A class of macrocyclic small molecule PCSK9 protein inhibitors

Abstract

The present invention relates to a macrocyclic small molecule PCSK9 protein inhibitor. Specifically, the PCSK9 protein inhibitor has a structure shown in Formula I. The compound of the present invention has a novel structure and is a class of PCSK9 protein inhibitors that can significantly enhance LDLR protein expression and reduce the biological activity of PCSK9 protease levels at lower concentrations.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and specifically relates to a macrocyclic small molecule PCSK9 protein inhibitor. Background Art

[0002] In today's society, cardiovascular disease and its related coronary artery disease (CAD) and atherosclerosis cause morbidity and mortality, which are the highest among various diseases. Recently, this trend has spread from developed countries to developing countries such as China and India. Complications caused by cardiovascular disease are the main cause of death worldwide, up to 14 million people per year. Among them, the most important factor leading to CAD and atherosclerosis is abnormal blood lipids, including elevated low-density lipoprotein cholesterol (LDL-C) levels or low high-density lipoprotein cholesterol (HDL-C).

[0003] One of the main challenges in the prevention of cardiovascular disease is how to effectively reduce lipid levels to prevent cardiovascular events. A large number of clinical trials have shown that LDL-C is a major biomarker for CAD and atherosclerosis.

[0004] In recent years, a protein called proprotein convertase subtilisin / kexin type 9 (PCSK9) discovered in 2003 has attracted great interest in the medical research community as a new target as one of the clinical means of lowering blood lipids. The function of PCSK9 is to destroy the low-density lipoprotein receptor (LDL-R) in the liver and degrade it, thereby increasing the level of LDL in plasma. The medical community has done a lot of research on the function and properties of PCSK9. The latest research shows that PCSK9 regulates the level of LDL-C in the blood by degrading the low-density lipoprotein receptor (LDLR). The first step for PCSK9 to cause LDLR degradation is the binding of two proteins. The binding site on LDLR is called EGF-A, and the corresponding binding region of PCSK9 is amino acids 367 to 381. The binding region of the two is approximately EGF-A binds to PCSK9 mainly at the amino terminus. Among them, the interaction between PCSK9Arg194, Asp238 and EGF-A is the most important. Secondly, the interaction between amino acids 153-155 of PCSK9 and EGF-A is also important. In addition, there are some polarization forces around the interface, such as PCSK9 Asp238 and Thr377 in PCSK9:EGF-A form hydrogen bonds with EGF-A Asn295 and Asn309 respectively; PCSK9 Ser153 forms a salt bridge with EGF-AAsp299. However, when binding to proteins such as LDLR-EGF-B, very low density lipoprotein receptor, and apolipoprotein E receptor 2, PCSK9 cannot form these hydrogen bonds and salt bridges. This ensures the specificity of PCSK9 when binding to LDLR. The next step is to pull this complex into the cell for elimination to achieve the purpose of controlling blood lipids. Studies on human genes have shown that loss-of-function (LOF) mutations in PCSK9 proteins can reduce plasma cholesterol concentrations and prevent CAD (carriers of such loss-of-function genes often have very low LDL-C and cardiovascular disease incidence rates); on the contrary, gain-of-function (GOF) mutations cause carriers of mutation genes to have high LDL-C and cardiovascular disease incidence rates. If these patients do not use drugs to control blood lipids, they will suffer from heart disease prematurely or even die. Studies have also shown that when patients are treated with statins, the drugs reduce the synthesis of cholesterol in the body, and the body responds by activating the SREBP-2 gene to secrete PCSK9 protein and low-density lipoprotein receptor LDLR. The main function of PCSK9 protein is to destroy the LDL receptor (LDL-R) in the liver and degrade it, thereby controlling changes in LDL-C cholesterol levels. The low-density lipoprotein receptor LDLR can reduce the level of LDL-C cholesterol in the blood. After degrading the low-density lipoprotein receptor LDLR, PCSK9 can increase the level of LDL-C.

[0005] Currently, statins are important and effective cholesterol-lowering drugs. Their mechanism of action is to inhibit a key enzyme responsible for cholesterol synthesis in the human body: hydroxymethylglutaryl coenzyme A reductase (HMG-CoA). Statins are the first-line treatment, which has the effect of lowering LDL-C for high LDL-C levels and dyslipidemia in diabetic patients. However, although the response rate of patients to statins is 60% to 70%, only 1 / 3 of patients treated with statins can achieve effective lipid reduction. Statins can only lower patients' LDL-C to a certain extent, but when LDL-C drops to a certain level, the drug stops working. For many patients who need to further lower LDL-C, finding other effective treatments has become a top priority.

[0006] Therefore, if we inhibit the function of PCSK9, we should be able to increase the low-density lipoprotein receptor LDLR, thereby lowering LDL-C. The results of studies on loss-of-function mutant genes are consistent with those of clinical trials of interfering antibodies, RNA interference agents, and antisense oligonucleotide inhibitors (ASO). In addition, the combination of PCSK9 inhibitors with statins can improve the therapeutic effect and further reduce LDL-C. However, it is reported that all PCSK9 inhibitors currently undergoing clinical trials are large molecules and must be injected. Low patient compliance and high treatment costs are real challenges facing current treatment. Another idea is to reduce the content of PCSK9 in the circulation system by interfering with the synthesis of PCSK9 in cells, thereby affecting LDL-C. A lot of work is still needed in this regard to answer many questions, such as what functions other than LDL-C will be affected after the reduction of PCSK9 synthesis, whether the interfering agent is toxic to cells, whether it is toxic to other organs, and so on.

[0007] In 2015, the FDA approved two large-molecule PCSK9 inhibitors (antibodies), Rapatha from Amgen and Praluent from Sanofi / Regeneron Pharmaceuticals. A single dose of Rapatha can reduce the LDL-C of volunteers by 70%. Praluent, a PCSK9 drug jointly developed by Regeneron and Sanofi, combined with a large dose of Lipitor can reduce cholesterol levels by 65%. At the same time, the LDL-C reduction rate was only 17% when only a large dose of Lipitor was used. The success of Rapatha is based on simulating the binding of EGF-A to PCSK9. There is a large contact area (a protein-protein interaction) between LDLR and PCSK9 protein and the surface is smooth. It is not easy for ordinary small molecules to stay and bind in this area. So far, there have been no successful reports of searching for ordinary small molecule PCSK9 inhibitors solely by traditional large-scale compound screening.

[0008] Therefore, it is extremely urgent to find more effective treatments. In particular, the incidence of cardiovascular diseases is expected to increase significantly in the next two decades, so the demand for more effective treatments will rise sharply. Summary of the invention

[0009] The present invention aims to provide a small molecule PCSK9 inhibitor targeting the LDLR and PCSK9 protein binding region. In a first aspect of the present invention, a compound of formula I or a stereoisomer or optical isomer, a pharmaceutically acceptable salt, a prodrug or a solvate thereof is provided.

[0010]

[0011] In the formula,

[0012] A is selected from the following group: -(CH2) n1 -, -C(=O)-, -C(=O)O-, -C(=O)NH-, SO, SO2;

[0013] B is selected from the following group: -(CH2) n2 -、-C(=O)-、NR b , -C(=O)O-, -C(=O)NH-, -O-, -S-, -SO-, -SO2-;

[0014] C is selected from the following group: -(CH2) n3 -、-C(=O)-、NR b , -C(=O)O-, -C(=O)NH-, -O-, -S-, -SO-, -SO2-;

[0015] D is selected from the following group: -(CH2) n4 -、-C(=O)-、NR b , -C(=O)O-, -O-, -S-, -SO-, -SO2-;

[0016] E is selected from the following group: -(CH2) n5 -、-C(=O)-、NR b , -C(=O)O-, -C(=O)NH-, -O-, -S-, -SO-, -SO2-;

[0017] or Part of Where p is 0, 1 or 2, R 11 Selected from the group consisting of H, D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl, and C6-C12 aryl;

[0018] M is selected from the group consisting of: -(CH2) n6 -, -C(=O)-, -C(=O)O-, -C(=O)NH-, -O-, -S-, -SO-, -SO2-;

[0019] Ring Q is independently selected from the group consisting of 3-12 membered heterocyclyl, C3-C12 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl, which are substituted or unsubstituted;

[0020] R b, R1 is independently selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 3-10 membered heterocyclyl, substituted or unsubstituted 5-12 membered heteroaryl and substituted or unsubstituted C6-C12 aryl; wherein, the substitution refers to substitution by one or more R4;

[0021] R2 is selected from the group consisting of H, D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl, C6-C12 aryl and -X1-Ar1;

[0022] R3 is selected from the group consisting of H, D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl, C6-C12 aryl and -X2-Ar2;

[0023] -(CH2) n1 -、-(CH2) n2 -、-(CH2) n3 -、-(CH2) n4 -、-(CH2) n5 -、-(CH2) n6 -H can be replaced by R c Replacement; wherein each R c independently selected from the following groups, substituted or unsubstituted: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl, C6-C12 aryl and -X3-Ar3;

[0024] X1, X2, X3 are each independently selected from the following group: -(CH2) n7 -, -C(=O)-, -C(=O)O-, -C(=O)NH-, -O-, -S-, -SO-, -SO2-; wherein n7 is 1, 2 or 3;

[0025] Ar1, Ar2, Ar3 are each independently selected from the following group of substituted or unsubstituted groups: 5-12 membered heteroaryl, C6-C12 aryl;

[0026] wherein R4 is selected from the group consisting of substituted or unsubstituted C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl;

[0027] wherein n1, n2, n3, n4, n5, and n6 are each independently 0, 1, 2, 3, 4, or 5;

[0028] Unless otherwise specified, the substitution refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0029] In another preferred embodiment, at most three of A, B, C, and D are -(CH2)-.

[0030] In another preferred embodiment, at most two of A, B, C, and D are -(CH2)-.

[0031] In another preferred embodiment, at most one of A, B, C, and D is -(CH2)-.

[0032] In another preferred embodiment, the compound of formula I or its stereoisomer or optical isomer, pharmaceutically acceptable salt, prodrug or solvate has a structure shown in formula II

[0033]

[0034] In the formula,

[0035] n is 0, 1, 2 or 3;

[0036] m is 0, 1, 2, 3 or 4;

[0037] Each R7 is independently selected from the group consisting of D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl, and C6-C12 aryl; or two R7 located at two adjacent carbon atoms together with the connected C atom form a substituted or unsubstituted 4-8 membered ring;

[0038] R1, R2, R3, A, B, C, D, E, and M are as defined above.

[0039] In another preferred embodiment, n is 0, 1 or 2.

[0040] Preferably, the compound of formula I or its stereoisomers or optical isomers, pharmaceutically acceptable salts, prodrugs or solvates has a structure shown in formula III

[0041]

[0042] In the formula,

[0043] R1, R2, R3, A, B, C, D, E, and M are as defined above.

[0044] In another preferred embodiment, C is -C(=O)-, NR b , -C(=O)O- or -C(=O)NH-, preferably, -C(=O)- or -C(=O)NH-.

