Targeted plasma protein degradation

Abstract

The present invention relates to bifunctional compounds and the use of such bifunctional compounds to reduce plasma levels of extracellular target molecules through lysosomal degradation. Such bifunctional compounds have a cell surface receptor ligand covalently linked to a ligand capable of binding to the extracellular target molecule (e.g., a ligand for a growth factor, cytokine, chemokine, hormone, neurotransmitter, capsid, soluble receptor, extracellular secreted protein, antibody, lipoprotein, exosome, virus, cell, or cell membrane protein), where the cell surface receptor is associated with receptor-mediated endocytosis, including asialoglycoprotein receptor (ASGPR)-mediated lysosomal degradation and mannose-6-phosphate (M6PR)-mediated lysosomal degradation. Pharmaceutical compositions containing such bifunctional compounds and methods for treating diseases or disorders mediated by extracellular molecules using such bifunctional compounds are also provided herein.

Description

[Technical Field]

[0001] The present invention relates to the field of receptor-mediated endocytosis or lysosomal degradation of target molecules located in or outside the cell membrane. [Background technology]

[0002] Conventional protein-targeted therapies, such as enzyme inhibitors and receptor antagonists, treat diseases by interfering with protein function or by recruiting immune effectors, as is the case with many monoclonal antibody drugs. However, potential therapeutic protein targets, such as transcription factors, scaffold proteins, aggregate-forming proteins, lipid carriers, mucins, orphan receptors, and multifunctional molecules, which have molecular functions that are not fully understood or readily inhibited, do not lead to the development of new drugs through conventional therapeutic approaches. Targeted proteolysis (TPD) is a therapeutic approach to treating these disease-causing proteins and signaling pathways that do not lead to the development of new drugs, by controlling the amount of target proteins through degradation of the target protein rather than inhibiting its function.

[0003] Examples of targeted proteolytic systems include proteolytic chimeras (PROTAC) (KMSakamoto et al., Proc. Natl. Acad. Sci. 98, 8554-8559, (2001) and GEWinter et al., Science. 348, 1376-1381 (2015)), dTAG (B. Nabet et al., Nat. Chem. Biol. 14, 431 (2018)), Trim-Away (D. Clift et al., Cell., 171, 1692-1706.e18 (2017)), chaperone-mediated autophagy targeting (X. Fan et al., Nat. Neurosci., 17, 471-480 (2014)), and SNIPER (M. Naito et al., Drug Discov. Today) Technol., (2019) is one example. PROTACs promote ubiquitination and proteasome degradation by forming crosslinks between E3 ubiquitin ligases and their target organisms (GMBurslem et.al., Chem. Rev., 117, 11269-11301 (2017)). These degradation systems utilize the proteasome pathway for degrading intracellular proteins. Furthermore, degradation systems utilizing the lysosomal pathway for degrading extracellular proteins (secretory and cell membrane proteins) have also been reported (S.Banik et al., ChemRxiv, 2019 and PCNRensen et). (al., J. Med. Chem., 47, 5798-5808, 2004), strategies for the degradation of extracellular targets such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, and cellular and cell membrane proteins remain unmet needs. [Overview of the project]

[0004] The present invention relates to the use of bifunctional compounds to reduce plasma levels of extracellular target molecules in patients by receptor-mediated endocytosis followed by lysosomal degradation, thereby enabling their use as pharmaceuticals in the treatment of disease conditions and / or conditions mediated by such extracellular molecules. Accordingly, the present invention provides bifunctional compounds and their use in the targeted degradation of extracellular target molecules such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, cells, and cell membrane proteins by lysosomal degradation. The present invention further provides bifunctional compounds and their use in the targeted degradation of extracellular target molecules such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, cells, and cell membrane proteins by asialoglycoprotein receptor (ASGPR)-mediated lysosomal degradation. The present invention also provides for the use of bifunctional compounds and their use in the targeted degradation of extracellular target molecules such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, cells, and cell membrane proteins by mannose-6-phosphate (M6PR)-mediated lysosomal degradation.

[0005] The bifunctional compounds of the present invention may provide patients with significant clinical utility, particularly for the treatment of disease states and conditions regulated by the extracellular target of interest.

[0006] The bifunctional compound of the present invention comprises a cell surface receptor ligand covalently bound to a ligand capable of binding to extracellular target molecules (such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, cells, or cell membrane proteins), wherein the cell surface receptor is associated with receptor-mediated endocytosis.

[0007] The present invention relates to formula (I): R L -L A -T L (I) (In the formula, R L This is the portion that binds to cell surface receptors associated with receptor-mediated endocytosis; L A is a linker; and T L (This is the part that binds to extracellular targets.) Further, we provide a bifunctional compound having the structure described above.

[0008] The present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of a bifunctional compound of formula (I) and a pharmaceutically acceptable carrier.

[0009] In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the compound of the present invention and one or more pharmaceutically acceptable carriers.

[0010] The present invention further provides pharmaceutical compositions comprising a bifunctional compound of formula (I) and a pharmaceutically acceptable carrier.

[0011] In another embodiment, the present invention provides a pharmaceutical composition comprising the compound of the present invention and one or more pharmaceutically acceptable carriers.

[0012] In another embodiment, the present invention provides a combination, particularly a combination pharmaceutical comprising a therapeutically effective amount of the compound of the present invention and one or more therapeutically active agents.

[0013] The present invention provides a method for targeted lysosomal degradation of extracellular target molecules such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secreted proteins, antibodies, lipoproteins, exosomes, viruses, cells, and cell membrane proteins by administration of the bifunctional compound of the present invention. The present invention also provides a method for targeted asialoglycoprotein receptor (ASGPR)-mediated lysosomal degradation of extracellular target molecules such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secreted proteins, antibodies, lipoproteins, exosomes, viruses, cells, and cell membrane proteins by administration of the bifunctional compound of the present invention. The present invention further provides a method for targeted mannose-6-phosphate (M6PR)-mediated lysosomal degradation of extracellular target molecules such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secreted proteins, antibodies, lipoproteins, exosomes, viruses, cells, and cell membrane proteins by administration of the bifunctional compound of the present invention.

[0014] These methods can be used in the treatment of various diseases, conditions, or clinical situations that are often treated via therapeutic apheresis, such as cardiovascular diseases, liver diseases, kidney diseases, autoimmune diseases, neurological diseases, hematological diseases, skin diseases, drug intoxication, and vasculitis. Examples of such diseases include hypercholesterolemia, familial hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, obstructive arteriosclerosis, coronary heart disease, peripheral vascular disease (including aortic and cerebrovascular diseases), peripheral artery disease, vasculitis, elevated Lp(a), elevated LDL, elevated TRL, elevated triglycerides, sepsis, xanthomas, fulminant hepatic failure, postoperative hepatic failure, acute hepatic failure, hepatitis C, hepatitis B, and chronic hepatitis C. Examples of conditions that may be considered invasive include, but are not limited to, inflammation, chronic hepatitis B, allogeneic liver transplantation, focal segmental glomerulosclerosis, allogeneic kidney transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, membranous nephropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood group incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, and Kawasaki disease.

[0015] These methods may also be used in the treatment of nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0016] In another aspect, the present invention further provides a method for treating a disease or condition modulated by an extracellular target molecule by administering a therapeutically effective amount of a bifunctional compound of formula (I) or a subformula thereof to a subject requiring it.

[0017] In another aspect, the present invention also provides the use of a bifunctional compound of formula (I) or a subformula thereof for treating a disease or condition regulated by a target extracellular molecule described herein.

[0018] In another aspect, the present invention also provides the use of a bifunctional compound of formula (I) or a subformula thereof in the manufacture of a pharmaceutical for treating a disease or condition regulated by a target extracellular molecule described herein.

[0019] In another embodiment, the present invention also provides a method for therapeutic plasmapheresis in vivo, the method comprising administering a bifunctional compound of formula (I) or a subformula thereof to a target. The present invention also provides a method for carrying out therapeutic plasmapheresis in vivo, the method comprising administering a bifunctional compound of the present invention to a target.

[0020] In another embodiment, the present invention also provides a method of in vivo therapeutic plasmapheresis for the treatment of cardiovascular disease, liver disease, kidney disease, autoimmune disease, neurological disease, hematological disease, skin disease, drug poisoning, or vasculitis, the method comprising administering the bifunctional compound of the present invention to a target. In certain embodiments, such diseases include hypercholesterolemia, familial hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, obstructive arteriosclerosis, coronary heart disease, peripheral vascular disease (including aortic disease and cerebrovascular disease), peripheral artery disease, vasculitis, elevated Lp(a), elevated LDL, elevated TRL, elevated triglycerides, sepsis, xanthomas, fulminant hepatic failure, postoperative hepatic failure, acute hepatic failure, hepatitis C, hepatitis B These include hepatitis, chronic hepatitis C, chronic hepatitis B, allogeneic liver transplantation, focal segmental glomerulosclerosis, allogeneic kidney transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, membranous nephropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood group incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, and Kawasaki disease.

[0021] In another aspect, the present invention also provides a method of in vivo therapeutic plasmapheresis for the treatment of nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0022] In another aspect, the present invention also provides a therapy based on the reduction of extracellular levels of extracellular target molecules by lysosomal degradation mediated by a bifunctional compound of formula (I) or a subformula thereof.

[0023] In another aspect, the present invention also provides a therapy for the treatment of cardiovascular disease based on a reduction in the extracellular level of the protein proprotein convertase subtilisin / kexin type 9 (PCSK9) by lysosomal degradation mediated by the bifunctional compound of formula (Ia).

[0024] In another aspect, the present invention also provides a therapy for treating diseases or disorders associated with the complement factor H-related protein 3 gene (CFHR3) based on a decrease in extracellular levels of protein complement factor H-related protein 3 (FHR3) by lysosomal degradation mediated by the bifunctional compound of formula (Ib).

[0025] In another aspect, the present invention also provides a therapy for treating diseases or disorders associated with complement factor H-related protein 3 (FHR3) based on a decrease in the level of extracellular complement factor H-related protein 3 (FHR3) due to lysosomal degradation mediated by the bifunctional compound of formula (Ib).

[0026] In another embodiment, the present invention also provides a bifunctional compound of formula (Ia) for use in the treatment of PCSK9-mediated diseases or disorders. In another embodiment, the present invention also provides a pharmaceutical composition comprising a bifunctional compound of formula (Ia) for use in the treatment of PCSK9-mediated diseases or disorders. In particular embodiments of such use, the PCSK9-mediated diseases or disorders are selected from hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral vascular disease, peripheral artery disease, vasculitis, elevated Lp(a), elevated LDL, triglyceride-rich lipoprotein (TRL), elevated triglycerides, sepsis, and xanthomas.

[0027] In another embodiment, the present invention also provides a bifunctional compound of formula (Ib) for use in the treatment of CFHR3-mediated diseases or disorders. In another embodiment, the present invention also provides a pharmaceutical composition comprising a bifunctional compound of formula (Ib) for use in the treatment of CFHR3-mediated diseases or disorders. In particular embodiments of such use, the CFHR3-mediated diseases or disorders are selected from nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0028] In another embodiment, the present invention also provides a bifunctional compound of formula (Ib) for use in the treatment of FHR3-mediated diseases or disorders. In another embodiment, the present invention also provides a pharmaceutical composition comprising a bifunctional compound of formula (Ib) for use in the treatment of FHR3-mediated diseases or disorders. In particular embodiments of such use, the FHR3-mediated diseases or disorders are selected from nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0029] In another aspect, the present invention also provides the use of the bifunctional compound of formula (Ia) in the treatment of PCSK9-mediated diseases or disorders. In another aspect, the present invention also provides the use of the bifunctional compound of formula (Ia) in the manufacture of a pharmaceutical for the treatment of PCSK9-mediated diseases or disorders. In another aspect, the present invention also provides the use of a pharmaceutical composition comprising the bifunctional compound of formula (Ia) in the treatment of PCSK9-mediated diseases or disorders. In particular embodiments of such use, the PCSK9-mediated diseases or disorders are selected from hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral vascular disease, peripheral artery disease, vasculitis, elevated Lp(a), elevated LDL, triglyceride-rich lipoprotein (TRL), elevated triglycerides, sepsis, and xanthomas.

[0030] In another aspect, the present invention also provides the use of the bifunctional compound of formula (Ib) in the treatment of CFHR3-mediated diseases or disorders. In another aspect, the present invention also provides the use of the bifunctional compound of formula (Ib) in the manufacture of a pharmacopoeia for the treatment of CFHR3-mediated diseases or disorders. In another aspect, the present invention also provides the use of a pharmaceutical composition comprising the bifunctional compound of formula (Ib) in the treatment of CFHR3-mediated diseases or disorders. In particular embodiments of such use, the CFHR3-mediated diseases or disorders are selected from nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0031] In another aspect, the present invention also provides the use of the bifunctional compound of formula (Ib) in the treatment of FHR3-mediated diseases or disorders. In another aspect, the present invention also provides the use of the bifunctional compound of formula (Ib) in the manufacture of a pharmacopoeia for the treatment of FHR3-mediated diseases or disorders. In another aspect, the present invention also provides the use of a pharmaceutical composition comprising the bifunctional compound of formula (Ib) in the treatment of FHR3-mediated diseases or disorders. In particular embodiments of such use, the FHR3-mediated diseases or disorders are selected from nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0032] In another embodiment, the present invention also provides a method for treating a PCSK9-mediated disease or disorder, comprising the step of administering a therapeutically effective amount of the bifunctional compound of formula (Ia) to a patient in need thereof. In a particular embodiment of this method, the PCSK9-mediated disease or disorder is selected from hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral vascular disease, peripheral artery disease, vasculitis, elevated Lp(a), elevated LDL, triglyceride-rich lipoprotein (TRL), elevated triglycerides, sepsis, and xanthomas.

[0033] In another embodiment, the present invention also provides a method for treating a CFHR3-mediated disease or disorder, comprising the step of administering a therapeutically effective amount of the bifunctional compound of formula (Ib) to a patient in need thereof. In a particular embodiment of this method, the CFHR3-mediated disease or disorder is selected from nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0034] In another embodiment, the present invention also provides a method for treating an FHR3-mediated disease or disorder, comprising the step of administering a therapeutically effective amount of the bifunctional compound of formula (Ib) to a patient in need thereof. In a particular embodiment of this method, the FHR3-mediated disease or disorder is selected from nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0035] In another aspect, the present invention also provides a method for targeted lysosomal degradation of an extracellular target molecule, comprising administering a bifunctional compound of formula (Ia), wherein the extracellular target molecule is PCSK9.

[0036] In another aspect, the present invention also provides a method for targeted lysosomal degradation of an extracellular target molecule, comprising administering a bifunctional compound of formula (Ib), wherein the extracellular target molecule is FHR3.

[0037] In another embodiment, the present invention also provides a method for removing an extracellular target molecule from the plasma of a patient requiring such removal, comprising administering a bifunctional compound of formula (Ia), wherein the extracellular target molecule is PCSK9.

[0038] In another aspect, the present invention also provides a method for removing an extracellular target molecule from the plasma of a patient requiring such removal, comprising administering a bifunctional compound of formula (Ib), wherein the extracellular target molecule is FHR3.

[0039] In another aspect, the present invention also provides a bifunctional compound of formula (Ia) for use in a therapy for the treatment of cardiovascular disease, the therapy based on a reduction in extracellular levels of PCSK9 by lysosomal degradation mediated by the bifunctional compound of formula (Ia).

[0040] In another aspect, the present invention also provides a bifunctional compound of formula (Ib) for use in therapies for the treatment of nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, or hepatocellular carcinoma (HCC), therapies based on a reduction in extracellular levels of FHR3 by lysosomal degradation mediated by the bifunctional compound of formula (Ib). [Brief explanation of the drawing]

[0041] [Figure 1A] Clearance of human FHR3 from transgenic mice expressing human FHR3 after intraperitoneal bolus administration of a solvent, 0.01 mg / kg of the bifunctional compound (BFC-13), and 0.1 mg / kg of the bifunctional compound (BFC-13). hFHR3 levels are relative to the FHR3 level before administration. [Figure 1B] Clearance of human FHR3 from transgenic mice expressing human FRH3 after intraperitoneal bolus administration of a solvent, 0.01 mg / kg of the bifunctional compound (BFC-15), and 0.1 mg / kg of the bifunctional compound (BFC-15). hFHR3 levels are relative to the FHR3 level before administration. [Figure 2A] Simultaneous administration study: Clearance of human PCSK9 from LDLR(- / -) mice after intravenous bolus administration of solvent + 3.3 μg of hPCSK9, 0.1 mg / kg of bifunctional compound (BFC-1) + 3.3 μg of hPCSK9, 0.05 mg / kg of PCSK9 ligand (C5), and 0.05 mg / kg of ASGPR ligand (int-CC2) + 3.3 μg of hPCSK9. [Figure 2B] Co-administration study: AUC plot of clearance data shown in Figure 2A. Statistics by standard one-way ANOVA with Dunnett's multiple comparison test. [Figure 3A]Simultaneous administration study: Clearance of human PCSK9 from LDLR(- / -) mice after intravenous bolus administration of solvent + 3.3 μg of hPCSK9, 0.1 mg / kg of bifunctional compound (BFC-2) + 3.3 μg of hPCSK9, 0.05 mg / kg of PCSK9 ligand (C5), and 0.05 mg / kg of M6PR ligand (int-CC6) + 3.3 μg of hPCSK9. [Figure 3B] Co-administration study: AUC plot of clearance data shown in Figure 3A. Statistics calculated using standard one-way ANOVA with Dunnett's multiple comparison test. [Figure 4A] Simultaneous administration study: Clearance of human PCSK9 from LDLR(- / -) mice after intravenous bolus administration of solvent + 3.3 μg of hPCSK9, 0.1 mg of bifunctional compound (BFC-1) + 3.3 μg of hPCSK9, 0.03 mg / kg of bifunctional compound (BFC-7) + 3.3 μg of hPCSK9, 0.1 mg / kg of bifunctional compound (BFC-7) + 3.3 μg of hPCSK9, and 0.3 mg / kg of bifunctional compound (BFC-7) + 3.3 μg of hPCSK9. [Figure 4B] Co-administration study: AUC plot of clearance data shown in Figure 4A. Statistics by standard one-way ANOVA for solvent: ***p=0.0001;****p<0.0001. [Figure 5A] Simultaneous administration study: Clearance of human PCSK9 from LDLR(- / -) mice after intravenous bolus administration of solvent + 3.3 μg of hPCSK9, 0.1 mg / kg of bifunctional compound (BFC-1) + 3.3 μg of hPCSK9, 0.1 mg / kg of bifunctional compound (BFC-5) + 3.3 μg of hPCSK9, and 1 mg / kg of bifunctional compound (BFC-5) + 3.3 μg of hPCSK9. [Figure 5B] Co-administration study: AUC plot of clearance data shown in Figure 5A. Statistics by standard one-way ANOVA for BFC-1: **p=0.0028. Statistics by standard one-way ANOVA for the solvent: ****p<0.0001. [Figure 6A]Simultaneous administration to pre-administration in the bifunctional compound test: Clearance of human PCSK9 from LDLR(- / -) mice after the following: i) IV bolus administration of solvent + 3.3 μg of hPCSK9 ii) IV bolus administration of 0.1 mg / kg of bifunctional compound (BFC-1) + 3.3 μg of hPCSK9 iii) IV bolus administration of 0.1 mg / kg of bifunctional compound (BFC-12) + 3.3 μg of hPCSK9 iv) IV bolus administration of 3.3 μg of hPCSK9 70 minutes after IV bolus administration of 0.1 mg / kg of bifunctional compound (BFC-12) v) IV bolus administration of 3.3 μg of hPCSK9 70 minutes after po administration of 30 mg / kg of bifunctional compound (BFC-12). [Figure 6B] Simultaneous administration versus pre-administration in the bifunctional compound test: AUC plot of clearance data shown in Figure 6A. Statistics by standard one-way ANOVA against solvent: ***p=0.0005;****p<0.0001;**p=0.0027. [Figure 7A] Simultaneous administration to pre-administration in the bifunctional compound test: Clearance of human PCSK9 from LDLR(- / -) mice after the following: i) IV bolus administration of solvent + 3.3 μg of hPCSK9 ii) IV bolus administration of 0.1 mg / kg of bifunctional compound (BFC-1) + 3.3 μg of hPCSK9 iii) IV bolus administration of 0.1 mg / kg of bifunctional compound (BFC-11) + 3.3 μg of hPCSK9 iv) IV bolus administration of 3.3 μg of hPCSK9 40 minutes after IV bolus administration of 0.1 mg / kg of bifunctional compound (BFC-11) v) IV bolus administration of 3.3 μg of hPCSK9 40 minutes after po administration of 30 mg / kg of bifunctional compound (BFC-11). [Figure 7B] Simultaneous administration versus pre-administration in bifunctional compound testing: AUC plot of clearance data shown in Figure 7A. Statistics by standard one-way ANOVA against solvent using Dunnett's multiple comparison test. [Figure 8A]Competitive study: Clearance of human PCSK9 from LDLR(- / -) mice after the following: i) IV bolus administration of solvent + 3.3 μg of hPCSK9 ii) IV bolus administration of 0.1 mg / kg of bifunctional compound (BFC-1) + 3.3 μg of hPCSK9 iii) IV bolus administration of 0.1 mg / kg of bifunctional compound (BFC-1) + 3.3 μg of hPCSK9 + 10 mg / kg of ASGPR ligand (int-CC2) iv) IV bolus administration of 0.1 mg / kg of bifunctional compound (BFC-1) + 3.3 μg of hPCSK9 + 10 mg / kg of PCSK9 ligand (C5). [Figure 8B] Competitive testing: AUC plot of clearance data shown in Figure 8A. Statistics by standard one-way ANOVA for the bifunctional compound (BFC-1): **p=0.0033; ***p=0.0003; ****p<0.0001. [Modes for carrying out the invention]

[0042] definition As used herein, the term "alkyl" refers to a linear or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms and containing no unsaturation. As used herein, the term "C1-C6 alkyl" refers to a linear or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms, containing no unsaturation, having 1 to 6 carbon atoms, and bonded to the remainder of the molecule by single bonds. Non-limiting examples of "C1-C6 alkyl" groups include methyl (C1 alkyl), ethyl (C2 alkyl), 1-methylethyl (C3 alkyl), n-propyl (C3 alkyl), isopropyl (C3 alkyl), n-butyl (C4 alkyl), isobutyl (C4 alkyl), sec-butyl (C4 alkyl), tert-butyl (C4 alkyl), n-pentyl (C5 alkyl), isopentyl (C5 alkyl), neopentyl (C5 alkyl), and hexyl (C6 alkyl).

[0043] As used herein, the term "alkenyl" refers to a radical group of a linear or branched hydrocarbon chain consisting only of carbon and hydrogen atoms and containing at least one double bond. e An "alkenyl" refers to a radical group of a linear or branched hydrocarbon chain consisting only of carbon and hydrogen atoms, containing at least one double bond, and having 2 to 6 carbon atoms, which are bonded to the rest of the molecule by single bonds. Non-restrictive examples of the "C2-C6 alkenyl" group include ethenyl (C2 alkenyl), propa-1-enyl (C3 alkenyl), buta-1-enyl (C4 alkenyl), penta-1-enyl (C5 alkenyl), penta-4-enyl (C5 alkenyl), penta-1,4-dienyl (C5 alkenyl), hexa-1-enyl (C6 alkenyl), hexa-2-enyl (C6 alkenyl), hexa-3-enyl (C6 alkenyl), hexa-1,4-dienyl (C6 alkenyl), hexa-1,5-dienyl (C6 alkenyl), and hexa-2,4-dienyl (C6 alkenyl). As used herein, the term "C2-C3 alkenyl" refers to a radical group of a linear or branched hydrocarbon chain consisting only of carbon and hydrogen atoms, containing at least one double bond and having 2-3 carbon atoms, with the remainder of the molecule bonded by a single bond. Non-limiting examples of "C2-C3 alkenyl" groups include ethenyl (C2 alkenyl) and propa-1-enyl (C3 alkenyl).

[0044] As used herein, the term "alkylene" refers to a divalent linear or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms and containing no unsaturation. As used herein, the term "C1-C6 alkylene" refers to a divalent linear or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms and containing no unsaturation, and having 1 to 6 carbon atoms. Non-limiting examples of the "C1-C6 alkylene" group include methylene (C1 alkylene), ethylene (C2 alkylene), 1-methylethylene (C3 alkylene), n-propylene (C3 alkylene), isopropylene (C3 alkylene), n-butylene (C4 alkylene), isobutylene (C4 alkylene), sec-butylene (C4 alkylene), tert-butylene (C4 alkylene), n-pentylene (C5 alkylene), isopentylene (C5 alkylene), neopentylene (C5 alkylene), and hexylene (C6 alkylene).

[0045] As used herein, the term "alkenylene" refers to a divalent linear or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms and containing at least one double bond. As used herein, the term "C2-C6 alkenylene" refers to a divalent linear or branched hydrocarbon chain radical group consisting only of carbon and hydrogen atoms, containing at least one double bond and having 2 to 6 carbon atoms. Non-limiting examples of the "C2-C6 alkenylene" group include etenylene (C2 alkenylene), propa-1-enylene (C3 alkenylene), buta-1-enylene (C4 alkenylene), penta-1-enylene (C5 alkenylene), penta-4-enylene (C5 alkenylene), penta-1,4-dienylene (C5 alkenylene), hexa-1-enylene (C6 alkenylene), hexa-2-enylene (C6 alkenylene), hexa-3-enylene (C6 alkenylene), hexa-1,4-dienylene (C6 alkenylene), hexa-1,5-dienylene (C6 alkenylene), and hexa-2,4-dienylene (C6 alkenylene). As used herein, the term "C2-C6 alkenylene" refers to a radical group of a divalent linear or branched hydrocarbon chain consisting only of carbon and hydrogen atoms, containing at least one double bond, and having 2-3 carbon atoms. Non-limiting examples of "C2-C3 alkenylene" groups include ethenylene (C2 alkenylene) and propa-1-enylene (C3 alkenylene).

[0046] As used herein, the term “alkoxy” refers to -O-alkyl or -alkyl-O-, and the “alkyl” group is as defined herein. In certain embodiments, the alkoxy group may be “C1-C2 alkoxy”, “C1-C3 alkoxy”, “C1-C4 alkoxy”, “C1-C5 alkoxy”, “C1-C6 alkoxy”, “C1-C7 alkoxy”, “C1-C8 alkoxy”, “C1-C9 alkoxy”, or “C1-C 10 The term is "alkoxy," and the terms are "C1-C3 alkoxy," "C1-C4 alkoxy," "C1-C5 alkoxy," "C1-C6 alkoxy," "C1-C7 alkoxy," "C1-C8 alkoxy," "C1-C9 alkoxy," and "C1-C10 "Alkoxy", as used herein, refers to -O-C1-C2 alkyl, -O-C1-C3 alkyl, -O-C1-C4 alkyl, -O-C1-C5 alkyl, -O-C1-C6 alkyl, -O-C1-C7 alkyl, -O-C1-C8 alkyl, -O-C1-C9 alkyl or -O-C1-C 10 alkyl. Non-limiting examples of "alkoxy" groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, isopentoxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, and the like.

[0047] As used herein, the term "aryl" refers to an aromatic monocyclic ring system having 6 carbon atoms as ring members, an aromatic fused bicyclic ring system having 9-10 carbon atoms as ring members or an aromatic fused tricyclic ring system having 14 carbon atoms as ring members. Non-limiting examples of aryl groups include phenyl, naphthalenyl, fluorenyl, indenyl, azulenyl, anthracenyl, phenanthrenyl, etc. as used herein. In certain embodiments, such aryl groups are optionally substituted. In preferred embodiments, the aryl group is phenyl.

[0048] As used herein, the term "cycloalkyl" or "C3-C8 cycloalkyl" refers to a saturated, monocyclic, fused bicyclic, fused tricyclic or bridged polycyclic ring system. Non-limiting examples of fused bicyclic or bridged polycyclic ring systems include bicyclo[1.1.1]pentane, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, bicyclo[3.2.1]octane, bicyclo[2.2.2]octane and adamantanyl. Non-limiting examples of monocyclic C3-C8 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl groups.

[0049] As used herein, the term “haloalkyl” means an alkyl group as defined herein, wherein at least one hydrogen atom of the alkyl group is replaced by a halo group as defined herein. A haloalkyl group may be a monohaloalkyl group, a dihaloalkyl group, a trihaloalkyl group, or a polyhaloalkyl group, including a perhaloalkyl group. A monohaloalkyl group may have one iodine, bromo, chloro, or fluoro group within the alkyl group. Dihaloalkyl groups and polyhaloalkyl groups may have two or more identical halo atoms or combinations of different halo groups within the alkyl group. Typically, a polyhaloalkyl group contains up to six, or four, or three, or two halo groups. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. Perhaloalkyl refers to an alkyl group in which all hydrogen atoms are replaced by halo atoms, such as trifluoromethyl. Typical haloalkyl groups, unless otherwise specified, include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl, which have at least one hydrogen atom substituted with a halogen, for example, fluorine and are: CF3CF2-, (CF3)2CH-, CH3-CF2-, CF3CF2-, CF3, CF2H-, CF3CF2CH(CF3)-, or CF3CF2CF2CF2-.

[0050] As used herein, the term “C1-C6 haloalkyl” refers to each “C1-C6 alkyl” as defined herein, wherein at least one hydrogen atom of the “C1-C6 alkyl” is replaced by a halo atom. A C1-C6 haloalkyl can be a mono-C1-C6 haloalkyl, such a C1-C6 haloalkyl having one iodine, one bromo, one chloro, or one fluoro. Furthermore, a C1-C6 haloalkyl can be a di-C1-C6 haloalkyl, such a C1-C6 haloalkyl may have two halo atoms independently selected from iodine, bromo, chloro, or fluoro. Furthermore, a C1-C6 haloalkyl can be a poly-C1-C6 haloalkyl, such a C1-C6 haloalkyl may have two or more identical halo atoms or a combination of two or more different halo atoms. Such polyC1-C6 haloalkyls may be perhalo-C1-C6 haloalkyls, in which all hydrogen atoms of each C1-C6 alkyl group are replaced by halo atoms, and the halo atoms may be the same or a combination of different halo atoms. Non-limiting examples of the "C1-C6 haloalkyl" group include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, fluoroethyl, difluoroethyl, trifluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl.

[0051] As used herein, the term “haloalkoxy” means an alkoxy as defined herein, wherein at least one hydrogen atom of the alkyl group is replaced by a halo group as defined herein. Haloalkyls may be polyhaloalkoxys, including monohaloalkoxys, dihaloalkoxys, trihaloalkoxys, or perhaloalkoxys. Monohaloalkoxys may have one iodine, bromo, chloro, or fluoro group within the alkyl group. Dihaloalkoxys and polyhaloalkoxy groups may have two or more identical halo atoms or combinations of different halo groups within the alkyl group. Typically, polyhaloalkoxys contain up to six, or four, or three, or two halo groups. Non-limiting examples of haloalkoxys include fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, pentafluoroethoxy, heptafluoropropoxy, difluorochloromethoxy, dichlorofluoromethoxy, difluoroethoxy, difluoropropoxy, dichloroethoxy, and dichloropropoxy. Perhalo-alkyloxy refers to an alkoxy in which all hydrogen atoms are replaced by halo atoms, such as trifluoromethoxy. Typical haloalkoxy groups, unless otherwise specified, include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, and tert-butoxy, which have at least one hydrogen substituted with a halogen, for example, fluorine and are: CF3CF2O-, (CF3)2CHO-, CH3-CF2O-, CF3CF2O-, -OCF3, -OCF2-, CF3CF2CH(CF3)O-, or CF3CF2CF2CF2O-.

[0052] As used herein, the term “C1-C6 haloalkoxy” refers to each “C1-C6 alkoxy” as defined herein, wherein at least one hydrogen atom of the “C1-C6 alkyl” is replaced by a halo atom. A C1-C6 haloalkoxy group may be mono-C1-C6 haloalkoxy, such a C1-C6 haloalkoxy group having one iodine, one bromo, one chloro, or one fluoro. Furthermore, a C1-C6 haloalkoxy group may be di-C1-C6 haloalkoxy, such a C1-C6 haloalkoxy group may have two halo atoms independently selected from iodine, bromo, chloro, or fluoro. Furthermore, a C1-C6 haloalkoxy group may be poly-C1-C6 haloalkoxy, such a C1-C6 haloalkoxy group may have two or more identical halo atoms or a combination of two or more different halo atoms. Such polyC1-C6 haloalkoxys may be perhalo-C1-C6 haloalkoxys, in which all hydrogen atoms of each C1-C6 alkyl group are replaced by halo atoms, and the halo atoms may be the same or a combination of different halo atoms. Non-limiting examples of "C1-C6 haloalkoxy" groups include fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, pentafluoroethoxy, heptafluoropropoxy, difluorochloromethoxy, dichlorofluoromethoxy, fluoroethoxy, difluoroethoxy, trifluoroethoxy, difluoropropoxy, dichloroethoxy, and dichloropropoxy.

[0053] As used herein, the term "halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromo (Br), or iodine (I).

[0054] As used herein, the term “heteroaryl” refers to an aromatic ring system containing one or more heteroatoms. A heteroaryl group containing two or more heteroatoms may contain different heteroatoms. A heteroaryl group may be optionally substituted with one or more substitutions as defined in formula (I). A heteroaryl group may be a monocyclic ring system or a fused bicyclic ring system. A monocyclic heteroaryl ring has 5 to 6 ring atoms. A bicyclic heteroaryl ring has 8 to 10 ring atoms. Bicyclic heteroaryl rings include these ring systems, in which the heteroaryl ring is fused to a phenyl ring. When used herein, non-limiting examples of heteroaryl groups include benzofuranyl, benzo[c]thiophenyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, cinnolinyl, flazanyl, furyl, imidazolyl, indolyl, indolidinyl, indazolyl, isoindolyl, isoquinolinyl, isoxazolyl, isothiazolyl, oxazolyl, oxaindolyl, oxadiazolyl (1,3,4-oxa Examples include diazolyl and 1,2,4-oxadiazolyl), prinyl, pyrazolyl, pyrrolyl, phthalazinyl, pyridinyl (including 2-, 3- and 4-pyridinyl), pyridadinyl, pyrazinyl, pyrimidinyl, quinoxalinyl, quinolinyl, quinazolinyl, tetradinyl, tetrazolyl, tetrazolo[1,5-a]pyridinyl, thiazolyl, thiadiazolyl (including 1,3,4-thiadiazolyl), thienyl, triazinyl, and triazolyl.

[0055] The term "5 or 6-membered heteroaryl containing 1 to 4 heteroatoms selected from N, O, and S" refers to an aromatic 5- to 6-membered monocyclic ring system having 1 to 4 heteroatoms independently selected from heteroatoms N, O, and S as ring members.

[0056] The term "5 or 6-membered heteroaryl containing 1 to 3 heteroatoms selected from N, O, and S" refers to an aromatic 5- to 6-membered monocyclic ring system having 1 to 3 heteroatoms independently selected from heteroatoms N, O, and S as ring members.

[0057] As used herein, the term "heteroatom" refers to a nitrogen, oxygen, or sulfur atom.

