.95 g, 0.22 mol) and 1,2-benzopyranone (100 g, 0.66 mol) were added and stirred at room temperature to dissolve. Then the reaction was stirred at 100 °C for 1 h. After monitoring the end of the reaction, the yellow filter cake was obtained as crude product P1 (96.5 g, yield 75.0%) after adding ice water and suction filtration. 1 H NMR (400 MHz, CDC13) δ 8.22 - 8.09 (m, 1H), 8.08 - 7.94 (m, 1H), 7.48 (s, 1H), 7.30 (d, J = 8.0 Hz, 1H), 6.95 - 6.77 (m, 1H), 3.82 (d, J = 1.5 Hz, 3H). ESI-MS calculated for [M+H] + : 196.0, found: 196.0.

[0070] Step 2: Synthesis of 2-methoxy-4-(2-nitroethyl)phenol (P2)

[0071] In 150 mL of tetrahydrofuran, P1 (20 g, 0.10 mol) was added, then sodium borohydride (11.35 g, 0.30 mol) was dissolved in 70 ml of tetrahydrofuran and 100 ml of ethanol under ice bath conditions, and the above sodium borohydride solution was added dropwise to the P1 solution, and the reaction was stirred at 25 °C for 1 h. After drying, purification (petroleum ether: ethyl acetate = 5:1) gave a light yellow oil compound P2 (9.9 g, yield 48.9%). ESI-MS calculated for [M-H] - : 196.0, found: 195.9.

[0072] Step 3: Synthesis of 4-(2-aminoethyl)-2-methoxyphenol (P3)

[0073] P2 (5 g, 0.025 mol) was dissolved in 25 ml of anhydrous tetrahydrofuran, and then 60 ml of lithium aluminum hydride (1 M) diluted in 50 ml of anhydrous THF was added. P2 was added dropwise into the above lithium aluminum hydride solution under ice bath condition, and then reacted at 60 °C for 4 hours. The reaction was monitored, and after completion, ice water was slowly added dropwise into the reaction under ice bath condition, and then the reaction solution was suction filtered, and the filter cake was the crude product P3 (5 g). ESI-MS calculated for [M+H]+: 168.1, found: 168.1. + :168.1, found:168.1.

[0074] Step 4: Synthesis of (E)-3-((4-hydroxy-3-methoxyphenethyl)amino)but-2-enoic acid ethyl ester (P4)

[0075] P3 (5 g, 0.03 mol), ethyl acetoacetate (3.9 g, 0.03 mol) and sodium sulfate (10 g) were added into anhydrous tetrahydrofuran (50 ml). The reaction was carried out at room temperature for 16 hours. After the reaction was completed, the filter cake was washed with methanol. P4 (2.71 g, yield 32.3%) was obtained by rotary evaporation and purification (petroleum ether: ethyl acetate = 5:1). ESI-MS calculated for [M+H]+: 280.1, found: 280.0. + :280.1, found:280.0.

[0076] Step 5: Synthesis of 2-(7-hydroxy-6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1- yl)acetic acid ethyl ester (P5)

[0077] A mixture of P2O5 (12.38 g, 87.21 mmol) and phosphoric acid (15.19 g, 155 mmol) was added to P4 (2.71 g, 9.69 mmol). The mixture was heated to 120 °C and stirred for 1 hour. After the reaction was completed, the mixture was cooled to 30 °C, adjusted to pH = 9-10 with 25% ammonia water, and extracted with dichloromethane. The crude product P5 (2.69 g) was obtained by rotary evaporation as a brownish yellow oil. ESI-MS calculated for [M+H]+: 280.2, found: 280.2.

[0078] Step 6: Synthesis of 1-(2-ethoxy-2-oxoethyl)-7-hydroxy-6-methoxy-1-methyl-3,4- dihydroisoquinoline-2(1H)-carboxylic acid tert-butyl ester (P6)

[0079] P5 (5.5 g, 19.64 mmol, prepared in batches multiple times) and (Boc)20 (4.23 g, 19.64 mmol) were dissolved in DCM (60 ml), followed by the addition of DIPEA (5.08 g, 39.28 mmol). The reaction was stirred at room temperature for 16 hours, after the reaction was completed by TLC monitoring, the reaction was rotary dried and purified (petroleum ether: ethyl acetate = 5: 1) to obtain yellow oil P6 (4.95 g, yield 66.3%). ESI-MS calculated for [M+H] + : 380.2, found: 379.9.

[0080] Step 7: Synthesis of 2-(2-(tert-butoxycarbonyl)-7-hydroxy-6-methoxy-1- methyl-1, 2, 3, 4-tetrahydroisoquinolin-1-yl) acetic acid (P7)

[0081] P6 (10 g, 26 mmol) and aqueous lithium hydroxide (66 ml, 1 N) were added to THF (90 ml). Heated to 90 °C, stirred for 16 hours, after the reaction was completed, the reaction PH was adjusted to 5 with dilute hydrochloric acid. Rotary dried after extraction with EA to obtain brown solid P7 (8.07 g, yield 88.2%). 1 H NMR (400 MHz, CDC13) δ 6.57 (m, 1H), 6.28 (m, 1H), 3.81 (d, J = 15.2 Hz, 1H), 3.62 (d, J = 14.5 Hz, 4H), 3.25 (s, 1H), 2.69 - 2.50 (m, 2H), 2.38 (d, J = 15.1 Hz, 1H), 1.43 (d, J = 14.6 Hz, 3H), 1.26 (d, J = 14.4 Hz, 8H). ESI-MS calculated for [M-H] - : 350.2, found: 349.8.

[0082] Step 8: Synthesis of tert-butyl 7-hydroxy-6-methoxy-1-methyl-1-(2-oxo-2- (thiazol-2-ylamino) ethyl)-3, 4-dihydroisoquinoline-2 (1H)-carboxylate

[0083] To P7 (8.07 g, 22.9 mmol) in 120 mL of N-methylpyrrolidone was added HATU (17.42 g, 45.8 mmol) and DIPEA (5.95 g, 45.8 mmol) and stirred at room temperature for 0.5 h before adding 2-aminothiazole (2.75 g, 27.48 mmol). After complete dissolution at room temperature, it was heated at 80 °C overnight and monitored by LC-MS until completion. Excess ammonia was neutralized by adding dilute hydrochloric acid, followed by extraction and purification (petroleum ether: ethyl acetate = 7:3) to give P8 (7.49 g, yield 75.5%). ESI-MS calculated for [M+H] + : 434.2, found: 433.9.

[0084] Step 9: Synthesis of 3-(benzyloxy)benzoyl chloride (M4-Cl)

[0085] Under nitrogen protection, oxalyl chloride (4.45 g, 35.1 mmol) was added dropwise to a solution of 3-(benzyloxy)benzoic acid (1 g, 4.4 mmol) in anhydrous DCM (10 mL) under ice bath conditions. After the dropwise addition was completed, a drop of DMF was added, and the reaction was allowed to proceed at room temperature for 0.5 h. After the reaction was completed, the reaction solution was rotary evaporated to give the crude acyl chloride, which was used directly in the next step without purification.