[0045] In another preferred embodiment, D is -C(=O)-, a bond or NR b , preferably a bond or NR b ; Among them, R b Selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 3-10 membered heterocyclyl, substituted or unsubstituted 5-12 membered heteroaryl and substituted or unsubstituted C6-C12 aryl; wherein the substitution refers to substitution by one or more R4; R4 is selected from the following group: substituted or unsubstituted: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl;

[0046] The substitution of R4 refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0047] In another preferred embodiment, -CD- is -C(=O)NR b -; where R b Selected from the following group: H, substituted or unsubstituted C1-C6 alkyl; wherein the substitution refers to substitution by one or more R4; R4 is selected from the following group: substituted or unsubstituted: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl;

[0048] The substitution of R4 refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0049] In another preferred embodiment, E is -C(=O)-, -C(=O)O- or -C(=O)NH-, preferably -C(=O)-.

[0050] In another preferred embodiment, the compound of formula I or its stereoisomer or optical isomer, pharmaceutically acceptable salt, prodrug or solvate has a structure shown in formula IV

[0051]

[0052] In the formula,

[0053] R1, R2, R3, R b , A, B, M, and Q are defined as above.

[0054] In another preferred embodiment, A is -(CH2) n1 -, -C(=O)-, -C(=O)O- or -C(=O)NH-, preferably, -(CH2) n1 -, -C(=O)-; wherein n1 is 0 or 1, -(CH2) n1 -H atom in may be replaced by Rc; Rc is selected from the group consisting of substituted or unsubstituted D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl, C6-C12 aryl and -X3-Ar3;

[0055] X3 is -(CH2)-;

[0056] Ar3 is selected from the group consisting of 5-12 membered heteroaryl, C6-C12 aryl, substituted or unsubstituted;

[0057] Unless otherwise specified, the substitution refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0058] In another preferred embodiment, B is NR b or -C(=O)NH-, wherein R b Selected from the following group: H, substituted or unsubstituted C1-C6 alkyl; wherein the substitution refers to substitution by one or more R4; R4 is selected from the following group: substituted or unsubstituted: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl;

[0059] The substitution of R4 refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0060] In another preferred embodiment, ring Q is independently a substituted or unsubstituted 3-12 membered heterocycloalkyl group, and the heterocycloalkyl group contains at least one N atom; wherein the substitution refers to substitution by one or more groups selected from the following group: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0061] In another preferred embodiment, the compound of formula I has a structure shown in formula V

[0062]

[0063] R1, R2, R3, R b , A, M, and Q are defined as above.

[0064] In another preferred embodiment, the compound of formula I or its stereoisomer or optical isomer, pharmaceutically acceptable salt, prodrug or solvate has a structure shown in formula VI

[0065]

[0066] In the formula,

[0067] n is 0, 1, 2 or 3;

[0068] m is 0, 1, 2, 3 or 4;

[0069] R7 is selected from the group consisting of D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamido, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl, and C6-C12 aryl;

[0070] R1, R2, R3, R b , A, B, and M are defined as above.

[0071] In another preferred embodiment, the compound of formula I or its stereoisomer or optical isomer, pharmaceutically acceptable salt, prodrug or solvate has a structure shown in formula VII

[0072]

[0073] In the formula,

[0074] n is 0, 1 or 2;

[0075] m is 0, 1, 2, 3 or 4;

[0076] each R7 is independently selected from the group consisting of D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl, and C6-C12 aryl;

[0077] R1, R2, R3, R b , A, and M are defined as above.

[0078] In another preferred embodiment, in Formula II, Formula VI-VII, Some selected from:

[0079] In another preferred embodiment, the compound of formula I has a structure shown in formula VIII or formula VIII'

[0080]

[0081] R1, R2, R3, M, R 11 The definition of is as above.

[0082] In another preferred embodiment, the compound of formula I has a structure shown in formula IX or formula IX'

[0083]

[0084] R1, R2, R3, R b , R c , M, R 11 The definition of is as above.

[0085] In another preferred embodiment, R3 is H or a substituted or unsubstituted C1-C6 alkyl group; wherein the substitution refers to substitution by one or more R4 groups; wherein R4 is selected from the following substituted or unsubstituted groups: a 5-12 membered heteroaryl group, a C6-C12 aryl group, preferably, R4 is a substituted or unsubstituted 5-12 membered heteroaryl group;

[0086] The substitution of R4 refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0087] In another preferred embodiment, the compound of formula I has a structure shown in formula X, formula X' or X".

[0088]

[0089] In the formula,

[0090] m' is 0, 1, 2, or 3;

[0091] R8 is selected from the group consisting of D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamido, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl, and C6-C12 aryl;

[0092] R1, R2, R b , Rc , and M are defined as above.

[0093] In another preferred embodiment, M is selected from the following group: -(CH2) n6 -, -C(=O)-; wherein, -(CH2) n6 -H can be replaced by R c replace;

[0094] Each R c independently selected from the following groups, substituted or unsubstituted: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl, C6-C12 aryl and -X3-Ar3;

[0095] X3 is selected from the group consisting of: -(CH2) n7 -, -C(=O)-, -C(=O)O-, -C(=O)NH-, -O-, -S-, -SO-, -SO2-;

[0096] Wherein, n7 is 1, 2 or 3;

[0097] Ar3 is selected from the group consisting of 5-12 membered heteroaryl, C6-C12 aryl, substituted or unsubstituted;

[0098] Unless otherwise specified, the substitution refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0099] In another preferred embodiment, n is 1 and m is 0.

[0100] In another preferred embodiment, R1 is H or substituted or unsubstituted C1-C6 alkyl; wherein the substitution refers to substitution by one or more R4; wherein R4 is selected from the following substituted or unsubstituted groups: 5-12 membered heteroaryl, C6-C12 aryl;

[0101] Unless otherwise specified, the substitution refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0102] In another preferred embodiment, R2 is a substituted or unsubstituted C1-C6 alkyl group, -X1-Ar1; wherein the substitution refers to substitution by one or more R4;

[0103] Wherein, R4 is selected from the following group which is substituted or unsubstituted: 5-12 membered heteroaryl, C6-C12 aryl;

[0104] X1 is -(CH2) n7 -; wherein n7 is 1, 2 or 3;

[0105] Ar1 is substituted or unsubstituted from the following groups: 5-12 membered heteroaryl, C6-C12 aryl;

[0106] Unless otherwise specified, the substitution refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0107] In another preferred embodiment, R b For H.

[0108] In another preferred embodiment, R1, R2, R3, A, B, C, D, E, M, and Q are specific groups corresponding to the specific compounds in the embodiments.

[0109] In another preferred embodiment, the compound of formula I or its stereoisomer or optical isomer, pharmaceutically acceptable salt, prodrug or solvate is selected from the following group:

[0110]

[0111]

[0112]

[0113] In another preferred embodiment, the compound or its stereoisomer or optical isomer, pharmaceutically acceptable salt, prodrug or solvate is selected from the compounds shown in the examples.

[0114] The second aspect of the present invention provides a pharmaceutical composition comprising the compound described in the first aspect or its stereoisomer or optical isomer, pharmaceutically acceptable salt, prodrug or solvate; and a pharmaceutically acceptable carrier.

[0115] In another preferred embodiment, the pharmaceutical composition further comprises a drug selected from the following group:

[0116] Atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, fibrates, niacin and its analogs, cholesterol absorption inhibitors, bile acid sequestrants, thyroid hormone mimetics, microsomal triglyceride transfer protein (MTP) inhibitors, diacylglycerol acyltransferase (DGAT) inhibitors, inhibitory nucleic acids targeting PCSK9, inhibitory nucleic acids targeting apoB100, or a combination thereof.

[0117] In another preferred embodiment, a method for preparing a pharmaceutical composition is provided, comprising the steps of: mixing a pharmaceutically acceptable carrier with the compound described in the first aspect of the present invention or its stereoisomer or optical isomer, pharmaceutically acceptable salt, prodrug or solvate to form a pharmaceutical composition.

[0118] In another preferred embodiment, the compound of the present invention can be prepared into powders, tablets, granules, capsules, solutions, emulsions, suspensions and the like.

[0119] In a third aspect, the present invention provides a use of the compound described in the first aspect or its stereoisomers or optical isomers, pharmaceutically acceptable salts, prodrugs or solvates for preparing a drug or pharmaceutical composition for inhibiting a disease associated with the activity or expression of the PCSK9 protein.

[0120] In another preferred embodiment, the compound is used to reduce the level of low-density lipoprotein cholesterol (LDL-C) or increase high-density lipoprotein cholesterol (HDL-C).

[0121] In another preferred embodiment, the disease is selected from: cardiovascular disease, inflammatory disease and infectious disease.

[0122] In another preferred embodiment, the disease is a low-density lipoprotein-cholesterol related disease or disorder.

[0123] In another preferred embodiment, the disease is hyperlipidemia, hypertriglyceridemia, hypercholesterolemia and / or atherosclerosis.

[0124] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 The Western-Blot detection of different compounds at different concentrations on LDLR protein expression is shown;

[0126] Among them, a. is compound 1; b. is compound 8; c. is compound 9; c. is compound 10; c. is compound 11; c. is compound 13. DETAILED DESCRIPTION

[0127] After extensive and in-depth research, the inventors have developed a small molecule PCSK9 inhibitor for the binding region of LDLR and PCSK9 proteins for the first time. The PCSK9 inhibitor of the present invention has the biological activity of enhancing LDLR protein expression and reducing PCSK9 protease levels. In addition, the compounds of the present invention have good pharmacodynamics / pharmacokinetic properties. On this basis, the present invention was completed.

[0128] the term

[0129] In the present invention, unless otherwise specified, the terms used have the general meanings well known to those skilled in the art.

[0130] When substituents are described by conventional chemical formulas written from left to right, the substituents also include chemically equivalent substituents that would result if the formula were written from right to left. For example, -CH2O- is equivalent to -OCH2-.

[0131] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0132] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0133] As used herein, the term "alkyl" includes straight or branched alkyl groups. For example, C1-C6 alkyl groups represent straight or branched alkyl groups having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, etc.

[0134] As used herein, the term "alkenyl" includes straight or branched alkenyls. For example, C2-C6 alkenyl refers to a straight or branched alkenyl having 2-6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.

[0135] As used herein, the term "alkynyl" includes straight or branched alkynyl groups. For example, C2-C6 alkynyl refers to a straight or branched alkynyl group having 2-6 carbon atoms, such as ethynyl, propynyl, butynyl, or the like.

[0136] As used herein, the term "cycloalkyl" refers to a cyclic alkyl group containing a specific number of C atoms, such as "C3-C10 cycloalkyl" refers to a cycloalkyl group having 3-10 (preferably 3, 4, 5, 6, 7 or 8) carbon atoms. It can be a monocyclic ring, such as a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or a similar group. It can also be a bicyclic form, such as a bridged ring or a spirocyclic form. In the present invention, cycloalkyl is intended to include substituted cycloalkyl.

[0137] As used herein, the term "C1-C6 alkoxy" refers to a straight or branched alkoxy group having 1 to 6 carbon atoms; it has the formula C1-C6 alkyl-O- or -C1-C5 alkyl-O-C1-C5 alkyl (e.g., -CH2-O-CH2CH3,

[0138] -CH2-O-(CH2)2CH3, -CH2CH2-O-CH2CH3) structure, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, etc.