[0058] As used herein, the term "heterocycloalkyl" means N, NH, N 12 , O or S (where R 12 NH refers to a cycloalkyl group as defined herein, having one or two carbon atoms in a ring structure that is replaced by one or two groups independently selected from H or C1-C6 alkyl groups. When used herein, the terms N, NH, NR 12 A "4-6 membered heterocycloalkyl having 1-2 ring members independently selected from O or S" refers to a 4-6 membered heterocycloalkyl having a fully saturated monocyclic hydrocarbon ring structure with 4-6 ring members, where 1-2 of the ring members are independently N, NH, or NR. 12 Selected from O or -S-, R 12The is H or C1-C6 alkyl. When used herein, non-limiting examples of heterocycloalkyl groups include azetadinyl, azetadin-1-yl, azetadin-2-yl, azetadin-3-yl, oxetanyl, oxetan-2-yl, oxetan-3-yl, oxetan-4-yl, thietanyl, thietan-2-yl, thietan-3-yl, thietan-4-yl, pyrrolidinyl, pyrrolidine-1-yl, pyrrolidine-2-yl, pyrrolidine-3-yl, pyrrolidine-4-yl, pyrrolidine-5-yl, tetrahydrofuranyl, tetrahydrofuran-2-yl, tetrahydrofuranyl Dilofuran-3-yl, tetrahydrofuran-4-yl, tetrahydrofuran-5-yl, tetrahydrothienyl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, tetrahydrothien-4-yl, tetrahydrothien-5-yl, piperidinyl, piperidine-1-yl, piperidine-2-yl, piperidine-3-yl, piperidine-4-yl, piperidine-5-yl, piperidine-6-yl, tetrahydropyranyl, tetrahydropyran-2-yl, tetrahydropyran-3-yl, tetrahydropyran-4-yl, tetra Hydropyran-5-yl, tetrahydropyran-6-yl, tetrahydrothiopyranil, tetrahydrothiopyran-2-yl, tetrahydrothiopyran-3-yl, tetrahydrothiopyran-4-yl, tetrahydrothiopyran-5-yl, tetrahydrothiopyran-6-yl, piperazinyl, piperazine-1-yl, piperazine-2-yl, piperazine-3-yl, piperazine-4-yl, piperazine-5-yl, piperazine-6-yl, morpholinyl, morpholin-2-yl, morpholin-3-yl, morpholin-4-yl, morpholin -5-yl, morpholin-6-yl, thiomorpholinyl, thiomorpholin-2-yl, thiomorpholin-3-yl, thiomorpholin-4-yl, thiomorpholin-5-yl, thiomorpholin-6-yl, oxatianyl, oxatian-2-yl, oxatian-3-yl, oxatian-5-yl, oxatian-6-yl, dithianyl, dithian-2-yl, dithian-3-yl, dithian-5-yl, dithian-6-yl, dioxolanyl, dioxolan-2-yl, dioxolan-4-yl, dioxolan-5-yl, thioxanyl,Examples include thioxan-2-yl, thioxan-3-yl, thioxan-4-yl, thioxan-5-yl, dithiolanyl, dithiolan-2-yl, dithiolan-4-yl, dithiolan-5-yl, pyrazolidinyl, pyrazolidine-1-yl, pyrazolidine-2-yl, pyrazolidine-3-yl, pyrazolidine-4-yl, and pyrazolidine-5-yl.

[0059] As used herein, the term "heterocyclyl" refers to a saturated (e.g., heterocycloalkyl ring) or partially unsaturated monocyclic or polycyclic ring containing carbon and at least one heteroatom selected from oxygen, nitrogen, or sulfur (O, N, or S), without delocalized n electrons (aromaticity) shared between ring carbons or heteroatoms.

[0060] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with one or more -OH groups. Examples of hydroxyalkyl groups include HO-CH2-, HO-CH2CH2-, and CH2-CH(OH)-.

[0061] As used herein, the terms “spirocycloalkyl” or “spirocyclyl” refer to a bicyclic ring system derived from carbon having both rings linked by a single atom. The rings may differ in size and properties, or may be identical in size and properties. Examples include spiropentane, spirohexane, spiroheptane, spirooctane, spirononane, or spirodecane. One or both rings in a spiro ring may be condensed to another carbocyclic, heterocyclic, aromatic, or heteroaromatic ring. (C3~C 12 Spirocycloalkyls are spiro rings containing 3 to 12 carbon atoms.

[0062] As used herein, the term “spiroheterocycloalkyl” or “spiroheterocyclyl” means a spiro ring in which at least one of the rings is a heterocycle (one or more carbon atoms may be substituted with heteroatoms (for example, one or more carbon atoms may be substituted with heteroatoms in at least one of the rings)). One or both of the rings in a spiroheterocycle may be condensed with another carbocyclic, heterocycle, aromatic ring, or heteroaromatic ring.

[0063] As used herein, the term “therapeutic plasmapheresis in the body” refers to the removal of unwanted extracellular target molecules from plasma in the body. Examples of such extracellular target molecules include growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, and cellular and cell membrane proteins. Specific examples of such target molecules include LDL(ApoB), Lp(a), ApoCIII, ANGPTL3, ANGPTL4, ANGPTL8, factor 11, GDF15, LPL, PCSK9, IL1β, IL17, complement factor B, complement factor D, MPO, IgE, IL7, IL12A, IL23, TNFA, CXCR4, MAPT, FHR3, TIMP1, Apelin, BMP6, BMP9 / GDF2, CSF-1, EPO, IL5, and MFGE. Examples of antibodies include, but are not limited to, 8, TSLP, TSP, C5, CXCL10, FGF23, IGF1, IL10, IL13, IL2, IL6, VEGFA, NKG2D, ZNFR3, ADA2, suPAR, TGF-β1, IL4 receptor, s-Tall receptor, histamine, tau, progranulin, alpha-synuclein, toxins, venom, HBV soluble antigen, viral antigen, prion protein, scFV, AAV, and anti-AAV antibodies.

[0064] As used herein, the terms "polyethylene glycol" or "PEG" refer to a linear, branched, or star-shaped structure composed of (OCH2CH2) groups. In certain embodiments, the polyethylene or PEG group is -(OCH2CH2) t*-, where t is 4-40, "-" indicates the end directed toward the self-sacrificing spacer, "*-" indicates the bond point to the terminal group R', where R' is OH, OCH3, or OCH2CH2C(=O)OH. In other embodiments, the polyethylene or PEG group is -(CH2CH2O) t *-, where t is 4 to 40, "-" indicates the end directed toward the self-sacrificing spacer, "*-" indicates the bond point to the terminal group R'', and R'' is H, CH3, or CH2CH2C(=O)OH.

[0065] As used herein, the term "polyalkylene glycol" means (O(CH2) m ) t This refers to a linear, branched, or star-shaped structure composed of groups. In certain embodiments, the polyethylene or PEG group is -(O(CH2) m ) t *-, where m is 1 to 10, t is 4 to 40, "-" indicates the end directed toward the self-sacrificing spacer, "*-" indicates the bond point to the terminal group R', where R' is OH, OCH3 or OCH2CH2C(=O)OH. In other embodiments, the polyethylene or PEG group is -((CH2) m O) t *-, where m is 1 to 10, t is 4 to 40, "-" indicates the end directed toward the self-sacrificing spacer, "*-" indicates the bond point to the terminal group R'', and R'' is H, CH3, or CH2CH2C(=O)OH.

[0066] As used herein, the term “extracellular” refers to the space outside the cell membrane of one or more cells.

[0067] As used herein, the terms “reduction in extracellular levels” or “reduction in multiple extracellular levels” refer to reducing or decreasing the concentration of a target molecule located in the space outside the cell membrane of one or more cells.

[0068] The terms "PCSK9," "hPCSK9," or "proprotein convertase subtilisin / kexin type 9" interchangeably refer to naturally occurring human proprotein convertases belonging to the proteinase K subfamily of the secreted subtilase family. PCSK9 is thought to be synthesized as a soluble enzyme precursor that undergoes autocatalytic intermolecular processing in the endoplasmic reticulum and functions as a proprotein convertase. PCSK9 plays a role in cholesterol homeostasis and may play a role in cortical neuron differentiation. Mutations in the PCSK9 gene are the cause of autosomal dominant familial hypercholesterolemia (Burnett and Hooper, Clin. Biochem. Rev. (2008) 29(1):11-26).

[0069] As used herein, the terms “PCSK9-mediated disease or disorder” or “PCSK9-associated disease or disorder” mean a disease or disorder associated with PCSK9 activity, and include hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral vascular disease (including aortic and cerebrovascular diseases), peripheral artery disease, vasculitis, elevated Lp(a), elevated LDL, elevated TRL, elevated triglycerides, sepsis, and xanthomas.

[0070] The terms "hypercholesterolemia" or "dyslipidemia" include, for example, familial and non-familial hypercholesterolemia. Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by elevated serum cholesterol that binds to low-density lipoprotein (LDL). Familial hypercholesterolemia includes both heterozygous and homozygous FH. Hypercholesterolemia (or dyslipidemia) is the presence of high levels of cholesterol in the blood. This can take the forms of hyperlipidemia (elevated levels of lipids in the blood) and hyperlipoproteinemia (elevated levels of lipoproteins in the blood).

[0071] Hyperlipidemia is characterized by elevated levels of lipids in the bloodstream. These lipids include cholesterol, cholesterol esters, phospholipids, and triglycerides. Hyperlipidemia includes, for example, types I, IIa, IIb, III, IV, and V.

[0072] Hypertriglyceridemia is characterized by high levels of triglycerides in the blood. Even in the absence of hypercholesterolemia, elevated triglyceride levels are associated with atherosclerosis and increase the risk of cardiovascular disease.

[0073] Sitosterolemia, or phytosterolemia, is a rare autosomal recessive lipid metabolism disorder characterized by excessive absorption of sitosterol from the gastrointestinal tract, decreased biliary excretion of dietary sterols (leading to hypercholesterolemia, tendon and nodular xanthomas, and early onset of atherosclerosis), and altered cholesterol synthesis.

[0074] Atherosclerosis is a condition characterized by the hardening of arteries, associated with the accumulation of fatty substances, cholesterol, cellular waste products, calcium, and fibrin in the inner lining of arteries. The resulting accumulation is called plaque.

[0075] Atherosclerosis, or atherosclerotic vascular disease (ASVD), is a specific form of arteriosclerosis characterized by thickening, hardening, and loss of elasticity of the arterial walls as a result of the invasion and accumulation of leukocytes, including cholesterol and triglycerides, and containing both living, active leukocytes (inflammation-generating) and dead cell remnants. Therefore, atherosclerosis is a syndrome affecting arterial blood vessels due to a chronic inflammatory response of leukocytes in the arterial walls.

[0076] Coronary heart disease, also known as atherosclerotic cardiovascular disease, coronary heart disease, or ischemic heart disease, is the most common type of heart disease and a cause of heart attacks. This disease is caused by plaque accumulating along the inner walls of the arteries of the heart, narrowing the lumen of the arteries and reducing blood flow to the heart.

[0077] Xanthomas are a skin manifestation of lipidosis, in which lipids accumulate in large foam cells within the skin. Xanthomas are associated with hyperlipidemia.

[0078] The term "elevated Lp(a) concentration," as used herein, refers to a serum Lp(a) concentration above 30 mg / dL (75 nmol / L). "Elevated serum Lp(a)" means a serum Lp(a) level above approximately 14 mg / dL. In certain embodiments, a patient is considered to have elevated serum Lp(a) if the serum Lp(a) level measured in the patient is higher than approximately 15 mg / dL, 20 mg / dL, 25 mg / dL, 30 mg / dL, 35 mg / dL, 40 mg / dL, 45 mg / dL, 50 mg / dL, 60 mg / dL, 70 mg / dL, 80 mg / dL, 90 mg / dL, 100 mg / dL, 20 mg / dL, 140 mg / dL, 150 mg / dL, 180 mg / dL, or 200 mg / dL. Serum Lp(a) levels may be measured in the patient after a meal. In some embodiments, Lp(a) levels are measured after a fasting period (e.g., after 8 hours, 8 hours, 10 hours, 12 hours, or longer). While any clinically acceptable diagnostic method may be used in connection with this disclosure, representative methods for measuring serum Lp(a) in patients include, but are not limited to, immunoturbidimetric analysis, ELISA, turbidimetric analysis, immunoturbidimetry, and dissociation-enhanced lantanide fluorescence immunoassays.

[0079] "Elevated triglyceride levels" or "ETL" means any level of triglycerides that is deemed undesirable or targeted for adjustment.

[0080] Sepsis is a systemic reaction characterized by arterial hypotension, metabolic acidosis, decreased systemic vascular resistance, tachypnea, and organ dysfunction. Sepsis can be caused by septismemia (i.e., organisms in the bloodstream, their metabolic end products, or toxins), including bacteremia (i.e., bacteria in the blood) and toxemia (i.e., toxins in the blood), including endotoxemia (i.e., endotoxins in the blood). The term sepsis also encompasses fungemia (i.e., fungi in the blood), viremia (i.e., viruses or viral particles in the blood), and parasitemia (i.e., parasitic or protozoan parasites in the blood). Therefore, sepsis and septic shock (acute circulatory failure due to sepsis, often accompanied by multiple organ failure and a high mortality rate) can be caused by many organisms.

[0081] The terms "CFHR3" or "complement factor H-related protein 3 gene" are interchangeable and refer to the gene that codes for human protein complement factor H-related protein 3 (FHR3).

[0082] The term "FHR3" or "complement factor H-related protein 3" is interchangeable and refers to the naturally occurring human complement factor H-related protein 3, a secreted protein belonging to the complement factor H-related protein family.

[0083] As used herein, the terms “CFHR3-mediated disease or disorder” or “CFHR3-related disease or disorder” refer to diseases or disorders associated with the non-adhesion activity of CFHR3, and include nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0084] As used herein, the terms “FHR3-mediated disease or disorder” or “FHR3-associated disease or disorder” refer to diseases or disorders associated with FHR3 activity, including nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0085] As used herein, the term "nephropathy" refers to a disease or disorder of the kidneys.

[0086] As used herein, the term “age-related macular degeneration” refers to an eye disease that affects patches of the eye that cause blindness over time.

[0087] As used herein, the term “atypical hemolytic uremic syndrome” refers to a disease that impairs kidney function due to abnormal blood clotting in the kidneys. Atypical hemolytic uremic syndrome is characterized by three main features related to abnormal clotting: hemolytic anemia, thrombocytopenia, and renal failure.

[0088] As used herein, the term "hemolytic anemia" refers to the premature breakdown of red blood cells.

[0089] As used herein, the term “thrombocytopenia” refers to a decrease in the level of circulating platelets, which are used to aid in clotting.

[0090] As used herein, the term “hepatocellular carcinoma (HCC)” refers to cancer of the liver.

[0091] As used herein, the term “reactive group” refers to a functional group capable of forming a covalent bond with a functional group of an antibody or antibody fragment. Non-limiting examples of such functional groups include the reactive groups provided herein in Table 1.

[0092] As used herein, the term “coupling group” refers to a divalent moiety that links a crosslinking spacer to an antibody or fragment thereof. A coupling group is a divalent moiety formed on an antibody or fragment thereof by the reaction of a reactive group with a functional group. Non-limiting examples of such divalent moieties include the divalent chemical moieties shown in Tables 1 and 2 provided herein.

[0093] When used in this specification, if a substructure of a compound is shown in the illustration, it will be indicated by a dashed line. [ka] This indicates the bonding point of the substructure to the rest of the molecule.

[0094] As used herein, the term “composition” or “pharmaceutical composition” means a mixture of the compound of the present invention with at least one, optionally two or more other pharmaceutically acceptable chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients.

[0095] As used herein, the terms “optical isomer” or “stereoisomer” refer to any of the various stereoisomer configurations that may exist with respect to a given compound of the present invention, including geometric isomers. Substituents are understood to be bonded at the chiral center of a carbon atom. The term “chiral” refers to a molecule that has the property of not being superimposed on its enantiomer partner, while the term “achiral” refers to a molecule that can be superimposed on its enantiomer partner. Accordingly, the present invention includes enantiomers, diastereomers, or racemates of a compound. An “enantiomer” is a pair of stereoisomers that are enantiomers of each other and cannot be superimposed on each other. A 1:1 mixture of a pair of enantiomers is a “racemic” mixture. This term is used, as necessary, to refer to a racemic mixture. A “diastereoisomer” is a stereoisomer that has at least two chiral atoms but is not enantiomer of each other. Absolute stereochemistry is specified according to the Cahn-Ingold-Prelog RS system. When a compound is a pure enantiomer, the stereochemistry at each chiral carbon may be designated as either R or S. Divided compounds whose absolute configuration is unknown may be indicated by (+) or (-) depending on the direction of rotation of plane polarization at the wavelength of the sodium D line (dextrorotatory or levorotatory). Certain compounds described herein contain one or more chiral centers or axes and may therefore give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined as (R)- or (S)- with respect to absolute stereochemistry.

[0096] As used herein, the term “pharmaceutically acceptable carrier” includes, as is well known to those skilled in the art, any solvent, dispersion medium, coating, surfactant, antioxidant, preservative (e.g., antibacterial, antifungal), isotonic agent, absorption retarder, salt, preservative, drug stabilizer, binder, excipient, disintegrant, lubricant, sweetener, flavoring agent, colorant, etc., and combinations thereof (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289–1329). Use in therapeutic or pharmaceutical compositions is considered unless any conventional carrier is incompatible with the active ingredient.

[0097] As used herein, the term “subject” encompasses both mammals and non-mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, apes, monkeys, cattle, horses, sheep, goats, pigs; rabbits, dogs, cats, rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds and fish. In many cases, the subject is humans.

[0098] The term "individuals requiring such treatment" refers to individuals who would benefit from such treatment in terms of biological, medical, or quality of life.

[0099] As used herein, the terms “to treat,” “to treat,” or “to cure” any disease or disorder mean, in one embodiment, improving the disease or disorder (i.e., slowing, preventing, or reducing the development of at least one of the disease or its clinical symptoms). In another embodiment, “to treat,” “to treat,” or “to cure” means reducing or improving at least one physical parameter, including one that may not be identifiable by the patient. In yet another embodiment, “to treat,” “to treat,” or “to cure” means modulating the disease or disorder either or both physically (e.g., stabilization of identifiable symptoms) or physiologically (e.g., stabilization of physical parameters).

[0100] As used herein, the terms “prevent,” “prevent,” or “prevention” with respect to any disease or disorder mean preventive treatment of the disease or disorder or delaying the onset or progression of the disease or disorder.

[0101] The term “therapeutic effective dose” or “therapeutic effective amount” interchangeably refers to an amount sufficient to produce a desired outcome (i.e., reduction or inhibition of enzyme or protein activity, improvement of symptoms, alleviation of symptoms or conditions, delay of disease progression, reduction of tumor size, inhibition of tumor growth, prevention of metastasis, inhibition or prevention of viral, bacterial, fungal or parasitic infections). In some embodiments, the therapeutic effective dose does not induce or cause undesirable side effects. In some embodiments, the therapeutic effective dose induces or causes side effects, but only to an amount acceptable to the healthcare provider considering the patient’s condition. The therapeutic effective dose can be determined by administering a low dose first and then gradually increasing the dose until the desired effect is achieved. The “preventive effective dose” or “preventive effective dose” of the molecule of the present invention can prevent the onset of disease symptoms, including cancer-related symptoms. The “therapeutic effective dose” or “therapeutic effective dose” of the molecule of the present invention can result in a reduction in the severity of disease symptoms, including cancer-related symptoms.

[0102] The compound names provided herein were obtained using ChemBioDraw Ultra version 14.0.

[0103] The terms “a,” “an,” and “it,” as used herein, and similar terms used in connection with the present invention (particularly in connection with the claims), should be construed to encompass both singular and plural forms unless otherwise specifically indicated herein or unless it is clearly inconsistent with the context.

[0104] Unless otherwise specified, the terms “the difunctional compounds of the present invention,” “the plurality of difunctional compounds of the present invention,” “the difunctional compounds of the present invention,” or “the plurality of difunctional compounds of the present invention” refer to one or more difunctional compounds of formula (I), their subformulas (such as formula (Ia) and formula (Ib)), and the exemplary compounds and their salts, as well as all stereoisomers (including diastereoisomers and enantiomers), rotational isomers, tautomers, and isotopically labeled compounds (including deuterium substitutions).

[0105] All formulas provided herein are intended to represent both the unlabeled and isotopically labeled forms of the compounds. Isotope-labeled compounds have the structure described by the formulas provided herein, except in which one or more atoms are substituted by atoms having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen.

[0106] As used herein, the terms “polypeptide” and “peptide” are used interchangeably to refer to two or more amino acids linked together. Except for the abbreviations for rare or unnatural amino acids shown in Table A below and the abbreviations for protected amino acids shown in Table B below, three-letter or one-letter abbreviations accepted in the art are used to represent the amino acid residues constituting the peptides and polypeptides of this disclosure. If “D” precedes an amino acid, it is a D-amino acid. If “L” precedes an amino acid, it is an L-amino acid. If a one-letter abbreviation is capitalized, it refers to an L-amino acid. If a one-letter abbreviation is lowercase, it refers to a D-amino acid. Groups or columns of amino acid abbreviations are used to indicate peptides. Peptides are shown with the N-terminus to the left, and the sequence is written from the N-terminus to the C-terminus.

[0107] The cyclic peptides described herein include non-natural amino acids (i.e., compounds not found in nature) and other amino acid analogs as known in the art, which may be used as alternatives.

[0108] Those skilled in the art will recognize that various amino acid substitutions, such as conservative amino acid substitutions, can be made in any order of the cyclic polypeptides described herein without necessarily reducing their activity. As used herein, “amino acids commonly used as substituents” includes conservative substitutions (i.e., substitutions with amino acids having equivalent chemical characteristics). Examples of nonpolar (hydrophobic) amino acids for conservative substitutions include alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan, and methionine. Examples of polar (hydrophilic) and neutral amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Examples of positively charged (basic) amino acids include arginine, lysine, and histidine. Examples of positively charged (acidic) amino acids include aspartic acid and glutamic acid. Examples of amino acid substitutions include substituting an L-amino acid with its corresponding D-amino acid, substituting cysteine ​​with homocysteine ​​or another unnatural amino acid having a thiol-containing side chain, substituting lysine with homolysine, diaminobutyric acid, diaminopropionic acid, ornithine or another unnatural amino acid having an amino acid-containing side chain, or substituting alanine with norvaline.

[0109] As used herein, the term “amino acid” refers to naturally occurring amino acids, unnatural amino acids, amino acid analogs, and amino acid mimes that function in a similar manner to naturally occurring amino acids, all of which are their D and L stereoisomers, where their structure allows for such stereoisomers. In this specification, amino acids are referred to by their names, their commonly known three-letter abbreviations, the codes listed in Table A or Table B, or the one-letter abbreviations recommended by the IUPAC-IUB Biochemical Nomenclature Committee.

[0110] The term "naturally occurring" refers to a substance found in nature and not artificially manipulated. Similarly, "not naturally occurring," "unnatural," etc., as used herein, refer to a substance that is not found in nature or that has been artificially modified or synthesized. When used in relation to amino acids, the term “naturally occurring” refers to the 20 conventional amino acids (i.e., alanine (A or Ala), cysteine ​​(C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), and tyrosine (Y or Tyr)).

[0111] As used herein, the terms “unnatural amino acids” and “unnatural amino acids” are interchangeable to refer to amino acid structures, whether the same or different, that cannot be produced by biosynthesis in any organism using unmodified or modified genes from any organism. These include, but are not limited to, modified amino acids and / or amino acid analogs that are not one of the 20 natural amino acids, selenocysteine, pyrrolidine (Pyl), or pyrroline-carboxylysine (e.g., Pcl as described in International Publication No. 2010 / 48582).

[0112] Modified coding amino acids include hydroxyproline, γ-carboxyglutamic acid, O-phosphoserine, azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutanoic acid, 4-aminobutanoic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutanoic acid, 3-aminoisobutanoic acid, 2-aminopimeric acid, tertiary butylglycine, 2,4-diaminoisobutanoic acid, desmosine, 2,2'-diaminopimeric acid, and 2,3-diamino Examples of amino acids include, but are not limited to, nopropionic acid, N-ethylglycine, N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthoalanine, norvaline, norleucine, ornithine, pentylglycine, pipecolic acid, and thioproline. The term "amino acid" refers to metabolites in certain organisms, but also includes naturally occurring amino acids that are not coded by the genetic code for incorporation into proteins. Examples of such amino acids include, but are not limited to, ornithine, D-ornithine, and D-arginine.

[0113] In this specification, a peptide is defined as an organic compound containing two or more amino acids covalently linked by a peptide bond. A peptide may be referred to in terms of the number of constituent amino acids; that is, a dipeptide or dimer contains two amino acid residues, a tripeptide or trimer contains three, and so on. A peptide containing 10 or fewer amino acids may be called an oligopeptide, while one containing more than 10 amino acid residues is a polypeptide.

[0114] As used herein, the term "peptide" means two or more amino acids linked together via a peptide bond.

[0115] [Table 1]

[0116] [Table 2]

[0117] The bifunctional compound of the present invention The bifunctional compounds of the present invention target extracellular targets such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, cells, or cell membrane proteins (T L This is a compound that contains a part that binds to the target molecule ligand (T). L ) is a cell surface receptor (R L The cell surface receptor is linked to the portion that binds to ), and is associated with receptor-mediated endocytosis. The bifunctional compound of the present invention is given by formula (I): R L -L A -T L (I) (In the formula, R L This is the portion that binds to cell surface receptors associated with receptor-mediated endocytosis; L A is a linker; and T L (This is the part that binds to extracellular targets.) It has the structure of [the object].

[0118] Specific embodiments and examples of the bifunctional compounds of the present invention are provided in the enumerated embodiments provided herein. It will be recognized that the features expressed in each embodiment, in combination with other expressed features, may provide further embodiments of the present invention.

[0119] A. Target binding moiety (T L ) The target binding moiety (T) of the bifunctional compound of the present invention LThe bifunctional compound of the present invention is a portion that binds to extracellular target molecules such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, cells, or cell membrane proteins, and can be used to induce extracellular target molecules into lysosomes for degradation. Examples of such target molecules that can be induced for degradation using the bifunctional compound of the present invention include LDL(ApoB), Lp(a), ApoCIII, ANGPTL3, ANGPTL4, ANGPTL8, factor 11, GDF15, LPL, PCSK9, IL1β, IL17, complement factor B, complement factor D, MPO, IgE, IL7, IL12A, IL23, TNFA, CXCR4, MAPT, FHR3, TIMP1, Apelin, BMP6, BMP9 / GDF2, and CSF-1. Examples of such compounds include, but are not limited to, EPO, IL5, MFGE8, TSLP, TSP, C5, CXCL10, FGF23, IGF1, IL10, IL13, IL2, IL6, VEGFA, NKG2D, ZNFR3, ADA2, suPAR, TGF-β1, IL4 receptor, s-Tall receptor, histamine, tau, progranulin, alpha-synuclein, toxins, venom, HBV soluble antigen, viral antigen, prion protein, scFV, AAV, and anti-AAV antibodies. In certain embodiments, extracellular target molecules that can be induced for degradation using the bifunctional compounds of the present invention are PCSK9 and FHR3.

[0120] Embodiment 1. T L However, it is a bifunctional compound of formula (I), where the part that binds to PCSK9 or FHR3 is the part that binds to PCSK9 or FHR3.

[0121] Embodiment 2. T L However, the part that binds to PCSK9 is the bifunctional compound of formula (I).

[0122] Embodiment 3. Equation (Ia): R L -L A -PCSK9 L (Ia) (In the formula, RL This is the portion that binds to cell surface receptors associated with receptor-mediated endocytosis; L A is a linker; and PCSK9 L (This is the part that connects to PCSK9.) A bifunctional compound having the structure of formula (I), or one of embodiments 1 to 2.

[0123] Embodiment 4. Target binding moiety (T L ) a compound of formula (A) that binds to PCSK9, or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] (In the formula, L A1 teeth, [ka] (In the formula, L A1 ** is linker (L A This indicates the connection point to L A1 The * is L A1 (Indicates the bond site to the -C(=O)- group that is bonded.) Selected from; (aa) 2 (aa) is an amino acid residue selected from L-proline residues and D-proline residues. 2 The C-terminus is a bond point to the -NH- group; (aa) 3 (aa) is an amino acid residue selected from L-arginine residues, D-arginine residues, L-serine residues, D-serine residues, L-histidine residues, D-histidine residues, L-alanine residues, and D-alanine residues. 3 The C-terminus is (aa) 2 It is a connection point to; (aa) 4(aa) is an amino acid residue selected from L-aspartic acid residue, D-aspartic acid residue, L-asparagine residue, D-asparagine residue, L-glutamic acid residue, D-glutamic acid residue, L-lysine residue, D-lysine residue, L-glutamine residue, D-glutamine residue, L-proline residue, D-proline residue, L-alanine residue, D-alanine residue, L-(N-Me)glutamic acid residue and D-(N-Me)glutamic acid residue, and 4 The C-terminus is (aa) 3 It is a connection point to; (aa) 5 (aa) is an amino acid residue selected from L-(N-Me)alanine residues, D-(N-Me)alanine residues, L-(N-Me)glutamic acid residues, and D-(N-Me)glutamic acid residues. 5 The C-terminus is (aa) 4 It is a connection point to; (aa) 6 (aa) is an amino acid residue selected from L-(4-phenyl-phenylalanine)(Bip) residue, D-(4-phenyl-phenylalanine)(Bip) residue, L-(4-trifluoromethyl-phenylalanine) residue, D-(4-trifluoromethyl-phenylalanine) residue, L-(3,4-dichloro-phenylalanine) residue, D-(3,4-dichloro-phenylalanine) residue, L-(3-fluoro-phenylalanine) residue, D-(3-fluoro-phenylalanine) residue, L-(4-chloro-phenylalanine) residue, and D-(4-chloro-phenylalanine) residue. 6 The C-terminus is (aa) 5 It is a connection point to; (aa) 7 (aa) is an amino acid residue selected from L-(N-Me)alanine residues, D-(N-Me)alanine residues, L-(N-Me)phenylalanine residues, and D-(N-Me)phenylalanine residues. 7 The C-terminus is (aa) 6 It is a connection point to; (aa) 8(aa) is an amino acid residue selected from L-(4-phenyl-phenylalanine)(Bip) residue, D-(4-phenyl-phenylalanine)(Bip) residue, L-serine residue, D-serine residue, L-tyrosine residue, D-tyrosine residue, L-(4-trifluoromethyl-phenylalanine) residue, D-(4-trifluoromethyl-phenylalanine) residue, L-alanine residue, D-alanine residue, L-phenylalanine residue, D-phenylalanine residue, L-valine residue, and D-valine residue. 8 The C-terminus is (aa) 7 It is a connection point to; (aa) 9 (aa) is an amino acid residue selected from L-threonine residues and D-threonine residues. 9 The C-terminus is (aa) 8 It is a connection point to; (aa) 10 (aa) is an amino acid residue selected from L-threonine residues, D-threonine residues, L-serine residues, and D-serine residues. 10 The C-terminus is (aa) 9 It is a connection point to; (aa) 11 (aa) is an amino acid residue selected from L-serine residues, D-serine residues, L-aspartic acid residues, D-aspartic acid residues, L-asparagine residues, D-asparagine residues, L-proline residues, D-proline residues, L-alanine residues, D-alanine residues, L-homoserine residues, and D-homoserine residues. 11 The C-terminus is (aa) 10 It is a connection point to; (aa) 12 (aa) is an amino acid residue selected from L-valine residues, D-valine residues, L-glutamic acid residues, and D-glutamic acid residues. 12 The C-terminus is (aa) 11 It is a connection point to; and (aa) 13 (aa) is an amino acid residue selected from L-phenylalanine residues and D-phenylalanine residues. 13 The C-terminus is (aa) 12 (It is the connection point to) The bifunctional compound of formula (I) or any one of Embodiments 1 to 3.

[0124] Embodiment 5. The bifunctional compound of Embodiment 4, wherein (aa) 2 is an amino acid residue selected from an L-proline residue and a D-proline residue, and the C-terminus of (aa) 2 is a bonding point to an -NH- group.

[0125] Embodiment 6. The bifunctional compound of Embodiment 4 or Embodiment 5, wherein (aa) 2 is an L-proline residue, and the C-terminus of (aa) 2 is a bonding point to the -NH- group shown in formula (A).

[0126] Embodiment 7. The bifunctional compound of any one of Embodiments 4 to 6, wherein (aa) 3 is an amino acid residue selected from an L-arginine residue, a D-arginine residue, an L-serine residue, a D-serine residue, an L-histidine residue, a D-histidine residue, an L-alanine residue and a D-alanine residue, and the C-terminus of (aa) 3 is a bonding point to (aa) 2 The bifunctional compound of any one of Embodiments 4 to 7, wherein

[0127] Embodiment 8. is an amino acid residue selected from an L-arginine residue, an L-serine residue, an L-histidine residue and an L-alanine residue, and the C-terminus of (aa) (aa) 3 is a bonding point to (aa) 3 The bifunctional compound of any one of Embodiments 4 to 8, wherein 2 is a bonding point to (aa)

[0128] Embodiment 9. is an amino acid residue selected from an L-arginine residue, an L-serine residue, an L-histidine residue and an L-alanine residue, and the C-terminus of (aa) (aa)3 is an L-alanine residue, (aa) 3 The C-terminus of (aa) 2 is the bonding point to (aa), a bifunctional compound.

[0129] Embodiment 10. A bifunctional compound according to any one of Embodiments 4 to 9, wherein in the formula (aa) 4 is an amino acid residue selected from an L-aspartic acid residue, a D-aspartic acid residue, an L-asparagine residue, a D-asparagine residue, an L-glutamic acid residue, a D-glutamic acid residue, an L-lysine residue, a D-lysine residue, an L-glutamine residue, a D-glutamine residue, an L-proline residue, a D-proline residue, an L-alanine residue, a D-alanine residue, an L-(N-Me) glutamic acid residue, and a D-(N-Me) glutamic acid residue, (aa) 4 The C-terminus of (aa) 3 is the bonding point to (aa), a bifunctional compound.

[0130] Embodiment 11. A bifunctional compound according to any one of Embodiments 4 to 10, wherein in the formula (aa) 4 is an amino acid residue selected from an L-aspartic acid residue, an L-asparagine residue, an L-glutamic acid residue, an L-lysine residue, an L-glutamine residue, an L-proline residue, an L-alanine residue, and an L-(N-Me) glutamic acid residue, (aa) 4 The C-terminus of (aa) 3 is the bonding point to (aa), a bifunctional compound.

[0131] Embodiment 12. A bifunctional compound according to any one of Embodiments 4 to 11, wherein in the formula (aa) 4 is an L-glutamic acid residue, (aa) 4 The C-terminus of (aa) 3 is the bonding point to (aa), a bifunctional compound.

[0132] Embodiment 13. A bifunctional compound according to any one of Embodiments 4 to 12, wherein in the formula (aa) 5 However, the amino acid residue is selected from L-(N-Me)alanine residues, D-(N-Me)alanine residues, L-(N-Me)glutamic acid residues, and D-(N-Me)glutamic acid residues, (aa) 5 The C-terminus is (aa) 4 A bifunctional compound that serves as a binding site to [a specific component].

[0133] Embodiment 14. A bifunctional compound, one of embodiments 4 to 13, wherein the formula is: (aa) 5 However, it is an amino acid residue selected from L-(N-Me)alanine residues and L-(N-Me)glutamic acid residues, (aa) 5 The C-terminus is (aa) 4 A bifunctional compound that serves as a binding site to [a specific component].

[0134] Embodiment 15. A bifunctional compound from any one of Embodiments 4 to 14, wherein the formula is: (aa) 5 However, it is an L-(N-Me)alanine residue, (aa) 5 The C-terminus is (aa) 4 A bifunctional compound that serves as a binding site to [a specific component].

[0135] Embodiment 16. A bifunctional compound, one of the embodiments 4 to 15, wherein the formula is: (aa) 6 However, the amino acid residue is selected from L-(4-phenyl-phenylalanine)(Bip) residue, D-(4-phenyl-phenylalanine)(Bip) residue, L-(4-trifluoromethyl-phenylalanine) residue, D-(4-trifluoromethyl-phenylalanine) residue, L-(3,4-dichloro-phenylalanine) residue, D-(3,4-dichloro-phenylalanine) residue, L-(3-fluoro-phenylalanine) residue, D-(3-fluoro-phenylalanine) residue, L-(4-chloro-phenylalanine) residue, and D-(4-chloro-phenylalanine) residue, (aa) 6 The C-terminus is (aa) 5 A bifunctional compound that serves as a binding site to [a specific component].

[0136] Embodiment 17. A bifunctional compound from any one of Embodiments 4 to 16, wherein the formula is: (aa) 6 However, the amino acid residue is selected from L-(4-phenyl-phenylalanine)(Bip) residue, L-(4-trifluoromethyl-phenylalanine) residue, L-(3,4-dichloro-phenylalanine) residue, L-(3-fluoro-phenylalanine) residue, and L-(4-chloro-phenylalanine) residue, (aa) 6 The C-terminus is (aa) 5 A bifunctional compound that serves as a binding site to [a specific component].

[0137] Embodiment 18. A bifunctional compound from any one of Embodiments 4 to 17, wherein, (aa) 6 However, it is an L-(4-phenyl-phenylalanine)(Bip) residue, and (aa) 6 The C-terminus is (aa) 5 A bifunctional compound that serves as a binding site to [a specific component].