[0086] Step 10: Synthesis of 8-(3-(benzyloxy)benzamido)octyl methanesulfonate (L1-S)

[0087] Under nitrogen protection, the above acyl chloride was added dropwise to a solution of Et3N (590 mg, 5.8 mmol) and 8-amino-1-octanol (420 mg, 2.9 mmol) in DCM (10 mL) under ice bath conditions. The reaction was allowed to proceed at room temperature for 0.5 h. After the reaction was completed, it was rotary evaporated to give a light yellow oil. Methanesulfonyl chloride (660 mg, 5.8 mmol) was added dropwise to a solution of Et3N (590 mg, 5.8 mmol) and the above oil in DCM (12 mL) at room temperature, and the reaction was allowed to proceed for 0.5 h. After the reaction was completed, it was rotary evaporated to give a yellow crude product. The crude product was purified by silica gel column chromatography (PE:EA = 1:3) to give L1-S (564 mg, yield 45.1%).

[0088] Step 11: Synthesis of tert-butyl 7-((8-(3-(benzyloxy)benzamido)octyl)oxy)-6-methoxy-1- methyl-1-(2-oxo-2-(thiazol-2-ylamino)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (L1-P8)

[0089] P8 (200 mg, 0.46 mmol) was dissolved in dry DMF (5 mL) under nitrogen at room temperature, and Cs2CO3 (300 mg, 0.92 mmol) was added. After 0.5 h, L1-S (239 mg, 0.55 mmol) was added, and the reaction mixture was stirred at 60 °C for 2 h. After the reaction was completed, the reaction mixture was quenched with ice water and extracted with EA. The crude product L1-P8 (150 mg) was obtained as a light yellow oil. ESI-MS calculated for [M+H]: 771.3, found: 771.3. + :771.3, found: 771.3.

[0090] Step 12: Synthesis of 3-(benzyloxy)-N-(8-((6-methoxy-1-methyl-1-(2-oxo-2-(thiazol-2-ylamino)ethyl)-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)octyl)benzamide (I-1)

[0091] L1-P8 (150 mg, 0.19 mmol) was dissolved in DCM (2 mL) and TFA (216 mg, 1.90 mmol) was added. The reaction mixture was stirred at room temperature for 0.5 h. The crude product was obtained by rotary evaporation. The crude product was purified by column chromatography (DCM:MeOH = 20:1) to give the target product I-1 (101 mg, yield 78%). 1 H NMR (400 MHz, CDCl3) δ 7.45-7.27 (m, 9H), 7.08 (d, J = 7.4 Hz, 1H), 6.89 (d, J = 3.4 Hz, 1H), 6.64 (s, 1H), 6.52 (s, 1H), 6.18 (s, 1H), 5.10 (s, 2H), 3.96 (s, 2H), 3.80 (s, 3H), 3.47 (d, J = 14.6 Hz, 2H), 3.30 (d, J = 13.2 Hz, 1H), 3.16 (s, 1H), 3.00 (m, 1H), 2.85 (d, J = 16.4 Hz, 1H), 2.76-2.53 (m, 2H), 1.81 (d, J = 9.5 Hz, 2H), 1.61 (s, 2H), 1.54 (s, 3H), 1.41-1.34 (m, 8H). 13C NMR (151 MHz, CDC13) δ 166.26, 157.92, 136.45, 135.60, 135.58, 135.30, 128.53, 127.58, 127.05, 126.53, 126.51, 118.04, 118.01, 117.98, 117.18, 117.16, 117.13, 112.33, 112.07, 111.09, 111.06, 69.11, 69.09, 69.07, 68.35, 68.28, 63.44, 59.37, 54.85, 54.82, 54.80, 53.79, 53.54, 45.61, 44.85, 39.12, 39.09, 39.07, 39.04, 37.78, 37.37, 28.61, 28.57, 28.23, 28.16, 27.51, 25.89, 25.87, 24.94, 24.79, 24.77. ESI-MS calculated for [M+H] + : 671.3, found: 671.3.

[0092] Example Two: Preparation of Compound I-2

[0093] Synthesis of 3-(benzyloxy)-N-(8-((6-methoxy-1-methyl-1-(2-oxo-2-(thiazol-2-ylamino)ethyl)-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)octyl)benzamide (I-2)

[0094] Reference to the synthetic route of I-1 in Example One. The starting materials were chosen from 3,4-bis(benzyloxy)benzoic acid and 8-amino-1-octanol, and the target product I-2 (42 mg, 8% overall yield for multi-step reactions) was purified by column chromatography (DCM:MeOH = 20:1). 1H NMR (600 MHz, CDC13-d) δ 7.49 (d, J = 2.1 Hz, 1H), 7.47 - 7.40 (m, 5H), 7.36 (td, J = 7.2, 1.0 Hz, 4H), 7.33 - 7.28 (m, 2H), 7.23 (dd, J = 8.4, 2.1 Hz, 1H), 6.92 - 6.86 (m, 2H), 6.52 (s, 1H), 6.07 (t, J = 5.7 Hz, 1H), 5.19 (d, J = 2.8 Hz, 4H), 3.96 (td, J = 6.9, 3.8 Hz, 2H), 3.80 (s, 3H), 3.41 (td, J = 7.2, 5.8 Hz, 2H), 3.30 (dt, J = 13.1, 5.3 Hz, 1H), 3.18 (td, J = 8.0, 3.9 Hz, 1H), 3.02 (d, J = 16.1 Hz, 1H), 2.86 (dt, J = 16.2, 5.3 Hz, 1H), 2.77 - 2.63 (m, 2H), 1.81 (p, J = 7.0 Hz, 2H), 1.58 (q, J = 6.8 Hz, 2H), 1.55 (s, 3H), 1.48 - 1.33 (m, 8H). 13 C NMR (151 MHz, CDC13) δ 165.95, 150.45, 147.74, 147.38, 146.32, 135.89, 135.71, 127.54, 127.50, 127.02, 126.92, 126.90, 126.43, 126.15, 118.89, 113.02, 112.70, 112.11, 111.02, 109.48, 76.21, 76.00, 75.79, 70.26, 69.98, 68.30, 54.88, 39.07, 37.31, 28.68, 28.62, 28.14, 28.04, 25.84, 24.77. ESI-MS calculated for [M+H] + : 777.4, found: 777.4.

[0095] Example Three: Preparation of Compound I-3

[0096] Synthesis of 3-((4-fluorobenzyl)oxy)-N-(2-(2-((6-methoxy-1-methyl-1-(2-oxo-2-(thiazol-2-ylamino)ethyl)-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)ethoxy)ethoxy)ethyl)benzamide (I-3)

[0097] The synthetic route of I-1 in Reference Example 1 was referred. The starting material was selected from 3-((4-fluorobenzyl)oxy)benzoic acid and 2-(2-(2-aminoethoxy)ethoxy)-1-ethanol. The target product I-3 (20 mg, total yield 12%) was purified by column chromatography (DCM:MeOH = 15:1). 1 H NMR (400 MHz, CDCl3) δ 7.49 (d, J = 2.2 Hz, 1H), 7.43-7.29 (m, 5H), 7.22 (t, J = 7.9 Hz, 1H), 7.09-6.98 (m, 3H), 6.86 (dt, J = 3.6, 0.7 Hz, 1H), 6.69 (s, 1H), 6.47 (s, 1H), 5.01 (s, 2H), 3.82 (dd, J = 6.0, 3.7 Hz, 2H), 3.75-3.56 (m, 13H), 3.26 (dt, J = 11.8, 5.5 Hz, 1H), 3.16 (ddd, J = 12.6, 7.2, 4.9 Hz, 1H), 2.98 (m, 1H), 2.85 (m, 5.7 Hz, 1H), 2.75-2.58 (m, 2H), 1.50 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 167.68, 166.31, 162.30, 160.67, 157.67, 156.93, 147.32, 145.76, 136.45, 135.15, 131.46, 131.44, 130.99, 128.44, 128.40, 128.34, 126.20, 118.41, 117.36, 114.53, 114.39, 112.37, 112.08, 111.07, 110.62, 69.67, 69.23, 68.91, 68.71, 68.37, 67.90, 59.38, 54.65, 53.68, 45.68, 38.91, 37.37, 28.38, 28.24. ESI-MS calculated for [M+H] + : 693.3, found: 693.3.