[0139] As used herein, "heterocyclyl" refers to a saturated or partially saturated cyclic group having a heteroatom selected from N, S and O, and "3-10 membered heterocyclyl" refers to a saturated or partially saturated cyclic group having 3-10 atoms and 1-3 atoms of which are heteroatoms selected from the following group N, S and O. It can be a monocyclic ring or a bicyclic form, such as a bridged ring or a spirocyclic form. The 3-10 membered heterocyclyl is preferably a 3-8 membered heterocyclyl, and more preferably a 6-8 membered heterocyclyl. Specific examples may be oxetane, azetidine, tetrahydro-2H-pyranyl, piperidinyl, piperazinyl, tetrahydrofuranyl, morpholinyl and pyrrolidinyl, etc.

[0140] As used herein, "aryl" refers to an aromatic ring group without heteroatoms on the ring, and "C6-C12 aryl" refers to an aromatic ring group with 6 to 12 carbon atoms without heteroatoms on the ring, and the aryl group may be fused to a heteroaryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring. Such as phenyl (i.e., a six-membered aromatic ring), naphthyl, etc., wherein the six-membered aryl group is also intended to include a six-membered aryl and a 5-6-membered cycloalkyl and a six-membered aryl and a 5-6-membered heterocyclic alkyl. C6-C12 aryl is preferably C6-C10 aryl. Aryl can be optionally substituted or unsubstituted.

[0141] As used herein, "heteroaryl" refers to a cyclic aromatic group having 1-3 atoms selected from the group consisting of N, S and O, and "5-12 membered heteroaryl" refers to a cyclic aromatic group having 5-12 atoms, of which 1-3 atoms are selected from the group consisting of N, S and O. It may be a monocyclic ring or a condensed ring. Specific examples may include pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3)-triazolyl and (1,2,4)-triazolyl, tetrazolyl, furanyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, and the like. The heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, deuterated alkyl, haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, alkylthio, alkylamino, halogen, amino, nitro, hydroxy, mercapto, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylthio, oxo, amide, sulfonamide, formyl, formamido, carboxyl and carboxylate groups, and the like.

[0142] As used herein, the term "4-8 membered ring" refers to a cycloalkyl, heterocyclyl, aryl, heteroaryl group containing 4-8 (i.e., 4, 5, 6, 7, 8) ring atoms, wherein the definitions of cycloalkyl, heterocyclyl, aryl, and heteroaryl groups are as described above. Examples of "4-8 membered rings" include, but are not limited to, cyclobutyl, cyclopentyl, cyclohexyl, oxetane, azetidine, tetrahydro-2H-pyranyl, piperidinyl, piperazinyl, tetrahydrofuranyl, morpholinyl, pyrrolidinyl, phenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3)-triazolyl and (1,2,4)-triazolyl, tetrazolyl, furanyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, and the like.

[0143] As used herein, the term "halogen" or "halogen atom" refers to F, Cl, Br, and I. More preferably, the halogen or halogen atom is selected from F, Cl and Br.

[0144] In the present invention, the term "amide group" refers to a group with the structure -CONRR', wherein R and R' can independently represent hydrogen, alkyl, cycloalkyl, aryl, heterocyclic, as defined above. R and R' can be the same or different in the dialkylamine fragment.

[0145] In the present invention, the term "sulfonamide" refers to a group with the structure -SO2NRR', wherein R and R' can independently represent hydrogen, alkyl, cycloalkyl, aryl, heterocyclic, as defined above. R and R' can be the same or different in the dialkylamine fragment.

[0146] In the present invention, the term "formyl" refers to a group containing -CHO.

[0147] In the present invention, the term "carboxamide group" refers to The carboxamide group is also intended to include substituted carboxamide groups having the formula Wherein, R each independently represents hydrogen, alkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, as defined above. Each R may be the same or different.

[0148] In the present invention, "amino" refers to a group having a -N-RR' structure, wherein R and R' independently represent hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclic group, as defined above, and R and R' may be the same or different.

[0149] In the present invention, "sulfoxide" refers to a group having -S(O)-R, wherein R independently represents hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclic group, as defined above.

[0150] In the present invention, "sulfone group" refers to a group having -S(O)2-R, wherein R independently represents hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclic group, as defined above.

[0151] In the present invention, "ester group" refers to a group having a -C(O)-OR or RC(O)-O- structure, wherein R independently represents hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclic group, as defined above.

[0152] In the present invention, the term "substituted" refers to one or more hydrogen atoms on a specific group being replaced by a specific substituent. The specific substituent is a substituent described above, or a substituent appearing in the embodiments. Unless otherwise specified, a substituted group may have a substituent selected from a specific group at any substitutable site of the group, and the substituent may be the same or different at each position. It will be appreciated by those skilled in the art that the combinations of substituents contemplated by the present invention are those that are stable or chemically feasible.

[0153] Unless otherwise specified as "substituted or unsubstituted", the groups described in the present invention may be substituted by substituents selected from the following groups: deuterium, halogen, cyano, nitro, hydroxyl, amino, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, 3-10 membered heterocycloalkyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0154] In the present invention, the term "plurality" independently refers to 2, 3, 4, or 5.

[0155] Unless otherwise specified, the structural formulas described in the present invention are intended to include all isomeric forms (such as enantiomers, diastereomers and geometric isomers (or conformational isomers)): for example, R, S configurations containing asymmetric centers, (Z), (E) isomers of double bonds, etc. Therefore, single stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers or geometric isomers (or conformational isomers) are all within the scope of the present invention.

[0156] As used herein, the term "tautomer" means that structural isomers with different energies can interconvert across a low energy barrier. For example, proton tautomers (i.e., prototropic) include interconversion via proton migration, such as 1H-indazole and 2H-indazole. Valence tautomers include interconversion via reorganization of some bonding electrons.

[0157] As used herein, the term "solvate" refers to a complex in which the compound of the present invention is coordinated with solvent molecules to form a specific ratio.

[0158] Active ingredients

[0159] As used herein, "compounds of the present invention" refers to compounds represented by Formula I, and also includes stereoisomers or optical isomers, pharmaceutically acceptable salts, prodrugs or solvates of the compounds of Formula I.

[0160] In the present invention, the compound of formula I has the following structure:

[0161]

[0162] R1, R2, R3, A, B, C, D, E, M, and Q are as defined above.

[0163] Preferably, the compound of formula I has the structure shown in formula II

[0164]

[0165] R1, R2, R3, A, B, C, D, E, M, R7, m and n are as defined above.

[0166] Preferably, in Formula II, n is 0 or 1.

[0167] Preferably, the compound of formula I has a structure shown in formula III

[0168]

[0169] In the formula,

[0170] R1, R2, R3, A, B, C, D, E, and M are as defined above.

[0171] Preferably, the compound of formula I has a structure shown in formula IV

[0172]

[0173] In the formula,

[0174] R1, R2, R3, R b , A, B, M, and Q are defined as above.

[0175] Preferably, the compound of formula I has a structure shown in formula V

[0176]

[0177] R1, R2, R3, R b , A, M, and Q are defined as above.

[0178] Preferably, the compound of formula I has the structure shown in formula VI

[0179]

[0180] R1, R2, R3, R b , A, B, M, R7, m and n are as defined above.

[0181] Preferably, the compound of formula I has the structure shown in formula VII

[0182]

[0183] R7, R1, R2, R3, R b , A, M, m and n are as defined above.

[0184] Preferably, in the above formula I-III, C is -C(=O)-, NR b , -C(=O)O- or -C(=O)NH-, preferably, -C(=O)- or -C(=O)NH-.

[0185] Preferably, in the above formula I-III, D is -C(=O)-, a bond or NRb , preferably a bond or NR b ; Among them, R b Selected from the following group: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 3-10 membered heterocyclyl, substituted or unsubstituted 5-12 membered heteroaryl and substituted or unsubstituted C6-C12 aryl; wherein the substitution refers to substitution by one or more R4; R4 is selected from the following group: substituted or unsubstituted: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl;

[0186] The substitution of R4 refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0187] Preferably, in the above formula I-III, -CD- is -C(=O)NR b -; where R b Selected from the following group: H, substituted or unsubstituted C1-C6 alkyl; wherein the substitution refers to substitution by one or more R4; R4 is selected from the following group: substituted or unsubstituted: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl;

[0188] The substitution of R4 refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0189] Preferably, in the above I-III, E is -C(=O)-, -C(=O)O- or -C(=O)NH-, more preferably -C(=O)-.

[0190] Preferably, in the above formulas I and VI-V, ring Q is independently a substituted or unsubstituted 3-12 membered heterocycloalkyl group, and the heterocycloalkyl group contains at least one N atom; wherein the substitution refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0191] Preferably, in the above formula I-VII, A is -(CH2) n1 -, -C(=O)-, -C(=O)O- or -C(=O)NH-, preferably, -(CH2) n1 -, -C(=O)-; wherein n1 is 0 or 1, -(CH2) n1 -H atom in may be replaced by Rc; Rc is selected from the group consisting of substituted or unsubstituted D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl, C6-C12 aryl and -X3-Ar3;

[0192] X3 is -(CH2)-;

[0193] Ar3 is selected from the group consisting of 5-12 membered heteroaryl, C6-C12 aryl, substituted or unsubstituted;

[0194] Unless otherwise specified, the substitution refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0195] Preferably, in the above formulas I-IV and VI, B is NR b or -C(=O)NH-, wherein R bSelected from the following group: H, substituted or unsubstituted C1-C6 alkyl; wherein the substitution refers to substitution by one or more R4; R4 is selected from the following group: substituted or unsubstituted: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl;

[0196] The substitution of R4 refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0197] Preferably, in Formula II, Formula VI-VII, Some selected from:

[0198] Preferably, the compound of formula I has a structure shown in formula VIII or VIII'

[0199]

[0200] R1, R2, R3, M, R 11 The definition of is as above.

[0201] Preferably, the compound of formula I has a structure shown in formula IX or formula IX'

[0202]

[0203] R1, R2, R3, R b , R c , M, R 11 The definition of is as above.

[0204] Preferably, the compound of formula I has a structure shown in formula X, formula X' or formula X".

[0205]

[0206] In the formula,

[0207] m' is 0, 1, 2, or 3;

[0208] R8 is selected from the group consisting of D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamido, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl, and C6-C12 aryl;

[0209] R1, R2, R b , R c , and M are defined as above.

