Targeting plasma protein degradation

Abstract

This invention relates to bifunctional compounds and the use of such bifunctional compounds to reduce plasma levels of extracellular target molecules via lysosomal degradation. These bifunctional compounds have cell surface receptor ligands covalently linked to ligands 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, cell or plasma membrane proteins), wherein said cell surface receptors are associated with receptor-mediated endocytosis, including lysosomal degradation mediated by desialylate glycoprotein receptor (ASGPR) and lysosomal degradation mediated by mannose-6-phosphate receptor (M6PR). Pharmaceutical compositions comprising such bifunctional compounds and methods of treating diseases or disorders mediated by extracellular molecules using such bifunctional compounds are also provided herein.

Description

Technical Field

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

[0002] Conventional protein-targeted therapies treat diseases by blocking protein function (e.g., enzyme inhibitors and receptor antagonists) 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 with incompletely understood or difficult-to-inhibit molecular functions, cannot be targeted with drugs using conventional therapies. Targeted protein degradation (TPD) is a therapeutic approach that treats these untargetable pathogenic proteins and signaling pathways by controlling the amount of the target protein through degradation rather than inhibiting its function.

[0003] Examples of targeted protein degradation systems include proteolytic targeting chimeras (PROTAC) (KMSakamoto et al., Proc. Natl. Acad. Sci. [PNAS] 98, 8554-8559, (2001) and GEWinter et al., Science. [Science] 348, 1376-1381 (2015)), dTAG (B. Nabet et al., Nat. Chem. Biol. [Nature Chemical Biology] 14, 431 (2018)), Trim-Away (D. Clift et al., Cell. [Cell], 171, 1692-1706, e18 (2017)), chaperone-mediated autophagy targeting (X. Fan et al., Nat. Neurosci. [Nature Neuroscience], 17, 471-480 (2014)), and SNIPER (M. Naito et al., Drug Discov. Today Technol. [Drug Discovery Today - Technology], (2019)). PROTACs form bridges between E3 ubiquitin ligases and their targets of interest, thereby facilitating ubiquitination and proteasome degradation (GM Burslem et al., Chem. Rev. [Chemical Reviews], 117, 11269-11301 (2017)). These degradation systems utilize the proteasome pathway to degrade intracellular proteins. In addition, degradation systems utilizing the lysosomal pathway to degrade extracellular proteins (secretory proteins and plasma membrane proteins) have been reported (S. Banik et al., ChemRxiv, 2019 and PCNRensen et al., J. Med. Chem. [Journal of Medicinal Chemistry], 47, 5798-5808, 2004), but strategies for degrading extracellular targets such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, cellular and plasma membrane proteins remain an unmet need. Summary of the Invention

[0004] This invention relates to bifunctional compounds and their use in reducing plasma levels of extracellular target molecules in patients via receptor-mediated endocytosis followed by lysosomal degradation, thereby discovering their use as pharmaceutical agents in treating disease states and / or conditions mediated by such extracellular molecules. Therefore, the 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 plasma membrane proteins, via lysosomal degradation. The invention also 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 plasma membrane proteins, via desialylate-glycoprotein receptor (ASGPR)-mediated lysosomal degradation. The present invention also 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, cellular and plasma membrane proteins, via mannose-6-phosphate receptor (M6PR)-mediated lysosomal degradation.

[0005] The bifunctional compounds of the present invention can provide important clinical benefits to patients, particularly for the treatment of disease states and conditions regulated by extracellular targets of interest.

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

[0007] The present invention also provides a bifunctional compound having the structure of formula (I):

[0008] R L -L A -T L (I)

[0009] in:

[0010] R L It is a portion of cell surface receptors that bind to receptor-mediated endocytosis;

[0011] L A It's a connector.

[0012] and

[0013] T L It is the part that binds to extracellular targets.

[0014] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a bifunctional compound having formula (I) and a pharmaceutically acceptable carrier.

[0015] On the other hand, 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.

[0016] The present invention also provides pharmaceutical compositions comprising a bifunctional compound having formula (I) and a pharmaceutically acceptable carrier.

[0017] In another aspect, the present invention provides pharmaceutical compositions comprising the compounds of the present invention and one or more pharmaceutically acceptable carriers.

[0018] In another aspect, the present invention provides combinations, particularly pharmaceutical combinations, comprising therapeutically effective amounts of the compounds of the present invention and one or more therapeutically active agents.

[0019] This invention provides a method for 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, and cell and plasma membrane proteins, by applying the bifunctional compounds of this invention. This invention also provides a method for targeting the desialylate glycoprotein receptor (ASGPR)-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, and cell and plasma membrane proteins, by applying the bifunctional compounds of this invention. The present invention also provides a method for targeting lysosomal degradation 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 plasma membrane proteins, by applying the bifunctional compounds of the present invention to target mannose-6-phosphate receptor (M6PR)-mediated extracellular target molecules.

[0020] These methods can be used to treat a variety of diseases, conditions, or clinical conditions that are typically treated with therapeutic apheresis, such as cardiovascular diseases, liver diseases, kidney diseases, autoimmune diseases, neurological diseases, blood diseases, skin diseases, drug poisoning, and vasculitis. For example, such diseases include, but are not limited to, hypercholesterolemia, familial hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, occlusive arteriosclerosis, coronary heart disease, peripheral vascular disease (including aortic and cerebrovascular diseases), peripheral artery disease, vascular inflammation, elevated Lp(a), elevated LDL, elevated TRL, elevated triglycerides, sepsis, xanthomas, fulminant hepatic failure, postoperative liver failure, acute liver failure, hepatitis C, hepatitis B, and chronic hepatitis C. Hepatitis, chronic hepatitis B, liver allogeneic transplantation, focal glomerulosclerosis, kidney allogeneic transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Gurney-Barth syndrome, chronic inflammatory demyelinating polyneuropathy, membranous nephropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood type incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, and Kawasaki disease.

[0021] These methods can also be used to treat kidney disease, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0022] In another aspect, the present invention also provides a method for treating a disease or condition regulated by an extracellular target molecule by administering a therapeutically effective amount of a bifunctional compound having formula (I) or a subformula thereof to a subject in need.

[0023] On the other hand, the present invention also provides the use of bifunctional compounds having formula (I) or its subforms for treating diseases or conditions regulated by targeted extracellular molecules as described herein.

[0024] On the other hand, the present invention also provides the use of bifunctional compounds having formula (I) or its subforms in the manufacture of medicaments for treating diseases or conditions regulated by extracellular molecules as described herein.

[0025] In another aspect, the present invention also provides an in vivo therapeutic plasma exchange method, wherein the method comprises administering to a subject a bifunctional compound having formula (I) or a subformula thereof. The present invention also provides a method for performing in vivo therapeutic plasma exchange, wherein the method comprises administering to a subject the bifunctional compound of the present invention.

[0026] On the other hand, the present invention also provides an in vivo therapeutic plasma exchange method for treating cardiovascular diseases, liver diseases, kidney diseases, autoimmune diseases, neurological diseases, blood diseases, skin diseases, drug poisoning, or vasculitis, wherein the method comprises administering the bifunctional compound of the present invention to a subject. In some embodiments, such diseases are hypercholesterolemia, familial hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, occlusive arteriosclerosis, coronary heart disease, peripheral vascular disease (including aortic disease and cerebrovascular disease), peripheral artery disease, vascular inflammation, elevated Lp(a), elevated LDL, elevated TRL, elevated triglycerides, sepsis, xanthomas, fulminant hepatic failure, postoperative liver failure, acute liver failure, hepatitis C, hepatitis B, chronic hepatitis C. Hepatitis, chronic hepatitis B, liver allogeneic transplantation, focal glomerulosclerosis, kidney allogeneic transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Gurney-Barth syndrome, chronic inflammatory demyelinating polyneuropathy, membranous nephropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood type incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, and Kawasaki disease.

[0027] On the other hand, the present invention also provides an in vivo therapeutic plasma exchange method for treating kidney disease, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0028] In another aspect, the present invention also provides a therapy based on inhibiting the extracellular level of extracellular target molecules by lysosomal degradation mediated by a bifunctional compound having formula (I) or a subformula thereof.

[0029] In another aspect, the present invention also provides a therapy for treating cardiovascular diseases, which is based on inhibiting the extracellular level of the proprotein convertase subtilisin / kexin9 type (PCSK9) by lysosomal degradation mediated by a bifunctional compound having formula (Ia).

[0030] In another aspect, the present invention also provides a therapy for treating diseases or disorders associated with complement factor H-related protein 3 (CFHR3) gene, the therapy being based on inhibiting extracellular levels of complement factor H-related protein 3 (FHR3) by lysosomal degradation mediated by a bifunctional compound having formula (Ib).

[0031] In another aspect, the present invention also provides a therapy for treating diseases or disorders associated with complement factor H-related protein 3 (FHR3), the therapy being based on inhibiting extracellular complement factor H-related protein 3 (FHR3) levels by lysosomal degradation mediated by a bifunctional compound having formula (Ib).

[0032] In another aspect, the present invention also provides a bifunctional compound having formula (Ia) for use in treating PCSK9-mediated diseases or disorders. In another aspect, the present invention also provides a pharmaceutical composition comprising a bifunctional compound having formula (Ia) for use in treating PCSK9-mediated diseases or disorders. In certain 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, vascular inflammation, elevated Lp(a), elevated LDL, triglyceride-rich lipoprotein (TRL), elevated triglycerides, sepsis, and xanthoma.

[0033] In another aspect, the present invention also provides a bifunctional compound having formula (Ib) for use in treating CFHR3-mediated diseases or disorders. In yet another aspect, the present invention also provides a pharmaceutical composition comprising a bifunctional compound having formula (Ib) for use in treating CFHR3-mediated diseases or disorders. In some embodiments of such use, the CFHR3-mediated diseases or disorders are selected from kidney disease, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0034] In another aspect, the present invention also provides a bifunctional compound having formula (Ib) for use in treating FHR3-mediated diseases or disorders. In yet another aspect, the present invention also provides a pharmaceutical composition comprising a bifunctional compound having formula (Ib) for use in treating FHR3-mediated diseases or disorders. In some embodiments of such use, the FHR3-mediated diseases or disorders are selected from kidney disease, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0035] On the other hand, the present invention also provides the use of a bifunctional compound having formula (Ia) in treating PCSK9-mediated diseases or disorders. On the other hand, the present invention also provides the use of a bifunctional compound having formula (Ia) in manufacturing a medicament for treating PCSK9-mediated diseases or disorders. On the other hand, the present invention also provides the use of a pharmaceutical composition comprising a bifunctional compound having formula (Ia) in treating PCSK9-mediated diseases or disorders. In some embodiments of such use, PCSK9-mediated diseases or disorders are selected from hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral vascular disease, peripheral artery disease, vascular inflammation, elevated Lp(a), elevated LDL, triglyceride-rich lipoprotein (TRL), elevated triglycerides, sepsis, and xanthoma.

[0036] In another aspect, the present invention also provides the use of a bifunctional compound having formula (Ib) in the treatment of CFHR3-mediated diseases or disorders. In another aspect, the present invention also provides the use of a bifunctional compound having formula (Ib) in the manufacture of a medicament for treating CFHR3-mediated diseases or disorders. In another aspect, the present invention also provides the use of a pharmaceutical composition comprising a bifunctional compound having formula (Ib) in the treatment of CFHR3-mediated diseases or disorders. In some embodiments of such use, the CFHR3-mediated diseases or disorders are selected from kidney disease, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0037] In another aspect, the present invention also provides the use of a bifunctional compound having formula (Ib) in treating FHR3-mediated diseases or disorders. In another aspect, the present invention also provides the use of a bifunctional compound having formula (Ib) in manufacturing a medicament for treating FHR3-mediated diseases or disorders. In another aspect, the present invention also provides the use of a pharmaceutical composition comprising a bifunctional compound having formula (Ib) in treating FHR3-mediated diseases or disorders. In some embodiments of such use, the FHR3-mediated diseases or disorders are selected from kidney disease, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0038] In another aspect, the present invention also provides a method for treating PCSK9-mediated diseases or disorders, the method comprising the step of administering a therapeutically effective amount of a bifunctional compound having formula (Ia) to a patient in need. In some embodiments of the method, the PCSK9-mediated diseases or disorders are selected from hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral vascular disease, peripheral artery disease, vascular inflammation, elevated Lp(a), elevated LDL, triglyceride-rich lipoprotein (TRL), elevated triglycerides, sepsis, and xanthoma.

[0039] In another aspect, the present invention also provides a method for treating CFHR3-mediated diseases or disorders, the method comprising the step of administering a therapeutically effective amount of a bifunctional compound having formula (Ib) to a patient in need. In some embodiments of the method, the CFHR3-mediated diseases or disorders are selected from nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0040] In another aspect, the present invention also provides a method for treating FHR3-mediated diseases or disorders, the method comprising the step of administering a therapeutically effective amount of a bifunctional compound having formula (Ib) to a patient in need. In some embodiments of the method, the FHR3-mediated disease or disorder is selected from nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0041] In another aspect, the present invention also provides a method for targeted lysosomal degradation of an extracellular target molecule, the method comprising administering a bifunctional compound having formula (Ia), wherein the extracellular target molecule is PCSK9.

[0042] In another aspect, the present invention also provides a method for targeted lysosomal degradation of an extracellular target molecule, the method comprising administering a bifunctional compound having formula (Ib), wherein the extracellular target molecule is FHR3.

[0043] In another aspect, the present invention also provides a method for removing extracellular target molecules from the plasma of a patient in need, the method comprising administering a bifunctional compound having formula (Ia), wherein the extracellular target molecule is PCSK9.

[0044] In another aspect, the present invention also provides a method for removing extracellular target molecules from the plasma of a patient in need, the method comprising administering a bifunctional compound having formula (Ib), wherein the extracellular target molecule is FHR3.

[0045] In another aspect, the present invention also provides a bifunctional compound having formula (Ia) for use in a therapy for treating cardiovascular diseases, wherein the therapy is based on inhibiting extracellular levels of PCSK9 by lysosomal degradation mediated by the bifunctional compound having formula (Ia).

[0046] In another aspect, the present invention also provides a bifunctional compound of formula (Ib) for use in a therapy for treating kidney disease, age-related macular degeneration, atypical hemolytic uremic syndrome, or hepatocellular carcinoma (HCC), wherein the therapy is based on inhibiting extracellular levels of FHR3 by lysosomal degradation mediated by the bifunctional compound of formula (Ib). Attached Figure Description

[0047] Figure 1A The clearance rate of human FHR3 from transgenic mice expressing human FHR3 after intraperitoneal bolus administration of the mediator, 0.01 mg / kg bifunctional compound (BFC-13), and 0.1 mg / kg bifunctional compound (BFC-13). hFHR3 level is relative to the FHR3 level before administration.

[0048] Figure 1B The clearance rate of human FHR3 from transgenic mice expressing human FHR3 after intraperitoneal bolus administration of the mediator, 0.01 mg / kg bifunctional compound (BFC-15), and 0.1 mg / kg bifunctional compound (BFC-15). hFHR3 level is relative to the FHR3 level before administration.

[0049] Figure 2A Co-administration study: Clearance of human PCSK9 from LDLR(- / -) mice after intravenous bolus administration of the mediator + 3.3 μg hPCSK9, 0.1 mg / kg bifunctional compound (BFC-1) + 3.3 μg hPCSK9, 0.05 mg / kg PCSK9 ligand (C5), and 0.05 mg / kg ASGPR ligand (int-CC2) + 3.3 μg hPCSK9.

[0050] Figure 2B Co-application study: Figure 2A The AUC plot depicts the clearance rate data. Statistics were obtained using ordinary one-way ANOVA with Dunnett's multiple comparison test.

[0051] Figure 3ACo-administration study: Clearance of human PCSK9 from LDLR(- / -) mice after intravenous bolus administration of the mediator + 3.3 μg hPCSK9, 0.1 mg / kg bifunctional compound (BFC-2) + 3.3 μg hPCSK9, 0.05 mg / kg PCSK9 ligand (C5), and 0.05 mg / kg M6PR ligand (int-CC6) + 3.3 μg hPCSK9.

[0052] Figure 3B Co-application study: Figure 3A The AUC plot depicts the clearance rate data. Statistics were obtained using ordinary one-way ANOVA with Dunnett's multiple comparison test.

[0053] Figure 4A Co-administration study: Clearance of human PCSK9 from LDLR(- / -) mice after intravenous bolus administration of the mediator + 3.3 μg hPCSK9, 0.1 mg / kg bifunctional compound (BFC-1) + 3.3 μg hPCSK9, 0.03 mg / kg bifunctional compound (BFC-7) + 3.3 μg hPCSK9, 0.1 mg / kg bifunctional compound (BFC-7) + 3.3 μg hPCSK9, and 0.3 mg / kg bifunctional compound (BFC-7) + 3.3 μg hPCSK9.

[0054] Figure 4B Co-application study: Figure 4A The AUC plot depicts the clearance rate data. Statistics are derived from ordinary one-way ANOVA relative to the medium:

[0055] ***p = 0.0001; ****p < 0.0001

[0056] Figure 5A Co-administration study: Clearance of human PCSK9 from LDLR(- / -) mice after intravenous bolus administration of the mediator + 3.3 μg hPCSK9, 0.1 mg / kg bifunctional compound (BFC-1) + 3.3 μg hPCSK9, 0.1 mg / kg bifunctional compound (BFC-5) + 3.3 μg hPCSK9, and 1 mg / kg bifunctional compound (BFC-5) + 3.3 μg hPCSK9.

[0057] Figure 5B Co-application study: Figure 5A The AUC plot depicts the clearance rate data.

[0058] Statistical analysis using ordinary one-way ANOVA relative to BFC-1: **p = 0.0028

[0059] Statistical analysis using ordinary one-way ANOVA relative to the medium: ****p<0.0001

[0060] Figure 6A Co-administration and pre-dose studies of bifunctional compounds: Clearance of human PCSK9 from LDLR(- / -) mice after the following treatments:

[0061] i) Intravenous bolus administration of the carrier + 3.3 μg hPCSK9

[0062] ii) Intravenous bolus administration of 0.1 mg / kg bifunctional compound (BFC-1) + 3.3 μg hPCSK9

[0063] iii) Intravenous bolus administration of 0.1 mg / kg bifunctional compound (BFC-12) + 3.3 μg hPCSK9

[0064] iv) Administer 0.1 mg / kg of the bifunctional compound (BFC-12) via intravenous bolus, followed by 3.3 μg of hPCSK9 via intravenous bolus 70 minutes later.

[0065] v) Oral administration of 30 mg / kg of the bifunctional compound (BFC-12), followed by an intravenous bolus of 3.3 μg / kg PCSK9 70 minutes later.

[0066] Figure 6B Studies on the co-administration and pre-drug administration of bifunctional compounds: Figure 6A The AUC plot depicts the clearance rate data.

[0067] Statistical analysis using ordinary one-way ANOVA relative to the medium:

[0068] ***p=0.0005; ****p<0.0001; **p=0.0027

[0069] Figure 7A Co-administration and pre-dose studies of bifunctional compounds: Clearance of human PCSK9 from LDLR(- / -) mice after the following treatments:

[0070] i) Intravenous bolus administration of the carrier + 3.3 μg hPCSK9

[0071] ii) Intravenous bolus administration of 0.1 mg / kg bifunctional compound (BFC-1) + 3.3 μg hPCSK9

[0072] iii) Intravenous bolus administration of 0.1 mg / kg bifunctional compound (BFC-11) + 3.3 μg hPCSK9

[0073] iv) Administer 0.1 mg / kg of the bifunctional compound (BFC-11) via intravenous bolus, followed by 3.3 μg of hPCSK9 via intravenous bolus 40 minutes later.

[0074] v) Oral administration of 30 mg / kg of the bifunctional compound (BFC-11), followed by an intravenous bolus of 3.3 μg / kg PCSK9 40 minutes later.

[0075] Figure 7B Studies on the co-administration and pre-drug administration of bifunctional compounds: Figure 7A The AUC plot depicts the clearance rate data.

[0076] Statistical analysis of ordinary one-way ANOVA relative to the medium using the Dunnett multiple comparison test.

[0077] Figure 8A Competitive study: Clearance of human PCSK9 from LDLR(- / -) mice after the following treatments:

[0078] i) Intravenous bolus administration of the carrier + 3.3 μg hPCSK9

[0079] ii) Intravenous bolus administration of 0.1 mg / kg bifunctional compound (BFC-1) + 3.3 μg hPCSK9

[0080] iii) Administer intravenous bolus of 0.1 mg / kg bifunctional compound (BFC-1) + 3.3 μg hPCSK9 + 10 mg / kg ASGPR ligand (int-CC2).

[0081] iv) Intravenous bolus administration of 0.1 mg / kg bifunctional compound (BFC-1) + 3.3 μg hPCSK9 + 10 mg / kg PCSK9 ligand (C5)

[0082] Figure 8B Competition Research: Figure 8A The AUC plot depicts the clearance rate data.

[0083] Statistical analysis using standard unidirectional ANOVA relative to the bifunctional compound (BFC-1):

[0084] **p=0.0033; ***p=0.0003; ****p<0.0001 Detailed Implementation

[0085] definition

[0086] As used herein, the term "alkyl" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, and said group is not unsaturated. As used herein, the term "C1-C6 alkyl" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, said group is not unsaturated, has one to six carbon atoms, and is attached to the rest 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).

[0087] As used herein, the term "alkenyl" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, wherein the group contains at least one double bond. As used herein, the term "C2-C" refers to... e "Alkenyl" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, containing at least one double bond, having two to six carbon atoms attached to the rest of the molecule by single bonds. Non-limiting examples of "C2-C6 alkenyl" groups include vinyl (C2 alkenyl), propenyl (C3 alkenyl), butenyl (C4 alkenyl), pentenyl (C5 alkenyl), pentenyl (C5 alkenyl), pentenyl (C5 alkenyl), pentenyl (C5 alkenyl), hexenyl (C6 alkenyl), and hexenyl (C6 alkenyl). Hex-3-enyl (C6-enyl), hex-1,4-dienyl (C6-enyl), hex-1,5-dienyl (C6-enyl), and hex-2,4-dienyl (C6-enyl). As used herein, the term "C2-C3-enyl" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, containing at least one double bond, having two to three carbon atoms attached to the remainder of the molecule by single bonds. Non-limiting examples of "C2-C3-enyl" groups include vinyl (C2-enyl) and propenyl (C3-enyl).

[0088] As used herein, the term "alkylene" refers to a divalent straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, and wherein the group is not unsaturated. As used herein, the term "C1-C6 alkylene" refers to a divalent straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, wherein the group is not unsaturated, and has one to six carbon atoms. Non-limiting examples of "C1-C6 alkylene" groups 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), secondary butylene (C4 alkylene), tert-butylene (C4 alkylene), n-pentylene (C5 alkylene), isopentylene (C5 alkylene), neopentylene (C5 alkylene), and hexylene (C6 alkylene).

[0089] As used herein, the term "alkenyl" refers to a divalent straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, and said group contains at least one double bond. As used herein, the term "C2-C6 alkenyl" refers to a divalent straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, said group contains at least one double bond, and has two to six carbon atoms. Non-limiting examples of “C2-C6 alkenyl” groups include vinylene (C2 alkenyl), propenyl-1-alkenyl (C3 alkenyl), butenyl-1-alkenyl (C4 alkenyl), pentylene-1-alkenyl (C5 alkenyl), pentylene-4-alkenyl (C5 alkenyl), pentylene-1,4-dienyl (C5 alkenyl), hexene-1-alkenyl (C6 alkenyl), hexene-2-alkenyl (C6 alkenyl), hexene-3-alkenyl (C6 alkenyl), hexene-1,4-dienyl (C6 alkenyl), hexene-1,5-dienyl (C6 alkenyl), and hexene-2,4-dienyl (C6 alkenyl). As used herein, the term “C2-C6 alkenyl” refers to a divalent straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, the group containing at least one double bond, and having two to three carbon atoms. Non-limiting examples of “C2-C3 imenyl” groups include vinylidene (C2 imenyl) and propylene-1-enyl (C3 imenyl).

[0090] As used herein, the term "alkoxy" refers to -O-alkyl or -alkyl-O-, wherein the "alkyl" group is as defined herein. In some embodiments, the alkoxy group is "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"Alkoxy", including the terms "C1-C3 alkoxy", "C1-C4 alkoxy", "C1-C5 alkoxy", "C1-C6 alkoxy", "C1-C7 alkoxy", "C1-C8 alkoxy", "C1-C9 alkoxy" and "C1-C 10 "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" include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, isopentoxy, hexoxy, heptoxy, octoxy, nonoxy, decoxy, etc.

[0091] As used herein, the term "aryl" refers to an aromatic monocyclic system having 6 carbon atoms as ring members, an aromatic fused bicyclic system having 9-10 carbon atoms as ring members, or an aromatic fused tricyclic system having 14 carbon atoms as ring members. Non-limiting examples of aryl groups as used herein include phenyl, naphthyl, fluorenyl, indenyl, azulel, anthraceneyl, phenanthryl, etc. In some embodiments, such aryl groups are optionally substituted. In a preferred embodiment, the aryl group is phenyl.

[0092] 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 adamantyl. Non-limiting examples of monocyclic C3-C8 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups.

[0093] As used herein, the term "haloalkyl" means an alkyl group as defined herein, wherein at least one of the hydrogen atoms of the alkyl group is replaced by a halogen group as defined herein. A haloalkyl group can be a monohaloalkyl, dihaloalkyl, trihaloalkyl, or polyhaloalkyl, including perhaloalkyl. A monohaloalkyl group may have an iodine, bromine, chlorine, or fluorine atom within the alkyl group. Dihaloalkyl and polyhaloalkyl groups may have two or more identical halogen atoms or combinations of different halogen groups within the alkyl group. Typically, a polyhaloalkyl group contains up to six, four, three, or two halogen groups. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. A perhaloalkyl group is an alkyl group in which all hydrogen atoms are replaced by halogen atoms, such as trifluoromethyl. Unless otherwise stated, representative haloalkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl groups in which at least one hydrogen atom is substituted by a halogen (e.g., where the halogen is fluorine): CF3CF2-, (CF3)2CH-, CH3-CF2-, CF3CF2-, CF3, CF2H-, CF3CF2CH(CF3)-, or CF3CF2CF2CF2-.

[0094] As used herein, the term "C1-C6 haloalkyl" refers to the corresponding "C1-C6 alkyl" as defined herein, wherein at least one of the hydrogen atoms of the "C1-C6 alkyl" is substituted with a halogen atom. The C1-C6 haloalkyl group can be a mono-C1-C6 haloalkyl group, wherein such a C1-C6 haloalkyl group has one iodine, one bromine, one chlorine, or one fluorine atom. Alternatively, the C1-C6 haloalkyl group can be a di-C1-C6 haloalkyl group, wherein such a C1-C6 haloalkyl group may have two halogen atoms independently selected from iodine, bromine, chlorine, or fluorine. Furthermore, the C1-C6 haloalkyl group can be a poly-C1-C6 haloalkyl group, wherein such a C1-C6 haloalkyl group may have two or more identical halogen atoms or a combination of two or more different halogen atoms. Such poly-C1-C6 haloalkyl groups can be perhalogenated C1-C6 haloalkyl groups, wherein all hydrogen atoms of the corresponding C1-C6 alkyl group have been substituted with halogen atoms, and these halogen atoms may be the same or a combination of different halogen atoms. Non-limiting examples of “C1-C6 haloalkyl” groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, fluoroethyl, difluoroethyl, trifluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl.

[0095] As used herein, the term "haloalkoxy" refers to an alkoxy group as defined herein, wherein at least one of the hydrogen atoms of the alkyl group is replaced by a halogen group as defined herein. The haloalkyl group can be a monohaloalkoxy, dihaloalkoxy, trihaloalkoxy, or polyhaloalkoxy, including perhaloalkoxy. A monohaloalkoxy group may have an iodine, bromine, chlorine, or fluorine group within the alkyl group. Dihaloalkoxy and polyhaloalkoxy groups may be compositions having two or more identical halogen atoms or different halogen groups within the alkyl group. Typically, polyhaloalkoxy groups contain up to 6, 4, 3, or 2 halogen groups. Non-limiting examples of haloalkoxy groups include fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, pentafluoroethoxy, heptafluoropropoxy, difluorochloromethoxy, dichlorofluoromethoxy, difluoroethoxy, difluoropropoxy, dichloroethoxy, and dichloropropoxy. A perhaloalkoxy group is an alkoxy group in which all hydrogen atoms are replaced by halogen atoms, such as trifluoromethoxy. Unless otherwise stated, representative haloalkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, and tert-butoxy groups in which at least one hydrogen atom is replaced by a halogen (e.g., where the halogen is fluorine): CF3CF2O-, (CF3)2CHO-, CH3-CF2O-, CF3CF2O-, -OCF3, -OCHF2-, CF3CF2CH(CF3)O-, or CF3CF2CF2CF2O-.

[0096] As used herein, the term "C1-C6 haloalkoxy" refers to the corresponding "C1-C6 3-alkoxy" as defined herein, wherein at least one hydrogen atom in the "C1-C6 alkyl" group is replaced by a halogen atom. The C1-C6 haloalkoxy group can be a mono-C1-C6 haloalkoxy group, wherein such a C1-C6 haloalkoxy group has one iodine, one bromine, one chlorine, or one fluorine atom. Alternatively, the C1-C6 haloalkoxy group can be a di-C1-C6 haloalkoxy group, wherein such a C1-C6 haloalkoxy group can have two halogen atoms independently selected from iodine, bromine, chlorine, or fluorine. Furthermore, the C1-C6 haloalkoxy group can be a poly-C1-C6 haloalkoxy group, wherein such a C1-C6 haloalkoxy group can have two or more identical halogen atoms or a combination of two or more different halogen atoms. Such polyC1-C6 haloalkoxy groups can be perhalogenated C1-C6 haloalkyl groups, wherein all hydrogen atoms of the corresponding C1-C6 alkyl group have been replaced by halogen atoms, and these halogen atoms can be the same or a combination of different halogen 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.

[0097] As used herein, the term “halogenated” or “halogen” refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

[0098] As used herein, the term "heteroaryl" refers to an aromatic ring system containing one or more heteroatoms. Heteroaryl groups containing more than one heteroatom may contain different heteroatoms. Heteroaryl groups may optionally be substituted by one or more substituents as defined in formula (I). Heteroaryl groups may be monocyclic or fused bicyclic ring systems. Monocyclic heteroaryl rings have 5-6 ring atoms. Bicyclic heteroaryl rings have 8-10 member atoms. Bicyclic heteroaryl rings include those ring systems in which the heteroaryl ring is fused to a phenyl ring. As used herein, non-limiting examples of heteroaryl groups include benzofuranyl, benzo[c]thiophenyl, benzothiophenyl, benzooxazolyl, benzothiazolyl, benzoimidazolyl, cenolinyl, furazonyl, furanyl, imidazolyl, indoleyl, indolyl, indololinyl, indazoleyl, isoindolyl, isoquinolinyl, isoxazolyl, isothiazolyl, oxazolyl, hydroxyindolyl, and oxadiazolyl (including 1,3,4-oxadiazolyl). (and 1,2,4-oxadiazolyl), purine, pyrazolyl, pyrroleyl, phthalazinyl, pyridyl (including 2-pyridyl, 3-pyridyl and 4-pyridyl), pyridazinyl, pyrazinyl, pyrimidinyl, quinoxalinyl, quinolinyl, quinazolinyl, tetrazinyl, tetrazolyl, tetrazono[1,5-a]pyridinyl, thiazolyl, thiadiazolyl (including 1,3,4-thiadiazolyl), thienyl, triazinyl and triazolyl.

[0099] The term "5- or 6-membered heteroaryl group containing 1 to 4 heteroatoms selected from N, O and S" refers to an aromatic 5- or 6-membered monocyclic ring system having 1 to 4 heteroatoms independently selected from heteroatoms N, O and S as ring members.

[0100] The term "5- or 6-membered heteroaryl group containing 1 to 3 heteroatoms selected from N, O, and S" refers to an aromatic 5- or 6-membered monocyclic ring system having 1 to 3 heteroatoms independently selected from heteroatoms N, O, and S as ring members.