[0098] Example Four: Preparation of compound I-4

[0099] Synthesis of 3-(benzyloxy)-N-(4-((6-methoxy-1-methyl-1-(2-oxo-2-(thiazol-2-ylamino)ethyl)-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)butyl)benzamide (I-4)

[0100] The synthetic route of I-1 in Reference Example 1 was referred. The starting material was selected from 3-(benzyloxy)benzoic acid and 4-amino-1-butanol. The target product I-4 (15 mg, total yield 7%) was purified by column chromatography (DCM:MeOH = 20:1). 1 H NMR (600 MHz, CDC13) δ 7.49 (dd, J = 2.6, 1.6 Hz, 1H), 7.40-7.33 (m, 6H), 7.31-7.27 (m, 1H), 7.14 (t, J = 5.9 Hz, 1H), 7.06 (ddd, J = 8.2, 2.6, 1.0 Hz, 1H), 6.86 (d, J = 3.6 Hz, 1H), 6.66 (s, 1H), 6.48 (s, 1H), 5.05 (s, 2H), 4.03 (td, J = 6.0, 4.7 Hz, 2H), 3.65 (s, 3H), 3.59-3.44 (m, 2H), 3.30-3.23 (m, 1H), 3.18 (ddd, J = 12.7, 6.7, 4.9 Hz, 1H), 3.00 (d, J = 15.9 Hz, 1H), 2.88 (m, 1H), 2.73-2.63 (m, 2H), 1.88 (q, J = 6.1 Hz, 2H), 1.81 (m, J = 6.9, 2.2 Hz, 2H), 1.52 (s, 3H). 13 C NMR (151 MHz, CDC13) δ 167.74, 166.40, 157.86, 156.91, 147.12, 145.72, 136.43, 135.66, 135.42, 130.72, 128.43, 127.59, 127.56, 127.01, 126.50, 125.67, 118.26, 117.26, 112.25, 112.09, 110.83, 109.45, 69.05, 67.97, 54.52, 53.84, 45.85, 38.32, 37.40, 28.32, 25.37, 25.05. ESI-MS calculated for [M+H] + : 615.3, found: 615.3.

[0101] Example Five: Preparation of compound I-5

[0102] Synthesis of 3-(benzyloxy)-N-(5-((6-methoxy-1-methyl-1-(2-oxo-2-(thiazol-2-ylamino)ethyl)-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)pentyl)benzamide (I-5)

[0103] The synthetic route of I-1 in Reference Example 1 was referred. The starting material was selected from 3-(benzyloxy)benzoic acid and 5-amino-1-pentanol. The target product I-5 (25 mg, total yield 18%) was purified by column chromatography (DCM:MeOH = 20:1). 1 H NMR (400 MHz, CDC13) δ 7.47 (t, J = 2.1 Hz, 1H), 7.41 - 7.26 (m, 7H), 7.09 - 7.02 (m, 1H), 6.85 (d, J = 3.6 Hz, 1H), 6.81 (t, J = 5.6 Hz, 1H), 6.63 (s, 1H), 6.48 (s, 1H), 5.03 (s, 2H), 3.98 (td, J = 6.5, 3.0 Hz, 2H), 3.72 (s, 3H), 3.56 - 3.38 (m, 2H), 3.25 (ddd, J = 12.0, 6.8, 4.8 Hz, 1H), 3.19 - 3.08 (m, 1H), 2.98 (d, J = 16.0 Hz, 1H), 2.86 (dt, J = 16.3, 5.8 Hz, 1H), 2.77 - 2.53 (m, 2H), 1.92 - 1.75 (m, 2H), 1.67 (q, J = 7.2 Hz, 2H), 1.51 (d, J = 14.2 Hz, 5H). 13 C NMR (151 MHz, CDC13) δ 167.78, 166.38, 157.87, 156.88, 147.27, 145.96, 136.46, 135.62, 135.34, 130.73, 128.49, 127.57, 127.03, 126.55, 125.59, 118.22, 117.16, 112.30, 112.07, 111.07, 109.75, 69.05, 68.08, 54.74, 53.73, 45.86, 38.92, 37.40, 28.33, 28.32, 28.08, 27.62, 22.47. ESI-MS calculated for [M+H] + : 629.3, found: 629.3.

[0104] Example Six: Preparation of compound I-6

[0105] Synthesis of 3-(benzyloxy)-N-(2-(2-((6-methoxy-1-methyl-1-(2-oxo-2-(thiazol-2-ylamino)ethyl)-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)ethoxy)ethoxy)ethyl)benzamide (I-6)

[0106] The synthetic route of I-1 in Reference Example 1 was referred. The starting material was selected from 3-(benzyloxy)benzoic acid and 2-(2-(2-aminoethoxy)ethoxy)-1-ethanol. The target product I-6 (48 mg, total yield 15%) was obtained by purification through column chromatography (DCM:MeOH = 15:1). 1 H NMR (400 MHz, CDC13) δ 7.49 (dd, J = 2.6, 1.6 Hz, 1H), 7.43 - 7.30 (m, 6H), 7.22 (d, J = 7.8 Hz, 1H), 7.16 (d, J = 5.7 Hz, 1H), 7.03 (m, 1H), 6.86 (d, J = 3.5 Hz, 1H), 6.70 (s, 1H), 6.48 (s, 1H), 5.05 (s, 2H), 4.10 (dd, J = 5.6, 4.1 Hz, 2H), 3.90 - 3.77 (m, 2H), 3.76 - 3.61 (m, 11H), 3.32 - 3.21 (m, 1H), 3.16 (ddd, J = 12.7, 7.2, 4.9 Hz, 1H), 3.00 (d, J = 16.0 Hz, 1H), 2.85 (dt, J = 16.3, 5.6 Hz, 1H), 2.74 - 2.58 (m, 2H), 1.50 (s, 3H). 13 C NMR (151 MHz, CDC13) δ 167.83, 157.02, 148.01, 147.46, 147.30, 145.79, 136.43, 136.34, 136.29, 131.19, 127.44, 127.40, 126.73, 126.71, 126.40, 126.35, 126.26, 120.64, 114.45, 114.09, 112.05, 111.24, 111.17, 70.36, 70.15, 69.64, 69.29, 68.74, 68.13, 54.76, 53.79, 51.80, 46.94, 45.55, 37.41, 28.42, 28.28. ESI-MS calculated for [M+H] + : 675.3, found: 675.3.