[0138] Embodiment 19. A bifunctional compound from any one of Embodiments 4 to 18, wherein the formula is: (aa) 7 However, the amino acid residue is selected from L-(N-Me)alanine residues, D-(N-Me)alanine residues, L-(N-Me)phenylalanine residues, and D-(N-Me)phenylalanine residues, (aa) 7 The C-terminus is (aa) 6 A bifunctional compound that serves as a binding site to [a specific component].

[0139] Embodiment 20. A bifunctional compound from any one of Embodiments 4 to 19, wherein the formula is: (aa) 7 However, it is an amino acid residue selected from L-(N-Me)alanine residues and L-(N-Me)phenylalanine residues, (aa) 7 The C-terminus is (aa) 6 A bifunctional compound that serves as a binding site to [a specific component].

[0140] Embodiment 21. A bifunctional compound from any one of Embodiments 4 to 20, wherein, (aa) 7 However, it is an L-(N-Me)alanine residue, (aa) 7 The C-terminus is (aa) 6 A bifunctional compound that serves as a binding site to [a specific component].

[0141] Embodiment 22. A bifunctional compound from any one of Embodiments 4 to 21, wherein the formula is: (aa) 8 However, the amino acid residue is selected from L-(4-phenyl-phenylalanine)(Bip) residues, D-(4-phenyl-phenylalanine)(Bip) residues, L-serine residues, D-serine residues, L-tyrosine residues, D-tyrosine residues, L-(4-trifluoromethyl-phenylalanine) residues, D-(4-trifluoromethyl-phenylalanine) residues, L-alanine residues, D-alanine residues, L-phenylalanine residues, D-phenylalanine residues, L-valine residues, and D-valine residues, (aa) 8 The C-terminus is (aa) 7 A bifunctional compound that serves as a binding site to [a specific component].

[0142] Embodiment 23. A bifunctional compound from any one of Embodiments 4 to 22, wherein, (aa) 8 However, the amino acid residue is selected from L-(4-phenyl-phenylalanine)(Bip) residues, L-serine residues, L-tyrosine residues, L-(4-trifluoromethyl-phenylalanine) residues, L-alanine residues, L-phenylalanine residues, and L-valine residues, (aa) 8 The C-terminus is (aa) 7 A bifunctional compound that serves as a binding site to [a specific component].

[0143] Embodiment 24. A bifunctional compound from any one of Embodiments 4 to 23, wherein the formula is: (aa) 8However, it is an L-(4-phenyl-phenylalanine)(Bip) residue, and (aa) 8 The C-terminus is (aa) 7 A bifunctional compound that serves as a binding site to [a specific component].

[0144] Embodiment 25. A bifunctional compound from any one of Embodiments 4 to 24, wherein the formula is: (aa) 9 However, it is an amino acid residue selected from L-threonine residues and D-threonine residues, (aa) 9 The C-terminus is (aa) 8 A bifunctional compound that serves as a binding site to [a specific component].

[0145] Embodiment 26. A bifunctional compound from any one of Embodiments 4 to 25, wherein the formula is: (aa) 9 However, it is an L-threonine residue, (aa) 9 The C-terminus is (aa) 8 A bifunctional compound that serves as a binding site to [a specific component].

[0146] Embodiment 27. A bifunctional compound from any one of Embodiments 4 to 26, wherein the formula is: (aa) 10 However, it is an amino acid residue selected from L-threonine residues, D-threonine residues, L-serine residues, and D-serine residues, (aa) 10 The C-terminus is (aa) 9 A bifunctional compound that serves as a binding site to [a specific component].

[0147] Embodiment 28. A bifunctional compound from any one of Embodiments 4 to 27, wherein, (aa) 10 However, it is an L-threonine residue, (aa) 10 The C-terminus is (aa) 9 A bifunctional compound that serves as a binding site to [a specific component].

[0148] Embodiment 29. A bifunctional compound of any one embodiment of Embodiments 4 to 28, wherein, (aa)11 However, the amino acid residue is selected from L-serine residues, D-serine residues, L-aspartic acid residues, D-aspartic acid residues, L-asparagine residues, D-asparagine residues, L-proline residues, D-proline residues, L-alanine residues, D-alanine residues, L-homoserine residues, and D-homoserine residues, (aa) 11 The C-terminus is (aa) 10 A bifunctional compound that serves as a binding site to [a specific component].

[0149] Embodiment 30. A bifunctional compound according to any one embodiment of Embodiments 4 to 29, wherein the formula is: (aa) 11 However, the amino acid residue is selected from L-serine residues, L-aspartic acid residues, L-asparagine residues, L-proline residues, L-alanine residues, and L-homoserine residues, (aa) 11 The C-terminus is (aa) 10 A bifunctional compound that serves as a binding site to [a specific component].

[0150] Embodiment 31. A bifunctional compound according to any one embodiment of Embodiments 4 to 30, wherein the formula is: (aa) 11 However, it is an L-proline residue, (aa) 11 The C-terminus is (aa) 10 A bifunctional compound that serves as a binding site to [a specific component].

[0151] Embodiment 32. A bifunctional compound from any one of Embodiments 4 to 31, wherein, (aa) 12 However, it is an amino acid residue selected from L-valine residues, D-valine residues, L-glutamic acid residues, and D-glutamic acid residues, (aa) 12 The C-terminus is (aa) 11 A bifunctional compound that serves as a binding site to [a specific component].

[0152] Embodiment 33. A bifunctional compound from any one of Embodiments 4 to 32, wherein the formula is: (aa) 12However, it is an amino acid residue selected from L-valine residues and L-glutamic acid residues, (aa) 12 The C-terminus is (aa) 11 A bifunctional compound that serves as a binding site to [a specific component].

[0153] Embodiment 34. A bifunctional compound from any one of Embodiments 4 to 33, wherein, (aa) 12 However, it is an L-valine residue, (aa) 12 The C-terminus is (aa) 11 A bifunctional compound that serves as a binding site to [a specific component].

[0154] Embodiment 35. A bifunctional compound from any one of Embodiments 4 to 34, wherein, (aa) 13 However, it is an amino acid residue selected from L-phenylalanine residues and D-phenylalanine residues, (aa) 13 The C-terminus is (aa) 12 A bifunctional compound that serves as a binding site to [a specific component].

[0155] Embodiment 36. A bifunctional compound from any one of Embodiments 4 to 35, wherein, (aa) 13 However, it is an L-phenylalanine residue, (aa) 13 The C-terminus is (aa) 12 A bifunctional compound that serves as a binding site to [a specific component].

[0156] Embodiment 37. The bifunctional compound of Embodiment 4, wherein T L However, a bifunctional compound which is a compound of formula (A) or a pharmaceutically acceptable salt or stereoisomer selected from the following:

[0157] [Table 3]

[0158] [Table 4]

[0159] Table 5

[0160] Table 6

[0161] Table 7

[0162] Table 8

[0163] Table 9

[0164] Table 10

[0165] Table 11

[0166] Table 12

[0167] Table 13

[0168] Table 14

[0169] [Table 15]

[0170] [Table 16]

[0171] [Table 17]

[0172] [Table 18]

[0173] [Table 19]

[0174] [Table 20]

[0175] During the ceremony, Ac is acetyl, and acetyl labeled with "*" and (L-Cys) labeled with "*" are linked via sulfide bonds formed through their side chains or terminals, and L A1 L is defined as described herein. A1 ** is linker (L A This indicates the connection point to ).

[0176] Embodiment 38. A bifunctional compound from any one of Embodiments 4 to 37, wherein L A1 but, [ka] (In the formula, L A1 ** is linker (L AThis indicates the connection point to L A1 (The asterisk indicates a bond site to a -C(=O)- group.) It is a bifunctional compound.

[0177] Embodiment 39. A bifunctional compound from any one of Embodiments 4 to 38, wherein L A1 but, [ka] (In the formula, L A1 ** is linker (L A This indicates the connection point to L A1 (The asterisk indicates a bond site to a -C(=O)- group.) It is a bifunctional compound.

[0178] Embodiment 40. A bifunctional compound from any one of Embodiments 4 to 38, wherein L A1 but, [ka] (In the formula, L A1 ** is linker (L A This indicates the connection point to L A1 (The asterisk indicates a bond site to a -C(=O)- group.) It is a bifunctional compound.

[0179] Embodiment 41. A bifunctional compound from any one of Embodiments 4 to 38, wherein T L However, the following: [ka] A bifunctional compound which is a compound of formula (A) selected from or a pharmaceutically acceptable salt or stereoisomer.

[0180] Embodiment 42. A bifunctional compound from any one of Embodiments 4 to 38, wherein T L but, [ka] It is a bifunctional compound.

[0181] Embodiment 43. A bifunctional compound of formula (I) or any one of the bifunctional compounds of embodiments 1 to 3, wherein T L However, compounds of formula (B) that bind to PCSK9, or their pharmaceutically acceptable salts or stereoisomers: [ka] (In the formula, X B1 is C or N; R B1 is H, (C1-C6) alkyl or (C1-C6) haloalkyl; or R B1 and R B11 Together with the atoms to which they are bonded, they form a 5-7 membered heterocyclyl ring containing 1-3 heteroatoms selected from N, O, and S, which are optionally substituted with one or more substituents independently selected from =(O), (C1-C6) alkyl, and (C1-C6) haloalkyl; R B2 (C1~C6)alkoxy, (C1~C6)alkyl, -L B1 -, (C1~C6)haloalkyl, (C1~C6)hydroxyalkyl, (C3~C7)cycloalkyl, or a 4-7 membered heterocyclyl containing 1-3 heteroatoms selected from N, O, and S, wherein the alkyl is optionally (C1~C6)alkoxy, (C1~C6)haloalkoxy, -C(=O)(C1~C6)alkyl, -C(=O)OH, -C(=O)O(C1~C6)alkyl, -OC(=O)(C1~C6)alkyl, -C(=O)NR B17 R B18 , -NR B17 C(=O)R B18 , (C6~C 10) Substituted with one or more substituents independently selected from aryls and 5- or 6-membered heteroaryls containing 1 to 4 heteroatoms selected from N, O, and S; R B3 is H, (C1-C6) alkyl, or (C1-C6) haloalkyl; R B4 is H, (C1-C6) alkyl, or (C1-C6) haloalkyl; R B5 is H, (C1-C6) alkyl, or (C1-C6) haloalkyl; R B6 H, (C1~C6) alkyl, -L B1 -, (C1~C6) haloalkyl or (C1~C6) hydroxyalkyl, wherein the alkyl is optionally (C1~C6) alkoxy, -C(=O)OH, -C(=O)O(C1~C6) alkyl, -NR B17 R B18 -C(=O)NR B17 R B18 , -NR B17 C(=O)R B18 , substituted with one or more substituents independently selected from (C3-C7) cycloalkyl groups and 4-7 membered heterocyclines containing 1-3 heteroatoms selected from N, O, and S; R B6’ H, (C1~C6) alkyl, -L B1 -, (C1~C6) haloalkyl or (C1~C6) hydroxyalkyl, wherein the alkyl is optionally (C1~C6) alkoxy, -C(=O)OH, -C(=O)O(C1~C6) alkyl, -NR B17 R B18 -C(=O)NR B17 R B18 , -NR B17 C(=O)R B18 , substituted with one or more substituents independently selected from (C3-C7) cycloalkyl groups and 4-7 membered heterocyclines containing 1-3 heteroatoms selected from N, O, and S; R B7 H, (C1~C6) alkyl, -L B1-, (C1~C6) haloalkyl or (C1~C6) hydroxyalkyl, wherein the alkyl is optionally (C1~C6) alkoxy, -C(=O)OH, -C(=O)O(C1~C6) alkyl, -NR B17 R B18 -C(=O)NR B17 R B18 , -NR B17 C(=O)R B18 , substituted with one or more substituents independently selected from (C3-C7) cycloalkyl groups and 4-7 membered heterocyclines containing 1-3 heteroatoms selected from N, O, and S; R B7’ H, (C1~C6) alkyl, -L B1 -, (C1~C6) haloalkyl or (C1~C6) hydroxyalkyl, wherein the alkyl is optionally (C1~C6) alkoxy, -C(=O)OH, -C(=O)O(C1~C6) alkyl, -NR B17 R B18 -C(=O)NR B17 R B18 , -NR B17 C(=O)R B18 , substituted with one or more substituents independently selected from (C3-C7) cycloalkyls and 4-7 membered heterocyclines containing 1-3 heteroatoms selected from N, O, and S; or R B6 and R B7 They, together with the carbon atoms to which they are bonded, form a 4-7 membered heterocyclyl ring containing (C3-C7) cycloalkyl or 1-3 heteroatoms selected from N, O, and S; or R B7 and R B7’ They, together with the carbon atoms to which they are bonded, form a 4-7 membered heterocyclyl ring containing (C3-C7) cycloalkyl or 1-3 heteroatoms selected from N, O, and S; or R B7 and R B9These, together with the atoms to which they are bonded, form a 5-7 membered heterocyclyl ring containing 1-3 heteroatoms selected from N, O, and S, which are optionally substituted with one or more substituents independently selected from (C1-C6) alkyl, (C1-C6) haloalkyl, and =(O); R B8 is H or (C1-C6) alkyl; R B9 The alkyl group is a 4-7 membered heterocyclyl containing 1-3 heteroatoms selected from H, (C1-C6) alkyl, (C2-C6) alkenyl, (C1-C6) haloalkyl, (C2-C6) haloalkenyl, (C1-C6) alkoxy, (C1-C6) haloalkoxy, (C1-C6) hydroxyalkyl, (C3-C7) cycloalkyl, or N, O, and S, wherein the alkyl group optionally contains one or more R B27 Replaced by; R B9’ The alkyl group is a 4-7 membered heterocyclyl containing 1-3 heteroatoms selected from H, (C1-C6) alkyl, (C2-C6) alkenyl, (C1-C6) haloalkyl, (C2-C6) haloalkenyl, (C1-C6) alkoxy, (C1-C6) haloalkoxy, (C1-C6) hydroxyalkyl, (C3-C7) cycloalkyl, or N, O, and S, wherein the alkyl group optionally contains one or more R B27 Replaced by; or R B9’ X B1 Does not exist when N; or R B9 and R B9’ They, together with the carbon atoms to which they are bonded, form a 4-7 membered heterocyclyl ring containing (C3-C7) cycloalkyl or 1-3 heteroatoms selected from N, O, and S; or R B7 and R B9 These, together with the atoms to which they are bonded, form a 5-7 membered heterocyclyl ring containing 1-3 heteroatoms selected from N, O, and S, which are optionally substituted with one or more substituents independently selected from (C1-C6) alkyl, (C1-C6) haloalkyl, and =(O); R B10(C6~C 10 ) A 5 or 6-membered heteroaryl, (C3-C7) cycloalkyl, or 4-7 membered heterocyclyl containing 1-3 heteroatoms selected from aryl, N, O, and S, wherein cycloalkyl, heterocyclyl, aryl, and heteroaryl are -OR B13 or -NR B23 R B13 Replaced by and optionally one or more R B14 Replaced by; R B11 is, -L B1 -, (C1~C6) alkyl, (C1~C6) haloalkyl, or (C1~C6) hydroxyalkyl, wherein the alkyl is optionally selected to have one or more R B15 Replaced by; or R B1 and R B11 Together with the atoms to which they are bonded, they form a 5-7 membered heterocyclyl ring containing 1-3 heteroatoms selected from N, O, and S, which are optionally substituted with one or more substituents independently selected from =(O), (C1-C6) alkyl, and (C1-C6) haloalkyl; R B12 These are halogens, (C1-C6) alkyls, (C1-C6) alkoxys, (C1-C6) haloalkyls, (C1-C6) haloalkoxys, -OH, or CN; R B13 (C6~C 10 ) A 5- or 6-membered heteroaryl containing aryl or 1 to 3 heteroatoms selected from N, O, and S, wherein the aryl and heteroaryl are R 16 Replaced by and optionally one or more R B16’ Replaced by; Each R B14 Independently in each presence, is a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, oxo, -OH, or CN; or R B10 When is a cycloalkyl or heterocyclyl, two R B14When bonded together to the same carbon atom, they form a single atom (O); Each R B15 These exist independently as (C1~C6)alkoxy, (C1~C6)haloalkoxy, and -C(=O)R in each respective entity. B19 , -S(O) q (C1~C6)alkyl, -C(=O)OH, -C(=O)O(C1~C6)alkyl, -OC(=O)(C1~C6)alkyl, -NR B17 R B18 -C(=O)NR B17 R B18 , -NR B17 C(=O)R B20 , -NR B17 C(=O)OR B18 , (C3~C7) cycloalkyl or 4~7 membered heterocycline containing 1~3 heteroatoms selected from N, O and S, wherein the cycloalkyl and heterocycline optionally contain one or more R B21 Replaced by; R B16 is -C(=O)NR B31 R B32 , (C6~C 10 ) A 5-7 membered heteroaryl containing aryl or 1-3 heteroatoms selected from N, O, and S, wherein the aryl and heteroaryl are optionally composed of one or more R B26 Replaced by; Each R B16’ Independently in each presence, is a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, or CN; or R B16 and R B16’ Together with the atoms to which they are bonded, they form (O) and R B34 Forms a 5-7 membered heterocyclyl ring optionally substituted with one or more substituents independently selected from; R B17 is a (C1-C6) alkyl group that is optionally substituted with one or more substituents independently selected from H or (C1-C6) alkoxy and -C(=O)O(C1-C6) alkyl groups; RB18 is a (C1-C6) alkyl group that is optionally substituted with one or more substituents independently selected from H or (C1-C6) alkoxy and -C(=O)O(C1-C6) alkyl groups; R B19 (C3~C7) cycloalkyl or one or more R B22 It is a 4-7 membered heterocycline containing 1-3 heteroatoms selected from N, O, and S, which are optionally substituted; R B20 is -(CH2CH2O) m CH2CH2ONH2, -(CH2CH2O) m CH2CH2ONH(C1~C6) alkyl or one or more -NR groups B23 C(=O)R B24 It is an alkyl group (C1-C6) that has been optionally substituted; Each R B21 Independently in each presence, these are (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, halogen, =(O), or -OH; Each R B22 Independently, in each presence, is (C1-C6)alkyl, (C1-C6)haloalkyl, halogen, or -OH; or Two R's 22 When they are on the same atom, together with the atom to which they are bonded, they form a 4-7 membered spiroheterocycline ring containing (C3-C7) spirocycloalkyl or 1-3 heteroatoms selected from N, O, and S; R B23 is H or (C1-C6) alkyl; R B24 is H or one or more R B25 It is an alkyl group (C1-C6) that has been optionally substituted; Each R B25Each is independently a 4-10 membered monocyclic or bicyclic heterocycline containing 1-4 heteroatoms selected from (C3-C7) cycloalkyl or N, O, and S, wherein the cycloalkyl or heterocycline is optionally substituted with one or more substituents independently selected from (C1-C6) alkyl, (C1-C6) haloalkyl, and =(O); Each R B26 Each entity independently has one or more R B29 (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C1-C6) alkoxy, (C1-C6) haloalkyl, (C1-C6) haloalkoxy, (C1-C6) hydroxyalkyl, -NR by optional substitution. B31 R B32 -C(=O)NR B31 R B32 , -C(=O)O(C1~C6)alkyl, (C3~C7)cycloalkyl, 4~7 membered heterocyclyl containing 1~3 heteroatoms selected from N, O and S, (C6~C 10 )A 5- or 6-membered heteroaryl containing aryl or 1 to 3 heteroatoms selected from N, O, and S, wherein the cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with one or more substituents independently selected from (C1-C6)alkyl, (C1-C6)haloalkyl, -NH2, -N(H)(C1-C6)alkyl, -N((C1-C6)alkyl)2, -N(H)(C1-C6)haloalkyl, -N((C1-C6)haloalkyl)2, halogen, and -OH; or Two R's B26 When they are on adjacent atoms, together with the atom to which they are bonded, they form a (C3-C7) cycloalkyl group or one or more R groups. B33 It forms a 4-7 membered heterocyclyl ring containing 1-3 heteroatoms selected from N, O, and S, which are optionally substituted; Each R B27 Each entity exists independently, with CN, (C6~C 10) A 5-7 membered heteroaryl, (C3-C7) cycloalkyl, or 4-7 membered heterocyclil containing 1-3 heteroatoms selected from aryl, N, O, and S, wherein the aryl, heteroaryl, cycloalkyl, and heterocyclil optionally contain one or more R B28 Replaced by; Each R B28 Independently, in each presence, are (C1-C6)alkyl, (C1-C6)haloalkyl, (C1-C6)alkoxy, (C1-C6)haloalkoxy, (C1-C6)hydroxyalkyl, halogen, oxo, or CN; or R B27 When is a cycloalkyl or heterocyclyl, two R B28 They, together with the atoms to which they are bonded, form a 4-7 membered heterocyclyl ring containing (C4-C7) cycloalkyl or 1-3 heteroatoms selected from N, O, and S; or R B27 When is a cycloalkyl or heterocyclyl, two R B28 When bonded together to the same carbon atom, they form a single atom (O); Each R B29 In each entity, -NR B31 R B32 or one or more R B30 It is a 4-7 membered heterocycline containing 1-3 heteroatoms selected from N, O, and S, which are optionally substituted; Each R B30 Independently, in each presence, is -OH, halogen, (C1-C6)alkyl or (C1-C6)haloalkyl; or Two R's B30 When they are on the same atom, together with the atom to which they are bonded, they form a 4-7 membered spiroheterocycline ring containing (C3-C7) spirocycloalkyl or 1-3 heteroatoms selected from N, O, and S; Each R B31The alkyl group is independently selected from a 4-7 membered heterocycline containing 1-3 heteroatoms selected from H, (C1-C6) alkyl, (C1-C6) hydroxyalkyl, (C3-C7) cycloalkyl, or N, O, and S, wherein the alkyl group is optionally substituted with one or more D atoms, and the cycloalkyl and heterocyclines are optionally substituted with one or more substituents independently selected from (C1-C6) alkyl, (C1-C6) haloalkyl, halogen, and -OH; Each R B32 The alkyl group is independently selected from a 4-7 membered heterocycline containing 1-3 heteroatoms selected from H, (C1-C6) alkyl, (C1-C6) hydroxyalkyl, (C3-C7) cycloalkyl, and N, O, and S, wherein the alkyl group is optionally substituted with one or more D atoms, and the cycloalkyl and heterocycline groups are optionally substituted with one or more substituents independently selected from (C1-C6) alkyl, (C1-C6) haloalkyl, halogen, and -OH; Each R B33 In each instance, is independently a (C1-C6)alkyl, a (C1-C6)haloalkyl, or -C(=O)R, where R is a (C1-C6)alkyl optionally substituted with one or more (C1-C6)alkoxys, or a 4-7 membered heterocyclyl containing 1-3 heteroatoms selected from (C1-C6)haloalkyl, (C3-C7)cycloalkyl, N, O, and S; or Two R's B33 When they are on the same atom, together with the atom to which they are bonded, they form a 4-7 membered spiroheterocycline ring containing (C3-C7) spirocycloalkyl or 1-3 heteroatoms selected from N, O, and S; Each R B34Each is independently a (C3-C7) cycloalkyl or a 4-10 member monocyclic or bicyclic heterocycline containing 1-4 heteroatoms selected from N, O, and S, wherein the cycloalkyl and heterocycline are optionally substituted with (C1-C6) alkyl groups, each optionally substituted with one or more substituents independently selected from the (C3-C7) cycloalkyl and the 4-10 member monocyclic or bicyclic heterocyclines containing 1-4 heteroatoms selected from N, O, and S; L B1 is, -(CH2) p It is NH-* and L B1 The * is a linker (L A The link point to R is shown, B11 , R B2 , R B6 or R B7 At least one of them is -L B1 -and; m is an integer selected from 1 to 13; n is 1, 2, 3, or 4; q is 0, 1 or 2, and p is 1, 2, 3, 4, 5, or 6. It is a bifunctional compound.

[0182] Embodiment 44. The bifunctional compound of Embodiment 43, wherein the formula is X B1 However, C is; R B1 However, H is; R B2 However, it is substituted with -C(=O)OH, (C1~C6)alkoxy, -L B1 - or (C1-C6) alkyl; R B3 However, it is H or (C1-C6) alkyl; R B4 However, it is H or (C1-C6) alkyl; R B5 However, it is H or (C1-C6) alkyl; R B6 However, H, (C1~C6) alkyl or -L B1 -and; R B6’ However, H is; R B7 However, H, (C1~C6) alkyl or -L B1 -and; R B7’ is H; or R B6 and R B7 However, together with the carbon atoms to which they are bonded, they form (C3-C7) cycloalkyl groups; R B8 However, it is H or (C1-C6) alkyl; R B9 However, H or one or more R B27 It is an alkyl group (C1-C6) that has been optionally substituted; R B9’ However, it is H or (C1-C6) alkyl; R B10 However, -OR B13 Replaced by and one or more R B14 Replaced by optional selection (C6~C 10 ) is an allele; R B11 However, -L B1 - or (C1-C6) alkyl; R B12 However, these are halogens, (C1-C6) alkyls, (C1-C6) alkoxys, (C1-C6) haloalkyls, (C1-C6) haloalkoxys, -OH, or CN; R B13 However, R 16 Replaced by and one or more R B16 Replaced by optional selection (C6~C 10 ) is an allele; Each R B14 However, each element independently is a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, oxo, -OH, or CN; R B16 However, one or more R B26 It is a 5-7 member heteroaryl compound containing 1-3 heteroatoms selected from N, O, and S, which are optionally substituted; Each R B16’ However, each element independently is a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, or CN; Each R B26 However, each entity independently has one or more R B29 It is an alkyl group (C1-C6) that has been optionally substituted; Each R B27 However, each entity exists independently, (C6~C 10 ) is an allele; Each R B29 However, -NR B31 R B32 Alternatively, a 4-7 membered heterocycline containing 1-3 heteroatoms selected from N, O, and S; Each R B31 However, independently selected from H and (C1-C6) alkyl groups; Each R B32 However, independently selected from H and (C1-C6) alkyl groups; L B1 However, -(CH2) p It is NH-* and L B1 The * is the linker (L A The link point to R is shown, B11 , R B6 or R B7 At least one of them is -L B1 -and; n is 1; and A bifunctional compound in which P is 1, 2, 3, 4, 5, or 6.

[0183] Embodiment 45. The compound of formula (B) is formula (B-1): [ka] The bifunctional compound of Embodiment 43, or having the structure of a pharmaceutically acceptable salt or stereoisomer thereof.

[0184] Embodiment 46. A bifunctional compound from any one of embodiments 43 to 45, wherein, RB1 However, H is; R B2 However, it is substituted with -C(=O)OH, (C1~C6)alkoxy, -L B1 - or (C1-C6) alkyl; R B3 However, it is H or (C1-C6) alkyl; R B6 However, H, (C1~C6) alkyl or -L B1 -and; R B7 However, H, (C1~C6) alkyl or -L B1 - or R B6 and R B7 These, together with the carbon atoms to which they are bonded, form (C3-C7) cycloalkyl groups; R B9 However, H or one or more R B27 It is an alkyl group (C1-C6) that has been optionally substituted; R B9’ However, it is H or (C1-C6) alkyl; R B10 However, -OR B13 Replaced by and one or more R B14 Replaced by optional selection (C6~C 10 ) is an allele; R B11 However, -L B1 - or (C1-C6) alkyl; R B12 However, these are halogens, (C1-C6) alkyls, (C1-C6) alkoxys, (C1-C6) haloalkyls, (C1-C6) haloalkoxys, -OH, or CN; R B13 However, R 16 Replaced by (C6~C 10 ) is an allele; Each R B14 However, each element independently is a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, oxo, -OH, or CN; R B16 However, one or more RB26 It is a 5-7 member heteroaryl compound containing 1-3 heteroatoms selected from N, O, and S, which are optionally substituted; Each R B26 However, each entity independently has one or more R B29 It is an alkyl group (C1-C6) that has been optionally substituted; Each R B27 However, each entity exists independently, (C6~C 10 ) is an allele; Each R B29 However, -NR B31 R B32 Alternatively, a 4-7 membered heterocycline containing 1-3 heteroatoms selected from N, O, and S; Each R B31 However, independently selected from H and (C1-C6) alkyl groups; Each R B32 However, independently selected from H and (C1-C6) alkyl groups; L B1 However, -(CH2) p It is NH-* and L B1 The * is the linker (L A The link point to R is shown, B11 , R B6 or R B7 At least one of them is -L B1 -and; n is 1; and A bifunctional compound in which P is 1, 2, 3, 4, 5, or 6.

[0185] Embodiment 47. T L but, [ka] or its pharmaceutically acceptable salt or stereoisomer (in the formula, L B1 is, -(CH2) p It is NH-* and L B1 The * is a linker (L A (Indicates a connection point to) A bifunctional compound of Embodiment 43, selected from the above.

[0186] Embodiment 48. A bifunctional compound from any one of embodiments 43 to 47, wherein T L but, [ka] A bifunctional compound which is a compound of formula (B) or a pharmaceutically acceptable salt or stereoisomer thereof, selected from the pharmaceutically acceptable salts or stereoisomers thereof.

[0187] Embodiment 49. A bifunctional compound from any one of embodiments 43 to 48, wherein T L but, [ka] A bifunctional compound, which is either a pharmaceutically acceptable salt or stereoisomer thereof.

[0188] Embodiment 50. A bifunctional compound of formula (I) or any one of the bifunctional compounds of embodiments 1 to 3, wherein T L However, compounds of formula (C) that bind to PCSK9, or their pharmaceutically acceptable salts or stereoisomers: [ka] (In the formula, X C1 is H or (C1-C6) alkyl; X C2 is H or (C1-C6) alkyl; or X C1 and X C2 These, together with the carbon atoms to which they are bonded, form = (O); X C1 and X C2 However, when each is independently H or (C1~C6) alkyl, X C3 is either -CH2- or X C1 and XC2 They, together with the carbon atoms to which they are bonded, form = (O); or X C1 and X C2 However, when they combine with the carbon atoms to which they are bonded, they form = (O), X C3 These are -O-, -NH-, or -N(C1~C6)alkyl-; R C1 (C6~C 10 ) A 5 or 6-membered heteroaryl containing aryl or 1 to 3 heteroatoms selected from N, O and S, wherein the aryl and heteroaryl are -OR C10 or -NR C21 R C10 Replaced by and one or more R C11 Replaced by optional selection; R C2 H, (C1~C6) alkyl, -L C1 -, (C2~C6) alkenyl, (C1~C6) haloalkyl, -NR C12 R C13 , (C3~C9)carbocyrill, (C3~C7)cycloalkenyl, 5-7 membered heterocyclyl containing 1-3 heteroatoms selected from N, O and S, (C6~C 10 ) A 5 or 6-membered heteroaryl containing aryl or 1 to 3 heteroatoms selected from N, O and S, wherein the alkyl is optionally composed of one or more R C18 Substituted with, and carbocyclyl, (C3-C7)cycloalkenyl, heterocyclyl, aryl, and heteroaryl are optionally one or more R C19 Replaced by; R C3 The alkyl group is H, D, (C1-C6) alkyl, (C1-C6) alkoxy, (C1-C6) haloalkyl, (C1-C6) haloalkoxy, or (C1-C6) hydroxyalkyl, and the alkyl group can be optionally selected from one or more R groups. C14 Replaced by; R C4 is H or (C1-C6) alkyl; or R C3 and R C4These, together with the atoms to which they are bonded, form a 5-7 membered heterocyclyl ring containing 1-3 heteroatoms selected from N, O, and S; R C5 is H, D, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, or (C1-C6)hydroxyalkyl, where (C1-C6)alkyl is optionally substituted with one or more D; R C6 (C1~C6) alkyl, (C1~C6) alkoxy, -L C1 -, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, or (C1-C6)hydroxyalkyl, wherein the alkyl is optionally substituted with one or more substituents independently selected from -OH, (C1-C6)alkoxy, (C1-C6)haloalkoxy, -C(O)(C1-C6)alkyl, -C(O)OH, and -C(O)O(C1-C6)alkyl; R C7 is H, D, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, or (C1-C6)hydroxyalkyl, where (C1-C6)alkyl is optionally substituted with one or more D; R C8 H, (C1~C6) alkyl, -L C1 - or (C1~C6) haloalkyl, where the alkyl is optionally a 4-7 membered heterocycline containing 1-3 heteroatoms selected from (C3~C7) carbocykyl, N, O, and S, -C(O)OH, -NR C16 R C17 and -C(O)NR C16 R C17 Substituted with one or more substituents independently selected from each; R C9 These are halogens, (C1-C6) alkyls, (C1-C6) alkoxys, (C1-C6) haloalkyls, (C1-C6) haloalkoxys, -OH, or CN; R C10 (C6~C 10) A 5- or 6-membered heteroaryl containing aryl or 1 to 3 heteroatoms selected from N, O, and S, wherein the aryl and heteroaryl are optionally composed of one or more R C22 Replaced by; Each R C11 These are, independently in each presence, halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, or CN; R C12 and R C13 Each of these is independently H or (C1-C6) alkyl; Each R C14 These exist independently in each entity, D, NR C15 R C15’ , (C3-C7) carbocyryl or 3-7 membered heterocyclyl containing 1-3 heteroatoms selected from N, O, and S, wherein the carbocyryl and heterocyclyl are optionally substituted with one or more substituents independently selected from halogens, (C1-C6) alkyl, (C1-C6) alkoxy, (C1-C6) haloalkyl, and (C1-C6) haloalkoxy; R C15 and R C15’ Each of these is independently H or (C1-C6) alkyl; R C16 and R C17 Each is independently either H or (C1-C6) alkyl; or R C16 and R C17 These, together with the nitrogen atom to which they are bonded, form a 4-7 membered heterocyclyl ring containing 1-2 additional heteroatoms selected from N, O, and S; Each R C18 Independently, each entity is a (C3-C7) carbocyrill, a 5-7 membered heterocycline containing 1-3 heteroatoms selected from N, O, and S, and (C6-C 10 ) A 5 or 6-membered heteroaryl containing aryl or 1 to 3 heteroatoms selected from N, O and S, and the carbocyclyl, heterocyclyl, aryl and heteroaryl may optionally contain one or more R C20Replaced by; Each R C19 Independently in each presence, is a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, or CN; or Two R's C19 When it is on an adjacent atom, (C6~C 10 ) Forms a 5 or 6-membered heteroaryl ring containing aryl or 1 to 3 heteroatoms selected from N, O, and S, wherein the aryl and heteroaryl are optionally substituted with one or more substituents independently selected from halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, and CN; Each R C20 Independently in each presence, is a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, oxo, -OH, or CN; or R C18 When is a carbocyclyl or heteroaryl, two R C20 When bonded to the same carbon atom, they together form (O); R C21 is H or (C1-C6) alkyl; Each R C22 These are, independently, halogens, (C1~C6)alkyls, (C1~C6)alkoxys, (C1~C6)haloalkyls, (C1~C6)haloalkoxys, -OH, CN, (C6~C 10 ) A 5- or 6-membered heteroaryl containing aryl or 1 to 3 heteroatoms selected from N, O, and S, wherein the aryl and heteroaryl are optionally composed of one or more R C23 Replaced by; Each R C23 These are, independently, halogens, (C1-C6) alkyls, (C1-C6) alkoxys, (C1-C6) haloalkyls, (C1-C6) haloalkoxys, and -CH2(OCH2CH2) in their respective forms. nOCH2CH3, -OH, CN, or a 4-7 membered heterocyclyl containing 1-3 heteroatoms selected from N, O, and S, wherein the heterocyclyl can optionally contain halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, or -C(O)R C24 R C25 , -NR C24 C(O)R C25 Substituting with one or more substituents independently selected from -NH2, -NH(C1~C6)alkyl, and -N((C1~C6)alkyl)2, wherein the alkyl group is optionally -NR C24 R C25 Alternatively, substituted with a 4-7 membered heterocycline containing 1-3 heteroatoms selected from N, O, and S, which is optionally substituted with one or more substituents independently selected from halogens, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, -NH2, -NH(C1-C6)alkyl, and -N((C1-C6)alkyl)2; R C24 is H, (C1-C6) alkyl, or (C3-C7) carbocyric; R C25 is H, (C1-C6) alkyl or (C3-C7) carbocyryl; and L C1 is, -(CH2) p It is NH-* and L C1 The * is a linker (L A The link point to R is shown, C2 , R C6 or R C8 At least one of them is -L C1 -is) It is a bifunctional compound.

[0189] Embodiment 51. The compound of formula (C) is formula (C-1): [ka] Or the bifunctional compound of Embodiment 50 having the structure of a pharmaceutically acceptable salt or stereoisomer thereof.