[0101] As used herein, the term "heteroatom" refers to a nitrogen, oxygen, or sulfur atom.

[0102] As used herein, the term "heterocyclic alkyl" refers to a cycloalkyl group as defined herein, in which one or two carbon atoms in the ring structure are independently selected from N, NH, N 12 Substitution of groups O or S, wherein R 12 It is an H or C1-C6 alkyl group. As used herein, the term "having one or two independently selected N, NH, NR" is used. 12 "4- to 6-membered heterocyclic alkyl group with ring members of 'O' or 'S'" refers to a 4- to 6-membered heterocyclic alkyl group, which is a fully saturated monocyclic hydrocarbon ring structure with 4 to 6 ring members, wherein one or two ring members are independently selected from N, NH, NR. 12 , O or -S-, where R 12It is an H or C1-C6 alkyl group. Non-limiting examples of heterocyclic alkyl groups as used herein include azirrobutane, azirrobutane-1-yl, azirrobutane-2-yl, azirrobutane-3-yl, oxacyclobutane, oxacyclobutane-2-yl, oxacyclobutane-3-yl, oxacyclobutane-4-yl, thiohexacyclobutane, thiohexacyclobutane-2-yl, thiohexacyclobutane-3-yl, thiohexacyclobutane-4-yl, pyrrolyl, pyrrolyl-1-yl, pyrrolyl-2-yl, pyrrolyl-3-yl, pyrrolyl-4-yl, pyrrolyl-5-yl, tetrahydrofuranyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrofuran-3-yl, etc. -yl, tetrahydrofuran-4-yl, tetrahydrofuran-5-yl, tetrahydrothiopheneyl, tetrahydrothiophene-2-yl, tetrahydrothiophene-3-yl, tetrahydrothiophene-4-yl, tetrahydrothiophene-5-yl, piperidinyl, piperidin-1-yl, piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, piperidin-5-yl, piperidin-6-yl, tetrahydropyranyl, tetrahydropyran-2-yl, tetrahydropyran-3-yl, tetrahydropyran-4-yl, tetrahydropyran-5-yl, tetrahydropyran-6-yl, tetrahydrothiopheneyl, tetrahydrothiophene-2-yl, tetrahydrothiophene-3-yl, tetrahydrothiophene-4-yl, tetrahydrothiophene-5-yl Tetrahydrothiaran-6-yl, piperazinyl, piperazin-1-yl, piperazin-2-yl, piperazin-3-yl, piperazin-4-yl, piperazin-5-yl, piperazin-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, oxothiohexane-2-yl, oxothiohexane-3-yl, oxothiohexane-5-yl, oxothiohexane-6-yl, dithiaalkyl, dithiaalkyl Alkyl-2-yl, dithiapan-3-yl, dithiapan-5-yl, dithiapan-6-yl, dioxopentyl, dioxopentyl-2-yl, dioxopentyl-4-yl, dioxopentyl-5-yl, oxothionecyclohexyl, oxothionecyclohexyl-2-yl, oxothionecyclohexyl-3-yl, oxothionecyclohexyl-4-yl, oxothionecyclohexyl-5-yl, dithionecyclopentyl, dithionecyclopentyl-2-yl, dithionecyclopentyl-4-yl, dithionecyclopentyl-5-yl, pyrazolyl, pyrazolidine-1-yl, pyrazolidine-2-yl, pyrazolidine-3-yl, pyrazolidine-4-yl, and pyrazolidine-5-yl.

[0103] As used herein, the term "heterocyclic group" refers to a saturated (e.g., heterocyclic alkyl ring) or partially unsaturated monocyclic or polycyclic ring containing a carbon and at least one heteroatom selected from oxygen, nitrogen, or sulfur (O, N, or S), wherein there is no shared, nonlocalized n electron (aromaticity) between the ring carbons or heteroatoms.

[0104] 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)-.

[0105] As used herein, the term "spirocycloalkyl" or "spirocycloyl" refers to a ring system having two rings connected by a single atom in a carbon-bicyclic ring. These rings may be of different sizes and properties, or they may be of the same size and properties. Examples include spiropentane, spirohexane, spiroheptane, spiroctane, spirononane, or spirodecane. One or both rings of the spirocyclic ring may be fused with another ring of carbocyclic, heterocyclic, aromatic, or heteroaromatic ring. (C3-C) 12 Spirocycloalkyl groups are spirocycles containing between 3 and 12 carbon atoms.

[0106] As used herein, the terms "spiroheteroalkyl" or "spiroheteroyl" refer to a spirocycle in which at least one ring is a heterocycle (one or more of the carbon atoms can be substituted by a heteroatom (e.g., one or more of the carbon atoms in at least one ring are substituted by a heteroatom)). One or both rings of a spiroheterocycle may be fused with another ring of carbocyclic, heterocyclic, aromatic, or heteroaromatic rings.

[0107] As used herein, the term "therapeutic plasma exchange in vivo" refers to the removal of unwanted extracellular target molecules from plasma in vivo. 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 plasma membrane proteins. Specific examples of such target molecules include, but are not limited to, 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, apralin, BMP6, BMP9 / GDF2, and CS. F-1, EPO, IL5, MFGE8, TSLP, TSP, C5, CXCL10, FGF23, IGF1, IL10, IL13, IL2, IL6, VEGFA, NKG2D, ZNFR3, ADA2, suPAR, TGF-β1, IL4 receptor, sToll receptor, histamine, Tau, granulin precursor, α-synuclein, toxin, venom, HBV soluble antigen, viral antigen, prion protein, scFV, AAV and anti-AAV antibody.

[0108] As used herein, the terms "polyethylene glycol" or "PEG" refer to a linear, branched, or star-shaped conformation consisting of (OCH2CH2) groups. In some embodiments, the polyethylene or PEG groups are -(OCH2CH2). t *-, where t is 4-40, and where "-" indicates the end of the suicide spacer and "*-" indicates the attachment 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 4-40, and where "-" indicates the end of the suicide spacer and "*-" indicates the attachment point to the terminal group R", where R" is H, CH3 or CH2CH2C(=O)OH.

[0109] As used herein, the term "polyethylene glycol" refers to polyethylene glycol composed of (O(CH2)2) m ) t The groups form linear chains, branched chains, or star-shaped conformations. In some embodiments, the polyethylene or PEG groups are -(O(CH2)). m ) t *-, where m is 1-10, t is 4-40, and where "-" indicates the end of the suicide spacer and "*-" indicates the attachment 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 1-10, t is 4-40, and where “-” indicates the end of the suicide spacer and “*-” indicates the attachment point to the terminal group R”, where R” is H, CH3 or CH2CH2C(=O)OH.

[0110] As used herein, the term “extracellular” refers to the space outside the plasma membrane of one or more cells.

[0111] As used herein, the term “extracellular level inhibition” refers to reducing or lowering the concentration of target molecules located in the space outside one or more cell membranes.

[0112] The terms “PCSK9,” “hPCSK9,” or “proprotein convertase subtilisin / kexin type 9” are interchangeable to refer to naturally occurring human proprotein convertase belonging to the proteinase K subfamily of the secretory subtilisin family. PCSK9 is synthesized as a lysozyme, undergoes autocatalytic intramolecular processing in the endoplasmic reticulum, and is thought to function as a proprotein convertase. PCSK9 plays a role in cholesterol homeostasis and may play a role in the differentiation of cortical neurons. Mutations in the PCSK9 gene are a cause of autosomal dominant familial hypercholesterolemia. (Burnett and Hooper, Clin. Biochem. Rev. [Clinical Biochemistry Review] (2008) 29(1):11-26)

[0113] As used herein, the terms "PCSK9-mediated disease or disorder" or "PCSK9-related disease or disorder" refer to diseases or disorders associated with PCSK9 activity, including hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral vascular disease (including aortic and cerebrovascular diseases), peripheral artery disease, vascular inflammation, elevated Lp(a), elevated LDL, elevated TRL, elevated triglycerides, sepsis, and xanthomas.

[0114] The terms "hypercholesterolemia" or "dyslipidemia" include, for example, familial and nonfamilial hypercholesterolemia. Familial hypercholesterolemia (FH) is an autosomal dominant inherited disorder characterized by elevated serum cholesterol bound to low-density lipoprotein (LDL). Familial hypercholesterolemia includes heterozygous FH and homozygous FH. Hypercholesterolemia (or dyslipidemia) is the presence of high levels of cholesterol in the blood. It is a form of hyperlipidemia (elevated levels of lipids in the blood) and hyperlipoproteinemia (elevated levels of lipoproteins in the blood).

[0115] Hyperlipidemia is an elevation of lipids in the blood. These lipids include cholesterol, cholesterol esters, phospholipids, and triglycerides. Hyperlipidemia includes types such as type I, IIa, IIb, III, IV, and V.

[0116] Hypertriglyceridemia indicates high levels of triglycerides in the blood. Elevated triglyceride levels are associated with atherosclerosis (even in the absence of hypercholesterolemia) and increase the risk of cardiovascular disease.

[0117] "Sitosterolemia" or "phytosterolemia" is a rare autosomal recessive inherited lipid metabolism disorder characterized by excessive absorption of sitosterol in the gastrointestinal tract and reduced bile excretion of dietary sterols (i.e., leading to hypercholesterolemia, tendon and nodular xanthomas, premature development of atherosclerosis) and altered cholesterol synthesis.

[0118] Atherosclerosis is a condition characterized by the deposition of fatty substances, cholesterol, cellular waste, calcium, and fibrin in the inner walls of arteries. The resulting accumulations are called plaques.

[0119] Atherosclerosis, or atherosclerotic vascular disease (ASVD), is a specific form of arteriosclerosis involving the thickening, hardening, and loss of elasticity of the arterial walls due to the invasion and accumulation of white blood cells (including live, active white blood cells that produce inflammation) and the residue of dead cells (including cholesterol and triglycerides). Therefore, atherosclerosis is a syndrome affecting arterial blood vessels due to the chronic inflammatory response of white blood cells in the arterial walls.

[0120] Coronary artery disease, also known as atherosclerotic heart disease, 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 buildup along the inner walls of the coronary arteries, which narrows the arteries and reduces the amount of blood flowing to the heart.

[0121] Xanthoma is a skin manifestation of lipid hyperplasia, in which lipids accumulate in large foam cells within the skin. Xanthoma is associated with hyperlipidemia.

[0122] As used herein, the term "elevated Lp(a) concentration" refers to a serum Lp(a) concentration above 30 mg / dL (75 nmol / L). "Elevated serum Lp(a)" means a serum Lp(a) level greater than approximately 14 mg / dL. In some embodiments, a patient's serum Lp(a) level is considered elevated if it is greater than about 15 mg / dL, about 20 mg / dL, about 25 mg / dL, about 30 mg / dL, about 35 mg / dL, about 40 mg / dL, about 45 mg / dL, about 50 mg / dL, about 60 mg / dL, about 70 mg / dL, about 80 mg / dL, about 90 mg / dL, about 100 mg / dL, about 20 mg / dL, about 140 mg / dL, about 150 mg / dL, about 180 mg / dL, or about 200 mg / dL. The patient's serum Lp(a) level can be checked after a meal. In some embodiments, Lp(a) levels are measured after a period of fasting (e.g., after 8 hours, 8 hours, 10 hours, 12 hours, or longer). Exemplary methods for measuring serum Lp(a) in patients include, but are not limited to, rate immunoturbidimetry, ELISA, turbidimetry, immunoturbidimetry, and dissociation-enhanced lanthanide fluorescence immunoassay, but any clinically acceptable diagnostic method may be used in the context of this disclosure.

[0123] "Elevated triglyceride levels" or "ETL" refers to any level of triglyceride levels that are identified as undesirable or targeted for regulation.

[0124] Sepsis is a systemic reaction characterized by hypotension, metabolic acidosis, decreased systemic vascular resistance, tachypnea, and organ dysfunction. Sepsis can produce both septicemia (i.e., organisms whose metabolic end products or toxins are in the blood) (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 "septicemia" also includes fungemia (i.e., fungi in the blood), viremia (i.e., viruses or viral particles in the blood), and parasitemia (i.e., worms or protozoan parasites in the blood). Therefore, sepsis and septic shock (acute circulatory failure caused by sepsis, often associated with multiple organ failure and high mortality) can be caused by many organisms.

[0125] The terms “CFHR3” or “complement factor H-related protein 3 gene” are interchangeable and refer to the gene that encodes the human protein complement factor H-related protein 3 (FHR3).

[0126] The terms “FHR3” or “complement factor H-associated protein 3” are interchangeable in referring to naturally occurring human complement factor H-associated protein 3, which is a secreted protein belonging to the complement factor H-associated protein family.

[0127] As used herein, the terms “CFHR3-mediated disease or disorder” or “CFHR3-associated disease or disorder” refer to diseases or disorders associated with abnormal activity of CFHR3, including nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0128] As used herein, the terms “FHR3-mediated disease or disorder” or “FHR3-associated disease or disorder” refer to diseases or disorders associated with the activity of FHR3, including nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0129] As used in this article, the term "nephropathy" refers to a disease or injury to the kidneys.

[0130] As used in this article, the term "age-related macular degeneration" refers to an eye disease that affects the macula of the eye and can lead to blindness over time.

[0131] As used in this article, the term "atypical hemolytic uremic syndrome" refers to a disease in which kidney function is impaired due to abnormal blood clotting in the kidneys. Atypical hemolytic uremic syndrome is characterized by three main features associated with abnormal blood clotting: hemolytic anemia, thrombocytopenia, and kidney failure.

[0132] As used in this article, the term "hemolytic anemia" refers to the premature breakdown of red blood cells.

[0133] As used in this article, the term "thrombocytopenia" refers to a decrease in the level of circulating platelets, which are used to aid in blood clotting.

[0134] As used in this article, the term "hepatocellular carcinoma (HCC)" refers to liver cancer.

[0135] As used herein, the term "reactive group" is 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 in Table 1 herein.

[0136] As used herein, the term "coupling group" refers to a divalent moiety that links a bridging spacer to an antibody or a fragment thereof. The coupling group is a divalent moiety formed by a reaction between a reactive group and a functional group of the antibody or a fragment thereof. Non-limiting examples of such divalent moieties include the divalent chemical moieties given in Tables 1 and 2 provided herein.

[0137] As used in this article, wavy lines are used when showing partial structures of compounds. Indicates the attachment points between the described partial structure and the rest of the molecule.

[0138] As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of the compound of the present invention with at least one and optionally more than one other pharmaceutically acceptable chemical component (such as a carrier, stabilizer, diluent, dispersant, suspending agent, thickener and / or excipient).

[0139] As used herein, the terms "optical isomer" or "stereoisomer" refer to any of the various stereoisomeric conformations that a given compound of the invention may exist in, including geometric isomers. It should be understood that substituents may be attached to the chiral center of a carbon atom. The term "chiral" refers to a molecule that has a non-overlapping property on its mirror-image partner, while the term "chiral" refers to a molecule that is overlapping on its mirror-image partner. Therefore, the invention includes enantiomers, diastereomers, or racemates of the said compounds. An "enantiomer" is a pair of stereoisomers that are non-overlapping mirror images of each other. A 1:1 mixture of a pair of enantiomers is a "racemic" mixture. This term is used to specify racemic mixtures where appropriate. A "diastereomer" is a stereoisomer having at least two asymmetric atoms and not being a mirror image of each other. Absolute stereochemistry is defined according to the Cahn-Lngold-Prelog RS system. When the compound is a pure enantiomer, the stereochemistry at each chiral carbon can be described by R or S. The resolution of compounds with unknown absolute configuration can be specified as (+) or (-) depending on the direction (right-handed or left-handed) in which they rotate plane-polarized light with wavelengths of the sodium D line. Some of the compounds described herein contain one or more asymmetric centers or axes and thereby can produce enantiomers, diastereomers, and other stereoisomers that can be defined according to absolute stereochemistry as (R)- or (S)-.

[0140] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and combinations thereof, as known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp. 1289-1329). Consider its use in therapeutic or pharmaceutical compositions, except where any conventional carrier is incompatible with the active ingredient.

[0141] 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, guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, etc. Typically, the subject is a human.

[0142] The term "subject in need of this treatment" refers to a subject who will benefit from this treatment biologically, medically, or in terms of quality of life.

[0143] As used herein, the term “treatment” for any disease or disorder, in one embodiment, means alleviating the disease or disorder (i.e., slowing or halting or reducing the development of the disease or at least one of its clinical symptoms). In another embodiment, “treatment” means relieving or reducing at least one bodily parameter, including those that cannot be identified by the patient. In yet another embodiment, “treatment” means regulating the disease or disorder in a physical aspect (e.g., stabilization of identifiable symptoms), a physiological aspect (e.g., stabilization of bodily parameters), or both.

[0144] As used herein, the term “prevention” for any disease or disorder means preventive treatment of the disease or disorder; or delaying the onset or progression of the disease or disorder.

[0145] The terms "therapeutic effective dose" or "therapeutic effective amount" are interchangeable in meaning to refer to an amount sufficient to achieve the desired outcome (i.e., reducing or inhibiting enzyme or protein activity, alleviating symptoms, relieving symptoms or condition, delaying disease progression, reducing tumor size, inhibiting tumor growth, preventing metastasis, inhibiting or preventing 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 those acceptable to a healthcare provider for the patient's condition. The therapeutic effective dose can be determined by initially administering a low dose and then incrementally increasing the dose until the desired effect is achieved. The "preventive effective dose" or "preventive effective amount" of the present invention molecules can prevent the onset of disease symptoms, including cancer-related symptoms. The "therapeutic effective dose" or "therapeutic effective amount" of the present invention molecules can lead to a reduction in the severity of disease symptoms, including cancer-related symptoms.

[0146] The compound names provided in this article were obtained using ChemBioDraw Ultra (version 14.0).

[0147] As used herein, the terms “a”, “the”, and similar terms used in the context of this invention (especially in the context of the claims) should be interpreted to cover both the singular and the plural, unless otherwise stated herein or clearly contradicted by the context.

[0148] Unless otherwise stated, the term "bifunctional compound of the present invention" refers to one or more bifunctional compounds having formula (I), its sub-formulas (e.g., formula (Ia) and formula (Ib)), exemplary compounds and their salts, as well as all stereoisomers (including diastereomers and enantiomers), rotational isomers, tautomers and isotopically labeled compounds (including deuterium-substituted compounds).

[0149] Any formulas given herein are also intended to represent both unlabeled and isotopically labeled forms of the compounds. Isotopically labeled compounds have the structures described by the formulas given herein, except that one or more atoms are replaced by atoms having selected atomic weights or mass numbers. Isotopes that can be incorporated into the compounds of the present invention include, for example, isotopes of hydrogen.

[0150] As used herein, the terms “polypeptide” and “peptide” are used interchangeably to refer to two or more amino acids linked together. In addition to the abbreviations for uncommon or non-natural amino acids listed in Table A below and the abbreviations for protected amino acids listed in Table B below, three-letter or single-letter abbreviations recognized in the art are used to denote the amino acid residues constituting the peptides and polypeptides disclosed herein. When preceded by a “D”, the amino acid is a D-amino acid. When preceded by an “L”, the amino acid is an L-amino acid. When a single-letter abbreviation is uppercase, it refers to an L-amino acid. When a single-letter abbreviation is lowercase, it refers to a D-amino acid. Groups or strings of amino acid abbreviations are used to denote peptides. Peptides are indicated by a left-hand N-terminus, with the sequence written from the N-terminus to the C-terminus.

[0151] The cyclic peptides described herein contain non-natural amino acids (i.e., compounds that do not exist in nature), and alternatively, other amino acid analogs known in the art can be used.

[0152] Those skilled in the art will understand that various amino acid substitutions, such as conserved amino acid substitutions, can be made in the sequence of any cyclic polypeptide described herein without necessarily reducing its activity. As used herein, “amino acids commonly used as substitutes” includes conserved substitutions (i.e., substitution with amino acids having comparable chemical properties). For the purpose of conserved substitution, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan, and methionine. Polar (hydrophilic) neutral amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Examples of amino acid substitution include replacing the corresponding D-amino acid with an L-amino acid, replacing homocysteine ​​or other non-natural amino acids with thiol side chains with cysteine, replacing homolysine, diaminobutyric acid, diaminopropionic acid, ornithine or other non-natural amino acids with amino side chains with lysine, or replacing valine with alanine, etc.

[0153] As used herein, the term "amino acid" refers to naturally occurring amino acids, non-natural amino acids, amino acid analogs, and amino acid mimics that function in a manner similar to that of naturally occurring amino acids, and, if their structure allows for such stereoisomers, their D and L stereoisomers. Amino acids are represented herein by their names, their commonly known three-letter symbols, the codes listed in Table A or Table B, or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee.

[0154] The term "naturally occurring" refers to materials found in nature and not manipulated by humans. Similarly, as used herein, "non-naturally occurring," "unnatural," etc., refer to materials not found in nature or that have been structurally modified or synthesized by humans. When used in conjunction with amino acids, the term "naturally occurring" refers to the 20 common 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)).

[0155] As used herein, the terms “non-natural amino acid” and “unnatural amino acid” are used interchangeably to refer to an amino acid structure that cannot be biosynthesized in any organism using an unmodified or modified gene (whether the same or different) 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 naturally occurring amino acids, selenocysteine, pyrrolysine (Pyl), or pyrrololine-carboxy-lysine (Pcl, e.g., as described in PCT Patent Publication WO 2010 / 48582).

[0156] The modified encoded amino acids include, but are not limited to, hydroxyproline, γ-carboxyglutamic acid, O-phosphoserine, azacyclobutanecarboxylic acid, 2-aminohexanoic acid, 3-aminohexanoic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, desmosine, and 2,2'-aminobutyric acid. -Diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesin, alloisoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthylalanine, n-valine, n-leucine, ornithine, pentylglycine, piperidinic acid, and thioproline. The term "amino acid" also includes naturally occurring amino acids that are metabolites in some organisms but are not encoded by the genetic code for incorporation into proteins. These amino acids include, but are not limited to, ornithine, D-ornithine, and D-arginine.

[0157] In this paper, a peptide is defined as an organic compound containing two or more amino acids covalently linked by peptide bonds. Peptides can be named according to the number of amino acids they make up: dipeptides or dimers contain two amino acid residues, tripeptides or trimers contain three, and so on. Peptides containing ten or fewer amino acids are called oligopeptides, while those with more than ten amino acid residues are called polypeptides.

[0158] As used in this article, the term "peptide" refers to two or more amino acids linked together by peptide bonds.

[0159] Table A: Examples of non-natural or non-natural amino acids as described in this disclosure:

[0160]

[0161]

[0162] Table B: Examples of protected amino acids as described in this disclosure:

[0163]

[0164] The bifunctional compound of the present invention

[0165] The bifunctional compound of the present invention contains a binding extracellular target (T) LCompounds that target extracellular targets such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, and cell or plasma membrane proteins. The target molecule ligand (T... L ) and binding to cell surface receptors (R L The bifunctional compound of the present invention has the structure of formula (I):

[0166] R L -L A -T L (I)

[0167] in:

[0168] R L It is a portion of cell surface receptors that bind to receptor-mediated endocytosis;

[0169] L A It's a connector.

[0170] and

[0171] T L It is the part that binds to extracellular targets.

[0172] Certain aspects and examples of the bifunctional compounds of the present invention are provided in the enumerated embodiments provided herein. It should be understood that the features specified in each embodiment may be combined with other specified features to provide further embodiments of the invention.

[0173] A. Target binding portion (T) L )

[0174] The target-binding portion (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, and cell or plasma membrane proteins. The bifunctional compound of the present invention can then be used to direct the extracellular target molecules to lysosomes for degradation. Examples of such target molecules that can be targeted for degradation using the bifunctional compound of the present invention include, but are not limited to, 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, apralin, BMP6, and BMP. 9 / GDF2, CSF-1, EPO, IL5, MFGE8, TSLP, TSP, C5, CXCL10, FGF23, IGF1, IL10, IL13, IL2, IL6, VEGFA, NKG2D, ZNFR3, ADA2, suPAR, TGF-β1, IL4 receptor, sToll receptor, histamine, Tau, granulin precursor, α-synuclein, toxin, venom, HBV soluble antigen, viral antigen, prion protein, scFV, AAV, and anti-AAV antibody. In some embodiments, the extracellular target molecules that can be directionally degraded using the bifunctional compounds of the present invention are PCSK9 and FHR3.

[0175] Example 1. A bifunctional compound having formula (I), wherein T L It is a part that combines PCSK9 or FHR3.

[0176] Example 2. A bifunctional compound having formula (I), wherein T L It is the part that combines PCSK9.

[0177] Example 3. A bifunctional compound having formula (I) or as described in any one of Examples 1 to 2, having the structure of formula (Ia):

[0178] R L -L A PCSK9 L (Ia)

[0179] in:

[0180] R L It is a portion of cell surface receptors that bind to receptor-mediated endocytosis;

[0181] L A It's a connector.

[0182] and

[0183] PCSK9 L It is the part that combines PCSK9.

[0184] Example 4. A bifunctional compound having formula (I) or as described in any one of Examples 1 to 3, wherein the target-binding moiety (T) L ) is a compound of formula (A) that is conjugated with PCSK9, or a pharmaceutically acceptable salt or stereoisomer thereof:

[0185]

[0186] in:

[0187] L A1 Selected from

[0188]

[0189] Where L A1 **Indicator and connector (L A ) attachment point and L A1 *Instructions and attachments to L A1 The attachment point of the -C(=O)- group;

[0190] (aa) 2 These are amino acid residues selected from L-proline and D-proline residues, where (aa) 2 The C-terminus is the attachment point for the -NH- group;

[0191] (aa) 3 It 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, wherein (aa) 3 The C-terminus is related to (aa) 2 Attachment point;

[0192] (aa) 4 It is an amino acid residue selected from L-aspartic acid residues, D-aspartic acid residues, L-asparagine residues, D-asparagine residues, L-glutamic acid residues, D-glutamic acid residues, L-lysine residues, D-lysine residues, L-glutamine residues, D-glutamine residues, L-proline residues, D-proline residues, L-alanine residues, D-alanine residues, L-(N-Me)glutamic acid residues, and D-(N-Me)glutamic acid residues, wherein (aa) 4 The C-terminus is related to (aa) 3Attachment point;

[0193] (aa) 5 It 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, wherein (aa) 5 The C-terminus is related to (aa) 4 Attachment point;

[0194] (aa) 6 It 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, wherein (aa) 6 The C-terminus is related to (aa) 5 Attachment point;

[0195] (aa) 7 It 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, wherein (aa) 7 The C-terminus is related to (aa). 6 Attachment point;

[0196] (aa) 8 It 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, wherein (aa) 8 The C-terminus is related to (aa) 7 Attachment point;

[0197] (aa) 9 These are amino acid residues selected from L-threonine and D-threonine residues, where (aa) 9 The C-terminus is related to (aa). 8 Attachment point;

[0198] (aa)10 It is an amino acid residue selected from L-threonine, D-threonine, L-serine, and D-serine residues, of which (aa) 10 The C-terminus is related to (aa) 9 Attachment point;

[0199] (aa) 11 It 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, wherein (aa) 11 The C-terminus is related to (aa) 10 Attachment point;

[0200] (aa) 12 It is an amino acid residue selected from L-valine, D-valine, L-glutamic acid, and D-glutamic acid residues, of which (aa) 12 The C-terminus is related to (aa) 11 The attachment point; and (aa) 13 These are amino acid residues selected from L-phenylalanine and D-phenylalanine residues, where (aa) 13 The C-terminus is related to (aa). 12 Attachment point.

[0201] Example 5. A bifunctional compound as described in Example 4, wherein...

[0202] (aa) 2 These are amino acid residues selected from L-proline and D-proline residues, where (aa) 2 The C-terminus is the attachment point for the -NH- group.

[0203] Example 6. A bifunctional compound as described in Example 4 or Example 5, wherein...

[0204] (aa) 2 It is an L-proline residue, of which (aa) 2 The C-terminus is the attachment point to the -NH- group depicted in formula (A).

[0205] Example 7. A bifunctional compound as described in any one of Examples 4 to 6, wherein

[0206] (aa) 3It 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, wherein (aa) 3 The C-terminus is related to (aa) 2 Attachment point.

[0207] Example 8. A bifunctional compound as described in any one of Examples 4 to 7, wherein

[0208] (aa) 3 It is an amino acid residue selected from L-arginine residues, L-serine residues, L-histidine residues, and L-alanine residues, of which (aa) 3 The C-terminus is related to (aa) 2 Attachment point.

[0209] Example 9. A bifunctional compound as described in any one of Examples 4 to 8, wherein

[0210] (aa) 3 It is an L-alanine residue, of which (aa) 3 The C-terminus is related to (aa) 2 Attachment point.

[0211] Example 10. A bifunctional compound as described in any one of Examples 4 to 9, wherein...

[0212] (aa) 4 It is an amino acid residue selected from L-aspartic acid residues, D-aspartic acid residues, L-asparagine residues, D-asparagine residues, L-glutamic acid residues, D-glutamic acid residues, L-lysine residues, D-lysine residues, L-glutamine residues, D-glutamine residues, L-proline residues, D-proline residues, L-alanine residues, D-alanine residues, L-(N-Me)glutamic acid residues, and D-(N-Me)glutamic acid residues, wherein (aa) 4 The C-terminus is related to (aa) 3 Attachment point.

[0213] Example 11. A bifunctional compound as described in any one of Examples 4 to 10, wherein...

[0214] (aa) 4 It is an amino acid residue selected from L-aspartic acid residues, L-asparagine residues, L-glutamic acid residues, L-lysine residues, L-glutamine residues, L-proline residues, L-alanine residues, and L-(N-Me)glutamic acid residues, wherein (aa) 4 The C-terminus is related to (aa) 3 Attachment point.

[0215] Example 12. A bifunctional compound as described in any one of Examples 4 to 11, wherein...

[0216] (aa) 4 It is an L-glutamic acid residue, of which (aa) 4 The C-terminus is related to (aa) 3 Attachment point.

[0217] Example 13. A bifunctional compound as described in any one of Examples 4 to 12, wherein

[0218] (aa) 5 It 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, wherein (aa) 5 The C-terminus is related to (aa) 4 Attachment point.

[0219] Example 14. A bifunctional compound as described in any one of Examples 4 to 13, wherein

[0220] (aa) 5 These are amino acid residues selected from L-(N-Me)alanine and L-(N-Me)glutamic acid residues, where (aa) 5 The C-terminus is related to (aa) 4 Attachment point.

[0221] Example 15. A bifunctional compound as described in any one of Examples 4 to 14, wherein

[0222] (aa) 5 It is an L-(N-Me) alanine residue, where (aa) 5 The C-terminus is related to (aa) 4 Attachment point.

[0223] Example 16. A bifunctional compound as described in any one of Examples 4 to 15, wherein...

[0224] (aa) 6 It 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, wherein (aa)6 The C-terminus is related to (aa) 5 Attachment point.

[0225] Example 17. A bifunctional compound as described in any one of Examples 4 to 16, wherein

[0226] (aa) 6 It is an amino acid residue 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, wherein (aa) 6 The C-terminus is related to (aa) 5 Attachment point.

[0227] Example 18. A bifunctional compound as described in any one of Examples 4 to 17, wherein

[0228] (aa) 6 It is an L-(4-phenyl-phenylalanine) (Bip) residue, where (aa) 6 The C-terminus is related to (aa) 5 Attachment point.

[0229] Example 19. A bifunctional compound as described in any one of Examples 4 to 18, wherein

[0230] (aa) 7 It 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, wherein (aa) 7 The C-terminus is related to (aa). 6 Attachment point.

[0231] Example 20. A bifunctional compound as described in any one of Examples 4 to 19, wherein...

[0232] (aa) 7 These are amino acid residues selected from L-(N-Me)alanine and L-(N-Me)phenylalanine residues, where (aa) 7 The C-terminus is related to (aa) 6 Attachment point.

[0233] Example 21. A bifunctional compound as described in any one of Examples 4 to 20, wherein...