[0210] Preferably, in the above formulae, M is selected from the following group: -(CH2) n6 -, -C(=O)-; wherein, -(CH2) n6 -H can be replaced by R c replace;

[0211] Each R c independently selected from the following groups, substituted or unsubstituted: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl, C6-C12 aryl and -X3-Ar3;

[0212] X3 is selected from the group consisting of: -(CH2) n7 -, -C(=O)-, -C(=O)O-, -C(=O)NH-, -O-, -S-, -SO-, -SO2-;

[0213] Wherein, n7 is 1, 2 or 3;

[0214] Ar3 is selected from the group consisting of 5-12 membered heteroaryl, C6-C12 aryl, substituted or unsubstituted;

[0215] Unless otherwise specified, the substitution refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0216] Preferably, in the above formulas, R1 is H or a substituted or unsubstituted C1-C6 alkyl group; wherein the substitution refers to substitution by one or more R4 groups; wherein R4 is selected from the following substituted or unsubstituted groups: 5-12 membered heteroaryl group, C6-C12 aryl group;

[0217] The substitution of R4 refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0218] Preferably, in the above formulas, R2 is a substituted or unsubstituted C1-C6 alkyl group, -X1-Ar1; wherein the substitution refers to substitution by one or more (such as 2, 3, 4) R4;

[0219] Wherein, R4 is selected from the following group which is substituted or unsubstituted: 5-12 membered heteroaryl, C6-C12 aryl;

[0220] X1 is -(CH2) n7 -; wherein n7 is 1, 2 or 3;

[0221] Ar1 is substituted or unsubstituted from the following groups: 5-12 membered heteroaryl, C6-C12 aryl;

[0222] Unless otherwise specified, the substitution refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0223] Preferably, in the above formulas, R3 is H or a substituted or unsubstituted C1-C6 alkyl group; wherein the substitution refers to substitution by one or more R4 groups; wherein R4 is selected from the following substituted or unsubstituted groups: 5-12 membered heteroaryl group, C6-C12 aryl group, preferably, R4 is a substituted or unsubstituted 5-12 membered heteroaryl group;

[0224] The substitution of R4 refers to substitution by one or more groups selected from the following groups: D, halogen, amino, amine, nitro, hydroxyl, thiol, cyano, carboxyl, sulfone, sulfoxide, amide, sulfonamide, ester, formyl, formamide, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered heterocyclyl, C3-C10 cycloalkyl, 5-12 membered heteroaryl and C6-C12 aryl.

[0225] The salts that may be formed by the compounds of the present invention also belong to the scope of the present invention. Unless otherwise specified, the compounds of the present invention are understood to include their salts. The term "salt" used herein refers to an acidic or basic salt formed with an inorganic or organic acid and a base. In addition, when the compound of the present invention contains a basic fragment, it includes but is not limited to pyridine or imidazole, and contains an acidic fragment, including but not limited to carboxylic acid, the zwitterions ("inner salts") that may be formed are included in the scope of the term "salt". Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful, for example, in separation or purification steps in the preparation process. The compounds of the present invention may form salts, for example, Compound I reacts with a certain amount of acid or base, such as an equivalent amount, and is salted out in a medium, or freeze-dried in an aqueous solution.

[0226] The compounds of the present invention contain basic fragments, including but not limited to amines or pyridine or imidazole rings, which may form salts with organic or inorganic acids. Typical acids that can form salts include acetates (such as acetic acid or trihaloacetic acid, such as trifluoroacetic acid), adipates, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphor salt, camphorsulfonate, cyclopentanepropionate, diglycolate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide salts, such as hydroxyethanesulfonates (e.g., 2-hydroxyethanesulfonate), lactates, maleates, methanesulfonates, naphthalenesulfonates (e.g., 2-naphthalenesulfonate), nicotinates, nitrates, oxalates, pectinates, persulfates, phenylpropionates (e.g., 3-phenylpropionate), phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates (e.g., formed with sulfuric acid), sulfonates, tartrates, thiocyanates, toluenesulfonates such as p-toluenesulfonates, dodecanoates, and the like.

[0227] Some compounds of the present invention may contain acidic fragments, including but not limited to carboxylic acids, which may form salts with various organic or inorganic bases. Typical base-formed salts include ammonium salts, alkali metal salts such as sodium, lithium, potassium salts, alkaline earth metal salts such as calcium, magnesium salts, and salts formed with organic bases (such as organic amines), such as benzathine, dicyclohexylamine, hepamine (salt formed with N,N-di(dehydroabietyl)ethylenediamine), N-methyl-D-glucamine, N-methyl-D-glucamide, tert-butylamine, and salts formed with amino acids such as arginine, lysine, etc. Basic nitrogen-containing groups can be combined with halide quaternary ammonium salts, such as small molecule alkyl halides (such as chlorides, bromides and iodides of methyl, ethyl, propyl and butyl), dialkyl sulfates (such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate and dipentyl sulfate), long chain halides (such as chlorides, bromides and iodides of decyl, dodecyl, tetradecyl and tetradecyl), aralkyl halides (such as benzyl and phenyl bromides), etc.

[0228] Prodrugs and solvates of the compounds of the present invention are also within the scope of the invention. The term "prodrug" herein refers to a compound that undergoes chemical transformation by metabolic or chemical processes to produce a compound, salt, or solvate of the present invention when treating a related disease. The compounds of the present invention include solvates, such as hydrates.

[0229] The compounds, salts or solvates of the present invention may exist in tautomeric forms (such as amides and imino ethers). All these tautomers are part of the present invention.

[0230] All stereoisomers of the compounds (e.g., those due to asymmetric carbon atoms that may exist for various substitutions), including enantiomeric and diastereomeric forms, are contemplated by the present invention. Individual stereoisomers of the compounds of the present invention may not exist simultaneously with other isomers (e.g., as a pure or substantially pure optical isomer having a particular activity), or may be mixtures, such as racemates, or mixtures with all other stereoisomers or portions thereof. The chiral centers of the present invention have two configurations, S or R, as defined by the 1974 recommendations of the International Union of Pure and Applied Chemistry (IUPAC). Racemic forms can be resolved by physical methods, such as fractional crystallization, or by crystallization of diastereoisomers derived therefrom, or by separation by chiral column chromatography. Individual optical isomers can be obtained from racemates by suitable methods, including but not limited to conventional methods, such as recrystallization after salt formation with an optically active acid.

[0231] The compounds of the present invention are prepared, separated and purified to obtain compounds with a weight content of equal to or greater than 90%, for example, equal to or greater than 95%, equal to or greater than 99% ("very pure" compounds), which are listed in the text description. Such "very pure" compounds of the present invention are also considered part of the present invention.

[0232] All configurational isomers of the compounds of the present invention are within the scope of the invention, whether in mixture, pure or very pure form. The definition of the compounds of the present invention includes both cis (Z) and trans (E) olefin isomers, as well as cis and trans isomers of carbocyclic and heterocyclic rings.

[0233] Throughout the specification, groups and substituents may be selected to provide stable fragments and compounds.

[0234] Specific functional groups and chemical term definitions are detailed below. For purposes of this invention, chemical elements are referred to as Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed. Definitions of specific functional groups are also described therein. In addition, the basic principles of organic chemistry as well as specific functional groups and reactivity are also described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, which is incorporated by reference in its entirety.

[0235] Certain compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention encompasses all compounds, including their cis and trans isomers, R and S enantiomers, diastereomers, (D) isomers, (L) isomers, racemic mixtures and other mixtures. In addition, asymmetric carbon atoms may represent substituents, such as alkyl. All isomers and their mixtures are included in the present invention.

[0236] According to the present invention, the ratio of isomers contained in the mixture of isomers can be various. For example, in the mixture of only two isomers, there can be the following combinations: 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0, and all ratios of isomers are within the scope of the present invention. Similar ratios that are easily understood by ordinary technicians in this profession, and ratios for more complex mixtures of isomers are also within the scope of the present invention.

[0237] The present invention also includes isotopically labeled compounds that are equivalent to the original compounds disclosed herein. However, in practice, it is common for one or more atoms to be replaced by atoms having a different atomic mass or mass number than the original atoms. Examples of isotopes of the compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine isotopes, such as 2 H. 3 H. 13 C. 11 C. 14 C. 15 N. 18 O. 17 O. 31 P. 32 P. 35 S. 18 F and 36 The compounds of the present invention, or enantiomers, diastereomers, isomers, or pharmaceutically acceptable salts or solvates, wherein the isotopes or other isotopic atoms of the above compounds are all within the scope of the present invention. Certain isotope-labeled compounds of the present invention, such as 3 H and 14 Radioisotopes of C are also included and are useful in tissue distribution experiments of drugs and substrates. 3 H and carbon-14, i.e. 14 C, their preparation and detection are relatively easy. It is the first choice among isotopes. In addition, heavier isotope substitutions such as deuterium, i.e. 2 H, due to its good metabolic stability in certain therapies has advantages, such as increasing half-life or reducing dosage in vivo, therefore, in some cases, it can be preferred. Isotope-labeled compounds can be prepared by general methods by replacing readily available isotope-labeled reagents with non-isotopic reagents using the schemes disclosed in the examples.

[0238] If a synthesis of a specific enantiomer of the compound of the present invention is to be designed, it can be prepared by asymmetric synthesis, or derivatized with a chiral auxiliary, the resulting diastereomeric mixture can be separated, and the chiral auxiliary can be removed to obtain the pure enantiomer. In addition, if the molecule contains a basic functional group, such as an amino acid, or an acidic functional group, such as a carboxyl group, a diastereomeric salt can be formed with a suitable optically active acid or base, and then separated by conventional means such as fractional crystallization or chromatography, and then the pure enantiomer can be obtained.

[0239] The metabolites of the compounds and pharmaceutically acceptable salts thereof involved in the present application, as well as prodrugs that can be converted into the structures of the compounds and pharmaceutically acceptable salts involved in the present application and in vivo, are also included in the claims of the present application.

[0240] Preparation method

[0241] The compounds of the present invention can be prepared according to conventional routes or methods, or can be obtained according to the methods or routes described herein.

[0242] The preparation method of the compounds of this invention is described in more detail below, but these specific methods do not constitute any limitation to the present invention. The compounds of this invention can also be prepared conveniently by combining various synthetic methods described in this specification or known in the art, and such combination can be easily carried out by a technician in the field of the present invention.

[0243] Usually, in the preparation process, each reaction is usually carried out in an inert solvent at room temperature to reflux temperature (such as 0°C to 150°C, preferably 10°C to 100°C). The reaction time is usually 0.1 hour to 60 hours, preferably 0.5 to 48 hours.

[0244] Preferably, the compounds of the present invention can be prepared by the following steps:

[0245] In an inert solvent and in the presence of a catalyst, compound A7 reacts to obtain compound I.

[0246]

[0247] In the formula,

[0248] B1 is selected from the group consisting of: carboxyl, ester, amide, amino, hydroxyl, and thiol;

[0249] B2 is selected from the group consisting of: carboxyl, ester, amide, amino, hydroxyl, and thiol;

[0250] R1, R2, R3, AC, D, E, M, and Q are as defined above.

[0251] The starting materials and reagents used in the synthesis method of the compounds of the present invention can be purchased commercially or synthesized by methods reported in the literature.

[0252] Pharmaceutical compositions and methods of administration

[0253] Since the compounds of the present invention have excellent inhibitory activity against PCSK9 protein, the compounds of the present invention or their stereoisomers or optical isomers, pharmaceutically acceptable salts, prodrugs or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to prevent and / or treat (stabilize, alleviate or cure) PCSK9 protein-related diseases (e.g., hyperlipidemia, hypertriglyceridemia, hypercholesterolemia and / or atherosclerosis).

[0254] The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention and a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, and more preferably, contains 10-200 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

[0255] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers Wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0256] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral administration (intravenous administration, intramuscular administration or subcutaneous administration).

[0257] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0258] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0259] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0260] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0261] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0262] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0263] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds or antibodies (eg, PCSK9 inhibitors, PCSK9 antibodies).