[0107] Example Seven: Preparation of compound I-7

[0108] Synthesis of 3-(benzyloxy)-N-(2-(2-(2-((6-methoxy-1-methyl-1-(2-oxo-2-(thiazol-2-ylamino)ethyl)-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)ethoxy)ethoxy)ethyl)benzamide (I-7)

[0109] The synthetic route of I-1 in Example One was referred. The starting materials were selected from 3-(benzyloxy)benzoic acid and 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-1-ethanol. The target product I-7 (15 mg, total yield 6%) was purified by column chromatography (DCM:MeOH=10:1). 1 H NMR (600 MHz, CDC13) δ 7.42 (t, J = 2.1 Hz, 1H), 7.34 - 7.26 (m, 5H), 7.24 - 7.16 (m, 2H), 7.06 (t, J = 5.3 Hz, 1H), 6.96 (dd, J = 8.2, 2.6 Hz, 1H), 6.78 (d, J = 3.5 Hz, 1H), 6.61 (s, 1H), 6.40 (s, 1H), 4.98 (s, 2H), 3.99 (t, J = 4.9 Hz, 2H), 3.71 - 3.67 (m, 2H), 3.65 (s, 3H), 3.57 (t, J = 10.6 Hz, 12H), 3.17 (dt, J = 11.8, 5.5 Hz, 1H), 3.07 (ddd, J = 12.7, 7.4, 4.9 Hz, 1H), 2.91 (d, J = 16.0 Hz, 1H), 2.76 (dt, J = 11.2, 5.7 Hz, 1H), 2.65 - 2.53 (m, 2H), 1.42 (s, 3H). ESI-MS calculated for [M+H] + : 719.3, found: 719.3.

[0110] Example Eight: Preparation of compound I-10

[0111] Synthesis of 3-(benzyloxy)-N-((4-(((6-methoxy-1-methyl-1-(2-oxo-2-(thiazol-2-ylamino)ethyl)-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)methyl)cyclohexyl)methyl)benzamide (I-10)

[0112] The synthetic route of I-1 in Example One was referred. The starting materials were selected from 3-(benzyloxy)benzoic acid and (4-(aminomethyl)cyclohexyl)methanol. The target product I-10 (43 mg, total yield 12%) was purified by column chromatography (DCM:MeOH=20:1). 1H NMR (400 MHz, CDC13) δ 7.47 - 7.29 (m, 9H), 7.09 (dt, J=7.4, 2.3 Hz, 1H), 6.89 (d, J=3.6 Hz, 1H), 6.64 (s, 1H), 6.52 (s, 1H), 6.24 (s, 1H), 5.10 (s, 2H), 3.79 (s, 3H), 3.76 (t, J=5.6 Hz, 2H), 3.32 (t, J=6.4 Hz, 2H), 3.20 (d, J=7.0 Hz, 1H), 3.06 (d, J=16.2 Hz, 1H), 2.97 (d, J=14.8 Hz, 1H), 2.87 (q, J=7.0 Hz, 1H), 2.73 (dd, J=16.6, 7.1 Hz, 2H), 1.98 (d, J=10.1 Hz, 2H), 1.87 (d, J=8.1 Hz, 5H), 1.57 (s, 3H), 1.44 - 1.17 (m, 1H). 13 C NMR (151 MHz, CDC13) δ 166.39, 157.97, 147.46, 146.56, 135.57, 135.35, 128.62, 127.62, 127.09, 126.55, 117.93, 117.18, 112.39, 112.10, 111.23, 109.62, 73.61, 69.17, 54.96, 45.15, 40.96, 37.28, 37.08, 36.53, 29.12, 29.10, 28.25, 28.22. ESI-MS calculated for [M+H] + : 669.3, found: 669.3.

[0113] Example Nine: Preparation of compound I-11

[0114] Synthesis of 2-(7-((7-(3-(benzyloxy)benzoyl)-7-azaspiro[3.5]nonan-2-yl)methoxy)-6- methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide (I-11)

[0115] Reference to the synthetic route of I-1 in Example One. The starting materials were chosen from 3-(benzyloxy)benzoic acid and (7-azaspiro[3.5]nonan-2-yl)methanol. The target product I-11 (18 mg, yield 8%) was purified by column chromatography (DCM:MeOH = 15:1). 1H NMR (400 MHz, CDC13) δ 7.47 - 7.34 (m, 5H), 7.36 - 7.27 (m, 2H), 7.00 (tdd, J = 7.6, 6.0, 1.9 Hz, 3H), 6.89 (d, J = 3.6 Hz, 1H), 6.66 (s, 1H), 6.53 (s, 1H), 5.09 (s, 2H), 3.80 (s, 5H), 3.36 - 3.25 (m, 1H), 3.20 (ddd, J = 12.5, 6.9, 4.8 Hz, 1H), 3.03 (d, J = 16.0 Hz, 2H), 2.89 (dt, J = 11.3, 5.6 Hz, 2H), 2.80 - 2.59 (m, 2H), 2.34 (t, J = 6.6 Hz, 1H), 2.19 - 2.08 (m, 2H), 1.97 (s, 1H), 1.92 - 1.77 (m, 2H), 1.77 - 1.68 (m, 1H), 1.56 (s, 3H). 13 C NMR (151 MHz, CDC13) δ 169.02, 167.64, 157.73, 156.97, 147.45, 146.07, 136.64, 136.40, 135.64, 130.85, 128.65, 127.60, 127.04, 126.46, 125.89, 118.22, 115.30, 112.07, 111.29, 69.09, 54.88, 53.73, 45.83, 40.96, 37.39, 35.15, 28.34, 28.32, 25.99, 23.97. ESI-MS calculated for [M+H] + : 641.3, found: 641.3.

[0116] Example Ten: Preparation of Compound I-12

[0117] Synthesis of 2-(7-((7-(3-(benzyloxy)benzoyl)-7-azaspiro[3.5]nonan-2-yl)methoxy)-6- methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide (I-12)

[0118] Reference to the synthetic route of I-1 in Example One. The starting materials were chosen from 3-(benzyloxy)benzoic acid and piperidin-4-ylmethanol. The target product I-12 (33 mg, yield 16%) was purified by column chromatography (DCM:MeOH = 15:1). 1H NMR (400 MHz, CDC13) δ 7.47 - 7.35 (m, 5H), 7.31 (tt, J = 7.2, 2.2 Hz, 2H), 7.00 (m 3H), 6.88 (d, J = 3.6 Hz, 1H), 6.65 (s, 1H), 6.53 (s, 1H), 5.08 (s, 2H), 3.79 (s, 5H), 3.30 (ddd, J = 11.7, 6.5, 4.8 Hz, 1H), 3.19 (m, 1H), 3.02 (d, J = 16.1 Hz, 2H), 2.89 (m, 2H), 2.78 - 2.63 (m, 2H), 2.33 (t, J = 6.6 Hz, 1H), 2.18 - 2.07 (m, 2H), 1.96 (s, 1H), 1.86 m, 2H), 1.77 - 1.69 (m, 1H), 1.55 (s, 3H). 13 C NMR (151 MHz, CDC13) δ 169.00, 167.73, 157.71, 147.49, 146.34, 136.72, 136.28, 135.66, 128.62, 127.61, 127.05, 126.46, 125.54, 118.22, 115.24, 112.09, 112.07, 111.26, 110.09, 73.06, 69.09, 54.91, 53.80, 45.64, 37.35, 33.66, 33.51, 28.68, 28.25, 28.18, 26.82. ESI-MS calculated for [M+H] + : 681.3, found: 681.3.