[0234] (aa) 7 It is an L-(N-Me) alanine residue, where (aa) 7 The C-terminus is related to (aa)6 Attachment point.

[0235] Example 22. A bifunctional compound as described in any one of Examples 4 to 21, wherein...

[0236] (aa) 8 It 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, wherein (aa) 8 The C-terminus is related to (aa) 7 Attachment point.

[0237] Example 23. A bifunctional compound as described in any one of Examples 4 to 22, wherein...

[0238] (aa) 8 It is an amino acid residue selected from L-(4-phenyl-phenylalanine)(Bip) residue, L-serine residue, L-tyrosine residue, L-(4-trifluoromethyl-phenylalanine) residue, L-alanine residue, L-phenylalanine residue, and L-valine residue, wherein (aa) 8 The C-terminus is related to (aa) 7 Attachment point.

[0239] Example 24. A bifunctional compound as described in any one of Examples 4 to 23, wherein

[0240] (aa) 8 It is an L-(4-phenyl-phenylalanine) (Bip) residue, where (aa) 8 The C-terminus is related to (aa) 7 Attachment point.

[0241] Example 25. A bifunctional compound as described in any one of Examples 4 to 24, wherein

[0242] (aa) 9 These are amino acid residues selected from L-threonine and D-threonine residues, where (aa) 9 The C-terminus is related to (aa). 8 Attachment point.

[0243] Example 26. A bifunctional compound as described in any one of Examples 4 to 25, wherein

[0244] (aa)9 It is an L-threonine residue, of which (aa) 9 The C-terminus is related to (aa) 8 Attachment point.

[0245] Example 27. A bifunctional compound as described in any one of Examples 4 to 26, wherein...

[0246] (aa) 10 It is an amino acid residue selected from L-threonine, D-threonine, L-serine, and D-serine residues, of which (aa) 10 The C-terminus is related to (aa) 9 Attachment point.

[0247] Example 28. A bifunctional compound as described in any one of Examples 4 to 27, wherein...

[0248] (aa) 10 It is an L-threonine residue, of which (aa) 10 The C-terminus is related to (aa) 9 Attachment point.

[0249] Example 29. A bifunctional compound as described in any one of Examples 4 to 28, wherein

[0250] (aa) 11 It 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, wherein (aa) 11 The C-terminus is related to (aa) 10 Attachment point.

[0251] Example 30. A bifunctional compound as described in any one of Examples 4 to 29, wherein

[0252] (aa) 11 It is an amino acid residue selected from L-serine residues, L-asparagine residues, L-proline residues, L-alanine residues, and L-homoserine residues, of which (aa) 11 The C-terminus is related to (aa) 10 Attachment point.

[0253] Example 31. A bifunctional compound as described in any one of Examples 4 to 30, wherein

[0254] (aa) 11 It is an L-proline residue, of which (aa)11 The C-terminus is related to (aa) 10 Attachment point.

[0255] Example 32. A bifunctional compound as described in any one of Examples 4 to 31, wherein...

[0256] (aa) 12 It is an amino acid residue selected from L-valine, D-valine, L-glutamic acid, and D-glutamic acid residues, of which (aa) 12 The C-terminus is related to (aa) 11 Attachment point.

[0257] Example 33. A bifunctional compound as described in any one of Examples 4 to 32, wherein...

[0258] (aa) 12 These are amino acid residues selected from L-valine and L-glutamic acid residues, where (aa) 12 The C-terminus is related to (aa) 11 Attachment point.

[0259] Example 34. A bifunctional compound as described in any one of Examples 4 to 33, wherein

[0260] (aa) 12 It is an L-valine residue, of which (aa) 12 The C-terminus is related to (aa) 11 Attachment point.

[0261] Example 35. A bifunctional compound as described in any one of Examples 4 to 34, wherein

[0262] (aa) 13 These are amino acid residues selected from L-phenylalanine and D-phenylalanine residues, where (aa) 13 The C-terminus is related to (aa). 12 Attachment point.

[0263] Example 36. A bifunctional compound as described in any one of Examples 4 to 35, wherein

[0264] (aa) 13 It is L-phenylalanine, of which (aa) 13 The C-terminus is related to (aa) 12 Attachment point.

[0265] Example 37. A bifunctional compound as described in Example 4, wherein T L It is a compound having formula (A) or a pharmaceutically acceptable salt or stereoisomer selected from the following:

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288] in:

[0289] Ac is an acetyl group, and wherein the acetyl group marked with "*" and the (L-cys) group marked with "*" are linked via a sulfur bond formed through their side chain or end, and

[0290] L A1As defined in this article, and L A1 **Indicator and connector (L A ) attachment point.

[0291] Example 38. A bifunctional compound as described in any one of Examples 4 to 37, wherein L A1 yes Where L A1 **Indicator and connector (L A ) attachment point and L A1 The * indicates the attachment point of the -C(=O)- group.

[0292] Example 39. A bifunctional compound as described in any one of Examples 4 to 38, wherein L A1 yes Where L A1 **Indicator and connector (L A ) attachment point and L A1 The * indicates the attachment point of the -C(=O)- group.

[0293] Example 40. A bifunctional compound as described in any one of Examples 4 to 38, wherein L A1 yes Where L A1 **Indicator and connector (L A ) attachment point and L A1 The * indicates the attachment point of the -C(=O)- group.

[0294] Example 41. A bifunctional compound as described in any one of Examples 4 to 38, wherein T L It is a compound having formula (A) or a pharmaceutically acceptable salt or stereoisomer selected from the following:

[0295]

[0296] Example 42. A bifunctional compound as described in any one of Examples 4 to 38, wherein T L yes

[0297]

[0298] Example 43. A bifunctional compound having formula (I) or as described in any one of Examples 1 to 3, wherein T L It is a compound of formula (B) that is conjugated with PCSK9, or a pharmaceutically acceptable salt or stereoisomer thereof:

[0299]

[0300] in:

[0301] XB1 It is C or N;

[0302] R B1 It is H, (C1-C6)alkyl, or (C1-C6)haloalkyl;

[0303] or R B1 and R B11 Together with the atoms to which they are attached, they form a 5- to 7-membered heterocyclic ring comprising 1 to 3 heteroatoms selected from N, O and S, which is optionally substituted by one or more substituents each independently selected from: (O), (C1-C6)alkyl and (C1-C6)haloalkyl;

[0304] R B2 It is (C1-C6)alkoxy, (C1-C6)alkyl, -L B1 -, (C1-C6) haloalkyl, (C1-C6) hydroxyalkyl, (C3-C7) cycloalkyl, or a 4- to 7-membered heterocyclic group comprising 1-3 heteroatoms selected from N, O, and S, wherein the alkyl group is optionally substituted by one or more substituents each independently selected from: (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 ) aryl, and 5- or 6-membered heteroaryl containing 1-4 heteroatoms selected from N, O and S;

[0305] R B3 It is H, (C1-C6)alkyl, or (C1-C6)haloalkyl;

[0306] R B4 It is H, (C1-C6)alkyl, or (C1-C6)haloalkyl;

[0307] R B5 It is H, (C1-C6)alkyl, or (C1-C6)haloalkyl;

[0308] R B6 It is H, (C1-C6)alkyl, -L B1 - (C1-C6) haloalkyl or (C1-C6) hydroxyalkyl, wherein the alkyl group is optionally substituted by one or more substituents each independently selected from: (C1-C6) alkoxy, -C(=O)OH, -C(=O)O(C1-C6)alkyl, -NR B17 R B18 -C(=O)NRB17 R B18 -NR B17 C(=O)R B18 (C3-C7)cycloalkyl groups, and 4- to 7-membered heterocyclic groups containing 1-3 heteroatoms selected from N, O and S;

[0309] R B6’ It is H, (C1-C6)alkyl, -L B1 - (C1-C6) haloalkyl or (C1-C6) hydroxyalkyl, wherein the alkyl group is optionally substituted by one or more substituents each independently selected from: (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 (C3-C7)cycloalkyl groups, and 4- to 7-membered heterocyclic groups containing 1-3 heteroatoms selected from N, O and S;

[0310] R B7 It is H, (C1-C6)alkyl, -L B1 - (C1-C6) haloalkyl or (C1-C6) hydroxyalkyl, wherein the alkyl group is optionally substituted by one or more substituents each independently selected from: (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 (C3-C7)cycloalkyl groups, and 4- to 7-membered heterocyclic groups containing 1-3 heteroatoms selected from N, O and S;

[0311] R B7’ It is H, (C1-C6)alkyl, -L B1 - (C1-C6) haloalkyl or (C1-C6) hydroxyalkyl, wherein the alkyl group is optionally substituted by one or more substituents each independently selected from: (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(C3-C7)cycloalkyl groups, and 4- to 7-membered heterocyclic groups containing 1-3 heteroatoms selected from N, O and S;

[0312] or R B6 and R B7 Together with the carbon atoms to which they are attached, they form (C3-C7) cycloalkyl groups or 4- to 7-membered heterocyclic rings containing 1-3 heteroatoms selected from N, O, and S;

[0313] or R B7 and R B7’ Together with the carbon atoms to which they are attached, they form (C3-C7) cycloalkyl groups or 4- to 7-membered heterocyclic rings containing 1-3 heteroatoms selected from N, O, and S; or

[0314] R B7 and R B9 Together with the atoms to which they are attached, they form a 5- to 7-membered heterocyclic ring comprising 1 to 3 heteroatoms selected from N, O and S, which is optionally substituted independently by one or more substituents selected from the following: (C1-C6)alkyl, (C1-C6)haloalkyl and =(O);

[0315] R B8 It is H or (C1-C6) alkyl;

[0316] R B9 It is an 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 a 4- to 7-membered heterocyclic group comprising 1 to 3 heteroatoms selected from N, O, and S, wherein the alkyl group is optionally separated by one or more R B27 replace;

[0317] R B9’ It is an 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 a 4- to 7-membered heterocyclic group comprising 1 to 3 heteroatoms selected from N, O, and S, wherein the alkyl group is optionally separated by one or more R B27 Replace; or when X B1 When it is N, R B9’ It does not exist;

[0318] or R B9 and R B9’ Together with the carbon atoms to which they are attached, they form (C3-C7) cycloalkyl groups or 4- to 7-membered heterocyclic rings containing 1-3 heteroatoms selected from N, O, and S;

[0319] or R B7 and R B9 Together with the atoms to which they are attached, they form a 5- to 7-membered heterocyclic ring comprising 1 to 3 heteroatoms selected from N, O and S, which is optionally substituted independently by one or more substituents selected from the following: (C1-C6)alkyl, (C1-C6)haloalkyl and =(O);

[0320] R B10 It is (C6-C) 10 aryl, a 5- or 6-membered heteroaryl group comprising 1-3 heteroatoms selected from N, O, and S, a (C3-C7) cycloalkyl group, or a 4- to 7-membered heterocyclic group comprising 1-3 heteroatoms selected from N, O, and S, wherein the cycloalkyl, heterocyclic, aryl, or heteroaryl group is determinated by -OR B13 or -NR B23 R B13 Replaced and optionally by one or more R B14 replace;

[0321] R B11 Yes -L B1 - (C1-C6)alkyl, (C1-C6)haloalkyl, or (C1-C6)hydroxyalkyl, wherein the alkyl group is optionally radically oxidized by one or more R groups. B15 replace;

[0322] or R B1 and R B11 Together with the atoms to which they are attached, they form a 5- to 7-membered heterocyclic ring comprising 1 to 3 heteroatoms selected from N, O and S, which is optionally substituted by one or more substituents each independently selected from: (O), (C1-C6)alkyl and (C1-C6)haloalkyl;

[0323] R B12 It is a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH or CN;

[0324] R B13 It is (C6-C) 10 ) aryl or 5- or 6-membered heteroaryl groups containing 1-3 heteroatoms selected from N, O, and S, wherein the aryl and heteroaryl groups are separated by R 16 Replaced and optionally by one or more R B16’ replace;

[0325] Each R B14 Each time it appears, it is independently a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, oxo, -OH or CN;

[0326] Or when R B10 When it is a cycloalkyl or heterocyclic group, the two Rs B14 When attached to the same carbon atom, they form =(O);

[0327] Each R B15 Each time it appears, it is independently (C1-C6)alkoxy, (C1-C6)haloalkoxy, -C(=O)R 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 a 4- to 7-membered heterocyclic group comprising 1-3 heteroatoms selected from N, O, and S, wherein the cycloalkyl and heterocyclic group are optionally separated by one or more R B21 replace;

[0328] R B16 It is -C(=O)NR B31 R B32 (C6-C 10 ) aryl, or a 5- to 7-membered heteroaryl comprising 1 to 3 heteroatoms selected from N, O, and S, wherein the aryl and heteroaryl are optionally separated by one or more R B26 replace;

[0329] Each R B16’ Each time it appears, it is independently a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, or CN;

[0330] or R B16 and R B16’ Together with the atoms to which they are attached, they form a 5- to 7-membered heterocyclic ring, which may optionally be independently substituted by one or more substituents selected from: = (O) and R B34 ;

[0331] R B17 It is an H or (C1-C6) alkyl group, which is optionally substituted by one or more substituents each independently selected from the following: (C1-C6) alkoxy and -C(=O)O(C1-C6) alkyl;

[0332] R B18It is an H or (C1-C6) alkyl group, which is optionally substituted by one or more substituents each independently selected from the following: (C1-C6) alkoxy and -C(=O)O(C1-C6) alkyl;

[0333] R B19 It is a (C3-C7) cycloalkyl group or a 4- to 7-membered heterocyclic group containing 1-3 heteroatoms selected from N, O, and S, optionally separated by one or more R groups. B22 replace;

[0334] R B20 It is -(CH2CH2O) m CH2CH2ONH2、-(CH2CH2O) m CH2CH2ONH(C1-C6)alkyl, or optionally with one or more -NR B23 C(=O)R B24 Substituted (C1-C6) alkyl groups;

[0335] Each R B21 Each time it appears, it is independently (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, halogen, =(O) or -OH;

[0336] Each R B22 Each time it appears, it is independently (C1-C6)alkyl, (C1-C6)haloalkyl, halogen, or -OH;

[0337] Or two Rs 22 When on the same atom, together with the atoms to which they are attached, they form (C3-C7) spirocyclic alkyl groups or 4- to 7-membered spiroheterocyclic rings containing 1-3 heteroatoms selected from N, O, and S;

[0338] R B23 It is H or (C1-C6) alkyl;

[0339] R B24 It is H or optionally by one or more R B25 Substituted (C1-C6) alkyl groups;

[0340] Each R B25 Each time it appears, it is independently a (C3-C7) cycloalkyl or a 4- to 10-membered monocyclic or bicyclic heterocyclic group containing 1-4 heteroatoms selected from N, O and S, wherein the cycloalkyl and heterocyclic group are optionally substituted by one or more substituents independently selected from the following: (C1-C6) alkyl, (C1-C6) haloalkyl and =(O);

[0341] Each R B26 Each time it appears, it is independently and optionally controlled by one or more R's.B29 Substituted (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, (C1-C6) hydroxyalkyl, -NR B31 R B32 -C(=O)NR B31 R B32 ,-C(=O)O(C1-C6)alkyl, (C3-C7)cycloalkyl, 4- to 7-membered heterocyclic groups containing 1-3 heteroatoms selected from N, O and S, (C6-C 10 ) aryl, or a 5- or 6-membered heteroaryl containing 1-3 heteroatoms selected from N, O and S, wherein the cycloalkyl, heterocyclol, aryl and heteroaryl are optionally substituted by one or more substituents each 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;

[0342] Or two Rs B26 When on adjacent atoms, together with the atoms to which they are attached, they form (C3-C7) cycloalkyl groups or 4- to 7-membered heterocyclic rings containing 1-3 heteroatoms selected from N, O, and S, optionally surrounded by one or more R B33 replace;

[0343] Each R B27 Each occurrence is independently CN, (C6-C) 10 aryl, a 5- to 7-membered heteroaryl group comprising 1-3 heteroatoms selected from N, O, and S, a (C3-C7) cycloalkyl group, or a 4- to 7-membered heterocyclic group comprising 1-3 heteroatoms selected from N, O, and S, wherein the aryl, heteroaryl, cycloalkyl, and heterocyclic group are optionally separated by one or more R B28 replace;

[0344] Each R B28 Each time it appears, it is independently (C1-C6)alkyl, (C1-C6)haloalkyl, (C1-C6)alkoxy, (C1-C6)haloalkoxy, (C1-C6)hydroxyalkyl, halogen, oxo or CN;

[0345] Or when R B27 When it is a cycloalkyl or heterocyclic group, the two Rs B28 Together with the atoms to which they are attached, they form (C4-C7) cycloalkyl groups or 4- to 7-membered heterocyclic rings containing 1-3 heteroatoms selected from N, O, and S;

[0346] Or when R B27When it is a cycloalkyl or heterocyclic group, the two Rs B28 When attached to the same carbon atom, they form =(O);

[0347] Each R B29 It is -NR independently each time it appears. B31 R B32 Or a 4- to 7-membered heterocyclic group comprising 1-3 heteroatoms selected from N, O, and S, optionally separated by one or more R B30 replace;

[0348] Each R B30 Each time it appears, it is independently -OH, halogen, (C1-C6)alkyl, or (C1-C6) haloalkyl;

[0349] Or two Rs B30 When on the same atom, together with the atoms to which they are attached, they form (C3-C7) spirocyclic alkyl groups or 4- to 7-membered spiroheterocyclic rings containing 1-3 heteroatoms selected from N, O, and S;

[0350] Each R B31 The alkyl group is independently selected from H, (C1-C6)alkyl, (C1-C6)hydroxyalkyl, (C3-C7)cycloalkyl, or a 4- to 7-membered heterocyclic group containing 1 to 3 heteroatoms selected from N, O and S, wherein the alkyl group is optionally substituted with one or more D, and the cycloalkyl and heterocyclic group are optionally substituted with one or more substituents independently selected from the following: (C1-C6)alkyl, (C1-C6)haloalkyl, halogen and -OH;

[0351] Each R B32 The alkyl group is independently selected from H, (C1-C6)alkyl, (C1-C6)hydroxyalkyl, (C3-C7)cycloalkyl, and 4- to 7-membered heterocyclic groups comprising 1 to 3 heteroatoms selected from N, O, and S, wherein the alkyl group is optionally substituted with one or more D atoms, and the cycloalkyl and heterocyclic groups are optionally substituted with one or more substituents independently selected from the following: (C1-C6)alkyl, (C1-C6)haloalkyl, halogen, and -OH;

[0352] Each R B33 Each time it appears independently, it is (C1-C6)alkyl, (C1-C6)haloalkyl, or -C(=O)R, wherein R is (C1-C6)haloalkyl, (C3-C7)cycloalkyl, a 4- to 7-membered heterocyclic group containing 1 to 3 heteroatoms selected from N, O, and S, or optionally a (C1-C6)alkyl group substituted with one or more (C1-C6)alkoxy groups;

[0353] Or two Rs B33When on the same atom, together with the atoms to which they are attached, they form (C3-C7) spirocyclic alkyl groups or 4- to 7-membered spiroheterocyclic rings containing 1-3 heteroatoms selected from N, O, and S;

[0354] Each R B34 Each time it appears independently, it is a (C3-C7) cycloalkyl or a 4- to 10-membered monocyclic or bicyclic heterocyclic group containing 1-4 heteroatoms selected from N, O and S, wherein the cycloalkyl and heterocyclic group are optionally substituted with a (C1-C6) alkyl group, which is optionally substituted with one or more substituents each independently selected from the following: (C3-C7) cycloalkyl and a 4- to 10-membered monocyclic or bicyclic heterocyclic group containing 1-4 heteroatoms selected from N, O and S;

[0355] L B1 It is -(CH2) p NH-*, where L B1 *Indicator and connector (L A The attachment point of ) and where R B11 R B2 R B6 or R B7 At least one of them is -L B1 -;

[0356] m is an integer selected from 1 to 13;

[0357] n is 1, 2, 3 or 4;

[0358] q is 0, 1, or 2, and

[0359] p is 1, 2, 3, 4, 5, or 6.

[0360] Example 44. A bifunctional compound as described in Example 43, wherein...

[0361] X B1 It is C;

[0362] R B1 It is H;

[0363] R B2 It is (C1-C6)alkoxy, -L B1 - or (C1-C6) alkyl groups substituted with -C(=O)OH;

[0364] R B3 It is H or (C1-C6) alkyl;

[0365] R B4 It is H or (C1-C6) alkyl;

[0366] R B5 It is H or (C1-C6) alkyl;

[0367] R B6 It is H, (C1-C6) alkyl or -L B1 -;

[0368] R B6’ It is H;

[0369] R B7 It is H, (C1-C6) alkyl or -L B1 -;

[0370] R B7’ It is H;

[0371] or R B6 and R B7 Together with the carbon atoms to which they are attached, they form (C3-C7) cycloalkyl groups;

[0372] R B8 It is H or (C1-C6) alkyl;

[0373] R B9 It is H or optionally by one or more R B27 Substituted (C1-C6) alkyl groups;

[0374] R B9’ It is H or (C1-C6) alkyl;

[0375] R B10 Is it -OR B13 Replaced and optionally by one or more R B14 Replacement (C6-C) 10 aryl;

[0376] R B11 Yes -L B1 -or (C1-C6) alkyl;

[0377] R B12 It is a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH or CN;

[0378] R B13 It was R 16 Replaced and optionally by one or more R B16 Replacement (C6-C) 10 aryl;

[0379] Each R B14 Each time it appears, it is independently a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, oxo, -OH or CN;

[0380] RB16 It is a 5- to 7-membered heteroaryl group containing 1 to 3 heteroatoms selected from N, O, and S, optionally separated by one or more R B26 replace;

[0381] Each R B16’ Each time it appears, it is independently a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, or CN;

[0382] Each R B26 Each time it appears, it is independently and optionally controlled by one or more R's. B29 Substituted (C1-C6) alkyl groups;

[0383] Each R B27 It is (C6-C) independently each time it appears. 10 aryl;

[0384] Each R B29 It is -NR independently each time it appears. B31 R B32 Or it may contain 4- to 7-membered heterocyclic groups with 1-3 heteroatoms selected from N, O, and S;

[0385] Each R B31 Independently selected from H and (C1-C6) alkyl groups;

[0386] Each R B32 Independently selected from H and (C1-C6) alkyl groups;

[0387] L B1 It is -(CH2) p NH-*, where L B1 *Indicator and connector (L A The attachment point of ) and where R B11 R B6 or R B7 At least one of them is -L B1 -;

[0388] n is 1;

[0389] and

[0390] p is 1, 2, 3, 4, 5, or 6.

[0391] Example 45. A bifunctional compound as described in Example 43, wherein the compound having formula (B) has the structure of formula (B-1):

[0392]

[0393] Or its pharmaceutically acceptable salts or stereoisomers.

[0394] Example 46. A bifunctional compound as described in any one of Examples 43 to 45, wherein...

[0395] R B1 It is H;

[0396] R B2 It is (C1-C6)alkoxy, -L B1 - or (C1-C6) alkyl groups substituted with -C(=O)OH;

[0397] R B3 It is H or (C1-C6) alkyl;

[0398] R B6 It is H, (C1-C6) alkyl or -L B1 -;

[0399] R B7 It is H, (C1-C6) alkyl or -L B1 -;

[0400] or R B6 and R B7 Together with the carbon atoms to which they are attached, they form (C3-C7) cycloalkyl groups;

[0401] R B9 It is H or optionally by one or more R B27 Substituted (C1-C6) alkyl groups;

[0402] R B9’ It is H or (C1-C6) alkyl;

[0403] R B10 Is it -OR B13 Replaced and optionally by one or more R B14 Replacement (C6-C) 10 aryl;

[0404] R B11 Yes -L B1 -or (C1-C6) alkyl;

[0405] R B12 It is a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH or CN;

[0406] R B13 It was R 16 Replacement (C6-C) 10 aryl;

[0407] Each R B14Each time it appears, it is independently a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, oxo, -OH or CN;

[0408] R B16 It is a 5- to 7-membered heteroaryl group containing 1 to 3 heteroatoms selected from N, O, and S, optionally separated by one or more R B26 replace;

[0409] Each R B26 Each time it appears, it is independently and optionally controlled by one or more R's. B29 Substituted (C1-C6) alkyl groups;

[0410] Each R B27 It is (C6-C) independently each time it appears. 10 aryl;

[0411] Each R B29 It is -NR independently each time it appears. B31 R B32 Or it may contain 4- to 7-membered heterocyclic groups with 1-3 heteroatoms selected from N, O, and S;

[0412] Each R B31 Independently selected from H and (C1-C6) alkyl groups;

[0413] Each R B32 Independently selected from H and (C1-C6) alkyl groups;

[0414] L B1 It is -(CH2) p NH-*, where L B1 *Indicator and connector (L A The attachment point of ) and where R B11 R B6 or R B7 At least one of them is -L B1 -;

[0415] n is 1;

[0416] and

[0417] p is 1, 2, 3, 4, 5, or 6.

[0418] Example 47. A bifunctional compound as described in Example 43, wherein T L Selected from:

[0419]

[0420] Or its pharmaceutically acceptable salt or stereoisomer, wherein:

[0421] L B1 It is -(CH2) p NH-*, where L B1 *Indicator and connector (L A ) attachment point.

[0422] Example 48. A bifunctional compound as described in any one of Examples 43 to 47, wherein T L It is a compound having formula (B) or a pharmaceutically acceptable salt or stereoisomer selected from:

[0423] Or its pharmaceutically acceptable salts or stereoisomers.

[0424] Example 49. A bifunctional compound as described in any one of Examples 43 to 48, wherein T L yes

[0425] Or its pharmaceutically acceptable salts or stereoisomers.

[0426] Example 50. A bifunctional compound having formula (I) or as described in any one of Examples 1 to 3, wherein T L It is a compound of formula (C) that is conjugated with PCSK9, or a pharmaceutically acceptable salt or stereoisomer thereof:

[0427]

[0428] in:

[0429] X C1 It is H or (C1-C6) alkyl;

[0430] X C2 It is H or (C1-C6) alkyl;

[0431] or X C1 and X C2 Together with the carbon atoms to which they are attached, they form =(O);

[0432] When X C1 and X C2 Each is independently H or (C1-C6) alkyl, or X C1 and X C2 When X forms =(O) together with the carbon atoms to which they are attached, C3 It is -CH2-;

[0433] Or when X C1 and X C2When X forms =(O) together with the carbon atoms to which they are attached, C3 It is -O-, -NH-, or -N(C1-C6)alkyl-;

[0434] R C1 It is (C6-C) 10 ) aryl or 5- or 6-membered heteroaryl groups containing 1-3 heteroatoms selected from N, O, and S, wherein the aryl and heteroaryl groups are separated by -OR C10 or -NR C21 R C10 Replaced and optionally by one or more R C11 replace;

[0435] R C2 It is H, (C1-C6)alkyl, -L C1 -, (C2-C6)alkenyl, (C1-C6) haloalkyl, -NR C12 R C13 (C3-C9) carbocyclic, (C3-C7) cycloalkenyl, 5- to 7-membered heterocyclic groups containing 1-3 heteroatoms selected from N, O, and S, (C6-C 10 aryl, or a 5- or 6-membered heteroaryl group comprising 1-3 heteroatoms selected from N, O, and S, wherein the alkyl group is optionally surrounded by one or more R C18 Substitution, and the carbocyclic, (C3-C7)cycloalkenyl, heterocyclic, aryl, and heteroaryl groups are optionally replaced by one or more R groups. C19 replace;

[0436] R C3 It is H, D, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, or (C1-C6)hydroxyalkyl, wherein the alkyl group is optionally radically oxidized by one or more R groups. C14 replace;

[0437] R C4 It is H or (C1-C6) alkyl;

[0438] or R C3 and R C4 Together with the atoms to which they are attached, they form 5- to 7-membered heterocyclic base rings containing 1-3 heteroatoms selected from N, O, and S;

[0439] R C5 It is H, D, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy or (C1-C6)hydroxyalkyl, wherein the (C1-C6)alkyl is optionally substituted by one or more D;

[0440] R C6It is (C1-C6)alkyl, (C1-C6)alkoxy, -L C1 - (C1-C6) haloalkyl, (C1-C6) haloalkoxy or (C1-C6) hydroxyalkyl, wherein the alkyl group is optionally substituted by one or more substituents each independently selected from the following: -OH, (C1-C6)alkoxy, (C1-C6) haloalkoxy, -C(O)(C1-C6)alkyl, -C(O)OH and -C(O)O(C1-C6)alkyl;

[0441] R C7 It is H, D, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy or (C1-C6)hydroxyalkyl, wherein the (C1-C6)alkyl is optionally substituted by one or more D;

[0442] R C8 It is H, (C1-C6)alkyl, -L C1 - or (C1-C6) haloalkyl, wherein the alkyl group is optionally substituted by one or more substituents each independently selected from: (C3-C7) carbocyclic, 4- to 7-membered heterocyclic group comprising 1-3 heteroatoms selected from N, O and S, -C(O)OH, -NR C16 R C17 , and -C(O)NR C16 R C17 ;

[0443] R C9 It is a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH or CN;

[0444] R C10 It is (C6-C) 10 ) aryl or a 5- or 6-membered heteroaryl group comprising 1-3 heteroatoms selected from N, O, and S, wherein the aryl and heteroaryl groups are optionally separated by one or more R C22 replace;

[0445] Each R C11 Each time it appears, it is independently a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, or CN;

[0446] R C12 and R C13 Each is independently an H or (C1-C6) alkyl group;

[0447] Each R C14 Each occurrence is independently D, NR C15 RC15’ (C3-C7) carbocyclic group, or a 3- to 7-membered heterocyclic group comprising 1-3 heteroatoms selected from N, O and S, wherein the carbocyclic group and the heterocyclic group are optionally substituted by one or more substituents each independently selected from: halogen, (C1-C6) alkyl, (C1-C6) alkoxy, (C1-C6) haloalkyl and (C1-C6) haloalkoxy;

[0448] R C15 and R C15’ Each is independently an H or (C1-C6) alkyl group;

[0449] R C16 and R C17 Each is independently an H or (C1-C6) alkyl group.

[0450] or R C16 and R C17 Together with the nitrogen atoms to which they are attached, they form 4- to 7-membered heterocyclic base rings containing 1-2 additional heteroatoms selected from N, O, and S;

[0451] Each R C18 Each time it appears, it is independently a (C3-C7) carbocyclic group, a 5- to 7-membered heterocyclic group containing 1-3 heteroatoms selected from N, O, and S, (C6-C 10 ) aryl, or a 5- or 6-membered heteroaryl group comprising 1-3 heteroatoms selected from N, O, and S, wherein the carbocyclic, heterocyclic, aryl, and heteroaryl group is optionally separated by one or more R C20 replace;

[0452] Each R C19 Each time it appears, it is independently a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, or CN;

[0453] Or two Rs C19 When they are on adjacent atoms, they form together (C6-C). 10 ) aryl or a 5- or 6-membered heteroaryl ring comprising 1-3 heteroatoms selected from N, O and S, wherein the aryl and heteroaryl are optionally substituted by one or more substituents each independently selected from: halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH and CN;

[0454] Each R C20 Each time it appears, it is independently a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, oxo, -OH, or CN; or

[0455] Or when R C18 When it is a carbocyclic or heterocyclic group, then the two Rs C20 When attached to the same carbon atom, they form =(O);

[0456] R C21 It is H or (C1-C6) alkyl;

[0457] Each R C22 Each occurrence is independently a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, CN, (C6-C 10 aryl, or a 5- or 6-membered heteroaryl group comprising 1-3 heteroatoms selected from N, O, and S, wherein the aryl and heteroaryl groups are optionally separated by one or more R atoms. C23 replace;

[0458] Each R C23 Each time it appears, it is independently a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -CH2(OCH2CH2). n OCH2CH3, -OH, CN, or a 4- to 7-membered heterocyclic group comprising 1-3 heteroatoms selected from N, O, and S, wherein the heterocyclic group is optionally substituted by one or more substituents each independently selected from: halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, -C(O)R C24 R C25 -NR C24 C(O)R C25 -NH2, -NH(C1-C6)alkyl and -N((C1-C6)alkyl)2, and the alkyl group is optionally replaced by -NR C24 R C25 Or it may contain a 4- to 7-membered heterocyclic group consisting of 1 to 3 heteroatoms selected from N, O and S, which may optionally be substituted by one or more substituents each independently selected from the following: halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, -NH2, -NH(C1-C6)alkyl and -N((C1-C6)alkyl)2;

[0459] R C24 It is H, (C1-C6)alkyl or (C3-C7)carbocyclic;

[0460] R C25 It is H, (C1-C6)alkyl, or (C3-C7)carbocyclic; and

[0461] L C1 It is -(CH2) p NH-*, where L C1 *Indicator and connector (L A The attachment point of ) and where R C2 R C6 or R C8 At least one of them is -L C1 -

[0462] Example 51. A bifunctional compound as described in Example 50, wherein the compound having formula (C) has the structure of formula (C-1):

[0463]

[0464] Or its pharmaceutically acceptable salts or stereoisomers.