[0264] When administered in combination, the pharmaceutical composition further comprises one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds (e.g., PCSK9 inhibitors). One or more (2, 3, 4, or more) of the other pharmaceutically acceptable compounds (e.g., PCSK9 inhibitors) can be used simultaneously, separately, or sequentially with the compounds of the present invention to prevent and / or treat diseases related to the activity or expression of PCSK9 kinase.

[0265] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 20 to 500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

[0266] The main advantages of the present invention are:

[0267] 1. The compounds of the present invention can significantly enhance LDLR protein expression at lower concentrations (less than 25 μM);

[0268] 2. The compounds of the present invention can significantly reduce the biological activity of PCSK9 protease level. Its IC 50 Can be as low as 0.17μM;

[0269] 3. The compounds of the present invention have novel structures and are a class of PCSK9 protein inhibitors.

[0270] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0271] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial channels.

[0272] Example

[0273] Preparative thin layer chromatography (PTLC) was performed on 20 x 20 cm plates (500 micron thick silica gel). Silica gel chromatography was performed using a Biotage flash chromatography system.

[0274] 1 H NMR was performed on a Bruker AscendTM400 spectrometer, 400 MHz, 298°K, and the chemical shifts (ppm) of the residual protons in the deuterated reagent are given as reference:

[0275] CHCl3δ=7.26ppm, CH3OH or CH3ODδ=3.30ppm, DMSO-d6δ=2.50ppm

[0276] LCMS chromatography was performed using an Agilent Technologies 1200 series or 6120 quadrupole spectrometer. For the LC mobile phase, acetonitrile (A) and water (B) with 0.01% formic acid, the eluent gradient was: 5-95% A in 6.0 minutes, 60-95% A in 5.0 minutes, 80-100% A in 5.0 minutes, and 85-100% A in 10 minutes, using a SBC1850 mm × 4.6 mm × 2.7 μm capillary column.

[0277] Mass spectra (MS) were measured by electrospray ionization mass spectrometry (ESI).

[0278] HPLC mass spectrometry analysis conditions:

[0279] LC1: Column: SB-C18 50mm×4.6mm×2.7μm

[0280] Temperature: 50℃

[0281] Eluent: 5:95 to 95:5 vol / vol acetonitrile / water + 0.01% formic acid, 6 min.

[0282] Flow rate: 1.51.5mL / min, injection 5μL

[0283] Detection: PDA, 200-600nm

[0284] MS: mass range 150-750 amu; positive ion electrospray ionization

[0285] LC2: Column: SB-C18 50mm×4.6mm×2.7μm

[0286] Temperature: 50℃

[0287] Eluent: 5:95 to 95:5 vol / vol acetonitrile / water + 0.05% TFA gradient over 3.00 min. Flow rate: 1.51.5 mL / min, 5 μL injection

[0288] Detection: PDA, 200-600nmMS: mass range 150-750amu; positive ion electrospray ionization

[0289] LC3 column: SB-C18 50mm×4.6mm×2.7μm

[0290] Temperature: 50℃

[0291] Eluent: 10:90 to 98:2 vol / vol acetonitrile / water + 0.05% TFA gradient over 3.75 min. Flow rate: 1.0 mL / min, 10 μL injection

[0292] Detection: PDA, 200-600nmMS: mass range 150-750amu; positive ion electrospray ionization

[0293] Abbreviations:

[0294] AcOH = acetic acid

[0295] Alk is an alkyl group

[0296] AR is aryl

[0297] Boc=tert-butyloxycarbonyl

[0298] bs = broad peak

[0299] CH2Cl2=dichloromethane

[0300] d = double peak

[0301] dd = doublet

[0302] DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene

[0303] DCM = dichloromethane

[0304] DEAD = diethyl azodicarboxylate

[0305] DMF=N,N-dimethylformamide

[0306] DMSO = dimethyl sulfoxide

[0307] EA = Ethyl acetate

[0308] ESI = Electrospray Ionization

[0309] Et=ethyl

[0310] EtOAc = Ethyl acetate

[0311] EtOH = ethanol

[0312] h = hours

[0313] HOAc=acetic acid

[0314] LiOH = Lithium Hydroxide

[0315] m = multiple

[0316] Me = methyl

[0317] MeCN = acetonitrile

[0318] MeOH = methanol

[0319] MgSO4 = Magnesium sulfate

[0320] min = minutes

[0321] MS = Mass Spectrometry

[0322] NaCl = Sodium Chloride

[0323] NaOH = Sodium Hydroxide

[0324] Na2SO4=Sodium sulfate

[0325] NMR = Nuclear Magnetic Resonance Spectroscopy

[0326] PE = Petroleum Ether

[0327] PG = Protecting group

[0328] Ph = Phenyl

[0329] rt = room temperature

[0330] s = single peak

[0331] t = triplet

[0332] TFA = trifluoroacetic acid

[0333] THF = Tetrahydrofuran

[0334] TS = p-toluenesulfonyl (tosyl)

[0335] Embodiment 1:

[0336]

[0337] (1) Synthesis of Compound 1-1a

[0338]

[0339] L-tyrosine (15 g, 82.9 mmol, 1.0 eq) and potassium carbonate (22.9 g, 165.7 mmol, 2 eq) were added to a mixed solution (50 mL: 200 mL) of methanol and water (1: 4). Di-tert-butyl dicarbonate (21.7 g, 99.5 mmol, 1.2 eq) was slowly added under stirring at room temperature. The reaction solution was stirred at room temperature for 3 hours. After the reaction was completed, 1N hydrochloric acid was slowly added to the reaction solution under an ice bath to adjust the pH to 5-6. It was extracted three times with ethyl acetate, and the organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was separated by column chromatography (petroleum ether: ethyl acetate = 3: 7) to obtain the product 1-1a (12.6 g, yield 54.5%).

[0340] 1H NMR (400MHz, CDCl3) δ: 7.01-7.00 (d, J = 8.4Hz, 2H), 6.74-6.72 (d, J = 8.4Hz, 1H), 5.02-5.01 (m, 1H), 3.05 (m, 2H), 1.43 (s, 9H).

[0341] LCMS: m / z=282[M+H] +.

[0342] (2) Synthesis of Compound 1-2a

[0343]

[0344] Compound 1-1a (12.6 g, 44.7 mmol, 1 eq), D-tryptophan methyl ester (9.7 g, 44.7 mmol, 1.0 eq) and triethylamine (9.0 g, 89.4 mmol, 2.0 eq) were dissolved in anhydrous DMF (200 mL). HATU (20.4 g, 53.6 mmol, 1.2 eq) was slowly added under stirring at room temperature, and then stirring was continued for 2 hours. After the reaction was complete, the reaction solution was added to a large amount of water to quench, and extracted with ethyl acetate three times. The organic phase was washed three times with a large amount of water, then washed with brine and dried over anhydrous sodium sulfate. After the organic phase was filtered and concentrated, column chromatography (petroleum ether: ethyl acetate = 4:6) gave the product 1-2a (16.2 g, yield 75.3%).

[0345] 1 H NMR (400MHz, DMSO) δ: 10.86 (s, 1H), 9.09 (s, 1H), 8.31-8.29 (d, J = 8.0Hz, 1H), 7.51-7.49 ( d,J=8.0Hz,1H),7.34-7.32(d,J=8.0Hz,1H),7.14(s,1H),7.07(m,1H),7.01-6.99(m,1H) ,6.90-6.88(m,1H),6.62-6.56(m,2H),4.55-4.50(m,1H),4.04-4.01(m,1H),3.62(s,3H) ,3.14-3.13(m,1H),2.74-2.69(m,1H),2.69-2.66(m,1H),2.49-2.45(m,1H),1.30(s,9H).

[0346] LCMS: m / z=482[M+H] +.

[0347] (3) Synthesis of Compound 1-3a

[0348]

[0349] Compound 1-2a (16.2 g, 33.6 mmol, 1.0 eq) was dissolved in a mixed solution (50 mL: 50 mL) of dioxane and 1N aqueous lithium hydroxide solution (1:1), and stirred at room temperature overnight. After the reaction was complete, the pH was slowly adjusted to 5-6 with 1N dilute hydrochloric acid under ice bath. It was extracted three times with ethyl acetate, washed with water and saturated brine, and dried over anhydrous sodium sulfate. The organic phase was filtered and concentrated to obtain a crude product, and column chromatography (dichloromethane: methanol = 30:1) was performed to obtain product 1-3a (12.5 g, yield 79.6%).

[0350] 1 H NMR (400MHz, DMSO) δ: 12.62 (br, 1H), 10.86 (s, 1H), 9.10 (s, 1H), 8.20-8.18 (d, J = 8.0Hz, 1H), 7.56- 7.54(d,J=7.9Hz,1H),7.33-7.31(d,J=8.4Hz,1H),7.14(s,1H),7.08-7.05(t,J=7.6Hz,1H),7.01- 6.97(t,J=7.6Hz,1H),6.88-6.86(d,J=8.0Hz,1H),6.63-6.54(m,2H),4.50-4.49(m,1H),1.14-4.1 3(m,1H),3.20-3.15(m,1H),3.05-2.99(m,1H),2.70-2.67(m,1H),2.49-2.43(m,1H),1.30(s.9H).

[0351] LCMS: m / z=468[M+H] +.

[0352] (1') Synthesis of Compound 1-1b

[0353]

[0354] The compound N-Boc-(s)-3-piperidine carboxylate (10.0 g, 43.7 mmol, 1 eq), dibenzylamine (8.6 g, 43.7 mmol, 1.0 eq) and triethylamine (8.9 g, 87.4 mmol, 2.0 eq) were dissolved in anhydrous DMF (200 mL). HATU (20.0 g, 52.4 mmol, 1.2 eq) was slowly added under stirring at room temperature, and then stirring was continued for 2 hours. After the reaction was complete, the reaction solution was added to a large amount of water to quench, and extracted with ethyl acetate three times. The organic phase was washed three times with a large amount of water, then washed with brine and dried over anhydrous sodium sulfate. After the organic phase was filtered and concentrated, column chromatography (petroleum ether: ethyl acetate = 2: 8) was used to obtain the product (14.5 g, yield 81.5%).

[0355] LCMS: m / z=409[M+H] + .

[0356] (2') Synthesis of Compound 1-2b

[0357]

[0358] Dissolve 1-1b (14.5 g, 35.5 mmol, 1.0 eq) in dichloromethane (100 ml), slowly add trifluoroacetic acid (20 mL), and stir at room temperature for 1 hour. After the reaction is complete, the reaction solution is concentrated to obtain the product 1-2b trifluoroacetate (10 g, crude product).

[0359] 1 H NMR(400MHz, CDCl3)δ:7.39-728(m,6H),7.18-7.12(m,4H),5.7(br,1H),4.74-4.70(d,J=14.8Hz,1 H),4.47-4.43(m,3H),3.22-3.07(m,4H),2.94-2.92(m,1H),1.84-1.80(m,3H),1.72-1.69(m,1H).

[0360] LCMS: m / z=309 [M+H] +.