[0190] Embodiment 52. A bifunctional compound of Embodiment 50 or Embodiment 51, wherein the formula is X C1 and X C2 These, together with the carbon atoms to which they are bonded, form = (O); X C3 However, it is -CH2-; R C1 However, -OR C10 and one or more R C11 Replaced by (C6~C 10 ) is an allele; R C2 However, H, (C1~C6) alkyl, -L C1 - or (C3~C9) carbocyric, where alkyl is one R C18 Substituted with, and the carbocyclyl is one or more R C19 Replaced by; R C3 However, it is H or (C1-C6) alkyl; R C4 is H or (C1-C6) alkyl; or R C3 and R C4 These, together with the atoms to which they are bonded, form a 5-7 membered heterocyclyl ring containing 1-3 heteroatoms selected from N, O, and S; R C5 However, it is H or (C1-C6) alkyl; R C6 However, (C1~C6) alkyl or -L C1 - and the alkyl group is optionally substituted with one or more substituents independently selected from -OH or (C1-C6) alkoxy groups; R C7 However, it is H or (C1-C6) alkyl; R C8 However, H, (C1~C6) alkyl or -L C1 -and; R C9 However, it is a halogen; R C10 However, one R C22 Replaced by (C6~C 10 ) is an allele; Each R C11 However, each element independently is a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, or CN; R C18 However, (C6~C 10 ) is an allele; Each R C19 However, each element independently is a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, or CN; R C22 However, one or more R C23 A 5- or 6-membered heteroaryl compound containing 1 to 3 heteroatoms selected from N, O, and S, which are substituted; Each R C23 However, each entity independently uses -NR C24 R C25 Alternatively, it is a (C1-C6) alkyl group optionally substituted with a 4-7 membered heterocycline containing 1-3 heteroatoms selected from N, O, and S; R C24 However, H, (C1~C6) alkyl; R C25 However, H, (C1~C6) alkyl, and L C1 However, -(CH2) p It is NH-* and L C1 The * is the linker (L A The link point to R is shown, C2 , R C6 or R C8 At least one of them is -L C1 -A bifunctional compound.

[0191] Embodiment 53. A bifunctional compound from any one of embodiments 50 to 52, wherein the formula is: R C1 However, -OR C10and one or more R C11 Replaced by (C6~C 10 ) is an allele; R C2 However, (C1~C6) alkyl, -L C1 - or (C3~C9) carbocyric, where alkyl is one R C18 Substituted with, and the carbocyclyl is one or more R C19 Replaced by; R C3 However, it is (C1~C6) alkyl; R C4 is H; or R C3 and R C4 These, together with the atoms to which they are bonded, form a 5-7 membered heterocyclyl ring containing 1-3 heteroatoms selected from N, O, and S; R C5 However, it is H or (C1-C6) alkyl; R C6 However, (C1~C6) alkyl or -L C1 - and the alkyl group is optionally substituted with one or more substituents independently selected from -OH or (C1-C6) alkoxy groups; R C7 However, H is; R C8 However, (C1~C6) alkyl or -L C1 -and; R C9 However, it is a halogen; R C10 However, one R C22 Replaced by (C6~C 10 ) is an allele; Each R C11 However, each entity independently contains halogens; R C18 However, (C6~C 10 ) is an allele; Each R C19 However, each entity independently is a (C1-C6) alkyl group; R C22 However, one or more R C23A 5- or 6-membered heteroaryl compound containing 1 to 3 heteroatoms selected from N, O, and S, which are substituted; Each R C23 However, each entity independently uses -NR C24 R C25 Alternatively, it is a (C1-C6) alkyl group optionally substituted with a 4-7 membered heterocycline containing 1-3 heteroatoms selected from N, O, and S; R C24 However, it is (C1~C6) alkyl; R C25 However, it is C1-C6 alkyl, and L C1 However, -(CH2) p It is NH-* and L C1 The * is the linker (L A The link point to R is shown, C2 , R C6 or R C8 At least one of them is -L C1 -A bifunctional compound.

[0192] Embodiment 54. T L but, [ka] Selected from, L C1 However, -(CH2) p It is NH-* and L C1 The * is the linker (L A A bifunctional compound of embodiment 50 showing a binding site to ).

[0193] Embodiment 55. A bifunctional compound from any one of embodiments 50 to 54, wherein T L but, [ka] A bifunctional compound which is a compound of formula (C) or a pharmaceutically acceptable salt or stereoisomer thereof, selected from the pharmaceutically acceptable salts or stereoisomers thereof.

[0194] Embodiment 56. Formula (Ib): R L -L A -FHR3 L (Ib) (In the formula, R L This is the portion that binds to cell surface receptors associated with receptor-mediated endocytosis; L A is a linker; and FHR3 L (This is the part that connects to FHR3.) A bifunctional compound having the structure of formula (I), or one of embodiments 1 to 2.

[0195] Embodiment 57. The part is coupled to FHR3, and [ka] (In the formula, L D1 is, -(CH2) p NH-*, where p is 1, 2, 3, 4, 5 or 6, L D1 The * is a linker (L A (Indicates a connection point to) A compound selected from the above, which is a bifunctional compound of formula (I) or formula (Ib), or one of embodiments 1 to 2.

[0196] Embodiment 58. The part that connects to FHR3 [ka] (In the formula, * represents the linker (L A (Indicates a connection point to) A bifunctional compound of Embodiment 56 or Embodiment 57, which is a compound selected from or a pharmaceutically acceptable salt or stereoisomer.

[0197] B. Receptor binding site (R L ) Receptor binding site (R L The ) is the portion that binds to cell surface receptors, which are associated with receptor-mediated endocytosis. Examples of such cell surface receptors include, but are not limited to, the asialoglycoprotein receptor (ASGPR), mannose-6-phosphate receptor (M6PR), insulin-like growth factor 2 receptor, mannose receptor system, Kupffer cell receptor, macrophage galactose lectin (MGL), scavenger receptor type C lectin (SRCL), EGF receptor, Fc receptor, lysosomal membrane endogenous protein receptor (LIMP-2), transferrin receptor, and sortilin and decoy receptors (CXCR7, DARC, D6, and CCX CKR, etc.).

[0198] Asialoglycoprotein receptor (ASGPR) The asialoglycoprotein receptor (ASGPR) is a type C lectin expressed on the surface of hepatocytes that regulates the levels of plasma glycoproteins terminated with galactose (Gal) or N-acetylgalactosamine (GalNAc) sugars. ASGPR binds to glycoproteins terminated with galactose (Gal) or N-acetylgalactosamine (GalNAc) sugars and is translocated internally via endocytosis, primarily mediated by receptors in covering pits on the basolateral membrane of hepatocytes. During internal translocation, the ligand-receptor complex is transported to a compartment within the lysosome. Calcium sequestration and subsequent acidification of the endosomal compartment promote the dissociation of the ligand-receptor complex, with the receptor returning to the cell membrane for reuse, while the cargo (ligand) is fractionated into the lysosome for degradation.

[0199] Because it has the ability to efficiently assist the delivery of glycoproteins terminated with galactose (Gal) or N-acetylgalactosamine (GalNAc) sugars to lysosomes, the asialoglycoprotein receptor (ASGPR) is used herein for the degradation of extracellular target molecules (such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, cells, and cell membrane proteins), and the extracellular target molecules are (T) of the bifunctional compound of the present invention. L ) is bound to the receptor binding site (R) of the bifunctional compound. L ) comprises one or more galactose (Gal) groups or one or more N-acetylgalactosamine (GalNAc) groups. Such receptor binding moieties (R L The ) group binds to the asialoclycoprotein receptor (ASGPR), thereby delivering the extracellular target molecule to the lysosome and degrading it via lysosomal degradation.

[0200] Embodiment 59. Receptor binding site (R L )but, [ka] [ka] (In the formula, R L The * is a linker (L A (Indicates a connection point to) A bifunctional compound of formula (I) or one of embodiments 1 to 58, selected from the above.

[0201] In other embodiments, the receptor-binding moiety (R) of the bifunctional compound of the present invention is used. L ) comprises one or more galactose (Gal) groups or one or more N-acetylgalactosamine (GalNAc) groups, and one or more galactose (Gal) groups or one or more N-acetylgalactosamine (GalNAc) groups comprises a cross-linked ketal moiety.

[0202] Embodiment 60. Receptor binding site (R L )but, [ka] (In the formula, R L The * is a linker (L A (Indicates a connection point to) A bifunctional compound of formula (I) or one of embodiments 1 to 58, selected from the above.

[0203] Mannose-6-phosphate receptor (M6PR), also known as insulin-like growth factor 2 receptor. Lysosomes are membrane-bound organelles in cells that possess a characteristic acidic pH and are involved in the degradation of many different substrates. This catabolic process is carried out by more than 60 soluble enzymes contained within the organelle, many of which belong to a broad category of hydrolases known as glycosidases, proteases, phosphatases, sulfatases, and lipases. These lysosomal hydrolases are first synthesized in the rough endoplasmic reticulum, specifically transported through the Golgi apparatus to the trans-Golgi network, and subsequently delivered to lysosomes by transport vesicles.

[0204] To ensure that lysosomal hydrolase is enriched and delivered to lysosomes, it is tagged with a unique marker: mannose-6-phosphate (M6P) group. The M6P group is exclusively attached to the N-linked oligosaccharide of lysosomal hydrolase as it moves through the cis-Golgi network. The M6P group is then recognized by two independent transmembrane M6P receptors (MPRs) present in the trans-Golgi network: the cation-independent M6P receptor (CI-MPR), also known as the insulin-like growth factor 2 receptor (IGF2R) and / or the cation-dependent M6P receptor (CD-MPR). In the trans-Golgi network, the M6P receptors bind to the M6P group on tagged lysosomal hydrolase at pH 6.5–6.7, subsequently helping to bundle the hydrolase into transport vesicles for delivery to late endosomes. The cation-independent M6P receptor (CI-MPR, also known as the insulin-like growth factor 2 receptor (IGF2R)) is also present on the cell surface, where it can bind to lysosomal enzymes that have escaped the cell and deliver them to late endosomes. Within endosomes, which are normally pH 6, lysosomal hydrolase dissociates from the MPR, and during the maturation of the endosome into a lysosome, the pH drops to pH 5, and the hydrolase begins digesting plasma-invaginated material delivered from the early endosome. The MPR is then reused from the endosome to the cell surface and subsequently returns to the Golgi complex.

[0205] Because it has the ability to efficiently assist the delivery of M6P-tagged proteins to lysosomes, the MP6 receptor is used herein for the degradation of extracellular target molecules (such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, cells, and cell membrane proteins), and the extracellular target molecules are (T) the bifunctional compounds of the present invention. L ) is bound to the receptor binding site (R) of the bifunctional compound. L ) contains one or more high-affinity ligands for the M6P receptor. Such receptor binding moieties (RL The ) group binds to the M6P receptor, thereby delivering the extracellular target molecule to the lysosome and degrading it via lysosomal degradation.

[0206] Embodiment 61. Receptor binding site (R L )but, [ka] [ka] (In the formula, R L The * is a linker (L A (Indicates a connection point to) A bifunctional compound of formula (I) or one of embodiments 1 to 58, selected from the above.

[0207] C. Linker (L A ) The linker portion of the bifunctional compound of the present invention, (L A ) is as follows: a) Alkylene group: -(CH2) n - This can be either linear or branched (in this example, n is 1 to 18); b) Alkenylene group; c) Alkynylene group; d) Alkenyl group; e) Alkynyl group; f) Ethylene glycol units: -OCH2CH2 or -CH2CH2O; g) Polyethylene glycol Unit: (-CH2CH2O-) x (In this example, x ranges from 2 to 20); h)-O; i)-S; j) Carbonyl: -C (=O); k) Ester: -C(=O)-O- or -OC(=O); l) Carbonate; -OC(=O)O; m) Amine: -NH; n) Tertiary amines o) Amide: -C(=O)-NH-, -NH-C(=O)- or -C(=O)N(C 1~6 Alkyl); p) Carbamates: -OC(=O)NH- or -NHC(=O)O; q) Urea:-NHC(=O)NH; r) Sulfonamide: -S(O)2NH- or -NHS(O)2; s) Ether: -CH2O- or -OCH2; t) Alkylenes substituted with one or more groups independently selected from carboxylates, sulfonates, hydroxyls, amines, amino acids, saccharides, phosphates, and phosphonates; u) Alkenylenes substituted with one or more groups independently selected from carboxylates, sulfonates, hydroxyls, amines, amino acids, saccharides, phosphates, and phosphonates; v) Alkynylenes substituted with one or more groups independently selected from carboxy, sulfonate, hydroxyl, amine, amino acid, saccharide, phosphate, and phosphonate; w) C1~C where one or more methylene groups are substituted by one or more -S-, -NH-, or -O- moieties 10 Alkylene; and x) Ring systems having two available bonding sites, such as divalent rings selected from phenyl (including 1,2-, 1,3-, and 1,4-disubstituted phenyl), C5-C6 heteroaryls, C3-C8 cycloalkyls (including 1,1-disubstituted cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl and 1,4-disubstituted cyclohexyl), and C4-C8 heterocycloalkyls. It is an uncuttable linker containing one or more linker components selected from the following.

[0208] In addition, Linker (L A The linker component of ) can be a chemical moiety readily formed by the reaction between two reactive groups. Non-limiting examples of such chemical moieties are provided in Table 1.

[0209] [Table 21]

[0210] [Table 22]

[0211] [Table 23]

[0212] [Table 24]

[0213] [Table 25]

[0214] In the formula, R in Table 1 32 H, C 1~4 Alkyl, phenyl, pyrimidine, or pyridine; R in Table 1 35 H, C 1~6 Alkyl, phenyl, or C substituted with 1-3 -OH groups 1~4 It is alkyl; each R in Table 1 7 H, C 1~6 Alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C substituted with -C(=O)OH 1~4 C substituted with alkoxy and -C(=O)OH 1~4 Selected independently of alkyl groups; R in Table 1 37 is independently selected from H, phenyl, and pyridine; q in Table 1 is 0, 1, 2, or 3.

[0215] In addition, Linker (L A The linker component of ) may be the groups provided in Table 2 below.

[0216] [Table 26]

[0217] [Table 27]

[0218] Embodiment 62. A bifunctional compound of formula (I) or one of embodiments 1 to 61, wherein the linker (L A )but, *-(CH2) n C(=O)NHNHC(=O)(CH2) n ON=CH2X1C(=O)-**;*-(CH2) n X3C(=O)-**;*-(CH2) n C(=O)-**;*-(CH2) n C(=O)NHNHC(=O)(CH2) n ON=CH2X1C(=O)NH(CH2) n CH(C=(O)NH2)-**;*-(CH2) n X3C(=O)NH(CH2) n CH(C=(O)NH2)-**;*-(CH2) n C(=O)NH(CH2) n CH(C=(O)NH2)-**, *-((CH2) n O) t (CH2) m C(=O)-**;*-((CH2) n O) t (CH2) m -**;*-(CH2) n C(=O)NH((CH2) n O) t (CH2) m -**;-(CH2) n -;*-(CH2) n NHC(=O)(CH2) m -**;*-(CH2) n NHC(=O)(CH2) n C(=O)NH(CH2) m -**;*-((CH2) n O) t (CH2)n NHC(=O)(CH2) m -**;*-((CH2) n O) t CH2) m C(=O)NH(CH2) m -**;*((CH2) n O) t (CH2) n NHC(=O)(CH2) m -**;*-(CH2) n O(CH2) m -**;*-(CH2) n NH(CH2) n -**;*-(CH2) n NH(CH2) m C(=O)-**;*-(CH2) n X3(CH2) m -**;*-((CH2) n O) t (CH2) n X3(CH2) m -**;*-(CH2) n NHC(=O)(CH2) n X3(CH2) m -**;*-((CH2) n O) t (CH2) n NHC(=O)(CH2) n X3(CH2) m -**;*-((CH2) n O) t (CH2) n C(=O)NH(CH2) m -**;*-(CH2) m NHC(=O)((CH2) n O) t (CH2) m -**;*-(CH2) n C(=O)NH(CH2) m -**;*-(CH2) n NHC(=O)((CH2) n O) t (CH2) m -**;*-(CH2) n NHC(=O)(CH2) n O(CH2) m-**;*-(CH2) n NH(CH2) m -**;*-((CH2) n O) t CH2) n C(=O)NH(CH2) m -**;*-(CH2) n NHC(=O(CH2) n X3(CH2) m -**;-C(=O)-;*-C(=O)(CH2) n C(=O)-**;*-C(=O)((CH2) n O) t (CH2) m C(=O)-**;*-C(=O)((CH2) n O) t (CH2) m -**;*-((CH2) n O) t (CH2) m X3(CH2) n O(CH2) n NHC(=O)((CH2) n O) t (CH2) m C(=O)-**,*-C(=O)(CH2) n C(=O)NH((CH2) n O) t (CH2) m -**;*-C(=O)NHNHC(=O)(CH2) n ON=CH2X1C(=O)NH(CH2) n CH(C=(O)NH2)-**;*-X3C(=O)NH(CH2) n CH(C=(O)NH2)-**;*-C(=O)(CH2) n C(=O)NHNHC(=O)(CH2) n ON=CH2X1C(=O)-**;*-C(=O)(CH2) n X3C(=O)-**;*-C(=O)(CH2) n NHC(=O)(CH2) n C(=O)NH(CH2) m -**;*-C(=O)((CH2) n O) t (CH2) nNHC(=O)(CH2) m -**;*-C(=O)((CH2) n O) t CH2) m C(=O)NH(CH2) m -**;*-C(=O)(CH2) n O(CH2) m -**;*-C(=O)(CH2) n -**;*-C(=O)NH((CH2) n O) t (CH2) m -**;*-C(=O)(CH2) n NH(CH2) n -**;*-C(=O)(CH2) n NH(CH2) m C(=O)-**;*-C(=O)(CH2) n X3(CH2) m -**;*-C(=O)((CH2) n O) t (CH2) n X3(CH2) m -**;*-C(=O)(CH2) n NHC(=O)(CH2) m -**;*-C(=O)(CH2) n NHC(=O)((CH2) n O) t (CH2) m -**;*-C(=O)(CH2) n NHC(=O)(CH2) n O(CH2) m -**;*-C(=O)(CH2) n NH(CH2) m -**;*-C(=O)((CH2) n O) t CH2) n C(=O)NH(CH2) m -**;*-C(=O)(CH2) n NHC(=O(CH2) n X3(CH2) m -**;*-C(=O)NH(CH2) n X3(CH2) m -**;*-C(=O)NH(CH2) nNHC(=O)(CH2) m -**;*-C(=O)NH(CH2) n NHC(=O)(CH2) n O(CH2) m -**;*-C(=O)NH(CH2) n NHC(=O)(CH2) n X3(CH2) m -**;*-C(=O)NH(CH2) n NHC(=O)-**;*-C(=O)NH((CH2) n O) t (CH2) n X3(CH2) m -**;*-C(=O)(CH2) n NHC(=O)(CH2) n X3(CH2) m -**;*-C(=O)((CH2) n O) t (CH2) n NHC(=O)(CH2) n X3(CH2) m -**;*-C(=O)((CH2) n O) t (CH2) n C(=O)NH(CH2) m -**;*-C(=O)(CH2) m NHC(=O)((CH2) n O) t (CH2) m -** or *-C(=O)(CH2) n C(=O)NH(CH2) m -** (In the formula, X1 [ka] and X3 [ka] And the * in X3 is R L The connection point to is shown, and ** of X3 is L A It shows the connection point to, L A 's * indicates the binding point to R L and L A 's ** indicates the binding point to T L (indicating) A bifunctional compound selected from

[0219] Embodiment 63. The bifunctional compound of formula (I) or any one of Embodiments 1 to 62, wherein the linker (L A ) is *-(CH2) n C(=O)NHNHC(=O)(CH2) n ON=CH2X1C(=O)-**; *-(CH2) n X3C(=O)-**; *-((CH2) n O) t (CH2) m C(=O)-**; *-(CH2) n C(=O)-**; *-(CH2) n C(=O)NHNHC(=O)(CH2) n ON=CH2X1C(=O)NH(CH2) n CH(C=(O)NH2)-**; *-(CH2) n X3C(=O)NH(CH2) n CH(C=(O)NH2)-**; -C(=O)-; *-C(=O)(CH2) n C(=O)-**; *-C(=O)((CH2) n O) t (CH2) m C(=O)-**; *-((CH2) n O) t (CH2) m X3(CH2) n O(CH2) n NHC(=O)((CH2) n O) t (CH2) m C(=O)-**; *-C(=O)((CH2) n O) t (CH2) m -**; or *-C(=O)(CH2) n C(=O)NH((CH2) n O)t (CH2) m -** (wherein X1 is

Chem.

Chem.

[0220] Embodiment 64. [[ID=4s6]]

Chem.

Chem.

Chem.

Chem.

Chem.

[0221] Embodiment 65.

Chem.

[0222] Process for preparing the compound of formula (I) For illustrative purposes, the general reaction schemes shown herein provide promising routes for synthesizing the compounds of the present invention. For further detailed descriptions of individual reaction steps, please refer to the Examples section below. In addition, many of the compounds prepared by the methods described below can be further modified using conventional chemical actions well known to those skilled in the art, in consideration of this disclosure. In the general schemes below, R L , L A and T L This is defined herein.

[0223] As an example, a general synthesis of the compound of formula (I) is shown in scheme I below, and the receptor ligand (R L The linker moiety (L) has a bound reactive group (e.g., RG1), and the target ligand has a pendant reactive group (e.g., RG2) that can react with the reactive group on the receptor ligand. A ') is linked to the linker (L) which then couples the receptor ligand to the target ligand. A ) forms a compound of formula (I). Scheme I R L -RG1+RG2-L A '-T L →R L -L A -T L

[0224] In Scheme I, RG1 is reactive group 1 in Table 1, RG2 is reactive group 1 in Table 1, and the reaction product of the reactive groups (as seen in Table 1) is linker L A It becomes a linker component.

[0225] Another common synthesis for the compound of formula (I) is shown in scheme II below, and the receptor ligand (R L ) is a linker moiety (L) having a pendant reactive group (e.g., RG1). A The target ligand is bound to a linker (L) that has a bound reactive group that can react with the reactive group of the receptor ligand, thereby coupling the receptor ligand to the target ligand. A ) forms a compound of formula (I). Scheme II R L -L A '-RG1+RG2-T L →R L -L A -T L

[0226] In Scheme II, RG1 is reactive group 1 in Table 1, RG2 is reactive group 1 in Table 1, and the reaction product of the reactive groups (as seen in Table 1) is linker L A It becomes a linker component.

[0227] Another common synthesis for the compound of formula (I) is shown in scheme III below, and the receptor ligand (R L ) is a linker moiety (L) having a pendant reactive group (e.g., RG1). A The target ligand is bound to a linker moiety (L) which has a pendant reactive group (e.g., RG2) that can react with the reactive group of the receptor ligand. A A linker (L) that binds to the receptor ligand and thereby couples the receptor ligand to the target ligand. A ) forms a compound of formula (I). Scheme III R L -L A ''-RG1+RG2-L A '-T L →R L -L A -T L

[0228] In Scheme II, RG1 is reactive group 1 in Table 1, RG2 is reactive group 1 in Table 1, and the reaction product of the reactive groups (as seen in Table 1) is linker L A It becomes a linker component.

[0229] Another common synthesis for the compound of formula (I) is shown in scheme IV below, with the receptor ligand (R L ) is the linker part (L A '') has a bonded reactive group (e.g., RG1) that can react with a reactive group (e.g., RG2) on the surface, thereby linking the linker moiety (L A '') to receptor ligand (R L ) is joined. Linker part (L A '') also has a protected reactive group (e.g., RG1-Prot) which can react with the reactive group on the target ligand (e.g., RG2) upon deprotection, thereby coupling the receptor ligand to the target ligand and forming the compound of formula (I). Scheme IV [ka]

[0230] In Scheme IV, RG1 is reactive group 1 in Table 1, RG2 is reactive group 1 in Table 1, and the reaction product of the reactive groups (as seen in Table 1) is linker L A It becomes a linker component.

[0231] Another common synthesis for the compound of formula (I) is shown in scheme V below, with the target ligand (T L ) is the linker part (L A '') has a bonded reactive group (e.g., RG1) that can react with a reactive group (e.g., RG2) on the surface, thereby linker moiety (L A '') target ligand (T L ) is joined. Linker part (L A'') also has a protected reactive group (e.g., RG1-Prot) which can react with the reactive group on the receptor ligand (e.g., RG2) upon deprotection, thereby coupling the receptor ligand to the target ligand and forming the compound of formula (I). Scheme V [ka]

[0232] In Scheme V, RG1 is reactive group 1 in Table 1, RG2 is reactive group 1 in Table 1, and the reaction product of the reactive groups (as seen in Table 1) is linker L A It becomes a linker component.

[0233] Pharmaceutical composition and route of administration For the therapeutic use of the bifunctional compounds of the present invention, such compounds are administered alone or as part of a pharmaceutical composition. Furthermore, for the therapeutic use of the bifunctional compounds of the present invention, such compounds are administered alone or as part of a pharmaceutical composition in a therapeutically effective amount. Accordingly, in another embodiment, the present invention provides a pharmaceutical composition comprising the bifunctional compound of the present invention and a pharmaceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.

[0234] The pharmaceutical compositions of the present invention may be prepared using a process that involves mixing the bifunctional compound of the present invention with one or more pharmaceutically acceptable carriers. For example, the pharmaceutical compositions of the present invention may be produced by mixing, granulating, and / or coating the bifunctional compound of the present invention in its free form in association with at least one pharmaceutically acceptable carrier.

[0235] The pharmaceutical compositions or combinations of the present invention may have a unit dose of about 0.1 to 100 mg of the active ingredient for a subject weighing about 50 to 70 kg. The therapeutically effective dose of the compound, pharmaceutical composition, or combination thereof depends on the species, weight, age, and individual condition of the subject, the disorder or disease being treated, or the severity thereof.

[0236] The dosage characteristics cited above can be advantageously demonstrated by in vitro and in vivo studies using mammals (e.g., mice, rats, dogs, monkeys) or their isolated organs, tissues, and preparations thereof. The compounds of the present invention can be applied in vitro in solution (e.g., in the form of aqueous solutions), and in vivo enterally, parenterally, subcutaneously, intravenously, for example, as a suspension or aqueous solution. The in vitro dose is approximately 10 -12 moles ~10 -6 The molar concentration may be within a range of approximately 0.01 to 10 mg / kg. The effective in vivo dose may range from approximately 0.01 to 10 mg / kg.

[0237] The activity of the compounds of the present invention can be evaluated by in vitro and in vivo methods described in the examples herein.

[0238] The bifunctional compound of the present invention may be the active ingredient in a pharmaceutical composition formulated for a specific route of enteral or parenteral administration.

[0239] Oral dosage form The pharmaceutical compositions of the present invention can be administered orally in separate dosage forms, including, but not limited to, capsules, gelatin capsules, caplets, tablets, chewable tablets, lozenges, powders, granules, syrups, flavored syrups, solutions or suspensions in aqueous or non-aqueous liquids, edible foams or whips, and oil-in-water or water-in-oil emulsions.

[0240] Accordingly, for oral administration, a pharmaceutical composition of the present invention containing an effective amount of the compound of the present invention may be prepared in solid form (including, but not limited to, capsules, gelatin capsules, hard or soft capsules, tablets, chewable tablets, lozenges, capsules, pills, granules, or powders) or in liquid form (including, but not limited to, solutions, aqueous or oily suspensions, syrups, elixirs, foams, whips, or emulsions). The pharmaceutical composition may be subjected to conventional pharmaceutical operations such as sterilization and / or may contain conventional inert diluents, lubricants, or buffers, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, and buffers.

[0241] Compositions intended for oral use are prepared according to any method known in the art for manufacturing pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweeteners, flavoring agents, coloring agents and preservatives in order to provide pharmaceutically simple and palatable preparations.

[0242] Generally, a pharmaceutical composition is a tablet or gelatin capsule containing an active ingredient in combination with one or more of the following: a) Diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine; b) Lubricants, such as silica, talcum, stearic acid, its magnesium or calcium salts and / or polyethylene glycol; also for tablets, c) Binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; if necessary, d) Disintegrants, such as starch, agar, alginic acid or its sodium salt or effervescent mixture; and e) Absorbents, colorants, flavorings, and sweeteners.

[0243] Tablets may contain the active ingredient in a mixture with non-toxic, pharmaceutically acceptable excipients suitable for tablet production. These excipients include, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granules and disintegrants such as corn starch or alginate; binders such as starch, gelatin, or acacia; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be film-coated or enterically coated by methods known in the art. Tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a longer-lasting effect. For example, time-delaying substances such as glyceryl monostearate or glyceryl distearate may be used. Formulations for oral use may be provided as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oily medium, such as peanut oil, liquid paraffin, or olive oil.

[0244] Parenteral dosage form In certain embodiments, the pharmaceutical compositions of the present invention are administered parenterally by various routes, including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intravitreous administration.

[0245] A specific injectable composition is an aqueous isotonic solution or suspension containing the bifunctional compound of the present invention. Such a composition may contain excipients such as preservatives, stabilizers, wetting agents or emulsifiers, dissolution accelerators, salts and / or buffers for adjusting osmotic pressure. Such a composition may be prepared according to conventional methods known in the art, and such a composition may be stabilized.

[0246] Combination therapy The compounds of the present invention and the pharmaceutical compositions provided herein are administered alone or in combination with one or more additional therapeutic agents.

[0247] In certain embodiments, the pharmaceutical composition of the present invention may optionally further comprise one or more additional therapeutic agents. Alternatively, the bifunctional compound of the present invention may be administered to a patient in need in combination with the administration of one or more other therapeutic agents.

[0248] The bifunctional compound of the present invention may be administered simultaneously with, before, or after, one or more other therapeutic agents. The bifunctional compound of the present invention may be administered separately by the same or different routes of administration, or together in the same pharmaceutical composition as the other agents. The therapeutic agent is, for example, a chemical compound, peptide, antibody, antibody fragment, or nucleic acid that is therapeutically active or enhances therapeutic activity when administered to a patient in combination with the bifunctional compound of the present invention.

[0249] In one embodiment, the present invention provides a product comprising the bifunctional compound of the present invention and at least one other therapeutic agent as a combination preparation for simultaneous, separate, or sequential use in a therapy, the treatment of a disease or condition as described herein by targeted lysosomal degradation of extracellular target molecules such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, cells, or cell membrane proteins. The product provided as a combination preparation comprises a composition comprising the bifunctional compound of the present invention and the other therapeutic agent combined in the same pharmaceutical composition, or a composition comprising the compound of the present invention and the other therapeutic agent in separate forms, for example, in the form of a kit.

[0250] In one embodiment, the present invention provides a pharmaceutical composition comprising the bifunctional compound of the present invention and another therapeutic agent. Optionally, the pharmaceutical composition may include a pharmaceutically acceptable carrier as described herein.

[0251] In one embodiment, the present invention provides a kit comprising two or more separate pharmaceutical compositions, at least one of which contains the bifunctional compound of the present invention. In one embodiment, the kit includes means for holding the compositions separately, such as a container, a divided bottle, or a divided metal foil bag. An example of such a kit is a blister pack, as is commonly used for packaging tablets, capsules, and the like.

[0252] The kits of the present invention may be used to administer different dosage forms, such as orally and parenterally, or to administer separate compositions at different dosing intervals, or to escalate separate compositions relative to each other. To assist in medication adherence, the kits of the present disclosure typically include instructions for administration.

[0253] In the combination therapy of the present invention, the bifunctional compound and other therapeutic agents of the present invention may be manufactured and / or formulated by the same or different manufacturers. Furthermore, the bifunctional compound and other therapeutic agents of the present invention may be taken (i) before the combination product is delivered to the physician (for example, in the case of a kit containing the compound and other therapeutic agents of the present invention); (ii) immediately before administration by the physician himself (or under the guidance of the physician); or (iii) by the patient himself, for example, during a series of administrations of the compound and other therapeutic agents of the present invention, in conjunction with the combination therapy.

[0254] Pharmacology and utility The bifunctional compounds of the present invention exhibit useful pharmacological properties based on the reduction of extracellular target molecules through lysosomal degradation, and are therefore designated for use as therapeutic or research chemicals (e.g., tool compounds).

[0255] Conventional therapeutic agents, such as those directed at proteins, treat diseases by interfering with protein function, for example, by inhibiting enzymes and receptors, or by recruiting immune effectors, as in the case of many monoclonal antibody drugs. Typically, due to the reversible nature of conventional drug / target interactions, the effectiveness of such conventional therapies requires hyperstoichiometric drug concentrations to maintain inhibition that may be lost over time as drug concentrations decrease. However, the methods described herein using the bifunctional compounds of the present invention may show improved efficacy at stoichiometric or quasi-stoichiometric concentrations, and the efficacy is limited not by drug concentration but by the resynthesis of the target molecule (e.g., protein).

[0256] Accordingly, the present invention provides bifunctional compounds for use in therapy by targeted lysosomal degradation of extracellular target molecules such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secreted proteins, antibodies, lipoproteins, exosomes, viruses, cells, or cell membrane proteins. In certain embodiments, the present invention also provides bifunctional compounds for use in therapy by targeted asialoglycoprotein receptor (ASGPR)-mediated lysosomal degradation of extracellular target molecules such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secreted proteins, antibodies, lipoproteins, exosomes, viruses, cells, and cell membrane proteins. In certain embodiments, the present invention also provides bifunctional compounds for use in therapies involving targeted mannose-6-phosphate (M6PR)-mediated lysosomal degradation of extracellular target molecules such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, cells, and cell membrane proteins. In certain embodiments, such therapies include the treatment of cardiovascular diseases, liver diseases, kidney diseases, autoimmune diseases, neurological diseases, hematological diseases, skin diseases, drug intoxication, or vasculitis. In certain embodiments, such therapies include the treatment of hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, obstructive arteriosclerosis, fulminant hepatic failure, postoperative hepatic failure, acute hepatic failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, allogeneic liver transplantation, focal segmental glomerulosclerosis, allogeneic kidney transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood group incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, or Kawasaki disease. In other embodiments, such therapies include the treatment of nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, or hepatocellular carcinoma (HCC).

[0257] In addition, the present invention provides a bifunctional compound for use in therapies, such as the treatment of cardiovascular diseases, liver diseases, kidney diseases, autoimmune diseases, neurological diseases, blood disorders, skin diseases, drug poisoning, or vasculitis. In certain embodiments, such therapies include the treatment of hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, obstructive arteriosclerosis, fulminant hepatic failure, postoperative hepatic failure, acute hepatic failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, allogeneic liver transplantation, focal segmental glomerulosclerosis, allogeneic kidney transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood group incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, or Kawasaki disease. In other embodiments, such therapies include the treatment of nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, or hepatocellular carcinoma (HCC).

[0258] The present invention further provides the use of the bifunctional compounds of the present invention for use in therapy, such as the treatment of cardiovascular disease, liver disease, kidney disease, autoimmune disease, neurological disease, hematological disease, skin disease, drug poisoning, or vasculitis. In certain embodiments, such therapies include the treatment of hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, obstructive arteriosclerosis, fulminant hepatic failure, postoperative hepatic failure, acute hepatic failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, allogeneic liver transplantation, focal segmental glomerulosclerosis, allogeneic kidney transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood group incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, or Kawasaki disease. In other embodiments, such therapies include the treatment of nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, or hepatocellular carcinoma (HCC).