[0465] Example 52. A bifunctional compound as described in Example 50 or Example 51, wherein...

[0466] X C1 and X C2 Together with the carbon atoms to which they are attached, they form =(O);

[0467] X C3 It is -CH2-;

[0468] R C1 Is it -OR C10 and one or more R C11 Replacement (C6-C) 10 aryl;

[0469] R C2 It is H, (C1-C6)alkyl, -L C1 - or (C3-C9) carbocyclic group, wherein the alkyl group is separated by an R C18 The carbocyclic group is replaced, and the carbocyclic group is replaced by one or more R... C19 replace;

[0470] R C3 It is H or (C1-C6) alkyl;

[0471] R C4 It is H or (C1-C6) alkyl;

[0472] or R C3 and R C4 Together with the atoms to which they are attached, they form 5- to 7-membered heterocyclic base rings containing 1-3 heteroatoms selected from N, O, and S;

[0473] R C5 It is H or (C1-C6) alkyl;

[0474] R C6 It is (C1-C6) alkyl or -L C1 - wherein the alkyl group is optionally substituted by one or more substituents, each independently selected from -OH or (C1-C6)alkoxy groups;

[0475] R C7 It is H or (C1-C6) alkyl;

[0476] R C8 It is H, (C1-C6) alkyl or -L C1 -;

[0477] R C9 It is halogen;

[0478] R C10 It was by an R C22 Replacement (C6-C) 10 aryl;

[0479] Each R C11 Each time it appears, it is independently a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, or CN;

[0480] R C18 It is (C6-C) 10 aryl;

[0481] Each R C19 Each time it appears, it is independently a halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -OH, or CN;

[0482] R C22 It is a 5- or 6-membered heteroaryl group containing 1-3 heteroatoms selected from N, O, and S, which is bonded by one or more R C23 replace;

[0483] Each R C23 Each time it appears, it is independently and optionally -NR C24 R C25 Substituted (C1-C6) alkyl groups or 4- to 7-membered heterocyclic groups containing 1-3 heteroatoms selected from N, O, and S;

[0484] R C24 It is H, (C1-C6) alkyl;

[0485] R C25 It is H, (C1-C6) alkyl, and

[0486] L C1 It is -(CH2)p NH-*, where L C1 *Indicator and connector (L A The attachment point of ) and where R C2 R C6 or R C8 At least one of them is -L C1 -

[0487] Example 53. A bifunctional compound as described in any one of Examples 50 to 52, wherein...

[0488] R C1 Is it -OR C10 and one or more R C11 Replacement (C6-C) 10 aryl;

[0489] R C2 It is (C1-C6) alkyl, -L C1 - or (C3-C9) carbocyclic group, wherein the alkyl group is separated by an R C18 The carbocyclic group is replaced, and the carbocyclic group is replaced by one or more R... C19 replace;

[0490] R C3 It is a (C1-C6) alkyl group;

[0491] R C4 It is H;

[0492] or R C3 and R C4 Together with the atoms to which they are attached, they form 5- to 7-membered heterocyclic base rings containing 1-3 heteroatoms selected from N, O, and S;

[0493] R C5 It is H or (C1-C6) alkyl;

[0494] R C6 It is (C1-C6) alkyl or -L C1 - wherein the alkyl group is optionally substituted by one or more substituents, each independently selected from -OH or (C1-C6)alkoxy groups;

[0495] R C7 It is H;

[0496] R C8 It is (C1-C6) alkyl or -L C1 -;

[0497] R C9 It is halogen;

[0498] R C10 It was by an R C22 Replacement (C6-C)10 aryl;

[0499] Each R C11 Each time it appears, it is a halogen independently;

[0500] R C18 It is (C6-C) 10 aryl;

[0501] Each R C19 Each time it appears, it is independently an (C1-C6) alkyl group;

[0502] R C22 It is a 5- or 6-membered heteroaryl group containing 1-3 heteroatoms selected from N, O, and S, which is bonded by one or more R C23 replace;

[0503] Each R C23 Each time it appears, it is independently and optionally -NR C24 R C25 Substituted (C1-C6) alkyl groups or 4- to 7-membered heterocyclic groups containing 1-3 heteroatoms selected from N, O, and S;

[0504] R C24 It is a (C1-C6) alkyl group;

[0505] R C25 It is a C1-C6 alkyl group, and

[0506] L C1 It is -(CH2) p NH-*, where L C1 *Indicator and connector (L A The attachment point of ) and where R C2 R C6 or R C8 At least one of them is -L C1 -

[0507] Example 54. A bifunctional compound as described in Example 50, wherein T L Selected from:

[0508] L C1 It is -(CH2) p NH-*, where L C1 *Indicator and connector (L A ) attachment point.

[0509] Example 55. A bifunctional compound as described in any one of Examples 50 to 54, wherein T LIt is a compound having formula (C) or a pharmaceutically acceptable salt or stereoisomer selected from:

[0510] Or its pharmaceutically acceptable salts or stereoisomers.

[0511] Example 56. A bifunctional compound having formula (I) or as described in any one of Examples 1 to 2, having the structure of formula (Ib):

[0512] R L -L A -FHR3 L (Ib)

[0513] in:

[0514] R L It is a portion of cell surface receptors that bind to receptor-mediated endocytosis;

[0515] L A It's a connector.

[0516] and

[0517] FHR3 L It is the part that combines FHR3.

[0518] Example 57. A bifunctional compound having formula (I), formula (Ib), or as described in any one of Examples 1 to 2, wherein the portion binding FHR3 is selected from compounds including:

[0519]

[0520]

[0521] Where L D1 It is -(CH2) p NH-*, p is 1, 2, 3, 4, 5 or 6, and where L D1 *Indicator and connector (L A ) attachment point.

[0522] Example 58. A bifunctional compound as described in Example 56 or Example 57, wherein the FHR3-binding moiety is selected from compounds or pharmaceutically acceptable salts or stereoisomers of:

[0523]

[0524] Among them, *indicator and connector (L) A ) attachment point.

[0525] B. Receptor-binding moiety (R) L )

[0526] Receptor-binding moiety (R) L These are the portions that bind to cell surface receptors, which are involved in receptor-mediated endocytosis. Examples of such cell surface receptors include, but are not limited to, desialylate glycoprotein receptor (ASGPR), mannose-6-phosphate receptor (M6PR), insulin-like growth factor 2 receptor, mannose receptor system, Kupffer cell receptor, macrophage galactose agglutinin (MGL), scavenger receptor C-type agglutinin (SRCL), EGF receptor, Fc receptor, lysosomal integrated membrane protein receptor (LIMP-2), transferrin receptor, sortilin, and decoy receptors (such as CXCR7, DARC, D6, and CCX CKR).

[0527] Asialic acid glycoprotein receptor (ASGPR)

[0528] The desialylate glycoprotein receptor (ASGPR) is a C-type lectin expressed on the surface of hepatocytes that regulates the levels of galactose (Gal) or N-acetylgalactosamine (GalNAc)-terminated plasma glycoproteins. ASGPR binds to galactose (Gal) or N-acetylgalactosamine (GalNAc)-terminated glycoproteins and is internalized via receptor-mediated endocytosis, primarily in capsule pits on the basolateral hepatocyte membrane. After internalization, the ligand-receptor complex is transported to the endosomal compartment. Calcium chelation and subsequent acidification of the endosomal compartment promote the dissociation of the ligand-receptor complex and recycle the receptor back to the plasma membrane, while simultaneously sorting the cargo (ligand) into the lysosome for degradation.

[0529] Given the ability of the desialylate glycoprotein receptor (ASGPR) to effectively facilitate the delivery of galactose (Gal) or N-acetylgalactosamine (GalNAc)-terminated glycoproteins to lysosomes, it is utilized herein to degrade extracellular target molecules (such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, cellular and plasma membrane proteins), wherein the extracellular target molecules are associated with the (T) of the bifunctional compounds of this invention. L ) group binding, and the receptor binding moiety of the bifunctional compound (R L It contains one or more galactose (Gal) groups or one or more N-acetylgalactosamine (GalNAc) groups. This type of receptor-binding moiety (R...) L The α group binds to the desialyl glycoprotein receptor (ASGPR), thereby delivering extracellular target molecules to lysosomes for degradation via lysosomal degradation.

[0530] Example 59. A bifunctional compound having formula (I) or as described in any one of Examples 1 to 58, wherein the receptor binding moiety (R) L (Selected from:)

[0531]

[0532] Where R L *Indicator and connector (L A ) attachment point.

[0533] In other embodiments, the receptor-binding portion (R) of the bifunctional compound of the present invention L It contains one or more galactose (Gal) groups or one or more N-acetylgalactosamine (GalNAc) groups, wherein the one or more galactose (Gal) groups or one or more N-acetylgalactosamine (GalNAc) groups contain a bridging ketal moiety.

[0534] Example 60. A bifunctional compound having formula (I) or as described in any one of Examples 1 to 58, wherein the receptor binding moiety (R) L (Selected from:)

[0535] Where R L *Indicator and connector (L A ) attachment point.

[0536] Mannose-6-phosphate receptor (M6PR) - also known as insulin-like growth factor 2 receptor

[0537] Lysosomes are membrane-bound organelles with a characteristic acidic pH, responsible for the degradation of many different substrates in the cell. This catabolic process is carried out by more than 60 soluble enzymes contained within the organelle, many of which belong to a broad class of hydrolases known as glycosidases, proteases, phosphatases, sulfatases, and lipases. These lysosomal hydrolases are initially synthesized in the rough endoplasmic reticulum and are specifically transported to the trans-Golgi network via the Golgi apparatus, and then delivered to the lysosome via transport vesicles.

[0538] To ensure the concentration and delivery of lysosomal hydrolases to lysosomes, lysosomal hydrolases are labeled with a unique marker: the mannose-6-phosphate (M6P) group. The M6P group is specifically added to the N-linked oligosaccharide of the lysosomal hydrolase as it moves through the cis-Golgi network. The M6P group is then recognized by two separate 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 receptor binds to the labeled M6P group on the lysosomal hydrolase at pH 6.5–6.7, thus aiding in the packaging of 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 binds to lysosomal enzymes that have escaped the cell, delivering them to late endosomes. Once inside the endosome, which is normally at pH 6, lysosomal hydrolases dissociate from the MPR, and during the maturation of the endosome into a lysosome, the pH drops to pH 5, where the hydrolases begin to digest the endocytic material delivered from the early endosome. Subsequently, the MPR recirculates from the endosome to the cell surface and then back to the Golgi complex.

[0539] Given that the M6P receptor effectively facilitates the delivery of M6P-tagged proteins to lysosomes, it is utilized herein to degrade extracellular target molecules (such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, cellular and plasma membrane proteins), wherein the extracellular target molecules are associated with the (T) group of the bifunctional compounds of this invention. L ) group binding, and the receptor binding moiety of the bifunctional compound (R L This type of receptor-binding moiety contains one or more high-affinity ligands for the M6P receptor. L The ) group binds to the M6P receptor, thereby delivering extracellular target molecules to the lysosome and degrading them via lysosomal degradation.

[0540] Example 61. A bifunctional compound having formula (I) or as described in any one of Examples 1 to 58, wherein the receptor binding moiety (R) L (Selected from:)

[0541]

[0542] Where R L *Indicator and connector (L A ) attachment point.

[0543] C. Connector (L) A )

[0544] The connector portion (L) of the bifunctional compound of the present invention A A joint is a non-cuttable joint that contains one or more joint components selected from the following:

[0545] a) Alkyl group: -(CH2) n -, which can be linear or branched (where in this case, n is 1-18);

[0546] b) Alkenyl group;

[0547] c) Alynyl group;

[0548] d) Alkenyl groups;

[0549] e) Acynyl group;

[0550] f) Ethylene glycol unit: -OCH2CH2 or -CH2CH2O;

[0551] g) Polyethylene glycol unit: (-CH2CH2O-) x (where x is 2-20 in this case);

[0552] h)-O;

[0553] i)-S;

[0554] j) Carbonyl group: -C (=O);

[0555] k) Ester: -C(=O)-O- or -OC(=O);

[0556] l) Carbonate: -OC(=O)O;

[0557] m)Amine: -NH;

[0558] n) Tertiary amines

[0559] o) Amides: -C(=O)-NH-, -NH-C(=O)- or -C(=O)N(C 1-6 alkyl);

[0560] p) Carbamate: -OC(=O)NH- or -NHC(=O)O;

[0561] q) Urea: -NHC(=O)NH;

[0562] r) Sulfonamides: -S(O)2NH- or -NHS(O)2;

[0563] s) Ethers: -CH2O- or -OCH2;

[0564] t) an alkylene group substituted with one or more groups independently selected from carboxyl, sulfonate, hydroxyl, amine, amino acid, sugar, phosphate and phosphonate groups;

[0565] u) an alkenyl group substituted by one or more groups independently selected from carboxyl, sulfonate, hydroxyl, amine, amino acid, sugar, phosphate and phosphonate groups;

[0566] v) An alkynyl group substituted by one or more groups independently selected from carboxyl, sulfonate, hydroxyl, amine, amino acid, sugar, phosphate and phosphonate groups;

[0567] w)C1-C 10 Alkylene, wherein one or more methylene groups are partially replaced by one or more -S-, -NH-, or -O- groups; and

[0568] x) A ring system having two available attachment sites, for example selected from divalent rings such as phenyl (including 1,2-, 1,3- and 1,4-disubstituted phenyl), C5-C6 heteroaryl, C3-C8 cycloalkyl (including 1,1-disubstituted cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl and 1,4-disubstituted cyclohexyl) and C4-C8 heterocycloalkyl.

[0569] Additionally, the connector (L) A The linker component can be a chemical motif that is readily formed by a reaction between two reactive groups. Non-limiting examples of such chemical motifs are given in Table 1.

[0570] Table 1

[0571]

[0572]

[0573]

[0574]

[0575]

[0576] Wherein: R in Table 1 32 It is H, C 1-4 Alkyl, phenyl, pyrimidine, or pyridine; R in Table 1 35 It is H, C 1-6 Alkyl, phenyl, or C substituted with 1 to 3 -OH groups 1-4 Alkyl; each R in Table 1 7 Independently selected from H and C 1-6Alkyl, fluorine, benzyloxy group substituted with -C(=O)OH, benzyl group substituted with -C(=O)OH, C group substituted with -C(=O)OH 1-4 Alkoxy groups and C groups substituted with -C(=O)OH 1-4 Alkyl; R in Table 1 37 The components are independently selected from H, phenyl, and pyridine; and q in Table 1 is 0, 1, 2, or 3.

[0577] In addition, the connector (L A The connector components can be the groups given in Table 2 below.

[0578] Table 2

[0579]

[0580]

[0581] Example 62. A bifunctional compound having formula (I) or as described in any one of Examples 1 to 61, wherein the connector (L) A (Selected from:)

[0582] *-(CH2) n C(=O)NHNHC(=O)(CH2) n ON=CH2X1C(=O)-**;*-(CH2) n X3C(=O)-**;*-(CH2) n C(=O)-**;

[0583] *-(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)-**;

[0584] *-((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) nC(=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) n NHC(=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 -**;

[0585] *-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 -**;

[0586] *-C(=O)NH(CH2) n X3(CH2) m -**; *-C(=O)NH(CH2) n NHC(=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 -**;

[0587] *-C(=O)NH(CH2) n NHC(=O)-**; *-C(=O)NH((CH2) n O) t (CH2) n X3(CH2) m -**;

[0588] *-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 -**;

[0589] in

[0590] X1 is and

[0591] X3 is The * indicator of X3 points towards R. L And the X3's **indicator attachment point faces L. A ,

[0592] And L A *Instructions and R L The attachment point, and L A **Instructions and T L Attachment point.

[0593] Example 63. A bifunctional compound having formula (I) or as described in any one of Examples 1 to 62, wherein the connector (L) A )yes:

[0594] *-(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)-**;

[0595] *-(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)-;

[0596] *-C(=O)(CH2) n C(=O)-**;*-C(=O)((CH2) n O) t (CH2) m C(=O)-**;*-((CH2) n O) t (CH2) mX3(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 -**;

[0597] in

[0598] X1 is and

[0599] X3 is The * indicator of X3 points towards R. L And the X3's **indicator attachment point faces L. A ,

[0600] And L A *Instructions and R L The attachment point, and L A **Instructions and T L Attachment point.

[0601] Example 64. A bifunctional compound having formula (I) or formula (Ia), selected from:

[0602]

[0603]

[0604]

[0605]

[0606]

[0607]

[0608] Example 65. A bifunctional compound having formula (I) or formula (Ib), selected from:

[0609]

[0610]

[0611] Method for preparing compounds having formula (I)

[0612] For illustrative purposes, the general reaction schemes described herein provide potential pathways for synthesizing the compounds of the present invention. Detailed descriptions of each reaction step are provided in the Examples section below. Furthermore, many compounds prepared by the methods described below can be further modified using conventional chemistry well known to those skilled in the art, according to this disclosure. In the following general scheme, R... L L A and T L As defined in this article.

[0613] For example, the general synthesis of compounds having formula (I) is shown in Scheme I below, wherein the receptor ligand (R) L The target ligand has an attached reactive group (e.g., RG1) and the target ligand is attached to a linker portion having a reactive side group (e.g., RG2). A This reactive side group can react with reactive groups on the receptor ligand, thereby forming a linker (L) that couples the receptor ligand to the target ligand. A ), thereby forming a compound having formula (I).

[0614] Option I

[0615] R L -RG1+RG2-L A ′-T L →R L -L A -T L

[0616] In scheme I, RG1 is reactive group 1 from Table 1 and RG2 is reactive group 1 from Table 1, wherein the reaction products of the corresponding groups (as seen in Table 1) become linker L. A The connector components.

[0617] Another general synthesis of a compound having formula (I) is shown in scheme II below, wherein the receptor ligand (R) L ) Attached to the connector portion (L) with reactive side groups (e.g., RG1) A Furthermore, the target ligand has an attached reactive group that can react with the reactive group leaving the receptor ligand, thereby forming a linker (L) that couples the receptor ligand to the target ligand. A ), thereby forming a compound having formula (I).

[0618] Option II

[0619] R L -L A ′-RG1+RG2-T L →R L-L A -T L

[0620] In scheme II, RG1 is reactive group 1 from Table 1 and RG2 is reactive group 1 from Table 1, wherein the reaction products of the corresponding groups (as seen in Table 1) become linker L. A The connector components.

[0621] Another general synthesis of a compound having formula (I) is shown in Scheme III below, wherein the receptor ligand (R) L ) Attached to the connector portion (L) with reactive side groups (e.g., RG1) A ") and the target ligand attaches to the junction portion with reactive side groups (e.g., RG2) (L A This reactive side group can react with reactive groups leaving the receptor ligand, thereby forming a linker (L) that couples the receptor ligand to the target ligand. A ), thereby forming a compound having formula (I).

[0622] Option III

[0623] R L -L A "-RG1+RG2-L A ′-T L →R L -L A -T L

[0624] In scheme II, RG1 is reactive group 1 from Table 1 and RG2 is reactive group 1 from Table 1, wherein the reaction products of the corresponding groups (as seen in Table 1) become linker L. A The connector components.

[0625] Another general synthesis of compounds having formula (I) is shown in scheme IV below, wherein the receptor ligand (R) L It has an attachment reactive group (e.g., RG1) that can interact with the connector portion (L). A The reactive groups (e.g., RG2) on the junction portion react with the reactive groups on the junction portion (L). A ) Attached to receptor ligand (R L ). Connector section (L) A It also has a protected reactive group (e.g., RG1-Prot) that, upon deprotection, can react with a reactive group (e.g., RG2) on the target ligand, thereby coupling the receptor ligand with the target ligand to form a compound having formula (I).

[0626] Option IV

[0627]

[0628] In scheme IV, RG1 is reactive group 1 from Table 1 and RG2 is reactive group 1 from Table 1, wherein the reaction products of the corresponding groups (as seen in Table 1) become linker L. A The connector components.

[0629] Another general synthesis of compounds having formula (I) is shown in scheme V below, wherein the target ligand (T) L It has an attachment reactive group (e.g., RG1) that can interact with the connector portion (L). A The reactive groups (e.g., RG2) on the junction portion react with the reactive groups on the junction portion (L). A ) Attached to the target ligand (T L ). Connector section (L) A It also has a protected reactive group (e.g., RG1-Prot) that, upon deprotection, can react with a reactive group (e.g., RG2) on the receptor ligand, thereby coupling the receptor ligand with the target ligand to form a compound having formula (I).

[0630] Option V

[0631]

[0632] In scheme V, RG1 is reactive group 1 from Table 1 and RG2 is reactive group 1 from Table 1, wherein the reaction products of the corresponding groups (as seen in Table 1) become linker L. A The connector components.

[0633] Pharmaceutical Compositions and Routes of Administration

[0634] 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. Therefore, in another aspect, the present invention provides a pharmaceutical composition comprising the bifunctional compound of the present invention and a pharmaceutically acceptable carrier. In further embodiments, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.

[0635] The pharmaceutical compositions of the present invention can be prepared using methods comprising mixing the bifunctional compound of the present invention with one or more pharmaceutically acceptable carriers. For example, the pharmaceutical compositions of the present invention are manufactured by mixing, granulating, and / or coating the free form of the bifunctional compound of the present invention with at least one pharmaceutically acceptable carrier.

[0636] The pharmaceutical compositions or combinations of the present invention may be administered at a unit dose of about 0.1-100 mg of active ingredient to a subject weighing about 50-70 kg. The therapeutically effective dose of the compound, pharmaceutical composition, or combination thereof depends on the species, weight, age, individual circumstances, disability or disease, or its severity, of the subject being treated.

[0637] The above-described dosage characteristics can be demonstrated in in vitro and in vivo studies using advantageous mammals, such as mice, rats, dogs, monkeys, or their isolated organs, tissues, and preparations. The compounds of the present invention can be applied in vitro in solution (e.g., aqueous solution) and in vivo, for example as a suspension or in aqueous solution, in the intestine, parenteral, subcutaneous, or intravenous systems. In vitro doses can be approximately 10... -12 molar concentration and 10 -6 The effective therapeutic dose in vivo ranges from approximately 0.01 to 10 mg / kg.

[0638] The activity of the compounds of the present invention can be assessed by the in vitro and in vivo methods described in the examples herein.

[0639] The bifunctional compound of the present invention may be an active ingredient in a pharmaceutical composition formulated for a specific route of oral or parenteral administration.

[0640] Oral dosage form

[0641] The pharmaceutical compositions of the present invention can be administered orally as discrete dosage forms, wherein such dosage forms include (but are not limited to) capsules, gelatin capsules, capsule tablets, tablets, chewable tablets, lozenges, dispersible powders, granules, syrups, flavored syrups, solutions or suspensions in aqueous or non-aqueous liquids, edible foams or foams, and oil-in-water or water-in-oil latexes.

[0642] Therefore, for oral administration, pharmaceutical compositions of the present invention containing an effective amount of the compounds of the present invention can be prepared in solid form (including but not limited to capsules, gelatin capsules, hard or soft capsules, tablets, chewable tablets, lozenges, capsule-type tablets, pills, granules, or dispersible powders) or in liquid form (including but not limited to solutions, aqueous or oily suspensions, syrups, elixirs, foams, foams, or emulsions). Conventional pharmaceutical processes, such as sterilization, can be performed on the pharmaceutical compositions, and / or they can be made to contain conventional inert diluents, lubricants, or buffers, as well as excipients (such as preservatives, stabilizers, wetting agents, emulsifiers, and buffers).

[0643] Compositions intended for oral use are prepared according to any method known in the art for manufacturing pharmaceutical compositions, and in order to provide a pharmaceutically refined and palatable formulation, such compositions may contain one or more agents selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives.

[0644] Typically, pharmaceutical compositions are tablets or gelatin capsules containing an active ingredient and one or more of the following:

[0645] a) Diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine;

[0646] b) Lubricants, such as silica, talc, stearic acid, their magnesium or calcium salts and / or

[0647] Polyethylene glycol; for tablets, it also contains

[0648] c) Adhesives, such as magnesium aluminum silicate, starch paste, gelatin, tragacanth gum,

[0649] Methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone; and if desired, also contains

[0650] d) Disintegrants, such as starch, agar, alginate or its sodium salt, or effervescent agents.

[0651] Mixtures; and

[0652] e) Adsorbents, colorants, flavoring agents and sweeteners.

[0653] Tablets may contain an active ingredient mixed with a non-toxic, pharmaceutically acceptable excipient suitable for tablet production. These excipients are, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrants such as corn starch or alginate; binders such as starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be film-coated or enteric-coated according to methods known in the art. Tablets may be uncoated or coated according to known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over a longer period of time. For example, time-delaying materials such as glyceryl monostearate or glyceryl distearate may be used. Formulations for oral use may be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate, or kaolin) or as soft gelatin capsules in which the active ingredient is mixed with an aqueous or oily medium (e.g., peanut oil, liquid paraffin, or olive oil).

[0654] Parenteral dosage form

[0655] In some embodiments, the pharmaceutical compositions of the present invention are administered parenterally via various routes, including but not limited to subcutaneous, intravenous (including bolus), intramuscular, and intravitreal administration.

[0656] Some injectable compositions are isotonic aqueous solutions or suspensions containing the bifunctional compounds of the present invention. Such compositions may contain excipients such as preservatives, stabilizers, wetting agents or emulsifiers, solution promoters, salts and / or buffers for adjusting osmotic pressure. Such compositions can be prepared according to conventional methods known in the art, and such compositions can be sterilized.

[0657] Combination therapy

[0658] The compounds of the present invention and the pharmaceutical compositions provided herein may be administered alone or in combination with one or more other therapeutic agents.

[0659] In some embodiments, the pharmaceutical compositions of the present invention optionally further comprise one or more additional therapeutic agents. Alternatively, the bifunctional compounds of the present invention may be combined with one or more other therapeutic agents and administered to patients in need.

[0660] The bifunctional compounds of the present invention can be administered simultaneously, before, or after one or more other therapeutic agents. The bifunctional compounds of the present invention can be administered separately via the same or different routes of administration as other pharmaceutical agents, or together in the same pharmaceutical composition. The therapeutic agents are, for example, chemical compounds, peptides, antibodies, antibody fragments, or nucleic acids, which, when administered to a patient in combination with the bifunctional compounds of the present invention, have therapeutic activity or enhance therapeutic activity.

[0661] In one embodiment, the present invention provides a product comprising the bifunctional compound of the present invention and at least one other therapeutic agent, which is intended as a combination formulation for simultaneous, separate, or sequential use in a therapy that treats a disease or condition as described herein by means of 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, cell or plasma membrane proteins. Products provided as combination formulations include compositions that collectively comprise the bifunctional compound of the present invention and other therapeutic agents in the same pharmaceutical composition, or that comprise the compound of the present invention and other therapeutic agents in a separate form (e.g., in a kit).

[0662] 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 comprise a pharmaceutically acceptable carrier as described above.

[0663] In one embodiment, the present invention provides a kit comprising two or more separate pharmaceutical compositions, wherein at least one pharmaceutical composition contains a bifunctional compound of the present invention. In one embodiment, the kit includes means for separately retaining the compositions (e.g., a container, separator bottle, or separator foil pack). An example of such a kit is blister packaging, such as blister packaging commonly used for packaging tablets, capsules, and the like.

[0664] The kits of the present invention can be used to administer different dosage forms (e.g., oral and parenteral), to administer individual compositions at different dose intervals, or to titrate individual compositions relative to each other. To aid in compliance, the kits of the present invention typically include instructions for use.

[0665] 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 combined to form a combination therapy: (i) before the combination product is released to a physician (e.g., in the case of a kit containing the compound and other therapeutic agents of the present invention); (ii) shortly before administration by the physician (or under the physician's guidance); (iii) in the patient, for example during the sequential administration of the compound and other therapeutic agents of the present invention.

[0666] Pharmacology and efficacy

[0667] The bifunctional compounds of the present invention exhibit valuable pharmacological properties based on the inhibition of extracellular target molecules through lysosomal degradation, and are therefore indicated for use in therapeutics or as research chemicals (e.g., as tool compounds).

[0668] Conventional therapeutic agents, such as protein-targeted therapies, treat diseases by blocking protein function (e.g., by inhibiting enzymes and receptors) or by recruiting immune effectors (as is the case with many monoclonal antibody drugs). Typically, due to the reversible nature of conventional drug / target interactions, the efficacy of such conventional therapies requires superstoichiometric drug concentrations to maintain inhibition, which can be lost over time as drug concentrations decrease. However, the methods described herein using the bifunctional compounds of the invention can exhibit improved efficacy at stoichiometric or substoichiometric concentrations, where efficacy is limited by the resynthesis of the target molecule (e.g., protein) rather than by drug concentration.

[0669] Therefore, the present invention provides bifunctional compounds for use in therapies involving 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, and cellular or plasma membrane proteins. In some embodiments, the present invention also provides bifunctional compounds for use in therapies involving targeted lysosomal degradation of extracellular target molecules mediated by the asialic acid glycoprotein receptor (ASGPR), such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, and cellular and plasma membrane proteins. In some embodiments, the present invention also provides bifunctional compounds for use in therapies involving lysosomal degradation mediated by the mannose-6-phosphate receptor (M6PR) targeting extracellular target molecules such as growth factors, cytokines, chemokines, hormones, neurotransmitters, capsids, soluble receptors, extracellular secretory proteins, antibodies, lipoproteins, exosomes, viruses, cellular and plasma membrane proteins. In some embodiments, such therapies include treatments for cardiovascular diseases, liver diseases, kidney diseases, autoimmune diseases, neurological diseases, blood disorders, skin diseases, drug poisoning, or vasculitis. In some embodiments, such therapies include treatment for hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, occlusive arteriosclerosis, fulminant hepatic failure, postoperative liver failure, acute liver failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, liver allogeneic transplantation, focal glomerulosclerosis, kidney allogeneic transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Grigor Gaucher-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood type incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, or Kawasaki disease. In other embodiments, such therapies include treatment for kidney disease, age-related macular degeneration, atypical hemolytic uremic syndrome, or hepatocellular carcinoma (HCC).

[0670] Furthermore, the present invention provides bifunctional compounds for use in therapies, wherein such therapies include the treatment of cardiovascular diseases, liver diseases, kidney diseases, autoimmune diseases, neurological diseases, blood diseases, skin diseases, drug poisoning, or vasculitis. In some embodiments, such therapies include treatment for hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, occlusive arteriosclerosis, fulminant hepatic failure, postoperative liver failure, acute liver failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, liver allogeneic transplantation, focal glomerulosclerosis, kidney allogeneic transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Grigor Gaucher-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood type incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, or Kawasaki disease. In other embodiments, such therapies include treatment for kidney disease, age-related macular degeneration, atypical hemolytic uremic syndrome, or hepatocellular carcinoma (HCC).