[0119] Example Eleven: Preparation of Compound I-8

[0120] Synthesis of 2-(7-((8-((3-(benzyloxy)benzyl)amino)octyl)oxy)-6-methoxy-1- methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide

[0121] Step 1-Step 8: P8 was synthesized according to the synthetic route of Example One, Steps 1 to 8 to synthesize I-1.

[0122] Step 9: Synthesis of 8-((3-(benzyloxy)benzyl)amino)octyl methanesulfonate (L8-S)

[0123] At room temperature, 3-(benzyloxy)benzaldehyde (500 mg, 2.36 mmol) and 8-aminooctane-1-ol (410 mg, 2.83 mmol) were dissolved in methanol (10 mL) and reacted at 25 °C for 5 hours. NaBH3CN (296 mg, 4.72 mmol) was added, and the reaction was continued overnight at 50 °C. After the reaction was complete, the reaction was quenched with ice water. The reaction mixture was extracted with EA and evaporated to dryness to obtain 598 mg of a pale yellow oil. The oil was dissolved in DCM (5 mL), and Boc2O (420 mg, 1.93 mmol) and DIPEA (453 mg, 3.51 mmol) were added. After stirring at room temperature for 3 hours, the mixture was extracted with DCM and evaporated to dryness. Under ice bath conditions, methanesulfonyl chloride (114 mg, 2.04 mmol) was added dropwise to a DCM (5 mL) solution containing the crude product (300 mg) and Et3N (206 mg, 2.04 mmol), and the reaction was carried out at room temperature for 0.5 h. After the reaction was complete, the solution was evaporated to dryness to give a yellow oil. The yellow oil was then purified by column chromatography (DCM:MeOH = 25:1) to give L8-S (197 mg, yield 16%).

[0124] Step 10: Synthesis of 7-((8-((3-(benzyloxy)benzyl)amino)octyl)oxy)-6-methoxy-1-methyl-1-(2-oxo-2-(thiazo-2-ylamino)ethyl)-3,4-dihydroisoquinoline-2(1H)-formate tert-butyl ester (L8-P8)

[0125] Under nitrogen protection and at room temperature, P8 (150 mg, 0.35 mmol) was dissolved in anhydrous DMF (8 mL), and Cs₂CO₃ (225 mg, 0.69 mmol) was added. After reacting for 0.5 hours, L8-S (218 mg, 0.42 mmol) was added, and the mixture was transferred to an oil bath at 60 °C for 2 hours. After the reaction was complete, the reaction was quenched with ice water, and the reaction solution was extracted with EA and evaporated to dryness to obtain a pale yellow oil. Subsequently, L8-P8 (75 mg, 25% yield) was purified by column chromatography (DCM:MeOH = 50:1). ESI-MS calculated for [M+H] + :857.4, found:858.0.

[0126] Step 11: Synthesis of 2-(7-((8-((3-(benzyloxy)benzyl)amino)octyl)oxy)-6-methoxy-1-methyl-1,2,3,4-tetrahydroisoquinoline-1-yl)-N-(thiazolyl-2-yl)acetamide (I-8)

[0127] L8-P8 (75 mg, 0.09 mmol) was dissolved in DCM (1 mL) and TFA (102 mg, 0.9 mmol) was added, and the reaction was stirred at room temperature for 0.5 h. The reaction was concentrated to give the crude product. The target product I-8 (38 mg, yield 64%) was obtained by column chromatography (DCM:MeOH = 10:1). 1 H NMR (400 MHz, CDC13) δ 7.46 - 7.27 (m, 6 H), 7.23 (d, J = 8.0 Hz, 1 H), 7.08 - 7.02 (m, 1 H), 6.96 (d, J = 7.5 Hz, 1 H), 6.92 - 6.85 (m, 2 H), 6.65 (s, 1 H), 6.51 (s, 1 H), 5.06 (s, 2 H), 3.95 (td, J = 7.0, 2.9 Hz, 2 H), 3.84 (s, 2 H), 3.80 (s, 3 H), 3.29 (dt, J = 13.0, 5.3 Hz, 1 H), 3.21 - 3.11 (m, 1 H), 3.02 (d, J = 16.1 Hz, 1 H), 2.84 (dt, J = 16.3, 5.2 Hz, 1 H), 2.75 - 2.61 (m, 4 H), 1.80 (p, J = 7.0 Hz, 2 H), 1.68 - 1.15 (m, 13 H). 13 C NMR (151 MHz, CDC13) δ 167.76, 158.02, 157.03, 147.26, 146.28, 136.40, 135.97, 131.09, 128.60, 127.53, 126.91, 126.52, 125.23, 120.24, 120.19, 113.93, 113.89, 113.16, 112.08, 111.03, 109.53, 68.94, 68.28, 61.87, 54.87, 53.62, 52.03, 47.49, 45.53, 37.38, 28.39, 28.20, 28.17, 28.16, 28.09, 26.04, 24.78. ESI-MS calculated for [M+H] + : 657.3, found: 657.3.

[0128] Example Twelve: Preparation of compound I-9

[0129] Synthesis of 2-(7-(2-(2-((3-(benzyloxy)benzyl)amino)ethoxy)ethoxy)ethoxy)-6-methoxy- 1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide (I-9)

[0130] The synthetic route of I-8 of Reference Example Eleven was referred to. The starting material was selected from 3-(benzyloxy)benzaldehyde and 2-(2-(2- aminoethoxy)ethoxy)-1-ethanol. The target product I-9 (35 mg, total yield 19%) was obtained by purification by column chromatography (DCM:MeOH=10:1). 1 H NMR (600 MHz, CDC13) δ 7.43 (d, J = 7.1 Hz, 2H), 7.41-7.36 (m, 3H), 7.34-7.30 (m, 1H), 7.22 (t, J = 7.8 Hz, 1H), 7.02 (t, J = 1.9 Hz, 1H), 6.94 (d, J = 7.5 Hz, 1H), 6.90-6.86 (m, 2H), 6.79 (s, 1H), 6.51 (s, 1H), 5.05 (s, 2H), 4.22-4.10 (m, 2H), 3.84 (dd, J = 9.4, 4.4 Hz, 4H), 3.77 (s, 3H), 3.72 (dd, J = 5.5, 3.2 Hz, 2H), 3.67 (tt, J = 6.7, 3.4 Hz, 4H), 3.28 (ddd, J = 12.9, 6.4, 4.9 Hz, 1H), 3.18 (m, 1H), 3.04 (d, J = 16.0 Hz, 1H), 2.90-2.82 (m, 3H), 2.78-2.62 (m, 2H), 1.53 (s, 3H). 13 C NMR (151 MHz, CDC13) δ 167.83, 158.00, 157.04, 147.45, 145.83, 136.40, 136.00, 131.12, 128.55, 127.53, 126.90, 126.52, 126.25, 120.10, 113.87, 112.97, 112.07, 111.17, 111.10, 69.66, 69.29, 68.92, 68.75, 68.52, 68.08, 54.77, 53.83, 52.10, 47.09, 45.51, 37.40, 28.38, 28.23. ESI-MS calculated for [M+H] + : 661.3, found: 661.3.