[0259] In another embodiment, the present invention provides a method for treating diseases associated with elevated levels of extracellular target molecules such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, cells, and cell membrane proteins, the method utilizing targeted lysosomal degradation of such extracellular target molecules. In a particular embodiment, the present invention also provides a method for treating diseases associated with elevated levels of extracellular target molecules such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, cells, and cell membrane proteins, the method utilizing targeted asialoglycoprotein receptor (ASGPR)-mediated lysosomal degradation of extracellular target molecules. In certain embodiments, the present invention also provides methods for treating diseases associated with elevated levels of extracellular target molecules such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, and cell and cell membrane proteins, the methods utilizing targeted mannose-6-phosphate (M6PR)-mediated lysosomal degradation of the extracellular target molecules. These methods may be useful in treating a variety of diseases, conditions, or clinical situations that are often treated via therapeutic apheresis, such as cardiovascular diseases, liver diseases, kidney diseases, autoimmune diseases, neurological diseases, hematological diseases, skin diseases, drug intoxication, and vasculitis. Examples of such diseases include, but are not limited to, hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, obstructive arteriosclerosis, fulminant hepatic failure, postoperative hepatic failure, acute hepatic failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, allogeneic liver transplantation, focal segmental glomerulosclerosis, allogeneic kidney transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood group incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, and Kawasaki disease.These methods may also be useful in the treatment of nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0260] In a further embodiment, the present invention provides a method for treating cardiovascular disease, liver disease, kidney disease, autoimmune disease, neurological disease, blood disorder, skin disease, drug poisoning and vasculitis, the method comprising administering a therapeutically effective amount of the bifunctional compound of the present invention to the subject in need. In certain embodiments, such diseases include, but are not limited to, hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, obstructive arteriosclerosis, fulminant hepatic failure, postoperative hepatic failure, acute hepatic failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, allogeneic liver transplantation, focal segmental glomerulosclerosis, allogeneic kidney transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood group incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, and Kawasaki disease. In further embodiments, such diseases include nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0261] In a further embodiment, the present invention provides a method for treating cardiovascular disease, liver disease, kidney disease, autoimmune disease, neurological disease, hematological disease, skin disease, drug poisoning, and vasculitis, the method comprising administering the bifunctional compound of the present invention to a subject in need. In certain embodiments, such diseases include, but are not limited to, hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, obstructive arteriosclerosis, fulminant hepatic failure, postoperative hepatic failure, acute hepatic failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, allogeneic liver transplantation, focal segmental glomerulosclerosis, allogeneic kidney transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood group incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, and Kawasaki disease. In further embodiments, such diseases include nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0262] In another aspect, the present invention provides the use of the bifunctional compound of the present invention in the manufacture of pharmaceuticals for the treatment of cardiovascular diseases, liver diseases, kidney diseases, autoimmune diseases, neurological diseases, blood disorders, skin diseases, drug poisoning and vasculitis, the method comprising administering a therapeutically effective amount of the bifunctional compound of the present invention to the subject in need. In certain embodiments, such diseases include, but are not limited to, hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, obstructive arteriosclerosis, fulminant hepatic failure, postoperative hepatic failure, acute hepatic failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, allogeneic liver transplantation, focal segmental glomerulosclerosis, allogeneic kidney transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood group incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, and Kawasaki disease. In further embodiments, such diseases include nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0263] The present invention also provides a method for therapeutic plasmapheresis in the body, the method comprising administering the bifunctional compound of the present invention to a target.

[0264] The present invention also provides a method of therapeutic plasmapheresis in the body for the treatment of cardiovascular disease, liver disease, kidney disease, autoimmune disease, neurological disease, hematological disease, skin disease, drug poisoning, or vasculitis, the method comprising administering the bifunctional compound of the present invention to a target. In certain embodiments, such diseases include hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, obstructive arteriosclerosis, fulminant hepatic failure, postoperative hepatic failure, acute hepatic failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, allogeneic liver transplantation, focal segmental glomerulosclerosis, allogeneic kidney transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, gestational adenomatous incompatibility, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, and Kawasaki disease.

[0265] The present invention also provides a method for therapeutic plasmapheresis in the body, the method comprising administering a bifunctional compound of formula (I) to a target. The present invention also provides a method for therapeutic plasmapheresis in the body, the method comprising administering a bifunctional compound of the present invention to a target.

[0266] The present invention also provides a method of therapeutic plasmapheresis in the body for the treatment of cardiovascular disease, liver disease, kidney disease, autoimmune disease, neurological disease, hematological disease, skin disease, drug poisoning, or vasculitis, the method comprising administering a bifunctional compound of formula (I) to the target. In certain embodiments, such diseases include hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, obstructive arteriosclerosis, fulminant hepatic failure, postoperative hepatic failure, acute hepatic failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, allogeneic liver transplantation, focal segmental glomerulosclerosis, allogeneic kidney transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, gestational adenomatous incompatibility, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, and Kawasaki disease.

[0267] In one embodiment of the present invention, the present invention provides a bifunctional molecule that utilizes receptor-mediated endocytosis to remove proprotein convertase subtilisin / kexin type 9 (PCSK9) from plasma or to reduce the level of PCSK9 in plasma.

[0268] PCSK9 significantly influences plasma low-density lipoprotein cholesterol (LDL-C) levels by regulating the hepatic low-density lipoprotein receptor (LDLR), a major pathway through which cholesterol is removed from circulation. PCSK9 binds to LDLR and directs it towards lysosomal degradation, thereby increasing plasma LDL-C levels and subsequently increasing the risk of coronary heart disease. (Maxwell KN, Proc. Natl. Acad. Sci., 101, 2004, 7100-7105; Park, SW, J. Biol. Chem. 279, 2004, 50630-50638; Lagace TA, et. al. J. Clin. Invest. 2006, 116(11):2995-3005). Overexpression of mouse or human PCSK9 in mice has been shown to increase overall and LDL-C levels and dramatically decrease hepatic LDLR protein levels without any observed effects on the nucleocytoplasmic ratio of mRNA, SREBP, or SREBP protein (Maxwell KN, Proc. Natl. Acad. Sci. 101, 2004, 7100-7105). Furthermore, mutations in PCSK9 that cause loss of PCSK9 function in mouse models have also been shown to decrease overall and LDL-C levels (Cohen, JC, et al., N. Engl. J. Med., 354, 2006, 1264-1272). Therefore, it is shown that regulation of PCSK9 leads to a decrease in LDLR protein levels.

[0269] Furthermore, PCSK9 gene deletion has also been performed in mice. PCSK9 knockout mice show approximately 50% lower plasma cholesterol levels and increased sensitivity to statins in plasma cholesterol reduction (Rashid.S., et al., (2005) Proc Natl Acad Sci 102:5374-5379). Human genetic data strongly support the role of PCSK9 in LDL homeostasis. The association between PCSK9 and plasma LDL-C levels was first established by the discovery of PCSK9 missense mutations in patients with autosomal dominant familial hypercholesterolemia (Abifadel M., et al., Nature Genetics, 2003;34:154-156). Patients with the PCSK9 gain-of-function allele have elevated plasma LDL-C levels and an increased risk of early-onset coronary heart disease, while patients with the PCSK9 loss-of-function allele have significantly lower plasma LDL-C levels and are protected from coronary heart disease.

[0270] PCSK9 also plays a role in lipoprotein (α) (Lp(α)) metabolism. Lp(α) is an atherogenic lipoprotein composed of LDL particles covalently bound to apoLp(α). Human genetic studies have shown that Lp(α) is necessarily associated with the risk of coronary heart disease. PCSK9 therapeutic antibodies have been shown to significantly reduce Lp(α) levels in patients with hypercholesterolemia (Desai, NR, et.al., Circulation. 2013; 128(9): 962-969; Lambert, G. et.al., Clinical Science, 2017, 131, 261-268). Patients receiving statin therapy with monoclonal antibodies against PCSK9 showed up to a 32% reduction in Lp(α) levels compared to placebo. (Desai NR,et.al.Circulation.2013;128(9):962-969).

[0271] In addition to its cardiovascular effects, PCSK9 plays a crucial role in sepsis, a life-threatening condition caused by the body's response to infection. Overexpression of PCSK9 in septic mice has been shown to exacerbate sepsis by increasing inflammation, while inhibition of PCSK9 has been shown to reduce mortality (Dwivedi, DJ, et al., Shock, 2016, 46(6), 672-680). Furthermore, flow cytometry studies in human HepG2 cells have shown that PCSK9 negatively modulates Gram-negative glycolipid (LPS) uptake by hepatocytes through the regulation of LDLR-mediated bacterial lipid uptake of lipoteichoic acid (LTA) and LPS via an LDL-dependent mechanism (Grin, PM, et al., Nature, 2018, 8(1):10496). Therefore, inhibition of PCSK9 may treat sepsis by reducing the body's immune response to infection.

[0272] Atherosclerotic cardiovascular disease is the leading cause of death worldwide, accounting for an estimated 7.4 million deaths globally in 2015. LDL, the primary carrier of cholesterol in the bloodstream, is the most extensively studied modifiable risk factor associated with ASCVD [Ference BA, et al 2017]. Prospective cohort studies, Mendelian randomized studies, and randomized clinical trials have demonstrated a log-linear association between absolute exposure to LDL cholesterol and the risk of ASCVD [Baigent C, et al 2005, Ference BA, et al 2017].

[0273] PCSK9 is a 692-amino acid serine protease that significantly influences LDL-C levels through the modulation of the hepatic LDLR receptor, a major pathway by which cholesterol is removed from circulation [Brown MS and Goldstein JL 1986]. PCSK9 binds to LDLR and directs it to lysosomal degradation, thereby increasing plasma LDL-C levels and subsequently increasing the risk of ASCVD. PCSK9 possesses exceptional target validation. Mice lacking PCSK9 exhibit decreased plasma cholesterol and increased hepatic LDLR expression compared to littermates. Mice in which PCSK9 is selectively inactivated in the liver lack detectable PCSK9 in the blood, suggesting that the liver is a major source of circulating PCSK9 [Zaid et al, 2008]. Patients with the gain-of-function PCSK9 allele have elevated plasma LDL-C levels and an increased risk of early-onset ASCVD, while patients with the loss-of-function PCSK9 allele have significantly lower plasma LDL-C levels and are protected from ASCVD [Cohen J, et al 2006]. Therefore, there is great interest in identifying novel therapies that mimic PCSK9 loss of function.

[0274] Clinical studies with PCSK9 blocking antibodies have demonstrated significant reductions in LDL in healthy volunteers and hypercholesterolemia patients, both with and without statins [Banerjee et al, 2012; Dias et al, 2012; Roth et al, 2012; Stein et al, 2012; Sullivan et al, 2012]. Statins increase PCSK9 levels and limit the usefulness of dose escalation [Careskey et al, 2008; Welder et al, 2010]. Data from several clinical studies performed with inclisiran have demonstrated that reducing plasma PCSK9 through inhibition of protein synthesis in hepatocytes significantly lowers circulating LDL-C levels [Fitzgerald et al, 2017; Ray et al, 2017; Nishikido and Ray, 2018; Ray et al, 2019].

[0275] The present invention relates to bifunctional compounds and compositions that can lower the plasma level of PCSK9 or remove PCSK9 circulating in the plasma. This disclosure features a method for treating, preventing or relieving a disease or disorder in which PCSK9 plays a role by administering a therapeutically effective dose of the bifunctional compound of formula (Ia) to a patient in need. The method of the present invention can be used in the treatment of a variety of PCSK9-dependent diseases and disorders by lowering the plasma level of PCSK9 or removing PCSK9 circulating in the plasma. Lowering the plasma level of PCSK9 or removing PCSK9 circulating in the plasma provides a novel approach to the treatment, prevention or remission of diseases including, but not limited to, hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral vascular disease (including aortic and cerebrovascular diseases), peripheral artery disease, vasculitis, elevated Lp(a), elevated LDL, elevated TRL, elevated triglycerides, sepsis, and xanthomas.

[0276] The bifunctional compound of formula (Ia) of the present invention is useful in the treatment of hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, coronary heart disease, peripheral vascular disease, peripheral artery disease, vasculitis, elevated Lp(a), elevated LDL, elevated TRL (e.g., elevated VLDL and / or chylomicrons), elevated triglycerides, sepsis, and xanthomas by lowering the plasma level of PCSK9 or removing PCSK9 circulating in the plasma.

[0277] For example, the bifunctional compound of formula (Ia) of the present invention binds to PCSK9 and directs it toward removal via receptor-mediated endocytosis, thereby reducing the plasma level of PCSK9 or removing PCSK9 circulating in the plasma. Consequently, PCSK9 is not available to bind to low-density lipoprotein receptors (LDLRs) or any other target receptors, resulting in the presence of more LDLRs on the cell surface and removal of LDL particles from the extracellular fluid. Therefore, by reducing the plasma level of PCSK9 or removing PCSK9 circulating in the plasma, the concentration of LDL particles in the blood can be reduced.

[0278] Accordingly, the bifunctional compound of formula (Ia) of the present invention may be potentially useful in the treatment, prevention, remission, or delay of progression of PCSK9-mediated diseases or disorders or diseases or disorders in which PCSK9 plays a role, as well as conditions, diseases and disorders that benefit from a decrease in plasma levels of PCSK9 or the removal of PCSK9 circulating in the plasma. Such diseases and disorders include, but are not limited to, hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, coronary heart disease, peripheral vascular disease, peripheral artery disease, vasculitis, elevated Lp(a), elevated LDL, elevated TRL (e.g., elevated VLDL and / or chylomicrons), elevated triglycerides, sepsis, and xanthomas.

[0279] In addition, the bifunctional compounds of formula (Ia) of the present invention may therefore be potentially useful in the treatment, prevention, remission, or delay of progression of diseases or disorders requiring a decrease in plasma levels of PCSK9 or removal of PCSK9 circulating in the plasma. Such diseases and disorders include those selected from hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, coronary heart disease, peripheral vascular disease, peripheral artery disease, vasculitis, elevated Lp(a), elevated LDL, elevated TRL (e.g., elevated VLDL and / or chylomicrons), elevated triglycerides, sepsis, and xanthomas.

[0280] Another aspect of the present invention is a bifunctional molecule that utilizes receptor-mediated endocytosis to remove or reduce the level of complement factor H-related protein 3 (FHR3) from plasma.

[0281] Complement-mediated immune responses are robustly regulated by several endogenously produced proteins that modulate activity and distinguish between healthy self (inactivated) and pathogenic activated cells that are damaged or non-self. These complement regulatory proteins range from those that bind to the cell surface (i.e., CR1, MCP, DAF) to circulating proteins (i.e., factor H and C4BP) that are recruited to the host self-surface by binding to polysaccharides such as glycosaminoglycans on the host self-surface and inactivate complement (Mol Immuno 47(13):2187-2197). Complement regulation is robustly controlled to maintain homeostasis, and its dysregulation and deficiency are associated with many diseases because they cause it to target host cells.

[0282] Factor H (FH), the primary negative regulator of alternative complement pathway activation, belongs to a family that also includes five other related family members thought to have arisen from non-alleletic homologous recombination and interlocal gene conversion: complement factor H-related protein 1 (FHR1), complement factor H-related protein 2 (FHR2), complement factor H-related protein 3 (FHR3), complement factor H-related protein 4 (with isoforms 4A and 4B, FHR4A and FHR4B), and complement factor H-related protein 5 (FHR5).

[0283] Due to the central role factor H plays in complement regulation, many clinical associations arise from abnormal FH activity. Loss-of-function mutations in factor H increase susceptibility to kidney disease, atypical hemolytic uremic syndrome (aHUS), and dense deposit disease (DDD), while polymorphic mutations in complement factor H are strongly associated with important human diseases, including age-related macular degeneration (AMD) and meningococcal sepsis (Clin Exp Immunol 151(2):210-230;Immunobiology 217(11):1034-1046).

[0284] Unlike factor H, FHR3 lacks the complement regulatory domain essential for complement inactivation and competes with factor H, leading to excessive complement activation. Therefore, the present invention provides a bifunctional compound for use in regulating the concentration of complement factor H protein, specifically FHR3, to eliminate a competitor of factor H and thereby restore factor H-mediated regulation that treats the damage caused by excessive complement activation.

[0285] The present invention also relates to bifunctional compounds and compositions that can reduce plasma levels of FHR3 or remove FHR3 circulating in the plasma. This disclosure features a method for treating, preventing or relieving FHR3-related diseases or disorders by administering a therapeutically effective dose of the bifunctional compound of formula (Ib) to a patient in need. The method of the present invention can be used in the treatment of various FHR3-related diseases or disorders by reducing plasma levels of FHR3 or removing FHR3 circulating in the plasma. Reducing plasma levels of FHR3 or removing FHR3 circulating in the plasma provides a novel approach to the treatment, prevention or remission of diseases including, but not limited to, nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome and hepatocellular carcinoma (HCC).

[0286] The bifunctional compound of formula (Ib) of the present invention is useful in the treatment of nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC) by reducing plasma levels of FHR3 or removing FHR3 circulating in the plasma.

[0287] For example, the bifunctional compound of formula (Ib) of the present invention binds to FHR3 and directs it toward removal via receptor-mediated endocytosis, thereby lowering plasma levels of FHR3 or removing FHR3 circulating in the plasma. Therefore, the bifunctional compound of formula (Ib) of the present invention may be potentially useful in the treatment, prevention, remission, or delay of progression of complement-mediated diseases or disorders such as nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC). [Examples]

[0288] The present invention is further described in the following embodiments, which are not intended to limit the scope of the invention as described in the claims.

[0289] Temperatures are expressed in Celsius. Unless otherwise specified, all evaporation is carried out under reduced pressure, typically at approximately 15 mmHg to 100 mmHg (= 20 to 133 mbar). The structures of the final product, intermediates, and starting materials are confirmed by standard analytical methods, e.g., trace analysis or spectroscopic properties, e.g., MS, IR, or NMR. Abbreviations used are those conventional in the art.

[0290] All starting materials, components, reagents, acids, bases, dehydrating agents, solvents, and catalysts used to synthesize the compounds of the present invention are commercially available, can be produced by organic synthesis methods known to those skilled in the art, or can be produced by organic synthesis methods as described herein.

[0291] The abbreviations used in the following examples and elsewhere in this specification are as follows: AA: Amino Acids Ac: Acetyl Ac2O: Acetic anhydride ACN: Acetonitrile aq.: water-based AM: Aminomethyl Boc:tert-butoxycarbonyl BnOH: Benzyl alcohol BSA: Bovine serum albumin DBU:1,8-Diazabicyclo[5.4.0]Undeca-7-En DCM: Dichloromethane DIC: N,N'-Diisopropylcarbodiimide DTT: Dithiothreitol DMA: Dimethylacetamide DMAP: 4-dimethylaminopyridine DMF: N,N-dimethylformamide DMSO: Dimethyl sulfoxide DIEA or DIPEA: N,N-diisopropylethylamine EA: Ethyl acetate EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EDT: Ethanediol eq.: equivalent ESI-MS: Electrospray Ionization Mass Spectrometry Et and chromate: ethyl and ethyl acetate Fmoc: Fluorenylmethyloxycarbonyl FRET: Fluorescence Resonance Energy Transfer HATU:O-(7-azobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HCTU:O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HEPES: 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid HFIP: Hexafluoroisopropanol HILIC: (Hydrophilic Interaction Liquid Chromatography) HOAt: 1-Hydroxy-7-Azabenzotriazole HOBt: Hydroxybenzotriazole HPLC: High-Pressure Liquid Chromatography h, hr: time HRMS: High resolution mass spectrometry IC50: 50% inhibitory concentration LC and LCMS: Liquid chromatography and liquid chromatography-mass spectrometry LDLR: Low-density lipoprotein receptor min: minutes Me: Methyl MS: Mass m / z: mass-to-charge ratio M and mM: Molar concentration and millimolar concentration mg: milligrams μL, mL, and L: microliter, milliliter, and liter N: Equivalent amount per liter NMP:N-methyl-2-pyrrolidone Oxima pure: 2-Cyano-2-(hydroxyimino)ethyl acetate, potassium salt, (hydroxyimino)cyanoethyl acetate potassium salt PBS: Phosphate-buffered saline PD: Pharmacodynamics PE: Petroleum ether PG: Protecting group PS: Polystyrene resin PyOxim:[ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl PCSK9: Proprotein convertase subtilisin / kexin type 9 Ph: Phenyl RP: Out of phase rpm: revolutions per minute rt: room temperature RU: Resonance Unit SPPS: Solid-phase peptide synthesis sat.: saturation tBu: Tertiary butyl TBAI: Tetrabutylammonium iodide TBTU:2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate TEA: Triethylamine THF: Tetrahydrofuran TentaGel(trademark) S RAM resin: N-Fmoc-4'-[poly(oxyethylene)carbamoylmethoxy]-2,4-dimethoxy-benzhydrylamine polymer bond, poly(oxyethylene)-RAM polymer bond TFA: Trifluoroacetic acid THPTA:tris-hydroxypropyltriazolylmethylamine TIS: Triisopropylsilane TLC: Thin-layer chromatography TMSOTF or TMSOTf: Trimethylsilyltrifluoromethanesulfonate tr: retention time TR: Time-decomposed TMSCl: Trimethylsilyl chloride Trt: Trichil TsOH: UPLC: Ultra-high-performance liquid chromatography UV: Ultraviolet light wt: weight

[0292] Analytical methods, materials, and measurement means Unless otherwise noted, reagents and solvents were used as received from the commercial suppliers. Proton nuclear magnetic resonance (NMR) spectra were obtained using a Varian spectrometer at 400 MHz and a Bruker spectrometer at 300 MHz or 400 MHz. Spectra are given in ppm(δ), and the coupling constant J is reported in Hertz. Tetramethylsilane (TMS) or solvent peaks were used as internal standards. Unless otherwise noted, purity and low-resolution mass spectrometry data were measured using a Thermo Finnigan Surveyor HPLC system with Surveyor photodiode array (PDA) detection and a Thermo LCQ Fleet™ ion trap mass spectrometer. Column: Synergi 4 micron, hydro-RP80A, 30 × 2.0 mm, flow rate: 0.500 mL / min; Solvent A (water + 0.1% formic acid), Solvent B (acetonitrile + 0.1% formic acid); Gradient: 2% B at t=0, 95% B at 3 min, and 95% B at 3.3 min.

[0293] General preparative HPLC purification procedure and mass spectrum Depending on the amount of crude peptide to be purified, different column sizes were used and the flow rate was varied to purify the crude peptide by preparative reverse-phase C18-HPLC. For example, 0.1% TFA(A) in water and 0.1% TFA(B) in acetonitrile were used as eluents. The product-containing fraction was collected and freeze-dried to obtain the purified product.

[0294] Condition E-1 (LCMS) - Column: Acquity UPLC(registered trademark) BEH C18, 300 Å, 1.7 μm 2.1 × 50 mm, 80°C; Flow rate: 1.0 mL / min; Mobile phase: (A) 0.5% TFA in water / (B) 0.4% TFA in acetonitrile; Gradient: 5% to 98% over 4.4 mins; Electrospray mass spectrum (+), DAD-UV chromatogram 214 nm.

[0295] Analysis method 1 Agilent 1100 / 1200 ALS System / Waters ZQD MS System Elution A: 0.05% trifluoroacetic acid in H2O Elutate B: Acetonitrile Column temperature: 40℃ Flow rate: 2.0mL / min Column: SunFire C18, 3.5 μm, 3.0 × 30 mm gradient:

[0296] [Table 28]

[0297] Analysis method 2 Waters Acquity UPLC System / Waters SQD MS System Elution A: 5 mM ammonium hydroxide in H2O Elution B: 5 mM ammonium hydroxide in acetonitrile Column temperature: 50℃ Flow rate: 1.0mL / min Column: Acquity UPLC BEH C18, 1.7 μm, 2.1 × 50 mm gradient:

[0298] [Table 29]

[0299] Analysis method 3 Waters Acquity UPLC System / Waters Xevo G2 Qtof MS System Elution A: 0.1% formic acid in H2O Elutate B: 0.1% formic acid in acetonitrile Column temperature: 50℃ Flow rate: 1.0mL / min Column: Acquity UPLC BEH C18, 1.7 μm, 2.1 × 50 mm gradient:

[0300] [Table 30]

[0301] Analysis method 5 Waters Acquity UPLC System / Waters SQD MS System Elution A: 5 mM ammonium hydroxide in H2O Elution B: 5 mM ammonium hydroxide in acetonitrile Column temperature: 50℃ Flow rate: 1.0mL / min Column: Acquity UPLC BEH C18, 1.7 μm, 2.1 × 30 mm gradient:

[0302] [Table 31]

[0303] Analysis method 7 Waters Acquity UPLC System / Waters SQD MS System Elution A: 0.1% formic acid in H2O Elutate B: 0.1% formic acid in acetonitrile Column temperature: 50℃ Flow rate: 1.0mL / min Column: Acquity UPLC BEH C18, 1.7 μm, 2.1 × 30 mm gradient:

[0304] [Table 32]

[0305] Analysis method 9 Waters Acquity UPLC / Waters QTof MS System Elution A: 0.05% trifluoroacetic acid in H2O Elutate B: 0.04% trifluoroacetic acid in acetonitrile Column temperature: 80℃ Flow rate: 0.5mL / min Column: AcQuity UPLC CSH C18, 1.7μm, 2.1mm x 100mm gradient:

[0306] [Table 33]

[0307] Analysis method 10 Waters Acquity UPLC / SQD MS System Elution A: 0.05% formic acid and 3.75 mM ammonium acetate in H2O Elutate B: 0.04% formic acid in acetonitrile Column temperature: 60℃ Flow rate: 1.0mL / min Column: Acquity UPLC HSS T3, 1.8 μm, 2.1 mm × 50 mm gradient:

[0308] [Table 34]

[0309] Analysis method 11 Waters Acquity UPLC / SQD MS System Elution A: 0.05% formic acid and 3.75 mM ammonium acetate in H2O Elutate B: 0.04% formic acid in acetonitrile Column temperature: 60℃ Flow rate: 1.0mL / min Column: Acquity UPLC HSS T3, 1.8 μm, 2.1 mm × 50 mm gradient:

[0310] [Table 35]

[0311] Analysis method 12 Waters Acquity UPLC / SQD MS System Elution A: 0.05% formic acid and 3.75 mM ammonium acetate in H2O Elutate B: 0.04% formic acid in acetonitrile Column temperature: 60℃ Flow rate: 1.0mL / min Column: Acquity UPLC HSS T3, 1.8 μm, 2.1 mm × 50 mm gradient:

[0312] [Table 36]

[0313] General procedure for cyclic peptide synthesis: The following general scheme can be used to obtain compound cyclic peptides such as compounds (C1) to (C4). [ka]

[0314] Step 1: Peptide synthesis Solid-phase peptide synthesis on the Liberty® peptide synthesizer from CEM, Inc. Synthesis Cycle A-1 The resin was washed with DMF and then deprotected by two cycles of treatment with 4-methylpiperidine / DMF (1:4) – the first cycle for 30 seconds and the second cycle for 3 minutes. Coupling was performed by adding Fmoc amino acids (4-5 equivalents; 0.2 M solution in DMF), HATU (4-5 equivalents; 0.5 M solution in DMF), and DIPEA (4-6 equivalents; 2 M solution in NMP). The coupling and deprotection steps were repeated until the desired cyclic polypeptide was obtained. All Fmoc amino acids were coupled at 75°C for 5 minutes, except for the amino acids shown in Table 3 below. After the final coupling was complete, Fmoc was removed by repeated treatment with 4-methylpiperidine / DMA (1:4) to obtain the deprotected peptide.

[0315] [Table 37]

[0316] Solid-phase peptide synthesis on the Prelude® peptide synthesizer from Gyros Protein Technologies AB. Instead, the peptides were synthesized on a Prelude® peptide synthesizer as described in synthesis cycle B-1 or synthesis cycle B-2.

[0317] Synthesis Cycle B-1 The resin was washed with DMA. Next, Fmoc was removed by repeated treatment of the resin with piperidine / DMA (1:4). Coupling was carried out by mixing the suspension with nitrogen for 15 minutes to 4 hours at room temperature, depending on the specific requirements, following the addition of Fmoc-amino acid (3 equivalents; 0.2 M solution in NMP), HCTU (3 equivalents; 0.3 M solution in NMP), and DIPEA (3-6 equivalents; 0.66-0.9 M solution in NMP). After washing with DMA, the coupling step was repeated. After washing with DMA, capping was carried out by adding a mixture of Ac2O / pyridine / DMA (1:1:8) and then mixing the suspension at room temperature. After the final coupling was completed, the peptide was obtained by removing Fmoc as described above in synthesis cycle A-1.

[0318] Synthesis Cycle B-2 The resin was washed with DMA. Fmoc was removed by repeated treatment with 4-methylpiperidine / DMA (1:4). Coupling was performed by adding a mixture of Fmoc-amino acids (3 equivalents; 0.2 M solution in NMP), Oxyma Pure (3 equivalents; 0.3 M solution in NMP), and DIPEA (6-7 equivalents; 0.66 M solution in NMP), followed by mixing of the suspension with nitrogen at room temperature for 15 minutes to 4 hours, depending on the specific requirements. After washing with DMA, the coupling step was repeated. After washing with DMA, capping was performed by adding a mixture of Ac2O / pyridine / DMA (1:1:8) and subsequently mixing of the suspension at room temperature. After the final coupling was completed, Fmoc was removed in synthesis cycle A-1 as described above to obtain the peptide.

[0319] Step 2: Peptide acylation The resin product obtained from step 1 was suspended in N-methylpyrrolidine, and 5 equivalents of N-succinimidyl 2-chloroacetate were added. The resulting resin mixture was shaken overnight at room temperature. Next, the resin was filtered and washed three times with dimethylformamide and dichloromethane, respectively, to obtain acylated peptide products.

[0320] Step 3: Cutting from the resin with or without simultaneous removal of the protective layer (PG). The resin product obtained from step 2 was shaken for 1–3 hours with one of the cleavage solutions listed below in this specification (1–5 mL per 0.1 mmol scale). The resin was filtered and treated again with fresh cleavage solution for 0.5–1.5 hours. The cleavage cycle was repeated as needed. Next, the resin was filtered and the combined filtrate was slowly poured onto a mixture of cold heptane / diethyl ether (1:1) to obtain a precipitate. The suspension containing the precipitate was centrifuged and the supernatant was discarded. The precipitate was suspended in cold ether, vortexed briefly, and then centrifuged. This washing process was repeated two more times. The crude peptide product was dried under high vacuum.

[0321] The following cutting solution was used: Cutting method 1: TFA / H2O / TIS / DTT(92.5:2.5:2.5:2.5) Cutting method 2: 95% aqueous TFA / EDT / TIS (95:2.5:2.5).

[0322] Step 4: Peptide cyclization The crude peptide product obtained from step 3 was dissolved in DMSO or DMA and treated with TEA or DIPEA. Next, the reaction mixture was shaken overnight at room temperature. The resulting reaction mixture containing the cyclized peptide was concentrated on a centrifugal evaporator to obtain the desired cyclic polypeptide.

[0323] Example 1: Synthesis of PCSK9 receptor ligand compounds (C1) to (C11) Example 1-1:3-((6S,9S,12S,15S,18S,21S,24S,27S,29aS,35S,38S,44R,46aS)-15,21-bis([1,1'-biphenyl]-4-ylmethyl)-38-benzyl-44-((2-(((S)-1,6-diamino-1-oxohexane-2-yl)amino)-2-oxoethyl)carbamoyl)-24,27-bis((R)-1-hydroxyethyl)-35- Synthesis of sopropyl-6,12,13,18,19-pentamethyl-5,8,11,14,17,20,23,26,29,34,37,40,46-tridecaoxotetratetracontahydro-5H-dipyrrolo[2,1-f:2',1'-g1][1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40]tridecazacyclodotetracontin-9-yl)propanoic acid (C1) [ka] Note: Compound (C1) has the following amino acid sequence. [ka] Step 1: The peptide sequence FVPTTB-(N-Me)AB-(N-Me)AEAPC(Trt)-GK-NH-resin (1-1b) was synthesized on Fmoc-RAM TentaGel® resin (1-1a, 0.22 mmol / g loading, 0.25 mmol scale) on a Liberty® peptide synthesizer. The resin was then filtered and washed with DMF (2×) and DCM (3×) to obtain FVPTTB-(N-Me)AB-(N-Me)AEAPC(Trt)-GK-NH-resin (1-1b).

[0324] Step 2:A solution of N-succinimidyl 2-chloroacetate (1-1c, 287 mg, 1.5 mmol) in NMP (8 mL) was added to peptide resin 1-1b (0.25 mmol) from step 1, and the resulting mixture was shaken overnight at room temperature. Next, the resin was drained, washed with DMF (3 ×) and DCM (4 ×), and dried to obtain tClCH2C(=O)-FVPTTB-(N-Me)AB-(N-Me)AEAPC(Trt)-GK-NH resin (1-1d).

[0325] Step 3: The peptide resin product 1-1d from step 2 was cleaved from the resin and simultaneously deprotected using cleavage method 1 described herein to obtain the crude peptide ClCH2C(O)-FVPTTB-(N-Me)AB-(N-Me)AEAPCGK-NH2(1-1e)(266 mg). Analytical method 1:t R =1.22 minutes; M+H+2 / 2 921.8.

[0326] Step 4:Crude peptide 1-1e (460 mg, 0.25 mmol) from step 3 was dissolved in DMSO (25.4 mL). A few drops of TEA were added to adjust the pH to 8-9. The resulting mixture was stirred overnight at room temperature. Next, the reaction mixture was concentrated on a centrifugal evaporator until it reached a few mL of DMSO. The crude cyclic peptide was subjected to preparative HPLC (Sunfire® Prep). The compound was purified by C18 column (130 Å, 5 μm, 30 × 50 mm, 15-40% at 6 min, 75 mL / min, ACN in water containing 0.1% TFA), followed by lyophilization to obtain the cyclic peptide compound 3-((6S,9S,12S,15S,18S,21S,24S,27S,29aS,35S,38S,44R,46aS)-15,21-bis([1,1'-biphenyl]-4-ylmethyl)-38-benzyl-44-((2-(((S)-1,6-diamino-1-oxohexane-2-yl)amino)-2-oxoethyl (Carbamoyl)-24,27-bis((R)-1-hydroxyethyl)-35-isopropyl-6,12,13,18,19-pentamethyl-5,8,11,14,17,20,23,26,29,34,37,40,46-tridecaoxotetratetracontahydro-5H-dipyrrolo[2,1-f:2',1'-g1][1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40]tridecazacyclodotetracontin-9-yl)propanoic acid (C1) (SEQ ID NO: 1) was obtained. Analytical method 3: t R =0.45 minutes, M+2 / 2 903.7.

[0327] Examples 1-2:2-((3R,9S,12S,15S,18S,21S,24S,27S,30S,33S,36S,39S,44aS)-30-([1,1'-biphenyl]-4-ylmethyl)-3-((2-amino-2-oxoethyl)carbamoyl)-36-(4-aminobutyl)-9-benzyl-18,21-bis((R)-1-hydroxyethyl)-24,39-bis(hydroxymethyl) Synthesis of -12-isopropyl-26,27,32,33-tetramethyl-1,7,10,13,16,19,22,25,28,31,34,37,40-tridecaoxodotetracontahydro-6H-pyrrolo[2,1-f][1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40]tridecazacyclodotetracontin-15-yl)acetic acid (C2) [ka] Note: Compound (C2) has the following amino acid sequence. [ka] Step 1: The peptide sequence FVDTTS-(N-Me)AB-(N-Me)AKSPC(Trt)-G-NH-resin (1-2b) was synthesized on Fmoc-Gly-RAM TentaGel® resin (1-2a, 0.22 mmol / g loading, 0.25 mmol scale) on a Liberty® peptide synthesizer. The resin was then filtered and washed with DMF (2×) and DCM (3×) to obtain FVDTTS-(N-Me)AB-(N-Me)AKSPC(Trt)-G-NH-resin (1-2b).

[0328] Step 2:A solution of N-succinimidyl 2-chloroacetate (1-2c, 287 mg, 1.5 mmol) in NMP (8 mL) was added to peptide resin 1-2b (0.25 mmol) from step 1, and the resulting mixture was shaken overnight at room temperature. Next, the resin was drained, washed with DMF (3 ×) and DCM (4 ×), and dried to obtain ClCH2C(=O)-FVDTTS-(N-Me)AB-(N-Me)AKSPC(Trt)-G-NH resin (1-2d).

[0329] Step 3: The peptide resin products 1-2d from step 2 were cleaved from the resin and simultaneously deprotected using cleavage method 1 described above herein to obtain the crude peptide ClCH2C(O)-FVDTTS-(N-Me)AB-(N-Me)AKSPCG-NH2(1-2e)(266 mg).