[0671] The present invention also provides the use of the bifunctional compounds of the present invention in therapies, wherein such therapies include the treatment of cardiovascular diseases, liver diseases, kidney diseases, autoimmune diseases, neurological diseases, blood diseases, skin diseases, drug poisoning, or vasculitis. In some embodiments, such therapies include treatment for hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, occlusive arteriosclerosis, fulminant hepatic failure, postoperative liver failure, acute liver failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, liver allogeneic transplantation, focal glomerulosclerosis, kidney allogeneic transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Grigor Gaucher-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood type incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, or Kawasaki disease. In other embodiments, such therapies include treatment for kidney disease, age-related macular degeneration, atypical hemolytic uremic syndrome, or hepatocellular carcinoma (HCC).

[0672] On the other hand, 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 plasma membrane proteins, wherein the method utilizes targeted lysosomal degradation of such extracellular target molecules. In some embodiments, 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 plasma membrane proteins, wherein the method utilizes lysosomal degradation of extracellular target molecules mediated by targeted desialylate glycoprotein receptor (ASGPR). In some 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, cellular and plasma membrane proteins, wherein the method utilizes lysosomal degradation mediated by the targeting of the extracellular target molecules to the mannose-6-phosphate receptor (M6PR). These methods can be used to treat a variety of diseases, conditions, or clinical conditions that are typically treated with therapeutic apheresis, such as cardiovascular diseases, liver diseases, kidney diseases, autoimmune diseases, neurological diseases, blood diseases, skin diseases, drug poisoning, and vasculitis. For example, such diseases include, but are not limited to, hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, occlusive arteriosclerosis, fulminant hepatic failure, postoperative liver failure, acute liver failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, liver allogeneic transplantation, focal glomerulosclerosis, kidney allogeneic transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Gurren Lahm-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood type incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, and Kawasaki disease. These methods can also be used to treat kidney disease, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0673] In another aspect, the present invention provides a method for treating cardiovascular diseases, liver diseases, kidney diseases, autoimmune diseases, neurological diseases, blood diseases, skin diseases, drug poisoning, and vasculitis, wherein the method comprises administering a therapeutically effective amount of the bifunctional compound of the present invention to a subject in need. In some embodiments, such diseases include, but are not limited to, hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, occlusive arteriosclerosis, fulminant hepatic failure, postoperative liver failure, acute liver failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, liver allogeneic transplantation, focal glomerulosclerosis, kidney allogeneic transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Gurren Lahm-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood type incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, and Kawasaki disease. As further embodiments, such diseases include nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0674] In another aspect, the present invention provides a method for treating cardiovascular diseases, liver diseases, kidney diseases, autoimmune diseases, neurological diseases, blood diseases, skin diseases, drug poisoning, and vasculitis, wherein the method comprises administering the bifunctional compound of the present invention to a subject in need. In some embodiments, such diseases include, but are not limited to, hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, occlusive arteriosclerosis, fulminant hepatic failure, postoperative liver failure, acute liver failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, liver allogeneic transplantation, focal glomerulosclerosis, kidney allogeneic transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Gurren Lahm-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood type incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, and Kawasaki disease. As further embodiments, such diseases include nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0675] In another aspect, the present invention provides the use of the bifunctional compound of the present invention in the manufacture of a medicament for treating cardiovascular diseases, liver diseases, kidney diseases, autoimmune diseases, neurological diseases, blood diseases, skin diseases, drug poisoning, and vasculitis, wherein the method comprises administering a therapeutically effective amount of the bifunctional compound of the present invention to a subject in need. In some embodiments, such diseases include, but are not limited to, hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, occlusive arteriosclerosis, fulminant hepatic failure, postoperative liver failure, acute liver failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, liver allogeneic transplantation, focal glomerulosclerosis, kidney allogeneic transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Gurren Lahm-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood type incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, and Kawasaki disease. As further embodiments, such diseases include nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0676] The present invention also provides a method for in vivo therapeutic plasma exchange, wherein the method includes administering the bifunctional compound of the present invention to a subject. The present invention further provides a method for performing in vivo therapeutic plasma exchange, wherein the method includes administering the bifunctional compound of the present invention to a subject.

[0677] The present invention also provides an in vivo therapeutic plasma exchange method for treating cardiovascular diseases, liver diseases, kidney diseases, autoimmune diseases, neurological diseases, blood diseases, skin diseases, drug poisoning, or vasculitis, wherein the method comprises administering the bifunctional compound of the present invention to a subject. In some embodiments, such diseases are hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, occlusive arteriosclerosis, fulminant hepatic failure, postoperative liver failure, acute liver failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, liver allogeneic transplantation, focal glomerulosclerosis, kidney allogeneic transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Gurren Lahm-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood type incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, and Kawasaki disease.

[0678] The present invention also provides a method for in vivo therapeutic plasma exchange, wherein the method comprises administering a bifunctional compound having formula (I) to a subject. The present invention further provides a method for performing in vivo therapeutic plasma exchange, wherein the method comprises administering the bifunctional compound of the present invention to a subject.

[0679] The present invention also provides an in vivo therapeutic plasma exchange method for treating cardiovascular diseases, liver diseases, kidney diseases, autoimmune diseases, neurological diseases, blood diseases, skin diseases, drug poisoning, or vasculitis, wherein the method comprises administering to a subject a bifunctional compound having formula (I). In some embodiments, such diseases are hypercholesterolemia, familial hypercholesterolemia, arteriosclerosis, occlusive arteriosclerosis, fulminant hepatic failure, postoperative liver failure, acute liver failure, hepatitis C, hepatitis B, chronic hepatitis C, chronic hepatitis B, liver allogeneic transplantation, focal glomerulosclerosis, kidney allogeneic transplantation, malignant rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Gurren Lahm-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, multiple myeloma, macroglobulinemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, blood type incompatibility during pregnancy, hemophilia, pemphigus, bullous pemphigoid, toxic epidermal necrosis, Stevens-Johnson syndrome, drug poisoning, and Kawasaki disease.

[0680] One aspect of the present invention is a bifunctional molecule that utilizes receptor-mediated endocytosis to eliminate or reduce the level of proprotein convertase subtilisin / kexin type 9 (PCSK9) in plasma.

[0681] PCSK9 significantly affects plasma low-density lipoprotein cholesterol (LDL-C) levels by modulating the hepatic low-density lipoprotein receptor (LDLR), the main pathway for cholesterol removal from the circulatory system. PCSK9 binds to LDLR and directs it to lysosomes for degradation, thereby increasing plasma LDL-C levels and consequently increasing the risk of coronary heart disease. (Maxwell KN, Proc. Natl. Acad. Sci., 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 PCSK9 in mice or humans has been shown to increase total LDL-C levels and significantly decrease hepatic LDLR protein, without affecting mRNA, SREBP, or the nucleocytoplasmic ratio of SREBP protein (Maxwell K.N., Proc. Natl. Acad. Sci. [Proceedings of the National Academy of Sciences] 101, 2004, 7100-7105). Furthermore, PCSK9 mutations that induce loss of PCSK9 function in mouse models have also shown reduced total and LDL-C levels (Cohen, JC et al., N. Engl. J. Med. [New England Journal of Medicine], 354, 2006, 1264-1272). Therefore, this indicates that PCSK9 regulation leads to decreased LDLR protein levels.

[0682] Furthermore, PCSK9 gene deletion has also been performed in mice. PCSK9 knockout mice showed approximately 50% reduction in plasma cholesterol levels and enhanced sensitivity to statins in lowering plasma cholesterol (Rashid.S. et al., (2005) ProcNatl Acad Sci [PNAS] 102:5374-5379). Human genetic data strongly support the role of PCSK9 in LDL balance. The link 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 [Nature Genetics], 2003, 34:154-156). Patients carrying the PCSK9 gain-of-function allele had increased plasma LDL-C levels and premature coronary heart disease, while patients carrying the PCSK9 loss-of-function allele had significantly reduced plasma LDL-C and were protected from coronary heart disease.

[0683] PCSK9 also plays a role in lipoprotein(a) (Lp(a)) metabolism. Lp(a) is a pro-atherosclerotic lipoprotein composed of LDL particles covalently linked to apoLp(a). Human genetic studies have shown a causal relationship between Lp(a) and the risk of coronary heart disease. Therapeutic antibodies against PCSK9 have been shown to significantly reduce Lp(a) 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). Compared with placebo, patients receiving statin therapy treated with monoclonal antibodies against PCSK9 showed a reduction in Lp(a) levels of up to 32%. (Desai, NR et al., Circulation. 2013; 128(9):962-969).

[0684] Besides its cardiovascular effects, PCSK9 plays a crucial role in sepsis, a life-threatening disease 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 regulates the uptake of Gram-negative lipopolysaccharide (LPS) by hepatocytes through an LDL-dependent mechanism by modulating LDLR-mediated bacterial lipid uptake of lipoteichoic acid (LTA) and LPS (Grin, PM et al., Nature, 2018, 8(1):10496). Therefore, inhibiting PCSK9 has the potential to treat sepsis by reducing the body's immune response to infection.

[0685] Atherosclerotic cardiovascular disease is a leading cause of death worldwide, estimated to have caused 7.4 million deaths globally in 2015. LDL cholesterol, 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 randomization studies, and randomized clinical trials have demonstrated a log-linear association between absolute LDL cholesterol exposure and ASCVD risk [Baigent C et al. 2005, Ference BA et al. 2017].

[0686] PCSK9 is a 692-amino acid serine protease that significantly influences LDL-C levels via its regulation of the hepatic LDLR receptor, a major pathway for cholesterol removal 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 consequently increasing the risk of ASCVD. PCSK9 has excellent target validation. Mice lacking PCSK9 showed decreased plasma cholesterol and increased hepatic LDLR expression compared to littermate controls. Mice in which PCSK9 was selectively inactivated in the liver had no detectable PCSK9 in the blood, indicating that the liver is the primary source of circulating PCSK9 [Zaid et al., 2008]. Patients carrying the PCSK9 gain-of-function allele had increased plasma LDL-C levels and premature ASCVD, while patients carrying the PCSK9 loss-of-function allele had significantly decreased plasma LDL-C and were protected against ASCVD [Cohen J et al., 2006]. Therefore, there is great interest in identifying novel therapies that mimic PCSK9 loss of function.

[0687] Clinical studies using PCSK9 blocking antibodies have demonstrated significant reductions in LDL in healthy volunteers and patients with hypercholesterolemia, 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, which limits the efficacy of dose escalation [Careskey et al., 2008; Welder et al., 2010]. Data from several clinical studies using inclisiran have shown that reducing PCSK9 levels by inhibiting its 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].

[0688] This invention relates to bifunctional compounds and compositions capable of lowering plasma PCSK9 levels or eliminating PCSK9 circulating in plasma. The disclosure is characterized by methods for treating, preventing, or improving diseases or disorders in which PCSK9 functions by administering a therapeutically effective amount of a bifunctional compound of formula (Ia) to a patient in need. The methods of this invention can be used to treat a variety of PCSK9-dependent diseases and disorders by lowering plasma PCSK9 levels or eliminating PCSK9 circulating in plasma. Lowering plasma PCSK9 levels or eliminating PCSK9 circulating in plasma provides novel methods for treating, preventing, or improving diseases including, but not limited to, hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral vascular disease (including aortic disease and cerebrovascular disease), peripheral artery disease, vascular inflammation, elevated Lp(a), elevated LDL, elevated TRL, elevated triglycerides, sepsis, and xanthomas.

[0689] The bifunctional compound of formula (Ia) of the present invention is useful in treating hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral vascular disease, peripheral artery disease, vascular inflammation, elevated Lp(a), elevated LDL, elevated TRL (e.g., elevated VLDL and / or elevated chylomicrons), elevated triglycerides, sepsis, and xanthomas by reducing plasma PCSK9 levels or eliminating PCSK9 circulating in plasma.

[0690] For example, the bifunctional compound of formula (Ia) of the present invention binds to PCSK9 and guides its elimination via receptor-mediated endocytosis, thereby reducing plasma PCSK9 levels or eliminating PCSK9 circulating in plasma. Therefore, PCSK9 cannot bind to the low-density lipoprotein receptor (LDLR) or any other target receptor, resulting in the presence of more LDLR on the cell surface to remove LDL particles from the extracellular fluid. Thus, reducing plasma PCSK9 levels or eliminating PCSK9 circulating in plasma reduces blood LDL-particle concentration.

[0691] Therefore, the bifunctional compounds of formula (Ia) of the present invention are potentially useful for treating, preventing, improving, or delaying the progression of PCSK9-mediated diseases or disorders, or diseases or disorders in which PCSK9 functions, and for conditions, diseases, and disorders that benefit from reduced plasma PCSK9 levels or elimination of PCSK9 circulating in the plasma. Such diseases and disorders include those selected from: hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral vascular disease, peripheral artery disease, vascular inflammation, elevated Lp(a), elevated LDL, elevated TRL (e.g., elevated VLDL and / or chylomicrons), elevated triglycerides, sepsis, and xanthomas.

[0692] Furthermore, the bifunctional compounds of formula (Ia) of the present invention are therefore potentially useful for treating, preventing, improving, or delaying the progression of diseases or disorders requiring a reduction in plasma PCSK9 levels or the elimination of PCSK9 circulating in the plasma. Such diseases and disorders include those selected from: hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral vascular disease, peripheral artery disease, vascular inflammation, elevated Lp(a), elevated LDL, elevated TRL (e.g., elevated VLDL and / or chylomicrons), elevated triglycerides, sepsis, and xanthomas.

[0693] Another aspect of the present invention is a bifunctional molecule that utilizes receptor-mediated endocytosis to eliminate or reduce the level of complement factor H-related protein 3 (FHR3) in plasma.

[0694] Complement-mediated immune responses are tightly regulated by a number of endogenously produced proteins that modulate activity and distinguish between healthy, self-activated (non-activated) and damaged or non-self-activated pathogenic cells. These complement-controlling proteins range from cell-surface-bound (i.e., CR1, MCP, DAF) to circulating proteins (i.e., factor H and C4BP), which are recruited to the host surface to inactivate complement by binding to polysaccharides such as glycosaminoglycans (Mol Immuno [Molecular Immunology] 47(13):2187-2197). Complement regulation is strictly controlled to maintain homeostasis, and its dysregulation and defects lead to its targeting of host cells, which is associated with many diseases.

[0695] Factor H (FH) is the major negative regulator of alternative complement pathway activation and belongs to a family that also includes five other related family members believed to arise from non-allelic homologous recombination and interlocular gene transfer: complement factor H-associated protein 1 (FHR1), complement factor H-associated protein 2 (FHR2), complement factor H-associated protein 3 (FHR3), complement factor H-associated protein 4 (FHR4A and FHR4B) with isotypes 4A and 4B, and complement factor H-associated protein 5 (FHR5).

[0696] Because of the central role of factor H in complement regulation, abnormal FH activity raises many clinical implications. Loss-of-function mutations in factor H increase susceptibility to kidney disease, atypical hemolytic uremic syndrome (aHUS), and dense deposit disease (DDD), while polymorphisms of 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).

[0697] Unlike factor H, FHR3 lacks the complement regulatory domain necessary for complement inactivation and also competes with factor H, leading to complement overactivation. Therefore, this invention provides a bifunctional compound for use in regulating the concentration of complement factor H proteins, particularly FHR3, to remove competitors to factor H and thereby restore factor H-mediated regulation to treat disorders caused by excessive complement activation.

[0698] This invention also relates to bifunctional compounds and compositions capable of reducing plasma FHR3 levels or eliminating FHR3 circulating in plasma. This disclosure is characterized by methods for treating, preventing, or improving FHR3-related diseases or disorders by administering a therapeutically effective amount of a bifunctional compound of formula (Ib) to a patient in need. The methods of this invention can be used to treat a variety of FHR3-related diseases or disorders by reducing plasma FHR3 levels or eliminating FHR3 circulating in plasma. Reducing plasma FHR3 levels or eliminating FHR3 circulating in plasma provides novel methods for treating, preventing, or improving diseases including, but not limited to, kidney disease, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0699] The bifunctional compound of formula (Ib) of the present invention is practical for treating kidney disease, age-related macular degeneration, atypical hemolytic uremic syndrome and hepatocellular carcinoma (HCC) by reducing plasma FHR3 levels or eliminating FHR3 circulating in plasma.

[0700] For example, the bifunctional compounds of formula (Ib) of the present invention bind to FHR3 and guide its elimination via receptor-mediated endocytosis, thereby reducing plasma FHR3 levels or eliminating circulating FHR3 in plasma. Therefore, the bifunctional compounds of formula (Ib) of the present invention are potentially useful for treating, preventing, improving, or delaying the progression of complement-mediated diseases or disorders such as nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

[0701] Example

[0702] The invention is further described in the following examples, which are not intended to limit the scope of the invention as described in the claims.

[0703] Temperatures are given in degrees Celsius. Unless otherwise specified, all evaporation is carried out under reduced pressure, typically between approximately 15 mmHg and 100 mmHg (=20–133 mbar). The structures of the final products, intermediates, and starting materials are confirmed by standard analytical methods (e.g., trace analysis and spectroscopic characterization (e.g., MS, IR, NMR)). Abbreviations used are those commonly used in the art.

[0704] All starting materials, structural units, reagents, acids, bases, dehydrating agents, solvents, and catalysts used to synthesize the compounds of this invention are commercially available or can be produced by organic synthesis methods known to those skilled in the art or by organic synthesis methods as described herein.

[0705] The following examples and abbreviations used elsewhere in this article are:

[0706] AA: Amino acid

[0707] Ac: Acetyl group

[0708] Ac2O: Acetic anhydride

[0709] ACN: Acetonitrile

[0710] aq.: water-based

[0711] AM: Aminomethyl

[0712] Boc: tert-butoxycarbonyl

[0713] BnOH: Benzyl alcohol

[0714] BSA: Bovine serum albumin

[0715] DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene

[0716] DCM: Dichloromethane

[0717] DIC: N,N'-Diisopropylcarbodiimide

[0718] DTT: Dithiothreitol

[0719] DMA: Dimethylacetamide

[0720] DMAP: 4-Dimethylaminopyridine

[0721] DMF: N,N-dimethylformamide

[0722] DMSO: Dimethyl sulfoxide

[0723] DIEA or DIPEA: N,N-Diisopropylethylamine

[0724] EA: Ethyl acetate

[0725] EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0726] EDT: Ethylene dithiol

[0727] eq.: equivalent

[0728] ESI-MS: Electrospray ionization mass spectrometry

[0729] Et and EtOAc: Ethyl and ethyl acetate

[0730] Fmoc: fluorenyl methoxy carbonyl

[0731] FRET: Fluorescence Resonance Energy Transfer

[0732] HATU: O-(7-azobenzotriazol-1-yl)-1,1,3,3-tetramethylureonium hexafluorophosphate

[0733] HCTU: O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate

[0734] HEPES: 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid

[0735] HFIP: Hexafluoroisopropanol

[0736] HILIC: Hydrophilic Interaction Liquid Chromatography

[0737] HOAt: 1-Hydroxy-7-azabenzotriazole

[0738] HOBt: Hydroxybenzotriazole

[0739] HPLC: High-performance liquid chromatography

[0740] h, hr: hours

[0741] HRMS: High-resolution mass spectrometry

[0742] IC50: Half-maximal inhibitory concentration

[0743] LC and LCMS: Liquid Chromatography and Liquid Chromatography-Mass Spectrometry

[0744] LDLR: Low-density lipoprotein receptor

[0745] min: minutes

[0746] Me: Methyl

[0747] MS: Quality

[0748] m / z: Mass-to-charge ratio

[0749] M and mM: mole and millimole

[0750] mg: milligram

[0751] μL, mL, and L: microliters, milliliters, and liters

[0752] N: Equivalent in liters

[0753] NMP: N-methyl-2-pyrrolidone

[0754] Oxima pure: Potassium salt of 2-cyano-2-(oxime)ethyl acetate, Potassium salt of (oxime)cyanoethyl acetate

[0755] PBS: Phosphate-buffered saline

[0756] PD: Pharmacodynamics

[0757] PE: Petroleum ether

[0758] PG: Protecting group

[0759] PS: Polystyrene resin

[0760] PyOxim: [Ethylcyano(oxime)acetate-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate

[0761] Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl

[0762] PCSK9: Preprotein convertase subtilisin / kexin type 9

[0763] Ph: Phenyl

[0764] RP: Inverse

[0765] rpm: revolutions per minute

[0766] rt: room temperature

[0767] RU: Resonance Unit

[0768] SPPS: Solid-phase peptide synthesis

[0769] sat.: saturated

[0770] tBu: tert-butyl

[0771] TBAI: Tetrabutylammonium iodide

[0772] TBTU: 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate

[0773] TEA: Triethylamine

[0774] THF: Tetrahydrofuran

[0775] TentaGel TM S RAM resin: A polymer-bonded N-Fmoc-4'-[poly(oxyethylene)carbamoylmethoxy]-2,4-dimethoxy-diphenylmethylamine, and a polymer-bonded poly(oxyethylene)-RAM polymer.

[0776] TFA: Trifluoroacetic acid

[0777] THPTA: Trihydroxypropyltriazolylmethylamine

[0778] TIS: Triisopropylsilane

[0779] TLC: Thin-layer chromatography

[0780] TMSOTF or TMSOTf: Trimethylsilyl trifluoromethanesulfonate

[0781] t r Retention time

[0782] TR: Time resolution

[0783] TMSCl: Trimethylchlorosilane

[0784] Trt: Triphenylmethyl

[0785] TsOH:

[0786] UPLC: Ultra-high performance liquid chromatography

[0787] UV: Ultraviolet light

[0788] wt: weight

[0789] Analytical methods, materials and instruments

[0790] Unless otherwise specified, use reagents and solvents obtained from commercial suppliers. Proton nuclear magnetic resonance (NMR) spectra were obtained using a Varian spectrometer at 400 MHz, or 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. 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 unless otherwise specified. Column: Synergi 4 μm, 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 to 95% B at 3 min to 95% B at 3.3 min.

[0791] General preparative HPLC purification procedures and mass spectrometry

[0792] Depending on the amount of crude peptide to be purified, the crude peptide was purified by preparative reversed-phase C18-HPLC using columns of different sizes and varying flow rates. For example, 0.1% TFA (A) in water and 0.1% TFA (B) in acetonitrile were used as eluents. Fractions containing the product were collected and lyophilized to obtain the purified product.

[0793] Condition E-1 (LCMS) -Column: Acquity BEH C18, 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 min; electrospray mass spectrometry (+), DAD-UV chromatogram 214 nm.

[0794] Analysis Method 1

[0795] Agilent 1100 / 1200ALS system / Waters ZQD MS system

[0796] Eluent A: 0.05% trifluoroacetic acid in H2O

[0797] Eluent B: Acetonitrile

[0798] Column temperature: 40℃

[0799] Flow rate: 2.0 mL / min

[0800] Column: SunFire C18, 3.5μm, 3.0×30mm

[0801] gradient:

[0802]

[0803] Analysis Method 2

[0804] Waters Acquity UPLC system / Waters SQD MS system

[0805] Eluent A: 5mM ammonium hydroxide in H2O

[0806] Eluent B: 5mM ammonium hydroxide in acetonitrile

[0807] Column temperature: 50℃

[0808] Flow rate: 1.0 mL / min

[0809] Column: Acquity UPLC BEH C18, 1.7μm, 2.1×50mm

[0810] gradient:

[0811]

[0812] Analysis Method 3

[0813] Waters Acquity UPLC system / Waters Xevo G2 Qtof MS system

[0814] Eluent A: 0.1% formic acid in H2O

[0815] Eluent B: 0.1% formic acid in acetonitrile

[0816] Column temperature: 50℃

[0817] Flow rate: 1.0 mL / min

[0818] Column: Acquity UPLC BEH C18, 1.7μm, 2.1×50mm

[0819] gradient:

[0820]

[0821] Analysis Method 5

[0822] Waters Acquity UPLC system / Waters SQD MS system

[0823] Eluent A: 5mM ammonium hydroxide in H2O

[0824] Eluent B: 5mM ammonium hydroxide in acetonitrile

[0825] Column temperature: 50℃

[0826] Flow rate: 1.0 mL / min

[0827] Column: Acquity UPLC BEH C18, 1.7μm, 2.1×30mm

[0828] gradient:

[0829]

[0830] Analysis Method 7

[0831] Waters Acquity UPLC system / Waters SQD MS system

[0832] Eluent A: 0.1% formic acid in H2O

[0833] Eluent B: 0.1% formic acid in acetonitrile

[0834] Column temperature: 50℃

[0835] Flow rate: 1.0 mL / min

[0836] Column: Acquity UPLC BEH C18, 1.7μm, 2.1×30mm

[0837] gradient:

[0838]

[0839]

[0840] Analysis Method 9

[0841] Waters Acquity UPLC / Waters QTof MS System

[0842] Eluent A: 0.05% trifluoroacetic acid in H2O

[0843] Eluent B: 0.04% trifluoroacetic acid in acetonitrile

[0844] Column temperature: 80℃

[0845] Flow rate: 0.5 mL / min

[0846] Column: Acquity UPLC CSH C18, 1.7μm, 2.1mm×100mm

[0847] gradient:

[0848]

[0849] Analysis Method 10

[0850] Waters Acquity UPLC / SQD MS System

[0851] Eluent A: 0.05% formic acid and 3.75mM ammonium acetate in H2O

[0852] Eluent B: 0.04% formic acid in acetonitrile

[0853] Column temperature: 60℃

[0854] Flow rate: 1.0 mL / min

[0855] Column: Acquity UPLC HSS T3, 1.8μm, 2.1mm×50mm

[0856] gradient:

[0857]

[0858] Analysis Method 11

[0859] Waters Acquity UPLC / SQD MS System

[0860] Eluent A: 0.05% formic acid and 3.75mM ammonium acetate in H2O

[0861] Eluent B: 0.04% formic acid in acetonitrile

[0862] Column temperature: 60℃

[0863] Flow rate: 1.0 mL / min

[0864] Column: Acquity UPLC HSS T3, 1.8μm, 2.1mm×50mm

[0865] gradient:

[0866]

[0867] Analysis Method 12

[0868] Waters Acquity UPLC / SQD MS System

[0869] Eluent A: 0.05% formic acid and 3.75mM ammonium acetate in H2O

[0870] Eluent B: 0.04% formic acid in acetonitrile

[0871] Column temperature: 60℃

[0872] Flow rate: 1.0 mL / min

[0873] Column: Acquity UPLC HSS T3, 1.8μm, 2.1mm×50mm

[0874] gradient:

[0875]

[0876] General procedure for cyclic peptide synthesis:

[0877] The following general procedures can be used to obtain cyclic peptides, such as compounds (C1) to (C4).

[0878]

[0879] Step 1: Peptide Synthesis

[0880] From CEM Corporation (CEM, Inc.) Solid-phase peptide synthesis on a peptide synthesizer

[0881] Synthesis Cycle A-1

[0882] The resin was washed with DMF and then deprotected in two cycles by treatment with 4-methylpiperidine / DMF (1:4) – 30 seconds for the first cycle and 3 minutes for the second. Coupling was completed 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 peptide was obtained. All Fmoc amino acids, except those shown in Table 3 below, were coupled at 75 °C for 5 min. After final coupling, Fmoc was removed by repeated treatment with 4-methylpiperidine / DMA (1:4) to obtain the deprotected peptide.

[0883] Table 3: AA Coupling Conditions for Synthesizing Cycle A-1

[0884]

[0885] From Gyros Protein Technologies AB Solid-phase peptide synthesis on a peptide synthesizer .

[0886] Alternatively, as described in synthetic cycle B-1 or synthetic cycle B-2, in Peptides are synthesized on a peptide synthesizer.

[0887] Synthetic cycle B-1

[0888] The resin was washed with DMA. Fmoc was then removed by repeated treatment of the resin with piperidine / DMA (1:4). Coupling was completed by adding Fmoc-amino acids (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), followed by mixing the suspension with nitrogen at room temperature for typically 15 min to 4 h, depending on the specific requirements. The coupling step was repeated after washing with DMA. After washing with DMA, the suspension was capped by adding a mixture of Ac₂O / pyridine / DMA (1:1:8) followed by mixing at room temperature. After final coupling, Fmoc was removed in synthesis cycle A-1 as described above to obtain the peptide.

[0889] Synthetic cycle B-2

[0890] The resin was washed with DMA. Fmoc was removed by repeated treatment with 4-methylpiperidine / DMA (1:4). Coupling was completed 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 the suspension with nitrogen at room temperature for 15 min to 4 h as required. After washing with DMA, the coupling step was repeated. After washing with DMA, the suspension was capped by adding a mixture of Ac2O / pyridine / DMA (1:1:8) followed by mixing at room temperature. After final coupling, Fmoc was removed in synthesis cycle A-1 as described above to obtain the peptide.

[0891] Step 2: Peptide acylation

[0892] The resin product obtained from step 1 was suspended in N-methylpyrrolidine, and N-succinimide-2-chloroacetate (5 equivalents) was added. The resulting resin mixture was shaken overnight at room temperature. The resin was then filtered and washed three times each with dimethylformamide and dichloromethane to obtain the acylated peptide product.

[0893] Step 3: Cutting from the resin with or without removal of the protecting group (PG).

[0894] The resin product obtained from step 2 is shaken together with one of the cleavage solutions listed below (1-5 mL, 0.1 mmol scale) for 1-3 h. The resin is filtered and treated again with fresh cleavage solution for 0.5-1.5 h. The cleavage cycle is repeated as needed. The resin is then filtered, and the combined filtrates are slowly poured into a mixture of cold heptane / diethyl ether (1:1) to obtain a precipitate. The suspension containing the precipitate is centrifuged and the supernatant is discarded. The precipitate is resuspended in cold diethyl ether, briefly vortexed, and then centrifuged. This washing process is repeated twice more. The crude peptide product is dried under high vacuum.

[0895] Use the following cutting solution:

[0896] Cutting method 1: TFA / H2O / TIS / DTT (92.5:2.5:2.5:2.5)

[0897] Cutting method 2: 95% TFA / EDT / TIS aqueous solution (95:2.5:2.5)

[0898] Step 4: Peptide cyclization

[0899] The crude peptide product obtained from step 3 was dissolved in DMSO or DMA and treated with TEA or DIPEA. The reaction mixture was then shaken overnight at room temperature. The resulting reaction mixture containing the cyclic peptide was concentrated on a centrifugal evaporator to obtain the desired cyclic peptide.

[0900] Example 1: Synthesis of PCSK9 receptor ligand compounds (C1) to (C11)

[0901] 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 Synthesis of 35-isopropyl-6,12,13,18,19-pentamethyl-5,8,11,14,17,20,23,26,29,34,37,40,46-tetraoxotetratetrahydro-5H-dipyrrolo[2,1-f:2',1'-g1][1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40]tetrazazoletetradodecene-9-yl)propionic acid (C1)

[0902]

[0903] Note: Compound (C1) has the following amino acid sequence:

[0904]

[0905]

[0906] Step 1: Peptide sequence FVPTTB-(N-Me)AB-(N-Me)AEAPC(Trt)-GK-NH- resin (1-1b) in Fmoc-RAM TentaGel TM On resin (1-1a, 0.22 mmol / g loading, 0.25 mmol scale) at The peptide was synthesized on a peptide synthesizer according to a general peptide synthesis cycle A-1 (Fmoc-amino acids (4 equivalents; 0.2M solution in DMF), HATU (4 equivalents; 0.5M solution in DMF), and DIPEA (4.4 equivalents; 2M solution in NMP)). The resin was then filtered and washed with DMF (2x) and DCM (3x) to obtain FVPTTB-(N-Me)AB-(N-Me)AEAPC(Trt)-GK-NH resin (1-1b).

[0907] Step 2: A solution of N-succinimide-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. The resin was then drained, washed with DMF (3x) and DCM (4x), and dried to obtain tClCH2C(=O)-FVPTTB-(N-Me)AB-(N-Me)AEAPC(Trt)-GK-NH resin (1-1d).

[0908] Step 3: The peptide resin product 1-1d from Step 2 was cleaved from the resin and simultaneously deprotected using cleavage method 1 described above, yielding the crude peptide ClCH2C(O)-FVPTTB-(N-Me)AB-(N-Me)AEAPCGK-NH2(1-1e) (266 mg). Analytical Method 1: t R =1.22min; M+H+2 / 2 921.8.