[0131] Example Thirteen: Preparation of Compound I-13

[0132] Synthesis of 2-(7-(2-(2-((3,4-bis(benzyloxy)benzyl)amino)ethoxy)ethoxy)ethoxy)-6- methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide (I-13)

[0133] The synthetic route of I-8 of Reference Example Eleven was referred to. The starting material was selected from 3,4-bis(benzyloxy)benzaldehyde and 2-(2-(2- aminoethoxy)ethoxy)-1-ethanol. The target product I-13 (55 mg, total yield 8%) was obtained by purification by column chromatography (DCM:MeOH = 10:1). 1 H NMR (600 MHz, CDC13) δ 7.46 - 7.41 (m, 4 H), 7.38 (d, J = 3.5 Hz, 1 H), 7.34 (ddt, J = 7.8, 6.3, 1.3 Hz, 4 H), 7.31 - 7.27 (m, 2 H), 7.04 (d, J = 1.9 Hz, 1 H), 6.89 - 6.83 (m, 3 H), 6.79 (s, 1 H), 6.49 (s, 1 H), 5.14 (s, 2 H), 5.12 (s, 2 H), 4.15 - 4.09 (m, 2 H), 3.82 (t, J = 5.0 Hz, 2 H), 3.78 (d, J = 1.9 Hz, 2 H), 3.74 (s, 3 H), 3.70 (dd, J = 6.1, 3.6 Hz, 2 H), 3.68 - 3.63 (m, 4 H), 3.26 (ddd, J = 12.9, 6.5, 4.8 Hz, 1 H), 3.17 (ddd, J = 12.6, 7.2, 4.9 Hz, 1 H), 3.03 (d, J = 15.9 Hz, 1 H), 2.89 - 2.78 (m, 3 H), 2.75 - 2.63 (m, 2 H), 1.52 (s, 3 H). 13 C NMR (151 MHz, CDC13) δ 167.83, 157.02, 148.01, 147.46, 147.30, 145.79, 136.43, 136.34, 136.29, 131.19, 127.44, 127.40, 126.73, 126.71, 126.40, 126.35, 126.26, 120.64, 114.45, 114.09, 112.05, 111.24, 111.17, 76.22, 76.01, 75.80, 70.36, 70.15, 69.64, 69.29, 68.74, 68.13, 54.76, 53.79, 51.80, 46.94, 45.55, 37.41, 28.42, 28.28. ESI-MS calculated for [M+H] + : 767.3473, found: 767.3492.

[0134] Example Fourteen: Preparation of Compound I-14

[0135] Synthesis of 2-(7-(2-(1-(3-(benzyloxy)benzoyl)piperidin-4-yl)ethoxy)-6- methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide (I-14)

[0136] Reference to the synthetic route of I-1 in Example one. The starting materials were chosen from 3-(benzyloxy)benzoic acid and 2-(piperidin-4-yl)ethanol. The target product I-14 (13 mg, total yield 11%) was purified by column chromatography (DCM:MeOH = 20:1). 1 HNMR (400 MHz, CDC13-d) δ 7.49-7.26 (m, 7H), 7.0-6.93 (m, 3H), 6.88 (d, J = 3.5 Hz, 1H), 6.65 (s, 1H), 6.52 (s, 1H), 5.07 (s, 2H), 4.02 (t, J = 6.2 Hz, 2H), 3.79 (d, J = 2.7 Hz, 3H), 3.37-3.25 (m, 1H), 3.18 (m, 1H), 3.01 (d, J = 16.0 Hz, 1H), 2.93-2.81 (m, 2H), 2.82-2.62 (m, 3H), 1.78 (d, J = 6.3 Hz, 5H), 1.67 (s, 1H), 1.54 (s, 3H). 13 C NMR (151 MHz, CDC13-d) δ 168.93, 167.64, 157.70, 156.99, 147.37, 146.01, 136.70, 136.39, 135.66, 130.91, 128.61, 127.61, 127.04, 126.46, 125.72, 118.24, 115.23, 112.10, 112.07, 111.08, 69.08, 65.96, 54.83, 53.70, 45.75, 37.38, 34.53, 32.11, 28.36, 28.26. ESI-MS calculated for [M+H] + : 670.3235, found: 670.3238.

[0137] Example Fifteen: Preparation of compound I-15

[0138] Synthesis of 2-(7-((2-(3-(benzyloxy)benzoyl)-2-azaspiro[3.3]heptan-6-yl)oxy)-6- methoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)-N-(thiazol-2-yl)acetamide (I-15)

[0139] The synthetic route of I-1 of Example 1 was referred. The starting material was selected from 3-(benzyloxy)benzoic acid and 2-azaspiro[3,3]heptan-6-ol. The target product I-15 (15 mg, total yield 16%) was obtained by purification with column chromatography (DCM:MeOH=20:1). 1 H NMR (400 MHz, CDC13-d) δ 7.50-7.46 (m, 1H), 7.26 (m, 8H), 7.09 (t, J = 4.2 Hz, 1H), 6.88-6.78 (m, 1H), 6.60-6.37 (m, 2H), 5.07 (d, J = 11.2 Hz, 2H), 4.73 (m, 1H), 3.79 (s, 2H), 3.74-3.61 (m, 5H), 3.27 (m, 2H), 2.94 (s, 2H), 2.87 (s, 2H), 2.78-2.53 (m, 2H), 2.42-1.99 (m, 2H), 1.51 (d, J = 13.6 Hz, 3H).13C NMR (151 MHz, CDC13-d) δ 167.70, 166.96, 161.52, 158.00, 157.94, 156.82, 143.68, 136.48, 135.56, 135.51, 134.95, 128.77, 128.60, 127.62, 127.60, 127.09, 127.07, 126.57, 126.53, 118.27, 117.62, 117.59, 112.43, 112.33, 112.11, 111.17, 69.18, 69.10, 67.47, 54.80, 54.69, 51.35, 50.29, 45.33, 37.35, 37.26, 35.46, 34.89, 34.61, 30.42.