[0330] Step 4:Crude peptides 1-2e (266 mg) from step 3 were dissolved in DMSO (20.5 mL). A few drops of TEA were added to adjust the pH to 8-9. The resulting mixture was stirred overnight at room temperature. Next, the reaction mixture was concentrated on a centrifugal evaporator until it reached a few mL of DMSO. The crude cyclic peptide was subjected to preparative HPLC (Sunfire® Prep). The compound was purified by C18 column (130 Å, 5 μm, 30 × 50 mm, 15-40% at 6 min, 75 mL / min, ACN in water containing 0.1% TFA), followed by lyophilization to obtain the cyclic peptide compound 2-((3R,9S,12S,15S,18S,21S,24S,27S,30S,33S,36S,39S,44aS)-30-([1,1'-biphenyl]-4-ylmethyl)-3-((2-amino-2-oxoethyl)carbamoyl)-36-(4-aminobutyl)-9-benzyl-18,21 -Bis((R)-1-hydroxyethyl)-24,39-bis(hydroxymethyl)-12-isopropyl-26,27,32,33-tetramethyl-1,7,10,13,16,19,22,25,28,31,34,37,40-tridecaoxodotetracontahydro-6H-pyrrolo[2,1-f][1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40]tridecazacyclodotetracontin-15-yl)acetic acid (C2) (SEQ ID NO: 2) was obtained. Analytical method 9:t R =7.88, m+1=1573.8;(m+2) / 2=787.3.

[0331] The cyclic peptide compounds (C3) and (C4) in Table 4 below were obtained using a similar method as described in Examples 1-2, except that the corresponding peptide sequences synthesized in Step 1 for the cyclic peptide compounds (C3) and (C4) are also given in Table 4, instead of the peptide sequences (1-2b) synthesized in Step 1.

[0332] [Table 38]

[0333] The analytical data for the cyclic peptide compounds (C3) and (C4) are summarized in Table 5 below and were obtained using analytical method E-1 as described herein.

[0334] [Table 39]

[0335] Examples 1-3: 3-((6S,9S,12S,15S,18S,21S,24S,27S,29aS,35S,38S,44R,46aS)-15,21-bis([1,1'-biphenyl]-4-ylmethyl)-44-((2-(((S)-1-amino-1-oxo-6-(4-oxopentanamide)hexane-2-yl)amino)-2-oxoethyl)carbamoyl)-38-benzyl-24,27-bis((R)-1-hydroxyethyl Synthesis of (L)-35-isopropyl-6,12,13,18,19-pentamethyl-5,8,11,14,17,20,23,26,29,34,37,40,46-tridecaoxotetratetracontahydro-5H-dipyrrolo[2,1-f:2',1'-g1][1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40]tridecazacyclodotetracontin-9-yl)propanoic acid (C5) [ka] To a room temperature solution of cyclic peptide (C1) (75 mg, 0.039 mmol) in DMSO (1 mL), DIPEA (0.020 mL, 0.117 mmol) and 2,5-dioxopyrrolidine-1-yl 4-oxopentanoate (0.130 mL, 0.117 mmol) were added in DMSO (1 mL). The reaction product was directly purified by C18 flash chromatography (30 g column, 15 minutes over 0-80% ACN / water), and the pure fraction was dried using Genevac to obtain cyclic peptide (C5). Analytical method 7:t R =1.07 minutes, M-1=951.3

[0336] Synthesis of intermediates Synthesis of (R)-2-benzyl-4-(tert-butoxy)-4-oxobutanoic acid (int-A1) [ka] Process 1. To a cold, stirred solution of (S)-4-benzyloxazolidine-2-one (500 g, 2.821 mol) in THF (9 L), n-BuLi (2.5 M in hexane) (1.24 L, 3.103 mol) was slowly added over 30 minutes at -78°C, and the resulting mixture was stirred at -78°C for 30 minutes. Next, a solution of 3-phenylpropanoyl chloride (571 g, 3.38 mol) in THF (1 L) was slowly added over 1 hour at -78°C to -60°C, and the reaction mixture was slowly warmed to room temperature for 2 hours. The reaction mixture was cooled to 0°C, quenched with saturated NH4Cl (500 mL), and extracted with dichloromethane (2 × 1.5 L). The combined organic layers were washed with 0.5 N NaOH (1 L) and brine (1 L), dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. Crude (S)-4-benzyl-3-(3-phenylpropanoyl)oxazolidine-2-one (int-A1-1) was triturated with petroleum ether (5 L) for 1 hour. The solid product was filtered, washed with petroleum ether (500 mL), and dried under vacuum to obtain (S)-4-benzyl-3-(3-phenylpropanoyl)oxazolidine-2-one (int-A1-1). Analytical method 7;t R =1.53 minutes;[M+H] + = 310.2.

[0337] Process 2.A cold stirred solution of (S)-4-benzyl-3-(3-phenylpropanoyl)oxazolidin-2-one (int-A1-1) (500 g, 1.616 mol) in THF (7 L) was slowly added dropwise with 1.0 M NaHMDS in THF (1.94 L, 1.939 mol) at -78 °C over 30 minutes. The resulting mixture was stirred at -78 °C for 1 hour, and then a solution of tert-butyl 2-bromoacetate (472.8 g, 2.424 mol) in THF (500 mL) was added dropwise at -78 °C over 30 minutes. The mixture was stirred for 2 hours, then quenched with saturated NH4Cl (500 mL) and extracted with ethyl acetate (2 × 1.5 L). The combined organic layers were washed with aqueous brine (2 L), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude material was triturated with methanol (800 mL) for 1 hour, then the solid product was filtered, washed with methanol (200 mL), and dried under vacuum to give (R)-tert-butyl 3-benzyl-4-((S)-4-benzyl-2-oxooxazolidin-3-yl)-4-oxobutanoate (int-A1-2). Analytical method 7;t R = 1.78 min; [M-tBu] + = 368.3.

[0338] Process 3.To a cold, stirred solution of 3-benzyl-4-((S)-4-benzyl-2-oxoxazolidine-3-yl)-4-oxobutanoic acid (R)-tert-butyl(int-A1-2) (250 g, 0.59 mol) in THF (9 L), 30% H2O2 (267 mL, 2.37 mol) was added at 0-5°C, and the reaction mixture was stirred at the same temperature for 30 minutes. Next, a solution of LiOH.H2O (49.5 g, 1.18 mol) in water (3 L) was added to the above reaction mixture at 0-5°C, and the mixture was stirred for 1 hour. The reaction mixture was quenched with saturated sodium sulfate (1.6 L) and saturated sodium bicarbonate (1.6 L). Next, the solvent was concentrated under reduced pressure, diluted with water (3 L), and washed with DCM (2 × 1 L) to remove impurities. Next, the aqueous layer was cooled to 5°C and acidified to approximately 1.5 pH with 6M HCl (1 L). The product was extracted with ethyl acetate (3 × 1 L). The combined organic layers were washed with a salt solution (1 L), dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain (R)-2-benzyl-4-(tert-butoxy)-4-oxobutanoic acid (int-A1). Analytical method 7;t R =1.78 minutes;[MH] - = 263.5. 1 ¹H NMR (400MHz, chloroform-d) δ: 1.42 (s, 9H), 2.36 (dd, J=16.93, 4.58Hz, 1H), 2.48-2.62 (m, 1H), 2.71-2.83 (m, 1H), 3.00-3.18 (m, 2H), 7.12-7.35 (m, 6H).

[0339] Synthesis of ((1S,2S)-2-(methylamino)cyclohexyl)carbamate tert-butyl(int-B1) [ka] Process 1.4-nitrobenzenesulfonyl chloride (23.29 g, 105 mmol) was added at 0°C to a solution of (1S,2S)-(+)-1,2-diaminocyclohexane (12 g, 105 mmol) and triethylamine (21.97 mL, 158 mmol) in DCM (200 mL), and the mixture was stirred at the same temperature for 30 minutes. The reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The progress of the reaction was monitored by TLC (80% ethyl acetate in petroleum ether). The reaction mixture was concentrated, diluted with water, and the solid was precipitated. The precipitate was filtered, washed with excess water and ethylacetate, and dried under vacuum to obtain N-((1S,2S)-2-aminocyclohexyl)-4-nitrobenzenesulfonamide (int-B1-1). Analytical method 7;t R =0.74 minutes;[M+H] + = 300.2. 1 HNMR(300MHz,CDCl3):δ 8.33-8.31(d,J=8.8Hz,2H),8.05-8.02(d,J=8.8Hz,2H),6.19-6.18(d,J=4.8Hz,1H),4.40-4.38(d,J=7.6Hz,1H),3.35-3.33(d,J=10 .4Hz,1H),2.97-2.91(m,1H),2.01-1.93(m,2H),1.73-1.68(m,2H),1.67-1.65(d,J=6.8Hz,1H),1.52-1.47(m,9H),1.29-1.16(m,4H).

[0340] Process 2. Boc-anhydrous (13.70 mL, 59.0 mmol) was added to a stirred solution of N-((1S,2S)-2-aminocyclohexyl)-4-nitrobenzenesulfonamide (int-B1-1) (17.66 g, 59.0 mmol) in DCM (200 mL), and the mixture was stirred for 3 hours. The progress of the reaction was monitored by TLC (50% ethyl acetate in petroleum ether). The reaction mixture was concentrated under reduced pressure to obtain ((1S,2S)-2-((4-nitrophenyl)sulfonamide)cyclohexyl)carbamate tert-butyl (int-B1-2). Analytical method 7;t R =1.14 minutes;[M-Boc+H] + = 299.9. 1HNMR(400MHz,CDCl3):δ 8.33-8.31(d,J=9.2Hz,2H),8.04-8.02(d,J=8.8Hz,2H),6.17-6.16(d,J=4.8Hz,1H),4.38-4.36(d,J=7.2Hz,1H),3.35-3.3 2(m,1H),2.96-2.91(m,1H),2.02-1.92(m,2H),1.73-1.65(m,2H),1.52-1.46(m,6H),1.36-1.27(m,9H),1.28-1.21(m,4H).

[0341] Process 3. Methyl iodide (18.19 mL, 294 mmol) was added to a stirred solution of ((1S,2S)-2-((4-nitrophenyl)sulfonamide)cyclohexyl)carbamate tert-butyl (int-B1-2) (23.5 g, 58.8 mmol) and Cs2CO3 (47.9 g, 147 mmol) in DMF (200 mL), and the mixture was stirred at the same temperature for 3 hours. The progress of the reaction was monitored by TLC (40% ethyl acetate in petroleum ether). The reaction mixture was diluted with water (300 mL) and extracted with ethyl acetate (300 mL). The organic layer was washed with water and brine and dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure to obtain the crude compound. The crude compound was purified by normal-phase chromatography using silica gel (100-200 mesh) column chromatography with elution in 0-30% ethyl acetate in petroleum ether as the solvent to obtain ((1S,2S)-2-((N-methyl-4-nitrophenyl)sulfonamide)cyclohexyl)carbamate tert-butyl (int-B1-3). Analytical method 7;t R =1.22 minutes;[M-Boc] + = 313.9. 1HNMR(300MHz,CDCl3):δ 8.36-8.35(d,J=6.8Hz,2H),8.00-7.97(d,J=8.8Hz,2H),4.48(s,1H),4.14-4.09(m,1H),3.56-3.54(d,J=6.4Hz ,2H),2.88(s,3H),2.12-2.10(m,1H),2.04(s,1H),1.72-1.70(d,J=7.6Hz,2H),1.41(s,9H),1.27-1.23(m,4H).

[0342] Step 4. A mixture of ((1S,2S)-2-((N-methyl-4-nitrophenyl)sulfonamide)cyclohexyl)carbamate tert-butyl (int-B1-3) (1.55 g, 3.75 mmol), Cs2CO3 (8.55 g, 26.2 mmol), and 2-mercaptoacetic acid (1.524 mL, 14.99 mmol) in a DMF:MeOH (1:1, 12 mL) mixture was stirred at room temperature for 1 hour. The progress of the reaction was monitored by TLC (10% MeOH in DMF). The reaction mixture was diluted with water (100 mL) and ethyl acetate (100 mL). The aqueous layer was extracted with ethyl acetate (100 mL). The organic layers were combined, washed with brine, and dried over Na2SO4. The organic layers were concentrated under reduced pressure to obtain the crude product. The crude product was purified by normal-phase chromatography using silica gel (100-200 mesh) column chromatography with elution using 0-5% MeOH in DCM as the eluent, to obtain ((1S,2S)-2-((methylamino)cyclohexyl)carbamate tert-butyl (int-B1). Analytical method 7;t R =0.75 minutes;[M+H] + = 229.3. 1 H NMR(300MHz,DMSO-d6):δ 6.26-6.18(m,1H),3.08-3.06(m,1H),2.35(s,3H),2.15-2.12(m,1H),1.93- 1.86(m,1H),1.77(m,1H),1.61-1.58(m,2H),1.37(s,9H),1.23-0.96(m,5H).

[0343] Synthesis of (S)-N-(2-(methylamino)propyl)-4-nitrobenzenesulfonamide (int-B2) [ka] Process 1. To a stirred solution of N-Me-Boc-Ala-OH (1000.0 g, 4.92 mol) in THF (6 L), DIEA (3179.0 g, 24.6 mol) was added at 0°C. After 5 minutes, HATU (2058.0 g, 294 mmol) was added all at once, and stirring was continued at the same temperature for 15 minutes. Next, solid ammonium chloride (1316.0 g, 24.6 mol) was added, and the mixture was stirred overnight at room temperature. A large precipitate formed, which was filtered through a disposable frit and washed multiple times with THF until most of the solid was dissolved. The filtrate was evaporated under reduced pressure to obtain the crude product. The crude mass was diluted with water (2 L) and extracted with petroleum ether (2 × 2.5 L) to remove nonpolar impurities. The water portion was then extracted multiple times with 40% siRNA in petroleum ether. Next, all the organic parts were combined, dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure to obtain (S)-(1-amino-1-oxopropan-2-yl)(methyl)carbamate tert-butyl(int-B2-1). 1 H NMR(400MHz,DMSO-d6)δ ppm 6.70-7.47(m,2H),4.48-4.49(m,1H),4.11-4.82(m,1H),3.08(s,1H),2.72(s,3H),1.39(br.s.,9H),1.18-1.29(m,3H),1.11(s,1H).

[0344] Process 2.To a stirred solution of (S)-(1-amino-1-oxopropan-2-yl)(methyl)carbamate tert-butyl (int-B2-1) (100 g, 0.49 mol.) in 1,4-dioxane (500 mL), NaBH4 (56.4 g, 1.48 mol.) was added under argon. Acetic acid (90 mL) in dioxane (200 mL) was added dropwise to the mixture using a bubbler fitted as a valve, maintaining a gentle foam. Upon completion of the acid addition, a reflux condenser was attached and the mixture was heated to 110 °C for 4 hours. The mixture was removed from the heat and allowed to cool to room temperature. The reaction mass was quenched with ice and acidified with 2N HCl. The solution was extracted with ELISA, and the aqueous portion was basicized to pH 13-14 with 50% NaOH. Next, the solution was extracted with diethyl ether, dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure to obtain (S)-(1-aminopropan-2-yl)(methyl)carbamate tert-butyl(int-B2-2). The crude compound was used for the next step without purification.

[0345] Process 3. To a stirred solution of crude (S)-(1-aminopropan-2-yl)(methyl)carbamate tert-butyl(int-B2-2) (300.0 g, 1.59 mol) in ACN (1500 mL), NaHCO3 (406.9 g, 4.78 mol) was added, followed by the addition of nosyl chloride (388.9 g, 1.75 mol) at 0°C. The mixture was stirred at room temperature for 2 hours. The reaction solution was slowly quenched with ice water (2000 mL) and stirred for 2 hours until a white precipitate formed. The solid was filtered, washed with water and petroleum ether, and dried under vacuum to obtain (S)-methyl(1-((4-nitrophenyl)sulfonamide)propan-2-yl)carbamate tert-butyl(int-B2-3). The crude compound was used for the next step without purification.

[0346] Step 4.To a stirred solution of crude (S)-methyl(1-(4-nitrophenyl)sulfonamide)propan-2-yl)carbamate tert-butyl (int-B2-3) (450.0 g, 1.2 mol) in 1,4-dioxane (2000 mL), HCl (4 M in 1,4-dioxane, 2000 mL) was added, and the reaction mixture was stirred at room temperature for 4 hours. The solid precipitated during the reaction was filtered, washed with diethyl ether, and dried under vacuum to obtain the amine as the HCl salt. The crude solid mass was washed multiple times with n-pentane and dried to obtain (S)-N-(2-(methylamino)propyl)-4-nitrobenzenesulfonamide (int-B2). Analytical method 7;t R =0.69 minutes;[M+H] + = 274.2. 1 H NMR(400MHz,DMSO-d6)δ ppm 1.18(d,J=6.57Hz,3H)2.47-2.55(m,4H)2.89-3.13(m,2H)3.14-3.26(m,1H)8.03-8.16(m,2H)8.37-8.52(m,2H).

[0347] Synthesis of (S)-(2-(methylamino)-6-((2-nitrophenyl)sulfonamide)hexyl)carbamate tert-butyl(int-B3) [ka] Process 1. To a suspension of Fmoc-Lys-OH HCl (4.05 g, 10 mmol) in DCM (80 mL), Me3SiCl (3.83 mL, 30.0 mmol) and DIEA (8.73 mL, 50.0 mmol) were added at 0°C. The resulting mixture was stirred at 0°C for 20 minutes until a clear solution was obtained. DIEA (1.747 mL, 10.00 mmol) and 2-nitrobenzene-1-sulfonyl chloride (2.327 g, 10.50 mmol) were added, and the reaction mixture was stirred at 0°C for 30 minutes, followed by concentration to dryness under vacuum. The resulting residue was partitioned between ₹ (150 mL) and 5% aq. KHSO4 (50 mL). The organic layer was washed with 5% aq. KHSO4 (3 × 50 mL) and brine (50 mL), dried over Na2SO4, filtered, and concentrated to dryness under vacuum. 2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N 6 -((2-nitrophenyl)sulfonyl)-L-lysine (int-B3-1) was obtained. The crude product was used in the next step without purification. Analytical method 10;t R =1.08min;[M+NH4] + = 571.3.

[0348] Process 2. N in DMF (80 mL) 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 6 A suspension of -((2-nitrophenyl)sulfonyl)-L-lysine (int-B3-1) (5.36 g, 9.68 mmol), NH4Cl (1.036 g, 19.36 mmol), and HOBt (1.483 g, 9.68 mmol) was mixed with DIEA (6.76 mL, 38.7 mmol) at 0°C. The resulting suspension was stirred at 0°C for 5 minutes, followed by the addition of TBTU (3.42 g, 10.65 mmol). After stirring at 0°C for 1 hour, the reaction mixture was partitioned between toluene (250 mL) and 5% aq. NaHCO3 (100 mL). The organic layer was washed with 5% aq. NaHCO3 (3 × 50 mL) and brine (25 mL), dried over Na2SO4, filtered, and concentrated to dryness under vacuum to obtain (S)-(1-amino-6-((2-nitrophenyl)sulfonamide)-1-oxohexane-2-yl)carbamate (9H-fluoren-9-yl)methyl(int-B3-2). The crude product was used in the next step without purification. Analytical method 19;t R =1.04 minutes;[M+H] + = 553.3.

[0349] Step 3-1:(S)-(1-amino-6-((2-nitrophenyl)sulfonamide)-1-oxohexan-2-yl)carbamate (9H-fluoren-9-yl)methyl(int-B3-2) (9.66 mmol) was dissolved in THF (60 mL), to which BH3-S(CH3)2 (5.50 mL, 58.0 mmol) was added. The resulting mixture was stirred at room temperature for 2 hours and 15 minutes, followed by 6.5 hours at 50°C. The reaction mixture was cooled to room temperature.

[0350] Step 3-2: H2O (1 mL) and 6 M aq. HCl (2 mL) were added, and the reaction mixture was stirred at room temperature for 14.5 hours.

[0351] Step 3-3: A solution of 0.5 M aq. Na2CO3 (48.3 mL, 24.15 mmol) and Boc2O (2.243 mL, 9.66 mmol) in THF (20 mL) was added. The resulting mixture was stirred at room temperature for 2 hours, quenched with 8 M MeNH2 in EtOH (1 mL), and stirred at room temperature for 30 minutes.

[0352] Step 3-4: 4M aq. NaOH (9.66 mL, 38.6 mmol) was added, and the reaction mixture was stirred at room temperature for 85 minutes. Next, 4-methylpiperidine (4 mL) was added, and the reaction mixture was stirred at room temperature for 40 minutes. Additional 4-methylpiperidine (8 mL) was added, and stirring at room temperature was continued for 30 minutes. Next, MeOH (10 mL) was added while stirring at room temperature for 25 minutes. Additional MeOH (20 mL) and 4-methylpiperidine (10 mL) were added, and the mixture was stirred for 30 minutes. Next, the reaction mixture was concentrated under vacuum, and the crude product was purified by flash chromatography on silica gel (eluent A: HCl / DIEA (98:2), eluent B: HCl / MeOH / DIEA (95:5:2)). The pure fractions were combined, concentrated under vacuum, and dried to obtain (S)-(2-amino-6-((2-nitrophenyl)sulfonamide)hexyl)carbamate tert-butyl (int-B3-3). Analytical method 10;t R =0.69 minutes;[M+H] + = 417.2.

[0353] Step 4. A mixture of formic acid (0.611 mL, 15.92 mmol) and Ac2O (1.502 mL, 15.92 mmol) was stirred at room temperature for 40 minutes, and then added to a solution of (S)-(2-amino-6-((2-nitrophenyl)sulfonamide)hexyl)carbamate tert-butyl (int-B3-3) (1.326 g, 3.18 mmol) in DCM (15 mL). The reaction mixture was stirred at room temperature for 15 minutes and concentrated to dryness under vacuum. The obtained residue was partitioned between SiO2 (80 mL) and 5% aq. NaHCO3 (10 mL). The organic layer was washed with 5% aq. NaHCO3 (4 × 10 mL) and brine (10 mL), dried over Na2SO4, filtered, and concentrated to dryness under vacuum to obtain (S)-(2-formamide-6-((2-nitrophenyl)sulfonamide)hexyl)carbamate tert-butyl(int-B3-4) (1.217 g, 2.74 mmol, yield 86%) as a yellow foam. The crude product was used in the next step without purification. Analytical method 10;t R =0.87 minutes;[M+H] + = 445.2.

[0354] Step 5-1: (S)-(2-formamide-6-((2-nitrophenyl)sulfonamide)hexyl)carbamate tert-butyl(int-B3-4) (1.217 g, 2.74 mmol) dissolved in THF (20 mL) was mixed with BH3-S(CH3)2 (1.300 mL, 13.69 mmol), and the resulting mixture was stirred at room temperature for 3 hours and 40 minutes.

[0355] Step 5-2: The reaction mixture was quenched by adding 2 mL of MeOH, and the resulting solution was stirred at room temperature for 125 minutes. 3 mL of MeOH was added, and stirring was continued at 60°C for 75 minutes. Next, the reaction mixture was concentrated to dryness under vacuum.

[0356] Step 5-3:The obtained residue was dissolved in MeOH (20 mL), and a suspension of 10% Pd / C (0.087 g, 0.082 mmol) in H2O (1 mL) was added. The resulting mixture was stirred at 60°C for 3.5 hours. An additional 10% Pd / C (0.087 g, 0.082 mmol) in H2O (1 mL) was added, and stirring at 60°C was continued for 2.5 hours. The reaction mixture was filtered on Hyflo (CAS No: 61790-53-2), and the filtrate was concentrated to dryness under vacuum to obtain (S)-(2-(methylamino)-6-((2-nitrophenyl)sulfonamide)hexyl)carbamate tert-butyl (int-B3). The crude product was used in the next step without purification. Analytical method 10;t R =0.71 min;[M+H] + = 431.3.

[0357] Synthesis of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-(4-chlorophenyl)butanoic acid (int-C1) [ka] Process 1. A 20 L four-necked round-bottom flask containing a solution of (3S)-3-[[(tert-butoxy)carbonyl]amino]-4-(4-chlorophenyl)butanoic acid (360 g, 1.15 mol) in THF (8 L) was purged and maintained under an inert nitrogen atmosphere. Sodium hydride (212 g, 5.74 mol, 65%) was added in small amounts at 0°C, and the resulting mixture was stirred at 0°C for 1 hour. Next, MeI (1633 g, 11.5 mol) was added dropwise while stirring at 0°C, and the resulting solution was stirred at 35°C for 4 hours. The reaction mixture was then quenched at -10°C by adding 300 g of water / ice, concentrated under vacuum, and subsequently diluted with 3 L of water. The aqueous phase was extracted with 3 × 1 L of ether. The pH of the aqueous phase was adjusted to pH 3 with HCl (2N) at 0°C, and the resulting solution was extracted with 3 × 2 L of ethyl acetate. The combined organic phases were washed with brine (1 × 2 L), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain (S)-3-((tert-butoxycarbonyl)(methyl)amino)-4-(4-chlorophenyl)butanoic acid (int-C1-1). Analytical method 7;t R =1.01 min;[M+H]+ = 328.1.

[0358] Process 2. A 5 L four-necked round-bottom flask containing a solution of (S)-3-((tert-butoxycarbonyl)(methyl)amino)-4-(4-chlorophenyl)butanoic acid (int-C1-1) (284.2 g, 866.98 mmol) in DCM (3 L) was purged under an inert nitrogen atmosphere and maintained. TFA (990.8 g, 8.77 mol) was added dropwise to the flask while stirring at 0°C. The resulting solution was stirred overnight at room temperature and then concentrated under vacuum to obtain (S)-4-(4-chlorophenyl)-3-(methylamino)butanoic acid (int-C1-2). Analytical method 1;t R =0.66 minutes;[M+H] + = 228.2.

[0359] Process 3. A 5 L four-necked round-bottom flask containing a solution of (S)-4-(4-chlorophenyl)-3-(methylamino)butanoic acid trifluoroacetate (int-C1-2) (320 g crude) in dioxane:H2O (5:1) (3.6 L), purged and maintained under an inert nitrogen atmosphere, was to be purged with sodium carbonate (249.1 g, 2.35 mol) in multiple batches. Next, Fmoc-Cl (242 g, 935.45 mmol) was added in multiple batches at 0°C. The resulting mixture was stirred overnight at room temperature, concentrated under vacuum, and then diluted with 3 L of water. The pH of the aqueous solution was adjusted to pH 5 with HCl (1 N). The aqueous phase was extracted with 3 × 1 L of ethyl acetate. The combined organic phase was washed with brine (1 × 1 L), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude residue was purified by silica gel column chromatography eluted with ethyl acetate / petroleum ether (1:5-1:3) to obtain (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-(4-chlorophenyl)butanoic acid (int-C1). Analytical method 1;t R =1.60 minutes;[M+H] + = 450.3. 1H NMR(300MHz,DMSO-d6,ppm):δ 12.09-12.45(br,1H),7.89(m,2H),7.21-7.69(m,8H),7.13-7.20(m,1H),6 .85-7.08(br,1H),4.04-4.55(m,4H),2.73-2.8(m,1H),2.11-2.85(m,6H).

[0360] Synthesis of (R)-3-amino-3-(4-chlorobenzyl)piperidine-1-carboxylate tert-butyl(int-C2) [ka] Process 1-1: 1-(tert-butoxycarbonyl)-3-(4-chlorobenzyl)piperidine-3-carboxylic acid (6.905 g, 19.51 mmol) dissolved in toluene (100 mL) and DIEA (5.11 mL, 29.3 mmol) was mixed with diphenyl phosphoryl azide (5.48 mL, 25.4 mmol), and the reaction mixture was stirred at room temperature for 2.5 hours, followed by 4 hours at 100 °C. The reaction mixture was partitioned between toluene (300 mL) and 5% aq. NaHCO3 (60 mL). The organic phase was washed with 5% aq. NaHCO3 (3 × 60 mL) and brine (50 mL), dried over Na2SO4, filtered, and concentrated to dryness under vacuum.

[0361] Step 1-2: To the residue from step 1-1, dissolved in dioxane (200 mL), 1 M NaOH (195 mL, 195 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour, and then concentrated to dryness under vacuum. The obtained residues were partitioned between siRNA (250 mL) and 5% aq. Na2CO3 (20 mL), and the aqueous phase was extracted with siRNA (70 mL). The combined organic phase was washed with 5% aq. Na2CO3 (40 mL) and brine (40 mL), dried over Na2SO4, filtered, and concentrated to dryness under vacuum to obtain a racemic tert-butyl 3-amino-3-(4-chlorobenzyl)piperidine-1-carboxylate, which was used in the next step without further purification. Analytical method 10;t R =0.80 minutes;[M+H] + = 325.2.

[0362] Process 2. Racemic 3-amino-3-(4-chlorobenzyl)piperidine-1-carboxylate tert-butyl (19.5 mmol) was separated under the following conditions: Column: ChiralPak AD, 300 × 50 mm ID, 10 μm; Elutate A: CO2; Elutate B: EtOH (0.1% NH4OH); Gradient: B 45%; Flow rate: 200 mL / min; Back pressure: 100 bar; Column temperature: 38 °C; Cycle time: approximately 9 minutes; The compound was dissolved in approximately 130 mL of MeOH; Injection: Separation was performed by preparative SFC (instrument: Thar 200 preparative SFC) using 10 mL per injection. (R)-3-amino-3-(4-chlorobenzyl)piperidine-1-carboxylate tert-butyl (int-C2) (a slower eluting isomer) and partial crystallization occurred during storage, allowing for structural confirmation by X-ray crystallography. Analytical method 10; t R =0.77 minutes;[M+H] + = 325.3.

[0363] Synthesis of (R)-3-(4-chlorobenzyl)piperidine-3-amine hydrochloride (int-C3) [ka] (R)-3-amino-3-(4-chlorobenzyl)piperidine-1-carboxylate tert-butyl (int-C2) (2.09 g, 6.43 mmol) was dissolved in dioxane (10 mL). 4 M HCl (50 mL) and H2O (5 mL) were added to the dioxane, and the solution was stirred at room temperature for 4 hours. The reaction mixture was concentrated to dryness under vacuum to obtain (R)-3-(4-chlorobenzyl)piperidine-3-amine hydrochloride (int-C3). Analytical method 10;t R =0.40 minutes;[M+H] + = 225.1.

[0364] Synthesis of 4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzaldehyde (int-F1) [ka] Process 1.In a 5 L three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, 216 g (1.14 mol, 1.00 equivalent) of 5-bromo-1-methyl-1H-imidazole-2-carbaldehyde and a 4 Å molecular sieve were added in 3 L of dichloromethane and 1 L of THF, followed by the addition of 862.5 mL (1.50 equivalent) of dimethylamine. The resulting mixture was stirred at room temperature for 30 minutes, and NaBH(OAc)3 (292.6 g, 1.38 mol, 1.20 equivalent) was added in a batch at 0°C. The reaction mixture was stirred overnight at room temperature, followed by quenching with 1 L of water. The organic phase was separated and washed with 2 × 2 L of H2O. The aqueous phase was then extracted with 2 × 1 L of DCM. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography eluted with dichloromethane / ethyl acetate (2:1) to obtain [(5-bromo-1-methyl-1H-imidazole-2-yl)methyl]dimethylamine (int-F1-1). Analytical method 5;t R =0.60 minutes;[M+H] + = 220.1.

[0365] Process 2.In a 3 L four-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, [(5-bromo-1-methyl-1H-imidazole-2-yl)methyl]dimethylamine (int-F1-1) (53.675 g, 246.11 mmol, 1.00 equivalent), (4-hydroxyphenyl)boronic acid (66.55 g, 482.49 mmol, 1.50 equivalent), Pd(dppf)Cl2 (11.75 g, 16.06 mmol, 0.05 equivalent), and potassium acetate (189.05 g, 1.93 mol, 6.00 equivalent) were added in N,N-dimethylformamide (1.3 L). The resulting solution was stirred in an oil bath at 90°C for 18 hours. The reaction was repeated three times on the same scale. The batches were combined, the mixture was cooled to room temperature, and then poured into 3.5 L of water / ice. The obtained solution was extracted with toluene (3 × 1.5 L) and the organic layer was combined. The mixture was diluted with water (1 L) and the pH of the solution was adjusted to 4-5 with 2 M aq. HCl. The aqueous phase was extracted with toluene (2 × 1 L) and the aqueous layers were combined. The pH of the solution was adjusted to 11 with NH4OH. The obtained solution was extracted with dichloromethane (6 × 1 L) and the organic layer was combined and concentrated under vacuum. The crude product was purified by silica gel column chromatography eluting with dichloromethane / acetic acid (8:1) to obtain 4-[2-[(dimethylamino)methyl]-1-methyl-1H-imidazole-5-yl]phenol (int-F1-2) (120 g, 53%) as purple oil. Analytical method 5;t R =0.55 minutes;[M+H] + = 232.1.

[0366] Process 3. In a 3L four-necked round-bottom flask, add 4-[2-[(dimethylamino)methyl] in N,N-dimethylformamide (2L). -1-Methyl-1H-imidazole-5-yl]phenol (int-F1-2) (120 g, 518.82 mmol, 1.00 equivalent) and potassium carbonate (214.9 g, 1.55 mol, 3.00 equivalent) were added. The resulting mixture was stirred at room temperature for 30 minutes, and 4-chloro-2-fluorobenzaldehyde (98.5 g, 621.23 mmol, 1.20 equivalent) was added. The reaction mixture was stirred in an oil bath at 90°C for 4 hours, then cooled to room temperature and diluted with water (3 L). The resulting solution was extracted with RINKAN (3 × 2 L), and the organic layers were combined. The mixture was diluted with water (1 L), and the pH of the solution was adjusted to 2 with 2 M aq. HCl. The aqueous phase was extracted with RINKAN (3 × 2 L), and the aqueous layers were combined. The pH of the solution was adjusted to 11 with NH₄OH, and then extracted with DCM (2 × 2 L). The combined organic phase is concentrated under vacuum and then purified by silica gel column chromatography eluting with dichloromethane / acetic acid (7:3) to obtain 4-chloro-2-(4-[2-[(dimethylamino)methyl] - We obtained 1-methyl-1H-imidazole-5-yl]phenoxy)benzaldehyde (int-F1). 1 H NMR:(300MHz,CDCl3,ppm):δ 10.47(s,1H),7.90(d,J=8.4Hz,1H),7.44(d,J=8.6Hz,1H),7.25-7.10(m, 3H),7.02(s,1H),6.94(d,J=1.9Hz,1H),3.71(s,3H),3.60(s,2H).Analysis method 5;t R =1.00 minutes;[M+H] + = 370.2.

[0367] Synthesis of 4-chloro-2-(4-(1-methyl-2-(pyrrolidine-1-ylmethyl)-1H-imidazole-5-yl)phenoxy)benzaldehyde (int-F2) [ka] Process 1.To a solution of 5-bromo-1-methyl-1H-imidazole-2-carbaldehyde (1.890 g, 10.0 mmol) in DCM (70 mL), pyrrolidine (1.643 mL, 20.0 mmol) was added. After stirring at room temperature for 25 minutes, NaBH(OAc)3 (8.48 g, 40.0 mmol) was added. The resulting mixture was stirred at room temperature for 105 minutes, then concentrated to dryness under vacuum and partitioned between siRNA (250 mL) and 1 M aq. NaOH (50 mL). The organic layer was washed with 1 M NaOH (2 × 40 mL) and brine (20 mL), dried over Na2SO4, filtered, and concentrated to dryness under vacuum to obtain 5-bromo-1-methyl-2-(pyrrolidine-1-ylmethyl)-1H-imidazole (int-F2-1). The crude product was used in the next step without purification. Analytical method 11;t R =0.66 minutes;[M+H] + =244.1.

[0368] Process 2.5-Bromo-1-methyl-2-(pyrrolidine-1-ylmethyl)-1H-imidazole (int-F2-1) (10 mmol), (4-hydroxyphenyl)boronic acid (2.76 g, 20.0 mmol), and [1,1'-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (0.978 g, 1.50 mmol) were mixed with dioxane (30 mL) and 1 M aq. Na2CO3 (30 mL). The reaction mixture was stirred at 100°C for 4 hours under an N2 atmosphere. An additional amount of (4-hydroxyphenyl)boronic acid (1.379 g, 10.0 mmol) was added, and stirring at 100°C was continued for 135 minutes. Further (4-hydroxyphenyl)boronic acid (1.379 g, 10.0 mmol) and [1,1'-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (0.244 g, 0.375 mmol) were added, and the mixture was stirred at 100°C for 18.75 hours. SiO (250 mL) and H₂O (50 mL) were added, and the mixture was filtered over Hyflo. The layers were separated, and the organic layer was washed with 5% aq. NaHCO₃ (3 × 40 mL) and brine (40 mL), dried over Na₂SO₄, filtered, and concentrated to dryness under vacuum. The crude product was purified by silica gel flash chromatography (eluent A: ethyl / MeOH / DIEA (95:5:2), eluent B: ethyl / MeOH / DIEA (85:15:2)) to obtain 4-(1-methyl-2-(pyrrolidine-1-ylmethyl)-1H-imidazole-5-yl)phenol (int-F2-2). Analytical method 11;t R =0.76 minutes;[M+H] + = 258.1.