[0909] Step 4: Dissolve the crude peptide 1-1e (460 mg, 0.25 mmol) from Step 3 in DMSO (25.4 mL). Add a few drops of TEA to adjust to pH 8-9. Stir the resulting mixture overnight at room temperature. Then concentrate the reaction mixture to a few mL of DMSO using a centrifugal evaporator. Analyze the crude cyclic peptide by preparative HPLC (Sunfire). TM Prep C18 column, 5 μm, 30 × 50 mm, 15%-40% within 6 min, 75 mL / min, in water with ACN (containing 0.1% TFA) purification 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-oxo (1-(R)-1-hydroxyethyl)-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-tetraoxotetratetrahydro-5H-dipyrrolo[2,1-f:2',1'-g1][1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40]tetrazazoletetradodecenoen-9-yl)propionic acid (C1) (SEQ ID NO:1). Analytical Method 3: t R =0.45min, M+2 / 2 903.7

[0910] Example 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(hydroxyethyl) Synthesis of (methyl)-12-isopropyl-26,27,32,33-tetramethyl-1,7,10,13,16,19,22,25,28,31,34,37,40-tetrazazoletetradodecanoyl-6H-pyrrolo[2,1-f][1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40]tetrazazoletetradodecenoen-15-yl)acetic acid (C2)

[0911]

[0912] Note: Compound (C2) has the following amino acid sequence:

[0913]

[0914] Step 1: Peptide sequence FVDTTS-(N-Me)AB-(N-Me)AKSPC(Trt)-G-NH- resin (1-2b) in Fmoc-Gly-RAM TentaGel TM On resin (1-2a, 0.22 mmol / g loading, 0.25 mmol scale) on The peptide was synthesized on a peptide synthesizer according to a general peptide synthesis cycle A-1 (Fmoc-amino acids (4 equivalents; 0.2M solution in DMF), HATU (4 equivalents; 0.5M solution in DMF), and DIPEA (4.4 equivalents; 2M solution in NMP)). The resin was then filtered and washed with DMF (2x) and DCM (3x) to obtain FVDTTS-(N-Me)AB-(N-Me)AKSPC(Trt)-G-NH resin (1-2b).

[0915] Step 2: A solution of N-succinimide-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. The resin was then drained, washed with DMF (3x) and DCM (4x), and dried to obtain ClCH2C(=O)-FVDTTS-(N-Me)AB-(N-Me)AKSPC(Trt)-G-NH resin (1-2d).

[0916] Step 3: Cut the peptide resin product 1-2d from Step 2 from the resin and simultaneously deprotect it using the cutting method 1 described above to obtain the crude peptide ClCH2C(O)-FVDTTS-(N-Me)AB-(N-Me)AKSPCG-NH2(1-2e) (266mg).

[0917] Step 4: Dissolve the crude peptide 1-2e (266 mg) from Step 3 in DMSO (20.5 mL). Add a few drops of TEA to adjust to pH 8-9. Stir the resulting mixture overnight at room temperature. Then concentrate the reaction mixture into a few mL of DMSO using a centrifugal evaporator. Analyze the crude cyclic peptide by preparative HPLC (Sunfire). TM Prep C18 column, 5 μm, 30 × 50 mm, 15%-40% in 6 min, 75 mL / min, in water with ACN (containing 0.1% TFA) purification 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-tetrazonetetradodecylhydro-6H-pyrrolo[2,1-f][1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40]tetrazonetetradodecyl-15-yl)acetic acid (C2) (SEQ ID NO:2). Analytical Method 9: t R =7.88, m+1=1573.8; (m+2) / 2=787.3.

[0918] The cyclic peptide compounds (C3) and (C4) in Table 4 below were obtained using a similar method as described in Examples 1-2; however, instead of the peptide sequences (1-2b) synthesized in step 1, the corresponding peptide sequences of the cyclic peptide compounds (C3) and (C4) synthesized in step 1 are also given in Table 4.

[0919] Table 4

[0920]

[0921]

[0922] The analytical data for cyclic peptide compounds (C3) and (C4) are summarized in Table 5 below and were obtained using analytical method E-1 described herein.

[0923] Table 5

[0924]

[0925] 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-oxopentamido)hex-2-yl)amino)-2-oxoethyl)carbamoyl)-38-benzyl-24,27-bis((R)-1 Synthesis of (-hydroxyethyl)-35-isopropyl-6,12,13,18,19-pentamethyl-5,8,11,14,17,20,23,26,29,34,37,40,46-tridecoxotetratetrahydro-5H-dipyrrolo[2,1-f:2',1'-g1][1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40]tetrazazoletetradodecenoen-9-yl)propionic acid (C5)

[0926]

[0927] A solution of cyclic peptide (C1) (75 mg, 0.039 mmol) in DMSO (1 mL) at room temperature was supplemented with DIPEA (0.020 mL, 0.117 mmol) and 2,5-dioxopyrrolidone-1-yl ester of 4-oxovaleric acid (0.130 mL, 0.117 mmol). The reaction was purified directly by C18 rapid chromatography (30 g column, 0%-80% ACN / water over 15 min), and the purified fraction was dried over Genevac to obtain cyclic peptide (C5). Analytical Method 7: t R =1.07min, M-1=951.3

[0928] Synthesis of intermediates

[0929] Synthesis of (R)-2-benzyl-4-(tert-butoxy)-4-oxobutyric acid (int-A1)

[0930]

[0931] Step 1. Over a 30-minute period, n-BuLi (2.5 M in hexane) (1.24 L, 3.103 mol) was slowly added to a cold-stirred solution of (S)-4-benzyloxazolidin-2-one (500 g, 2.821 mol) in THF (9 L) at -78 °C, and the resulting mixture was stirred at -78 °C for 30 minutes. Then, over a 1-hour period, a solution of 3-phenylpropionyl chloride (571 g, 3.38 mol) in THF (1 L) was slowly added, and the reaction mixture was slowly heated 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-phenylpropionyl)oxazolidin-2-one (int-A1-1) was ground with petroleum ether (5 L) for 1 h. The solid product was filtered, washed with petroleum ether (500 mL), and dried under vacuum to obtain (S)-4-benzyl-3-(3-phenylpropionyl)oxazolidin-2-one (int-A1-1). Analytical Method 7; t R = 1.53 min; [M+H] + =310.2.

[0932] Step 2. Over a 30-minute period, slowly add 1.0 M NaHMDS in THF (1.94 L, 1.939 mol) to a cold-stirred solution of (S)-4-benzyl-3-(3-phenylpropionyl)oxazolidin-2-one (int-A1-1) (500 g, 1.616 mol) in THF (7 L) at -78 °C. Stir the resulting mixture at -78 °C for 1 h, then add dropwise over a 30-minute period of tert-butyl 2-bromoacetate (472.8 g, 2.424 mol) in THF (500 mL) at -78 °C. Stir the mixture for 2 h, then quench with saturated NH4Cl (500 mL) and extract with ethyl acetate (2 × 1.5 L). Wash the combined organic layers with brine (2 L), dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure. The crude material was ground with methanol (800 mL) for 1 h. The solid product was then filtered, washed with methanol (200 mL), and dried under vacuum to obtain (R)-3-benzyl-4-((S)-4-benzyl-2-oxooxazolidine-3-yl)-4-oxobutyric acid tert-butyl ester (int-A1-2). Analytical Method 7; t R = 1.78 min; [M-tBu] + =368.3.

[0933] Step 3. Add 30% H₂O₂ (267 mL, 2.37 mol) to a cold-stirred solution of (R)-3-benzyl-4-((S)-4-benzyl-2-oxooxazolidine-3-yl)-4-oxobutyrate tert-butyl ester (int-A1-2) (250 g, 0.59 mol) in THF (9 L) at 0–5 °C and stir the reaction at the same temperature for 30 min. Then add a solution of LiOH·H₂O (49.5 g, 1.18 mol) in water (3 L) to the above reaction mixture at 0–5 °C and stir the mixture for 1 h. Quench the reaction mixture with saturated sodium sulfite (1.6 L) and saturated sodium bicarbonate (1.6 L). Then concentrate the solvent under reduced pressure, dilute with water (3 L), and wash with DCM (2 × 1 L) to remove impurities. Then cool the aqueous layer to 5 °C and acidify with 6 M HCl (1 L) to pH approximately 1.5. The product was extracted with ethyl acetate (3 × 1 L). The combined organic layers were washed with a brine solution (1 L), dried over anhydrous sodium sulfate, and concentrated under vacuum to give (R)-2-benzyl-4-(tert-butoxy)-4-oxobutyric acid (int-Al). Analytical Method 7; t R = 1.78 min; [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).

[0934] Synthesis of ((1S,2S)-2-(methylamino)cyclohexyl)carbamate tert-butyl ester (int-B1)

[0935]

[0936] Step 1. 4-Nitrobenzenesulfonyl chloride (23.29 g, 105 mmol) was added to a solution of (1S,2S)-(+)-1,2-diaminocyclohexane (12 g, 105 mmol) and triethylamine (21.97 mL, 158 mmol) in DCM (200 mL) at 0 °C and stirred for 30 min at the same temperature. The reaction was slowly heated to room temperature and stirred for 16 h. The progress of the reaction was monitored by TLC (80% ethyl acetate in petroleum ether). The reaction mixture was concentrated and diluted with water, and a solid precipitated. The precipitate was filtered, washed with excess water and EtOAc, and dried under vacuum to give N-((1S,2S)-2-aminocyclohexyl)-4-nitrobenzenesulfonamide (int-B1-1). Analytical Method 7; t R = 0.74 min; [M+H]+ =300.2. 1 HNMR (300MHz, CDCl3): δ8.33-8.31(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).

[0937] Step 2. Boc-anhydride (13.70 mL, 59.0 mmol) was added to N-((1S,2S)-2-aminocyclohexyl)-4-nitrobenzenesulfonamide (int-B1-1) (17.66 g, 59.0 mmol) in a stirred solution in DCM (200 mL) and stirred for 3 h. The reaction progress was monitored by TLC (50% ethyl acetate in petroleum ether). The reaction mixture was concentrated under reduced pressure to give tert-butyl ((1S,2S)-2-((4-nitrophenyl)sulfonylamino)cyclohexyl)carbamate (int-B1-2). Analytical Method 7; t R = 1.14 min; [M-Boc+H] + =299.9. 1 HNMR (400MHz, CDCl3): δ8.33-8.31(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 ),3.35-3.32(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).

[0938] Step 3. Methyl iodine (18.19 mL, 294 mmol) was added to a stirred solution of ((1S,2S)-2-((4-nitrophenyl)sulfonylamino)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 stirred at the same temperature for 3 h. The reaction progress 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 give the crude compound. The crude compound was purified by normal-phase chromatography using a silica gel (100-200 mesh) column, eluted with 0-30% ethyl acetate in petroleum ether, to give ((1S,2S)-2-((N-methyl-4-nitrophenyl)sulfonylamino)cyclohexyl)tert-butyl carbamate (int-B1-3). Analytical Method 7; t R = 1.22 min; [M-Boc] + =313.9. 1 HNMR (300MHz, CDCl3): δ8.36-8.35(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).

[0939] Step 4. The mixture of ((1S,2S)-2-((N-methyl-4-nitrophenyl)sulfonamide)cyclohexyl)tert-butyl carbamate (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 mixture of DMF:MeOH (1:1, 12 mL) was stirred at room temperature for 1 h. The reaction progress was monitored by TLC (10% MeOH in DCM). 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 give the crude product. The crude product was purified by normal-phase chromatography using silica gel (100-200 mesh) column chromatography, eluting with 0-5% MeOH in DCM to obtain tert-butyl ((1S,2S)-2-(methylamino)cyclohexyl)carbamate (int-B1). Analytical Method 7; t R =0.75min; [M+H] + =229.3. 1 H NMR (300MHz, DMSO-d6): δ6.26-6.18(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).

[0940] Synthesis of (S)-N-(2-(methylamino)propyl)-4-nitrobenzenesulfonamide (int-B2)

[0941]

[0942] Step 1. DIEA (3179.0 g, 24.6 mol) was added to a stirred solution of N-Me-Boc-Ala-OH (1000.0 g, 4.92 mol) in THF (6 L) at 0 °C. After 5 min, HATU (2058.0 g, 294 mmol) was added in one go, and stirring was continued for 15 min at the same temperature. Then, solid ammonium chloride (1316.0 g, 24.6 mol) was added, and the mixture was stirred overnight at room temperature. A large amount of precipitate formed, which was filtered through a disposable sieve (frit) and washed several times with THF until most of the solid dissolved. The filtrate was evaporated under reduced pressure to obtain the crude product. The crude product was diluted with water (2 L) and extracted with petroleum ether (2 × 2.5 L) to remove nonpolar impurities. The aqueous portion was then extracted several times with 40% EtOAc in petroleum ether. Then all organic parts were combined, dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure to obtain (S)-(1-amino-1-oxopropane-2-yl)(methyl)carbamate tert-butyl ester (int-B2-1). 1 H NMR(400MHz,DMSO-d6)δppm 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).

[0943] Step 2. Under argon atmosphere, add NaBH4 (56.4 g, 1.48 mol) to a stirred solution of (S)-(1-amino-1-oxopropane-2-yl)(methyl)carbamate tert-butyl (int-B2-1) (100 g, 0.49 mol) in 1,4-dioxane (500 mL). Add acetic acid (90 mL) in 200 mL of dioxane dropwise to the mixture, maintaining gentle bubbling with a bubbler fitted with a vent. After complete acid addition, attach a reflux condenser and heat the mixture to 110 °C for 4 h. Remove the mixture while hot and allow it to reach room temperature. Quench the reactants with ice and acidify with 2N HCl. Extract the solution with EtOAc and alkalize the aqueous portion to pH 13-14 with 50% NaOH. The solution was then extracted with diethyl ether, dried over anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure to obtain (S)-(1-aminopropyl-2-yl)(methyl)carbamate tert-butyl ester (int-B2-2). The crude compound was used in the next step without purification.

[0944] Step 3. At 0°C, NaHCO3 (406.9 g, 4.78 mol) was added to a stirred solution of crude (S)-(1-aminopropyl-2-yl)(methyl)carbamate (int-B2-2) (300.0 g, 1.59 mol) in 1500 mL of ACN, followed by the addition of p-nitrobenzenesulfonyl chloride (388.9 g, 1.75 mol). The mixture was stirred at room temperature for 2 h. The reaction solution was slowly quenched with ice water (2000 mL) and stirred for 2 h until a white precipitate formed. The solid was filtered, washed with water and petroleum ether, and dried under vacuum to give (S)-methyl(1-((4-nitrophenyl)sulfonylamino)propyl-2-yl)carbamate (int-B2-3). The crude compound was used directly in the next step without purification.

[0945] Step 4. HCl (4M in 2000 mL, 1.2 mol) was added to a stirred solution of crude (S)-methyl(1-((4-nitrophenyl)sulfonylamino)propyl-2-yl)carbamate tert-butyl (int-B2-3) (450.0 g, 1.2 mol) in 1,4-dioxane (2000 mL), and the reaction mixture was stirred at room temperature for 4 h. A solid precipitated during the reaction was filtered, washed with diethyl ether, and dried under vacuum to give an amine as an HCl salt. The crude solid was washed several times with n-pentane and dried to give (S)-N-(2-(methylamino)propyl)-4-nitrobenzenesulfonamide (int-B2). Analytical Method 7; t R = 0.69 min; [M+H] + =274.2. 1 H NMR (400MHz, DMSO-d6) δppm1.18 (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).

[0946] Synthesis of (S)-(2-(methylamino)-6-((2-nitrophenyl)sulfonylamino)hexyl)carbamate tert-butyl ester (int-B3)

[0947]

[0948] Step 1. Me3SiCl (3.83 mL, 30.0 mmol) and DIEA (8.73 mL, 50.0 mmol) were added to a suspension of Fmoc-Lys-OH HCl (4.05 g, 10 mmol) in DCM (80 mL) at 0 °C, and the resulting mixture was stirred at 0 °C for 20 min 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 min, then concentrated to dryness under vacuum. The resulting residue was partitioned between EtOAc (150 mL) and 5% KHSO4 aqueous solution (50 mL). The organic layer was washed with 5% KHSO4 aqueous solution (3 × 50 mL) and brine (50 mL), dried over Na2SO4, filtered, and concentrated to dryness under vacuum to obtain N. 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 6 -((2-nitrophenyl)sulfonyl)-L-lysine (int-B3-1). The crude product was used directly in the next step without purification. Analytical Method 10; t R = 1.08 min; [M+NH4] + =571.3.

[0949] Step 2. At 0℃, add N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 6 DIEA (6.76 mL, 38.7 mmol) was added to 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) in DMF (80 mL). The resulting suspension was stirred at 0 °C for 5 min, and then TBTU (3.42 g, 10.65 mmol) was added. After stirring at 0 °C for 1 h, the reaction mixture was partitioned between EtOAc (250 mL) and 5% NaHCO3 aqueous solution (100 mL). The organic layer was washed with 5% NaHCO3 aqueous solution (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)sulfonylamino)-1-oxohexane-2-yl)carbamate (9H-fluorene-9-yl)methyl ester (int-B3-2). The crude product was used in the next step without purification. Analytical Method 19; t R = 1.04 min; [M+H] + =553.3.

[0950] Step 3-1: Add BH3-S(CH3)2 (5.50 mL, 58.0 mmol) to (S)-(1-amino-6-((2-nitrophenyl)sulfonylamino)-1-oxohexane-2-yl)carbamate (9H-fluorene-9-yl)methyl ester (int-B3-2) (9.66 mmol) dissolved in THF (60 mL) and stir the resulting mixture at room temperature for 2 h 15 min, then at 50 °C for 6.5 h. Allow the reaction mixture to cool to room temperature.

[0951] Step 3-2: Add H2O (1 mL) and 6M HCl aqueous solution (2 mL) and stir the reaction mixture at room temperature for 14.5 h.

[0952] Step 3-3: Add 0.5M Na2CO3 aqueous solution (48.3 mL, 24.15 mmol) and Boc2O (2.243 mL, 9.66 mmol) to a THF solution (20 mL). Stir the resulting mixture at room temperature for 2 h, quench it with 8 M MeNH2 in EtOH (1 mL), and stir at room temperature for 30 min.

[0953] Steps 3-4: Add 4M NaOH aqueous solution (9.66 mL, 38.6 mmol) and stir the reaction mixture at room temperature for 85 min. Then add 4-methylpiperidine (4 mL) and stir the reaction mixture at room temperature for 40 min. Add another 4-methylpiperidine (8 mL) and continue stirring at room temperature for 30 min. Then add MeOH (10 mL) and stir at room temperature for 25 min. Add another MeOH (20 mL) and 4-methylpiperidine (10 mL) and stir for 30 min. Then concentrate the reaction mixture under vacuum and purify the crude product by rapid silica gel chromatography (eluent A: EtOAc / DIEA (98:2), eluent B: EtOAc / MeOH / DIEA (95:5:2)). Combine the pure fractions and concentrate to dryness under vacuum to give (S)-(2-amino-6-((2-nitrophenyl)sulfonylamino)hexyl)carbamate tert-butyl ester (int-B3-3). Analytical Method 10; t R = 0.69 min; [M+H] + =417.2.

[0954] 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 min, and then added to a solution of (S)-(2-amino-6-((2-nitrophenyl)sulfonylamino)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 min, and then concentrated to dryness under vacuum. The resulting residue EtOAc (80 mL) and 5% NaHCO₃ aqueous solution (10 mL) were partitioned between the two solutions. The organic layer was washed with 5% NaHCO3 aqueous solution (4 × 10 mL) and brine (10 mL), dried over Na2SO4, filtered, and concentrated under vacuum to dryness to give (S)-(2-formamido-6-((2-nitrophenyl)sulfonylamino)hexyl)carbamate tert-butyl ester (int-B3-4) (1.217 g, 2.74 mmol, 86% yield, as a yellow foam). The crude product was used in the next step without purification. Analytical method 10; t R = 0.87 min; [M+H] + =445.2.

[0955] Step 5-1: Add BH3-S(CH3)2 (1.300 mL, 13.69 mmol) to (S)-(2-formamido-6-((2-nitrophenyl)sulfonylamino)hexyl)carbamate tert-butyl ester (int-B3-4) (1.217 g, 2.74 mmol) dissolved in THF (20 mL) and stir the resulting mixture at room temperature for 3 h 40 min.

[0956] Step 5-2: Quench the reaction by adding MeOH (2 mL) and stir the resulting solution at room temperature for 125 min. Add MeOH (3 mL) and continue stirring at 60 °C for 75 min. Then concentrate the reaction mixture to dryness under vacuum.

[0957] 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 h. Another 10% Pd / C (0.087 g, 0.082 mmol) in H2O (1 mL) was added and stirring was continued at 60 °C for 2.5 h. The reaction mixture was filtered through a Hyflo filter (CAS No.: 61790-53-2), and the filtrate was concentrated to dryness under vacuum to give (S)-(2-(methylamino)-6-((2-nitrophenyl)sulfonylamino)hexyl)carbamate tert-butyl ester (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.

[0958] Synthesis of (S)-3-((((9H-fluorene-9-yl)methoxy)carbonyl)(methyl)amino)-4-(4-chlorophenyl)butyric acid (int-C1)

[0959]

[0960] Step 1. Sodium hydride (212 g, 5.74 mol, 65%) was added dropwise to a 20 L 4-necked round-bottom flask (purged and maintained under an inert nitrogen atmosphere) containing (3S)-3-[[(tert-butoxy)carbonyl]amino]-4-(4-chlorophenyl)butyric acid (360 g, 1.15 mol) in THF (8 L) at 0 °C, and the resulting mixture was stirred at 0 °C for 1 h. Then, MeI (1633 g, 11.5 mol) was added dropwise with stirring at 0 °C, and the resulting solution was stirred at 35 °C for 4 h. The reaction mixture was then quenched at -10 °C by adding 300 g of water / ice, concentrated under vacuum, and then 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 give (S)-3-((tert-butoxycarbonyl)(methyl)amino)-4-(4-chlorophenyl)butyric acid (int-C1-1). Analytical Method 7; t R = 1.01 min; [M+H] + =328.1.

[0961] Step 2. TFA (990.8 g, 8.77 mol) was added dropwise to a 5 L 4-necked round-bottom flask (purged and maintained under an inert nitrogen atmosphere) containing a solution of (S)-3-((tert-butoxycarbonyl)(methyl)amino)-4-(4-chlorophenyl)butyric acid (int-C1-1) (284.2 g, 866.98 mmol) in DCM (3 L), 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)butyric acid (int-C1-2). Analytical Method 1; t R = 0.66 min; [M+H] + =228.2.

[0962] Step 3. Sodium carbonate (249.1 g, 2.35 mol) was added in portions to a 5 L 4-necked round-bottom flask (purged and maintained under an inert nitrogen atmosphere) containing a solution of (S)-4-(4-chlorophenyl)-3-(methylamino)butyric acid trifluoroacetate (int-C1-2) (320 g crude) in dioxane:H2O (5:1) (3.6 L). Then, Fmoc-Cl (242 g, 935.45 mmol) was added in portions 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 phases were 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 give (S)-3-((((9H-fluorene-9-yl)methoxy)carbonyl)(methyl)amino)-4-(4-chlorophenyl)butyric acid (int-C1). Analytical Method 1; t R = 1.60 min; [M+H] + =450.3. 1 H NMR (300MHz, DMSO-d6, ppm): δ12.09-12.45(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).

[0963] Synthesis of (R)-3-amino-3-(4-chlorobenzyl)piperidine-1-carboxylic acid tert-butyl ester (int-C2)

[0964]

[0965] Step 1-1: Diphenylphosphoazide (5.48 mL, 25.4 mmol) was added to 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), and the reaction was stirred at room temperature for 2.5 h, then stirred at 100 °C for 4 h. The reaction mixture was partitioned between EtOAc (300 mL) and 5% NaHCO3 aqueous solution (60 mL). The organic phase was washed with 5% NaHCO3 aqueous solution (3 × 60 mL) and brine (50 mL), dried over Na2SO4, filtered, and concentrated to dryness under vacuum.

[0966] Steps 1-2: Add 1M NaOH (195 mL, 195 mmol) to the residue from Step 1-1 dissolved in dioxane (200 mL). Stir the resulting mixture at room temperature for 1 h, then concentrate to dryness under vacuum. Partition the resulting residue between EtOAc (250 mL) and 5% Na2CO3 aqueous solution (20 mL), and extract the aqueous phase with EtOAc (70 mL). Wash the combined organic phases with 5% Na2CO3 aqueous solution (40 mL) and brine (40 mL), dry to Na2SO4, filter, and concentrate to dryness under vacuum to give the 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 min; [M+H] + =325.2.

[0967] Step 2. The racemic tert-butyl 3-amino-3-(4-chlorobenzyl)piperidine-1-carboxylate (19.5 mmol) was separated by a preparative SFC (instrument: Thar 200 preparative SFC) under the following conditions: column: ChiralPak AD, 300 × 50 mm I.D., 10 μm; eluent A: CO2; eluent 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 min; the compound was dissolved in approximately 130 mL MeOH; injection: 10 mL per injection. The structure of (R)-3-amino-3-(4-chlorobenzyl)piperidine-1-carboxylate (int-C2) (slower elution isomer) and partial crystallization occurring during storage were allowed for structural confirmation by X-ray crystallography. Analytical Method 10; t R =0.77min; [M+H] + =325.3.

[0968] Synthesis of (R)-3-(4-chlorobenzyl)piperidine-3-amine hydrochloride (int-C3)

[0969]

[0970] (R)-3-amino-3-(4-chlorobenzyl)piperidine-1-carboxylic acid tert-butyl ester (int-C2) (2.09 g, 6.43 mmol) was dissolved in dioxane (10 mL). 4M HCl was added to dioxane (50 mL) and H2O (5 mL), and the solution was stirred at room temperature for 4 h. The reaction mixture was concentrated to dryness under vacuum to give (R)-3-(4-chlorobenzyl)piperidine-3-amine hydrochloride (int-C3). Analytical Method 10; t R = 0.40 min; [M+H] +=225.1.

[0971] Synthesis of 4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzaldehyde (int-F1)

[0972]

[0973] Step 1. Place 216 g (1.14 mol, 1.00 equivalent) of 5-bromo-1-methyl-1H-imidazol-2-carboxaldehyde in a 5-L 3-necked round-bottom flask (purged and maintained under an inert nitrogen atmosphere) and in dichloromethane (3 L) and THF (1 L). Molecular sieves were used, followed by dimethylamine (862.5 mL, 1.50 equivalents). The resulting mixture was stirred at room temperature for 30 min, and NaBH(OAc)3 (292.6 g, 1.38 mol, 1.20 equivalents) was added in portions at 0 °C. The reaction mixture was stirred overnight at room temperature and then quenched with water (1 L). The organic phase was separated and washed with H2O (2 × 2 L). The aqueous phase was then extracted with DCM (2 × 1 L). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography, eluting with dichloromethane / ethyl acetate (2:1) to give [(5-bromo-1-methyl-1H-imidazol-2-yl)methyl]dimethylamine (int-F1-1). Analytical Method 5; t R = 0.60 min; [M+H] + =220.1.

[0974] Step 2. Place [(5-bromo-1-methyl-1H-imidazol-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) in N,N-dimethylformamide (1.3 L). Stir the resulting solution in an oil bath at 90 °C for 18 h. Repeat the reaction three times on the same scale. Combine the batches and cool the mixture to room temperature before pouring it into 3.5 L of water / ice. The resulting solution was extracted with EtOAc (3 × 1.5 L) and the organic layers were combined. The mixture was diluted with water (1 L) and the pH of the solution was adjusted to 4–5 with 2 M HCl aqueous solution. The aqueous phase was extracted with EtOAc (2 × 1 L) and the aqueous layers were combined. The pH of the solution was then adjusted to 11 with NH4OH. The resulting solution was extracted with DCM (6 × 1 L), and the organic layers were combined and concentrated under vacuum. The crude product was purified by silica gel column chromatography, eluting with dichloromethane / methanol (8:1) to give 4-[2-[(dimethylamino)methyl] - 1-Methyl-1H-imidazol-5-yl]phenol (int-F1-2) (120 g, 53%) is a purple oil. Analytical method 5; t R =0.55min; [M+H] + =232.1.

[0975] Step 3. Place 4-[2-[(dimethylamino)methyl] in N,N-dimethylformamide (2 L) into a 3-L 4-necked round-bottom flask. -1-Methyl-1H-imidazol-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). The resulting mixture was stirred at room temperature for 30 min 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 h, then cooled to room temperature and diluted with water (3 L). The resulting solution was extracted with EtOAc (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 HCl aqueous solution. The aqueous phase was extracted with EtOAc (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 phases were concentrated under vacuum and then purified by silica gel column chromatography, eluting with dichloromethane / ethyl acetate (7:3) to give 4-chloro-2-(4-[2-[(dimethylamino)methyl) - 1-Methyl-1H-imidazol-5-yl]phenoxy)benzaldehyde (int-F1). 1 ¹H 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). Analytical method 5; t R = 1.00 min; [M+H] + =370.2.

[0976] Synthesis of 4-chloro-2-(4-(1-methyl-2-(pyrrolidone-1-ylmethyl)-1H-imidazol-5-yl)phenoxy)benzaldehyde (int-F2)

[0977]

[0978] Step 1. Pyrrolidine (1.643 mL, 20.0 mmol) was added to a solution of 5-bromo-1-methyl-1H-imidazolium-2-carboxaldehyde (1.890 g, 10.0 mmol) in DCM (70 mL). After stirring for 25 min, NaBH(OAc)3 (8.48 g, 40.0 mmol) was added at room temperature. The resulting mixture was stirred at room temperature for 105 min, then concentrated to dryness under vacuum and partitioned between EtOAc (250 mL) and 1M NaOH aqueous solution (50 mL). The organic layer was washed with 1M NaOH (2 × 40 mL) and brine (20 mL), dried over Na2SO4, filtered, and concentrated to dryness under vacuum to give 5-bromo-1-methyl-2-(pyrrolidine-1-ylmethyl)-1H-imidazolium (int-F2-1). The crude product was used in the next step without purification. Analytical Method 11; t R = 0.66 min; [M+H] + =244.1.

[0979] Step 2. Dioxane (30 mL) and 1 M Na₂CO₃ aqueous solution (30 mL) were added to 5-bromo-1-methyl-2-(pyrrolidone-1-ylmethyl)-1H-imidazolium (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). The reaction mixture was stirred at 100 °C under a N₂ atmosphere for 4 h. An additional amount of (4-hydroxyphenyl)boronic acid (1.379 g, 10.0 mmol) was added and stirring was continued at 100 °C for 135 min. More (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 h. EtOAc (250 mL) and H2O (50 mL) were added and the mixture was filtered through a Hyflo filter. The layers were separated, and the organic layer was washed with 5% NaHCO3 aqueous solution (3 × 40 mL) and brine (40 mL), dried over Na2SO4, filtered, and concentrated to dryness under vacuum. The crude product was purified by rapid silica gel chromatography (eluent A: EtOAc / MeOH / DIEA (95:5:2), eluent B: EtOAc / MeOH / DIEA (85:15:2)) to obtain 4-(1-methyl-2-(pyrrolidone-1-ylmethyl)-1H-imidazol-5-yl)phenol (int-F2-2). Analytical Method 11; t R = 0.76 min; [M+H] + =258.1.

[0980] Step 3. Dissolve 4-(1-methyl-2-(pyrrolidone-1-ylmethyl)-1H-imidazol-5-yl)phenol (int-F2-2) (1.029 g, 4 mmol) and 4-chloro-2-fluorobenzaldehyde (0.824 g, 5.20 mmol) in NMP (20 mL) and add K2CO3 (1.437 g, 10.40 mmol). Stir the reaction mixture at 80 °C for 18 h, then partition it between EtOAc (125 mL) and H2O (20 mL). Wash the organic layer with 5% NaHCO3 aqueous solution (3 × 10 mL) and brine (10 mL), dry with Na2SO4, filter, and concentrate to dryness under vacuum. The crude product was purified by rapid silica gel chromatography (eluent A: EtOAc / DIEA (98:2), eluent B: EtOAc / MeOH / DIEA (90:10:2)) to obtain 4-chloro-2-(4-(1-methyl-2-(pyrrolidone-1-ylmethyl)-1H-imidazol-5-yl)phenoxy)benzaldehyde (int-F2). Analytical Method 10; t R = 0.79 min; [M+H] + =396.2.