[0140] Example Six: Preparation of Compound I-16

[0141] Synthesis of 3-(benzyloxy)-N-(2-(2-(2-((6-methoxy-1-methyl-1-(2-oxo-2-(pyridazin-3-ylamino)ethyl)-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)ethoxy)ethoxy)ethyl)benzamide (I-16)

[0142] The synthetic route of I-1 of Example 1 was referred. The starting material was selected from 3-(benzyloxy)benzoic acid and 2-azaspiro[3,3]heptan-6-ol. The target product I-15 (15 mg, total yield 16%) was obtained by purification with column chromatography (DCM:MeOH=20:1). 1H NMR (400 MHz, CDC13-d) δ 8.83 (dd, J = 4.8, 1.5 Hz, 1H), 8.27 (d, J = 9.0 Hz, 1H), 7.49 (t, J = 2.1 Hz, 1H), 7.37 (m, 8H), 7.13 (s, 1H), 7.03 (dd, J = 8.2, 2.6 Hz, 1H), 6.75 (s, 1H), 5.07 (s, 2H), 4.10 (dd, J = 5.9, 3.9 Hz, 2H), 3.82 (t, J = 4.8 Hz, 2H), 3.75 - 3.60 (m, 14H), 3.30 (s, 2H), 3.01 (d, J = 11.0 Hz, 1H), 2.72 (d, J = 9.6 Hz, 2H), 1.58 (s, 4H).13C NMR (101 MHz, CDC13-d) δ 167.44, 158.91, 155.38, 148.41, 148.15, 136.71, 136.16, 129.49, 128.64, 128.09, 127.59, 119.35, 118.89, 118.35, 113.48, 112.03, 111.47, 70.77, 70.31, 70.13, 69.99, 69.80, 68.95, 55.72, 39.97, 38.42, 29.74. ESI-MS calculated for [M+H]+: 655.2949, found: 655.2943.

[0143] Cellular assay to measure the targeted degradation activity of the compounds on PCSK9.

[0144] Western Blot and immunofluorescence experiments were performed to measure the targeted degradation of PCSK9 by the compounds. The targeted degradation was measured by the maximum degradation of PCSK9 protein and the optimal degradation concentration of the compounds.

[0145] Cell lines and reagents:

[0146] LDLR (cat# ab240748), P62 antibody (cat# ab314504) were purchased from Abeam. PCSK9 (cat# 55723), LC3B antibody (cat# 3868) were purchased from Cell Signaling Technologies. GAPDH antibody was purchased from Bioworld (cat# MB66349). All other reagents were purchased from Sigma Aldrich.

[0147] Except for palmitic acid solution, other drug solutions were stored in DMSO or PBS at 1000 times the actual use concentration.

[0148] Preparation of linker compounds:

[0149] Synthesis of 3-(benzyloxy)-N-methylbenzamide (M4)

[0150] M4-Cl (713 mg, 2.9 mmol) was added dropwise to a solution of Et3N (590 mg, 5.8 mmol) and methylamine (90 mg, 2.9 mmol) in DCM (10 mL) under nitrogen protection with ice-bath cooling. The reaction was allowed to proceed at room temperature for 0.5 h. After completion of the reaction, the reaction mixture was concentrated to give a light yellow oil. The crude product was purified by column chromatography on silica gel (PE:EA = 3:1) to give M4 75 mg in 68% yield.

[0151] Synthesis of 2-(7-hydroxy-6-methoxy-l-methyl-l,2,3,4-tetrahydroisoquinolin-l- yl)-N-(thiazol-2-yl)acetamide (P9)

[0152] P8 (150 mg, 0.35 mmol) was dissolved in DCM (2 mL) and TFA (200 mg, 1.75 mmol) was added. The reaction was allowed to proceed at room temperature for 0.5 h. The reaction mixture was concentrated to give the crude product. The crude product was purified by column chromatography (DCM:MeOH = 30:1) to give the target product P9 (91 mg, 78% yield).

[0153] Experimental methods:

[0154] HepG2 cells (ATCC) were cultured in high glucose DMEM medium (Hyclone) supplemented with 10% fetal bovine serum (BI) and 1% penicillin and 1% streptomycin (Sigma) in an incubator environment of 37 °C, 95% air and 5% carbon dioxide.

[0155] HepG2 cells were dosed directly or after modeling when they were grown to about 70% density. High-fat inflammatory cell model modeling: when HepG2 cells were grown to the appropriate concentration, the cells were co-treated with 500 μΜ palmitic acid and 1 μg / mL LPS for 24 hours.

[0156] After drug treatment, HepG2 cells were lysed on ice for 30 minutes by RIPA (Beyotime), and the supernatant was obtained by high-speed centrifugation. The protein concentration was determined by BCA method (Beyotime) and balanced to the same concentration. Then 5xLoading Buffer was added, and the protein sample solution was prepared by boiling for 5 minutes for Western blot experiment. Western blot experiment mainly includes polyacrylamide gel electrophoresis separation of protein, PVDF membrane transfer, 5% skim milk powder-containing TBST solution blocking for 30 minutes, primary antibody solution incubation at 4°C overnight, secondary antibody incubation at room temperature for 60 minutes, development with developing solution (Millipore), and recording using gel imaging system. In this experiment, PCSK9 protein bands use PCSK9 primary antibody solution and rabbit anti-secondary antibody solution. GAPDH protein bands use GAPDH primary antibody solution and mouse anti-secondary antibody solution. LDLR protein bands use LDLR primary antibody solution and rabbit anti-secondary antibody solution.

[0157] Figures 1-6 show the Western Blot experiment results of the compounds prepared according to the embodiments of the present application. It can be seen that the compounds I-6 can all produce the effect of reducing PCSK9 from the protein level on HepG2 cell lines, and have obvious hook effect. The Western Blot experiment of co-incubation with linker compounds proves that the linker compounds M4 and P9 do not have the activity of reducing PCSK9 protein alone. It is proved that the compounds of the present application have good degradation effect on PCSK9 protein.

[0158] Table 1 shows the statistical results of Western Blot experiment data of compounds I-1 to I-16 prepared according to the embodiments of the present application. Optimal concentration degradation rate (%) = 1-dose group Western Blot experiment band gray value / Control group band gray value. Among them, the operation of the Control group and the dose group is basically the same, and the only difference is that the concentration of the test compound for the Control group is 0nM. The administration time of I-1 to I-16 groups is 24 hours.

[0159] Table 1

[0160] Under the same other conditions, the administration time of I-11 is adjusted to 20 hours, and the degradation rate (%) (optimal concentration) is 43.44 ** (10nM).

[0161] Timed quantitative PCR experiment: The primers for real-time quantitative PCR were synthesized by Sangon Inc, TRIZOL was purchased from TAKARA, and the rest of the real-time quantitative PCR related reagents were purchased from Yeason. The real-time quantitative PCR results were homogenized according to GAPDH. The primer sequences are as follows: PCSK9: forward primer, 5'-AGGGGAGGACATCATTGGTG-3'(SEQ ID NO: 1); reverse primer, 5'-CAGGTTGGGGGTCAGTACC-3'(SEQ ID NO: 2); GAPDH: forward primer, 5'-ATGGGGAAGGTGAAGGTCG-3'(SEQ ID NO: 3); reverse primer, 5'-GGGGTCATTGATGGCAACAATA-3'(SEQ ID NO: 4).

[0162] Figure 7 shows the qPCR experimental results of compound I-6 prepared according to the embodiment of the present application. It can be seen that compound I-6 has no obvious effect on the expression level of PCSK9 mRNA, and only has an effect on PCSK9 at the protein level.

[0163] Cell MTT toxicity test: HepG2 cells were treated with drugs for 24h, then the culture medium was discarded, 20μl MTT was added per well and incubated for 4 hours, then the supernatant was discarded, 150μl DMSO was added, and after shaking for 10 minutes, it was tested in the enzyme marker instrument (absorbance value was 490nm and 570nm).

[0164] Figure 8 shows the MTT toxicity test determination results of compound I-6 prepared according to the embodiment of the present application. It can be seen that compound I-6 has no obvious cytotoxicity.