[0361] (3') Synthesis of Compound 1-3b

[0362]

[0363] The obtained crude trifluoroacetate of 1-2b (10 g, 32.5 mmol, 1.0 eq) was dissolved in anhydrous tetrahydrofuran (150 mL) and ice-bathed to 0°C. Lithium aluminum tetrahydride (3.1 g, 81.2 mmol, 2.5 eq) was slowly added to the reaction solution. After the addition was completed, the reaction solution was slowly warmed to room temperature and stirred overnight. After the reaction was complete, water (2 mL) was slowly added dropwise under an ice bath, stirred for 10 minutes, and then 3N sodium hydroxide solution (3 mL) and water (3 mL) were added dropwise and stirred for 30 minutes. A large amount of anhydrous sodium sulfate was added and stirred for 30 minutes. Filtration and concentration were performed to obtain a crude product (8.4 g, yield 87.6%).

[0364] 1 H NMR(400MHz, CDCl3)δ:7.36-7.20(m,10H),3.62-3.58(d,J=13.6Hz,2H),3.43-3.99(d,J=13.6Hz,2H),3.40-3.21(m,1H),2.96-2.93 (d,J=12.0Hz,2H),2.47-2.42(m,1H),2.21-2.04(m,3H),1.86-1.77(m,2H),1.61-1.56(m,2H),1.42-139(m,1H),0.91-0.89(m,1H).

[0365] LCMS: m / z=295[M+H] + .

[0366] (4) Synthesis of Compound 1-4

[0367]

[0368] 1-3b (2.3 g, 7.9 mmol, 1.0 eq), 1-3a (3.7 g, 7.9 mmol, 1.0 eq) and diisopropylethylamine (2.0 g, 15.8 mmol, 2.0 eq) were dissolved in anhydrous DMF (100 mL), and then PyBOP (4.5 g, 8.7 mmol, 1.2 eq) was slowly added to the reaction solution and stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was quenched by adding a large amount of water and extracted three times with ethyl acetate. The organic phase was washed with a large amount of water, then washed with saturated brine, dried over anhydrous sodium sulfate, and the concentrated crude product was filtered and concentrated, and the product (5.0 g, yield 85.1%) was obtained by column chromatography (dichloromethane: methanol = 20: 1).

[0369] 1H NMR(400MHz, DMSO)δ:10.83-10.79(d,J=15.6Hz,1H),9.13-9.11(d,J=8.0Hz,1H),8.40-8.23(m,1H),7.54-7.53(m,1H),7.34-7.22(m, 10H),7.06-6.95(m,4H),6.90-6.74(m,1H),6.63-6.57(m,2H),5.13-4.98(m,1H),4.56-4.53(m,0.5H),4.21-4.01(m,2H),3.88-3.78(m ,1H),3.64-3.56(m,2.5H),3.33-3.31(m,2H),3.22-2.99(m,1.5H),2.84-2.79(m,1H),2.74-2.60(m,1.5H),2.41-2.37(m,0.5H),2.28 -2.16(m,1.5H),2.14-2.06(m,0.5H),2.03-2.00(m,0.5H),1.81(m,1H),1.71-1.62(m,1.5H),1.33-1.25(m,10H),0.90-0.76(m,1.5H).

[0370] LCMS: m / z=744[M+H] +.

[0371] (5) Synthesis of Compound 1-5

[0372]

[0373] 1-4 (5.0 g, 6.7 mmol, 1.0 eq) and Pd / C (500 mg, 10%) were dissolved in anhydrous methanol (100 mL), and the reaction solution was stirred at room temperature overnight under a hydrogen atmosphere. After the reaction was complete, the product (3.6 g, yield 95.5%) was obtained by filtration and concentration.

[0374] 1H NMR (400MHz, DMSO) δ: 10.85-10.81 (d, J=15.6Hz, 1H), 8.36-8.16 (m, 1H), 7.64-7.54 (m, 1H), 7.3 5-7.30(t,J=8.4Hz,1H),7.07-6.97(m,5H),6.78-6.77(m,1H),6.62-6.58(m,2H),5.10-4.99(m ,1H),4.25-4.13(m,3H),3.75-3.62(m,1H),3.08-2.99(m,2H),2.90-2.68(m,3H),2.39-2.35(m ,2H),2.11-1.97(m,1H),1.82-1.68(m,1H),1.48(m,1H),1.31-1.22(m,9H),1.06-0.86(m,2H).

[0375] LCMS: m / z=564[M+H] +.

[0376] (6) Synthesis of Compounds 1-6

[0377]

[0378] 1-5 (3.6 g, 6.4 mmol, 1.0 eq), 2-methoxy-4-trifluoromethylbenzaldehyde (1.3 g, 6.4 mmol, 1.0 eq) and acetic acid (3 drops) were dissolved in a mixed solution (30 mL: 15 mL) of dichloromethane and ethanol (2: 1). Sodium acetate borohydride (2.7 g, 12.8 mmol, 2.0 eq) was slowly added to the reaction solution at low temperature and stirred at room temperature overnight. After the reaction was complete, saturated sodium bicarbonate solution was added to quench, and dichloromethane was extracted three times. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the concentrated crude product was filtered and concentrated. The product (1.6 g, yield 33.3%) was obtained by column chromatography (dichloromethane: methanol = 15: 1).

[0379] LCMS: m / z=752[M+H] +.

[0380] (7) Synthesis of Compound 1-7

[0381]

[0382] Prepared according to the method of intermediate 1-2b, replacing intermediate 1-1b with 1-5 (1.6 g, 2.1 mmol, 1.0 eq) to obtain intermediate 1-7 (1.7 g, crude trifluoroacetate salt) as a yellow solid.

[0383] LCMS: m / z=652[M+H] +.

[0384] (8) Compound 1: Synthesis of (3R,6S,11S)-3-((1-H indol-3-yl)-methyl)-6-(4-hydroxybenzyl)-9-(2-methoxy-4-(trifluoromethyl)benzyl)-1,4,7,9-tetraazabicyclo[9,3,1]-pentadecan-2,5,8-trione

[0385]

[0386] 1-7 (200 mg, 0.3 mmol, 1.0 eq) and triethylamine (0.5 mL) were dissolved in anhydrous dichloromethane (100 mL), and carbonyl diimidazole (200 mg, 1.2 mmol, 4.0 eq) was slowly added and stirred at room temperature for 1 hour. The reaction solution was dried on a rotary evaporator, the crude product was dissolved in dichloromethane, the organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, the concentrated crude product was filtered and separated by HPLC, and after freeze-drying, compound 1 (24 mg, yield 11.8%) was obtained.

[0387] 1 H NMR(400MHz, CD3OD)δ:7.64-7.62(d,J=8.0Hz,1H),7.50-7.48(d,J=8.0Hz,1H),7.37-7.30(m,4H),7.09-7 .05(t,J=8.0Hz,1H),7.01-6.95(m,4H),6.68-6.66(d,J=8.0Hz,2H),4.29(s,2H),4.03(s,3H),3.96-3.91 9m,1.5H),3.66-3.64(m,1H),3.45-3.39(m,3H),3.17-3.11(m,1.5H),3.01 -2.86(m,4H),2.76-2.72(m,2H),1.96(m,1H),1.79(m,1.5H),1.34(m,2H).

[0388] LCMS: m / z=678[M+H] +.

[0389] Embodiment 2:

[0390]

[0391] (1) Synthesis of Compound 2-3

[0392]

[0393] Compound 2-1 (synthesis method is similar to 1-2a) (200 mg, 0.35 mmol, 1 eq), 2-2 (107 mg, 0.42 mmol, 1.2 eq) and triethylamine (140 mg, 1.4 mmol, 4.0 eq) were dissolved in dichloromethane (20 mL), and then PyBOP (270 mg, 0.52 mmol, 1.5 eq) was added and stirred at room temperature for 4 hours. The reaction solution was washed with water and brine, dried over anhydrous sodium sulfate, and the concentrated crude product was filtered and concentrated by column chromatography (dichloromethane: methanol = 20: 1) to obtain 240 mg of the product, with a yield of 87.9%).

[0394] LCMS: m / z=782[M+H] +.

[0395] (2) Synthesis of Compound 2-4

[0396]

[0397] Compound 2-3 (240 mg) was dissolved in 3M hydrochloric acid dioxane solution (20 mL) and stirred at room temperature for 2 hours. After the reaction was complete, the product hydrochloric acid was directly concentrated and carried out to the next step.

[0398] LCMS: m / z=626[M+H] +.

[0399] (3) Synthesis of Compound 2

[0400]

[0401] The hydrochloride of compound 2-4 (250 mg, 0.4 mmol, 1.0 eq) and triethylamine (0.5 mL) were dissolved in dichloromethane (200 mL). A mixture of HOBt (108 mg, 0.8 mmol, 2.0 eq) and EDCI (154 mg, 0.8 mmol, 2.0 eq) in dichloromethane (5 mL) was slowly added dropwise under stirring. After the addition was complete, stirring was continued for three days. The reaction solution was washed with water and brine, dried over anhydrous sodium sulfate, and the concentrated product was filtered and separated using a large plate (dichloromethane: methanol: 15:1) to obtain the product (2.3 mg, yield 0.9%).

[0402] 1H NMR (400MHz, CDCl3) δ: 8.14 (s, 1H), 7.82-7.90 (m, 1H), 7.71-7.69 (d, J = 7.6Hz, 1 H),7.53-7.58(m,1H),7.18-6.94(m,6H),6.76-6.70(m,3H),5.34-5.30(m,2H), 4.65-4.63(m,1H),4.55(m,0.5H),4.17-4.06(m,0.5H),3.74-7.65(m,0.5H),3. 18-3.00(m,7.5H),2.66-2.45(m,2),2.24-2.20(m,1.5H),2.02-2.00(m,1.5H).

[0403] LCMS: m / z=608[M+H] +.

[0404] According to the method of Example 1 and Example 2, Examples 3-14 were prepared with different starting materials or intermediates as shown in Table 1 below:

[0405] Table 1

[0406]

[0407]

[0408]

[0409]

[0410] Example 15: ELISA activity test

[0411] Experimental materials: 20xPBS (Sangon Biotechnology, B548117-0500), skim milk powder (BD, 232100), Anti-6XHistag primary antibody (abcam, ab27025), HRP secondary antibody (Vectorlabs, SA-5014), TMB single-component colorimetric reagent (Solaibo Biotechnology, PR1200), Strip TM well Microplate (Corning, 42592), concentrated sulfuric acid (Kermiou), DMSO (Sangon Biotechnology, A600163-0500), DPBS (HyClone, SH30028.02), Tween 20 (Aladdin, T104863-500ml), ddH2O (prepared by Milli-Q) and other necessary consumables.

[0412] Experimental equipment: 4℃ refrigerator, Multimode Plate Reader EnVision (PerkinElmer), Constant temperature incubator (Shanghai Yiheng), Oscillating shaker (Colin Bell).

[0413] 1. Solution preparation

[0414] 1.1 Preparation of PBST Concentration Components: 1x PBS, 0.05% Tween20

[0415] Configuration volume: 1L Configuration method:

[0416] a) Take a clean 1L beaker and add about 600mL of ddH2O;

[0417] b) Add the following reagents in sequence and stir until dissolved;

[0418] 20xPBS 50mL

[0419] Tween20 500μL

[0420] c) Add 3 mL of 1 M NaOH to adjust the pH to about 7.5;

[0421] d) Adjust the volume to 1 L, filter with a 0.22 μm filter, and store at 4°C.