[0981] Synthesis of methyl (R)-4-((R)-3-amino-3-(4-chlorobenzyl)piperidin-1-yl)-3-benzyl-4-oxobutyrate (int-F3)

[0982]

[0983] Step 1. Add DIPEA (0.323 mL, 1.848 mmol) and HATU (358 mg, 0.942 mmol) in several batches to a vial containing (R)-2-benzyl-4-(tert-butoxy)-4-oxobutyric acid (int-A1) (244 mg, 0.924 mmol) in DMA (4 mL). Once the addition is complete, stir the resulting mixture at room temperature for another 15 min, then add dropwise to another vial containing (R)-3-(4-chlorobenzyl)piperidin-3-amine (int-C3) (275 mg, 0.924 mmol) in DMA (1.5 mL) and DIPEA (0.807 mL, 4.62 mmol). Stir the reaction mixture overnight at room temperature, then transfer it to a separatory funnel, dilute with EtOAc, and wash with saturated sodium bicarbonate solution and saline (x3). The organic phase was dried over sodium sulfate, filtered, and concentrated to obtain tert-butyl (R)-4-((R)-3-amino-3-(4-chlorobenzyl)piperidin-1-yl)-3-benzyl-4-oxobutyrate, which was then carried into the next step without purification.

[0984] Step 2. Add thionyl chloride (1.35 mL, 18.47 mmol) dropwise to a round-bottom flask containing (R)-4-((R)-3-amino-3-(4-chlorobenzyl)piperidin-1-yl)-3-benzyl-4-oxobutyrate tert-butyl ester (435 mg, 0.924 mmol) in anhydrous methanol (18 mL) and cooled in an ice bath. After the addition is complete, gradually heat the resulting mixture to room temperature and then stir overnight to complete the reaction. Concentrate the reaction mixture to dryness under reduced pressure in a 30 °C water bath. Dissolve the crude oil in EtOAc, wash with a semi-saturated aqueous sodium bicarbonate solution, and then wash with brine. Dry the separated organic phase over sodium sulfate, filter and concentrate to give methyl (R)-4-((R)-3-amino-3-(4-chlorobenzyl)piperidin-1-yl)-3-benzyl-4-oxobutyrate (int-F3).

[0985] Synthesis of (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)butyric acid (int-G1)

[0986]

[0987] NaOH (4.67 mL, 4.67 mmol) was added to a suspension of (S)-2-amino-4-((tert-butoxycarbonyl)amino)butyric acid (1.04 mg, 4.77 mmol) in MeOH (10 mL) and water (0.46 mL) at room temperature. The resulting mixture was stirred at room temperature for 1 hour, and then 4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzaldehyde (int-F1) (1.6 g, 4.33 mmol) was added and 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 portions, keeping the internal reaction temperature below 0 °C. The reaction mixture was stirred at -5 °C for 30 minutes, and then stirred at room temperature for 2 hours. The reaction mixture was quenched by adding water dropwise until gas escape stopped. The reaction mixture was then concentrated to remove MeOH and 60 mL of water was added. The aqueous mixture was extracted with EtOAc (150 mL), and the organic layer was washed with 40 mL of NaHCO3 solution. After cooling, 1.0 N HCl was added to adjust the pH to approximately 8. 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 give (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)butyric acid (int-G1). Analytical Method 7: t R = 0.66 min; [M+H] + =572.0

[0988] Synthesis of (S)-5-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)valerate (int-G2)

[0989]

[0990] (S)-5-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)valerate (int-G2) was obtained using a procedure similar to that used in the synthesis of (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)butyric acid (int-G1), except that (S)-2-amino-4-((tert-butoxycarbonyl)amino)butyric acid was substituted with (S)-2-amino-4-((tert-butoxycarbonyl)amino)butyric acid. Analytical Method 7: t R = 0.67 min; [M+H] + =586.2.

[0991] Synthesis of (S)-5-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(1-methyl-2-(pyrrolidone-1-ylmethyl)-1H-imidazol-5-yl)phenoxy)benzyl)amino)valerate (int-G3)

[0992]

[0993] The procedure used in the synthesis of (S)-5-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(1-methyl-2-(pyrrolidone-1-ylmethyl)-1H-imidazol-5-yl)phenoxy)benzyl)amino)valerate (int-G3) is similar to that used in the synthesis of (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)butyric acid (int-G1). The method obtained by this procedure differs in that (S)-2-amino-4-((tert-butoxycarbonyl)amino)butyric acid is replaced with (S)-2-amino-5-((tert-butoxycarbonyl)amino)valeric acid, and 4-chloro-2-(4-(1-methyl-2-(pyrrolidone-1-ylmethyl)-1H-imidazol-5-yl)phenoxy)benzaldehyde (int-F2) is replaced with 4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzaldehyde (int-F1). Analytical Method 7: t R = 1.46 min; [M+H] + =612.6.

[0994] Synthesis of PS-(2-chlorotriphenylmethyl)(R)-4-(((S)-1-aminopropyl-2-yl)(methyl)amino)-3-benzyl-4-oxobutyrate (AB1)

[0995]

[0996] Step 1. DIPEA (388 mL, 2.227 mol) was added 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-oxobutyric acid (int-A1) (186.4 g, 0.705 mol) in DMF (460 mL) at 5-10 °C, followed by HATU (310.3 g, 0.816 mol). The resulting mixture was removed from the cooling bath and stirred at room temperature for 4 h. 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 brine (2 L), dried over anhydrous Na2SO4, and concentrated under vacuum. The crude product was purified by 230-400 mesh silica gel column chromatography, eluted with 20% ethyl acetate in petroleum ether, to give (R)-3-benzyl-4-(methyl((S)-1-((4-nitrophenyl)sulfonylamino)propyl-2-yl)amino)-4-oxobutyrate tert-butyl ester (AB1-1). Analytical Method 7; t R = 1.40 min; [M+H] + =520.3.

[0997] Step 2. Cesium carbonate (1.995 kg, 6.125 mol) was added to a cold-stirred solution of (R)-3-benzyl-4-(methyl((S)-1-((4-nitrophenyl)sulfonylamino)prop-2-yl)amino)-4-oxobutyrate tert-butyl ester (AB1-1) (455 g, 0.875 mol) in acetonitrile (3.5 L) and methanol (3.5 L) at below 10 °C, and the resulting mixture was stirred for 15 min. Then 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 h and concentrated under reduced pressure to remove the solvent. 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 brine (2 L), dried over anhydrous Na2SO4, filtered and concentrated under vacuum to obtain (R)-4-(((S)-1-aminopropyl-2-yl)(methyl)amino)-3-benzyl-4-oxobutyrate tert-butyl ester (AB1-2), which was used in the next step without purification.

[0998] Step 3. Fmoc-Cl (209.1 g, 0.807 mol) was added in portions over 30 min to a cold-stirred solution of (R)-4-(((S)-1-aminopropyl-2-yl)(methyl)amino)-3-benzyl-4-oxobutyrate tert-butyl ester (AB1-2) (270 g, 0.807 mol) in THF (1.5 L). This was followed by the addition of saturated sodium bicarbonate solution (3.3 L) over 30 min intervals at the same temperature. The mixture was then stirred at room temperature for 2 h. The reaction mixture was diluted with water (2 L) and extracted with ethyl acetate (2 × 1 L). The combined organic layers were washed with a brine 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 and eluted with 20% ethyl acetate in petroleum ether to give (R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propyl-2-yl)(methyl)amino)-3-benzyl-4-oxobutyrate tert-butyl ester (AB1-3). Analytical Method 7; t R = 1.49 min; [M+H] + =557.3.

[0999] Step 4. At room temperature, 4M HCl in dioxane (2.65 L) was added to a solution of (R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propyl-2-yl)(methyl)amino)-3-benzyl-4-oxobutyric acid tert-butyl ester (AB1-3) (265 g, 0.476 mol) in 1,4-dioxane (530 mL). The resulting mixture was stirred for 16 h and then concentrated under reduced pressure. The crude product was purified on a 230-400 mesh silica gel column and eluted with 30% ethyl acetate in petroleum ether to give (R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propyl-2-yl)(methyl)amino)-3-benzyl-4-oxobutyric acid (AB1-4). Analytical Method 7; t R = 1.48 min; [M+H] + =501.4.

[1000] Step 5. Pre-wash the 2-chlorotriphenylmethyl chloride resin (AB-1-4A, 4.27 g, 4.27 mmol) with DCM (3 × 20 mL). Add (R)-4-(((S)-1-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propyl-2-yl)(methyl)amino)-3-benzyl-4-oxobutyric acid (AB1-4) (1.9 g, 3.80 mmol) dissolved in DCM (20 mL) and DIPEA (1.5 mL, 8.59 mmol) to the resin. Shake the resulting mixture at room temperature for 16 h, then wash with DCM (3 × 40 mL) and shake in DCM / MeOH (50 mL / 20 mL) for 30 min to end-cap the resin. The resin was then filtered, washed with DMF (2 × 50 mL) and DCM (2 × 50 mL), and dried under vacuum to obtain PS-(2-chlorotriphenylmethyl)(R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propyl-2-yl)(methyl)amino)-3-benzyl-4-oxobutyric acid (AB1-5) resin (6.13 g, crude). The resin was carried to the next step without purification.

[1001] Step 6. Add 20% 4-methylpiperidine in DMF (10 mL) to PS-(2-chlorotriphenylmethyl)(R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propyl-2-yl)(methyl)amino)-3-benzyl-4-oxobutyric acid (AB1-5) (285 mg, 1.283 mmol) and shake the resulting mixture at room temperature for 2 h. Then filter the resin, wash with DMF (2 × 10 mL) and DCM (2 × 10 mL), and dry under vacuum. This gives the resin PS-(2-chlorotriphenylmethyl)(R)-4-(((S)-1-aminopropyl-2-yl)(methyl)amino)-3-benzyl-4-oxobutyric acid (AB1), which can be used directly without purification.

[1002] Synthesis of PS-(2-chlorotriphenylmethyl)(R)-4-(((1S,2S)-2-aminocyclohexyl)(methyl)amino)-3-benzyl-4-oxobutyrate (AB2)

[1003]

[1004] PS-(2-chlorotriphenylmethyl)(R)-4-(((1S,2S)-2-aminocyclohexyl)(methyl)amino)-3-benzyl-4-oxobutyrate (AB2) was obtained using the procedure used in the synthesis of PS-(2-chlorotriphenylmethyl)(R)-4-(((S)-1-aminopropyl-2-yl)(methyl)amino)-3-benzyl-4-oxobutyrate (AB1), except that (S)-N-(2-(methylamino)propyl)-4-nitrobenzenesulfonamide (int-B2) was replaced with ((1S,2S)-2-(methylamino)cyclohexyl)carbamate tert-butyl ester (int-B1).

[1005] Synthesis of PS-(2-chlorotriphenylmethyl)(R)-4-(((S)-1-amino-6-((2-nitrophenyl)sulfonylamino)hex-2-yl)(methyl)amino)-3-benzyl-4-oxobutyrate (AB3)

[1006]

[1007] Step 1. (R)-2-benzyl-4-(tert-butoxy)-4-oxobutyric 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 min. (S)-(2-(methylamino)-6-((2-nitrophenyl)sulfonylamino)hexyl)carbamate tert-butyl ester (1.049 g, 2.436 mmol) was added to the solution in DCM (20 mL) and the reaction was stirred at room temperature for 2 h 10 min. Another DIEA (0.390 mL, 2.233 mmol) was added and stirring continued for 44 h. H2O (1 mL) was added and DCM was removed under vacuum. The residue was partitioned between EtOAc (60 mL) and 5% NaHCO3 aqueous solution (15 mL). The organic layer was washed with 5% NaHCO3 aqueous solution (3 × 10 mL) and brine (10 mL), dried over Na2SO4, filtered, and concentrated under vacuum to dryness to give (1.535 g, 2.030 mmol, 100% yield) a brown oil. Crude (R)-3-benzyl-4-(((S)-1-((tert-butoxycarbonyl)amino)-6-((2-nitrophenyl)sulfonylamino)hex-2-yl)(methyl)amino)-4-oxobutyrate tert-butyl ester was used in the next step without purification. Analytical Method 11; t R = 1.31 min; [M+H] + =677.5.

[1008] Step 2. Dissolve tert-butyl (R)-3-benzyl-4-(((S)-1-((tert-butoxycarbonyl)amino)-6-((2-nitrophenyl)sulfonamide)hex-2-yl)(methyl)amino)-4-oxobutyrate (1.535 g, 2.020 mmol) in TFA (95% aqueous solution, 20 mL), stir at room temperature for 1 h, and then concentrate to dryness to obtain (R)-4-(((S)-1-amino-6-((2-nitrophenyl)sulfonamide)hex-2-yl)(methyl)amino)-3-benzyl-4-oxobutyric acid, which is directly used in the next reaction.

[1009] Step 3. The crude (R)-4-(((S)-1-amino-6-((2-nitrophenyl)sulfonamide)hex-2-yl)(methyl)amino)-3-benzyl-4-oxobutyric acid obtained from Step 2 was dissolved in dioxane (20 mL) and then treated with a solution of 0.5 M Na₂CO₃ aqueous solution (12.18 mL, 6.09 mmol) and (2,5-dioxopyrrolidone-1-yl)carbonate (9H-fluorene-9-yl)methyl ester (0.685 g, 2.030 mmol) in dioxane (20 mL). The reaction was maintained at room temperature for 90 min, then quenched by adding 2.0 M HCl aqueous solution (15 mL) and concentrated under vacuum to about half volume. The residue was partitioned between EtOAc (100 mL) and 5% KHSO₄ (15 mL). The organic layer was washed with 5% KHSO4 aqueous solution (3 × 15 mL) and brine (15 mL), dried over Na2SO4, filtered, and concentrated to dryness under vacuum. The crude product was purified by silica gel rapid chromatography. The purified fractions were combined and concentrated to dryness under vacuum. The residue was partitioned between EtOAc (80 mL) and 5% NaHCO3 aqueous solution (7 mL). The organic layer was washed with 5% NaHCO3 aqueous solution (3 × 7 mL), 5% KHSO4 aqueous solution (15 mL), and brine (10 mL), dried over Na2SO4, filtered, and concentrated under vacuum to dryness to obtain (R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((2-nitrophenyl)sulfonylamino)hex-2-yl)(methyl)amino)-3-benzyl-4-oxobutyric acid (900 mg, 1.212 mmol, 60% yield, as white foam, analytical method 11; t R = 1.21 min; [M+H] + =743.15.

[1010] Step 4. Prewash the 2-chlorotriphenylmethyl chloride resin (1.250 mg, 2.00 mmol) with DCM (3 × 30 mL). Add (R)-4-(((S)-1-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-((2-nitrophenyl)sulfonylamino)hex-2-yl)(methyl)amino)-3-benzyl-4-oxobutyrate tert-butyl ester (AB3-3) (900 mg, 1.21 mmol) in DCM (30 mL) and DIEA (1.69 mL, 9.69 mmol) to the resin. Shake the resulting mixture at room temperature for 16 h, wash with DCM (3 × 50 mL), and then shake in DCM / MeOH (5:2) (20 mL) to end-cap the resin for 30 min. Then filter the resin, wash with DCM / MeOH / DIEA (17:2:1) (3 × 15 mL) and vacuum dry. This yields the resin PS-(2-chlorotriphenylmethyl)-(R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((2-nitrophenyl)sulfonylamino)hex-2-yl)(methyl)amino)-3-benzyl-4-oxobutyrate (AB3-4), which is then carried into the next step without purification.

[1011] Step 5. Add 20% 4-methylpiperidine in DMF (10 mL) to PS-(2-chlorotriphenylmethyl)-(R)-4-(((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((2-nitrophenyl)sulfonamide)hex-2-yl)(methyl)amino)-3-benzyl-4-oxobutyrate (AB3-4) (266 mg, 0.336 mmol) and shake the resulting mixture at room temperature for 2 h. Then filter the resin, wash with DMF (2 × 10 mL) and DCM (2 × 10 mL), and dry under vacuum. This yields resin PS-(2-chlorotriphenylmethyl)-(R)-4-(((S)-1-amino-6-((2-nitrophenyl)sulfonamide)hex-2-yl)(methyl)amino)-3-benzyl-4-oxobutyrate (AB3), which can be used directly without purification.

[1012] Synthesis of PS-(2-chlorotriphenylmethyl)(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-ester (AB4)

[1013]

[1014] Step 1-1. A solution of (S)-3-((((9H-fluorene-9-yl)methoxy)carbonyl)(methyl)amino)-4-(4-chlorophenyl)butyric 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 min, and then added to PS-(2-chlorotriphenylmethyl)(R)-4-(((S)-1-aminopropyl-2-yl)(methyl)amino)-3-benzyl-4-oxobutyrate (AB1) (6.00 mmol) that had been washed with NMP (3x). The resulting suspension was shaken at room temperature for 20 h, then filtered, and the resin was washed with DMA (3x). For end capping, Ac2O / pyridine / DMA (1:1:8) (70 mL) was added, and the resulting suspension was shaken at room temperature for 15 min. Drain the resin and then wash with DMA (3x).

[1015] Steps 1-2. Fmoc-deprotection was performed by repeated treatment with 4-methylpiperidine / DMA (1:4) (5 × 70 mL, shaking for 5 min each time at room temperature). The cleavage solution was collected and used to determine the resin loading via UV spectroscopy. After Fmoc removal, the resin was washed with DMA (3x), DCM (3x), DMA (3x), and DCM (5x) and vacuum dried to obtain PS-(2-chlorotriphenylmethyl)(R)-3-benzyl-4-(((S)-1-((S)-4-(4-chlorophenyl)-3-(methylamino)butamido)-propyl-2-yl)(methyl)amino)-4-oxobutyrate (AB4-1).

[1016] Step 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 min, and then added to PS-(2-chlorotriphenylmethyl)(R)-3-benzyl-4-(((S)-1-((S)-4-(4-chlorophenyl)-3-(methylamino)butamido)-propyl-2-yl)(methyl)amino)-4-oxobutyrate (AB4-1) (4.477 mmol), which had been washed with NMP (3x). The resulting suspension was shaken at room temperature for 6 h, 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 min and then added to the resin. The resulting suspension was shaken at room temperature for 16 h and filtered, and the resin was washed with DMA (3x). For end capping, Ac2O / pyridine / DMA (1:1:8) (40 mL) was added and the resulting suspension was shaken at room temperature for 15 min. The resin was drained and then washed with DMA (3x).

[1017] Step 2-2. Fmoc-deprotection was performed by repeated treatment with 4-methylpiperidine / DMA (1:4) (3 × 30 mL, shaking for 15 min at room temperature each time). After Fmoc- removal, the resin was washed with DMA (3x) and DCM (5x) to obtain PS-(2-chlorotriphenylmethyl)(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-ester (AB4).

[1018] Synthesis of PS-(2-chlorotriphenylmethyl)(R)-4-(((1S,2S)-2-((S)-3-((S)-2-amino-3-methoxy-N-methylpropionamido)-4-(4-chlorophenyl)butamido)cyclohexyl)(methyl)amino)-3-benzyl-4-oxobutyrate (AB5)

[1019]

[1020] PS-(2-chlorotriphenylmethyl)(R)-4-(((1S,2S)-2-((S)-3-((S)-2-amino-3-methoxy-N-methylpropionamido)-4-(4-chlorophenyl)butamido)cyclohexyl)(methyl)amino)-3-benzyl-4-oxobutyrate (AB5) uses PS-(2-chlorotriphenylmethyl)(4S,7S,12S,15R)-4-amino-15-benzyl-7-(4-chlorobenzyl)-6,12,13-trimethyl-5,9,14-trimethyl-5,9,14-trimethyl-4-oxobutyrate. The procedure used in the synthesis of oxo-2-oxa-6,10,13-triazaheptadecane-17-ester (AB4) was used to obtain it, except that PS-(2-chlorotriphenylmethyl)(R)-4-(((1S,2S)-2-aminocyclohexyl)(methyl)amino)-3-benzyl-4-oxobutyrate (AB2) was used instead of PS-(2-chlorotriphenylmethyl)(R)-4-(((S)-1-aminopropyl-2-yl)(methyl)amino)-3-benzyl-4-oxobutyrate (AB1).

[1021] Synthesis of N-(4-((2S,5S,8S,13S,16R)-16-benzyl-1-(4-chloro-2-(4-(1-methyl-2-(pyrrolidone-1-ylmethyl)-1H-imidazol-5-yl)phenoxy)benzyl)-8-(4-chlorobenzyl)-5-(methoxymethyl)-2,7,14-trimethyl-3,6,10,15,18-pentoxo-1,4,7,11,14-pentazacyclooctadecane-13-yl)butyl)-2-nitrobenzenesulfonamide (AB6)

[1022]

[1023] Step 1-1. A solution of (S)-3-((((9H-fluorene-9-yl)methoxy)carbonyl)(methyl)amino)-4-(4-chlorophenyl)butyric 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 min, and then added to PS-(2-chlorotriphenylmethyl)-(R)-4-(((S)-1-amino-6-((2-nitrophenyl)sulfonamide)hex-2-yl)(methyl)amino)-3-benzyl-4-oxobutyrate (AB3) (1.113 mmol), which had been washed with NMP (3x). The resulting suspension was shaken at room temperature for 20 h, then filtered, and the resin was washed with DMA (3x). For end capping, add Ac2O / pyridine / DMA (1:1:8) (70 mL) and shake the resulting suspension at room temperature for 20 h. Drain the resin and then wash with DMA (3x).

[1024] Steps 1-2. Fmoc-deprotection was performed by repeated treatment with 4-methylpiperidine / DMA (1:4) (5 × 20 mL, shaking for 15 min each time at room temperature). The cleavage solution was collected and used to determine the resin loading via UV spectroscopy. After Fmoc removal, the resin was washed with DMA (3x), DCM (3x), DMA (3x), and DCM (5x) and vacuum dried to obtain PS-(2-chlorotriphenylmethyl)(R)-3-benzyl-4-(((S)-1-((S)-4-(4-chlorophenyl)-3-(methylamino)butamido)-6-((2-nitrophenyl)sulfonamide)hex-2-yl)(methyl)amino)-4-oxobutyrate.

[1025] Step 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 min. This solution was then added to PS-(2-chlorotriphenylmethyl)(R)-3-benzyl-4-(((S)-1-((S)-4-(4-chlorophenyl)-3-(methylamino)butamido)-6-((2-nitrophenyl)sulfonamide)hex-2-yl)(methyl)amino)-4-oxobutyrate (1.113 mmol), which had been washed with NMP (3x). The resulting suspension was shaken at room temperature for 3 h, filtered, and the resin was washed with DMA (3x). For end-capping, Ac2O / pyridine / DMA (1:1:8) (40 mL) was added, and the resulting suspension was shaken at room temperature for 15 min. Drain the resin and then wash with DMA (3x).

[1026] Step 2-2. Fmoc-deprotection was performed by repeated treatment with 4-methylpiperidine / DMA (1:4) (3 × 15 mL, shaking for 15 min each time at room temperature). After Fmoc- removal, the resin was washed with DMA (3x) and DCM (5x) to obtain PS-(2-chlorotriphenylmethyl)(4S,7S,12S,15R)-4-amino-15-benzyl-7-(4-chlorobenzyl)-6,13-dimethyl-12-(4-((2-nitrophenyl)sulfonylamino)butyl)-5,9,14-trioxo-2-oxa-6,10,13-triazaheptadecane-17-ester, which was then carried to the next step.

[1027] Step 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 min. This solution was then added to PS-(2-chlorotriphenylmethyl)(4S,7S,12S,15R)-4-amino-15-benzyl-7-(4-chlorobenzyl)-6,13-dimethyl-12-(4-((2-nitrophenyl)sulfonylamino)butyl)-5,9,14-trioxo-2-oxa-6,10,13-triazaheptadecane-17-ester (1.113 mmol), which had been washed with NMP (3x). The resulting suspension was shaken at room temperature for 3 h, then filtered, and the resin was washed with DMA (3x). For end capping, add Ac2O / pyridine / DMA (1:1:8) (20 mL) and shake the resulting suspension at room temperature for 15 min. Drain the resin and then wash with DMA (3x).

[1028] Step 3-2. Fmoc-deprotection was performed by repeated treatment with 4-methylpiperidine / DMA (1:4) (3 × 20 mL, shaking for 15 min each time at room temperature). After Fmoc- removal, the resin was washed with DMA (3x) and DCM (5x) to obtain PS-(2-chlorotriphenylmethyl)(2S,5S,8S,13S,16R)-2-amino-16-benzyl-8-(4-chlorobenzyl)-5-(methoxymethyl)-7,14-dimethyl-13-(4-((2-nitrophenyl)sulfonylamino)butyl)-3,6,10,15-tetraoxo-4,7,11,14-tetraazaoctadecane-18-ester, which was then carried to the next step.

[1029] Step 4. At room temperature, add HFIP / DCM (1:3) (20 mL) to PS-(2-chlorotriphenylmethyl)(2S,5S,8S,13S,16R)-2-amino-16-benzyl-8-(4-chlorobenzyl)-5-(methoxymethyl)-7,14-dimethyl-13-(4-((2-nitrophenyl)sulfonylamino)butyl)-3,6,10,15-tetraoxo-4,7,11,14-tetraazaoctadecane-18-ester (1.113 mmol) and shake the resulting suspension for 20 min. Then filter and collect (3x). Wash the resin with DCM (2x) and collect the wash. Concentrate the combined cutting solution and wash to dryness under vacuum. The crude residue was lyophilized by passing it through tBuOH / H2O (4:1) to give (2S,5S,8S,13S,16R)-2-amino-16-benzyl-8-(4-chlorobenzyl)-5-(methoxymethyl)-7,14-dimethyl-13-(4-((2-nitrophenyl)sulfonylamino)butyl)-3,6,10,15-tetraoxo-4,7,11,14-tetraazaoctadecane-18-acid. Analytical Method 12: t R =0.87min; [M+H]+ =902.7

[1030] Step 5. Dissolve (2S,5S,8S,13S,16R)-2-amino-16-benzyl-8-(4-chlorobenzyl)-5-(methoxymethyl)-7,14-dimethyl-13-(4-((2-nitrophenyl)sulfonylamino)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-(pyrrolidone-1-ylmethyl)-1H-imidazol-5-yl)phenoxy)benzaldehyde (int-F2) (238 mg, 0.6 mmol) in DCM (30 mL) and AcOH (0.114 mL, 2 mmol), and stir the resulting solution at room temperature for 1.5 h. Then, NaBH(OAc)3 (530 mg, 2.5 mmol) was added and the reaction mixture was stirred at room temperature for 18 h. MeOH (2 mL) was added and the reaction mixture was concentrated to dryness under vacuum. The crude product was purified by preparative reversed-phase HPLC (eluent A: 0.1% TFA in H2O and eluent B: ACN). The pure fractions were combined and freeze-dried to give (3S,6S,9S,14S,17R)-17-benzyl-1-(4-chloro-2-(4-(1-methyl-2-(pyrrolidone-1-ylmethyl)-1H-imidazol-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-pentazanonadecan-19-acid. Analytical Method 9: t R =4.05min; [M+H]+ =1281.5

[1031] Step 6. To (3S,6S,9S,14S,17R)-17-benzyl-1-(4-chloro-2-(4-(1-methyl-2-(pyrrolidone-1-ylmethyl)-1H-imidazol-5-yl)phenoxy)phenyl)-9-(4-chlorobenzyl)-6-(methoxymethyl)-3,8,15-trimethyl-14-(4-((2-nitrophenyl)sulfonamide)butyl)-4,7,11,16-tetraoxy 2,6-Rutidine (0.946 mL, 8.13 mmol) was added to a solution of 2,6-pentazine-19-acid (440 mg, 271 μmol), HATU (412 mg, 1.083 mmol), and HOAt (55.3 mg, 0.406 mmol) in DCM (100 mL), and the resulting mixture was stirred at room temperature for 18.5 h. The reaction mixture was concentrated to dryness under vacuum, and the resulting residue was partitioned between EtOAc (100 mL) and 5% NaHCO3 aqueous solution (15 mL). The organic layer was washed with 5% NaHCO3 aqueous solution (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-(pyrrolidone-1-ylmethyl)-1H-imidazol-5-yl)phenoxy)benzyl)-8-(4-chlorobenzyl)-5-(methoxymethyl)-2,7,14-trimethyl-3,6,10,15,18-pentoxo-1,4,7,11,14-pentazacyclooctadecane-13-yl)butyl)-2-nitrobenzenesulfonamide (AB6). Analytical Method 10: t R = 1.1 min; [M+2H] 2+ =633.6.

[1032] 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-imidazol-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecylbenz[l][1,4,7,11,14]pentazacyclooctadecyne-2,6,9,12,15(3H)-pentanone (C6)

[1033]

[1034] Step 1. Add DIPEA (0.587 mL, 3.36 mmol) to (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)butyric acid (int-G1) (1.000 g, 1.680 mmol) in DMF (20 mL), followed by HATU (0.639 g, 1.680 mmol) and stir the resulting mixture until completely homogeneous. The solution was then added to PS-(2-chlorotriphenylmethyl)(R)-4-(((1S,2S)-2-((S)-3-((S)-2-amino-3-methoxy-N-methylpropionamido)-4-(4-chlorophenyl)butamido)cyclohexyl)(methyl)amino)-3-benzyl-4-oxobutyrate (AB5) (2.8 g, 0.840 mmol) in 10 mL of DMF in a shake 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-imidazol-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-acylamino)cyclohexyl)(methyl)amino)-4-oxobutyric acid PS-2-chlorotriphenylmethyl ester, which was carried to the next step without purification.

[1035] Step 2. Cut (R)-3-benzyl-4-(((1S,2S)-2-((8S,11S,14S)-8-((4-chloro-2-(4-(2-((dimethylamino)-methyl)-1-methyl-1H-imidazol-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-acylamino)cyclohexyl)(methyl)amino)-4-oxobutyric acid PS-2-chlorotriphenylmethyl ester (2.9 g, 0.87 mmol) from the resin by shaking with 75 mL of 20% HFIP / DCM at room temperature for 20 min. Filter the resin and collect the filtrate. Repeat both steps three more times to ensure that all product is cut from the resin. The combined filtrates were concentrated to obtain a crude oil (1.8 g), which was purified by reversed-phase chromatography (eluting with MeCN / H2O containing 0.1% NH4OH) to give 1(R)-3-benzyl-4-(((1S,2S)-2-((8S,11S,14S)-8-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-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-acylamino)cyclohexyl)(methyl)amino)-4-oxobutyric acid. Analytical Method 7; t R = 1.77 min; [M+H] + =1182.7.

[1036] Step 3. Add (R)-3-benzyl-4-(((1S,2S)-2-((8S,11S,14S)-8-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)-14-(4-chlorobenzyl)-11-(methoxymethyl)-2,2,13-trimethyl-4,9,12-trioxo HATU (148 mg, 0.389 mmol), 2,6-rutidine (0.340 mL, 2.92 mmol), and HOAt (13.23 mg, 0.097 mmol) were added to 3-oxa-5,10,13-triazahexadecane-16-acylamino)cyclohexyl)(methyl)amino)-4-oxobutyric acid (115 mg, 0.097 mmol), and the resulting mixture was stirred overnight at 45 °C. The reaction mixture was then concentrated to dryness and partitioned between EtOAc (100 mL) and 5% NaHCO3 aqueous solution (100 mL). The organic layer was washed with 5% NaHCO3 aqueous solution (2 × 50 mL) and brine (50 mL), dried over Na2SO4, filtered, and concentrated to dryness by rotary evaporation. The residue was then absorbed into EtOAc and washed with 1M HCl (x2). Brine was added to the combined 1M HCl layers and back-extracted with EtOAc (x2). The combined EtOAc layers were dried over sodium sulfate, filtered, and concentrated under vacuum. The product was purified by reversed-phase chromatography (eluting with MeCN / H2O at a gradient of 20%-65%, containing 0.1% NH4OH) to give (2-((4S,7S,10S,14R,16aS,20aS)-14-benzyl-11-(4-chloro-2-(4-(2-((dimethylamino)-methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethyl-2,6,9,12,15-pentaoxododocos[l][1,4,7,11,14]pentazonooctadecyn-10-yl)ethyl)carbamate tert-butyl trifluoroacetate. Analytical Method 2; t R = 3.17 min; [M+H] + =1163.6.