[0369] Process 3.4-(1-methyl-2-(pyrrolidine-1-ylmethyl)-1H-imidazole-5-yl)phenol (int-F2-2) (1.029 g, 4 mmol) and 4-chloro-2-fluorobenzaldehyde (0.824 g, 5.20 mmol) were dissolved in NMP (20 mL), and K2CO3 (1.437 g, 10.40 mmol) was added. The reaction mixture was stirred at 80°C for 18 hours, and then partitioned between siRNA (125 mL) and H2O (20 mL). The organic layer was washed with 5% aq. NaHCO3 solution (3 × 10 mL) and brine (10 mL), dried over Na2SO4, filtered, and concentrated to dryness under vacuum. The crude product was purified by silica gel flash chromatography (eluent A: siRNA / DIEA (98:2), eluent B: siRNA / MeOH / DIEA (90:10:2)) to obtain 4-chloro-2-(4-(1-methyl-2-(pyrrolidine-1-ylmethyl)-1H-imidazole-5-yl)phenoxy)benzaldehyde (int-F2). Analytical method 10;t R =0.79 minutes;[M+H] + = 396.2.

[0370] Synthesis of (R)-4-((R)-3-amino-3-(4-chlorobenzyl)piperidine-1-yl)-3-benzyl-4-oxobutanoate methyl (int-F3) [ka] Process 1.To a vial containing (R)-2-benzyl-4-(tert-butoxy)-4-oxobutanoic acid (int-A1) (244 mg, 0.924 mmol) in DMA (4 mL), DIPEA (0.323 mL, 1.848 mmol) and HATU (358 mg, 0.942 mmol) were added in multiple portions at room temperature. After the addition was complete, the resulting mixture was stirred at room temperature for a further 15 minutes, and then added dropwise to another vial containing DMA (1.5 mL) and (R)-3-(4-chlorobenzyl)piperidine-3-amine (int-C3) (275 mg, 0.924 mmol) in DIPEA (0.807 mL, 4.62 mmol). The reaction mixture was stirred at room temperature overnight, then transferred to a separatory funnel, diluted with ethyl acetate, and washed with saturated sodium bicarbonate solution and brine (×3). The organic phase was dried over sodium sulfate, filtered, and concentrated to obtain (R)-4-((R)-3-amino-3-(4-chlorobenzyl)piperidine-1-yl)-3-benzyl-4-oxobutanoate tert-butyl, which was then proceeded to the next step without further purification.

[0371] Process 2. A round-bottom flask containing (R)-4-((R)-3-amino-3-(4-chlorobenzyl)piperidine-1-yl)-3-benzyl-4-oxobutanoate tert-butyl (435 mg, 0.924 mmol) in anhydrous methanol (18 mL), cooled in an ice bath, was to be added dropwise by thionyl chloride (1.35 mL, 18.47 mmol). After the addition was complete, the resulting mixture was gradually warmed to room temperature, followed by stirring overnight to complete the reaction. The reaction mixture was concentrated to dryness under reduced pressure while heating in a water bath at 30°C. The crude oil was dissolved in toluene, washed with a semisaturated aqueous solution of sodium bicarbonate, and then washed with brine. The separated organic phase was dried over sodium sulfate, filtered, and concentrated to obtain (R)-4-((R)-3-amino-3-(4-chlorobenzyl)piperidine-1-yl)-3-benzyl-4-oxobutanoate methyl (int-F3).

[0372] Synthesis of (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)butanoic acid (int-G1) [ka] To a suspension of (S)-2-amino-4-((tert-butoxycarbonyl)amino)butanoic acid (1.04 mg, 4.77 mmol) in MeOH (10 mL) and water (0.46 mL), NaOH (4.67 mL, 4.67 mmol) was added at room temperature. The resulting mixture was stirred at room temperature for 1 hour, then 4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzaldehyde (int-F1) (1.6 g, 4.33 mmol) was added, the reaction mixture was stirred for 15 minutes, cooled to -5°C, and then stirred for 1 hour. NaBH4 (65 mg, 1.73 mmol) was added in small amounts while maintaining the internal reaction temperature below 0°C. The reaction mixture was stirred at -5°C for 30 minutes, and then at room temperature for 2 hours. The reaction mixture was quenched by adding water dropwise until the release of gas ceased. Next, the reaction mixture was concentrated to remove MeOH, and 60 mL of water was added. The aqueous mixture was extracted with HCl (150 mL), the organic layer was washed with 40 mL of NaHCO3 solution, cooled, and the pH was adjusted to approximately 8 by adding 1.0 N HCl. The resulting precipitate was filtered, washed with water, and dried. The filtrate was extracted with DCM (4 × 200 mL). The organic matter was concentrated, and the residue was combined with the precipitate. The solid was dried under vacuum to obtain (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)butanoic acid (int-G1). Analytical method 7:t R =0.66 minutes;[M+H] + =572.0

[0373] Synthesis of (S)-5-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)pentanoic acid (int-G2) [ka] (S)-5-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)pentanoic acid (int-G2) was obtained using a method similar to that used in the synthesis of (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)butanoic acid (int-G1), except that (S)-2-amino-4-((tert-butoxycarbonyl)amino)butanoic acid was replaced with (S)-2-amino-5-((tert-butoxycarbonyl)amino)pentanoic acid. Analytical method 7:t R =0,67 minutes;[M+H] + = 586.2.

[0374] Synthesis of (S)-5-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(1-methyl-2-(pyrrolidine-1-ylmethyl)-1H-imidazole-5-yl)phenoxy)benzyl)amino)pentanoic acid (int-G3) [ka] (S)-5-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(1-methyl-2-(pyrrolidin-1-ylmethyl)-1H-imidazole-5-yl)phenoxy)benzyl)amino)pentanoic acid (int-G3) is formed when (S)-2-amino-4-((tert-butoxycarbonyl)amino)butanoic acid is replaced by (S)-2-amino-5-((tert-butoxycarbonyl)amino)pentanoic acid, and 4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl) It was obtained using a method similar to that used in the synthesis of (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)butanoic acid (int-G1), except that phenoxy)benzaldehyde (int-F1) was replaced with 4-chloro-2-(4-(1-methyl-2-((pyrroridine-1-ylmethyl)-1H-imidazole-5-yl)phenoxy)benzyl)amino)butanoic acid (int-G1). Analytical method 7:t R =1.46 minutes;[M+H] + = 612.6.

[0375] Synthesis of (R)-4-(((S)-1-aminopropan-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoic acid PS-(2-chlorotrityl)(AB1) [ka] Process 1.To a cold, stirred solution of (S)-N-(2-(methylamino)propyl)-4-nitrobenzenesulfonamide (int-B2) (230 g, 0.742 mol) and (R)-2-benzyl-4-(tert-butoxy)-4-oxobutanoic acid (int-A1) (186.4 g, 0.705 mol) in DMF (460 mL), DIPEA (388 mL, 2.227 mol) was added, followed by HATU (310.3 g, 0.816 mol) at 5-10°C. The resulting mixture was removed from the cold bath and stirred at room temperature for 4 hours. The reaction mixture was then poured into ice-cold water (5 L) and extracted with ethyl acetate (2 × 2 L). The combined organic layers were washed with aqueous salt solution (2 L), dried over anhydrous Na₂SO₄, and concentrated under vacuum. The crude product was purified by silica gel column chromatography using 230-400 mesh, eluting with 20% ethyl acetate in petroleum ether, to obtain (R)-3-benzyl-4-(methyl((S)-1-((4-nitrophenyl)sulfonamide)propan-2-yl)amino)-4-oxobutanoate tert-butyl (AB1-1). Analytical method 7;t R =1.40 minutes;[M+H] + = 520.3.

[0376] Process 2.To a cold, stirred solution of (R)-3-benzyl-4-(methyl((S)-1-((4-nitrophenyl)sulfonamide)propan-2-yl)amino)-4-oxobutanoate tert-butyl (AB1-1) (455 g, 0.875 mol) in acetonitrile (3.5 L) and methanol (3.5 L), cesium carbonate (1.995 kg, 6.125 mol) was added at less than 10°C, and the resulting mixture was stirred for 15 minutes. Next, 2-mercaptoacetic acid (322.7 g, 3.50 mol) was added at the same temperature. The resulting mixture was stirred at room temperature for 1 hour, concentrated under reduced pressure, and the solvent was removed. The crude product was dissolved in water (3 L), and the aqueous phase was extracted with dichloromethane (2 × 2 L). The combined organic layers were washed with saturated sodium bicarbonate (3 L) and salt solution (2 L), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to obtain (R)-4-(((S)-1-aminopropan-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoate tert-butyl (AB1-2), which was used in the next step without further purification.

[0377] Process 3. To a cold, stirred solution of (R)-4-(((S)-1-aminopropan-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoate tert-butyl (AB1-2) (270 g, 0.807 mol) in THF (1.5 L), Fmoc-Cl (209.1 g, 0.807 mol) was added in small amounts over 30 minutes at 10°C, followed by the addition of saturated sodium bicarbonate solution (3.3 L) over 30 minutes at the same temperature. The mixture was then stirred at room temperature for 2 hours. The reaction mixture was diluted with water (2 L) and extracted with ethyl acetate (2 × 1 L). The combined organic layers were washed with aqueous salt solution (2 L), dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude material was purified on a 230-400 mesh silica gel column eluted with 20% ethyl acetate in petroleum ether to obtain (R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propan-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoate tert-butyl (AB1-3). Analytical method 7;t R =1.49 minutes;[M+H] += 557.3.

[0378] Step 4. To a solution of (R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propan-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoic acid tert-butyl (AB1-3) (265 g, 0.476 mol) in 1,4-dioxane (530 mL), 4 M HCl (2.65 L) in the dioxane was added at room temperature. The resulting mixture was stirred for 16 hours and then concentrated under reduced pressure. The crude product was purified on a 230-400 mesh silica gel column eluted with 30% ethyl acetate in petroleum ether to obtain (R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propan-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoic acid (AB1-4). Analysis method 7;t R =1.48 minutes;[M+H] + = 501.4.

[0379] Process 5. 2-Chlorotrityl chloride (AB-1-4A), 4.27 g, 4.27 mmol) was pre-washed with DCM (3 × 20 mL). (R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propan-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoic acid (AB1-4) (1.9 g, 3.80 mmol), dissolved in DCM (20 mL) and DIPEA (1.5 mL, 8.59 mmol), was added to the resin. The resulting mixture was shaken at room temperature for 16 hours, then washed with DCM (3 × 40 mL), shaken in DCM / MeOH (50 mL / 20 mL) for 30 minutes, and the resin was capped. Next, the resin was filtered, washed with DMF (2 × 50 mL) and DCM (2 × 50 mL), and dried under vacuum to obtain PS-(2-chlorotrityl)(R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propan-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoic acid (AB1-5) resin (6.13 g, crude product). The resin was subjected to the next step without purification.

[0380] Process 6. PS-(2-chlorotrityl)(R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propan-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoic acid (AB1-5) (285 mg, 1.283 mmol) was mixed with 20% 4-methylpiperidine in DMF (10 mL), and the resulting mixture was shaken at room temperature for 2 hours. The resin was then filtered, washed with DMF (2 × 10 mL) and DCM (2 × 10 mL), and dried under vacuum. This yielded PS-(2-chlorotrityl)(R)-4-(((S)-1-aminopropan-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoic acid (AB1), which was used without further purification.

[0381] Synthesis of (R)-4-(((1S,2S)-2-aminocyclohexyl)(methyl)amino)-3-benzyl-4-oxobutanoic acid PS-(2-chlorotrityl)(AB2) [ka] (R)-4-(((1S,2S)-2-aminocyclohexyl)(methyl)amino)-3-benzyl-4-oxobutanoic acid PS-(2-chlorotrityl)(AB2) was obtained using a method similar to that used in the synthesis of (R)-4-(((S)-1-aminopropan-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoic acid PS-(2-chlorotrityl)(AB1), except that (S)-N-(2-(methylamino)propyl)-4-nitrobenzenesulfonamide (int-B2) was replaced with ((1S,2S)-2-(methylamino)cyclohexyl)carbamate tert-butyl (int-B1).

[0382] Synthesis of (R)-4-(((S)-1-amino-6-((2-nitrophenyl)sulfonamide)hexane-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoic acid PS-(2-chlorotrityl)(AB3) [ka] Process 1.(R)-2-benzyl-4-(tert-butoxy)-4-oxobutanoic acid (0.537 g, 2.03 mmol) and TBTU (0.717 g, 2.233 mmol) were suspended in DCM / DMF (3:1) (20 mL), and DIEA (0.390 mL, 2.233 mmol) was added. The mixture was stirred at room temperature for 25 minutes. A solution of (S)-(2-(methylamino)-6-((2-nitrophenyl)sulfonamide)hexyl) tert-butyl carbamate (1.049 g, 2.436 mmol) in DCM (20 mL) was added, and the reaction was stirred at room temperature for 2 hours and 10 minutes. Additional DIEA (0.390 mL, 2.233 mmol) was added, and stirring was continued for 44 hours. H2O (1 mL) was added, and DCM was removed under vacuum. The residue was partitioned between ethyl acetate (60 mL) and 5% aq. NaHCO3 (15 mL). The organic layer was washed with 5% aq. NaHCO3 (3 × 10 mL) and brine (10 mL), dried over Na2SO4, filtered, and concentrated to dryness under vacuum to obtain brown oil (1.535 g, 2.030 mmol, yield 100%). Crude (R)-3-benzyl-4-(((S)-1-((tert-butoxycarbonyl)amino)-6-((2-nitrophenyl)sulfonamide)hexane-2-yl)(methyl)amino)-4-oxobutanoate tert-butyl was used in the next step without purification. Analytical method 11;t R =1.31 minutes;[M+H] + = 677.5.

[0383] Process 2. (R)-3-benzyl-4-(((S)-1-((tert-butoxycarbonyl)amino)-6-((2-nitrophenyl)sulfonamide)hexane-2-yl)(methyl)amino)-4-oxobutanoic acid tert-butyl (1.535 g, 2.020 mmol) was dissolved in TFA (95% aq., 20 mL), stirred at room temperature for 1 hour, and then concentrated to dryness to obtain (R)-4-(((S)-1-amino-6-((2-nitrophenyl)sulfonamide)hexane-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoic acid, which was used directly in the following reaction.

[0384] Process 3. Crude (R)-4-(((S)-1-amino-6-((2-nitrophenyl)sulfonamide)hexane-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoic acid obtained from step 2 was dissolved in dioxane (20 mL) and subsequently treated with a solution of (2,5-dioxopyrrolidine-1-yl)carbonate (9H-fluoren-9-yl)methyl (0.685 g, 2.030 mmol) in 0.5 M aq. Na2CO3 (12.18 mL, 6.09 mmol) and dioxane (20 mL). The reaction mixture was maintained at room temperature for 90 minutes, then quenched by adding 2.0 M aq. HCl (15 mL), and concentrated under vacuum to approximately half the volume. The residue was partitioned between siRNA (100 mL) and 5% KHSO4 (15 mL). The organic layer was washed with 5% aq. KHSO4 (3 × 15 mL) and brine (15 mL), dried over Na2SO4, filtered, and concentrated to dryness under vacuum. The crude product was purified by flash chromatography on silica gel. The pure fractions were combined and concentrated to dryness under vacuum. The residue was partitioned between toluene (80 mL) and 5% aq. NaHCO3 (7 mL). The organic layer was washed with 5% aq. NaHCO3 (3 × 7 mL), 5% aq. KHSO4 (15 mL), and brine (10 mL), dried over Na2SO4, filtered, and concentrated to dryness under vacuum to obtain (R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((2-nitrophenyl)sulfonamide)hexane-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoic acid (900 mg, 1.212 mmol, yield 60%) as a white foam. Analytical method 11;t R =1.21 minutes;[M+H] + = 743.15.

[0385] Step 4.2-Chlorotritylchloride resin (1.250 mg, 2.00 mmol) was pre-washed with DCM (3 × 30 mL). (R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((2-nitrophenyl)sulfonamide)hexane-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoate tert-butyl (AB3-3) (900 mg, 1.21 mmol) was added to the resin in DCM (30 mL) and DIEA (1.69 mL, 9.69 mmol). The resulting mixture was shaken at room temperature for 16 hours, washed with DCM (3 × 50 mL), and then shaken in DCM / MeOH (5:2) (20 mL) for 30 minutes before capping the resin. Next, the resin was filtered, washed with DCM / MeOH / DIEA (17:2:1) (3 × 15 mL), and dried under vacuum. This yielded the resin PS-(2-chlorotrityl)-(R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((2-nitrophenyl)sulfonamide)hexane-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoate (AB3-4), which was then removed without further purification and proceeded to the next step.

[0386] Process 5. PS-(2-chlorotrityl)-(R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((2-nitrophenyl)sulfonamide)hexane-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoate (AB3-4) (266 mg, 0.336 mmol) was mixed with 20% 4-methylpiperidine in DMF (10 mL), and the resulting mixture was shaken at room temperature for 2 hours. The resin was then filtered, washed with DMF (2 × 10 mL) and DCM (2 × 10 mL), and dried under vacuum. This yielded the resin PS-(2-chlorotrityl)-(R)-4-(((S)-1-amino-6-((2-nitrophenyl)sulfonamide)hexane-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoate (AB3), which was used without purification.

[0387] Synthesis of (4S,7S,12S,15R)-4-amino-15-benzyl-7-(4-chlorobenzyl)-6,12,13-trimethyl-5,9,14-trioxo-2-oxa-6,10,13-triazaheptadecane-17-acid PS-(2-chlorotrityl)(AB4) [ka] Process 1-1. A solution of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-(4-chlorophenyl)butanoic acid (int-C1) (4.05 g, 9.00 mmol), TBTU (2.89 g, 9.00 mmol), and DIEA (1.729 mL, 9.90 mmol) in NMP (70 mL) was shaken at room temperature for 2 minutes, and then added to (R)-4-(((S)-1-aminopropan-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoic acid PS-(2-chlorotrityl)(AB1) (6.00 mmol) which had been washed with NMP (3 ×). The resulting suspension was shaken at room temperature for 20 hours, then filtered, and the resin was washed with DMA (3 ×). To cap the resin, Ac2O / pyridine / DMA (1:1:8) (70 mL) was added, and the resulting suspension was shaken at room temperature for 15 minutes. The resin was drained and then washed with DMA (3 ×).

[0388] Process 1-2. Fmoc deprotection was performed by repeated treatment with 4-methylpiperidine / DMA (1:4) (5 × 70 mL, shaken for 5 minutes at room temperature each time). The cleavage solution was collected and used to determine the resin loading by UV spectroscopy. After Fmoc removal, the resin was washed with DMA (3x), DCM (3x), DMA (3x), and DCM (5x), dried in vacuum, to obtain (R)-3-benzyl-4-(((S)-1-((S)-4-(4-chlorophenyl)-3-(methylamino)butanamide)-propan-2-yl)(methyl)amino)-4-oxobutanoic acid PS-(2-chlorotrityl)(AB4-1).

[0389] Process 2-1.A solution of Fmoc-O-methyl-L-serine (2.292 g, 6.72 mmol), PyOxim (3.54 g, 6.72 mmol), and DIEA (2.346 mL, 13.43 mmol) in NMP (55 mL) was shaken at room temperature for 2 minutes and then added to (R)-3-benzyl-4-(((S)-1-((S)-4-(4-chlorophenyl)-3-(methylamino)butanamide)-propan-2-yl)(methyl)amino)-4-oxobutanoic acid PS-(2-chlorotrityl)(AB4-1) (4.477 mmol) which had been washed with NMP (3×). The resulting suspension was shaken at room temperature for 6 hours and then filtered. A solution of Fmoc-O-methyl-L-serine (1.528 g, 4.48 mmol), PyOxim (2.361 g, 4.48 mmol), and DIEA (1.564 mL, 8.95 mmol) in NMP (35 mL) was stirred at room temperature for 2 minutes, and then added to the resin. The resulting suspension was shaken at room temperature for 16 hours, filtered, and the resin was washed with DMA (3 ×). For capping, Ac2O / pyridine / DMA (1:1:8) (40 mL) was added, and the resulting suspension was shaken at room temperature for 15 minutes. The resin was drained and then washed with DMA (3 ×).

[0390] Step 2-2. Fmoc-deprotection was performed by repeated treatment with 4-methylpiperidine / DMA (1:4) (3 × 30 mL, shaken for 15 minutes at room temperature each time). After Fmoc-removal, the resin was washed with DMA (3 ×) and DCM (5 ×) to obtain (4S,7S,12S,15R)-4-amino-15-benzyl-7-(4-chlorobenzyl)-6,12,13-trimethyl-5,9,14-trioxo-2-oxa-6,10,13-triazaheptadecane-17-acid PS-(2-chlorotrityl)(AB4).

[0391] Synthesis of (R)-4-(((1S,2S)-2-((S)-3-((S)-2-amino-3-methoxy-N-methylpropanamide)-4-(4-chlorophenyl)butanamide)cyclohexyl)(methyl)amino)-3-benzyl-4-oxobutanoic acid PS-(2-chlorotrityl)(AB5) [ka] (R)-4-(((1S,2S)-2-((S)-3-((S)-2-amino-3-methoxy-N-methylpropanamide)-4-(4-chlorophenyl)butanamide)cyclohexyl)(methyl)amino)-3-benzyl-4-oxobutanoic acid PS-(2-chlorotrityl)(AB5) is (R)-4-(((S)-1-aminopropan-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoic acid PS-(2-chlorotrityl)(AB1) is (R)-4-(((1S,2S)- It was obtained using a method similar to that used in the synthesis of (4S,7S,12S,15R)-4-amino-15-benzyl-7-(4-chlorobenzyl)-6,12,13-trimethyl-5,9,14-trioxo-2-oxa-6,10,13-triazaheptadecane-17-acid PS-(2-chlorotrityl)(AB4), except that it was replaced with 2-aminocyclohexyl)(methyl)amino)-3-benzyl-4-oxobutanoic acid PS-(2-chlorotrityl)(AB2).

[0392] Synthesis of N-(4-((2S,5S,8S,13S,16R)-16-benzyl-1-(4-chloro-2-(4-(1-methyl-2-(pyrroridine-1-ylmethyl)-1H-imidazole-5-yl)phenoxy)benzyl)-8-(4-chlorobenzyl)-5-(methoxymethyl)-2,7,14-trimethyl-3,6,10,15,18-pentaoxo-1,4,7,11,14-pentazacyclooctadecane-13-yl)butyl)-2-nitrobenzenesulfonamide (AB6) [ka] Process 1-1.A solution of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-(4-chlorophenyl)butanoic acid (int-C1) (0.601 g, 1.336 mmol), TBTU (0.429 g, 1.339 mmol), and DIEA (0.257 mL, 1.4690 mmol) in NMP (20 mL) was shaken at room temperature for 2 minutes and then added to PS-(2-chlorotrityl)-(R)-4-(((S)-1-amino-6-((2-nitrophenyl)sulfonamide)hexane-2-yl)(methyl)amino)-3-benzyl-4-oxobutanoate (AB3) (1.113 mmol) washed with NMP (3 ×). The resulting suspension was shaken at room temperature for 20 hours, then filtered, and the resin was washed with DMA (3 ×). To cap the resin, Ac2O / pyridine / DMA (1:1:8) (70 mL) was added, and the resulting suspension was shaken at room temperature for 20 hours. The resin was drained and then washed with DMA (3×).

[0393] Process 1-2. Fmoc deprotection was performed by repeated treatment with 4-methylpiperidine / DMA (1:4) (5 × 20 mL, shaken for 15 minutes at room temperature each time). The cleavage solution was collected and used to determine the resin loading by UV spectroscopy. After Fmoc removal, the resin was washed with DMA (3x), DCM (3x), DMA (3x), and DCM (5x), dried in vacuum, to obtain (R)-3-benzyl-4-(((S)-1-((S)-4-(4-chlorophenyl)-3-(methylamino)butanamide)-6-((2-nitrophenyl)sulfonamide)hexane-2-yl)(methyl)amino)-4-oxobutanoic acid PS-(2-chlorotrityl).

[0394] Process 2-1.A solution of Fmoc-O-methyl-L-serine (0.57 g, 1.67 mmol), PyOxim (0.88 g, 1.67 mmol), and DIEA (0.583 mL, 3.339 mmol) in NMP (15 mL) was shaken at room temperature for 2 minutes, and then added to (R)-3-benzyl-4-(((S)-1-((S)-4-(4-chlorophenyl)-3-(methylamino)butanamide)-6-((2-nitrophenyl)sulfonamide)hexane-2-yl)(methyl)amino)-4-oxobutanoic acid PS-(2-chlorotrityl) (1.113 mmol) which had been washed with NMP (3 ×). The resulting suspension was shaken at room temperature for 3 hours, filtered, and the resin was washed with DMA (3 ×). To cap the resin, 40 mL of Ac2O / pyridine / DMA (1:1:8) was added, and the resulting suspension was shaken at room temperature for 15 minutes. The resin was drained and then washed with DMA (3×).

[0395] Step 2-2. Fmoc-deprotection was performed by repeated treatment with 4-methylpiperidine / DMA (1:4) (3 × 15 mL, shaken at room temperature for 15 minutes each time). After Fmoc-removal, the resin was washed with DMA (3 ×) and DCM (5 ×) to obtain (4S,7S,12S,15R)-4-amino-15-benzyl-7-(4-chlorobenzyl)-6,13-dimethyl-12-(4-((2-nitrophenyl)sulfonamide)butyl)-5,9,14-trioxo-2-oxa-6,10,13-triazaheptadecane-17-acid PS-(2-chlorotrityl), which was then used in the next step.

[0396] Process 3-1.A solution of Fmoc-Ala-OH (1.04 g, 3.339 mmol), PyOxim (1.761 g, 3.339 mmol), and DIEA (1.166 mL, 6.678 mmol) in NMP (25 mL) was shaken at room temperature for 2 minutes, and then added to (4S,7S,12S,15R)-4-amino-15-benzyl-7-(4-chlorobenzyl)-6,13-dimethyl-12-(4-((2-nitrophenyl)sulfonamide)butyl)-5,9,14-trioxo-2-oxa-6,10,13-triazaheptadecane-17-acid PS-(2-chlorotrityl) (1.113 mmol) which had been washed with NMP (3 ×). The resulting suspension was shaken at room temperature for 3 hours, then filtered, and the resin was washed with DMA (3 ×). To cap the resin, Ac2O / pyridine / DMA (1:1:8) (20 mL) was added, and the resulting suspension was shaken at room temperature for 15 minutes. The resin was drained and then washed with DMA (3 ×).

[0397] Step 3-2. Fmoc-deprotection was performed by repeated treatment with 4-methylpiperidine / DMA (1:4) (3 × 20 mL, shaken for 15 minutes at room temperature each time). After Fmoc-removal, the resin was washed with DMA (3 ×) and DCM (5 ×) to obtain (2S,5S,8S,13S,16R)-2-amino-16-benzyl-8-(4-chlorobenzyl)-5-(methoxymethyl)-7,14-dimethyl-13-(4-((2-nitrophenyl)sulfonamide)butyl)-3,6,10,15-tetraoxo-4,7,11,14-tetraazaoctadecane-18-acid PS-(2-chlorotrityl), which was then used in the next step.

[0398] Step 4.(2S,5S,8S,13S,16R)-2-amino-16-benzyl-8-(4-chlorobenzyl)-5-(methoxymethyl)-7,14-dimethyl-13-(4-((2-nitrophenyl)sulfonamide)butyl)-3,6,10,15-tetraoxo-4,7,11,14-tetraazaoctadecane-18-acid PS-(2-chlorotrityl) (1.113 mmol) was mixed with HFIP / DCM (1:3) (20 mL), and the resulting suspension was shaken at room temperature for 20 minutes. Next, the cutting solution was filtered off and recovered (3×). The resin was washed with DCM (2×), and the washing solution was recovered. The combined cutting solution and washing solution were concentrated to dryness under vacuum. The crude residue was freeze-dried in tBuOH / H2O (4:1) to obtain (2S,5S,8S,13S,16R)-2-amino-16-benzyl-8-(4-chlorobenzyl)-5-(methoxymethyl)-7,14-dimethyl-13-(4-((2-nitrophenyl)sulfonamide)butyl)-3,6,10,15-tetraoxo-4,7,11,14-tetraazaoctadecane-18-acid. Analytical method 12:t R =0.87 minutes;[M+H]+=902.7

[0399] Process 5.(2S,5S,8S,13S,16R)-2-amino-16-benzyl-8-(4-chlorobenzyl)-5-(methoxymethyl)-7,14-dimethyl-13-(4-((2-nitrophenyl)sulfonamide)butyl)-3,6,10,15-tetraoxo-4,7,11,14-tetraazaoctadecane-18-acid (456 mg, 0.5 mmol) and 4-chloro-2-(4-(1-methyl-2-(pyrroridine-1-ylmethyl)-1H-imidazole-5-yl)phenoxy)benzaldehyde (int-F2) (238 mg, 0.6 mmol) were dissolved in DCM (30 mL) and AcOH (0.114 mL, 2 mmol), and the resulting solution was stirred at room temperature for 1.5 hours. Next, NaBH(OAc)3 (530 mg, 2.5 mmol) was added, and the reaction mixture was stirred at room temperature for 18 hours. MeOH (2 mL) was added, and the reaction mixture was concentrated to dryness under vacuum. The crude product was purified by preparative reverse-phase HPLC (eluent A: 0.1% TFA in H2O and eluent B: ACN). The pure fractions were combined and freeze-dried to obtain (3S,6S,9S,14S,17R)-17-benzyl-1-(4-chloro-2-(4-(1-methyl-2-(pyrroridine-1-ylmethyl)-1H-imidazole-5-yl)phenoxy)phenyl)-9-(4-chlorobenzyl)-6-(methoxymethyl)-3,8,15-trimethyl-14-(4-((2-nitrophenyl)sulfonamide)butyl)-4,7,11,16-tetraoxo-2,5,8,12,15-pentazanonadecane-19-acid. Analytical method 9:t R =4.05 minutes;[M+H]+=1281.5

[0400] Process 6.(3S,6S,9S,14S,17R)-17-benzyl-1-(4-chloro-2-(4-(1-methyl-2-(pyrroridine-1-ylmethyl)-1H-imidazole-5-yl)phenoxy)phenyl)-9-(4-chlorobenzyl)-6-(methoxymethyl)-3,8,15-trimethyl-14-(4-((2-nitrophenyl)sulfonamide in DCM (100mL) To a solution of do)butyl)-4,7,11,16-tetraoxo-2,5,8,12,15-pentazanonadecane-19-acid (440 mg, 271 μmol), HATU (412 mg, 1.083 mmol), and HOAt (55.3 mg, 0.406 mmol), 2,6-lutidine (0.946 mL, 8.13 mmol) was added, and the resulting mixture was stirred at room temperature for 18.5 hours. The reaction mixture was concentrated to dryness under vacuum, and the resulting residue was partitioned between toluene (100 mL) and 5% aq. NaHCO3 (15 mL). The organic layer was washed with 5% aq. NaHCO3 (3 × 15 mL) and brine (10 mL), dried over Na2SO4, filtered, and evaporated to dryness to obtain N-(4-((2S,5S,8S,13S,16R)-16-benzyl-1-(4-chloro-2-(4-(1-methyl-2-(pyrroridine-1-ylmethyl)-1H-imidazole-5-yl)phenoxy)benzyl)-8-(4-chlorobenzyl)-5-(methoxymethyl)-2,7,14-trimethyl-3,6,10,15,18-pentaoxo-1,4,7,11,14-pentazacyclooctadecane-13-yl)butyl)-2-nitrobenzenesulfonamide (AB6). Analytical method 10:t R =1.1min;[M+2H] 2+ = 633.6.

[0401] Examples 1-4:Synthesis of (4S,7S,10S,14R,16aS,20aS)-10-(2-aminoethyl)-14-benzyl-11-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecahydrobenzo[l][1,4,7,11,14]pentazacyclooctadecine-2,6,9,12,15(3H)-pentaone(C6) [ka] Process 1.(S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)butanoic acid (int-G1) (1.000 g, 1.680 mmol) in DMF (20 mL) was mixed with DIPEA (0.587 mL, 3.36 mmol), then HATU (0.639 g, 1.680 mmol) was added, and the resulting mixture was stirred until completely homogenized. Next, this solution was added to 10 mL of DMF containing (R)-4-(((1S,2S)-2-((S)-3-((S)-2-amino-3-methoxy-N-methylpropanamide)-4-(4-chlorophenyl)butanamide)cyclohexyl)(methyl)amino)-3-benzyl-4-oxobutanoic acid PS-(2-chlorotrityl)(AB5) (2.8 g, 0.840 mmol) in a shaking flask. The reaction mixture was shaken overnight at room temperature. The resin was filtered, washed with DMF (3x) and DCM (3x), and dried under vacuum to obtain (R)-3-benzyl-4-(((1S,2S)-2-((8S,11S,14S)-8-((4-chloro-2-(4-(2-((dimethylamino)-methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)-14-(4-chlorobenzyl)-11-(methoxymethyl)-2,2,13-trimethyl-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecane-16-amide)cyclohexyl)(methyl)amino)-4-oxobutanoic acid PS-2-chlorotrityl, which was then removed without purification and proceeded to the next step.

[0402] Process 2.(R)-3-benzyl-4-(((1S,2S)-2-((8S,11S,14S)-8-((4-chloro-2-(4-(2-((dimethylamino)-methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)-14-(4-chlorobenzyl)-11-(methoxymethyl)-2,2,13-trimethyl-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecane-16-amide)cyclohexyl)(methyl)amino)-4-oxobutanoic acid PS-2-chlorotrityl (2.9 g, 0.87 mmol) was cleaved from the resin by shaking with 75 mL of 20% HFIP / DCM at room temperature for 20 minutes. The resin was filtered and the filtrate was collected. Both processes were repeated three more times to ensure that all of the product was cleaved from the resin. The combined filtrate was concentrated to obtain crude oil (1.8 g), which was purified by reverse-phase chromatography (eluting with MeCN / H2O along with 0.1% NH4OH) to obtain 1(R)-3-benzyl-4-(((1S,2S)-2-((8S,11S,14S)-8-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)-14-(4-chlorobenzyl)-11-(methoxymethyl)-2,2,13-trimethyl-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecane-16-amide)cyclohexyl)(methyl)amino)-4-oxobutanoic acid. Analysis method 7;t R =1.77 minutes;[M+H] + = 1182.7.

[0403] Process 3.(R)-3-benzyl-4-(((1S,2S)-2-((8S,11S,14S)-8-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)-14-(4-chlorobenzyl)-11-(methoxymethyl)-2,2,13-trimethyl-4,9,12-trio Xo-3-oxa-5,10,13-triazahexadecane-16-amide)cyclohexyl)(methyl)amino)-4-oxobutanoic acid (115 mg, 0.097 mmol) was mixed with HATU (148 mg, 0.389 mmol), 2,6-lutidine (0.340 mL, 2.92 mmol), and HOAt (13.23 mg, 0.097 mmol), and the resulting mixture was stirred overnight at 45°C. Next, the reaction mixture was concentrated to dryness and then partitioned between HCl (100 mL) and 5% aq. NaHCO3 (100 mL). The organic layer was washed with 5% aq. NaHCO3 (2 × 50 mL) and brine (50 mL), dried over Na2SO4, filtered, and concentrated to dryness by rotovap. Next, the residue was dissolved in HCl and washed with 1 M HCl (× 2). Saltwater was added to the combined 1M HCl layer and back-extracted with butyl(×2). The combined butyl layer was dried with sodium sulfate, filtered, and concentrated in vacuum. The product was purified by reverse-phase chromatography (eluting with MeCN / H2O, gradient 20-65% with 0.1% NH4OH) to obtain (2-((4S,7S,10S,14R,16aS,20aS)-14-benzyl-11-(4-chloro-2-(4-(2-((dimethylamino)-methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethyl-2,6,9,12,15-pentaoxodocosahydrobenzo[l][1,4,7,11,14]pentazacyclooctadecine-10-yl)ethyl)carbamate trifluoroacetate tert-butyl. Analytical method 2;t R =3,17 minutes;[M+H] + = 1163.6.