[0422] 1.2 Blocking solution

[0423] Component concentration: 5% (W / V) skimmed milk powder

[0424] Configuration volume: 50mL

[0425] Preparation method: a) weigh 2.5g skim milk powder into a 50mL centrifuge tube;

[0426] b) Add 30 mL of 1× PBST and shake to dissolve; c) Add 1× PBST to make up to 50 mL.

[0427] 1.3 Color stop solution

[0428] Component concentration: 2M H2SO4

[0429] Configuration volume: 100mL

[0430] Preparation method: a) Add 70 mL of pure water into a beaker;

[0431] b) Add 10.9 mL of concentrated sulfuric acid with stirring, mix well and make up to 100 mL, and store at room temperature.

[0432] 2. EGFAB protein coating

[0433] 2.1 Adjust the EGFAB protein concentration to 20 μg / mL with DPBS and the total volume of the solution to 10 mL.

[0434] 2.2 Select 3 wells of the 96-well plate and add 100 μL of DPBS, and add 100 μL of EGFAB protein solution to each of the remaining 93 wells.

[0435] 2.3 Seal the plate and incubate at 37°C for 2 hours or at 4°C for 16 hours.

[0436] 2.4 After incubation, discard the solution, tap dry, add 375 μL of PBST wash solution to each well, shake and wash on a shaker at room temperature for 5 minutes, discard the solution and tap dry. Wash once in total.

[0437] 3. Closure

[0438] 3.1 Add 375 μL of the prepared blocking solution to each well of a 96-well plate.

[0439] 3.2 Seal the plate and incubate at 37°C for 2 hours.

[0440] 3.3 After incubation, discard the solution, tap dry, add 375 μL of PBST solution to each well, shake and wash on a shaker at room temperature for 5 minutes, discard the solution and tap dry. Wash twice in total.

[0441] 3.4 (If the subsequent steps are carried out on the same day of the experiment, this step can be ignored) Dry the sealed plate at 37°C for 1 hour, seal it with sealing paper and store it at 4°C.

[0442] 4. Determination of PCSK9 Concentration

[0443] 4.1 Use 1 mg / mL PCSK9 as the stock concentration, 20 μg / mL as the starting concentration, and PBS as the diluent to decrease the concentration by 1 / 2. Take 8 1.5 mL centrifuge tubes, mark them, and prepare the samples according to the table below.

[0444] PCSK9 PBS 20μg / mL 12μL 1mg / mL 588μL 10μg / mL 300μL 20μg / mL 300μL 5μg / mL 300μL 10μg / mL 300μL 2.5μg / mL 300μL 5μg / mL 300μL 1.25μg / mL 300μL 2.5μg / mL 300μL 0.625μg / mL 300μL 1.25μg / mL 300μL 0μM 0μL 300μL 0μM 0μL 300μL

[0445] After the preparation is completed, add 1.5 μL of DMSO to each centrifuge tube.

[0446] 4.2 All samples were pre-incubated at room temperature for 1 hour with shaking.

[0447] 4.3 Each sample was added to each well of the blocked ELISA plate at 100 μL, and each sample was repeated in triplicate; one of the blank samples should be added to three blank wells that have not been coated with EGFAB.

[0448] 4.4 Incubate with shaking at room temperature for 2 hours.

[0449] 4.5 After incubation, discard the solution, tap dry, add 375 μL PBST wash solution to each well, shake and wash on a shaker at room temperature for 5 minutes, discard the solution and tap dry. Wash three times in total.

[0450] 4.6 Primary Antibody Dilution:

[0451] 4.6.1 Take 10 mL of the working solution per plate as the base volume, take 10 mL of the blocking solution into a 15 mL centrifuge tube, add 5 μL of the primary antibody, and mix to form the working solution (1:2000).

[0452] 4.6.2 Add 100 μL of the working solution to each well of a 96-well plate.

[0453] 4.6.3 Incubate with shaking at room temperature for 2 hours.

[0454] 4.6.4 After incubation, discard the solution, tap dry, add 375 μL of PBST wash solution to each well, shake and wash on a shaker at room temperature for 5 minutes, discard the solution and tap dry. Wash four times in total.

[0455] 4.7 Secondary antibody dilution:

[0456] 4.7.1 Take 10mL of blocking solution into a 15mL centrifuge tube, add 2μL of secondary antibody, and mix to make working solution (1:5000).

[0457] 4.7.2 Add 100 μL of the working solution to each well of a 96-well plate.

[0458] 4.7.3 Incubate with shaking at room temperature for 1 hour.

[0459] 4.7.4 After incubation, discard the solution, tap dry, add 375 μL of PBST wash solution to each well, shake and wash on a shaker at room temperature for 5 minutes, discard the solution and tap dry. Wash four times in total.

[0460] 4.8 Color Development & Termination

[0461] 4.8.1 Use the equilibrated colorimetric solution to cool to room temperature for half an hour.

[0462] 4.8.2 Add 100 μL of 10 mL of color development solution to each test well and incubate at room temperature in the dark for 10 minutes (the incubation time can be appropriately extended or shortened depending on the color development status).

[0463] 4.8.3 Add 50 μL of 5 mL stop solution to each well to stop color development.

[0464] 4.9. Reading:

[0465] 4.9.1 Use PlateReader to read the OD value at 450nm, and select the corresponding concentration between OD values ​​1.2-0.9 as the test concentration of PCSK9 compound.

[0466] 5. Treatment and loading of test compounds

[0467] 5.1 Use 20mM of the compound as the stock solution concentration, 50μM as the starting concentration, and DMSO as the diluent to decrease the gradient by 1 / 4 for 6 steps. Take 9 1.5mL centrifuge tubes, mark them, and prepare the samples according to the table below.

[0468]

[0469]

[0470] After the preparation is completed, except for the two blank centrifuge tubes, each of which is added with 300 μL of PBS, the remaining tubes are added with 300 μL of PCSK9 diluted with PBS at an appropriate concentration. (For the specific appropriate concentration, please refer to the results after step 4.8.4)

[0471] 5.2 For subsequent sample processing and sample addition, please refer to steps 4.2-4.6

[0472] 6. Primary Antibody Incubation

[0473] 6.1 For primary antibody operation, please refer to steps 4.6.1-4.6.4

[0474] 7. Secondary Antibody Incubation

[0475] 7.1 For secondary antibody operation, please refer to steps 4.7.1-4.7.4

[0476] 8. Color development & termination

[0477] 8.1 Operations are detailed in steps 4.8.1-4.8.3

[0478] 9. Reading

[0479] 9.1 Use PlateReader to read the OD value at 450nm, and calculate the EGFAB and PCSK9 binding inhibition rate and IC50 of the test compound based on the data.

[0480] Experimental results:

[0481]

[0482] Example 16: Western-Blot cell function activity test (LDLR)

[0483] Experimental subjects: HepG2 cell lysate / mouse liver tissue extract

[0484] Experimental materials: Tris (Sinopharm), aminoacetic acid (Kermiou), methanol (Sinopharm), NaCl (Sinopharm), Tween 20 (Aladdin), skimmed milk powder (BD, REF232100), MOPS (Genscript), 4%-12% gradient gel (Genscript), LDLR primary antibody (abcam, ab52818), α-Tubulin primary antibody (abcam, ab176560), goat anti-rabbit secondary antibody (ABclonal Cat. NO. AS014), HRP luminescent liquid (millipore, Cat. NO. WBKLS0500), PVDF membrane (0.45μm, millipore, Cat. NO. IPVH00010), filter paper (BIO-RAD), and Yijie membrane.

[0485] Experimental equipment: vertical electrophoresis instrument (BIO-RAD, Tetra System), wet transfer instrument (BIO-RAD, BioSurplus), chemiluminescence imaging system (Cree Innovation, K3000 mini), constant temperature shaker (Ningbo Jiangnan Instrument Factory), and flat shaker (VWR).

[0486] 1. Solution preparation

[0487] 1.1 10×Towbin transfer buffer (methanol-free)

[0488] Component concentration: 250mM Tris, 1920mM glycine

[0489] Configuration volume: 2L

[0490] Preparation method: a) Take a clean 2L beaker and add about 1200mL of ultrapure water;

[0491] b) Add the following reagents in sequence and stir until dissolved;

[0492]

[0493] c) The solution was diluted to 2 L;

[0494] d) Filter with a 0.22 μm filter and store in a refrigerator at 4°C.

[0495] Note: The pH of the solution should be between 8.1 and 8.5.

[0496] 1.2 1×Towbin transfer buffer

[0497] Component concentration: 25mM Tris, 192mM glycine, 20% (V / V) methanol

[0498] Configuration volume: 2L

[0499] Preparation method: a) Take a clean 2L beaker and add about 800mL of ultrapure water;

[0500] b) Add the following reagents in sequence and stir evenly;

[0501]

[0502] c) Make the solution to 2L and pre-cool in a 4℃ refrigerator;

[0503] Note: The pH of the solution should be between 8.1 and 8.5.

[0504] 1.3 5×TBS

[0505] Component concentration: 50 mM Tris-Cl, 4.5% (W / V) NaCl

[0506] Configuration volume: 2L

[0507] Preparation method: a) Take a clean 2L beaker and add about 1600mL of ultrapure water;

[0508] b) Add the following reagents in sequence and stir until dissolved;

[0509]

[0510] c) adjust the pH of the solution to 7.5 with 2M HCl (about 30 mL of 2M HCl);

[0511] d) The solution was diluted to 2 L;

[0512] e) Filter the solution through a 0.22 μm filter and store at room temperature.

[0513] 1.4 1× TBST

[0514] Component concentration: 10 mM Tris-cl, 0.9% (W / V) NaCl, 0.02% (V / V) Tween-20

[0515] Configuration volume: 2L

[0516] Preparation method: a) Take a clean 2L beaker and add about 1600mL of ultrapure water;

[0517] b) Add the following reagents in sequence and stir until dissolved;

[0518]

[0519] c) Make the solution up to 2 L and store at room temperature.

[0520] 1.5 Blocking solution

[0521] Component concentration: 5% (W / V) skimmed milk powder

[0522] Configuration volume: 50mL

[0523] Preparation method: a) weigh 2.5g skim milk powder into a 50mL centrifuge tube;

[0524] b) Add 30 mL 1× TBST and shake to dissolve;

[0525] c) Add 1× TBST to make up to 50 mL.

[0526] 1.6 5×SDS-PAGE Loading Buffer

[0527] Component concentration: 250 mM Tris-Hcl (pH 6.8), 10% (W / V) SDS, 0.5% (W / V) BPB, 50% (V / V) glycerol, 5% (V / V) β-mercaptoethanol (2-ME)

[0528] Configuration volume: 10mL

[0529] Preparation method: a) Weigh the following reagents into a 50 mL centrifuge tube;

[0530]

[0531] b) Add pure water to the container, dilute to 10 mL after dissolution, and store at room temperature;

[0532] c) Aliquot into small portions (500 μL / portion) and store at room temperature;

[0533] d) Add 25 μL of 2-ME to each aliquot before use;

[0534] e) Loading Buffer with 2-ME can be stored at room temperature for about one month.