[1037] Step 4. At 0°C, HCl (4M in dioxane) (0.67 mL, 2.40 mmol) was added dropwise to a solution of (2-((4S,7S,10S,14R,16aS,20aS)-14-benzyl-11-(4-chloro-2-(4-(2-((dimethylamino)-methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethyl-2,6,9,12,15-pentoxododocosidine-10-yl)ethyl)carbamate tert-butyl trifluoroacetate (40 mg, 0.034 mmol) in anhydrous dioxane (1.72 mL) (0.67 mL, 2.40 mmol). The reaction mixture became turbid. After 15 min, the ice bath was removed and the mixture was stirred at room temperature for 1 hr. The reaction mixture was then concentrated and the resulting residue was dried under vacuum. The crude material was dissolved in EtOAc and washed with a saturated aqueous sodium bicarbonate solution. The aqueous layer was then extracted with fresh EtOAc. The combined organic matter was dried over sodium sulfate, filtered, and concentrated under vacuum to give (4S,7S,10S,14R,16aS,20aS)-10-(2-aminoethyl)-14-benzyl-11-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecylhydrobenzo[l][1,4,7,11,14]pentazacyclooctadecyne-2,6,9,12,15(3H)-pentanone (C6). Analytical Method 7; t R = 2.36 min; [M+H] + =1063.7.

[1038] 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-imidazol-5-yl)phenoxy)benzyl)-8-(4-chlorobenzyl)-5-(methoxymethyl)-7,13,14-trimethyl-1,4,7,11,14-pentazoctadecane-3,6,10,15,18-pentanone (C7)

[1039]

[1040] (2S,5S,8S,13S,16R)-2-(3-aminopropyl)-16-benzyl-1-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)-8-(4-chlorobenzyl)-5-(methoxymethyl)-7,13,14-trimethyl-1,4,7,11,14-pentazoctadecane-3,6,10,15,18-pentanone (C7) using (4S,7S,10S,14R,16aS,20aS) The method used in the synthesis of 10-(2-aminoethyl)-14-benzyl-11-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecylhydrobenzo[l][1,4,7,11,14]pentazoneoctadecyn-2,6,9,12,15(3H)-pentanone (C6) was obtained, except that PS-(2-chlorotriphenylmethyl) was used. (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-ester (AB4) replaces PS-(2-chlorotriphenylmethyl)(R)-4-(((1S,2S)-2-((S)-3-((S)-2-amino-3-methoxy-N-methylpropionamido)-4-(4-chlorophenyl)butamido)cyclohexyl)(methyl)amino)-3-benzyl 4-O-butanoic acid ester (AB5) was replaced with (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)valerate (int-G2), replacing (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)butanoic acid (int-G1). Analytical Method 7; t R = 1.18 min; [M+H] + =1038.4.

[1041] Examples 1-6: Synthesis of (4S,7S,10S,14R,16aS,20aS)-10-(3-aminopropyl)-14-benzyl-11-(4-chloro-2-(4-(1-methyl-2-(pyrrolidone-1-ylmethyl)-1H-imidazol-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecylhydrobenzo[l][1,4,7,11,14]pentazacyclooctadecyne-2,6,9,12,15(3H)-pentanone (C8)

[1042]

[1043] (4S,7S,10S,14R,16aS,20aS)-10-(3-aminopropyl)-14-benzyl-11-(4-chloro-2-(4-(1-methyl-2-(pyrrolidone-1-ylmethyl)-1H-imidazol-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecylbenzyl[l][1, [4,7,11,14] Pentaazacyclooctadecyn-2,6,9,12,15(3H)-pentanone (C8) using (4S,7S,10S,14R,16aS,20aS)-10-(2-aminoethyl)-14-benzyl-11-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl The 4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecylbenz[l][1,4,7,11,14]pentazonyl octadecyn-2,6,9,12,15(3H)-pentanone (C6) was obtained by the procedure used in the synthesis of 4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecylbenzyl[l][1,4,7,11,14]pentazonyl octadecyn-2,6,9,12,15(3H)-pentanone (C6), except that (S)-5-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)valerate (int-G3) was replaced by (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)butyric acid (int-G1). Analytical Method 7; t R = 1.35 min; [M+H] + =1102.2.

[1044] 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-imidazol-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecylhydrobenzo[l][1,4,7,11,14]pentazacyclooctadecyne-2,6,9,12,15(3H)-pentanone (C9)

[1045]

[1046] ((4S,7S,10S,14R,16aS,20aS)-10-(3-aminopropyl)-14-benzyl-11-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)-4-(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecylbenzo[l][1,4] [7,11,14] Pentaazacyclooctadecyn-2,6,9,12,15(3H)-pentanone (C9) using (4S,7S,10S,14R,16aS,20aS)-10-(2-aminoethyl)-14-benzyl-11-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)-4 -(4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecylhydrobenzo[l][1,4,7,11,14]pentazonyl octadecyn-2,6,9,12,15(3H)-pentanone (C6) was obtained using the procedure described in the synthesis of 1,4-chlorobenzyl)-7-(methoxymethyl)-5,16-dimethylhexadecylhydrobenzo[l][1,4,7,11,14]pentazonyl octadecyn-2,6,9,12,15(3H)-pentanone (C6), except that (S)-5-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)pentanolic acid (int-G2) was used instead of (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)butyric acid (int-G1). Analytical Method 7; t R = 1.23 min; [M+H] + =1078.7.

[1047] Examples 1-8: Synthesis of (2S,5S,8S,13S,16R)-13-(4-aminobutyl)-16-benzyl-1-(4-chloro-2-(4-(1-methyl-2-(pyrrolidone-1-ylmethyl)-1H-imidazol-5-yl)phenoxy)benzyl)-8-(4-chlorobenzyl)-5-(methoxymethyl)-2,7,14-trimethyl-1,4,7,11,14-pentazoctadecane-3,6,10,15,18-pentanone trifluoroacetate (C10)

[1048]

[1049] To N-(4-((2S,5S,8S,13S,16R)-16-benzyl-1-(4-chloro-2-(4-(1-methyl-2-(pyrrolidone-1-ylmethyl)-1H-imidazol-5-yl)phenoxy)benzyl)-8-(4-chlorobenzyl)-5-(methoxymethyl)-2,7,14-trimethyl-3,6,10,15,18-pentoxo-1,4,7,11,14-pentazacyclooctadecane-13- (B)-Butyl)-2-nitrobenzenesulfonamide (AB6) (532 mg, 0.271 mmol, 64.5%) was added to a solution of DMF (15 mL) with 2-mercaptoethanol (0.115 mL, 1.628 mmol) and DBU (0.082 mL, 0.543 mmol). The resulting mixture was stirred at room temperature for 30 min, then quenched by adding AcOH (0.4 mL) and concentrated to dryness under vacuum. The crude product was purified by preparative reversed-phase HPLC (eluent A: 0.1% TFA in H2O and eluent B: ACN). The pure fractions were combined and lyophilized to give (2S,5S,8S,13S,16R)-13-(4-aminobutyl)-16-benzyl-1-(4-chloro-2-(4-(1-methyl-2-(pyrrolidone-1-ylmethyl)-1H-imidazol-5-yl)phenoxy)benzyl)-8-(4-chlorobenzyl)-5-(methoxymethyl)-2,7,14-trimethyl-1,4,7,11,14-pentazolidinyl octadecane-3,6,10,15,18-pentanone trifluoroacetate (C10). Analytical Method 9; t R = 3.85 min; [M+H] + =1078.5.

[1050] 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-imidazol-5-yl)phenoxy)benzyl)-13-(4-chlorobenzyl)-10-(hydroxymethyl)-1,6,9,12-tetraazabicyclo[11.3.1]heptadecane-2,5,8,11-tetraone (C11)

[1051]

[1052] Step 1: Add HATU (455 mg, 1.197 mmol) and DIPEA (0.789 mL, 4.52 mmol) to a solution of Fmoc-Ser(OtBMe2Si)OH (548 mg, 1.242 mmol) in DMA (5 mL). Stir the resulting mixture at room temperature for 2 min, then add methyl (R)-4-((R)-3-amino-3-(4-chlorobenzyl)piperidin-1-yl)-3-benzyl-4-oxobutyrate (int-F3) (484 mg, 1.129 mmol) to a solution of DMA (3 mL). Stir the reaction mixture at room temperature for 6 h. Add additional Fmoc-Ser(OtBMe2Si)OH (88 mg, 0.200 mmol) and HATU (76 mg, 0.20 mmol), and continue stirring overnight at room temperature. Add 4-methylpiperidine (0.8 mL, 6.77 mmol), and continue stirring for 30 min at room temperature. The reaction mixture was concentrated under reduced pressure (bath temperature 50°C), and the residue was purified by reversed-phase rapid column chromatography (eluting with 5%-90% water / ACN containing 0.1% NH4OH) to give methyl (R)-4-((R)-3-((S)-2-amino-3-((tert-butyldimethylsilyl)oxy)propamido)-3-(4-chlorobenzyl)piperidin-1-yl)-3-benzyl-4-oxobutyrate. Analytical Method 7, t R = 1.41 min, [M+H] + =630.5.

[1053] Step 2: Add DIPEA (0.374 mL, 2.142 mmol) to a solution of (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-(((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)butyric acid (int-G1) (449 mg, 0.785 mmol) and (R)-4-((R)-3-((S)-2-amino-3-((tert-butyldimethylsilyl)oxy)propamido)-3-(4-chlorobenzyl)piperidin-1-yl)-3-benzyl-4-oxobutyrate (450 mg, 0.714 mmol) in DMA (5 mL). Stir the resulting solution at room temperature for 2 min, then add HATU (299 mg, 0.785 mmol) to the solution of DMA (3 mL). Stir the reaction mixture at room temperature for 3 h. Add additional (S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)butyric acid (int-G1) (88 mg, 0.14 mmol) and HATU (76 mg, 0.20 mmol) and continue stirring overnight at room temperature. The resulting mixture was concentrated under reduced pressure, and the residue was purified by rapid column chromatography (eluting with 98 / 2 to 85 / 15 DCM / MeOH containing 0.3% triethylamine) to give methyl (R)-3-benzyl-4-((R)-3-((S)-2-((S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)butamido)-3-((tert-butyldimethylsilyl)oxy)propamido)-3-(4-chlorobenzyl)piperidin-1-yl)-4-oxobutyrate. Analytical Method 7, t R = 1.55 min, [M+H] + =1184.1.

[1054] Step 3: Add water (1 mL) and THF (4 mL) 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-imidazol-5-yl)phenoxy)benzyl)amino)butamido)-3-((tert-butyldimethylsilyl)oxy)propamido)-3-(4-chlorobenzyl)piperidin-1-yl)-4-oxobutyrate (770 mg, 0.65 mmol) in DMA (5 mL). Stir the resulting mixture at room temperature, then add a solution of LiOH (1.300 mL, 1.300 mmol). Stir the reaction mixture at room temperature for 3 h. Add additional LiOH (1.300 mL, 1.300 mmol) and continue stirring overnight at room temperature (LCMS showed the desired product and a mixture of demethylated silyl alcohols R=H). Cool the reaction mixture in an ice bath, neutralize the pH (pH=7) by adding 1 N HCl, and then concentrate under reduced pressure (bath maintained at 30 °C). Absorb the residue in 250 mL of EtOAc. The organic phase was washed with water and brine, dried over Na2SO4, filtered, and concentrated to give (R)-3-benzyl-4-((R)-3-((S)-2-((S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)butamido)-3-((tert-butyldimethylsilyl)oxy)propamido)-3-(4-chlorobenzyl)piperidin-1-yl)-4-oxobutyric acid. Analytical Method 5, t R = 1.97 min, [M+H] + =1055.1.

[1055] Step 4: Add 2,6-rutidine (2.3 mL, 19.74 mmol), HOAt (107 mg, 0.790 mmol), and HATU (1001 mg, 2.63 mmol) to a 1 L round-bottom flask containing (R)-3-benzyl-4-((R)-3-((S)-2-((S)-4-((tert-butoxycarbonyl)amino)-2-((4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-5-yl)phenoxy)benzyl)amino)butamido)-3-((tert-butyldimethylsilyl)oxy)propamido)-3-(4-chlorobenzyl)piperidin-1-yl)-4-oxobutyric acid (770 mg, 0.658 mmol) in DCM (700 mL). Stir the resulting mixture at 38 °C for 16 hr. The reaction mixture was then concentrated to dryness under reduced pressure, and the residue was partitioned between EtOAc (400 mL) and 5% NaHCO3 aqueous solution (30 mL). The organic phase was washed with 5% NaHCO3 aqueous solution (2 × 25 mL) and brine (30 mL), dried over Na2SO4, filtered, and concentrated. The crude substance was purified by reversed-phase rapid column chromatography (eluting with 5%-60% water / AcN containing 0.1% trifluoroacetic acid) to obtain tert-butyl (2-((3R,7S,10S,13R)-3-benzyl-6-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-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. Analytical method 2, t R = 3.22 min, [M+H] + =1039.4.

[1056] Step 5: Add 4.0N hydrogen chloride in dioxane (2 mL, 8.00 mmol) to a round-bottom flask containing (320 mg, 0.308 mmol)-3-benzyl-6-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-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 carbamate (2 mL, 8.00 mmol) in an ice bath. Then remove the ice bath and stir the resulting mixture overnight at room temperature. The reaction mixture was concentrated under reduced pressure to give a grayish-white solid, which was purified by reversed-phase rapid column chromatography (eluting with 5%-50% water / ACN containing 0.1% trifluoroacetic acid) to give (3R,7S,10S,13R)-7-(2-aminoethyl)-3-benzyl-6-(4-chloro-2-(4-(2-((dimethylamino)methyl)-1-methyl-1H-imidazol-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 min, [M+H] + =937.6.

[1057] Example 2: Synthesis of FHR3 receptor ligand compounds (C12) to (C16)

[1058] 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-guanidinylpropyl)-33-(4-hydroxybenzyl)-24-(hydroxymethyl)-5,20,35-trimethyl-1,4,7,13,16,19,22,25,28,31,34,37,40,43-tetradecanoic acid Synthesis of oxo-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-tetratetradecano-2H-[1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40,43]tetradecazine tetrapentaeno[13,12-b]isoquinoline-42-yl)acetic acid (C12)

[1059]

[1060] Note: Compound (C12) has the following amino acid sequence:

[1061]

[1062] 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)

[1063] Peptide sequence 2-1b in Fmoc-Gly-RAM TentaGel TM On resin (2-1a, 0.22 mmol / g loading, 0.25 mmol scale) at The peptide was synthesized on a peptide synthesizer according to a standard peptide synthesis cycle A-1 (Fmoc-amino acids (4 equivalents; 0.2 M solution in DMF), HATU (4 equivalents; 0.5 M solution in DMF), and DIPEA (4.4 equivalents; 2 M solution in NMP)). The product was then filtered through resin and washed with DMF (2x) and DCM (3x) to obtain the desired product 2-1b.

[1064] 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)

[1065] A solution of N-succinimide-2-chloroacetate (2-1c, 287 mg, 1.5 mmol) in NMP (8 mL) was added to peptide resin 2-1b (0.25 mmol) from step 1, and the resulting mixture was shaken overnight at room temperature. The resin was then drained, washed with DMF (3x) and DCM (4x), and dried to give the desired product 2-1d.

[1066] Step 3: ClCH2C(O)-FRF(N-Me)-SKNYG(N-Me)-RD-Tic-NF(N-Me)-CG-NH2(1-1e)

[1067] The peptide resin product 2-1d from step 2 is cleaved from the resin and simultaneously deprotected using the cleavage method 1 described above to obtain crude peptide 2-1e.

[1068] 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-guanidinylpropyl)-33-(4-hydroxybenzyl)-24-(hydroxymethyl)-5,20,35-trimethyl-1,4,7,13,16,19,22,25,28,31,34,37,40,43-tetradecanoic acid oxo-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-tetratetradecano-2H-[1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40,43]tetradecazine tetradecano[13,12-b]isoquinoline-42-yl)acetic acid (C12)

[1069] The crude peptide 1-2e (266 mg) from step 3 was dissolved in DMSO (20.5 mL). A few drops of TEA were added to obtain pH 8-9. The resulting mixture was stirred overnight at room temperature. The reaction mixture was then concentrated to a few mL of DMSO using a centrifugal evaporator. The crude cyclic peptide was analyzed by preparative HPLC (Sunfire). TM Prep C18 column, Purification was performed using ACN (containing 0.1% TFA) in water at a depth of 5 μm, 30 × 50 mm, for 6 min at a rate of 15%-40%, 75 mL / min, followed by lyophilization to obtain the target cyclic peptide compound (C12) (SEQ ID NO: 5). Analytical Method 9: t R =3.24, m+1 = 1951.20

[1070] Example 2-2: 2-((3S,6S,9R,15S,18S,21S,24S,27S,30S,33S,36S,39S,44aS)-30-((1H-imidazol-5-yl)methyl)-33-((1H-indol-3-yl)methyl)-9-((2-amino-2-oxoethyl)carbamoyl)-18-(4-aminobutyl)-15-benzyl-6,39-bis(3-guanidinopropyl)-24 Synthesis of -(hydroxymethyl)-27-isobutyl-20,21,35,36-tetramethyl-1,4,7,13,16,19,22,25,28,31,34,37,40-tetrazoloxytetradodecano-12H-pyrrolo[1,2-e1][1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40]tetrazazoletetradodecenoen-3-yl)acetic acid (C13)

[1071]

[1072] Note: Compound (C13) has the following amino acid sequence:

[1073]

[1074] The cyclic peptide compound (C13) (SEQ ID NO:6) was obtained using a method similar to that described in Example 2-1, but in step 1, instead of peptide sequences 2-4b, it was synthesized and attached to the resin. The peptide sequence on the [top / side]. Analysis method 9: t R =3.01, m+1=1710.98

[1075] Example 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-guanidinylpropyl)-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-tetradecano-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-tetradecanohydro-2H-[1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40,43]tetradecazine tetrapentaeno[13,12-b]isoquinoline-42-yl)acetic acid (C14)

[1076]

[1077] Note: Compound (C14) has the following amino acid sequence:

[1078]

[1079] The cyclic peptide compound (C14) (SEQ ID NO:7) was obtained using a method similar to that described in Example 2-1, but in step 1, instead of peptide sequences 2-4b, it was synthesized and attached to the resin. The peptide sequence on the [top / side]. Analysis method 9: t R =3.35, m+1=1979.25

[1080] Example 2-4: 2-((3R,6S,9S,12S,15S,18S,21S,24S,27S,30S,33S,36S,39S)-24-((1H-imidazol-5-yl)methyl)-21-((1H-indol-3-yl)methyl)-3-((2-amino-2-oxoethyl)carbamoyl)-12-(4-aminobutyl)-36,39-dibenzyl-6,15-bis(3 Synthesis of (-guanidinopropyl)-30-(hydroxymethyl)-27-isobutyl-18,19,33,34,37-pentamethyl-5,8,11,14,17,20,23,26,29,32,35,38,41-tridecano-1-thia-4,7,10,13,16,19,22,25,28,31,34,37,40-tetrazazole tetradodecane-9-yl)acetic acid (C15)

[1081]

[1082] Note: Compound (C15) has the following amino acid sequence:

[1083]

[1084] The cyclic peptide compound (C15) (SEQ ID NO:8) was obtained using a method similar to that described in Example 2-1, but in step 1, instead of peptide sequences 2-4b, it was synthesized and attached to the resin. The peptide sequence on the [top / side]. Analysis method 9: t R =3.35, m+1=1775.07

[1085] Example 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-tetradecoxo-1,3,4 Synthesis of ,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-tetratetradecano-2H-[1]thia[4,7,10,13,16,19,22,25,28,31,34,37,40,43]tetradecazine tetrapentadecene[13,12-b]isoquinoline-3,30-diyl)diacetamide (C16)

[1086]

[1087] Note: Compound (C16) has the following amino acid sequence:

[1088]

[1089] The cyclic peptide compound (C16) (SEQ ID NO:9) was obtained using a method similar to that described in Example 2-1, but in step 1, instead of peptide sequences 2-4b, it was synthesized and attached to the resin. The peptide sequence on the [top / side]. Analysis method 9: t R =3.45, m+1=1935.24

[1090] Example 3: Synthesis of ASGPR receptor ligand and M6P receptor ligand

[1091] Synthesis of intermediates

[1092] Type AA:

[1093] Synthesis of benzyl 5-hydroxyvalerate (int-AA1)

[1094]

[1095] Step 1: At 0°C, DMAP (0.32 g, 2.62 mmol) and Et3N (29 mL, 210.5 mmol) were added 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). The reaction mixture was heated to room temperature and stirred for two days. The reaction mixture was evaporated to dryness and the resulting residue was dissolved in DCM (200 mL) and washed with 3M HCl (100 mL × 2). The organic layer was dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (elution: PE:EA = 20:1-10:1) to give 5-(benzyloxy)-5-oxovaleric acid as a colorless oil. 1 H NMR (400MHz, CDCl3) δppm 7.38-7.28(m,5H),5.12(s,2H),2.46-2.40(m,4H),2.00-1.93(m,2H).

[1096] Step 2: BH3-S(CH3)2 (20.2 mL, 202.7 mmol) was added dropwise to a solution of 5-(benzyloxy)-5-oxovalerate (30 g, 135.1 mmol) in THF (200 mL) under N2 protection at 0 °C. The mixture was heated to room temperature and stirred for 16 hours. TLC showed that the starting material was completely consumed. The reaction was carefully quenched with H2O (8 mL). The resulting mixture was filtered and the filtrate was concentrated. The residue was eluted by silica gel column chromatography (elution: PE:EA = 20:1-2:1) to give benzyl 5-hydroxyvalerate (int-AA1). 1 H NMR (400MHz, CDCl3) δppm 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).

[1097] Synthesis of 3-((6-azidohexyl)oxy)-2-hydroxy-3-((1-hydroxy-3-oxopropane-2-yl)oxy)propanal (int-AA2)

[1098]

[1099] Sodium periodate (420 mg, 0.197 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, No. 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 reversed-phase rapid chromatography to obtain 3-((6-azidohexyl)oxy)-2-hydroxy-3-((1-hydroxy-3-oxopropane-2-yl)oxy)propanal (int-AA2). Analytical Method 7: t r =0.80min, MS m / z 326.2[M+Na]+.

[1100] BB type:

[1101] Synthesis of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2,5-dioxopyrrolidone-1-yl)oxy)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (int-BB1)

[1102]

[1103] Step 1: Acetic anhydride (473 g, 4.64 mol) was added 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) at 0 °C. The reaction mixture was stirred at room temperature for 72 hours. The resulting precipitate was collected, washed with H2O (200 mL × 2), and dried under vacuum to give (3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-trimethyltriacetate. 1H NMR (400MHz, CDCl3) δppm 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).

[1104] Step 2: TMSOTF (85.5 g, 0.385 mol) was added to a solution of (3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-trimethyltriacetate (100 g, 0.257 mol) in 1,2-dichloroethane (500 mL) cooled to 0 °C. The mixture was stirred for 10 min, then heated to 50 °C and stirred for 3 h. TLC showed that the starting material was completely consumed. After cooling, the resulting mixture was treated with a saturated aqueous solution of NaHCO3 (1000 mL) at 0 °C and extracted with DCM (500 mL × 2). The combined organic layers were dried over Na2SO4 and concentrated. The residue was dried overnight under high vacuum to give (3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6,7-dimethyldiacetate. 1 H NMR (400MHz, CDCl3) δppm 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).

[1105] Step 3: (3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6,7-dimethyldiacetate (65 g, 197.4 mmol) and benzyl 5-hydroxyvalerate (int-AA1) (41 g, 197.4 mmol) were dissolved in DCM (600 mL). Molecular sieve (50 g) was added, the reaction was stirred for 30 min, and TMSOTF (6.5 g, 29.6 mmol) was added. The reaction mixture was then stirred overnight at room temperature. TLC showed that the starting material was completely consumed. The reaction mixture was filtered to remove the molecular sieve. The filtrate was treated with a saturated aqueous solution of NaHCO3 (500 mL) and extracted with DCM (500 mL × 2). The combined organic layers were dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (elution: PE:EA = 2:1-1:2) to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-(benzyloxy)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate. 1 H NMR (400MHz, CDCl3) δppm7.37-7.32(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).

[1106] Step 4: Dissolve (2R,3R,4R,5R,6R)-5-acetamamido-2-(acetoxymethyl)-6-((5-(benzyloxy)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetic acid ester (90 g, 167.4 mmol) in a mixture of EtOAc (250 mL) and MeOH (250 mL), then add wet Pd / C (4.5 g, 10%). Degas the reaction mixture and refill it with H2 using a balloon, then stir overnight. TLC showed that the starting material was completely consumed. Filter the reaction mixture and concentrate the filtrate to dryness to give 5-(((2R,3R,4R,5R,6R)-3-acetamamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid. 1H NMR (400MHz, CDCl3) δppm 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).

[1107] Step 5: Add DIC (19.4 g, 154.2 mmol) and DMAP (36 mg, 0.29 mmol) to a solution of 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)valerate (69 g, 154.2 mmol) and NHS-OH (19.5 g, 169.62 mmol) in DCM (600 mL). Stir the reaction mixture at room temperature for 3 hours. TLC showed that the starting material was completely consumed. Filter the resulting mixture and concentrate the filtrate. The residue was purified by silica gel column chromatography (elution: PE:EA = 2:1-1:4) to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2,5-dioxopyrrolidone-1-yl)oxy)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (int-BB1). 1 H NMR (400MHz, CDCl3) δppm 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).

[1108] Synthesis of (1S,2R,3R,4R,5S)-4-amino-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octane-2,3-diol (int-BB2)

[1109]

[1110] 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 min. The mixture was then cooled to 0 °C, and Ac₂O (1.12 L, 11.97 mol) was added dropwise while maintaining the temperature between 5 °C and 15 °C. The reaction mixture was further stirred at 20 °C under N₂ for 2 h, cooled to 0 °C, quenched with ice-water (1 L), and then extracted first with MTBE (3 × 1.2 L), followed by extraction with EA (2 × 1 L). The combined organic layers were washed with 0.5 N HCl (3 × 1 L), saturated NaHCO₃ (1 L), and then brine (1 L). The combined aqueous layers were then extracted with EtOAc (3 L), and the organic layers were washed with 0.5 N HCl (3 × 1 L), saturated NaHCO3 (1 L), and then brine (1 L). All organic layers were combined, dried over Na2SO4, filtered, and concentrated under vacuum at 35 °C to give (2R,3R,4R)-2-(acetoxymethyl)-3,4-dihydro-2H-pyran-3,4-dimethyldiacetate. 1 H NMR: (CDCl3400MHz)δ1.98-2.14(m,9H)4.17-4.33(m,3H)4.71(ddt,J=5.00,2.61,1.2 7,1.27Hz,1H)5.41(dd,J=4.34,1.65Hz,1H)5.51-5.57(m,1H)6.45(d,J=6.24Hz,1H).

[1111] Step 2: A solution of (2R,3R,4R)-2-(acetoxymethyl)-3,4-dihydro-2H-pyran-3,4-diyldiacetate (900 g, 3.31 mol, 1 equivalent) dissolved in MeCN (2 L) was added to MeCN (16 L) with stirring (300 rpm) under a flow of N2. The mixture was then cooled to -15 °C under N2. NaN3 (429.82 g, 6.61 mol, 2 equivalents) was added in portions to the reaction mixture while maintaining the temperature between -15 °C and -10 °C under a gentle flow of N2. Cerium ammonium nitrate (5.44 kg, 9.92 mol) was added in 6 portions over 3 hours with stirring (350 rpm) while maintaining the temperature between -15 °C and -10 °C under a gentle flow of N2. The reaction mixture was then stirred under N2 at a temperature between -15°C and -10°C for 4 hours, followed by the addition of MTBE (10 L) in two batches. H2O (10 L) was carefully added to the reaction mixture under N2 at a temperature between -5°C and 0°C, and the mixture was stirred at 0°C for 30 min, then allowed to stand at room temperature (25°C) for 16 hours. The mixture was separated, and the organic layer was washed with H2O (8 × 10 L), dried over Na2SO4, filtered, and concentrated under vacuum at 20–25°C to give (2R,3R,4R,5R,6R)-2-(acetoxymethyl)-5-azido-6-(nitrooxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate, which was used directly in the next step.

[1112] Step 3: Cool a solution of (2R,3R,4R,5R,6R)-2-(acetoxymethyl)-5-azido-6-(nitrooxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (1140 g, 3.03 mol) in MeOH (8 L) to 0 °C, then add NaOMe (1.1 M, 1.60 L) while maintaining the temperature between 0 °C and 5 °C. Stir the reaction mixture between 0 °C and 5 °C for 2 hours. Then add resin (H+) (500 g) and stir the reaction mixture further for 30 min. Filter the reaction mixture and wash the filter cake with MeOH (2 L). Grind with MeOH (2 L) at room temperature (25 °C) for 30 min and filter (repeat 4 times). All filtrates were combined and concentrated under vacuum at 35°C to obtain the residue, which was purified by silica gel column chromatography (DCM:MeOH = 50:1 to 30:1) to obtain (2R,3R,4R,5R)-5-azido-2-(hydroxymethyl)-6-methoxytetrahydro-2H-pyran-3,4-diol.

[1113] Step 4: At 25°C, (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). The mixture was cooled to 0°C and stirred for 30 min between 0°C and 5°C. Then, TMSCl (634.42 g, 5.84 mol, 741.14 mL, 4 equivalents) was added dropwise, and the resulting white suspension was stirred for 1 hour between 5°C and 10°C. The slurry was quenched with saturated NH4Cl (1.5 L), stirred for 10 min, allowed to stand for 5 min, and then separated. The DCM layer was washed with NH4Cl (1.5 L × 4) and H2O (1.5 L × 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 directly used in the next step.

[1114] Step 5: At a temperature between -10°C and -5°C under N2, K2CO3 (1.84 g, 13.31 mmol, 0.01 equivalent) dissolved in MeOH (160 mL) 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 (3.5 L), and the reaction mixture was stirred for 30 min. The reaction mixture was then acidified to pH 6 with AcOH (1.84 g), concentrated, and then EtOAc (6 L) was added. The mixture was washed with H2O (3L×2) and ...

Claims

1. Bifunctional compounds, selected from: ; ; ; ; ; ; ; ; ; ; ;and 。 2. Bifunctional compounds, selected from: ； ； ； ;and 。 3. A pharmaceutical composition comprising a bifunctional compound as described in claim 1 or 2 and one or more pharmaceutically acceptable carriers.

4. Use of the bifunctional compound of claim 1 in the preparation of a medicament for treating PCSK9-mediated diseases, wherein the PCSK9-mediated diseases are selected from hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, sitosterolemia, coronary heart disease, peripheral artery disease, sepsis, and xanthomas.

5. The use as described in claim 4, wherein the PCSK9-mediated disease is a peripheral vascular disease.

6. The use as claimed in claim 4, wherein the PCSK9-mediated disease is atherosclerosis.

7. The use as claimed in claim 4, wherein the PCSK9-mediated disease is arteriosclerosis.

8. The use as claimed in claim 4, wherein the PCSK9-mediated disease is vascular inflammation.

9. Use of the bifunctional compound of claim 2 in the preparation of a medicament for treating FHR3-mediated diseases, wherein the FHR3-mediated diseases are selected from nephropathy, age-related macular degeneration, atypical hemolytic uremic syndrome, and hepatocellular carcinoma (HCC).

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