[0165] High-fat inflammation cell experiment: HepG2 cells were incubated with 500μM PA & 1μg / mL LPS (PA: palmitic acid; LPS: lipopolysaccharide) for 24 hours in advance to simulate a high-fat and inflammatory environment, and then different concentration gradient groups (10nM, 50nM and 100nM) of I-6 were added and incubated with HepG2 cells for 24 hours.

[0166] Figure 9 shows the Western Blot experimental results of compound I-6 prepared according to the embodiment of the present application in a high-fat model. It can be seen that in the high-fat and inflammatory environment simulated by palmitic acid and LPS, PCSK9 is elevated, and compound I-6 drug can reduce the content of PCSK9 and increase LDLR, and the experimental results show the possibility of compound I-6 drug as a therapeutic drug for patients with hyperlipidemia.

[0167] Cell heat shift assay: to explore the change of protein thermal stability when ligand binds, compared with the protein alone, the protein-ligand complex is less likely to unfold and subsequently aggregate as the temperature increases. First, total proteins extracted from HepG2 cells were co-incubated with I-6 and DMSO, respectively, and after heating the proteins at different temperatures for a period of time, the levels of PCSK9 and P62 proteins were tested and analyzed by Western Blot, which can establish the relationship between the level of protein aggregation and temperature in different experimental groups.

[0168] Figure 10 shows the Western Blot experimental results of the binding ability of compound I-6 prepared according to the embodiment of the present application to PCSK9 and P62 proteins. It can be seen that the compound I-6 drug can target PCSK9 protein and P62 protein, and has good affinity with the two proteins.

[0169] Chloroquine (Chq) as a common autophagy inhibitor can change the PH value in lysosomes, so that autophagosomes and lysosomes cannot fuse, and a large number of LC3-II cannot be degraded. Carfilzomib (Car) is a commonly used proteasome inhibitor that can irreversibly bind to proteasomes to inhibit the ubiquitination degradation process. By adopting the ubiquitination inhibition and autophagy inhibition schemes respectively, the influence of the change of autophagy intensity on the degradation activity of I-6 is detected. First, chloroquine or carfilzomib is added to HepG2 cells for pre-incubation, and a 4-hour starvation treatment is performed. Subsequently, 10 nM of I-6 is added and co-incubated for 24 hours. Finally, the level of PCSK9 after administration is analyzed and evaluated by Western Blot technology.

[0170] Figure 11 shows the Western Blot experimental results of compound I-6 prepared according to the embodiment of the present application in the autophagy and ubiquitination inhibition model. It can be seen that in the autophagy inhibition environment simulated by chloroquine, the degradation effect of compound I-6 drug on PCSK9 is inhibited; in the ubiquitination inhibition environment simulated by carfilzomib, compound I-6 drug still has degradation activity on PCSK9. The experimental results show that compound I-6 drug reduces PCSK9 through the autophagy pathway.

[0171] The above detailed description of the application has further detailed the purposes, technical solutions and beneficial effects of the application. It should be understood that the above is only one specific embodiment of the application and is not limited to the protection scope of the application. Without departing from the basic characteristics of the application, the application can be embodied in various forms. Therefore, the embodiments in the application are used for illustration and not limitation. Since the scope of the application is defined by the claims and not by the specification, and all changes falling within the scope defined by the claims, or within the equivalent scope of the defined scope, should be understood as included in the claims. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the application should be included in the protection scope of the application.

Claims

1. A PCSK9 small molecule autophagy degrading agent, which is a tetrahydroisoquinoline compound according to the general chemical structure I: ###0001### or a pharmaceutically acceptable salt or solvate thereof. wherein, X is -CH2- or -C(=O)-; R1is hydrogen or C1-C 20 alkyl; Linker group A is selected from: a C1-C 20 alkyl chain, ether chain, oxa chain, thia chain, aza chain, C2-C 20 alkenyl chain, C2-C 20 alkynyl chain, -C(=O)NH(C1-C 19 alkyl chain, ether chain, oxa chain, thia chain, aza chain, C2-C 19 alkenyl chain, C2-C 19 alkynyl chain), or any substituents on linker group A are attached to R1 and / or are linked between any substituents on linker group A to make the linking fragment forming a linker fragment with a monocyclic, polycyclic, spirocyclic or bridged ring structure; R2and R3are independently hydrogen or R8, R9and R 10 independently selected from hydrogen, halogen, hydroxyl, nitro, C1-C3alkyl, C1-C3alkoxy, amino, C1-C3alkylamino, carboxyl, cyano, ester, halogen substituted C1-C3alkyl, halogen substituted C1-C3alkoxy, halogen substituted C1-C3alkylamino; R5 is selected from hydrogen, C1-C3 alkyl, C1-C3 alkoxy, halogen, hydroxyl, nitro, amino, C1-C3 alkylamino, carboxyl, cyano, ester; R6 is selected from hydrogen, C1-C3 alkyl, C1-C3 alkoxy, halogen, hydroxyl, nitro, amino, C1-C3 alkylamino, carboxyl, cyano, ester; R7 is an aromatic ring group having 0-3 substituents selected from C1-C3 alkyl, C1-C3 alkoxy, halogen, hydroxyl, nitro, amino, C1-C3 alkylamino, carboxyl, cyano, ester, or an aromatic heterocyclic group having 0-3 substituents selected from C1-C3 alkyl, C1-C3 alkoxy, halogen, hydroxyl, nitro, amino, C1-C3 alkylamino, carboxyl, cyano, ester and having 0-3 heteroatoms. 2.The PCSK9 small molecule autophagy degrading agent of claim 1, characterized in that, Linking fragments selected from the group consisting of: wherein, A1, A2 and A3 are independently selected from methylene, O, S and NH, a, b, c are independently selected from an integer between 0-5; or A1, A2 and A3 are absent. 3.The PCSK9 small molecule autophagy degrading agent of claim 1, characterized in that, Linking fragments selected from the group consisting of:

4. The PCSK9 small molecule autophagy degrader of any one of claims 1 to 3, wherein, said R7 is selected from 5. The PCSK9 small molecule autophagy degrader of any one of claims 1 to 4, wherein said R2is hydrogen, or 6. The PCSK9 small molecule autophagy degrader of any one of claims 1 to 5, wherein, The R6 is methyl.

7. The PCSK9 small molecule autophagy degrader of any one of claims 1 to 6, wherein, The tetrahydroisoquinoline compounds of the chemical structure general formula I are selected from the following compounds:

8. A pharmaceutical composition, characterized by, The PCSK9 small molecule autophagy degrading agent according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

9. Use of the PCSK9 small molecule autophagy degrading agent according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for preventing and / or treating a disease associated with inhibiting PCSK9 activity.

10. Use according to claim 9, characterized in that, The disease is selected from at least one 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.

11. Use according to claim 10, characterized in that, The cardiovascular and cerebrovascular disease is selected from at least one of cholesterol-related diseases, hyperlipidemia and atherosclerosis; the malignant tumor is selected from at least one of leukemia, prostate cancer, thyroid cancer, liver cancer, ovarian cancer, breast cancer, esophageal cancer, adenocarcinoma, gastric cancer, lung cancer and colorectal cancer.

Images