[0535] 2.SDS-PAGE

[0536] 2.1 Sample preparation

[0537] 2.1.1 Adjust the concentration of protein extract to 0.5-2 mg / mL, add the corresponding amount of 5× Loading-buffer, mix well, and incubate in a water bath at 90°C or above for 10 min.

[0538] 2.1.2 Cool the sample to room temperature and centrifuge at 3000 rpm for 2 min.

[0539] 2.2 Electrophoresis

[0540] 2.2.1 Protein samples were added to each lane with 10-40 μg of protein and 5 μL of protein marker;

[0541] 2.2.2 Insert the electrodes correctly and turn on the power;

[0542] 2.2.3 Run the gel at 80-140 V until bromophenol blue completely runs out of the gel.

[0543] 3. Electroblotting

[0544] 3.1 Cut one 7.2*8.6cm PVDF membrane and two 7*8.4cm filter papers.

[0545] 3.2 Equilibration of gel, PVDF membrane and filter paper

[0546] 3.2.1 Soak the PVDF membrane completely in 100% methanol for 1 minute, rinse it with ultrapure water, and then equilibrate it in the transfer solution for 10 to 15 minutes;

[0547] 3.2.2 After electrophoresis, remove the gel, rinse it with ultrapure water, and then equilibrate it in transfer buffer for 10 to 15 minutes;

[0548] 3.2.3 Place the filter paper directly into the transfer solution and equilibrate for 10 to 15 minutes.

[0549] 3.3 Assembly of sandwich structure of gel, PVDF membrane and filter paper

[0550] 3.3.1 Place the gel holder in the dish, place a fiber pad on the black gel holder, and add transfer solution to completely soak the fiber pad;

[0551] 3.3.2 Place a piece of filter paper on the fiber pad and drop the transfer solution onto the filter paper;

[0552] 3.3.3 Gently place the gel on the filter paper and use a moistened gloved finger to gently dislodge any bubbles that have accumulated under the gel;

[0553] 3.3.4 Add transfer solution to the gel, and place the front of the PVDF membrane close to the surface of the gel. Try not to move the PVDF membrane after placement. Add transfer solution to the PVDF membrane, and use a roller to drive out bubbles and excess transfer solution to ensure that the gel and PVDF membrane are in complete contact;

[0554] 3.3.5 Add transfer solution to the PVDF membrane, place another piece of filter paper on the PVDF membrane, and use a roller to remove bubbles;

[0555] 3.3.6 Soak another fiber pad and place it on the filter paper.

[0556] 3.3.7 Fasten the gel transfer holder clamp and insert it vertically into the transfer tank, making sure the black side of the transfer holder corresponds to the black electrode.

[0557] 3.4 Electrotransfer process

[0558] 3.4.1 Fill the transfer tank with transfer solution, add a stirring bar into the transfer tank, and seal it with sealing film;

[0559] 3.4.2 Place the transfer tank into the ice-water mixture and start stirring on a magnetic stirrer;

[0560] 3.4.3 Insert the electrodes correctly and turn on the power supply;

[0561] 3.4.4 Constant voltage 100V, electroblotting for 60min;

[0562] 3.4.5 Record the real-time current at the beginning, 10 minutes and end of the electrotransfer into the corresponding table.

[0563] 3.5 After the transfer is completed, take out the blotting membrane, cut the upper left corner of the blotting membrane to identify the direction, and rinse the blotting membrane slightly in ultrapure water.

[0564] 4. Closure

[0565] 4.1 Place the blot membrane in 30 mL TBST and shake for 3 minutes;

[0566] 4.2 Take out the blotting membrane and put it into the Yijie membrane, add 20mL of blocking solution;

[0567] 4.3 Incubate at 37°C, 120 rpm, shake for 60 min.

[0568] 5. Primary Antibody Incubation

[0569] 5.1 Primary Antibody Dilution

[0570] Take 3 mL of blocking solution into a 5 mL centrifuge tube, add 3 μL of LDLR primary antibody, and mix to form LDLR antibody working solution (LDLR: blocking solution = 1:1000);

[0571] Take 3 mL of blocking solution into a 5 mL centrifuge tube, add 3 μL of α-Tubulin primary antibody, and mix to form α-Tubulin antibody working solution (α-Tubulin: blocking solution = 1:1000);

[0572] 5.2 After blocking, pour off the blocking solution and cut the LDLR and internal reference α-Tubulin on the blot membrane at the protein red marker position;

[0573] 5.3 Place the blotting membrane into the Yijie membrane and add the corresponding primary antibody working solution;

[0574] 5.4 Incubate at room temperature with shaking for 60 min or at 4°C with shaking overnight;

[0575] 5.5 After incubation, remove the blot membrane and place it in 30 mL TBST. Wash the membrane by shaking for 5 minutes. Replace TBST and repeat the washing 6 times.

[0576] 6. Secondary Antibody Incubation

[0577] 6.1 Secondary Antibody Dilution

[0578] Take 10mL of blocking solution into a 15mL centrifuge tube, add 2μL of HRP secondary antibody, and mix to make HRP secondary antibody working solution (HRP secondary antibody: blocking solution = 1:5000);

[0579] 6.2 Place the blotting membrane into the Yijie membrane and add 5 mL of secondary antibody working solution to each half of the blotting membrane;

[0580] 6.3 Incubate at room temperature with shaking for 60 min;

[0581] 6.4 After incubation, remove the blot membrane and place it in 30 mL TBST. Wash the membrane by shaking for 5 minutes. Replace the TBST and repeat the washing 6 times.

[0582] 7. Color rendering

[0583] 7.1 Prepare the developer solution according to the developer solution instructions. Prepare 6 mL for each blotting membrane (7.2 cm*8.6 cm).

[0584] Color solution, mix well, and equilibrate at room temperature for 10 min;

[0585] 7.2 Rinse the blotted membrane briefly in ultrapure water;

[0586] 7.3 Drain excess water from the blotting membrane, quickly add 3 mL of color developing solution to the front of the blotting membrane, and let stand at room temperature for 5 minutes;

[0587] 7.4 Take out the blot membrane and put it into the Yijie membrane, and take pictures with a chemiluminescence instrument.

[0588] The experimental results are as follows Figure 1 As shown, the results show that the compounds of the present invention can significantly enhance the expression of LDLR protein at a lower concentration (such as 25 μM), such as compound 1, compound 8, compound 9, etc.

[0589] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A compound represented by formula II or a stereoisomer or optical isomer, or a pharmaceutically acceptable salt thereof, In the formula, n is 0, 1, 2 or 3; m is 0; A is selected from the group consisting of -CH2-, -C(=O)-; B is selected from the group consisting of: -NH-, -C(=O)NH-; C is selected from the group consisting of: -C(=O)-; D is selected from the following group: -NR b -; R b is H; E is selected from the group consisting of: -C(=O)-; M is selected from the group consisting of -CH2-, -C(=O)-, -C(=O)NH-; R1 is a substituted C1-C6 alkyl group; wherein, The substitution refers to substitution by one or more R4; R4 is selected from the group consisting of substituted or unsubstituted 5-12 membered heteroaryl, C6-C12 aryl; wherein the substitution refers to substitution by one or more groups selected from the group consisting of D, halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C6-C12 aryl; R2 is -X1-Ar1; Wherein, the X1 is -(CH2) n7 -; n7 is 1, 2 or 3; Ar1 is substituted or unsubstituted by the following group: 5-12 membered heteroaryl, C6-C12 aryl; wherein the substitution refers to substitution by one or more groups selected from the following group: D, halogen, amino, nitro, hydroxyl, mercapto, cyano, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl; R3 is a substituted C1-C6 alkyl; wherein the substitution refers to substitution by one substituted or unsubstituted 5-12 membered heteroaryl; wherein the substitution refers to substitution by one or more groups selected from the following group: D, halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl.

2. The compound of formula II or its stereoisomers or optical isomers, or pharmaceutically acceptable salts as claimed in claim 1, characterized in that: The n is 0, 1 or 2.

3. The compound of formula II or its stereoisomers or optical isomers, or pharmaceutically acceptable salts as claimed in claim 1, characterized in that: Having the structure shown in formula III In the formula, R1, R2, R3, A, B, C, D, E, and M are as defined in claim 1.

4. The compound of formula II or its stereoisomers or optical isomers, or pharmaceutically acceptable salts as claimed in claim 1, characterized in that: Having the structure shown in Formula VI In the formula, n is 0, 1, 2 or 3; m is 0; R1, R2, R3, R b , A, B, and M are defined as in claim 1.

5. The compound of formula II or its stereoisomers or optical isomers, or pharmaceutically acceptable salts as claimed in claim 1, characterized in that: Having the structure shown in Formula VII In the formula, n is 0, 1 or 2; m is 0; R1, R2, R3, R b , A, and M are defined as in claim 1.

6. The compound of formula II or its stereoisomers or optical isomers, or pharmaceutically acceptable salts as claimed in claim 1, characterized in that: Some selected from:

7. The compound of formula II or its stereoisomers or optical isomers, or pharmaceutically acceptable salts as claimed in claim 1, characterized in that: The n is 1, and m is 0.

8. The compound of formula II or its stereoisomers or optical isomers, or pharmaceutically acceptable salts as claimed in claim 1, characterized in that: Having the structure shown in Formula VIII R1, R2, R3, M, R 11 The definition as in claim 1.

9. The compound of formula II or its stereoisomers or optical isomers, or pharmaceutically acceptable salts as claimed in claim 1, characterized in that: Having the structure shown in formula X In the formula, m' is 0; R1, R2, R b , M is defined as in claim 1.

10. The compound according to claim 1 or its stereoisomer or optical isomer, or pharmaceutically acceptable salt, characterized in that: The compound is selected from the group consisting of:

11. A pharmaceutical composition, characterized in that Comprising the compound according to any one of claims 1 to 10 or its stereoisomer or optical isomer, a pharmaceutically acceptable salt; and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to claim 11, characterized in that It contains a drug selected from the group consisting of: Atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, fibrates, niacin and its analogs, cholesterol absorption inhibitors, bile acid sequestrants, thyroid hormone mimetics, microsomal triglyceride transfer protein (MTP) inhibitors, diacylglycerol acyltransferase (DGAT) inhibitors, inhibitory nucleic acids targeting PCSK9, inhibitory nucleic acids targeting apoB100, or a combination thereof.

13. Use of a compound according to any one of claims 1 to 10 or its stereoisomers or optical isomers, or pharmaceutically acceptable salts, characterized in that: Used for preparing drugs or pharmaceutical compositions for inhibiting diseases related to PCSK9 protein activity or expression.

14. The use according to claim 13, characterized in that Used for preparing a medicine or a pharmaceutical composition for lowering the level of low-density lipoprotein cholesterol (LDL-C) or increasing the level of high-density lipoprotein cholesterol (HDL-C).

15. The use according to claim 13, characterized in that The disease is selected from cardiovascular disease, inflammatory disease or infectious disease.

16. The use according to claim 13, characterized in that The disease is a low-density lipoprotein-cholesterol related disease or disorder.

17. The use according to claim 13, characterized in that The diseases are hyperlipidemia, hypertriglyceridemia, hypercholesterolemia and / or atherosclerosis.

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