[0404] Step 4.To a solution of (2-((4S,7S,10S,14R,16aS,20aS)-14-benzyl-11-(4-chloro-2-(4-(2-((dimethylamino)-methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethyl-2,6,9,12,15-pentaoxodocosahydrobenzo[l][1,4,7,11,14]pentazacyclooctadecine-10-yl)ethyl)carbamate trifluoroacetate tert-butyl (40 mg, 0.034 mmol) in anhydrous dioxane (1.72 mL), HCl (4 M in dioxane) (0.67 mL, 2.40 mmol) was added dropwise at 0°C. The reaction mixture became turbid. After 15 minutes, the ice bath was removed, and the mixture was stirred at room temperature for 1 hour. Next, the reaction mixture was concentrated, and the resulting residue was dried under vacuum. The crude material was dissolved in phenylethylamine and washed with a saturated sodium bicarbonate solution. The aqueous layer was then extracted with fresh phenylethylamine. The combined organic matter was dried with sodium sulfate, filtered, and concentrated in vacuum to obtain (4S,7S,10S,14R,16aS,20aS)-10-(2-aminoethyl)-14-benzyl-11-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecahydrobenzo[l][1,4,7,11,14]pentazacyclooctadecine-2,6,9,12,15(3H)-pentaone(C6). Analytical method 7;t R =2.36 minutes;[M+H] + = 1063.7.

[0405] Examples 1-5: Synthesis of (2S,5S,8S,13S,16R)-2-(3-aminopropyl)-16-benzyl-1-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)-8-(4-chlorobenzyl)-5-(methoxymethyl)-7,13,14-trimethyl-1,4,7,11,14-pentazacyclooctadecane-3,6,10,15,18-pentaone (C7)

change

[0406] Examples 1-6: Synthesis of (4S,7S,10S,14R,16aS,20aS)-10-(3-aminopropyl)-14-benzyl-11-(4-chloro-2-(4-(1-methyl-2-(pyrroridine-1-ylmethyl)-1H-imidazole-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecahydrobenzo[l][1,4,7,11,14]pentazacyclooctadecine-2,6,9,12,15(3H)-pentaone(C8) [ka] (4S,7S,10S,14R,16aS,20aS)-10-(3-aminopropyl)-14-benzyl-11-(4-chloro-2-(4-(1-methyl-2-(pyrroridine-1-ylmethyl)-1H-imidazole-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecahydrobenzo[l][1,4,7,11, 14] Pentaazacyclooctadecine-2,6,9,12,15(3H)-pentaone(C8) is (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)butanoic acid (int-G1), (S)-5-((tert-butoxycarbon Except for being replaced by (4S,7S,10S,14R,16aS,20aS)-10-(2-aminoethyl)-14-benzyl-11-(4-chloro-2-(4-(2-((dimethyl))(4-aminoethyl)(4-(2-((dimethyl1-methyl-2-(pyrroridine-1-ylmethyl)-1H-imidazole-5-yl)phenoxy)(4-aminoethyl)(4-aminoethyl)(4-(2-aminoethyl)-10-(2-aminoethyl)-14-benzyl-11-(4-aminoethyl)(4-(2-(2-((dimethyl)(4-aminoethyl)(4-(2-(2-(2-((dimethyl)(4-aminoethyl)(4-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2 It was obtained using a method similar to that used in the synthesis of tylaminomethyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecahydrobenzo[l][1,4,7,11,14]pentazacyclooctadecine-2,6,9,12,15(3H)-pentaone (C6). Analytical method 7;t R =1.35 minutes;[M+H] + = 1102.2.

[0407] Examples 1-7:Synthesis of (4S,7S,10S,14R,16aS,20aS)-10-(3-aminopropyl)-14-benzyl-11-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecahydrobenzo[l][1,4,7,11,14]pentazacyclooctadecine-2,6,9,12,15(3H)-pentaone(C9) [ka] ((4S,7S,10S,14R,16aS,20aS)-10-(3-aminopropyl)-14-benzyl-11-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecahydrobenzo[l][1,4,7,11,14 Pentaazacyclooctadecine-2,6,9,12,15(3H)-pentaone(C9) is (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)butanoic acid (int-G1), (S)-5-((tert-butoxycarbonyl (4S,7S,10S,14R,16aS,20aS)-10-(2-aminoethyl)-14-benzyl-11-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)pentanoic acid (int-G2), except that it has been replaced by (4S,7S,10S,14R,16aS,20aS)-10-(2-aminoethyl)-14-benzyl-11-(4-chloro-2-(4-(2-((dimethylamino It was obtained using a method similar to that used in the synthesis of )methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecahydrobenzo[l][1,4,7,11,14]pentazacyclooctadecine-2,6,9,12,15(3H)-pentaone (C6). Analytical method 7;t R =1.23 minutes;[M+H] + = 1078.7.

[0408] Examples 1-8:Synthesis of (2S,5S,8S,13S,16R)-13-(4-aminobutyl)-16-benzyl-1-(4-chloro-2-(4-(1-methyl-2-(pyrroridine-1-ylmethyl)-1H-imidazole-5-yl)phenoxy)benzyl)-8-(4-chlorobenzyl)-5-(methoxymethyl)-2,7,14-trimethyl-1,4,7,11,14-pentazacyclooctadecane-3,6,10,15,18-pentaonetrifluoroacetate (C10) [ka] N-(4-((2S,5S,8S,13S,16R)-16-benzyl-1-(4-chloro-2-(4-(1-methyl-2-(pyrroridine-1-ylmethyl)-1H-imidazole-5-yl)phenoxy)benzyl)-8-(4-chlorobenzyl)-5-(methoxymethyl)-2,7,14-trimethyl-3,6,10,15,18-pentaoxo-1,4,7,11,14-penta To a solution of (532 mg, 0.271 mmol, 64.5%) of (zacyclooctadecane-13-yl)butyl)-2-nitrobenzenesulfonamide (AB6), 0.115 mL, 1.628 mmol and DBU (0.082 mL, 0.543 mmol) were added. The resulting mixture was stirred at room temperature for 30 minutes, quenched by the addition of AcOH (0.4 mL), and concentrated to dryness under vacuum. The crude product was purified by preparative reverse-phase HPLC (eluent A: 0.1% TFA in H2O and eluent B: ACN). The pure fractions were combined and freeze-dried to obtain (2S,5S,8S,13S,16R)-13-(4-aminobutyl)-16-benzyl-1-(4-chloro-2-(4-(1-methyl-2-(pyrroridine-1-ylmethyl)-1H-imidazole-5-yl)phenoxy)benzyl)-8-(4-chlorobenzyl)-5-(methoxymethyl)-2,7,14-trimethyl-1,4,7,11,14-pentazacyclooctadecane-3,6,10,15,18-pentaonetrifluoroacetate (C10). Analytical method 9;t R =3.85 minutes;[M+H] + = 1078.5.

[0409] Examples 1-9: Synthesis of (3R,7S,10S,13R)-7-(2-aminoethyl)-3-benzyl-6-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)-13-(4-chlorobenzyl)-10-(hydroxymethyl)-1,6,9,12-tetraazabicyclo[11.3.1]heptadecane-2,5,8,11-tetraone (C11) [ka] Step 1: To a solution of Fmoc-Ser(OtBMe2Si)OH (548 mg, 1.242 mmol) in DMA (5 mL), HATU (455 mg, 1.197 mmol) and DIPEA (0.789 mL, 4.52 mmol) were added. The resulting mixture was stirred at room temperature for 2 minutes, and then added to a solution of (R)-4-((R)-3-amino-3-(4-chlorobenzyl)piperidine-1-yl)-3-benzyl-4-oxobutanoate methyl (int-F3) (484 mg, 1.129 mmol) in DMA (3 mL). The reaction mixture was stirred at room temperature for 6 hours. Additional Fmoc-Ser(OtBMe2Si)OH (88 mg, 0.200 mmol) and HATU (76 mg, 0.20 mmol) were added, and stirring was continued overnight at room temperature. 4-methylpiperidine (0.8 mL, 6.77 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure (bath temperature 50°C), and the residue was purified by reverse flash column chromatography (eluting with ACN containing 5-90% / 0.1% NH4OH) to obtain (R)-4-((R)-3-((S)-2-amino-3-((tert-butyldimethylsilyl)oxy)propanamide)-3-(4-chlorobenzyl)piperidine-1-yl)-3-benzyl-4-oxobutanoate methyl. Analytical method 7, t R =1.41 minutes;[M+H] + = 630.5.

[0410] Step 2:DIPEA (0.374 mL, 2.142 mmol) was added to a solution of (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)butanoic acid (int-G1) (449 mg, 0.785 mmol) and (R)-4-((R)-3-((S)-2-amino-3-((tert-butyldimethylsilyl)oxy)propanamide)-3-(4-chlorobenzyl)piperidine-1-yl)-3-benzyl-4-oxobutanoate methyl (450 mg, 0.714 mmol) in DMA (5 mL). The resulting solution was stirred at room temperature for 2 minutes, followed by the addition of a solution of HATU (299 mg, 0.785 mmol) in DMA (3 mL). The reaction mixture was stirred at room temperature for 3 hours. Additional (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)butanoic acid (int-G1) (88 mg, 0.14 mmol) and HATU (76 mg, 0.20 mmol) were added, and stirring was continued overnight at room temperature. The resulting mixture was concentrated under reduced pressure, and the residue was purified by flash column chromatography (eluting with MeOH with 98 / 2~85 / 15 DCM / 0.3% triethylamine) to obtain (R)-3-benzyl-4-((R)-3-((S)-2-((S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)butanamide)-3-((tert-butyldimethylsilyl)oxy)propanamide)-3-(4-chlorobenzyl)piperidine-1-yl)-4-oxobutanoate methyl. Analytical method 7, t R =1.55 minutes;[M+H] + = 1184.1.

[0411] Step 3:To a solution of (R)-3-benzyl-4-((R)-3-((S)-2-((S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)butanamide)-3-((tert-butyldimethylsilyl)oxy)propanamide)-3-(4-chlorobenzyl)piperidine-1-yl)-4-oxobutanoate methyl (770 mg, 0.65 mmol) in DMA (5 mL), water (1 mL) and THF (4 mL) were added. The resulting mixture was stirred at room temperature, and then a solution of LiOH (1,300 mL, 1,300 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. An additional 1,300 mL of LiOH (1,300 mmol) was added, and the mixture was stirred overnight at room temperature (LC-MS showed a mixture of the desired product and desilyl alcohol R=H). The reaction mixture was cooled in an ice bath, the pH was neutralized by adding 1N HCl (pH=7), and then concentrated under reduced pressure (the bath was maintained at 30°C). The residue was dissolved in 250 mL of ethyl acetate. The organic phase was washed with water and brine, dried over Na2SO4, filtered, and concentrated to obtain (R)-3-benzyl-4-((R)-3-((S)-2-((S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)butanamide)-3-((tert-butyldimethylsilyl)oxy)propanamide)-3-(4-chlorobenzyl)piperidine-1-yl)-4-oxobutanoic acid. Analytical method 5, t R =1.97 minutes;[M+H] + = 1055.1.

[0412] Step 4:To a 1 L round-bottom flask containing a solution of (R)-3-benzyl-4-((R)-3-((S)-2-((S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)amino)butanamide)-3-((tert-butyldimethylsilyl)oxy)propanamide)-3-(4-chlorobenzyl)piperidine-1-yl)-4-oxobutanoic acid (770 mg, 0.658 mmol) in DCM (700 mL), 2,6-lutidine (2.3 mL, 19.74 mmol), HOAt (107 mg, 0.790 mmol), and HATU (1001 mg, 2.63 mmol) were added. The resulting mixture was stirred at 38°C for 16 hours. Next, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was partitioned between toluene (400 mL) and 5% aq. NaHCO3 (30 mL). The organic phase was washed with 5% aq. NaHCO3 (2 × 25 mL) and brine (30 mL), dried over Na2SO4, filtered, and concentrated. The crude material was purified by reverse flash column chromatography (eluting with AcN in 5-60% water / 0.1% trifluoroacetic acid) to obtain (2-((3R,7S,10S,13R)-3-benzyl-6-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)-13-(4-chlorobenzyl)-10-(hydroxymethyl)-2,5,8,11-tetraoxo-1,6,9,12-tetraazabicyclo[11.3.1]heptadecan-7-yl)ethyl)carbamate tert-butyl. Analytical method 2, t R =3.22 minutes, [M+H] + =1039.4).

[0413] Step 5:(2-((3R,7S,10S,13R)-3-benzyl-6-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)-13-(4-chlorobenzyl)-10-(hydroxymethyl)-2,5,8,11-tetraoxo-1,6,9,12-tetraazabicyclo[11.3.1]heptadecane-7-yl)ethyl)carbamate tert-butyl (320 mg, 0.308 mmol) was added to a round-bottom flask containing (2 mL, 8.00 mmol) 4.0 N hydrogen chloride in dioxane (6 mL), which had been cooled in an ice bath. The ice bath was then removed, and the resulting mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure to obtain a grayish-white solid, which was purified by reverse flash column chromatography (eluting with ACN in 5-50% water / 0.1% trifluoroacetic acid) to obtain (3R,7S,10S,13R)-7-(2-aminoethyl)-3-benzyl-6-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazole-5-yl)phenoxy)benzyl)-13-(4-chlorobenzyl)-10-(hydroxymethyl)-1,6,9,12-tetraazabicyclo[11.3.1]heptadecane-2,5,8,11-tetraone (C11). Analytical method 3, t R =1.23 minutes;[M+H] + = 937.6.

[0414] Example 2: Synthesis of FHR3 receptor ligand compounds (C12) to (C16) Example 2-1:2-((3S,6S,9R,15S,18S,21S,24S,27S,30S,33S,39S,42S,50aS)-3,30-bis(2-amino-2-oxoethyl)-9-((2-amino-2-oxoethyl)carbamoyl)-27-(4-aminobutyl)-6,15,21-tribenzyl-18,39-bis(3-guanidinopropyl)-33-(4-hydroxybenzyl)-24-(hydroxymethyl)-5,20,35-trimethyl-1,4,7,13,16,19,22,25,28,31,34,37,40,43-tetra Synthesis of decaoxo-1,3,4,5,6,7,8,9,10,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,45,50,50a-tetratetracontahydro-2H-[1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40,43]tetradecazacyclopentatetracontino[13,12-b]isoquinoline-42-yl)acetic acid (C12) [ka] Note: Compound (C12) has the following amino acid sequence. [ka] Step 1: FRF(N-Me)-S(tBu)-KNY(tBu)-G(N-Me)-RD(tBu)-Tic-NF(N-Me)-C(Trt)-G-NH resin (1-1b) Peptide sequence 2-1b was synthesized on Fmoc-Gly-RAM TentaGel® resin (2-1a, 0.22 mmol / g loading, 0.25 mmol scale) on a Liberty® peptide synthesizer. The resin was then filtered and washed with DMF (2×) and DCM (3×) to obtain the desired product 2-1b.

[0415] Step 2: ClCH2C(=O)-FRF(N-Me)-S(tBu)-KNY(tBu)-G(N-Me)-RD(tBu)-Tic-NF(N-Me)-C(Trt)-G-NH resin (1-1d) A solution of N-succinimidyl 2-chloroacetate (2-1c, 287 mg, 1.5 mmol) in NMP (8 mL) was added to the peptide resin 2-1b (0.25 mmol) from step 1, and the resulting mixture was shaken overnight at room temperature. Next, the resin was drained, washed with DMF (3 ×) and DCM (4 ×), and dried to obtain the desired product 2-1d.

[0416] Step 3: ClCH2C(O)-FRF(N-Me)-SKNYG(N-Me)-RD-Tic-NF(N-Me)-CG-NH2(1-1e) The peptide resin product 2-1d from step 2 was cleaved from the resin and simultaneously deprotected using cleavage method 1 described above herein to obtain crude peptide 2-1e.

[0417] Step 4:2-((3S,6S,9R,15S,18S,21S,24S,27S,30S,33S,39S,42S,50aS)-3,30-bis(2-amino-2-oxoethyl)-9-((2-amino-2-oxoethyl)carbamoyl)-27-(4-aminobutyl)-6,15,21-tribenzyl-18,39-bis(3-guanidinopropyl)-33-(4-hydroxybenzyl)-24-(hydroxymethyl)-5,20,35-trimethyl-1,4,7,13,16,19,22,25,28,31,34,37,40,43-teto Radecaoxo-1,3,4,5,6,7,8,9,10,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,45,50,50a-Tetratetracontahydro-2H-[1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40,43]tetradecazacyclopentatetracontino[13,12-b]isoquinoline-42-yl)acetic acid (C12) Crude peptide 1-2e (266 mg) from step 3 was dissolved in DMSO (20.5 mL). A few drops of TEA were added to adjust the pH to 8-9. The resulting mixture was stirred overnight at room temperature. Next, the reaction mixture was concentrated on a centrifugal evaporator until it reached a few mL of DMSO. The crude cyclic peptide was purified by preparative HPLC (Sunfire® Prep C18 column, 130 Å, 5 μm, 30 × 50 mm, 15-40% in 6 min, 75 mL / min, ACN in water with 0.1% TFA), followed by lyophilization to obtain the title cyclic peptide compound (C12) (SEQ ID NO: 5). Analytical method 9:t R =3.24, m+1=1951.20

[0418] Example 2-2:2-((3S,6S,9R,15S,18S,21S,24S,27S,30S,33S,36S,39S,44aS)-30-((1H-imidazole-5-yl)methyl)-33-((1H-indole-3-yl)methyl)-9-((2-amino-2-oxoethyl)carbamoyl)-18-(4-aminobutyl)-15-benzyl-6,39-bis(3-guanidinopropyl)-24-(Hyd Synthesis of Roxymethyl)-27-Isobutyl-20,21,35,36-Tetridecaoxodotetracontahydro-12H-Pyrrolo[1,2-e1][1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40]tridecazacyclodotetracontin-3-yl)acetic acid (C13) [ka] Note: Compound (C13) has the following amino acid sequence. [ka] The cyclic peptide compound (C13) (SEQ ID NO: 6) was obtained using a peptide sequence bound to resin in step 1, in a similar manner to that described in Example 2-1. [ka] This was synthesized in place of peptide sequences 2-4b. Analytical method 9:t R =3.01, m+1=1710.98

[0419] Examples 2-3:2-((3S,6S,9R,15S,18S,21S,24S,27S,30S,33S,36S,39S,42S,50aS)-3,30-bis(2-amino-2-oxoethyl)-9-((2-amino-2-oxoethyl)carbamoyl)-36-(4-aminobutyl)-6,15,21-tribenzyl-18,39-bis(3-guanidinopropyl)-33-(4-hydroxybenzyl)-24-(hydroxymethyl)-27-isopropyl-5,20-dimethyl-1,4,7,13,16,19,22,25,28,31,34,37,40,4 Synthesis of 3-tetradecaoxo-1,3,4,5,6,7,8,9,10,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,45,50,50a-tetratetracontahydro-2H-[1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40,43]tetradecazacyclopentatetracontino[13,12-b]isoquinoline-42-yl)acetic acid (C14) [ka] Note: Compound (C14) has the following amino acid sequence. [ka] The cyclic peptide compound (C14) (SEQ ID NO: 7) was obtained using a peptide sequence bound to resin in step 1, in a similar manner to that described in Example 2-1. [ka] This was synthesized in place of peptide sequences 2-4b. Analytical method 9:t R =3.35, m+1=1979.25

[0420] Examples 2-4:2-((3R,6S,9S,12S,15S,18S,21S,24S,27S,30S,33S,36S,39S)-24-((1H-imidazole-5-yl)methyl)-21-((1H-indole-3-yl)methyl)-3-((2-amino-2-oxoethyl)carbamoyl)-12-(4-aminobutyl)-36,39-dibenzyl-6,15-bis(3-guanidinopropyl) Synthesis of (Pyr)-30-(hydroxymethyl)-27-isobutyl-18,19,33,34,37-pentamethyl-5,8,11,14,17,20,23,26,29,32,35,38,41-tridecaoxo-1-thia-4,7,10,13,16,19,22,25,28,31,34,37,40-tridecazacyclodotetracontancontan-9-yl)acetic acid (C15) [ka] Note: Compound (C15) has the following amino acid sequence. [ka] The cyclic peptide compound (C15) (SEQ ID NO: 8) was obtained using a peptide sequence bound to resin in step 1, in a similar manner to that described in Example 2-1. [ka] This was synthesized in place of peptide sequences 2-4b. Analytical method 9:t R =3.35, m+1=1775.07

[0421] Examples 2-5:2,2'-((3S,6S,9R,15S,18S,21S,24S,27S,30S,33S,39S,42S,50aS)-9-((2-amino-2-oxoethyl)carbamoyl)-42-(4-aminobutyl)-6,15,21-tribenzyl-18,39-bis(3-guanidinopropyl)-33-(4-hydroxybenzyl)-24-(hydroxymethyl)-27-isopropyl-5,20,35-trimethyl-1,4,7,13,16,19,22,25,28,31,34,37,40,43-tetradecaoxo-1,3, Synthesis of 4,5,6,7,8,9,10,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,45,50,50a-Tetratetracontahydro-2H-[1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40,43]tetradecazacyclopentatetracontino[13,12-b]isoquinoline-3,30-diyl)diacetamide (C16) [ka] Note: Compound (C16) has the following amino acid sequence. [ka] The cyclic peptide compound (C16) (SEQ ID NO: 9) was obtained using a peptide sequence bound to resin in step 1, in a similar manner to that described in Example 2-1. [ka] This was synthesized in place of peptide sequences 2-4b. Analytical method 9:t R =3.45, m+1=1935.24

[0422] Example 3: Synthesis of ASGPR receptor ligand and M6P receptor ligand Synthesis of intermediates Type AA: Synthesis of benzyl 5-hydroxypentanoate (int-AA1) [ka] Step 1: To a solution of dihydro-2H-pyran-2,6(3H)-dione (20 g, 175.44 mmol) and BnOH (20.8 g, 192.98 mmol) in DCM (150 mL), DMAP (0.32 g, 2.62 mmol) and Et3N (29 mL, 210.5 mmol) were added at 0°C. The reaction mixture was warmed to room temperature and stirred for 2 days. The reaction mixture was evaporated to dryness, and the resulting residue was dissolved in DCM (200 mL) and washed with 3 M HCl (100 mL x 2). The organic layer was dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (eluate:PE:EA = 20:1 to 10:1) to obtain 5-(benzyloxy)-5-oxopentanoic acid as a colorless oil. 1 H NMR(400MHz,CDCl3)δ ppm 7.38-7.28(m,5H),5.12(s,2H),2.46-2.40(m,4H),2.00-1.93(m,2H).

[0423] Step 2: The starting material was added dropwise at 0°C under N2 protection to a solution of 5-(benzyloxy)-5-oxopentanoic acid (30 g, 135.1 mmol) in THF (200 mL)BH3-S(CH3)2 (20.2 ml, 202.7 mmol). The mixture was warmed to room temperature and stirred for 16 hours. TLC showed that the starting material was completely consumed. The reaction product was carefully quenched with H2O (8 mL). The resulting mixture was filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (eluate:PE:EA = 20:1~2:1) to obtain benzyl 5-hydroxypentanoate (int-AA1). 1 H NMR(400MHz,CDCl3)δ ppm 7.41-7.33(m,5H),5.14(s,2H),3.66(t,2H,J=6Hz),2.43(t,2H,J=7.2Hz),1.80-1.73(m,2H),1.65-1.58(m,2H).

[0424] Synthesis of 3-((6-azidohexyl)oxy)-2-hydroxy-3-((1-hydroxy-3-oxopropan-2-yl)oxy)propanal (int-AA2) [ka] (3R,4R,5R,6R)-2-((6-azidohexyl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (see Hwu,Jih Ru;Hsu,Chuan-I;Hsu,ming-Hua;Liang,Yu-Chuan;Huang,Ru Chih C.;Lee,Yuan C. Bioorganic and Medicinal Chemistry Letters, 2011, vol.21, #1, pp.380-382) (40 mg, 0.131 mmol) was added to (3R,4R,5R,6R)-2-((6-azidohexyl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol) (see Hwu,Jih Ru;Hsu,Chuan-I;Hsu,ming-Hua;Liang,Yu-Chuan;Huang,Ru Chih C.;Lee,Yuan C. Bioorganic and Medicinal Chemistry Letters, 2011, vol.21, #1, pp.380-382) (40 mg, 0.131 mmol) on silica. The mixture was stirred at room temperature for 4 hours, filtered, and then purified by reverse-phase flash chromatography to obtain 3-((6-azidohexyl)oxy)-2-hydroxy-3-((1-hydroxy-3-oxopropan-2-yl)oxy)propanal (int-AA2). Analytical method 7:t r =0.80 min, MS m / z 326.2[M+Na]+.

[0425] Type BB: Synthesis of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2,5-dioxopyrrolidine-1-yl)oxy)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl(int-BB1) [ka] Step 1: To a solution of (2R,3R,4R,5R)-2-amino-3,4,5,6-tetrahydroxyhexanal hydrochloride (100.0 g, 0.132 mol) in pyridine (1 L), acetic anhydride (473 g, 4.64 mol) was added at 0°C. The reaction mixture was stirred at room temperature for 72 hours. The resulting precipitate was collected, washed with H2O (200 mL x 2), and dried under vacuum to obtain triacetic acid (3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl.1 H NMR(400MHz,CDCl3)δ ppm 5.68(d,1H,J=8.8Hz),5.46(d,1H,J=9.2Hz),5.35(d,1H,J=3.2Hz),5.07(dd,1H,J1=11.2Hz,J2=3.2Hz),4.47-4. 39(m,1H),4.18-4.07(m,2H),4.02-3.98(m,1H),2.16(s,3H),2.11(s,3H),2.03(s,3H),2.00(s,3H),1.93(s,3H).

[0426] Step 2: To a solution of (3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetic acid (100 g, 0.257 mol) in 1,2-dichloroethane (500 mL) cooled to 0°C, TMSOTF (85.5 g, 0.385 mol) was added, and the mixture was stirred for 10 minutes, then cooled to 50°C and stirred for 3 hours. TLC showed that the starting material was completely consumed. After cooling, the resulting mixture was treated with saturated aqueous solution of NaHCO3 (1000 mL) at 0°C and extracted with DCM (500 mL x 2). The combined organic layers were dried over Na2SO4 and concentrated. The residue was dried overnight under high vacuum to obtain (3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazole-6,7-diyl diacetic acid. 1 H NMR(400MHz,CDCl3)δ ppm 6.00(d,1H,J=2.8Hz),5.47-5.46(m,1H),4.93-4.90(m,1H),4.27-4.18(m,2H) ,4.13-4.09(m,1H),4.02-3.98(m,1H),2.13(s,3H),2.07(s,6H),2.06(s,3H).

[0427] Step 3:(3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazole-6,7-diyl diacetic acid (65 g, 197.4 mmol) and benzyl 5-hydroxypentanoate (int-AA1) (41 g, 197.4 mmol) were dissolved in DCM (600 mL). Molecular sieves (50 g) were added, and the reaction mixture was stirred for 30 minutes, after which TMSOTF (6.5 g, 29.6 mmol) was added. The reaction mixture was then stirred overnight at room temperature. TLC showed that the starting materials were completely consumed. The reaction mixture was filtered to remove the molecular sieves. The filtrate was treated with saturated aqueous solution of NaHCO3 (500 ml) and extracted with DCM (500 mL x 2). The combined organic layers were dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (eluate:PE:EA = 2:1 to 1:2) to obtain diacetic acid (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-(benzyloxy)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl. 1 H NMR(400MHz,CDCl3)δ ppm 7.37-7.32(m,5H),5.60(d,1H,J=8.4Hz),5.35(d,1H,J=2.4Hz),5.25(dd,1H,J1=11.6Hz,J2=3.6Hz),5.11(s,2H),4.63(d,1H,J1=8.4Hz),4.15 -4.11(m,2H),3.98-3.86(m,3H),3.55-3.45(m,1H),2.41-2.36(m,2H), 2.14(s,3H),2.03(s,3H),2.00(s,3H),1.91(s,3H),1.72-1.55(m,4H).

[0428] Step 4:(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-(benzyloxy)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetic acid (90 g, 167.4 mmol) was dissolved in a mixture of ELISA (250 mL) and MeOH (250 mL), followed by the addition of Pd / C (4.5 g, 10%). The reaction mixture was degassed, refilled with H2 by balloon, and then stirred overnight. TLC showed that the starting material had been completely consumed. The reaction mixture was filtered, and the filtrate was concentrated to dryness to obtain 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid. 1 H NMR(400MHz,CDCl3)δ ppm 5.95(d,1H,J=8.4Hz),5.35(d,1H,J=2.4Hz),5.25(dd,1H,J1=11.6Hz,J2=3.6Hz),4.66(d,1H,J1=8Hz),4.16-4.11(m,2H),4. 01-3.90(m,3H),3.56-3.49(m,1H),2.40-2.34(m,2H),2.16(s,3H),2.06(s,3H),2.01(s,3H),1.98(s,3H),1.72-1.55(m,4H).

[0429] Step 5:To a solution of 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (69 g, 154.2 mmol) and NHS-OH (19.5 g, 169.62 mmol) in DCM (600 mL), DIC (19.4 g, 154.2 mmol) and DMAP (36 mg, 0.29 mmol) were added. The reaction mixture was stirred at room temperature for 3 hours. TLC showed that the starting materials were completely consumed. The resulting mixture was filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (eluate:PE:EA = 2:1 to 1:4) to obtain diacetic acid (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2,5-dioxopyrrolidine-1-yl)oxy)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl (int-BB1). 1 H NMR(400MHz,CDCl3)δ ppm 5.83(d,1H,J=8.4Hz),5.33(d,1H,J=2.4Hz),5.25(dd,1H,J1=11.6Hz,J2=3.6Hz),4.67(d,1H,J1=8Hz),4.13-4.07(m,2H),4. 00-3.87(m,3H),2.86-2.82(m,4H),2.73-2.55(m,2H),2.14(s,3H),2.02(s,3H),1.97(s,3H),1.91(s,3H),1.72-1.55(m,4H).

[0430] Synthesis of (1S,2R,3R,4R,5S)-4-amino-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octane-2,3-diol (int-BB2) [ka] Step 1:A mixture of (2R,3R,4R)-2-(hydroxymethyl)-3,4-dihydro-2H-pyran-3,4-diol (500 g, 3.42 mol) and pyridine (1.93 L, 23.95 mol) was stirred at 20°C for 30 minutes, then cooled to 0°C. Ac2O (1.12 L, 11.97 mol) was added dropwise while maintaining a temperature of 5°C to 15°C. The reaction mixture was further stirred under N2 at 20°C for 2 hours, cooled to 0°C, quenched with ice water (1 L), then extracted with MTBE (3 × 1.2 L), followed by extraction with EA (2 × 1 L). The combined organic layers were washed with 0.5N HCl (3 × 1 L), saturated NaHCO3 (1 L), and then brine (1 L). Next, the combined aqueous layer was extracted with toluene (3 L), and the organic layer was washed with 0.5 N HCl (3 × 1 L), saturated NaHCO3 (1 L), and then brine (1 L). All the organic layers were combined, dried over Na2SO4, filtered, and concentrated under vacuum at 35°C to obtain (2R,3R,4R)-2-(acetoxymethyl)-3,4-dihydro-2H-pyran-3,4-diyl diacetic acid. 1 H NMR:(CDCl3400MHz)δ 1.98-2.14(m,9H)4.17-4.33(m,3H)4.71(ddt,J=5.00,2.61,1.27,1.27Hz, 1H)5.41(dd,J=4.34,1.65Hz,1H)5.51-5.57(m,1H)6.45(d,J=6.24Hz,1H).

[0431] Step 2:A solution of (2R,3R,4R)-2-(acetoxymethyl)-3,4-dihydro-2H-pyran-3,4-diyl diacetic acid (900 g, 3.31 mol, 1 equivalent) dissolved in MeCN (2 L) was added to MeCN (16 L) with stirring (300 rpm) under a stream of N2, and the mixture was then cooled to -15°C under N2. NaN3 (429.82 g, 6.61 mol, 2 equivalents) was added to the reaction mixture in small amounts under a gentle stream of N2 while maintaining a temperature of -15°C to -10°C. Cerium ammonium nitrate (5.44 kg, 9.92 mol) was added to the reaction mixture in six portions over 3 hours with stirring (350 rpm) under a gentle stream of N2 while maintaining a temperature of -15°C to -10°C. Next, the reaction mixture was stirred under N2 at a temperature of -15°C to -10°C for 4 hours, and MTBE (10 L) was added in two batches. H2O (10 L) was carefully added to the reaction mixture under N2 flow at a temperature of -5°C to 0°C, the mixture was stirred at 0°C for 30 minutes, and then allowed to stand at room temperature (25°C) for 16 hours. The mixture was separated, the organic layer was washed with H2O (8 × 10 L), then dried over Na2SO4, filtered, and concentrated under vacuum at 20-25°C to obtain (2R,3R,4R,5R,6R)-2-(acetoxymethyl)-5-azido-6-(nitrooxy)tetrahydro-2H-pyran-3,4-diyl diacetic acid, which was used directly in the next step.

[0432] Step 3:A solution of (2R,3R,4R,5R,6R)-2-(acetoxymethyl)-5-azido-6-(nitrooxy)tetrahydro-2H-pyran-3,4-diyl diacetic acid (1140g, 3.03mol.) in MeOH (8L) was cooled to 0°C, and NaOMe (1.1M, 1.60L) was added while maintaining a temperature of 0°C to 5°C. Next, the reaction mixture was stirred at a temperature of 0°C to 5°C for 2 hours. Then, resin (H+) (500g) was added, and the reaction mixture was stirred for a further 30 minutes. The reaction mixture was filtered, and the filtrate was rinsed with MeOH (2L). The mixture was triturated with MeOH (2L) at room temperature (25°C) for 30 minutes and filtered (4 repetitions). All the filtrates were combined and concentrated under vacuum at 35°C to obtain the residue, which was purified by column chromatography (DCM:MeOH = 50:1 to 30:1) on silica gel to obtain (2R,3R,4R,5R)-5-azido-2-(hydroxymethyl)-6-methoxytetrahydro-2H-pyran-3,4-diol.

[0433] Step 4: (2R,3R,4R,5R)-5-azido-2-(hydroxymethyl)-6-methoxytetrahydro-2H-pyran-3,4-diol (320 g, 1.46 mol) in pyridine (589.17 mL, 7.30 mol) was added to DCM (3.2 L) at 25°C. The mixture was cooled to 0°C and stirred at a temperature of 0°C to 5°C for 30 minutes. Next, TMSCl (634.42 g, 5.84 mol, 741.14 mL, 4 equivalents) was added dropwise, and the resulting white suspension was stirred at a temperature of 5°C to 10°C for 1 hour. The slurry was quenched with saturated NH4Cl (1.5 L), stirred for 10 minutes, allowed to stand for 5 minutes, and then separated. The DCM layer was washed with NH4Cl (1.5 L x 4) and H2O (1.5 L x 5), dried with Na2SO4, filtered, and concentrated under vacuum at 35°C to obtain (((2R,3S,4R,5R)-5-azido-6-methoxy-2-(((trimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3,4-diyl)bis(oxy))bis(trimethylsilane), which was used directly for the next step.

[0434] Step 5:((((2R,3S,4R,5R)-5-azido-6-methoxy-2-(((trimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3,4-diyl)bis(oxy))bis(trimethylsilane) (580.00 g, 1.33 mol) was added dropwise to (((2R,3S,4R,5R)-5-azido-6-methoxy-2-(((trimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3,4-diyl)bis(oxy))bis(trimethylsilane) (580.00 g, 1.33 mol) in MeOH (160 mL) at a temperature of -10 °C to 5 °C, and the reaction mixture was stirred for 30 minutes. Next, the reaction m...

Claims

【Request Item 1】 【Chemistry 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 A bifunctional compound selected from among them.

2. A pharmaceutical composition comprising the bifunctional compound described in claim 1 and one or more pharmaceutically acceptable carriers.

3. The bifunctional compound according to claim 1, for use in the treatment of PCSK9-mediated diseases or disorders, wherein the PCSK9-mediated disease or disorder is selected from hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral vascular disease, peripheral artery disease, vasculitis, elevated Lp(a), elevated LDL, triglyceride-rich lipoprotein (TRL), elevated triglycerides, sepsis, and xanthomas.

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